Diketopiperazine Alkaloids and Bisabolene Sesquiterpenoids from Aspergillus versicolor AS-212, an Endozoic Fungus Associated with Deep-Sea Coral of Magellan Seamounts

Two new quinazolinone diketopiperazine alkaloids, including versicomide E (2) and cottoquinazoline H (4), together with ten known compounds (1, 3, and 5–12) were isolated and identified from Aspergillus versicolor AS-212, an endozoic fungus associated with the deep-sea coral Hemicorallium cf. imperiale, which was collected from the Magellan Seamounts. Their chemical structures were determined by an extensive interpretation of the spectroscopic and X-ray crystallographic data as well as specific rotation calculation, ECD calculation, and comparison of their ECD spectra. The absolute configurations of (−)-isoversicomide A (1) and cottoquinazoline A (3) were not assigned in the literature reports and were solved in the present work by single-crystal X-ray diffraction analysis. In the antibacterial assays, compound 3 exhibited antibacterial activity against aquatic pathogenic bacteria Aeromonas hydrophilia with an MIC value of 18.6 μM, while compounds 4 and 8 exhibited inhibitory effects against Vibrio harveyi and V. parahaemolyticus with MIC values ranging from 9.0 to 18.1 μM.


Introduction
Marine-derived fungi living under extreme survival conditions are considered as abundant sources of structurally diverse and biologically active compounds [1,2]. In the deep-sea habitats, seamounts are regarded locations for a wide variety of current-topography interactions and biophysical coupling which have large biomass and higher biodiversity than their surrounding deep-sea floors [3,4]. Endozoic fungi surviving in deep-sea seamounts are a promising new source to mining bioactive secondary metabolites owing to their unique habitats. To date, only three papers investigating bioactive secondary metabolites of fungi derived from deep-sea seamounts have been published [5][6][7]. Therefore, a study on the chemical diversity of deep-sea seamount-derived endozoic fungi is warranted.
In our continuous efforts to explore bioactive metabolites from deep-sea seamountderived fungi [5][6][7], chemical investigation of the endozoic fungus Aspergillus versicolor AS-212 associated with the deep-sea coral, Hemicorallium cf. imperiale, which was collected from the Magellan Seamounts in the Western Pacific Ocean was carried out due to its unique HPLC profiles. As a result, two new quinazolinone diketopiperazine alkaloids, namely, versicomide E (2) and cottoquinazoline H (4), together with five known related analogs (1, 3, 5-7) as well as four known bisabolene derivatives (8)(9)(10)(11) and a bisabolene dimer (12), have been isolated and identified. Herein, we report the isolation and structure elucidation as well as the antimicrobial activities of compounds 1-12 ( Figure 1).

Structure Elucidation of the Isolated Compounds
Compound 1 was isolated as colorless crystals, and the molecular formula was established as C19H25N3O3 by analysis of the HRESIMS data. The 1 H and 13 C NMR of 1 (DMSO-d6, Table 1) extremely resembled those of versicomide A, a quinazoline-containing compound isolated from the crab-derived fungus Aspergillus versicolor XZ-4 which was collected from hydrothermal vent [8]. Further analysis of the 2D NMR spectra (Figure 2) indicated the same planar structure of 1 as that of versicomide A ( Figure 1). However, a strong NOE cross-peak of H-3/H-20 was in favor of the structure with the 3S*-and 14R*relative configuration rather than a 3S* and 14S* configuration ( Figure 3). Single-crystal X-ray diffraction analysis with Cu Kα radiation further demonstrated its structure and absolute configurations (Figure 4). A Flack parameter of 0.0(2) enabled the definition of

Structure Elucidation of the Isolated Compounds
Compound 1 was isolated as colorless crystals, and the molecular formula was established as C 19 H 25 N 3 O 3 by analysis of the HRESIMS data. The 1 H and 13 C NMR of 1 (DMSO-d 6 , Table 1) extremely resembled those of versicomide A, a quinazoline-containing compound isolated from the crab-derived fungus Aspergillus versicolor XZ-4 which was collected from hydrothermal vent [8]. Further analysis of the 2D NMR spectra (Figure 2) indicated the same planar structure of 1 as that of versicomide A ( Figure 1). However, a strong NOE cross-peak of H-3/H-20 was in favor of the structure with the 3S*-and 14R*-relative configuration rather than a 3S* and 14S* configuration ( Figure 3). Singlecrystal X-ray diffraction analysis with Cu Kα radiation further demonstrated its structure and absolute configurations ( Figure 4). A Flack parameter of 0.0(2) enabled the definition of its absolute configuration as 3S, 14R, and 15S, indicating that 1 was the 14-epimer of versicomide A. sicomide A.        Compound 1 was initially treated as a new quinazoline alkaloid during the preparation of this manuscript, while Tasdemir and co-workers recently reported a new quinazoline-containing diketopiperazine (−)-isoversicomide A from the deep-sea sediment-de- Compound 1 was initially treated as a new quinazoline alkaloid during the preparation of this manuscript, while Tasdemir and co-workers recently reported a new quinazolinecontaining diketopiperazine (−)-isoversicomide A from the deep-sea sediment-derived fungus Aspergillus versicolor PS108-62 [13]. Notably, compound 1 shared the same planar structure and virtually similar optical rotation value ([α] 25 D −30 vs. [α] 20 D −25) as that of (−)-isoversicomide A, in which the stereogenic centers at C-3 and C-14 showed the same relative configurations with that of compound 1. However, the configuration at C-18 on the short flexible aliphatic chain and the absolute configuration of (−)-isoversicomide A were not assigned due to the limited sample available [13]. Considering their similar rotation values and same relative configuration at C-3 and C-14, we assumed that compound 1 and (−)-isoversicomide A are the same compound. As the reported evidence to determine the absolute configuration of versicomide A does not seem entirely solid and in view of the highly similar NMR data of those isomers with multi-chiral centers, it is necessary to clarify the absolute configuration of 1. The results from the X-ray diffraction analysis of compound 1 unambiguously determined its absolute configuration as 3S, 14R, and 15S. This is likely the first time the configuration of isoleucine in a quinazoline-containing diketopiperazine skeleton with a Val-Ile cyclic dipeptide moiety was unambiguously defined by X-ray crystallography analysis.
Versicomide E (2) was obtained as a colorless amorphous solid with the molecular formula C 19 H 23 N 3 O 3 based on the HRESIMS data. Its NMR data (CDCl 3 , Table 1) were similar to those of 1, which indicated that 2 possessed the same quinazoline backbone as 1. The obvious difference was the absence of signals for two methines at δ C 58.1/δ H 4.70 (CH-3) and δ C 36.1/δ H 2.62 (CH-15) in the NMR spectra of 1, whereas additional resonances corresponding to a tetra-substituted double bond at δ C 121.3 (C-3) and δ C 135.0 (C-15) were found in that of 2 (CDCl 3 , Table 1), which were further confirmed by COSY and HMBC correlations ( Figure 2). The geometry of the double bond between C-3 and C-15 was determined as Z-configuration by key NOE correlations from NH-2 (δ H 8.02) to H-16 (δ H 2.29) and H 3 -17 (δ H 1.15) ( Figure 3). Compound 2 has the same planar structure as that of versicomide B (Figure 1), which was also isolated from hydrothermal vent crab-derived fungus Aspergillus versicolor XZ-4 by Wu and co-workers in 2017 [8], with the exception of the geometry of the double bond at C3(15) (Z in 2 vs. E in versicomide B) and the absolute configuration of C-14 (R in 2 vs. S in versicomide B) as well. To clarify the stereochemistry of compound 2, calculations of specific rotation (SR) were carried out for 14R-2 and 14S-2, and the calculated SR value for 14R-2 (+59.8) at CAM-B3LYP/TZVP level was compatible with the experimental SR value [α] 25 D +112.0 (c 0.08, MeOH), contrary to that of versicomide B ([α] 20 D −23.4) [8], which allowed the assignment of absolute configuration of C-14 in 2 as 14R (Table S2). To further verify the absolute configuration of C-14 in 2, the time-dependent density functional (TDDFT)-ECD calculation was performed on 14R-2 and 14S-2 at the CAM-B3LYP/TZVP level in Gaussian 09. The experimental curve matched that of the calculated ECD spectrum for 14R-2 and also assigned the absolute configuration of C-14 in 2 as 14R ( Figure S27).
Compound 3 was obtained as colorless prisms and was identified as cottoquinazoline A by comparing its NMR data (measured in DMSO-d 6 , Table S3) with those previously reported in the literature [10]. Cottoquinazoline A is a 16-nor analog of the known fumiquinazoline D and was first isolated from a marine-derived fungal strain of A. versicolor (MST-MF495) by Capon and co-workers in 2009, with a partial stereostructure assigned [10]. Considering the complexity of the structure of 3 and the presence of many stereoisomers, it is important to clarify the assignment of the absolute configurations of 3 [10,14]. Fortunately, a suitable crystal of 3 was picked out from DMSO-MeOH (1:1) and subjected to X-ray crystallographic analysis to assign its absolute configurations of the stereogenic centers in 3 as 3S, 14S, 16R, 17S, and 19S ( Figure 4).

Antimicrobial Assays
The antimicrobial activity evaluation of all the isolated compounds was performed against human pathogenic bacterium (Escherichia coli), marine-derived aquatic pathogenetic bacteria (Aeromonas hydrophila, Edwardsiella ictarda, Micrococcus luteus, Pseudomonas aeruginosa, Vibrio harveyi, V. parahemolyticus, V. vulnificus), and plant-pathogenic fungi (Colletotrichum gloeosporioides, Curvularia spicifera, Epicoccum sorghinum, Fusarium oxysporum, F. proliferatum, and Penicillium digitatum) ( Table 3 However, neither the quinazoline-containing diketopiperazine derivatives (1 and 2) nor the oxepine-containing diketopiperazine analogs (5-7) showed any activity against all the tested pathogenic bacteria. These data suggested that the 16-nor-methyl fumiquinazoline alkaloids generally showed higher antimicrobial activity than that of quinazolinone alkaloids (3 and 4 vs. 1 and 2) and the oxepine congeners (5)(6)(7). A comparison of the antimicrobial results of 3 and 4 revealed that different substituent groups at C-20 could influence the inhibitory potency against the pathogenic bacteria. Concerning bisabolene derivatives, the antimicrobial results revealed that compound 12, a dimeric bisabolene analog, showed weaker antimicrobial activities than that of the monomeric bisabolenes (8-11) against M. luteus and V. harveyi. In addition, hydroxylation at C-10 or C-11 likely decreased the activity against V. harveyi, V. parahaemolyticus, and C. gloeosporioides (8 vs . 10 and 11).  The above results showed that compounds 4, 8, and 9 were found to be efficient in suppressing the growth of aquatic pathogenic bacteria V. parahaemolyticus and V. harveyi. To a great degree, the endozoic fungus A. versicolor AS-212 which is associated with the deep-sea coral Hemicorallium cf. imperiale may provide a chemical defense to help its host to fight off the aquatic pathogenic bacteria by producing an array of antimicrobial secondary metabolites.

General Experimental Procedures
The general experimental procedures, apparatus, and solvents/reagents used in this work were the same as those described in our previous reports [5][6][7].

Fungal Material
The endophytic fungus Aspergillus versicolor AS-212 associated with deep-sea coral, Hemicorallium cf. imperial, was collected from the Magellan Seamounts (depth 1420 m) in May 2018. By comparing its ITS region sequence with that of A. versicolor (accession no. MT582751.1) in the GenBank database, the sequence data of strain AS-212 were identical (100%) to those of A. versicolor and subsequently uploaded in GenBank with accession no. OP009765.1. The fungus AS-212 has been conserved at the Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences (IOCAS).

Fermentation, Extraction, and Isolation
The fungal strain AS-212 was cultivated on potato dextrose agar (PDA) plates at 28 • C for 7 days to generate spores. The fresh mycelia were transferred into 1 L Erlenmeyer flasks, each containing 300 mL potato-dextrose broth (PDB) medium, which was reported in our previous publication [5], and fermented under static conditions for 30 days at room temperature. After 30 days of incubation, a total of 33 L cultures were filtered and collected to separate the broth and mycelia. The broth was adequately extracted three times with EtOAc, while the mycelia were mechanically crushed and then extracted three times with 80% volume aqueous acetone. Acetone was removed in vacuo to afford an aqueous solution, which was successively extracted with EtOAc. Based on their virtually similar TLC and HPLC profiles ( Figure S26), both EtOAc extracts from broth and mycelia were combined and evaporated under a vacuum to render the EtOAc extract (61 g).

X-ray Crystallographic Analysis of Compounds 1 and 3
Suitable crystals were picked out to obtain crystallographic data using a Bruker Smart-1000 or Bruker D8 VENTURE CCD diffractometer with Cu Kα radiation (λ = 1.54178 Å). Absorption correction was applied using the program SADABS [20]. The structures were solved by direct methods with the SHELXTL software package [21,22]. All non-hydrogen atoms were refined anisotropically. The absolute structures were determined by refinement of the Flack parameter [23]. The structures were optimized by full-matrix least-squares techniques. Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre with deposition numbers CCDCs 2192654 and 2192653 for 1 and 3, respectively. Crystal data and structure refinements for 1 and 3 are listed in Table S1.
Crystal data for compound 1:

Specific Rotation and ECD Calculations
General computational procedures were consistent with our previous reports [5,25].

Conclusions
In conclusion, two new quinazolinone derivatives, versicomide E (2) and cottoquinazoline H (4), along with ten known compounds (1, 3, and 5-12), were isolated and identified from the deep-sea coral-derived Aspergillus versicolor AS-212. This marks the first time that the absolute configurations of all the stereogenic centers in (−)-isoversicomide A (1) and cottoquinazoline A (3), which were not assigned in the previous literature, were accurately solved in the present work by X-ray crystallographic analysis. Compound 3 exhibited activity against aquatic pathogenic bacteria A. hydrophilia with an MIC value of 18.6 µM, while compounds 4 and 8-10 exhibited inhibitory effects against V. harveyi with MIC values ranging from 15.0 to 28.4 µM. In addition, compounds 4 and 8 exhibited potent inhibitory effects against V. parahaemolyticus with MIC values of 9.0 and 15.0 µM, which might have the potential to be developed as leading compounds in discovering aquatic antibiotics.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/md21050293/s1, Figures S1-S27: The analyzed data of MS, 1D and 2D NMR spectra of compounds 1-4, crystal packing of compounds 1 and 3, HPLC analysis of mycelia extract, broth extract, and compounds 1-12 of Aspergillus versicolor AS-212, and experimental and calculated ECD spectra of compound 2 at the CAM-B3LYP/TZVP level. Table S1: Crystal data and structure refinement for compounds 1 and 3. Table S2: Calculated specific rotation values at 589.44 nm for the enantiomers 14R-2 and 14S-2 at the CAM-B3LYP/TZVP level.