Four New Highly Oxygenated Eremophilane Sesquiterpenes from an Endophytic Fungus Boeremia exigua Isolated from Fritillaria hupehensis

Four new eremophilane-type sesquiterpenes, boeremialanes A–D (1–4) were obtained from solid substrate cultures of Boeremia exigua (Didymellaceae), an endophytic fungus isolated from Fritillaria hupehensis (Liliaceae). Boeremialanes A–C (1–3) are highly oxygenated eremophilanes with a benzoate unit attached at the C-13 position and are rarely found in nature. Their structures and absolute configurations were determined by extensive spectroscopic methods, electronic circular dichroism (ECD), and nuclear magnetic resonance (NMR) calculations with DP4+ analysis. Boeremialane D (4) potently inhibited nitric oxide production in lipopolysaccharide-treated RAW264.7 macrophages with an IC50 of 8.62 μM and was more potent than the positive control, pyrrolidinedithiocarbamate (IC50 = 23.1 μM).


Introduction
Eremophilane-type derivatives are structurally irregular and bicyclic natural products belonging to a small sesquiterpene family [1,2]. These eremophilane sesquiterpenes are biogenetically derived from farnesyl diphosphate in association with a methyl migration [3] and consist of three isoprene subunits [4]. The structural diversity of eremophilane analogs is due to oxidation occurring at different sites along the isopropyl side chain and bicyclic backbone to generate alcohol [5], acid [6], ester [7][8][9], furan [10,11], and lactone functionalities, with some of the alcohols further glycosylated [12]. Since the first eremophilane-type sesquiterpene was isolated from the wood oil of Eremophila mitchellii in 1932 [13], more than 650 biologically active eremophilane derivatives have been obtained [2,14]. In addition to the related analogs obtained from terrestrial plants [15,16] and marine fungi [17,18], plant endophytic fungi are recognized as a new source of derivatives eremophilane [19,20]. Due to their special structural features and various functional groups, eremophilane-type sesquiterpenes possess a lot of biological activities such as anti-inflammatory [21], antitumor [10], and antibacterial [22,23] activities, which have received increasing interest in the recent years. As part of our ongoing efforts to discover bioactive terpenoids derived from endophytic fungi [24][25][26][27], a chemical investigation on the cultural broth of B. exigua in rice medium was carried out. As a result, four new highly oxygenated eremophilane-type sesquiterpenes, boeremialanes A-D (1)(2)(3)(4), were isolated from cultures of the fungus B. exigua. The new structures were established by extensive spectroscopic methods, ECD and NMR calculations, as well as DP4+ analysis. All compounds were tested for their antiinflammatory activities on nitric oxide production in LPS-induced RAW264.7 macrophages. Herein, details of the isolation, structural elucidation and bioactivities of the compounds are reported.

Culture and Fermentation of Fungal Material
The strain B. exigua was isolated from the healthy leaf tissue of Fritillaria hupehensis Hsiao. It was identified by Dr. Hong-Lian Ai (South-Central MinZu University). The ITS sequence of this strain is almost identical to the strain deposited in Genbank with accession number MT154621.1 (max identity: 100%, query cover: 100%). The fungal specimen is deposited at South-Central MinZu University, China. The strain was cultured on PDA medium for 8 days, and then was cut into small pieces to incubate solid rice medium to culture for further 30 days at 25 • C (50 g rice, 50 mL water, in each 500 mL Erlenmeyer flask, the total weight of rice was 17 kg).

Quantum Chemical Calculations
The initial conformational analysis of compounds 1-4 was performed using the Monte Carlo search algorithm via the MMFF94 molecular mechanics force field [28], with the aid of the Spartan 16 program package that resulted in some relatively favorable conformations with an energy range of 3 kcal/mol above the global minimum. The minimum energy conformers of the resulting force field were optimized in vacuum with the M06-2X/def2-SVP level, and implemented in the Gaussian 09 software package by the Density functional theory [29]. At the same time, harmonic vibrational frequencies were also measured to confirm the lack of imaginary frequencies of the finally optimized conformers. These primary conformations were subjected to theoretical calculations of ECD utilizing timedependent density functional theory (TDDFT) calculations at the M06-2X/def2-SVP level in MeOH using the polarizable continuum model (PCM) solvent model. The energies, oscillator strengths, and rotational strengths of each conformation were determined with the Gaussian 09 software package. Theoretical calculations of ECD spectra for each part were then approximated by the Gaussian distribution. The final ECD spectrum of the individual conformers was summed up on the basis of the Boltzmann-weighed population contribution by the SpecDisv1.71 [30]. DFT GIAO 13 C NMR calculations were performed on the mPW1PW91/6-31 + G(d,p)//M06-2X/def2-SVP level of theory [31]. The solvent effect was accounted for by using methanol in the calculations to mimic the experimental conditions. The 13 C NMR chemical shifts in compound 1 were considered the average values of the same atoms in the different conformers. We took the relative Gibbs free energy as the weighting factor and used the Boltzmann distribution to find the average values. The overall theoretical NMR data were analyzed using DP4+ probability [32].

Nitric Oxide Production Inhibitory Assay
The anti-inflammatory effect of Raw264.7 macrophages was studied and cultured in Dulbecco's modified eagle medium (DMEM, HyClone, Logan, UT, USA) with 10% fetal bovine serum (FBS, PAN, Aidenbach, Germany) in a humidified incubator (5% CO 2 , 37 • C). RAW264.7 cells (5 × 10 4 cells/well) were seeded into a 96-well multiplate for 12 h. After 12 h of incubation, the cells were treated with LPS (1 µg/mL) and different concentrations of the tested compounds (1-4, 20 µM) for 18 h. A Griess reagent kit (Promega, Madison, WI, USA) was used to measure the amount of nitrite, a stable metabolite of Nitric Oxide (NO), in the supernatants. Briefly, 50 µL of each culture medium was added to a 96-well plate, and then the same volume of sulfanilamide solution was added. After incubation at room temperature for 5 min, 50 µL of N-1-naphthylethylenediamine dihydrochloride solution was added to all wells. The absorption at 540 nm was measured by a microplate reader after 10 min incubation at room temperature [33]. The IC 50 values were calculated by GraphPad Prism 6 software. Cell viability was determined with the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Pyrrolidine dithiocarbamate (PDTC, Sigma−Aldrich, St Louis, MO, USA) was used as a positive control.
Boeremialane B (2) was obtained as a yellowish oil, and the molecular formula was determined to be C 22 Figures S12-S17). The 1 H and 13 C NMR data of compound 2 were structurally similar to those of compound 1, except for the absence of a carbomethoxy group at δ C 168.9 and 53.5 in compound 1 and the presence of an additional hydroxy group in compound 2. The hydroxyl group at C-4' was evident from the downfield shift of C-4' (δ C 158.8) as well as the HMBC correlations from H-13 to the sp2 quaternary carbon (C-4') ( Figure 2). The relative configuration of compound 2 was the same as that found in compound 1 based on the ROESY correlations of H-3/H 3 Figure 3). Finally, the absolute configuration of 2 was determined by ECD calculations on the M06-2X/def2-SVP (IEFPCM, MeOH) level of theory. The experimental ECD spectrum of compound 2 fits well with the calculated spectrum of 3R, 4R, 5R, 6R, 7S, and 11S-2 ( Figures 5 and S2). Therefore, the structure of compound 2 was determined, and it was given the name boeremialane B.     The configuration of boeremialane A (1) was established by ROESY experiments and quantum chemistry calculations. The ROESY correlations of H-3/H3-15, H-3/H3-14, H-6/H3-14, and H-6/H3-15 suggested that they were β-oriented ( Figure 3). In addition, to determine the configuration of C-11 in the flexible bond, nuclear magnetic resonance (NMR) calculations of two epimers, 11S-1 and 11R-1, were carried out. The two epimers were subjected to a strict conformational screening procedure; then, the NMR chemical shifts were calculated at the mPW1PW91/6-31 + G(d,p)//M06-2X/def2-SVP level of theory with the PCM solvent in methanol. The DP4+ analysis identified 11S-1 as the most likely structure of compound 1 with 100.00% DP4+ probability (all data) (Figure 4 and Table S1). Finally, the absolute configuration of compound 1 was resolved by comparing the calculated and experimental ECD data using time-dependent density-functional theory (TDDFT). The theoretical spectrum of compound 1 showed an excellent fit with the experimental plot recorded in MeOH ( Figure 5 and S1), which supported an absolute configuration of 3R, 4R, 5R, 6R, 7S, and 11S. Thus, the structure of compound 1 was determined, and it was named boeremialane A.

-15, H-3/H 3 -14, H-6/H 3 -14, and H-6/H 3 -15 (
Boeremialane B (2) was obtained as a yellowish oil, and the molecular formula was determined to be C22H26O8 from the HRESI mass spectrum data ([M + Na] + , found 441.15195, calcd. 441.15199). The 1 H and 13 C NMR data of compound 2 indicated the presence of two methyl groups (δC 11.6 and 19.0), four methylene groups (δC 31.  (Table 1 and Figure S12-S17). The 1 H and 13 C NMR data of compound 2 were structurally similar to those of compound 1, except for the absence of a carbomethoxy group at δC 168.9 and 53.5 in compound 1 and the presence of an additional hydroxy group in compound 2. The hydroxyl group at C-4' was evident from the downfield shift of C-4' (δC 158.8) as well as the HMBC correlations from H-13 to the sp2 quaternary carbon (C-4') ( Figure 2). The relative configuration of compound 2 was the same as that found in compound 1 based on the ROESY correlations of H-3 /H3-15, H-3/H3-14, H-6/H3-14, and H-6/H3-15 (Figure 3). Finally, the absolute configuration of 2 was determined by ECD calculations on the M06-  Figure  S33, Supporting Information), which indicated that they have an the identical absolute configuration. In addition, the identity of the measured ECD and calculated ECD spectrum of compound 4 further confirmed this conclusion ( Figure 5 and S4). Therefore, the structure of compound 4 was determined, and it was given the name boeremialane D. All compounds were evaluated for their inhibition of NO production in LPS-treated RAW264.7 macrophages. As a result, compound 4 showed certain inhibitory activity with IC50 values of 8.62 μM, which was more potent than the positive control, pyrrolidinedithiocarbamate (IC50 = 23.1 μM) ( Figure 6).    (Table 2 and Figures S19-S24) were structurally similar to those of compound 2, except for the presence of a para-substituted benzene ring of the benzoate unit. This difference was supported by the HMBC correlations from H-3' (7') (δ H 7.81) to C-1' (δ C 168.4) and C-5' (δ C 165.9) along with the COSY correlations between H-3' (7')/H-4' (6') (δ H 6.74) (Figure 2). The ECD spectrum of compound 3 was similar to that of compound 1 with negative exciton coupling at 211 nm and positive exciton coupling at 241 nm ( Figure S33), which indicated that they share the identical absolute configuration. Therefore, the absolute configuration of 3 was defined as 3R, 4R, 5R, 6R, 7S, and 11S. This presumption was confirmed by comparative analysis of calculated and experimental ECD spectra. The experimental ECD spectrum of 3 fits well with the calculated spectrum of 3R, 4R, 5R, 6R, 7S, and 11S-3 ( Figures 5 and S3). Thus, the structure of 3 was determined and named boeremialane C.  Figures S26-S31). The 1 H and 13 C NMR data of compound 4 were structurally similar to those of phaseolinone [35], except for the appearance of an additional acetyl group in compound 4. The attachment of this acetyl group at C-3 was supported by the HMBC correlation from the H-3 to the ester carbonyl carbons (δ C 172.4). The relative configuration of compound 4 was the same as that found in compound 1 based on the ROESY correlations of H-3/H 3 -15, H-3/H 3 -14, H-6/H 3 -14, and H-6/H 3 -15 ( Figure 3). Similar to compound 3, the tendencies of the ECD curves of compounds 4 and 1 with negative exciton coupling at 225 nm and positive exciton coupling at 250 and 337 nm were relatively consistent ( Figure S33, Supporting Information), which indicated that they have an the identical absolute configuration. In addition, the identity of the measured ECD and calculated ECD spectrum of compound 4 further confirmed this conclusion (Figures 5 and S4). Therefore, the structure of compound 4 was determined, and it was given the name boeremialane D.
All compounds were evaluated for their inhibition of NO production in LPS-treated RAW264.7 macrophages. As a result, compound 4 showed certain inhibitory activity with IC 50 values of 8.62 µM, which was more potent than the positive control, pyrrolidinedithiocarbamate (IC 50 = 23.1 µM) ( Figure 6).

Conclusions
In summary, the structures of four new eremophilane-type sesquiterpenes (1-4) were unambiguously determined by analyses of their HRESI and NMR spectroscopic data, with the absolute configuration being determined by quantum chemistry calculations. Boeremialanes A-C (1-3) are highly oxygenated eremophilanes with the benzoate unit attached at the C-13 position, and only one such natural compound has been discovered to date [35]. Compound 4 exhibited potent inhibition against NO production in LPS-activated RAW 264.7 macrophages, suggesting that it is a new chemical entity for anti-inflammatory effects. The present research provides new insights into understanding the structural diversity and interesting biological activities of eremophilane sesquiterpenes.

Conclusions
In summary, the structures of four new eremophilane-type sesquiterpenes (1-4) were unambiguously determined by analyses of their HRESI and NMR spectroscopic data, with the absolute configuration being determined by quantum chemistry calculations. Boeremialanes A-C (1-3) are highly oxygenated eremophilanes with the benzoate unit attached at the C-13 position, and only one such natural compound has been discovered to date [35]. Compound 4 exhibited potent inhibition against NO production in LPS-activated RAW 264.7 macrophages, suggesting that it is a new chemical entity for anti-inflammatory effects. The present research provides new insights into understanding the structural diversity and interesting biological activities of eremophilane sesquiterpenes.