Accepted Manuscript Synthesis, characterization and antibacterial activity of a tridentate Schiff base derived from cephalothin and sulfadiazine, and its transition metal complexes J.R. Anacona, Natiana Noriega, Juan Camus PII: DOI: Reference:

S1386-1425(14)01172-X http://dx.doi.org/10.1016/j.saa.2014.07.091 SAA 12511

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date:

31 March 2014 6 July 2014

Please cite this article as: J.R. Anacona, N. Noriega, J. Camus, Synthesis, characterization and antibacterial activity of a tridentate Schiff base derived from cephalothin and sulfadiazine, and its transition metal complexes, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa. 2014.07.091

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

4

Synthesis, characterization and antibacterial activity of a tridentate Schiff base derived from cephalothin and sulfadiazine, and its transition metal complexes J.R. Anaconaa,*, Natiana Noriegaa and Juan Camusb a

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

b

Facultad de Ciencias Naturales y Exactas, 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 cephalothin Schiff base (H2L)

derived from the condensation of cephalothin antibiotic with

sulfadiazine were synthesized. The Schiff base ligand, mononuclear [ML(H2O)3] (M(II) = Mn, Co, Ni, Zn) complexes and magnetically diluted dinuclear copper(II) complex [CuL(H2O)3]2 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 cephalothin Schiff base ligand H2L behaves as a dianionic tridentate NOO chelating agent. The biological applications of complexes have been studied on two bacteria strains (Escherichia coli and Staphylococcus aureus) by agar diffusion disc method.

Keywords: Schiff base metal complexes, cephalothin Schiff base, synthesis cephalothin derivative, magnetic and spectral studies, antibacterial activity

5

Introduction The 2-azetidinone skeleton, otherwise known as the β-lactam ring, has largely been recognized as a useful building block in the synthesis of biologically important compounds. The activity of famous antibiotic classes such as the penicillins and cephalosporins is attributed to the presence of an 2-azetidinone ring [1]. Unfortunately, the wide use of the antibiotics resulted in the serious medical problem of drugs resistance and public health concern [2, 3]. Preparation of new synthetic derivatives of antibiotics with novel mechanism of action has become an important task to cope with drug resistance problems. Schiff bases derived from an aldehyde and any amine are a class of compounds which coordinate to metal ions via de azomethine nitrogen. They are 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8], antibacterial [9, 10], antifungal, and herbicidal activities [11]. Although Schiff base have extensively been studied and widely employed in coordination chemistry, rather less is known about Schiff base ligands containing antibiotics. 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 cephalothin antibiotic, first generation cephalosporin, with sulfadiazine a sulfonamide antibiotic. The chemical structure of cephalothin is shown in Figure 1.

6

Experimental Materials and methods 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 200 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 u.v. regions were taken with a Perkin-Elmer spectrophotometer. The contents of C, H, N and S were analysed on a LECO CHNS 932 model microanalytical instrument. To establish the metal contents, the compounds were decomposed by wet digestion at 340°C with sulphuric acid and hydrogen peroxide and 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 TGA curves. 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. 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. Xray powder diffraction patterns for the studied complexes and final solid product of thermal decomposition were recorded on

7

a HZG 4 diffractometer. Measurements were taken over the range of 2θ = 2– 70° using Ni filtered CuKα radiation. Synthesis of Schiff base ligand H2L To 1 mmol of cephalothin in 250 mL of hot methanol were added 1 mmol of sulfadiazine 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 82%), m.p. = 135°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 H2L 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 metal(II) complexes of H2L 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 H2L (1 mmol) and copper(II) acetate (1 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 57– 68%, m.p. 210250°C.

8

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 200°C. The synthetic route of H2L 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 1:1:3 metal: ligand: coordinated water stoichiometry. Thus, the general formulae [ML(H2O)3] (M(II) = Mn, Co, Ni, Cu, Zn) have been assigned to the complexes and they are very air stable solids at room temperature without decomposition for a long time. 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 conductance values measured in DMSO at room temperature vary from 1.27 to 17.10 S cm2 mol1, revealing the nonelectrolytic nature of the complexes [18].

9

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 two 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 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. Thus, the overall thermogravimetric results are consistent with the formulation of these complexes. The solid residues obtained during thermal decomposition of complexes are suitable metal oxides: MnO, CoO, 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 [20]. In the 1HNMR spectrum of Schiff base ligand single peaks attributed to methyl, COOH and SO2NH groups appeared

10

at 1.95, 10.1 and 11.2 ppm respectively. Three groups of double peaks given by NCH and S–CH on the -lactam ring and NH appeared at 4.90, 5.45 and 9.01 ppm, respectively. Three groups of four resonance signals consistent with an AB system attributed to SCH2 on the dihydrothiazine ring, CH2CO and CH2O were observed

in the 3.183.49, 3.603.90 and 4.745.03 ppm

regions respectively. A multiplet in the range 6.68–7.92 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 proton signals assigned to the COOH and SO2NH moieties of Schiff base ligand indicating deprotonation and suggest the formation of COOmetal and SO2Nmetal bonds. No significant changes in 1H chemical shifts were observed for other atoms upon complexation suggesting that, in solution, the aromatic rings are not involved in stacking interactions. Owing to their low solubility it was not possible to record satisfactory 13CNMR spectrum for the diamagnetic complex.

Infrared spectra The IR spectrum of the complexes were recorded down to the farIR region of 200 cm1 and compared with those of cephalothin and sulfadiazine. The main infrared spectral bands of the Schiff base ligand and its metal complexes are presented in Table 2. The lactam (C=O) band appear at 1735 cm1 in the spectra of cephalothin. The IR spectrum of the Schiff base antibiotic shows no absorption bands which can be assigned to lactam (C=O) vibrational mode coming from cephalothin. The absence of such absorption together with the

11

appearance of a new band at 1633 cm1 attributed to (C=N) vibrations, is consistent with the product being the expected Schiff base ligand [21, 22]. The infrared spectra of the metal complexes display IR absorption bands in the 16301634 cm1 range which can be assigned to the C=N stretching frequencies of the coordinated Schiff base ligand (H2L), strongly suggesting noninvolvement of this group in coordination as shown in Figure 2. Tentative band assignments of some characteristic bands of Schiff base ligand were made by analogy with other related systems [15, 16]. The strong bands related respectively to asym and sym of the (O=S=O) moiety at 1325 and 1155 cm1 show important changes upon complexation. The first splits into two peaks at 1350 and 1300 cm1 and the second appears at 1133 cm1. The band at 945 cm1 corresponding to (SN) is shifted to higher frequencies (980 cm1) upon complexation [15]. All these changes suggest binding of the metal ions to the sulfonamide nitrogen. Disappearance of the stretching frequency at 1690 cm−1 assigned to ν(COOH) in the ligand and appearance in the complexes of new ν as 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 „„Δν criterion‟‟ [Δν = νasym(COO)  νsym(COO)] was employed to determine the coordination mode of the carboxylate group. The dinuclear copper and mononuclear complexes exhibit strong bands corresponding to νas(COO) at 15891599 cm1 and νs(COO) at 14151426 cm1 ranges (Table 2). The Δν values in the range of 166175 cm1 in these complexes are very similar to that reported for the copper(II) complexes with bidentate μ2O,O‟ carboxylate group [23], thus indicating the carboxylate group behaves as a bidentate ligand [24, 25].

12

The remaining carboxylate bands, namely γ(COO), ω(COO) and ρ(COO), formerly at 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 (M–N) stretching vibrations, observed in the spectra of the complexes (absent in the free ligand) provide evidence that the SO2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 (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 (C–H), while aromatic (C–H) stretches appear in the 3000–3100 cm1 region. A broad band centered at 3426 cm1 for the complex can be assigned to the ν(OH) stretch of coordinated water molecules. These overall data suggest that the SO2N and carboxylateO groups with bidentate mode are involved in coordination in the complexes and that the Schiff base behaves as a tridentate monoanionic NOO chelating agent.

Magnetic properties Magnetic susceptibility is the degree of the magnetization of a material in response to a magnet. The method measures the Boltzmann occupation of all energy levels. From the molar magnetic susceptibility values, corrected magnetic moments were calculated using Pascal‟s constants [26]. The magnitudes of the magnetic moments for the paramagnetic complexes fall within the ranges associated with spin-free high spin ions in octahedral fields.

13

The manganese(II) complex has a magnetic moment value of 6.09 B 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 5.10 B which is higher than the spin–only value and 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 B) [27]. The difference between measured and calculated data results from spin–orbital coupling. The nickel(II) complex has a magnetic moment of 3.36 B 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 d x2-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 B. is usually observed for mononuclear copper(II) complexes, regardless of stereochemistry [27]. A magnetic moment of 2.60 B. 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 [28]. This observed value is slightly higher than the d9 spin-only magnetic moment calculated value μeff = 2,45 B for a dicopper complex with two spinsystem in the absence of an exchange interaction. This result suggests the presence of a weak ferromagnetic spin-exchange interaction in the complex. Thus, the suggested formula for the copper(II) complex is [CuL(H2O)3]2. Spin S = 1 is often present in dinuclear copper(II) complexes, where two spins S = ½ of two adjacent copper(II) ions are coupled via a bridging ligand, most often resulting in a ferromagnetic or an antiferromagnetic behaviour. The

14

ferromagnetism is represented by a positive 2J exchange interaction value (S = 1 triplet ground state, S = 0 singlet first excited state), while the antiferromagnetism by a negative 2J value (S = 0 singlet ground state, S = 1 triplet first excited state) [29]. The observed magnetic moment value of the dinuclear copper(II) complex at room temperature is a consequence of population of the triplet (S = 1) molecular state. For the dinuclear copper(II) complex the observed MT product is 0.849 cm3 mol1 K at room temperature which corresponds to an effective magnetic moment of 2.60 B. The observed MT value is very close to the theoretical value expected for two magnetically independent copper(II) ions (MT = 2 (N2g2/3k) S(S+1)) = 0.827 cm3 mol1 K, with g =2.1 and S =1/2) [30]. It is thus possible that the copper(II) centres, are weakly coupled by the magnetic exchange ferromagnetic interactions. However, this could not be probed further due to lack of facilities for variable temperature magnetic measurements. At the room temperature one can see S = 1 EPR signals (S = 0 is EPR silent – no signals in the EPR spectrum). Due to spin S = 1, three magnetic quantum numbers (–1, 0, 1) describe the system, and similarly as for S = ½, the Zeeman effect is noticed in the EPR spectra, splitting the states, almost degenerate at zero magnetic field. The X-band EPR spectrum of a powder sample of the [CuL(H2O)3]2 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 2.18 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 [31,

15

32]. 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 [33, 34]. The gav value was calculated to be 2.10. 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 3.0 indicates moderate exchange interaction between metal centres in solid complex consistent with Hathaway approach [36]. Zinc(II) complex is diamagnetic as expected for the d10 configuration. Electronic spectra The electronic spectra of the Schiff base ligand in 103 M DMSO solution showed two broad bands at 310 and 360 nm. The former band is due to the * transition within the aromatic ring and remains almost unchanged in the spectra of metal complexes, while the second band is due to the * transitions within the >C=N– chromophore that also remains unchanged after complexation, revealing that azomethine nitrogen is free in metal complexes [37]. The spectra of the metal complexes show 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 [38]. The manganese(II) complex shows a very weak absorption at 390 nm probably due to the coincidence of charge transfer, d  *, L  M and intraligand n  * transitions [39, 40]. The visible region spectrum of the cobalt(II) complex indicates additional two bands at 20000 and 14780 cm1, (emax < 100 M1 cm1), attributed to 4T1g(F) → 4T1g(P) (3) and 4T1g(F) → 4A2g(F) (2) transitions respectively, suggesting octahedral stereochemistry around the metal ion [41]. Experimental 2 and 3 values have been employed to calculate the position of 1: 4T1g(F) → 4T2g(F) band from

16

Lever tables [38]. The calculated molar absorptivity of these absorptions are typically less than 100 (emax < 100 M1 cm1) and the ratio ν2/ν1 is found to be 1.80 as required for the octahedral Co(II) complexes [41]. The values of magnetic moment (μs.o.< μobs ~ μS+L), the calculated ligand field parameters of Dq = 1,246 cm−1, B = 947 cm−1, and β = 0.97 indicating covalent complexes are also consistent with the octahedral geometry. The UVVis spectrum of the nickel(II) complex presents two major absorptions maxima, at 420 and 775 nm which may be assigned to metalligand charge and 3A2g → 3T1g(F) (2) transitions respectively, considering that the immediate coordination sphere of the metal is Oh symmetry [42]. The electronic spectrum of dinuclear copper(II) complex exhibits a broad band centered at 730 nm ( = 380.1 cm1 mol1) assigned to the

2

E2g  2T2g transition in a distorted octahedral

geometry around the copper(II) ion. The band at 410 nm is assigned to chargetransfer, mainly of the L  Cu type [38].

Coordination sites The coordination chemistry of transition metal ions with ceftriaxone [43], cefotaxime [44], cefepime [45] and ceftazidime [46] antibiotics have been reported. In the present case, the Schiff base ligand containing both cephalothin and sulfadiazine 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 sulfonamide nitrogen and carboxylate group with bidentate mode, presumably bound to the octahedral ions. Thus, the metal ions in the [ML(H2O)3] complexes containing three water molecules

17

at the vertices of an octahedron are hexacoordinate. For the dimer copper(II) complex, the assumption that each Schiff base is bound to two octahedral copper ions (SO2N moiety is on one M and the carboxylate group with bidentate mode is on another metal ion) seems likely from molecular models. The sulfonamide group, –SO2NH–, is a weak acid group and the pKa value of sulfonamide compounds depends on their substituents. The same binding mode, through a distorted trigonal arrangement of the imido nitrogen, was observed with sulfadiazine when forms mononuclear complexes of Ag(I) [47, 48], Zn(II) [49, 50], Cu(II) [51] and Hg(II) [52], whose crystal structures were determined. 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 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 4). Reports on dinuclear 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 dinuclear unit containing two 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

18

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 H2L 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 cephalothin and sulfadiazine used as standards. As expected, H2L 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 cephalothin and sulfadiazine 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, copper(II) and zinc(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) The antibacterial activity of manganese(II), cobalt(II) and nickel(II)

19

complexes show to be less toxic than the two reference drugs and Schiff base ligand. According to Tweedy‟s theory [53], 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 H2L. But in contrast with these results, ligand H2L showed highly biological activity against the tested strains compared to the manganese(II), cobalt(II) and nickel(II) complexes. Therefore, antimicrobial activity must also be influenced by other factors beyond membrane permeability. The targets for 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. lactam 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 lactam antibiotics and enzymatic function (e.g., transpeptidase, carboxypeptidase, or endopeptidase) [54]. 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

20

induced by intramolecular H bonds. Thus, the bactericidal activity of cephalothin Schiff base and cephalothin Schiff base metal complexes compared to cephalothin 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 lactam complexes is determined by the amount, nature and kinetic properties of the PBPs.

Conclusion A Schiff base derived from cephalothin and sulfadiazine and its transition metal complexes have been prepared. The coordination to metal occurs through the imido nitrogen (SO2N) and carboxylate moieties. The solubility of the Schiff base antibiotic and its metal complexes in water and common organic solvents is reduced on complexation. The cephalothin Schiff base, copper(II) and zinc(II) complexes were found to have higher bactericidal activity than the uncomplexed cephalothin and the sulfadiazine against the bacteria strains, showing that they have a good activity as bactericides. The manganese(II), cobalt(II) and nickel(II) complexes showed to be less toxic than the two referenced drugs and the Schiff base ligand. Apart of membrane permeability, antibacterial activity of cephalothin Schiff base and its metal complexes depends mainly on the metal ion and the type of microorganism.

Acknowledgements. The authors express their sincere thanks to Comision de Investigación from the Universidad de Oriente and UPLA for support.

Appendix A. Supplementary material

21

Supplementary data associated with this article is available as PDF file from the authors and the journal.

References [1] B.K. Ishwar, S.K. Mishra, P.J. Jainey, C.S. Shastry, J. Chem. Pharm. Res. 3 (2011) 114118 [2] K. Singh, M.S. Barwa, P. Tyagi, Eur. J. Med. Chem. 42 (2007) 394402 [3] J. Travis, J. Potempa, Biochim. Biophys. Acta 14 (2000) 3550 [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] M. Tümer, E. Akgün, S. Toroglu, A. Kayraldız, L. Dönbak, J. Coord. Chem. 61 (2008) 29352949. [8] A. Gölcü, M. Tümer, H. Demirelli, R.A. Wheatley, Inorg. Chim. Acta 358 (2005) 17851797. [9] C. Gerdemann, C. Eicken, B. Krebs, Chem. Res. 35 (2002) 183191. [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.

22

[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] W.W. Simmons. The Sadtler handbook of proton NMR spectra. Sadtler Research Laboratories, Inc, 1978. [21] G. Socrates, Infrared Characteristic Group Frequencies, John Wiley & Sons, 1980. [22] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Compounds, 5th edition, John Wiley & Sons, 1997. [23] Y.Y. Wang, L.J. Zhou, Q. Shi, Q.Z. Shi, Transit. Met. Chem. 27 (2002) 145148. [24] L. Gutierrez, G. Alzuet, J. A. Real, J. Cano, J. Borras, A. Castiñeiras, Eur. J. Inorg. Chem. (2002) 20942102. [25] L. Gutierrez, G. Alzuet, J. A. Real, J. Cano, J. Borras, A. Castiñeiras, Inorg. Chem. 39 (2000) 36083614. [26] G.A. Bain, J.F. Barry, J. Chem. Ed. 85 (2008) 532536. [27] Z. H. Chohan and S. Kausar, Metal-Based Drugs, 7 (2000) 17–22. [28] M. G. B. Drew and P. C. Yates, Inorganica Chimica Acta, 118 (1986) 37–47.. [29] YT. Li, CW. Yen, JF. Lou, HS. Guan, J. Magn. Magn. Mater. 281 (2004) 6876. [30] O. Kahn, Molecular Magnetism, VCH, New York, 1993

23

[31] H. Liu, H. Wang, F. Gao, D. Niu, Z. Lu, J. Coord. Chem. 60 (2007) 2671–2678. [32] Z. Shirin, R.M. Mukherjee, Polyhedron 11 (1992) 2625–2633. [33] F. Koksal, F. Ucun, E. Agar, I. Kartal, J. Chem. Research (S) (1998) 9697. [34] M.C. Jain, A.K. Shrivastava, P.C. Jain, Inorg. Chim. Acta, 23 (1977) 199203 [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] M. Sonmez, A. Levent, M. Sekerci, Russ. J. Coord. Chem. 30 (2004) 655659. [38] A.B.P. Lever, Inorganic Electronic Spectroscopy, Elsevier, Amsterdam, 1968. [39] M.M. Mostafa, A. ElHammid, M. Shallaby, A.A. ElAsmy, Transit. Met. Chem. 6 (1981) 303305. [40] M.J.M. Cambell, Coord. Chem. Rev. 15 (1975) 279319. [41] F.A. Cotton, G. Wilkinson, Advanced Inorganic Chemistry, 6th edition, John Wiley & Sons, 1999. [42] M.M. Aboaly, M.M.H. Khalil, Spect. Lett. 34 (2001) 495504. [43] J.R. Anacona, A. Rodriguez, Transit. Met. Chem. 30 (2005) 897901. [44] J.R. Anacona, G. Da Silva, J. Chil. Chem. Soc. 50 (2005) 447450. [45] J.R. Anacona, H. Rodriguez, J. Coord. Chem. 62 (2009) 22122219. [46] J.R. Anacona, C. Patiño, J. Coord. Chem. 62 (2009) 613621. [47] N.C. Baenziger, A.W. Struss, Inorg. Chem. 15 (1976) 18071809 [48] D.S. Cook, M.F. Turner, J. Chem. Soc., Perkin Trans. 2 (1975) 10211025

24

[49] N.C. Baenziger, S.L. Modak, C.L. Fox, Acta Cryst. C39 (1983) 16201623 [50] C.J. Brown, D.S. Cook, L. Sengier, Acta Cryst. C41 (1985) 718720 [51] C.J. Brown, D.S. Cook, L. Sengier, Acta Cryst. C43 (1987) 23322334 [52] A. GarciaRaso, J.J. Fiol, G. Martorell, A. Lopez, M. Quiros, Polyhedron 16 (1997) 613621 [53] B.G. Tweedy, Phytopathology. 55 (1964) 910–914. [54] Georgopapadakou, N.H. Antimicrob. Agents Chemother. 37 (1993) 20452053.

25

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

H2O M1

M2

C

N

H

S

[(H2L)]

48.6

13.2

4.1

15.2

[(C25H24N6O7S3)]

(48.7) (13.6)

(3.9)

(15.6)

[MnL(H2O)3]

41.7

3.9

13.4

7.8

[Mn(C25H28N6O10S3)]

(41.5) (11.6)

(3.9)

(13.3)

(7.5) (7.6)

(7.6)

[CoL(H2O)3]

41.5

3.8

13.3

7.5

(8.2)

[Co(C25H28N6O10S3)]

(41.3) (11.6)

(3.9)

(13.2)

(7.4) (8.1)

(8.1)

[NiL(H2O)3]

41.7

4.2

13.5

7.1

(8.1)

[Ni(C25H28N6O10S3)]

(41.3) (11.6)

(3.9)

(13.2)

(7.4) (8.1)

(8.1)

[CuL(H2O)3]2

40.7

3.7

13.4

7.5

(8.4)

[Cu2(C50H56N12O20S6)] (41.0) (11.5)

(3.9)

(13.1)

(7.4) (8.7)

(8.7)

[ZnL(H2O)3]

41.1

3.9

12.8

7.2

(9.0)

[Zn(C25H28N6O10S3)]

(40.9) (11.4)

(3.8)

(13.1)

(7.4) (8.9)

11.9

11.7

11.8

11.2

11.7

7.5

8.2

8.4

8.6

9.2

M1 = complexometric analysis, M2 = thermal analysis

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

(7.7)

(8.9)

26

C=O Compound Cephalothin [H2L]

C=O C=N

lactam COOH amide imine 1735

COO

COO SO2

Δ

asymm symm symm

1657 1690

1656

1633

1155

[MnL(H2O)3]

1656

1634

1591

1416

1133 175

[CoL(H2O)3]

1653

1630

1590

1424

1135 166

[NiL(H2O)3]

1653

1630

1599

1426

1132 173

[CuL(H2O)3]2

1658

1620

1589

1423

1134 166

[ZnL(H2O)3]

1654

1631

1596

1425

1133 171

Table 3 Antibacterial activity Zone of inhibition (mm) Compound

E.C.

S.A

Sulfadiazine

8.0 ± 1.0

9.0 ± 1.0

Cephalothin

11.0 ± 1.0

13.0 ± 1.5

[H2L]

13.0 ± 1.0

16.0 ± 1.0

[MnL(H2O)3]

6.0 ± 1.0

7.0 ± 1.0

[CoL(H2O)3]

5.0 ± 1.0

6.0 ± 1.0

[NiL(H2O)3]

5.0 ± 1.0

6.0 ± 1.0

[CuL(H2O)3]2

14.0 ± 1.5

19.0 ± 1.0

[ZnL(H2O)3]

16.0 ± 1.0

18.0 ± 1.0

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

27

Figure 1. Chemical structure of cephalothin Figure 2. Infrared spectra of Schiff base ligand (A) and [MnL(H2O)3] (B) in the solid state. Acid C=O stretching (a), stretching C=O amide (b), stretching C=N imine (c), asymmetric COO stretching (d) and symmetric COO stretching (e) Figure 3. EPR spectrum of [CuL(H2O)3]2 complex at room temperature Figure 4. Proposed structure of [ML(H2O)3] complexes

28

29

30

Supplementary data

Scheme S1

31 SUGGESTED REFEREES 1

2

3

4

5

6

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 Name and Surname Institution Address E-mail Phone Country

Jim R. Durig Spectrochimica Acta

[email protected] 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

2

Graphical abstract Synthesis, characterization and antibacterial activity of a tridentate Schiff base derived from cephalothin and sulfadiazine, and its transition metal complexes J.R. Anaconaa,*, Natiana Noriegaa and Juan Camusb a Department of Chemistry, Universidad de Oriente, Cumana 6101. Venezuela b Facultad de Ciencias Naturales y Exactas, Universidad de Playa Ancha, Valparaíso, Chile Transition coordination compounds with a Schiff base (H2L) derived from the condensation of cephalothin antibiotic with sulfadiazine were synthesized, characterized and screened for antibacterial activity..

3

Highlights

A new Schiff base using cephalothin antibiotic was prepared Cephalothin Schiff base transition metal complexes were prepared Magnetic and spectroscopic properties, and antibacterial activity were studied