Seven naphtho-γ-pyrones from the marine-derived fungus Alternaria alternata: structure elucidation and biological properties

Eight bioactive pyrone derivatives were identified from the culture of Alternaria alternata strain D2006, isolated from the marine soft coral Denderonephthya hemprichi, which was selected as its profound antimicrobial activities. The compounds were assigned as pyrophen (1), rubrofusarin B (2), fonsecin (3), and fonsecin B (5) beside to the four dimeric naphtho-γ-pyrones; aurasperone A (6), aurasperone B (7), aurasperone C (8), and aurasperone F (9). Structures of the isolated compounds were identified on the basis of 1D and 2D NMR spectroscopy and mass (EI, ESI, HRESI) data, and by comparison with the literature. Configuration of the four dimeric naphtho-γ-pyrones 6-9 was analyzed by CD spectra, exhibiting an identical stereochemistry.


Background
Infectional diseases and drug resistance phenomena are the most effective reasons for the death of ca. 20 millions yearly. For example, tuberculosis (TB) was the leading cause of ca. two million deaths due to a bacterial pathogen, Mycobacterium tuberculosis, among them more than 80% of TB patients living in sub-Africa and Asia [1][2][3][4]. Thus, new and more-powerful drugs are necessary to solve these problems. Marine microorganisms, especially fungi, are still a less investigated resource of bioactive substances [5,6]; recent investigations indicated their tremendous potential as source of new drugs [7][8][9][10][11][12][13].
In this article, a report on the antimicrobial activity of naphtho-γ-pyrones (naphthopyran-4-ones) attracted our interest [14]. During the investigation of fungal strains for the production of structurally novel active compounds from marine microorganisms, we found that the EtOAc extract of the marine-derived fungal strain Alternaria alternata D2006 (isolated from a red soft coral, Denderonephthya hemprichi, collected from the Red Sea at Safaga coasts, Egypt) was selected due to its distinctive features in the chemical and biological assays. We therefore performed a bioassay-guided fractionation.

Taxonomy and characterization
The fungal isolate was identified as A. alternata (Dematiaceae) according to Barnett [17]. Microscopically, the conidiophores were dark, simple, rather short or elongate and contained simple or branched chains of conidia. Conidia were dark, typically with both cross and longitudinal septa, with various shapes, obclavate to elliptical or ovoid. The fungal spores were multicellular, dark and having thick cell walls.

Results and discussion
The fungal extract showed several UV absorbing (254 nm) yellow bands, exhibiting yellowish-green UV fluorescence at 366 nm. On spraying with anisaldehyde/sulphuric acid and heating they turned orange to dark red, but showed no colour change with sodium hydroxide, thus excluding peri-hydroxyquinones.
The molecular formula of compound 1 was determined by HRMS as C 16 H 17 NO 4 ; the 1 H NMR spectrum revealed signals for a phenyl residue, an amino NH doublet, and two m-coupled methines (δ 5.90, 5.43). Further signals were a methine quartet, a methylene 2H multiplet and two methyl singlets. The 13 C NMR/ HMQC spectra indicated the existence of 16 carbons corresponding to a phenyl residue, 2 up-field sp 2 methines (δ100.6, 88.0), 4 quaternary sp 2 atoms (δ171.0-161.9), representing carbonyls or phenolic carbons, and 4 sp 3 carbon signals (δ55. 7-22.3). According to these data, compound 1 was identified as pyrophen (1) [5], which was isolated and reported previously from Aspergillus niger [18,19] and elucidated by crystal structure analysis. Here, we report the full NMR assignments data for 1 using the 2D NMR experiments for the first time ( Figure 1 and  [20], 5 non-oxygenated sp 2 , 2 aromaticattached methyl ethers (δ 56.0, 55.4) and 1 sp 2 -attached methyl (δ 20.6). Full assignment of the 2D NMR experiments ( Figure 2 and Table 2) established the structure of 2 as rubrofusarin B, and excluded the structure of the isomeric asperxanthon (11) in the same way [21]. Structure of 2 was not fully assigned using 2D NMR before, which we report her to first time (see Additional file 2).
The closely related compound 3 afforded a molecular weight of 290 Da (C 15 H 12 O 5 by HRESI MS); EI MS gave easily an ion peak at m/z 272 by expulsion of water molecule. The 1 H NMR spectrum exhibited aromatic m-coupled doublets (δ 6.47, 6.31, J~1.1 Hz) and a methine singlet (δ 6.41), but in contrast to 2, two phenolic hydroxy signals (δ 14. 19, 10.18), and only one methoxy signal (δ 3.84). In addition, an AB signal of diastereotopic methylene protons (δ 3.14, 2.72, J~16.8) and a methyl singlet (δ 1.60) were visible. Based on 13 C/ HMQC spectra (Table 2) and HMBC experiment (as it was not fully assigned before using 2D NMR) (  Compound 5 displayed similar chromatographic properties and the same 1 H NMR pattern as 3. The molecular weight of 5 was deduced as 304 Da, which is 14 amu higher than that of 3, attributing to the methylation of the phenolic hydroxyl group at 8-position, hence compound 5 was identified as fonsecin B [22] (see Additional file 4)

Aurasperones A-C and F
Compound 6 was obtained from fraction II as middle polar yellow solid, displaying a molecular weight at m/z 570. The expectation of a dimeric rubrofusarin B (2) was confirmed by 1 H NMR spectra, where six sp 2 methine protons were visible, which were classified into two m-coupled protons, two α-methines of the consequent γ -pyrones (δ 6.15, 6.08) and two singlet methines (δ 7.35 and 7.24), together with six methyls, among them four methoxy signals. Based on these data and search in literature, compound 6 was identified as aurasperone A [22] (see Additional file 5) Compound 7 exhibited a close structural similarity with fonsecin B (5); the molecular weight was determined as 606 Da, corresponding to the molecular formula C 32 H 30 O 12 (HRESI MS). EI MS of 7 displayed an ion signal at m/z 570 as base peak, resulting from the expulsion of two water molecules, affording the molecular weight of aurasperone A (6). The 1 H NMR spectrum established a dimeric pattern of fonsecin B (5), where four sp 2 methines protons being of two m-coupled protons and two singlet methines; two methylene signals (δ 3.02 and 2.89) instead of the two α-methines of the consequent-γpyrones shown in 6, along with six methyls, among them four methoxy signals and two sp 3 -bounded methyl signals (δ 1.79, 1.46). In accordance, structure of 7 was assigned as aurasperone B (7) [22]. (see Additional file 6) A third dimer 8 had a molecular weight of 592 Da and a corresponding molecular formula C 31 H 28 O 12 . Three consecutive fragment ions (m/z 574, 556 and 525) on EI MS corresponded to the expulsion of one H 2 O molecule (to afford aurasperone F, 9), two H 2 O (dianhydroaurasperone C, 10) and 2H 2 O + OCH 3 , respectively. The 1 H NMR spectrum displayed the same pattern as in aurasperone B (7), except that the methoxy signal (δ 3.78) of 8-OCH 3 in 7 was replaced by a phenolic hydroxyl group, pointing to aurasperone C (8) [23]. (see Additional file 7) Compound 9 was a fourth dimer with a molecular formula C 31 H 26 O 11 ; on EI MS, it displayed a fragment ion at m/z 556 corresponding to an aromatized structural analogue (dianhydroaurasperone C, 10), and a further fragment at m/z 286 corresponded to rubrofusarin B (2). The 1 H NMR spectra displayed five sp 2 methines (δ 6.87-6.08), one less than in 6, replaced by an AB signal of a methylene group (δ 3.35-3.25). Accordingly, one of the β-bounded methyls of the lactones was up-field shifted (δ 1.65), while the other one was retained at δ 2.16 as in 6. In contrast to 6, only three methoxy signals (δ 3.95-3.43) were visible, while the fourth one was replaced by a phenolic OH. Based on these spectroscopic features, structure 9 was confirmed as aurasperone F [24] (see Additional file 8) The four dimeric naphtho-g-pyrones (6-9) were presently constructed from two naphtho-g-pyrone units, which are not symmetrically linked; i.e. the first pyrone (above) unit is linked via a middle aromatic moiety (10'position) to a terminal aromatic residue (7-position) of the second pyrone (down) unit.  The optical rotations of the dimers had the same negative sign and similar values indicating that the optical rotation value was dominated by the chiral axes between the two naphthopyranone moieties (atropisomerism). The absolute configurations of dimeric naphtho-g-pyrones have been determined by circulardichroism (CD). According to the literature [25], (S)configured dimeric naphtho-g-pyrones exhibit a first positive Cotton Effect in the long-wavelength region, a negative Cotton Effect at middle wavelength and then a positive Cotton Effect at shorter one. In our experimental data, the CD spectra for three representative dimeric naphtho-g-pyrones (6)(7)(8) showed closely related values with pronounced Cotton Effects, recognizing them to have the same patterns. In accordance, the ellipticity of aurasperones A-C (6-8) showed three Cotton Effects, one peak was shown firstly in the region of [θ] 284-285 +359274-22843.4, then one trough between [θ] 270-267 -151670-339938 and the last elliptical peak was shown at [θ] 227-219 +107899-5629. As the dimer 6 has no further chiral elements, the chiral axis is dominating the absolute configuration. Based on the revealed features from the CD spectroscopic data, the four dimeric compounds (6-9) have identical (S)-configurations around their corresponding axis between C-10' and C-7 (see Additional file 9)

Biological activities
The antibiotic activity of compounds 1-8 was examined against 11 microbial test organisms using the agar diffusion method (40 μg/disc) ( Table 3). According to the antimicrobial assay, the crude extract of the fungal strain exhibited high activity against bacteria and yeasts (Table 4). Nevertheless, only three of the isolated metabolites were found to exhibited activity: pyrophen (1) and rubrofusarin B (2) displayed high (28 mm) and moderate (12 mm) activity against C. albicans, respectively, while aurosperone A (6) was active (13 mm) against the plant pathogenic fungi, Rhizoctonia solani. In the brine shrimp assay (10 μg/mL), all studied compounds here showed weak cytotoxicity (approx. 4-11%).

Experimental
The NMR spectra were measured on a Bruker AMX 300 (300.

Sampling and isolation of the fungal strain
The reddish soft coral D. hemprichi was collected from the Red Sea; approx. 30 km offshore from Safaga (east Egypt) at a depth of approx. 30 m. Pieces of the coral were rinsed three times with sterile seawater and then aseptically cut into smaller pieces and shaken for 2 h. The aqueous supernatant was serially diluted, and each 200 μL were inoculated onto 15-cm Petri dishes, each containing 50 mL of yeast extract/starch agar (yeast extract 0.2 g/L, soluble starch 1.0 g/L, agar 20 g/L, chloramphenicol 50 mg/L natural seawater at pH 6.0) [7]. The black single colonies were picked from the plates after inoculation for 25 days at 30°C and subcultured on the same medium without chloramphenicol. The strain is deposited in the culture collection of the Department of Microbial Chemistry, NRC, Cairo, Egypt.

Fermentation and working up
The well-grown single colonies of A. alternata were inoculated in subculture agar slants containing malt extract medium: malt extract (30 g/L), peptone 5 g/L), agar (20 g/L), natural sea water (1000 mL); at pH approx. 5.5 for 7 days at 30°C). The obtained grown agar slants were served to inoculate 500-mL Erlenmeyer flasks, each containing 100 mL of GYMP medium (g/L): malt extract (3), yeast extract (3), peptone (5), glucose (10) and 1000 mL natural seawater at pH approx. 6.5 at 30°C. The culture media was in turn applied to cultivation on a rotary shaker (10 days). After harvesting, the afforded black broth was centrifuged (7,000 rpm for 15 min), and the obtained two phases, mycelial cake and supernatant, were individually extracted with ethyl acetate. The obtained unique black organic extracts were applied to biological and chemical screenings. The well-grown agar slants of the fungal strain D2006 were served to inoculate 60 of 1-L Erlenmeyer flasks, each containing 300-mL of GYMP medium (g/L): malt extract (3), yeast extract (3), peptone (5), glucose (10), agar (20) and 1000 mL of 100% seawater at pH approx. 6.5. The inoculated media was applied to additional cultivation using a rotary shaker (150 rpm) for 10 days. After harvesting, the obtained black culture broth was mixed with celite (approx. 1.5 kg) and then filtered in vacuo. The afforded two phases, filtrate and mycelium, were applied to exhaustive extraction by ethyl acetate. TLC of both organic extracts recognized their unique, and they were combined therefore, and concentrated in vacuo, affording 5.5 g as black crude extract.

Biological activities Antimicrobial activity
Compounds 1-8 were dissolved in CH 2 Cl 2 /10% MeOH at a concentration of 1 mg/mL. Aliquots of 40 μL were soaked on filter paper discs (9 mm ∅, no. 2668, Schleicher & Schüll GmbH, Germany) and dried for 1 h at room temperature under sterilized conditions. The paper discs were placed on inoculated agar plats and incubated for 24 h at 38°C for bacterial and 48 h (30°C) for the fungal isolates, while the algal test strains were incubated at room temperature in day light.
For the fungal extract examination, representative test microbes; P.aeruginosa, S. aureus, C. albicans and A. niger were used. Both bacterial and yeast strains were grown on nutrient agar medium (g/L): Beef extract 3; peptone, 10; and agar, 20. The pH was adjusted to 7.2. The fungal strain was grown on Czapek-Dox medium (g/l): Sucrose, 30; NaNO 3 , 3; MgSO 4 .7H 2 O, 0.5l; KCl, 0.5; FeSO 4 , 0.01; K 2 HPO 4 , 1; and agar, 20. The pH was maintained at 6.0. The disc diffusion test has been done according to Collins and Lyne [26]. Filter paper discs (5 mm diameter) were saturated with 200 μg from the culture extract, and located on the surface of the agar plates (150 mm diameter containing 50 mL of solidified media). The paper discs were placed on inoculated agar plats and incubated for 24 h at 38°C (bacteria and yeast) and 48 h at 30°C (fungi).