Protoilludene and Alkenoic Acid Derivatives from the European Polypore Fomitiporia hartigii

Chemical investigation of the solid-state rice culture of the endangered European polypore Fomitiporia hartigii (Hymenochaetaceae) afforded a previously undescribed protoilludene derivative (1) in addition to six known compounds (2–7). Chemical structures of the isolated compounds were established based on HR-ESI-MS, comprehensive 1D/2D NMR spectroscopic analyses, and comparisons with the literature. All isolated compounds were assessed for their cytotoxic and antimicrobial activities. Among the tested compounds, hymeglusin (3) revealed potent cytotoxic activity against all tested cell lines with IC50 values between 0.3 and 6.8 μM. Compound 3 and fusaridioic acid A (4) revealed weak to moderate antimicrobial activities with its most potent effect against Candida albicans (minimum inhibitory concentration of 4.2 μg/mL).

Most species of the family Hymenochaetaceae are forest pathogens, with considerable economic significance.Some species have been widely studied for their interesting therapeutic properties, e.g., Inonotus obliquus ("Chaga") 5 and Sanghuangporus sanghuang ("Sanghuang"). 6Nevertheless, the genus Fomitiporia has not been studied extensively for its secondary metabolites.Despite that, most species of the genus have been implicated as causal agents for the Esca disease of grapevines. 7,8Our previous exploration of the Kenyan Fomitiporia aethiopica led to the discovery of previously unprecedented steroids with moderate cytotoxic activities. 9ecently, we encountered F. hartigii, an endangered European species known to be closely associated with fir (Abies spp.).Thus, inspired by our previous success with the related F. aethiopica, we chemically explored the axenic solid-state rice culture of F. hartigii.Herein, we report the results obtained from the chemical and pharmacological prospections.
is known since the 1960s to harbor this German red listed fungus (i.e., category: near threatened). 10A copy of the mycelial culture of the fungus was deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany, under no.STMA 23102.
Structure Elucidation of Secondary Metabolites.Dereplication study of EtOAc extract derived from the solidstate rice culture of the European polypore F. hartigii was conducted implementing HPLC−DAD−MS supported by secondary metabolite database searches (https://dnp.chemnetbase.com/chemical/ChemicalSearch.xhtml?dswid=-621), and it revealed the presence of one previously undescribed protoilludene derivative and six known compounds (Figure 1).Chemical structures of the isolated compounds were elucidated, based on HR-ESI-MS, 1D/2D NMR spectroscopic analyses and the comparisons with the reported literature.The known compounds were determined as tricoprotoilludene A (2), 11 hymeglusin (3), 12 fusaridioic acid A (4), 12 neovasinin (5), 13 and neovasiopyrones A (6) and B (7). 14 All of the isolated compounds were assessed for their cytotoxic and antimicrobial activities against a panel of cell lines and microbes, respectively.
Compound 1 was purified as a white amorphous inseparable solid mixture with tricoprotoilludene A (2). HR-ESI-MS established its molecular formula as C 14 H 20 O 3 indicating five degrees of unsaturation by revealing a protonated molecular ion peak at m/z 237.1480 [M + H] + (calculated 237.1485) and a sodium adduct at m/z 259.1303 [M + Na] + (calculated 259.1305).The 13 C NMR spectral data of 1 (Table 1) revealed the presence of two carbonyl carbon atoms differentiated into one ketocarbonyl carbon (δ C 211.4) and one carboxylic acid carbon (δ C 182.0) accounting for two degrees of unsaturation and thus suggesting a tricyclic sesquiterpenoidal structure for 1.In addition, the 13 C NMR and HSQC spectra of 1 (Table 1, Figures S7, and S10) revealed the presence of 12 sp 3 carbon atoms recognized into one unprotonated (δ C 31.8), five methines (δ C 69.4, 44.9, 42.1, 40.5, and 32.2), four methylenes (δ C 57.5, 37.9, 35.0, and 31.7), and two methyl groups (δ C 27.5 and 22.3).A literature search of 1 based on the obtained results suggested it to comprise a protoilludene sesquiterpenoidal skeleton closely related to tricoprotoilludenes and compounds that were given the trivial names "phellinharts A− C", recently reported from two basidiomycetous fungi Daedaleopsis tricolor 7 and "P.hartigii", 15 respectively.A detailed comparison of both 1 H and 13 C NMR data of 1 to those reported for tricoprotoilludenes and phellinharts revealed its close resemblance to "phellinhart C 15" except for the absence of the methyl ester group.In addition, we compared the solvents used during our extraction, isolation, and purification schemes to those adopted to attain phellinharts.In our scheme where we avoided any exposure to methanol, only the free carboxylic acid form of phellinhart C was purified and identified as compound 1, whereas the scheme followed by Zheng et al. involved several column chromatographic separations using mobile phases including methanol as a solvent and they purified phellinhart C as a methyl ester together with another sesquiterpene lactone methyl ester, phellinhart A. 15 These notions might suggest that phellinharts A and C as methyl esters perhaps emerged from an esterification reaction that took place during chromatographic workups.To affirm or negate this assumption, a backward tracing of phellinharts A and C to the crude extract of their fungal source might be requested.Further confirmation of the depicted structure of 1 (Figure 1) was obtained by the HMBC spectrum (Figures 2  and S9) that revealed key correlations from a diastereotopic methylene protons at δ H 2.45/3.22(H 2 -4) to a ketocarbonyl carbon at δ C 211.4 (C-5) and a methyl carbon at δ H 27.5 (C-12).In addition, key correlations have also been revealed from two methyl groups (H 3 -12 and H 3 -13) to a methine carbon at δ H 32.2 (C-7) and from a proton signal at δ H 2.92 (dddd, J = 10.8, 9.2, 7.4, 3.5 Hz, H-11) to a terminal carboxylic acid carbon at δ C 182.0 (C-14).The relative configuration of 1 was determined by the ROESY spectrum (Figure 2) that revealed key ROE correlations between two methyl groups H 3 -12/H 3 -13 and two methine protons H-6/H-11 indicating four of them to be directed toward the same face of the molecule while H-2, H-7, and H-9 are directed toward the opposite side of the  molecule.By comparing the 1 H and 13 C NMR spectral data of 1 (Table 1) and "phellinhart C 15" whose absolute configuration was determined by X-ray crystallography and taking in consideration their common biosynthetic origin, the absolute configuration of 1 was conclusively determined as (2S,3R,6S,7R,9R,11R).The obtained results confirmed that compound 1 is a previously undescribed protoilludene derivative that was trivially named tricoprotoilludene C. Biological Activity of Compounds 1−7.Using our standardized protocols, 16−18 all isolated compounds were subjected to cytotoxic and antimicrobial activity assays against two cell lines, namely, mouse fibroblasts (L929) and human endocervical adenocarcinoma (KB3.1)whereas the antimicrobial activity was determined against 12 different bacterial and fungal pathogens (Table 2).For compounds featuring potent cytotoxic activity, a further assessment against five additional cell lines was conducted.
The obtained results (Table 2) revealed that among the tested compounds, hymeglusin (3) induced significant cytotoxic activity against all tested cell lines except human lung carcinoma (A549) with IC 50 values ranging from 0.3 to 6.8 μM.In the antimicrobial assay (Table 2), hymeglusin (3) also revealed potent antifungal activity against C. albicans at MIC of 4.2 μg/mL whereas it revealed together with fusaridioic acid A (4) weak antimicrobial activities against few bacterial and fungal strains with MIC values of 33.3−66.6 μg/mL (Table 2).

■ CONCLUSIONS
In this study, an EtOAc crude organic extract of the European polypore F. hartigii was chemically explored, implementing a dereplication scheme followed by chromatographic workup to purify the potentially new natural products.Results unveiled the presence of one previously undescribed protoilludene derivative, tricoprotoilludene C (1) in addition to six known metabolites (2−7).In the cytotoxic and antimicrobial activity assays, hymeglusin (3) revealed potent cytotoxic activity against most of the tested cell lines and antifungal activity against C. albicans that could be attributed to the β-lactone functionality in its structure which is opened in fusaridioic acid A (4) lacking activity in these assays.This study valorizes rare Basidiomycota and fungi in general as a prolific source of bioactive natural products that could be of potential applications.

General Experimental Procedures. HPLC−DAD/MS
samples were analyzed on an amaZon speed ETD ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) in positive and negative ionization modes.The HPLC system consisted of a Dionex UltiMate 3000 UHPLC (Thermo Fisher Scientific Inc., Waltham, MA, USA), equipped with a C 18 Acquity UPLC BEH column (Waters, Milford, USA).The mobile phase consisted of solvent A [deionized water +0.1% formic acid (FA)] and solvent B [acetonitrile (MeCN) + 0.1% FA].The gradient used was as follows: 5% B for 0.5 min, 100% B over a period of 20 min, and then holding the 100% B for 10 min, at a flow rate of 0.6 mL/min.UV−vis detections were obtained at 190−600 and 210 nm wavelengths.HR-(+)ESI-MS measurements were performed in the positive ionization mode using the timsTOF Pro 2 mass spectrometer (Bruker Daltonics, Bremen, Germany) connected to an Agilent 1290 series HPLC-UV system (Agilent Technologies, Santa Clara, CA, USA) equipped with a C 18 Acquity UPLC BEH column (Waters, Milford, USA).The used eluent were solvents A (deionized water + 0.1% FA) and B (MeCN +0.1% FA).The gradient of separation was as follows: 5% B for 0.5 min, 100% B over a period of 20 min, and keeping the 100% B for 5 min.The flow rate was 0.6 mL/min (40 °C) and UV−vis detections were made at 200−600 nm.Molecular formulas of the secondary metabolites were obtained using the Smart Formula  Fungal Specimen and Preparation of Cultures.A fruiting body of F. hartigii (IHI 750) was collected, and pieces of the pore region of the underside were cut and placed on water agar.After a successful growth, the culture was transferred on malt agar plates.The isolate generally showed a slow growing behavior.
Fermentation of Cultures and Extraction of Metabolites.YMG (4 g of yeast extract, 10 g of malt extract, 4 g of glucose, and 20 g of agar in 1 L deionized water) medium plates were prepared and used to inoculate 6 × 500 mL Erlenmeyer culture flasks, containing sterile rice medium as previously described. 14Subsequently, secondary metabolites were extracted after static incubation at 24 °C in the dark for 28 days to yield a crude extract (1.2 g) as initially reported. 14solation of Compounds and Their Physico-Chemical Properties.The aforementioned EtOAc extract (1.2 g) was purified on a preparative HPLC system (PLC 2020; Gilson, Middleton, WI, USA).The solvent system consisted of deionized water + 0.1% FA (solvent A) and MeCN + 0.1% FA (solvent B) using a VP NUCLEODUR 100−5C 18 column (250 × 40 mm, 7 μm: Macherey-Nagel, Duren, Germany).
The gradient implemented for elution of the compounds was as follows: an initial isocratic start condition at 5% solvent B for 10 min, followed by an increase from 5 to 25% of solvent B within 3 min, thereafter another increase from 25 to 65% solvent B in 120 min, and a final holding of the gradient at 100% solvent B for 10 min.The flow rate was 40 mL/min and UV detections were made at 190, 210, and 280 nm wavelengths.The separation yielded 6 and 7 (2.58 mg, t R = 31 min), 4 (9.73 mg, t R = 47 min), 1 and 2 (11.45 mg, t R = 57−59 min), 5 (5.48 mg, t R = 61 min), and 3 (13.40mg, t R = 70−72 min).

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