Butenolides from the Coral-Derived Fungus Aspergillius terreus SCSIO41404

Five undescribed butenolides including two pairs of enantiomers, (+)-asperteretal G (1a), (−)-asperteretal G (1b), (+)-asperteretal H (2a), (−)-asperteretal H (2b), asperteretal I (3), and para-hydroxybenzaldehyde derivative, (S)-3-(2,3-dihydroxy-3-methylbutyl)-4-hydroxybenzaldehyde (14), were isolated together with ten previously reported butenolides 4–13, from the coral-derived fungus Aspergillus terreus SCSIO41404. Enantiomers 1a/1b and 2a/2b were successfully purified by high performance liquid chromatography (HPLC) using a chiral column, and the enantiomers 1a and 1b were new natural products. Structures of the unreported compounds, including the absolute configurations, were elucidated by NMR and MS data, optical rotation, experimental and calculated electronic circular dichroism, induced circular dichroism, and X-ray crystal data. The isolated butenolides were evaluated for antibacterial, cytotoxic, and enzyme inhibitory activities. Compounds 7 and 12 displayed weak antibacterial activity, against Enterococcus faecalis (IC50 = 25 μg/mL) and Klebsiella pneumoniae (IC50 = 50 μg/mL), respectively, whereas 6 showed weak inhibitory effect on acetylcholinesterase. Nevertheless, most of the butenolides showed inhibition against pancreatic lipase (PL) with an inhibition rate of 21.2–73.0% at a concentration of 50 μg/mL.


Results and Discussion
The wheat culture of Aspergillus sp. SCSIO41404 was extracted with EtOAc. Several chromatographic methods, including silica gel column and semi-preparative HPLC with octadecylsilyl (ODS) column, were used for isolation of the 16 compounds ( Figure 1).

Fermentation and Extraction
The seed medium (malt extract 15 g, sea salt 10 g, H 2 O 1 L, pH 7.4-7.8) in 500 mL Erlenmeyer flasks (150 mL/flask) was incubated at 28 • C for 3 days on a rotating shaker (180 rpm). The seed medium was added to the wheat fermentation medium (wheat 200 g, sea salt 10g, H 2 O 200 mL) in a 1000 mL Erlenmeyer flask. In total, 40 Erlenmeyer flasks were incubated for 30 days at 25 • C without shaking. The whole wheat cultures were crushed and extracted with EtOAc three times to afford an organic extract.

X-ray Crystallographic Analysis
Colorless crystals of 1a were obtained in MeOH/CHCl 3 (1:2) followed by slow evaporation at 4 • C and the crystals' data were collected on a XtalLAB PRO single-crystal diffractometer using Cu Kα radiation. The X-ray crystal structure of 1a was solved using SHELXS97, expanded by difference Fourier techniques, and refined by full-matrix least-squares calculation finally. All non-hydrogen atoms were refined anisotropically, and hydrogen atoms were fixed at calculated positions. Crystallographic data of 1a (Table S1) have been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 2077455). These data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB21EZ, UK [fax: +44(0)-1223-336033 or e-mail: deposit@ccdc.cam.ac.uk].

Mo 2 (AcO) 4 -Induced Circular Dichroism
Mo 2 (AcO) 4 (1.0 mg) and 14 (1.0 mg) were dissolved in dimethyl sulfoxide (DMSO) (1 mL) to use as stock solutions, which were mixed by 1:2 v/v. After mixing, the CD spectrum was recorded immediately and scanned every 5 min, until a stationary ICD spectrum (the CD of 14 in Mo 2 (AcO) 4 solution subtracted from inherent CD of 14 in DMSO) was observed. The sign of the diagnostic band at around 310 nm in the ICD spectrum was correlated to the absolute configuration of C-9 of 14 [22].

ECD Calculation
The structures of 1a, 1b, 2a, and 2b were subjected to random conformational searches using the Spartan'14 software with the MMFF method, as used previously [23]. The conformers with a Boltzmann population of over 5% were chosen for ECD calculations using the Gaussian 09 software [24], and the stable conformers were initially optimized at the B3LYP/6-31+G(d,p) level in MeOH using the CPCM model. The overall theoretical calculation of ECD was achieved in MeOH using time-dependent density functional theory at the B3LYP/6-31+G (d, p) level. The ECD spectra were generated using the SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA) software with a half-bandwidth of 0.3-0.4 eV, according to the Boltzmann-calculated contribution of each conformer after UV correction.

Bioassay
The cytotoxic activity of the obtained compounds was evaluated by the MTT method as reported in our previous study [25]. The antibacterial activity against five bacterial strains, Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Klebsiella pneumoniae ATCC 13883, methicillin-resistant S. aureus (MRSA, clinical strain), and methicillin-resistant S. epidermidis (MRSE, clinical strain) was evaluated using a modified broth microdilution method [25,26]. The acetylcholinesterase and PL inhibitory activities were evaluated according to the methods used in our previous study [26,27].

Conclusions
We describe here the isolation and structure elucidation of five undescribed and ten previously reported butenolides from the coral-derived fungus Aspergillus terreus SCSIO41404, together with an undescribed para-hydroxybenzaldehyde derivative (S)-3-(2,3-dihydroxy-3methylbutyl)-4-hydroxybenzaldehyde. Two pairs of enantiomers were separated by HPLC using a chiral column, and enantiomers (+)-asperteretal G (1a) and (−)-asperteretal G (1b) were new natural products. After preliminary antibacterial, cytotoxic and enzyme inhibitory bioassays of these compounds, several natural butenolides showed inhibition against PL. This is the first report of the PL inhibitor activity of butenolides, and the effects of these butenolides on the regulation of lipid metabolism deserve further study [28].