New Meroterpenoids from the Endophytic Fungus Aspergillus flavipes AIL8 Derived from the Mangrove Plant Acanthus ilicifolius

Four new meroterpenoids (2–5), along with three known analogues (1, 6, and 7) were isolated from mangrove plant Acanthus ilicifolius derived endophytic fungus Aspergillus flavipes. The structures of these compounds were elucidated by NMR and MS analysis, the configurations were assigned by CD data, and the stereochemistry of 1 was confirmed by X-ray crystallography analysis. A possible biogenetic pathway of compounds 1–7 was also proposed. All compounds were evaluated for antibacterial and cytotoxic activities.

In the course of our study on marine derived microbes, several active metabolites were isolated [13][14][15][16]. In our previous study, two aromatic butyrolactones were reported from the same fungus, A. flavipes [17]. In a continuing study, four new and three known meroterpenoids were isolated from the same fungus. Herein, the isolation, structural elucidation, biological activities, and plausible biogenetic pathway of compounds 1-7 are reported ( Figure 1).

Structure Elucidation
Compound 1 was isolated as colorless crystals. The molecular formula of 1 was established as C17H22O4 according to MS and NMR data. The NMR and optical rotation data were in agreement with those previously published, guignardone A [18]; the absolute configuration was confirmed by X-ray crystallography ( Figure 2).   [19], with the only obvious difference being the presence of a ketone group (IR νmax 1608, δC 212.0) instead of a terminal double bond and the presence of a methyl ketone singlet downfield replacing the original vinyl methyl singlet observed in guignardone H. A small coupling constant of 3.0 Hz between H-6 and H-5 placed H-6 in a pseudoequatorial orientation of the cyclohexanone ring. Hence, H-6 was deduced to be β-oriented. Since the specific rotation and CD spectrum of compound 2 has the same sign as guignardone A (1) [18], and on biogenetic grounds, compound 2 was tentatively assigned as having the same absolute configuration as guignardone A.
Compound 3 was obtained as a colorless oil.  [19], with the only obvious difference being the presence of a methyl group replacing a hydroxymethylene group (δC 65.3). A small coupling constants of 3.0 Hz between H-6 and H-5 indicated β-oriented of H-6. The configuration of 3 was tentatively established as same as that of 2 for the biogenetic pathway consideration. The optical rotation exhibited the same sign with 2, and the CD spectrum of 3 showed the same profile with 2 ( Figure 3), and thus absolute stereochemistry of 3 was the same as 2, and named guignardone K (3 , seven methine (one oxygenated), and six quaternary carbon (including two α, β-unsaturated carbonyl and two oxygenated). Comparison of the 1 H NMR and 13 C NMR spectroscopic data with guignardone H (6) [19] revealed that 4 had the same tricyclic moiety as 6, but different side chain. The geometry of the disubstituted double bond (C-18) was determined to be E on the basis of the large coupling constants of the respective protons (J = 15.0 Hz). The presence of a methyl ketone group (δH 2.27, s; δC 198.7 and 27.7) at the end of the side chain of 4, which was confirmed by the HMBC correlations, the side chain was connected to the tricyclic moiety at C-5 in the ring. The absolute configuration of 4 was assumed to be the same as that of 2, and named guignardone L (4). Compound 5 was isolated as a yellow oil. Its molecular formula was determined as C16H23O4 by HRESIMS [M + Na] + 301.1407 and [M + H] + 279.1590. Comparison of the 1 H NMR and 13 C NMR spectroscopic data with guignardone G [19], revealed that 5 had the same structure as guignardone G, but different stereochemistry at C-6. H-6 was deduced to be β-oriented by a small coupling constant of 3.0 Hz between H-6 and H-5. The configuration of 5 was established as same as that of 2, and named guignardone M (5).
Compounds 6 and 7 were identified as guignardones H [19] and I [19,20], respectively, by comparison of the 1 H and 13 C NMR with those reported data.

Plausible Biosynthetic Pathway
As compounds 1-7 were co-metabolites, we propose that they are on the same biosynthetic pathway. A possible biogenetic pathway of compounds 1-7 was proposed as shown in Figure 4. As suggested in pathways 1 and 2, we hypothesized the alkylation of precursor 1,5-dihydroxy-2,4-dioxocyclohexyl carboxylic acid by monoterpenoids or sesquiterpenoids to give the intermediates. Further modification leads to the generation of compounds 1-7.   Figure 4. Plausible biosynthetic pathway of compounds 1-7.

Fungal Material
The endophytic fungus A. flavipes AIL8 was isolated from the inner leaves of mangrove plant A. ilicifolius collected at Daya Bay, Shenzhen, China, in 2011. This fungus was deposited at 4 °C on potato dextrose agar (PDA) slants in CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China.
The strain stored on PDA slants was inoculated and cultured on PDA agar plates for 7 days. Seed medium (potato 200 g, dextrose 20 g, distilled water 1000 mL) in 100 mL × 10 Erlenmeyer flasks was inoculated with fungus and incubated at 25 °C for 48 h on a rotating shaker (180 rpm). Production medium of solid rice in 1000 mL flasks (rice 200 g, distilled water 210 mL) was inoculated with seed solution (10 mL) one for one. Flasks were incubated at 25 °C under still culture and fermented for 40 days, cultures from 30 flasks were harvested for the isolation of substances. Fungal identification was carried out by using the method previously reported [13].

Antimicrobial Activity
Antibacterial activity was tested on strains of Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, Candida albicans, and Micrococcus luteus. MIC values of the compounds were determined by the modified 0.5 Mcfarland standard method. Two-fold dilutions of the compounds in the range of (40-0.31 mg/mL) were prepared in 0.5% MeOH. Compounds and positive control (Penicillin) were similarly diluted in 0.5% MeOH to generate a series of concentration, ranging from 40 to 0.31 mg/mL per well. The turbidity of the bacterial suspensions was measured at 600 nm, and adjusted with a medium to match the 0.5 McFarland standard (10 5 -10 6 colony forming units/mL). Subsequently, 180 μL of bacterial culture was inoculated into each well, and the test solutions (20 μL) were added to 96-well plates. Finally, the plates were incubated at 36 °C for 24 h, and the MIC values were determined in triplicates and re-examined at appropriate times. To ensure that these vehicles had significant effect on the bacterial growth, each of the bacterial species was additionally cultured in a blank solution containing LB broth media at concentrations equivalent to those of test solutions [36].

Cytotoxicity
Proliferation of PC-3 cells was evaluated by using a cell counting kit (CCK-8, Ddojindo, Japan) following the manufacturer's protocol. Cells were routinely grown and maintained in mediums RPMI or DMEM with 10% FBS and with 1% penicillin/streptomycin. All cell lines were incubated in a Thermo/Forma Scientific CO2 Water Jacketed Incubator with 5% CO2 in air at 37 °C. Cell viability assay was determined by the CCK8 assay. Three-fold dilutions of the compounds in the range of (10-5.08 × 10 5 μM) were prepared in DMSO. Cells were seeded at a density of 400-800 cells/ well in 96 well plates and treated with various concentration of compounds, positive control (Trichostatin A and Taxol) or solvent control. After 72 h incubation, CCk8 reagent was added, and absorbance was measured at 450 nm using Envision 2104 multi-label Reader (Perkin Elmer, USA). Dose response curves were plotted to determine the IC50 values using Prism 5.0 (GraphPad Software Inc., San Diego CA, USA). The inhibitory rate was calculated using the formula: (ODcontrol cells − ODtreated cells)/ODcontrol cells × 100% [37,38].

Conclusions
Four new meroterpenoids, along with three known analogues, were isolated from marine-derived endophytic fungus A. flavipes AIL8 derived from the mangrove plant Acanthus ilicifolius. A possible biogenetic pathway of compounds 1-7 was proposed. All compounds were evaluated for antibacterial activity and cytotoxicity; none of them are active. Comparison with those reported tricycloalternarenes, the structural differences mainly occur in the isoprenoid side chains, this maybe the reason for their lack of antibacterial and cytotoxic activities.