Polyhedron 79 (2014) 197–206

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Synthesis and biological evaluation of ternary silver compounds bearingN,N-chelating ligands and thiourea: X-ray structure of [{Ag(bpy)(l-tu)}2](NO3)2 (bpy = 2,20-bipyridine; tu = thiourea)

http://dx.doi.org/10.1016/j.poly.2014.05.0040277-5387/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding authors. Tel.: +55 16 3301 9626; fax: +55 16 3322 7932(A.V.G. Netto and D.F. Segura).

E-mail addresses: daniel@iq.unesp.br (D.F. Segura), adelino@iq.unesp.br(A.V.G. Netto).

Daniel F. Segura a,⇑, Adelino V.G. Netto a,⇑, Regina C.G. Frem a, Antonio E. Mauro a, Patrícia B. da Silva a,José A. Fernandes b, Filipe A.A. Paz b, Amanda L.T. Dias c, Naiara C. Silva c, Eduardo T. de Almeida c,Marcos J. Marques c, Letícia de Almeida c, Karina F. Alves c, Fernando R. Pavan d, Paula C. de Souza d,Heloisa B. de Barros d, Clarice Q.F. Leite d

a Departamento de Química Geral e Inorgânica, Instituto de Química de Araraquara, UNESP – Univ Estadual Paulista, P.O. Box 355, Araraquara, São Paulo 14801–970, Brazilb Department of Chemistry, CICECO, Campus Universitário de Santiago, University of Aveiro, 3810-193 Aveiro, Portugalc UNIFAL/MG-Universidade Federal de Alfenas, CEP 37130-000 Alfenas, MG, Brazild Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas de Araraquara, UNESP – Univ Estadual Paulista, P.O. Box 502, Araraquara, São Paulo 14801–902, Brazil

a r t i c l e i n f o a b s t r a c t

Article history:Received 27 January 2014Accepted 5 May 2014Available online 11 May 2014

Keywords:Silver(I)1,10-PhenanthrolineAntileishmanial activityTuberculosisAntifungal and antibacterial activity

Compounds [{Ag(phen)(l-tu)}2](NO3)2 (1), [{Ag(phen)(l-tu)}2](CF3SO3)2 (2), [{Ag(bpy)(l-tu)}2](NO3)2 (3)(where phen = 1,10-phenanthroline; bpy = 2,20-bipyridine; tu = thiourea) were prepared by reacting theappropriate AgX salt (X� = NO3

�, CF3SO3�), the N,N-chelating ligand (phen or bpy) and thiourea in a ca.

1:1:2 M ratio, respectively. The silver(I) complexes were characterized by elemental analysis, infrared(IR), 1H and 13C NMR spectroscopies, MS/ESI and conductivity measurements. The IR and NMR data wereconsistent with the presence of chelating phen (1 and 2) and bpy (3) ligands and demonstrated theS-coordination mode of thiourea. The crystal and molecular structures of compound [{Ag(bpy)(l-tu)}2](NO3)2 (3) were determined by single-crystal X-ray diffraction. The complexes 1–3 were screened fortheir in vitro antimycobacterial (Mycobacterium tuberculosis), antileishmanial (Leishmania (L.) amazonensis),antibacterial (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa), antifungal activities(Candida albicans, Candida tropicalis, Candida krusei).

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

An important medical problem to be overcome is the resistanceof pathogenic microorganisms to classical antibiotics, leading to aworldwide demand for the discovery of new antimicrobial agents[1]. For instance, Tuberculosis (TB) is a highly contagious anddeadly disease caused by the bacillus Mycobacterium tuberculosis(MTB). This disease is spread in the air when people infected withactive pulmonary TB expel bacteria via droplets from their throatand lungs [2]. This disease is still a serious worldwide public healthproblem and infects approximately 30% of the global population aswell as a leading cause of morbidity and mortality in developingcountries [3]. In 2011, the World Health Organization (WHO) esti-mated 8.7 million incident cases and a total of approximately

1.4 million people died of TB globally [2]. Despite the advances inthe field of the antitubercular chemotherapy, the recent outbreakof multidrug-resistant strains and its opportunistic coinfectionwith the human immunodeficiency virus (HIV) highlights theurgent need for new effective anti-TB drugs and for alternativechemotherapy regimens [4,5]. Besides Tuberculosis, Leishmaniasisrepresents one of the neglected tropical diseases (NTDs), caused byintracellular protozoan parasites from the genus Leishmania, thataffect million people in wide world [6,7]. The digenetic life cycleof Leishmania consists of motile, flagellated, extracellular prom-astigotes form in the gut of sand fly vector that infects mammalianhost and transform into nonmotile, nonflagellated amastigotesform, which survive and multiply within phagolysosomal compart-ment of macrophages [8]. Moreover, the leishmaniasis treatmentremains difficult, since the available drugs have shown to be highlytoxic and cases of resistance have emerged.

Within this context, significant efforts aimed at designing newmetal-based compounds for diagnostic and/or therapeutic useshave been stimulated by the success of the anticancer agent

198 D.F. Segura et al. / Polyhedron 79 (2014) 197–206

cisplatin [9]. Particularly, silver and its derivatives possess remark-able antimicrobial properties and for this reason they have longbeen utilized for medicinal purposes with no known effect uponthe mammalian cell membrane and limited toxicity to humans[10]. For instance, silver nitrate has been used to prevent ophthal-mianeonatorum in newborns or to treat skin ulcers, postsurgicalwounds, and suppurating wounds [11]. Silver sulfadiazine,introduced in the late 1960s, is still one of the most effective top-ical burn treatments. It has been observed that the silver ions arethe responsible for the bactericidal activity [12–14]. Despite thefact that the mechanisms of its antimicrobial action are notcompletely understood, some possible mechanisms for inhibitionby the aqueous silver(I) ion have been suggested: (i) interferencewith electron transport, (ii) binding to DNA, and (iii) interactionwith cell membrane [15].

Silver coordination compounds have attracted considerableinterest since the antimicrobial activity and other desirable proper-ties can be fine-tuned by varying the number and type of ligandspresent on the coordination sphere [10,16–18]. According toNomiya et al. [19–23], the main targets for the inhibition of bacte-ria and yeast by silver(I) complexes are proteins bearing sulfurdonor atoms. In fact it is well known that N,S-donors ligands playan important role in the coordination of metals at the active sites ofseveral metallobiomolecules [24]. In addition, the type of the coor-dinated donor atoms and the ease of ligand replacement appear tobe the key factors to be considered during the molecular design ofactive silver(I) complexes. Moreover, if the ligands themselvesexhibit antimicrobial activities, these biological properties can beenhanced with the coordination to the metal centre [25].

Among the N-donor ligands suitable to afford new activesilver(I) complexes, 1,10-phenanthroline (phen) and its derivativesrepresent a good choice because they possess the ability to act on awide variety of biological functions [26]. In particular, silver com-plexes incorporating this chelating ligand were found to be extre-mely active in vitro against pathogenic microbes. Compounds[Ag(phen)2]ClO4 and [Ag2(phen)3(mal)]�2H2O (malH2 = malonicacid) inhibited the growth of C. albicans by ca. 95% at aconcentration of 5 lg�mL�1 by damaging mitochondrial functionand uncoupling respiration [26]. Likewise, Coyle et al. [27]reported that the complex [Ag(phendio)2]ClO4 (phendio = 1,10-phenanthroline-5,6-dione) displayed a MIC (minimum inhibitoryconcentration) value of 0.5 lg mL�1 against this same fungus,causing extensive, non-specific DNA cleavage, disrupting celldivision and inducing gross distortions in fungal cell morphology.Silver complexes of the type [Ag(L)2]NO3 (L = polypyridyl ligands)have also been shown to be biologically active against Leishmaniamexicana by interacting with DNA [28].

Following our interest on the synthesis and biological activity ofmetal-based compounds [29–36], we report the preparation andspectroscopic characterization (IR, 1H and 13C NMR) of thecomplexes [{Ag(phen)(l-tu)}2](NO3)2 (1), [{Ag(phen)(l-tu)}2](CF3SO3)2 (2), [{Ag(bpy)(l-tu)}2](NO3)2 (3) (where phen = 1,10-phenanthroline; bpy = 2,20-bipyridine; tu = thiourea). Single crystalX-ray diffraction studies of compound 3 are reported herein. Allcomplexes were evaluated in vitro for their antimycobacterial(Mycobacterium tuberculosis H37Rv ATCC – 27194), leishmanicidal(Leishmania (L.) amazonensis) antibacterial (Staphylococcus aureus,Escherichia coli, Pseudomonas aeruginosa), and antifungal (Candidaalbicans, Candida tropicalis, Candida krusei) activities.

Our original interest in thiourea-type ligands arose as thesecompounds display remarkable activity against several pathogenicmicroorganisms [37]. For instances, thiourea derivatives are wellknown to possess antibacterial and antifungal activities. Dogrueret al. [38] have reported that some of thiourea-based moleculesexhibited not only promising inhibitory activity against S. aureus(MIC ranging from 2 to 4 lg mL�1) and E. coli (MIC ranging from

4 to 16 lg mL�1), but also antifungal activity against both C. albi-cans and C. parapsilosis, with a MIC value of 8 lg mL�1. Similarly,silver(I)–chitosan–thiourea compound showed a wide spectrumof effective antimicrobial activities against six species of bacteriaand molds [39]. We assumed that the incorporation of thioureain the molecular structures of silver(I) compounds may increasetheir biological activities as we observed in other thiourea–metalbased systems [40–43]. Despite the intensive work devoted tothe investigation on antimicrobial activities of silver(I) phenan-throline-based compounds, less attention has been paid to theirinhibitory effect on the growth of M. tuberculosis (MTB) and Leish-mania (L.) amazonensis.

2. Results and discussion

2.1. Synthesis considerations

The ternary silver(I) complexes [{Ag(phen)(l-tu)}2](NO3)2 (1)and [{Ag(phen)(l-tu)}2](CF3SO3)2 (2), represented in Scheme 1,have been isolated from methanol–acetonitrile mixtures byreacting the appropriate AgX salt {X� = NO3

� (1), CF3SO3� (2)},

1,10-phenanthroline (phen) and thiourea (tu) in a 1:1:2 M ratio,respectively.

Compound [{Ag(bpy)(l-tu)}2](NO3)2 (3) was obtained using asimilar procedure to that described for 1 by employing 2,20-bipyr-idine (bpy) instead of 1,10-phenanthroline. Unlike the reaction ofAgCF3SO3, phen, and tu, we could not isolate the di-metallic com-pound [Ag(bpy)(l-tu)]2(CF3SO3)2. Several attempts to synthesizethis complex were unsuccessful, yielding a clear solution with aprecipitate of Ag0.

The concentrated solutions of 1 and 2 in methanol were left atroom temperature for 24 h protected from light, providing micro-crystalline powders that were further purified by repeated wash-ing with methanol. In order to obtain suitable crystals for X-raydiffraction the supernatant solvent was submitted to further evap-oration. However, this second harvest of crystals presented differ-ent NMR spectra from compounds 1 and 2, being secondaryproducts. Ultimately, these new products were identified by theconjugation of powder and single crystal X-ray diffraction data.While the crystals from the supernatant of 1 were found to be amixture of [Ag(tu)2(l-tu)]2(NO3)2 [44] and the new compound[Ag4(phen)(l-tu)11](NO3)4�7phen [45], that from the supernatantof 2 was identified as being [Ag(phen)2](CF3SO3) [46].

Complexes are air and light stable solids and exhibit color thatvaries from white to light brown. Compounds are soluble in DMSO,sparingly soluble in CH3CN and CH3OH, and insoluble in water. Ele-mental analysis of the silver(I) complexes 1–3 showed that theircompositions have molar ratios of Ag+:L:tu:X� = 1:1:1:1 (L = bpy,phen; X = NO3

�, CF3SO3�).

2.2. Crystal description of [{Ag(bpy)(l-tu)}2](NO3)2 (3)

Compound [{Ag(bpy)(l-tu)}2](NO3)2 (3) crystallizes in the cen-trosymmetric triclinic space group Pı̄ as a dinuclear cation withtwo charges balancing nitrate ions (see Fig. 1).

The cation is composed by two crystallographically indepen-dent [Ag(bpy)(tu)] moieties, with two S-bridging thiourea ligands.Both of the silver atoms have distorted tetrahedral environmentswith angles, at the metal, ranging from 70.88(10)� to 137.33(7)�.The tetrahedral coordination consists of two nitrogen atoms (frombpy ligand), and two sulfur atoms from bridging thiourea ligands.The bridging sulfur atoms and the two silver atoms form a lozengeshaped planar Ag2S2 metallocycle, in which the medium planes ofthe organic ligands are almost perpendicular to the {Ag1–S1–Ag2–S2} central medium plane [values of 72.85(9)� and 79.44(8)� for

Scheme 1. Proposed structural units for the silver complexes 1–3.

Fig. 1. Asymmetric unit of compound 3 with most of the non-hydrogen atomsbeing represented as thermal ellipsoids drawn at the 30% probability level.Hydrogen atoms are represented as small spheres with arbitrary radii. Two oxygenatoms are disordered over three sites, being represented as thermal spheres drawnat the 30% probability level. For a detailed description of the bond lengths andangles see Table S1 in Supplementary information.

D.F. Segura et al. / Polyhedron 79 (2014) 197–206 199

bpy and 88.55(11)� and 86.48(14)� for tu]. The tu ligands areplaced at opposite sites of the {Ag1–S1–Ag2–S2} plane withC–S� � �S angles of 108.66(12)� and 110.60(11)�. The Ag2S2 core in3, as well as in other thiourea-based Ag binuclear compounds[44,47–50], is characterized by short and long Ag-S distances,narrow angles at the bridging sulfur atoms, somewhat largerangles at the silver atoms, together with a relatively large separa-tion between the bridging sulfur atoms and moderate separationbetween the silver atoms.

According to Stocker et al. [47], there is no obvious pattern inthe behavior of these Ag(l-tu)Ag bridges: by one side, the bridgingcan be made by one or two sulfur atoms, and by the other, theangle Ag–S–Ag and the Ag� � �Ag distance can vary considerably(64–140� and 2.85–5.06 Å, respectively). Since then, several other

Ag(I) compounds bearing thiourea-type ligands have beendescribed [49,51–53], but there was no change on the given limits.In compound 3 the short values for Ag–S–Ag angles of 76.01(2)�and 70.10(2)� at atoms S1 and S2, respectively, decrease theAg� � �Ag distance to 3.0910(4) Å, which is considerably shorter thantwice the van der Waals radius for silver (3.44 Å), suggesting a cer-tain degree of metal–metal interaction [51–54].

The Ag–S bonds involving the atom Ag2 are similar [Ag2–S22.5554(8) Å; Ag2–S1 2.5573(8) Å] whereas those involving atomAg1 are somewhat different [Ag1–S1 2.4611(2) Å; Ag1–S22.8140(9) Å]. The Ag1–S2 bond distance, although longer thanthe sum of the corresponding tetrahedral covalent radii (2.515 Å)[55], lies within the typical range of other dimeric or polymericsilver–thione/thionate compounds like [(AgCN)(tu)2]n [2.505(2)–2.885(3) Å] (tu = thiourea) [47], [(AgCN)2(dmtu)2]n [2.504(1)–3.143(1) Å] (dmtu = N,N0-dimethylthiourea) [47], [(tu)2Ag(l-tu)2Ag(tu)2]Cl2 [2.5235(5)–2.7926(5) Å] [49], {[Ag6(l3-pyS)4

(l4-pyS)2]n} [2.456(5)–2.959(5) Å] (pyS = pyridine-2-thiolate)[56], {[Ag5(pyS)4(pySH)BF4]n} [2.45–2.90 Å] [57], [Ag6(l2-Br)6

(l2-StpmH2)4(l3-StpmH2)2]n [2.4832(2)–2.949(2) Å] (StpmH2 =2-mercapto-3,4,5,6-tetrahydropyrimidine) [58]. The Ag–N bonddistances are in the range of 2.315(3)–2.351(3) Å and agree wellwith other silver compounds bearing chelating 2,20-bipyridine[59]. The tu ligands are placed at opposite sites of the Ag2S2 planewith C–S� � �S angles of 108.66(12)� and 110.60(11)�. All C–S bondlengths in 3 have nearly the same magnitude and are comparableto the reported mean C@S distance of 1.725 Å for metal–thioureacomplexes [60].

Hydrogen bonds in the crystal of 3 share the remarkable featureof being disposed into a layer very close to the (022) plane of theunit cell. This plane is also very close to the medium plane of the tuligands and passes through the nitrate ions. Other supramolecularinteraction present in the crystal is a weak p� � �p stacking of thebpy ligands, with distances between centromers of 3.919(2) and4.025(3) Å (See Fig. 2).

2.3. IR spectroscopy

The thiourea ligand displays the ability of bonding to transitionmetal ions via sulfur or nitrogen atoms [61,62]. Although a varietyof physical techniques have been utilized to infer the coordination

Fig. 2. Schematic representation of the supramolecular interactions present in compound 3: (a) Hydrogen bonding network as seen from the [011] direction of the unit cell.O� � �H and S� � �H hydrogen bonds are represented as pink and blue dashed lines, respectively. Some graph set motifs are highlighted as orange, green and yellow rings. Forclarity, only the A component of the disordered nitrate ion is shown, and the bpy ligands have been omitted. For geometrical details on the represented supramolecularinteractions see Table S2 in Supplementary material. (b) p� � �p stacking between adjacent organic ligands with the interactions being represented as mauve dashed lines.

200 D.F. Segura et al. / Polyhedron 79 (2014) 197–206

mode of the thiourea, IR spectroscopy continues to be one of themost widely used methods. The most important IR frequencies ofthe new silver(I) complexes along with their assignments are pre-sented in Table 1.

Table 1Selected vibrational data (cm�1) for [{Ag(phen)(l-tu)}2](NO3)2 (1), [{Ag(phen)(l-tu)}2](CF3SO3)2 (2), [{Ag(bpy)(l-tu)}2](NO3)2 (3), where phen = 1,10-phenanthro-line; bpy = 2,20-bipyridine; tu = thiourea).

1 2 Assignment 3 Assignment

3339 br 3390 br msNH2 3399 s msNH2

1643 s 1610 s dNH2 1610 s dNH2

1512 m 1513 w mCN 1564 m mCN1325 m msNO2 1322 m msNO2

1268 m mSO3

1226 m mCF3

1171 m mCF3

1086 w 1084 w mCN 1095 w mCN846 s 857 s cCH(ar) 1000 w cCH(ar)

826 w – dNO2 827 w dNO2

703 m 716 m mC@S 717 m mC = S

m = stretching; d = in-plane bending; c = out-of-plane bending; w = wagging;s = strong, m = medium, w = weak, sh = shoulder; br = broad.

It is well established that mNH2, mCN and mCS bands are diagnos-tic of the coordination mode of thiourea [52]. The mCS band at730 cm�1, observed in the IR spectrum of the tu ligand, shiftedslightly towards lower frequency region on complexation(703 cm�1, 1; 716 cm�1, 2; 717 cm�1, 3). The intense mCN absorp-tion at 1473 cm�1 observable in the free ligand decreased in inten-sity and shifted to 1512 cm�1 (1), 1513 cm�1 (2) and 1511 cm�1 (3)after coordination. Symmetric and asymmetric NH2 stretchingmodes, which appeared as four intense absorptions over the3370–3100 cm�1 range in the IR spectrum of the free thiourea,are detected as two broad bands centered at ca. 3390 cm�1 andca. 3280 cm�1 in the IR spectra of compounds 1–3. All these mod-ifications are consistent with S-bonding of thiourea to the silveratom in 1–3.

The IR spectra of the nitrato complexes 1 and 3 showed thecharacteristic IR frequencies for uncoordinated NO3

� group by theappearance of a strong band at ca. 1324 cm�1 (m1) and 826 cm�1

(m2) [63]. The presence of ionic trifluoromethanesulfonate groupis detected in the IR spectrum of 2 by the appearance of its typicalbands at 1268 (mSO3), 1226 (mCF3), 1171 (mCF3) and 1028 cm�1

(mSO3). The band positions found for 2 fall into the region observedfor the CF3SO3

� ion in AgCF3SO3 salt [64]. The IR absorptions of phenat 1505 cm�1 (mring) and 1090 cm�1 (dCH) shift to higher

D.F. Segura et al. / Polyhedron 79 (2014) 197–206 201

frequencies upon coordination in 1 (1512 and 1100 cm�1) and 2(1513 and 1100 cm�1). The out-of-plane CH bending absorptionat 739 cm�1 shifted to lower frequencies in 1 (728 cm�1) and 2(729 cm�1) [65,66]. The mCC and mCN bands of bpy in the1600–1400 cm�1 range are sensitive to chelation [67,68]. Thebands at 1556, 1452 and 1414 cm�1 of free bpy shifted to higherfrequencies in 3 (1564, 1472 and 1435 cm�1), being consistentwith the chelating coordination mode of bpy. It is worth mention-ing that IR spectra of 1 and 3 are strikingly similar in relativepositions and intensities of the typical bands originated fromvibrational modes of thiourea and nitrato groups, suggesting aclose structural relationship between them.

2.4. Solution studies

1H and 13C NMR spectroscopic data and assignment for the sil-ver(I) compounds and the free ligands were collected in Table S3and S4, respectively (Supplementary material). NMR spectra inDMSO-d6 of the isolated crystalline products reveal them to becompletely pure. Although the overall pattern of the 1H NMR spec-tra of 1–3 resemble very closely to that of the phen and bpyligands, all the signals have been shifted upon coordination. Forcompounds 1 and 2, only one set of resonances are observed intheir 1H NMR spectra, with the H2, H3, H4, and H5 phenanthrolineprotons equivalent to the H9, H8, H7, and H6 atoms, respectively(see numbering Scheme in the Supplementary material). We havealso found that the NMR spectra of nitrato (1) and triflato (2) com-plexes with the same stoichiometric ratio are strikingly similar. Asan indicative example, the 1H NMR spectrum of complex 1 showedtwo double doublet resonances at 9.11 and 8.71 ppm assigned asH2/H9 and H4/H7 protons, respectively, and one singlet at8.15 ppm attributed to H5/H6 nuclei. One doublet of doublets reso-nance is observed at 7.98 ppm assigned as H3/H8 atoms. The singletattributed to the H5/H6 protons indicated their magnetic equiva-lence. The 1,10-phenanthroline proton signals H3/H8, H4/H7, andH5/H6 in 1 are 0.19, 0.21 and 0.15 ppm shifted to downfield,respectively, as compared to the corresponding protons of the freeligand. For compound 2, the H2/H9 resonance changed from9.11 ppm (free ligand) to 9.07 ppm whereas H3/H8 signal was dis-placed 0.04 ppm to downfield. 1H NMR spectrum of 3 also showedonly one set of the expected signals from bpy ligands, indicatingthe symmetric nature of the complex in solution. The H3/H3

0, H4/

H40, and H5/H5

0resonances in 1H NMR spectrum of 3 experienced

a downfield shift of 0.52, 0.16 and 0.13 ppm, respectively, as com-pared to the corresponding hydrogen atoms of the bpy ligand.With regard to the coordinated thiourea, all 1H NMR spectra exhib-ited two broadened signals, attributed to the NH2 protons, over the7.7–8.2 ppm range, which are shifted to downfield in comparisonwith the corresponding protons of the free ligand [69].

13C NMR spectra of 1–3 exhibited the characteristic signals ofthe coordinated phen and bpy ligands (Table S4). An upfield shiftof ca. 4 ppm of the 13C@S resonance in the 13C NMR spectra of thecomplexes indicates a p-back bonding from the silver to the thionesulfur atom and gives a clear evidence of Ag–S bond formation [69].

It is important to emphasize that NMR spectra of the complexesshowed no changes after storage at room temperature for 24 hwhich could indicate their stability in dmso solution (see Supple-mentary material, Figs. S1–S3). Although the NMR studies give animportant indication about the ligand coordination, it does not giveany information on the exact composition and the nature of thespecies in solution (monomers, dimers, etc.). Conductivity mea-surements can provide useful data to infer whether these com-plexes remain as dimers or possess a mononuclear structure insolution. The expected behavior of complexes [{Ag(L)(l-tu)}2]X2

in solution would be 1:2 electrolytes, [{Ag(L)(l-tu)}2]+2 + 2X�.However, the molar conductivities of all complexes in dmso are

comparable to those of 1:1 electrolytes (KM = 61–67 X�1cm2

mol�1) [70]. This finding may indicate the breaking of the sulfurbridge of the dinuclear structure in dmso solution, resultingin monomeric species such as [Ag(L)(tu)]+ or [Ag(L)(tu)(dmso)]+.The ESI/MS spectrum for a representative complex [{Ag(phen)(l-tu)}2](NO3)2 (1) (Fig. S4) was fully consistent with molarconductivity results. Although the most abundant signal correspondto [Ag(phen)]+ ion (m/z 287.8), it was also detected the presence ofthe mononuclear [Ag(phen)(tu)]+ at m/z 362.9.

2.5. Antimycobacterial activities

All synthesized silver(I) compounds were evaluated for theirantiproliferative activities (MIC) against M. tuberculosis and werefurther examined for their toxicity (IC50) in J774 macrophages.The minimum inhibitory concentration (MIC) and IC50 values aresummarized in Table 2. For comparison purposes, the activity offree ligands (phen, bpy, and tu) and the precursor salts AgNO3

and AgCF3SO3 were also evaluated in the same experimentalconditions.

With regard to the free ligands (tu, bpy and phen), the 1,10-phenanthroline demonstrated to be most active with a MIC valueof 12.8 ± 6.27 lM. Such observation would appear to substantiatethe hypothesis that the bioactivity of 1,10-phenanthroline is attrib-uted to its ability to sequester specific transition metals and that itis the resulting metal chelate complex that is the active specie [26].On the other hand, 2,20-bipyridine exhibited a poor inhibitoryeffect on M. tuberculosis (119 ± 0.00 lM). Probably, the differencebetween the inhibitory activity values of 1,10-phenanthrolineand 2,20-bipyridine could be associated to the enhanced hydropho-bic nature of 1,10-phenanthroline, favoring the efficient penetra-tion across the lipoid bacterial membrane. The antiproliferativeactivity of AgNO3 is comparable to that observed for AgCF3SO3,suggesting that the Ag(I) ion itself is the active specie. Compounds[{Ag(phen)(l-tu)}2](NO3)2 (1) and [{Ag(phen)(l-tu)}2](CF3SO3)2 (2)also displayed comparable MIC values, indicating that thesubstitution of nitrato by trifluoromethanesulfonato groups doesnot affect the bioactivity. On the other hand, the antitubercularactivity of the Ag(I) compounds was deeply affected by the coordi-nated N,N-chelating ligand. The replacement of 2,20-bipyridine by1,10-phenanthroline in [{Ag(L)(l-tu)}2](NO3)2 resulted in a signif-icant increase in the antitubercular activity by a factor of ca. 6.The 1,10-phenanthroline-based derivatives 1 and 2 were moreactive than thiourea and precursor salts AgNO3 and AgCF3SO3,implying that a synergistic effect of both the silver cation andthe phen ligand clearly has a role in the activity of 1 and 2. It isworth to emphasize that the antiproliferative activities of 1(11.0 ± 0.99 lM) and 2 (14.2 ± 2.81 lM) are superior than thosefound for other silver(I) complexes, such as [Ag(6-mercaptopur-ine)]�H2O (93.2 lM) [71], [Ag(tartarate)] (31 lM) and silversulfadiazine (22 lM) [72].

Compounds 1 and 2 displayed a higher inhibitory activity thanpyrazinamide (MIC value of 406–812 lM) used for tuberculosistreatment but they were less effective than the first-line antituber-cular drug isoniazid (MIC = 0.22 lM) [30,42].

Compounds with MIC < 10 lg mL�1 (silver salts, phen, 1 and 2)were also evaluated for cytotoxicity (IC50) towards J774 macro-phages. It is important to emphasize that compounds withMICs 6 6.25 lg mL�1 and SIs P 10 are suitable candidates for fur-ther advanced screening in order to investigate the antimycobacte-rial properties more extensively [73]. The substitution of nitrato bytrifluoromethanesulfonato groups in [{Ag(phen)(l-tu)}2](X)2

decreased significantly the cytotoxicity towards macrophages. Thisfinding suggestes that the anionic groups are supposed to play animportant role in modulating the toxicity in this class ofcompounds. It seems possible that the solubility of the complexes,

Table 2Tuberculosis inhibition activity (MIC), cytotoxic effect to J774 macrophages (IC50)a and selectivity indexes (SI)a of the AgNO3 AgCF3SO3 1,10-phenanthroline, 2,2-bipyridine,thiourea, [{Ag(phen)(l-tu)}2](NO3)2 (1), [{Ag(phen)(l-tu)}2](CF3SO3)2 (2), [{Ag(bpy)(l-tu)}2](NO3)2 (3) against M. tuberculosis H37Rv.

Compound MIC IC50 SI

lg mL�1 lM lg mL�1 lM IC50/MIC

AgNO3 4.60 ± 2.06 27.1 ± 12.1 4.90 ± 0.00 28.8 ± 0.00 0.73–1.92AgCF3SO3 8.80 ± 3.93 34.2 ± 15.3 4.90 ± 0.00 19.1 ± 0.00 0.39–1.011,10-Phenanthroline 2.30 ± 1.13 12.8 ± 6.27 14.6 ± 6.86 81.0 ± 38.1 6.24–6.562,2-Bipyridine 18.5 ± 0.00 119 ± 0.00 – – –Thiourea >25 – – – –1 4.70 ± 0.42 11.0 ± 0.99b 5.70 ± 3.76 13.4 ± 8.82b 0.46–1.852 7.30 ± 1.44 14.2 ± 2.81b 39.1 ± 0.00 76.2 ± 0.00b 4.48–6.693 >25 – – – –

a MIC = minimum inhibitory concentration; IC50 = concentration that inhibited in 50% the cellular proliferation of J774 macrophages; SI = selectivity index: defined as theratio of IC50 to MIC.

b Concentration calculated by moles of [Ag(L)(tu)]X L�1.

202 D.F. Segura et al. / Polyhedron 79 (2014) 197–206

and consequently, their ability to penetrate the cell wall is affectedby the nature of counteranions. However, structure–activity rela-tionships proposed in this study are only very preliminary sincethey were based on only three Ag(I) compounds. Despite its lowin vitro cytotoxicity to macrophages cells, compound 2 displayedan unfavourable selectivity index (SI) range of 4.48–6.69.

2.6. Antileishmanial activity

Antileishmanial activities of complexes 1–3 and Amphotericin B(AmpB) against both forms of the parasite was estimated by theinhibitory concentration of growth at 50% (IC50) and are listed inTable 3. The cytotoxic effect of these compounds to murine perito-neal macrophages (CC50) is also listed in Table 3.

As observed previously, the antiproliferative activity of Ag(I)compounds towards L. amazonensis promastigotes was also depen-dent to the coordinated N,N-chelating ligand. The antileishmanialactivity of the 1,10-phenanthroline-based derivatives (1 and 2)was comparable to that observed for the reference drug Amphoter-icin B. Compounds 1 and 2 were less toxic to murine peritonealmacrophages than Amphotericin B. Interestingly, compound 1and 2 were also effective against some fungi tested in this work.These findings support the idea that Ag(I) complex leads to areduction of the amount of ergosterol in the cell membrane andto a subsequent increase in its permeability [74], since bothLeishmania and fungi express ergosterol in their cell membrane.Particularly, the antiproliferative activity against intracellularamastigote of L. amazonensis (IC50 = 5.81 ± 0.45 lM) and selectiveindex displayed by compound 2 deserves further comment.According to literature, SI values > 1 is considered more selectivefor activity against parasites, and a value less than 1 is more selec-tive for activity against normal cells [75]. Complex 2 displayed notonly a comparable effect to that observed for Amphotericin B(IC50 = 4.77 ± 0.33 lM), but also exhibited an excellent biologicalprofile, with a selectivity index (SI) range of 15.5–13.8, which isconsiderably superior to the standard drugs Amphotericin B

Table 3Antileishmanial activity against promastigote and intracellular amastigotes forms of Leishmperitoneal macrophages (CC50 given as lg mL�1 and lM) by the complexes [Ag(phen)(l-tu)drug – amphotericin B (AmpB).

Compound IC50 (promastigote) CC50

lg mL�1 lM lg mL�1

1 2.42 ± 0.06* 5.68 ± 0.14a 29.11 ± 1.832 2.52 ± 0.08* 4.91 ± 0.16a 45.20 ± 1.64*

3 3.97 ± 0.39 9.87 ± 0.97a 19.80 ± 0.95*

AmpB 4.71 ± 0.57 5.10 ± 0.62 25.03 ± 2.45

a Concentration calculated by moles of [Ag(L)(tu)]X L�1. Selectivity index (SI) is expre* Values of biological activity that differ statistically from reference drug when p < 0.05

(SI = 4.40) and pentamidine (SI = 0.58) [76]. These results suggestthat the type of the anionic group (X) in [{Ag(phen)(l-tu)}2](X)2

complexes may affect their solubility, and consequently, their per-meability towards the cell membrane of macrophages as well asthe intracellular amastigote. However, further experiments shouldbe undertaken in order to evaluate the possible potentiating effectof anionic group in this series of complexes.

2.7. Antifungal and antibacterial activity

Antibacterial and antifungal activities of complexes 1–3,together with the organic ligands and its silver salts, are listed inTables 4 and 5, respectively, as estimated by the inhibitory concen-tration of microbial growth at 50% (MIC50).

The thiourea and 2,20-bipyridine ligands showed no drugresponse at drug concentrations <100 lg mL�1 in all the tested cul-tures, and thus were considered inactive whereas 1,10-phenan-throline exhibited only significant activity against C. tropicalis(7.5 lM). With regard to silver salts, AgNO3 demonstrated to beactive against C. krusei (MIC = 11 lM) and moderately activeagainst the other microorganisms, with a MIC50 value of 44 lM.The AgCF3SO3 displayed poor inhibitory activity towards the testedcultures with MIC50 values ranging from 117–233 lM.

Interestingly, the 1,10-phenanthroline based Ag(I) derivatives(1 and 2) showed a wide spectra of effective activities againstGram-negative (E. coli) and -positive (S. aureus) bacteria and yeast(C. albicans and C. tropicalis). The complex [{Ag(phen)(l-tu)}2](NO3)2 (1) showed significant MIC50 values against E. coli (17.6 lM)and C. tropicalis (17.6 lM) and displayed moderate effects onthe growth of S. aureus (70.4 lM) and C. albicans (70.4 lM).Compound [{Ag(phen)(l-tu)}2](CF3SO3)2 (2) demonstrated to beactive only against S. aureus (29.2 lM) and C. albicans (29.2 lM).Analogously to the results obtained from M. tuberculosisassays, the replacement of nitrato by trifluoromethanesulfonatogroups in [{Ag(phen)(l-tu)}2](X)2 lowered the activity by a factorof ca. 2.

ania (L.) amazonensis (IC50 given as lg mL�1 and lM) and cytotoxic effect to murine]2(NO3)2 (1), [Ag(phen)(l-tu)]2(CF3SO3)2 (2), [Ag(bpy)(l-tu)]2(NO3)2 (3) and reference

IC50 (amastigote) SI

lM lg mL�1 lM

68.30 ± 4.30a 4.95 ± 0.38 11.6 ± 0.90a 5.81–5.9888.06 ± 3.19a 3.10 ± 0.30 6.04 ± 0.58a 13.8–15.549.23 ± 2.36a 8.85 ± 1.31* 22.0 ± 3.26a 2.04–2.5027.09 ± 2.65 4.41 ± 0.31 4.77 ± 0.33 5.50–5.83

ssed by the ratio between cytotoxicity (CC50) and anti-amastigote potency (IC50).by Tukey’s test.

Table 4Antibacterial activities {MIC50 given as lg mL�1 (lMa)} of compounds [{Ag(phen)(l-tu)}2](NO3)2 (1), [{Ag(phen)(l-tu)}2](CF3SO3)2 (2), [{Ag(bpy)(l-tu)}2](NO3)2 (3)against bacteria (S. aureus, E. coli, P. aeruginosa).

Compound S. aureus E. coli P. aeruginosa

AgNO3 7.5 (44) 7.5 (44) 7.5 (44)AgCF3SO3 60 (233) 30 (117) 60 (233)1,10-Phenanthroline inactive inactive inactive2,2-Bipyridine – – –Thiourea inactive inactive inactive1 30 (70.4a) 7.5 (17.6a) inactive2 60 (117a) 15 (29.2a) inactive3 inactive inactive inactiveChloramphenicol 0.975 (3.0) 0.975 (3.0) 31.2 (96.5)

a Concentration calculated by moles of [Ag(L)(tu)]X L�1.

Table 5Antifungal activities {MIC50 given as lg mL�1 (lMa)} of compounds [{Ag(phen)(l-tu)}2](NO3)2 (1), [{Ag(phen)(l-tu)}2](CF3SO3)2 (2), [{Ag(bpy)(l-tu)}2](NO3)2 (3)against fungi (C. albicans, C. tropicalis, C. krusei).

Compound C. albicans C. tropicalis C. krusei

AgNO3 7.5 (44) 7.5 (44) 1.9 (11)AgCF3SO3 60 (233) 30 (117) 60 (233)1,10-Phenanthroline 15 (83) 7.5 (41.5) inactive2,2-Bipyridine – – –Thiourea inactive inactive inactive1 30 (70.4a) 7.5 (17.6a) inactive2 60 (117a) 15 (29.2a) inactive3 inactive inactive inactiveFluconazole 1.0 (3.3) 1.0 (3.3) 32 (105)

a Concentration calculated by mols of [Ag(L)(tu)]X L�1.

D.F. Segura et al. / Polyhedron 79 (2014) 197–206 203

On comparing the MIC50 values found for 1–3, it was noticedthat compound [{Ag(bpy)(l-tu)}2](NO3)2 (3) exhibited no activity(IC50 > 100 lg mL�1) in all the tested cultures. This finding suggeststhat the presence of phen ligand in the molecular structure of 1 and2 may improve general membrane permeability via enhanced lipo-philicity and, as a consequence, result in an enhanced activity thanits inactive bpy analogue 3.

The molecular basis for the activity of the complexes 1 and 2tested in this work remains unknown. Previous studies on theanti-fungal activity on 1,10-phenanthroline and its silver complex[Ag2(phen)3(mal)]�2H2O (malH2 = malonic acid) demonstrated thatthey disturb mitochondrial function, retard the synthesis of cyto-chromes b and c and uncouple respiration [77]. In addition, theexposition of fungal cells with the Ag(I) complex leads to a reduc-tion of the amount of ergosterol in the cell membrane and to a sub-sequent increase in its permeability. This silver complex inducesapoptosis in fungal and mammalian cells as a direct result of itsaction on the cell or a secondary effect originated from its reduc-tion in respiration [74].

Some authors have also considered that the potential targetsites for inhibition of bacterial and fungal growth by silver com-plexes might be the sulfur containing residues of proteins andnot nucleic acids [19–23]. Therefore, the ease with which thesecompounds participate in ligand exchange reactions with biologi-cal ligands plays a vital role in determining their antimicrobialactivities [78]. If the free phen ligand becomes available from sub-stitution reactions involving compounds 1 and 2 and S-donor bio-logical ligands, it can act as a chelator for other metal ions essentialto the physiology of microorganisms [77].

3. Conclusions

The synthesis, characterization (in solution and solid state) andbiological (antitubercular, antileishmanial, antibacterial, andantifungal) activity evaluation of three silver(I) compounds

[{Ag(phen)(l-tu)}2](NO3)2 (1), [{Ag(phen)(l-tu)}2](CF3SO3)2 (2),and [{Ag(bpy)(l-tu)}2](NO3)2 (3), (where phen = 1,10-phenanthro-line; bpy = 2,20-bipyridine; tu = thiourea) were reported in thiswork. Although this is a relatively small investigation, employinga limited number of microorganisms and silver compounds, ourresults indicated that activity of this class of compounds may bemodulated by the N,N-chelating ligand and, in a lesser extent, bythe nature of oxyanion group (X). Nevertheless, it must be empha-sized that the activity data described in this work cannot be rigor-ously interpreted to mean that the silver complexes remain intactduring the experiments and that the MIC values reflect exactly theeffects of either the free Ag(I) ion or the silver complexes.

Further investigations on this type of compounds are underwayin our laboratories in order to rationalize the MIC values in termsof structure–activity relationship. Despite the interesting biologicalprofile of [{Ag(phen)(l-tu)}2](CF3SO3)2 (2) towards L. (L.) amazon-ensis amastigotes and macrophages, it is important to point outthat the antileishmanial evaluation described here is only the firstin a long series of assays that would have to be employed to estab-lish safety and efficacy.

4. Experimental

4.1. General methods

Syntheses were performed at room temperature protected fromlight. Reagents and solvents were all analytically pure andemployed without further purification. Silver salts and organicligands were purchased from Sigma Aldrich, Merck or Fluka.

4.2. Synthesis

4.2.1. Compound [{Ag(phen)(l-tu)}2](NO3)2 (1)1,10-phenanthroline (0.58 mmol; 116.7 mg) dissolved in 10 mL

of CH3OH was added to a 15 mL CH3CN solution containing AgNO3

(0.58 mmol; 100.0 mg) leading to a yellow suspension. Afterstirring for 30 min, thiourea (1.16 mmol; 89.6 mg) dissolved in10 mL of CH3OH was added, affording a light brown solid whichwas isolated by simple filtration and washed with methanol. Yield70%. Anal. Calc. for C26H24Ag2N10O6S2: C, 36.64; H, 2.84; N, 16.43.Found: C, 37.03; H, 2.75; N, 16.52%. KM = 67.2 X�1 cm2 mol�1.

4.2.2. Compound [{Ag(phen)(l-tu)}2](CF3SO3)2 (2)1,10-phenanthroline (0.58 mmol; 116.7 mg) dissolved in 10 mL

of CH3OH was added to a 15 mL CH3CN solution containingAgCF3SO3 (0.58 mmol; 151.2 mg) leading to a yellow suspension.After stirring for 30 min, thiourea (1.16 mmol; 89.6 mg) dissolvedin 10 mL of CH3OH was added, giving rise to a clear solution. Slowevaporation of the solvent afforded white crystals which wereseparated and washed with methanol. Yield 70%. Anal. Calc. forC28H24Ag2F6N8O6S4: C, 34.52; H, 2.69; N, 11.67. Found: C, 33.76;H, 2.36; N, 11.92%. KM = 61.2 X�1 cm2 mol�1.

4.2.3. Compound [{Ag(bpy)(l-tu)}2](NO3)2 (3)2,20-bipyridine (0.58 mmol; 92.0 mg) dissolved in 10 mL of

CH3OH was added to a 15 mL CH3CN solution containing AgNO3

(0.58 mmol; 100.0 mg) leading to a clear solution. After stirring for30 min thiourea (1.16 mmol; 89.6 mg) dissolved in 10 mL of CH3OHwas added. Slow evaporation of the solvent from the reaction mediaafforded white crystals of good quality for single-crystal X-raydiffraction studies. Yield 60%. Anal. Calc. for C22H24Ag2N10O6S2: C,32.85; H, 3.01; N, 17.41. Found: C, 32.76; H, 2.86; N, 16.92%.KM = 64.4 X�1 cm2 mol�1.

Table 6Crystal and structure refinement data for [{Ag(bpy)(l-tu)}2](NO3)2 (3).

Formula C22H24Ag2N10O6S2

Formula weight 804.37Crystal system monoclinicSpace group P�1a (Å) 8.0435(5)b (Å) 13.5107(9)c (Å) 14.3983(8)a (�) 92.937(3)b (�) 100.246(2)c (�) 106.444(2)V (Å3) 1468.20(16)Z 2Dcalc (g cm�3) 1.819l (Mo Ka) (mm�1) 1.532Crystal size (mm) 0.09 � 0.09 � 0.009Crystal type Colourless blockh range (�) 2.25–29.13Index ranges �11 6 h 6 10,

�18 6 k 6 18,0 6 l 6 19

Reflections collected 62069Independent reflections 7865 [Rint = 0.0915]Completeness to h = 29.13� 99.6%Final R indices [I > 2r(I)]a,b R1 = 0.0409

wR2 = 0.0829Final R indices (all data)a,b R1 = 0.621

wR2 = 0.0905Weighting schemec m = 0.0292

n = 1.8741Largest difference in peak and hole (e �3) 0.964 and �1.303

a R1 ¼PjjFoj � jFc jj=

PjFoj.

b w ¼ 1=½r2ðF2oÞ þ ðmPÞ2 þ nP�.

c w ¼ 1=½r2ðF2oÞ þ ðmPÞ2 þ nP� where P ¼ ðF2

o þ 2F2c Þ=3.

204 D.F. Segura et al. / Polyhedron 79 (2014) 197–206

4.3. Physical measurements

C, H, N, and S analyses were performed on a Leco InstrumentsLTDA – TruSpec CHNS. The electrical conductivity measurements(KM, reported as X�1cm2mol�1) of the three complexes in dmsosolutions (c = 1.0 � 10�3 mol L�1) were taken with a Digimed-DM-31 conductometer. Electrospray mass spectrometric analyseswere performed on a LCP Fleet – Thermo Scientific Electrospray,operating in positive and negative-ion modes (sheath gas flowN2: 8 a.u.; capillary voltage: in positive ion mode 20 V; ion transfercapillary temperature: 250 �C). Sample solutions (0.1 mg cm�3 inCH3OH) were directly injected into ESI source by use of a syringepump at a flow rate of 20 mL min�1. Infrared spectra were recordedas KBr pellets on a Spectrum 2000 Perkin Elmer spectrophotometerin the spectral range 4000–400 cm�1 with resolution of 2 cm�1. 1Hand 13C NMR spectra were obtained as dmso-d6 solutions, on a Var-ian INOVA 500 spectrometer.

4.4. Single-crystal X-ray diffraction studies

Single-crystal X-ray diffraction data for [{Ag(bpy)(l-tu)}2](NO3)2 (3) were collected on a Bruker X8 Kappa APEX II charge-coupled device (CCD) area-detector diffractometer (Mo Kagraphite-monochromated radiation, k = 0.71073 Å) controlled bythe APEX2 software package. Images were processed using the soft-ware package SAINT+ [79], and data were corrected for absorptionby the multi-scan semi-empirical method implemented in SADABS.The crystal structure of 3 was solved using the Patterson synthesisalgorithm implemented in SHELXS-97, which allowed the immediatelocation of the crystallographically independent silver and sulfuratoms. All remaining non-hydrogen atoms were located from dif-ference Fourier maps calculated from successive full-matrix leastsquares refinement cycles on F2 using SHELXL-97 [80,81]. Aromatichydrogen atoms bound to carbon atoms were placed at their ideal-ized positions using appropriate HFIX 43 instructions in SHELXL.Amino hydrogen atoms were located from difference Fourier mapsand included in the final structural models with the N–H and H� � �Hdistances restrained to 0.84(1) and 1.55(1) Å, respectively, in orderto ensure a chemically reasonable geometry for these moieties. Allhydrogen atoms were included in subsequent refinement cycleswith isotropic thermal displacement parameters (Uiso) fixed at1.2 or 1.5 � Ueq, of the parent carbon or nitrogen atoms, respec-tively. All non-hydrogen atoms, except for two oxygen atoms inone nitrate anion, were refined anisotropically. One nitrate anionwas found highly disordered and a complex model of disorderwas used (please see the Electronic Supporting information foradditional details). The last difference Fourier map synthesisshowed the highest peak (0.964 e Å�3) and deepest hole(�1.303 e Å�3) located at 0.79 and 0.75 Å from Ag1, respectively.Details of the crystal data and structure refinement parametersfor 3 are summarized in Table 6.

4.5. Antimycobacterial assays

The anti-M. tuberculosis activity of the ligands and the silvercomplexes was determined by the Resazurin Microtiter Assay(REMA) [82,83]. Stock solutions of the test compounds were pre-pared in DMSO and diluted in Middlebrook 7H9 broth (Difco), sup-plemented with oleic acid, albumin, dextrose and catalase (OADCenrichment – BBL/Becton Dickinson, Sparks, MD, USA), to obtainfinal drug concentration ranging from 0.15 to 250 lg mL�1. Theserial dilutions were performed in a Precision XS Microplate Sam-ple Processor (Biotek™). The isoniazid was dissolved in distilledwater, according to the manufacturers’ recommendations (Difcolaboratories, Detroit, MI, USA), and used as a standard drug. M.tuberculosis H37Rv ATCC 27294 was grown for 7–10 days in

Middlebrook 7H9 broth supplemented with OADC, plus 0.05%Tween 80 to avoid clumps. Suspensions were prepared and theirturbidities matched to the optical density of the McFarland No. 1standard. After a further dilution of 1:25 in Middlebrook 7H9 brothsupplemented with OADC, 100 lL of the culture were transferredto each well of a 96-well microtiter plate (NUNC), together withthe test compounds. Each test was set up in triplicate. Microplateswere incubated for 7 days at 37 �C, after which resazurin wasadded for the reading. Wells that turned from blue to pink, withthe development of fluorescence, indicated growth of bacterialcells; maintenance of the blue color indicated bacterial inhibition.The fluorescence was read (530 nm excitation filter and 590 nmemission filter) in a SPECTRAfluor Plus (Tecan) microfluorimeter.The MIC was defined as the lowest concentration resulting in90% inhibition of growth of M. tuberculosis. As a standard test,the MIC of isoniazid was determined on each microplate. Theacceptable range of isoniazid MIC is from 0.015 to 0.05 lg mL�1

[83,84].

4.6. Cytotoxicity to J774 assays

In vitro cytotoxicity assays (IC50) were performed on the J774(ATCC TIB-67) cell line, as recommended by Ahmed et al. [83]and modified by us [84]. The cells were routinely maintained inComplete Medium (RPMI-1640 supplemented with 10% heat-inac-tivated fetal bovine serum (FBS); 100 U/mL penicillin and 100lgmL�1 streptomycin), at 37 �C, in a humidified 5% CO2 atmo-sphere. After reaching confluence, the cells were detached andcounted. For the cytotoxicity assay, 1 � 105 cells mL�1 were seededin 200 lL of Complete Medium in 96- well plates (NUNC). Theplates were incubated at 37 �C under a 5% CO2 atmosphere for24 h, to allow cell adhesion prior to drug testing. Compounds weredissolved in DMSO and subjected to twofold serial dilution from

D.F. Segura et al. / Polyhedron 79 (2014) 197–206 205

1250 to 3.9 lg mL�1. Cells were exposed to the compounds at var-ious concentrations for a 24 h-period. Resazurin solution was thenadded to the cell cultures and incubated for 6 h. Cell respirationwas followed as an indicator of cell viability and was detected byreduction of resazurin to resorufin, whose pink color and fluores-cence indicates cell viability. A persistent blue color of resazurinis a sign of cell death. The fluorescence measurements (530 nmexcitation filter and 590 nm emission filter) were performed in aSPECTRAfluor Plus (Tecan) microfluorimeter. The IC50 value wasdefined as the highest drug concentration at which 50% of the cellsare viable relative to the control. A selectivity index (SI) was thencalculated by dividing the IC50 by the MIC.

4.7. Antipromastigote activity evaluation

Promastigote forms of L. (L.) amazonensis (MHOM/BR/71973/M2269) were grown on a 24-wells plate in Schneider’s Drosophilamedium (Sigma, USA) supplemented with 10.0% (v/v) heat-inacti-vated fetal bovine serum and 1.0% penicillin (10000 UI mL�1)/streptomycin (10.0 mg mL�1) (Sigma, USA). Compounds 1–3 solu-bilized in dimethylsulfoxide (DMSO) (in the range of 0.05–40.0 lg mL�1) were added to promastigote cultures, at 1 � 106

cells mL�1, and incubated at 25 �C. After 72 h of incubation, thesurviving parasites were counted in a Neubauer’s chamber andcompared with controls and DMSO in a concentration of 0.6% v/v,for the determination of 50.0% inhibitory growth concentration(MIC50). All tests were performed in triplicate and AmphotericinB (Eurofarma) was used as the reference drug [85].

4.8. Cytotoxicity to murine peritoneal macrophages assays

For the cytotoxicity assay a suspension of 8 � 105 cells mL�1 ofmurine peritoneal macrophages, in RPMI-1640 medium, supple-mented with 10.0% heat-inactivated fetal bovine serum and 1.0%penicillin (10000 UI mL�1)/streptomycin (10 mg mL�1) wereadded to each well in 24-well plates. The plates were incubatedin a 5.0% CO2 air mixture at 37 �C to adhesion of the cells. After24 h, the non-adherent cells were removed by washing with themedium. Thus, several concentrations of compounds 1–3 (in therange of 0.05–160.0 lg mL�1) were added to the wells containingthe cells. All target compounds were solubilized in DMSO at a finalconcentration of 0.6% v/v and the plates were incubated for more72 h. Then, the medium was removed and 50.0 lL of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)was added to each well at concentration of 5.0 mg mL�1, followedby incubation for more 4 h. After this, 1 mL of DMSO was added toeach well and it was homogenized for 15 min. Next, the absor-bance of each individual well, minus the control value, was calcu-lated in according to the next formula at 570 nm (OD = opticaldensity) [86].

% inhibition ¼ ODcontrol � ODdrugs

ODcontrol

� �� 100

4.9. Antiamastigote activity evaluation

Murine peritoneal macrophages were maintained in RPMI-1640medium, supplemented with 10.0% heat-inactivated fetal bovineserum and 1.0% penicillin (10000 UI mL�1)/streptomycin(10 mg mL�1). Cells, at concentration 8 � 105 cells mL�1, were cul-tivated in 24-well plates on the glass slides of 13 mm (Nunc, USA).After 30 min to adhesion, the cells were infected with L. (L.) ama-zonensis promastigotes at a multiplicity of infection of 10:1 (para-site/macrophage). The plates were incubated in a 5.0% CO2 airmixture at 37 �C for 24 h. Then, nonphagocytosed promastigotes

were removed by washing; the compounds (in the range of 0.10–40.00 lg mL�1) were added to each well. After 72 h, chamber slideswere fixed in absolute methanol, stained with Giemsa and exam-ined under an oil immersion objective of the light microscope. Atleast 200 macrophages were counted per well for calculating thepercent inhibition for the determination of IC50 value. All testswere performed in triplicate on three different occasions andAmphotericin B (Eurofarma) was used as the reference drug [86].The selectivity index (SI) was established by the relationshipbetween the CC50 value and amastigote IC50.

4.10. Antibacterial and antifungal activity evaluation

Compounds 1–3 were evaluated in vitro for their antimicrobialactivities against fungi through a Mueller Hinton broth microdilu-tion method and with the methodology and interpretative criteriaproposed by document M27A3 [87] and through a standard Muel-ler Hinton broth microdilution method for bacteria proposed bydocument M7A6 [88]. The standard pathogenic/opportunistic fungiwere Candida albicans (ATCC 10231), Candida krusei (ATCC 6258),Candida tropicalis (ATCC 750) and bacteria, the Gram positiveStaphylococcus aureus (ATCC 6538) and the Gram negative Esche-richia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC27853). The stock solutions of all the compounds were preparedin DMSO 1% at final concentration and tested at concentrations(lg mL�1) 100; 60; 30; 15; 7.5; 3.75; 1.875; 0.468; 0.23; 0.06.The standard drug fluconazole was applied as control of fungistaticaction at concentration (lg mL�1) 64; 32; 16; 8; 4; 2; 1; 0.5; 0.25;0.125; 0.0625; 0.03125 and the standard drug chloramphenicolwas applied as a control of bacteriostatic action at concentrations(lg mL�1) 8; 4; 2; 1; 0.5; 0.25; 0.12; 0.06; 0.03; 0.015. The micro-plates were incubated at 35 �C for 24 h for bacteria and 37 �C andfor 24 h for fungi. Results were visualized and analyzed by spectro-photometry. The inhibitory concentration of microbial growth wasdetermined at 50% (MIC50) in lg mL�1 and compared for each com-pound and microorganism. Tests have been performed in duplicateand the results obtained from the replicas were coincident.

Acknowledgements

This research was supported by CNPq 487092/2012-0, FAPESP,CAPES and FAPEMIG. Some of the authors are grateful to Fundaçãopara a Ciência e a Tecnologia (FCT, Portugal), FEDER (Portugal) andCOMPETE for the financial support towards the purchase of the sin-gle-crystal diffractometer, for the post-doctoral research grant No.SFRH/BPD/63736/2009 (to JAF), and for funding the R&D projectPTDC/QUI-QUI/098098/2088 (FCOMP-01-0124-FEDER-010785).

Appendix A. Supplementary data

CCDC 931693 contains the supplementary crystallographic datafor [{Ag(bpy)(l-tu)}2](NO3)2 (3). These data can be obtained free ofcharge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, orfrom the Cambridge Crystallographic Data Centre, 12 Union Road,Cambridge CB2 1EZ, UK; fax: +44 1223-336-033; or e-mail: depos-it@ccdc.cam.ac.uk. Supplementary data contains a detaileddescription of the crystallographic modeling of the disorderednitrate anion, selected bond length distances and angles, geometri-cal features of the hydrogen bonding interactions (Tables S1 andS2), and an additional Figure depicting the crystal packing of com-pound 3. 1H and 13C NMR spectroscopic data and assignment forthe silver(I) compounds and the free ligands were collected inTables S3 and S4. Supplementary data associated with this articlecan be found, in the online version, at http://dx.doi.org/10.1016/j.poly.2014.05.004.

206 D.F. Segura et al. / Polyhedron 79 (2014) 197–206

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