Eutypenoids A–C: Novel Pimarane Diterpenoids from the Arctic Fungus Eutypella sp. D-1

Eutypenoids A–C (1–3), pimarane diterpenoid alkaloid and two ring A rearranged pimarane diterpenoids, were isolated from the culture of Eutypella sp. D-1 obtained from high-latitude soil of the Arctic. Their structures, including absolute configurations, were authenticated on the basis of the mass spectroscopy (MS), nuclear magnetic resonance (NMR), X-ray crystallography, and electronic circular dichroism (ECD) analysis. The immunosuppressive effects of eutypenoids A–C (1–3) were studied using a ConA-induced splenocyte proliferation model, which suggested that 2 exhibited potent immunosuppressive activities.


Introduction
Marine-sourced fungi are, increasingly, a rich source of novel and bioactive compounds [1][2][3], but natural products from Arctic fungi are rarely studied. Arctic fungi are abundant and functionally important in the Arctic, where they drive mineral and energy cycles, and influence the occurrence of other organisms as mutualists, decomposers, and pathogens [4]. Meanwhile, Arctic fungi living in low temperatures, strong ultraviolet radiation, low nutrition, etc., might have evolved specific physiological and biochemical pathways to produce structurally novel and biological active metabolites, which can provide the opportunity for the discovery of new natural medicines.
Eutypella (Diatrypaceae) species, from the South Sea in China and Thailand, have been widely investigated in recent years. Most of metabolites, including pimarane diterpenoids, cytochalasin derivatives, γ-lactones, sesquiterpenoids, polyketides, and cytosporin-related compounds, display moderate or significant cytotoxic and antimicrobial activities [5][6][7][8][9][10][11][12][13]. However, no such study has been carried out on Eutypella species from the Polar region. Eutypella sp. D-1 was isolated from high-latitude soil of the Arctic. Previously, we have reported six pimarane diterpenoids and four tyrosine-derived cytochalasins from the culture of E. sp. D-1 [14][15][16]. In the current study on the crude extract of E. sp. D-1, we reported the isolation, structure elucidation, and immunosuppressive effects of three novel

Results and Discussion
Under bioassay guidance, the chemical constituents of E. sp. D-1 have been extensively investigated further. In this paper, three new novel rearranged pimarane diterpenoids (1)(2)(3) were isolated from the EtOAc extract of its culture broth.
Mar. Drugs 2016, 14, x 2 of 9 structure type of diterpenoid, respectively. Further, these compounds were evaluated for their immunosuppressive effects.

Results and Discussion
Under bioassay guidance, the chemical constituents of E. sp. D-1 have been extensively investigated further. In this paper, three new novel rearranged pimarane diterpenoids (1-3) were isolated from the EtOAc extract of its culture broth.
Mar. Drugs 2016, 14, x 3 of 9 The relative configuration of 1 was deduced from nuclear overhauser effect spectroscopy (NOESY) analysis ( Figure 3). The correlations of H-18 with H-20 indicated β-orientations. Finally, the proposed structure of 1 was confirmed by X-ray crystallography analysis using anomalous scattering of Cu Kα radiation ( Figure 4). Accordingly, the absolute configuration of 4R, 10R was established based on the value of the Flack absolute structure parameter −0.12 (10).   (Table 1), and HSQC spectroscopic data showed the presence of a ketone carbon (δ 181.3), a ketoxime carbon (δ 153.5), eight olefinic carbons, including one methylene and three methine, three sp 3 quaternary carbons, one oxymethine carbon, two methylene carbons, and four methyl carbons. Specifically, the HSQC did not provide the correction between the carbons and protons (δ 6.76, s; 7.94, s), which were indicative of two active hydrogens. The planar structure of 2 was established by extensive analyses of its 1 H-1 H COSY and HMBC spectra ( Figure 2). Key HMBC correlations from H3-18, 19 to C-3, 5, from H3-20 to C-1, 5, 9, 10, 11, from H-15, H3-17 to C-12, 13, 14, allowed the structure of 2 to be assigned as a pimarane diterpenoid. Additionally, HMBC corrections from 6-OH to C-5, 6, 7, from N-OH to C-11 established the enol moiety at C-6 and ketoxime moiety at C-11. 1 H-1 H COSY spectrum of 2 revealed the partial structure H-1 to H-3 via H-2, and the connectivity between H-15 and H-16. The relative configuration of 2 was established from the nuclear overhauser effect (NOE) effects observed in the NOESY experiment ( Figure 3). The NOE correlation revealed the relative configuration of 2 partially, in which the cross peaks of H-14/ H3-17 indicated that H-14 and H3-17 possessed the same The relative configuration of 1 was deduced from nuclear overhauser effect spectroscopy (NOESY) analysis ( Figure 3). The correlations of H-18 with H-20 indicated β-orientations. Finally, the proposed structure of 1 was confirmed by X-ray crystallography analysis using anomalous scattering of Cu Kα radiation ( Figure 4). Accordingly, the absolute configuration of 4R, 10R was established based on the value of the Flack absolute structure parameter −0.12 (10).   (Table 1), and HSQC spectroscopic data showed the presence of a ketone carbon (δ 181.3), a ketoxime carbon (δ 153.5), eight olefinic carbons, including one methylene and three methine, three sp 3 quaternary carbons, one oxymethine carbon, two methylene carbons, and four methyl carbons. Specifically, the HSQC did not provide the correction between the carbons and protons (δ 6.76, s; 7.94, s), which were indicative of two active hydrogens. The planar structure of 2 was established by extensive analyses of its 1 H-1 H COSY and HMBC spectra ( Figure 2). Key HMBC correlations from H3-18, 19 to C-3, 5, from H3-20 to C-1, 5, 9, 10, 11, from H-15, H3-17 to C-12, 13, 14, allowed the structure of 2 to be assigned as a pimarane diterpenoid. Additionally, HMBC corrections from 6-OH to C-5, 6, 7, from N-OH to C-11 established the enol moiety at C-6 and ketoxime moiety at C-11. 1 (Table 1), and HSQC spectroscopic data showed the presence of a ketone carbon (δ 181.3), a ketoxime carbon (δ 153.5), eight olefinic carbons, including one methylene and three methine, three sp 3 quaternary carbons, one oxymethine carbon, two methylene carbons, and four methyl carbons. Specifically, the HSQC did not provide the correction between the carbons and protons (δ 6.76, s; 7.94, s), which were indicative of two active hydrogens. The planar structure of 2 was established by extensive analyses of its 1 H-1 H COSY and HMBC spectra ( Figure 2). Key HMBC correlations from H 3 -18, 19 to C-3, 5, from H 3 -20 to C-1, 5, 9, 10, 11, from H-15, H 3 -17 to C-12, 13, 14, allowed the structure of 2 to be assigned as a pimarane diterpenoid. Additionally, HMBC corrections from 6-OH to C-5, 6, 7, from N-OH to C-11 established the enol moiety at C-6 and ketoxime moiety at C-11. 1 H-1 H COSY spectrum of 2 revealed the partial structure H-1 to H-3 via H-2, and the connectivity between H-15 and H-16. The relative configuration of 2 was established from the nuclear overhauser effect (NOE) effects observed in the NOESY experiment ( Figure 3). The NOE correlation revealed the relative configuration of 2 partially, in which the cross peaks of H-14/ H 3 -17 indicated that H-14 and H 3 -17 possessed the same orientation. The absolute configuration of 2 was established by comparison experimental and calculated electronic circular dichroism (ECD) spectra using the time-dependent density functional theory (TD-DFT) method at the B3LYP/6-31G (d,p) level in methanol with the conductor-like polarizable continuum model (CPCM), the E-isomer of ketoxime possessed the lower energy. The overall pattern of calculated ECD spectrum of (E,10R,13S,14R)-2b was in accordance with the experimental data of 2 ( Figure 5A). Therefore, the absolute configuration of 2 was established as E, 10R, 13S, 14R.  1 (s)). In addition to these two substituents, the 1 H NMR spectrum of 3 also exhibited the characteristic pattern for a terminal vinyl group (δ 5.33, d, J = 17.7 Hz, 5.36, d, J = 10.7 Hz; 5.92, dd, J = 17.7/10.7 Hz), the 13 C NMR spectrum of 3 in combination with HSQC data revealed two methyls and eight olefinic carbons. These data suggested that compound 3 is a disubstituted tetracyclic pimarane diterpenoid. The basic carbon skeleton of 3 was established by comprehensive analysis of the 2D NMR spectroscopic data, particularly the 1 H-1 H COSY and HMBC correlations (Figure 2). Correlations were detected for H 2 -20/H-1/H-2/H-3 by COSY, indicating C-20 was attached to C-1, the HMBC correlations from H 2 -20 to C-1, C-2, C-10, C-11, and from H-11 to C-8, C-9, indicating a pyranoid ring was formed through C-20/C-1/C-10/C-9/C-11. The acetylated and isobutyrylated positions were determined to be C-3 and C-19 based on the HMBC correlations from H-3 to δ 170.6, and from H-19 to δ 177.1, respectively. The relative configuration of 3 was established using NOESY (  Pimarane diterpenoids with anti-inflammtory effects have been reported [17,18]. In our bioassay of eutypenoids A-C with several anti-inflammatory models, we found that eutypenoid B (2) had an immunosuppressive effect. The immunosuppressive effects of eutypenoids A-C (1-3) were examined on splenocyte proliferation induced by concanavalin A (ConA) using a method described in the literature [19]. The results showed that compounds 1-3 had no cytotoxic effect on splenocytes at concentrations from 1.6 μmol to 40 μmol. Within the concentration range, eutypenoid B (2) exhibited significant inhibition of splenocyte proliferation under ConA induction, while eutypenoids A and C has no significant effects ( Figure 6). Our findings, from an antiproliferation assay, propose that compound 2, not only has no cytotoxic effect on splenocytes, but exhibited significant inhibition of splenocyte proliferation under ConA induction. Further study is needed to confirm the effect and pursue the mechanisms of action. Pimarane diterpenoids with anti-inflammtory effects have been reported [17,18]. In our bioassay of eutypenoids A-C with several anti-inflammatory models, we found that eutypenoid B (2) had an immunosuppressive effect. The immunosuppressive effects of eutypenoids A-C (1-3) were examined on splenocyte proliferation induced by concanavalin A (ConA) using a method described in the literature [19]. The results showed that compounds 1-3 had no cytotoxic effect on splenocytes at concentrations from 1.6 µmol to 40 µmol. Within the concentration range, eutypenoid B (2) exhibited significant inhibition of splenocyte proliferation under ConA induction, while eutypenoids A and C has no significant effects ( Figure 6). Our findings, from an antiproliferation assay, propose that compound 2, not only has no cytotoxic effect on splenocytes, but exhibited significant inhibition of splenocyte proliferation under ConA induction. Further study is needed to confirm the effect and pursue the mechanisms of action.

General Experimental Procedures
Optical rotations were determined using a Perkin-Elmer 341 polarimeter. CD spectra were obtained on a Chirascan spectrometer (Applied Photophysics Ltd., Leatherhead, UK). The NMR spectra were recorded on a Bruker AM-400 spectrometer at 400 MHz for 1 H and 100 MHz for 13 C in CDCl3 or Bruker AVANCE-III instrument operating at 600 MHz for 1 H and 150 MHz for 13 C. ECD spectra were recorded in EtOH with a Chirascan CD spectrometer. ESIMS and HRESIMS were obtained using an Esquire 3000 plus and a Q-TOF-Ultima mass spectrometer, respectively. Silica gel (200 mesh to 300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), C18 reversed phase silica gel (150 to 200 mesh, Fuji Silysia Chemical, Ltd., Aichi, Japan), MCI gel (CHP20P, 75 μm to 150 μm, Mitsubishi Chemical Industries, Ltd., Tokyo, Japan), and Sephadex LH-20 gel (Pharmacia Biotech AB, Uppsala, Sweden) were used for column chromatography (CC). High performance liquid chromatography was performed on an Angilent 1200 HPLC apparatus with an Eclipse XDB-C18 column (250 × 9.4 mm, 5 μm).

Fungal Strain
The fungus was isolated from the soil of London Island of Kongsfjorden of Ny-lesund District (altitude of 100 m) in the Arctic. It was isolated in potato dextrose agar (PDA) medium with incubation at 20 °C. Due to its chemical and morphological features, as well as the 18S rDNA

General Experimental Procedures
Optical rotations were determined using a Perkin-Elmer 341 polarimeter. CD spectra were obtained on a Chirascan spectrometer (Applied Photophysics Ltd., Leatherhead, UK). The NMR spectra were recorded on a Bruker AM-400 spectrometer at 400 MHz for 1 H and 100 MHz for 13 C in CDCl 3 or Bruker AVANCE-III instrument operating at 600 MHz for 1 H and 150 MHz for 13 C. ECD spectra were recorded in EtOH with a Chirascan CD spectrometer. ESIMS and HRESIMS were obtained using an Esquire 3000 plus and a Q-TOF-Ultima mass spectrometer, respectively. Silica gel (200 mesh to 300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), C 18 reversed phase silica gel (150 to 200 mesh, Fuji Silysia Chemical, Ltd., Aichi, Japan), MCI gel (CHP20P, 75 µm to 150 µm, Mitsubishi Chemical Industries, Ltd., Tokyo, Japan), and Sephadex LH-20 gel (Pharmacia Biotech AB, Uppsala, Sweden) were used for column chromatography (CC). High performance liquid chromatography was performed on an Angilent 1200 HPLC apparatus with an Eclipse XDB-C 18 column (250ˆ9.4 mm, 5 µm).

Fungal Strain
The fungus was isolated from the soil of London Island of Kongsfjorden of Ny-lesund District (altitude of 100 m) in the Arctic. It was isolated in potato dextrose agar (PDA) medium with incubation at 20˝C. Due to its chemical and morphological features, as well as the 18S rDNA (GenBank accession No. FJ430580), the strain was assigned to the genus Eutypella. The strain was deposited in PDA medium with the Second Military Medical University, Xiangyin Road 800, 200433, Shanghai, P. R. China. Eutypella sp. D-1 was cultured in potato dextrose broth (PDB; potato 1%, glucose 2%, dist. H 2 O 1000 mL).

Culture Condition
The fungus was maintained in PDA medium at 20˝C for 7 days, and then three pieces (0.5ˆ0.5 cm 2 ) of mycelial agar plugs were inoculated into 60ˆ250 mL Erlenmeyer flasks, each containing 70 mL of PDB. After 5 d of incubation at 20˝C on a rotary shaker at 180 rpm, 70 mL of seed cultures were transferred into a total of 250 flasks (2.0 L) containing 700 mL of PDB. The liquid cultivation that followed was kept for 9 days at 20˝C and 180 rpm on a rotary shaker.

Material and Method
Electronic circular dichroism (ECD) spectrum, associated ab initio (TD) DFT calculations, is a reliable spectroscopic tool for determining the absolute configuration of chiral compounds [20,21]. Firstly, conformation searches were carried out using a conformational search module in Schrodinger, with the OPLS_2005 Force field and the torsional sampling (MCMM) method. Then, the conformers were optimized using the DFT calculation. Frequency calculations were also performed to confirm that the geometries obtained correspond to energetic minima. The geometries of the optimized conformers were provided in the Supporting Information. Calculation of ECD spectra were performed using the TDDFT calculation. The ECD spectra were obtained by weighing the Boltzmann distribution rate of each conformer with the software SpecDis1.62 [22].

Antiproliferation Assay
Cell viability was assessed by performing an MTT assay [17]. In brief, splenocytes (4ˆ10 5 cells/well) were cultured in triplicate with or without compounds 1-3 in a 96 well plate at 37˝C in a 5% CO 2 atmosphere for 48 h. MTT was then added to the medium (0.5 mg/mL) and incubated for 4 h before the end of the incubation period. The medium was removed, and the cells were diluted in dimethyl sulphoxide. The relative formazan concentration was measured by the optical density at 570 nm (OD570 nm) using a microplate reader (BioTek, PowWave XS2, Winooski, VT, USA). Compounds 1-3 did not react with MTT. Splenic lymphocytes (4ˆ10 5 cells/well) and compounds 1-3 in 96 well plates were cultured in triplicate for 48 h by using ConA (2 µg/mL). The cells were pulsed at 0.25 µCi/well of [ 3 H]-thymidine for 8 h before the end of the culture period, and then harvested onto glass fiber filters. [ 3 H]-thymidine incorporation was measured by counts per minute (cpm) using a beta scintillation counter (MicroBeta Trilux, PerkinElmer Life Sciences, Boston, MA, USA).

Conclusions
In conclusion, through the chemical investigation of Eutypella sp. D-1 from the Arctic, Eutypenoid B (2) a pimarane diterpenoid alkaloid was firstly isolated from natural metabolites, Eutypenoids A and C (1 and 3) were two unusual ring A rearranged pimarane diterpenoids with a new carbon skeleton, respectively. Among of them, Eutypenoid B (2) exhibited potent immunosuppressive activities.