Complexes of Th ( IV ) , Zr ( IV ) and V ( IV ) with Norfloxacin : Synthesis , Spectroscopic , Thermal , Kinetic Measurements and Antimicrobial Evaluation

M(IV) complexes of thorium, zirconium, and vanadium with norfloxacin were synthesized and characterized with physical and spectroscopic techniques, including elemental analysis (C,H,N), IR, electronic, H NMR spectral methods, as well as thermogravimetric (TG) and differential thermogravimetric (DTG) analyses. Based on these studies, the proposed structure of the obtained complexes are [Th(NFX)2Cl4], [Zr(NFX)2Cl2]Cl2 and [VO(NFX)2]SO4 (where, NFX: norfloxacin). In all complexes, norfloxacin acts as a bidentate deprotonated ligand bound to the metal through the pyridone and one carboxylate oxygen atoms, forming eight, six, five atoms ring with Th (IV), Zr(IV) and V(IV) metal ions, respectively. The kinetic thermodynamic parameters, such as, activation energy, E, enthalpy, H, entropy, S, and Gibbs free energy, G, have been calculated from the TG and DTG curves, using Coats-Redfern and Horowitz-Metzger methods. Norfloxacin as well as their metal complexes were also evaluated for their antibacterial activity against several bacterial species, such as Bacillus subtilis, Pseudomonas aeruginosa (P. aeruginosa), and Escherichia coli (E. coli) and antifungal screening was studied against three species: Aspergills flavus, Fusarium solani, and Penicillium verrcosum (P. verrcosum). The results show significant increase in antibacterial and antifungal activity of metal complexes as compared to the NFX itself.


Introduction
Introduction of nalidixic acid during 1962 has shown the new avenue for patients with the bacterial infections and the dramatic impact in altering morbidity and mortality rate compared with the chemotherapy during 1940s.The continuous effort to increase the efficacy against bacteria has led to identify new prototype quinolone, which is active only against Gram-negative bacteria, similar to that of nalidixic acid (Sunduru et al., 2011).However, during 1980s, the introduction of fluoroquinolone drugs has changed the landscape of antibacterial chemotherapy, which was active against both Gram-negative and Gram-positive bacteria pathogens (Koga et al., 1980).Fluoroquinolones are broad-spectrum antibiotics widely used for the treatment of numerous diseases (Reynolds, 1993;Naumann & Dopp, 1989).The most active fluoroquinolone drugs are Norfloxacin, Ciprofloxacin, Sparfloxacin, and Trovafloxacin.The antibacterial activity of fluoroquinolones depends not only on the bicyclic heteroaromatic pharmacophore but also on the nature of the peripheral substituent's and their spatial relationship (Koga et al., 1980;, Fang et al., 2000;Efthimiadou et al., 2007).These substituents exert their influence on antibacterial activity by providing additional affinity for the bacterial enzymes, enhancing the cell penetration, or altering the pharmacokinetics (Chu et al, 1986;Domagala et al, 1988).Norfloxacin (NFX, Fig. 1) (1-ethyl -6fluoro -4-oxo-7-(1-piperazinyl) -1,4-dihydroquinoline-3-carboxylic acid) is a second generation fluoroquinolone, and widely used representative member of this family (Dhaneshwar et al, 2001).It is antibiotic agent that extensively used in both human and veterinary medicine (Cui et al., 2011).Norfloxacin (NFX) is active against a wide variety of aerobic Gram-negative and Gram-positive bacteria but specifically, active against aminoglycoside-resistant Pseudomonas aeruginosa and betalactamase producing organisms (Shaikh et al., 2007).When dealing with the interaction between drugs and metal ions in living systems, a particular interest has been given to the interaction of metal ions with antibiotics.Presence of metal ions considerably alters the activity of fluoroquinolones against potentially susceptible bacteria (Shaikh et al., 2007).A number of works have been reported about the synthesis and characterization of new metal complexes with fluoroquinolone antibacterial agents (Shaikh et al., 2007;Turel et al., 1997a;Turel et al., 1998;Wu et al., 2003;Refat, 2007;Al-Mustafa, 2002;Sadeek et al., 2006;Polk & Pharm, 1989).
In this paper, the interaction of norfloxacin (NFX) with tetraverse transition metal ions in an attempt to examine the mode of norfloxacin coordination and the biological properties of the resulting complexes.In particular, complexes of NFX with the Th(IV), Zr(IV), and V(IV) have been synthesized and characterized with elemental analysis, infrared (IR), 1H NMR and electronic spectra.The thermal behavior of the obtained complexes has also been studied as well as their biological activity against several microorganisms.The antibacterial activity was tested against Bacillus subtilis, Pseudomonas aeruginosa (P.aeruginosa), and Escherichia coli (E.coli) and antifungal screening was studied against three species: Aspergills flavus, Fusarium solani, and Penicillium verrcosum (P.verrcosum).

Materials
All chemicals used were of analytical reagent grade, commercially available, and were used without further purification.Egyptian Interactional Pharmaceutical Industrial Company (EIPICO) supplied Norfloxacin used in this work.Th(NO 3 ) 4 , ZrCl 4 .xH 2 O, and VOSO 4 .5H 2 O were purchased from Aldrich Chemical Company.

Synthesis of the metal complexes
Norfloxacin (2 mmol) in mixed solvent (50/50 %) of methanol/acetone was stirred at room temperature for 20 min.A solution of 1.0 mmol of Th(NO 3 ) 4 , ZrCl 4 , and VOSO 4 in 5 ml of methanol was added to the norfloxacin solution with constant stirring.The resulting mixture was heated  50 C under reflux on a water bath for about 12 h and then cooled.The resulted solid complexes were separated from the reaction mixture by filtration, washed with methanol and dried under vacuum to a constant weight.The yields were found around 70% based on the metal salts.The compounds resulted have low solubility in water and in common organic solvents.The obtained complexes were characterized by their elemental analysis, infrared, electronic, and 1 H NMR, as well as thermal analysis.

Elemental analysis
Elemental analysis was performed using a Perkin-Elmer CHN 2400, and the metal contents were found gravimetrically by ignition-weighted samples in atmospheric air to constant weight and definite structure.
Decomposition of complexes was performed in concentrated HNO 3 .After decomposition, the sample was diluted with water to 100 ml, and qualitative presence of Clions (in Th(IV) Zr(IV) complexes), and SO 4ions (in V(IV) complex) were determined by means of AgNO 3 and BaCl 2 , respectively.

Molar conductivity
Molar conductivities of freshly prepared 10 -3 mol dm -3 DMSO solutions were measured on a Jenway 4010 conductivity meter.

Electronic spectra
Electronic spectra were recorded in the region of (200-800 nm) for DMSO solution (10 -3 ) of the NFX and their three complexes with Shimadzu UV-spectrophotometer model 1601 PC with a 1 cm quartz cell.

Infrared spectra
Infrared (IR) measurements were carried out on a Genesis II FT-IR spectrophotometer using KBr pellets in the = 400-4000 cm -1 range.

1 H NMR spectra
1 H NMR spectra of Thorium complex was obtained on a Varian Gemini 200 MHz spectrometer using DMSO-d6 as solvent and TMS as an internal reference. 1H NMR data are expressed in parts per million (ppm), referenced internally to the residual proton impurity in DMSO solvent.

Thermal analysis
Thermogravimetric measurements (TG and DTG) were carried out in dynamic nitrogen atmosphere (30 ml/min.) between room temperature and 900 C with a heating rate of 10 C/min.using a Shimadzu TGA -50H thermal analyzers.

Antimicrobial investigation
Antibacterial activity of the Th(IV), Zr(IV), and V(IV) complexes, and the NFX ligand, as well as the pure solvent was investigated by a previously reported modified method of Beecher and Wong (Beecher & Wong, 1994), against different bacterial species, such as Escherichia coli (E.coli), Bacillus subtilis, and Pseudomonas aeruginosa (P.aeruginosa), and antifungal screening was studied against three species, Aspergills flavus, Fusarium solani, and Penicillium verrcosum (P.verrcosum).The microorganisms were purchased from the laboratory of (Microbiology) in the Egyptian International Pharmaceutical Industrial Company (EIPICO).

Results and Discussion
The results of the elemental analysis and some physical characteristics of NFX ligand and the obtained complexes are given in Table 1 (Note 1.).The complexes were synthesized using 1:2 (metal: ligand) mole ratio of all reactants.The elemental analysis (Table 1) of the complexes indicates a 1:2 metal to ligand stoichiometry, too.All complexes are stable in air, hygroscopic, with high melting points, insoluble in water, but partly soluble in dimethylformamide; DMF.All complexes have been prepared at high yield (73-76%), and melting points of the complexes are higher than that of the ligand revealing that the complexes are much more stable than ligand.The molar conductance values (Table 1) of the complexes in DMF solvent (10 -3 mol dm -3 ) lay in the range of 73.00-87.20  -1 cm 2 mol -1 (at 25 C), which indicates that the complexes are electrolytes, and the complexes are of much increasing in electrolytic nature than NFX ligand.The low conductivity values are in agreement with the low solubility of NFX complexes in water, ethanol, chloroform, acetone and most organic solvents.On the other hand, they are soluble in DMSO and DMF.The proposed structures of the obtained complexes are shown in Fig. 2.These structures were confirmed by its analytical data.

Infrared spectra
The IR spectra of quinolones are most representative in the region 1800-1300 cm -1 (Turel et al., 1997b).The tentative assignments of IR bands of Th(IV), Zr(IV), and V(IV) complexes are presented in Table 2 (Note 2. and 3.) and Fig. 3, and have been compared with those of the free ligand norfloxacin, in order to determine the site of coordination that may be involved in chelation.
(ii) Th(IV), Zr(IV), V(IV) complexes shows no absorption band at 1700 cm -1 (C=O), that is indicative of the deprotonation of -COOH group and involvement of the carboxyl group in the formation of Th-O, Zr-O, and V-O bonds.
(iii) The (C=O) p peak (of pyridone keto) is shifted from 1630 cm -1 in the ligand to lower frequencies in the complexes ( 1610-1617 cm -1 ) upon bonding.
(iv) The spectra of the three complexes shows two very strong to strong characteristic bands (1575 cm -1 and 1380) at 1576-1380, 1579-1382, 1583-1381 cm -1 for Th(IV), Zr(IV), V(IV) complexes, respectively, assigned as (O-C-O) asymmetric and symmetric stretching vibrations of the ligated carboxylate anion, respectively.These bands are weak in the spectrum of NFX.These complexes also have medium intensity peak around 1450 cm -1 that correspond to asymmetrical and symmetrical carboxylate anion stretching.Deacon and Phillips, 1980, have studied the criteria that can be used to distinguish between the three binding states of the carboxylate complexes.These criteria are: (a) 200 cm -1 (where =[as(COO -)-s(COO -)]) this relation was found in case of monodentate carboxylato complexes, (b) bidentate or chelating carboxylato complexes exhibit  significantly smaller than ionic values (100 cm -1 ), and finally, (c) bridging complexes show  comparable to ionic values (150 cm -1 ).Therefore, the difference value  is a useful characteristic for determining the coordination mode of the carboxylate group of the ligands.The observed  for Th(IV), Zr(IV), and V(IV) complexes (Table 3, Note 4. and 5.) fall in the range 196-202 cm -1 indicating a monodentate coordination mode of the carboxylato group (Dendrinou-Samara et al., 1998;Nakamoto, 1986) .
(v) The IR spectral data of the three complexes show medium-to-medium-weak bands around 3390 cm -1 , and weak to very weak band at 2933-2375, 2845-2356, and 2883-2344 for Th(IV), Zr(IV), and V(IV) complexes, respectively.These bands can be assigned to the vibrations of the quaternized nitrogen (-+ NH 2 ) of the piperazinyl group, which indicated that the zwitterionic form of NFX is involved in the coordination of the Th, Zr, and V ion investigated (Silverstein et al., 1991).
(vi) The vibrations of the free sulphate group in V(IV) complex, [VO(NFX) 2 ]SO 4 , can be assigned as follows: two weak intensities bands occur in the region above 1000 cm -1 at 1195 and 1036 cm -1 , are assigned to the different symmetric and antisymmetric bond vibrations, (SO 4 2-), and medium strong band at 689 cm -1 is assigned to the bending motion of (SO 4 2-).
(vii) The (V=O) stretching vibration in the vanadyl complex is observed as expected as a medium band at 956 cm -1 , which is a good agreement with those known for many dioxovanadium (IV) complexes.
According to the IR spectral data, the NFX is coordinated to the metal ions as a bidentate ligand via one-carboxylato oxygen atoms and the oxygen atom of the pyridine carbonyl group (Turel, 2002).

Electronic absorption spectra
Electronic spectra of the NFX complexes were recorded in the 200-800 nm region in DMSO (Fig. 4) There are two detected absorption bands at 285 and 335 nm in electronic spectrum of NFX, assigned to n-* and -* transitions.These transitions occur in case of unsaturated hydrocarbons, which contain carbon atom attached with oxygen atoms as in carboxylic and ketone groups (Refat, 2007).The electronic spectra of Th(IV), Zr(IV), and V(IV) complexes show two absorption bands at 250 and 285 nm.The band at 335 nm is absent in the electronic spectra of the complexes which may indicate that the carboxylic group and ketone groups are involved in the complexation.

1 H NMR spectra
The 1 H NMR spectral data of the [Th(NFX) 2 Cl 4 ] complexes and the free ligand (NFX) are represented in Table 4, and all assignments.The spectrum of the complex is shown in Fig. 5.The literature values of the 1 H NMR chemical shifts (, ppm) of NFX are included in Table 4 for comparison purposes.Upon comparison with the free ligand, the absent of the characteristic peak for hydrogen of -COOH at  11.00 ppm in Th(IV) complex indicates coordination of NFX ligand to Th(VI) through the deprotonated carboxylic group (Refat, 2007;Sadeek, 2005).The spectrum also show characteristic three peaks for quaternized nitrogen (-+ NH 2 ) at  2.10, 2.50, and 3.9 ppm (Sadeek et al., 2009).The broad peak at  3.9 ppm, which was not detected in the spectra of free NFX ligand, confirmed the deprotonation of the two -COOH groups in NFX.The overall changes of the 1 H NMR spectrum of the Th (IV) complex is indicative of coordination of NFX ligand to the metal via the pyridone and one carboxylate oxygen atoms (Riley et al., 1993).This suggested coordination is in agreement with that obtained by elemental analysis and IR spectra.

Thermogravimetric analysis
The Norfloxacin of T(IV), Zr(IV), and V(IV) complexes are stable at room temperature and can be stored for several months without any changes.The obtained complexes were studied by thermogravimetric (TG) and differential thermogravimetric (DTG) analysis from ambient temperature to 900 C N 2 atmospheres.The TG curves were redrawn as mg mass loss versus temperature (TG) curves and as the rate of loss of mass versus temperature (DTG) curves.Typical TG and DTG curves are presented in Fig. 6, and the thermoanalytical results are summarized in Thermal decomposition of [VO(NFX) 2 ]SO 4 proceeds in three stages.The first stage is occurs at maximum temperature at 60 C (DTG), in the temperature range 25-210 C.The mass loss in this stage is found to be 14.81%, corresponding to the thermal decomposition of the complex by the loss of 2C 2 H 2 + SO 2 .This is followed by another mass loss (68.38%) in the temperature range 210-520 C, by giving an endothermic effect (DTG max , 260, 374, 445 C).In the final stage, the complex decomposes in consecutive steps in the 520-900 C temperature range to give VO 2.5 with some carbon atoms.
Reported data on thermal analysis studies in the nitrogen atmosphere indicate that the Th(IV), Zr(IV), V(IV) complexes decompose to give metal oxide as final product, with few carbon atoms (except Zr(IV) complex), indicates that no sufficiently of oxygen atoms helps to evolved carbon as carbon monoxide or dioxide.The found weight loss associated with each step of decomposition for each complex agrees well with the calculated weight loss (Table 5).The final products of the complexes obtained at 900 C were confirmed with infrared spectra.

Coats-Redfern equation
The Coats-Redfern equation ( 1), which is atypical integral method, can be represented as: (1) For convenience of integration, the lower limit T1 is usually taken as zero.This equation on integration gives:

ln [-ln (1-)/T 2 ] = -E*/RT + ln [AR/E]
(2) A plot of left-hand side (LHS) against 1/T was drawn.E * is the energy of activation in KJ mol -1 and calculated from the slop and A in (s -1 ) from the intercept.The entropy of activation S * in (JK -1 mol -1 ) was calculated by using the equation: where k is the Boltzmann constant, h is the Plank's constant and T s is the DTG peak temperature.

Horowitz-Metzger equation
The  was written in the form as follows: Where  = T -T s , w  = w  -w, w  = mass loss at the completion of the reaction; w = mass loss up to time t.
The plot of Log [log (w  /w  )] versus  was drawn and found to be linear from the slope E * was calculated.The pre-exponential factor, A, was calculated from the Eg. ( 5): From the TG curves, the activation energy, E * , entropy of activations The activation energy of Th(IV), Zr(IV) and V(IV) complexes is expected increase in relation with decrease in their radius (Avsar et al., 2002).The E * values calculated with the method of Coats-Redfern for the first decomposition stage of the complexes are given below: rV(IV) = 72 pm  rZr(IV) = 86 pm  rTh(IV) = 108 pm

Antimicrobial activities
Applying the nutrient filter paper disc method, all of the newly synthesized NFX complexes and NFX were screened in vitro for antibacterial and antifungal activity.The activity was determined against three bacterial species, Bacillus subtilis, Pseudomonas aeruginosa (P.aeuginosa), and Escherichia coli (E.coli), and antifungal screening was studied against three species, Aspergills flavus, Fusarium solani, and Penicillium verrcosum.The activity was performed by measuring the diameter of the inhabitation zone, and the screening results are given in Table 7 (Note 7.).The results show that the metal complexes are more effective as antibacterial and antifungal agents as compared to the uncomplexed ligand.This increase in the activity is being considered due to increased bioavailability of the metal drug complexes and the aqueous solubility of NFX.Th(IV) complex exhibit higher activity than other complexes.All the three complexes exhibited no inhibitory activates against Bacillus subtilis species.

Table 5 .
(Shaikh et al., 2007)ass from the TG curves is 97.74% for NFX, 68.64% for [Th(NFX) 2 Cl 4 ], 86.28% for [Zr(NFX) 2 Cl 2 ]Cl 2 , and 85.12% for [VO(NFX) 2 ]SO 4 .All the complexes show two or three stages of mass loss in their TG/DTG curves, and all mass loss in these stages is due to the decomposition of counter ions and NFX molecules.3.4.1 Norfloxacin ligandThe data obtained indicate that the NFX is thermally stable in the temperature range 25-50 C.Decomposition of NFX start at 50 C and finished at 726 C with three stages.The first stage of decomposition occurs between 25 and 270 C, at maximum temperature of 125 C, and is accompanied by weight loss of 8.38%, corresponding exactly to the loss of ethylene molecule (C 2 H 2 ).The second stage of decomposition starts at 270 C and end at 575 C, showing an endothermic peak at 335 C.This stage is accompanied by a weight loss of 69.80%, corresponding to the loss of 4C 2 H 2 + HF + N 2 + CO + CO 2 .The final decomposition step occurs in the range 575-720 C with a maximum at 650 C, and is accompanied by a weight loss of 19.56%, and my be attributed to the loss of pyrrole ring, 2C 2 H 2 + 1/2N 2 , 1/2H 2 , in reasonable agreement with the theoretical value of 21.0%.The weight loss in this step is 37.69%, corresponding to the loss of 5C 2 H 2 + Cl 2 + 2HF + 2N 2 + 2NO + 2CO.The final step is associated with the loss of 7C 2 H 2 + CO + 4H 2 forming ThO 2 + 4C as a final product.3.4.3 [Zr(NFX) 2 Cl 2 ]Cl 2 complexThe thermal analysis curves of [Zr(NFX) 2 Cl 2 ]Cl 2 complex show that decomposition takes places in two stages.The first stage is exothermic stage occurs at two maximum temperature at 60 and 298 C.The broader exothermic peak at 60 C indicate a slow in heat capacity(Shaikh et al., 2007).
The thermal decomposition of [Th(NFX) 2 Cl 4 ] complex proceeds approximately with three main degradation stages in 25-900 C temperature range.The first stage of decomposition is exothermic stage occurs at three maximum temperature of 50, 108, and 218 C, and is accompanied by a weight loss of 11.84%, corresponding to the loss of 2C 2 H 2 + Cl 2 .The second stage of decomposition occurs at three maximum temperatures at 308, 377, and 455 C.

Table 1 .
Elemental analysis and physical properties of the compounds

Table 3 .
Characteristic absorptions of carboxylate group (in cm -1 ) and  values

Table 5 .
Thermo analytical results for the investigated compounds

Table 7 .
Antibacterial and antifungal activities Note 7. data for NFX and its compounds