Aculeaxanthones A–E, new xanthones from the marine-derived fungus Aspergillus aculeatinus WHUF0198

Introduction Dimeric natural products are widespread in plants and microorganisms, which usually have complex structures and exhibit greater bioactivities than their corresponding monomers. In this study, we report five new dimeric tetrahydroxanthones, aculeaxanthones A−E (4−8), along with the homodimeric tetrahydroxanthone secalonic acid D (1), chrysoxanthones B and C (2 and 3), and 4−4’-secalonic acid D (9), from different fermentation batches of the title fungus. Methods A part of the culture was added to a total of 60 flasks containing 300 ml each of number II fungus liquid medium and culture 4 weeks in a static state at 28˚C. The liquid phase (18 L) and mycelia was separated from the fungal culture by filtering. A crude extract was obtained from the mycelia by ultrasound using acetone. To obtain a dry extract (18 g), the liquid phase combined with the crude extract were further extracted by EtOAc and concentrated in vacuo. The MIC of anaerobic bacteria was examined by a broth microdilution assay. To obtain MICs for aerobic bacteria, the agar dilution streak method recommended in Clinical and Laboratory Standards Institute document (CLSI) M07-A10 was used. Compounds 1−9 was tested against the Bel-7402, A-549 and HCT-116 cell lines according to MTT assay. Results and Discussion The structures of these compounds were elucidated on the base of 1D and 2D NMR and HR-ESIMS data, and the absolute configurations of the new xanthones 4−8 were determined by conformational analysis and time-dependent density functional theory-electronic circular dichroism (TDDFT-ECD) calculations. Compounds 1–9 were tested for cytotoxicity against the Bel-7402, A549, and HCT-116 cancer cell lines. Of the dimeric tetrahydroxanthone derivatives, only compound 6 provided cytotoxicity effect against Bel-7402 cell line (IC50, 1.96 µM). Additionally, antimicrobial activity was evaluated for all dimeric tetrahydroxanthones, including four Gram-positive bacteria including Enterococcus faecium ATCC 19434, Bacillus subtilis 168, Staphylococcus aureus ATCC 25923 and MRSA USA300; four Gram-negative bacteria, including Helicobacter pylori 129, G27, as well as 26,695, and multi drug-resistant strain H. pylori 159, and one Mycobacterium M. smegmatis ATCC 607. However, only compound 1 performed activities against H. pylori G27, H. pylori 26695, H. pylori 129, H. pylori 159, S. aureus USA300, and B. subtilis 168 with MIC values of 4.0, 4.0, 2.0, 2.0, 2.0 and 1.0 μg/mL, respectively.

In the process of our ongoing screening for new biologically active natural products from marine-derived fungi, we discovered that the fungus Aspergillus aculeatinus WHUF0198 contained an assortment of chemically diverse metabolites revealed by LC-ESIMS (UV-vis) profiles, and displayed potent antibacterial and antitumor properties during our preliminary screening of bioassays. We previously reported one new norditerpene, one new indone, and one paraherquamide alkaloid, along with 13 known compounds from the culture of this fungus (Wu et al., 2021(Wu et al., , 2022. In this study, we report five new dimeric tetrahydroxanthones, aculeaxanthones A-E (4-8), along with the homodimeric tetrahydroxanthone secalonic acid D (1), chrysoxanthones B and C (2 and 3), and 4-4′-secalonic acid D (9), from different fermentation batches of the title fungus. In contrast to the reported dimeric tetrahydroxanthones, compounds 4-8 have many kinds of dimeric patterns, covering the common 2-2′ linkage (5-7) and the less prevalent 2-4′ linkage (4 and 8). Employing NMR and ECD spectroscopy and TDDFT calculations, the absolute configurations of these tetrahydroxanthones were investigated. What's more, the cytotoxicity and antibacterial evaluation of all the isolates were also discussed herein.

General experimental procedures
A PerkinElmer Model 341 polarimeter was applied to measure optical rotations. ECD spectra were acquired using a Chirascan V100 spectropolarimeter. NMR data were recorded at 400 or 600 MHz (Bruker AVANCE). HRESIMS spectra were obtained on a ThermoFisher mass spectrometer (LTQ Orbitrap XL). Size-exclusion chromatography was conducted with Sephadex LH-20. Column chromatography (CC) was applied using silica gel which was produced by Anhui Liangchen Co., Ltd.

Fungal material and mass culture
A specimen of A. aculeatinus was identified using the ITS sequences and the morphological characteristics (Wu et al., 2021). A voucher specimen (WHUF0198) has been preserved in School of Pharmaceutical Sciences, Wuhan University. The fungus was precultivated on number II fungus medium (Wu et al., 2022) and incubated at 28°C for a week. After that, a part of the culture was added to a total of 60 flasks containing 300 ml each of number II fungus liquid medium and culture 4 weeks in a static state at 28°C.

Computational analysis
The conformational majorization of the stereoisomers was achieved using computational TDDFT calculations. To perform the conformational analysis, MMFF94 molecular mechanics was carried out. The ground-state geometries of those stereoisomers were further optimized using Gaussian 09 (Frisch et al., 2009). Vibrational evaluation was finished using TDDFT calculations at the B3LYP/6-311G (2d, p) level to determine minima. The Boltzmann distribution law (Eq. 1) was used to calculate the equilibrium populations at roomtemperature. The overall theoretical ECD spectra were simulated with a Gaussian function and then acquired according to the Boltzmann weighting.
In this case, N i represents the number of conformers i with degeneracy g i and energy E i at temperature T, and k B is Boltzmann constant. Structures of dimeric tetrahydroxanthones 1-9.

Antibacterial assay
All the isolates were tested against Gram-positive bacteria including Enterococcus faecium ATCC 19434, Bacillus subtilis 168, Staphylococcus aureus ATCC 25923 and MRSA USA300, Gram-negative bacteria including Helicobacter pylori 129, G27, as well as 26695, and multi drugresistant strain H. pylori 159, and one Mycobacterium M. smegmatis ATCC 607. The MIC of anaerobic bacteria was examined by a broth microdilution assay. Briefly, twofold serial dilutions of compounds 1-9 were prepared in 96-well microtiter plates. H. pylori liquid cultures was also diluted with BHI broth and was inoculated into each well to get a final concentration of 5 × 10 5 CFU/ml. After incubation in a microaerophilic atmosphere at 37°C for 72 h, the MIC was confirmed to be the lowest concentration which resulted in no turbidity. Metronidazole was used as a positive control. To obtain MICs for aerobic bacteria, the agar dilution streak method recommended in Clinical and Laboratory Standards Institute document (CLSI) M07-A10 was used. The broth was diluted with saline and applied to plates, delivering a final concentration of approximately 10 5 CFU/spot.

Cytotoxicity assay
Compounds 1-9 was tested against the Bel-7402, A-549 and HCT-116 cell lines according to MTT assay. All the isolates were dissovled and diluted using dimethyl sulfoxide (DMSO). Cells were seeded at 4000 cells in 96-well microplates and incubated for 24 h and spent with the isolates for 72 h. After that, each well was treat for 4 h with MTT reagent. By operating a microplate reader, absorbance at 570 nm was measured after replacing the medium with 100 μl of DMSO. All compounds were tested three times independently (n = 3). 5-Fluorouracil was applied to positive control. Finally, the Logit method was applied to caculate IC 50 values.
Aculeaxanthone A (4) was obtained as an orange powder. Compound 4 provided the near-identical NMR data to those of 2. The significant difference appeared in 4 was the HMBC correlations from the H-2 and hydroxyl proton 1-OH to C-9a, which suggested the C-4-C-2′ linkage for 4 instead of C-2-C-2′ linkage in 2. The tetrahydroxanthone and the chromanone monomers in 4 connected with a C-4-C-2′ linkage was determined by the COSY correlations of H-2/H-3 instead of H-4/H-3, and HMBC correlations of H-3 with C-2′ and C-4a and H-3′ with C-4. The relative configuration of the two monomers in 4 was the same to those in 2, as indicated by the coupling constants (Table 1) and the interpretation of the NOE signals, together with the biogenetic consideration. To confirm the absolute configuration of 4, the calculated ECD spectrum of 4a were acquired according to the TDDFT calculations (Grkovic et al., 2007;Bringmann et al., 2009). The Molecular Operating Environment (MOE) was performed to conduct the systematic conformational analysis for 4a (5R, 6S, 10aR, 5'R, 6'R, 10a'R)  (Grimme, 2006) level. These were further filtered to gain the principal conformer on the base of the Boltzmann distribution. Finally, Gaussian broadening was used to provide the complete calculated ECD spectrum of 4a. Obviously, the experimental and calculated ECD spectra for 4 was in great agreement (Figure 2), indicating that an 5R, 6S, 10aR, 5'R, 6'R, 10a'R absolute configuration could be assigned to 4. Compound 4 was found unstable in DMSO-d 6 , consistent with the findings of Wu et al. (2015). Then the central chirality elements of 2 and 4 was assigned by chemical conversions. The conversion was monitored by 1 H-NMR spectra and the product was isolated using a shimodzu HPLC system. The Wessely-Moser rearrangement between 2 (2′-2 linkage) and 4 (2′-4 linkage) was represented in Figure 3, which further confirmed the absolute configuration of 4.
Frontiers in Microbiology 06 frontiersin.org methines (δ C 82.7 and 33.5), two methylenes (δ C 36.5 and 39.6), one methyl (δ C 14.8), and one methoxyl (δ C 53.6). Analysis of 1 H NMR spectrum indicated one aromatic ring, two methylenes, one oxymethine, one methine, one methyl, and one methoxyl (Table 2). These evidence indicated that 5 must be a symmetric homodimer of two chromanone lactone monomers. The HMBC correlations of H-5 with C-10a and C-12 determined the connection between the lactone moiety and the chromanone monomeric unit (Figure 4). The 2-2′ linkage of 5 was established by the HMBC correlations of 1-OH with C-2 and C-9a, H-3 (H-3′) with C-1 (C-1′), C-4a (C-4a'), and C-2′ (C-2) (Figure 4). The NOESY spectrum was used to provide the relative configuration of 4 ( Figure 5). The strong NOESY correlations from H-5 (H-5′) to H-6 (H-6′) and H α -8a (H α -8a') suggested that these protons provided the co-facial orientation, which was also determined by the evidence of the coupling constant ( 3 J H-5,H-6 = 6.9 Hz) with analogues in literatures (Zhang et al., 2008;El-Elimat et al., 2015;Wu et al., 2015). For 5S, 6R, 10aR, 5'S, 6'R, 10a'R, the experimental spectrum agreed well with the calculated one, which unequivocally assigns the absolute configuration of 5 ( Figure 6). Aculeaxanthone C (6) was also found to possess the identical molecular formula (C 32 H 30 O 14 ) to 2-5, as suggested by the HR-ESIMS ions observed at m/z 661.1526 (calcd for C 32 H 30 O 14 Na + , 661.1533). A scrupulous analysis of the 1D NMR data of 6 and 5 (Table 2) indicated 6 to be a heterodimer of two different chromanone lactone monomers. Subtraction of the signals of aculeaxanthone B (5) subunit confirmed the near-identical remaining NMR data with those of the chromanone lactone of 2. The relative configuration of one chromanone lactone in 6 was determined by the NOESY correlation of H-5/H-6 ( Figure 5). The configuration of H-5′ and H-6′ in another chromanone lactone monomer was determined as the same as that of 2, confirmed by the NOESY correlation of H-5′ with H-11′ ( Figure 5). To establish the absolute configuration of 6, the lowest energy conformer was calculated. Distinctly, the experimental ECD spectrum for 6 and the calculated one for 6a can Proposed interconversion mechanism between 2 and 4. Frontiers in Microbiology 07 frontiersin.org be found a great fit (Figure 7). Finally, the 5S, 6R, 10aR, 5'R, 6'R, 10a'R configuration could be assigned to 6 ( Figure 1). Aculeaxanthone D (7) was derived as a yellow powder. Its molecular formula was deduced as C 33 H 34 O 15 from the HR-ESIMS ions at m/z [M + Na] + 693.1789, indicating that 7 presented one more carbon and one less unsaturation degree than compounds 1-6. The 1D NMR data (Table 2) displayed that the chromanone lactone monomer of 7 was identical to that of 2 and 6. Compound 7 was determined to possess 2-2′ linkage by the HMBC correlations from H-3 (δ H 7.49) to C-2′ (δ C 117.4) and H-3′ (δ H 7.52) to C-2 (δ C 117.4).
The distinction difference was that a side chain in 7 replaced the cyclohexene moiety in 2, determined by the HMBC correlations of the methoxyl H 3 -14 (δ H 3.70) with C-8 (δ C 173.0). The relative configurations of the chromanone monomer in 7 were identical to those in 2 and 6 from the NOESY correlations between H-5′ with H-11′ (Figure 5), the chemical shifts, and biogenetic grounds. The anti relationship between H-5 with H-6 in 7 were proposed to be the same as those in 2, deduced from the coupling constants ( 3 J H-5,H-6 = 6.7 Hz) and biogenetic consideration. To gain the absolute configuration of 7, the ECD spectrum for the lowest energy conform 7a (5R, 6S, 10aR, 5'R, 6'R, 10a'R), was calculated and compared with the experimental one. Notably, the calculated ECD spectrum showed good fitting with the experimental one (Figure 7), determining that an 5R, 6S, 10aR, 5'R, 6'R, 10a'R absolute configuration could be defined to 7.
Compound 9 possessed the same molecular formula (C 32 H 30 O 14 ) as secalonic acid D (1) which was based on its HR-ESIMS analysis (m/z 661.1530; [M + Na] + , calcd for 661.1533), suggesting it to be an isomer of 1. Similar to 5, 9 displayed half of the expected carbon signals, suggesting a structurally symmetrical. Careful analysis of the NMR spectra, 9 displayed identical 1D NMR data to those of 4-4′-secalonic acid D, 4-4′-secalonic acid A (Chen L. et al., 2019), and talaroxanthone (Koolen et al., 2013), suggesting that 9 shared the same planar structure and relative configurations as those compounds. Compound 9 was finally determined to be 4-4′-secalonic acid D by comparison their specific rotation values, of which the absolute configuration was further determined by the TDDFT-ECD calculation ( Figure S52). However, after comparison of the 1 D NMR data of talaroxanthone with those of 4-4′-secalonic acid A, the structure of talaroxanthone should be revised to 4-4′-secalonic acid A (Koolen et al., 2013), due to their identical NMR data, particularly the large 3 J H-5, H-6 (12.0 Hz) value, and the specific rotation values (Koolen et al., 2013;Qin and Porco, 2015).
Compounds 1-9 were tested for cytotoxicity against the Bel-7402, A549, and HCT-116 cancer cell lines. Of the dimeric tetrahydroxanthone derivatives, only compound 6 provided cytotoxicity effect against Bel-7402 cell line (IC 50

Conclusion
In summary, five new dimeric tetrahydroxanthones (4-8) with a high degree of structural complexity and diversity were separated from the culture of the marine-derive fungus A. aculeatinus Experimental ECD spectra of 6 and 7 and calculated ECD spectra of 6a and 7a. Experimental and calculated ECD spectra of compound 8.
Frontiers in Microbiology 09 frontiersin.org WHUF0198. Compound 4 represented the chemical conversion product of 2, indicating the possibility that some dimeric tetrahydroxanthones might produce spontaneously from the natural dimers in the process of the fermentation or extraction. Compounds 5 and 6 contained two chromanone monomers coupled by a 2-2′ linkage to form a symmetric homodimer and an asymmetric dimer, respectively. Compound 7 included a common chromanone lactone unit and a ring-opened tetrahydroxanthone monomer, which might derive from 2 instead of the methanolysis product of 6. Thus, compound 8 represented the fifth dimeric hexahydroxanthones, of which the common tetrahydroxanthone monomer and the hexahydroxanthone monomer were connected by a 2-4′ linkage. Furthermore, the structure of talaroxanthone should be revised to 4-4′-secalonic acid A based on their identical NMR data and specific rotation values. The absolute configurations of all dimeric tetrahydroxanthones were determined by a combination of ECD calculation, chemical conversions, specific rotations, and biogenetic consideration.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

Author contributions
Y-SC, KH, and HB: conceptualization, methodology, and writing-review and editing. JW, HS, and K-KZ: data curation. Y-SC, HS, and MeZ: funding acquisition. K-KZ: software. JW, YZ, and HS: chemical investigation. MiZ, S-BW, and HB: bioactivity assays. KH: fungal resources. JW, HS, and Y-SC: data analysis. JW, MeZ, and HS: writing-original draft preparation. All authors contributed to the article and approved the submitted version.