Abstract
Two new highly acetylated steroids, named dysiroid A (1) and dysiroid B (2), together with a known compound (3) were isolated from the marine sponges Dysidea sp. The chemical structures of 1 and 2 were elucidated by spectroscopic analyses including 1D and 2D NMR experiments and high-resolution electrospray impact mass spectroscopy. Their in vitro antimicrobial activities against a panel of bacterial strains were evaluated. Compounds 1 and 2 showed potent activity against some of the strains with minimum inhibitory concentrations ranging from 4 to 8 μg mL−1.
1 Introduction
Sponges belonging to the genus Dysidea have been well recognized as a rich source of secondary metabolites endowed with a range of structural diversity and various biological activities. Members of this genus have been widely studied, leading to the identification of unusual sesquiterpene quinone [1], [2], [3], [4], diterpene [5], sesterterpene [6], and steroids [7, 89]. Some of these metabolites are of considerable interest and merit continuous attention due to their unique structures and significant biological activities, including antitumor [2], protein tyrosine phosphatase 1B inhibitory activity [3], and 2,2-diphenyl-1-picryhydrazyl radical scavenging activity [4]. As part of our efforts to discover structurally intriguing biologically significant metabolites from natural resources, we undertook the chemical analysis of Dysidea sp., which led to the isolation of two new highly acetylated steroids (1 and 2), together with a known compound (3). Herein, the details of the isolation, structure elucidation, and antibacterial activities of these compounds are described.
2 Results and discussion
Compound 1, a colorless powder, had the molecular formula C37H56O12, as established by high-resolution electrospray impact mass spectroscopy (HRESIMS) at m/z=691.3698 [M−H]− (calcd. 691.3694). The IR spectrum exhibited the absorption bands for carbonyl (1737 cm−1) and hydroxy (3420 cm−1) functionalities. The 1H NMR and heteronuclear single-quantum coherence (HSQC) spectra exhibited signals for six methyl singlets (δH=0.68, 2.00, 2.03, 2.05, 2.10, and 2.14 ppm) including five acetyl groups, three methyl doublets [δH=0.90 (d, J=5.9 Hz) and 0.85×2 ppm (d, J=6.6 Hz)], an oxygenated methylene [δH=4.61 (d, J=13.6 Hz) and 3.77 ppm (d, J=13.6 Hz)], four oxymethines (δH=5.31, 5.03, 4.67, 4.97 ppm), an olefinic proton [δH=5.48 ppm (dd, J=5.6, 2.0 Hz)], and a series of aliphatic methylene multiplets. The 13C NMR spectrum, in combination with distortionless enhancement by polarization transfer experiments, resolved 37 carbon resonances attributable to five carbonyl groups, a double bond (δC=119.0 and 142.8 ppm), nine methyls, eight sp3 methines (four oxygenated), nine sp3 methylenes (one oxygenated), and four sp3 quaternary carbons (two oxygenated). As six of the ten degrees of unsaturation were consumed by a double bond and five carbonyls, the remaining four degrees of unsaturation required that 1 was tetracyclic. The above-mentioned data were similar to those of 5α-cholest-7-ene-2α,3β,5α,6β,9α,11α,19-heptol 11,19-diacetate [9] except for the presence of three more acetyl groups. The five acetoxy groups were located at C-2, C-3, C-7, C-11, and C-19 by the heteronuclear multiple-bond correlations (HMBCs) of H-2, H-3, H-7, H-11, and H-19 to the carbonyls δC=170.7, 170.3, 169.9, 171.4, 171.4 ppm, respectively. Detailed 2D analyses [HSQC, 1H–1H correlation spectroscopy (COSY), and HMBC] further secured the planar structure of 1 (Fig. 1). The relative configuration of 1 was established by the analysis of the nuclear Overhauser effect spectroscopy (NOESY) correlations. The interactions of H-2/H-4β, H2-19, H-19/H-11, H-4β, H-11/H3-18, and H3-18/H-20 indicated that these protons were cofacial and were arbitrarily assigned β-oriented. The interactions of H-4α/H-7 and H-14/H-16α indicated that these protons were α-oriented (Fig. 2). Compound 1 was given the trivial name dysiroid A.
Compound 2, a colorless powder, had a molecular formula of C39H58O13 as determined by HRESIMS at m/z=351.1810 [M+H]+ (calcd. 351.1808). The 1H and 13C NMR spectra of 2 were similar to those of 1 except for the presence of one more acetyl group [δH=2.13 (s), δC=22.5 and 167.5 ppm], indicating that 2 was an acetylated derivative of 1. The additional acetoxy group was located at C-5 according to HMBC correlations of H-3 and H-4 to the downfield-shifted carbon at δC 86.5. The planar structure was confirmed by the further analysis of its 2D NMR data. The relative configuration of 2 was assigned to be the same as that of 1 by comparing their NMR data and the analysis of its NOESY spectrum. Finally, the chemical transformation of 1 to 2 by acetylation confirmed the structure of 2 as depicted in Fig. 3. Compound 2 was given the trivial name dysiroid B.
The known compound (5R)-3,15,27-triacontatriene-1,29-diyn-5-ol (3) was identified by comparison of its NMR data, MS data, and specific rotation with those in the literature [10].
The in vitro antimicrobial activities of compounds 1−3 against a panel of bacterial strains were evaluated. The results (Table 1) revealed that all three compounds possessed potent activity to some of the strains with minimum inhibitory concentrations (MICs) ranging from 4 to 8 μg mL−1.
Compounds | MIC (μg mL−1) | |||
---|---|---|---|---|
Staphylococcus aureus ATCC 29213 | Staphylococcus aureus ATCC 43300 | Enterococcus faecalis ATCC 29212 | Bacillus licheniformis ATCC 10716 | |
1 | 4 | 8 | 4 | 16 |
2 | 4 | 4 | 4 | 8 |
3 | 8 | 128 | 4 | 64 |
Penicillin | <0.5 | <0.5 | 1 | 64 |
3 Experimental section
3.1 General methods
Optical rotations were measured on a Rudolph Autopol I automatic polarimeter. IR spectra were determined on a Bruker Tensor 37 infrared spectrophotometer. NMR spectra were measured on a Bruker AM-400 spectrometer at 25°C. ESIMS was measured on a Finnigan LCQ Deca instrument, and HRESIMS was performed on a Waters Micromass Q-TOF instrument. Silica gel (300−400 mesh, Qingdao Haiyang Chemical Co., Ltd.), C18 reversed-phase silica gel (12 nm, S-50 μm, YMC Co., Ltd.), and Sephadex LH-20 gel (Amersham Biosciences) were for column chromatography.
3.2 Animal material
Specimens of the sponge Dysidea sp. were collected from Dongshan Island, Guangdong Province, PR China, in October 2013. The biological material was frozen immediately until used and was identified by associate Prof. Wei Wang from East China Sea Fisheries Research Institute. A voucher specimen (accession number: Dysid201310) has been deposited at the Jiangsu Xuyi People’s Hospital.
3.3 Extraction and isolation
The frozen sample (0.2 kg, wet weight) was chopped and exhaustively extracted with CH2Cl2–MeOH (1:1) (3×0.5 L) at room temperature. After removal of solvent in vacuo, the residue (3 g) was suspended in H2O (120 mL) and then partitioned sequentially with EtOAc (3×200 mL) and n-BuOH (3×200 mL). The separation of the EtOAc extract (0.8 g) by Sephadex LH-20 eluted with EtOH led to three fractions (fr. I–III). Fr. I (300 mg) was chromatographed over silica gel CC (PE-acetone, 20:1 → 3:1) to give three fractions (fr. Ia−Ie). Fr. Ib (64 mg) was purified on a semi-preparative reversed-phase high-performance liquid chromatography system equipped with a YMC column (MeCN–H2O, 95:5, 2 mL min−1) to give 1 (6 mg, tR=12.7 min) and 2 (2 mg, tR=15.5 min). Fr. Ia (38 mg) was purified on a silica gel (CH2Cl2, 2 mL min−1) to afford 3 (5 mg).
3.4 Dysiroid A (1)
Colorless powder.−
No. | 1 | 2 | ||
---|---|---|---|---|
δH (J in Hz) | δC | δH (J in Hz) | δC | |
1 | 1.98 m | 35.1 | 3.23 m 2.05 m | 29.4 |
2 | 5.31 dt (10.3, 6.7) | 70.2 | 4.99 m | 68.8 |
3 | 4.67 | 71.9 | 4.66 m | 71.5 |
4 | 2.65 dd (12.9, 4.5) 2.14 m | 27.3 | 2.85 dd (13.6, 4.4) 2.14 m | 27.7 |
5 | 74.6 | 86.5 | ||
6 | 4.97 dd (5.5, 1.9) | 71.4 | 6.03 dd (5.7, 1.8) | 64.6 |
7 | 5.48 dd (5.6, 2.0) | 119.0 | 5.48 (5.8, 2.2) | 117.2 |
8 | 142.8 | 144.7 | ||
9 | 75.5 | 75.1 | ||
10 | 47.1 | 48.8 | ||
11 | 5.03 dd (11.3, 4.8) | 74.6 | 4.97 m | 74.7 |
12 | 2.33 dd (12.0, 4.8) 1.59 m | 40.5 | 2.32 m 1.69 m | 40.3 |
13 | 42.3 | 42.5 | ||
14 | 2.57 m | 50.6 | 2.64 m | 50.5 |
15 | 1.58 m 1.40 m | 22.8 | 1.54 m 1.41 m | 22.8 |
16 | 1.94 m 1.33 m | 27.7 | 1.95 m 1.35 m | 27.7 |
17 | 1.33 m | 56.0 | 1.36 m | 55.9 |
18 | 0.68 s | 12.4 | 0.68 s | 12.4 |
19 | 4.61 d (13.6) 3.77 d (13.6) | 62.9 | 4.69 d (13.8) 3.75 d (13.8) | 62.5 |
20 | 1.33 m | 35.9 | 1.35 m | 36.0 |
21 | 0.90 d | 18.7 | 0.91 s | 18.7 |
22 | 1.33 m 0.97 m | 35.8 | 1.35 m 1.00 m | 35.9 |
23 | 1.31 m 1.12 m | 23.8 | 1.33 m 1.14 m | 23.8 |
24 | 1.11 m | 39.4 | 1.11 m | 39.5 |
25 | 1.50 m | 28.0 | 1.51 m | 28.0 |
26 | 0.85 d (6.8) | 22.8 | 0.86 d (6.8) | 22.8 |
27 | 0.85 d (6.8) | 22.5 | 0.86 d (6.8) | 22.5 |
2-OAc | 2.03 s | 21.1 | 2.03 s | 21.0 |
170.7 | 169.8 | |||
3-OAc | 2.00 s | 21.0 | 2.00 s | 20.9 |
170.3 | 170.2 | |||
5-OAc | 2.13 s | 22.5 | ||
167.5 | ||||
6-OAc | 2.10 s | 21.2 | 2.11 s | 21.0 |
169.9 | 169.1 | |||
11-OAc | 2.06 s | 21.7 | 2.09 s | 21.9 |
171.4 | 169.7 | |||
19-OAc | 2.14 s | 20.7 | 2.16 s | 20.7 |
171.4 | 171.4 |
3.5 Dysiroid B (2)
Colorless powder. −
3.6 Chemical transformation of 1 to 2
Acetic anhydride (50 μL) was added to a stirred solution of compound 1 (1 mg) in pyridine (0.5 mL). The mixture was stirred at room temperature for 2 h and then evaporated under vacuum. The residue was purified by Sephadex LH-20 eluted with CH2Cl2–MeOH (1:1) to afford 2 (0.6 mg), which was identified by 1H NMR spectroscopy and
3.7 Antibacterial testing
The strains used in antimicrobial tests were obtained from ATCC. Enterococcus faecalis was cultured in brain heart infusion medium (Oxoid, Thermo Fisher Scientific Inc.), other bacterial strains were grown on Mueller–Hinton (MH, Oxoid) medium. The MIC was determined using the microtiter dilution method according to Clinical and Laboratory Standards Institute guidelines. For all experiments, the compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1280 μg mL−1. The cultures were inoculated with 5×105 cfu mL−1 (cfu, colony-forming units) in a final volume of 200 μL in 96-well tissue culture-treated microtiter plates (Greiner Bio-One, CELLSTAR) and incubated at 37°C. Each microtiter plate contained a positive control (Penicillin), a negative control (bacteria grown in the presence of DMSO), and a contamination control (medium only). The bacterial growth was determined turbidimetrically (OD600) using a BIO-RAD iMark microplate Reader. The MIC value was obtained after 24 h for E. faecalis and 18 h for the other pathogens. The resulting values were compared with the values for a positive control under the same conditions.
4 Supplementary information
1D NMR and 2D NMR data of Dysidea A (1) and Dysidea B (2) are given as Supplementary Information available online (DOI: 10.1515/znb-2016-0156).
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Supplemental Material:
The online version of this article (DOI: 10.1515/znb-2016-0156) offers supplementary material, available to authorized users.
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