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Silver sulfadoxinate: Synthesis, structural and spectroscopic characterizations, and preliminary antibacterial assays in vitro
Journal of Molecular Structure 1082 (2015) 180–187
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Silver sulfadoxinate: Synthesis, structural and spectroscopic characterizations, and preliminary antibacterial assays in vitro 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, Brazil Biological and Health Sciences Department – UNIARA, 14801-320 Araraquara, SP, Brazil Department of Chemistry, Exact Sciences Institute – UFJF, Juiz de Fora, MG, Brazil d Dipartimento di Scienza e Alta Tecnologia, Università degli Studi dell’Insubria, 22100 Como, Italy b c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A novel silver(I) complex with
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.
sulfadoxine. IR and NMR data indicate coordination of the ligand to Ag(I) by the nitrogen and oxygen atoms. Structural characterization of the complex was based on X-ray powder diffraction data. Antibacterial activities of the complex were observed over Gram-negative and Gram-positive strains.
a r t i c l e
i n f o
Article history: Received 4 September 2014 Received in revised form 2 November 2014 Accepted 2 November 2014 Available online 13 November 2014 Dedicated to Professor Antonio C. Massabni in the occasion of his 70th birthday. Keywords: Silver complex Sulfadoxine ESI-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) through the nitrogen atoms of the (deprotonated) sulfonamide group and by the pyrimidine ring, as well as through oxygen atom(s) of the sulfonamide group. These hypotheses were corroborated by 13C and 15 N SS-NMR spectroscopy and by an unconventional structural characterization based on X-ray powder diffraction data. The latter showed that AgSFX crystallizes as centrosymmetric dimers with a strong Ag Ag interaction of 2.7435(6) Å, induced by the presence of exo-bidentate N,N0 bridging ligands and the formation of an eight-membered ring of [AgNCN]2 sequence, nearly planar. Participation of oxygen atoms of the sulfonamide residues generates in the crystal a 1D coordination polymer, likely responsible for its very limited solubility in all common solvents. Besides the analytical, spectroscopic and structural
Abbreviations: SFX, Sulfadoxine (4-Amino-N-(5,6-dimethoxy-4-pyrimidinyl)benzenesulfonamide); AgSFX, Ag(I) complex with sulfadoxine; AgSFD, Ag(I) complex with sulfadiazine; NMR, nuclear magnetic resonance; HSQC, Heteronuclear Single Quantum Coherence; HMBC, Heteronuclear Multiple Bond Coherence; SS-NMR, Solid State NMR spectroscopy; ESI-TOF-MS, Electrospray Ionization Time-of-flight Mass Spectrometry; IR, Infrared Spectroscopy; TGA/DTA, Thermogravimetric and Differential Thermal Analysis; XRPD, X-ray Powder Diffraction; ATCC, American Type Collection Cell; MH, Mueller-Hinton agar; BHI, Brain-Heart Infusion Medium; CFU, Colony Forming Unit. ⇑ Corresponding author. Tel.: +55 19 35213130; fax: +55 19 35213023. E-mail address: [email protected] (P.P. Corbi). http://dx.doi.org/10.1016/j.molstruc.2014.11.004 0022-2860/Ó 2014 Elsevier B.V. All rights reserved.
N.T. Zanvettor et al. / Journal of Molecular Structure 1082 (2015) 180–187 X-ray powder diffraction Antibacterial activities
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description, the antibacterial properties of AgSFX were assayed using disc diffusion methods against Escherichia coli and Pseudomonas aeruginosa (Gram-negative), and Staphylococcus aureus (Gram-positive) bacterial strains. The AgSFX complex showed to be active against Gram-positive and Gram-negative bacterial strains, being comparable to the activities of silver sulfadiazine. Ó 2014 Elsevier B.V. All rights reserved.
Introduction Metal complexes have been widely used in pharmacology and medicine worldwide for the treatment of many diseases. The medicinal 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 millennia later, in the 19th century (well before the advent of antibiotics), silver compounds were popular drugs [2]. The main use of silver was for the treatment of burns and wounds, efficiently limiting bacterial infection. Initially, silver dosage was made through solid silver nitrate by the use of instruments as the so-called lapis infernalis, followed by the use of solutions and foils [3]. Recently, the advent of penicillin and sulfa drugs made silver-based antibacterial agents fall into disuse. The interest in silver was recovered by Moyer, who proposed the use of silver nitrate solutions to treat burns [4]. Unfortunately, the fast delivery of silver(I) ions into the blood system causes severe toxicity, and it is accompanied by the inhibition of the epithelial growth. In the 1970’s, Fox studied [5] the combination of different sulfonamides with silver nitrate, aiming for improving the antibacterial activity, while diminishing the undesired effects of silver nitrate solutions. It was then demonstrated [6] that Fox’s antibacterial agent was a silver complex with sulfonamide. Among the many sulfonamides, silver sulfadiazine (AgSFD) was the one that showed the highest activity against bacteria, and was soon introduced in the clinic for the treatment of burn infections and skin ulcers as a topical cream [2,4–8]. The good performance of silver sulfadiazine is mainly attributed to the presence of slowly released Ag+ ions [9–11], with no sulfadiazine molecules being ever found inside of the bacterial cells. In this case, sulfadiazine acts as a carrier of silver ions, and avoids the readily precipitation of Ag+ as silver chloride, or oxide/hydroxide, maintaining the electrolyte levels of the body fluids as well as the antibacterial activity due to the constant concentration gradient of Ag+ [9]. Nowadays silver is known as a broad-spectrum antimicrobial agent and diverse formulations are commercially available. For
example, colloidal silver, silver salts, silver complexes, nanocrystalline silver, silver oxide, and silver zeolite formulations are known, all possessing good antimicrobial activity [2,8]. However, the widespread use of silver has caused the isolation of some resistant bacterial strains. The studies on silver resistance are sparse, but they suggest that the resistance is mediated by plasmid. These evidences increased the concerns about the misuse of silver and the development of novel antibacterial silver compounds became of great interest [9]. For this reason, novel silver complexes with enhanced, or tailored, antimicrobial activity are continuously being investigated. For example, a silver complex with N-acetyl-L-cysteine was recently synthesized, and showed a broad spectrum of activity against microorganisms [12]. In addition, a silver complex with the antiinflammatory drug nimesulide (another sulfonamide) [13] was shown to be active against Gram-positive and Gram-negative bacteria. Moreover, the Ag(I) derivatives are currently being investigated as anticancer drugs [12,14]. Sulfadoxine (SFX, C12H14N4SO4, Fig. 1) is a sulfonamide widely used 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 antimalarial activity, the sulfadoxine-pyrimethamine hybrid is also active against the parasite Toxoplasma gondii [15] and is considered as primary prophylaxis of pneumonia caused by Pneumocyctis carinii [16–18] and toxoplasmatic encephalitis in patients infected with HIV [17,18]. Sulfadoxine is also used in combination with trimethoprim to treat different bacterial infections in animals as horses and calves, being active against Gram-negative and Gram-positive bacteria [19–21]. Metal complexes of sulfadoxine were also recently studied and Fe(II), Fe(III), Co(II), Cu(II), Cr(III) derivatives were synthesized [22–24]. Ogunniran et al. synthesized Cu and Fe complexes with sulfadoxine and pyrimethamine, and showed that these species are active against E. coli, S. aureus, P. aureginosa and S. typhi. The complexes have shown to have an enhanced activity in comparison with the free ligands [23]. In the current manuscript, inspired by these works and by the desirable properties of many silver derivatives, we present the synthesis, spectroscopic and structural characterization, and the antibacterial activity of the novel silversulfadoxine (AgSFX) complex. Experimental Materials and methods
Fig. 1. Sketch of SFX molecule. The SFX molecular conformation was defined by eight torsion (si) angles indicated by arrows and used in the powder diffraction study (vide infra).
Sulfadoxine (SFX 95%) and silver 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 CHNS/O Perkin Elmer 2400 Analyzer. Infrared (IR) spectra from 4000 cm1 to 400 cm1 of SFX and the AgSFX complex were measured using a Bomen MB Series Model B100 spectrometer with resolution of 4 cm1; samples were prepared as KBr pellets. The 1H nuclear magnetic resonance (NMR) spectrum of SFX was recorded on a Bruker Avance 400 MHz spectrometer operating at 400.1 MHz. The 13C
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NMR spectrum of SFX was also recorded on a Bruker Avance 400 MHz operating at 100.6 MHz. The heteronuclear [1HA13C] single quantum coherence (HSQC), heteronuclear [1HA13C] multiple bond coherence (HMBC) and the heteronuclear [1HA15N] multiple bond coherence (HMBC) spectra of SFX were acquired on a Bruker Avance 400 MHz, using a 5-mm probe at 303 K, operating at 40.5 MHz for 15N; samples were was prepared in deuterated dimethylsulfoxide solutions. The solid state nuclear magnetic resonance (SS-NMR) 13C spectrum of SFX an AgSFX was recorded on a Bruker 400 MHz Avance II (9.395T) operating at 100 MHz using cross polarization, proton decoupling and magic angle spinning (CP/MAS). The solid-state 15N{1H} nuclear magnetic resonance spectra were recorded on a Bruker 400 MHz, using the combination of 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 Micro API instrument: methanol was added to a sample of 1.5 mg AgSFX, the supernatant was separated and formic acid was added until concentration of 1%; the resulting solution was directly infused into the instrument’s ESI source with capillary potential of 3.00 kV, cone potential of 30 kV, trap potential of 2 kV, source temperature of 150 °C and nitrogen gas for desolvation. Synthesis of the complex The silver(I) complex with sulfadoxine (AgSFX) was synthesized by the reaction of 1.0 103 mol of a freshly prepared aqueous AgNO3 solution (2 mL) with a basic aqueous solution of SFX containing 1.1 103 mol of potassium hydroxide and 1.0 103 mol of SFX (12.5 mL). After one hour of constant stirring at room temperature, a white solid precipitated and was collected by filtration, washed with cold water, and dried in a desiccator with P4O10. Yield 71.9%. 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 in water, DMSO, DMF, chloroform, acetonitrile, hexane, ethanol and methanol. Accordingly, in the absence of single crystals of suitable size and quality, and being impossible to grow suitable samples by recrystallization from solution, we adopted the powder diffraction route to structural characterization [25] which, in through the years [26–28], became a viable and efficient tool to obtain otherwise inaccessible structural information of metal complexes of limited structural complexity. Structural analysis of AgSFX by X-ray powder diffraction data The polycrystalline AgSFX material was gently ground in an agate mortar and the powder was deposited in a sample holder equipped with a silicon zero-background plate. Diffraction data were collected at room temperature by an overnight scan in the 2h range of 6–105° with steps of 0.02° using a Bruker AXS D8 Advance diffractometer equipped with Ni-filtered Cu Ka radiation (k = 1.5418 Å) and Lynxeye linear position-sensitive detector. The
following optics were set up: primary beam Soller slits: 2.3°, fixed divergence slit: 0.5°, and receiving slit: 8 mm. The generator was set at 40 kV and 40 mA. Standard peak search and profile fitting, followed by indexing through the single-value decomposition approach implemented in TOPAS [29] allowed detection of the approximate monoclinic unit cell parameters (GOF: 3.04 from Indexing and 3.78 from LeBail). Density considerations suggested Z = 4 and the space group P21/c was chosen. Structure solution was performed by the simulated annealing technique implemented in TOPAS. A (partially flexible) rigid body for SFX was used with free molecular location and orientation within the unit cell; the SFX rigid body was defined by Cartesian coordinates, obtained by molecular mechanics optimization of the bond distances and angles, using the ChemSketch freeware program, and by resizing the CAH bond distances down to the common ‘‘X-ray’’ value of 0.95 Å. In addition, s1 to s5 torsion angles (see Fig. 1) were freed in the simulated annealing step, while a single silver(I) ion was left to freely float in the cell. The final refinement was carried out by the Rietveld method maintaining the rigid body introduced at the simulated annealing stage. Additionally, a total of eight torsion angles were refined in the final cycles. The background was modelled by a Chebyshev polynomial function. An isotropic thermal parameter was assigned to the metal (BAg) and refined; lighter atoms were given a Biso = BAg + 2.0 Å2 thermal parameter. The final Rietveld refinement plot is supplied as Fig. 2.
Antibacterial assays Three referenced bacterial strains: Escherichia coli – ATCC 25,922, Pseudomonas aeruginosa – ATCC 27,853 and Staphylococcus aureus – 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 separate test tubes containing 5.0 mL of sterile brain heart infusion (BHI) medium and incubated for 18 h at 35–37 °C. Sufficient inocula were added in new tubes until the turbidity equaled 0.5 McFarland (1.5 108 CFU/cm3). The bacterial inocula diluted with BHI (McFarland standard) were uniformly spread using sterile cotton swabs on sterile Petri dishes containing MH agar. Sterile filter paper discs (10 mm diameter) were aseptically impregnated with 800 lg of SFX and AgSFX according to the following procedure: 20 mg of the compounds were suspended in 1000 lL of distilled water, homogenized in a vortex, and 50 lL of the suspension were collected with a micropipette and transferred to the paper discs. Sterile discs impregnated with 1000 lg of pure SFX were used as a negative control. Discs impregnated with the AgSFX complex or with SFX were dried and sterilized in a vertical laminar flow under UV radiation for 45 min before the experiment. The impregnated discs were placed on the surfaces of the solid agar. The plates were incubated for 18 h at 35–37 °C and examined thereafter. Clear zones of
Fig. 2. Final Rietveld refinement plot for AgSFX complex, with difference plot and peak markers at the bottom. The high angle region is shown as inset.
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inhibition around the discs were measured and the complex sensitivity was assayed from the diameter of the inhibition zones (in millimeters). The observed results were compared to discs with the standard antibiotic levofloxacin, and with discs impregnated with 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 powder X-ray diffraction analysis on laboratory data and, despite of the high number of torsion angles of SFX to be determined, relevant crystallochemical information (stoichiometry, crystal packing, conformation) could be obtained. Crystal data for AgSFX are summarized in Table 1, while relevant bond distances and angles are given in Table 2. The centrosymmetric dimer found in the crystal structure of AgSFX is shown in Fig. 3, where only atoms of the asymmetric unit were labelled. As pictorially shown therein, the crystals of AgSFX structure contain dinuclear moieties centered about the strong Ag Ag argentophilic interaction (2.744(6) Å). The eight-membered ring of the [ANCNAgA]2 type present in the dimer is further bound to oxygen atoms of S@O group (with AgAO interactions in the 2.6–2.7 Å range), eventually connecting the different moieties in a 1D polymer, stretching along the a axis. The proposed structure of AgSFX requires some further comments. Apparently, the refined distances for the AgAN1 and AgAN3 bonds (2.256(2) and 2.533(1) Å, respectively, similar to the typical values found for other Ag(I) complexes containing N-donor ligands [32] possess rather distinct values, and suggested a deeper look into the problem, aiming at detecting if, for any reason, this discrepancy is real. This doubt is even more pronounced for XRPD structure determinations of moderately complex systems, which are known
Table 1 Crystallographic data of AgSFX. AgSFX Empirical formula Formula weight T(K) k(Cu Ka) (Å) Crystal system Space group a (Å) b (Å) c (Å) b (°) V (Å3) Z dcalc (g cm3) l (mm1) F (0 0 0) Number of parameters RBragg, Rwp
C12H13AgN4O4S 417.18 298 1.5418 Monoclinic P21/c 5.4448(5) 19.226(2) 13.852(1) 102.502(5) 1415.7(2) 4 1.9573 13.09 800 34 0.044/0.080
Bond distances (Å) Ag–N1 Ag–N3i Ag–O12i Ag–O11ii Ag Agi
Angles (°) 2.26(2) 2.53(1) 2.60(2) 2.71(2) 2.744(6)
N1–Ag–N3i N1–Ag–O12i N1–Ag–O11ii N3i–Ag–O12i N3i–Ag–O11ii O12i–Ag–O11ii
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, the Cambridge Structural Database (CSD) was searched for cyclic eight-membered ring [AAgNCNA]2 type, and a statistical average of 2.241 Å for bonded AgAN was found, with a broad distribution reaching values higher than 2.6 Å. At the same time, the Ag Ag distance distribution showed highly probable intermetallic interactions near, or slightly below, 2.80 Å. Several restrained Rietveld refinements were also performed, on the same original XRPD data, by setting equal AgAN distances at 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 well defined minimum. These data were subjected to a parabolic fit, which gave the minimum Rwp value (0.083) for a bond distance of 2.542 Å. Needless to say, on relaxing the two AgAN distances, a slightly lower Rwp value (0.080) is obtained. Thus, the close matching of (i) the statistically derived AgAN average and the observed AgAN1 values; (ii) the typically observed Ag Ag distances and the 2.744(6) Å value here determined, and (iii) the distance determined for minimum Rwp value in the restrained refinements and the observed AgAN3 bond, significantly suggest that the proposed structural model cannot be too far from reality. Note that the final comment is based on two statistically independent observations: our experimental XRPD data and a complete statistical analysis of CSD. The bond distances of AgAO are, normally, larger than AgAN [34] accordingly, also in the AgSFX complex, long(er) AgAO bond distances 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 the highest abundance at m/z 419 assigned to [AgC12H13N4O4S + H+]. An adduct of the molecular ion with methanol is observed at m/z 451. The free ligand, C12H14N4O4S, is also observed as singly charged [C12H14N4O4S + H+] ions of m/z 311, respectively. The silver ion is observed at 109 m/z and a peak at 156 m/z is assigned as the breakage of the ligand molecule, forming the ion [C6H6NSO+2]. 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 structure previously observed in the powder diffraction data, but only more soluble fragments were detected in the analysis. An isotope pattern for the singly charged [AgSFX + H+] ion is also shown on Fig. 5. Solid-state
Table 2 Relevant bond distances (Å) and angles (°) for AgSFX.
170.9(4) 123.1(5) 83.6(5) 58.6(3) 105.4(4) 78.7(4)
183
13
C and
15
N spectroscopic measurements
The SFX 13C NMR spectrum in solution was assigned based on the NMR by experiments of [13CA1H] HSQC and HMBC and [15NA1H] HMBC. The spectrum in solution was later used to perform the assignment of the SS-NMR 13C spectra of SFX and AgSFX. The spectra of SFX in solution are provided as Supplementary material. The AgSFX 13C SS-NMR spectrum was analysed in comparison to that of SFX. The 13C SS-NMR spectra of AgSFX and SFX are provided in Fig. 6. The structure of SFX with the carbon atoms numbering is provided on Fig. 1. The spectrum of the complex shows more resolved peaks than the free ligand, which is probably due to the presence of different polymorphs of the ligand as previously reported [35]. As demonstrated by powder diffraction analysis, only one polymorph was isolated for the complex. Besides the change in resolution, significant variations of the chemical shifts are observed when comparing the two spectra. The 4 and 7 carbon atoms signals shift to lower field by 3.5 and 3.3 ppm, respectively due to the coordination. This
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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. The atoms 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 figure legend, the reader is referred to the web version of this article.)
10 Rwp
R wp , %
9.5
"Final Model"
9 8.5 8 7.5 7 2
2.2
2.4
2.6
2.8
Ag-N, Å Fig. 4. Plot of the standard Rietveld refinement Rwp value (%) vs. restrained (and equal) Ag–N bond distances in AgSFX (black dots). A deep minimum near 2.54 Å is clearly seen. The empty circles are positioned at Rwp 8.02 (the absolute minimum in the 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 CSD search and the minimum of the Rwp curve, respectively.
observation agrees with the powder diffraction result that the coordination of the ligand to the metal occurs through the nitrogen of the sulfonamide group and the amine group is involved in a series of hydrogen bonds with the oxygen of the sulphonamide group of the next dimer of AgSFX. Other shifts are observed for the signals of carbon atoms 5, 2 and 3 by 2.5, 2.1 and 3.7 ppm respectively upon the coordination. These results also agree with previous powder diffraction data that the coordination of sulfadoxine to Ag(I) is also through nitrogen 1 of the pyrimidine ring, changing the electronic density of all the carbons of the ring. This mode of coordination, through the sulphonamide and the nitrogen of the pyrimidine ring is common and is also observed for silver sulfadiazine [36]. No significant variations of the chemical shifts of the methoxy group were observed, which indicates that this functional group 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 comparison to that of SFX. The 15N SS-NMR spectra of AgSFX and SFX are provided in Fig. 7, while the numbered structure of SFX is provided in Fig. 1.
Four signals assigned to the four nitrogen atoms are observed in the spectrum of the SFX molecule, the two nitrogen atoms of the pyrimidine ring, N2 and N1, at 251.0 and 246.7 ppm, respectively, the sulfonamide group nitrogen at 121.5 ppm and the amine group nitrogen at 71.7 ppm. Nevertheless, the signal of the sulfonamide group nitrogen is not observed in the complex 15N spectrum. Since the cross polarization NMR technique requires the existence of a hydrogen atom near to this nitrogen for the polarization transfer [37] it indicates, that the coordination of the ligand to the silver atom is accompanied by the loss of the hydrogen and the coordination of the silver ion to the nitrogen atom of sulphonamide group. The signals of the nitrogen atoms of the pyrimidine ring are observed at 206.3 and 188.1 ppm in the spectrum of the complex, which represents a shift of 44.7 ppm for N2 and 58.6 ppm for N1 to higher field, respectively, when compared to the ligand spectrum. This is consistent with the participation of N1 in the coordination. The shift observed for N2 is probably due to changes in the electron density of the whole ring, caused by the coordination to N1. The nitrogen of the amino group is observed at 20.2 ppm in the spectrum of the complex with a shift of 51.5 ppm to higher field when compared to the ligand spectrum. The shift observed for the amine group can be attributed to hydrogen bonding to the oxygen atom of the sulphonamide group of the adjacent dimer of AgSFX. The NMR data is consistent to the structure proposed by the powder diffraction. The chemical shifts of all the carbon and nitrogen NMR signals are provided in Table 3. IR spectroscopic data The AgSFX IR spectrum was analysed in comparison to that of SFX. The IR spectra of AgSFX and SFX of the region from 4000 to 2500 cm1 are provided in Fig. 8. The spectrum of SFX shows three absorption bands at 3240, 3377 and 3465 cm1, assigned as the NAH stretches of the sulphonamide group and the symmetric and asymmetric NAH stretches of the amine group, respectively. In the spectrum of the complex the NAH stretch of the sulphonamide group is observed as a very weak broad shoulder, whereas the stretches of the amine group do not change considerably as it can be observed in Fig. 8. This result indicates that the ligand coordinates to the metal through
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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 AgSFX complex.
the nitrogen atom of the sulfonamide as the hydrogen atom bonded to the sulphonamide group is no longer present, consequently the NAH stretching is also absent. This result is consistent with the SS-NMR results and the powder diffraction data. The comparison of the two spectra also shows a shift of the C@N stretching mode of the pyrimidine ring from 1650 cm1 in the SFX spectrum to 1618 cm1 in the complex which can also indicate coordination of the ligand to the metal by the nitrogen atoms of the pyrimidine ring. In the spectrum of the ligand the O@S@O asymmetric and symmetric stretching modes of the sulphonamide group also shifts from 1319 cm1 and 1157 cm1 in the ligand to 1236 cm1 and 1115 cm1 in the complex, respectively. The changes of O@S@O stretching frequencies suggest the ligand coordination to the metal through this group as well, and the decrease in frequency indicates the coordination by the oxygen atom [38], as shown by the powder diffraction data, where one oxygen atom of the sulphonamide coordinates to the silver ion and the other one interacts weakly with the silver ion of the adjacent dimer and also forms hydrogen bonds with the amine group of the adjacent dimer. This observation is also in agreement with the reported structure of silver sulfadiazine, which presents the oxygen of sulphonamide as a coordination site in the polymeric structure [34]. The full IR spectrum of the complex and the ligand are provided as Supplementary material.
Fig. 7. 15N solid-state nuclear magnetic resonance spectra of SFX and AgSFX complex.
Table 3 Solid-state
13
C and
15
N NMR data for SFX and AgSFX.
Assignment
C7 C8, C12 C9, C11 C10 C4 C5 C2 C3 2OCH3 3OCH3 N2 N1 NH–SO2 NH2
Chemical shift (ppm) SFX
AgSFX
152.9 129.5 114.7, 113.3 125.9 150.5 150.5 162.5 125.9 53.8 60.0 251.0 245.7 121.5 71.7
156.2 132.6 115.6, 113.7 127.9 154.0 148.0 164.6 129.6 55.4 59.4 206.3 188.1 – 20.2
Antibacterial assays Antibacterial sensitivity profiles of bacterial strains demonstrate the antibacterial activity of the silver(I) complex with SFX against Gram-negative (E. coli and P. aeruginosa) and Gram-positive
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Conclusion
Fig. 8. Infrared vibrational spectra of SFX and AgSFX for the region 4000– 2500 cm1.
Table 4 Antibiotic sensitivity profiles of bacterial strains against the Ag(I) complex with sulfadoxine (AgSFX), pure SFX (sulfadoxine), AgNO3, Ag(I) complex with sulfadiazine (AgSFD) and the standard antibiotic levofloxacin. Compound
AgSFX SFX AgSFD AgNO3 Levofloxacin
Inhibition zone (mm) (±0.1 mm) P. aeruginosa
S. aureus
E. coli
16 0 18 18 12
16 0 14 16 30
14 0 14 14 30
(S. aureus) microorganisms, as observed by the disc diffusion method. It was found that paper discs impregnated with the AgSFX complex 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, respectively. The inhibition zones for E. coli, P. aeruginosa and S. aureus indicates that these bacterial strains are sensitive to the silver(I) complex. The activity of the pure silver(I) ion was also evaluated by the same analysis performed with AgNO3 salt. Due to the poor solubility of the AgSFX complex, the AgSFD complex was also used to a better comparison. The qualitative results of the AgSFX complex are very similar to the results for the AgSFD complex, as it can be seen on Table 4. The AgSFX complex was more active against S. aureus than the AgSFD complex and less active against P. aeruginosa; the activity against E. coli was qualitatively the same. These results are interesting because the prevalent bacteria to colonize the burn wound in the first days are Staphylococci, being considered a significant pathogen and the commonest colonizing organism in burn patients [39]. Methicillin-resistant S. aureus is also a significant problem in burn wound infection [39]. The results indicate that the AgSFX complex could be a viable alternative to AgSFD in medical clinic, and further tests are necessary to confirm this hypothesis. It is important to note that pure SFX did not exhibit antibacterial activity against the considered bacterial strains under the same experimental conditions, while discs impregnated with silver(I) nitrate also exhibited clear inhibition zones for the bacterial strains similar to the AgSFX complex, as observed in Table 4. These results indicate that the antibacterial activity of the AgSFX is most probably due to the Ag(I) ions, as it also occur with the AgSFD complex [6,9–10]. The minimum inhibitory concentration could not be determined, as the complex is insoluble.
The molar composition of the silver(I) complex with SFX was found to be 1:1 (metal:ligand). Mass spectrometric measurements showed 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. Only the smaller and more soluble fragments were detected. The powder X-ray diffraction analysis showed the coordination of pyrimidine ring nitrogen and the nitrogen of sulphonamide to Ag(I). The oxygen atom of sulphonamide participates in interactions with metal, and a strong silver–silver interaction is present. The nitrogen of the amine group is not coordinated; however interactions in the solid state through hydrogen bonds are consistent with SS-NMR and XRPD analyses. The IR, 13C and 15N SS-NMR confirms coordination of the ligand to Ag(I) through the nitrogen atoms of the sulphonamide group and the nitrogen N1 of the pyrimidine ring, as well as the oxygen atom of the sulphonamide group, which is consistent with the powder diffraction data. The compound showed antibacterial activity against E. coli, P. aeruginosa, and S. aureus microorganisms as observed by antibiogram assays, which is a common assay for the evaluation of the antibacterial activity of insoluble complexes [12,40,41]. The minimum inhibitory concentration (MIC) could not be determined due to the low solubility of the complex. The antibacterial assays showed the AgSFX complex has a similar activity as the commercial antibacterial agent 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 Agencies FAPESP (São Paulo State Research Council, Grant 2011/02452-0 and 2012/08230-2), CNPq (National Council of Scientific and Technological 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 for fruitful discussions. Appendix A. Supplementary material Crystal data, fractional atomic coordinates, and displacement parameters of structures described in this article are supplied in standard CIFs deposited in the Cambridge Crystallographic Data Center (1012191). The data can be obtained free of charge at http://www.ccdc.cam.ac.uk/conts/retrieving.html [or from Cambridge Crystallographic Data Center (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 (0)1223-336033; E-mail: [email protected]]. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.molstruc.2014.11.004. References [1] [2] [3] [4] [5] [6] [7]
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Journal of Molecular Structure journal homepage: www.elsevier.com/locate/molstruc
Silver sulfadoxinate: Synthesis, structural and spectroscopic characterizations, and preliminary antibacterial assays in vitro 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, Brazil Biological and Health Sciences Department – UNIARA, 14801-320 Araraquara, SP, Brazil Department of Chemistry, Exact Sciences Institute – UFJF, Juiz de Fora, MG, Brazil d Dipartimento di Scienza e Alta Tecnologia, Università degli Studi dell’Insubria, 22100 Como, Italy b c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A novel silver(I) complex with
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.
sulfadoxine. IR and NMR data indicate coordination of the ligand to Ag(I) by the nitrogen and oxygen atoms. Structural characterization of the complex was based on X-ray powder diffraction data. Antibacterial activities of the complex were observed over Gram-negative and Gram-positive strains.
a r t i c l e
i n f o
Article history: Received 4 September 2014 Received in revised form 2 November 2014 Accepted 2 November 2014 Available online 13 November 2014 Dedicated to Professor Antonio C. Massabni in the occasion of his 70th birthday. Keywords: Silver complex Sulfadoxine ESI-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) through the nitrogen atoms of the (deprotonated) sulfonamide group and by the pyrimidine ring, as well as through oxygen atom(s) of the sulfonamide group. These hypotheses were corroborated by 13C and 15 N SS-NMR spectroscopy and by an unconventional structural characterization based on X-ray powder diffraction data. The latter showed that AgSFX crystallizes as centrosymmetric dimers with a strong Ag Ag interaction of 2.7435(6) Å, induced by the presence of exo-bidentate N,N0 bridging ligands and the formation of an eight-membered ring of [AgNCN]2 sequence, nearly planar. Participation of oxygen atoms of the sulfonamide residues generates in the crystal a 1D coordination polymer, likely responsible for its very limited solubility in all common solvents. Besides the analytical, spectroscopic and structural
Abbreviations: SFX, Sulfadoxine (4-Amino-N-(5,6-dimethoxy-4-pyrimidinyl)benzenesulfonamide); AgSFX, Ag(I) complex with sulfadoxine; AgSFD, Ag(I) complex with sulfadiazine; NMR, nuclear magnetic resonance; HSQC, Heteronuclear Single Quantum Coherence; HMBC, Heteronuclear Multiple Bond Coherence; SS-NMR, Solid State NMR spectroscopy; ESI-TOF-MS, Electrospray Ionization Time-of-flight Mass Spectrometry; IR, Infrared Spectroscopy; TGA/DTA, Thermogravimetric and Differential Thermal Analysis; XRPD, X-ray Powder Diffraction; ATCC, American Type Collection Cell; MH, Mueller-Hinton agar; BHI, Brain-Heart Infusion Medium; CFU, Colony Forming Unit. ⇑ Corresponding author. Tel.: +55 19 35213130; fax: +55 19 35213023. E-mail address: [email protected] (P.P. Corbi). http://dx.doi.org/10.1016/j.molstruc.2014.11.004 0022-2860/Ó 2014 Elsevier B.V. All rights reserved.
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description, the antibacterial properties of AgSFX were assayed using disc diffusion methods against Escherichia coli and Pseudomonas aeruginosa (Gram-negative), and Staphylococcus aureus (Gram-positive) bacterial strains. The AgSFX complex showed to be active against Gram-positive and Gram-negative bacterial strains, being comparable to the activities of silver sulfadiazine. Ó 2014 Elsevier B.V. All rights reserved.
Introduction Metal complexes have been widely used in pharmacology and medicine worldwide for the treatment of many diseases. The medicinal 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 millennia later, in the 19th century (well before the advent of antibiotics), silver compounds were popular drugs [2]. The main use of silver was for the treatment of burns and wounds, efficiently limiting bacterial infection. Initially, silver dosage was made through solid silver nitrate by the use of instruments as the so-called lapis infernalis, followed by the use of solutions and foils [3]. Recently, the advent of penicillin and sulfa drugs made silver-based antibacterial agents fall into disuse. The interest in silver was recovered by Moyer, who proposed the use of silver nitrate solutions to treat burns [4]. Unfortunately, the fast delivery of silver(I) ions into the blood system causes severe toxicity, and it is accompanied by the inhibition of the epithelial growth. In the 1970’s, Fox studied [5] the combination of different sulfonamides with silver nitrate, aiming for improving the antibacterial activity, while diminishing the undesired effects of silver nitrate solutions. It was then demonstrated [6] that Fox’s antibacterial agent was a silver complex with sulfonamide. Among the many sulfonamides, silver sulfadiazine (AgSFD) was the one that showed the highest activity against bacteria, and was soon introduced in the clinic for the treatment of burn infections and skin ulcers as a topical cream [2,4–8]. The good performance of silver sulfadiazine is mainly attributed to the presence of slowly released Ag+ ions [9–11], with no sulfadiazine molecules being ever found inside of the bacterial cells. In this case, sulfadiazine acts as a carrier of silver ions, and avoids the readily precipitation of Ag+ as silver chloride, or oxide/hydroxide, maintaining the electrolyte levels of the body fluids as well as the antibacterial activity due to the constant concentration gradient of Ag+ [9]. Nowadays silver is known as a broad-spectrum antimicrobial agent and diverse formulations are commercially available. For
example, colloidal silver, silver salts, silver complexes, nanocrystalline silver, silver oxide, and silver zeolite formulations are known, all possessing good antimicrobial activity [2,8]. However, the widespread use of silver has caused the isolation of some resistant bacterial strains. The studies on silver resistance are sparse, but they suggest that the resistance is mediated by plasmid. These evidences increased the concerns about the misuse of silver and the development of novel antibacterial silver compounds became of great interest [9]. For this reason, novel silver complexes with enhanced, or tailored, antimicrobial activity are continuously being investigated. For example, a silver complex with N-acetyl-L-cysteine was recently synthesized, and showed a broad spectrum of activity against microorganisms [12]. In addition, a silver complex with the antiinflammatory drug nimesulide (another sulfonamide) [13] was shown to be active against Gram-positive and Gram-negative bacteria. Moreover, the Ag(I) derivatives are currently being investigated as anticancer drugs [12,14]. Sulfadoxine (SFX, C12H14N4SO4, Fig. 1) is a sulfonamide widely used 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 antimalarial activity, the sulfadoxine-pyrimethamine hybrid is also active against the parasite Toxoplasma gondii [15] and is considered as primary prophylaxis of pneumonia caused by Pneumocyctis carinii [16–18] and toxoplasmatic encephalitis in patients infected with HIV [17,18]. Sulfadoxine is also used in combination with trimethoprim to treat different bacterial infections in animals as horses and calves, being active against Gram-negative and Gram-positive bacteria [19–21]. Metal complexes of sulfadoxine were also recently studied and Fe(II), Fe(III), Co(II), Cu(II), Cr(III) derivatives were synthesized [22–24]. Ogunniran et al. synthesized Cu and Fe complexes with sulfadoxine and pyrimethamine, and showed that these species are active against E. coli, S. aureus, P. aureginosa and S. typhi. The complexes have shown to have an enhanced activity in comparison with the free ligands [23]. In the current manuscript, inspired by these works and by the desirable properties of many silver derivatives, we present the synthesis, spectroscopic and structural characterization, and the antibacterial activity of the novel silversulfadoxine (AgSFX) complex. Experimental Materials and methods
Fig. 1. Sketch of SFX molecule. The SFX molecular conformation was defined by eight torsion (si) angles indicated by arrows and used in the powder diffraction study (vide infra).
Sulfadoxine (SFX 95%) and silver 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 CHNS/O Perkin Elmer 2400 Analyzer. Infrared (IR) spectra from 4000 cm1 to 400 cm1 of SFX and the AgSFX complex were measured using a Bomen MB Series Model B100 spectrometer with resolution of 4 cm1; samples were prepared as KBr pellets. The 1H nuclear magnetic resonance (NMR) spectrum of SFX was recorded on a Bruker Avance 400 MHz spectrometer operating at 400.1 MHz. The 13C
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NMR spectrum of SFX was also recorded on a Bruker Avance 400 MHz operating at 100.6 MHz. The heteronuclear [1HA13C] single quantum coherence (HSQC), heteronuclear [1HA13C] multiple bond coherence (HMBC) and the heteronuclear [1HA15N] multiple bond coherence (HMBC) spectra of SFX were acquired on a Bruker Avance 400 MHz, using a 5-mm probe at 303 K, operating at 40.5 MHz for 15N; samples were was prepared in deuterated dimethylsulfoxide solutions. The solid state nuclear magnetic resonance (SS-NMR) 13C spectrum of SFX an AgSFX was recorded on a Bruker 400 MHz Avance II (9.395T) operating at 100 MHz using cross polarization, proton decoupling and magic angle spinning (CP/MAS). The solid-state 15N{1H} nuclear magnetic resonance spectra were recorded on a Bruker 400 MHz, using the combination of 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 Micro API instrument: methanol was added to a sample of 1.5 mg AgSFX, the supernatant was separated and formic acid was added until concentration of 1%; the resulting solution was directly infused into the instrument’s ESI source with capillary potential of 3.00 kV, cone potential of 30 kV, trap potential of 2 kV, source temperature of 150 °C and nitrogen gas for desolvation. Synthesis of the complex The silver(I) complex with sulfadoxine (AgSFX) was synthesized by the reaction of 1.0 103 mol of a freshly prepared aqueous AgNO3 solution (2 mL) with a basic aqueous solution of SFX containing 1.1 103 mol of potassium hydroxide and 1.0 103 mol of SFX (12.5 mL). After one hour of constant stirring at room temperature, a white solid precipitated and was collected by filtration, washed with cold water, and dried in a desiccator with P4O10. Yield 71.9%. 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 in water, DMSO, DMF, chloroform, acetonitrile, hexane, ethanol and methanol. Accordingly, in the absence of single crystals of suitable size and quality, and being impossible to grow suitable samples by recrystallization from solution, we adopted the powder diffraction route to structural characterization [25] which, in through the years [26–28], became a viable and efficient tool to obtain otherwise inaccessible structural information of metal complexes of limited structural complexity. Structural analysis of AgSFX by X-ray powder diffraction data The polycrystalline AgSFX material was gently ground in an agate mortar and the powder was deposited in a sample holder equipped with a silicon zero-background plate. Diffraction data were collected at room temperature by an overnight scan in the 2h range of 6–105° with steps of 0.02° using a Bruker AXS D8 Advance diffractometer equipped with Ni-filtered Cu Ka radiation (k = 1.5418 Å) and Lynxeye linear position-sensitive detector. The
following optics were set up: primary beam Soller slits: 2.3°, fixed divergence slit: 0.5°, and receiving slit: 8 mm. The generator was set at 40 kV and 40 mA. Standard peak search and profile fitting, followed by indexing through the single-value decomposition approach implemented in TOPAS [29] allowed detection of the approximate monoclinic unit cell parameters (GOF: 3.04 from Indexing and 3.78 from LeBail). Density considerations suggested Z = 4 and the space group P21/c was chosen. Structure solution was performed by the simulated annealing technique implemented in TOPAS. A (partially flexible) rigid body for SFX was used with free molecular location and orientation within the unit cell; the SFX rigid body was defined by Cartesian coordinates, obtained by molecular mechanics optimization of the bond distances and angles, using the ChemSketch freeware program, and by resizing the CAH bond distances down to the common ‘‘X-ray’’ value of 0.95 Å. In addition, s1 to s5 torsion angles (see Fig. 1) were freed in the simulated annealing step, while a single silver(I) ion was left to freely float in the cell. The final refinement was carried out by the Rietveld method maintaining the rigid body introduced at the simulated annealing stage. Additionally, a total of eight torsion angles were refined in the final cycles. The background was modelled by a Chebyshev polynomial function. An isotropic thermal parameter was assigned to the metal (BAg) and refined; lighter atoms were given a Biso = BAg + 2.0 Å2 thermal parameter. The final Rietveld refinement plot is supplied as Fig. 2.
Antibacterial assays Three referenced bacterial strains: Escherichia coli – ATCC 25,922, Pseudomonas aeruginosa – ATCC 27,853 and Staphylococcus aureus – 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 separate test tubes containing 5.0 mL of sterile brain heart infusion (BHI) medium and incubated for 18 h at 35–37 °C. Sufficient inocula were added in new tubes until the turbidity equaled 0.5 McFarland (1.5 108 CFU/cm3). The bacterial inocula diluted with BHI (McFarland standard) were uniformly spread using sterile cotton swabs on sterile Petri dishes containing MH agar. Sterile filter paper discs (10 mm diameter) were aseptically impregnated with 800 lg of SFX and AgSFX according to the following procedure: 20 mg of the compounds were suspended in 1000 lL of distilled water, homogenized in a vortex, and 50 lL of the suspension were collected with a micropipette and transferred to the paper discs. Sterile discs impregnated with 1000 lg of pure SFX were used as a negative control. Discs impregnated with the AgSFX complex or with SFX were dried and sterilized in a vertical laminar flow under UV radiation for 45 min before the experiment. The impregnated discs were placed on the surfaces of the solid agar. The plates were incubated for 18 h at 35–37 °C and examined thereafter. Clear zones of
Fig. 2. Final Rietveld refinement plot for AgSFX complex, with difference plot and peak markers at the bottom. The high angle region is shown as inset.
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inhibition around the discs were measured and the complex sensitivity was assayed from the diameter of the inhibition zones (in millimeters). The observed results were compared to discs with the standard antibiotic levofloxacin, and with discs impregnated with 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 powder X-ray diffraction analysis on laboratory data and, despite of the high number of torsion angles of SFX to be determined, relevant crystallochemical information (stoichiometry, crystal packing, conformation) could be obtained. Crystal data for AgSFX are summarized in Table 1, while relevant bond distances and angles are given in Table 2. The centrosymmetric dimer found in the crystal structure of AgSFX is shown in Fig. 3, where only atoms of the asymmetric unit were labelled. As pictorially shown therein, the crystals of AgSFX structure contain dinuclear moieties centered about the strong Ag Ag argentophilic interaction (2.744(6) Å). The eight-membered ring of the [ANCNAgA]2 type present in the dimer is further bound to oxygen atoms of S@O group (with AgAO interactions in the 2.6–2.7 Å range), eventually connecting the different moieties in a 1D polymer, stretching along the a axis. The proposed structure of AgSFX requires some further comments. Apparently, the refined distances for the AgAN1 and AgAN3 bonds (2.256(2) and 2.533(1) Å, respectively, similar to the typical values found for other Ag(I) complexes containing N-donor ligands [32] possess rather distinct values, and suggested a deeper look into the problem, aiming at detecting if, for any reason, this discrepancy is real. This doubt is even more pronounced for XRPD structure determinations of moderately complex systems, which are known
Table 1 Crystallographic data of AgSFX. AgSFX Empirical formula Formula weight T(K) k(Cu Ka) (Å) Crystal system Space group a (Å) b (Å) c (Å) b (°) V (Å3) Z dcalc (g cm3) l (mm1) F (0 0 0) Number of parameters RBragg, Rwp
C12H13AgN4O4S 417.18 298 1.5418 Monoclinic P21/c 5.4448(5) 19.226(2) 13.852(1) 102.502(5) 1415.7(2) 4 1.9573 13.09 800 34 0.044/0.080
Bond distances (Å) Ag–N1 Ag–N3i Ag–O12i Ag–O11ii Ag Agi
Angles (°) 2.26(2) 2.53(1) 2.60(2) 2.71(2) 2.744(6)
N1–Ag–N3i N1–Ag–O12i N1–Ag–O11ii N3i–Ag–O12i N3i–Ag–O11ii O12i–Ag–O11ii
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, the Cambridge Structural Database (CSD) was searched for cyclic eight-membered ring [AAgNCNA]2 type, and a statistical average of 2.241 Å for bonded AgAN was found, with a broad distribution reaching values higher than 2.6 Å. At the same time, the Ag Ag distance distribution showed highly probable intermetallic interactions near, or slightly below, 2.80 Å. Several restrained Rietveld refinements were also performed, on the same original XRPD data, by setting equal AgAN distances at 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 well defined minimum. These data were subjected to a parabolic fit, which gave the minimum Rwp value (0.083) for a bond distance of 2.542 Å. Needless to say, on relaxing the two AgAN distances, a slightly lower Rwp value (0.080) is obtained. Thus, the close matching of (i) the statistically derived AgAN average and the observed AgAN1 values; (ii) the typically observed Ag Ag distances and the 2.744(6) Å value here determined, and (iii) the distance determined for minimum Rwp value in the restrained refinements and the observed AgAN3 bond, significantly suggest that the proposed structural model cannot be too far from reality. Note that the final comment is based on two statistically independent observations: our experimental XRPD data and a complete statistical analysis of CSD. The bond distances of AgAO are, normally, larger than AgAN [34] accordingly, also in the AgSFX complex, long(er) AgAO bond distances 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 the highest abundance at m/z 419 assigned to [AgC12H13N4O4S + H+]. An adduct of the molecular ion with methanol is observed at m/z 451. The free ligand, C12H14N4O4S, is also observed as singly charged [C12H14N4O4S + H+] ions of m/z 311, respectively. The silver ion is observed at 109 m/z and a peak at 156 m/z is assigned as the breakage of the ligand molecule, forming the ion [C6H6NSO+2]. 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 structure previously observed in the powder diffraction data, but only more soluble fragments were detected in the analysis. An isotope pattern for the singly charged [AgSFX + H+] ion is also shown on Fig. 5. Solid-state
Table 2 Relevant bond distances (Å) and angles (°) for AgSFX.
170.9(4) 123.1(5) 83.6(5) 58.6(3) 105.4(4) 78.7(4)
183
13
C and
15
N spectroscopic measurements
The SFX 13C NMR spectrum in solution was assigned based on the NMR by experiments of [13CA1H] HSQC and HMBC and [15NA1H] HMBC. The spectrum in solution was later used to perform the assignment of the SS-NMR 13C spectra of SFX and AgSFX. The spectra of SFX in solution are provided as Supplementary material. The AgSFX 13C SS-NMR spectrum was analysed in comparison to that of SFX. The 13C SS-NMR spectra of AgSFX and SFX are provided in Fig. 6. The structure of SFX with the carbon atoms numbering is provided on Fig. 1. The spectrum of the complex shows more resolved peaks than the free ligand, which is probably due to the presence of different polymorphs of the ligand as previously reported [35]. As demonstrated by powder diffraction analysis, only one polymorph was isolated for the complex. Besides the change in resolution, significant variations of the chemical shifts are observed when comparing the two spectra. The 4 and 7 carbon atoms signals shift to lower field by 3.5 and 3.3 ppm, respectively due to the coordination. This
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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. The atoms 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 figure legend, the reader is referred to the web version of this article.)
10 Rwp
R wp , %
9.5
"Final Model"
9 8.5 8 7.5 7 2
2.2
2.4
2.6
2.8
Ag-N, Å Fig. 4. Plot of the standard Rietveld refinement Rwp value (%) vs. restrained (and equal) Ag–N bond distances in AgSFX (black dots). A deep minimum near 2.54 Å is clearly seen. The empty circles are positioned at Rwp 8.02 (the absolute minimum in the 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 CSD search and the minimum of the Rwp curve, respectively.
observation agrees with the powder diffraction result that the coordination of the ligand to the metal occurs through the nitrogen of the sulfonamide group and the amine group is involved in a series of hydrogen bonds with the oxygen of the sulphonamide group of the next dimer of AgSFX. Other shifts are observed for the signals of carbon atoms 5, 2 and 3 by 2.5, 2.1 and 3.7 ppm respectively upon the coordination. These results also agree with previous powder diffraction data that the coordination of sulfadoxine to Ag(I) is also through nitrogen 1 of the pyrimidine ring, changing the electronic density of all the carbons of the ring. This mode of coordination, through the sulphonamide and the nitrogen of the pyrimidine ring is common and is also observed for silver sulfadiazine [36]. No significant variations of the chemical shifts of the methoxy group were observed, which indicates that this functional group 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 comparison to that of SFX. The 15N SS-NMR spectra of AgSFX and SFX are provided in Fig. 7, while the numbered structure of SFX is provided in Fig. 1.
Four signals assigned to the four nitrogen atoms are observed in the spectrum of the SFX molecule, the two nitrogen atoms of the pyrimidine ring, N2 and N1, at 251.0 and 246.7 ppm, respectively, the sulfonamide group nitrogen at 121.5 ppm and the amine group nitrogen at 71.7 ppm. Nevertheless, the signal of the sulfonamide group nitrogen is not observed in the complex 15N spectrum. Since the cross polarization NMR technique requires the existence of a hydrogen atom near to this nitrogen for the polarization transfer [37] it indicates, that the coordination of the ligand to the silver atom is accompanied by the loss of the hydrogen and the coordination of the silver ion to the nitrogen atom of sulphonamide group. The signals of the nitrogen atoms of the pyrimidine ring are observed at 206.3 and 188.1 ppm in the spectrum of the complex, which represents a shift of 44.7 ppm for N2 and 58.6 ppm for N1 to higher field, respectively, when compared to the ligand spectrum. This is consistent with the participation of N1 in the coordination. The shift observed for N2 is probably due to changes in the electron density of the whole ring, caused by the coordination to N1. The nitrogen of the amino group is observed at 20.2 ppm in the spectrum of the complex with a shift of 51.5 ppm to higher field when compared to the ligand spectrum. The shift observed for the amine group can be attributed to hydrogen bonding to the oxygen atom of the sulphonamide group of the adjacent dimer of AgSFX. The NMR data is consistent to the structure proposed by the powder diffraction. The chemical shifts of all the carbon and nitrogen NMR signals are provided in Table 3. IR spectroscopic data The AgSFX IR spectrum was analysed in comparison to that of SFX. The IR spectra of AgSFX and SFX of the region from 4000 to 2500 cm1 are provided in Fig. 8. The spectrum of SFX shows three absorption bands at 3240, 3377 and 3465 cm1, assigned as the NAH stretches of the sulphonamide group and the symmetric and asymmetric NAH stretches of the amine group, respectively. In the spectrum of the complex the NAH stretch of the sulphonamide group is observed as a very weak broad shoulder, whereas the stretches of the amine group do not change considerably as it can be observed in Fig. 8. This result indicates that the ligand coordinates to the metal through
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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 AgSFX complex.
the nitrogen atom of the sulfonamide as the hydrogen atom bonded to the sulphonamide group is no longer present, consequently the NAH stretching is also absent. This result is consistent with the SS-NMR results and the powder diffraction data. The comparison of the two spectra also shows a shift of the C@N stretching mode of the pyrimidine ring from 1650 cm1 in the SFX spectrum to 1618 cm1 in the complex which can also indicate coordination of the ligand to the metal by the nitrogen atoms of the pyrimidine ring. In the spectrum of the ligand the O@S@O asymmetric and symmetric stretching modes of the sulphonamide group also shifts from 1319 cm1 and 1157 cm1 in the ligand to 1236 cm1 and 1115 cm1 in the complex, respectively. The changes of O@S@O stretching frequencies suggest the ligand coordination to the metal through this group as well, and the decrease in frequency indicates the coordination by the oxygen atom [38], as shown by the powder diffraction data, where one oxygen atom of the sulphonamide coordinates to the silver ion and the other one interacts weakly with the silver ion of the adjacent dimer and also forms hydrogen bonds with the amine group of the adjacent dimer. This observation is also in agreement with the reported structure of silver sulfadiazine, which presents the oxygen of sulphonamide as a coordination site in the polymeric structure [34]. The full IR spectrum of the complex and the ligand are provided as Supplementary material.
Fig. 7. 15N solid-state nuclear magnetic resonance spectra of SFX and AgSFX complex.
Table 3 Solid-state
13
C and
15
N NMR data for SFX and AgSFX.
Assignment
C7 C8, C12 C9, C11 C10 C4 C5 C2 C3 2OCH3 3OCH3 N2 N1 NH–SO2 NH2
Chemical shift (ppm) SFX
AgSFX
152.9 129.5 114.7, 113.3 125.9 150.5 150.5 162.5 125.9 53.8 60.0 251.0 245.7 121.5 71.7
156.2 132.6 115.6, 113.7 127.9 154.0 148.0 164.6 129.6 55.4 59.4 206.3 188.1 – 20.2
Antibacterial assays Antibacterial sensitivity profiles of bacterial strains demonstrate the antibacterial activity of the silver(I) complex with SFX against Gram-negative (E. coli and P. aeruginosa) and Gram-positive
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Conclusion
Fig. 8. Infrared vibrational spectra of SFX and AgSFX for the region 4000– 2500 cm1.
Table 4 Antibiotic sensitivity profiles of bacterial strains against the Ag(I) complex with sulfadoxine (AgSFX), pure SFX (sulfadoxine), AgNO3, Ag(I) complex with sulfadiazine (AgSFD) and the standard antibiotic levofloxacin. Compound
AgSFX SFX AgSFD AgNO3 Levofloxacin
Inhibition zone (mm) (±0.1 mm) P. aeruginosa
S. aureus
E. coli
16 0 18 18 12
16 0 14 16 30
14 0 14 14 30
(S. aureus) microorganisms, as observed by the disc diffusion method. It was found that paper discs impregnated with the AgSFX complex 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, respectively. The inhibition zones for E. coli, P. aeruginosa and S. aureus indicates that these bacterial strains are sensitive to the silver(I) complex. The activity of the pure silver(I) ion was also evaluated by the same analysis performed with AgNO3 salt. Due to the poor solubility of the AgSFX complex, the AgSFD complex was also used to a better comparison. The qualitative results of the AgSFX complex are very similar to the results for the AgSFD complex, as it can be seen on Table 4. The AgSFX complex was more active against S. aureus than the AgSFD complex and less active against P. aeruginosa; the activity against E. coli was qualitatively the same. These results are interesting because the prevalent bacteria to colonize the burn wound in the first days are Staphylococci, being considered a significant pathogen and the commonest colonizing organism in burn patients [39]. Methicillin-resistant S. aureus is also a significant problem in burn wound infection [39]. The results indicate that the AgSFX complex could be a viable alternative to AgSFD in medical clinic, and further tests are necessary to confirm this hypothesis. It is important to note that pure SFX did not exhibit antibacterial activity against the considered bacterial strains under the same experimental conditions, while discs impregnated with silver(I) nitrate also exhibited clear inhibition zones for the bacterial strains similar to the AgSFX complex, as observed in Table 4. These results indicate that the antibacterial activity of the AgSFX is most probably due to the Ag(I) ions, as it also occur with the AgSFD complex [6,9–10]. The minimum inhibitory concentration could not be determined, as the complex is insoluble.
The molar composition of the silver(I) complex with SFX was found to be 1:1 (metal:ligand). Mass spectrometric measurements showed 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. Only the smaller and more soluble fragments were detected. The powder X-ray diffraction analysis showed the coordination of pyrimidine ring nitrogen and the nitrogen of sulphonamide to Ag(I). The oxygen atom of sulphonamide participates in interactions with metal, and a strong silver–silver interaction is present. The nitrogen of the amine group is not coordinated; however interactions in the solid state through hydrogen bonds are consistent with SS-NMR and XRPD analyses. The IR, 13C and 15N SS-NMR confirms coordination of the ligand to Ag(I) through the nitrogen atoms of the sulphonamide group and the nitrogen N1 of the pyrimidine ring, as well as the oxygen atom of the sulphonamide group, which is consistent with the powder diffraction data. The compound showed antibacterial activity against E. coli, P. aeruginosa, and S. aureus microorganisms as observed by antibiogram assays, which is a common assay for the evaluation of the antibacterial activity of insoluble complexes [12,40,41]. The minimum inhibitory concentration (MIC) could not be determined due to the low solubility of the complex. The antibacterial assays showed the AgSFX complex has a similar activity as the commercial antibacterial agent 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 Agencies FAPESP (São Paulo State Research Council, Grant 2011/02452-0 and 2012/08230-2), CNPq (National Council of Scientific and Technological 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 for fruitful discussions. Appendix A. Supplementary material Crystal data, fractional atomic coordinates, and displacement parameters of structures described in this article are supplied in standard CIFs deposited in the Cambridge Crystallographic Data Center (1012191). The data can be obtained free of charge at http://www.ccdc.cam.ac.uk/conts/retrieving.html [or from Cambridge Crystallographic Data Center (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 (0)1223-336033; E-mail: [email protected]]. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.molstruc.2014.11.004. References [1] [2] [3] [4] [5] [6] [7]
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