DPPH scavenging activity of some Bis-benzimidazole derivatives

As part of our research on substituted benzimidazoles, we are interested in the synthesis of new heterocyclic molecules. This new organic molecule is a subclass of quinolines with a wide variety of biological properties. In order to affect the binding of quinoline to our bis-benzimidazole derivatives, we have chosen the "azo" bond as a means of attachment. To achieve our goal, we investigated different parameters for the reactions to determine the conditions to obtain the best results. This article discusses the antioxidant activity of our molecules using the DPPH method.


Introduction
Heterocyclic molecules belong to a class of cyclic compounds harboring one or more heteroatoms, containing heteroatoms of the same element or even different ones different 1,2 .The role of heterocyclic compounds has become more and more important in recent years particularly in the design of new classes of compounds with demonstrated activities (corrosion inhibitors, dyes, and stabilizers…), as well in the medicinal field (vitamins, hormones, antibiotics, and antineoplastics …) 3,4 .Among these heterocyclic compounds are the benzimidazoles, the series of benzimidazole which is one of the most versatile in therapeutic chemistry.
Benzimidazole is an important pharmacophore and a privileged structure in medicinal chemistry.This bicyclic compound consists of an imidazole ring, containing two nitrogen atom at adjacent position fused to a benzene ring [5][6][7] .

Figure 2. Bis(1H-benzimidazole)
These heterocycles can be prepared by different synthetic methods [10][11][12][13][14][15] .Recent studies have shown that these heterocycles have several biological applications in chemistry 16 and in biology 17,18 .This work discusses the antioxidant activity of our compounds.Antioxidants are chemical compounds capable of effectively minimizing retentions, a retardant of lipid peroxidation, with no effect on the sensorial and nutritional properties of the food product 19 .They help to retain the quality and increase the shelf life of the product.
Several in vitro and in vivo methods are used to evaluate, in vitro and in vivo, the antioxidant activity by trapping different radicals, such as peroxides (ROO•) by the methods ORAC (Oxygen Radical Absorbance Capacity) and TRAP (Total Radical Trapping Antioxidant Parameter); Ferric ions by the FRAP method (Ferricion Reducing Antioxidant Parameter) or the ABTS• (ammonium salt of 2,2'azinobis-3-ethylbenzothiazoline-6-sulfonic acid), as well as the method using the free radical DPPH• (diphenyl-picrylhydrazyl).
This work discusses the antioxidant activity of a set of Bis-benzimidazoles developed in our research group using the DPPH method.

General procedure for the synthesis
In the first Time, the unsubstituted bis(benzimidazole) derivatives are synthesized (Fig. 3) with a condensation reaction of 1,2-phenylenediamine a with diacids: Sebacic acid and Terephthalic acid.The nitration of our molecules is carried out according to the procedure described in the experimental part 1 .By nitrating the unsubstituted Bisbenzimidazoles such that this nitration reaction is carried out at 0-5 °C with HNO3 (65%) in solution with H2SO4 (92%).The reduction was carried out by the use of tin chloride in the presence of acetic acid and hydrochloric acid according to a standard procedure 20 .These compounds are excellent synthetic precursors of other multicyclic heterocycles of imidazobenzodiazepine and imidazoquinolinone types.

Antioxidant activity
The measurement of the antiradical activity was tested according to the method developed by Blois 21 as described by Brand-Williams et al. 22 with some modifications.The principle of this method is based on the measurement of the free radical scavenging of DPPH (diphenyl picrylhydrazyl) dissolved in methanol (MeOH).The antioxidant activity of our samples and the standard antioxidant (ascorbic acid) vis-à-vis the DPPH radical was evaluated using a UV spectrophotometer following the reduction of this radical (Fig. 7) which was accompanied by its change from violet (DPPH•) to yellow (DPPH-H) measurable at 517nm.This reduction capacity is determined by a decrease in the absorbance induced by antiradical substances 2 3 .
The DPPH solution is obtained by dissolving 4 mg of the powder in 100 ml of methanol.Bisbenzimidazole samples were prepared by dissolving in DMSO at final concentration of 0.2 mg/mL.The test is carried out by mixing 4 mL of the previous solution of DPPH with 1 mL of the sample to be tested at different concentrations.
At the end of the incubation period, the absorbance at 517 nm is read and the antioxidant activity is calculated according to the following formula: AControl: is the absorbance of the control (containing the different concentrations of the reagent) Atest: is the absorbance of the samples.AA: is the antioxidant activity

Result and discussion
After 30 minutes of incubation of the DPPHsample, the solution changes from purple color to yellow in most samples, this change in color is due to the reduction of DPPH, which shows that the samples have a scavenger effect of radical DPPH.The results of the antiradical power by the DPPH of the different samples are gathered in Table 1.We note from the previous results that the antioxidant activity of the compounds coupled with the 8-Hydroxyquinoline molecule is significantly greater compared to the unsubstituted Bisbenzimidazole, bis(nitrobenzimidazole) and bis(aminobenzimidazole) but the antioxidant activity of the ascorbic acid of 60,57% remains high as that of Bis-benzimidazole products.It has been clearly demonstrated that the parent molecules have no significant antioxidant activity while its derivatives have an important antioxidant activity.A. Ouaket et al.  106   After this step, the compounds which have an DPPH inhibition rate of more than 40% (highlighted in yellow in Table 1, compounds 4b, 5a and 5b), were selected to study their antioxidant activity versus concentration (0; 0,05; 0,1; 0,2; 0,3 and 0,4 mg / mL).The data obtained is summarized in Table 2 and Fig. 8.

Figure 8. Percentage inhibition curve as a function of the concentration
The inhibition percentages are 46,39%, 49,92% and 48,05% for 5b.It is noted that the antioxidant efficiency increases with the concentration.

Conclusion
This article aims to evaluate and compare the antioxidant efficacy of benzimidazole molecules.The results of the DPPH test revealed that our molecules have very good anti-radical activities.
This study contributes to the knowledge of the antioxidant potentials of our molecules in vitro, it would also be interesting to carry out other studies to evaluate the antioxidant potential in vivo to correlate the results observed in both cases, and publish them soon.

Figure 7 .
Figure 7.The reaction of an antioxidant with the radical DPPH

Table 2 .
The percentage inhibition of different concentrations of our samples.