Pleosporalone A, the first azaphilone characterized with aromatic A-ring from a marine-derived Pleosporales sp. fungus

Abstract A fungal strain, Pleosporales sp. CF09-1, was isolated from marine sediment collected from the Bohai Sea. A novel azaphilone derivative, named pleosporalone A (1), along with two known analogues, cohaerins A and B (2 and 3), were obtained and identified from the culture extract of Pleosporales sp. CF09-1. Their planar structures were elucidated by detailed analysis of spectroscopic data and by comparison with related known compounds. Pleosporalone A (1) represents the first azaphilone derivative characterised with A-ring aromatisation. Compound 1 showed strong antifungal activity against three plant pathogenic fungi Botrytis cinerea, Rhizopus oryzae and Phytophthora capsici with the MIC values of 0.39, 0.78 and 0.78 μM, respectively.


Introduction
Azaphilones are a class of bioactive natural products obtained exclusively from marine and terrestrial fungal sources, whose names arose as a result of their affinity for ammonia: the pigments react with amines -such as proteins, amino acids and nucleic acids -to form red or purple vinylogous γ-pyridones due to the exchange of pyran oxygen for nitrogen (Osmanova et al. 2010;Gao et al. 2011;Cheng et al. 2012;Xiao et al. 2015;Zhao et al. 2015). According to the literature, over 230 different azaphilones with unique chemical scaffolds possessing a variety of potent biological activities were produced by 23 genera of fungi (Gao et al. 2013). All of these azaphilones had a highly oxygenated bicyclic core and a chiral quaternary centre (Osmanova et al. 2010;Gao et al. 2011Gao et al. , 2013Cheng et al. 2012;Xiao et al. 2015;Zhao et al. 2015). during our ongoing search for bioactive metabolites from fungi (Cao et al. 2015;He et al. 2015;Yu et al. 2015), a marine sediment-derived fungus collected from the Bohai Sea, Pleosporales sp. CF09-1, attracted our attention because an etOAc extract of the fungal culture exhibited antifungal activity. Bioassay-guided fractionation of the active extract led to the isolation of a novel azaphilone derivative, pleosporalone A (1), which represents the first azaphilone derivative characterised with A-ring aromatisation, along with two known analogues, cohaerins A and B (2 and 3) (Quang et al. 2005) (Figure 1). Herein, we report the isolation, structure elucidation and bioactivity of these compounds.

Results and discussion
Pleosporalone A (1) was obtained as a yellow, amorphous powder. The molecular formula C 17 H 14 O 5 results from the Hr-eSI mass spectrum at m/z = 299.0919, [M + H] + (calcd. 299.0914) and indicates eleven degrees of unsaturation. In the 1 H NMr spectrum of 1, an aromatic ABX spin system at δ H 7.14 (1H, dd, J = 8.0, 7.5 Hz, H-13), 6.77 (1H, d, J = 7.5 Hz, H-12) and 6.73 (1H, d, J = 8.0 Hz, H-14) suggested a trisubstituted aromatic ring (ring-C). In the 13 C NMr spectrum of 1, twelve olefinic carbon signals in the region of δ C 99.7-165.2 indicated the existence of the other aromatic ring (ring-A) in 1. The NMr data also exhibited the resonances assignable to an ester carbonyl (δ C 168.8), and a trisubstituted double bond [δ H 6.50 (1H, s, H-4); δ C 151.5 (C-3) and 110.3 (C-4); HMBC from H-4 to C-3 and C-10]. These function groups accounted for ten of the eleven degrees of unsaturation, and the remaining one thus required another ring (ring-B) of 1. Careful analysis of the 1 H NMr and 13 C NMr data of 1 together with the literature search revealed that 1 shares the similar nucleus structure as that of thunberginol B, previous isolated from Hydrangeae dulcis folium the fermented leaves of Hydrangea macrophylla SerINGe var. thunbergii MAKINO (Matsuda et al. 1998). The most obvious difference was the presence of the two methyl groups at C-7 and C-11 in 1. The HMBC correlations ( Figure S6) from CH 3 -9 (δ H 2.12) to the C-6 (δ C 165.2) and C-8 (δ C 162.0) and from CH 3 -16 (δ H 2.26) to the C-10 (δ C 121.7) and C-12 (δ C 122.3) demonstrated that the CH 3 -9 and CH 3 -16 were attached at C-7 and C-11, respectively. The HMBC correlations from H-13 to the C-11 (δ C 140.1) and C-15 (δ C 151.7) and the 1 H-1 H COSY correlations of H-12/H-13/H-14 indicated that 15-OH was attached at C-15. detailed analysis of the HMQC, COSY and HMBC spectra allowed the assignment for all proton and carbon resonances of 1. Thus, the structure of 1 was assigned completely.
Although the structure of 1 was similar to that of thunberginol B, the structure classification of them was different. Compound 1 belonged to an azaphilone derivative; however, thunberginol B belonged to a flavanone derivative. So, pleosporalone A (1) was not the analogue of thunberginol B. Careful comparison of the structure of 1 with that of cohaerins A (2), an azaphilone derivative obtained from fungus Hypoxylon cohaerens (Quang et al. 2005), revealed that 1 shared the same azaphilone nucleus structure as 2. In the past decades, a large amounts of azaphilones classified into ten different subtypes were isolated from 23 genera of fungi (Osmanova et al. 2010). However, pleosporalone A (1) was the first azaphilone derivative characterised with A-ring aromatisation. The plausible pathway of 1 was probably derived from the normal azaphilone by multisteps oxidation reactions.
The antifungal activities of 1-3 were evaluated against three plant pathogenic fungi, including Botrytis cinerea, Rhizopus oryzae and Phytophthora capsici. Among them, 1 showed the potential activity against B. cinerea, R. oryzae and P. capsici with the MIC values of 0.39, 0.78 and 0.78 μM, respectively, stronger than that of the positive control carbendazim (0.78, 1.56 and 1.56 μM, respectively). Compounds 2 and 3 displayed no antifungal activity (MICs > 25.0 μM).

General experimental procedures
Optical rotations were measured on a JASCO P-1020 digital polarimeter. NMr spectra were acquired using a JeOL JeM-eCP NMr spectrometer (500 MHz for 1 H and 125 MHz for 13 C), using TMS as an internal standard. eSIMS spectra were obtained on a Micromass Q-TOF spectrometer. HPLC separation was performed in a Waters 1525 prep-HPLC system coupled with Waters 2996 photodiode array detector. A Kromasil C 18 semi-preparative HPLC column (250 × 10 mm, 5 μm) was used. Silica gel (Qing dao Hai Yang Chemical Group Co.; 200-300 mesh), Sephadex LH-20 (Amersham Biosciences) and octadecylsilyl silica gel (unicorn; 45-60 μm) were used for column chromatography. Precoated silica gel GF254 plates (Yantai Zifu Chemical Group Co.) were used for analytical TLC.

Fungus material
The fungus Pleosporales sp. CF09-01 was isolated from marine sediment, which was collected from the Huanghua in the Bohai Sea in June 2015. The strain (CF09-01) was deposited at the Key Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmaceutical Sciences, Hebei university, Baoding, People's republic of China.

Extraction and isolation
Ten erlenmeyer flasks of the fungal strain were cultivated in solid medium (erlenmeyer flasks each containing rice 80 g, water 100 mL, sea salt 3.0 g) at 28 °C for four weeks. The fermented solid medium was extracted three times with etOAc. The combined etOAc layers were evaporated to dryness under reduced pressure to give an etOAc extract. The etOAc extract (2.0 g) was subjected to silica gel column chromatography using a step gradient elution with etOAcpetroleum ether and then with MeOH-CHCl 3 to provide four fractions (Fr.1-Fr.4). Fr.3 was separated by CC on silica gel (Pe/etOAc = 1:1) and repeated chromatography over Sephadex LH-20 and finally purified by semi-preparative HPLC to yield 1 (10.0 mg), 2 (3.0 mg) and 3 (2.0 mg).

Biological assays
Antifungal activity was evaluated by the conventional broth dilution assay. Compounds were dissolved in dMSO. Carbendazim was used as the positive control, and dMSO (25 μg/mL) was used as the negative control. Those fungi were cultivated on PYG medium (peptone 1.0%, yeast extract 1.0%, glucose 2.0%) and incubated at 28 °C for 48 h. The seed culture was diluted in infusion broth and delivered into presterilised 96-well plates and adopted double dilution method to conduct. The plates were incubated for 48 h in a humidified incubator at 28 °C. The MIC was defined as the lowest concentration of the pure compound/antibiotics showing no visible growth after the incubation time. Three plant pathogenic fungi, B. cinerea, R. oryzae and P. capsici, were used, and carbendazim was used as a positive control.

Conclusion
Three azaphilone derivatives (1-3) were obtained from the Bohai Sea marine-derived fungus Pleosporales sp. CF09-01. Compounds 1-3 represent the first examples of azaphilone derivatives obtained from the fungus of the genus Pleosporales. Among them, pleosporalone A (1) represents the first azaphilone derivative characterised with A-ring aromatisation and also showed pharmaceutical potential as antifungal agents.

Supplementary material
The 1 H NMr, 13 C NMr, HMQC, COSY, HMBC, and MS spectra of 1 were available.

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

Funding
This work was supported by the Science and Technology Support Project of Baoding [grant number 15ZN020], Scientific research Foundation of Hebei educational committee [grant number QN2016177] and the Financial Support from the Hebei university.