Antimicrobial Secondary Metabolites from the Seawater-Derived Fungus Aspergillus sydowii SW9

Marine-derived fungi are considered to be valuable producers of bioactive secondary metabolites used as lead compounds with medicinal importance. In this study, chemical investigation of the seawater-derived fungus Aspergillus sydowii SW9 led to the isolation and identification of one new quinazolinone alkaloid, 2-(4-hydroxybenzyl)-4-(3-acetyl)quinazolin-one (1), one new aromatic bisabolene-type sesquiterpenoid, (2) and one new chorismic acid analogue (3), as well as two known alkaloids (compounds 4 and 5). Their structures were determined by extensive 1D/2D NMR and mass spectrometric data, and the absolute configurations of 2 and 3 were assigned by the analysis of ECD spectra aided by quantum chemical computations. Compounds 1, 2, and 4 exhibited selective inhibitory activities against the human pathogenic bacteria Escherichia coli, Staphylococcus aureus, S. epidermidis, and Streptococcus pneumoniae, with MIC values ranging from 2.0 to 16 μg/mL.

With the purpose of further searching for new antibiotic active metabolites from A. sydowii, we continued our studies, and five compounds, including three previously undescribed compounds (1-3) along with two known alkaloids (4, 5) (Figure 1), were obtained and identified from the culture extract of A. sydowii SW9. Their structures were identified by extensive 1D/2D NMR and mass spectrometric data, and the absolute configurations of the new compounds 2 and 3 were assigned by the analysis of ECD spectra aided by quantum chemical computations. All of these compounds were tested for antimicrobial activities against four human pathogenic bacterial strains.
With the purpose of further searching for new antibiotic active metabolites from A. sydowii, we continued our studies, and five compounds, including three previously undescribed compounds (1−3) along with two known alkaloids (4, 5) (Figure 1), were obtained and identified from the culture extract of A. sydowii SW9. Their structures were identified by extensive 1D/2D NMR and mass spectrometric data, and the absolute configurations of the new compounds 2 and 3 were assigned by the analysis of ECD spectra aided by quantum chemical computations. All of these compounds were tested for antimicrobial activities against four human pathogenic bacterial strains.

Results and Discussion
Compound 1 was obtained as white powder. Its molecular formula was determined as C17H14N2O3 by the HRESIMS at m/z 295.1079 [M + H] + ( Figure S1 in the Supplementary File), implying 12 degrees of unsaturation. The 1 H-NMR spectrum (Table 1 and Figure S2) displayed signals for eight aromatic protons in the downfield region. The 13 C-NMR and DEPT data (Table 1 and Figure S3) exhibited 17 carbon signals classified as one methyl, one methylene, eight methines, and seven quaternary carbons. Analysis of the 1 H-1 H coupling patterns (Figures 2 and S4) revealed an osubstituted aryl ring, and the remaining four aromatic protons were attributed to a p-substituted aryl ring with two sets of doublets (J = 7.8 Hz each) at δH 6.70 and 7.16 ppm, respectively. The HMBC correlation (Figures 2 and S6) from H-5 to C-4 revealed the connection of C-4a to C-4, whereas those from H2-9 to C-2, C-11/C-15 indicated that C-9 was attached to C-10 and the remaining sp 2 carbon C-2 (δC 157.7). The NMR data of 1 (Table 1) displayed similar signals to those of 2-(4hydroxybenzyl)quinazolin-4(3H)-one, a quinazolinone isolated from a Cordyceps-colonizing fungus Isaria farinose [16]. However, an extra methyl signal (δH 1.67 and δC 25.3) and a quaternary carbon (δC 175.8) were present in compound 1. The key HMBC correlation from H-2′ to C-1′ suggested that compound 1 was the acetyl derivative of 2-(4-hydroxybenzyl)quinazolin-4(3H)-one.

Results and Discussion
Compound 1 was obtained as white powder. Its molecular formula was determined as C 17 (Table 1 and Figure S2) displayed signals for eight aromatic protons in the downfield region. The 13 C-NMR and DEPT data (Table 1 and Figure S3) exhibited 17 carbon signals classified as one methyl, one methylene, eight methines, and seven quaternary carbons. Analysis of the 1 H-1 H coupling patterns (Figures 2 and S4) revealed an o-substituted aryl ring, and the remaining four aromatic protons were attributed to a p-substituted aryl ring with two sets of doublets (J = 7.8 Hz each) at δ H 6.70 and 7.16 ppm, respectively. The HMBC correlation (Figures 2 and S6) from H-5 to C-4 revealed the connection of C-4a to C-4, whereas those from H 2 -9 to C-2, C-11/C-15 indicated that C-9 was attached to C-10 and the remaining sp 2 carbon C-2 (δ C 157.7). The NMR data of 1 (Table 1) displayed similar signals to those of 2-(4-hydroxybenzyl)quinazolin-4(3H)-one, a quinazolinone isolated from a Cordyceps-colonizing fungus Isaria farinose [16]. However, an extra methyl signal (δ H 1.67 and δ C 25.3) and a quaternary carbon (δ C 175.8) were present in compound 1.
The key HMBC correlation from H-2 to C-1 suggested that compound 1 was the acetyl derivative of 2-(4-hydroxybenzyl)quinazolin-4(3H)-one.   The molecular formula of compound 2 was determined to be C16H22O4 based on its HRESIMS The molecular formula of compound 2 was determined to be C 16 Figure S7), indicating six degrees of unsaturation. The 1 H and 13 C-NMR spectroscopic data (Table 1 and Figures S8 and S9) along with HSQC correlations revealed the presence of 16 carbon atoms, which were assigned as six quaternary carbons, four methines including two aromatic methines at δ 119.1 (C-2) and 120.5 (C-3), two methylenes, and four methyls (with one oxygenated at δ 51.1, C-1 ). The 1 H-1 H COSY correlation (Figures 2 and S10) between H-2 (δ 6.28) and H-3 (δ 6.49) as well as the HMBC correlations (Figures 2 and S12) between H-2/C-6 and C-4, H-3/C-1 and C-5 revealed a 1,4,5,6-tetrasubstituted aryl ring. The HMBC correlations from H-3 to C-13, and from H-13 to C-3 and C-5 assigned the link of Me-13 and C-4. There should be hydroxyl groups bonded to C-5 and C-6, respectively, according to the downfield shifts of C-5 (δ 143.2) and C-6 (δ 142.1). The HMBC correlations from H-2 to C-7, and from H-14 to C-1 revealed the connectivity between C-7 and C-1. The HMBC correlations from H-14 to C-8, and from H-8 to C-1 and C-14 assigned the link of Me-14 and C-7. Moreover, this moiety was further extended from C-8 to C-11 and from C-11 to C-15 by 1 H-1 H COSY correlations, where C-8 was connected to C-7 on the basis of the HMBC correlations of H-8/C-14 and H-14/C-8. On the other hand, the HMBC correlations from H-10, H-15 to C-12 combined with the downfield chemical shift of C-12 indicated that an ester carbonyl (δ 176.1, C-12) was bonded to C-11. The HMBC correlations from H-1 to C-12 indicated the connectivity between them through an ester bond. Thus, the structure of compound 2 was assigned as a bisabolene-type sesquiterpenoid. The key NOE correlation (Figures 3 and S13) between H-8 and H-14 assigned the Z-double bond in C-7/C-8 (7Z). The experimental ECD spectra of compound 2 matched well with that calculated for (11S)-2 (Figures 4 and S14), leading to the absolute configuration of compound 2 being determined as 11S.
Molecules 2019, 24, x FOR PEER REVIEW 4 of 8 and S13) between H-8 and H-14 assigned the Z-double bond in C-7/C-8 (7Z). The experimental ECD spectra of compound 2 matched well with that calculated for (11S)-2 ( Figures 4 and S14), leading to the absolute configuration of compound 2 being determined as 11S.   (Figures 2 and S20) from H-3′′ and H-5′′ to C-1′′. Combined with the key HMBC correlations from H-3 and H-4 to C-1′′, the structure of compound 3 was definitively established.
The relative configuration of compound 3 was assigned by the analysis of NOESY data ( Figures  3 and S21). The key NOE correlations of H-3′ with H-3 and H-4 indicated that they were on the same side of the molecule, while the NOE correlation between H-5 and H-1′ revealed them on the other     (Figure S15), indicating six degrees of unsaturation. Detailed analysis of the 1D and 2D NMR spectra data ( Table 2 and Figures S16 and S17) showed that the signals of 3 were similar to those of methyl[(lR),lα,5β,6α]-5-(l-methyl-2-methoxy-2-oxoethyl)-8,8-dimethyl-7,9dioxabicyclo[4.3.0]non-2-ene-3-carboxylate, a biosynthetically interrelated intermediate which deals with synthesis of 5-enolpyruvylshikimic acid-3-phosphate (EPSP) analogues [17], revealing that compound 3 may also be produced by the same shikimic acid pathway. The main differences between them were the presence of five methylene groups at δH/C 1.46/37.  (Figures 2 and S20) from H-3′′ and H-5′′ to C-1′′. Combined with the key HMBC correlations from H-3 and H-4 to C-1′′, the structure of compound 3 was definitively established.
The relative configuration of compound 3 was assigned by the analysis of NOESY data ( Figures  3 and S21). The key NOE correlations of H-3′ with H-3 and H-4 indicated that they were on the same side of the molecule, while the NOE correlation between H-5 and H-1′ revealed them on the other   (Figure S15), indicating six degrees of unsaturation.
The obtained compounds 1-5 were tested for antimicrobial activities against four human pathogenic bacterial strains ( Table 3). The new compound 1 exhibited obvious inhibitory activity against S. epidermidis, with an MIC value of 4.0 µg/mL. Compound 2 showed significant inhibitory activity on E. coli, with an MIC value of 2.0 µg/mL, comparable to that of the positive control chloramphenicol (MIC 2.0 µg/mL). Moreover, compound 2 also exhibited potent activity against S. pneumoniae, with an MIC value of 4.0 µg/mL. Table 3. Antimicrobial activities of compounds 1-5 (MIC, µg/mL) a .

Fungal Material
The fungus Aspergillus sydowii SW9 was isolated from a seawater sample collected in August 2016, from Yangma Island, Yantai, China. The fungus was identified by morphological observation and analysis of the Internal Transcribed Spacer (ITS) regions of its rDNA, whose sequence data were deposited at GenBank with the accession number MN696205.

Extraction and Isolation
The mycelia were separated from the culture broth by filtration, and they were dried in the shade and exhaustively extracted with a mixture of ethyl alcohol and H 2 O (95:5, v/v). After removing organic solvents by evaporation under vacuum, the residue was partitioned between EtOAc and H 2 O to produce an EtOAc soluble extract. The filtrate was directly extracted with EtOAc and then concentrated to afford an extract. Since the TLC and HPLC profiles of the two EtOAc extracts were almost identical, they were combined and concentrated under reduced pressure to produce an extract (27.2 g) for further separation.

Antimicrobial Assays
Antimicrobial evaluation against pathogenic bacteria (Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pneumoniae) was assayed as described previously [20], with chloramphenicol as the positive control. The specific experiment was as follows: the initial cultures were maintained on Luria Broth (LB) agar plates for each pathogenic strain, picking bacterial colonies, and suspended in Mueller-Hinton broth to approximately 5 × 10 5 CFU/mL. Aliquots of bacterial suspension (95 µL) and compound dilution (5 µL) were then added to each well (to give final compound concentrations of 128 to 0.125 µg/mL in 2.0% DMSO), and the plate was incubated at 37 • C aerobically for 24 h. After that, the optical density of each well at 600 nm was measured using a Tecan GENios multifunctional microplate reader (infinite M1000 PRO, Männedorf, Switzerland). Then, MIC values were defined as the minimum concentration of compound that inhibited visible bacterial growth. The pathogenic bacterial strains were provided by the Key Laboratory of Marine Biotechnology in Universities of Shandong, Ludong University.

Computational Section
The conformational searches of compounds 2 and 3 were performed via molecular mechanics using the MM+ method in HyperChem software (Version 8.0, Hypercube, Inc., Gainesville, FL, USA), and the geometries were further optimized at the B3LYP/6-31G(d) level via Gaussian 09 software (Version D.01; Gaussian, Inc., Wallingford, CT, USA) [21] to give the energy-minimized conformers. Then, the optimized conformers were subjected to the calculation of ECD by using TDDFT at B3LYP/6-31G level. Solvent effects of the MeOH solution were evaluated at the same DFT level using the SCRF/PCM method.

Conclusions
One new alkaloid, 2-(4-hydroxybenzyl)-4-(3-acetyl)quinazolin-one (1), one new aromatic bisabolene-type sesquiterpenoid (2) and one new chorismic acid analogue (3), along with two known quinazolinone alkaloids (4, 5), were isolated and identified from the culture extract of Aspergillus sydowii SW9, a seawater-derived fungus. The structures of the new compounds were elucidated using NMR and HRESIMS data analysis as well as quantum chemical ECD calculations. All of the isolated compounds were tested for antimicrobial activities against four human pathogenic bacterial strains. The new compounds 1 and 2, and the known compound 4 exhibited obvious inhibitory activities against the human pathogenic bacterial strains tested.