We screened a library constructed by the advanced compound-identification system based on the accumulated HPLC-MS profiling data combined with strain information designated as ‘MBJ’s special selection’ for bioactive substances, and have isolated five novel eremophilane sesquiterpenoids MBJ-0009 and -0010 from the saprobic fungus Nectria sp. and MBJ-0011, -0012 and -0013 from the endophytic fungus Apiognomonia sp.1, 2 Further screening for cytotoxic compounds led to the identification of MBJ-0038 (1), -0039 (2) and -0040 (3), together with the known compounds chaetoglobosins C and F3, 4 from Chaetomium sp. f24230. Here, we describe the fermentation, isolation, structure elucidation, and, in brief, the cytotoxic activity of 1–3.
Chaetomium sp. f24230 was isolated from a soil sample collected in the Okinawa Prefecture, Japan. The strain was cultivated in 250-ml Erlenmeyer flasks, each containing 25 ml of a seed medium consisting of 2% potato starch (Tobu Tokachi Nosan Kako Agricultural Cooperative Association, Hokkaido, Japan), 1% glucose (Junsei Chemical, Tokyo, Japan), 2% soybean powder (Honen SoyPro, J-Oil Mills, Tokyo, Japan), 0.1% KH2PO4 and 0.05% MgSO4·7H2O. The flasks were shaken on a rotary shaker (220 r.p.m.) at 25 °C for 3 days. Aliquots (0.5 ml) of the broth were transferred to 500-ml Erlenmeyer flasks containing 50 ml of a production medium consisting of 2% potato starch (Tobu Tokachi Nosan Kako Agricultural Cooperative Association), 1% glucose (Junsei Chemical), 2% soybean powder (Honen SoyPro, J-Oil Mills), 0.1% KH2PO4 and 0.05% MgSO4·7H2O, and were cultured on a rotary shaker (220 r.p.m.) at 25 °C for 4 days.
The whole-culture broth (2 l) was extracted with an equal volume of n-BuOH and, following concentration, was successively partitioned between ethyl acetate (350 ml × 3) and water (350 ml). The ethyl acetate extract (4.56 g) was subjected to silica gel medium-pressure liquid chromatography (MPLC; Purif-Pack SI-30, Shoko Scientific Co., Yokohama, Japan) with a gradient system of n-hexane–ethyl acetate (0–25% ethyl acetate) followed by CHCl3–methanol (MeOH) with stepwise increases from 0 to 100% MeOH with monitoring by UV at 254 nm. The active 5% MeOH fraction (190.1 mg) was separated by gel filtration on a Sephadex LH-20 column (1:1, CHCl3/MeOH, GE Healthcare BioSciences AB, Uppsala, Sweden) followed by reversed phase-MPLC (Purif-Pack ODS-30; 70–100% aq. MeOH with 10% stepwise increments in the MeOH concentration) purification to yield 3 (90% MeOH, 6.1 mg). The active 10% MeOH fraction (563.9 mg) was subjected to Sephadex LH-20 column chromatography (1:1, CHCl3/MeOH) to yield a crude material containing 2 (98.9 mg). This material was purified using ODS MPLC (Purif-Pack ODS-30; 50–100% aq. MeOH, 10% stepwise) to yield 2 (80% MeOH, 12.3 mg). Another active fraction from the 10% MeOH eluate (1.70 g) was further purified using an ODS MPLC (Purif-Pack ODS-100; 20–100% aq. MeOH in 20% stepwise increments), and an 80% MeOH eluate (940 mg) was re-chromatographed using an ODS MPLC (Purif-Pack ODS-30; 50–100% aq. MeOH, 10% stepwise) to yield crude 1 (80% MeOH, 119.9 mg). Compound 1 (55.8 mg) was finally purified using Sephadex LH-20 column chromatography (1:1, CHCl3/MeOH).
MBJ-0038 (1) was isolated as a colorless amorphous powder: [α]25D –66 (MeOH; c 0.7); UV λmax (ɛ) in MeOH: 281 (7700) nm; IR (ATR) νmax: 3400 (hydroxy) and 1689 (carbonyl) cm−1. The molecular formula of 1 was established as C41H44N2O9 by HR-ESI-MS; (m/z 709.3122 [M+H]+, calcd. for C41H45N2O9 m/z 709.3125). The 13C and 1H NMR data for 1 are listed in Table 1. Structural information on 1 was obtained by a series of 2D NMR analyses such as double quantum-filtered COSY (DQF-COSY), HSQC and constant-time (CT)-HMBC.5
An indole moiety in 1 was identified based on 1H couplings from a doublet aromatic proton H-4′ (δH 7.53) through a doublet of doublets aromatic protons H-5′ (δH 7.03) and H-6′ (δH 7.10) to a doublet aromatic proton H-7′ (δH 7.34), and by following 1H–13C long-range couplings from a singlet aromatic proton H-2′ (δH 7.07) to aromatic quaternary carbon C-3′ (δC 111.2), C-3a′ (δC 128.7) and C-7a′ (δC 138.2); from H-4′ to C-3′ and C-7a′; from H-5′ to C-3a′; from H-6′ to C-7a′; and from H-7′ to C-3a′, together with their typical 13C NMR chemical shifts (Figure 1c).
The DQF-COSY spectrum revealed the following proton spin networks, from a methyl proton H3-11 (δH 0.62) through methine protons H-5 (δH 1.75), H-4 (δH 2.64) and H-3 (δH 3.85) to methylene protons H2-10 (δH 3.06 and 2.85); from a methine proton H-7 (δH 2.72) through a methine proton H-8 (δH 2.30), olefinic methine protons H-13 (δH 6.04) and H-14 (δH 5.15), methylene protons H2-15 (δH 2.14 and 1.72) and a methine proton H-16 (δH 2.42) to an olefinic proton H-17 (δH 4.89); and from a methine proton H-22 (δH 5.78) to a methine proton H-28 (δH 3.68) through a methine proton H-21 (δH 4.34). The presence of a six-membered ring (C-4 to C-9) was identified from HMBC correlations from a singlet methyl proton H3-12 (δH 1.18) to a methine carbon C-5 (δC 37.9), a quaternary carbon C-6 (δC 58.9) and a methine carbon C-7 (δC 63.3); and from the methine proton H-8 to a methine carbon C-4 (δC 52.8) and a quaternary carbon C-9 (δC 64.8). The high-field shift for the C-6 signal supported the presence of an epoxide ring between C-6 and C-7. HMBC correlations from H-4, H-8 and the nitrogen-substituted methine proton H-3 (δC 54.9) to a carbonyl carbon C-1 (δC 176.6) suggested the presence of a γ-lactam ring moiety, as shown in Figure 1c. Furthermore, the presence of a 13-membered carbon ring structure was revealed by following 1H–13C long-range couplings, from a methyl proton H3-30 (δH 0.89), which exhibited 1H spin coupling to H-16, to an olefinic methine carbon C-17 (δC 137.5); from a methyl proton H3-31 (δH 1.55) to C-17, an olefinic quaternary carbon C-18 (δC 131.8) and an oxymethine carbon C-19 (δC 82.2); from an oxymethine proton H-19 (δH 3.96) together with H-21 and H-22 to a ketone carbonyl carbon C-20 (δC 209.1); and from H-4, H-8, H-21 and H-28 to another ketone carbonyl carbon C-29 (δC 209.0). The direct connectivity between C-10 and C-3′ was evident from HMBC correlations from H2-10 to C-2′ (δC 124.6), C-3′ and C-3a′. Thus, a chaetoglobosin skeleton was established. The structure of the remaining part of the molecule was determined as follows. The structure of a tetrahydrofuran ring moiety was determined from long-range couplings from an oxymethine proton H-27 (δH 5.81, δC 82.7) to methine carbons C-21 (δC 51.2), C-28 (δC 58.2) and an oxymethine carbon C-22 (δC 82.1). HMBC correlations from an allylic methyl proton H3-32 (δH 2.28) to aromatic quaternary carbons C-25 (δC 145.0), C-26 (δC 111.3), C-26a (δC 135.1) and additionally to C-24 (δC 133.8) weakly; from H-27 to an aromatic quaternary carbon C-22a (δC 121.6) and C-26; from H-22 to aromatic quaternary carbons C-22a, C-23 (δC 138.8), C-24 and C-26a; and from H-28 to C-26a indicated the presence of an 8-methyl-1,2,3,4-tetrahydro-1,4-epoxynaphthalene-5,6,7-triol substructure. The proton chemical shift values at H-22 and H-27, together with low-field shifted 13C chemical shift values at C-22 and C-27 because of the presence of an ether bond agreed well with the corresponding proton chemical shifts of 1,4-epoxy-1,2,3,4-tetrahydronaphthalene-endo,cis-2,3-dicarboxylic acid (δH 5.48 and 5.44, in dimethyl sulfoxide-d6).6 A NOESY correlation between H-27 and H3-32 also supported these assignments. On the basis of the chemical shifts of C-23, C-24 and C-25, and the index of hydrogen deficiency deduced from the molecular formula, all three of these carbons were attached to phenolic hydroxy groups. An E geometry for the double bond at C-13–C-14 was deduced from a large coupling constant between H-13 and H-14 (15.0 Hz). The geometry of the position at C-17 was assigned for E based on the high-field shift for C-31 signal (δC 11.8) owing to a γ-effect. Therefore, the gross structure of 1 was identified to be that shown in Figure 1c.
MBJ-0039 (2) was obtained as a colorless amorphous powder: [α]24D –115 (MeOH; c 0.6). The UV absorption maxima in MeOH at 281 nm (ɛ 6300) and the IR spectrum (νmax: 3400 and 1689 cm−1) of 2 resembled those of 1. Furthermore, the molecular formula of 2 (C41H44N2O9) established based on HR-ESI-MS data (m/z 709.2923 [M+H]+) was the same as that of 1. Thus, 2 was considered to be a structural isomer of 1. The 1H and 13C NMR spectroscopic data of 2 were very similar to those of 1 (Table 1). Detailed analyses of the 2D NMR data revealed a chaetoglobosin skeleton identical to 1, including the exact assignments for the oxymethine protons H-22 (δH 5.63, δC 83.4) and H-27 (δH 5.97, δC 82.1). The structure of the 8-methyl-1,2,3,4-tetrahydro-1,4-epoxynaphthalene-5,6,7-triol moiety of 2 differed from that in 1. Observation of a NOESY correlation between an oxymethine proton H-22 and an allylic methyl proton H3-32 (δH 2.00) suggested that 2 had an inverted arrangement of 1 for this region. In addition, COSY and HMBC data supported these assignments as shown in Figure 1d. Thus, the structure determination of 2 was accomplished as shown in Figure 1a.
MBJ-0040 (3) features the following properties: [α]24D –110 (MeOH; c 0.3); UV λmax (ɛ) in MeOH: 281 (6000) nm; HR-ESI-MS: m/z 691.2986 [M–H]–, calcd. for C41H43N2O8 m/z 691.3019; and IR absorption (νmax) 3400 and 1689 cm−1. Analysis of NMR spectra revealed that the partial structure of 3 was the same as that of 2, including the geometries of 13E and 17E, with the exception of the cyclohexane ring moiety (C-4 to C-9) as described below. The DQF-COSY spectrum showed sequences from a methyl proton H3-11 (δH 0.94) through methine protons H-5 (δH 2.53) and H-4 (δH 2.62) to a nitrogen-substituted methine proton H-3 (δH 3.41) and from an olefinic methine proton H-7 (δH 5.36, δC 126.8) to a methine proton H-8 (δH 3.03). Furthermore, the CT-HMBC spectrum showed 1H–13C long-range correlations from H3-11 to methine carbons C-4 (δC 55.2), C-5 (δC 36.6) and an olefinic quaternary carbon C-6 (δC 141.7); from an allylic methyl proton H3-12 (δH 1.74) to C-5, C-6 and an olefinic methine carbon C-7; and from H-5 and H-7 to a quaternary carbon C-9 (δC 67.7), which revealed a 1,6-dimethylcyclohex-1-ene moiety (Figure 1e). Thus, the gross structure of 3 was determined as shown in Figure 1b.
The cytotoxic activities of novel compounds 1–3 and chaetoglobosins C and F against human ovarian adenocarcinoma SKOV-3 cells were tested using the WST-8 ((2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) colorimetric assay (Cell Counting Kit; Dojindo, Kumamoto, Japan). After 72 h of treatment, all compounds exhibited moderate cytotoxic activity against SKOV-3 cells (IC50; 1: 14 μM, 2: 11 μM, 3: 14 μM, and chaetoglobosins C and F: 8 μM and 2 μM, respectively).
Kawahara, T. et al. Cytotoxic sesquiterpenoids MBJ-0009 and MBJ-0010 from a saprobic fungus Nectria sp. f26111. J. Antibiot. (e-pub ahead of print 8 May 2013; doi:10.1038/ja.2013.45).
Kawahara, T. et al. Three eremophilane derivatives, MBJ-0011, MBJ-0012 and MBJ-0013, from an endophytic fungus Apiognomonia sp. f24023. J. Antibiot. 66, 299–302 (2013).
Sekita, S., Yoshihira, K. & Natori, S. Chaetoglobosins, cytotoxic 10-(indol-3-yl)-cytochalasans from Chaetomium spp. IV. 13C-Nuclear magnetic resonance spectra and their application to a biosynthetic study. Chem. Pharm. Bull. 31, 490–498 (1983).
Springer, J. P. et al. Isolation and structure determination of the mycotoxin chaetoglobosin C, a new  cytochalasin. Tetrahedron Lett. 17, 1355–1358 (1976).
Furihata, K. & Seto, H. Constant time HMBC (CT-HMBC), a new HMBC technique useful for improving separation of cross peaks. Tetrahedron. Lett. 39, 7337–7340 (1998).
Smith, J. G. & Dibble, P. W. 2-(Dimethoxymethyl)benzyl alcohol: a convenient isobenzofuran precursor. J. Org. Chem. 48, 5361–5362 (1983).
Rateb, M. E. & Ebel, R. Secondary metabolites of fungi from marine habitats. Nat. Prod. Rep. 28, 290–344 (2011).
This work was supported by a grant from the New Energy and Industrial Technology Department Organization (NEDO) of Japan and a Grant-in-Aid for Scientific Research (23380067 to KS) from the Japan Society for the Promotion of Science (JSPS).
About this article
Cite this article
Kawahara, T., Itoh, M., Izumikawa, M. et al. New chaetoglobosin derivatives, MBJ-0038, MBJ-0039 and MBJ-0040, isolated from the fungus Chaetomium sp. f24230. J Antibiot 66, 727–730 (2013). https://doi.org/10.1038/ja.2013.75
This article is cited by
Applied Microbiology and Biotechnology (2021)
The Journal of Antibiotics (2017)
The Journal of Antibiotics (2016)
The Journal of Antibiotics (2015)
The Journal of Antibiotics (2014)