2.3.2. ABTS Radical-Scavenging Assay

For the determination of the ABTS radical cation (ABTS+) scavenging activity, we used our implementation of the Re et al. and Kuskoski et al. [17,18] method. ABTS+ was produced by reacting an ABTS stock solution (7 mM in water) with 2.45 mM potassium persulfate. The resulting mixture was kept at room temperature in the dark for 16–18 h before its use. Methanol was used to dilute the ABTS+ solution (150 μL) to give an absorbance of 0.7 ± 0.02 at 754 nm. This value was taken as the initial absorbance (A1). For each sample, aliquots were prepared mixing 980 μL of the ABTS+ methanolic solution with 20 μL of the samples at diverse concentrations. Each mixture was stirred, incubated at room temperature for 7 min, and its absorbance was read (754 nm). This value was considered as the final absorbance (A2). To calculate the radical-scavenging activity as a percentage of ABTS decoloration, the employed formula was:

$$\% \text{ of inhibition} = \left[ (\text{A}\_1 - \text{A}\_2)/\text{A}\_1 \right] \times 100$$

All determinations were performed in triplicate. Results were expressed as EC50, which represents the required concentration to diminish the absorbance of ABTS by 50%. Quercetin was used as the reference compound.

### *2.4. In Vitro Acetylcholinesterase Inhibitory Assay*

The determination of acetylcholinesterase activity was done using our implementation of the methodology reported by Adewusi et al. [19]. Employing a 96-well plate, 75 μL of Trizma-HCl bu ffer (50 mM, pH 8) was added along with 75 μL of the synthesized compound diluted, obtaining a 150 μg/mL concentration (0.15% for the dimethyl sulfoxide DMSO) at the end. Subsequently, 25 μL of a bu ffer solution of 15 mM acetylthiocholine chloride (ATCl) was added to each well with 125 μL of a 3 mM bu ffer solution of Ellman's reagen<sup>t</sup> (DTNB) (Ellman's reagent), giving both of them concentrations of 1.5 mM at the end. Employing a microplate reader every 45 s, the absorbance was measured at a wavelength of 405 nm, for three consecutive times. After these lectures, to each well 25 μL of an enzyme bu ffer solution with a concentration 2 U/mL of acetylcholinesterase was supplied, enriched with 0.1 mg/mL bovine serum albumin, obtaining an enzyme 0.2 U/mL final concentration. Five consecutive lectures were taken every 45 s. Of each plate, six wells served as the control for the acetylcholinesterase 100% activity, having no tested compound on them. Galantamine was used as the positive control. A correction for the substrate's spontaneous hydrolysis was made by subtracting the absorbance from before the addition of the enzyme from the enzyme containing wells. Using the equation:

$$\text{Inhhibition } \%= 1 - (\text{A }\_{\text{sample}}/\text{A }\_{\text{control}}) \times 100$$

we obtained the percentage of acetylcholinesterase inhibition, where the absorbances were the 0 and 225 s di fferences of the sample evaluated and for the enzyme 100% activity control previously described. All experiments were performed in triplicate.

### *2.5. In Vitro Xanthine Oxidase Inhibitory Assay*

The XO inhibition activity was evaluated using our implementation of the protocol reported by Almada-Taylor et al. [20]. To a volume of 0.33 mL of a xanthine 150 mM solution, phosphate buffer 120 mM with a pH of 7.8 was added (0.4 mL) and mixed with 0.25 mL of a solution of the compound to be analyzed. The reaction was started with the addition of a 0.5 U/mL solution of xanthine oxidase enzyme (0.02 mL). This was allowed to incubate for 3 min at 24 ◦C, followed by absorbance lecture at 295 nm (*A*295) for the measurement of the formation of uric acid. As a reference, allopurinol was employed, and the control was an absorbance lecture without an inhibitor. Employing the formula:

#### % of Xanthine Oxidase inhibition = [1 − (A*S* /A*C*)] × 100,

the percentage of xanthine oxidase inhibition activity was determined. A*S* indicates the initial velocity of reaction of the sample, and A*C* indicates that for the control. All determinations were made in duplicate, and repeated at least three times. Using interpolation from a linear regression analysis, the required concentration to diminish the XO activity by 50% (IC50) was calculated.

### *2.6. Cell Lines and Culture Conditions*

The in vitro antiproliferative activity of the investigated compounds was evaluated against six human solid tumor cell lines: A549 (non-small cell lung), HBL-100 (breast), HeLa (cervix), and SW1573 (non-small cell lung) as drug sensitive lines; and T-47D (breast) and WiDr (colon) as drug resistant lines. These cell lines were a kind gift from Prof. G. J. Peters (VU Medical Center, Amsterdam, The Netherlands). Cells were maintained in 25 cm<sup>2</sup> culture flasks in Roswell Park Memorial Institute (RPMI) 1640 media enriched with 5% FCS (Fetal Calf Serum) and 2 mM L-glutamine in a 37 ◦C, 5% CO2, and 95% humidified air incubator.

### *2.7. In vitro Antiproliferative Assay*

Cells were trypsinized, resuspended in medium containing 5% FCS and antibiotics (100 U/mL of penicillin G and 0.1 mg/mL of streptomycin), counted (Moxi Z automated cell counter), and diluted to reach the appropriate cell densities (2500 cells/well A549, HBL-100, HeLa and SW1573, and 5000 cell/well for T-47D and WiDr) for inoculation onto 96-well plates. Twenty-four hours later, compounds were added at concentrations in the range 0.01–100 μM. Cisplatin and etoposide were used as the positive control and DMSO (0.25% v/v) was used as the negative control. Drug incubation times were 48 h. Then, cells were fixed using 25 μL ice-cold trichloroacetic acid (TCA) solution (50% w/v) for 60 min at 4 ◦C, after which time the plates were rinsed with water. Next, 25 μL of a sulforhodamine B (SRB) solution (0.4% w/v in 1% acetic acid) was added for 15 min. Unbound SRB was rinsed with 1% acetic acid. The remaining dye was dissolved with 150 μL of Tris solution (10 mM, pH 10.5). The optical density of each well was determined at 530 and 620 nm using a microplate reader. The anti-proliferative activity, expressed as 50% growth inhibition (GI50), was calculated according to NCI formulas [21].
