Dentifragilones A–B and Other Benzoic Acid Derivatives from the European Basidiomycete Dentipellis fragilis

A chemical and biological exploration of the European polypore Dentipellis fragilis afforded two previously undescribed natural products (1 and 2), together with three known derivatives (3–5). Chemical structures of the isolated compounds were confirmed through 1D/2D NMR spectroscopic analyses, mass spectrometry, and by comparison with the reported literature. The relative and absolute configurations of 1 were determined according to the ROESY spectrum and time-dependent density functional theory electronic circular dichroism (TDDFT-ECD), respectively. Furthermore, the absolute configuration of dentipellinol (3) was revisited and revealed to be of (R) configuration. All the isolated compounds were assessed for their cytotoxic and antimicrobial activities, with some being revealed to have weak to moderate antimicrobial activity, particularly against Gram-positive bacteria.


Introduction
Fungal-based natural products have made an immense contribution to the modern-day medical and agrochemistry sectors, with their vast chemical novelties unmatched by many sources.Over the last two centuries, regions rich in biodiversity have undoubtedly proved to be invaluable sources of therapeutic targets useful for the global pharmaceutical industries [1].Nonetheless, Basidiomycota of the temperate zones, assumed to be well sampled, still offer great opportunities for novelty [2].The rare Basidiomycota of Europe in particular have been neglected, but constitute potential reservoirs of new pharmacotherapeutic agents [3].
During our investigations of the seldom-found Basidiomycetes of Germany, we encountered Dentipellis fragilis, a red-listed member of the wood-rot fungi of the Hericiaceae family.The fungal genus Dentipellis was coined by Anton Donk in 1962 and, thus far, D. fragilis is the only studied species of the genus with regards to its secondary metabolites.It is worth mentioning that D. fragilis has been demonstrated to be quite 'talented' in relation to the diversity of its produced natural compounds, with bioactive phthalide, cyathane, benzofuranone, and drimane derivatives having recently been reported from the fungus [4][5][6][7][8][9].In particular, the discovery of cyathanes is in strong accordance with the placement of Dentipellis in the Hericiaceae family, since the same compound class is also characteristic of cultures of other genera in this family like Hericium and Laxitextum [2,6].The current paper is dedicated to describing additional secondary metabolites (SMs) of the strain studied by Sum et al. [6] that were obtained upon modifying the culture conditions.
Molecules 2024, 29, x FOR PEER REVIEW        A literature search of 1 revealed its common structural features of strobiloscyphones [10], pestallic acids [11], dentipellin [4], and the recently reported lachnoic acids [12], where they shared the presence of a 2-cyclohexenone moiety in their structures.A careful interpretation of the obtained 1D and 2D NMR spectral data of 1 (Table 1, Figure 2) suggested the structure depicted in Figure 1, with a 1,1-dimethyl-2-propenyl functionality being attached at C-6.The relative configuration of 1 at C-5 and C-6 was determined by its ROESY spectrum, which revealed an ROE correlation between two exchangeable broad singlet proton signals at δ H 3.44 and 2.37 assigned to 5-OH and 6-OH, respectively.This thus indicated their cofacial orientation, while the 1,1-dimethyl-2-propenyl moiety is projected toward the opposite face of the molecule.The ROESY spectrum of 1 (Figures 2 and S8) also revealed key ROE correlations between the two diastereotopic methyl groups, H 3 -11 and H 3 -12, and H-4β, H-5, H-8, and H 2 -9, confirming the depicted structure of 1. Accordingly, the ROESY spectrum suggested the relative configurations at C-5 and C-6 were either (5S*,6S*) or (5R*,6R*).To determine the absolute configuration of 1, its ECD spectrum was acquired and, hence, compared to the calculated TDDFT-ECD spectra of (5S,6S) and (5R,6R) enantiomers.The obtained results (Figure 3) revealed a coherence between both the experimental and calculated ECD spectra of (5S,6S) configuration.Based on the obtained results, compound 1 was identified as a previously undescribed natural product named dentifragilone A.  Further characterization for the suggested structure of 2 was obtained via the HMBC spectrum (Figures 2 and S13), which revealed key correlations between two aromatic protons assigned to H-3 (δH 7.75) and H-5 (δH 7.49), the hydoxymethylene carbon at δC 64.2 (C-7), and an oxygenated aromatic carbon at δC 162.3 (C-1), indicating that the hydroxymethylene group is bound at C-4.The HMBC spectrum of 2 also revealed key correlations from two magnetically equivalent methyl groups (H3-11/H3-12) to C-10 (δC 70.0) and C-9 (δC 48.1), whereas the methylene group at δH 3.20 (H2-9) and H-3 revealed corre-  2, Figure S12) revealed three aromatic proton signals at δ H 7.75 (d, J = 2.1 Hz), 7.49 (dd, J = 8.5, 2.1 Hz), and 7.00 (d, J = 8.5 Hz) that were correlated as one spin system, suggesting their presence on a 1,2,4-trisubstituted aromatic ring.In addition, the 1 H NMR spectral data of 2 (Table 2) revealed the presence of three singlet proton resonances that were categorized, according to their integration indices, into two methylene groups at δ H 4.66 and δ H 3.20 (each of an integration index of two), together with two magnetically equivalent methyl groups at δ H 1.37 integrated for six protons.The 13 C NMR spectral data and DEPTQ spectrum (Table 2, Figure S16) revealed the presence of five unprotonated carbon signals resolved into one carbonyl carbon at δ C 207.2, three sp 2 carbon atoms (δ C 162.3, 131.4,and 119.2), and one oxygenated sp 3 carbon (δ C 70.0).A literature search for 2 revealed its close resemblance to methyl 4-hydroxy-3-(3-methylbutanoyl) benzoate, a fungal metabolite that has been previously reported from scrap cultivation beds of Hericium erinaceus (published by Ueda et al. [13] under the grammatically incorrect name "H. erinaceum").Further characterization for the suggested structure of 2 was obtained via the HMBC spectrum (Figures 2 and S13), which revealed key correlations between two aromatic protons assigned to H-3 (δ H 7.75) and H-5 (δ H 7.49), the hydoxymethylene carbon at δ C 64.2 (C-7), and an oxygenated aromatic carbon at δ C 162.3 (C-1), indicating that the hydroxymethylene group is bound at C-4.The HMBC spectrum of 2 also revealed key correlations from two magnetically equivalent methyl groups (H 3 -11/H 3 -12) to C-10 (δ C 70.0) and C-9 (δ C 48.1), whereas the methylene group at δ H 3.20 (H 2 -9) and H-3 revealed correlations to a carbonyl carbon (C-8), indicating the presence of a 3-hydroxy-3-methylbutanoyl moiety at C-2 on the aromatic ring.The ROESY spectrum of 2 (Figure 2) revealed key correlations from H-3 to both H 2 -7 and H 2 -9, confirming the depicted arrangement of substituents on the aromatic ring at C-1, C-2, and C-4.A literature search revealed that compound 2 revealed a reduced primary alcohol derivative related to crassinervic acid, an antifungal metabolite from Piper crassinervium [14].Based on the obtained results, compound 2 was identified as a previously undescribed natural product and it was given a trivial name, dentifragilone B.
Compound 3 was obtained as an off-white amorphous solid.Its molecular formula was established to be C 12 H 14 O 5 , indicating six degrees of unsaturation.A literature search of 3, based on its molecular formula and the 1D (1H and 13 C) NMR spectral data (Table S1), revealed that the measured values were in close accordance with those recently reported for dentipellinol [5].Although the absolute configuration of dentipellinol (3) was reported to be (S) configuration [5], herein its structure was revisited and determined by comparing its experimental and calculated ECD spectra (Figure S4).The results in Figure S4 indicate a closer coherence to the calculated ECD spectrum of (R) configuration than that of (S).Moreover, the 3D coordinates provided in the Supplementary Material of Ki et al.'s study confirmed the (R) configuration of dentipellinol (3), which was misinterpreted as (S) [5].

Biological Assays
To assess the antimicrobial activity of compounds 1-4, a serial dilution assay was conducted against several Gram-positive and Gram-negative bacteria as well as fungal strains.Compound 5 was not tested, since similar activities had been reported by our group in a recent study [15].Notably, compound 4 demonstrated moderate or weak antibiotic effects against Staphylococcus aureus with a MIC value of 66.6 µg/mL.Compounds 1-3 were inactive in the antimicrobial tests.
An evaluation of the cytotoxic activities of the isolated compounds was first conducted against the two most sensitive cell lines, namely mouse fibroblast (L929) and human endocervical adenocarcinoma (KB3.1).The compounds had no apparent cytotoxic effects; hence, further tests were not conducted.

Fungal Material
The fungus was collected on a decaying beech (Fagus sylvatica) in the Bavarian Forest National Park (49.098387N, 13.246003 E) in August 2015 [16] and cultured by one of the authors (HK).The mycelial culture was deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, designated as DSM 105465.Its identification was reported in our previous study [15] and an ITS-nRDNA sequence of the strain is deposited at the GenBank under the accession number MK463979.We would, however, like to point out that ITS sequences are unreliable for fungal identification and the morphological characters of the specimen already allowed for an unambiguous assignment to the taxon Dentipellis fragilis.

Fermentation and Extraction
The fungal strain was cultured in Erlenmeyer flasks containing either YMG or rice media.For submerged YMG cultivation, fermentation was carried out in 18 × 1 L shaker flasks containing 400 mL of medium (10 g/L malt extract, 4 g/L D-glucose, 4 g/L yeast extract, pH 6.3 before autoclaving), as previously described [6], with each inoculum consisting of 10 well-grown mycelial plugs.The cultures were incubated under shaking conditions in the dark at 140 rpm and 23 • C, and the fermentation process was monitored by checking the concentration of free glucose with Medi-Test glucose (Macherey-Nagel, Düren, Germany).The free glucose was fully consumed after 45 days, and extraction was performed after 3 days of glucose depletion.Alternatively, the rice substrate cultures were cultivated as previously reported [15].Basically, 10 × 500 mL Erlenmeyer flasks consisting of 90 mg of rice in 90 mL distilled water were prepared and autoclaved.These were used to inoculate fungal plugs, as similarly carried out for the YMG cultures.However, the rice medium cultures were cultivated under static conditions at 23 • C, and the cultures were extracted after 30 days.
To extract the secondary metabolites from the liquid cultures, the supernatant and mycelia were first separated by vacuum filtration.The supernatant was decanted with an equal amount of EtOAc in a separatory funnel.The obtained organic phase was filtered through anhydrous sodium sulfate and the filtrate was evaporated to dryness under a vacuum at 40 • C with a rotary evaporator (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany; pump: Vacuubrand GmbH & Co. KG, Wertheim am Main, Germany) in order to produce a solid residue of the total extract.The secondary metabolites from the mycelia (from either submerged or rice medium cultures) were extracted by initial soaking the mycelia in acetone, followed by immediate sonication for 30 min at 40 • C using an ultrasonic bath (Sonorex Digital 10 P, Bandelin Electronic GmbH & Co. KG, Berlin, Germany).The acetone was evaporated under reduced pressure at 40 • C, the resulting aqueous phase was decanted with an equal amount of ethyl acetate, and the total extract was obtained, as previously described for the supernatant phase.The overall process yielded 881 mg, 367 mg, and 1.6 g of supernatant, mycelia, and rice crude extracts, respectively.

Isolation of Compounds 1-5
To further separate the compounds, the mycelial and supernatant crude extracts were first combined due to their similar chemical profiles.The total extract (1.25 g) was dissolved in methanol (MeOH) and pre-fractionated using a Reveleris X2 flash chromatography system (W.R. Grace and Co., Columbia, MD, USA) equipped with a 40 g silica pre-packed column (Reveleris ® ).Dichloromethane (CH 2 Cl 2 ) (solvent A) and CH 2 Cl 2 :MeOH (ratio 8:2) (solvent B) were used as eluents, with a flow rate of 60 mL/min.The gradient of separation started with 0% to 30% B in 30 min, isocratic holding at 30% B for 2 min, 30% to 60% B in 15 min, isocratic holding at 60% B for 2 min, and 60% to 100% B in 10 min.UV detections were obtained at 190, 210, and 280 nm, and several fractions were obtained from this separation and further purified on the Gilson preparative reversed-phase HPLC (PLC 2020, Gilson, Middleton, WI, USA).A Synergi TM 10 µm Polar-RP 80 Å (250 × 50 mm) AXIA™ packed column (Phenomenex Inc., Aschaffenburg, Germany) was used as the stationary phase.Deionized H 2 O + 0.1% formic FA (v/v) (solvent A) and acetonitrile (MeCN) + 0.1% FA (v/v) (solvent B) were used as the mobile phase with a flow rate of 40 mL/min.Separation was carried out with an elution gradient beginning isocratically at 5% solvent B for 10 min, followed by a gradient increase to 65% B in 30 min, then an increase from 65% B to 100% B in 10 min, and ending with an isocratic hold at 100% B for 15 min.UV detection was performed at 190, 210, and 280 nm to yield compounds 1 (t R = 14.0 min), 3 (t R = 12.0 min), and 2 (t R = 16.0 min).The purity of the fractions was checked using HPLC-DAD-ESI-MS.
The rice crude extract was divided into two portions.One portion was fractionated directly on the Gilson system after initially passing it through a RP solid-phase cartridge (Strata-X 33 µm Polymeric Reversed Phase; Phenomenex, Aschaffenburg, Germany) to remove fatty acids.A similar solvent system and UV detections were used on the instrument as mentioned for the YMG extract.However, the stationary phase in this case was a C 18 VP-Nucleodur column 100-5 (250 × 40 mm, 7 µm: Machery-Nagel, Düren, Germany).The gradient was operated with isocratic conditions at 5% B for 10 min, followed by an increase from 5% B to 10% B in 10 min, from 10% B to 80% B in 40 min, from 80% to 100% B in 5 min, and a final isocratic step at 100% B for 10 min.This yielded compounds 4 (t R = 41.0 min) and 5 (t R = 55.0 min).

Antimicrobial Assay
The Minimum Inhibitory Concentration (MIC) of the isolated compounds was determined following the method previously described [16].Accordingly, the compounds were tested against bacteria and fungi on a serial dilution assay performed on 96-well microtiter plates.YMG medium was used to culture the yeasts and filamentous fungi, whereas MHB media (Müller-Hinton Broth: SNX927.1,Carl Roth GmbH, Karlsruhe, Germany) was used for bacteria.

Cytotoxicity Assay
The in vitro cytotoxicity (IC 50 ) of isolated compounds was evaluated against an array of mammalian cell lines using a colorimetric tetrazolium dye MTT assay with epothilone B as a positive control.The cell lines, L929 (mouse fibroblasts) and KB3.1 (human endocervical adenocarcinoma), were employed, following established methodologies [16].

Density Functional Theory Calculations
In order to elucidate the electronic circular dichroism (ECD) spectra, a conformational analysis was principally executed to extract all possible conformations of compounds 1 and 2, employing Omega2 software 2.5.1.4[17] within an energy window value of 10 kcal/mol [18].The resulting configurations were set to the geometry optimization process and then frequency computations at the B3LYP/6-31G* level of theory.The time-dependent density functional theory (TDDFT) calculations were then performed in methanol to determine the first fifty excitation states.The solvent effect was incorporated using the polarizable continuum model (PCM).The calculated ECD spectra were graphed

Compound 1
was isolated as a white amorphous solid.The HR-ESI-MS of 1 revealed a protonated molecular ion and sodium adduct peaks at m/z 227.1277 [M+H] + (calculated 227.1278) and 249.1098 [M+Na] + (calculated 249.1097), respectively, determining its molecular formula as C 12 H 18 O 4 and hence indicating four degrees of unsaturation.The 13 C NMR and HSQC spectral data of 1 ( ) unveiled the presence of twelve carbon resonances that can be classified into five unprotonated carbon atoms separated into one carbonyl carbon at δ C 195.8 (C-1), two olefinic carbon atoms at δ C 144.0 (C-2), 130.0 (C-3), and two sp 3 carbon atoms at δ C 44.8 (C-7) and 80.4 (C-6).In addition, the 13 C NMR spectral data revealed one methylene sp 3 carbon atom at δ C 36.7 (C-4) and one methylidene sp 2 carbon atom at δ C 113.2 (C-9).The 1D ( 1 H and 13 C) NMR data and HSQC spectrum of 1 (

Figure 3 .
Figure 3. Experimental and calculated ECD spectra of 1. Compound 2 was purified as an off-white amorphous solid.The HR-ESI-MS revealed a protonated molecular ion peak and a sodium adduct peak at m/z 225.1117 [M+H] + (calculated 225.1121) and 247.0937 [M+Na] + (calculated 247.0941), respectively, determining its molecular formula as C 12 H 16 O 4 and indicating five degrees of unsaturation.The 1 H NMR spectral data and the 1 H-1 H COSY spectrum of 2 (Table2, FigureS12) revealed three aromatic proton signals at δ H 7.75 (d, J = 2.1 Hz), 7.49 (dd, J = 8.5, 2.1 Hz), and 7.00 (d, J = 8.5 Hz) that were correlated as one spin system, suggesting their presence on a 1,2,4-trisubstituted aromatic ring.In addition, the 1 H NMR spectral data of 2 (Table2) revealed the presence of three singlet proton resonances that were categorized, according to their integration indices, into two methylene groups at δ H 4.66 and δ H 3.20 (each of an integration index of two), together with two magnetically equivalent methyl groups at δ H 1.37 integrated for six protons.The 13 C NMR spectral data and DEPTQ spectrum (Table2, FigureS16) revealed the presence of five unprotonated carbon signals resolved into one carbonyl carbon at δ C 207.2, three sp 2 carbon atoms (δ C 162.3, 131.4,and 119.2), and one oxygenated sp 3 carbon (δ C 70.0).A literature search for 2 revealed its close resemblance to methyl 4-hydroxy-3-(3-methylbutanoyl) benzoate, a fungal metabolite that has been previously reported from scrap cultivation beds of Hericium erinaceus (published by Ueda et al.[13] under the grammatically incorrect name "H. erinaceum").
Measured in chloroform-d a at 125 MHz/ b at 500 MHz.Measured in methanol-d 4 c at 125 MHz/ d at 500 MHz.e Assignment confirmed by HMBC and HSQC spectra.