Download (III) COMPLEX OF 4-VINYL PYRIDINE LIGAND

CHARACTERIZATION AND BIOLOGICAL ACTIVITIES OF POLYMER
Fe (III) COMPLEX OF 4-VINYL PYRIDINE LIGAND.
Ahmed G. Ibrahim a, Mahmoud M. Elaasserb , Alaa Fahmy a, Ibrahim Osman a, Hussein H.
El-Shiekhc , Farag Abd El-Haia and Ahmed M. Salah. b
a
Department of Chemistry, Faculty of science, Al-Azhar University, Nasr City, Cairo, Egypt
E-mail: [email protected]
b
Regional Center for Mycology and Biotechnology, Al-Azhar University, Nasr City, Cairo, Egypt
E-mail: [email protected]
c Department
of Botany and Microbiology, Faculty of Science, Al-Azhar University Nasr City, Cairo, Egypt
E-mail: [email protected]
ABSTRACT
Reaction of Fe (III) with 4-vinyl pyridine in non aqueous medium led to the formation of metal complex. This complex
reacted with methyl methacrylate by using AIBN as initiator to form the polymer metal complex. This metal complex and
polymer metal complex have been characterized by elemental analyses, molar conductance, IR, 1H-NMR, Mass spectra
and thermal analyses (DTA and TGA). The molar conductance of the complex indicating that, the complex is not
electrolytes. This confirms that, the anion is coordinated to the metal ion. The IR data show that the metal ion is
coordinated via the nitrogen atom of 4-VP. Metal VP-Fe complex and Polymer Fe Complex have been tested in vitro
against number of tumor and number of microorganisms in order to assess their anti tumor and antimicrobial properties.
The activity against HCT-116 cells was detected for compound VP-Fe 17.8±1.3, compared with reference standard
(24.6±0.3 µg/ml) followed by MMA-VP-Fe (88.3±1.2 µg/ml), respectively. The obtained results revealed the moderate
biological activities of the synthesized VP-Fel complex and polymer Fe complex.
Keywords: Metal complexes, Polymer metal complexes, Spectral studies, anti tumor and antimicrobial activity.
INTRODUCTION
Vinyl pyridine is a heterocyclic six-member cyclic aromatic molecule with binding capabilities through its nitrogen electron
lone pair which has inspired considerable interests in its bonding with metals [1–5]. Iron pyridine complexes are being
studied intensely by many researchers as the constituents of catalytic systems for the polymerization of ethylene and other
olefins [6-7]. The Fe(II) and Fe(III) complexes have recently received considerable attention in chelation therapy and are
cytotoxic to tumor cells in mice [8]. Recently we have reported the antiproliferative activity of Fe(III) complexes of a novel
nitrogen donor ligand [9]. 4-vinyl pyridine complexes played a vital role in the development of coordination chemistry [10].
It also plays an important role in biological process as exemplified in many instances in which enzymes are known to be
activated by metal ions [11]. These complexes have been occupied in the strongest and transport of active substances
through membrane [12]. Many metal complexes are resulting application in the microelectronic industry, chemical vapor
deposition of metals, and drugs [13]. Coordination compounds display diverse characteristic properties which depend on
the metal ion to which they are bound. On the basis of nature of the metal as well as the type of ligand, these metal
complexes have wide applications in different fields of human curiosity [14, 15].
2. EXPERIMENTAL SECTION
2.1. Materials
The monomers, 4-vinyl pyridine (4-VP) and Methyl methacrylate (MMA), were used as provided by Sigma-Aldrich
Company Ltd. 2,2ʹ -Azobis (isobutyronitrile) (AIBN; initiator) was supplied by Merck. FeCl 3.6H2O (III) was used as
received. Ethanol (Aldrich) was distilled from anhydrous stannous chloride. All other reagents were of analytical reagent
grade and used as purchased without further purification.
2.2. Preparation of Fe complex.
0.5 gm (0.0018 mol) FeCl3.6H2O was dissolved in 100 ml filtered ethanol with stirring. In this solution 0.582gm (0.0055
mol) of 4-vinyl pyridine was added dropwise with stirring [1M: 3L] (VP-Fe) Complex. The mixture was refluxed with stirring
on a magnetic stirrer for one hour at 60 oC. Then the mixture is cooled to room temperature. The brown complex
Precipitated and filtered off, washed several times with ethanol and dried under vacuum over P 4O10. The possible
mechanism of the produced complex was represented in Fig 1. The analytical and physical characteristics was determined
(Table1).
Table 1: The analytical and physical data of Fe complex
Elemental analysis
complex
4-VP-Fe
Yield
Molecular
%
Formula
80
C21H33Cl3FeN3O6
%C
m.p
M.Wt
Color
(oC)
586
285
%H
%N
conductivity
Calcd
found
Calcd
found
.
Brown
Molar
43.06
43.0
Calcd
found
.
5.68
5.59
7.17
7.13
9.8
2.3. Preparation of polymer Fe complex.
1.0 g of VP-Fe complex and 4.0 g of Methyl methacrylate were mixed in dimethylsulfoxide (DMSO; 50 mL). Then, the
flask of reaction mixture was refluxed with stirring for 3 h and immersed in a pre-heated oil bath at 70±1◦C. During the
reaction, a small amount of azobisisobutyronitrile (AIBN; 100 mg, 0.6 mmol)
initiator was added to flask. The reaction
mixture was precipitated in methanol as brownish bowder. The product was filtered, washed with methanol and dried
using a vacuum at 60 ◦C for 2 h. The product will be referred here in as MMA-VP-Fe. The possible mechanism of the
produced polymer complex is represented in Figure 2.
2.4. Test solubility
Solubility of the synthesized vinyl pyridine Fe complex and the synthesized polymer Fe complex were tested in various
polar and non-polar solvents. About 5–10 mg of the sample was added to about 2 ml of the respective solvent in a test
tube and kept overnight with the tube tightly closed. The solubility of the compounds was noted after 24 hours.
2.5. Physical and spectroscopic techniques
The Fourier transform infrared (FT-IR) spectroscopy of the prepared samples were performed on a Perkin–Elmer 683
spectrophotometer (4000–200 cm-1) using KBr pellets. The elemental analysis for the synthesized complex was
undertaken at the National Research Center, Micro analytical Centre, Giza, Egypt. The molar conductivity of (10-3 M)
solutions of the complex in DMSO were measured at 25 oC with a Bibby conductimeter type MCl. 1H-NMR spectra were
obtained with Perkin–Elmer R32-90-MHz spectrophotometer using TMS as internal standard. Mass spectra of the metal
complex were recorded using JEULJMS-AX-500 mass spectrometer provided with data system. The thermal analyses
(DTA and TGA) were carried out on a Shimadzu (TGA-50H) thermal analyzer, the temperature range covered was 27–
1000 oC and the scanning rate 10 oC per minute, under nitrogen atmosphere.
2.6. Antimicrobial and antitumor studies
2.6.1. Antimicrobial activity
Antibacterial activity of the prepared compunds were measured against four bacterial strains (Escherichia coli,
Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus pneumoniae) and the antifungal activity of the compounds
were measured against two fungal strains (Aspergillus fumigatus and Candida albicans) by disc diffusion method by using
approved standard method (M38-A & M44-P, respectively) developed by the National Committee for Clinical Laboratory
Standards [16]. The agar plates were prepared and the homogenous inocula of bacteria and fungus (in log phase) was
made, then spreaded by swabbing on the solidified agar media (nutrient agar for tested bacteria and malt extract agar for
tested fungi), in Petri-dishes (150 mm x 20 mm). Under aseptic condition, the plates were then punched with a 5 mm
diameter cork-borer to create wells. The tested samples were suspended in DMSO at 10 mg/mL, and 100 µl of each
suspended sample was added into each well. A well loaded with the solvent was used as negative control. The agar
plates were incubated for 48 h at 28±2°C and 24 h at 37±2°C for fungi and bacteria, respectively. All experiments were
carried out in triplicate. The antimicrobial activity of the tested substances was determined by measuring the sizes of
inhibitory zones in millimeter (including the diameter of wells) on the agar surface around the wells. The results are
reported as the mean of zones of inhibition ± standard deviation calculated from the triplicate samples for each test.
2.6.2. Antitumor activity
The antitumor activity was measured for the synthesized compounds according to sulfo-rhodamine-B-stain (SRB). The
prepared compounds were tested against two tumor cell lines i.e., Cervical cancer cell line (HeLa) and Colon carcinoma
cell line (HCT-116) using crystal violet viability assay [17]. All the experiments concerning the cytotoxicity evaluation were
performed and analyzed by tissue culture unit at the Regional Center for Mycology and Biotechnology, Al-Azhar
University, Cairo, Egypt. In brief, cells were plated in 96-well plate in 100µl of growth medium at a cell concentration of
and 50 µg /ml) were 1×104 cells per well. Different concentrations of the complexes in DMSO (1.56, 3.125, 6.25, 12.5, 25
added to the cell monolayer triplicate. Monolayer cells were incubated with the complexes for 48 h at 37°C under
atmosphere of 5% CO2. After 48 h, cells were fixed, washed and stained with Sulfo-Rhodamine-B-stain. Excess stain was
wash with acetic acid and attached stain was recovered with tris EDTA buffer (10 m M tris HCl and 1 m M disodium EDTA,
pH 7.5-8.0). Color intensity was measured by ELISA reader. The relation between surviving fraction and drug is plotted to
get the survival curve of each tumor cell line. Imitanib is used as a standard drug.
3. RESULTS AND DISCUSSION
The VP-Fe complex and polymer Fe complex are stable at room temperature, non hydroscopic, insoluble in water and
common solvent, viz: MeOH, EtOH, CHCl3, CCl4 , (CH3)2CO and DMF but soluble in DMSO. The analytical and physical
data of the complex are given in Table (1), spectral data; (Tables 2 and 3) are compatible with the proposed structure,
Figure (1). Reaction of 4vinyl pyridine with metal salt in ethanol gives complex 4-VP-Fe. The reaction leading to the
complex is represented schematically in Figure (1).
HC
FeCl3.6H2O
+
CH2
3
N
Cl

N
N
Fe
N
.6H2O
Cl
Cl
Figure (1): Preparation of Fe complex
Polymer Fe complex was synthesized by polymerizing monomeric metal complex with methyl methacrylate in DMSO
gives MMA-VP-Fe. Illustrated in Figure (2).
CH3
H 2C
C
Cl
+
N
COOCH3
N
N
Fe
Cl
Cl
AIBN

O
O
Cl
N
O
N
Cl
.6H2O
N
Fe
Cl
O
O
O
Figure (2): Preparation of polymer metal complex
3.1. Molar conductivity
The molar conductance values of the complex in DMSO (10-3 M) is 9.8 Ω mol-1cm2 , indicating that, the complex is not
electrolytes. This confirms that, the anion is coordinated to the metal ion .
3.2. Infrared spectra
The mode of bonding between the ligand and the metal and the monomer can be revealed by comparing the IR spectra
of the solid metal complex and ligand with that of the polymer metal complex. The IR spectrum of the 4-VP-Fe and MMAVP-Fe compounds are presented in figures 3 and 4, respectively. In the spectra of 4-vinyl pyridine, bands in the region
1600-1380 cm-1 were attributed to C=C, C=N stretching and ring vibration [18]. On complexation these frequencies are
shifted to the higher wave numbers, clearly indicating that the ligand is bonded to the central metal atom through the
hetero N-atom. This is also confirmed by the appearance of new bands at 261 and 265 cm_1 this have been assigned to
the M-N in 4-VP-Fe and MMA-VP-Fe, respectively [19]. The peaks at 1632,1520 and 1456 cm_1 are attributed to
characteristic vibration of the pyridine ring on 4-VP/Fe [20] and the intensity of the beaks in polymer complex MMA-VP-Fe
decrease. The beaks of the C=N stretching band of the pyridine ring displaced to 1618 and 1402 [21] .The absorption
band at 1068 cm−1 is assigned to the C- H bending in the in-plane rings in Ligand and it shifted to 1170 cm−1 in 4-VP-Fe
indicating that the ligand is bonded to the central metal atom through the hetero N-atom [22]. The absorption band at
1726 cm_1 is assigned to the C=O in MMA-VP-Fe and this band not found in 4-VP-Fe [23]. The absorption band at 1021
cm_1 is assigned to the C-O in MMA-VP-Fe [24]. We have found that the absorption band at 465 and 544cm_1 is due to the
stretching vibration of Fe-Cl bond in 4-VP-Fe and it shifted to 478 and 599 cm_1 due to polymerization in MMA-VP-Fe [20].
Figure3: FTIR spectrum of the Fe(III) complex
Figure4 : FTIR spectrum of the polymer Fe(III) complex.
3.3. 1H – NMR studies
1H-NMR
spectra of the 4VP, 4-VP-Fe and MMA-VP-Fe in deuterated DMSO show signals consistent with the proposed
structure. For Ligand , the peak at 8.3 ppm are assigned to proton of CH=N . The aromatic proton observed at 7.1 ppm.
The peak at 6.5 ppm is assigned to proton of CH=C which attach to pyridine ring. The peaks at 5.8 and 5.3 ppm are
assigned to protons of C=CH2. For 4-VP-Fe, the peak at 8.9 ppm is assigned to protons of CH=N. The aromatic protons
observed in 6.65-7.6 ppm range. The peak at 6.19 ppm is assigned to proton of CH=C which attach to pyridine ring. The
peak at 5.55 is assigned to proton of C=CH2. However, for MMA-VP-Fe, the peak at 8.56 ppm is assigned to proton of
CH=N. aromatic proton observed at 7.5 ppm [24]. The peak at 3.295 ppm is assigned to proton of C–CH-C which attached
to pyridine ring. The peaks at 2.529 and 2.495 ppm are assigned to protons of C-CH2-C-C=O and the peak at 1.737 and
1.708 ppm is assigned to proton of C-CH2-C [25].
3.4. Mass spectra of metal complex studies
The mass spectra of Fe (III) complex 4-VP-Fe confirmed their proposed formulations. It reveals the molecular ion peaks
(m/z) at 586 amu, consistent with the molecular weight of the Fe(III) complex. Furthermore, the fragments observed at
105,108, 162, 267, 372, 478 and 481 are due to C7H7N, H12O6, FeCl3, C7H7 Cl3FeN , C14 H14Cl3FeN2, C21H21Cl3FeN3 and
C14H26Cl3FeN2O6, moieties respectively. The spectral data of the Fe(III) Complex (4-VP-Fe) are presented in table 2.
Table 2 : Mass fragmentation of metal complex
Fragment
m/z
Rel. Int.
C7H7N
105
100
H12O6
108
2.62
FeCl3
162
1.06
C7H7 Cl3FeN
267
0.57
C14 H14Cl3FeN2
372
0.49
C21H21Cl3FeN3
478
1.4
C14H26Cl3FeN2O6
481
1.6
C21H33Cl3FeN3O6
586
3.2
3.5. DTA and TGA (Thermal analyses)
Since the IR spectra indicate the presence of water molecule, thermal analyses (DTA and TGA) were carried out to a
certain their nature. The thermal curves in the temperature 27-1000°C range for metal complex and polymer complex are
thermally stable up to 60°C. Dehydration is characterized by endothermic peak within the temperature 85 -122°C range.
The decomposition step for 4-VP-Fe shows endothermic peak at 85°C with 11.76 % weight loss is due to loss of six
hydrated water molecules [26]. , however, endothermic peak observed at 257°C with 22.68 % weight loss, is correspond
to the loss of VP unit [19] . However, the endothermic peak appears at 285°C with 23.53% weight loss may be assigned to
the melting point. Oxidative thermal decomposition occurs at 578°C with exothermic peaks, leaving Fe2O3 with 15.87%
weight loss. MMA-VP-Fe shows endothermic peak at 122°C
with 4.77% weight loss, are assigned to elimination of six
hydrated water molecules. The endothermic peak observed at 238.3°C with 17.5 % weight loss may be assigned to the
loss of VP unit [27]. The endothermic peak observed at 267°C with 19.25 % weight loss may be assigned to the melting
point. Oxidative thermal decomposition occurs at 468°C with exothermic peaks, leaving Fe2O3 with 16.73 % weight loss.
The thermal data are shown in table 3 and the thermograms of 4-VP-Fe and MMA-VP-Fe are shown in figures 5 and 6,
respectively.
Table 3: DTA and TGA data for metal complex and polymer metal complex
TGA
Compound
Temp.
(oC)
Assignment
DTA (Peak)
(Wt.loss%)
Found
85
Loss of six hydrated water
Endo
11.76
metal
257
Loss of VP unit
Endo
22.68
complex
578
Thermal decomposition with the formation of
Exo
15.87
Loss of six hydrated water
Endo
4.77
238
Loss of VP unit
Endo
17.5
468
Thermal decomposition with the formation
Exo
16.73
(VP-Fe)
Polymer
complex
(MMA-VP-Fe)
Fe2O3
122
Fe2O3
Figure 5: Thermogram of the (4-VP-Fe).
Figure 6: Thermogram of the (MMA-VP-Fe).
3.6 Antimicrobial activity
The antibacterial activity was tested against four pathogenic bacterial strains (Escherichia coli, Pseudomonas aeruginosa,
Bacillus subtilis and Streptococcus pneumoniae). The results compared with standard drugs (Ampicillin and Gentamicin).
The compounds were also subjected to antifungal activity against two pathogenic fungal strains (Aspergillus fumigatus and
Candida albicans) using the agar well-diffusion method. The results of the antimicrobial activities showed variations in
activities among the tested compounds. Interestingly, some of the tested compounds also exhibited highest tendency to
inhibit Gram positive bacteria than Gram negative bacteria. This activity can be explained on the basis of chelation theory
[28]. All compounds were found with no antibacterial activity against the Psedomonas aeruginosa under the screening
conditions. Additionally, none of the tested compounds exhibited antifungal activity against the Candida albicans under the
screening conditions. On the other hand, the tested compound
VP-Fe was found with no antibacterial or antifungal
activities under the screening conditions (Table 4 and 5).The mode of action of the compounds may involve the formation
of a coordinate bond groups with the active centers of the cell constituents resulting in an interference with the cell
process.
Table 4: Antibacterial activity of the compounds at 10 mg/ml
Gram-negative bacteria
compounds
Gram-positive bacteria
Escherichia coli
Psedomonas
aeruginosa
Staphylococcus
pneumoniae
Bacillus subtilis
0
0
0
0
17.3 ± 0.63
0
17.4 ± 0.64
20.2 ± 0.58
*Ampicillin
--
--
23.8 ± 0.2
32.4 ± 0.3
*Gentamicin
19.9 ± 0.3
17.3 ± 0.1
--
--
metal complex
(VP-Fe)
Polymer complex
(MMA-VP-Fe)
* Ampicillin and Gentamicin were used as standard antibacterial drugs against Gram positive and Gram negative
bacteria respectively.
Table 5. Antifungal activity of the compounds at 10 mg/ml
Filamentous fungi
Compounds
Candida albicans
Aspergillus fumigatus
0
0
0
15.2 ± 0.58
25.4 ± 0.1
23.7 ± 0.1
metal complex
(VP-Fe)
Polymer complex
(MMA-VP-Fe)
Amphotricin B*
*Amphotricin B was used as standard antifungal drug
3.7. Antitumor activities
The activity of the compounds was performed against tumor cell line using viability assay on two carcinoma cells (i.e.
cervical cancer cell line (HeLa cells) and human colon carcinoma cell line (HCT-116 cells). The in vitro growth inhibitory
rates (%) and inhibitory growth activity (as measured by IC50) of the synthesized compounds were investigated in
comparison with the well-known anticancer standard drug Imatinib (2-substituted aminopyrimidine derivative; Gleevec®).
Data generated were used to plot dose response curves and presented in Fig. (7a,b). However, the results revealed that
the tested compounds showed high variation in the inhibitory growth rates and activities against the tested tumor cell lines
in a concentration dependent manner (Fig. 7 a,b). The difference between inhibitory activities of all compounds with
different concentrations was statistically significant P < 0.001. Furthermore, the activity against HCT-116 cells was
detected for compound VP-Fe (with IC50 value 17.8±1.3 µg/ml), compared with reference standard (24.6±0.3 µg/ml)
followed by MMA-VP-Fe (88.3±1.2 µg/ml). Lower sensitivity was detected for HeLa cell line showing higher IC 50 values in
the same trend of activity measured for HCT-116.This can be explained as Metal binds to DNA. It seems that, change the
anion and the nature of the metal ion has effect on the biological behavior, due to alter binding ability of the DNA binding.
Antitumor effect of the compounds may be attributed to the central metal atom which was explained by Tweedy's chelation
theory [29]. Also, the positive charge of the metal increases the acidity of coordinated ligand that bears protons, leading to
stronger hydrogen bonds which enhance the biological activity. Also, metal could act as a double-edged, sword by
inducing DNA damage and also by inhibiting their repair. The OH radical reacts with DNA sugars and bases and the most
significant and well-characterized of the OH reactions is hydrogen atom abstraction from the C 4 on the deoxyribo unit to
yield sugar radicals with subsequent β-elimination, by this mechanism strand breakage occurs as well as the release of
the free bases. Another form of attack on the DNA bases is by solvated electrons, probably via a similar reaction to those
discussed below for the direct effects of radiation on DNA [29].
Figure (7a,b): Activity of compounds against (HCT-116) cell line and (HeLa) cell line
Table 6: Activities of the compounds with reference standard drug evaluated on colon and cervical cancer cell lines
compounds
Metal complex
(VP-Fe)
Polymer complex
(MMA-VP-Fe)
Imitanib
IC50 values (µg/ml)
HCT-116
HeLa
17.8±1.3
21.5±1.4
88.3±1.2
135.9±3.4
24.6±0.3
30.1±0.7
CNCLUSION
We synthesized the metal Fe complex and polymer Fe complex by using AIBN as initiator. Suggested structure of
complex and polymer complex confirmed by using spectroscopic measurements. Prepared compounds have been tested
against number of tumor and number of microorganisms.
REFERENCES
[1] Chaubey, A. and Pandeya, S. Pyridine: a versatile nucleuse in pharmaceutical field. Asian Journal of Pharmaceutical
and Clinical Research. 4(2011), 5–8.
[2] Hsu, H. C., Lin, F. W., Lai, C. C., Sua, P. H. and Yeh, C. S. Photodissociation and theoretical studies of the
Au+.(C5H5N) Complex. New Journal of Chemistry. 26(2002), 481–484.
[3] Guo, W., Liu, H. and Yang, S. Photodissociation spectroscopy of Mg+-pyridine complex. International Journal of Mass
arylimino)pyrrolide ligand. Organometallics. 28 (2009), 3100–3104.
[4] Wu, D. Y., Hayashi, M., Chang, C. H., Liang, K. K. and Lin, S. H. Bonding interaction, low-lying states and excited
chargetransfer states of pyridine-metal clusters: pyridine-Mn (M = Cu, Ag, Au; n=2–4). The Journal of Chemical Physics.
118 (2003), 4073–4085.
[5] Guo, C., Cao, Z. and Zhang, Q. Theoretical study of dissociative potential energy curves and photodissociation
mechanisms of the Mg+- pyridine complex in the low-lying states. Chemical Physics Letters. 386 (2004) , 448–453.
[6] Britovsek, G. J. P., Bruce, M. and Gibson, V.C. Iron and Cobalt Ethylene Polymerization Catalysts Bearing 2, 6Bis(Imino) Pyridyl Ligands: Synthesis, Structures, and Polymerization Studies. Journal of American Chemical Society. 121
(1999), 8728-8740.
[7] Ittel, S. D., Johnson, L. K. and Brookhart, M. Late-metal catalysts for ethylene homo- and copolymerization. Chem.Rev.
100 (2000),1169–1213.
[8] Singh, N. K., Shrivastva, A. and Singh, S. M. Synthesis, characterization and antitumor studiesof Mn(II), Fe(III), Co(II),
Ni(II), Cu(II) and Zn(II) complexes of N-salicyloyl-N_-ohydroxythiobenzhydrazide. Bioorg. Med. Chem. 10 (2002), 887–
895.
[9] Singh, N. K., Srivastava, A. K., Tripathi, P., Shrivastva, A. and Sharma, R. K.: Bioactivity of novel transition metal
complexes of N_-[(4-methoxy)thiobenzoyl]benzoic acid hydrazide. Eur. J. Med.
Chem. 43(2008), 577–583.
[10] Raman, N. and Ravichandran, S. Effect of substituents on (1- piperidinobenzyl) acetamide and N-(1morpholinobenzyl) acetamide and their antimicrobial activity. Asian Journal of Chemistry. 15 (2003), 1848–1850.
[11] Bell, G., Davidson, J. and Scarborough, H. Textbook of Physiology and Biochemistry, Churchill Livingstone,
Edinburgh, UK, 7th edition, (1968).
.
[12] Baran, E. J. Metal complexes of carnosine. Biochemistry. 65(2000), 789–797.
[13] Prasad, R. N., Sharma, K. M. and Agrawal, A. Mixed ligand complexes of Co(II) containing 5-bromosalicylaldehyde
and diketones, hydroxyaryl aldehydes or ketones. Indian Journal of Chemistry, Section A: Inorganic, Physical, Theoretical
and Analytical Chemistry. 46 (2007), 600–604.
[14] Johari, R., Kumar, G. and Singh, S. Synthesis and anti bacterial activity of M (II) schiff-base complex. Journal of the
Indian Chemical Council. 26 (2009) , 23–27.
[15] Mittal, P. and Uma, V. Synthesis, spectroscopic and cytotoxic studies of biologically active new Co (II),Ni (II),Cu (II)
and Mn II) complexes of Schiff base hydrazones. Der Chemica Sinica. 1(2010), 124–137.
[16] National Committee for Clinical Laboratory Standards, Reference Method for Broth Dilution Antifungal Susceptibility
Testing of Conidium Forming Filamentous Fungi; Proposed Standard, NCCLS Document M38‐A; Wayne, PA, USA,
(2002).
[17] Wilson AP In: Masters JRW (ed) Cytotoxicity and viability assays in animal cell culture: A Practical Approach, 3rd edn.
Oxford University Press, Oxford (2000).
[18] Srivastava, T. N., Tandon, S. K. and Bhakru, N. O. complexes of zirconium and thorium perchlorates with some
heterocyclic bases. Inorg.Nucl. Chem. 40(1978), 1180.
[19] Agarwal, R. K., Kishor, A., Himanshu, A. and Sarin , R. K . Synthesis and structural investigations of thorium (IV) and
dioxouranium (VI) complexes of 4-vinyl pyridine. Synth. React. Inorg. Met-Org. Cem. 25(6) (1995), 899-913.
[20] Vahid, A., Nasser, S., Hamid, R. K. and Peiman, M. Iron(III) mixed-ligand complexes: synthesis, characterization and
crystal structure determination of iron(III) hetero-ligand complexes containing 1,10-phenanthroline, 2,2′-bipyridine, chloride
and dimethyl sulfoxide, [Fe(phen)Cl3(DMSO)] and [Fe(bipy)Cl3(DMSO)]. Polyhedron. 26 ( 2007), 4908–4914
[21] Drelinkiewicza, A., Hasikb, M., Quillardc, S. and Paluszkiewicz, C. Infrared and Raman studies of palladium–
nitrogen-containing polymers interactions. J. Molecular Structure. 511 (1999), 205–215.
[22] Somashekarappa, M. P ., Keshavayya, J . and Sherigara, B. S. Synthesis and spectroscopic investigations of
iron(III) complexes with chlorides and dianionic, symmetrically halogen substituted phthalocyanines as ligands.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy . 59 (2003), 883-893.
[23] Liqin, C., Chuang, M., Jide, W. and Pei, C. Synthesis of polymer–copper(II) complexes in supercritical carbon dioxide.
The Journal of Supercritical Fluids. 75 (2013), 152– 158.
[24] Nurulsaidah, A. R., Fabrice, A., Brendan, T., Johannes, G. V. and Andreas, H. Synthesis of poly(4-vinyl pyridine-bmethyl methacrylate) by MAMA-SG1 initiated sequential polymerization and formation of metal loaded block copolymer
inverse micelles. European Polymer Journal. 48 (2012), 990 – 996.
[25] Nugay, N. and Nugay, T. Inter polymer complexes of poly (methyl methacrylate)-block-(4-vinylpyridine) with
polyacrylic acid and polyacrylic acid –block-poly(methyl methacrylate). European polymer journal. 36 (2000), 1027-1033.
[26] Alvaro, J., Alcides, D., Jorge, E., Ya´ne, z. and Paulino, B. Spectroscopic characterization of coordination complexes
based on dichlorocopper(II) and poly(4-vinylpyridine): Application in catalysis. Polyhedron. 24 (2005), 511–519.
[27] Hafida, H. F., Kamel, O., and Said, D. Synthesis, characterization and thermal behavior of poly (methyl methacrylate),
copolymers of methylmethacrylate with 4-vinyl pyridine and maghnia bentonite nanocomposites initiated by Ni (II)benzoinoxim complex. Polymer Preprints. 50 (2) (2009), 250-251.
[28] El –Tabl , A. S., El-Said, F. A. and Al-Hakimi, A. N. Synthesis, spectroscopic investigation and biological activity of
metal complexes with ONO trifunctionalalized hydrazone ligand. Transition Metal Chemistry. 32 (2007), 689-701.
[29] El –Tabl, A. S., Abd El-Waed, M. M. and Rezk , A. M. S. M. Cytotoxic behavior and spectroscopic characterization
of metal complexes of ethylacetoacetate bis(thiosemicarbazone)ligand. Spectrochimica acta part A: Molecular and
Biomolecular Spectroscopy.117 (2014), 772-788.