Territrem and Butyrolactone Derivatives from a Marine-Derived Fungus Aspergillus Terreus

Seventeen lactones including eight territrem derivatives (1–8) and nine butyrolactone derivatives (9–17) were isolated from a marine-derived fungus Aspergillus terreus SCSGAF0162 under solid-state fermentation of rice. Compounds 1–3 and 9–10 were new, and their structures were elucidated by spectroscopic analysis. The acetylcholinesterase inhibitory activity and antiviral activity of compounds 1–17 were evaluated. Among them, compounds 1 and 2 showed strong inhibitory activity against acetylcholinesterase with IC50 values of 4.2 ± 0.6, 4.5 ± 0.6 nM, respectively. This is the first time it has been reported that 3, 6, 10, 12 had evident antiviral activity towards HSV-1 with IC50 values of 16.4 ± 0.6, 6.34 ± 0.4, 21.8 ± 0.8 and 28.9 ± 0.8 μg·mL−1, respectively. Antifouling bioassay tests showed that compounds 1, 11, 12, 15 had potent antifouling activity with EC50 values of 12.9 ± 0.5, 22.1 ± 0.8, 7.4 ± 0.6, 16.1 ± 0.6 μg·mL−1 toward barnacle Balanus amphitrite larvae, respectively.


Introduction
Alzheimer's disease (AD) is a neurodegenerative disorder that is the most common cause of dementia among the elderly. Recent studies demonstrated that cholinergic neurodegeneration could be a major pathologic feature of AD [1,2]. Thus, enhancement of the central cholinergic neurotransmission has been regarded as one of the most promising strategies for the symptomatic treatment of AD. Accordingly, acetylcholinesterase (AChE) inhibitors are currently the most effective treatment targets for the design of anti-Alzheimer drug candidates [3].

Results and Discussion
Compound 1 has a molecular formula of C28H32O9 as determined by HRESIMS (m/z 535.1919 [M + Na] + ). The 1 H NMR spectrum of 1 showed the presence of six singlet methyl signals and five olefinicmethines. The 13 C NMR spectrum showed the presence of 28 carbons, including six methyls, three methylenes, five olefinicmethines, and 14 quaternary carbons.These data showed close similarity to those of 6 [15], which suggested that 1 had a territrem skeleton. Comparison of the NMR data of 1 and 6 showed that the only obvious difference between them was the additional appearance of one low-field quaternary carbon (δC 150.7) and the absence of one aromatic methine in 1. In the HMBC spectrum, correlations of H-2′ with C-3′/C-4′/C-6′/C-9, H-6′ with C-2′/C-4′/C-5′/C-9, 4′-OCH3 with C-4′, and 5′-OCH3 with C-5′, suggested that C-3′ at the benzene ring was oxygenated. The relative configuration of 1 was confirmed by the NOESY spectrum ( Figure 2). NOESY correlations of H-5β with 4β-CH3/12b-CH3, and H-12β with 6a-CH3/12b-CH3 suggested that 4β-CH3, H-5β, 6a-CH3, 12b-CH3 and H-12β were in β-oriented, while the NOESY correlations of 4a-OH with 4α-CH3/12a-OH indicated 4α-CH3, 4a-OH and 12a-OH were in α-oriented. Its relative configuration was identical to that of 6 according to their identical NOESY data. The specific optical rotation value of 1 ([α] 25 D +110 (c 0.24, CH3OH)) was also similar to that of 6 ([α] 25 D +144 (c 0.10, CHCl3)) [15]. These data suggested that the absolute configuration of 1 was the same as that of 6 and determined to be 4aR, 6aR, 12aS, 12bS. So, the structure of 1 was elucidated as shown and named territrem D. Compound 2 has a molecular formula of C27H30O8 as determined by its HRESIMS (m/z 505.1822 [M + Na] + ). Its 1 H and 13 C NMR data showed close similarity to those of 6 [15], and the only obvious difference between them was the absence of one oxymethyl group in 2. The HMBC spectrum showed correlations of H-2′ with C-4′/C-6′/C-9, H-5′ with C-1′/C-3′, H-6′ with C-2′/C-4′/C-9, and 3′-OCH3 with C-3′, suggesting that C-3′ was methyloxygenated and C-4′ was hydroxylated. The relative configuration of 2 was identical to those of 1 and 6 [15] according to their identical NOESY data, which suggested that the absolute configuration of 2 was the same as those of 1 and 6 and assigned to be 4aR, 6aR, 12aS, 12bS. So, the structure of 2 was elucidated as shown and named territrem E.
Compound 9 has a molecular formula of C22H22O6 as determined by its HRESIMS (m/z 405.1310 [M + Na] + ). Analysis of the 1 H and 13 C NMR spectra showed the presence of one 1,4-disubstitued aromatic ring, one 1,4,5-trisubstitued aromatic ring, two methyl groups, one methylene, two oxymethines, one tetra-substituted double bond and one ester carbonyl group. These data showed close similarity to those of 11 8 , 12 9 , and 13 17 , which suggested that 9 also had a butyrolactone skeleton. Comparison of the NMR data of 9 and 11 showed that the only obvious difference between them was the lack of one esterified carboxyl group substituent at C-10 in 9. This was proved by the HMBC spectrum showing correlations of H-10 with C-7/C-9/C-11/C-12. Thus, the planar structure of 9 was assigned. The coupling constant observed for protons at C-19 and C-18 (J18,19 = 5.0, 7.5 Hz) suggested that H-19 was equatorial, which demonstrated the configuration of C-19 was S. [18] And the similar coupling constant for H-19 in 9 and 11 (J18,19 = 5.5, 8.4 Hz) further proved the 19S-configuration in 9 was as the same as that in 11. In addition, the β-configuration of H-10 and corresponding 10R-configuration of C-10 in 9 was speculated from the proposed biogenetic pathway that 9 was derived from the decarboxylation of 11, which was further supported by the comparison of CD spectra of 9, 11 and 12 (see Supplementary Figure S39). In the CD spectra, the experimental data of 9 revealed a good agreement with that of 10 and 11. So, the structure of 9 was established and named isobutyrolactone V.
Compound 10 has a molecular formula of C18H16O6 as determined by its HRESIMS (m/z 351.0842 [M + Na] + ). Its 1 H and 13 C NMR data showed close similarity to those of 14-17 [10][11][12]17,19] and butyrolactone II [17], which suggested that 10 had a butyrolactone skeleton. Comparison of the NMR data of 10 and butyrolactone II [17]showed that the obvious difference between them was the substituent at C-10. In the HMBC spectrum, correlations of 10-OCH3 (δH 3.10, s) with C-10 (δC 108.3) suggested an oxymethyl group attached at C-10. Compound 10 was inferred as a racemic mixture because its specific rotation was recorded as zero and its CD spectrum did not show a cotton effect. So, 10 was named isobutyrolactone II.
The AChE inhibitory activities of 1-17 were evaluated by the modified Ellman method [20]. The results showed that 1, 2, 4-6, and 8 exhibited strong inhibiting activity with IC50 values of 4.2 ± 0.6, 4.5 ± 0.6, 4.2 ± 0.6, 20.1 ± 3.3, 11.9 ± 2.1, and 50.0 ± 1.5 nM, respectively, compound 7 displayed medium inhibiting activity with IC50 value of 5.7 ± 0.8 μM, while other compounds had weak or no activity (see Table 1). The inhibiting activities of 1 and 2 were stronger than the positive control huperzine A (IC50 = 39.3 ± 7.6 nM), which indicated that 1 and 2 were new potent AChE inhibitors. The IC50 values of 4, 5, 6 and 8 were consistent with the literature data [5,21]. The results further proved the conclusion that the enone group at the A-ring was responsible for the AChE inhibition capacity of these territrems, as it involved in binding to the active site of AChE [22,23].

General Experimental Procedure
Optical rotations were measured with an Anton Paar MCP 500 polarimeter (Anton Paar GmbH, Graz, Austria). UV spectra were obtained using a Shimadzu UV-2600 UV−vis spectrophotometer (Shimadzu, Tokyo, Japan). CD spectra were measured with a Chirascan circular dichroism spectrometer (Applied Photophysics Ltd, London, UK). IR spectra were measured with a Shimadzu IR Affinity-1 Fourier transform infrared spectrophotometer (Shimadzu, Tokyo, Japan). 1 H, 13

Fungal Material
The fungal strain SCSGAF0162 (GenBank access number JN851044) was isolated from the South China Sea (18°11′ N, 109°25′ E) gorgonian corals Echinogorgia aurantiaca, and identified as Aspergillus terreus SCSGAF0162 by a molecular biological protocol calling for DNA amplification and ITS region sequence comparison with GenBank database, sharing a similarity of 99% with Aspergillus niger EIODSF002 (GenBank access number KJ173525), which was deposited in RNAM center, South China Sea Institute of Oceanology, Chinese Academy of Sciences.

Fermentation and Extraction
The fungal strain was inoculated in PDA liquid medium (containing 20 g/L glucose, 200 g/L potato and 30 g/L sea salt) in 500 mL shake flask loading 120 mL as seed culture and incubated on a rotary shaker (200 rpm) at 28 °C for 3 days. Fermentation of the strain was carried out in 5000 mL Erlenmeyer flasks containing solid-state rice medium (each flask contained 500 g of commercially available rice, yeast extracts 32 g, 24.0 g of sea salt, and 800 mL of water). Then, each of the seed cultures (10 mL) was transferred into autoclaved 5000 mL Erlenmeyer flasks that contained solid-state rice medium. After that, the flasks were incubated at 26 °C as static cultures for 42 days. The total 2 kg of rice culture was crushed and extracted with 80% acetone three times. The acetone extract was evaporated under reduced pressure to afford an aqueous solution, and then the aqueous solution was extracted with EtOAc to yield 30 g of a crude gum.

Purification
The crude extract was subjected to silica gel CC using gradient elution with a CHCl3/CH3OH solvent system at the ratios of 100:0, 98:2, 95:5, 90:10, 80:20, 50:50, and 0:100 (v/v) to give eight fractions (Fr.1-Fr.8). And the compounds 1-17 were isolated and purified by using silica gel CC, MPLC with an ODS column and SP-RP HPLC from the above fractions (more detail see Supplementary   For determining the blank value, the 20 μL of test compound solution was instead of 20 μL of DMSO solution. Each concentration was analyzed in triplicate. The inhibition of the enzyme was calculated from the slope of the linear part of the enzyme reaction (absorption vs time) in relation to controls (no inhibition, 100% activity). IC50 values were determined graphically from the regression analysis of concentration-inhibition curves. The IC50 values are the mean ± standard deviations of three independent experiments. The inhibitory effects are represented as compounds concd (nM) giving 50% inhibition on AChE activity (IC50).

Plaque Reduction Assay
Cytotoxic activity was evaluated using Vero cell lines by the MTT method. Anti-HSV-1 activity was determined by plaque assay using monolayer cultures of Vero cells in 24-well culture plates (Corning, New York, NY, USA). Virus suspension containing HSV-1 (30 plaque forming units (PFU)/well) was added to the cell wells and incubated at 37 °C with 5% CO2 for 2 h. The virus inoculum was then removed and overlay medium (maintenance medium containing 1% methylcellulose and various concentrations of tested compound) was added to each well. After another 72 h of incubation, the cell monolayers were fixed with 10% formalin and stained with 1% crystal violet. Plaques were counted and the percentage of inhibition was calculated according to literature [25]. The concentration reducing plaque numbers by 50% was calculated by regression analysis of the dose-response curves generated from the plaque assay and was defined as 50% inhibitory concentration (IC50).

Barnacle Balanus Amphitrite Larval Settlement Bioassays
Larval settlement bioassays were performed using sterile 24-well polystyrene plates. Tested samples were dissolved in DMSO to a concentration of 50 μg· mL −1 for preliminary bioassay. To define the EC50 values of anti-larval compounds found in the preliminary bioassay, different dilutions of the tested compounds were further prepared to the concentrations ranging from 0.1 to 200 μg· mL −1 in autoclaved FSW. About 20 competent larvae were added to each well in 1 mL of the test solution. The experiment was repeated twice with four replicateseach time. Wells containing only FSW with DMSO served as the controls. The plates were incubated at 27 °C for 24 h. The percentage of larval settlement was determined by counting the settled, live individuals under a dissecting microscope and expressing the result as a proportion of the total number of larvae in the well. Statistical calculations were performed with the SPSS software package. EC50 (inhibits 50% of settlement of B. amphitrite larvae in comparison with the control) levels of tested compounds were calculated by using the Probit software program [26].