Optical Response of Two Azo Ligands Containing Salicyaldimine-based Ligand as Side Chains Towards Some Divalent Metal Ions and Their Antioxidant Behavior

According to applicability of azo-azomethine compounds in chemical sensors and biological activities, two receptors: 1,2-[1-(3-imino-4-hydroxophenylazobenzene)]-4-nitrobenzene (1) and 1,2-[1-(3-imino-4-hydroxophenylazo-4-nitrobenzene)]-4-nitrobenzene (2) are investigated for detection of nickel, cobalt, copper, lead, mercury, zinc and cadmium divalent metal ions by UV-vis spectroscopy. With the addition of all metal ions to the DMSO solution of ligands, the peaks at 558 and 549 nm increase in intensity with hypsochromic or bathochromic shifts except Zn2+ ions and 2, while the peaks at 388 and 391 nm dramatically decrease in intensity. In both cases, the largest shift is observed after addition of copper ions. In solution, both receptors produce a cation blue shift from 558 and 549 nm to 503 and 497 nm with the sensible color change of solutions from purple-red to orange. Therefore, both compounds can highly recognize copper ions in DMSO solution. In the next step, Benesi-Hildebrand plot and Job’s method are used for determination of binding constant (Ka) and stoichiometry of formed complexes, respectively. Also, the investigation of solvent effect in the UV-vis spectra of ligands shows that the generation of hydrazine and enaminone tautomers increases in highly polar solvents such as DMF and DMSO. Finally, the antioxidant activity of ligands is studied by DPPH method. The results show that NO2 withdrawing groups in 1,2-[1-(3-imino-4-hydroxophenylazo-4-nitrobenzene)]-4-nitrobenzene probably affect keto−enol equilibrium. As a result, this ligand reduces free radicals to non-reactive species by donating hydrogen.


Introduction
Schiff bases are common organic compounds which can be easily synthesized.Among Schiff base derivatives, azo dyes are very important.Azo-azomethine compounds contain both azo and imine units.These compounds are produced by condensation of an azo dye containing aldehyde groups with primary amines. 1 Schiff bases and azo dyes have found applications in several fields such as medicinal, pharmaceutical and coordination chemistry.][14] Additionally, azomethine compounds can be used as chemosensors for metal ions and anions.][17][18] Copper ions are important in metabolic processes, but in excess they can cause the imbalance of homeostasis leading to severe diseases such as Alzheimer's, Parkinson's, Mekne's, and Wilson's diseases. 19,20Also copper ions are one of the materials that pollute environment and produce some problems in industry.Cobalt ions are dangerous pollutants.Cobalt can irritate respiratory system and cause lung diseases. 21Therefore, the development of simple and selective chemosensors for copper and cobalt ions is necessary. 22,23Previously, we reported easy methods for designing low cost sensors based on azo-azomethine ligands for recognition of copper and cobalt ions. 24,25In progress, here we report synthesis, characterization and optical response of one new azo-salicyaldimine based ligand 1 (1,2-[1-(3-imino-4-hydroxophenylazobenzene)]-4-ni-trobenzene) for detection of copper, lead, mercury, cobalt, nickel, cadmium and zinc divalent metal ions by spectrophotometry technique.Then, the response of azo-azomethine groups of compound 1 is compared with compound 2 (1,2-[1-(3-imino-4-hydroxophenylazo-4-nitrobenzene)] -4-nitrobenzene).Moreover, the antioxidant activity of both ligands is investigated against DPPH method.

3. Measurement of Radical Scavenging Activity
The ability of compounds 1 and 2 was investigated for removing free radicals by DPPH (1,1-diphenyl-2-picrylhydrazyl) using the method of litrature. 28The solutions of 1 or 2 in DMSO with concentrations of 10 to 60μg/mL were added to a methanol solution of DPPH (0.1 mM).The mixtures were shaken seriously.Then the absorption of solutions was measured at λ = 517 nm after 10 minutes.Finally, the percentage of radical scavenging was determined by the following equation: (A C is the absorbance of free DPPH and A S is the absorbance of DPPH after reaction with 1 or 2.)

1. 1. FT-IR and 1 H NMR Spectra
In the IR spectra of ligands, imine stretching vibration appears at 1617 cm -1 for 1 and 1609 cm -1 for 2. The NO 2 symmetric and asymmetric stretching vibrations occur as sharp and strong bands at 1313 cm -1 and 1343 cm -1 .Moreover, the vibrations of (-N=N-) groups as cis and trans forms are present at 1519 cm -1 and 1489 cm -1 respectively.
The structure of 1 and 2 are fully characterized by 1 H NMR spectroscopy.The formation of imine group is established by the appearance of the signal at 8.56 ppm in 1 and 8.54 ppm in 2, depending on the substituent attached to the imine nitrogen atom.The OH proton signals appear as one singlet at 12.64 in 1 and 12.61 ppm in 2. The aromatic proton signals appear in the range of 6.76-9.09ppm (Scheme 1 and Table 1).
An antioxidant acts via two mechanisms: one of them depends on the benzyl hydrogen atom and other follows the route of keto-enol form. 29As shown in Fig. 1, compound 2 reveals high antioxidant activity against DPPH method (78% for 60 μg/mL), while compound 1 display low activity (less than 13% for 20 μg/mL).It seems that the possible mechanism for both compounds is the keto-enol route (Scheme 3).Several factors such as structure, temperature and solvent can affect the keto-enol equilibrium.The structure factors involve steric bulk, conjugation, electron-withdrawing/ donating groups and resonance. 30The structure of both compounds is similar (the only difference between two structures is the existence of NO 2 groups at the para position of azo units in 2) (Scheme 1).These NO 2 withdrawing groups in compound 2 probably affect keto-enol equilibrium.As a result, ligand 2 reduces free radicals to non-reactive species by donating hydrogen.Scheme 2: DPPH radical and its stable form (DPPH= 1,1-diphenyl-2-picrylhydrazyl)

2. Antioxidant Activity
The antioxidant activity of compounds 1 and 2 is investigated by DPPH radical scavenging method.In this method, radical DPPH reduces to its non-radical form in the presence of hydrogen-donating material that named antioxidant (Scheme 2).

1. UV-vis Spectra of Ligands
Figs. 2 and 3 show the UV-vis spectra of receptors 1 and 2 in DMSO solution.Both compounds show one strong absorption band at λ = 278 nm corresponding to the π→π ✳ transition of aromatic rings, one broad absorption band at λ = 388 nm for 1 and 391 nm for 2 attributable to the π→π ✳ transition of azo groups and π→π ✳ or n→π ✳ transition of imine groups 31 and the strong and broad absorption band at λ = 558 nm for 1 and 549 nm for 2 corresponding to the n →π ✳ transition of (-N=N) units. 32e UV-vis absorption spectra of azo Schiff-base ligands 1 and 2 in CH 2 Cl 2 , CHCl 3 , CH 3 OH and C 2 H 5 OH show main band at 364-382 nm which can be assigned to π→π* transition of azo groups.However, in DMSO and DMF solution, the first band that located at 388-391 nm, similar to other solvents, is because of π→π* transition of azo groups, while the second one which appeared at 549-558 nm can be assigned to an intramolecular charge transfer n→π* transition of azo-aromatic chromophore (Fig. 4). 33,34In general, the absorption bands of 1 and 2 at 364-  35,36 Also, the solvatochromism that exhibited by azo ligands may be to the effect of proton transfer or dipole moment changes in various solvents (in DMSO and DMF an additional absorption maximum is observed at 549-558 nm.This absorption is attributed to the existence of tautomeric form on highly polar solvents). 37,38

2. Cation Binding Studies
The optical response of 1 (2 × 10 -5 mol L -1 ) for Cu 2+ , Cd 2+ , Co 2+ , Zn 2+ , Ni 2+ , Pb 2+ and Hg 2+ as their acetate salts (2 × 10 -5 mol L -1 ) in DMSO is studied.As shown in Fig. 2, the broad bond at 558 nm rises in intensity after addition of Pb 2+ , Zn 2+ , Hg 2+ and Cd 2+ ions to the DMSO solution of 1 with a bathochromic shift (+29 nm).The intensity of the band at 558 nm increases as fellow: Pb 2+ > Zn 2+ > Hg 2+ > Cd 2+ .Upon the addition of Cu 2+ and Co 2+ ions to 1 the peak at 558 nm increases in intensity and shits to shorter wavelengths.The largest hypsochromic shift is seen after addition of copper ions (-55 nm) with the sensible color change of solution to orange.The peak at 388 nm shrinks in intensity with the addition of all studied cations.As shown in Fig. 2, no significant change is observed in the UV-vis spectrum of 1 after adding nickel ions.
The recognition ability of 2 (2 × 10 -5 mol L -1 ) for Cu 2+ , Cd 2+ , Co 2+ , Zn 2+ , Ni 2+ , Pb 2+ and Hg 2+ as acetate salts (2 × 10 -5 mol L -1 ) in DMSO is shown in Fig. 3. Upon addition of Pb 2+ , Cd 2+ and Hg 2+ ions to 2, the peak at 549 nm increases in intensity with a bathochromic shift (almost +20 nm).Addition of Cu 2+ , Co 2+ and Ni 2+ ions to the DMSO solution of 2 exhibits significant increase in peak intensity at 549 nm with a hypsochromic shift.Similarity to 1, the largest blue shift is obtained after addition of Cu 2+ ions (-52 nm).As expected, addition of all metal ions to 2 shows decrease in peak intensity at 391 nm.It is notable that no significant change is observed in the UV-vis spectra of 2 after addition of Zn 2+ ions.
In both cases, the changing in the UV-vis Spectra can be explained that: there is likely a fine balance between enol and keton forms of ligands in DMSO solution and the complexion of ligands with metal ions probably affect it.[41] Fig. 3. UV-vis spectra of ligand 2 (0.01 mM) before and after adding a 0.01 mM concentration of various metal acetates in a DMSO solution.

3. 3. Titrations with UV-vis Spectroscopy
Upon gradual addition of Cu 2+ ions to DMSO solution of 1, the absorption at 558 nm gradually increases in intensity with hypsochromic shift to 503 nm and the peak at 388 nm strongly decreases in intensity (Fig. 5).Similarity to 1, with the progressive addition of Cu 2+ ions to 2, the peak at 549 nm shifts to 497 nm and increases in intensity, while the peak at 391 nm dramatically decreases and finally disappears (Fig. 7).In both case, the color solution change from purple-red to orange after addition of copper ions.ally increases in intensity while the peak at 391 nm decreases in intensity (Fig. 8).
The Job's plot results show a 1:1 binding stoichiometry for 1 and 2 with Cu 2+ .(the proposed structure of 1 and 2 with Cu 2+ is shown in Scheme 4) while the 2:1 binding stoichiometry for 1 and 2 with Co 2+ is determined by Job's plot experiments (Fig. 9).
In the next step, 1:1 association constants of 1 and 2 with Cu 2+ are determined on the Benesi-Hildebrand plots 42 at λ = 503 and 497 nm, respectively (Figs. 5 and 7).Correspondingly, assuming a 2:1 (1 or 2: cobalt ion) complex, the binding constants (K a ) are also calculated using the Benesi-Hildebrand method (Figs. 6 and 8).The resulting values are summarized in Tables 3 and 4. As shown, the ability of both receptors for recognition of Cu 2+ metal ion is similar while the tendency of 1 for detection of Co 2+ ion is higher than 2.   Upon successive addition of Co 2+ to 1, a hypsochromic shift is observed from 558 to 525 nm.The peak at 525 nm rises with the gradual addition of cobalt ions to 1.The peak at 388 nm dramatically decreases in intensity and disappears after extra addition of Co 2+ ions (Fig. 6).Finally, upon incremental addition of Co 2+ to 2, n→π ✳ transition shifts from 549 nm to 537 nm.The peak at 537 nm gradu-

Conclusion
At first new azo-azomethin derivative 1 was synthesized and characterized with some standard methods.Then the optical response of azo units of the synthesized compound was investigated for detection of some divalent met-  between two structures is the existence of nitro groups at the para positions of azo units in 2).Study revealed the ability of both receptors for recognition of Cu 2+ metal ion to be similar while the tendency of 1 for detection of Co 2+ ion is higher than 2 (the binding constant of 1 with copper and cobalt ions is larger than 2).Also, the investigation of solvent effect in the UV-vis spectra shows that the generation of hydrazine and enaminone tautomers increase in highly polar solvents such as DMF and DMSO.Moreover, the investigation of antioxidant activity of ligands with DPPH method indicates NO 2 withdrawing groups in compound 2 probably affect keto−enol equilibrium.As a result, ligand 2 strongly reduced free radicals to non-reactive species while compound 1 showed low activity.

Appendix
FT-IR and 1 H NMR spectra of compounds 1 and 2 are available in Appendix.

Fig. 6 .
Fig. 6.Changes in the UV-vis spectra of 1 upon titration by Co(CH 3 COO) 2 in a DMSO solution, where the concentration of Co(CH 3 COO) 2 varies from 0.004-0.2mM.Insets: above: Absorption at selected wavelength versus equivalents of cation added, down; Benesi-Hildebrand plot of the receptor with Co 2+ ion.

Table 2 :
UV-vis spectra data upon titration of compound 1 with cations in DMSO

Table 3 :
UV-vis spectra data upon titration of compound 2 with cations in DMSO