Production of New Antibacterial 4-Hydroxy-α-Pyrones by a Marine Fungus Aspergillus niger Cultivated in Solid Medium

Four 4-hydroxy-α-pyrones including three new ones named nipyrones A–C (1–3) together with one known analogue germicidin C (4) were discovered from a marine sponge-derived fungus Aspergillus niger cultivated in a solid rice culture. Their structures and absolute configurations were elucidated through a combination of spectroscopic data and electronic circular dichroism (ECD) calculations as well as comparison with literature data. Compounds 1–4 were evaluated for their antibacterial activities against five pathogenic bacteria (Staphylococcus aureus, Escherichia coli, Bacillus subtilis, methicillin-resistant Staphylococcus aureus, and Mycobacterium tuberculosis). Compound 3 showed promising activity against S. aureus and B. subtilis, with minimum inhibitory concentration (MIC) values of 8 μg/mL and 16 μg/mL, respectively, and displayed weak antitubercular activities against M. tuberculosis, with MIC value of 64 μg/mL, while compounds 1 and 2 exhibited moderate antibacterial efficacy against four pathogenic bacteria with MIC values of 32–64 μg/mL.

Members of the genus Aspergillus are well known to produce chemically diverse secondary metabolites, many of which have been developed to therapeutic leads for human health [16][17][18][19]. During our ongoing search for sponge-derived fungi capable of producing antibiotics, a sponge-derived fungus Aspergillus niger LS24 showed antimicrobial activities. HPLC-UV profile of crude extract of A. niger LS24 grown on solid rice medium indicated the presence of various α-pyrone derivatives with the characteristic UV absorption similar to that of germicidin C [20]. Antibacterial-guided guided fractionation of the EtOAc extract from a scale-up culture led to the isolation and identification of three new 4-hydroxy-α-pyrones, named nipyrones A-C (1-3) and one known analogue, germicidin C (4) (Figure 1). Compounds 1-4 were evaluated for their antibacterial activities against five pathogenic bacteria. Herein, the isolation, structure elucidation, and antibacterial evaluation of these α-pyrones are described.

Structure Elucidation
Nipyrone A (1) was obtained as a colorless oil. The molecular formula of 1 was assigned as C 13 H 20 O 3 by HRESIMS data and gave an [M + H] + peak at m/z 225.1487, suggesting four degrees of unsaturation. Its UV absorption at 290 nm indicated the presence of a conjugated α-pyrone chromophore [21] ( Figure S2). The 1 H-NMR spectrum ( Table 1)  3), C-3 (δ C 98.7), and C-4 (δ C 168.7) as well as H-5/C-3 (δ C 98.7) in the HMBC spectrum suggested the existence of the 4-hydroxy-3-methyl α-pyrone skeleton ( Figure 2 and Figure S7). The structure of a 1,3-dimethylpentyl group was further verified by the COSY correlations (H-7/H 2 -8/H 3 -13, H-9/H 3 -12, and H-10/H 3 -11) and HMBC correlations from H 3 -12/H 3 -13 to C-8 (δ C 41.7) and H 3 -11 to C-9 (δ C 32.1). The 1,3-dimethylpentyl group was connected to C-6 (δ C 167.3) of 4-hydroxy-3-methyl α-pyrone moiety, supported by the HMBC correlations of H-7/C-5 (δ C 100.0) and C-6 together with H 3 -13/C-6 ( Figure 2). Thus, the planar structure of 1 was established as 4-hydroxy-3-methyl-6-(-4-methylhexan-2-yl)-2H-pyran-2-one. The relative configuration of 1 was established by the NOESY data ( Figure S9). The NOE correlation between H 3 -12/H 3 -13 clarified H 3 -12 and H 3 -13 to be cofacial of the side chain ( Figure 2). Thus, the absolute configuration at C-7 and C-9 of 1 was identified as 7S,9S or 7R,9R. The absolute configuration of 1 was further determined by comparing the experimental electronic circular dichroism (ECD) spectrum of 1 with the correspondingly time-dependent density functional theory (TDDFT)-calculated one. The Cotton effects of the experimental ECD spectrum of 1 matched very well with the calculated ECD spectrum for the model molecule of 7S,9S at the B3LYP/6-311 + G(d, p) level ( Figure 3 and see Supplementary Materials), thus confirming its absolute configuration.   Nipyrone B (2) was also obtained as a colorless oil with a molecular formula of C14H22O3 as determined by HRESIMS data with one CH2 more than 1. The spectroscopic data of 2 (Table 1) were highly identical to that of 1 except for the absence of one olefinic proton and an additional methyl singlet. The additional methyl group was located at C-5 (δC 106.8, Table 1) by HMBC correlation from H3-14 to C-6 ( Figure 2 and see Supplementary Materials). The absolute configuration of 2 tentatively led to deduction of the same as 1 based on the biosynthetic consideration and specific rotation.
Nipyrone C (3) was isolated a as a colorless oil, and its molecular formula was assigned as C14H23O4 based on the HRESIMS ([M + H] + m/z 255.1591) and 13 C NMR data. The 1D and 2D NMR   Nipyrone B (2) was also obtained as a colorless oil with a molecular formula of C14H22O3 as determined by HRESIMS data with one CH2 more than 1. The spectroscopic data of 2 (Table 1) were highly identical to that of 1 except for the absence of one olefinic proton and an additional methyl singlet. The additional methyl group was located at C-5 (δC 106.8, Table 1) by HMBC correlation from H3-14 to C-6 ( Figure 2 and see Supplementary Materials). The absolute configuration of 2 tentatively led to deduction of the same as 1 based on the biosynthetic consideration and specific rotation.
Nipyrone C (3) was isolated a as a colorless oil, and its molecular formula was assigned as C14H23O4 based on the HRESIMS ([M + H] + m/z 255.1591) and 13 C NMR data. The 1D and 2D NMR  Nipyrone B (2) was also obtained as a colorless oil with a molecular formula of C 14 H 22 O 3 as determined by HRESIMS data with one CH 2 more than 1. The spectroscopic data of 2 (Table 1) were highly identical to that of 1 except for the absence of one olefinic proton and an additional methyl singlet. The additional methyl group was located at C-5 (δ C 106.8, Table 1) by HMBC correlation from H 3 -14 to C-6 ( Figure 2 and see Supplementary Materials). The absolute configuration of 2 tentatively led to deduction of the same as 1 based on the biosynthetic consideration and specific rotation. Nipyrone C (3) was isolated a as a colorless oil, and its molecular formula was assigned as C 14 H 23 O 4 based on the HRESIMS ([M + H] + m/z 255.1591) and 13 C NMR data. The 1D and 2D NMR spectroscopic data for 3 (Table 1) closely resembled those of 1. Significant differences in the NMR data for 3 were only found in the resonances assigned to an additional hydroxy group and an additional methoxy group. Moreover, the C-9 position in 1 replaced by a hydroxy group in 3 was confirmed by chemical shift C-9 (δ C 72.9). The attachment of the methoxy group (C-14, δ C 56.1) at C-4 was supported by the HMBC correlation of H 3 -14/C-4 (see Supplementary Materials). Based on the biosynthetic point of view and specific rotation, the absolute configuration of 3 might be assigned to be 7S,9R.
In addition, compound 4 was identified as germicidin C by comparison of its spectral data with those reported in [20]. Many α-pyrone-based secondary metabolites biosynthesized by polyketide synthetase (PKS) pathway have been widely reported, uncovering their biosynthetic gene clusters [7,22,23]. We proposed that the polyketide chain primed with acetyl-CoA and malonyl-CoA was elongated, enolized, cyclized, methylated, hydroxylated, and released as the corresponding four 4-hydroxy-α-pyrones. A probable biosynthesis pathway of 1-4 is illustrated in Scheme 1.  (Table 1) closely resembled those of 1. Significant differences in the NMR data for 3 were only found in the resonances assigned to an additional hydroxy group and an additional methoxy group. Moreover, the C-9 position in 1 replaced by a hydroxy group in 3 was confirmed by chemical shift C-9 (δC 72.9). The attachment of the methoxy group (C-14, δC 56.1) at C-4 was supported by the HMBC correlation of H3-14/C-4 (see Supplementary Materials). Based on the biosynthetic point of view and specific rotation, the absolute configuration of 3 might be assigned to be 7S,9R. In addition, compound 4 was identified as germicidin C by comparison of its spectral data with those reported in [20]. Many α-pyrone-based secondary metabolites biosynthesized by polyketide synthetase (PKS) pathway have been widely reported, uncovering their biosynthetic gene clusters [7,22,23]. We proposed that the polyketide chain primed with acetyl-CoA and malonyl-CoA was elongated, enolized, cyclized, methylated, hydroxylated, and released as the corresponding four 4hydroxy-α-pyrones. A probable biosynthesis pathway of 1-4 is illustrated in Scheme 1.

Biological Activities
The antibacterial activities of 1-4 were evaluated against four bacteria, including Gram-positive and Gram-negative bacteria, using broth micro-dilution method within a concentration range of 256-1 μg/mL. Four pathogenic bacteria, including S. aureus, E. coli, B. subtilis, and methicillin-resistant S. aureus (MRSA) were performed. The results are shown in Table 2

Fungal Material
Marine sponge Haliclona sp. was collected at Lingshui, Hainan Province, China. The sponge tissue was cut into small pieces of about 0.1 cm 3 each, which were homogenized with sterile seawater. 20 µL of the diluted homogenate (1:100, sterile seawater) was inoculated in PDA agar plates, which were incubated for periods of 3 days to 4 weeks for purifying fungal colonies. 15 fungal isolates were obtained. Among them, the EtOAc extract of the fungal strain LS24 showed antimicrobial activities. Surprisingly, its extract showed stronger antimicrobial effects (MICs, 32-128 µg/mL against different pathogenic bacteria) when grown on solid rice medium in comparison to liquid medium (MICs, >128 µg/mL against different pathogenic bacteria). The fungal strain LS24 was identified using morphological studies, DNA amplification, and the internal transcribed spacer region (ITS) sequencing (GenBank accession ID: KX290301, 100% similarity). The isolate was stored on PDA medium (potato 200 g, dextrose 20 g, sea salt 35 g and agar 15 g in 1.0 L of H 2 O, pH 7.4-7.8) slants at 4 • C. A voucher strain was preserved at College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo, China.

Fermentation
The fungus A. niger was cultured on PDA agar plate at 28 • C for 7 days. The fungal colony was further inoculated into the PDB medium (potato 200 g, dextrose 20 g, and sea salt 35 g in 1.0 L of H 2 O, pH 7.4-7.8) at 28 • C for 3 days on a rotating shaker (180 rpm). Then, a large-scale fermentation of the strain was performed. The fungal seed broth (20 mL) was added to 10 flasks (1000 mL), each containing 100 g rice and 160 mL water. These flasks were incubated at 28 • C for 30 days under static conditions.

Extraction and Isolation
All the fermented materials were extracted with 5 L EtOAc three times to afford a brown extract (8 g

Antibacterial Assay
The antibacterial effects of compounds 1-4 were evaluated against Gram-positive bacteria S. aureus ATCC 25923, B. subtilis ATCC 6633, methicillin-resistant S. aureus ATCC 43300 (MRSA), and Gram-negative bacterium E. coli ATCC 25922 according to a previously described method [24]. The test compounds were dissolved in DMSO (1 mg/mL for 1-4). The minimum inhibitory concentration (MIC) values were defined as the lowest concentration of test compound that inhibited the growth of more than 99% of the bacterial population after overnight incubation in 96-well microtiter plates, as detected by eye. Briefly, 100 µL of each bacterial solution was inoculated in each well (10 5 CFU/mL) and added with 100 µL of each compound solution and control in triples. Microplates were incubated for 24 h at 37 • C. The final concentrations of each test compound in the wells were in the range of 256-1 µg/mL using sequential 2-fold serial dilutions. The final DMSO concentration was maintained at 0.5% by adding the medium. Chloramphenicol and DMSO were used as the positive control and the negative control, respectively. The detailed method of the antitubercular activity of compounds 1-4 against M. tuberculosis H37Rv was described by the agar proportion method based on the previous report [25]. The blank control was DMSO. Ethambutol was used as a positive control.

Conclusions
In summary, three new 4-hydroxy-α-pyrone derivatives, nipyrones A-C (1-3) along with one known analogue germicidin C (4) were isolated from a marine sponge-derived fungus A. niger grown on a solid rice culture. These 4-hydroxy-α-pyrone derivatives have in common the differences in functional group substitution and side chain length. Biogenetically, nipyrones A-C (1-3) are presumably originated from the polyketide pathway. This study further expanded the structural diversity of naturally occurring α-pyrone derivatives. Notably, compound 3 displayed significant inhibitory effects on two pathogenic bacteria, S. aureus and E. coli and may be considered to have potential as an antibiotic agent.