2017 Volume 65 Issue 6 Pages 593-597
Fifteen steroids, including two new compounds, leptosteroid (1) and 5,6β-epoxygorgosterol (2), were isolated and structurally elucidated from the Vietnamese soft coral Sinularia leptoclados. Their cytotoxic effect against a panel of eight human cancer cell lines was evaluated using sulforhodamine B (SRB) method. Significant cytotoxicity against hepatoma cancer (HepG2, IC50=21.13±0.70 µM) and colon adenocarcinoma (SW480, IC50=28.65±1.53 µM) cell lines were observed for 1 and against acute leukemia (HL-60, IC50=20.53±2.26 µM) and SW480 (IC50=26.61±1.59 µM) for ergost-5-en-3β,7β-diol (8). In addition, 3β,7β-dihydroxyergosta-5,24(28)-diene (13) showed significant cytotoxic activity on all tested cell lines with IC50 values ranging from 13.45±1.81 to 29.01±3.21 µM.
The phylum Cnidaria includes about 10000 species which are traditionally divided into three classes: Anthozoa, Hydrozoa, and Scyphozoa. The best-known representatives of the class Anthozoa are the gorgonians and soft corals among the subclass Octocorallia and the sea anemones and stony corals among the subclass Hexacorallia. Soft corals attracted considerable attention because of the wide range of their bioactive secondary metabolites. In these marine invertebrates, genus Sinularia (phylum Cnidaria, class Anthozoa, subclass Octocorallia, order Alcyonacea) is one of the most widely distributed soft corals and constitutes a dominant portion of the biomass in the tropical reef environment.1,2) Sinularia soft corals are a rich source of steroids and terpenoids.2–5)
As a part of our ongoing investigations on cytotoxic steroids from Vietnamese Sinularia soft corals,6–8) this paper addressed the isolation, structure elucidation, and cytotoxic evaluation of fifteen steroids, including two new compounds, leptosteroid (1) and 5,6β-epoxygorgosterol (2), from the soft coral S. leptoclados.
Using various chromatographic separations, fifteen steroids (Fig. 1) were isolated from a methanol extract of the soft coral S. leptoclados. The known compounds, 7-oxogorgosterol (3),6) crassumsterol (4),9) 7β-hydroxygorgosterol (5),10) sarcophytosterol (6),11) 3β-hydroxyergosta-5-ene-7-one (7),12) ergost-5-en-3β,7β-diol (8),13) ergost-5-en-3β,7α-diol (9),14,15) ergosta-5,24(28)-diene-3β-ol (10),16) 3β-hydroxyergosta-5,24(28)-diene-7-one (11),17,18) ergosta-5,22,24(28)-trien-3β-ol (12),19) 3β,7β-dihydroxyergosta-5,24(28)-diene (13),20) 3β,7α-dihydroxyergosta-5,24(28)-diene (14),20) and 3β,4α-dihydroxyergosta-5,24(28)-diene (15),7) were identified by detailed analysis of their one and two dimensional (1- and 2D)-NMR and MS data as well as comparison of them with those reported in the literatures.
Leptosteroid (1) was isolated as a white powder with molecular formula of C29H48O3, determined by high-resolution electrospray ionization (HR-ESI)-MS at m/z 445.36819 [M+H]+.The NMR features (Table 1) indicated a C29 steroid containing two oxymethines [δC 71.6 (C-3) and 73.2 (C-16)/δH 3.53 (1H, m, H-3) and 4.04 (1H, t, J=7.5 Hz, H-16)], one trisubstituted double bond [δC 140.7 (C, C-5) and 121.0 (CH, C-6)/δH 5.38 (1H, t, J=2.5 Hz, H-6)], one ketone [δC 218.0 (C-17)], three singlet-methyls [δC 17.6 (C-18), 19.4 (C-19), and 22.6 (C-21)/δH 0.96 (H-18), 1.00 (H-19), and 1.23 (H-21), each 3H, s], and four doublet-methyls [δC 21.0 (C-26), 21.5 (C-27), 11.5 (C-28), and 15.4 (C-29)/δH 0.85 (H-26), 0.87 (H-27), 0.73 (H-28), and 0.78 (H-29), each 3H, d, J=6.5 Hz]. The 1H–1H correlation spectroscopy (COSY) cross-peaks of H2-1/H2-2/H-3/H2-4 as well as heteronuclear multiple bond correlation (HMBC) cross-peaks of H-19 (δH 1.00) with C-1 (δC 36.9), C-5 (δC 140.7), C-9 (δC 49.4), and C-10 (δC 36.7) and H-6 (δH 5.38) with C-4 (δC 42.0) and C-10 (δC 36.7), indicated positions of the oxymethine at C-3 and double bond at C-5/C-6. Detailed analysis of other COSY and HMBC correlations (Fig. 2) clearly confirmed the planar structure of 1. The relative configurations of 1 were assigned by comparison of the 1H- and 13C-NMR data with the literature values and by nuclear Overhauser spectroscopy (NOESY). The signals of H-3 at δH 3.53 (1H, m) and C-3 at δC 71.6 are indicative for a common α-orientation of H-3.6) The oxymethine proton H-16 (δH 4.04) had NOESY correlations with the methyl protons H-18 (δH 0.96)/H-21 (δH 1.23) and no correlation with H-14 (δH 1.71) suggesting a β-orientation of H-16. The relative configurations for side chain of 1 were assigned to be identical to those of sarcophytosterol (6)11) by an agreement of their 1H- and 13C-NMR data and coexistence in S. leptoclados.
| Pos. | 1 | 2 | ||
|---|---|---|---|---|
| δC | δH (J in Hz) | δC | δH (J in Hz) | |
| 1 | 36.9 | 1.08 ddd (3.5, 13.0, 14.5)1.88 m | 37.2 | 1.25 m/1.96 m |
| 2 | 31.5 | 1.52 m/1.86 m | 31.1 | 1.40 m/1.80 m |
| 3 | 71.6 | 3.53 m | 69.5 | 3.70 m |
| 4 | 42.0 | 2.23 m2.33 ddd (2.0, 4.5, 12.5) | 42.3 | 1.42 m/2.03 m |
| 5 | 140.7 | — | 62.9 | — |
| 6 | 121.0 | 5.38 t (2.5) | 63.7 | 3.06 br s |
| 7 | 31.3 | 1.55 m/2.20 m | 32.6 | 1.20 m/2.08 m |
| 8 | 33.2 | 1.48 m | 29.9 | 1.48 m |
| 9 | 49.4 | 0.96 m | 51.4 | 0.61 m |
| 10 | 36.7 | — | 34.9 | — |
| 11 | 19.8 | 1.45 m/1.64 m | 22.1 | 1.38 m/1.41 m |
| 12 | 33.7 | 1.23 m2.06 dt (13.5, 3.5) | 39.9 | 1.08 m/1.98 m |
| 13 | 46.1 | — | 42.8 | — |
| 14 | 41.8 | 1.71 m | 56.1 | 0.87 m |
| 15 | 31.5 | 1.89 m | 24.4 | 1.06 m/1.60 m |
| 16 | 73.2 | 4.04 t (7.5) | 28.2 | 1.30 m/2.00 m |
| 17 | 218.0 | — | 58.0 | 1.18 m |
| 18 | 17.6 | 0.96 s | 11.8 | 0.62 s |
| 19 | 19.4 | 1.00 s | 17.0 | 1.00 s |
| 20 | 53.0 | — | 35.2 | 1.00 m |
| 21 | 22.6 | 1.23 s | 21.1 | 0.98 br s |
| 22 | 38.9 | 1.40 m/1.70 m | 32.1 | 0.15 m |
| 23 | 29.3 | 1.73 m | 25.8 | — |
| 24 | 45.9 | 0.88 m | 50.8 | 0.23 m |
| 25 | 31.0 | 1.41 m | 32.0 | 1.57 m |
| 26 | 21.0 | 0.85 d (6.5) | 21.3 | 0.85 d (6.5) |
| 27 | 21.5 | 0.87 d (6.5) | 22.2 | 0.95 d (6.5) |
| 28 | 11.5 | 0.73 d (6.5) | 15.4 | 0.93 d (6.5) |
| 29 | 15.4 | 0.78 d (6.5) | 14.3 | 0.89 s |
| 30 | 21.5 | β-0.13 t (4.0)α 0.44 dd (4.0, 8.5) | ||
Assignments were confirmed by HSQC, HMBC, COSY, and NOESY experiments.
) Correlations of 1 and 2The molecular formula of 5,6β-epoxygorgosterol (2) was determined as C30H50O2 by HR-ESI-MS with a quasi-molecular ion peak at m/z 443.38907 [M+H]+. The 1H-NMR spectrum revealed four high-field signals at δH 0.15 (1H, m, H-22), 0.23 (1H, m, H-24), −0.13 (1H, t, J=4.0 Hz, Hβ-30), and 0.44 (1H, dd, J=4.0, 8.5 Hz, Hα-30) confirming a gorgosterol derivative possessing a cyclopropane ring in side chain.6,21) In addition, signals of one oxymethine [δC 69.5 (C-3)/δH 3.70 (1H, m, H-3)], one epoxy [δC 62.9 (C, C-5) and 63.7 (CH, C-6)/δH 3.06 (1H, br s, H-6)], three singlet-methyl [δC 11.8 (C-18), 17.0 (C-19), and 14.3 (C-29)/δH 0.62 (H-18), 1.00 (H-19), and 0.89 (H-29), each 3H, s], and three doublet-methyl [δC 21.3 (C-26), 22.2 (C-27), and 15.4 (C-28)/δH 0.85 (H-26), 0.95 (H-27), and 0.93 (H-28), each 3H, d, J=6.5 Hz] groups were also observed in the 1H- and 13C-NMR spectra of 2. The fourth sec-methyl signal appeared as a broad singlet at δH 0.98, assigned to H-21 by heteronuclear single quantum coherence (HSQC), HMBC, and COSY experiments, is also characteristic of a gorgosterol-type side chain possessing a cyclopropane ring.21,22) Positions of the oxymethine group at C-3 and epoxy group at C-5/C-6 were identified by COSY cross-peaks of H2-1/H2-2/H-3/H2-4 as well as HMBC cross-peaks of H-19 (δH 1.00) with C-1 (δC 37.2), C-5 (δC 62.9), C-9 (δC 51.4), and C-10 (δC 34.9) and Hb-4 (δH 2.03) with C-5 (δC 62.9) and C-6 (δC 63.7). Detailed analysis of other COSY and HMBC correlations (Fig. 2) clearly confirmed the planar structure of 2. Comparison of the 13C-NMR chemical shifts at C-3 (δC 69.5), C-4 (δC 42.3), C-5 (δC 62.9), C-6 (δC 63.7), and C-7 (δC 32.6) of 2 with those of aragusterol L23) at δC 69.4 (C-3), 42.2 (C-4), 62.9 (C-5), 63.7 (C-6), and 32.6 (C-7) as well as 5,6α-epoxypetrosterol24) at δC 68.8 (C-3), 39.8 (C-4), 65.7 (C-5), 59.3 (C-6), and 28.8 (C-7), respectively, indicated that the oxymethine and epoxy groups are both in β-orientation. The relative configurations for side chain of 2 were assigned to be identical to those of 7-oxogorgosterol (3),6) crassumsterol (4),9) and 7β-hydroxygorgosterol (5),10) by agreement of their 1H- and 13C-NMR data and coexistence in S. leptoclados as well as a NOESY experiment (see Supplementary materials, Fig. S12).
The sulforhodamine B (SRB) method25) was used to evaluate cytotoxic activity of isolated compounds 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). As the obtained results (Table 2), leptosteroid (1) exhibited significant cytotoxic effect against HepG2 (IC50=21.13±0.70 µM) and SW480 (IC50=28.65±1.53 µM) cell lines and moderate effect on the remaining cell lines (IC50 values ranging from 34.95±4.21 to 64.12±1.71 µM), relative to that of the positive control, ellipticine (IC50 values ranging from 1.26±0.28 to 2.03±0.16 µM on all tested cell lines). Significant cytotoxicity against HL-60 (IC50=20.53±2.26 µM) and SW480 (IC50=26.61±1.59 µM) and moderate effect on the remaining cell lines (IC50 values ranging from 32.86±3.46 to 49.13±4.74 µM) were observed for ergost-5-en-3β,7β-diol (8) whereas 3β,7β-dihydroxyergosta-5,24(28)-diene (13) showed significant activity on all eight tested cell lines (IC50 values ranging from 13.45±1.81 to 29.01±3.21 µM). Ergosta-5,22,24(28)-trien-3β-ol (12) only showed weak cytotoxicity against HL-60 (IC50=92.96±8.29 µM), SW480 (IC50=83.84±3.72 µM), and LNCaP (IC50=89.80±3.34 µM) cell lines and compounds 2, 4, 5, 7, 9, and 10 showed less activity against all tested cell lines (IC50>100 µM). Previously, compounds 3, 6, 11, 14, and 15 were found to have weak or less cytotoxicity against the tested cancer cell lines.6,7)
| Comp. | IC50 values (µM) | |||||||
|---|---|---|---|---|---|---|---|---|
| KB | HepG2 | MCF7 | LU-1 | HL-60 | SW480 | SK-Mel2 | LNCaP | |
| 1 | 34.95±4.21 | 21.13±0.70 | 38.92±6.26 | 51.80±31.22 | 62.07±10.88 | 28.65±1.53 | 59.35±4.23 | 64.12±1.71 |
| 2 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 |
| 4 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 |
| 5 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 |
| 7 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 |
| 8 | 32.86±3.46 | 39.54±4.90 | 36.97±2.24 | 49.13±4.74 | 20.53±2.26 | 26.61±1.59 | 33.87±3.82 | 40.55±3.63 |
| 9 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 |
| 10 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 |
| 12 | >100 | >100 | >100 | >100 | 92.96±8.29 | 83.84±3.72 | >100 | 89.80±3.34 |
| 13 | 19.03±2.92 | 21.79±2.20 | 17.29±1.91 | 21.21±1.47 | 13.45±1.81 | 14.42±1.88 | 29.01±3.21 | 17.13±1.81 |
| Ellip.a) | 1.79±0.28 | 1.38±0.28 | 1.34±0.16 | 1.26±0.28 | 1.91±0.12 | 2.03±0.16 | 1.91±0.20 | 1.95±0.20 |
a) Ellipticine was used as positive control. Results are the mean±standard deviation (S.D.) of triplicate experiments.
Optical rotations were determined on a JASCO P-2000 polarimeter (Hachioji, Tokyo, Japan). 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 F254S plates (1.15685.0001, Merck), and compounds were visualized by spraying with aqueous 10% H2SO4 and heating for 3–5 min.
Biological MaterialsThe samples of S. leptoclados (Ehrenberg, 1834) were collected near the Con Co island, Quangtri, Vietnam, in May 2016, and identified by Prof. Do Cong Thung, Institute of Marine Environment and Resources, VAST. A voucher specimen (SLE052016) was deposited at the Institute of Marine Environment and Resources and Institute of Marine Biochemistry, VAST, Vietnam.
Extraction and IsolationDried bodies of the soft coral S. leptoclados (3.0 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 (M, 200.0 g), which was suspended in water and extracted in turn with n-hexane and CH2Cl2 resulting in extracts of n-hexane (H, 110.0 g), CH2Cl2 (D, 10.0 g), and water layer. Extract H (110.0 g) was crude separated on silica gel MPLC (column: Biotage® SNAP Cartridge, KP-SIL, 340 g) using the mobile phase of n-hexane–acetone (gradient 50 : 1→1 : 1, v/v) to obtain eight fractions, H1−H8. Fraction H3 (30.0 g) was further separated into eight subfractions, H3A–H3H, by silica gel MPLC (column: Biotage® SNAP Cartridge, KP-SIL, 340 g) using the mobile phase of n-hexane–EtOAc (gradient 40 : 1→1 : 1, v/v). Subfraction H3B (8.0 g) was separated by RP-18 MPLC (column: Biotage® SNAP Cartridge, KP-C18-HS, 120 g) using the mobile phase of increasing concentrations of MeOH in H2O (gradient 60→100%) to obtain thirteen smaller fractions, H3B1−H3B13. Fraction H3B13 (0.4 g) was further separated into six fractions, H3B13A−H3B13F, by silica gel CC (ϕ2.0 cm, L 80 cm) eluting with CH2Cl2−EtOAc (8 : 1, v/v, 0.7 L). Purification of the H3B13C fraction (60.0 mg) by YMC RP-18 CC (ϕ0.8 cm, L 80 cm) using eluent of acetone−H2O (4.5 : 1, v/v, 0.3 L), followed by silica gel CC with hexane−EtOAc (4 : 1, v/v, 0.5 L) to give compounds 10 (5.0 mg) and 12 (3.0 mg). Fraction H3B13F (20 mg) was purified by YMC CC (ϕ0.8 cm, L 80 cm) eluting with MeOH−H2O (5 : 1, v/v, 0.3 L) to give compounds 2 (1.0 mg) and 15 (2.5 mg). Subfraction H3E (6.0 g) was further separated into six smaller fractions, H3E1−H3E6, by RP-18 MPLC (column: Biotage® SNAP Cartridge, KP-C18-HS, 120 g) using the mobile phase of increasing concentrations of MeOH in H2O (gradient 60→100%). Fraction H3E6 (1.5 g) was separated by silica gel MPLC (column: Biotage® SNAP Cartridge, KP-SIL, 100 g) using mobile phase of CH2Cl2−EtOAc (4 : 1, v/v, 1.5 L) to obtain six fractions, H3E6A−H3E6F. Fraction H3E6C (101.0 mg) was purified by YMC CC (ϕ0.8 cm, L 80 cm) eluting with MeOH−H2O (5 : 1, v/v, 0.5 L) to give compounds 3 (13.0 mg), 7 (15.0 mg), and 11 (10.0 mg). Fraction H3E6E (300 mg) was separated on silica gel CC (ϕ2.0 cm, L 80 cm) eluting with n-hexane−acetone (6 : 1, v/v, 0.6 L) to obtain two fractions, H3E6E1A (180.0 mg) and H3E6E1B (100.0 mg). H3E6E1A (180.0 mg) was purified by YMC (ϕ0.8 cm, L 80 cm) using eluent of acetone−H2O (3 : 1, v/v, 0.5 L) to give compounds 4 (6.0 mg), 8 (2.5 mg), and 13 (7.0 mg). Compounds 5 (2.5 mg), 9 (1.2 mg), and 14 (3.5 mg) were obtained from fraction H3E6E1B (100.0 mg) after subjecting it on YMC CC (ϕ0.8 cm, L 80 cm) eluted with acetone−H2O (3 : 1, v/v, 0.5 L). Finally, subfraction H3G (0.5 g) was purified by YMC CC (ϕ2.0 cm, L 80 cm) eluting with acetone−H2O (3 : 1, v/v, 0.6 L), followed by silica gel CC (ϕ0.8 cm, L 80 cm) with n-hexane–EtOAc (3 : 1, v/v, 0.5 L) to furnish compounds 1 (2.5 mg) and 6 (4.5 mg).
Leptosteroid (1):White powder, [α]D25−25.5 (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 445.36819 [M+H]+ (Calcd for C29H49O3+, 445.36762).
5,6β-Epoxygorgosterol (2):White powder, [α]D25−6.5 (c=0.05, CHCl3); 1H-NMR (CDCl3, 500 MHz) and 13C-NMR (CDCl3, 125 MHz) are given in Table 1; HR-ESI-MS m/z 443.38907 [M+H]+ (Calcd for C30H51O2+, 443.38836).
Cytotoxic AssaysCytotoxic evaluations were performed by following the previously described protocols.26,27)
This study was financially supported by a Grant from Vietnam Academy of Science and Technology (VAST.TÐ.DLB.02/16-18) and VAST-FEB RAS in Akademik Oparine expedition (VAST.HTQT.NGA15-04/16-17). The authors are grateful to the Institute of Chemistry, VAST for the provision of the NMR spectroscopic instrument.
The authors declare no conflict of interest.
The online version of this article contains supplementary materials, 1D- and 2D-NMR spectra for the new compounds 1 and 2.
