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New silver complexes with levofloxacin: Synthesis, characterization and microbiological studies
Journal of Molecular Structure 1123 (2016) 384e393
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Journal of Molecular Structure journal homepage: http://www.elsevier.com/locate/molstruc
New silver complexes with levofloxacin: Synthesis, characterization and microbiological studies } To th b, Szende Vancea c, Felicia Toma d, Aura Rusu a, Gabriel Hancu a, *, Gergo Anca Delia Mare d, Adrian Man d, George Mihai Nit¸ulescu e, Valentina Uivarosi f a
University of Medicine and Pharmacy from Tîrgu Mures¸, Faculty of Pharmacy, Pharmaceutical Chemistry Department, Romania Semmelweis University, Budapest, Department of Pharmaceutical Chemistry, Hungary University of Medicine and Pharmacy from Tîrgu Mures¸, Faculty of Pharmacy, Physical Chemistry Department, Romania d University of Medicine and Pharmacy from Tîrgu Mures¸, Faculty of Medicine, Microbiology, Virology, and Parasitology Department, Romania e University of Medicine and Pharmacy Carol Davila, Bucharest, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Romania f University of Medicine and Pharmacy Carol Davila, Bucharest, Faculty of Pharmacy, General and Inorganic Chemistry Department, Romania b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 April 2016 Received in revised form 27 June 2016 Accepted 10 July 2016 Available online 12 July 2016
Levofloxacin is a third generation fluoroquinolone antibiotic with a broad-spectrum including both Gram positive bacteria and Gram negative bacteria, as well atypical bacteria. In order to extend the spectrum of activity and to add new biological effects, several metal complexes of levofloxacin have been obtained and reported recently. The aim of our study was to obtain new silver complexes with levofloxacin with potential broad spectrum antibacterial and antifungal activity. Therefore three new silver complexes of levofloxacin with the proposed chemical structures (levofloxacin)2Ag(NO3), (levofloxacin)2Ag(NO3)(CH3OH) and (levofloxacin)Ag(C6H6O7)$3H2O were synthesized. In order to characterize the obtained complexes elemental analysis, conductivity measurement, spectroscopic, and thermal methods were used. Optimized molecular structures were determined using DFT (density functional theory) analysis. The antibacterial activity against Gram negative and Gram positive bacteria and antifungal activity against Candida spp of the complexes was tested by determination of minimum inhibitory concentration through microtitre broth dilution method. © 2016 Elsevier B.V. All rights reserved.
Keywords: Silver Levofloxacin Fluoroquinolones Metalcomplex Silver nitrate Silver citrate
1. Introduction Antibacterial fluoroquinolones are chemotherapeutic compounds commonly used in therapy to treat bacterial infections. As in the case of most classes of antibiotic compounds, the development of bacterial resistance represents a major challenge and a serious threat to therapists. Therefore, the development of metal complexes with fluoroquinolones had captivated attention of many researchers recently, taking into account the potential of broadening the activity spectrum of the ligand or the emergence of
* Corresponding author. Faculty of Pharmacy, Department of Pharmaceutical Chemistry, University of Medicine and Pharmacy of Tîrgu Mures¸, 38 Gheorghe Marinescu Street, Tîrgu Mures¸, 540139, Romania. E-mail addresses: [email protected] (A. Rusu), [email protected] th), [email protected] (G. Hancu), [email protected] (G. To (S. Vancea), [email protected] (F. Toma), [email protected] (A.D. Mare), [email protected] (A. Man), [email protected] (G.M. Nit¸ulescu), [email protected] (V. Uivarosi). http://dx.doi.org/10.1016/j.molstruc.2016.07.035 0022-2860/© 2016 Elsevier B.V. All rights reserved.
possible new biological effects. Many studies report the excellent complexing capacity of fluoroquinolones particularly due to the chemical structure [1e3]. Among the large variety of metal cations which can be used in the production of metal complexes of the fluoroquinolones, silver appears to be adequate, mainly due to its antibacterial, antifungal and cytotoxic activity [4e6]. Thus, taking in account the biological effects of silver ions and the activity spectrum of fluoroquinolones, it makes sense to study, if there is a possible summation and potentiation of these properties in case of metal complexes which contain silver and a fluoroquinolone. Some studies have approached this line of research using as ligands first generation quinolones (nalidixic acid, norfloxacin, pefloxacin, and ciprofloxacin) or a modern fluoroquinolone (moxifloxacin) [7e16]. From a structural point of view, silver complexes with fluoroquinolones have a particular aspect, namely silver binding that may occur at the nitrogen atom of the ligand N4’-piperazine, different from most of fluoroquinolone metal complexes which have the complexing location 3-carboxyl and 4-oxo groups [3]. Levofloxacin (LEV) or (S)-9-fluoro-2,3-dihydro-3-methyl-10-(4-
A. Rusu et al. / Journal of Molecular Structure 1123 (2016) 384e393
methylpiperazin-1-yl)-7-oxo-7H-pyrido [1,2,3-de]-1,4benzoxazine-6-carboxylic acid is a third generation fluoroquinolone, being the levorotatory isomer of racemic ofloxacin (Fig. 1). As mechanism of action, LEV directly inhibits DNA synthesis by acting on two target enzymes, respectively DNA gyrase and topoisomerase IV. LEV has a broad-spectrum, acting on the Gram positive bacteria and Gram negative bacteria, and atypical bacteria, as well [17e21]. Thus far several metal complexes of LEV were synthesized and studied. Among targeted cations can be found Mg2þ, Ca2þ, Zn2þ, Pd2þ, Pt2þ, Au3þ, Cu2þ, Fe3þ, MoO2þ 2 , and the antimicrobial activity, effect on the leukemia HL-60 and HePG-2 cell line, human colon carcinoma HCT-116 and human hepatocellular carcinoma HepG-2 tumor cell lines, and interaction with DNA were tackled in different studies [22e31]. Due to an intensive worldwide use, an increase of bacterial resistance to fluoroquinolones appeared. Thus, the need to improve the antibacterial potential of existing antimicrobial drugs is more challenging, adding the concept that metal complexes could be an alternative to conventional drugs [32]. As far as we known no reported data regarding silver complex of LEV has been described in the literature. Our study reports reliable methods for preparation of silver metal complexes of LEV which will possess the biological effects of the two constituent components. In order to determine the most probable structures of the obtained silver complexes elemental analysis, conductivity measurement, spectroscopic, thermal and density functional theory (DFT) methods were used. The antimicrobial and antifungal activity of the complexes has been evaluated. 2. Methods 2.1. Materials LEV was purchased from Sigma, silver nitrate from SC UTCHIM SRL (Rom^ ania), silver citrate hydrate from Aldrich; all other chemicals used were of analytical reagent grade.
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spectrometer equipped with electrospray ionization (ESI) ion source in positive ion mode. The recorded data were processed using MassHunter software. Conditions of the ionization source were: gas flow 8 L/min, 40 psi, 300 C, 4000 V, with a full scan on the field 100e1500 amu data acquisition module. NMR measurements were carried out on a Varian Unity Inova DDR spectrometer (599 MHz for 1H) with a 5 mm inverse-detection gradient probehead using dimethylsulfoxide-d6 (DMSO-d6) as solvent. Standard pulse sequences and processing routines available in VnmrJ 2.2C/Chempack 4.0 were used. The chemical shifts were referenced to the solvent signal of internal DMSO-d6 (2.500 ppm). For Differential Scanning Calorimetry (DSC) analysis we used a DSC 60 Shimadzu apparatus. The weight of the samples was 3 mg. The DSC curves were recorded in the range of 40e400 C, with a temperature increase rate of 10 C/ min. UV spectra were recorded on a T70 þ UV/VIS Spectrometer (PG Instruments Ltd). Electronic spectra by diffuse reflectance technique were recorded in the range 200e800 nm with spectralon as standard, on a Jasco V650 spectrophotometer. 2.3. Computational study Full geometry optimizations of the ligand and complexes (1) and (3) were carried out using the DFT method in Gaussian09 [33]. The split valence plus polarization basis set 6-31G(d) was applied for any atom that is not involved in coordination. 6-31G(d0 ,p’) basis set was used for the fluorine atoms. To give a better description of the Agþ e ligand interactions, basis sets with additional diffuse functions were added for the piperazine nitrogen and the oxygen atoms involved in coordination (6-311Gþ(d,p)). The Stuttgart-Cologne small-core quasi-relativistic pseudopotential ECP28MWB (28 electrons in the core) [34,35] was used for the Agþ ion in conjunction with its optimized valence basis set 8s7p6d 2f1g. DFT/ B3LYP/(MWB28 for Ag) was proven to give good results for prediction of coordination polyhedral and geometry parameters of the Agþ complexes in other [36,37]. 2.4. Experimental procedures
2.2. General Elemental analyses of C, H and N were carried out on a Perkin Elmer PE 2400 analyzer. The metal ion content was determined by flame atomic absorption spectrometry (FAAS) using a Shimadzu AA 6300 spectrometer after destruction of the complex in a Berghof microwave digestion system. The molar conductance was determined for 103 M solution of complex in dimethyl sulfoxide (DMSO) with an analyzer inoLab® pH/Cond 740. To record the FT-IR spectra we used a FT-IR Thermo Nicolet (USA) spectrometer with samples prepared as KBr pellets in the range of 4000e400 cm1, 32 scans at 4 cm1 resolution, and processed using Omnic V.6 software. Mass spectra of the obtained compounds were analysed with an Agilent 6410 Triple Quadrupole (Agilent Technologies, USA) mass
Fig. 1. The chemical structure and numbering of LEV.
In order to obtain silver metal complexes of LEV we considered several possibilities. Although in previously reported articles regarding synthesis of silver complexes with fluoroquinolones only silver nitrate was used, we tried to synthesize silver complexes starting with another silver compound in this case silver citrate. In the preliminary study we tried to adapt several methods described in the literature which used as reaction temperature 80e100 C [8,14e16]. These methods were not successful due to the fact that the Agþ ion can easily generate Ag2O (black-brown precipitate), which partially decomposed to Ag and O2 (Ag2O / 2Ag þ 1/2O2) when heated. Thus, we opted for a “cold” method that does not use heat. Also for drying the obtained substance we choose to avoid as much as possible the high temperatures and direct exposure to light [38,39]. 2.4.1. Synthesis of complex (1) A solution obtained from 1.38 mmol AgNO3 and 50 mL of water have been added into a mixture of 2.76 mmol of LEV and 75 mL methanol (2:1 molar ratio LEV:AgNO3). After mixing the solutions 25 mL of concentrated ammonia was added. The entire mixture was stirred for 4 h with a magnetic stirrer, in a sealed flat-bottom flask, protected from light. A pale yellowish clear solution was obtained, which was left overnight at room temperature. The obtained solution was concentrated with a rotary evaporator at 40 C under vacuum and the last 20 mL solution was slowly dried in a normal oven set at 40 C for 24 h. The solid compound was kept in desiccator over anhydrous CaCl2, protected from light.
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2.4.2. Synthesis of complex (2) A solution obtained from 1.38 mmol AgNO3 and 50 mL of water has been added into a mixture of 2.76 mmol of LEV and 100 mL methanol (2:1 molar ratio LEV:AgNO3). The entire mixture was stirred for 8 h on a magnetic stirrer, in a sealed flat-bottom flask, protected from light. This mixture was concentrated with a rotary evaporator at 40 C under vacuum to obtain a suspension with a minimal volume of about 20 mL. The suspension was filtered to obtain a white precipitate, and washed with distilled water. The precipitate was slowly dried in a normal oven set at 40 C for 24 h, than the solid compound was kept in desiccator over anhydrous CaCl2, protected from light.
presented no bacterial growth. The DMSO solvent was used as a sterility control (25 mL solvent and 175 mL Muller Hinton broth). Muller Hinton broth alone was used as a negative control. The positive growth control consisted in a mixture of 25 mL Muller Hinton broth and 175 mL bacterial suspension [44]. 2.5.2. Antifungal activity screening Antifungal activity was assessed following the same protocol, as for bacterial MIC, excepting the fact that the Muller-Hinton broth was supplemented with 2% glucose and that the plates were incubated for 48 h. All experiments were carried out in triplicate. 3. Results and discussion
2.4.3. Synthesis of complex (3) A solution obtained from 1 mmol silver citrate previously dissolved in 25 mL 4 M citric acid aqueous solution has been added into a mixture of 2 mmol of LEV and 100 mL methanol (2:1 molar ratio LEV:silver citrate) resulting a greenish-yellow solution. The entire mixture was stirred for 8 h on a magnetic stirrer, in a sealed flat-bottom flask, protected from light, at room temperature. The solution was kept at room temperature overnight. The solution was concentrated with a rotary evaporator at 45 C under vacuum to obtain a viscous yellow liquid with a minimal volume of about 20 mL. This liquid was slowly dried in a normal oven set at 40 C for 48 h. The resulted solid crystalline compound was kept in desiccator over anhydrous CaCl2, protected from light. In order to eliminate the excess of citric acid, the resulting compound was dissolved in acetone (or ethanol) and the resulting suspension was filtered and slowly dried in an oven set at 40 C and kept in desiccator over anhydrous CaCl2, protected from light.
The physical properties of the resulted complexes, including their melting point and solubility are shown in Table 1. The complexes were soluble in DMSO and DMF similar to other metal complexes of fluoroquinolones. The molar conductivity data reveal that the complexes (1e3) are well below for an electrolyte 1:1 [24,45]. 3.1. Elemental analysis The results of elemental analysis and the content of silver in the obtained compounds are shown in Table 2. The results of elemental analysis indicated that complexation occurs through our selected methods, as complexes with molar ratio metal:ligand of 1:2 for complexes (1) and (2), and 1:1 for complex (3) are obtained. 3.2. FT-IR spectroscopy
2.5. Microbiological studies 2.5.1. Antibacterial activity screening The antibacterial activity of LEV and the synthesized complexes was tested on six bacterial strains, three Gram positive (Staphylococcus aureus ATCC 29213, Staphylococcus aureus MRSA ATCC 43300, Enterococcus faecalis ATCC 29212) and three Gram negative (Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 27853). Antimicrobial activity was analysed according to the standard CLSI (Clinical and Laboratory Standards Institute) microdilution method. In 96-well microtiter plates, binary dilutions were performed for each tested complex, to a final volume of 25 mL [40e43]. Approximately 1.5 104 CFU (colony forming unit) of each bacterial strain and 175 mL were inoculated into the corresponding wells. The plates were incubated at 35 C for 24 h. For all samples, the minimum inhibitory concentration (MIC) was interpreted at the last dilution which
The FT-IR spectra of LEV and the complexes (1e3) were registered and compared (Supplementary Information File) [10,14,28,46e51]. The most significant assignments are presented in Table 3. In the most of the metallic complexes with fluoroquinolone derivatives reported previously in literature, the ligands coordinate in a bidentate manner via the carboxylic oxygen and the carbonyl oxygen [3]. In the case of LEV the bands at 3258e2800 cme 1 correspond to the vCeH stretching vibrations of a methyl radical at the N40 nitrogen atom in the piperazinyl moiety, and/or to the vCeH vibrations of methylene groups in ReOeAr [47]. In our silver complexes (1e2) these weak bands appear shifted at 3043/ 3044 cm1 (very weak) and around 2839 cm1(shoulder); for complex (3) these weak bands appear shifted at 3380 cm1, 3082 cm1 and 2847 cm1 (Fig. 2). These shifts could suggest involvement the N40 nitrogen atom in complexation of silver ion.
Table 1 Physical properties of obtained silver complexes. Complex
Appearance
Color
Melting point
Molar conductance LM (U1 cm2 mol1)
Solubility (at 1 mg mL1) in different solvents
(1)
amorphous
beige-white
240e242 C with decomposition
25.2
(2)
amorphous
white
205e223 C with decomposition
19.9
(3)
amorphous
yellowishwhite
205e208 C with decomposition
24.3
very soluble in DMSO, dimethylformamide (DMF), concentrated ammonia, and in 10% NaOH with formation of a black precipitate (Ag2O); slightly soluble in methanol, ethanol, acetonitrile, dichloromethane and 10% HCl, insoluble in water, acetone and glacial acetic acid very soluble in DMSO, DMF, concentrated ammonia; slightly soluble in acetonitrile and 10% HCl, insoluble in water, methanol, ethanol, dichloromethane, and acetone; insoluble in glacial acetic acid and in 10% NaOH very soluble in DMSO, concentrated ammonia, 10% NaOH with formation of a black precipitate (Ag2O); slightly soluble in DMF and 10% HCl, insoluble in water, methanol, ethanol, dichloromethane, acetone, and acetonitrile; insoluble in glacial acetic acid
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Table 2 Elemental analysis and silver concentrations of the complexes. Element (%)
Complex (1)
(2)
C36H40AgF2N7O11
C37H44AgF2N7O12
C24H33AgFN3O14
M ¼ 892
M ¼ 924
M ¼ 714
LEV2Ag(NO3)
C H N Ag
LEV2Ag(NO3)(CH3OH)
LEV (1) (2) (3)
Found
Calculated
Found
Calculated
Found
48.44 4.52 10.98 12.08
47.94 4.84 10.64 12,29
48.06 4.8 10.6 11.67
47.64 4.66 10.04 11,19
40.35 4.66 5.88 15.10
40.56 4.69 5.54 15.46
Assignments/Band position (cm1)
n(OeH) (COOH; MeOH; H2O)
n(C]O) carb
n(C]O) pyridone
(carboxyl)
(pyridone)
3258 vw
1720 1711 1711 1704
1618 1621 1620 1622
3658 vw 3380 vw
LEVAg(C6H6O7)$3H2O
Calculated
Table 3 The most important FT-IR band assignments for LEV and its silver complexes. Compound
(3)
m m m s
vs s s m
(Fig. 3) demonstrate clearly differences comparing to the ligand. In the electronic spectrum of LEV in solid state, a main peak situated at 386 nm and few shoulders at 339.5, 303.5, and 266 nm, assigned to p-p* and n-p* transitions are observed. The hypsochromic and hyperchromic shift of the main band of LEV and the appearance of a new band at 440e450 nm for all complexes are attributed to complexation process. The new is assigned to metal to ligand charge transfer (MLCT), since no ded transitions are expected for a d10 complex [60].
s, strong; w, weak; m, medium; sh, shoulder; v, very; br, broad.
3.4. MS spectra analysis The coordination of the piperazine ring are relatively rare comparably to other reported complexes [3,52]. LEV shows characteristic absorption bands for stretching vibrations of the carboxyl groups v(C]O)carb at 1720 cm1 and the pyridone v(C]O)py at 1618 cm1 [47,53]. The decrease in the v(C] O)carb wavenumber in carboxyl group can be related to the formation of dimers and/or to the participation of carboxyl groups in complex conjugated systems. The shift of the absorption bands for stretching vibrations of the carboxyl groups v(C]O)carb are reported in other metal complexes that involve the carboxyl group in complexation [28,53,54]. Although there is weak shift in our complexes this particular aspect can be explained with the intramolecular H-bond which stabilizes the protonated form of the carboxyl group [55e58]. In our records for complexes (1e3) the characteristic absorption bands for stretching vibrations of the pyridone groups v(C]O)py are very close to LEV record which shows that this group does not directly participate in the chelation of silver. The stretching vibration for the pyridone group v(C]O)py is assigned to 1618 cm1. These data suggest that the pyridone and carboxyl group are not involved in the formation of the silver complex.
The ESI-MS is considered the most sensitive and specific method for the analysis of fluoroquinolones, which usually shows only
3.3. UVeVIS spectroscopy UV spectra of the complexes and LEV were registered in DMSO using 2.5 103 mM concentrations. Spectra show two absorption maxima. LEV and complexes have a maximum increase of the intensity at lmax 303e304 nm, characteristic to the quinolone structure obtained through a condensation of a benzene with dihydro-4-pyridone heterocycle, corresponding to the carbonyl group in position 4 and is due to p/p* and n/p* electronic transitions [59]. Using equimolar solutions obtained from ligand and complexes, it was clearly evidenced the molar ratio metal:ligand of 1:2 for complexes (1) and (2), and 1:1 for complex (3) (Supplementary Information File). UV spectra of complexes are almost similar with the LEV spectrum in DMSO solution and suggests that the complexes keep their integrity in solution [54]. Instead, the electronic spectra of complexes registered in solid state
Fig. 2. IR spectra (4000e2000 cm1): silver complexes (1e3) and LEV.
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Fig. 3. Electronic spectra in solid state of LEV, and its complexes (1), (2), and (3).
protonated molecule ions and/or molecule adduct ions. In the first step fluoroquinolones lose CO2 and H2O from the 3-carboxyl group. Than in the next step the fluoroquinolones show the loss of peripheral groups: -CH2CHNH2 or CH3CH2NCH2 from the piperazine substituent and also HF and CO followed by losses of H2O [49]. Until now the main fragmentation pattern of ofloxacin (racemic) and LEV
(pure levogir enantiomer) was published with reported molecular ions: [M þH]þ ¼ 362, [M -H2O þH]þ ¼ 344, [M -CO2 þH]þ ¼ 318, [M -CO2, eHF þH]þ ¼ 297, and [M eCH2-CH2NeCH3 þH]þ ¼ 261 [61e63]. Mass spectra analysis is a method less often used for characterization of metal complexes of fluoroquinolones. The bonds participating in complexation of silver ions are less stable under ESI mass spectra technique, even the ESI continues to be the method of first choice for analyzing thermolabile chemicals [26], and [53]. Metal complexes of fluoroquinolones could be insoluble in all tested solvents suitable for mass spectrometry. Generally, ESIMS technique intercepted cationic species corresponding to the protonated molecular ion. These species form fragment ions of m/z correspond to the loss of ligand molecules [53]. The peak observed for complex (1) (Supplementary Information File) at m/z 831 corresponds to the [(LEV)2Ag]þ complex, which clearly indicate the complexation process. The peak observed at m/z 785 corresponds to the fragment produced by the loss of one of HF and two eCH3 group from piperazine substituent. In the next step the compound lose two eCH3 group from piperazine substituent and eCH2CH(CH3)- from the last oxazine ring which corresponding to m/z 723 fragment [61,63]. This latter fragment is also present in the mass spectra of complex (2) (Supplementary Information File). Also the peak at m/z 776 corresponds to the fragment produced on removal of one oxazine ring from the complex. In addition, peaks m/z 573 and m/z 501 were also observed due to the fragmentation of non-aromatic core of LEV (Fig. 4). The LEV appeared at 362 m/z [Mþ1] in all complexes spectra. Note that peaks fragments are very low in abundance compared to that of LEV, probably because bonds participating in complexation of silver ions are broken [61,63]. In the mass spectra of complex (3) only appropriate ligand fragment is
Fig. 4. Proposed scheme of stepwise source fragmentation pathway for the complex (1).
A. Rusu et al. / Journal of Molecular Structure 1123 (2016) 384e393
found, and this can be correlated with a reduced stability of the complex in DMSO.
389
corresponding to a dehydration process, followed by an exothermic peak at 187.05 C, and two endothermic peaks at 195.68 C and 207.52 C, and began to decompose steadily up to 400 C.
3.5. NMR analysis 3.7. DFT calculations In the 1H NMR spectra of complexes only small chemical shifts were observed comparing to the LEV spectrum. The major shifts observed for the protons of piperazine ring; appear most probably as a consequence of the involvement of the piperazinic nitrogen atoms N40 or N10 in the coordination process. The value of the complexation-induced chemical shifts 1H NMR is summarized in (Supplementary Information File) [31]. 3.6. DSC analysis The DSC curves of LEV and the complexes were registered and compared (Supplementary Information File). The melting point interval of LEV (hemihydrate) is 222e236 C with decomposition [64]. Through DSC method we obtained a melting point of 228.93 C for LEV (an endothermic phenomenon) followed by a decomposition process, which correspond to the value reported in ^ form of anhydrous LEV. On the DSC thermogram literature for the a of LEV also a dehydration process around 98.83 C can be observed. This phenomenon can be attributed to the fact that pharmaceutical substances are often processed under conditions that result in the formation of crystalline hydrates, solvates, and mixtures of these forms. Dehydration of the hemihydrate form of LEV produces a mixture of physical forms, including amorphous LEV. Drying of LEV hemihydrate could generate amorphous material, which is chemically or physically unstable. The rehydration of the crystalline samples seems to be reversible [65e67]. The obtained silver complexes are stable at room temperature and can be stored for several months protected from humidity and light without any changes. All the complexes show from two to four phases of DSC curves. A phase corresponds to the formation of the anhydrous compound is present on the DSC thermogram of complex (3). Other phases that are present on DSC curves of all complexes correspond to the decomposition of LEV molecules. This thermal behavior is similar with the one of metal complexes of other fluoroquinolones [10,68]. Complex (1) start to decompose around 240e245 C with a pronounced exothermic peak, followed by others with low height up to 400 C. Complex (2) start to decompose at 206.24 C with an exothermic peak, followed by a pronounced exothermic peak at 233.4 C and a lower one at 249.84 C. The both complexes (1) and (2) show a very different behavior from the LEV DSC thermogram. Instead, the complex (3) has a different behavior than complexes (1) and (2) with a small endothermic phenomenon at 122.94 C,
The fully optimized geometries of the ligand and complexes (1) and (3) and the symbols and the labels of atoms are shown in Figs. 5 and 6. Selected bond angles, charge density, total energy and total dipole moment are presented in (Supplementary Information File). In the optimized structure of complex (1) (Fig. 5), the piperazine N22 and N48 are bound to Agþ, as well as O94, O95 (belonging to the nitrate anion). The coordination sphere around the metal centre in complex (3) (Fig. 6) is made up of the following atoms: N22 (piperazine nitrogen), O19, O20 (belonging to the citrate anion). The organic moiety (not involved in coordination) is bent away to relieve the steric hindrance. Based on data provided by elemental analysis and silver content combined with the DFT calculations and used analytical methods were outlined proposals chemical structures of the obtained complexes (Fig. 7). The proposed structure for the complex (1) consists of two ligand molecules which act as a neutral monodentate to coordinate to Agþ by N40 piperazinic atom, similar to the silver complex of norfloxacin obtained by Y.X. Li et al. [8] as a coordinating mode. The 4-oxo and 3-carboxyl group appear not to be involved in the coordination of silver. This bonding mode was unexpected for LEV that contains a methyl piperazine group in chemical structure. For the complex (2) we proposed a similar chemical structure which has a methanol molecule in addition according with CHN elemental analysis. The similarity of the two complexes is derived from the obtaining methods that use silver nitrate as a silver source. The use of ammonia basic medium conferred no interference on the chemical structure of silver complex (1) comparative with complex (2). According to the results of the analysis the complex (3) probably corresponds to a silver complex with LEV acting as a monodentate ligand by the N40 piperazinic atom. In the same time a citric acid molecule is acting a bidentate ligand by the oxygen atoms of the carboxyl group [69]. In all synthesized complexes silver is coordinated through nitrogen atoms of piperazinic moiety of LEV, similar to other reported silver complexes of fluoroquinolones [3,10,12]. 3.8. Microbiological studies The susceptibility of bacteria against tested compounds is presented in Table 4. All the three silver complexes of LEV have shown similar antibacterial activity with LEV against selected Gram positive and Gram negative bacteria.
Fig. 5. The optimised structure of the electronic ground state of LEV at the B3LYP level of theory (6-31G(d) basis). Selected calculated bond lengths (Å) in the gas phase are given above.
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Fig. 6. The optimised structure of the electronic ground state of complexes (1) and (3) at the B3LYP level of theory (6-31G(d) basis set for all atoms that are not involved in coordination; 6-31G(d0 ,p’) for the F atoms; 6-311Gþ(d,p) for N, O atoms that are bound to Ag; MWB28 for Ag). Selected calculated bond lengths (Å) in the gas phase are given above.
Fig. 7. The proposed structure for silver complexes of LEV (1e3).
A. Rusu et al. / Journal of Molecular Structure 1123 (2016) 384e393 Table 4 MIC for antibacterial and antifungal activity of LEV, and the investigated complexes (1e3). Selected bacteria and fungi
Gram positive Staphylococcus aureus 29213 Staphylococcus aureus MRSA 43300 Enterococcus faecalis 29212 Gram negative Escherichia coli 30041 Klebsiella pneumoniae 700603 Pseudomonas aeruginosa 27853 Candida spp. Candida albicans Candida guilliermondii Candida parapsilosis
MIC (mg mL1) LEV
Complex (1)
Complex (2)
Complex (3)
0.12 0.12
0.25 0.25
0.25 0.25
0.25 0.25
0.5
0.5
1
0,5
0.25 0.5 1
0.5 0.5 1
1 1 2
0.5 0.5 1
64 64 64
64 64 64
64 64 64
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silver complexes we used spectroscopic methods, including FT-IR, ESI-MS, 1H NMR, and UV spectrometry, DSC and DFT analysis. In the proposed structure for the complex (LEV)2Ag(NO3) (1), which is similar with that of complex (LEV)2Ag(NO3)(CH3OH) (2), LEV is coordinated to silver ion through the N40 piperazine atom. The complex LEVAg(C6H6O7)(3H2O (3) presents a similar coordination of LEV. The antibacterial screening shows no differences between all three complexes and ligand. Moreover antifungal activity of obtained silver complexes is modest. Acknowledgements The research was supported by the a project funded through Internal Research Grants by the University of Medicine and Phar^nia (grant contract for execution of macy of Tîrgu Mures¸, Roma research projects nr. 2/23.12.2014). Appendix A. Supplementary data
The antifungal activity of synthesized complexes was analysed on four yeast species, namely Candida albicans, Candida krusei, Candida guilliermondii and Candida parapsilosis. The results of the antifungal tests are illustrated in Table 4. The MIC values for Candida krusei were not conclusive. Antibacterial and antifungal properties of the synthesized metal complexes of LEV reported so far are varying most of them being equal as LEV activity. For a newly synthesized complex of cobalt with LEV it has been demonstrated increased activity on Escherichia coli [70]. Two zinc complexes with N-donor heterocyclic ligands are more active against Escherichia coli and Staphylococcus aureus. Regarding activity against Pseudomonas aeruginosa there is no differences comparing with LEV [54]. In the case of new copper complexes of LEV it was reported the same MIC as the LEV against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae by the in vitro doubling dilutions method; Cu2þ did not influence the antibacterial activity of fluoroquinolone [23]. For the [Cu(LEV)(phen)(H2O)](NO3)$2H2O (phen ¼ phenanthroline) the results obtained for various E. coli strains clearly showed that the ternary complex has an antimicrobial effect comparable to that of the free LEV [28]. Antibacterial results demonstrated that the effects of molybdenum complexes of LEV were comparable to the ones of LEV against Staphylococcus aureus and Escherichia coli [30]. Regarding antifungal activity of the synthesized silver complexes against the three species of Candida these are relatively modest then expected taking in consideration significant activity reported in the analysis of other silver organic compounds [71e74]. Through the microbiological screening results it is more than evident that supplementary silver antibacterial and antifungal properties are not present although the complexes contain a high percentage of silver, especially complex (3). We consider the possibility that DMSO solvent could negatively influence the stability of silver complexes. On the other hand the results of in vitro microbiological studies may be different from those in vivo. In addition it is very important the type of the bond in the structure of complexes for obtaining antimicrobial and antifungal activity [73]. Through the assumptions listed previously further studies are needed to obtain more reliable data regarding silver complexes of LEV.
4. Conclusion Using simple methods that avoid high temperatures we obtained three silver complexes of LEV. The molecular formulas of the complexes were determined by elemental analysis (CHN) and FAAS (%Ag). In order to elucidate the chemical structures of the obtained
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New silver complexes with levofloxacin: Synthesis, characterization and microbiological studies } To th b, Szende Vancea c, Felicia Toma d, Aura Rusu a, Gabriel Hancu a, *, Gergo Anca Delia Mare d, Adrian Man d, George Mihai Nit¸ulescu e, Valentina Uivarosi f a
University of Medicine and Pharmacy from Tîrgu Mures¸, Faculty of Pharmacy, Pharmaceutical Chemistry Department, Romania Semmelweis University, Budapest, Department of Pharmaceutical Chemistry, Hungary University of Medicine and Pharmacy from Tîrgu Mures¸, Faculty of Pharmacy, Physical Chemistry Department, Romania d University of Medicine and Pharmacy from Tîrgu Mures¸, Faculty of Medicine, Microbiology, Virology, and Parasitology Department, Romania e University of Medicine and Pharmacy Carol Davila, Bucharest, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Romania f University of Medicine and Pharmacy Carol Davila, Bucharest, Faculty of Pharmacy, General and Inorganic Chemistry Department, Romania b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 April 2016 Received in revised form 27 June 2016 Accepted 10 July 2016 Available online 12 July 2016
Levofloxacin is a third generation fluoroquinolone antibiotic with a broad-spectrum including both Gram positive bacteria and Gram negative bacteria, as well atypical bacteria. In order to extend the spectrum of activity and to add new biological effects, several metal complexes of levofloxacin have been obtained and reported recently. The aim of our study was to obtain new silver complexes with levofloxacin with potential broad spectrum antibacterial and antifungal activity. Therefore three new silver complexes of levofloxacin with the proposed chemical structures (levofloxacin)2Ag(NO3), (levofloxacin)2Ag(NO3)(CH3OH) and (levofloxacin)Ag(C6H6O7)$3H2O were synthesized. In order to characterize the obtained complexes elemental analysis, conductivity measurement, spectroscopic, and thermal methods were used. Optimized molecular structures were determined using DFT (density functional theory) analysis. The antibacterial activity against Gram negative and Gram positive bacteria and antifungal activity against Candida spp of the complexes was tested by determination of minimum inhibitory concentration through microtitre broth dilution method. © 2016 Elsevier B.V. All rights reserved.
Keywords: Silver Levofloxacin Fluoroquinolones Metalcomplex Silver nitrate Silver citrate
1. Introduction Antibacterial fluoroquinolones are chemotherapeutic compounds commonly used in therapy to treat bacterial infections. As in the case of most classes of antibiotic compounds, the development of bacterial resistance represents a major challenge and a serious threat to therapists. Therefore, the development of metal complexes with fluoroquinolones had captivated attention of many researchers recently, taking into account the potential of broadening the activity spectrum of the ligand or the emergence of
* Corresponding author. Faculty of Pharmacy, Department of Pharmaceutical Chemistry, University of Medicine and Pharmacy of Tîrgu Mures¸, 38 Gheorghe Marinescu Street, Tîrgu Mures¸, 540139, Romania. E-mail addresses: [email protected] (A. Rusu), [email protected] th), [email protected] (G. Hancu), [email protected] (G. To (S. Vancea), [email protected] (F. Toma), [email protected] (A.D. Mare), [email protected] (A. Man), [email protected] (G.M. Nit¸ulescu), [email protected] (V. Uivarosi). http://dx.doi.org/10.1016/j.molstruc.2016.07.035 0022-2860/© 2016 Elsevier B.V. All rights reserved.
possible new biological effects. Many studies report the excellent complexing capacity of fluoroquinolones particularly due to the chemical structure [1e3]. Among the large variety of metal cations which can be used in the production of metal complexes of the fluoroquinolones, silver appears to be adequate, mainly due to its antibacterial, antifungal and cytotoxic activity [4e6]. Thus, taking in account the biological effects of silver ions and the activity spectrum of fluoroquinolones, it makes sense to study, if there is a possible summation and potentiation of these properties in case of metal complexes which contain silver and a fluoroquinolone. Some studies have approached this line of research using as ligands first generation quinolones (nalidixic acid, norfloxacin, pefloxacin, and ciprofloxacin) or a modern fluoroquinolone (moxifloxacin) [7e16]. From a structural point of view, silver complexes with fluoroquinolones have a particular aspect, namely silver binding that may occur at the nitrogen atom of the ligand N4’-piperazine, different from most of fluoroquinolone metal complexes which have the complexing location 3-carboxyl and 4-oxo groups [3]. Levofloxacin (LEV) or (S)-9-fluoro-2,3-dihydro-3-methyl-10-(4-
A. Rusu et al. / Journal of Molecular Structure 1123 (2016) 384e393
methylpiperazin-1-yl)-7-oxo-7H-pyrido [1,2,3-de]-1,4benzoxazine-6-carboxylic acid is a third generation fluoroquinolone, being the levorotatory isomer of racemic ofloxacin (Fig. 1). As mechanism of action, LEV directly inhibits DNA synthesis by acting on two target enzymes, respectively DNA gyrase and topoisomerase IV. LEV has a broad-spectrum, acting on the Gram positive bacteria and Gram negative bacteria, and atypical bacteria, as well [17e21]. Thus far several metal complexes of LEV were synthesized and studied. Among targeted cations can be found Mg2þ, Ca2þ, Zn2þ, Pd2þ, Pt2þ, Au3þ, Cu2þ, Fe3þ, MoO2þ 2 , and the antimicrobial activity, effect on the leukemia HL-60 and HePG-2 cell line, human colon carcinoma HCT-116 and human hepatocellular carcinoma HepG-2 tumor cell lines, and interaction with DNA were tackled in different studies [22e31]. Due to an intensive worldwide use, an increase of bacterial resistance to fluoroquinolones appeared. Thus, the need to improve the antibacterial potential of existing antimicrobial drugs is more challenging, adding the concept that metal complexes could be an alternative to conventional drugs [32]. As far as we known no reported data regarding silver complex of LEV has been described in the literature. Our study reports reliable methods for preparation of silver metal complexes of LEV which will possess the biological effects of the two constituent components. In order to determine the most probable structures of the obtained silver complexes elemental analysis, conductivity measurement, spectroscopic, thermal and density functional theory (DFT) methods were used. The antimicrobial and antifungal activity of the complexes has been evaluated. 2. Methods 2.1. Materials LEV was purchased from Sigma, silver nitrate from SC UTCHIM SRL (Rom^ ania), silver citrate hydrate from Aldrich; all other chemicals used were of analytical reagent grade.
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spectrometer equipped with electrospray ionization (ESI) ion source in positive ion mode. The recorded data were processed using MassHunter software. Conditions of the ionization source were: gas flow 8 L/min, 40 psi, 300 C, 4000 V, with a full scan on the field 100e1500 amu data acquisition module. NMR measurements were carried out on a Varian Unity Inova DDR spectrometer (599 MHz for 1H) with a 5 mm inverse-detection gradient probehead using dimethylsulfoxide-d6 (DMSO-d6) as solvent. Standard pulse sequences and processing routines available in VnmrJ 2.2C/Chempack 4.0 were used. The chemical shifts were referenced to the solvent signal of internal DMSO-d6 (2.500 ppm). For Differential Scanning Calorimetry (DSC) analysis we used a DSC 60 Shimadzu apparatus. The weight of the samples was 3 mg. The DSC curves were recorded in the range of 40e400 C, with a temperature increase rate of 10 C/ min. UV spectra were recorded on a T70 þ UV/VIS Spectrometer (PG Instruments Ltd). Electronic spectra by diffuse reflectance technique were recorded in the range 200e800 nm with spectralon as standard, on a Jasco V650 spectrophotometer. 2.3. Computational study Full geometry optimizations of the ligand and complexes (1) and (3) were carried out using the DFT method in Gaussian09 [33]. The split valence plus polarization basis set 6-31G(d) was applied for any atom that is not involved in coordination. 6-31G(d0 ,p’) basis set was used for the fluorine atoms. To give a better description of the Agþ e ligand interactions, basis sets with additional diffuse functions were added for the piperazine nitrogen and the oxygen atoms involved in coordination (6-311Gþ(d,p)). The Stuttgart-Cologne small-core quasi-relativistic pseudopotential ECP28MWB (28 electrons in the core) [34,35] was used for the Agþ ion in conjunction with its optimized valence basis set 8s7p6d 2f1g. DFT/ B3LYP/(MWB28 for Ag) was proven to give good results for prediction of coordination polyhedral and geometry parameters of the Agþ complexes in other [36,37]. 2.4. Experimental procedures
2.2. General Elemental analyses of C, H and N were carried out on a Perkin Elmer PE 2400 analyzer. The metal ion content was determined by flame atomic absorption spectrometry (FAAS) using a Shimadzu AA 6300 spectrometer after destruction of the complex in a Berghof microwave digestion system. The molar conductance was determined for 103 M solution of complex in dimethyl sulfoxide (DMSO) with an analyzer inoLab® pH/Cond 740. To record the FT-IR spectra we used a FT-IR Thermo Nicolet (USA) spectrometer with samples prepared as KBr pellets in the range of 4000e400 cm1, 32 scans at 4 cm1 resolution, and processed using Omnic V.6 software. Mass spectra of the obtained compounds were analysed with an Agilent 6410 Triple Quadrupole (Agilent Technologies, USA) mass
Fig. 1. The chemical structure and numbering of LEV.
In order to obtain silver metal complexes of LEV we considered several possibilities. Although in previously reported articles regarding synthesis of silver complexes with fluoroquinolones only silver nitrate was used, we tried to synthesize silver complexes starting with another silver compound in this case silver citrate. In the preliminary study we tried to adapt several methods described in the literature which used as reaction temperature 80e100 C [8,14e16]. These methods were not successful due to the fact that the Agþ ion can easily generate Ag2O (black-brown precipitate), which partially decomposed to Ag and O2 (Ag2O / 2Ag þ 1/2O2) when heated. Thus, we opted for a “cold” method that does not use heat. Also for drying the obtained substance we choose to avoid as much as possible the high temperatures and direct exposure to light [38,39]. 2.4.1. Synthesis of complex (1) A solution obtained from 1.38 mmol AgNO3 and 50 mL of water have been added into a mixture of 2.76 mmol of LEV and 75 mL methanol (2:1 molar ratio LEV:AgNO3). After mixing the solutions 25 mL of concentrated ammonia was added. The entire mixture was stirred for 4 h with a magnetic stirrer, in a sealed flat-bottom flask, protected from light. A pale yellowish clear solution was obtained, which was left overnight at room temperature. The obtained solution was concentrated with a rotary evaporator at 40 C under vacuum and the last 20 mL solution was slowly dried in a normal oven set at 40 C for 24 h. The solid compound was kept in desiccator over anhydrous CaCl2, protected from light.
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2.4.2. Synthesis of complex (2) A solution obtained from 1.38 mmol AgNO3 and 50 mL of water has been added into a mixture of 2.76 mmol of LEV and 100 mL methanol (2:1 molar ratio LEV:AgNO3). The entire mixture was stirred for 8 h on a magnetic stirrer, in a sealed flat-bottom flask, protected from light. This mixture was concentrated with a rotary evaporator at 40 C under vacuum to obtain a suspension with a minimal volume of about 20 mL. The suspension was filtered to obtain a white precipitate, and washed with distilled water. The precipitate was slowly dried in a normal oven set at 40 C for 24 h, than the solid compound was kept in desiccator over anhydrous CaCl2, protected from light.
presented no bacterial growth. The DMSO solvent was used as a sterility control (25 mL solvent and 175 mL Muller Hinton broth). Muller Hinton broth alone was used as a negative control. The positive growth control consisted in a mixture of 25 mL Muller Hinton broth and 175 mL bacterial suspension [44]. 2.5.2. Antifungal activity screening Antifungal activity was assessed following the same protocol, as for bacterial MIC, excepting the fact that the Muller-Hinton broth was supplemented with 2% glucose and that the plates were incubated for 48 h. All experiments were carried out in triplicate. 3. Results and discussion
2.4.3. Synthesis of complex (3) A solution obtained from 1 mmol silver citrate previously dissolved in 25 mL 4 M citric acid aqueous solution has been added into a mixture of 2 mmol of LEV and 100 mL methanol (2:1 molar ratio LEV:silver citrate) resulting a greenish-yellow solution. The entire mixture was stirred for 8 h on a magnetic stirrer, in a sealed flat-bottom flask, protected from light, at room temperature. The solution was kept at room temperature overnight. The solution was concentrated with a rotary evaporator at 45 C under vacuum to obtain a viscous yellow liquid with a minimal volume of about 20 mL. This liquid was slowly dried in a normal oven set at 40 C for 48 h. The resulted solid crystalline compound was kept in desiccator over anhydrous CaCl2, protected from light. In order to eliminate the excess of citric acid, the resulting compound was dissolved in acetone (or ethanol) and the resulting suspension was filtered and slowly dried in an oven set at 40 C and kept in desiccator over anhydrous CaCl2, protected from light.
The physical properties of the resulted complexes, including their melting point and solubility are shown in Table 1. The complexes were soluble in DMSO and DMF similar to other metal complexes of fluoroquinolones. The molar conductivity data reveal that the complexes (1e3) are well below for an electrolyte 1:1 [24,45]. 3.1. Elemental analysis The results of elemental analysis and the content of silver in the obtained compounds are shown in Table 2. The results of elemental analysis indicated that complexation occurs through our selected methods, as complexes with molar ratio metal:ligand of 1:2 for complexes (1) and (2), and 1:1 for complex (3) are obtained. 3.2. FT-IR spectroscopy
2.5. Microbiological studies 2.5.1. Antibacterial activity screening The antibacterial activity of LEV and the synthesized complexes was tested on six bacterial strains, three Gram positive (Staphylococcus aureus ATCC 29213, Staphylococcus aureus MRSA ATCC 43300, Enterococcus faecalis ATCC 29212) and three Gram negative (Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 27853). Antimicrobial activity was analysed according to the standard CLSI (Clinical and Laboratory Standards Institute) microdilution method. In 96-well microtiter plates, binary dilutions were performed for each tested complex, to a final volume of 25 mL [40e43]. Approximately 1.5 104 CFU (colony forming unit) of each bacterial strain and 175 mL were inoculated into the corresponding wells. The plates were incubated at 35 C for 24 h. For all samples, the minimum inhibitory concentration (MIC) was interpreted at the last dilution which
The FT-IR spectra of LEV and the complexes (1e3) were registered and compared (Supplementary Information File) [10,14,28,46e51]. The most significant assignments are presented in Table 3. In the most of the metallic complexes with fluoroquinolone derivatives reported previously in literature, the ligands coordinate in a bidentate manner via the carboxylic oxygen and the carbonyl oxygen [3]. In the case of LEV the bands at 3258e2800 cme 1 correspond to the vCeH stretching vibrations of a methyl radical at the N40 nitrogen atom in the piperazinyl moiety, and/or to the vCeH vibrations of methylene groups in ReOeAr [47]. In our silver complexes (1e2) these weak bands appear shifted at 3043/ 3044 cm1 (very weak) and around 2839 cm1(shoulder); for complex (3) these weak bands appear shifted at 3380 cm1, 3082 cm1 and 2847 cm1 (Fig. 2). These shifts could suggest involvement the N40 nitrogen atom in complexation of silver ion.
Table 1 Physical properties of obtained silver complexes. Complex
Appearance
Color
Melting point
Molar conductance LM (U1 cm2 mol1)
Solubility (at 1 mg mL1) in different solvents
(1)
amorphous
beige-white
240e242 C with decomposition
25.2
(2)
amorphous
white
205e223 C with decomposition
19.9
(3)
amorphous
yellowishwhite
205e208 C with decomposition
24.3
very soluble in DMSO, dimethylformamide (DMF), concentrated ammonia, and in 10% NaOH with formation of a black precipitate (Ag2O); slightly soluble in methanol, ethanol, acetonitrile, dichloromethane and 10% HCl, insoluble in water, acetone and glacial acetic acid very soluble in DMSO, DMF, concentrated ammonia; slightly soluble in acetonitrile and 10% HCl, insoluble in water, methanol, ethanol, dichloromethane, and acetone; insoluble in glacial acetic acid and in 10% NaOH very soluble in DMSO, concentrated ammonia, 10% NaOH with formation of a black precipitate (Ag2O); slightly soluble in DMF and 10% HCl, insoluble in water, methanol, ethanol, dichloromethane, acetone, and acetonitrile; insoluble in glacial acetic acid
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Table 2 Elemental analysis and silver concentrations of the complexes. Element (%)
Complex (1)
(2)
C36H40AgF2N7O11
C37H44AgF2N7O12
C24H33AgFN3O14
M ¼ 892
M ¼ 924
M ¼ 714
LEV2Ag(NO3)
C H N Ag
LEV2Ag(NO3)(CH3OH)
LEV (1) (2) (3)
Found
Calculated
Found
Calculated
Found
48.44 4.52 10.98 12.08
47.94 4.84 10.64 12,29
48.06 4.8 10.6 11.67
47.64 4.66 10.04 11,19
40.35 4.66 5.88 15.10
40.56 4.69 5.54 15.46
Assignments/Band position (cm1)
n(OeH) (COOH; MeOH; H2O)
n(C]O) carb
n(C]O) pyridone
(carboxyl)
(pyridone)
3258 vw
1720 1711 1711 1704
1618 1621 1620 1622
3658 vw 3380 vw
LEVAg(C6H6O7)$3H2O
Calculated
Table 3 The most important FT-IR band assignments for LEV and its silver complexes. Compound
(3)
m m m s
vs s s m
(Fig. 3) demonstrate clearly differences comparing to the ligand. In the electronic spectrum of LEV in solid state, a main peak situated at 386 nm and few shoulders at 339.5, 303.5, and 266 nm, assigned to p-p* and n-p* transitions are observed. The hypsochromic and hyperchromic shift of the main band of LEV and the appearance of a new band at 440e450 nm for all complexes are attributed to complexation process. The new is assigned to metal to ligand charge transfer (MLCT), since no ded transitions are expected for a d10 complex [60].
s, strong; w, weak; m, medium; sh, shoulder; v, very; br, broad.
3.4. MS spectra analysis The coordination of the piperazine ring are relatively rare comparably to other reported complexes [3,52]. LEV shows characteristic absorption bands for stretching vibrations of the carboxyl groups v(C]O)carb at 1720 cm1 and the pyridone v(C]O)py at 1618 cm1 [47,53]. The decrease in the v(C] O)carb wavenumber in carboxyl group can be related to the formation of dimers and/or to the participation of carboxyl groups in complex conjugated systems. The shift of the absorption bands for stretching vibrations of the carboxyl groups v(C]O)carb are reported in other metal complexes that involve the carboxyl group in complexation [28,53,54]. Although there is weak shift in our complexes this particular aspect can be explained with the intramolecular H-bond which stabilizes the protonated form of the carboxyl group [55e58]. In our records for complexes (1e3) the characteristic absorption bands for stretching vibrations of the pyridone groups v(C]O)py are very close to LEV record which shows that this group does not directly participate in the chelation of silver. The stretching vibration for the pyridone group v(C]O)py is assigned to 1618 cm1. These data suggest that the pyridone and carboxyl group are not involved in the formation of the silver complex.
The ESI-MS is considered the most sensitive and specific method for the analysis of fluoroquinolones, which usually shows only
3.3. UVeVIS spectroscopy UV spectra of the complexes and LEV were registered in DMSO using 2.5 103 mM concentrations. Spectra show two absorption maxima. LEV and complexes have a maximum increase of the intensity at lmax 303e304 nm, characteristic to the quinolone structure obtained through a condensation of a benzene with dihydro-4-pyridone heterocycle, corresponding to the carbonyl group in position 4 and is due to p/p* and n/p* electronic transitions [59]. Using equimolar solutions obtained from ligand and complexes, it was clearly evidenced the molar ratio metal:ligand of 1:2 for complexes (1) and (2), and 1:1 for complex (3) (Supplementary Information File). UV spectra of complexes are almost similar with the LEV spectrum in DMSO solution and suggests that the complexes keep their integrity in solution [54]. Instead, the electronic spectra of complexes registered in solid state
Fig. 2. IR spectra (4000e2000 cm1): silver complexes (1e3) and LEV.
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Fig. 3. Electronic spectra in solid state of LEV, and its complexes (1), (2), and (3).
protonated molecule ions and/or molecule adduct ions. In the first step fluoroquinolones lose CO2 and H2O from the 3-carboxyl group. Than in the next step the fluoroquinolones show the loss of peripheral groups: -CH2CHNH2 or CH3CH2NCH2 from the piperazine substituent and also HF and CO followed by losses of H2O [49]. Until now the main fragmentation pattern of ofloxacin (racemic) and LEV
(pure levogir enantiomer) was published with reported molecular ions: [M þH]þ ¼ 362, [M -H2O þH]þ ¼ 344, [M -CO2 þH]þ ¼ 318, [M -CO2, eHF þH]þ ¼ 297, and [M eCH2-CH2NeCH3 þH]þ ¼ 261 [61e63]. Mass spectra analysis is a method less often used for characterization of metal complexes of fluoroquinolones. The bonds participating in complexation of silver ions are less stable under ESI mass spectra technique, even the ESI continues to be the method of first choice for analyzing thermolabile chemicals [26], and [53]. Metal complexes of fluoroquinolones could be insoluble in all tested solvents suitable for mass spectrometry. Generally, ESIMS technique intercepted cationic species corresponding to the protonated molecular ion. These species form fragment ions of m/z correspond to the loss of ligand molecules [53]. The peak observed for complex (1) (Supplementary Information File) at m/z 831 corresponds to the [(LEV)2Ag]þ complex, which clearly indicate the complexation process. The peak observed at m/z 785 corresponds to the fragment produced by the loss of one of HF and two eCH3 group from piperazine substituent. In the next step the compound lose two eCH3 group from piperazine substituent and eCH2CH(CH3)- from the last oxazine ring which corresponding to m/z 723 fragment [61,63]. This latter fragment is also present in the mass spectra of complex (2) (Supplementary Information File). Also the peak at m/z 776 corresponds to the fragment produced on removal of one oxazine ring from the complex. In addition, peaks m/z 573 and m/z 501 were also observed due to the fragmentation of non-aromatic core of LEV (Fig. 4). The LEV appeared at 362 m/z [Mþ1] in all complexes spectra. Note that peaks fragments are very low in abundance compared to that of LEV, probably because bonds participating in complexation of silver ions are broken [61,63]. In the mass spectra of complex (3) only appropriate ligand fragment is
Fig. 4. Proposed scheme of stepwise source fragmentation pathway for the complex (1).
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found, and this can be correlated with a reduced stability of the complex in DMSO.
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corresponding to a dehydration process, followed by an exothermic peak at 187.05 C, and two endothermic peaks at 195.68 C and 207.52 C, and began to decompose steadily up to 400 C.
3.5. NMR analysis 3.7. DFT calculations In the 1H NMR spectra of complexes only small chemical shifts were observed comparing to the LEV spectrum. The major shifts observed for the protons of piperazine ring; appear most probably as a consequence of the involvement of the piperazinic nitrogen atoms N40 or N10 in the coordination process. The value of the complexation-induced chemical shifts 1H NMR is summarized in (Supplementary Information File) [31]. 3.6. DSC analysis The DSC curves of LEV and the complexes were registered and compared (Supplementary Information File). The melting point interval of LEV (hemihydrate) is 222e236 C with decomposition [64]. Through DSC method we obtained a melting point of 228.93 C for LEV (an endothermic phenomenon) followed by a decomposition process, which correspond to the value reported in ^ form of anhydrous LEV. On the DSC thermogram literature for the a of LEV also a dehydration process around 98.83 C can be observed. This phenomenon can be attributed to the fact that pharmaceutical substances are often processed under conditions that result in the formation of crystalline hydrates, solvates, and mixtures of these forms. Dehydration of the hemihydrate form of LEV produces a mixture of physical forms, including amorphous LEV. Drying of LEV hemihydrate could generate amorphous material, which is chemically or physically unstable. The rehydration of the crystalline samples seems to be reversible [65e67]. The obtained silver complexes are stable at room temperature and can be stored for several months protected from humidity and light without any changes. All the complexes show from two to four phases of DSC curves. A phase corresponds to the formation of the anhydrous compound is present on the DSC thermogram of complex (3). Other phases that are present on DSC curves of all complexes correspond to the decomposition of LEV molecules. This thermal behavior is similar with the one of metal complexes of other fluoroquinolones [10,68]. Complex (1) start to decompose around 240e245 C with a pronounced exothermic peak, followed by others with low height up to 400 C. Complex (2) start to decompose at 206.24 C with an exothermic peak, followed by a pronounced exothermic peak at 233.4 C and a lower one at 249.84 C. The both complexes (1) and (2) show a very different behavior from the LEV DSC thermogram. Instead, the complex (3) has a different behavior than complexes (1) and (2) with a small endothermic phenomenon at 122.94 C,
The fully optimized geometries of the ligand and complexes (1) and (3) and the symbols and the labels of atoms are shown in Figs. 5 and 6. Selected bond angles, charge density, total energy and total dipole moment are presented in (Supplementary Information File). In the optimized structure of complex (1) (Fig. 5), the piperazine N22 and N48 are bound to Agþ, as well as O94, O95 (belonging to the nitrate anion). The coordination sphere around the metal centre in complex (3) (Fig. 6) is made up of the following atoms: N22 (piperazine nitrogen), O19, O20 (belonging to the citrate anion). The organic moiety (not involved in coordination) is bent away to relieve the steric hindrance. Based on data provided by elemental analysis and silver content combined with the DFT calculations and used analytical methods were outlined proposals chemical structures of the obtained complexes (Fig. 7). The proposed structure for the complex (1) consists of two ligand molecules which act as a neutral monodentate to coordinate to Agþ by N40 piperazinic atom, similar to the silver complex of norfloxacin obtained by Y.X. Li et al. [8] as a coordinating mode. The 4-oxo and 3-carboxyl group appear not to be involved in the coordination of silver. This bonding mode was unexpected for LEV that contains a methyl piperazine group in chemical structure. For the complex (2) we proposed a similar chemical structure which has a methanol molecule in addition according with CHN elemental analysis. The similarity of the two complexes is derived from the obtaining methods that use silver nitrate as a silver source. The use of ammonia basic medium conferred no interference on the chemical structure of silver complex (1) comparative with complex (2). According to the results of the analysis the complex (3) probably corresponds to a silver complex with LEV acting as a monodentate ligand by the N40 piperazinic atom. In the same time a citric acid molecule is acting a bidentate ligand by the oxygen atoms of the carboxyl group [69]. In all synthesized complexes silver is coordinated through nitrogen atoms of piperazinic moiety of LEV, similar to other reported silver complexes of fluoroquinolones [3,10,12]. 3.8. Microbiological studies The susceptibility of bacteria against tested compounds is presented in Table 4. All the three silver complexes of LEV have shown similar antibacterial activity with LEV against selected Gram positive and Gram negative bacteria.
Fig. 5. The optimised structure of the electronic ground state of LEV at the B3LYP level of theory (6-31G(d) basis). Selected calculated bond lengths (Å) in the gas phase are given above.
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Fig. 6. The optimised structure of the electronic ground state of complexes (1) and (3) at the B3LYP level of theory (6-31G(d) basis set for all atoms that are not involved in coordination; 6-31G(d0 ,p’) for the F atoms; 6-311Gþ(d,p) for N, O atoms that are bound to Ag; MWB28 for Ag). Selected calculated bond lengths (Å) in the gas phase are given above.
Fig. 7. The proposed structure for silver complexes of LEV (1e3).
A. Rusu et al. / Journal of Molecular Structure 1123 (2016) 384e393 Table 4 MIC for antibacterial and antifungal activity of LEV, and the investigated complexes (1e3). Selected bacteria and fungi
Gram positive Staphylococcus aureus 29213 Staphylococcus aureus MRSA 43300 Enterococcus faecalis 29212 Gram negative Escherichia coli 30041 Klebsiella pneumoniae 700603 Pseudomonas aeruginosa 27853 Candida spp. Candida albicans Candida guilliermondii Candida parapsilosis
MIC (mg mL1) LEV
Complex (1)
Complex (2)
Complex (3)
0.12 0.12
0.25 0.25
0.25 0.25
0.25 0.25
0.5
0.5
1
0,5
0.25 0.5 1
0.5 0.5 1
1 1 2
0.5 0.5 1
64 64 64
64 64 64
64 64 64
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silver complexes we used spectroscopic methods, including FT-IR, ESI-MS, 1H NMR, and UV spectrometry, DSC and DFT analysis. In the proposed structure for the complex (LEV)2Ag(NO3) (1), which is similar with that of complex (LEV)2Ag(NO3)(CH3OH) (2), LEV is coordinated to silver ion through the N40 piperazine atom. The complex LEVAg(C6H6O7)(3H2O (3) presents a similar coordination of LEV. The antibacterial screening shows no differences between all three complexes and ligand. Moreover antifungal activity of obtained silver complexes is modest. Acknowledgements The research was supported by the a project funded through Internal Research Grants by the University of Medicine and Phar^nia (grant contract for execution of macy of Tîrgu Mures¸, Roma research projects nr. 2/23.12.2014). Appendix A. Supplementary data
The antifungal activity of synthesized complexes was analysed on four yeast species, namely Candida albicans, Candida krusei, Candida guilliermondii and Candida parapsilosis. The results of the antifungal tests are illustrated in Table 4. The MIC values for Candida krusei were not conclusive. Antibacterial and antifungal properties of the synthesized metal complexes of LEV reported so far are varying most of them being equal as LEV activity. For a newly synthesized complex of cobalt with LEV it has been demonstrated increased activity on Escherichia coli [70]. Two zinc complexes with N-donor heterocyclic ligands are more active against Escherichia coli and Staphylococcus aureus. Regarding activity against Pseudomonas aeruginosa there is no differences comparing with LEV [54]. In the case of new copper complexes of LEV it was reported the same MIC as the LEV against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae by the in vitro doubling dilutions method; Cu2þ did not influence the antibacterial activity of fluoroquinolone [23]. For the [Cu(LEV)(phen)(H2O)](NO3)$2H2O (phen ¼ phenanthroline) the results obtained for various E. coli strains clearly showed that the ternary complex has an antimicrobial effect comparable to that of the free LEV [28]. Antibacterial results demonstrated that the effects of molybdenum complexes of LEV were comparable to the ones of LEV against Staphylococcus aureus and Escherichia coli [30]. Regarding antifungal activity of the synthesized silver complexes against the three species of Candida these are relatively modest then expected taking in consideration significant activity reported in the analysis of other silver organic compounds [71e74]. Through the microbiological screening results it is more than evident that supplementary silver antibacterial and antifungal properties are not present although the complexes contain a high percentage of silver, especially complex (3). We consider the possibility that DMSO solvent could negatively influence the stability of silver complexes. On the other hand the results of in vitro microbiological studies may be different from those in vivo. In addition it is very important the type of the bond in the structure of complexes for obtaining antimicrobial and antifungal activity [73]. Through the assumptions listed previously further studies are needed to obtain more reliable data regarding silver complexes of LEV.
4. Conclusion Using simple methods that avoid high temperatures we obtained three silver complexes of LEV. The molecular formulas of the complexes were determined by elemental analysis (CHN) and FAAS (%Ag). In order to elucidate the chemical structures of the obtained
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