Synthesis and characterization and biological study of pyridylazo ligand and its compounds of Co, Ni and Cu divalent ions

In this work, we synthesized and characterized an azo dye of amino pyridine namely 6-[3- pyridylazo]-2,4-dichlorophenol (PADCP) furthermore its complexes of cobalt, nickel and copper divalent ions. The infrared spectra refereed that PADCP ligand coordinates by oxygen and nitrogen atoms to the metals in the complexes as bidentate ligand. The synthesized complexes have general formula [M(PADCP)2] which confirmed by elemental analysis, magnetic moments, molar conductivity and electronic spectra. The synthesized complexes of PADCP have tetrahedral shape and non-ionic. The PADCP dye exhibit clear color changes under pH changes. The PADCP ligand and its complexes shown notable biological activity against E.coli, Staph.aureus and Aspergillus Niger


Introduction
Azo dyes are organic compounds and the azo group considered the main component of azo dyes, as they represent the largest part of all types of dyes used at the present time [1]. This type of compounds discovered by Greiss and named in 1860. He indication to the double bound of (-N = N-) with hybridized (sp 2 ) where both nitrogen atoms linked to aromatic system which is more stable than aliphatic system [2][3][4].
This type of dyes widely use as organic reagents due to its versatile properties like high stability and speed of interaction with metal ions with high sensitivity and selectivity under complexation [5][6][7]. The direct dye (Congo red dye) is a sulfonated azo pigment used on a large scale in the biological field, where it was the focus of many attention due to its effect on the growth of viruses and it showed interest effect as an anti-HIV and anti-Alzheimer agent [8][9][10].
The azo-ruthenium (II) dye of bipyridyl was used as a sensitive dye in the photochemical treatment technique, it was characterized by high accumulation in the mitochondria of HeLa cells and it was 2 non-toxic in dark conditions. It also showed high absorption capacity for two-photon at 810 nm with high killing for cancer cell [12,13]. The azo dyes of pyridine exhibited large interesting like reagent for metal ions, indicator, and in biology [14][15][16][17], therefore we synthesis azo dye 6-[3-pyridylazo]-2,4dichlorophenol and its complexes of Co, Ni and Cu.
We took 0.5g of 3-aminopridine and dissolve it in a mixture containing 3mL of concentrated HCl acid with 7mL of distilled water and place the mixture in an ice bath at 5 ° C. Then we prepared another solution by dissolving 0.37g of sodium nitrite in 5mL of distilled water and placing the solution in an ice bath at 5 ° C. The diazonium salt was prepared by mixing the solution of acidic amino aromatic with the solution of sodium nitrite slowly drop by drop while keeping the solution cool. 0.8g of sodium hydroxide dissolved in 6mL of distilled water then mixed with 0.83g of 2,4dichloro phenol dissolving in 12 mL of alcohol, the new solution was put under cooling. The diazonium salt was added slowly with continuous stirring to the basic solution of 2,4-dichlor phenol while continuing with cooling. The coloration of the solution was observed in a yellowish brown color. When the addition process is completed, leaves the solution for the next day, filter the precipitate and wash with distilled water several times.

Preparation of metal ion complexes with ligand (PADCP):
0.1g of (PADCP) dissolved in 10mL of alcohol then mixed with 3mL of aqueous solution of potassium hydroxide (0.02g). A hot solution of MCl 2 (0.044 g, 0.0314g and 0.044g, of CoCl 2 .6H 2 O, CuCl 2 .2H 2 O, and NiCl 2 .6H 2 O respectively dissolving in 4mL distilled water) was mixed with hot solution of PADCP, the mixture solution was heated under 60-80 ° C for 30 minutes. The mixture was left for the next day. The precipitate is filtered and washed with water.
We used Muller-Hinton agar to growth the bacteria and we use diffusion method with four halls for each Petri dish while in case of fungi we used potato dextrose agar to growth the fungi.

Scheme 1 Preparation steps of PADCP dye
The mass spectrum of PADCP dye ( Figure 1) exhibited important fragments such as 268.5 as mother fragment and 233 as M-Cl with high abundance. The PADCP dye showed high red shift in basic aqueous with hyperchromic effect comparing to the dye in neutral aqueous. The PADCP dye exhibited red shift in acidic aqueous with little bit hypochromic effect these changes are represented in (Figure 3) [18,19,7]. The physical properties and elemental analysis of The PADCP ligand and its complexes are incorporated in Table 1. The PADCP ligand reacts with metals ions of Co, Ni and Cu under mole ratio equal to 2:1 as ligand : metal as shown in (Figure 4)  The infrared spectra of PADCP dye and its complexes ( Figure 5-8) exhibited important characteristic peaks for example, CH aromatic, C=N stretching, C=C aromatic, N=N stretching, C-Cl and C-H bending [20] which are depicted in (Table 2). We noticed that frequencies of N=N in complexes exhibited in red shift and we did not see the stretching frequency of O-H that means that the azo group and oxygen atom of phenoxide ion coordinated to metals in complexes.  Electronic spectra (Figure 9) refer that PADCP ligand exhibited a band at 319 for π-π* and large band at 390 nm for π-π* overlap with n-π*. The cobalt complex exhibited a band at 314 nm and 390 nm for electronic transition of PADCP ligand in the complex and a band at 570 nm due to 4 A 2 → 4 T 1 (F) with magnetic susceptibility 4.56 B.M. in agreement with tetrahedral complexes of cobalt. Nickel complex exhibited three bands, at 319 for π-π* of PADCP ligand in the complex, at 405 nm for 3 T 1 (F) → 3 A 2 electronic transition, and at 560 nm for 3 T 1 (F)→ 3 T 2 (F) electronic transition of d-d and magnetic susceptibility equal to 3.54 B.M. which is in agreement with tetrahedral complexes of nickel. Copper complex exhibited band at 319 nm for π-π* of PADCP dye and band at 392 nm due to charge transfer from copper to ligand with magnetic susceptibility 1.92 B.M. in agreement with tetrahedral complexes of copper [21]. On the other hand, the molar conductivity of synthesized complexes of Co, Ni and Cu in DMSO solvent at room temperature indicated that the complexes are not ionic [22]. The PADCP dye and its complexes shown interesting biological activity against Aspergillus Niger as shown in Table 3. The PADCP dye exhibited excellent biological activity against Aspergillus Niger, it was 6.5 cm as shown in Figure 10. The complexes of Ni, Co and Cu exhibited good biological activity against Aspergillus Niger, it was 2-3.3 cm respectively. The compounds exhibited the better role against fungi at 100 mg/mL.  Figure 10 Biological activity of PADCP dye against Aspergillus Niger fungi We tested our prepared materials against E. coli bacteria. The materials showed high biological efficacy (Table 4), ranging from 3.5 to 4 cm for the PADCP dye and cobalt complex respectively. Whereas, the nickel and copper complexes did not show biological efficacy against E. coli bacteria, noting that substances did not show biological efficacy at high concentration. The PADCP dye and its complex of Ni exhibited inhibition against Staph.aureus ( Figure 11) from 2.5-1.5 cm respectively while other complexes did not show biological activity against Staph.aureus.

Conclusion
The spectra of PADCP dye exhibited perfect agreement between the changes of color and the shift of spectra in different values of pH. The PADCP dye in its electronic spectra showed red shift in acid and basic aqueous. Infrared spectra confirmed that the PADCP dye is bidentate ligand. The behaviour of PADCP ligand is bidentate. The synthesized complexes of PADCP dye have tetrahedral environment. The PADCP dye and its synthesized compounds are consider a candidate as antibiotic against E.coli, Staph.aureus and Aspergillus Niger.