The Scavenging of DPPH, Galvinoxyl and ABTS Radicals by Imine Analogs of Resveratrol

Resveratrol (3,5,4′-trihydroxystilbene) is a phytoalexin produced by plants. Resveratrol is known for its anti-cancer, antiviral and antioxidant properties. We prepared imine analogs of resveratrol ((hydroxyphenyliminomethyl)phenols) and tested their antioxidant activity. All prepared resveratrol analogs were able to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH), galvinoxyl radical (GOR) and 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS) radicals. The antioxidant activity efficiency correlated with the number and position of hydroxyl groups. The most effective antioxidants were resveratrol analogs containing three hydroxyl groups in the benzylidene part of their molecules. These results provide new insights into the relationship between the chemical structure and biological activity of resveratrol analogs.


Introduction
The structure of (hydroxyphenyliminomethyl)phenols ( Figure 1) is similar to the structure of resveratrol (3,5,4 1 -trihydroxystilbene), which exhibits various biological activities. There are two geometric isomers of resveratrol-the trans configuration undergoes isomerisation to the cis form following exposure to ultraviolet light. Resveratrol is a phytoalexin produced by several plants that was initially identified in the flowering plant Veratrum grandiflorum. Relatively high concentrations of resveratrol are also found in grape skins and seeds, as well as red wine. Resveratrol has gained widespread attention due to its ability to extend the lifespan of various organisms, to protect against age-related diseases such as cancer and a broad range of biological activities including antiviral and antioxidant properties [1][2][3][4]. However, it is not clear whether the health benefits associated with wine drinking ("French paradox", the observation of reduced incidence of cardiovascular disease in regions of France where red wine and fats are consumed in large quantities) are due to resveratrol [5]. Molecular mechanisms underlying the health-enhancing effects of resveratrol are not well understood but some of them may be due to its antioxidant properties [6]. Although basal level of oxidative stress is important for normal cell function, severe oxidative stress often leads to oxidative damage, cell death and various diseases [7]. Resveratrol supplementation decreases the oxidative stress caused by Previous studies showed that modification of resveratrol could enhance its biological activities [10][11][12]. It is therefore important to synthesize resveratrol analogs and study their physico-chemical and biological properties. Imines are often used as ligands for the complexation of metals; they are used for the preparation of liquid crystals and are also used in analytical and pharmaceutical chemistry [13]. The traditional method of the imine preparation is based on heating aldehydes with amines in an organic solvent (benzene or toluene) [13][14][15]. Synthesis in which the organic solvent is replaced with water [16] or other recycle medium such as polypropylene glycol [17] has been described as "green" alternative to these preparations.
The goal of this work was to prepare various imine analogs of resveratrol (some of which have not been described in the literature yet) and to test their antioxidant activity. We analyzed antioxidant properties of twenty-one (hydroxyphenyliminomethyl)phenols ( Figure 1) where R i = OH or H.

Chemistry
The starting compounds for preparation of (hydroxyphenyliminomethyl)phenols were mono aminophenols (APs) and mono-, di-and trihydroxybenzaldehydes (HBAs). Twenty-one (hydroxyphenyliminomethyl)phenols (compounds 1-21, for full names see Section 3.2. Synthesis) were prepared by condensation reactions where the first step was addition of amino group on carbonyl group followed by elimination of water according to protocols of Tanaka and Shiraishi [16] and Lu et al. [29] (Scheme 1). Briefly, equimolar amounts of appropriate AP and HBA were stirred in a small amount of distilled water at room temperature (25 °C) for 2 h. Obtained products were filtered, washed several times with distilled water and dried at 45 °C in vacuum. Yields of (hydroxyphenyliminomethyl)phenols ranged between 26% and 95%. The compounds were characterized by melting point, elemental analysis, 1 H-and 13   Previous studies showed that modification of resveratrol could enhance its biological activities [10][11][12]. It is therefore important to synthesize resveratrol analogs and study their physico-chemical and biological properties. Imines are often used as ligands for the complexation of metals; they are used for the preparation of liquid crystals and are also used in analytical and pharmaceutical chemistry [13]. The traditional method of the imine preparation is based on heating aldehydes with amines in an organic solvent (benzene or toluene) [13][14][15]. Synthesis in which the organic solvent is replaced with water [16] or other recycle medium such as polypropylene glycol [17] has been described as "green" alternative to these preparations.
The goal of this work was to prepare various imine analogs of resveratrol (some of which have not been described in the literature yet) and to test their antioxidant activity. We analyzed antioxidant properties of twenty-one (hydroxyphenyliminomethyl)phenols ( Figure 1) where R i = OH or H.

Chemistry
The starting compounds for preparation of (hydroxyphenyliminomethyl)phenols were mono aminophenols (APs) and mono-, di-and trihydroxybenzaldehydes (HBAs). Twenty-one (hydroxy phenyliminomethyl)phenols (compounds 1-21, for full names see Section 3.2. Synthesis) were prepared by condensation reactions where the first step was addition of amino group on carbonyl group followed by elimination of water according to protocols of Tanaka and Shiraishi [16] and Lu et al. [29] (Scheme 1). Briefly, equimolar amounts of appropriate AP and HBA were stirred in a small amount of distilled water at room temperature (25˝C) for 2 h. Obtained products were filtered, washed several times with distilled water and dried at 45˝C in vacuum. Yields of (hydroxyphenyliminomethyl)phenols ranged between 26% and 95%. The compounds were characterized by melting point, elemental analysis, 1 H-and 13 C-NMR, IR and MS spectra. carbonyl group followed by elimination of water according to protocols of Tanaka and Shiraishi [16] and Lu et al. [29] (Scheme 1). Briefly, equimolar amounts of appropriate AP and HBA were stirred in a small amount of distilled water at room temperature (25 °C) for 2 h. Obtained products were filtered, washed several times with distilled water and dried at 45 °C in vacuum. Yields of (hydroxyphenyliminomethyl)phenols ranged between 26% and 95%. The compounds were characterized by melting point, elemental analysis, 1 H-and 13 C-NMR, IR and MS spectra.
We found that the efficiency of the radical scavenging correlated with the position and number of hydroxyl groups in prepared compounds. Regarding the position of the hydroxyl group in hydroxyphenylimino parts of prepared compounds, most (hydroxyphenyliminomethyl)phenols with the OH group in ortho position of the ring A were the most potent scavengers of DPPH and galvinoxyl radicals. (Hydroxyphenyliminomethyl)phenols with three OH groups in the ring A were the most potent scavengers of ABTS radicals. (Hydroxyphenyliminomethyl)phenols 2, 4, 6, 7, 11, 16 which contained one or two OH groups in positions 3 and 4 of the ring A exhibited relatively low ability to scavenge DPPH or GOR radicals (their values of SC 50 ranged from 38 to 3075 µmol/dm 3 ). However, compounds 3 and 5, which have OH group in the ortho position of the benzene ring A, exhibited low antioxidant activity. On the other hand, compunds 17 and 21, which do not have OH groups in the ortho position of the phenyl ring A, exhibited high antioxidant activity.
Based on the 1 H-NMR spectra analyses, we assumed that proton could be most easily dissociation from the OH group in the ortho position of the ring A. (Hydroxyphenyliminomethyl)phenols with R 1 = OH could form intramolecular hydrogen bond with the nitrogen of imine group. Due to this interaction acidity of hydrogen at this position increased, which was confirmed by the high chemical shift (δ = 12.25 to 14.19 ppm). On the other hand, the chemical shift of the OH protons bound in the meta or para position were <8.10 ppm.
It is generally assumed that the mechanism of radical scavenging by phenolic compounds is associated with the ability to release hydrogen atom, proton or electron from the antioxidant molecule. We speculated that releasing of the proton from OH group could be the main mechanism respossible for the antioxidant activity of studied (Hydroxyphenyliminomethyl)phenols. Consistent with this assumption were the SC 50 values of studied compounds with higher δ which exhibited higher antioxidant activity as compared to other (Hydroxyphenyliminomethyl)phenols.
To find a possible correlation between the antioxidant activity of (hydroxyphenyliminome thyl)phenols and a position of the hydroxyl groups, we used the method PM6 (see the Experimental Section) to calculate the energy associated with the release of hydrogen (bond dissociation enthalpy, BDE); the proton relaxation (PDE); release of electron (IP ionization potential) and the energy associated with combined transfer of the electron and proton (ETE and PA, respectively) for the studied (Hydroxyphenyliminomethyl)phenols. The values of these energies in methanol or in water are presented in Tables S1-S8.
The antioxidant mechanism associated with the release of a hydrogen atom can be described by the Equation (1). From the point of view of thermodynamics the strength of the antioxidant effect in the model describes BDE (bond dissociation enthalpy, Equation (2)): The other antioxidant mechanism of (hydroxyphenyliminomethyl)phenols may be associated with combined transfer of electrons and protons to radical (R ‚ ). In the first step, the electron is transferred to the radical (Equation (3)). The possibility of such transition is determined by the size of ionization potential of IP, which describes the Equation (4): In the second step, a proton is transferred to the anion radical R´(Equation (5)). The possibility of such a transfer is characterized by the value of PDE (proton dissotiation enthalpy, Equation (6)): Another possible mechanism of the antioxidant effect of (hydroxyphenyliminomethyl)phenols is the gradual loss of a proton followed by electron transfer (Equations (7) and (8)): Molecules 2016, 21, 127 The reaction enthalpy of the first step (Equation (7)) corresponds to the proton affinity PA (Equation (10)) whereas the reaction enthalpy of the second step (Equation (8)) describes enthalpy of electron transfer (ETE, Equation (11)): Comparison of enthalpies required for the above mentioned antioxidant reactions suggests that the preferred mechanism seems to be the one described by Equations (7)- (11), those characterized by the values of PA + ETE. Based on the above mentioned ideas, we speculated that the lower PA + ETE values of studied (hydroxyphenyliminomethyl)phenols may imply their higher antioxidant activity, i.e., lower value of SC 50 . Figure 2 shows the dependence of SC 50 scavenging DPPH radicals on the sum of PA + ETE enthalpy. As can be seen from these figures, there is no obvious relationship between these parameters. This discrepancy could be caused by forming of (hydroxyphenyliminomethyl)phenols self-aggregates which could reduce the antioxidant activity of original compounds. Comparison of enthalpies required for the above mentioned antioxidant reactions suggests that the preferred mechanism seems to be the one described by Equations (7)- (11), those characterized by the values of PA + ETE. Based on the above mentioned ideas, we speculated that the lower PA + ETE values of studied (hydroxyphenyliminomethyl)phenols may imply their higher antioxidant activity, i.e., lower value of SC50. Figure 2 shows the dependence of SC50 scavenging DPPH radicals on the sum of PA + ETE enthalpy. As can be seen from these figures, there is no obvious relationship between these parameters. This discrepancy could be caused by forming of (hydroxyphenyliminomethyl)phenols self-aggregates which could reduce the antioxidant activity of original compounds. To test this assumption we performed the following experiment. According to the work of Cigáň et al. [32], creation of self-aggregates can be determined from concentration-dependence of absorption or fluorescence spectra. The formation of self-aggregates should be manifested by a shift of emission maxima or by a shape change in absorption/fluorescence spectra with an increase of concentration of To test this assumption we performed the following experiment. According to the work of Cigáň et al. [32], creation of self-aggregates can be determined from concentration-dependence of absorption or fluorescence spectra. The formation of self-aggregates should be manifested by a shift of emission maxima or by a shape change in absorption/fluorescence spectra with an increase of concentration of the studied compound. We observed that the corresponding changes in absorption spectra were very small. Also in fluorescence emission spectra, relatively small batochromic shifts were observed with increasing concentrations. Compound 5 with lower antioxidant activity exhibited approximately 11 nm, whereas compound 14 with higher antioxidant activity showed only 4 nm (data not shown). The formation of self-aggregates was apparent from the fluorescence excitation spectra. We observed changes in shapes of these spectra with the increasing concentrations of (Hydroxyphenyliminomethyl)phenols (Figure 3). These results suggest that one possible explanation for the lower antioxidant activity of some of the studied (Hydroxyphenyliminomethyl)phenols is formation of self-aggregates. A possible structure of aggregates for compound 3 is presented in Figure S1.  Numerous studies showed that various modifications of resveratrol molecule enhance its biological activities including the antioxidant properties [10][11][12]33]. It is therefore important to synthesize new resveratrol analogs and study their physico-chemical and biological properties. We aimed to identify novel analogues of resveratrol that could potently scavenge radicals and serve as leads for the development of future drugs. To find out more about potential pharmacological and nutritional relevance of (Hydroxyphenyliminomethyl)phenols synthesized in our current study, it will be important to test in vivo activities of these compounds. However, given their unstability in water, additional chemical modifications may be required to achieve high in vivo activity. Our current study brings new insights into the relationship between the chemical structure and antioxidant properties of resveratrol analogs.

DPPH Assay
Scavenging of DPPH radicals by prepared (hydroxyphenyliminomethyl)phenols was performed according to our previous work Šeršeň et al. [34]. Briefly, various amounts of tested (Hydroxy phenyliminomethyl)phenols were added into methanol solution of DPPH and the final DPPH concentration was kept constant (c = 10´4 mol¨dm´3). 20 min after the addition of the tested substance, absorbance was measured at 517 nm. The antioxidant activity was evaluated using the values SC 50 , i.e., concentration of the studied compound, which causes a 50% decrease in absorbance at 517 nm as compared to the control sample. Methanol was used as a blank.

Scavenging of Galvinoxyl Radicals
Various amounts of (hydroxyphenyliminomethyl)phenols were added into methanol solution of galvinoxyl radical such that the final galvinoxyl concentration was c = 10´4 mol¨dm´3. 20 min after the addition of (hydroxyphenyliminomethyl)phenols, absorbance was measured at 862 nm. SC 50 values were calculated from the absorbance vs. the concentration dependence. Methanol was used as a blank.

ABTS Assay
ABTS cation radicals were prepared according to Kurin et al. [35] with minor adaptations. Briefly 7 mM aqueous solution of ABTS was mixed in equimolar ratio with 2.45 mM K 2 S 2 O 8 , and let 24 h to stand in the dark at room temperature. After 24 h 1.1 mL of concentrated solution of the radical was diluted with 50 mL of phosphate buffer (solution A: 900 mg of Na 2 HPO 4 in 500 mL H 2 O, the solution B: 340 mg of KH 2 HPO 4 in 500 mL H 2 O, we combined 9 parts of solution A and 1 part of solution B, the pH we set to 7.4). To a solution of 1.8 mL of ABTS we added 0, 25, 50, 75, 100, 125, 150, 175 or 200 mL of the test substance and completed with 200, 175, 150, 125, 100, 75, 50, 25 or 0 mL of ethanol (to a final volume of 2 mL. The absorbance at 734 nm of prepared solutions was measured after 6 min. We calculated the appropriate values of the SC 50 from the linear part of the absorbance vs. the concentration of (hydroxyphenyliminomethyl)phenols. The water was used as blank.

Molecular Calculations
The prepared (hydroxyphenyliminomethyl)phenols, their anions and radicals were studied using the quantum chemical method PM6 [36], which is part of the program MOPAC2012 [37]. Optimal structures of compunds were calculated (keyword PRECISE). The effect of solvents on the above mentioned compounds were studied by COSMO-method [38], which is also part of the MOPAC2012 [39]. Ionization potentials and heats of formations used for the calculation of PDE, BDE, PA and ETE according to the Equations (2), (6), (10) and (11) were obtained using the method PM6.

Conclusions
In this work we prepared twenty-one (hydroxyphenyliminomethyl)phenols, which were synthesized by condensation reactions of the corresponding aminophenols and hydroxybenzaldehydes. The chemical structures of these compounds were confirmed by 1 H-NMR, IR spectroscopy, elemental analysis and GCMS. All prepared (hydroxyphenyliminomethyl)phenols were able to scavenge DPPH, GOR and ABTS radicals. Some of the (hydroxyphenyliminomethyl)phenols were more potent scavengers of DPPH (compounds: 8, 10, 12-14, 11-27) and GOR (compounds: 2, 9, 12, 15, 17-21) radicals as compared to resveratrol. The efficiency of antioxidant activity correlated with the number and position of hydroxyl groups. The most efficient antioxidants were (hydroxyphenyliminomethyl)phenols containing three hydroxyl groups in the benzylidene part of molecules.