Polyketones from the endophytic fungus Cytospora rhizophorae

Abstract Cytosporaphenones D (1) and E (2), two new polyketones, and one new natural product 1,7-dihydroxy-6-methyl-8-formylxanthone (3), along with four known compounds, were isolated from Cytospora rhizophorae, an endophytic fungus from Morinda officinalis. Their structures were elucidated by extensive spectroscopic analyses and X-ray diffraction technique. Graphical Abstract


Introduction
The genus Cytospora is considered as a prolific source of structurally unique and biologically diverse metabolites (Sadorn et al. 2018;Narmani et al. 2019). Most of the secondary metabolisms from Cytospora showed a variety of bioactivities including cytotoxic, antimicrobial, antifungal and antiviral activity (Brady et al. 2000b;Kokubun et al. 2003;Singh et al. 2007;Lu et al. 2011;Li et al. 2012;Sadorn et al. 2018). In our continuing screen program toward the discovery of structurally unprecedented and privileged constituents with biologically potentials from endophytic fungal strains (Liu et al. 2017;2019a;2019b;2019c;2019d;Chen et al. 2019), two new polyketones, named cytosporaphenones D (1) and E (2), and one new natural product 1,7-dihydroxy-6-methyl-8-formylxanthone (3), along with four known compounds, were isolated from C. rhizophorae, an endophytic fungus from Morinda officinalis ( Figure 1). Herein, the details of the isolation, structural interpretation, and biological assays of these compounds were described.
Compound 1 was obtained as a colorless crystal. In its HR-ESI-MS analysis in positive-ion mode, 1 displayed an [M þ H] þ ion peak at m/z 283.0969, consistent with C 17 H 14 O 4 . The 13 C NMR data (Table S3) revealed 17 carbon signals, which was determined from the HSQC spectrum corresponded to two carbonyl, two methyl, one methylene, five methine, as well as seven quaternary carbon functionalities. The 1 H NMR signals d H 7.75 (1H, t, J ¼ 8.3 Hz, H-2), 7.05 (1H, dd, J ¼ 8.3, 0.8 Hz, H-3), 6.81 (1H, dd, J ¼ 8.3, 0.8 Hz, H-1)] suggested the presence of a trisubstituted benzene ring A. The 1 H-1 H COSY and HSQC spectra clearly revealed the presence of the following two spin coupling systems: a (C-1/C-2/C-3), and b (C-15/C-16). As referring to fragment a, a 4,5,6-trisubstituted benzene ring A was successfully established on the basis of the HMBC correlations from H-2 to C-4 and C-6 as well as H-3 to C-1 and C-5 ( Figure S32). Moreover, the HMBC correlations from H-12 to C-8 and C-10 as well as H 3 -17 to C-10, C-11, and C-12 suggested a second 1,3,5,6-tetrasubstituted benzene ring B. A propionyl group was deduced to be located at the C-13 position of ring B, and it was further evidenced by the HMBC correlations form H-16 to C-14 and H-15 to C-13, along with the 1 H-1 H COSY fragment b. The structure of 1 and the combination of subunits A and B was further established by the X-ray diffraction analysis with CuKa ( Figure   Figure 1. Structures of compounds 1-7. S33). Therefore, the structure of compound 1 was finally determined and given the trivial name as cytosporaphenone D.
Compound 2 was isolated as yellowish powder. Its molecular formula of C 17 H 14 O 4 was deduced from its HRESIMS m/z 283.0969 [M þ H] þ , indicating eleven degrees of unsaturation. The 1 H NMR spectroscopic data [d H 6.43 (1H, d, J ¼ 8.2 Hz, H-1,3) and 7.05 (1H, d, J ¼ 8.2 Hz, H-2)] together with the HSQC spectra of 2 indicated the presence of a symmetric benzene ring A. This conclusion was also confirmed by the HMBC correlations from H-1 to C-3 and C-5, H-2 to C-4 and C-6, as well as the 1 H-1 H COSY fragment H-1/H-2/H-3. Likewise, the ring B was established by the key HMBC correlations form the cross-peaks of H-11/C-13 and C-15, H-13/C-11 and C-15 ( Figure S32). Furthermore, the HMBC correlations from H 3 -16 to C-11, C-12, and C-13 evidenced that the methyl group C-16 was connected to C-12. The HMBC correlations from H-11 to C-9, H-17 to C-7, C-8, and C-9 confirmed the ring C. The HMBC correlations from H-1/H-3 to C-7 indicating the ring A was connected to the C-7 of ring C. Therefore, the structure of compound 2 was finally determined and given the trivial name as cytosporaphenone E.
Compound 3 was obtained as a yellowish crystal. Its molecular formula was consistent as C 15 H 10 O 5 by HRESIMS of the pseudomolecular ion peak at m/z 271.0605 [M þ H] þ (cacld. For C 15 H 11 O 5 , 271.0601). The 1 H-1 H COSY spin system C-1/C-2/C-3 and the 1 H NMR signals for a trisubstituted benzene ring [d H 7.54 (1H, t, J ¼ 8.2 Hz, H-2), 6.87 (1H, dd, J ¼ 8.2, 0.9 Hz, H-3), 6.76 (1H, dd, J ¼ 8.2, 0.9 Hz, H-1)], suggested that the presence of the same 4,5,6-trisubstituted benzene ring A in compound 3. Moreover, the HMBC correlations from H-10 to C-8, and C-12, H-15 to C-9 and C-11 attributed a penta-substituted phenyl ring B ( Figure S32). The key HMBC cross peak from the methyl group [d H 2.38 (3H, s)] to C-10, C-11 and C-13 assumed that the methyl group was located at the C-11 (ring B). An aldehyde group was deduced to be located at position C-13 based on the HMBC correlations from H-14 to C-8, C-13, and C-12. Finally, a single crystal of compound 3 was obtained. The X-ray crystallographic measurement was completed on the CuKa with a Flack parameter of -0.11(16), clarifying the structure of 3 without any ambiguity ( Figure S33). In light of the aforementioned evidence, the structure of 3 was concluded as shown in Figure 1. Compound 3 was previously reported as a biosynthetic product by Li's group (Pockrandt et al. 2012).
Compounds 1-3 were evaluated for antimicrobial activity against the bacteria Escherichia coli and Staphylococcus aureus (Drummond and Waigh 2000) and cytotoxic activity (Skehan et al. 1990), with vancomycin and adriamycin as the positive controls, respectively. However, all of them were found to be devoid of significant activity even at a concentration of 100 mg/mL.

General experimental procedures
The melting point was measured by SGW X-4 micro melting point apparatus; Optical rotation was measured on an Anton Paar MCP-500 spectropolarimeter (Anton Paar, Graz, Austria). UV spectra were measured on a SHIMADZU UV-2600 UV-visible spectrophotometer (Shimadzu, Kyoto, Japan). 1D and 2D NMR spectra were recorded on a Bruker Avance-500 spectrometer (Bruker, F€ allanden, Switzerland) with TMS as internal standard, d in ppm, J in Hz. HREIMS was measured on a Thermo MAT95XP high resolution mass spectrometer and EIMS on a Thermo DSQ EI mass spectrometer (Thermo Scientific, Massachusetts, USA). All solvents were analytical grade (Guangzhou Chemical Plant, Guangzhou, China). Silica gel (200-300 mesh) was used for column chromatography, and precoated silica gel GF 254 plates (Qingdao Marine Chemical Inc., Qingdao, China) were used for TLC spotting. C 18 reversed-phase silica gel (40-63 mm, Merck, German), and Sephadex LH-20 gel (Pharmacia Fine Chemical Co. Ltd., Sweden) were also used for column chromatography. TLC spots were visualized under UV light and by dipping into 10% H 2 SO 4 in alcohol followed by heating.

Fungal material and identification
The strain A761 was isolated from the plant Morinda officinalis, which was collected from Gaoyao city of Guangdong province in January 2015. The strain was identified by sequence analysis of rDNA ITS (internal transcribed spacer) region. The sequence of ITS region of the fungus A761 has been submitted to GenBank (Accession No. KU529867). By using BLAST (nucleotide sequence comparison program) to search the GenBank database, A761 has 99.5% similarity to Cytospora rhizophorae M225 (Accession No. KR056292). The strain is preserved at the Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Institute of Microbiology, Guangdong Academy of Sciences.

Fermentation, extraction, and isolation
Cytospora rhizophorae A761 was cultured in potato dextrose broth (potato 20%, glucose 2%, K 2 HPO 4 0.3%, MgSO 4 7H 2 O 0.15%, vitamin B 10 mg/L). The fungus C. rhizophorae A761 was maintained on potato dextrose agar (PDA) medium at 28 C for 5 days, and then three pieces (0.5 Â 0.5 cm 2 ) of mycelial agar plugs were inoculated into 20 Â 500 mL Erlenmeyer flasks, each containing 250 mL potato dextrose broth. After 4 days of incubation at 28 C on a rotary shaker at 120 r/m, 25 mL seed cultures were aseptically transferred into each of a total of 150 flasks (1000 mL) containing 500 mL of potato dextrose broth. The liquid cultivation that followed was kept for 7 days at 28 C and 120 r/m on a rotary shaker.
The culture (70 L) was centrifuged to give the broth and mycelia. The broth was exhaustively extracted with EtOAc for four times, and then the EtOAc layers were combined and evaporated under reduced pressure at a temperature not exceeding 40 C to yield a dark brown gum (26 g). The crude EtOAc extract was subjected to reversedphase silica gel C 18 (MeOH/H 2 O, 30%!100%) column chromatography to afford 6 fractions (Fr.1-Fr.6).

Antibacterial assay
Compounds 1-3 were dissolved in DMSO at a final concentration of 10 mg/mL. The antibacterial activity evaluation was carried out in triplicate according to the standard microdilution method and expressed as minimum inhibitory concentration (MIC), with vancomycin as the positive control. The compound was tested at concentrations ranging from 0.53 to 100 lg/mL. Test strains were S. aureus (CMCC 26003) and E. coli (ATCC 8739).

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
Financial support for this research was provided by Guangdong Special Support Program (2019TQ05Y375).