A new resorcylic acid lactone from the endophytic fungus Chaetosphaeronema sp.

Abstract A new 14-membered resorcylic acid lactone (RAL14), chaetolactone A (1), along with three known ones (2−4), was obtained from the fermentation of the soil-derived fungus Chaetosphaeronema sp. SSJZ001. Their structures were established based on extensive spectroscopic data analyses (UV, IR, HRESIMS, 1D, and 2D NMR),13C NMR chemical shifts calculations coupled with the DP4+ probability method, theoretical calculations of ECD spectra, as well as X-ray diffraction analysis. All compounds were evaluated for their cytotoxic effects against A549, HO-8910, and MCF-7 cell lines.


Results and discussion
Chaetosphaeronema sp.SSJZ001 was fermented, and the resulting cultures were extracted three times with EtOAc.The organic extract was then fractionated using silica gel column chromatography (CC), followed by Sephadex LH-20, led to the RALs containing fractions FC2, FC3, and FC5, showing characteristic HPLC profiles with PDA data.Thus, these fractions were subjected to further purifications using preparative and semipreparative HPLC to yield a new RAL 14 and three known ones.The known compounds, zearalenone (2) [24], α-zearalenol (3) [25], and 4-methoxy-α-zearalenol (4) [26], were characterized by comparing their spectroscopic data with the published values.Furthermore, the structure and the absolute configuration of zearalenone (2) was confirmed by X-ray crystallography using Cu Kα radiation (Figure 2).
The new RAL 14 , chaetolactone A (1), obtained as a white amorphous powder, has a molecular formula of C 19 H 26 O 6 as determined by (+)-HRESIMS data, requiring 7 degrees of unsaturation.The UV spectrum of 1 showed absorptions at 203, 233, 272, and 316 nm, respectively.The 1 H and 13 C NMR spectroscopic data (Table 1), with the aid of the HSQC data, displayed signals for two aromatic methines [δ H 6.46 and 6.38 (each 1H, d, J = 2.7 Hz); δ C 109.4 and 100.9], an aromatic methoxy group [δ H 3.81 (3H, s); δ C 55.9], an ester carbonyl (δ C 173.0), and four sp 2 carbons (δ C 167.1, 165.6, 145.1, and 104.2), which are characteristic of a 5-O-methyl-resorcylic acid moiety [26].The remaining proton and carbon resonances were ascribed to three oxygenated methines, five methylenes, one methyl group, and a trans double bond [δ H 7.11 and 5.71, with the coupling constant of 15.2 Hz (J 8,9 ); δ C 134.2 and 134.6].The aforementioned information implied that 1 was a RAL 14 derivative that was supported by the key HMBC correlations from H-17 to C-1/C-15/C-16/C-18, from H-8 to C-10/C-6/C-7/C-2  (Figure 3).The 1 H and 13 C NMR data of 1 showed similarity to those of 4, suggesting the structural analogs of these two compounds.The major difference between the NMR data of 1 and 4 was the absence of a methylene group in 1 which was present in 4. A new oxygenated methine was observed at δ H 3.58 and δ C 66.0 in 1.With the help of HSQC spectroscopic data, the proton and hydrogen-bearing carbon resonances in the NMR data of 1 were assigned unambiguously.Combined with the HRESIMS data, it could be inferred that there was an additional hydroxyl group at C-15 in 1 compared to 4, which was confirmed by the 1 H−  3).Based on these results, the complete planar assignment for compound 1 was confirmed.
In the NOESY data, NOE interactions were observed between H 3 -18/H-16β, H-16β/H-13, H-13/H-10β, H-17/H-15, H-17/H-16α, and H-15/H-12α, suggested the α-orientation of OH-13 and β-orientation of OH-15, respectively (Figure 3).In order to confirm the relative configuration of 1, the computationally calculated 13 C NMR chemical shifts of four diastereomers (13S*, 15R*-1, 13 R*, 15R*-1, 13 R*, 15S*-1, and 13S*, 15S*-1) were compared to the experimental data for 1.The DP4 + probability analysis was conducted, and the results indicate that 13 R*, 15S*-1 appeared consistent with the experimental data with the probability of 100%.Because the carbon backbones of 1-4 are likely to be formed by the same biosynthetic pathway, the absolute configuration of the Me-18 of these compounds is assumed to be the same.Hence, the absolute configuration of 1 was identified as 13 R, 15S, 17S which was supported by the comparation of calculated and experimental ECD data (Figure 4).Therefore, the structure of chaetolactone A (1) was elucidated as shown in Figure 1.

Fungal material
The fresh healthy roots of Gymnadenia orchidis Lindl.were collected from Huangnan Tibetan autonomous prefecture, Qinghai Province, China, in October 2018.The selective roots were cut into pieces about 5 cubic millimeters and surface sterilized by soaking in 75% ethanol for 30 s, followed by placing in 5% NaOCl for 10 min.Then the root pieces were rinsing in sterile water and then cultured in Potato Dextrose Agar (PDA) plate culture-medium at 20 °C without light.One of the dominant fungal isolations, Chaetosphaeronema sp. was identified by DNA amplification and sequencing of ITS region.A voucher strain (no.SSJZ001) was deposited at College of Pharmaceutical Sciences, Qinghai University for Nationalities.

Extraction and isolation
The fungus Chaetosphaeronema sp.SSJZ001 was inoculated in the solid medium of sterile rice in 30 × 1 L conical flasks (the culture-medium was prepared as described previously [19]).After incubation at 28 °C for 30 d, the fermented material was extracted with EtOAc by using ultrasonic, and the solution was evaporated to dryness under reduced pressure to afford an EtOAc extract (43.2 g).

Cytotoxicity assay
The cytotoxic activities were determined using the MTT colorimetric method as previously described [9].Compounds in DMSO were serially diluted with culture medium.Each compound was prepared at final concentrations of 50, 25, 10, 5, 1, and 0.5 μM, respectively.Triplicate experiments were conducted for each concentration.Adriamycin was used as a positive control.The inhibitory rate of cell growth was calculated according to the following formula: Inhibition rate (%) = [1 − (OD treated − OD control )/(OD control − OD blank )] × 100%.IC 50 values were determined by nonlinear regression analysis of logistic dose − response curves (SPSS version 23.0 statistic software; SPSS Inc., Chicago, IL).

Computational methods
See Supplementary information.

Disclosure statement
No potential conflict of interest was reported by the authors.

Table 1 .
nMr data (δ) were measured at 400 Mhz for 1 h nMr and at 100 Mhz for 13 c nMr. Proton coupling constants (J) in hz are given in parentheses.the assignments were based on 1 h-1 h cosY, hsQc, hMBc, and noesY experiments.