Note pubs.acs.org/jnp
Iminolactones from Schizophyllum commune Xuemei Liu,†,‡ Karla Frydenvang,† Huizhen Liu,† Lin Zhai,§ Ming Chen,§ Carl Erik Olsen,⊥ and Søren Brøgger Christensen*,† †
Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark ‡ Chemistry Department, Guangxi Medical University, Shuang Yong Road, Nan Ning, Guang Xi, People’s Republic of China § Department of Clinical Microbiology 7602, Rigshospitalet, Tagensvej 20, DK-2200 Copenhagen N, Denmark ⊥ Department of Basic Sciences and Environment, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark S Supporting Information *
ABSTRACT: Schizines A (1) and B (2), the first naturally occurring iminolactones (3,6-dihydro-2H-1,4-oxazin-2-one derivatives) to be reported, have been isolated from the fruiting bodies of Schizophyllym commune. In principle the 2oxazinone moiety might have been formed by a reaction between the amino acid phenylalanine or tryptophan and an 2α-hydroxy-1-ketomarasmone. The alkaloids are unusual in that the carboxyl group of the amino acid precursor is preserved during the biosynthesis. The compounds showed some inhibition of the growth of cancer cells.
B
B, epischizine A, and ergosterol peroxide. Schizine A (1) crystallized, and the structure was elucidated by X-ray crystallography (Figures 1 and 2). Comparison of the spectra of shizines A and B (2) (Table 1) revealed that the only difference was the presence of a phenylalanine moiety in schizine A and a tryptophan moiety in schizine B. Together with schizines A and B a small amount of an isomer of 1, epischizine A (3), was found. Comparison of the spectra of compounds 1 and 3 reveals that no significant difference is found in the 13C NMR spectra, but the signals of H-2 to H-6 varied considerably. In particular, a dramatic shift is observed for the α-protons attached to C-3. This observation together with the presence of a NOE in 3 between H-2 and H-3′ but no similar NOE in the spectrum of 1 suggests that 3 is an isomer of 1 epimerized at C-2′. The suggested epimerization also explains the shift of H-3α since this proton now will be outside the anisotropic field of the aromatic ring. To reflect this epimerization, compound 3 is named epischizine A. Since 1 easily epimerizes into 3 in a slightly basic solution, compound 3 might be an artifact. As a part of the biosynthesis of the alkaloids, a reaction of the precursor amino acid with 2α-hydroxy-1-ketomarasmone (4, Scheme 1) might have occurred. The proposed drimane precursor 4 is not known, but 1α,2β-dihydroxymarasmone has recently been described.10 In plants alkaloids are biosynthesized using amino acids as precursors. No examples are known of alkaloids in which the carboxyl group from the precursor amino acid has been
asidomycetes and other fungi have played a minor role in European traditional medicine1 even though they have been the source of important drugs such as penicillins (Penicillium species), cephalosporins (Cephalosporium species),2 the ergot alkaloids (Claviceps purpurea),3 the statins (Pecicillium species),2 and many other examples.2 A few basidomycetes, for example species belonging to the genus Psilocybe3 and the fly agaric mushroom (Amanita muscaria),4 have been used as recreational drugs. In contrast basidomycetes have been used extensively in traditional medicine in East Asia.1,5 Inspired by the Chinese use of basidiomycetes and other fungi, a screening of Danish species was initiated in order to find compounds with some selective toxicity toward malignant cells. An extract from Schizophyllum commune (Schizophyllaceae) was observed to reduce the proliferation of cancer cells more efficiently than the growth of benign cells. Inspired by this observation a chemical investigation of this extract was performed. S. commune is used for food in Asia.6 In addition the mushroom has attracted some attention because of the presence of schizophyllan, a (1→3)-β-D-glucan, which is used as a biological response modifier in combination with chemoand radiation therapy.6 Other chemical studies of S. commune have revealed the presence of a potent squalene synthase inhibitor, schizostatin A.7 In addition a number of phenolic esters8 and steroids9 have been described from the fruiting bodies. Our approach involved screening of an ethanolic extract of the fruiting bodies for the ability to reduce the growth of various cancer cell lines including a leukemic cell line (EL4), a human breast cancer cell line (MCF7), and a human prostate cancer cell line (PC3). Fractions showing activity were further purified to give four homogeneous compounds, schizines A and © XXXX American Chemical Society and American Society of Pharmacognosy
Received: October 30, 2014
A
DOI: 10.1021/np500836y J. Nat. Prod. XXXX, XXX, XXX−XXX
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chemistry. A number of 3,6-dihydro-2H-1,4-oxazin-2-one derivatives have been obtained by reacting glycine and a chiral α-hydroxyketone derived from camphor or pinane. The methylene group of the glycine iminolactones was stereoselectively alkylated. After hydrolysis the alkylated glycine is obtained in a high enantiomeric excess.12 This principle has been used for other stereoselective reactions.13 One of the protons attached to C-3 of 1 and 2 is resonating at a remarkably high field. Inspection of the X-ray structure reveals that this proton experiences a very strong anisotropic effect from the aromatic ring of phenylalanine or tryptophan, respectively. In the X-ray structure of 1 the distances from C-5′ and C9′ to the H-3α are 3.1 Å, revealing that the proton is located in the shielding field of the aromatic nucleus. Force field calculations suggest that the gauche (−70°) conformation of the C-2′−C-3′ bond found in the crystalline state also is a dominating conformation in solution, explaining the negative δvalue of H-3α in the spectra of schizines A and B. The schizines were found in the fractions inhibiting the growth of a series of cancer cell lines. The IC50 values are given in Table 2 together with the IC50 values toward some benign cell lines. In contrast, epischizine A showed no activity up to 200 μM. Workup of the residue of the aqueous fraction afforded an additional cytotoxic compound, ergosterol peroxide, the spectra and biological effects of which were similar to those previously reported.14
Figure 1. Configuration of schizines A (1) and B (2) and epischizine A (3).
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotation was measured on a PerkinElmer 241 polarimeter. IR was recorded neat on a PerkinElmer Spectrum One Fourier-transform (FT)-IR spectrometer with a universal ATR accessory. The NMR spectra were recorded on a Varian Mercury spectrometer at 300 (1H) or 75 (13C) MHz, respectively, using standard pulse sequences. Accurate mass measurements were determined on a Bruker micrOTOF-Q instrument (Bruker Daltonics, Bremen, Germany). TLC was performed on aluminum sheets covered with TLC silica gel 60 F254 (Merck 1.05549). Column chromatography was performed over silica gel 60A 35−70 μm Davisil (Fischer Scientific S/0693/60). Biological Material. The fruit bodies of Schizophyllum commune were collected in Dyrehaven, Copenhagen. The material was identified by Professor Henning Knudsen, University of Copenhagen. A voucher specimen is deposited as C-F-84677 at the University of Copenhagen. Extraction and Isolation. The dried fruit bodies (206 g) were powdered and extracted twice with ethanol (96%, 3 L) for 24 h. The mixtures were filtered, and the combined filtrates concentrated in vacuo to give 4.85 g. The residue was dissolved in 100 mL of water and extracted first with heptane, sequentially with toluene, and finally with ethyl acetate (200 mL). The water phase afforded 4 g of a residue. Similar TLC patterns of the toluene, ethyl acetate, and heptane extracts led to a combination, and concentration in vacuo gave 406 mg of a residue. The constituents of the residue were separated by CC (50 g of silica gel 60) using the following eluents: petroleum ether−ethyl acetate−acetic acid (90:10:1) 570 mL, (75:25:1) 720 mL, (50:50:1) 225 mL, (30:70:1) 180 mL and ethyl acetate−acetic acid (100:1) 405 mL. Fractions of 15 mL with similar TLC profiles (fractions 46−50) were combined and concentrated, and the residue was purified by CC (silica gel 60) using the following eluents: dichloromethane 110 mL, dichloromethane−ethyl acetate (100:0.5) 110 mL, (100:2) 150 mL, and (50:50) 180 mL. Fractions (10 mL) with similar TLC profiles (fractions 14−71) were combined and concentrated to give compound 1 (29 mg, schizine A). The residues of fractions 51−68 were fractionated by CC (36 g of silica gel 60) using dichloromethane 230 mL, dichloromethane−ethyl
Figure 2. Perspective drawing (ORTEP-3)22 of schizine A (1). Displacement ellipsoids of the non-hydrogen atoms are shown at the 50% probability level. Hydrogen atoms are represented by spheres of arbitrary size. Oxygen atoms are colored red, and the nitrogen atom is colored blue.
preserved in the formed alkaloid. In alkaloids having phenylalanine as a precursor only a C6C2N moiety remains in the formed alkaloid, the carboxyl group being lost by decarboxylation during biosynthesis. However, in mushrooms several examples exist in which amino acids are assumed to react with a precursor to form a specialized product preserving the carboxyl group. Several examples can be found among the hydrazine alkaloids.11 The formation of 3,6-dihydro-2H-1,4-oxazin-2-one derivatives (iminolactones) is unprecedented among natural products. This heterocycle has been heavily used in synthetic B
DOI: 10.1021/np500836y J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. 13C (100 MHz) and 1H (400 MHz) NMR Data of Schizines A (1) and B (2) and Epischizine A (3) no.
1, δC
1 2 3
163.4, C 75.7, CH 47.9, CH2
4 5 6 7 8 9 10 11 12
33.6, 46.7, 24.9, 125.6, 131.0, 49.2, 59.7, 105.4, 72.3,
C CH CH2 CH C CH C CH CH2
13 14 15 1′ 2′ 3′
31.0, 22.0, 171.0, 166.3, 59.5, 39.5,
CH3 CH3 C C CH CH2
4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′
135.7, 128.5, 130.4, 127.5, 130.4, 128.5,
C CH CH CH CH CH
δH (J in Hz) 4.99, ddd (13.3, 2.3, 2.0) α −0.22, t (13.0) β 1.56, overlaid dd (13.0, 5.0) 1.56 (overlaid) 2.27 (overlaid) 5.95 br s 3.60, br s 6.12, d (4.1) α 4.59, d br m (10.5) β 4.50, d br m (10.5) 0.70, s 1.24, s
4.69, ddd (4.7, 4.3, 2.3) a 3.54, dd (12.6, 4.3) b 3.31, dd (12.6, 4.7) 7.09, m 7.35−7.20 (overlaid) 7.35−7.20 (overlaid) 7.35−7.20 (overlaid) 7.09, m
2, δC
δH (J in Hz)
162.4, C 75.1, CH 48.0, CH2 33.4, 45.6, 24.7, 125.2, 130.6, 48.3, 59.6, 105.4, 72.0,
C CH CH2 CH C CH C CH CH2
31.0, 22.1, 171.1, 167.0, 61.2, 29.3,
CH3 CH3 C C CH CH2
110.4, 123.7, 119.2, 119.6, 122.3, 111.3, 135.4,
C C CH CH CH CH C
4.92, ddd (13.5, 8.2, 5.0) α −0.43, t (12.6) β 1.56, dd (12.6, 5.0) 0.72, dd (11.1, 6.15) 2.07 (overlaid) 5.87, br s 3.76, br s 6.15, d (5.6) α 4.65, d br m (10.5) β 4.48, d br m (10.5) 0.40, s 1.14, s
4.77, dd (4.1, 7.6) a 3.68, dd (14.4, 4.3) b 3.58, dd (14.4, 4.7)
7.60, 7.05, 7.16, 7.35,
br br br br
d (7.9) t (7.9) t (7.0) d (7.9)
3, δC 164.1, C 74.8, CH 48.1, CH2 33.3, 46.5, 24.6, 126.1, 131.1, 50.5, 59.5, 105.6, 72.1,
C CH CH2 CH C CH C CH CH2
31.6, 21.6, 170.2, 166.9, 60.3, 39.9,
CH3 CH3 C C CH CH2
134.4, 129.4, 129.0, 127.5, 129.0, 129.4,
C CH CH CH CH CH
δH (J in Hz) 4.11, ddd (13.5, 5.9, 1.8) α 2.01, dd (12.9 5.9) β 1.57 (overlaid) 1.89, dd (10.6, 6.5) 2.33 (overlaid) 5.98, br s 3.42, br s 6.27, d (4.1) α 4.53, br d (10.5) β 4.47, br d (10.5) 1.02, s 1.21, s
4.85, m a 3.36, dd (13.8, 5.9) b 3.20, dd (13.8, 4.1) 7.02, m 7.32−7.22 (overlaid) 7.32−7.22 (overlaid) 7.35−7.20 (overlaid) 7.02, m
8.20, br s 7.04, br d (1.5)
128.0, CH
1680 m cm−1; NMR data (CDCl3) see Table 1; HRMS m/z 430.1634 (calcd for C24H25NNaO5+ 430.1625). Schizine B (2): colorless powder; [α]25D +51.5 (c 6.0, CHCl3); NMR data (CDCl3) see Table 1; HRMS m/z 447.1908 (calcd for C26H27N2O5+ 447.1914). Epischizine A (3): colorless powder; [α]25D +63.1 (c 0.2, CHCl3); NMR data (CDCl3) see Table 1; HRMS m/z 430.1621 (calcd for C24H25NNaO5+ 430.1625). Conversion of Schizine A (1) into Epischizine A (3). To a solution of schizine A (0.5 mg) in dichloromethane was added triethylamine (50 μL). After stirring for 15 min a spot of epischizine A could be visualized on the TLC using toluene−ethyl acetate (8:2) as an eluent. No epimerization of schizine A was observed if a solution in dichloromethane with added glacial acetic acid (50 μL) was left for 48 h. X-ray Crystallographic Analysis of Schizine A (1). Crystal data: Colorless single crystals suitable for X-ray diffraction studies were grown from a solution in methanol: orthorhombic, space group P212121, a = 8.7130(6) Å, b = 11.5100(8) Å, λ = 20.2530(13) Å, V = 2031.2(2) Å3, Z = 4, Dc = 1.332 Mg/m3, F(000) = 864, μ(Mo Kα) = 0.093 mm−1, crystal size: 0.4 × 0.32 × 0.06 mm. Data collection and reduction: A single crystal was mounted and immersed in a stream of nitrogen gas [T = 122(1) K]. Data were collected using a graphitemonochromated Mo Kα radiation source (λ = 0.710 73 Å) on a KappaCCD diffractometer. Data collection and cell refinement were performed using COLLECT and DIRAX.15 Data reduction was performed using EvalCCD.16 Correction for absorption was performed using Gaussian integration.17 Structure solution and refinement: The positions of all non-hydrogen atoms were found by directs methods (SHELX).18 Full-matrix least-squares refinements (SHELX97)18 were performed on F2, minimizing squares ∑w(Fo2 − kFc2)2, with anisotropic displacement parameters of the non-hydrogen
Scheme 1. Possible Formation of 1 or 2 from 4 and the Corresponding Amino Acid Precursor (Phenylalanine and Tryptophan, Respectively)
acetate (100:0.1) 140 mL, (100:0.2) 550 mL, and (100:0.5) 130 mL. Fractions 24−38 (each 10 mL) were combined and concentrated to give compound 3 (7 mg, epischizine A). Fractions of 15 mL with similar TLC profiles from CC 1 (fractions 69−80) were combined and concentrated. The residue was purified by CC (22 g of silica gel 60) using dichloromethane 190 mL, dichloromethane−ethyl acetate (155:0.2) 150 mL, (100:0.4) 100 mL, (99:1) 110 mL, (98:2) 380 mL, (96:4) 120 mL, (92:8) 100 mL, (90:10) 140 mL, and (50:50) 150 mL. Fractions 68−98 (10 mL) with similar TLC profiles were combined to give schizine B (17 mg). Schizine A (1): colorless crystals; mp 188−189 °C (MeOH); [α]25D +141.3 (c 6.0, CHCl3); IR (KBr) νmax 3030 w, 2940 m, 1770 s, 1750 s, C
DOI: 10.1021/np500836y J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 2. In Vitro Cytotoxicity Effects of Schizines A and B on Three Cancer Cell Lines, EL4 (Leukemic Cell Line), MCF7 (Human Breast Cancer Cell Line), and PC3 (Human Prostate Cancer Cell Line), and Three Benign Cell Lines, McCoy Cell, 3T3, and MCF 10A compound
EL4a
MCF7a
PC3a
McCoya
3T3a
MCF 10Aa
schizine A schizine B thapsigargin methotrexate paclitaxel
± ± ± ± ±
4.9 ± 1.8 6.7 ± 2.6 2.7 ± 0.8 ND ND
14.7 ± 3.6 13.4 ± 3.5 2.4 ± 0.6 ND ND
b
c
>1230d >1230 2.4 ± 0.8 ND ND
3.7 4.0 1.4 5.1 1.7
1.4 1.6 0.5 1.5 0.7
>1230 >1230 2.0 ± 0.7 ND ND
>1230 >1230 1.8 ± 0.6 347 ± 42 196 ± 20
Data are given as mean IC50 (μM) from four experiments. Thapsigargin, methotrexate, and paclitaxel are used as positive controls. bAt 1230 μM, schizine A gave 30% inhibition and schizine B gave 41% inhibition to McCoy cells. cAt 1230 μM, schizine A gave 20% inhibition and schizine B gave 32% inhibition to 3T3 cells. dAt 1230 μM, schizine A gave 22% inhibition and schizine B gave 20% inhibition to MCF 10A cells. a
(2) Butler, M. S.; Newman, D. J.. In Natural Compounds as Drugs; Petersen, F.; Amstutz, R., Eds.; Birkhäuser: Basel, 2008; Chapter 2, pp 1−44. (3) Dewick, P. M. Medicinal Natural Products, 3rd ed.; John Wiley and Sons Ltd: Chichester, UK, 2009. (4) Wasson, V. P.; Wasson, R. G. Mushrooms, Russia and History; Pantheon: New York, 1957. (5) (a) Meletis, C. D.; Barker, N. D. Altern. Complementary Ther. 2005, 141−145. (b) Huang, K. C. The Pharmacology of Chinese Herbs, 2d ed.; CRC Press: Boca Raton, 1999. (6) Hobbs, C. R. Int. J. Med. Mushrooms 2005, 7, 127−139. (7) Tanimoto, T.; Tsujita, Y.; Hamano, K.; Haruyama, H.; Kinoshita, T.; Hosoya, T.; Kaneko, S.; Tago, K.; Kogen, H. Tetrahedron Lett. 1995, 36, 6301−6304. (8) Tripathi, A. M.; Tiwary, B. N. World J. Microbiol. Biotechnol. 2013, 29, 1431−1442. (9) Mao, S.-c.; Li, Z.-y.; Li, C. Tianran Chanwu Yanjiu Yu Kaifa 2007, 19, 610−613. (10) Zheng, Y.-b.; Lu, C.-h.; Xu, L.; Su, W.-j.; Shen, Y. Chem. Res. Chin. Univ. 2012, 28, 976−979. (11) Antkowiak, R.; Antkowiak, W. Z., In Alkaloids; Brossi, A., Ed.; Academic Press, 1991; Vol. 40, pp 189−340. (12) (a) Xu, P. F.; Chen, Y. S.; Lin, S. I.; Lu, T. J. J. Org. Chem. 2002, 67, 2309−2314. (b) Xu, P. F.; Li, S.; Lu, T. J.; Wu, C. C.; Fan, B. T.; Golfis, G. J. Org. Chem. 2006, 71 (12), 4364−4373. (c) Wang, H. F.; Ma, G. H.; Yang, S. B.; Han, R. G.; Xu, P. F. Tetrahedron: Asymmetry 2008, 19, 1630−1635. (13) (a) Iwama, S.; Gao, W. G.; Shinada, T.; Ohfune, Y. Synlett 2000, 1631−1633. (b) Wang, P. F.; Gao, P.; Xu, P. F. Synlett 2006, 1095− 1099. (14) (a) Gonzalez, A. G.; Barrera, J. B.; Marante, F. J. T. Phytochemistry 1983, 22, 1049−1050. (b) Bok, J. W.; Lermer, L. Phytochemistry 1999, 51, 891−898. (c) Nam, K. S.; Jo, Y. S.; Kim, Y. H.; Hyun, J. W.; Kim, H. W. Life Sci. 2001, 69, 229−237. (15) (a) Duisenberg, A. J. M. J. Appl. Crystallogr. 1992, 25, 92−96. (b) Nonius COLLECT; Nonius BV: Delft, The Netherlands, 1999. (16) Duisenberg, A. J. M. EvalCCD. Ph.D. Thesis, University of Utrecht, The Netherlands 1998. (17) Coppens, P. In Crystallographic Computing; Ahmed, F. R.; Hall, S. R.; Huber, C. P., Eds.; Munksgaard: Copenhagen, 1970; pp 255− 270. (18) Sheldrick, G. M. Acta Crystallogr. Sect. A: Found. Crystallogr. 2008, 64, 112−122. (19) Flack, H. D. Acta Crystallogr. Sect. A: Found. Crystallogr. 1983, 39, 876−881. (20) Wilson, A. J. C. International Tables for Crystallography; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1995; Vol. C: Tables 4.2.6.8 and 6.1.1.4. (21) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; Mcmahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107−1112. (22) Farrugia, L. J. Appl. Chrystallogr. 1997, 30, 565.
atoms. The positions of the hydrogen atoms were located in subsequent difference electron density maps and refined with fixed isotropic displacement parameters (Uiso = 1.2Ueq for CH and CH2, Uiso = 1.5Ueq for OH and CH3, methyl and aromatic hydrogens are refined riding on the parent atoms). Refinement (321 paramters, 4674 unique reflections) converged at RF = 0.035, wRF2 = 0.078 [4201 reflections with Fo > 4σ(Fo); w−1 = (σ2(Fo2) + (0.0379P)2 + 0.5019P), where P = (Fo2 + 2Fc2)/3; S = 1.092. The residual electron density varied between −0.21 and 0.22 e Å−3 [non-centrosymmetric space group, but the absolute configuration cannot be determined (Flack = −0.5(8)].19 Complex scattering factors for neutral atoms were taken from International Tables for Crystallography as incorporated in SHELX97.18,20 Fractional atomic coordinates, lists of anisotropic displacement parameters, and a complete list of geometrical data have been deposited in the Cambridge Crystallographic Data Centre (CCDC 1020196). Cytotoxic Assay. The mouse leukemic cell line (EL4), human breast cancer cell line (MCF7), and human prostate cancer cell line (PC3), purchased from National Cancer Institute, were tested by a high-flux anticancer-drug screening method.21 Briefly, cancer cells were incubated with the test compound in different concentrations at 37 °C for 48 h. Cultures were fixed with trichloroacetic acid, then stained with sulforhodamine B and read at 490 nm by an ELISA reader.
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ASSOCIATED CONTENT
S Supporting Information *
1
H and 13C NMR, DEPT, COSY, NOESY, HSQC, and HMBC spectra of compounds 1−3 and 5 and crystallographic data of compound 1. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ np500836y.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +45-35336253. Fax: +45 35336041. E-mail: soren.
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Prof. H. Knudsen, University of Copenhagen, for identifying the mushrooms and Mr. F. Hansen and Mr. N. V. Holst, Department of Chemistry, University of Copenhagen, for collecting X-ray data. We thank Fondation Idella, Familien Erchsens Mindefond, and Aase og Ejnar Danielsens Fond for financial support.
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REFERENCES
(1) Lindequist, U.; Rausch, R.; Fuessel, A.; Hanssen, H. P. Med. Monatsschr. Pharm. 2010, 33, 40−48. D
DOI: 10.1021/np500836y J. Nat. Prod. XXXX, XXX, XXX−XXX
