New Isocoumarins from the Marine Fungus Phaeosphaeriopsis sp. WP-26

Five new isocoumarins, phaeosphaerins A–E (1–5), were isolated from the fermentation broth of the marine fungus Phaeosphaeriopsis sp. WP-26, along with one known isocoumarin, 6,8-dihydroxy-7-methoxy-3-methylisocoumarin (6), and two known pimarane-type diterpenes, diaportheins A (7) and B (8). Their structures were elucidated via NMR experiments, X-ray diffraction analysis, and comparison of the experimental and computed ECD curves. Compounds 1–7 displayed weak neuroprotective effects against H2O2-induced damage in SH-SY5Y cells. Moreover, compound 8 showed cytotoxicity against BEL-7402, SGC-7901, K562, A549, and HL-60 cell lines.


Introduction
Natural products are still currently considered as the best options for finding novel agents/active templates [1]. As an important source of natural products, marine fungi have provided a great deal of new active compounds [2][3][4]. Phaeosphaeriopsis sp. was first described as a new genus of fungi in 2003 [5], being obtained from marine and plant resources [6,7]. This genus of fungi has mainly been investigated for its plantprotective effects [8,9] and microbial taxonomy [5,10,11]. However, their metabolites are rarely studied, and up to now there has only been one paper about the metabolites of Phaeosphaeriopsis sp. [7].
In our ongoing investigation into the chemical diversity of marine fungi for marine drug discovery, a marine-derived fungus identified as Phaeosphaeriopsis sp. WP-26 was isolated from Strombus luhuanus Linnaeus. We studied the active metabolites of the marinederived fungus Phaeosphaeriopsis sp. WP-26. During initial chemical screening of this fungus via HPLC-UV analysis, the ethyl acetate extract of fermentation broth of this strain exhibited a series of peaks showing attractive structure diversity, with UV absorptions different to those of the compounds reported in reference [7]. A detailed chemical investigation of this strain led to the isolation of five new isocoumarins (1)(2)(3)(4)(5) (Figure 1), along with one known isocoumarin, 6,8-dihydroxy-7-methoxy-3-methylisocoumarin (6) [12], and two known pimarane-type diterpenes, diaportheins A (7) and B (8) [13,14]. Hence, their isolation, structure determination, and biological activities are reported.
Compound 2, a brown powder, gave a [M + Na] + peak at m/z 297.1035 and a [M + 2 + Na] + peak at m/z 299.0108 in the ratio of 3:1 in HRESIMS, indicating the presence of one chlorine atom in 2. So, the molecular formula of 2 was determined as C 11 H 11 ClO 6 . The 1 H NMR, 13 C NMR, and HSQC spectra of 2 revealed resonances for a methyl (δ C/H 16.5/1.53), a methoxy (δ C/H 61.1/3.87), two oxygenated methines (δ C/H 79.6/4.62 and δ C/H 64.7/4.81), six aromatic quaternary carbons (δ C 156.0, δ C 154.9, δ C 137.0, δ C 135.6, δ C 112.5, and δ C 101.9), and an ester carbonyl carbon (δ C 171.0), a phenolic hydroxyl chelated to the lactone carbonyl (δ H 11.4). A detailed comparison of the aforesaid data and those of compound 1 suggested they had similar NMR data, except for the fact that an sp 2 quaternary carbon (δ C 112.5) in 2 replaced an sp 2 methine (δ C/H 108.5/6.49) in 1. The above data combined with the COSY correlations ( Figure 2) from H-4 to H 3 -11 through H-3, together with HMBC correlations (Figure 2) from H-4 to C-5 (δ C 112.5), C-9 (δ C 101.9), C-10 (δ C 135.6), from H 3 -11 to C-1 (δ C 171.0), H 3 -12 to C-7 (δ C 137.0), and 8-OH to C-7, C-8 (δ C 156.0) and C-9, suggested 2 had a similar structure to 1 and their only difference was that an aromatic proton in 1 was substituted by a Cl atom in 2. The above assignment was further confirmed by a single-crystal X-ray diffraction pattern obtained using the anomalous scattering of Mo Kα radiation ( Figure 3). The absolute configuration of 2 was determined as 3R and 4R according to the single-crystal X-ray crystallographic analysis, which was consistent with the result of ECD calculation ( Figure 4). Hence, the structure of compound 2 was identified as that shown in Figure 1 and it was named as phaeosphaerin B.
Compound 3 was obtained as a brown oil. It had the same molecular formula as 2, which was determined as C 11 H 11 ClO 6 , based on the characteristic protonated molecular ions at m/z 297.0147 [M + Na] + (calcd. for C 11 H 11 ClO 6 Na + : 297.0136) and 299.0121 [M + Na + 2] + in the ratio of 3:1 in the HRESIMS spectrum. A comparison of the NMR data of 3 (Table 1 and Figure 2) and 2 suggested they had the same planar structure but significant chemical shift differences in C-  Figure S19 in Supplementary Information), indicating that they were a pair of epimers. So, the relative configuration of 3 was determined as 3R* and 4S*. The absolute configuration of 3 was identified via a comparison of experimental and calculated ECD spectra (Figure 4), and the well-matched calculated and experimental ECD spectra of 3 suggested the 3R, 4S configurations of 3. Thus, the structure of compound 3 was identified as that shown in Figure 1, and it was named phaeosphaerin C.  Compound 4 was also obtained as a brown powder. Its molecular formula was identified as C12H14O6 according to HRESIMS with a peak at m/z 277.0692 [M + Na] + (calcd. for C12H14NaO6 + :277.0683). The NMR data of 4 ( Table 2) were very similar to those of 1 except for the presence of an extra methoxy group (δC/H 56.8/3.28) in 4. The aforementioned data indicated that the difference between the planar structure of 4 and that of 1 was a hydroxyl group in 1 substituted by a methoxy in 4, as evidenced by the COSY correlations ( Figure  2) from H-4 to H3-11 through H-3, together with HMBC correlations from H-5 to C-1 (δC 171.1), C-4 (δC 76.3), C-7 (δC 136.6), and C-9 (δC 101.4), from H-4 to C-9 and C-10 (δC 135.6), from H3-12 to C-7, from H3-11 to C-1 (weak correlation), and from H3-13 to C-4. The above assignment was further confirmed via a single-crystal X-ray diffraction pattern obtained using the anomalous scattering of Cu Kα radiation (Figure 3), which also led to an unambiguous assignment of the absolute configuration of 4 as 3R and 4R. The absolute configuration of 4 was also verified by its well-matched calculated and experimental ECD spectra ( Figure 4). So, the structure of compound 4 was identified as that shown in Figure 1 and it was named phaeosphaerin D.   (Table 2 and Figure 2) of 5 and 4 suggested that they have the sa planar structure, while the significant chemical shift differences in C-1 (1.4), C-4 (1.9), 10 (1.2), and C-11 (1.4) between them and the 1D NOE correlation from H-4 (δH 4.11) H3-11 (δH 1.30) indicated that they were also a pair of epimers, just like 2 and 3. So, relative configuration of 5 was determined as 3R* and 4S*. Then, the calculated ECD cur for 5 was found to match well with the experimental one ( Figure 4), and so we assign the absolute configurations of 5 as 3R and 4S. Thus, the structure of compound 5 w identified as that shown in Figure 1, and it was named phaeosphaerin E.   Figure 2) of 5 and 4 suggested that they have th planar structure, while the significant chemical shift differences in C-1 (1.4), C-4 10 (1.2), and C-11 (1.4) between them and the 1D NOE correlation from H-4 (δH H3-11 (δH 1.30) indicated that they were also a pair of epimers, just like 2 and 3. relative configuration of 5 was determined as 3R* and 4S*. Then, the calculated EC for 5 was found to match well with the experimental one ( Figure 4), and so we a the absolute configurations of 5 as 3R and 4S. Thus, the structure of compound identified as that shown in Figure 1, and it was named phaeosphaerin E.  Besides the above new compounds 1-5, three known compounds were also and indentified as isocoumarins 6,8-dihydroxy-7-methoxy-3-methylisocoumarin and two known pimarane-type diterpenes, diaportheins A (7) and B (8) [13,14] paring their NMR spectroscopic (Tables S1-S3 in Supplementary Information) an ical data with literature values. Compound 4 was also obtained as a brown powder. Its molecular formula was identified as C 12 (Table 2) were very similar to those of 1 except for the presence of an extra methoxy group (δ C/H 56.8/3.28) in 4. The aforementioned data indicated that the difference between the planar structure of 4 and that of 1 was a hydroxyl group in 1 substituted by a methoxy in 4, as evidenced by the COSY correlations ( Figure 2) from H-4 to H 3 -11 through H-3, together with HMBC correlations from H-5 to C-1 (δ C 171.1), C-4 (δ C 76.3), C-7 (δ C 136.6), and C-9 (δ C 101.4), from H-4 to C-9 and C-10 (δ C 135.6), from H 3 -12 to C-7, from H 3 -11 to C-1 (weak correlation), and from H 3 -13 to C-4. The above assignment was further confirmed via a single-crystal X-ray diffraction pattern obtained using the anomalous scattering of Cu Kα radiation (Figure 3), which also led to an unambiguous assignment of the absolute configuration of 4 as 3R and 4R. The absolute configuration of 4 was also verified by its well-matched calculated and experimental ECD spectra ( Figure 4). So, the structure of compound 4 was identified as that shown in Figure 1 and it was named phaeosphaerin D.  Figure 2) of 5 and 4 suggested that they have the same planar structure, while the significant chemical shift differences in C-1 (1.4), C-4 (1.9), C-10 (1.2), and C-11 (1.4) between them and the 1D NOE correlation from H-4 (δ H 4.11) to H 3 -11 (δ H 1.30) indicated that they were also a pair of epimers, just like 2 and 3. So, the relative configuration of 5 was determined as 3R* and 4S*. Then, the calculated ECD curve for 5 was found to match well with the experimental one (Figure 4), and so we assigned the absolute configurations of 5 as 3R and 4S. Thus, the structure of compound 5 was identified as that shown in Figure 1, and it was named phaeosphaerin E.

Collection and Phylogenetic Analysis
The fungus strain, Phaeosphaeriopsis sp. WP-26, was isolated from a Strombus luhuanus Linnaeus collected from Yagong Island of the Xisha Islands in the South China Sea in August 2020. The sample (1.0 g) was diluted to 10 −2 g/mL with sterile H2O after grinding, and then a 100 μL supernate was deposited on a Potato Dextrose Agar (PDA) (200.0 g of potato, 20.0 g of glucose, 20.0 g of agar per liter, 33.0g of NaCl, 1.0 L of water) plate containing chloramphenicol (100 μg/mL) as a bacterial inhibitor. A reference culture was maintained in our laboratory at −80 °C. Working stocks were prepared on PDA slants stored at 4 °C.
The fungus was identified based on the DNA sequences, which were deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in the Na-

Collection and Phylogenetic Analysis
The fungus strain, Phaeosphaeriopsis sp. WP-26, was isolated from a Strombus luhuanus Linnaeus collected from Yagong Island of the Xisha Islands in the South China Sea in August 2020. The sample (1.0 g) was diluted to 10 −2 g/mL with sterile H 2 O after grinding, and then a 100 µL supernate was deposited on a Potato Dextrose Agar (PDA) (200.0 g of potato, 20.0 g of glucose, 20.0 g of agar per liter, 33.0g of NaCl, 1.0 L of water) plate containing chloramphenicol (100 µg/mL) as a bacterial inhibitor. A reference culture was maintained in our laboratory at −80 • C. Working stocks were prepared on PDA slants stored at 4 • C.
The fungus was identified based on the DNA sequences, which were deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in the National Genomics Data Center (Nucleic Acids Res 2022), the China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA009121. The ITS gene sequence data are provided in the Supplementary Materials), and are publicly accessible at https://ngdc.cncb.ac.cn/gsa (accessed on 2 December 2022). The mycelium was ground to a fine powder in liquid N 2 ; then, genomic DNA was extracted, and the TIS region was amplified via PCR using primers ITS1 (GTAG TCATAT-GCTTGTCTC) and ITS4 (GCATCACAG ACCTG TTATTGCCTC). PCR products were sequenced on an Applied Biosystems 3730 XL Genetic Analyzer (Applied Biosystems Inc., Foster City, CA, USA).

Cultivation and Extraction
The spores of Phaeosphaeriopsis sp. WP-26 were directly transferred to 150 mL of a liquid medium (the potato liquid media consisting of 200.0 g/L potato, 20.0 g/L glucose, and 1000 mL deionized water) in Erlenmeyer flasks (500 mL) and shaken for 48 h (28 ± 0.5 • C, 180 rpm). Further, 5 mL of seed broth was transferred aseptically to 1000 mL Erlenmeyer flasks (160 flasks), each containing rice medium (80 g of rice and 120 mL of water). The flasks were incubated at room temperature under static conditions for 30 days. Then, the cultures were extracted three times via EtOAc and concentrated in vacuo to obtain a 138.5 g EtOAc extract.

Purification
The EtOAc extract (138.5 g) was suspended in 90% MeOH aqueous solution, and then the suspension was extracted three times using petroleum ether. The remaining MeOH aqueous part was concentrated in vacuo to obtain a 70.5 g extract.

ECD Calculation
The preliminary conformational search for 1-5 was carried out in Confab [16] using an MMFF94 molecular mechanics force field. The Gaussian 16 package was used to carry out the calculations using the density functional theory (DFT) [17]. The obtained conformers were optimized at the B3LYP/6-31G (d) level, and frequency analysis was also performed at the same level. More accurate energies of optimized conformers were evaluated at the M06-2X/Def2-TZVP level in the methanol, and were then added to the thermal correction of Gibbs free energies obtained with frequency analyses to afford the Gibbs free energies of each conformer. Then, the ECD spectrum was calculated for the optimized conformers using the TDDFT method at the APFD/6-311+G (2d) level with the IEFPCM model in MeOH. The ECD spectra were simulated using the versatile web server provided by Yinfo Information Technology Co., Ltd. (https://cloud.yinfotek.com, accessed on 1 February 2023) with a half-bandwidth of 0.29-0.50 eV and a UV shift (−17, −1, 9, 0, 3) for 1-5. Finally, the calculated ECD spectra were compared with the experimental data.

Neuroprotective Properties against H 2 O 2 -Induced Damage In Vitro
The neuroprotective properties of compounds 1-8 against H 2 O 2 -induced damage in SH-SY5Y cells were assayed using the MTT method [18]. The SH-SY5Y cells were cultured in 96-well plates for 24 h. Next, the cells were treated with the tested compounds at the test concentrations (50, 25, and 12.5 µM) for 12 h, and then exposed to 1000 µM H 2 O 2 for 12 h. After adding 20 µL of MTT (5 mg/mL) to each well for 4 h, 150 µL of DMSO was added to dissolve the formazan crystals. Finally, absorbance was read at 490 nm with a Synergy H1 microplate reader (BioTek, Winooski, VT, USA). The cell viability was expressed as a percentage with the control group as 100%.

Cytotoxicity Assay
The MTT method optimized by Mosmann et al. [19] was performed in vitro to test the cytotoxic activity of compounds 1-8. Adriamycin was used as a positive control with IC 50 values of 3.0, 4.1, 4.2, 2.0, and 11 µM for the cell lines K562, BEL-7402, SGC-7901, A549, and Hela, respectively. Additionally, the medium without the test compound was used as a negative control in the bioassay.