New Antibacterial Phenone Derivatives Asperphenone A–C from Mangrove-Derived Fungus Aspergillus sp. YHZ-1

Marine fungi are a promising source of novel bioactive natural products with diverse structure. In our search for new bioactive natural products from marine fungi, three new phenone derivatives, asperphenone A–C (1–3), have been isolated from the ethyl acetate extract of the fermentation broth of the mangrove-derived fungus, Aspergillus sp. YHZ-1. The chemical structures of these natural products were elucidated on the basis of mass spectrometry, one- and two-dimensional NMR spectroscopic analysis and asperphenone A and B were confirmed by single-crystal X-ray crystallography. Compounds 1 and 2 exhibited weak antibacterial activity against four Gram-positive bacteria, Staphylococcus aureus CMCC(B) 26003, Streptococcus pyogenes ATCC19615, Bacillus subtilis CICC 10283 and Micrococcus luteus, with the MIC values higher than 32.0 µM.

In the primary screen for antibacterial compounds, compounds 1 and 2 were tested against four Gram-positive bacteria, Staphylococcus aureus CMCC(B) 26003, Streptococcus pyogenes ATCC19615, Bacillus subtilis CICC 10283 and Micrococcus luteus. As summarized in Table 2, Both of these two compounds showed weak activity against the tested bacteria, Staphylococcus aureus CMCC(B) 26003, Streptococcus pyogenes ATCC19615, Bacillus subtilis CICC 10283 and Micrococcus luteus, with the MIC values higher than 32.0 µM.

General Experimental Procedures
The

Strain Isolation and Cultivation
The fungus Aspergillus sp. YHZ-1 was isolated by one of the authors (Y.-Q.Z.) from an unidentified mangrove plant from Hainan Island, China, in October 2015. The isolate was identified as Aspergillus sp. by its morphological characteristics and the voucher specimen (IFB-YHZ-1) was deposited in the Institute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University. The strain was cultivated on MEA agar plate (consisting of 20 g/L malt extract, 20 g/L sucrose, 1 g/L peptone, 20 g/L agar and deionized water) at 30 • C for 5 days. Then small agar plugs with mycelia were inoculated into five 1 L-Erlenmeyer flasks, each containing 400 mL MEA liquid medium, which were cultivated at 28 • C with 108 rpm/min. After 3 days of fermentation, 10 mL of the seed cultures were inoculated into 200 flasks with 250 mL ME liquid medium and fermented on a rotary shaker with 120 rpm/min at 28 • C for 14 days.

Extraction and Isolation
The entire filtrate of the fermented broth (50 L) was harvested and extracted three times with an equivalent volume of ethyl acetate at room temperature. The organic solvent was evaporated in vacuo to yield 25 g of crude extract.

Crystal Data of 1
The single crystal X-ray diffraction data of compound 1 was collected on a Bruker APEX-II diffractometer at 130 K with Cu Kα radiation (λ = 1.54178 Å). The structure was solved using the program SHELXS-97 and refined by full-matrix least-squares on F 2 . Crystal data of compound 1 have

Crystal Data of 2
The single crystal data of compound 2 was collected on a Bruker APEX-II diffractometer at 130 K with Cu Kα radiation (λ = 1.54178 Å). The structure was solved using the program SHELXS-97 and refined by full-matrix least-squares on

Antibacterial Activity Assay
The in vitro antibacterial activity of compounds 1 and 2 were evaluated against four bacteria including Staphylococcus aureus CMCC(B) 26003, Streptococcus pyogenes ATCC19615, Bacillus subtilis CICC 10283 and Micrococcus luteus in accordance with previously reported methods [14,15]. In the assays, the medium used in the test was Müller-Hinton (MH) broth. All test compounds and the positive control ampicillin were dissolved in dimethyl sulfoxide (DMSO). The minimum inhibitory concentration (MIC) values were determined in the 96-well plates (triplicate) and determined as the lowest sample concentration exhibiting no bacterial growth.

Conclusions
Three new phenone derivatives, asperphenone A-C (1-3), were isolated from the ethyl acetate extract of the fermentation broth of the mangrove-derived fungus Aspergillus sp. YHZ-1. The chemical structures of these compounds were elucidated on the basis of HR-ESI-MS, 1D and 2D NMR spectroscopic analysis, as well as X-ray crystallographic data. Both of the tested compounds 1 and 2 displayed weak antibacterial activity against four Gram-positive bacteria, Staphylococcus aureus CMCC(B) 26003, Streptococcus pyogenes ATCC19615, Bacillus subtilis CICC 10283 and Micrococcus luteus, indicating that the mangrove-associated fungi are still a rich source for discovering diverse new bioactive natural products which could be used as lead compounds in drug development.