New chlamydosporol derivatives from the endophytic fungus Pleosporales sp. Sigrf05 and their cytotoxic and antimicrobial activities

Five new chlamydosporol derivatives, named pleospyrones A-E (1–5), together with one known congener (6), were isolated from the culture of the endophytic fungus Pleosporales sp. Sigrf05, obtained from the medicinal plant Siraitia grosvenorii. The structures of the new compounds were elucidated mainly by analysis of the HRESIMS, and (1D, 2D) NMR data, while ECD and optical rotation calculations were used to assign the absolute configurations. The plausible biosynthetic pathway of these compounds were proposed. The isolated compounds were evaluated for their cytotoxicity, antifungal and antibacterial activities. Compounds 1, and 4–6 were cytotoxic against the tested cancer cells with IC50 values of 1.26~47.5 μM. Compounds 1–3 showed moderate antifungal activities against Magnaporthe oryzae, while compound 5 displayed weak antibacterial activity.

carbons), two oxygenated methines (δ C 94.8, 65.7), two methoxyl groups (δ C 57.5, 56.0), and one methyl group (δ C 18.1), by the aid of HMQC experiment. Inspection of the 1 H NMR spectrum (Table 1), revealed the presence of three olefinic protons at δ H 5.66 (s), 5.95 (br. s), 5.47 (br. s), respectively, with the latter two protons attached to one carbon (δ C 114.6, CH 2 ), as indicated by the HMQC spectrum. In addition, one dioxygenated methine (δ H 5.38, s), one oxygenated methine appeared as a quartet (δ H 4.76, q) that obviously bonded to a methyl group (δ H 1.47, d), along with two methoxyl groups (δ H 3.90, 3.47) were discerned. The aforementioned functionalities only account for four degrees of unsaturation, meaning that the structure must be bicyclic to fulfill the remaining degrees of unsaturation.
The planar structure of 1 was established by analysis of the HMBC spectrum ( Fig. 2A). A 2-pyrone moiety was constructed firstly, as correlations were seen from H-3 (δ H 5.66, s) to C-2 (δ C 165.1), C-4 (δ C 170.4), and C-5 (δ C 109.9), from H-11 (δ H 5.38, s) to C-4, C-5, and C-6 (δ C 154.0), as well as from 4-OMe (δ H 3.90, s) to C-4. The correlations from H 2 -10 (δ H 5.95, 5.47) to C-6, C-7 (δ C 138. 3), and C-8 (δ C 65.7) were used to piece the exocyclic double bond and the pyrone ring together, while correlations from terminal methyl group (CH 3 -9, δ H 1.47, d) to C-7 and C-8, further extend the above moiety to C-9. Another methoxyl group (δ H 3.47, s; δ C 56.0) was positioned at C-11 as it showed correlation to C-11 (δ C 94.8). Additional key correlations of H-11/C-8, and H-8 (δ H 4.76, q)/C-11, were used to establish the gross structure of 1 as shown in Fig. 1. This was corroborated by the NOESY correlations of H-3/4-OMe, and H-10b (δ H 5.47, br. s)/CH 3 -9. The correlation between H-8 and 11-OMe (δ H 3.47, s) indicated that these protons were directed to a same face (Fig. 2B). Thus, the relative configuration of 1 was established.    The absolute configuration was determined by TDDFT ECD calculation, which was a powerful tool in solving the stereochemistry of complex natural products 19 . A randomly selected structure of 1 (8 S, 11 R) was subjected to conformational search using the Merck Molecular Force Field (MMFF), followed by geometry optimization at B3LYP/6-31 G (d) level, which resulted in only one predominant conformer (Fig. 2B). In this conformer, the dihydropyran ring adopted a half-chair conformation, in which H-8 was pseudo-axial, while H-11 was pseudo-equatorial. Subsequent TDDFT ECD calculation of this conformer was carried out at PBE0/TZVP level with the PCM solvent model in MeOH. The measured CD spectrum of 1 displayed strong positive Cotton effects at 210 and 220 nm, and a weak positive at 250 nm, while a strong negative peak at 231 nm, reflecting a strong interaction between the intrinsic chromophores. The calculated spectrum reproduced well these peaks as shown in Fig. 3, therefore, the absolute configuration of pleospyrone A (1) was established as 8 S, 11 R.
Pleospyrone B (2) was isolated as a pale-yellow oil. Its molecular formula was determined as C 11 H 12 O 5 by HRESIMS. The NMR data of 2 were similar to those of 1 (Table 1), indicating it to be a 2-pyrone derivative. Detailed comparison of the data revealed that the exocyclic double bond of 1 was missing in 2, instead, one methyl group (δ H 1.31, d; δ C 10.4, CH 3 -10) and a methine group (δ H 2.76, qd; δ C 37.0, C-7) were present, suggesting that the C-7/C-10 double bond was reduced in 2. In addition, the acetal group in 1 was replaced by an ester group (δ C 159.4, C-11) in 2, which could explain the significant downfield shift of C-6 (+21.4 ppm), while C-5 was upfield shifted (-8.9 ppm). This was proved by analysis of the HMBC spectrum, in which correlations from CH 3 -10 to C-6 (δ C 175.4), C-7, and C-8 (δ C 73.9), from H 3 -9 (δ H 1.43, d) to C-7, and C-8, from H-8 (δ H 4.69, qd) to C-11, and long-range correlation from H-3 (δ H 5.53, s) to C-11 were observed (Fig. 4). The small coupling constant (3.0 Hz) between H-7 and H-8 indicated the cis relationship for both protons, which was similar to that of inflatin C 2 . The absolute configuration of 2 was also assigned by quantum chemical ECD calculations. A randomly selected configuration of 2 (7 R, 8 S) was used for the calculation. Geometry optimization of the MMFF conformers at B3LYP/6-31 G(d) in vacuo resulted in two major conformers (2a, 94.6%, and 2b, 5.3%, see Supplementary Fig. S1), in which one methyl was pseudo-equatorial, while the other was pseudo-axial with regard to the half-chair conformation of the 2-dihydropyrone ring. Subsequent TDTFT ECD calculations of both conformers at the CAMB3LYP/TZVP (PCM = MeOH) level gave an almost opposite ECD spectrum except in the long-wavelength region (ca. 273~310 nm, Supplementary Fig. S2), and the resulting Boltzmann-averaged spectrum was opposite to the experimental spectrum of 2 ( Supplementary Fig. S2). On the contrary, the spectrum for (7 S, 8 R)-2 matched well the experimental one (Fig. 5). Therefore, the absolute configuration of 2 was assigned as 7 S, 8 R.
Pleospyrone C (3) was isolated as an isomer of 2, and both have a same molecular formula. Inspection of the NMR data (Table 1) revealed their great similarities, and the differences were only ascribed to the different  www.nature.com/scientificreports www.nature.com/scientificreports/ orientation of the methyl groups. The large coupling constant (9.1 Hz) between H-7 and H-8 was reminiscent of their trans relationship, similar to that of isochlamydosporol 2 . With this relative configuration in hand, the absolute configuration of 3 was then assigned by comparing the calculated and experimental ECD spectra. An arbitrary input structure of 3 (7 S, 8 S) was subjected to conformational search and geometry optimization at the B3LYP/6-31 G(d) level, which resulted in two predominant conformers (3a, 24.7%, and 3b, 75.2%, see Supplementary Fig. S3), where both methyl groups were pseudo-equatorial in 3b, while pseudo-axial in 3a. These two conformers also give an opposite spectrum except in the high wavelength range (ca. 277~310 nm, Supplementary Fig. S4). The Boltzmann-averaged spectrum of (7 S, 8 S)-3 gave an opposite image to the recorded spectrum of 3 ( Supplementary Fig. S4). Then, the absolute configuration of 3 was determined to be 7 R, 8 R (Fig. 5) by comparing the spectrum of (7 R, 8 R)-3 with the experimental spectrum. Compound 3 was thus determined to be a 7-epimer of 2. Interestingly, their ECD spectra were quite similar, while only opposite in the long-wavelength range (ca.270-320 nm) (Fig. 5). Such differences were attributed to a change in the orientation of CH 3 -10 (axial in 2a, vs equatorial in 3b).
[α] 24 D -21.8 (c 0.50, MeOH)). Pleospyrone E (5) was determined to be the 8-acetyl derivative of the co-isolated clearanol A (6) 18 , by comparison of their NMR data and analysis of the HMBC spectrum. The key HMBC correlation from H-8 (δ H 5.47, q) to the carbonyl (δ C 170.3) of the acetyl group was seen. The absolute configuration of 5 was determined to be the same as that of clearanol A (6) by the observation of a similar CD profile between both compounds ( Supplementary Fig. S6). Clearanol A was previously isolated from the fungus derived from a microbial mat collected from an iron-rich natural spring 18 .
In this study, six polyketides (1-6) with an α-pyrone motif were isolated, and their plausible biosynthetic pathways were proposed (Fig. 7). The PKS could incorporate one acetyl CoA, three malonyl CoA, and two S-adenosyl methionines (SAM) to build up the tetraketide (S1), which was released by esterification to give the α-pyrone, followed by O-methylation at C-4 to produce S2. Oxidation at C-11 should give the oxygenated derivatives (S3), and reduction of the 8-keto group would give the 8-hydroxy derivatives. Lactonization of the 8-hydroxylated S 3 c should give the lactone-pyrones (2 and 3), while dehydration of the 8-hydroxylated S 3 a and S 3 b would give the Δ 7 derivatives (S4), which could be epoxidized to produce the epoxides S5. Reduction of the epoxide would give  www.nature.com/scientificreports www.nature.com/scientificreports/ 4, while the nucleophilic attack of H 2 O to the epoxy ring could produce the vicinal diols S6, which were then dehydrated to give 6 and 1a. Further acetylation of 6 should give rise to 5. The attack from 8-OH to the aldehyde group in 1a, followed by O-methylation could produce 1.
cytotoxic and antimicrobial activities. The six isolated α-pyrone derivatives (1-6) were structurally related to chlamydosporol, a mycotoxin widely occurred in Fusarium spp 1 ., and they differed only in the eastern part (i.e. non-α-pyrone part). It was found that chlamydosporol displayed cytotoxicity to mouse and human fibroblast cells, caused feed refusal and weight loss of rats, and mortality to chick embryos 5 , and also exhibited toxicity to Artemia salina larvae 20 . Hence, the cytotoxicities of compounds 1-6 were also evaluated ( Table 2). Compounds 1, and 4-6 were active against the tested cancer cell lines including HCT-116, HepG2, BGC-823, NCI-H1650 and Daoy, with IC 50 values in the range of 1.26~47.5 μM, though not as active as the positive control (taxol), while 2 and 3 were inactive (IC 50 > 50 μM). Among them, compound 5 was active against all the five tested cell lines, with IC 50 s of 1.17~20.7 μM, compound 1 was effective against all the cell lines except HCT-116 (IC 50s 1.26~15.1 μM), whereas compounds 4 and 6 were selectively cytotoxic against NCI-H1650 cells with IC 50 values of 29.6, and 47.5 μM, respectively. It seemed that the substituents at C-7 had strong influence to the activity, when C-7 was a methine as in 2 and 3, the compounds were inactive, while when it was a quaternary carbon, as in 1, and 4-6, the cytotoxicity was increased. In addition, the most potent compounds (i.e, 1 and 5) all had a C7/C10 exocyclic double bond. However, the loss of one acetyl group, as in the case of 6 vs 5, the activity was significantly decreased, meaning that a suitable lipophilicity was also important for the cytotoxicity. More structures should be evaluated to get a better understanding of the structure-activity-relationship. And the mechanism of action towards these cells is yet to be revealed.
The isolated compounds were further evaluated for their antifungal activities (Table 3). Among them, compounds 1-3 showed moderate inhibition against the spore germination of the rice blast fungus Magnaporthe oryzae with IC 50 value of 98.73, 47.77, and 51.08 µg/mL, respectively, while the other compounds did not display any significant activity when tested at a concentration of 200 µg/mL. It was interesting that all the active compounds were bicyclic, whereas the monocyclic compounds (i.e., 4-6) were inactive. More derivatives should be evaluated to get a better structure-activity-relationship.  conclusion Six chlamydosporol congeners, including five new pleospyrones A-E (1-5), and the known clearanol A (6) were isolated from the endophytic fungus Pleosporales sp. Sigrf05 that resided inside the medicinal plant Siraitia grosvenorii. The absolute configuration of the new compounds was unambiguously determined by applying the quantum chemical calculations (ECD and optical rotation). The occurrence of these polyketides added a new diversity to the α-pyrone class of natural products, and also reflected the presence of abundant post-PKS tailoring enzymes in their biosynthesis, which merited further study. Compounds 1, and 4-6 were cytotoxic, while compounds 1-3 displayed moderate antifungal activities. A preliminary structure-activity-relationship analysis revealed that the substituents at C-7 and the lipophilicity were relevant to the cytotoxicity, while bicyclic structures were important for the antifungal activities. These polyketides could be interesting candidates for the development of cytotoxic and antimicrobial agents.
The fungus was grown on potato dextrose agar (PDA) plates at 25 °C for 8~10 days. Then, four to five plugs of agar medium (0.5 cm × 0.5 cm) with fungal hyphae were transferred to several 250 mL Erlenmeyer flasks each containing 100 mL potato dextrose broth (PDB) medium to prepare the seed culture, and incubated on a rotary shaker at 150 rpm and 25 °C for 7 days. The scale-up fermentation was carried out in 1 L-Erlenmeyer flasks, each containing 150 g rice and 150 mL distilled water. Each flask was inoculated with a seed culture. The static fermentation was kept at 25 °C for approximately 45 days before harvest. extraction and isolation. The fermented rice substrates in 25 flasks were combined, and extracted with MeOH by exhaustive maceration (3 × 10 L) at room temperature. After filtration, the filtrate was concentrated under vacuum at 40 °C. The brown residue was suspended in water and sequentially partitioned with petroleum ether, EtOAc, and n-BuOH to give their corresponding fractions. Then, the EtOAc extracts were combined and concentrated under vacuum at 40 °C to obtain a brownish residue (40.8 g).
Fraction D (5.5 g) was processed in the same manner as that of subfraction C4 using MeOH-H 2 O (38:62) as eluent to afford compound 6 (4.5 mg).
Pleospyrone E (5 Antifungal assay. The antifungal activities of 1-6 were tested against the rice blast pathogen M. oryzae using the spore germination assay 21 . Briefly, the tested compounds were dissolved in 10% aqueous ethanol (25 μL) and mixed with a same volume of spore suspension (2 × 10 6 spores/mL) that prepared from the 7-day old cultures of M. oryzae, on a concave glass slide. Slides containing the spores were incubated in a dark moist chamber at 25 °C for 8 h, then the germination status was inspected under a microscope. 10% ethanol was used as the blank control. Carbendazim was used as the positive control. The percentage (%) of inhibition was determined as [(Gb − Gt)/ Gb] × 100, where Gb is the average numbers of the germinated spores in the blank control (n = 3), and Gt is the average numbers of those in the treated sets (n = 3). The half-inhibition concentration (IC 50 ) of each sample was then calculated by linear regression.
Antibacterial assay. The antibacterial activities of 1-6 were tested against four pathogenic bacteria including B. subtilis, A. tumefaciens, R. solanacearum, and X. vesicatoria using the modified broth micro-dilution-MTT assay as described previously 21 . The bacteria were grown in liquid LB medium overnight at 28 °C, and the diluted bacterial suspension (10 6 cfu/mL) was used for the assay. Streptomycin sulfate was used as the positive control, which showed minimum inhibition concentration (MIC) of 12.5, 25.0, 12.5, and 25.0 µM, respectively, towards these bacteria. www.nature.com/scientificreports www.nature.com/scientificreports/ at 1200 cells/well. After 24 h, the tested compounds were added to the cells. Cell viability was determined after a further treatment of 96 h by using the MTT assay 22 . The UV absorption of each well at 570 nm were read using a plate reader. Compounds dissolved in DMSO (final DMSO concentration of 0.1% in each well), were tested in five concentrations with each concentration tested in triplicate. IC 50 values were then calculated using Microsoft Excel software. Taxol was used as the positive control. ecD calculation. The Molecular Merck force field (MMFF) conformational search, geometry optimization and frequency calculations at the B3LYP/6-31 G(d) level in vacuo, and TDDFT ECD calculations of the dominant conformers (>1%) at different levels (PBE0/TZVP and CAMB3LYP/TZVP) with the polarizable continuum model (PCM) for MeOH, were performed as described previously 23 . ECD spectrum of each conformer was plotted with the program SpecDis 24 using the dipole-length computed rotational strengths with Gauss curve and exponential half-width (σ) of 0.16 ev for 1, and 0.3 eV for 2-4, respectively. The equilibrium population of each conformer at 298.15 K was calculated from its relative Gibbs free energies using Boltzmann statistics. The Boltzmann-averaged ECD spectra for (8 S, 11 R)-1, (7 R, 8 S)-2, (7 S, 8 S)-3, and (7 S)-4 were generated according to the equilibrium population of the lowest energy conformers of each structure, while the calculated spectra for (7 S, 8 R)-2 and (7 R, 8 R)-3 were obtained by mirroring those of the enantiomers. The calculated spectra were then compared with the experimental spectra to determine the absolute configuration. The calculated ECD spectra were scaled (y-axes) (by 0. 5 for 1 and 4, 0.05 for 2, and 0.1 for 3) for a better comparison with the experimental data.
optical rotation calculation. The b3lyp/6-31 g(d)-optimized conformers of (7 S)-4 were used to calculate the optical rotations (OR). The OR calculations were carried out using the time-dependent DFT methods at the B3LYP/6-31 + G(d,p) level, with the PCM model for methanol, as described previously 23 . Boltzmann statistics analysis was employed to calculate the overall OR.