Six Unprecedented Cytochalasin Derivatives from the Potato Endophytic Fungus Xylaria curta E10 and Their Cytotoxicity

Six previously undescribed cytochalasins, Curtachalasins X1–X6 (1–6), together with six known compounds (7–12) were isolated from the endophytic fungus Xylaria curta E10 harbored in the plant Solanum tuberosum. The structures were elucidated by the interpretation of HRESIMS, UV, and NMR data. The absolute configurations of Curtachalasins X1–X6 were determined by comparison of their experimental and calculated electronic circular dichroism (ECD) spectra. In bioassays, Curtachalasin X1 (1) and X5 (5) showed cytotoxic activity against the MCF-7 cell line with IC50 values of 2.03 μM and 0.85 μM, respectively.


Introduction
Cytochalasins are a class of cell membrane permeability mycotoxins, and they can lead to disruption of the filament mesh structure, preventing cell movement and changing cell morphology by binding to intracellular filaments and inhibiting actin polymerization at this point [1]. Meanwhile, they are also a large group of fungal polyketide nonribosomal peptide products with remarkable biological activities and structural diversity [2]. Recently, bioactive cytochalasins with various skeletons keep springing up, indicating they are a hot spot of natural product research [3][4][5].
Xylaria was the largest genus of the family Xylariaceae. In the traditional view of this genus, they were saprotrophic fungi that usually appeared on deadwood, participated in the decomposition of organic waste, and even destructed the growth of plants [6]. At the same time, members of this genus are also commonly found in endophytes of vascular plants, so there are many types and contents of cytochalasin in this genus [7,8]. In addition, the type of bioactive compounds in the genus Xylaria also include polyketones [9], alkaloids [10], diphenyl ethers [11], diketopiperazines [12], triterpenoid glycosides [13], alkyl aromatics [14], and cyclic depsipeptides [15], with cytotoxic [16] and antithrombotic activity, acetylcholinesterase (AChE) inhibition, antibacterial activity, and phytotoxic activities [17]. In our previous study on Xylaria curta, a series of cytochalasins including Curtachalasins A-P were characterized [18][19][20]. Of these, Curtachalasins A and B possess an unprecedented pyrolidine/perhydroanthracene (5/6/6/6 tetracyclic skeleton) fusedring system [18]. Considering the potential of Xylariales species to produce undescribed specialized metabolites, we were inspired to further investigate the traceable cytochalasins produced in different fermentation conditions. As a result, six unprecedented cytochalasins, Curtachalasins X1-X6, together with six known compounds were isolated from X. curta E10 (Figure 1). The undescribed structures were established by extensive spectroscopic methods and ECD calculations. All compounds were evaluated by cytotoxicity assay against MCF-7 specialized metabolites, we were inspired to further investigate the traceable cytochalasins produced in different fermentation conditions. As a result, six unprecedented cytochalasins, Curtachalasins X1-X6, together with six known compounds were isolated from X. curta E10 (Figure 1). The undescribed structures were established by extensive spectroscopic methods and ECD calculations. All compounds were evaluated by cytotoxicity assay against MCF-7 cell lines. Herein, we report the isolation, structural determination, and bioactivity of these compounds.

Results
Compound (1) was obtained as a colorless amorphous powder. The molecular formula was deduced as C28H37NO7 from the quasimolecular ion at m/z 522.2463 [M + Na] + (calcd for C28H37NO7Na + , 522.2462) in HRESIMS, indicating eleven degrees of unsaturation. Analysis of the 1 H and 13 C NMR data (Tables 1 and 2)

Results
Compound (1) was obtained as a colorless amorphous powder. The molecular formula was deduced as C 28 H 37 NO 7 from the quasimolecular ion at m/z 522.2463 [M + Na] + (calcd for C 28 H 37 NO 7 Na + , 522.2462) in HRESIMS, indicating eleven degrees of unsaturation. Analysis of the 1 H and 13 C NMR data (Tables 1 and 2) Figure 2. The HBC correlations from H-23 to C-17, from H-19 to C-17 and C-16, and from H-16 to C-19, as well as in combination with the 1 H-1 H COSY correlations, established a six-membered ring D. Moreover, the HMBC correlations from H-21 to C-8, C-14, and C-19 and from H-13 to C-9, C-15 and C-20 established a six-membered ring C, which was fused to ring D via C-14 and C-20. According to the HMBC correlations from H-3 to C-1 and C-9, H-11 to C-4, C-5 and C-6, H-8 to C-4, and C-6 and C-9, the fusing pattern of rings A and B can be determined. In addition, the rings B and C of 1 were suggested to be fused through C-9 and C-8 based on the HMBC correlations between H-20 and C-9, between H-21 and C-4, and between H-13 and C-7. Locations of the acetyl group (δ C 214.8 and 26.7) at C-17 (δ C 84.6) and the phenyl group at C-10 in 1 were determined by HMBC correlations from CH 3 -22 to C-17 and C-18, and from H-10 to C-24 and C-25/29.  H-16 implied that they were cofacial and assigned as β-orientated. In contrast, the NOE correlations of H-15b/H-20 and H-15b/17-OH suggested that they were on the same side, with an α-orientation. Therefore, the relative configuration was determined as shown in Figure 4. Compound 2 was obtained as a colorless powder and had the same molecular formula (C28H37NO7) as compound 1, according to HRESIMS. Their NMR (Tables 1 and 2) signals were almost identical, except that the coupling constant of H-7/H-8 (3.0 Hz) in 2 The absolute configuration of 1 was determined by ECD calculation (Figure 3) on B3LYP-D3(BJ)/6-311G* (IEFPCM, MeOH) level of theory. The calculated ECD curve of the conformers of 1 with 3S, 4R, 7S, 8S, 9R, 13S, 14R, 16S, 17S, 19R, 20R, and 21R-1 matched the experimental ECD well. Therefore, compound 1 can be fully assigned to Curtachalasin X1.   Compound 2 was obtained as a colorless powder and had the same molecular formula (C 28 H 37 NO 7 ) as compound 1, according to HRESIMS. Their NMR (Tables 1 and 2)  confirmed that H-7 should be β-orientated. Finally, to assign the absolute configuration, ECD calculation of 2 was performed at the B3LYP-D3(BJ)/6-311G* (IEFPCM, MeOH) level of theory, and the absolute configuration was deduced as 3S, 4R, 7R, 8S, 9R, 13S, 14R, 16S, 17S, 19R, 20R, and 21R-2 by comparison of the experimental and calculated ECD data ( Figure 3). Therefore, compound 2 can be fully assigned to Curtachalasin X2.   Compound 3 was obtained as a colorless powder with the molecular formula determined to be C 30 H 39 NO 8 by HRESIMS at m/z 564.2585 [M + Na] + (calcd for C 30 H 39 NO 8 Na + , 564.2568). A comparison of the NMR data (Tables 1 and 2) of 3 with 2 indicated that both compounds share the same skeleton, with the only difference between the two compounds being the presence of an acetyl moiety in 3 instead of a hydroxy in 2. In the HMBC experiment, the correlation between H-21 (δ H 5.50) and acetyl group (δ C 172.5) was observed, which suggested that the acetyl group connects to the oxygen at position 21, as shown in Figure 1. The absolute configuration of 3 was assigned as 3S, 4R, 7R, 8S, 9R, 13S, 14R, 16S, 17S, 19R, 20S, and 21R by comparison of the calculated and experimental ECD data (Figure 3), Finally the structure of 3 was established and named as Curtachalasin X3.
Compound 4 was isolated as a colorless amorphous powder and given a molecular formula of C 31 H 41 NO 8  above data revealed that 4 was a tetracyclic cytochalasin bearing two acetyl groups, highly similar to those of 3. The only difference is that the hydroxyl group in 3 is replaced by a methoxyl group in 4, which was supported by HMBC correlations from -OCH 3 (δ H 3.29) to C-7 (δ C 78.1) (Figure 2). By analysis on the 2D NMR spectra, the coupling constants of H-7/H-8 and NOE for the correlation pattern of 4 are similar to 3. Thus, the relative configuration of 4 can be deduced, as shown in Figure 4. The absolute configuration of 4 was determined to be 3S, 4R, 7R, 8S, 9R, 13S, 14R, 16S, 17S, 19R, 20S, and 21R by ECD calculation on the same level as for compound 1 (Figure 3).
Compound 5 was isolated as a colorless powder and given a molecular formula of C 32 H 43 NO 8 on the basis of the HRESIMS at m/z 592.2893 [M + Na] + (calcd for C 32 H 43 NO 8 Na + , 592.2881). The NMR spectra of 5 highly matched with those of 4 (Tables 1 and 3). The only difference is the presence of a methoxyl group at C-13 in 5 instead of 13-OH in 4, as evidenced by the key HMBC correlations from -OCH 3 (δ H 3.27) to C-13 (δ C 80.4). The similar experimental CD spectra ( Figure 3) and the coupling constants between H-7 and H-8 in 4 and 5 indicated that they share the identical absolute configuration. The absolute configuration of 5 was determined to be 3S, 4R, 7R, 8S, 9R, 13S, 14R, 16S, 17S, 19R, 20S, and 21R. The identity of the measured circular dichromism (CD) and the calculated ECD spectra of 5 ( Figure 3) supported this prediction.
All compounds were evaluated for their cytotoxicity. As a result, compounds 1 and 5 showed powerful inhibitory activities with IC 50 values of 2.03 and 0.85 µM, which were more potent than the positive control, cisplatin (IC 50 = 9.12 µM). Comparing the structures of compounds 1 and 2, the configuration of the hydroxyl group at the C-7 position may jointly affect the cytotoxic activity. Comparing compounds 4 and 5, we found that a methoxyl group at C-13 may be a key factor for cytotoxic activity. In addition, the acetylation of OH-21 was also essential for the cytotoxicity of this structure class based on the comparison of 2 with 3 (Table 4). A similar biological property regarding cytotoxic activities has been observed on cytochalasin derivatives previously. For example, xylarichalasin A exhibited moderate cytotoxicity against human cancer cell lines MCF-7 and SMMC-7721 with IC 50 values of 6.3 and 8.6 µM, respectively. Cytochalasin P1 was also found to have strong cytotoxicity against MCF-7 (IC 50 0.71 µM) and SF-268 (1.37 µM) [21].

Culture and Fermentation of Fungal Material
Xylaria curta E10 was isolated from the healthy stem tissues of potato (Solanum tuberosum), which were collected from Dali, Yunnan, China. This isolate was identified according to the ITS sequence (GenBank Accession No. KJ883611.1, query cover 100%, maximum identity 99%). The fungal specimen is deposited at the School of Pharmaceutical Sciences, South-Central Minzu University, Wuhan, China. The strain was fermented by cooked rice medium. The preparation of rice media was 50 g rice with 50 mL water each in 500 mL Erlenmeyer flasks. The fermentation was kept in a dark environment for 30 days at 25 • C (the total weight of rice was 5 kg).

Extraction and Isolation
The rice cultures of X. curta E10 (5 kg) were collected and extracted with methanol at room temperature to yield a crude extract after evaporation under vacuum. The crude extract was partitioned between H 2 O and ethyl acetate three times to give an EtOAc extract. The EtOAc extract was concentrated under reduced pressure to give an organic extract (125 g), which was subjected to silica gel column flushing with  Tables 1 and 2

Quantum Chemical Calculations
Theoretical calculation of ECD spectra for compounds 1-6 were performed with the Gaussian 16 program package. Conformational analysis was initially performed using Spartan 14. The optimized conformation geometries, thermodynamic parameters, and populations of all conformations are provided in Tables S7-S18 in the Supporting Information. The conformers were first identified using the time-dependent density functional theory (TDDFT) method at the B3LYP-D3(BJ)/6-311G* level, and the frequency was calculated at the same level of theory. Then, the theoretical calculations of ECD were performed using TDDFT at B3LYP-D3(BJ)/6-311G* level with PCM in methanol. The final ECD spectra were obtained according to the Boltzmann calculated contribution of each conformer after UV correction.

Cytotoxicity Assay against MCF-7
The cytotoxicity actions of the pure isolated compounds were tested on the breast cancer MCF-7 cell lines using the MTS assay. For this, cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum FBS, 100 U/mL penicillin, and 100 U/mL streptomycin (Invitrogen, Carlsbad, CA, USA), and incubated at 37 • C, 5% CO 2 until 70-80% coverage. Cells were then transferred into 96-well culture plates with appropriate density. The plate was incubated for another 24 h at 37 • C, 5% CO 2 for cell growth and adhesion. Then, the cells were treated with test samples prepared in culture media at different concentrations (0.08, 0.16, 0.31, 0.62, 1.25, 2.5, 5, 10, 20, and 40 µM) for 48 h. The blank control (wells with MTS, without cells) and the negative control (wells with solution, without samples) were performed simultaneously. After 48 h, 20 µL of MTS reagent was added into each well and optical density at 490 nm was read using a Spectra Max M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA) after 1 h incubation at 37 • C, 5% CO 2 , in the dark. The percentage of cell inhibition was calculated based on the equation below. % Inhibition = 1 − Absorbance (sample) − Absorbance (blank) Absorbance (negative) − Absorbance (blank) × 100%

Conclusion
In summary, the structures of six undescribed cytochalasins with a tetracyclic skeleton (1-6) were determined unambiguously by extensive spectroscopic analysis, with the absolute configuration being determined by quantum chemistry calculations. In the cytotoxicity assay, compounds 1 and 5 showed activity against MCF-7 cell lines with outstandingly low IC 50 values (2.03 and 0.85 µM) compared to the positive anti-cancer drug cisplatin. Our studies have certain significances in exploring the structural diversity of cytochalasins.