Mono- and Dimeric Xanthones with Anti-Glioma and Anti-Inflammatory Activities from the Ascidian-Derived Fungus Diaporthe sp. SYSU-MS4722

Seven new xanthones, diaporthones A−G (1−7), together with 13 known analogues, including five mono- (8−14) and six dimeric xanthones (15−20), were obtained from the ascidian-derived fungus Diaporthe sp. SYSU-MS4722. Their planar structures were established by extensive spectroscopic analyses, including 1D and 2D NMR and high-resolution mass spectrometry (HR-ESIMS). The absolute configurations of 1−7 were clearly identified by X-ray crystallographic analysis and calculation of the ECD Spectra. Compounds 15−20 showed significant anti-inflammatory activity with IC50 values between 6.3 and 8.0 μM. In addition, dimeric xanthones (15−20) showed selective cytotoxicity against T98G cell lines with IC50 values ranging from 19.5 to 78.0 μM.


Introduction
Glioma is a fatal disease of the central nervous system having an incidence rate of 3.20 per 100,000 people [1][2][3]. Due to limitations of the blood-brain barrier, only four drugs, including lomustine, carmustine, temozolomide, and bevacizumab, have been approved by FDA to treat glioma in the past four decades [4,5]. Temozolomide (TMZ) as the first-line therapy drug is used for the treatment of glioma; however, at least 50% of TMZ-treated patients and more than 90% of recurrent gliomas show no response to TMZ [6,7]. Thus, it is an urgent need to find more anti-glioma agents for the treatment of glioma. In recent years, anti-glioma molecules of marine origin have attracted many scientific research institutes or pharmaceutical companies' attention. For example, marizomib (salinosporamide A) received orphan drug designation for glioblastoma in the United States [8,9], which was discovered from the marine actinomycete Salinispora tropica [10] and Salinispora Arenicola [11] and is an irreversible proteasome inhibitor with a nanomolar range IC 50 value [11]. Depatuxizumab vedotin was an antibody-drug conjugate (ADC) in phase 3 trials to treat newly diagnosed glioblastoma with EGFR amplification [8], which was developed from pentapeptide dolastatin 10 that was produced from the sea hare Dolabella Auricularia [12].

Results and Discussion
The EtOAc extract of marine-derived fungus Diaporthe sp. SYSU-MS4722 was performed on the repeated silica gel and Sephadex LH-20 column chromatography, followed by semipreparative HPLC to afford seven new xanthones, diaporthones A−H (1−7) along

Results and Discussion
The EtOAc extract of marine-derived fungus Diaporthe sp. SYSU-MS4722 was performed on the repeated silica gel and Sephadex LH-20 column chromatography, followed by semipreparative HPLC to afford seven new xanthones, diaporthones A−H (1−7) along with 13 known analogues including seven mono- (8−14) and six dimeric xanthones (15−20). Diaporthone A (1) was obtained as yellow crystal, and its molecular formula was established as C 15   The planar structure of 1 was identified based on the extensive 2D NMR ( 1 H-1 H COSY, HSQC, and HMBC) (Figures S2-S7) spectroscopic data ( Figure 2). The HMBC correlations from H-7 to C-5 and C-8a, from H-8 to C-4a, from H-13 to C-2 and C-3, and 1 H-1 H COSY of H-6/H-7/H-8, as well as the NMR chemical shifts, complete a 4-hydroxylchromone skeleton with a hydroxymethyl group at C-2. The γ-lactone moiety with a methyl group was assigned by HMBC correlations from H-9 to ester carbonyl C-12, from H-11 to C-9 and C-12, from H-14 to C-9, C-10 and C-11, and the 1 H-1 H COSY correlations of H-9/H-10/H-11 and H-10/H-14. The γ-lactone moiety was finally linked to C-2 of the chromone skeleton, supported by HMBC correlations from H-13 to C-9 and H-9 to C-2.  The planar structure of 1 was identified based on the extensive 2D NMR ( 1 H-1 H COSY, HSQC, and HMBC) (Figures S2-S7) spectroscopic data ( Figure 2). The HMBC correlations from H-7 to C-5 and C-8a, from H-8 to C-4a, from H-13 to C-2 and C-3, and 1 H-1 H COSY of H-6/H-7/H-8, as well as the NMR chemical shifts, complete a 4-hydroxylchromone skeleton with a hydroxymethyl group at C-2. The γ-lactone moiety with a methyl group was assigned by HMBC correlations from H-9 to ester carbonyl C-12, from H-11 to C-9 and C-12, from H-14 to C-9, C-10 and C-11, and the 1 H-1 H COSY correlations of H-9/H-10/H-11 and H-10/H-14. The γ-lactone moiety was finally linked to C-2 of the chromone skeleton, supported by HMBC correlations from H-13 to C-9 and H-9 to C-2.  The NOESY spectrum of 1 showed a correlation from H-9 to H-14, indicating that H-9 and H-14 were on the same side of the γ-lactone (Figure 3). The X-ray structure (Figure 4) showed that the protons H-13 and H-9 were located on the close position of the axis of rotation C2-C9, indicating that the relative configuration of 1 was 2S*, 9S*, and 10S*. In addition, the ECD spectrum of (2S, 9S, 10S)-1 calculated by a quantum chemical method at the [B3LYP/6-311 + G(2d,p)] was in good agreement with that of the experimental one ( Figure 5). Thus, the absolute configuration of 1 was identified as 2S, 9S, and 10S.
The NOESY spectrum of 1 showed a correlation from H-9 to H-14, indicating that H-9 and H-14 were on the same side of the γ-lactone ( Figure 3). The X-ray structure ( Figure   4) showed that the protons H-13 and H-9 were located on the close position of the axis of rotation C2-C9, indicating that the relative configuration of 1 was 2S*, 9S*, and 10S*. In addition, the ECD spectrum of (2S, 9S, 10S)-1 calculated by a quantum chemical method at the [B3LYP/6-311 + G(2d,p)] was in good agreement with that of the experimental one ( Figure 5). Thus, the absolute configuration of 1 was identified as 2S, 9S, and 10S.
It can be seen that the proton H2-3 of 1 was the α-H atom of ketone with weak acid, whose α-hydrogen exchange (α-deuterodeprotonatioion reaction) could be found in CD3OD. The signal of H2-3 was observed as a weak and small peak, and the integrals were much less than two in the 1 H NMR spectrum. The natural product with the α-H atom of ketone moiety would be better for acquiring the NMR data under the deuterated solvents without exchangeable deuterium.   9 and H-14 were on the same side of the γ-lactone ( Figure 3). The X-ray structure ( Figure   4) showed that the protons H-13 and H-9 were located on the close position of the axis of rotation C2-C9, indicating that the relative configuration of 1 was 2S*, 9S*, and 10S*. In addition, the ECD spectrum of (2S, 9S, 10S)-1 calculated by a quantum chemical method at the [B3LYP/6-311 + G(2d,p)] was in good agreement with that of the experimental one ( Figure 5). Thus, the absolute configuration of 1 was identified as 2S, 9S, and 10S.
It can be seen that the proton H2-3 of 1 was the α-H atom of ketone with weak acid, whose α-hydrogen exchange (α-deuterodeprotonatioion reaction) could be found in CD3OD. The signal of H2-3 was observed as a weak and small peak, and the integrals were much less than two in the 1 H NMR spectrum. The natural product with the α-H atom of ketone moiety would be better for acquiring the NMR data under the deuterated solvents without exchangeable deuterium.    It can be seen that the proton H 2 -3 of 1 was the α-H atom of ketone with weak acid, whose α-hydrogen exchange (α-deuterodeprotonatioion reaction) could be found in CD 3 OD. The signal of H 2 -3 was observed as a weak and small peak, and the integrals were much less than two in the 1 H NMR spectrum. The natural product with the α-H atom of ketone moiety would be better for acquiring the NMR data under the deuterated solvents without exchangeable deuterium.
Diaporthone B (2) was obtained as yellow crystal, and its molecular formula was established as C 15 Figure 2). The HMBC correlations from H-10 to C-5a and C-8a, from H-3 to C-1 and C-4a, and 1 H-1 H COSY of H-2/H-3/H-4, as well as the NMR chemical shifts, suggested the presence of a hydroxylchromone skeleton with a hydroxymethyl group at C-5a and a hydroxyl at C-8a. The remaining ring was assigned to be 4-hydroxyl-5-methyl cyclohexenol on the basis of 1 H-1 H COSY of H-6/H-7/H-8/H-9 and H-6/H-11, and key HMBC correlations from H-8 to C-9 and C-5a, H-5 to C-8a. Finally, the structure and absolute configuration (5S, 5aS, 6R, 8aR, 8S) were distinctly demonstrated by X-ray crystallographic analysis ( Figure 4) from Cu Kα data with a Flack parameter of −0.01(14) [17] and a Hooft parameter of 0.06(7) [18].
Diaporthone C (3) was obtained as a colorless oil, whose molecular formula was determined as C 15 (Table 2) suggested that 3 was similar to 2 belonging to the xanthone class, except for the presence of an additional double bond (δ C 140.1 and 122.1; δ H 5.60) in 3. The key HMBC correlations from methyl protons H-11 to olefinic carbons C-6 and C-7 were allowed to assign the location of the double bond in 3. The gross structure of 3 was identified by the 2D NMR spectroscopy ( Figure 2). NOE correlations of H-5 with H-10 and H-10 with H-8 were not observed, and the relative configuration of compound 3 should be 5R*, 5aS*, 8aR*, and 8S* (Figure 3). The predicted ECD curve of 3 matched well with the experimental one ( Figure 5). Hence, the absolute configuration of 3 was identified as 5R, 5aS, 8aR, and 8S.
Diaporthone D (4) was obtained as a colorless oil, and its molecular formula C 15 (Table 1)  , and H-10 were on the same side. The relative configuration of compound 4 was assigned as 5R*, 5aR*, 6R*, 8aR*, and 8R*. The absolute configuration of 4 was assigned by comparing the experimental and calculated ECD spectra, and the calculated ECD spectrum was agreed with that of the experimental one ( Figure 6). Therefore, the absolute configuration of 4 was assigned as 5R, 5aR, 6R, 8aR, and 8R.   S39) indicated that compound 5 shared the same xanthone skeleton as penexanthone B (9). The only difference between them was that compound 5 was the absence of an additional acetyl group (10-Ac) compared to 6. Compound 5 was a precursor of biosynthesis of 12-deacetylphomoxanthone A (15), and there is reason to believe that they share the same absolute configuration of 5S, 5aS, and 6S. The predicted ECD spectrum of (5S, 5aS, 6S)-5 showed well fit with that of the experimental one ( Figure 6). Thus, compound 5 was identified as de-10-acetylpenexanthone B.
Diaporthone F (6) was obtained as a yellow crystal and had the same molecular formula (C15H16O7) as 8 established by the HR-ESIMS ions at m/z 307.0828 [M − H] − (calculated for C15H15O7, 307.0823). Compound 6 shared the same planar structure as phomoxanthone The only difference between them was that compound 5 was the absence of an additional acetyl group (10-Ac) compared to 6. Compound 5 was a precursor of biosynthesis of 12-deacetylphomoxanthone A (15), and there is reason to believe that they share the same absolute configuration of 5S, 5aS, and 6S. The predicted ECD spectrum of (5S, 5aS, 6S)-5 showed well fit with that of the experimental one ( Figure 6). Thus, compound 5 was identified as de-10-acetylpenexanthone B.
Diaporthone F (6) was obtained as a yellow crystal and had the same molecular formula (C 15 (8), which was further identified by 1 H-1 H COSY, HSQC, and HMBC spectroscopies (Figures S42-S47). Detailed analysis of their NMR (Table 3), diaporthone F (6) and phomoxanthone G should be a pair of epimers. The structure of 6 ( Figure 7) and configuration (5R, 5aR, 6S, 8aS, 8R) were identified by X-ray crystallographic analysis from Cu Kα data with a Flack parameter of -0.13 (15) [17] and a Hooft parameter of 0.16(9).   (9). The only difference between them was that compound 5 was the absence of an additional acetyl group (10-Ac) compared to 6. Compound 5 was a precursor of biosynthesis of 12-deacetylphomoxanthone A (15), and there is reason to believe that they share the same absolute configuration of 5S, 5aS, and 6S. The predicted ECD spectrum of (5S, 5aS, 6S)-5 showed well fit with that of the experimental one ( Figure 6). Thus, compound 5 was identified as de-10-acetylpenexanthone B.

Fungal Material
The experimental strain SYSU-MS4722 was isolated from the ascidian Styela plicata that was obtained by Professor Lan Liu from the Bay of Da'ao, Shenzhen City, Guangdong, Province, China, in April 2016. The standard protocol [29] was used for the isolation of fungus. The molecular biological protocol that included 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).

Extraction and Isolation
The strain Diaporthe sp. SYSU-MS4722 was fermented on a solid medium in a 1 L culture flask (containing 50 g of rice and 50 mL of H 2 O with 3% sea salt) with a total of 120 flasks incubating at room temperature for 30 days. The solid fermentation was extracted with MeOH four times to afford a crude extract, and then the crude was dissolved in H 2 O and continuously was extracted four times with EtOAc. The EtOAc extract (42 g) was subjected to a silica gel column eluting with gradient petroleum ether/EtOAc (from 8:2 to 0:1) to obtain six fractions (A-F).

Calculation of the ECD Spectra
Merck molecular force field (MMFF) and DFT/TD-DFT calculations were carried out with the Spartan'14 software package (Wavefunction Inc., Irvine, CA, USA) and the Gaussian 09 program, respectively. MMFF conformational search generated low-energy conformers within a 10 kcal·mol −1 energy window and optimized using PM6 semi-empirical optimizations. Then, each conformer was optimized with HF/6-31G(d) method in Gaus-sian09. Further optimization was performed at the b3lyp/6-311g** level. The frequency was calculated at the same level to confirm each optimized conformer with the true minimum and to estimate their relative thermal free energies (∆G) at 298.15 K. The optimized conformers were continually used for the ECD calculations in methanol, which were carried out with Gaussian09 (b3lyp/6-311g**). Solvent effects were taken into account by using the polarizable continuum model (PCM). The ECD data were generated by the program SpecDis using a Gaussian band shape with 0.30 eV exponential half-width from dipolelength dipolar and rotational strengths, and the final ECD spectrum was drawn by Origin 2018. All calculations were performed by Tianhe-2 of the National Super Computer Center in Guangzhou.
Cell proliferation was analyzed by MTT according to the manufacturer's instructions. Briefly, T98G, U87MG, and U251 were digested and seeded at 1 × 10 3 cells/well in 96-well plates and cultured in 100 µL medium overnight. The cells were treated by 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
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.

Conclusions
The chemical investigation of the ascidian-derived fungus Diaporthe sp. SYSU-MS4722 afforded seven new polyketides, diaporthones A−G (1−7), together with 13 known analogues, including five mono- (8−14) and six dimeric xanthones (15−20). The absolute configurations of 1−7 were identified by X-ray crystallographic analysis and Calculation of the ECD Spectra. Compounds 11 and 15−20 showed significant anti-inflammatory activity with inhibition of nitric oxide (NO) production in RAW264.7 cells activated by lipopolysaccharide with IC 50 values between 6.3 and 8.0 µM. At the same time, dimeric xanthones (15−20) exhibited selectively growth-inhibitory effects on T98G cell lines with IC 50 values ranging from 19.5 to 78.0 µM.