Alkaloids isolated from the endophytic fungus Fusarium concentricum

Abstract Endophytic fungi are an important resource for bioactive natural products. In this study, a new tryptophan derivative fusaconate A (1) and three pyridone alkaloids, including one new pyridone derivative 1′-methoxy-6′-epi-oxysporidinone (2) and two known ones (3−4), were identified from the endophytic fungus Fusarium concentricum which was isolated from the medicinal plant Anoectochilus roxburghii. Their structures were elucidated through extensive spectroscopic analysis, including HR-ESI-MS, 1 D and 2 D NMR. Compound 4 exhibited moderate cytotoxicities against HT29 and PC3 cells with IC50 values of 7.60 and 4.99 μM, respectively.


Introduction
Secondary metabolites from fungi have gradually become one of the key sources of bioactive natural small molecules (Carroll et al. 2020;Wei and Wu 2020;Zheng et al. 2021).Plant endophytic fungi coevolved with the host organisms in the long-term evolution process, leading to unique structures and remarkable bioactivities of secondary metabolites (Zhao et al. 2011), such as the well-known anticancer agent taxol produced by an endophytic fungus isolated from Taxus brevifolia (Nisa et al. 2015).The genus Fusarium provided all kinds of natural products for drug innovation, such as beauvericin with neurotoxic properties in mice (Li et al. 2020;Urbaniak et al. 2020).
Increasing studies have recognized the diverse structure and broad bioactivity of natural pyridone alkaloids.Pyridone alkaloids with a 2-pyridone ring in their structures are important microbial secondary metabolites with cytotoxic, antifungal, and antibacterial effects (Jessen and Gademann 2010;Wang et al. 2015).Fusarium concentricum is an endophytic fungus isolated from the medicinal plant Anoectochilus roxburghii which exhibited a group of pharmacological activities, such as anti-inflammatory and immunomodulatory effects (Deng et al. 2020).In our ongoing search for new bioactive compounds from endophytic fungi, two new alkaloids (1 and 2) together with two known compounds (3 and 4) were isolated from F. concentricum, including a tryptophan derivative (1) and three pyridone alkaloids (2À4) (Figure 1).Their cytotoxic activities were also evaluated.
Compound 2 was isolated as a white powder solid.The molecular formula C 29 H 45 NO 6 was established on the basis of the HR-ESI-MS ion at m/z 504.3320Additionally, the 1 H-1 H COSY correlations of H-2 0 /H-3 0 and H-5 0 /H-6 0 and the HMBC correlations from H-6 0 to C-4 0 /C-2 0 /C-5 and from H-2 0 to C-4 0 /C-6 0 suggested that 2 possessed a cyclohexanone ring (moiety A), which linked with C-5 of pyridone ring (moiety B).Bearing on the chemical shift of C-6 0 (d C 87.0), the hydroxy group was substituted at C-6 0 .Consequently, we speculated that 2 was a pyridone alkaloid derivative.Detailed comparison of NMR data of 2 with known compound 6 0 -epi-Oxysporidinone (Breinholt et al. 1997;Jayasinghe et al. 2006), which was isolated from Fusarium oxysporum, suggested that an additional O-CH 3 [d H 3.25 (3H, s, H-24), d C 51.1] existed in 2. In the HMBC spectrum, the correlation from H-24 to C-1 0 and the chemical shift of C-1 0 (d C 83.5) proved that the additional O-CH 3 attached to C-1 0 in cyclohexanone moiety.Thus, the planar structure of 2 could be assigned as shown in Figure 1.
The relative configuration of was determined by the NOESY data analysis (Figure S2).The NOESY correlation of H-24 with H-6 0 disclosed that the hydroxy group at C-6 0 and the methoxy group at C-1 0 were on the opposite side of the ring.In the NOESY spectrum, both H-7 and H-19 were correlated with H-11, which suggested that H-11, H-7 and the methyl group at C-10 were located on the same side of the ring.The stereochemistry of the double bond in 2 was determined as 12E based on the NOESY correlation of H-20 with H-14.Unfortunately, the NOE correlation was not observed between the protons in ring A and those in ring C, thus the complete relative stereochemistry of 2 could not be determined at this stage.As a result, compound 2 was identified as 1 0 -methoxy-6 0 -epi-oxysporidinone.
The MTT method was used to test the cytotoxicities of compounds 1À4 against six cancer cell lines (A172, A549, HepG2, Bel7402, HT29, PC3).Compound 4 exhibited moderate cytotoxicties against HT29 and PC3 cells with IC 50 values of 7.60 and 4.99 lM, respectively, which were weaker than those of the positive control drug doxorubicin (IC 50 0.13 and 0.21 lM), while the other compounds were inactive (IC 50 > 10 lM) (Table S3).

General experimental procedures
Optical rotations were measured with a MCP 200 polarimeter (Anton Paar GmbH, Graz, Austria).UV spectra were determined with JASCO V-650 instrument.IR spectra were recorded using a Thermo Nicolet 5700 FT-IR spectrophotometer (Waltham, MA, USA).HR-ESI-MS analysis was performed with Bruker micro TOF-Q mass spectrometer in m/z mode.The NMR data were obtained from Bruker-AV-600 spectrometer (Bruker Corporation, Bremen, Germany) using TMS as an internal standard.Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden), silica gel (Qingdao Haiyang Chemical Co.Ltd.) and ODS (50 lm, YMC Co. Ltd., Kyoto, Japan) were used for column chromatography (CC).Semipreparative HPLC separations were performed on a YMC-pack ODS-A column (250 Â 10 mm, 5 mm) with a Shimadzu SPD-20 A UV/vis detector and a Shimadzu LC-6AD liquid chromatography series pumping system (Tokyo, Japan).All chemical reagents were acquired from Tianjin Concord Technology Co. Ltd., Tianjin, China.

Fungal material and fermentation
The fungal strain was isolated from an endangered plant A. roxburghii in Fujian Province, China, by Bingguo Lu from Fujian Normal University.The fungus was identified as F. concentricum by analysis of its internal transcribed spacers (ITS) rDNA sequence with GenBank accession number OP646479.A voucher sample (No.FNE055) was deposited in our laboratory at À80 C. The fungus F. concentricum was cultured on potato dextrose agar (PDA) medium at room temperature for 4 days.It was inoculated into a 500 mL Erlenmeyer flask containing 200 mL potato dextrose broth (PDB) medium and cultured for 4 days in a ambient temperature incubator with shaking at 220 rmp to obtain the seed solution.After aspirating 5 mL of seed solution with a pipette, it was inoculated into rice medium (100 bottles, 500 mL Erlenmeyer flasks, 100 g rice) and incubated at ambient temperature for 35 days.

Extraction and isolation
The fermented material was extracted with ethyl acetate (4 Â 5 L), and the whole extract was combined and concentrated by rotary evaporator to obtain a brownishred extract (54.6 g).The extract was dissolved with methanol and extracted by equal volume of petroleum ether to obtain the methanol layer extract.The methanol extract (21.6 g) was subjected to silica gel column chromatography (200 À 300 mesh) and gradient elution with a solvent system of dichloromethane À methanol (100:0 $ 1:1, v/v) to give twelve subfractions (M1$M12).

Cytotoxicity assays
The cytotoxicity activities against six cancer cell lines, including A172, A549, HepG2, Bel7402, HT29, and PC3, were evaluated using the MTT method as described in the literatures (Wang et al. 2018;Zhang et al. 2021).Doxorubicin (Sigma-Aldrich; St. Louis, MO, USA) was used as a positive control.

Conclusions
In summary, two new alkaloids, including a tryptophan derivative (1) and a pyridone alkaloid (2), along with two known pyridone analogues (3À4) were isolated from the endophytic fungus F. concentricum, which was obtained from the endangered medicinal plant A. roxburghii.Compound 4 showed potent cytotoxicities against HT29 and PC3 cells.These results further present the potential of novel and bioactive natural products discovery from endophtyic fungi.

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