2016 Volume 64 Issue 10 Pages 1519-1522
A new 10-demethylated steroid, nephtheasteroid A (1), a new 19-oxygenated steroid, nephtheasteroid B (2) as well as five known steroids 3–7 were isolated from the organic extract of a Taiwanese soft coral Nephthea erecta. The structure was determined by means of IR, MS, and NMR techniques. Among these metabolites, 1 is rarely found in steroids possessing a 19-norergostane skeleton. In vitro cytotoxicity study using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that compounds 3 and 4 exhibited cytotoxicity against human chronic myelogenous leukemia (K562), human acute lymphoblastic leukemia (Molt-4), human T lymphoblastoid (Sup-T1), and human leukemic monocyte lymphoma (U937), with IC50 of 6.5–14.0 µM.
Steroids are some of the most frequently encountered natural products in soft corals of the genus Nephthea.1–19) Of particular interest, steroids have been reported to show diverse biological properties, including antitumor,1–4,19) anti-inflammatory,7,9) and antifouling.2) Furthermore, in our previous research on natural products, we have discovered series of steroids from the soft coral Nephthea chabrolii, and some of these have been found to possess cytotoxic activity.10,14) Our recent study on the bioactive matabolites derived from the soft coral Nephthea erecta, resulted in the isolation of a new 10-demethylated steroid, nephtheasteroid A (1), a new 19-oxygenated steroid, nephtheasteroid B (2), as well as five known metabolites, 3–7. The structures of the new compounds were determined using one dimensional (1D)- and two dimensional (2D)-NMR analyses and comparison of the NMR data with those of known analogues. Steroids 1–7 were tested for cytotoxic activity to inhibit the growth of four cancer cell lines (K562, Molt-4, Sup-T1, U937). The structural determination and cytotoxic activities, in addition to the structure–activity relationships (SAR), of the isolates are reported herein.
The frozen bodies of Nephthea erecta were minced and extracted exhaustively with ethyl acetate (EtOAc). The EtOAc extract was filtered and concentrated under reduced pressure. The crude extract was fractionated by silica gel column chromatography and resolved fractions were further purified by normal and reverse phase HPLC to yield compounds 1–7. The new natural products were given the trivial names nephtheasteroid A (1) and B (2). The known steroids were identified as erectasteroid F (3),12) (3β,7β)-ergost-5,24(28)-diene-3β,7β,19-triol 7,19-diacetate (4),20) 24-methyl-cholesta-5,24(28)-diene-3β,19-diol-7β-monoacetate (5),19) armatinol B (6),15) and ergosta-5,24(28)-dien-3β-ol (7).21)
The high resolution-electrospray ionization (HR-ESI)-MS of 1 revealed a pseudomolecular ion peak at m/z 383.3311 [M+H]+, corresponding to the molecular formula C27H42O (calcd for C27H43O m/z 383.3308). IR absorptions showed the presence of hydroxy group (3342 cm−1). The 1H-NMR spectrum (Table 1) revealed four methyl signals [δ 1.03 (d, J=7.0 Hz), 1.02 (d, J=7.0 Hz), 0.97 (d, J=6.5 Hz), and 0.69 (s)], one oxymethine signal [δ 4.06 (m)], two olefinic methine protons signal [δ 5.56 (d, J=4.5 Hz) and 5.36 (s)], and two olefinic methylene protons signal (δ 4.72 and 4.66, each s). In the 13C-NMR spectrum (Table 1), compound 1 showed 27 carbon resonances, with multiplicities determined by distortionless enhancement by polarization transfer (DEPT) 90 and 135 experiments. The olefinic carbon signals appearing at δC 138.2 (C), 130.1 (C), 126.0 (CH), and 115.2 (CH) corresponded to two trisubstituted double bonds. Moreover, one 1,1-disubstituted carbon double bond was also identified from NMR signals appearing at δ 105.9, (CH2) and 156.9, (C). The resonance appearing at δ 66.0 (CH) confirmed the presence of one oxymethine carbon. On the basis of the above results and by the assistance of 1H–1H correlation spectroscopy (COSY) and heteronuclear multiple bond connectivity (HMBC) experiments (Fig. 1), the molecular framework of 1 could be established. As the new compound 1 was isolated together with 3–7 from the same species and possesses a similar molecular skeleton, it was proposed that the six compounds are synthesized through a common biosynthetic pathway and thus have the same absolute configurations at C-3, C-8, C-9, C-13, C-14, C-17, and C-20. Moreover, the very similar NMR data observed for protons within ring D and side chain in compounds 1–7 reflected the α orientation of the methyl at C-20 in 1. Furthermore, the configuration at C-3 was determined by comparison of the NMR data of A ring of 1 with those of two related synthetic compounds, 19-norcholesta-l(10),5-dien-3β-ol and 3β-hydroxy-19-norandrosta-l(10),5-dien-17-one.22) On the basis of above analysis, the structure of 1 was established as (3S)-19-norergost-1(10),5,24(28)-trien-3β-ol.
| Position | 1 | 2 | ||
|---|---|---|---|---|
| δH (J in Hz)a) | δC (mult.)b) | δH (J in Hz)a) | δC (mult.)b) | |
| 1 | 5.36 s | 115.2 (CH) | 2.08 m; 1.10 m | 33.7 (CH2) |
| 2 | 2.46 m; 2.25 m | 34.8 (CH2) | 1.87 m; 1.42 m | 31.8 (CH2) |
| 3 | 4.06 m | 66.0 (CH) | 3.56 m | 71.5 (CH) |
| 4 | 2.50 d (15.0); 2.33 dd (15.0, 7.0) | 39.1 (CH2) | 2.26 m; 2.36 m | 42.4 (CH2) |
| 5 | 138.2 (C) | 135.5 (C) | ||
| 6 | 5.56 d (4.5) | 126.0 (CH) | 5.60 d (5.0) | 125.9 (CH) |
| 7 | 2.16 m; 1.75 m | 31.7 (CH2) | 2.03 m; 1.52 m | 31.3 (CH2) |
| 8 | 1.40 m | 38.2 (CH) | 1.70 m | 32.9 (CH) |
| 9 | 1.76 m | 42.5 (CH) | 0.95 m | 50.2 (CH) |
| 10 | 130.1 (C) | 39.5 (C) | ||
| 11 | 1.86 m; 1.47 m | 24.5 (CH2) | 1.60 m; 1.50 m | 21.7 (CH2) |
| 12 | 2.07 m; 1.27 m | 39.4 (CH2) | 2.07 m; 1.27 m | 39.9 (CH2) |
| 13 | 42.6 (C) | 42.5 (C) | ||
| 14 | 1.14 m | 56.4 (CH) | 0.94 m | 57.3 (CH) |
| 15 | 1.88 m; 1.30 m | 28.2 (CH2) | 1.86 m; 1.28 m | 28.2 (CH2) |
| 16 | 1.60 m; 1.10 m | 23.8 (CH2) | 1.60 m; 1.10 m | 24.2 (CH2) |
| 17 | 1.16 m | 56.0 (CH) | 1.14 m | 55.9 (CH) |
| 18 | 0.69 s | 11.9 (CH3) | 0.70 s | 11.9 (CH3) |
| 19 | 4.45 d (12.0); | 64.7 (CH2) | ||
| 3.98 d (12.0) | ||||
| 20 | 1.43 m | 35.7 (CH) | 1.42 m | 35.7 (CH) |
| 21 | 0.97 d (6.5) | 18.7 (CH3) | 0.95 d (7.0) | 18.7 (CH3) |
| 22 | 1.54 m; 1.16 m | 34.6 (CH2) | 1.55 m; 1.14 m | 34.7 (CH2) |
| 23 | 2.11 m; 1.90 m | 30.9 (CH2) | 2.10 m; 1.88 m | 31.0 (CH2) |
| 24 | 156.9 (C) | 156.9 (C) | ||
| 25 | 2.25 m | 33.8 (CH) | 2.24 m | 33.8 (CH) |
| 26 | 1.02 d (7.0) | 21.9 (CH3) | 1.02 d (7.0) | 21.9 (CH3) |
| 27 | 1.03 d (7.0) | 22.0 (CH3) | 1.03 d (7.0) | 22.0 (CH3) |
| 28 | 4.72 s; 4.66 s | 105.9 (CH2) | 4.72 s; 4.66 s | 105.9 (CH2) |
| OAc | 2.05 s | 21.1 (CH3) | ||
| 170.8 (C) | ||||
a) 500 MHz in CDCl3. b) 125 MHz in CDCl3.
Nephtheasteroid B (2) was found to have the [M+Na]+ ion at m/z 479 in its ESI-MS. The molecular formula was assigned as C30H48O3 from HR-ESI-MS and NMR data (Table 1). The 1H- and 13C-NMR spectral data of A–D rings and side chain in 2 were nearly identical with those of 7 except for the replacement of a methyl substitution at C-10 in 7 by an acetoxymethyl group [δH 4.45 (d, J=12.0 Hz), 3.98 (d, J=12.0 Hz); δC 64.7 (CH2)] in 2. This was further confirmed by the HMBC correlations from both H2-19 and the acetoxyl methyl to the ester carbonyl carbon appeared at δC 170.8 (C). Thus, the structure of steroid 2 was established as (3S)-ergost-5,24(28)-diene-3β-ol-19-acetate.
To the best of our knowledge, the C10 demethylation of steroids in marine natural products is rarely found previously.3,6) A possible biosynthetic pathway of 1 was proposed as illustrated in Fig. 2. Nephtheasteroid A (1) might be derived from a normal steroid, ergosta-5,24(28)-dien-3β-ol (7) through the intermediates A and B. This pathway involves oxidation, decarboxylation and dehydration to produce 1. The cytotoxicity of compounds 1–7 against the proliferation of a limited panel of cancer cell lines, including human chronic myelogenous leukemia (K562), human acute lymphoblastic leukemia (Molt-4), human T lymphoblastoid (Sup-T1), and human leukemic monocyte lymphoma (U937) carcinoma cell lines was evaluated. The results presented in Table 2 show that compounds 3–5 were found to exhibit cytotoxicity against all or part of the above carcinoma cell lines, while compound 4 (IC50 values of 7.9, 7.3 and 6.8 µM against Molt-4, Sup-T1, and U937 cancer cell lines, respectively) was the most potent one. Furthermore, compound 3 exhibited significant cytotoxic activity against K562, Molt-4, and Sup-T1 cancer cell lines (IC50 values of 6.5, 8.0, and 8.0 µM). Preliminary SAR in oxygenated steroid may be deduced from cytotoxicity bioassays. In the bioassays, the presence of the acetoxy or hydroxy functional group at C-7 is critical for the cytotoxicity action of this class of compounds.
| Compound | K562 | Molt-4 | Sup-T1 | U937 |
|---|---|---|---|---|
| 1 | >20 | >20 | >20 | >20 |
| 2 | >20 | >20 | >20 | >20 |
| 3 | 6.5 | 8.0 | 8.0 | 12.9 |
| 4 | 14.0 | 7.9 | 7.3 | 6.8 |
| 5 | 11.2 | 19.9 | 16.3 | >20 |
| 6 | >20 | >20 | >20 | >20 |
| 7 | >20 | >20 | >20 | >20 |
| Doxorubicin | 0.20 | 0.02 | 0.042 | 0.42 |
Infrared (IR) spectra were obtained on a Fourier-transform (FT) IR spectrophotometer (Varian Digilab FTS 1000). Ultraviolet spectra were recorded on a JASCO V-650 spectrophotometer. 1H- and 13C-NMR spectra were recorded on a Varian Unity INOVA 500 FT-NMR at 500 and 125 MHz, respectively. Optical rotations were determined by a digital polarimeter (Jasco P-1010). ESI-MS analyses were performed on an APEX II Instrument (Bruker). Gravity column chromatography was performed with 230–400 mesh silica gel (Merck). TLC analyses were conducted on 0.2-mm-thick pre-coated Kieselgel 60 F254 plates (Merck), and visualization of TLC spots was carried out by spraying the plate with 10% aqueous H2SO4 solution followed by heating. High-performance liquid chromatography (HPLC) was performed using a system consisting of a Hitachi L-7100 pump and a Rheodyne 7725 injection port. A preparative normal phase column (ϕ 21.2 mm×25 cm, silica gel 60, 5 µm) and a Supelco C18 column (ϕ 21.2 mm×25 cm, 5 µm) were used for HPLC.
Animal MaterialSoft coral Nephthea erecta (specimen no. 20150603-1) was collected by scuba divers at a depth of around 10 m off the coast of Pingtung, southern Taiwan in June 2015. A voucher specimen was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.
Extraction and SeparationThe frozen bodies of N. erecta material (1.6 kg, wet wt) was freeze-dried, and the resulting material (280 g) was then minced and extracted exhaustively with EtOAc (2 L ×5). The EtOAc crude extract was evaporated under reduced pressure to afford a residue (20.5 g), and the residue was subjected to column chromatography on silica gel, using n-hexane, an n-hexane and EtOAc mixture of increasing polarity, and finally acetone to yield 15 fractions: Fr-1 (eluted by n-hexane), Fr-2 (eluted by n-hexane–EtOAc, 100 : 1), Fr-3 (eluted by n-hexane–EtOAc, 50 : 1), Fr-4 (eluted by n-hexane–EtOAc, 25 : 1), Fr-5 (eluted by n-hexane–EtOAc, 15 : 1), Fr-6 (eluted by n-hexane–EtOAc, 10 : 1), Fr-7 (eluted by n-hexane–EtOAc, 8 : 1), Fr-8 (eluted by n-hexane–EtOAc, 5 : 1), Fr-9 (eluted by n-hexane–EtOAc, 3 : 1), Fr-10 (eluted by n-hexane–EtOAc, 2 : 1), Fr-11 (eluted by n-hexane–EtOAc, 3 : 2), Fr-12 (eluted by n-hexane–EtOAc, 1 : 1), Fr-13 (eluted by n-hexane–EtOAc, 1 : 2), Fr-14 (eluted by EtOAc), and Fr-15 (eluted by acetone). Fraction 8 was further purified with silica gel (n-hexane–acetone=8 : 1) to afford nine subfractions (8A−I). Subfraction 8C was then separated by normal-phase HPLC (n-hexane–acetone=8 : 1) to obtain 1 (2.1 mg) and 7 (500 mg). Fraction 9 was further purified with silica gel (n-hexane–EtOAc–acetone=8 : 1 : 1) to afford eight subfractions (9A−9I). Subfraction 9I was then separated by reversed phase HPLC (pure methanol) to obtain 2 (3.0 mg). Fraction 10 was separated by normal-phase HPLC with gradient elution (n-hexane–acetone=3 : 1 to 2 : 1) to yield eight subfractions (10A–H). Subfraction 10H was separated by normal-phase HPLC (n-hexane–acetone=4 : 1) to afford 4 (6.5 mg). Fraction 11 was purified by normal-phase HPLC (n-hexane–EtOAc=2 : 1) to afford 5 (15.6 mg) and 3 (16.7 mg). Compound 6 (5.4 mg) was obtained from fraction 12 using normal-phase HPLC (n-hexane–EtOAc=3 : 2).
Nephtheasteroid A (1): white solid; [α]D25 +58.8 (c 0.18, CHCl3); UV (MeOH) λmax (log ε) 240 (3.96) nm; IR (neat) vmax 3342, 2957, 2870, 1641, 1458, 1380 cm−1; 13C- and 1H-NMR data (125, 500 MHz; CDCl3), see Table 1; ESI-MS m/z 383 [M+H]+; HR-ESI-MS m/z 383.3311 [M+H]+ (calcd for C27H43O, 383.3308).
Nephtheasteroid B (2): white solid; [α]D25 −22.8 (c 0.45, CHCl3); IR (neat) vmax 3390, 2932, 2867, 1742, 1469, 1380 cm−1; 13C- and 1H-NMR data (125 and 500 MHz; CDCl3), see Table 1; ESI-MS m/z 479 [M+Na]+; HR-ESI-MS m/z 479.3493 [M+Na]+ (calcd for C30H48O3Na, 479.3495).
Cytotoxicity TestingThe 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was performed as described previously with some modifications.23) Cytotoxicity assays of compounds 1–7 were conducted against K562, Molt-4, Sup-T1, and U937 carcinoma cell lines using a MTT colorimetric method.
This research was supported by Grants from the National Museum of Marine Biology & Aquarium and the Ministry of Science and Technology (MOST 103-2320-B-291-001-MY3), Taiwan, awarded to J.-H. Su.
The authors declare no conflict of interest.
The online version of this article contains supplementary materials.
