Synthesis of some New Azo Compounds of Salicylic Acid Derivatives and Determination of their In vitro Anti-inflammatory Activity

This study included the preparation of a series of some new azo compounds by diazo coupling aromatic amines with salicylic acid derivatives. The prepared compounds identified using precise elemental analysis (C.H.N.), the results supported the structure of concerned compounds. The synthesized azo compounds also identified by using infrared spectroscopy and 1H-NMR spectroscopy. Anti-inflammatory activity of the compounds were determined in-vitro by human red blood cell (HRBC) membrane stability method, the compounds showed a significant activity to protection of the cell membrane. Other compounds show moderate to low activity, sodium diclofenac was used as positive control.


INTRODUCTION
Compounds containing in their structure a group or more of the AZO groups (-N=N-) called azo compounds 1 , in which the nitrogen atom hybridization is sp 3 2 .Because of their physical and chemical properties as well as their biological efficacy, they possess several important applications in pharmaceuticals, cosmetics, textile industry, analytical chemistry and food 1 Azo compounds are well known to have medical importance, they are recognized in many applications such as antidiabetics 1 , and they involved in many biological reactions such as inhibition RNA, DNA, protein synthesis carcinogenesis, and nitrogen fixation 3 .Azo compounds are studied as HIV inhibitors of viral replications 4 .Because of the presence of the azo moiety make these compounds possess biological efficacy as anti-bacterial 5 , antitumor 6 insecticide and pesticidal 7,8 activities.Salicylate compounds are widely valued because of their antipyretic, pain killing and anti-inflammation properties.2-Hydroxybenzoic acid (called also salicylic acid) is most commonly used, and known in salicylates class compounds 9 .
The aim of study synthesis of some new azo compounds of salicylic acid derivatives by the diazo coupling reaction and determine their In-vitro anti-inflammatory activity by human red blood cell (HRBC) membrane stability method.

MATERIAL AND METHODS
p-Nitroaniline, p-aminobenzoic acid, m-nitroaniline, 3-methoxysalicylic acid, 3-methylsalicylic acid and 4-methylsalicylic acid and solvents were used in this study sourced from Sigma-Aldrich company and Merck & Co.The purity of prepared compounds was checked by thin layer chromatography.Melting points recorded by using Gallenkamp apparatus.FT-IR spectra (KBr) of prepared compounds determined on Shimadzu spectrometer (400-4000 cm -1 ).The proton nuclear magnetic resonance ( 1 H-NMR) spectra determined

General method for synthesis of diazonium salts
A solution of an aromatic amine (5 mmol), 1.5 ml of water and 1.5 ml concentrated HCl kept cooled in an ice-salt bath (0 o C).A solution of sodium nitrite (5.5 mmol) in 1.5 ml of water added slowly with stirring.The mixture kept at 0 o C. for the next step 10,11 .The other diazonium salts synthesized in a similar procedure.

General method for synthesis of azo compounds
The prepared solution of diazonium salt was added portion wise to a solution prepared from salicylic acid derivatives (5.4 mmol) and 10 ml of 2.5 M aq.Sodium hydroxide.The mixture kept with stirring at (0-5 o C) for 3-5 hours.The mixture then acidified with conc.HCl (1.5 ml) up to pH ≈ 3. The precipitated compound separated and washed with H 2 O.The desired product dried and recrystallized with glacial acetic acid 10,11 .Fig. (2) show synthesis of azo compounds.The melting points, names and the percentage of yield are given in Table (1).
In vitro anti-inflammatory activity 12 HRBC method was used to estimation In vitro anti-inflammatory activity.Blood was collected from healthy volunteers; the blood was mixed with equal volume of sterilized Alsever's solution.The blood solution centrifuged at 3000 rpm and the packed cells were separate.The packed cells were washed with isosaline solution and 10% v/v suspension was prepared by complete the volume with isosaline.Alsever's solution were prepared of 2.05% glucose, 0.42% NaCl, 0.8% trisodium citrate, 0.055% citric acid, all dissolved in water.This solution was using for storage RBC.Other solution were using in this method Hyposaline (0.7% NaCl), Isosaline (0.9% NaCl), phosphate buffer (pH 7.4) and ethanol.

Fig. 2. Synthesis of azo compounds
Tested compounds (75 mg) was dissolve in 1 ml of ethanol.Samples of each compound, control and sodium diclofenac were separately mixed with (1 ml) phosphate buffer, (2 ml) of hyposaline and (0.5 ml) of HRBC suspension.All the assay mixtures were incubate at 36.5 O C for 30 min and centrifuged at 3000 rpm for 10 minutes.The supernatant liquid was decanted and haemoglobin content was estimate by spectrophotometer at 560 nm.The percentage of haemolysis protection was estimate by assuming the haemolysis produced in the control as 100%, according to following equation.

Percentage protection=100-(Ac-As/Ac)
Were Ac= Absorption of control and As= Absorption of sample

Statistical analysis
The results of anti-inflammatory activity were analysis in one way analysis of variance (ANOVA).Value with probability (p<0.01) was considered significant.

RESULTS AND DISCUSSION
Table (2) show CHN analysis of synthesized azo compounds (A-I) and the practical results support the structure of synthesized compounds.

H-NMR spectra
The proton-NMR analysis of prepared compounds performed by using deuterated dimethyl sulfoxide as a solvent.The 1 H-NMR spectra of all prepared azo compounds showed a singlet signal within the ranges (2.264-2.296ppm), (3.918-3.931ppm) and (2.681-2.725ppm) for the groups 3-CH 3 , 3-OCH 3 and 4-CH 3 respectively.Compounds A, B and C showed a singlet signals at (7.785-8.042ppm) and (8.132-8.507ppm) which attributed to protons H-4 and H-6 respectively.The 1 H-NMR spectra of the synthesized compounds for compounds D, E and F showed singlet signals at ranges (7.675-7.732ppm) and (8.050-8.444ppm) for protons H-4 and H-6 respectively with 4J= 2.1.In addition to that compounds G, H and I appeared singlet signals at (7.013-7.072ppm).Proton (H-6) for compounds G and H appeared at 8.171 ppm and 8.175 ppm respectively [15][16][17] , the general structure of azo compounds was shown in Fig. (3).Other aromatic protons of all azo synthesized compounds summarized in Table (4).The data of 1 H-NMR spectra of synthesized compounds reported in Figures (4 -12).From the results, compounds B, E and H show high activity compared with other compounds.The active compounds B, E, and H contain a nitro group at meta position.Replacing nitro group with carboxyl group will be minimize the activity.Therefore, the activity is the best when a nitro group located at meta position.In salicylic acid moiety, the activity of protection was found increased when methyl group located at para-position compared with the metamethyl and meta-methoxy groups.Compound E contain meta nitro group and meta methoxy group, it is the compound with meta-methoxy group has high activity than other methoxy derivatives.The results showed compounds A, D, F and I have less activity.The action of the azo compounds could be related to the binding of compounds with erythrocyte membrane, especially phospholipids.Since the compounds has polar and nonpolar groups can bonded with same groups of phospholipids.This prevents membrane damage by physical interaction of osmotic pressure differences, which is causative to hemolysis of red blood cells 19 .

CONCLUSION
In this study, three series of azo were prepared using three different of salicylic acid derivatives.The prepared compounds identified using the element analysis (CHN) as well as infrared and 1 H-NMR spectroscopy.The results supported the structures of prepared compounds.All compounds showed significant In vitro anti-inflammatory activity.Compounds B, E and H showed high antiinflammatory activity compared with other prepared azo compounds.