Furoic acid derivatives from the endophytic fungus Coniothyrium sp.

The endophytic fungus Coniothyrium sp. was isolated from leaves of Quercus robur. Fermentation of this fungus on solid rice medium yielded two new furoic acid derivatives ( 1 and 2 ) and two additional known compounds. The structures of the new compounds were determined by extensive analysis of 1D and 2D nuclear magnetic resonance spectra as well as high-resolution mass spectrometry data. Compound 1 , containing three aromatic chromophores attached by rotatable sigma bonds and a chirality center in benzylic position, was found to be a scalemic mixture with an excess of the (S) enantiomer, the absolute configuration of which was elucidated as by the solution time-dependent density functional theory-electronic circular dichroism approach. The ω B97X/TZVP PCM/MeCN and SOGGA11-X/TZVP SMD/MeCN methods were used for geometry reoptimization to reproduce the solution conformational ensemble. All isolated compounds were tested for their cytotoxicity but proved to be inactive.


| INTRODUCTION
Endophytic fungi are a prominent source for the discovery of new compounds. 1,2 There are an estimated 1.5 million fungal species on Earth, of which only about 70 000 are currently described. 3 Some endophytic fungal genera such as Aspergillus and Penicillium are well investigated, and their metabolic patterns are well known, even though new compounds are still reported. [4][5][6] In this study, the endophytic fungus Coniothyrium sp. was isolated from leaves of the deciduous tree Quercus robur. Literature search for natural products from Coniothyrium species revealed several bioactive compounds. Conioimide that was isolated from the Baltic sea alga-derived fungus Coniothyrium cereale showed prominent and selective inhibitory activity towards the protease human leukocyte elastase with an IC 50 value of 0.2 μg/mL. 7 Conioscleroderolide, a phenalenone derivative, displayed [This article is part of the Special Issue: In honor and memory of Prof. Koji Nakanishi. See the first articles for this special issue previously published in Volumes 31:12, 32:3, and 32:4. More special articles will be found in this issue as well as in those to come.] antibacterial activity against Staphylococcus aureus SG511 with an MIC value of 24 μM. 8 Coniothyrione, a chlorocyclopentandienylbenzopyrone, was shown to be a bacterial protein synthesis inhibitor and was isolated from Coniothyrium cerealis MF7209. 9 In this study, fermentation of Coniothyrium sp. on solid rice medium yielded two new furoic acid derivatives (1 and 2) and two additional known compounds 2,3-dihydroxy-2,4-diphenylcyclopent-4-en-1-one (3) 10 and 2-anhydromevalonic acid (4). 11 The structure elucidation of the new compounds and the results of cytotoxicity assay are described in this paper ( Figure 1).

| General experimental procedures
Optical rotations were measured on a PerkinElmer-241 MC polarimeter. 1D and 2D nuclear magnetic resonance (NMR) spectra were recorded with Bruker ARX 300 or AVANCE DMX 600 NMR spectrometers. Mass spectra were obtained from a Finnigan LCQ Deca XP mass spectrometer, while high-resolution mass spectra were recorded by a FTHRMS-Orbitrap (Thermo-Finnigan) mass spectrometer. A Dionex P580 system was used for high-performance liquid chromatography (HPLC) separations in combination with a diode array detector (UVD340S) and an Eurosphere 10 C 18 column (125 × 4 mm). Semipreparative HPLC was conducted on a Lachrom-Merck Hitachi system (pump L7100, ultraviolet (UV) detector L7400, Eurosphere 100 C 18 column, 300 × 8 mm, Knauer Germany). Merck MN silica gel 60M (0.04-0.063 mm) was used as stationary phase for column chromatography. TLC plates precoated with silica gel 60 F254 were used for monitoring separation. UV and electronic circular dichroism (ECD) spectra were recorded on a J-810 spectropolarimeter. The HRESIMS, UV, and NMR spectra were included in the Supporting Information.

| Fungal material and fermentation
Leaves of Quercus robur were collected in 2017 in Juelich, Germany. The fresh sample was washed by sterilized water, surface sterilized with 70% ethanol for 1 minute, and cut into small pieces (around 1 × 1 × 1 cm 3 ) using a flame sterilized blade. These pieces were put on malt agar plates (15 g/L malt extract, 15 g/L agar, and 0.2 g/L chloramphenicol in distilled water, pH 7.4-7.8), and then incubated at room temperature for several days. The purified fungus was later transferred to solid rice medium for fermentation. All steps were conducted in an aseptic environment. The identification of the fungus was done using a molecular protocol as described previously. 12 Sequence data were submitted to GenBank with the accession number MN043344. A voucher strain (2BEY) is kept in the Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine University, Düsseldorf, Germany.
Large-scale fermentation of this fungus was conducted in five Erlenmeyer flasks on solid rice medium (100 g rice in 110-mL water and autoclaved) at 20 C under static condition. After 20 days, every flask was treated with 600-mL EtOAc and left overnight. Then the EtOAc extract was evaporated under vacuum to obtain the crude extract.

| Cytotoxicity assay
Cytotoxicity against the L5178Y mouse lymphoma cell line was measured using the MTT assay. 13 Kahalalide F and 0.1% ethylene glycol monomethyl ether in DMSO were used as positive and negative control, respectively. preceding DFT reoptimization step. ECD spectra were generated as the sum of Gaussians 18 with 3000 cm −1 halfheight width, using dipole-velocity-computed rotational strengths. Boltzmann distributions were estimated from the ωB97X and the SOGGA11-X energies. The MOLEKEL software package was used for visualization of the results. 19 3 | RESULTS AND DISCUSSION Compound 1 was isolated as a white amorphous solid. Its molecular formula was determined as C 18 (Figure 2). The attachment of an oxygenated methine at C-7 was deduced from HMBC correlations from H-6 to C-7 and C-8 (12). In addition, HMBC correlations from H-4 to C-2 (δ C 141.0), C-3 (δ C 135.5), C-5 (δ C 160.8), and C-13; from H-6 to C-4 and C-5 (δ C 112.1); and from H-14(18) to C-3 indicated the presence of a furan ring and the linkages between C-5/C-6 and C-3/C-13. The location of a carboxy group at C-2 was suggested by the molecular formula of 1. Thus, the planar structure of 1 was elucidated for which the trivial name coniofuroic acid A is suggested.

| Computational methods
For the configurational assignment of 1, the solution TDDFT-ECD method was applied on the arbitrarily chosen (S) stereoisomer. 20,21 Since 1 is conformationally flexible and the relative orientation of the three aromatic chromophores produced by rotation along the C-3-C-13 biaryl axis and the sigma bonds of the C-6 chirality center  is expected to be fundamental for the sign and shape of the ECD transitions, a thorough conformational search is inevitable. [22][23][24][25] Consequently, the initial 37 MMFF conformers of (S)-1 were reoptimized at both the ωB97X/TZVP 15 PCM/MeCN and the SOGGA11-X/ TZVP 16 SMD/MeCN levels of theory. 26,27 (see Supporting Information) ECD calculations were performed for both sets of conformers at the B3LYP/TZVP, BH&HLYP/TZVP, CAM-B3LYP/TZVP, and the PBE0/ TZVP levels with the same solvent model as applied for the preceding DFT reoptimization level. Although as expected the individual conformers gave rather diverse ECD spectra, the Boltzmann-averaged spectra obtained at all applied combinations of levels reproduced well the experimental ECD allowing elucidation of the absolute configuration as (S) with high confidence (Figures 3 and  4). The low-energy conformers could be classified into six groups ( Figure 5) according to the relative orientation of the three aromatic chromophores, ie, by rotation around the C-3-C-13, C-5-C-6, and C-6-C-7 bonds. The contribution of conformer ingroups A and C with a sum Boltzmann population of 56.6% at the SOGGA11-X/TZVP SMD/MeCN level reproduced already the overall shape of the experimental spectrum ( Figure 4). It is also worth to note that the averaged computed ECD spectra obtained at any combinations were much more intense than the experimental one. The experimental ECD spectrum had to be multiplied by 20 to be comparable with the calculated ECD spectra, which implies about more than one order of magnitude difference. This indicated that the sample is probably a scalemic mixture with slight excess of the (S) stereoisome. [28][29][30] Partial racemization of the chirality center is certainly aided by the benzylic position, since the C-6 chirality center is α position to both a  benzene and a furan ring. For the (S) enantiomer of 1, a relatively strong negative Cotton effect is expected at 215 nm and a strong positive one at around 265 nm. Although separation of the enantiomers on various chiral HPLC columns was attempted, no successful separation could be achieved with the tested conditions. The molecular formula of 2 was determined as C 18 H 12 O 4 based on the HRMS data, lacking two protons when compared to 1. The 1 H NMR data of 2 were similar to those of 1 except for the disappearance of the oxygenated methine in 2 (Table 1). In addition, H-4 (δ H 7.49) and H-8(12) (δ H 8.11) in 2 were deshielded (δ H 6.41 and 7.49, respectively in 1). The above data suggested that the oxygenated methine was replaced by a ketone group in 2, which was further confirmed by HMBC correlations from H-8 (12) to the carbon of this ketone group (δ C 184.0). Detailed analysis of 2D NMR spectra of 2 revealed that the remaining substructures of 2 were identical to that of 1. The trivial name coniofuroic acid B is given to compound 2.
All isolated compounds were submitted for bioassay against the L5178Y mouse lymphoma cell line but proved to be inactive when tested at an initial concentration of 10 μM.