Journal of Molecular Structure 1082 (2015) 180–187

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Journal of Molecular Structure

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Silver sulfadoxinate: Synthesis, structural and spectroscopiccharacterizations, and preliminary antibacterial assays in vitro

http://dx.doi.org/10.1016/j.molstruc.2014.11.0040022-2860/� 2014 Elsevier B.V. All rights reserved.

Abbreviations: SFX, Sulfadoxine (4-Amino-N-(5,6-dimethoxy-4-pyrimidinyl)benzenesulfonamide); AgSFX, Ag(I) complex with sulfadoxine; AgSFD, Ag(I) compsulfadiazine; NMR, nuclear magnetic resonance; HSQC, Heteronuclear Single Quantum Coherence; HMBC, Heteronuclear Multiple Bond Coherence; SS-NMR, Solid Stspectroscopy; ESI-TOF-MS, Electrospray Ionization Time-of-flight Mass Spectrometry; IR, Infrared Spectroscopy; TGA/DTA, Thermogravimetric and DifferentialAnalysis; XRPD, X-ray Powder Diffraction; ATCC, American Type Collection Cell; MH, Mueller-Hinton agar; BHI, Brain-Heart Infusion Medium; CFU, Colony Formi⇑ Corresponding author. Tel.: +55 19 35213130; fax: +55 19 35213023.

E-mail address: ppcorbi@iqm.unicamp.br (P.P. Corbi).

Nina T. Zanvettor a, Camilla Abbehausen a, Wilton R. Lustri b, Alexandre Cuin c, Norberto Masciocchi d,Pedro P. Corbi a,⇑a Institute of Chemistry, University of Campinas – UNICAMP, P.O. Box 6154, 13083-970 Campinas, SP, Brazilb Biological and Health Sciences Department – UNIARA, 14801-320 Araraquara, SP, Brazilc Department of Chemistry, Exact Sciences Institute – UFJF, Juiz de Fora, MG, Brazild Dipartimento di Scienza e Alta Tecnologia, Università degli Studi dell’Insubria, 22100 Como, Italy

h i g h l i g h t s

� A novel silver(I) complex withsulfadoxine.� IR and NMR data indicate

coordination of the ligand to Ag(I) bythe nitrogen and oxygen atoms.� Structural characterization of the

complex was based on X-ray powderdiffraction data.� Antibacterial activities of the complex

were observed over Gram-negativeand Gram-positive strains.

g r a p h i c a l a b s t r a c t

Structure of the AgSFX dimer. Colour code: silver in pink, nitrogen, oxygen, sulphur, carbon and hydrogenatoms in blue, red, yellow, black and white, respectively.

a r t i c l e i n f o

Article history:Received 4 September 2014Received in revised form 2 November 2014Accepted 2 November 2014Available online 13 November 2014

Dedicated to Professor Antonio C. Massabniin the occasion of his 70th birthday.

Keywords:Silver complexSulfadoxineESI-TOF-MS

a b s t r a c t

The sulfa drug sulfadoxine (SFX) reacted with Ag+ ions in aqueous solution, affording a new silver(I)complex (AgSFX), which was fully characterized by chemical, spectroscopic and structural methods.Elemental, ESI-TOF mass spectrometric and thermal analyses of AgSFX suggested a [Ag(C12H13N4O2S)]empirical formula. Infrared spectroscopic measurements indicated ligand coordination to Ag(I) throughthe nitrogen atoms of the (deprotonated) sulfonamide group and by the pyrimidine ring, as well asthrough oxygen atom(s) of the sulfonamide group. These hypotheses were corroborated by 13C and15N SS-NMR spectroscopy and by an unconventional structural characterization based on X-ray powderdiffraction data. The latter showed that AgSFX crystallizes as centrosymmetric dimers with a strongAg� � �Ag interaction of 2.7435(6) Å, induced by the presence of exo-bidentate N,N0 bridging ligands andthe formation of an eight-membered ring of [AgNCN]2 sequence, nearly planar. Participation of oxygenatoms of the sulfonamide residues generates in the crystal a 1D coordination polymer, likely responsiblefor its very limited solubility in all common solvents. Besides the analytical, spectroscopic and structural

lex withate NMRThermal

ng Unit.

N.T. Zanvettor et al. / Journal of Molecular Structure 1082 (2015) 180–187 181

X-ray powder diffractionAntibacterial activities

Fig. 1. Sketch of SFX molecule. The SFX molecular ceight torsion (si) angles indicated by arrows and usstudy (vide infra).

description, the antibacterial properties of AgSFX were assayed using disc diffusion methods against Esch-erichia coli and Pseudomonas aeruginosa (Gram-negative), and Staphylococcus aureus (Gram-positive) bac-terial strains. The AgSFX complex showed to be active against Gram-positive and Gram-negative bacterialstrains, being comparable to the activities of silver sulfadiazine.

� 2014 Elsevier B.V. All rights reserved.

Introduction

Metal complexes have been widely used in pharmacology andmedicine worldwide for the treatment of many diseases. Themedicinal use of metals in China dates back to ca. 2500 b.C [1],being silver one of the most used metals. As early as 1000 b.C.,silver was used to make water potable and, nearly two millennialater, in the 19th century (well before the advent of antibiotics),silver compounds were popular drugs [2]. The main use of silverwas for the treatment of burns and wounds, efficiently limitingbacterial infection. Initially, silver dosage was made through solidsilver nitrate by the use of instruments as the so-called lapisinfernalis, followed by the use of solutions and foils [3]. Recently,the advent of penicillin and sulfa drugs made silver-based antibac-terial agents fall into disuse.

The interest in silver was recovered by Moyer, who proposedthe use of silver nitrate solutions to treat burns [4]. Unfortunately,the fast delivery of silver(I) ions into the blood system causessevere toxicity, and it is accompanied by the inhibition of theepithelial growth. In the 1970’s, Fox studied [5] the combinationof different sulfonamides with silver nitrate, aiming for improvingthe antibacterial activity, while diminishing the undesired effectsof silver nitrate solutions. It was then demonstrated [6] that Fox’santibacterial agent was a silver complex with sulfonamide. Amongthe many sulfonamides, silver sulfadiazine (AgSFD) was the onethat showed the highest activity against bacteria, and was soonintroduced in the clinic for the treatment of burn infections andskin ulcers as a topical cream [2,4–8]. The good performance of sil-ver sulfadiazine is mainly attributed to the presence of slowlyreleased Ag+ ions [9–11], with no sulfadiazine molecules beingever found inside of the bacterial cells. In this case, sulfadiazineacts as a carrier of silver ions, and avoids the readily precipitationof Ag+ as silver chloride, or oxide/hydroxide, maintaining the elec-trolyte levels of the body fluids as well as the antibacterial activitydue to the constant concentration gradient of Ag+ [9].

Nowadays silver is known as a broad-spectrum antimicrobialagent and diverse formulations are commercially available. For

onformation was defined byed in the powder diffraction

example, colloidal silver, silver salts, silver complexes, nanocrystal-line silver, silver oxide, and silver zeolite formulations are known,all possessing good antimicrobial activity [2,8]. However, thewidespread use of silver has caused the isolation of some resistantbacterial strains. The studies on silver resistance are sparse, butthey suggest that the resistance is mediated by plasmid. These evi-dences increased the concerns about the misuse of silver and thedevelopment of novel antibacterial silver compounds became ofgreat interest [9].

For this reason, novel silver complexes with enhanced, ortailored, antimicrobial activity are continuously being investigated.For example, a silver complex with N-acetyl-L-cysteine was recentlysynthesized, and showed a broad spectrum of activity againstmicroorganisms [12]. In addition, a silver complex with the anti-inflammatory drug nimesulide (another sulfonamide) [13] wasshown to be active against Gram-positive and Gram-negative bacte-ria. Moreover, the Ag(I) derivatives are currently being investigatedas anticancer drugs [12,14].

Sulfadoxine (SFX, C12H14N4SO4, Fig. 1) is a sulfonamide widelyused in association with pyrimethamine as an antimalarial drug.The associated drug is indeed active against Plasmodium falciparum,the main human malaria parasite [15–18]. Besides the antimalarialactivity, the sulfadoxine-pyrimethamine hybrid is also activeagainst the parasite Toxoplasma gondii [15] and is considered as pri-mary prophylaxis of pneumonia caused by Pneumocyctis carinii[16–18] and toxoplasmatic encephalitis in patients infected withHIV [17,18]. Sulfadoxine is also used in combination with trimeth-oprim to treat different bacterial infections in animals as horses andcalves, being active against Gram-negative and Gram-positive bac-teria [19–21].

Metal complexes of sulfadoxine were also recently studied andFe(II), Fe(III), Co(II), Cu(II), Cr(III) derivatives were synthesized[22–24]. Ogunniran et al. synthesized Cu and Fe complexes withsulfadoxine and pyrimethamine, and showed that these speciesare active against E. coli, S. aureus, P. aureginosa and S. typhi. Thecomplexes have shown to have an enhanced activity in comparisonwith the free ligands [23]. In the current manuscript, inspired bythese works and by the desirable properties of many silverderivatives, we present the synthesis, spectroscopic and structuralcharacterization, and the antibacterial activity of the novel silver-sulfadoxine (AgSFX) complex.

Experimental

Materials and methods

Sulfadoxine (SFX 95%) and silver nitrate (AgNO3 99%) were pur-chased from Sigma Aldrich Laboratories. Potassium hydroxide(85%) was obtained from Fluka. Elemental analyses for carbon,hydrogen, and nitrogen were performed using a CHNS/O PerkinElmer 2400 Analyzer. Infrared (IR) spectra from 4000 cm�1 to400 cm�1 of SFX and the AgSFX complex were measured using aBomen MB Series Model B100 spectrometer with resolution of4 cm�1; samples were prepared as KBr pellets. The 1H nuclear mag-netic resonance (NMR) spectrum of SFX was recorded on a BrukerAvance 400 MHz spectrometer operating at 400.1 MHz. The 13C

182 N.T. Zanvettor et al. / Journal of Molecular Structure 1082 (2015) 180–187

NMR spectrum of SFX was also recorded on a Bruker Avance400 MHz operating at 100.6 MHz. The heteronuclear [1HA13C] sin-gle quantum coherence (HSQC), heteronuclear [1HA13C] multiplebond coherence (HMBC) and the heteronuclear [1HA15N] multiplebond coherence (HMBC) spectra of SFX were acquired on a BrukerAvance 400 MHz, using a 5-mm probe at 303 K, operating at40.5 MHz for 15N; samples were was prepared in deuterateddimethylsulfoxide solutions. The solid state nuclear magnetic reso-nance (SS-NMR) 13C spectrum of SFX an AgSFX was recorded on aBruker 400 MHz Avance II (9.395T) operating at 100 MHz usingcross polarization, proton decoupling and magic angle spinning(CP/MAS). The solid-state 15N{1H} nuclear magnetic resonancespectra were recorded on a Bruker 400 MHz, using the combinationof cross-polarization, proton decoupling and magic angle spinning(CP/MAS) at 6 kHz. Electrospray ionization mass spectrometry(ESI-MS) measurements were carried out in a Waters Quattro MicroAPI instrument: methanol was added to a sample of 1.5 mg AgSFX,the supernatant was separated and formic acid was added untilconcentration of 1%; the resulting solution was directly infused intothe instrument’s ESI source with capillary potential of 3.00 kV, conepotential of 30 kV, trap potential of 2 kV, source temperature of150 �C and nitrogen gas for desolvation.

Synthesis of the complex

The silver(I) complex with sulfadoxine (AgSFX) was synthesizedby the reaction of 1.0 � 10�3 mol of a freshly prepared aqueousAgNO3 solution (2 mL) with a basic aqueous solution of SFX con-taining 1.1 � 10�3 mol of potassium hydroxide and 1.0 � 10�3 molof SFX (12.5 mL). After one hour of constant stirring at room tem-perature, a white solid precipitated and was collected by filtration,washed with cold water, and dried in a desiccator with P4O10. Yield71.9%. Anal. Calcd. for [Ag(C12H13N4O4S)] (%): C, 34.5; H, 2.64; N,13.4. Found: C, 34.3; H, 2.52; N, 13.0. AgSFX is poorly soluble inwater, DMSO, DMF, chloroform, acetonitrile, hexane, ethanol andmethanol. Accordingly, in the absence of single crystals of suitablesize and quality, and being impossible to grow suitable samples byrecrystallization from solution, we adopted the powder diffractionroute to structural characterization [25] which, in through theyears [26–28], became a viable and efficient tool to obtain other-wise inaccessible structural information of metal complexes oflimited structural complexity.

Structural analysis of AgSFX by X-ray powder diffraction data

The polycrystalline AgSFX material was gently ground in anagate mortar and the powder was deposited in a sample holderequipped with a silicon zero-background plate. Diffraction datawere collected at room temperature by an overnight scan in the2h range of 6–105� with steps of 0.02� using a Bruker AXS D8Advance diffractometer equipped with Ni-filtered Cu Ka radiation(k = 1.5418 Å) and Lynxeye linear position-sensitive detector. The

Fig. 2. Final Rietveld refinement plot for AgSFX complex, with difference plot

following optics were set up: primary beam Soller slits: 2.3�, fixeddivergence slit: 0.5�, and receiving slit: 8 mm. The generator wasset at 40 kV and 40 mA. Standard peak search and profile fitting,followed by indexing through the single-value decompositionapproach implemented in TOPAS [29] allowed detection of theapproximate monoclinic unit cell parameters (GOF: 3.04 fromIndexing and 3.78 from LeBail). Density considerations suggestedZ = 4 and the space group P21/c was chosen. Structure solutionwas performed by the simulated annealing technique imple-mented in TOPAS. A (partially flexible) rigid body for SFX was usedwith free molecular location and orientation within the unit cell;the SFX rigid body was defined by Cartesian coordinates, obtainedby molecular mechanics optimization of the bond distances andangles, using the ChemSketch freeware program, and by resizingthe CAH bond distances down to the common ‘‘X-ray’’ value of0.95 Å. In addition, s1 to s5 torsion angles (see Fig. 1) were freedin the simulated annealing step, while a single silver(I) ion was leftto freely float in the cell. The final refinement was carried out bythe Rietveld method maintaining the rigid body introduced atthe simulated annealing stage. Additionally, a total of eight torsionangles were refined in the final cycles. The background was mod-elled by a Chebyshev polynomial function. An isotropic thermalparameter was assigned to the metal (BAg) and refined; lighteratoms were given a Biso = BAg + 2.0 Å2 thermal parameter. The finalRietveld refinement plot is supplied as Fig. 2.

Antibacterial assays

Three referenced bacterial strains: Escherichia coli – ATCC25,922, Pseudomonas aeruginosa – ATCC 27,853 and Staphylococcusaureus – ATCC 25,923 were used for the antibacterial experiments.The antibiogram assay was performed by the disc diffusion method[30,31]. The sensitivity of SFX and AgSFX was tested in Mueller–Hinton (MH) agar. The microorganisms were transferred to sepa-rate test tubes containing 5.0 mL of sterile brain heart infusion(BHI) medium and incubated for 18 h at 35–37 �C. Sufficient inoc-ula were added in new tubes until the turbidity equaled 0.5 McFar-land (�1.5 � 108 CFU/cm3). The bacterial inocula diluted with BHI(McFarland standard) were uniformly spread using sterile cottonswabs on sterile Petri dishes containing MH agar.

Sterile filter paper discs (10 mm diameter) were asepticallyimpregnated with 800 lg of SFX and AgSFX according to the fol-lowing procedure: 20 mg of the compounds were suspended in1000 lL of distilled water, homogenized in a vortex, and 50 lL ofthe suspension were collected with a micropipette and transferredto the paper discs. Sterile discs impregnated with 1000 lg of pureSFX were used as a negative control.

Discs impregnated with the AgSFX complex or with SFX weredried and sterilized in a vertical laminar flow under UV radiationfor 45 min before the experiment. The impregnated discs wereplaced on the surfaces of the solid agar. The plates were incubatedfor 18 h at 35–37 �C and examined thereafter. Clear zones of

and peak markers at the bottom. The high angle region is shown as inset.

N.T. Zanvettor et al. / Journal of Molecular Structure 1082 (2015) 180–187 183

inhibition around the discs were measured and the complex sensi-tivity was assayed from the diameter of the inhibition zones (inmillimeters). The observed results were compared to discs withthe standard antibiotic levofloxacin, and with discs impregnatedwith silver nitrate (AgNO3) and silver sulfadizine (AgSFD).

Results and discussion

Structural analysis by X-ray powder diffraction data

The crystal structure of AgSFX has been derived from powderX-ray diffraction analysis on laboratory data and, despite of thehigh number of torsion angles of SFX to be determined, relevantcrystallochemical information (stoichiometry, crystal packing, con-formation) could be obtained.

Crystal data for AgSFX are summarized in Table 1, while relevantbond distances and angles are given in Table 2. The centrosymmet-ric dimer found in the crystal structure of AgSFX is shown in Fig. 3,where only atoms of the asymmetric unit were labelled. As pictori-ally shown therein, the crystals of AgSFX structure containdinuclear moieties centered about the strong Ag� � �Ag argentophilicinteraction (2.744(6) Å). The eight-membered ring of the[ANCNAgA]2 type present in the dimer is further bound to oxygenatoms of S@O group (with AgAO interactions in the 2.6–2.7 Årange), eventually connecting the different moieties in a 1Dpolymer, stretching along the a axis.

The proposed structure of AgSFX requires some further com-ments. Apparently, the refined distances for the AgAN1 and AgAN3bonds (2.256(2) and 2.533(1) Å, respectively, similar to the typicalvalues found for other Ag(I) complexes containing N-donor ligands[32] possess rather distinct values, and suggested a deeper look intothe problem, aiming at detecting if, for any reason, this discrepancyis real. This doubt is even more pronounced for XRPD structuredeterminations of moderately complex systems, which are known

Table 1Crystallographic data of AgSFX.

AgSFX

Empirical formula C12H13AgN4O4SFormula weight 417.18T(K) 298k(Cu Ka) (Å) 1.5418Crystal system MonoclinicSpace group P21/ca (Å) 5.4448(5)b (Å) 19.226(2)c (Å) 13.852(1)b (�) 102.502(5)V (Å3) 1415.7(2)Z 4dcalc (g cm�3) 1.9573l (mm�1) 13.09F (000) 800Number of parameters 34RBragg, Rwp 0.044/0.080

Table 2Relevant bond distances (Å) and angles (�) for AgSFX.

Bond distances (Å) Angles (�)

Ag–N1 2.26(2) N1–Ag–N3i 170.9(4)Ag–N3i 2.53(1) N1–Ag–O12i 123.1(5)Ag–O12i 2.60(2) N1–Ag–O11ii 83.6(5)Ag–O11ii 2.71(2) N3i–Ag–O12i 58.6(3)Ag� � �Agi 2.744(6) N3i–Ag–O11ii 105.4(4)

O12i–Ag–O11ii 78.7(4)

Symmetry codes: i (1 � x, �y, �z) and ii (�x, �y, �z).

to be not as accurate than conventional single-crystal methods,but – and the question is relevant – to what extension?

Yet, in order to double check our results on AgSFX, theCambridge Structural Database (CSD) was searched for cycliceight-membered ring [AAgNCNA]2 type, and a statistical averageof 2.241 Å for bonded AgAN was found, with a broad distributionreaching values higher than 2.6 Å. At the same time, the Ag� � �Agdistance distribution showed highly probable intermetallic inter-actions near, or slightly below, 2.80 Å.

Several restrained Rietveld refinements were also performed,on the same original XRPD data, by setting equal AgAN distancesat values ranging from 2.10 to 2.75 Å (in 0.05 Å steps). Fig. 4 shows(black dots) the pertinent Rwp vs. AgAN curve, exhibiting a welldefined minimum. These data were subjected to a parabolic fit,which gave the minimum Rwp value (0.083) for a bond distanceof 2.542 Å. Needless to say, on relaxing the two AgAN distances,a slightly lower Rwp value (0.080) is obtained. Thus, the closematching of (i) the statistically derived AgAN average and theobserved AgAN1 values; (ii) the typically observed Ag� � �Agdistances and the 2.744(6) Å value here determined, and (iii) thedistance determined for minimum Rwp value in the restrainedrefinements and the observed AgAN3 bond, significantly suggestthat the proposed structural model cannot be too far from reality.Note that the final comment is based on two statistically indepen-dent observations: our experimental XRPD data and a completestatistical analysis of CSD.

The bond distances of AgAO are, normally, larger than AgAN[34] accordingly, also in the AgSFX complex, long(er) AgAO bonddistances fall in the 2.60(2) to 2.71(2) Å range.

Mass spectrometric measurements

The AgSFX complex was analysed by mass spectrometry (Fig. 5).The results show the molecular ion as singly charged peak with thehighest abundance at m/z 419 assigned to [AgC12H13N4O4S + H+].An adduct of the molecular ion with methanol is observed at m/z451. The free ligand, C12H14N4O4S, is also observed as singlycharged [C12H14N4O4S + H+] ions of m/z 311, respectively. The silverion is observed at 109 m/z and a peak at 156 m/z is assigned as thebreakage of the ligand molecule, forming the ion [C6H6NSO2

+].Peaks at m/z 525, 729 and 834 are assigned as [Ag2(C12H13N4

O4S) + H+], [Ag(C12H13N4O4S)2+H+] and [Ag2(C12H13N4O4S)2+H+],respectively. This result is consistent with a polymeric structurepreviously observed in the powder diffraction data, but only moresoluble fragments were detected in the analysis. An isotope patternfor the singly charged [AgSFX + H+] ion is also shown on Fig. 5.

Solid-state 13C and 15N spectroscopic measurements

The SFX 13C NMR spectrum in solution was assigned based onthe NMR by experiments of [13CA1H] HSQC and HMBC and[15NA1H] HMBC. The spectrum in solution was later used to per-form the assignment of the SS-NMR 13C spectra of SFX and AgSFX.The spectra of SFX in solution are provided as Supplementarymaterial. The AgSFX 13C SS-NMR spectrum was analysed in com-parison to that of SFX. The 13C SS-NMR spectra of AgSFX and SFXare provided in Fig. 6. The structure of SFX with the carbon atomsnumbering is provided on Fig. 1.

The spectrum of the complex shows more resolved peaks thanthe free ligand, which is probably due to the presence of differentpolymorphs of the ligand as previously reported [35]. As demon-strated by powder diffraction analysis, only one polymorph wasisolated for the complex. Besides the change in resolution, signifi-cant variations of the chemical shifts are observed when comparingthe two spectra. The 4 and 7 carbon atoms signals shift to lowerfield by 3.5 and 3.3 ppm, respectively due to the coordination. This

Fig. 3. Structure of the AgSFX dimer. Colour code: silver in pink; nitrogen, oxygen, sulphur, carbon and hydrogen atoms in blue, red, yellow, black and white, respectively. Theatoms of the asymmetric unit (except hydrogen ones) were labelled. The dimeric atoms were generated applying 1 � x, �y, �z symmetry code and the long O11–Ag bond,promoting the dimer-to-polymer formation, is not shown. This molecular sketch was drawn using SCHAKAL [33]. (For interpretation of the references to colour in this figurelegend, the reader is referred to the web version of this article.)

7

7.5

8

8.5

9

9.5

10

2 2.2 2.4 2.6 2.8

Rw

p, %

Ag-N, Å

Rwp

"Final Model"

Fig. 4. Plot of the standard Rietveld refinement Rwp value (%) vs. restrained (andequal) Ag–N bond distances in AgSFX (black dots). A deep minimum near 2.54 Å isclearly seen. The empty circles are positioned at Rwp 8.02 (the absolute minimum inthe final refinement) and refer to the observed Ag–N distances of 2.26 and 2.53 Å,closely matching the expected (statistically averaged) value determined from a CSDsearch and the minimum of the Rwp curve, respectively.

184 N.T. Zanvettor et al. / Journal of Molecular Structure 1082 (2015) 180–187

observation agrees with the powder diffraction result that the coor-dination of the ligand to the metal occurs through the nitrogen ofthe sulfonamide group and the amine group is involved in a seriesof hydrogen bonds with the oxygen of the sulphonamide group ofthe next dimer of AgSFX. Other shifts are observed for the signalsof carbon atoms 5, 2 and 3 by �2.5, 2.1 and 3.7 ppm respectivelyupon the coordination. These results also agree with previouspowder diffraction data that the coordination of sulfadoxine toAg(I) is also through nitrogen 1 of the pyrimidine ring, changingthe electronic density of all the carbons of the ring. This mode ofcoordination, through the sulphonamide and the nitrogen of thepyrimidine ring is common and is also observed for silver sulfadia-zine [36]. No significant variations of the chemical shifts of themethoxy group were observed, which indicates that this functionalgroup probably do not participate on the coordination to silver ions,as shown by powder diffraction data.

The AgSFX 15N SS-NMR spectrum was analysed in comparisonto that of SFX. The 15N SS-NMR spectra of AgSFX and SFX areprovided in Fig. 7, while the numbered structure of SFX is providedin Fig. 1.

Four signals assigned to the four nitrogen atoms are observed inthe spectrum of the SFX molecule, the two nitrogen atoms of thepyrimidine ring, N2 and N1, at 251.0 and 246.7 ppm, respectively,the sulfonamide group nitrogen at 121.5 ppm and the amine groupnitrogen at 71.7 ppm.

Nevertheless, the signal of the sulfonamide group nitrogen isnot observed in the complex 15N spectrum. Since the cross polari-zation NMR technique requires the existence of a hydrogen atomnear to this nitrogen for the polarization transfer [37] it indicates,that the coordination of the ligand to the silver atom is accompa-nied by the loss of the hydrogen and the coordination of the silverion to the nitrogen atom of sulphonamide group. The signals of thenitrogen atoms of the pyrimidine ring are observed at 206.3 and188.1 ppm in the spectrum of the complex, which represents ashift of 44.7 ppm for N2 and 58.6 ppm for N1 to higher field,respectively, when compared to the ligand spectrum. This is con-sistent with the participation of N1 in the coordination. The shiftobserved for N2 is probably due to changes in the electron densityof the whole ring, caused by the coordination to N1. The nitrogen ofthe amino group is observed at 20.2 ppm in the spectrum of thecomplex with a shift of 51.5 ppm to higher field when comparedto the ligand spectrum. The shift observed for the amine groupcan be attributed to hydrogen bonding to the oxygen atom of thesulphonamide group of the adjacent dimer of AgSFX. The NMR datais consistent to the structure proposed by the powder diffraction.The chemical shifts of all the carbon and nitrogen NMR signalsare provided in Table 3.

IR spectroscopic data

The AgSFX IR spectrum was analysed in comparison to that ofSFX. The IR spectra of AgSFX and SFX of the region from 4000 to2500 cm�1 are provided in Fig. 8.

The spectrum of SFX shows three absorption bands at 3240,3377 and 3465 cm�1, assigned as the NAH stretches of the sulph-onamide group and the symmetric and asymmetric NAH stretchesof the amine group, respectively. In the spectrum of the complexthe NAH stretch of the sulphonamide group is observed as a veryweak broad shoulder, whereas the stretches of the amine groupdo not change considerably as it can be observed in Fig. 8. Thisresult indicates that the ligand coordinates to the metal through

Theoretical [AgSFX +H+]

Fig. 5. ESI(+)-TOF mass spectrum of AgSFX from m/z 100 to 1000 and theoretical isotope pattern for the monoprotonated complex of m/z 419.

Fig. 6. 13C solid-state nuclear magnetic resonance spectra of SFX and AgSFXcomplex.

Fig. 7. 15N solid-state nuclear magnetic resonance spectra of SFX and AgSFXcomplex.

Table 3Solid-state 13C and 15N NMR data for SFX and AgSFX.

Assignment Chemical shift (ppm)

SFX AgSFX

C7 152.9 156.2C8, C12 129.5 132.6C9, C11 114.7, 113.3 115.6, 113.7C10 125.9 127.9C4 150.5 154.0C5 150.5 148.0C2 162.5 164.6C3 125.9 129.62OCH3 53.8 55.43OCH3 60.0 59.4N2 251.0 206.3N1 245.7 188.1NH–SO2 121.5 –NH2 71.7 20.2

N.T. Zanvettor et al. / Journal of Molecular Structure 1082 (2015) 180–187 185

the nitrogen atom of the sulfonamide as the hydrogen atombonded to the sulphonamide group is no longer present, conse-quently the NAH stretching is also absent. This result is consistentwith the SS-NMR results and the powder diffraction data.

The comparison of the two spectra also shows a shift of the C@Nstretching mode of the pyrimidine ring from 1650 cm�1 in the SFXspectrum to 1618 cm�1 in the complex which can also indicatecoordination of the ligand to the metal by the nitrogen atoms ofthe pyrimidine ring.

In the spectrum of the ligand the O@S@O asymmetric and sym-metric stretching modes of the sulphonamide group also shiftsfrom 1319 cm�1 and 1157 cm�1 in the ligand to 1236 cm�1 and1115 cm�1 in the complex, respectively. The changes of O@S@Ostretching frequencies suggest the ligand coordination to the metalthrough this group as well, and the decrease in frequency indicatesthe coordination by the oxygen atom [38], as shown by the powderdiffraction data, where one oxygen atom of the sulphonamidecoordinates to the silver ion and the other one interacts weaklywith the silver ion of the adjacent dimer and also forms hydrogenbonds with the amine group of the adjacent dimer. This observa-tion is also in agreement with the reported structure of silversulfadiazine, which presents the oxygen of sulphonamide as acoordination site in the polymeric structure [34]. The full IR spec-trum of the complex and the ligand are provided as Supplementarymaterial.

Antibacterial assays

Antibacterial sensitivity profiles of bacterial strains demon-strate the antibacterial activity of the silver(I) complex with SFXagainst Gram-negative (E. coli and P. aeruginosa) and Gram-positive

Fig. 8. Infrared vibrational spectra of SFX and AgSFX for the region 4000–2500 cm�1.

Table 4Antibiotic sensitivity profiles of bacterial strains against the Ag(I) complex withsulfadoxine (AgSFX), pure SFX (sulfadoxine), AgNO3, Ag(I) complex with sulfadiazine(AgSFD) and the standard antibiotic levofloxacin.

Compound Inhibition zone (mm) (±0.1 mm)

P. aeruginosa S. aureus E. coli

AgSFX 16 16 14SFX 0 0 0AgSFD 18 14 14AgNO3 18 16 14Levofloxacin 12 30 30

186 N.T. Zanvettor et al. / Journal of Molecular Structure 1082 (2015) 180–187

(S. aureus) microorganisms, as observed by the disc diffusionmethod. It was found that paper discs impregnated with the AgSFXcomplex exhibited inhibition zones for E. coli, P. aeruginosa and S.aureus of 14.0 ± 0.1 mm, 16.0 ± 0.1 mm and 16.0 ± 0.1 mm, respec-tively. The inhibition zones for E. coli, P. aeruginosa and S. aureusindicates that these bacterial strains are sensitive to the silver(I)complex. The activity of the pure silver(I) ion was also evaluatedby the same analysis performed with AgNO3 salt. Due to the poorsolubility of the AgSFX complex, the AgSFD complex was also usedto a better comparison. The qualitative results of the AgSFX com-plex are very similar to the results for the AgSFD complex, as itcan be seen on Table 4. The AgSFX complex was more activeagainst S. aureus than the AgSFD complex and less active againstP. aeruginosa; the activity against E. coli was qualitatively the same.

These results are interesting because the prevalent bacteria tocolonize the burn wound in the first days are Staphylococci, beingconsidered a significant pathogen and the commonest colonizingorganism in burn patients [39]. Methicillin-resistant S. aureus isalso a significant problem in burn wound infection [39]. The resultsindicate that the AgSFX complex could be a viable alternative toAgSFD in medical clinic, and further tests are necessary to confirmthis hypothesis.

It is important to note that pure SFX did not exhibit antibacte-rial activity against the considered bacterial strains under the sameexperimental conditions, while discs impregnated with silver(I)nitrate also exhibited clear inhibition zones for the bacterial strainssimilar to the AgSFX complex, as observed in Table 4. These resultsindicate that the antibacterial activity of the AgSFX is most proba-bly due to the Ag(I) ions, as it also occur with the AgSFD complex[6,9–10]. The minimum inhibitory concentration could not bedetermined, as the complex is insoluble.

Conclusion

The molar composition of the silver(I) complex with SFX wasfound to be 1:1 (metal:ligand). Mass spectrometric measurementsshowed species of metal: ligand (M:L) in proportions of 1 M:1L,2 M:1L, 1 M:2L and 2 M:2L, suggesting a polymeric structure. Onlythe smaller and more soluble fragments were detected.

The powder X-ray diffraction analysis showed the coordinationof pyrimidine ring nitrogen and the nitrogen of sulphonamide toAg(I). The oxygen atom of sulphonamide participates in interac-tions with metal, and a strong silver–silver interaction is present.The nitrogen of the amine group is not coordinated; however inter-actions in the solid state through hydrogen bonds are consistentwith SS-NMR and XRPD analyses.

The IR, 13C and 15N SS-NMR confirms coordination of the ligandto Ag(I) through the nitrogen atoms of the sulphonamide groupand the nitrogen N1 of the pyrimidine ring, as well as the oxygenatom of the sulphonamide group, which is consistent with thepowder diffraction data.

The compound showed antibacterial activity against E. coli, P.aeruginosa, and S. aureus microorganisms as observed by antibio-gram assays, which is a common assay for the evaluation of the anti-bacterial activity of insoluble complexes [12,40,41]. The minimuminhibitory concentration (MIC) could not be determined due to thelow solubility of the complex. The antibacterial assays showed theAgSFX complex has a similar activity as the commercial antibacterialagent silver sulfadiazine, being a viable alternative to this drug,although further tests are necessary to confirm this hypothesis.

Acknowledgments

This study was supported by grants from the Brazilian AgenciesFAPESP (São Paulo State Research Council, Grant 2011/02452-0 and2012/08230-2), CNPq (National Council of Scientific and Techno-logical Development, Grant No. 240094/2012-3) and FAPEMIG(Minas Gerais State Research Council, Grant No. CEX-APQ-00525/14). The authors are grateful to MSc. Fernando R.G. Bergamini forfruitful discussions.

Appendix A. Supplementary material

Crystal data, fractional atomic coordinates, and displacementparameters of structures described in this article are supplied instandard CIFs deposited in the Cambridge Crystallographic DataCenter (1012191). The data can be obtained free of charge athttp://www.ccdc.cam.ac.uk/conts/retrieving.html [or from Cam-bridge Crystallographic Data Center (CCDC), 12 Union Road,Cambridge CB2 1EZ, UK; Fax: +44 (0)1223-336033; E-mail:deposit@ccdc.cam.ac.uk]. Supplementary data associated with thisarticle can be found, in the online version, at http://dx.doi.org/10.1016/j.molstruc.2014.11.004.

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