Theoretical insights into the antiradical activity and copper-catalysed oxidative damage of mexidol in the physiological environment

Mexidol (MD, 2-ethyl-6-methyl-3-hydroxypyridine) is a registered therapeutic agent for the treatment of anxiety disorders. The chemical structure suggests that MD may also act as an antioxidant. In this study, the hydroperoxyl radical scavenging activity of MD was studied to establish baseline antioxidant activity, followed by an investigation of the effect of MD on the copper-catalysed oxidative damage in biological systems, using computational methods. It was found that MD exhibits moderate radical scavenging activity against HOO• in water and pentyl ethanoate solvents following the single electron transfer and formal hydrogen transfer mechanisms, respectively. MD can chelate Cu(II), forming complexes that are much harder to reduce than free Cu(II): MD chelation completely quenches the Cu(II) reduction by ascorbic acid and suppresses the rate of reduction reaction by O2⋅− that are the main reductants of Cu(II) in biological environments. Therefore, MD exerts its anti-HO• activity primarily as an OIL-1 inhibitor.


Introduction
Mexidol (MD, emoxypine, 2-ethyl-6-methyl-3-hydroxypyridine) is a drug used primarily for the treatment of anxiety. It is known to have antiischemic, antihypoxic, neuroprotective, antistress, nootropic and geroprotective properties [1,2]. MD is also used as an antioxidant for reducing tissue damage by reactive oxygen species [1,3]. For this reason, the radical scavenging activity of MD was assessed in experimental studies [3,4]. The rate constant of MD reaction with peroxyl radical in methyl oleate was measured as k = 2.8 × 10 4 M −1 s −1 [4], while that for 1,4-dioxane solvent was k = 2.1 ± 0.3 × 10 4 M −1 s −1 [3]. However, organic solutions such as methyl oleate and 1,4dioxane are not suitable model environments to assess in vivo activity; in the physiologically relevant aqueous environment MD may exhibit very different behaviour due to dissociation of the OH moiety. Thus there is an impetus to study the antiradical activity of MD in physiological media.
In evaluating the antioxidant activity of MD, one has to also consider that pyridines are very good coordinating ligands and therefore it is highly likely that MD forms chelates with trace metals [5,6]. Previous studies showed that the presence of transition metals such as Cu(I) can produce hydroxyl radicals via the Fenton-like reaction (1.1) [7][8][9][10][11]: Cu þ þ H 2 O 2 ! Cu 2þ þ HO Á :

ð1:3Þ
Chelation of Cu(II) has the potential to inhibit or suppress reactions (1.2) and (1.3) and therefore indirectly inhibit reaction (1.1). Thus, the capacity of MD to inhibit Cu(II) reduction by chelation is also of interest (figure 1).
Previous studies showed that computational method offers the most convenient path for studying structure-activity relationships in radical reactions to guide the design of novel antioxidants with enhanced activity [12][13][14][15][16][17][18][19]. In several prior studies, the radical scavenging activity of organic compounds in physiological environments was successfully evaluated by quantum chemistry calculations [20][21][22][23]. Thus, in this study, the HOO • radical scavenging activity of MD was assessed in lipid and polar media using the quantum mechanics-based test for overall free radical scavenging activity (QM-ORSA) protocol [13,20]. Cu(II) chelation ability was also assessed and the ability of MD to act as an OIL-1 inhibitor of the copper-catalysed oxidative damage in biological systems was investigated.
The stability of Cu(II) chelates was compared by calculating the Gibbs free energy of formation for all possible chelates that were first constructed with MD based on [Cu(H 2 O) 4 ] 2+ geometry, optimized by molecular mechanics calculations using the Spartan software [31], then energy-minimized with DFT as per above.

Gas phase evaluation
To reduce computing time, the radical scavenging activity of MD was first evaluated in the gas phase following the liturature [16,40], according to the three main reaction pathways: formal hydrogen transfer (FHT), single electron transfer followed by proton transfer (SETPT) and sequential proton loss electron transfer (SPLET) [41,42]. Radical adduct formation (RAF) is another common pathway that however requires a localized C=C bond; the aromatic ring of MD cannot support this mechanism [21,43,44]. The probabilities of the three feasible antioxidant mechanisms (FHT, SETPT and SPLET) were first evaluated by computing the main thermodynamic parameters associated with these mechanisms: bond dissociation enthalpy (BDE), ionization energy (IE) and proton affinity (PA), respectively. The calculated BDE, IE and PA values of MD are shown in electronic supplementary material, table S3.

The HOO • radical scavenging activity of MD in physiological environments
In aqueous environment, the antiradical activity of acidic species is typically dominated by the ionic form [26,30,48]. Therefore, the protonation state of MD was first evaluated at physiological pH to find the most likely radical scavenging reactions. The MD structure allows protonation at the N1−H and O3−H bonds (figure 3); thus the pK a values of MD were calculated based on the literature [48,49] and are shown in figure 3.
The calculated pK a values for the amine were pK a1 = 7.17 (for N−H bond of cation form) and pK a2 = 9.79 (for O3−H bond). Therefore, in pH = 7.4 aqueous solution, MD exists in three states: the cation (H 2 A + , 37.0%), neutral (HA, 62.7%) and anion (A − , 0.3%) states. Therefore, all three states were used in the kinetic evaluation of HOO • radical removal of MD in water at physiological pH = 7.4.
The preferred radical scavenging pathways of the neutral and anionic states have been established; the cationic state requires initial evaluation. In the aqueous solution  royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 9: 211239 royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 9: 211239 5 nonpolar media, respectively) [40], the HOO • radical scavenging activity of MD is lower in all of the studied environments. However, according to the empirical test for radical scavenging (activity should exceed 10 3 ) [20,50] MD can be considered an antioxidant, albeit a weak one.  figure 4) at −82.5 kcal mol −1 that is more than two times lower than the second lowest value (reaction 8, N site, electronic supplementary material, table S2). The equilibrium constant for reaction 14 is K = 3.01 × 10 60 M −1 and this chelate will make up 100% of the possible complexes, based on Maxwell-Boltzmann calculations. The mono-, tri-and tetra-coordinate Cu-MD complexes were also investigated; however, these reactions were not favoured for the MD +      2 ] + are only solvating the system. That is consistent with previous studies of the coordination numbers and geometries of Cu(II) and Cu(I) complexes [9,53].

OIL-1 inhibition of copper-catalysed oxidative damage in biological systems
To evaluate the capacity of MD to reduce the copper-induced oxidative stress following the OIL-1 process in water at pH = 7.4, reduction reactions of the most stable complex ([Cu(HA) 2

Conclusion
The antioxidant activity of MD was investigated by evaluating the radical scavenging activity and the OIL-1 suppression of copper-catalysed oxidative damage in biological systems using computer calculations. It was found that MD exhibits moderate hydroperoxyl radical scavenging activity in both lipid and polar media. The antiradical activity in non-polar environments follows the FHT mechanism at the O3−H bond, whereas in aqueous solution, it follows the SET pathway of the anion state.
Chelation with MD could suppress the Cu(II) reduction by O ÁÀ