Cytotoxic polyketides from the deep-sea-derived fungus Aspergillus fischeri FS452

Abstract Six globoscin derivatives (1‒6) including two new ones fischerins A (1) and B (2) were isolated from the deep-sea-derived fungus Aspergillus fischeri FS452. Their structures were elucidated by comprehensive spectroscopic analysis and the absolute configurations were determined by the quantum chemical ECD calculations. The in vitro cytotoxicity assays indicated that fischerin B (2) exhibited potential activities against the four tested human cancer cell lines (SF-268, MCF-7, HepG-2 and A549) with the IC50 values in the range of 7–10 µM. Graphical Abstract


Introduction
Globoscin derivatives are a small class of polyketides constructing a phenylbutyrolactone or phenylbutyric acid core with different chiral center at C-4. The first examples discovered are antafumicins A and B, which were isolated from Aspergillus niger in 1993 with antimicrobial activity (Fujimoto et al. 1993). Then, another two analogues, globoscin and globoscinic acid were discovered from Xylaria globosa and X. obovate (Adeboya et al. 1995), and eight new analogues were reported from fungi Neosartorya spinosa and Aspergillus sp. (Rajachan et al. 2016;Wang et al. 2018). However, none of them exhibited significant bioactivities.
Marine fungus-derived natural products are considered as a great potential resource for new drug development in the 21st century, of which the marine-derived fungi are recognized to be the promising source for the discovery of novel biological active agents such as anticancer, anti-inflammatory and antitubercular (Carroll et al. 2021(Carroll et al. , 2020(Carroll et al. , 2019 during the past years. Moreover, the genera Penicillium and Aspergillus are considered to be the main contributors of bioactive metabolites (Wang et al. 2019;Amin et al. 2021;Salendra et al. 2021). In our previously searching for bioactive metabolites from deep-sea-derived fungi, the strain Aspergillus fischeri FS452 isolated from Indian Ocean sludge (81 0.00 0 N, 1 59.987 0 E; depth 3000 m) attracted our attention since its ability to produce the novel polypropionates with MptpB inhibitory activity when fermented in rice medium (Liu et al. 2019a). In order to dig out the chemical diversity of this strain, the fermentation in potato dextrose broth (PDB) medium was carried out in this study and two new polyketides belonging to globoscin family were obtained together with four known analogues. Herein, the details of the isolation, structure identification and cytotoxic activities of 1-6 are discussed.

Results and discussions
The methanol extract of the deep-sea derived-fungus Aspergillus fischeri FS452 was concentrated under reduced pressure and the obtained crude extract was further subjected to repeated column chromatography and semi-preparative HPLC to afford compounds 1-6 ( Figure 1).
Fischerin A (1) was obtained as a colorless oil, of which the molecular formula was deduced to be C 14 H 18 O 7 based on the deprotonated molecule ion peak at m/z 297.0979 [M À H] À . The 1 H NMR spectrum collected in chloroform-d (Table S1) indicated the presence of a chelating proton at d H 13.13, three methyls including two methoxyl at d H 3.51/3.46 (Me-13/14, respectively), one AB coupling methylene group at d H 2.28 (ddd, J ¼ 14.6, 10.4, 3.1) and 2.09 d H 2.28 (ddd, J ¼ 14.6, 10.5, 3.1), four methine groups including two aromatic ones (d H 7.59/6.41, H-8/H-9) and two O-bearing ones (d H 4.11/5.14, H-2/H-4). The 13 C NMR spectrum resolved 14 signals composing of two carbonyl carbons (d C 175.3 and 202.8, C-1 and C-11. respectively), six aromatic carbons and six sp3 ones. By comparing the 1D NMR data to those of the known compounds spinoates suggested that compound 1 was a demethylation product of spinosate.
The COSY correlations ( Figure S1) from H-2 to H-4 indicated the only coupling unit of C-2/C-3/C-4 and the HMBC correlations from H-2/H 3 -2 to C-1 located the carboxyl group at C-2, which constructed a C 4 aliphatic chain. The correlations from H-4 to C-5/ C-6/C-10 suggested that the side chain was linked to C-5 of the benzene ring. Furthermore, the substitutions of the chelating hydroxyl and acetyl groups at C-6 and C-7 were elucidated through HMBC correlations ( Figure S1) from H 3 -12 to C-7/C-11 and from 6-OH to C-5/C-6/C-7, respectively. Finally, based on the correlations from H-13 to C-2 and from H-14 to C-4, the methoxy groups Me-13 and Me-14 should be connected to C-2 and C-4, respectively. Hence, the planar structure was confirmed.
Fischerin B (2) was also obtained as a colorless oil and gave a molecular formula of C 14 H 18 O 7 based on HRESIMS, The 1 H/ 13 C NMR data (Table S1) were nearly identical to those of 1 and one of the obvious differences detected was that the coupling constant between H 2 -3 and H-2/H-4 (J H-2/H-3l ¼ 10.4 Hz, J H-4/H-3h ¼ 10.5 Hz in 1 vs. J H-2/H-3h ¼ 5.9 Hz, J H-4/H-3l ¼ 9.9 Hz in 2), which suggested that they were isomers at C-2 or C-4. Analysis of the 2D NMR spectra ( Figure S1) confirmed the planar structure of 2.
As to fischerins A (1) and B (2), the relative configuration between C-2 and C-4 was hard to be determined through the NOESY spectrum since they were in the flexible side chain. Thus, there are four possible absolute configurations: (2S,4R), (2S,4S), (2R,4R), (2R,4S). Since 1 and 2 were epimers but not enantiomers, they should present (2S,4R)/(2S,4S)-1/2 or (2R,4R)/(2R,4S)-1/2. According to the same biosynthetic pathway as those of compound 3-6, all of which constructed a 2S configuration, the absolute configuration of C-2 in 1 and 2 could be proposed to be S. In order to further confirm the stereochemistry, the theoretical ECD plots of the two possible conformers were calculated at b3lyp/6-311 þ g(d,p) level. As a result, the calculated plot of (2S,4R)-candidate exhibited a good fit to the experimental spectrum of 1 while the calculated plot of (2S,4S)-candidate fit well with the experimental spectrum of 2 ( Figure S2). The above evidence suggested that the absolute configuration of 1 and 2 were 2S, 4R and 2S, 4S, respectively.
Compounds 3 and 4 were identified to be (2S,4R)-and (2S,4S)-spinosate, respectively (Rajachan et al. 2016). Compound 5 and 6 were identified to be antafumicins A and B, which were previously isolated from the fungus Aspergillus niger NH-401 in 1993. In this study, the single crystal of the mixture of 5 and 6 was obtained for the first time ( Figure S3).
In the bioassays, compounds 1-6 were tested for their in vitro cytotoxicity against four human cancer cell lines (Table S2) and only fischerin B (2) showed potential activities against cell lines SF-268, MCF-7, HepG-2 and A549 with the IC 50 values of 7.56, 8.45, 9.03 and 9.98 mM, respectively. Since no obvious cytotoxicity were detected for fischerin A (1) when compared to fischerin B (2), it could be concluded that the chiral center at C-4 might make a contribution to the cytotoxicity. Besides, a comparison of the cytotoxic activities results between compounds 2 and 4 (both of which presented a 4S configuration) indicated that the terminal carboxyl group at C-1 might also make a difference to the cytotoxic activities.

General experimental procedures
The optical rotation data were collected on an Anton Paar MCP-500 (Anton Paar, Graz, Austria). The circular dichroism (ECD) and the UV spectra were collected on Jasco 820 spectropolarimeter. IR spectra were recorded through Shimadzu IR Affinity-1 spectrophotometer. The 1D and 2D NMR spectra were recorded on a 600 MHz Bruker Avance-III HD spectrometer referenced to the signals of tetramethylsilane as an internal standard. HR-ESI-MS was measured on a Bruker maXis high-resolution mass spectrometer. A Shimadzu LC-20 AT with an SPD-M20A PDA detector was used for HPLC analysis and preparative separations. The ACE 5 AR-C 18 column (250 Â 10.0 mm, 5 lm, 12 nm) and the CHIRAL-MD (2)-RH column (250 Â 10.0 mm, 5 lm) were used for semipreparative separation and chiral resolution, respectively (Guangzhou FLM Scientific Instrument Co., Ltd). Column chromatography material contained commercial silica gel (SiO 2 ; 200-300 mesh; Qingdao Marine Chemical Plant), Sephadex LH-20 gel (Amersham Biosciences) and different analytical grade solvents (Guangzhou Chemical Regents Company, Ltd.). The natural sea salt was purchased from Guangdong Yueyan saltern.

Fungal material
The strain Aspergillus fischeri FS452 was isolated from the deep-sea sludge in the Indian Ocean (81 0.00 0 N, 1 59.987 0 E; depth 3000 m) in March 2016, which was identified according to morphological traits and ITS rDNA sequence analysis. The sequence data have been submitted to GenBank (accession number KF294264). The strain was deposited at the Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Institute of Microbiology, Guangdong Academy of Science. Working stocks were prepared on PDA slants at 4 C.

Fermentation, extraction, and isolation
A grown plate culture of A. fischeri FS452 was used for the preparation of the seed cultures. Mycelia were inoculated in PDB culture in a rotary shaker at 28 C for 4 days, which was next transferred into large-scale fermentation PDB medium (seven Erlenmeyer flasks each containing 1 L PDB and incubated in a rotary shaker at 28 C for 15 days. Then, the fermented liquid and mycelia were extracted with EtOAc and methanol for three times, respectively. A total of 84.5 g dark brown oily residue was obtained. After being subjected to silica gel column chromatography eluting with petroleum ether/EtOAc in a linear gradient (10:1 to 1:2), eight fractions (Fr.1-Fr.8) were obtained. Fr.7 was subjected to silica gel column eluting with MeOH/CH 2 Cl 2 (5:95, v/v) to give the mixtures of 1 and 2, which was further purified by HPLC with CHIRAL-MD (2)-RH column (MeCN/H 2 O, 65:35, 2 mL/min) to give 1 (7.3 mg, t R ¼ 9.9 min), 2 (7.1 mg, t R ¼ 12.7 min). The Fr. 4 was subjected to silica gel column eluting with MeOH/CH 2 Cl 2 (2:98, v/v) to give the mixture of 3 and 4, which was purified by HPLC with same chiral column (MeCN/H 2 O, 75:25, 2 mL/min; 3: 19.1 mg, t R ¼ 8.7 min; 4: 12.2 mg, t R ¼ 11.5 min). The mixture of compounds 5 and 6 were recrystallized from the purified fragment of Fr. 5 by Sephadex LH-20 column, which were further separated by HPLC with CHIRAL-MD (2)-RH column (MeCN/H 2 O, 60:40, 2 mL/min) to give the pure 5 (27.1 mg, t R ¼ 10.9 min) and 6 (16.5 mg, t R ¼ 13.1 min).

Details of quantum chemical calculations
The pre-optimization through Merck molecular force field (MMFF) and DFT/TD-DFT calculations at b3lyp/6-311 þ g(d,p) was proceeded by the Spartan'14 software (Wavefunction Inc.) and the Gaussian 09 program, respectively (Frisch et al. 2013). Pre-optimized conformers with an energy window lower than 5 kcal mol À1 were generated and optimized using at the b3lyp/6-31 þ g(d,p) level. The frequency calculations were performed at the same level to confirm that each optimized conformer was a true minimum and to estimate their relative thermal free energy (DG) at 298.15 K. Then, conformers with the Boltzmann distribution over 10% were chosen for energy calculations at the b3lyp/6-311 þ g(d,p) level. The rotatory strengths for a total of 50 excited states were collected according to their Boltzmann distribution. The solvent effects were considered based on the self-consistent reaction field (SCRF) method with the polarizable continuum model (PCM). Finally, the spectra were generated by the SpecDis program (Bruhn et al. 2013) with a Gaussian band shape with 0.35 eV exponential half-width from dipole-length dipolar and rotational strengths.

Cytotoxicity assay
The in vitro cytotoxicity assays were carried out according to our previously reported method (Liu et al. 2019b). Human cancer cells (SF-268, MCF-7, HepG-2, A549) purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were selected to be the targets. The tested cell lines were injected into 96-well plates and incubated at 37 C under 5% CO 2 protection. After 24 h, different concentrations of the inhibitors were added and further co-incubated for another 72 h. Then, cell monolayers were fixed with 50 lL trichloroacetic acid (wt/v: 50%) and stained with 0.4% SRB (dissolved in 1% CH 3 COOH) for 30 min, which were washed by 1% CH 3 COOH three times to remove the unbound dye. Cisplatin was used as a positive control possessing potent cytotoxic activity. All data were obtained in triplicate experiment.

Conclusion
In conclusion, two new polyketides fischerins A (1) and B (2) together with four known analogues were isolated from the deep-sea-derived fungus Aspergillus fischeri FS452. In the bioassays, compounds 2 exhibited potential in vitro cytotoxicity against four human cancer cell lines (SF-268, MCF-7, HepG-2 and A549) with the IC 50 values in the range of 7-10 mM. We hope this study will pave the way for the discovery of anti-cancer polyketides from deep-sea-derived fungi.