20-Nor-Isopimarane Epimers Produced by Aspergillus wentii SD-310, a Fungal Strain Obtained from Deep Sea Sediment

Four new uncommon 20-nor-isopimarane diterpenoid epimers, aspewentins I−L (1–4), together with a new methylated derivative of 3, aspewentin M (5), were isolated from the deep sea sediment-derived fungus Aspergillus wentii SD-310. The very similar structures of these epimers made the separation and purification procedures difficult. The structures of compounds 1–5 were illustrated based on spectroscopic analysis, and the absolute configurations of compounds 1–5 were unambiguously determined by the combination of NOESY, time-dependent density functional (TDDFT)-ECD calculations, and X-ray crystallographic analysis. These metabolites represented the rare examples of 20-nor-isopimarane analogues possessing a cyclohexa-2,5-dien-1-one moiety. These compounds were tested for antimicrobial activities against human and aquatic pathogenic bacteria, as well as plant-pathogenic fungi. While compounds 1 and 2 exhibited inhibitory activities against zoonotic pathogenic bacteria such as Escherichia coli, Edwardsiella tarda, Vibrio harveyi, and V. parahaemolyticus, compound 5 showed potent activity against the plant pathogen Fusarium graminearum.

In the process of our excavation to identify new bioactive compounds from marine-derived fungi [7][8][9][10][11], the fungus A. wentii SD-310, which was isolated from a deep sea sediment sample and which produces a series of 20-nor-isopimarane diterpenoids [12][13][14], was selected for further study. As a result, five new 20-nor-isopimarane diterpenoids-including a set of diastereoisomers, namely aspewentins I−L (1-4), and aspewentin M (5), a methylated derivative of 3 ( Figure 1)-were isolated and characterized from the dynamical culture extract. The very similar structures of these epimers made the separation and purification procedures difficult. The mixture of compounds 1 and 2 as well as 3 and 4 displayed one dot on thin-layer chromatography (TLC) but was proven to contain two compounds by 1 H and 13 C NMR spectra. Compounds 1-4 were finally purified by semipreparative HPLC after many attempts at gradient optimization. The structures of the isolated compounds were established through detailed interpretation of the NMR and mass spectrometric data, and the absolute configurations were determined by the combination of NOESY, quantum chemical ECD calculations, and X-ray crystallographic analysis. Notably, compounds 1- 5 represented the rare examples of 20-nor-isopimarane analogues possessing a cyclohexa-2,5-dien-1-one moiety at ring B. All of these compounds were examined for antimicrobial activities against human and aquatic pathogenic bacteria as well as plant-pathogenic fungi. Herein, details of the isolation, structure elucidation, and biological activities of compounds 1-5 are described.
Mar. Drugs 2018, 16, x FOR PEER REVIEW 2 of 10 compounds by 1 H and 13 C NMR spectra. Compounds 1-4 were finally purified by semipreparative HPLC after many attempts at gradient optimization. The structures of the isolated compounds were established through detailed interpretation of the NMR and mass spectrometric data, and the absolute configurations were determined by the combination of NOESY, quantum chemical ECD calculations, and X-ray crystallographic analysis. Notably, compounds 1- 5 represented the rare examples of 20-nor-isopimarane analogues possessing a cyclohexa-2,5-dien-1-one moiety at ring B. All of these compounds were examined for antimicrobial activities against human and aquatic pathogenic bacteria as well as plant-pathogenic fungi. Herein, details of the isolation, structure elucidation, and biological activities of compounds 1-5 are described.

Structure Elucidation of the New Compounds
Compound 1, named as aspewentin I, was isolated as white amorphous powder. The molecular formula was determined to be C19H26O3 by HRESIMS ( Figure S1), requiring seven degrees of unsaturation. In the 1 H NMR spectrum (Table 1) ; and a broad singlet for exchangeable protons (δH 5.24, brs, 10-OH) were also observed. The 13 C NMR data (Table 2) revealed the presence of 19 carbon signals, and were classified into three methyls, six methylenes (with one olefinic), three methines (with one oxygenated and two olefinic), and seven quaternary carbons by DEPT and HSQC experiments. A detailed analysis of the 1D NMR data revealed that 1 might be a 14-hydroxylated derivative of aspewentin C, a 20-norisopimarane diterpenoid identified from the algicolous strain A. wentii na-3 [6]. The observed HMBC correlations ( Figure 2) from H-14 to C-7, C-9, and C-12 supported this conduction. On the basis of the above analysis, the planar structure of 1 was determined as shown in Figure 1. The relative configuration of aspewentin I (1) was proposed through analysis of its NOESY data ( Figure 3). The observed NOEs from the proton of 10-OH to H-11α, from H-15 to H-11α and H-12α, and from 12α to H-14 assigned them on the same face of the molecule, whereas NOEs from the proton of 14-OH to H-12β and H3-17 located them on the other face ( Figure 3). The absolute configuration of 1 was established by the time-dependent density functional (TDDFT)-ECD calculation in Gaussian 09 [15]. We obtained the minimum energy conformers by geometry optimization of each possible isomer of 1, and then employed the TDDFT method at three different levels to get calculated ECD

Structure Elucidation of the New Compounds
Compound 1, named as aspewentin I, was isolated as white amorphous powder. The molecular formula was determined to be C 19 H 26 O 3 by HRESIMS ( Figure S1), requiring seven degrees of unsaturation. In the 1 H NMR spectrum (Table 1) ; one singlet for olefinic methine (δ H 5.95, s, H-6); and a broad singlet for exchangeable protons (δ H 5.24, brs, 10-OH) were also observed. The 13 C NMR data ( Table 2) revealed the presence of 19 carbon signals, and were classified into three methyls, six methylenes (with one olefinic), three methines (with one oxygenated and two olefinic), and seven quaternary carbons by DEPT and HSQC experiments. A detailed analysis of the 1D NMR data revealed that 1 might be a 14-hydroxylated derivative of aspewentin C, a 20-nor-isopimarane diterpenoid identified from the algicolous strain A. wentii na-3 [6]. The observed HMBC correlations ( Figure 2) from H-14 to C-7, C-9, and C-12 supported this conduction. On the basis of the above analysis, the planar structure of 1 was determined as shown in Figure 1.
Mar. Drugs 2018, 16, x FOR PEER REVIEW 2 of 10 compounds by 1 H and 13 C NMR spectra. Compounds 1-4 were finally purified by semipreparative HPLC after many attempts at gradient optimization. The structures of the isolated compounds were established through detailed interpretation of the NMR and mass spectrometric data, and the absolute configurations were determined by the combination of NOESY, quantum chemical ECD calculations, and X-ray crystallographic analysis. Notably, compounds 1-5 represented the rare examples of 20-nor-isopimarane analogues possessing a cyclohexa-2,5-dien-1-one moiety at ring B. All of these compounds were examined for antimicrobial activities against human and aquatic pathogenic bacteria as well as plant-pathogenic fungi. Herein, details of the isolation, structure elucidation, and biological activities of compounds 1-5 are described.

Structure Elucidation of the New Compounds
Compound 1, named as aspewentin I, was isolated as white amorphous powder. The molecular formula was determined to be C19H26O3 by HRESIMS ( Figure S1), requiring seven degrees of unsaturation. In the 1 H NMR spectrum (Table 1) ; one singlet for olefinic methine (δH 5.95, s, H-6); and a broad singlet for exchangeable protons (δH 5.24, brs, 10-OH) were also observed. The 13 C NMR data (Table 2) revealed the presence of 19 carbon signals, and were classified into three methyls, six methylenes (with one olefinic), three methines (with one oxygenated and two olefinic), and seven quaternary carbons by DEPT and HSQC experiments. A detailed analysis of the 1D NMR data revealed that 1 might be a 14-hydroxylated derivative of aspewentin C, a 20-norisopimarane diterpenoid identified from the algicolous strain A. wentii na-3 [6]. The observed HMBC correlations ( Figure 2) from H-14 to C-7, C-9, and C-12 supported this conduction. On the basis of the above analysis, the planar structure of 1 was determined as shown in Figure 1. The relative configuration of aspewentin I (1) was proposed through analysis of its NOESY data ( Figure 3). The observed NOEs from the proton of 10-OH to H-11α, from H-15 to H-11α and H-12α, and from 12α to H-14 assigned them on the same face of the molecule, whereas NOEs from the proton of 14-OH to H-12β and H3-17 located them on the other face ( Figure 3). The absolute configuration of 1 was established by the time-dependent density functional (TDDFT)-ECD calculation in Gaussian 09 [15]. We obtained the minimum energy conformers by geometry optimization of each possible isomer of 1, and then employed the TDDFT method at three different levels to get calculated ECD The relative configuration of aspewentin I (1) was proposed through analysis of its NOESY data ( Figure 3). The observed NOEs from the proton of 10-OH to H-11α, from H-15 to H-11α and H-12α, and from 12α to H-14 assigned them on the same face of the molecule, whereas NOEs from the proton of 14-OH to H-12β and H 3 -17 located them on the other face ( Figure 3). The absolute configuration of 1 was established by the time-dependent density functional (TDDFT)-ECD calculation in Gaussian 09 [15]. We obtained the minimum energy conformers by geometry optimization of each possible isomer of 1, and then employed the TDDFT method at three different levels to get calculated ECD spectra of 1. The experimental ECD spectrum of 1 displayed excellent accordance with that calculated for (10R, 13R, 14R)-1 at all of the three levels, which allowed unambiguous assignment of its absolute configuration ( Figure 4).

Mar. Drugs 2018, 16, x FOR PEER REVIEW
3 of 10 spectra of 1. The experimental ECD spectrum of 1 displayed excellent accordance with that calculated for (10R, 13R, 14R)-1 at all of the three levels, which allowed unambiguous assignment of its absolute configuration ( Figure 4).    Mar. Drugs 2018, 16, x FOR PEER REVIEW 3 of 10 spectra of 1. The experimental ECD spectrum of 1 displayed excellent accordance with that calculated for (10R, 13R, 14R)-1 at all of the three levels, which allowed unambiguous assignment of its absolute configuration ( Figure 4).     Aspewentin J (2) was obtained as white amorphous powder. The molecular formula was also determined as C 19 H 26 O 3 , the same as that of 1, based on HRESIMS data ( Figure S9). The 1 H, 13 C, and DEPT NMR data (Tables 1 and 2) of 2 showed almost identical patterns to those of 1, with some minor variations for the chemical shifts of C-8, C-9, C-12, C-15, C-16, and C-17. Inspection of the NMR data suggested that 2 is a diastereomer of 1, epimeric at C-14. This was supported by the observed NOEs from the proton of 10-OH to H-11α and H 3 -18, from H-12α to H-15, from H 3 -17 to H-11β and H-14, and from H-12β to H-14 ( Figure 3). The absolute configuration of 2 was also confirmed by ECD calculation, in which the experimental ECD spectrum of 2 matched well with that calculated for (10R, 13R, 14S)-2.
The molecular formula of aspewentin K (3) was also determined to be C 19 H 26 O 3 , the same as 1 and 2, on the basis of HRESIMS ( Figure S17). Detailed analysis of the spectroscopic data (Tables 1 and 2) indicated that compound 3 was a diastereomer of 1 and 2. Some minor variations for the chemical shifts of C-1, C-5, and C-9, as in 2, suggested the epimeric at C-10. This was supported by the NOE correlations from H-14 to H-12β and H 3 -17, from H 3 -17 to H-11β, and from H-12α to H-15. The absolute configuration of 3 was also verified by TDDFT-ECD calculation. The experimental ECD spectrum of 3 was opposite to that of 1 and matched well with that calculated for (10S, 13R, 14S)-3 ( Figure 4).
Aspewentin L (4) was isolated as white amorphous powder. HRESIMS data ( Figure S25) gave the molecular formula C 19 H 26 O 3 , same as that of compounds 1-3. The general features of the 1 H and 13 C NMR data (Tables 1 and 2) resembled compound 1, and a minor difference was found in the chemical shifts of C-9, C-10, C-12, C-14, and C-17, suggesting that 4 was a diastereomer of 1. The relative configuration of 4 was assigned through analysis of NOESY data ( Figure 3). The key NOE correlations from the proton of 10-OH to H-11β and H-12β indicated that these groups were on the same side of the molecule, while the NOEs from H-11α to H-15 and from H-14 to H-12α suggested they were on the other face. On the basis of the above evidence, the relative configuration of 4 was determined. The experimental ECD spectrum for 4 was in accordance with that calculated for (10S, 13R, 14R)-4 ( Figure 4).  Aspewentin M (5) was obtained as a colorless crystal, and was found to have the molecular formula C 20 H 28 O 3 on the basis of HRESIMS data ( Figure S33), with a CH 2 unit more than those of 1-4. The 1 H and 13 C NMR data for 5 (Tables 1 and 2) matched well with the data for 3. However, one of the two exchangeable protons at δ H 4.55 (14-OH) in 3 was not detected in 5, whereas the additional resonances for a methoxy group at δ H 3.23 (s)/δ c 59.4 (14-OMe) were shown in the NMR spectra of 5. Compared to compound 3, an upfield shift for H-14 and downfield shift for C-14 in 5 was detected. The above observation suggested that compound 5 was a 14-methoxylated derivative of 3. These inferences were further supported by the HMBC correlations from protons of 14-OMe to C-14 ( Figure 2). The NOEs for 5 were consistent with the relative configuration as described for 3 ( Figure 3). To confirm the absolute configuration of compound 5, we tried to crystallize it for an X-ray single crystallographic analysis. After many attempts, single crystals that were suitable for X-ray analysis were obtained through slow evaporation of a solution of 5 in MeOH. Once the X-ray crystallographic experiment was conducted, the absolute configuration of 5 was unambiguously assigned as (10S, 13R, 14S)-5 ( Figure 5). The absolute configuration of 5 was further studied with the TDDFT-ECD calculation. The ECD spectrum of 5 exhibited positive Cotton Effect (CE) at 240 nm and weak negative CE at 355 nm, which was the same as with 3 ( Figure 4). The experimental ECD spectrum of 5 also matched well with that of (10S, 13R, 14S)-5 (Figure 4), in accordance with the result of the X-ray. Aspewentin M (5) was obtained as a colorless crystal, and was found to have the molecular formula C20H28O3 on the basis of HRESIMS data ( Figure S33), with a CH2 unit more than those of 1-4. The 1 H and 13 C NMR data for 5 (Tables 1 and 2) matched well with the data for 3. However, one of the two exchangeable protons at δH 4.55 (14-OH) in 3 was not detected in 5, whereas the additional resonances for a methoxy group at δH 3.23 (s)/δc 59.4 (14-OMe) were shown in the NMR spectra of 5. Compared to compound 3, an upfield shift for H-14 and downfield shift for C-14 in 5 was detected. The above observation suggested that compound 5 was a 14-methoxylated derivative of 3. These inferences were further supported by the HMBC correlations from protons of 14-OMe to C-14 ( Figure  2). The NOEs for 5 were consistent with the relative configuration as described for 3 ( Figure 3). To confirm the absolute configuration of compound 5, we tried to crystallize it for an X-ray single crystallographic analysis. After many attempts, single crystals that were suitable for X-ray analysis were obtained through slow evaporation of a solution of 5 in MeOH. Once the X-ray crystallographic experiment was conducted, the absolute configuration of 5 was unambiguously assigned as (10S, 13R, 14S)-5 ( Figure 5). The absolute configuration of 5 was further studied with the TDDFT-ECD calculation. The ECD spectrum of 5 exhibited positive Cotton Effect (CE) at 240 nm and weak negative CE at 355 nm, which was the same as with 3 ( Figure 4). The experimental ECD spectrum of 5 also matched well with that of (10S, 13R, 14S)-5 (Figure 4), in accordance with the result of the X-ray.

Biological Activities of the Isolated Compounds
The obtained compounds 1-5 were tested for antimicrobial activities against seven zoonotic pathogenic bacteria and one plant-pathogenic fungus (Table 3). Compound 1 displayed activity against E. coli with MIC 32 μg/mL, while compounds 1 and 2 exhibited inhibitory activities against E. tarda, V. harveyi, and V. parahaemolyticus, each with an MIC value of 8.0 μg/mL. Compounds 1 and 2 were more active toward bacteria than compounds 3-5 were, suggesting that compounds with R absolute configuration at C-10 were more active than those that were S-configured. Moreover, compound 5 exhibited activity against the plant pathogen F. graminearum with an MIC value of 4.0 μg/mL, which was comparable to the positive control amphotericin B (MIC 4.0 μg/mL). These data indicated that the methoxylation at C-14 increased the activity against F. graminearum (5 vs. 1-4).

Biological Activities of the Isolated Compounds
The obtained compounds 1-5 were tested for antimicrobial activities against seven zoonotic pathogenic bacteria and one plant-pathogenic fungus (Table 3). Compound 1 displayed activity against E. coli with MIC 32 µg/mL, while compounds 1 and 2 exhibited inhibitory activities against E. tarda, V. harveyi, and V. parahaemolyticus, each with an MIC value of 8.0 µg/mL. Compounds 1 and 2 were more active toward bacteria than compounds 3-5 were, suggesting that compounds with R absolute configuration at C-10 were more active than those that were S-configured. Moreover, compound 5 exhibited activity against the plant pathogen F. graminearum with an MIC value of 4.0 µg/mL, which was comparable to the positive control amphotericin B (MIC 4.0 µg/mL). These data indicated that the methoxylation at C-14 increased the activity against F. graminearum (5 vs. 1-4). Aspewentins I−M (1−5) were also evaluated for lethal activity against brine shrimp Artemia salina, but none of these compounds showed obvious activity (LD 50 > 20 µg/mL). Table 3. Antimicrobial activities of compounds 1-5 (MIC, µg/mL) a .

Fungal Material
The isolation and identification of the fungal material were the same as those reported in our previous publications [12][13][14]. The strain was preserved at the Key Laboratory of Experimental Marine Biology, Institute of Oceanology of the Chinese Academy of Sciences, with accession number SD-310.

Fermentation
For the secondary metabolites study, the fungal strain was dynamically fermented in a 500 L fermentor preloaded with 300 L of sterilized liquid medium containing 50% (v/v) sea water collected from Hui Quan Bay (20% potato, 2% glucose, 0.5% peptone, and 0.3% yeast extract, pH 6.0) for 7 days at room temperature.

Extraction and Isolation
The whole fermented cultures were filtered to separate the broth from the mycelia. The former was extracted three times with EtOAc, while the latter was extracted three times with a mixture of acetone and H 2 O (80:20, v/v). The acetone solution was evaporated under reduced pressure to afford an aqueous solution, which was then extracted with EtOAc three times. Because the TLC and HPLC profiles of the two EtOAc solutions from the broth and mycelia were almost identical, they were combined and concentrated under reduced pressure to give an extract (34.7 g) for further separation.

Antimicrobial and Brine Shrimp Lethality Assays
Antimicrobial evaluation against seven zoonotic pathogenic bacteria between human and aquatic animals (E. coli QDIO-1, Aeromonas hydrophilia QDIO-3, E. tarda QDIO-4, Pseudomonas aeruginosa QDIO-6, V. anguillarum QDIO-8, V. harveyi QDIO-9, and V. parahaemolyticus QDIO-10), as well as one plant-pathogenic fungus (F. graminearum QDIO-13), were carried out with a microplate assay with three repetitions [16]. The pathogenic bacteria and aquatic pathogen strains were provided by the Institute of Oceanology, Chinese Academy of Sciences, while the plant pathogenic fungi strains were provided by Qingdao Agricultural University. Chloramphenicol and amphotericin B were used as positive controls against the bacteria and fungi, respectively. Evaluation of brine shrimp lethality against A. salina was performed as previously reported [17].

X-ray Crystallographic Analysis
A colorless crystal of compound 5 was obtained from a solution of MeOH. Crystallographic data were collected on an Agilent Xcalibur Eos Gemini CCD plate diffractometer (Agilent Technologies, Santa Clara, CA, USA), equipped with graphite-monochromatic Cu Kα radiation (λ = 1.54178 Å) at 293(2) K [18]. The data were corrected for absorption by using the program SADABS [19]. The structure was solved through direct methods and subsequent difference Fourier synthesis and refined by full-matrix least-squares techniques with the SHELXTL software package (Version 6.10, Sheldrick G.M., University of Göttingen, Germany) [20]. All nonhydrogen atoms were refined anisotropically. The H atoms belonging to C atoms were calculated theoretically, and those belonging to O atoms were determined by difference Fourier maps [21]. Crystal

Computational Section
Conformational searches were performed via molecular mechanics using the MM+ method in HyperChem software (Version 8.0, Hypercube, Inc., Gainesville, FL, USA), and the geometries were further optimized at the B3LYP/6-31G(d) PCM/MeCN level via Gaussian 09 software (Version D.01; Gaussian, Inc.: Wallingford, CT, USA) [15] to give the energy-minimized conformers. Then, the optimized conformers were subjected to the calculations of ECD spectra using TDDFT at PBE0/TZVP, CAM-B3LYP/TZVP, and BH&HLYP/TZVP. Solvent effects of the MeCN solution were evaluated at the same DFT level using the SCRF/PCM method.

Conclusions
In summary, we have isolated and characterized five new compounds, aspewentins I−M (1-5), which are new members of 20-nor-isopimarane diterpenoids. These compounds contained the rare examples of 20-nor-isopimarane analogues possessing a cyclohexa-2,5-dien-1-one moiety. Compound 1, which may prove useful as an antifungal agent, exhibited potent antimicrobial activities against some plant pathogenic fungi. Compounds 1 and 2 showed inhibitions against zoonotic pathogenic bacteria between human and aquatic animals.

Conflicts of Interest:
The authors declare no conflicts of interest.