Two new chromone derivatives from the rhizosphere soil fungus Ilyonectria robusta

Abstract Two new chromone derivatives (1 and 2), and two known compounds (3 and 4) were isolated from the rhizosphere soil fungus Ilyonectria robusta. Their planar structures and absolute configurations were determined by extensive spectroscopic analysis and electronic circular dichroism (ECD) calculations. Additionally, all the isolated compounds were evaluated for their antibacterial activity against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli, but no obvious activity was observed at a concentration of 128 μg/mL. Graphical Abstract


Introduction
Chromones are a very abundant class of secondary metabolites in nature, whose parent nucleus is benzo-c-pyrone (Keri et al. 2014).Chromone derivatives are widely distributed and have various structures, which have been concerned by researchers all the time (Mohsin et al. 2020).Numerous reports show that it has a variety of significant biological activities, including antimicrobial (Du et al. 2013;Cano et al. 2015;Reis et al. 2017), antioxidant (Phosrithong et al. 2012;Ibrahim 2014;Yadav et al. 2014), anti-inflammatory (Huo et al. 2017;Khan et al. 2018;Zhang et al. 2022) and anti-tumor (Duan et al. 2019;Patil et al. 2021), etc. Natural chromones are generally distributed in Asteraceae, Compositae, Guttiferae, Thymelaeaceae, Umbelliferae and so on (Keri et al. 2014;Duan et al. 2019).Besides plants, chromones derived from microorganisms also deserve further exploration and enrichment.Our team has been engaged in the discovery of natural products for a long time, mainly focusing on plants in the early stage, and paying more and more attention to plant-related microorganisms in recent years, and isolated multiple types of compounds such as biflavonoids, neolignan, etc (Zou et al. 2018;Dong et al. 2019;Cheng et al. 2020;Kang et al. 2021;Liu et al. 2021;Zhang, Chen, et al. 2021;Zhang, Wang, et al. 2021).
In this study, the fungus Ilyonectria robusta was isolated from the rhizosphere soil of Phyllagathis caualeriei.Ilyonectria, as a kind of soil fungus, having a broad geographic distribution, can infect a wide range of host plants to cause root rot.In the past, researchers usually focused on the morphology, genetics and ecology of Ilyonectria and made some great progress (Seifert et al. 2003;Cabral et al. 2012;Farh et al. 2018;Yang et al. 2019;Zhu et al. 2019;Bischoff Nunes and Goodwin 2022), but the chemical composition of Ilyonectria was rarely studied.To the best of our knowledge, there are a few investigations on the metabolites of the genus Ilyonectria, only involving resorcinol acid lactones and several terpenoids, and no report on natural chromone derivatives (Zhou et al. 2020;Ye et al. 2021;Gao et al. 2022;Walsh et al. 2022).Herein, we systematically investigated the secondary metabolites of Ilyonectria robusta, from which we isolated two new chromone derivatives (1 and 2), and 2 was a chlorochromone.

Chemistry
Compound 1 was obtained as white amorphous powder.Its molecular formula was defined as C 13 H 14 O 4 , according to the HRESIMS ion peak at m/z 235.0973 The UV spectrum exhibited absorption maxima at 212, 237, 250 and 291 nm, corresponding to a chromone core.(Basudan et al. 2005;Zhao et al. 2011; Figure S5).The IR absorption bands indicated the existence of hydroxyl (3426 cm À1 ), strongly conjugated carbonyl (1645 cm À1 ), aromatic ring (1610, 1560, 1486 cm À1 ), methyl and methylene (2969, 2924, and 2822 cm À1 ) (Figure S6).The 1 H NMR spectrum (Table S1 3), indicated that the 7-position on the benzene ring was substituted by a methyl group (Figure S1).Apart from the planar structure, the absolute configuration was confirmed by ECD.Compound 1 has only one chiral carbon atom, thus, by comparing the measured electronic circular dichroism spectrum with the calculated result, we found that the experimental spectrum was in high agreement with the calculated ECD spectrum of (2'S), directly indicating the absolute configuration of compound 1 behaved as 2'S (Figure S2).To sum up, compound 1 was identified as (2'S)-5-hydroxy-2-(2-hydroxypropyl)-7-methyl-4H-chromen-4-one.See the Figure 1 for the structure and the Table S1 for more detailed NMR information.
Compound 2 was obtained as white amorphous powder.Its molecular formula was defined as C 13 H 13 ClO 4 , according to the HRESIMS ion peak at m/z 269.0584 The UV and IR spectra of 2 showed a high degree of similarity to those of 1, indicating that they shared the same chromone nucleus.By comparing the similar NMR data of compounds 1 and 2, it was found that compound 2 was the chloride of compound 1.This was evidenced by the following points.On the one hand, in the nuclear magnetic resonance spectra, the most prominent difference between two compounds was that the chemical shift of the 8-position carbon of 2 was smaller than 1, and compound 2 lacked an aromatic hydrogen proton at 8-position (Table S1).On the other hand, the isotopic peaks and molecular weight in the high-resolution mass spectrometry inferred the existence of the chlorine atom (Figure S13).The absolute configuration of compound 2 was also determined as 2'S by ECD through the same procedures (Figure S3).Ultimately, compound 2 was resolved as (2'S)-8-chloro-5-hydroxy-2-(2-hydroxypropyl)-7-methyl-4H-chromen-4-one and shown in Figure 1.For more details see Table S1.By comparing with previously reported HRESIMS and NMR spectra in the literatures, it was found that compound 3 was also a chromone derivative, identified as altechromone A (K€ onigs et al. 2010;Zhang et al. 2017), while compound 4 was an anthraquinone called (11S)-1,3,6-trihydroxy-7-(1-hydroxyethyl) anthracene-9,10-dione (Zhao et al. 2018).Figure 1 displayed the structures and Table S2 showed the details.

Antibacterial activity
After massive literature investigations, it was found that some chromone derivatives have antibacterial potential, especially those substituted by halogen atoms (Trujillo-Esquivel et al. 2015;Lei et al. 2018;Mohsin et al. 2020;Liu et al. 2021).The four compounds in this study were tested for inhibition against four common clinical pathogens including S. aureus, E. faecalis, P. aeruginosa and E. coli.Unfortunately, in preliminary assessments of activity, none of the four compounds showed inhibition at the highest test concentration (Table S3).

General experimental procedures
NMR data were provided by a Bruker AVIII-600 spectrometer analyser (Bruker, Germany), with TMS as reference and deuterated methanol and dimethyl sulfoxide as solvent; Mass spectra from an Agilent 6500 series Q-TOF mass spectrometer analyser (Agilent, Singapore) in positive mode; The instrument used for HPLC preparation was Agilent 1100 (Agilent Technologies, America), and a reversed-phase column (Agilent, ZORBAX SB-C 18 , 5 lm, 9.4 Â 250 mm); UV and experimental CD data from an Applied PhotoPhysics Chiascan circular dichroism spectrometer (APL, Britain); Optical rotation data from an automatic polarimeter (Rudolph Research Analytical, America); IR data from a Shimadzu IR Affinity-1 spectrometer (Shimadzu, Japan).

Fungal material
The fungal strain Ilyonectria robusta was isolated from the rhizosphere soil of the Phyllagathis caualeriei plant in the Sanfenshi Scenic Area of the Jiuyi Mountain, Ningyuan, Hunan Province.ITS sequence data are deposited in GeneBank along with accession information.(JF735266.1).It is now preserved in the Xiangya School of Pharmaceutical Sciences, Central South University.

Cultivation, extraction and isolation
The strain was inoculated into PDA medium from 30% glycerol aqueous solution in a À80 C refrigerator, and then inoculated into 4 L of sterile liquid medium, and cultured with shaking at 150 r/min in a constant temperature shaker at 28 C.For 7 d, the seed solution was poured into 40 L of sterilised rice solid medium and cultivated at room temperature for 28 d.

ECD calculation
The ECD spectra of compounds 1 and 2 were calculated by using of the Gaussian09 package (Frisch et al. 2016).Their each configuration was optimised at the B3LYP/SVP level of theory (O'Boyle et al. 2011).The theoretic ECD spectra were calculated on mPW1PW91/SVP level of theory and were Boltzmann averaged according to Gibbs free energy.SpecDis v1.71 was used to simulate the ECD curve of compounds 1 and 2, with sigma/gamma value of 0.3 eV (Bruhn et al. 2013).The calculated ECD curves of compounds 1 and 2 were red and blue shifted by 10 nm and 25 nm, respectively.

Antibacterial activity assay
The inhibitory activities of each compound against four common pathogenic bacteria (S. aureus ATCC29213, E. faecalis ATCC29212, P. aeruginosa ATCC27853 and E. coli ATCC25922) were tested by 96-well plate microdilution method.Firstly, four bacterial solutions with a concentration of 1 Â 10 6 CFU/mL were prepared.Simultaneously, dissolve the compounds to a certain concentration.Nutrient broth medium, bacterial liquid and sample solution were added to make the concentration of the mixture in the first well 128 lg/mL, followed by doubling dilution to make the concentration of the seventh well 2 lg/mL.Commonly, set a positive control (vancomycin hydrochloride and amikacin sulfate), a negative control (same concentration of DMSO), a blank control (100% nutrient broth medium) and repeated experiments.At last, placed in a 37 C constant temperature incubator for 8 h and visually judged the degree of bacterial turbidity to read the MIC values.

Conclusions
In this study, two new chromone derivatives and two known compounds were reported from the rhizosphere soil fungus Ilyonectria robusta, and their inhibitory activity against four common pathogenic bacteria was tested, although the results showed no significant inhibitory effect.This study not only enriches the chemical composition of Ilyonectria robusta, which has rarely reported in secondary metabolites until now, but also contributes to the diversity of naturally derived chromone resources.

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