Synthesis, Characterization and Biological Evaluation of Some Azo and Azo-Schiff Compounds

This work involves the preparation of some new azo and azo-Schiff compounds derived from benzothiophene, dibenzothiophene, and diamines (1, 5-diaminonaphthalene, and thiocarbohydrazide). Azo compounds of benzothiophene and dibenzothiophene have been prepared using different solvents to enhance the miscibility of the polar aqueous solution of the diazonium salt and the slightly polar benzothiophene and dibenzothiophene. The Schiff’s bases have been synthesized by the condensation reaction of carbonyl compound with the diamines. FT-IR has been used to identify the prepared compounds, whereas 1 H-NMR and 13 C-NMR were used to identify some others.


Introduction:
Aromatic azo dyes, which are characterized by the functional group (-N=N-), are considered the largest group of organic dyes as they have widespread applications in many areas of textile and medicine [1,2]. The dyes are stabilized by incorporating the aromatic group through conjugation, which very often absorbs visible light frequencies yielding colored compounds [3].
Dyes are normally large aromatic molecules that are consisting of more than one linked ring [4]; the more stable derivatives are containing two aryl groups [5]. Prontosil is an azo dye that acts as an antibacterial drug, it is metabolized in vivo to produce the active amine part (sulfanilamide) [6]. Schiff bases resulted from salicylaldehyde condensation have arouse the researchers' interest because of their ability to act as bidentate ligands for transitional metal ions [7,11]. Studies concerning quantitative structure-antitumor activity relationship of a series of Schiff bases derived from variously substituted aromatic amines and aldehydes, it has been shown that azomethines from salicylaldehydes gave the best correlation [12,13]. The reported biological activity of azo dyes and the pharmacological activity of Schiff bases mentioned above were the motive to conduct this research.

Experimental:
The chemicals used in the synthesis of the studied molecules were of the brands (Aldrich, Alfa aesar, BDH, Merck and Sharlau) and they were used as they received from suppliers.
Melting points have been determined by the use of open-ended capillary tube instrument and are uncorrected. Infrared spectra were recorded on Thermo Fischer Scientific Nicolet IR100 spectrometer, 1 H-NMR and 13 C-NMR were recorded on a Bruker 300 MHz ultra-shield with TMS as internal reference using CDCl 3, DMSO-d6 and D 2 O as solvents

Preparation of 1-(4-substituted phenyl)-2-(dibenzothiophen-2-yl) diazenes (H 1 -H 6 ), and 1-(benzothiophen-3-yl)-2-(4-substituted phenyl) diazenes (H 7 -H 11 ):
Substituted anilines 0.002 mol were dissolved in 4 ml of 3M hydrochloric acid in a round bottom flask equipped with magnetic stirrer in an ice bath, then an icy solution of sodium nitrite (0.002 mol, 0.138 gm in 2 ml of water) was added dropwise keeping the temperature in the range of 0-5 o C. The resultant diazonium salt kept in ice bath. Then an icy solution of [(0.002 mol, 0.368 gm) of dibenzothiophene (for compounds (H 1 -H 6 )) or (0.002 mol, 0.268 gm) of benzothiophene (for compounds (H 7 -H 11 )] in 4 ml of acetic acid was added to the diazonium salt solution and the resultant mixture was vigorously stirred for 30 minutes. A solution of 2.5 M of sodium hydroxide was added slowly keeping the temperature under 5 o C until the pH level was above 7. The reaction mixture was kept under stirring in a cold medium for another 30 minutes. The resultant solid was filtered in a Buchner funnel and washed with water.
Recrystallization of the products with a mixture of 90% Ethanol in water yielded the following derivatives and their data:

Biological screening of some of the synthesized compounds:
The biological activities of the some of the synthesized compounds have been examined against Staphylococcus aureus and E.coli bacteria. Bacteria were cultured on an Agar plate and a solution of 1% (w/v) of each of the compounds in DMSO was used to prepare the sampling discs. The results were recorded depending on the diameter of inhibition of bacterial growth in mm as shown in Table 1. Highly reactive: (inhibition zone < 30mm), Active (inhibition zone 20mm -30mm) Slightly active (inhibition zone 14mm -20mm) [22].
The new Azo dyes and Azo-Schiff compounds prepared were identified by FT-IR, and some of them by 1 H NMR. The synthesized compounds were screened against two types of bacteria for the evaluation of their biological activity.
The 1 H NMR spectrum of compound H 26 showed a single peak at 1.28 ppm which was assigned to the methyl groups, while the thiocarbohydrazide moiety N-H was recorded as singlet at 2.2 ppm. The multiplet peaks within the range (6.9 -7.9) ppm were assigned to the aromatic protons.
The protons of CH=N group were recorded as singlet at 7.25 ppm, while the protons of the two OH groups shown single peak at 9.74 ppm. For the compound H 29 , the 1 H NMR spectrum showed a multiplet in the range (6.9 -8.6) ppm that is attributed to the aromatic. A single peak showed at 9.2 ppm that is attributed to the imine group proton. And for the OH group it was assigned 10.1. Due to the deshielding effect of internal hydrogen bonding with the imine group, both the OH and the imine groups are shifted towards downfield.

Conclusion:
The aim of this work is to synthesize, study spectroscopically, and evaluate the Anti-bacteria activity of some new azo dyes containing benzothiophene and dibenzothiophene and Schiff's bases containing more than one functional group. These compounds are very stable which makes them suitable to be used in pharmacological and biological researches. Having more than one functional group makes them versatile molecules to be used in different reaction types and a good candidate for stable active photosensitizers [15].
The use of 4-aminoacetophenone as a building block in the synthesis of macro molecular Schiff bases made it possible to attach additional phenyl groups to the diamines, i.e. 1,5diaminonaphthalene and thiocarbohydrazide. Compound H 2 showed the highest value of biological activity among the other compounds studied. This anti-bacterial activity might be due to the effect of the azo group in the compound, although it has been reported that the activity of the azo compound prontosil against bacteria is due to its biodegradation product sulfanilamide which is liberated due to the action of the enzymes inside human body [24].