Pregnane steroids from a gorgonian coral Subergorgia suberosa with anti-flu virus effects

Steroids 108 (2016) 99–104

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Pregnane steroids from a gorgonian coral Subergorgia suberosa with anti-flu virus effects Wei Cheng a, Jinwei Ren b, Qixi Huang a, Hailin Long a, Hongwei Jin a, Liangren Zhang a, Huagang Liu c, Leen van Ofwegen d, Wenhan Lin a,⇑ a

State Key Laboratory of Natural and Biomimetic Drugs, Institute of Ocean Research, Peking University, Beijing 100191, PR China State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, PR China Guangxi University of Chinese Medicine, Nanning 530001, PR China d National Museum of Natural History Naturalis, 2300 RA Leiden, The Netherlands b c

a r t i c l e

i n f o

Article history: Received 20 November 2015 Received in revised form 1 February 2016 Accepted 3 February 2016 Available online 4 February 2016 Keywords: Gorgonian coral Subergorgia suberosa Subergorgols T–X Structural elucidation Anti-virus effects

a b s t r a c t Five new pregnane-type steroids namely subergorgols T–X (1–5) and three known analogues (6–8) were isolated from a gorgonian coral Subergorgia suberosa. The structures of new compounds were determined on the basis of extensive spectroscopic (IR, MS, 1D and 2D NMR) data analyses, in association with photochemical transformation and ECD methods for the configurational assignment. Compounds 1–8 were evaluated for the inhibitory effects against H1N1 virus infected in MDCK cells, while subergorgols T–U and 1,2-dehydroprogesterone exerted potent inhibition against A/WSN/33 virus. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction Marine invertebrates are recognized to be the rich source of steroids with structural diversity [1–3]. Apart from steroid glycosides and sulfated polyoxide steroids obtained from starfishes [4–7], the steroids with unique scaffolds such as dimeric steroid (crellastatin A) [8], hemiketal steroid (cladiellin A) [9], 24-N-imidazolyl steroidal alkaloid (amaranzole A) [10], and steroid glycosides with an isopropyl side chain (sokodosides) [11] were generated by marine sponges and soft corals. In addition, gorgonian corals appear to be the prolific sources to produce steroids. For instance, the gorgonian coral Subergorgia suberosa has been investigated extensively, while a profile of sterol derivatives including 9,11-secosteroids [12–15], pregnane steroids [16,17], and polyhydroxy steroids were isolated [18,19]. It was noted that the structural variety of S. suberosa was related to their ecological locations. Among the abundance of marine sterols, pregnane steroids are a rare group in the marine environment. Although the ecological role of pregnane sterols derived from marine organisms is unclear, some analogues exhibiting cytotoxicity against tumor cell lines and antibacterial activity [20] conducted to be the potential leads for

⇑ Corresponding author. E-mail address: [email protected] (W. Lin). http://dx.doi.org/10.1016/j.steroids.2016.02.003 0039-128X/Ó 2016 Elsevier Inc. All rights reserved.

pharmaceutical usage. In the course of our discovery for bioactive metabolites derived from marine benthic organisms, a gorgonian coral S. suberosa was collected from the coral reef near Yongxin island of South China Sea. Analyses of the HPLC chromatographic spectrum [18,19] of the EtOAc extract in association with the NMR and MS features revealed that the EtOAc extract contained a number of components with unreported structures. In addition, the EtOAc extract exhibited the inhibitory effect against A/ WSN/33 virus. Thus, a separation protocol was designed for the isolation and purification of the bioactive compounds. Chromatographic separation of the EtOAc extract resulted in the isolation of eight pregnane sterols including five new compounds (Fig. 1). This paper reports the structural elucidation of the new compounds and their antiviral effects.

2. Experimental 2.1. General methods Optical rotations were measured on a Rudolph IV Autopol automatic polarimeter. IR spectra were recorded on a Thermo Nicolet Nexus 470 FT-IR spectrometer. 1H and 13C NMR, and 2D NMR spectra were recorded on Bruker Advance 400 NMR spectrometer (400 MHz for 1H and 100 MHz for 13C, respectively). Chemical

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W. Cheng et al. / Steroids 108 (2016) 99–104

1 O

3

21 20 O 18 17

11

H

9

10 H 5

19

6

18 11

R1 1 10

R2

14

H

8H

R3

4 R4

1 2. 3. 4. 5. 6. 7.

O

H

H H

H

6

R1 = R3 = R1 = R3 = R2 = R4 = R3 = R4 = R2 = R4 = R2 = R3 =

which was further partitioned between H2O and EtOAc to obtain an EtOAc extract (10.0 g). The EtOAc extract (2.0 g) was subjected to column chromatography (2.5  20 cm) using silica gel (160– 200 mesh, 50 g) with a gradient of petroleum ether PE/acetone (gradient from 20:1 to 1:1) to obtain seven fractions (FA–FG). FC (50 mg) was further separated on ODS column eluting with MeOH/H2O (7:3) as a mobile to yield compounds 3 (10.0 mg), 4 (2.0 mg), and 5 (2.0 mg). FD (100 mg) was chromatographed by semipreparetive HPLC (C18) using MeOH/H2O (82:18) as a mobile phase to yield compounds 2 (5.4 mg), 6 (24.60 mg), and 7 (3.5 mg). FE (619 mg) was purified on an ODS column eluting with MeOH/H2O (68:32) as a mobile phase to yield compounds 1 (27 mg) and 8 (582 mg).

O

17

O

H 8

H, R 2 = OH, R4 = Me H, R 2 = Me, R 4 = OH H, R 1 = OH, R3 = Me H, R 1 = Me, R 2 = OH H, R 1 = Me, R 3 = OH H, R 1 = OH, R4 = Me

Fig. 1. Structures of compounds 1–8.

shifts are expressed in d (ppm) referenced to the solvent peaks at dH 7.28 and dC 77.0 for CDCl3, and coupling constants are in Hz. HREIMS spectra were obtained from a Autospee Ultima-TFO spectrometer. Silica gels (160–200 and 200–300 mesh, Qingdao Marine Chemistry Co. Ltd.) and ODS (50 lm, YMC) were used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) were used for TLC analysis. Semi-preparative HPLC chromatography was performed on an Alltech instrument (426HPLC pump) equipped with an Alltech uvis-200 detector at 210 nm, and semi-preparative reversed-phase columns (YMCpacked C18, 5 lm, 250 mm  10 mm) were purchased from YMC Co. 2.2. Animal material The gorgonian coral S. suberosa was collected from the coral reef at a depth of around 8 m near Yongxin island of South China Sea, in May 2013. Samples were frozen immediately after collection. The specimen was identified by Leen van Ofwegen (National Museum of National History Naturalis). The voucher specimens (YXQ-07 (9)) are deposited at the State Key Laboratory of Natural and Biomimetic Drugs, Peking University, China.

2.3.1. Subergorgol T (1) White amorphous powder; [a]24 D +84.0 (c 0.10, CH2Cl2); UV (MeOH) kmax (log e) 283 (2.85) nm; IR (KBr) mmax 3428, 1700, 1659 cm 1; 1H and 13C NMR data, see Table 1; HREIMS m/z 312.2083 [M]+ (calcd for C21H28O2, 312.2089). 2.3.2. Subergorgol U (2) White amorphous powder; [a]24 D +37.6 (c 0.54, CH2Cl2); UV (MeOH) kmax (log e) 283 (3.47) nm; IR (KBr) mmax 3428, 1697, 1611 cm 1; 1H and 13C NMR data, see Table 1; HREIMS m/z 312.2087 [M]+ (calcd for C21H28O2, 312.2089). 2.3.3. Subergorgol V (3) White amorphous powder; [a]24 D + 51.9 (c 0.32, CH2Cl2); UV (MeOH) kmax (log e) 283 (3.19) nm; IR (KBr) mmax 3444, 1683 cm 1; 1H and 13C NMR data, see Table 1; HREIMS m/z 312.2088 [M]+ (calcd for C21H28O2, 312.2089).

2.3. Extraction and isolation

2.3.4. Subergorgol W (4) White amorphous powder; [a]24 D + 93.0 (c 0.20, CH2Cl2); UV (MeOH) kmax (log e) 283 (3.53) nm; IR (KBr) mmax 3444, 1683 cm 1; 1H and 13C NMR data, see Table 1; HREIMS m/z 312.2075 [M]+ (calcd for C21H28O2, 312.2089);

Gorgonian coral S. suberosa (2.3 kg) was homogenized and extracted with 95% EtOH (7 L 3). The concentrated extract (39.6 g) was desalted by dissolving in MeOH to obtain a residue,

2.3.5. Subergorgol X (5) White amorphous powder; [a]24 D +60.0 (c 0.20, CH2Cl2); UV (MeOH) kmax (log e) 283 (3.38) nm; IR (KBr) mmax 3399, 1690,

Table 1 H NMR data of compounds 1–5 (CDCl3, 400 MHz).a

1

Position dC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 a

154.7 126.6 186.6 128.6 163.3 34.8 30.4 42.3 56.2 52.5 21.9 38.4 45.1 56.6 24.8 23.3 62.8 13.6 19.5 209.3 31.4

d(ppm), J (Hz).

1 dH

dC

6.88 (d, 10.0) 6.23 (d, 10.0) 6.20 (s) 2.01 2.01 1.82 1.59

(m), 1.80 (m) (m), 1.39 (m) (m) (ddd, 4.5, 12.0, 12.0)

1.27 (m), 1.19 (m) 1.98 (m), 1.33 (m) 1.33 1.72 2.22 2.54 0.62 1.99

(m) (m), 1.36 (m) (m), 1.75 (m) (t, 9.6) (s) (s)

2.12 (s)

109.8 153.3 114.5 137.9 127.2 26.5 27.9 37.9 44.4 141.6 26.8 39.1 44.2 55.9 24.1 23.0 63.9 13.4 19.8 209.6 31.5

2 dH 6.71 (d, 1.5) 6.56 (d, 1.5)

2.72 (m), 2.57 (m) 1.99 (m), 1.36 (m) 1.40, m 2.28, m 2.32 (m), 1.55 (m) 2.19 (m), 1.65 (m) 1.38, m 1.82 (m), 1.36 (m) 2.24 (m), 1.75 (m) 2.63, t, (9.6) 0.67, s 2.19, s 2.18, s

dC 118.4 136.2 113.1 153.4 119.7 22.8 27.2 38.0 44.2 141.6 26.6 39.1 44.3 56.0 24.2 22.9 64.0 13.4 21.2 210.1 31.5

3 dH 6.76, s 6.51, s

2.82 (m), 2.59 (m) 2.01 (m), 1.38 (m) 1.41, m 2.28, m 2.38 (m), 1.56 (m) 2.18 (m), 1.66 (m) 1.40, m 1.83 (m), 1.38 (m) 2.25 (m), 1.74 (m) 2.64, t, (9.6) 0.68, s 2.29, s 2.18, s

dC 154.7 114.2 136.4 122.7 140.1 31.4 26.4 40.8 44.3 124.1 26.4 39.8 44.1 55.9 24.2 22.7 64.1 13.9 20.6 209.5 31.4

4 dH 6.36, s 6.54, s 2. 84 (m), 2.72 (m) 1.78 (m), 1.26 (m) 1.48, m 2.44, m 3.10 (m), 1.35 (m) 2.09 (m), 1.72 (m) 1.52, m 1.78 (m), 1.36 (m) 2.24 (m), 1.72 (m) 2.65, t, (9.6) 0.70, s 2.23, s 2.16, s

dC 122.6 152.4 112.5 127.4 130.6 31.7 26.1 41.5 47.1 139.9 28.4 39.8 45.2 56.2 24.0 22.9 63.9 14.3 14.7 209.5 31.4

5 dH

6.61,d (8.2) 6.85,d (8.2) 2.84 (m), 2.69 (m) 1.75 (m), 1.23 (m) 1.51, m 2.49, m 2.42 (m), 1.35 (m) 2.09 (m), 1.77 (m) 1.59, m 1.78 (m), 1.38 (m) 2.26 (m), 1.73 (m) 2.67, t, (9.6) 0.72, s 2.24, s 2.17, s

W. Cheng et al. / Steroids 108 (2016) 99–104

1585 cm 1; 1H and 13C NMR data, see Table 1; HREIMS m/z 312.2075 [M]+ (calcd for C21H28O2, 312.2089). 2.4. ECD calculation Conformational searches were carried out by means of the Powell methods using MMFF94s force field in the SYBYL-X software package. Geometry optimizations were calculated at TD-DFT/ B3LYP-6-31+G(d) level to select lowest energy conformers. Subsequently, the conformer were re-optimized using DFT at the B3LYP/6-31+G(d) level in gas phase by the GAUSSIAN 09 program. The B3LYP/6-31+G(d) harmonic vibrational frequencies were also calculated to confirm their stability. The energies, oscillator strengths, and rotational strengths (velocity) of the conformers were calculated using the TD-DFT methods at the B3LYP/6-31+G (d) level in vacuum. The ECD spectra were simulated by the Gaussian function according to the Boltzmann distribution theory and its relative Gibbs free energy (DG). The oretical ECD spectrum of (8S, 9S, 10R, 13R, 14S, 17S)-1 was obtained following the same procedure as that of (8R, 9R, 10S, 13S, 14R, 17R)-1. By comparison of the calculated ECD spectra with the experimental CD spectra of 1, the absolute configuration of 1 was resolved. 2.5. Photochemical conversion A solution of compound 8 (10.0 mg, 0.032 mmol) in 1.5 mL of anhydrous dioxane was irradiated with a 400w medium-pressure mercury lamp for 1.0 h. The solvent was removed in vacuo, while the residue was chromatographed by semipreparetive HPLC using MeOH/H2O (68:32) as a mobile phase to yield compounds 1 (2.0 mg, 20% yield) and 9 (4.0 mg, 40% yield). By the same protocol, compound 8 (10.0 mg) was irradiated under a 400w medium-pressure mercury lamp to extend the illumination time to 7.5 h, and then the solvent was removed in vacuo. Column chromatography (silica gel 200 mesh) eluting with PE–EtOAc (20:1) to obtain compounds 3 (6.0 mg, 60% yield) and 6 (1.3 mg, 13% yield). Compound 9: 1H NMR (CDCl3, 400 MHz) dH 7.25 (1H, d, J = 5.2 Hz, H-1), 5.88 (1H, d, J = 5.2 Hz, H-2), 2.56 (1H, t, J = 9.6 Hz, H-17), 2.34 (1H, dd, J = 14.4, 7.2 Hz, H-6a), 2.20 (1H, m, H-16a), 2.14 (3H, s, H3-21), 2.07 (1H, m, H-6b), 2.06 (1H, m, H-12a), 1.92 (1H, s, H-4), 1.87 (1H, m, H-11a), 1.72(1H, m, H-15a), 1.70(1H, m, H-16b), 1.59(1H, m, H-7a), 1.50(1H, m, H-12b), 1.33(1H, m, H11b), 1.26(1H, m, H-9), 1.25(3H, s, H3-19), 1.23(1H, m, H-15b), 1.21(1H, m, H-14), 1.05(1H, m, H-8), 0.97(1H, m, H-7b), 0.64 (3H, s, H3-18). 13C NMR (CDCl3, 100 MHz) dC 209.2 (C-20), 206.4 (C-3), 165.4 (C-1), 131.5 (C-2), 63.8 (C-17), 57.1 (C-5), 55.0 (C-14), 50.8 (C-9), 44.3 (C-13), 41.4 (C-10), 39.5 (C-4), 38.8 (C-12), 35.0 (C-8), 31.5 (C-21), 26.6 (C-7), 25.3 (C-11), 25.0 (C-6), 24.3 (C-15), 22.8 (C-16), 14.3 (C-19), 13.6 (C-18). HRESIMS m/z 313.2162 [M+H]+ (calcd for C21H29O2, 313.2168). 2.6. Antiviral assay Madin-Darby canine kidney (MDCK) cells (ATCC) were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 lg/mL). The influenza virus strains A/WSN/33(H1N1) were propagated in MDCK cells in the presence of 2 lg/ml trypsin TPCK treated from bovine pancreas (Sigma–Aldrich, Lot#031M7358V). After incubation at 37 °C for 2 days, the supernatant was centrifuged at 1000 rpm  3 min and the progeny virus was harvested and used for the infection. MDCK cells were seeded into 96-well plates at 1  105 cells per well 24 h prior to infection and incubated at 37 °C in 5% CO2. Compounds were mixed with virus and incubated at room temperature for 15 min. The original medium in 96-well plate was removed and the mixed culture con-

101

taining compound and virus was added to the cells. At 1.5 days post-infection, microscopy was performed to determine the antiviral activity, and the data were confirmed by CellTiter-Glo Luminescent Cell Viability Assay (Promega, Aat#G7570). In addition, the cytotoxicity of the compounds was determined in uninfected MDCK cells, which were incubated with indicated concentrations of compounds for 1.5 days. Then microscopy was performed to determine the cytopathic effect, and the data were confirmed by CellTiter-Glo Luminescent Cell Viability Assay (Promega, Aat#G7570).

3. Results and discussion Subergorgol T (1) had a molecular formula of C21H28O2 as determined by the HREIMS (m/z 312.2083 [M]+) and NMR data, requiring eight degrees of unsaturation. The IR absorptions at 1700 and 1659 cm 1 suggested the presence of a saturated and an unsaturated carbonyl groups, respectively. The 13C NMR and APT spectra displayed a total of 21 carbon resonances, which were classified to three methyl, six methylene, seven methine and five quaternary carbons. In addition, the NMR resonances for two carbonyl groups and four olefinic carbons bearing two double bonds were recognized. The 1H NMR spectrum exhibited three methyl singlets (dH 0.62, 1.99 and 2.12 ppm) and three olefinic protons at dH 6.88 (d, J = 10.0 Hz, H-1), 6.23 (d, J = 10.0 Hz, H-2) and 6.20 (s, H-4). The similar NMR data between 1 and pregna-1,4-diene-3,20-dione (8) [21], which was isolated from the same fraction, indicated 1 to be a C21-pregnane analogue. However, the HMBC correlation in ring A from the quaternary carbon C-10 (dC 52.5) to H-1, H-2, H4 and H3-19 (dC 19.5), and from C-3 (dC 186.6) to H-1, H-2 and H-4, assigned a 5-methylcyclohexa-1,4-diene-3-one resided at C10 to form a spirodienone ring A. The COSY relationships connected ring B from H2-6 (dH 2.01, 1.80) to H-9 (dH 1.59), while the HMBC interactions from H2-6 to C-9 (dC 56.2) and C-10 and from H-9 to C-6 (dC 34.8) and C-10, defined ring B to be a cyclopentane. In addition, the HMBC correlations from H-1 to C-9 and C-6 and from H-9 to C-1 (dC 154.7) and C-5 (dC 163.3) further connected rings A and B through C-10 as the spirocyclic center. The 2D NMR data (COSY, HMBC and HMQC) established the remaining rings C and D and their substitution to be the same as that of 8. Thus, the structure of 1 was determined to be a spiropregnane. The relative configuration was determined by NOESY data. The NOE observed between H-1/H-8, H-8/H3-18, H3-19/H-9, and H-9/ H-14 assigned the same orientation of H-8 and H3-18, whereas H-9 and H-14 were in opposite face toward H-8. These findings also assigned H3-19 of ring A to be spatially closed to H-9. Additional NOE interaction between H-14 and H-17 determined the b-orientation of the acetyl group (Fig. 2). The absolute configuration of the stereogenic centers was determined by the comparison of the experimental CD curve of 1 with the computed ECD spectra of the model molecules for the enantiomers of 1 (Fig. 3), revealing 8S, 9S, 10R, 13R, 14S, and 17S configurations. A conversion of 8 to generate 1 by the photochemical rearrangement (Fig. 4), further confirmed the configurational assignment. The molecular formula of subergorgol U (2) was established as C21H28O2 on the basis of the HREIMS (m/z 312.2087 [M]+) and NMR data. The IR absorption bands at 3428, 1697 and 1611 cm 1 suggested the presence of hydroxy, carbonyl and phenyl groups. The 1 H NMR spectrum of 2 displayed three methyl singlets (dH 0.67, 2.18, 2.19). In the 13C NMR (APT) spectrum, a total of 21 carbon resonances were observed. Comparison of the NMR data revealed the structures of 2 and 8 to be closely related. The distinction was attributed to ring A, in which an aromatic ring was recognized by the presence of six aromatic resonances ranging from 109.8 to 153.3 ppm in the 13C NMR spectrum. The presence of two aromatic

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W. Cheng et al. / Steroids 108 (2016) 99–104

Fig. 2. Key NOE interactions of 1 and 9.

homologue of 2, with the difference of the aromatic substitution. The meta-coupling aromatic protons at dH 6.76 (d, J = 1.5 Hz) and 6.51 (d, J = 1.5 Hz) were assigned to H-1 and H-3, based on the HMBC interactions from H-1 to C-3 (dC 113.1), C-5 (dC 119.7) and C-9 (dC 44.2) and H-3 to C-1 (dC 118.4), C-4 (dC 153.4) and C-5. Additional HMBC interactions from H3-19 (dH 2.29, s) to C-1, C-2 and C-3 confirmed the methyl substitution at C-2. Thus, C-4 was positioned by a hydroxyl group. Subergorgol W (4) had a molecular formula to be the same as that of 2 as determined by the HRESIMS and NMR data. Comparison of the NMR data (Table 1) revealed the structure of 4 to be closely related to 2 with the distinction of the aromatic substitution. The meta-coupling aromatic protons at dH 6.36 (d, J = 1.5 Hz) and 6.54 (d, J = 1.5 Hz) were assigned to H-2 and H-4, respectively, according to the HMBC correlations such as H-2 correlated to C-4 (dC 122.7), C-10 (dC 124.1) an C-1 (dC 154.7), and H-4 to C-2 (dC 114.2), C-10 and C-6 (dC 31.4). Additional HMBC relationships of the methyl protons H3-19 (dH 2.23, s) with C-2, C-3 and C-4 determined the methyl group to be positioned at C-3 (dC 136.4), thus C-1 (dC 154.7) was substituted by a hydroxyl group. The HRESIMS data provided the molecular formula of subergorgol X (5) to be the same as that of 2–4, while the NMR data of both 4 and 5 (Table 1) were comparable. These findings suggested both 5 and 4 to be homologues. The distinction was attributed to the NMR resonances at ring A, where the ortho-coupling of aromatic protons at dH 6.61 (d, J = 8.2 Hz, H-3) and 6.85 (d, J = 8.2 Hz, H-4) was observed in the COSY spectrum. The HMBC correlations from H-4 to C-2 (dC 152.4), C-6 (dC 31.7) and C-10 (dC 139.9), H-3

Fig. 3. CD curve of 1 and the computed ECD spectra of enantiomers of 1.

protons for a meta-coupling at dH 6.71 (d, J = 1.5 Hz, H-1) and 6.56 (d, J = 1.5 Hz, H-3) in the 1H NMR spectrum indicated the aromatic ring to be tetra-substituted. In the HMBC spectrum, the long range correlations of methyl protons at dH 2.19 (s) to C-3 (dC 114.5), C-4 (dC 137.9) and C-5 (dC 127.2), H-3 to C-1 (dC 109.8), C-2 (dC 153.3), C-5 and C-19 (dC 19.8), and H-1 to C-2, C-3, C-5 and C-9 (dC 44.4), confirmed 2-hydroxy and 4-methyl substitution at the aromatic ring A. The similar NOE correlations of both 2 and 8 indicated both compound sharing the same configurations. The HREIMS (m/z 312.2088 [M]+) and NMR data provided the molecular formula of subergorgol V (3) to be the same as that of 2. Analyses of 2D NMR data established the structure of 3 to be a

1

19

10

O 3

H H

1

H

H

5

O

8

hv

10

19

O

4

O

8a

10

H

5

H

10 5

O

H

19

6

1

H

1

H

10 5

H 6 19

O

H

O

2 10

5

O H H HO

H 6

H

1

2 1

2

H 1b

H

H

H

1

H

O H

H

H O

H 1d

Fig. 4. Photochemical transformation of 8 and 1.

H

2

O

H

H H

H 1e

HO

1a

2 10

H

O

H

O

1c

9

5

H 5

1

O

1

1

O O

H

10

H

5

O hv

hv O

10

O

O

8d

H

4

H

8b

H

8c

O

H

O

H

5H

1

H

O

O

H

H

hv

5

O

O

O

H OH

H 3

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to C-5 (dC 130.6), C-1 (dC 122.6) and C-2, in association with the HMBC correlation from H3-19 (dH 2.24, s) to C-1, C-2 and C-10, clarified the linkage of a methyl group at C-1 and a hydroxyl group at C-2. In order to establish the structural relationship among the isolated compounds, a protocol for the photochemical conversion starting from 8 was designed. Irradiation of compound 8 under a 400w medium-pressure mercury lamp for 1.0 h generated two products, of which one was identical to 1 on the basis of the comparison of their NMR data and specific rotation. The second product was determined to be an enone intermediate (9) according to the analyses of 2D NMR and MS data. The photochemical pathway for the generation of 9 was suggested through a lumiketone rearrangement [22,23], in which the formation of a dipolar intermediate 8a was considered to arise by electron excitation. This intermediate can undergo an internal C-1/C-5 bond formation to generate 8b, while further formation of C-4/C-10 bond and cleavage of C-1/C-10 bond in 8b yielded the product of 9 (Fig. 4). Photolysis of 8 under the same condition with the extension of time to 7.5 h derived two products, which were identical to 3 and 6, respectively (Fig. 4). Mechanistic study led to the postulation that a dipolar intermediate 8c as derived by electron excitation was undertaken a methyl migration from C-10 to C-5 to generate 8d, while methylene migration from C-5 to C-10 in 8d yielded a spirodienone 1. The photoconversion of spirodienone 1 to the analogues with aromatic ring A such as 2, 3 and 6 was processed via dipolar intermediate 1a, which can undergo the cleavage of C-5/C-10 bond to form a cyclopropane intermediate 1b. The latter was converted to 1c or 1d through the cleavage of C-1/C-2 bond [23]. Methine migration from C-10 to C-1 in 1c generated compound 6, while methylene migration from C-10 to C-2 yielded compound 3 (Fig. 4). It was noted that compound 1 was able to convert to 2 under UV detector (254 nm) during the purification of 1 by HPLC separation, indicating that compound 1 is sensitive to UV light. Therefore, the separation of 1 using other monitor instead of UV detector is recommended. The pathway for this conversion was postulated to be achieved by methine migration to C-1 in the dipolar intermediate 1e (Fig. 4). Thus, compound 1 was suggested to be the intermediate to derive 3 and 6 through additional Wagner– Meerwein reaction [24,25]. Compounds 6 and 7 (1-hydroxy-4-methyl-19-norpregna-1,3,5 (10)-trien-20-one) were previously reported to be derived from 1,2-dehydroprogesterone (8) by chemical conversion [26,27]. The fact that the catalysts used for chemical conversion in the literature were excluded from the separation process, compounds 6 and 7 were thus assumed to be generated from coral origin. Compounds 1–8 were evaluated for the inhibitory effects against influenza virus strain A/WSN/33 (H1N1) that was propagated in MDCK cells by cytopathic effect (CPE) reduction assay and CellTiter-Glo assay [28–30]. The CellTiter-Glo assay revealed that all tested compounds exhibited no cytotoxic activity against uninfected MDCK cells at concentration of 100 lM. The CPE reduction assay revealed that compounds 1, 2, 7, 8 exhibited potent inhibitory activities with IC50 values ranging from 35.64 to 50.95 lM, respectively, whereas compounds 3–6 were inactive (IC50 > 100 lM). In the parallel experiment, the positive control, oseltamivir, exerted the inhibitory effect with IC50 value of 46.5 lM (Table 2). In summary, present work uncovered a group of new pregnane sterols derived from the soft coral S. suberosa, while the pregnanes with an aromatic ring-A were rarely found from nature. Photolysis experiments revealed 1,2-dehydroprogesterone (8) to be the precursor to generate some of the aromatic pregnanes. Examination of the HPLC chromatographic spectrum of the EtOAc extract indicated the isolated compounds to be genesis of coral origin. The potent inhibitory effects of compounds 1, 2 and 8 against influenza virus strain A/WSN/33 (H1N1) without cytotoxicity suggested

Table 2 Inhibitory effects of 1–8 against Influenza virus stain A/WSN/33 (H1N1).a Comps 1 2 3 4 5 6 7 8 OSV-P

R1

R2

R3

R4

H H OH CH3 CH3 OH

OH CH3 H OH H H

H H CH3 H OH H

CH3 OH H H H CH3

IC50 (lM) 35.64 37.73 >100 >100 >100 >100 50.95 41.6 46.5

OSV-P: oseltamivir, positive control. a Measured in virus infected MDCK cells.

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