Cytotoxic anthrasteroid glycosides, malsterosides A–C, from Malbranchea filamentosa

Malbranchea species belong to the family Onygenaceae and are taxonomically close to human and animal pathogenic fungi.1 The fact prompted us to investigate the chemical constituents of Malbranchea fungi. We already have reported the isolation and structural characterization of 4-benzyl-3-phenyl-5H-furan-2-one as a vasodilator, malfilanols A and B as antifungal and cytotoxic sesquiterpenes, malbrancheosides A–D as triterpene glycosides and malfilamentosides A and B as furanone glycosides, from the fungus Malbranchea filamentosa IFM41300.2, 3, 4, 5 Further purification of extracts of rice cultivated by the above fungus allowed us to isolate three new cytotoxic anthrasteroid glycosides, designated malsterosides A (1), B (2) and C (3) (Figure 1). This paper deals mainly with the structure determination and cytotoxic activity of 13.

Figure 1
figure1

Structure of malsterosides A (1)–C (3).

The fungus M. filamentosa Sigler and Carmichael IFM41300 was provided from Medical Mycology Research Center, Chiba University in Japan.

The fungus was cultivated on moisturized rice (1120 g) using 8 Roux flasks (Kimura laboratory glass, Tokyo, Japan) at 25 °C for 21 days. The cultivated rice was extracted with methanol after extracted with acetone and the solvent was evaporated in vacuo. The residue was chromatographed on DIAION HP20 (Mitsubishi chemical, Tokyo, Japan) with H2O, 20% MeOH, 40% MeOH, 60% MeOH, 80% MeOH, MeOH and acetone, in turn. The evaporated acetone elute was purified by centrifugal partition chromatography (Senshu Scientific, Tokyo, Japan) using the solvent system of CH2Cl2–MeOH–H2O (5:6:4) to obtain seven fractions. The second fraction (169 mg) was purified using HPLC on ODS-4 (5 μm, 10 × 250 mm, GL Sciences, Tokyo, Japan) (70% MeOH, CH3CN–MeOH–H2O (7:7:6), in turn) to obtain malsteroside B (2: 10 mg) and malsteroside C (3: 30 mg). The sixth fraction was purified using HPLC on ODS-4 (85% MeOH) and Inertsil SIL 100 A (5 μm, 10 × 250 mm, GL Sciences) (CH2Cl2–MeOH (7:1)) to obtain malsteroside A (1: 37 mg).

Malsteroside A (1) was obtained as a colorless crystalline powder (m.p. 129.5 °C from MeOH); molecular formula C34H52O7 by direct ion analysis in real time-time of flight-MS (m/z: 573.3762 [M+H]+, calculated for C34H53O7, m/z: 573.3791); −12° (c 1.0, MeOH); UV λmax nm (log ɛ): 263.5 (3.47), 254.5 (3.46), 227.0 (sh, 4.07), 213.0 (4.29); IR cm−1: 3360, 1650, 1450. 1H-NMR spectrum showed three singlet methyl protons (δ 0.43, δ 1.80 and δ 2.06), three doublet methyl protons (δ 0.78 (d, 7.0), δ 0.85 (d, 6.7) and δ 0.92 (d, 6.9)), two methine protons (δ 5.34 (d, 9.3), 6.56 (s)), nine oxygenated protons including an anomeric proton (δ 4.54 (bs)) (Table 1). 13C-NMR spectrum showed 6 methyl carbons, 8 sp3 methylene carbons including an oxygenated methylene (δ 62.5), 11 sp3 methine carbons, 6 of which were bearing oxygen functions (δ 67.9, δ 68.9, δ 72.7, δ 75.3, δ 77.8 and δ 79.7) and 1 of which was anomeric carbon (δ 100.9), an sp3 quaternary carbon (δ 44.1), 2 sp2 methine carbons (δ 124.5 and δ 126.2) and 6 sp2 quaternary carbons (Table 1). The contiguous 2D-INADEQUATE correlations from C-1 to C-21, C-22 to C-28 and C-1′ to C′-6 of 1 suggested that structure of 1 consisted of a hexose moiety, a 1(10→6)abeo-5,7,9-androstatriene skeleton and a 2,3-dimethylpentane unit (Figure 2a). The HMBC correlations from 21-H3 to C-22 suggested that C-20 position of the tetracyclic structure and C-22 position of side chain were linked by a carbon double bond, and 1′-H to C-23 also suggested that the C-1 position of sugar was attached to C-23 position of side chain in ether linkage. These results suggested that 1 was an anthrasteroid monoglycoside, which should be rearranged such that the C-1–C-10 linkage of ergostane skeleton cleavaged, followed by the C-1–C-6 linkage was newly formed by ring-closing reaction. The relative stereochemistry of 1 was confirmed by the analysis of ROESY spectrum (Figure 2b). The ROESY correlations between the methyl group of 18-H3 and methine proton of 16α-H, between the methine proton of 14-H and the methine proton of 16β-H and 17-H suggested that the stereochemistry between C and D ring was trans-form, and two protons of 14-H and 17-H were on same side. The geometric position at olefinic bond of C-20 and C-22 was confirmed to E-configuration by the ROESY correlations from 21-H3 to 18-H3 and 23-H, and from 22-H to 28-H3. Acid hydrolysis of 1 gave the sugar moiety of 1, but the genin could not be obtained for the production of complicated compounds unfortunately. The sugar moiety was determined D-mannose as by the comparison to commercial chemical with 1H-NMR spectrum and the value of [α]D (D-mannose, +15.3° (H2O)). The glycoside linkage of D-mannose in 1 was confirmed to β-anomer from the ROESY correlation between 1′-H and 5′-H. Given that biosynthetic intermediate of 1 is an ergosterol, it is supposed that the stereochemistry of C-13 and C-17 is ‘R’ and ‘R’ configuration, respectively.

Table 1 1H- and 13C-NMR data of malsterosides A (1)–C (3) in methanol-d4
Figure 2
figure2

2D-NMR correlations of malsteroside A (1). (a) Correlations of 2D-INADEQUATE and selected HMBC spectra. (b) Correlations of NOESY spectrum.

Malsteroside B (2) was obtained as a colorless crystalline powder: molecular formula C42H65NO12 by direct ion analysis in real time-time of flight-MS (m/z: 776.4615 [M+H]+, calculated for 776.4585); +56° (c 1.0, MeOH); UV λmax nm (log ɛ): 271.0 (3.14), 249.0 (3.03), 226.5 (sh, 3.93); m.p. 160.8 °C (from MeOH); IR cm−1: 3330, 1645, 1550, 1430. Malsteroside B (2) showed two anomeric protons (δ 4.54 (bs) and δ 5.07 (d, 3.7)) in the 1H-NMR spectrum (Table 1), and the contiguous 1H–1H COSY correlations from 1′-H to 6′-H2 and 1′′-H to 6′′-H2 of 2. Therefore, malsteroside B (2) should be a diglycoside. Observation of HMBC correlations from 1-H′′ to C-5′′ and from 2′′-H to carbonyl carbon of acetylated group, NOESY correlation between 3′′-H and 5′′-H, and the J value of 11.0 Hz between 2′′-H and 3′′-H and between 3′′-H and 4′′-H in 2 suggested that one sugar contained within 2 was N-acetyl-D-glucosamine (Figure 3). Acid hydrolysis of 2 gave one sugar and complicated decomposed products. This sugar was determined D-mannose from the comparison with its standard chemical. N-acetyl-D-glucosamine, which was another sugar part of 2, was not detected in hydrolysate of 2. The reasons for this are not clear, but we suppose that N-acetyl-D-glucosamine was degraded by hydrolysis procedure. The 1H- and 13C-NMR spectra of genin part in 2 were similar to that of 1, except for the downfield shift of C-3 (1: δ 68.9, 2: δ 74.0). From the analysis of 1H–1H COSY and HMBC correlations in 2, the genin of 2 was determined to be the same as the anthrasteroid skeleton of 1 (Figure 3a). Observation of the HMBC correlations from the anomeric proton (1′-H) of D-mannose to C-23 and anomeric proton (1′′-H) of N-acetyl-D-glucosamine to C-3 in 2 suggested that the D-mannose and the N-acetyl-D-glucosamine linked at C-23 and C-3 in the genin of 2, respectively. The glycoside linkage of N-acetyl-D-glucosamine was determined as α-configuration from the J of the anomeric proton (3.7 Hz for 1′′-H), and that of D-mannose was determined as β-configuration from the NOESY correlation between 1′′-H and 5′′-H (Figure 3b).

Figure 3
figure3

2D-NMR correlations of malsteroside B (2). (a) Selected correlations of 1H–1H COSY and HMBC spectra. (b) Correlations of selected NOESY spectrum.

Malsteroside C (3) was obtained as a colorless crystalline powder (m.p. 137.9 °C from MeOH): molecular formula C34H52O8 by direct ion analysis in real time-time of flight-MS (m/z: 589.3778 [M+H]+, calculated for 589.3740); −33° (c 1.0, MeOH); UV λmax nm (log ɛ): 272.0 (3.00), 246.0 (2.49), 226.0 (sh, 4.04), 214.0 (4.25); IR cm−1: 3280, 1645, 1500. The 1H- and 13C-NMR spectra of genin part in 3 were similar to that of 1, except for the downfield shift of C-11 (1: δ 26.7, 3: δ 67.7). From the results of MS spectra, it was clear that malsteroside C (3) has more than one oxygen atom than malsteroside A (1). These results revealed that the genin of 3 was 11-oxygenated of that of 1. This was confirmed by the detail analysis of 2D-INADEQUATE and HMBC spectra (Figure 4a). Acid hydrolysis of 3 gave D-mannose, which was determined by the comparison to standard compound, and analysis of 2D-INADEQUATE and NOESY spectra of 3 (Figure 4b). Observation of the NOESY correlation between methyl group of 18-H3 and oxygenated methine proton of 11-H in 3 suggested that the stereochemistry of C-11 is ‘R’ configuration.

Figure 4
figure4

2D-INADEQUATE and HMBC correlations of malsteroside C (3). (a) Correlations of 2D-INADEQUATE and selected HMBC spectra. (b) Selected correlations of NOESY spectrum.

Malsterosides A (1)–C (3) were tested for cytostatic activity against human malignant epithelial cells (Hela) and human lung cancer cells (A549) using a modified method.3 Malsteroside A (1) inhibited the cell proliferation of Hela and A549 with IC50 values of 28.1 and 38.6 μM, respectively. Malsteroside C (3) showed moderate cytostatic activity for Hela cell line (IC50 value: 76.9 μM) and malsteroside B (2) showed no inhibition of these cell growths.

Compounds with anthrasteroidal skeletons are rare in nature. Isolation of anthrasteroids has been first reported by Hussler and Albrecht6 from Cretaceous black shale. Then, citreoanthrasteroids A–B from fungi7 and other anthrasteroidal structure from sheep tick8 have been isolated. From our knowledge, malsterosides A (1)–C (3) are the first examples of anthrasteroidal glycosides.

Acid hydrolysis of malsterosides A (1)-C (3) were carried out as follows. Malsterosides A (1)–C (3) (each 10 mg) were dissolved in 4 M HCl (10 ml) and the solution was refluxed for 2 h. After cooling, the reaction mixture was extracted with CHCl3 and then concentrated in vacuo. The aqueous layer obtained after extraction of reaction mixture was chromatographed using HPLC on amino column with 60% CH3CN to give D-mannose (1: 2 mg, 2: 1 mg and 3: 2 mg).

References

  1. 1

    Udagawa, S. Taxonomical outline of dermatophytes in the Ascomycetes. Jpn J. Med. Mycol. 38, 1–4 (1997).

    Article  Google Scholar 

  2. 2

    Hosoe, T. et al. 4-benzyl-3-phenyl-5H-furan-2-one, a vasodilator isolated from Malbranchea filamentosa IFM 41300. Phytochemistry 66, 2776–2779 (2005).

    CAS  PubMed  Article  Google Scholar 

  3. 3

    Wakana, D. et al. The cytotoxic and antifngal activities of two new sesquiterpenes, malfilanol A and B, derived from Marblanchea filamentosa. J. Antibiot. 62, 217–219 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 4

    Wakana, D. et al. Structures of new triterpene glycosides, malbrancheosides A-D, from Malbranchea filamentosa. Heterocycles 75, 1109–1122 (2008).

    CAS  Article  Google Scholar 

  5. 5

    Wakana, D., Hosoe, T., Itabashi, T., Fukushima, K. & Kawai, K. Structures of two new glycosides of furanone derivatives, malfilamentosides A and B, isolated from Malbranchea filamentosa. Mycotoxins 58, 1–7 (2008).

    CAS  Article  Google Scholar 

  6. 6

    Hussler, G. & Albrecht, P. C27-C29 monoaromatic anthrasteroid hydrocarbons in Cretaceous black shales. Nature 304, 262–263 (1983).

    CAS  Article  Google Scholar 

  7. 7

    Nakada, T. & Yamamura, S. Three new metabolites of hybrid strain KO 0231, derived from Penicillium citreo-viride IFO 6200 and 4692. Tetrahedron 56, 2595–2602 (2000).

    CAS  Article  Google Scholar 

  8. 8

    Saman, D, Cvacka, J., Svatos, A., Bouman, E. A. P. & Kalinova, B. Structure identification of an anthrasteroid hydrocarbon from the Sheep tick Ixodes ricinus. J. Nat. Prod. 69, 1203–1205 (2006).

    CAS  PubMed  Article  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to Tomoo Hosoe.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wakana, D., Itabashi, T., Kawai, Ki. et al. Cytotoxic anthrasteroid glycosides, malsterosides A–C, from Malbranchea filamentosa. J Antibiot 67, 585–588 (2014). https://doi.org/10.1038/ja.2014.43

Download citation

Further reading

Search

Quick links