Cordypyridones E−J: Antibiofilm 2‑Pyridone Alkaloids from the Nematode Antagonistic Fungus Laburnicola nematophila

Cordypyridones E−J: Antibiofilm 2‑Pyridone Alkaloids from the Nematode Antagonistic Fungus Laburnicola nematophila Jan-Peer Wennrich, Caren Holzenkamp, Sara Fushimi, Mahmoud A. A. Ibrahim, Samad Ashrafi, Wolfgang Maier, Hedda Schrey, Sherif S. Ebada,*and Marc Stadler* Cite This: https://doi.org/10.1021/acs.jnatprod.5c00768 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: In the course of biochemical prospection of Laburnicola nematophila isolated from eggs of the plant-parasitic cyst nematode Heterodera filipjevi, eight 2-pyridone alkaloids were isolated from its solid-state BRFT cultures and identified as six previously undescribed, cordypyridones E−J (1−6), and two known congeners, cordypyridones C (7) and D (8). The structures and absolute configurations of all isolated compounds were elucidated through HR-ESI−MS, 1D/2D NMR spectroscopy, and TDDFT−ECD calculations. Compound 4, bearing a rare Nhydroxy-2-pyridone moiety structurally related to that of PF1140, exhibited broad-spectrum bioactivity in cytotoxicity and antimicrobial assays. Compounds 5and 6demonstrated potent antibiofilm activities against Staphylococcus aureus, reducing its biofilm formation by almost 50% at 0.25 and 7.8 μg/mL, respectively. Fungi are an established source of structurally diverse and biologically active natural products, many of which have found applications as pharmaceuticals, agrochemicals, or research tools. 1 Despite decades of intensive research, fungal secondary metabolism continues to yield novel scaffolds and bioactivities. 2 Continuous exploration of underexplored fungal niches remains a key strategy for the discovery of novel secondary metabolites. 1,3 Among these, cyst nematodes provide a promising source for isolating their associated fungi many of which produced a diverse array of bioactive compounds. 4−8 These fungi live in close association with nematodes, which are among the most abundant and ecologically significant invertebrates. 9 The interaction between fungi and nematodes has likely driven the evolution of specialized metabolites with roles in chemical defense, parasitism, or interspecies communication. 4 Concurrently, exploring new biofilm inhibitors has become a priority in antimicrobial research. 10 Biofilms, structured microbial communities encased in an extracellular matrix, confer enhanced tolerance to antibiotics/host defenses and are a major factor in chronic infections and medical-deviceassociated complications. 9,11,12 The development of compounds that can inhibit biofilm formation or disrupt mature biofilms, ideally without promoting resistance or causing cytotoxicity, is urgently needed. Natural products offer an attractive starting point for such agents, given their structural diversity and evolutionary optimization for biological activity. In this context, we investigated the secondary metabolome of Laburnicola nematophila, a fungus derived from infected cysts of the plant-parasitic nematode Heterodera filipjevi, 13 which was reported as the producer of diverse classes of bioactive metabolites including the potent antifungal polyalcohol α-pyrone dactylfungins, tetralones and peptides. 5−7 In this study, the chemical investigation led to the identification of six previously undescribed pyridone alkaloids (1−6) and the known compounds cordypyridones C (7) and D (8). 14 Their structures were determined by a combination of HR-ESI−MS, NMR spectroscopy, and TDDFT−ECD calculations. The antimicrobial, cytotoxic, nematicidal, and biofilm inhibitory properties of the isolated compounds were assessed. This study describes the chromatographic separation, comprehensive structure elucidation, and biological evaluation of the isolated natural products. Received: June 24, 2025 Revised: August 26, 2025 Accepted: September 9, 2025 Articlepubs.acs.org/jnp © XXXX The Authors. Published by American Chemical Society and American Society of Pharmacognosy A https://doi.org/10.1021/acs.jnatprod.5c00768 J. Nat. Prod. XXXX, XXX, XXX−XXX This article is licensed under CC-BY 4.0 Downloaded via 91.60.222.145 on October 14, 2025 at 16:00:19 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. Table 1. 1H and 13C NMR Data of 1−4 1 2 3 4 position δC, a type δH a multi [J(Hz)] δC, a type δH a multi [J(Hz)] δC, b type δH b multi [J(Hz)] δC, a type δH a multi [J(Hz)] 2 159.7, CO 160.0, CO 164.6, CO 159.3, CO 3 110.8, C 110.8, C 109.3, C 109.5, C 4 165.7, C 165.7, C 167.7, C 164.5, C 5 100.6, CH 5.89 d (7.7) 100.9, CH 5.91 d (7.7) 101.7, CH 5.90 d (7.0) 99.1, CH 5.89 d (7.5) 6 134.6, CH 7.63 dd (7.7, 0.8) 134.9, CH 7.65 dd (7.7, 0.8) 133.6, CH 7.14 dd (7.0) 133.1, CH 7.62 d (7.5) 7 50.8, CH 2.05 d (11.5) 51.2, CH 2.07 d (11.4) 50.2, CH 2.02 d (11.5) 50.4, CH 2.03 d (11.3) 8 37.0, C 37.2, C 36.9, C 37.3, C 9 47.8, CH2α1.09 d (13.2) 41.1, CH2α0.71 m (overlapped) 47.7, CH2α1.09 d (13.7) 46.5, CH2α0.70 t (13.1) β1.74 dd (13.2, 2.8) β1.76 dt (12.0, 2.5) β1.74 dd (13.7, 2.8) β1.62 m (overlapped) 10 69.7, C 35.3, CH 1.70 dqd (12.4, 6.1, 3.1) 69.6, C 27.0, CH 1.64 m (overlapped) 11 49.0, CH2α1.04 dd (14.0, 1.9) 40.3, CH2α0.65 m (overlapped) 49.0, CH2α1.04 dd (14.1, 12.2) 46.1, CH2α0.63 dd (13.1) β1.85 ddd (14.0, 4.3, 2.8) β1.91 ddt (13.2, 4.8, 2.9) β1.84 ddd (14.0, 4.3, 2.8) β1.84 ddt (13.1, 4.9, 2.9) 12 25.2, CH 2.84 dqd (12.0, 6.0, 4.3) 28.9, CH 2.61 dqd (12.0, 5.9, 4.8) 25.0, CH 2.79 dqd (11.9, 5.9, 4.3) 28.9, CH 2.56 tq (11.3, 5.7) 13 88.3, CH 4.10 q (6.5) 87.7, CH 4.16 q (6.5) 88.0, CH 4.09 q (6.5) 87.3, CH 4.13 q (6.5) 14 15.8, CH31.19 d (6.5) 16.0, CH31.23 d (6.5) 15.8, CH31.19 d (6.5) 15.7, CH31.21 d (6.5) 15 16.4, CH30.82 s 14.9, CH30.69 s 16.2, CH30.83 s 14.7, CH30.66 s 16 32.7, CH31.21 s 68.6, CH2α3.34 dd (10.6, 4.1) 32.7, CH31.20 s 22.9, CH30.91 d (6.4) β3.40 dd (10.6, 6.0) 17 24.9, CH31.15 d (6.0) 25.6, CH31.16 d (5.8) 24.7, CH31.14 d (5.9) 25.3, CH31.15 d (5.7) 18 65.0, CH33.93 s 65.2, CH33.93 s a Measured in methanol-d4at 500 MHz for 1H and 125 MHz for 13C. b Measured in methanol-d4at 700 MHz for 1H and 175 MHz for 13C. Journal of Natural Products pubs.acs.org/jnp Article https://doi.org/10.1021/acs.jnatprod.5c00768 J. Nat. Prod. XXXX, XXX, XXX−XXX B ■RESULTS AND DISCUSSION Isolation and Identification of Compounds 1−8. Compound 1was obtained as a yellow amorphous solid. Its molecular formula was established as C17H25NO4based on HR-ESI−MS (Figure S1), which showed a protonated molecular ion peak at m/z308.1857 [M + H]+(calculated 308.1856), indicating six degrees of unsaturation. The 13C NMR and HSQC spectral data of 1(Table 1 and Figures S4 and S7) revealed 17 carbon signals, categorized into five unprotonated, including a carbonyl carbon, and five methines accounting for three degrees of unsaturation together with two methylenes and five methyls including a methoxy group. Based on the 13C NMR spectral data analysis, compound 1was deduced to comprise a tricyclic structure. The 1H NMR and 1H−1H COSY spectra of 1(Table 1,Figure 1, and Figures S3 and S5) revealed the presence of three spin systems corresponding to those revealed by the known compound cordypyridone C (7). 12 A literature search of 1revealed its structural resemblance to cordypyridones A−D, N-hydroxyand N-methoxy-2-pyridone alkaloids previously reported from the entomopathogenic fungus Pleurocordyceps nipponica. 14 The structure of 1appeared to be different from cordypyridone C (7) only in the presence of a tertiary alcohol moiety at C-10. Its HMBC spectrum was acquired and the obtained results (Figure 1 and Figure S6) revealed key correlations that confirmed the depicted structure of 1. The relative configuration of 1was determined via its ROESY spectrum (Figure 2 and Figure S8) that revealed key ROE correlations between H3-17/H-7/H-13/Hα-9/Hα-11 indicating their projection toward the same face of the molecule whereas key ROE correlations were noted between H-12/H3-15/H3-14/Hβ-9/ Hβ-11 indicating that they are facing the opposite side of the molecule. The absolute configuration of 1was determined based on the similarity between its measured and calculated TDDFT−ECD spectra (Figure 3). As can be seen from Figure 3, a close coherence was observed between the experimental ECD spectrum and that predicted for the (7R,8R,10S,12S,13S) configuration over the whole range. Based on the obtained results, compound 1was identified as a previously undescribed N-methoxy-2-pyridone alkaloid named cordypyridone E. Compound 2was purified as a yellow amorphous solid whose HR-ESI−MS spectrum (Figure S10) determined its molecular formula as C17H25NO4by revealing a protonated molecular ion peak at m/z308.1855 [M + H]+(calculated 308.1856) indicating six degrees of unsaturation similar to 1. The 13C and 1H NMR spectral data of 2(Table 1) revealed an obvious resemblance to 1despite having clearly different retention times in their HPLC chromatograms (Figures S1 and S2 for 1and Figures S9 and S10 for 2). A comparison of the 1H/13C NMR spectral data of 1and 2(Table 1) revealed the replacement of a nonprotonated sp3carbon at δC69.7 (C-10) and a singlet methyl group at δH1.21 (H3-16; δC32.7) in 1by an aliphatic methine at δH1.70 (dqd, J= 12.4, 6.1, 3.1 Hz, H10; δC35.3) coupled to a diastereotopic oxymethylene group at δH3.34/3.40 (H2-16; δC68.6). Apart from these differences, the 1H/13C NMR spectral data of 1and 2(Table 1) are quite comparable. To confirm the depicted structure of 2, its 1H−1H COSY and HMBC data (Figure 1 and Figures S13 and S14) were acquired confirming the connection of CH2−16 and CH10. The relative configuration of 2was deduced through its ROESY spectrum (Figure 2 and Figure S16) that revealed key ROE correlations from H-12 to H-10 and H3-15, suggesting their projection toward the same face of the molecule while H7 was correlated to H-13 and H3-17 indicating that they are facing the opposite side of the molecule. Regarding the absolute configuration of 2, a good fit was observed between the experimental ECD and the calculated TDDFT−ECD for the (7R,8R,10R,12S,13S) configuration (Figure 4). Accordingly, compound 2was deduced to be a previously undescribed N-methoxy-2-pyridone alkaloid that was given the trivial name cordypyridone F. Compound 3was isolated as a yellow amorphous solid with its HR-ESI−MS spectrum (Figure S18), determining its molecular formula as C16H23NO3by revealing a protonated molecular ion peak at m/z278.1748 [M + H]+(calculated 278.1751) and thus indicating six degrees of unsaturation equal to those in 1and 2. By comparing the molecular formulas of 1−3, the latter was found to lack the CH2O moiety, accounting for its smaller molecular weight. By comparing the 13C, 1H, and 2D NMR spectral data of 1−3(Table 1,Figures 1 and 2, and Figures S20−S23), it was clearly observed that compound 3is closely similar to 1, lacking one methoxy group bound to the nitrogen in 1. The absolute configuration of 3was confirmed by comparing its experimental and calculated ECD spectra (Figure 5), revealing a similar pattern with the conformer featuring the (7R,8R,10S,12S,13S) configuration. Therefore, compound 3was identified as shown and trivially named cordypyridone G. Compound 4was obtained as a yellow amorphous solid with its molecular formula established as C16H23NO3by revealing a protonated molecular ion peak at m/z278.1751 [M + H]+ (calculated 278.1751) and thus indicating six degrees of unsaturation as 3. By comparing the 13C and 1H NMR spectral Figure 1. Key 1H−1H COSY and HMBC correlations of 1−6. Journal of Natural Products pubs.acs.org/jnp Article https://doi.org/10.1021/acs.jnatprod.5c00768 J. Nat. Prod. XXXX, XXX, XXX−XXX C data of 2and 4(Table 1), it was clear that the oxymethylene moiety in 2is replaced by a doublet methyl group in 4. In addition, a second difference between 2and 4was noticed, namely the absence of the methoxy group. Based on the obtained results and a literature search of 4, it was suggested to be a demethylated derivative of cordypyridone C (7), first reported from the insect pathogenic fungus P. nipponica, 14 fusaricide, a reported cytotoxic metabolite from Fusarium sp., 15 and asperpyridone A from an endophytic Aspergillus sp. 16 The structure of 4was ascertained through comprehensive 2D NMR spectral analyses (Figures 1 and 2and Figures S27−S30) and comparison with the reported literature, 16 which revealed it as an analogue of 7with a hydroxyl replacing the methoxy group at the nitrogen. The relative configuration of 4was identified through its ROESY spectrum (Figure 2 and Figure S30) that revealed comparable key ROE correlations to those of compounds 1−3(Figure 2). The absolute configuration of 4 was determined based on the comparison between its experimental and calculated TDDFT−ECD spectra (Figure 6), and the obtained results revealed a close coherence of the measured ECD spectrum to that calculated for the conformer adopting the (7R,8R,10R,12S,13S) configuration. According to Figure 2. Key ROESY correlations of 1−6. Figure 3. Measured and calculated ECD spectra of 1in MeOH. Figure 4. Measured and calculated ECD spectra of 2in MeOH. Figure 5. Measured and calculated ECD spectra of 3in MeOH. Journal of Natural Products pubs.acs.org/jnp Article https://doi.org/10.1021/acs.jnatprod.5c00768 J. Nat. Prod. XXXX, XXX, XXX−XXX D the obtained results, compound 4was identified as depicted and named cordypyridone H. Compound 5was obtained as a yellow amorphous solid. The HR-ESI−MS spectrum (Figure S32) revealed its protonated molecular ion peak at m/z354.2061 [M + H]+ (calculated 354.2064) and thus established its molecular formula as C22H27NO3indicating ten degrees of unsaturation. The 1H NMR and HSQC spectral data of 5(Table 2 and Figures S33 and S36) unraveled the presence of a deshielded singlet aromatic proton at δH7.13 (H-6) directly correlated to a carbon atom at δC132.0 in addition to two doublet proton signals each integrated for two hydrogen atoms at δH7.20 and 6.78 with a coupling constant (Jvalue) of 8.6 Hz. The obtained results denoted the presence of a 1,4-disubstituted aromatic ring in 5compared to compounds 1−4and suggested its position at C-5 whose corresponding proton signal disappeared in 5. To ascertain the suggested structure modification in 5compared to 1−4, 2D NMR spectral analyses were acquired, including 1H−1H COSY, HMBC and ROESY spectra (Figures 1 and 2and Figures S34,S35, and S37). In addition to the featured spin systems in 1−4, the 1H−1H COSY data of 5(Figure 1 and Figure S34) revealed a spin system between H2-2′,6′and H2-3′,5′. Further confirmation to the depicted structure of 5was obtained through its HMBC spectrum (Figure 1 and S35) that revealed key correlations from H2-2′,6′at δH7.20 (d, J= 8.6 Hz) to a carbon resonance at δC116.4 (C-5) confirming the presence of 4′-hydroxyphenyl moiety at C-5 of the 2-pyridone nucleus. The relative configuration of the 2-pyridone nucleus in 5was determined based on its ROESY spectrum (Figure 2 and Figure S37), which revealed similar key ROE correlations to those in 4. The absolute configuration of 5was established based on the comparison of its experimental and calculated TDDFT−ECD spectra. The obtained results revealed a cohering pattern of the measured ECD spectrum to that calculated for the conformer with the configuration of (7R,8R,10R,12S,13S) (Figure 7). A literature search of 5 revealed that it is closely related to several 2-pyridone fungal metabolites despite adopting different stereochemistry including epipyridone A, 17 trichodin A 18 and chaunolidone A. 19 It is noteworthy to mention that a literature search of 5revealed Figure 6. Measured and calculated ECD spectra of 4in MeOH. Table 2. 1H and 13C NMR Data of 5 and 6 5 6 position δC, a type δH a multi [J(Hz)] δC, b type δH b multi [J(Hz)] 2 163.9, CO 163.9, CO 3 109.5, C 109.7, C 4 165.3, C 165.5, C 5 116.4, C 116.8, C 6 132.0, CH 7.13 s 132.0, CH 7.11 d (0.8) 7 50.0, CH 2.02 d (11.3) 50.1, CH 2.02 d (11.4) 8 37.6, C 37.7, C 9 46.9, CH2α0.72 m (overlapped) 47.0, CH2α0.72 m (overlapped) β1.63 dt (12.6, 2.7) β1.64 m 10 27.1, CH 1.65 m (overlapped) 27.2, CH 1.64 m (overlapped) 11 46.2, CH2α0.64 q (12.6) 46.2, CH2α0.64 q (12.3) β1.84 ddt (13.2, 4.8, 2.5) β1.84 m 12 28.9, CH 2.65 tq (11.2, 5.5) 29.0, CH 2.66 m 13 87.9, CH 4.19 q (6.5) 88.0, CH 4.19 q (6.5) 14 15.7, CH31.16 d (6.5) 15.9, CH31.18 d (6.5) 15 15.0, CH30.72 s 15.1, CH30.72 s 16 22.9, CH30.91 d (6.2) 23.0, CH30.91 d (6.3) 17 25.1, CH31.14 d (5.8) 25.2, CH31.14 d (5.9) 18 1′126.5, C 127.1, C 2′131.0, CH 7.20 d (8.6) 117.4, CH 6.84 d (2.1) 3′115.6, CH 6.78 d (8.6) 145.5, C 4′157.6, C 145.8, C 5′115.6, CH 6.78 d (8.6) 115.8, CH 6.75 d (8.2) 6′131.0, CH 7.20 d (8.6) 121.6, CH 6.69 dd (8.2, 2.1) a Measured in methanol-d4at 700 MHz for 1H and 175 MHz for 13C. b Measured in methanol-d4at 500 MHz for 1H and 125 MHz for 13C. Journal of Natural Products pubs.acs.org/jnp Article https://doi.org/10.1021/acs.jnatprod.5c00768 J. Nat. Prod. XXXX, XXX, XXX−XXX E that it was reported earlier this year in a Chinese patent as a fermentation product from axenic or co-cultures of a marinederived Aspergillus aculeatinus strain for treating acute liver injury. 20 Based on the obtained results, compound 5is reported here for the first time from the nematode-cyst-derived fungus L. nematophila and was trivially named cordypyridone I. Compound 6was isolated as a yellow amorphous solid. The HR-ESI−MS spectrum of 6(Figure S39) revealed a protonated molecular ion peak at m/z370.2013 [M + H]+ (calculated 370.2013) that determined its molecular formula as C22H27NO4indicating ten degrees of unsaturation equal to those in 5despite having an additional oxygen atom. The 1H/13C NMR spectral data of 6(Table 2) were close to identical in comparison with their respective values in 5. The only difference that could be noticed is the replacement of the two doublet aromatic proton resonances in 5by three aromatic proton signals at δH6.84 (d, J= 2.1 Hz, H-2′), 6.75 (d, J= 8.2 Hz, H-5′) and 6.69 (dd, J= 8.2, 2.1 Hz, H-6′) that were directly correlated via the HSQC spectrum (Figure S43) to three methine sp2carbon atoms at δC117.4 (C-2′), 115.8 (C-5′) and 121.6 (C-6′), respectively. The obtained results suggested that the additional oxygen atom in 6afforded a 1,3,4-trisubstituted aromatic ring. Further confirmation was obtained via the HMBC spectrum (Figure S42) that revealed key correlations from H-2′and H-6′to C-5, C-3′and C-4′ indicating that the 3,4-dihydroxyphenyl moiety is bound to C5 of the 2-pyridone core nucleus. The ROESY spectrum of 6 (Figure S44) revealed similar key ROE correlations to those observed for 5indicating that they both have a similar spatial orientation of substituents. The absolute configuration of 6was established based on comparing its experimental and the calculated TDDFT−ECD spectra that revealed similar Cotton effect patterns for the experimental ECD spectrum of 6and that calculated for the conformer with the configuration of (7R,8R,10R,12S,13S) (Figure 8). Accordingly, compound 6 was recognized as shown and it was named cordypyridone J. Compounds 7and 8were isolated as yellow amorphous solids. Their HR-ESI−MS spectra (Figures S46 and S55) revealed their molecular formulas to be C17H25NO3and C17H25NO4, respectively. Their structures were elucidated based on comprehensive 1D/2D spectroscopic analyses in addition to comparison with the reported literature. 14 Accordingly, compounds 7and 8were identified as cordypyridones C and D, respectively, which were previously reported from the entomopathogenic fungus Pleurocordyceps nipponica. 14 Biological Assays. Among the tested compounds, cordypyridone H (4), a N-hydroxy-2-pyridone structurally related to PF1140, 21 exhibited the most potent pancytotoxic activity (Table 3) across all tested cell lines (IC50: 0.08− 0.35 μM) and broad antimicrobial activity, with low MIC values against Gram-positive and Gram-negative bacteria (Bacillus subtilis,Staphylococcus aureus, and Escherichia coli) and several fungi (Candida albicans,Rhodotorula glutinis, and Schizosaccharomyces pombe). No nematicidal effects (see Table S15) were observed for any of the tested compounds. This profile is consistent with reported activities of N-hydroxy-2pyridones, known to interfere with microbial and eukaryotic targets. 22 In contrast, compounds 1,2,5,6, and 7exhibited limited cytotoxic activity. Only compound 5showed moderate antibacterial effects (Table 3), while compound 7displayed weak cytotoxicity against KB3.1 cells. In the antibiofilm assay, compounds 1−3and 5−8exhibited varying levels of inhibitory activity against the formation of S. aureus biofilms (Figure 9 and Table S1), with 5being the most effective. Compounds 5−7inhibited the biofilm formation by more than 75% at a concentration of 125 μg/mL. In particular, cordypyridone I (5) revealed biofilm inhibitory activity by ca. 43% even at a low concentration of 0.25 μg/mL, indicating a strong dose-dependent effect. Additionally, cordypyridones J (6) and C (7) displayed significant biofilm inhibition by 54 and 51% at 7.8 and 15.6 μg/mL, respectively. ■EXPERIMENTAL SECTION General Experimental Procedures. A Shimadzu UV−vis spectrophotometer UV-2450 (Shimadzu, Kyoto, Japan) was used to record the UV−vis spectra. An Anton Paar MCP-150 polarimeter (Anton Paar, Graz, Austria) was used for measuring optical rotation values at 20 °C. A Jasco J-815 spectropolarimeter (Jasco, Pfungstadt, Germany) was used to acquire the ECD spectra. An amaZon speed ETD ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) was used to conduct the HPLC−DAD−MS analysis in positive and negative ionization modes. As a stationary phase, a C18 Acquity UPLC BEH column (50 ×2.1 mm, 1.7 μm Waters, MA, U.S.A.) connected to the HPLC system (Dionex UltiMate 3000 Figure 7. Measured and calculated ECD spectra of 5in MeOH. Figure 8. Measured and calculated ECD spectra of 6in MeOH. Journal of Natural Products pubs.acs.org/jnp Article https://doi.org/10.1021/acs.jnatprod.5c00768 J. Nat. Prod. XXXX, XXX, XXX−XXX F UHPLC, Thermo Scientific, Inc., Waltham, MA, U.S.A.) was employed. Analysis was performed applying the following conditions: solvent A [deionized H2O + 0.1% formic acid (FA)], solvent B [acetonitrile (MeCN) + 0.1% FA], gradient: starting at 5% B for 0.5 min increasing to 100% B in 19.5 min then holding 100% B for 5 min, flow rate 0.6 mL min−1, UV−vis detection 190−600 nm. A maXis ESI−time-of-flight (TOF) mass spectrometer (Bruker Daltonics, Bremen, Germany) was used to acquire the HR-ESI−MS data. It was equipped with an Agilent 1200 Infinity Series HPLC−UV system (Agilent Technologies, Santa Clara, CA, U.S.A.) using the same column and separation gradient as for the HPLC−DAD−MS analysis. Additional parameters: scan range, m/z100−2500; rate, 2 Hz; capillary voltage, 4500 V; and dry temperature, 200 °C. The 1D/2D NMR spectra of the isolated compounds were recorded on a Bruker Avance III 500 MHz spectrometer equipped with BBGO (Plus) Smartprobe (1H, 500 MHz; 13C, 125 MHz) and/or a Bruker Avance III 700 MHz spectrometer utilizing a 5 mm TCI cryoprobe (1H, 700 MHz; 13C, 175 MHz). Compounds were dissolved in methanol-d4 (δH3.310; δC49.00) or chloroform-d(δH7.260; δC77.160). Fungal Material and Identification. Two strains of Laburnicola nematophila, namely, 20AD (DSM 112866) and K01 (DSM 112867); DSMZ−German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany), were isolated from the infected eggs of cereal cyst nematode Heterodera filipjevi collected in n Yozgat, Turkey. 13 Molecular phylogenies using combined sequence data were conducted. The acquired sequences for L. nematophila 20AD and K01 strains using four genome loci markers were registered on the GenBank database with respective accession numbers. 13 The isolates were cultured on 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, adjusted to pH 6.3, before autoclaving) in the dark. Cultivation and Metabolite Extraction. Seed culture of individual strains, each containing 200 mL of Q6/2 medium (Dglucose, 2.5 g L−1; glycerol, 10 g L−1; cottonseed flour, 5 g L−1, pH 7.2) in a 500 mL Erlenmeyer flask, were inoculated with 5 ×25 mm2 sections of mycelium grown on YM6.3 agar and cultivated at 23 °C with shaking at 140 rpm in the dark. After reaching sufficient biomass, the culture broth was homogenized using an 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 served as the inoculum for subsequent cultivations on BRFT medium (100 mL of a solution of K2HPO4, 0.5 g L−1; sodium tartrate, 0.5 g L−1; yeast extract, 1 g L−1, added to 28 g of brown rice and autoclaved) which was inoculated with 6 mL of the Q6/2 media seed culture. Solid-State Fermentation. An initial cultivation of L. nematophila 20AD (DSM 112866) was performed as described above using six Erlenmeyer flasks. Two flasks were harvested after 2, 3, and 4 weeks, respectively, following incubation in the dark at room temperature. After the respective incubation periods, each flask was extracted with 3 ×250 mL of acetone, mixed thoroughly, and processed according to a previously described protocol. 5 The resulting crude extracts were defatted by liquid−liquid partitioning between nheptane and methanol. Both solvent fractions were evaporated to dryness and analyzed by HPLC−DAD−MS. Table 3. Cytotoxicity (μM) and Antimicrobial Activity Assay (MIC) of 1, 2, and 4−7 a IC50 (μM) positive control test cell line 1 2 4 5 6 7 epothilone B (nM) mouse fibroblast (L929) * * 0.28 ** ** ** 0.65 human endocervival adenocarcinoma (KB3.1) * * 0.35 ** ** 61.86 0.17 human prostate carcinoma (PC-3) n.d. n.d. 9.37 n.d. n.d. n.d. 0.09 human breast adenocarcinoma (MCF-7) n.d. n.d. 0.10 n.d. n.d. n.d. 0.07 human ovarian cancer (SKOV-3) n.d. n.d. 0.08 n.d. n.d. n.d. 0.09 human epidermoid carcinoma (A431) n.d. n.d. 0.08 n.d. n.d. n.d. 0.06 human lung carcinoma (A549) n.d. n.d. 0.25 n.d. n.d. n.d. 0.05 MIC (μg/mL) test microorganism 1 2 4 5 6 7 positive control (μg/mL) Bacillus subtilis (DSM 10) n.i. n.i. 8.3 33.3 n.i. 66.6 0.42G Mycobacterium smegmatis (ATCC 700084) n.i. n.i. 66.6 n.i. n.i. n.i. 0.83G Staphylococcus aureus (DSM 346) n.i. n.i. 8.3 66.6 n.i. n.i. 16.6O Acinetobacter baumannii (DSM 30008) n.i. n.i. 33.3 n.i. n.i. n.i. 0.42G Chromobacterium violaceum (DSM 30191) n.i. n.i. 16.6 n.i. n.i. n.i. 8.30N Escherichia coli (DSM 1116) n.i. n.i. 33.3 n.i. n.i. n.i. 8.30N Pseudomonas aeruginosa (PA14) n.i. n.i. 66.6 n.i. n.i. n.i. 1.04C Candida albicans (DSM 1665) n.i. n.i. 4.2 n.i. n.i. n.i. 1.67G Mucor hiemalis (DSM 2656) n.i. n.i. 8.3 n.i. n.i. 66.6 8.30N Rhodotorula glutinis (DSM 10134) n.i. n.i. 4.2 33.3 n.i. 66.6 8.30N Schizosaccharomyces pombe (DSM 70572) n.i. n.i. 4.2 n.i. n.i. n.i. 4.20N Wickerhamomyces anomalus (DSM 6766) n.i. n.i. 16.6 n.i. n.i. n.i. 1.70K a (∗) Slight inhibition of cell proliferation. (∗∗) No cytotoxic activity observed. n.d., not determined; n.i., no inhibition up to 67 μg mL−1; C, ciprofloxacin; G, gentamicin; K, kanamycin; N, nystatin; and O, oxytetracycline. Figure 9. Inhibitory activity of compounds 1−3and 5−8against biofilm formation of S. aureus. The solvent control (MeOH) was used as the baseline, and its average value was set as 0% inhibition. Microporenic acid A (MAA) was used as a positive control. Error bars indicate SD of duplicates in two biological repeats; pvalues: (∗∗∗)p < 0.001, (∗∗)p< 0.01, and (∗)p< 0.05. Journal of Natural Products pubs.acs.org/jnp Article https://doi.org/10.1021/acs.jnatprod.5c00768 J. Nat. Prod. XXXX, XXX, XXX−XXX G Based on these results, a scale-up cultivation of strain 20AD was conducted, with 12 flasks harvested after 4 weeks and 8 flasks after 6 weeks under the same conditions. In parallel, strain K01 (DSM 112867) was cultivated in 35 and 15 flasks, respectively, using the same parameters as described above. Isolation of Compounds 1−8. The screening cultivation of L. nematophila 20AD (DSM 112866) on BRFT medium yielded 1.3 g of crude methanol extract and 2.2 g of n-heptane extract. A subsequent scale-up cultivation on BRFT medium produced an additional 2.2 g of methanol extract. Strain K01 was cultivated on BRFT medium affording 3.8 g of the crude methanol extract. Purification of all crude extracts was performed according to the workflow shown in Figures S62−S63 for 20AD and Figure S64 for K01 and the conditions are described Tables S2−S14, respectively. Initial fractionation was carried out using a FlashPure ID silica cartridge on a Grace Reveleris X2 flash chromatography system. The resulting fractions were further purified by reversed-phase preparative HPLC. This process yielded 1 (1.2 mg), 2(3.1 mg), 3(1.1 mg), 5(1.5 mg), 7(2.6 mg), 8(1.2 mg) from methanol extracts and 4(13.1 mg) from n-heptane extract of strain 20AD, whereas strain K01 afforded 6(2.1 mg). Cordypyridone E (1): Yellow amorphous solid; [α]D 20 +106 (c0.07, MeOH); UV/vis (MeOH): λmax (log ε) = 284 (4.1), 224 (4.2) nm; ECD (c= 8.14 ×10−4M; MeOH): λ[nm] (Δε) 246 (+1.6), 218 (+36.1) nm; NMR data (1H NMR: 500 MHz, 13C NMR: 125 MHz, methanol-d4), see Table 1; HR-(+)ESI−MS: m/z290.1749 [M − H2O + H]+(calcd. 290.1751 for C17H24NO3+), 308.1857 [M + H]+ (calcd. 308.1856 for C17H26NO4+), 330.1674 [M + Na]+(calcd. 330.1676 for C17H25NNaO4+); tR= 7.56 min (LC−ESI−MS). Cordypyridone F (2): Yellow amorphous solid; [α]D 20 +153 (c0.1, MeOH); UV/vis (MeOH): λmax (log ε) = 289 (3.8), 216 (4.4) nm; ECD (c= 8.14 ×10−4M; MeOH): λ[nm] (Δε) 292 (+0.5), 270 (+0.4), 218 (+56.1) nm; NMR data (1H NMR: 500 MHz, 13C NMR: 125 MHz, methanol-d4), see Table 1; HR-(+)ESI−MS: m/z308.1855 [M + H]+(calcd. 308.1856 for C17H26NO4+), 330.1672 [M + Na]+ (calcd. 330.1676 for C17H25NNaO4+); tR= 6.86 min (LC−ESI−MS). Cordypyridone G (3): Yellow amorphous solid (limited purity); [α]D 20 −492 (c0.08, MeOH); UV/vis (MeOH): λmax (log ε) = 285 (3.5), 258 (3.4), 212.5 (4.2) nm; ECD (c= 9.03 ×10−4M; MeOH): λ[nm] (Δε) 294 (+0.6), 266 (+0.3), 218 (+28.1) nm; NMR data (1H NMR: 700 MHz, 13C NMR: 175 MHz, methanol-d4), see Table 1; HR-(+)ESI−MS: m/z278.1748 [M + H]+(calcd. 278.1751 for C16H24NO3+), tR= 6.88 min (LC−ESI−MS). Cordypyridone H (4): Yellow amorphous solid; [α]D 20 +106 (c0.04, MeOH); UV/vis (MeOH): λmax (log ε) = 290.5 (3.0), 218.5 (3.7) nm; ECD (c= 4.51 ×10−4M; MeOH): λ[nm] (Δε) 263 (+0.3), 251 (−0.3), 216 (+17.3) nm; NMR data (1H NMR: 500 MHz, 13C NMR: 125 MHz, methanol-d4), see Table 1;m/z278.1751 [M + H]+(calcd. 278.1751 for C16H24NO3+), 300.1567 [M + Na]+(calcd. 300.1570 for C16H23NNaO3+); tR= 11.26 min (LC−ESI−MS). Cordypyridone I (5): Yellow amorphous solid; [α]D 20 +88 (c0.1, MeOH); UV/vis (MeOH): λmax (log ε) = 250.5 (3.8), 212 (4.0), 204 (4.0) nm; ECD (c= 14.16 ×10−4M; MeOH): λ[nm] (Δε) 227 (−2.6), 198 (+26.0) nm; NMR data (1H NMR: 700 MHz, 13C NMR: 175 MHz, methanol-d4), see Table 2; HR-(+)ESI−MS: m/z354.2061 [M + H]+(calcd. 354.2064 for C22H28NO3+), tR= 10.19 min (LC− ESI−MS). Cordypyridone J (6): Yellow amorphous solid (limited purity); [α]D 20 +16 (c0.1, MeOH); UV/vis (MeOH): λmax (log ε) = 223 (3.7), 206 (3.9) nm; ECD (c= 13.55 ×10−4M; MeOH): λ[nm] (Δε) 277 (+2.8), 220 (+8.0), 198 (+12.3); NMR data (1H NMR: 500 MHz, 13C NMR: 125 MHz, methanol-d4), see Table 2; HR-(+)ESI−MS: m/z370.2013 [M + H]+(calcd. 370.2013 for C22H28NO4+); tR= 9.25 min (LC−ESI−MS). Cordypyridone C (7): Yellow amorphous solid; [α]D 20 +176 (c0.09, MeOH); UV/vis (MeOH): λmax (log ε) = 292 (3.2), 217 (3.9) nm; ECD (c= 4.30 ×10−4M; MeOH): λ[nm] (Δε) 256 (+0.8), 239 (+1.2), 218 (+27.5) nm (Figure S53); NMR data (1H NMR: 500 MHz, 13C NMR: 125 MHz, methanol-d4) comparable to those reported in literature; 14 HR-(+)ESI−MS: m/z292.1912 [M + H]+ (calcd. 292.1907 for C17H26NO3+), 314.1723 [M + Na]+(calcd. 314.1727 for C17H25NNaO3+); tR= 11.23 min (LC−ESI−MS). Cordypyridone D (8): Yellow amorphous solid; [α]D 20 +106 (c0.07, MeOH); UV/vis (MeOH): λmax (log ε) = 291.5 (3.2), 217.5 (3.9) nm; ECD (c= 16.29 ×10−4M; MeOH): λ[nm] (Δε) 242 (+3.4), 220 (+72.1), 211 (+42.1) nm (Figure S61); NMR data (1H NMR: 500 MHz, 13C NMR: 125 MHz, methanol-d4) comparable to those reported in literature; 14 HR-(+)ESI−MS: m/z290.1746 [M −H2O + H]+(calcd. 290.1751 for C17H24NO3+), 308.1856 [M + H]+(calcd. 308.1856 for C17H26NO4+), 330.1672 [M + Na]+(calcd. 330.1676 for C17H25NNaO4+); tR= 7.04 min (LC−ESI−MS). Antimicrobial Assay. The antimicrobial activity assay was performed applying a serial dilution methodology. The minimum inhibitory concentrations (MIC) of the isolated metabolites were determined against a panel of Gram-positive, Gram-negative bacteria and fungi applying our previously described protocol. 23 Cytotoxicity Assay. Compounds (1,2, and 4−7) were evaluated for their cytotoxic activity against seven different cell lines using the MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], as previously described. 24 Epothilone B served as a positive control. Nematicidal Assay. Nematicidal effects were assessed using Caenorhabditis elegans in a 48-well flat-bottom plate. Metabolites (1, 2,4, and 6−8) were tested at the concentrations 100, 50, and 10 μg mL−1in biological triplicates as described by Phutthacharoen et al. 24 Ivermectin was used as a positive control at the concentration of 1 μg mL−1and methanol was used as a negative control. The percentage of mortality was corrected by applying the Schneider−Orelli formula, which accounts for the observed baseline mortality in the negative control. 25 Biofilm formation. The inhibitory activity against biofilm formation of Staphylococcus aureus was assessed by applying the same protocol as previously described. 26 Briefly, S. aureus DSM 1104 was revived from a −20 °C stock in 25 mL of CASO medium at 37 °C in a 250 mL flask for 20 h. The resulting culture was adjusted to an OD600 equivalent to 0.001 McFarland standard, and 150-μL aliquots were transferred to 96-well tissue culture plates (TPP, reference number 92196, Switzerland) containing CASO medium supplemented with 4% glucose. Serial dilutions of compounds 1−3and 6− 8: ranging from 125−1μg/mL; 5: ranging from 125 to 0.004 μg/mL) were added to the wells. Plates were incubated at 37 °C with shaking at 150 rpm for 20 h. Following incubation, wells were washed and stained with crystal violet to quantify biofilm biomass. Methanol (2.5%) was used as the solvent control, and microporenic acid A (MAA; 125−1μg/mL) served as the positive control. All experiments were performed in at least two independent biological replicates, each with technical duplicates. Statistical significance was evaluated using Student’s t-test (two-tailed, unpaired), and pvalues categorized as follows: (∗∗∗)p< 0.001, (∗∗)p< 0.01, and (∗)p< 0.05. Error bars represent standard deviation (SD). Computational Section. Within the realm of electronic circular dichroism (ECD) spectra elucidation, all possible conformations of compounds 1−8were obtained by performing a conformational analysis by means of Omega2 software 27 (with an energy window of 10 kcal/mol). The obtained conformations were subjected to geometrical optimization followed by frequency calculations at the B3LYP/631+G*level of theory. Upon frequency calculations, Gibbs free energies were calculated. Based on the optimized geometries, the time-dependent density functional theory (TDDFT) computations were performed at the CAM-B3LYP/TZVP level of theory to identify the first 50 excitation states. According to the reported literature, the employed level of theory is appropriate for TDDFT−ECD calculations. 28,29 For each compound, the ECD spectra of the conformers were Boltzmann averaged and graphed by adopting the SpecDis 1.71 using Gaussian band shapes with a sigma value of 0.20− 30 eV. 30,31 All quantum mechanics calculations were conducted using Gaussian09 software. 32 All calculations, including geometry optimization and TDDFT computations, were carried out in methanol Journal of Natural Products pubs.acs.org/jnp Article https://doi.org/10.1021/acs.jnatprod.5c00768 J. Nat. Prod. XXXX, XXX, XXX−XXX H solvent employing the integral equation formalism variant (IEFPCM) model. 33,34 ■ASSOCIATED CONTENT * sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jnatprod.5c00768. LR-ESI−MS, HR-ESI−MS, 1D (1H/13C), 2D (1H−1H COSY, HMBC, HSQC, and ROESY) NMR, and ECD spectra of compounds 1−8(PDF) ■AUTHOR INFORMATION Corresponding Authors Sherif S. Ebada −Department of Microbial Drugs, Helmholtz Centre for Infection Research GmbH (HZI) and German Centre for Infection Research (DZIF), 38124 Braunschweig, Germany; Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, 11566 Cairo, Egypt; orcid.org/0000-0002-2753-0031; Email: [email protected],sherif_elsayed@ pharma.asu.edu.eg Marc Stadler −Department of Microbial Drugs, Helmholtz Centre for Infection Research GmbH (HZI) and German Centre for Infection Research (DZIF), 38124 Braunschweig, Germany; Institute of Microbiology, Technische Universität Braunschweig, 38106 Braunschweig, Germany; orcid.org/ 0000-0002-7284-8671; Phone: +49-531-6181-424; Email: [email protected] Authors Jan-Peer Wennrich −Department of Microbial Drugs, Helmholtz Centre for Infection Research GmbH (HZI) and German Centre for Infection Research (DZIF), 38124 Braunschweig, Germany; Institute of Microbiology, Technische Universität Braunschweig, 38106 Braunschweig, Germany; Laboratory of Fungal Genetics and Metabolism, Institute of Microbiology of the Czech Academy of Sciences, 14220 Prague, Czechia; orcid.org/0000-0002-43097290 Caren Holzenkamp −Department of Microbial Drugs, Helmholtz Centre for Infection Research GmbH (HZI) and German Centre for Infection Research (DZIF), 38124 Braunschweig, Germany; Institute of Microbiology, Technische Universität Braunschweig, 38106 Braunschweig, Germany; orcid.org/0009-0008-8439-3230 Sara Fushimi −Department of Microbial Drugs, Helmholtz Centre for Infection Research GmbH (HZI) and German Centre for Infection Research (DZIF), 38124 Braunschweig, Germany; Institute of Microbiology, Technische Universität Braunschweig, 38106 Braunschweig, Germany; Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, Imizu, Toyama 939-0398, Japan Mahmoud A. A. Ibrahim −Computational Chemistry Laboratory, Chemistry Department, Faculty of Science, Minia University, Minia 61519, Egypt; Department of Engineering, College of Engineering and Technology, University of Technology and Applied Sciences, Nizwa 611, Sultanate of Oman; School of Health Sciences, University of KwaZuluNatal, Durban 4000, South Africa; orcid.org/00000003-4819-2040 Samad Ashrafi −Institute for Epidemiology and Pathogen Diagnostics, Julius Kuhn Institute (JKI)−Federal Research Centre for Cultivated Plants, 38104 Braunschweig, Germany; Institute for Crop and Soil Science, Julius Kuhn Institute (JKI)−Federal Research Centre for Cultivated Plants, 38116 Braunschweig, Germany; Department of Zoology and Entomology, University of the Free State, Bloemfontein 9300, South Africa; orcid.org/0000-0003-0426-7039 Wolfgang Maier −Institute for Epidemiology and Pathogen Diagnostics, Julius Kuhn Institute (JKI)−Federal Research Centre for Cultivated Plants, 38104 Braunschweig, Germany Hedda Schrey −Department of Microbial Drugs, Helmholtz Centre for Infection Research GmbH (HZI) and German Centre for Infection Research (DZIF), 38124 Braunschweig, Germany; Institute of Microbiology, Technische Universität Braunschweig, 38106 Braunschweig, Germany Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jnatprod.5c00768 Funding The authors benefited from the H2020-RISE Project Mycobiomics−Joining Forces to Exploit the Mycobiota of Asia, Africa and Europe for Beneficial Metabolites and Potential Biocontrol Agents, Using -OMICS Techniques (101008129). This work was supported by funds of the Landwirtschaftliche Rentenbank, Germany. The Alexander von Humboldt (AvH) Foundation funded Sherif S. Ebada through the Georg-Forster Fellowship for Experienced Researchers (Reference 3.4-1222288-EGY-GF-E). Work was also supported by the project Southeast Asia−Europe Joint Funding Scheme (SEA−Europe Grant JFS20ST-127, Acronym: Antiviralfun). C. elegans strain N2 was provided by the Caenorhabditis Genetics Center (CGC), which is funded by the National Institute of Health (NIH) Office of Research Infrastructure Programs (P40 OD010440). Notes The authors declare no competing financial interest. ■ACKNOWLEDGMENTS All authors gratefully acknowledge Wera Collisi for assistance with the cytotoxicity and antimicrobial assays. Also, the authors are indebted to Kirsten Harmrolfs and Esther Surges for performing the NMR spectroscopic measurements as well as Aileen Gollasch for acquiring the HR-ESI−MS samples. ■REFERENCES (1) Schrey, H.; Lambert, C.; Stadler, M. IMA Fungus 2025,16, No. e142462. (2) Bills, G. F.; Gloer, J. B. Microbiol. Spectr. 2016,4(6), FUNK0009-2016. (3) Karwehl, S.; Stadler, M. Exploitation of Fungal Biodiversity for Discovery of Novel Antibiotics. In How to Overcome the Antibiotic Crisis; Stadler, M., Dersch, P., Eds.; Springer: Cham, Switzerland, 2016; Current Topics in Microbiology and Immunology, Vol. 398, pp 303−338, DOI: 10.1007/82_2016_496. (4) Degenkolb, T.; Vilcinskas, A. Appl. Microbiol. Biotechnol. 2016, 100 (9), 3799−3812. (5) Holzenkamp, C.; Wennrich, J.-P.; Muema, J. M.; Ashrafi, S.; Maier, W.; Stadler, M.; Ebada, S. S. ACS Omega 2024,9, 21658− 21667. (6) Wennrich, J.-P.; Holzenkamp, C.; Ashrafi, S.; Maier, W.; Wang, H.; Ibrahim, M. A. A.; Ebada, S. S.; Stadler, M. Chem. 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