Synthesis, Characterization and Biological Evaluation of Ciprofloxacin N-N Donor Metal Complex

 

Prafulla M Sabale*, Dhiraj S Bhagwat, Karan Parwe

Department of Pharmaceutical Sciences, RashtrasantTukadoji Maharaj Nagpur University, Nagpur-440033 M.S.

*CorrespondingAuthorE-mail:prafullasable@yahoo.com

The development of metal based drugs with promising pharmacological application and may offer unique therapeutic opportunities. The advances in inorganic chemistry provide better opportunities to use metal complexes as therapeutic agents as the mode of action of metal complexes on living organism is differing from non metals. The lipophilicity of the drug was found to increase through the formation of chelates by which drug action is significantly increased due to the effective permeability of the drug into the site of action. The fluoroquinolones are synthetic antibiotics, active against aerobic Gram (-ve) and Gram (+ve) microorganisms. Fluoroquinolones possess 3-carboxylate group and 4-keto group which are essential for formation of metal complex. Fe(III) complex of  ciprofloxacin in presence of neutral bidentate ligand (N-N) donors 1,10-Phenanthroline had been synthesized. The structure of metal complex was confirmed by physiochemical parameters and spectroscopy techniques such as Quantitative UV, Mass Spectrometry, FT-IR spectroscopy and ¹H NMR spectroscopy. Through UV studies the log ε value and molar absorptivity (ε) were compared indicating the attachment of only one mole of ciprofloxacin and 1,10-phenanthroline donor to the central Fe(III) metal ion. The stability study of complex was evaluated as per ICH guidelines and the complex was found to be a stable. Cipofloxacin complex was evaluated for its antibacterial activity against two different microorganisms (Escherichia coli and Staphylococcus aureus). Antibacterial activity of ciprofloxacin complex showed better activity as compared to ciprofloxacin alone.

 

 

INTRODUCTION:

Metal complexes are known as co-ordination compounds, consisting of a central atom or ion (metal) bonded with anions (ligands). The environment around the metal center, co-ordination geometry, number of co-ordinated ligands and their donor group is the key factor for metalloprotein to carry out a specific physiological function.

 

Metal ions can interact with many different kinds of biomolecules including DNA, RNA, proteins, receptors, and lipids, rendering their unique and specific bioactivities.The lipophilicity of the drug is increased through the formation of chelate and drug action is significantly increased due to effective permeability of the drug into the site of action. Interaction of various metal ions with antibiotics may enhance their antimicrobial activity as compared to that of free ligands^1,2.Metal ions play a key role in the action of synthetic and natural antibiotics and improve specific interactions of these antibiotics with enzyme and biomolecules³.The presence of metal ions results in a higher uptake of quinolones by bacterial cells compared to that of the drug alone⁴. Bidentate Nitrogen donors like 1,10-phenanthroline forms strong complex with most metal ions. In the presence of ligand such as 1,10-phenanthroline, chelation was through the 3-carboxylate and the 4-oxo group⁵.

 

Fluoroquinolones are important group of synthetic antibacterial compounds having a fluorine atom at position 6 and a piperazine ring at position 7 of quinolone-3-carboxylic acid⁶. Fluoroquinolone, such as cipofloxacin,sparfloxacin, ofloxacin, levofloxacin, pefloxacin, norfloxacin and gatifloxacin possesses broad spectrum antibiotic activity and capable of forming a well-defined metal-fluoroquinolone complex due to the presence of vicinal oxo and carboxyl groups. The complex has been prepared between a nitrogen donor atom, quinolone moiety and metal salt. Quinoline moiety is used for preparing complex with known drugs and extension of the work can be done by changing metal having its physiological importance in body which may be any inorganic or organic acid salt of zinc, manganese, magnesium, copper, ferrous and bismuth etc⁷.

 

Our research group is working on synthesis, characterizations, analytical method development and validation of metalloantibiotics. We have synthesized Fe (III) cipofloxacin complex with N-N donors 1,10-phenanthroline and characterized on the basis of spectral interpretations. Ciprofloxacin complex was also evaluated for antimicrobial activity which exhibited better activity against E. Coli and S. Aureus as compared to parent ciprofloxacin.

 

MATERIALS AND METHODS:

Instrumentation:

Melting points (m.p) were determined using a VEEGO make silicon oil bath-type melting point apparatus and are   uncorrected. Thin layer chromatography (TLC) was performed on silica gel plates, visualizing with ultraviolet light and/or iodine vapors. Ultraviolet spectra were obtained on Shimadzu 1700 UV-Visible spectrophotometer using methanol as a solvent. Karl-Fischer titrimeter (Aqua Cal) was used for the determination of water contents. The reagent was calibrated using standard sodium tartrate dihydrate. IR spectra (wave numbers in cm-1) were recorded on a BRUKER ALPHA FT-IR spectrophotometer using potassium bromide discs.¹H NMR spectra were recorded on BRUKER AVANCE II 500 MHz   instrument in DMSO using TMS as an internal standard. Chemical shift values are mentioned in δ, ppm, where s, d, t, m and bs designate singlet, doublet, triplet, multiplet and broad singlet, respectively.

 

Chemicals and reagents:

All reagents used were of analytical reagents grade, obtained from s. d. Fine chemicals and Loba Chemicals and all solvents were purified by general laboratory techniques before use. All moisture-free operations were performed in oven dried glassware.

 

Scheme of Synthesis:

Fig. 1: Synthetic scheme

 

Fe(III)-Ciprofloxacin-1,10-Phenanthroline complex:

Ciprofloxacin (150 mg) drug was dissolved in sufficient quantity of methanol in presence of sodium methoxide (150 mg) and stirred for 30 minutes. A methanolic solution of Ferric chloride (150 mg) (dry) was added to methanolic solution of 1,10-phenanthroline (Phen) (150 mg) followed by addition of a previously prepared solution of ciprofloxacin in methanol. The pH was adjusted to 6.2-6.7 using dilute solution of sodium methoxide. The resulting solution was refluxed for 5 hours on a steam bath followed by concentrating it to half of its volume. The solution was left for slow evaporation. The progress of the reaction was monitored by TLC in solvent Chloroform:Methanol(7:3) with R_f 0.17. A reddish brown crystalline product was collected after 2 days and washed with ether/hexane and dried in vacuum desiccators, the % yield was obtained 62%. The melting point of the complex was found to be 199-203 °C. The moisture content was determined by KFR titrimetry, showed 0.31% water in the metal complex.:

 

RESULTS AND DISCUSSION:

Characterization of the synthesized complex

Quantitative UV (Molar absorptivity) Spectrometric Studies.:

The molar absorptivity (ε) of a molecule remains constant under identical conditions and this parameter can be used to find out the number of moles of that compound in a complex. In order to find out the number of moles of ciprofloxacin and 1,10-phenanthroline attached to the central Fe(III) metal atom in the structure of synthesized compound, quantitative UV studies were preformed. (Fig.2) The complex was dissolved in methanol. The solutions were diluted to get the absorbance in the range of 0.2-0.8. The molar absorptivity (ε) and log molar absorptivity (log ε) values obtained for the complex were compared with the molar absorptivity and log ε values of ciprofloxacin to get the mole ratio of ciprofloxacin: Fe(III)(Cipro). The number of moles of 1,10-phenanthroline attached to the central  metal Fe(III) were also determined by comparing the molar absorptivity (ε) and log ε values of complex with 1,10-phenanthroline.(Table-1)

Table-1 Molar absorptivity of ciprofloxacin and its metal complex

Compounds	Molar absorptivity (ε)	Log ε
Ciprofloxacin	40897.98	4.61
1,10-Phenanthroline	14598.56	4.16
Metal Complex	26419.26	4.42

 

Fig.2:Overlaid UVspectrum of Ciprofloxacin, and metal complex.

 

FT-IR spectroscopic studies:

The IR spectra of the synthesized complex was [Fe(III)(Cipro)(Phen)Cl] compared with that of the IR spectra of ciprofloxacin (Fig.3). There are two very strong absorption peaks in the spectra of the original drug (ciprofloxacin) of 1716 cm^-1 and 1621 cm^-1, assigned to ν_COOH and ν_C=O stretching vibrations respectively. On comparison it was observed that the band in the region 1710-1725 cm^-1 completely disappeared in the spectra of complex. In spectra of complex, the band of carboxylic group have been replaced by 1640 cm^-1  and 1262 cm^-1, and the Δ value fall at 200 cm^-1 which was greater than 257 cm^-1. In the spectra of complex, band observed at 1620 cm^-1 for 4-oxo group. There was no shifting in wave number compared to the spectra of parent drug, This complex was further confirmed by mass spectroscopy. The result showed that the ciprofloxacin was acted as bidentate ligands wherein the carboxylates was provided an ionic bond while the 4-oxo group was afforded a co-ordinate bond to metal ion.

 

 

Fig.No.3: Overlaid FT-IR spectrum of Ciprofloxacin and its complex

IR (KBr cm^-1 ):  1640 (C=O), 1262= COO_sy, 1141= C-F, 1260 = C-N.

¹H-NMR Spectroscopy:

               

Fig.4: ¹H-NMR spectrum of complex

NMR (δ ppm): 0.50-0.26 [d, (CH)], 1.35 [s, (CH)], 2.0 [s, (CH)], 2.50-2.78 [d,(CH)],  2.47 [s, (CH)], 5.93[s, (CH)₃],

7.0 [s(CH)], 7.12-7.35 [d, (CH)₂], 7.96 [s,(CH)₂],  8.0  [s, (CH)], 8.81 [s, (CH)₃], 11.0[s, (CH)₃]

 

Microbiological Studies:

The antibacterial evaluation of the synthesized complex has been carried out against Gram (+ve) S.aureus and Gram (-ve) E.coli microorganisms by cup plate method. The result represented that the synthesized Fe(III)(cipro)(phen)Cl complex showed better antibacterial activity against E. coli and S. aureus s compared to parent drug  at same concentration. The comparison of antibacterial activity of ciprofloxacin and its complex was shown in Table-2 and the graphical representation for comparison of antibacterial activity of drug and its complex was showed in Fig.5.

 

 

 

Table-2 Comparison of antibacterial activity of Ciprofloxacin and its complex

E. Coli			S. Aureus
Concentration (μg/ml)	Zone of Inhibition (mm)		Concentration (μg/ml)	Zone of Inhibition (mm)
	Ciprofloxacin	Complex		Ciprofloxacin	Complex
100	22	26	100	25	27
200	28	30	200	30	33

 

 

 

Stability Studies:

According to ICH guidelines, a synthesised complex was stored at 40^○C temperature and 75 % relative humidity (RH) for a period of 1 month. Complex was evaluated for physical appearance and UV spectrometry. Evaluation parameters did not show any significant change after 1 month. Slight colour change was observed in Fe(III)(cipro)(phen)Cl complex.(Table-3)

 

Fig: 5 Comparison of activity of ciprofloxacin and its complex

 

Table-3 Accelerated Stability Study of metal complex at 40°C temperature and 75% RH for 1 month

Compounds	Parameters
	PhysicalappearanceColor	UV λmax (CH3OH)
Fe(III)(cipro)(phen)Cl	Slight change	No change

 

CONCLUSION:

The synthesis and the characterization of Fe(III)(cipro)(phen)Cl was carried out   in presence of nitrogen-donor heterocyclic ligand 1,10-Phenanthroline. Physicochemical properties and spectroscopic studies of the complex confirms the ciprofloxacin ligand was bound to Fe(III) via the pyridone oxygen and one carboxylate oxygen. The antimicrobial activity of the complex has been tested on two different microorganisms and the results have showed better antimicrobial activity in comparison to the ciprofloxacin as parent drug.

 

AKNOWLEDGEMENT:

We are thankful to the director, SAIF, Punjab University, Chandigarh and Oxygen healthcare private ltd. Ahmedabad for providing spectroscopic analysis of the compounds. The authors are thankful to All India Council for Technical Education, New Delhi for the financial assistance in the form of fellowship. We would like to thank University Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur for providing necessary infrastructure and facility.

 

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Received on 03.03.2018         Modified on 20.03.2018

Accepted on 19.04.2018         © AJRC All right reserved