Introduction

Heterocyclic azo compounds have a lot of scientific attention especially in the last five decades because of their applications in different of applied and academic fields such as Analytical reagents (Cheira, Khawassek, & Hussein, 2012), antibacterial (Witwit, Motaweq, & Mubark, 2018), antifungal (Slassi, Fix-Tailler, Larcher, Amine, & El-Ghayoury, 2019) anticancer (Vernieuwe et al., 2017) and Optical electrical switching of liquid crystals (Oh, Baek, Kim, & Yoon, 2017). Imidazole azo ligands considered as an essential type of heterocyclic azo ligands chiefly in coordination chemistry due to their ability to form stable complexes with metal ions in various of oxidation states (Al-Adilee & Kyhoiesh, 2017), formation of stable five-member ring with each ion through (N3) atom of imidazole ring and one of nitrogen atoms of azo group (Al-Muhanaa & Al-Khafagy, 2018). As well as the contribution of imidazole molecule in preparation of many ligands which have π – conjugated system that increases their stability and follows the colour change of them before and after the coordination with metal ions (Erbaş & Gülle, 2018). The goal of this research is representing by preparation and characterization of new ligand as a derivative of 4,5-diphenylimidazole, studying it's coordination behavior with Mn(II) , Co(II) , Ni(II) , Cu(II) , Zn(II) , Cd(II) , and Hg(II), and experienced their biological activity against two types of multi-drug resistant aerobic pathogenic bacteria have isolated from burn patients.

Materials and Methods

Chemicals and Instruments

All chemicals and solvents were equipped with high purity from Sigma Aldrich, BDH and Merck companies. Mass Spectrum was measured using AB SCIEX 3200 QTRAP Mass analyzer , FT-IR carried out by Shimadzu FTIR8400 using KBr disks from (400-4000)cm^-, Electronic spectrum measured by Shimadzu UV-1650 UV-Vis Spectrophotometer , The element analysis performed on Costech ECS Elemental 4010, magnetic measurements of prepared complexes recorded by Balance Magnetic Susceptibility Model –M.S.B Auto , Molar conductivity menstruated via 720(WTW), and ¹HNMR carried out by Bruker Avance-111 300 MHz NMR Spectrometer.

Preparation of (DPIDA) ligand

Two and thirty-one gram of N1, N1-dimethylbenzene1,4-diamine dihydrochloride was dissolved in twenty-five ml of distilled water than one ml of hydrochloric acid added gradually to this solution which cooled in ice bath 0-5 ^oC, the formation of diazonium salt occurred by addition the solution of sodium nitrate which prepared by dissolved 0.70 gm of it in 10 ml of distilled water drop by drop with stirring, This solution leaved in the ice bath for 30 minute then coupled with alcoholic solution of 4,5- diphenyl imidazole which prepared by dissolving 2.21 gm of imidazole derivative and 0.44 gm of sodium hydroxide in 25 ml of ethanol, orange precipitate was appeared after the completing of addition, filtered and dried then recrestalysid from ethanol yield percentage 71 % as shown in Figure 1.

Preparation of complexes (general method)

All complexes were prepared with mole ratio 1:2 (metal: ligand) by mixing 1 mmole of metal chlorides in twenty-five ml of distilled water with 2 m moles of (DPIDA) in twenty-five ml of ethanol with stirring until the precipitations of the complexes were appeared, filtered and dried the yield percentage of them were shown in Table 1.

Biological Activity

Biological activity testing was done to detect the antibacterial activity of four synthesized derivatives compounds against two multi-drug resistant aerobic pathogenic bacteria isolated from patients with burn infection; Staphylococcus aureus (S.aureus) is a gram-positive bacteria and pseudomonas aeruginosa (P.aeruginosa) as a gram harmful bacteria. The two pathogenic bacteria were provided with kindly from the university of Kufa, Faculty of science, department of microbiology, Iraq. Antibacterial activity test was done according to the agar well diffusion method (Aljanaby, 2013; Aljanaby, 2018). Three concentrations were selected (50, 100, 150) mg/ml for each crud synthesized derivative compounds. Four wells were made by crock-poorer (Oxoid, UK) in Muller-Hinton agar surface (Oxoid, UK) and swabbed with two pathogenic bacteria with turbidity according to 0.5 McFarland tube. Fifty µl of each dilution was transferred to each well and left at (20)^oC for 3 hours and incubated at (37)^oC for 24 hours. Four replicates were done for each test. The inhibition zone around each well was measured in millimetres (Adam, Al-Labban, Aljanaby, & Abbas, 2019; Aljanaby & Alhasnawi, 2017).

Statically analysis

Graph pad prism V.6 windows soft were been used in statically analysis to compare between diameters of inhibition zone (mm) according to T-test. P-value < 0.05 was considered indicative of statistically significant (Adam et al., 2019).

Results and Discussion

¹HNMR spectra of free ligand (DPIDA) in (d⁶ DMSO) inhibit a singlet signal in (3.03) ppm due to the protons of (N-CH₃) groups, whilst the siglet signal of (N-H) for imidazole ring (Rehab, Al-Hassani, & Journal, 2016) appeared in (12.63) ppm, this spectrum confirms the number of protons in the molecular structure, as shown in Figure 2.

Mass spectrum of (DPIDA) ligand showed molecular ion peak(M+1) at m/e (368) , the initial fragmentation started by losing (-N₂) molecule at m/e (340) , while the base peak appeared at ( e/z= 221) corresponding to 4,5- diphenylimidazole fragment ( C₁₅H₁₂N₂) (Mehdi & Ali, 2005). The spectrum of Mn(DPIDA)₂Cl₂ complex exhibited molecular peak at (e/z = 860) that affirmed the molecular weight of this complex, the fragmentation also started by losing the nitrogen's of the two coordinated azo ligands at (e/z=804) and continued to the last step which showed the fragment of 4,5- diphenylimidazole as base peak, theFigure 5; Figure 3 and Figure 6; Figure 4 illustrated the fragmentation of ligand, and it's complicated.

Uv-Vis spectrum of free ligand (DPIDA) show up three bands at 284, 257, 238 nm which attributed to (π – π*) transitions of aromatic rings which shifted to higher wavelengths with little changes of values in complexes spectrums , while the bands at 466 nm of (n-π*) transitions that exhibited redshift in the ranges of complexes as a result of charge transfer transitions after coordination as shown in Table 2.

IR spectra of ligand (DPIDA) showed v(N-H) peak of imidazole ring at (3400) cm^- (Abbas & Kadhim, 2016) two peeks at (3034) and (3061) cm- due to the vibrations of aromatic v(C-H) , and one peak at (2991) cm- of aliphatic v(C-H) that showed no significant changes in the spectra of the complexes frequencies. The peaks at (1588) cm- and (1315) cm- exhibited a stretching of v(C=N) and v(C-N) (Al-Hasani & Almaliky, 2015) respectively of imidazole ring that proceed a changing in position and intensity in complexes which is an indicate the participation of (N3) atom in coordination, also the values of v(N=N) (Jarad & Kadhim, 2018; Slassi, Fix-Tailler, Larcher, Amine, & El-Ghayoury, 2019) peak at (1498) cm- shifted to lower values in the complexes that’s considered as an evidence on coordination proceed through one nitrogen atom of azo group, new values of v(M-N) frequencies appeared between (543 -511) cm^- in the complexes that consider as additional evidence on coordination process as shown in Table 3 and Figure 9; Figure 8; Figure 7.

Conductivity measurements at 25^oC in both of DMF and DMSO solvents for (10^-3) M encouraged non- ionic character of all complexes , The values of molar conductivity ranged between 23.6-15.4 S.cm².mole in DMF , while their values within 20.6-11.7 S.cm².mole, as well no white precipitate of AgCl observed when a drops of 0.1 N from AgNO₃ solution to metal complexes solutions added which also confirms the absence of counter ion outside the coordination sphere (Hayder & Aljanaby, 2019; Reddy, Reddy, & Raju, 1997) as apparent in Table 2. The suggested structure of the complexes showed the octrahyral geometry that two (DPIDA) ligands coordinated with central metal ion as bidentate through nitrogen atom number 3 of imidazole ring and one nitrogen atom of the azo group as explained in Figure 10.

The results of biological activity demonstrated that most the ligand and complexes have good antibacterial activity against two pathogenic bacteria with inhibition zones in three concentrations Table 4 andFigure 11 and Figure 12 While, the derivative compound 4,5-diphenyl imidazol with concentration 150mg/ml had excellent antibacterial effect against S.aureus and P.aeruginosa with inhibition zone 21.83 ± 0.1764 mm and 24.30 ± 0.4163 mm respectively.

Conclusion

Seven complexes of Mn(II) , Co(II) , Ni(II) , Cu(II) , Zn(II) , Cd(II) , and Hg(II) ions with new imidazole azo ligand (DPIDA) were prepared as an octahedral geometry of them with bidentate ligand with general formal [M (DPIDA)₂Cl₂] the prepared compounds showed excellent biological activity against two types of bacteria S.aureus and P.aeruginosa.