Polyhedron 63 (2013) 28–35

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Polyhedron

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Investigation on the bioactivities of clioquinol and its bismuth(III)and platinum(II, IV) complexes

0277-5387/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.poly.2013.07.008

⇑ Corresponding authors. Tel.: +55 31 34095740; fax: +55 31 34095700(H. Beraldo).

E-mail addresses: baran@quimica.unlp.edu.ar (E.J. Baran), hberaldo@ufmg.br(H. Beraldo).

Karina S.O. Ferraz a, Débora C. Reis a, Jeferson G. Da Silva a, Elaine M. Souza-Fagundes b, Enrique J. Baran c,⇑,Heloisa Beraldo a,⇑a Departamento de Química, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazilb Departamento de Fisiologia, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazilc Universidad Nacional de La Plata, C. Correo 962, 1900 La Plata, Argentina

a r t i c l e i n f o

Article history:Received 23 May 2013Accepted 8 July 2013Available online 15 July 2013

Keywords:ClioquinolBismuth(III) complexPlatinum(II, IV) complexesLeukemia cellsAntimicrobial activity

a b s t r a c t

Complexes [Bi(HCQ)2(H2O)Cl3] (1), [Pt(CQ)2]�2KCl (2) and [Pt(CQ)2Cl2]�KCl (3) were obtained with 5-chloro-7-iodo-8-hydroxyquinoline, ‘‘clioquinol’’, HCQ. Upon coordination to bismuth(III) the antimicro-bial activity improved. Complex (1) was 70-fold more active than fluconazole against Candida albicans.HCQ proved to be cytotoxic to HL-60 and Jurkat human leukemia cells. Although coordination to bis-muth(III) did not result in significant modification of HCQ’s cytotoxic effect, on coordination to plati-num(II, IV) cytotoxicity improved against both cell lines. Complexes (2) and (3) were more active thanHCQ against HL-60 cells. Complex (2) also revealed to be the most cytotoxic compound against Jurkatcells, being fivefold more active than cisplatin. Although HCQ and 1 did not show a pro-apoptotic effect,2 and 3 presented moderate pro-apoptotic activity.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction Platinum-based compounds are widely used as chemothera-

8-Hydroxyquinoline and its derivatives are systems of largeinterest in the field of medicinal inorganic chemistry due to theirmetal-binding properties and wide bioactivity profile [1].Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline, HCQ, Fig. 1),was clinically used as an antibiotic for the treatment of diarrheaand skin infection. Topical formulations of the compound are stillavailable for the treatment of fungal and parasitic infections [2].Recently, clioquinol has reemerged for the treatment of non-infec-tious indications including malignancy and Alzheimer’s disease [3].Clioquinol presents the potential to become an antitumor drugcandidate [4]. Studies have shown that the treatment with clio-quinol at low doses reduced the cell viability of different humantumor cell lines [5]. Since clioquinol can act as a chelating agentits metal complexes are also of great pharmacological interest.

Bismuth compounds have demonstrated activity as antimicro-bial and antitumor agents and have been used to treat a varietyof medical disorders. Most evident has been the widespread useof bismuth compounds in the treatment of duodenal ulcers, gastri-tis and chronic diarrhea [6,7]. The cytotoxic and antiproliferativeactivities of bismuth(III) complexes have been investigated [7,8].

peutics for the treatment of a variety of cancers. The ability of plat-inum complexes to significantly inhibit tumor growth is the resultof a combination of factors, including diffusion across the cellularmembrane, accumulation in cells, and reactivity against nuclearDNA [9]. Cis-diamminedichloroplatinum(II) (cisplatin) is currentlyused clinically and is one of the most effective anticancer drugsin the treatment of a variety of human tumors [10]. However, theuse of cisplatin to treat malignancies is limited because of sideeffects and acquired resistance. The employment of inert plati-num(IV) compounds has been explored as a tool to overcome tu-mor cell resistance and toxicity to normal tissues. Platinum(IV)complexes could act as pro-drugs and release clinically effectivelevels of platinum(II) compounds, following cellular uptake [11].

In the present work bismuth(III) and platinum(II, IV) complexeswith clioquinol were obtained. The antimicrobial activity of thebismuth(III) complex was investigated against the growth ofGram-positive Staphylococcus aureus and Gram-negative Pseudomo-nas aeruginosa bacteria and Candida albicans fungi. The cytotoxiceffects of the bismuth(III) and platinum(II, IV) complexes againstHL-60 and Jurkat leukemia cells were evaluated.

2. Experimental

2.1. Materials and methods

All common chemicals were purchased from Aldrich and usedwithout further purification. Partial elemental analyses were

9

10

4

N1

3

2

6

7

5

8

OH

Cl

I

Fig. 1. Structure of clioquinol (HCQ).

K.S.O. Ferraz et al. / Polyhedron 63 (2013) 28–35 29

performed on a Perkin Elmer CHN 2400 analyzer. An YSI model 31conductivity bridge was employed for molar conductivity mea-surements. Infrared spectra were recorded on a Perkin Elmer FT-IR Spectrum GX spectrometer using KBr pellets (4000–400 cm�1)and Nujol mulls between CsI plates (400–200 cm�1). NMR spectrawere obtained with a Bruker DPX-200 Avance (200 MHz) spec-trometer using DMSO-d6 as the solvent and TMS as internal refer-ence. Splitting patterns are designated as follows: s, singlet; d,doublet; t, triplet; q, quartet; m, multiplet. Single crystal X-ray dif-fraction measurements were carried out on an Oxford-DiffractionGEMINI-Ultra diffractometer (LabCri-UFMG) using graphite-Enhance Source Mo Ka radiation (k = 0.71073 Å). Data collection,cell refinements, and data reduction were performed using the CRY-

SALISPRO software [12,13]. The analytical absorption correctionmethod was applied [12]. The structures were solved by directmethods using SHELXS-97 [14]. Full-matrix least-squares refinementprocedure on F2 with anisotropic thermal parameters was carriedout using SHELXL-97 [14]. Positional and anisotropic atomic dis-placement parameters were refined for all non-hydrogen atoms.Hydrogen atoms were placed geometrically and the positionalparameters were refined using a riding model.

2.2. Syntheses of the bismuth(III) and platinum(II, IV) complexes

The bismuth(III) complex was obtained by treating an ethanolsolution of clioquinol with BiCl3 in 3:1 ligand-to-metal molar ratioat room temperature with stirring for 24 h. The platinum(II) andplatinum(IV) complexes were prepared by treating an ethanolsolution of clioquinol with an aqueous solution of K2PtCl4 or K2-

PtCl6 in 1:1 ligand-to-metal molar ratio. The reaction mixturewas left under reflux for 2 h in the case of the platinum(II) complexand under stirring at room temperature for a week in that of theplatinum(IV) complex. The resulting solids were filtered, washedwith ethanol followed by diethyl ether, and dried in vacuo.

2.2.1. Aquatrichloro [bis(5-chloro-7-iodo-8-hydroxyquinoline)]bismuth(III)[Bi(HCQ)2(H2O)Cl3] (1)

Yellow solid. Anal. Calc. for (C18H12BiCl5I2N2O3): C, 22.87; H,1.27; N, 2.91. Found: C, 22.89; H, 1.15; N, 2.97%. FW:944.35 g mol�1. IR (KBr, cm�1): m(H2O) 3460m, m(O–H) 3046m,m(M–O2H) 1622m, m(C@N) 1543m, m(C–O) 1080w, q(py) 653m. IR(CsI/Nujol, cm�1): m(M–Npy) 268w, m(M–Cl) 222w. 1H NMR[200 MHz, DMSO-d6,((ppm), J (Hz)] main signals: 9.43–9.41 [d,1H, H(2), 3.28], 8.23–8.17 [m, 1H, H(3), 4.24], 8.95–8.90 [d, 1H,H(4), 8.45], 8.42 [s, 1H, H(6)]. 13C NMR [50 MHz, DMSO-d6, d(ppm)] main signals: 149.55 [C(2)], 123.36 [C(3)], 133.20 [C(4)],118.86 [C(5)], 135.04 [C(6)], 48.63 [C(7)], 206.47 [C(8)], 137.72[C(9)], 125.93 [C(10)]. KM = 7.27 X�1 cm2 mol�1 in DMF. Meltingpoint: 168.3–169.0 �C.

2.2.2. Bis(5-chloro-7-iodo-8-hydroxyquinolinato)platinum(II),[Pt(CQ)2]�2KCl (2)

[Pt(CQ)2] has been prepared by other authors [15]. Unlike ourprocedure, in which the reaction mixture was kept under reflux

in ethanol/water for 2 h, with no addition of acid, the other authorsreport that drops of acetic acid were added to the reaction mixturewhich was kept under reflux in ethanol/water during 24 h.

Brown solid. Anal. Calc. for (C18H8Cl4I2K2N2O2Pt): C, 22.68; H,0.85; N, 2.98. Found: C, 22.95; H, 0.99; N, 2.58%. FW:953.16 g mol�1. IR (KBr, cm�1): m(C@N) 1552s, m(C–O) 1114 m,q(py) 676w. IR (CsI/Nujol, cm�1): m(M–Npy) 345 m, m(M–O)338 m, m(M–Cl) n/o. 1H NMR [200 MHz, DMSO-d6, d (ppm), J(Hz)]main signals: 9.49–9.46 [d,1H, H(2), 4.04], 7.87–7.84 [m, 1H,H(3), 4.16], 9.01–8.99 [d, 1H, H(4), 8.49], 8.06 [s, 1H, H(6)]. 13CNMR [50 MHz, DMSO-d6, d (ppm)] main signals: 149.10 [C(2)],123.40 [C(3)], 136.94 [C(4)], 127.47 [C(5)], 136.34 [C(6)], 81.69[C(7)], n/o [C(8)], 141.42 [C(9)], 115.53 [C(10)]. KM = 27 X�1 cm2

mol�1 in DMF. Melting point: decomposes at 222 �C. n/o = non-observed

2.2.3. Dichloro[bis(5-chloro-7-iodo-8 hydroxyquinolinato)]platinum(IV),[Pt(CQ)2Cl2]�KCl (3)

Red solid. Anal. Calc. (C8H10Cl6I2N2O2Pt): C, 22.81; H, 1.06; N,2.96. Found: C, 22.71; H, 0.91; N, 2.79%. FW: 947.89 g mol�1. IR(KBr, cm�1): m(C@N) 1552 m, m(C–O) 1115 m, q(py) 687 m. IR(CsI/Nujol, cm�1): m(M–Npy) 346 m, m(M–O) 339 m, m(M–Cl) n/o,g(M-O) n/o. 1H NMR [-d6, d (ppm), J (Hz)] main signals: 8.97–8.95 [d, 1H, H(2), 4.04], 7.79–7.73 [m, 1H, H(3), 4.16], 8.50–8.46[d, 1H, H(4), 8.49], 7.98 [s, 1H, H(6)], 11.04 [s, 1H, (OH)]. 13CNMR [50 MHz, DMSO-d6, d (ppm)] main signals: 149.61 [C(2)],123.38 [C(3)], 134.90 [C(4)], 125.63 [C(5)], 132.97 [C(6)], 78.92[C(7)], 153.51 [C(8)], 137.50 [C(9)], 119.37 [C(10)]. KM = 16.6 X�1 -cm2 mol�1 in DMF. Melting point: >300 �C. n/o = non-observed

2.3. X-ray crystallography

Upon slow evaporation of [Bi(HCQ)2(H2O)Cl3] (1) and [Pt(CQ)2]-�2KCl (2) in 9:1 acetone/DMSO crystals of [Bi(CQ)3] (1a) and[Pt(CQ)(DMSO)Cl] (2a) were obtained. The crystal structures of1a and 2a were determined using single-crystal X-raydiffractometry.

A summary of the crystal data, data collection details andrefinement results for 1a and 2a is listed in Table 1. Moleculargraphics and packing figures were prepared using ORTEP [16] andMERCURY [17], respectively.

2.4. Antimicrobial activity

Antibacterial activity was evaluated by minimum inhibitoryconcentration (MIC) using the macrodilution test [18]. S. aureusATCC 6538 and P. aeruginosa ATCC 27853, stored in Mueller Hiltonbroth were sub cultured for testing in the same medium and grownat 37 �C. Then the bacterial cells were suspended, according to theMcFarland protocol [19], in saline solution, to produce a suspen-sion of about 105 CFU mL�1 (colony-forming units per mL). Serialdilutions of the test compounds, previously dissolved in dimethyl-sulfoxide (DMSO), were prepared in test tubes to final concentra-tions of 512, 256, 128, 64, 32, 16, 8, 4, 2, and 1 lg mL�1; 100 lLof a 24 h old inoculum were added to each tube. The MIC, definedas the lowest concentration of the test compound which inhibitsthe visible growth after 20 h, was determined visually after incuba-tion for 20 h at 37 �C. Tests using tetracycline as reference andDMSO as negative control were carried out in parallel. All testswere performed in triplicate with full agreement between results.

Antifungal activity was also evaluated by minimum inhibitoryconcentration (MIC) using the macrodilution test [20]. C. albicansATCC 10231, stored in Sabouraud broth, was sub cultured for test-ing in the same medium and grown at 35 �C. Then the yeast cellswere suspended, according to the McFarland protocol [20], in sal-ine solution, to produce a suspension of about 105 CFU mL�1. Serial

Table 1Crystal data and structure refinement for [Bi(CQ)3] (1a) and [Pt(CQ)(DMSO)Cl] (2a).

Identification code (1a) (2a)

Empirical formula C27H12BiCl3I3N3O3 C11H10Cl2INO2PtSFormula weight (g mol�1) 1122.43 613.15T (K) 150(2) 270(2)Wavelength (Å) 0.71073 0.71073Crystal system monoclinic monoclinicSpace group P21/c P21/cUnit cell dimensionsa (Å) 12.3214(2) 7.23850(10)b (Å) 27.2835(5) 20.4599(4)c (Å) 17.9583(3) 20.2428(3)a (�) 90 90b (�) 102.753(2) 93.078(2)c (�) 90 90V (Å3) 5888.13(17) 2993.61(8)F(000) 4096 2240Z 8 8Dcalc (Mg m�3) 2.532 2.721Absorption coefficient (mm�1) 9.442 11.928Theta range for data collection (�) 2.76–26.37 2.82–26.37Index ranges �15 6 h 6 15,

�34 6 k 6 34,�22 6 l 6 22

�9 6 h 6 9,�25 6 k 6 25,�25 6 l 6 25

Reflections collected 112788 45129Independent reflections 12031

[R(int) = 0.0740]6092[R(int) = 0.0555]

Completeness to theta = 26.37� 99.9% 99.9%Data/restraints/parameters 12031/0/721 6092/0/347Goodness-of-fit (GOF) on F2 1.022 1.187Final R indices [I > 2r(I)] R1 = 0.0365,

wR2 = 0.0756R1 = 0.0466;wR2 = 0.0912

R indices (all data) R1 = 0.0534,wR2 = 0.0819

R1 = 0.0577;wR2 = 0.0946

Largest difference peak and hole(e �3)

2.652 and �2.460 1.777 and �1.318

30 K.S.O. Ferraz et al. / Polyhedron 63 (2013) 28–35

dilutions of the test compounds, previously dissolved in DMSO,were prepared in test tubes to final concentrations of 512, 256,128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.3 and 0.1 lg mL�1. 100 lL of a24 h old inoculum were added to each tube. The MIC was deter-mined visually after incubation for 24 h at 35 �C. Tests using fluco-nazole as reference and DMSO as negative control were carried outin parallel. All tests were performed in triplicate.

2.5. Cytotoxic activity

2.5.1. MaterialsCisplatin, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazo-

lium bromide) (MTT), RPMI-1460, and L-glutamine were purchasedfrom Sigma, and Antibiotic/Antimicotic Solution and fetal calf ser-um were purchased from Gibco (Grand Island, NY).

2.5.2. Cell linesJurkat (human immortalized line of T lymphocyte) and HL-60

(wild type human promyelocytic leukemia) cell lines were kindlygiven by Dr. Gustavo Amarante-Mendes (São Paulo University,Brazil). All lineages were maintained in the logarithmic phase ofgrowth in RPMI 1640 medium supplemented with 100 U mL�1

penicillin and 100 lg mL�1 streptomycin (GIBCO BRL, Grand Island,NY) enriched with 2 mM of L-glutamine and 10% of fetal bovineserum. All cultures were maintained at 37 �C in a humidified incu-bator with 5% CO2 and 95% air. The media were changed twiceweekly and they were regularly examined.

2.5.3. Evaluation of the cytotoxic effect against human leukemia celllines

HL-60 cell lineage was inoculated at 50,000 cells per well whileJurkat cell lineage was inoculated at 100,000 cells per well. The

plates were pre-incubated for 24 h at 37 �C to allow adaptationof cells prior to the addition of the test compounds. Freshly pre-pared solutions of the different compounds were tested at10 lmol L�1. Subsequently, the plates were inoculated for 48 h inan atmosphere of 5% CO2 and 100% relative humidity. Controlgroups included treatment with 0.1% DMSO (negative control)and 10 lmol L�1 of cisplatin (positive control). Cell viability wasestimated by measuring the rate of mitochondrial reduction ofMTT. All substances were dissolved in DMSO, prior to dilution.All compounds were tested in triplicate, in three independentexperiments.

2.5.4. Evaluation of the cytotoxic effect against human peripheralblood mononuclear cells

Human peripheral blood mononuclear cells (PBMC) were sepa-rated according to the method described by Gazzinelli et al. [21]. Inbrief, PBMC samples were obtained through agreement with MinasGerais Hematology and Hemotherapy Center Foundation –HEMOMINAS (protocol No. 105/2004) from healthy adult volun-teers of both sexes by centrifugation of heparinized venous bloodover Ficoll cushion (Sigma–Aldrich, St. Louis, MO). Mononuclearcells were collected from the interphase after Ficoll separationand washed three times in RPMI-1640 before further processing.The cells were washed and the cell density was adjusted to2.5 � 106 cells mL�1. 100 lL of this suspension (250,000 cells) wereadded to a 96-well plate and incubated for 24 h in the presence of2.5 lg mL�1 of phytohemaglutinin (PHA) for stabilization. Afterthis period, cells were incubated in the presence of different con-centrations of selected compounds (from 100 to 0.00001 lM) for48 h, at 37 �C in a humidified atmosphere containing 5% CO2. Thecells were maintained in culture medium containing RPMI (Sigma)supplemented with 5% normal human serum AB Rh+, previouslyinactivated, 2 mM L-glutamine and an antibiotic/anti-mycotic solu-tion containing 1000 U mL�1 penicillin, 1000 lg mL�1 streptomy-cin; 25 lg mL�1 fungisone (GIBCO/BRL, Grand Island, NY) wereadded to control fungal and bacterial contamination.

All experiments were performed in triplicate using cisplatin aspositive control. We conducted a solvent control (DMSO) at thesame concentration of the tested samples (less than 0.5%). Prolifer-ation and viability were evaluated by the MTT assay.

2.5.5. In vitro cell viability assay — MTT assayThe MTT assay is a standard colorimetric assay, in which mito-

chondrial activity is measured by splitting tetrazolium salts withmitochondrial dehydrogenases in viable cells only [22]. Briefly,after 4 h of the end of incubation of cells with different compounds,20 lL of MTT solution (2.5 mg mL�1 in phosphate-buffered saline)were added to each well, the supernatant was removed and 200 lLof 0.04 M HCl in isopropyl alcohol were added to dissolve the for-mazan crystals. The optical densities (OD) were measured in aspectrophotometer at 570 nm. Controls included drug-containingmedium (background) and drug-free complete medium. Drug-freecomplete medium was used as control (blank) and was treated inthe same way as the drug-containing media. Results were ex-pressed as percentage of cell proliferation, comparing with 0.1%DMSO control and were calculated as follows: viability (%) = (meanOD treated – mean OD background)/mean OD untreated cultured,i.e. 0.1% DMSO – mean OD blank wells) � 100. Interactions of com-pounds and media were estimated on the basis of the variationsbetween drug-containing medium and drug-free medium to es-cape from false-positive or false-negative [23].

2.5.6. DNA fragmentation assayThe sub-diploid DNA content was determined for the quantifi-

cation of cellular DNA fragmentation, which is characteristic ofapoptosis. This experiment was used as a predictive method of

K.S.O. Ferraz et al. / Polyhedron 63 (2013) 28–35 31

the pro-apoptotic potential of the compounds. The sub-diploidDNA content was evaluated in HL-60 and Jurkat cells subjectedto different treatments with selected compounds, according tothe method described by Nicoletti et al. [24]. For this study,200,000 cells in suspension treated or not with compounds werecentrifuged for 5 min at 200 g, 4 �C. After centrifugation cells werere-suspended in hypotonic fluorochrome solution-HFS[50 lg mL�1 propidium iodide – PI (Sigma), 0.1% sodium citrate(Sigma) and 0.1% Triton X-100 (Sigma)]. The samples in HFS wereincubated at 8 �C for 4–8 h and immediately taken to the flowcytometer. Incubation of cells with a hypotonic fluorochrome solu-tion (HFS) leads to the weakening of the cell membrane by the ac-tion of Triton-X100 and hypotonic shock causes its lysis. Thenuclear material becomes accessible to PI, which will intercalatein nuclear DNA. Normal cells have DNA content equal to 2n or4n, depending on the phase of the cell cycle. In apoptotic cellsthe DNA content is lower than 2n, because the fragments of smallmolecular weight leave the inner core, which is the sub-diploidphase. The sub-diploid DNA content was determined by the Cell-Quest program (Becton Dickinson).

2.5.7. Statistical analysisAll experiments were performed in at least three replicates per

compound and results shown are the average of three independentexperiments. Data are represented as mean ± SEM. Significancewas tested by the Student’s t-test.

2.6. Studies of interactions with supercoiled plasmid DNA

Studies of interactions with supercoiled plasmid DNA by aga-rose gel electrophoresis were carried out to identify the modifica-tions resulting from the interaction of complexes with DNA. Thus,423 ng of purified plasmid DNA-pUC 19 from Escherichia coli(Sigma) were incubated with compounds (HCQ, 1, 2, 3, K2PtCl4,K2PtCl6 and cisplatin) at 50 lM in Tris–HCl buffer (NaCl 50 mM,Tris–HCl 5 mM, pH 7.2). The mixture was incubated at 37 �C for24 h. Thereafter, the reactions were quenched by adding 5 lL ofthe loading buffer solution (50 mmol L�1 Tris, pH 7.2, 0.01% bromo-phenol blue, 50% glycerol, and 250 mmol L�1 EDTA). The sampleswere analyzed by 1% agarose gel electrophoresis in 0.5X TBE bufferfor 1 h 30 min at 75 mV. The gel was stained after electrophoresisin 0.5X TBE buffer with 2.5 lg mL�1 ethidium bromide for 15 minand visualized by UV light.

3. Results and discussion

3.1. Characterization of the bismuth(III) and platinum(II, IV) complexes

Microanalyses and molar conductivity data are compatible withthe formation of [Bi(HCQ)2(H2O)Cl3] (1), [Pt(CQ)2]�2KCl (2) and[Pt(CQ)2Cl2]�KCl (3). Molar conductivity data suggest that the com-pounds are non-electrolytes [complex(1)] or weak electrolytes[complexes (2–3)]. The presence of KCl in 2 and 3 could have arisenfrom the precursor salts (see Section 2.2), contributing to the molarconductivity. In fact the presence of KCl in a palladium(II) complexwith D-penicilamine had been previously reported [25].

In complex (1) two neutral ligands, a water molecule and threechloride ions are attached to the bismuth(III) center. The infraredspectrum of 1 supported the presence of a coordination water mol-ecule (see Section 2.2.1). In complex (2) two anionic ligands are at-tached to a platinum(II) center while in complex (3) two anionicligands and two chloride ions are attached to a platinum(IV) cen-ter. In both complexes co-crystallized KCl was observed.

3.2. Spectroscopic characterization

The vibration attributed to m(O–H) at 3070 cm�1 in the spec-trum of free clioquinol shifts to 3046 cm�1 in the spectrum of com-plex (1), suggesting the presence of a neutral ligand [26]. Thisabsorption is absent in the spectrum of 2 and 3. In addition, anew absorption at 1622 cm�1 was observed in the spectrum ofcomplex (1), which is compatible with the presence of a coordina-tion water molecule.

The absorption at 1084 cm�1 attributed to the m(C–O) vibrationin the spectrum of the free base shifts to 1080 cm�1 in complex (1)and to 1114 and 1115 cm�1 in the spectra of complexes (2) and (3),indicating that the oxygen is probably not involved in coordinationin 1 and that in 2 and 3 coordination through the oxygen occurs[26–28].

The vibration attributed to m(C@N) at 1576 cm�1 in the infraredspectrum of free clioquinol shifts to 1552–1543 cm�1 in the spec-tra of complexes (1–3), indicating coordination through the nitro-gen [7,29]. The in-plane deformation mode of the pyridine ring at616 cm�1 in the spectrum of free clioquinol shifts to 653–687 incomplexes (1–3) in agreement with coordination of the nitrogen[7,29].

Absorptions at 268 and 222 cm�1 in the spectrum of complex(1) were attributed to the m(Bi–N) and m(Bi–Cl) vibrations, respec-tively [30]. In the spectrum of 2 m(Pt–N) and m(Pt–O) vibrations ap-peared at 345 and 338 cm�1 respectively while in the spectrum of3 these vibrations were observed at 346 and 339 cm�1, respec-tively [30]. Hence the infrared spectra indicate coordinationthrough the nitrogen in 1 and through the N–O chelating systemin 2 and 3.

The NMR spectra of the compounds were recorded in DMSO-d6.The 1H resonances were assigned on the basis of chemical shiftsand multiplicities. The carbon type (C, CH) was determined byusing distortionless enhancement by polarization transfer (DEPT135) experiments. The assignments of the protonated carbonswere made by 2D hetero-nuclear multiple quantum coherenceexperiments (HMQC).

In the 1H NMR spectra of 1–2 the signals of all hydrogens under-go significant shifts in relation to their positions in free clioquinolwhile in 3 small shifts were observed upon coordination. The broadsignal of O–H was observed at d 11.07 ppm in the spectrum ofcomplex (1). This signal is absent in the spectra of complexes (2)and (3). Hence the ligand is protonated at the oxygen in 1 anddeprotonated in 2 and 3.

The 1H NMR spectrum of 1 obtained in DMSO-d6 after 2 weeksdoes not show the signal of O–H, indicating deprotonation withtime in this solvent. Hence release of three HCl molecules probablyoccurred in the DMSO-d6 solution. The spectrum of 2 and 3 do notchange with time.

In the 13C NMR spectra of 1–2 the signals of the pyridine car-bons and those of the phenol ring undergo significant shifts uponcomplexation, while in 3 only small shifts were noticed uponcoordination.

3.3. X-ray crystallography

The crystal structures of [Bi(CQ)3] (1a) and [Pt(CQ)(DMSO)Cl](2a) were determined. As mentioned before 1a and 2a were ob-tained from slow evaporation of [Bi(HCQ)2(H2O)Cl3] (1) and[Pt(CQ)2]�2KCl (2) in 1:9 DMSO:acetone. During crystallizationcomplexes (1) and (2) underwent reactions in solution leading tothe formation of 1a and 2a. As suggested by the NMR data depro-tonation of clioquinol in 1 occurs in DMSO solution, with the re-lease of three molecules of HCl. Upon re-crystallization of 2release of a clioquinol ligand takes place upon coordination ofDMSO to the platinum(II) center, together with complexation of

32 K.S.O. Ferraz et al. / Polyhedron 63 (2013) 28–35

a chloride from KCl. Hence the potassium salt of clioquinolate isprobably formed.

ORTEP drawings of 1a and 2a are shown in Fig. 2. Selected bondlengths and angles are given in Table 2, as well as bond distancesand angles in the crystal structure of clioquinol [31] for the sakeof comparison.

Both 1a and 2a crystallize in the monoclinic system, P21/c, withtwo molecules of the complex in the asymmetric unit. In 1a threeanionic clioquinol ligands are coordinated to bismuth(III), while in2a only one anionic clioquinol ligand is attached to the platinum(II)center, together with an S-atom of a DMSO molecule and a chlorideanion occupying the third and fourth coordination positions,respectively. The presence of anionic clioquinol in the complexesis confirmed by the shortening of the C8–O bond from 1.372(2) Åin HCQ to 1.314(8) Å in 1a and 1.317(11) Å in 2a.

Fig. 2. Molecular structure of [Bi(CQ)3] (1a) and [Pt(CQ)(DMSO)Cl] (2a) showing the lprobability level.

In both complexes clioquinol is attached to the metal centerthrough the N–O chelating system (see Fig. 2). As a consequencemodifications were observed in the bond angles involving the C8atom. The N1–C9–C8, C8–C7–I1 and C7–C8–C9 angles decreasefrom 118.37�, 121.55� and 120.15� respectively, in HCQ, to116.0(6)�, 116.6(5)� and 116.2(6)� (in 1a) and 115.7(9)�, 119.2(8)�and 116.0(9)� (in 2a), respectively. This coordination mode is sim-ilar to that observed in other complexes of clioquinol [15].

In 1a the bismuth(III) center is hexa-coordinated in a distortedpentagonal pyramid geometry. Due to the Lewis acidic character ofbismuth(III), additional intermolecular contacts can be establishedin the presence of donor atoms (D), which occur when the Bi� � �Ddistance is shorter than the sum of the van der Waals radii (dvWr)of these atoms [32]. Actually this effect was observed for Bi1� � �O62[d = 3.162 (5) Å], Bi1� � �O42 [d = 3.160 (5) Å], Bi2� � �O11 [d = 3.008

abelling scheme of the non-H atoms and their displacement ellipsoids at the 50%

Table 2Bond lengths [Å] and angles [�] for clioquinol (HCQ), [Bi(CQ3] (1a) and [Pt(CQ)(DMSO)Cl] (2a).

Atoms HCQ Atoms (1a) Atoms (2a)

Bond lengths (Å)N(1)–C(2) 1.3092(9) N(11)–C(12) 1.317(9) C(2)–N(1) 1.314(14)N(1)–C(9) 1.360(0) N(11)–C(19) 1.364(9) C(9)–N(1) 1.388(13)C(8)–C(9) 1.3986(9) C(18)–C(19) 1.444(9) C(8)–C(9) 1.421(14)C(7)–C(8) 1.358(1) C(17)–C(18) 1.378(9) C(7)–C(8) 1.380(14)Cl(1)–C(5) 1.754(3) Cl(1)–C(15) 1.738(7) C(5)–Cl(1) 1.757(11)I(1)–C(7) 2.103(2) I(1)–C(17) 2.095(7) C(7)–I(1) 2.092(10)O(1)–C(8) 1.372(2) O(11)–C(18) 1.314(8) C(8)–O(1) 1.317(11)– – Bi(1)–O(11) 2.302(4) N(1)–Pt(1) 2.026(9)– – Bi(1)–O(21) 2.312(5) O(1)–Pt(1) 2.024(7)– – Bi(1)–O(31) 2.289(5) S(1)–Pt(1) 2.185(3)– – Bi(1)–N(11) 2.612(5) Cl(2)–Pt(1) 2.342(3)– – Bi(1)–N(21) 2.333(6) – –– – Bi(1)–N(31) 2.540(6) – –Angles (�)N(1)–C(9)–C(8) 118.37 N(11)–C(19)–C(18) 116.0(6) N(1)–C(9)–C(8) 115.7(9)C(2)–N(1)–C(9) 119.16 C(12)–N(11)–C(19) 118.6(6) C(2)–N(1)–C(9) 118.9(9)C(8)–C(7)–I(1) 121.55 C(18)–C(17)–I(1) 116.6(5) C(8)–C(7)–I(1) 119.2(8)C(7)–C(8)–C(9) 120.15 C(17)–C(18)–C(19) 116.2(6) C(7)–C(8)–C(9) 116.0(9)

– C(12)–N(11)–Bi(1) 128.3(5) C(2)–N(1)–Pt(1) 130.0(8)– C(18)–O(11)–Bi(1) 123.1(4) C(8)–O(1)–Pt(1) 112.1(6)– O(11)–Bi(1)–N(11) 66.61(16) O(1)–S(1)–Pt(1) 177.7(2)– O(21)–Bi(1)–N(21) 70.80(18) N(1)–Pt(1)–O(1) 82.2(3)– O(31)–Bi(1)–N(31) 67.69(18) N(1)–Pt(1)–S(1) 98.0(3)– O(31)–Bi(1)–O(11) 72.68(16) O(1)–Pt(1)–S(1) 177.7(2)– O(11)–Bi(1)–N(31) 139.99(17) N(1)–Pt(1)–Cl(2) 172.3(3)– O(11)–Bi(1)–O(21) 134.62(16) O(1)–Pt(1)–Cl(2) 90.2(2)– O(31)–Bi(1)–N(21) 76.35(18) S(1)–Pt(1)–Cl(2) 89.66(10)

Fig. 3. (A) View of the crystal structure of [Bi(CQ)3] (1a) showing the non-centrosymmetric dimmer. (B) Crystal packing of [Pt(CQ)Cl(DMSO)] (2a) along the crystallographica-axis. The intermolecular interactions are indicated by dashed lines.

Table 3Minimum inhibitory concentration (MIC) values for clioquinol and its complex withbismuth(III) (1).

Compound S. aureus (lM) P. aeruginosa (lM) C. albicans (lM)

Clioquinol 23.7 33.7 4.3(1) 7.3 110.7 1.4Fluconazole – – 72.7Tetracicline 0.5 66.9 –

K.S.O. Ferraz et al. / Polyhedron 63 (2013) 28–35 33

(5)] and Bi2� � �I1 [d = 3.6599(6) Å] (the values for the sum of vander Waals radii for Bi–O and Bi–I are 3.9 and 4.5 Å, respectively)[33]. These, together with the Bi1� � �Bi2 interaction [d = 3.9701(3) Å], sum of van der Waals radii = 4.8 Å] lead to the formationof a non-centrosymmetric dimmer in the asymmetric unit of 1a[33].

It is worth noticing that p–p stacking interactions play animportant role in the crystal packing of the complexes. Suchinteractions can be observed in 1a between the aromatic rings[centroid–centroid c.a. 3.5 Å] favoring the formation of thenon-centrosymmetric dimmer (see Fig. 3). This also occurs be-tween clioquinol ligands along the crystallographic a-axis in 2awith values of the centroid–centroid of c.a. 3.65 Å which makesthe two platinum(II) centers relatively near to each other[d(Pt� � �Pt) = 4.0132(6) Å].

3.4. Antimicrobial activity

Antimicrobial activity of complex (1) was evaluated against S.aureus, P. aeruginosa and C. albicans (see Table 3).

Table 4Cytotoxic activity (IC50) of clioquinol and its bismuth(III) and platinum(II, IV)complexes on HL60 and Jurkat leukemia cells.

Compound IC50 (lM) 95% Confidence interval in parenthesis

HL60 Jurkat

HCQ 14.1 (11.3–17.6) 20.2 (13.7–29.6)1 13.7 (10.9–17.1) 8.9 (2.8–27.9)2 5.9 (4.4–7.9) 3.7 (2.3–6.1)3 6.1 (5.1–7.4) 10.8 (5.7–20.3)K2PtCl4 >100 >100K2PtCl6 >100 >100Cisplatin 0.06 (0.04–0.12) 17.5 (8.9–34.5)

Table 5Inhibition of viability (%) of human PBMC cells of the cytotoxic compounds.

Compound % inhibition of viability Concentration (lM)

HCQ 5.8 (3.7 to 15.4) 14(1) 56 (52.8 to 59.2) 9(2) 40.8 (18.9 to 52.8) 4(3) 20.4 (14.1 to 26.8) 6Cisplatin 37 (35.8 to 38.2) 0.06

34 K.S.O. Ferraz et al. / Polyhedron 63 (2013) 28–35

Clioquinol was more active against P. aeruginosa and C. albicansthan tetracycline and fluconazole used as controls. Upon coordina-tion to bismuth(III) the antimicrobial activity improved against S.aureus and C. albicans. Complex (1) was seventy-fold more active

Fig. 4. Agarose gel electrophoresis of plasmid DNA pUC-19 treated without (control) an(3), K2PtCl4, K2PtCl6 and cisplatin (Cisp) at 50 lM in Tris–HCl buffer (NaCl 50 mM, Tris–

Fig. 5. DNA fragmentation induced by HCQ, its complexes (1–3) and cisplatin (Cisp) at 1DNA fragmentation compared to control (cells treated with DMSO 0.1%).

than fluconazole against C. albicans. Hence, complexation reveledto be a good tool to make clioquinol more active in this case.

3.5. Cytotoxic activity against leukemia cells

Table 4 shows the IC50 values of clioquinol and its bismuth(III)and platinum(II, IV) complexes against HL60 and Jurkat leukemiacells. Clioquinol proved to be cytotoxic to the studied leukemiacells. Upon coordination to platinum(II, IV) cytotoxicity improvedagainst both cell lines. Coordination to bismuth(III) did not resultin significant modification of clioquinol’s cytotoxic effect.

Complexes (2) and (3) were more active than clioquinol againstHL-60 cells. Complex (2) also revealed to be the most cytotoxiccompound against Jurkat cells being almost fivefold more activethan cisplatin in the assayed conditions.

The platinum(II, IV) precursors (K2PtCl4 and K2PtCl6) were notcytotoxic against both cell lineages. However, coordination of clio-quinol to platinum(II, IV) showed to be a good strategy to increaseits cytotoxic activity against the studied leukemia cell lineages.Coordination to platinum(II) seemed to be better than to plati-num(IV) probably because to exhibit cytotoxicity platinum(IV)has to be reduced to platinum(II) inside the cell.

The cytotoxicity of the studied compounds was also evaluatedin normal cells (human PBMC cells). We used the lowest concen-tration of each compound that showed cytotoxic effect against leu-kemia cells to evaluate the percentage of proliferation inhibition inPBMC cells (see Table 5). Complex (2) presented high cytotoxicity

d with compounds HCQ, [Bi(HCQ)2(H2O)Cl3] (1), [Pt(CQ)2]�2KCl (2), [Pt(CQ)2Cl2]�KClHCl 5 mM, pH 7.2) incubated at 37 �C for 24 h.

0 lM in HL-60 and Jurkat cells. Data are expressed as mean ± SEM of percentage of

K.S.O. Ferraz et al. / Polyhedron 63 (2013) 28–35 35

against both investigated leukemia cell lines, showing greatercytotoxic effect than cisplatin against Jurkat cells. However itproved to be very toxic to PBMC cells. Complex (3) revealed tobe less cytotoxic than 2 against Jurkat leukemia cells and as cyto-toxic as 2 against HL-60 cells. Complex (3) was the least toxicamong the tested complexes (including cisplatin) against PBMCcells.

3.6. Studies of interactions with supercoiled plasmid DNA

The effect of HCQ, 1–3, K2PtCl4, K2PtCl6 and cisplatin on DNAconformation was evaluated by the electrophoretic mobility ofplasmid DNA after association with the compounds. At the em-ployed concentration (50 lM) the compounds did not alter theelectrophoretic mobility of DNA, except cisplatin that interactedsignificantly with DNA (Fig. 4). Clioquinol did not modify the elec-trophoretic mobility of DNA as well. Hence our investigation on theinteraction of 1–3 with DNA revealed either that these compoundsdo not interact with the DNA bases or that interaction, if it occurs,does not lead to stable adducts at 50 lM.

3.7. Investigation on the pro-apoptotic potential of clioquinol andcomplexes (1–3)

Induction of apoptosis, or programmed cell death in cancer cellsis thought to be fundamental to the success of treatments for can-cer [34]. To determine if the observed cytotoxic effects of clio-quinol and its complexes were associated with other cell deathmechanisms, we evaluated the potential of these compounds to in-duce cell death by apoptosis. Cells with an increase of subdiploidDNA content, thus with fragmented DNA, were classified as apop-totic cells. Fig. 5 shows the subdiploid DNA content for both celllineages after incubation with clioquinol and its complexes.

Clioquinol and complex (1) did not exert a strong effect on DNAfragmentation in HL-60 cells, while complexes (2) and (3) inducedmoderate effect in comparison with cisplatin used as positive con-trol. None of the studied compounds showed a pro-apoptotic effectin Jurkat cells.

These results suggest that coordination of clioquinol to bis-muth(III) did not significantly alter clioquinol’s cytotoxic activityin HL-60 cells while coordination to platinum(II, IV) resulted in in-creased cytotoxic effect. Although clioquinol and 1 did not show apro-apoptotic effect, complexes (2) and (3) presented moderatepro-apoptotic activity in HL-60 cells. Apoptosis induction is proba-bly not the main mode of cytotoxic activity of clioquinol and itscomplexes (1–3) against HL-60 and Jurkat cells.

Acknowledgement

This work was supported by Fapemig, Capes, CNPq and INCT-INOFAR (Proc. CNPq 573.364/2008-6).

Appendix A. Supplementary data

CCDC 929498 and 929502 contain the supplementary crystallo-graphic data for [Bi(CQ)3] (1a) and [Pt(CQ)(DMSO)Cl] (2a), respec-tively. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the CambridgeCrystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ,UK; fax: +44 1223 336 033; or e-mail: deposit@ccdc.cam.ac.uk.

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