Bioactive Naphtho-α-Pyranones from Two Endophytic Fungi of the Genus Polyphilus

In the course of our survey to study the metabolic potential of two species of a new helotialean genus Polyphilus, namely P. frankenii and P. sieberi, their crude extracts were obtained using different cultivation techniques, which led to the isolation and characterization of two new naphtho-α-pyranone derivatives recognized as a monomer (1) and its 6,6′-homodimer (2) together with two known diketopiperazine congeners, outovirin B (3) and (3S,6S)-3,6-dibenzylpiperazine-2,5-dione (4). The structures of isolated compounds were determined based on extensive 1D and 2D NMR and HRESIMS. The absolute configuration of new naphtho-α-pyranones was determined using a comparison of their experimental ECD spectra with those of related structural analogues. 6,6′-binaphtho-α-pyranone talaroderxine C (2) exhibited potent cytotoxic activity against different mammalian cell lines with IC50 values in the low micromolar to nanomolar range. In addition, talaroderxine C unveiled stronger antimicrobial activity against Bacillus subtilis rather than Staphylococcus aureus with MIC values of 0.52 µg mL−1 (0.83 µM) compared to 66.6 µg mL−1 (105.70 µM), respectively.

A rare 5,8 -linkage was also recently reported in lulworthinone obtained from a marinederived fungus: Lulworthia medusa [16].Several members of the dimer class have been reported to exhibit potential antibacterial activity.In particular, viriditioxin has been reported to inhibit FtsZ, which is essential for bacterial cell division [17].Based on the fact that antibiotic resistance is an exacerbating problem, the need for new antimicrobial agents remains an unmet demand.
During our ongoing research targeting the discovery of new fungal metabolites with potential antimicrobial activities, in this study, we investigated the root endophytic representatives of the recently described fungal genus Polyphilus, namely P. frankenii and P. sieberi [18].The chemical exploration resulted in the isolation and identification of a monomer (1) and its symmetric 6,6 -homodimer (2) (Figure 1) together with two known metabolites identified as outovirin B (3) [19] and (3S,6S)-3,6-dibenzylpiperazine-2,5-dione (4) [20].In this study, we report the isolation and structure elucidation of two new naphtoα-pyranone derivatives together with their antimicrobial and cytotoxic activity, which were recorded in our routine assays.
Antibiotics 2023, 12, x FOR PEER REVIEW 2 of 9 been reported to exhibit potential antibacterial activity.In particular, viriditioxin has been reported to inhibit FtsZ, which is essential for bacterial cell division [17].Based on the fact that antibiotic resistance is an exacerbating problem, the need for new antimicrobial agents remains an unmet demand.
During our ongoing research targeting the discovery of new fungal metabolites with potential antimicrobial activities, in this study, we investigated the root endophytic representatives of the recently described fungal genus Polyphilus, namely P. frankenii and P. sieberi [18].The chemical exploration resulted in the isolation and identification of a monomer (1) and its symmetric 6,6′-homodimer (2) (Figure 1) together with two known metabolites identified as outovirin B (3) [19] and (3S,6S)-3,6-dibenzylpiperazine-2,5-dione (4) [20].In this study, we report the isolation and structure elucidation of two new naphtoα-pyranone derivatives together with their antimicrobial and cytotoxic activity, which were recorded in our routine assays.

Isolation and Identification of Compounds (1 and 2)
Compound 1 was purified as a white solid powder, and its molecular formula was established to be C 18 H 20 O 5 based on its HRESIMS spectrum, which revealed pseudomolecular ion peaks at m/z 317.1386 [M+H] + (calculated for 317.1384) and at m/z 339.1202 [M+Na] + (calculated for 339.1203), indicating the existence of nine degrees of unsaturation.The 13 C NMR spectral data of 1 (Table 1) displayed the presence of fifteen carbon resonances that can be differentiated into eight quaternary carbon atoms recognized as one carbonyl at δ C 170.7 (C-1) and seven olefinic (two oxygenated) carbon atoms at δ C 162.6 (C-10), 161.0 (C-7), 160.7 (C-9), 140.6 (C-5a), 133.9 (C-4a), 107.2 (C-9a), and 98.8 (C-10a).In addition, the 13 C NMR spectrum of 1 also revealed the presence of four tertiary (one aliphatic and three olefinic) carbon atoms at δ C 114.5 (C-5), 101.5 (C-8), 101.4 (C-6), and 79.3 (C-3) along with four methylenes (δ C 34.0 (C-11), 32.3 (C-4), 31.0 (C-13), and 22.0 (C-14)) and one methyl carbon atom (δ C 13.7 (C-15)).By comparing the obtained results with the reported literature, compound 1 was suggested to be a naphthopyrone derivative related to those reported as fungal metabolites, such as penicitor A [4,5], semiviriditoxin [1,2], and semivioxanthin [3].Further structural features of 1 were concluded using 2D NMR spectra including 1 H-1 H COSY, HMBC, and HSQC.The 1 H-1 H COSY spectrum of 1 revealed two main spin systems, with one extending over two meta-positioned aromatic protons at δ H 6.31 (H-6) and 6.47 (H-8) while the second spin system was found to begin at a methylene group at δ H 2.88/δ H 3.00 (H 2 -4) and extended to an aliphatic oxygenated methine group at δ H 4.58 (H-3), thus extending over four methylenes, forming an aliphatic side chain at δ H 1.68/δ H 1.74 (H 2 -11), δ H 1.41/δ H 1.46 (H 2 -12), and δ H 1.31 (H 2 -14 and H 2 -13), and ending with a terminal triplet methyl group at δ H 0.88 (H 3 -15) to confirm the presence of the n-pentyl side chain.The HMBC spectrum of 1 (Figure 2) revealed key correlations from H 2 -4 to four carbon atoms ascribed to C-10a (δ C 98.8), C-4a (δ C 133.9), C-3 (δ C 79.3), and C-11 (δ C 34.0), whereas additional HMBC correlations from H 2 -11 to C-3 confirmed that the n-pentyl side chain was present at C-3.Further key HMBC correlations (Figure 2) were also observed in the aromatic protons from H-5, H-6, and H-8 to C-9a (δ C 107.2) and from H-5 and H 2 -4 to C-10a, which confirmed the depicted structure of 1 as a naphthopyrone derivative.The ECD spectrum of 1 (see Supplementary Materials: Figure S9) showed negative Cotton effects (CEs) at 219 nm and 267 nm, a weaker positive one at 242 nm, and a broad positive plateau within the range of 280-400.This ECD spectrum was a near mirror image of that of (S)-7-O-methylpenicitor A [5], which differed only in the C-9 and C-14 methoxy groups, and its absolute configuration (AC) was determined using ECD calculations and single-crystal X-ray diffraction analysis.Due to the mirror image ECD curves, the AC of 1 was assigned as (R), and as a new naphthopyrone derivative, it was given the trivial name semitalaroderxine C. nm and 267 nm, a weaker positive one at 242 nm, and a broad positive plateau within the range of 280-400.This ECD spectrum was a near mirror image of that of (S)-7-Omethylpenicitor A [5], which differed only in the C-9 and C-14 methoxy groups, and its absolute configuration (AC) was determined using ECD calculations and single-crystal Xray diffraction analysis.Due to the mirror image ECD curves, the AC of 1 was assigned as (R), and as a new naphthopyrone derivative, it was given the trivial name semitalaroderxine C.   Compound 2 was isolated as a white solid powder that revealed in a pseudomolecular ion peak at m/z 631.2541 [M+H] + (calculated for 631.2538) its HRESIMS spectrum, confirming its molecular formula as C 36 H 38 O 10 and indicating its inclusion of eighteen degrees of unsaturation.Intriguingly, by comparing the molecular formulas of 1 and 2, it could be obviously observed that 2 is a symmetric dimer of the two monomers of 1.This assumption was further confirmed using 13 C NMR spectral data (Table 1), which revealed only eighteen carbon resonances; thus, each was assigned to two electromagnetically equivalent carbon atoms in the two monomers.The 1 H NMR spectral data of 2 (Table 1) displayed an identical set of proton resonances to those of 1, except in the absence of one aromatic proton at δ H 6.31 (H-6) in 1 and hence suggesting that compound 2 is a 6,6 -binaphthopyrone dimer of semitalaroderxine C (1).Based on the obtained results and by searching the reported literature, compound 2 was found to be related to the previously reported 6,6 -binaphthopyrone dimers, talaroderxines A and B, that were reported from a soil-derived fungus Talaromyces derxii [8] and pigmentosins A/B [10,21].
The major structural difference between 2 and talaroderxines A/B was the presence of n-pentyl in 2 instead of the n-propyl side chain in talaroderxines A/B.Since compound 2 is the 6,6 -linked axially chiral homodimer of 1, the (3S,3 S) absolute configuration of the central chirality elements was deduced on the basis of their common biosynthetic origin.The (aS) axial chirality of 2, arising from the hindered rotation around the C-6-C-6 biaryl axis, was determined by comparing its experimental ECD spectrum (see Supplementary Materials Figure S18) with those of related 6,6 -linked bis-naphthopyrone pigmentosins A and B [10] and talaroderxine A [8]. Compound 2 showed an intense positive excitoncoupled couplet centered at 260 nm (268 nm (∆ε: +16.58); 252 nm (∆ε: −14.15)), which was a mirror image of the negative couplet of (aR)-pigmentosins A and B and (aR)-talaroderxine B [8][9][10].Compound 2 was identified as a new 6,6 -linked bis-naphthopyrone homodimer with (aS) axial chirality, which was named talaroderxine C.

Fermentation, Extraction, and Isolation
The two fungal species explored in this study namely, P. frankenii strain V16 (DSM 106521) and P. sieberi strain Ref052 (DSM 106515) were recognized as endophytic fungi associated with the roots of Pinus sylvetris and Asclepias syriaca collected from Villerupt (France) and Bugac (Hungary), respectively.The fungi were maintained on a YM6.3 agar (D-glucose: 4 g L −1 ; malt extract: 10 g L −1 : yeast extract: 4 g L −1 ; agar: 20 g L −1 ; pH adjusted to 6.3 and sterilized by autoclaving) at 23 • C. To prepare seed cultures for the following larger-scale cultivation, five mycelia plugs measuring 25 mm 2 were transferred to a 500 mL Erlenmeyer shaking flask containing 200 mL of Q6/2 media (D-glucose: 2.5 g L −1 ; glycerine: 10 g L −1 ; cotton seed flour: 5 g L −1 ; pH: 7.2) and incubated at 23 • C and 140 min −1 .After achieving a sufficient amount of biomass, the culture broth was homogenized using Ultra-Turrax (T25 easy clean digital, IKA) equipped with an S25 N-25F dispersing tool at 10,000 rpm for 10 s.This seed culture was used for all following cultivations as an inoculum for YM6.3, BRFT, and WOFT media (K 2 HPO 4 : 0.5 g L −1 ; sodium tartrate: 0.5 g L −1 ; yeast extract: 1 g L −1 ; 100 mL of solution was added to 28 g brown rice or whole oat and autoclaved).

Solid State Fermentation
Six Erlenmeyer culture flasks with BRFT and WOFT were inoculated with 6 mL of homogenized seed culture, mixed under sterile conditions, and incubated at room temperature for two, three, and four weeks in the dark.After the planned incubation time, the cultures were stopped by adding 250 mL of acetone, mixed with a spatula, and kept in an ultrasonic bath for 30 min at 40 • C for extraction.The liquid was separated from the solid phase via filtration.The extraction was repeated twice using fresh acetone.The filtrate was evaporated under a vacuum at 40 • C in a rotary evaporator, and the remaining aqueous phase was filled up to 50 mL with distilled water and extracted with EtOAc (1:1) in a separatory funnel three times.The organic phase (EtOAc) was separated, evaporated under reduced pressure, and then dissolved again in 5 mL of MeOH and extracted again with 45 mL of heptane.Extraction was performed in a separatory funnel twice with fresh heptane.Both fractions (heptane and methanol) were evaporated to dryness.

Liquid Fermentation
In this study, 0.5% of the homogenized inoculum was transferred into twelve 2 L Erlenmeyer culture flasks containing 400 mL of YM6.3 medium.The content of glucose was monitored using test stripes (Medi-Test Glucose, Machery-Nagel ® , Düren, Germany).Incubation was terminated after five days of glucose depletion.Mycelia and supernatant were separated using a Büchner funnel and a vacuum pump.The extraction of metabolites of the mycelia followed the procedure of solid-state fermentation.The culture broth was mixed with 2% AmberLite TM XAD TM 16N polymeric absorbent and stirred for 4 h on a magnetic stirrer.The extraction of metabolites was carried out with acetone under stirring.Afterward, the purification scheme followed the same steps as described above for solid-state extraction.

Analytical HPLC
The extracts obtained were dissolved in Acetone:MeOH (1:1) and adjusted to a concentration of 4.5 mg mL −1 .An injection volume of 2 µL was applied to an UltiMate ® 3000 Series uHPLC (Thermo Fisher Scientific ® , Waltman, MA, USA).Mass spectrometry was performed with a connected amaZon ® speed ESI Iontrap MS (Amazon, Bruker).HRESIMS measurements were performed with sample concentrations of 1 mg mL −1 with an Agilent 1200 series HPLC-UV system in combination with an ESI-TOF-MS (Maxis, Bruker).The conditions were identical to the methods described before [26].

Isolation of Compounds
The mycelial extract of Polyphilus sieberi Ref052 (DSM 106515) cultivated in YM6.3 media (86 mg) was separated with a Reveleris ® X2 flash chromatography system using a FlashPure ID Silica 12 g cartridge.The mobile phase consisted of three solvent mixtures supplemented with 0.1% formic acid: solvent A (heptane 100%), solvent B (heptane 58%, TBME 40%, and MeOH 2%), and solvent C (Acetone 37.5%, DCM 37.5%, and MeOH 25%); flow rate: 30 mL min −1 ; gradient: 3 min B at 0%, increasing to 100% B in 10 min, maintaining 100% B for 5 min, switching to solvent mixture B and C, starting from 0% C and increasing to 100% C in 10 min, and maintaining 100% C for 10 min.All the following flash chromatographic separations were performed, implementing the same conditions.The separation led to the purification of 1 (1.1 mg; t R = 8.5 min).
The strains of the genus Polyphilus arose from an attempt to find nematode antagonistic fungi that can be used as ecologically friendly alternatives to toxic chemicals and soil fumigants.Such biocontrol agents may turn out to be a valid alternative, especially as they are supposed to be host-specific and do not kill harmless organisms in the soil.However,

Table 1 .
1H and 13 C NMR data of 1 and 2.
Measured in DMSO-d 6a at 500 MHz/ b at 125 MHz.c Assigned based on HMBC and HSQC spectra.

Table 1 .
1H and 13 C NMR data of 1 and 2