Carneusones A-F, Benzophenone Derivatives from Sponge-Derived Fungus Aspergillus carneus GXIMD00543

Six benzophenone derivatives, carneusones A-F (1–6), along with seven known compounds (7–13) were isolated from a strain of sponge-derived marine fungus Aspergillus carneus GXIMD00543. Their chemical structures were elucidated by detailed spectroscopic data and quantum chemical calculations. Compounds 5, 6, and 8 exhibited moderate anti-inflammatory activity on NO secretion using lipopolysaccharide (LPS)-induced RAW 264.7 cells with EC50 values of 34.6 ± 0.9, 20.2 ± 1.8, and 26.8 ± 1.7 μM, while 11 showed potent effect with an EC50 value of 2.9 ± 0.1 μM.


Introduction
Marine microorganisms, including fungi, are widely distributed in various marine environments, such as the deep sea, polar regions, even in the body of the host organisms including marine animals and plants [1].As a result of their long-term existence in extreme survival environments, marine fungi have evolved unique metabolic methods and could produce abundant structurally diverse secondary metabolites [2,3].Compounds isolated from marine fungi have also attracted considerable attention for their broad range of biological activities and have become important sources of novel drugs [4].In the last five years, over one third of bioactive natural marine products were obtained from marine fungi and mangrove fungi [5].
Natural benzophenone derivatives, or dipenyl ketone analogues, are a class of compounds widely distributed in nature with a phenol-carbonyl-phenol skeleton.More than 300 benzophenone derivatives have been isolated from plants and fungi.They exhibit great structural diversity, which is attributed to protons replaced by hydroxyl, alkyl, alkyloxy groups, or halogen atoms [6,7].Owing to their variable substituents, many benzophenone derivatives show a range of biological activities including anticancer, anti-inflammatory, antimicrobial, and antiviral effects [8].
As part of our ongoing search for bioactive compounds from marine fungi, Aspergillus carneus GXIMD00543, which is associated with sponge sample obtained from Weizhou Island, Beibu Gulf, was selected for further studies.Chemical investigation of the fungal extract led to the isolation of six benzophenone derivatives (1-6), along with seven known compounds (7-13) (Figure 1).The anti-inflammatory potential of the compounds was assayed.Herein, we reported the details of the isolation, structural elucidation, and determination of the anti-inflammatory effect of those compounds.

Strain Isolation and Species Identification
The fungus was identified as Aspergillus carneus based on its morphological features and the sequence analysis of the ITS region of the rRNA gene (Figure S1), which exhibited 99.81% similarity with fungal strain A. carneus, with GenBank accession number MH777426.1.

Anti-Inflammatory Activity Test
All the compounds were determined for inhibition on NO secretion using LPS induced RAW 264.7 cells.Compounds 5, 6, and 8 exhibited a moderate effect with EC 50 values of 34.6 ± 0.9, 20.2 ± 1.8, and 26.8 ± 1.7 µM, while 11 showed a potent effect with an EC 50 value of 2.9 ± 0.1 µM.The positive control dexamethasone had an EC 50 value of 2.9 ± 0.1 µM (Figure 4).

Isolation and Species Identification of Marine Fungus
The fungal strain GXIMD00543 was isolated from a sponge tissue sample that was collected from the Weizhou islands coral reef, Beibu Gulf, in December 2019.The sponge was collected and identified as Haliclona sp. by Dr. Xin-Ming Liu, Institute of Marine Drugs, Guangxi University of Chinese Medicine (Nanning, China) (Figure S1).
The strain was deposited in the Institute of Marine Drugs, Guangxi University of Chinese Medicine, Nanning, China.The fungus was identified by its morphological features and the sequence analysis of the internally transcribed spacer (ITS) region of the rRNA gene.The ITS sequence was amplified from the genome DNA via PCR with primers (ITS1: 5 ′ TCCGTAGGTGAACCTGCGG3 ′ and ITS4: 5 ′ TCCTCCGCTTATTGATATGC3 ′ ).ITS se-quences (see Supplementary Materials) were then uploaded to the National Center of Biotechnology Information (NCBI) for BLAST analysis (GenBank accession number: OR501447).

Fungal Fermentation
The fungal strain was statically cultivated in one hundred and fifty 1000 mL Erlenmeyer flasks, each containing modified solid rice medium (80 g of rice, 0.4 g of yeast extract, 0.4 g of glucose, 3.6 g of artificial sea salt, and 120 mL of H 2 O) for 35 days at room temperature.Then, the fermented cultures were extracted with EtOAc three times and concentrated in vacuo to obtain extract (230 g).

Computational Methods
MMFF and DFT/TDDFT calculations were performed with CONFLEX 8.5 (Conflex Corp., Tokyo, Japan) and Gaussian16 program package (Gaussian Inc., Wallingford, CT, USA), respectively.The MMFF94s conformational search-generated low-energy conformers within a 10 kcal/mol energy window were subjected to further geometry optimization and frequency calculation using the B3LYP/6-31G(d) method.The single-point energy of the optimized conformers was recalculated at the M06-2X/def2TZVP level.Thus, the Gibbs free energy, obtained by the sum of the single-point energy and the thermal correction was used for the relative thermal free energy (DG) calculation and following Boltzmann population analysis at 298.15 K.The TDDFT-calculated conformers were performed using the cam-B3LYP functional with def2TZVP basis set.The number of excited states per each molecule was set to 20.The CD spectra were generated by the program SpecDis V1.71 [24] using a Gaussian band shape from dipole-length dipolar and rotational strengths.The calculated spectra were finally generated from the Boltzmann weighting of each conformer.The Grimme's dispersion (D3 version) was used for empirical dispersion correction.Solvent effects (in MeOH) were taken into account by using the default SCRF method integral equation formalism variant (IEFPCM) for the whole calculation.
The B3LYP/6-31G(d) optimized geometries of 5 were adopted for further NMR computation.GIAO calculations of the 1 H and 13 C NMR chemical shifts were accomplished by DFT at the mPW1PW91/6-311+G(d,p) level in DMSO solution.The calculated NMR spectroscopic data were averaged according to the Boltzmann distribution by the program Multiwfn 3.7 [25].

Anti-Inflammatory Activity Test
The inhibition of the NO production assay was performed according to the reported procedures [26].The mouse macrophage RAW264.7 cell lines used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 ug/L of streptomycin, and 100 IU/mL of penicillin at 37 • C and 5% CO 2 atmosphere (PHCbi, Minato-ku, Tokyo, Japan).The viability of the RAW264.7 cells was determined by MTT assay.The RAW264.7 cells were seeded at a density of 5 × 10 5 cells/well in 96-well plates and incubated for 24 h at 37 • C and 5% CO 2 .Then, the media in each well was replaced using fresh FBS-free DMEM media.Different concentrations of compounds (2.5, 5, 10, 20, 40 µM) were prepared in FBS-free DMEM to give a total volume of 100 µL in each well of a microtiter plate.After 1 h of treatment, the cells were stimulated with 1 µg/mL of LPS for 24 h.The presence of nitrite was determined in the cell culture media using a commercial Griess reagent kit (Thermo Fisher Scientific, Waltham, MA, USA).Protocols supplied with assay kit were used for the application of the assay procedure.Briefly, 100 µL of cell culture medium with an equal volume of Griess reagent in a 96-well plate was incubated at room temperature for 10 min.Then, the absorbance was measured at 540 nm in a microplate reader (PerkinElmer, Waltham, MA, USA).The amount of nitrite in the media was calculated from the standard curve of sodium nitrite (NaNO 2 ).Dexamethasone (DXM) was used as a positive control.

Figure 4 .
Figure 4.The inhibition of NO secretion in the RAW 264.7 cell line by the compounds 5, 6, 8, and 11.The inhibitory rate of NO in RAW 264.7 cells with different concentrations of compounds.Data were presented as mean ± SD of the experiments (n = 3).#### p < 0.0001 compared with the blank control.* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared with the LPS model group.No cytotoxicity of compounds on RAW 264.7 cells was observed at 40 µM.