research article cytotoxicity and in vitro antileishmanial...

Hindawi Publishing CorporationBioinorganic Chemistry and ApplicationsVolume 2013 Article ID 961783 7 pageshttpdxdoiorg1011552013961783

Research ArticleCytotoxicity and In Vitro Antileishmanial Activity ofAntimony (V) Bismuth (V) and Tin (IV) Complexes of Lapachol

Marcele Neves Rocha1 Paula Monalisa Nogueira1 Cynthia Demicheli2

Ludmila Gonccedilalvez de Oliveira2 Meiriane Mariano da Silva2 Freacutedeacuteric Freacutezard3

Maria Norma Melo4 and Rodrigo Pedro Soares1

1 Centro de Pesquisas Rene Rachou Fundacao Oswaldo CruzFIOCRUZ 30190-002 Belo Horizonte MG Brazil2 Departamento de Quımica Instituto de Ciencias Exatas Universidade Federal de Minas Gerais 31270-901 Belo HorizonteMG Brazil

3 Departamento de Fisiologia e Biofısica Instituto de Ciencias Biologicas Universidade Federal de Minas Gerais 31270-901 BeloHorizonte MG Brazil

4Departamento de Parasitologia Instituto de Ciencias Biologicas Universidade Federal de Minas Gerais31270-901 Belo Horizonte MG Brazil

Correspondence should be addressed to Rodrigo Pedro Soares rsoarescpqrrfiocruzbr

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

Academic Editor Patrick Bednarski

Copyright copy 2013 Marcele Neves Rocha et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Leishmania amazonensis is the etiologic agent of the cutaneous and diffuse leishmaniasis often associated with drug resistanceLapachol [2-hydroxy-3-(31015840-methyl-2-butenyl)-14-naphthoquinone] displays a wide range of antimicrobial properties againstmanypathogens In this study using the classicmicroscopic in vitromodel we have analyzed the effects of a series of lapachol and chloridescomplexes with antimony (V) bismuth (V) and tin (IV) against L amazonensis All seven compounds exhibited antileishmanialactivity but most of the antimony (V) and bismuth (V) complexes were toxic against humanHepG2 cells andmurinemacrophagesThe best IC

50values (017 plusmn 003 and 010 plusmn 011 120583gmL) were observed for Tin (IV) complexes (3) [(Lp)(Ph

3Sn)] and (6) (Ph

3SnCl2)

respectively Their selective indexes (SIs) were 7065 and 12035 for HepG2 cells respectively However while analyzing murinemacrophages the SI decreased Those compounds were moderately toxic for HepG2 cells and toxic for murine macrophages stillunderlying the need of chemical modification in this class of compounds

1 Introduction

Leishmania amazonensis a New World species has beenidentified as a dermotropic species often associated with drugresistance [1] Current antileishmanial therapies are toxic tohuman and some simply fail [2 3] In the Americas forover six decades parenteral administrations of pentavalentantimonials (Sb-V) sodium stibogluconate (Pentostam) andmeglumine antimoniate (Glucantime) have been used fortreating leishmaniasis In places where resistance to anti-monials is common such as India other chemotherapeutictreatments include amphotericin B and pentamidine [2 4]Therefore the absence of a low toxic and safe oral drug stillunderlines the need for new antileishmanial compounds

Lapachol [2-hydroxy-3-(31015840-methyl-2-butenyl)-14-naph-thoquinone] (Figure 1) is a natural compound extractedfrom the core of Bignoniaceae trees In Leishmania lapacholanalogues derivatives and complexes have been tested byseveral groups Lapachol isolapachol and some of theirderivatives were active in vitro and in vivo against Leishma-nia braziliensis and L amazonensis respectively [5] Bismuth(III) antimony (V) and tin (IV) complexes were activeagainst Helicobacter pylori Leishmania major and Leishma-nia donovani respectively [6ndash8]

The design of bifunctionalmetal complex where both theligand and the metal exert pharmacological activity repre-sents a promising strategy for achieving more effective andselective drugs In the present study lapachol was coupled

2 Bioinorganic Chemistry and Applications

Bi

(1) (2) (3)

(4) (5) (6)

Bi

HO

OH

Sb

Bi SbCl Cl Cl

Cl

Cl

Sn

Sn

O

O

O

O O

O

O

O

OO

O

O

O

O

O

Lapachol metal complexes

Chloride metal complexes

(7) Lapachol

Figure 1 Structures of lapachol metal (Bi Sb and Sn) complexes (1ndash3) and chloride metal (Bi Sb and Sn) compounds (4ndash6) and lapachol(7) Legend Bi = bismuth Sb = antimony and Sn = tin

with three different metals triphenyltin (IV) triphenylbis-muth (V) and triphenylantimony (V) We have tested the invitro activity and cytotoxicity of synthesized antimony (V)bismuth (V) and tin (IV) lapachol and chloride complexesagainst intracellular L amazonensis HepG2 cells andmurinemacrophages

2 Materials and Methods

21 Synthesis of the Lapachol Metal Complexes andTested Metal Chlorides The (Lp)(Ph

3Bi)O05(1) 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 (70120583L) wasadded to a mixture of lapachol (0121 g 05mmol) andtriphenyltin (IV) chloride (193mg 05mmol) in chloroform(20mL) The resulting mixture was stirred for 4 h at roomtemperature Removal of the solvent under vacuum yieldeda solid material The material was subsequently dissolved inacetone and precipitated in water The triethylammoniumhydrochloride formed during the reaction was dissolved andremoved by water Elemental analyses were carried out usinga Perkin-Elmer 240 Elemental Analyzer Atomic absorptionanalyses 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 theformation of (Lp)(Ph

3Sb)OH complex as follows

LpH + Et3NH THF997888997888997888rarr Lpminus + [Et

3NH]+

Lpminus + Ph3SbCl2+ [Et3NH]+

THF997888997888997888rarr LpSbPh

3Cl + [Et

3NH]Cl

LpSbPh3Cl + [Et

3NH]Cl +H

2O

997888rarr LpSbPh3OH + [Et

3NH] +HCl

(1)

The same process can be proposed for all complexes Theyields melting points and elemental analyses of the com-pounds prepared are given in Table 1

Triphenylbismuth dichloride (4) triphenylantimonydichloride (5) triphenyltin chloride (6) and lapachol (7)were obtained from Aldrich Triethylamine was obtainedfrom SigmaThe predicted structures of all tested compoundsare shown in Figure 1

22 Parasites The World Health Organization (WHO) ref-erence strain L amazonensis (IFLABR1967PH8) was usedand typed as previously described [10] Promastigote forms

Bioinorganic Chemistry and Applications 3

Table 1 Yields and elemental analyses of the compounds

Compound Yield () Mp (∘C)a C found (calc) () H found (calc) () Metal found (calc) () Formula for calc(1) 79 126ndash129 5731 (5740) 409 (423) 2903 (2998) (Lp)(Ph3Bi)O05(2) 76 154ndash156 6530 (6482) 452 (478) 2064 (1919) (Lp)(Ph3Sb)OH(3) 79 107ndash109 6641 (6703) 440 (477) 2174 (2007) (Lp)(Ph3Sn)aMp melting point

were grown at 25∘C in M199 medium (Sigma) supple-mented with 10 heat-inactivated fetal calf serum (Culti-lab) 40mmolL HEPES (Amersham) 01mmolL adenine(Sigma) 00005 hemin (Sigma) 00002 biotin (Sigma)50 unitsmL penicillin and 50mgmL streptomycin (Invitro-gen) [11]

23 In Vitro Classic Microscopic Tests Animals were keptin the Animal Facility of the Centro de Pesquisas ReneRachouFIOCRUZ in strict accordance to the Guide for theCare and Use of Experimental Animals [12] The procedureswere approved by the Internal Ethics Committee in Ani-mal Experimentation (CEUA) of Fundacao Oswaldo Cruz(FIOCRUZ) Brazil (Protocol L-04208) Mice were eutha-nizedwithCO

2in an induction chamber prior tomacrophage

removal Balbc mice were injected intraperitoneally with2mL of 3 sodium thioglycollate medium After 72 h peri-toneal macrophages were removed by washing with coldRPMI 1640 medium and enriched by adherence to roundglass coverslips (13mm) placed in a 4-well culture plate Cells(2 times 105 cellswell) were cultured (37∘C 5 CO

2 18 h) in

RPMI supplemented with 10 heat-inactivated FBS (fetalbovine serum) prior to infection with parasites Macrophageswere exposed to stationary phase promastigotes (2times 106well)at a final ratio of 1 10 The plates were incubated at 37∘C 5CO2for 5 h in BOD to allow internalization of parasites [13]

Then the medium was removed for the remaining noninter-nalized parasites Negative control included only infectedmacrophages and medium Incubations were tested in dupli-cate in two independent experiments [14 15] The sub-stances were serial diluted with RPMI 1640 medium sup-plemented with 10 FBS at five different concentrations(50 rarr 31 120583gmL) For compounds (3) and (6) the dilutionwas 10 rarr 0016 120583gmL Amphotericin B was used as refer-ence drug Infected macrophages were exposed daily to thecompounds for 3 consecutive days After this period cover-slips were collected stained with Panoptic (Laborclin) andsubsequently mounted with Entellan (Merck) on glass slides

24 Cytotoxicity Tests The cell lineage HepG2 A16 wasderived from a human hepatocellular carcinoma cell lineHepG2 (ATCC HB-8065) and obtained from AmericaType Culture Collection line (ATCC) [16] Balbc murineperitoneal macrophages were obtained as described aboveCytotoxicity was determined using theMTTmethod (3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide)(Sigma) HepG2 cells were kept in RPMI medium supplem-ented with 10 FBS and confluent monolayers were trypsi-nized washed in RPMI and transferred to 96-well microtiterplates (4 times 104 cellswell) for 16ndash18 h Murine macrophages

were used in the concentration 2 times 105 cellswell in 96-well microtiter plates The compounds were serial dilutedin different concentrations (10 rarr 016mgmL) In bothtests the medium was removed and the compounds wereincubated for 24 h (37∘C 5 CO

2) Colorimetric reaction

was developed following the incubation with MTT (37∘C5 CO

2 4 h) and addition of acidified isopropanol [17] The

reaction was read spectrophotometrically (Spectramax M5Molecular Devices San Francisco CA) with a 570 nm filterand a background of 670 nm Incubations were tested intriplicate in two independent experiments The minimumdose that killed 50of the cells (MLD

50)was determined [18]

and the values were plotted to generate dose-response curvesusing Microcal Origin Software (Northampton MA USA)[15 19] The selective indexes (SIs) of compounds werecalculated using the MLD

50IC50

ratios to HepG2 andperitoneal macrophages [20 21]

3 Results

The in vitro classicmicroscopic test enables direct counting todetermine the percentage of infected cells andor the numberof amastigotes [22] Here the IC

50values were calculated

based on the percentage of infected macrophages [15] Thein vitro antileishmanial activities cytotoxicity and selectiveindexes (SIs) of lapachol metal complexes and chlorides (1ndash6) lapachol (7) and amphotericin B are shown in Table 2Lapachol and compounds (1) (2) and (5) were consideredinactive (IC

50gt 10120583gmL) and toxic (SIlt 20) forHepG2 cells

and macrophages [20 21] The tin (IV) lapachol complex (3)and chloride (6) were active against intracellular amastigoteforms of L amazonensis (Figures 2(a) and 2(b)) and lesstoxic for HepG2 cells (SIs ranging from 7065 to 12035)(Figures 2(d) and 2(e)) (Table 2) One triphenyl bismuthchloride (4) (Figure 2(c)) was also active and a little moretoxic for HepG2 cells (Figure 2(f)) than (3) (Figure 2(d))and (6) (Figure 2(e)) (SI = 3403) All compounds weretoxic for murine macrophages (SI lt 20) Amphotericin Ban antileishmanial reference drug exhibited an IC

50value

approximately fourfold higher than (3) and (6) (073 plusmn060 120583gmL) (Table 2)

4 Discussion

Leishmaniases are considered by the WHO as one of themajor six important infectious diseases worldwide Overthe past years the absence of research and developmentfor new medicines targeting diseases affecting people indeveloping countries has become a global concern [23]


100

80

60

40

20

0

0 101 10

Mac

roph

ages

infe

cted

()

IC50 = 015 120583gmL

3

(a)

IC50 = 018 120583gmL

100

80

60

40

20

0

6

0 101 10

Mac

roph

ages

infe

cted

()

(b)

IC50 = 551120583gmL

100

100

80

60

40

20

0

Mac

roph

ages

infe

cted

()

0 1 10

4

(c)

Cel

l via

bilit

y (

)

100

80

60

40

20

0

MLD50 = 119 120583gmL

1000 1 10 1000

3

(d)

Cel

l via

bilit

y (

)

100

80

60

40

20

0

1000 1 10 1000

MLD50 = 18120583gmL

6

(e)

MLD50 = 1871 120583gmL

Cel

l via

bilit

y (

)

100

80

60

40

20

0

1000 1 10 1000

4

(f)

Figure 2 In vitro antileishmanial activity of compounds (3) (4) and (6) against intracellular L amazonensis ((a) (b) and (c)) and cytotoxicityagainst hepatoma HepG2 cell ((d) (e) and (f)) Curves were obtained using Microcal Origin Software IC

50= half-maximal inhibitory

response MLD50= the minimum lethal dose Figures are a representation of one experiment

Currently the development of new drugs combinations orprotocols against tropical and neglected diseases is of greatimportance in public health [24ndash27] However side effectstreatment failure due to parasite resistance HIV coinfectionand intravenous administration are the major concernshindering leishmaniasis chemotherapy [2 3]

Lapachol derivatives and complexes have exhibited anti-tumor anti-inflammatory antiangiogenic analgesic andantimicrobial properties [6 28ndash32] Lapachol and some of itsanalogues demonstrated activity in vitro againstL braziliensisand L amazonensis [5] The use of metal complexes againstLeishmania may represent a potential alternative against


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

Compound Formula IC50

a HepG2 MacrophagesMLD50

b SIc MLD50b SIc

(1) (Lp)(Ph3Bi)O05 2905 plusmn 1845 5838 plusmn 847 201 324 plusmn 820 111(2) (Lp)(Ph3Sb)OH 1827 plusmn 558 32522 plusmn 8940 1781 13065 plusmn 4052 715(3) (Lp)(Ph3Sn) 017 plusmn 003 1201 plusmn 017 7065 16 plusmn 057 941(4) Ph3BiCl2 540 plusmn 016 18375 plusmn 477 3403 2515 plusmn 049 467(5) Ph3SbCl2 1161 plusmn 785 15746 plusmn 3713 1356 3075 plusmn 601 265(6) Ph3SnCl2 010 plusmn 011 1204 plusmn 842 12035 073 plusmn 013 730(7) Lpd 1548 plusmn 523 20177 plusmn 532 1303 18465 plusmn 658 1192Amphotericin B 073 plusmn 060 64459 plusmn 12657 88300 17995 plusmn 884 24651aIC50 the inhibitory concentration that killed 50 of the L amazonensis in 120583gmLbMLD50 the minimum lethal dose that killed 50 of the cells in 120583gmLcSI selective index calculated based on the MLD50IC50 ratiosdLp Lapachol

the disease since antimony-based regimens tend to be verytoxic In this context we have explored the use of lapacholand 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 againstL amazonensis (1548 plusmn 523 versus 52 plusmn 070 120583gmL) [5]This IC

50value is close to that observed for L braziliensis

(119 plusmn 69 120583gmL) This discrepancy could be attributed tothe strain of L amazonensis used (MHOMBR77LTB0016)and experimental conditions The highest antiproliferativeactivity against intracellular L amazonensis was observed fortin (IV) lapachol and chloride complexes (3) and (6) (Figures2(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 amongall substances tested while using HepG2 cells (SIs of 7065and 12035 resp) Interestingly the resulting compound oflapachol and tin (IV) showed a marked decrease in metaltoxicity than lapachol alone (SIs of 7065 versus 1303 resp)One of the possibilities that could justify such phenomenoncould be due to an increase in the lipophilicity of the lapachol-complexed molecule Another hypothesis is that lapacholcomplexation could affect the REDOX potential of the com-pound thus consequently changing its activity Consistentwith this idea themechanisms underlying those activities arerelated to the generation of reactive oxygen radicals (ROSs)induced by the bioreduction of its quinonoid nucleus throughspecific enzymes and oxygen [33ndash35] ROS mechanismsinduced by lapachol have been implicated in the chemothera-peutic activities against many protozoa such as Trypanosomacruzi [30] and also tumor cells [31] Similarly among allmetal chloride substances the triphenyl tin (IV) chloridecompound exhibited lower toxicity compared to bismuth(V) and antimony (V) chloride ones Finally compound (4)exhibited moderate toxicity (SI = 3403) with an IC

50value 7-

fold higher than amphotericin B However when cytotoxicitywas tested against murine macrophages the host cells forLeishmania all compounds were toxic Those data indicatethe need of chemicalmodifications in this class of compoundsin the search of novel antileishmanial molecules

5 Conclusions

Lapachol and a series of six lapachol and chloride metalcomplexes have been evaluated for their in vitro activityagainst intracellular amastigote forms of L amazonensis Thetin (IV) lapachol and chloride complexes (3 and 6) exhibitedhigher antileishmanial activity compared to amphotericinB The triphenyl bismuth (V) compound (4) also exhibitedantileishmanial activity with moderate cytotoxicity Lapacholcompounds with bismuth (V) and tin (IV) were less toxicwhen compared with lapachol alone for HepG2 cells Inconclusion tin and in a less extent bismuth complexes weremoderately toxic forHepG2 cells and toxic formurinemacro-phages

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 Frezard and M N Melo aresupported by the National Council for the Development ofResearch of Brazil (CNPq) (3050422010-6 3038662010-1and 3030462009-1) M N Rocha is supported by CNPq(1423612009-7) This work was supported by Fundacao deAmparo a Pesquisa do Estado de Minas Gerais (FAPEMIG)(APQ-00197-11 APQ-01123-09 PRONEX PPMndash00382-11)The authors would like to thank Dr Deborah Sullivan-Davisfor reviewing the paper

References

[1] A Bittencourt A Barral A R de Jesus R P de Almeidaand G Grimaldi Junior ldquoIn situ identification of Leishmaniaamazonensis associated with diffuse cutaneous leishmaniasis in


Bahia BrazilrdquoMemorias do Instituto Oswaldo Cruz vol 84 no4 pp 585ndash586 1989

[2] S L Croft S Sundar and A H Fairlamb ldquoDrug resistance inleishmaniasisrdquo Clinical Microbiology Reviews vol 19 no 1 pp111ndash126 2006

[3] S L Croft K Seifert and V Yardley ldquoCurrent scenario ofdrug development for leishmaniasisrdquo Indian Journal of MedicalResearch vol 123 no 3 pp 399ndash410 2006

[4] J Mishra A Saxena and S Singh ldquoChemotherapy of leishma-niasis past present and futurerdquo Current Medicinal Chemistryvol 14 no 10 pp 1153ndash1169 2007

[5] N M F Lima C S Correia L L Leon et al ldquoAntileishmanialactivity of lapachol analoguesrdquo Memorias do Instituto OswaldoCruz vol 99 no 7 pp 757ndash761 2004

[6] P C Andrews R L Ferrero P C Junk et al ldquoBismuth(iii) com-plexes derived fromnon-steroidal anti-inflammatory drugs andtheir activity against Helicobacter pylorirdquo Dalton Transactionsvol 39 no 11 pp 2861ndash2868 2010

[7] B Raychaudhury S Banerjee S Gupta R V Singh and S CDatta ldquoAntiparasitic activity of a triphenyl tin complex againstLeishmania donovanirdquo Acta Tropica vol 95 no 1 pp 1ndash8 2005

[8] P C Andrews R Frank P C Junk L Kedzierski I Kumarand J G MacLellan ldquoAnti-Leishmanial activity of homo- andheteroleptic bismuth(III) carboxylatesrdquo Journal of InorganicBiochemistry vol 105 no 3 pp 454ndash461 2011

[9] L G Oliveira M M Silva F C Paula et al ldquoAntimony(V) andbismuth(V) complexes of lapachol synthesis crystal structureand cytotoxic activityrdquoMolecules vol 16 no 12 pp 10314ndash103232011

[10] MN Rocha CMargonari I M Presot and R P Soares ldquoEval-uation of 4 polymerase chain reaction protocols for culturedLeishmania spp typingrdquoDiagnostic Microbiology and InfectiousDisease vol 68 no 4 pp 401ndash409 2010

[11] R P P Soares M E Macedo C Ropert et al ldquoLeishmaniachagasi lipophosphoglycan characterization and binding to themidgut of the sand fly vector Lutzomyia longipalpisrdquoMolecularand Biochemical Parasitology vol 121 no 2 pp 213ndash224 2002

[12] D O D V M Ernest M C D V M Brenda and A AMcWilliamGuide to the Care and Use of Experimental AnimalsCanadian Council on Animal Care 1993

[13] M N Rocha C M Correa M N Melo et al ldquoAn alternative invitro drug screening test using Leishmania amazonensis trans-fected with red fluorescent proteinrdquo Diagnostic Microbiologyand Infectious Diseases vol 75 no 3 pp 282ndash291 2013

[14] J D Berman and L S Lee ldquoActivity of antileishmanial agentsagainst amastigotes in human monocyte-derived macrophagesand in mouse peritoneal macrophagesrdquo Journal of Parasitologyvol 70 no 2 pp 220ndash225 1984

[15] A C Pinheiro M N Rocha P M Nogueira et al ldquoSynthesiscytotoxicity and in vitro antileishmanial activity of mono-t-butyloxycarbonyl-protected diaminesrdquoDiagnosticMicrobiologyand Infectious Disease vol 71 no 3 pp 273ndash278 2011

[16] G J Darlington J H Kelly and G J Buffone ldquoGrowth andhepatospecific gene expression of human hepatoma cells in adefined mediumrdquo In Vitro Cellular amp Developmental Biologyvol 23 no 5 pp 349ndash354 1987

[17] F Denizot and R Lang ldquoRapid colorimetric assay for cellgrowth and survivalmdashmodifications to the tetrazolium dyeprocedure giving improved sensitivity and reliabilityrdquo Journalof Immunological Methods vol 89 no 2 pp 271ndash277 1986

[18] M CMadureira A PMartinsMGomes J Paiva A P Cunhaand V Rosario ldquoAntimalarial activity of medicinal plants usedin traditionalmedicine in S Tome and Prıncipe islandsrdquo Journalof Ethnopharmacology vol 81 pp 23ndash29 2002

[19] I Oliveira A Sousa J S Morais et al ldquoChemical compositionand antioxidant and antimicrobial activities of three hazelnut(Corylus avellana L) cultivarsrdquo Food and Chemical Toxicologyvol 46 no 5 pp 1801ndash1807 2008

[20] S Nwaka and A Hudson ldquoInnovative lead discovery strategiesfor tropical diseasesrdquoNature Reviews Drug Discovery vol 5 no11 pp 941ndash955 2006

[21] J R Ioset R Brun TWenzler M Kaiser and V Yardley ldquoDrugscreening for kinetoplastids diseases a training manual forscreening in neglected diseasesrdquoDNDi and Pan-Asian ScreeningNetwork pp 1ndash74 2009

[22] D Sereno A Cordeiro da Silva F Mathieu-Daude and AOuaissi ldquoAdvances and perspectives in Leishmania cell baseddrug-screening proceduresrdquo Parasitology International vol 56no 1 pp 3ndash7 2007

[23] P Chirac and E Torreele ldquoGlobal framework on essential healthRampDrdquoThe Lancet vol 367 no 9522 pp 1560ndash1561 2006

[24] R Pink A Hudson M A Mouries and M Bendig ldquoOppor-tunities and challenges in antiparasitic drug discoveryrdquo NatureReviews Drug Discovery vol 4 no 9 pp 727ndash740 2005

[25] M Moran J Guzman A L Ropars et al ldquoNeglected diseaseresearch and development how much are we really spendingrdquoPLoS Medicine vol 6 no 2 Article ID e1000030 2009

[26] J L Siqueira-Neto O R Song H Oh et al ldquoAntileishmanialhigh-throughput drug screening reveals drug candidates withnew scaffoldsrdquo PLOS Neglected Tropical Diseases vol 4 no 5article e675 2010

[27] G de Muylder K K H Ang S Chen M R Arkin J CEngel and J H McKerrow ldquoA screen against Leishmania intra-cellular amastigotes comparison to a promastigote screen andidentification of a host cell-specific hitrdquo PLoS Neglected TropicalDiseases vol 5 no 7 Article ID e1253 2011

[28] F G de Miranda J C Vilar I A Alves S C Cavalcanti andA R Antoniolli ldquoAntinociceptive and antiedematogenic prop-erties and acute toxicity of Tabebuia avellanedae Lor ex Grisebinner bark aqueous extractrdquo BMC Pharmacology vol 1 no 1article 6 2001

[29] V F Andrade-Neto M G L Brandao F Q Oliveira et alldquoAntimalarial activity of Bidens pilosa L (Asteraceae) ethanolextracts fromwild plants collected in various localities or plantscultivated in humus soilrdquo Phytotherapy Research vol 18 no 8pp 634ndash639 2004

[30] A V Pinto and S L de Castro ldquoThe trypanocidal activity ofnaphthoquinones a reviewrdquoMolecules vol 14 no 11 pp 4570ndash4590 2009

[31] J M Mates and F M Sanchez-Jimenez ldquoRole of reactiveoxygen species in apoptosis implications for cancer therapyrdquoThe International Journal of Biochemistry amp Cell Biology vol 32no 2 pp 157ndash170 2000

[32] G L Parrilha R P Vieira P P Campos et al ldquoCoordinationof lapachol to bismuth(III) improves its anti-inflammatory andanti-angiogenic activitiesrdquo BioMetals vol 25 no 1 pp 55ndash622012

[33] J Tonholo L R Freitas F C de Abreu et al ldquoElectrochemicalproperties of biologically active heterocyclic naphthoquinonesrdquoJournal of the Brazilian Chemical Society vol 9 no 2 pp 163ndash169 1998


[34] J Benites J A Valderrama F Rivera et al ldquoStudies on quin-onesmdashpart 42 synthesis of furylquinone and hydroquinoneswith antiproliferative activity against human tumor cell linesrdquoBioorganic and Medicinal Chemistry vol 16 no 2 pp 862ndash8682008

[35] E A Hillard F C de Abreu D C M Ferreira G Jaouen M OF Goulart and C Amatore ldquoElectrochemical parameters andtechniques in drug development with an emphasis on quinonesand related compoundsrdquo Chemical Communications no 23 pp2612ndash2628 2008

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Applied ChemistryJournal of



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Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


Bi

(1) (2) (3)

(4) (5) (6)

Bi

HO

OH

Sb

Bi SbCl Cl Cl

Cl

Cl

Sn

Sn

O

O

O

O O

O

O

O

OO

O

O

O

O

O

Lapachol metal complexes

Chloride metal complexes

(7) Lapachol

Figure 1 Structures of lapachol metal (Bi Sb and Sn) complexes (1ndash3) and chloride metal (Bi Sb and Sn) compounds (4ndash6) and lapachol(7) Legend Bi = bismuth Sb = antimony and Sn = tin

with three different metals triphenyltin (IV) triphenylbis-muth (V) and triphenylantimony (V) We have tested the invitro activity and cytotoxicity of synthesized antimony (V)bismuth (V) and tin (IV) lapachol and chloride complexesagainst intracellular L amazonensis HepG2 cells andmurinemacrophages

2 Materials and Methods

21 Synthesis of the Lapachol Metal Complexes andTested Metal Chlorides The (Lp)(Ph

3Bi)O05(1) 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 (70120583L) wasadded to a mixture of lapachol (0121 g 05mmol) andtriphenyltin (IV) chloride (193mg 05mmol) in chloroform(20mL) The resulting mixture was stirred for 4 h at roomtemperature Removal of the solvent under vacuum yieldeda solid material The material was subsequently dissolved inacetone and precipitated in water The triethylammoniumhydrochloride formed during the reaction was dissolved andremoved by water Elemental analyses were carried out usinga Perkin-Elmer 240 Elemental Analyzer Atomic absorptionanalyses 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 theformation of (Lp)(Ph

3Sb)OH complex as follows

LpH + Et3NH THF997888997888997888rarr Lpminus + [Et

3NH]+

Lpminus + Ph3SbCl2+ [Et3NH]+

THF997888997888997888rarr LpSbPh

3Cl + [Et

3NH]Cl

LpSbPh3Cl + [Et

3NH]Cl +H

2O

997888rarr LpSbPh3OH + [Et

3NH] +HCl

(1)

The same process can be proposed for all complexes Theyields melting points and elemental analyses of the com-pounds prepared are given in Table 1

Triphenylbismuth dichloride (4) triphenylantimonydichloride (5) triphenyltin chloride (6) and lapachol (7)were obtained from Aldrich Triethylamine was obtainedfrom SigmaThe predicted structures of all tested compoundsare shown in Figure 1

22 Parasites The World Health Organization (WHO) ref-erence strain L amazonensis (IFLABR1967PH8) was usedand typed as previously described [10] Promastigote forms








2 18 h) in











50IC50


3 Results






50value


4 Discussion



100

80

60

40

20

0

0 101 10

Mac

roph

ages

infe

cted

()

IC50 = 015 120583gmL

3

(a)

IC50 = 018 120583gmL

100

80

60

40

20

0

6

0 101 10

Mac

roph

ages

infe

cted

()

(b)

IC50 = 551120583gmL

100

100

80

60

40

20

0

Mac

roph

ages

infe

cted

()

0 1 10

4

(c)

Cel

l via

bilit

y (

)

100

80

60

40

20

0

MLD50 = 119 120583gmL

1000 1 10 1000

3

(d)

Cel

l via

bilit

y (

)

100

80

60

40

20

0

1000 1 10 1000

MLD50 = 18120583gmL

6

(e)

MLD50 = 1871 120583gmL

Cel

l via

bilit

y (

)

100

80

60

40

20

0

1000 1 10 1000

4

(f)










b SIc MLD50b SIc






50value 7-


5 Conclusions




Financial Support


Acknowledgments


References
















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of








2 18 h) in











50IC50


3 Results






50value


4 Discussion



100

80

60

40

20

0

0 101 10

Mac

roph

ages

infe

cted

()

IC50 = 015 120583gmL

3

(a)

IC50 = 018 120583gmL

100

80

60

40

20

0

6

0 101 10

Mac

roph

ages

infe

cted

()

(b)

IC50 = 551120583gmL

100

100

80

60

40

20

0

Mac

roph

ages

infe

cted

()

0 1 10

4

(c)

Cel

l via

bilit

y (

)

100

80

60

40

20

0

MLD50 = 119 120583gmL

1000 1 10 1000

3

(d)

Cel

l via

bilit

y (

)

100

80

60

40

20

0

1000 1 10 1000

MLD50 = 18120583gmL

6

(e)

MLD50 = 1871 120583gmL

Cel

l via

bilit

y (

)

100

80

60

40

20

0

1000 1 10 1000

4

(f)










b SIc MLD50b SIc






50value 7-


5 Conclusions




Financial Support


Acknowledgments


References
















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


100

80

60

40

20

0

0 101 10

Mac

roph

ages

infe

cted

()

IC50 = 015 120583gmL

3

(a)

IC50 = 018 120583gmL

100

80

60

40

20

0

6

0 101 10

Mac

roph

ages

infe

cted

()

(b)

IC50 = 551120583gmL

100

100

80

60

40

20

0

Mac

roph

ages

infe

cted

()

0 1 10

4

(c)

Cel

l via

bilit

y (

)

100

80

60

40

20

0

MLD50 = 119 120583gmL

1000 1 10 1000

3

(d)

Cel

l via

bilit

y (

)

100

80

60

40

20

0

1000 1 10 1000

MLD50 = 18120583gmL

6

(e)

MLD50 = 1871 120583gmL

Cel

l via

bilit

y (

)

100

80

60

40

20

0

1000 1 10 1000

4

(f)










b SIc MLD50b SIc






50value 7-


5 Conclusions




Financial Support


Acknowledgments


References
















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





b SIc MLD50b SIc






50value 7-


5 Conclusions




Financial Support


Acknowledgments


References
















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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