Accepted Manuscript Synthesis, characterization and antibacterial activity of a Schiff base derived from cephalexin and sulphathiazole and its transition metal complexes J.R. Anacona, Juan Luis Rodriguez, Juan Camus PII: DOI: Reference:

S1386-1425(14)00430-2 http://dx.doi.org/10.1016/j.saa.2014.03.019 SAA 11852

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Accepted Date:

1 December 2013 15 March 2014

Please cite this article as: J.R. Anacona, J.L. Rodriguez, J. Camus, Synthesis, characterization and antibacterial activity of a Schiff base derived from cephalexin and sulphathiazole and its transition metal complexes, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa. 2014.03.019

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Dear Editor We would apréciate if you consider the attached manuscript to be published in Spectrochimica Acta. The present study is of interest considering that the emergence of resistant human pathogens is a major problem in current antimicrobial therapy, encouraging efforts to develop novel drugs. In recent years, intensive research has been carried out in order to obtain modified cephalosporins with improved antimicrobial properties. Metal complexes were found to be particularly useful in this matter, extending the landscape of drug design and enabling novel mechanisms of action. The transport properties and other biological and physiological properties of the complexes may be of greater importance. Sincerely,

J.R. Anacona

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Graphical abstract Synthesis, characterization and antibacterial activity of a Schiff base derived from cephalexin and sulphathiazole and its transition metal complexes J.R. Anaconaa,*, Juan Luis Rodrigueza and Juan Camusb a Department of Chemistry, Universidad de Oriente, Cumana 6101. Venezuela b Facultad de Ciencias, Universidad de Playa Ancha, Valparaíso, Chile Transition coordination compounds with a Schiff base (HL) derived from the condensation of cephalexin antibiotic with sulphathiazole were synthesized, characterized and screened for antibacterial activity..

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Spectrochimica Acta Part A Highlights

We prepare a new Schiff base using cephalexin antibiotic We prepare cephalexin Schiff base transition metal complexes. We study magnetic, spectroscopic properties and antibacterial activity

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Synthesis, characterization and antibacterial activity of a Schiff base derived from cephalexin and sulphathiazole and its transition metal complexes J.R. Anaconaa,*, Juan Luis Rodrigueza and Juan Camusb a

Department of Chemistry, Universidad de Oriente, Cumana 6101. Venezuela

b

Facultad de Ciencias, Universidad de Playa Ancha, Valparaíso, Chile

*Corresponding author at: Department of Chemistry, Universidad de Oriente, Cumana 6101. Venezuela Tel.: +(58)(293)4002405 Fax: +(58)(293)4002405 E-mail address: [email protected] (J. Anacona)

Abstract: Metal(II) coordination compounds of a cephalexin Schiff base (HL) derived from the condensation of cephalexin antibiotic with sulphathiazole were synthesized. The Schiff base ligand, mononuclear [ML(OAc)(H2O)2] (M(II) = Mn, Co, Ni, Zn) complexes and magnetically diluted trinuclear copper(II) complex [Cu3L(OH)5] were characterized by several techniques, including elemental and thermal analysis, molar conductance and magnetic susceptibility measurements, electronic, FT−IR, EPR and 1H NMR spectral studies. The analytical and molar conductance values indicated that the acetate ions coordinate to the metal ions. The Schiff base ligand HL behaves as a monoanionic tridentate NNO and tetradentate NNOO chelating agent in the mono and trinuclear complexes respectively.

Keywords: Schiff base metal complexes, Schiff base containing cephalexin, synthesis cephalexin derivative, magnetic studies, spectroscopic studies, synthesis

Introduction

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The Schiff bases are widely employed as ligands in coordination chemistry [1, 2]. Schiff base ligands containing an amine group (–RC=N–) are usually formed by the condensation of a primary amine with an active carbonyl. They are readily available, versatile and depending on the nature of the starting materials they exhibit various denticities and functionalities. Moreover, the nature, number, and the relative position of the donor atoms of a Schiff base ligand allow a good control over the stereochemistry of the metallic centers, as well as over the number of the metal ions within homo and heteropolynuclear complexes [3]. All these advantages make Schiff bases very good candidates in the effort to synthesize metal complexes of interest in bioinorganic chemistry, catalysis, encapsulation, transport and separation processes [3]. Schiff bases form an important class of organic compounds with a wide variety of biological properties [4]. Development of a new chemotherapeutic Schiff base is now attracting the attention of medicinal chemist [5]. Many studies have been reported regarding the biological activities of Schiff bases, including their anticancer [6], antibacterial [7], antifungal, and herbicidal activities. Schiff bases derived from various amine and carbonyl derivatives were reported to possess genotoxicity [8, 9], antimicrobial [10], and antifungal activities [11]. Continuing with metal-based antibiotics studies in order to establish whether complexation affects the pharmacological properties of the ligand and to derive additional fundamental knowledge about antibiotic action [12−16], we report here the isolation and characterization of metal(II) complexes containing a Schiff base ligand derived from the condensation of cephalexin antibiotic, first generation cephalosporin, with sulphathiazole. The chemical structure of cephalexin is shown in Figure 1. Experimental Materials and methods

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All necessary precautions were taken to exclude oxygen and moisture during the synthesis and handling of the compounds. Analytical grade chemicals were used as received for all experiments. Fourier transform infrared (FTIR) spectra of the ligand and its metal complexes as KBr pellets were recorded in the spectral range 4,000–400 cm−1 with a Perkin-Elmer Series 2000 apparatus. FT−IR spectra as polyethylene pellets were recorded between 450 and 120 cm−1 using a Bruker IFS 66V spectrophotometer. EPR spectra were recorded on a Bruker ECS 106 spectrometer operating in the X−band (9.76 GHz). DPPH free radical was used as the g marker. Measurements of d–d transitions in the visible and near infrared region were taken with a Cary Recording Spectrophotometer Model 17D, while a Perkin-Elmer spectrophotometer was used for recording the visible and u.v. regions. The contents of C, H, N and S were analysed on a LECO CHNS 932 model microanalytical instrument. The compounds were decomposed by wet digestion at 340°C with sulphuric acid and hydrogen peroxide. The metal ion contents in this solution were determined by normal complexometric titration procedures with standard 0.01 mol L-1 EDTA solution using xilenol orange as an indicator [17]. The metal contents as well as the coordinated water were also obtained from the TG curves. Magnetic susceptibilities were measured on a Johnson Matthey Magnetic Susceptibility balance at room temperature using HgCo(NCS)4 as calibrant. 1H NMR spectra were run at 80 MHz on a Varian spectrometer in DMSO against tetramethylsilane (TMS) as internal reference. Thermograms were recorded on a simultaneous thermal analyzer, STA−6000 (Perkin Elmer) instrument at a heating rate of 10ºC min−1 up to 800°C. X-ray powder diffraction patterns for the studied complexes and final solid product of thermal decomposition were recorded on a HZG 4 diffractometer.

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Measurements were taken over the range of 2θ=2–70° using Ni filtered CuKα radiation. Synthesis of Schiff base ligand HL To 1 mmol of cephalexin in 250 mL of hot methanol were added 1 mmol of sulphathiazole The solution was refluxed under nitrogen atmosphere at 70°C for 3 h to give a dark yellow precipitate. This material was filtered off and washed with methanol and ether, and dried under reduced pressure. The product was purified by recrystallization from the same solvent (yield 72%), m.p. = 165°C. Synthesis of Schiff base complexes. Manganese(II), cobalt(II), nickel(II) and zinc(II) complexes were prepared by the same general method. To a solution of 1 mmol of the appropriate M(OAc)2 metal salts in 20 mL of water was slowly added with stirring a solution of 1 mmol of HL in 10 mL of ethanol. To this solution KOH (0.1% in methanol) was added to adjust the pH of the solution at 7–8 and the mixture was then refluxed for 4 h. and colored precipitates formed. The acetate metal(II) complexes of HL were separated from the reaction mixture as amorphous solids and washed several times with water, methanol and ether and dried under reduced pressure at room temperature. Copper(II) complex was prepared by mixing HL (1 mmol) and copper(II) acetate (3 mmol) in methanol (20 mL), then pH of the solution was adjusted to 8.0 with KOH solution and the reaction mixture was stirred at room temperature for about 4 h and then left to stand overnight. A green polymeric complex was formed but on adding ethanol and scratching, the polymeric substance changed to a powder. Complexes were purified by recrystallization from dimethylsulfoxide/water mixture. Yield 55–75%, m.p. > 250°C.

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Antibacterial activity The antibacterial activity of Schiff base ligand and metal complexes was tested against Staphylococcus aureus as a Gram-positive bacterium, and Escherichia coli as a Gram-negative bacterium according to a modified Kirby−Bauer disc diffusion method under standard conditions using Mueller−Hinton agar medium, as previously reported [12−15].

Results and discussion The ligand and the metal(II) complexes were isolated pure in very good yields and they are of various colours. The ligand, manganese(II) and zinc(II) complexes are yellow, cobalt(II) complex is red wine, nickel(II) and copper(II) complexes are green in colour. All the complexes did not melt/decompose when heated up to 250°C. The synthetic route of HL ligand is given in Scheme S1. The elemental analyses of the ligand and complexes are contained in Table 1 and they agree well with a 1:1:1:2 metal: ligand: acetate: coordinated water stoichiometry for the mononuclear complexes. Thus, the general formulae [ML(OAc)(H2O)2] (M(II) = Mn, Co, Ni, Zn) have been assigned to the mononuclear complexes and they are very air stable solids at room temperature without decomposition for a long time. The trinuclear copper(II) compound of formula [Cu3L(OH)5] was obtained by reaction of HL with either copper(II) acetate or chloride in a 1:3 molar ratio. The complexes are insoluble in water and other common organic solvents such as ethanol, chloroform, benzene, acetone, dichloromethane, DMF, acetonitrile and ether but soluble in DMSO. Attempts to form complexes of a well−defined stoichiometry, under the above-mentioned conditions, with chromium(III), iron(II), copper(I) and

mercury(II) ions were unsuccessful. The molar

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conductance values measured in DMSO at room temperature vary from 1.20 to 20.2 S cm2 mol-1, revealing the non-electrolytic nature of the complexes [18] and suggesting that the acetate ion in [ML(OAc)(H2O)2] complexes is coordinated to the metal(II) ions.

Thermal analysis By thermal analysis, information on their properties, nature of intermediate and final products of their thermal decomposition can be obtained [19]. From TGA curves, the mass loss was calculated for the different steps and compared with those theoretically calculated for the suggested formulae based on the results of elemental analyses as well as molar conductance measurements. TGA indicated the formation of metal oxide as the end product from which the metal content could be calculated and compared with that obtained from analytical determination. Thermograms of the hydrated metal complexes indicate endothermic decompositions in three steps and also reveal that the complexes are stable with no hydration water and solvent molecules. The first step in the 158 to 175°C range is assigned to loss of coordinated water molecules (Table 1). The second step at 210–395°C corresponds to removal of acetate as acetic acid, which is well known in the literature [20]. The final decomposition step includes complete evaporation of the ligand as well as formation of metal oxide as final product from which the metal content was found to be in very good agreement with the data obtained from complexometric analyses. Thermal degradation of hydrated complexes are given as follows: [ML(OAc)(H2O)2] → [ML(OAc)] [ML(OAc)] [ML]

→

→ [MO]

[ML]

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Thus, the overall thermogravimetric results are consistent with the formulation of these complexes. The degradation process of the trinuclear copper(II) complex is intricate and is not possible to distinguish intermediate solid products. The solid residues obtained during thermal decomposition of complexes are suitable metal oxides: MnO, Co3O4, NiO, CuO and ZnO. Their compositions have been confirmed by X-ray diffraction measurement. The diffraction patterns of obtained residues have been compared with reference patterns. 1

H− −NMR

The values of the chemical shifts obtained were similar to those of Schiff base ligands reported in the literature [21]. In the 1H−NMR spectrum of Schiff base ligand single peaks attributed to methyl, COOH and SO2NH groups appeared at 1.95, 10.1 and 12.4 ppm respectively. Three groups of double peaks given by N−CH and N=C–CH on the β-lactam ring and NH appeared at 4.90, 5.45 and 9.01 ppm, respectively. One group of four resonance signals consistent with an AB system attributed to S–CH2 on the dihydrothiazine ring was observed in the 3.15–3.45 ppm region with coupling constant 16.9 Hz for JAB. Furthermore, coupling between NH2 and the adjacent CH could not be distinguished and a broad single signal due to NH2 protons was observed at 4.97 ppm. A multiplet in the range 6.58–8.02 ppm due to aromatic ring protons was also present. All the complexes are paramagnetic with the exception of zinc(II) complex, therefore the

1

H−NMR spectra of the

complexes could not be obtained Comparison of the 1H−NMR spectrum of Schiff base with that of the diamagnetic zinc(II) complex, shows the absence of the signal assigned to the COOH proton of Schiff base ligand indicating deprotonation and suggests the formation of a metal–COO bond. Owing to

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their low solubility it was not possible to record satisfactory

13

C−NMR

spectrum for the diamagnetic complex.

Infrared spectra The main infrared spectral bands of the ligand and its metal complexes are presented in Table 2. The lactam ν(C=O) band appear at 1750 cm−1 in the spectra of cephalexin. The IR spectrum of the Schiff base ligand shows no absorption bands which can be assigned to lactam ν(C=O) vibrational mode coming from cephalexin. The absence of such absorption together with the appearance of a new band at 1630 cm−1 attributed to ν(C=N) vibrations, is consistent with the product being the expected Schiff base ligand [22, 23]. The infrared spectra of the metal complexes display IR absorption bands in the 1640-1645 cm−1 range which can be assigned to the C=N stretching frequencies of the coordinated ligand (HL). The shift of this band on complexation towards higher wave numbers indicate coordination of the azomethine nitrogen to the metal centre [23]. Three bands of the free Schiff base ligand at 3340−3200 cm−1 due to ν(NH) and ν(NH2) are still present in the complexes, strongly suggesting noninvolvement of these groups in coordination. Also, disappearance of the stretching frequency at 1690 cm−1 assigned to ν(COOH) in the ligand and appearance in the complexes of new νas and νs modes of the (COO−) group indicates that the Schiff base has reacted. Since the carboxylate group can coordinate to the metal ion in either bidentate or monodentate fashion, the ‘‘∆ν criterion’’ [∆ν = νasym(COO) − νsym(COO)] was employed to determine the coordination mode of the carboxylate group. The tricopper(II) and mononuclear complexes exhibit strong bands corresponding to νas(COO) at 1595 and 1580 cm−1, and νs(COO) at 1425 and

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1370 cm−1 respectively (Table 1). The ∆ν value of 170 and 210 cm−1 in these complexes are very similar to that reported for the copper(II) complexes with both bidentate µ2-O,O’ and monodentate carboxylate groups [24], thus indicating the carboxylate group behaves both as a bridging bidentate ligand between copper(II) ions [25, 26], and as monodentate ligand in the mononuclear complexes. A broad band centered at 3426 cm−1 for the complex can be assigned to the ν(OH) stretch of coordinated water molecules. The remaining carboxylate bands, namely νsym(COO), γ(COO), ω(COO) and ρ(COO), formerly at 1400, 785, 610 and 530 cm−1, respectively, also change as a result of coordination. Furthermore, the appearance of new bands in the 450–490 cm−1 ranges attributed to ν(M–N) stretching vibrations, observed in the spectra of the complexes (absent in the free ligand) provide evidence that the C=N moiety could be bonded to the metal ion through the nitrogen atom. The bands in the 350–400 cm−1 region observed in the complexes, and absent in the free Schiff base ligand, are tentatively assigned to ν(M–O) vibrations. The metal(II) complexes also show bands in the 1420–1460, 1070–1100 and 720–740 cm−1 ranges which can be assigned to phenyl ring vibrations. Medium intensity band appearing in the 2830–2950 cm−1 region corresponds to aliphatic ν(C–H), while aromatic ν(C–H) stretches appear in the 3000–3100 cm−1 region. The usual modes of sulphathiazol moiety are also present, as expected, the bands at 1310, 1150, and 560 cm−1 attributed to an O=S=O group remain unchanged with respect to those of the ligand, excluding the coordination of the SO2 group. These overall data suggest that the azomethine−N, thiazole−N and carboxylate−O group with monodentate mode are involved in coordination in the mononuclear complexes and that the Schiff base behaves as a tridentate monoanionic NNO chelating agent. In the

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trinuclear copper(II) complex the Schiff base behaves as a tetradentate monoanionic NNOO ligand having a carboxylate group with bidentate mode.

Magnetic properties From the molar magnetic susceptibility values, corrected magnetic moments were calculated using Pascal’s constants [27]. The magnitudes of the magnetic moments for the paramagnetic complexes fall within the ranges associated with spin-free high spin ions in octahedral fields. The manganese(II) complex has a magnetic moment value of 5.83 µB which is typical of high spin d5 systems with five unpaired electrons and S = 5/2 ground state. The cobalt(II) complex has a magnetic moment of 5.13 µB which is a typical value of a d7 system with three unpaired electrons indicating a quartet state in an octahedral arrangement around the metal, as compared with the reported values for octahedral complexes of cobalt(II) (4.7-5.2 µB) [28]. The nickel(II) complex has a magnetic moment of 3.30 µB characteristic of two unpaired electrons and greater than the spin-only value, presumably due to the orbital contribution resulting from the transfer of an electron from the dx2-y2 orbital to the dxy orbital. The complex therefore probably has distorted octahedral geometry. At room temperature a magnetic moment of 1.9–2.2 µB. is usually observed for mononuclear copper(II) complexes, regardless of stereochemistry [28]. A magnetic moment of 2.84 µB. is observed for the copper(II) compound in the solid state, indicative of the presence of a polynuclear complex with some ferro/antiferromagnetic interactions, operating through Cu–Cu interactions [29]. This observed value is slightly lower than the d9 spin-only magnetic moment calculated value µeff = 3,00 µB for a tricopper complex with three

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spin-system in the absence of an exchange interaction. This result suggests the presence of a weak antiferromagnetic spin-exchange interaction in the complex. Thus, the suggested formula for the copper(II) complex is [Cu3L(OH)5]. According to Kambe’s approach [30], in trinuclear copper(II) system, first couple the spins of the two copper(II) ions (SCu1 = SCu2 = 1/2) to give two spin states of S’ = 1 and 0. Then couple S’ to the third copper(II) ion (SCu3 = 1/2) to yield three total spin states of ST = 3/2, 1/2, 1/2. Thus for the case of copper(II) trinuclear complex three different spin configurations can be found: the high spin quartet, with a total spin of 3/2, and two degenerate doublets, with total spin 1/2 separated by exchange coupling constants J. The exchange coupling constant between adjacent metal ions comes from the contributions of the sum of both ferromagnetic (JF) and antiferromagnetic (JAF) interactions. The spin−exchange interaction will be antiferromagnetic (J < 0) if S=1/2 it is the ground state; on the contrary if S=3/2 is the ground state, the spin−exchange interaction will be ferromagnetic (J > 0) [31]. The observed magnetic moment value of the tricopper(II) complex at room temperature is a consequence of population of the doublet (S = 1/2) molecular state. For the tricopper(II) complex the observed χMT product is 1.007 cm3 mol−1 K at room temperature which corresponds to an effective magnetic moment of 2.84 µB. The observed χMT value is very close to the theoretical value expected for three magnetically independent copper(II) ions (χMT = 3 (Nβ2g2/3kT) S(S+ 1)) = 1.24 cm3 mol−1 K, with g =2.1 and S =1/2) [32]. It is thus possible that the copper(II) centres, are weakly coupled by the magnetic exchange antiferromagnetic interactions. However, this could not be probed

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further due to lack of facilities for variable temperature magnetic measurements. The X-band EPR spectrum of a powder sample of the Cu3L(OH)5 complex at room temperature showed a single broad signal with poor resolution of the hyperfine structure (Figure 2). The analysis of spectrum gives g|| value of 2.21 and g⊥ value of 2.06. The trend g|| > g⊥ > 2.0023 observed for the complex indicate that the unpaired electron is localized in the dx2−y2 orbital of the Cu(II) ion and is characteristic of the axial symmetry [33, 34]. The gav value was calculated to be 2.11. The deviation of gav from that of the free electron (2.0023) is due to covalence character as per Kivelson and Neiman [35]. The parameter ‘G’ is calculated by using the expression, i.e., G = (g|| − 2) / (g⊥−2). The G value of 3.5 indicates moderate exchange interaction between metal centres in solid complex consistent with Hathaway approach [36]. The powder EPR spectrum recorded at liquid nitrogen temperature does not show any change compared to that of room temperature spectrum. The linewidth does not change very much with decreasing temperature and should be the result of the dipolar and exchange interactions between the copper ions [37]. In general, polynuclear Cu(II) complexes give broad EPR peaks and the broadening is assigned to a dipolar interaction [38]. The greater value of g║ compared to g⊥ proposes a distorted tetrahedral-square pyramidal structure and rules out the possibility of a trigonal bipyramidal structure which is expected to have g⊥ > g║. Also, the observed g║ value of less than 2.3 provide evidence for the covalent character of bonding between Cu(II) ion and the ligand [37, 38]. Electronic spectra A long-term UV–Vis study was carried out to verify the stability of new complexes in DMSO solution. Compared with ligand, it is significant to note

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that the absorption wavelengths of new complexes hardly varied for up to 1 month, meaning that new complexes were stable in DMSO solution. The electronic spectra of the Schiff base ligand in 10-3 M DMSO solution showed three broad bands at 250, 310 and 360 nm. The former two bands are due to the π−π* transitions within the aromatic ring and remain almost unchanged in the spectra of metal complexes while the third band is due to the π−π* transitions within the >C=N– chromophore and shows a bathochromic shift in the spectra of metal complexes due to the coordination of azomethine nitrogen to the metal atom [39]. This band shifts slightly to the higher energy region in the spectra of metal complexes due to the polarization within the >C=N chromophore caused by the metal-ligand electron interaction. The spectra of the metal complexes shows that the absorptions around 400– 800 nm is due to ligand to metal charge transfer and d–d transition bands of the metal in the complexes [40]. The manganese(II) complex shows a very weak absorption at 380 nm probably due to the coincidence of charge transfer, d → π*, L → M and intraligand n → π* transitions [41, 42]. The visible region spectrum of the cobalt(II) complex indicates additional two bands at 420 and 460 nm attributed to metal-ligand charge and 4T1g(F) → 4T2g(F) transition respectively, suggesting octahedral stereochemistry around the metal ion [43]. The UV−Vis spectrum of the nickel(II) complex presents two major absorptions maxima, at 420 and 480 nm due to d−d bands which may be assigned, considering that the immediate coordination sphere of the metal is Oh symmetry, to the transitions

3

A2g →

3

T1g(P) and

3

A2g →

3

T1g(F)

respectively [44]. The electronic spectrum of trinuclear copper(II) complex shows a broad band centered at 620 nm (ε = 560.1 cm−1 mol−1) assigned to the 2

T2g → 2E2g transition in a distorted tetrahedral/planar geometry around the

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copper(II) ion. According to the crystal field theory, the d-d transition absorption band envelope shifts to lower energy as the planar copper(II) complexes are twisted toward the distorted tetrahedral structure [40]. The bands at 390 and 470 nm are assigned to charge-transfer, mainly of the L → Cu type [40]. Coordination sites The coordination chemistry of transition metal ions with ceftriaxone [45], cefotaxime [46], cefepime [47] and ceftazidime [48] antibiotics have been reported. In the present case, the Schiff base ligand containing cephalexin has a number of potential donor atoms in various positions which can bind to the metal ions forming multinuclear chelates. From the data it appears that each metal ion lies in a distorted octahedron coordination sphere and the Schiff base would act as an efficient pseudo-encapsulating ligand, with N−thiazole ring, C=N and carboxylate group with monodentate mode, presumably bound to the octahedral ions. Thus, the metal ions in the [ML(OAc)(H2O)2] complexes containing one coordinated acetate anion and two water molecules at the vertices of an octahedron are hexacoordinate. For the trinuclear copper(II), the adjacent coppers would be connected by hydroxo bridges and a bidentate carboxylato group. The assumption that each Schiff base is bound to three tetrahedral copper ions (with N−thiazole ring, C=N and the carboxylate group with bidentate mode) seems likely from molecular models. We have attempted to grow single crystals of the metal chelates but in no case have we had any success, due to their insolubility in common organic solvents. The complexes only form amorphous materials as revealed by their XRD patterns. Up to now no crystal structures containing cephalosporin Schiff base complexes have been reported. These studies represent a contribution to future

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crystallographic analyses, which are complicated by the difficulties in obtaining X-ray quality crystals of cephalosporin derived complexes. Although crystal structure of the complexes are not known, the coordination environment of mononuclear complexes, may tentatively be proposed (Figure 3). Reports on trinuclear and higher nuclearity copper compounds are rather scarce. In the present case, the magnetic and spectroscopic data are sufficient to deduce the ocurrence of a magnetically coupled trinuclear unit containing three copper(II) ions.

Antibacterial activity A long-term UV–Vis study was carried out to verify the stability of new complexes in DMSO solution. Compared with ligands, it is significant to note that the absorption wavelengths of new complexes hardly varied for up to 1 month, meaning that new complexes were stable in DMSO solution. Preliminary screening for antimicrobial activities of the stock solutions at 20 mg/mL were performed qualitatively using the disc diffusion assay. In vitro antimicrobial activities were measured from the diameter of clear inhibition zones caused by samples against the same bacteria and under the identical experimental conditions. As assessed by colour, the complexes remain intact during biological testing. In order to clarify role of DMSO any participating and metal(II) acetate salts in the biological screening, separate studies were carried out with the solutions alone of DMSO and the free metal salt and they have been found that they have no effect on the growth of any microorganisms taken. The antibacterial activity of Schiff base ligand HL as well as its metal(II) complexes were tested on against S. aureus as a Gram-positive and E. coli as a Gram-negative microorganism and compared to cephalexin and sulphathiazol

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used as standards. As expected, HL is significantly more toxic against Grampositive than Gram-negative bacteria, which may be due to the different cell wall structure of the tested microorganisms, while the reference compounds cephalexin and sulphathiazol show almost equal activity against both strains tested. The average results are shown in Table 3 where can be appreciated that the Schiff base ligand and its metal complexes have different behaviour compared with standard antibiotics against the same bacteria. Thus: (1) The Schiff base ligand, zinc(II) and copper(II) complexes were found to have higher activity than the two established drugs against the bacteria strains studied under the test conditions, showing that they have a good activity as bactericides. (2) While both cobalt(II) and nickel(II) complexes are inactive, the antibacterial activity of manganese(II) complex shows to be less toxic than the two referenced drugs and the Schiff base ligand. According to Tweedy’s theory [49], chelation could enhance the lipophilic character of the central metal atom, which subsequently favors its permeation through the lipid layers of the cell membrane and blocking the metal binding sites on enzymes of microorganism. In the present case, the in vitro antibacterial activities demonstrated that copper(II) and zinc(II) complexes have higher antimicrobial activity in comparison with that of the ligand HL, but, ligand HL also showed highly biological activity against the tested strains compared to the manganese(II), cobalt(II) and nickel(II) complexes. Therefore, antimicrobial activity can also be influenced by other factors beyond membrane permeability. The targets for β-lactam antibiotics are cell wall-synthesizing enzymes (penicillin binding proteins, PBPs) which are found as both membrane-bound and cytoplasmic enzymes that catalyze cross-linking reactions. β−lactam

20

antibiotics, interfere with cell wall synthesis by binding covalently to the PBPs catalytic site. PBPs are present in almost all bacteria, but they vary from species to species differing in amount, molecular weight, affinity for β-lactam antibiotics and enzymatic function (e.g., transpeptidase, carboxypeptidase, or endopeptidase) [50]. The results in Table 3 can be understood considering that the enzyme probably serves primarily to hold catalytic groups or the substrate in the proper positions and is possible to expect that Schiff base metal complexes may change the stereochemistry required in solvolytic reactions on an enzyme surface. The obtained results may highlight that the activity of the compounds is most probably related to their conformational adaptability, depending on the size and nature of the metal complexes and the geometrical constraints induced by intramolecular H bonds. Thus, the bactericidal activity of cephalexin Schiff base and cephalexin Schiff base metal complexes compared to cephalexin antibacterial activity may reflect a different mechanistic pathway by which they react with the PBP active sites to achieve formation of a stable PBP−inhibitor adduct. The level of resistance to β−lactam complexes is determined by the amount, nature and kinetic properties of the PBPs.

Conclusion A Schiff base derived from cephalexin and sulphathiazole and its transition metal complexes have been prepared. The coordination to metal occurs through the carboxylate, imine and N-thiazole moieties. The solubility of the Schiff base antibiotic and its metal complexes in water and common organic solvents is reduced on complexation. The cephalexin Schiff base, zinc(II) and copper(II) complexes were found to have higher bactericidal activity than the uncomplexed cephalexin and the sulphathiazole against the bacteria strains,

21

showing that they have a good activity as bactericides. The cobalt(II) and nickel(II) complexes show no activity at all against the bacteria while the manganese(II) complex showed to be less toxic than the two referenced drugs and the Schiff base ligand. Apart of membrane permeability, antibacterial activity of cephalexin Schiff base and its metal complexes depends mainly on the metal ion and the type of microorganism.

Supplementary data Scheme S1 is available as PDF file from the authors and the journal. Acknowledgements. The authors express their sincere thanks to Comision de Investigación from the Universidad de Oriente for financial support.

References [1] R. Hernandez-Molina, A. Mederos, in: J.A. McCleverty, T.J. Meyer (Eds.), Comprehensive Coordination Chemistry II, vol. 1, Elsevier, Amsterdam, 2004, p. 241. [2] M. Andruh, F. Tuna, in: M.A. Cato (Ed.), Focus on Organometallic Chemistry Research, Nova Publishers, Hauppauge, 2005, p. 144. [3] A.J. Atkins, D. Black, A.J. Blake, A. Marin-Bocerra, S. Parsons, L. RuizRamirez, M. Schröder, Chem. Commun. (1996) 457–458. [4] B. Witkop, L.K. Ramachandran, Metabolism 13 (1964) 1016−1025. [5] K. Bernardo, S. Leppard, A. Robert, G. Commenges, F. Dahan, B. Meunier, Inorg. Chem. 35 (1996) 387−396. [6] E.I. Solomon, M.D. Lowery, Science 259 (1993) 1575−1581. [7] C. Gerdemann, C. Eicken, B. Krebs, Chem. Res. 35 (2002) 183−191.

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[8] M. Tümer, E. Akgün, S. Toroglu, A. Kayraldız, L. Dönbak, J. Coord. Chem. 61 (2008) 2935−2949. [9] A. Gölcü, M. Tümer, H. Demirelli, R.A. Wheatley, Inorg. Chim. Acta 358 (2005) 1785−1797. [10] M. Tümer, N. Deligonul, A. Gölcü, E. Akgün, M. Dolaz, H. Demirelli, M. Dıgrak, Transit. Met. Chem. 31 (2006) 1−12. [11] M. Tümer, H. Köksal, S. Serin, M. Dıgrak, Transit. Met. Chem. 24 (1999) 13−17. [12] J.R. Anacona, H. Rodriguez. J. Coord. Chem. 62 (2009) 2212-2219. [13] J. R. Anacona, L. Brito, W. Peña, Synth. React. Inorg. Met.−Org. Nano−Met. Chem. 42 (2012) 1278-1284. [14] J.R. Anacona, M. López. Int. J. Inorg. Chem. (2012) doi:10.1155/2012/106187. [15] J.R. Anacona, J. Calvo, O. Almanza. Int. J. Inorg. Chem. (2013) doi: 10.1155/2013/108740. [16] J.R. Anacona, J.J. Santaella, Spectrochim. Acta A 115 (2013) 800−804. [17] H.A. Flaschka, EDTA Titrations, Pergamon Press, New York, 1964. [18] W. Geary, Coord. Chem. Rev. 7 (1971) 81−122. [19] M. Badea, A. Emandi, D. Marinescu, E. Cristurean, R. Olar, A. Braileanu, P. Budrugeac, E. Segal. J. Therm. Anal. Calorim. 72 (2003) 525−531. [20] R.M. Issa, S.A. Amer, I.A. Mansour, A.I. Abdel-Monsef. J. Therm. Anal. Cal. 90 (2007) 261−267. [21] W.W. Simmons. The Sadtler handbook of proton NMR spectra. Sadtler Research Laboratories, Inc, 1978. [22] G. Socrates, Infrared Characteristic Group Frequencies, John Wiley & Sons, 1980.

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[23] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Compounds, 5th edition, John Wiley & Sons, 1997. [24] Y.-Y. Wang, L.-J. Zhou, Q. Shi, Q.-Z. Shi, Transit. Met. Chem. 27 (2002) 145−148. [25] L. Gutierrez, G. Alzuet, J. A. Real, J. Cano, J. Borras, A. Castiñeiras, Eur. J. Inorg. Chem. (2002) 2094−2102. [26] L. Gutierrez, G. Alzuet, J. A. Real, J. Cano, J. Borras, A. Castiñeiras, Inorg. Chem. 39 (2000) 3608−3614. [27] G.A. Bain, J.F. Barry, J. Chem. Ed. 85 (2008) 532−536. [28] Z. H. Chohan and S. Kausar, Metal-Based Drugs, 7 (2000) 17–22. [29] M. G. B. Drew and P. C. Yates, Inorganica Chimica Acta, 118 (1986) 37–47.. [30] K. Kambe, J. Phys. Soc. Jpn. 5 (1950) 48−51. [31] Y-T. Li, C-W. Yen, J-F. Lou, H-S. Guan, J. Magn. Magn. Mater. 281 (2004) 68−76. [32] O. Kahn, Molecular Magnetism, VCH, New York, 1993 [33] H. Liu, H. Wang, F. Gao, D. Niu, Z. Lu, J. Coord. Chem. 60 (2007) 2671–2678. [34] Z. Shirin, R.M. Mukherjee, Polyhedron 11 (1992) 2625–2633. [35] D. Kivelson, R. Neiman, J. Chem. Phys. 35 (1961) 149–155. [36] B.J. Hathaway, D.E. Billing, Coord. Chem. Rev. 5 (1970) 143–207. [37] F. Koksal, F. Ucun, E. Agar, I. Kartal, J. Chem. Research (S) (1998) 96−97. [38] M.C. Jain, A.K. Shrivastava, P.C. Jain, Inorg. Chim. Acta, 23 (1977) 199−203 [39] M. Sonmez, A. Levent, M. Sekerci, Russ. J. Coord. Chem. 30 (2004)

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655−659. [40] A.B.P. Lever, Inorganic Electronic Spectroscopy, Elsevier, Amsterdam, 1968. [41] M.M. Mostafa, A. El-Hammid, M. Shallaby, A.A. El-Asmy, Transit. Met. Chem. 6 (1981) 303−305. [42] M.J.M. Cambell, Coord. Chem. Rev. 15 (1975) 279−319. [43] F.A. Cotton, G. Wilkinson, Advanced Inorganic Chemistry, 6th edition, John Wiley & Sons, 1999. [44] M.M. Aboaly, M.M.H. Khalil, Spect. Lett. 34 (2001) 495−504. [45] J.R. Anacona, A. Rodriguez, Transit. Met. Chem. 30 (2005) 897−901. [46] J.R. Anacona, G. Da Silva, J. Chil. Chem. Soc. 50 (2005) 447−450. [47] J.R. Anacona, H. Rodriguez, J. Coord. Chem. 62 (2009) 2212−2219. [48] J.R. Anacona, C. Patiño, J. Coord. Chem. 62 (2009) 613−621. [49] B.G. Tweedy, Phytopathology. 55 (1964) 910–914. [50] Georgopapadakou, N.H. Antimicrob. Agents Chemother. 37 (1993) 2045-2053.

Table 1. Analytical and thermoanalytical (TG) results Found (Calcd.) %

25

H2O

M1

13.4

4.8

7.5

(4.1)

(13.1)

(4.9) (7.5)

(7.5)

11.7

3.8

13.2

4.5

(8.2)

(44.0)

(11.4)

(4.1)

(13.0)

(4.9) (8.0)

(8.0)

[NiL(OAc)(H2O)2]

44.2

11.6

3.9

13.5

5.1

(8.1)

[Ni(C27H30N6O9S3)]

(44.0)

(11.4)

(4.1)

(13.0)

(4.9) (8.0)

[Cu3L(OH)5]

34.7

9.9

3.6

11.6

4.5

[Cu3(C25H29N6O10S3)]

(34.9)

(9.8)

(3.4)

(11.2)

(4.2) (22.2) (22.2)

[ZnL(OAc)(H2O)2]

43.8

11.7

3.9

13.4

4.4

[Zn(C27H30N6O9S3)]

(43.6)

(11.3)

(4.1)

(13.0)

(4.8) (8.8)

Compound

C

N

H

S

[(HL)]

51.6

14.1

4.4

16.2

[(C25H24N6O5S3)]

(51.4)

(14.4)

(4.1)

(16.5)

[MnL(OAc)(H2O)2]

44.3

11.9

3.9

[Mn(C27H30N6O9S3)]

(44.2)

(11.5)

[CoL(OAc)(H2O)2]

44.3

[Co(C27H30N6O9S3)]

8.2

8.4

22.6

9.2

M2

(7.7)

(8.0) (22.4)

(9.0) (8.8)

M1 = complexometric analysis, M2 = thermal analysis

Table 2. Main vibrational wavenumbers of the metal complexes (cm-1)

Compound

νC=O

νC=O

νC=N

νCOO νCOO

lactam

amide

imino

asymm symm

∆ν

26

Cephalexin

1750

1690

[HL]

1690

1635

[MnL(OAc)(H2O)2]

1690

1620

1580

1370

210

[CoL(OAc)(H2O)2]

1690

1620

1580

1370

210

[NiL(OAc)(H2O)2]

1690

1620

1580

1370

210

[Cu3L(OH)5]

1690

1615

1595

1425

170

[ZnL(OAc)(H2O)2]

1690

1620

1580

1370

210

Table 3 Antibacterial activity Zone of inhibition (mm) Compound

E.C.

S.A

7.0 ± 1.0

8.0 ± 1.0

Cephalexin

10.0 ± 1.0

12.0 ± 1.5

[HL]

12.0 ± 1.0

15.0 ± 1.0

[MnL(OAc)(H2O)2]

6.0 ± 1.0

7.0 ± 1.0

[CoL(OAc)(H2O)2]

0.0

0.0

[NiL(OAc)(H2O)2]

0.0

0.0

Sulphathiazole

[CuL(OH)5]

13.0 ± 1.5

18.0 ± 1.0

[ZnL(OAc)(H2O)2]

15.0 ± 1.0

17.0 ± 1.0

a

S.A. Staphylococcus aureus ATCC 25923, E.C. Escherichia coli 35939, All doses were 400 µg /disc. Values are the mean ± Standard deviation of the mean. FIGURE CAPTIONS Figure 1. Chemical structure of cephalexin

27

Figure 2. EPR spectrum of [Cu3L(OH)5] complex at room temperature Figure 3. Proposed structure of [ML(OAc)(H2O)2] complexes

28

29

Supplementary data

Scheme S1

SUGGESTED REFEREES 1

Name and Surname Institution Address E-mail Phone

Jim R. Durig Spectrochimica Acta

[email protected]

30

2

3

4

5

6

Country Name and Surname Institution Address E-mail Phone Country Name and Surname Institution Address E-mail Phone Country Name and Surname Institution Address E-mail Phone Country Name and Surname Institution Address E-mail Phone Country Name and Surname Institution Address E-mail Phone Country

Dinorah Gambino Universidad La Republica [email protected] Uruguay Luca Fadini Universidad Nacional [email protected] Colombia Ana Burgos Universidad Nacional [email protected] Colombia V.E. Marquez Instituto Universitario Tecnología [email protected] venezuela Luigi Messori University of Florence [email protected] Italy