*2.3. Biological Activities*

Compound 2-hydroxy-4-methoxychalcone **8** was provided by the Laboratory of Organic Chemistry of the University of Aveiro, and the reactional conditions, yield and spectroscopic data are reported by Silva et al. [35,36].

#### 2.3.1. DPPH Scavenging Activity

Antioxidant activity was assayed by the DPPH (1,1-diphenyl-2-picryl-hydrazyl) radical scavenging assay [37]. Serial dilutions of studied or reference compounds (Trolox and quercetin) were carried out in 96-well microplates, at different concentrations, ranging between 0.148 μg/mL and 150 μg/mL in methanol. DPPH dissolved in methanol was added to the microwells, yielding a final concentration of 45 μg/mL, and the absorbance at 515 nm was measured in a Bio Rad Model 680 Microplate Reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA), after 30 min in the dark. In each assay, a control was prepared, in which the sample or standard was substituted by the same amount of solvent. Percentage of antioxidant activity (%AA) was calculated as:

$$\% \text{AA} = 100 \left[ 1 - (\text{A}\_{\text{control}} - \text{A}\_{\text{sample}}) / \text{A}\_{\text{control}} \right]$$

where Acontrol is the absorbance of the control, and Asample is the absorbance of the chalcone/flavanone or standard. All assays were carried out in triplicate and results expressed as EC50, i.e., as the concentration yielding 50% scavenging of DPPH, calculated by interpolation from the %AA vs concentration curve.

#### 2.3.2. ABTS Scavenging Activity

To determine ABTS radical scavenging, the method of Re et al. [38] was adopted. The stock solutions included 7 mM ABTS solution and 2.4 mM potassium persulfate solution. The working solution was prepared by mixing the two stock solutions in equal quantities and allowing them to react for 12–16 h at room temperature in the dark. The solution was then diluted by mixing 1 mL ABTS solution with the amount of methanol necessary to obtain an absorbance of 0.7 at 734 nm. Serial dilutions of studied or reference compounds (trolox, quercetin) were carried out in 96-well microplates, at different concentrations, ranging between 0.146 μg/mL and 150 μg/mL in methanol. ABTS solution was then added to the microwells, and after 8 min of incubation the absorbance was taken at 405 nm in a Bio Rad Model 680 Microplate Reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). In each assay, a control was prepared, in which the sample or standard was substituted by the same amount of solvent. Percentage of antioxidant activity (%AA) was calculated as:

$$\mathbf{(\%)} = \mathbf{[(Abs\_{control} - \text{Abs\_{sample}})]} \text{[/(Abs\_{control})]} \times 100$$

where Abscontrol is the absorbance of ABTS radical + methanol; Abssample is the absorbance of ABTS radical + sample/standard. All assays were carried out in triplicate and results expressed as EC50, i.e., as the concentration yielding 50% scavenging of ABTS, calculated by interpolation from the %AA vs concentration curve.

## 2.3.3. Anticholinesterasic Activity

The assay for measuring AChE and BuChE activity was modified from the assay described by Ellman et al. [39] and Arruda et al. [40]. Briefly, 3 mM 5,5-dithiobis [2-nitrobenzoic acid] (DTNB, 5 μL), 75 mM acetylthiocholine iodide (ATCI, 5 μL) or butyrylthiocoline iodide (BuTCI, 5 μL), and sodium phosphate buffer 0.1 mol dm−<sup>3</sup> (pH 8.0, 110 μL), and sample or standard (quercetin or donepezil) dissolved in buffer containing no more than 2.5% DMSO were added to the wells, and serial dilutions were carried out to obtain concentrations ranging between 0.293 μg/mL and 150 μg/mL (0.098 and 50 μg/mL for galantamine and berberine; 0.010 and 5 μg/mL for donepezil; 0.195 and 100 μg/mL for quercetin), followed by 0.25 U/mL AChE or BuChE (10 μL). The microplate was then read at 415 nm every 2.5 min for 7.5 min in a Bio Rad Model 680 Microplate Reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). For each concentration, enzyme activity was calculated as a percentage of the velocities compared to that of the assay using buffer without any inhibitor. Every experiment was done in triplicate.

## 2.3.4. Antimicrobial Activity

Antibacterial activity against Gram-positive *Bacillus subtilis* DSM10 and *Micrococcus luteus* DSM 20030 and Gram-negative *Escherichia coli* DSM498 was assessed by the broth microdilution method, as described by De León et al. [41]. The bacteria cultures were developed in nutrient broth (NB) at 30 ◦C for *B. subtilis* and *M. luteus* and at 37 ◦C for *E. coli*. Briefly, compounds were added to the microplates at a concentration of 200 μg/mL, and serial dilutions in NB were made until the concentration of 0.391 μg/mL. Then, the starting inoculum (1 × 10<sup>5</sup> CFU/mL) was added, and the bacterial growth was measured by the increase in optical density at 550 nm with a Bio Rad Model 680 Microplate Reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA), after 24 h of growth (48 h for *M. luteus*) at the above-mentioned temperatures for each bacterial strain. Penicillin and streptomycin were used as reference compounds. The IC50 was calculated as the concentration of compound that inhibits 50% of bacterial growth by interpolation from the % of growth inhibition vs. concentration curve.

## 2.3.5. Antitumor Activity

Antitumor activity was determined by the method described in Moujir et al. [42]. A-549 (human lung carcinoma) cell line, obtained from ATCC-LGC (American Type Culture Collection), was grown as a monolayer in DMEM (Sigma) supplemented with 2% fetal bovine serum (Sigma), 1% penicillin–streptomycin mixture (10,000 UI/mL), p-hydroxybenzoic acid (2 × 10<sup>4</sup> mg/mL) and L-glutamine (200 mM). Cells were maintained at 37 ◦C in 5% CO2 and 80% humidity in a SANYO CO2 Incubator. Cytotoxicity was assessed using the colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] reduction assay. Cell suspension (2 × 10<sup>4</sup> cells/well) in the lag phase of growth was incubated in a 96-well microplate with the compounds or the standards (colchicine and paclitaxel) dissolved in medium, with the concentrations ranging between 0.195 μg/mL and 200 μg/mL (0.010 μg/mL and 10 μg/mL for standards). After 48 h, MTT was added to the cells, which were then allowed to incubate for 3–4 h, and the optical density was measured using a Bio Rad Model 680 Microplate Reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 550 nm after dissolving the MTT formazan with DMSO (100 μL). The percentage viability (IC50) was calculated from the % of inhibition vs. concentration curve. All the experiments were repeated three times.

#### **3. Results and Discussion**

#### *3.1. Chalcones and Flavanones Synthesis*

The compounds obtained in this work were synthesized by aldol condensation between 4- and/or 6-substituted 2-hydroxyacetophenones and either 4-hydroxy or 4-methoxybenzaldehydes, using NaH and LiHMDS (Scheme 1).

**Scheme 1.** General procedure for the synthesis of chalcones and flavanones.

Compounds **1** and **3** were synthesized, as far as we could confirm, using for the first time in the aldol condensation the strong non-nucleophilic base NaH, which was used with success in similar condensations [43,44]. The experimental results in the synthesis of chalcones **1**–**3**, with very good yields (~80%) and relatively low reaction times (3 to 4 h), proves that NaH is an effective and efficient base in the aldol condensation when it is desired to obtain non-hydroxylated chalcones, other than at the C-2 position.

As referred above (Introduction), the hydroxyl groups present in the reagents require protection before the aldol condensation because in the strong basic conditions used, the phenoxide ions can undergo transformation into quinones and consequently prevent the formation of the desired chalcones. So, in order to obtain the desired compounds (the ones bearing hydroxyl and/or methoxyl groups), chalcones **2** and **3** need to be treated with acid to cleave the benzyl groups. The mixture used (HCl/AcOH) is one of the less harsh conditions and, in fact, removed the benzyl groups, but the obtained products were, in fact, flavanones **4** and **5** (Scheme 1). This can be explained by the acidic conditions used and the fact that those conditions favor the chalcones' isomerization into flavanones [45]. On the other hand, these extra steps, the protection and deprotection of hydroxyl groups, contribute to decrease the overall yield. For example, if we consider the synthesis of flavanones **4** and **5**, their precursors' chalcones **2** and **3** have excellent yields (80%), and the deprotection step, which produces the flavanones, is less efficient. This demonstrates that, due to this step, the flavanones' overall yield will be below 50%. At the same time, the yields (Scheme 1) allowed the assumption that the deprotection of the ring A hydroxyl groups is easier.

Since our purpose was the synthesis of chalcones bearing hydroxyl and methoxyl groups, taking into consideration that these substituents might improve the activity, and that the synthesis of flavanones **4** and **5** involves three steps, namely, (i) protection of the reagent's hydroxyl groups, (ii) aldol condensation and (iii) cleavage of the protecting groups, we envisaged the synthesis of the desired chalcones using another base. Knowing that LiHMDS was used in the synthesis of hydroxylated flavones without the protection step and with good results [46,47], we tested its use in the synthesis of chalcone **6** (Scheme 1). The experimental results showed that, using LiHMDS as the base, it is possible to synthesize polyhydroxylated chalcones in a one-pot process. However, although the desired chalcone was obtained, the yield was very low and its isomeric form, flavanone **7**, was also obtained. It should be highlighted that this reaction was accomplished at room temperature, controlled by TLC and finished after 5 days, when no more conversion was detected and 80% of the starting acetophenone was recovered. These data sugges<sup>t</sup> that the conversion rate is high and that the reaction does not occur due to a lack of energy. We may sugges<sup>t</sup> that using other sources of energy, such as a microwave, could originate higher yields.

The synthesized compounds' structures were confirmed by detailed analysis of their 1D and 2D NMR spectra, MS spectra (e.g., in Figures S1–S9, supplementary material) and available literature data; moreover, their purity was confirmed by UHPLC. Herein, chalcones' and flavanones' characterization is briefly discussed.

The 1H NMR spectra of compounds **1**–**3** and **6** present the signal characteristics of chalcone structures: (i) the resonance of the AB system assigned to the olefinic protons; (ii) doublets at δH 7.7–7.9 ppm with *J* ~ 15 Hz, which confirms the *E* configuration of the <sup>α</sup>,β double bond; (iii) the signal at δC ~192 ppm assigned to the carbonyl group; (iv) the singlet at δH 14.4–13.7 ppm assigned to 2-OH proton signal involvement in a hydrogen bond with the carbonyl oxygen atom; (v) two sets of doublets, with *J* = 6.8 Hz, at δH 6.9–7.8 ppm, assigned to the protons of the *para*-substituted aromatic ring B (Figure 1).

The 1H, 13C and HSQC NMR spectra of chalcone **1** also display two sets of doublets at δH 5.9–6.1 ppm, with a *meta*-coupling constant (*J* = 2.4 Hz), that exhibit correlation with the signals at 93.8 and 91.2 ppm, which indicates the presence of a tetra-substituted aromatic ring (ring A, Figure 1). The three sets of singlets at δH 3.83–3.92 ppm, showing correlation with three signals at δC 55.4–55.8 ppm, are characteristic of three methoxyl groups. The MS spectrum of chalcone **1** showed a signal at *m*/*z* 315 correspondent to [M+H]<sup>+</sup>, which agrees with the molecular formula C18H18O5. All the spectroscopic data and the melting point are in agreemen<sup>t</sup> with previously published data [31], and additionally, the connectivities found in the HMBC NMR spectrum confirm compound **1** as <sup>2</sup>-hydroxy-4,4,6-trimethoxychalcone, also named flavokawain A.

The 1H and 13C NMR spectra of chalcone **6** show three sets of signals in the range of δC 6.38–8.12 ppm, characteristic of the a trisubstituted aromatic ring (ring A, Figure 1). The MS spectrum showed a signal at *m*/*z* 257 corresponding to [M+H]<sup>+</sup>, which agrees with the molecular formula C15H12O4. These data and the connectivities found in the HMBC spectrum confirm compound **6** as <sup>2</sup>,4,4-tri-hydroxychalcone, also known as isoliquiritigenin [48].

Compounds **4**, **5** and **7** are flavanones, and this fact is well confirmed by the presence of the signals characteristic of ring C (Scheme 1): two double doublets at δH 2.68–3.10 and δH 2.68–2.78 assigned to the protons H-3, and the double doublet at δH 5.34–5.46 ppm assigned to H-2 in coupling with H-3; the signal at δ 196 ppm characteristic of the carbonyl group (C=O). Two sets of doublets at δH 7.29–7.42 (H-2 and H-6) and 6.86–6.95 ppm (H-3 and H-5), with *orto*-coupling constant of *J* = 8.7 Hz, indicate the presence of a *para*-substituted aromatic ring (ring B).

The presence of the methoxyl group in the ring B of compound **4** is deduced from the singlet at δ 3.83, correlating with the signal at δC 55.4 ppm. The MS data showed a signal at *m*/*z* 287 corresponding to the protonated molecule [M+H]<sup>+</sup> that is in accordance with the molecular formula C16H14O5. The spectroscopic data and the melting point are consistent with the published data for isosakuranetin [33,49], and together with the connectivities found in HMBC spectrum, confirm that compound **4** is 5,7-dihydroxy-4-methoxyflavanone.

The signals found in the 1H and 13C NMR spectra of compound **5** are very similar to the ones found for compound **4**. The differences are the non-appearance of the signal at δH 12.05 ppm, which confirms the absence of a 5-O*H* group; and the appearance of two singlets at δH 3.86 and 3.81 ppm, which means that compound **5** has two methoxyl groups. The location of the two methoxyl groups and the hydroxyl group were confirmed by the connectivities found in the HMBC spectrum of compound **5**. All the spectroscopic data are compatible with the structure of 4-hydroxy-5,7-dimethoxyflavanone, also named naringenin 5,7-dimethyl ether. Although compound **5** is not new, [50,51], to the best of our knowledge, the full spectroscopic data are reported here for the first time.

The quasi-molecular ion at *m*/*z* 257, compatible with molecular formula C15H12O4, confirms that compound **7** is an isomeric form of compound **6**. The presences of two signals at δH 9.53 and 8.60 ppm are assigned to 7-O*H* and 4-O*H*, respectively, by the connectivities found in the HMBC spectrum. Moreover, the spectroscopic data are identical to that previously published for <sup>4</sup>,7-dihydroxyflavanone, also named liquiritigenin [52].
