New Guaiane-Type Sesquiterpenoids Biscogniauxiaols A–G with Anti-Fungal and Anti-Inflammatory Activities from the Endophytic Fungus Biscogniauxia Petrensis

Seven undescribed guaiane-type sesquiterpenoids named biscogniauxiaols A–G (1–7) were isolated from the endophytic fungus Biscogniauxia petrensis on Dendrobium orchids. Their structures were determined by extensive spectroscopic analyses, electronic circular dichroism (EC) and specific rotation (SR) calculations. Compound 1 represented a new family of guaiane-type sesquiterpenoids featuring an unprecedented [5/6/6/7] tetracyclic system. A plausible biosynthetic pathway for compounds 1–7 was proposed. The anti-fungal, anti-inflammatory and multidrug resistance reversal activities of the isolates were evaluated. Compounds 1, 2 and 7 exhibited potent inhibitory activities against Candida albicans with MIC values ranging from 1.60 to 6.30 μM, and suppressed nitric oxide (NO) production with IC50 ranging from 4.60 to 20.00 μM. Additionally, all compounds (100 μg/mL) enhanced the cytotoxicity of cisplatin in cisplatin-resistant non-small cell lung cancer cells (A549/DDP). This study opened up a new source for obtaining bioactive guaiane-type sesquiterpenoids and compounds 1, 2, and 7 were promising for further optimization as multifunctional inhibitors for anti-fungal (C. albicans) and anti-inflammatory purposes.

In our endeavor to search for new bioactive compounds from the endophytic fungi of medicinal plants, the fungus Biscogniauxia petrensis MFLUCC 14-0151 isolated from the Dendrobium orchids was investigated [16], leading to the identification of one unusual iridoid In our endeavor to search for new bioactive compounds from the endophytic fungi of medicinal plants, the fungus Biscogniauxia petrensis MFLUCC 14-0151 isolated from the Dendrobium orchids was investigated [16], leading to the identification of one unusual iridoid biscogniauxiaol A (1), which possesses an unprecedented [5/6/6/7] tetracyclic system and six new [5/7] bicyclic guaiane-type sesquiterpenoids biscogniauxiaol B-G (2-7) (Figure 1). Herein, the details of the isolation, a discussion of their structural characterization, a plausible biosynthetic pathway for 1-7 and their bioactivities were reported.

General Experimental Procedures
The optical rotations and CD spectra were measured on Autopol VI in MeOH (Rudolph Research Analytical, Hackettstown, NJ, USA) and Chirascan CD spectrophotometer in MeOH (Applied Photophysics, Leatherhead, UK), respectively. The IR spectra via KBr pellets and UV spectra were recorded on Nicolet iS10 spectrometer (Thermo Fisher Scientific, Madison, WI, USA) and Shimadzu UV2401PC (Shimadzu, Kyoto, Japan), respectively. NMR spectra (500 MHz for 1 H and 125 MHz for 13 C) were recorded using Avance III 500 MHz equipment (Bruker, Bremerhaven, Germany). HRFABMS data and HRESIMS data were determined using Fast-atom-bombardment mass spectrometry DFS (Thermo Fisher Scientific, Madison, WI, USA) and Shimadzu LC/MS-IT-TOF mass instrument (Shimadzu, Kyoto, Japan), respectively. Column chromatography was conducted on silica gel (200-300 mesh, Qingdao Puke Abruption Materials Co., Ltd. Qingdao, China) and Sephadex LH-20 (Shanghai Yuanye Bio-Technology Co., Ltd. Shanghai, China). ODS column chromatography was performed using C18 silica gel (Fuji Silysia Chemical Ltd. Kasugai, Japan). TLC was carried out on precoated glass silica gel GF254 plates and compounds were visualized under UV light or via heating silica gel plates sprayed with 5% H2SO4/EtOH.

Fungal Material
The fungus B. petrensis MFLUCC14-0151 was isolated and identified by our research group [16], and preserved at the China General Microbiological Culture Collection Center (CGMCC 40341), Beijing.

General Experimental Procedures
The optical rotations and CD spectra were measured on Autopol VI in MeOH (Rudolph Research Analytical, Hackettstown, NJ, USA) and Chirascan CD spectrophotometer in MeOH (Applied Photophysics, Leatherhead, UK), respectively. The IR spectra via KBr pellets and UV spectra were recorded on Nicolet iS10 spectrometer (Thermo Fisher Scientific, Madison, WI, USA) and Shimadzu UV2401PC (Shimadzu, Kyoto, Japan), respectively. NMR spectra (500 MHz for 1 H and 125 MHz for 13 C) were recorded using Avance III 500 MHz equipment (Bruker, Bremerhaven, Germany). HRFABMS data and HRESIMS data were determined using Fast-atom-bombardment mass spectrometry DFS (Thermo Fisher Scientific, Madison, WI, USA) and Shimadzu LC/MS-IT-TOF mass instrument (Shimadzu, Kyoto, Japan), respectively. Column chromatography was conducted on silica gel (200-300 mesh, Qingdao Puke Abruption Materials Co., Ltd. Qingdao, China) and Sephadex LH-20 (Shanghai Yuanye Bio-Technology Co., Ltd. Shanghai, China). ODS column chromatography was performed using C18 silica gel (Fuji Silysia Chemical Ltd. Kasugai, Japan). TLC was carried out on precoated glass silica gel GF254 plates and compounds were visualized under UV light or via heating silica gel plates sprayed with 5% H 2 SO 4 /EtOH.

Fungal Material
The fungus B. petrensis MFLUCC14-0151 was isolated and identified by our research group [16], and preserved at the China General Microbiological Culture Collection Center (CGMCC 40341), Beijing.

Fermentation, Extraction and Isolation
Martin modified (MM) medium was inoculated with the aforementioned fungus and incubated in a constant-temperature incubator at 28 • C for 5 d to obtain seed culture. Fermentation was carried out in a conical flask (1 L) containing 200 g of rice and 150 mL of distilled water, and then autoclaving at 120 • C for 30 min. Each flask was inoculated with 10 mL of seed culture and incubated at 28 • C for two months.

Quantum Chemical Calculation (ECD)
The systematic random conformational analyses were performed in the GMMX program by using a MMFF94 molecular force field, which afforded a few conformers each, with an energy cutoff of 10 kcal/mol to the global minima. All of the obtained conformers were further optimized using DFT at the B3LYP/6-31G(d) level in CH 3 OH by using Gaussian 09 software [17]. The optimized stable conformers were used for TDDFT [B3LYP/6-311G(2d,p)] computations, with the consideration of the first 20 excitations. The overall ECD curves were all weighted by the Boltzmann distribution. The calculated ECD spectra were subsequently compared with the experimental ones. The ECD spectra were produced by SpecDis 1.70.1 software [18].

Specific Rotation Calculation (SRC)
Pcmodel program (version 10.075) was used to generate conformers at the MMFF94 force field [18]. Then, the isomers were preoptimized with the molculs program (version 1.9.9) by invoking xTB at GFN2-xTB level [18][19][20][21]. The clusters were optimized at B3LYP/def2-TZVP level with ORCA program and ensured the optimized structures have no imaginary frequency. After all the Boltzmann population properties were obtained from the Gibbs free energy calculated at PWPB95/def2-QZVPP with thermal corrections in methanol with the SMD solvation model [22]. Next, the most populated conformations obtained were used for specific optical rotations calculation at the B3LYP/6-311 + g(2d,p) level with the Gaussian 09 program package and integrated according to Boltzmann weighting proportions [17].

Anti-Fungal Assay
The minimal inhibitory concentration (MIC) values of each compound against Candida albicans (336485) (BeNa Culture Collection, Henan, China), were determined using the broth microdilution method [23]. Briefly, the screened compounds were 2-fold serially diluted in cell suspensions in RPMI 1640 medium. Then, 100 µL aliquots were added to 96-well plates. The plates were incubated at 35 • C for 24 h. The commercial amphotericin B (AMB) and fluconazole (FCZ) were used as the positive controls. Zero visible growth was considered as the endpoint value according to the guidelines (M27-A3) (CLSI 2008) [24]. All experiments were carried out in triplicate. To determine the anti-inflammatory activities of compounds 1-7, the cells were pretreated with fresh DMEM medium (100 µL/well) containing the tested compounds at various final concentrations (0-100 µg/mL) for 2 h. Then, the lipopolysaccharide (LPS, 1 µg/mL) was added and cultured for another 24 h. NO production in the supernatant was assessed using Griess reagents [4]. The absorbance at 540 nm was measured on a microplate reader. Indomethacin was used as the positive control. Meanwhile, the viability of RAW264.7 cells were evaluated by MTT assay to exclude the interference of the cytotoxicity of the test compounds. All the tests were repeated three times.

Cytotoxicity and MDR Reversal Assay
The cisplatin (DDP) sensitive A549 and resistant A549/DDP cells purchased from IMMOCELL (Xiamen, Fujian, China) were cultured in Roswell Park Memorial Institute (RPMI-1640) or DMEM supplemented with 10% FBS. The tested compounds were prepared at 100 µg/mL DMSO stocks and diluted with fresh RPMI-1640 medium to final concentrations at 50 µg/mL and 100 µg/mL. Before determining MDR reversal activity, the viability of A549 and A549/DDP cells were evaluated by MTT assay to exclude the interference of the cytotoxicity of compounds 1-7. A549 and A549/DDP cells were seeded in 96-well plates (5 × 10 4 cells/well) for 24 h, and then the medium containing the tested compounds was added (100 µL/well) and cultured for another 24 h. The absorbance at 490 nm was measured. The MDR reversal activities were assayed via combining cisplatin (20 µg/mL) and the tested compounds. The verapamil was used as a positive control. All experiments were performed in parallel three times.

Statistical Analysis
All experiments were performed in triplicates and expressed as mean ± standard deviation (SD). An unpaired t-test was performed for data analyses using the GraphPad Prism software (version 5). p < 0.05 indicated a significant difference.

Structure Identification of Compounds 1-7
Biscogniauxiaol A (1) was isolated as a colorless solid with the molecular formula C 15 H 22 O 3 determined by the HRFABMS (Figures S7 and S8), indicating five degrees of unsaturation. The infrared (IR) (Figures S9 and S10) spectrum of 1 displayed characteristic absorption bands for hydroxy (3438 cm −1 ) and double bond (1620 cm −1 ) functionalities. The 1 H nuclear magnetic resonance (NMR) ( Figure S1) data ( Table 1) Figure S2) spectrum (Tables S1-S12), fifteen carbon resonances included three methyls, four methylenes (one ether oxygen at δ C 71.1), four methines (two olefinic carbons at δ C 130.7, 147.0) and four oxy-nonprotonated carbons. The presence of one double bond accounted for one of the five degrees of unsaturation, suggesting that compound 1 required a tetracyclic skeleton (rings A-D).
The relative configuration of 1 was established by analysis of the rotating-frame nuclear Overhauser effect spectroscopy (ROESY) ( Figure S6) spectrum ( Figure 3). The ROESY interactions of H-7/H-9/H 3 -12/H 3 -15 and H-9/10-OH indicated that these protons had the same orientation. The absolute configuration of 1 was determined by the time-dependent density-functional theory (TD-DFT) quantum calculation of two possible isomers. As shown in Figure 4, the calculated electronic circular dichroism (ECD) spectrum of 3R,4S,7R,9S,10R,13R-1 matched well with the experimental one, which assigned its absolute configuration. Finally, compound 1 was named Biscogniauxiaol A, which was the first reported example of a [5/7] bicyclic sesquiterpenoid with an unprecedented [5/6/6/7] ring system.     Biscogniauxiaol B (2) was obtained as a colorless solid with the molecular formula C 15 H 28 O 3 determined by the HRESIMS (Figures S11 and S12), and a corresponding two degrees of unsaturation. The 1 H NMR data ( Table 1)  Biscogniauxiaol B (2) was obtained as a colorless solid with the molecular formula C15H28O3 determined by the HRESIMS (Figure S11-S12), and a corresponding two degrees of unsaturation. The 1 H NMR data ( Table 1) (Figure 4), which determined its absolute configuration.
Biscogniauxiaol C (3) was purified as a colorless solid with the molecular formula C15H28O4 determined by the HRESIMS (Figure S29-S31), suggesting two degrees of unsaturation. A detailed comparison of its 1 H ( Figure S23) and 13 C NMR ( Figure S24) spectroscopic data (Table 1) with that reported for bicyclic sesquiterpene [25] indicated that 3 was tetrahydroxy derivatives of guaiane-type sesquiterpene. The 13 C NMR (DEPT) ( Figure S24) spectra of 3 displayed fifteen carbons consisting of three methyls, five methylenes of which Biscogniauxiaol C (3) was purified as a colorless solid with the molecular formula C 15 H 28 O 4 determined by the HRESIMS (Figures S29-S31), suggesting two degrees of unsaturation. A detailed comparison of its 1 H ( Figure S23) and 13 C NMR ( Figure S24) spectroscopic data (Table 1) with that reported for bicyclic sesquiterpene [25] indicated that 3 was tetrahydroxy derivatives of guaiane-type sesquiterpene. The 13 C NMR (DEPT) ( Figure S24) spectra of 3 displayed fifteen carbons consisting of three methyls, five methylenes of which one was oxygenated at δ C 68.61, five methines of which one was oxygenated at δ C 74. 26 and two quaternary carbons that were oxygenated at δ C 75.37 and 76.59. The analyses of COSY ( Figures S25 and S26) and HMBC spectra ( Figure S27) confirmed that 3 contained a guaiane-type sesquiterpene skeleton. The connection between C-11 and C-7 was confirmed by the HMBC correlations from H-7 to C-11. These data indicated that 3 shared the same planar structure as that of 1R,3S,4R,5S,7R,10R,11S-guaiane-3,10,11,12-tetraol [25]. However, comparison of their optical rotations and NMR data suggested that they were not identical but stereoisomeric. The relative configuration of 3 was assigned by the ROESY (Figure S28 (Figures S33 and S34) spectra of 4 (Table 2) revealed the presence of fifteen carbons consisting of three methyls, five methylenes of which one was oxygenated at δ C 68.55, five methines of which one was oxygenated at δ C 75.33 and two quaternary carbons that were oxygenated at δ C 75.60 and δ C 76.47. Its 1 H and 13 C NMR spectroscopic data were similar to that of compound 3, indicating that compound 4 was a tetrahydroxy derivative of guaiane-type sesquiterpenes. The COSY ( Figure S35) correlations of H-1 to oxidic H-2 and H-4 to H-3 suggested that the C-2 rather than C-3 was oxidized. In the analyses in combination with HMBC ( Figures S36 and S37) and COSY spectrum correlations, the planar structure of compound 4 was elucidated as shown ( Figure 1). The relative configuration of 4 was assigned by the ROESY (Figure S38 (Figures S40-S42). Thus, its absolute configuration was determined as 1R,2R,4R,5R,7S, and 10S.
Biscogniauxiaol E (5) was isolated as a colorless solid and had the molecular formula C 15 H 28 O 4 established by the HRESIMS (Figure S49), indicating 2 degrees of unsaturation. Its 1 H and 13 C NMR spectroscopic data were similar to that of compound 3, suggesting that compound 5 was a tetrahydroxy derivative of guaiane-type sesquiterpenes. The 13 C NMR (DEPT) (Figures S43 and S44) spectra of 5 (Table 2) had fifteen carbons consisting of three methyls, five methylenes of which one was oxygenated at δ C 68.77, five methines of which one was oxygenated at δ C 81.10 and two oxygenated carbons at δ C 75.90 and 78.17. The COSY ( Figure S45) correlations of H-9 to H-8 and the HMBC (Figures S46 and S47) correlations of H-14 to C-10 and H-13 to C-11 and C-12 confirmed that four hydroxyls were located at C-9, C-10, C-11 and C-12. The relative configuration of 5 was assigned by the ROESY ( Figure S48) spectrum ( Figure 3). The ROESY correlations H-1/H-9 and H-9/H-7 established that they were cofacial. Meanwhile, the ROESY correlations H 3 -14/H 3 -15/H-5 indicated that they were cofacial. The absolute configuration of 5 was determined by the time-dependent density-functional theory quantum calculation of two possible isomers. As shown in Figure 4, the calculated electronic circular dichroism (ECD) spectrum of the 1S,4R,5S,7R,9R,10R-5 matched well with the experimental one, which assigned its absolute configuration.
Biscogniauxiaol 6 (F) was isolated as a colorless solid and its molecular formula C 15 H 28 O 4 was established by the HRESIMS (Figure S60), corresponding to two degrees of unsaturation. The 13 C NMR (DEPT) ( Figure S55) spectra of 6 ( Table 2) had fifteen carbons consisting of three methyls, five methylenes of which one was oxygenated at δ C 68.54, five methines of which one was oxygenated at δ C 73.90 and two quaternary carbons oxygenated at δ C 74.62 and 75.82. Careful analyses of the 1 H NMR (Figures S50-S54) and HSQC ( Figure S57) data revealed that 6 had three methyl groups (δ H 1.01, δ C 10.01; δ H 1.04, δ C 18.43; δ H 1.19, δ C 30.92), five methylene groups of which one was oxygenate (δ H 3.38 and δ H 3.56, δ C 68.54), and five methine groups of which one was oxygenate (δ H 4.15, δ C 73.90). The COSY ( Figure S56) correlations of H-2 to H-1/H-3 and the HMBC ( Figure S58) correlations of H-14 to C-10 and H-13 to C-11 and C-12 confirmed that four hydroxyls were located at C-2, C-9, C-11 and C-12 ( Figure 2). The relative configuration of 6 was assigned by the ROESY (Figure S59 Figures S61-S63), which assigned its absolute configuration.
Biscogniauxiaol G (7) was obtained as a colorless solid with the molecular formula C 15 H 26 O 3 deduced by the HRESIMS (Figures S70-S75), indicating three degrees of unsaturation. Its 1 H ( Figure S64) and 13 C NMR ( Figure S65) spectroscopic data were similar to those of compounds 2 and 3. The 13 C NMR (DEPT) ( Figure S65) spectra of 7 (Table 2) had fifteen carbons consisting of two methyls, six methylenes of which one was oxygenated at δ C 65.09, five methines of which one was oxygenated at δ C 78.75. The HMBC (Figures S66-S68) correlations H 3 -14 to C-10 and H-12 to C-11/C-13 and their chemical shift confirmed that three hydroxyls were located at C-3, C-10 and C-12 ( Figure 2). The 13 C NMR data and HMBC correlations demonstrated the presence of two sp 2 carbons (δ C 107.51 and 157.27) at C-11 and C-13. The relative configuration of 7 was assigned by the ROESY (Figure S69) spectrum ( Figure 3). The ROESY correlations of H-1/H-3, H-3/H 3 -15, H-7/H-5 and H-5/H 3 -15 indicated that they were cofacial. The absolute configuration of 7 was determined by the time-dependent density-functional theory quantum calculation of two possible isomers. As shown in Figure 4, the ECD spectrum of the 1S,3R,4R,5R,7S,10S-7 matched with the experimental one, which allowed the assignment of the absolute configuration of 7 as 1S,3R,4R,5R,7S, and 10S.

Anti-Fungal Evaluation of Compounds
In consideration of the previously discovered anti-fungal activities for guaiane-type sesquiterpenoids [28], the inhibitory effects of compounds 1-7 against C. albicans (336485) were evaluated. As shown in Table 3, all compounds had inhibitory activities against C. albicans. Among them, compounds 1, 2, and 7 exhibited as potent with MICs of 1.60, 6.25 and 6.30 µM, respectively (Amphotericin B and Fluconazole with MICs of 0.43 and 2.61 µM, respectively).

Anti-Fungal Evaluation of Compounds
In consideration of the previously discovered anti-fungal activities for guaiane-type sesquiterpenoids [28], the inhibitory effects of compounds 1-7 against C. albicans (336485) were evaluated. As shown in Table 3, all compounds had inhibitory activities against C. albicans. Among them, compounds 1, 2, and 7 exhibited as potent with MICs of 1.60, 6.25 and 6.30 µM, respectively (Amphotericin B and Fluconazole with MICs of 0.43 and 2.61 µM, respectively).

Anti-Inflammatory Activities of Compounds
The anti-inflammatory is one of the most important biological activities for guaianetype sesquiterpenoids [29]. The inhibitory effects of compounds 1-7 on the LPS-induced production of NO in the RAW264.7 cell line were evaluated in vitro. The results showed that compounds 1, 2, and 7 suppressed the NO production with IC 50 values of 4.60 ± 0.42, 20.00 ± 1.54 and 18.38 ± 1.12 µM, respectively (indomethacin, IC 50 = 22.94 ± 1.42 µM), and none of the compounds exhibited cytotoxicity (Table 4).

Anti-cancer and MDR Reversal Effects of Compounds
According to literature reports, guaiane-type sesquiterpenoids had anticancer and MDR reversal activities [30,31]. Thus, the anti-cancer and reversal activities of compounds 1-7 in the cisplatin sensitive A549 cells and resistant A549/DDP cells were evaluated in vitro, respectively. The data displayed that all compounds showed no cytotoxicity to the cisplatin sensitive A549 cells and resistant A549/DDP cells at concentration of 100 µg/mL ( Figure 6). However, they had weak reversal activities to the cisplatin resistant A549/DDP cells at concentrations of 50 µg/mL and 100 µg/mL compared to the control group DDP (Figures 7 and 8).

Anti-cancer and MDR Reversal Effects of Compounds.
According to literature reports, guaiane-type sesquiterpenoids had anticancer and MDR reversal activities [30,31]. Thus, the anti-cancer and reversal activities of compounds 1-7 in the cisplatin sensitive A549 cells and resistant A549/DDP cells were evaluated in vitro, respectively. The data displayed that all compounds showed no cytotoxicity to the cisplatin sensitive A549 cells and resistant A549/DDP cells at concentration of 100 µg/mL ( Figure 6). However, they had weak reversal activities to the cisplatin resistant A549/DDP cells at concentrations of 50 µg/mL and 100 µg/mL compared to the control group DDP (Figures 7 and 8).

Conclusions
In summary, seven new bioactive sesquiterpenoids including an unprecedented [5/6/6/7] tetracyclic system iridoid were obtained from the endophytic fungus viz B. petrensis on D. orchids. Their structures including absolute stereochemistry were determined. A possible biosynthetic pathway for them was proposed, which may promote further chemical synthesis efforts. Compounds 1, 2, 7 showed potent inhibitory effects on C. albicans with MIC values of 1.60, 6.25 and 6.30 µM, respectively. Meanwhile, they exhibited prominent inhibitory activities against the NO production in the RAW264.7 cells with IC 50 values of 4.60 ± 0.42, 20.00 ± 1.54, 18.38 ± 1.12 µM, respectively. Moreover, all the isolated compounds had weak reversal activities to the cisplatin resistant A549/DDP cells. These data confirmed that the endophytic fungus B. petrensis is a new source of bioactive guaiane-type sesquiterpenoids that can replace the plants, and compounds 1, 2 and 7 (biscogniauxiaols A, B and G) were promising for further optimization as multifunctional inhibitors for anti-fungal (C. albicans) and anti-inflammatory purposes.