Insights into the Discovery of Novel Neuroprotective Agents: A Comparative Study between Sulfanylcinnamic Acid Derivatives and Related Phenolic Analogues

Exogenous antioxidants may be beneficial therapeutic tools to tackle the oxidative damage in neurodegenerative diseases by regulation of the redox state that is critical for cell viability and organ function. Inspired by natural plant polyphenols, a series of cinnamic acid-based thiophenolic and phenolic compounds were synthesized and their antioxidant and neuroprotective properties were studied. In general, our results showed that the replacement of the hydroxyl group (OH) by a sulfhydryl group (SH) increased the radical scavenging activity and enhanced the reaction rate with 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•) and galvinoxyl radical (GO•). These results correlated well with the lower oxidation potential (Ep) values of thiophenols. However, a lower peroxyl radical (ROO•) scavenging activity was observed for thiophenols in oxygen radical absorbance capacity (ORAC-FL) assay. Furthermore, the introduction of 5-methoxy and 5-phenyl groups in the aromatic ring of 4-thioferulic acid (TFA) 2 and ferulic acid (FA) 1 did not significantly improve their antioxidant activity, despite the slight decrease of Ep observed for compounds 5, 6, and 9. Concerning cinnamic acid amides, the antioxidant profile was similar to the parent compounds. None of the compounds under study presented significant cytotoxic effects in human differentiated neuroblastoma cells. Thiophenolic amide 3 stands out as the most promising thiophenol-based antioxidant, showing cellular neuroprotective effects against oxidative stress inducers (hydrogen peroxide and iron).

The .
where f0 = initial fluorescence reading at 0 min and fi = fluorescence reading at time i. study (10 mM, 100 μL) in the supporting electrolyte (10 mL) to obtain a final concentration of 100 μM.
The scan rates used in differential pulse voltammetry (DPV) was 5 mV.s −1 .

SH-SY5Y cell culture
Human neuroblastoma SH-SY5Y cells were routinely cultured into 75 cm 3

MTT reduction assay
In the MTT reduction assay, the water-soluble MTT tetrazolium salt is reduced inside viable cells with active metabolism. The resulting purple colored formazan product accumulates as an insoluble precipitate inside the cells. The amount of formazan formed, presumably directly proportional to the number of viable cells, can be quantified spectrophotometrically after solubilization in an organic solvent [9,10]. Briefly, after the incubation periods with test compounds or the oxidative stressors, the cell culture medium was removed and a solution of MTT 0.5 mg/mL in fresh culture medium was added. Cells were incubated at 37 °C in a humidified, 5% CO2−95% air atmosphere for 1 h. Then, the cell culture medium was removed, and the formed formazan crystals were dissolved in 100 % DMSO. The absorbance was measured at 550 nm in a multiwell microplate reader (PowerWave XS from BioTek Instruments, Vermont, US). The results were expressed as the percentage of MTT reduction relative to that of the control [MTT reduction (% of control)] ± standard error mean (SEM) of at least three independent experiments.

NR uptake assay
The neutral red (NR) uptake assay is based on the ability of viable cells to incorporate the supravital dye NR and retain it inside the lysosomes [11]. NR uptake assay was performed as previously described by Fernandes et al. [12] Briefly, after the 24 h incubation periods with the test compounds, the cell culture medium was removed and a solution of NR (50 mg/mL) in fresh cell culture medium was added.
Differentiated SH-SY5Y cells were incubated for 1 h at 37 °C in a humidified, 5% CO2−95% air atmosphere. Then, the cell culture medium was removed and replaced by ethanol absolute/distilled water

Statistical analysis
The data obtained are expressed as mean ± standard error mean (SEM) of at least three independent experiments. All statistical analyses were performed using GraphPad PRISM version 6 for Windows . After a pre-treatment with CA (50 µM) for 24 h, cells were exposed to H2O2 1.5 mM and FeNTA 4 mM for additional 24 h. Cellular viability was evaluated by the MTT reduction assay. Results are expressed as the mean % of untreated controls ± SEM (n = 3). Statistical comparisons were estimated using the parametric method of one-way ANOVA, followed by the Dunnett's multiple comparisons test (**** p < 0.001 compared with cells treated only with FeNTA or H2O2; $$$$ p < 0.0001 compared with CTRL).