Highly oxygenated caryophyllene-type and drimane-type sesquiterpenes from Pestalotiopsis adusta , an endophytic fungus of Sinopodophyllum hexandrum

Four new highly oxidized caryophyllene-type sesquiterpenes ( 1 – 4 ), one new caryophyllene-type natural product ( 5 ), and two new drimane-type sesquiterpenes ( 6 , 7 ), along with eight known compounds have been isolated from an endophytic fungus Pestalotiopsis adusta . Their structures were elucidated on the basis of HRESIMS, 1D and 2D NMR spectroscopic data analyses. The absolute con ﬁ gurations for 1 and 5 were determined by single-crystal X-ray crystallographic analysis using Cu K a radiation. The absolute con ﬁ guration of 6 was determined by CD spectrum associated with TD-DFT calculation of its benzoylated derivative 6a . The in vitro bioassay revealed that 8 and 9 presented cytotoxicity against cancer cell lines A549, HeLa, and SMMC-7721, in which 8 showed comparable activity (IC 50 ¼ 28.3 m M) to the positive control etoposide (IC 50 ¼ 23.2 m M) against SMMC-7721.


Introduction
Plant endophytic fungi are well known for their potential to produce abundant secondary metabolites and contribute greatly to the discovery of new medicines. 1,2The fungal genus of Pestalotiopsis is one of the most commonly found endophytes on the planet, 234 species of which are listed in Index Fungorum. 3Since a broad-spectrum antitumor drug taxol identied from P. microspora, a plant endophyte of Taxus wallachiana, exploring for the bioactive secondary metabolites from endophytes of the Pestalotiopsis genus of different plants has attracted much attention. 4Species of Pestalotiopsis are proved to be a rich source for natural products, and the chemical investigations of Pestalotiopsis spp.[9][10] b-Caryophyllene, a sesquiterpene characteristic of a strained four-membered ring and a exible nine-membered macrocyclic moiety, exists popularly in the essential oils from medicinal plants.It acts as a key precursor in nature to form tricyclic and tetracyclic highly oxygenated caryophyllene-type sesquiterpenes by transannular rearrangements. 11Its highly oxygenated derivatives were proved to possess signicant bioactivities, including immunosuppressive activity, 7 affecting growth and proliferation of numerous cancer cells, 12 antibacterial activity, 13 and anti-inammatory activity. 14Although b-caryophyllene is widely distributed, its highly oxygenated derivatives are not common. 76][17][18][19][20][21][22][23][24] The highly oxygenated caryophyllenetype sesquiterpenes also attracted the interests of synthetic chemists due to their structural characteristics and special bioactivities. 25n our continuous screening for bioactive natural products from plant endophytes, [26][27][28] we investigated an endophytic fungus Pestalotiopsis adusta collected from the stem bark of wild rare medicinal plant Sinopodophyllum hexandrum (Royle) Ying in Qinling Mountains.The chemical investigation on the solid culture of P. adusta led to the isolation of four new highly oxidized caryophyllene-type sesquiterpenes pestalotiopsins D-G (1-4), two new drimane-type sesquiterpenes pestalotiophol A and B (6, 7), along with nine known compounds pestalotiopsin H (5), 25 pestalotiopsin C (8), 29 pestalotiopsin A (9), 29 pestalotiopsin B (10), 29 punctaporonin H (11), 30 punctatin A (12), 31 punctaporonin B (13), 30 2a-hydroxydimeninol ( 14), 32 and 7-hydroxy-5-methoxy-4,6-dimethylphthalide (15), 33 of which 5 was a new natural product (Fig. 1).Details of the isolation, structure elucidation, and cytotoxicity of these secondary metabolites are reported herein.
This journal is © The Royal Society of Chemistry 2017 ray diffraction, a suitable low Flack parameter 1.1(10) effectively suggested 1 as 1S, 2S, 4R, 5R, 6R, 7R, 8S, 9R, 14S (Fig. 3).Therefore, 1 was elucidated and named as pestalotiopsin D. Pestalotiopsin E (2), obtained as a colorless oil, had a molecular formula of C 20 H 32 O 6 with ve degrees of unsaturation based on its HRESIMS analysis (m/z 391.2090 [M + Na] + , calcd for 391.2096).The 1 H NMR (Table 1) spectrum revealed that 2 had three methoxyls  2), along with DEPT 135 and HSQC, revealed the existence of twenty carbon atoms including one acetoxyl (for its methyl and carbonyl), three methoxyls, three tertiary methyls, two methylenes, seven methines (one olenic and four oxygenated), and three quaternary carbons (one olenic).One acetoxyl and one double bond accounting for two of the ve indices of unsaturation illustrated the presence of a tricyclic ring system.All the proton signals were well assigned to their relevant carbons by HSQC experiment.The 1 H-1 H COSY spectrum established two isolated systems (Fig. 2).Comparing the NMR data of 2 with those of 1, revealed that they possessed identical carbon skeleton, with the only difference for C-4 12, H 3 -13, and H 3 -15 were also located by HMBC correlations (Fig. 2).ROESY correlations of H-2 with H 3 -13/H-9/H 3 -15, and H 3 -15 with H-6 suggested that H-2, H-6, H-9, H 3 -13 and H 3 -15 had the relative conguration of a. ROESY correlations of H-14 with H-7/H-8 revealed that H-7, H-8 and H-14 had the relative conguration of b.No observation of ROESY correlation between H-5 with H 3 -15 indicated a E-geometry for the double bond 7 (Fig. 2).At last, 2 was elucidated and named as pestalotiopsin E.
Pestalotiopsin F (3), obtained as a colorless oil, had a molecular formula of C 19 H 28 O 5 with six degrees of unsaturation based on its HRESIMS analysis (m/z 359.1834 [M + Na] + , calcd for 359.1834), which was 18 atomic mass units less than that of pestalotiopsin D (1).Comparing the NMR data of 3 (Tables 1 and 2) with those of 1 and on the basis of its 1 H-1 H COSY and HMBC (Fig. 2) revealed that they possessed identical carbocyclic cores.The difference was the one side oxygenated single bond between C-4 (d C 74.6) and C-5 (d C 56.8 and d H 2.88) in 1 transferred to a non-oxygenated double bond (d C 129.7 for C-4 and 133.8 for C-5) in 3, it was clear that 1 experienced the loss of a H 2 O, this was also supported by the molecular weight difference between 1 and 3. ROESY correlations of H-2 with H 3 -13/H-9 and H 3 -15 with H-6 but no correlation of H-6 with H-7 suggested that H-2, H-6, H-9, H 3 -13 and H-15 had the relative conguration of a. ROESY correlations of H-14 with H-7/H-8 revealed that H-7, H-8 and H-14 had the relative conguration of b (Fig. 2).Finally, 3 was elucidated and named as pestalotiopsin F.
Pestalotiopsin G (4), obtained as white power.Based on the HRESIMS analysis (m/z 359.1833 [M + Na] + , calcd for 359.1834) and the 13 C NMR data, the molecular formula of was established as C 19 H 28 O 5 with six degrees of unsaturation.The NMR data of 4 (Tables 1 and 2) revealed that it had the similar structure with 3, the largest variation being at C-5 (from d C 133.8 in 3 to d C 56.9 in 4) and C-15 (from d C 22.1 for a methyl in 3 to d C 118.3 for a terminal vinyl in 4), implying that the double bond between C-4 and C-5 in 3 fused to a terminal double bond between C-4 and C-15 in 4 (Fig. 2), this was also supported by HMBC correlations (Fig. 2).The NOESY experiment analysis (Fig. 2) indicated that H-2, H-9, H-6 and H 3 -13 had the relative conguration of a, whereas H-5, H-7, H-8 and H-14 had the relative conguration of b.Finally, 4 was elucidated and named as pestalotiopsin G.
Pestalotiopsin H (5) was isolated as colorless oil, its molecular formula was established as C 18 H 26 O 5 with six degrees of unsaturation by the quasi-molecular ion at m/z 345.1674 [M + Na] + (calcd for 345.1678) in the HRESIMS, which was 32 atomic mass units fewer than 1.The 1 H and 13 C NMR spectroscopic data (Tables 1 and 2) revealed 5 had a very similar structural feature to 1.The difference of the downeld shi for C-4 (from d C 74.6 in 1 to d C 79.8 in 5), the upeld shi for C-7 (from d C 85.9 in 1 to d C 77.0 in 5), and the downeld shi for H-7 (from d H 3.66 in 1 to d H 3.97 in 5) were observed.For 5, the HMBC (Fig. 2) correlation between H-7 and C-4, and 32 atomic mass units less than 1, indicated that 1 experienced the loss of a CH 3 OH molecule to produce 5.The relative conguration of 5 was assigned by NOESY experiment (Fig. 2).The structure and spectral data of 5 were consistent with a synthetic intermediate in the literature, 25 and therefore it was deduced as a new natural product and named as pestalotiopsin H. Its absolute conguration (1S, 2S, 4S, 5R, 6R, 7R, 8S, 9R, 14S) was identied by single-crystal X-ray crystallographic analysis with a suitable low Flack parameter 0.02(11) (Fig. 3).
Pestalotiophol A (6), obtained as a colorless oil, had a molecular formula of C 15 H 26 O 3 with three degrees of unsaturation based on its HRESIMS analysis (m/z 277.1779 [M + Na] + , calcd for 277.1779) and 13 C NMR data.The 1 H NMR (   Fig. 4 The 1 H-1 H COSY, key HMBC and NOESY/ROESY correlations of compounds 6 and 7 and the structure of compound 6a. of b.NOESY signals (Fig. 4) of H-9 with H-5/H 3 -14 indicated that H-9, H-5 and H 3 -14 had the relative conguration of a.To determine the absolute conguration of 6, its fully benzoylated derivative 6a (Fig. 4) was prepared.The absolute conguration of 6a was determined to be 2R, 5R, 9S and 10R by its CD spectrum (Fig. 5) associated with TD-DFT calculation.So, the absolute conguration of 6 was 2R, 5R, 9S and 10R, same to 6a.Thus, 6 was elucidated and named as pestalotiophol A. Pestalotiophol B ( 7) was isolated as colorless oil.The quasimolecular ion at m/z 273.1464 [M + Na] + (calcd for 273.1466) by HRESIMS indicated the molecular formula of 7 was C 15 H 22 O 3 with ve degrees of unsaturation.The 13 C NMR (Table 3), with DEPT 135 together, showed een carbons including one carbonyl, two olenic carbons, four aliphatic methylenes, three aliphatic methines, and three methyl carbons.The 1 H NMR spectrum (Table 3) revealed that 7 had one olenic proton  13 C NMR data indicated that 7 was characteristic of the drimane-type sesquiterpene with 6/6/5-fused ring system. 34The 1 H-1 H COSY (Fig. 4) showed two isolated spin systems (Fig. 4).The HMBC correlations of H-9 with C-7/C-8, and H-7 with C-8/C-9, located a double bond of C-7/C-8, a hydroxyl was located at C-3 by considering the molecular formula and the downeld chemical shi of C-3.OH-3 and double bond of C-7 and C-8 in 7 were two differences between 7 and one reported drimane-type sesquiterpene in the literature. 34ROESY correlations (Fig. 4) of H 3 -15 with H-3/H 3 -13 indicated that H 3 -15, H 3 -13 and H-3 had the relative conguration of b.ROESY correlation (Fig. 4) of H-9 with H-5 indicated that H-9 and H-5 had the relative conguration of a. Finally, 7 was elucidated and named as pestalotiophol B.
Compounds 1-15 were evaluated in vitro against cancer cell lines A549, HeLa, and SMMC-7721 by MTT assay.Compound 8 exhibited the strongest cytotoxicity against SMMC-7721 with an IC 50 value of 28.3 mM, which is comparable to that of the positive control etoposide (IC 50 of 23.2 mM), while it displayed moderate activity against A459 and Hela with IC 50 values of 49.3 and 33.3 mM, respectively.Compound 9 showed the moderate activity against all three cell lines of A549, HeLa, and SMMC-7721 with IC 50 values of 61.9, 42.9, and 44.7 mM, respectively.Unfortunately, the remaining compounds were inactive.

Experimental
General experimental procedures 1 H, 13 C, and 2D NMR data were measured on an Agilent DD2 400-MR NMR spectrometer (Agilent Technologies Inc, Santa Clara, United States), and the chemical shis were referenced to the residual solvent peaks.IR spectra were measured with a Bruker Tensor 27 spectrophotometer in KBr pellets.HR-ESI-MS data were obtained by an AB Sciex Triple TOF® 4600 system.UV spectra were recorded on an UV-2550 UV/Vis spectrophotometer (Shimadzu Corporation, Tokyo, Japan).CD spectra were measured on a JASCO J-810 spectrometer in nhexane and CD calculation was carried out by TD-DFT method with cam-B3LYP/TZVP level.The analytical and semipreparative HPLC were performed on a Waters HPLC system, which was equipped with a W1525 Binary pump, a W2998 photodiode array detector, a W1500-CH column compartment, and a W2707 auto sampler using a Waters XBridge C18 column (4.6 Â 250 mm, 5 mm) and Waters XBridge C18 column (10 Â 250 mm, 5 mm) (Waters Corporation, Milford, MA, USA).Preparative HPLC was performed on an Agilent 1260 series HPLC (Agilent Technologies Inc, Santa Clara, United States), which was equipped with a 1260 pump and a 1260 diode array detector using a Waters XBridge C18 column (19 mm Â 250 mm, 5 mm) eluted by MeCN/H 2 O system, ow rate was 15 mL min À1 .The medium-pressure liquid chromatography (MPLC) apparatus used was equipped with a dual-pump gradient system, and a UV preparative detector (Buchi Inc, Swiss Confederation).Silica gels (200-300, 300-400 and 230-400 mesh) used for column chromatographies were from Qingdao Haiyang Chemical Company Ltd., Qingdao, China, and from Merck, Darmstadt, Germany.Sephadex LH-20 used for column chromatographies was from GE Healthcare Life Sciences (GE).

Fungal material
The strain numbered HM-5 was isolated from stem bark of Sinopodophyllum hexandrum (Royle) Ying.collected from Qinling mountains, and was deposited at Shaanxi Key Laboratory of Phytochemistry, Baoji University of Arts and Sciences.The strain was identied as Pestalotiopsis adusta based on the morphological characteristics.

Fermentation and cultivation
The strain was grown on a plate of potato dextrose medium consisting of 20 g glucose, 200 g potato, and 20 g agar in 1000 mL distilled water.Aer 7 days inoculation at 28 C, a mycelium was inoculated into each of the 100 mL erlenmeyer asks containing 30 mL of liquid PD medium consisting of 20 g glucose and 200 g potato in 1000 mL distilled water to obtain the seed strain.The asks were incubated at 28 C on a rotary shaker (120 rpm) for 3 days.A suspension (500 mL) of the seed liquid was inoculated aseptically to 500 mL Erlenmeyer asks each containing 40 g rice and 60 mL distilled water, a total of 200 asks.The asks were incubated at 28 C for 30 days.

X-ray single-crystal diffraction
Crystals of compounds 1 and 5 were grown in methanol at room temperature.Crystallographic data for 1 were collected on a SuperNova Eos diffractometer with graphite monochromatic Cu Ka radiation at 297 (2) K.All calculations were performed using SHELXL-97 program.Crystallographic data for 5 were collected on a SuperNova Eos diffractometer with graphite monochromatic Cu Ka radiation at 293 K.All calculations were performed using SHELXL-97 program. 35Parameters in CIF format are available as ESI † from Cambridge Crystallographic Data Centre (pestalotiopsin D (1), CCDC1534582; pestalotiopsin H (5), CCDC1534584).

CD spectrum associated with TD-DFT calculation analysis
A preliminary conformational search was performed in Con-ex6.7 using MMFF94s forceeld. 36Conformers were saved and further optimized using the density functional theory (DFT) method and CPCM solvent model at B3LYP/6-31+G (d,p) level in Gaussian 09 soware package. 37Frequency was calculated at the same level of theory to check optimized results.The stable conformers with populations greater than 1% and without imaginary frequencies were submitted to ECD calculation by the TDDFT method associated at cam-B3LYP/TZVP level.The excitation energies (E), oscillator strength (f), rotatory strength in velocity form (R vel ), and rotatory strength in length form (R len ) of the lowest 32 excited states were calculated.ECD spectra of different conformers were summated in SpecDis according to their Boltzmann-calculated distributions. 38

Cytotoxicity assay
The cell viability was determined by the MTT assay.8 Â 10 3 cells were incubated with the tested compounds in triplicate in 96well plates for 68 h at 37 C in a nal volume of 100 mL.Cells treated with DMSO alone were set as controls.At the end of the treatment, 10 mL of MTT (5 mg mL À1 ) was added to each well and incubated for an additional 4 h.An extraction buffer (100 mL, 10% SDS, 5% iso-butanol, 0.1% HCl) was added, and the cells were incubated overnight at 37 C.The viability was calculated by measuring the absorbance at 570 nm using a microplate reader (BioTek Powerwave XS2, USA).

1 H- 1 H
COSY correlations of H-14 with H-8 and H-8 with H-9, HMBC correlations of H-8 with C-9/C-1 and H-14 with C-1, and consideration of the downeld chemical shis of C-1 and C-14, constructed a ve membered tetrahydrofuran ring of C-8/ C-9/C-1/O/C-14. 1 H-1 H COSY correlation of H-9 with H-10, HMBC correlations of H-9 with C-1/C-10/C-11, and both H 3 -12 and H 3 -13 with C-1/C-10/C-11, produced a four membered carbon ring of C-1/C-9/C-10/C-11 with two tertiary methyls attached.The last ring, the nine-membered macrocyclic moiety for caryophyllene-type sesquiterpene, was deduced by 1 H-1 H COSY correlation of H-2 with H-3, HMBC correlations of H-9 with C-2 and H-3 with C-4/C-5, and some of the above 1 H-1 H COSY and HMBC signals.Subsequently, an acetoxyl was attached to C-2 by H-2 correlating with carbonyl at d C 170.4 in HMBC.A methyl was attached to C-4 by H 3 -15 correlating with C-3/C-4/C-5.The HMBC correlations of methoxyl at d H 3.36 with C-6 and methoxyl at d H 3.27 with C-7 located two methoxyls at C-6 and C-7, respectively.Taking into account the molecular formula and the chemical shi of C-4, there should be a hydroxyl at C-4.The relative conguration of 1 was determined by ROESY experiment (Fig.

Fig. 5
Fig. 5 Experimental and calculated CD spectra of compound 6a.

Table 2
13C NMR data for compounds 1-5 at 100 MHz in CDCl 3 (d in ppm)