Modification and Study Biological Activity of Chitosan with Compounds Containing Azo Group

safe and antibacterial polymer 9,10 . Due to its ability Abstract In the present research synthesis and study of biological activity a series of new polymers modified of chitosan with compounds containing azo group. Beginning diazonium salt produced from 3,3'-dimethyl- [1,1'-biphenyl]-4,4'-diamine reacted with concentrated HCl acid and sodium nitrite. The coupling reaction between diazonium salt with substituted aromatic aldehyde to produce Azo derivatives )1-6(. Azo Schiff bases Chitosan )7-12 ( were synthesized by condensation of Chitosan with Azo derivatives )1-6( in ethanol with some drops of glacial acetic acid. The structural modifications of Chitosan ring (linked to a bioactive azo moiety) were expected to give new derivatives )7-12( with a diverse range of biological functions. These compounds' structures have been determined using FT-IR, 1 H-NMR spectroscopic and Field Emission Scanning Electron Microscopy studies. Additionally, two other kinds of bacteria: Staphlococcus aureus and E. coli were tested for possible antibacterial properties utilizing some new compounds. Modified Chitosan (7-10) showed high activity comparable to a penicillin (used as the reference antibiotic), Especially the modified polymer(7), which showed high inhibition against both types of bacteria . The anticancer activity of modified chitosan (7) against MCF-7 (human breast carcinoma cells) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to determine and compare with normal cells WRL-68(the human hepatic cell line). Polymer (7) exhibited a high cancer cell inhibition rate and less toxicity to normal cells.


Introduction
Azo compounds are very important organic compounds having a wide spectrum of biological activities.Diazonium coupling reactions are typical electrophilic aromatic substitutions in which the positively charged diazonium ion in the electrophile interact with the electron-rich ring of substituted benzaldehyde 1,2 .Schiff bases, having imine groups (C= N) are formed by nucleophile addition (condensation reaction of (NH2) primary amines with carbonyl groups (C=O) of aldehydes or ketones.These compounds have been used for industrial purposes such as pigments, catalysts, liquid-liquid extraction, active transport, intermediates in organic synthesis and as polymer stabilizers [3][4][5][6] .Azo compounds, such as the azobisisobutylonitrile (AIBN) may be utilized as radical initiators in the polymerization of the alkenes for making plastics 7 .The aromatic azo compounds have been utilized as acid-base indicators like the methyl orange, methyl red and Congo red 8 .Chitosan Published Online First: August, 2024 https://doi.org/10.21123/bsj.2024.9453P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal to produce bio functional materials, it has a wide range of biological activities including anti-diabetic, anti-oxidant, anti-bacterial activities, antimicrobial, antitumor and is used pharmaceutically as an anticoagulant agent [11][12][13] .It can also be helpful in several fields including textiles, environmental protection, water treatment, cosmetics and biotechnology.Due to the presence of -OH and NH2 groups, which separate chitosan from cellulose, its structure is easily changed to produce various derivatives [14][15][16] .The modified chitosan exhibits new properties like biological activity and biocompatibility [17][18][19] .Because of all the above datum facts, This study is aimed at the achievement of the syntheses, characterization and study of the biological activities of some synthesized new polymers modified of chitosan based to Schiff bases containing azo groups, whose molecules include the three moieties , chitosan , imine group (-N=CH-)and azo groups together 20 , since the combination of all these biologically active moieties in one molecule may increase the likelihood of producing more potent newly-developed prodrugs with a wide range of diverse biological activities (antibacterial and anticancer), as an attempt to correlate the biological results with their structural characteristics.

Materials and Methods
All chemicals have been supplied from CDH , SCR and BDH .The FT-IR Spectra have been registered on Shimadzu FT-IR-8400 s, ranging between 400-4000cm -1 , using the potassium bromide disk.Company: Ultra Shield 400MHz, Bruker, University of Basrah, Iraq performed the 1  3,3'-Dimethylbiphenyl-4,4'-diamine (2.12g.,0.01mole) has been dissolved in 2mL of the 2N hydrochloric acid and 20mL of the distilled water.This solution has been cold at 0-3 o C in an ice-water bath.Sodium nitrite (1.38g.,0.02mole) has been dissolved in 10mL of the distilled water and added dropwise to a cold solution while stirring.The mixture ) diazonium solution ( is stable for a few minutes.The above cold diazonium solution was added slowly to a well stirred solution to 0.02 mole from different substituted benzaldehydes : (2.98g.) of N,N-dimethylbenzaldehyde , or (2.81g.) of 2chlorobenzaldehyde, or (2.44gm) of 4hydroxybenazldehyde, or (3.13g.) of 5-chloro-2hydroxybenzaldehyde, or (3.04g.) of 2-hydroxy-3methoxybenzaldehyde or (3g.) of 4-Ethoxybenzaldehyde) in 20mL of absolute ethanol and 5mL of 10 % sodium hydroxide and the mixture was cooled to a temperature of 0-5 o C with stirring for 2hrs in order to obtain coupling agent.The progress of the reaction was monitored by TLC.When the reaction was completed, the orange to red compound result was precipitated, then filtered and recrystallized from absolute ethanol 21,22 .Physical properties of compounds(1-6) are listed in Table .1

Synthesis of Azo Schiff bases Chitosan (7-12)
A mixture of ethanol (10 mL) and glacial acetic acid (5 mL) was added to chitosan (0.5 g.), which was then dissolved and stirred for 30 minutes at room temperature, then add one of azo derivatives(1-6) (0.01mol).The mixture was heated by stirring for 24 hours in a water bath at a 60°C.The reaction mixture was cooled , and the residue produced was filtered, washed with EtOH, dried at room temperature for 24 hours 23 Biological Activity : Antibacterial Activity Some of the synthesized compounds and modified polymers have been screened for antibacterial activities against (Staphylococcus and Escherichia coli) using cup-plate agar diffusion method 24 .Penicillin (50µg /ml) was used as a standard drug for antibacterial activity.These sterilized agar media were poured into petri dishes and allowed to solidify.Some of the synthesized compounds (50µg /ml) were placed serially in the cavities with the help of a micropipette and allowed to diffuse for 1 hr.DMSO was used as a solvent for all the compounds and as a control.These plates were incubated at 37 o C for 24 hr for antibacterial activities.The zone of inhibition observed around the cups after respective incubation was measured in mm .Anticancer Activity 25,26 The cytotoxic effect of modified chitosan (7) against MCF-7 (human breast carcinoma cells) was studied and compared with normal cell line WRL-68(the human hepatic cell line).The anti-proliferative activity of modified polymer (7) was tested by studying their ability to inhibit the proliferation of human breast carcinoma cells (MCF-7).The MTT test was used in 96-well plates to investigate the cytotoxic impact of polymer (7).Cells were treated with polymer (7) after 24 hours or when a confluent monolayer was established.After 24 hours of treatment, cell viability was determined by removing the medium, µl/well solutions of MTT and incubating for 4 hr.at 37°C.The crystals in the wells were solubilized after the MTT solution was removed by adding 200 mL of DMSO (Dimethyl Sulphoxide) and incubated at 37 °C for 15 minutes while shaking.with the use of a microplate reader, the absorbency was determined at 620 nm.

Results and Discussion
Azo aldehyde derivatives )1-6(, Scheme.1 were obtained by coupling reaction between diazonium salt with two moles of substituted aldehydes.The new compounds (1-6) were identified by FTIR and 1 HNMR spectroscopy obtained for elegant compounds are listed in Tables 2& 4 respectively.
The novel polymers modified of chitosan Scheme 2, were identified by FTIR and 1 HNMR spectroscopy 23 .
The FT-IR spectral data are given in Table 3. 1     Antibacterial Activity [27][28][29] Anti-bacterial activities of some of the synthesized compounds and modified chitosan (azo schiff bases chitosan) were observed (in vitro) against E. coli (G-) and (G+) Staphylococcus aureus, based upon agar diffusion approach.A standard drug (50μg/mL of Penicillin) has been utilized for comparison with synthesized azo compounds and modified chitosan.
The reason for the increased effectiveness is the presence of chitosan (biopolymer), which has biological activity against bacteria because it contains amino groups with positive charges that bind with the negative charges present on the surface of the bacterial cell, which causes a change in its properties.Such as the permeability of its cell membrane and an imbalance in osmosis processes, https://doi.org/10.21123/bsj.2024.9453P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal which results in preventing the growth of bacteria, Figs. 1, 2. All of the compounds and their antibacterial activities have been listed in Table 5.
Table 5. zone of inhibition (in mm) of some synthesized azo compounds (1-4) and modified chitosan (7-10) .FESEM micrographs have been utilized to study changes in surface morphology for prepared polymers( 7) and ( 12).Figs.3,4 shows the modified chitosan's surface morphology.In FESEM images, it can be noticed increasing average size of the pores in comparison with chitosan pore size.Variations of surface morphology result from new bonds in the polymer that have been prepared.Anticancer Activity [35][36][37][38] : The results demonstrated the ability of prepared modified chitosan to destroy and kill cancer cells as shown in Figs.5-7.Table 6 shows the inhibition ratio of the modified polymer(7) equal is 84.65 while table 7 shows cell viability% is 15.35 .Table 8 show the activity of polymer( 7) against cancer cells is dependent on concentration ,the inhibition rate at the concentration (10, 25,  Chitosan or its derivatives selectively penetrate tumor cells and exhibit anti proliferative activities via antiangiogenic, immunoenhancing, antioxidant defense, apoptosis and enzymatic regulation possibly because azo molecules are involved in the inhibition of DNA, RNA and protein synthesis, as well as hindering carcinogenesis.In addition, the presence of (N=N) in the azo molecular structure is accountable for the interaction with the active site of the target protein https://doi.org/10.21123/bsj.2024.9453P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal

Figure 7 .
Figure 7. (a) Image of the MCF-7 well before staining (b) Image of the MCF-7 well after Staining