Pestalotiones A–D: Four New Secondary Metabolites from the Plant Endophytic Fungus Pestalotiopsis Theae

Two new xanthone derivatives, pestalotiones A (1) and B (2), one new diphenyl ketone riboside, pestalotione C (7), and one new diphenyl ether, pestalotione D (8), along with five known compounds isosulochrin dehydrate (3), 3,8-dihydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate (4), isosulochrin (5), chloroisosulochrin (6), and pestalotether D (9), were isolated from the crude extract of the plant endophytic fungus Pestalotiopsis theae (N635). The structures of the new compounds were unambiguously deduced by HRESIMS and 1D/2D-NMR spectroscopic data. Compound 6 showed modest cytotoxicity against the HeLa cell line with an IC50 value of 35.2 μM. Compound 9 also showed cytotoxic to the HeLa and MCF-7 cell lines, with IC50 values of 60.8 and 22.6 μM, respectively. Additionally, compounds 1 and 2 exhibited antioxidant activity in scavenging DPPH radical with IC50 values of 54.2 and 59.2 μg/mL, respectively.


Isolation and Structure Elucidation
Pestalotione A (1) was assigned the molecular formula C 17 H 12 O 8 (12 degrees of unsaturation) on the basis of HRESIMS data. The UV spectrum of the yellow powder showed four maxima (234, 254, 310, and 369 nm), suggesting a xanthone chromophore [24]. Analysis of its NMR data (Table 1) revealed the presence of one exchangeable proton (δ H 12.3), two methoxy groups, 12 aromatic carbons (the carbon C-6 signal resonated at δ C 139.4 was determined by HMBC correlations (Figure 2) from H-5 and H-7 to C-6), including four protonated, two carboxylic carbons (δ C 165.8, 167.9), and one conjugated ketone carbon (δ C 179.5). These data accounted for all the NMR resonances of 1 except for one unobserved exchangeable proton and required 1 to be a tricyclic compound. A detailed NMR data comparison with xanthone derivative isosulochrin dehydrate (3) revealed the similarity of them. The 1 H-1 H coupling patterns for the four aromatic protons also revealed two m-substituted aryl rings. HMBC correlations ( Figure 2) from H-2 to C-3, C-4, C-9a, and the carboxylic carbon C-12 (δ C 167.9), H-4 to C-2, C-3, C-4a, the ketone carbon C-9 (δ C 179.5) and C-9a, H-5 to C-6, C-7, C-9, C-10a, and the carboxylic carbon C-11 (δ C 165.8), and from H-7 to C-5, C-6, C-8, C-8a, C-9, and C-11 permitted completion of the xanthone core structure with two carboxylic carbons C-11 and C-12 located at C-6 and C-1, respectively. The cross-peaks from the phenolic proton OH-8 (δ H 12.3) to C-7, C-8, and C-8a led to the attachment of the hydroxy groups to C-8. HMBC correlations from two methoxy groups to C-3 and C-12, established the locations of these methoxy groups. The remaining one exchangeable proton was located at C-11 by default. Therefore, the planar structure of compound 1 was established as 1-hydroxy-6-methoxy-8-(methoxycarbonyl)-9-oxo-9H-xanthene-3-carboxylic acid, named pestalotione A ( Figure 1).
The molecular formula of pestalotione B (2) was established as C 19 H 18 O 7 (11 degrees of unsaturation) on the basis of the HRESIMS. The 1 H and 13 C-NMR spectrum (Table 1) of 2 exhibited one exchangeable proton at δ H 12.3, two oxygenated methyls, one methyl, two methylenes (one oxygenated), twelve olefinic/aromatic carbons (three of which were protonated), one carboxylic carbon (δ C 168.4), and one conjugated ketone carbon (δ C 180.3). These data accounted for all the resonances observed in the NMR spectra of 2 except for one unobserved exchangeable proton. The 1 H-and 13 C-NMR spectra of 2 displayed signals for structural features similar to 1, except that the aromatic proton H-2 (δ H 7.13) and the carboxylic carbon (δ C 165.8) in 1 were replaced by the 2-hydroxyethyl unit (δ H 2.87, 3.00, 3.50, 3.56, δ C 38.0, 61.5, respectively) and a methyl group (δ H/C 2.43/22.4), respectively. This was further confirmed by HMBC correlations (Figure 2) from H 3 -11 to C-5, C-6, and C-7, H 2 -13 to C-2 and C-14, and from H 2 -14 to C-13. Accordingly, compound 2, namely pestalotione B, was identified as methyl 8-hydroxy-2-(2-hydroxyethyl)-3-methoxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate. Table 1. NMR data for compounds 1 (DMSO-d 6 ) and 2 (acetone-d 6 ).  The molecular formula of pestalotione C (7) was established as C 22 H 24 O 11 (11 degrees of unsaturation) on the basis of HRESIMS data. Its 1 H, 13 C, and HSQC NMR spectroscopic data (Table 2) showed resonances for two exchangeable protons (δ H 10.9 and 3.3, respectively), three methyl groups with two oxygenated, one methylene unit, four oxymethines, twelve olefinic/aromatic carbons including four protonated, one carboxylic carbon (δ C 167.7), and one conjugated ketone carbon (δ C 201.1). Interpretation of these data revealed structural features similar to those presented in isosulochrin (5) [23] except for the presence of a furanose unit. Interpretation of the 1 H-1 H COSY NMR data (Figure 2) led to the identification of one isolated proton spin-system corresponding to the C-1 -C-2 -C-3 -C-4 -C-5 subunit of structure 7. The ribose residue was confirmed by comparing the 13 C-NMR data with those of several furanoside, such as isotorachrysone-6-O-α-d-ribofuranoside, and asperflavin ribofuranoside [26,27]. The sugar moiety was further determined as α-form by comparison of the coupling constant (J 1 2 = 4.4 Hz) of the anomeric proton with those of the methyl-α-d-ribofuranoside (J 1,2 = 4.3 Hz) and methyl-β-d-ribofuranoside (J 1,2 = 1.2 Hz) [28]. The key HMBC correlations (Figure 2) from the anomeric proton H-1 to C-4 (δ C 88.1) and C-5 (δ C 155.7) determined the ribose moiety, which was linked to C-5 through oxygen bond. Upon acid hydrolysis of 7 with methanol/HCl, the liberated sugar was identified as d-ribose by measurement of its specific rotation [29]. Thus, compound 7 was elucidated as isosulochrin-5 -O-α-d-ribofuranoside, named pestalotione C (7). The molecular formula of pestalotione D (8) was established as C 19 H 20 O 8 (10 degrees of unsaturation) on the basis of HRESIMS data. The overall appearance of 1 H and 13 C-NMR spectra ( Table 3)   Biogenetically, emodin, biosynthesized from one molecule of acetyl-CoA and seven molecules of malonyl-CoA [30], could be the biosynthetic precursor not only for compounds 1-4, but also for 5-9, first via oxidation and methylation to form the key intermediate b, and then followed by a series of reactions through different routes to form 1-9. The proposed precursor and the reaction cascades leading to the generation of these metabolites are illustrated in Figure 3.  Meanwhile, their antioxidant activity was also evaluated by the DPPH (2,2-diphenyl-1-picrylhydrazyl radical) scavenging method with ascorbic acid as positive control (IC 50 = 6.0 µg/mL). Only compounds 1 and 2 exhibited weak DPPH scavenging activity with respective IC 50 values of 54.2 and 59.2 µg/mL. Xanthone derivatives were found to display diverse activities, such as tumor cytotoxic activity, antivirus, antibacterial, antifungal, and antimalaria activities [31][32][33][34]. Pestalotiopsis sp. was an interesting producer of bioactive metabolites. In our previous study, two spiroketals chlorotheolides A and B from P. theae showed an antiproliferative effect against the human tumor cell lines HeLa and MCF-7 [22]. In the current study, new structural metabolites with cytotoxic and antioxidant activities were identified from the same fungus. This highlighted the high potential of bioprospecting larvicides from the endophytic fungi.

General Experimental Procedures
Optical rotations were measured on an Anton Paar MCP 200 Automatic Polarimeter and UV data were obtained on a Thermo Genesys-10S UV/Vis spectrophotometer. IR data were recorded using a Nicolet IS5 FT-IR spectrophotometer. 1 H and 13 C-NMR data were acquired with Bruker Avance-400 and -500 spectrometer using solvent signals (acetone-d 6 : δ H 2.05/δ C 29.8, 206.3; DMSO-d 6 : δ H 2.50/δ C 39.5; methanol-d 4 : δ H 3.31/δ C 49.0; CDCl 3 : δ H 7.26/δ C 77.2 ppm) as references. The HSQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. ESIMS and HRESIMS data were obtained using an Agilent Accurate-Mass-Q-TOF LC/MS 6520 instrument equipped with an electrospray ionization (ESI) source. The fragmentor and capillary voltages were kept at 125 and 3500 V, respectively. Nitrogen was supplied as the nebulizing and drying gas. The temperature of the drying gas was set at 300 • C. The flow rate of the drying gas and the pressure of the nebulizer were 10 L/min and 10 psi, respectively. All MS experiments were performed in positive ion mode. Full-scan spectra were acquired over a scan range of m/z 100-1000 at 1.03 spectra/s. HPLC separations were performed on an Agilent 1260 instrument equipped with a variable-wavelength UV detector.

Fungal Material
The culture of P. theae (N635) was isolated from Camellia sinensis (Theaceae) in Hangzhou, People's Republic of China. The isolate was identified based on sequence analysis of the ITS region of the rDNA (GenBank Accession No. KF641183). Firstly, the strain was cultured on potato dextrose agar (PDA) at 25 • C for 10 days. Secondly, agar plugs were cut into small pieces (about 0.5 × 0.5 × 0.5 cm 3 ) under aseptic conditions, and every five pieces were inoculated into an Erlenmeyer flask (250 mL) containing 50 mL of media (0.4% glucose, 1% malt extract, and 0.4% yeast extract) with final pH 6.5. The flasks inoculated with the media were used as seed cultures after incubating at 25 • C on a rotary shaker at 170 rpm for 5 days. Spore inoculum was prepared by suspension in sterile, distilled H 2 O, resulting in a final spore/cell suspension of 1 × 10 6 /mL. Thirdly, each Fernbach flask (500 mL) containing 80 g of rice and 120 mL of distilled H 2 O was then sealed, soaked overnight, and autoclaved at 15 psi for 30 min. After cooling to room temperature, 5.0 mL of the spore inoculum obtained from liquid phase cultivation was added to each flask and incubated at 25 • C for 40 days.

MTS Assay
The assay plate was read at 490 nm using a microplate reader. The assay was run in triplicate. In a 96-well plate, each well was plated with (2-5) × 10 3 cells (depending on the cell multiplication rate). After cell attachment overnight, the medium was removed, and each well was treated with 100 µL of medium containing 0.1% DMSO, or appropriate concentrations of the test compounds and the positive control cisplatin (100 mM as stock solution of a compound in DMSO and serial dilutions; the test compounds showed good solubility in DMSO and did not precipitate when added to the cells). The plate was incubated at 37 • C for 48 h in a humidified, 5% CO 2 atmosphere. Proliferation was assessed by adding 20 µL of MTS (Promega) to each well in the dark, followed by incubation at 37 • C for 90 min. The assay plate was read at 490 nm using a microplate reader. The assay was run in triplicate [36].

Antioxidant Assay
The DPPH scavenging assay was performed according to the former reported method [37]. The DPPH radical scavenging test was conducted in a 96-well plate. The tested compounds 1-9 were added to 50 µL (0.34 mmol/L) DPPH solution in ethanol solutions at a range of 50 µL solutions of different concentrations (12.5, 25, 50, 100, and 200 µM). After 30 min of incubation at 37 • C in the dark environment, the absorbance was read at 517 nm using a microplate reader, employing distilled water as a blank for baseline correction. The data that represent three independent experiments was calculated, and ascorbic acid was used as a positive control.

Conclusions
In summary, nine polyketides including four new ones were isolated from the crude extract of the fungus P. theae. Compounds 1 and 2 exhibited antioxidant activity, while compounds 6 and 9 showed moderate cytotoxic to the human tumor cells. The discovery of these secondary metabolites further expanded the structural diversity of the natural products produced by the fungal genus Pestalotiopsis.