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Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Two new secondary metabolites from the marine-derived fungus Nigrospora sphaerica a
b
a
a
Qi-Hui Zhang , Li Tian , Zong-Liang Sun , Shuai Fang , Gong-Li a
a
Cai , Yu-Jia Wang & Yue-Hu Pei
c
a
School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044, China b
The First Institute of Oceanography SOA, Qingdao 266061, China
c
Click for updates
Shenyang Pharmaceutical University, Shenyang 110016, China Published online: 23 Mar 2015.
To cite this article: Qi-Hui Zhang, Li Tian, Zong-Liang Sun, Shuai Fang, Gong-Li Cai, Yu-Jia Wang & Yue-Hu Pei (2015): Two new secondary metabolites from the marine-derived fungus Nigrospora sphaerica, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2015.1009899 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1009899
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Journal of Asian Natural Products Research, 2015 http://dx.doi.org/10.1080/10286020.2015.1009899
Two new secondary metabolites from the marine-derived fungus Nigrospora sphaerica Qi-Hui Zhanga, Li Tianb, Zong-Liang Suna, Shuai Fanga, Gong-Li Caia, Yu-Jia Wanga and Yue-Hu Peic* a
School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044, China; b The First Institute of Oceanography SOA, Qingdao 266061, China; cShenyang Pharmaceutical University, Shenyang 110016, China
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(Received 23 October 2014; final version received 15 January 2015) Two new secondary metabolites, (2S)-5-acetamidopentyl-2-hydroxypropanoate (1) and 2, 5, 7-trihydroxy-4-(30 -methylbut-20 -en-10 -yl) oxy-2H-naphtho [1, 8-b c] furan-9one (2) were isolated from the marine-derived fungus Nigrospora sphaerica. The structures were established on the basis of their spectroscopic data, including 1D NMR and 2D NMR, HR-TOF-MS, and the absolute configuration of compound 1 was determined by the Mosher method. Keywords: marine natural product; Nigrospora sphaerica; (2S)-5-acetamidopentyl-2hydroxypropanoate; phenalenone derivative
1.
Introduction
Published reviews have shown that marine microorganisms are an important source of pharmacologically active metabolites [1 – 4]. Nigrospora sphaerica is the bestknown species of the genus Nigrospora, a filamentous dematiaceous fungus, widely distributed in soil, decaying plants, and seeds. Previous chemical studies of N. sphaerica in plants have been reported [5 – 15]. In the continuous search for novel and bioactive substances from the marinederived fungi N. sphaerica, two new derivatives were isolated. (2S)-5-Acetamidopentyl-2-hydroxypropanoate (1) is an acetamidopentane derivative, and 2, 5, 7trihydroxy-4-(3 0 -methylbut-2 0 -en-1 0 -yl) oxy-2H-naphtho [1, 8-b c] furan-9-one (2) is a new phenalenone derivative (Figure 1). Compound 1 was different from the previously reported analogs, of which the carbon skeleton was made up of a pentyl ester group and a propionic acid group. Compound 2 was distinguished with other
phenalenone derivatives by a characteristic five-membered lactone ring between C-1 and C-8 and a phenolic hydroxyl at C-5. In addition, compounds 1 and 2 were tested by using MTT assay for their cytotoxic activities against PC3 and HepG2 cancer cell lines. Only compound 1 exhibited remarkable cytotoxicity with IC50 values of 5.25 and 3.51 mg/ml, respectively. 2.
Compound 1 was isolated as white amorphous powder. The molecular formula of 1, determined as C10H19NO4, was deduced from the observed [M þ Na]þ at m/z 240.1205 in HR-TOF-MS, which requires 2 degrees of unsaturation. The IR absorption spectrum of 1 showed a hydroxy stretching vibration at 3365 cm21 and absorption bands at 1742 and 1650 cm21 corresponding to an ester carbonyl and an amide carbonyl, which was confirmed by the presence of carbon singlets at dC 174.7 (the ester carbonyl)
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
Results and discussion
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Figure 1. Structures of compounds 1 and 2.
and dC 169.1 (the amide carbonyl) in the13 C NMR spectrum. Inspection of the 1H spectrum of 1 (Table 1) revealed the presence of two methyls (dH 1.77 and dH 1.24), a triplet for oxygen-substituted methylene (dH 4.04, J ¼ 6.3 Hz, H-4), a quartet for oxygensubstituted methenyl (dH 4.09, J ¼ 6.9 Hz, H-2), as well as two broad singlets for ZNH (dH 7.82) and ZOH (dH 4.65). The13 C spectrum (Table 1) disclosed 10 resonances, two for methyl groups, one for oxygen-substituted methenyl, one for ester carbonyl and one for amide carbonyl. From the 13C, DEPT and 1H – 13C HMQC spectra, the remaining carbon resonances were five methylenes. Table 1. J in Hz).
1
The propionic acid group in 1 can be proven as follows: in the 1H – 13C HMBC spectrum, H-1 showed correlation with C-3, whereas H-2 had correlation with C-3. The1H – 1H COSY spectrum showed crosspeak for a 1H – 1H-spin system between H-1 and H-2. The OH was assigned to C-2 according to the key HMBC cross-peaks (between OH and C-1 or 3) and the chemical shift value of C-2. The acetamido group was proven by the cross-correlation between H-10 and C-9, and the key correlation between H-N and C-10 in the 1 H – 13C HMBC spectrum. The oxygensubstituted CH2-4 showed correlations with C-5 and C-6, CH2-5 showed correlations with C-6 and C-7, CH2-6 showed
H (600 MHz) and 13C (150 MHz) NMR spectral data for compounds 1 and 2 (d in ppm, 1 (in DMSO-d6)
Position 1 2 3 4 5 6 7 8 9 10 NH OH-2
2 (in CDCI3)
d(H)
d(C)
Position
1.24 (d, J ¼ 6.9) 4.09 (q, J ¼ 6.9)
20.5 66.1 174.7 63.9 27.9 22.9 28.8 38.4 169.1 22.6
1 2 3 4 4a 5 6 7 8 8a 9 10 20 30 40 50 OH-2 OH-5 OH-7
4.04 (t, J ¼ 6.3) 1.53 – 1.55 (m) 1.29 – 1.31 (m) 1.40 – 1.42 (m) 3.00 – 3.02 (m) 1.77 (s) 7.82 (br. s) 4.65 (br. s)
d(H)
6.47 (s)
6.29 (s)
4.71 (d, J ¼ 6.6) 5.54 (t, J ¼ 6.6) 1.76 (s) 1.79 (s) 11.35 (s) 8.93 (s) 9.89 (s)
d(C) 93.5 164.4 98.5 160.1 106.6 147.9 104.8 137.2 122.3 132.8 162.5 65.8 118.8 138.5 18.2 25.5
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Journal of Asian Natural Products Research correlations with C-7 and C-8, CH2-7 showed a correlation with C-8. The 1 H – 1H COSY spectrum showed a 1H1Hspin system ranging from the oxygensubstituted methylene H-4 via H-5, H-6 and H-7 to H-8. In conclusion, the pentyl group can be concluded. The a-OH propionic acid was connected to the pentyl group due to the long-range correlation between H-4 and C-3 in the 1H – 13C HMBC spectrum. The acetamido was connected to pentyl group based on the correlations between HN and C-7/8, and the correlation between H-8 and C-9 as shown in Figure 2. Interpretation of these results suggested that the planar structure of compound 1 was confirmed to be 5-acetamidopentyl-2hydroxypropanoate. The absolute stereochemistry of 1 was determined by the Mosher method [16]. Figure 3 shows the selected Dd S 2 R values of the corresponding bis-(S) and (R)-MTPA esters derived from 1 by the standard procedures [17]. The sign at the C-1 position showed negative, and the C-4 and C-5 positions exhibited positive. Therefore, the absolute configuration of 1 was assigned to be (2S) as shown in Figure 3. The molecular formula of compound 2 was deduced from accurate mass measurements to be C16H14O6 (m/z 325.0683 [M þ Na] þ; calc. 325.0682), which requires 10 degrees of unsaturation. The UV absorption maxima at 262 and 340 nm suggested a strongly conjugated function in the molecule. The IR absorption spectrum of 2 showed an OH stretching vibration at 3247 cm21 which corresponds
Figure 2. Key 1H– 1H COSY (C
3
to hydroxy groups in the molecule and an absorption band at 1717 cm21 which corresponds to a lactone carbonyl. Inspection of the 1H spectrum of 2 (Table 1) revealed that a 1H NMR resonance was due to an oxygen-substituted methylene moiety (dH 4.71, d, J ¼ 6.6 Hz, H-10 ), whereas two 1H NMR singlet resonance signals arose from aryl protons (dH 6.29, H-6 and dH 6.47, H-3). Two aryl protons (H-3 and H-6) each had a distinctive set of correlations in the 1H – 13C HMBC spectrum, suggesting that each of two protons is attached to a different benzene ring. Resonances for an olefinic proton (dH 5.54, t, J ¼ 6.6 Hz, H-20 ) and three downfield shifted 1H NMR signals due to hydroxy groups (dH 8.93 for OH-5; dH 9.89 for OH7 and dH 11.35 for OH-2) were also detected. The13C NMR spectrum (Table 1) disclosed 16 resonances, 10 of them for quaternary carbons. Carbons C-1, C-4a and C-8a are sp2 quaternary aromatic carbon atoms not attached to electronegative substituents, whereas carbons C-2, C4, C-5, C-7 and C-8 are sp2 quaternary aromatic carbons attached to oxygen as judged from their chemical shifts. The remaining quaternary carbons were accounted for one carbonyl, C-9, and C30 being part of an olefinic double bond. In the 1H – 13C HMBC spectrum, H-3 showed correlations with C-1, C-2, C-4 and C-4a, whereas H-6 had correlations with C-4a, C-7 and C-8. OH-2 showed correlations with C-1, C-2 and C-3; OH-5 showed correlations with C-4a and C-6; OH-7 showed correlations with C-6, C-7
C) and HMBC correlations (H
C) of compounds 1 and 2.
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Figure 3. Dd values (dS 2 dR) obtained from (S) - and (R)-MTPA esters (1a and 1b, respectively) of compound 1.
and C-8. This pattern of heteronuclear correlations, together with the UV and 1H NMR data, indicated two connected pentasubstituted benzene rings. The naphthalene-type ring was substituted at C-2, C-5 and C-7 with hydroxy groups. The quaternary aromatic carbon C-4 was connected to an oxygen containing substituent according to its 13C NMR chemical shift. The isoprenyl group in 2 can be proven as follows: the 1H NMR spectrum contained two singlet resonances (dH 1.76 and 1.79) due to two methyl groups, they were attached to C-30 , by the 1 13 H C HMBC correlations between CH350 / CH3-40 and C-30 . The1H – 1H COSY spectrum showed correlations for a 1H-1Hspin system between H-20 and H-10 . The isoprenyl unit is attached to C-4 through oxygen due to a correlation in the 1H – 13C HMBC spectrum between H-10 and C-4 as shown in Figure 2. Further structural confirmation was achieved by comparison of the NMR data of 2 with analogous compounds [18]. Thus, the structure of compound 2 was elucidated as 2, 5, 7trihydroxy-4-(3 0 -methylbut-2 0 -en-1 0 -yl) oxy-2H-naphtho [1, 8-b c] furan-9-one.
3. Experimental 3.1 General experimental procedures Optical rotation was measured with a JASCO P-2000 Series (Jasco Co., Ltd, Tokyo, Japan). Melting points were measured on a Buchi 535 apparatus (Buchi, Haverhill, MA, USA). UV spectra were recorded on a UV-VIS-260 spectro-
photometer (Shimadzu, Kyoto, Japan). IR spectra were recorded on an FT-IR Bruker instrument using KBr disks over the range 400 – 4000 cm21 (Bruker Optik BmbH, Ettlingen, Germany). The 1D and 2D NMR data (1H, 13C, 1H – 1H COSY, HMQC, and HMBC) were acquired in DMSO-d6 and CDCl3, respectively, on Bruker Avance DRX-600 150 and 600 MHz spectrometer with TMS as internal standard (Bruker BioSpin GmbH, Rheinstetten, Germany). 1H chemical shifts (dH) were measured in ppm, relative to TMS, and 13C chemical shifts (dC) were measured relative to DMSO-d6/CDCl3 and converted to TMS scale. HR-TOF-MS were measured on a microTOFQ Bruker mass spectrometer (Bruker Daltonics, Billerica, MA, USA), Column chromatography (CC) was performed on silica gel (300 –500 mesh, Qingdao Marine Chemical Co., Qingdao, China) and Sephadex LH20 (Pharmacia, Uppsala, Sweden). Analytical and preparative thin-layer chromatographies (TLC) were performed on silica gel plates (GF254 10 – 40 mm, Qingdao Marine Chemical Co., China). Analytical TLC was used to follow the separation and check the purity of isolated compounds. Spots on the plates were observed under UV light and visualized by spraying 5% H2SO4 in EtOH (v/v), followed by heating. HPLC was performed on an Agilent 1260 liquid chromatograph with a ZORBAXSB-C18 column (4.6 mm £ 250 mm, 5 mm) and a Shimadzu LC-10 AVP liquid chromatograph using a Shim-pack GPC-2002 C18 preparative column (20 mm £ 300 mm, 10 mm). 3.2
Microorganism
The fungus N. sphaerica was obtained from the mangrove plant Xylocarpus granatum in the intertidal zone of the China’s Sea of Huanghai and was identified by comparing the ITS sequences by Prof. Li Tian, The First institute of Oceanography, Qingdao. The voucher
Journal of Asian Natural Products Research specimen (HTTM-Y05012) has been deposited in the Idioplasm Bank of Pharmaceutical Microorganism, the First Institute of Oceanography State Oceanic Administration (GenBank accession numbers FJ594991).
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3.3
Fermentation
The fungus was cultivated and maintained on slants of yeast malt glucose (YMG) agar (yeast extract 0.4%, malt extract 0.4%, glucose 1%, and 1.5% agar, pH 7.0) at 288C. Then, the fresh mycelium was inoculated into 20 £ 500 ml Erlenmeyer flasks each containing 120 ml of YMG medium (yeast extract 0.4%, glucose 1%, malt extract 0.4% and 0.5% agar, pH 7.0 in 1L of H2O). After 4 d of the incubation at 28 ^ 18C on a rotary shaker at 200 r.p.m., a 20 ml culture liquid was transferred as seed into each of a total of 150 1 L Erlenmeyer flasks containing 250 ml of YMG medium. The following cultivation was kept for 11 d at 28 ^ 18C at 200 r.p.m. on a rotary shaker. The mycelia were separated from the culture fluid (36 L) by filtration. 3.4 Extraction and isolation Mycelial mass was extracted with methanol four times at room temperature. The extracts were evaporated under reduced pressure to yield crude extract (32.6 g). The extract was subjected to CC on silica gel columns using a stepwise solvent gradient method with petroleum ether (PE, 60 –908C)/EtOAc (100:0, 100:5, 100:10, 100:15, 100:25, 100:35, 100:50, 100:100) to give eight fractions (F1 – F8) and EtOAc /MeOH (100:0, 100:5, 100:10, 100:15, 100:35) to give five fractions (F9 – F13). The F2 (1.6 g) was subjected to CC on a silica gel column (PE/Acetone, 100:5) to give four sub-fractions (F2a –F2d). The F2c (220 mg) was subjected to Sephadex LH20 column (CH2Cl2/MeOH, 2:1), followed by semi-preparative HPLC using MeOH/H2O (65:35, v/v) as the mobile
5
phase to yield 2 (flow rate 8 ml/min, Rt 17.33 min; 7.1 mg). The F9 (3.1 g) was subjected to Sephadex LH20 column (CH2Cl2/MeOH, 1:1), followed by semipreparative HPLC using MeOH/H 2O (35:65, v/v) as the mobile phase to yield 1 (flow rate 8 ml/min, Rt 8.25 min; 12.1 mg).
3.4.1 (2S)-5-Acetamidopentyl-2hydroxypropanoate (1) Amorphous white powder from MeOH, ½a22 D 2 18.51 (C 0.26, MeOH); UV (MeOH) lmax (log 1): 275 (2.69), 245 (2.17) nm; IR (KBr) nmax: 3365 (OH), 2953 (CH), 1742 (lactone CvO), 1650 (amide CvO), 1358 and 1283(CH) cm21. 1 H and 13C NMR spectral data, see Table 1. HR-TOF-MS: m/z 240.1205 [M þ Na]þ (calcd for C10H19NNaO4, 240.1206).
3.4.2 2,5,7-Trihydroxy-4-(3 0 -methylbut2 0 -en-1 0 -yl) oxy-2H-naphtho [1, 8-bc] furan-9-one (2) Yellow microcrystal from MeOH, mp 215 –2168C; UV (MeOH) lmax (log 1): 340 (4.23), 262 (4.18) nm; IR (KBr) nmax: 3247 (OH), 2924(CH), 1717 (lactone CvO), 1666 (aromatic CZH), 1447 and 1382 (CH), 1218 and 1182 (CZO) cm21. 1 H and 13C NMR spectral data, see Table 1. HR-TOF-MS: m/z 325.0683 [M þ Na]þ (calcd for C16H14NaO6, 325.0682).
3.5
Cell lines and cell culture
PC3 and HepG2 cells were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum. The cells were maintained in 5% CO2 at 378C until reaching approximately 50 – 70% confluence, and then treated with different amounts of chemicals. DMSO was used as a control vehicle.
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3.6 Cell proliferation assays MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide, Sigma, San Francisco, CA, USA) assay [19] was used to measure the proliferation of the cells treated with compounds and positive control in 96-well plates. After treatment with vehicle or tested compounds for 48 h, cells were incubated with 10 ml MTT for 4 h and the cell growth response to the compounds was detected by measuring the absorbance at 570 nm on a plate reader (Bio-Rad, Boston, MA, USA). All tests were performed in triplicate, and results are expressed as IC50 values (concentration resulting in 50% inhibition of cell growth) for compounds 1 and 2. Paclitaxel was used as the positive control, and the IC50 values were calculated from the plotted results using untreated cells as 100%, and the IC50 values were 5.25 and 3.51 mg/ml, respectively [20]. Funding This work was financially supported by the Fundamental and Frontier Research Fund of Chongqing under [grant number cstc2014jcyjA10108]; Fundamental Research Funds for the Central Universities under [grant number CQDXWL-2014-Z007]; Special Fund for Basic Scientific Research of Central Colleges, Chongqing University under [grant number 201310611045] and Fundamental Research Funds for the Central Universities under [grant number CQDXWL2012-031].
Disclosure statement No potential conflict of interest was reported by the authors.
References [1] T.S. Bugni and C.M. Ireland, Nat. Prod. Rep. 21, 143 (2004). doi:10.1039/ b301926h. [2] J.Q. Dai, K. Krohn, D. Gehle, I. Kock, U. Flo¨rke, H.J. Aust, S. Draeger, B. Schulz, and J. Rheinheimer, Eur. J. Org. Chem. 2005, 4009 doi:10.1002/ejoc.200500290.
[3] S. Wang, X.M. Li, F. Teuscher, D. Li, A. Diesel, R. Ebel, P. Proksch, and B.G. Wang, J. Nat. Prod. 69, 1622 (2006). doi:10.1021/np060248n. [4] L.J. Fremlin, A.M. Piggott, E. Lacey, and R.J. Capon, J. Nat. Prod. 72, 666 doi:10. 1021/np800777f. [5] H.G. Cutler, K. Hoogsteen, R.H. Littrell, and H. Arison, Agric. Biol. Chem. 55, 2037 (1991). doi:10.1271/bbb1961.55.2037. [6] M. Tanaka, T. Fukushima, Y. Tsujino, and T. Fujimori, Biosci. Biotechnol. Biochem. 61, 1848 (1997). doi:10.1271/bbb.61. 1848. [7] T. Fukushima, M. Tanaka, M. Gohbara, and T. Fujimori, Phytochemistry 48, 625 (1998). doi:10.1016/S0031-9422(97) 01023-6. [8] J.C. Kim, G.J. Choi, J.H. Park, H.T. Kim, and K.Y. Cho, Pest Manag. Sci. 57, 554 (2001). doi:10.1002/ps.318. [9] K. Trisuwan, V. Rukachaisirikul, Y. Sukpondma, S. Preedanon, S. Phongpaichit, N. Rungjindamai, and J. Sakayaroj, J. Nat. Prod. 71, 1323 (2008). doi:10.1021/np8002595. [10] M.B.C. Gallo, F.O. Chagas, M.O. Almeida, C.C. Macedo, B.C. Cavalcanti, F.W.A. Barros, M.O. De moraes, L.V. Costa-Lotufo, C. Pessoa, J.K. Bastos, and M.T. Pupo, J. Basic. Microbiol. 49, 142 (2009). doi:10.1002/jobm.200800093. [11] K. Trisuwan, V. Rukachaisirikul, Y. Sukpondma, S. Preedanon, S. Phongpaichit, and J. Sakayaroj, Phytochemistry 70, 554 (2009). doi:10.1016/j. phytochem.2009.01.008. [12] D.T. Wicklow and S.M. Poling, Phytopathology 99, 109 (2009). doi:10.1094/ PHYTO-99-1-0109. [13] S.H. Wu, Y.W. Chen, S.C. Shao, L.D. Wang, Y. Yu, Z.Y. Li, L.Y. Yang, S.L. Li, and R. Huang, Chem. Biodivers. 6, 79 (2009). doi:10.1002/cbdv. 200700421. [14] Y.N. Wang, C.L. Shao, C.J. Zheng, Y.Y. Chen, and C.Y. Wang, Marine drugs 9, 1379 (2011). doi:10.3390/ md9081379. [15] S. Zhou, X.M. Lin, C.S. Chen, and B.G. Chen, Chem. Biodivers. 9, 1125 (2012). doi:10.1002/cbdv.201100183. [16] J.A. Dale and H.S. Mosher, J. Am. Chem. Soc. 95, 512 (1973). doi:10.1021/ ja00783a034.
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[17] I. Ohtani, T. Kusumi, Y. Kashman, and H. Kakisawa, J. Am. Chem. Soc. 113, 4092 (1991). doi:10.1021/ja00011a006. [18] M.F. Elsebai, S. Kehraus, U. Lindequist, F. Sasse, E. Shaaban, M. Gu¨tschow, M. Josten, H.G. Sahl, and G.M. Ko¨nig, Org. Biomol. Chem. 9, 802 (2011). doi:10.1039/c0ob00625d.
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[19] M. Johansson, B. Ko¨pcke, H. Anke, and O. Sterner, J. Antibiot. 55, 104 (2002). doi:10.7164/antibiotics.55.104. [20] J.A. Manthey, K. Grohmann, A. Montanari, K. Ash, and C.L. Manthey, J. Nat. Prod. 62, 441 (1999). doi:10.1021/ np980431j.
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Two new secondary metabolites from the marine-derived fungus Nigrospora sphaerica a
b
a
a
Qi-Hui Zhang , Li Tian , Zong-Liang Sun , Shuai Fang , Gong-Li a
a
Cai , Yu-Jia Wang & Yue-Hu Pei
c
a
School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044, China b
The First Institute of Oceanography SOA, Qingdao 266061, China
c
Click for updates
Shenyang Pharmaceutical University, Shenyang 110016, China Published online: 23 Mar 2015.
To cite this article: Qi-Hui Zhang, Li Tian, Zong-Liang Sun, Shuai Fang, Gong-Li Cai, Yu-Jia Wang & Yue-Hu Pei (2015): Two new secondary metabolites from the marine-derived fungus Nigrospora sphaerica, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2015.1009899 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1009899
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2015 http://dx.doi.org/10.1080/10286020.2015.1009899
Two new secondary metabolites from the marine-derived fungus Nigrospora sphaerica Qi-Hui Zhanga, Li Tianb, Zong-Liang Suna, Shuai Fanga, Gong-Li Caia, Yu-Jia Wanga and Yue-Hu Peic* a
School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044, China; b The First Institute of Oceanography SOA, Qingdao 266061, China; cShenyang Pharmaceutical University, Shenyang 110016, China
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(Received 23 October 2014; final version received 15 January 2015) Two new secondary metabolites, (2S)-5-acetamidopentyl-2-hydroxypropanoate (1) and 2, 5, 7-trihydroxy-4-(30 -methylbut-20 -en-10 -yl) oxy-2H-naphtho [1, 8-b c] furan-9one (2) were isolated from the marine-derived fungus Nigrospora sphaerica. The structures were established on the basis of their spectroscopic data, including 1D NMR and 2D NMR, HR-TOF-MS, and the absolute configuration of compound 1 was determined by the Mosher method. Keywords: marine natural product; Nigrospora sphaerica; (2S)-5-acetamidopentyl-2hydroxypropanoate; phenalenone derivative
1.
Introduction
Published reviews have shown that marine microorganisms are an important source of pharmacologically active metabolites [1 – 4]. Nigrospora sphaerica is the bestknown species of the genus Nigrospora, a filamentous dematiaceous fungus, widely distributed in soil, decaying plants, and seeds. Previous chemical studies of N. sphaerica in plants have been reported [5 – 15]. In the continuous search for novel and bioactive substances from the marinederived fungi N. sphaerica, two new derivatives were isolated. (2S)-5-Acetamidopentyl-2-hydroxypropanoate (1) is an acetamidopentane derivative, and 2, 5, 7trihydroxy-4-(3 0 -methylbut-2 0 -en-1 0 -yl) oxy-2H-naphtho [1, 8-b c] furan-9-one (2) is a new phenalenone derivative (Figure 1). Compound 1 was different from the previously reported analogs, of which the carbon skeleton was made up of a pentyl ester group and a propionic acid group. Compound 2 was distinguished with other
phenalenone derivatives by a characteristic five-membered lactone ring between C-1 and C-8 and a phenolic hydroxyl at C-5. In addition, compounds 1 and 2 were tested by using MTT assay for their cytotoxic activities against PC3 and HepG2 cancer cell lines. Only compound 1 exhibited remarkable cytotoxicity with IC50 values of 5.25 and 3.51 mg/ml, respectively. 2.
Compound 1 was isolated as white amorphous powder. The molecular formula of 1, determined as C10H19NO4, was deduced from the observed [M þ Na]þ at m/z 240.1205 in HR-TOF-MS, which requires 2 degrees of unsaturation. The IR absorption spectrum of 1 showed a hydroxy stretching vibration at 3365 cm21 and absorption bands at 1742 and 1650 cm21 corresponding to an ester carbonyl and an amide carbonyl, which was confirmed by the presence of carbon singlets at dC 174.7 (the ester carbonyl)
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
Results and discussion
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Figure 1. Structures of compounds 1 and 2.
and dC 169.1 (the amide carbonyl) in the13 C NMR spectrum. Inspection of the 1H spectrum of 1 (Table 1) revealed the presence of two methyls (dH 1.77 and dH 1.24), a triplet for oxygen-substituted methylene (dH 4.04, J ¼ 6.3 Hz, H-4), a quartet for oxygensubstituted methenyl (dH 4.09, J ¼ 6.9 Hz, H-2), as well as two broad singlets for ZNH (dH 7.82) and ZOH (dH 4.65). The13 C spectrum (Table 1) disclosed 10 resonances, two for methyl groups, one for oxygen-substituted methenyl, one for ester carbonyl and one for amide carbonyl. From the 13C, DEPT and 1H – 13C HMQC spectra, the remaining carbon resonances were five methylenes. Table 1. J in Hz).
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The propionic acid group in 1 can be proven as follows: in the 1H – 13C HMBC spectrum, H-1 showed correlation with C-3, whereas H-2 had correlation with C-3. The1H – 1H COSY spectrum showed crosspeak for a 1H – 1H-spin system between H-1 and H-2. The OH was assigned to C-2 according to the key HMBC cross-peaks (between OH and C-1 or 3) and the chemical shift value of C-2. The acetamido group was proven by the cross-correlation between H-10 and C-9, and the key correlation between H-N and C-10 in the 1 H – 13C HMBC spectrum. The oxygensubstituted CH2-4 showed correlations with C-5 and C-6, CH2-5 showed correlations with C-6 and C-7, CH2-6 showed
H (600 MHz) and 13C (150 MHz) NMR spectral data for compounds 1 and 2 (d in ppm, 1 (in DMSO-d6)
Position 1 2 3 4 5 6 7 8 9 10 NH OH-2
2 (in CDCI3)
d(H)
d(C)
Position
1.24 (d, J ¼ 6.9) 4.09 (q, J ¼ 6.9)
20.5 66.1 174.7 63.9 27.9 22.9 28.8 38.4 169.1 22.6
1 2 3 4 4a 5 6 7 8 8a 9 10 20 30 40 50 OH-2 OH-5 OH-7
4.04 (t, J ¼ 6.3) 1.53 – 1.55 (m) 1.29 – 1.31 (m) 1.40 – 1.42 (m) 3.00 – 3.02 (m) 1.77 (s) 7.82 (br. s) 4.65 (br. s)
d(H)
6.47 (s)
6.29 (s)
4.71 (d, J ¼ 6.6) 5.54 (t, J ¼ 6.6) 1.76 (s) 1.79 (s) 11.35 (s) 8.93 (s) 9.89 (s)
d(C) 93.5 164.4 98.5 160.1 106.6 147.9 104.8 137.2 122.3 132.8 162.5 65.8 118.8 138.5 18.2 25.5
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Journal of Asian Natural Products Research correlations with C-7 and C-8, CH2-7 showed a correlation with C-8. The 1 H – 1H COSY spectrum showed a 1H1Hspin system ranging from the oxygensubstituted methylene H-4 via H-5, H-6 and H-7 to H-8. In conclusion, the pentyl group can be concluded. The a-OH propionic acid was connected to the pentyl group due to the long-range correlation between H-4 and C-3 in the 1H – 13C HMBC spectrum. The acetamido was connected to pentyl group based on the correlations between HN and C-7/8, and the correlation between H-8 and C-9 as shown in Figure 2. Interpretation of these results suggested that the planar structure of compound 1 was confirmed to be 5-acetamidopentyl-2hydroxypropanoate. The absolute stereochemistry of 1 was determined by the Mosher method [16]. Figure 3 shows the selected Dd S 2 R values of the corresponding bis-(S) and (R)-MTPA esters derived from 1 by the standard procedures [17]. The sign at the C-1 position showed negative, and the C-4 and C-5 positions exhibited positive. Therefore, the absolute configuration of 1 was assigned to be (2S) as shown in Figure 3. The molecular formula of compound 2 was deduced from accurate mass measurements to be C16H14O6 (m/z 325.0683 [M þ Na] þ; calc. 325.0682), which requires 10 degrees of unsaturation. The UV absorption maxima at 262 and 340 nm suggested a strongly conjugated function in the molecule. The IR absorption spectrum of 2 showed an OH stretching vibration at 3247 cm21 which corresponds
Figure 2. Key 1H– 1H COSY (C
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to hydroxy groups in the molecule and an absorption band at 1717 cm21 which corresponds to a lactone carbonyl. Inspection of the 1H spectrum of 2 (Table 1) revealed that a 1H NMR resonance was due to an oxygen-substituted methylene moiety (dH 4.71, d, J ¼ 6.6 Hz, H-10 ), whereas two 1H NMR singlet resonance signals arose from aryl protons (dH 6.29, H-6 and dH 6.47, H-3). Two aryl protons (H-3 and H-6) each had a distinctive set of correlations in the 1H – 13C HMBC spectrum, suggesting that each of two protons is attached to a different benzene ring. Resonances for an olefinic proton (dH 5.54, t, J ¼ 6.6 Hz, H-20 ) and three downfield shifted 1H NMR signals due to hydroxy groups (dH 8.93 for OH-5; dH 9.89 for OH7 and dH 11.35 for OH-2) were also detected. The13C NMR spectrum (Table 1) disclosed 16 resonances, 10 of them for quaternary carbons. Carbons C-1, C-4a and C-8a are sp2 quaternary aromatic carbon atoms not attached to electronegative substituents, whereas carbons C-2, C4, C-5, C-7 and C-8 are sp2 quaternary aromatic carbons attached to oxygen as judged from their chemical shifts. The remaining quaternary carbons were accounted for one carbonyl, C-9, and C30 being part of an olefinic double bond. In the 1H – 13C HMBC spectrum, H-3 showed correlations with C-1, C-2, C-4 and C-4a, whereas H-6 had correlations with C-4a, C-7 and C-8. OH-2 showed correlations with C-1, C-2 and C-3; OH-5 showed correlations with C-4a and C-6; OH-7 showed correlations with C-6, C-7
C) and HMBC correlations (H
C) of compounds 1 and 2.
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Figure 3. Dd values (dS 2 dR) obtained from (S) - and (R)-MTPA esters (1a and 1b, respectively) of compound 1.
and C-8. This pattern of heteronuclear correlations, together with the UV and 1H NMR data, indicated two connected pentasubstituted benzene rings. The naphthalene-type ring was substituted at C-2, C-5 and C-7 with hydroxy groups. The quaternary aromatic carbon C-4 was connected to an oxygen containing substituent according to its 13C NMR chemical shift. The isoprenyl group in 2 can be proven as follows: the 1H NMR spectrum contained two singlet resonances (dH 1.76 and 1.79) due to two methyl groups, they were attached to C-30 , by the 1 13 H C HMBC correlations between CH350 / CH3-40 and C-30 . The1H – 1H COSY spectrum showed correlations for a 1H-1Hspin system between H-20 and H-10 . The isoprenyl unit is attached to C-4 through oxygen due to a correlation in the 1H – 13C HMBC spectrum between H-10 and C-4 as shown in Figure 2. Further structural confirmation was achieved by comparison of the NMR data of 2 with analogous compounds [18]. Thus, the structure of compound 2 was elucidated as 2, 5, 7trihydroxy-4-(3 0 -methylbut-2 0 -en-1 0 -yl) oxy-2H-naphtho [1, 8-b c] furan-9-one.
3. Experimental 3.1 General experimental procedures Optical rotation was measured with a JASCO P-2000 Series (Jasco Co., Ltd, Tokyo, Japan). Melting points were measured on a Buchi 535 apparatus (Buchi, Haverhill, MA, USA). UV spectra were recorded on a UV-VIS-260 spectro-
photometer (Shimadzu, Kyoto, Japan). IR spectra were recorded on an FT-IR Bruker instrument using KBr disks over the range 400 – 4000 cm21 (Bruker Optik BmbH, Ettlingen, Germany). The 1D and 2D NMR data (1H, 13C, 1H – 1H COSY, HMQC, and HMBC) were acquired in DMSO-d6 and CDCl3, respectively, on Bruker Avance DRX-600 150 and 600 MHz spectrometer with TMS as internal standard (Bruker BioSpin GmbH, Rheinstetten, Germany). 1H chemical shifts (dH) were measured in ppm, relative to TMS, and 13C chemical shifts (dC) were measured relative to DMSO-d6/CDCl3 and converted to TMS scale. HR-TOF-MS were measured on a microTOFQ Bruker mass spectrometer (Bruker Daltonics, Billerica, MA, USA), Column chromatography (CC) was performed on silica gel (300 –500 mesh, Qingdao Marine Chemical Co., Qingdao, China) and Sephadex LH20 (Pharmacia, Uppsala, Sweden). Analytical and preparative thin-layer chromatographies (TLC) were performed on silica gel plates (GF254 10 – 40 mm, Qingdao Marine Chemical Co., China). Analytical TLC was used to follow the separation and check the purity of isolated compounds. Spots on the plates were observed under UV light and visualized by spraying 5% H2SO4 in EtOH (v/v), followed by heating. HPLC was performed on an Agilent 1260 liquid chromatograph with a ZORBAXSB-C18 column (4.6 mm £ 250 mm, 5 mm) and a Shimadzu LC-10 AVP liquid chromatograph using a Shim-pack GPC-2002 C18 preparative column (20 mm £ 300 mm, 10 mm). 3.2
Microorganism
The fungus N. sphaerica was obtained from the mangrove plant Xylocarpus granatum in the intertidal zone of the China’s Sea of Huanghai and was identified by comparing the ITS sequences by Prof. Li Tian, The First institute of Oceanography, Qingdao. The voucher
Journal of Asian Natural Products Research specimen (HTTM-Y05012) has been deposited in the Idioplasm Bank of Pharmaceutical Microorganism, the First Institute of Oceanography State Oceanic Administration (GenBank accession numbers FJ594991).
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3.3
Fermentation
The fungus was cultivated and maintained on slants of yeast malt glucose (YMG) agar (yeast extract 0.4%, malt extract 0.4%, glucose 1%, and 1.5% agar, pH 7.0) at 288C. Then, the fresh mycelium was inoculated into 20 £ 500 ml Erlenmeyer flasks each containing 120 ml of YMG medium (yeast extract 0.4%, glucose 1%, malt extract 0.4% and 0.5% agar, pH 7.0 in 1L of H2O). After 4 d of the incubation at 28 ^ 18C on a rotary shaker at 200 r.p.m., a 20 ml culture liquid was transferred as seed into each of a total of 150 1 L Erlenmeyer flasks containing 250 ml of YMG medium. The following cultivation was kept for 11 d at 28 ^ 18C at 200 r.p.m. on a rotary shaker. The mycelia were separated from the culture fluid (36 L) by filtration. 3.4 Extraction and isolation Mycelial mass was extracted with methanol four times at room temperature. The extracts were evaporated under reduced pressure to yield crude extract (32.6 g). The extract was subjected to CC on silica gel columns using a stepwise solvent gradient method with petroleum ether (PE, 60 –908C)/EtOAc (100:0, 100:5, 100:10, 100:15, 100:25, 100:35, 100:50, 100:100) to give eight fractions (F1 – F8) and EtOAc /MeOH (100:0, 100:5, 100:10, 100:15, 100:35) to give five fractions (F9 – F13). The F2 (1.6 g) was subjected to CC on a silica gel column (PE/Acetone, 100:5) to give four sub-fractions (F2a –F2d). The F2c (220 mg) was subjected to Sephadex LH20 column (CH2Cl2/MeOH, 2:1), followed by semi-preparative HPLC using MeOH/H2O (65:35, v/v) as the mobile
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phase to yield 2 (flow rate 8 ml/min, Rt 17.33 min; 7.1 mg). The F9 (3.1 g) was subjected to Sephadex LH20 column (CH2Cl2/MeOH, 1:1), followed by semipreparative HPLC using MeOH/H 2O (35:65, v/v) as the mobile phase to yield 1 (flow rate 8 ml/min, Rt 8.25 min; 12.1 mg).
3.4.1 (2S)-5-Acetamidopentyl-2hydroxypropanoate (1) Amorphous white powder from MeOH, ½a22 D 2 18.51 (C 0.26, MeOH); UV (MeOH) lmax (log 1): 275 (2.69), 245 (2.17) nm; IR (KBr) nmax: 3365 (OH), 2953 (CH), 1742 (lactone CvO), 1650 (amide CvO), 1358 and 1283(CH) cm21. 1 H and 13C NMR spectral data, see Table 1. HR-TOF-MS: m/z 240.1205 [M þ Na]þ (calcd for C10H19NNaO4, 240.1206).
3.4.2 2,5,7-Trihydroxy-4-(3 0 -methylbut2 0 -en-1 0 -yl) oxy-2H-naphtho [1, 8-bc] furan-9-one (2) Yellow microcrystal from MeOH, mp 215 –2168C; UV (MeOH) lmax (log 1): 340 (4.23), 262 (4.18) nm; IR (KBr) nmax: 3247 (OH), 2924(CH), 1717 (lactone CvO), 1666 (aromatic CZH), 1447 and 1382 (CH), 1218 and 1182 (CZO) cm21. 1 H and 13C NMR spectral data, see Table 1. HR-TOF-MS: m/z 325.0683 [M þ Na]þ (calcd for C16H14NaO6, 325.0682).
3.5
Cell lines and cell culture
PC3 and HepG2 cells were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum. The cells were maintained in 5% CO2 at 378C until reaching approximately 50 – 70% confluence, and then treated with different amounts of chemicals. DMSO was used as a control vehicle.
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3.6 Cell proliferation assays MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide, Sigma, San Francisco, CA, USA) assay [19] was used to measure the proliferation of the cells treated with compounds and positive control in 96-well plates. After treatment with vehicle or tested compounds for 48 h, cells were incubated with 10 ml MTT for 4 h and the cell growth response to the compounds was detected by measuring the absorbance at 570 nm on a plate reader (Bio-Rad, Boston, MA, USA). All tests were performed in triplicate, and results are expressed as IC50 values (concentration resulting in 50% inhibition of cell growth) for compounds 1 and 2. Paclitaxel was used as the positive control, and the IC50 values were calculated from the plotted results using untreated cells as 100%, and the IC50 values were 5.25 and 3.51 mg/ml, respectively [20]. Funding This work was financially supported by the Fundamental and Frontier Research Fund of Chongqing under [grant number cstc2014jcyjA10108]; Fundamental Research Funds for the Central Universities under [grant number CQDXWL-2014-Z007]; Special Fund for Basic Scientific Research of Central Colleges, Chongqing University under [grant number 201310611045] and Fundamental Research Funds for the Central Universities under [grant number CQDXWL2012-031].
Disclosure statement No potential conflict of interest was reported by the authors.
References [1] T.S. Bugni and C.M. Ireland, Nat. Prod. Rep. 21, 143 (2004). doi:10.1039/ b301926h. [2] J.Q. Dai, K. Krohn, D. Gehle, I. Kock, U. Flo¨rke, H.J. Aust, S. Draeger, B. Schulz, and J. Rheinheimer, Eur. J. Org. Chem. 2005, 4009 doi:10.1002/ejoc.200500290.
[3] S. Wang, X.M. Li, F. Teuscher, D. Li, A. Diesel, R. Ebel, P. Proksch, and B.G. Wang, J. Nat. Prod. 69, 1622 (2006). doi:10.1021/np060248n. [4] L.J. Fremlin, A.M. Piggott, E. Lacey, and R.J. Capon, J. Nat. Prod. 72, 666 doi:10. 1021/np800777f. [5] H.G. Cutler, K. Hoogsteen, R.H. Littrell, and H. Arison, Agric. Biol. Chem. 55, 2037 (1991). doi:10.1271/bbb1961.55.2037. [6] M. Tanaka, T. Fukushima, Y. Tsujino, and T. Fujimori, Biosci. Biotechnol. Biochem. 61, 1848 (1997). doi:10.1271/bbb.61. 1848. [7] T. Fukushima, M. Tanaka, M. Gohbara, and T. Fujimori, Phytochemistry 48, 625 (1998). doi:10.1016/S0031-9422(97) 01023-6. [8] J.C. Kim, G.J. Choi, J.H. Park, H.T. Kim, and K.Y. Cho, Pest Manag. Sci. 57, 554 (2001). doi:10.1002/ps.318. [9] K. Trisuwan, V. Rukachaisirikul, Y. Sukpondma, S. Preedanon, S. Phongpaichit, N. Rungjindamai, and J. Sakayaroj, J. Nat. Prod. 71, 1323 (2008). doi:10.1021/np8002595. [10] M.B.C. Gallo, F.O. Chagas, M.O. Almeida, C.C. Macedo, B.C. Cavalcanti, F.W.A. Barros, M.O. De moraes, L.V. Costa-Lotufo, C. Pessoa, J.K. Bastos, and M.T. Pupo, J. Basic. Microbiol. 49, 142 (2009). doi:10.1002/jobm.200800093. [11] K. Trisuwan, V. Rukachaisirikul, Y. Sukpondma, S. Preedanon, S. Phongpaichit, and J. Sakayaroj, Phytochemistry 70, 554 (2009). doi:10.1016/j. phytochem.2009.01.008. [12] D.T. Wicklow and S.M. Poling, Phytopathology 99, 109 (2009). doi:10.1094/ PHYTO-99-1-0109. [13] S.H. Wu, Y.W. Chen, S.C. Shao, L.D. Wang, Y. Yu, Z.Y. Li, L.Y. Yang, S.L. Li, and R. Huang, Chem. Biodivers. 6, 79 (2009). doi:10.1002/cbdv. 200700421. [14] Y.N. Wang, C.L. Shao, C.J. Zheng, Y.Y. Chen, and C.Y. Wang, Marine drugs 9, 1379 (2011). doi:10.3390/ md9081379. [15] S. Zhou, X.M. Lin, C.S. Chen, and B.G. Chen, Chem. Biodivers. 9, 1125 (2012). doi:10.1002/cbdv.201100183. [16] J.A. Dale and H.S. Mosher, J. Am. Chem. Soc. 95, 512 (1973). doi:10.1021/ ja00783a034.
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[17] I. Ohtani, T. Kusumi, Y. Kashman, and H. Kakisawa, J. Am. Chem. Soc. 113, 4092 (1991). doi:10.1021/ja00011a006. [18] M.F. Elsebai, S. Kehraus, U. Lindequist, F. Sasse, E. Shaaban, M. Gu¨tschow, M. Josten, H.G. Sahl, and G.M. Ko¨nig, Org. Biomol. Chem. 9, 802 (2011). doi:10.1039/c0ob00625d.
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[19] M. Johansson, B. Ko¨pcke, H. Anke, and O. Sterner, J. Antibiot. 55, 104 (2002). doi:10.7164/antibiotics.55.104. [20] J.A. Manthey, K. Grohmann, A. Montanari, K. Ash, and C.L. Manthey, J. Nat. Prod. 62, 441 (1999). doi:10.1021/ np980431j.
