Metal Complexes of Enrofloxacin Part I: Preparation, Spectroscopic, Thermal Analyses Studies and Antimicrobial Evaluation

Abstract

The interaction of titanium (IV), yttrium (III), zirconium (IV), palladium (II) and cerium (IV) with deprotonated enrofloxacin leads to the formation of the neutral or cationic mononuclear complexes. The isolated solid complexes have been characterized with physicochemical and spectroscopic techniques and thermogravimeteric analyses. The spectroscopic data indicate that the enrofloxacin ligand is on the deprotonated mode acting as bidentate ligand coordinated to the metal ions through the ketone oxygen and a carboxylato oxygen and the metal ions completed the coordination number with water molecules. The thermal decomposition mechanisms proposed for enrofloxacin and their metal complexes were discussed. The activation energies, $E^*$, enthalpies, ${\Delta}H^*$, entropies, ${\Delta}S^*$ and Gibbs free energies, ${\Delta}G^*$, of the thermal decomposition reactions have been derived from thermogravimetric (TG) and differential thermogravimetric (DTG) curves, using Coats-Redfern (CR) and Horowitz-Metzeger (HM) methods. The antimicrobial activity has been evaluated against six different microorganisms.

INTRODUCTION

Enrofloxacin is a synthetic chemotherapeutic agent from a class of the fluoroquinolone carboxylic acid derivatives under the trade name Baytril. In September 2005, the FDA withdraw approval of Baytril for use in water to treat flocks of poultry. Enrofloxacin (= HErx Scheme 1), is a second generation quinolone antibacterial drug1 and the first developed drug for many veterinary applications. It has excellent activity against a broad spectrum of both Gram-negative and Gram-positive pathogens.2,3 Its mechanism of action is not thoroughly understood, but it is believed to act by inhibiting bacterial DNA gyrase (a type II topoisomerase), thereby preventing DNA supercoiling and DNA synthesis.

Scheme 1.Chemical structure for enrofloxacin (HErx = 1-cyclopropyl-7-(4-ethylpiperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid).

The coordination chemistry of fluoroquinolones drugs with metal ions of biological and pharmaceutical importance is of considerable interest. There have been several reports about the synthesis and crystal structure of metal complexes.4-7 The proposed mechanism of the interaction between quinolone and metal cations was chelation between the metal and the 4-oxo and adjacent carboxyl group.8-16 The coordination of the fluoroquinolones with metallic ions by piperazine nitrogen is much less common.17-23 The crystal structure of enrofloxacin complexes indicates that enrofloxacin is reacted with the metal ions as bidentate ligand through the pyridine oxygen and one carboxylate oxygen atom when coordinated to metal atoms.24-30

The study of the interaction of some fluoroquinolones with metal ions has been initiated in an attempt to examine the mode of binding and the effect of metal ions on the biological activity of fluoroquinolone against some of Gram-negative and Gram-positive bacteria.31,32 In this context, the synthesis, the characterization and the antimicrobial activity of the novel mononuclear Ti(IV), Y(III), Zr(IV), Pd(II) and Ce(IV) complexes with enrofloxacin [Ti(Erx)2(H2O)2]SO4. 7H2O, [Y(Erx)2(H2O)Cl].9H2O, [ZrO(Erx)2(H2O)].6H2O, [Pd(Erx)2(H2O)2].4H2O and [Ce(Erx)2(H2O)2]SO4.4H2O are presented.

 

EXPERIMENTAL

Materials and Reagents

All the chemicals and reagents for the synthesis of the complexes were used as purchased without further purification. Enrofloxacin was purchased from Egyptian Company for Chemicals & Pharmaceuticals (ADWIA). Ti(SO4)2, NaOH and all solvents were from Aldrich Chemical Co., YCl3, ZrOCl2.8H2O, PdCl2 and Ce(SO4)2 were from Fluka Chemical Co.

Instrumentation

Elemental C, H, N and halogen analyses were carried out on a Perkin Elmer CHN 2400. The percentage of the metal ions was determined gravimetrically by transforming the solid products into oxide or sulphate, and also determined by using atomic absorption method. Spectrometer model PYE-UNICAM SP 1900 fitted with the corresponding lamp was used for this purpose. IR spectra were recorded on FTIR 460 PLUS (KBr discs) in the range from 4000−400 cm−1, 1H NMR spectra were recorded on Varian Mercury VX-300 NMR Spectrometer by using DMSO-d6 as solvent. TGA-DTG measurements were carried out under N2 atmosphere within the temperature range from room temperature to 800 °C using TGA-50H Shimadzu. Electronic spectra were obtained using UV-3101PC Shimadzu. The solid reflection spectra were recorded with KBr pellets. Magnetic measurements were carried out on a Sherwood scientific magnetic balance by using Gouy method and Hg[Co(SCN)4] as calibrant. Molar conductivities of the solution of the ligand and metal complexes in DMSO at 6.5×10−4 M were measured on CONSORT K410. All measurements were carried out at ambient temperature with freshly prepared solution.

Synthesis of the Complexes

[Ti(Erx)2(H2O)2]SO4.7H2O

Enrofloxacin (359.4 mg, 1 mmol) and NaOH (40 mg, 1 mmol) were dissolved in methanol (50 ml). After 40 min of stirring we dropping wisely 0.5 mmol of Ti(SO4)2 (119.95 mg, 1.3 mL) to the mixture. The reaction mixture was stirred for one day at room temperature. The solution was reduced in volume and left for slow evaporation. The reddish brown precipitate was filtered off and dried in vacuum over CaCl2.

[Y(Erx)2(H2O)Cl].9H2O

The white complex [Y(Erx)2(H2O)Cl].9H2O was prepared by dropping wisely of YCl3 (195.4 mg, 0.5 mmol) in 15 ml ethanol to a stirred solution of HErx (359.4 mg, 1 mmol) and NaOH (40 mg, 1 mmol) in 45 ml ethanol. The reaction mixture was stirred for 20 h at room temperature. The white precipitate was filtered off and dried in vacuum over CaCl2.

[ZrO(Erx)2(H2O)].6H2O

Enrofloxacin (359.4 mg, 1 mmol) and NaOH (40 mg, 1 mmol) were dissolved in methanol (50 ml). After 30 min of stirring we dropping wisely of ZrOCl2.8H2O (161.11 mg, 0.5 mmol) to the mixture. The reaction mixture was stirred for 15 h at room temperature. The solution was reduced in volume and left for slow evaporation. The yellow precipitate was filtered off and dried in vacuum over CaCl2.

[Pd(Erx)2(H2O)2].4H2O

The faint-brown complex [Pd(Erx)2(H2O)2].4H2O was prepared by dropping wisely of PdCl2 (88.71mg, 0.5mmol) in 10 ml methanol to a stirred solution of HErx (359.4 mg, 1 mmol) and NaOH (40 mg, 1 mmol) in 50 ml ethanol. The reaction mixture was stirred for three days at room temperature. The faint-brown precipitate was filtered off and dried in vacuum over CaCl2.

[Ce(Erx)2(H2O)2]SO4.4H2O

The pale-orange solid complex [Ce(Erx)2(H2O)2]SO4.4H2O was prepared by dropping wisely of cerium(IV) sulphate Ce(SO4)2 (166.06 mg, 0.5 mmol) in 20 ml ethanol to a stirred solution of HErx (359.4 mg, 1 mmol) and NaOH (40 mg, 1 mmol) in 50 ml ethanol. The reaction mixture was stirred for two days at 50 °C in water bath. The paleorange precipitate was filtered off and dried in vacuum over CaCl2.

We did not manage to obtain a crystal of the complexes suitable for the structure determination with X-ray crystallography, although diverse crystallization techniques were used.

Antimicrobial Investigation

Antibacterial activity of the ligand and its metal complexes was investigated by a previously reported modied method of Beecher and Wong,33 against different bacterial species, such as S. aureus K1, B. subtilis K22, Br. otitidis K76, E. Coli K32, P. aeruginosa SW1 and K. oxytoca K42. The tested microorganisms isolates were isolated from Egyptian soil and identied according to the standard bacteriological keys for identication of bacteria as stock cultures in the Microbiology Laboratory, Faculty of Science, Zagazig University. The Müller–Hington agar (30.0% beef extract, 1.75% Casein hydrolysate, 0.15% starch and 1.7% agar) was prepared and then cooled to 47 °C and seeded with tested microorganisms. After solid-ication 5 mm diameter holes were punched by a sterile cork-borer. The investigated compounds, i.e., ligand and their complexes, were introduced in holes (only 100 μL) after being dissolved in DMSO at 10−4 M. These culture plates were then incubated at 37 °C for 20 h. The activity was determined by measuring the diameter of the inhibition zones (in mm). Growth inhibition was calculated with reference to the positive control, i.e., enrooxacin.

 

RESULTS AND DISCUSSION

The enrofloxacin (HErx) form complexes with Ti(SO4)2, PdCl2, ZrOCl2.8H2O and YCl3, Ce(SO4)2 in methanol and ethanol, respectively. All the Ti(IV), Y(III), Zr(IV), Pd(II) and Ce(IV) are colored and stable at room temperature (Table 1). These complexes are nearly insoluble in common organic solvents but soluble in DMSO and DMF. Table 1 summarizes the percentage of carbon, hydrogen, nitrogen, halogen and sulpher and metal contents in all compounds as well as color, melting points, magnetic properties and molar conductance of the isolated complexes. The elemental analyses show that, all the five complexes have 1:2 stoichiometery of the type ML2, where L stands for deprotonated ligand (Scheme 1). The molar conductance value (20.23 S cm2 mol−1) for free ligand and the corresponding values (50.77, 43.71 and 23.78 S cm2 mol−1) for Y(III), Zr(IV) and Pd(II) in DMSO are low to account for any dissociation of the complexes, indicating the nonelectrolytic nature of the complexes in DMSO. The molar conductance values for Ti(IV) and Ce(IV) in the same solvent (DMSO) are 99.2 and 95.69 S cm2 mol−1 indicating the electrolytic nature of the complexes and the presence of sulphate as counter ion. Also, the qualitative reactions revealed the presence of sulphate as counter ion (outside the complex sphere) and the chloride inside the complex sphere (no precipitate formed with AgNO3).

The magnetic moments (as B.M) of five complexes at room temperature were measured. The magnetic measurement of Ti(IV), Y(III), Zr(IV), Pd(II) and Ce(IV) complexes found in diamagnetic character and octahedral geometry around the metal ion.

Table 1.Elemental analysis and Physico-analytical data for enrofloxacin(HErx) and its metal complexes

IR Spectral Studies

The structures of the complexes were characterized by FTIR. In a comparison of the IR spectra of enrofloxacin ligand and their metal complexes. Therewere some obvious changes (Fig. 1). The enrofloxacin have two characteristic absorption peaks, 1736 cm−1 and 1628 cm−1; the first is the C=O vibration absorbtion peak from carboxylic acid oxygen, and the second was assigned to keto C=O peak from the ring of enrofloxacin. However, the 1736 cm−1 peak disappeared in the complexes, and instead, two very strong characteristic bands are present around 1630 cm−1 and 1390 cm−1 (Table 2) that could be assigned as ν(O−C−O) asymmetric and symmetric stretching vibrations, respectively.

At the same time, the keto oxygen was coordinated with metal ions, the peak of which shifted from 1628 cm−1 to a value around 1575 cm−1, these changes of the IR spectra suggest that the enrofloxacinato ligand is coordinated to the metal ions via one oxygen of carboxylic group and oxygen of pyridone group.34−38

The bands in the range 3467−3210 cm−1 in the spectra of the complexes can be attributed to the ν(O−H) vibration of the water molecules and carboxylic group.37 The absorption peaks at 2787−2453 cm−1 should hydrogen bonds in molecule or between molecules.39 Also, the data given in Table 2 show that ν(Zr=O) is a very strong band at 815 cm−1.32,40 The spectra of the isolated solid complexes show a group of bands with different intensities which characteristics for ν(M−O), the ν(Ti−O) bands observed at 660, 590 and 490 cm−1 and at 629, 540 and 463 cm−1 for ν(Y−O) and at 667, 617 and 532 cm−1 for ν(Zr−O) and at 629 and 490 cm−1 for ν(Pd−O) and at 617, 502 and 480 cm−1 for ν(Ce−O) which are absent in the spectrum of enrofloxacin. The overall changes and the presence of new of the infrared spectra suggest that enrofloxacin is coordinated to the metal ion via the pyridone and one carboxylate oxygen atoms.

Fig. 1.Infrared spectra of (A) enrofloxacin, (B) [Ti(Erx)2(H2O)2] SO4.7H2O, (C) [Y(Erx)2(H2O)Cl].9H2O, (D) [ZrO(Erx)2(H2O)].6H2O, (E)[Pd(Erx)2(H2O)2].4H2O and (F) [Ce(Erx)2(H2O)2]SO4.4H2O.

Electronic Spectroscopy of the Complexes

The UV-Vis. Spectra of the ligand and their metal complexes has been recorded as electronic solid reflection spectra from 200 nm to 800 nm and listed in Table 3 and shown in Fig. 2. Free enrofloxacin shows bands at 216, 257 and 300 nm assigned to π-π* and n-π* transitions. The bathochromic shift of the reflectance band and appearance of new bands at higher wavelength for the complexes indicative of coordination through the pyridone and one carboxylate oxygen.37

All complexes exhibit a ligand-metal charge transfer bands at 487−534 nm and also exhibit a new bands at 620 nm for Y(III) and at 632 nm for Zr(IV) and at 615 nm for Pd(II) and at 620 nm for Ce(IV) which may be assigned to d-d transition.

The 1H NMR Spectra

The 1H NMR spectra of free enrofloxacin and their complexes of Ti(IV), Y(III), Zr(IV) and Ce(IV) in DMSO were obtained and illustrated in Fig. 3, and all the observed signals are assigned in Table 4. Using the 1H NMR technique, we can identify the nature of the complexes resulted from the interaction between ligand and metal ions.

Concerning the complexes, [Ti(Erx)2(H2O)2]SO4.7H2O, [Y(Erx)2(H2O)Cl].9H2O, [ZrO(Erx)2(H2O)].6H2O and [Ce(Erx)2(H2O)2]SO4.4H2O, we find out that the characteristic signal of –COOH group, which lies at the values 15.12 ppm in the enrofloxacin (HErx), disappears and has no existence in the interaction outcomes. The absence of this peak is justified that the coordination of enrofloxacin via the carboxylic group.32,33 The previous discussion has been proved through the use of the IR analysis, where the –COOH group at the values 1736 cm−1 in the infrared spectrum of free enrofloxacin disappears and two new peaks around 1630 and 1390 cm−1 are formed. Also the 1H NMR spectra for complexes exhibit new peak at 3.9, 4.41−5.06, 3.87−4.49 and 3.82 ppm, due to the presence of water molecules in the complexes.

On comparing enrofloxacin with its complexes, all peaks of the free ligand are present in spectra of the complexes with some shifts from binding of the ligand to the metal and also the complexes exhibit new resonances (Table 4) due to the presence of water in the complexes.30,41

Thermal Studies

Thermogravimetric analysis (TGA) and differential thermogravimetric (DTG) analyses were carried out for enrofloxacin ligand, [Y(Erx)2(H2O)Cl].9H2O, [Pd(Erx)2(H2O)2].4H2O and [Ce(Erx)2(H2O)2]SO4.4H2O complexes in the 700 °C range and for [Ti(Erx)2(H2O)2]SO4.7H2O and [ZrO(Erx)2(H2O)].6H2O in the 800 °C range under nitrogen atmosphere with rate flow 20 mL/min. and temperature program rate 10 °C/min. The TGA and DTG curves are presented in Fig. 4. Thermal data of the complexes are given in Table 5. The correlation between the different decomposition steps of the complexes with corresponding weight losses is discussed in terms of the proposed formula of the complexes.

Table 2.as=strong, w=weak, sh=shoulder, v=very, br=broad, bν=stretching and δ=bending.

Table 3.UV-Vis. spectra of enrofloxacin (HErx) and its metal complexes

Fig. 2.Electronic reflection spectra of (A) enrofloxacin, (B) [Ti(Erx)2(H2O)2]SO4.7H2O, (C) [Y(Erx)2(H2O)Cl].9H2O, (D) [ZrO(Erx)2(H2O)]. 6H2O, (E) [Pd(Erx)2(H2O)2].4H2O and (F) [Ce(Erx)2(H2O)2]SO4.4H2O.

Table 4.1H NMR values (ppm) and tentative assignments for (A) enrofloxacin, (B) [Ti(Erx)2(H2O)2]SO4.7H2O, (C) [Y(Erx)2(H2O)Cl]. 9H2O, (D)[ZrO(Erx)2(H2O)].6H2O, (E) [Pd(Erx)2(H2O)2].4H2O and (F) [Ce(Erx)2(H2O)2]SO4.4H2O

The TGA curve of enrofloxacin compound with molecular formula (C19H22N3O3F) shows one stage of decomposition within the temperature range 22−700 °C with a mass lose 100% corresponds to loss of 9C2H2+CO2+HF+NH3+N2O.

The TGA curve of [Ti(Erx)2(H2O)2]SO4.7H2O with the general formula (TiC38H60N6O19F2S) displays an initial mass loss in the temperature range 42−235 °C, corresponding to the decomposition of the complex to anhydrous complex by the loss of seven uncoordinated water molecules. The low value of temperature of this step may indicate that these water molecules undergoes less H-bonding. This is followed by another mass loss 71.46% (calculated mass loss=71.29%) in the temperature range 236−635 °C with three maxima at 321, 400 and 565 °C corresponding to the decomposition of the organic part with a final titanium sulphate residue and a total mass loss 83.71% (calc.= 83.62%).

Fig. 3.1H NMR spectra for (A) enrofloxacin; (B) [Ti(Erx)2 (H2O)2]SO4.7H2O, (C) [Y(Erx)2(H2O)Cl].9H2O, (D) [ZrO(Erx)2(H2O)]. 6H2O, (E) [Pd(Erx)2(H2O)2].4H2O and (F) [Ce(Erx)2(H2O)2]SO4. 4H2O in DMSO, δTM.

The thermal decomposition of [Y(Erx)2(H2O)Cl].9H2O, [Pd(Erx)2(H2O)2].4H2O and [Ce(Erx)2(H2O)2]SO4.4H2O complexes with the chemical formula (YC38H62N6O16F2Cl), (PdC38H54N6O12F2) and (CeC38H54N6O16F2S) takes place in two steps as indicated by DTG peaks around 50−170 °C and 170−650 °C corresponding to the mass loss of lattic water molecules and two coordinated water molecules with enrofloxacin, respectively. The nature of proposed chemical change with the temperature range and the percentage of yttrium oxide, palladium oxide or cerium sulphate obtained as a final residue are given in Table 5.

The TGA and DTG curves of [ZrO(Erx)2(H2O)].6H2O are shown in Fig. 4 and Table 5. The TGA shows three stages of decomposition within temperature range 25−800 °C. The first step of decomposition in the temperature range 50−120 °C shows that a mass loss 5.63% (calc.=5.68%) with a maximum temperature 81°C corresponds to loss of 3H2O. The second step occurs within temperature range 120−208 °C with mass loss 5.65% which could be attributed to the liberation of the residue lattic water molecules. The thrid step is reasonably accounted for the organic enrofloxacin ligand and the one coordinated water with estimated mass loss 74.63%. The total mass loss 85.91% (calc.= 85.79%) up to 750 °C is in agreement with the formation ZrO2+C as a final residue.

According to these conculsions, the decomposition mechansims proposed for HErx and thier complexes are summarized as follows:

Fig. 4.TGA/DTG plot for the thermal behavior of (A) enrofloxacin, (B) [Ti(Erx)2(H2O)2]SO4.7H2O, (C) [Y(Erx)2(H2O)Cl].9H2O, (D) [ZrO(Erx)2(H2O)].6H2O, (E) [Pd(Erx)2(H2O)2].4H2O and (F) [Ce(Erx)2(H2O)2]SO4.4H2O.

Table 5.Thermogravimetric data of HErx and their metal complexes

The proposed structure7,26,30 formula on the basis of the results discussed in this paper located in Formula (I):

Formula (I).The coordination mode of Ti(IV), Y(IV), Zr(IV), Pd(II) and Ce(IV) with enrofloxacin.

Biological Activities Test

The efficiencies of the ligand, complexes and the metal salts have been tested against three Gram (+ve), S. aureus K1, B. subtilis K22, Br. otitidis K76 and three Gram (−ve), E. coli K32, P. aeruginosa SW1 and K. oxytoca K42, microorganisms. The ligand and all their complexes have inhibitory action against all three Gram (−ve) and two Gram (+ve), B. subtilis K22, Br. otitidis K76 (Table 6). All the metal complexes exhibits higher inhibition against all microorganisms tested except S. aureus K1 compared to free enrofloxacin. The biological activity of many fluoroquinolone increased after the coordination with metal12,14,22,31 and the rate of antimicrobial activity of the metal complexes depending on the following five principal factors,42-45 (i) the chelate effect, (ii) the nature of coordinated ligands, (iii) the total charge of the complex, (iv) the nature of the ion neutralizing the ionic complex, (v) the nuclearity of the metal center in the complex. According to the overtone concept of all permeability, the lipid membrane that surrounds the cell favors the passage of only lipid soluble materials in which liposolubility is an important factor that controls the antimicrobial activity. On chelation the polarity of the metal ion will be reduced to a greater extent due to overlap of ligand orbital and partial sharing of the positive charge of the metal ion with donor groups. Further it increases the delocalization of π-electrons over the whole chelate ring and enhances the lipophilicity of the complexes.46 This increased lipophilicity enhances the penetration of the complexes into the lipid membranes and blocks the metal binding sites in enzymes of microorganisms. The antimicrobial activity of Ti(SO4)2, PdCl2, YCl3, ZrOCl2.8H2O and Ce(SO4)2 have also been investigated. It has been found that it did not have major exhibition of antibacterial activity with some bacteria compared with free enrofloxacin and metal complexes, where as, it did not have any antibacterial activity exhibition to other bacteria compared with metal complexes and free enrofloxacin at the concentration used to assay the activity of the complexes in this work.

 

CONCLUSION

The five new metal complexes of enrofloxacin with Ti(IV), Y(III), Zr(IV), Pd(II) and Ce(IV) complexes were prepared and isolated as solids and formulated as [Ti(Erx)2(H2O)2]SO4.7H2O, [Y(Erx)2(H2O)Cl].9H2O, [ZrO(Erx)2(H2O)]. 6H2O, [Pd(Erx)2(H2O)2].4H2O and [Ce(Erx)2(H2O)2]SO4. 4H2O. The five complexes have been characterized using melting point, molar conductance, magnetic properties, elemental analysis, IR, UV-Vis., 1H NMR and thermal analyses. From these techniques, it may observed that the carbonyl and carboxylic groups in all complexes shifted to lower values in IR spectra support the chelation effect via carboxylic oxygen and ketone oxygen and also the nondetectable of signals for carboxylic acid proton in the 1H NMR spectra of four complexes support the chelation of carboxylic oxygen. The complexes generated are effective antibacterial agents compared with free enrofloxacin against most of used bacterial strain (Table 6). This study displays very interesting group of potential antibacterial agents, which may further expand their uses as potential broad spectrum therapeutic antibacterial drugs and pave the way to prepared useful compounds with specific biomedical applications.

Table 6.ND: non-detectable. i.e., the inhibition zones exceeds the plate diameter. (−): no activity observed aginst microbial bacteria species. Statistical significance PNS P not significant, P<0.05; P+1 P significant, P > 0.05; P+2 P highly significant, P > 0.01; P+3 P very highly significant, P > 0.001; student’st-test (Paired).