Platinum(II) metal complexes as potentialanti-Trypanosoma cruzi agents

Marisol Vieites a Lucıa Otero a Diego Santos a Jeannette Toloza bRoberto Figueroa b Ester Norambuena c Claudio Olea-Azar b Gabriela Aguirre d

Hugo Cerecetto d Mercedes Gonzalez d Antonio Morello e Juan Diego Maya eBeatriz Garat f Dinorah Gambino a

a Catedra de Quımica Inorganica Facultad de Quımica Universidad de la Republica Gral Flores 2124 CC 1157 11800 Montevideo Uruguayb Departamento de Quımica Inorganica y Analıtica Facultad de Ciencias Quımicas y Farmaceuticas Universidad de Chile Casilla 233 Santiago Chile

c Departamento de Quımica Universidad Metropolitana de Ciencias de la Educacion Santiago Chiled Laboratorio de Quımica Organica Facultad de Quımica Facultad de Ciencias Universidad de la Republica Igua 4225 11400 Montevideo Uruguay

e Programa de Farmacologıa Molecular y Clınica ICBM Facultad de Medicina Universidad de Chile Independencia 1027 Santiago Chilef Laboratorio de Interacciones Moleculares Facultad de Ciencias Universidad de la Republica Igua 4225 11400 Montevideo Uruguay

Abstract

In the search for new therapeutic tools against Chagasrsquo disease (American Trypanosomiasis) two series of new platinum(II) complexeswith bioactive 5-nitrofuryl containing thiosemicarbazones as ligands were synthesized characterized and in vitro evaluated Most of thecomplexes showed IC50 values in the lM range against two different strains of Trypanosoma cruzi causative agent of the disease being asactive as the anti-trypanosomal drug Nifurtimox In particular the coordination of L3 (4-ethyl-1-(5-nitrofurfurylidene)thiosemicarba-zide) to Pt(II) forming [Pt(L3)2] lead to almost a five-fold activity increase in respect to the free ligand Trying to get an insight intothe trypanocidal mechanism of action of these compounds DNA and redox metabolism (intra-parasite free radical production) wereevaluated as potential parasite targets Results suggest that the complexes could inhibit parasite growth through a dual mechanism ofaction involving production of toxic free radicals by bioreduction and DNA interaction

Keywords Chagasrsquo disease Platinum 5-Nitrofuryl containing thiosemicarbazones Free radical production NS ligands

1 Introduction

According to WHO infectious and parasitic diseases aremajor causes of human disease worldwide [12] Althoughrepresenting a tremendous burden when compared to othercommunicable diseases that receive a high level of attention

Corresponding author Tel +5982 9249739 fax +5982 9241906E-mail address dgambinofqeduuy (D Gambino)

from health systems a group of parasitic and infectiousdiseases called neglected diseases has been characterizedby historically low investment by the pharmaceuticalindustry Often most affected populations are also thepoorest and the most vulnerable and they are found mainlyin tropical and subtropical areas of the world Among theseneglected diseases Chagasrsquo disease (American Trypanoso-miasis) is the largest parasitic disease burden in the Amer-ican continent affecting approximately 20 million peoplefrom southern United States to southern Argentina Themorbidity and mortality associated with this disease inAmerica are more than one order of magnitude higher thanthose caused by malaria schistosomiasis or leishmaniasis

[13] The disease caused by the flagellate protozoan para-site Trypanosoma cruzi is mainly transmitted to humans intwo ways either by blood-sucking reduviid insects ofTriatominae family which deposit their infective faeces onthe skin of the host at the time of biting or directly bytransfusion of infected blood Humans and a large numberof species of domestic and wild animals constitute the res-ervoir of the parasite

Despite the progress achieved in the study of T cruzirsquosbiochemistry and physiology in which several crucialenzymes for parasite survival absent in the host have beenidentified as potential new drug targets the chemotherapyof this parasitic infection remains undeveloped and noneffective method of immune prophylaxis is available Thetreatment has been based on old and quite unspecificnitroaromatic drugs that have significant activity only inthe acute phase of the disease and when associated withlong term treatments give rise to severe side effects[456ndash8]

In the search for a pharmacological control of Chagasrsquodisease metal complexes appear to be a promising newapproach [59ndash12] In this sense the design of complexescombining ligands bearing anti-trypanosomal activity andpharmacologically active metals has been successfullydeveloped This strategy takes advantage of the medicinalchemistry emerging drug discovery paradigm of developingagents that could modulate multiple targets simultaneouslywith the aim of enhancing efficacy or improving safety rel-ative to drugs that address only a single target [13] Thiscurrent approach based on the development of a singlechemical entity as a dual inhibitor capable to modulatemultiple targets simultaneously has the advantage of lowerrisk of drug-drug interactions compared to cocktails ormulticomponent drugs [1314] In the case of the metalcomplexation approach the obtained metal compoundscould act through dual or even multiple mechanisms ofaction by combining the pharmacological properties ofboth the ligand and the metal or at least lead to an addi-tive effect [1112] The development of single agents thatprovide maximal anti-protozoal activity by acting againstmultiple parasitic targets could diminish host toxic effectsby lowering therapeutic dose andor circumvent the devel-opment of drug resistance [15] Leading work performed bySanchez-Delgado et al drove to metal complexes of clo-trimazole and ketoconazole intended for anti-trypanosometherapy Synergistic effects were observed in most of thecases [1112] We have also been successfully working onthe development of potential anti-trypanosome agentsthrough this approach A series of vanadyl bioactive com-pounds of aromatic amine N-oxides has been developedand exhaustively studied [16] The anti-T cruzi activity oftwo novel series of palladium compounds of bioactive 5-nitrofuryl containing thiosemicarbazones has been evalu-ated in vitro and some aspects related to their possiblesynergistic effect and dual or even multiple mechanismsof action have been investigated These ligands had shownhigher in vitro activity against T cruzi than Nifurtimox the

nitrofuran drug used in the past Their main mode ofaction as for other nitroheterocyclic antiparasitic agentscould be related to the intracellular reduction of the nitromoiety followed by redox cycling yielding reactive oxygenspecies (ROS) known to cause cellular damage [17] Resultsstrongly suggested that the palladium complexes retainedthe mechanism of action of the thiosemicarbazone ligandsrelying their trypanocidal action mainly on the productionof oxidative stress as a result of their bioreduction to toxicfree radicals and extensive redox cycling Moreover thecomplexes strongly interacted with DNA and were foundto be irreversible inhibitors of trypanothione reductase aparasite-specific enzyme with an essential role in thedefense of trypanosomatids against oxidative stress [18]Having these results in mind platinum(II) coordinationof this last series of bioactive ligands seemed interestingbecause of the postulated metabolic similarities betweentumor cells and T cruzirsquos [11] Platinum compounds haveproven their effect on tumor cells and their ability to bindDNA as main anti-tumoral mechanism of action [19ndash21]In addition some Pt compounds have shown anti-T cruzi

activity acting through different mechanisms like DNAinteraction and irreversible inhibition of T cruzirsquos trypano-thione reductase [522ndash24]

Based on this approach in this work eight new plati-num(II) complexes of the bioactive thiosemicarbazonesshown in Fig 1 have been synthesized and characterizedTwo different compounds of the formula [PtCl2(HL)] and[Pt(L)2] were obtained for each ligand Compounds werestudied by elemental analyses conductometry electrosprayionization mass spectrometry (ESI-MS) and vibrational(IR and Raman) and 1H NMR spectroscopies Due to itspotential relevance in determining biological activitythrough generation of toxic free radicals by intra-parasitebioreduction electrochemical behavior was investigatedby cyclic voltammetry In addition the electrochemical freeradicals production was studied by electronic spin reso-nance technique (ESR) The in vitro anti-T cruzi activityof most of the compounds has been stated against two dif-ferent strains of the parasite Furthermore to get an insightinto the probable mechanism of anti-trypanosomal actionthe capacity to produce free radicals that could lead to par-asite death was evaluated by ESR experiments in the para-site and by respiration measurements In additioncompounds were tested for their DNA interaction abilityResults were compared with those previously reported forthe free ligands and the analogous palladium(II) com-pounds [18]

2 Experimental

21 Materials

All common laboratory chemicals were purchasedfrom commercial sources and used without furtherpurification K2[PtCl4] was commercially available All

NNH

S

NHROO2N

L1 R = HL2 R = methylL3 R = ethylL4 R = phenyl

NNH

S

NHROO2N

Cl

ClPt

NN

S

NHROO2N

NN

S

RHN ONO2

Pt

[PtCl2(HL)] [Pt(L)2]

a

b

Fig 1 Scheme showing (a) the 5-nitrofuryl containing thiosemicarbazones selected as ligands and (b) the two series of platinum(II) complexes

thiosemicarbazone ligands were synthesized using the pre-viously reported technique [17]

22 Syntheses of the complexes

221 [PtCl2(HL)] complexes L L1-L4K2[PtCl4] (50 mg 0120 mmol) and the corresponding

ligand (0120 mmol) were heated under reflux in methanol(10 mL) during 6 h after which a solid precipitated

222 [Pt(L)2] complexes L L1-L4

K2[PtCl4] (50 mg 0120 mmol) and the correspondingligand (0240 mmol) were heated under reflux in methanol(10 mL) during 6 h after which a solid precipitated

In all cases the obtained solid was filtered off andwashed with hot methanol followed by warm water Com-pounds were recrystallized by slow diffusion of water into adimethylformamide (DMF) solution of each compound

[PtCl2(HL1)] Brown solid yield 30 mg 52 Analcalc for C6H6Cl2N4O3SPt C 1501 H 126 N 1167 S668 Found C 1518 H 125 N 1170 S 667 ESI-MS (DMSO) mz 47896 (M) 44298 (MHCl)40898 (M2Cl)

[PtCl2(HL2)] Red-brown solid yield 32 mg 54 Analcalc for C7H8Cl2N4O3SPt C 1701 H 164 N 1134 S649 Found C 1681 H 167 N 1122 S 635 ESI-MS (DMSO) mz 49199 (MH) 45701 (MHCl)

[PtCl2(HL3)] Brown solid yield 27 mg 44 Analcalc for C8H10Cl2N4O3SPt C 1890 H 198 N 1102S 630 Found C 1902 H 202 N 1124 S 642

[PtCl2(HL4)] Red-orange solid yield 28 mg 42Anal calc for C12H10Cl2N4O3SPt C 2591 H 181 N1007 S 576 Found C 2601 H 180 N 1029 S 582

[Pt(L1)2] Dark brown solid yield 39 mg 52 Analcalc for C12H10N8O6S2Pt C 2319 H 162 N 1803 S1031 Found C 2308 H 168 N 1801 S 1008 ESI-MS (DMSO) mz 62196 (M+H)+ 62007 (MH)

[Pt(L2)2] Brown solid yield 28 mg 36 Anal calc forC14H14N8O6S2Pt C 2588 H 217 N 1725 S 987Found C 2579 H 213 N 1693 S 965

[Pt(L3)2] Red-orange solid yield 48 mg 78 Analcalc for C16H18N8O6S2Pt C 2836 H 267 N 1653 S946 Found C 2806 H 260 N 1639 S 929

[Pt(L4)2] Red-orange solid yield 43 mg 46 Analcalc For C24H18N8O6S2Pt C 3726 H 234 N 1448S 828 Found C 3683 H 235 N 1428 S 824

23 Physicochemical characterization

C H N and S analyses were performed with a CarloErba Model EA1108 elemental analyzer Conductimetricmeasurements were performed at 25 C in 103 M DMFsolutions using a Conductivity Meter 4310 Jenway [25]Electrospray ionization mass spectra (ESI-MS) of dimethylsulfoxide (DMSO) solutions of the complexes wererecorded on a TSQ Thermo Finnigan equipment with aHESI probe at the analytical services of the Instituto Ven-ezolano de Investigaciones Cientıficas (Caracas Venezuela)and the quoted mz values are for the major peaks in theisotope distribution FTIR spectra (4000ndash400 cm1) ofthe complexes and the free ligand were measured as KBrpellets with a Bomen FTIR model MB102 instrumentRaman spectra were scanned with the FRA 106 accessoryof a Bruker IF 66 FTIR spectrophotometer The 1064 nmradiation of a NdYAG laser was used for excitation and50ndash60 scans were routinely accumulated 1H NMR spectraof the complexes were recorded on a Bruker DPX-400instrument at 400 MHz Experiments were performed at30 C in acetone-d6 Tetramethylsilane was used as theinternal standard Cyclic voltammetry (CV) was carriedout on ca 10 103 M DMSO (spectroscopic grade) solu-tions using a Metrohm 693 VA instrument with a 694 VAStand convertor and a 693 VA Processor and a three-elec-trode cell under nitrogen atmosphere at room temperaturewith tetrabutyl ammonium perchlorate (TBA) (ca 01 M)as supporting electrolyte A hanging drop mercury elec-trode (HDME) was used as the working electrode a plati-num wire as the auxiliary electrode and saturated calomel(SCE) as the reference electrode ESR spectra of the freeradicals obtained by electrolytic reduction were recorded

in the X band (985 GHz) using a Bruker ECS 106 spec-trometer with a rectangular cavity and 50 kHz field modu-lation Radicals of the Pt complexes were generated byelectrolytic reduction in situ in 103 M DMSO solutionsat room temperature under nitrogen atmosphere and theconditions previously established by cyclic voltammetrySimulations of the ESR spectra were made using the soft-ware WINEPR Simphonia 125 version The hyper-finesplitting constants were estimated to be accurate within005 G [26]

24 In vitro anti-T cruzi activity

Compounds were tested against epimastigote form oftwo strains of T cruzi namely Tulahuen 2 and Dm28cHandling of live T cruzi was done according to establishedguidelines [27]

241 Tulahuen 2 strain epimastigotes

The epimastigote form of the parasite Tulahuen 2 strainwas grown at 28 C in an axenic medium (BHI-tryptose)complemented with 5 fetal calf serum Cells from a 5days-old culture were inoculated into 50 mL of fresh cul-ture medium to give an initial concentration of 1 106

cellsmL Cell growth was followed by daily measuringthe absorbance A of the culture at 600 nm for 11 daysBefore inoculation the media was supplemented with10 lM or 25 lM of compounds from a stock DMSO solu-tion The final DMSO concentration in the culture medianever exceeded 04 (volvol) and had no effect by itselfon the proliferation of the parasites (no effect on epimasti-gote growth was observed by the presence of up to 1DMSO in the culture media) The compounds ability toinhibit growth of the parasite was evaluated in triplicatein comparison to the control (no drug added to the media)The control was run in the presence of 04 DMSO and inthe absence of any drug The percentage of growth inhibi-tion (PGI) was calculated as follows = 1[(ApA0p)(AcA0c)] 100 where Ap = A600 of the culture contain-ing the drug at day 5 A0p = A600 of the culture containingthe drug just after addition of the inocula (day 0)Ac = A600 of the culture in the absence of any drug (con-trol) at day 5 A0c = A600 in the absence of the drug atday 0 Nifurtimox and Benznidazol were used as the refer-ence trypanocidal drugs After stating activity at the stud-ied doses dose-response curves were recorded and the IC50

(50 inhibitory concentration) values were assessed [18]Reported values are mean of three independentexperiments

242 Dm28c strain epimastigotes

T cruzi epimastigotes Dm28c strain from our own col-lection (Programa de Farmacologıa Molecular y ClınicaFacultad de Medicina Universidad de Chile) were grownat 28 C in Diamondrsquos monophasic medium as reportedearlier [28] but replacing blood by 4 lM hemin Fetal calfserum was added to a final concentration of 4 Com-

pounds dissolved in DMSO (1 final concentration) wereadded to a suspension of 3 106 epimastigotesmL Para-site growth was followed by nephelometry for 10 days Notoxic effect of DMSO alone was observed at the final con-centration From the epimastigote exponential growthcurve the culture growth constant (kc) for each compoundconcentration treatment and for controls were calculated(regression coefficient gt09 P lt 005) This constant corre-sponds to the slope resulting from plotting the natural log-arithm (Ln) of nephelometry lecture versus time [29]ICkc50 is the drug concentration needed to reduce the kcin 50 and it was calculated by lineal regression analysisfrom the kc values and the concentrations used at theemployed concentrations Reported values are mean of atleast three independent experiments

25 Calf thymus DNA interaction experiments

Complexes were tested for their DNA interaction abilityusing native calf thymus DNA (CT DNA) (Type I) by amodification of a previously reported procedure [1830]CT DNA (50 mg) was dissolved in water (30 mL) (over-night) Solutions of the complexes in DMSO (spectroscopygrade) (1 mL 103 M) were incubated at 37 C with solu-tion of CT DNA (1 mL) during 96 h DNAcomplexesmixtures were exhaustively washed to eliminate the un-reacted complex Quantification of bound metal was doneby atomic absorption spectroscopy on a Perkin Elmer5000 spectrometer Standards were prepared by diluting ametal standard solution for atomic absorption spectros-copy Final DNA concentration per nucleotide was deter-mined by UV absorption spectroscopy using molarabsorption coefficient of 6000 M1 cm1 at 260 nm

26 Free radicals production in T cruzi (Dm28c strain)

The free radical production capacity of the new com-plexes was assessed in the parasite by ESR using 55-dimethyl-1-pirroline-N-oxide (DMPO) for spin trappingEach tested compound was dissolved in DMF (spectros-copy grade) (ca 1 mM) and the solution was added to amixture containing the epimastigote form of T cruzi

(Dm28c strain final protein concentration 4ndash8 mgmL)and DMPO (final concentration 250 mM) The mixturewas transferred to a 50 lL capillary ESR spectra wererecorded in the X band (985 GHz) using a Bruker ECS106 spectrometer with a rectangular cavity and 50 KHzfield modulation All the spectra were registered in thesame scale after 15 scans [17]

27 Oxygen uptake

Dm28c strain T cruzi epimastigotes were harvested by500 g centrifugation followed by washing and re-suspen-sion in 005 M sodium phosphate buffer pH 74 andcontaining 0107 M sodium chloride Respiration measure-ments were carried out polarographically with a Clark

electrode (Yellow Springs Instruments 53 YSI model)[1831] An amount of parasites equivalent to 10 mg ofproteinmL was added to a 06 mL chamber and oxygenconsumption was recorded at 28 C Control respirationrate corresponded to 28 natoms gram of oxygenminmgprotein In order to evaluate redox cycling mitochondrialrespiration was inhibited with 2 mM potassium cyanideFor comparative purposes and in order to maintain a sim-ilar parasite to drug mass ratio in growth inhibition and inoxygen uptake experiments metal compounds were used ina 125 mM final concentration in the oxygraph chamberResults were corrected according to the observed effectproduced by DMSO alone

3 Results and discussion

Two new series of platinum(II) complexes [PtCl2(HL)]and [Pt(L)2] with bioactive 5-nitrofuryl containing thio-semicarbazones (L) as ligands have been synthesized withhigh purities and good yields All of them are neutralnon conducting compounds and analytical and ESI massspectrometry results are in agreement with the proposedformula As shown below NS bidentate coordinationwas observed in all cases remaining the ligand non depro-tonated at the NH group for the [PtCl2(HL)] compoundsand deprotonated for the [Pt(L)2] compounds

31 IR and Raman spectroscopic studies

Based on experimental spectra and DFT studies wehave previously defined a characteristic vibrational (IRand Raman) spectroscopic pattern for 5-nitrofuryl thio-semicarbazones and their Pd(II) complexes that allowed

Table 1Tentative assignment of the main characteristic IR and Raman bands of the p

Compound IRcm1

m(CN) ms(NO2) m(CndashOndashC) + CndashCcontracta

m(NndashN)b m(Cndash

L1 1602 s 1356 s na 1108 846[PtCl2(HL1)] 1570 vw 1347 s na 1170[Pt(L1)2] 1557 w 1352 s 1335 s 1157L2 1599 w 1354 s na 1114 820PtCl2(HL2)] 1560 s 1349 s na 1173[Pt(L2)2] 1579 w 1350 s 1338 m 1169L3 1602 w 1352 s na 1104 823[PtCl2(HL3)] 1590 w 1344 s na 1175[Pt(L3)2] 1577 w 1350 s 1333 s 1156L4 1595 m 1344 s na 1104 822[PtCl2(HL4)] 1600 m 1347 s na 1175[Pt(L4)2] 1584 vw 1353 s 1339 sh 1084

m stretching d bending s strong m medium w weakna non assignedBands previously reported for the free ligands are included for comparison [3

a Furan CndashOndashC in-phase stretching + CndashC contractb Mediumc Weak to very weakd d(NO2) + furan modes or furan hydrogen wagging symmetric modese Furan in-phase CC stretching

us to analyze the spectroscopic behavior of the eight newPt(II) complexes holding the 5-nitrofuran thiosemicarba-zone moiety IR and Raman spectra of the latter were com-pared with those previously reported for the free ligandsand their Pd(II) analogues and main bands were tentativelyassigned (Table 1) [32]

Although thiosemicarbazone ligands are potentiallycapable of interacting with metal centers in different waysthey usually coordinate to metals as NS bidentate ligandsThere are at least three major stretching vibrations thatpresent strong diagnostic value in relation to the bindingmode of these ligands m(CN) m(CS) and m(NndashN) Aspreviously reported for the selected 5-nitrofuryl containingthiosemicarbazones these bands are located in spectralregions showing a complicated signals pattern that hasmade their assignment difficult [32] In particular thiosem-icarbazone CN and CS stretchings are located in wavenumber regions where vibrations of other portions of theligands occur namely mas (NO2) and 2-substituted furansout of-phase m(CC) occurring in the CN stretchingregion (1650ndash1500 cm1) and d (NO2) furan scissoringvibrations and furan hydrogen wagging symmetric modesand combination of them occurring in the CS stretchingregion (850ndash700 cm1) The combination of experimentaland DFT theoretical methods had previously allowed usto explain the complexity of the observed spectral patternof these ligands and their Pd(II) complexes and to performan assignment of the CN and NndashN stretchings Althoughclear changes were observed in the spectral region around750ndash900 cm1 the CS stretching that is usually used asprobe of the coordination of the metal to the thiocarbonylsulfur could not be undoubtably assigned for the palladiumcomplexes due to the high complexity of the spectra [32]

latinum complexes

Ramancm1

S)c d(NO2)+ furand

m(CN) m(CC)e ms (NO2) m(CndashOndashC) + CndashCcontracta

811 1592 m 1455 s 1351 m 1339 m812 1568 m 1454 s 1354 w na812 1563 s 1450 s 1348 m 1329 m808 1585 br 1474 s 1347 s 1312 m812 1554 sh 1470 s na 1335 s811 1580 s 1469 s 1343 sh 1332 m805 1601 m 1470 s 1351 s 1331 m811 1594 m 1470 s 1355 sh 1324 m810 1575 s 1471 s 1349 m 1330 s811 1596 m 1474 s 1346 m 1321 m811 1599 s 1466 s 1340 s 1319 sh812 1578 s 1472 s 1333 s 1312 sh

2]

Table 21H NMR chemical shift values (d) in ppm of L2 [18] and [PtCl2(HL2)] at30 C

OO2NN

NH

N

SCH3

H

1 2

3

4 5

6

1H NMR L2 Dda

H dLigandb dComplex

c

1 779 767 0122 730 754 0243 798 784 0144 1187 890 2975 852 840 0146 303 310 007

a Dd = (dComplex dLigand)b DMSO-d6c Acetone-d6

The vibrational spectra of the newly developed platinumcomplexes showed a very similar pattern to that of the cor-responding palladium complexes Although the complexityof the IR spectra of the ligands and concomitantly of theirmetal complexes increased from L1 to L4 as the R-substi-tuent complexity increased a common spectral patterncould be observed for each series of platinum compounds([PtCl2(HL)] and [Pt(L)2]) As shown in Table 1 after coor-dination m(CN) bands of the free thiosemicarbazoneligands shifted in almost all cases to lower wave numbersThis modification is consistent with coordination of the thi-osemicarbazone ligands through the azomethyne nitrogenOn the other hand the shift to higher wave numbers of them(NndashN) band observed for the platinum complexes hasalso been previously related to the electronic delocalizationproduced as a consequence of coordination through theazomethine nitrogen atom andor deprotonation of the thi-osemicarbazone ligands [32] The m(CS) bands shouldshift to lower wave numbers when thiosemicarbazonescoordinate through the thiocarbonyl sulfur For the Ptcomplexes weak to very weak sets of bands were detectedin the 700ndash850 cm1 region some of them probably associ-ated with m(CS) vibrations As it was the case for the Pdanalogues the complexity of the spectra in this regiondue to vibrations assigned to the nitrofuran moiety andmixing of vibrations superimposed to a very significantintensity decrease of the CS bands experimentallyobserved after coordination of thiosemicarbazones to ametal made the unambiguos assignment of the CS bandsdifficult Nevertheless clear changes observed in this spec-tral region agree with the coordination of the thiocarbonylsulfur to platinum [32] In agreement with the reported for-mulae of the complexes the m(NH) band at approximately3120ndash3150 cm1 is present in all [PtCl2(HL)] complexesindicating that the ligand is non deprotonated in theseneutral complexes In contrast m(NH) band is not observedin all [Pt(L)2] complexes due to deprotonation of theligands Taking into account the crystal structure of[Pd(L5)2] 3DMSO previously reported [18] and the greatsimilarities in vibrational spectral pattern shown by thePd and Pt analogous compounds a trans configuration isproposed for the [Pt(L)2] complexes (Fig 1) Calculationspreviously performed for the [Pd(L)2] complexes are alsoin agreement with this proposal [32] Vibrations associatedto those portions of the ligands that are not involved in thecoordination to the metal were also recognized in the spec-tra of the complexes and are also shown in Table 1 nitromoiety symmetric stretching (ms(NO2)) furan combinationmode involving furan CndashOndashC in-phase stretching and CndashCcontract (m(CndashOndashC) + CndashC contract) and d(NO2) + furanmodes or furan hydrogen wagging symmetric modes

32 NMR studies

The complexes were characterized by 1H NMR spec-troscopy The low solubility of many of the complexesdid not allow to acquire the complete series of 1H NMR

spectra Platinum complexes and their palladium analoguesshowed similar NMR spectra and behavior [18] Results ofthe performed NMR experiments are in agreement with theproposed structures and with the results of the other spec-troscopies 1H NMR integrations and signal multiplicitiesare in agreement with the proposed formula As an exam-ple results for PtCl2(HL2) are shown in Table 2 1H NMRchemical shifts (d) of the ligand and the complex and thechemical shift differences between complex and ligandexpressed as Dd are shown The attached figure showsthe numbering scheme of the free ligand mentioned in theTable and the text Pt and Pd complexes showed similar1H chemical shifts of the nitrofurylthiosemicarbazone com-mon portion of their molecules [18] When the ligand iscoordinated the effect of the metal is apparent for the pro-tons that are located close to the coordinating atoms theazomethyne nitrogen (H3 according to Table 2) and theNH exchangeable protons (H4 and H5 see Table 2) Thepresence of a signal corresponding to proton H4 is inaccordance with the coordination of the ligand in a nondeprotonated form Furthermore the largest Dd areobserved for this proton

33 Cyclic voltammetry

Voltammetric studies were mainly performed to deter-mine the effect of platinum complexation on the peakpotential of the nitro moiety due to the biological signifi-cance of this potential in relation to the capability of thecompounds to be bioreduced in the parasite leading tothe toxic nitro anion radical species

Fig 2 shows as examples the cyclic voltammograms for[PtCl2(HL3)] and [Pt(L3)2] at 2000 mVs (a) and for[PtCl2(HL3)] when scan rate is changed in the range 100ndash2000 mVs (b) All Pt complexes displayed comparable vol-tammetric behavior showing three well-defined reductionwaves in DMSO as shown by the free ligands [1826]

00 -05 -10 -15 -2015x10-5

10x10-5

50x10-6

00

-50x10-6

-10x10-5

IV

II III[PtL32]

Cur

rent

A

PotentialV

[PtCl2HL3]

I

00 -05 -10 -15 -2050x10-6

40x10-6

30x10-6

20x10-6

10x10-6

00

-10x10-6

-20x10-6

-30x10-6

Cur

rent

A

PotentialV

2000 mVs --- 1000 mVshellip 500 mVs minusminus 250 mVsminus 100 mVs

a b

Fig 2 Cyclic voltammograms of 1 mM DMSO solutions 01 M TBAP of (a) [PtCl2(HL3)] and [Pt(L3)2] at 2000 mVs and (b) [PtCl2(HL3)] when scanrate is changed in the range 100ndash2000 mVs

Table 3Cyclic voltammetric parameters for the reduction of the platinumcomplexes corresponding to the couple II peak Ic IIIc and IVc measuredin DMSO at 2000 mVs

Compound EpIca EpIIc

a EpIIab EpIIIc

a EpIVca

[PtCl2(HL1)] 043 070 066 1209 151[Pt(L1)2] ndash 069 056 122 149[PtCl2(HL2)] 049 073 060 117 137[Pt(L2)2] ndash 068 070 134 165[PtCl2(HL3)] 058 084 058 125 146[Pt(L3)2] ndash 077 061 122 149[PtCl2(HL4)] na 078 056 117 137[Pt(L4)2] ndash 072 062 109 126Nfx [18] 091 085

Nfx Nifurtimox na not assignedPotentials are reported in volts versus saturated calomel electrode

a Epc cathodic peak potentialb Epa anodic peak potential

The first wave for all the Pt compounds corresponded to aquasireversible process involving a one-electron transfer(couple II) This wave around 080 V versus SCE corre-sponds to the generation of the anion radical RNO2 byreduction of the nitro group [1833] The reverse scanshowed the anodic counterpart of the reduction waveAccording to the standard reversibility criteria this couplecan be attributed to a diffusion-controlled one-electrontransfer For some complexes sharp peaks around thiscouple could be observed as a result of adsorption phenom-ena in the electrode surface due to the presence in the mol-ecules of the thiocarbonyl group and the metal Thisphenomenon has been also observed for the Pd analogues[33] Next two couples (III and IV) are assigned to thereduction of the platinum complex nitro anion radicalgenerated in the first couple Subsequent less negativethree-electron irreversible cathodic peak (IIIc Fig 2a) isirreversible in the whole range of sweep rates used (100ndash2000 mVs) and can be attributed to the production ofthe hydroxylamine derivative [2634] Peak IVc is presumedto belong to the reduction of the imine moiety (CHN) ofthe thiosemicarbazone group [35]

Small differences were found between [PtCl2(HL)] and[Pt(L)2] complexes as previously observed for the Pd analo-gous compounds [33] The voltammogram of [PdCl2HL]complexes showed a prepeak (Ic Fig 2a) that appeared evenbefore the reduction of the nitro group meaning that thenitro group follows another reaction path besides the knownelectron-transfer mechanism of the nitroaromatic com-pounds in aprotic media This prepeak would correspondto the four-electron reduction of a small portion of the mol-ecules reaching the electrode surface while the remainingportion would supply the protons required for this reduc-tion This is a typical behavior of a self-protonation phenom-enon displayed by nitrocompounds with acidic moieties intheir structures [263334] The presence of the nitro group

increases the acidity of the NH moiety of the thiosemicarba-zone group present only in the [PtCl2(HL)] complexes (L nondeprotonated) which becomes capable of protonating thenitro group of a minor part of the molecules in the solutionresulting into a lower intensity of these signals [Pt(L)2] com-plexes did not show the Ic prepeak because they do not havethe capability to protonate the nitro group since the NH pro-ton was lost as a consequence of coordination of the ligand toplatinum

Table 3 lists the values of voltammetric peaks for allstudied compounds The potentials of the voltammetricpeaks corresponding to the nitro moiety of the free ligandsslightly changed as a consequence of platinum complexa-tion being the latter only slightly more favourable thanthe former and than those previously reported for the pal-ladium analogues [1826] However it should be stated thatall studied compounds showed under the same conditions ahigher capacity to be reduced than Nifurtimox (E12

[103]a

088 V Table 3) and therefore a better ability to generate

radical species that could be toxic for the parasite [36]

[G]

[G]

3420 3430 3440 3450 3460 3470-25-20-15-10-5

05

10152025

[103]

3420 3430 3440 3450 3460 3470

-40-30-20-10

01020304050

b

Fig 3 (a) ESR experimental spectrum of [Pt(L2)2] Spectrometer condi-tions microwave frequency 971 GHz microwave power 20 mW modu-lation amplitude 02 G scan rate 125 Gs time constant 05 s number ofscans 15 (b) Simulated spectrum of [Pt(L2)2]

34 Free radicals production studied by ESR spectroscopy

The complexes were tested for their capability to pro-duce free radicals in reductive conditions The freeradicals characterized by ESR were prepared lsquolsquoin siturdquo

by electrochemical reduction in DMSO applying apotential corresponding to the first monoelectronic wave(IIcIIa) obtained from the cyclic voltammetric experi-ments The interpretation of the ESR spectra by meansof a simulation process has led to the determination ofthe hyperfine coupling constants for all the magneticnuclei The obtained hyperfine constants are listed inTable 4

The ESR spectra of the [PtCl2(HL)] complexes were sim-ulated in terms of one triplet corresponding to the nitrogennucleus belonging to the nitro group and one triplet to thenitrogen of the CN thiosemicarbazone group and threedoublets due to non-equivalent hydrogens belonging tothe side chain Other nuclei presented hyperfine constantsmaller than the line width

The ESR spectra of the [Pt(L)2] complexes were ana-lyzed in terms of one triplet due to the nitrogen of the nitrogroup two doublets due to hydrogens corresponding to thefuran ring and one triplet due to hydrogens having verysimilar hyperfine constants belonging to the thiosemicar-bazone chains Other hyperfine constants resulted smallerthan the line width and they were not observed in theexperimental spectrum Fig 3 shows the ESR spectrumof complex [Pt(L2)2]

Different substituents in the thiosemicarbazone chain ofthe coordinated ligands did not seem to affect the hyperfinepattern of platinum complexes as it had been previouslyobserved for the corresponding palladium ones Howeverthe number of ligands coordinated per Pt central atomseemed to determine spin density distribution and so thehyperfine pattern of the complexes For the [Pt(L)2] com-plexes the spin density was more located on the nitro furanring while other observed couplings could be related to noncompletely equivalent thiosemicarbazone ligands coordi-nated to the platinum atom For the [PtCl2(HL)] com-

Table 4Hyperfine splittings (gauss) for the anion radical of the platinumcomplexes

aN aN aH aH aH aH aH

[PtCl2(HL1)] 695 41 128 14 112 lt06 lt06[Pt(L1)2] 68 lt06 08 40 lt06 245 235[PtCl2(HL2)] 697 35 12 15 100 lt06 lt06[Pt(L2)2] 70 lt06 12 40 lt06 31 33[PtCl2(HL3)] 690 37 115 16 128 lt06 lt06[Pt(L3)2] 70 lt06 124 40 lt06 30 33[PtCl2(HL4)] 687 40 117 125 13 lt06 lt06[Pt(L4)2] 72 lt06 110 38 lt06 298 310

pounds the spin density was more delocalized on theentire radical

The effect of changing the metal atom on the hyperfinepattern of the ESR spectra of the complexes should benoted For palladium complexes no differences have beenobserved between [PdCl2(HL)] and [Pd(L)2] ones Both ser-ies of palladium compounds presented a quite similarhyperfine pattern to that observed for the [PtCl2(HL)] com-plexes The different observed ESR pattern for [M(L)2]complexes when changing palladium by platinum couldbe related to small differences in complexes structure Thisfact would also agree with a quite different biologicalbehavior

35 In vitro anti T cruzi activity

The existence of the epimastigote form of T cruzi as anobligate mammalian intracellular stage has been confirmedrecently [3738] Therefore compounds were tested in vitro

against epimastigote form of the parasite The complexeswere evaluated for their anti-T cruzi activities against epim-astigotes of Tulahuen 2 strain in order to compare with pre-viously performed biological evaluation of the free ligandsand the analogous Pd complexes Fifty percent of inhibitoryconcentrations (IC50) obtained from dose ndash response curvesare depicted in Table 5 In addition some complexes wereevaluated against a second strain of T cruzi epimastigotesnamely Dm28c

For comparative purposes Table 5 also includes IC50

values for the four free ligands and the Pd complexes

Table 5In vitro biological activity of the Pt complexes

Compound T cruzi (Tulahuen 2) T cruzi (Dm28c) Compound T cruzi (Tulahuen 2)PGIa IC50

b ICkc50b IC50

c

10 lM 25 lM

L1 27[PtCl2(HL1)] 109 382 gt25 nd [PdCl2(HL1)] 24[Pt(L1)2] 34 208 gt25 nd [Pd(L1)2] 45

L2 50[PtCl2(HL2)] 415 605 131 367 [PdCl2(HL2)] 43[Pt(L2)2] 684 100 69 102 [Pd(L2)2] 47

L3 49[PtCl2(HL3)] 269 441 275 nd [PdCl2(HL3)] 59[Pt(L3)2] 100 100 08 288 [Pd(L3)2] gt25

L4 gt25[PtCl2(HL4)] 338 720 15 284 [PdCl2(HL4)] gt25[Pt(L4)2] 189 121 gt25 413 [Pd(L4)2] gt25Benznidazol 74 380Nifurtimox 61 [17] 228

IC50 values of the free ligand and their Pd complexes on T cruzi (Tulahuen 2 strain) have been included for comparison [18]nd not determined

a PGI percentage of growth inhibition of T cruzi epimastigote cells at the specified doseb plusmn10c [18]

3430 3440 3450 3460 3470 3480 3490 3500 3510 3520 3530

-8

-6

-4

-2

0

2

4

6

8[103]

previously determined through the same technique on epi-mastigote form of T cruzi Tulahuen 2 strain [18] Most ofthe Pt complexes were active against epimastigotes of T

cruzi Tulahuen 2 strain showing some of them IC50 valuesof the same order than Nifurtimox and the correspondingfree ligands According to the IC50 values [Pt(L3)2] and[Pt(L2)2] were the most active Pt complexes against thisparasite strain being [Pt(L3)2] significantly more activethan L3 and than Nifurtimox

When the results are compared with those previouslyreported for the free ligands and the Pd analogous com-pounds it can be stated that the activity pattern is signifi-cantly modified by changing the central atom The activityof the Pd complexes followed the general trend[PdCl2(HL)] gt free ligand gt [Pd(L)2] This regular trendwas not observed for the Pt analogues This differentbehavior could be related with differences in bioavailabil-ity unspecific toxicity andor mechanism of action of bothseries of metal complexes

Similar general conclusions can be obtained from theanalysis of the activity results on the T cruzi Dm28c strainalthough some strain susceptibility differences can beobserved Dm28c is a parasite clone that had previouslyshown to be less susceptible to Nifurtimox and Ben-znidazol trypanocidal effect than Tulahuen strain whichis mediated by a Dm28c higher thiol content of the former[39]

[G]

Fig 4 ESR spectrum of [PtCl2(HL2)] obtained when [PtCl2(HL2)] wasincubated with T cruzi epimastigotes in the presence of DMPOSpectrometer conditions microwave frequency 968 GHz microwavepower 20 mW modulation amplitude 02 G scan rate 125 Gs timeconstant 05 s number of scans 15 signals of DMPO-nitro anionradical adduct

36 Capacity of derivatives to generate free radical species

into T cruzi

The complexes were incubated with intact T cruzi

(Dm28c strain) epimastigotes in the presence of DMPO

as spin trapping agent in order to detect possible intra-celullar free radical species produced by bioreduction andhaving short half-lives The biological free radical produc-tion capacity of the new compounds was assessed by ESRAll the compounds were capable to produce free radicals inthe intact parasite giving an ESR spectrum Fig 4 showsas an example the ESR spectrum obtained when[PtCl2(HL2)] was incubated with T cruzi in the presenceof DMPO The typical hyperfine pattern of adductsobtained when DMPO traps radicals centered in C atom

[Pt(L2)2]

1 minute

10 O2

[Pt(L2)2]KCN

a

b

Fig 5 (a) Effect of compound [Pt(L)2] on the parasite (T cruzi Dm28cstrain) oxygen consumption related to control (dotted line) (b) Effect ofcompound [Pt(L)2] on the parasite (T cruzi Dm28c strain) oxygenconsumption after inhibition of the respiration with KCN related tocontrol (dotted line)

Table 6Effect of the addition of Pt complexes on oxygen uptake by T cruzi

epimastigotes (Dm28c strain)

Compound Respiration (nanoatom-gramof Ominmg protein)

Increase on oxygenconsumption ()a

Control 280 plusmn 20[PtCl2(HL2)] 322 plusmn 32 15[Pt(L2)2] 313 plusmn 17 12[Pt(L3)2] 392 plusmn 42 40[PtCl2(HL4)] 355 plusmn 32 27[Pt(L4)2] 320 plusmn 13 14Nifurtimox 356 plusmn 08 27

a increase on oxygen consumption after addition of 125 mM ofcompounds with respect to control (no added compound)

Table 7Interaction of Pt complexes with CT DNA after 96 h of incubation at37 C

Compound nmol Ptmg DNA Metalbasea Basemetal

[PtCl2(HL1)] 209 0069 14[Pt(L1)2] 390 0129 8[PtCl2(HL2)] 236 0078 13[Pt(L2)2] 224 0074 14[PtCl2(HL3)] 191 0063 16[Pt(L3)2] 527 0174 6[PtCl2(HL4)] 170 0056 18[Pt(L4)2] 170 0056 18

a Platinum (mol) per DNA base (mol)

(aN = 167 G and aH = 225 G) was observed [18] A trip-let produced by DMPO decomposition was also detected[40]

ESR studies showed that the newly developed Pt thio-semicarbazone complexes were bioreduced in the parasiteindicating that they could maintain the mode of action ofthe 5-nitrofuryl pharmacophore

37 Oxygen uptake

The involvement of the platinum complexes in redoxcycling processes should increase the parasite oxygen con-sumption Thus the effect of the compounds on parasiteoxygen uptake was measured The effect of complex[Pt(L2)2] on T cruzi epimastigotes oxygen consumptionwith and without inhibition of the respiration with KCNis shown in Fig 5 Both series of complexes modified oxy-gen uptake by the parasite increasing oxygen consumption(Table 6) These results are in accordance with a bioreduc-tive and redox cycling mechanism of action as previouslyreported for the free 5-nitrofuryl containing thiosemicarba-zones their Pd complexes and other nitrofuran derivativeslike Nifurtimox [1841] For both series of compoundseffect on parasite respiration seemed to correlate with theIC50 values For instance [Pt(L3)2] being the most activePt compound showed the highest increase on oxygenconsumption

38 Calf thymus DNA interaction

In order to address if interaction with DNA could bepart of the mode of action of the complexes experimentswith CT DNA were carried out Binding of the Pt com-plexes to DNA was studied by combining atomic absorp-tion determinations (for the metal) and electronicabsorption measurements for DNA quantification Thenew complexes are good binding agents for CT DNA

(Table 7) Although the determined platinum to DNAbinding levels were similar to those of known anti-tumormetal complexes they resulted significantly lower thanthose of the palladium analogous compounds [1819] Nogeneral pattern could be detected for both series of com-pounds being binding levels almost not affected by thenature of the thiosemicarbazone ligand [PtCl2(HL)] com-plexes having labile chloride ligands could probably inter-act with DNA through a mechanism similar to thatpreviously reported for cisplatin becoming activatedthrough aquation and reacting with nucleophilic DNAbases [42] [Pt(L)2] complexes cannot act in this way butthey could intercalate DNA or interact through othermechanisms Due to the planar motive contained in the[PdCl2(HL)] complexes intercalation could also be possiblefor these compounds Further studies are under progress inorder to get an insight into the mechanism of this interac-tion It is interesting to note that [Pt(L3)2] being the mostpotent anti-T cruzi compound of the series showed thehighest interaction with DNA Nevertheless [Pt(L1)2]being almost non active strongly interacts with DNAshowing that the mechanism of trypanocidal activity couldnot involve only DNA as target

4 Conclusions

Pt(II) complexes with 5-nitrofuryl containing thiosemi-carbazones as bioactive ligands showed good anti-trypan-osomal activity In particular the coordination of L3 to

Pt(II) forming [Pt(L3)2] lead to almost a five-fold activityincrease in respect to the free ligand Possible targetsnamely reductive metabolism (intra-parasite free radicalproduction) and DNA were screened trying to get insightinto the mechanism of action of these metal compoundsResults showed that some of the compounds could act asdual inhibitors in the parasite through production of toxicfree radicals and interaction with DNA

Results of this work show that the approach of coordi-nating anti-trypanosomal organic compounds with phar-macologically interesting metals could be a suitablestrategy to develop novel therapeutic tools against tropicaldiseases produced by trypanosomatids particularly Amer-ican Trypanosomiasis

Acknowledgments

This work was partially supported by PEDECIBA andUniversidad de La Republica (CSIC project 36406) ofUruguay Prosul-CNPq project 4902092005-0 SIBAS-0807 UMCE FONDECYT Chile 1061072 and CONI-CYT-PBCT Anillo ACT 29 We wish to thank Dr EJBaran CEQUINOR UNLP Argentina for Raman spec-tra facilities and Matilde Gomez Instituto Venezolano deInvestigaciones Cientıficas Caracas Venezuela for per-forming ESI-MS experiments

References

[1] httpwwwwhoint lthttpwwwwhointctdchagasgt[2] D Engels L Savioli Trend Parasitol 22 (2006) 363ndash366[3] J Urbina Expert Opin Ther Pat 13 (2003) 661ndash669[4] H Cerecetto M Gonzalez Curr Topics Med Chem 2 (2002) 1185ndash

1190[5] RL Krauth-Siegel H Bauer RH Schirmer Angew Chem Int Ed

44 (2005) 690ndash715[6] S Croft M Barret J Urbina Trend Parasitol 21 (2005) 508ndash512[7] M Ceaser The Lancet Infect Dis 5 (8) (2005) 470ndash471[8] Y Yamagata J Nakagawa Adv Parasitol 61 (2006) 129ndash165[9] C Zhang S Lippard Curr Opin Chem Biol 7 (2003) 481ndash489

[10] NP Farrell Metal Complexes as Drugs and ChemotherapeuticAgents in JM McCleverty TJ Meyer (Eds) ComprehensiveCoordination Chemistry II vol 9 Elsevier 2003 pp 809ndash840

[11] RA Sanchez-Delgado A Anzellotti L Suarez Metal complexes aschemotherapeutic agents against tropical diseases malaria trypano-somiasis and leishmaniasis in H Sigel A Sigel (Eds) Metal Ionsin Biological Systems vol 41 Marcel Dekker New York 2004 pp379ndash419

[12] RA Sanchez-Delgado A Anzellotti Mini-Rev Med Chem 4(2004) 23ndash30

[13] R Morphy Z Rankovic J Med Chem 48 (2005) 6523ndash6543[14] Z Wang EM Bennett DJ Wilson C Salomon R Vince J Med

Chem 50 (2007) 3416ndash3419[15] K Chibale Towards broad spectrum antiprotozoal agents ARKI-

VOC IX 2002 pp 93ndash98

[16] C Urquiola M Vieites G Aguirre A Marın B Solano GArrambide ML Lavaggi MH Torre M Gonzalez A Monge DGambino H Cerecetto Bioorg Med Chem 14 (2006) 5503ndash5509

[17] G Aguirre H Cerecetto M Gonzalez D Gambino L Otero COlea-Azar C Rigol A Denicola Bioorg Med Chem 12 (2004)4885ndash4893

[18] L Otero M Vieites L Boiani A Denicola C Rigol L Opazo COlea-Azar JD Maya A Morello RL Krauth-Siegel OE Piro ECastellano M Gonzalez D Gambino H Cerecetto J Med Chem49 (2006) 3322ndash3331

[19] A Gomez-Quiroga C Navarro-Ranninger Coord Chem Rev 248(2004) 119ndash133

[20] MD Hall TW Hambley Coord Chem Rev 232 (2002) 49ndash67[21] TW Hambley Coord Chem Rev 166 (1997) 181ndash223[22] S Bonse JM Richards SA Ross G Lowe RL Krauth-Siegel J

Med Chem 43 (2000) 4812ndash4821[23] G Lowe AS Droz T Vilaiyan GW Weaver L Tweedale JM

Pratt P Rock V Yardley SL Croft J Med Chem 42 (1999) 999ndash1006

[24] SL Croft Mem Inst Oswaldo Cruz 94 (1999) 215ndash220[25] WJ Geary Coord Chem Rev 7 (1971) 81ndash91[26] C Rigol C Olea-Azar F Mendizabal L Otero D Gambino M

Gonzalez H Cerecetto Spectrochim Acta A 61 (2005) 2933ndash2938

[27] L Huang A Lee JA Ellman J Med Chem 45 (2002) 676ndash684[28] JD Maya A Morello Y Repetto R Tellez A Rodriguez U

Zelada P Puebla M Bonta S Bollo A San Feliciano CompBiochem Phys C 125 (2000) 103ndash109

[29] MA Cuellar C Salas MJ Cortes A Morello JD Maya MDPreitea Bioorg Med Chem 11 (2003) 2489ndash2497

[30] RE Mahnken MA Billadeau EP Nikonowicz H Morrison JAm Chem Soc 114 (1992) 9253ndash9265

[31] ME Letelier E Rodrıguez A Wallace M Lorca Y Repetto AMorello J Aldunate Exp Parasitol 71 (1990) 357ndash363

[32] D Gambino L Otero M Vieites M Boiani M Gonzalez EJBaran H Cerecetto Spectrochim Acta A 68 (2007) 341ndash348

[33] L Otero C Folch G Barriga C Rigol L Opazo M Vieites DGambino H Cerecetto E NorambuenaC Olea-Azar SpectrochimActa A lthttpdxdoiorg101016jsaa200707045gt

[34] C Olea-Azar C Rigol F Mendizabal A Morello JD Maya CMoncada E Cabrera R Di Maio M Gonzalez H Cerecetto FreeRad Res 37 (2003) 993ndash1001

[35] S Bollo E Soto-Bustamante LJ Nunez-Vergara JA Squella JElectroanal Chem 492 (2000) 54ndash62

[36] C Olea-Azar AM Atria F Mendizabal R Di Maio G Seoane HCerecetto Spectrosc Lett 31 (1998) 99ndash109

[37] VJ Aran C Ochoa L Boiani P Buccino H Cerecetto A GerpeM Gonzalez D Montero JJ Nogal A Gomez-Barrio A AzquetaA Lopez de Ceraın OE Piro EE Castellano Bioorg Med Chem13 (2005) 3197ndash3207

[38] KM Tyler DM Engman Int J Parasitol 31 (2001) 472ndash480[39] Y Repetto E Opazo JD Maya M Agosin A Morello Comp

Biochem Phys B 115 (1996) 281ndash285[40] N Veerapen SA Taylor CJ Walsby B Mario Pinto J Am

Chem Soc 128 (2006) 227ndash239[41] JD Maya S Bollo LJ Nunez-Vergara JA Squella Y Repetto

A Morello J Perie G Chauviere Biochem Pharmacol 65 (2003)999ndash1006

[42] V Brabec Prog Nucl Acid Res Mol Biol 71 (2002) 1ndash68

in the X band (985 GHz) using a Bruker ECS 106 spec-trometer with a rectangular cavity and 50 kHz field modu-lation Radicals of the Pt complexes were generated byelectrolytic reduction in situ in 103 M DMSO solutionsat room temperature under nitrogen atmosphere and theconditions previously established by cyclic voltammetrySimulations of the ESR spectra were made using the soft-ware WINEPR Simphonia 125 version The hyper-finesplitting constants were estimated to be accurate within005 G [26]

24 In vitro anti-T cruzi activity

Compounds were tested against epimastigote form oftwo strains of T cruzi namely Tulahuen 2 and Dm28cHandling of live T cruzi was done according to establishedguidelines [27]

241 Tulahuen 2 strain epimastigotes

The epimastigote form of the parasite Tulahuen 2 strainwas grown at 28 C in an axenic medium (BHI-tryptose)complemented with 5 fetal calf serum Cells from a 5days-old culture were inoculated into 50 mL of fresh cul-ture medium to give an initial concentration of 1 106

cellsmL Cell growth was followed by daily measuringthe absorbance A of the culture at 600 nm for 11 daysBefore inoculation the media was supplemented with10 lM or 25 lM of compounds from a stock DMSO solu-tion The final DMSO concentration in the culture medianever exceeded 04 (volvol) and had no effect by itselfon the proliferation of the parasites (no effect on epimasti-gote growth was observed by the presence of up to 1DMSO in the culture media) The compounds ability toinhibit growth of the parasite was evaluated in triplicatein comparison to the control (no drug added to the media)The control was run in the presence of 04 DMSO and inthe absence of any drug The percentage of growth inhibi-tion (PGI) was calculated as follows = 1[(ApA0p)(AcA0c)] 100 where Ap = A600 of the culture contain-ing the drug at day 5 A0p = A600 of the culture containingthe drug just after addition of the inocula (day 0)Ac = A600 of the culture in the absence of any drug (con-trol) at day 5 A0c = A600 in the absence of the drug atday 0 Nifurtimox and Benznidazol were used as the refer-ence trypanocidal drugs After stating activity at the stud-ied doses dose-response curves were recorded and the IC50

(50 inhibitory concentration) values were assessed [18]Reported values are mean of three independentexperiments

242 Dm28c strain epimastigotes

T cruzi epimastigotes Dm28c strain from our own col-lection (Programa de Farmacologıa Molecular y ClınicaFacultad de Medicina Universidad de Chile) were grownat 28 C in Diamondrsquos monophasic medium as reportedearlier [28] but replacing blood by 4 lM hemin Fetal calfserum was added to a final concentration of 4 Com-

pounds dissolved in DMSO (1 final concentration) wereadded to a suspension of 3 106 epimastigotesmL Para-site growth was followed by nephelometry for 10 days Notoxic effect of DMSO alone was observed at the final con-centration From the epimastigote exponential growthcurve the culture growth constant (kc) for each compoundconcentration treatment and for controls were calculated(regression coefficient gt09 P lt 005) This constant corre-sponds to the slope resulting from plotting the natural log-arithm (Ln) of nephelometry lecture versus time [29]ICkc50 is the drug concentration needed to reduce the kcin 50 and it was calculated by lineal regression analysisfrom the kc values and the concentrations used at theemployed concentrations Reported values are mean of atleast three independent experiments

25 Calf thymus DNA interaction experiments

Complexes were tested for their DNA interaction abilityusing native calf thymus DNA (CT DNA) (Type I) by amodification of a previously reported procedure [1830]CT DNA (50 mg) was dissolved in water (30 mL) (over-night) Solutions of the complexes in DMSO (spectroscopygrade) (1 mL 103 M) were incubated at 37 C with solu-tion of CT DNA (1 mL) during 96 h DNAcomplexesmixtures were exhaustively washed to eliminate the un-reacted complex Quantification of bound metal was doneby atomic absorption spectroscopy on a Perkin Elmer5000 spectrometer Standards were prepared by diluting ametal standard solution for atomic absorption spectros-copy Final DNA concentration per nucleotide was deter-mined by UV absorption spectroscopy using molarabsorption coefficient of 6000 M1 cm1 at 260 nm

26 Free radicals production in T cruzi (Dm28c strain)

The free radical production capacity of the new com-plexes was assessed in the parasite by ESR using 55-dimethyl-1-pirroline-N-oxide (DMPO) for spin trappingEach tested compound was dissolved in DMF (spectros-copy grade) (ca 1 mM) and the solution was added to amixture containing the epimastigote form of T cruzi

(Dm28c strain final protein concentration 4ndash8 mgmL)and DMPO (final concentration 250 mM) The mixturewas transferred to a 50 lL capillary ESR spectra wererecorded in the X band (985 GHz) using a Bruker ECS106 spectrometer with a rectangular cavity and 50 KHzfield modulation All the spectra were registered in thesame scale after 15 scans [17]

27 Oxygen uptake

Dm28c strain T cruzi epimastigotes were harvested by500 g centrifugation followed by washing and re-suspen-sion in 005 M sodium phosphate buffer pH 74 andcontaining 0107 M sodium chloride Respiration measure-ments were carried out polarographically with a Clark

electrode (Yellow Springs Instruments 53 YSI model)[1831] An amount of parasites equivalent to 10 mg ofproteinmL was added to a 06 mL chamber and oxygenconsumption was recorded at 28 C Control respirationrate corresponded to 28 natoms gram of oxygenminmgprotein In order to evaluate redox cycling mitochondrialrespiration was inhibited with 2 mM potassium cyanideFor comparative purposes and in order to maintain a sim-ilar parasite to drug mass ratio in growth inhibition and inoxygen uptake experiments metal compounds were used ina 125 mM final concentration in the oxygraph chamberResults were corrected according to the observed effectproduced by DMSO alone

3 Results and discussion

Two new series of platinum(II) complexes [PtCl2(HL)]and [Pt(L)2] with bioactive 5-nitrofuryl containing thio-semicarbazones (L) as ligands have been synthesized withhigh purities and good yields All of them are neutralnon conducting compounds and analytical and ESI massspectrometry results are in agreement with the proposedformula As shown below NS bidentate coordinationwas observed in all cases remaining the ligand non depro-tonated at the NH group for the [PtCl2(HL)] compoundsand deprotonated for the [Pt(L)2] compounds

31 IR and Raman spectroscopic studies

Based on experimental spectra and DFT studies wehave previously defined a characteristic vibrational (IRand Raman) spectroscopic pattern for 5-nitrofuryl thio-semicarbazones and their Pd(II) complexes that allowed

Table 1Tentative assignment of the main characteristic IR and Raman bands of the p

Compound IRcm1

m(CN) ms(NO2) m(CndashOndashC) + CndashCcontracta

m(NndashN)b m(Cndash

L1 1602 s 1356 s na 1108 846[PtCl2(HL1)] 1570 vw 1347 s na 1170[Pt(L1)2] 1557 w 1352 s 1335 s 1157L2 1599 w 1354 s na 1114 820PtCl2(HL2)] 1560 s 1349 s na 1173[Pt(L2)2] 1579 w 1350 s 1338 m 1169L3 1602 w 1352 s na 1104 823[PtCl2(HL3)] 1590 w 1344 s na 1175[Pt(L3)2] 1577 w 1350 s 1333 s 1156L4 1595 m 1344 s na 1104 822[PtCl2(HL4)] 1600 m 1347 s na 1175[Pt(L4)2] 1584 vw 1353 s 1339 sh 1084

m stretching d bending s strong m medium w weakna non assignedBands previously reported for the free ligands are included for comparison [3

a Furan CndashOndashC in-phase stretching + CndashC contractb Mediumc Weak to very weakd d(NO2) + furan modes or furan hydrogen wagging symmetric modese Furan in-phase CC stretching

us to analyze the spectroscopic behavior of the eight newPt(II) complexes holding the 5-nitrofuran thiosemicarba-zone moiety IR and Raman spectra of the latter were com-pared with those previously reported for the free ligandsand their Pd(II) analogues and main bands were tentativelyassigned (Table 1) [32]

Although thiosemicarbazone ligands are potentiallycapable of interacting with metal centers in different waysthey usually coordinate to metals as NS bidentate ligandsThere are at least three major stretching vibrations thatpresent strong diagnostic value in relation to the bindingmode of these ligands m(CN) m(CS) and m(NndashN) Aspreviously reported for the selected 5-nitrofuryl containingthiosemicarbazones these bands are located in spectralregions showing a complicated signals pattern that hasmade their assignment difficult [32] In particular thiosem-icarbazone CN and CS stretchings are located in wavenumber regions where vibrations of other portions of theligands occur namely mas (NO2) and 2-substituted furansout of-phase m(CC) occurring in the CN stretchingregion (1650ndash1500 cm1) and d (NO2) furan scissoringvibrations and furan hydrogen wagging symmetric modesand combination of them occurring in the CS stretchingregion (850ndash700 cm1) The combination of experimentaland DFT theoretical methods had previously allowed usto explain the complexity of the observed spectral patternof these ligands and their Pd(II) complexes and to performan assignment of the CN and NndashN stretchings Althoughclear changes were observed in the spectral region around750ndash900 cm1 the CS stretching that is usually used asprobe of the coordination of the metal to the thiocarbonylsulfur could not be undoubtably assigned for the palladiumcomplexes due to the high complexity of the spectra [32]

latinum complexes

Ramancm1

S)c d(NO2)+ furand

m(CN) m(CC)e ms (NO2) m(CndashOndashC) + CndashCcontracta

811 1592 m 1455 s 1351 m 1339 m812 1568 m 1454 s 1354 w na812 1563 s 1450 s 1348 m 1329 m808 1585 br 1474 s 1347 s 1312 m812 1554 sh 1470 s na 1335 s811 1580 s 1469 s 1343 sh 1332 m805 1601 m 1470 s 1351 s 1331 m811 1594 m 1470 s 1355 sh 1324 m810 1575 s 1471 s 1349 m 1330 s811 1596 m 1474 s 1346 m 1321 m811 1599 s 1466 s 1340 s 1319 sh812 1578 s 1472 s 1333 s 1312 sh

2]

Table 21H NMR chemical shift values (d) in ppm of L2 [18] and [PtCl2(HL2)] at30 C

OO2NN

NH

N

SCH3

H

1 2

3

4 5

6

1H NMR L2 Dda

H dLigandb dComplex

c

1 779 767 0122 730 754 0243 798 784 0144 1187 890 2975 852 840 0146 303 310 007

a Dd = (dComplex dLigand)b DMSO-d6c Acetone-d6

The vibrational spectra of the newly developed platinumcomplexes showed a very similar pattern to that of the cor-responding palladium complexes Although the complexityof the IR spectra of the ligands and concomitantly of theirmetal complexes increased from L1 to L4 as the R-substi-tuent complexity increased a common spectral patterncould be observed for each series of platinum compounds([PtCl2(HL)] and [Pt(L)2]) As shown in Table 1 after coor-dination m(CN) bands of the free thiosemicarbazoneligands shifted in almost all cases to lower wave numbersThis modification is consistent with coordination of the thi-osemicarbazone ligands through the azomethyne nitrogenOn the other hand the shift to higher wave numbers of them(NndashN) band observed for the platinum complexes hasalso been previously related to the electronic delocalizationproduced as a consequence of coordination through theazomethine nitrogen atom andor deprotonation of the thi-osemicarbazone ligands [32] The m(CS) bands shouldshift to lower wave numbers when thiosemicarbazonescoordinate through the thiocarbonyl sulfur For the Ptcomplexes weak to very weak sets of bands were detectedin the 700ndash850 cm1 region some of them probably associ-ated with m(CS) vibrations As it was the case for the Pdanalogues the complexity of the spectra in this regiondue to vibrations assigned to the nitrofuran moiety andmixing of vibrations superimposed to a very significantintensity decrease of the CS bands experimentallyobserved after coordination of thiosemicarbazones to ametal made the unambiguos assignment of the CS bandsdifficult Nevertheless clear changes observed in this spec-tral region agree with the coordination of the thiocarbonylsulfur to platinum [32] In agreement with the reported for-mulae of the complexes the m(NH) band at approximately3120ndash3150 cm1 is present in all [PtCl2(HL)] complexesindicating that the ligand is non deprotonated in theseneutral complexes In contrast m(NH) band is not observedin all [Pt(L)2] complexes due to deprotonation of theligands Taking into account the crystal structure of[Pd(L5)2] 3DMSO previously reported [18] and the greatsimilarities in vibrational spectral pattern shown by thePd and Pt analogous compounds a trans configuration isproposed for the [Pt(L)2] complexes (Fig 1) Calculationspreviously performed for the [Pd(L)2] complexes are alsoin agreement with this proposal [32] Vibrations associatedto those portions of the ligands that are not involved in thecoordination to the metal were also recognized in the spec-tra of the complexes and are also shown in Table 1 nitromoiety symmetric stretching (ms(NO2)) furan combinationmode involving furan CndashOndashC in-phase stretching and CndashCcontract (m(CndashOndashC) + CndashC contract) and d(NO2) + furanmodes or furan hydrogen wagging symmetric modes

32 NMR studies

The complexes were characterized by 1H NMR spec-troscopy The low solubility of many of the complexesdid not allow to acquire the complete series of 1H NMR

spectra Platinum complexes and their palladium analoguesshowed similar NMR spectra and behavior [18] Results ofthe performed NMR experiments are in agreement with theproposed structures and with the results of the other spec-troscopies 1H NMR integrations and signal multiplicitiesare in agreement with the proposed formula As an exam-ple results for PtCl2(HL2) are shown in Table 2 1H NMRchemical shifts (d) of the ligand and the complex and thechemical shift differences between complex and ligandexpressed as Dd are shown The attached figure showsthe numbering scheme of the free ligand mentioned in theTable and the text Pt and Pd complexes showed similar1H chemical shifts of the nitrofurylthiosemicarbazone com-mon portion of their molecules [18] When the ligand iscoordinated the effect of the metal is apparent for the pro-tons that are located close to the coordinating atoms theazomethyne nitrogen (H3 according to Table 2) and theNH exchangeable protons (H4 and H5 see Table 2) Thepresence of a signal corresponding to proton H4 is inaccordance with the coordination of the ligand in a nondeprotonated form Furthermore the largest Dd areobserved for this proton

33 Cyclic voltammetry

Voltammetric studies were mainly performed to deter-mine the effect of platinum complexation on the peakpotential of the nitro moiety due to the biological signifi-cance of this potential in relation to the capability of thecompounds to be bioreduced in the parasite leading tothe toxic nitro anion radical species

Fig 2 shows as examples the cyclic voltammograms for[PtCl2(HL3)] and [Pt(L3)2] at 2000 mVs (a) and for[PtCl2(HL3)] when scan rate is changed in the range 100ndash2000 mVs (b) All Pt complexes displayed comparable vol-tammetric behavior showing three well-defined reductionwaves in DMSO as shown by the free ligands [1826]

00 -05 -10 -15 -2015x10-5

10x10-5

50x10-6

00

-50x10-6

-10x10-5

IV

II III[PtL32]

Cur

rent

A

PotentialV

[PtCl2HL3]

I

00 -05 -10 -15 -2050x10-6

40x10-6

30x10-6

20x10-6

10x10-6

00

-10x10-6

-20x10-6

-30x10-6

Cur

rent

A

PotentialV

2000 mVs --- 1000 mVshellip 500 mVs minusminus 250 mVsminus 100 mVs

a b

Fig 2 Cyclic voltammograms of 1 mM DMSO solutions 01 M TBAP of (a) [PtCl2(HL3)] and [Pt(L3)2] at 2000 mVs and (b) [PtCl2(HL3)] when scanrate is changed in the range 100ndash2000 mVs

Table 3Cyclic voltammetric parameters for the reduction of the platinumcomplexes corresponding to the couple II peak Ic IIIc and IVc measuredin DMSO at 2000 mVs

Compound EpIca EpIIc

a EpIIab EpIIIc

a EpIVca

[PtCl2(HL1)] 043 070 066 1209 151[Pt(L1)2] ndash 069 056 122 149[PtCl2(HL2)] 049 073 060 117 137[Pt(L2)2] ndash 068 070 134 165[PtCl2(HL3)] 058 084 058 125 146[Pt(L3)2] ndash 077 061 122 149[PtCl2(HL4)] na 078 056 117 137[Pt(L4)2] ndash 072 062 109 126Nfx [18] 091 085

Nfx Nifurtimox na not assignedPotentials are reported in volts versus saturated calomel electrode

a Epc cathodic peak potentialb Epa anodic peak potential

The first wave for all the Pt compounds corresponded to aquasireversible process involving a one-electron transfer(couple II) This wave around 080 V versus SCE corre-sponds to the generation of the anion radical RNO2 byreduction of the nitro group [1833] The reverse scanshowed the anodic counterpart of the reduction waveAccording to the standard reversibility criteria this couplecan be attributed to a diffusion-controlled one-electrontransfer For some complexes sharp peaks around thiscouple could be observed as a result of adsorption phenom-ena in the electrode surface due to the presence in the mol-ecules of the thiocarbonyl group and the metal Thisphenomenon has been also observed for the Pd analogues[33] Next two couples (III and IV) are assigned to thereduction of the platinum complex nitro anion radicalgenerated in the first couple Subsequent less negativethree-electron irreversible cathodic peak (IIIc Fig 2a) isirreversible in the whole range of sweep rates used (100ndash2000 mVs) and can be attributed to the production ofthe hydroxylamine derivative [2634] Peak IVc is presumedto belong to the reduction of the imine moiety (CHN) ofthe thiosemicarbazone group [35]

Small differences were found between [PtCl2(HL)] and[Pt(L)2] complexes as previously observed for the Pd analo-gous compounds [33] The voltammogram of [PdCl2HL]complexes showed a prepeak (Ic Fig 2a) that appeared evenbefore the reduction of the nitro group meaning that thenitro group follows another reaction path besides the knownelectron-transfer mechanism of the nitroaromatic com-pounds in aprotic media This prepeak would correspondto the four-electron reduction of a small portion of the mol-ecules reaching the electrode surface while the remainingportion would supply the protons required for this reduc-tion This is a typical behavior of a self-protonation phenom-enon displayed by nitrocompounds with acidic moieties intheir structures [263334] The presence of the nitro group

increases the acidity of the NH moiety of the thiosemicarba-zone group present only in the [PtCl2(HL)] complexes (L nondeprotonated) which becomes capable of protonating thenitro group of a minor part of the molecules in the solutionresulting into a lower intensity of these signals [Pt(L)2] com-plexes did not show the Ic prepeak because they do not havethe capability to protonate the nitro group since the NH pro-ton was lost as a consequence of coordination of the ligand toplatinum

Table 3 lists the values of voltammetric peaks for allstudied compounds The potentials of the voltammetricpeaks corresponding to the nitro moiety of the free ligandsslightly changed as a consequence of platinum complexa-tion being the latter only slightly more favourable thanthe former and than those previously reported for the pal-ladium analogues [1826] However it should be stated thatall studied compounds showed under the same conditions ahigher capacity to be reduced than Nifurtimox (E12

[103]a

088 V Table 3) and therefore a better ability to generate

radical species that could be toxic for the parasite [36]

[G]

[G]

3420 3430 3440 3450 3460 3470-25-20-15-10-5

05

10152025

[103]

3420 3430 3440 3450 3460 3470

-40-30-20-10

01020304050

b

Fig 3 (a) ESR experimental spectrum of [Pt(L2)2] Spectrometer condi-tions microwave frequency 971 GHz microwave power 20 mW modu-lation amplitude 02 G scan rate 125 Gs time constant 05 s number ofscans 15 (b) Simulated spectrum of [Pt(L2)2]

34 Free radicals production studied by ESR spectroscopy

The complexes were tested for their capability to pro-duce free radicals in reductive conditions The freeradicals characterized by ESR were prepared lsquolsquoin siturdquo

by electrochemical reduction in DMSO applying apotential corresponding to the first monoelectronic wave(IIcIIa) obtained from the cyclic voltammetric experi-ments The interpretation of the ESR spectra by meansof a simulation process has led to the determination ofthe hyperfine coupling constants for all the magneticnuclei The obtained hyperfine constants are listed inTable 4

The ESR spectra of the [PtCl2(HL)] complexes were sim-ulated in terms of one triplet corresponding to the nitrogennucleus belonging to the nitro group and one triplet to thenitrogen of the CN thiosemicarbazone group and threedoublets due to non-equivalent hydrogens belonging tothe side chain Other nuclei presented hyperfine constantsmaller than the line width

The ESR spectra of the [Pt(L)2] complexes were ana-lyzed in terms of one triplet due to the nitrogen of the nitrogroup two doublets due to hydrogens corresponding to thefuran ring and one triplet due to hydrogens having verysimilar hyperfine constants belonging to the thiosemicar-bazone chains Other hyperfine constants resulted smallerthan the line width and they were not observed in theexperimental spectrum Fig 3 shows the ESR spectrumof complex [Pt(L2)2]

Different substituents in the thiosemicarbazone chain ofthe coordinated ligands did not seem to affect the hyperfinepattern of platinum complexes as it had been previouslyobserved for the corresponding palladium ones Howeverthe number of ligands coordinated per Pt central atomseemed to determine spin density distribution and so thehyperfine pattern of the complexes For the [Pt(L)2] com-plexes the spin density was more located on the nitro furanring while other observed couplings could be related to noncompletely equivalent thiosemicarbazone ligands coordi-nated to the platinum atom For the [PtCl2(HL)] com-

Table 4Hyperfine splittings (gauss) for the anion radical of the platinumcomplexes

aN aN aH aH aH aH aH

[PtCl2(HL1)] 695 41 128 14 112 lt06 lt06[Pt(L1)2] 68 lt06 08 40 lt06 245 235[PtCl2(HL2)] 697 35 12 15 100 lt06 lt06[Pt(L2)2] 70 lt06 12 40 lt06 31 33[PtCl2(HL3)] 690 37 115 16 128 lt06 lt06[Pt(L3)2] 70 lt06 124 40 lt06 30 33[PtCl2(HL4)] 687 40 117 125 13 lt06 lt06[Pt(L4)2] 72 lt06 110 38 lt06 298 310

pounds the spin density was more delocalized on theentire radical

The effect of changing the metal atom on the hyperfinepattern of the ESR spectra of the complexes should benoted For palladium complexes no differences have beenobserved between [PdCl2(HL)] and [Pd(L)2] ones Both ser-ies of palladium compounds presented a quite similarhyperfine pattern to that observed for the [PtCl2(HL)] com-plexes The different observed ESR pattern for [M(L)2]complexes when changing palladium by platinum couldbe related to small differences in complexes structure Thisfact would also agree with a quite different biologicalbehavior

35 In vitro anti T cruzi activity

The existence of the epimastigote form of T cruzi as anobligate mammalian intracellular stage has been confirmedrecently [3738] Therefore compounds were tested in vitro

against epimastigote form of the parasite The complexeswere evaluated for their anti-T cruzi activities against epim-astigotes of Tulahuen 2 strain in order to compare with pre-viously performed biological evaluation of the free ligandsand the analogous Pd complexes Fifty percent of inhibitoryconcentrations (IC50) obtained from dose ndash response curvesare depicted in Table 5 In addition some complexes wereevaluated against a second strain of T cruzi epimastigotesnamely Dm28c

For comparative purposes Table 5 also includes IC50

values for the four free ligands and the Pd complexes

Table 5In vitro biological activity of the Pt complexes

Compound T cruzi (Tulahuen 2) T cruzi (Dm28c) Compound T cruzi (Tulahuen 2)PGIa IC50

b ICkc50b IC50

c

10 lM 25 lM

L1 27[PtCl2(HL1)] 109 382 gt25 nd [PdCl2(HL1)] 24[Pt(L1)2] 34 208 gt25 nd [Pd(L1)2] 45

L2 50[PtCl2(HL2)] 415 605 131 367 [PdCl2(HL2)] 43[Pt(L2)2] 684 100 69 102 [Pd(L2)2] 47

L3 49[PtCl2(HL3)] 269 441 275 nd [PdCl2(HL3)] 59[Pt(L3)2] 100 100 08 288 [Pd(L3)2] gt25

L4 gt25[PtCl2(HL4)] 338 720 15 284 [PdCl2(HL4)] gt25[Pt(L4)2] 189 121 gt25 413 [Pd(L4)2] gt25Benznidazol 74 380Nifurtimox 61 [17] 228

IC50 values of the free ligand and their Pd complexes on T cruzi (Tulahuen 2 strain) have been included for comparison [18]nd not determined

a PGI percentage of growth inhibition of T cruzi epimastigote cells at the specified doseb plusmn10c [18]

3430 3440 3450 3460 3470 3480 3490 3500 3510 3520 3530

-8

-6

-4

-2

0

2

4

6

8[103]

previously determined through the same technique on epi-mastigote form of T cruzi Tulahuen 2 strain [18] Most ofthe Pt complexes were active against epimastigotes of T

cruzi Tulahuen 2 strain showing some of them IC50 valuesof the same order than Nifurtimox and the correspondingfree ligands According to the IC50 values [Pt(L3)2] and[Pt(L2)2] were the most active Pt complexes against thisparasite strain being [Pt(L3)2] significantly more activethan L3 and than Nifurtimox

When the results are compared with those previouslyreported for the free ligands and the Pd analogous com-pounds it can be stated that the activity pattern is signifi-cantly modified by changing the central atom The activityof the Pd complexes followed the general trend[PdCl2(HL)] gt free ligand gt [Pd(L)2] This regular trendwas not observed for the Pt analogues This differentbehavior could be related with differences in bioavailabil-ity unspecific toxicity andor mechanism of action of bothseries of metal complexes

Similar general conclusions can be obtained from theanalysis of the activity results on the T cruzi Dm28c strainalthough some strain susceptibility differences can beobserved Dm28c is a parasite clone that had previouslyshown to be less susceptible to Nifurtimox and Ben-znidazol trypanocidal effect than Tulahuen strain whichis mediated by a Dm28c higher thiol content of the former[39]

[G]

Fig 4 ESR spectrum of [PtCl2(HL2)] obtained when [PtCl2(HL2)] wasincubated with T cruzi epimastigotes in the presence of DMPOSpectrometer conditions microwave frequency 968 GHz microwavepower 20 mW modulation amplitude 02 G scan rate 125 Gs timeconstant 05 s number of scans 15 signals of DMPO-nitro anionradical adduct

36 Capacity of derivatives to generate free radical species

into T cruzi

The complexes were incubated with intact T cruzi

(Dm28c strain) epimastigotes in the presence of DMPO

as spin trapping agent in order to detect possible intra-celullar free radical species produced by bioreduction andhaving short half-lives The biological free radical produc-tion capacity of the new compounds was assessed by ESRAll the compounds were capable to produce free radicals inthe intact parasite giving an ESR spectrum Fig 4 showsas an example the ESR spectrum obtained when[PtCl2(HL2)] was incubated with T cruzi in the presenceof DMPO The typical hyperfine pattern of adductsobtained when DMPO traps radicals centered in C atom

[Pt(L2)2]

1 minute

10 O2

[Pt(L2)2]KCN

a

b

Fig 5 (a) Effect of compound [Pt(L)2] on the parasite (T cruzi Dm28cstrain) oxygen consumption related to control (dotted line) (b) Effect ofcompound [Pt(L)2] on the parasite (T cruzi Dm28c strain) oxygenconsumption after inhibition of the respiration with KCN related tocontrol (dotted line)

Table 6Effect of the addition of Pt complexes on oxygen uptake by T cruzi

epimastigotes (Dm28c strain)

Compound Respiration (nanoatom-gramof Ominmg protein)

Increase on oxygenconsumption ()a

Control 280 plusmn 20[PtCl2(HL2)] 322 plusmn 32 15[Pt(L2)2] 313 plusmn 17 12[Pt(L3)2] 392 plusmn 42 40[PtCl2(HL4)] 355 plusmn 32 27[Pt(L4)2] 320 plusmn 13 14Nifurtimox 356 plusmn 08 27

a increase on oxygen consumption after addition of 125 mM ofcompounds with respect to control (no added compound)

Table 7Interaction of Pt complexes with CT DNA after 96 h of incubation at37 C

Compound nmol Ptmg DNA Metalbasea Basemetal

[PtCl2(HL1)] 209 0069 14[Pt(L1)2] 390 0129 8[PtCl2(HL2)] 236 0078 13[Pt(L2)2] 224 0074 14[PtCl2(HL3)] 191 0063 16[Pt(L3)2] 527 0174 6[PtCl2(HL4)] 170 0056 18[Pt(L4)2] 170 0056 18

a Platinum (mol) per DNA base (mol)

(aN = 167 G and aH = 225 G) was observed [18] A trip-let produced by DMPO decomposition was also detected[40]

ESR studies showed that the newly developed Pt thio-semicarbazone complexes were bioreduced in the parasiteindicating that they could maintain the mode of action ofthe 5-nitrofuryl pharmacophore

37 Oxygen uptake

The involvement of the platinum complexes in redoxcycling processes should increase the parasite oxygen con-sumption Thus the effect of the compounds on parasiteoxygen uptake was measured The effect of complex[Pt(L2)2] on T cruzi epimastigotes oxygen consumptionwith and without inhibition of the respiration with KCNis shown in Fig 5 Both series of complexes modified oxy-gen uptake by the parasite increasing oxygen consumption(Table 6) These results are in accordance with a bioreduc-tive and redox cycling mechanism of action as previouslyreported for the free 5-nitrofuryl containing thiosemicarba-zones their Pd complexes and other nitrofuran derivativeslike Nifurtimox [1841] For both series of compoundseffect on parasite respiration seemed to correlate with theIC50 values For instance [Pt(L3)2] being the most activePt compound showed the highest increase on oxygenconsumption

38 Calf thymus DNA interaction

In order to address if interaction with DNA could bepart of the mode of action of the complexes experimentswith CT DNA were carried out Binding of the Pt com-plexes to DNA was studied by combining atomic absorp-tion determinations (for the metal) and electronicabsorption measurements for DNA quantification Thenew complexes are good binding agents for CT DNA

(Table 7) Although the determined platinum to DNAbinding levels were similar to those of known anti-tumormetal complexes they resulted significantly lower thanthose of the palladium analogous compounds [1819] Nogeneral pattern could be detected for both series of com-pounds being binding levels almost not affected by thenature of the thiosemicarbazone ligand [PtCl2(HL)] com-plexes having labile chloride ligands could probably inter-act with DNA through a mechanism similar to thatpreviously reported for cisplatin becoming activatedthrough aquation and reacting with nucleophilic DNAbases [42] [Pt(L)2] complexes cannot act in this way butthey could intercalate DNA or interact through othermechanisms Due to the planar motive contained in the[PdCl2(HL)] complexes intercalation could also be possiblefor these compounds Further studies are under progress inorder to get an insight into the mechanism of this interac-tion It is interesting to note that [Pt(L3)2] being the mostpotent anti-T cruzi compound of the series showed thehighest interaction with DNA Nevertheless [Pt(L1)2]being almost non active strongly interacts with DNAshowing that the mechanism of trypanocidal activity couldnot involve only DNA as target

4 Conclusions

Pt(II) complexes with 5-nitrofuryl containing thiosemi-carbazones as bioactive ligands showed good anti-trypan-osomal activity In particular the coordination of L3 to

Pt(II) forming [Pt(L3)2] lead to almost a five-fold activityincrease in respect to the free ligand Possible targetsnamely reductive metabolism (intra-parasite free radicalproduction) and DNA were screened trying to get insightinto the mechanism of action of these metal compoundsResults showed that some of the compounds could act asdual inhibitors in the parasite through production of toxicfree radicals and interaction with DNA

Results of this work show that the approach of coordi-nating anti-trypanosomal organic compounds with phar-macologically interesting metals could be a suitablestrategy to develop novel therapeutic tools against tropicaldiseases produced by trypanosomatids particularly Amer-ican Trypanosomiasis

Acknowledgments

This work was partially supported by PEDECIBA andUniversidad de La Republica (CSIC project 36406) ofUruguay Prosul-CNPq project 4902092005-0 SIBAS-0807 UMCE FONDECYT Chile 1061072 and CONI-CYT-PBCT Anillo ACT 29 We wish to thank Dr EJBaran CEQUINOR UNLP Argentina for Raman spec-tra facilities and Matilde Gomez Instituto Venezolano deInvestigaciones Cientıficas Caracas Venezuela for per-forming ESI-MS experiments

References

[1] httpwwwwhoint lthttpwwwwhointctdchagasgt[2] D Engels L Savioli Trend Parasitol 22 (2006) 363ndash366[3] J Urbina Expert Opin Ther Pat 13 (2003) 661ndash669[4] H Cerecetto M Gonzalez Curr Topics Med Chem 2 (2002) 1185ndash

1190[5] RL Krauth-Siegel H Bauer RH Schirmer Angew Chem Int Ed

44 (2005) 690ndash715[6] S Croft M Barret J Urbina Trend Parasitol 21 (2005) 508ndash512[7] M Ceaser The Lancet Infect Dis 5 (8) (2005) 470ndash471[8] Y Yamagata J Nakagawa Adv Parasitol 61 (2006) 129ndash165[9] C Zhang S Lippard Curr Opin Chem Biol 7 (2003) 481ndash489

[10] NP Farrell Metal Complexes as Drugs and ChemotherapeuticAgents in JM McCleverty TJ Meyer (Eds) ComprehensiveCoordination Chemistry II vol 9 Elsevier 2003 pp 809ndash840

[11] RA Sanchez-Delgado A Anzellotti L Suarez Metal complexes aschemotherapeutic agents against tropical diseases malaria trypano-somiasis and leishmaniasis in H Sigel A Sigel (Eds) Metal Ionsin Biological Systems vol 41 Marcel Dekker New York 2004 pp379ndash419

[12] RA Sanchez-Delgado A Anzellotti Mini-Rev Med Chem 4(2004) 23ndash30

[13] R Morphy Z Rankovic J Med Chem 48 (2005) 6523ndash6543[14] Z Wang EM Bennett DJ Wilson C Salomon R Vince J Med

Chem 50 (2007) 3416ndash3419[15] K Chibale Towards broad spectrum antiprotozoal agents ARKI-

VOC IX 2002 pp 93ndash98

[16] C Urquiola M Vieites G Aguirre A Marın B Solano GArrambide ML Lavaggi MH Torre M Gonzalez A Monge DGambino H Cerecetto Bioorg Med Chem 14 (2006) 5503ndash5509

[17] G Aguirre H Cerecetto M Gonzalez D Gambino L Otero COlea-Azar C Rigol A Denicola Bioorg Med Chem 12 (2004)4885ndash4893

[18] L Otero M Vieites L Boiani A Denicola C Rigol L Opazo COlea-Azar JD Maya A Morello RL Krauth-Siegel OE Piro ECastellano M Gonzalez D Gambino H Cerecetto J Med Chem49 (2006) 3322ndash3331

[19] A Gomez-Quiroga C Navarro-Ranninger Coord Chem Rev 248(2004) 119ndash133

[20] MD Hall TW Hambley Coord Chem Rev 232 (2002) 49ndash67[21] TW Hambley Coord Chem Rev 166 (1997) 181ndash223[22] S Bonse JM Richards SA Ross G Lowe RL Krauth-Siegel J

Med Chem 43 (2000) 4812ndash4821[23] G Lowe AS Droz T Vilaiyan GW Weaver L Tweedale JM

Pratt P Rock V Yardley SL Croft J Med Chem 42 (1999) 999ndash1006

[24] SL Croft Mem Inst Oswaldo Cruz 94 (1999) 215ndash220[25] WJ Geary Coord Chem Rev 7 (1971) 81ndash91[26] C Rigol C Olea-Azar F Mendizabal L Otero D Gambino M

Gonzalez H Cerecetto Spectrochim Acta A 61 (2005) 2933ndash2938

[27] L Huang A Lee JA Ellman J Med Chem 45 (2002) 676ndash684[28] JD Maya A Morello Y Repetto R Tellez A Rodriguez U

Zelada P Puebla M Bonta S Bollo A San Feliciano CompBiochem Phys C 125 (2000) 103ndash109

[29] MA Cuellar C Salas MJ Cortes A Morello JD Maya MDPreitea Bioorg Med Chem 11 (2003) 2489ndash2497

[30] RE Mahnken MA Billadeau EP Nikonowicz H Morrison JAm Chem Soc 114 (1992) 9253ndash9265

[31] ME Letelier E Rodrıguez A Wallace M Lorca Y Repetto AMorello J Aldunate Exp Parasitol 71 (1990) 357ndash363

[32] D Gambino L Otero M Vieites M Boiani M Gonzalez EJBaran H Cerecetto Spectrochim Acta A 68 (2007) 341ndash348

[33] L Otero C Folch G Barriga C Rigol L Opazo M Vieites DGambino H Cerecetto E NorambuenaC Olea-Azar SpectrochimActa A lthttpdxdoiorg101016jsaa200707045gt

[34] C Olea-Azar C Rigol F Mendizabal A Morello JD Maya CMoncada E Cabrera R Di Maio M Gonzalez H Cerecetto FreeRad Res 37 (2003) 993ndash1001

[35] S Bollo E Soto-Bustamante LJ Nunez-Vergara JA Squella JElectroanal Chem 492 (2000) 54ndash62

[36] C Olea-Azar AM Atria F Mendizabal R Di Maio G Seoane HCerecetto Spectrosc Lett 31 (1998) 99ndash109

[37] VJ Aran C Ochoa L Boiani P Buccino H Cerecetto A GerpeM Gonzalez D Montero JJ Nogal A Gomez-Barrio A AzquetaA Lopez de Ceraın OE Piro EE Castellano Bioorg Med Chem13 (2005) 3197ndash3207

[38] KM Tyler DM Engman Int J Parasitol 31 (2001) 472ndash480[39] Y Repetto E Opazo JD Maya M Agosin A Morello Comp

Biochem Phys B 115 (1996) 281ndash285[40] N Veerapen SA Taylor CJ Walsby B Mario Pinto J Am

Chem Soc 128 (2006) 227ndash239[41] JD Maya S Bollo LJ Nunez-Vergara JA Squella Y Repetto

A Morello J Perie G Chauviere Biochem Pharmacol 65 (2003)999ndash1006

[42] V Brabec Prog Nucl Acid Res Mol Biol 71 (2002) 1ndash68

electrode (Yellow Springs Instruments 53 YSI model)[1831] An amount of parasites equivalent to 10 mg ofproteinmL was added to a 06 mL chamber and oxygenconsumption was recorded at 28 C Control respirationrate corresponded to 28 natoms gram of oxygenminmgprotein In order to evaluate redox cycling mitochondrialrespiration was inhibited with 2 mM potassium cyanideFor comparative purposes and in order to maintain a sim-ilar parasite to drug mass ratio in growth inhibition and inoxygen uptake experiments metal compounds were used ina 125 mM final concentration in the oxygraph chamberResults were corrected according to the observed effectproduced by DMSO alone

3 Results and discussion

Two new series of platinum(II) complexes [PtCl2(HL)]and [Pt(L)2] with bioactive 5-nitrofuryl containing thio-semicarbazones (L) as ligands have been synthesized withhigh purities and good yields All of them are neutralnon conducting compounds and analytical and ESI massspectrometry results are in agreement with the proposedformula As shown below NS bidentate coordinationwas observed in all cases remaining the ligand non depro-tonated at the NH group for the [PtCl2(HL)] compoundsand deprotonated for the [Pt(L)2] compounds

31 IR and Raman spectroscopic studies

Based on experimental spectra and DFT studies wehave previously defined a characteristic vibrational (IRand Raman) spectroscopic pattern for 5-nitrofuryl thio-semicarbazones and their Pd(II) complexes that allowed

Table 1Tentative assignment of the main characteristic IR and Raman bands of the p

Compound IRcm1

m(CN) ms(NO2) m(CndashOndashC) + CndashCcontracta

m(NndashN)b m(Cndash

L1 1602 s 1356 s na 1108 846[PtCl2(HL1)] 1570 vw 1347 s na 1170[Pt(L1)2] 1557 w 1352 s 1335 s 1157L2 1599 w 1354 s na 1114 820PtCl2(HL2)] 1560 s 1349 s na 1173[Pt(L2)2] 1579 w 1350 s 1338 m 1169L3 1602 w 1352 s na 1104 823[PtCl2(HL3)] 1590 w 1344 s na 1175[Pt(L3)2] 1577 w 1350 s 1333 s 1156L4 1595 m 1344 s na 1104 822[PtCl2(HL4)] 1600 m 1347 s na 1175[Pt(L4)2] 1584 vw 1353 s 1339 sh 1084

m stretching d bending s strong m medium w weakna non assignedBands previously reported for the free ligands are included for comparison [3

a Furan CndashOndashC in-phase stretching + CndashC contractb Mediumc Weak to very weakd d(NO2) + furan modes or furan hydrogen wagging symmetric modese Furan in-phase CC stretching

us to analyze the spectroscopic behavior of the eight newPt(II) complexes holding the 5-nitrofuran thiosemicarba-zone moiety IR and Raman spectra of the latter were com-pared with those previously reported for the free ligandsand their Pd(II) analogues and main bands were tentativelyassigned (Table 1) [32]

Although thiosemicarbazone ligands are potentiallycapable of interacting with metal centers in different waysthey usually coordinate to metals as NS bidentate ligandsThere are at least three major stretching vibrations thatpresent strong diagnostic value in relation to the bindingmode of these ligands m(CN) m(CS) and m(NndashN) Aspreviously reported for the selected 5-nitrofuryl containingthiosemicarbazones these bands are located in spectralregions showing a complicated signals pattern that hasmade their assignment difficult [32] In particular thiosem-icarbazone CN and CS stretchings are located in wavenumber regions where vibrations of other portions of theligands occur namely mas (NO2) and 2-substituted furansout of-phase m(CC) occurring in the CN stretchingregion (1650ndash1500 cm1) and d (NO2) furan scissoringvibrations and furan hydrogen wagging symmetric modesand combination of them occurring in the CS stretchingregion (850ndash700 cm1) The combination of experimentaland DFT theoretical methods had previously allowed usto explain the complexity of the observed spectral patternof these ligands and their Pd(II) complexes and to performan assignment of the CN and NndashN stretchings Althoughclear changes were observed in the spectral region around750ndash900 cm1 the CS stretching that is usually used asprobe of the coordination of the metal to the thiocarbonylsulfur could not be undoubtably assigned for the palladiumcomplexes due to the high complexity of the spectra [32]

latinum complexes

Ramancm1

S)c d(NO2)+ furand

m(CN) m(CC)e ms (NO2) m(CndashOndashC) + CndashCcontracta

811 1592 m 1455 s 1351 m 1339 m812 1568 m 1454 s 1354 w na812 1563 s 1450 s 1348 m 1329 m808 1585 br 1474 s 1347 s 1312 m812 1554 sh 1470 s na 1335 s811 1580 s 1469 s 1343 sh 1332 m805 1601 m 1470 s 1351 s 1331 m811 1594 m 1470 s 1355 sh 1324 m810 1575 s 1471 s 1349 m 1330 s811 1596 m 1474 s 1346 m 1321 m811 1599 s 1466 s 1340 s 1319 sh812 1578 s 1472 s 1333 s 1312 sh

2]

Table 21H NMR chemical shift values (d) in ppm of L2 [18] and [PtCl2(HL2)] at30 C

OO2NN

NH

N

SCH3

H

1 2

3

4 5

6

1H NMR L2 Dda

H dLigandb dComplex

c

1 779 767 0122 730 754 0243 798 784 0144 1187 890 2975 852 840 0146 303 310 007

a Dd = (dComplex dLigand)b DMSO-d6c Acetone-d6

The vibrational spectra of the newly developed platinumcomplexes showed a very similar pattern to that of the cor-responding palladium complexes Although the complexityof the IR spectra of the ligands and concomitantly of theirmetal complexes increased from L1 to L4 as the R-substi-tuent complexity increased a common spectral patterncould be observed for each series of platinum compounds([PtCl2(HL)] and [Pt(L)2]) As shown in Table 1 after coor-dination m(CN) bands of the free thiosemicarbazoneligands shifted in almost all cases to lower wave numbersThis modification is consistent with coordination of the thi-osemicarbazone ligands through the azomethyne nitrogenOn the other hand the shift to higher wave numbers of them(NndashN) band observed for the platinum complexes hasalso been previously related to the electronic delocalizationproduced as a consequence of coordination through theazomethine nitrogen atom andor deprotonation of the thi-osemicarbazone ligands [32] The m(CS) bands shouldshift to lower wave numbers when thiosemicarbazonescoordinate through the thiocarbonyl sulfur For the Ptcomplexes weak to very weak sets of bands were detectedin the 700ndash850 cm1 region some of them probably associ-ated with m(CS) vibrations As it was the case for the Pdanalogues the complexity of the spectra in this regiondue to vibrations assigned to the nitrofuran moiety andmixing of vibrations superimposed to a very significantintensity decrease of the CS bands experimentallyobserved after coordination of thiosemicarbazones to ametal made the unambiguos assignment of the CS bandsdifficult Nevertheless clear changes observed in this spec-tral region agree with the coordination of the thiocarbonylsulfur to platinum [32] In agreement with the reported for-mulae of the complexes the m(NH) band at approximately3120ndash3150 cm1 is present in all [PtCl2(HL)] complexesindicating that the ligand is non deprotonated in theseneutral complexes In contrast m(NH) band is not observedin all [Pt(L)2] complexes due to deprotonation of theligands Taking into account the crystal structure of[Pd(L5)2] 3DMSO previously reported [18] and the greatsimilarities in vibrational spectral pattern shown by thePd and Pt analogous compounds a trans configuration isproposed for the [Pt(L)2] complexes (Fig 1) Calculationspreviously performed for the [Pd(L)2] complexes are alsoin agreement with this proposal [32] Vibrations associatedto those portions of the ligands that are not involved in thecoordination to the metal were also recognized in the spec-tra of the complexes and are also shown in Table 1 nitromoiety symmetric stretching (ms(NO2)) furan combinationmode involving furan CndashOndashC in-phase stretching and CndashCcontract (m(CndashOndashC) + CndashC contract) and d(NO2) + furanmodes or furan hydrogen wagging symmetric modes

32 NMR studies

The complexes were characterized by 1H NMR spec-troscopy The low solubility of many of the complexesdid not allow to acquire the complete series of 1H NMR

spectra Platinum complexes and their palladium analoguesshowed similar NMR spectra and behavior [18] Results ofthe performed NMR experiments are in agreement with theproposed structures and with the results of the other spec-troscopies 1H NMR integrations and signal multiplicitiesare in agreement with the proposed formula As an exam-ple results for PtCl2(HL2) are shown in Table 2 1H NMRchemical shifts (d) of the ligand and the complex and thechemical shift differences between complex and ligandexpressed as Dd are shown The attached figure showsthe numbering scheme of the free ligand mentioned in theTable and the text Pt and Pd complexes showed similar1H chemical shifts of the nitrofurylthiosemicarbazone com-mon portion of their molecules [18] When the ligand iscoordinated the effect of the metal is apparent for the pro-tons that are located close to the coordinating atoms theazomethyne nitrogen (H3 according to Table 2) and theNH exchangeable protons (H4 and H5 see Table 2) Thepresence of a signal corresponding to proton H4 is inaccordance with the coordination of the ligand in a nondeprotonated form Furthermore the largest Dd areobserved for this proton

33 Cyclic voltammetry

Voltammetric studies were mainly performed to deter-mine the effect of platinum complexation on the peakpotential of the nitro moiety due to the biological signifi-cance of this potential in relation to the capability of thecompounds to be bioreduced in the parasite leading tothe toxic nitro anion radical species

Fig 2 shows as examples the cyclic voltammograms for[PtCl2(HL3)] and [Pt(L3)2] at 2000 mVs (a) and for[PtCl2(HL3)] when scan rate is changed in the range 100ndash2000 mVs (b) All Pt complexes displayed comparable vol-tammetric behavior showing three well-defined reductionwaves in DMSO as shown by the free ligands [1826]

00 -05 -10 -15 -2015x10-5

10x10-5

50x10-6

00

-50x10-6

-10x10-5

IV

II III[PtL32]

Cur

rent

A

PotentialV

[PtCl2HL3]

I

00 -05 -10 -15 -2050x10-6

40x10-6

30x10-6

20x10-6

10x10-6

00

-10x10-6

-20x10-6

-30x10-6

Cur

rent

A

PotentialV

2000 mVs --- 1000 mVshellip 500 mVs minusminus 250 mVsminus 100 mVs

a b

Fig 2 Cyclic voltammograms of 1 mM DMSO solutions 01 M TBAP of (a) [PtCl2(HL3)] and [Pt(L3)2] at 2000 mVs and (b) [PtCl2(HL3)] when scanrate is changed in the range 100ndash2000 mVs

Table 3Cyclic voltammetric parameters for the reduction of the platinumcomplexes corresponding to the couple II peak Ic IIIc and IVc measuredin DMSO at 2000 mVs

Compound EpIca EpIIc

a EpIIab EpIIIc

a EpIVca

[PtCl2(HL1)] 043 070 066 1209 151[Pt(L1)2] ndash 069 056 122 149[PtCl2(HL2)] 049 073 060 117 137[Pt(L2)2] ndash 068 070 134 165[PtCl2(HL3)] 058 084 058 125 146[Pt(L3)2] ndash 077 061 122 149[PtCl2(HL4)] na 078 056 117 137[Pt(L4)2] ndash 072 062 109 126Nfx [18] 091 085

Nfx Nifurtimox na not assignedPotentials are reported in volts versus saturated calomel electrode

a Epc cathodic peak potentialb Epa anodic peak potential

The first wave for all the Pt compounds corresponded to aquasireversible process involving a one-electron transfer(couple II) This wave around 080 V versus SCE corre-sponds to the generation of the anion radical RNO2 byreduction of the nitro group [1833] The reverse scanshowed the anodic counterpart of the reduction waveAccording to the standard reversibility criteria this couplecan be attributed to a diffusion-controlled one-electrontransfer For some complexes sharp peaks around thiscouple could be observed as a result of adsorption phenom-ena in the electrode surface due to the presence in the mol-ecules of the thiocarbonyl group and the metal Thisphenomenon has been also observed for the Pd analogues[33] Next two couples (III and IV) are assigned to thereduction of the platinum complex nitro anion radicalgenerated in the first couple Subsequent less negativethree-electron irreversible cathodic peak (IIIc Fig 2a) isirreversible in the whole range of sweep rates used (100ndash2000 mVs) and can be attributed to the production ofthe hydroxylamine derivative [2634] Peak IVc is presumedto belong to the reduction of the imine moiety (CHN) ofthe thiosemicarbazone group [35]

Small differences were found between [PtCl2(HL)] and[Pt(L)2] complexes as previously observed for the Pd analo-gous compounds [33] The voltammogram of [PdCl2HL]complexes showed a prepeak (Ic Fig 2a) that appeared evenbefore the reduction of the nitro group meaning that thenitro group follows another reaction path besides the knownelectron-transfer mechanism of the nitroaromatic com-pounds in aprotic media This prepeak would correspondto the four-electron reduction of a small portion of the mol-ecules reaching the electrode surface while the remainingportion would supply the protons required for this reduc-tion This is a typical behavior of a self-protonation phenom-enon displayed by nitrocompounds with acidic moieties intheir structures [263334] The presence of the nitro group

increases the acidity of the NH moiety of the thiosemicarba-zone group present only in the [PtCl2(HL)] complexes (L nondeprotonated) which becomes capable of protonating thenitro group of a minor part of the molecules in the solutionresulting into a lower intensity of these signals [Pt(L)2] com-plexes did not show the Ic prepeak because they do not havethe capability to protonate the nitro group since the NH pro-ton was lost as a consequence of coordination of the ligand toplatinum

Table 3 lists the values of voltammetric peaks for allstudied compounds The potentials of the voltammetricpeaks corresponding to the nitro moiety of the free ligandsslightly changed as a consequence of platinum complexa-tion being the latter only slightly more favourable thanthe former and than those previously reported for the pal-ladium analogues [1826] However it should be stated thatall studied compounds showed under the same conditions ahigher capacity to be reduced than Nifurtimox (E12

[103]a

088 V Table 3) and therefore a better ability to generate

radical species that could be toxic for the parasite [36]

[G]

[G]

3420 3430 3440 3450 3460 3470-25-20-15-10-5

05

10152025

[103]

3420 3430 3440 3450 3460 3470

-40-30-20-10

01020304050

b

Fig 3 (a) ESR experimental spectrum of [Pt(L2)2] Spectrometer condi-tions microwave frequency 971 GHz microwave power 20 mW modu-lation amplitude 02 G scan rate 125 Gs time constant 05 s number ofscans 15 (b) Simulated spectrum of [Pt(L2)2]

34 Free radicals production studied by ESR spectroscopy

The complexes were tested for their capability to pro-duce free radicals in reductive conditions The freeradicals characterized by ESR were prepared lsquolsquoin siturdquo

by electrochemical reduction in DMSO applying apotential corresponding to the first monoelectronic wave(IIcIIa) obtained from the cyclic voltammetric experi-ments The interpretation of the ESR spectra by meansof a simulation process has led to the determination ofthe hyperfine coupling constants for all the magneticnuclei The obtained hyperfine constants are listed inTable 4

The ESR spectra of the [PtCl2(HL)] complexes were sim-ulated in terms of one triplet corresponding to the nitrogennucleus belonging to the nitro group and one triplet to thenitrogen of the CN thiosemicarbazone group and threedoublets due to non-equivalent hydrogens belonging tothe side chain Other nuclei presented hyperfine constantsmaller than the line width

The ESR spectra of the [Pt(L)2] complexes were ana-lyzed in terms of one triplet due to the nitrogen of the nitrogroup two doublets due to hydrogens corresponding to thefuran ring and one triplet due to hydrogens having verysimilar hyperfine constants belonging to the thiosemicar-bazone chains Other hyperfine constants resulted smallerthan the line width and they were not observed in theexperimental spectrum Fig 3 shows the ESR spectrumof complex [Pt(L2)2]

Different substituents in the thiosemicarbazone chain ofthe coordinated ligands did not seem to affect the hyperfinepattern of platinum complexes as it had been previouslyobserved for the corresponding palladium ones Howeverthe number of ligands coordinated per Pt central atomseemed to determine spin density distribution and so thehyperfine pattern of the complexes For the [Pt(L)2] com-plexes the spin density was more located on the nitro furanring while other observed couplings could be related to noncompletely equivalent thiosemicarbazone ligands coordi-nated to the platinum atom For the [PtCl2(HL)] com-

Table 4Hyperfine splittings (gauss) for the anion radical of the platinumcomplexes

aN aN aH aH aH aH aH

[PtCl2(HL1)] 695 41 128 14 112 lt06 lt06[Pt(L1)2] 68 lt06 08 40 lt06 245 235[PtCl2(HL2)] 697 35 12 15 100 lt06 lt06[Pt(L2)2] 70 lt06 12 40 lt06 31 33[PtCl2(HL3)] 690 37 115 16 128 lt06 lt06[Pt(L3)2] 70 lt06 124 40 lt06 30 33[PtCl2(HL4)] 687 40 117 125 13 lt06 lt06[Pt(L4)2] 72 lt06 110 38 lt06 298 310

pounds the spin density was more delocalized on theentire radical

The effect of changing the metal atom on the hyperfinepattern of the ESR spectra of the complexes should benoted For palladium complexes no differences have beenobserved between [PdCl2(HL)] and [Pd(L)2] ones Both ser-ies of palladium compounds presented a quite similarhyperfine pattern to that observed for the [PtCl2(HL)] com-plexes The different observed ESR pattern for [M(L)2]complexes when changing palladium by platinum couldbe related to small differences in complexes structure Thisfact would also agree with a quite different biologicalbehavior

35 In vitro anti T cruzi activity

The existence of the epimastigote form of T cruzi as anobligate mammalian intracellular stage has been confirmedrecently [3738] Therefore compounds were tested in vitro

against epimastigote form of the parasite The complexeswere evaluated for their anti-T cruzi activities against epim-astigotes of Tulahuen 2 strain in order to compare with pre-viously performed biological evaluation of the free ligandsand the analogous Pd complexes Fifty percent of inhibitoryconcentrations (IC50) obtained from dose ndash response curvesare depicted in Table 5 In addition some complexes wereevaluated against a second strain of T cruzi epimastigotesnamely Dm28c

For comparative purposes Table 5 also includes IC50

values for the four free ligands and the Pd complexes

Table 5In vitro biological activity of the Pt complexes

Compound T cruzi (Tulahuen 2) T cruzi (Dm28c) Compound T cruzi (Tulahuen 2)PGIa IC50

b ICkc50b IC50

c

10 lM 25 lM

L1 27[PtCl2(HL1)] 109 382 gt25 nd [PdCl2(HL1)] 24[Pt(L1)2] 34 208 gt25 nd [Pd(L1)2] 45

L2 50[PtCl2(HL2)] 415 605 131 367 [PdCl2(HL2)] 43[Pt(L2)2] 684 100 69 102 [Pd(L2)2] 47

L3 49[PtCl2(HL3)] 269 441 275 nd [PdCl2(HL3)] 59[Pt(L3)2] 100 100 08 288 [Pd(L3)2] gt25

L4 gt25[PtCl2(HL4)] 338 720 15 284 [PdCl2(HL4)] gt25[Pt(L4)2] 189 121 gt25 413 [Pd(L4)2] gt25Benznidazol 74 380Nifurtimox 61 [17] 228

IC50 values of the free ligand and their Pd complexes on T cruzi (Tulahuen 2 strain) have been included for comparison [18]nd not determined

a PGI percentage of growth inhibition of T cruzi epimastigote cells at the specified doseb plusmn10c [18]

3430 3440 3450 3460 3470 3480 3490 3500 3510 3520 3530

-8

-6

-4

-2

0

2

4

6

8[103]

previously determined through the same technique on epi-mastigote form of T cruzi Tulahuen 2 strain [18] Most ofthe Pt complexes were active against epimastigotes of T

cruzi Tulahuen 2 strain showing some of them IC50 valuesof the same order than Nifurtimox and the correspondingfree ligands According to the IC50 values [Pt(L3)2] and[Pt(L2)2] were the most active Pt complexes against thisparasite strain being [Pt(L3)2] significantly more activethan L3 and than Nifurtimox

When the results are compared with those previouslyreported for the free ligands and the Pd analogous com-pounds it can be stated that the activity pattern is signifi-cantly modified by changing the central atom The activityof the Pd complexes followed the general trend[PdCl2(HL)] gt free ligand gt [Pd(L)2] This regular trendwas not observed for the Pt analogues This differentbehavior could be related with differences in bioavailabil-ity unspecific toxicity andor mechanism of action of bothseries of metal complexes

Similar general conclusions can be obtained from theanalysis of the activity results on the T cruzi Dm28c strainalthough some strain susceptibility differences can beobserved Dm28c is a parasite clone that had previouslyshown to be less susceptible to Nifurtimox and Ben-znidazol trypanocidal effect than Tulahuen strain whichis mediated by a Dm28c higher thiol content of the former[39]

[G]

Fig 4 ESR spectrum of [PtCl2(HL2)] obtained when [PtCl2(HL2)] wasincubated with T cruzi epimastigotes in the presence of DMPOSpectrometer conditions microwave frequency 968 GHz microwavepower 20 mW modulation amplitude 02 G scan rate 125 Gs timeconstant 05 s number of scans 15 signals of DMPO-nitro anionradical adduct

36 Capacity of derivatives to generate free radical species

into T cruzi

The complexes were incubated with intact T cruzi

(Dm28c strain) epimastigotes in the presence of DMPO

as spin trapping agent in order to detect possible intra-celullar free radical species produced by bioreduction andhaving short half-lives The biological free radical produc-tion capacity of the new compounds was assessed by ESRAll the compounds were capable to produce free radicals inthe intact parasite giving an ESR spectrum Fig 4 showsas an example the ESR spectrum obtained when[PtCl2(HL2)] was incubated with T cruzi in the presenceof DMPO The typical hyperfine pattern of adductsobtained when DMPO traps radicals centered in C atom

[Pt(L2)2]

1 minute

10 O2

[Pt(L2)2]KCN

a

b

Fig 5 (a) Effect of compound [Pt(L)2] on the parasite (T cruzi Dm28cstrain) oxygen consumption related to control (dotted line) (b) Effect ofcompound [Pt(L)2] on the parasite (T cruzi Dm28c strain) oxygenconsumption after inhibition of the respiration with KCN related tocontrol (dotted line)

Table 6Effect of the addition of Pt complexes on oxygen uptake by T cruzi

epimastigotes (Dm28c strain)

Compound Respiration (nanoatom-gramof Ominmg protein)

Increase on oxygenconsumption ()a

Control 280 plusmn 20[PtCl2(HL2)] 322 plusmn 32 15[Pt(L2)2] 313 plusmn 17 12[Pt(L3)2] 392 plusmn 42 40[PtCl2(HL4)] 355 plusmn 32 27[Pt(L4)2] 320 plusmn 13 14Nifurtimox 356 plusmn 08 27

a increase on oxygen consumption after addition of 125 mM ofcompounds with respect to control (no added compound)

Table 7Interaction of Pt complexes with CT DNA after 96 h of incubation at37 C

Compound nmol Ptmg DNA Metalbasea Basemetal

[PtCl2(HL1)] 209 0069 14[Pt(L1)2] 390 0129 8[PtCl2(HL2)] 236 0078 13[Pt(L2)2] 224 0074 14[PtCl2(HL3)] 191 0063 16[Pt(L3)2] 527 0174 6[PtCl2(HL4)] 170 0056 18[Pt(L4)2] 170 0056 18

a Platinum (mol) per DNA base (mol)

(aN = 167 G and aH = 225 G) was observed [18] A trip-let produced by DMPO decomposition was also detected[40]

ESR studies showed that the newly developed Pt thio-semicarbazone complexes were bioreduced in the parasiteindicating that they could maintain the mode of action ofthe 5-nitrofuryl pharmacophore

37 Oxygen uptake

The involvement of the platinum complexes in redoxcycling processes should increase the parasite oxygen con-sumption Thus the effect of the compounds on parasiteoxygen uptake was measured The effect of complex[Pt(L2)2] on T cruzi epimastigotes oxygen consumptionwith and without inhibition of the respiration with KCNis shown in Fig 5 Both series of complexes modified oxy-gen uptake by the parasite increasing oxygen consumption(Table 6) These results are in accordance with a bioreduc-tive and redox cycling mechanism of action as previouslyreported for the free 5-nitrofuryl containing thiosemicarba-zones their Pd complexes and other nitrofuran derivativeslike Nifurtimox [1841] For both series of compoundseffect on parasite respiration seemed to correlate with theIC50 values For instance [Pt(L3)2] being the most activePt compound showed the highest increase on oxygenconsumption

38 Calf thymus DNA interaction

In order to address if interaction with DNA could bepart of the mode of action of the complexes experimentswith CT DNA were carried out Binding of the Pt com-plexes to DNA was studied by combining atomic absorp-tion determinations (for the metal) and electronicabsorption measurements for DNA quantification Thenew complexes are good binding agents for CT DNA

(Table 7) Although the determined platinum to DNAbinding levels were similar to those of known anti-tumormetal complexes they resulted significantly lower thanthose of the palladium analogous compounds [1819] Nogeneral pattern could be detected for both series of com-pounds being binding levels almost not affected by thenature of the thiosemicarbazone ligand [PtCl2(HL)] com-plexes having labile chloride ligands could probably inter-act with DNA through a mechanism similar to thatpreviously reported for cisplatin becoming activatedthrough aquation and reacting with nucleophilic DNAbases [42] [Pt(L)2] complexes cannot act in this way butthey could intercalate DNA or interact through othermechanisms Due to the planar motive contained in the[PdCl2(HL)] complexes intercalation could also be possiblefor these compounds Further studies are under progress inorder to get an insight into the mechanism of this interac-tion It is interesting to note that [Pt(L3)2] being the mostpotent anti-T cruzi compound of the series showed thehighest interaction with DNA Nevertheless [Pt(L1)2]being almost non active strongly interacts with DNAshowing that the mechanism of trypanocidal activity couldnot involve only DNA as target

4 Conclusions

Pt(II) complexes with 5-nitrofuryl containing thiosemi-carbazones as bioactive ligands showed good anti-trypan-osomal activity In particular the coordination of L3 to

Pt(II) forming [Pt(L3)2] lead to almost a five-fold activityincrease in respect to the free ligand Possible targetsnamely reductive metabolism (intra-parasite free radicalproduction) and DNA were screened trying to get insightinto the mechanism of action of these metal compoundsResults showed that some of the compounds could act asdual inhibitors in the parasite through production of toxicfree radicals and interaction with DNA

Results of this work show that the approach of coordi-nating anti-trypanosomal organic compounds with phar-macologically interesting metals could be a suitablestrategy to develop novel therapeutic tools against tropicaldiseases produced by trypanosomatids particularly Amer-ican Trypanosomiasis

Acknowledgments

This work was partially supported by PEDECIBA andUniversidad de La Republica (CSIC project 36406) ofUruguay Prosul-CNPq project 4902092005-0 SIBAS-0807 UMCE FONDECYT Chile 1061072 and CONI-CYT-PBCT Anillo ACT 29 We wish to thank Dr EJBaran CEQUINOR UNLP Argentina for Raman spec-tra facilities and Matilde Gomez Instituto Venezolano deInvestigaciones Cientıficas Caracas Venezuela for per-forming ESI-MS experiments

References

[1] httpwwwwhoint lthttpwwwwhointctdchagasgt[2] D Engels L Savioli Trend Parasitol 22 (2006) 363ndash366[3] J Urbina Expert Opin Ther Pat 13 (2003) 661ndash669[4] H Cerecetto M Gonzalez Curr Topics Med Chem 2 (2002) 1185ndash

1190[5] RL Krauth-Siegel H Bauer RH Schirmer Angew Chem Int Ed

44 (2005) 690ndash715[6] S Croft M Barret J Urbina Trend Parasitol 21 (2005) 508ndash512[7] M Ceaser The Lancet Infect Dis 5 (8) (2005) 470ndash471[8] Y Yamagata J Nakagawa Adv Parasitol 61 (2006) 129ndash165[9] C Zhang S Lippard Curr Opin Chem Biol 7 (2003) 481ndash489

[10] NP Farrell Metal Complexes as Drugs and ChemotherapeuticAgents in JM McCleverty TJ Meyer (Eds) ComprehensiveCoordination Chemistry II vol 9 Elsevier 2003 pp 809ndash840

[11] RA Sanchez-Delgado A Anzellotti L Suarez Metal complexes aschemotherapeutic agents against tropical diseases malaria trypano-somiasis and leishmaniasis in H Sigel A Sigel (Eds) Metal Ionsin Biological Systems vol 41 Marcel Dekker New York 2004 pp379ndash419

[12] RA Sanchez-Delgado A Anzellotti Mini-Rev Med Chem 4(2004) 23ndash30

[13] R Morphy Z Rankovic J Med Chem 48 (2005) 6523ndash6543[14] Z Wang EM Bennett DJ Wilson C Salomon R Vince J Med

Chem 50 (2007) 3416ndash3419[15] K Chibale Towards broad spectrum antiprotozoal agents ARKI-

VOC IX 2002 pp 93ndash98

[16] C Urquiola M Vieites G Aguirre A Marın B Solano GArrambide ML Lavaggi MH Torre M Gonzalez A Monge DGambino H Cerecetto Bioorg Med Chem 14 (2006) 5503ndash5509

[17] G Aguirre H Cerecetto M Gonzalez D Gambino L Otero COlea-Azar C Rigol A Denicola Bioorg Med Chem 12 (2004)4885ndash4893

[18] L Otero M Vieites L Boiani A Denicola C Rigol L Opazo COlea-Azar JD Maya A Morello RL Krauth-Siegel OE Piro ECastellano M Gonzalez D Gambino H Cerecetto J Med Chem49 (2006) 3322ndash3331

[19] A Gomez-Quiroga C Navarro-Ranninger Coord Chem Rev 248(2004) 119ndash133

[20] MD Hall TW Hambley Coord Chem Rev 232 (2002) 49ndash67[21] TW Hambley Coord Chem Rev 166 (1997) 181ndash223[22] S Bonse JM Richards SA Ross G Lowe RL Krauth-Siegel J

Med Chem 43 (2000) 4812ndash4821[23] G Lowe AS Droz T Vilaiyan GW Weaver L Tweedale JM

Pratt P Rock V Yardley SL Croft J Med Chem 42 (1999) 999ndash1006

[24] SL Croft Mem Inst Oswaldo Cruz 94 (1999) 215ndash220[25] WJ Geary Coord Chem Rev 7 (1971) 81ndash91[26] C Rigol C Olea-Azar F Mendizabal L Otero D Gambino M

Gonzalez H Cerecetto Spectrochim Acta A 61 (2005) 2933ndash2938

[27] L Huang A Lee JA Ellman J Med Chem 45 (2002) 676ndash684[28] JD Maya A Morello Y Repetto R Tellez A Rodriguez U

Zelada P Puebla M Bonta S Bollo A San Feliciano CompBiochem Phys C 125 (2000) 103ndash109

[29] MA Cuellar C Salas MJ Cortes A Morello JD Maya MDPreitea Bioorg Med Chem 11 (2003) 2489ndash2497

[30] RE Mahnken MA Billadeau EP Nikonowicz H Morrison JAm Chem Soc 114 (1992) 9253ndash9265

[31] ME Letelier E Rodrıguez A Wallace M Lorca Y Repetto AMorello J Aldunate Exp Parasitol 71 (1990) 357ndash363

[32] D Gambino L Otero M Vieites M Boiani M Gonzalez EJBaran H Cerecetto Spectrochim Acta A 68 (2007) 341ndash348

[33] L Otero C Folch G Barriga C Rigol L Opazo M Vieites DGambino H Cerecetto E NorambuenaC Olea-Azar SpectrochimActa A lthttpdxdoiorg101016jsaa200707045gt

[34] C Olea-Azar C Rigol F Mendizabal A Morello JD Maya CMoncada E Cabrera R Di Maio M Gonzalez H Cerecetto FreeRad Res 37 (2003) 993ndash1001

[35] S Bollo E Soto-Bustamante LJ Nunez-Vergara JA Squella JElectroanal Chem 492 (2000) 54ndash62

[36] C Olea-Azar AM Atria F Mendizabal R Di Maio G Seoane HCerecetto Spectrosc Lett 31 (1998) 99ndash109

[37] VJ Aran C Ochoa L Boiani P Buccino H Cerecetto A GerpeM Gonzalez D Montero JJ Nogal A Gomez-Barrio A AzquetaA Lopez de Ceraın OE Piro EE Castellano Bioorg Med Chem13 (2005) 3197ndash3207

[38] KM Tyler DM Engman Int J Parasitol 31 (2001) 472ndash480[39] Y Repetto E Opazo JD Maya M Agosin A Morello Comp

Biochem Phys B 115 (1996) 281ndash285[40] N Veerapen SA Taylor CJ Walsby B Mario Pinto J Am

Chem Soc 128 (2006) 227ndash239[41] JD Maya S Bollo LJ Nunez-Vergara JA Squella Y Repetto

A Morello J Perie G Chauviere Biochem Pharmacol 65 (2003)999ndash1006

[42] V Brabec Prog Nucl Acid Res Mol Biol 71 (2002) 1ndash68

Table 21H NMR chemical shift values (d) in ppm of L2 [18] and [PtCl2(HL2)] at30 C

OO2NN

NH

N

SCH3

H

1 2

3

4 5

6

1H NMR L2 Dda

H dLigandb dComplex

c

1 779 767 0122 730 754 0243 798 784 0144 1187 890 2975 852 840 0146 303 310 007

a Dd = (dComplex dLigand)b DMSO-d6c Acetone-d6

The vibrational spectra of the newly developed platinumcomplexes showed a very similar pattern to that of the cor-responding palladium complexes Although the complexityof the IR spectra of the ligands and concomitantly of theirmetal complexes increased from L1 to L4 as the R-substi-tuent complexity increased a common spectral patterncould be observed for each series of platinum compounds([PtCl2(HL)] and [Pt(L)2]) As shown in Table 1 after coor-dination m(CN) bands of the free thiosemicarbazoneligands shifted in almost all cases to lower wave numbersThis modification is consistent with coordination of the thi-osemicarbazone ligands through the azomethyne nitrogenOn the other hand the shift to higher wave numbers of them(NndashN) band observed for the platinum complexes hasalso been previously related to the electronic delocalizationproduced as a consequence of coordination through theazomethine nitrogen atom andor deprotonation of the thi-osemicarbazone ligands [32] The m(CS) bands shouldshift to lower wave numbers when thiosemicarbazonescoordinate through the thiocarbonyl sulfur For the Ptcomplexes weak to very weak sets of bands were detectedin the 700ndash850 cm1 region some of them probably associ-ated with m(CS) vibrations As it was the case for the Pdanalogues the complexity of the spectra in this regiondue to vibrations assigned to the nitrofuran moiety andmixing of vibrations superimposed to a very significantintensity decrease of the CS bands experimentallyobserved after coordination of thiosemicarbazones to ametal made the unambiguos assignment of the CS bandsdifficult Nevertheless clear changes observed in this spec-tral region agree with the coordination of the thiocarbonylsulfur to platinum [32] In agreement with the reported for-mulae of the complexes the m(NH) band at approximately3120ndash3150 cm1 is present in all [PtCl2(HL)] complexesindicating that the ligand is non deprotonated in theseneutral complexes In contrast m(NH) band is not observedin all [Pt(L)2] complexes due to deprotonation of theligands Taking into account the crystal structure of[Pd(L5)2] 3DMSO previously reported [18] and the greatsimilarities in vibrational spectral pattern shown by thePd and Pt analogous compounds a trans configuration isproposed for the [Pt(L)2] complexes (Fig 1) Calculationspreviously performed for the [Pd(L)2] complexes are alsoin agreement with this proposal [32] Vibrations associatedto those portions of the ligands that are not involved in thecoordination to the metal were also recognized in the spec-tra of the complexes and are also shown in Table 1 nitromoiety symmetric stretching (ms(NO2)) furan combinationmode involving furan CndashOndashC in-phase stretching and CndashCcontract (m(CndashOndashC) + CndashC contract) and d(NO2) + furanmodes or furan hydrogen wagging symmetric modes

32 NMR studies

The complexes were characterized by 1H NMR spec-troscopy The low solubility of many of the complexesdid not allow to acquire the complete series of 1H NMR

spectra Platinum complexes and their palladium analoguesshowed similar NMR spectra and behavior [18] Results ofthe performed NMR experiments are in agreement with theproposed structures and with the results of the other spec-troscopies 1H NMR integrations and signal multiplicitiesare in agreement with the proposed formula As an exam-ple results for PtCl2(HL2) are shown in Table 2 1H NMRchemical shifts (d) of the ligand and the complex and thechemical shift differences between complex and ligandexpressed as Dd are shown The attached figure showsthe numbering scheme of the free ligand mentioned in theTable and the text Pt and Pd complexes showed similar1H chemical shifts of the nitrofurylthiosemicarbazone com-mon portion of their molecules [18] When the ligand iscoordinated the effect of the metal is apparent for the pro-tons that are located close to the coordinating atoms theazomethyne nitrogen (H3 according to Table 2) and theNH exchangeable protons (H4 and H5 see Table 2) Thepresence of a signal corresponding to proton H4 is inaccordance with the coordination of the ligand in a nondeprotonated form Furthermore the largest Dd areobserved for this proton

33 Cyclic voltammetry

Voltammetric studies were mainly performed to deter-mine the effect of platinum complexation on the peakpotential of the nitro moiety due to the biological signifi-cance of this potential in relation to the capability of thecompounds to be bioreduced in the parasite leading tothe toxic nitro anion radical species

Fig 2 shows as examples the cyclic voltammograms for[PtCl2(HL3)] and [Pt(L3)2] at 2000 mVs (a) and for[PtCl2(HL3)] when scan rate is changed in the range 100ndash2000 mVs (b) All Pt complexes displayed comparable vol-tammetric behavior showing three well-defined reductionwaves in DMSO as shown by the free ligands [1826]

00 -05 -10 -15 -2015x10-5

10x10-5

50x10-6

00

-50x10-6

-10x10-5

IV

II III[PtL32]

Cur

rent

A

PotentialV

[PtCl2HL3]

I

00 -05 -10 -15 -2050x10-6

40x10-6

30x10-6

20x10-6

10x10-6

00

-10x10-6

-20x10-6

-30x10-6

Cur

rent

A

PotentialV

2000 mVs --- 1000 mVshellip 500 mVs minusminus 250 mVsminus 100 mVs

a b

Fig 2 Cyclic voltammograms of 1 mM DMSO solutions 01 M TBAP of (a) [PtCl2(HL3)] and [Pt(L3)2] at 2000 mVs and (b) [PtCl2(HL3)] when scanrate is changed in the range 100ndash2000 mVs

Table 3Cyclic voltammetric parameters for the reduction of the platinumcomplexes corresponding to the couple II peak Ic IIIc and IVc measuredin DMSO at 2000 mVs

Compound EpIca EpIIc

a EpIIab EpIIIc

a EpIVca

[PtCl2(HL1)] 043 070 066 1209 151[Pt(L1)2] ndash 069 056 122 149[PtCl2(HL2)] 049 073 060 117 137[Pt(L2)2] ndash 068 070 134 165[PtCl2(HL3)] 058 084 058 125 146[Pt(L3)2] ndash 077 061 122 149[PtCl2(HL4)] na 078 056 117 137[Pt(L4)2] ndash 072 062 109 126Nfx [18] 091 085

Nfx Nifurtimox na not assignedPotentials are reported in volts versus saturated calomel electrode

a Epc cathodic peak potentialb Epa anodic peak potential

The first wave for all the Pt compounds corresponded to aquasireversible process involving a one-electron transfer(couple II) This wave around 080 V versus SCE corre-sponds to the generation of the anion radical RNO2 byreduction of the nitro group [1833] The reverse scanshowed the anodic counterpart of the reduction waveAccording to the standard reversibility criteria this couplecan be attributed to a diffusion-controlled one-electrontransfer For some complexes sharp peaks around thiscouple could be observed as a result of adsorption phenom-ena in the electrode surface due to the presence in the mol-ecules of the thiocarbonyl group and the metal Thisphenomenon has been also observed for the Pd analogues[33] Next two couples (III and IV) are assigned to thereduction of the platinum complex nitro anion radicalgenerated in the first couple Subsequent less negativethree-electron irreversible cathodic peak (IIIc Fig 2a) isirreversible in the whole range of sweep rates used (100ndash2000 mVs) and can be attributed to the production ofthe hydroxylamine derivative [2634] Peak IVc is presumedto belong to the reduction of the imine moiety (CHN) ofthe thiosemicarbazone group [35]

Small differences were found between [PtCl2(HL)] and[Pt(L)2] complexes as previously observed for the Pd analo-gous compounds [33] The voltammogram of [PdCl2HL]complexes showed a prepeak (Ic Fig 2a) that appeared evenbefore the reduction of the nitro group meaning that thenitro group follows another reaction path besides the knownelectron-transfer mechanism of the nitroaromatic com-pounds in aprotic media This prepeak would correspondto the four-electron reduction of a small portion of the mol-ecules reaching the electrode surface while the remainingportion would supply the protons required for this reduc-tion This is a typical behavior of a self-protonation phenom-enon displayed by nitrocompounds with acidic moieties intheir structures [263334] The presence of the nitro group

increases the acidity of the NH moiety of the thiosemicarba-zone group present only in the [PtCl2(HL)] complexes (L nondeprotonated) which becomes capable of protonating thenitro group of a minor part of the molecules in the solutionresulting into a lower intensity of these signals [Pt(L)2] com-plexes did not show the Ic prepeak because they do not havethe capability to protonate the nitro group since the NH pro-ton was lost as a consequence of coordination of the ligand toplatinum

Table 3 lists the values of voltammetric peaks for allstudied compounds The potentials of the voltammetricpeaks corresponding to the nitro moiety of the free ligandsslightly changed as a consequence of platinum complexa-tion being the latter only slightly more favourable thanthe former and than those previously reported for the pal-ladium analogues [1826] However it should be stated thatall studied compounds showed under the same conditions ahigher capacity to be reduced than Nifurtimox (E12

[103]a

088 V Table 3) and therefore a better ability to generate

radical species that could be toxic for the parasite [36]

[G]

[G]

3420 3430 3440 3450 3460 3470-25-20-15-10-5

05

10152025

[103]

3420 3430 3440 3450 3460 3470

-40-30-20-10

01020304050

b

Fig 3 (a) ESR experimental spectrum of [Pt(L2)2] Spectrometer condi-tions microwave frequency 971 GHz microwave power 20 mW modu-lation amplitude 02 G scan rate 125 Gs time constant 05 s number ofscans 15 (b) Simulated spectrum of [Pt(L2)2]

34 Free radicals production studied by ESR spectroscopy

The complexes were tested for their capability to pro-duce free radicals in reductive conditions The freeradicals characterized by ESR were prepared lsquolsquoin siturdquo

by electrochemical reduction in DMSO applying apotential corresponding to the first monoelectronic wave(IIcIIa) obtained from the cyclic voltammetric experi-ments The interpretation of the ESR spectra by meansof a simulation process has led to the determination ofthe hyperfine coupling constants for all the magneticnuclei The obtained hyperfine constants are listed inTable 4

The ESR spectra of the [PtCl2(HL)] complexes were sim-ulated in terms of one triplet corresponding to the nitrogennucleus belonging to the nitro group and one triplet to thenitrogen of the CN thiosemicarbazone group and threedoublets due to non-equivalent hydrogens belonging tothe side chain Other nuclei presented hyperfine constantsmaller than the line width

The ESR spectra of the [Pt(L)2] complexes were ana-lyzed in terms of one triplet due to the nitrogen of the nitrogroup two doublets due to hydrogens corresponding to thefuran ring and one triplet due to hydrogens having verysimilar hyperfine constants belonging to the thiosemicar-bazone chains Other hyperfine constants resulted smallerthan the line width and they were not observed in theexperimental spectrum Fig 3 shows the ESR spectrumof complex [Pt(L2)2]

Different substituents in the thiosemicarbazone chain ofthe coordinated ligands did not seem to affect the hyperfinepattern of platinum complexes as it had been previouslyobserved for the corresponding palladium ones Howeverthe number of ligands coordinated per Pt central atomseemed to determine spin density distribution and so thehyperfine pattern of the complexes For the [Pt(L)2] com-plexes the spin density was more located on the nitro furanring while other observed couplings could be related to noncompletely equivalent thiosemicarbazone ligands coordi-nated to the platinum atom For the [PtCl2(HL)] com-

Table 4Hyperfine splittings (gauss) for the anion radical of the platinumcomplexes

aN aN aH aH aH aH aH

[PtCl2(HL1)] 695 41 128 14 112 lt06 lt06[Pt(L1)2] 68 lt06 08 40 lt06 245 235[PtCl2(HL2)] 697 35 12 15 100 lt06 lt06[Pt(L2)2] 70 lt06 12 40 lt06 31 33[PtCl2(HL3)] 690 37 115 16 128 lt06 lt06[Pt(L3)2] 70 lt06 124 40 lt06 30 33[PtCl2(HL4)] 687 40 117 125 13 lt06 lt06[Pt(L4)2] 72 lt06 110 38 lt06 298 310

pounds the spin density was more delocalized on theentire radical

The effect of changing the metal atom on the hyperfinepattern of the ESR spectra of the complexes should benoted For palladium complexes no differences have beenobserved between [PdCl2(HL)] and [Pd(L)2] ones Both ser-ies of palladium compounds presented a quite similarhyperfine pattern to that observed for the [PtCl2(HL)] com-plexes The different observed ESR pattern for [M(L)2]complexes when changing palladium by platinum couldbe related to small differences in complexes structure Thisfact would also agree with a quite different biologicalbehavior

35 In vitro anti T cruzi activity

The existence of the epimastigote form of T cruzi as anobligate mammalian intracellular stage has been confirmedrecently [3738] Therefore compounds were tested in vitro

against epimastigote form of the parasite The complexeswere evaluated for their anti-T cruzi activities against epim-astigotes of Tulahuen 2 strain in order to compare with pre-viously performed biological evaluation of the free ligandsand the analogous Pd complexes Fifty percent of inhibitoryconcentrations (IC50) obtained from dose ndash response curvesare depicted in Table 5 In addition some complexes wereevaluated against a second strain of T cruzi epimastigotesnamely Dm28c

For comparative purposes Table 5 also includes IC50

values for the four free ligands and the Pd complexes

Table 5In vitro biological activity of the Pt complexes

Compound T cruzi (Tulahuen 2) T cruzi (Dm28c) Compound T cruzi (Tulahuen 2)PGIa IC50

b ICkc50b IC50

c

10 lM 25 lM

L1 27[PtCl2(HL1)] 109 382 gt25 nd [PdCl2(HL1)] 24[Pt(L1)2] 34 208 gt25 nd [Pd(L1)2] 45

L2 50[PtCl2(HL2)] 415 605 131 367 [PdCl2(HL2)] 43[Pt(L2)2] 684 100 69 102 [Pd(L2)2] 47

L3 49[PtCl2(HL3)] 269 441 275 nd [PdCl2(HL3)] 59[Pt(L3)2] 100 100 08 288 [Pd(L3)2] gt25

L4 gt25[PtCl2(HL4)] 338 720 15 284 [PdCl2(HL4)] gt25[Pt(L4)2] 189 121 gt25 413 [Pd(L4)2] gt25Benznidazol 74 380Nifurtimox 61 [17] 228

IC50 values of the free ligand and their Pd complexes on T cruzi (Tulahuen 2 strain) have been included for comparison [18]nd not determined

a PGI percentage of growth inhibition of T cruzi epimastigote cells at the specified doseb plusmn10c [18]

3430 3440 3450 3460 3470 3480 3490 3500 3510 3520 3530

-8

-6

-4

-2

0

2

4

6

8[103]

previously determined through the same technique on epi-mastigote form of T cruzi Tulahuen 2 strain [18] Most ofthe Pt complexes were active against epimastigotes of T

cruzi Tulahuen 2 strain showing some of them IC50 valuesof the same order than Nifurtimox and the correspondingfree ligands According to the IC50 values [Pt(L3)2] and[Pt(L2)2] were the most active Pt complexes against thisparasite strain being [Pt(L3)2] significantly more activethan L3 and than Nifurtimox

When the results are compared with those previouslyreported for the free ligands and the Pd analogous com-pounds it can be stated that the activity pattern is signifi-cantly modified by changing the central atom The activityof the Pd complexes followed the general trend[PdCl2(HL)] gt free ligand gt [Pd(L)2] This regular trendwas not observed for the Pt analogues This differentbehavior could be related with differences in bioavailabil-ity unspecific toxicity andor mechanism of action of bothseries of metal complexes

Similar general conclusions can be obtained from theanalysis of the activity results on the T cruzi Dm28c strainalthough some strain susceptibility differences can beobserved Dm28c is a parasite clone that had previouslyshown to be less susceptible to Nifurtimox and Ben-znidazol trypanocidal effect than Tulahuen strain whichis mediated by a Dm28c higher thiol content of the former[39]

[G]

Fig 4 ESR spectrum of [PtCl2(HL2)] obtained when [PtCl2(HL2)] wasincubated with T cruzi epimastigotes in the presence of DMPOSpectrometer conditions microwave frequency 968 GHz microwavepower 20 mW modulation amplitude 02 G scan rate 125 Gs timeconstant 05 s number of scans 15 signals of DMPO-nitro anionradical adduct

36 Capacity of derivatives to generate free radical species

into T cruzi

The complexes were incubated with intact T cruzi

(Dm28c strain) epimastigotes in the presence of DMPO

as spin trapping agent in order to detect possible intra-celullar free radical species produced by bioreduction andhaving short half-lives The biological free radical produc-tion capacity of the new compounds was assessed by ESRAll the compounds were capable to produce free radicals inthe intact parasite giving an ESR spectrum Fig 4 showsas an example the ESR spectrum obtained when[PtCl2(HL2)] was incubated with T cruzi in the presenceof DMPO The typical hyperfine pattern of adductsobtained when DMPO traps radicals centered in C atom

[Pt(L2)2]

1 minute

10 O2

[Pt(L2)2]KCN

a

b

Fig 5 (a) Effect of compound [Pt(L)2] on the parasite (T cruzi Dm28cstrain) oxygen consumption related to control (dotted line) (b) Effect ofcompound [Pt(L)2] on the parasite (T cruzi Dm28c strain) oxygenconsumption after inhibition of the respiration with KCN related tocontrol (dotted line)

Table 6Effect of the addition of Pt complexes on oxygen uptake by T cruzi

epimastigotes (Dm28c strain)

Compound Respiration (nanoatom-gramof Ominmg protein)

Increase on oxygenconsumption ()a

Control 280 plusmn 20[PtCl2(HL2)] 322 plusmn 32 15[Pt(L2)2] 313 plusmn 17 12[Pt(L3)2] 392 plusmn 42 40[PtCl2(HL4)] 355 plusmn 32 27[Pt(L4)2] 320 plusmn 13 14Nifurtimox 356 plusmn 08 27

a increase on oxygen consumption after addition of 125 mM ofcompounds with respect to control (no added compound)

Table 7Interaction of Pt complexes with CT DNA after 96 h of incubation at37 C

Compound nmol Ptmg DNA Metalbasea Basemetal

[PtCl2(HL1)] 209 0069 14[Pt(L1)2] 390 0129 8[PtCl2(HL2)] 236 0078 13[Pt(L2)2] 224 0074 14[PtCl2(HL3)] 191 0063 16[Pt(L3)2] 527 0174 6[PtCl2(HL4)] 170 0056 18[Pt(L4)2] 170 0056 18

a Platinum (mol) per DNA base (mol)

(aN = 167 G and aH = 225 G) was observed [18] A trip-let produced by DMPO decomposition was also detected[40]

ESR studies showed that the newly developed Pt thio-semicarbazone complexes were bioreduced in the parasiteindicating that they could maintain the mode of action ofthe 5-nitrofuryl pharmacophore

37 Oxygen uptake

The involvement of the platinum complexes in redoxcycling processes should increase the parasite oxygen con-sumption Thus the effect of the compounds on parasiteoxygen uptake was measured The effect of complex[Pt(L2)2] on T cruzi epimastigotes oxygen consumptionwith and without inhibition of the respiration with KCNis shown in Fig 5 Both series of complexes modified oxy-gen uptake by the parasite increasing oxygen consumption(Table 6) These results are in accordance with a bioreduc-tive and redox cycling mechanism of action as previouslyreported for the free 5-nitrofuryl containing thiosemicarba-zones their Pd complexes and other nitrofuran derivativeslike Nifurtimox [1841] For both series of compoundseffect on parasite respiration seemed to correlate with theIC50 values For instance [Pt(L3)2] being the most activePt compound showed the highest increase on oxygenconsumption

38 Calf thymus DNA interaction

In order to address if interaction with DNA could bepart of the mode of action of the complexes experimentswith CT DNA were carried out Binding of the Pt com-plexes to DNA was studied by combining atomic absorp-tion determinations (for the metal) and electronicabsorption measurements for DNA quantification Thenew complexes are good binding agents for CT DNA

(Table 7) Although the determined platinum to DNAbinding levels were similar to those of known anti-tumormetal complexes they resulted significantly lower thanthose of the palladium analogous compounds [1819] Nogeneral pattern could be detected for both series of com-pounds being binding levels almost not affected by thenature of the thiosemicarbazone ligand [PtCl2(HL)] com-plexes having labile chloride ligands could probably inter-act with DNA through a mechanism similar to thatpreviously reported for cisplatin becoming activatedthrough aquation and reacting with nucleophilic DNAbases [42] [Pt(L)2] complexes cannot act in this way butthey could intercalate DNA or interact through othermechanisms Due to the planar motive contained in the[PdCl2(HL)] complexes intercalation could also be possiblefor these compounds Further studies are under progress inorder to get an insight into the mechanism of this interac-tion It is interesting to note that [Pt(L3)2] being the mostpotent anti-T cruzi compound of the series showed thehighest interaction with DNA Nevertheless [Pt(L1)2]being almost non active strongly interacts with DNAshowing that the mechanism of trypanocidal activity couldnot involve only DNA as target

4 Conclusions

Pt(II) complexes with 5-nitrofuryl containing thiosemi-carbazones as bioactive ligands showed good anti-trypan-osomal activity In particular the coordination of L3 to

Pt(II) forming [Pt(L3)2] lead to almost a five-fold activityincrease in respect to the free ligand Possible targetsnamely reductive metabolism (intra-parasite free radicalproduction) and DNA were screened trying to get insightinto the mechanism of action of these metal compoundsResults showed that some of the compounds could act asdual inhibitors in the parasite through production of toxicfree radicals and interaction with DNA

Results of this work show that the approach of coordi-nating anti-trypanosomal organic compounds with phar-macologically interesting metals could be a suitablestrategy to develop novel therapeutic tools against tropicaldiseases produced by trypanosomatids particularly Amer-ican Trypanosomiasis

Acknowledgments

This work was partially supported by PEDECIBA andUniversidad de La Republica (CSIC project 36406) ofUruguay Prosul-CNPq project 4902092005-0 SIBAS-0807 UMCE FONDECYT Chile 1061072 and CONI-CYT-PBCT Anillo ACT 29 We wish to thank Dr EJBaran CEQUINOR UNLP Argentina for Raman spec-tra facilities and Matilde Gomez Instituto Venezolano deInvestigaciones Cientıficas Caracas Venezuela for per-forming ESI-MS experiments

References

[1] httpwwwwhoint lthttpwwwwhointctdchagasgt[2] D Engels L Savioli Trend Parasitol 22 (2006) 363ndash366[3] J Urbina Expert Opin Ther Pat 13 (2003) 661ndash669[4] H Cerecetto M Gonzalez Curr Topics Med Chem 2 (2002) 1185ndash

1190[5] RL Krauth-Siegel H Bauer RH Schirmer Angew Chem Int Ed

44 (2005) 690ndash715[6] S Croft M Barret J Urbina Trend Parasitol 21 (2005) 508ndash512[7] M Ceaser The Lancet Infect Dis 5 (8) (2005) 470ndash471[8] Y Yamagata J Nakagawa Adv Parasitol 61 (2006) 129ndash165[9] C Zhang S Lippard Curr Opin Chem Biol 7 (2003) 481ndash489

[10] NP Farrell Metal Complexes as Drugs and ChemotherapeuticAgents in JM McCleverty TJ Meyer (Eds) ComprehensiveCoordination Chemistry II vol 9 Elsevier 2003 pp 809ndash840

[11] RA Sanchez-Delgado A Anzellotti L Suarez Metal complexes aschemotherapeutic agents against tropical diseases malaria trypano-somiasis and leishmaniasis in H Sigel A Sigel (Eds) Metal Ionsin Biological Systems vol 41 Marcel Dekker New York 2004 pp379ndash419

[12] RA Sanchez-Delgado A Anzellotti Mini-Rev Med Chem 4(2004) 23ndash30

[13] R Morphy Z Rankovic J Med Chem 48 (2005) 6523ndash6543[14] Z Wang EM Bennett DJ Wilson C Salomon R Vince J Med

Chem 50 (2007) 3416ndash3419[15] K Chibale Towards broad spectrum antiprotozoal agents ARKI-

VOC IX 2002 pp 93ndash98

[16] C Urquiola M Vieites G Aguirre A Marın B Solano GArrambide ML Lavaggi MH Torre M Gonzalez A Monge DGambino H Cerecetto Bioorg Med Chem 14 (2006) 5503ndash5509

[17] G Aguirre H Cerecetto M Gonzalez D Gambino L Otero COlea-Azar C Rigol A Denicola Bioorg Med Chem 12 (2004)4885ndash4893

[18] L Otero M Vieites L Boiani A Denicola C Rigol L Opazo COlea-Azar JD Maya A Morello RL Krauth-Siegel OE Piro ECastellano M Gonzalez D Gambino H Cerecetto J Med Chem49 (2006) 3322ndash3331

[19] A Gomez-Quiroga C Navarro-Ranninger Coord Chem Rev 248(2004) 119ndash133

[20] MD Hall TW Hambley Coord Chem Rev 232 (2002) 49ndash67[21] TW Hambley Coord Chem Rev 166 (1997) 181ndash223[22] S Bonse JM Richards SA Ross G Lowe RL Krauth-Siegel J

Med Chem 43 (2000) 4812ndash4821[23] G Lowe AS Droz T Vilaiyan GW Weaver L Tweedale JM

Pratt P Rock V Yardley SL Croft J Med Chem 42 (1999) 999ndash1006

[24] SL Croft Mem Inst Oswaldo Cruz 94 (1999) 215ndash220[25] WJ Geary Coord Chem Rev 7 (1971) 81ndash91[26] C Rigol C Olea-Azar F Mendizabal L Otero D Gambino M

Gonzalez H Cerecetto Spectrochim Acta A 61 (2005) 2933ndash2938

[27] L Huang A Lee JA Ellman J Med Chem 45 (2002) 676ndash684[28] JD Maya A Morello Y Repetto R Tellez A Rodriguez U

Zelada P Puebla M Bonta S Bollo A San Feliciano CompBiochem Phys C 125 (2000) 103ndash109

[29] MA Cuellar C Salas MJ Cortes A Morello JD Maya MDPreitea Bioorg Med Chem 11 (2003) 2489ndash2497

[30] RE Mahnken MA Billadeau EP Nikonowicz H Morrison JAm Chem Soc 114 (1992) 9253ndash9265

[31] ME Letelier E Rodrıguez A Wallace M Lorca Y Repetto AMorello J Aldunate Exp Parasitol 71 (1990) 357ndash363

[32] D Gambino L Otero M Vieites M Boiani M Gonzalez EJBaran H Cerecetto Spectrochim Acta A 68 (2007) 341ndash348

[33] L Otero C Folch G Barriga C Rigol L Opazo M Vieites DGambino H Cerecetto E NorambuenaC Olea-Azar SpectrochimActa A lthttpdxdoiorg101016jsaa200707045gt

[34] C Olea-Azar C Rigol F Mendizabal A Morello JD Maya CMoncada E Cabrera R Di Maio M Gonzalez H Cerecetto FreeRad Res 37 (2003) 993ndash1001

[35] S Bollo E Soto-Bustamante LJ Nunez-Vergara JA Squella JElectroanal Chem 492 (2000) 54ndash62

[36] C Olea-Azar AM Atria F Mendizabal R Di Maio G Seoane HCerecetto Spectrosc Lett 31 (1998) 99ndash109

[37] VJ Aran C Ochoa L Boiani P Buccino H Cerecetto A GerpeM Gonzalez D Montero JJ Nogal A Gomez-Barrio A AzquetaA Lopez de Ceraın OE Piro EE Castellano Bioorg Med Chem13 (2005) 3197ndash3207

[38] KM Tyler DM Engman Int J Parasitol 31 (2001) 472ndash480[39] Y Repetto E Opazo JD Maya M Agosin A Morello Comp

Biochem Phys B 115 (1996) 281ndash285[40] N Veerapen SA Taylor CJ Walsby B Mario Pinto J Am

Chem Soc 128 (2006) 227ndash239[41] JD Maya S Bollo LJ Nunez-Vergara JA Squella Y Repetto

A Morello J Perie G Chauviere Biochem Pharmacol 65 (2003)999ndash1006

[42] V Brabec Prog Nucl Acid Res Mol Biol 71 (2002) 1ndash68

00 -05 -10 -15 -2015x10-5

10x10-5

50x10-6

00

-50x10-6

-10x10-5

IV

II III[PtL32]

Cur

rent

A

PotentialV

[PtCl2HL3]

I

00 -05 -10 -15 -2050x10-6

40x10-6

30x10-6

20x10-6

10x10-6

00

-10x10-6

-20x10-6

-30x10-6

Cur

rent

A

PotentialV

2000 mVs --- 1000 mVshellip 500 mVs minusminus 250 mVsminus 100 mVs

a b

Fig 2 Cyclic voltammograms of 1 mM DMSO solutions 01 M TBAP of (a) [PtCl2(HL3)] and [Pt(L3)2] at 2000 mVs and (b) [PtCl2(HL3)] when scanrate is changed in the range 100ndash2000 mVs

Table 3Cyclic voltammetric parameters for the reduction of the platinumcomplexes corresponding to the couple II peak Ic IIIc and IVc measuredin DMSO at 2000 mVs

Compound EpIca EpIIc

a EpIIab EpIIIc

a EpIVca

[PtCl2(HL1)] 043 070 066 1209 151[Pt(L1)2] ndash 069 056 122 149[PtCl2(HL2)] 049 073 060 117 137[Pt(L2)2] ndash 068 070 134 165[PtCl2(HL3)] 058 084 058 125 146[Pt(L3)2] ndash 077 061 122 149[PtCl2(HL4)] na 078 056 117 137[Pt(L4)2] ndash 072 062 109 126Nfx [18] 091 085

Nfx Nifurtimox na not assignedPotentials are reported in volts versus saturated calomel electrode

a Epc cathodic peak potentialb Epa anodic peak potential

The first wave for all the Pt compounds corresponded to aquasireversible process involving a one-electron transfer(couple II) This wave around 080 V versus SCE corre-sponds to the generation of the anion radical RNO2 byreduction of the nitro group [1833] The reverse scanshowed the anodic counterpart of the reduction waveAccording to the standard reversibility criteria this couplecan be attributed to a diffusion-controlled one-electrontransfer For some complexes sharp peaks around thiscouple could be observed as a result of adsorption phenom-ena in the electrode surface due to the presence in the mol-ecules of the thiocarbonyl group and the metal Thisphenomenon has been also observed for the Pd analogues[33] Next two couples (III and IV) are assigned to thereduction of the platinum complex nitro anion radicalgenerated in the first couple Subsequent less negativethree-electron irreversible cathodic peak (IIIc Fig 2a) isirreversible in the whole range of sweep rates used (100ndash2000 mVs) and can be attributed to the production ofthe hydroxylamine derivative [2634] Peak IVc is presumedto belong to the reduction of the imine moiety (CHN) ofthe thiosemicarbazone group [35]

Small differences were found between [PtCl2(HL)] and[Pt(L)2] complexes as previously observed for the Pd analo-gous compounds [33] The voltammogram of [PdCl2HL]complexes showed a prepeak (Ic Fig 2a) that appeared evenbefore the reduction of the nitro group meaning that thenitro group follows another reaction path besides the knownelectron-transfer mechanism of the nitroaromatic com-pounds in aprotic media This prepeak would correspondto the four-electron reduction of a small portion of the mol-ecules reaching the electrode surface while the remainingportion would supply the protons required for this reduc-tion This is a typical behavior of a self-protonation phenom-enon displayed by nitrocompounds with acidic moieties intheir structures [263334] The presence of the nitro group

increases the acidity of the NH moiety of the thiosemicarba-zone group present only in the [PtCl2(HL)] complexes (L nondeprotonated) which becomes capable of protonating thenitro group of a minor part of the molecules in the solutionresulting into a lower intensity of these signals [Pt(L)2] com-plexes did not show the Ic prepeak because they do not havethe capability to protonate the nitro group since the NH pro-ton was lost as a consequence of coordination of the ligand toplatinum

Table 3 lists the values of voltammetric peaks for allstudied compounds The potentials of the voltammetricpeaks corresponding to the nitro moiety of the free ligandsslightly changed as a consequence of platinum complexa-tion being the latter only slightly more favourable thanthe former and than those previously reported for the pal-ladium analogues [1826] However it should be stated thatall studied compounds showed under the same conditions ahigher capacity to be reduced than Nifurtimox (E12

[103]a

088 V Table 3) and therefore a better ability to generate

radical species that could be toxic for the parasite [36]

[G]

[G]

3420 3430 3440 3450 3460 3470-25-20-15-10-5

05

10152025

[103]

3420 3430 3440 3450 3460 3470

-40-30-20-10

01020304050

b

Fig 3 (a) ESR experimental spectrum of [Pt(L2)2] Spectrometer condi-tions microwave frequency 971 GHz microwave power 20 mW modu-lation amplitude 02 G scan rate 125 Gs time constant 05 s number ofscans 15 (b) Simulated spectrum of [Pt(L2)2]

34 Free radicals production studied by ESR spectroscopy

The complexes were tested for their capability to pro-duce free radicals in reductive conditions The freeradicals characterized by ESR were prepared lsquolsquoin siturdquo

by electrochemical reduction in DMSO applying apotential corresponding to the first monoelectronic wave(IIcIIa) obtained from the cyclic voltammetric experi-ments The interpretation of the ESR spectra by meansof a simulation process has led to the determination ofthe hyperfine coupling constants for all the magneticnuclei The obtained hyperfine constants are listed inTable 4

The ESR spectra of the [PtCl2(HL)] complexes were sim-ulated in terms of one triplet corresponding to the nitrogennucleus belonging to the nitro group and one triplet to thenitrogen of the CN thiosemicarbazone group and threedoublets due to non-equivalent hydrogens belonging tothe side chain Other nuclei presented hyperfine constantsmaller than the line width

The ESR spectra of the [Pt(L)2] complexes were ana-lyzed in terms of one triplet due to the nitrogen of the nitrogroup two doublets due to hydrogens corresponding to thefuran ring and one triplet due to hydrogens having verysimilar hyperfine constants belonging to the thiosemicar-bazone chains Other hyperfine constants resulted smallerthan the line width and they were not observed in theexperimental spectrum Fig 3 shows the ESR spectrumof complex [Pt(L2)2]

Different substituents in the thiosemicarbazone chain ofthe coordinated ligands did not seem to affect the hyperfinepattern of platinum complexes as it had been previouslyobserved for the corresponding palladium ones Howeverthe number of ligands coordinated per Pt central atomseemed to determine spin density distribution and so thehyperfine pattern of the complexes For the [Pt(L)2] com-plexes the spin density was more located on the nitro furanring while other observed couplings could be related to noncompletely equivalent thiosemicarbazone ligands coordi-nated to the platinum atom For the [PtCl2(HL)] com-

Table 4Hyperfine splittings (gauss) for the anion radical of the platinumcomplexes

aN aN aH aH aH aH aH

[PtCl2(HL1)] 695 41 128 14 112 lt06 lt06[Pt(L1)2] 68 lt06 08 40 lt06 245 235[PtCl2(HL2)] 697 35 12 15 100 lt06 lt06[Pt(L2)2] 70 lt06 12 40 lt06 31 33[PtCl2(HL3)] 690 37 115 16 128 lt06 lt06[Pt(L3)2] 70 lt06 124 40 lt06 30 33[PtCl2(HL4)] 687 40 117 125 13 lt06 lt06[Pt(L4)2] 72 lt06 110 38 lt06 298 310

pounds the spin density was more delocalized on theentire radical

The effect of changing the metal atom on the hyperfinepattern of the ESR spectra of the complexes should benoted For palladium complexes no differences have beenobserved between [PdCl2(HL)] and [Pd(L)2] ones Both ser-ies of palladium compounds presented a quite similarhyperfine pattern to that observed for the [PtCl2(HL)] com-plexes The different observed ESR pattern for [M(L)2]complexes when changing palladium by platinum couldbe related to small differences in complexes structure Thisfact would also agree with a quite different biologicalbehavior

35 In vitro anti T cruzi activity

The existence of the epimastigote form of T cruzi as anobligate mammalian intracellular stage has been confirmedrecently [3738] Therefore compounds were tested in vitro

against epimastigote form of the parasite The complexeswere evaluated for their anti-T cruzi activities against epim-astigotes of Tulahuen 2 strain in order to compare with pre-viously performed biological evaluation of the free ligandsand the analogous Pd complexes Fifty percent of inhibitoryconcentrations (IC50) obtained from dose ndash response curvesare depicted in Table 5 In addition some complexes wereevaluated against a second strain of T cruzi epimastigotesnamely Dm28c

For comparative purposes Table 5 also includes IC50

values for the four free ligands and the Pd complexes

Table 5In vitro biological activity of the Pt complexes

Compound T cruzi (Tulahuen 2) T cruzi (Dm28c) Compound T cruzi (Tulahuen 2)PGIa IC50

b ICkc50b IC50

c

10 lM 25 lM

L1 27[PtCl2(HL1)] 109 382 gt25 nd [PdCl2(HL1)] 24[Pt(L1)2] 34 208 gt25 nd [Pd(L1)2] 45

L2 50[PtCl2(HL2)] 415 605 131 367 [PdCl2(HL2)] 43[Pt(L2)2] 684 100 69 102 [Pd(L2)2] 47

L3 49[PtCl2(HL3)] 269 441 275 nd [PdCl2(HL3)] 59[Pt(L3)2] 100 100 08 288 [Pd(L3)2] gt25

L4 gt25[PtCl2(HL4)] 338 720 15 284 [PdCl2(HL4)] gt25[Pt(L4)2] 189 121 gt25 413 [Pd(L4)2] gt25Benznidazol 74 380Nifurtimox 61 [17] 228

IC50 values of the free ligand and their Pd complexes on T cruzi (Tulahuen 2 strain) have been included for comparison [18]nd not determined

a PGI percentage of growth inhibition of T cruzi epimastigote cells at the specified doseb plusmn10c [18]

3430 3440 3450 3460 3470 3480 3490 3500 3510 3520 3530

-8

-6

-4

-2

0

2

4

6

8[103]

previously determined through the same technique on epi-mastigote form of T cruzi Tulahuen 2 strain [18] Most ofthe Pt complexes were active against epimastigotes of T

cruzi Tulahuen 2 strain showing some of them IC50 valuesof the same order than Nifurtimox and the correspondingfree ligands According to the IC50 values [Pt(L3)2] and[Pt(L2)2] were the most active Pt complexes against thisparasite strain being [Pt(L3)2] significantly more activethan L3 and than Nifurtimox

When the results are compared with those previouslyreported for the free ligands and the Pd analogous com-pounds it can be stated that the activity pattern is signifi-cantly modified by changing the central atom The activityof the Pd complexes followed the general trend[PdCl2(HL)] gt free ligand gt [Pd(L)2] This regular trendwas not observed for the Pt analogues This differentbehavior could be related with differences in bioavailabil-ity unspecific toxicity andor mechanism of action of bothseries of metal complexes

Similar general conclusions can be obtained from theanalysis of the activity results on the T cruzi Dm28c strainalthough some strain susceptibility differences can beobserved Dm28c is a parasite clone that had previouslyshown to be less susceptible to Nifurtimox and Ben-znidazol trypanocidal effect than Tulahuen strain whichis mediated by a Dm28c higher thiol content of the former[39]

[G]

Fig 4 ESR spectrum of [PtCl2(HL2)] obtained when [PtCl2(HL2)] wasincubated with T cruzi epimastigotes in the presence of DMPOSpectrometer conditions microwave frequency 968 GHz microwavepower 20 mW modulation amplitude 02 G scan rate 125 Gs timeconstant 05 s number of scans 15 signals of DMPO-nitro anionradical adduct

36 Capacity of derivatives to generate free radical species

into T cruzi

The complexes were incubated with intact T cruzi

(Dm28c strain) epimastigotes in the presence of DMPO

as spin trapping agent in order to detect possible intra-celullar free radical species produced by bioreduction andhaving short half-lives The biological free radical produc-tion capacity of the new compounds was assessed by ESRAll the compounds were capable to produce free radicals inthe intact parasite giving an ESR spectrum Fig 4 showsas an example the ESR spectrum obtained when[PtCl2(HL2)] was incubated with T cruzi in the presenceof DMPO The typical hyperfine pattern of adductsobtained when DMPO traps radicals centered in C atom

[Pt(L2)2]

1 minute

10 O2

[Pt(L2)2]KCN

a

b

Fig 5 (a) Effect of compound [Pt(L)2] on the parasite (T cruzi Dm28cstrain) oxygen consumption related to control (dotted line) (b) Effect ofcompound [Pt(L)2] on the parasite (T cruzi Dm28c strain) oxygenconsumption after inhibition of the respiration with KCN related tocontrol (dotted line)

Table 6Effect of the addition of Pt complexes on oxygen uptake by T cruzi

epimastigotes (Dm28c strain)

Compound Respiration (nanoatom-gramof Ominmg protein)

Increase on oxygenconsumption ()a

Control 280 plusmn 20[PtCl2(HL2)] 322 plusmn 32 15[Pt(L2)2] 313 plusmn 17 12[Pt(L3)2] 392 plusmn 42 40[PtCl2(HL4)] 355 plusmn 32 27[Pt(L4)2] 320 plusmn 13 14Nifurtimox 356 plusmn 08 27

a increase on oxygen consumption after addition of 125 mM ofcompounds with respect to control (no added compound)

Table 7Interaction of Pt complexes with CT DNA after 96 h of incubation at37 C

Compound nmol Ptmg DNA Metalbasea Basemetal

[PtCl2(HL1)] 209 0069 14[Pt(L1)2] 390 0129 8[PtCl2(HL2)] 236 0078 13[Pt(L2)2] 224 0074 14[PtCl2(HL3)] 191 0063 16[Pt(L3)2] 527 0174 6[PtCl2(HL4)] 170 0056 18[Pt(L4)2] 170 0056 18

a Platinum (mol) per DNA base (mol)

(aN = 167 G and aH = 225 G) was observed [18] A trip-let produced by DMPO decomposition was also detected[40]

ESR studies showed that the newly developed Pt thio-semicarbazone complexes were bioreduced in the parasiteindicating that they could maintain the mode of action ofthe 5-nitrofuryl pharmacophore

37 Oxygen uptake

The involvement of the platinum complexes in redoxcycling processes should increase the parasite oxygen con-sumption Thus the effect of the compounds on parasiteoxygen uptake was measured The effect of complex[Pt(L2)2] on T cruzi epimastigotes oxygen consumptionwith and without inhibition of the respiration with KCNis shown in Fig 5 Both series of complexes modified oxy-gen uptake by the parasite increasing oxygen consumption(Table 6) These results are in accordance with a bioreduc-tive and redox cycling mechanism of action as previouslyreported for the free 5-nitrofuryl containing thiosemicarba-zones their Pd complexes and other nitrofuran derivativeslike Nifurtimox [1841] For both series of compoundseffect on parasite respiration seemed to correlate with theIC50 values For instance [Pt(L3)2] being the most activePt compound showed the highest increase on oxygenconsumption

38 Calf thymus DNA interaction

In order to address if interaction with DNA could bepart of the mode of action of the complexes experimentswith CT DNA were carried out Binding of the Pt com-plexes to DNA was studied by combining atomic absorp-tion determinations (for the metal) and electronicabsorption measurements for DNA quantification Thenew complexes are good binding agents for CT DNA

(Table 7) Although the determined platinum to DNAbinding levels were similar to those of known anti-tumormetal complexes they resulted significantly lower thanthose of the palladium analogous compounds [1819] Nogeneral pattern could be detected for both series of com-pounds being binding levels almost not affected by thenature of the thiosemicarbazone ligand [PtCl2(HL)] com-plexes having labile chloride ligands could probably inter-act with DNA through a mechanism similar to thatpreviously reported for cisplatin becoming activatedthrough aquation and reacting with nucleophilic DNAbases [42] [Pt(L)2] complexes cannot act in this way butthey could intercalate DNA or interact through othermechanisms Due to the planar motive contained in the[PdCl2(HL)] complexes intercalation could also be possiblefor these compounds Further studies are under progress inorder to get an insight into the mechanism of this interac-tion It is interesting to note that [Pt(L3)2] being the mostpotent anti-T cruzi compound of the series showed thehighest interaction with DNA Nevertheless [Pt(L1)2]being almost non active strongly interacts with DNAshowing that the mechanism of trypanocidal activity couldnot involve only DNA as target

4 Conclusions

Pt(II) complexes with 5-nitrofuryl containing thiosemi-carbazones as bioactive ligands showed good anti-trypan-osomal activity In particular the coordination of L3 to

Pt(II) forming [Pt(L3)2] lead to almost a five-fold activityincrease in respect to the free ligand Possible targetsnamely reductive metabolism (intra-parasite free radicalproduction) and DNA were screened trying to get insightinto the mechanism of action of these metal compoundsResults showed that some of the compounds could act asdual inhibitors in the parasite through production of toxicfree radicals and interaction with DNA

Results of this work show that the approach of coordi-nating anti-trypanosomal organic compounds with phar-macologically interesting metals could be a suitablestrategy to develop novel therapeutic tools against tropicaldiseases produced by trypanosomatids particularly Amer-ican Trypanosomiasis

Acknowledgments

This work was partially supported by PEDECIBA andUniversidad de La Republica (CSIC project 36406) ofUruguay Prosul-CNPq project 4902092005-0 SIBAS-0807 UMCE FONDECYT Chile 1061072 and CONI-CYT-PBCT Anillo ACT 29 We wish to thank Dr EJBaran CEQUINOR UNLP Argentina for Raman spec-tra facilities and Matilde Gomez Instituto Venezolano deInvestigaciones Cientıficas Caracas Venezuela for per-forming ESI-MS experiments

References

[1] httpwwwwhoint lthttpwwwwhointctdchagasgt[2] D Engels L Savioli Trend Parasitol 22 (2006) 363ndash366[3] J Urbina Expert Opin Ther Pat 13 (2003) 661ndash669[4] H Cerecetto M Gonzalez Curr Topics Med Chem 2 (2002) 1185ndash

1190[5] RL Krauth-Siegel H Bauer RH Schirmer Angew Chem Int Ed

44 (2005) 690ndash715[6] S Croft M Barret J Urbina Trend Parasitol 21 (2005) 508ndash512[7] M Ceaser The Lancet Infect Dis 5 (8) (2005) 470ndash471[8] Y Yamagata J Nakagawa Adv Parasitol 61 (2006) 129ndash165[9] C Zhang S Lippard Curr Opin Chem Biol 7 (2003) 481ndash489

[10] NP Farrell Metal Complexes as Drugs and ChemotherapeuticAgents in JM McCleverty TJ Meyer (Eds) ComprehensiveCoordination Chemistry II vol 9 Elsevier 2003 pp 809ndash840

[11] RA Sanchez-Delgado A Anzellotti L Suarez Metal complexes aschemotherapeutic agents against tropical diseases malaria trypano-somiasis and leishmaniasis in H Sigel A Sigel (Eds) Metal Ionsin Biological Systems vol 41 Marcel Dekker New York 2004 pp379ndash419

[12] RA Sanchez-Delgado A Anzellotti Mini-Rev Med Chem 4(2004) 23ndash30

[13] R Morphy Z Rankovic J Med Chem 48 (2005) 6523ndash6543[14] Z Wang EM Bennett DJ Wilson C Salomon R Vince J Med

Chem 50 (2007) 3416ndash3419[15] K Chibale Towards broad spectrum antiprotozoal agents ARKI-

VOC IX 2002 pp 93ndash98

[16] C Urquiola M Vieites G Aguirre A Marın B Solano GArrambide ML Lavaggi MH Torre M Gonzalez A Monge DGambino H Cerecetto Bioorg Med Chem 14 (2006) 5503ndash5509

[17] G Aguirre H Cerecetto M Gonzalez D Gambino L Otero COlea-Azar C Rigol A Denicola Bioorg Med Chem 12 (2004)4885ndash4893

[18] L Otero M Vieites L Boiani A Denicola C Rigol L Opazo COlea-Azar JD Maya A Morello RL Krauth-Siegel OE Piro ECastellano M Gonzalez D Gambino H Cerecetto J Med Chem49 (2006) 3322ndash3331

[19] A Gomez-Quiroga C Navarro-Ranninger Coord Chem Rev 248(2004) 119ndash133

[20] MD Hall TW Hambley Coord Chem Rev 232 (2002) 49ndash67[21] TW Hambley Coord Chem Rev 166 (1997) 181ndash223[22] S Bonse JM Richards SA Ross G Lowe RL Krauth-Siegel J

Med Chem 43 (2000) 4812ndash4821[23] G Lowe AS Droz T Vilaiyan GW Weaver L Tweedale JM

Pratt P Rock V Yardley SL Croft J Med Chem 42 (1999) 999ndash1006

[24] SL Croft Mem Inst Oswaldo Cruz 94 (1999) 215ndash220[25] WJ Geary Coord Chem Rev 7 (1971) 81ndash91[26] C Rigol C Olea-Azar F Mendizabal L Otero D Gambino M

Gonzalez H Cerecetto Spectrochim Acta A 61 (2005) 2933ndash2938

[27] L Huang A Lee JA Ellman J Med Chem 45 (2002) 676ndash684[28] JD Maya A Morello Y Repetto R Tellez A Rodriguez U

Zelada P Puebla M Bonta S Bollo A San Feliciano CompBiochem Phys C 125 (2000) 103ndash109

[29] MA Cuellar C Salas MJ Cortes A Morello JD Maya MDPreitea Bioorg Med Chem 11 (2003) 2489ndash2497

[30] RE Mahnken MA Billadeau EP Nikonowicz H Morrison JAm Chem Soc 114 (1992) 9253ndash9265

[31] ME Letelier E Rodrıguez A Wallace M Lorca Y Repetto AMorello J Aldunate Exp Parasitol 71 (1990) 357ndash363

[32] D Gambino L Otero M Vieites M Boiani M Gonzalez EJBaran H Cerecetto Spectrochim Acta A 68 (2007) 341ndash348

[33] L Otero C Folch G Barriga C Rigol L Opazo M Vieites DGambino H Cerecetto E NorambuenaC Olea-Azar SpectrochimActa A lthttpdxdoiorg101016jsaa200707045gt

[34] C Olea-Azar C Rigol F Mendizabal A Morello JD Maya CMoncada E Cabrera R Di Maio M Gonzalez H Cerecetto FreeRad Res 37 (2003) 993ndash1001

[35] S Bollo E Soto-Bustamante LJ Nunez-Vergara JA Squella JElectroanal Chem 492 (2000) 54ndash62

[36] C Olea-Azar AM Atria F Mendizabal R Di Maio G Seoane HCerecetto Spectrosc Lett 31 (1998) 99ndash109

[37] VJ Aran C Ochoa L Boiani P Buccino H Cerecetto A GerpeM Gonzalez D Montero JJ Nogal A Gomez-Barrio A AzquetaA Lopez de Ceraın OE Piro EE Castellano Bioorg Med Chem13 (2005) 3197ndash3207

[38] KM Tyler DM Engman Int J Parasitol 31 (2001) 472ndash480[39] Y Repetto E Opazo JD Maya M Agosin A Morello Comp

Biochem Phys B 115 (1996) 281ndash285[40] N Veerapen SA Taylor CJ Walsby B Mario Pinto J Am

Chem Soc 128 (2006) 227ndash239[41] JD Maya S Bollo LJ Nunez-Vergara JA Squella Y Repetto

A Morello J Perie G Chauviere Biochem Pharmacol 65 (2003)999ndash1006

[42] V Brabec Prog Nucl Acid Res Mol Biol 71 (2002) 1ndash68

[103]a

088 V Table 3) and therefore a better ability to generate

radical species that could be toxic for the parasite [36]

[G]

[G]

3420 3430 3440 3450 3460 3470-25-20-15-10-5

05

10152025

[103]

3420 3430 3440 3450 3460 3470

-40-30-20-10

01020304050

b

Fig 3 (a) ESR experimental spectrum of [Pt(L2)2] Spectrometer condi-tions microwave frequency 971 GHz microwave power 20 mW modu-lation amplitude 02 G scan rate 125 Gs time constant 05 s number ofscans 15 (b) Simulated spectrum of [Pt(L2)2]

34 Free radicals production studied by ESR spectroscopy

The complexes were tested for their capability to pro-duce free radicals in reductive conditions The freeradicals characterized by ESR were prepared lsquolsquoin siturdquo

by electrochemical reduction in DMSO applying apotential corresponding to the first monoelectronic wave(IIcIIa) obtained from the cyclic voltammetric experi-ments The interpretation of the ESR spectra by meansof a simulation process has led to the determination ofthe hyperfine coupling constants for all the magneticnuclei The obtained hyperfine constants are listed inTable 4

The ESR spectra of the [PtCl2(HL)] complexes were sim-ulated in terms of one triplet corresponding to the nitrogennucleus belonging to the nitro group and one triplet to thenitrogen of the CN thiosemicarbazone group and threedoublets due to non-equivalent hydrogens belonging tothe side chain Other nuclei presented hyperfine constantsmaller than the line width

The ESR spectra of the [Pt(L)2] complexes were ana-lyzed in terms of one triplet due to the nitrogen of the nitrogroup two doublets due to hydrogens corresponding to thefuran ring and one triplet due to hydrogens having verysimilar hyperfine constants belonging to the thiosemicar-bazone chains Other hyperfine constants resulted smallerthan the line width and they were not observed in theexperimental spectrum Fig 3 shows the ESR spectrumof complex [Pt(L2)2]

Different substituents in the thiosemicarbazone chain ofthe coordinated ligands did not seem to affect the hyperfinepattern of platinum complexes as it had been previouslyobserved for the corresponding palladium ones Howeverthe number of ligands coordinated per Pt central atomseemed to determine spin density distribution and so thehyperfine pattern of the complexes For the [Pt(L)2] com-plexes the spin density was more located on the nitro furanring while other observed couplings could be related to noncompletely equivalent thiosemicarbazone ligands coordi-nated to the platinum atom For the [PtCl2(HL)] com-

Table 4Hyperfine splittings (gauss) for the anion radical of the platinumcomplexes

aN aN aH aH aH aH aH

[PtCl2(HL1)] 695 41 128 14 112 lt06 lt06[Pt(L1)2] 68 lt06 08 40 lt06 245 235[PtCl2(HL2)] 697 35 12 15 100 lt06 lt06[Pt(L2)2] 70 lt06 12 40 lt06 31 33[PtCl2(HL3)] 690 37 115 16 128 lt06 lt06[Pt(L3)2] 70 lt06 124 40 lt06 30 33[PtCl2(HL4)] 687 40 117 125 13 lt06 lt06[Pt(L4)2] 72 lt06 110 38 lt06 298 310

pounds the spin density was more delocalized on theentire radical

The effect of changing the metal atom on the hyperfinepattern of the ESR spectra of the complexes should benoted For palladium complexes no differences have beenobserved between [PdCl2(HL)] and [Pd(L)2] ones Both ser-ies of palladium compounds presented a quite similarhyperfine pattern to that observed for the [PtCl2(HL)] com-plexes The different observed ESR pattern for [M(L)2]complexes when changing palladium by platinum couldbe related to small differences in complexes structure Thisfact would also agree with a quite different biologicalbehavior

35 In vitro anti T cruzi activity

The existence of the epimastigote form of T cruzi as anobligate mammalian intracellular stage has been confirmedrecently [3738] Therefore compounds were tested in vitro

against epimastigote form of the parasite The complexeswere evaluated for their anti-T cruzi activities against epim-astigotes of Tulahuen 2 strain in order to compare with pre-viously performed biological evaluation of the free ligandsand the analogous Pd complexes Fifty percent of inhibitoryconcentrations (IC50) obtained from dose ndash response curvesare depicted in Table 5 In addition some complexes wereevaluated against a second strain of T cruzi epimastigotesnamely Dm28c

For comparative purposes Table 5 also includes IC50

values for the four free ligands and the Pd complexes

Table 5In vitro biological activity of the Pt complexes

Compound T cruzi (Tulahuen 2) T cruzi (Dm28c) Compound T cruzi (Tulahuen 2)PGIa IC50

b ICkc50b IC50

c

10 lM 25 lM

L1 27[PtCl2(HL1)] 109 382 gt25 nd [PdCl2(HL1)] 24[Pt(L1)2] 34 208 gt25 nd [Pd(L1)2] 45

L2 50[PtCl2(HL2)] 415 605 131 367 [PdCl2(HL2)] 43[Pt(L2)2] 684 100 69 102 [Pd(L2)2] 47

L3 49[PtCl2(HL3)] 269 441 275 nd [PdCl2(HL3)] 59[Pt(L3)2] 100 100 08 288 [Pd(L3)2] gt25

L4 gt25[PtCl2(HL4)] 338 720 15 284 [PdCl2(HL4)] gt25[Pt(L4)2] 189 121 gt25 413 [Pd(L4)2] gt25Benznidazol 74 380Nifurtimox 61 [17] 228

IC50 values of the free ligand and their Pd complexes on T cruzi (Tulahuen 2 strain) have been included for comparison [18]nd not determined

a PGI percentage of growth inhibition of T cruzi epimastigote cells at the specified doseb plusmn10c [18]

3430 3440 3450 3460 3470 3480 3490 3500 3510 3520 3530

-8

-6

-4

-2

0

2

4

6

8[103]

previously determined through the same technique on epi-mastigote form of T cruzi Tulahuen 2 strain [18] Most ofthe Pt complexes were active against epimastigotes of T

cruzi Tulahuen 2 strain showing some of them IC50 valuesof the same order than Nifurtimox and the correspondingfree ligands According to the IC50 values [Pt(L3)2] and[Pt(L2)2] were the most active Pt complexes against thisparasite strain being [Pt(L3)2] significantly more activethan L3 and than Nifurtimox

When the results are compared with those previouslyreported for the free ligands and the Pd analogous com-pounds it can be stated that the activity pattern is signifi-cantly modified by changing the central atom The activityof the Pd complexes followed the general trend[PdCl2(HL)] gt free ligand gt [Pd(L)2] This regular trendwas not observed for the Pt analogues This differentbehavior could be related with differences in bioavailabil-ity unspecific toxicity andor mechanism of action of bothseries of metal complexes

Similar general conclusions can be obtained from theanalysis of the activity results on the T cruzi Dm28c strainalthough some strain susceptibility differences can beobserved Dm28c is a parasite clone that had previouslyshown to be less susceptible to Nifurtimox and Ben-znidazol trypanocidal effect than Tulahuen strain whichis mediated by a Dm28c higher thiol content of the former[39]

[G]

Fig 4 ESR spectrum of [PtCl2(HL2)] obtained when [PtCl2(HL2)] wasincubated with T cruzi epimastigotes in the presence of DMPOSpectrometer conditions microwave frequency 968 GHz microwavepower 20 mW modulation amplitude 02 G scan rate 125 Gs timeconstant 05 s number of scans 15 signals of DMPO-nitro anionradical adduct

36 Capacity of derivatives to generate free radical species

into T cruzi

The complexes were incubated with intact T cruzi

(Dm28c strain) epimastigotes in the presence of DMPO

as spin trapping agent in order to detect possible intra-celullar free radical species produced by bioreduction andhaving short half-lives The biological free radical produc-tion capacity of the new compounds was assessed by ESRAll the compounds were capable to produce free radicals inthe intact parasite giving an ESR spectrum Fig 4 showsas an example the ESR spectrum obtained when[PtCl2(HL2)] was incubated with T cruzi in the presenceof DMPO The typical hyperfine pattern of adductsobtained when DMPO traps radicals centered in C atom

[Pt(L2)2]

1 minute

10 O2

[Pt(L2)2]KCN

a

b

Fig 5 (a) Effect of compound [Pt(L)2] on the parasite (T cruzi Dm28cstrain) oxygen consumption related to control (dotted line) (b) Effect ofcompound [Pt(L)2] on the parasite (T cruzi Dm28c strain) oxygenconsumption after inhibition of the respiration with KCN related tocontrol (dotted line)

Table 6Effect of the addition of Pt complexes on oxygen uptake by T cruzi

epimastigotes (Dm28c strain)

Compound Respiration (nanoatom-gramof Ominmg protein)

Increase on oxygenconsumption ()a

Control 280 plusmn 20[PtCl2(HL2)] 322 plusmn 32 15[Pt(L2)2] 313 plusmn 17 12[Pt(L3)2] 392 plusmn 42 40[PtCl2(HL4)] 355 plusmn 32 27[Pt(L4)2] 320 plusmn 13 14Nifurtimox 356 plusmn 08 27

a increase on oxygen consumption after addition of 125 mM ofcompounds with respect to control (no added compound)

Table 7Interaction of Pt complexes with CT DNA after 96 h of incubation at37 C

Compound nmol Ptmg DNA Metalbasea Basemetal

[PtCl2(HL1)] 209 0069 14[Pt(L1)2] 390 0129 8[PtCl2(HL2)] 236 0078 13[Pt(L2)2] 224 0074 14[PtCl2(HL3)] 191 0063 16[Pt(L3)2] 527 0174 6[PtCl2(HL4)] 170 0056 18[Pt(L4)2] 170 0056 18

a Platinum (mol) per DNA base (mol)

(aN = 167 G and aH = 225 G) was observed [18] A trip-let produced by DMPO decomposition was also detected[40]

ESR studies showed that the newly developed Pt thio-semicarbazone complexes were bioreduced in the parasiteindicating that they could maintain the mode of action ofthe 5-nitrofuryl pharmacophore

37 Oxygen uptake

The involvement of the platinum complexes in redoxcycling processes should increase the parasite oxygen con-sumption Thus the effect of the compounds on parasiteoxygen uptake was measured The effect of complex[Pt(L2)2] on T cruzi epimastigotes oxygen consumptionwith and without inhibition of the respiration with KCNis shown in Fig 5 Both series of complexes modified oxy-gen uptake by the parasite increasing oxygen consumption(Table 6) These results are in accordance with a bioreduc-tive and redox cycling mechanism of action as previouslyreported for the free 5-nitrofuryl containing thiosemicarba-zones their Pd complexes and other nitrofuran derivativeslike Nifurtimox [1841] For both series of compoundseffect on parasite respiration seemed to correlate with theIC50 values For instance [Pt(L3)2] being the most activePt compound showed the highest increase on oxygenconsumption

38 Calf thymus DNA interaction

In order to address if interaction with DNA could bepart of the mode of action of the complexes experimentswith CT DNA were carried out Binding of the Pt com-plexes to DNA was studied by combining atomic absorp-tion determinations (for the metal) and electronicabsorption measurements for DNA quantification Thenew complexes are good binding agents for CT DNA

(Table 7) Although the determined platinum to DNAbinding levels were similar to those of known anti-tumormetal complexes they resulted significantly lower thanthose of the palladium analogous compounds [1819] Nogeneral pattern could be detected for both series of com-pounds being binding levels almost not affected by thenature of the thiosemicarbazone ligand [PtCl2(HL)] com-plexes having labile chloride ligands could probably inter-act with DNA through a mechanism similar to thatpreviously reported for cisplatin becoming activatedthrough aquation and reacting with nucleophilic DNAbases [42] [Pt(L)2] complexes cannot act in this way butthey could intercalate DNA or interact through othermechanisms Due to the planar motive contained in the[PdCl2(HL)] complexes intercalation could also be possiblefor these compounds Further studies are under progress inorder to get an insight into the mechanism of this interac-tion It is interesting to note that [Pt(L3)2] being the mostpotent anti-T cruzi compound of the series showed thehighest interaction with DNA Nevertheless [Pt(L1)2]being almost non active strongly interacts with DNAshowing that the mechanism of trypanocidal activity couldnot involve only DNA as target

4 Conclusions

Pt(II) complexes with 5-nitrofuryl containing thiosemi-carbazones as bioactive ligands showed good anti-trypan-osomal activity In particular the coordination of L3 to

Pt(II) forming [Pt(L3)2] lead to almost a five-fold activityincrease in respect to the free ligand Possible targetsnamely reductive metabolism (intra-parasite free radicalproduction) and DNA were screened trying to get insightinto the mechanism of action of these metal compoundsResults showed that some of the compounds could act asdual inhibitors in the parasite through production of toxicfree radicals and interaction with DNA

Results of this work show that the approach of coordi-nating anti-trypanosomal organic compounds with phar-macologically interesting metals could be a suitablestrategy to develop novel therapeutic tools against tropicaldiseases produced by trypanosomatids particularly Amer-ican Trypanosomiasis

Acknowledgments

This work was partially supported by PEDECIBA andUniversidad de La Republica (CSIC project 36406) ofUruguay Prosul-CNPq project 4902092005-0 SIBAS-0807 UMCE FONDECYT Chile 1061072 and CONI-CYT-PBCT Anillo ACT 29 We wish to thank Dr EJBaran CEQUINOR UNLP Argentina for Raman spec-tra facilities and Matilde Gomez Instituto Venezolano deInvestigaciones Cientıficas Caracas Venezuela for per-forming ESI-MS experiments

References

[1] httpwwwwhoint lthttpwwwwhointctdchagasgt[2] D Engels L Savioli Trend Parasitol 22 (2006) 363ndash366[3] J Urbina Expert Opin Ther Pat 13 (2003) 661ndash669[4] H Cerecetto M Gonzalez Curr Topics Med Chem 2 (2002) 1185ndash

1190[5] RL Krauth-Siegel H Bauer RH Schirmer Angew Chem Int Ed

44 (2005) 690ndash715[6] S Croft M Barret J Urbina Trend Parasitol 21 (2005) 508ndash512[7] M Ceaser The Lancet Infect Dis 5 (8) (2005) 470ndash471[8] Y Yamagata J Nakagawa Adv Parasitol 61 (2006) 129ndash165[9] C Zhang S Lippard Curr Opin Chem Biol 7 (2003) 481ndash489

[10] NP Farrell Metal Complexes as Drugs and ChemotherapeuticAgents in JM McCleverty TJ Meyer (Eds) ComprehensiveCoordination Chemistry II vol 9 Elsevier 2003 pp 809ndash840

[11] RA Sanchez-Delgado A Anzellotti L Suarez Metal complexes aschemotherapeutic agents against tropical diseases malaria trypano-somiasis and leishmaniasis in H Sigel A Sigel (Eds) Metal Ionsin Biological Systems vol 41 Marcel Dekker New York 2004 pp379ndash419

[12] RA Sanchez-Delgado A Anzellotti Mini-Rev Med Chem 4(2004) 23ndash30

[13] R Morphy Z Rankovic J Med Chem 48 (2005) 6523ndash6543[14] Z Wang EM Bennett DJ Wilson C Salomon R Vince J Med

Chem 50 (2007) 3416ndash3419[15] K Chibale Towards broad spectrum antiprotozoal agents ARKI-

VOC IX 2002 pp 93ndash98

[16] C Urquiola M Vieites G Aguirre A Marın B Solano GArrambide ML Lavaggi MH Torre M Gonzalez A Monge DGambino H Cerecetto Bioorg Med Chem 14 (2006) 5503ndash5509

[17] G Aguirre H Cerecetto M Gonzalez D Gambino L Otero COlea-Azar C Rigol A Denicola Bioorg Med Chem 12 (2004)4885ndash4893

[18] L Otero M Vieites L Boiani A Denicola C Rigol L Opazo COlea-Azar JD Maya A Morello RL Krauth-Siegel OE Piro ECastellano M Gonzalez D Gambino H Cerecetto J Med Chem49 (2006) 3322ndash3331

[19] A Gomez-Quiroga C Navarro-Ranninger Coord Chem Rev 248(2004) 119ndash133

[20] MD Hall TW Hambley Coord Chem Rev 232 (2002) 49ndash67[21] TW Hambley Coord Chem Rev 166 (1997) 181ndash223[22] S Bonse JM Richards SA Ross G Lowe RL Krauth-Siegel J

Med Chem 43 (2000) 4812ndash4821[23] G Lowe AS Droz T Vilaiyan GW Weaver L Tweedale JM

Pratt P Rock V Yardley SL Croft J Med Chem 42 (1999) 999ndash1006

[24] SL Croft Mem Inst Oswaldo Cruz 94 (1999) 215ndash220[25] WJ Geary Coord Chem Rev 7 (1971) 81ndash91[26] C Rigol C Olea-Azar F Mendizabal L Otero D Gambino M

Gonzalez H Cerecetto Spectrochim Acta A 61 (2005) 2933ndash2938

[27] L Huang A Lee JA Ellman J Med Chem 45 (2002) 676ndash684[28] JD Maya A Morello Y Repetto R Tellez A Rodriguez U

Zelada P Puebla M Bonta S Bollo A San Feliciano CompBiochem Phys C 125 (2000) 103ndash109

[29] MA Cuellar C Salas MJ Cortes A Morello JD Maya MDPreitea Bioorg Med Chem 11 (2003) 2489ndash2497

[30] RE Mahnken MA Billadeau EP Nikonowicz H Morrison JAm Chem Soc 114 (1992) 9253ndash9265

[31] ME Letelier E Rodrıguez A Wallace M Lorca Y Repetto AMorello J Aldunate Exp Parasitol 71 (1990) 357ndash363

[32] D Gambino L Otero M Vieites M Boiani M Gonzalez EJBaran H Cerecetto Spectrochim Acta A 68 (2007) 341ndash348

[33] L Otero C Folch G Barriga C Rigol L Opazo M Vieites DGambino H Cerecetto E NorambuenaC Olea-Azar SpectrochimActa A lthttpdxdoiorg101016jsaa200707045gt

[34] C Olea-Azar C Rigol F Mendizabal A Morello JD Maya CMoncada E Cabrera R Di Maio M Gonzalez H Cerecetto FreeRad Res 37 (2003) 993ndash1001

[35] S Bollo E Soto-Bustamante LJ Nunez-Vergara JA Squella JElectroanal Chem 492 (2000) 54ndash62

[36] C Olea-Azar AM Atria F Mendizabal R Di Maio G Seoane HCerecetto Spectrosc Lett 31 (1998) 99ndash109

[37] VJ Aran C Ochoa L Boiani P Buccino H Cerecetto A GerpeM Gonzalez D Montero JJ Nogal A Gomez-Barrio A AzquetaA Lopez de Ceraın OE Piro EE Castellano Bioorg Med Chem13 (2005) 3197ndash3207

[38] KM Tyler DM Engman Int J Parasitol 31 (2001) 472ndash480[39] Y Repetto E Opazo JD Maya M Agosin A Morello Comp

Biochem Phys B 115 (1996) 281ndash285[40] N Veerapen SA Taylor CJ Walsby B Mario Pinto J Am

Chem Soc 128 (2006) 227ndash239[41] JD Maya S Bollo LJ Nunez-Vergara JA Squella Y Repetto

A Morello J Perie G Chauviere Biochem Pharmacol 65 (2003)999ndash1006

[42] V Brabec Prog Nucl Acid Res Mol Biol 71 (2002) 1ndash68

Table 5In vitro biological activity of the Pt complexes

Compound T cruzi (Tulahuen 2) T cruzi (Dm28c) Compound T cruzi (Tulahuen 2)PGIa IC50

b ICkc50b IC50

c

10 lM 25 lM

L1 27[PtCl2(HL1)] 109 382 gt25 nd [PdCl2(HL1)] 24[Pt(L1)2] 34 208 gt25 nd [Pd(L1)2] 45

L2 50[PtCl2(HL2)] 415 605 131 367 [PdCl2(HL2)] 43[Pt(L2)2] 684 100 69 102 [Pd(L2)2] 47

L3 49[PtCl2(HL3)] 269 441 275 nd [PdCl2(HL3)] 59[Pt(L3)2] 100 100 08 288 [Pd(L3)2] gt25

L4 gt25[PtCl2(HL4)] 338 720 15 284 [PdCl2(HL4)] gt25[Pt(L4)2] 189 121 gt25 413 [Pd(L4)2] gt25Benznidazol 74 380Nifurtimox 61 [17] 228

IC50 values of the free ligand and their Pd complexes on T cruzi (Tulahuen 2 strain) have been included for comparison [18]nd not determined

a PGI percentage of growth inhibition of T cruzi epimastigote cells at the specified doseb plusmn10c [18]

3430 3440 3450 3460 3470 3480 3490 3500 3510 3520 3530

-8

-6

-4

-2

0

2

4

6

8[103]

previously determined through the same technique on epi-mastigote form of T cruzi Tulahuen 2 strain [18] Most ofthe Pt complexes were active against epimastigotes of T

cruzi Tulahuen 2 strain showing some of them IC50 valuesof the same order than Nifurtimox and the correspondingfree ligands According to the IC50 values [Pt(L3)2] and[Pt(L2)2] were the most active Pt complexes against thisparasite strain being [Pt(L3)2] significantly more activethan L3 and than Nifurtimox

When the results are compared with those previouslyreported for the free ligands and the Pd analogous com-pounds it can be stated that the activity pattern is signifi-cantly modified by changing the central atom The activityof the Pd complexes followed the general trend[PdCl2(HL)] gt free ligand gt [Pd(L)2] This regular trendwas not observed for the Pt analogues This differentbehavior could be related with differences in bioavailabil-ity unspecific toxicity andor mechanism of action of bothseries of metal complexes

Similar general conclusions can be obtained from theanalysis of the activity results on the T cruzi Dm28c strainalthough some strain susceptibility differences can beobserved Dm28c is a parasite clone that had previouslyshown to be less susceptible to Nifurtimox and Ben-znidazol trypanocidal effect than Tulahuen strain whichis mediated by a Dm28c higher thiol content of the former[39]

[G]

Fig 4 ESR spectrum of [PtCl2(HL2)] obtained when [PtCl2(HL2)] wasincubated with T cruzi epimastigotes in the presence of DMPOSpectrometer conditions microwave frequency 968 GHz microwavepower 20 mW modulation amplitude 02 G scan rate 125 Gs timeconstant 05 s number of scans 15 signals of DMPO-nitro anionradical adduct

36 Capacity of derivatives to generate free radical species

into T cruzi

The complexes were incubated with intact T cruzi

(Dm28c strain) epimastigotes in the presence of DMPO

as spin trapping agent in order to detect possible intra-celullar free radical species produced by bioreduction andhaving short half-lives The biological free radical produc-tion capacity of the new compounds was assessed by ESRAll the compounds were capable to produce free radicals inthe intact parasite giving an ESR spectrum Fig 4 showsas an example the ESR spectrum obtained when[PtCl2(HL2)] was incubated with T cruzi in the presenceof DMPO The typical hyperfine pattern of adductsobtained when DMPO traps radicals centered in C atom

[Pt(L2)2]

1 minute

10 O2

[Pt(L2)2]KCN

a

b

Fig 5 (a) Effect of compound [Pt(L)2] on the parasite (T cruzi Dm28cstrain) oxygen consumption related to control (dotted line) (b) Effect ofcompound [Pt(L)2] on the parasite (T cruzi Dm28c strain) oxygenconsumption after inhibition of the respiration with KCN related tocontrol (dotted line)

Table 6Effect of the addition of Pt complexes on oxygen uptake by T cruzi

epimastigotes (Dm28c strain)

Compound Respiration (nanoatom-gramof Ominmg protein)

Increase on oxygenconsumption ()a

Control 280 plusmn 20[PtCl2(HL2)] 322 plusmn 32 15[Pt(L2)2] 313 plusmn 17 12[Pt(L3)2] 392 plusmn 42 40[PtCl2(HL4)] 355 plusmn 32 27[Pt(L4)2] 320 plusmn 13 14Nifurtimox 356 plusmn 08 27

a increase on oxygen consumption after addition of 125 mM ofcompounds with respect to control (no added compound)

Table 7Interaction of Pt complexes with CT DNA after 96 h of incubation at37 C

Compound nmol Ptmg DNA Metalbasea Basemetal

[PtCl2(HL1)] 209 0069 14[Pt(L1)2] 390 0129 8[PtCl2(HL2)] 236 0078 13[Pt(L2)2] 224 0074 14[PtCl2(HL3)] 191 0063 16[Pt(L3)2] 527 0174 6[PtCl2(HL4)] 170 0056 18[Pt(L4)2] 170 0056 18

a Platinum (mol) per DNA base (mol)

(aN = 167 G and aH = 225 G) was observed [18] A trip-let produced by DMPO decomposition was also detected[40]

ESR studies showed that the newly developed Pt thio-semicarbazone complexes were bioreduced in the parasiteindicating that they could maintain the mode of action ofthe 5-nitrofuryl pharmacophore

37 Oxygen uptake

The involvement of the platinum complexes in redoxcycling processes should increase the parasite oxygen con-sumption Thus the effect of the compounds on parasiteoxygen uptake was measured The effect of complex[Pt(L2)2] on T cruzi epimastigotes oxygen consumptionwith and without inhibition of the respiration with KCNis shown in Fig 5 Both series of complexes modified oxy-gen uptake by the parasite increasing oxygen consumption(Table 6) These results are in accordance with a bioreduc-tive and redox cycling mechanism of action as previouslyreported for the free 5-nitrofuryl containing thiosemicarba-zones their Pd complexes and other nitrofuran derivativeslike Nifurtimox [1841] For both series of compoundseffect on parasite respiration seemed to correlate with theIC50 values For instance [Pt(L3)2] being the most activePt compound showed the highest increase on oxygenconsumption

38 Calf thymus DNA interaction

In order to address if interaction with DNA could bepart of the mode of action of the complexes experimentswith CT DNA were carried out Binding of the Pt com-plexes to DNA was studied by combining atomic absorp-tion determinations (for the metal) and electronicabsorption measurements for DNA quantification Thenew complexes are good binding agents for CT DNA

(Table 7) Although the determined platinum to DNAbinding levels were similar to those of known anti-tumormetal complexes they resulted significantly lower thanthose of the palladium analogous compounds [1819] Nogeneral pattern could be detected for both series of com-pounds being binding levels almost not affected by thenature of the thiosemicarbazone ligand [PtCl2(HL)] com-plexes having labile chloride ligands could probably inter-act with DNA through a mechanism similar to thatpreviously reported for cisplatin becoming activatedthrough aquation and reacting with nucleophilic DNAbases [42] [Pt(L)2] complexes cannot act in this way butthey could intercalate DNA or interact through othermechanisms Due to the planar motive contained in the[PdCl2(HL)] complexes intercalation could also be possiblefor these compounds Further studies are under progress inorder to get an insight into the mechanism of this interac-tion It is interesting to note that [Pt(L3)2] being the mostpotent anti-T cruzi compound of the series showed thehighest interaction with DNA Nevertheless [Pt(L1)2]being almost non active strongly interacts with DNAshowing that the mechanism of trypanocidal activity couldnot involve only DNA as target

4 Conclusions

Pt(II) complexes with 5-nitrofuryl containing thiosemi-carbazones as bioactive ligands showed good anti-trypan-osomal activity In particular the coordination of L3 to

Pt(II) forming [Pt(L3)2] lead to almost a five-fold activityincrease in respect to the free ligand Possible targetsnamely reductive metabolism (intra-parasite free radicalproduction) and DNA were screened trying to get insightinto the mechanism of action of these metal compoundsResults showed that some of the compounds could act asdual inhibitors in the parasite through production of toxicfree radicals and interaction with DNA

Results of this work show that the approach of coordi-nating anti-trypanosomal organic compounds with phar-macologically interesting metals could be a suitablestrategy to develop novel therapeutic tools against tropicaldiseases produced by trypanosomatids particularly Amer-ican Trypanosomiasis

Acknowledgments

This work was partially supported by PEDECIBA andUniversidad de La Republica (CSIC project 36406) ofUruguay Prosul-CNPq project 4902092005-0 SIBAS-0807 UMCE FONDECYT Chile 1061072 and CONI-CYT-PBCT Anillo ACT 29 We wish to thank Dr EJBaran CEQUINOR UNLP Argentina for Raman spec-tra facilities and Matilde Gomez Instituto Venezolano deInvestigaciones Cientıficas Caracas Venezuela for per-forming ESI-MS experiments

References

[1] httpwwwwhoint lthttpwwwwhointctdchagasgt[2] D Engels L Savioli Trend Parasitol 22 (2006) 363ndash366[3] J Urbina Expert Opin Ther Pat 13 (2003) 661ndash669[4] H Cerecetto M Gonzalez Curr Topics Med Chem 2 (2002) 1185ndash

1190[5] RL Krauth-Siegel H Bauer RH Schirmer Angew Chem Int Ed

44 (2005) 690ndash715[6] S Croft M Barret J Urbina Trend Parasitol 21 (2005) 508ndash512[7] M Ceaser The Lancet Infect Dis 5 (8) (2005) 470ndash471[8] Y Yamagata J Nakagawa Adv Parasitol 61 (2006) 129ndash165[9] C Zhang S Lippard Curr Opin Chem Biol 7 (2003) 481ndash489

[10] NP Farrell Metal Complexes as Drugs and ChemotherapeuticAgents in JM McCleverty TJ Meyer (Eds) ComprehensiveCoordination Chemistry II vol 9 Elsevier 2003 pp 809ndash840

[11] RA Sanchez-Delgado A Anzellotti L Suarez Metal complexes aschemotherapeutic agents against tropical diseases malaria trypano-somiasis and leishmaniasis in H Sigel A Sigel (Eds) Metal Ionsin Biological Systems vol 41 Marcel Dekker New York 2004 pp379ndash419

[12] RA Sanchez-Delgado A Anzellotti Mini-Rev Med Chem 4(2004) 23ndash30

[13] R Morphy Z Rankovic J Med Chem 48 (2005) 6523ndash6543[14] Z Wang EM Bennett DJ Wilson C Salomon R Vince J Med

Chem 50 (2007) 3416ndash3419[15] K Chibale Towards broad spectrum antiprotozoal agents ARKI-

VOC IX 2002 pp 93ndash98

[16] C Urquiola M Vieites G Aguirre A Marın B Solano GArrambide ML Lavaggi MH Torre M Gonzalez A Monge DGambino H Cerecetto Bioorg Med Chem 14 (2006) 5503ndash5509

[17] G Aguirre H Cerecetto M Gonzalez D Gambino L Otero COlea-Azar C Rigol A Denicola Bioorg Med Chem 12 (2004)4885ndash4893

[18] L Otero M Vieites L Boiani A Denicola C Rigol L Opazo COlea-Azar JD Maya A Morello RL Krauth-Siegel OE Piro ECastellano M Gonzalez D Gambino H Cerecetto J Med Chem49 (2006) 3322ndash3331

[19] A Gomez-Quiroga C Navarro-Ranninger Coord Chem Rev 248(2004) 119ndash133

[20] MD Hall TW Hambley Coord Chem Rev 232 (2002) 49ndash67[21] TW Hambley Coord Chem Rev 166 (1997) 181ndash223[22] S Bonse JM Richards SA Ross G Lowe RL Krauth-Siegel J

Med Chem 43 (2000) 4812ndash4821[23] G Lowe AS Droz T Vilaiyan GW Weaver L Tweedale JM

Pratt P Rock V Yardley SL Croft J Med Chem 42 (1999) 999ndash1006

[24] SL Croft Mem Inst Oswaldo Cruz 94 (1999) 215ndash220[25] WJ Geary Coord Chem Rev 7 (1971) 81ndash91[26] C Rigol C Olea-Azar F Mendizabal L Otero D Gambino M

Gonzalez H Cerecetto Spectrochim Acta A 61 (2005) 2933ndash2938

[27] L Huang A Lee JA Ellman J Med Chem 45 (2002) 676ndash684[28] JD Maya A Morello Y Repetto R Tellez A Rodriguez U

Zelada P Puebla M Bonta S Bollo A San Feliciano CompBiochem Phys C 125 (2000) 103ndash109

[29] MA Cuellar C Salas MJ Cortes A Morello JD Maya MDPreitea Bioorg Med Chem 11 (2003) 2489ndash2497

[30] RE Mahnken MA Billadeau EP Nikonowicz H Morrison JAm Chem Soc 114 (1992) 9253ndash9265

[31] ME Letelier E Rodrıguez A Wallace M Lorca Y Repetto AMorello J Aldunate Exp Parasitol 71 (1990) 357ndash363

[32] D Gambino L Otero M Vieites M Boiani M Gonzalez EJBaran H Cerecetto Spectrochim Acta A 68 (2007) 341ndash348

[33] L Otero C Folch G Barriga C Rigol L Opazo M Vieites DGambino H Cerecetto E NorambuenaC Olea-Azar SpectrochimActa A lthttpdxdoiorg101016jsaa200707045gt

[34] C Olea-Azar C Rigol F Mendizabal A Morello JD Maya CMoncada E Cabrera R Di Maio M Gonzalez H Cerecetto FreeRad Res 37 (2003) 993ndash1001

[35] S Bollo E Soto-Bustamante LJ Nunez-Vergara JA Squella JElectroanal Chem 492 (2000) 54ndash62

[36] C Olea-Azar AM Atria F Mendizabal R Di Maio G Seoane HCerecetto Spectrosc Lett 31 (1998) 99ndash109

[37] VJ Aran C Ochoa L Boiani P Buccino H Cerecetto A GerpeM Gonzalez D Montero JJ Nogal A Gomez-Barrio A AzquetaA Lopez de Ceraın OE Piro EE Castellano Bioorg Med Chem13 (2005) 3197ndash3207

[38] KM Tyler DM Engman Int J Parasitol 31 (2001) 472ndash480[39] Y Repetto E Opazo JD Maya M Agosin A Morello Comp

Biochem Phys B 115 (1996) 281ndash285[40] N Veerapen SA Taylor CJ Walsby B Mario Pinto J Am

Chem Soc 128 (2006) 227ndash239[41] JD Maya S Bollo LJ Nunez-Vergara JA Squella Y Repetto

A Morello J Perie G Chauviere Biochem Pharmacol 65 (2003)999ndash1006

[42] V Brabec Prog Nucl Acid Res Mol Biol 71 (2002) 1ndash68

[Pt(L2)2]

1 minute

10 O2

[Pt(L2)2]KCN

a

b

Fig 5 (a) Effect of compound [Pt(L)2] on the parasite (T cruzi Dm28cstrain) oxygen consumption related to control (dotted line) (b) Effect ofcompound [Pt(L)2] on the parasite (T cruzi Dm28c strain) oxygenconsumption after inhibition of the respiration with KCN related tocontrol (dotted line)

Table 6Effect of the addition of Pt complexes on oxygen uptake by T cruzi

epimastigotes (Dm28c strain)

Compound Respiration (nanoatom-gramof Ominmg protein)

Increase on oxygenconsumption ()a

Control 280 plusmn 20[PtCl2(HL2)] 322 plusmn 32 15[Pt(L2)2] 313 plusmn 17 12[Pt(L3)2] 392 plusmn 42 40[PtCl2(HL4)] 355 plusmn 32 27[Pt(L4)2] 320 plusmn 13 14Nifurtimox 356 plusmn 08 27

a increase on oxygen consumption after addition of 125 mM ofcompounds with respect to control (no added compound)

Table 7Interaction of Pt complexes with CT DNA after 96 h of incubation at37 C

Compound nmol Ptmg DNA Metalbasea Basemetal

[PtCl2(HL1)] 209 0069 14[Pt(L1)2] 390 0129 8[PtCl2(HL2)] 236 0078 13[Pt(L2)2] 224 0074 14[PtCl2(HL3)] 191 0063 16[Pt(L3)2] 527 0174 6[PtCl2(HL4)] 170 0056 18[Pt(L4)2] 170 0056 18

a Platinum (mol) per DNA base (mol)

(aN = 167 G and aH = 225 G) was observed [18] A trip-let produced by DMPO decomposition was also detected[40]

ESR studies showed that the newly developed Pt thio-semicarbazone complexes were bioreduced in the parasiteindicating that they could maintain the mode of action ofthe 5-nitrofuryl pharmacophore

37 Oxygen uptake

The involvement of the platinum complexes in redoxcycling processes should increase the parasite oxygen con-sumption Thus the effect of the compounds on parasiteoxygen uptake was measured The effect of complex[Pt(L2)2] on T cruzi epimastigotes oxygen consumptionwith and without inhibition of the respiration with KCNis shown in Fig 5 Both series of complexes modified oxy-gen uptake by the parasite increasing oxygen consumption(Table 6) These results are in accordance with a bioreduc-tive and redox cycling mechanism of action as previouslyreported for the free 5-nitrofuryl containing thiosemicarba-zones their Pd complexes and other nitrofuran derivativeslike Nifurtimox [1841] For both series of compoundseffect on parasite respiration seemed to correlate with theIC50 values For instance [Pt(L3)2] being the most activePt compound showed the highest increase on oxygenconsumption

38 Calf thymus DNA interaction

In order to address if interaction with DNA could bepart of the mode of action of the complexes experimentswith CT DNA were carried out Binding of the Pt com-plexes to DNA was studied by combining atomic absorp-tion determinations (for the metal) and electronicabsorption measurements for DNA quantification Thenew complexes are good binding agents for CT DNA

(Table 7) Although the determined platinum to DNAbinding levels were similar to those of known anti-tumormetal complexes they resulted significantly lower thanthose of the palladium analogous compounds [1819] Nogeneral pattern could be detected for both series of com-pounds being binding levels almost not affected by thenature of the thiosemicarbazone ligand [PtCl2(HL)] com-plexes having labile chloride ligands could probably inter-act with DNA through a mechanism similar to thatpreviously reported for cisplatin becoming activatedthrough aquation and reacting with nucleophilic DNAbases [42] [Pt(L)2] complexes cannot act in this way butthey could intercalate DNA or interact through othermechanisms Due to the planar motive contained in the[PdCl2(HL)] complexes intercalation could also be possiblefor these compounds Further studies are under progress inorder to get an insight into the mechanism of this interac-tion It is interesting to note that [Pt(L3)2] being the mostpotent anti-T cruzi compound of the series showed thehighest interaction with DNA Nevertheless [Pt(L1)2]being almost non active strongly interacts with DNAshowing that the mechanism of trypanocidal activity couldnot involve only DNA as target

4 Conclusions

Pt(II) complexes with 5-nitrofuryl containing thiosemi-carbazones as bioactive ligands showed good anti-trypan-osomal activity In particular the coordination of L3 to

Pt(II) forming [Pt(L3)2] lead to almost a five-fold activityincrease in respect to the free ligand Possible targetsnamely reductive metabolism (intra-parasite free radicalproduction) and DNA were screened trying to get insightinto the mechanism of action of these metal compoundsResults showed that some of the compounds could act asdual inhibitors in the parasite through production of toxicfree radicals and interaction with DNA

Results of this work show that the approach of coordi-nating anti-trypanosomal organic compounds with phar-macologically interesting metals could be a suitablestrategy to develop novel therapeutic tools against tropicaldiseases produced by trypanosomatids particularly Amer-ican Trypanosomiasis

Acknowledgments

This work was partially supported by PEDECIBA andUniversidad de La Republica (CSIC project 36406) ofUruguay Prosul-CNPq project 4902092005-0 SIBAS-0807 UMCE FONDECYT Chile 1061072 and CONI-CYT-PBCT Anillo ACT 29 We wish to thank Dr EJBaran CEQUINOR UNLP Argentina for Raman spec-tra facilities and Matilde Gomez Instituto Venezolano deInvestigaciones Cientıficas Caracas Venezuela for per-forming ESI-MS experiments

References

[1] httpwwwwhoint lthttpwwwwhointctdchagasgt[2] D Engels L Savioli Trend Parasitol 22 (2006) 363ndash366[3] J Urbina Expert Opin Ther Pat 13 (2003) 661ndash669[4] H Cerecetto M Gonzalez Curr Topics Med Chem 2 (2002) 1185ndash

1190[5] RL Krauth-Siegel H Bauer RH Schirmer Angew Chem Int Ed

44 (2005) 690ndash715[6] S Croft M Barret J Urbina Trend Parasitol 21 (2005) 508ndash512[7] M Ceaser The Lancet Infect Dis 5 (8) (2005) 470ndash471[8] Y Yamagata J Nakagawa Adv Parasitol 61 (2006) 129ndash165[9] C Zhang S Lippard Curr Opin Chem Biol 7 (2003) 481ndash489

[10] NP Farrell Metal Complexes as Drugs and ChemotherapeuticAgents in JM McCleverty TJ Meyer (Eds) ComprehensiveCoordination Chemistry II vol 9 Elsevier 2003 pp 809ndash840

[11] RA Sanchez-Delgado A Anzellotti L Suarez Metal complexes aschemotherapeutic agents against tropical diseases malaria trypano-somiasis and leishmaniasis in H Sigel A Sigel (Eds) Metal Ionsin Biological Systems vol 41 Marcel Dekker New York 2004 pp379ndash419

[12] RA Sanchez-Delgado A Anzellotti Mini-Rev Med Chem 4(2004) 23ndash30

[13] R Morphy Z Rankovic J Med Chem 48 (2005) 6523ndash6543[14] Z Wang EM Bennett DJ Wilson C Salomon R Vince J Med

Chem 50 (2007) 3416ndash3419[15] K Chibale Towards broad spectrum antiprotozoal agents ARKI-

VOC IX 2002 pp 93ndash98

[16] C Urquiola M Vieites G Aguirre A Marın B Solano GArrambide ML Lavaggi MH Torre M Gonzalez A Monge DGambino H Cerecetto Bioorg Med Chem 14 (2006) 5503ndash5509

[17] G Aguirre H Cerecetto M Gonzalez D Gambino L Otero COlea-Azar C Rigol A Denicola Bioorg Med Chem 12 (2004)4885ndash4893

[18] L Otero M Vieites L Boiani A Denicola C Rigol L Opazo COlea-Azar JD Maya A Morello RL Krauth-Siegel OE Piro ECastellano M Gonzalez D Gambino H Cerecetto J Med Chem49 (2006) 3322ndash3331

[19] A Gomez-Quiroga C Navarro-Ranninger Coord Chem Rev 248(2004) 119ndash133

[20] MD Hall TW Hambley Coord Chem Rev 232 (2002) 49ndash67[21] TW Hambley Coord Chem Rev 166 (1997) 181ndash223[22] S Bonse JM Richards SA Ross G Lowe RL Krauth-Siegel J

Med Chem 43 (2000) 4812ndash4821[23] G Lowe AS Droz T Vilaiyan GW Weaver L Tweedale JM

Pratt P Rock V Yardley SL Croft J Med Chem 42 (1999) 999ndash1006

[24] SL Croft Mem Inst Oswaldo Cruz 94 (1999) 215ndash220[25] WJ Geary Coord Chem Rev 7 (1971) 81ndash91[26] C Rigol C Olea-Azar F Mendizabal L Otero D Gambino M

Gonzalez H Cerecetto Spectrochim Acta A 61 (2005) 2933ndash2938

[27] L Huang A Lee JA Ellman J Med Chem 45 (2002) 676ndash684[28] JD Maya A Morello Y Repetto R Tellez A Rodriguez U

Zelada P Puebla M Bonta S Bollo A San Feliciano CompBiochem Phys C 125 (2000) 103ndash109

[29] MA Cuellar C Salas MJ Cortes A Morello JD Maya MDPreitea Bioorg Med Chem 11 (2003) 2489ndash2497

[30] RE Mahnken MA Billadeau EP Nikonowicz H Morrison JAm Chem Soc 114 (1992) 9253ndash9265

[31] ME Letelier E Rodrıguez A Wallace M Lorca Y Repetto AMorello J Aldunate Exp Parasitol 71 (1990) 357ndash363

[32] D Gambino L Otero M Vieites M Boiani M Gonzalez EJBaran H Cerecetto Spectrochim Acta A 68 (2007) 341ndash348

[33] L Otero C Folch G Barriga C Rigol L Opazo M Vieites DGambino H Cerecetto E NorambuenaC Olea-Azar SpectrochimActa A lthttpdxdoiorg101016jsaa200707045gt

[34] C Olea-Azar C Rigol F Mendizabal A Morello JD Maya CMoncada E Cabrera R Di Maio M Gonzalez H Cerecetto FreeRad Res 37 (2003) 993ndash1001

[35] S Bollo E Soto-Bustamante LJ Nunez-Vergara JA Squella JElectroanal Chem 492 (2000) 54ndash62

[36] C Olea-Azar AM Atria F Mendizabal R Di Maio G Seoane HCerecetto Spectrosc Lett 31 (1998) 99ndash109

[37] VJ Aran C Ochoa L Boiani P Buccino H Cerecetto A GerpeM Gonzalez D Montero JJ Nogal A Gomez-Barrio A AzquetaA Lopez de Ceraın OE Piro EE Castellano Bioorg Med Chem13 (2005) 3197ndash3207

[38] KM Tyler DM Engman Int J Parasitol 31 (2001) 472ndash480[39] Y Repetto E Opazo JD Maya M Agosin A Morello Comp

Biochem Phys B 115 (1996) 281ndash285[40] N Veerapen SA Taylor CJ Walsby B Mario Pinto J Am

Chem Soc 128 (2006) 227ndash239[41] JD Maya S Bollo LJ Nunez-Vergara JA Squella Y Repetto

A Morello J Perie G Chauviere Biochem Pharmacol 65 (2003)999ndash1006

[42] V Brabec Prog Nucl Acid Res Mol Biol 71 (2002) 1ndash68

Pt(II) forming [Pt(L3)2] lead to almost a five-fold activityincrease in respect to the free ligand Possible targetsnamely reductive metabolism (intra-parasite free radicalproduction) and DNA were screened trying to get insightinto the mechanism of action of these metal compoundsResults showed that some of the compounds could act asdual inhibitors in the parasite through production of toxicfree radicals and interaction with DNA

Results of this work show that the approach of coordi-nating anti-trypanosomal organic compounds with phar-macologically interesting metals could be a suitablestrategy to develop novel therapeutic tools against tropicaldiseases produced by trypanosomatids particularly Amer-ican Trypanosomiasis

Acknowledgments

This work was partially supported by PEDECIBA andUniversidad de La Republica (CSIC project 36406) ofUruguay Prosul-CNPq project 4902092005-0 SIBAS-0807 UMCE FONDECYT Chile 1061072 and CONI-CYT-PBCT Anillo ACT 29 We wish to thank Dr EJBaran CEQUINOR UNLP Argentina for Raman spec-tra facilities and Matilde Gomez Instituto Venezolano deInvestigaciones Cientıficas Caracas Venezuela for per-forming ESI-MS experiments

References

[1] httpwwwwhoint lthttpwwwwhointctdchagasgt[2] D Engels L Savioli Trend Parasitol 22 (2006) 363ndash366[3] J Urbina Expert Opin Ther Pat 13 (2003) 661ndash669[4] H Cerecetto M Gonzalez Curr Topics Med Chem 2 (2002) 1185ndash

1190[5] RL Krauth-Siegel H Bauer RH Schirmer Angew Chem Int Ed

44 (2005) 690ndash715[6] S Croft M Barret J Urbina Trend Parasitol 21 (2005) 508ndash512[7] M Ceaser The Lancet Infect Dis 5 (8) (2005) 470ndash471[8] Y Yamagata J Nakagawa Adv Parasitol 61 (2006) 129ndash165[9] C Zhang S Lippard Curr Opin Chem Biol 7 (2003) 481ndash489

[10] NP Farrell Metal Complexes as Drugs and ChemotherapeuticAgents in JM McCleverty TJ Meyer (Eds) ComprehensiveCoordination Chemistry II vol 9 Elsevier 2003 pp 809ndash840

[11] RA Sanchez-Delgado A Anzellotti L Suarez Metal complexes aschemotherapeutic agents against tropical diseases malaria trypano-somiasis and leishmaniasis in H Sigel A Sigel (Eds) Metal Ionsin Biological Systems vol 41 Marcel Dekker New York 2004 pp379ndash419

[12] RA Sanchez-Delgado A Anzellotti Mini-Rev Med Chem 4(2004) 23ndash30

[13] R Morphy Z Rankovic J Med Chem 48 (2005) 6523ndash6543[14] Z Wang EM Bennett DJ Wilson C Salomon R Vince J Med

Chem 50 (2007) 3416ndash3419[15] K Chibale Towards broad spectrum antiprotozoal agents ARKI-

VOC IX 2002 pp 93ndash98

[16] C Urquiola M Vieites G Aguirre A Marın B Solano GArrambide ML Lavaggi MH Torre M Gonzalez A Monge DGambino H Cerecetto Bioorg Med Chem 14 (2006) 5503ndash5509

[17] G Aguirre H Cerecetto M Gonzalez D Gambino L Otero COlea-Azar C Rigol A Denicola Bioorg Med Chem 12 (2004)4885ndash4893

[18] L Otero M Vieites L Boiani A Denicola C Rigol L Opazo COlea-Azar JD Maya A Morello RL Krauth-Siegel OE Piro ECastellano M Gonzalez D Gambino H Cerecetto J Med Chem49 (2006) 3322ndash3331

[19] A Gomez-Quiroga C Navarro-Ranninger Coord Chem Rev 248(2004) 119ndash133

[20] MD Hall TW Hambley Coord Chem Rev 232 (2002) 49ndash67[21] TW Hambley Coord Chem Rev 166 (1997) 181ndash223[22] S Bonse JM Richards SA Ross G Lowe RL Krauth-Siegel J

Med Chem 43 (2000) 4812ndash4821[23] G Lowe AS Droz T Vilaiyan GW Weaver L Tweedale JM

Pratt P Rock V Yardley SL Croft J Med Chem 42 (1999) 999ndash1006

[24] SL Croft Mem Inst Oswaldo Cruz 94 (1999) 215ndash220[25] WJ Geary Coord Chem Rev 7 (1971) 81ndash91[26] C Rigol C Olea-Azar F Mendizabal L Otero D Gambino M

Gonzalez H Cerecetto Spectrochim Acta A 61 (2005) 2933ndash2938

[27] L Huang A Lee JA Ellman J Med Chem 45 (2002) 676ndash684[28] JD Maya A Morello Y Repetto R Tellez A Rodriguez U

Zelada P Puebla M Bonta S Bollo A San Feliciano CompBiochem Phys C 125 (2000) 103ndash109

[29] MA Cuellar C Salas MJ Cortes A Morello JD Maya MDPreitea Bioorg Med Chem 11 (2003) 2489ndash2497

[30] RE Mahnken MA Billadeau EP Nikonowicz H Morrison JAm Chem Soc 114 (1992) 9253ndash9265

[31] ME Letelier E Rodrıguez A Wallace M Lorca Y Repetto AMorello J Aldunate Exp Parasitol 71 (1990) 357ndash363

[32] D Gambino L Otero M Vieites M Boiani M Gonzalez EJBaran H Cerecetto Spectrochim Acta A 68 (2007) 341ndash348

[33] L Otero C Folch G Barriga C Rigol L Opazo M Vieites DGambino H Cerecetto E NorambuenaC Olea-Azar SpectrochimActa A lthttpdxdoiorg101016jsaa200707045gt

[34] C Olea-Azar C Rigol F Mendizabal A Morello JD Maya CMoncada E Cabrera R Di Maio M Gonzalez H Cerecetto FreeRad Res 37 (2003) 993ndash1001

[35] S Bollo E Soto-Bustamante LJ Nunez-Vergara JA Squella JElectroanal Chem 492 (2000) 54ndash62

[36] C Olea-Azar AM Atria F Mendizabal R Di Maio G Seoane HCerecetto Spectrosc Lett 31 (1998) 99ndash109

[37] VJ Aran C Ochoa L Boiani P Buccino H Cerecetto A GerpeM Gonzalez D Montero JJ Nogal A Gomez-Barrio A AzquetaA Lopez de Ceraın OE Piro EE Castellano Bioorg Med Chem13 (2005) 3197ndash3207

[38] KM Tyler DM Engman Int J Parasitol 31 (2001) 472ndash480[39] Y Repetto E Opazo JD Maya M Agosin A Morello Comp

Biochem Phys B 115 (1996) 281ndash285[40] N Veerapen SA Taylor CJ Walsby B Mario Pinto J Am

Chem Soc 128 (2006) 227ndash239[41] JD Maya S Bollo LJ Nunez-Vergara JA Squella Y Repetto

A Morello J Perie G Chauviere Biochem Pharmacol 65 (2003)999ndash1006

[42] V Brabec Prog Nucl Acid Res Mol Biol 71 (2002) 1ndash68