Coumarin N-Acylhydrazone Derivatives: Green Synthesis and Antioxidant Potential—Experimental and Theoretical Study

Coumarin N-acylhydrazone derivatives were synthesized in the reaction of 3-acetylcoumarin and different benzohydrazides in the presence of molecular iodine as catalyst and at room temperature. All reactions were rapidly completed, and products were obtained in good to excellent yields. It is important to emphasize that four products were reported for the first time in this study. The obtained compounds were subjected to evaluation of their in vitro antioxidative activity using DPPH, ABTS, and FRAP methods. It was shown that products with a catechol moiety in their structure are the most potent antioxidant agents. The thermodynamic parameters and Gibbs free energies of reactions were used to determine the most probable mechanism of action. The results of in silico examination emphasize the need to take solvent polarity and free radical species into account when examining antiradical action. It was discovered by using computational approaches that HAT and SPLET are competitive molecular pathways for the radical scavenging activity of all compounds in polar mediums, while the HAT is the dominant mechanism in non-polar environments.


Introduction
Coumarins constitute a large group of natural compounds that are extensively prevalent in nature [1,2].In addition to fruits, they are most commonly found in the roots, stems, and leaves of higher plants.Some essential oils, such as lavender oil, cinnamon bark oil, and cassia leaf oil, contain large amounts of different coumarin derivatives.Certain varieties of cinnamon are the most common source of coumarins for humans [3].Coumarins are relatively simple compounds that basically consist of fused α-pyrone and a benzene ring.Coumarins that occur naturally exhibit a wide range of pharmacological activities and are frequently employed by researchers in the design and development of novel synthetic coumarins [4].The significance of coumarin hybrids in the fields of medicine and pharmacy is noteworthy due to their relatively low level of adverse effects on living things, as reported in the literature [5].Several pharmacological activities, such as antioxidant [6][7][8], anticancer [9,10], antitubercular [11], antibacterial [12][13][14], antifungal [15], antiviral [16], and anticoagulant [17][18][19][20][21], have been exhibited by many of the obtained hybrid compounds.Chemically modified coumarins have great potential as drugs for the treatment of various diseases.Several coumarin derivatives, including warfarin (I), dicoumarol (II), phenprocoumon (III), armilarizine A (IV), carbochromen (V) and chlorochromen (VI), have found significant clinical application, Figure 1.
Antioxidants 2023, 12, x FOR PEER REVIEW 2 of 17 dicoumarol (II), phenprocoumon (III), armilarizine A (IV), carbochromen (V) and chlorochromen (VI), have found significant clinical application, Figure 1.Hydrazone compounds are a well-known group of compounds with specific structural units (R2C=N-NH2).These compounds are of great significance in organic synthesis and in the creation of compounds that exhibit a wide range of biological activities.The literature describes the coumarin compounds with various hydrazide-hydrazone pharmacophores attached to position three in the chromen ring [22][23][24][25][26].The synthesis of coumarin hydrazone derivatives involves the condensation of diverse 3-formylcoumarin with different hydrazides in the presence of acid as a catalyst [22,23].According to the literature, a method is known for the synthesis of a series of 3-acylhydrazono-4-hydroxycoumarins using the reaction of 3-acetyl-4-hydroxycoumarin with the corresponding hydrazides [24][25][26].However, it is worth pointing out that the reactions between 3-acetylcoumarin and various benzoyl hydrazides have been described in only one article [27].Pangal et al. conducted a study wherein they successfully synthesized three derivatives of coumarin N-acylhydrazone through the reaction of 3-acetylcoumarin with appropriate hydrazides under reflux in methanol.
In this paper, we report a green methodology for the synthesis of coumarin N-acylhydrazone derivatives in ethanol and in the presence of molecular iodine as an efficient, relatively nontoxic, and inexpensive catalyst.Additionally, this study involves the assessment of the antioxidative potential of the obtained products by using both experimental and theoretical approaches.

Materials and Methods
All chemicals used for the synthesis were purchased from Sigma Aldrich, Darmstadt, Germany with purities above 98%.The IR spectra were obtained on a PerkinElmer Spectrum One FT-IR spectrometer using the KBr disc.The NMR spectra were recorded on a Varian Gemini spectrometer (200 MHz for 1 H and 50 MHz for 13 C) by using DMSO-d6 as solvent.All UV-Vis determinations were carried out using a PerkinElmer, Lambda 365 UV/Vis Spectrophotometer.Elemental microanalysis for carbon, hydrogen, and nitrogen was performed at the Institute for Information Technologies, University of Kragujevac.Elemental (C, H, N) analysis of the samples was carried out on an Elemental analysis system NC Technologies, ECS 8020 CHNOS, model-dual furnace-NC.

Synthesis of Coumarin N-Acylhydrazone Derivatives
Coumarin N-acylhydrazone derivatives 3 were obtained in the reaction of 3-acetylcoumarin (0.001 mol) and corresponding benzohydrazide 2 (0.001 mol) in presence of molecular iodine as catalyst (10 mol%) in absolute ethanol at room temperature.The progress of the reaction was monitored using thin-layer chromatography (TLC).Upon completion of the reaction, the purified product was isolated by filtration.All coumarin N- Hydrazone compounds are a well-known group of compounds with specific structural units (R 2 C=N-NH 2 ).These compounds are of great significance in organic synthesis and in the creation of compounds that exhibit a wide range of biological activities.The literature describes the coumarin compounds with various hydrazide-hydrazone pharmacophores attached to position three in the chromen ring [22][23][24][25][26].The synthesis of coumarin hydrazone derivatives involves the condensation of diverse 3-formylcoumarin with different hydrazides in the presence of acid as a catalyst [22,23].According to the literature, a method is known for the synthesis of a series of 3-acylhydrazono-4-hydroxycoumarins using the reaction of 3-acetyl-4-hydroxycoumarin with the corresponding hydrazides [24][25][26].However, it is worth pointing out that the reactions between 3-acetylcoumarin and various benzoyl hydrazides have been described in only one article [27].Pangal et al. conducted a study wherein they successfully synthesized three derivatives of coumarin Nacylhydrazone through the reaction of 3-acetylcoumarin with appropriate hydrazides under reflux in methanol.
In this paper, we report a green methodology for the synthesis of coumarin Nacylhydrazone derivatives in ethanol and in the presence of molecular iodine as an efficient, relatively nontoxic, and inexpensive catalyst.Additionally, this study involves the assessment of the antioxidative potential of the obtained products by using both experimental and theoretical approaches.

Materials and Methods
All chemicals used for the synthesis were purchased from Sigma Aldrich, Darmstadt, Germany with purities above 98%.The IR spectra were obtained on a PerkinElmer Spectrum One FT-IR spectrometer using the KBr disc.The NMR spectra were recorded on a Varian Gemini spectrometer (200 MHz for 1 H and 50 MHz for 13 C) by using DMSO-d 6 as solvent.All UV-Vis determinations were carried out using a PerkinElmer, Lambda 365 UV/Vis Spectrophotometer.Elemental microanalysis for carbon, hydrogen, and nitrogen was performed at the Institute for Information Technologies, University of Kragujevac.Elemental (C, H, N) analysis of the samples was carried out on an Elemental analysis system NC Technologies, ECS 8020 CHNOS, model-dual furnace-NC.

Synthesis of Coumarin N-Acylhydrazone Derivatives
Coumarin N-acylhydrazone derivatives 3 were obtained in the reaction of 3-acetylcoumarin (0.001 mol) and corresponding benzohydrazide 2 (0.001 mol) in presence of molecular iodine as catalyst (10 mol%) in absolute ethanol at room temperature.The progress of the reaction was monitored using thin-layer chromatography (TLC).Upon completion of the reaction, the purified product was isolated by filtration.All coumarin N-acylhydrazone derivatives (3a-f) were characterized with 1 H NMR, 13 C NMR, IR, and UV-Vis spectra.In addition, elemental analysis confirmed the purity of the newly synthesized compounds 3b-f.

DPPH Radical Scavenging Assay
The free radical scavenging activities of referent compounds and synthesized products were determined using a 1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay [28].The tested samples (20 µL of different concentrations dissolved in DMSO and 980 µL of methanol) were mixed with the same volume of the solution of DPPH in methanol (0.05 mM).The prepared samples were shaken well and left at room temperature in the dark for 20 min and 60 min.After the incubation period, absorbance was determined at 517 nm by using the methanol as a blank control.All tests were run in triplicate and averaged.The results are presented as mean value ± standard deviation.Nordihydroguaiaretic acid (NDGA) and quercetin were used as positive controls.For products that exert good activity, IC 50 values were determined, and the corresponding concentrations used for this determination are given in Table S1.The stoichiometric factor (SF) was calculated using next equation:

ABTS Radical Cation Scavenging Assay
The antioxidant capacity was measured using the 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) (ABTS) assay according to the modified procedure of Pontiki et al. [29].In this assay, stock solutions of ABTS (7 mM) and potassium persulfate (2.45 mM) were first prepared.The working solution was then prepared by mixing the two stock solutions in equal quantities and allowing them to react for 12-16 h at room temperature in the dark.The ABTS +• radical cation is diluted with methanol to obtain the solution with an absorbance of 0.70 units at 734 nm.Different concentrations of samples were prepared in DMSO.Next, 20 µL of sample and 980 µL of methanol were mixed, then an equal amount of ABTS was added and the absorbance was measured at 734 nm.IC 50 values were determined for products with noticeable activity and the concentrations used for this determination are given in Table S2.

Ferric Ion Reducing Capacity Assay
The ferric ion reducing capacity (FRAP) assay is a direct method for measuring the combined antioxidant activity of reductive antioxidants in a substance under investigation.In this study, the FRAP assay was conducted with some modifications to the method described by Pownall et al. [30].The stock solutions of tested compounds were prepared in DMSO, and then sample dilutions were prepared in phosphate buffer, pH 7.4.After that, 500 µL of diluted samples were mixed with 250 µL of 1% potassium ferricyanide solution followed by incubation for 20 min at 50 • C. Following the incubation period, a mixture was prepared by combining 500 µL of the sample with 500 µL of 10% trichloroacetic acid, 100 µL of 0.1% ferric chloride, and 500 µL of deionized water.The mixture was subjected to an incubation period of 10 min at room temperature, after which, the absorbance was measured at the wavelength of 700 nm.Concentrations of investigated compounds and referent standard ascorbic acid in the sample were 10 µM.The results of reducing the power of compounds were expressed as % Activity of Ascorbic acid (% AAa) [31].

% AAa =
A sample A re f erence × 100

Computational Methodology
The Gaussian09 software package was used to carry out all calculations [32].Quantum chemical calculations based on density functional theory, specifically M06-2X functional in conjunction with 6-311++G(d,p) basis set (with polarization and diffuse functions included), were used to optimize the structures of parent molecules as well as of the corresponding radicals, anions, and radical cations [33,34].Previous research has demonstrated that the M062-X method is suitable for thermodynamic and kinetic investigation of reaction mechanisms of examined compounds with free radicals and that it describes short-and medium-range interatomic interactions well (500 pm) [35].The SMD continuum solvation model [36] was used to simulate the structures in benzene (ε = 2.27) and methanol (ε = 32.61) with no geometrical restrictions.These solvents were chosen to replicate both polar and nonpolar settings as well as the environment in which experimental measurements were conducted.
For the assessment of the antioxidant activity, three main radical scavenging mechanisms-Hydrogen Atom Transfer (HAT), Single-Electron Transfer Followed by Proton Transfer (SET-PT), and Sequential Proton Loss-Electron Transfer (SPLET) were used [37][38][39][40].In the first reaction mechanism, the O-H bond is homolytic cleavage, resulting in the separation of a hydrogen atom and a radical species (A-O • ) (Equation ( 1)).In the first step of the SET-PT mechanism, a molecule loses an electron, which leads to the formation of the radical cation (A-OH +• ), which loses a proton in the second step (Equations ( 2) and ( 3)).Another two-step mechanism is the SPLET mechanism.The antioxidant molecule is used to create an anion (A-O − ) in the first stage, and the matching radical is created in the second step following an electron transfer (Equations ( 4) and ( 5)).
The solvated electron and proton enthalpies for the M062-X technique in benzene (−877.4 and −10.5 kJ mol −1 ) and methanol (−1065.4 and −61.4 kJ mol −1 ) were obtained from the available data in the literature [43].The Natural Bond Orbital (NBO) method and NBO 6.0 package were used to conduct the population study [44].The intermolecular orbitals' role in the examined compounds' anions and radicals is highlighted using NBO analysis.

Results and Discussion
Our recent results related to the preparation of coumarin-hydroxybenzohydrazide derivatives prompted us to carry out the synthesis of structurally similar compounds starting from 3-acetylcoumarin [26].The synthesis of various coumarin hydrazones (3) from 3-acetylcoumarin (1) and the corresponding benzoyl hydrazide (2) was presented in Scheme 1.The reaction between 1 and 4-hydroxybenzohydrazide (2a) in ethanol was used as a model for optimizing reaction conditions (Table 1).Initially, the reaction was conducted at room temperature and without a catalyst, but only trace amounts of the reaction product were obtained.The moderate yield of the product was observed after 10 h of heating the reaction mixture under reflux.In order to attain a higher product yield, acetic acid was used as a catalyst.In this instance, an adequate yield of 3a was obtained by increasing the catalyst concentration to 20 mol% and heating the mixture for 10 h.However, the catalyst had a substantial effect on both the reaction rate and yield.Specifically, the use of molecular iodine as a catalyst allowed the desired product 3a to be obtained in only 20 min and without heating (Table 1).The optimal conditions for carrying out these reactions were 10 mol% I 2 as a catalyst, room temperature, and 20 min.
was used as a catalyst.In this instance, an adequate yield of 3a was obtained by increasing the catalyst concentration to 20 mol% and heating the mixture for 10 h.However, the catalyst had a substantial effect on both the reaction rate and yield.Specifically, the use of molecular iodine as a catalyst allowed the desired product 3a to be obtained in only 20 min and without heating (Table 1).The optimal conditions for carrying out these reactions were 10 mol% I2 as a catalyst, room temperature, and 20 min.The results indicate that the applied reaction conditions result in the outstanding dissolution of the reactants, rapid precipitation of the product, and simple isolation of the reaction products.Table 2 shows that all products were obtained with yields ranging from outstanding to very good.All reactions were completed very rapidly, except for compounds 3d and 3e, for which a slight extension of the reaction time was required.It is essential to note that four obtained products have been reported for the first time in this study (3c-f).The isolated products' structures were characterized using NMR, IR, and Scheme 1. Synthesis of coumarin N-acylhydrazone derivatives: 3-acetylcoumarin (1), benzoyl hydrazide (2), iodine as catalyst (10 mol%), absolute ethanol, and room temperature (RT).

Entry
Catalyst (mol%) Yield (%) The results indicate that the applied reaction conditions result in the outstanding dissolution of the reactants, rapid precipitation of the product, and simple isolation of the reaction products.Table 2 shows that all products were obtained with yields ranging from outstanding to very good.All reactions were completed very rapidly, except for compounds 3d and 3e, for which a slight extension of the reaction time was required.It is essential to note that four obtained products have been reported for the first time in this study (3c-f).The isolated products' structures were characterized using NMR, IR, and UV-Vis spectroscopies.Additionally, elemental analysis was performed on newly synthesized compounds.

Structural Characterization of Coumarin N-Acylhydrazone Derivatives
The signals in the 1 H NMR spectra of derivatives 3 were assigned to three groups of protons.Singlets originating from the protons of the methyl group were observed at approximately 2.30 ppm in the 1 H NMR spectra of all the compounds.The aromatic protons of coumarin and hydrazide moiety were in a region of 6.64-8.01ppm.The hydrogen atoms of NH and phenolic OH groups were observed in the regions of 10.53-11.79ppm and 9.29-11.41ppm as broad singlets.In the spectra of compounds 3e and 3f, singlets originating from protons of methoxy groups attached to the aromatic ring at about 3.85 ppm were observed.In the 13 C NMR spectra of all the products, the carbon atom of the methyl group appears around 17 ppm.Signals in the range of 105-145 ppm were attributed to aromatic carbon atoms.Peaks detected at around 150, 153, and 160 ppm originate from C-O, C-N, and C=O carbon atoms.The additional peaks at about 60 ppm were observed in the spectra of compounds 3e and 3f because of the presence of methoxy groups.
The vibration of OH and NH groups corresponds to regions at approximately 3400 and 3250 cm −1 in the IR spectra of investigated compounds.The peaks in region 1650-1725 cm −1 are assigned to the C=O of hydrazone group and lactone ring, while bands present at lower values are attributed to the stretching vibration of C-N, C-O, and aromatic rings.In the UV-Vis spectra of all the compounds, two major absorption bands appear around 270 and 330 nm.

The Proposed Mechanism for the Synthesis of Coumarin N-Acylhydrazone Derivatives 3
The suggested mechanism for the synthesis of coumarin N-acylhydrazone derivatives 3 was presented in Scheme 2. In this reaction, molecular iodine most likely acts only as a precatalytic.In the first step, as a solvent, ethanol reacts with molecular iodine to give the Brønsted acid HI, which further acts as a catalyst, as shown in Scheme 2. In the subsequent step, the carbonyl group of 3-acetylcoumarin is protonated with Brønsted acid HI, followed by the nucleophilic attack of hydrazide's nitrogen atom to the carbonyl group, and the deprotonation of the same nitrogen atom.In this manner, an appropriate hemiaminal is formed as an intermediate.The resulting intermediate undergoes dehydration in a similar way.During this stage, the HI species facilitates the protonation of the hydroxyl group, leading to the formation of a water molecule, with simultaneous deprotonation of the nitrogen atom.In this way, corresponding coumarin N-acylhydrazone derivative 3 is formed.

Antioxidant Activity of Coumarin N-Acylhydrazone Derivatives
The DPPH and ABTS scavenging ability, as well as the ferric ion reducing capacity, of the synthesized coumarin N-acylhydrazone derivatives (3a-f) were evaluated to determine their in vitro antioxidant properties.The results are presented in Tables 3 and 4. In this study, NDGA, quercetin, (S)-6-methoxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), and ascorbic acid were used as referent compounds.

Antioxidant Activity of Coumarin N-Acylhydrazone Derivatives
The DPPH and ABTS scavenging ability, as well as the ferric ion reducing capacity, of the synthesized coumarin N-acylhydrazone derivatives (3a-f) were evaluated to determine their in vitro antioxidant properties.The results are presented in Tables 3 and 4. In this study, NDGA, quercetin, (S)-6-methoxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), and ascorbic acid were used as referent compounds.
The results obtained from the DPPH assay revealed that compounds 3c and 3d expressed the best antioxidant activity with IC50 values of 2.1 µM and 3.2 µM.The activity of these compounds is slightly lower than the activity of the positive controls, quercetin and NDGA.In order to determine whether the most active compounds belong to the group of good antioxidants, the SF was determined [26,45].It is known that compounds with an SF greater than 2 are good antioxidants.Namely, the tested compounds 3c and 3d, with SF values 6 and 3.9, belong to the group of excellent antioxidants.Other tested compounds showed extremely low activity toward the DPPH radical even at significantly higher concentrations, Table 3.The ABTS assay, which is a commonly employed technique for evaluating in vitro antioxidant capacity, was utilized to examine the radical scavenging potential of the investigated compounds, as presented in Table 4.The results obtained from this study indicate that compounds 3c and 3d exhibit significant scavenging activity against ABTS radical cation, with IC50 values of 2.2 µM and 3.0 µM, respectively.This finding is consistent with previous DPPH assay results.Compound 3f exhibited better activity relative to the DPPH results, exhibiting an efficacy of roughly 50% at a concentration of 100 µM.The results obtained from the DPPH assay revealed that compounds 3c and 3d expressed the best antioxidant activity with IC 50 values of 2.1 µM and 3.2 µM.The activity of these compounds is slightly lower than the activity of the positive controls, quercetin and NDGA.In order to determine whether the most active compounds belong to the group of good antioxidants, the SF was determined [26,45].It is known that compounds with an SF greater than 2 are good antioxidants.Namely, the tested compounds 3c and 3d, with SF values 6 and 3.9, belong to the group of excellent antioxidants.Other tested compounds showed extremely low activity toward the DPPH radical even at significantly higher concentrations, Table 3.
The ABTS assay, which is a commonly employed technique for evaluating in vitro antioxidant capacity, was utilized to examine the radical scavenging potential of the investigated compounds, as presented in Table 4.The results obtained from this study indicate that compounds 3c and 3d exhibit significant scavenging activity against ABTS radical cation, with IC 50 values of 2.2 µM and 3.0 µM, respectively.This finding is consistent with previous DPPH assay results.Compound 3f exhibited better activity relative to the DPPH results, exhibiting an efficacy of roughly 50% at a concentration of 100 µM.
The results of the FRAP assay revealed that all the examined compounds exhibited higher capacities for Fe 3+ reduction than ascorbic acid, which was used as the standard.It should be emphasized that the obtained results are in good agreement with the activities of these compounds against DPPH and ABTS radicals.Namely, compound 3c showed the greatest reducing ability, followed by compounds 3d and 3f.All the obtained results point out the compounds 3c and 3d, with two neighboring hydroxyl groups, i.e., compounds with a catechol fragment, as the best antioxidants.In addition, moderate activity against the ABTS radical cation is observed for compound 3f with vanillic fragment.The reason for this is the resonance and electron donating effects of these groups, as well as the very effective stabilization of the formed phenoxy radical by an intramolecular hydrogen bond.

Determination of the Plausible Mechanisms
The optimized geometries of the newly synthesized compounds, as shown in Figure 2, were obtained using the M06-2X/6-311++G(d,p) theoretical model.The geometries of the compounds under investigation exhibited a correlation with the geometries of structurally analogous compounds obtained in previous research [26,46,47].An examination of the optimal molecular structure of the six coumarin derivatives revealed the existence of a partly double N-N bond, which facilitated the delocalization of π-electrons between the coumarin base and the fused aromatic ring.Based on the observed value of the dihedral angle C2-C3-C1 -N1, which is around ~130 • , it is apparent that all the structures display deviations from planarity.Moreover, the determined N2-C7 -C1 -C6 dihedral angles within the range of 152-179 • for all the examined compounds suggest a deviation from planarity and a reduction in the electronic distribution within the compounds, which significantly affects their antioxidant activity.The obtained geometries were used for a deeper study of the mechanisms of the antioxidant action.

HAT Mechanism
The analysis of the BDE values for homolytic O-H cleavage showed that the reactivity decreased in the following order: 3d > 3c > 3f > 3b > 3a > 3e (Table 5).The reactivity of a compound is correlated with the stability of the newly formed radical species.Therefore, the NBO spin density of the studied radical species was analyzed.Figure S1 shows that the unpaired electron was mainly delocalized over the oxygen undergoing hydrogen ab-

HAT Mechanism
The analysis of the BDE values for homolytic O-H cleavage showed that the reactivity decreased in the following order: 3d > 3c > 3f > 3b > 3a > 3e (Table 5).The reactivity of a compound is correlated with the stability of the newly formed radical species.Therefore, the NBO spin density of the studied radical species was analyzed.Figure S1 shows that the unpaired electron was mainly delocalized over the oxygen undergoing hydrogen abstraction and the ortho and para carbon atoms.Obviously, the better the delocalization of the unpaired electron, the more stable the obtained radical.The radicals formed from the oand p-hydroxyl groups are the most stable because their unpaired electrons are delocalized over the benzene ring.The spin density values of the newly formed radicals increased in the following order: 3d-2O • (0.284e) > 3f (0.306e) > 3c-4O • (0.312e) > 3b (0.327e) > 3d-3O • (0.330e) > 3c-3O • (0.341e) > 3a (0.358e).The oxygen atom of the strongest 3d radical scavenger has the lowest spin density.The lower BDE value for 3d can be attributed to the stability of the radical species due to the free rotation of the C7 -C1 bond, as well as the formation of an intramolecular hydrogen bond.Since the BDE values in benzene are comparatively lower than the PA and ETE values, it can be concluded that HAT is likely to be the dominant mechanism in nonpolar solvents.On the other hand, the calculated BDE values for the homolytic cleavage of N-H bonds exhibited an increasing trend in the following order: 3e > 3b > 3d> 3f > 3c > 3a.The limited delocalization of the electron and the reduced stability of radicals arising from the homolytic cleavage of the N2-H bond can be attributed to the higher spin density values observed for N-centered radicals compared with O-centered radicals (Figure S2).This discrepancy is exemplified by the higher BDE values associated with N-centered radicals.

SET-PT Mechanism
The SET-PT mechanism is often thermodynamically least likely due to high IP values [44].However, it is important to note that there exist certain instances when the SET-PT mechanism might exhibit dominance [54][55][56][57][58][59][60].In methanol, the first step of the SET-PT mechanism for all investigated compounds, which is described by IP values, is extremely endothermic, whereas the second step is significantly exothermic in methanol (Table 5).As anticipated, the IP values were approximately 100 kJ mol −1 greater in the non-polar solvent than in methanol.Due to their high values, however, it can be inferred that the SET-PT mechanism is not a viable reaction pathway in either solvent.The theoretically predicted reactivity order was 3f > 3e > 3a, 3c > 3b > 3d.Although the studied compounds had lower and even exergonic PDE values, the initial step of the SET-PT pathway was the limiting factor for the antioxidant activity of these compounds via this mechanism.

SPLET Mechanism
The heterolytic cleavage of the O-H and N-H bonds of investigated compounds is the first step of the SPLET mechanism, which leads to the formation of the corresponding anions.The calculated thermodynamic parameters for the heterolytic cleavage of the O-H and N-H groups are shown in Table 5.The PA values of the O-H group were lower than those of the N-H group.The order of activity based on the PA value for the O-H group was 3d > 3b > 3c > 3a > 3f.The difference in reactivity was a consequence of the stabilization of the newly formed anionic species (Figures S3 and S4).According to the PA values in methanol (Table 5), the heterolytic cleavage of the O2 -H bond in 3d is favourable.The negative charge of this anion is distributed over O7 , O2 , O3 , and C5 .This anion is additionally stabilized with the hydrogen bonds.As expected, the stabilization of the anionic species in the polar solvent was much more pronounced, which is reflected in the lower PA values.Because the BDE values in benzene are lower than the PA and ETE values, it is plausible that HAT predominates in nonpolar solvents.The PA values in methanol were considerably lower than the BDE values; however, the ETE values were higher, but still comparable with the BDEs.Therefore, it is plausible to assume that HAT and SPLET compete within polar media.

Radical Scavenging Activity toward DPPH Radical
Potential free radical scavenging mechanisms are discussed based on the reaction free energies (∆ r G) following the HAT and SET-PT mechanisms (Figure 3).Calculations were performed using methanol as the solvent, mimicking the in vitro DPPH test conditions.The difference in energies between the reactants and products serves as the primary criterion for determining the probability of the occurrence of the reaction.The lower value of thermodynamic parameters describing HAT and SET mechanisms indicates the favorability of the pathway by which reaction occurs.The optimized geometries of the reactants involved in the reaction, namely DPPH • , DPPH 2 , and DPPH − are presented in Figure S5.
An examination of the results presented in Table 6 shows that all the investigated compounds exhibited positive ∆ r G SET values (>132 kJ mol −1 ).This implies that the reactions involving the inactivation of the DPPH radical through the SET-PT mechanism are characterized by endergonic values.Based on the observed data, it can be concluded that the reactivity of the compounds decreased in the following order: 3f > 3e > 3a, 3c > 3b > 3d.The significant values of ∆ r G SET clearly demonstrate that the SET-PT mechanism can be disregarded considering DPPH radical scavenging, in agreement with the thermodynamic data presented in Table 6.
∆ r G HAT values have been identified as a reliable measure for assessing the ability of coumarin derivatives to inactivate DPPH • .The observed antioxidant activity, as assessed by in vitro measurements, exhibited the same trend as the ∆ r G HAT values obtained in methanol: 3d > 3c > 3f > 3b > 3a > 3e.This finding further substantiates the notion that both this parameter and spin density possess the capacity to elucidate the reactivity with respect to DPPH • .The enhanced antioxidant activity of compounds 3b and 3f has been previously highlighted because of the intramolecular stabilization of the radical species produced.Based on the aforementioned information, it is reasonable to anticipate that the DPPH • present in methanol will undergo scavenging via the HAT mechanistic pathway.
were performed using methanol as the solvent, mimicking the in vitro DPPH test co tions.The difference in energies between the reactants and products serves as the prim criterion for determining the probability of the occurrence of the reaction.The lower v of thermodynamic parameters describing HAT and SET mechanisms indicates the fa ability of the pathway by which reaction occurs.The optimized geometries of the react involved in the reaction, namely DPPH • , DPPH2, and DPPH − are presented in Figure An examination of the results presented in Table 6 shows that all the investig compounds exhibited positive ∆rGSET values (>132 kJ mol -1 ).This implies that the react involving the inactivation of the DPPH radical through the SET-PT mechanism are c acterized by endergonic values.Based on the observed data, it can be concluded tha reactivity of the compounds decreased in the following order: 3f > 3e > 3a, 3c > 3b > The significant values of ∆rGSET clearly demonstrate that the SET-PT mechanism ca disregarded considering DPPH radical scavenging, in agreement with the therm namic data presented in Table 6.
ΔrGHAT values have been identified as a reliable measure for assessing the abilit coumarin derivatives to inactivate DPPH • .The observed antioxidant activity, as asse by in vitro measurements, exhibited the same trend as the ΔrGHAT values obtained in m anol: 3d > 3c > 3f > 3b > 3a > 3e.This finding further substantiates the notion that both parameter and spin density possess the capacity to elucidate the reactivity with respe DPPH • .The enhanced antioxidant activity of compounds 3b and 3f has been previo highlighted because of the intramolecular stabilization of the radical species produ Based on the aforementioned information, it is reasonable to anticipate that the DP present in methanol will undergo scavenging via the HAT mechanistic pathway.ABTS •+ inactivation was evaluated using two different reaction pathways, namely HAT and SET-PT.Methanol was used as the solvent to replicate the experimental conditions (Figure 4).Table 7 lists the estimated values of the thermodynamic parameters.The optimized geometries of ABTS •+ , ABTS + , and ABTS are shown in Figure S6.
The transfer of a hydrogen atom from the -OH groups of A-OH to ABTS •+ , with the formation of A-O • and ABTS + , is a thermodynamically favored reaction.The exergonic ∆ r G HAT values of compound 3d (−13 kJ mol −1 ) demonstrated the highest efficiency in neutralizing radical species, which aligns with the findings from the experimental data.
On the other hand, electron transfer from A-OH to ABTS •+ with the formation of A-OH + and ABTS + thermodynamics was favored in all the investigated compounds.Based on an analysis of the ∆ r G SET values, it can be concluded that compound 3f (−155 kJ mol −1 ) exhibits the highest electron transfer ability.However, endergonic ∆ r G PT values (>118 kJ mol −1 ) represent a limiting factor for the manifestation of antioxidant activity through the SET-PT mechanism.Based on the sum ∆ r G SET + ∆ r G PT , it can be concluded that the reactivity of the compounds under investigation follows a decreasing trend in the sequence 3d > 3c > 3f > 3b > 3a > 3e, which aligns with the experimental results.In summary, it can be concluded that HAT and SET-PT are in competition when it comes to the inactivation of the ABTS

Radical Scavenging Activity toward ABTS Radical Cation
ABTS •+ inactivation was evaluated using two different reaction pathways, HAT and SET-PT.Methanol was used as the solvent to replicate the experimenta tions (Figure 4).Table 7 lists the estimated values of the thermodynamic paramet optimized geometries of ABTS •+ , ABTS + , and ABTS are shown in Figure S6.The transfer of a hydrogen atom from the -OH groups of A-OH to ABTS •+ , formation of A-O • and ABTS + , is a thermodynamically favored reaction.The ex ΔrGHAT values of compound 3d (−13 kJ mol -1 ) demonstrated the highest efficiency tralizing radical species, which aligns with the findings from the experimental da On the other hand, electron transfer from A-OH to ABTS •+ with the formati OH + and ABTS + thermodynamics was favored in all the investigated compound on an analysis of the ΔrGSET values, it can be concluded that compound 3f (−155 k exhibits the highest electron transfer ability.However, endergonic ΔrGPT values mol -1 ) represent a limiting factor for the manifestation of antioxidant activity thro SET-PT mechanism.Based on the sum ΔrGSET + ΔrGPT, it can be concluded

Conclusions
In this study, six coumarin N-acylhydrazone derivatives have been synthesized in high yields and characterized using NMR and IR spectroscopy, as well as elemental analysis.Also, the mechanism for the synthesis of coumarin N-acylhydrazone derivatives 3 in the

Figure 1 .
Figure 1.Structures of some coumarin derivatives with clinical application.

Figure 1 .
Figure 1.Structures of some coumarin derivatives with clinical application.
Reaction conditions: 1 (1 mmol), 2a (1 mmol), 3 mL EtOH; a Reaction performed at the room temperature; b Reaction performed by heating under reflux; c Reaction finished for 10 h; d Reaction finished for 20 min.

Scheme 2 .
Scheme 2. The suggested mechanism for formation of coumarin N-acylhydrazone derivatives.

Scheme 2 .
Scheme 2. The suggested mechanism for formation of coumarin N-acylhydrazone derivatives.

Table 1 .
Optimization of reaction conditions.

Table 3 .
DPPH scavenging activity of products 3 and referent compounds.

Table 3 .
DPPH scavenging activity of products 3 and referent compounds.

Table 4 .
ABTS radical cation scavenging activity and ferric ion reducing capacity of products 3 and corresponding referent compounds.

r G HAT ∆ r G SET ∆ r G PT
•+ radical in methanol.