New cytotoxic indole derivatives with anti-FADU potential produced by the endophytic fungus Penicillium oxalicum 2021CDF-3 through the OSMAC strategy

Fungi possess well-developed secondary metabolism pathways that are worthy of in-depth exploration. The One Strain Many Compounds (OSMAC) strategy is a useful method for exploring chemically diverse secondary metabolites. In this study, continued chemical investigations of the marine red algae-derived endophytic fungus Penicillium oxalicum 2021CDF-3 cultured in PDB media yielded six structurally diverse indole derivatives, including two new prenylated indole alkaloids asperinamide B (1) and peniochroloid B (5), as well as four related derivatives (compounds 2–4 and 6). The chemical structures of these compounds, including the absolute configurations of 1 and 5, were determined by extensive analyses of HRESIMS, 1D and 2D NMR spectroscopic data, and TDDFT-ECD calculations. Compound 1 was found to possess an unusual 3-pyrrolidone dimethylbenzopyran fused to the bicyclo[2.2.2]diazaoctane moiety, which was rare in previously reported prenylated indole alkaloids. In vitro cytotoxic experiments against four human tumor cell lines (HeLa, HepG2, FADU, and A549) indicated that 1 strongly inhibited the FADU cell line, with an IC50 value of 0.43 ± 0.03 μM. This study suggested that the new prenylated indole alkaloid 1 is a potential lead compound for anti-FADU drugs.


Introduction
Secondary metabolism in filamentous fungi is well-developed (Gerke and Braus, 2014).These organisms are prolific producers of structurally diverse secondary metabolites that exhibit various promising biological properties (Zhang et al., 2019;Li et al., 2021).In the study of natural products, many useful methods, including genetic and cultivation-based strategies, have been developed to activate silent or cryptic secondary metabolites (Yuan et al., 2020a;Pinedo-Rivilla et al., 2022).Among them, the One Strain Many Compounds (OSMAC) approach, which is conceptualized as a single strain that can produce different metabolites Song et al. 10.3389/fmicb.2024.1400803Frontiers in Microbiology 02 frontiersin.orgwhen cultured under different conditions, is among the most effective tools for regulating microbial secondary metabolism (Romano et al., 2018;Ying et al., 2018).Indole alkaloids have a bicyclic structure that consists of a six-membered benzene ring fused to a five-membered nitrogencontaining pyrole ring (Hu et al., 2021;Umer et al., 2022).Indole alkaloids are among the most important secondary metabolites for drug developments (Zhang et al., 2020).Prenylated indole alkaloids with a bicyclo[2.2.2]diazaoctane ring system are well known for their chemical, biosynthetic, and biological interests (Zhao et al., 2023).Structurally, prenylated indole alkaloids contain a bicyclo[2.2.2] diazaoctane framework and densely functionalized indole-derived subunits.These alkaloids represent a large and highly structurally diverse group of secondary metabolites that exhibit numerous potent pharmaceutical properties (Zhang et al., 2019).It has been reported that prenylated indole alkaloids possess anticancer, antimalarial, antimicrobial, anti-inflammatory, anti-diabetic, and immuneregulatory activities (Zhang et al., 2019;Zhao et al., 2023).

General experimental procedures
Optical rotations were measured with a JASCO P-1020 digital polarimeter (Tokyo, Japan) in MeOH.UV spectra were obtained with a Lambda 35 UV/Vis spectrophotometer (Perkin Elmer, Waltham, USA). 1 H and 13 C NMR data were acquired with an Agilent DD2 spectrometer (500 MHz for 1 H and 125 MHz for 13 C) (Santa Clara, CA, United States).Chemical shifts (δ) are referenced using residual deuterium reagent signals as an internal standard.The 1D NMR assignments were confirmed by the following 1 H-1 H COSY, HSQC, and HMBC experiments.HRESIMS spectra were taken with a scientific LTQ Orbitrap XL spectrometer (Thermo Scientific, Waltham, United States).Preparative HPLC separations were conducted with an Agilent 1,260 system.Commercial silica gel (200-300 mesh, Qingdao Marine Chemical Factory, Qingdao, China), octadecylsilyl reversed-phase gel (30-50 μm, YMC Co., Ltd., Japan), and Sephadex LH-20 (GE Healthcare, United States) were purchased and subjected to column chromatography.

Isolation and identification of P. Oxalicum 2021CDF-3
The fungus P. oxalicum 2021CDF-3 used in this study was previously isolated from the inner tissue of the marine red alga R. confervoides and was obtained from Lianyungang, Jiangsu, China.The internal transcribed spacer sequence of 2021CDF-3 displayed 99% identity to that of the reported P. oxalicum species.This sequence has been submitted to the GenBank database with no.OP349593.This fungus was preserved at the School of Food and Pharmacy, Zhejiang Ocean University.

Fermentation, extraction, and isolation
Fermentation: Previously, the fungal strain P. oxalicum 2021CDF-3 was fermented on solid rice media, which yielded ten structurally diverse polyketides (Weng et al., 2022).To explore the metabolic potential of this strain, the OSMAC approach was used to cultivate this fungus in PDB medium.Mycelia of P. oxalicum 2021CDF-3 grown on PDA media (Solarbio Life Sciences Co., Ltd., Beijing, China) were inoculated into a 1 L Erlenmeyer flask containing 300 mL of PDB media (Solarbio Life Sciences Co., Ltd.).Afterwards, the flask was incubated at 28°C at 200 rpm for five days.The whole culture medium was then transferred into 100 × 1 L Erlenmeyer flasks containing PDB medium.Finally, all flasks were fermented statically at 28°C for 30 days.

Computational section
The computational details are shown in Supplementary material.

Cytotoxic assay
The cytotoxicity of isolated compounds 1-6 was determined in vitro against HeLa, HepG2, FADU, and A549 cells by the CCK8 colorimetric method (Yuan et al., 2020b).Doxorubicin was used as a positive control.

Structural elucidation
The EtOAc crude extracts of P. oxalicum 2021CDF-3 were initially chromatographed on a silica gel column, and then fractionated by gel chromatography on Sephadex LH-20 to yield the following compounds 1 and 5.
The relative configurations of 1 were determined based on its NOESY relationships (Figure 4).The observed NOE interactions of H-20a with H 3 -23, and of H-20b with H 3 -24, suggested that H-20a and H 3 -23 were oriented in the same direction, tentatively assigned as β, while H-20b and H 3 -24 were oriented in the α direction.Further, the NOE interactions of H-10/H-21/H 3 -23 indicated that H-10 and H-21 were β-oriented.Similarly, NOE interactions of H 3 -30 with H 3 -24 suggested that H 3 -30 was α-oriented.By comparing the chemical shifts with those of compound 2, the chemical shifts from C-11 to C-21 were very close, indicating a shared relative configuration.In summary, the relative configuration of compound 1 was determined as 2S*, 10S*, 11R*, 17S*, 21S*.The absolute configurations of 1 were initially determined by comparing its ECD spectrum with those of previously known compounds.Previous studies revealed that the arrestive Cotton effect at 200-250 nm caused by an n-π* transition of the amide bond was responsible for the bicyclo[2.2.2]diazaoctane framework (Kato et al., 2007).The ECD spectrum of 1 displayed a positive Cotton effect at 230 nm, which was similar to that of notoamides (Kato et al., 2007).Therefore, the absolute stereochemistry of 1 was deduced to be 2S,10S,11R,17S,21S.Meanwhile, compound 1 was also subjected to TDDFT-ECD calculations at the CAM-B3LYP/6-311G(d) level.As expected, the good agreement of the high-energy ECD transitions (Figure 5) allowed the determination of the absolute configuration of 1.The positive CE at ~230 nm was ascribed to the electron transition from MO131 (HOMO) to MO133 (LUMO +1).The positive CE at ~330 nm was caused by the electron transition from MO131 (HOMO) to MO132 (LUMO) (Figure 6).This approach solidified the determination of the stereochemistry and highlighted the role of ECD spectroscopy and TDDFT calculations in the structural analysis of complex molecules.Peniochroloid B (5) was isolated as a colorless oil (MeOH).Its molecular formula was assigned as C 17 H 23 NO 6 according to the primary HRESIMS peak generated at m/z 336.1458 [M − H] − (calcd for C 17 H 22 NO 6 , 336.1447).The UV peaks at 214, 258, and 296 nm indicated the presence of an indolinone subunit, which was identical to that previously reported for the compound sclerotiamide (Whyte et al., 1996).The 1 H NMR data of 5 (Table 1) showed three aromatic methines at δ H 7.15 (m, H-4), 6.95 (m, H-5), and 7.14 (m, H-6), one oxygenated methine at δ H 3.61 (dd, J = 8.6, 2.3 Hz, H-11), two oxygenated methylenes at δ H 3.19 (dd, J = 16.9, 7.7 Hz, H-12α), 2.88 (dd, J = 11.0,8.6 Hz, H-12β), 3.95 (m, H-16α), and 3.73 (m, H-16β), and three methyl groups at δ H 1.38 (s, H 3 -13), 1.19 (s, H 3 -14), and 1.83 (s, H 3 -19).The 13 C NMR spectra of 5 revealed two ester/amide carbonyls at δ C 178.5 (C-2) and 170.6 (C-18), four methines including three aromatic at δ C 122.3 (C-4), 122.2 (C-5), and 127.6 (C-6), and one oxygenated at δ C 79.6 (C-11), three methylenes including two oxygenated at δ C 63.6 (C-12) and 60.1 (C-16), three methyls, and five quaternary carbons.Analysis of the UV and NMR data led to the identification of an indolinone framework.The relevant 1 H and 13 C NMR data (Table 1) for 5 are consistent with those for peniochroloid A (6) (Liu et al., 2023).The presence of an isopentene group was determined by the COSY correlation between H-11 (δ H 3.61, dd, J = 8.6 and 2.3 Hz) and H 2 -12 (δ H 3.19, dd, J = 16.9 and 7.7 Hz; 2.88, dd, J = 11.0 and 8.6 Hz) and correlative HMBC correlations from H-11 to C-13/C-14 and from H 3 -13 to C-7 (Figure 3), as is found in peniochroloid A. The COSY correlation of the methylene protons at δ H 2.19 and 2.08 (H 2 -15) with oxygenated methylene protons at δ H 3.95 and 3.73 (H 2 -16), together with additional HMBC correlations observed from H 2 -15 to C-2, C-3, and C-9, indicated that both oxygenated quaternary carbon C-3 and OCH 2 CH 2 -unit methylene C-15 were linked.The remaining methyl proton signal at δ H 1.83 (H 3 -19) correlated with the ester carbonyl C-18, requiring the connection of H 3 -19 with C-18 to form the acetyl group and completing the assignment of the planar structure of 5, as shown in Figure 2.
Mosher's method is considered as a useful tool to determine the absolute configuration of C-11 in compound 5.However, hampered by a deficiency in sample quantity, compound 5 was unable to undergo Mosher's experiment.The structures of compounds 5 and 6 were closely similar.In their biosynthetic pathways, the segment from C-10 to C-14 was derived from a prenyl group attached to C-7.Compound 6, a known compound, exhibited an R configuration at C-11.A comparison of the chemical shifts for C-10 to C-14 in both compounds revealed their structural similarity and identical chirality at C-11.By carefully comparing the chemical shifts of C-11 and adjacent carbons, and considering that compounds 5 and 6 shared the same biosynthetic pathway (specifically the addition of an isopentenyl unit at C-7 via an isopentenyl transferase), it was inferred that the chirality at C-11 for both compounds should be consistent.The configuration at C-11 in compound 5 was supposed to be R.The side chains at positions C-5 and C-7, alongside the side chain at C-3,  Experimental and calculated ECD spectra of 1. et al. 10.3389/fmicb.2024.1400803Frontiers in Microbiology frontiersin.orgcontributed minimally to the Cotton effects observed in the ECD spectra.Consequently, we simplified these side chains to methyl groups, yielding 3-hydroxy-3,7-dimethylindolin-2-one, featuring a sole chiral center at C-3.By computing the ECD spectra for both the 3S and 3R configurations and comparing these with the experimental ECD spectra of compound 5, we were able to definitively ascertain the absolute configuration of C-3 in compound 5. Upon comparison with the measured spectra (Figure 7), we concluded that the stereochemistry of the C-3 position was S.

Cytotoxic activity
Prenylated indole alkaloids containing the bicyclo[2.2.2] diazaoctane framework have been reported to possess remarkable biological activities, including antitumor, antibacterial, antiinflammatory, anthelmintic, and insecticidal activities.Based on the same structural characteristics between asperinamide B (1) and other this kind of compounds, the new prenylated indole alkaloid 1 was supposed to possess high potential biological significance.The cytotoxicities of compounds 1-6 against four human tumor cell lines (HeLa, HepG2, FADU, and A549) were measured.The cells were treated with compounds 1-6 at concentrations of 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 50 μM, 100 μM, and 200 μM for 48 h.The CCK-8 assay results are shown in Table 2. Compound 1 demonstrated dose-dependent cytotoxicity against the human pharyngeal squamous cell line FADU, with an IC 50 value of 0.43 ± 0.03 μM.This result indicated that the unusual 3-pyrrolidone moiety in 1 may play an important role in cytotoxic activity.Moreover, compound 6 showed higher activity (IC 50 = 15.30± 0.13 μM) against the A549 cell line than compound 5 (IC 50 = 29.84 ± 0.21 μM), suggesting that the ester carbonyl group in 6 may enhance cytotoxic activity.Head and neck squamous cell carcinoma (HNSCC) is one of the six major malignant tumors worldwide.Hypopharyngeal squamous cell carcinoma (HSCC), accounting for 3 to 5% of all HNSCC cases, has become a current research hotspot due to its high incidence and mortality rates (Chen et al., 2017).Despite significant progress in the treatment of HSCC in recent years, the use of conventional chemotherapy drugs is limited by drug resistance and side effects in tumor drug therapy.Therefore, there is an urgent need to discover alternative antitumor drugs.Compound 1 showed high inhibitory activity against the FADU cell line, comparable to the positive control doxorubicin.Further pharmacological studies will provide evidences to reveal this compound as a potential lead compound for anti-FADU drugs.

Conclusion
In summary, continued chemical investigation of the marine red alga-derived endophytic fungus P. oxalicum 2021CDF-3 cultured in PDB media yielded six structurally diverse indole derivatives, including two new prenylated indole alkaloids asperinamide B (1) and peniochroloid B (5). Compound 1 was characterized as possessing an unusual 3-pyrrolidone dimethylbenzopyran fused to the bicyclo[2.2.2] diazaoctane moiety, which was rare in previously reported prenylated indole alkaloids.In vitro cytotoxic assays revealed that 1 strongly inhibited the growth of the FADU cell line, indicating that this compound could be a potential lead compound for anti-FADU drugs.This study reported a new prenylated indole alkaloid featuring a 6/6/5/6/6/6/5 heptacyclic scaffold, which added the structural diversity of these kinds of compounds.In addition, the new prenylated indole alkaloid showed promising cytotoxic activity, which will receive more and more attention from natural product chemists for the further pharmacological and biosynthetic/synthetic interests.29.84 ± 0.21

FIGURE 1 HPLC
FIGURE 1 HPLC profile of crude extracts of P. oxalicum 2021CDF-3 in different cultural conditions.

TABLE 1
NMR spectroscopic data for compounds 1 and 5 ( 1 H at 500 MHz and 13 C at 125 MHz).
a Measured in CD3OD; b Measured in DMSO-d6.