Synthesis, antibacterial and cytotoxic activities of new biflorin-based hydrazones and oximes

Accepted Manuscript Synthesis, antibacterial and cytotoxic activities of new biflorin-based hydrazones and oximes Luciana G.da S. Souza, Macia C.S. Almeida, Telma L.G. Lemos, Paulo R.V. Ribeiro, Edy S.de Brito, Vera L.M. Silva, Artur M.S. Silva, Raimundo BrazFilho, José G.M. Costa, Fábio F.G. Rodrigues, Francisco S. Barreto, Manoel O. de Moraes PII: DOI: Reference:

S0960-894X(15)30303-6 http://dx.doi.org/10.1016/j.bmcl.2015.11.095 BMCL 23351

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Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

6 October 2015 24 November 2015 26 November 2015

Please cite this article as: Souza, L.G., Almeida, M.C.S., Lemos, T.L.G., Ribeiro, P.R.V., Brito, E.S., Silva, V.L.M., Silva, A.M.S., Braz-Filho, R., Costa, J.G.M., Rodrigues, F.F.G., Barreto, F.S., de Moraes, M.O., Synthesis, antibacterial and cytotoxic activities of new biflorin-based hydrazones and oximes, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.11.095

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Synthesis, antibacterial and cytotoxic activities of new biflorin-based hydrazones and oximes

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Luciana G.da S. Souza, Macia C. S. Almeida, Telma L. G. Lemos, Paulo R. V. Ribeiro, Edy S.de Brito, Vera L. M. Silva, Artur M. S. Silva, Raimundo Braz-Filho, José G. M. Costa, Fábio F. G. Rodrigues, Francisco S. Barreto, Manoel O. de Moraes

Bioorganic & Medicinal Chemistry Letters

Synthesis, antibacterial and cytotoxic activities of new biflorin-based hydrazones and oximes Luciana G. da S. Souzaa, Macia C. S. Almeidaa, Telma L. G. Lemosa,*, Paulo R. V. Ribeirob, Edy S.de Britob, Vera L. M. Silva c, Artur M. S. Silva c,*, Raimundo Braz-Filhod, José G. M. Costae, Fábio F. G. Rodriguese, Francisco S. Barretof, Manoel O. de Moraesf a

Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, Campus do Pici 60451-970 - Fortaleza, CE - Brazil Embrapa Agroindustria Tropical, R Dra Sara Mesquita, 2270, 60511-110, Fortaleza, CE, Brazil c Department of Chemistry & QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal d Laboratório de Ciências Químicas, Universidade Estadual do Norte Fluminense Darcy Ribeiro, 28013-602 Campos dos Goytacazes – RJ, Brazil e Laboratório de Pesquisa de Produtos Naturais, Universidade Regional do Cariri, 63105-000, Crato, CE, Brazil f Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, 60430-270 Fortaleza, CE, Brazil b

A RT I C L E I N F O

A BS T RA C T

Article history: Received Revised Accepted Available online

Biflorin 1 is a biologically active quinone, isolated from Capraria biflora. Five new biflorinbased nitrogen derivatives were synthesized, of which two were mixtures of (E)- and (Z)isomers: (Z)-2a, (Z)-2b, (Z)-3a, (Z)- and (E)-3b, (Z)- and (E)-3c. The antibacterial activity was investigated using the microdilution method for determining the minimum inhibitory concentration (MIC) against six bacterial strains. Tests have shown that these derivatives have potential against all bacterial strains. The cytotoxic activity was also evaluated against three strains of cancer cells, but none of the derivatives showed activity.

Keywords: Biflorin Hydrazones Oximes Antibacterial Antitumoral

Quinones represent a wide and varied family of secondary metabolites of natural occurrence. The interest in these substances has been intensified in recent years due to their pharmacological importance and great structural variety. Many natural and synthetic quinone derivatives possess potent and varied pharmacological effects such as antitumor,1-3 antiinflammatory,4,5 analgesic,6 antifungal,7,8 and trypanocidal9,10 activities. Biflorin [6,9-dimethyl-3-(4-methylpent-3-en-1-yl)benzo[de] chromene-7,8-dione] 1 is a prenylated ortho-naphthoquinone isolated from the roots of Capraria biflora L. species. There two naphthoquinone isomers, ortho-naphthoquinones and paranaphthoquinones. They are widespread in the plant kingdom and, due to its redox properties they can interfere in different biological oxidative processes.11 Several naphthoquinones were found to exhibit interesting range of pharmacological properties such as antimicrobial,12,13 antiviral,14 antifungal,15 trypanocidal,16 antimalarial,17,18 and anticancer19 activities. Some orthonaphthoquinones, are trypanosomatid growth inhibitors with high cytotoxic activity.20 Specifically, biflorin has shown antitumor3,21 and antibiotic activities.22,23 Recent studies have shown this

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compounds also possess anticancer melanoma type activity24 as well as anti-metastatic potential,25 being able to inhibit tumor colonization. Herein we present our results on the study of the reactivity of biflorin 1, towards different nucleophiles (hydrazines and hydroxylamines) (Scheme 1). Novel nitrogenated derivatives of biflorin 1 were obtained, aiming to study its chemistry and the potential pharmacological activities of the new derivatives 2 and 3.

Scheme 1. Synthesis of biflorin derivatives: modification in carbonyl C-7.

——— *

Corresponding authors: Tel.: +351 234 370 714; fax: +351 234 370 084; e-mail address: [email protected] (A.M.S. Silva). Tel.: +55 85 3366 9369; fax: +55 85 3366 9782; e-mail address: [email protected] (T.L.G. Lemos)

First efforts were focused on the reaction of biflorin 1 with arylhydrazine derivatives (Scheme 2).26,27 The reaction of biflorin 1 with phenylhydrazine hydrochloride resulted in the formation of the corresponding hydrazone 2a in moderate yield (42%).27 When using 2,4-dinitrophenylhydrazine, the corresponding hydrazone 2b was obtained in good yield (63%), thus confirming the more nucleophilic character of the terminal nitrogen of 2,4dinitrophenylhydrazine. 27 Despite the possibility of the nucleophilic attack of hydrazine derivatives on any of the carbonyl carbons C-7 or C-8 of 1, only hydrazone compounds 2 were formed, resulting from the nucleophilic addition to the more electrophilic carbonyl group (C-7).

ppm (OCH2CH3), assigned to the (Z)-3c and (E)-3c isomers, respectively. 30 These mixture of isomers can be easily separated by column chromatography, but they are gradually converted into the mixture immediately after its separation into individual isomers.31

Figure 1. Hydrogen bond forming the six-membered ring in (Z)-isomers 2a, 2b and 3a.

All derivatives, including biflorin 1,32,33 were tested for antibacterial activity against six bacterial strains, Gram-positive and Gram-negative, by employing the microdilution method.34,35 The corresponding minimum inhibitory concentration (MIC) values are shown in Table 1. Scheme 2. Synthesis of biflorin-based hydrazone derivatives 2a,b.

Then the reaction of biflorin 1 with substituted amine hydrochlorides28-30 was carried out and led to the formation of oxime derivatives 3, in good yields (51-63 %),30 also resulting from the nucleophilic addition to the more electrophilic carbonyl group (C-7) (Schemes 1 and 3). The structures of all compounds were confirmed by NMR spectroscopic techniques (1H, 13C, COSY, HSQC and HMBC) as well as by high resolution mass spectrometry (HRMS) analysis.27,30

Previous studies reported the biflorin 1 antibacterial activity against Gram-positive and Gram-negative bacteria.22,23 In this work it was possible to prove this activity with very satisfactory MIC values (in terms of clinical MIC ≤ 1.024 μg/mL),36 for all strains of bacteria, using as positive control the antibiotics Amikacin (Ami), Gentamicin (Gen) and Neomycin (Neo). The most satisfactory results were observed against strains of Proteus vulgaris (ATCC 13315), with a MIC value of 16 µg/mL, showing to be more efficient than antibiotics used as positive control, and for Enterococcus faecalis (ATCC 4083), with values MIC 32 µg/mL, similar to that of Gentamicin (Gen). The hydrazones were susceptible to all strains, yielding the best result for the hydrazone 2a, with the MIC value of 256 µg/mL for Staphylococcus aureus (ATCC 25923), similar to CIM value of Amikacin (Ami). The MIC values for hydrazones were well above the values of biflorin 1 which was used as a standard.

Scheme 3. Synthesis of biflorin-based oxime derivatives 3a-c.

In this type of reactions it is possible to obtain a mixture of the (Z)- and (E)-diastereomers. The configuration of the obtained products was confirmed by NMR. The 1H NMR spectra of the hydrazones 2a and 2b (Scheme 2) indicated that only the (Z)isomer was formed. The (Z)-configuration of these hydrazones was established by observation of an intramolecular hydrogen bond forming a six membered ring (Fig. 1), evidenced by the signals at δ 17.23 and 17.42 ppm (N-H---O) in the 1H NMR spectra of hydrazones 2a and 2b respectively.27 The same applies to the oxime derivative 3a, where it is observed a signal at δ 19.91 ppm (O-H---O)30 related to the hydrogen bond forming the six-membered ring, in which only the (Z)- isomer is formed (Fig. 1). In the synthesis of methyloxime 3b and ethyloxime 3c it was observed the formation of a mixture of the two (Z)- and (E)isomers (Scheme 3). The 1H NMR spectrum of the mixture showed the presence of both (Z)- and (E)-isomers in the ratio 2:3 in both cases, with methyloxime and ethyloxime. These mixtures were evidenced by the presence of pairs of singlets at δ 4.17 (OCH3) and 4.18 ppm (OCH3) assigned to the (Z)-3b and (E)-3b isomers, and the pair of quartets at δ 4.42 (OCH2CH3) and 4.46

The oximes showed more satisfactory results for all strains of bacteria, among which ethyloxime and methyloxime showed greater sensitivity against Enterococcus faecalis (ATCC 4083), with MIC values of 32 µg/mL and 16 µg/mL respectively, and Staphylococcus aureus (ATCC 25923) with MIC values 32 µg/mL, both. The MIC values of ethyloxime and methyloxime were similar or even better than those of biflorin 1, and antibiotics used as positive control, suggesting that the modifications made on the biflorin structure led to an increase of the antibacterial potential for these derivatives. Cytotoxic activities in vitro of novel derivatives of biflorin 1 were evaluated using three human cancer cell lines, SF-295 (glioblastoma), OVCAR-8 (breast cancer) and HCT-116 (colon).37,38 An intensity scale was used to assess the cytotoxic potential of the samples tested; being considered samples without activity, with little (cell growth inhibition 1-50%), moderate (cell growth inhibition 50-75%) and a high activity (growth inhibition 75-100%). The experiments were analysed according to the mean ± standard deviation of the percentage of inhibition of cell growth using the GraphPad Prism program. Doxorubicin (Dox) was used as a positive control, in addition to biflorin 1 which has a high cytotoxic activity. The activity analysis was performed by the MTT method used in the

screening program at the National Cancer Institute of the United States (NCI)39 and allows to easily set the cytotoxicity of the substances.40 The results obtained indicate that these derivatives showed no activity (Fig. 2), suggesting that the carbonyl modification caused inactivation of the substances against cancer cells.

the FCT/MEC (Portugal) for the financial support to the QOPNA research Unit (FCT UID/QUI/00062/2013), through national funds and where applicable to those co-financed by the FEDER, within the PT2020 Partnership Agreement, and the Portuguese NMR Network. Finally, they thank Embrapa Agroindustria Tropical, Ceará, for the high-resolution mass spectrometry analysis.

Acknowledgments The authors thank CNPq and CAPES (Brasil) for financial support and study grants and also to the University of Aveiro and

Table 1. Values of the minimal inhibitory concentration (MIC) of biflorin 1 and derivatives 2a,b and 3a-c against six bacterial strains. MIC (µg/mL) Bacterial strains 1

2a

2b

3a

3b(Z)/(E)

3c(Z)/(E)

Ami

Neo

Gen

Enterococcus faecalis (ATCC 4083)

32

512

512

256

32

16

256

128

32

Proteus vulgaris (ATCC 13315)

16

≥ 1024

512

512

64

512

512

512

64

Escherichia coli (27)

256

≥ 1024

≥ 1024

512

128

128

128

128

512

Escherichia coli (ATCC 10536)

128

≥ 1024

512

≥ 1024

≥ 1024

512

512

512

128

Staphylococcus aureus (ATCC 25923)

64

256

512

256

32

32

256

32

32

Staphylococcus aureus (358)

128

≥ 1024

512

≥ 1024

64

64

256

32

8

SF-295

100

% growth inhibition

75 50 25

100 75 50 25

0

ox

1

D

3c

3b

3a

2a

ox D

1

3c

3b

3a

2b

2a

0 2b

% growth inhibition

HCT-116

% growth inhibition

OVCAR-8 100 75 50 25

ox D

1

3c

3b

3a

2b

2a

0

Figure 2. Cell growth inhibition percentage (% CI) of the samples 1, 2a,b and 3a-c in three tumor cell lines. Values are means ± standard deviation.

References and notes

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Acosta, S. L.; Muro, L. V.; Sacerio, A. L.; Monteagudo, G. L.; Penã, A. R.; Okwei, S. N. Acta Farm. Bonaerense, 2003a, 22, 53. Acosta, S. L.; Muro, L. V.; Sacerio, A. L.; Penã, A. R.; Okwei, S. N. Fitoterapia 2003, 74, 686. Gafner, S.; Wolfender, J.-L.; Nianga, M.; Stoeckli-Evans, H.; Hostettmann, K. Phytochemistry 1996, 42, 1315. Tandon, V. K.; Maurya, H. K.; Mishra, N. N.; Shukla P. K. Eur. J. Med. Chem. 2009, 44, 3130. Ferreira, V. F.; Jorqueira, A.; Souza, A. M. T.; Silva, M. N.; Souza, M. C. B. V.; Gouvêa, R. M.; Rodrigues,C. R.; Pinto, A. V.; Castro, H. C.; Santos, D. O.; Araújo, H. P.; Bourguignon, S. C. Bioorg. Med. Chem. 2006, 14, 5459.

10. Menna-Barreto, R. F. S.; Beghini, D. G.; Ferreira, A. T. S.; Pinto, A. V.; Castro, S. L.; Perales, J. J. Proteomics 2010, 73, 2306. 11. Tonholo, J.; Freitas, L. R.; De Abreu, F. C.; Azevedo, D. C.; Zani, C. L.; De Oliveira, A. B.; Goulart, M. O. F., J. Braz. Chem. Soc.1998, 9, 163. 12. Didry, N.; Pinkas, M.; Dubreuil, L. Ann. Pharm. Fr. 1986, 44, 73. 13. Riffel, A.; Medina, L. F.; Stefani, V.; Santos, R. C.; Bizani, D.; Brandelli, A. Braz. J. Med. Biol. Res. 2002, 35, 811. 14. Sendl, A.; Chen, J. L.; Jolad, S. D.; Stoddart, C.; Rozhon, E.; Kernan, M.; Nanakorn, W.; Balick M. J. Nat. Prod. 1996, 59, 808. 15. Ferreira, M. P. S. B. C.; Cardoso, M. F. C.; Silva, F. de C., Ferreira, V. F.; Lima, E. S.; Souza, J. V. B. Ann. Clin. Microbiol. Antimicrob. 2014, 13, 26. 16. Pinto, A. V.; Castro, S. L. Molecules 2009, 14, 4570. 17. Sharma, A.; Santos, I. O.; Gaur, P.; Ferreira, V. F.; Garcia, C. R.; da Rocha, D. R. Eur. J. Med. Chem. 2013, 59, 48. 18. Rezende, L. C.; Fumagalli, F.; Bortolin, M. S.; Oliveira, M. G.; Paula, M. H.; Andrade-Neto, V. F.; Emery F. S. Bioorg. Med. Chem. Lett. 2013, 23, 4583. 19. Shukla, S.; Srivastava, R. S.; Shrivastava, S. K.; Sodhi, A.; Kumar, P. Appl. Biochem. Biotechnol. 2012, 167, 1430. 20. Paulino, M.; Alvareda, E. M.; Denis, P. A.; Barreiro, E. J.; Sperandio da Silva, G. M.; Dubin, M.; Gastellu, C.; Aguilera, S.; Tapia O. Eur. J. Med. Chem. 2008, 43, 2238. 21. Wisintainer, G. G. N. S.; Simões E. R. B.; Lemos, T. L. G.; Moura, S.; Souza, L. G. S.; Fonseca, A. M.; Moraes, M. O.; Pessoa, C.; Roesch-Ely, M.; Henriques, J. A. P. An. Acad Bras Cienc 2014, 86, 1907. 22. (a) Gonçalves de Lima, O.; D'Albuquerque, I. L.; Loureiro, P.; Carmona, C. L.; Bernard, M. Z. Rev. Quim. Ind., 1953, 22, 2. (b) Gonçalves de Lima, O.; D’Albuquerque, I. L.; Loureiro, P.; Carmona, C. L.; Bernard, M. Z. Rev. Quím. Ind. 1954, 249, 28. 23. Santana, E. R. B.; Ferreira-Neto, J. P.; Yara, R.; Sena, K. X. F. R.; Fontes, A.; Lima, C. S. A. Molecules, 2015, 20, 8595. 24. Montenegro, R. C.; Vasconcellos, M. C.; Barbosa, G. S.; Burbano, R. M. R.; Souza, L. G. S.; Lemos, T. L. G.; Costa-Lotufo, L. V.; Moraes, M. O. Toxicol. in Vitro, 2013, 27, 2076. 25. Carvalho, A. A.; Costa, P. M.; Souza, L. G. S.; Lemos, T. L. G.; Alves, A. P. N. N.; Pessoa, C.; Moraes, M. O. Life Sci. 2013, 93, 201. 26. Campos, V. R.; Santos, E. A.; Ferreira, V. F.; Montenegro, R. C.; Souza, M. C. B. V.; Costa-Lotufo, L. V.; Moraes, M. O.; Regufe, A. K. P.; Jordão, A. K.; Pinto, A. C.; Resende, J. A. L. C.; Cunha, A. C. RSC Adv, 2012, 2, 11438. 27. General procedure for the synthesis of hydrazones 2a,b. Hydrazones 2a,b were synthesized following the methodology described by Campos and coworkers.26 The appropriate arylhydrazine hydrochloride (0.2 mmol) was added to a solution of biflorin 1 (0.1 mmol, 30.8 mg) in MeOH (2 mL). The mixture was stirred for 24 h at room temperature. After this period, the reaction mixture was concentrated under reduced pressure and the resulting residue was taken in dichloromethane and purified by TLC, using hexane and dichloromethane (4:6 v/v) as the eluent. The reaction of biflorin 1 (0.1 mmol, 30.8 mg) with phenylhydrazine hydrochloride (0.2 mmol, 29.0 mg) yielded (16.7 mg, 42 %) of an orange solid (Z)-6,9-dimethyl-3-(4-methylpent-3en-1-yl)-7-(2-phenylhydrazono)benzo[de]chromen-8(7H)-one (2a): mp 115-117 ºC; IR (KBr): 3400, 2925, 1600, 1495, 1470, 1190 cm-1. 1H NMR (500.13 MHz, CDCl3) δ = 1.58 (s, 3 H, H-14), 1.72 (s, 3H, H-15), 2.10 (s, 3H, H-17), 2.30 (q, J = 7.1 Hz, 2 H, H11), 2.46 (t, J = 7.1 Hz, 2 H, H-10), 2.83 (s, 3H, H-16), 5.19 (m, 1 H, H-12), 6.94 (s, 1 H, H-2), 7.12-1.16 (m, 2 H, H-4 and H-4’), 7.38-7.49 (m, 5 H, H-5, H-2’, H-3’, H-5’ and H-6’), 17.23 (s, 1 H, N-H) ppm. 13C NMR (125.77 MHz, CDCl3) δ = 7.4 (C-17), 17.9 (C-14), 25.7 (C-15), 26.7 (C-11), 27.6 (C-10), 27.9 (C-16), 111.7 (C-9), 116.3 (C-2’ and C-6’), 116.5 (C-3), 117.7 (C-4’), 119.3 (C9b), 123.1 (C-12), 124.5 (C-4), 128.3 (C-3a), 129.5 (C-5’ and C3’), 130.2 (C-6a), 131.5 (C-7), 133.0 (C-13), 134.8 (C-6), 135.1 (C-5), 139.3 (C-2), 143.1 (C-1’), 160.0 (C-9a), 179.2 (C-8) ppm. ESI+ -HRMS: calculated for C26H27N2O2 [M+H]+: 399.2073; found: 399.2071 [M+H]+. The reaction of biflorin 1 (0.1 mmol, 30.8 mg) with 2,4dinitrophenylhydrazine hydrochloride (67 %) (0.1 mmol, 29.0 mg) yielded (30.8 mg, 63 %) of a brown solid (Z)-7-(2-(2,4dinitrophenyl)hydrazono)-6,9-dimethyl-3-(4-methylpent-3-en-1yl)benzo[de]chromen-8(7H)-one (2b): mp 246-248 ºC; IR (KBr): 3425, 2920, 1600, 1430 cm-1. 1H NMR (500.13 MHz, CDCl3) δ = 1.60 (s, 3 H, H-14), 1.81 (s, 3 H, H-15), 2.11 (s, 3 H, H-17), 2.33 (q, J= 7.2 Hz, 2 H, H-11), 2.55 (t, J = 7.2 Hz, 2 H, H-10), 2.86 (s,

3 H, H-16), 5.19 (t, J = 7.2 Hz, 1 H, H-12), 7.09 (s, 1 H, H-2), 7.39 (d, J = 8.1 Hz, 1 H, H-4), 7.56 (d, J = 8.1 Hz, 1 H, H-5), 8.26 (d, J = 9.4 Hz, 1 H, H-6’), 8.48 (dd, J = 2.4 and 9.4 Hz, 1 H, H5’), 9.21 (d, J = 2.4 Hz, 1 H, H-3’), 17.42 (s, 1 H, N-H) ppm. 13C NMR (125.77 MHz, CDCl3) δ = 7.5 (C-17), 17.9 (C-14), 25.7 (C15), 26.9 (C-11), 27.5 (C-10 and C-16), 112.5 (C-9), 116.5 (C-3), 117.4 (C-6’), 120.7 (C-9b), 121.8 (C-4), 122.7 (C-12), 123.2 (C3’), 128.9 (C-6a), 129.2 (C-3a), 129.6 (C-5’) 133.0 (C-2’), 133.4 (C-13), 136.4 (C-5), 137.0 (C-6), 138.5 (C-7), 140.0 (C-2), 140.5 (C-4’), 144.2 (C-1’), 161.4 (C-9a), 180.3 (C-8) ppm. ESI+ -HRMS: calculated for C26H25N4O6 [M+H]+: 489.1774; found: 489.1770 [M+H]+. 28. Oliveira, M. F. Contribuição ao Conhecimento Químico das Espécies Tabebuia serratifolia Nichols e Tabebuia rosea Bertol. PhD thesis, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil, 2000, pp.1-236. 29. Silva, A. R.; Herbst, M. H.; Ferreira, A. B. B.; Silva, A. M.; Visentin, L. C. Molecules, 2011, 16, 1192. 30. General procedure for the synthesis of oximes 3a-c. The oximes were synthesized following the methodology proposed by Oliveira and coworkers.28,29 Biflorin 1 (0.1-0.16 mmol) was added to a methanolic solution (1-2 mL) of the appropriate amine hydrochloride (0.1-0.16 mmol) under magnetic stirring. The reaction mixture was left at reflux (70°C) for a period of 6 h. After this period the reaction mixture was evaporated to the dryness. The obtained residue was taken in dichloromethane and purified by TLC using hexane: ethyl acetate (6: 4 v/v). The reaction of biflorin 1 (0.16 mmol, 50.0 mg) with hydroxylamine hydrochloride (0.16 mmol, 11.1 mg) yielded (31.9 mg, 58 %) of an orange residue (Z)-7-(hydroxyimino)-6,9dimethyl-3-(4-methylpent-3-en-1-yl)benzo[de]chromen-8(7H)-one (3a): IR (KBr): 3400, 2920, 1600, 1530, 1030 cm-1. 1H NMR (500.13 MHz, CDCl3) δ = 1.59 (s, 3 H, H-14), 1.72 (s, 3 H, H-15), 2.03 (s, 3 H, H-17), 2.32 (q, J = 7.2 Hz, 2 H, H-11), 2.54 (t, J = 7.2 Hz, 2 H, H-10), 2.70 (s, 3 H, H-16), 5.18 (t, J = 7.2 Hz, 1 H, H-12), 7.09 (s, 1 H, H-2), 7.36 (d, J = 8.1 Hz, 1 H, H-4), 7.53 (d, J = 8.1 Hz, 1 H, H-5), 19.91 (s, 1 H, N-OH) ppm. 13C NMR (125.77 MHz, CDCl3) δ = 6.9 (C-17), 18.0 (C-14), 25.1 (C-15), 26.9 (C11), 26.8 (C-16), 27.5 (C-10), 110.7 (C-9), 117.8 (C-3), 119.7 (C9b), 121.3 (C-4), 122.8 (C-12), 128.0 (C-6a), 128.9 (C-3a), 133.5 (C-13), 136.9 (C-5), 138.5 (C-6), 147.4 (C-7), 139.8 (C-2), 163.2 (C-9a), 179.5 (C-8) ppm. ESI+ -HRMS: calculated for C20H21NO3 [M+H]+: 324.1601; found: 324.1600. The reaction between biflorin 1 (0.1 mmol, 30.8 mg) and Omethylhydroxylamine hydrochloride (0.1 mmol, 8.35 mg) yielded (21.3 mg, 63 %) of an orange residue, consisting of a mixture of (Z)-7-(methoxyimino)-6,9-dimethyl-3-(4-methylpent-3-en-1yl)benzo[de]chromen-8(7H)-one (Z-3b) and (E)-7(methoxyimino)-6,9-dimethyl-3-(4-methylpent-3-en-1yl)benzo[de]chromen-8(7H)-one (E-3b): IR (KBr): 3410, 2930, 1600, 1195, 1015 cm-1. (Z-3b): 1H NMR (500.13 MHz, CDCl3) δ = 1.59 (s, 3 H, H-14), 1.71 (s, 3 H, H-15), 1.95 (s, 3 H, H-17), 2.25 (m, 2 H, H-11), 2.47 (m, 2 H, H-10), 2.61 (s, 3 H, H-16), 4.18 (s, 3 H, OCH3), 5.18 (m, 1 H, H-12), 6.93 (s, 1 H, H-2), 7.24 (d, J = 8.6 Hz, 1 H, H-4), 7.35 (d, J = 8.6 Hz, 1 H, H-5) ppm. 13C NMR (125.77 MHz, CDCl3) δ = 7.5 (C-17), 17.9 (C-14), 24.0 (C-16), 25.7 (C-15), 27.0 (C-11), 27.1 (C-10), 64.8 (OCH3), 112.4 (C-9), 115.7 (C-3), 121.2 (C-4), 121.4 (C-9b), 122.9 (C-12), 128.0 (C6a), 128.8 (C-3a), 133.1 (C-13), 134.9 (C-5), 138.2 (C-6), 139.7 (C-2), 147.3 (C-7), 160.1 (C-9a), 178.9 (C-8) ppm. (E-3b): 1H NMR (500.13 MHz, CDCl3) δ = 1.59 (s, 3 H, H-14), 1.71 (s, 3 H, H-15), 1.96 (s, 3 H, H-17), 2.25 (m, 2 H, H-11), 2.27 (s, 3 H, H16), 2.47 (m, 2 H, H-10), 4.18 (s, 3 H, OCH3), 5.18 (m, 1 H, H12), 6.47 (s, 1 H, H-2), 7.33 (s, 1 H, H-4), 7.33 (s, 1 H, H-5) ppm. 13 C NMR (125.77 MHz, CDCl3) δ = 7.6 (C-17), 17.9 (C-14), 23.0 (C-16), 25.7 (C-15), 27.0 (C-11), 27.1 (C-10), 63.7 (OCH3), 110.5 (C-9), 115.5 (C-3), 121.3 (C-9b), 122.8 (C-12), 123.2 (C-4), 126.3 (C-6a), 127.6 (C-3a), 133.2 (C-13), 133.5 (C-5), 140.1 (C-2), 140.7 (C-6), 151.5 (C-7), 160.5 (C-9a), 185.2 (C-8) ppm. ESI+ HRMS: calculated for C21H24NO3 [M + H]+: 338.1756; found: 338.1753 [M+H]+. The reaction of biflorin 1 (0.1 mmol, 30.8 mg) with Oethylhydroxylamine hydrochloride (0.1 mmol, 9.75 mg) yielded (17.9 mg, 51 %) of an orange residue, consisting of a mixture of (Z)-7-(ethoxyimino)-6,9-dimethyl-3-(4-methylpent-3-en-1yl)benzo[de]chromen-8(7H)-one (Z-3c) and (E)-7-(ethoxyimino)6,9-dimethyl-3-(4-methylpent-3-en-1-yl)benzo[de]chromen8(7H)-one (E-3c).; IR (KBr): 3395, 2915, 1600, 1185, 1050 cm-1. (Z-3c): 1H NMR (500.13 MHz, CDCl3) δ = 1.43 (m, 3 H, CH3),

31.

32.

33.

34.

35.

1.59 (s, 3 H, H-14), 1.71 (s, 3 H, H-15), 1.95 (s, 3 H, H-17), 2.25 (m, 2 H, H-11), 2.47 (m, 2 H, H-10), 2.61 (s, 3 H, H-16), 4.42 (q, J = 7.1 Hz, 2 H, OCH2), 5.16 (m, 1 H, H-12), 6.93 (s, 1H, H-2), 7.24 (d, J = 8.2 Hz, 1 H, H-4), 7.35 (d, J = 8.2 Hz, 1 H, H-5) ppm. 13 C NMR (125.77 MHz, CDCl3) δ = 7.5 (C-17), 14.8 (CH3), 17.9 (C-14), 24.3 (C-16), 25.7 (C-15), 27.0 (C-11), 27.4 (C-10), 72.8 (OCH2), 112.5 (C-9), 115.6 (C-3), 121.4 (C-9b), 121.5 (C-4), 122.9 (C-12), 128.8 (C-6a), 128.0 (C-3a), 133.1 (C-13), 134.9 (C5), 138.0 (C-6), 139.7 (C-2), 147.0 (C-7), 159.9 (C-9a), 178.7 (C8) ppm. (E-3c): 1H NMR (500.13 MHz, CDCl3) δ = 1.35 (t, J =7.1 Hz, 3 H, CH3), 1.59 (s, 3 H, H-14), 1.71 (s, 3 H, H-15), 1.96 (s, 3 H, H17), 2.25 (m, 2 H, H-11), 2.27 (s, 3 H, H-16), 2.46 (m, 2 H, H-10), 4.46 (q, J = 7.1 Hz, 2 H, OCH2), 5.16 (m, 1 H, H-12), 6.96 (s, 1 H, H-2), 7.33 (s, 1 H, H-4), 7.33 (s, 1 H, H-5) ppm. 13C NMR (125.77 MHz, CDCl3) δ = 7.6 (C-17), 14.6 (CH3), 17.9 (C-14), 23.3 (C16), 25.7 (C-15), 27.1 (C-11), 27.4 (C-10), 72.2 (OCH2), 110.5 (C-9), 110.6 (C-3), 121.3 (C-9b), 122.8 (C-12), 123.0 (C-4), 126.4 (C-6a), 127.6 (C-3a), 133.2 (C-13), 133.5 (C-5), 140.0 (C-2), 140.6 (C-6), 150.7 (C-7), 160.5 (C-9a), 185.4 (C-8) ppm. ESI+ HRMS: calculated for C22H26NO3 [M + H]+: 352.1919; found: 352.1913 [M+H]+. Nicolaides, D. N.; Gautam, D. R.; Litinas, K. E.; HadjipavlouLitina, D. J.; Kontogiorgis, C. A. J. Heterocyclic Chem., 2004, 41, 605. Souza, L. G. S; Almeida, M. C. S.; Monte, F. J. Q.; Santiago, G. M. P.; Braz-Filho, R.; Lemos, T. L. G. Quim. Nova, 2012, 35, 2258. Plant Material: The species Capraria biflora L. (Scrophulariaceae) was collected in Itapiúna, Ceará, Brazil and was identified by Dr. Edson Nunes. A voucher specimen (No. 30848) was deposited in the Herbarium Prisco Bezerra of the Biology Department of the Federal University of Ceará. Isolation of biflorin: The isolation of biflorin 1 was performed as described by Souza et al.32 Air-dried powdered roots (700 g) were extracted with petroleum ether and the solvent was evaporated under reduced pressure to yield 1.7 g of extract. The extract (1.0 g) was chromatographed on silica gel by elution using binary mixtures of hexane/EtOAc and EtOAc/MeOH, with increasing polarity. Fourteen fractions were collected with a volume of 200 ml. Fractions 8-10 were pooled according to thin-layer chromatographic (TLC) analysis, yielding 58 mg of biflorin 1. NCCLS (2006): Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. 16th Informational Supplement. CLSI document M100-S16. Wayne, PA: CLSI, 2006. Antibacterial Activity Evaluation: The antibacterial activity of the samples was evaluated using the broth microdilution method based on the document M100-S16 (CLSI, 2006)34 for bacteria. In the tests, six strains of Gram-positive, Enterococcus faecalis (ATCC 4083), Staphylococcus aureus (ATCC 25923), and multidrug-resistant Staphylococcus aureus (358) and Gramnegative, Escherichia coli (ATCC 10536), Proteus vulgaris (ATCC 13315) and multidrug-resistant Escherichia coli (27) were

36.

37. 38.

39.

40.

used. The Oswaldo Cruz Foundation-FIOCRUZ provided the six standard bacteria strains used. The bacterial strains were activated in the midst Brain Heart Infusion Broth (BHI) for 24 h at 35 ± 2 °C. Next, a bacterial suspension was prepared in BHI at 3.8%, corresponding to the turbidity of 0.5 McFarland scale (1 x 10 8 CFU/mL). Then the suspension was diluted to 1 x 10 6 CFU/mL in BHI broth at 10%, and volume of 100 µL, and homogenized in microdilution 96 well plates, plus different concentrations of the samples resulting in a final inoculum of 5 x 10 5 CFU/mL. The samples were solubilized in distilled water and DMSO to obtain a stock solution of 1024 µg/mL. The final concentrations of the samples were in the culture medium 512-8 µg/mL. The tests were performed in triplicate. The plates were incubated at 35 ± 2 °C for 24 h. The antibacterial activity was detected using a colorimetric method by adding 25 μL of the resauzurin staining (0.01%) aqueous solution to each one of the wells at the end of the incubation period. The minimal inhibitory concentration (MIC) was defined as the lowest extract concentration able to inhibit the bacteria growth, as indicated by resauzurin staining (dead bacterial cells are not able to change the staining color by visual observation—blue to red). The antibiotics Amikacin, Gentamicin and Neomycin were utilized as a positive control. Nascimento, P. G. G.; Lemos, T. L. G.; Bizerra, A. M. C.; Arriaga, A. M. C.; Ferreira, D. A.; Santiago, G. M. P.; Braz-Filho, R.; Costa, J. G. M. Molecules, 2014, 19, 1317. Mosmann, T. J. Immunol. Methods, 1983, 65, 55. Cytotoxicity activity: The assays for cytotoxic activity were performed using tumor cell lines, SF-295 (glioblastoma - human), OVCAR-8 (breast cancer) and HCT-116 (colon) (National Cancer Institute, USA). The cell lines were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 1% antibiotic, which were incubated at 37 ºC and in an atmosphere containing 5% CO2. Samples were diluted in DMSO and tested at a concentration of 5 µg/mL. Cells were plated at a concentration of 0.1 x 10 6 cells/mL for strains SF-295 and OVCAR-8 and 0.7 x 105 cells/mL for the strain HCT-8 and incubated for 72 hours in an oven. Subsequently the samples were centrifuged and the supernatant was removed. Then 150 µL of 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2Htetrazolium bromide (MTT) solution were added and the plates were incubated for 3h. The absorbance was measured after dissolution of the precipitate with 150 µL of DMSO in plate spectrophotometer at 595 nm. Cell viability was evaluated by reduction of the yellow dye (MTT) to a blue product as described by Mosmann.37 Biflorin 1 was utilized as a positive control. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst., 1990, 82, 1107. Berridge, M. V.; Tan, A. S.; McCoy, K. D.; Wang, R. Biochemica, 1996, 4, 14.