Bioinorganic Chemistry and Applications

Volume 2013 (2013), Article ID 961783, 7 pages

http://dx.doi.org/10.1155/2013/961783

Research Article

Cytotoxicity and In Vitro Antileishmanial Activity of Antimony (V), Bismuth (V), and Tin (IV) Complexes of Lapachol

Marcele Neves Rocha,¹ Paula Monalisa Nogueira,¹ Cynthia Demicheli,² Ludmila Gonçalvez de Oliveira,² Meiriane Mariano da Silva,² Frédéric Frézard,³ Maria Norma Melo,⁴ and Rodrigo Pedro Soares¹

¹Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz/FIOCRUZ, 30190-002 Belo Horizonte, MG, Brazil

²Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil

³Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil

⁴Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil

Received 30 January 2013; Revised 26 April 2013; Accepted 6 May 2013

Academic Editor: Patrick Bednarski

Copyright © 2013 Marcele Neves Rocha et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Leishmania amazonensis is the etiologic agent of the cutaneous and diffuse leishmaniasis often associated with drug resistance. Lapachol [2-hydroxy-3-(3′-methyl-2-butenyl)-1,4-naphthoquinone] displays a wide range of antimicrobial properties against many pathogens. In this study, using the classic microscopic in vitro model, we have analyzed the effects of a series of lapachol and chlorides complexes with antimony (V), bismuth (V), and tin (IV) against L. amazonensis. All seven compounds exhibited antileishmanial activity, but most of the antimony (V) and bismuth (V) complexes were toxic against human HepG2 cells and murine macrophages. The best IC₅₀ values (0.17 0.03 and 0.10 0.11 g/mL) were observed for Tin (IV) complexes (3) [(Lp)(Ph₃Sn)] and (6) (Ph₃SnCl₂), respectively. Their selective indexes (SIs) were 70.65 and 120.35 for HepG2 cells, respectively. However, while analyzing murine macrophages, the SI decreased. Those compounds were moderately toxic for HepG2 cells and toxic for murine macrophages, still underlying the need of chemical modification in this class of compounds.

1. Introduction

Leishmania amazonensis, a New World species, has been identified as a dermotropic species often associated with drug resistance [1]. Current antileishmanial therapies are toxic to human and some simply fail [2, 3]. In the Americas, for over six decades, parenteral administrations of pentavalent antimonials (Sb-V), sodium stibogluconate (Pentostam), and meglumine antimoniate (Glucantime) have been used for treating leishmaniasis. In places where resistance to antimonials is common, such as India, other chemotherapeutic treatments include amphotericin B and pentamidine [2, 4]. Therefore, the absence of a low toxic and safe oral drug still underlines the need for new antileishmanial compounds.

Lapachol, [2-hydroxy-3-(3′-methyl-2-butenyl)-1,4-naphthoquinone] (Figure 1) is a natural compound extracted from the core of Bignoniaceae trees. In Leishmania, lapachol analogues, derivatives, and complexes have been tested by several groups. Lapachol, isolapachol, and some of their derivatives were active in vitro and in vivo against Leishmania braziliensis and L. amazonensis, respectively [5]. Bismuth (III), antimony (V), and tin (IV) complexes were active against Helicobacter pylori, Leishmania major, and Leishmania donovani, respectively [6–8].

Figure 1: Structures of lapachol metal (Bi, Sb, and Sn) complexes (1–3) and chloride metal (Bi, Sb, and Sn) compounds (4–6) and lapachol (7). Legend: Bi = bismuth, Sb = antimony, and Sn = tin.

The design of bifunctional metal complex, where both the ligand and the metal exert pharmacological activity, represents a promising strategy for achieving more effective and selective drugs. In the present study, lapachol was coupled with three different metals: triphenyltin (IV), triphenylbismuth (V), and triphenylantimony (V). We have tested the in vitro activity and cytotoxicity of synthesized antimony (V), bismuth (V), and tin (IV) lapachol and chloride complexes against intracellular L. amazonensis, HepG2 cells, and murine macrophages.

2. Materials and Methods

2.1. Synthesis of the Lapachol Metal Complexes and Tested Metal Chlorides

The (Lp)(Ph₃Bi)O0.5 and (Lp)(Ph₃Sb)OH (2) complexes were synthesized by following the procedure described by [9]. To prepare (Lp)(Ph₃Sn) (3) the same procedure was used. Triethylamine (70 μL) was added to a mixture of lapachol (0.121 g, 0.5 mmol) and triphenyltin (IV) chloride (193 mg, 0.5 mmol) in chloroform (20 mL). The resulting mixture was stirred for 4 h at room temperature. Removal of the solvent under vacuum yielded a solid material. The material was subsequently dissolved in acetone and precipitated in water. The triethylammonium hydrochloride formed during the reaction was dissolved and removed by water. Elemental analyses were carried out using a Perkin-Elmer 240 Elemental Analyzer. Atomic absorption analyses of bismuth, antimony and tin contents were carried out on a Hitachi Atomic Absorption Spectrophotometer (Model 8200).

The following equations can be proposed to illustrate the formation of (Lp)(Ph₃Sb)OH complex as follows:

The same process can be proposed for all complexes. The yields, melting points, and elemental analyses of the compounds prepared are given in Table 1.

Table 1: Yields and elemental analyses of the compounds.

Compound
Yield (%)
M.p. (°C)a
C found (calc.) (%)
H found (calc.) (%)
Metal found (calc.) (%)
Formula for calc.

79
126–129
57.31 (57.40)
4.09 (4.23)
29.03 (29.98)
(Lp)(Ph₃Bi)O0.5

76
154–156
65.30 (64.82)
4.52 (4.78)
20.64 (19.19)
(Lp)(Ph₃Sb)OH

79
107–109
66.41 (67.03)
4.40 (4.77)
21.74 (20.07)
(Lp)(Ph₃Sn)

M.p.: melting point.

Triphenylbismuth dichloride (4), triphenylantimony dichloride (5), triphenyltin chloride (6), and lapachol (7) were obtained from Aldrich. Triethylamine was obtained from Sigma. The predicted structures of all tested compounds are shown in Figure 1.

2.2. Parasites

The World Health Organization (WHO) reference strain L. amazonensis (IFLA/BR/1967/PH8) was used and typed as previously described [10]. Promastigote forms were grown at 25°C in M199 medium (Sigma) supplemented with 10% heat-inactivated fetal calf serum (Cultilab), 40 mmol/L HEPES (Amersham), 0.1 mmol/L adenine (Sigma), 0.0005% hemin (Sigma), 0.0002% biotin (Sigma), 50 units/mL penicillin, and 50 mg/mL streptomycin (Invitrogen) [11].

2.3. In Vitro Classic Microscopic Tests

Animals were kept in the Animal Facility of the Centro de Pesquisas René Rachou/FIOCRUZ in strict accordance to the Guide for the Care and Use of Experimental Animals [12]. The procedures were approved by the Internal Ethics Committee in Animal Experimentation (CEUA) of Fundação Oswaldo Cruz (FIOCRUZ), Brazil (Protocol L-042/08). Mice were euthanized with CO₂ in an induction chamber prior to macrophage removal. Balb/c mice were injected intraperitoneally with 2 mL of 3% sodium thioglycollate medium. After 72 h, peritoneal macrophages were removed by washing with cold RPMI 1640 medium and enriched by adherence to round glass coverslips (13 mm) placed in a 4-well culture plate. Cells (2 × 10⁵ cells/well) were cultured (37°C, 5% CO₂, 18 h) in RPMI supplemented with 10% heat-inactivated FBS (fetal bovine serum) prior to infection with parasites. Macrophages were exposed to stationary phase promastigotes (2 × 10⁶/well) at a final ratio of 1 : 10. The plates were incubated at 37°C, 5% CO2, for 5 h in BOD to allow internalization of parasites [13]. Then, the medium was removed for the remaining noninternalized parasites. Negative control included only infected macrophages and medium. Incubations were tested in duplicate in two independent experiments [14, 15]. The substances were serial diluted with RPMI 1640 medium supplemented with 10% FBS at five different concentrations (50 → 3.1 μg/mL). For compounds (3) and (6), the dilution was 10 → 0.016 μg/mL. Amphotericin B was used as reference drug. Infected macrophages were exposed daily to the compounds for 3 consecutive days. After this period, coverslips were collected, stained with Panoptic (Laborclin), and subsequently mounted with Entellan (Merck) on glass slides.

2.4. Cytotoxicity Tests

The cell lineage HepG2 A16 was derived from a human hepatocellular carcinoma cell line HepG2 (ATCC HB-8065) and obtained from America Type Culture Collection line (ATCC) [16]. Balb/c murine peritoneal macrophages were obtained as described above. Cytotoxicity was determined using the MTT method (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma). HepG2 cells were kept in RPMI medium supplemented with 10% FBS, and confluent monolayers were trypsinized, washed in RPMI, and transferred to 96-well microtiter plates (4 × 10⁴ cells/well) for 16–18 h. Murine macrophages were used in the concentration 2 × 10⁵ cells/well in 96-well microtiter plates. The compounds were serial diluted in different concentrations (10 → 0.16 mg/mL). In both tests, the medium was removed, and the compounds were incubated for 24 h (37°C, 5% CO₂). Colorimetric reaction was developed following the incubation with MTT (37°C, 5% CO₂, 4 h) and addition of acidified isopropanol [17]. The reaction was read spectrophotometrically (Spectramax M5, Molecular Devices, San Francisco, CA) with a 570 nm filter and a background of 670 nm. Incubations were tested in triplicate in two independent experiments. The minimum dose that killed 50% of the cells (MLD₅₀) was determined [18], and the values were plotted to generate dose-response curves using Microcal Origin Software (Northampton, MA, USA) [15, 19]. The selective indexes (SIs) of compounds were calculated using the MLD₅₀/IC₅₀ ratios to HepG2 and peritoneal macrophages [20, 21].

3. Results

The in vitro classic microscopic test enables direct counting to determine the percentage of infected cells and/or the number of amastigotes [22]. Here, the IC₅₀ values were calculated based on the percentage of infected macrophages [15]. The in vitro antileishmanial activities, cytotoxicity and selective indexes (SIs) of lapachol metal complexes and chlorides (1–6), lapachol (7) and amphotericin B are shown in Table 2. Lapachol and compounds , (2), and (5) were considered inactive (IC₅₀ > 10 μg/mL) and toxic (SI 20) for HepG2 cells and macrophages [20, 21]. The tin (IV) lapachol complex (3) and chloride (6) were active against intracellular amastigote forms of L. amazonensis (Figures 2(a) and 2(b)) and less toxic for HepG2 cells (SIs ranging from 70.65 to 120.35) (Figures 2(d) and 2(e)) (Table 2). One triphenyl bismuth chloride (4) (Figure 2(c)) was also active and a little more toxic for HepG2 cells (Figure 2(f)) than (3) (Figure 2(d)) and (6) (Figure 2(e)) (SI = 34.03). All compounds were toxic for murine macrophages (SI < 20). Amphotericin B, an antileishmanial reference drug, exhibited an IC₅₀ value approximately fourfold higher than (3) and (6) ( μg/mL) (Table 2).

Table 2: Antileishmanial activity, cytotoxicity, and selective indexes of tested compounds for HepG2 cells and murine macrophages.

Compound
Formula

HepG2
Macrophages

SIc

SIc

(Lp)(Ph₃Bi)O0.5

2.01

1.11

(Lp)(Ph₃Sb)OH

17.81

7.15

(Lp)(Ph₃Sn)

70.65

9.41

Ph₃BiCl₂

34.03

4.67

Ph₃SbCl₂

13.56

2.65

Ph₃SnCl₂

120.35

7.30

Lpd

13.03

11.92

Amphotericin B

883.00

246.51

IC₅₀: the inhibitory concentration that killed 50% of the L. amazonensis in μg/mL.

bMLD₅₀: the minimum lethal dose that killed 50% of the cells in μg/mL.

cSI: selective index, calculated based on the MLD₅₀/IC₅₀ ratios.

dLp: Lapachol.

(a)

(b)

(c)

(d)

(e)

(f)

Figure 2: In vitro antileishmanial activity of compounds (3), (4), and (6) against intracellular L. amazonensis ((a), (b), and (c)) and cytotoxicity against hepatoma HepG2 cell ((d), (e), and (f)). Curves were obtained using Microcal Origin Software. IC₅₀ = half-maximal inhibitory response; MLD₅₀ = the minimum lethal dose. Figures are a representation of one experiment.

4. Discussion

Leishmaniases are considered by the WHO as one of the major six important infectious diseases worldwide. Over the past years, the absence of research and development for new medicines targeting diseases affecting people in developing countries has become a global concern [23]. Currently, the development of new drugs, combinations, or protocols against tropical and neglected diseases is of great importance in public health [24–27]. However, side effects, treatment failure due to parasite resistance, HIV coinfection, and intravenous administration are the major concerns hindering leishmaniasis chemotherapy [2, 3].

Lapachol derivatives and complexes have exhibited antitumor, anti-inflammatory, antiangiogenic, analgesic, and antimicrobial properties [6, 28–32]. Lapachol and some of its analogues demonstrated activity in vitro against L. braziliensis and L. amazonensis [5]. The use of metal complexes against Leishmania may represent a potential alternative against the disease since antimony-based regimens tend to be very toxic. In this context, we have explored the use of lapachol and chloride metal complexes with antimony (V), bismuth (V), and tin (IV)] against L. amazonensis.

In contrast to data from previous studies, lapachol (7) did not exhibit significant antileishmanial activity against L. amazonensis ( versus  μg/mL) [5]. This IC₅₀ value is close to that observed for L. braziliensis ( μg/mL). This discrepancy could be attributed to the strain of L. amazonensis used (MHOM/BR/77/LTB0016) and experimental conditions. The highest antiproliferative activity against intracellular L. amazonensis was observed for tin (IV) lapachol and chloride complexes (3) and (6) (Figures 2(a) and 2(b)) and one bismuth (V) chloride compound (4) (Figure 2(c)). More importantly, compounds (3) and (6) were more active than amphotericin B and less toxic among all substances tested while using HepG2 cells (SIs of 70.65 and 120.35, resp.). Interestingly, the resulting compound of lapachol and tin (IV) showed a marked decrease in metal toxicity than lapachol alone (SIs of 70.65 versus 13.03, resp.). One of the possibilities that could justify such phenomenon could be due to an increase in the lipophilicity of the lapachol-complexed molecule. Another hypothesis is that lapachol complexation could affect the REDOX potential of the compound, thus, consequently changing its activity. Consistent with this idea, the mechanisms underlying those activities are related to the generation of reactive oxygen radicals (ROSs) induced by the bioreduction of its quinonoid nucleus through specific enzymes and oxygen [33–35]. ROS mechanisms induced by lapachol have been implicated in the chemotherapeutic activities against many protozoa such as Trypanosoma cruzi [30] and also tumor cells [31]. Similarly, among all metal chloride substances, the triphenyl tin (IV) chloride compound exhibited lower toxicity compared to bismuth (V) and antimony (V) chloride ones. Finally, compound (4) exhibited moderate toxicity (SI = 34.03) with an IC₅₀ value 7-fold higher than amphotericin B. However, when cytotoxicity was tested against murine macrophages, the host cells for Leishmania, all compounds were toxic. Those data indicate the need of chemical modifications in this class of compounds in the search of novel antileishmanial molecules.

5. Conclusions

Lapachol and a series of six lapachol and chloride metal complexes have been evaluated for their in vitro activity against intracellular amastigote forms of L. amazonensis. The tin (IV) lapachol and chloride complexes (3 and 6) exhibited higher antileishmanial activity compared to amphotericin B. The triphenyl bismuth (V) compound (4) also exhibited antileishmanial activity with moderate cytotoxicity. Lapachol compounds with bismuth (V) and tin (IV) were less toxic when compared with lapachol alone for HepG2 cells. In conclusion, tin, and in a less extent, bismuth complexes were moderately toxic for HepG2 cells and toxic for murine macrophages.

Conflict of Interests

The authors have declared that no conflict of interests exists.

Financial Support

The authors received the following funds.

Acknowledgments

R. P. Soares, C. Demicheli, F. Frézard, and M. N. Melo are supported by the National Council for the Development of Research of Brazil (CNPq) (305042/2010-6, 303866/2010-1, and 303046/2009-1). M. N. Rocha is supported by CNPq (142361/2009-7). This work was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) (APQ-00197-11, APQ-01123-09, PRONEX, PPM–00382-11). The authors would like to thank Dr. Deborah Sullivan-Davis for reviewing the paper.

References

A. Bittencourt, A. Barral, A. R. de Jesus, R. P. de Almeida, and G. Grimaldi Júnior, “In situ identification of Leishmania amazonensis associated with diffuse cutaneous leishmaniasis in Bahia, Brazil,” Memórias do Instituto Oswaldo Cruz, vol. 84, no. 4, pp. 585–586, 1989.

S. L. Croft, S. Sundar, and A. H. Fairlamb, “Drug resistance in leishmaniasis,” Clinical Microbiology Reviews, vol. 19, no. 1, pp. 111–126, 2006.

S. L. Croft, K. Seifert, and V. Yardley, “Current scenario of drug development for leishmaniasis,” Indian Journal of Medical Research, vol. 123, no. 3, pp. 399–410, 2006.

J. Mishra, A. Saxena, and S. Singh, “Chemotherapy of leishmaniasis: past, present and future,” Current Medicinal Chemistry, vol. 14, no. 10, pp. 1153–1169, 2007.

N. M. F. Lima, C. S. Correia, L. L. Leon et al., “Antileishmanial activity of lapachol analogues,” Memórias do Instituto Oswaldo Cruz, vol. 99, no. 7, pp. 757–761, 2004.

P. C. Andrews, R. L. Ferrero, P. C. Junk et al., “Bismuth(iii) complexes derived from non-steroidal anti-inflammatory drugs and their activity against Helicobacter pylori,” Dalton Transactions, vol. 39, no. 11, pp. 2861–2868, 2010.

B. Raychaudhury, S. Banerjee, S. Gupta, R. V. Singh, and S. C. Datta, “Antiparasitic activity of a triphenyl tin complex against Leishmania donovani,” Acta Tropica, vol. 95, no. 1, pp. 1–8, 2005.

P. C. Andrews, R. Frank, P. C. Junk, L. Kedzierski, I. Kumar, and J. G. MacLellan, “Anti-Leishmanial activity of homo- and heteroleptic bismuth(III) carboxylates,” Journal of Inorganic Biochemistry, vol. 105, no. 3, pp. 454–461, 2011.

L. G. Oliveira, M. M. Silva, F. C. Paula et al., “Antimony(V) and bismuth(V) complexes of lapachol: synthesis, crystal structure and cytotoxic activity,” Molecules, vol. 16, no. 12, pp. 10314–10323, 2011.

M. N. Rocha, C. Margonari, I. M. Presot, and R. P. Soares, “Evaluation of 4 polymerase chain reaction protocols for cultured Leishmania spp. typing,” Diagnostic Microbiology and Infectious Disease, vol. 68, no. 4, pp. 401–409, 2010.

R. P. P. Soares, M. E. Macedo, C. Ropert et al., “Leishmania chagasi: lipophosphoglycan characterization and binding to the midgut of the sand fly vector Lutzomyia longipalpis,” Molecular and Biochemical Parasitology, vol. 121, no. 2, pp. 213–224, 2002.

D. O. D. V. M. Ernest, M. C. D. V. M. Brenda, and A. A. McWilliam, Guide to the Care and Use of Experimental Animals, Canadian Council on Animal Care, 1993.

M. N. Rocha, C. M. Correa, M. N. Melo et al., “An alternative in vitro drug screening test using Leishmania amazonensis transfected with red fluorescent protein,” Diagnostic Microbiology and Infectious Diseases, vol. 75, no. 3, pp. 282–291, 2013.

J. D. Berman and L. S. Lee, “Activity of antileishmanial agents against amastigotes in human monocyte-derived macrophages and in mouse peritoneal macrophages,” Journal of Parasitology, vol. 70, no. 2, pp. 220–225, 1984.

A. C. Pinheiro, M. N. Rocha, P. M. Nogueira et al., “Synthesis, cytotoxicity, and in vitro antileishmanial activity of mono-t-butyloxycarbonyl-protected diamines,” Diagnostic Microbiology and Infectious Disease, vol. 71, no. 3, pp. 273–278, 2011.

G. J. Darlington, J. H. Kelly, and G. J. Buffone, “Growth and hepatospecific gene expression of human hepatoma cells in a defined medium,” In Vitro Cellular & Developmental Biology, vol. 23, no. 5, pp. 349–354, 1987.

F. Denizot and R. Lang, “Rapid colorimetric assay for cell growth and survival—modifications to the tetrazolium dye procedure giving improved sensitivity and reliability,” Journal of Immunological Methods, vol. 89, no. 2, pp. 271–277, 1986.

M. C. Madureira, A. P. Martins, M. Gomes, J. Paiva, A. P. Cunha, and V. Rosário, “Antimalarial activity of medicinal plants used in traditional medicine in S. Tomé and Príncipe islands,” Journal of Ethnopharmacology, vol. 81, pp. 23–29, 2002.

I. Oliveira, A. Sousa, J. S. Morais et al., “Chemical composition, and antioxidant and antimicrobial activities of three hazelnut (Corylus avellana L.) cultivars,” Food and Chemical Toxicology, vol. 46, no. 5, pp. 1801–1807, 2008.

S. Nwaka and A. Hudson, “Innovative lead discovery strategies for tropical diseases,” Nature Reviews Drug Discovery, vol. 5, no. 11, pp. 941–955, 2006.

J. R. Ioset, R. Brun, T. Wenzler, M. Kaiser, and V. Yardley, “Drug screening for kinetoplastids diseases: a training manual for screening in neglected diseases,” DNDi and Pan-Asian Screening Network, pp. 1–74, 2009.

D. Sereno, A. Cordeiro da Silva, F. Mathieu-Daude, and A. Ouaissi, “Advances and perspectives in Leishmania cell based drug-screening procedures,” Parasitology International, vol. 56, no. 1, pp. 3–7, 2007.

P. Chirac and E. Torreele, “Global framework on essential health R&D,” The Lancet, vol. 367, no. 9522, pp. 1560–1561, 2006.

R. Pink, A. Hudson, M. A. Mouriès, and M. Bendig, “Opportunities and challenges in antiparasitic drug discovery,” Nature Reviews Drug Discovery, vol. 4, no. 9, pp. 727–740, 2005.

M. Moran, J. Guzman, A. L. Ropars et al., “Neglected disease research and development: how much are we really spending?” PLoS Medicine, vol. 6, no. 2, Article ID e1000030, 2009.

J. L. Siqueira-Neto, O. R. Song, H. Oh et al., “Antileishmanial high-throughput drug screening reveals drug candidates with new scaffolds,” PLOS Neglected Tropical Diseases, vol. 4, no. 5, article e675, 2010.

G. de Muylder, K. K. H. Ang, S. Chen, M. R. Arkin, J. C. Engel, and J. H. McKerrow, “A screen against Leishmania intracellular amastigotes: comparison to a promastigote screen and identification of a host cell-specific hit,” PLoS Neglected Tropical Diseases, vol. 5, no. 7, Article ID e1253, 2011.

F. G. de Miranda, J. C. Vilar, I. A. Alves, S. C. Cavalcanti, and A. R. Antoniolli, “Antinociceptive and antiedematogenic properties and acute toxicity of Tabebuia avellanedae Lor. ex Griseb. inner bark aqueous extract,” BMC Pharmacology, vol. 1, no. 1, article 6, 2001.

V. F. Andrade-Neto, M. G. L. Brandão, F. Q. Oliveira et al., “Antimalarial activity of Bidens pilosa L. (Asteraceae) ethanol extracts from wild plants collected in various localities or plants cultivated in humus soil,” Phytotherapy Research, vol. 18, no. 8, pp. 634–639, 2004.

A. V. Pinto and S. L. de Castro, “The trypanocidal activity of naphthoquinones: a review,” Molecules, vol. 14, no. 11, pp. 4570–4590, 2009.

J. M. Matés and F. M. Sánchez-Jiménez, “Role of reactive oxygen species in apoptosis: implications for cancer therapy,” The International Journal of Biochemistry & Cell Biology, vol. 32, no. 2, pp. 157–170, 2000.

G. L. Parrilha, R. P. Vieira, P. P. Campos et al., “Coordination of lapachol to bismuth(III) improves its anti-inflammatory and anti-angiogenic activities,” BioMetals, vol. 25, no. 1, pp. 55–62, 2012.

J. Tonholo, L. R. Freitas, F. C. de Abreu et al., “Electrochemical properties of biologically active heterocyclic naphthoquinones,” Journal of the Brazilian Chemical Society, vol. 9, no. 2, pp. 163–169, 1998.

J. Benites, J. A. Valderrama, F. Rivera et al., “Studies on quinones—part 42: synthesis of furylquinone and hydroquinones with antiproliferative activity against human tumor cell lines,” Bioorganic and Medicinal Chemistry, vol. 16, no. 2, pp. 862–868, 2008.

E. A. Hillard, F. C. de Abreu, D. C. M. Ferreira, G. Jaouen, M. O. F. Goulart, and C. Amatore, “Electrochemical parameters and techniques in drug development, with an emphasis on quinones and related compounds,” Chemical Communications, no. 23, pp. 2612–2628, 2008.