Hypoxylonone, a new oxa-bridged seven-membered ring analog from fungus Hypoxylon cf. subgilvum SWUF15-004

Abstract A new oxa-bridged seven-membered ring analog, hypoxylonone (1), and thirteen known compounds (2–14) were isolated from fungus Hypoxylon cf. subgilvum SWUF15-004. The structures were elucidated by the analysis of spectroscopic (IR, 1 D and 2 D NMR), HRESIMS and X-ray diffraction (MoKα) data. Several isolated compounds were evaluated for cytotoxicity against four human cancer cell lines (HeLa, HT29, MCF-7, A549). Compound 1 exhibited weak inhibitory effects of the nitric oxide production in RAW264.7 cells. Compounds 8 and 9 exhibited slight cytotoxicity. GRAPHIC ABSTRACT


Introduction
The genus Hypoxylon belongs to the family of Hypoxylaceae (Wendt et al. 2018), which contains more than 200 species and is widely distributed throughout tropical regions of the world (Hyde 2020).There have been numerous reports on diverse chemical structures and biologically active secondary metabolites from this genus, e.g. the antibacterial azaphilones (Surup et al. 2018;Becker et al. 2021), the cytotoxic cytochalasans (Kretz et al. 2019), the cytotoxic terphenylquinones (Kuhnert et al. 2015;Intaraudom et al. 2017), and the antimalarial drimane sesquiterpenoids (Kuhnert et al. 2015;Intaraudom et al. 2019).This is why the exploration of undescribed active metabolites from this genus continues to be of interest to medicinal and natural products researchers.
In this research, the chemical constituents from Hypoxylon cf.subgilvum SWUF15-004, were studied.The isolation and purification of crude extracts were achieved by chromatographic techniques to yield fourteen compounds.The analysis of IR, NMR, HRESIMS and X-ray crystallographic data as well as a comparison of those data with literature revealed one rare-tricyclic compound (1), bearing an oxabicyclo[3.2.1]octane scaffold and thirteen known compounds.The seven-membered 8-oxabicyclo[3.2.1]octane ring is a unique core structure, found in several classes of natural products.Some of these have exhibited potent biological activities and because of their remarkable biological activities, its analogues have attracted a great deal of attention from organic synthetic researchers (Fu et al. 2015;Gao et al. 2019;Wang et al. 2019Wang et al. , 2021)).The cytotoxicities of several isolated compounds from this fungus were evaluated against cervical epithelial carcinoma (HeLa), colon carcinoma (HT29), breast carcinoma (MCF-7), lung carcinoma (A549) and prostate carcinoma (PC-3) cells.The new compound (1) was also tested for its inhibitory activity on nitric oxide (NO) production in RAW264.7 cells.Herein, the results of the isolation, purification, structure elucidation and bioactivity evaluation are presented.

General experimental procedures
IR spectra were recorded on a Bruker TENSOR27 FT-IR spectrometer.A JASCO J-810 circular dichroism spectropolarimeter was used to determine ECD and UV spectra.Melting points were recorded by a Sanyo/Gallenkamp MPD350.BM3.5 melting point apparatus.Optical rotations were recorded by a JASCO DIP-1000 digital polarimeter.NMR spectra were recorded on Varian Mercury Plus 400 and Bruker 400 NMR spectrometers (400 MHz).The X-ray crystal data were collected on a Bruker D8 Quest PHOTON100 CMOS diffractometer with graphite-monochromated MoKa radiation (k ¼ 0.71073).High-resolution electrospray ionization mass spectra (HRESIMS) were measured on a micrOTOF Bruker mass spectrometer.For chromatographic separation, silica gel 60 (0.063-0.200 mm and 0.040-0.063mm, Merck, Germany) were used for column chromatography (CC) and flash column chromatography (FCC), respectively.Sephadex LH-20 (Sigma-Aldrich, United States) was used as the stationary phase for gel filtration chromatography.Silica gel PF254 (Merck, Germany) was used as the stationary phase for preparative thin-layer chromatography (PLC).

Fungal material
Fungal specimens were collected from Ratchaburi Province, Thailand and were identified based on morphological characteristics and nucleotide sequences of 5 loci, internal transcribed spacers (ITS), large subunit (LSU), a-actin gene (act), b-tubulin gene (tub) and RNA polymerase II gene (rpb2).The accession numbers of all loci were submitted to GenBank database as OM978240 (ITS), ON222526 (LSU), ON286890 (act), ON286891 (tub) and ON286892 (rpb2).The morphological characteristics of this fungus were similar to H. subgilvum Berk.& Broome, except that the tissue below the perithecial layer was a little bit thicker (1.0-1.5 mm high) and the perithecial size was slightly larger (0.3-0.5 mm in diam Â 0.5-0.8mm high) than the species description by Ju and Rogers (Ju and Rogers 1996).These differences corresponded to the nucleotide sequences of 5 loci results that were different from H. subgilvum.This led to the possibility of the fungus being a cryptic species.Therefore, this fungus was named as Hypoxylon cf.subgilvum SWUF15-004.The specimen was deposited at the Srinakharinwirot University Fungal Herbarium (SWUF) with the associated number of SWUF15-004.

Fermentation, extraction, and isolation
The fungus stroma was initially cultivated on potato dextrose agar (PDA) for 7 days.The pure cultures were cut into small cubes and transferred into YM medium starters (50 mL/flask) that contained yeast extract (3 g/L), malt extract (3 g/L), peptone (5 g/L) and dextrose (10 g/L).After a week, the starter culture was transferred to 1000 mL Erlenmeyer flask of YM medium for scale-up cultivation.The culture solution was filtered after 45 days to obtain the mycelium (173.02g) and broth (48 L).The broth was extracted 3 times with EtOAc (300 mL/time) to yield crude EtOAc extract (4.95 g).The mycelium was dried at 60 C in the oven for 2 days, and further ground by a blender.The dried mycelium (173.02g) was extracted 3 times with EtOAc (700 mL/time), and the solvent was removed to give crude EtOAc extract (2.63 g).Following this, the EtOAc extract's marc was repeatedly extracted with MeOH to obtain crude MeOH extract (26.64 g).Then it was dissolved with MeOH: water (1:1) and partitioned with EtOAc to obtain 6.95 g of EtOAc-partitioned extract.

X-ray crystallographic analysis
The crystals of 1 were analyzed by X-ray diffraction.The SAINT (Bruker-AXS 2000) was used to integrate diffraction data.The multiscan absorption corrections were conducted by the SADABS program (Sheldrick 2000).The structure was solved by intrinsic phasing using SHELXT (Apex2 program suite) (Sheldrick 2015) and refined by the fullmatrix least-squares methods using SHELXTL (Sheldrick 2008), with the ShelXle user interface (Hubschle et al. 2011).All non-hydrogen atoms were refined anisotropically while the H-atoms were located on calculated positions.The crystallographic data were deposited with CCDC No. 2121656.These data can be obtained free of charge from the Cambridge Crystallographic Data Centre (CCDC) via http://www.ccdc.cam.ac.uk/data_request/cif.

Cytotoxicity against cancer cell lines
The toxicity of the isolated compounds against HeLa, HT29, HCT116, MCF-7 and Vero cells was evaluated by MTT colorimetric assay.Briefly, the cells were seeded onto the 96-well plate at approximately 8 Â 10 4 cells/well.The compounds were added to the cell culture and then incubated for 72 h.The cell culture was treated with 10 lL of MTT (3-(4,5-dimethyltriazol-2-yl)-2,5-diphenyltetrazolium bromide) solution at 37 C for 2 h.The formazan dye was dissolved by DMSO, and the UV absorbance of the solution was recorded at 550 nm.