Note pubs.acs.org/jnp
Structure and Absolute Configuration of Fumiquinazoline L, an Alkaloid from a Gorgonian-Derived Scopulariopsis sp. Fungus Chang-Lun Shao,† Ru-Fang Xu,† Mei-Yan Wei,†,‡ Zhi-Gang She,*,§ and Chang-Yun Wang*,† †
Key Laboratory of Marine Drugs, The Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China ‡ School of Pharmacy, Guangdong Medical College, Dongguan 523808, People’s Republic of China § School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, People’s Republic of China S Supporting Information *
ABSTRACT: Fumiquinazoline L (1), an alkaloid with a heptacyclic skeleton formed via a bridging hemiaminal linkage, was isolated from a gorgonian-derived Scopulariopsis sp. fungus. The structure and absolute configuration of the new compound were identified by comprehensive spectroscopic data and X-ray diffraction analysis. During acid hydrolysis of 1, the isomerization of the valine residue was observed and also studied in different conditions. Fumiquinazoline L (1) showed no cytotoxic or antibacterial activities.
S
Chemical investigation of the bioactive extracts led to the discovery of fumiquinazoline L (1), together with the known cytotoxic metabolite sterigmatocystin.13 The planar structure and absolute configuration of 1 were established by a combination of spectroscopic methods, acid hydrolysis, and X-ray diffraction analysis. Compound 1 was obtained as optically active colorless crystals. Its molecular formula of C26H25N5O4 (17 degrees of unsaturation) was determined by HREIMS. The IR absorption bands at 1721 and 1698 cm−1 suggested the presence of amide groups. The 1H NMR spectrum showed signals of eight aromatic protons due to two 1,2-disubstituted benzene rings (δH 7.00−8.29), one N−H (δH 7.29), two doublet methyls (δH 1.07), one singlet methyl (δH 2.04), and other methine and methylene groups. The 13C NMR spectrum contained 26 carbon resonances, including signals resulting from three methyls, one methylene group, four sp3 methine groups, eight sp2 methine groups, and another 10 signals for quaternary carbon atoms (including three carbonyl groups). The COSY and key HMBC correlations are illustrated in Figure 1. Detailed analysis of the 1H NMR and 13C NMR data of 1 (Table 1) together with a literature search revealed that 1 was closely
econdary metabolites from marine-derived fungi have proven to be rich sources of structurally novel and biologically active compounds that have become significant chemical entities for drug discovery.1,2 Among them are alkaloids that often possess novel structural skeletons and biological activities. For example, a series of interesting alkaloids, fumiquinazolines A−G, were reported from a strain of Aspergillus f umigatus isolated from the marine fish Pseudolabrus japonicus in 1992.3,4 Eight years later, two related metabolites, fumiquinazolines H and I, were separated from the organic extract of Acremonium sp., a fungus obtained from the surface of the Caribbean tunicate Ecteinascidia turbinata, and their structures were established by spectroscopic analysis and acid hydrolysis.5 Fumiquinazoline J was first reported as a synthetic compound,6 then isolated from A. f umigatus in 2007.7 Recently, fumiquinazoline K was reported from A. f umigatus KMM4631 isolated from the soft coral Sinularia sp.8 As part of our ongoing investigation of new biologically active natural products from marine invertebrate-derived fungi in the South China Sea,9,10 the EtOAc extracts of both the fermentation broth and mycelia of a Scopulariopsis sp. fungus, which was isolated from a gorgonian coral Carijoa sp. collected from the Weizhou coral reef, exhibited strong cytotoxic activity. Moreover, literature research revealed that there are limited reports on the metabolites of the genus Scopulariopsis sp.11,12 © XXXX American Chemical Society and American Society of Pharmacognosy
Received: March 11, 2013
A
dx.doi.org/10.1021/np4002042 | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
Also, in a selective NOE experiment, the irradiation of the signal at δH 3.62 (H-20) resulted in the enhancement of the signal at δH 2.06 (H-15b), which indicated that they are on the same side of the structure. To determine the absolute configuration of the valine residue, 1 was hydrolyzed with 6.0 N HCl at 105 °C for 14 h and derivatived with Marfey’s reagent.14 An L:D valine ratio of 1.6:1 was observed. Because the NMR spectra did not suggest a mixture of C-20 epimers, a series of acid hydrolysis conditions were applied and hydrolysis results are summarized in Table 2. Both L- and D-valine residues were observed under Table 2. Ratio of L-/D-Val FDAA Derivatives from 1 under Different Conditions entry 1 2 3 4 5 6 7 8 9 10 11 12 13
Figure 1. COSY and key HMBC correlations for compound 1 and structures of fumiquinazoline L (1), fumiquinazoline C (2), and fumiquinazoline H (3).
Table 1. NMR Data for 1 (ppm)a position 1 2-NH 3 4 6 7 8 9 10 11 12 14 15a 15b 16 17 18 19-NH 20 21 23 24 25 26 27 28 29 30/31 a
δC, type
δH (J in Hz)
169.7, C 7.29, d (2.4) 84.7, 151.1, 147.1, 128.4, 135.1, 128.1, 127.2, 120.7, 159.6, 53.1, 35.0,
C C C CH CH CH CH C C CH CH2
24.8, CH3 87.4, C 89.3, CH 70.0, CH 171.7, C 136.7, C 115.6, CH 130.4, CH 125.4, CH 126.0, CH 137.5, C 32.5, CH 18.8/18.2, CH3
7.74, 7.79, 7.54, 8.29,
d (7.2) ddd (8.4, 7.2, 1.2) ddd (8.4, 7.8, 1.2) dd (7.8, 1.2)
5.71, 3.10, 1.90, 2.04,
m dd (15.0, 6.0) dd (15.0, 1.8) s
concn of HCl 6 6 6 6 6 6 6 6 6 6 5 3 3
M M M M M M M M M M M M M
HCl HCl HCl HCl HCl HCl HCl HCl HCl HCl HCl HCl HCl
time (h)
temp (°C)
14 10 5 1 0.5 0.5 0.5 0.5 48 0.25 1 1 0.25
105 105 105 105 105 80 40 35 r.t. r.t. r.t. r.t. r.t.
L/D
ratio
1.6 1.6 1.6 1.5 1.2 1.9 2.0 2.5 2.4 3.2 2.6 2.1 3.4
all conditions, with the highest ratio of approximately 3.4:1 being attained with the mildest conditions used (3 N HCl at room temperature for 15 min). The above results indicated either partial racemization of the valine under acidic conditions or compound 1 was isolated as a mixture of enantiomers. Both possibilities have precedence in studies of natural compounds. It was reported that acid hydrolysis of the cyclic hexapeptide mollamide B obtained from the Indonesian tunicate Didemnum molle resulted in partial racemization of the L-valine from the Lvalinythiazoline moiety.15 Conversely, several natural products including sporothrins A and B16 and (±)-pestalachloride D17 have been found as racemic mixtures. The possibility of 1 containing a mixture of enantiomers was excluded by analysis of the specific rotation, CD, and chiral-phase HPLC data (Supporting Information). Finally, by slow crystallization from MeOH, single crystals of 1 suitable for X-ray diffraction analysis using Cu Kα radiation were obtained, allowing the structure of 1 to be fully established (Figure 2). The possible mixture of isomers of 1 was further excluded by X-ray data. The absolute configuration of 1 was also determined as 3R,14R,17R,18R,20S by Flack’s method.18 There are only three fumiquinazoline alkaloids, fumiquinazolines C (2), H (3), and L (1), with a seven-membered ring formed between C-3 and the oxygen on C-17. Interestingly, they were all isolated from marine fungi derived from marine animals.3−5 A key difference for the three is that 1 and 3 have the C-18 R configuration, while 2 has the C-18 S configuration. This difference is reflected in the NMR data for 1 and 2 [C-18 (δC 89.3) in 1 and C-18 (δC 87.1) in 2; H-18 in 1 (δH 5.77) and 2 (δH 5.34)]. Compound 1 was inactive against the human lung cancer cell line A549 (IC50 > 10 μM) and did not exhibit any activity against the human lung adenocarcinoma cell line SPCA1, the
5.77, dd (6.0, 1.8) 2.27, d (5.4) 3.75, dd (4.8, 1.8)
7.50, d (7.8) 7.26, ddd (7.8, 7.8, 1.8) 7.00, ddd (7.8, 7.2, 1.2) 7.00 (overlapped) 2.14, m 1.07, d (6.6)/1.07, d (7.2)
600 MHz for 1H NMR and 150 MHz for 13C NMR in CDCl3.
related to two alkaloids, fumiquinazolines C (2) and H (3). The most obvious differences were the presence of the different amino acid residues, valine, alanine, and leucine in compounds 1, 2, and 3, respectively (Figure 1). The relative configuration of 1 was determined by 1D NOE and 2D NOESY experiments. The NOESY spectrum of 1 suggested a syn orientation of H-18 with H-16 and H-30/H-31. B
dx.doi.org/10.1021/np4002042 | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, PR China. Fermentation and Extraction. The fungal strain was cultivated in 20 L of liquid medium (200 g/L cooked and sliced potatoes, 20 g/L glucose in artificial seawater, in 1 L Erlenmeyer flasks each containing 400 mL of culture broth) at 25.0 °C without shaking for 8 weeks. The culture was filtered to separate the culture broth from the mycelia. The fermentation broth was extracted three times with an equal volume of EtOAc, and the fungal mycelia were extracted three times with CHCl3/MeOH (v/v, 1:1). The organic extracts were combined and concentrated under vacuum to afford a dry extract (30.210 g). Isolation. The resulting extract (30.210 g) was subjected to silica gel column chromatography (CC) (petroleum ether−EtOAc, v/v, gradient) to afford seven fractions (Fr. 1−Fr. 7). Fr. 5 was subjected to Sephadex LH-20 CC eluting with mixtures of petroleum ether/ CHCl3/MeOH (2:1:1). Further purification by semipreparative HPLC using a C18 (Kromasil 7 μm, 10 × 250 mm) column eluting with 75% of MeOH/H2O at a flow rate of 2.0 mL/min yielded fumiquinazoline L (25.0 mg) together with sterigmatocystin (30.2 mg).13 Fumiquinazoline L: colorless crystals; mp 202 °C; [α]24D = −46 (c 0.25, CHCl3); UV (MeOH) λmax (log ε) 225 (2.67), 257 (1.22), 266 (1.13), 279 (0.88), 304 (0.36), and 317 (0.30) nm; CD (21.2 mM, MeOH), λmax (Δε) 314 (−2.48), 302 (−2.94), 256 (−4.55), 235 (−5.56), and 215 (22.99) nm; IR (KBr) νmax 3438, 3373, 2869, 2960, 1721, 1698, 1608, and 1388 cm−1; 1H NMR (CDCl3, 600 MHz) and 13 C NMR (CDCl3, 150 MHz), see Table 1; EIMS m/z 471 [M]+; HREIMS m/z 471.1905 [M]+ (calcd for C26H25N5O4, 471.1901). Marfey’s Analysis of Fumiquinazoline L (1). Fumiquinazoline L (1, 0.5 mg) was hydrolyzed for 15 min to 14 h in 3−6 M HCl under different temperatures (ranging from room temperature to 105 °C). After cooling to room temperature, the solution was evaporated to dryness and redissolved in H2O (50 μL). To the acid hydrolysate was added, 200 μL of 1% (w/v) FDAA (Marfey’s reagent; 1-fluoro-2,4dinitrophenyl-5-L-alanine amide) in acetone solution. After the addition of NaHCO3 solution (1 M, 40 μL), the mixture was incubated at 45 °C for 1 h. The reactions were stopped by addition of 20 μL of 2 N HCl. Separately, L-Val and D-Val were derivatived with FDAA in the same manner as that of 1. The standard amino acid derivatives were analyzed by HPLC (Kromasil 10 × 250 mm, 7 μm, 1.0 mL/min) with 35% CH3CN in H2O (0.05% trifluoroacetic acid) for 0−30 min to 45% CH3CN in H2O (0.05% trifluoroacetic acid) for 30−90 min detected at 340 nm. The retention times of the FDAA derivatives of L-Val and D-Val were 58 and 73 min, respectively. X-ray Crystallographic Analysis of 1. Colorless crystals of 1 were obtained from MeOH. The crystal data were recorded at 293 K on an Agilent Gemini Ultra diffractometer with Cu Kα radiation (λ = 1.54718 Å). The structure was solved by direct methods (SHELXS97) and refined using full-matrix least-squares difference Fourier techniques. All non-hydrogen atoms were refined anisotropically, and all hydrogen atoms were placed in idealized positions and refined as riding atoms with the relative isotropic parameters. The crystallographic data for 1 have been deposited at the Cambridge Crystallographic Data Centre with the deposition number 888494. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB21EZ, UK [fax: +44(0)-1233-336033 or e-mail:
[email protected]]. Crystal data for 1: C27H29N5O5, Mr = 503.55, monoclinic, space group P2(1) with a = 11.3275(2) Å, b = 7.81270(10) Å, c = 13.6034(2) Å, β = 90.9540(10), V = 1203.71(3) Å3, Z = 2, Dx = 1.389 mg/m3, μ(Cu Kα) = 0.803 mm−1, and F(000) = 532. Crystal dimensions: 0.30 × 0.20 × 0.20 mm3. Independent reflections: 4271 (Rint = 0.0219). The final R1 values were 0.0281, Flack parameter = −0.02(13), wR2 = 0.0749 (I >2σ(I)). Cytotoxicity Assays. The cytotoxic activities were evaluated against the human lung cancer cell line A549, the human lung adenocarcinoma cell line SPCA1, the human hepatoma cell line BEL7402, the human gastric cancer cell line SGC7901, and the human erythromyeloblastoid leukemia cell line K562 by the MTT method as described previously.19 Mitomycin was used as a positive control.
Figure 2. Perspective ORTEP drawing for 1.
human hepatoma cell line BEL7402, the human gastric cancer cell line SGC7901, and the human erythromyeloblastoid leukemia cell line K562 at a concentration of 100 μM.19 Compound 1 was also found to have weak antibacterial activity against pathogenic bacteria Bacillus subtilis, Staphylococcus albus, and Vibrio parahemolyticus with MIC values of 50 μM, while it did not show any antibacterial activity against Bacillus cereus, Micrococcus luteus, Micrococcus tetragenus, Staphylococcus aureus, Escherichia coli, and Vibrio anguillarum at a concentration of 50 μM.20 In summary, an alkaloid with a heptacyclic skeleton formed via a bridging hemiaminal linkage, fumiquinazoline L (1), was isolated from a gorgonian-derived fungus Scopulariopsis sp. Its structure and absolute configuration were determined by a combination of extensive spectroscopic methods, acid hydrolysis, and X-ray diffraction analysis. The racemization of the valine residue under room-temperature acid hydrolysis conditions was observed and studied.
■
EXPERIMENTAL SECTION
General Experimental Procedures. The melting point was determined on an X-4 micromelting point apparatus and is uncorrected. Optical rotations were measured on a JASCO P-1020 digital polarimeter. UV spectra were recorded on a UV-2501PC spectrophotometer. The CD spectrum was aquired on a J-810 circular dichroism spectrometer. IR spectra were taken on a Bruker EQUINOX 55 spectrometer using KBr pellets. 1H and 13C NMR spectra were recorded on a JEOL JEM-ECP (600 and 150 MHz) or Varian Mercury-Plus 300 (300 and 75 MHz) NMR spectrometer in CDCl3 or acetone-d6. Chemical shifts (δ) were reported in ppm, using TMS as an internal standard. The EIMS spectrum was measured on a Thermo DSQ EI-mass spectrometer, and HREIMS data were measured on a Thermo MAT95XP high-resolution mass spectrometer. Single-crystal data were obtained on an Agilent Gemini Ultra diffractometer (Cu Kα radiation). Semipreparative HPLC was performed on an ODS C18 column [HPLC (Kromasil 250 × 10 mm, 7 μm, 2.0 mL/min)]. Silica gel (Qing Dao Hai Yang Chemical Group Co.; 200−300 mesh), octadecylsilyl silica gel (Unicorn; 45−60 μm), and Sephadex LH-20 (GE Healthcare) were used for column chromatography (CC). Precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co.; G60, F-254) were used for thin layer chromatography. Fungal Material. The fungal strain (TA01-33) was isolated from a piece of fresh tissue from the inner part of the gorgonian Carijoa sp. (GX-WZ-2010001), collected from the Weizhou coral reef in the South China Sea in April 2010. The strain was identified as Scopulariopsis sp. according to morphologic traits and molecular identification. Its 444 base pair ITS sequence had 99% sequence identity to that of Scopulariopsis sp. 2468 (EU821474). The sequence data have been submitted to GenBank, accession number JX993828. The strain was deposited at the Key Laboratory of Marine Drugs, the C
dx.doi.org/10.1021/np4002042 | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
Antibacterial Assays. The antibacterial activities were evaluated against nine bacterial strains, Gram-positive B. subtilis (ATCC 6633), B. cereus (ACCC 11077), M. luteus (ATCC 49732), M. tetragenus (ATCC 13623), S. albus (ATCC 8799), and S. aureus (ATCC 27154) and Gram-negative E. coli (ATCC 25922), V. anguillarum (ATCC 19019), and V. parahemolyticus (ATCC 17802), and the minimum inhibitory concentration (MIC, μM) values were determined by a serial dilution technique using 96-well microtiter plates.20 The compound was dissolved in DMSO to give a stock solution. Bacterial species were cultured overnight at 37 °C in LB broth and diluted to 106 cfu/mL when used. Ciprofloxacin, DMSO, and LB broth were used as positive control, negative control, and blank control, respectively. The plates were incubated at 37 °C for 24 h. The results were observed with a Multiskan Mk3 (Thermo Labsystems) at 630 nm.
■
(10) Shao, C.-L.; Wang, C.-Y.; Wei, M.-Y.; Gu, Y.-C.; She, Z.-G.; Qian, P.-Y.; Lin, Y.-C. Bioorg. Med. Chem. Lett. 2011, 21, 690−693. (11) Yu, Z. G.; Lang, G.; Kajahn, I.; Schmaljohann, R.; Imhoff, J. F. J. Nat. Prod. 2008, 71, 1052−1054 and references therein. (12) Proksch, P.; Ebel, R.; Edrada, R. A.; Riebe, F.; Liu, H.; Diesel, A.; Bayer, M.; Li, X.; Lin, W. H.; Grebenyuk, V.; Müller, W. E. G.; Draeger, S.; Zuccaro, A.; Schulz, B. Bot. Mar. 2008, 51, 209−218. (13) Bullock, E.; Kirkaldy, D.; Roberts, J. C.; Underwood, J. G. J. Chem. Soc. 1963, 829−835. (14) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591−596. (15) Donia, M. S.; Wang, B.; Dunbar, D. C.; Desai, P. V.; Patny, A.; Avery, M.; Hamann, M. T. J. Nat. Prod. 2008, 71, 941−945. (16) Wen, L.; Cai, X.; Xu, F.; She, Z.; Chan, W. L.; Vrijmoed, L. L.; Jones, E. B.; Lin, Y. J. Org. Chem. 2009, 74, 1093−1098. (17) Wei, M.-Y.; Li, D.; Shao, C.-L.; Deng, D.-S.; Wang, C.-Y. Mar. Drugs 2013, 11, 1050−1060. (18) Flack, H. D. Acta Crystallogr. 1983, A39, 876−881. (19) Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks, A.; Tierney, S.; Nofziger, T. H.; Currens, M. J.; Seniff, D.; Boyd, M. R. Cancer Res. 1988, 48, 4827−4833. (20) Pierce, C. G.; Uppuluri, P.; Teistan, A. R.; Wormley, J. F. L.; Mowat, E.; Ramage, G.; Lopez-ribot, J. L. Nat. Protoc. 2008, 3, 1494− 1500.
ASSOCIATED CONTENT
S Supporting Information *
1
H NMR, 13C NMR, HMQC, HMBC, NOESY, 1D NOE, CD, and MS spectra of compound 1; HPLC profiles of chiral-phase Crownpak CR (+) column analysis and Marfey’s method analysis; LC-MS trace of acidic hydrolysates of 1. This material is available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: 86-532-82031536 (C.-Y.W.). Tel/Fax: 86-2084034096 (Z.-G.S.). E-mail:
[email protected] (C.-Y.W.);
[email protected] (Z.-G.S.). Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS C.-L.S. thanks Dr. A. M. Fenner (SIO, UCSD) and Prof. Y.-C. Gu (Syngenta) for their proofreading of the manuscript. We thank Drs. Z.-Y. Guo (cytotoxic activity) and D.-S. Deng (crystal data) and acknowledge funding from the Program of National Natural Science Foundation of China (Nos. 41130858, 81172977, and 41176121), the Natural Science Foundation of Shandong Province (No. ZR2011DQ019), and the Program for New Century Excellent Talents in University, Ministry of Education of China grant to C.-L.S. (No. NCET11-0472).
■
REFERENCES
(1) Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.; Prinsep, M. R. Nat. Prod. Rep. 2013, 30, 237−323. (2) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2012, 75, 311−335. (3) Numata, A.; Takahashi, C.; Matsushita, T.; Miyamoto, T.; Kawai, K.; Usami, Y.; Matsumura, E.; Inoue, M.; Ohishi, H.; Shingu, T. Tetrahedron Lett. 1992, 33, 1621−1624. (4) Takahashi, C.; Matsushita, T.; Doi, M.; Minoura, K.; Shingu, T.; Kumeda, Y.; Numata, A. J. Chem. Soc., Perkin Trans. 1 1995, 2345− 2353. (5) Belofsky, G. N.; Anguera, M.; Jensen, P. R.; Fenical, W.; Köck, M. Chem.Eur. J. 2000, 6, 1355−1360. (6) Heredia, M. L.; Cuesta, E.; Avendaño, C. Tetrahedron 2002, 58, 6163−6170. (7) Han, X. X.; Xu, X. Y.; Cui, C. B.; Gu, Q. Q. Chin. J. Med. Chem. 2007, 17, 232−237. (8) Afiyatullov, S. S.; Zhuravleva, O. I.; Antonov, A. S.; Kalinovsky, A. I.; Pivkin, M. V.; Menchinskaya, E. S.; Aminin, D. L. Nat. Prod. Commun. 2012, 7, 497−500. (9) Shao, C.-L.; Wu, H.-X.; Wang, C.-Y.; Liu, Q.-A.; Xu, Y.; Wei, M.Y.; Qian, P.-Y.; Gu, Y.-C.; Zheng, C.-J.; She, Z.-G.; Lin, Y.-C. J. Nat. Prod. 2011, 74, 629−633. D
dx.doi.org/10.1021/np4002042 | J. Nat. Prod. XXXX, XXX, XXX−XXX
