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THERAPEUTIC APPLICATION OF HEAVY METALS NI CU AND FE WITH SCHIFF BASE DERIVED FROM SALICYLALDEHYDE


Imran Ullah1, Najeeb Ullah*1, Farhan Haider2, Sajid Iqbal3, Aqal Badshah4
1. Department of Biochemistry, Hazara University Mansehra, Khyber Pakhtunkhwa, Pakistan.
2. Department of Oral microbiology and immunology, Kyungpook National Univesrity, South Korea.
3. Department of Pharmacy, Kohat University of Science and University, kohat, Khyber Pakhtunkhwa, Pakistan.
4. Department of Microbiology, Kohat University of Science and University, kohat, Khyber Pakhtunkhwa, Pakistan.

Abstract

The synthesis and characterization of new coordination compounds of Ni (II), Cu (II) and Fe (II) with Schiff base derived from salicylaldehyde and aniline is reported. The ligand molecule appears to be bound to the transition metals through oxygen and nitrogen atoms. Their structures have been characterized by elemental analysis and bonding in these complexes is discussed in terms of IR Spectral study. The spectroscopic results obtained are in full agreement with the proposed 2:1 and 1:2 stoichiometries. The infrared spectra of complexes display the complexation behavior of the Schiff base towards Ni (II), Cu(II) and Fe (II) ions and shows that they possesses square planar and trigonalbipyramidal geometry. The metal complexes soluble in DMSO have been tested against various Gram Positive and Gram negative bacteria. The results revealed that the complexes show promising to significant activity against Gram positive as well as Gram negative bacteria. The synthesized Schiff base and complexes were also exposed to different fungi in order to establish their antifungal activity. The copper complex exhibit promising activity against all fungi, while the complexes of Ni(II) and Fe(II) shows significant activity.

Keywords: Heavy metals, Schiff base, Antibacterial activity, Antifungal activity.

 

INTRODUCTION

Metal complexes of Schiff bases have occupied a central role in the development of coordination chemistry after the work of Jorgensen and Werner 1. This situation is manifested by the huge number of publications ranging from the purely synthetic to modem physiochemical to biochemically relevant studies of these complexes. A tremendous variety of stable chemical species have been synthesized containing both transition and non-transition metals and multifarious ligand systems 2. Schiff bases and their biologically active complexes have been studied extensively over the past decade 3. A large number of Schiff bases and their complexes have been studied for their interesting and important properties. Catalytic activity in hydrogenation of olefins 4.

Transfer of an amino group 5. Photo chromic properties 6. Complexing ability towards some toxic metals 7. The high affinity for the chelation of the Schiff bases towards the transition metal ions is utilized in preparing their solid complexes. Preparative accessibility, diversity and structural variability. Schiff bases appear to be important in a number of enzymatic reactions involving interaction of an enzyme with an amino or a carbonyl group of the substrate  8. It has been found that activity of the biometals is attained through the formation of complexes with different bio-ligands and the thermodynamic and kinetic properties of the complexes govern the mode of biological action. Sometimes, the permeability, i.e. lipophilicity of drugs is increased through the formation of chelates in vivo and the drugs action is significantly increased due to much more effective penetration of the drug into the site of action. The knowledge of drug action in   vivo is extremely important in designing more potential drugs 9. Tetracycline forms an important group of antibiotics. Their activity appears to result from their ability to chelate metals. The extent of antibacterial activity parallels the ability to form stable chelates. In the study it has been shown that tetracycline and cycloserine bindings to metal ions suppress their antimicrobial activity because the associated ph changes alter the intra and inter-molecular interactions. A similar correlation has been drawn between active tetracycline and the ability to form 2:1 complexes with Cu (II), Ni (II), and Zn (II) 10.

MATERIALS AND METHODS 

Synthesis of Schiff Base Derived from Salicylaldehyde and Aniline

For the preparation of ligand 50mM (5.23ml) of salicylaldehyde was added to 50mM (4.5ml) of aniline in 2-neck flask fitted with a reflux condenser. To this mixture 50ml of methanol was added and refluxed for 3 hrs with constant stirring. The water formed during the reaction was removed azeotropically by adding 50ml of benzene and Dean Stalk funnel was fitted with reflux assembly. The reaction mixture was refluxed until all the water formed was drained out. The remaining solution was concentrated under vacuum. The yellow solid obtained was recrystallized from chloroform. The melting point was found to be 47°C. The ligand was used without further purification.

Synthesis of Ni (Ii) Complex

50ml of methanol was taken in a 2-neck flask fitted with a reflux condenser. 10mM (1.97g) of ligand and 5mM (O.64Sg) of nickel chloride were added in it. Then the reaction mixture was refluxed for 3 hrs at 120°C. The ppts formed were filtered, washed properly with chloroform and dried at room temperature and light green solid was obtained.

Synthesis of Cu (Ii) Complex

50ml of methanol was taken in a 2-neck flask fitted with a reflux condenser. 5mM (O.985g) of ligand and 5mM (1.247Sg) of copper sulfate pentahydrate were added in it. Then the reaction mixture was refluxed for 3 hrs at 120°C. The water formed during the reaction was removed azeotropically by adding 20ml of benzene in the reaction mixture through Dean Stalk funnel. The remaining solution was concentrated under vacuum and the dark green solid obtained was recrystallized from chloroform.  

Synthesis of Fe (II) Complex

20ml of methanol was taken in a 2-neck flask fitted with a reflux condenser. 5mM (0.985g) of ligand and 5mM (l.39g) of ferrous sulfate heptahydrate were added in it. The reaction mixture was refluxed for 3 hrs at 120°C. The water formed during the reaction was removed azeotropically by adding 10ml of benzene in the reaction mixture through Dean Stalk apparatus. The remaining solution was concentrated under vacuum and the blackish brown crystals obtained were recrystallized from chloroform.

Antifungal Activity

The dilution plate method 11 was used for isolation of fungi. Selected and isolated fungi were maintained on potato dextrose agar plates at 4°C for further experimental work. The antifungal activities of the ligands, mixed ligand complexes, metal nitrates, fungicides (bavistin and emcarb), and the control (dimethylsulfoxide) were screened using the plate poison technique 12. Seven day- old cultures of Aspergillus Niger, Fusariumoxysporum, and Aspergillusflavuswere used as test organisms. A stock solution of 500 r g/ml was made by dissolving 50 mg of each compound in dimethylsulphoxide (100 ml). The sterilized medium with the added stock solution was poured into 90 mm sterile petri plates and allowed to solidify. They were inoculated with a 5 mm actively growing mycelial disc and incubated at 27°C for 72 h. After 72 h of inoculation, the percent reduction in the radial growth diameter over the control was calculated. The growth was compared with dimethylsulfoxide as the control.

Antibacterial activity

The antibacterial activities were investigated using agar well diffusion method 13. The activity was performed on Staphylococcus aureusand Bacillus subtillis(as gram positive bacteria) and Pseudomonas aereuguinosa, Escherichia coli and Salmonella typhi(as gram negative bacteria). Two milligrams of the complexes were dissolved in 1 mL of DMSO. Centrifuged pellets of bacteria from a 24-h-old culture containing approximately104–106 colony forming unit (CFU) per mL was spread on the surface of Muller Hinton Agar (MHA) plates. Wells were created in medium with the help of a sterile metallic borer and nutrients agar medium were prepared by suspending nutrient agar (Merck) 20 g in 1000 mL of distilled water (pH 7.0), autoclaved and cooled down to 45 ºC. Then it was seeded with 10 mL of prepared inocula to have 106 CFU/mL. Peter plates were prepared by pouring 75 mL of seeded nutrients agar. The activity was determined by measuring the diameter of the inhibition zone (in mm). Growth inhibition was calculated with reference.

RESULT AND DISCUSSION

IR Spectral Analysis of Ligand and Complexes  

The results of IR Spectral analysis of ligand Schiff base derived from aniline and salicylaldehyde and its metal complexes are shown in table 2. The most important bands are v (M-N), v (M-O), v (C=N) and v (C-O) where M may be Ni, Cu, Fe. In the IR spectrum of ligand the broad band of OH at 3650 cm-1 is absent in the spectra of metal complexes showing complex formation. The new bands which are not present in the spectrum of Schiff base derived from salicylaldehyde and aniline appeared at 500cm-1 and 700cm-1 respectively are attributed to v (M-N) and v (M-O) vibrations. The appearance of these vibrations supports the involvement of nitrogen and oxygen atoms in the complexation with metal ions under investigation. The broad band for C-H bond lies between 3100cm-1 and 3500cm-1.The band at 1600cm-1 is assigned to v (C-O) stretching frequency which appeared at 1400cm-1 in the spectrum of the Ligand. The shifting of this band indicates the involvement of oxygen atom of hydroxyl group of salicylaldehyde in bonding with metal ions. The band between 1100cm-1 and 1400cm-1is assigned to v (C=N) which appeared at 1600cm-1 in the spectrum of Schiff base showing the coordination of metal ions with nitrogen.

Table 1: Observed infrared frequencies (cm-1) and assignment of Schiff base ligand and its complexes.

Complex v (C=N) v (M-N) v (M-O) v (C-O) v (C-H) v (OH)
Ligand 1600 1400 3650
Ni (II) Complex 1400 500 700 1610 3200
Cu (II) Complex 1100 600 700 1600 3500
Fe (II) Complex 1200 600 710 1600 3400

Solubility-Solubility of ligand and metal complexes was checked by using different solvents and is given in table1. All the solvents were used as such.

Table 2: Solubility of Schiff base ligand and metal complexes in various solvents.

Complexes Methanol Ethanol DMF DMSO Ethyl Acetate
Ligand S S S S S
Ni (II) Complex S S S S IN
Cu (II) Complex S S S S S
Fe (II) Complex S S IN S IN

IN = Insoluble S = Soluble

Antifungal Activity 

The results of the antifungal studies are given in Table 4. The results show that the mixed-ligand complexes are more toxic than their parent ligands against the same microorganisms. The increase in the antifungal activity of the mixed-ligand complexes may be due to the effect of the metal ion on the normal cell processes. A possible mode for the toxicity increase may be considered in light of Tweedy’s chelation theory 14. Chelation considerably reduced the polarity of the metal ion because of the partial sharing of its positive charge with the donor groups and the n-electron delocalization over the whole chelate ring. Such chelation could enhance the lipophilic character of the central metal atom, which subsequently favors its permeation through the lipid layer of the cell membrane. Although there is a sufficient increase in the fungicidal activity of the mixed-ligand complexes as compared to the free ligands (metal nitrate) and the control (dimethylsulfoxide), they cannot attain the effectiveness of the conventional fungicides (bavistin and emcarb).

Table 3: Antifungal Activity of Ligand and metal complexes

Compounds % Inhibition (Growth Diameter In Mm)
  A.     niger F. oxysporum A. flavus
Ni Complex 33(52) 21(41) 29(55)
Copper complex 51 (78) 43 (69) 38 (59)
Zinc Complex 20 (33) 18 (32) 17 (30)
*Bavistin 100(100) 100(100) 100(100)
*Emcarb 100(100) 100(100) 100(100)

* standards (commercial fungicides)  

Table 4: Antibacterial Activity of Ligand and metal complexes. 

MICROORGANISMS L NI COMPLEX COPPER COMPLEX ZINC COMPLEX STANDARD DRUG
Gram-negative  
 

Bacillus subtilis

 

+ + ++++ + ++++
Staphylococcus aureus

 

n.c + ++++ + ++++
Gram-negative  
Escherichia coli

 

+ + +++ ++ ++++
Salmonella typhi + n.a +++ + ++++
Pseudomonas aeruginosa n.c + +++ + ++++

L = Schiff base; ++++ = Excellent activity (100% inhibition), +++ = good activity (60-70% inhibition), ++ = significant activity (30-50% inhibition), + = negligible activity (10-20% inhibition), n.a = no activity, n.c = not checked, Size of well: 6mm (diameter).

Antibacterial activity

Almost all antibiotics have tendency to prevent the growth and multiplication of susceptible bacterial, microbial inhibition. This is not surprising as the term antibiotics brings together a diverse group of chemical compounds with little in common excepting antimicrobial activity.The biological activity of the Schiff base ligand with its transition metal complexes and Imipinem (as a standard compound) were tested against bacteria. The microorganisms used in the present research included Staphylococcus aureusand Bacillus subtillis(as gram positive bacteria) and Pseudomonas aereuguinosaand Escherichia coli (as gram negative bacteria). The diffusion agar technique was used to assess the antibacterial activity of the synthesized ligands and mixed ligand complexes 15. The data obtained specify that the ligand and its transition metal (Ni, Cu, Zn) complexes has significant to promising bactericidal activity with all Gram positive and Gram negative bacteria used table 4. Especially the copper complex of the Schiff base exhibit promising activity, this may be due to copper metal which combines with the bacterial and ultimately results in the killing of bacteria.

REFERENCES 

  • Vanden BH, Koymans L, Moereels H: P450 inhibitors of use in medical treatment: focus on mechanisms of action. PharmacolTher 1995; 7:100.
  • Asai K, Tsuchimori N, OkonogiK, Perfect JR., Gotoh O, Yoshida Y: Formation of azole-resistant Candida albicans by mutation of sterol 14-demethylase P450.Antimicrob. Agents Chemother 1999; 13:1163-1169.
  • Lewiński J, Zachara J, Justyniak L,Dranka M: Hydrogen-bond supramolecular structure of group Schiff base complexes.Coord.Chem. Rev.2005;9:1185–1199.
  • Shiotsuka M, Okaue Y, Matsumoto N. Okawa M, Isobe T: Crystal structures and single-crystal electron spin resonance spectra of p···p type molecular complexes of bis(1 methyliminomethyl-2-naphtholato) copper(II).Chem. Soc1940;26:2065–2070.
  • Rajsekhar G, Rao CP, Saarenketo P, Nattinen, K:Rissanen. Complexationbehaviour of hexadentate ligands possessing N2O4 and N2O2S2 cores: differential reactivity towards Co(II), Ni(II) and Zn(II) salts and structures of the products. NewChem 2004;8:75–84.
  • Kamenar B, Kaitner B, Stefanović A: Structures of three bis(3-ethoxy-N-R-salicylideneaminato) nickel(II) complexes (R = H, methyl, ethyl). ActaCrystallogr,2004; 35:1627–1631.
  • Blagus A, Kaitner B: Interactions between dimmers of {1,1’-[o-phenylenebis(nitrilomethylidyne)]di-2-naphtholato-κ4O,N,N’,O’}nickel(II).ActaCrystallogr,2006;17:455–458.
  • Dey M, Rao CP, Saarenketo KP, Rissanen K,Kolehmainen E, Guionneau P:Mn(IV) and Co(III)- complexes of –OH-rich ligands possessing O2N, O3N and O4N cores: syntheses, characterization and crystal structures.Polyhedron 2003;55:3515–3521.
  • Filarowski A, Koll A, Głowiak T: Structure and hydrogen bonding in ortho-hydroxyKetimines. Mol. Struct 2003;8:187–195.
  • C. Jones, T. N. Waters, B. Kaitner, B. Kamenar (1986), The crystal structure and conformation of bis (Nmethyl- 5-chlorosalicylideneaminato)nickel(II) and Bis(N-ethyl-5-chlorosalicylideneaminato)nickel (II), Croat. Chem. Acta 1996; 8:825–831.
  • Gabud S, B. Sc. Thesis, Faculty of Science, University of Zagreb, 1997.
  • Yaghi OM, Li H, Davis C, Richardson D,Groy TL:Synthetic Strategies, Structure Patterns, and Emerging Properties in the Chemistry of Modular Porous Solids. Chem. Res. 1998;31:474-480.
  • A. Shally, A. Lotzen, M. Albrect, Chem. Eur. J. 2004; 10 : 1072.
  • Rehman w. Badshah A. Baloch MK: Synthesis, spectral characterization and bio-analysis some organotin(IV) complexes. Eur J Med Chem 2008;43:2380-2385.
  • Murukan B, Mohanan K: Studies on some trivalent transition metal complexes bishydrazone. J. Saudi Chem. Soc 2006; 10: 261-270.