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Extraction and isolation of ganoderic acid Σ from Ganoderma lucidum
Tetrahedron Letters 57 (2016) 5368–5371
Contents lists available at ScienceDirect
Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Extraction and isolation of ganoderic acid R from Ganoderma lucidum Chihiro Murata a, Quang Thuong Tran a,b, Shingo Onda a, Toyonobu Usuki a,⇑ a b
Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioicho, Chiyoda-ku, Tokyo 102-8554, Japan Department of Organic Chemistry, Hanoi University of Science and Technology (HUST), 1 Dai Co Viet Road, Hanoi, Viet Nam
a r t i c l e
i n f o
Article history: Received 25 September 2016 Revised 16 October 2016 Accepted 20 October 2016 Available online 21 October 2016
a b s t r a c t A new lanostane triterpene was extracted from Ganoderma lucidum using cellulose-dissolving ionic liquids. The structure of the isolated compound was elucidated by spectroscopic analysis, including NMR, MS, and a modified Mosher’s method. The newly isolated lanostane natural product was named ganoderic acid R. Ó 2016 Elsevier Ltd. All rights reserved.
Keywords: Ganoderma lucidum Lanostane Ganoderic acid Extraction Ionic liquid
Ganoderma lucidum is a fungus historically used in traditional herb medicines and supplements in China and other parts of East-Asia. Its pharmaceutical effects include enhanced health and longevity.1 The major secondary metabolites of G. lucidum are lanostane triterpenoid natural products, and more than 150 lanostanes, including ganoderic acids, ganolucidic acids, and lucidic acids have been isolated from Ganoderm spp.2 These compounds exhibit various biological activities such as anti-HIV activity,3–5 angiotensin converting enzyme inhibitory effect,6,7 cholesterol synthesis inhibitory effect,8,9 anti-histamine releasing activity,10 and potential anticancer activities.2,11–14 One Ganoderma triterpenoid was shown to specifically interact with tubulin proteins, which play an important role in cell growth and differentiation.15 It is also reported that G. lucidum polysaccharides could potentially prevent cancer.16,17 Ionic liquids are organic salts designed to melt below 100 °C. Compared to typical organic solvents, they have various advantages such as low volatility, low flammability, and excellent solubility for organic compounds.18,19 1-Butyl-3-methylimidazolium chloride ([C4mim]Cl, Fig. 1)20 and 1-ethyl-3-methylimidazolium methylphosphonate ([C2mim][(MeO)(H)PO2], Fig. 1)21 are wellknown ionic liquids that dissolve cellulose to 10 wt % at 100 °C and 10 wt % at 45 °C, respectively. The extraction of natural products using ionic liquids has recently attracted attention due to ⇑ Corresponding author. Tel.: +81 3 3238 3446; fax: +81 3 3238 3361. E-mail address: [email protected] (T. Usuki). http://dx.doi.org/10.1016/j.tetlet.2016.10.072 0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.
growing interest in green chemistry and the potential of ionic liquids for the development of new and useful technologies.22 We have developed an effective method for obtaining the natural products shikimic acid and bilobalide from Ginkgo biloba leaves using [C4mim]Cl.23,24 The cell walls in leaves consist of cellulose and hemicellulose, whereas the cell wall of G. lucidum consists of polysaccharides such as (1?3) and (1?6)-b-D-glucan.25 Thus, we focused on extracting compounds from G. lucidum to expand the range of application for our ionic liquid-mediated method because the composition of the cell wall of G. lucidum is different from that of plant leaves. Herein we report the extraction of a new natural product from G. lucidum using ionic liquids and determination of the compound’s structure. First, the extraction of triterpenoids from G. lucidum was attempted using H2O or EtOH as a control experiment (Fig. 2). G. lucidum was collected in a forest in Vietnam. Samples (0.5 g each) were crushed using a mixer, added to flasks. 16 g of H2O or EtOH was added to the flasks, and each mixture was subjected to one of the temperature conditions shown in Figure 2 for 1 h. Each solution was filtered through Celite, the solvent was evaporated, then the crude extract was dissolved in MeOH/H2O (7:3), and washed with hexane three times. The solvent was evaporated and the residue was analyzed by reversed-phase high performance liquid chromatography (RP-HPLC). Quantitative analysis of the main peak, corresponding to unknown 1, was performed by HPLC using a calibration curve (y = 4.135 1089 x 9.951 10 7 (y: amount of 1 (mg), x: peak area), R2 = 0.9999). The extraction yield of 1 was
C. Murata et al. / Tetrahedron Letters 57 (2016) 5368–5371
Figure 1. Structures of [C4mim]Cl and [C2mim][(MeO)(H)PO2].
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1.56% for H2O and 1.74% for EtOH. In the same manner, [C4mim]Cl, [C4mim]Cl/MeOH (1:1), [C4mim]Cl/EtOH (1:1) and [C4mim]Cl/H2O (1:1) were used as the extraction solvent and subjected to the temperature conditions shown in Figure 2; the extraction yields were 1.40%, 1.42%, 1.33%, and 1.35%, respectively. Thus, [C4mim]Cl was not more effective for extracting compound 1 compared with H2O or EtOH (Fig. 3). Extraction with [C2mim][(MeO)(H)PO2] was
Figure 2. Extraction protocol (IL layer: ionic liquid layer).
Figure 3. Extraction yield of 1 from G. lucidum (0.5 g scale) using [C4mim]Cl, [C4mim]Cl mixed solvents, or neat water or ethanol.
Figure 4. Extraction yield of 1 from G. lucidum (0.15 g scale) using [C2mim][(MeO)(H)PO2], C2mim][(MeO)(H)PO2 mixed solvents, or neat water or ethanol.
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C. Murata et al. / Tetrahedron Letters 57 (2016) 5368–5371
Figure 6. NOE correlations of the relative structure of 1. Figure 5. Key 1H–1H COSY and HMBC of the planar structure of 1.
Table 1 Assignments of the
13
C and 1H NMR spectral signals of 1a
C
dC
DEPT
dH
Multiplicity
1a 1b 2a 2b 3 4 5 6a 6b 7 8 9 10 11 12a 12b 13 14 15 16a 16b 17 18 19 20 21 22 23 24 25 26 27 28a 28b 29 30
36.7
CH2
36.1
CH2
1.57–1.46 3.16–3.11 2.47–2.42 2.62–2.54
m m m m
J (Hz)
218.9 54.3 54.8 19.5
C C CH CH2 CH2 C C C C CH2
d m m m
12.3
31.3 167.2 139.3 38.4 200.9 53.5
1.68 1.89–1.86 1.65–1.59 2.64–2.54
2.84 2.47–2.42
d m
16.5
55.1 49.8 73.1 33.1
C C CH CH2 CH CH3 CH3 C CH3 CH2 CH2 CH C C CH3 CH2
dd m m m s s
9.3, 5.5
52.0 19.1 19.7 75.1 25.4 43.2 24.5 143.8 128.8 171.6 12.4 65.6
4.39 2.47–2.42 1.65–1.59 2.25–2.21 1.07 1.25 1.24 1.57–1.46 2.25–2.21 6.78
s m m t
21.7 19.9
CH3 CH3
1.83 3.55 4.00 1.21 1.19
s d d s s
7.4
11.5 11.5
a Spectra were recorded in CD3OD at 125 MHz (13C) and 500 MHz (1H) with dC and dH in ppm, respectively.
conducted using 0.15 g of G. lucidum and 5 g of [C2mim][(MeO)(H) PO2],26 but again the extraction yield of 1 was not improved (Fig. 4).27 These results suggest that the branched structure of bD-glucan caused relatively low solubility than in ionic liquids than cellulose from plant leaves, although lignin can dissolve in ionic liquids.28,29 Although we were unable to efficiently extract unknown compound 1, we set about to determine its structure, including its absolute configuration. The structural elucidation of 1, which was isolated from the water extract of G. lucidum by RP-HPLC, was performed by spectroscopic analysis, including 1H NMR, 13C NMR, DEPT, HMQC, HMBC, NOESY, and HRMS.12,30 The molecular formula was assigned as C30H44O7, as deduced from the mass spectra (ESI-HRMS (m/z) calcd for C30H44NaO7 [M+Na]+ 539.2985, found
539.3001). The 1H NMR spectrum of 1 showed peaks assigned to six tertiary methyl groups (dH 1.07, 1.19, 1.21, 1.24, 1.25, and 1.83 each 3H, s), one oxygenated methylene (dH 4.00, d, J = 11.5 Hz, and 3.6, d, J = 11.5 Hz), one oxygenated methine (dH 4.39, dd, J = 9.5, 5.5 Hz), and one olefinic proton (dH 6.78, t, J = 7.4 Hz). The 13C NMR spectrum in combination with the distortionless enhancement by polarization transfer (DEPT 135 and 90) of 1 exhibited 30 carbons corresponding to six methyls, nine methylenes (one oxygenated carbon at dC 65.1), four methines (one oxygenated carbon at dC 73.1), four quaternary carbons, two pairs of olefinic carbons (dC 167.2, 143.8, 139.3, and 128.9), one oxygenated carbon (dC 75.1), and two carbonyl carbons (dC 218.9 and 200.9). The structure was thus estimated to have a lanostane triterpenoid skeleton, as shown in Figure 5. On the basis of spectroscopic evidence obtained by 1H–1H COSY and HMQC, all protons and carbons were assigned as shown in Table 1. These assignments were further supported by long-range correlations between signals in the key HMBC spectrum from H3-18 to C-12 and C-17, from H319 to C-1, C-5, and C-9, from H2-28 to C-3, C-5, and C-29, from H329 to C-3, C-4, C-5, and C-28, from H-5 to C-1, C-6, C-7, C-10, and C28, from H2-7 to C-5, C-6, and C-8, from H2-12 to C-11, C-13, and C18, from H3-21 to C-17, and C-20, from H-24 to C-22, C-23, C-25, and C-26, from H3-27 to C-24, C-25, and C-26, and from H3-30 to C-8, C-13, and C-15 (Fig. 5). The relative configuration of 1 was then determined by NOESY experiments. Key NOE correlations were observed between H-12a and H3-30, H-15b and H3-18, and H-16a and H-17a (Fig. 6). Although an NOE correlation between H2-28 and H3-29 was also observed, the stereochemistry at C-4 remained unclear. A modified Mosher’s method31 was used to assign the absolute configuration of 1. As shown in Scheme 1, methoxy-trifluoromethylphenylacetic acid (MTPA) esterification with (S)-(+)- or (R)-( )- MTPA chloride and DMAP was carried out to afford ester 2 or 3 in 71% or 86% yield, respectively. Esterification was achieved on only OH-28 and not on OH-15. The (R)-MTPA ester 2 showed two doublet peaks, at 4.68 and 4.36 ppm (Dd 0.32), which correspond to the carbinolic protons, whereas the corresponding peaks were found at 4.51 and 4.37 ppm (Dd 0.14) in the (S)-MTPA ester 3. A comparative analysis of the signals in these spectra indicated that the absolute configuration at C-4 is R,32 allowing the chemical structure of 1, called ganoderic acid R, to be determined as shown in Scheme 1. The obtained lanostane-type triterpenoid 1 was biologically evaluated to determine its utility as a possible anticancer agent.2,11–14 However, compound 1 did not show any activity, including no inhibitory activity of histone deacetylases (HDAC1, DDAC6, and SIRT1) at 10 lM, no inhibitory activity of androgen receptor at 10 lM, no inhibitory activity of protein kinase at 10 lM, and no inhibitory activity of hypoxia-inducible factor (HIF) at 10 lM. In summary, unknown compound 1 was extracted from G. lucidum using ionic liquids, and the structure of ganoderic acid R (1) was determined by MS and NMR analysis together with a modified
C. Murata et al. / Tetrahedron Letters 57 (2016) 5368–5371
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Scheme 1. Synthesis of 2 and 3. Reagents and conditions: (a) (S)-(+)- or (R)-( )-MTPA chloride, DMAP, pyridine, rt, 24 h, 71% (2), 86% (3).
Mosher’s method. Newly isolated lanostane triterpenoid natural product 1 did not show any activity in the assays as a possible anticancer agent. Acknowledgements We thank Prof. Masahiro Yoshizawa-Fujita (Sophia University) for valuable comments, The Hitachi Scholarship Foundation for The Hitachi Research Fellowship (to Q.T.T.), and the Screening Committee of Anticancer Drugs supported by Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Support Programs for Cancer Research, from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan for performing the biological assays. Supplementary data Supplementary data (experimental procedures, HPLC chromatograms, 1H, 13C, and 2D NMR spectra, and UV–Vis spectra of 1. 1H NMR spectra of compounds 2 and 3) associated with this article can be found, in the online version, at http://dx.doi.org/10. 1016/j.tetlet.2016.10.072. References and notes 1. Baby, S.; Johnson, A. J.; Govindan, B. Phytochemistry 2015, 114, 66–101. 2. Rios, J.-L.; Andújar, I.; Recio, M.-C.; Giner, R.-M. J. Nat. Prod. 2012, 75, 2016– 2044. 3. El-Mekkawy, S.; Meselhy, M. R.; Nakamura, N.; Tezuka, Y.; Hattori, M.; Kakiuchi, N.; Shimotohno, K.; Kawahata, T.; Otake, T. Phytochemistry 1998, 49, 1651–1657. 4. Min, B.-S.; Nakamura, N.; Miyashiro, H.; Bae, K.-W.; Hattori, M. Chem. Pharm. Bull. 1998, 46, 1607–1612. 5. Sato, N.; Zhang, Q.; Ma, C.-M.; Hattori, M. Chem. Pharm. Bull. 2009, 57, 1076– 1080. 6. Morigiwa, A.; Kitabatake, K.; Fujimoto, Y.; Ikekawa, N. Chem. Pharm. Bull. 1986, 34, 3025–3028. 7. Hai-Bang, T.; Shimizu, K. Phytochem. Lett. 2015, 12, 243–247. 8. Komoda, Y.; Shimizu, M.; Sonoda, Y.; Sato, Y. Chem. Pharm. Bull. 1989, 37, 531– 533.
9. Hajjaj, H.; Macé, C.; Roberts, M.; Niederberger, P.; Fay, L. B. Appl. Environ. Microbiol. 2005, 71, 3653–3658. 10. Kohda, H.; Tokumoto, W.; Sakamoto, K.; Fujii, M.; Hirai, Y.; Yamasaki, K.; Komoda, Y.; Nakamura, H.; Ishihara, S.; Uchida, M. Chem. Pharm. Bull. 1985, 33, 1367–1374. 11. Liu, J.; Tamura, S.; Kurashiki, K.; Shimizu, K.; Noda, K.; Konishi, F.; Kumamoto, S.; Kondo, R. Chem. Biodiversity 2009, 6, 231–243. 12. Min, B.-S.; Gao, J.-J.; Nakamura, N.; Hattori, M. Chem. Pharm. Bull. 2000, 48, 1026–1033. 13. Gao, J.-J.; Min, B.-S.; Ahn, E.-M.; Nakamura, N.; Lee, H.-K.; Hattori, M. Chem. Pharm. Bull. 2002, 50, 837–840. 14. Liu, J.; Kurashiki, K.; Shimizu, K.; Kondo, R. Bioorg. Med. Chem. 2006, 14, 8654– 8660. 15. Liu, J.; Shimizu, K.; Tanaka, A.; Shinobu, W.; Ohnuki, K.; Nakamura, T.; Kondo, R. Sci. Rep. 2012, 905, 1–5. 16. Zhu, X.-L.; Chen, A.-F.; Lin, Z.-B. J. Ethnopharmacol. 2007, 111, 219–226. 17. Chien, C. M.; Cheng, J.-L.; Chang, W.-T.; Tien, M.-H.; Tsao, C.-M.; Chang, Y.-H.; Chang, H.-Y.; Hsieh, J.-F.; Wong, C.-H.; Chen, S.-T. Bioorg. Med. Chem. 2004, 12, 5603–5609. 18. Ohno, H.; Fukaya, Y. Chem. Lett. 2009, 38, 2–7. 19. Sun, N.; Rodríguez, H.; Rahman, M.; Rogers, R. D. Chem. Commun. 2011, 47, 1405–1421. 20. Swatloski, R. P.; Spear, S. K.; Holbrey, J. D.; Rogers, R. D. J. Am. Chem. Soc. 2002, 124, 4974–4975. 21. Fukaya, Y.; Hayashi, K.; Wada, M.; Ohno, H. Green Chem. 2008, 10, 44–46. 22. Dai, Y.; van Spronsen, J.; Witkamp, G.-J.; Verpoorte, R.; Choi, Y. H. J. Nat. Prod. 2013, 76, 2162–2173. 23. Usuki, T.; Yasuda, N.; Yoshizawa-Fujita, M.; Rikukawa, M. Chem. Commun. 2011, 47, 10560–10562. 24. Onda, S.; Usuki, T.; Yoshizawa-Fujita, M.; Rikukawa, M. Chem. Lett. 2015, 44, 1461–1463. 25. Hung, W.-T.; Wang, S.-H.; Chen, C.-H.; Yang, W.-B. Molecules 2008, 13, 1538– 1550. 26. This is due to the higher price of [C2mim][(MeO)(H)PO2]. 27. In the control experiments (H2O and EtOH), the extraction yields were slightly different in Figures 3 and 4, probably due to the different amount of G. lucidum used in the various experiments. 28. Abe, M.; Yamanaka, S.; Yamada, H.; Yamada, T.; Ohno, H. Green Chem. 2015, 17, 4432–4438. 29. Yan, P.; Xu, Z.; Zhang, C.; Liu, X.; Xu, W.; Zhang, Z. C. Green Chem. 2015, 17, 4913–4920. 30. Wang, K.; Bao, L.; Xiong, W.; Ma, K.; Han, J.; Wang, W.; Yin, W.; Liu, H. J. Nat. Prod. 2015, 78, 1977–1989. 31. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113, 4092–4096. 32. Tsuda, M.; Toriyabe, Y.; Endo, T.; Kobayashi, J. Chem. Pharm. Bull. 2003, 51, 448– 451.
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Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Extraction and isolation of ganoderic acid R from Ganoderma lucidum Chihiro Murata a, Quang Thuong Tran a,b, Shingo Onda a, Toyonobu Usuki a,⇑ a b
Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioicho, Chiyoda-ku, Tokyo 102-8554, Japan Department of Organic Chemistry, Hanoi University of Science and Technology (HUST), 1 Dai Co Viet Road, Hanoi, Viet Nam
a r t i c l e
i n f o
Article history: Received 25 September 2016 Revised 16 October 2016 Accepted 20 October 2016 Available online 21 October 2016
a b s t r a c t A new lanostane triterpene was extracted from Ganoderma lucidum using cellulose-dissolving ionic liquids. The structure of the isolated compound was elucidated by spectroscopic analysis, including NMR, MS, and a modified Mosher’s method. The newly isolated lanostane natural product was named ganoderic acid R. Ó 2016 Elsevier Ltd. All rights reserved.
Keywords: Ganoderma lucidum Lanostane Ganoderic acid Extraction Ionic liquid
Ganoderma lucidum is a fungus historically used in traditional herb medicines and supplements in China and other parts of East-Asia. Its pharmaceutical effects include enhanced health and longevity.1 The major secondary metabolites of G. lucidum are lanostane triterpenoid natural products, and more than 150 lanostanes, including ganoderic acids, ganolucidic acids, and lucidic acids have been isolated from Ganoderm spp.2 These compounds exhibit various biological activities such as anti-HIV activity,3–5 angiotensin converting enzyme inhibitory effect,6,7 cholesterol synthesis inhibitory effect,8,9 anti-histamine releasing activity,10 and potential anticancer activities.2,11–14 One Ganoderma triterpenoid was shown to specifically interact with tubulin proteins, which play an important role in cell growth and differentiation.15 It is also reported that G. lucidum polysaccharides could potentially prevent cancer.16,17 Ionic liquids are organic salts designed to melt below 100 °C. Compared to typical organic solvents, they have various advantages such as low volatility, low flammability, and excellent solubility for organic compounds.18,19 1-Butyl-3-methylimidazolium chloride ([C4mim]Cl, Fig. 1)20 and 1-ethyl-3-methylimidazolium methylphosphonate ([C2mim][(MeO)(H)PO2], Fig. 1)21 are wellknown ionic liquids that dissolve cellulose to 10 wt % at 100 °C and 10 wt % at 45 °C, respectively. The extraction of natural products using ionic liquids has recently attracted attention due to ⇑ Corresponding author. Tel.: +81 3 3238 3446; fax: +81 3 3238 3361. E-mail address: [email protected] (T. Usuki). http://dx.doi.org/10.1016/j.tetlet.2016.10.072 0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.
growing interest in green chemistry and the potential of ionic liquids for the development of new and useful technologies.22 We have developed an effective method for obtaining the natural products shikimic acid and bilobalide from Ginkgo biloba leaves using [C4mim]Cl.23,24 The cell walls in leaves consist of cellulose and hemicellulose, whereas the cell wall of G. lucidum consists of polysaccharides such as (1?3) and (1?6)-b-D-glucan.25 Thus, we focused on extracting compounds from G. lucidum to expand the range of application for our ionic liquid-mediated method because the composition of the cell wall of G. lucidum is different from that of plant leaves. Herein we report the extraction of a new natural product from G. lucidum using ionic liquids and determination of the compound’s structure. First, the extraction of triterpenoids from G. lucidum was attempted using H2O or EtOH as a control experiment (Fig. 2). G. lucidum was collected in a forest in Vietnam. Samples (0.5 g each) were crushed using a mixer, added to flasks. 16 g of H2O or EtOH was added to the flasks, and each mixture was subjected to one of the temperature conditions shown in Figure 2 for 1 h. Each solution was filtered through Celite, the solvent was evaporated, then the crude extract was dissolved in MeOH/H2O (7:3), and washed with hexane three times. The solvent was evaporated and the residue was analyzed by reversed-phase high performance liquid chromatography (RP-HPLC). Quantitative analysis of the main peak, corresponding to unknown 1, was performed by HPLC using a calibration curve (y = 4.135 1089 x 9.951 10 7 (y: amount of 1 (mg), x: peak area), R2 = 0.9999). The extraction yield of 1 was
C. Murata et al. / Tetrahedron Letters 57 (2016) 5368–5371
Figure 1. Structures of [C4mim]Cl and [C2mim][(MeO)(H)PO2].
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1.56% for H2O and 1.74% for EtOH. In the same manner, [C4mim]Cl, [C4mim]Cl/MeOH (1:1), [C4mim]Cl/EtOH (1:1) and [C4mim]Cl/H2O (1:1) were used as the extraction solvent and subjected to the temperature conditions shown in Figure 2; the extraction yields were 1.40%, 1.42%, 1.33%, and 1.35%, respectively. Thus, [C4mim]Cl was not more effective for extracting compound 1 compared with H2O or EtOH (Fig. 3). Extraction with [C2mim][(MeO)(H)PO2] was
Figure 2. Extraction protocol (IL layer: ionic liquid layer).
Figure 3. Extraction yield of 1 from G. lucidum (0.5 g scale) using [C4mim]Cl, [C4mim]Cl mixed solvents, or neat water or ethanol.
Figure 4. Extraction yield of 1 from G. lucidum (0.15 g scale) using [C2mim][(MeO)(H)PO2], C2mim][(MeO)(H)PO2 mixed solvents, or neat water or ethanol.
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C. Murata et al. / Tetrahedron Letters 57 (2016) 5368–5371
Figure 6. NOE correlations of the relative structure of 1. Figure 5. Key 1H–1H COSY and HMBC of the planar structure of 1.
Table 1 Assignments of the
13
C and 1H NMR spectral signals of 1a
C
dC
DEPT
dH
Multiplicity
1a 1b 2a 2b 3 4 5 6a 6b 7 8 9 10 11 12a 12b 13 14 15 16a 16b 17 18 19 20 21 22 23 24 25 26 27 28a 28b 29 30
36.7
CH2
36.1
CH2
1.57–1.46 3.16–3.11 2.47–2.42 2.62–2.54
m m m m
J (Hz)
218.9 54.3 54.8 19.5
C C CH CH2 CH2 C C C C CH2
d m m m
12.3
31.3 167.2 139.3 38.4 200.9 53.5
1.68 1.89–1.86 1.65–1.59 2.64–2.54
2.84 2.47–2.42
d m
16.5
55.1 49.8 73.1 33.1
C C CH CH2 CH CH3 CH3 C CH3 CH2 CH2 CH C C CH3 CH2
dd m m m s s
9.3, 5.5
52.0 19.1 19.7 75.1 25.4 43.2 24.5 143.8 128.8 171.6 12.4 65.6
4.39 2.47–2.42 1.65–1.59 2.25–2.21 1.07 1.25 1.24 1.57–1.46 2.25–2.21 6.78
s m m t
21.7 19.9
CH3 CH3
1.83 3.55 4.00 1.21 1.19
s d d s s
7.4
11.5 11.5
a Spectra were recorded in CD3OD at 125 MHz (13C) and 500 MHz (1H) with dC and dH in ppm, respectively.
conducted using 0.15 g of G. lucidum and 5 g of [C2mim][(MeO)(H) PO2],26 but again the extraction yield of 1 was not improved (Fig. 4).27 These results suggest that the branched structure of bD-glucan caused relatively low solubility than in ionic liquids than cellulose from plant leaves, although lignin can dissolve in ionic liquids.28,29 Although we were unable to efficiently extract unknown compound 1, we set about to determine its structure, including its absolute configuration. The structural elucidation of 1, which was isolated from the water extract of G. lucidum by RP-HPLC, was performed by spectroscopic analysis, including 1H NMR, 13C NMR, DEPT, HMQC, HMBC, NOESY, and HRMS.12,30 The molecular formula was assigned as C30H44O7, as deduced from the mass spectra (ESI-HRMS (m/z) calcd for C30H44NaO7 [M+Na]+ 539.2985, found
539.3001). The 1H NMR spectrum of 1 showed peaks assigned to six tertiary methyl groups (dH 1.07, 1.19, 1.21, 1.24, 1.25, and 1.83 each 3H, s), one oxygenated methylene (dH 4.00, d, J = 11.5 Hz, and 3.6, d, J = 11.5 Hz), one oxygenated methine (dH 4.39, dd, J = 9.5, 5.5 Hz), and one olefinic proton (dH 6.78, t, J = 7.4 Hz). The 13C NMR spectrum in combination with the distortionless enhancement by polarization transfer (DEPT 135 and 90) of 1 exhibited 30 carbons corresponding to six methyls, nine methylenes (one oxygenated carbon at dC 65.1), four methines (one oxygenated carbon at dC 73.1), four quaternary carbons, two pairs of olefinic carbons (dC 167.2, 143.8, 139.3, and 128.9), one oxygenated carbon (dC 75.1), and two carbonyl carbons (dC 218.9 and 200.9). The structure was thus estimated to have a lanostane triterpenoid skeleton, as shown in Figure 5. On the basis of spectroscopic evidence obtained by 1H–1H COSY and HMQC, all protons and carbons were assigned as shown in Table 1. These assignments were further supported by long-range correlations between signals in the key HMBC spectrum from H3-18 to C-12 and C-17, from H319 to C-1, C-5, and C-9, from H2-28 to C-3, C-5, and C-29, from H329 to C-3, C-4, C-5, and C-28, from H-5 to C-1, C-6, C-7, C-10, and C28, from H2-7 to C-5, C-6, and C-8, from H2-12 to C-11, C-13, and C18, from H3-21 to C-17, and C-20, from H-24 to C-22, C-23, C-25, and C-26, from H3-27 to C-24, C-25, and C-26, and from H3-30 to C-8, C-13, and C-15 (Fig. 5). The relative configuration of 1 was then determined by NOESY experiments. Key NOE correlations were observed between H-12a and H3-30, H-15b and H3-18, and H-16a and H-17a (Fig. 6). Although an NOE correlation between H2-28 and H3-29 was also observed, the stereochemistry at C-4 remained unclear. A modified Mosher’s method31 was used to assign the absolute configuration of 1. As shown in Scheme 1, methoxy-trifluoromethylphenylacetic acid (MTPA) esterification with (S)-(+)- or (R)-( )- MTPA chloride and DMAP was carried out to afford ester 2 or 3 in 71% or 86% yield, respectively. Esterification was achieved on only OH-28 and not on OH-15. The (R)-MTPA ester 2 showed two doublet peaks, at 4.68 and 4.36 ppm (Dd 0.32), which correspond to the carbinolic protons, whereas the corresponding peaks were found at 4.51 and 4.37 ppm (Dd 0.14) in the (S)-MTPA ester 3. A comparative analysis of the signals in these spectra indicated that the absolute configuration at C-4 is R,32 allowing the chemical structure of 1, called ganoderic acid R, to be determined as shown in Scheme 1. The obtained lanostane-type triterpenoid 1 was biologically evaluated to determine its utility as a possible anticancer agent.2,11–14 However, compound 1 did not show any activity, including no inhibitory activity of histone deacetylases (HDAC1, DDAC6, and SIRT1) at 10 lM, no inhibitory activity of androgen receptor at 10 lM, no inhibitory activity of protein kinase at 10 lM, and no inhibitory activity of hypoxia-inducible factor (HIF) at 10 lM. In summary, unknown compound 1 was extracted from G. lucidum using ionic liquids, and the structure of ganoderic acid R (1) was determined by MS and NMR analysis together with a modified
C. Murata et al. / Tetrahedron Letters 57 (2016) 5368–5371
5371
Scheme 1. Synthesis of 2 and 3. Reagents and conditions: (a) (S)-(+)- or (R)-( )-MTPA chloride, DMAP, pyridine, rt, 24 h, 71% (2), 86% (3).
Mosher’s method. Newly isolated lanostane triterpenoid natural product 1 did not show any activity in the assays as a possible anticancer agent. Acknowledgements We thank Prof. Masahiro Yoshizawa-Fujita (Sophia University) for valuable comments, The Hitachi Scholarship Foundation for The Hitachi Research Fellowship (to Q.T.T.), and the Screening Committee of Anticancer Drugs supported by Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Support Programs for Cancer Research, from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan for performing the biological assays. Supplementary data Supplementary data (experimental procedures, HPLC chromatograms, 1H, 13C, and 2D NMR spectra, and UV–Vis spectra of 1. 1H NMR spectra of compounds 2 and 3) associated with this article can be found, in the online version, at http://dx.doi.org/10. 1016/j.tetlet.2016.10.072. References and notes 1. Baby, S.; Johnson, A. J.; Govindan, B. Phytochemistry 2015, 114, 66–101. 2. Rios, J.-L.; Andújar, I.; Recio, M.-C.; Giner, R.-M. J. Nat. Prod. 2012, 75, 2016– 2044. 3. El-Mekkawy, S.; Meselhy, M. R.; Nakamura, N.; Tezuka, Y.; Hattori, M.; Kakiuchi, N.; Shimotohno, K.; Kawahata, T.; Otake, T. Phytochemistry 1998, 49, 1651–1657. 4. Min, B.-S.; Nakamura, N.; Miyashiro, H.; Bae, K.-W.; Hattori, M. Chem. Pharm. Bull. 1998, 46, 1607–1612. 5. Sato, N.; Zhang, Q.; Ma, C.-M.; Hattori, M. Chem. Pharm. Bull. 2009, 57, 1076– 1080. 6. Morigiwa, A.; Kitabatake, K.; Fujimoto, Y.; Ikekawa, N. Chem. Pharm. Bull. 1986, 34, 3025–3028. 7. Hai-Bang, T.; Shimizu, K. Phytochem. Lett. 2015, 12, 243–247. 8. Komoda, Y.; Shimizu, M.; Sonoda, Y.; Sato, Y. Chem. Pharm. Bull. 1989, 37, 531– 533.
9. Hajjaj, H.; Macé, C.; Roberts, M.; Niederberger, P.; Fay, L. B. Appl. Environ. Microbiol. 2005, 71, 3653–3658. 10. Kohda, H.; Tokumoto, W.; Sakamoto, K.; Fujii, M.; Hirai, Y.; Yamasaki, K.; Komoda, Y.; Nakamura, H.; Ishihara, S.; Uchida, M. Chem. Pharm. Bull. 1985, 33, 1367–1374. 11. Liu, J.; Tamura, S.; Kurashiki, K.; Shimizu, K.; Noda, K.; Konishi, F.; Kumamoto, S.; Kondo, R. Chem. Biodiversity 2009, 6, 231–243. 12. Min, B.-S.; Gao, J.-J.; Nakamura, N.; Hattori, M. Chem. Pharm. Bull. 2000, 48, 1026–1033. 13. Gao, J.-J.; Min, B.-S.; Ahn, E.-M.; Nakamura, N.; Lee, H.-K.; Hattori, M. Chem. Pharm. Bull. 2002, 50, 837–840. 14. Liu, J.; Kurashiki, K.; Shimizu, K.; Kondo, R. Bioorg. Med. Chem. 2006, 14, 8654– 8660. 15. Liu, J.; Shimizu, K.; Tanaka, A.; Shinobu, W.; Ohnuki, K.; Nakamura, T.; Kondo, R. Sci. Rep. 2012, 905, 1–5. 16. Zhu, X.-L.; Chen, A.-F.; Lin, Z.-B. J. Ethnopharmacol. 2007, 111, 219–226. 17. Chien, C. M.; Cheng, J.-L.; Chang, W.-T.; Tien, M.-H.; Tsao, C.-M.; Chang, Y.-H.; Chang, H.-Y.; Hsieh, J.-F.; Wong, C.-H.; Chen, S.-T. Bioorg. Med. Chem. 2004, 12, 5603–5609. 18. Ohno, H.; Fukaya, Y. Chem. Lett. 2009, 38, 2–7. 19. Sun, N.; Rodríguez, H.; Rahman, M.; Rogers, R. D. Chem. Commun. 2011, 47, 1405–1421. 20. Swatloski, R. P.; Spear, S. K.; Holbrey, J. D.; Rogers, R. D. J. Am. Chem. Soc. 2002, 124, 4974–4975. 21. Fukaya, Y.; Hayashi, K.; Wada, M.; Ohno, H. Green Chem. 2008, 10, 44–46. 22. Dai, Y.; van Spronsen, J.; Witkamp, G.-J.; Verpoorte, R.; Choi, Y. H. J. Nat. Prod. 2013, 76, 2162–2173. 23. Usuki, T.; Yasuda, N.; Yoshizawa-Fujita, M.; Rikukawa, M. Chem. Commun. 2011, 47, 10560–10562. 24. Onda, S.; Usuki, T.; Yoshizawa-Fujita, M.; Rikukawa, M. Chem. Lett. 2015, 44, 1461–1463. 25. Hung, W.-T.; Wang, S.-H.; Chen, C.-H.; Yang, W.-B. Molecules 2008, 13, 1538– 1550. 26. This is due to the higher price of [C2mim][(MeO)(H)PO2]. 27. In the control experiments (H2O and EtOH), the extraction yields were slightly different in Figures 3 and 4, probably due to the different amount of G. lucidum used in the various experiments. 28. Abe, M.; Yamanaka, S.; Yamada, H.; Yamada, T.; Ohno, H. Green Chem. 2015, 17, 4432–4438. 29. Yan, P.; Xu, Z.; Zhang, C.; Liu, X.; Xu, W.; Zhang, Z. C. Green Chem. 2015, 17, 4913–4920. 30. Wang, K.; Bao, L.; Xiong, W.; Ma, K.; Han, J.; Wang, W.; Yin, W.; Liu, H. J. Nat. Prod. 2015, 78, 1977–1989. 31. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113, 4092–4096. 32. Tsuda, M.; Toriyabe, Y.; Endo, T.; Kobayashi, J. Chem. Pharm. Bull. 2003, 51, 448– 451.