Abstract
Methanolic extracts of the Brazilian endemic ascidian Eudistoma vannamei have been extensively studied for their cytotoxic activity against several human cancer cell lines. Previous work reported the occurrence of purine derivatives and staurosporine alkaloids as the major nitrogen-containing compounds. In this study, we report the occurrence of three pyrimidine alkaloids in addition to cholesterol, sitosterol and stigmasterol.
Keywords
Pyrimidine; Tunicate; Steroids; Nucleosides
Introductory remarks
The colonial ascidian Eudistoma vannamei Millar, 1977, an endemic species to the Brazilian Northeast coast, has long been a subject of our marine natural product research group. This, such as many tropical ascidian species, has shown to be a rich and prolific source of biologically active compounds, and publications have been spanning through the specialized literature for over 10 years.
We have previously reported cytotoxicity in the crude extract (Jimenez et al., 2003Jimenez, P.C., Fortier, S.C., Lotufo, T.M.C., Pessoa, C., Moraes, M.E.A., Moraes, M.O., Costa-Lotufo, L.V., 2003. Biological activity in extracts of ascidians (Tunicata, Ascidiacea) from the northeastern Brazilian coast. J. Exp. Mar. Biol. Ecol. 287, 93-101.), induction of cellular apoptosis by derived fractions (Jimenez et al., 2008Jimenez, P.C., Wilke, D.V., Takeara, R., Lotufo, T.M.C., Pessoa, C.O., Moraes, M.O., Lopes, N.P., Costa-Lotufo, L.V., 2008. Preliminary studies on the cytotoxic activity of a dichloromethane extract and fractions obtained from Eudistoma vannamei (Tunicata: Ascidiacea). Comp. Biochem. Physiol. 151A, 391-398.), and the isolation of novel highly cytotoxic staurosporine derivatives - 2-hydroxy-7-oxostaurosporine (9) and 3-hydroxy-7-oxostaurosporine (10) (Jimenez et al., 2012Jimenez, P.C., Wilke, D.V., Ferreira, E.G., Takeara, R., Moraes, M.O., Silveira, E.R., Lotufo, T.M.C., Lopes, N.P., Costa-Lotufo, L.V., 2012. Structure elucidation and anticancer activity of 7-oxostaurosporine derivatives from the Brazilian endemic Tunicate Eudistoma vannamei. Mar. Drugs 10, 1092-1102.). Applying directly crude extracts analysis in electrospray ionization tandem mass spectrometry (ESI-MS/MS) method allowed the identification of four purine derivatives - adenine (4), 2'-deoxyadenosine (5), 2'-deoxynosine (6), and deoxyguanosine (7) (Takeara et al., 2007aTakeara, R., Jimenez, P.C., Costa-Lotufo, L.V., Lopes, J.L.C., Lopes, N.P., 2007. Sample optimization for rapid identification of nucleosides and bases from ascidian extracts using ESI/MS-MS. J. Braz. Chem. Soc. 18, 1054-1060.) and the continuous chemical investigation of this extract afforded other new and unusual amino acid bearing molecules - 9-[N-(leucyl)-isoleucyl]-adenine (1), 8-hydroxy-8-isopentyl-7,8-dihydroadenine (2) and N-[N-(leucyl)-isoleucyl]phenethylamine (3) (Pimenta et al., 2014Pimenta, A.T., Jimenez, P.C., Costa-Lotufo, L.V., Braz-Filho, R., Lima, M.A., 2014. New unusual alkaloids from the ascidian Eudistoma vannamei. Nat. Prod. Commun. 9, 1713-1715.). Moreover, we have prospected the cultivable microorganisms associated to E. vannamei for producers of biologically active compounds and described the recovery of cytotoxic fungi strains, among which an Aspergillus sp. yielding mellein derivatives and penicillic acid (Montenegro et al., 2012Montenegro, T.G.C., Rodrigues, F.A.R., Jimenez, P.C., Angelim, A.L., Maciel, V.M.M., Rodrigues-Filho, E., Oliveira, M.C.F., Costa-Lotufo, L.V., 2012. Cytotoxic activity of fungal strains isolated from the Ascidian. Chem. Biodiver. 9, 2203-2209.). Regarding the bacteria, we have reported some bioactive strains of actinomycetes (Jimenez et al., 2013Jimenez, P.C., Ferreira, E.G., Araújo, L.A., Guimarães, L.A., Sousa, T.S., Pessoa, O.D.L., Lotufo, T.M.C., Costa-Lotufo Leticia, V., 2013. Cytotoxicity of actinomycetes associated with the ascidian Eudistoma vannamei (Millar 1977), endemic of northeastern coast of Brazil. Lat. Am. J. Aquat. Res. 41, 335-343.), a staurosporine-producing Streptomyces sp. (8) which can support the structure block for the final products 9 and 10 isolated in E. vannamei (Andréo et al., 2012Andréo, M.A., Jimenez, P.C., Siebra, J.B.C.N., Costa-Lotufo, L.V., Vessecchi, R., Niehues, M., Lopes, J.L.C., Lopes, N.P., 2012. Systematic UPLC–ESI-MS/MS study on the occurrence of staurosporine and derivatives in associated marine microorganisms from Eudistoma vannamei. J. Braz. Chem. Soc. 23, 335-343.), the isolation of novel anthracyclinones from a strain of Micromonospora sp. (Sousa et al., 2012Sousa, T.S., Jimenez, P.C., Ferreira, E.G., Silveira, E.R., Braz-Filho, R., Pessoa, O.D.L., Costa-Lotufo, L.V., 2012. Anthracyclinones from Micromonospora sp. J. Nat. Prod. 75, 489-493.), and the isolation of an anti-cytokinesis dithiolpyrrolone from Streptomyces sp. (Abreu et al., 2014Abreu, P.A., Sousa, T.S., Jimenez, P.C., Wilke, D.V., Rocha, D.D., Freitas, H.P.S., Pessoa, O.D.L., La Clair, J.J., Costa-Lotufo, L.V., 2014. Identification of pyrroloformamide as a cytokinesis modulator. ChemBioChem 15, 501-506.). In the present short communication we are reporting a continuation to the chemical characterization of active extracts of E. vannamei collected on the coast of Ceará State, Brazil, where this species is highly abundant.
Materials and methods
The NMR spectra were obtained in a Bruker Avance DRX 400. Samples were dissolved in deuterated DMSO and transferred to 5 mm tubes. ESI-MS analysis was carried out in Quatro LC machine (Micromass - Waters). Solutions were infused into the ESI source at 5 μl min−1 using a Harvard Apparatus model 1746 (Holliston, MA) syringe pump. CID fragmentation was performed using argon as collision gas at 25 eV. For steroids and triterpene analysis, a gas chromatograph (GC-MS-QP-2010, Shimadzu) coupled to a quadruple mass spectrometer with electron impact (EI) ionization at 70 eV was applied. Samples were infused to EI machine through the gas chromatograph equipment furnished with an auto sampler AOC-20i and 1 μl split injections 1/10 were performed at 250 °C. Separations were carried out on an HP1 column (30 m × 0.25 mm × 0.25 μm film thickness). The oven temperature program for separation conditions was 200 °C for 5 min followed by temperature increases to 280 °C at 5 °C min−1 and 280 °C for 30 min. The carrier gas was ultrapure helium (grade 5.5) at constant flow rates of 1.10 ml min−1. The spectra of all metabolites were compared with Wiley Mass Spectral Database and with authentic samples from NPPNS (Núcleo de Pesquisas em Produtos Naturais e Sintéticos) standards. For classical CC (column chromatography) and TLC (thin layer chromatography), silica gel 60 (70-230 mesh ASTM, Sigma) and silica gel GF254 (Merck) were used, respectively. Finally, for pyrimidine semi-preparative purification in HPLC (Shimadzu LC-6A, with UV-detector SPD-6A) was applied Shimpack ODS-C column (20 mm × 250 mm) and the solvent system flow was 9.7 min/ml in MeOH/H2O gradiente.
Results and discussion
Chromatography fractionation of the methanolic extracts afforded several of the secondary metabolites previously isolated and structures were confirmed by TLC or ESI-MS data in negative mode. Fraction 16 (from a total of twenty polled fractions) showed signals relative to [M−H]− of 11 (m/z 241), 12 (m/z 227) and 13 (m/z 243) in ESI-MS. Our previous investigation with the ascidian Didemnum psammatodes resulted in the isolation and identification of same three nucleosides in addition to 2'-deoxyinosine and 2'-deoxyguanosine (Takeara et al., 2007bTakeara, R., Lopes, J.L.C., Lopes, N.P., Jimenez, P.C., Costa-Lotufo, L.V., Lotufo, T.M.C., 2007. Constituintes químicos da ascídia Didemnum psammatodes (Sluiter, 1895) coletada na costa cearense. Quim. Nova 30, 1179-1181.). Seeing confirmation of such structures, fraction 16 (8 mg) was submitted to purification by HPLC and pure samples were submitted to NMR analysis. 1H NMR and 13C NMR (DMSO, 400 MHz) spectra of 11 exhibited the signals: δH 7.7 (brd, H-6), 6.16 (s, H-1'), 4.20 (s, H-3'), 3.76 (m, H-4'), 3.56 (m, H-5'), 2.07 (m, H-2'), 1.77 (s, CH3) and δC 164.0 (C-4), 151.2 (C-2), 136.4 (C-6), 109.4 (C-5), 88.5 (C-4'), 84.4 (C-1'), 71,3 (C-3'), 62.1 (C-5'), 40.0 (C-2'), 12.7 (s, CH3). This data, in addition with the deprotonated ion, are in agreement with thymidine (Pouchert and Behnke, 1993Pouchert, C.J., Behnke, J., 1993. The Aldrich Library of 13C and 1H FT NMR Spectra . Aldrich Chemical Company vol. 3). 1H NMR and 13C NMR (DMSO, 400 MHz) spectra of 12 exhibited the signals: δH 7.86 (d, J = 8.0, H-6), 6.16 (brd, H-1'), 5.63 (d, J = 8.0, H-5), 4.21 (s, H-3'), 3.77 (m, H-4'), 3.55 (m, H-5'), 2.07 (m, H-2') and δC 163.0 (C-4), 150.0 (C-2), 139.0 (C-6), 101.4 (C-5), 86.5 (C-4'), 83.0 (C-1'), 70,3 (C-3'), 61.0 (C-5'), 40.0 (C-2'). This data, in addition with the deprotonated ion are, in agreement with NMR data from Pouchert and Behnke (1993)Pouchert, C.J., Behnke, J., 1993. The Aldrich Library of 13C and 1H FT NMR Spectra . Aldrich Chemical Company vol. 3 for 2'-deoxyuridine. Compound 13, uridine, also has similar 1H NMR (DMSO, 400 MHz) spectra to our previous work with D. psammatodes (Takeara et al., 2007a) and previously published data (Kitajima et al., 1999Kitajima, J., Ishikawa, T., Tanaka, Y., Ida, Y., 1999. Water-soluble constituents of fennel. IX. glucosides and nucleosides. Chem. Pharm. Bull. 47, 988-992.): δH 7.88 (d, J = 8.1, H-6), 5.77 (d, J = 5.2, H-1'), 5.64 (d, J = 8.1, H-5), 3.96 (m, H-3'), 3.84 (m, H-4'), 3.58 (m, H-5'), 4.02 (m, H-2'). Finally, CG-MS analysis of apolar fractions allowed the identification of a mixture of steroids containing cholesterol, sitosterol and stigmasterol. The occurrence of plant steroids in the extract of this filter-feeding invertebrate can be related with the presence of plankton in the ascidian's diet (Takeara et al., 2007aTakeara, R., Jimenez, P.C., Costa-Lotufo, L.V., Lopes, J.L.C., Lopes, N.P., 2007. Sample optimization for rapid identification of nucleosides and bases from ascidian extracts using ESI/MS-MS. J. Braz. Chem. Soc. 18, 1054-1060.).
In this work, pyrimidine compounds were reported for the first time in E. vannamei, however they have been previously reported in the following ascidians: Aplidium pantherinum (Kim et al., 1993Kim, J., Pordesimo, E.O., Toth, S.I., Schmitz, F.J., Altena, I.V., 1993. Pantherinine, a cytotoxic aromatic alkaloid, and 7-deazainosine from the Ascidian Aplidium pantherinum. J. Nat. Prod. 56, 1813-1816.); Atriolum robustum (Kehraus et al., 2004Kehraus, S., Gorzalka, S., Hallmen, C., Iqbal, J., Müller, C.E., Wright, A.D., Wiese, M., König, G.M., 2004. Novel amino acid derived natural products from the ascidian Atriolum robustum: identification and pharmacological characterization of a unique adenosine derivative. J. Med. Chem. 47, 2243-2255.); an unidentified Eudistoma sp. (Schupp et al., 2003Schupp, P., Pochner, T., Edrada, R., Ebel, R., Berg, A., Wray, V., Proksch, P., 2003. Eudistomins W and X, two new beta-carbolines from the micronesian tunicate Eudistoma sp. J. Nat. Prod. 66, 272-275.); Didemnun spp. (Takeara et al., 2007aTakeara, R., Jimenez, P.C., Costa-Lotufo, L.V., Lopes, J.L.C., Lopes, N.P., 2007. Sample optimization for rapid identification of nucleosides and bases from ascidian extracts using ESI/MS-MS. J. Braz. Chem. Soc. 18, 1054-1060.; Mitchell et al., 1996Mitchell, S.S., Pomerantz, S.C., Concepción, G.P., Ireland, C.M., 1996. Tubercidin analogs from the ascidian Didmnum voeltzkowi. J. Nat. Prod. 59, 1000-1001.); Trididemnum cereum (Demattè et al., 1985Demattè, N., Guerriero, A., De Clauser, R., De Stanchina, G., Lafargue, F., Cuomo, V., Pietra, F., 1985. A screening of some colonial Ascidiacea of Banyuls-sur-Mer for antibacterial and antifungal activities and, preliminarily, for natural products: 2′-deoxyribonucleosides from Trididemnum cereum (Giard, 1872). Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 81, 479-484.). The occurrence of thymidine, 2'-deoxyuridine and uridine are in agreement with observed bioactivity for the extract of E. vannamei. As a matter of fact, marine nucleosides are well known for their cytotoxic activity and have been engineered into drugs to treat viral and parasitical infections, as well as cancer (Huang et al., 2014Huang, R.-M., Chen, Y.-N., Zeng, Z., Gao, C.-H., Su, X., Peng, Y., 2014. Marine nucleosides: structure, bioactivity, synthesis and biosynthesis. Mar. Drugs 12, 5817-5838.). Notably, sponge pyrimidine arabinonucleosides isolated from Crytothetya crypta (Bergmann and Feeney, 1951Bergmann, W., Feeney, R.J., 1951. Contributions to the study of marine products. XXXII. The nucleosides of sponges. J. Org. Chem. 116, 981-987.) were developed into cytarabine (ARA-C) (Evans et al., 1961Evans, J.S., Musser, E.A., Mengel, G.D., Forsblad, K.R., Hunter, J.H., 1961. Antitumor activity of 1-beta-D-arainofuranosylcytosine hydrochloride. Exp. Biol. Med. 106, 350-353.) and vidarabine (ARA-A) (Privat de Garilhe and De Rudder, 1964Privat de Garilhe, M., De Rudder, J., 1964. Effect of 2 arabinose nucleosides on the multiplication of herpes virus and vaccine in cell culture. C. R. Hebd. Seances Acad. Sci. 259, 2725-2728.) in the early 1970s while transforming the existing logic applied to rational design of unnatural nucleosides. Cytotoxicity of such molecules are mostly a consequence of their capacity of misleading DNA-polymerase in to inserting a false, although similar, substrate in the newly synthetized DNA chain.
Naturally occurring nucleosides remain of great interest to scientists and industry alike, as these compounds have served as a prototype to a considerable number of pharmaceuticals, while enlightening the mechanisms of fundamental cellular processes. Marine organisms, including the ascidian E. vannamei chemically addressed herein, may be a promising source of lead molecules to new bioactive compounds of this class.
Acknowledgements
This work was supported by grants from the Brazilian funding agencies CNPq and FAPESP.
References
- Abreu, P.A., Sousa, T.S., Jimenez, P.C., Wilke, D.V., Rocha, D.D., Freitas, H.P.S., Pessoa, O.D.L., La Clair, J.J., Costa-Lotufo, L.V., 2014. Identification of pyrroloformamide as a cytokinesis modulator. ChemBioChem 15, 501-506.
- Andréo, M.A., Jimenez, P.C., Siebra, J.B.C.N., Costa-Lotufo, L.V., Vessecchi, R., Niehues, M., Lopes, J.L.C., Lopes, N.P., 2012. Systematic UPLC–ESI-MS/MS study on the occurrence of staurosporine and derivatives in associated marine microorganisms from Eudistoma vannamei J. Braz. Chem. Soc. 23, 335-343.
- Bergmann, W., Feeney, R.J., 1951. Contributions to the study of marine products. XXXII. The nucleosides of sponges. J. Org. Chem. 116, 981-987.
- Demattè, N., Guerriero, A., De Clauser, R., De Stanchina, G., Lafargue, F., Cuomo, V., Pietra, F., 1985. A screening of some colonial Ascidiacea of Banyuls-sur-Mer for antibacterial and antifungal activities and, preliminarily, for natural products: 2′-deoxyribonucleosides from Trididemnum cereum (Giard, 1872). Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 81, 479-484.
- Evans, J.S., Musser, E.A., Mengel, G.D., Forsblad, K.R., Hunter, J.H., 1961. Antitumor activity of 1-beta-D-arainofuranosylcytosine hydrochloride. Exp. Biol. Med. 106, 350-353.
- Huang, R.-M., Chen, Y.-N., Zeng, Z., Gao, C.-H., Su, X., Peng, Y., 2014. Marine nucleosides: structure, bioactivity, synthesis and biosynthesis. Mar. Drugs 12, 5817-5838.
- Jimenez, P.C., Ferreira, E.G., Araújo, L.A., Guimarães, L.A., Sousa, T.S., Pessoa, O.D.L., Lotufo, T.M.C., Costa-Lotufo Leticia, V., 2013. Cytotoxicity of actinomycetes associated with the ascidian Eudistoma vannamei (Millar 1977), endemic of northeastern coast of Brazil. Lat. Am. J. Aquat. Res. 41, 335-343.
- Jimenez, P.C., Fortier, S.C., Lotufo, T.M.C., Pessoa, C., Moraes, M.E.A., Moraes, M.O., Costa-Lotufo, L.V., 2003. Biological activity in extracts of ascidians (Tunicata, Ascidiacea) from the northeastern Brazilian coast. J. Exp. Mar. Biol. Ecol. 287, 93-101.
- Jimenez, P.C., Wilke, D.V., Ferreira, E.G., Takeara, R., Moraes, M.O., Silveira, E.R., Lotufo, T.M.C., Lopes, N.P., Costa-Lotufo, L.V., 2012. Structure elucidation and anticancer activity of 7-oxostaurosporine derivatives from the Brazilian endemic Tunicate Eudistoma vannamei Mar. Drugs 10, 1092-1102.
- Jimenez, P.C., Wilke, D.V., Takeara, R., Lotufo, T.M.C., Pessoa, C.O., Moraes, M.O., Lopes, N.P., Costa-Lotufo, L.V., 2008. Preliminary studies on the cytotoxic activity of a dichloromethane extract and fractions obtained from Eudistoma vannamei (Tunicata: Ascidiacea). Comp. Biochem. Physiol. 151A, 391-398.
- Kehraus, S., Gorzalka, S., Hallmen, C., Iqbal, J., Müller, C.E., Wright, A.D., Wiese, M., König, G.M., 2004. Novel amino acid derived natural products from the ascidian Atriolum robustum: identification and pharmacological characterization of a unique adenosine derivative. J. Med. Chem. 47, 2243-2255.
- Kim, J., Pordesimo, E.O., Toth, S.I., Schmitz, F.J., Altena, I.V., 1993. Pantherinine, a cytotoxic aromatic alkaloid, and 7-deazainosine from the Ascidian Aplidium pantherinum J. Nat. Prod. 56, 1813-1816.
- Kitajima, J., Ishikawa, T., Tanaka, Y., Ida, Y., 1999. Water-soluble constituents of fennel. IX. glucosides and nucleosides. Chem. Pharm. Bull. 47, 988-992.
- Mitchell, S.S., Pomerantz, S.C., Concepción, G.P., Ireland, C.M., 1996. Tubercidin analogs from the ascidian Didmnum voeltzkowi J. Nat. Prod. 59, 1000-1001.
- Montenegro, T.G.C., Rodrigues, F.A.R., Jimenez, P.C., Angelim, A.L., Maciel, V.M.M., Rodrigues-Filho, E., Oliveira, M.C.F., Costa-Lotufo, L.V., 2012. Cytotoxic activity of fungal strains isolated from the Ascidian. Chem. Biodiver. 9, 2203-2209.
- Pimenta, A.T., Jimenez, P.C., Costa-Lotufo, L.V., Braz-Filho, R., Lima, M.A., 2014. New unusual alkaloids from the ascidian Eudistoma vannamei Nat. Prod. Commun. 9, 1713-1715.
- Pouchert, C.J., Behnke, J., 1993. The Aldrich Library of 13C and 1H FT NMR Spectra . Aldrich Chemical Company vol. 3
- Privat de Garilhe, M., De Rudder, J., 1964. Effect of 2 arabinose nucleosides on the multiplication of herpes virus and vaccine in cell culture. C. R. Hebd. Seances Acad. Sci. 259, 2725-2728.
- Schupp, P., Pochner, T., Edrada, R., Ebel, R., Berg, A., Wray, V., Proksch, P., 2003. Eudistomins W and X, two new beta-carbolines from the micronesian tunicate Eudistoma sp. J. Nat. Prod. 66, 272-275.
- Sousa, T.S., Jimenez, P.C., Ferreira, E.G., Silveira, E.R., Braz-Filho, R., Pessoa, O.D.L., Costa-Lotufo, L.V., 2012. Anthracyclinones from Micromonospora sp. J. Nat. Prod. 75, 489-493.
- Takeara, R., Jimenez, P.C., Costa-Lotufo, L.V., Lopes, J.L.C., Lopes, N.P., 2007. Sample optimization for rapid identification of nucleosides and bases from ascidian extracts using ESI/MS-MS. J. Braz. Chem. Soc. 18, 1054-1060.
- Takeara, R., Lopes, J.L.C., Lopes, N.P., Jimenez, P.C., Costa-Lotufo, L.V., Lotufo, T.M.C., 2007. Constituintes químicos da ascídia Didemnum psammatodes (Sluiter, 1895) coletada na costa cearense. Quim. Nova 30, 1179-1181.
Publication Dates
-
Publication in this collection
Dec 2015
History
-
Received
2 July 2015 -
Accepted
9 Aug 2015