Chemical Investigation of Endophytic Diaporthe unshiuensis YSP3 Reveals New Antibacterial and Cytotoxic Agents

Chemical investigation of the plant-derived endophytic fungus Diaporthe unshiuensis YSP3 led to the isolation of four new compounds (1–4), including two new xanthones (phomopthane A and B, 1 and 2), one new alternariol methyl ether derivative (3) and one α-pyrone derivative (phomopyrone B, 4), together with eight known compounds (5–12). The structures of new compounds were interpreted on the basis of spectroscopic data and single-crystal X-ray diffraction analysis. All new compounds were assessed for their antimicrobial and cytotoxic potential. Compound 1 showed cytotoxic activity against HeLa and MCF-7 cells with IC50 values of 5.92 µM and 7.50 µM, respectively, while compound 3 has an antibacterial effect on Bacillus subtilis (MIC value 16 μg/mL).


Introduction
Microorganisms produce a wide range of secondary metabolites (SM), also known as natural products, which have an incredible and dignified success history concerning pharmaceutical potential and structural diversity [1]. Metabolites produced by endophytic fungal isolates are not only renowned for providing protection in the survival of their host but also have a phenomenal contribution to agriculture, medicine, and modern industry [2]. For pathogenic fungi, some natural products were used as chemical weapons to facilitate their invasion. For example, fusaoctaxin A was recently characterized as a virulence factor during the infection progress of Fusarium graminearum, which is a destructive wheat pathogen [3]. Moreover, for endophytic fungi, their metabolites can provide benefits to residing hosts by functioning as antibacterials, nutrition transporting agents, and plant growth regulators [3,4].
As part of our continuous endeavor to search for structurally intriguing and/or bioactive natural products from endophytic fungi, an attempt was made to investigate the fungus Diaporthe unshiuensis YSP3 isolated from the leaves of Caesalpinia sepiaria. This work led to the isolation of two new xanthone derivatives (1)(2), one new alternariol derivative (3), one new pyrone derivative (4), and eight known compounds. Herein, the isolation, structural elucidation, and bioactivity evaluation of metabolites 1-12 are described in detail.

General Experimental Procedures
Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III 400 and 600 MHz NMR spectrometer (Bruker, Rheinstetten, Germany) in CDCl 3 and acetone-d 6 with TMS as an internal standard at room temperature. Ultraviolet (UV) spectra were recorded on a Hitachi U-3000 spectrophotometer (Hitachi, Tokyo, Japan). Optical rotations were measured in MeOH solution on a Rudolph Autopol III automatic polarimeter (Rudolph Research Analytical, NJ, USA). High-resolution-electrospray ionization-mass spectrometry (HR-ESI-MS) spectra were obtained on an Agilent 6210 TOF LC-MS spectrometer (Agilent, CA, USA). X-ray data were obtained on a Bruker APEX-II CCD diffractometer (Bruker, MA, USA). Column chromatography was performed on silica gel (200−300 mesh, Qingdao Marine Chemical Inc., Qingdao, China) and Sephadex LH-20 (Pharmacia Biotech, Uppsala, Sweden). High-performance liquid chromatography (HPLC) was performed on a Shimadzu LC-20AT instrument with an SPD-20A detector (Agilent, CA, USA) using an ODS column (ODS-2 HYPERSIL, 250 × 10 mm, 5 µm, Thermo Scientific, Shanghai, China). All chemicals used in this study were of analytical or HPLC grade.

Fungal Materials
The fungal strain YSP3 was isolated from the leaves of C. sepiaria collected in August 2016 from Nanjing Botanical Garden, Mem. Sun Yat-Sen, Nanjing, Jiangsu Province, People's Republic of China. The strain was identified as D. unshiuensis (GenBank OP804247) based on a morphological characterization as well as phylogenetic analysis using five molecular markers (ITS, TEF1, HIS, CAL, and TUB) ( Figure S1) [16]. The strain YSP3 was deposited in the culture collection bank of the Laboratory of Natural Products and Pesticide Chemistry, Nanjing Agricultural University (NAU).

X-ray Crystallographic Analysis
The single colorless crystals of compounds 1 and 2 were shaped from the MeOH + CH 2 Cl 2 mixture solvent (v/v, 1:1/2) at 4 • C after 25 or 27 days of slow solvent evaporation. Crystal diffraction data were collected on a Bruker APEX-II CCD diffractometer using Cu Kα radiation (λ = 1.5418 Å). These structures were solved by direct methods in OLEX2-1.3 software, followed by the refinement method of full-matrix least-squares calculations on F2 using SHELXL-2018 [17,18]. All crystal data of these compounds have been deposited in the Cambridge Crystallographic Data Centre (CCDC).

Antimicrobial Assays
All isolated new compounds were evaluated for their antibacterial potencies against four bacterial strains (Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, Bacillus subtilis, and Ralstonia solanacearum) in sterile 96-well plates by a broth dilution method [19]. Antifungal potencies were performed against Rhizoctonia solani, Fusarium solani, F. graminearum, Botrytis cinerea, Sclerotinia sclerotiorum, Alternaria solani, and Phytophthora capsici referred to the method reported [20]. All strains were provided by the Laboratory of Natural Products and Pesticide Chemistry, Nanjing Agricultural University, Nanjing, Jiangsu, China.

Cytotoxic Assays
The inhibitory effects of isolated compounds on the HeLa and MCF-7 cells were assessed in vitro by using the MTT assay on a 96-well plate. Experimental details of test used in this study are summarized in previous reports [21,22]. The toxicities against normal LO2 and HaCaT cells were tested via the cell counting kit-8 (CCK-8) method, similar to the reported reference [23].

Statistical Analysis
All tests were repeated thrice to remove the experimental error, and the quantitative data were presented as mean values ± standard deviation. The data were analyzed using IBM SPSS 22.0 with the probit analysis, and the Duncan statistical test was used for variance analysis between means. The value p ≤ 0.05 was considered statistically significant.

Results and Discussion
Phomopthane A (1) was obtained as colorless crystals with the molecular formula C 15 H 16 O 7 as inferred from its HRESIMS, (m/z 331.0789 [M + Na] + , cacld for C 15 H 16 O 7 Na, 331.0788), indicating eight degrees of unsaturation. The 13 C and DEPT NMR spectra exhibited 15 resonances resulting from one methyl, two methylenes, five methines, and seven quaternary carbons, including two carbonyl groups (δ C 204.5 and δ C 196.9). The 1 H NMR spectrum revealed the presence of characteristic signals for three aromatic protons from a 1,2,3-trisubstituted benzene at δ H 6.54 (d, J = 8.  (Table 1) were closely similar to those of mangrovamide K [24], a xanthone derivative isolated from Penicillium sp.. The difference was attributed to the methyl group in mangrovamide K being changed to the oxygenated methylene at C-14 in structure 1 (Figure 1), which was further supported by the HMBC correlations from H 3 -15 to C-7, C-8, and C-9; H-14 to C-6 and C-7; and H-9 to C-7, C-11, and C-10 ( Figure 2). Thus, the planar structure of 1 was proposed, as shown in Figure 1. The relative configuration of 1 was partially determined by NOESY and 3JHH coupling data. H-10 showed a small coupling constant (2.5 Hz) to H-9, suggesting H-10 has an equatorial orientation. The NOESY correlation between H-8 and H-14 indicates that they both possess an axial position (Figure 3). There was no sufficient data to deduce the configuration at C-11. Fortunately, a single crystal X-ray study not only confirmed the planar structure but also determined the relative configuration of 1 (Figure 4).   The relative configuration of 1 was partially determined by NOESY and 3JHH coupling data. H-10 showed a small coupling constant (2.5 Hz) to H-9, suggesting H-10 has an equatorial orientation. The NOESY correlation between H-8 and H-14 indicates that they both possess an axial position ( Figure 3). There was no sufficient data to deduce the configuration at C-11. Fortunately, a single crystal X-ray study not only confirmed the planar structure but also determined the relative configuration of 1 (Figure 4).   The relative configuration of 1 was partially determined by NOESY and 3JHH coupling data. H-10 showed a small coupling constant (2.5 Hz) to H-9, suggesting H-10 has an equatorial orientation. The NOESY correlation between H-8 and H-14 indicates that they both possess an axial position (Figure 3). There was no sufficient data to deduce the configuration at C-11. Fortunately, a single crystal X-ray study not only confirmed the planar structure but also determined the relative configuration of 1 (Figure 4). The relative configuration of 1 was partially determined by NOESY and 3JHH coupling data. H-10 showed a small coupling constant (2.5 Hz) to H-9, suggesting H-10 has an equatorial orientation. The NOESY correlation between H-8 and H-14 indicates that they both possess an axial position (Figure 3). There was no sufficient data to deduce the configuration at C-11. Fortunately, a single crystal X-ray study not only confirmed the planar structure but also determined the relative configuration of 1 (Figure 4).    The absolute configuration of 1 was assigned by comparing its electronic circular dichroism (ECD) spectrum with the structurally similar mangrovamide K, of which the absolute configuration has been established. Compound 1 contains the same chromophoric system as mangrovamide K but shows nearly a mirror image on ECD (  Phomopthane B (2) was isolated as colorless crystals. Its molecular formula, C15H18O7, was deduced by HR-ESI-MS (m/z 333.0950, [M + Na] + , calcd for C15H18O7Na, 333.0945), which was two more mass units than 1. The close comparison of the 1D NMR data between 2 and 1 showed a general similarity (Table 1), except that the ketone at the C-7 position in 1 was replaced by an oxygenated methine at δH 4.42 (H-7) in 2. This assumption was supported by the key HMBC correlations of H3-15 to C-7, C-8, and C-9; H-14 to C-6 and C-7; and H-9 to C-7 and C-11 ( Figure 2). H-7 was suggested to have an equatorial position, evidenced by its small J value (2.8 Hz). The relative configuration of the rest chiral centers in 2 was the same as in 1, based on their similar NOESY correlations and coupling constants (Figure 3 and Table 1). The above-mentioned structural elucidation was confirmed by a single X-ray analysis. Unlike 1, 2 crystallized in the monoclinic P21/c space group and has a near-zero optical rotation, suggesting that 2 was a racemic mixture [25,26].
Compound 3 was obtained as a gray powder with a molecular formula of C20H20O9 determined by the HR-ESI-MS spectrum (m/z 427.1006 [M + Na] + ; calcd for C20H20O9Na, 427.1000), indicating 11 degrees of unsaturation. The 13 C NMR, DEPT, and HSQC spectra The absolute configuration of 1 was assigned by comparing its electronic circular dichroism (ECD) spectrum with the structurally similar mangrovamide K, of which the absolute configuration has been established. Compound 1 contains the same chromophoric system as mangrovamide K but shows nearly a mirror image on ECD (  The absolute configuration of 1 was assigned by comparing its electronic circular dichroism (ECD) spectrum with the structurally similar mangrovamide K, of which the absolute configuration has been established. Compound 1 contains the same chromophoric system as mangrovamide K but shows nearly a mirror image on ECD (  Phomopthane B (2) was isolated as colorless crystals. Its molecular formula, C15H18O7, was deduced by HR-ESI-MS (m/z 333.0950, [M + Na] + , calcd for C15H18O7Na, 333.0945), which was two more mass units than 1. The close comparison of the 1D NMR data between 2 and 1 showed a general similarity (Table 1), except that the ketone at the C-7 position in 1 was replaced by an oxygenated methine at δH 4.42 (H-7) in 2. This assumption was supported by the key HMBC correlations of H3-15 to C-7, C-8, and C-9; H-14 to C-6 and C-7; and H-9 to C-7 and C-11 ( Figure 2). H-7 was suggested to have an equatorial position, evidenced by its small J value (2.8 Hz). The relative configuration of the rest chiral centers in 2 was the same as in 1, based on their similar NOESY correlations and coupling constants (Figure 3 and Table 1). The above-mentioned structural elucidation was confirmed by a single X-ray analysis. Unlike 1, 2 crystallized in the monoclinic P21/c space group and has a near-zero optical rotation, suggesting that 2 was a racemic mixture [25,26].
Compound 3 was obtained as a gray powder with a molecular formula of C20H20O9 determined by the HR-ESI-MS spectrum (m/z 427.1006 [M + Na] + ; calcd for C20H20O9Na, 427.1000), indicating 11 degrees of unsaturation. The 13 C NMR, DEPT, and HSQC spectra Phomopthane B (2) was isolated as colorless crystals. Its molecular formula, C 15 H 18 O 7 , was deduced by HR-ESI-MS (m/z 333.0950, [M + Na] + , calcd for C 15 H 18 O 7 Na, 333.0945), which was two more mass units than 1. The close comparison of the 1D NMR data between 2 and 1 showed a general similarity (Table 1), except that the ketone at the C-7 position in 1 was replaced by an oxygenated methine at δ H 4.42 (H-7) in 2. This assumption was supported by the key HMBC correlations of H 3 -15 to C-7, C-8, and C-9; H-14 to C-6 and C-7; and H-9 to C-7 and C-11 ( Figure 2). H-7 was suggested to have an equatorial position, evidenced by its small J value (2.8 Hz). The relative configuration of the rest chiral centers in 2 was the same as in 1, based on their similar NOESY correlations and coupling constants ( Figure 3 and Table 1). The above-mentioned structural elucidation was confirmed by a single X-ray analysis. Unlike 1, 2 crystallized in the monoclinic P2 1 /c space group and has a near-zero optical rotation, suggesting that 2 was a racemic mixture [25,26].
Compound 3 was obtained as a gray powder with a molecular formula of C 20 H 20 O 9 determined by the HR-ESI-MS spectrum (m/z 427.1006 [M + Na] + ; calcd for C 20 H 20 O 9 Na, 427.1000), indicating 11 degrees of unsaturation. The 13 C NMR, DEPT, and HSQC spectra revealed the presence of 20 carbons signals resulting from for one methoxy, one methyl, one methylene, eight methines, and nine quaternary carbons, including one lactonic ester group at δ C 165.7 (C-9). The 1 H-NMR data (Table 3) (Figure 2) from H 3 -15 to C-2 and C-6; H 3 -14 to C-10, C-7, and C-11; H-13 to C-8 and C-11 suggested the presence of alternariol methyl ether in the chemical structure of 3 [27]. The NMR data (Table 3) 13 C and DEPT NMR data demonstrated 11 resonances resulting from two methyls, one methoxy, two methylenes, two methines, and four quaternary carbons signals, including an ester carbonyl group at (δ C 164.9, C-2). The 1 H-NMR spectral data (Table 3) 63 (m, H-7). The α-pyrone ring structure was confirmed by HMBC correlations (Figure 2) from H-6 to C-2, C-4, and C-5. HMBC correlations from H-7 to C-8, C-9, and COSY correlations of H-7/H-8/H-9/H-10 determined the presence of a butyl side chain. The attachment of the butyl side chain at C-5 was confirmed by HMBC correlation from H-7 to C-4, C-5, and C-6. The attachment of the methoxy group (C-12) was also confirmed by the HMBC correlation between H-12 and C-4. Thus, the planar structure of 4 was deduced, as shown in Figure 1.
Compound 4 was isolated as a pale yellow oil. Its molecular form deduced by HR-ESI-MS (m/z 213.1129, [M + H] + , calcd for C11H17O4, 21 four degrees of unsaturation. The 13 C and DEPT NMR data demonstr resulting from two methyls, one methoxy, two methylenes, two meth ternary carbons signals, including an ester carbonyl group at (δC 164.9, spectral data ( Table 2)  . The α-pyrone ring structure was confirmed by H ( Figure 2) from H-6 to C-2, C-4, and C-5. HMBC correlations from H COSY correlations of H-7/H-8/H-9/H-10 determined the presence of The attachment of the butyl side chain at C-5 was confirmed by HMB H-7 to C-4, C-5, and C-6. The attachment of the methoxy group (C-12) w by the HMBC correlation between H-12 and C-4. Thus, the planar stru duced, as shown in Figure 1.
To fully evaluate the antimicrobial potencies, newly isolated compounds (1-4) were screened for their antibacterial effect against four bacterial strains (three Gram-negative phytopathogenic bacteria and one Gram-positive bacterium) as well as for antifungal activities against seven fungal strains, respectively. Compound 3 showed the bactericidal effect against B. subtilis with an MIC value of 16 µg/mL, which was better than the positive control (streptomycin sulfate 64 µg/mL) ( Table 4). The compounds (1)(2)(3)(4) were also tested for their cytotoxic activities by the MTT test method. Compound 1 was active against HeLa and MCF-7 cell lines with IC 50 values of 5.92 ± 0.04 µM and 7.50 ± 0.02 µM, respectively. Colchicine was used as a positive control (0.36 ± 0.07 µM for Hela and 0.44 ± 0.18 µM for MCF-7 cell lines) ( Table 5). The other compounds were found to be ineffective (IC 50 values > 20 µM). To explore the side effects of compound 1 on normal cells, 1 was tested for the toxicities towards normal LO2 and HaCaT cells using the CCK-8 assay. As shown in Table S3, compound 1 showed little effect on the viability of both LO2 and HaCat cells, which maintained high cell viability (~90 %) even at the compound concentration of 100 µM. These results indicated that 1 has a selective cytotoxic effect on tumor cell lines.

Conclusions
The current investigation reported the isolation and structural elucidation of twelve secondary metabolites, including two new xanthone derivatives (1 and 2), one new alternariol derivative (3), one new pyrone derivative (4), along with eight known compounds (5)(6)(7)(8)(9)(10)(11)(12). All new compounds were evaluated for their bioactivities (antifungal, antibacterial, and cytotoxic). Compound 1 revealed potent cytotoxic potencies against human cancer cell lines HeLa and MCF-7, while compound 3 showed a bactericidal effect on B. subtilis. Additionally, 1 exhibited low toxicity to normal cells (LO2 and HaCaT). Thus, taking the importance of natural products, these findings enriched the structural diversity of secondary metabolites from Diaporthe species and highlighted their values for pharmaceutical or bactericidal applications.

Data Availability Statement:
The data presented in the manuscript are available on request from the corresponding authors.

Conflicts of Interest:
The authors declare no competing financial interests.