Acetylcholinesterase Inhibitory Meroterpenoid from a Mangrove Endophytic Fungus Aspergillus sp. 16-5c

One new meroterpenoid, named 2-hydroacetoxydehydroaustin (1), together with nine known meroterpenoids, 11-acetoxyisoaustinone (2), isoaustinol (3), austin (4), austinol (5), acetoxydehydroaustin (6), dehydroaustin (7), dehydroaustinol (8), preaustinoid A2 (9), and 1,2-dihydro-acetoxydehydroaustin B (10), were isolated from the mangrove endophytic fungus, Aspergillus sp. 16-5c. These structures were characterized by spectroscopic analysis, further the absolute configurations of stereogenic carbons for Compounds 1, 3, 4, 6, 7, 8, 9, and 10 were determined by single crystal X-ray diffraction analysis using Cu Kα radiation. Moreover, the absolute configurations of stereogenic carbons for Known Compounds 3, 7, 8, and 9 are identified here for the first time. Compounds 3, 7, and 8 showed acetylcholinesterase (AchE) inhibitory activity with IC50 values of 2.50, 0.40, and 3.00 μM, respectively.


Results and Discussion
Compound

Results and Discussion
Compound 1 was obtained as colorless crystal, m.p. >300  The above spectroscopic features suggested that 1 belonged to the austin analogue [16], whose planar structure is similar to the 1,2-dihydro-acetoxydehydroaustin B. However, the carbon chemical shifts values of C-1 (δ C 37.1) and C-2 (δ C 64.8) were different from those of 1,2-dihydro-acetoxydehydroaustin B. The detailed comparison of the NMR data of 1 with those of 1,2-dihydro-acetoxydehydroaustin B clearly confirmed that 1 was a new structure ( Figure 3). The absolute configurations of strereogenic carbons for 1 were determined by single-crystal X-ray diffraction using Cu Kα radiation [21]. Therefore, Compound 1 was named 2-hydroacetoxydehydroaustin. Its absolute configurations were 1S, 7R, 8S, 9R, 11S, 3 R, 5 R, 7 R. Compound 2 was obtained as a colorless powder. Its molecular formula was assigned to be C27H32O8 from the HRESIMS molecular ion peak at m/z 507.1988 [M + Na] + . Comparison of the NMR data revealed that the structure of 2 resembled that of 3 except for the presence of an acetyl group. A carbon signal at δC 74.1 (C-11) was observed in 2 compared to that of 3 [7]. It was deduced that the acetyl group was connected to C-11 via the hydroxyl group in 2 supported by the HMBC correlation from H-11 (δH 5.74, 1H, s) to 11-CH3CO (δC 171.5). The relative configuration was confirmed by the NOE correlations, and the correlation signals between 9-CH3 (δH 1.25), 11-OCOCH3 (δH 2.04), and 12-CH3  Compound 2 was obtained as a colorless powder. Its molecular formula was assigned to be C 27 H 32 O 8 from the HRESIMS molecular ion peak at m/z 507.1988 [M + Na] + . Comparison of the NMR data revealed that the structure of 2 resembled that of 3 except for the presence of an acetyl group. A carbon signal at δ C 74.1 (C-11) was observed in 2 compared to that of 3 [7]. It was deduced that the acetyl group was connected to C-11 via the hydroxyl group in 2 supported by the HMBC correlation from H-11 (δ H 5.74, 1H, s) to 11-CH 3 CO (δ C 171.5). The relative configuration was confirmed by the NOE correlations, and the correlation signals between 9-CH 3 (δ H 1.25), 11-OCOCH 3 (δ H 2.04), and 12-CH 3 (δ H 1.57) showed that the 11-acetoxyl, 12 and 9'-CH 3 were oriented at the same side. Furthermore, the NOE correlations between 10'-CH 3 (δ H 1.30) and 6'-OH (δ H 2.88) supported that the methyl and hydroxyl groups were at the same side. The NOE correlations of 2 were the same as 3, in addition to their positive optical rotation values. Finally, the absolute configurations of stereogenic carbons for 2 were assigned as 5R, 8S, 11S, 3 R, 5 R, 6 R, 7 R. These results confirmed that 2 was consistent with 11-acetoxylisoaustinone [22].

General
Melting points were determined on an X-4 micromelting point apparatus and are uncorrected. Optical rotations were measured on a Polartronic HHW5 digital polarimeter. IR spectra were measured on a Bruker Vector 22 spectrophotometer (Bruker, Billerica, MA, USA) using KBr pellets. The NMR spectra were recorded on a Bruker Avance 400 spectrometer at 400 MHz for 1 H and 100 MHz for 13 C in CDCl 3 . All chemical shifts (δ) are given in ppm with reference to the solvent signal (CDCl 3 , δ H 7.26 for 1 H, δ C 77.23 for 13 C; DMSO, δ H 2.50 for 1 H, δ C 39.52 for 13 C), and coupling constants (J) are given in Hz. HRESIMS spectra were recorded on a Finnigan LCQ-DECA mass spectrometer (Thermo Scientific, shanghai, China). ESIMS spectra were recorded on a Shimadzu LCMS-IT-TOF mass spectrometer (Shimadzu, Taiwan). Single-crystal data were measured on an Oxford Gemini S Ultra diffractometer (Oxford Instrument, Oxfordshire, UK). Column chromatography (CC) was performed on silica gel (200-300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 (Amersham Pharmacia, Piscataway, NJ, USA).

Fungal Material
The fungus used in this study was isolated from a mangrove, leaves of S. apetala, which were collected in Hainan Island, China. The fungus was identified as Aspergillus sp. by the ITS region (deposited in GenBank, accession number JX993829). A voucher strain was deposited in the China Center for Type Culture Collection under patent depository number CCTCC M 2012358.

Extraction and Isolation
The fungus Aspergillus sp. 16-5c was fermented on autoclaved rice solid-substrate medium for 28 days at room temperature. The mycelia and solid rice medium were extracted with MeOH. Then, the MeOH layer was dried in vacuo to yield 6.8 g of organic extract. The extract was separated by column chromatography (CC) over silica gel eluting with a gradient of CHCl 3 /MeOH from 1:0 to 1:45 to yield five fractions (Fractions 1-5). Fraction 3 (120 mg) was applied to Sephadex LH-20 CC, eluting with CHCl 3 /MeOH (1:1) to obtain Compound 1 (0.8 mg), 2 (1.  Analysis of 1, 3, 4, 6, 7, 8, 9 and 10 All single crystal X-ray diffraction data were collected at 150(2) 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. Hydrogen atoms bonded to carbons were placed on the geometrically ideal positions by the "ride on" method. Hydrogen atoms bonded to oxygen were located by the difference Fourier method and were included in the calculation of structure factors with isotropic temperature factors. Crystallographic data for 1, 3,

Assays for Enzyme Inhibiting Activities and Cytotoxic Activities
These two experiments were conducted according to reference procedures [14,23].

Conflicts of Interest:
The authors declare no conflict of interest.