Arthrinins E–G, Three Botryane Sesquiterpenoids from the Plant Endophytic Fungus Arthrinium sp. HS66

Arthrinins E–G (1–3), three new sesquiterpenoids possessing non-isoprenoid botryane skeleton, were isolated from the fermentation of an endophytic fungus named Arthrinium sp. HS66 which colonized in the stems of Isodon xerophilus. Their structures were determined by extensive spectroscopic methods. Furthermore, the structure of 1 was unambiguously confirmed by X-ray diffraction, while those of 2 and 3 were verified through quantum chemical calculation of NMR data and ECD spectra. Electronic supplementary material The online version of this article (doi:10.1007/s13659-020-00248-y) contains supplementary material, which is available to authorized users.

Fungal endophytes have become an important source for discovering structurally novel and biologically active secondary metabolites [17]. Over the past several years, our groups have made great efforts to study the secondary metabolites from endophytic fungi inhabiting the Isodon species. As a result, isopenicin A, a potent inhibitor of Wnt signaling [18], as well as several antineoplastic compounds like phomopchalasins A and B [19] have been successfully obtained. In the present research, an endophytic fungus colonizing in the stems of Isodon xerophilus was discovered and identified as Arthrinium sp. HS66. Subsequent large fermentation and chemical investigation on this strain resulted in the isolation of three new botryane sesquiterpenoids named arthrinins E-G (1)(2)(3). Notably, compound 2 possesses uncommon 15-nor-botryane skeleton. Herein, details of the isolation, structure elucidation, and cytotoxicity of these compounds were reported (Fig. 1). the existence of three singlet methyls (δ H 0.97, 1.18 and 1.38), one doublet methyl (δ H 1.10, d, J = 6.9 Hz), a pair of nonequivalent oxygenated methylene protons at δ H 4.04 (dd, J = 11.9, 2.1 Hz)/4.33 (d, J = 11.9 Hz), one hydroxylated methylene signal at δ H 3.30 (overlap), and one hydroxylated methine proton at δ H 3.63 (ddd, J = 11.9, 9.6, 3.6 Hz). The analysis of its 13 C NMR and DEPT spectra revealed 15 carbon resonances which were assigned as four methyls, four sp 3 methylenes, three sp 3 methines, two sp 3 quaternary carbons and two olefinic carbons (Table 2). These data suggested that compound 1 might be a botryane sesquiterpenoid [20].
The relative configuration of 1 was deduced by analyses of ROESY and 1 H NMR data. Randomly assigning H-2   as α-oriented, the cross peaks of H-4/H-2 and H-5/H 2 -15, together with the coupling constant (J = 9.6 Hz) between H-4 and H-5 indicated that both H-4 and CH 3 -14 adopted α-orientation, while both H-5 and CH 2 OH-15 adopted β-orientation (Fig. 2). Fortunately, colorless square crystals of 1 were eventually obtained through slow evaporation of methanol. Then a single-crystal X-ray diffraction experiment with Cu Kα radiation of 1 [Flack parameter = 0.07 (3)] unambiguously verified the aforementioned deduction and assigned the absolute configuration of 1 as 2R,4S,5S, and 8S (Fig. 3). Arthrinins A-D from the sponge derived fungus Arthrinium sp. has been reported, so we gave compound 1 the trivial name from arthrinin E [21]. Compound 2 was obtained as colorless oil. Its molecular formula C 14 H 24 O 3 was ascertained by the positive HRESIMS ion peak (m/z 263.1618, [M + Na] + , calcd for 263.1618), indicating three degrees of unsaturation. Comparison of the 1 H NMR spectrum of 2 with that of 1 disclosed that 2 was structurally analogous to compound 1 (Table 1). Furthermore, the 13 C NMR and DEPT spectra, which demonstrated 14 carbons resonances involving three methyls, four sp 3 methylenes, four sp 3 methines, one quaternary carbon and two olefinic carbons ( Table 2), suggested that the structure of 2 was highly similar to that of boledulin C [22], a 15-nor-botryane sesquiterpenoid.
As for the stereochemistry of 3, randomly assigning H-7a as α-oriented, according to the ROESY correlations observed from H-7α to both H 3 -13 and H 3 -14, as well as from H-7β to both H 2 -12 and H 2 -15, it can be concluded that both CH 3 -13 and CH 3 -14 adopted α-orientation. Then, the NMR chemical shifts of (6R*,8S*)-3 were calculated at mPW1PW91-SCRF/6-31 + G(d,p)//B3LYP-D3BJ-SCRF/6-31G(d) level of theory with methanol as solvent and SMD solvent model, the predicted chemical shifts matched their experimental counterparts very well (Table 3), which supported the above deduction concerning the relative configuration of 3. Furthermore, the theoretical ECD spectrum of (6R,8S)-3 was obtained at CAM-B3LYP-SCRF/def2-SVP// B3LYP-D3BJ-SCRF/6-31G(d) level of theory in MeOH with SMD solvent model, and the calculated curve matched the experimental one very well and thus supported the absolute configuration of 3 to be 6R,8S. (Fig. 5). Compound 3 was given the trivial name arthrinin G.

General Experimental Procedures
Optical rotations were measured with a JASCO P-1020 polarimeter. UV spectra were obtained using a Shimadzu UV-2401 PC spectrophotometer. A Tensor 27 spectrophotometer was used for scanning IR spectroscopy with KBr pellets. 1D and 2D NMR spectra were recorded on Bruker DRX-600 and 500 spectrometers with TMS as internal standard. Chemical shifts (δ) are expressed in parts per million (ppm) with reference to the solvent signals. HRESIMS was performed on an API QSTAR spectrometer. Semipreparative HPLC was performed on an Agilent 1200 liquid chromatograph with a Zorbax SB-C18 (9.4 mm × 250 mm) column. LC-MS/MS was performed on Agilent 6530 Accurate-Mass Q-TOF spectrometer coupled to an Agilent 1290 LC system with Zorbax SB-C18 (9.4 mm × 250 mm) column. Column chromatography was performed with silica gel (100-200 mesh, Qingdao Marine Chemical, Inc., Qingdao, People's Republic of China). Fractions were monitored by TLC, and spots were visualized by heating silica gel plates sprayed with 10% H 2 SO 4 in EtOH.

Fungal Material, Identification and Fermentation
The fungal strain of Arthrinium sp. HS66 was isolated from the fresh stems of Isodon xerophilus that was collected from Kunming Botanical Garden, Kunming City, Yunnan Province, People's Republic of China, in August 2018. The isolate was identified based on sequence (GenBank Accession No. MT355097) analysis of the ITS region of the rDNA. The fungal strain was cultured on slants of potato dextrose agar at 28 °C for 7 days. Agar plugs were cut into small pieces (about 0.5 × 0.5 × 0.5 cm 3 ) under aseptic conditions, and 15 pieces were used to inoculate three Erlenmeyer flasks (500 mL), each containing 200 mL of media (0.4% glucose, 1% malt extract, and 0.4% yeast extract); the final pH of the media was adjusted to 7.0, and the flasks were sterilized by autoclave. Three flasks of the inoculated media were incubated at 28 °C on a rotary shaker at 170 rpm for 7 days to prepare the seed culture. The detailed lager fermentation procedure was as following: Fermentation was carried out on solid rice medium in 125 Fernbach flasks (500 mL, 90 mL distilled water was added to 80 g rice and kept overnight before autoclaving). Each flask was inoculated with 5.0 mL of the spore inoculum and incubated for 30 days at 28 °C in a static incubator.

Cytotoxicity Assay
Five human cancer cell lines, human myeloid leukemia HL-60, lung cancer A-549 cells, hepatocellular carcinoma SMMC-7721, breast cancer MCF-7, and colon cancer SW480, were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured according to the manufacturer' recommendations. All mediums were supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin G sodium and 100 μg/ml streptomycin (HyClone). All the cells were incubated at 37 ℃, 5% CO 2 in a humidified atmosphere. Cytotoxicity of compounds was determined by MTS method. Briefly, 5 × 10 3 cells were plated in 96-well plates 12 h before treatment and continuously exposed to test compounds for 48 h. Then MTS (Promega) was added to each well. The samples were incubated at 37 ℃ for 1-4 h and the optical density (OD) was measured at 490 nm using a microplate reader (Bio-Rad Laboratories). The IC 50 values were calculated by Reed and Muench's method [24].

Extraction and Isolation of Compounds 1-3
The culture medium was overlaid and extracted with MeOH by maceration. With filtration and concentration, the resultant extract was partitioned with EtOAc. Then the solvent was evaporated in vacuo to afford a crude extract (130 g).
The extraction was subjected to column chromatography on silica gel with a CHCl 3 /Me 2 CO gradient system (1:0

Computational Method
Conformational searching of (1R*,2R*,4S*,5S*)-2 and (6R*,8S*)-3 were undertaken with the CREST code (version 2.8) using the default iMTD-GC procedure [25]. The first 20 conformers of (1R*,2R*,4S*,5S*)-2 and (6R*,8S*)-3 were subjected to DFT geometry optimization at B3LYP-D3BJ-SCRF/6-31G(d) level of theory (with MeOH as solvent and SMD solvent model). Frequency analyses of all optimized conformers were undertaken at the same level of theory to ensure that no imaginary frequency exists. Then, thermal correction to Gibbs free energies obtained by frequency analyses were added to the electronic energies obtained at B3LYP-D3BJ-SCRF/6-311 + G(d,p) level of theory (with MeOH as solvent and SMD solvent model) to get the Gibbs free energies of each conformer. Subsequently, Room-temperature (298.15 K) equilibrium populations were calculated according to Boltzmann distribution law: where P i is the population of the ith conformer; n i the number of molecules in ith conformer; ΔG is the relative Gibbs free energy (kcal/mol); T is room temperature (298.15 K) here; R is the ideal gas constant (0.0019858995). Those conformers with a population of over 2% were subjected to subsequent NMR and ECD calculations.
NMR shielding constants were calculated with the GIAO method at mPW1PW91-SCRF/6-31 + G(d,p) level (with MeOH and SMD solvent model). The obtained shielding constants were converted into chemical shifts by referencing to TMS at 0 ppm (δ cal = σ TMSσ cal ), where the σ TMS was the shielding constant of TMS calculated at the same level of theory. The parameters a and b of the linear regression δ cal = aδ exp + b; the correlation coefficient, R 2 ; the mean absolute error (MAE) defined as Σ n |δ calδ exp |/n; the corrected mean absolute error (CMAE) defined as Σ n |δ corr δ exp |/n, where δ corr = (δ calb)/a were calculated [26,27]. Calculation of coupling constants were run at B972/pcJ-1 level of theory (with MeOH as solvent and SMD solvent model) [28].
TDDFT ECD calculations were run at CAM-B3LYP/ def2-SVP level of theory (with MeOH as solvent and SMD solvent model) [29]. For each conformer, 30 excited states were calculated. The calculated ECD curves were generated using the Multiwfn software (version 3.7) [30].
The geometry optimization, single-point energy calculation, NMR shielding constant calculation, coupling constant calculation, and TDDFT ECD calculation were all completed in Gaussian 09 program [31].