*3.2. Antioxidant Activity*

Both in the DPPH and ABTS assays, imidazole presented EC50 of >15 and >10 mg/mL, respectively (Table 1), which compared to most of the results shown by its derivatives, suggests that the 2,4,5-triphenyl substitution in the imidazole heterocyclic is relevant for the antioxidant activity of these compounds, where the e ffect of their substitutions on their A ring is further developed below.

The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging method is widely used to evaluate antioxidant activities in a relatively short period of time compared to other methods. The results of this assay are shown in Table 1, comparing the synthesized products with the standard quercetin, where the most active synthesized imidazole derivatives were **3**, **10**, **7**, and **2** with values of EC50 of 0.141, 0.174, 0.341, and 1.389 mg/mL, respectively. These results show that the presence of electron donating groups such as hydroxy and *p*-dimethylamino on an aromatic ring bonded to imidazole are essential in the antioxidant activity. The consulted literature indicates that this could be due to the free pair of electrons in nitrogen or in the oxygen of the hydroxy group, which can react with free radicals, being favored due to their aromatic ring stabilization [29]. The rest of the compounds presented low activity in this assay, mainly because of their lack of acidic hydrogen in the aromatic system of position two (A ring); instead, compounds **4**, **5**, **6** and **8** bear methoxy groups, there is a chlorine atom in **9** (EC50 of 5.62 mg/mL), an electron attractor e ffect of the NO2 group in products **11** and **12**, and an anthracene group in **13**.

It is interesting to point out the di fference in antioxidant activity between isomers **2** and **3**, where it is shown from the last one that there was a higher oxidative inhibitory potential in both employed techniques (DPPH and ABTS). It is well known that the antioxidant mechanisms of phenolic compounds are hydrogen atom transfer and single electron transfer, in order to inhibit free radicals, which are the expected mechanisms for the phenolic hydroxyls present in these isomers. These di fferent results could be due to the fact that even though both compounds can transfer their hydrogens because of their high acidity, hydroxyl in **2** is in an *ortho* position, favoring the formation of a hydrogen bond along with a nitrogen of the imidazole nucleus, and forming a 6-membered stable ring. It is referenced that these hydrogen bond interactions can diminish the hydrogen dissociation and therefore the antioxidant ability of these groups [30].

In 2015, Hemalatha et al. [31] evaluated the antioxidant activity with a DPPH assay of compounds **2**, **3**, and **10**, reporting IC50 values of 0.003, 0.0037, and 0.0031 mg/mL, respectively, while the IC50 values for the same compounds in our analysis were 1.389, 0.141 and 0.174 mg/mL, respectively. Even though there were notorious di fferences between both results, establishing a direct comparation was complicated due to di fferences in the methodologies employed for the assay, as in [31], a higher concentration of the DPPH radical was employed, and the incubation times for the reactions were not stated.

In a similar way to the DPPH methodology, the ABTS radical-scavenging assay showed that compounds **10**, **3**, **2**, and **7** with EC50 values of 0.162, 0.168, 0.188 and 0.199 mg/mL, respectively, were the most active products, however, compound **13** showed moderate activity, while products **1**, **4**, **5**, **6**, **8**, **11**, and **12** presented low activity, as can be seen in Table 1. With these compounds, once again, it is important to emphasize that the participation of hydroxyl and dimethylamino groups play an important role as free radical scavengers. Several reports have discussed the possible mechanisms involved in ABTS+ quenching, suggesting the mixed hydrogen atom transfer/single electron transfer reaction mechanisms [32], and some groups have these properties of chemical reactivity such as *<sup>N</sup>*,*<sup>N</sup>*-dimethylaniline derivatives, which can generate e fficient and stable radicals [33].
