Preparation, Characterization and Analytical Studies of novel Azo dyes and Diazo dyes

The amino {4-hydroxy-3-[(E)-{4-[(5-methylisoxazol-3-yl) sulfamoyl] phenyl} diazenyl] phenyl} acetic acid (1) and the amino {4-hydroxy-3,5-bis[(E)-{4-[(5-methylisoxazol-3-yl) sulfamoyl] phenyl} diazenyl] phenyl} acetic acid (2) were Prepared. The resulting azo and diazo dyes were then characterized using m.p., IR, UV-visible, mass spectrum and 1H-NMR spectrum. Analytical studies were carried on the azo dye (1) and the diazo dye (2). The best solubility of (1) and (2) was in methanol and ethyl acetate respectively, with no deviation from the linear relationship in each dye, which is due to the fact that the effect of the dielectric constant is the main factor that can control the shift of the absorption beaks. Though, the pH effects of (1) and (2) in a range of buffer solution were showed one and two isopiestic points respectively. Calculation of the ionization (pKa) and the protonation (pKp) constants by using the half height method was associated to the nitrogen atom and the OH-groups respectively. The pKp1, pKa1 and pKa2 were also intended. But, the value of the pKp2 was absent in the azo dye (1) and seems to be equal to 2.5 in the diazo dye (2). These results were indicated the suggested ionization scheme in acidic and basic medium of each dye. Furthermore, the dyes with different concentrations have harmless, nontoxic and no haemolysis effects.


Introduction
Scientific research are regarding the azo dyes [1] [2] [3] [4], besides expended a great rank in chemical analysis. the powerfully coloured dyes, depending on their exact molecule structure were created as very significant compounds as pigments for an extensive period [5]. The structural features in the organic compounds, that typically display colour are C=C, N=O, N=N, aromatic rings, C=O and NO 2 . However, the azo (-N=N-) and nitroso (-N=O) groups can confer colour, while the other groups actually dose in convinced conditions [6]. one or more azo groups, that are linked to SP 2 hybridized carbon atoms also founded on same dyes [7]. dyes can hold an extra active group able to express chelatic coordinational complexes with metal ions. Essentially reactive dyes, that reported for their pharmaceutical significance as antidiabetic [8], antineoplastic [9], antibacterial [10], and anticancer agent [11]. Analytical studies were carried on some diazo dyes [12]. The solvents and the pH effects of these dyes displayed high solubility in ethanol and distil water, and appropriate values, ( pH= 12) in each dye were established. Three isopiestic points were gained in these dyes owing to envision their pK a and pK b constants. But, the pK a1 was disappeared. Theoretical studies were also carried on the structure of each synthetic dye in order to explain the relation between the structure and their properties. The structure of each synthetic diazo dye was accepted by means of the internal coordinate mechanics (ICM), the conformational analysis and the molecular mechanics (MM2) studies [12]. Furthermore, the best minimization of these dyes was attended successfully.

Experimental section
The melting points of azo and diazo dyes were attended using Buchi B190K. The IR spectrum was carried out on aFT-IR-8400S.Fourier Transform Infrared Spectrophotometer Shimadzu (Japan) by using a KBr disc in the range (600 -4000) cm -1 . The UV-Visible spectrum was done using ethanol (1 x 10 -4 M). The IR, UV-Visible spectrophotometer and melting point completed by Chemistry Department-Education College of pure science-Basrah University, Iraq. Correct mass spectrum and the 1 H NMR were measured in Tehran University.

The Solution of the azo and diazo dye in ethanol
The stock solution of each azo dye and diazo dye was prepared by dissolving weights (0.0215 g) and (0.0347 g) respectively in (50 mL) of ethanol to give the resulting concentration (1 x 10 -3 M) from each dye. Then, the (0.5 mL) of each dye was took from their stock solution, (1 x 10 -3 M) and diluted with (5 mL) of ethanol, to give (1x10 -4 M) concentration.

The solvent effect
The stock solution of each azo dye and diazo dye was prepared by dissolving weights (0.0215 g) and (0.0347 g) respectively in (50 mL) of solvents, (ethanol, methanol, water, DMSO, DMF, chloroform and ethyl acetate) to give the resulting concentration (1 x 10 -3 M) from each dye in each solvent. Then, the (0.5 mL) of each dye was took from their stock solution, (1 x 10 -3 M) and diluted with (5 mL) of each solvent, to give (1x10 -4 M) concentration.

The toxic effect of azo and diazo dyes in the haemolytic red blood cells
A stock solution (200 mg / mL) was prepared, then a series of diluted (1000, 500, 250, 125,62.25) ⁄ solutions were setup individually (0.8 mL) with red blood cells (0.2 mL) in Eppendorf tubes. Two tubes then equipped. At that point, Ringer solution (0.8 mL) was added to the first tube as a negative control, then the tap water used as a positive control in the second tube. Followed by adding 0.2 mL of red blood cells to each tube. These tubes kept for 3 hours at 37⁰C in a special incubator, and the results recorded.

Result and Discussion
The azo dye (1) and the diazo dye, (2) were prepared as seen in Scheme (1) below.
Conc.HCl / aq.NaNO 2 The synthetic dyes, (1) and (2) were derived from the amoxicillin drug using with optimize stoichiometry and conditions of the reactions. Then, the azo and diazo dyes were characterized by IR spectrum, mass spectrum, 1 H NMR spectrum and the UV-visible spectrum. The IR spectrum of the (1) and (2), (Figures 1 and 2) were showed the stretching vibration of the ν (O-H) and (N-H) groups in the regions 3223.05 cm -1 and 3442.94 cm -1 respectively. But, the (N-H) groups of NH 2 were in the regions 3157.05 cm -1 and 3161.33 cm -1 respectively. the stretching vibration band of ν (N=N) was looked at 1465.90 cm -1 and 1465.90 cm -1 respectively [2] [3]. Additional bands considered as skeletal vibrations, the (C=C) seems at 1506.41 cm -1 and 1494.83 cm -1 respectively[2] [3]. The (C=O) and the aromatic CH bands were appeared in the regions (1614.42 and 3092.83) cm -1 and (1616.35 and 3091.89) cm -1 respectively [3]. Though, the C-N bands were appeared in the regions1168.86 cm -1 and 1168.86 cm -1 respectively. The (O=S=O) asymmetric w. and symmetric s. bands were appeared in the regions (1334.74 and 1168.86) cm -1 and (1334.74 and 1168.86) cm -1 respectively.    Therefore, the mass spectrum was showed that the peak of (1) and (2) at m/z were equal to 431 and 695 respectively as seen in figures (3) and (4).      The UV-visible spectrum was documented at the range (320-520) nm in ethanol for each synthetic dye. The absorption spectrum of synthetic azo dye (1) and diazo dye (2) were showed bands at (360 nm and 410 nm) and (330 nm and 380 nm) related to (π -π*) and (n-π*) respectively. Analytical studies were also curried on (1) and (2), first of all, the solvent effect of (1) and (2) were intended, (Figures 1 and 2) using set of different solvents. The results displayed that the best solubility of (1) in methanol and (2)    Each of azo dye (1) and diazo dye (2) was gave different values of λ max , (Table 1) attributed to π -π* transition of the azo group and indicates the absence of the hydrazone formula within the studied region.  The table shows the ‫ג‬ max (nm) in the absorption spectrum for each dye. The azo dye (1) was displayed a slight red shift towards a higher wavelength using methanol and ethyl acetate in contrast with the diazo dye (2). These results indicated that the synthetic dyes were affected by the solubility and dielectric constant (D), which can be expressed in relation to Gati and Szalay [15] as follows: The F(D) and Φ(D) were also calculated, ( Table 2) which gave a linear relationship when the dielectric constant is the only effect controlling the beak shift.  The results, (figures 9 and 10) also indicated, that there is no deviation from the linear relationship, which is due to the fact that the effect of the dielectric constant can control the shift of the absorption beaks.  Figure 9. The relation between the dipole moment constants and the maximum wavelengths using different solvent.
1-Ethanol , 2-Methanol , 3-Water , 4-DMSO , 5-DMF , 6-Chloroform , 7-Ethyl acetate . Figure 10. The relation between the dipole moment constants and the maximum wavelengths using different solvent. Further, the pH effect in the range of (320-620) nm was also studied for each dye (1) and (2) in pH (1-12) using M concentration as seen in figures (11) and (12) below.  The results revealed suitable pH values at pH12 in each dye with one and two isopiestic points as seen in Figures (11 and 12) above respectively. Due to calculate the pK a and pK b in each of (1) and (2) applying half height method [16], (Figures13 and 14).   The following equilibrium schemes, (2 and 3) displays the suggested ionization of azo and diazo dye respectively in acidic and basic medium.     The method [14] applied for toxity measurement using haemolytic red blood cells in vitro. The results were showed that all prepared compounds harmless and didn't display haemolysis effect in the cells, using different concentrations.