New Monoterpenoids and Polyketides from the Deep-Sea Sediment-Derived Fungus Aspergillus sydowii MCCC 3A00324

Chemical study of the secondary metabolites of a deep-sea-derived fungus Aspergillus sydowii MCCC 3A00324 led to the isolation of eleven compounds (1–11), including one novel (1) and one new (2) osmane-related monoterpenoids and two undescribed polyketides (3 and 4). The structures of the metabolites were determined by comprehensive analyses of the NMR and HRESIMS spectra, in association with quantum chemical calculations of the 13C NMR, ECD, and specific rotation data for the configurational assignment. Compound 1 possessed a novel monoterpenoid skeleton, biogenetically probably derived from the osmane-type monoperpenoid after the cyclopentane ring cleavage and oxidation reactions. Additionally, compound 3 was the first example of the α-pyrone derivatives bearing two phenyl units at C-3 and C-5, respectively. The anti-inflammatory activities of 1–11 were tested. As a result, compound 6 showed potent inhibitory nitric oxide production in lipopolysaccharide (LPS)-activated BV-2 microglia cells with an inhibition rate of 94.4% at the concentration of 10 µM. In addition, a plausible biosynthetic pathway for 1 and 2 was also proposed.

Compound 1 was a novel chained monoterpenoid. The plausible biosynthetic pathway for 1 and 2 was proposed (Scheme 1). Starting from the GPP, hydrosis, oxygenation, and cyclization reaction occurrence constructed the monocyclic ring monoterpenoid osmane. The osmane might undergo carbon-carbon bond cleavage to form a key intermediate A, which was further oxygenated to yield 1. Additionally, the osmane might also undergo oxygenation to form 2.
Compound 1 was a novel chained monoterpenoid. The plausible biosynthetic pathway for 1 and 2 was proposed (Scheme 1). Starting from the GPP, hydrosis, oxygenation, and cyclization reaction occurrence constructed the monocyclic ring monoterpenoid osmane. The osmane might undergo carbon-carbon bond cleavage to form a key intermediate A, which was further oxygenated to yield 1. Additionally, the osmane might also undergo oxygenation to form 2. Scheme 1. Hypothetical biogenetic pathways for compounds 1 and 2.
As terpenoids usually possess the anti-inflammatory activities, all isolated metabolites were evaluated for their inhibitory effects against NO secretion in LPS-activated BV-2 microglia cells. As a result, none of them showed obvious cytotoxic activities against BV-2 microglia cells at the concentration of 20 µM by the CCK-8 Kit, and all compounds exhibited dose-dependent inhibitory effects against NO production induced by the LPS at the concentrations of 20 and 10 µM, respectively (Table 3). Interestingly, compound 6 (10 µM) showed potent anti-inflammatory activities with the inhibition rate of 94.4%. As terpenoids usually possess the anti-inflammatory activities, all isolated metabolites were evaluated for their inhibitory effects against NO secretion in LPS-activated BV-2 microglia cells. As a result, none of them showed obvious cytotoxic activities against BV-2 microglia cells at the concentration of 20 µM by the CCK-8 Kit, and all compounds exhibited dose-dependent inhibitory effects against NO production induced by the LPS at the concentrations of 20 and 10 µM, respectively (Table 3). Interestingly, compound 6 (10 µM) showed potent anti-inflammatory activities with the inhibition rate of 94.4%. Table 3. Inhibitory effects of 1-11 against NO production in LPS-activated BV-2 microglia cells and their cytotoxicities against BV-2 microglia cells.

Compounds
Anti

General Experimental Procedures
UV spectra were recorded using a UV8000 UV/Vis spectrophotometer (Shanghai Metash Instruments Inc., Shanghai, China). Optical rotation data were measured on the basis of the Rudolph Autopol IV automatic polarimeter (Rudolph Reaearch Analytical, Newburgh, NY, USA). ECD spectra were measured using a Chirascan spectrometer (Applied Photophysics inc., Leatherhead, Surrey, UK). HRESIMS data were measured using a Xevo G2 Q-TOF mass spectrometer (Waters, Milford, MA, USA). The 1 H, 13 C, HSQC, COSY, HMBC, and NOESY spectra were measured on the basis of the Bruker Avance 400 FT NMR spectrometer (Bruker Company, Fällanden, Switzerland) with tetramethylsilicane (TMS) as an internal standard. Semi-preparative HPLC was performed using an Alltech LS class pump with a model 201 variable wavelength UV/Vis detector, and a YMC packed ODS-A (250 × 10 mm, 5 µm) column (YMC Co., Ltd. Kyoto, Japan) was used for the purification. Column chromatography (CC) Mar. Drugs 2020, 18, 561 8 of 12 was carried out using a Sephadex LH-20 (Amersham Biosciences, San Francisco, CA, USA), ODS-A-HG (YMC Co., Ltd. Kyoto, Japan), and silica gel (Qingdao Marine Chemistry Co., Ltd., Qingdao, China). The TLC analyses were carried out with the precoated silica gel plates by heating after spraying with vanillin sulfuric acid chromogenic reagent (Xilong Scientific Co., Ltd., Shantou, China).

Fungal Material and Identifiation
The fungus was isolated from the deep-sea sediment at the depth of 2246 m sampling from the South Atlantic Ocean (W13.6639 • , S14.2592 • ), and it was identified to be Aspergillus sydowii on the basis of the morphology and the internal transcribed spacer (ITS) region of the rDNA sequence. The ITS gene sequence was deposited in GenBank and assigned the accession no. MN918102. The fungus was preserved at the Marine Culture Collection of China (MCCC), and assigned the accession no. MCCC 3A00324. Therefore, the producing fungus was named Aspergillus sydowii MCCC 3A00324.

Fermentation, Extraction, and Isolation
The fungus was cultivated in a potato dextrose agar (PDA) plate under 25 • C for four days, and then the fresh mycelia and spores were inoculated into 500 mL Erlenmeyer flasks (×2), each containing 100 mL potato dextrose broth (PDB) medium and followed by cultivation in a rotary shaker under 25 • C at 200 rpm for four days. The seed cultures were subsequently inoculated to 30 Erlenmeyer flasks (1 L) (each containing 80 g rice and 120 mL sea water) after autoclaving at 121 • C for 22 min. The fermentation was performed under static conditions at 25 • C for 26 days.

BV-2 Cell Culture and Treatment
The BV-2 microglia cells were cultured in DMEM medium containing 10% fetal bovine serum and antibiotics (100 units/mL of penicillin and 100 g/mL of streptomycin) and maintained in a humidified 5% CO 2 incubator (Beijing Luxi Technology Co., Ltd., Beijing, China) at 37 • C. For the experiment, cells were seeded into 24-well plates (2 × 10 4 cells/well) overnight. Next day, cells were incubated with fresh culture medium containing indicated concentration of the tested compounds for half an hour and following LPS treatment (1 µg/mL). Cells were treated vehicle (DMSO, 0.1%) as control.

Nitrite Quantification
The concentration of nitrite in culture medium was determined using a Griess Reagent Kit (Thermo Fisher, Shanghai, China). Briefly, 75 µL of cell culture supernatants were reacted with an equal volume of Griess Reagent Kit for 30 min at room temperature, and absorbance of diazonium was obtained at a wavelength of 560 nm. Nitrite production by vehicle stimulation was designated as 100% inhibition compared to LPS stimulation for the experiment.

13 C NMR Calculation of 2
Conformational searches were carried out using the Maestro 10.2 program (Schrödinger Inc., NY, USA) at the OPLS3 molecular mechanics force field within an energy window of 3.0 kcal/mol. The results exhibited 8 conformers for (4S*,5R*,6S*)-2 and (4S*,5R*,6R*)-2, respectively. The conformers were further optimized at the B3LYP/6-31+G(d,p) level in gas phase using Gaussian 09 (Gaussian, Inc., Wallingford, CT, USA) [38]. The conformers with a Boltzmann population over 1% were selected for the NMR calculations. The NMR data were calculated by the GIAO method at the mPW1PW91/6-311+G(2d,p) level with the IEFPCM model in methanol. Finally, the calculated NMR data were averaged according to the Boltzmann distribution for each conformer and then fitted to the experimental values by linear regression.

ECD Calculation of 2
The eight conformers of (4S*,5R*,6S*)-2 were optimized by density functional theory (DFT) calculations at the B3LYP/6-31+G(d,p) level in gas phase using Gaussian 09. The energies, oscillator strengths, and rotational strengths of the first 60 electronic excitations were calculated by the TDDFT method at the B3LYP/6-311+G(2d,p) level in methanol. The ECD spectrum was simulated using SpecDis (version1.70, Berlin, Germany) by applying the Gaussian band shapes with sigma = 0.3 eV. Finally, the calculated ECD data were weighted and then summed up each stable conformer on the basis of the Boltzmann population.

Conclusions
In summary, four new compounds, including one novel (1) and one new (2) monoterpenoids and two new polyketides (3 and 4), were obtained from the EtOAc extract of the deep-sea-derived fungus Aspergillus sydowii MCCC 3A00324, together with seven known compounds (5)(6)(7)(8)(9)(10)(11). The structures of metabolites were determined by comprehensive analyses of the NMR and HRESIMS spectra, in association with quantum chemical calculations of the ECD, 13 C NMR, and specific rotation data for their configurational assignment. Compound 1 possessed a novel monoterpenoid skeleton, biogenetically probably derived from the osmane-type monoperpenoid after the cyclopentane ring cleavage and oxidation reactions. Additionally, 2 was the first osmane-type monoterpenoid representative discovered from the fungi, while 3 was the first example of the α-pyrone derivatives bearing two phenyl units at C-3 and C-5, respectively, indicating that the deep-sea-derived fungi are a unique source of the structurally novel compounds. Compound 6 exhibited significant inhibitory effects against NO secretion in LPS-activated BV-2 microglia cells (94.4% inhibition rate, 10 µM), suggesting the potential application for the anti-inflammatory agents.

Conflicts of Interest:
The authors declare no conflict of interest.