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New glucosidated pyrazinoquinazoline indole alkaloids from fungus Aspergillus fumigatus derived of a jellyfish
Tetrahedron xxx (2014) 1e5
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New glucosidated pyrazinoquinazoline indole alkaloids from fungus Aspergillus fumigatus derived of a jellyfish Juan Liu a, Xiaoyi Wei b, Eun La Kim c, Xiuping Lin a, Xian-Wen Yang a, Xuefeng Zhou a, Bin Yang a, Jee H. Jung c, *, Yonghong Liu a, * a Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China b Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China c College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 September 2014 Received in revised form 15 November 2014 Accepted 23 November 2014 Available online xxx
Three new pyrazinoquinazoline indole glucosides (1e3) were isolated from the fungus Aspergillus fumigatus derived from the jellyfish Nemopilema nomurai. Compounds 1e3 represent the first examples of glycosidated fumiquinzoline-type alkaloids from nature. The structures of the compounds were elucidated by spectroscopic methods and ECD/TDDFT calculations. A possible biogenetic pathway was proposed. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Pyrazinoquinazoline indole glucosides Aspergillus fumigatus Jellyfish-derived ECD/TDDFT
1. Introduction The sea is richly biodiverse, and numerous pharmaceutical leads have been obtained from it; the field of marine natural products has progressed over the past decades.1 Marine microorganisms are considered an important source of bioactive secondary metabolites. These metabolites can consistently be obtained from fungi. Thus, interest in endozoic microorganisms continually increases.2e4 Twelve fungal strains were isolated from the jellyfish Nemopilema nomurai collected off the southern coast of Korea in June 2007. These strains were fermented on a small scale to obtain ethyl acetate extracts. These extracts were screened for antibacterial and antitumor activities. After screening, the bioactive strains were analyzed further for causative secondary metabolites. We previously isolated four new polyketides from the jellyfish-derived fungus Paecilomyces variotii, namely, paecilocins BeD. These polyketides have unique chain lengths and/or carbon skeletons.5 As part of our progressive program to explore the biomedical potential of endozoic fungi obtained from jellyfish, the secondary metabolites of the strain J08NF-8 were examined. The large-scale
fermentation and isolation of these fungi resulted in the discovery of three new quinazoline glucosides, namely, fumigatosides BeD. These glucosides are the first examples of natural glycosidated alkaloids of the fumiquinzoline type. The strain J08NF-8 was identified by analyzing the gene sequence and morphology of the internal gene spacer. The strain is closely related to the type strain of Aspergillus fumigatus, a common Aspergillus species that induces disease in individuals with immunodeficiencies.
* Corresponding authors. Tel./fax: þ86 (0)20 8902-3244 (Y.L.); tel.: þ82 51 510 2803; fax: þ82 51 513 6754 (J.H.J.); e-mail addresses: [email protected] (J.H. Jung), [email protected] (Y. Liu). http://dx.doi.org/10.1016/j.tet.2014.11.063 0040-4020/Ó 2014 Elsevier Ltd. All rights reserved.
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2. Result and discussion Compound 1 was isolated as a yellow, amorphous powder. Its molecular formula was expressed as C30H33N5O10 based on highresolution FABMS and NMR data. The exact mass of the [MþH]þ ion at m/z 624.2305 matched well with the formula C30H34N5O10 (D 0.1 mmu). The IR spectrum displayed absorption bands at 3371, 1647, and 1608 cm1, which indicated the signals for an alcohol, two amides, and an aromatic ring. The NMR data of 1H and 13C are listed in Table 1.
C inside the fungus A. fumigatus. The C-16 protons of compound 1 resonated as a singlet signal, and the carbon signals for C-3 and C-15 revealed a difference in chemical shift relative to that of compound 2. A correlation between H-29 (dH 1.37) and Ha-15 (dH 2.14) was observed through the NOESY spectrum, indicating that the C18eN19, N22eC21, and C17eC15 bonds had a cisoid orientation. The methyl group at C-20 was on the same side as the C17eC15 bond. The ether linkage between C-3 and C-17 confirmed that the relative configuration of the spiroquinazoline moiety was deduced as 14R*, 17S*, 18S*, and 20S* (Fig. 1).
Table 1 1 H and13C NMR data of compounds 1e3 (in CD3OD) Position
Fumigatoside B (1)
Fumigatoside C (2)
Fumigatoside D (3)
dH
dC
dH
dH
1 3 4 6 7 8
7.76, d (8.0) 7.84, t (7.0)
173.3 84.7 149.6 146.5 127.5 134.6
7.73, d (8.0) 7.82, t (7.5)
170.9 48.9 152.4 147.2 127.2 134.5
9 10
7.54, m 8.10, d (7.0)
127.5 125.9
7.51, m 8.09, d (7.5)
126.9 126.1
11 12 14 15a 15b 16 17 18 20 21 23 24 25 26 27 28 29 10 20 30 40 50 60a 60b
5.62, dd (10.0, 4.0) 2.14, dd (15.0, 4.0) 2.85, dd (14.5, 10.0) 1.94, s 5.57, s 4.55, q (6.5)
7.54, 7.43, 7.27, 7.91,
m t (7.5) t (7.5) d (7.5)
1.35, 4.26, 3.42, 3.36, 3.36, 3.37, 3.72,
d (7.0) d (9.0) t (8.5) m m m m
3.92, d (11.5)
120.2 160.8 53.1 38.1
19.0 80.5 88.8 57.8 170.6 135.9 115.3 129.0 125.2 126.1 138.4 19.0 90.4 71.5 78.0 70.0 78.3 61.4
dC
5.02, q (6.5)
5.72, dd (10.5, 4.5) 1.91, dd (15.0, 5.5) 2.78, dd (14.5, 10.5) 1.76, d (6.5) 5.54, s 4.57, q (6.5)
7.51, 7.43, 7.30, 8.00,
m t (7.5) t (7.5) d (7.5)
1.35, d (7.0) 4.30, d (9.0) 3.47, t (8.5) 3.34, m 3.34, m 3.43, t (8.0) 3.73, dd (12.0, 5.5) 3.92, dd (12.0, 1.5)
119.9 160.7 52.3 36.0
15.4 80.2 88.9 57.9 170.5 135.9 115.2 129.1 125.2 126.2 138.0 18.8 90.2 71.6 77.9 70.0 78.3 61.4
dC
4.73, q (7.0)
7.64, d (8.5) 7.81, td (7.5, 1.0) 7.46, t (8.0) 8.04, dd (8.0, 1.0)
5.47, dd (11.5, 3.0) 2.11, dd (15.0, 3.0) 2.63, dd (15.0,11.5) 1.83, d (7.5) 5.51, s 4.54, q (7.0)
7.54, 7.43, 7.30, 7.94,
d (8.0) t (7.5) t (8.0) d (7.5)
1.30, d (6.5) 4.17, d (9.0) 3.41, t (8.0) 3.34, m 3.34, m 3.37, m 3.73, dd (11.5, 4.5) 3.91, m
171.0 52.3 152.4 147.0 126.1 134.7 126.8 126.2 119.8 160.3 51.6 38.5
23.5 80.4 88.3 57.8 170.5 136.0 115.3 129.1 125.1 126.2 138.1 18.8 90.1 71.4 78.2 70.0 78.3 61.4
Fig. 1. Key HMBC (half arrows) and NOESY (dashed arrows) correlations of compound 1.
The absolute configuration of the aglycone moiety was determined by ECD/TDDFT calculations using the truncated structure (the sugar moiety was replaced by a methyl), which afforded the predicted gas phase and solution ECD spectra consistent with the experimental one (Fig. 2). Thus, the absolute configuration was established as 3R, 14R, 17S, 18S, and 20S. Six additional resonances in the NMR spectrum of 13C were detected, which revealed a hexose moiety in the structure. The hexose moiety was verified as a glucose by HSQC, HMBC, and COSY correlations. Further comparison with previous NMR data confirmed that this moiety is a b-D-glucoside.8,9 The NOESY spectra showed that H-10 was correlated with H-30 (dH 3.36) and H-50 (dH 3.37), thereby proving the configuration of b-D-glucoside. The HMBC correlation of the anomeric proton H-10 (dH 4.26) with C-18 (dC 88.8) and C-20 (dC 57.8) established the glucosidic linkage at position N-19. This linkage was supported by the NOESY correlations between H-18 (dH 5.57) and H-10 (dH 4.26), which were also correlated with H-30 (dH 3.36) and H-50 (dH 3.37). Compound 1 was defined as a fumiquinazoline-19-b-D-glucoside based on these data. Therefore, the structure of compound 1 was identified and given the trivial name fumigatoside B.
The subscripts ‘a’ and ‘b’, indicate axial and equatorial bond, respectively.
The 1H and 13C NMR data for compound 1 revealed spectroscopic and structural features that are comparable with those of the fumiquinazoline class of fungal metabolites belonging to the spiroquinazoline family of alkaloids. These metabolites were previously obtained from a marine-derived strain of A. fumigatus isolated from the gastrointestinal tract of a marine fish and an Acremonium sp. fungus isolated from the surface of the Caribbean tunicate Ecteinascidia turbinata.6,7 The NMR data for compound 1 suggested that its spiroquinazoline moiety was similar to that of the known demethyl fumiquinazoline E, except that compound 1 possessed one hydroxy group at C-3 instead of the methoxy proton. Fumiquinazoline E was previously formed by treating fumiquinazoline C with 2% HCl in MeOH.5 Consequently, the quinazoline moiety in compound 1 was proposed to be derived through enzyme hydrolysis of fumiquinazoline
Fig. 2. Comparison of the calculated and the experimental ECD spectra of compound 1.
Fumigatoside C (2) was isolated as a white, amorphous powder. Its spectroscopic data were similar to those of compound 1. This result suggests that the two compounds have the same molecular skeleton. Its molecular formula was expressed as C30H33N5O9 based
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on HRFABMS and NMR data. The exact mass of the [MþNa]þ ion at m/z 630.2151 matched well with the formula C30H33N5O9Na (D 2.5 mmu), thereby indicating the loss of one oxygen equivalent as compared with the formula of compound 1. The IR spectrum depicted absorption bands at 3368, 1663, and 1609 cm1. These bands were very similar to those of fumigatoside B. The 1H, 13C NMR, and DEPT spectra of the spiroquinazoline moiety in compound 2 were similar to those of 1, except that the C-3 hydroxyl group in compound 1 was replaced in 3 by a proton signal resonating at dH 5.02 (dC 48.9) ppm (3-H). The HMBC correlation of the anomeric proton H-10 (dH 4.30) to C-18 (dC 88.9) and C-20 (dC 57.9) also established the glucosidic linkage at position N-19. The hexose moiety was verified as a b-D-glucoside by HSQC, HMBC, and COSY correlations. The relative configurations were determined by NOESY correlation. With respect to corresponding CD transitions, the pattern exhibited by compound 2 was similar to that of compound 1 (Fig. 3). Meanwhile, the absolute configuration of compound 2 was determined as 3R, 14R, 17S, 18S, and 20S by ECD/ TDDFT calculations, which were carried out with the lowest-energy conformer of the truncated structure and provided simulated ECD spectra closely similar to the measured one (see Supplementary data). Therefore, the structure of compound 2 was identified and given the trivial name fumigatoside C.
3
glucosides fumigatosides BeD were isolated from a natural source for the first time. Fumigatosides BeD are supposed precursors of fumiquinazolines prior to enzymolysis. However, the hexose moiety may have been biotransformed from rice solid media; thus, a possible biogenetic pathway is proposed (see Supplementary data). The spiroquinazoline family of alkoloids contains a pyrazino[2,1-b] quinazoline-3,6-dione moiety and an indole ring. In all members, anthranilic acid and tryptophan substructures were detected. Therefore, fumigatosides BeD may have also been derived from anthranilic acid, tryptophan, and alanine.17,18 Upon the introduction of L-alanine, fungi produce fumigatosides B and C but not fumigatoside D (Fig. 4). To address the indistinct biosynthesis of this class of compounds, however, further study is necessary.19
Fig. 3. CD spectra of compounds 1e3.
The molecular formula of fumigatoside D (3) was similar to that of compound 2. The general features of the spectra of compound 3 closely resembled those of compound 2 except for the signals of C-1, C-3, C-15, and C-16 in the 13C NMR spectrum. This result implies that compound 3 is a stereoisomer of compound 2 at either C-3, C-14, or both. In the NOE experiments, compound 2 exhibited NOEs between 3-H and 15-Hb, whereas compound 3 displayed NOEs between 16-H and 15-Hb. The relative configurations were determined by NOESY correlation. The transition pattern of the CD spectrum of compound 3 was similar to those of the other compounds (Fig. 3). The CD Cotton effects induced by compound 3 were also quite similar to those of 3-hydroxyglyantrypine at 230 (D3 þ3.98) and 217 nm (D3 11.84). This result implies that the R-configuration at C-14 of compound 3 is identical to that of compound 2. Thus, compounds 3 and 2 are stereoisomers at C-3.10 The ECD/TDDFT calculations also confirmed that the absolute configuration of spiroquinazoline moiety in compound 3 was 3S, 14R, 17S, 18S, and 20S (see Supplementary data). The results of 1D and 2D NMR proved that the hexose moiety was a b-D-glucoside and that the glucosidic linkage position in compound 3 was the same as that in the other compounds. To determine the inhibitors of surfactant protein binding to the human NK-1 receptor, spiroquinazoline, which is the first member of the spiroquinazoline family of alkaloids, was isolated from the fungus Aspergillus flavipes, whose carbon skeleton was new at the time.11 Although more than 20 natural products closely resembling spiroquinazoline have been developed, analogs were isolated from a variety of fungi, particularly A. fumigatus.12e16 The fumiquinazoline
Fig. 4. Plausible biogenetic pathways of compounds 1e3.
The various biological activities of fumiquinazolines have attracted the attention of researchers interested in analgesics or insecticides. Fumiquinazolines are moderately cytotoxic and reportedly exhibit antitumor activity against several cancer cell lines.15 Thus, the antibacterial activities of compounds 1e3 were evaluated against a panel of strains, including Staphyloccocus aureus ATCC 29213, Escherichia coli ATCC 25922, Klebsiella pneumonia ATCC 13883, Acinetobacter baumannii ATCC 19606, Aeromonas hydrophila ATCC 7966, and Enterococcus faecalis ATCC 29212. The cytotoxicities of the compounds were also assessed against 10 cell lines of solid human tumors (K562, A549, Huh-7, H1975, MCF-7, U937, BGC823,
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HL60, Hela, and MOLT-4). However, neither antibacterial activity nor cytotoxicity was observed. 3. Experimental 3.1. General experimental procedures The NMR spectra were recorded on a Bruker AC 500 NMR spectrometer with TMS as an internal standard. HRESIMS data were measured on a Bruker microTOF-QII mass spectrometer. IR spectra were measured on JASCO FT/IR-480 plus spectrometer with KBr pellets. Optical rotation values were measured with an Anton Paar MCP500 polarimeter. ECD spectra of 1e3 in MeOH were recorded on a Jasco J-810 CD spectrometer (Jasco Inc., Japan) using 50 nm/min scanning speed,1 nm bandwidth, and three accumulations. CD spectra were measured with a Chirascan circular dichroism spectrometer (Applied Photophysics). YMC gel (ODS-A,12 nm, S-50 mm) was used for column chromatography; D3 values were expressed in L mol1 cm1. HPLC was carried on Hitachi L-2400 with YMC ODS column. 3.2. Fungal material The fungal strain A. fumigatus was isolated from the jellyfish Nemopilema nomurai collected off the southern coast of Korea in June 2007 and identified by Dr. K.S. Bae. The specimen was identified by Dr. Xiuping Lin. A voucher specimen was deposited at the Marine Natural Product Laboratory, PNU. The fungus was cultured under static conditions at 25 C in 1 L Erlenmeyer flasks containing rice (200 g each) and artificial sea water (200 mL per each) for 30 days, in total of 4.4 kg. 3.3. Details of the isolation procedures The culture medium and mycelia were extracted with EtOAc at room temperature. Guided by the antibacterial activity against S. aureus, the EtOAc extract was subjected to silica gel column chromatography, using a gradient of MeOH in CDCl3, to afford 10 fractions. Fraction 7 (294.7 mg) was subjected to silica gel column chromatography, using a gradient of MeOH in CDCl3, to give five subfractions. Compounds 1 (3.7 mg), 2 (5.2 mg), and 3 (8.1 mg) were purified from subfraction 3 by reversed-phase HPLC (YMC gel (ODS-A, 12 nm, S-50 mm)) eluting with 58% MeOH. 3.4. Computational details Molecular Merck force field (MMFF) and DFT/TDDFT calculations were performed with Spartan’14 software package (Wavefunction Inc., Irvine, CA, USA) and Gaussian09 program package, respectively, using default grids and convergence criteria.20 In order to save computation time, the structures of compounds 1e3 were truncated by replacement of the sugar moiety with a methyl and the truncated structures were used in all calculations. MMFF conformational search generated low-energy conformers within a 10 kcal/mol energy window were subjected to geometry optimization using DFT method at the B3LYP/6-31G (d) level. Frequency calculations were run at the same level to verify that each optimized conformer was a true minimum and to estimate their relative thermal free energies (DG) at 298.15 K. Solvent effects were taken into account by using polarizable continuum model (PCM). The TDDFT calculations were performed using the long-range corrected hybrid CAM-B3LYP and the hybrid B3LYP functionals, and Ahlrichs’ basis sets SVP (split valence plus polarization) and TZVP (triple zeta valence plus polarization).21,22 The number of excited states per each molecule was 60. In a preliminary set of calculations with compound 3, the SVP basis set gave consistent results with the TZVP one; therefore, the smaller SVP basis set was used in all the
subsequent calculations. The functionals CAM-B3LYP and B3LYP were evaluated in each compound using the conformer of absolute minimum. Because of the better performance in the evaluation, the former was used in TDDFT calculations of compound 1 and the latter was used in those of compounds 2 and 3. The CD spectra were generated by the program SpecDis23 using a Gaussian band shape with 0.25 eV exponential half-width from dipole-length dipolar and rotational strengths; the difference with dipole-velocity values was negligible (<10%) for most transitions. Equilibrium population of each conformer at 298.15 K was calculated from its relative free energies using Boltzmann statistics. The calculated CD spectra of 1e3 were generated from the lowest-energy conformers, which all accounted for more than 90% in equilibrium population of the compounds in MeOH solution. 3.5. Characteristics of compounds 3.5.1. Fumigatoside B (1). Yellow, amorphous powder; [a]25 D 74.1 (c 0.30, MeOH); UV (MeOH) lmax (log 3 ) 204 (4.62); IR (KBr) nmax: 3372, 1647, 1608 cm-1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 646.2120 [MþNa]þ, 624.2305 [MþH]þ (calcd for C30H33N5O10Na, 646.2125 and C30H34N5O10, 624.2306). 3.5.2. Fumigatoside C (2). White, amorphous powder; [a]25 D 134.0 (c 0.48, MeOH); UV (MeOH) lmax (log 3 ) 205 (4.77), 225 (4.65), 255 (4.31); IR (KBr) nmax: 3368, 1663, 1609, 1489, 1392, 1080, 1018, 764, 756 cm-1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 630.2151 [MþNa]þ, 608.2347 [MþH]þ (calcd for C30H33N5O9Na, 630.2176 and C30H34N5O9, 608.2357). 3.5.3. Fumigatoside D (3). Yellow, amorphous powder; [a]25 D 118.8 (c 0.75, MeOH); UV (MeOH) lmax (log 3 ) 203 (4.57), 226 (4.37), 255 (4.03); IR (KBr) nmax: 3341, 1663, 1605, 1481, 1408, 1076, 1015, 764 cm-1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 630.2171 [MþNa]þ, 608.2359 [MþH]þ (calcd for C30H33N5O9Na, 630.2176 and C30H34N5O9, 608.2357). 3.6. Antibacterial assay MIC values of the compounds were determined by the modified 0.5 Mcfarland standard method. Two-fold dilutions of the compounds in the range of (40e0.31 mg/mL) were prepared in 0.5% MeOH. Compounds and positive control (Penicillin) were similarly diluted in 0.5% MeOH to generate a series of concentration, ranging from 40 to 0.31 mg/mL per well. The turbidity of the bacterial suspensions was measured at 600 nm, and adjusted with a medium to match the 0.5 McFarland standard (105e106 colony forming units/mL). Subsequently, 180 mL of bacterial culture was inoculated into each well, and the test solutions (20 mL) were added to 96-well plates. Finally, the plates were incubated at 36 C for 24 h, and the MIC values were determined in triplicates and re-examined at appropriate times. To ensure that these vehicles had significant effect on the bacterial growth, each of the bacterial species was additionally cultured in a blank solution containing LB broth media at concentrations equivalent to those of test solutions. 3.7. Cytotoxicity assay Cytotoxicity was assayed with the CCK8 (DOjinDo, Japan) method.21 Cell lines, K562, A549, Huh-7, H1975, MCF-7, U937, BGC823, HL60, Hela, and MOLT-4 were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. Cells were routinely grown and maintained in mediums RPMI or DMEM with 10% FBS and with 1% penicillin/streptomycin. All cell lines were incubated in a Thermo/Forma Scientific CO2 Water Jacketed Incubator with 5% CO2 in air at 37 C. Cell viability assay was determined by the CCK8
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(DOjinDo, Japan) assay. Three-fold dilutions of the compounds in the range of (10e5.08105 mM) were prepared in DMSO. Cells were seeded at a density of 400e800 cells/well in 384 well plates and treated with various concentration of compounds, positive control (Trichostatin A and Taxol) or solvent control. After 72 h incubation, CCk8 reagent was added, and absorbance was measured at 450 nm using Envision 2104 multi-label Reader (Perkin Elmer, USA). Doseeresponse curves were plotted to determine the IC50 values using Prism 5.0 (GraphPad Software Inc., USA), the highest concentration of samples tested in the CCK8 assay was set at 50 mM. Acknowledgements This work was supported financially by the National Key Basic Research Program of China (973)’s Project (2011CB915503), the National High Technology Research and Development Program (863 Program, 2012AA092104), the National Natural Science Foundation of China (Nos. 21202177, 31270402, 21172230, 20902094, 41176148, and 21002110), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA11030403), and Guangdong Marine Economic Development and Innovation of Regional Demonstration Project (GD2012-D01-001). The authors acknowledge Dr. W.D. Yoon (National Fisheries Research & Development Institute) for the collection of the jellyfish Nemopilema nomurai. Supplementary data The ECD/TDDFT computational details, full spectroscopic data, and other derivatives for all new compounds. Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.tet.2014.11.063. References and notes 1. Munro, M. H.; Blunt, J. W.; Dumdei, E. J.; Hickford, S. J.; Lill, R. E.; Li, S.; Battershill, C. N.; Duckworth, A. R. J. Biotechnol. 1999, 70, 15e25.
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2. Mille-Lindblom, C.; Fischer, H.; Tranvik, L. J. OIKOS 2006, 113, 233e242. 3. Zhang, H. W.; Song, Y. C.; Tan, R. X. Nat. Prod. Rep. 2006, 23, 753e771. 4. Yang, B.; Dong, J.; Lin, X.; Zhou, X. F.; Zhang, Y.; Liu, Y. Tetrahedron 2014, 70, 3859e3863. 5. Liu, J.; Li, F.; Kim, E. L.; Li, J. L.; Hong, J.; Bae, K. S.; Chung, H. Y.; Kim, H. S.; Jung, J. H. J. Nat. Prod. 2011, 74, 1826e1829. 6. Takahashi, C.; Matsushita, T.; Doi, M.; Minoura, K.; Shingu, T.; Kumeda, Y.; Numata, A. J. Chem. Soc., Perkin Trans. 2 1995, 2345e2353. € ck, M. Chem.dEur. J. 7. Belofsky, G. N.; Anguera, M.; Jensen, P. R.; Fenical, W.; Ko 2000, 6, 1355e1360. 8. Yu, Z. G.; Rateb, M. E.; Smanski, M. J.; Peterson, R. M.; Shen, B. J. Antibiot. 2013, 66, 291e294. 9. Kobayashi, S.; Toshio, M. T.; Noguchi, H. J. Nat. Prod. 2002, 65, 319e328. 10. Peng, J.; Lin, T.; Wang, W.; Xin, Z.; Zhu, T.; Gu, Q.; Li, D. J. Nat. Prod. 2013, 76, 1133e1140. 11. Barrow, C. J.; Sun, H. H. J. Nat. Prod. 1994, 57, 471e476. 12. Yamazaki, M.; Fujimoto, H.; Okuyama, E. Tetrahedron Lett. 1976, 33, 2861e2864. 13. Frisvad, J. C.; Rank, C.; Nielsen, K. F.; Larsen, T. O. Med. Mycol. 2009, 47, 53e71. 14. Anisimov, M. M.; Chaikina, E. L.; Afiyatullov, S. S.; Klykov, A. G. IJRRAS 2012, 13, 326e329. 15. Han, X. X.; Xu, X. Y.; Cui, C. B.; Gu, Q. Q. Chin. J. Med. Chem. 2007, 17, 232e237. 16. Numata, A.; Takahashi, C.; Matsushita, T.; Miyamoto, T.; Kawai, K.; Usami, Y.; Matsumura, E.; Inoue, M.; Ohishi, H.; Singu, T. Tetrahedron Lett. 1992, 33, 1621e1624. 17. Ames, B. D.; Liu, X.; Walsh, C. T. Biochemistry 2010, 49, 8564e8576. 18. Ames, B. D.; Haynes, S. W.; Gao, X.; Evans, B. S.; Kelleher, N. L.; Tang, Y.; Walsh, C. T. Biochemistry 2011, 50, 8756e8769. 19. Hart, D. J. ARKIVOC 2010, 3, 32e65. 20. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Keith, T.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision C.01; Gaussian: Wallingford, CT, 2010. €fer, A.; Horn, H.; Ahlrichs, R. J. Chem. Phys. 1992, 97, 2571e2577. 21. Scha €fer, A.; Huber, C.; Ahlrichs, R. J. Chem. Phys. 1994, 100, 5829e5835. 22. Scha €ffel, A.; Hemberger, Y.; Bringmann, G. SpecDis Version 1.60; 23. Bruhn, T.; Schaumlo University of Wuerzburg: Germany, 2012.
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New glucosidated pyrazinoquinazoline indole alkaloids from fungus Aspergillus fumigatus derived of a jellyfish Juan Liu a, Xiaoyi Wei b, Eun La Kim c, Xiuping Lin a, Xian-Wen Yang a, Xuefeng Zhou a, Bin Yang a, Jee H. Jung c, *, Yonghong Liu a, * a Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China b Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China c College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 September 2014 Received in revised form 15 November 2014 Accepted 23 November 2014 Available online xxx
Three new pyrazinoquinazoline indole glucosides (1e3) were isolated from the fungus Aspergillus fumigatus derived from the jellyfish Nemopilema nomurai. Compounds 1e3 represent the first examples of glycosidated fumiquinzoline-type alkaloids from nature. The structures of the compounds were elucidated by spectroscopic methods and ECD/TDDFT calculations. A possible biogenetic pathway was proposed. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Pyrazinoquinazoline indole glucosides Aspergillus fumigatus Jellyfish-derived ECD/TDDFT
1. Introduction The sea is richly biodiverse, and numerous pharmaceutical leads have been obtained from it; the field of marine natural products has progressed over the past decades.1 Marine microorganisms are considered an important source of bioactive secondary metabolites. These metabolites can consistently be obtained from fungi. Thus, interest in endozoic microorganisms continually increases.2e4 Twelve fungal strains were isolated from the jellyfish Nemopilema nomurai collected off the southern coast of Korea in June 2007. These strains were fermented on a small scale to obtain ethyl acetate extracts. These extracts were screened for antibacterial and antitumor activities. After screening, the bioactive strains were analyzed further for causative secondary metabolites. We previously isolated four new polyketides from the jellyfish-derived fungus Paecilomyces variotii, namely, paecilocins BeD. These polyketides have unique chain lengths and/or carbon skeletons.5 As part of our progressive program to explore the biomedical potential of endozoic fungi obtained from jellyfish, the secondary metabolites of the strain J08NF-8 were examined. The large-scale
fermentation and isolation of these fungi resulted in the discovery of three new quinazoline glucosides, namely, fumigatosides BeD. These glucosides are the first examples of natural glycosidated alkaloids of the fumiquinzoline type. The strain J08NF-8 was identified by analyzing the gene sequence and morphology of the internal gene spacer. The strain is closely related to the type strain of Aspergillus fumigatus, a common Aspergillus species that induces disease in individuals with immunodeficiencies.
* Corresponding authors. Tel./fax: þ86 (0)20 8902-3244 (Y.L.); tel.: þ82 51 510 2803; fax: þ82 51 513 6754 (J.H.J.); e-mail addresses: [email protected] (J.H. Jung), [email protected] (Y. Liu). http://dx.doi.org/10.1016/j.tet.2014.11.063 0040-4020/Ó 2014 Elsevier Ltd. All rights reserved.
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2. Result and discussion Compound 1 was isolated as a yellow, amorphous powder. Its molecular formula was expressed as C30H33N5O10 based on highresolution FABMS and NMR data. The exact mass of the [MþH]þ ion at m/z 624.2305 matched well with the formula C30H34N5O10 (D 0.1 mmu). The IR spectrum displayed absorption bands at 3371, 1647, and 1608 cm1, which indicated the signals for an alcohol, two amides, and an aromatic ring. The NMR data of 1H and 13C are listed in Table 1.
C inside the fungus A. fumigatus. The C-16 protons of compound 1 resonated as a singlet signal, and the carbon signals for C-3 and C-15 revealed a difference in chemical shift relative to that of compound 2. A correlation between H-29 (dH 1.37) and Ha-15 (dH 2.14) was observed through the NOESY spectrum, indicating that the C18eN19, N22eC21, and C17eC15 bonds had a cisoid orientation. The methyl group at C-20 was on the same side as the C17eC15 bond. The ether linkage between C-3 and C-17 confirmed that the relative configuration of the spiroquinazoline moiety was deduced as 14R*, 17S*, 18S*, and 20S* (Fig. 1).
Table 1 1 H and13C NMR data of compounds 1e3 (in CD3OD) Position
Fumigatoside B (1)
Fumigatoside C (2)
Fumigatoside D (3)
dH
dC
dH
dH
1 3 4 6 7 8
7.76, d (8.0) 7.84, t (7.0)
173.3 84.7 149.6 146.5 127.5 134.6
7.73, d (8.0) 7.82, t (7.5)
170.9 48.9 152.4 147.2 127.2 134.5
9 10
7.54, m 8.10, d (7.0)
127.5 125.9
7.51, m 8.09, d (7.5)
126.9 126.1
11 12 14 15a 15b 16 17 18 20 21 23 24 25 26 27 28 29 10 20 30 40 50 60a 60b
5.62, dd (10.0, 4.0) 2.14, dd (15.0, 4.0) 2.85, dd (14.5, 10.0) 1.94, s 5.57, s 4.55, q (6.5)
7.54, 7.43, 7.27, 7.91,
m t (7.5) t (7.5) d (7.5)
1.35, 4.26, 3.42, 3.36, 3.36, 3.37, 3.72,
d (7.0) d (9.0) t (8.5) m m m m
3.92, d (11.5)
120.2 160.8 53.1 38.1
19.0 80.5 88.8 57.8 170.6 135.9 115.3 129.0 125.2 126.1 138.4 19.0 90.4 71.5 78.0 70.0 78.3 61.4
dC
5.02, q (6.5)
5.72, dd (10.5, 4.5) 1.91, dd (15.0, 5.5) 2.78, dd (14.5, 10.5) 1.76, d (6.5) 5.54, s 4.57, q (6.5)
7.51, 7.43, 7.30, 8.00,
m t (7.5) t (7.5) d (7.5)
1.35, d (7.0) 4.30, d (9.0) 3.47, t (8.5) 3.34, m 3.34, m 3.43, t (8.0) 3.73, dd (12.0, 5.5) 3.92, dd (12.0, 1.5)
119.9 160.7 52.3 36.0
15.4 80.2 88.9 57.9 170.5 135.9 115.2 129.1 125.2 126.2 138.0 18.8 90.2 71.6 77.9 70.0 78.3 61.4
dC
4.73, q (7.0)
7.64, d (8.5) 7.81, td (7.5, 1.0) 7.46, t (8.0) 8.04, dd (8.0, 1.0)
5.47, dd (11.5, 3.0) 2.11, dd (15.0, 3.0) 2.63, dd (15.0,11.5) 1.83, d (7.5) 5.51, s 4.54, q (7.0)
7.54, 7.43, 7.30, 7.94,
d (8.0) t (7.5) t (8.0) d (7.5)
1.30, d (6.5) 4.17, d (9.0) 3.41, t (8.0) 3.34, m 3.34, m 3.37, m 3.73, dd (11.5, 4.5) 3.91, m
171.0 52.3 152.4 147.0 126.1 134.7 126.8 126.2 119.8 160.3 51.6 38.5
23.5 80.4 88.3 57.8 170.5 136.0 115.3 129.1 125.1 126.2 138.1 18.8 90.1 71.4 78.2 70.0 78.3 61.4
Fig. 1. Key HMBC (half arrows) and NOESY (dashed arrows) correlations of compound 1.
The absolute configuration of the aglycone moiety was determined by ECD/TDDFT calculations using the truncated structure (the sugar moiety was replaced by a methyl), which afforded the predicted gas phase and solution ECD spectra consistent with the experimental one (Fig. 2). Thus, the absolute configuration was established as 3R, 14R, 17S, 18S, and 20S. Six additional resonances in the NMR spectrum of 13C were detected, which revealed a hexose moiety in the structure. The hexose moiety was verified as a glucose by HSQC, HMBC, and COSY correlations. Further comparison with previous NMR data confirmed that this moiety is a b-D-glucoside.8,9 The NOESY spectra showed that H-10 was correlated with H-30 (dH 3.36) and H-50 (dH 3.37), thereby proving the configuration of b-D-glucoside. The HMBC correlation of the anomeric proton H-10 (dH 4.26) with C-18 (dC 88.8) and C-20 (dC 57.8) established the glucosidic linkage at position N-19. This linkage was supported by the NOESY correlations between H-18 (dH 5.57) and H-10 (dH 4.26), which were also correlated with H-30 (dH 3.36) and H-50 (dH 3.37). Compound 1 was defined as a fumiquinazoline-19-b-D-glucoside based on these data. Therefore, the structure of compound 1 was identified and given the trivial name fumigatoside B.
The subscripts ‘a’ and ‘b’, indicate axial and equatorial bond, respectively.
The 1H and 13C NMR data for compound 1 revealed spectroscopic and structural features that are comparable with those of the fumiquinazoline class of fungal metabolites belonging to the spiroquinazoline family of alkaloids. These metabolites were previously obtained from a marine-derived strain of A. fumigatus isolated from the gastrointestinal tract of a marine fish and an Acremonium sp. fungus isolated from the surface of the Caribbean tunicate Ecteinascidia turbinata.6,7 The NMR data for compound 1 suggested that its spiroquinazoline moiety was similar to that of the known demethyl fumiquinazoline E, except that compound 1 possessed one hydroxy group at C-3 instead of the methoxy proton. Fumiquinazoline E was previously formed by treating fumiquinazoline C with 2% HCl in MeOH.5 Consequently, the quinazoline moiety in compound 1 was proposed to be derived through enzyme hydrolysis of fumiquinazoline
Fig. 2. Comparison of the calculated and the experimental ECD spectra of compound 1.
Fumigatoside C (2) was isolated as a white, amorphous powder. Its spectroscopic data were similar to those of compound 1. This result suggests that the two compounds have the same molecular skeleton. Its molecular formula was expressed as C30H33N5O9 based
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on HRFABMS and NMR data. The exact mass of the [MþNa]þ ion at m/z 630.2151 matched well with the formula C30H33N5O9Na (D 2.5 mmu), thereby indicating the loss of one oxygen equivalent as compared with the formula of compound 1. The IR spectrum depicted absorption bands at 3368, 1663, and 1609 cm1. These bands were very similar to those of fumigatoside B. The 1H, 13C NMR, and DEPT spectra of the spiroquinazoline moiety in compound 2 were similar to those of 1, except that the C-3 hydroxyl group in compound 1 was replaced in 3 by a proton signal resonating at dH 5.02 (dC 48.9) ppm (3-H). The HMBC correlation of the anomeric proton H-10 (dH 4.30) to C-18 (dC 88.9) and C-20 (dC 57.9) also established the glucosidic linkage at position N-19. The hexose moiety was verified as a b-D-glucoside by HSQC, HMBC, and COSY correlations. The relative configurations were determined by NOESY correlation. With respect to corresponding CD transitions, the pattern exhibited by compound 2 was similar to that of compound 1 (Fig. 3). Meanwhile, the absolute configuration of compound 2 was determined as 3R, 14R, 17S, 18S, and 20S by ECD/ TDDFT calculations, which were carried out with the lowest-energy conformer of the truncated structure and provided simulated ECD spectra closely similar to the measured one (see Supplementary data). Therefore, the structure of compound 2 was identified and given the trivial name fumigatoside C.
3
glucosides fumigatosides BeD were isolated from a natural source for the first time. Fumigatosides BeD are supposed precursors of fumiquinazolines prior to enzymolysis. However, the hexose moiety may have been biotransformed from rice solid media; thus, a possible biogenetic pathway is proposed (see Supplementary data). The spiroquinazoline family of alkoloids contains a pyrazino[2,1-b] quinazoline-3,6-dione moiety and an indole ring. In all members, anthranilic acid and tryptophan substructures were detected. Therefore, fumigatosides BeD may have also been derived from anthranilic acid, tryptophan, and alanine.17,18 Upon the introduction of L-alanine, fungi produce fumigatosides B and C but not fumigatoside D (Fig. 4). To address the indistinct biosynthesis of this class of compounds, however, further study is necessary.19
Fig. 3. CD spectra of compounds 1e3.
The molecular formula of fumigatoside D (3) was similar to that of compound 2. The general features of the spectra of compound 3 closely resembled those of compound 2 except for the signals of C-1, C-3, C-15, and C-16 in the 13C NMR spectrum. This result implies that compound 3 is a stereoisomer of compound 2 at either C-3, C-14, or both. In the NOE experiments, compound 2 exhibited NOEs between 3-H and 15-Hb, whereas compound 3 displayed NOEs between 16-H and 15-Hb. The relative configurations were determined by NOESY correlation. The transition pattern of the CD spectrum of compound 3 was similar to those of the other compounds (Fig. 3). The CD Cotton effects induced by compound 3 were also quite similar to those of 3-hydroxyglyantrypine at 230 (D3 þ3.98) and 217 nm (D3 11.84). This result implies that the R-configuration at C-14 of compound 3 is identical to that of compound 2. Thus, compounds 3 and 2 are stereoisomers at C-3.10 The ECD/TDDFT calculations also confirmed that the absolute configuration of spiroquinazoline moiety in compound 3 was 3S, 14R, 17S, 18S, and 20S (see Supplementary data). The results of 1D and 2D NMR proved that the hexose moiety was a b-D-glucoside and that the glucosidic linkage position in compound 3 was the same as that in the other compounds. To determine the inhibitors of surfactant protein binding to the human NK-1 receptor, spiroquinazoline, which is the first member of the spiroquinazoline family of alkaloids, was isolated from the fungus Aspergillus flavipes, whose carbon skeleton was new at the time.11 Although more than 20 natural products closely resembling spiroquinazoline have been developed, analogs were isolated from a variety of fungi, particularly A. fumigatus.12e16 The fumiquinazoline
Fig. 4. Plausible biogenetic pathways of compounds 1e3.
The various biological activities of fumiquinazolines have attracted the attention of researchers interested in analgesics or insecticides. Fumiquinazolines are moderately cytotoxic and reportedly exhibit antitumor activity against several cancer cell lines.15 Thus, the antibacterial activities of compounds 1e3 were evaluated against a panel of strains, including Staphyloccocus aureus ATCC 29213, Escherichia coli ATCC 25922, Klebsiella pneumonia ATCC 13883, Acinetobacter baumannii ATCC 19606, Aeromonas hydrophila ATCC 7966, and Enterococcus faecalis ATCC 29212. The cytotoxicities of the compounds were also assessed against 10 cell lines of solid human tumors (K562, A549, Huh-7, H1975, MCF-7, U937, BGC823,
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HL60, Hela, and MOLT-4). However, neither antibacterial activity nor cytotoxicity was observed. 3. Experimental 3.1. General experimental procedures The NMR spectra were recorded on a Bruker AC 500 NMR spectrometer with TMS as an internal standard. HRESIMS data were measured on a Bruker microTOF-QII mass spectrometer. IR spectra were measured on JASCO FT/IR-480 plus spectrometer with KBr pellets. Optical rotation values were measured with an Anton Paar MCP500 polarimeter. ECD spectra of 1e3 in MeOH were recorded on a Jasco J-810 CD spectrometer (Jasco Inc., Japan) using 50 nm/min scanning speed,1 nm bandwidth, and three accumulations. CD spectra were measured with a Chirascan circular dichroism spectrometer (Applied Photophysics). YMC gel (ODS-A,12 nm, S-50 mm) was used for column chromatography; D3 values were expressed in L mol1 cm1. HPLC was carried on Hitachi L-2400 with YMC ODS column. 3.2. Fungal material The fungal strain A. fumigatus was isolated from the jellyfish Nemopilema nomurai collected off the southern coast of Korea in June 2007 and identified by Dr. K.S. Bae. The specimen was identified by Dr. Xiuping Lin. A voucher specimen was deposited at the Marine Natural Product Laboratory, PNU. The fungus was cultured under static conditions at 25 C in 1 L Erlenmeyer flasks containing rice (200 g each) and artificial sea water (200 mL per each) for 30 days, in total of 4.4 kg. 3.3. Details of the isolation procedures The culture medium and mycelia were extracted with EtOAc at room temperature. Guided by the antibacterial activity against S. aureus, the EtOAc extract was subjected to silica gel column chromatography, using a gradient of MeOH in CDCl3, to afford 10 fractions. Fraction 7 (294.7 mg) was subjected to silica gel column chromatography, using a gradient of MeOH in CDCl3, to give five subfractions. Compounds 1 (3.7 mg), 2 (5.2 mg), and 3 (8.1 mg) were purified from subfraction 3 by reversed-phase HPLC (YMC gel (ODS-A, 12 nm, S-50 mm)) eluting with 58% MeOH. 3.4. Computational details Molecular Merck force field (MMFF) and DFT/TDDFT calculations were performed with Spartan’14 software package (Wavefunction Inc., Irvine, CA, USA) and Gaussian09 program package, respectively, using default grids and convergence criteria.20 In order to save computation time, the structures of compounds 1e3 were truncated by replacement of the sugar moiety with a methyl and the truncated structures were used in all calculations. MMFF conformational search generated low-energy conformers within a 10 kcal/mol energy window were subjected to geometry optimization using DFT method at the B3LYP/6-31G (d) level. Frequency calculations were run at the same level to verify that each optimized conformer was a true minimum and to estimate their relative thermal free energies (DG) at 298.15 K. Solvent effects were taken into account by using polarizable continuum model (PCM). The TDDFT calculations were performed using the long-range corrected hybrid CAM-B3LYP and the hybrid B3LYP functionals, and Ahlrichs’ basis sets SVP (split valence plus polarization) and TZVP (triple zeta valence plus polarization).21,22 The number of excited states per each molecule was 60. In a preliminary set of calculations with compound 3, the SVP basis set gave consistent results with the TZVP one; therefore, the smaller SVP basis set was used in all the
subsequent calculations. The functionals CAM-B3LYP and B3LYP were evaluated in each compound using the conformer of absolute minimum. Because of the better performance in the evaluation, the former was used in TDDFT calculations of compound 1 and the latter was used in those of compounds 2 and 3. The CD spectra were generated by the program SpecDis23 using a Gaussian band shape with 0.25 eV exponential half-width from dipole-length dipolar and rotational strengths; the difference with dipole-velocity values was negligible (<10%) for most transitions. Equilibrium population of each conformer at 298.15 K was calculated from its relative free energies using Boltzmann statistics. The calculated CD spectra of 1e3 were generated from the lowest-energy conformers, which all accounted for more than 90% in equilibrium population of the compounds in MeOH solution. 3.5. Characteristics of compounds 3.5.1. Fumigatoside B (1). Yellow, amorphous powder; [a]25 D 74.1 (c 0.30, MeOH); UV (MeOH) lmax (log 3 ) 204 (4.62); IR (KBr) nmax: 3372, 1647, 1608 cm-1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 646.2120 [MþNa]þ, 624.2305 [MþH]þ (calcd for C30H33N5O10Na, 646.2125 and C30H34N5O10, 624.2306). 3.5.2. Fumigatoside C (2). White, amorphous powder; [a]25 D 134.0 (c 0.48, MeOH); UV (MeOH) lmax (log 3 ) 205 (4.77), 225 (4.65), 255 (4.31); IR (KBr) nmax: 3368, 1663, 1609, 1489, 1392, 1080, 1018, 764, 756 cm-1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 630.2151 [MþNa]þ, 608.2347 [MþH]þ (calcd for C30H33N5O9Na, 630.2176 and C30H34N5O9, 608.2357). 3.5.3. Fumigatoside D (3). Yellow, amorphous powder; [a]25 D 118.8 (c 0.75, MeOH); UV (MeOH) lmax (log 3 ) 203 (4.57), 226 (4.37), 255 (4.03); IR (KBr) nmax: 3341, 1663, 1605, 1481, 1408, 1076, 1015, 764 cm-1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 630.2171 [MþNa]þ, 608.2359 [MþH]þ (calcd for C30H33N5O9Na, 630.2176 and C30H34N5O9, 608.2357). 3.6. Antibacterial assay MIC values of the compounds were determined by the modified 0.5 Mcfarland standard method. Two-fold dilutions of the compounds in the range of (40e0.31 mg/mL) were prepared in 0.5% MeOH. Compounds and positive control (Penicillin) were similarly diluted in 0.5% MeOH to generate a series of concentration, ranging from 40 to 0.31 mg/mL per well. The turbidity of the bacterial suspensions was measured at 600 nm, and adjusted with a medium to match the 0.5 McFarland standard (105e106 colony forming units/mL). Subsequently, 180 mL of bacterial culture was inoculated into each well, and the test solutions (20 mL) were added to 96-well plates. Finally, the plates were incubated at 36 C for 24 h, and the MIC values were determined in triplicates and re-examined at appropriate times. To ensure that these vehicles had significant effect on the bacterial growth, each of the bacterial species was additionally cultured in a blank solution containing LB broth media at concentrations equivalent to those of test solutions. 3.7. Cytotoxicity assay Cytotoxicity was assayed with the CCK8 (DOjinDo, Japan) method.21 Cell lines, K562, A549, Huh-7, H1975, MCF-7, U937, BGC823, HL60, Hela, and MOLT-4 were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. Cells were routinely grown and maintained in mediums RPMI or DMEM with 10% FBS and with 1% penicillin/streptomycin. All cell lines were incubated in a Thermo/Forma Scientific CO2 Water Jacketed Incubator with 5% CO2 in air at 37 C. Cell viability assay was determined by the CCK8
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(DOjinDo, Japan) assay. Three-fold dilutions of the compounds in the range of (10e5.08105 mM) were prepared in DMSO. Cells were seeded at a density of 400e800 cells/well in 384 well plates and treated with various concentration of compounds, positive control (Trichostatin A and Taxol) or solvent control. After 72 h incubation, CCk8 reagent was added, and absorbance was measured at 450 nm using Envision 2104 multi-label Reader (Perkin Elmer, USA). Doseeresponse curves were plotted to determine the IC50 values using Prism 5.0 (GraphPad Software Inc., USA), the highest concentration of samples tested in the CCK8 assay was set at 50 mM. Acknowledgements This work was supported financially by the National Key Basic Research Program of China (973)’s Project (2011CB915503), the National High Technology Research and Development Program (863 Program, 2012AA092104), the National Natural Science Foundation of China (Nos. 21202177, 31270402, 21172230, 20902094, 41176148, and 21002110), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA11030403), and Guangdong Marine Economic Development and Innovation of Regional Demonstration Project (GD2012-D01-001). The authors acknowledge Dr. W.D. Yoon (National Fisheries Research & Development Institute) for the collection of the jellyfish Nemopilema nomurai. Supplementary data The ECD/TDDFT computational details, full spectroscopic data, and other derivatives for all new compounds. Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.tet.2014.11.063. References and notes 1. Munro, M. H.; Blunt, J. W.; Dumdei, E. J.; Hickford, S. J.; Lill, R. E.; Li, S.; Battershill, C. N.; Duckworth, A. R. J. Biotechnol. 1999, 70, 15e25.
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