*3.2. Biological Evaluations*

The antioxidant, anticholinesterasic, antimicrobial and antitumor activities of the compounds bearing hydroxyl and methoxyl groups were studied, and the results obtained are presented and discussed in the following paragraphs. Compound **8**, 2-hydroxy-4-methoxychalcone, was not synthesized in this work, but was included in the bioactivities study because it is, in structural terms, one of the simplest 2-hydroxychalcones and can be used as a reference and as a starting point to analyze structure/activity relationships of the compounds that were synthesized.

#### 3.2.1. Antioxidant Activity

In the DPPH (1,1-diphenyl-2-picryl-hydrazyl) scavenging test, the capacity of the compounds to neutralize the DPPH radical via electron transfer is measured [53,54].

Nearly all the compounds had some antioxidant activity, which was concentration-dependent (see Supplementary Materials, Figure S10), except for compounds **4** and **8**, which only had a residual activity at the highest concentration tested (150 μg/mL). Chalcone **6**, the most active compound, presented higher antioxidant activity than the isomeric flavanone **7** (Table 1), confirming that the conjugated double bond improves the molecule's ability to scavenge the DPPH radical [55].


**Table 1.** Antioxidant 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).

The results also confirm that the number of free hydroxyl groups influences antioxidant activity, since compounds with a higher number of these groups present better activity [55], a fact that is also confirmed by the lower activity of the compounds with more methoxyl groups (Figure 2).

**Figure 2.** Structure/activity relationships established for DPPH scavenging activity.

A previous work reported that compound **6** presented a low antioxidant activity in the DPPH scavenging test [56] (86.92 ± 0.43% of DPPH inhibition at 150 μg/mL); however, since the maximum concentration used in that work was about six times lower than the maximum tested concentration in the present work, it does not conflict with the results herein reported.

Another test used was the ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) scavenging assay. This assay measures the ability of compounds to neutralize the ABTS radical by radical quenching via hydrogen atom transfer. However, it can also be neutralized by electron transfer on some occasions, resulting in a higher sensitivity of this method when compared with the DPPH assay [53,57]. Again, compound **6** was the one presenting the best activity, with an IC50 of 12.72 μg/mL (Table 1), which is another result that confirms the importance of the hydroxyl groups in the chalcone sca ffold for their ability to reduce and/or eliminate free radicals. This conclusion is confirmed by the lower activity of chalcones **1** and **8**, which have only one hydroxyl group. Compound **5** presents better activity than **4**, which means that the relative position between the hydroxyl and methoxyl groups is more important than the number of hydroxyl groups on the flavanone sca ffold. This is confirmed by the results obtained for compound **7**, which has two hydroxyl groups, but only one substituent in ring A, and presents lower activity than compounds **4** and **5** (Figure 3). It was observed that the ABTS scavenging activity of the tested compounds is dose-dependent (Figure S11, supplementary material).

**Figure 3.** Structure/activity relationships established for ABTS scavenging activity.
