Bioactive Monoterpenes and Polyketides from the Ascidian-Derived Fungus Diaporthe sp. SYSU-MS4722

There has been a tremendous increase in the rate of new terpenoids from marine-derived fungi being discovered, while new monoterpenes were rarely isolated from marine-derived fungi in the past two decades. Three new monoterpenes, diaporterpenes A–C (1–3), and one new α-pyrones, diaporpyrone A (6), along with nine known polyketides 4, 5, and 7–13 were isolated from the ascidian-derived fungus Diaporthe sp. SYSU-MS4722. Their planar structures were elucidated based on extensive spectroscopic analyses (1D and 2D NMR and HR-ESIMS). The absolute configurations of 1 and 3 were identified by an X-ray crystallographic diffraction experiment using Cu-Ka radiation, and those of compound 2 were assigned by calculating NMR chemical shifts and ECD spectra. It afforded an example of natural epimers with different physical properties, especially crystallization, due to the difference in intermolecular hydrogen bonding. Compounds 9, 10, and 13 showed moderate total antioxidant capacity (0.82 of 9; 0.70 of 10; 0.48 of 13) with Trolox (total antioxidant capacity: 1.0) as a positive control, and compounds 5 and 7 showed anti-inflammatory activity with IC50 values of 35.4 and 40.8 µM, respectively (positive control indomethacin: IC50 = 35.8 µM).

The genus Diaporthe (asexual state: Phomopsis) comprises pathogenic, endophytic and saprobic species with both temperate and tropical distributions [12,13]. Chemical investigation of secondary metabolites and the discovery of novel bioactive natural products have been extensively researched because of their importance as plant pathogens [12]. For example, the mangrove-derived fungus Diaporthe sp. SCSIO 41011 produced six new highly oxygenated chloroazaphilone derivatives, isochromophilones A-F, with cytotoxicity [14]. Diaporisoindoles A-C were isolated from the mangrove endophytic fungus Diaporthe sp. SYSU-HQ3, which showed significant inhibitory activity against Mycobacterium tuberculosis highly oxygenated chloroazaphilone derivatives, isochromophilones A-F, with cytotoxicity [14]. Diaporisoindoles A-C were isolated from the mangrove endophytic fungus Diaporthe sp. SYSU-HQ3, which showed significant inhibitory activity against Mycobacterium tuberculosis protein tyrosine phosphatase B [15]. Dihydroisocoumarin, diaporone A, with antibacterial activity, was obtained from an endophytic plant fungus Diaporthe sp. [16].
Recently, our research group has focused on the secondary metabolites of ascidianderived fungi isolated from the South China Sea [17][18][19][20]. Twenty mono-and dimeric xanthones with anti-glioma and anti-inflammatory activities have been obtained from the ascidian-derived fungus Diaporthe sp. SYSU-MS4722 [21]. Continuous chemical investigation of the EtOAc extract of marine fungus Diaporthe sp. SYSU-MS4722 led to the isolation of three new monoterpenes, diaporterpenes A-C (1-3), and one new α-pyrone, diaporpyrone A (6), along with nine known compounds 4, 5, and 7-13 ( Figure 1) Herein, the details of the isolation, structural elucidation, and bioactivity of compounds 1-13 are reported.
Diaporterpene A (1) was obtained as a colourless crystal with a melting point of 126-127 °C. The molecular formula was determined as C10H16O3 on the basis of the positive HR-ESIMS ions at m/z 185.1172 [M + H] + (calculated for 185.1172, C10H17O3), implying three degrees of unsaturation. The IR spectrum (Supplementary Figure S8)  . These spectroscopic features suggested that 4 belongs to the family of monoterpenoids and is very similar to 2-thujanone, except for the presence of additional two oxygen-bearing non-hydrogen carbons (δC 68.9, 73.9) and the absence of two methines.

H-2
H-10 H-4 H-5   The monoterpenes, diaporterpenes A and B, were a pair of epimers with different physical properties, especially crystallization and solid-liquid states. Diaporterpene A was a solid state and had easy crystallization properties, while diaporterpene B was a liquid state and had difficult crystallization properties. Analysis of single crystal structure and molecular packing properties of 1 (Figure 7), intermolecular hydrogen bonds were observed. The 3-OH and 7-OH were the key functional groups for forming the intermolecular hydrogen bonds, while the O atom of the ketone does not make contributions. One molecule and four molecules around it together form five hydrogen bonding and look like a door handle. This is the main reason for the easy crystallization property for 1. However, the absolute configuration of 3-OH in 2 was S with opposite orientation compared to 1, which led to difficulty forming intermolecular hydrogen.  The monoterpenes, diaporterpenes A and B, were a pair of epimers with different physical properties, especially crystallization and solid-liquid states. Diaporterpene A was a solid state and had easy crystallization properties, while diaporterpene B was a liquid state and had difficult crystallization properties. Analysis of single crystal structure and molecular packing properties of 1 (Figure 7), intermolecular hydrogen bonds were observed. The 3-OH and 7-OH were the key functional groups for forming the intermolecular hydrogen bonds, while the O atom of the ketone does not make contributions. One molecule and four molecules around it together form five hydrogen bonding and look like a door handle. This is the main reason for the easy crystallization property for 1. However, the absolute configuration of 3-OH in 2 was S with opposite orientation compared to 1, which led to difficulty forming intermolecular hydrogen. The monoterpenes, diaporterpenes A and B, were a pair of epimers with different physical properties, especially crystallization and solid-liquid states. Diaporterpene A was a solid state and had easy crystallization properties, while diaporterpene B was a liquid state and had difficult crystallization properties. Analysis of single crystal structure and molecular packing properties of 1 (Figure 7), intermolecular hydrogen bonds were observed. The 3-OH and 7-OH were the key functional groups for forming the intermolecular hydrogen bonds, while the O atom of the ketone does not make contributions. One molecule and four molecules around it together form five hydrogen bonding and look like a door handle. This is the main reason for the easy crystallization property for 1. However, the absolute configuration of 3-OH in 2 was S with opposite orientation compared to 1, which led to difficulty forming intermolecular hydrogen.    1.42, s). The 13 C NMR and DEPT data (Table 1) of 5 revealed the presence of 10 carbons which were assigned with the help of an HSQC experiment. Except for seven sp 2 hybridized carbons (δ C 108.9, 130.4, 126.7, 152.9, 126.7, 130.4, 170.1) belonging to one benzoic acid, the remaining three sp 3 hybridized carbons were sorted as methyl (δ C 26.0), methylene (δ C 71.6), and an oxygen-bearing non-hydrogen carbon (δ C 75.7). The HMBC correlations from methyl H-10 and hydroxylmethyl H-9 to C-8 and C-5 of benzoic acid indicated a methyl, a hydroxylmethyl, and a hydroxyl were all connected to C-7 that was directly linked to C-5 of benzoic acid. Finally, the absolute configuration of 3 was assigned as S by a single-crystal X-ray diffraction experiment using Cu-Kα radiation (Flack parameter = −0.08 (15) (Figure 3).
Diaporpyrone A (6) was obtained as a white powder, and its molecular formula C 10  The key HMBC correlations of H-10 to C-2, C-3, and C-4, and H-5 to C-3, C-4, and C-6 suggested the presence of an α-pyrone moiety with a methyl substituent at C-3 ( Figure 8). The HMBC correlations from H-11 to C-7 and H-8 to C-9 assigned a but-2-enoic acid moiety, which was connected to α-pyrone moiety via C-6. Key NOE correlation between H-5 with H-11 indicated that H-5 and H-11 were on the same side, while NOE correlation between H-8 with H-11 was not observed, suggesting that H-8 with H-11 were in the trans position. Hence, compound 6 was identified as shown in Figure 8.  (Table 1) revealed resonances for four para-substituted aromatic protons (δH 7.49 (2H, dd, J = 8.4 Hz); 7.88 (2H, d, J = 8.4 Hz)), one hydroxylmethyl (δH 3.52 (2H, q, J = 11.2 Hz)), and one tertiary methyl (δH 1.42, s). The 13 C NMR and DEPT data (Table 1) of 5 revealed the presence of 10 carbons which were assigned with the help of an HSQC ex periment. Except for seven sp 2 hybridized carbons (δC 108.9, 130.4, 126.7, 152.9, 126.7 130.4, 170.1) belonging to one benzoic acid, the remaining three sp 3 hybridized carbon were sorted as methyl (δC 26.0), methylene (δC 71.6), and an oxygen-bearing non-hydrogen carbon (δC 75.7). The HMBC correlations from methyl H-10 and hydroxylmethyl H-9 to C 8 and C-5 of benzoic acid indicated a methyl, a hydroxylmethyl, and a hydroxyl were al connected to C-7 that was directly linked to C-5 of benzoic acid. Finally, the absolute con figuration of 3 was assigned as S by a single-crystal X-ray diffraction experiment using Cu-Kα radiation (Flack parameter = −0.08 (15) (Figure 3).
Phenolic compounds (including cinnamic acids, benzoic acids, flavonoids, proanthocyanidins, coumarins, stilbenes, lignans, and lignins) are the most widespread class of metabolites in nature and are proven to be effective antioxidants [30]. Compounds 1-13 were evaluated using the total antioxidant capacity assay kit with a rapid ABTS method. Only compounds 9, 10, and 13 showed moderate total antioxidant capacity (0.82 of 9; 0.70 of 10; 0.48 of 13) with Trolox as a positive control. Anti-inflammatory activities were performed for compounds 1-13 with the inhibition of nitric oxide (NO) production in RAW264.7 cells activated by lipopolysaccharides, and compounds 5 and 7 showed antiinflammatory activity with IC 50 values 35.4 and 40.8 µM, respectively, with indomethacin (IC 50 = 35.8 µM) as a positive control. All isolates were evaluated for anti-glioma activity using T98G human cell line with temozolomide as the positive control, and none of them showed significant cytotoxicity against T98G human cell line.

Fungal Material
The detail of the fungus was described previously [21]. The molecular biological protocol, including DNA amplification and sequencing of the ITS region, was used for fungal identification. The sequence data of the fungal strain have been deposited at GenBank with accession no. OK623372. A BLAST search result suggested that the sequence was most similar (100%) to the sequence of Diaporthe sp. NFIF-2-6 (compared to MW202988.1). The strain was preserved at the School of Marine Sciences, Sun Yat-Sen University.

Extraction and Isolation
The strain Diaporthe sp. SYSU-MS4722 was grown on a solid rice medium in a 1000 mL culture flask containing 50g of rice and 50 mL of 3% artificial seawater after sterilization. A total of 120 flasks of fungal incubation were cultivated at room temperature for 30 days. The solid fermented substrate was extracted with MeOH four times to obtain a crude extract, then dissolved in H 2 O and continuously extracted four times with EtOAc solvent. The EtOAc extract (42 g) was subjected to a silica gel column eluting with linear gradient petroleum ether/EtOAc (from 8:2 to 0:1) to obtain six fractions (A-F).  9.0 mg). Fr.C.5 was subjected to silica gel chromatography to afford 6 (11 mg) and Fr.C.5.2, which was further purified by RP-HPLC (MeOH/H 2 O, 60:40 flow rate 2 mL/min, ACE-C18-PFP column 10 × 250 mm, 5 µm) to give 1 (5.0 mg, t R =14.6 min). Fr.C.4.5 was further fractionated on a silica gel column and RP-HPLC to afford 3 (12 mg, t R =19.5 min).

X-ray Crystallographic Analysis
Compounds 1 and 3 were obtained as colorless crystals using the vapor diffusion method. The single crystal X-ray diffraction data were recorded on a Rigaku Oxford Diffraction with Cu-Kα radiation (λ = 1.54178A). The structures were solved by direct methods (SHELXS-97 and Olex2-1.2) and refined using full-matrix least-squares difference measured (R int = 0.0387, R sigma = 0.0581), which were used in all calculations. The final R 1 was 0.0402 (I ≥ 2u(I)), and wR 2 was 0.1031. The Flack parameter was 0.09 (19). The goodness of fit on F 2 was 1.053. CCDC 1997240.

Calculation of the ECD Spectra
Merck molecular force field (MMFF) and DFT/TD-DFT calculations were performed with the Spartan'14 software package (Wavefunction Inc., Irvine, CA, USA) and the Gaussian 09 program (Gaussian, Inc., Wallingford, CT, USA), respectively [31]. MMFF conformational search generated low-energy conformers within a 10 kcal·mol −1 energy window. All the lowest energy conformers were subjected to DFT geometry optimizations at the ωB97X-D [32]/TZVP [33] level with the solvation model PCM for methanol. Frequency calculations were performed at the same level to confirm that each optimized conformer was at its true minimum and to estimate their relative thermal free energies (∆G) at 298.15 K. Single-point energies were calculated at the M06-2X [34]/def2-TZVP [35]/SDM(MeOH) level of theory. The population of each conformer was calculated by Boltzmann distribution based on Gibbs free energy with the Shermo [36]. Finally, a free energy correction +1.89 kcal·mol −1 was applied to all free energies to consider the conversion from the gas phase (1 atm) to the liquid phase (1 M). Then, conformers with distributions higher than 1% were chosen for the TDDFT calculations at the PBE1PBE/TZVP [33] level in methanol. The ECD spectrum was generated by the program SpecDis [37] using a Gaussian band shape with 0.20 eV exponential half-width from dipole-length dipolar and rotational strengths. NMR calculations used the gauge-including atomic orbitals (GIAO) method at the mPW1PW91/6-311+G(d,p) level in MeOH simulated by the IEFPCM model. The TMScorrected NMR chemical shift values were averaged according to Boltzmann distribution and fitted to the experimental values by linear regression. To confirm the conclusions of NMR calculations, DP4+ analysis was also performed. All calculations were performed by Tianhe-2 in National Super Computer Center in Guangzhou.

Anti-Glioma Activity
The assays for anti-glioma activity were evaluated as described previously [21]. The human glioma cell line, T98G, was purchased from the Cell Bank of the Chinese Academy of Sciences. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Carlsbad, CA, USA), containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 µg/mL streptomycin (all from Gibco, Carlsbad, CA, USA) in a cell incubator with 5% CO 2 at 37 • C.
Cell proliferation was analyzed by MTT according to the manufacturer's instructions. Briefly, T98G was digested and seeded at 1 × 10 3 cells/well in 96-well plates and cultured in a 100 µL medium overnight. The cells were treated with tested compounds with gradient concentrations for 48 h. At each indicated time point, MTT solution (10 µL/well) was added and then incubated at 37 • C for 2 h. The optical density (OD) at 450 nm was recorded by a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA). Each experiment was performed three times.

Anti-Inflammatory Activity
The assays for anti-inflammatory activity were performed as described previously [38]. RAW 264.7 cells were seeded in 96-well plates at a density of 5 × 10 5 cells/ mL. After 12 h, the cells were treated with 1 µg/mL of LPS and tested samples, followed by additional incubation for 24 h at 37 • C. MTT stock solution (2 mg/mL) was added to wells for a total reaction volume of 100 µL. After 4 h incubation, the supernatants were aspirated. The formazan crystals in each well were dissolved in DMSO (100 µL), and the absorbance was measured with the wavelength of 490 nm by a microplate reader (Multiskan GO, Thermo Scientific). The data were expressed as mean percentages of the viable cells compared to the respective control. After pre-incubation of RAW 264.7 cells (1.5 × 10 5 cells/mL) with 1 µg/mL LPS and samples at 37 • C for 24 h, the quantity of nitrite accumulated in the culture medium was measured as an indicator of NO production. Briefly, cell culture medium (50 µL) was added with Griess reagent (100 µL) and incubated at room temperature for 10 min. The absorbance was measured by a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA) at 540 nm wavelength.

Total Antioxidant Capacity Assay
The assays for total antioxidant capacity were carried out as described previously [18]. A total antioxidant capacity assay kit with a rapid ABTS method (Beyotime Institute of Biotechnology, Shanghai, China) was used to evaluate the total antioxidant capacity based on the manufacturer's instructions. Samples were incubated at 25 • C for 6 min and then were recorded at 414 nm using a multimode reader (Thermo Fisher Scientific, Waltham, MA, USA).

Conclusions
It is a challenging thing to discover new monoterpenes from marine-derived fungi. Continuous chemical investigation of the EtOAc extract of marine fungus Diaporthe sp. SYSU-MS4722 afforded three new monoterpenes, diaporterpenes A-C (1-3), and one new α-pyrone, diaporpyrone A (6), along with nine known polyketides. Compounds 9, 10, and 13 showed moderate total antioxidant capacity (0.82 of 9; 0.70 of 10; 0.48 of 13) with Trolox as a positive control, and compounds 5 and 7 showed anti-inflammatory activity with IC 50 values of 35.4 and 40.8 µM, respectively. It afforded an example of natural epimers with different physical properties, especially crystallization, due to the difference in intermolecular hydrogen bonding.
Author Contributions: Conceptualization, S.C. and L.L.; methodology, S.C. and H.G.; data analysis, M.J., H.G. and S.C.; experiment, H.S. and G.Z.; resources, writing-original draft preparation, S.C. and G.Z.; writing-review and editing, S.C.; funding acquisition, S.C. and L.L. All authors have read and agreed to the published version of the manuscript.