Using 5-(4-aminoantipyrineazo)-8-hyrroxyquinoline as Complexometry Reagent with Cupper (II) and Zinc (II) Ions and its Biological Activity

The complexes of azo dye 5-(4-aminoantipyrineazo)-8-hyroxyquinoline (L) with cupper (II) and zinc (II) Ions were synthesized. Characterization of these azo dyes complexes have been done on the basis of elemental analysis and IR techniques. The work involves a study of optimum conditions (time, pH, sequence of addition and temperature effects) for forming the complexes. The spectra of the complexes have been studied for a range of concentrations which Lambert – Beer's law were obeyed with sensitivity of the spectrophotometric method in terms of molar absorptivity 1.6×10 4 and 1.2×10 4 l .mol -1 .cm -1 for zinc and cupper azo dyes complexes respectively .The stoichiometry of the complexes has been found to be 1:2 (metal : ligand). The overall stability constants were determined by the corresponding solutions method. The interferences effects of the foreign ions were studied. The biological activity of two complexes with two type of Bacteria (Clostridium botulinum and Escherichia Coli) were also studied.


Introduction
Azo compounds are a very important class of chemical compounds receiving attention in scientific research. They are highly colored and have been used as dyes and pigments for a long time. Furthermore, they have been studied widely because of their excellent thermal and optical properties in applications [1]. Azo derivatives complex combinations have been widely used as dyes for synthetic polyamide supports and as pigments [2]. Azo derivatives and their metal complexes are very important pigments for synthetic leather and vinyl polymers and used as analytical reagent of many metals ions [3]. On the other hand, azo compounds are known to be involved in a number of biological reactions, such as inhibition of DNA, RNA, and protein synthesis, nitrogen fixation [4,5]. Furthermore metal-azo complex dyes are used in the recording layer of DVD-R ( Digital Versatile Disc-Recordable) discs . In comparison with the dyes themselves, metalazo dyes are more light stable, allow for easier control of the wavelength by selection of the appropriate substituent groups and have good thermal stability [6,7]. The systematic name of 4-aminoantipyrine is 4-amino-2,3-dimethyl-1-phenyl-3pyrazolin-5-one or 4-amino-phenazone which is widely used in medicine and pharmaceutical fields [8]. The antimicrobial activity of the 4-aminoantipyrine and its complexes ( Cu 2+ and Zn 2+ ) have been extensively studied on microorganisms such as Staphylococcus aureus, Klebsiela pneumonia, Bacillus subtillis, Escherichia coli, Citrobacter ferundii and Salmonella typhi , most of the complexes have higher activities than that of the free ligand [9] . The azo dyes compounds have been used as antiseptic and antibiotic [10] and also used as drugs , like 3-phenylazopyridine-2,6-diyldiamine [ 11] and salicylic azo sulfopyridine [12]. The sulfa azo dyes derived from 1-hydroxy-2-naphthoic acid were prepared as antibiotic and complexing reagents [ 13,14] . A azo compound, 8-quinolinol-5-azoantipyrine, was studied as an inhibitor for the corrosion of mild steel in 1M HCl [15]. The 7-arylazo-8-hydroxyquinoline is a good reagent for forming a stable complexes with dioxouranium (VI) [ 16] and Cu(II) [17] . Zinc (II) ions were determined spectrophotometrically with 7-(4-Nitro phenylazo )-8-hydroxyquinoline-5-sulfonic acid ) [ 18] .
The present work involves the use of azo dye 5-(4-aminoantipyrineazo)-8-hyroxyquinoline ( which was prepared previously in part I (Scheme 1 )) [19] for the formation of complexes with cupper (II) and zinc (II) ions under optimum conditions (time , pH , sequence of addition and temperature effects ) . Study the ability of these complexes as biological reagents for two types of Bacteria ( Clostridium botulinum and Escherichia Coli).

Scheme 1 Experimental
Double distilled water and all the reagents and solvents were of reagent-grad quality, The progress of reaction was monitored by TLC using silica gel coated plate sand spots were visualized under UV radiation. Infrared spectra ( in K Br pellets) were recorded on IR-8400S shimadzu , Melting points were determined on melting point apparatus , Element analysis (C.H.N.) were carried out by Perkin element 2400 clement analysis and UV/V is absorption spectra studies of the dyes were recorded using Perkin Elmer Lampda EZ 210 UV/V Spectrophotometer. The pH measurements were made with pH-Meter (H. Jurgons Co. Bremen,L. Puls Munchen15). The Cu(II) and Zn(II) ions were determined by atomic absorption spectrophotometer U.K., Pg. instruments AA500 .

Solutions
-A stock solution of (1 x 10 -3 M) of azo dye L were prepared by dissolving an accurately weighed amount of the compounds in the ethanol and more dilute solution were obtained by accurate dilution.
-A stock solution of (1 x 10 -3 M) of each Cu(NO3)2.3 H2O and Zn(NO3)2.6 H2O were prepared by dissolving the accurate weights in required volume of distilled water , and more dilute solution were obtained by accurate dilution.

Synthesis of the solid complexes
Azodyes complexes of Cu(II) and Zn(II) were prepared in a similar manner as described [21]. Thus, (0.004 mol) of each metal ions was added to a hot solution of the dyes (0.008 mol) in ethanol (70 ml) and the resulting mixture was refluxed for 1 h. A dark violet precipitates separated, which were collected by filtration, and recrystallized from mixture of ethanol : chloroform (1:3 v/v) . D e c e m b e r 2 9 , 2 0 1 4

Results and Discussion
The analytical and physical data of the L-Cu 2+ and L-Zn 2+ complexes are presented in Table 1. Elemental analysis and continues variation method of the complexes indicate the stoichiometry to be 1: 2 (metal: ligand). The IR has proven to be, in this particular case, a suitable technique to give enough information to elucidate the way of bonding of the ligands. Thus a detailed interpretation of IR spectra of these and the effect of binding of metal ions on the vibration frequencies of the free dye ( Table 2).

Table -2: The I.R spectral data of ( KBr disk )
The IR spectrum of the ligand (Fig.(1)) exhibited broad band at (3402 cm-1) was assigned to the stretching vibration of υ(OH) of the carboxyl and phenol [22,]. Strong band in the ligand spectrum was observed at (1660 cm-1) ascribed to the υ(C=O) for the carboxyl group. Significant change in the position to higher frequency was also observed on complexation with metal ion. The strong band in the free ligand spectrum at (1604 cm-1) due to υ(CO) asymmetric vibration, significant change in the intensity and in position to lower frequency was observed on complexation with metal ion.
The IR spectra showed that values of -N=Nstretching frequency (1501 cmP-1P) in dye LR1R and its synthesized complexes and (1504 cmP-1P) in dye LR2R and its synthesized complexes remain practically unchanged indicating that -N=N-group have no part in coordination whereas the carbonyl group C=O stretching frequency (1668 cmP-1P) in dye LR2R have unchanged values as well, indicating that carbonyl group is not taking part in coordination. However, the high changing in the shape and

Complex formation studies
The complexes of Cu 2+ and Zn 2+ ions with L solutions were always performed . These complexes were identified optimum conditions for the composition of these complexes ( time , pH , temperature and sequence of addition effects ) .

pHeffect
The absorption spectra of L-Cu 2+ and L-Zn 2+ were studied at pH values range (0.65 -12) (Fig. 2), by using acetate and universal buffer solutions in the range of wavelength 250 -600 nm. It was found the pH 12 is the suitable value for giving highest absorbance due to the formation of basic form (anionic form) which gives essayer condition for attraction with cations.
The interaction of Cu 2+ and Zn 2+ ions with the dye ( L) manifest itself in the absorption spectra by the appearance of a peak in the range ( 510 and 480 nm) for L-Cu 2+ and L-Zn 2+ complexes respectively. A great red shift in the visible region was detected in the complex solutions spectra with respect to that of the free dye ( λmax = 390 nm.). The shift in the (λmax) gave a good indication for complex formation. (Fig.-3 ). D e c e m b e r 2 9 , 2 0 1 4

Time effect
It was found from Fig. (4) the absorbencies are constant from 15 min up to 48 hrs. that means the complex is of high stability .

Squence of additions
It was found from Table -3, that the sequence (Dye + pH12 + M 2+ ) having heighest absorbance value of the azo dyes complexes . This attributed to the effect of alkaline medium of buffer solution ( anionic form ) , which makes the dye suitable for accepting metal ions .

The composition of the complexes (stoichiometry)
The composition of the complexes formed has been established by continues variation and mole ratio methods [23]. From Figs.- 6 & 7 , it was found that the ( M 2+ : azo dye) equal to (1 : 2) .

Beer's law and sensitivity
To get better results for beer's law , the optimum blank composition technique [ 23 ] was applied ( i.e. using unreacted dye as blank solution from knowledgement of stoichiometry of complexes ) The calibration curve ( Fig.-8 ) shows that Beer's law is obeyed up to concentration range 8.87 and 7.80 µg ml -1 for Cu(II) and Zn(II) in L-Cu 2+ and L-Zn 2+ complexes respectively . Table ( 4 ) shows the data obtained , that represented by the molar absorbitivity coefficient ( ε , L.mol -1 .cm -1 ) and sensitive index ( S , μg.cm -2 ) , and the detection limit ( DL μg.ml ) of the complexes. With high precision that represented by the results of standard deviation (S.D), and high linearity of Beer's law which represent by the correlation coefficient ( r ) which is nearly to unity. From a table, it was found the method of high precision and high sensitivity. D e c e m b e r 2 9 , 2 0 1 4 ,Fe +3 and EDTA ) were interfered only in 5-fold and 10fold concentrations .   The absorbance of L -Cu (II) complex in absence of foreign ions = 0.820 The absorbance of L -Zn (II) complex in absence of foreign ions = 1.105
It was found that the absorbance of L-Cu 2+ and L-Zn 2+ complexes were affected by ± 0.009 and ± 0.007 respectively.

The stability of the complexes
The stability constants (formation constant) of complexes Cu 2+ and Zn 2+ ions with L were determined by using Corresponding solutions method [25] , which depends on aid of half-value method [26]. This method required two series of solutions of total metal ion concentration C1M = 8 Then ñ -pL was plotted (Fig.-10) and by using the half value method, the log β1 and log β2 are obtained (Table -6). When ñ = 0.5 gives log β 1 (log K1) and ñ =1.5 gives log β2 (log K1+ log K2) (Where K and β are step stability and overall stability constants).  It was found from Table -6 , the sequence of azo dyes complexes stability are L-Zn(II) > L -Cu(II) and also found that log K1 is nearly equal to the log K2 , that means the azo dyes complex reaction is spontaneous and happened in one step .

Structural interpretation
From the C.H.N. analysis, Atomic analysis for Cu and Zn, IR technique and conductivity measurements, the structure of complexes L-Zn(II) and L -Cu(II) were suggested . The Zn 2+ and Cu 2+ ions have been coordinated through phenolic O atom and N atom of pyridine of 8-hydroxyquinoline of the azo dye L by forming square planer structure with respect to Cu 2+ ions and octahedral structure with respect to Zn 2+ ions ( in case of L-Zn complex two molecules of ethanol have been coordinated with metal ion ). Consequently, the proposed general structures are shown in Schemes 2.
Scheme 2 D e c e m b e r 2 9 , 2 0 1 4  Fig. (11) show the comparison of biological activities of the dye ( L ) and its complexes with Cu 2+ and Zn 2+ ions on two types of bacteria (Escherichia Coli and Clostridium Botulinum). It was found that the dye and its complexes having only biological activity on Clostridium Botulinum ,while no effect on Escherichia Coli, this because may be thickness of cell's wall slightly hard ( high lipidely ) that resists the chemical substances to enter the cell. It was also found that quince of biological activity of free dye, L-Cu(II) complex and L-Zn(II) complex as; L -Cu(II) > L -Zn(II) > Dye ( L ) in two concentrations 1 and 5 mg.ml -1 . Table -