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Synthesis, Characterization, and Spectroscopic Studies for New Cu(II), Co(II), Zn(II), Fe(III) And Zr (II) Complexes of Oxytetracycline Antibiotic, In vitro Antimicrobial Assessment Studies

Ragab R Amin1*, Mohammed Abo-Elkassem2 and Alaa Anwar3

1Basic Science Department, Faculty of Engineering, Nahda University, Egypt

2Department of Chemistry, Faculty of Science, Beni Sueff University, Egypt

3Faculty of Pharmacy, Nahda University, Egypt

*Corresponding Author:
Ragab R Amin
Head of Basic Science Department Faculty of Engineering, Nahda University, Egypt
E-mail: [email protected]

Received date: 16-01-2017 Revised date: 30-01-2017 Accepted date: 05-02-2017

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The 1:1 and 2:1 M ratio metal complexes of new Cu(II), Co(II), Fe(III), Zn(II) and Zr(II) complexes of Oxytetracycline hydrochloride have been synthesized. Attempts have been made to ascertain their probable structures based on elemental analysis, spectra (IR, Electronic) and NMR studies. The ligand form a complex in a molar ratio 1:1 having an empirical formula (MLSO4.nH2O) and 2:1 molar ratio having an empirical formula (M2LSO4.nH2O) where M=Cu(II), Co(II), Fe(III), Zn(II) and Zr(II), L=Oxytetracycline and n=2-6. The infrared spectral data and NMR studies were support the postulate that oxytetracycline reacts with metal ions as an ionic bidentate ligand through its carboxylate oxygen and the amide carbonyl oxygen. The antibacterial evaluation of the Oxytetracycline drug and their complexes were also performed against some gram positive and negative bacteria as well as fungi.


Spectroscopic studies, Oxytetracycline, Antibacterial evaluation, Negative bacteria


Metals and metal complexes have played a key role in the development of modern chemotherapy [1,2]. For example, complexation of non-steroidal anti-inflammatory drugs to copper overcomes some of the gastric side effects of these drugs [37]. A number of drugs and potential pharmaceutical agents also contain metal-binding or metal-recognition sites, which can bind or interact with metal ions and potentially influence their bioactivities and might also cause damages on their target biomolecules. Numerous examples of these metallodrugs and metallopharmaceuticals and their actions can be found in the literature, for instance: (a) several anti-inflammatory drugs, such as aspirin and its metabolite salicylglycine [811], suprofen [12], and paracetamol [13] are known to bind metal ions and affect their antioxidant and anti- inflammatory activities; (b) the potent histamine-H2-receptor antagonist cimetidine [14] can form complexes with Cu2+ and Fe3+, and the histidine blocker antiulcer drug famotidine can also form stable complex with Cu2+ [15,16] (c) the anthelmintic and fungi static agent thiabendazole, which is used for the treatment of several parasitic diseases, forms a Co2+ complex of 1:2 metal to drug ratio [17] and (d) the Ru2+ complex of the anti-malaria agent chloroquine exhibits an activity two to five times higher than the parent drug against drug resistant strains of Plasmodium falciparum [18]. However, it is known that some drugs act via chelation or by inhibiting metalloenzymes but most of the drugs act as potential ligands, a lot of studies are being carried out to ascertain how metal binding influences the activities of the drugs [19]. Metal complexes are gaining increasing importance in the design of drugs on coordination with a metal. The therapeutic importance of oxytetracycline drug was behind the development of numerous methods for its determination.

The different method techniques adapted to the analysis of oxytetracycline drug have been reported [20-33]. Literature survey fell to reveal any previous work or literature regarding the complexation of oxytetracycline drug with some transition metals [34,35]. An attempt of synthesizing, characterization, and biological screening of oxytetracycline drug ligand and their metal complexes of Calcium and Magnesium have been successfully achieved. The characteristic structure of the molecule is apparent in the existence of the carboxylic and carbonyl electron-rich ligand. However, literature survey has revealed that no attempt has been made to study the complexes of some alkali earth metal ions with the above-mentioned drug ligand compounds. It is a thought of interest to study the synthesis and characterization and biological screening of metal complexes of new Cu(II), Co(II), Fe(III), Zn(II) and Zr(II) complexes of oxytetracycline drug. Drug compounds are biologically active, these compounds have become of interest to be studied biologically, and compared their activities against four species of bacteria (Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa), and two fungal species (Aspergillus flavus and Candida albicans).

Materials and Methods

The chelating agent oxytetracycline complexes was prepared by mixing ethanolic solution of oxytetracycline drug (1 gm) and Metal salts (0.5 gm) in the same solvent. the reaction mixture was boiled under reflux in water bath for 3 hours then left overnight until precipitated yields 70%. Identification of New complexes was carried out by elemental analysis and IR Spectral.

The complexes were prepared by adding EtOH solutions of equimolar amounts of oxytetracycline and metal (II) salts. The mixture was heated on a water bath for 3 hrs. And allowed to evaporate slowly prior to filtration. The precipitates were washed with EtOH and left overnight until dried yield 70% to 80%.

Results and Discussion

Transition elements were found to form stable complexes with many ligands containing heteroatoms. there is preference for amines, halogens, CN-, tertiary phosphrines and sulfides.

Nichel (II) is one of the transition elements was found to form complex of square shape with the general formula MLX2 where L is neutral ligand and X Non-Negative Ion [9].

Nickel(II) was found to form stable complexes with many drugs acetylsalicylic acid [10], fluoxetine [11], vitamin C [12], and ephedrine [13]. Theoretically and from what was mentioned above Oxytetracycline could form chelate, through its nitrogen and oxygen atoms, with Nickel(II). Addition of NiSO4 to oxytetracycline produced black complex that is soluble in Dimethyl sulfoxide (DMSO).

Solution Studies

Effect of pH

The influence pH was studied over the range (1-10) on the absorbance of complex at 354.3 nm, the results were evaluated as shown in Figure 1. The shape of the absorption spectrum, the position of the absorption maximum and the apparent molar absorptivity of oxytetracycline-Nickel (II) complex do vary with pH, where the maximum absorbance obtained at pH [4], but a neutral and basic media the oxytetracycline-Nickel(II) complex was precipitate.


Figure 1. Effect of pH on the absorbance of oxytetracycline-Nickel(II) complex.

Effect of reaction time

The stability of the absorbance with the time was studied from 1-60 min. Figure 2 shows the relation between absorbance and the time, where the maximum absorbance was reached at the 6 and 7 min. after the addition of NiSO4 to oxytetracycline, the absorbance after this optimal time decreased at 8 min. decreased and then the absorbance was almost stable till 60 min.


Figure 2. Effect of time on the absorbance of oxytetracycline-Ni(II) complex.

Effect of temperature

The effect of temperature was studied in the range of (5°C to 60°C) on the produce of complex of oxytetracycline with Ni (II) as shown in Figure 3, the absorbance was reduced as the temperature was increased.


Figure 3. Effect of temperature on the absorbance of oxytetracycline-Ni(II) complex.

Effect of volume of Ni (II) Salt

The volume of Ni(II) has a great effect on the formation of oxytetracycline-Ni(II) complex. Figure 4 shows that as the volume of Ni(II) increase the absorbance slightly decrease.


Figure 4. Effect of volume of 1 × 10-4 M NiSO4 on the absorbance.

Solid Studies

Elemental analysis

The elemental analysis is applied for oxytetracycline complexes to suggest the structure of oxytetracycline complexes, carbon and hydrogen percentage shown in Table 1. The proposal structure for oxytetracycline complexes shown in Schemes 1-5.


Scheme 1. Oxytetracycline antibiotic structure.


Scheme 2. The Proposal structure of oxytetracycline-Nickel(II) (1:1) complex.


Scheme 3. The proposal structure of oxytetracycline-Nickel(II) (2:1) complex.


Scheme 4. The proposal structure of oxytetracycline-Cupper(II) (1:1) complex.


Scheme 5. The proposal structure of oxytetracycline-Cupper(II) (2:1) complex.

Color Oxy.Complexes formula .Wt(g/mol) Elemental analysis %
Found Calculated
H % C % H % C %
Black crystalline powder {Ni(L)SO4}. H2O
5.29 38.26 4.24 38.31
Black crystalline powder Ni2(L)(SO4)2
5.29 32.92 3.12 32.68
Black Cu(L)SO4}.5H2O }
3.95 37.32 4.4 38.04
Black {Cu2(L)(SO4)2}.6 H2O
4.67 28.22 4.09 28.47
Brown near to be Orange Zr(L)(Cl)}. H2O }
3.86 41.62 4.2 41.14
Black Fe(L) (H2O)
4.89 46.02 4.77 46.28
Black {Fe2(L)(Cl3)} (2H20)
4.27 34.52 3.9 35.2

Table 1. Elemental analysis of oxytetracycline and their metal complexes.

IR Spectral Studies

The infrared absorption bands were one of the important tools of the analyses used for determining the mode of chelation. Oxytetracycline HCl behaves as an ionic mono-dentate molecule and was coordinated to the metals through its amide carbonyl oxygen. Therefore, in these complexes one metal ion was coordinated to one molecule of the Oxytetracycline. In comparison with the published spectra [14-16] of the free Oxytetracycline spectra, bands found are:

• The band at 3000 cm-1 to 3700 cm-1 in all complexes was broad. This was due to the presence of the hydroxyl group. The ligand band at 3080 cm-1 assigned to the secondary amide NH2 [17] was either absent or very weak in all complexes, indicating the interference of the OH and NH2 bands in this region (3000 cm-1 to 3700 cm-1).

• The resolved bands at 3020 cm-1 and 3000 cm-1 in the free Ligand and copper complex due to the aromatic CH.

• The next diagnostic band in the free ligand was that of the amide carbonyl group which appears at 1583.27 cm-1. This band C=O is slightly positively shifted in the spectra of the complexes due to the coordination between amide and carbonyl group (tautomeric), indicating the involvement of the C=O of the amide in the chelating process [18].

• The appearance of an acetate bands at 676 cm-1 to 640 cm-1 and 601 cm-1 to 525 cm-1 can be attributed to the carbonyl, these bands support the chelation through the O atoms [19] (Table 2).

  Assignments The compounds
L(Ligand) Cu (1:1) Cu (2:1) Ni (1:1) Ni (2:1) Fe (1:1) Fe (2:1)
υ(OH) 3382.5 3418.2 3424.9 3381.5 3410.4 3411.4 3418.2
υ(NH2) 3080 3100 3100 3250 3250 3220 3220
CH-aliph 2926.4 2362.3 2337.3 2362.3   2928.3  
υ (C=O) 1583.2 1559.1 1580 1600 1560 1623.7 1580
C=N 1622.8 1621.8 1621.8 1636.3 1639.2 1580 1624.7

Table 2. Significant IR spectral bands (cm-¹) of the ligand of oxytetracycline HCl and their metal complexes.

Biological Screening

The oxytetracycline HCl ligand and their metal complexes were tested for their antimicrobial activity against some species of bacteria (S. aureus, B. subtilis, E. coli, P. aeruginosa, N. gonorrhoeae and S. faecalis) using modified Kirby-Bauer disc diffusion method (20) Briefly, 100 ml of the test bacteria/Fungi were grown in 10 ml of fresh media until they reached account of approximately 108 cells/ml for bacteria or 105 cells/ml for fungi [21]. 100 ml of microbial suspension was spread onto agar plates corresponding to the broth in which they were maintained.

• Isolated colonies of each organism that might be playing pathogenic role should be selected from primary agar plates and tested for susceptibility by disc diffusion method.

• Of the many media, available, NCCLS recommends Mueller- Hinton agar due to: it results in good batch-to-batch reproducibility.

• Disc diffusion method for filamentous fungi tested by using approved standard method (M38-A) developed by the (NCCLS, 2002) for evaluating the susceptibilities of filamentous fungi to antifungal agents.

• Disc diffusion method for yeasts developed by using approved standard method (M44-P) by the (NCCLS, 2003).

• Plates inoculated with filamentous fungi as Aspergillus flavus at 25°C for 48 hours Gram (+) bacteria as Staphylococcus aureus, Bacillus subtilis: Gram(-) bacteria as Escherichia coli, Pseudomonas aeruginosa they were incubated at 35°C to 37°C for 24-48 hours and yeast as Candida albicans incubated at 30°C for 24-48 hours and then the diameters of the inhibition zones were measured in millimeters.

• Standard discs of Ampicillin (Antibacterial agent), Amphotericin B (Antifungal agent) served as positive controls for antimicrobial activity but filter discs impregnated with 10 μl of solvent (distilled water, chloroform, DMSO) were used as negative control.

• The agar used is Muller-Hinton agar that is rigorously tested for composition and pH. Further the depth of the agar in the plate is a factor to be considered in the disc diffusion method. this method is well documented and standard zones of inhibition have been determined for susceptible and resistant values.

• Blank paper disks (Schleicher & Schuell, Spain) with a diameter of 8 mm were impregnated 10 μl of tested concentration of the stock solutions.

• When a filter paper disc impregnated with a tested chemical is placed on agar the chemical will diffuse from the disc into the agar. This diffusion will place the chemical in the agar only around the disc. The solubility of the chemical and its molecular size will determine the size of the area of chemical infiltration around the disc. If an organism is placed on the agar it will not grow in the area around the disc if it is susceptible to the chemical. This area of no growth around the disc is known as a “Zone of inhibition “or “Clear Zone”.

• For the disc diffusion, the zone diameters were measured with slipping calipers of the National Committee for Clinical Laboratory Standards (NCCLS, 1993).

• Agar-based methods such as E-test and disk diffusion can be good alternatives because they are simpler and faster than broth-based methods (Schemes 2-5) [24].


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