Omnipolyphilins A and B: Chlorinated Cyclotetrapeptides and Naphtho-α-pyranones from the Plant Nematode-Derived Fungus Polyphilus sieberi

Chemical exploration for two isolates of the recently described ascomycete species Polyphilussieberi, derived from the eggs of the plant parasitic nematode Heterodera filipjevi, afforded the identification of many compounds that belong to various metabolite families: two previously undescribed chlorinated cyclotetrapeptides, omnipolyphilins A (1) and B (2), one new pyranonaphthoquinone, ventiloquinone P (3), a 6,6′-binaphto-α-pyranone dimer, talaroderxine D (4) in addition to nine known metabolites (5–13) were isolated from this biocontrol candidate. All isolated compounds were characterized by comprehensive 1D, 2D NMR, and HR-ESI-MS analyses. The absolute configurations of the cyclotetrapeptides were determined by a combination of advanced Marfey’s method, ROE correlation aided by conformational analysis, and TDDFT-ECD calculations, while ECD calculations, Mosher’s method, and experimental ECD spectra were used for ventiloquinone P (3) and talaroderxine D (4). Among the isolated compounds, talaroderxine D (4) showed potent antimicrobial activities against Bacillus subtilis and Staphylococcus aureus with MIC values of 2.1 and 8.3 μg mL–1, respectively. Additionally, promising inhibitory effects on talaroderxine D (4) against the formation of S. aureus biofilms were observed up to a concentration of 0.25 μg mL–1. Moreover, ophiocordylongiiside A (10) showed activity against the free-living nematode Caenorhabditis elegans.


INTRODUCTION
Plant parasitic nematodes (PPNs) are known to reduce the yield and health of their host plants, making them agricultural pathogens of global importance.The annual cost is estimated to be around 80 billion USD. 1 Due to their complex biotrophic parasitism and multiple developmental stages, cyst-forming nematodes, such as Heterodera and Globodera spp., are among the most destructive plant parasitic nematodes. 2Based on their sedentary lifestyle, cyst nematodes are susceptible to fungal infections. 3Cyst parasitizing fungi were first described by Julius Kuḧn, who isolated Catenaria auxiliaris, first described as a Tarichium species, from the sugar beet cyst nematode Heterodera schachtii. 4Since then, a variety of different fungi associated with plant parasitic nematodes were found. 5They include but are not limited to the following: Ijuhya vitellina, 6 Niesslia gamsii, 7 Polydomus karssenii, 8,9 and Pochonia chlamydosporia. 10Studies of the secondary metabolites of these fungi showed that they can be prolific producers of biologically active secondary metabolites.As they are about to be studied for their potential as biocontrol agents, they must be checked carefully for production of potentially detrimental toxins before further development, such as a scale-up of production, formulation, and registration.For instance, Ijuhya vitellina produces cytotoxic cytochalasans of the chaetoglobosin type and the oligopeptides named leucinostatins with nematicidal activity against Caenorhabditis elegans. 6,11Arthrichitins A and B produced by P. karssenii showed moderate activity against the host cyst nematode Heterodera filipjevi. 8,9The present study deals with the secondary metabolism of two further, hitherto unexplored strains of the recently described species Polyphilus sieberi that were isolated from eggs of the cereal cyst nematode H. filipjevi collected from wheat fields in Turkey. 12Herein, we describe the purification and structure elucidation up to the absolute configurations of two previously undescribed chlorinated cyclotetrapeptides (1−2), a new pyranonaphthoquinone (3) and a new dimeric 6,6′-binaphtho-α-pyranone derivative (4) together with nine known metabolites, namely, talaroderxine C (5), 13 gliovirin-like compounds outovirins A (6) and C (7) 14 along with trichodermamide C (8), 15 peniciadametizine B (9), 16 ophiocordylongiiside A (10), 17 two fusidane triterpenes fusidic acid (11), 18,19 3-ketofusidic acid (12), 18,19 and lumichromone (13)  20 from different total extracts of the two nematode eggs-associated strains P. sieberi 17A and 17C.Selected compounds were subjected to antimicrobial, antibiofilm, cytotoxicity, and nematicidal activities, and herein we report their results.
2.9.Preparation of (R)-and (S)-MTPA Ester Derivatives of 4.An aliquot of compound 4 (0.4 mg) was dissolved in 220 μL of deuterated pyridine.Afterwards, 4 μL of (R)-(−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride were added to the solution and incubated for 6 h at 23 °C.After conversion into the corresponding (S)-Mosher ester, the sample was transferred to a 3.0-mm NMR tube and subjected to 1 H and 1 H− 1 H COSY NMR measurements (Figures S92−S97).The same procedure was followed using the (S)-(+)-αmethoxy-α-(trifluoromethyl)phenylacetyl chloride to produce (R)-Mosher ester.The Δδ SR values (Table S1) were calculated, and the configuration was assigned as previously described. 22.10.Maintaining and Synchronization of Caenorhabditis elegans Population.C. elegans was kept on nematode growth media (NGM).For the preparation of NGM plates, the additional ingredients were added when cooled down to 55 °C after autoclaving as previously described. 23As a food source for nematodes, Escherichia coli plated on NGM media was used.For the synchronization of the nematode population, a time with high egg density (∼120 h) was chosen to bleach them.NGM plates were washed three times with 4 mL of 0.9% NaCl solution and transferred to a 15 mL conical centrifuge tube.The nematode suspension was centrifuged at 1100 g for 2 min and the supernatant was discarded.The procedure was repeated several times until a clear nematode suspension was obtained.After the last washing step, 3 mL of the suspension was kept and mixed with 3 mL of bleaching solution (1 mL of NaClO, 1 mL of 5 M NaOH, 6.25 mL of ultrapure H 2 O).Digestion of C. elegans was controlled under the microscope and stopped after disappearance of hatched nematodes.The bleaching was stopped by adding 0.9% NaCl solution, centrifuged, and washed as mentioned above.After the last washing step, the supernatant was removed and replaced by 7 mL of 0.9% NaCl solution.The tube was incubated at 23 °C on a rotary shaker with 80 rpm to hatch the eggs.The hatched nematodes were transferred to fresh NGM plates with E. coli lawn after 18 h.After 50− 70 h, the J4 and adult C. elegans were washed from the plate, centrifuged, and washed as mentioned above.The amount of nematodes was determined and diluted to the desired concentration.
2.11.Biological Assays.Isolated natural products were assessed for their effects on different organisms and cell lines including Grampositive, Gram-negative bacteria, filamentous fungi, yeasts, Caenorhabditis elegans, Staphylococcus aureus biofilm formation, and mammalian cell lines (Tables S2−S5).The effect on microbial growth was assessed by performing a serial dilution assay, which resulted in the determination of their minimum inhibitory concentration (MIC).A detailed description of the antimicrobial and cytotoxicity assays can be found in the Supporting Information.
The cytotoxic effect of isolated compounds was tested against the cell lines KB3.1 (human endocervical adenocarcinoma), L929 (mouse fibroblasts), A431 (human epidermoid carcinoma), A549 (human lung carcinoma), PC-3 (human prostate adenocarcinoma), and MCF-7 (human breast adenocarcinoma).The nematicidal activity of the isolated compounds (Table S4) was evaluated against C. elegans in a 48-well flat-bottom plate.The methanol solutions of the tested compounds were filled in the wells, and subsequently, the solvent was evaporated and 300 μL of 0.9% NaCl solution with 1000 J4 and adult nematodes per mL was added per well.After 15 min, the wells were checked for immediate effects.Finally, the mortality rate was evaluated after 18 h of incubation on a plate shaker with 150 rpm at 23 °C.Biofilm inhibition activities of omnipolyphilin A (1) and talaroderxines C (5) and D (4) were evaluated against Staphylococcus aureus (DSM 1104) according to a previously described protocol. 24riefly, a preculture of S. aureus (DSM 1104) was made from stock kept at −20 °C.After 18 h incubation, a media suspension for biofilm assay was made and OD 600 of culture was adjusted to 0.001 McFarland standard.S. aureus was then incubated along with the serially diluted compounds (omnipolyphilin A (1): 125−0.9μg mL −1 ; talaroderxine D (4): 33−0.001μg mL −1 ; talaroderxine C (5): 125− 0.003 μg mL −1 ) to produce biofilms in 96-well microtiter plates for 24 h.The biofilm inhibition activity of compounds 1, 4, and 5 was assessed by crystal violet staining with subsequent washing steps and analyzing the stained biomass with a microtiter plate reader (Synergy

Journal of Agricultural and Food Chemistry
2, BioTek).Methanol was used as a solvent control and taken as 100% of no inhibition against biofilm formation of S. aureus.Microporenic acid A (MAA) was used as a positive control. 24,25All experiments were performed twice with duplicates.Differences between samples and control group were determined by two-tailed Student's t-test.Statistical significance was defined as p < 0.05.Analysis was carried out using Excel 2016 (Microsoft).2.12.Computational Section.Mixed torsional/low-mode conformational searches were carried out by means of the Macromodel 10.8.011 software using the MMFF with an implicit solvent model for CHCl 3 applying a 21 kJ mol −1 energy window. 26eometry reoptimizations of the resultant conformers [ωB97X/ TZVP with the PCM solvent model for MeCN or MeOH] and TDDFT-ECD calculations were performed with Gaussian 16. 27 For TDDFT-ECD, various functionals (B3LYP, BH&HLYP, CAM-B3LYP, PBE0) and the TZVP basis set were used with the same solvent model as in the preceding DFT optimization step.The sTDA spectra were calculated with the CAM-B3LYP, LC-BLYP, and ωB97X functionals and the sTDA 1.6 package. 28ECD spectra were generated as the sum of Gaussians with 2400−2700 cm −1 half-height widths, using dipole-velocity computed rotational strengths. 29Boltzmann distributions were estimated from the ωB97X energies.The MOLEKEL program was used for visualization of the results. 30

Journal of Agricultural and Food Chemistry
presumably incorporates two identical substituents at 6″,8″positions on its aromatic ring imparting a deshielding effect on their carbon atoms that appeared at δ C 130.0 instead of δ C 116.0 ppm in Tyr 1 and Tyr 2 .These findings suggested that the third tyrosine moiety is probably seen as 6,8-dichlorotyrosine residue (diClTyr).In addition, the 13 C NMR spectrum of 1 disclosed further unassigned carbon resonances together with the insufficient degrees of unsaturation (15 from three Tyr residues) provided some clues for the existence of another highly conjugated functionality in 1 that comprises the 1,2disubstituted aromatic ring.To elucidate its structure, the HMBC spectrum of 1 was measured revealing key correlations that helped together with the 1 H− 1 H COSY spectrum (Figure 2) to elucidate 3a-hydroxypyrroloindolinone (HPI) moiety fulfilling the lacking seven degrees of unsaturation and characterizing 1 as a cyclotetrapeptide comprising two tyrosines (Tyr 1 and Tyr 2 ), one dichlorotyrosine (diClTyr), and 3a-hydroxypyrroloindolinone (HPI) residues.The HMBC spectrum of 1 (Figure 2) has also defined the amino acid sequence through the key correlations from the α-proton of Tyr 1 at δ H 4.43 (dd, J = 9.8, 7.0 Hz, H-2′) to the adjacent carbonyl carbon of HPI at δ C 174.3 (C-1) and to its own carbonyl carbon (δ C 175.0, C-1′).The α-proton of dichlorotyrosine moiety at δ H 4.54 (dd, J = 13.7,7.6 Hz, H-2″) in turn revealed key correlations to its own carbonyl carbon (δ C 173.6, C-1″) in common with the second tyrosine amino acid α-proton at δ H 5.02 (dd, J = 8.8, 6.6 Hz, H-2‴).Finally, H-2‴ showed key correlations in the HMBC spectrum to its own carbonyl carbon at δ C 175.4 (C-1‴).These key correlations suggested the cyclotetrapeptide to be HPI-Tyr 1 -diClTyr-Tyr 2 , as depicted in Figure 1.Further confirmation for the depicted amino acid arrangement in 1 was provided by ROESY spectral analysis (Figure 2) that unveiled key ROE correlations from NH protons of the amino acid residues, Tyr 1 at δ H 7.07 (d, J = 8.3 Hz) and diClTyr at δ H 7.38 (d, J = 9.6 Hz) to H-2″ that in turn revealed key ROE correlations with NH proton of Tyr 2 amino acid residue at δ H 7.14 (d, J = 8.4 Hz).The H-8a in HPI at δ H 5.85 (s) revealed key ROE correlations to H-2‴ of Tyr 2 at δ H 5.02 (dd, J = 8.8, 6.6 Hz) and the OH-3a proton at δ H 6.31 (br s) indicating cofacial orientation of H-2‴, H-8a and OH-3a.As a conclusion, the obtained results indicated the amino acid sequence in 1 to be HPI-Tyr 1 -diClTyr-Tyr 2 .A literature search on cyclic peptides featuring the HPI moiety revealed the plant-derived cyclopentapeptide melicoptelines D and E comprising HPI residues with (2S,3aS,8aR) and (2S,3aR,8aS) absolute configurations, respectively, that were recently reported as potent antiviral derivatives against influenza A virus. 31 The HPI moiety can be only produced with cis ring junction to afford either (3aR,8aS) or (3aS,8aR) absolute configuration. 32By comparing 1 H and 13 C NMR data of HPI in 1 to those reported for melicoptelines D and E, 31 it could be deduced that HPI moiety in 1 adopts the cis-(3aR*,8aS*) relative configuration.The H-8a/OH-3a ROE correlation of 1 observed in DMSO-d 6 (Figures 2 and  S8a) confirmed their cis orientation, while ROE correlation of H-8a and the NH proton of the C-2 carboxamide group suggested the (2S*,3aR*,8aS*) relative configuration.In addition, four stereoisomers of the HPI residue including the (2S,3aR,8aS) and (2S,3aS,8aR) ones were previously synthesized and their chemical shifts of H-2 and H-8a revealed clear differences appearing at δ H 3.86 (q, J = 12.0, 7.0 Hz) and 5.40 (s) rather than at δ H 4.34 (t, J = 7.0 Hz) and 5.31 (s), respectively. 33Reported ECD spectra of the four synthetic stereoisomeric HPI revealed that the configuration of the chiral carbon atoms C-3a and C-8a of the cis-annulated 2,3-dihydro-1H-indole governs the ECD spectrum. 33Stereoisomers of HPI with (3aR,8aS) absolute configuration showed two negative Cotton effects (CEs) at about 300 and 240 nm deriving from the π−π* transitions of the substituted benzene chromophore.The ECD spectra of compound 1 in acetonitrile and methanol (Figure S9) also showed two negative transitions in that region, on the basis of which (3aR,8aS) absolute configuration was assigned to the HPI residue.Considering the known relative configuration, this allowed determining (2S,3aR,8aS) absolute configuration for HPI residue in 1, implying that it was derived from L-tryptophan amino acid.The absolute configuration of α-carbon atoms for each amino acid was unambiguously determined using the advanced Marfey's method (see Supporting Information Figures S83 and  S84). 21Based on the comparison of retention times between authentic amino acid standards and the hydrolyte of 1, the results disclosed that the dichlorotyrosine residue had Lconfiguration and there were one D-and one L-tyrosine amino acid residues that could be distinguished on the basis of ROE correlations between the α-proton of Tyr 2 and H-8a suggesting (L-Tyr 1 ,D-Tyr 2 ) assignment.In order to confirm the proposed positions of L-and D-tyrosine, the characteristic ROE correlation between H-8a and H-2‴ of Tyr 2 was checked on the computed low-energy MMFF and DFT-optimized conformers of (L-Tyr 1 ,D-Tyr 2 )-1, in which the two protons were found to be on the same face and their interatomic distance was 2.20 Å (Figure S98a).Moreover, the low-energy MMFF conformers of the diastereomeric (D-Tyr 1 ,L-Tyr 2 )-1 were also computed, in which the H-8a and H-2‴ of Tyr 2 are facing the opposite direction with a distance of 4.12 Å (Figure S98b), which would not enable the characteristic ROE correlation.In

Journal of Agricultural and Food Chemistry
order to confirm further the configurational assignment of the HPI, L-Tyr 1 , and D-Tyr 2 residues, TDDFT-ECD spectrum of 1 was computed with different methods.Since the initial conformational search resulted in a large number of conformer clusters in the applied 21 kJ mol −1 energy window, the 959 MMFF conformers of 1 (Figure S99) were optimized first at the B3LYP/6-31G(d) PCM/MeCN level, reclustered and the resulting 219 conformers were reoptimized at the ωB97X/ TZVP PCM/MeCN level.In the 13 low-energy ωB97X/ TZVP PCM/MeCN conformers, the characteristic H-8a and H-2‴ of Tyr 2 had the same orientation as those in the lowestenergy MMFF conformer (Figure S99).
TDDFT-ECD 34 and sTDA-ECD 35 calculations were performed on the 13 low-energy (≥1% Boltzmann population) conformers, which gave good agreements with the experimental ECD spectrum allowing verification of the absolute configuration (Figures 3 and S100).Since there are only a few examples of sTDA calculations on natural products, 36 this may serve as a further example of the successful application of a natural cyclic peptide derivative.Based on the obtained results, compound 1 was unambiguously confirmed to represent a previously undescribed dichlorinated cyclotetrapeptide with the amino acid sequence of L-Tyr 1 -L-diClTyr-D-Tyr 2 -HPI, and it was given a trivial name omnipolyphilin A.
Compound 2 was obtained as a colorless amorphous solid that revealed a characteristic pattern of a halogenated compound in its LR-ESI-MS with a pseudomolecular ion peak cluster of m/z 726.  1) and HSQC spectrum (Figure S15) of 2 revealed a close similarity to 1 with one major difference is the presence of an additional aromatic proton on the chlorinated tyrosine residue and hence turned it into 1,3,4-trisubstituted aromatic ring bearing three protons at δ H 6.81 (d, J = 8.3 Hz, H-6″; δ C 117.7), δ H 6.96 (dd, J = 8.3, 2.4 Hz, H-5″; δ C 129.5), and δ H 7.20 (d, J = 2.4 Hz, H-9″; δ C 131.5).Apart from that difference, compound 2 revealed a close resemblance to 1 in both its 1D ( 1 H/ 13 C NMR) and 2D ( 1 H− 1 H COSY, HMBC, HSQC, and ROESY) spectral data (Table 1, Figure 2) that suggested compound 2 to comprise three amino acids recognized into two tyrosines (Tyr 1 /Tyr 2 ) and one chlorotyrosine (ClTyr) along with an HPI moiety.−33 Based on the close coherence between 1 and 2 in their depicted structures, spectral data, and biosynthetic origin, the absolute configuration of the three amino acids in 2 were concluded to be identical to those in 1 namely, L-Tyr 1 -L-ClTyr-D-Tyr 2 -HPI, and it was identified as a previously undescribed monochlorinated cyclotetrapeptide that was trivially named as omnipolyphilin B.
Compound 3 was purified as an orange solid powder whose molecular formula was determined to be C 18 H 18 O 6 based on the HR-ESI-MS spectrum that revealed a pseudomolecular ion peak at m/z 331.1173 [M + H] + (calculated 331.1176) and a sodium adduct at m/z 353.0993 [M + Na] + (calculated 353.0996) indicating the presence of ten degrees of unsaturation.The 13 C NMR and HSQC spectra of 3 (Table 2) revealed the presence of 18 different carbon resonances that can be differentiated into nine quaternary carbon atoms including three carbonyl carbons (δ C 190.0, 181.0, and 167.9), two oxygenated olefinic carbon atoms (δ C 161.8 and 160.7) and four olefinic carbon atoms (δ C 148.4, 133.6, 117.8, and 114.0) in addition to three tertiary carbon atoms (δ C 116.3, 110.3, and 77.3), five secondary carbon atoms (δ C 33.8, 33.6, 31.0,24.0, and 22.0), and one primary carbon (δ C 13.9).The 1 H NMR, 1 H− 1 H COSY, and HSQC spectra of 3 (Table 2, Figure 4) revealed the presence an extended spin system beginning at two diastereotopic methylene protons (H 2 -4) at δ H 2.96 (dd, J = 16.8, 11.4 Hz) and δ H 3.17 (dd, J = 16.8, 2.8 Hz) both directly correlated to a secondary sp 3   13.9)indicating the presence of npentyl aliphatic side chain as a substituent on a 6-membered lactone ring.According to the obtained results, compound 3 was deduced to have a naphthoquinone moiety in its structure supported by the key HMBC correlations (Figure 4) from aromatic protons H-5 at δ H 7.42 (s; δ C 116.3) and H-8 at δ H 6.06 (d, J = 3.3 Hz; δ C 110.3) to two naphthoquinone carbonyl at δ C 190.0 (C-9) and δ C 181.0 (C-6).−39  Further confirmation of the suggested structure of compound 3 (Figure 1) was provided by its HMBC spectral results (Figure 4) that revealed key correlations from H-3 to C-4a (δ C 148.4) and C-12 (δ C 24.0) that confirmed the n-pentyl to be present as a substituent at C-3 on an α-pyranone ring that is fused to a naphthoquinone moiety.
Compound 3, containing a hydroxylated 1,4-naphthoquinone subunit, is the oxidized analogue of the related semitalaroderxine C, 13 which has a trihydroxynaphthalene moiety.Based on the structural similarity and the common biosynthetic origin between 3 and (R)-semitalaroderxine C, recently reported from different strains of the genus Polyphilus, the absolute configuration of C-3 was suggested to be (R). 13he absolute configuration of 3 was determined to be (R) based on the negative and positive CEs observed in the ECD spectrum (Figure S85) at 301 and 266 nm, which were in accordance with those revealed by the structurally related derivative, (R)-semixanthomegnin at 305 and 272 nm, respectively. 40n order to confirm the configurational assignment independently, TDDFT-ECD calculations were performed on the 30 low-energy ωB97X/TZVP PCM/MeOH conformers of (R)-3 (Figure S101) obtained by the reoptimization of the 205 initial MMFF conformers.The Boltzmann-weighted CAM-B3LYP/TZVP PCM/MeOH ECD spectrum of (R)-3 gave a good agreement with the experimental ECD spectrum of 3 (Figure 5) confirming our assignment.Based on the obtained results, compound 3 was determined to be a new natural compound, and it was named ventiloquinone P.
By comparing the spectral data of 4 and the reported literature, it was obvious to be structurally comprising 6,6′binaphtho-α-pyranone skeleton similar to that found in talaroderxines A-C, 13,41 pigmentosins, 24,42 and asteromine. 43he closest similarity in spectral data was found between 4 and talaroderxine C (5) 13 where the only difference was the presence of a sec-hydroxyl group at C-14 in 4 rather than being a methylene group in 5.
The 6,6′-linked biaryl-type heterodimer 4 had three isolated blocks of chirality, which could not be correlated by experimental NMR methods: (i) axial chirality due to the stereogenic ortho-tetrasubstituted 6,6′-biaryl axis, (ii) C-3 chirality centers of the naphthopyrone subunits, and (iii) C-14 chirality center of the flexible 4-hydroxypent-1-yl side chain.−47 The sign and magnitude of the biaryl dihedral angle of the two aryl units in axially chiral biaryls are reflected in the interaction of the two aromatic chromophores, giving rise to exciton-coupled ECD bands.The signs of the exciton-coupled ECD couplets (Figure S86) are characteristic of the axial chirality as exemplified by the related 6,6′-linked bis-naphthopyrones pigmentosins A and B 24 and talaroderxine A. 41 Due to the intense positive exciton couplet of 4 centered at 260 nm, the axial chirality was determined as (aS).On the basis of common biosynthetic origin with the related monomeric units semitalaroderxine C 13 and ventiloquinone P, (3R,3′R) absolute configuration was assigned to the naphthopyrone subunits.The absolute configuration of the secondary 14-OH group of the side chain was established by implementing the Mosher's method, which implied the synthesis of (S)-and (R)-MTPA esters using (R)-and (S)-MTPA acid chloride reagents, respectively. 22The measured consistent chemical shift differences (Figure 6, Table S1) exhibited positive Δδ SR values for H 2 -13, whereas negative one was recorded for H 3 -15 that determined the absolute configuration of 4 as (14R).Based on the obtained results, compound 4 was identified as a new 6,6′-binaphtho-αpyranone derivative, (14R)-OH-talaroderxine C, that was given a trivial name talaroderxine D.
Based on the isolated amounts, compounds 1, 3-5, 8, 10, and 11 were evaluated for their cytotoxic activity against a panel of six different cell lines (Table S2), antimicrobial and nematicidal activities against twelve microorganisms consisting of Gram-positive, Gram-negative bacteria, fungi (Table S3), and C. elegans (Table S4), respectively.Additionally, omnipolyphilin A (1) and talaroderxines D (4) and C (5) were evaluated for their inhibitory effects on S. aureus biofilm formation.
Furthermore, talaroderxines C (5) and D (4) significantly inhibited the formation of S. aureus biofilms up to 70% by concentrations of 3.9 μg mL −1 and 0.25 μg mL −1 , respectively.However, omnipolyphilin A (1) was slightly active against S. aureus biofilms with significant inhibitory effects of 30% at a concentration of 62.5 μg mL −1 compared to microporenic acid that inhibited biofilm formation by >50% at 7.8 μg mL −1 .In nematicidal activity assay, only ophiocordylongiiside A (10)  showed significant effects on C. elegans with a mortality rate of 69% at 100 μg mL −1 .No significant effect against tested cell lines or microorganisms was observed for compounds 3, 8, and 10.
In the course of our ongoing search for anti-infectives, this study introduces a follow-up exploration of secondary metabolites from the fungal genus Polyphilus.In conclusion, thirteen metabolites were isolated from P. sieberi including two new cyclotetrapeptides, omnipolyphilins A (1) and B (2), a naphthoquinopyranone, ventiloquinone P (3), two naptho-αpyranone dimers, talaroderxines D (4) and C (5), 13 together with other known metabolites, outovirins A (6) and C (7), 14 trichodermamide C (8), 15 peniciadametizine B (9), 16 ophiocordylongiiside A (10), 17 two fusidane triterpenes, fusidic acid (11), 18,19 3-ketofusidic acid (12), 18,19 and lumichromone (13). 20The isolated talaroderxines C (5) and D (4) exhibited significant inhibitory effects on the formation of S. aureus biofilms far below their MIC values (Table S5, Figures S88-S89).The same properties were also described for the structure-related pigmentosins A and B. 24 In comparison to the latter, talaroderxines C (5) and D (4) exhibited not only pronounced effects on S. aureus biofilm formation but also stronger cytotoxicity on the tested cell lines.However, talaroderxine D (4) showed a 7-fold higher biofilm inhibitory effect compared to its highest cytotoxicity.Future studies may attempt to reduce the cytotoxicity of these natural products while retaining or increasing their potency against biofilms.Ophiocordylongiiside A (10) was active against C. elegans and should also be tested in the future against plant pathogenic nematodes to establish a possible ecological role.Furthermore, the effects of P. sieberi on the actual infestation of H. filipjevi in agricultural crops could be evaluated in greenhouse experiments.Within these investigations, studies of the production of additional mycotoxins in the natural environment as well as their ecological impact should be conducted.For this purpose, it might even be feasible to use modern metabolomics techniques as recently employed for a strain of the basidiomycete Armillaria. 48Polyphilus spp., however, should perhaps not be further pursued with priority for biocontrol purposes because several of the metabolites we have encountered may be potentially harmful for beneficial organisms, and even the production of the marketed antibiotic drugs of the fusidic acid type, which were finally identified as major antibacterial principles, may pose a risk that can lead to antimicrobial resistance against the antibiotic among soil bacteria.
On the other hand, this study also shows the huge potential of the genus Polyphilus to produce a variety of different natural product classes.
28 and 728.24 [M + H] + in relative abundance of 3:1 suggesting the compound to possess one chlorine atom in its structure that explains its smaller molecular weight by 35 atomic units compared to 1.The HR-ESI-MS spectrum of 2 confirmed this notion by establishing its molecular formula as C 38 H 36 ClN 5 O 8 based on its pseudomolecular ion peaks at m/z 726.2310 [M + H] + (calculated 726.2325) and m/z 748.2133 [M + Na] + (calculated 748.2145) indicating the existence of twenty-three degrees of unsaturation equal to those in 1.The 1D ( 1 H and 13 C) NMR data (Table

Table 1 .
1H and 13 C NMR Data of 1 and 2 a Measured in methanol-d 4 : a At 125 MHz.b At 500 MHz.c At 700 MHz.d Assignment confirmed by HMBC and HSQC spectra.e Measured in DMSO-d 6 at 500 MHz.

Table 2 .
1 H and 13 C NMR Data of 3 and 4 Measured in DMSO-d 6 at 175 MHz b at 700 MHz.c Assignment confirmed by HMBC and HSQC spectra. a