Synthesis, crystallographic studies, high resolution mass spectrometric analyses and antibacterial assays of silver(I) complexes with sulfisoxazole and sulfadimethoxine

Accepted Manuscript Synthesis, crystallographic studies, high resolution mass spectrometric analyses and antibacterial assays of silver(I) complexes with sulfisoxazole and sulfadimethoxine Ana Thereza M. Fiori, Douglas H. Nakahata, Alexandre Cuin, Wilton R. Lustri, Pedro P. Corbi PII: DOI: Reference:

S0277-5387(16)30476-4 http://dx.doi.org/10.1016/j.poly.2016.09.046 POLY 12234

To appear in:

Polyhedron

Received Date: Revised Date: Accepted Date:

24 August 2016 22 September 2016 26 September 2016

Please cite this article as: A.T.M. Fiori, D.H. Nakahata, A. Cuin, W.R. Lustri, P.P. Corbi, Synthesis, crystallographic studies, high resolution mass spectrometric analyses and antibacterial assays of silver(I) complexes with sulfisoxazole and sulfadimethoxine, Polyhedron (2016), doi: http://dx.doi.org/10.1016/j.poly.2016.09.046

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Synthesis, crystallographic studies, high resolution mass spectrometric analyses and antibacterial assays of silver(I) complexes with sulfisoxazole and sulfadimethoxine Ana Thereza M. Fioria,1, Douglas H. Nakahataa,1, Alexandre Cuinb, Wilton R. Lustric, Pedro P. Corbia,* a

Institute of Chemistry, University of Campinas –UNICAMP, P.O. Box 6154, 13083-970 Campinas, SP, Brazil.

b

Bioinorganic Chemistry Research Laboratory, Federal University of Juiz de Fora, UFJF, 36036-330 Juiz de Fora, MG, Brazil.

c

Biological and Health Sciences Department – University of Araraquara-

UNIARA, 14801-320 Araraquara, SP, Brazil. 1

These authors contributed equally to the manuscript.

* Corresponding author: Telephone: + 5519 35213420 / FAX: + 5519 35213023 E-mail addresses: [email protected] and [email protected]

Dedicatory This work is dedicated to the memory of Professor Douglas Wagner Franco from the Institute of Chemistry of the University of São Paulo-USP, Brazil

2 Abstract

The present work describes the synthesis, structural characterization and antibacterial assays of two silver(I) complexes with the sulfa drugs sulfisoxazole (SIZH) and sulfadimethoxine (SDMXH). Chemical analyses and high resolution mass spectrometric studies led to 1:1 metal/ligand compositions, suggesting the minimal formulas AgC11H12O 3N3S·H2O for Ag-SIZ and AgC12H13O4N4S for AgSDMX. Infrared and solution state NMR spectroscopic measurements indicated ligand coordination to Ag(I) by the nitrogen atoms of the SO2N and NH2 groups in Ag-SIZ, whereas for Ag-SDMX the coordination of the ligand to Ag(I) occurs by the SO2N group and one of the nitrogen atoms of the pyrimidine ring. The proposed coordination modes were confirmed by X-ray diffraction studies and showed the formation of dimeric structures in both cases. The Ag-SDMX complex shows the classical bridging mode of coordination, in which the silver ions bridge between the nitrogen atom of the sulfonamide group of one ligand and the nitrogen atom of the pyrimidine ring of the second ligand, while for the Ag-SIZ complex coordination occurs by the nitrogen atoms of the NH2 and SO2N groups. Antibacterial assays indicated that Ag-SIZ and Ag-SDMX complexes are effective against Gram-negative (E. coli and P. aeruginosa) and Gram-positive (S. aureus) bacterial strains, being the Ag-SDMX complex more active than the Ag-SIZ one. These results are an indicative that the ligand structures and dissimilar coordination modes of the sulfonamides may be associated to the antibacterial activities of the complexes.

Keywords Sulfisoxazole; Sulfadimethoxine; Silver; X-Ray diffraction; Antibacterial agents.

3 Abbreviation list (in alphabetical order)

Ag-SDMX, Ag(I) complex with sulfadimethoxine; Ag-SIZ, Ag(I) complex with sulfisoxazole; ATR, Attenuated Total Reflectance; ATCC, American Type Culture Collection; BHI, Brain Heart Infusion; CFU, Colony Forming Unit. DMSO, Dimethylsulfoxide; ESI-QTOF-MS,

Electrospray

Ionization

Quadrupole

Time-of-flight

Spectrometry; SIZH, Sulfisoxazole; SDMXH, Sulfadimethoxine; IR, Infrared vibrational spectroscopy; MIC, Minimum Inhibitory Concentration; NMR, Nuclear Magnetic Resonance; SDMX, anionic form of sulfadimethoxine; SSD, Silver-sulfadiazine; Sulfa, Sulfonamide drugs; SIZ, anionic form of sulfisoxazole; TG/DTA, Thermogravimetric and Differential Thermal Analyses; TMS, Tetramethylsilane

Mass

4 Introduction The pharmacological activities of metals have been described for a long time. Copper was used in Egypt in 3000 b.C to sterilize water, while gold was applied for medicinal purposes by the Chinese and Arabs [1,2]. Mercury chloride was used as a diuretic in the Renaissance and arsenic was applied to treat syphilis in the early twentieth century [2]. Nowadays, metal ions are used in a wide range of medical applications. Gold compounds, for example, are used as anti-inflammatory drugs to treat rheumatoid arthritis. Platinum(II) complexes, such as cisplatin, carboplatin and oxaliplatin, are effective in treating many types of tumors [1–8]. Furthermore, iron(III) complexes have shown efficacy in the treatment of hypertension, while lithium compounds are used to treat psychiatric disorders [1]. Such applications have stimulating the search for new inorganic compounds as therapeutic agents [2]. More specifically, silver(I), gold(I) and palladium(II) complexes have shown promising results as antimicrobial agents [7,9–16]. Silver is used as antiseptic, antibacterial and anti-inflammatory agent. Recently, the antitumor activity of silver compounds have also been described in the literature [17]. The coordination of drugs with metals, both showing recognized antibacterial activities, has been an alternative in the search to overcome antibiotic-resistant bacteria, which is one of the most important challenges in pharmacology and medicine [1,18–20]. One of the examples of silver compounds in medicine is silver-sulfadiazine (SSD, see the structure of sulfadiazine in Scheme 1), which is used worldwide in the treatment of skin infections in burns and wounds [21,22]. The use of SSD brought attention to the class of sulfonamides, or sulfa drugs, as possible ligands in the synthesis of new metallopharmaceutical agents [23,24]. The mechanism of antimicrobial action of sulfonamides involves competitive inhibition of the enzyme dihydropteroate synthase, preventing the synthesis of folic acid, a cofactor essential for bacterial growth [2,3,25–27]. Silver(I) complexes with the sulfonamides sulfamethoxazole and sulfathiazole were synthesized in our laboratories and tested for their antibacterial activities. These complexes have shown antibacterial activities in vitro on Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella

5 enterica bacterial strains [28]. Other complexes using silver(I) ions and sulfonamides, such as nimesulide, sulfadoxine and sulfamoxole also exhibits biological activities against strains of P. aeruginosa, S. aureus and E. coli [24,29–31]. The complex with sulfamoxole has also shown to have activity against 10 fungi strains [31].

Scheme 1. Chemical structures of (A) sulfadiazine, (B) sulfisoxazole and (C) sulfadimethoxine. Sulfisoxazole yl)benzenesulfonamide)

(SIZH, and

4-amino-N-(3,4-dimethyl-1,2-oxazol-5-

sulfadimethoxine

(SDMXH,

4-amino-N-(2,6-

dimethoxy-4-pyrimidinyl)benzenesulfonamide) are representative molecules of the sulfa drugs [32]. The structures of these compounds are shown in Scheme 1. Sulfisoxazole is a short-acting antibacterial agent with activity against a broad range of Gram-positive and Gram-negative organisms. It is used to treat acute urinary tract infections, acute otitis and prophylaxis of chronic otitis. This compound has also been used to treat infections caused by the microorganism Chlamydia trachomatis, which causes urethritis and other urinary tract infections [33,34]. Sulfadimethoxine, on the contrary, is a long-acting sulfonamide, which is specially used for the treatment of coccidiosis, a parasitic disease of animals. In some countries, it is also used for the treatment of bacterial infections in humans [35,36]. Synthesis of a silver(I) complex with sulfadimethoxine was first described by Bult and Klasen. However, only infrared spectroscopy was used for the proposition of the coordination mode of SDMX to Ag(I) [37]. No biological studies were reported. In this paper, we describe the synthesis, spectroscopic characterization, crystallographic studies and antibacterial assays in vitro of silver(I) complexes with sulfisoxazole and sulfadimethoxine.

6 Materials and methods Sulfisoxazole (SIZH ≥ 99%), sulfadimethoxine (SDMXH ≥ 98.5 %) and silver(I)

nitrate

(AgNO3 ≥

99%)

were purchased from Sigma-Aldrich

Laboratories. Potassium hydroxide (≥ 85%) was obtained from Fluka. Elemental analyses for carbon, hydrogen and nitrogen were performed using a Perkin Elmer 2400 CHNS/O Analyzer. Thermogravimetric and differential thermal analyses (TG/DTA) were performed on a simultaneous TGA/DTA SEIKO EXSTAR 6000 thermoanalyzer, using the following conditions: synthetic air, flow rate of 50 cm3 min-1 and heating rate of 10°C min-1, from 25°C to 900°C. Infrared (IR) spectroscopic analyses were performed in an Agilent Cary 630 FTIR spectrometer, using the Attenuated Total Reflectance (ATR) method, in the range from 4000-400 cm-1 and with resolution of 4 cm-1. Electronic absorption spectra were recorded on a HP Agilent 8453 spectrophotometer, using a quartz cuvette of 1.0 cm optical path length and equipped with a HP 89090A Peltier. The measurements were performed at 37 ºC. Solution state 1H and

13

C spectra were recorded in DMSO-d6 in a Bruker AVANCE III 500 MHz

spectrometer. The chemical shifts were given relative to tetramethylsilane (TMS). Electrospray ionization quadrupole time-of-flight mass spectrometric (ESI-QTOF-MS) measurements were carried out in a Waters Synapt HDMS instrument (Manchester, UK). Samples of the Ag(I) complexes were dissolved in a 50:50 H2O/MeCN solution containing 0.1% formic acid (v/v) and 10 µL of DMSO, at concentration of 1.0 mg·mL-1, and then further diluted 100-fold. Synthesis of the Ag-SIZ complex The silver(I) complex with sulfisoxazole (Ag-SIZ) was synthesized by the reaction of 5.0 x 10-4 mol (0.0846 g) of a freshly prepared aqueous AgNO3 solution (3.0 mL) with an aqueous solution (8.0 mL) containing 5.0 x 10-4 mol (0.1338 g) of SIZH and 1.0 x 10-3 mol (0.0574 g) of potassium hydroxide. The synthesis was carried out under stirring at room temperature for 2 hours. A white solid was obtained and vacuum filtered, washed with cold water and dried in a desiccator over P4O10. Elemental analysis led to a 1:1 Ag:SIZ composition. Anal. Calc. for C11H12AgN3O3S·H2O (%): C, 33.7; H, 3.60; N, 10.7. Experimental (%): C, 34.4; H, 3.18; N, 10.8. The yield of the synthesis was 90%. The Ag-SIZ

7 complex is insoluble in ethanol, methanol, hexane, acetonitrile and water. It is soluble in dimethylsulfoxide (DMSO). Synthesis of the Ag-SDMX complex The silver(I) complex with sulfadimethoxine (Ag-SDMX) was synthesized by the reaction of 5.1 x 10-4 mol (0.0860 g) of a freshly prepared aqueous solution of AgNO3 (3.0 mL) with an aqueous solution (2.0 mL) of SDMXH containing 5.1 x 10-4 mol (0.1589 g) of the ligand and 1.1 x 10-3 mol (0.0598 g) of potassium hydroxide. The synthesis was carried out under stirring at room temperature for 1 hour. A white solid was obtained and vacuum filtered, washed with cold water and dried in a desiccator over P4O10. Anal. Calc. for C12H13Ag N4O4S (%): C, 34.6; H, 3.14; N, 13.4. Experimental (%): C, 34.5; H, 2.82; N, 13.1. The yield of the synthesis was 95%. As observed for the Ag-SIZ complex, Ag-SDMX is also insoluble in ethanol, methanol, hexane, acetonitrile and water. It is soluble in DMSO. Structural analysis of Ag-SIZ and Ag-SDMX by X-ray powder diffraction Polycrystalline Ag-SIZ and Ag-SDMX samples were grounded in an agate mortar and then each powder was deposited in a very thin glass sampleholder plate. The diffraction data were collected by overnight scans in the 2θ range of 5-105° with steps of 0.02° using a Bruker AXS D8 da Vinci diffractometer, equipped with Ni-filtered Cu Kα radiation (λ=1.5418 Å), a Lynxeye linear position-sensitive detector – PDS and the following optics: primary beam Soller slits (2.94°), fixed divergence slit (0.3°) and receiving slit (5 mm for Ag-SIZ and 8 mm for Ag-SDMX). The generator was set at 40 kV and 40 mA. Standard peak search, followed by indexing through the single-value decomposition approach implemented in TOPAS [TOPAS-R, v.4.2, 2009, Bruker AXS, Karlsruhe, Germany] allowed the determination of approximate unit cell parameters. In the Ag-SIZ case, the C2 space group was chosen and the structure solution process was performed by the simulated annealing (SA) technique [38], implemented in TOPAS, employing a rigid body model for the full ligand (in the

8 Z matrix formalism), with free rotations, translation and torsion angles (Fig. 1 (A)). In the same way, the Ag(I) ion as well water molecule were left free of translation and rotation. For Ag-SDMX the space group assignment was troublesome since the a axis (22.4 Å) is nearly double than b axis (11.15 Å) and several Bragg peaks overlapped. In the present case, LeBail refinement in the space groups P21/a and P21/n afforded close Rwp values (6.52% and 6.94%) and even careful inspection of systematic absences did not resolve the space group ambiguity. Hence, the structure solutions, performed by the SA technique, were attempted in both space groups described above, eventually leading to a robust crystalchemically plausible model only when P21/a space group was chosen. In the structure solution process, a rigid body model for organic ligand was idealized as Cartesian coordinates data based on literature [39] and the rigid body was left free in translation and rotation and only flexible torsion angle, highlighted as blue arrows in Fig. 1, were left free. Also, free translations (x, y, z) were set up to Ag(I) ion. The final Rietveld refinement plots are shown in Supplementary Material S1, Fig. S1 and S2.

Fig. 1. Structure of anionic forms of (A) sulfisoxazole (SIZ) and (B) sulfadimethoxine (SDMX) based on literature data [39]. For the powder diffraction studies, six torsion angles were used and were assigned in the SIZ drawn molecule. The blue highlighted torsion angles in (B) define the conformation of the organic moiety in simulated annealing and all torsion angles were refined in the Rietveld refinement routine. In the final refinement, carried out by the Rietveld method [40], 54 parameters were refined for Ag-SIZ and 37 parameters for Ag-SDMX. The rigid

9 body description introduced at the solution stage was maintained for each structure. For Ag-SIZ, the background was modeled by 11 Chebyshev polynomial functions and the peak width anisotropy described by a spherical harmonics model was also refined in the present stage. An isotropic thermal parameter was assigned to all atoms and refined given a common isotropic thermal parameter, set at 10.67 Å2 for Ag-SIZ and 5.30 Å2 for Ag-SDMX. A summary of crystal data and data collection parameters is presented in Table 1. Table 1. Crystallographic data of Ag-SIZ and Ag-SDMX. Ag-SIZ·H2O

Ag-SDMX

Empirical formula

AgC11H14N3O4S

AgC12H13N4O 4S

Formula weight

392.18

417.19

T(K)

298

298

λ(CuKα) (Å)

1.5418

1.5418

Crystal System

Monoclinic

Monoclinic

Space Group

C2

P21/a

a (Å)

12.913(7)

22.421(3)

b (Å)

15.730(9)

11.138(1)

c (Å)

7.413(4)

5.941(5)

β (o)

110.46(3)

93.62(1)

V (Å3)

1410.8(1)

1480.8(3)

Z

4

4

dcalc (g cm-3)

1.825

2.079

µ (mm-1)

13.0

12.5

F(000)

776

832

Number of Parameters

54

37

RBragg,Rwp

0.017 / 0.071

0.063 / 0.10

10 Antibacterial assays In this study, three referenced bacterial strains (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 25923) were chosen to analyze the antibacterial activity of the Ag-SIZ and Ag-SDMX complexes. The microorganisms were cultured in separate test tubes containing 2.0 mL of sterile brain heart infusion (BHI) and incubated for 18 h at 35-37 °C, following the recommendations of the Clinical and Laboratory Standards Institute [41]. An inoculum from each culture was added to new tubes with sterile BHI until the turbidity measured was1.0 McFarland (≈ 3.0·108 CFU·mL-1; CFU = Colony Forming Units). Stock solutions of the free sulfonamides and silver complexes were prepared in DMSO (20.0 mg·mL-1). The tested compounds were sequentially diluted with BHI medium in 96 multiwell plate for a final volume of 100 µL/well. An inoculum of 100 µL of bacterial in BHI suspension at 1.0 McFarland scale was added to each serial dilution reaching turbidimetric 0.5 McFarland (≈ 1.5·108 CFU·mL-1) in a final volume of 200 µL/well. The multi-well plate was incubated for 18 h at 35-37 °C [41]. A negative control was obtained by leaving one of the wells of each bacterial strain with no addition of the considered compounds, only in DMSO, while silver nitrate was used as a positive control. The minimum inhibitory concentrations (MIC) were estimated as reported in the literature [41]. Results and discussion

Structural analysis by X-ray powder diffraction data The structures of the two silver complexes were solved by the X-ray powder diffraction technique using the Rietveld method for data refinement in the final stage. Both Ag-SIZ and Ag-SDMX structures belong to the monoclinic crystal system and to the C2 and P21/a space groups, respectively. The structures of both complexes are shown in Fig. 2 and selected bond lengths and angles are given in Table 2. In both structures the Ag(I) ions adopted nearly linear geometry and the deprotonation of the nitrogen atom from the sulfonamide group leads to its

11 coordination to Ag(I), as in other complexes with sulfonamides [28,29,31]. Molecular packing of both structures is presented in Supplementary Material S1, Fig. S3 and S4. The Ag-SDMX coordination sphere is consistent with other silversulfonamide complexes reported in the literature, where the classical eightmembered planar ring [AgNCN]2 is formed. In the Ag-SDMX structure, each silver ion bridges between the pyrimidinyl nitrogen atom of one ligand and the sulfonamide nitrogen atom of the second one [28,29,31]. This complex also shows an argentophilic interaction [42] of length 2.737(9) Å, being consistent with the values found for other silver complexes with sulfonamides [28,29,31]. However, the structure of Ag-SIZ differs from the classical mode of coordination. In this case, the dimer is formed by coordination of one silver ion to the nitrogen from the sulfonamide group and also to the nitrogen of the amino group of an adjacent sulfonamide molecule. The structure of Ag-SIZ does not have an argentophilic interaction due to the large distance (7.292 Å) between the two silver ions. In Ag-SIZ, the nitrogen atom of the sulfonamide group is coordinated to Ag(I) with a bond length of 2.270(2) Å (Ag-N7, see atom numbering in Fig. 2 (A)), whereas it is slightly shorter than Ag-N9, 2.408(6) Å in Ag-SDMX. Both values are similar to the distances found in literature for Ag(I) complexes with sulfonamides, as sulfathiazole (2.184(7) Å) [28] and sulfamoxole (2.152(4) Å) [31]. The Ag-N6 bond length (2.235(6) Å, Fig. 2(B)) in Ag-SDMX is also consistent with values found for pyrimidinyl nitrogen atoms coordinated to silver(I). The nitrogen atom of the NH2 group is coordinated to another Ag(I) in Ag-SIZ with the bond length of 2.018(8) Å (Ag-N12), forming a dimer with two sulfisoxazole and two silver atoms. The coordination of the amino group of sulfa drugs is more common in copper(II) complexes [43,44]. This interaction probably occurs in Ag-SIZ due to the position of the nitrogen atom of the isoxazole ring, which does not allow the formation of the common bridged [AgNCN]2 eight-membered ring of the silver-sulfonamide complexes.

12

Fig. 2. Dimers of (A) Ag-SIZ and (B) Ag-SDMX complexes obtained by X-ray powder diffraction. Silver is pink colored, while carbon, hydrogen, nitrogen, oxygen and sulfur are black, white, blue, red and yellow, respectively. Symmetry codes: i = 1 – x, y, 2-z, ii = -x, -y, -z. In the Ag-SDMX structure Ag···O interactions were also identified, which are in the limit of a weak bond and a strong interaction (see Fig. 2(B) and Table 2). The interactions of silver with the oxygen atom of the sulfonamide group were also observed for the silver complexes with sulfadiazine [45], sulfathiazole [28] and sulfadoxine [29]. Table 2. Main bond lengths and angles of Ag-SIZ and Ag-SDMX. For atoms numbering, see Fig. 2. Compounds

Lengths / Å

Ag-SIZ

Ag-N7

2.270(2)

Ag-N12

2.018(8)

Ag-N6

2.235(6)

Ag-SDMX

Ag-N9

Angle / ° N7-Ag-N12i

138.2(2)

N6ii-Ag-N9

167.9(3)

N6-Ag-Agii

87.3(3)

2.408(6) ii

Ag···Ag

2.737(9)

Ag···O15

2.806(7)

Ag···O16

2.899(6)

i = 1 – x, y, 2-z symmetry code. ii = -x, -y, -z symmetry code.

Thermal analysis In order to determine the thermal stability and to confirm the proposed compositions of the silver(I) complexes, thermogravimetric analysis (TGA) and

13 differential thermal analysis (DTA) studies were performed. The experimental TGA and DTA curves for the ligands and complexes are presented in Supplementary Material S2, Fig. S1-S4. The thermal decomposition of SIZH starts at 180 °C and no residue was observed at 900 °C. The oxidation of the Ag-SIZ complex occurred in three stages. The first step (endothermic event) is consistent with the loss of one water molecule of hydration. The second and third steps (exothermic) are consistent with the loss of one ligand (C11H12N3O3S) Anal. Calcd. for organic mass loss of AgC11H12O3N3S·H2O: 72.5%. Found: 71.2%. The final residue is consistent with the formation of metallic silver. Anal. Calcd. for Ag⁰ residue: 27.5%. Found: 28.8%. For SDMXH, the thermal decomposition starts at 190 oC and ends at 620 oC. Coordination of SDMX to silver(I) leads to a slight change in the profile of the thermal decomposition of the ligand (see Supplementary Material S2, Fig. S4). Anal. Calcd. for organic mass loss of AgC12H13O4N4S: 74.1%. Found: 72.9%. Anal. Calcd. for Ag⁰ residue: 25.9%. Found: 27.1%. These results agree with the proposed compositions by elemental analysis. Mass spectrometric measurements The Ag-SIZ and Ag-SDMX complexes were analyzed by ESI(+)-QTOFMS and the corresponding full spectra are presented in Fig. 3(A) and (B). The isotopic patterns (experimental and expected) are presented in Supplementary Material S3, Figure S1 (A-D). For Ag-SIZ, the peak at m/z 374.30 in Fig. 3(A) refers to the molecular ion [Ag(C11H12N3O3S) + H]+ and its isotopic pattern can be observed in Supplementary Material S3, Figure S1 (C). This result confirms the 1:1 metal/ligand composition. The peak at m/z 268.33 corresponds to the free ionized SIZH [C11H13N3O3S + H]+. The mass spectrum of the Ag-SDMX complex, Fig. 3(B), shows a peak corresponding to the [AgC12H13O4N4S + H]+ ion at m/z 416.98. One species containing one silver ion and two sulfonamide ligands and two protons is observed at m/z 729.05 [Ag(C12H13O 4N4S)2 + 2H]+, while a dimeric ion, consistent with the molecular structure determined from the X-ray powder diffraction data, is seen at m/z 870.93, corresponding to [Ag2(C12H13O 4N4S)2 +

14 2H + Cl]+. The presence of the chloride ion in this species is confirmed from the greater intensity of the isotope peak (see isotopic pattern in Supplementary Material S3, Figure S1 (D)) at m/z 872.92 when compared to a model without an added chloride. The presence of chloride ions may be originated from the solvent used in the experiment. The signal at m/z 311.07 is attributed to the protonated sulfonamide, [C12H14O4N4S + H]+.

Fig. 3. ESI(+)-QTOF mass spectrum for (A) the Ag-SIZ complex from m/z 100 to 500 and (B) the Ag-SDMX complex from m/z 100 to 900. NMR spectroscopic measurements Solution state 1H and

13

C NMR spectra were obtained in deuterated

DMSO solution in order to confirm the coordination sites of the sulfonamides to Ag(I). The spectra of the complexes were analyzed by comparison with the NMR spectrum of the corresponding ligands. Atoms numbering for signal attributions are given in Fig. 4.

15 As observed in Fig. 4, the peaks corresponding to the hydrogen from the sulfonamide group, H7 and H9 in the SIZH and SDMXH spectra, respectively, do not appear in the spectra of their corresponding Ag(I) complexes. This result confirms the loss of the hydrogen atom and reinforces the coordination of the nitrogen atom of the sulfonamide group to Ag(I) in both complexes. The hydrogen 12 in the SIZH spectrum is observed at 6.12 ppm, while in the spectrum of the complex this signal is observed at 5.64 ppm, showing a ∆δ (δ complex – δ ligand) of -0.48 ppm. This chemical shift difference also suggests that the nitrogen atom of the amino group is directly coordinated to Ag(I) in Ag-SIZ, forming a dimer as already observed in the crystallographic studies. For SDMXH, H14 is observed at 6.09 ppm, whereas in Ag-SDMX it appears at 5.83 ppm, with ∆δ of -0.26 ppm. This difference is also observed in other silver(I) complexes with sulfonamides, and it is an indicative of interactions of the NH2 group in solution, but probably not in direct coordination [28,29]. Table 3 presents the hydrogen and carbon atom assignments and the chemical shift differences for the sulfonamides and their corresponding Ag(I) complexes. Coordination of SDMX to Ag(I) via one of the nitrogen atoms of the pyrimidine ring is also evidenced by the greater difference of chemical shifts from the signals of H1 and H2. This may be originated by the alteration of the electronic structure of the pyrimidine ring after coordination of SDMX to Ag(I) by N6. These

proposals

are also verified by

13

C

NMR

spectroscopic

measurements and the recorded spectra are given in Supplementary Material S4, Fig. S1 and S2. The main chemical shifts occurred at carbons 3, 4, 5, 8 and 11 for SIZH and Ag-SIZ. For SDMXH and Ag-SDMX, the most significant shifts are observed for carbon atoms from the pyrimidine ring (C1, C2, C5 and C8), which is proposed to be directly involved in coordination.

16

Fig. 4. 1H NMR spectra of (A) SIZH, (B) Ag-SIZ, (C) SDMXH and (D) Ag-SDMX in DMSO-d6. Table 3. 1H and

13

C NMR assignments and chemical shifts for SIZH and Ag-SIZ, and

for SDMXH and Ag-SDMX. For hydrogen and carbon atoms numbering, see Fig. 4. Assignment

SIZH

Ag-SIZ

∆δ (ppm)

δ (ppm)

δ (ppm)

H1

2.09

2.01

-0.08

H2

1.63

1.72

H7

10.51

H9, H9a

Assignment

SDMXH

Ag-SDMX

∆δ (ppm)

δ (ppm)

δ (ppm)

H1

3.78

3.82

0.04

0.09

H2

3.79

3.89

0.10

−

−

H8

5.93

5.99

0.06

7.38

7.49

0.11

H9

11.09

-

-

H10, H10a

6.61

6.54

-0.07

H11, H11a

7.55

7.57

0.02

H12

6.12

5.64

-0.48

H12, H12a

6.59

6.56

-0.03

C1

10.82

11.27

0.45

H14

6.09

5.83

-0.26

C2

6.31

7.435

1.123

C1

53.69

54.55

0.86

C3

161.7

165.7

4.0

C2

54.40

55.42

1.02

C4

104.8

93.95

-10.85

C3

171.60

171.89

0.29

C5

156.8

161.2

4.4

C5

160.18

163.80

3.62

C8

124.9

131.2

6.3

C7

164.40

164.93

0.53

C9, C9a

129.2

128.6

-0.6

C8

84.21

85.70

1.49

C10, C10a

113.1

112.9

-0.2

C10

153.35

152.63

-0.72

C11

153.8

151.7

2.1

C11, C11a

129.34

129.37

0.03

C12, C12a

112.43

113.02

0.59

C13

124.16

126.71

2.55

17 Infrared absorption spectroscopy The IR spectra of the silver complexes were analyzed in comparison to the spectrum of the free sulfonamides. The IR spectra are provided in Supplementary Material S5, Fig. S1 and S2. The spectrum of SIZH has two medium intensity absorptions in the region of 3500-3200 cm-1, which are assigned to the asymmetrical stretching (ν as) of the NH2 group in 3485 cm-1 and to the stretching of the N-H group of sulfonamide at 3377 cm-1 [46,47]. The symmetrical stretching (νs) of the amino group could not be identified since this band is possibly overlapped by the ν(N-H) band of the sulfonamide group. For SDMXH, the νas and the νs bands appear at 3449 and 3345 cm-1, respectively, whereas the band at 3223 cm-1 can be attributed to the ν(N-H) of the sulfonamide group. In the IR spectra of the Ag-SIZ and Ag-SDMX complexes the characteristic medium intensity absorption band of ν(N-H) of sulfonamide group is not observed, confirming the loss of the hydrogen atom and coordination to Ag(I) by this group, as observed previously from X-ray diffraction and NMR spectroscopic data. Asymmetric and symmetric stretching of NH2 group in Ag-SIZ appear at 3569 and 3357 cm-1, respectively, with a ∆ν of 84 cm-1, also confirming coordination of Ag(I) by the nitrogen atom of the NH2 group as observed in the X-ray crystallographic studies. For Ag-SDMX, however, the ∆ν is of only 27 cm-1, showing once again the involvement of the NH2 group in weaker interactions and not in direct coordination to Ag(I) [28,31]. Minimum inhibitory concentration (MIC) The determination of the minimum inhibitory concentration (MIC) was used to evaluate the antibacterial activity of the Ag-SIZ and Ag-SDMX complexes over Gram-positive (S. aureus) and Gram-negative (E. coli and P. aeruginosa) bacterial strains, some of the most common microorganisms responsible for wounds colonization [48]. The results are presented in Table 4. The Ag-SDMX complex was shown to be more active when compared to the Ag-SIZ one. For the Ag-SIZ complex, the MIC value for E. coli was 1597.8 µmol·L-1, while for P. aeruginosa, the MIC was 798.9 µmol·L-1. For S. aureus

18 the MIC was 399.4 µmol·L-1. For Ag-SDMX, the lowest MIC found was of 93.6 µmol·L-1 for both E. coli and P. aeruginosa. For S. aureus, the MIC value was 187.3 µmol·L-1. These results also indicate that Ag-SDMX is twice more effective against Gram-negative bacteria than the positive one (S. aureus). The obtained results for the free SIZH showed that it did not exhibit antibacterial activities in any concentration used in the assay. For free SDMXH the MIC value was 16.1 mmol·L-1, which shows its low activity over the considered strains. The different activity profile observed for both complexes may be related to the dissimilar coordination modes of the two sulfonamides to silver(I), which may result in different degrees of silver release. As suggested by Fox, silver complexes with sulfonamides are active due to a slow silver release, whereas the ligand role is mainly of a carrier [49]. The stability of the complexes in solution was evaluated by UV-Vis spectroscopic measurements over 22 h. The UV-Vis spectra revealed a slight hypochromism of the main absorption band of Ag-SIZ (see Supplementary Material S6, Fig. S1). The spectra of Ag-SDMX showed, in addition to the slight hypochromism, a minor batochromic shift of the main absorption band, with the presence of an isosbestic point (Supplementary Material S6, Fig. S2). The observed results indicate a greater dissociation with the release of silver ions in Ag-SDMX when compared to Ag-SIZ. The different degree of dissociation of the complexes may be related to the greater antibacterial activities of Ag-SDMX. The MIC values found for the Ag-SDMX complex, which was shown to be the most active one, are comparable to the activity of silver sulfadiazine, which shows MIC values from 180 to 360 µmol·L-1 for S. aureus and from 45 to 90 µmol·L-1 for E. coli and P. aeruginosa bacterial strains [50]. The antibacterial activities of the Ag-SDMX complex are also comparable to the activities of a silver(I) complex with sulfameter (Ag-SMTR) recently published in the literature, which presents MIC values of 40.9 µmol L-1 for E. coli and P. aeruginosa bacteria [51]. The MIC values found for the Ag-SDMX complex also indicate that this compound is more active in vitro than the first published silver complexes with the sulfonamides sulfathiazole (Ag-SFT) and sulfamethoxazole (Ag-SFM) over the same bacterial strains. The MIC values for Ag-SFT and Ag-SFM were 6.90

19 mmol L-1 and 13.90 mmol L-1 over S. aureus, respectively, while for P. aeruginosa, the MIC values for Ag-SFT and Ag-SFM were 3.45 mmol L-1 and 1.74 mmol L-1, respectively [28]. In the case of silver nitrate, the MIC value found for S. aureus, E. coli and P. aeruginosa bacterial strains was 0.724 µmol L-1 [51]. The MIC values of standard antibiotics for the same bacterial strains are reported in CLSI [41]. Although silver nitrate exhibits MIC values lower than those observed for the complexes, silver nitrate in concentrations higher than 1% are toxic to tissues. Also, the instability in the presence of light and the fast release of silver ions from AgNO3 have limited its application in medicine [49,52]. Silver complexes, such as silver sulfadiazine, can provide a slow and sustained release of silver ions expanding its applications. In addition, an early reported copper(II) complex with SIZ did not show any selectivity over either S. aureus or E. coli strains and the free SIZH was also found to be only weakly active under the tested conditions [27]. However, the copper complex with SDMX already described in the literature was 6 times more active against S. aureus than E.coli [43]. Table 4. Minimum inhibitory concentration (MIC) values of the antibacterial activities of the Ag-SIZ and Ag-SDMX complexes compared to free SIZH, SDMXH and AgNO3. Compounds

MIC (µmol·L-1) Gram-positive

Gram-negative

S. aureus

E. coli

P. aeruginosa

SIZH

R

R

R

Ag-SIZ

399.4

1597.8

798.9

SDMXH

16.1 x 103

16.1 x 103

16.1 x 103

Ag-SDMX

187.3

93.6

93.6

AgNO3(a)

72.4 x 10-2

72.4 x 10-2

72.4 x 10-2

R: resistant, aRef. [51]. Conclusions Silver(I) complexes with sulfisoxazole and sulfadimethoxine were synthesized. Molar composition was 1:1 metal/ligand. Chemical analyses confirmed the minimal formulas AgC11H12N3O3S·H2O and AgC12H13O4N4S for

20 the Ag-SIZ and Ag-SDMX complexes, respectively. The mass spectrometric analysis permitted identification of the representative ions [Ag(C11H12N3O3S) + H)]+ and [Ag2(C12H13O 4N4S)2 + 2H + Cl]+. Crystallographic studies indicated an unusual structure for Ag-SIZ complex, where the coordination of the ligand to the silver atom occurs by the nitrogen atoms of the sulfonamide and amino groups. The Ag-SDMX structure is consistent with classical coordination of the nitrogen of the sulfonamide group and one nitrogen from the pyrimidine ring to the Ag(I) ions, with the formation of a dimer with an argentophilic interaction. Infrared and

1

H and

13

C NMR spectroscopies confirmed the proposed

coordination sites of the ligands to Ag(I). The MIC values showed that both complexes are effective against Gram-positive and Gram-negative bacterial strains. The Ag-SDMX complex was more active over the considered bacteria. This behavior may be related to the different coordination modes of the sulfonamides to the silver ions, which can affect the silver release. Acknowledgements This study was supported by grants from the Brazilian Agencies FAPESP (São Paulo State Research Council, Grants # 2015/20882-3, 2015/09833-0 and 2015/25114-4), CNPq (National Council of Scientific and Technological Development, Grant # 442123/2014-0) and Rede Mineira de Química (RQ-MG) supported by FAPEMIG (Project: REDE-113/10; Project: CEX - RED-0001014). Supplementary Material Crystal data, fractional atomic coordinates and displacement parameters of Ag-SIZ and Ag-SDMX crystal structures described in manuscript are supplied in standard CIFs deposited in the Cambridge Crystallographic Data Centre (#1499483 and #1499482). The data can be obtained free of charge at http://www.ccdc.cam.ac.uk/conts/retrieving.html

[or

from

Cambridge

Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 (0) 1223-336033; e-mail: [email protected]].

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25

Figure Captions Fig. 1. Structure of anionic forms of (A) sulfisoxazole (SIZ) and (B) sulfadimethoxine (SDMX) based on literature data [39]. For the powder diffraction studies, six torsion angles were used and were assigned in the SIZ drawn molecule. The blue highlighted torsion angles in (B) define the conformation of the organic moiety in simulated annealing and all torsion angles were refined in the Rietveld refinement routine. Fig. 2. Dimers of (A) Ag-SIZ and (B) Ag-SDMX complexes obtained by X-ray powder diffraction. Silver is pink colored, while carbon, hydrogen, nitrogen, oxygen and sulfur are black, white, blue, red and yellow, respectively. Symmetry codes: i = 1 – x, y, 2-z, ii = -x, -y, -z. Fig. 3. ESI(+)-QTOF mass spectrum for (A) the Ag-SIZ complex from m/z 100 to 500 and (B) the Ag-SDMX complex from m/z 100 to 900. Fig. 4. 1H NMR spectra of (A) SIZH, (B) Ag-SIZ, (C) SDMXH and (D) Ag-SDMX in DMSO-d6.

26 Fig. 1- Manuscript Fiori et al.

27 Fig. 2- Manuscript Fiori et al.

28 Fig. 3- Manuscript Fiori et al.

Fig. 3. ESI(+)-QTOF mass spectrum for (A) the Ag-SIZ complex from m/z 100 to 500 and (B) the Ag-SDMX complex from m/z 100 to 900.

29 Fig. 4- Manuscript Fiori et al.

30 Graphical Abstract – Manuscript Fiori et al.

Graphical Abstract - synopsis Molecular view of the Ag-SIZ and Ag-SDMX dimers obtained by X-ray powder diffraction. Silver is pink colored, while carbon, hydrogen, nitrogen, oxygen and sulfur are black, white, blue, red and yellow, respectively.