Synthesis and evaluation of the antioxidant activity of 3-pyrroline-2-ones: experimental and theoretical insights

The heterocyclic γ-lactam ring 2-pyrrolidinone has four carbon atoms and one nitrogen atom. Among the group of derivatives of 2-pyrrolidinones, 1,5-dihydro-2H-pyrrol-2-ones, also known as 3-pyrroline-2-ones, play a significant structural role in a variety of bioactive natural compounds. In this study, three-component reactions were used to successfully synthesize six polysubstituted 3-hydroxy-3-pyrroline-2-one derivatives. The antioxidant activity of the compounds was tested by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, identifying 4-ethoxycarbonyl-3-hydroxy-5-(4-methylphenyl)-1-phenyl-3-pyrroline-2-one (4b) as the most promising radical scavenger. Quantum chemistry calculations of the thermodynamics and kinetics of the radical scavenging activity also suggest that 4b is an effective HO˙ radical scavenger, with koverall values of 2.05 × 109 and 1.54 × 1010 M−1 s−1 in pentyl ethanoate and water, respectively. On the other hand, 4b could not scavenge hydroperoxyl radicals in either media. The ability of 4b to scavenge hydroxyl radicals in polar and non-polar environments is comparable to that of conventional antioxidants such as melatonin, gallic acid, indole-3-carbinol, ramalin, or Trolox. Thus 4b may be classed as a promising HO˙ radical scavenger in the physiological environment.

A key bioactivity described above is the potential antioxidant activity of this family of compounds. 2,[14][15][16][17][18]31 The imbalance between the production and consumption of oxidants in biological systems leads to oxidative stress, that is, chemical breakdown/damage of various biologically important molecules. 32 Although there are natural oxidants of various chemical characteristics, free radicals are the main cause of oxidative stress. They are highly reactive and capable of initiating chain reactions, hence propagating molecular damage. A representative reactive oxygen species is the hydroxyl radical. 33 It is so reactive that it would effectively assault nearly every organic molecule around the place of its production. This radical is held accountable for the majority of ionizing radiation-induced tissue damage 34 and is the most signicant cause of oxidative DNA damage. [35][36][37] Inhibiting OHc generation would therefore be an effective means of reducing oxidative stress. However, studies of antioxidant activity of 3-hydroxy-3-pyrroline-2-ones are thus far limited: there is no information on the mechanism and kinetics of the antiradical activity, particularly in physiological environments.
The use of computational approaches to investigate the structure-activity relationship and to direct the design of novel medications with enhanced activity is well established, [38][39][40][41][42] making it possible to perform the evaluation of the radical (i.e., HOc and HOOc) scavenging activity in silico, benchmarked against experimental data.
Thus, in this study, three-component reactions of aromatic aldehydes, amines and sodium diethyl oxalacetate will be optimized in order to shorten reaction time, increase the yields and eliminate the need for elevated temperature. Consecutively, the 1,1-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging capacity of six polysubstituted 3-hydroxy-3-pyrroline-2ones will be measured. Finally, the interaction of the best performing 3-hydroxy-3-pyrroline-2-one with HOc and HOOc radicals will be evaluated using well-established model chemistry based on the quantum mechanics-based test for the overall free radical scavenging activity (QM-ORSA) protocol. 40 Melting points (not corrected) were determined using a Büchi Melting Point B-545 apparatus. NMR spectra were acquired on Bruker Avance II+ 500 MHz or Bruker Avance II+ 600 MHz instruments and chemical shis (d) are reported in parts per million (ppm) referenced to tetramethylsilane (TMS) or the internal (NMR) solvent signals. Exact mass measurements were acquired on a quadrupole orthogonal acceleration time-of-ight mass spectrometer (Synapt G2 HDMS, Waters, Milford, MA). Samples were infused at 3 mL min À1 and spectra were obtained in positive (or negative) ionization mode with a resolution of 15 000 (FWHM) using leucine enkephalin as lock mass. Highresolution mass measurements were recorded on a SCIEX X500 QTOF with an electrospray ionization source in a positive ion mode. The temperatures of the source were set at 300 C. Curtain gas chambers were lled with high-purity nitrogen (25 psi). The capillary voltage was constantly kept at 5500 V. The collision energy was set at 10 V and zero collision energy spread. IDA mode was used to nd mass in the range (100 to 1000).
2.1.2. General procedure for the synthesis of 1,5-disubstituted-4-ethoxycarbonyl-3-hydroxy-3-pyrroline-2-one. Aromatic aldehyde (1 equiv.), amine (1 equiv.), citric acid (2 equiv.) and absolute ethanol (1.0 mL) were mixed in a round bottom ask. The mixture was magnetically stirred at room temperature under an argon atmosphere for 1 hour. Subsequently, sodium diethyl oxalacetate (2 equiv.) was added and the mixture was stirred vigorously at room temperature under Ar atmosphere for 8 hours and the formation of 1,5-disubstituted-4ethoxycarbonyl-3-hydroxy-3-pyrroline-2-one was followed by TLC (hexane/EtOAc ¼ 5 : 1 and hexane/EtOAc ¼ 5 : 3.5). Then, CH 2 Cl 2 and HCl (5%) were added and the resulting mixture was stirred vigorously for 15 minutes. The organic layer was separated, then washed three times with distilled water and dried over MgSO 4 . The crude product was puried via recrystallization in the solvent mixture of CH 2 Cl 2 and absolute ethanol or CH 2 Cl 2 and ethylacetate to obtain a pure product.  Radical-scavenging properties of polysubstituted 3-hydroxy-3-pyrroline-2-ones were evaluated against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. 44-48 DPPH solution (1 mM) was prepared in methanol and solutions of each polysubstituted 3-hydroxy-3-pyrroline-2-one were prepared in DMSO at various concentrations (128, 32 and 8 mg mL À1 ). Then, 200 mL DPPH solution was added to 1.28 mL samples at each tested concentration and the free radical scavenging reactions were carried out on a 96-well plate at 37 C for 30 minutes. The absorbance was measured at 517 nm wavelength by a BioTek Epoch 2 Microplate Spectrophotometer. The percentage of free radical scavenging was calculated as SP(%) ¼ [(OD 0 À OD 1 )/ OD 0 ] Â 100, where OD 0 was dened as the nal absorbance of the control reaction with quercetin as the reference antioxidant, and OD 1 stands for the absorbance in the presence of the sample. Each experiment was repeated three times and quercetin was used as the positive control.

Synthesis of polysubstituted 3-pyrroline-2-ones
The reaction between benzaldehyde (1a), aniline (2a) and sodium diethyl oxalacetate (3) in the presence of citric acid (2 equiv.) as the catalyst was used as the starting point to optimize the reaction conditions. Equimolar amounts of 1a, 2a and 3 at 0.5 M concentrations in absolute ethanol at room temperature turned over to 1,5-diphenyl-4-ethoxycarbonyl-3-hydroxy-3-pyrroline-2-one (4a) with 42% yield. This result is consistent with prior reports. 29,30 There was a slight decrease in the yield of 4a (35%) when the concentration of starting materials in solvent was reduced to 0.34 M. The yield was only 37-38% when the concentration of 1a or 2a was increased to 0.75 M in absolute ethanol. However, there was a dramatic increase in the yield of 4a to 86% when 2 equiv of sodium diethyl oxalacetate (3) was used (Table 1). Therefore, the ratio 1 : 1 : 2 of reactants aromatic aldehyde, amine and sodium diethyl oxalacetate, respectively, was used to synthesize other polysubstituted 3-hydroxy-1,5-dihydro-2Hpyrrol-2-ones in absolute ethanol (1 mL) ( Table 2).
In the structure of polysubstituted 3-hydroxy-1,5-dihydro-2Hpyrrol-2-ones, the -C(O)OCH 2 CH 3 group is almost coplanar with the plane of the 3-pyrroline-2-one. 21,22 As a consequence, two protons of methylene group are diastereotopic and they will show geminal coupling in 1 H NMR. In addition, methylene protons (CH 2 ) will also couple with protons of methyl group (CH 3 ) separated by three sigma bonds. Therefore, two protons of methylene group should be represented by two doublet of quartet (dq) instead of a simple quartet. For instance, 1

Calculations of the HOOc and HOc radical scavenging activity of the most active compound (4b)
3.2.2.1 The antiradical activity in the gas phase. The antioxidant properties of 4b were initially tested in the gas phase in order to identify the predominant antioxidant mechanism(s) for the more complex calculations in physiological environments. As previously demonstrated, this approach decreases computation time while giving accurate and reliable data. 55 In the rst step, the principal thermodynamic properties (proton affinity (PA), ionization energy (IE) and bond dissociation enthalpy (BDE)) that dene the radical scavenging activity mechanisms (sequential proton loss electron transfer (SPLET), single electron transfer proton transfer (SETPT) and formal hydrogen transfer (FHT)), 67,68 of 4b were computed ( Table 4).
The lowest calculated BDE was observed at the C5-H bond at 76.0 kcal mol À1 , whereas those of the other C(O)-H bonds were higher by about 13.7-25.5 kcal mol À1 . Thus, data implies that the C5-H bond dominates the antioxidant activity of 4b following the FHT pathway. On the other hand, the radical scavenging activity of 4b according to either the SPLET or SETPT would be challenging due to the high PA and IE values (PA ¼ 326.6 kcal mol À1 and IE ¼ 186.4 kcal mol À1 ) in comparison to the BDEs.
particularly in the HOc antiradical action, as demonstrated by prior research 62,69-71 and hence this pathway should be also examined. Gibbs free energy changes (DG ) for the HOc and HOOc radical scavenging reactions of 4b via the FHT, SET and RAF mechanisms were calculated in the gas phase and is presented in Table 5. With the exception of the SET reaction (DG ¼ 158.8 kcal mol À1 ), it was found that the HOc radical scavenging reactions are spontaneous (DG < 0) for all positions in 4b; however, the HOOc radical scavenging is spontaneous only at the C5-H bond (DG ¼ À10.2 kcal mol À1 ) according to the FHT mechanism. Consistently all positions (DG < 0, Table 5) were evaluated kinetically for the radical scavenging of 4b against the HOc radical in a vacuum, while only the H-abstraction of the C5-H bond was estimated for the HOOc radical. The results are presented in Table 6 and Fig. 2.
The overall rate constant (k overall ) for HOc radical scavenging in the gas phase was 5.77 Â 10 10 M À1 s À1 , however only 5.48 Â 10 1 M À1 s À1 for HOOc antiradical activity ( Table 6). The hydroxyl antiradical activity was dened by a combination of the RAF mechanism (at the C3 (G ¼ 3.3%) and C4 (G ¼ 55.1%)) and the FHT mechanism (at the C5-H (G ¼ 37.9%) and C7-H (G ¼ 2.4%)). The H-abstraction of the C5-H bond determined the HOOc antiradical activity while contributing 37.9% to the total HOc radical scavenging. Thus these reactions should be used for further kinetic evaluation in the physiological environments.
3.2.2.2. The antiradical activity in the physiological environments 3.2.2.2.1. Acid base equilibrium. In order to account for the inuence of physiological settings, the radical scavenging of 4b against HOc and HOOc radicals was simulated in water at pH ¼ 7.4 for the aqueous solution and in pentyl ethanoate for lipid medium. 40,55 Following the literature, 72,73 the acid-base equilibria of 4b at the O3-H bond were computed to estimate the state of 4b in aqueous solution at pH ¼ 7.4 (Fig. 3). The pK a value was 5.40. Fig. 3 shows that 4b consistently exists both in the neutral state (HA, 1.0%) and the monoanion state (A À , 99.0%) at physiological pH (7.40). These states are employed for further investigation in the polar medium.
The QM-ORSA protocol was used to evaluate the kinetics of the HOc and HOOc scavenging reactions in physiological environments. 40 The results are shown in Table 7. The results showed that in pentyl ethanoate and water solvents, the k overall values for the 4b + HOc + reaction were 2.05 Â 10 9 and 1.54 Â 10 10 M À1 s À1 , respectively, while those were 6.90 Â 10 À1 and 1.82 Â 10 3 M À1 s À1 for the 4b + HOOc reaction. The HOc antiradical activity in lipid medium was dened by the RAF mechanism at the C4 position (G ¼ 57.1%) and the FHT pathway at C5-H bond (G ¼ 30.6%), whereas that for the aqueous solution was characterized by the SET reaction of the anion state (G ¼ 56.7%). The RAF and FHT reactions of the anion state contributed more than 40% in the hydroxyl antiradical activity in water at pH ¼ 7.40, however these reactions of the neutral state had no contribution to the activity. At the same time, the FHT (C5-H, G ¼ 59.7%) and RAF (C4, G ¼ 40.2%) reactions of the anion state played a decisive role in the HOOc antiradical activity of 4b in water at pH ¼ 7.40. The ability of 4b to scavenge hydroxyl radical in polar and non-polar environments is comparable to that of common antioxidants such melatonin, 74 gallic acid, 75 indole-3-carbinol, 71 ramalin, 70 and Trolox. 40 However, 4b exhibited low hydroperoxyl radical scavenging activity in both the lipid and polar media. Thus 4b is a good but not exceptional hydroxyl radical scavenger in the physiological  environment with targeted activities that may invite further studies into its activity against specic compounds.

Conclusion
Six polysubstituted 3-hydroxy-3-pyrroline-2-ones were successfully synthesized by using three-component reactions at room temperature. The DPPH assay indicated that 4b exhibits the highest DPPH radical scavenging activity. The thermodynamic and kinetic calculations also showed that 4b is a potent HOc radical scavenger with k overall ¼ 2.05 Â 10 9 and 1.54 Â 10 10 M À1 s À1 in pentyl ethanoate and water, respectively. However, 4b exhibited only low hydroperoxyl radical scavenging activity in either of the lipid and polar media. Compared to typical antioxidants such as melatonin, gallic acid, indole-3-carbinol, ramalin, and Trolox, the ability of 4b to scavenge hydroxyl radicals in polar and non-polar environments is similar to that of these compounds. Thus 4b is a promising HOc radical scavenger in physiological environments.

Conflicts of interest
There are no conicts to declare. Table 7 Gibbs free energies of activation (DG s , kcal mol À1 ), rate constants (k app , k f , M À1 s À1 ) and branching ratios (G, %) at 298.15 K, in the NPY oxidation by HOc/HOOc radicals in the studied environments