Maciej
Spiegel
a,
Tiziana
Marino
a,
Mario
Prejanò
b and
Nino
Russo
*a
aDipartimento di Chimica e Tecnologie Chimiche, Università della Calabria, I-87136 Rende, CS, Italy. E-mail: nrusso@unical.it
bDepartment of Organic Chemistry, Arrhenius Laboratory, Stockholm University, Stockholm, SE-10691, Sweden
First published on 13th June 2022
In this study, the scavenging activity against OOH radicals and the copper-chelating ability of two new synthesized molecules (named L1 and L2) that can act as multiple target agents against Alzheimer's disease have been investigated at the density functional theory level. The pKa and molar fractions at physiological pH have been predicted. The main antioxidant reaction mechanisms in lipid-like and water environments have been considered and the relative rate constants determined. The copper-chelating ability of the two compounds has also been explored at different coordination sites and computing the complexation kinetic constants. Results show the L1 compound is a more effective radical scavenging and copper-chelating agent than L2.
The pathology of AD is essentially characterized by the accumulation of senile plaques and neurofibrillary tangles and by abnormal levels of neurotransmitters.3–5 The senile plaques are formed by insoluble peptide segments composed of 39–43 amino acids (Aβ), and their aggregation (amyloid cascade hypothesis) and the possibility of the formation of small and soluble oligomers (oligomer hypothesis) are considered important phenomena and the basis of the disease.6,7 Despite the great amount of research carried out over the past twenty years, the exact cause of AD is still poorly understood and an effective cure has not yet been proposed.3,4,8,9 The few drugs approved by the US FDA and other European and Asian agencies essentially serve to alleviate the symptoms of the disease and improve the quality of life of patients, but they are ineffective in slowing down its evolution.10 For these reasons, the development of research in the field of AD is fundamental to both a better understanding of the mechanisms underlying its onset and the proposal of new, more effective and targeted drugs.
Autopsy examination of the brain tissues of patients who died due to AD has shown the presence of high and abnormal concentrations of metals such as copper, zinc and iron ions (about six and three times the normal quantity in the human brain, for Cu, and for Zn and Fe, respectively).11–13 It has been shown that this metal ion dyshomeostatis can play an important role since the formation of toxic metal–amyloid complexes promotes Aβ aggregation.13 Furthermore, owing to the redox nature of the metal ions involved, the presence of molecular oxygen in the brain tissue leads to the formation of reactive oxygen species (ROS) through Fenton-like reactions with a consequent increase in oxidative stress. In fact, due to the occurrence of high levels of unsaturated fatty acids (the preferred target of attack for ROS), the brain is particularly sensitive to oxidative stress, as has been proved in different neurodegenerative diseases.13–15
Among the three metal ions that undergo dyshomeostatis in the brains of AD patients, the most involved ions seem to be Cu2+ and Zn2+, which form stable complexes with the Aβ16 proteins, with different and much higher stability constants in the case of Cu2+.17,18
A strategy to limit ROS damage is based on the use of chelator agents that are able to control the increase in the concentration of metals, and to increase the level of antioxidant substances that are not usually present in the brain in sufficient quantities so as to counteract the effects caused by the rise in concentration of free radicals.19,20
On the basis of the observations reported above, the proposal of new drugs endowed with multiple actions in the latest research can contribute to increasing our knowledge not only about degenerative diseases, including Alzheimer's, but also to their treatment using multiple actions.21–23
Very recently, Cho et al.24 proposed some multifunctional molecular systems whose structure provided an antioxidant action together with a chelating action, to reduce the concentration of the copper ions in the brain (L1 and L2 of Scheme 1). The first action is due to the presence of a guaiacyl group, while some nitrogenous chelating groups, of different sizes that can coordinate the Cu2+ ion, express the second. Furthermore, some of their derivatives form stable Cu-radiolabeled complexes.25 These systems, which show values of lipophilicity such that their brain barrier penetration is possible, have been chemically characterized, and in vivo tests have suggested them as promising drugs against AD.
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Scheme 1 Chemical structure of the bifunctional chelators investigated. The atoms coloured represent the metal chelating sites considered in the present work. |
A better knowledge of their structural and electronic properties, as well as of their biological mechanisms, is very useful for their proposition as lead compounds for the development of new therapies in the treatment of AD. For these reasons, we have undertaken a detailed theoretical study using the density functional theory (DFT), which has proved to be particularly useful in the study and prediction of various molecular properties and reaction mechanisms, even in systems containing transition metals.26 In particular, we have considered different antioxidant reaction paths to establish what kind of scavenging mechanism these molecules follow and the possible chelating sites for copper-ion coordination.
The computational procedure previously described36 has been applied to obtain the molar fraction and pKa values. Numerous previous studies have demonstrated, in fact, that this computational protocol gives reliable results for thermochemistry and kinetics parameters relative to reactions involving free radicals and metals.37–40
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Fig. 1 Optimized structures, dissociation constants and molar distribution at pH = 7.4 for L1 and L2 molecules. |
Based on our previous experience42,43 and that of other research groups,39,40,44–46 the following redox and non-redox pathways were explored:
Hydrogen atom transfer (HAT): HX + R˙ → X˙ + RH |
Single electron transfer (SET): HX + R˙ → HX+˙ + R– |
Radical adduct formation (RAF): HX + R˙ → [HX−R]˙ |
The obtained Gibbs free energies of reaction are collected in Table 1. In both the considered environments and for both the considered species, the RAF mechanism generates endergonic reactions with Gibbs free energies higher than 10 kcal mol−1, suggesting that they do not take place. In addition, the SET mechanism in a lipid-like environment appears to be highly unlikely given the high endothermic values of the reaction Gibbs free energies. In the aqueous phase, calculations for both neutral and deprotonated forms confirm the feasibility of the SET mechanism. In fact, for the former we find ΔG values of 9.6 and 7.5 kcal mol−1 for L1 and L2, respectively, while for the latter they become −0.6 kcal mol−1 for L1 and 0.1 kcal mol−1 for L2.
Mechanism | L 1(PE) (L2(PE)) | L 1(W) (L2(W)) | L 1 − (W) (L2−(W)) |
---|---|---|---|
HAT | −4.1 (−3.5) | −9.6 (−6.9) | — |
RAF-C1 | 18.2 (19.0) | 14.2 (16.9) | 18.3 (17.5) |
RAF-C2 | 24.8 (31.0) | 24.7 (30.3) | 21.5 (21.5) |
RAF-C3 | 18.0 (14.9) | 16.7 (17.5) | 18.9 (18.5) |
RAF-C4 | 25.5 (24.8) | 24.0 (24.0) | 21.9 (21.2) |
RAF-C5 | 22.2 (22.5) | 20.3 (21.6) | 19.4 (19.5) |
RAF-C6 | 20.0 (22.8) | 18.8 (22.0) | 11.7 (16.4) |
RAF-C2′ | 20.6 (18.2) | 15.9 (17.3) | 17.9 (19.9) |
RAF-C3a′ | 32.9 (33.8) | 29.2 (30.5) | 27.6 (29.5) |
RAF-C4′ | 17.9 (17.8) | 15.3 (16.4) | 15.2 (13.9) |
RAF-C5′ | 22.3 (22.2) | 20.6 (21.2) | 20.4 (19.7) |
RAF-C6′ | 18.3 (18.7) | 17.5 (18.4) | 16.8 (16.4) |
RAF-C7′ | 18.3 (18.6) | 16.9 (17.9) | 17.5 (17.0) |
RAF-C7a′ | 27.9 (27.7) | 25.9 (27.1) | 24.4 (25.1) |
SET | 45.8 (67.3) | 9.6 (7.5) | −0.6 (0.1) |
Analyzing the data for the HAT mechanism, we note that, in both environments, the ΔG values indicate exergonic reactions for both the studied compounds. In particular, those related to the aqueous medium assume exothermic values (−9.6 and −6.9 kcal mol−1 for L1 and L2, respectively).
Based on the ΔG values obtained for the various reaction mechanisms, we proceeded to determine the potential energy surfaces by characterizing the transition states and the products for those processes whose reaction energies result in being lower than 10 kcal mol−1. The results, reported in Table 2 and Fig. 2, indicate that in the lipid-like phase, for both systems, only the HAT mechanism is possible with energy barriers of 15.0 and 19.1 kcal mol−1, for L1 and L2, respectively. In the aqueous environment, for the neutral forms of both compounds, the reactions that occur with SET and HAT mechanisms are energetically feasible. In particular, the energy barriers for L1 turn out to be 11.9 (SET) and 15.4 kcal mol−1 (HAT). Those found for L2 are 11.2 and 22.4 kcal mol−1 for the SET and HAT channels, respectively. Finally, the only possible path for both the anionic compounds results in being the SET mechanism, for which a barrier of 3.3 kcal mol−1 for L1 (3.6 kcal mol−1 for L2) must be overcome. The transition-state structures for the HAT mechanism, reported in Fig. 2, show similar geometrical parameters for the H transfer from the OH–L1 (L2) to the OOH radical in both the considered environments. The analysis of the computed negative frequency accounts for this process satisfactorily.
Mechanism | L 1(PE) (L2(PE)) | L 1(W) (L2(W)) | L 1 − (W) (L2−(W)) | |||
---|---|---|---|---|---|---|
ΔG | ΔG‡ | ΔG | ΔG‡ | ΔG | ΔG‡ | |
HAT | −4.1 (−3.5) | 15.0 (19.1) | −9.6 (−6.9) | 15.4 (22.4) | — | |
SET | 45.8 (67.3) | 52.3 (194.0) | 9.6 (7.5) | 11.9 (11.2) | −0.6 (0.1) | 3.3 (3.6) |
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Fig. 2 L 1 and L2 transition-state structures for the HAT mechanism with the corresponding frequencies, main bond lengths (Å), and dihedral angles (degrees). |
The obtained kinetic rate constants (Table 3) in the PE solvent for the HAT path indicate that the L1 system is a better antioxidant than the L2 system, with their k values being 1.89 × 104 and 7.43 × 101 M−1 s−1, respectively. Comparison with the corresponding k value (3.40 × 103 M−1 s−1) of Trolox (generally used as reference antioxidant)31,47 suggests that L1 is a more efficient OOH scavenger than L2.
Mechanism | k (M−1 s−1) | Γ (%) | k (M−1 s−1) | Γ (%) | k (M−1 s−1) | Γ (%) |
---|---|---|---|---|---|---|
L 1(PE) | L 1(W) | L 1 − (W) | ||||
HAT | 1.89 × 104 | 100.00 | 1.02 × 105 | 89.87 | ||
SET | 1.15 × 104 | 10.13 | 2.51 × 1010 | 100.00 | ||
F | 100% | 93.7% | 6.3% | |||
k total | 1.89 × 104 | 1.14 × 105 | 2.51 × 1010 | |||
fk total | 1.07 × 105 | 1.58 × 109 | ||||
L 2(PE) | L 2(W) | L 2 − (W) | ||||
HAT | 7.43 × 101 | 100.00 | 1.22 × 102 | 3.39 | ||
SET | 3.59 × 104 | 96.61 | 1.49 × 1010 | 100.00 | ||
F | 100% | 98.5% | 1.5% | |||
k total | 7.43 × 101 | 3.60 × 104 | 1.49 × 1010 | |||
fk total | 3.55 × 104 | 2.24 × 108 |
In water and at physiological pH (7.4) the situation is different. In particular, we underline that, in the neutral form, which has a higher molar fraction (93.7 and 98.5% for L1 and L2, respectively), the HAT mechanism in L1 (k = 1.02 × 105 M−1 s−1) is preferred, while the SET mechanism dominates for the L2 species (k = 3.59 × 104 M−1 s−1). Looking at the ktotal values, our data indicate that L1 is a better radical scavenger than L2 with a difference of one order of magnitude. The comparison with Trolox in the aqueous solvent and under physiological pH conditions (k = 8.96 × 104 M−1 s−1)46 shows that L1 is more efficient, while L2 has almost the same antioxidant power.
Our results well agree with the experimental measurements that reveal L1 to be 1.6 times more efficient than Trolox as a radical-scavenging system.25
Cu(H2O)62+ + L → [Cu(H2O)6 L]2+ |
Cu(H2O)62+ + L− → [Cu(H2O)6 L]+ |
For both the ligands we have considered three possible coordination sites of the Cu2+ ion (Scheme 1): coordination with the O1 and O6 oxygen atoms linked in the phenolic cycle, with the nitrogen atoms of the peripheral macrocycle (N3 or N4 for L1 and L2, respectively) and with both sites (the oxygen atoms of the phenyl ring and the nitrogen atoms of the macrocycle (N3 or N4 for L1 and L2)). This topology will hereafter be referred to as O,O, N3 (or N4), and N,O. The energetic values (ΔG and ΔΔG) and the main geometrical parameters around the copper ion are reported in Table 4 and Table S1 (ESI†), respectively. The optimized structures are given in Fig. 3 (for L1) and Fig. 4 (for L2).
Coordination site | ΔGf (kcal mol−1) | ΔΔGf (kcal mol−1) | K f | ΣKf (KIIf) |
---|---|---|---|---|
L 1 (93.7%) | ||||
N3 | −18.4 | 1.2 | 2.88 × 1013 | |
N,O | −19.6 | 0.0 | 2.24 × 1014 | |
O,O | −5.8 | 13.8 | 1.61 × 104 | |
2.53 × 1014 (2.37 × 1014) | ||||
L 1 − (6.3%) | ||||
N3 | −25.2 | 11.2 | 2.81 × 1018 | |
N,O | −36.4 | 0.0 | 5.01 × 1026 | |
O,O | −16.2 | 20.2 | 7.11 × 1011 | |
5.01 × 1026 (3.16 × 1025) | ||||
K appf (L1) = 3.16 × 1025 | ||||
L 2 (98.5%) | ||||
N4 | −13.7 | 0.0 | 1.21 × 1010 | |
O,O | 4.4 | 18.1 | 6.38 × 104 | |
1.21 × 1010 (1.19 × 1010) | ||||
L 2 − (1.5%) | ||||
N4 | −11.5 | 1.6 | 2.68 × 108 | |
O,O | −13.1 | 0.0 | 4.58 × 109 | |
6.44 × 1018 (9.66 × 1016) | ||||
K appf (L2) = 9.66 × 1016 |
For L1, Table 4 shows that the preferred coordination site is N,O followed by N3 at only 1.2 kcal mol−1. The O,O topology results in being at a higher energy (13.8 kcal mol−1). For N,O coordination, analysis of the Cu–X bond lengths (Fig. 3 and 4 and Table S1, ESI†) reveals that the Cu ion interacts with the three nitrogen atoms of the peripheral macrocycle, while the bond with the O1 oxygen is weak since Cu–O1 assumes a value of 2.812 Å. In addition, the Cu2+ interacts with the two water molecules at distances of 2.084 and 2.355 Å. The situation is slightly different for the interaction with the anionic ligand. In fact, although the preferred coordination continues to be N,O, the other two are at higher energies (11.2 and 20.2 kcal mol−1 for N3 and O,O, respectively). In the absolute minimum, the Cu–O1 bond is strong (Cu–O distance is 1.957 Å) due to the presence of a more negative charge on the interacting oxygen atom, and the solvation water molecules are away from the metal center (more than 3.1 Å).
Analysis of the spin density maps reveals that in the N,O coordination mode, for both the Cu–L1 and Cu–L1− systems (see Fig. S1 and S2, ESI†), the spin density is almost distributed on the entire molecule, enhancing the stability of this conformation.
To discuss the complexation process for the L2 ligand, we note that for both the considered species, the N,O coordination is not a minimum in the potential energy surfaces. For the L2 neutral species the N4 topology is preferred over the O,O topology by 18.1 kcal mol−1 (see Table 4). From Table S1 (ESI†) and Fig. 4 it is possible to see that the Cu2+ ion is bonded with the N4 and N10 nitrogen atoms of the pyridinophane ring (2.085 and 2.073 Å for Cu–N4 and Cu–N10 bonds, respectively) and with the oxygen atoms of two water molecules with bond distances of about 2.0–2.1 Å. For the anionic form of L2, the preferred coordination site results in being the O,O topology while the N4 lies at only 1.6 kcal mol−1. In the O,O mode, the copper ion forms two bonds with the O1 and O6 atoms of the phenyl ring, assuming the values of 1.918 Å (Cu–O1) and 2.343 Å (Cu–O6). There are two other strong interactions with the oxygen atoms of two water molecules with bond distances of 2.031 and 2.244 Å.
For both anionic species (L1− and L2−) in the O,O topology the copper ion shows an octahedral coordination geometry.
Furthermore, the spin density behavior reveals in the neutral complex (Fig. S1, ESI†) that the resulting distribution with N4 coordination being shared in the two molecular fragments, whereas with O,O chelation it is more localized. In the charged complexes (Fig. S2, ESI†) spin delocalization is found, for both coordination modes, in only one segment, in agreement with the small energy difference between the complexes.
Comparing the molecular electrostatic potential of the L1 and L2 complexes (Fig. S3, ESI†) it is possible to note that in the L1 ligand the negative charge is mainly concentrated on the N3 site while in the L2 ligand, albeit to only a minor extent, the other fragment is also implicated. This distribution can be correlated with the fact that in L1 the preferred coordination site is the N,O.
According to the data gathered in Table 4, for the chelation processes predicted to be viable from a thermochemical point of view (exergonic or slightly endergonic reaction pathways) we computed the complexation kinetic constants according to a previously suggested procedure48 and taking into account the populations of the complexes estimated through the Maxwell–Boltzmann distribution (see Table 4).
The obtained apparent equilibrium constants (Kappf) for the L1 and L2 ligands were found to be 3.16 × 1025 and 9.66 × 1016 M−1, respectively, indicating that the former is predicted to be a stronger Cu(II)-chelating agent that can reduce the Cu(II) free ions produced by the Fenton reactions associated with Alzheimer's disease.
The computed pKa values of the methoxyphenol group present in the 2-phenylbenzothiazole moiety show a higher value for the L2 species. The molar distribution at physiological pH indicates that for both molecules the neutral form is dominant. However, the anionic species, although present at a small concentration, should be considered for their contribution to both the antioxidant and Cu-chelating properties.
In lipid-like and water environments, the HAT mechanism results in being exergonic.
The obtained rate constants clearly indicate that L1 is much more efficient than L2 as an OOH scavenger in both the studied environments and that L1 is more efficient than Trolox, which is generally used as a reference antioxidant.
The Cu2+ preferred coordination sites have been determined. The computed apparent equilibrium constants clearly show that the L1 ligand is much more efficient as a chelating agent.
Our results well agree with the available experimental indications.
We hope that our results can further stimulate other investigations devoted to the possible use of these systems as multiple target drugs against Alzheimer's disease.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d2cp01918c |
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