synthesis, characterisation, antimicrobial and

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Keywords: fluoroquinolone, metal complexes, norfloxacin, ofloxacin. 1. .... 400mg, all per 70kg body weight, twice daily for seven days) [5]. The drugs.
Centrepoint (Science Edition).(2009) Vol 16.. 37 - 56

SYNTHESIS, CHARACTERISATION, ANTIMICROBIAL AND TOXICOLOGICAL STUDIES OF SOME METAL COMPLEXES OF NORFLOXACIN AND OFLOXACIN 1

OBALEYE, J. A., 2AKINREMI, C. A., 3BALOGUN, E. A., 3 ADEBAYO, J. O. and 4OMOTOWA, B.

1. Department of Chemistry, University of Ilorin, Ilorin, Kwara State, Nigeria. 2. Department of Chemistry, University of Agriculture, Abeokuta, Ogun State, Nigeria. 3. Department of Biochemistry, University of Ilorin, Kwara state, Nigeria 4. Fluorine Products Division, International Isotopes Inc., Idaho Falls, USA

Abstract The synthesis of some copper(II), cobalt(II), nickel(II) and iron(III) complexes of norfloxacin (Nor) and ofloxacin (Ofl) are reported here. Their structures were elucidated by elemental analysis, ultraviolet and infrared spectroscopy. It was observed from the study that norfloxacin and ofloxacin molecule coordinated to metals through the two oxygen of the 3- carboxylate and 4-keto groups. Antimicrobial and toxicological studies carried out on some of the products showed a level of antimicrobial activity and toxicity comparable with their respective parent drug. Keywords: fluoroquinolone, metal complexes, norfloxacin, ofloxacin 1. Introduction Many drugs possessed modified pharmacological and toxicological properties when administered in the form of metallic complexes. Complexes of fluoroquinolones had been synthesized and extensive works carried out in elucidating their structure. Depending on the medium of preparation of the complexes, different products can be obtained [1-4]. Fluoroquinolones have a broad spectrum of antimicrobial activity, bioavailability, penetration into tissues, long serum half-life and safety which have made them very attractive agents for treating numerous infectious diseases [5]. The site of action of fluoroquinolones has been pinpointed to a subunit of that remarkable enzyme, DNA gyrase which unwinds the super coiled DNA helix prior to replication and transcription. In this study, some copper(II), cobalt(II), nickel(II) and iron(III) complexes of ofloxacin and norfloxacin were synthesized and the results of the microanalysis were discussed. The antimicrobial and toxicological properties of some of the complexes were investigated. 37

Obaleye J.A. et al O F

F

O N

N CH3N

O

OH

O

N HN

CH3

OH O

N C2H5

Norfloxacin

Ofloxacin

Fig. 1: .The structures of Ofloxacin and Norfloxacin Fluoroquinolone Antibiotics 2 Experimental 2.1 Materials and methods Norfloxacin and Ofloxacin were purchased from Sigma-Aldrich Chemie, Germany. All reagents and solvents were reagent grade. The pH was measured on a Crison model pH meter. Melting points and decomposition temperatures were determined using the Gallenkamp melting point apparatus in open capillary tubes. Elemental analysis (for C, H, and N) was carried out at the laboratory of Desert Analytics, Tucson, Arizona, USA. The metal concentration was determined on an alpha 4 atomic absorption spectrophotometer at the central laboratory, O.A.U., Ile-Ife, Nigeria. The UVVis spectra between 190-800nm were obtained on Aquamate UV-Vis spectrometer V.4.60 at the Department of Chemistry, University of Ilorin. The sample solutions were prepared in distilled water. The I.R. spectra were recorded using KBr pellets with Buck scientific infrared spectrometer M500 at the range of 4000-600cm-1. 2.2

Synthesis of complexes Solutions of the ligands (norfloxacin and ofloxacin) were prepared in distilled water, basic medium (in NaOH or NH4OH) and acid medium (HCl). A mixture of aqueous solutions of the metal salts (CuCl2.2H2O, CoCl2.6H2O, NiCl2.6H2O and FeCl3.6H2O) with the ligands in 1:1 and 1:2 metal:ligand molar ratios were prepared and stirred with a magnetic stirrer(1-2hrs). Similar synthetic procedures had been reported elsewhere [2, 3]. The products were obtained by filtration, washed with water and dried over CaCl2 in a dessicator. 2.2.1 N1-[Co(Nor)2Cl2].5H2O. The solution of norfloxacin prepared in distilled water gave a light pink amorphous product, 21.0%. M.p. 258ºC. Found: C, 45.01; H, 5.57; N, 9.68%. Calc.(for (C32H46N6F2O11Co Cl2):C, 44.72; H, 5.39; N, 9.78, Me, 6.97%. Insoluble in H2O, MeOH and EtOH. ; UV(slightly acidified H2O) λ/nm: 281.5 and 328.0; IR(KBr)Vmax/cm-1 : 3424& 3376 (OH for water molecule) 1623 (C=O)p, 1588, (OCO)as, 1493 (C-C +C-N), 1378 (OCO)s. 2.2.2 N2-Ni(Nor)2Cl2].4H2O The solution of norfloxacin prepared in NH4OH gave a light green amorphous product, 83.7%. M.p. >400ºC. Found: C, 46.16; H, 5.14; N, 9.99%. Calc.(for C32H44N6F2O10Ni Cl2): C, 45.79; H, 5.28; N, 10.00, Me, 6.99%.

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Insoluble in H2O, MeOH and EtOH. ; UV(slightly acidified H2O) λ/nm: 276.0 and 329.5; IR(KBr)Vmax/cm-1: 3500-3300 (OH for water molecule) 1614 (C=O)p, 1580, (OCO)as, 1484 (C-C +C-N), 1392 (OCO)s. 2.2.3 N3-(H2Nor)2[NiCl4] The solution of norfloxacin prepared in HCl gave a light yellow amorphous product, 96.3%. M.p. 310-312ºC; Found: C, 46.65; H, 5.07; N, 10.08%. Calc.(for C32H38N6F2O6Ni Cl4): C, 45.69; H, 4.55; N, 9.99, Me, 6.98%. Soluble in H2O, Insoluble in MeOH and EtOH; UV(H2O) λ/nm: 276.5.and 329.5.; IR(KBr)Vmax/cm-1: 3392 (OH for water molecule), 1738,1704 (C=O)c, 1632 (C=O)p, 1470 (C-C +C-N). 2.2.4 N4-[Fe(HNor)2Cl2]Cl.9H2O The solution of norfloxacin prepared in distilled water gave a deep orange amorphous product, 69.8%. M.p. >400ºC; Found: C, 40.17; H, 5.53; N, 8.54; Me, 6.70%. Calc.(for C32H54N6F2O14Fe Cl3): C, 39.91; H, 5.65; N, 8.73, Me, 5.80%. Soluble in H2O and MeOH, Insoluble in EtOH; UV(H2O) λ/nm: 276.5 and 326.0.; IR(KBr)Vmax/cm-1 : 3500-3300 (OH for water molecule) 1738, 1684 (C=O)c, 1636 (C=O)p, 14750 (C-C +C-N). 2.2.5 N5-Fe(Nor)2Cl3.5H2O The solution of norfloxacin prepared in NH4OH gave a orange amorphous product, 56.2%. M.p. >400ºC. Found: C, 42.87; H, 4.92; N, 9.14; Me, 5.80%. Calc.(for C32H46N6F2O11Fe Cl3): C, 43.14; H, 5.20; N, 9.43, Me, 6.27 %. Soluble in H2O and MeOH, Insoluble in EtOH; UV(H2O) λ/nm: 275.5 and 329.0; IR(KBr)Vmax/cm-1: 3500-3300 (OH for water molecule) 1631 (C=O)p, 1577, (OCO)as, 1465 (C-C +C-N), 1383 (OCO)s. 2.2.6 O1-[Cu(Ofl)2(H2O)].2H2O The solution of ofloxacin prepared in NH4OH gave a green amorphous product, 88.3%. M.p. 280-281ºC. Calc.(for C36H44N6F2O11Cu): C, 51.57; H, 5.29; N, 10.02, Me, 7.58%. Soluble in MeOH, Insoluble in H2O and EtOH; UV(MeOH) λ/nm: 299.5 and 327.0; IR(KBr)Vmax/cm-1 : 3518&3401 (OH for water molecule) 1592 (C=O)p, 1524, , (OCO)as, 1446 (C-C +C-N), 1398 (OCO)s. 2.2.7 O2-(H3Ofl)[CuCl4].0.5H2O The solution of ofloxacin prepared in HCl gave a yellow amorphous product, 99.5 %. M.p. 200-202 ºC. Found: C, 37.32; H, 3.64; N, 7.27%. Calc.(for C18H23N3FO4.5Cl4Cu): C, 37.40; H, 4.01; N, 7.27, Me, 11.00 %. Soluble in H2O, Insoluble in MeOH and EtOH; UV(H2O) λ/nm: 292.5 and 325.5; IR(KBr)Vmax/cm-1 : 3400&3303 (OH for water molecule), 1705 (C=O)c, 1619 (C=O)p, 1479 (C-C +C-N). 2.2.8 O3-Cu(Ofl)2.5H2O The solution of ofloxacin prepared in distilled water gave a light green amorphous product, 88.3%. M.p. 277-278ºC. Found: C, 49.70; H4.95; N, 9.54; 39

Obaleye J.A. et al

Calc.(for C36H48N6F2O13Cu): C, 49.45; H, 55.53; N, 9.61, Me, 7.27%. Insoluble in H2O, MeOH and EtOH; UV(MeOH) λ/nm: 302.0 and 327.0; IR(KBr)Vmax/cm-1 : 3538& 3363 (OH for water molecule) 1612 (C=O)p, 1470 (C-C +C-N), 1398 (OCO)s. 2.2.9 O4-[Co(Ofl)2]. 4H2O The solution of ofloxacin prepared in NH4OH gave a peach amorphous product, 90.1 %. M.p. 270ºC. Found: C, 50.95; H, 5.31; N, 9.77%. Calc.(for C36H46N6F2O12Co): C, 50.70; H, 5.39; N, 9.86, Me, 7.03 %. Soluble in MeOH but sparingly in EtOH, Insoluble in H2O; UV(MeOH) λ/nm: 297.0 and 327.0; IR(KBr)Vmax/cm-1 : 3376 (OH for water molecule) 1609 (C=O)p, 1570, (OCO)as, 1474 (C-C +C-N), 1402 (OCO)s. 2.2.10 O5-[Ni(Ofl)2]. 4H2O The solution of ofloxacin prepared in NH4OH gave a light green amorphous product, 40.4 %. M.p. >400 ºC. Found: C, 50.60; H, 5.28; N, 9.70 %. Calc.(for C36H46N6F2O12Ni): C, 50.78; H, 5.40; N, 9.87, Me, 6.89 %. Soluble in MeOH, Insoluble in H2O and EtOH; UV(MeOH) λ/nm: 295.5 and 329.0; IR(KBr)Vmax/cm-1: 3354 (OH for water molecule) 1619 (C=O)p, 1580, (OCO)as, 1469 (C-C +C-N), 1407 (OCO)s. 2.2.11 O6-Fe(Ofl)Cl..3H2O The solution of ofloxacin prepared in distilled water gave a red crystalline product, 86.4%. M.p. 288-290ºC. Found: C43.39; H, 4.95; N, 8.37%. Calc.(for C18H25N3FO7Fe): C, 42.75; H, 4.97; N, 8.31, Me, 11.04%. Soluble in MeOH, H2O and EtOH; UV(H2O) λ/nm: 292.5 and 327.0; IR(KBr)Vmax/cm-1 : 327.0 (OH for water molecule) 1621 (C=O)p, 1466 (C-C +C-N), 1367 (OCO)s 2.2.12 O7-Fe(HOfl)(H2O)2Cl2.H2O The solution of ofloxacin prepared in NaOH gave a deep red crystalline product, 84.4 %. M.p. >400 ºC. Found: C, 40.53; H, 4.26; N, 7.80 %. Calc.(for C18H25N3FO7Cl2Fe): C, 39.95; H, 4.62; N, 7.76, Me, 10.32 %. Soluble in H2O, MeOH and EtOH; UV(H2O) λ/nm: 293.5; IR(KBr)Vmax/cm1 : 3435 (OH for water molecule), 1750, 1694 (C=O)c, 1650 (C=O)p, 1475 (C-C +C-N). 2.2.13 O8-(H3Ofl)[FeCl4]Cl.H2O The solution of ofloxacin prepared in HCl gave a light orange amorphous product, 71.3%. M.p. 258-260 ºC. Found: Me 10.4 %. Calc.(for C18H24N3FO5Cl5Fe): C, 35.17; H, 3.93; N, 6.84, Me, 9.08 %. Soluble in H2O, sparingly in MeOH, Insoluble in EtOH; UV(H2O) λ/nm: 293.5 and 326.0; IR(KBr)Vmax/cm-1 : 3397 (OH for water molecule), 1709 (C=O)c, 1621 (C=O)p, 1465 (C-C +C-N). 40

Centrepoint (Science Edition).(2009) Vol 16.. 37 - 56

2.3

Antimicrobial Studies The Minimum Inhibitory Concentration and Minimum Bactericidal Concentration were determined using method described elsewhere [2]. The microorganism isolates were obtained from the University Teaching Hospital, Ilorin. The clinical cultures of Staphylococcus. aureus, Klebsiella specie, Eschgerichia coli, Pseudomonas aeruginosa, Neissera gonorrhoea, Salmonella typhi and Shigella dysentria were used. The test organisms were individually grown overnight on nutrient agar slant at 35 oC. The suspension was then prepared by washing the growth off with the normal saline solution and further serially diluting with sterile 20mM phosphate buffer to give counts of factor of 1x 10 6. The test sample stock solution for each metal complex and ligand were prepared by placing 0.05g in an amber coloured bottle and the appropriate solvent (either sterile 0.01M HCl or distilled water) was added to get a solution of 100ug/ml. 2.4 Toxicological Studies Toxicological studies were carried out as described elsewhere [2] on some selected products using male albino rats. The test solutions were also prepared based on therapeutic dose (Ofloxacin-250mg and Norfloxacin400mg, all per 70kg body weight, twice daily for seven days) [5]. The drugs were dissolved in distilled water and administered orally (by forced feeding) to the rats in proportion to their body weight. The control groups were given distilled water only. The serum and homogenates of the heart, kidney, liver and small intestine were studied for enzyme activities. All assays were carried out at 37oC and optimal condition was employed for serum, kidney, liver, heart and small intestine using enzyme kits prepared by Randox Laboratories Ltd. 3. 3.1

Results and Discussion Syntheses and Physico-analytical data The different conditions used in the syntheses have been reported to lead to differences in bonding or complex formation of fluoroquinolone complexes [1-4,7-10]. Differences were observed in the amount of water molecules, anions present in or outside the coordination sphere, protonation of the piperazinyl or carboxylic groups etc. The ratio of metal to ligand for most of the complexes was 1:2 for single ligand metal complexes though few had a 1:1 ratio. The chloride ions from the metal salts appear in the complexes either within or outside the coordination spheres as shown by the elemental analysis and test with silver nitrate. The terminal nitrogen on the 7-piperazinyl group may be protonated where chloride ions are present to balance the charge on the complex.[1]. Cobalt and Nickel complexes of norfloxacin showed poor solubility in most solvents. The ofloxacin complexes showed the best solubility in polar solvents like water and methanol. All the ionic compounds (that is complexes with protonated fluoroquinolone) and some iron complexes were soluble in water due to their strong ionic nature.

41

Obaleye J.A. et al

3.2

Ultraviolet-visible Spectra The metal complexes of norfloxacin and ofloxacin showed bands at similar regions since they have similar chromophores. The intense UV bands at 260- 340nm were assigned to intraligand π- π* transitions. The strong absorption peak corresponds to the chromophore involving the nitrogen atom at position 1 to carboxyl group. The weak absorption peak at the long wavelength side corresponds to the chromophore involving the nitrogen of the piperazinyl group attached to the 7-carbon to the keto group [11]. No absorption bands are seen in the visible region even at high sample concentration. This is attributed to the low intensity of the d-d transitions. From past work, no d-d transitions in the visible region have been reported for norfloxacin and ciprofloxacin metal complexes. However, d-d transitions have been reported for ofloxacin metal complexes [7] and were obtained using solid-state diffuse reflectance. 3.3 Infrared Spectra 3.3.1 Norfloxacin Metal Complexes In the infrared spectrum of Norfloxacin, the ligand exists as a zwitterion since no band exists for V(C=O)c (stretching vibration frequency of carbonyl in carboxyl group) at 1720cm-1 as reported for H-Nor [6,9]. However, peaks appear for Vas(OCO) and Vs(OCO). A strong peak also appears around 1490cm-1 assigned to vibrations for C-C, and C-N, which appears at a lower frequency on complexation. The infrared spectrum of Norfloxacin showed an unexpected broad band between 3600-3000cm-1, which was assigned to absorbed moisture and not OHstr since the carboxylic group is deprotonated. The V(C=O)c vibration was absent in the IR spectra of the compounds [Co(Nor)2Cl2].5H2O-N1 [Ni(Nor)2Cl2].4H2O-N2 and Fe(Nor)2Cl3.5H2O –N5 as present in the free ligand, indicating that the carboxyl group is deprotonated and complexed to the metal. The V(C=O)p (stretching vibration frequency of pyridone group) absorption appears strong for the compounds. However, the IR spectrum of [Fe(HNor)2Cl2]Cl.3H2O-N4 shows bands at 1738.2 cm-1 and 1684 .9cm-1 which is similar to that reported for ionic norfloxacin complexes [6]. Strong bands assigned to V(C=O)c were also seen in the IR spectra of (H2Nor)2[NiCl4 ]-N3 indicating the ligand is the ligand is not directly coordinated to the metal ion. The IR spectra of N1, N2, and N5 showed absorptions for Vas(OCO) and Vs(OCO) at 1390-1410 cm-1 and 1580-1610cm-1 of resonance stabilized carboxylate anion indicating that coordination is through the carboxyl group. The bands for Vas(OCO) and Vs(OCO) present in the free ligand disappears in the IR spectra of N3 and N4. This is indicative of the ionic nature of the complexes since the carboxyl group exists as the free (-COOH) form and the ligand protonated at the 7-piperazinyl group [6]. 3.3.2 Ofloxacin Metal Complexes The infrared spectrum of ofloxacin showed bands at 1714.1cm-1 assigned to V(C=O)c and at 1626.4cm-1 assigned to V(C=O)p. This shows that 42

Centrepoint (Science Edition).(2009) Vol 16.. 37 - 56

the carboxyl group in the ligand is protonated, therefore the ligand exists in its nonionised neutral form as for ciprofloxacin hydrochloride [1]. The medium band at 3421.2 cm-1 is assigned to OH str of the carboxylic group, thus further confirming the presence of the protonated carboxyl group. The band for V(C=O)c at 1714.1cm-1 in the ligand disappeared in the IR spectra of [Cu(Ofl)2(H2O)].2H2O-O1,Co(Ofl)2].4H2O-O4, [Ni(Ofl)2].4H2O-O5, and Fe(Ofl)Cl..3H2O-O6 indicating that the carboxyl moiety is involved in complexation. The bands for V(C=O) absorption still appeared in the IR spectra of (H3Ofl)[CuCl4 ].0.5H2O-O2,. Fe(HOfl)(H2O)2Cl2.H2O-O7 and (H3Ofl)[FeCl4]Cl.H2O-O8. The presence of the V(C=O)c indicates that the carboxyl group is protonated which shows the ionic nature of the compound [6,9]. The appearance of bands for Vas(OCO) and Vs(OCO) vibrations in O1, O4,O5, O6 shows that the carboxyl group is deprotonated and involved in complexation. The peak assigned to Vas(OCO) absorption was however not seen but bands appeared at 1398cm-1 for O3 assigned to Vs(OCO). It is possible that the peaks for Vas(OCO) appeared at a higher frequency which may intercept with the peaks of V(C=O)p as in some earlier studies where the peaks at 1600cm-1 were assigned to Vas(OCO) [7]. The Vas(OCO) and Vs(OCO) absorptions were absent in compounds O2, O7 and O8 which agreed with the study on ionic quinolones [9]. In general, most of the fluoroquinolone metal complexes showed very broad bands between 3600-3000cm-1 which was assigned to V(-OH) for water molecules with hydrogen bonding. The water molecule band could be as a result of lattice or coordinated water. Scheme 1 shows the proposed form of the norfloxacin and ofloxacin ions when in coordination to the metal either as a zwitterion as in products N1, N2, N5 and O6 or as a basic anion as in products O1, O3, O4 and O5. Scheme 2 shows how they behave when the ligand exist as singly (+1) or doubly (+2) protonated cations in ionic complexes as in products N3, N4, O2, O7 and O8. The positive charge on the FQ ion is neutralized by the tetrachlorometalate anion and/or any extra chloride ion. M O F

M O

O

(-) O

F

O

(-) O

or N RN H +

N

N

R

RN

N R

Scheme 1 shows proposed mode of coordination the resonance stabilized carboxylate anion 43

Obaleye J.A. et al

O

OH

F

OH

O

F

O

OH

or N

+ RN H

N

+ RN H

R

N

N R

Scheme 2 shows structures of singly(+1) and doubly (+2) protonated cation. for the ionic fluoroquinolone complexes

3.4 Results on Biological Studies 3.4.1 Antimicrobial Studies The results of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) determinations of the fluoroquinolones and their metal complexes are presented in Tables 1 and 2. The ligands and metal complexes also showed antimicrobial activity against the tested organisms except against the molds of Penicillin and Aspergillus as has been earlier reported [2]. Some of the metal complexes showed comparable activity or greater activity against some of the microorganisms in comparison to the parent compounds. The MIC of the samples against the various isolates ranged from 50ug/ml to 600ug/ml of the antimicrobial dilutions, while that of the MBC ranged from 100ug/ml to 700ug/ml. These concentrations in comparison to reported MIC90 of the ligands are seemingly high [5, 10, 12]. Increasing the concentrations of the samples led initially to increased bactericidal effect to a certain point, after which the reverse effect occurred, that is increasing concentration leading to decreasing sensitivity. Table 1. Minimum Inhibitory Concentration and Minimum Bactericidal Concentration (ug/ml) of some norfloxacin metal complexes. Bacteria

Minimum Inhibitory concentration (ug/ml)

Minimum Bactericidal concentration(ug/ml)

Norfloxacin 200

N4 150

N5 300

Norfloxacin 300

N4 250

N5 450

Shigella spp

100

100

100

200

200

200

E.coli

500

500

500

600

600

650

Klebsiella spp

500

450

400

500

550

500

S.aureus

500

500

400

600

600

600

Pseudomonas spp.

100

500

500

200

600

200

N.gonorrhoea

200

300

300

300

400

500

S.Typhi

44

Centrepoint (Science Edition).(2009) Vol 16.. 37 - 56

Table 2 Minimum inhibitory concentration and Minimum bactericidal concentration (ug/ml) of some fluoroquinolone metal

complexes.

Minimum Inhibitory concentration (ug/ml)

Minimum Bactericidal concentration (ug/ml)

Bacteria Ofloxacin

O1

O2

O3

O4

O5

O6

O8

Ofloxacin

O1

O2

O3

O4

O5

O6

O8

S.Typhi

300

300

100

100

400

300

400

500

400

400

150

200

500

400

500

600

Shigella spp

200

300

450

200

200

100

400

350

300

500

550

300

300

200

500

450

E.coli

500

400

450

400

500

400

350

550

600

500

550

500

600

500

450

550

Klebsiella spp

300

300

400

300

400

200

500

450

400

400

500

400

500

300

500

550

S.aureus

350

300

100

300

500

400

550

500

450

400

200

400

600

500

650

600

Pseudomonas spp.

400

400

300

100

200

550

200

350

500

500

400

200

300

650

300

450

N.gonorrhoea

350

500

50

100

200

400

200

300

450

600

100

200

300

500

300

400

45

Obaleye J.A. et al

3.4.2 Toxicological Studies Figures 2-11 show the effect of norfloxacin, ofloxacin and their metal complexes on some enzymes and tissue body weight ratio in some of the rat tissues. Values with different superscripts are significantly different at P