Fungi have been regarded as an attractive source for discovering new bioactive compounds.1, 2 Several bioactive metabolites from fungi, such as penicillins, cephalosporins, mevastatin and lovastatin, have been the important products of pharmaceutical industry.3 With the aim of searching for bioactive metabolites from fungi, our studies of fungi imperfeci from a variety of ecological groups have resulted in the discovery of several kinds of new natural products with antitumor activity.4, 5, 6 As part of our continuous screening for more active secondary metabolites from soil-derived fungi imperfeci, a new stachybotrin congener, named stachybotrin G (1) and a known metabolite, stachybotrin (2) were obtained from Stachybotrys parvispora strain HS-FG-843. Here, the report details the fermentation, isolation, structure elucidation and bioactivity of the new compound.

The S. parvispora strain HS-FG-843 was isolated from a soil sample collected from the peak of a bamboo forest of Hunan province, China. It was provided and identified as S. parvispora by Professor Tianyu Zhang at the Shandong Agricultural University, China. The strain HS-FG-843 has been deposited in the Pharmaceutical Research Culture Collection, Zhejiang Hisun Group Co., Ltd with accession No. HS-FG-843.

The strain was grown and maintained on potato dextrose agar slant and incubated for 6–7 days at 24 °C. The stock culture was transferred into 1 l Erlenmeyer flasks containing 250 ml of the seed medium and incubated at 24 °C for 24 h, shaken at 150 r.p.m. Then, 1 l of the culture was transferred into a 50-l fermentor containing 30 l of producing medium consisting of peptone 0.5%, potato starch 0.5%, yeast extract 0.2%, NaCl 0.4%, KH2PO4 0.1%, MgSO4·7H2O 0.05%, CaCO3 0.2% (pH 6.2–6.4). The fermentation was carried out at 24 °C for 7 days stirred at 100 r.p.m. with an aeration rate of 900 l of air per hour.

The final 30 l of broth from 50 l fermentor was filtered and the resulting cake was washed with water (3 l) and subsequently extracted with MeOH (3 l). The supernatant and the wash water were subjected to a Diaion HP-20 resin (Mitsubushi Chemical Co., Ltd., Tokyo, Japan) column eluting with 95% EtOH (5 l). The MeOH extract and the EtOH eluents were evaporated under reduced pressure to a volume of 1 l at 50 °C and the resulting concentrate was extracted three times using an equal volume of EtOAc. The combined EtOAc phase was concentrated under reduced pressure to yield a mixture (15 g). The mixture was subjected to a Sephadex LH-20 gel (GE Healthcare, Glies, UK) column eluted with CHCl3/MeOH (1:1, v/v) and detected by TLC to give four fractions (Fr.1 to Fr.4). The Fr.2 was chromatographed on a silica gel (Qingdao Haiyang Chemical Group, Qingdao, Shandong, China; 100–200 mesh) column and successively eluted with a stepwise gradient of CHCl3/MeOH (100:0–50:50, v/v) to obtain three fractions Fr.2-1 to Fr.2-3 based on the TLC profiles. The Fr.2-2 was subjected to another silica gel column eluted with CHCl3/MeOH (90:10–70:30, v/v) to give three fractions (Fr.2-2-1, Fr.2-2-2 and Fr.2-2-3). Fr.2-2-3 was further isolated by semi-preparative HPLC (Agilent 1100, Zorbax SB-C18, 5μm, 250 × 9.4 mm i.d; 1.5 ml min−1; 220 nm; Agilent, Palo Alto, CA, USA) eluting with MeOH/H2O (78:22, v/v) to obtain compound 1 (tR 23.4 min, 3.6 mg). The Fr.2-2-1 was purified by semi-preparative HPLC eluting with MeOH/H2O (74:26) to yield stachybotrin (2) (tR 23.2 min, 23 mg). 1H and 13C NMR spectra were measured with a Bruker DRX-400 (400 MHz for 1H and 100 MHz for 13C) spectrometer (Bruker, Rheinstetten, Germany). The ESIMS and high-resolution electrospray ionization MS (HRESIMS) spectra were taken on a Q-TOF Micro LC-MS-MS mass spectrometer (Milford, MA, USA).

Compound 1 was isolated as a white amorphous powder with + 50 (c 0.036, EtOH) and UV (EtOH) λmax nm (log ɛ): 338 nm (4.89), 285 nm (4.95), 228 nm (5.08). Its molecular formula was determined as C28H38N2O5 according to HRESIMS (found m/z 483.2846 [M+H]+, calcd 483.2853). The IR spectrum of 1 showed absorption bands at 3414 cm−1 (OH), 1650 (C=O), 1610 cm−1 (C=O) in the functional group region. 1H NMR spectrum of 1 showed an aromatic proton singlet at δH 6.93 (1H, s), three tertiary methyl signals at δH 0.85 (3H, s), 0.96 (3H, s) and 0.98 (3H, s), one aliphatic methyl doublet at δH 0.69 (3H, d, J=6.5 Hz). Its 13C NMR and DEPT spectra displayed 28 carbon resonances, including two amide or ester carbonyl carbons at δC 169.4 (s, 2C), one sp2 methine, five sp2 quaternary carbons (two oxygenated), three sp3 quaternary carbons at δC 98.6 (s), 42.2 (s) and 37.6 (s), four sp3 methines at δC 75.7 (d), 52.3 (d), 39.9 (d) and 37.2 (d), and four methyls in addition to nine methylenes.

The proton signal at δH 6.93 (1H, s) in the 1H NMR spectrum and the carbon resonances at δC 156.2 (s), 152.7 (s), 133.7 (s), 117.3 (s) and 102.3 (s) revealed the presence of a penta-substituted benzene moiety in 1. Two protons of an AB system at δH 2.82 (d, J=16.8 Hz) and 3.19 (d, J=16.8 Hz), and the similar two protons of an AB system at δH 4.28 (d, J=16.1 Hz) and 4.39 (d, J=16.11 Hz) observed in 1H NMR spectrum in connection with HMQC spectrum were assigned to the protons of two isolated methylene groups. Comparison of the NMR data (Table 1) of 1 with those of stachybotrin (2) isolated from this strain and stachybotrins D-F obtained from the sponge-derived fungus Stachybotrys chartarum MXH-X737 suggested that 1 belonged to the stachybotrin series containing a combination of a spirobenzofuran system with a sesquiterpenoid drimane nucleus.

Table 1 1H and 13C NMR data for compounds 1 (in CDCl3) and 2 (in DMSO-d6) (δ, p.p.m)
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Further detailed analysis of the NMR data revealed that the differences between 1 and stachybotrin (2) were that the isolated C-24 and C-25 methylene groups in 2 were replaced by the five carbon signals at δC 169.4 (s), 52.3 (d), 42.4 (t), 25.0 (t), and 22.1 (t). Considering the molecular formula C28H38N2O5 of 1, the above five carbon signals were assigned to a six-membered lactam subunit as shown in Figure 1. This result was supported by the 1H NMR and 13C NMR chemical shifts of C-24 (δH 4.83, δC 52.3) and C-27 (δH 3.39, δC 42.4) and the HMBC correlation from δH 3.39 to δC 169.4. The connection of C-24 with C-18 and C-19 through a nitrogen atom was confirmed by the HMBC correlations from δH 4.83 to δC 169.4 (C-19) and from δH 4.39, 4.28 to δC 52.3 (C-24). The signal of H-24 was observed in 1H NMR spectrum of 1 at δH 4.83 (dd, J=11.4, 6.5 Hz) and those constants showed the axial orientation of H-24. Biogenetically, the relative configuration of 1 was assigned by analogy with 2 and stachybotrins D-F. Therefore, the structure of 1 was determined (Figure 1), and named stachybotrin G.

Figure 1
figure 1

Structures of 1, 2 and key HMBC correlations of 1.

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Compound 2 was also obtained as a white amorphous powder. Its structure was elucidated as stachybotrin by the comparison of the 1H and 13C NMR data (Table 1) of 2 with the NMR data reported by Kamalov et al.8, 9

The cytotoxicity of compounds 1 and 2 were assayed in vitro against the human lung carcinoma A549 cell lines by the CCK8 colorimetric method as described in our previous papers.10, 11 As a result, compounds 1 and 2 exhibited cytotoxic activity with IC50 values of 17.4 and 9.5 μg ml−1, respectively.