Synthesis, In Vitro Antioxidant Properties and Distribution of a New Cyanothiophene-Based Phenolic Compound in Olive Oil-In-Water Emulsions

We synthesized and determined the antioxidant activity and distribution of a new cyanothiophene-based compound, N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3,5-dihydroxybenzamide (SIM-53B), in intact stripped olive oil-in-water emulsion. The in vitro antioxidant properties of SIM-53B were evaluated and compared to those for Trolox and resveratrol. Addition of an emulsifier (Tween 20) creates a narrow region, the aqueous–oil interface, and the distribution of SIM-53B can be described by two partition constants: PWI (between aqueous/interfacial regions) and POI (between oil/interfacial regions). The effects of emulsifier concentration expressed in terms of the volume fraction, ΦI, and O/W ratio were also evaluated on its distribution. SIM-53B is predominantly distributed (>90%) in the interfacial region of 1:9 (O/W) olive oil-in-water emulsions at the lowest emulsifier volume fraction (ΦI = 0.005) and only a small fraction is located in the aqueous (<5%) and the oil (<5%) regions. Besides, the concentration of SIM-53B in the interfacial region of the emulsions is ~170–190-fold higher than the stoichiometric concentration, emphasizing the compartmentalization effects. Results suggest that the emulsifier volume fraction is a key parameter that may modulate significantly its concentration in the interface. Our study suggests that cyanothiophene-based compounds may be interesting additives for potential lipid protection in biomembranes or other lipid-based systems.


Introduction
Drug-resistant bacteria emerge as an increasing threat to human health and new antibacterials are needed to treat bacterial infections [1,2]. Bacterial cell wall biosynthesis involves enzymes which have been engaged as targets for developing potential antibacterial candidates [3,4]. In this sense, cyanothiophene scaffold has been used in medicinal chemistry for the preparation of biologically active compounds and for this reason, the isolation and the structural characterization of innovative derivatives, the improvement of new synthetic methods and the evaluation of biological properties are topics of growing interest [2,[5][6][7][8]. A broad series of cyanothiophene-based inhibitors Scheme 1. Distribution of SIM-53B between the different regions of the emulsified system, aqueous (W), oil (O) and interfacial (I) regions. Φ is the volume fraction of a particular region (Φ = Vregion/Vtotal). PW I and PO I are the partition constants between the aqueous and interfacial regions and between the oil and interfacial regions, respectively. kI is the rate constant for the reaction between the chemical probe (hexadecylbenzenediazonium ion, 16ArN2 + ) and SIM-53B in the interfacial reaction of the O/W emulsion.

Chemicals and Reagents
All chemicals and reagents were of the highest purity available and were used as received. They were purchased from Acros Organics, Carlo Erba reagents, Apollo Scientrific, Fluka, Sigma-Aldrich, TCI and Merck. 2,2-Diphenyl-1-picrylhydrazyl (DPPH • ), neocuproine and polyoxyethylene (20) sorbitan monolaurate (Tween 20) were obtained from Aldrich, Merck and Acros Organics, respectively. 2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) (ABTS), Trolox and resveratrol were obtained from TCI. The synthesis of N- (3-cyano-4,5,6,7tetrahydrobenzo[b]thiophen-2-yl)-3,5-dihydroxybenzamide (SIM-53B) was monitored using thinlayer chromatography on silica-gel plates (Merck DC Fertigplatten Kieselgel 60 GF254) and visualized by UV light and/or stained with the relevant reagents (ninhydrin and FeCl3 solution). Flash column chromatography was completed on Merck silica gel 60 (mesh size, 70-230). A Reichert hot-stage microscope was employed to measure melting points. IR spectra were recorded on a Perkin-Elmer FTIR 1600 spectrometer. 1 H and 13 C NMR spectra were acquired on a Bruker AVANCE III 400 MHz spectrometer in DMSO-d6, with TMS as the internal standard. IR, 1 H and/or 13 C NMR spectra of compounds 2, 3 and SIM-53B can be found in the Suplementary material ( Figures S1-S8). Highresolution mass spectra were acquired with a Micromass ® Q-Tof Premier™ mass spectrometer (Micromass, Waters, Manchester, UK). The purity of SIM-53B was determined by reversed-phase high-performance liquid chromatography (HPLC) analysis on an Agilent 1100 system (Agilent Technologies, Santa Clara, CA, USA) equipped with a quaternary pump and a multiple-wavelength detector G1365 UV-VIS. Agilent Eclipse Plus C18 (5 μm, 4.6 × 150 mm) was used as a column, with a flow rate of 1.0 mL/min, detection at 210, 220, 254 and 280 nm and an eluent system of: A = 0.1% TFA in H2O; B = acetonitrile. The following gradient was applied: 0-19 min, 20% B to 90% B in A; 19-20 Scheme 1. Distribution of SIM-53B between the different regions of the emulsified system, aqueous (W), oil (O) and interfacial (I) regions. Φ is the volume fraction of a particular region (Φ = V region /V total ). P W I and P O I are the partition constants between the aqueous and interfacial regions and between the oil and interfacial regions, respectively. k I is the rate constant for the reaction between the chemical probe (hexadecylbenzenediazonium ion, 16ArN 2 + ) and SIM-53B in the interfacial reaction of the O/W emulsion.
Stripped olive oil was used in the preparation of emulsions. Endogenous tocopherols and phenols were removed by washing olive oil (~700 mL) with a 0.5 M NaOH solution (4 × 300 mL), followed by 0.5 HCl solution (2 × 200 mL) and passing it twice through an aluminum oxide column. Total elimination of tocopherols was checked by HPLC according to the IUPAC method 2.432 [27].

Emulsion Preparation
Olive oil-in-water emulsions at different ratios of oil (O) and water (W)-4:6 and 1:9, O/W-were produced by mixing stripped olive oil, acidic water and Tween 20 as the emulsifier. The pH of the aqueous phase was adjusted to 3.65 employing 0.04 M citric acid/citrate buffer. The emulsifier volume fractions Φ I ranging from 0.5% to 4% were used. The mixture was blended for 1 min using a Polytronic PT-1600 homogenizer at room temperature. The required amount of SIM-53B was added to the emulsion at a final concentration of 1 mM.

Antioxidant Activity Determined by DPPH • , ABTS and CUPRAC Assays
Free-radical scavenging activity was evaluated employing the DPPH • and ABTS assays [31]. 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH • ) was dissolved in ethanol (150 µL, 140 µM) and added to 150 µL ethanol solution of the test sample (62.5-500 µM of SIM-53B) or ethanol (negative control) in each well of a flat-bottomed 96-well microliter plate (TPP, Tissue Culture Test Plates). The reaction between DPPH • and SIM-53B was then monitored at λ = 517 nm by using a Synergy H4 Hybrid Multi-Mode Microplate Reader (Bio-Tek Instruments, Inc) at T = 20 • C in the dark for 180 min. Each set of experiments was performed in triplicate. For the reduction kinetics of the DPPH • , absorbance was measured using an Agilent Cary 3500 UV-Vis spectrophotometer with the Compact Peltier UV-Vis Module, set at a wavelength of 517 nm. Spectrophotometric disposable (2 mL capacity and 1 cm path-length) cuvettes were used. The absorbance of a sample and a blank were measured simultaneously at a sampling rate of one point per second. Automatic acquisition of data was set For the ABTS assay, a slightly modified procedure described in [32] was used. To 10 mL of 7 mM stock solution of 2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) (ABTS) was added 178 µL 140 mM solution of potassium persulfate. Working solution was allowed to react for 16 h at room temperature in the dark. The solution was than diluted by mixing 1 mL ABTS •+ with 64 mL of ethanol (96%) to obtain an absorbance of 1.1 units at 734 nm. The solution of SIM-53B and solutions of standards (Trolox and resveratrol) were freshly prepared in 96% ethanol at 1 mM concentration. To a test tube were added ethanol [(2.00−x) mL] and solution of SIM-53 (or standard) (x mL) and finally 2 mL of ABTS •+ so as to make the final volume 4.0 mL. The tubes were closed by parafilm, and the mixtures were vortexed and incubated for 90 min at room temperature in a dark condition. Absorbance at λ = 734 nm was recorded against a blank using the mentioned UV-Vis spectrophotometer. The calibration curve obtained can be found in the Supplementary material.
Trolox equivalent antioxidant capacity (TEAC CUPRAC ) of SIM-53B was determined using its Cu 2+ reducing capability in the presence on neocuproine by the CUPRAC method [33]. The solution of SIM-53B and solutions of standards (Trolox and resveratrol) were freshly prepared in 96% ethanol at 1 mM concentration. To a test tube were added 1 mL each of CuCl 2 (10 mM in water), neocuproine (7.5 mM in 96% ethanol) and ammonium acetate buffer (pH 7, 1 mM in water) solutions. SIM-53 (or standard) solution (x mL) and water [(1.10−x) mL] were added to the mixture so as to make the final volume 4.1 mL. The tubes were closed by parafilm, and the mixtures were vortexed and incubated for 60 min at room temperature (or in a water bath at T = 50 • C for 20 min). Absorbance at 450 nm was recorded against a reagent blank using the mentioned UV-Vis spectrophotometer. The molar absorptivity (ε) for each antioxidant was calculated from the slope of the calibration line by plotting absorbance versus concentration (the calibration curve obtained can be found in the Supplementary material). TEAC CUPRAC was calculated by dividing the molar absorptivity of SIM-53B or resveratrol by that of Trolox.

Pseudophase Kinetic Model Applied to Emulsions: Partition Constants and Distribution of SIM-53B
In an emulsified system, SIM-53B can be transferred between the oil, aqueous and interfacial regions, Scheme 1. Thus, the distribution of SIM-53B can be defined by two partition constants, that between the aqueous and interfacial regions, P W I (Equation (1)), and that between the oil and interfacial regions, P O I (Equation (2)). The partition constants were assessed in the intact emulsions by making use of a chemical kinetic method that grounded the reaction between a chemical probe (4-hexadecylbenzenediazonium ion, 16-ArN 2 + ) and SIM-53B in the interfacial region and the experimental data were interpreted on the basis of the pseudophase kinetic model. In brief, the suppositions of the pseudophase kinetic model are that the partitioning of a compound is in dynamic equilibrium in an emulsified system and it depends on its relative solubility in the aqueous, oil and interfacial regions. Details of the method can be found elsewhere [22].
Equation (3) has been derived elsewhere [22] and describes the relationship between the observed rate constant k obs for the reaction between 16-ArN 2 + and SIM-53B and the partition constants, P O Equation (4) is equivalent to Equation (3) and the parameters a and b are defined by Equations (5) and (6), respectively. The parameters a and b can be determined by linear least squares fitting of the (1/k obs , Φ I ) pairs of data, Equation (7). Therefore, P O I and P W I values can be calculated from the variations in k obs with Φ I in two set of kinetics experiments for two different oil-in-water ratios by solving a system of two equations and two unknowns (Equation (6) for two different Φ O and Φ W values).
Once the partition constants are known, the percentages of SIM-53B in the different regions of the emulsion are obtained by using Equations (8)(9)(10). Details of the calculations are provided elsewhere [22].

Determination of k obs in Opaque Emulsion by Spectrophotometry
The reaction between 16-ArN 2 + and SIM-53B was followed spectrophotometrically in opaque olive oil-in-water emulsions by employing a colorimetric assay (derivatization reaction) described in detail elsewhere [34]. The derivatization reaction was grounded in the speedy reaction between the chemical probe, 16-ArN 2 + ions, and an appropriate coupling agent (N-(1-naphthyl)ethylenediamine dihydrochloride, NED) producing an instant and stable azo dye. In a common experiment, a SIM-53B-loaded emulsion was located in a stirred and thermostated (T = 25 • C) water-jacketed cell. Aliquots of 200 µL of emulsion were removed at selected time intervals and added to 15 test tubes each containing 2.5 mL of the reagent solution (alcoholic NED solution 19 mM, 50:50 (v:v) BuOH/EtOH). The optimal absorption wavelength was found to be 572 nm and was selected for azo dye determination. Auxiliary experiments confirmed that the absorption of the azo dye was proportional to the concentration of 16-ArN 2 + ions that do not react with SIM-53B.
The absorbance values obtained were plotted against the time and the observed first order rate constants, k obs , were determined from the fitting absorbance-time data to the integrated first order rate equation (Equation (11)), where A t , A o and A ∞ are the measured absorbance at any time, at t = 0 and at infinite time, respectively [34].

Statistical Analysis
Each set of kinetic experiments was performed in triplicate giving observed rate constants k obs values within ± 8%. Uncertainty in the P O I and P W I values was calculated by propagation of the standard error of the slope and intercept of the equation fit of the linear plots illustrated in Figure 1. In all cases, results are expressed as average ± standard deviation (n = 3). Figure 1 shows a typical kinetic plot illustrating the changes in the absorbance of the azo dye with time (circles) and the fitting curve to the integrated and linearized first-order equation (Equation 11, squares) for the reaction between 16-ArN2 + and SIM-53B in 1:9 (O/W) olive oil-in-water emulsions. The slopes of the linear fits of ln (At -A∞) vs. time were used to calculate the kobs values ( Figure 1B). Figure 1B illustrates the variations in kobs with ΦI for SIM-53B in 1:9 and 4:6 (O/W) olive oil-in water emulsions. In both kinetics experiments, kobs decreases asymptotically with increasing ΦI by a factor ∼6 on going from ΦI = 0.005 to ΦI = 0.04 for both emulsions. The excellent fits of Equations (4) and (7) to the kobs vs. ΦI plots and their reciprocals in Figure 1B show that the hypotheses considered in the derivation of Equation (3) are met [22]. The partition constants PW I and PO I and the kI values were determined from the slopes and intercepts of the linear fits of plots of 1/kobs vs. ΦI and values are included in Table 1. An initial analysis of the PW I and PO I values obtained for SIM-53B, Table 1, indicates that both values are quite high and positive, suggesting the tendency of SIM-53B to be distributed in the interfacial region of the olive oil-in-water emulsions. This fact means that the processes of transfer of SIM-53B from the oil to the interfacial and from the aqueous to the interfacial regions are spontaneous, with PW I >>> PO I reflecting the relative higher solubility of SIM-53B in olive oil than in the aqueous solution.

Partition Constants Values PW I and PO I for SIM-53B
For comparative purposes, note that the value of the partition constant between the oil and aqueous phase in the absence of an emulsifier, PW O , for SIM-53B can be defined as the ratio between  (4)) and to its reciprocal, Equation (7). The slopes of the linear fits of ln (A t -A ∞ ) vs. time were used to calculate the k obs values ( Figure 1B). Figure 1B illustrates the variations in k obs with Φ I for SIM-53B in 1:9 and 4:6 (O/W) olive oil-in water emulsions. In both kinetics experiments, k obs decreases asymptotically with increasing Φ I by a factor 6 on going from Φ I = 0.005 to Φ I = 0.04 for both emulsions. The excellent fits of Equations (4) and (7) to the k obs vs. Φ I plots and their reciprocals in Figure 1B show that the hypotheses considered in the derivation of Equation (3) are met [22]. The partition constants P W I and P O I and the k I values were determined from the slopes and intercepts of the linear fits of plots of 1/k obs vs. Φ I and values are included in Table 1. An initial analysis of the P W I and P O I values obtained for SIM-53B, Table 1, indicates that both values are quite high and positive, suggesting the tendency of SIM-53B to be distributed in the interfacial region of the olive oil-in-water emulsions. This fact means that the processes of transfer of SIM-53B from the oil to the interfacial and from the aqueous to the interfacial regions are spontaneous, with P W I >>> P O I reflecting the relative higher solubility of SIM-53B in olive oil than in the aqueous solution. For comparative purposes, note that the value of the partition constant between the oil and aqueous phase in the absence of an emulsifier, P W O , for SIM-53B can be defined as the ratio between the P W I and P O I values and calculated from Equation (12). A value of P W O = 8.6 ± 1.6 (Log P W O = 0.9 ± 0.2) was calculated at T = 25 • C, indicating its higher oil solubility in keeping with its hydrophobic nature. Note that the Log P W O value is~2.9 times lower than that calculated in n-octanol/water mixtures (Log P W OCT , Table 1), reflecting that SIM-53B is less soluble in olive oil than in octanol, questioning if the octanol/water system is the best model system for analyzing the hydrophobic effect in more complex biologic systems.

Partition Constants Values P W I and P O I for SIM-53B
In parallel, the rate constant for the reaction between the chemical probe and SIM-53B, k I , was also determined ( Table 1). This value is not necessary to describe how SIM-53B partitions between the different regions but its knowledge may be important because it delivers insights into the aspects of the reaction mechanism for the reaction between the chemical probe and SIM-53B. The k I value is comparable to that of the value obtained for chlorogenic acid (k I = 15 mM −1 s −1 ) [26] but lower for that of, for example, gallic acid (k I = 53 mM −1 s −1 ) [36], hydroxytyrosol (k I = 117 mM −1 s −1 ) [29] or α-tocopherol (k I = 190 mM −1 s −1 ) (unpublished results).

Distribution of SIM-53B and Its Local Effective Concentrations: Effect of Emulsifier Volume Fraction and O/W Ratio
Once partition constants P O I and P W I were known, the percentage of SIM-53B in each region of the olive oil-in-water emulsions was determined by employing Equations (8)(9)(10) in order to analyze its availability in the different regions of the emulsions, Figure 2. Details of the determination of the percentages of SIM-53B are given in Section 2.5.
Antioxidants 2020, 9, x FOR PEER REVIEW 10 of 16  (15) [ ]( ) % ( )  Figure 3 highlight three relevant points. First, from the comparison of the results in Figure 2, Figure 3, it can be seen that the (%SIM I ) increases upon increasing Φ I (Figure 2), whereas it has the opposite effect on the interfacial concentrations of SIM-53B ( Figure 3). This fact can be explained on the basis of the Equations (13)- (15). As described in Equation (14), the interfacial concentration of SIM-53B depends not only on the percentage of SIM-53B in the interfacial region but also on the interfacial volume. In this particular case, the increase in the incorporation of SIM-53B to the interfacial region of the emulsion does not compensate the increase in the interfacial volume resulting in the corresponding decrease in the interfacial concentration of SIM-53B. Second, the interfacial concentrations of SIM-53B are~20-200 times (depending on the Φ I value) higher than the stoichiometric concentration of SIM-53B ([SIM T ] = 1 mM), while the concentrations of SIM-53B in the oil and aqueous regions become much smaller than the stoichiometric concentration of SIM-53B. Notice that this observation may produce a significant change in its potential biological activity by amplifying it since the rate of any reaction depends on the concentrations of reagents at the reaction site.

Results in
Third, changes in the local concentrations of SIM-53B with O/W ratio from 1:9 to 4:6 (O/W) used in the preparation of the emulsion are not significant. For example, at Φ I = 0.005, (SIM I ) changes from 0.17 to 0.2 M upon decreasing the O/W ratio (differences less than 15%), and the differences are much less at higher emulsifier volume fractions (Φ I = 0.045) than at lower emulsifier volume fractions (Φ I = 0.005). Therefore, changes in the oil-in-water ratio should have predictably an almost negligible effect on the antioxidant efficiency of SIM-53B in inhibiting lipid oxidation since the effectiveness of the series of homologous AOs is directly related to their distribution according to our previous work [25][26][27][28][29].

Radical Scavenging Activity of SIM-53B
The antioxidant efficiency of a compound to inhibit oxidative damage caused by free radicals depends not only on its concentration at the reaction site but also its reactivity against free radicals. The radical scavenging activity of SIM-53B was assessed by employing the stable free radical 2,2-diphenyl-1-pycrilhydrazyl (DPPH • ) and 2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Antioxidant capacity was expressed in terms of the EC 50 value, labelled as the concentration of SIM-53B needed to decrease the DPPH • or ABTS •+ radical concentration by 50%. Figure 4A shows a typical kinetic plot obtained for the variation in the concentration of the DPPH • radical in the presence of different concentrations of SIM-53B at T = 20 • C. The percentage of the remaining DPPH • at a certain time of the reaction was obtained by using Equation (16), where A 0 is the absorbance of the negative control and A 1 is the absorbance of the test sample, and it was plotted against the concentration of SIM-53B ( Figure 4B). Trolox and resveratrol were used as positive controls in this assay with EC 50 12.4 ± 0.3 and 50.1 ± 4.6 µM, respectively [31]. The EC 50 value obtained for SIM-53B is listed in Table 1.
Antioxidants 2020, 9, x FOR PEER REVIEW 12 of 16 The EC50 value determined by the DPPH method for SIM-53B (440 μM, Table 1) is around 80 times higher than that observed for phenolic compounds such as gallic acid (3,4,5-trihidroxybenzoic acid, EC50 = 5.1 μM [37]) or catechin (EC50 = 6 μM [37]), suggesting a much lower reactivity of resorcinol derivatives vs. catechols towards DPPH • radicals. This can be explained by the presence of a second hydroxyl group in the meta position which enables intramolecular hydrogen bonding and more potent antioxidant activity [38,39]. Indeed, the antiradical efficiency of dihydroxybenzenes was determined in the following order: catechol > hydroquinone > resorcinol [40]. Furthermore, a similar pattern was observed in the series of dihydroxybenzaldehydes, where 2,3 or 3,4dihydroxybenzaldehyde exhibited the strongest antioxidant activities (EC50 between 2.3 and 15.7 μM in the ABTS and DPPH • assays), whereas 3,5-dihydroxybenzaldehyde was less potent in antioxidant activities with an EC50 of 20.59 and >100 μM in the ABTS and DPPH • assays, respectively [41]. The reduction kinetics of DPPH • caused by SIM-53B are typical for slow-kinetics polyphenols like the flavanone hesperetin [42]. On the contrary to the DPPH method, the EC50 value determined by the ABTS method (Supplementary material, Figure S10) for SIM-53B (6.45 ± 0,60 μM,  Figure S11) reflects the number of phenolic -OH groups in a molecule and depends on temperature which is again typical for slowkinetics polyphenols [33]

Prediction of Dug-Like Properties Pharmacokinetic Profile.
The pharmacokinetic profile of SIM-53B was evaluated by employing the SwissADME (absorption, distribution, metabolism and excretion of drugs) tool, a computer-designed model based on Lipinski's rule of five [43,44]. This rule states that high absorption or permeation of a compound is more probable when its chemical structure fulfils two or more of the following conditions: molecular weight is less than 500 Da; no more than five hydrogen bond donors (-NH-, -OH); no more than ten hydrogen bond acceptors (-N=, -O-); and octanol-water partition coefficient not greater than The EC 50 value determined by the DPPH method for SIM-53B (440 µM, Table 1) is around 80 times higher than that observed for phenolic compounds such as gallic acid (3,4,5-trihidroxybenzoic acid, EC 50 = 5.1 µM [37]) or catechin (EC 50 = 6 µM [37]), suggesting a much lower reactivity of resorcinol derivatives vs. catechols towards DPPH • radicals. This can be explained by the presence of a second hydroxyl group in the meta position which enables intramolecular hydrogen bonding and more potent antioxidant activity [38,39]. Indeed, the antiradical efficiency of dihydroxybenzenes was determined in the following order: catechol > hydroquinone > resorcinol [40]. Furthermore, a similar pattern was observed in the series of dihydroxybenzaldehydes, where 2,3 or 3,4-dihydroxybenzaldehyde exhibited the strongest antioxidant activities (EC 50 between 2.3 and 15.7 µM in the ABTS and DPPH • assays), whereas 3,5-dihydroxybenzaldehyde was less potent in antioxidant activities with an EC 50 of 20.59 and >100 µM in the ABTS and DPPH • assays, respectively [41]. The reduction kinetics of DPPH • caused by SIM-53B are typical for slow-kinetics polyphenols like the flavanone hesperetin [42]. On the contrary to the DPPH method, the EC 50 value determined by the ABTS method (Supplementary material, Figure S10) for SIM-53B (6.45 ± 0,60 µM, Table 1) is between those of Trolox (17.55 ± 0.36 µM) and resveratrol (5.69 ± 0.47 µM). The Trolox equivalent antioxidant capacity (TEAC CUPRAC ) of SIM-53B (1.04 at r.t., 1.81 at 50 • C) and resveratrol (1.55 at r.t., 2.31 at 50 • C) determined by the CUPRAC method (Supplementary material, Figure S11) reflects the number of phenolic -OH groups in a molecule and depends on temperature which is again typical for slow-kinetics polyphenols [33]

Prediction of Dug-Like Properties Pharmacokinetic Profile
The pharmacokinetic profile of SIM-53B was evaluated by employing the SwissADME (absorption, distribution, metabolism and excretion of drugs) tool, a computer-designed model based on Lipinski's rule of five [43,44]. This rule states that high absorption or permeation of a compound is more probable when its chemical structure fulfils two or more of the following conditions: molecular weight is less than 500 Da; no more than five hydrogen bond donors (-NH-, -OH); no more than ten hydrogen bond acceptors (-N=, -O-); and octanol-water partition coefficient not greater than 5. This study was carried out to forecast the pharmacokinetic profile, potential biological activities and toxicity of SIM-53B before it is assessed as a potential oral active drug [45]. Poor pharmacokinetic profile and toxicity are the main reasons for failures in drug discovery. Some parameters such as blood-brain barrier (BBB) permeation, human drug-likeness, interaction with cytochrome P450 (isoenzymes that play an important role in drug elimination through metabolic transformation) and bioavailability score were predicted, Table 1 [43].
The calculated "drug-likeness" value and the Abbot bioavailability score (defined as the probability of a compound to have at least 10% oral bioavailability in rat or measurable Caco-3 permeability, respectively) predict that SIM-53B may become a potential oral drug candidate since no violations to the Lipinski's rule of five were found, Table 1 [43].
Values predict a high probability of passive absorption by the gastrointestinal tract but not brain penetration. The interaction of SIM-53B with cytochrome P450 isoforms plays an important role in SIM-53B elimination through metabolic biotransformation. SIM-53B may inhibit some of the cytochrome P450 isoforms such as CYP1A2, CYP2C9 and CYP3A4. SIM-53B is not a substrate for p-glycoprotein (P-gp), a protein located in the intestinal epithelium where it pumps drugs back into the intestinal lumen and in the capillary endothelial cells composing the blood-brain barrier where it pumps them back into the capillaries. The skin permeate value suggests that SIM-53B would have low skin permeability.

Conclusions
We describe the synthesis of a new cyanothiophene-based compound, N-(3-cyano-4,5,6,7tetrahydrobenzo[b]thiophen-2-yl)-3,5-dihydroxybenzamide (SIM-53B), and evaluation of its antioxidant activity and distribution in intact stripped olive oil-in-water emulsions. The radical scavenging activity and reductive properties of SIM-53B were assessed by DPPH, ABTS and CUPRAC assays. The reduction kinetics of DPPH • caused by SIM-53B are typical for slow-kinetics polyphenols. Regarding the distribution, results show that SIM-53B has a natural tendency to be incorporated into the interfacial region of olive oil-in-water emulsions and its interfacial concentration is much higher (~20-200-fold) than the total added concentration due to the smaller interfacial volume in comparison with that of the total volume of the emulsion. The interfacial percentage of SIM-53B depends on both the O/W ratio and the emulsifier volume fraction (Φ I ). An increase in the emulsifier volume fraction increases its percentage from 91% (Φ I = 0.005) to 98% (Φ I = 0.04) for 1:9 (O/W) emulsions but decreases its interfacial concentration~9 times as a consequence of the higher increase in the interfacial volume than in the percentage of SIM-53B in the interface. Changing the O/W ratio from 4:6 (O/W) to 1:9 (O/W) has a minor effect on the interfacial concentration of SIM-53B due to its high accumulation in the interface at any Φ I value. Taken together, results show that the key parameter that controls the distribution of SIM-53B is the emulsifier volume fraction, suggesting that emulsions with low emulsifier volume fractions may enhance its bioavailability to attain the targets sites.
In addition, the pharmacokinetic properties predicted for this compound are in accordance with the general requirements for potential drugs and its interfacial location makes this new compound a very interesting scaffold for the development of new synthetic compounds that may be effective candidates for preventing or inhibiting oxidation in biomembranes or in other types of lipidic systems.