Experimental and theoretical study on structure-tautomerism among edaravone, isoxazolone, and their heterocycles derivatives as antioxidants.

Edaravone is a heterocyclic pyrazolone compound. It has pronounced effect against free radicals, however renal and hepatic disorders have been reported. Isoxazolones are considered bioisosteric analogues of pyrazolones and may have comparable properties. Thus, we investigated the structural and electronic influences for edaravone, isoxazolone, and their tautomers on antioxidant process. Structure and tautomerism study among edaravone, isoxazolone and their heterocycles derivatives were related to antioxidant mechanisms by using the hybrid DFT method B3LYP with the basis sets 6-31++G(2d,2p). The C-H tautomer was the most stable and energetically favored among them. Intramolecular N-H-N hydrogen bonds and polar medium were responsible for the low energy differences among all possible tautomers. N-H tautomers in both systems proved to be better antioxidant by SET (single electron transfer), while O-H tautomers were better antioxidant on HAT (homolytic hydrogen atom transfer) mechanism. Theoretical calculation showed that edaravone is more potent than phenylisoxazolone, however, both has similar antioxidant scavenging on experimental DPPH. The carbonyliminic system played a very important role in the antioxidant activity for both studied classes.

Introduction

Heterocyclic stn class="Chemical">ructures are present in >68% of the drugs therapeutically used today. They are available in pharmaceutical market and used in several therapeutic action, as well as in drug discovery and development (Parlapalli and Manda, 2015). They are increasingly responsible for many advances in this area (Pitt et al., 2009).

Pyrazoles and n class="Chemical">isoxazoles are heterocyclic compounds belonging to the azol family, containing nitrogen and oxygen arranged at position 1 and 2 of the five-membered ring, which, although rare in natural products, have significantly contributed to the development of effective and alternative bioactive agents, especially replacing pyrrol and imidazole in drug design and discovery approaches (Kumar and Kaur, 2014, Taylor et al., 2016). Their oxidized derivatives, i.e. pyrazolone and isoxazolone, have different molecular properties, especially due to keto-enol or imine-enamine tautomerization, which gives it a free radical scavenging capacity that may be greater than or equal to vitamin C with less toxicity when compared to phenols (Borges et al., 2012, Brígida et al., 2013, Queiroz et al., 2010).

Among their biological effects, n class="Chemical">pyrazolones and isoxazolones have antioxidant (Dinesha et al., 2015, Padmaja et al., 2009), anti-inflammatory (Bandgar et al., 2009, Joy et al., 2018, Shivkumar and Nargund, 1998) and anticancer activities (Lv et al., 2010, Sadashiva et al., 2012). Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one, MCI-186, Radicut) is the representative lead of this class and has shown effectiveness against the stroke effects and amyotrophic lateral sclerosis (Watanabe et al., 2018). Its antioxidant mechanism is due to its relative rates in the scavenging reaction of various free radical species such as hydroxyl radical, superoxide, alkoxyl, tert-butylperoxyl, methyl radical and singlet oxygen, very superior when compared to uric acid, glutathione and trolox, showing high elimination measurements in all these studied species (Kamogawa and Sueishi, 2014). In addition, the edaravone protection against oxidative stress has been demonstrated in other previous studies, however some negative aspects have been reported, including renal and hepatic disorders maybe due to toxic intermediates (Lapchak and Zivin, 2009, Wang et al., 2018, Watanabe et al., 2018, Wu et al., 2014). Carvalho et al., 2017 have demonstrated the existence of two metabolic hydroxylation mechanisms of edaravone using Density Functional Theory (DFT), which the generation of hydroxy pyrazole or hydroxy aniline intermediates, attributed as responsible for the toxic properties of edaravone by redox mechanism.

In this work, the antioxidant capacity and compan class="Species">rative study of derivative tautomerism between pyrazolones, isoxazolones, and their heterocycles derivatives by using DFT methods were approached to establish the influence on chemical stability of tautomers and preferential antioxidant mechanism, i.e. single electron transfer (SET) and hydrogen atom transfer (HAT). These theoretical calculations were supported by DPPH (2,2-Diphenyl-1-picrylhydrazyl radical) evaluation.

Materials and methods

Computational details

All the calculations were performed with Gaussian 09 molecular package (Frisch et al., 2009). A conformational search was performed by using PM3 semiempirical method (Patterson, 1989, Stewart, 1989). In addition, all geometries of the tautomers a, b, and c (DXH) for edaravone or pyrazolone 1–3 and isoxazolone 4–6 derivatives (Fig. 1) and their heterocycles derivatives 7 and 8 (Fig. 2) can be exploited. The torsion angles were chosen between both rings, i.e. pyrazolone or isoxazolone and phenyl or pyridyl rings. The structures of low energies were reoptimized at B3LYP hybrid density functional theory (Becke, 1988, Lee et al., 1988), with 6-31++G(2d,2p) bases (Hehre et al., 1986). These conformations of low energies are close to planar conformations and dihedral angles between 0 and 15° θ. The restricted B3LYP functional was used for the neutral tautomers, and the unrestricted (U)B3LYP functional was applied for all cation free radicals and semiquinones. The frequencies were calculated to prove that the neutral and charged structures are minima at the level of theory.

Fig. 1

Edaravone (1–3) and isoxazolone derivatives (4–6) and their tautomers (a-c), where R is H, CH or C and X  = O or N.

Fig. 2

Heterocycle derivatives of edaravone (7) and phenylisoxazolone (8) and their tautomers a (C—H), b (N—H) and c (O—H).

Edaravone (1–3) and n class="Chemical">isoxazolone derivatives (4–6) and their tautomers (a-c), where R is H, CH or C and X  = O or N.

Heterocycle den class="Chemical">rivatives of edaravone (7) and phenylisoxazolone (8) and their tautomers a (C—H), b (N—H) and c (O—H).

In orden class="Chemical">r to understand the mechanisms involved in the scavenging of these compounds, electronic and theoretical parameters were obtained by theoretical values involved on redox process such as the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), neutral energies (E), adiabatic ionization potential (AIP), bond dissociation energies (BDEXH) of hydroxyl (OH), amine (NH) or methylene (CH) moieties and GapL-H (LUMO-HOMO) (Horton et al., 2019). AIP was calculated as the difference between the optimized neutral molecule energy and the optimized cation energy (Eq. (1)). It was assumed that the specific equilibrium geometry of each specie do not affect the AIP values due to the minimal structural deformation on dihedral angles between both rings with the loss of an electron due to the planar conformation and wide electronic delocalization of the evaluated compounds. The bond dissociation energies (BDE) were calculated as the energy differences between a neutral molecule and the respective semiquinone plus hydrogen radical (Eq. (2)). In order to examine the reactivity of the calculated compounds, GapL-H was obtained by determining the difference in energy between the LUMO and HOMO (Eq. (3)).

All calculations were pen class="Chemical">rformed in gas phase seeking the intrinsic properties of the studied tautomers. In addition, the solvent effect calculation was implemented in chloroform, ethanol and water by using polarizable continuum model (PCM) (Cossi and Barone, 1998, Miertuš et al., 1981) in the GAUSSIAN 09 W package.

Reagents

All chemicals, i.e. edaravone, n class="Chemical">phenylisoxazolone, 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH), and methanol were purchased from Sigma-Aldrich, St. Louise, MO, USA. The chemicals used were of analytical grades.

DPPH scavenging activity

A methanolic solution (25 μM) of the n class="Chemical">radical DPPH• was prepared and stored at 10 °C in the dark. All compounds were properly dissolved in methanol using the procedure described previously (Borges et al., 2013a, Borges et al., 2013b, Lôbo et al., 2009, Minnelli et al., 2019). Samples of different methanolic solutions of edaravone and related derivative were added to DPPH• methanolic solutions. Absorbance was recorded at 517 nm wavelength at different time intervals until the reaction reached an equilibrium. The DPPH radical absorbance without antioxidant was measured also as control and used 95% methanol as blank. All measurements were performed in triplicate. Nine different concentrations (250 at 1 μM) of each azolic derivative studied were assayed in order to check the linearity of response and to establish the values of antioxidant activity at proper linear range. Results were presented as inhibition % of the DPPH radicals.

Results

Tautomerism study

The relative enen class="Chemical">rgy differences between edaravone 1–3 or isoxazolone 4–6 tautomers and their corresponding heterocyclic analogs 7 and 8 in gas phase and under solvent effects (PCM method) were calculated and are presented in Table 1. The results demonstrated that the most stable tautomer in compounds 1–3 and 4–6 is C—H (a).

Table 1

Energy differences for edaravone and isoxazolone tautomers in different media.

Tautomers	ΔEgas phase (kcal.mol−1)	ΔECHCl3 (kcal.mol−1)	ΔEEtOH (kcal.mol−1)	ΔEH2O (kcal.mol−1)
1a	0	0	0	0
1b	4.14	2.43	1.70	1.56
1c	4.40	4.36	4.23	4.20
2a	0	0	0	0
2b	5.03	3.44	2.77	2.64
2c	6.30	6.68	6.68	6.67
3a	0	0	0	0
3b	5.19	3.61	2.95	2.82
3c	6.96	6.97	6.81	6.77
4a	0	0	0	0
4b	2.17	1.46	1.26	1.22
4c	6.02	6.47	6.59	6.61
5a	0	0	0	0
5b	2.96	2.31	2.16	2.14
5c	8.53	9.23	9.41	9.44
6a	0	0	0	0
6b	2.51	1.78	1.63	1.61
6c	8.08	8.36	8.33	8.32
7	0	0	0	0
7a	0	0	0	0
7b	−4.04	−3.37	−2.97	−2.88
7c	−9.04	−5.80	−4.44	−4.18
8	0	0	0	0
8a	0	0	0	0
8b	−0.88	−0.02	0.72	0.89
8c	7.73	7.79	7.66	7.63

Energy diffen class="Chemical">rences for edaravone and isoxazolone tautomers in different media.

The energy diffen class="Chemical">rences for tautomerization was influenced by bulky substituents on position 3 on pyrazolone or isoxazolone rings, showing an increase of energy values as follows: C. The N—H form (b) is more favored than enolic (c) form (O—H). These differences decreased in polar medium (PCM), where edaravone 2 presented for N—H tautomers (b) and O—H (c) energy differences values in nonpolar solvent (chloroform) 3.44 and 6.68 kcal.mol−1, in ethanol of 2.77 and 6.68 kcal.mol−1 and in polar solvent (water) values of 2.64 and 6.67 kcal.mol−1, respectively.

The isoxazolonic den class="Chemical">rivatives 4–6 showed higher relative energy differences values when the substituents are in position 3. The following group in this order was: CH. However, N—H (b) isoxazole tautomers 4–6 have the lowest average values when compared to the respective pyrazolone tautomers 1 to 3. Regarding derivative 6, the solvent effect reduced the differences of N—H or O—H tautomerization to 1.63 kcal.mol−1 in ethanol and 1.61 kcal.mol−1 in water, respectively, but the solvent effect did not show significant influence on the energy values among the O—H tautomers of the isoxazolone derivatives. These values show that N—H tautomers are favored, but their energy differences can decrease in polar medium.

Nevertheless, the changes of n class="Chemical">benzene ring to pyridinyl group at position 1 of edaravone and position 3 of the isoxazolone moiety to give 7 and 8 derivatives, respectively, and also their related isomers at 7a and 8a positions show different performance.

It was found that among edaravone analogues the most stable tautomen class="Chemical">rs are 7b (N—H) and 7c (O—H) for compound 7, due to the favoring of intramolecular hydrogen bond between the amine nitrogen at position 2 of the pyrazolone moiety and the nitrogen of the pyridinyl ring. Enolic oxygen at position 6 of the pyrazolone moiety with isopyridinil followed the same tendency. The relative energy differences are lower for enol form, favoring compound 7c mainly in nonpolar solvents as chloroform (ΔECHCl3 = −5.80 kcal.mol−1) and disfavored on the phenyl ring analogue, the energy difference for formation of tautomers 7c as well as 7b increases in polar medium.

For the heten class="Chemical">rocyclic derivative of isoxazolone 8, the pyridinyl ring showed an unclear tautomeric behavior. In nonpolar medium, the formation of the 8b tautomer prevailed (N—H) (ΔECHCl3 = −0.02 kcal.mol−1), and with increasing solvent polarity, the most stable and favored tautomer was 8 (C—H), which is also the prevalent form in its corresponding position isomer 8a.

These tautomers 7b, 7c, and 8b an class="Chemical">re influenced mainly by intramolecular hydrogen bond on pyridyl ring followed by steric hindrance (Hatanaka, 2015). The interaction distances on tautomers 7b, 7c, and 8b are 2.25, 1.76, and 2.50 Å, respectively. Therefore pyrazolone moieties are more favored than isoxazolone rings due to their fewer distances.

Electron abstraction

The antioxidant capacity by means of electron tn class="Chemical">ransfer among C—H (a), N—H (b) and O—H (c) tautomers for edaravone derivatives 1–3 and 7 and isoxazolones 4–6 and 8, were theoretically predicted using HOMO and adiabatic ionization potential (AIP) values. HOMO and ionization potential values are important properties associated to nucleophilicity and single electron tansfer capacity.

Also, the HOMO topology for any molecule may show thein class="Chemical">r active sites for qualitative quenching mechanism of free-radical elimination ability. The HOMO and AIP values for each tautomer studied here are shown in Table 2.

Table 2

Theoretical properties of edaravone and isoxazolone tautomeric forms in gas phase calculated at the B3LYP 6-31++G(2d, 2p) level.

Tautomers	HOMO (eV)	LUMO (eV)	GAP (eV)	AIP (kcal.mol−1)	BDEXH (kcal.mol−1)
1a	−6.19	−1.40	4.78	179.83	81.93
1b	−6.08	−1.20	4.88	176.48	77.93
1c	−6.32	−0.78	5.54	181.33	77.71
Average	−6.19	−1.13	5.06	179.21	79.19
2a	−6.03	−1.22	4.81	175.03	82.88
2b	−5.97	−1.09	4.88	172.70	78.17
2c	−6.12	−0.68	5.45	175.80	76.91
Average	−6.04	−1.00	5.04	174.51	79.32
3a	−5.95	−2.01	3.95	168.78	82.92
3b	−6.00	−1.88	4.12	170.25	77.96
3c	−5.90	−1.23	4.67	167.25	76.92
Average	−5.95	−1.71	4.24	168.76	79.27
4a	−8.05	−1.64	6.41	238.43	86.13
4b	−7.01	−1.38	5.63	205.62	84.29
4c	−6.91	−0.86	6.05	210.55	80.34
Average	−7.33	−1.29	6.04	218.20	83.59
5a	−7.87	−1.39	6.48	225.78	87.21
5b	−6.47	−0.74	5.73	199.20	84.77
5c	−6.73	−0.79	5.94	203.18	79.11
Average	−7.03	−0.97	6.06	209.39	83.70
6a	−7.11	−2.19	4.92	198.90	87.16
6b	−6.83	−2.09	4.75	193.25	84.93
6c	−6.65	−1.37	5.28	189.33	79.31
Average	−6.86	−1.88	4.98	193.82	83.80
7	−6.28	−1.18	5.09	181.65	83.39
7a	−6.22	−1.24	4.98	178.33	81.24
7b	−6.01	−1.32	4.68	173.32	85.95
7c	−6.06	−1.50	4.56	164.04	90.80
Average	−6.14	−1.31	4.82	174.33	85.34
8	−7.40	−2.42	4.98	205.84	86.60
8a	−7.35	−2.39	4.96	204.84	87.47
8b	−6.26	−2.30	3.96	189.38	87.88
8c	−6.48	−1.29	5.19	194.40	79.24
Average	−6.87	−2.10	4.77	198.61	85.29

Theoretical pn class="Chemical">roperties of edaravone and isoxazolone tautomeric forms in gas phase calculated at the B3LYP 6-31++G(2d, 2p) level.

Substitution at position 3 of pyrazolone and n class="Chemical">isoxazolone rings have direct impact on electron donating capability of the 1–3 and 4–6 derivatives by increasing the HOMO values and decreasing the AIP values in the following order: C > CH > H. In general, pyrazolone tautomers had higher HOMO values on gas phase than isoxazolone tautomers. The average values for the most stable tautomeric forms were: −6.19 eV for compound 1a, −6.04 eV for 2a and −5.95 eV for 3a. However, on isoxazolone derivatives, compound 6 showed different behavior and its average HOMO value was −6.86 eV. This value is higher when compared to average values of 4 (−7.33 eV) and 5 (7.03 eV). The HOMO topologies (Fig. 3) of most stable tautomers show all nucleophilic positions for free radical scavenger reactions and it also highlights the influence in the electron donation capacity of heterocyclic moieties on pyrazolone and isoxazolone derivatives.

Fig. 3

HOMO topology of the most stable tautomers in gas phase calculated at the B3LYP 6-31++G(2d,2p) level.

Comparing the two n class="Chemical">rings, there was a difference in terms of hetreroatomic arrangement, between N1-N2 and O1-N2, whereby the contributions of free electron pairs of the two nitrogen increased the antioxidant capacity due to higher electron donation mechanism of the pyrazolone tautomers, as observed in edaravone 2a and phenylisoxazolone 6a.

From n class="Chemical">edaravone pyridinyl tautomers, the most stable forms 7b (N—H) and 7c (O—H), showed HOMO values of −6.01 and −6.06 eV, where hydrogen bonds amplified the resonance effect on pyrazolone moiety, increasing their electron donating ability when compared to the 7 and 7a forms. On the contrary, the change of pyridinyl to phenyl moieties at position 3, in tautomers 6a and 8b, increased the resonance effects on the isoxazolone moiety.

Previous studies showed a good pen class="Chemical">rformance between HOMO and ionization potential (Antonczak, 2008, Lôbo et al., 2009), having similar results. The AIP values on gas phase for edaravone and isoxazolone tautomers matched the HOMO values.

Tautomers 3a (C—H) and 7c (O—H) had lowen class="Chemical">r AIP values (168.78 and 164.04 kcal.mol−1), when compared to edaravone 2 (174.51 kcal.mol−1). The tautomers 6a and 8b are the most stable isoxazolone ones and had AIP values of 198.90 kcal.mol−1 and 189.38 kcal.mol−1, respectively, and N—H tautomers of the compounds 1, 2, 4 and 5 showed better electron donation properties. Phenyl substitution was used in order to give the compounds the best theoretical electron donating capacity. Accordingly, in compound 3, the most active tautomers are 3c (O—H) and 3a (C—H), and their AIP values are 167.25 and 168.78 kcal.mol−1, respectively. On isoxazolones, the tautomer 6c has the best electron donating capacity and the lower AIP value of 189.33 kcal.mol−1.

The tautomers 7b and 7c an class="Chemical">re the most stable tautomeric forms when a pyridinyl ring is on position 1. They have more electron donating capacity than edaravone. Their HOMO and AIP values are −6.01 and −6.06 eV, 173.32 and 164.04 kcal.mol−1, respectively. On the contrary, the pyridinyl on the isoxazolone, tautomer 8b show the lesser capacity and the AIP value of 189.38 kcal.mol−1 is approximately 10 kcal.mol−1 less when compared to another tautomer 6a of the same related compound.

HOMO and LUMO values are impon class="Chemical">rtant parameters of the molecular structure related to the reactivity studied in this work. They show all nucleophilic and electrophilic regions around the molecule (Lôbo et al., 2009). The GAPL-H value, that is, the difference between the LUMO and HOMO values of these structures may indicate their chemical reactivity in terms of electron transfer, where the lowest values are related to the high chemical reactivity. In fact, studies show that, in general, it is possible to relate HOMO energy values and scavenging activities of compounds with antioxidant properties (Queiroz et al., 2009).

Thus, our n class="Chemical">results showed that the most reactive tautomers of edaravone derivatives 1 to 3 were the C—H and N—H forms. This result has a direct influence of the phenyl moiety on the tautomer 3a, with the lowest GAP value. Similar tendency was observed in isoxazolone tautomers 6a and 6b, with the low GAP values and the phenyl group at position 3 of the heterocyclic ring. The replacement of the phenyl group by pyridinyl revealed more reactive tautomers by the low GAP values due to the available electrons in the nitrogen atom of the pyridinyl ring, especially in the isoxazolone tautomer 8b, with a GAP value of 3.96 eV.

Hydrogen elimination

Bond dissociation energies (BDEXH) fon class="Chemical">r edaravone, isoxazolone, their heterocycles derivatives, and their tautomers were calculated, being X substituted with C, N and O. This value represents the easiness of hydrogen donation among tautomer derivatives 1 to 8 for formation of their semiquinone forms. Homolytic hydrogen atom transfer (HAT) is one of the main antioxidant mechanisms (Alves et al., 2006, Cao et al., 2003, Diniz et al., 2004, Sens et al., 2018). Therefore, molecules with lower BDEXH values are more active.

According to Table 2, the best BDEXH values fon class="Chemical">r tautomers of edaravone derivatives 1, 2 and 3 are for O—H (enolic form), with values of 77.71, 76.91 and 76.92 kcal.mol−1, respectively. For isoxazolone derivatives 4, 5 and 6, the most active was enol tautomer in terms of hydrogen donation. Their respective values are 80.34, 79.11 and 79.31 kcal.mol−1. In addition, the heterocyclic pyridinyl in edaravone tautomers 7b and 7c had the highest BDEXH values of 85.95 and 90.80 kcal.mol−1, due to hydrogen bonds, decreasing their HAT capacity.

Except for tautomen class="Chemical">r 7c, O—H tautomers have better predisposition to perform a HAT mechanism, especially on C—H and N—H tautomers. The substitution of the phenyl ring by pyridinyl ring favor the formation of the tautomers 7b and 7c and increases their stability. This property is favored in polar medium. The formation of tautomer 8b (N—H) in nonpolar medium is favored. However, tautomer 8a (C—H) in polar medium is the most stable form. There may be some relationship between tautomers (7 and 8) with respect to their reactivity properties, antioxidant capacity or even an equilibrium which require more studies.

Chemical stability

Spin density contributions an class="Chemical">re related to chemical stability and depend on the number of resonance structures. It is an important parameter for the understanding of the preferential free radical scavenging mechanisms and it is often estimated as the stability of the free radicals produced after an electron or hydrogen transfer, which can be influenced by the molecular substituents that can shift their electron density to different intramolecular positions (Reis et al., 2007).

Therefon class="Chemical">re, the most stable tautomers of edaravone 2a and isoxazolonic 6a, as well as their respective pyridinyl tautomeric forms (7b, 7c, and 8b) are shown in Fig. 4. They are generated after an electron abstraction with the respective cation radical forms. In Fig. 5, these tautomers are observed after the elimination of a hydrogen resulting in their respective semiquinone free radicals.

Fig. 4

Contribution of free radical cation spin density among the most stable tautomers of edaravone (2a), pyridinyl edaravone (7b and 7c) and isoxazolones (6a and 8b).

Fig. 5

Contribution of spin density of semiquinones among the most stable tautomers of edaravone (2a), pyridinyl edaravone (7b and 7c) and isoxazolones (6a and 8b).

Contribution of n class="Chemical">free radical cation spin density among the most stable tautomers of edaravone (2a), pyridinyl edaravone (7b and 7c) and isoxazolones (6a and 8b).

Contribution of spin density of n class="Chemical">semiquinones among the most stable tautomers of edaravone (2a), pyridinyl edaravone (7b and 7c) and isoxazolones (6a and 8b).

Spin density distributions fon class="Chemical">r their cation free radical showed significant contributions from the heterocyclic rings. The spin density in 2a (C—H) was particularly on N1, with a value of 0.29 and a total contribution on pyrazolone ring of 0.51. A larger contribution was detected on phenyl ring at ortho (0.15) and para (0.31) positions. An increase was observed in pyridinyl, mainly on the cabonylimine system, especially in 7b and 7c tautomers and these results can be involved for chemical stability of cation free radical. Isoxazolone tautomer 6a, showed a greater participation in heteroatoms O1 and N2. The total spin density contribution in isoxazolone moiety was 0.53, which is a lower contribution on phenyl ring at position 3, when compared to the pyridinyl ring of 8b (N—H). The spin density contribution on isoxazolone moiety is 1.22 and a preeminent spin density contribution on carbon methylene at position 4 is 0.50.

Spin density calculations of the initial hydrogen elimination fon class="Chemical">r edaravone tautomer 2a and the isoxazolone tautomer 6a showed a high spin density contribution on the carbonyliminic group. These values are 0.41, 0.22, 0.29 (0.92 total) and 0.53, 0.36, 0.26 (total of 1.15), respectively. These results show the important participation of these rings in the semiquinone stabilization. Isoxazolone moiety has a more spin density contribution than the pyrazolone ring. The replacement of the phenyl ring to pyridinyl ring shows that the contribution of position 4 of the heterocyclic rings had expressive impact on BDECH values for tautomeric forms 2a, 7b, and 7c.

The contribution values wen class="Chemical">re 0.22, 0.24 and 0.27, and the BDECH values were 82.88, 85.95, and 90.80 kcal.mol−1, respectively. These values are similar to those of isoxazolone tautomers 6a and 8b. For these compounds, the spin density contributions were 0.36 and 0.38, and the BDEXH values were 87.16 (BDECH) and 87.88 (BDENH) kcal.mol−1, respectively.

Scavenging activity against DPPH

The antioxidant capacity of isoxazolone 6 was compan class="Chemical">red with edaravone 2 using the reaction with the 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH) at the same concentrations in methanolic medium. Nine dilutions at doses of 250 to 1 μM were used. The test was performed in triplicate and the results presented as the mean and standard deviations as shown in Fig. 6.

Fig. 6

Evaluation of scavenging capacity of edaravone (2) and isoxazolone (6) at different concentrations (250-1 μM) in methanolic solution against DPPH radical (25 μM).

Evaluation of scavenging capacity of edaravone (2) and n class="Chemical">isoxazolone (6) at different concentrations (250-1 μM) in methanolic solution against DPPH radical (25 μM).

Despite the results of n class="Chemical">DPPH inhibition between the compounds edaravone (2) and isoxazolone (6) did not demonstrate statistical significance in the highest concentrations from 250 to 62.5 μM there was a close relationship in the free-radical inhibition where edaravone achieved values from 88 to 90% and isoxazolone from 78 to 82%. However, in the lowest concentrations of 16 to 4 μM, the radical inhibition was greater for the isoxazolone. Therefore, the EC50 was inferior to 16 μM for both edaravone (2) and isoxazolone (6). This result shows a similarity in the DPPH free-radical scavenging behavior between the phenylisoxazolone derivative and edaravone.

Discussion

Studies of chemical structun class="Chemical">re-anti-ischemic activity relationship showed that edaravone biological effects can be caused by its tautomeric forms (Queiroz et al., 2010). In spite of the balance between neutral and anionic forms, as well as their respective keto-enolic and imine-enamine tautomers, the tautomerization energy barriers of these molecules are reduced by the solvent effect (Queiroz et al., 2010, Watanabe et al., 2018, Zhao and Truhlar, 2008). Our study, show that edaravone derivatives with two methyl on 4-position did not show inhibitory activity on lipid peroxidation, presumably because keto-enolic and imine-enamine tautomerization was blocked (Watanabe et al., 2018).

Our theon class="Chemical">retical study among tautomers of pyrazolone 1–3 or isoxazolone 4–6, shows the keto or CH (a) is the most thermodynamically stable form between these two classes. Substitutions in position 3 of these heterocycles and chemical similarity have great influence on the HOMO, AIP, and BDEXH energy differences. Changes in polar medium have no significant impact, even so PCM medium decreases the energy differences for the formation of the NH (b) tautometer. The tautomeric forms presented the lower values of the HOMO, AIP, and BDEXH energy differences and are formed more easily in the compounds of 4–6 especially due to their electron transfer mechanisms in isoxazolone compounds.

A thermodynamic equilibn class="Chemical">rium between tautomers of isoxazolone 8 and edaravone 7 can be shown when the substitution on position 3 (Queiroz et al., 2010) favored intramolecular interactions. The pyridinyl ring in position 1 of the pyrazolone moiety favored hydrogen bond on N2 when compared with the isoxazolone moiety. In fact, intramolecular bonds between heteroatoms of the pyridinyl ring and their tautomeric forms 7b and 7c are favored in pyrazolone, thus, the predominance of these forms is energetically favored. Tautomerism of these compounds may contribute significantly to the understanding of pharmacological and biological activities related to their antioxidant properties.

All compounds with the highest energy of HOMO and lowen class="Chemical">r AIP values have potent electron donating ability. These values are in agreement with antioxidant activities (Antonczak, 2008, Lôbo et al., 2009, Queiroz et al., 2009). Any electron donating group (EDG) on position 3 of pyrazolone or isoxazolone tautomers increases HOMO values and decreases AIP values for tautomers of low energies, following the order 7c > 3a > 2a for pyrazolones and 8b > 6a > 5a for isoxazolones. Therefore, EDG and resonance effects can be linked to heterocyclic rings, increasing the nucleophilicity and the carbonyliminic system involvement, favoring a greater stabilization of the cation free-radical related to the HOMO topology. These properties have great impact on the antioxidant process by SET mechanism for these molecules.

All molecular substitutions on 3-position of n class="Chemical">pirazolone or isoxazolone rings by hydrogen, methyl, phenyl group and pyridinyl ring did not show significant change on BDEXH values. The BDECH for edaravone 2a of 82.88 kcal.mol−1 shows a difference of 4.28 kcal.mol−1 when compared to the more stable isoxazolone tautomer 6a (pheniyisoxazolone) with BDECH of 87.21 kcal.mol−1, showing consequently low influence on antioxidant activity by HAT mechanism. These values were confirmed by the chemical similarity and stability of the semiquinone free-radical generated after hydrogen elimination in the most stable tautomers of both classes and high contribution on carbon methylene at 4 position. Thus, the high spin density contribution on methylenic carbon at position 4 is in accordance with the lowest average BDEXH values. In fact, the differences between edaravone 2 and 6 can be an important strategy for new derivatives with better antioxidant activity by HAT mechanism.

Previous studies (Lôbo et al., 2009; Bon class="Chemical">rges et al., 2013a; Borges et al., 2013b) have demonstrated a direct correlation between theoretical and experimental evaluation in scavenger of free radicals. The antioxidant activity against DPPH free-radical can be directly related to the hydrogen atom transfer mechanism (HAT). Indeed, HAT calculation is a theoretical parameter to predict the antioxidant capacity for many phenolic and heterocyclic derivatives (Al-Salahi et al., 2018, Almehizia et al., 2019, Borges et al., 2013b, Xue et al., 2014). The experimental results in the DPPH free-radical scavenging showed that edaravone 2 had a higher activity at almost all evaluated concentrations and the DPPH activity was accompanied by the isoxazolone 6 mainly from the concentration of 16 µM.

Our n class="Chemical">results have showed that derivatives containing isoxazolone ring have similar antioxidant activity to edaravone. This result is confirmed to their BDECH of 87.21 and 82.88 kcal.mol−1. These results match the values related of electron and hydrogen donations, since edaravone presented the lowest values for these properties. Further studies are in progress in our research group looking for a possible prediction of the structure development of new analogous of isoxazolone derivative from the edaravone.

Conclusions

The association between DFT methods and DPPH evaluation showed the pn class="Chemical">resence of similar electronic performance between pyrazolone and isoxazolone derivatives. The tautomerism is essentially important for the choise of more related stable compounds. Keto or C—H forms were the most related stable and thermodynamically favored. The polar medium values decrease the energy differences forN—H and O—H tautomer formations. A hydrogen bond changes the preferential tautomers. The electron donating capacity was influenced by the modification in position-3. Phenyl and pyridinyl groups can provide the most potent derivatives for free-radical scavenging. N—H tautomers showed the best HOMO and AIP values by the SET mechanism. BDEXH values of O—H tautomers were the forms with the best antioxidant capacity through HAT mechanism. A structural similarity was observed between pyrazolone and isoxazolone systems for SET and HAT as antioxidant mechanisms. The carbonylinic system is the essential chemical structure for the antioxidant activity of these compounds. Theoretical calculation showed that edaravone (2) is more potent than phenylisoxazolone (6), however, both has similar antioxidant scavenging on experimental DPPH. Thus, isoxazolone derivatives may be promising candidates as new analogue drugs of the edaravone to act as antioxidant, and perhaps anti-inflammatory, anticancer and neuroprotective agents.

Funding

This resean class="Chemical">rch received no external funding.