Bioactive α-pyrone meroterpenoids from mangrove endophytic fungus Penicillium sp.

Abstract Five α-pyrone meroterpenoids, including one new 3-epiarigsugacin E (1) and four known compounds, arisugacin D (2), arisugacin B (3), territrem C (4) and terreulactone C (5) were obtained from the marine fungus Penicillium sp. SK5GW1L. Their structures were identified by MS and NMR experiments, and the absolute configuration of compound 1 was further confirmed by low temperature (150 K) single crystal X-ray diffraction with Cu Kα radiation. Compounds 3, 4 and 5 showed strong inhibitory activities against acetylcholinesterase (AchE) with IC50 values of 3.03, 0.23 and 0.028 μM, respectively.


Introduction
Endophytic fungi have long been known as a rich source of biologically active secondary metabolites. Pencillium species, one of these extensive endophytic fungi, is a main focus of bioactive natural products research. Recently, diverse structures and interesting biological activities were isolated from these species (Huang et al. 2012;Yang et al. 2013;Chen et al. 2014;Li et al. 2014;Zheng et al. 2015;Yang et al. 2016).
The term meroterpenoids, one of the important secondary metabolites from endophytic fungi, was initially proposed to describe the mixed biosynthetic origins, which was grouped into two major classes: polyketide-terpnoids and non-polyketideterpenoids (Birch 1967;Geris & Simpson 2009). α-pyrone meroterpenoids containing triketide terpenoid moieties were discovered from the fungi with acetylcholinesterase inhibitors (Sunazuka & Ōmura 2005). So these α-pyrone meroterpenoids have been attracting chemists and pharmacologists' considerable attention.
In our group, research on novel bioactive secondary metabolites from mangrove endophytic fungi, which were collected from the South China Sea in the last few years (Xu et al. 2008;Wen et al. 2009;Shao et al. 2010;Huang et al. 2011;Yuan et al. 2012;) is ongoing. Three meroterpenoid compounds, arisugacin I, arisugacin F and territrem B have been isolated from the mangrove endophytic fungus, Penicillium sp. SK5GW1L in our previous research ). With our intensive study, five other α-pyrone meroterpenoids, including one new 3-epiarigsugacin E (1) and four known compounds, arisugacin D (2), arisugacin B (3), territrem C (4) and terreulactone C (5) were isolated from this fungus. Their structures were identified through MS and NMR experiments, and the absolute configuration of 1 was further confirmed by low temperature (150 K) single crystal X-ray diffraction with Cu Kα radiation ( Figure 1). Herein, details of the isolation, structure elucidation as well as evaluation of biological activities against AchE about these compounds are reported.

Results and discussion
The fungus Penicillium sp. SK5GW1L was fermented on autoclaved rice solid-substrate medium and then extracted with ethyl acetate. The extract was subjected to silica gel column chromatography (CC) using gradient elution to afford 15 fractions ). Due to the ultraviolet absorption peaks of α-pyrone meroterpenoids at 252.3 and 330.7 nm , the isolation of these α-pyrone meroterpenoids was guided by high performance liquid chromatography in combination with ultraviolet and photo diode array (HPLC-UV/PDA). Finally, the other five α-pyrone meroterpenoids, including one new compound 3-epiarigsugacin E (1), and four known compounds were obtained by semi-preparative HPLC-UV. The structures of arisugacin D (2) (otoguro et al. 2000), arisugacin B (3) (Kuno et al. 1996), territrem C (4) (Kuno et al. 1996) and terreulactone C (5) (Cho et al. 2003) were established by the comparison of their 1 H and 13 C NMR and MS data with literature values.
The NMR data of 1 were compared with that of arigsugacin E's in the literature (Figure 1), which revealed that the structure of 1 was similar to arigsugacin E (otoguro et al. 2000). However, the 1 H NMR, and 13 C NMR signals of CH-3 at δ H 3.88 (dd, J = 11.0 Hz, 5.7 Hz, H-3), δ C 73.8 (C-3) about the compound 1, while the arisugacin E's at δ H 3.62 (dd, J = 2.9 Hz, 2.9 Hz, H-3), δ C 77.7 (C-3) in the same solvent, which indicated that the configuration of C-3 would be different. The elucidated structure of 1 was further confirmed by a single crystal X-ray diffraction analysis using Cu Kα radiation ( Figure S1), which confirmed and determined the absolute configuration as 3S, which is the 3-C-epimer of arigsugacin E, named as 3-epiarigsugacin E.
The anti-acetylcholinesterase inhibitory activities of all the five compounds were investigated in vitro using the modified Ellman method (Ellman et al. 1961). Displayed inhibitory IC 50 values of these α-pyrone meroterpenoids are shown in Table 1. The new compound 1 showed weak inhibitory activity, whereas Arisugacin B, territrem C and terreulactone C displayed IC 50 values of 3.03, 0.23 and 0.028 μM, respectively.

General experimental procedures
Melting points were determined on a Fisher-Johns hot-stage apparatus and are uncorrected. optical rotations were measured on a MCP 300 (Anton Paar, Graz, Austria) polarimeter at 25 °C. IR spectra were measured on a Nicolet 5DX-FTIR spectrophotometer (Nicolet, USA). UV spectra were measured on the HPLC (Waters 1525, Westbrook, CT, USA) with PDA detection (Waters 2998, Greenville SC, USA). Methanol used for HPLC was of chromatographic grade (Welch, Shanghai, China), water used was ultrapure water. NMR data were recorded on a Bruker Avance 400, 500 and 600 spectrometers using TMS as an internal standard, and CDCl 3 or CD 3 oD as the solvent. HRMS data were measured on LTQ Qrbitrap LC-MS (Thermo, Rockford, IL, USA). CC was performed using silica gel (200-300 mesh, Qingdao Marine Chemicals, Qingdao, China). Single crystal data were measured on an oxford Gemini S Ultra diffractometer. The single crystal X-ray diffraction data were collected at 150 K on an oxford Gemini S Ultra diffractometer with Cu Kα radiation (λ = 1.54178 Å). The structures were solved by direct methods (SHELXS-97) and refined using full-matrix least squares difference Fourier techniques.
The analysis of the crude extract was performed on a Waters 1525 liquid chromatograph system with a Waters 2998 PDA detector (Waters, USA), coupled to a Ultimate XB-C18 column (250 × 4.6 mm, 5 μM; Welch, Shanghai, China) eluted with a gradient of methanol: water (from 5:95 to 100:0 (volume ratio) in 60 min or from 60:40 to 100:0 in 25 min). PDA detector was recorded between 200 and 400 nm. The crude extract was separated by CC over silica gel to get 15 fractions. The three and eight fractions were analysed to find the α-pyrone meroterpenoids with HPLC-PDA (detection was performed at 330 nm) and isolated by semi-preparative HPLC-UV, which was operated on a Waters 1525 LC using an Ultimate XB-C18 column (250 × 10 mm, 5 μM). All structures were identified through MS, NMR and single crystal X-ray diffraction experiments.

Fungal material
The endophytic fungus was isolated from the leaves of Kandelia candel, collected from the town of Shankou, Guangxi province, China in September 2008. The fungus was identified as Penicillium sp. SK5GW1L (GenBank accession number: KC545798) and deposited in the school of chemistry and chemical engineering, Sun Yat-sen University.

Arisugacin D (2)
A colourless crystal. The 1 H and 13 C NMR data were as following: 1 H NMR (

Terreulactone C (5)
A white powder. The 1 H, 13 C NMR data were as following: 1 H NMR (500 MHz

Anti-acetylcholinesterase activity in vitro
All the α-pyrone meroterpenoids were tested for acetylcholinesterase inhibitory activity in vitro. The experiment was employed based on the literature method (otoguro et al. 2000).

Conclusion
A new α-pyrone meroterpenoid, named 3-epiarigsugacin E (1), was obtained from marine fungus Penicillium sp. SK5GW1L. Moreover, the four known α-pyrone meroterpenoid were also isolated from this fungus. All the α-pyrone meroterpenoids were tested for acetylcholinesterase inhibitory activity, and some showed strong inhibitory activities.

Supplementary material
UV, IR, MS, 1D and 2D NMR spectra of compounds 1; CIF file with the X-ray crystallographic data for 1. These materials are available free of charge via the internet at http://www.tandfonline.com.

Disclosure statement
No potential conflict of interest was reported by the authors.