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

molecules Article Insights into the Discovery of Novel Neuroprotective Agents: A Comparative Study between Sulfanylcinnamic Acid Derivatives and Related Phenolic Analogues Daniel Chavarria 1,2,†, Carlos Fernandes 1,†, Brandon Aguiar 1, Tiago Silva 1,2, Jorge Garrido 3, Fernando Remião4, Paulo J. Oliveira 2, Eugenio Uriarte 5,6 and Fernanda Borges 1,* 1 CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal; [email protected] (D.C.); [email protected] (C.F.); [email protected] (B.A.); [email protected] (T.S.) 2CNC–Center for Neuroscience and Cell Biology, University of Coimbra, UC Biotech, Biocant Park, 3060-197 Cantanhede, Portugal; [email protected] 3Department of Chemical Engineering, School of Engineering (ISEP), Polytechnic of Porto, 4200-072 Porto, Portugal; [email protected] 4UCIBIO-REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal; [email protected] 5Departamento de Química Orgánica, Facultad de Farmacia, 15782 Santiago de Compostela, Spain; [email protected] 6Instituto de Ciencias Químicas Aplicadas, Universidad Autónoma de Chile, Santiago 8900000, Chile *Correspondence: fbor[email protected] †Both authors contributed equally to this work. Academic Editor: Dimitra Hadjipavlou-Litina Received: 23 October 2019; Accepted: 28 November 2019; Published: 2 December 2019   Abstract: 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 (E p ) 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 E p 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). Keywords: cinnamic acid; 4-thioferulic acid derivatives; ferulic acid derivatives; antioxidant activity; cytotoxicity; neuroprotection Molecules 2019,24, 4405; doi:10.3390/molecules24234405 www.mdpi.com/journal/molecules Molecules 2019,24, 4405 2 of 16 1. Introduction Oxidative stress is currently defined as the deregulation of the balance between the oxidants and antioxidants in favor of the former, with consequential disruption of the redox signaling and/or molecular damage [ 1 ]. The harmful effects on human cells associated with the oxidative damage inflicted by reactive species (RS) may ultimately lead to programmed cell death [ 2 ]. Several lines of evidence implicate oxidative stress on the neurodegeneration process observed in patients with Alzheimer’s disease (AD) and Parkinson’s disease (PD) [ 3 ]. Actually, markers of lipid peroxidation and protein oxidation were detected in individuals with AD and PD [ 4 – 8 ]. Both disorders were also associated with a decline of the pool of antioxidants, and a decrease of the activity of antioxidant enzymes [9–12]. Given the role of oxidative stress on the pathogenesis of neurodegenerative diseases, the use of exogenous antioxidants to assist the antioxidant defense system in upstream (prevention of RS generation) or downstream (radical scavenging) antioxidant pathways may help to maintain neuronal integrity and delay the neurodegenerative processes [13,14]. Ferulic acid (3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid, FA, compound 1 , Figure 1) is a naturalhydroxycinnamicacid(HCA)abletoactdirectlybyscavengingharmfuloxidants,orindirectlyby downregulation of pathways involved in cell-death and upregulation of the expression of antioxidant enzymes [ 15 ]. Neuroprotective properties in both in vitro and in vivo models, associated with antioxidant and anti-inflammatory activities, were ascribed to FA [ 16 – 18 ]. However, the therapeutic use of FA is hampered by its poor bioavailability, as a consequence of its low absorption and rapid metabolism and excretion [19,20]. Figure 1. Rational design of new chemical entities based on FA (compound 1 ) and TFA (compound 2) scaffolds. Ferulic acid has been used as a scaffold for the design and development of novel antioxidants with potential therapeutic interest [ 21 – 23 ]. Within this framework, 4-thioferulic acid (TFA, compound 2 , Figure 1), a new antioxidant obtained by the isosteric replacement of the hydroxyl group (OH) by a sulfhydryl group (SH) in FA, was reported by our group [24]. To gain insight on the advantages of thiophenol systems, herein we report the synthesis and the biological screening of novel TFA derivatives (compounds 3 – 6 , Figure 1). The corresponding hydroxycinnamic acid derivatives (compounds 7 – 10 , Figure 1) were synthesized to directly compare the effect of the OH to SH replacement. Following the studies of the antioxidant activity, we assessed the cytotoxicity of the new derivatives in differentiated SH-SY5Y cells and their protective properties against oxidative stress insults (H2O2, iron) in the same cellular model. Molecules 2019,24, 4405 3 of 16 2. Results and Discussion 2.1. Chemistry The synthesis of TFA (compound 2 ) [ 24 ] and derivatives thereof (compounds 3 – 6 ) was performed following the strategy depicted in Scheme 1. First, the reaction between 5-bromovanillin (compound 11 ) and phenylboronic acid in the presence of Pd(OAc) 2 and K 2 CO 3 under microwave (MW) irradiation yielded 5-phenylvanillin (compound 12 ) (Scheme 1, step a). Then, the thiocarbamoylation of 4-hydroxybenzaldehyde derivatives 12 – 14 with dimethylthiocarbamoyl chloride (DMTCl) under alkaline conditions afforded compounds 15 – 17 (Scheme 1, step b). A MW-assisted Newman-Kwart rearrangement under inert atmosphere of compounds 15 – 17 yielded compounds 18 – 20 (Scheme 1, step c). Cinnamic acid derivatives 21 – 23 were obtained via Knoevenagel-Doebner condensation between aldehydes 18 – 20 and malonic acid (Scheme 1, step d). The PyBOP-mediated amidation of compound 21 with benzylamine or phenethylamine led to the obtainment of the amides 24 and 25 , respectively (Scheme 1, step e). Finally, the alkaline hydrolysis of compounds 21 – 25 afforded thiophenol derivatives 2–6(Scheme 1, step f). Scheme 1. Synthetic strategy followed for the synthesis of compounds 2 – 6 . ( a ) phenylboronic acid, K 2 CO 3 , Pd(OAc) 2 , TBAB, DMF, 150 ◦ C (microwave (MW) irradiation), 15 min; ( b ) DMTCl, DABCO, DMF, 70 ◦ C, 3 h; ( c ) diphenyl ether, argon, 240 ◦ C (MW irradiation), 20 min; ( d ) malonic acid, anhydrous pyridine, piperidine, 70 ◦ C, 4–5 h; ( e ) 1. DMF, DIPEA, PyBOP, CH 2 Cl 2 , 0 ◦ C, 30 min; 2. benzylamine or 2-phenylethylamine, rt, overnight; (f) MeOH, aqueous solution of NaOH 2 M, reflux, 1.5–16 h. Molecules 2019,24, 4405 4 of 16 The corresponding hydroxycinnamic acid derivatives (compounds 7 – 10 , Figure 1) were synthesized according to procedures previously reported [21,25]. 2.2. Evaluation of Antioxidant Activity Following the synthesis of TFA and FA derivatives (compounds 1 – 10 ), we evaluated their radical scavenging activity against three synthetic radicals: 1,1-diphenyl-2-picrylhydrazyl radical (DPPH • ), 2,2 0 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS •+ ) and galvinoxyl (GO • ). In these assays, after the generation of the synthetic colored radical, the radical scavenging activity is evaluated by spectrophotometry [ 26 ]. Trolox was used as a reference antioxidant. The results are presented as mean IC50 ±SD (n=3) values (Table 1). Table 1. Radical scavenging activities and redox potentials (E p ) of thiophenols 2 – 6 and phenols 1 , 7 – 10 . The results obtained in DPPH • , ABTS •+ , GO • assays are expressed as mean IC 50 standard deviation (n=3). Compound IC50 (µM) ORAC-FL Index Ep(mV) R1R2R3DPPH•ABTS•+GO• 2 (TFA) 47.8 ±0.9a19.9 ±1.0a 4.25 ± 0.10 a 0.34 ± 0.04 a271 349.8 ±0.7 20.6 ±0.7 4.50 ±0.08 0.12 ±0.02 198 447.3 ±0.7 19.9 ±0.3 4.41 ±0.15 0.15 ±0.01 186 542.7 ±2.1 22.9 ±0.6 6.70 ±0.07 0.30 ±0.03 218 650.7 ±1.6 27.0 ±1.8 6.66 ±0.13 0.34 ±0.01 245 1(FA) 64.6 ±1.9a16.8 ±0.4a37.5 ±2.5a3.2 ±0.1a344 b 761.7 ±0.8 25.6 ±1.8 76.4 ±3.6 4.0 ±0.4 327 861.2 ±0.7 26.0 ±1.7 70.7 ±2.5 3.7 ±0.3 338 943.9 ±1.6 16.6 ±0.3 3.90 ±0.1 3.2 ±0.3 291 10 69.7 ±3.8 19.8 ±0.8 31.2 ±3.9 2.7 ±0.1 351 Trolox —- —- —- 24.6 ±0.9 18.2 ±0.5 2.98 ±0.15 1.00 ±0.02 110 aData from Chavarria et al. [24]. bData from Garrido et al. [23]. In general, thiophenols 2 – 6 showed higher scavenging of DPPH • and GO • radicals than phenols 1, 7 – 10 , while the scavenging of ABTS •+ radical was similar. The introduction of an additional 5-methoxy group (compound 5 ) was proven to be a structural modification with limited benefits, since it only caused slight changes in the antioxidant activity of TFA (compound 2 ) (a modest improvement in the antioxidant activity was only observed in DPPH • assay). Conversely, the presence of an additional aromatic ring (compound 6 ) decreased the antioxidant activity of TFA. Although an additional aromatic ring can increase the electron delocalization and stabilize the generated thiophenoxyl radical by resonance, it may reduce the accessibility of the compound to the center of the radicals and thereby decrease the radical scavenging activity [ 27 ]. The same tendency was observed with the phenolic analogues. Considering that both 5-methoxy and 5-phenyl groups did not significantly increase the radical scavenging activity of TFA, we then evaluated the effect of the introduction of substituents on the carboxylic function of TFA. As shown in Table 1, benzyl and phenethyl amides of TFA (compounds 3 and 4, respectively) displayed similar IC 50 values to the free carboxylic acid. The same tendency was observed for the phenolic analogues (compounds 7 and 8 ). Therefore, the carboxylic acid amidation with lipophilic substituents maintained the antioxidant activity of TFA. Molecules 2019,24, 4405 5 of 16 The kinetic data obtained in DPPH • , ABTS •+ , and GO • assays can provide information concerning the reactivity of the compounds under study (Figures S1–S3). As previously reported for TFA [ 24 ], thiophenols 3 – 6 reacted almost instantaneously with the radicals, leading to fast absorbance decays and reaching the steady state within the first minutes of reaction (Figures S1A–E, S2A–E and S3A–E). These results pointed out that the reaction rate was increased by the OH to SH replacement. Compared to TFA, the reactions of compounds 5 and 6 with the radicals occurred slower, requiring more time to reach the steady state (Figures S1D,E, Figures S2D,E and Figures S3D,E). Therefore, the presence of 5-methoxy and 5-phenyl groups in thiophenols delayed the reaction progress, possibly due to increased steric hindrance. On the other hand, compounds 3 and 4 displayed the same kinetic profile of TFA (Figures S1B,C, Figures S2B,C and Figures S3B,C), indicating that the reactivity of TFA was preserved with the functionalization of the carboxylic acid with lipophilic groups. Like ferulic acid 1 [ 24 ], phenols 7 – 10 displayed hyperbolic kinetic curves indicating moderate reactivity towards DPPH • , ABTS •+ , and GO•(Figures S1F–J, S2F–J and S3F–J). We also studied the antioxidant activity toward peroxyl radicals (ROO • ) using the oxygen radical absorbance capacity (ORAC-FL) assay. In this assay, ROO • radicals are scavenged by hydrogen atom transfer (HAT) or by radical addition processes [ 27 ]. Phenols (compounds 1 , 7 – 10 ) showed ORAC-FL indexes ranging between 2.7–4.0 (Table 1) and displayed higher ORAC-FL indexes than thiophenols (compounds 2 – 6 ). Thiophenols presented ORAC-FL indexes below 1 (Table 1). Therefore, contrary to what was observed in DPPH • , ABTS •+ and GO • assays, these results show that phenols are better ROO • radical scavengers than thiophenols. The dissimilarities between the two sets of compounds may be related to the differences on the acidity of the phenol and the thiophenol moieties. Since the thiophenol group of TFA 1 is significantly more acidic than the phenol group of FA (pK a thiophenol =5.6; pK a phenol =9.1) [ 24 ], there is a high amount of thiophenolate anions at pH 7.3 while the phenol group remains undissociated. Therefore, in opposition to phenols 1 and 7 – 10 , the deprotonated thiophenols 2–6were poor scavengers of ROO•radicals. 2.3. Electrochemical Studies Voltammetric measurements are particularly useful to estimate the redox properties of bioactive systems, thus enabling a prompt screening of their antioxidant capacity [ 28 ]. In this context, to obtain complementary information concerning the antioxidant activity of the compounds under study, differential pulse voltammetry (DPV) experiments were performed to study the electrochemical properties of compounds 2 – 6 and 7 – 10 . Experiments were carried out at a physiological pH of 7.4 using a glassy carbon-working electrode. The oxidation potential (E p ) values obtained are depicted in Table 1. All compounds showed well-defined anodic waves at physiological pH, which may be attributed to the oxidation of the thiophenol or the phenol groups. Thiols and phenols can undergo one-electron oxidation processes with the formation of thiyl and phenoxyl radicals, respectively [ 29 , 30 ]. The E p values of thiol-containing compounds 2 – 6 ranged between +186 mV and +271 mV, while phenols 1 and 7 – 10 displayed higher E p values, ranging between +291 mV and +351 mV (Table 1 ) . The results obtained showed that the replacement of the OH by an SH group shifted the peak potentials toward less positive values, suggesting that thiophenols are more prone to oxidation than phenols. The introduction of 5-methoxy (compounds 5 and 9 ) or 5-phenyl (compounds 6 and 10 ) groups did not significantly change the E p values when compared to FA and TFA, although a slight decrease was observed for compounds 5 , 6, and 9 . The lower potential values may result from the increased stabilization of the thiophenoxyl/phenoxyl radicals, which is associated with the electron-donating properties of the methoxy group [ 31 ] (compounds 5 and 9 ) or the increased electron delocalization provided by the additional aromatic ring (compound 6). The voltammetric behavior of the cinnamic acid amides 3 , 4 , 7 , and 8 was similar to that observed for the parent compounds (TFA and FA). The introduction of lipophilic groups at the side chain did Molecules 2019,24, 4405 6 of 16 not significantly affect the redox properties of phenol and thiophenol groups and, therefore, no major changes in the redox potentials were observed. 2.4. Cellular Studies 2.4.1. Evaluation of Cytotoxicity Profile Thecytotoxicityprofile of thiophenols 3 – 6 andphenols 7 – 10 wasassessedinhumanneuroblastoma SH-SY5Y cells differentiated into a dopaminergic phenotype [ 32 ]. After 24 h of exposure with the compounds at three different concentrations (1, 10 and 50 µ M), the cell viability was evaluated using two methods: the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction assay, a method that relies on the reduction of MTT into the respective formazan by cells with active metabolism [33], and the neutral red (NR) uptake assay, which is based on the lysosomal uptake of the dye NR [ 34 ]. The results, expressed as mean MTT reduction (% of control) SEM and NR uptake (% of control) ± SEM (n=3), are depicted in Figure 2. Figure 2. Cellular viability of differentiated SH-SY5Y cells after a 24 h treatment with thiophenols 3 – 6 and phenols 7 – 10 at three different concentrations (1, 10 and 50 µ M). Cellular viability was evaluated using two methods: MTT reduction ( A , C ) and NR uptake ( B , D ) assays. Results are expressed as the mean % of untreated controls ± SEM (n=3). Statistical comparisons were performed using the parametric method of one-way ANOVA, followed by the Dunnett’s multiple comparisons test. In all cases, pvalues lower than 0.05 were considered significant (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 vs untreated cells). Thiophenols 3 – 6 (Figure 2A,B) and phenols 7 – 10 (Figure 2C,D) did not cause marked decreases in MTT reduction and in NR uptake at concentrations up to 50 µ M (MTT reduction and NR uptake were >85%), which is in accordance with the previous data obtained for FA 1 and TFA 2 [ 24 ]. Based on these results, we can conclude that the cytotoxicity profile within the tested concentration range was upheld with the OH to SH replacement, the introduction of 5-methoxy and 5-phenyl substituents, and the functionalization of the carboxylic acid group (benzyl or phenethyl amides). Molecules 2019,24, 4405 7 of 16 2.4.2. Protection against H2O2and Ferric Iron-Induced Oxidative Damage The protective properties of compounds 1 – 10 against oxidative stress in the same cellular model were also examined. Hydrogen peroxide (H 2 O 2 ) and ferric nitrilotriacetate (FeNTA) were used as inducers of oxidative stress [ 32 ]. After the pre-treatment with the test compounds at 10 and 50 µ M for 24 h, differentiated SH-SY5Y cells were exposed to H 2 O 2 (1.5 mM) or FeNTA (4 mM) for 30 min and 24 h, respectively. The cellular viability was assessed using the MTT reduction assay. The results obtained are illustrated in Figures 3and 4. Figure 3. Protection against H 2 O 2 -induced damage in differentiated SH-SY5Y cells by thiophenols 2 – 6 ( A ) and phenols 1 , 7 – 10 ( B ). After a pre-treatment with the compounds under study (10 and 50 µ M) for 24 h, cells were exposed H 2 O 2 1.5 mM for additional 30 min. 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 nonparametric method of Kruskal–Wallis (one-way ANOVA on ranks), followed by Dunn’s post hoc test (* p<0.05, ** p<0.01, compared with cells treated only with H 2 O 2 ; $$ p<0.01, $$$ p<0.001; $$$$ p<0.0001 compared with CTRL). Positive control with caffeic acid (50 µ M) was performed under the same conditions described above. The result is depicted in Supplementary Information (Figure S4A). Figure 4. Protection against FeNTA-induced damage in differentiated SH-SY5Y cells by thiophenols 2 – 6 ( A ) and phenols 1 , 7 – 10 ( B ). After a pre-treatment with the compounds under study (10 and 50 µ M) for 24 h, cells were exposed FeNTA 4 mM for additional 24 h. Cellular viability was evaluated by the MTT reduction assay. Results are expressed as the mean % of untreated controls ± SEM (n=3). Statistical comparisons were estimated using the nonparametric method of Kruskal–Wallis (one-way ANOVA on ranks), followed by Dunn’s post hoc test (* p<0.05 compared with cells treated only with FeNTA; $ p<0.05, $$ p<0.01, $$$ p<0.001; $$$$ p<0.0001 compared with CTRL). Positive control with caffeic acid (50 µ M) was performed under the same conditions described above. The result is depicted in Supplementary Information (Figure S4B). The incubation of differentiated SH-SY5Y cells with H 2 O 2 (1.5 mM) decreased the MTT reduction to 50.7 ± 1.9% (p<0.0001) in comparison to control cells (Figure 3A,B). The damage inflicted by H 2 O 2 Molecules 2019,24, 4405 8 of 16 was attenuated by thiophenols and phenols containing benzyl amide (compounds 3 and 7 ,p<0.05) and 5-methoxy groups (compound 5 ,p<0.05, and compound 9 ,p<0.01) at 10 µ M (Figure 3A,B). A mild protection against H 2 O 2 -induced cell death was also observed with pre-treatment of neuroblastoma cells with TFA (10 µ M, p<0.01; 50 µ M, p<0.05, Figure 3A) and compound 10 (p<0.05, Figure 3A) at both tested concentrations. The data obtained for TFA 2 and FA 1 at 50 µ M were previously reported by Chavarria et al. [24]. Transition metals like iron accumulate with age in specific brain areas, such as the substantia nigra and the hippocampus [ 35 ]. FeNTA was used to mimic iron overload conditions in SH-SY5Y cells. Treatment with FeNTA 4 mM decreased the MTT reduction to 47.2 ± 1.2% (p<0.0001) in comparison to control cells (Figure 4A,B). Remarkably, TFA derivative 3 significantly protected neuroblastoma cells from the deleterious effects of FeNTA at both tested concentrations (p<0.05, Figure 4A). The FeNTA-induced decrease in MTT reduction was also attenuated in neuroblastoma cells pre-treated with TFA 2(p<0.05, Figure 4A) and compound 10 (p<0.05, Figure 4B) at 50 µM. Taking these data together, we concluded that TFA 2 , TFA benzyl amide 3, and FA derivative 10 significantly protected neuroblastoma cells against oxidative stress, both by the direct action of H 2 O 2 and by high concentrations of iron. In addition, 5-methoxy derivatives (compounds 5 and 9 ) and FA benzyl amide (compound 7 ) were able to prevent the damage induced by H 2 O 2 , but were ineffective against the harmful effects caused by high concentrations of iron. 2.5. Estimation of Drug-Like Properties Inearlydrugdiscovery, thebalancingofkeyphysicochemicalpropertieswithinoptimalrangesmay improve the compounds’ quality and thereby increase the likelihood of therapeutic success [ 36 ]. In this context, we calculated several physicochemical parameters of TFA (compound 2 ) and compounds 3 and 10 to predict their “drug-likeness”. These compounds were considered the most promising cinnamic acid derivatives because they displayed significant neuroprotective activity against two oxidative stressors in neuroblastoma cells. The predicted physicochemical properties included topological polar surface area (TPSA in Å), number of hydrogen acceptors (HBA), number of hydrogen donors (HBD), number of rotatable bonds (RB), and logarithm of the ratio of the concentration of a drug in the brain and in the blood (logBB). The results obtained are presented in Table 2. Table 2. Predicted physicochemical properties of TFA (compound 2) and compounds 3and 10. Compound MW acLogPbTPSA aHBA aHBD aRB LogBB 2210.25 1.97 46.53 4 2 3 −0.9373 3299.39 3.25 38.33 4 2 6 0.02948 10 270.28 2.76 66.76 4 2 4 -1.511 CNS+drugs <500 c2–5 c<90 c<7d<3d<8d<−1 a Properties predicted using the Stardrop software. MW: molecular weight; TPSA: topological polar surface area; HBA: number of H-bond acceptor atoms; HBD: number of H-bond donor atoms; RB: number of rotatable bonds; logBB: logarithm of the ratio of the concentration of a drug in the brain and in the blood. b Properties predicted using the SwissADME (http://swissadme.ch/index.php); clogP: logarithm of the octanol/water partition coefficient. cData from Hitchcock et al. [37]. dData from Pajouhesh et al. [38]. The values obtained for compound 3 are within the optimal ranges of drugs able to act in the central nervous system (CNS). On the other hand, TFA and compound 10 presented cLogP and LogBB values below or close to the recommended limits. Therefore, TFA 2 and compound 10 are less likely to cross the blood-brain barrier by passive diffusion and thereby attain the CNS. The presence of a free carboxylic acid may restrict the access to the brain, as previously observed for similar systems [21]. Overall, these results suggest that TFA benzyl amide (compound 3 ) presents the most favorable drug-like properties among the selected candidates. Molecules 2019,24, 4405 9 of 16 3. Materials and Methods 3.1. Chemistry 3.1.1. Synthesis I. Synthesis of 6-Hydroxy-5-methoxy-[1,10-biphenyl]-3-carbaldehyde (12) Synthesis and structural characterization are described in literature [21]. II. Thiocarbamoylation of Phenols In a bottom flask with the phenol (1 mmol) dissolved in DMF, DABCO (4 mmol), and DMTCl (2 mmol) were added. The mixture was heated at 70 ◦ C for 3 h. Upon completion, the mixture was poured onto water (15 mL) cooled on an ice bath and acidified with HCl 6 M. The product was collected by filtration under reduced pressure and washed with water. The general procedure was adapted from Nowakowska et al. [39]. O-(4-Formyl-2-methoxyphenyl) dimethylcarbamothioate ( 15 ). Synthesis and structural analysis are described in literature [24,39]. O-(4-Formyl-2,6-dimethoxyphenyl) dimethylcarbamothioate ( 16 ). The reaction mixture was poured onto water (15 mL) cooled on an ice bath and acidified with HCl 6 M. The product was collected by filtration under reduced pressure and washed with water. η =93%. 1 H NMR (CDCl 3 -d 1 ): δ (ppm) = 3.40 (s, 3H, NC H3 ), 3.49 (s, 3H, NC H3 ), 3.93 (s, 6H, 2 × OC H3 ), 7.19 (s, 2H, H2, H6), 9.94 (s, 1H, C H O). 13 C NMR (CDCl 3 -d 1 ): δ (ppm) = 38.9 (N C H 3 ), 43.5 (N C H 3 ), 56.5 (2 × O C H 3 ), 106.3 (C2, C6), 134.2 (C1), 136.9 (C4), 153.4 (C3, C5), 186.7 ( C SO), 191.1 ( C HO). EI/MS m/z(%): 269.0 (M •+ , 69), 88.0 (99), 72.0 (100). O-(5-Formyl-3-methoxy-[1,1 0 -biphenyl]-2-yl) dimethylcarbamothioate ( 17 ). The reaction mixture was poured onto water (15 mL) cooled on an ice bath and acidified with HCl 6 M. The product was collected by filtration under reduced pressure and washed with water. η =75%. 1 H NMR (CDCl 3 -d 1 ): δ (ppm) =3.17 (s, 3H, NC H3 ), 3.34 (s, 3H, NC H3 ), 3.94 (s, 3H, OCH3 ), 7.39 (m, 3H, H3 0 , H4 0 , H5 0 ), 7.52 (m, 4H, H2, H6, H2 0 , H6 0 ), 9.98 (s, 1H, C H O). 13 C NMR (CDCl 3 -d 1 ): δ (ppm) = 38.7 (N C H 3 ), 43.3 (N C H 3 ), 56.4 (O C H 3 ), 109.7 (C2), 126.6 (C6), 128.0 (C4 0 ), 128.2 (C2 0 , C6 0 ), 129.1 (C3 0 , C5 0 ), 134.5 (C5), 136.4 (C1), 137.2 (C1 0 ), 145.0 (C4), 153.1 (C3), 186.4 ( C SO), 191.2 ( C HO). EI/MS m/z(%): 315.1 (M •+ , 38), 243.0 (29), 88.0 (100), 72.0 (89). III. Microwave-Assisted Newman-Kwart Rearrangement O-phenyl-N,N-dimethylthiocarbamates 15 – 17 were placed onto a 5 mL microwave glass vial. The vial was sealed with a septum and the content was maintained under an argon atmosphere for 30 min at room temperature. After the addition of diphenyl ether, the vial was placed into the microwave cavity. The reaction took place under microwave irradiation at 240 ◦C for 20 min. S-(4-Formyl-2-methoxyphenyl) dimethylcarbamothioate ( 18 ). Synthesis and structural analysis are described in the literature [24]. S-(4-Formyl-2,6-dimethoxyphenyl) dimethylcarbamothioate ( 19 ). Compound 19 was isolated by flash column chromatography (ethyl acetate/dichloromethane (3:7)) and recrystallized in dichloromethane/petroleum ether. η =91%. 1 H NMR (CDCl 3 -d 1 ): δ (ppm) =3.00 (bs, 3H, NC H3 ), 3.18 (bs, 3H, NC H3 ), 3.94 (s, 6H, 2 × OC H3 ), 7.16 (s, 2H, H2, H6), 9.97 (s, 1H, C H O). 13 C NMR (CDCl 3 -d 1 ): δ (ppm) = 37.1 (2 × N C H 3 ), 56.7 (2 × O C H 3 ), 105.0 (C2, C6), 112.7 (C4), 138.7 (C1), 161.8 (C3, C5), 164.5 (COS), 191.5 (CHO). EI/MS m/z(%): 269.0 (M•+, 48), 72.1 (100). S-(5-Formyl-3-methoxy-[1,1 0 -biphenyl]-2-yl) dimethylcarbamothioate ( 20 ). Compound 20 was isolated by flash column chromatography (ethyl acetate/dichloromethane (3:7)) and recrystallized in dichloromethane/petroleum ether. η =83%. 1 H NMR (DMSO-d 6 ): δ (ppm) =2.96 (bs, 6H, 2 × NC H3 ), 3.98 (s, 3H, OC H3 ), 7.38 (m, 5H, H2 0 , H3 0 , H4 0 , H5 0 , H6 0 ), 7.46 (d,J=1.6 Hz, 1H, H2), 7.48 (d,J=1.6 Hz, 1H, H6), 10.01 (s, 1H, C H O). 13 C NMR (DMSO-d 6 ): δ (ppm) = 37.2 (2 × N C H 3 ), 56.8 (O C H 3 ), 108.2 Molecules 2019,24, 4405 16 of 16 30. Teixeira, J.; Gaspar, A.; Garrido, E.M.; Garrido, J.; Borges, F. Hydroxycinnamic acid antioxidants: An electrochemical overview. Biomed. Res. Int. 2013,2013, 1–13. [CrossRef] 31. Brand-Williams, W.; Cuvelier, M.E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. LWT - Food Sci. Technol. 1995,28, 25–30. [CrossRef] 32. Fernandes, C.; Pinto, M.; Martins, C.; Gomes, M.J.; Sarmento, B.; Oliveira, P.J.; Remi ã o, F.; Borges, F. Development of a PEGylated-Based Platform for Efficient Delivery of Dietary Antioxidants Across the Blood-Brain Barrier. Bioconjug. Chem. 2018,29, 1677–1689. [CrossRef] [PubMed] 33. Belyanskaya, L.; Manser, P.; Spohn, P.; Bruinink, A.; Wick, P. The reliability and limits of the MTT reduction assay for carbon nanotubes-cell interaction. Carbon 2007,45, 2643–2648. [CrossRef] 34. Repetto, G.; del Peso, A.; Zurita, J.L. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat. Protoc. 2008,3, 1125–1131. [CrossRef] 35. Nunez, M.T.; Gallardo, V.; Munoz, P.; Tapia, V.; Esparza, A.; Salazar, J.; Speisky, H. Progressive iron accumulation induces a biphasic change in the glutathione content of neuroblastoma cells. Free Radic. Biol. Med. 2004,37, 953–960. [CrossRef] 36. Arnott, J.A.; Planey, S.L. The influence of lipophilicity in drug discovery and design. Expert Opin. Drug Discov. 2012,7, 863–875. [CrossRef] 37. Hitchcock, S.A.; Pennington, L.D. Structure-brain exposure relationships. J. Med. Chem. 2006 ,49, 7559–7583. [CrossRef] 38. Pajouhesh, H.; Lenz, G.R. Medicinal Chemical Properties of Successful Central Nervous System Drugs. NeuroRx 2005,2, 541–553. [CrossRef] 39. Nowakowska, Z. The conversion of stilbenols to stilbenethiols via N,N-dimethylthiocarbamates. Phosphorus Sulfur 2006,181, 707–715. [CrossRef] 40. Teixeira, J.; Silva, T.; Benfeito, S.; Gaspar, A.; Garrido, E.M.; Garrido, J.; Borges, F. Exploring nature profits: Development of novel and potent lipophilic antioxidants based on galloyl-cinnamic hybrids. Eur. J. Med. Chem. 2013,62, 289–296. [CrossRef] 41. Gaspar, A.; Silva, T.; Yanez, M.; Vina, D.; Orallo, F.; Ortuso, F.; Uriarte, E.; Alcaro, S.; Borges, F. Chromone, a privileged scaffold for the development of monoamine oxidase inhibitors. J. Med. Chem. 2011 ,54, 5165–5173. [CrossRef] [PubMed] 42. Tai, A.; Sawano, T.; Ito, H. Antioxidative properties of vanillic acid esters in multiple antioxidant assays. Biosci. Biotechnol. Biochem. 2012,76, 314–318. [CrossRef] [PubMed] 43. Feng, J.Y.; Liu, Z.Q. Phenolic and enolic hydroxyl groups in curcumin: Which plays the major role in scavenging radicals? J. Agric. Food Chem. 2009,57, 11041–11046. [CrossRef] [PubMed] 44. Yang, Y.; Song, Z.G.; Liu, Z.Q. Synthesis and antioxidant capacities of hydroxyl derivatives of cinnamoylphenethylamine in protecting DNA and scavenging radicals. Free Radic. Res. 2011 ,45, 445–453. [CrossRef] 45. Ou, B.; Hampsch-Woodill, M.; Prior, R.L. Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. J. Agric. Food Chem. 2001 ,49, 4619–4626. [CrossRef] 46. Mura, F.; Silva, T.; Castro, C.; Borges, F.; Zuniga, M.C.; Morales, J.; Olea-Azar, C. New insights into the antioxidant activity of hydroxycinnamic and hydroxybenzoic systems: Spectroscopic, electrochemistry, and cellular studies. Free Radic. Res. 2014,48, 1473–1484. [CrossRef] 47. Fernandes, C.; Martins, C.; Fonseca, A.; Nunes, R.; Matos, M.J.; Silva, R.; Garrido, J.; Sarmento, B.; Remiao, F.; Otero-Espinar, F.J.; et al. PEGylated PLGA Nanoparticles As a Smart Carrier to Increase the Cellular Uptake of a Coumarin-Based Monoamine Oxidase B Inhibitor. ACS Appl. Mater. Interfaces 2018 ,10, 39557–39569. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).