3.2.2. Anticholinesterasic Activity

The results for acetylcholinesterase (AChE) inhibitory activity showed that compound **7** is the only one which presents some activity at the maximum concentration tested (47.1 ± 3.6%, at 150 μg/mL). This activity is less pronounced than the inhibition obtained for control compound donepezil (95.2 ± 0.4% at 50 μg/mL), a pure competitive inhibitor of AChE used clinically in early stages of Alzheimer's disease. The results sugges<sup>t</sup> that the cyclization seems to increase the inhibitory activity of the compound, since compound **7**, which is a flavanone, is more active than compound **6**, the respective chalcone. Also, the existence of free hydroxyl groups in both A and B rings seems to be important because, out of the three compounds with a flavanone structure (compounds **4**, **5** and **7**), only the one with a free hydroxyl group in both C-7 and C-4 (compound **7**) shows activity. This is in agreemen<sup>t</sup> with the strong activity revealed by quercetin, whose structure also possesses free hydroxyl groups in both rings A and B, like compound **7**. So, it can be concluded that the presence of hydroxyl groups in rings A and B of the flavanone scaffold is crucial for the inhibition of AChE activity (Figure 4).

**Figure 4.** Structure/activity relationships established for antioxidant activity.

In terms of butyrylcholinesterase inhibition, compound **6** presented excellent activity, with a percentage of inhibition of 96.0 ± 1.1% and IC50 of 26.55 ± 0.55 μg/mL, which are similar values to the ones presented by donepezil (IC50 of 28.94 ± 1.76 μg/mL) and better than quercetin at the same concentrations. This shows that, unlike what happens with acetylcholinesterase, the presence of three hydroxyl groups in the chalcone scaffold highly favors the inhibition of butyrylcholinesterase activity. The results presented by chalcones **1** (12.51 ± 0.82%) and **8** (0%) also confirm that, like with acetylcholinesterase, the presence of methoxyl groups does not confer inhibitory activity to chalcones.

Compound **7** presents an activity very similar to the one observed for acetylcholinesterase inhibition (46.26 ± 1.27%), meaning that it is a dual inhibitor, a much-appreciated feature in the search for compounds with therapeutic potential towards Alzheimer's disease. The presence of a hydroxyl group in ring B of the flavanone scaffold increases its inhibitory effect, since flavanones **5** (36.30 ± 0.20%) and **7** present higher activity than flavanone **4** (9.00 ± 1.30%), which has a methoxyl group in said position (Figure 4). The compounds inhibited butyrylcholinesterase in a dose-dependent manner (Figure S12, supplementary material).

#### 3.2.3. Antimicrobial Activity

As observable in Table 2, most of the compounds tested inhibited the growth of gram-positive bacteria, but none was effective against the gram-negative strain tested. This inhibition was found to be concentration-dependent (Figures S13 and S14).

The most efficient compounds against *Micrococcus luteus* were **6** and **7**, both with similar IC50, which suggests that cyclization does not influence the toxicity of these molecules against this species. These results are concordant with the literature, since it is described that these compounds presented similar activity against *Mycobacterium tuberculosis* [58]. Although some compounds tested exhibit an IC50 value greater than 200 μg/mL, the percent of growth inhibition exhibited at the maximum concentration tested allows one to deduce some interesting structure/activity relationships, which are discussed below. The chalcone **1** had no effect against *M. luteus*, while chalcone **8** presents 41.71% of growth inhibition at 200 μg/mL. These results sugges<sup>t</sup> that the presence of methoxyl groups at ring A reduces its activity against this species. The same effect is observed when comparing the activity of compounds **4** and **5** (Figure 5).


**Table 2.** Antimicrobial activity of synthesized compounds **1**, **4**–**8**.

In each column, the letters a, b, c, d, and e indicate significant differences (*p* < 0.05).

**Figure 5.** Structure/activity relationships established for antibacterial effect against *M. luteus*.

Compound **5** inhibits 43.08% of bacterial growth at 200 μg/mL, while this value is almost double for compound **4** (74.43 ± 1.95%), which confirms that the methoxyl groups at ring A decrease the molecule's antibacterial effect.

The strongest activity against *Bacillus subtilis* was displayed by compound **6** (IC50 = 9.33 μg/mL), presenting even better activity than streptomycin (Table 2). The corresponding flavanone **7** has a higher IC50 (20.48 μg/mL), which leads to the conclusion that the <sup>α</sup>,β unsaturated carbonyl bridge linking ring A to ring B is important in the mode of action of the compounds against this bacterial strain. The molecules with the higher numbers of hydroxyl groups are the ones with better activity, showing that these groups also play an important role in the molecule's mode of action against *B. subtilis*. Again, the compounds with two methoxyl groups in *meta* positioning (**1** and **5**) were the ones with lower or none antimicrobial activity. Comparing the results from compounds **4** and **7** (both flavanones with two hydroxyl groups), one can conclude that the presence of the methoxyl group in ring B (compound **4**) reduces the compound's activity against this bacterial strain (Figure 6).

**Figure 6.** Structure/activity relationships established for antibacterial effect against *B. subtilis*.

#### 3.2.4. Antitumor Activity

Considering the antitumor activity of the studied compounds, it can be noticed that compounds **5** and **8** were the most active, presenting very similar IC50 values. Compounds **1** and **4** showed some activity, although lower than the ones mentioned above (Table 3). However, none of the compounds tested were as active as colchicine or paclitaxel, the reference compounds used (Table 3). It was also observed that the inhibition of tumor cell growth is dose-dependent for all the compounds tested (Figure S15, supplementary material).


**Table 3.** Antitumor activity of the synthesized compounds **1**, **4**–**8**.

In each column the letters a, b, c, d, and e indicate significant differences (*p* < 0.05).

Results for the activity of compounds **6** and **7** against the A549 cell line have already been reported in the literature [7,59–61]; however, their activity was tested in order to facilitate the comparison of the results, since all the compounds were tested in the same conditions. The results for compound **7** are concordant with the literature, since it is reported that the effects of this compound in A549 cells is limited to the inhibition of cell migration, without any effects on growth or cytotoxicity level [7,60]. Compound **6** did not present any activity, which was unexpected, since it is reported to possess antitumor activity against several cell lines, including A549, by inhibiting proliferation and inducing tumor cell apoptosis [59,61]. This can be explained by the fact that the authors used a much lower cell concentration than the one used in the present work, and it has been proved that lower cell concentrations are correlated with higher activities of the compounds tested [62].

From the results obtained, the only conclusions that can be drawn regarding structure/activity relationships are that neither the <sup>α</sup>,β unsaturated carbonyl bridge nor the cyclization of chalcone into flavanone have an influence in the inhibition of tumor-cell growth, since both chalcones and flavanones presented interesting activities (Figure 7). Also, the presence of methoxyl groups has some importance in the antitumor effect of the compounds, since compounds **6** and **7**, which do not have methoxyl groups, did not present any activity against the A549 cell line (Table 3). The influence of a methoxyl group in synthetic polyphenolic compounds' antitumor activity was not a surprise because it was previously detected in our group [63].

**Figure 7.** Structure/activity relationships established for antitumor activity.
