versión impresa ISSN 0103-5053
J. Braz. Chem. Soc. vol.22 no.6 São Paulo jun. 2011
Arturo CanoI; María Teresa Ramírez-ApanII; Guillermo DelgadoII,*
IFacultad de Estudios Superiores Zaragoza, Universidad Nacional Autónoma de México, Av. Guelatao nº. 66 (Eje 7 Oriente), Col Ejército de Oriente, Iztapalapa 09230, Mexico, D.F
IIInstituto de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, Circuito Exterior, Coyoacán 04510, Mexico, D.F
Sclareolide (1) was incubated with eight different species of filamentous fungi conventionally used for bio-oxidations. Compound 1 was metabolized with Aspergillus niger in medium A to yield 3-ketosclareolide (2) and 3β-hydroxysclareolide (4), while in medium B (containing major number of nutrients with respect to medium A), compounds 2, 4, 3α,6β-dihydroxysclareolide (16), 1-ketosclareolide (17), 3-keto-15-hydroxysclareolide (18) and 3β,15-dihydroxysclareolide (19) were obtained. The biotransformation products 16-19 were found to be new substances. Fermentation of 1 with Cunninghamella blackesleeana using medium A afforded 2 and 4, while using medium B yielded 2, 4, 16 and 17. Compounds 2, 4 and 17 were also obtained with Curvularia lunata. Biotransformation of 1 with Beauveria bassiana yielded 4 in satisfactory yield, with Rhizopus oligosporus and Mucor miehei afforded 2 and 4, while with R. nigricans and Fusarium moliniforme yielded 2, 4 and 16. Cytotoxic evaluation of 1 and the obtained products against selected human cancer cell lines (U251, PC-3, K562, HCT-15, MCF-7 and SKUL-1) indicated that 16 (3a,6b-dihydroxysclareolide) displayed moderate cytotoxic (IC50 < 100 mM) against U251, PC-3, HCT-15 and MCF-7.
Keywords: biotransformation, sclareolide, filamentous fungi, microbiological oxidation
O esclareolido (1) foi incubado com oito diferentes espécies de fungos filamentosos usados convencionalmente para bio-oxidações. O composto 1 metabolizado pelo fungo Aspergilus niger em um meio de cultura A forneceu o 3-cetoesclareolido (2) e 3β-hidroxiesclareolido (4). Quando em um meio de cultura B (mais rico em nutrientes em relação ao meio de cultura A), foram obtidos os compostos 2, 4, e ainda 3α,6β-diidroxiesclareolido (16), 1-cetoesclareolido (17), 3-ceto-15-hidroxiesclareolido (18) e 3β,15-diidroxiesclareolido (19). Os produtos 16-19 resultantes da biotransformação de 1 são relatados como substâncias inéditas. A fermentação de 1 com Cunninghamella blackesleeana usando o meio de cultura A forneceu os compostos 2 e 4, enquanto que empregando o meio de cultura B, forneceu os compostos 2, 4, 16 e 17. Os compostos 2, 4 e 17 foram obtidos também com Curvularia lunata. A biotransformação de 1 com Beauveria bassiana forneceu o composto 4 com rendimento satisfatório; com Rhizopus oligosporus e com Mucor miehei forneceu os compostos 2 e 4, enquanto que com R. nigricans and Fusarium moliniforme os compostos 2, 4 e 16 foram obtidos. A avaliação dos efeitos citotóxicos do composto 1 e dos produtos obtidos frente as linhagens de células cancerosas humanas selecionadas (U251, PC-3, K562, HCT-15, MCF-7 e SKUL-1) indicaram que o composto 16 (3α,6β- diidroxiesclareolido) apresenta um efeito citotóxico moderado (IC50 < 100 mM) contra a U251, a PC-3, a HCT-15 e a MCF-7.
The use of natural catalysts is now considered one of the most valuable routes for the synthesis of fine chemicals employing ecologically competitive procedures, particularly at industrial scale.1-3 The selectivity and mildness of the biotransformations can be considered as advantages to similar, chemical-based methods4-6 and this is evident for the selective oxidation of non-activated carbon atoms which is difficult to achieve by classical organic chemistry. Biotransformations are typically carried out using either whole cells or isolated enzymes. Although in recent years some enzymes responsible for fungal hydroxylation have been isolated, whole-cell fermentation is the technique most often employed.7-9 A major challenge for the transformations using biocatalytic methods is to determine the appropriate microorganism and conditions, so, we proceeded with the screening using different fungal strains and media, with sclareolide (1, Figure 1) as substrate and then the processes were scaled up. In addition and in the search of new bioactive agents from natural products,10,11 the cytotoxic evaluation against several human tumor cell lines of 1 and the obtained products (2, 4, 16-19) were carried up. Here we report our results.
Sclareolide (1) is a natural product isolated from several plant species which displays antifungal,12 phytotoxic13 and cytotoxic14 activities against several human tumor cell lines. This compound has also been used as starting material for the synthesis of various bioactive natural products.15,16 Regarding the biotransformation of sclareolide (1) it has been previously reported that the incubation of 1 with Mucor plumbeus afforded 3-ketosclareolide (2), 1β-hydroxysclareolide (3) and 3β-hydroxysclareolide (4).17 The bioconversion of 1 with Cephalosporium aphidicola gave 2, 4 and 3b,6b-dihydroxysclareolide (5).18 The microbial transformation of 1 by Curvularia lunata yielded 2-4, 1α,3β-hydroxysclareolide (6) and 1β,3β‑dihydroxysclareolide (7).19 The incubation of sclareolide with Cunninghamella elegans afforded 2, 4, 6 and 8-10.20 Cunninghamella blackesleeana metabolized compound 1 to afford 2, 5, 7 and 11-13. Biotransformation of 1 with C. echinulata yielded 5-hydroxysclareolide (14) and 7β-hydroxysclareolide (15).21
Results and Discussion
A preliminary screening and preparative bioconversion of 1 with eight fungal species showed a widespread capacity to transform the starting material into more polar products in different yields (Table 1), which were separated by vacuum column chromatography.22,23
The crude organic extract obtained from incubation of sclareolide (1) with A. niger in culture medium A was separated by silica gel chromatographed to yield the known metabolites 2 and 4. However, when the same microorganism was grown in culture medium B (containing major number of nutrients with respect medium A), compounds 2, 4 and four new metabolites: 16-19 were obtained. Compound 2 was identified as the 3-ketosclareolide by comparison of its spectral data with those reported in the literature.17,19 The 1H NMR spectrum of 4 (Table 2) revealed a signal at δH 3.27 (dd, 3J 11, 5 Hz, 1H,) indicative of an axial proton at C-3 (δC 78.6). The location of the hydroxyl group at C-3 was confirmed on the basis of HMBC experiments and this compound was identified as 3β-hydroxysclareolide (4).17,19,20 Unambiguous and detailed 1H NMR assignments (in comparison with previous reports17) are shown in Table 2.
For compound 16, the HRFABMS established a molecular formula of C16H26O4 [M+H]+, at m/z 283.1915, calculated for (C16H26O4 + H) 283.1909. The 1H NMR spectrum revealed two signals at δH 3.37 (dd, 3J 3, 3 Hz, 1H) and δH 4.70 (ddd, 3J 3, 3, 3 Hz, 1H), indicative of two equatorial hydrogens at C-3 (δC 72.6) and C-6 (δC 69.0), respectively. The locations of the hydroxyl groups at C-3 and C-6 in 16 were determined on the basis of HMBC experiments, in particular by the 16CH3 (δH 1.21) and the 15CH3 (δH 1.04) crosspeaks with C(3) (δC 72.6) and the crosspeaks between H-6 (δH 4.70) and C-4(δC 34.0), C-5 (δC 50.3), C-10 (δC 39.7), C-7 (δC 46.7), and C-8 (δC 86.3). COSY, HMQC and NOESY experiments allowed complete assignments for all protons and carbons (Table 2) and the structure was confirmed as 3α,6β-dihydroxysclareolide (16). The epimer at C(3) has been reported previously.18
The new metabolite 17 was obtained as a white crystalline solid. Its HRFABMS exhibited [M+H]+ at m/z 265.1805, corresponding to the molecular formula C16H25O3 (calculated for C16H24O3 + H: 265.1804). The IR spectral data displayed absorptions for γ-lactone (1774 cm-1) and ketone (1708 cm‑1). These data were similar to those for compound 2.17,18 The 13C NMR data (Table 2) showed resonances for sixteen carbons and the DEPT experiments established the multiplicity for each carbon signal and they revelead the presence of four methyls, five methylenes, two methines and five quaternary carbons. The 1H NMR (500 MHz) data of 17 (Table 2) showed singlets at δH 1.35, 1.19, 1.03 and 1.06 which were assigned to 13CH3, 14CH3, 15CH3 and 16CH3, respectively, by direct comparison with similar compounds. In particular, 13CH3 is located downfield (δH 1.35) due to it is linked to the carbon closing the γ-lactone (C-8) and the signal at δH 1.19, which showed NOESY crosspeak with H-11β (δH 2.96) and HMBC crosspeak with the signal at δC 52.00 (C-9) was assigned to 14CH3. The location of the ketone at C-1 was determined by the HMBC crosspeak of the signal at δH 1.19 (14CH3) with the resonance at δC 214.1. Assignments for all hydrogens and carbons were done by COSY, HMQC, HMBC and NOESY experiments.
Compound 18 was obtained as white crystalline solid and its IR spectral data displayed absorptions at 3459 (O-H), 1773 (C=O) and 1174 (C-O) cm-1. The HREIMS exhibited [M+H]+ at m/z 281.1751 establishing the molecular formula C16H24O4 (calculated for C16H24O4 + H: 281.1753). The 13C NMR spectrum of 18 (DEPT experiment) displayed resonances for sixteen carbon atoms, including three methyls, six methylenes, two methines and five quaternary carbons. Its 1H NMR spectrum (CDCl3, 500 MHz) showed three singlets at δH 1.41, 1.13 and 1.00 due the H-13, H-14 and H-16 methyls, respectively, due to the observed NOESY crosspeaks between C-13 and C-14 and between C-14 with C-16. A notable difference with the 4,4'-dimethyl products was the absence of one methyl group and the presence of an AB system (δH 3.76 and 3.39, 2J 11 Hz), which was assigned to the methylene protons at C-15 (δC 66.6). The signal at δC 216.1 corresponds to a ketone carbonyl located at C-3 due to HMBC correlations of this carbon with the AB system (H-15a and H-15b), H-2bax (δH 2.67), H-2aec (δH 2.42), H-1bec (δH 1.74) and with 16CH3 (δH 1.00). The 1H and 13C NMR chemical shifts assigments for compound 18 (Table 2), confirmed this compound as 3-keto-15-hydroxysclareolide.
The other novel biotransformation product, 19, was a crystalline solid. The IR spectral data displayed absorptions at 3489 (O-H) and 1767 (C=O) cm-1. The HREIMS of 19 exhibited a molecular ion m/z 283.1908 corresponding to the molecular formula C16H26O4 (calculated for C16H26O4 + H: 283.1909). The 13C NMR spectrum of 19 showed resonances for sixteen carbons including three methyls, six methylenes, three methines and four quaternary carbons. The 1H NMR data of 19 (Table 2) showed singlets at δH 1.34, 0.97 and 0.88, assigned to 13CH3, 14CH3, 16CH3, respectively, by comparison with the above described substances. Additionally, the signals at δH 3.72 and 3.43 (2J 10, 5 Hz) established the presence of an AB system for the hydroxymethylene protons located at C-15 (δC 70.9) which overlapped with a doublet of doublet signal corresponding to H-3aax (δH 3.70, 3J 10, 5 Hz). The signal at δC 75.4, assigned to C-3, showed HMBC correlations with the AB system for H-15a and H-15b, and with 16CH3 (δH 0.88). Assignments for all hydrogens and carbons for compound 19 were done by COSY, HMQC, HMBC and NOESY experiments and verified its structure as 3β,15-dihydroxysclareolide (Table 2).
An interesting variety of products and yields were obtained by the biotransformation of sclereolide (1) with different fungi. The transformation of 1 with A. niger in medium A (containing minor number of nutrients) produced 2 and 4, while using medium B yielded six compounds (2, 4, 16-19) in variable yields (from 5.9% to 27.1%), affording a keto group at C-1 (17) for the first time. Using medium B with A. niger, C. blackesleeana and C. lunata yielded various products in relatively variable yields, while medium A produced fewer products, with better yields though (see Table 1). The transformation of 1 with B. bassiana afforded exclusively product 4 in acceptable yield (51.3%), in comparison with other biotransformations.24 These results gave additional evidences of the importance of the species, strains and media employed in the structural diversity of the products and the yields of the biotransformations.
Sclareolide (1) and derivatives 2, 4, 16-19 were tested for in vitro cytotoxic activity against human cancer cell lines U251 (central nervous system), PC-3 (prostate cancer), K562 (leukemia), HCT-15 (colon), MCF-7 (breast) and SKUL-1 (lung) following standard procedures.25 Compound 1 displayed activity against PC-3 tumor cell line (IC50 71.12 ± 4.7 µM) and 16 displayed activity against U251 (IC50 87.40 ± 5.4 µM), PC-3 (IC50 34.47 ± 7.4 µM), HCT-15 (IC50 92.23 ± 4.2 µM), MCF-7 (IC50 89.11 ± 2.4 µM). All the other IC50 values were above a 100 µM and were considered not active.25 It is interesting that the presence of the hydroxyl groups at C-3α and C-6β increased the cytotoxicity with respect to the starting material (1).
General experimental procedures
Melting points were determined on a Fischer-Jones apparatus. Optical rotation were measured on a Perkin-Elmer 341 polarimeter. Infrared spectra are registred Nicolet Magna FT-IR 750 spectrometer. 1H and 13C NMR spectra were taken on a Varian Unity-plus 500 (at 500 and 125 MHz) instrument. EI-MS: Jeol JMS-AX505HA mass spectrometer and Jeol JMS-SX 102 A) for HREIMS. TLC spots were revealed by spraying with ceric ammonium sulfate, followed by heating. Vacuum column chromatographies were done following the reported procedures.22,23 Column chromatography (CC) were performed using silica gel 70-230, TLC using silica gel 60F254 (Merck) plates and preparative TLC using silica gel 60F254 (Merck) Plates. Sclareolide was purchased from Sigma-Aldrich.
A. niger (ATCC 16404), B. bassiana (ATCC 13144), C. blackesleeana (ATCC 8688a), C. lunata (ATCC 13432), F. moliniforme (ATCC 10209), M. miehei (ATCC 16457), R. oligosporus (ATCC 22959), R. nigricans (ATCC 6227b), were obtained from the Instituto de Biotecnología, Universidad Nacional Autónoma de México, maintained on potato dextrose agar (PDA) and stored at 4 ºC.
Media and culture conditions
The media were prepared by mixing the following ingredients in 1 L of distilled water. Medium A (YEPGA): (10 g) peptone, (10 g) yeast extract, (10 g) beef extract and (50 g) glucose. The pH was adjusted to 7 (NaOH 1 mol L-1) before autoclaving. Medium B was prepared by mixing the following ingredients: glucose (10 g), glycerol (10 g), peptone (5 g), yeast extract (5 g), KH2PO4 (5 g) and NaCl (5 g) at pH 7 (adjusted with NaOH 1 mol L-1) for A. niger, C. blackesleeana, C. lunata.
Firstly, the eight fungi were tested for their ability to metabolize 1 on the analytical scale (10 to 15 mg of substrate, 25 mL medium culture), and then the same biotransformation experiments were carried out on preparative scale (150 to 310 mg). Both analytical and preparative biotransformations were conducted following similar procedures. Erlenmeyer flasks (250 mL) containing 125 mL of medium A or the medium B were inoculated with a dense suspension (2 mL) of the corresponding fungi. Incubations were maintained at 25 ºC with gyratory shaking (125 rpm) for 24 h (A. niger, R. nigricans, R. oligosporus, M. miehei), 48 h (F. moniliforme) or 72 h (C. blackesleeana, C. lunata, B. bassiana). Then, the substrates in acetone (5-10 mL) were added and the process continued for 14 days. These biotransformation experiments were monitored by TLC, including two controls, a "culture control" and a "substrate control" to eliminate the possibility that the isolated products were microbial secondary metabolites and/or that the culture media did not perform any chemical transformation on the substrate.
Recovery and purification of metabolites
Cultures were filtered and fungal cells were washed thoroughly with water and the filtrate and washings were combined, saturated with NaCl and extracted with CH2Cl2 (three times). The organic extracts were combined, dried with Na2SO4 and evaporated under reduced pressure. The organic residues were subjected to chromatography by VCC using gradient elution system with hexane/EtOAc. CC and preparative TLC allowed the final purification of the compounds.
Biotransformation of 1 by A. niger in medium A
After 24 h of inoculation with a dense suspension of the spores of A. niger, 20 erlenmeyer flask cultures (YEPGA) received 204.3 mg of sclareolide (1) in 10 mL of acetone. After incubation for 14 days the cultures were processed as indicated above to yield a crude dark oily residue (194.3 mg). Column chromatography of the organic extract yielded 3-ketosclareolide (2, 44.2 mg, 22.7%), 3b-hydroxysclareolide (4, 72.7 mg, 37.4%) and sclareolide (1, 12.6 mg, 6.5%).
Biotransformation of 1 by A. niger in medium B
The substrate 1 (210 mg) was dissolved in acetone (10 mL), distributed among 20 erlenmeyer flask cultures (medium B), previously (24 h) inoculated with a dense suspension of spores of A. niger. The biotransformation was allowed to proceed for 14 days and the cultures were processed as indicated above to obtain starting material 1 (6 mg, 2.8%), 2 (24.5 mg, 11.7%), 4 (58.2 mg, 27.7%), 16 (22.4 mg, 5.9%), 17 (21.2 mg, 10.1%) 18 (26 mg, 12.4%) and 19 (19.7 mg, 9.4%).
3α,6β-Dihydroxysclareolide (16): mp.188-190ºC; [a]25D + 30.3 (c 0.30, CHCl3); IR nmax/ cm-1 (CHCl3): 3618, 2933, 1758; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz): see Table 2; HRFABMS [M+H]+ Found: 283.1915. Calc. for C16H26O4+H: 283.1909; EIMS m/z 267 (24%), 249 (56), 169 (83), 43 (100).
1-Ketosclareolide (17): mp. 152-153 ºC; [α]25D + 83.8 (c 0.11, CHCl3); IR νmax/cm-1 (CHCl3): 2958, 1774, 1708, 1232, 1199, 925. 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz): see Table 2; HRFABMS [M+H]+ Found: 265.1805. Calc. for C16H24O3+H: 265.1804; EIMS m/z 264 (17%), 205 (86), 55(69), 43 (100).
3-Keto-15-hydroxysclareolide (18): mp. 169-171 ºC; [α]25D + 29.3 (c 0.20, MeOH); IR νmax/cm-1 (CHCl3): 3459, 2985, 2952, 1773, 1698, 1431, 1234, 1174, 920; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz): see Table 2; HRFABMS [M+H]+ Found: 281.1751. Calc. for C16H24O4+H: 281.1753); EIMS m/z 280 (12%), 278 (36), 277 (11), 250 (67), 235 (48), 81 (41), 43 (100).
3β-15-Dihydroxysclareolide (19): mp. 157-158 ºC; [α]25D + 49.2 (c 0.13, MeOH); IR νmax/cm-1 (CHCl3): 3489, 2944, 1767, 1603,1457, 1349, 1243, 920. 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz): see Table 2. HRFABMS [M+H]+ Found: 283.1908. Calc. for C16H26O4+H: 283.1909; EIMS m/z 282 [M+, 20%], 264 [M+H2O, 41], 251 (57), 233 (56), 181 (12), 173 (30) 147 (69), 121 (64), 93 (52), 81 (41), 43 (100), 18 (36).
Biotransformation of 1 by C. blackesleeana in medium B
The substrate 1 (307.5 mg) was dissolved in acetone (15 mL), distributed among 20 erlenmeyer flask cultures (medium B), previously inoculated (72 h) with a dense suspension of spores of C. blackesleeana. The fermentation was allowed to proceed for 14 days, the cultures were processed as indicated above to obtain starting material 1 (16.3 mg, 5.3%), 2 (22.3, 7.2%), 4 (63.4 mg, 20.6%), 3α,6β-dihydroxysclareolide (16, 86.9 mg, 28.3%) and 1-ketosclareolide (17, 26.7 mg, 8.7%).
Biotransformation of 1 by C. lunata, B. bassiana, R. oligosporus, R. nigricans, M. miehei and F. moliniforme
Compound 1 (150 mg) was reacted with the microorganisms following the procedure described above, obtaining the results shown in Table 1.
Human tumor cell lines of central nervous system (U251), prostate cancer (PC-3), leukemia (K562), colon (HCT‑15), breast (MCF-7), lung (SKLU-1) were supplied by the National Cancer Institute (NCI). The cytotoxic activities of 1, 2, 4, 16-19 were determined using the protein-binding dye sulforhodamine B in a microculture assay to measure cell growth, following the protocols described in the literature.18 Results were expressed as concentration giving 50% inhibition (IC50). The IC50 values (mean ± standard error) were 100 mM, and those with minor values are reported in the text. The positive control was adriamycin (IC50 = 0.32 ± 0.02 mM against U251).
Supplementary information data are available free of charge at http://jbcs.org.br as a PDF file.
The autors thank Rocío Patiño, Beatriz Quiroz, Angeles Peña, María Isabel Chávez, Luis Velasco, Javier Pérez and Antonio Nieto from the Instituto de Química, UNAM for technical assistence. Financial supports from Consejo Nacional de Ciencia y Tecnología (Project 102158), Dirección General de Asuntos del Personal Académico, UNAM and Carrera de Biología, Facultad de Estudios Superiores Zaragoza, UNAM, are gratefully acknowledged.
1. Panke, S.; Wubbolts, M.; Curr. Opin. Chem. Biol. 2005, 9, 188. [ Links ]
2. Huisman, G. W.; Gray, D.; Curr. Opin. Biotechnol. 2002, 13, 352. [ Links ]
3. Straathof, A. J. J.; Panke, S.; Schmid, A.; Curr. Opin. Biotechnol. 2002, 13, 548. [ Links ]
4. Davis, B. G.; Boyer, V.; Nat. Prod. Rep. 2001, 18, 1669. [ Links ]
5. De Raadt, A.; Herfried, G.; Curr. Opin. Biotechnol. 2002, 13, 537. [ Links ]
6. Zaks, A.; Curr. Opin. Chem. Biol. 2001, 5, 130. [ Links ]
7. Lehman, L. R.; Stewart, J. D.; Curr. Org. Chem. 2001, 5, 439. [ Links ]
8. Urlacher, V.; Schmid, R. D.; Curr. Opin. Biotechnol. 2002, 13, 557. [ Links ]
9. Van Beilen, J. B.; Duetz, W. A.; Schmid, A.; Whitholt, B.; Trends Biotechnol. 2003, 4, 170. [ Links ]
10. Lee, K. H.; J. Nat. Prod. 2010, 73, 500. [ Links ]
11. León, A.; Reyes, B. M.; Chávez, M. I.; Toscano, R. A.; Delgado, G.; J. Mex. Chem. Soc. 2009, 53, 193. [ Links ]
12. Upar, K. B.; Mishra, S. J.; Nalawade, S. P.; Singh, S. A.; Khandare, R. P.; Bhat, S. V.; Tetrahedron: Asymmetry 2009, 20, 1637. [ Links ]
13. Atta-ur-Rahman; Farooq, S.; Choudhary, M. I.; Curr. Org. Chem. 1999, 3, 309. [ Links ]
14. Rodríguez, E.; Towers, G. H. N.; Mitchell, J. C.; Phytochemistry 1976, 15, 1573. [ Links ]
15. Oh, S.; Jeong, I. H.; Shim, W. S.; Wang, Q. S.; Lee, S.; Bioorg. Med. Chem. Lett. 2006, 16, 1656. [ Links ]
16. Zoretic, P. A.; Fang, H.; Ribeiro, A. A.; Dubay, G.; J. Org. Chem. 1998, 63, 1156. [ Links ]
17. Aranda, G.; El-Korbi, M. S.; Lallermand, J. V.; Neuman, A.; Tetrahedron 1981, 47, 8339. [ Links ]
18. Hanson, J. R.; Truneh, A.; Phytochemistry 1996, 42, 1021. [ Links ]
19. Atta-ur-Rahman; Farooq, A.; Choudhary, M. I.; J. Nat. Prod. 1997, 60, 1038. [ Links ]
20. Choudhary, M. I.; Musharraf, S. G.; Sami, A.; Atta-ur-Rahman; Helv. Chem. Acta 2004, 87, 2685. [ Links ]
21. Ata A.; Conci, L. J.; Betteridge, J.; Orhan, I.; Sener, B.; Chem. Pharm. Bull. 2007, 55, 118. [ Links ]
22. Pelletier, S. W.; Chokshi, H. P.; Desai, H. K.; J. Nat. Prod. 1986, 49, 892. [ Links ]
23. Coll, J. C.; Bowden, B. F.; J. Nat. Prod. 1986, 49, 934. [ Links ]
24. García Granados, A.; Martínez, A.; Parra, A.; Rivas, F.; Curr. Org. Chem. 2007, 11, 679. [ Links ]
25. Monks, A.; Seudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A.; Gray-Goodrich, M.; Campbell, H.; Mayo, J.; Boyd, M.; J. Nat. Cancer Inst. 1991, 83, 753. [ Links ]
Submitted: March 30, 2010
Published online: February 24, 2011