Accepted Manuscript

Synthesis and Biological Evaluation of ternary silver compounds bearingN,N-chelating ligands and thiourea: X-ray structure of [Ag(bpy)(μ-tu)2](NO3)2

(bpy = 2,2’-bipyridine; tu = thiourea)

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

PII: S0277-5387(14)00300-3DOI: http://dx.doi.org/10.1016/j.poly.2014.05.004Reference: POLY 10716

To appear in: Polyhedron

Received Date: 27 January 2014Accepted Date: 5 May 2014

Please cite this article as: D.F. Segura, A.V.G. Netto, R.C.G. Frem, A.E. Mauro, P.B. da Silva, J.A. Fernandes, F.A.Almeida Paz, A.L.T. Dias, N.C. Silva, E.T. de Almeida, M.J. Marques, L. de Almeida, K.F. Alves, F.R. Pavan, P.C.de Souza, H.B. de Barros, C.Q.F. Leite, Synthesis and Biological Evaluation of ternary silver compoundsbearingN,N-chelating ligands and thiourea: X-ray structure of [Ag(bpy)(μ-tu)2](NO3)2 (bpy = 2,2’-bipyridine; tu= thiourea), Polyhedron (2014), doi: http://dx.doi.org/10.1016/j.poly.2014.05.004

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1

Synthesis and Biological Evaluation of ternary silver compounds bearing

N,N-chelating ligands and thiourea: X-ray structure of [Ag(bpy)(-tu)2](NO3)2

(bpy = 2,2’-bipyridine; tu = thiourea)

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. Almeida 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, Brazil.

Phone: ++ 55 16 3301-9626; FAX: ++ 55 16 3322-7932

Corresponding authors: D. F. Segura: daniel@iq.unesp.br; A. V. G. Netto:

adelino@iq.unesp.br.

[b] Department of Chemistry, CICECO, Campus Universitário de Santiago, University of Aveiro,

3810-193 Aveiro, Portugal

[c] UNIFAL/MG-Universidade Federal de Alfenas, CEP 37130-000, Alfenas, MG, Brazil

[d] Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas de Araraquara,

UNESP – Univ Estadual Paulista, P.O. Box 502, Araraquara, São Paulo 14801–902, Brazil.

Compounds [Ag(phen)(-tu)2](NO3)2 (1), [Ag(phen)(-tu)2](CF3SO3)2 (2),

[Ag(bpy)(-tu)2](NO3)2 (3) (where phen = 1,10-phenanthroline; bpy = 2,2’-bipyridine; tu =

thiourea) were prepared by reacting the appropriate AgX salt (X- = NO3

-, CF3SO3

-), the N,N-

chelating ligand (phen or bpy) and thiourea in a ca. 1:1:2 molar 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 were consistent

with the presence of chelating phen (1 and 2) and bpy (3) ligands and demonstrated the S-

coordination mode of thiourea. The crystal and molecular structures of compound [Ag(bpy)(-

tu)2](NO3)2 (3) were determined by single-crystal X-ray diffraction. The complexes 1-3 were

screened for their in vitro antimycobacterial (M. tuberculosis), antileishmanial (Leishmania (L.)

amazonensis), antibacterial (S. aureus, E. coli, P. aeruginosa), antifungal activities (C. albicans, C.

tropicalis, C. krusei).

Keywords: silver(I); 1,10-phenanthroline; thiourea; antileishmanial activity; tuberculosis; antifungal

and antibacterial activity

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2

1. Introduction

An important medical problem to be overcome is the resistance of pathogenic

microorganisms to classical antibiotics, leading to a worldwide demand for the discovery of new

antimicrobial agents [1]. For instance, Tuberculosis (TB) is a highly contagious and deadly disease

caused by the bacillus Mycobacterium tuberculosis (MTB). This disease is spread in the air when

people infected with active pulmonary TB expel bacteria via droplets from their throat and lungs

[2]. This disease is still a serious worldwide public health problem and infects approximately 30%

of the global population as well as a leading cause of morbidity and mortality in developing

countries [3]. In 2011, the World Health Organization (WHO) estimated 8.7 million incident cases

and a total of approximately 1.4 million people died of TB globally [2]. Despite the advances in the

field of the antitubercular chemotherapy, the recent outbreak of multidrug-resistant strains and its

opportunistic coinfection with the human immunodeficiency virus (HIV) highlights the urgent need

for new effective anti-TB drugs and for alternative chemotherapy regimens [4-5]. Besides

Tuberculosis, Leishmaniasis represents one of the neglected tropical diseases (NTDs), caused by

intracellular protozoan parasites from the genus Leishmania, that affect million people in wide

world [6-7]. The digenetic life cycle of Leishmania consists of motile, flagellated, extracellular

promastigotes form in the gut of sand fly vector that infects mammalian host and transform into

nonmotile, nonflagellated amastigotes form, which survive and multiply within phagolysosomal

compartment of macrophages [8]. Moreover, the leishmaniasis treatment remains difficult, since the

available drugs have shown to be highly toxic and cases of resistance have emerged.

Within this context, significant efforts aimed at designing new metal-based compounds for

diagnostic and/or therapeutic uses have been stimulated by the success of the anticancer agent

cisplatin [9]. Particularly, silver and its derivatives possess remarkable antimicrobial properties and

for this reason they have long been utilized for medicinal purposes with no known effect upon the

mammalian cell membrane and limited toxicity to humans [10]. For instance, silver nitrate has been

used to prevent ophthalmianeonatorum in newborns or to treat skin ulcers, postsurgical wounds, and

suppurating wounds [11]. Silver sulfadiazine, introduced in the late 1960s, is still one of the most

effective topical burn treatments. It has been observed that the silver ions are the responsible for the

bactericidal activity [12-14]. Despite the fact that the mechanisms of its antimicrobial action are not

completely understood, some possible mechanisms for inhibition by the aqueous silver(I) ion have

been suggested: (i) interference with electron transport, (ii) binding to DNA, and (iii) interaction

with cell membrane [15].

Silver coordination compounds have attracted considerable interest since the antimicrobial

activity and other desirable properties can be fine-tuned by varying the number and type of ligands

present on the coordination sphere [10, 16-18]. According to Nomiya et al. [19-23], the main targets

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3

for the inhibition of bacteria and yeast by silver(I) complexes are proteins bearing sulfur donor

atoms. In fact it is well known that N,S-donors ligands play an important role in the coordination of

metals at the active sites of several metallobiomolecules [24]. In addition, the type of the

coordinated donor atoms and the ease of ligand replacement appear to be the key factors to be

considered during the molecular design of active silver(I) complexes. Moreover, if the ligands

themselves exhibit antimicrobial activities, these biological properties can be enhanced with the

coordination to the metal centre [25].

Among the N-donor ligands suitable to afford new active silver(I) complexes, 1,10-

phenanthroline (phen) and its derivatives represent a good choice because they possess the ability to

act on a wide variety of biological functions [26]. In particular, silver complexes incorporating this

chelating ligand were found to be extremely active in vitro against pathogenic microbes.

Compounds [Ag(phen)2]ClO4 and [Ag2(phen)3(mal)]·2H2O (malH2 = malonic acid) inhibited the

growth of C. albicans by ca. 95% at a concentration of 5 μg·mL-1

by damaging mitochondrial

function and 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

inhibitory concentration) value of 0.5 μg·mL-1

against this same fungus, causing extensive, non-

specific DNA cleavage, disrupting cell division 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 Leishmania mexicana by interacting with DNA [28].

Following our interest on the synthesis and biological activity of metal-based compounds

[29-36], we report the preparation and spectroscopic characterization (IR, 1H and

13C NMR) of the

complexes [Ag(phen)(-tu)2](NO3)2 (1), [Ag(phen)(-tu)2](CF3SO3)2 (2), [Ag(bpy)(-

tu)2](NO3)2 (3) (where phen = 1,10-phenanthroline; bpy = 2,2’-bipyridine; tu = thiourea). Single

crystal X-ray diffraction studies of compound 3 are reported herein. All complexes were evaluated

in vitro for their antimycobacterial (M. tuberculosis H37Rv ATCC – 27194), leishmanicidal

(Leishmania (L.) amazonensis) antibacterial (S. aureus, E. coli, P. aeruginosa), and antifungal (C.

albicans, C. tropicalis, C. krusei) activities.

Our original interest in thiourea-type ligands arose as these compounds display remarkable

activity against several pathogenic microorganisms [37]. For instances, thiourea derivatives are well

known to possess antibacterial and antifungal activities. Dogruer et al. [38] have reported that some

of thiourea-based molecules exhibited not only promising inhibitory activity against S. aureus (MIC

ranging from 2 to 4 μg·mL-1

) and E. coli (MIC ranging from 4 to 16 μg·mL-1

), but also antifungal

activity against both C. albicans and C. parapsilosis, with a MIC value of 8 μg·mL-1

. Similarly,

silver(I)-chitosan-thiourea compound showed a wide spectrum of effective antimicrobial activities

against six species of bacteria and molds [39]. We assumed that the incorporation of thiourea in the

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4

molecular structures of silver(I) compounds may increase their biological activities as we observed

in other thiourea-metal based systems [40-43]. Despite the intensive work devoted to the

investigation on antimicrobial activities of silver(I) phenanthroline-based compounds, less attention

has been paid to their inhibitory effect on the growth of Mycobacterium tuberculosis (MTB) and

Leishmania (L.) amazonensis.

2. Results and discussion

2.1 Synthesis Considerations

The ternary silver(I) complexes [Ag(phen)(-tu)2](NO3)2 (1) and [Ag(phen)(-

tu)2](CF3SO3)2 (2), represented in Scheme 1, have been isolated from methanol-acetonitrile

mixtures by reacting the appropriate AgX salt X- = NO3

- (1), CF3SO3

- (2), 1,10-phenanthroline

(phen) and thiourea (tu) in a 1:1:2 molar ratio, respectively.

(Insert Scheme 1)

Compound [Ag(bpy)(-tu)2](NO3)2 (3) was obtained using a similar procedure to that

described for 1 by employing 2,2´-bipyridine (bpy) instead of 1,10-phenanthroline. Unlike the

reaction of AgCF3SO3, phen, and tu, we could not isolate the di-metallic compound [Ag(bpy)(-

tu)]2(CF3SO3)2. Several attempts to synthesize this complex were unsuccessful, yielding a clear

solution with a precipitate of Ag0.

The concentrated solutions of 1 and 2 in methanol were left at room temperature for 24 h

protected from light, providing microcrystalline powders that were further purified by repeated

washing with methanol. In order to obtain suitable crystals for X-ray diffraction the supernatant

solvent was submitted to further evaporation. However, this second harvest of crystals presented

different NMR spectra from compounds 1 and 2, being secondary products. Ultimately, these new

products were identified by the conjugation of powder and single crystal X-ray diffraction data.

While the crystals from the supernatant of 1 were found to be a mixture of [Ag(tu)2(-tu)]2(NO3)2

[44] and the new compound [Ag4(phen)(-tu)11](NO3)4·7phen [45], that from the supernatant of 2

was identified as being [Ag(phen)2](CF3SO3) [46].

Complexes are air and light stable solids and exhibit color that varies from white to light

brown. Compounds are soluble in DMSO, sparingly soluble in CH3CN and CH3OH, and insoluble

in water. Elemental analysis of the silver(I) complexes 1-3 showed that their compositions have

molar ratios of Ag+: L : tu : X

- = 1:1:1:1 (L = bpy, phen; X = NO3

-, CF3SO3

-).

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5

2.2 Crystal description of [Ag(bpy)(-tu)2](NO3)2 (3)

Compound [Ag(bpy)(-tu)2](NO3)2 (3) crystallizes in the centrosymmetric triclinic space

group Pī as a dinuclear cation with two charges balancing nitrate ions (see Fig.1).

(Insert Figure 1)

The cation is composed by two crystallographically independent [Ag(bpy)(tu)] moieties,

with two S-bridging thiourea ligands. Both of the silver atoms have distorted tetrahedral

environments with angles, at the metal, ranging from 70.88(10) to 137.33(7)º. The tetrahedral

coordination consists of two nitrogen atoms (from bpy ligand), and two sulfur atoms from bridging

thiourea ligands. The bridging sulphur atoms and the two silver atoms form a lozenge shaped planar

Ag2S2 metallocycle, in which the medium planes of the organic ligands are almost perpendicular to

the Ag1-S1-Ag2-S2 central medium plane [values of 72.85(9) and 79.44(8)º for bpy and

88.55(11) and 86.48(14)º for tu]. The tu ligands are placed at opposite sites of the Ag1-S1-Ag2-

S2 plane with C–S···S angles of 108.66(12) and 110.60(11)º. The Ag2S2 core in 3, 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 sulphur atoms, somewhat larger angles at the silver atoms,

together with a relatively large separation between the bridging sulphur atoms and moderate

separation between the silver atoms.

According to Stocker et al. [47], there is no obvious pattern in the behavior of these Ag(-

tu)Ag bridges: by one side, the bridging can be made by one or two sulfur atoms, and by the other,

the angle Ag–S–Ag and the Ag···Ag distance can vary considerably (64–140º and 2.85–5.06 Å,

respectively). Since then, several other Ag(I) compounds bearing thiourea-type ligands have been

described [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)º and 70.10(2) at atoms S1 and S2, respectively, decrease the

Ag···Ag distance to 3.0910(4) Å, which is considerably shorter than twice the van der Waals radius

for silver (3.44 Å), suggesting a certain degree of metal-metal interaction [51-54].

The Ag–S bonds involving the atom Ag2 are similar [Ag2-S2 2.5554(8) Å; Ag2-S1

2.5573(8) Å] whereas those involving atom Ag1 are somewhat different [Ag1-S1 2.4611(2) Å;

Ag1-S2 2.8140(9) Å]. The Ag1-S2 bond distance, although longer than the sum of the

corresponding tetrahedral covalent radii (2.515 Å) [55], lies within the typical range of other

dimeric or polymeric silver-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,N’-dimethylthiourea)

[47], [(tu)2Ag(μ-tu)2Ag(tu)2]Cl2 [2.5235(5)-2.7926(5) Å] [49], [Ag6(μ3-pyS)4(μ4-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]

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6

[57], [Ag6(μ2-Br)6(μ2-StpmH2)4(μ3-StpmH2)2]n [2.4832(2)- 2.949(2) Å] (StpmH2 = 2-mercapto-

3,4,5,6-tetrahydropyrimidine) [58]. The Ag–N bond distances are in the range of 2.315(3)- 2.351(3)

Å and agree well with other silver compounds bearing chelating 2,2’-bipyridine [59]. The tu ligands

are placed at opposite sites of the Ag2S2 plane with C–S···S angles of 108.66(12) and 110.60(11)º.

All C-S bond lengths in 3 have nearly the same magnitude and are comparable to the reported mean

C=S distance of 1.725 Å for metal-thiourea complexes [60].

Hydrogen bonds in the crystal of 3 share the remarkable feature of being disposed into a

layer very close to the (022) plane of the unit cell. This plane is also very close to the medium plane

of the tu ligands and passes through the nitrate ions. Other supramolecular interaction present in the

crystal is a weak π···π stacking of the bpy ligands, with distances between centromers of 3.919(2)

and 4.025(3) Å (See Fig.2).

(Insert Figure 2)

2.3 IR spectroscopy

The thiourea ligand displays the ability of bonding to transition metal ions via sulfur or

nitrogen atoms [61-62]. Although a variety of physical techniques have been utilized to infer the

coordination mode of the thiourea, IR spectroscopy continues to be one of the most widely used

methods. The most important IR frequencies of the new silver(I) complexes along with their

assignments are presented in Table 1.

(Insert Table 1)

It is well established that NH2, CN and CS bands are diagnostic of the coordination

mode of thiourea [52]. The CS band at 730 cm-1

, observed in the IR spectrum of the tu ligand,

shifted slightly towards lower frequency region on complexation (703 cm-1

, 1; 716 cm-1

, 2; 717 cm-

1, 3). The intense CN absorption at 1473 cm

-1 observable in the free ligand decreased in intensity

and shifted to 1512 cm-1

(1), 1513 cm-1

(2) and 1511 cm-1

(3) after coordination. Symmetric and

asymmetric NH2 stretching modes, which appeared as four intense absorptions over the 3370 - 3100

cm-1

range in the IR spectrum of the free thiourea, are detected as two broad bands centered at ca.

3390 cm-1

and ca. 3280 cm-1

in the IR spectra of compounds 1-3. All these modifications are

consistent with S-bonding of thiourea to the silver atom in 1-3.

The IR spectra of the nitrato complexes 1 and 3 showed the characteristic IR frequencies for

uncoordinated NO3- group by the appearance of a strong band at ca. 1324 cm

-1 (1) and 826 cm

-1

(2) [63]. The presence of ionic trifluoromethanesulfonate group is detected in the IR spectrum of 2

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7

by the appearance of its typical bands at 1268 (νSO3), 1226 (νCF3), 1171 (νCF3) and 1028 cm-1

(νSO3). The band positions found for 2 fall into the region observed for the CF3SO3- ion in

AgCF3SO3 salt [64]. The IR absorptions of phen at 1505 cm-1

(νring) and 1090 cm-1

(CH) shift to

higher frequencies upon coordination in 1 (1512 and 1100 cm-1

) and 2 (1513 and 1100 cm-1

). The

out-of-plane CH bending absorption at 739 cm-1

shifted to lower frequencies in 1 (728 cm-1

) and 2

(729 cm-1

) [65-66]. The νCC and νCN bands of bpy in the 1600-1400 cm-1

range are sensitive to

chelation [67-68]. The bands at 1556, 1452 and 1414 cm-1

of free bpy shifted to higher frequencies

in 3 (1564, 1472 and 1435 cm-1

), being consistent with the chelating coordination mode of bpy. It is

worth mentioning that IR spectra of 1 and 3 are strikingly similar in relative positions and

intensities of the typical bands originated from vibrational modes of thiourea and nitrato groups,

suggesting a close structural relationship between them.

2.4 Solution studies

1H and

13C NMR spectroscopic data and assignment for the silver(I) compounds and the free

ligands were collected in Table S3 and S4, respectively (Supplementary Material). NMR spectra in

DMSO-d6 of the isolated crystalline products reveal them to be completely pure. Although the

overall pattern of the 1H NMR spectra of 1–3 resemble very closely to that of the phen and bpy

ligands, all the signals have been shifted upon coordination. For compounds 1 and 2, only one set of

resonances are observed in their 1H-NMR spectra, with the H2, H3, H4, and H5 phenanthroline

protons equivalent to the H9, H8, H7, and H6 atoms, respectively (see numbering Scheme in the

Supplementary Material). We have also found that the NMR spectra of nitrato (1) and triflato (2)

complexes with the same stoichiometric ratio are strikingly similar. As an indicative example, the

1H NMR spectrum of complex 1 showed two double doublet resonances at 9.11 and 8.71 ppm

assigned as H2/H9 and H4/H7 protons, respectively, and one singlet at 8.15 ppm attributed to H5/H6

nuclei. One doublet of doublets resonance is observed at 7.98 ppm assigned as H3/H8 atoms. The

singlet attributed to the H5/H6 protons indicated their magnetic equivalence. The 1,10-

phenanthroline proton signals H3/H8, H4/H7, and H5/H6 in 1 are 0.19, 0.21 and 0.15 ppm shifted to

downfield, respectively, as compared to the corresponding protons of the free ligand. For compound

2, the H2/H9 resonance changed from 9.11 ppm (free ligand) to 9.07 ppm whereas H3/H8 signal was

displaced 0.04 ppm to downfield. 1

H-NMR spectrum of 3 also showed only one set of the expected

signals from bpy ligands, indicating the symmetric nature of the complex in solution. The H3/H3’,

H4/H4’, and H5/H5

’ resonances in

1H-NMR spectrum of 3 experienced a downfield shift of 0.52, 0.16

and 0.13 ppm, respectively, as compared to the corresponding hydrogen atoms of the bpy ligand.

With regard to the coordinated thiourea, all 1H-NMR spectra exhibited two broadened signals,

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8

attributed to the NH2 protons, over the 7.7-8.2 ppm range, which are shifted to downfield in

comparison with the corresponding protons of the free ligand [69].

13C NMR spectra of 1-3 exhibited the characteristic signals of the coordinated phen and bpy

ligands (Table S4). An upfield shift of ca. 4 ppm of the 13

C=S resonance in the 13

C NMR spectra of

the complexes indicates a -back bonding from the silver to the thione sulfur atom and gives a clear

evidence of Ag–S bond formation [69].

It is important to emphasize that NMR spectra of the complexes showed no changes after

storage at room temperature for 24 h which could indicate their stability in dmso solution (see

Supplementary Material, Figures S1-S3). Although the NMR studies give an important indication

about the ligand coordination, it does not give any information on the exact composition and the

nature of the species in solution (monomers, dimers, etc.). Conductivity measurements can provide

useful data to infer whether these complexes remain as dimers or possess a mononuclear structure

in solution. The expected behavior of complexes [Ag(L)(-tu)2]X2 in solution would be 1:2

electrolytes, [Ag(L)(-tu)2]+2

+ 2X-. However, the molar conductivities of all complexes in dmso

are comparable to those of 1:1 electrolytes (M = 61-67 -1

cm2mol

-1) [70]. This finding may

indicate the breaking of the sulphur bridge of the dinuclear structure in dmso solution, resulting in

monomeric species such as [Ag(L)(tu)]+ or [Ag(L)(tu)(dmso)]

+. The ESI/MS spectrum for a

representative complex [Ag(phen)(-tu)2](NO3)2 (1) (Figure S4) was fully consistent with molar

conductivity results. Although the most abundant signal correspond to [Ag(phen)]+ ion (m/z 287.8),

it was also detected the presence of the mononuclear [Ag(phen)(tu)]+ at m/z 362.9.

2.5. Antimycobacterial activities

All synthesized silver(I) compounds were evaluated for their antiproliferative activities

(MIC) against M. tuberculosis and were further examined for their toxicity (IC50) in J774

macrophages. The minimum inhibitory concentration (MIC) and IC50 values are summarized in

Table 2. For comparison purposes, the activity of free ligands (phen, bpy, and tu) and the precursor

salts AgNO3 and AgCF3SO3 were also evaluated in the same experimental conditions.

(Insert Table 2)

With regard to the free ligands (tu, bpy and phen), the 1,10-phenanthroline demonstrated to

be most active with a MIC value of 12.8±6.27 µM. Such observation would appear to substantiate

the hypothesis that the bioactivity of 1,10-phenanthroline is attributed to its ability to sequester

specific transition metals and that it is the resulting metal chelate complex that is the active specie

[26]. On the other hand, 2,2’-bipyridine exhibited a poor inhibitory effect on M. Tuberculosis

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9

(119±0.00 µM). Probably, the difference between the inhibitory activity values of 1,10-

phenanthroline and 2,2’-bipyridine could be associated to the enhanced hydrophobic nature of 1,10-

phenanthroline, favoring the efficient penetration across the lipoid bacterial membrane. The

antiproliferative activity of AgNO3 is comparable to that observed for AgCF3SO3, suggesting that

the Ag(I) ion itself is the active specie. Compounds [Ag(phen)(-tu)2](NO3)2 (1) and

[Ag(phen)(-tu)2](CF3SO3)2 (2) also displayed comparable MIC values, indicating that the

substitution of nitrato by trifluoromethanesulfonato groups does not affect the bioactivity. On the

other hand, the antitubercular activity of the Ag(I) compounds was deeply affected by the

coordinated N,N-chelating ligand. The replacement of 2,2’-bipyridine by 1,10-phenanthroline in

[Ag(L)(-tu)2](NO3)2 resulted in a significant increase in the antitubercular activity by a factor of

ca. 6. The 1,10-phenanthroline-based derivatives 1 and 2 were more active than thiourea and

precursor salts AgNO3 and AgCF3SO3, implying that a synergistic effect of both the silver cation

and the phen ligand clearly has a role in the activity of 1 and 2. It is worth to emphasize that the

antiproliferative activities of 1 (11.0±0.99 µM) and 2 (14.2±2.81 µM) are superior than those found

for other silver(I) complexes, such as [Ag(6-mercaptopurine)]H2O (93.2 µM) [71], [Ag(tartarate)]

(31 µM) and silver sulfadiazine (22 µM) [72].

Compounds 1 and 2 displayed a higher inhibitory activity than pyrazinamide (MIC value of

406-812 µM) used for tuberculosis treatment but they were less effective than the first-line

antitubercular drug isoniazid (MIC = 0.22 M) [30, 42].

Compounds with MIC < 10 g mL-1

(silver salts, phen, 1 and 2) were also evaluated for

cytotoxicity (IC50) towards J774 macrophages. It is important to emphasize that compounds with

MICs 6.25 g mL-1

and SIs 10 are suitable candidates for further advanced screening in order to

investigate the antimycobacterial properties more extensively [73]. The substitution of nitrato by

trifluoromethanesulfonato groups in [Ag(phen)(-tu)2](X)2 decreased significantly the

cytotoxicity towards macrophages. This finding suggestes that the anionic groups are supposed to

play an important role in modulating the toxicity in this class of compounds. It seems possible that

the solubility of the complexes, and consequently, their ability to penetrate the cell wall is affected

by the nature of counteranions. However, structure-activity relationships proposed in this study are

only very preliminary since they were based on only three Ag(I) compounds. Despite its low in

vitro cytotoxicity to macrophages cells, compound 2 displayed an unfavourable selectivity index

(SI) range of 4.48-6.69.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

10

2.6 Antileishmanial activity

Antileishmanial activities of complexes 1–3 and Amphotericin B (AmpB) against both

forms of the parasite was estimated by the inhibitory concentration of growth at 50% (IC50) and are

listed in Table 3. The cytotoxic effect of these compounds to murine peritoneal macrophages (CC50)

is also listed in Table 3.

(Insert Table 3)

As observed previously, the antiproliferative activity of Ag(I) compounds towards L.

amazonensis promastigotes was also dependent to the coordinated N,N-chelating ligand. The

antileishmanial activity of the 1,10-phenanthroline-based derivatives (1 and 2) was comparable to

that observed for the reference drug Amphotericin B. Compounds 1 and 2 were less toxic to murine

peritoneal macrophages than Amphotericin B. Interestingly, compound 1 and 2 were also effective

against some fungi tested in this work. These findings support the idea that Ag(I) complex leads to a

reduction of the amount of ergosterol in the cell membrane and to a subsequent increase in its

permeability [74], since both Leishmania and fungi express ergosterol in their cell membrane.

Particularly, the antiproliferative activity against intracellular amastigote of L. amazonensis (IC50 =

5.81±0.45 µM) and selective index displayed by compound 2 deserves further comment. According

to literature, SI values > 1 is considered more selective for activity against parasites, and a value

less than 1 is more selective for activity against normal cells [75]. Complex 2 displayed not only a

comparable effect to that observed for Amphotericin B (IC50 = 4.77±0.33 µM), but also exhibited an

excellent biological profile, with a selectivity index (SI) range of 15.5-13.8, which is considerably

superior to the standard drugs Amphotericin B (SI = 4.40) and pentamidine (SI = 0.58) [76]. These

results suggest that the type of the anionic group (X) in [Ag(phen)(-tu)2](X)2 complexes may

affect their solubility, and consequently, their permeability towards the cell membrane of

macrophages as well as the intracellular amastigote. However, further experiments should be

undertaken in order to evaluate the possible potentiating effect of 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 in Table 4 and 5, respectively, as estimated by the inhibitory

concentration of microbial growth at 50% (MIC50).

(Insert Table 4)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

11

(Insert Table 5)

The thiourea and 2,2’-bipyridine ligands showed no drug response at drug concentrations <

100 μgmL-1

in all the tested cultures, and thus were considered inactive whereas 1,10-

phenanthroline exhibited only significant activity against C. tropicalis (7.5 μM). With regard to

silver salts, AgNO3 demonstrated to be active against C. krusei (MIC = 11 μM) and moderately

active against the other microorganisms, with a MIC50 value of 44 μM. The AgCF3SO3 displayed

poor inhibitory activity towards the tested cultures with MIC50 values ranging from 117-233 μM.

Interestingly, the 1,10-phenanthroline based Ag(I) derivatives (1 and 2) showed a wide

spectra of effective activities against Gram-negative (E. coli) and -positive (S. aureus) bacteria and

yeast (C. albicans and C. tropicalis). The complex [Ag(phen)(-tu)2](NO3)2 (1) showed

significant MIC50 values against E. coli (17.6 μM) and C. tropicalis (17.6 μM) and displayed

moderate effects on the growth of S. aureus (70.4 μM) and C. albicans (70.4 μM). Compound

[Ag(phen)(-tu)2](CF3SO3)2 (2) demonstrated to be active only against S. aureus (29.2 μM) and

C. albicans (29.2 μM). Analogously to the results obtained from M. tuberculosis assays, the

replacement of nitrato by trifluoromethanesulfonato groups in [Ag(phen)(-tu)2](X)2 lowered the

activity by a factor of ca. 2.

On comparing the MIC50 values found for 1-3, it was noticed that compound [Ag(bpy)(-

tu)2](NO3)2 (3) exhibited no activity (IC50 > 100 μgmL-1

) in all the tested cultures. This finding

suggests that the presence of phen ligand in the molecular structure of 1 and 2 may improve general

membrane permeability via enhanced lipophilicity and, as a consequence, result in an enhanced

activity than its inactive bpy analogue 3.

The molecular basis for the activity of the complexes 1 and 2 tested in this work remains

unknown. Previous studies on the anti-fungal activity on 1,10-phenanthroline and its silver complex

[Ag2(phen)3(mal)]2H2O (malH2 =malonic acid) demonstrated that they disturb mitochondrial

function, retard the synthesis of cytochromes b and c and uncouple respiration [77]. In addition, the

exposition of fungal cells with the Ag(I) complex leads to a reduction of the amount of ergosterol in

the cell membrane and to a subsequent increase in its permeability. This silver complex induces

apoptosis in fungal and mammalian cells as a direct result of its action on the cell or a secondary

effect originated from its reduction in respiration [74].

Some authors have also considered that the potential target sites for inhibition of bacterial

and fungal growth by silver complexes might be the sulfur containing residues of proteins and not

nucleic acids [19-23]. Therefore, the ease with which these compounds participate in ligand

exchange reactions with biological ligands plays a vital role in determining their antimicrobial

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

12

activities [78]. If the free phen ligand becomes available from substitution reactions involving

compounds 1 and 2 and S-donor biological ligands, it can act as a chelator for other metal ions

essential to the physiology of microorganisms [77].

3. Conclusions

The synthesis, characterization (in solution and solid state) and biological (antitubercular,

antileishmanial, antibacterial, and antifungal) activity evaluation of three silver(I) compounds

[Ag(phen)(-tu)2](NO3)2 (1), [Ag(phen)(-tu)2](CF3SO3)2 (2), and [Ag(bpy)(-tu)2](NO3)2

(3), (where phen = 1,10-phenanthroline; bpy = 2,2’-bipyridine; tu = thiourea) were reported in this

work. Although this is a relatively small investigation, employing a limited number of

microorganisms and silver compounds, our results indicated that activity of this class of compounds

may be modulated by the N,N-chelating ligand and, in a lesser extent, by the nature of oxyanion

group (X). Nevertheless, it must be emphasized that the activity data described in this work cannot

be rigorously interpreted to mean that the silver complexes remain intact during the experiments

and that the MIC values reflect exactly the effects of either the free Ag(I) ion or the silver

complexes.

Further investigations on this type of compounds are underway in our laboratories in order

to rationalize the MIC values in terms of structure-activity relationship. Despite the interesting

biological profile of [Ag(phen)(-tu)2](CF3SO3)2 (2) towards L. (L.) amazonensis amastigotes

and macrophages, it is important to point out that the antileishmanial evaluation described here is

only the first in a long series of assays that would have to be employed to establish safety and

efficacy.

Appendix A. Supplementary data

CCDC 931693 contains the supplementary crystallographic data for [Ag(bpy)(-

tu)2](NO3)2 (3). These data can be obtained free of charge via

http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data

Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail:

deposit@ccdc.cam.ac.uk.

Supplementary data contains a detailed description of the crystallographic modeling of the

disordered nitrate anion, selected bond length distances and angles, geometrical features of the

hydrogen bonding interactions (Tables S1 and S2), and an additional Figure depicting the crystal

packing of compound 3. 1H and

13C NMR spectroscopic data and assignment for the silver(I)

compounds and the free ligands were collected in Tables S3 and S4.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

13

Acknowledgements

This research was supported by CNPq 487092/2012-0, FAPESP, Capes and FAPEMIG.

Some of the authors are grateful to Fundação para a Ciência e a Tecnologia (FCT, Portugal),

FEDER and COMPETE for the financial support towards the purchase of the single-crystal

diffractometer, for the post-doctoral research grant No. SFRH/BPD/63736/2009 (to JAF), and for

funding the R&D project PTDC/QUI-QUI/098098/2088 (FCOMP-01-0124-FEDER-010785).

4. Experimental

4.1. General methods

Syntheses were performed at room temperature protected from light. Reagents and solvents

were all analytically pure and employed without further purification. Silver salts and organic

ligands were purchased from Sigma Aldrich, Merck or Fluka.

4.2. Synthesis

Compound [Ag(phen)(-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. After

stirring for 30 min, thiourea (1.16 mmol; 89.6 mg) dissolved in 10 mL of CH3OH was added,

affording a light brown solid which was isolated by simple filtration and washed with methanol.

Yield 70%. Anal. Calcd. for C26H24Ag2N10O6S2 (%): C: 36,64; H: 2.84; N: 16.43. Found (%): C:

37.03; H: 2.75; N: 16.52. M = 67.2 -1

cm2mol

-1.

Compound [Ag(phen)(-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 containing AgCF3SO3 (0.58 mmol; 151.2 mg) leading to a yellow suspension.

After stirring for 30 min, thiourea (1.16 mmol; 89.6 mg) dissolved in 10 mL of CH3OH was added,

giving rise to a clear solution. Slow evaporation of the solvent afforded white crystals which were

separated and washed with methanol. Yield 70%. Anal. Calcd. for C28H24Ag2F6N8O6S4 (%): C:

34.52; H: 2.69; N: 11.67. Found (%): C: 33.76; H: 2.36; N: 11.92. M = 61.2 -1

cm2mol

-1.

Compound [Ag(bpy)(-tu)2](NO3)2 (3)

2,2'-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 for 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

14

min thiourea (1.16 mmol; 89.6 mg) dissolved in 10 mL of CH3OH was added. Slow evaporation of

the solvent from the reaction media afforded white crystals of good quality for single-crystal X-ray

diffraction studies. Yield 60%. Anal. Calcd. for C22H24Ag2N10O6S2 (%): C: 32.85; H: 3.01; N:

17.41. Found (%): C: 32.76; H: 2.86; N: 16.92. M = 64.4 -1

cm2mol

-1.

4.3. Physical measurements

C, H, N, and S analyses were performed on a Leco Instruments LTDA - TruSpec CHNS.

The electrical conductivity measurements (M, reported as -1

cm2mol

-1) of the three complexes in

dmso solutions (c = 1.0 x 10-3

molL-1

) were taken with a Digimed-DM-31 conductometer.

Electrospray mass spectrometric analyses were performed on a LCP Fleet – Thermo Scientific

Electrospray, operating in positive and negative-ion modes (sheath gas flow N2: 8 a.u.; capillary

voltage: in positive ion mode 20 V; ion transfer capillary temperature: 250oC). Sample solutions

(0.1 mg cm-3

in CH3OH) were directly injected into ESI source by use of a syringe pump at a flow

rate of 20 mL min-1

. Infrared spectra were recorded as KBr pellets on a Spectrum 2000 Perkin

Elmer spectrophotometer in the spectral range 4000-400 cm-1

with resolution of 2 cm-1

. 1H and

13C

NMR spectra were obtained as dmso-d6 solutions, on a Varian INOVA 500 spectrometer.

4.4 Single-Crystal X-ray diffraction studies

Single-crystal X-ray diffraction data for [Ag(bpy)(-tu)2](NO3)2 (3) were collected on a

Bruker X8 Kappa APEX II charge-coupled device (CCD) area-detector diffractometer (Mo K

graphite-monochromated radiation, λ=0.71073 Å) controlled by the APEX2 software package.

Images were processed using the software package SAINT+ [79], and data were corrected for

absorption by the multi-scan semi-empirical method implemented in SADABS. The crystal

structure of 3 was solved using the Patterson synthesis algorithm implemented in SHELXS-97,

which allowed the immediate location of the crystallographically independent silver and sulphur

atoms. All remaining non-hydrogen atoms were located from difference Fourier maps calculated

from successive full-matrix least squares refinement cycles on F2 using SHELXL-97 [80-81].

Aromatic hydrogen atoms bound to carbon atoms were placed at their idealized positions using

appropriate HFIX 43 instructions in SHELXL. Amino hydrogen atoms were located from difference

Fourier maps and included in the final structural models with the N–H and H···H distances

restrained to 0.84(1) and 1.55(1) Å, respectively, in order to ensure a chemically reasonable

geometry for these moieties. All hydrogen atoms were included in subsequent refinement cycles

with isotropic thermal displacement parameters (Uiso) fixed at 1.2 or 1.5×Ueq, of the parent carbon

or nitrogen atoms, respectively. All non-hydrogen atoms, except for two oxygen atoms in one

nitrate anion, were refined anisotropically. One nitrate anion was found highly disordered and a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

15

complex model of disorder was used (please see the Electronic Supporting Information for

additional details). The last difference Fourier map synthesis showed the highest peak (0.964 eÅ-3

)

and deepest hole (-1.303 eÅ-3

) located at 0.79 Å and 0.75 Å from Ag1, respectively. Details of the

crystal data and structure refinement parameters for 3 are summarized in Table 6.

(Insert Table 6)

Antimycobacterial assays

The anti-M. tuberculosis activity of the ligands and the silver complexes was determined by

the Resazurin Microtiter Assay (REMA) [82-83]. Stock solutions of the test compounds were

prepared in DMSO and diluted in Middlebrook 7H9 broth (Difco), supplemented with oleic acid,

albumin, dextrose and catalase (OADC enrichment - BBL/Becton Dickinson, Sparks, MD, USA), to

obtain final drug concentration ranging from 0.15 to 250 µg·mL-1

. The serial dilutions were

performed in a Precision XS Microplate Sample Processor (Biotek™). The isoniazid was dissolved

in distilled water, according to the manufacturers' recommendations (Difco laboratories, Detroit,

MI, USA), and used as a standard drug. M. tuberculosis H37Rv ATCC 27294 was grown for 7 to 10

days in Middlebrook 7H9 broth supplemented with OADC, plus 0.05% Tween 80 to avoid clumps.

Suspensions were prepared and their turbidities matched to the optical density of the McFarland no.

1 standard. After a further dilution of 1:25 in Middlebrook 7H9 broth supplemented with OADC,

100 μL of the culture were transferred to each well of a 96-well microtiter plate (NUNC), together

with the test compounds. Each test was set up in triplicate. Microplates were incubated for 7 days at

37 °C, after which resazurin was added for the reading. Wells that turned from blue to pink, with

the development of fluorescence, indicated growth of bacterial cells; maintenance of the blue color

indicated bacterial inhibition. The fluorescence was read (530 nm excitation filter and 590 nm

emission filter) in a SPECTRAfluor Plus (Tecan) microfluorimeter. The MIC was defined as the

lowest concentration resulting in 90% inhibition of growth of M. tuberculosis. As a standard test,

the MIC of isoniazid was determined on each microplate. The acceptable range of isoniazid MIC is

from 0.015 to 0.05 µg·mL-1

[83-84].

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 in

Complete Medium (RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS);

100 U/mL penicillin and 100 µgmL-1

streptomycin), at 37°C, in a humidified 5% CO2 atmosphere.

After reaching confluence, the cells were detached and counted. For the cytotoxicity assay, 1×105

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

16

cells mL-1

were seeded in 200 μL of Complete Medium in 96- well plates (NUNC). The plates were

incubated at 37 °C under a 5% CO2 atmosphere for 24 h, to allow cell adhesion prior to drug

testing. Compounds were dissolved in DMSO and subjected to two-fold serial dilution from 1250 to

3.9 µg·mL-1

. Cells were exposed to the compounds at various concentrations for a 24 h-period.

Resazurin solution was then added to the cell cultures and incubated for 6 h. Cell respiration was

followed as an indicator of cell viability and was detected by reduction of resazurin to resorufin,

whose pink color and fluorescence indicates cell viability. A persistent blue color of resazurin is a

sign of cell death. The fluorescence measurements (530 nm excitation filter and 590 nm emission

filter) were performed in a SPECTRAfluor Plus (Tecan) microfluorimeter. The IC50 value was

defined as the highest drug concentration at which 50% of the cells are viable relative to the control.

A selectivity index (SI) was then calculated by dividing the IC50 by the MIC.

Antipromastigote activity evaluation

Promastigote forms of L. (L.) amazonensis (MHOM/BR/71973/M2269) were grown on a

24-wells plate in Schneider's Drosophila medium (Sigma, USA) supplemented with 10.0% (v/v)

heat-inactivated fetal bovine serum and 1.0% penicillin (10000 UI mL-1

)/streptomycin (10.0

mg.mL-1

) (Sigma, USA). Compounds 1-3 solubilized in dimethylsulfoxide (DMSO) (in the range of

0.05 to 40.0 μg mL-1

) were added to promastigote cultures, at 1x106 cells mL

-1, and incubated at

25ºC. After 72h of incubation, the surviving parasites were counted in a Neubauer's chamber and

compared 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 Amphotericin B

(Eurofarma) was used as the reference drug [85].

Cytotoxicity to murine peritoneal macrophages assays

For the cytotoxicity assay a suspension of 8x105 cells mL

-1 of murine peritoneal

macrophages, in RPMI-1640 medium, supplemented with 10.0% heat-inactivated fetal bovine

serum and 1.0% penicillin (10000 UI mL-1

)/streptomycin (10 mg mL-1

) were added to each well in

24-well plates. The plates were incubated in a 5.0% CO2 air mixture at 37 ºC to adhesion of the

cells. After 24 h, the non-adherent cells were removed by washing with the medium. Thus, several

concentrations of compounds 1-3 (in the range of 0.05 to 160.0 μg mL-1

) were added to the wells

containing the cells. All target compounds were solubilized in DMSO at a final concentration of

0.6% v/v and the plates were incubated for more 72 h. Then, the medium was removed and 50.0 μL

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

, followed by incubation for more 4 h. After this, 1 mL of DMSO

was added to each well and it was homogenized for 15 min. Next, the absorbance of each individual

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

17

well, minus the control value, was calculated in according to the next formula at 570 nm (OD =

optical density) [86].

Antiamastigote activity evaluation

Murine peritoneal macrophages were maintained in RPMI-1640 medium, supplemented

with 10.0% heat-inactivated fetal bovine serum and 1.0% penicillin (10000 UI mL-1

)/streptomycin

(10 mg mL-1

). Cells, at concentration 8x105 cells mL

-1, were cultivated 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.)

amazonensis promastigotes at a multiplicity of infection of 10:1 (parasite/macrophage). The plates

were incubated in a 5.0% CO2 air mixture at 37 ºC for 24h. Then, nonphagocytosed promastigotes

were removed by washing; the compounds (in the range of 0.10 to 40.00 µg mL-1

) were added to

each well. After 72 h, chamber slides were fixed in absolute methanol, stained with Giemsa and

examined under an oil immersion objective of the light microscope. At least 200 macrophages were

counted per well for calculating the percent inhibition for the determination of IC50 value. All tests

were performed in triplicate on three different occasions and Amphotericin B (Eurofarma) was used

as the reference drug [86]. The selectivity index (SI) was established by the relationship between

the CC50 value and amastigote IC50.

Antibacterial and antifungal activity evaluation

Compounds 1-3 were evaluated in vitro for their antimicrobial activities against fungi

through a Mueller Hinton broth microdilution method and with the methodology and interpretative

criteria proposed by document M27A3 [87] and through a standard Mueller Hinton broth

microdilution method for bacteria proposed by document M7A6 [88]. The standard

pathogenic/opportunistic fungi were Candida albicans (ATCC 10231), Candida krusei (ATCC

6258), Candida tropicalis (ATCC 750) and bacteria, the Gram positive Staphylococcus aureus

(ATCC 6538) and the Gram negative Escherichia coli (ATCC 25922) and Pseudomonas

aeruginosa (ATCC 27853). The stock solutions of all the compounds were prepared in DMSO 1%

at final concentration and tested at concentrations (µg·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 fungistatic action at

concentration (µg·mL-1

) 64; 32; 16; 8; 4; 2; 1; 0.5; 0.25; 0.125; 0.0625; 0.03125 and the standard

drug chloramphenicol was applied as a control of bacteriostatic action at concentrations (µg·mL-1

)

8; 4; 2; 1; 0.5; 0.25; 0.12; 0.06; 0.03; 0.015. The microplates were incubated at 35oC for 24 h for

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

18

bacteria and 37oC and for 24 h for fungi. Results were visualized and analyzed by

spectrophotometry. The inhibitory concentration of microbial growth was determined at 50%

(MIC50) in µg·mL-1

and compared for each compound and microorganism. Tests have been

performed in duplicate and the results obtained from the replicas were coincident.

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23

Table 1 Selected vibrational data (cm-1

) for [Ag(phen)(-tu)2](NO3)2 (1), [Ag(phen)(-

tu)2](CF3SO3)2 (2), [Ag(bpy)(-tu)2](NO3)2 (3), where phen = 1,10-phenanthroline; bpy = 2,2’-

bipyridine; tu = thiourea).

1 2 assignment 3 assignment

3339 br 3390 br νsNH2 3399 s νsNH2

1643 s 1610 s δNH2 1610 s δNH2

1512 m 1513 w νCN 1564 m νCN

1325 m sNO2 1322 m sNO2

1268 m νSO3

1226 m νCF3

1171 m νCF3

1086 w 1084 w νCN 1095 w νCN

846 s 857 s γCH(ar) 1000 w γCH(ar)

826 w - NO2 827 w NO2

703 m 716 m νC=S 717 m νC=S = stretching

; = in-plane bending; = out-of-plane bending; w = wagging; s = strong, m = medium, w = weak, sh =

shoulder; br = broad.

Table 2 Tuberculosis 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)(-tu)2](NO3)2 (1), [Ag(phen)(-tu)2](CF3SO3)2 (2), [Ag(bpy)(-tu)2](NO3)2 (3)

against M. tuberculosis H37Rv.

Compound MIC IC50 SI

gmL–1

M gmL–1

M IC50/MIC

AgNO3 4.60±2.06 27.1±12.1 4.90±0.00 28.8±0.00 0.73-1.92

AgCF3SO3 8.80±3.93 34.2±15.3 4.90±0.00 19.1±0.00 0.39-1.01

1,10-phenanthroline 2.30±1.13 12.8±6.27 14.6±6.86 81.0±38.1 6.24-6.56

2,2-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±8.82

b 0.46-1.85

2 7.30±1.44 14.2±2.81b 39.1±0.00 76.2±0.00

b 4.48-6.69

3 > 25 - - - - a MIC = minimum inhibitory concentration; IC50 = concentration that inhibited in 50% the cellular proliferation of J774

macrophages; SI = selectivity index: defined as the ratio of IC50 to MIC. b concentration calculated by moles of [Ag(L)(tu)]X L

-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

24

Table 3 Antileishmanial activity against promastigote and intracellular amastigotes forms of

Leishmania (L.) amazonensis (IC50 given as g mL–1

and M) and cytotoxic effect to murine

peritoneal macrophages (CC50 given as g mL–1

and M) by the complexes [Ag(phen)(-

tu)]2(NO3)2 (1), [Ag(phen)(-tu)]2(CF3SO3)2 (2), [Ag(bpy)(-tu)]2(NO3)2 (3) and reference drug -

amphotericin B (AmpB).

Compound IC50 (promastigote) CC50 IC50 (amastigote) SI

g mL–1

M g mL–1

M g mL–1

M

1 2.42 ± 0.06* 5.68±0.14

a 29.11 ± 1.83 68.30±4.30

a 4.95 ± 0.38 11.6±0.90

a 5.81-5.98

2 2.52 ± 0.08* 4.91±0.16

a 45.20 ± 1.64

* 88.06±3.19

a 3.10 ± 0.30 6.04±0.58

a 13.8-15.5

3 3.97 ± 0.39 9.87±0.97a 19.80 ± 0.95

* 49.23±2.36

a 8.85 ± 1.31

* 22.0±3.26

a 2.04-2.50

AmpB 4.71 ± 0.57 5.10±0.62 25.03 ± 2.45 27.09±2.65 4.41 ± 0.31 4.77±0.33 5.50-5.83 a

concentration calculated by moles of [Ag(L)(tu)]X L-1

. Selectivity index (SI) is expressed by the ratio between

cytotoxicity (CC50) and anti-amastigote potency (IC50). *Values of biological activity that differ statistically from

reference drug when p < 0.05 by Tukey’s test.

Table 4 Antibacterial activities MIC50 given as g mL–1

(Ma) of compounds [Ag(phen)(-

tu)2](NO3)2 (1), [Ag(phen)(-tu)2](CF3SO3)2 (2), [Ag(bpy)(-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 inactive

2,2-bipyridine - - -

thiourea inactive inactive inactive

1 30 (70.4a) 7.5 (17.6

a) inactive

2 60 (117a) 15 (29.2

a) inactive

3 inactive inactive inactive

Chloramphenicol 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 5 Antifungal activities MIC50 given as g mL–1

(Ma) of compounds [Ag(phen)(-

tu)2](NO3)2 (1), [Ag(phen)(-tu)2](CF3SO3)2 (2), [Ag(bpy)(-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) inactive

2,2-bipyridine - - -

thiourea inactive inactive inactive

1 30 (70.4a) 7.5 (17.6

a) inactive

2 60 (117a) 15 (29.2

a) inactive

3 inactive inactive inactive

Fluconazole 1.0 (3.3) 1.0 (3.3) 32 (105) a concentration calculated by mols of [Ag(L)(tu)]X L

-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

25

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

Formula C22H24Ag2N10O6S2

Formula weight 804.37

Crystal system Monoclinic

Space group Pī

a/Å 8.0435(5)

b/Å 13.5107(9)

c/Å 14.3983(8)

α/º 92.937(3)

β/º 100.246(2)

γ/º 106.444(2)

Volume/Å3 1468.20(16)

Z 2

Dc/g cm-3

1.819

(Mo-K)/mm-1

1.532

Crystal size/mm 0.090.090.009

Crystal type Colourless block

range (º) 2.25 to 29.13

Index ranges -11 h 10

-18 k 18

0 l 19

Reflections collected 62069

Independent reflections 7865 [Rint = 0.0915]

Completeness to θ = 29.13º 99.6%

Final R indices [I>2(I)]a,b

R1 = 0.0409

wR2 = 0.0829

Final R indices (all data)a,b

R1 = 0.621

wR2 = 0.0905

Weighting schemec m = 0.0292

n = 1.8741

Largest diff. peak and hole 0.964 and -1.303 eÅ-3

a) 1 /o c oR F F F ; b)

22 21/ ow F mP nP

c)

22 21/ ow F mP nP where

2 22 /3o cP F F

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

26

FIGURE CAPTIONS

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

Figure 1. Asymmetric unit of compound 3 with most of the non-hydrogen atoms being represented

as thermal ellipsoids drawn at the 30% probability level. Hydrogen atoms are represented as small

spheres with arbitrary radii. Two oxygen atoms are disordered over three sites, being represented as

thermal spheres drawn at the 30% probability level. For a detailed description of the bond lengths

and angles see Table S1 in the Supplementary Information.

Figure 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. For clarity, only the A component of the

disordered nitrate ion is shown, and the bpy ligands have been omitted. For geometrical details on

the represented supramolecular interactions see Table S2 in the Supplementary Material. (b) π···π

stacking between adjacent organic ligands with the interactions being represented as mauve dashed

lines.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

27

Scheme 1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

28

Figure 1. Asymmetric unit of compound 3 with most of the non-hydrogen atoms being represented

as thermal ellipsoids drawn at the 30% probability level. Hydrogen atoms are represented as small

spheres with arbitrary radii. Two oxygen atoms are disordered over three sites, being represented as

thermal spheres drawn at the 30% probability level. For a detailed description of the bond lengths

and angles see Table S1 in the Supplementary Information.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

29

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. For clarity, only the A component of the

disordered nitrate ion is shown, and the bpy ligands have been omitted. For geometrical details on

the represented supramolecular interactions see Table S2 in the Supplementary Material. (b) π···π

stacking between adjacent organic ligands with the interactions being represented as mauve dashed

lines.

1

Synthesis and Biological Evaluation of ternary silver compounds bearing

N,N-chelating ligands and thiourea: X-ray structure of [Ag(bpy)(-tu)2](NO3)2

(bpy = 2,2’-bipyridine; tu = thiourea)

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. Almeida 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, Brazil.

Phone: ++ 55 16 3301-9626; FAX: ++ 55 16 3322-7932

Corresponding authors: D. F. Segura: daniel@iq.unesp.br; A. V. G. Netto: adelino@iq.unesp.br.

[b] Department of Chemistry, CICECO, Campus Universitário de Santiago, University of Aveiro, 3810-193 Aveiro, Portugal

[c] UNIFAL/MG-Universidade Federal de Alfenas, CEP 37130-000, Alfenas, MG, Brazil

[d] Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas de Araraquara, UNESP – Univ Estadual Paulista, P.O. Box

502, Araraquara, São Paulo 14801–902, Brazil.

Pictogram

1

Synthesis and Biological Evaluation of ternary silver compounds bearing

N,N-chelating ligands and thiourea: X-ray structure of [Ag(bpy)(-tu)2](NO3)2

(bpy = 2,2’-bipyridine; tu = thiourea)

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. Almeida 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, Brazil.

Phone: ++ 55 16 3301-9626; FAX: ++ 55 16 3322-7932

Corresponding authors: D. F. Segura: daniel@iq.unesp.br; A. V. G. Netto: adelino@iq.unesp.br.

[b] Department of Chemistry, CICECO, Campus Universitário de Santiago, University of Aveiro, 3810-193 Aveiro, Portugal

[c] UNIFAL/MG-Universidade Federal de Alfenas, CEP 37130-000, Alfenas, MG, Brazil

[d] Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas de Araraquara, UNESP – Univ Estadual Paulista,

P.O. Box 502, Araraquara, São Paulo 14801–902, Brazil.

Synopsis

Three new ternary silver(I) compounds were synthesised, characterized and had their

antimycobacterial, antileishmanial, antifungal and antibacterial activities evaluated. Compound

[Ag(phen)(-tu)2](CF3SO3)2 (2) was very effective against intracellular amastigote of L. amazonensis

(IC50 = 4.77±0.33 µM) and exhibited an excellent biological profile, with a selectivity index (SI) range

of 15.5-13.8.