*3.3. Acetylcholinesterase Inhibitory Assay*

In this assay, galantamine was more active than the products evaluated. Nevertheless, as an initial screening a structure activity relationship is attempted to obtain valuable information for future research.

Among the synthesized products, compound **1** showed the best activity with 25.8% of inhibition (Figure 1). AChE inhibitors bond with the enzyme in a well-known gorge, which in its bottom presents a Trp residue (Trp84 for *Tetronarce californica* AChE, the enzyme used for the in vitro assay). This residue is of crucial importance for ligand interaction by means of a π–cation interaction [34,35]; however, it can also have purely hydrophobic interactions. In the case of galantamine and donepezil [34,36] this residue presents classical π–π stacking with a galantamine double bond, and with the benzyl ring in donepezil. In a similar way, compound **1** could adopt a similar position against AChE, presenting a π–π interaction with Trp84 through its A ring, which has no substitutions that could a ffect the π electron cloud in the ring, thus explaining the result shown. Hydrophobic and π–π interactions tend to be the most observed ones between AChE and the sca ffolds of di fferent inhibitors [37–39].

The next compounds with high inhibition percentages were compounds **11** and **12**, which presented a nitro functionality in their *p*- and *o*- positions. The nitrogen atom in this group is positively charged; in this manner, these compounds could have π–cation interactions with Trp84, or even with Phe330, which is another residue that commonly has this interaction. This could explain why **11** and **12** followed compound **1** with the best results.

Some tendencies seen in the results when comparing **2** (which has an *o*-OH substitution) against **6** (which presents an *o*-OMe one), we can see that the inhibition activity diminishes; the same pattern was observed with **7** and its methoxy version **8**, although the comparison between **3** and **4** appeared as the exception of this behavior. Compound **9** had only 5.9% inhibition activity; as π–π interactions with AChE are important, the chloride presence in **9** could alter the electron cloud from the A ring, disturbing the π–π interactions that can be made.


**Table 1.** Antioxidant activity (EC50) of synthesized compounds **1**–**13.**

\* Served as the reference compound. Values are mean ± SD, DPPH n = 2, ABTS n = 3. ND = Non-detected in the evaluated concentrations. EC50 = Concentration required to decrease the absorbance by 50%.

**Figure 1.** Percentage of acetylcholinesterase inhibition of triphenyl imidazole compounds **1**–**13** (150 μg/mL). Galantamine served as the reference compound.

### *3.4. Xanthine Oxidase Assay*

Although not being as active as the positive control allopurinol, some tendencies in the structure activity relationship of the synthesized compounds can be noticed, as seen in Figure 2.

Comparing compounds **2**–**6** where hydroxy and methoxy substitutions are present, the *p*- substitution can be inferred as a significant requirement for this products, as only *p*-OH and *p*-OMe products showed activity. This was also the case for compounds **7** and **8**, with hydroxy and methoxy groups as substitutions, while having a *para* substitution besides a *meta* one, allowed them to show activity.

It appears that not only the *p*- position is of importance, but also that the functionality in these synthesized compounds must be of -OH or -OMe type, bearing an oxygen as a heteroatom bonded to the aromatic ring. Products **10** and **11** also have substitutions in this position, but with nitrogen as the heteroatom (an amine and nitro group, respectively) and in their case, the *para* position with a nitrogenated group showed no activity. For the synthesized products, the interaction with xanthine oxidase, instead of being similar to the one for allopurinol, which interacts with one of its aromatic nitrogen to bond with molybdenum in the catalytic site of the enzyme [40], could be similar to the topiroxostat one. This inhibitor interacts with the xanthine oxidase molybdenum with its oxygen in a covalent bond [41]. While compound **10** has its nitrogen in a tertiary amine, and **11** in a nitro group, it could be more difficult for them to bond with the Mo center of the enzyme, favoring in our products the presence of oxygen over nitrogen.

Product **3**, having a *p*-OH group and no other substitution that diminishes its activity, resulted in the most active compound from the synthesized ones. Between the hydroxy and methoxy substitutions, it appeared as the first one favored the inhibition activity over xanthine oxidase. Compound **3** with a *p*-OH substitution showed an IC50 of 85.8 μg/mL, while **4**, which has a *p*-OMe, showed almost double the IC50; again, between **7** and **8**, we could see that the methoxy version was less potent than the hydroxy one. This can be related with the observation made for different products with alcohol groups in their structure such as polyphenols, which can form hydrogen bonds with XO via their hydroxyl groups [14,42].

However, the exception to the structure activity relationship discussed was **12**, having an *o*-NO2, which lacked a *para* position and oxygen heteroatom functionality. This compound was achieved as one of the few products with xanthine oxidase inhibition, although it showed the second lowest activity. Further *ortho* nitrogen containing products must be synthesized to expand this analysis.

**Figure 2.** Xantine oxidase inhibitory activity of synthesized compounds **1**–**13**. Allopurinol served as the reference compound. Bars are mean ± SD, n = 3.
