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Gymnopilin K: a new cytotoxic gymnopilin from Gymnopilus spectabilis

In our continuing search for structurally interesting and bioactive metabolites from Korean wild mushrooms,1, 2, 3, 4, 5 we have collected scores of endemic Korean mushroom species in the mountainous areas during the hot humid summer and prepared MeOH extracts from them for antitumor activity. Among the collected wild mushrooms, the extract of Gymnopilus spectabilis showed significant cytotoxicity against some human tumor cell lines using a sulforhodamine B bioassay. The hallucinogenic mushroom G. spectabilis (Cortinariaceae) is widely known as big laughter mushroom (Ohwaraitake in Japanese) as it can cause excessive laughing in those who consume it.6 This hallucinogenic mushroom has been a rich source of unique metabolites. Chemical constituents of this mushroom have been reported to have gymnoprenols possessing the structure of a novel type of polyisoprenepolyol with 45–60 carbon atoms as major substances.7, 8, 9 Some gymnopilins showing depolarizing activity as bitter principles, were also isolated.10 In addition, a cytotoxic fatty acid (ostopanic acid),11 antioxidant phenolics (bisnoryangonin and hispidin)12 and a hallucinogenic alkaloid (psilocybin)13 have been isolated from this source. Column chromatographic separation of its MeOH extract resulted in the isolation of a new gymnopilin named gymnopilin K (1), together with four known compounds, including gymnopilin A9 (2),10 gymnopilin A10 (3),10 gymnopilene (4)9 and gymnoprenol A9 (5)10 (Figure 1). The structure of 1 was elucidated on the basis of 1D and 2D NMR spectroscopic data analysis as well as chemical reactions, and the known compounds 25 were identified by comparison of physical and spectroscopic data with literature values. Here, we describe the isolation and structural elucidation of 1 as well as the cytotoxic activities of compounds 15.

Figure 1
figure1

Structures of compounds 15.

The air-dried and powdered fruiting bodies of G. spectabilis (153 g) were extracted with 80% aqueous MeOH two times at room temperature and then filtered. The filtrate was evaporated under vacuum to afford a MeOH extract (20 g), which was partitioned with n-hexane, CHCl3 and n-BuOH subsequently using H2O, yielding n-hexane (100 mg), CHCl3 (2.3 g) and n-BuOH fractions (1.9 g). Each fraction was evaluated for cytotoxicity against A549, SK-OV-3, SK-MEL-2 and HCT-15 cell lines using a sulforhodamine B bioassay. We selected the CHCl3 soluble fraction for current phytochemical investigation because the CHCl3 soluble fraction had significant cytotoxicity against the tested tumor cell lines. The active CHCl3 soluble fraction (2.3 g) was separated on a silica gel column with CHCl3-MeOH (5:1) to yield seven fractions (G1–G7). Fraction G3 (250 mg) was separated on a Sephadex LH-20 (Pharmacia, Uppsala, Sweden) column with CH2Cl2-MeOH (1:1) and then purified by RP-C18 preparative HPLC (Econosil RP-18 10 μ column (Alltech, Nicholasville, KY, USA), 250 × 10 mm) using a solvent of MeCN-MeOH-H2O (9:1:0.3) to yield compounds 4 (4 mg) and 5 (5 mg). Fraction G5 (420 mg) was separated on a Sephadex LH-20 column with CH2Cl2-MeOH (1:1) to afford two subfractions (G51–G52). Fraction G51 (170 mg) was subjected to passage over a Waters Sep-Pak Vac 6 cc (Waters, Milford, MA, USA) (100% MeOH) and then purified by RP-C18 preparative HPLC (80% MeOH) to yield compound 3 (5 mg). Fraction G6 (780 mg) was separated on a Sephadex LH-20 column with CH2Cl2-MeOH (1:1) to obtain two subfractions (G61–G62). Fraction G62 (450 mg) was separated on a RP-C18 silica gel column (75% MeOH) to yield six subfractions (G621–G626). Fraction G621 (27 mg) was purified by RP-C18 preparative HPLC (80% MeOH) to yield compound 2 (4 mg) and fraction G622 (35 mg) was purified by RP-C18 preparative HPLC (70% MeOH) to afford compound 1 (7 mg).

Compound 1 was obtained as a colorless gum with a negative specific rotation value [α]25D−8.7 (c 0.35, MeOH). Its molecular formula was determined to be C51H98O14 from the [M+Na]+ peak at m/z 957.6859 (calcd for C51H98NaO14, 957.6854) in the positive-ion high resolution (HR)-ESI-MS spectrum. The IR spectrum of 1 showed a broad hydroxyl band at 3440 cm−1 and a carbonyl absorption band at 1715 cm−1. The physicochemical properties of 1 are summarized in Supplementary Information. The 1H- and 13C-NMR spectral data of 1 are shown in Table 1.

Table 1 1H- and 13C-NMR data of compounds 1 and 2*

The 1H- and 13C-NMR spectra (Table 1) of 1 were very similar to those of 2, with an apparent difference being the absence of signals for cis-vinyl methyls at δH 1.63 (3H, s, H-18) and 1.62 (3H, s, H-18); δC 14.8 (C-18) and 14.8 (C-18), allylic methylenes at δH 2.15-1.95 (8H, m, H-5, H-8); δC 26.6 (C-5), 22.5 (C-5), 41.1 (C-8), and 40.1 (C-8), and double bonds at δH 5.12 (2H, m, H-6); δC 124.6 (C-6) and 134.9 (C-7) in 2.10 The intensity of the 1H-NMR signal at δH 5.12 (1H, m, H-14) and two vinyl methyls at δH 1.67 (3H, s, H-16) and 1.61 (3H, s, H-20), which were correlated with H-14 in the HMBC spectrum, indicated that compound 1 has only one isoprene unit with a trisubstituted double bond at the terminal position of 1. Compound 1 was assigned to be a successively hydrated isoprenoid structure, which had the same number of repeated isoprene units compared to those of 2 by the molecular formula C51H98O14 obtained from the analysis of the HR-ESI-MS data. This suggested that compound 1 may be produced by the saturation of C-6/7 double bond in 2 by the addition of tertiary alcohols. The full NMR assignments of 1 were performed by the analysis of the 1H-1H COSY, DEPT, HMQC and HMBC spectroscopic data (Table 1).

The absolute configurations of C-2 and C-3 of 1 were established on the basis of the modified Mosher's method in combination with the NOESY experiment.14 Treatment of 1 with (S)-(+)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride [(S)-MTPA-Cl] and 4-(dimethylamino)pyridine (DMAP) in pyridine gave the (R)-MTPA esters 1r. Similar treatment of 1 with (R)-(-)-MTPA-Cl afforded the (S)-MTPA esters 1s. Analysis of the 1H-NMR chemical shift differences (ΔδS−R; see Supplementary Information) of the two MTPA esters allowed the assignment of the absolute configuration of C-2 as R (Figure 2). In the NOESY spectrum of 1, H2-1 and H-2 showed consistent correlations with H3-17 and H2-4, respectively. However, there was no apparent correlation between H-2 and H3-17 in its 1D NOE spectrum (Figure 2), which indicated that H-2 and H3-17 in 1 were in the anti configuration. These NOESY data, in combination with the result from the modified Mosher's method, allowed us to assign the absolute configuration of C-3 as S. Finally, the absolute configuration of C-3′ of 1 was determined by the alkaline methanolysis of 1. Treatment of 1 with 3% sodium methoxide (NaOMe) in MeOH yielded methyl (S)-3-hydroxy-3-methylglutarate, which was identified by comparison of its 1H-NMR, specific rotation [α]25D+6.1 (c 0.04, CHCl3), and MS data (see Supplementary Information).15, 16 Thus, the structure of 1 was established as shown in Figure 1 and the compound was named gymnopilin K.

Figure 2
figure2

Δδ Values (δSδR) in p.p.m. of the two MTPA esters derived from 1 (a) and Key NOE correlations in the NOESY spectrum of 1 (b).

Compounds 1-5 were evaluated for their antiproliferative activities against four human cancer cell lines including A549, SK-OV-3, SK-MEL-2, and HCT-15 using the sulforhodamine B bioassay.17 All tested compounds showed inhibitory activity against proliferation of the tested cell lines with inhibitory concentration (IC)50 values ranging between 3.26-26.30 μM (Table 2). In particular, all compounds exhibited significant cytotoxicity against the SK-MEL-2 cell line with IC50 values in the range of 3.26-14.40 μM (Table 2). In the structure-activity relationship (SAR), it appears that the methyl (S)-3-hydroxy-3-methylglutarate moiety in the molecule improves cytotoxicity, as compounds 1–3 (IC50 values ranging between 3.26 and 16.41 μM) with the above moiety were more active than compounds 4 and 5 (IC50 values ranging between 12.80 and 26.30 μM). This SAR study provides valuable data for future synthetic and pharmacological studies with the aim of obtaining more potent cytotoxic compounds.

Table 2 Cytotoxic activities of compounds 15 isolated from Gymnopilus spectabili s

References

  1. 1

    Kim, K.H., Choi, S.U., Park, K.M., Seok, S.J. & Lee, K.R. Cytotoxic constituents of Amanita subjunquillea. Arch. Pharm. Res. 31, 579–586 (2008).

    CAS  Article  Google Scholar 

  2. 2

    Kim, K.H., Choi, S.U. & Lee, K.R. Neomastoidin A, a novel monoacylglycerol with an amino acid moiety from Macrolepiota neomastoidea. Chem. Lett. 38, 894–895 (2009).

    CAS  Article  Google Scholar 

  3. 3

    Kim, K.H., Park, K.M., Choi, S.U. & Lee, K.R. Macrolepiotin, a new indole alkaloid from Macrolepiota neomastoidea. J. Antibiot. 62, 335–338 (2009).

    CAS  PubMed  Article  Google Scholar 

  4. 4

    Kim, K.H. et al. Russulfoen, a new cytotoxic marasmane sesquiterpene from Russula foetens. J. Antibiot. 63, 575–577 (2010).

    CAS  PubMed  Article  Google Scholar 

  5. 5

    Kim, K.H. et al. Lactarane sesquiterpenoids from Lactarius subvellereus and their cytotoxicity. Bioorg. Med. Chem. Lett. 20, 5385–5388 (2010).

    CAS  PubMed  Article  Google Scholar 

  6. 6

    Hashimoto, M., Yanagiya, M., Okuno, T. & Shirahama, H. The absolute configuration of 4,6-decadiyne-1,3,8-triol isolated from Gymnopilus spectabilis. Bull. Chem. Soc. Jpn. 62, 2751–2752 (1989).

    CAS  Article  Google Scholar 

  7. 7

    Nozoe, S., Koike, Y., Ito, N. & Kusano, G. Isolation and structure of gymnoprenol D, a homologous series of fully hydrated polyisoprenepolyol from Gymnopilus spectabilis. Chem. Lett. 6, 1001–1002 (1984).

    Article  Google Scholar 

  8. 8

    Nozoe, S., Koike, Y., Tsuji, E., Kusano, G. & Seto, H. Isolation and structure of gymnoprenols, a novel type of polyisoprenepolyols from Gymnopilus spectabilis. Tetrahedron Lett. 24, 1731–1734 (1983).

    CAS  Article  Google Scholar 

  9. 9

    Findlay, J.A. & He, Z.Q. Minor constituents of Gymnopilus spectabilis. J. Nat. Prod. 54, 184–189 (1991).

    CAS  Article  Google Scholar 

  10. 10

    Tanaka, M., Hashimoto, K., Okuno, T. & Shirahama, H. Neurotoxic oligoisoprenoids of the hallucinogenic mushroom, Gymnopilus spectabilis. Phytochemistry 34, 661–664 (1993).

    CAS  Article  Google Scholar 

  11. 11

    Nunez-Alarcon, J., Paredes, J. C. C., Carmona, M. T. & Quinones, M. Ostopanic acid, a cytotoxic fatty acid from Gymnopilus spectabilis. Bol. Soc. Chil. Quim. 46, 227–229 (2001).

    CAS  Google Scholar 

  12. 12

    Lee, I.K., Cho, S.M., Seok, S.J. & Yun, B.S. Chemical constituents of Gymnopilus spectabilis and their antioxidant activity. Mycobiology 36, 55–59 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. 13

    Hatfield, G. M., Valdes, L. J. & Smith, A. H. The occurrence of psilocybin in Gymnopilus species. Lloydia 41, 140–144 (1978).

    CAS  PubMed  Google Scholar 

  14. 14

    Kim, K.H., Choi, S.U., Kim, Y.C. & Lee, K.R. Tirucallane triterpenoids from Cornus walteri. J. Nat. Prod. 74, 54–59 (2011).

    CAS  PubMed  Article  Google Scholar 

  15. 15

    Morikawa, T., Yamaguchi, I., Matsuda, H. & Yoshikawa, M. A New amide, piperchabamide F, and two new phenylpropanoid glycosides, piperchabaosides A and B, from the fruit of Piper chaba. Chem. Pharm. Bull. 57, 1292–1295 (2009).

    CAS  PubMed  Article  Google Scholar 

  16. 16

    Nozoe, S., Takahashi, A. & Ohta, T. Chirality of the 3-hydroxy-3-methylglutaric acid moiety of fasciculic acid A, a calmodulin antagonist isolated from Naematoloma fasciculare. Chem. Pharm. Bull. 41, 1738–1742 (1993).

    CAS  Article  Google Scholar 

  17. 17

    Skehan, P. et al. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 82, 1107–1112 (1990).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0004672). We thank Drs EJ Bang, SG Kim and JJ Seo at the Korea Basic Science Institute for the NMR and MS spectra measurements.

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Correspondence to Kang Ro Lee.

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Kim, K., Choi, S. & Lee, K. Gymnopilin K: a new cytotoxic gymnopilin from Gymnopilus spectabilis. J Antibiot 65, 135–137 (2012). https://doi.org/10.1038/ja.2011.122

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Keywords

  • Gymnopilus spectabilis
  • Cortinariaceae
  • gymnopilin K
  • cytotoxicity

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