Investigation of 4-Hydrazinobenzoic Acid Derivatives for Their Antioxidant Activity: In Vitro Screening and DFT Study

Abstract

Hydrazinobenzoic acid derivatives with isothiocyanate, benzylidene, and acid anhydride core units (1–13) were previously synthesized and fully characterized. Targets 1–13 were investigated for their antioxidant activities using different in vitro assays such as 1,1-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), ferric reducing antioxidant power (FRAP), and reducing power capability. All derivatives showed antioxidant properties in relation to the standard butylated hydroxylanisole (BHA). Superior antioxidant activities was observed for compounds 3 and 5–9 at a concentration of 20 μg/mL (70–72%) when tested by the DPPH method in comparison to BHA (92%), and compounds 1–10 showed the highest free radical quenching activity (80–85%) when examined by ABTS at 20 μg/mL in relation to BHA (85%). Density function theory (DFT) studies were carried out using the B3LYP/6-311G(d,p) level of theory. Several antioxidant descriptors were calculated for targets 1–13 compared with BHA. Targets 1–13 were proposed to exhibit their antioxidant activities via the following three proposed antioxidant mechanisms: single electron transfer (SET), hydrogen atom transfer (HAT), and sequential proton loss electron transfer (SPLET). The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energies and electron levels for 1–13 were also determined.

1. Introduction

The development of several organic heterocyclic compounds, owing to their diverse biological properties, has gained much attention. Among them, hydrazine-, hydrazone-, and isothiocyanate-based compounds are found to have various applications in organic synthesis, medicinal chemistry, combinatorial chemistry, and supramolecular chemistry.¹⁻³ Concerning organic synthesis, such compounds were proved to participate in the construction of different heterocyclic scaffold classes such as azetidines, coumarins, oxadiazolines, diazoles, triazoles, quinazolines, thiazolidines benzothiazines, and triazoloquinazolines.¹⁻¹⁰ Their distinct biological significance was explained on the basis of the specific structural combination for a (−NH–N=CH−) or (N–N) group with a carbonyl or thiocarbonyl group and their reactivities toward both electrophiles and nucleophiles. Therefore, they are present in many bioactive compounds and demonstrate a wide variety of biological activities including anticancer, antioxidant, antimicrobial, antifungal, anti-inflammatory, anticonvulsant, antiviral, and antiprotozoal effects.⁹⁻¹⁶ The process of discovering antioxidants has been gaining growing interest due to their valuable protective roles in both pharmaceutical and food industries. In human physiology, they play an essential role against oxidative deterioration and oxidative stress-mediated mechanisms, whereas in the food system, antioxidants retard lipid peroxidation, prevent the formation of secondary lipid peroxidation products, maintain the flavor, color, and texture of the food product during storage, and reduce the interaction of lipid-derived carbonyls with proteins that is accompanied by alteration in protein function.^17,18 There has been renewed interest to explore new antioxidant molecules either synthetically or naturally, so as to utilize them as the main agents to counter toxicity mediated by radicals.

Further evidence suggests that age-related human illnesses like heart disease, cancer, neurodegenerative, arthritis, immunosuppression, cerebral dysfunction, cataracts, and inflammation are the consequences of cellular damage by free radicals.¹⁹ Based on the aforementioned facts and our interest to discover novel synthetic antioxidants capable of preventing free radical-mediated damage and toxicity, we embarked on this experiment to evaluate the previously synthesized 4-hydrazinobenzoic acid derivatives 1–13 for their antioxidant activities using different in vitro assays such as DPPH, FRAP, ABTS, and reducing power capability. Moreover, the electronic and structural features of the investigated 1–13 have been evaluated by DFT.²⁰ The bond dissociation enthalpy (BDE), proton dissociation enthalpy (PDE), ionization potential (IP), and proton affinity (PA) descriptors have been computed at the B3LYP/6-311G(d,p) level of theory to explain antioxidant mechanisms. For more clarification, the calculated reactivity descriptors such as dipole moment, electron affinity (EA), softness (S), hardness (η), electrophilicity (ω), electronegativity (χ), and chemical potential (μ) have been taken into account for targets 1–13 and also for BHA. In addition, the HOMO and LUMO energies were calculated.

2. Results and Discussion

2.1. DPPH Radical Scavenging Activity

In the published literature, several compounds incorporating hydrazine, hydrazone, and isothiocyanate units have been described as potential antioxidants. For instance, hydrazone heterocycles bearing an imidazole or benzimidazole entity showed the highest DPPH radical scavenging (IC₅₀ = 22.4 ± 3.2 μM) and comparable ABTS scavenging (IC₅₀ = 7.3 ± 0.8 μM) in relation to the reference BHT (70.8 ± 6.6 and 7.2 ± 1.7 μM) as a standard substance.¹⁶ Moreover, in comparison with quercetin as a reference drug, veratric acid (3,4-dimethoxybenzoic acid) derivatives incorporating benzylidene-hydrazine moieties were reported as promising tyrosinase inhibitors and potent free radical scavengers, giving an EC₅₀ value of 0.0097 ± 0.0011 mM.²¹ In addition, triazoloquinazoline compounds resulting from a combination of hydrazinobenzoic acid with cyanoimdiocarbaonte demonstrated the highest capacity to deplete DPPH and free radicals (85–91%), in relation to BHT (93%), as a synthetic antioxidant substance.²² Introduction of the alkyl(aralkyl)isothiocyanate unit to the naphthaoic or anthranilic acid scaffold afforded promising antioxidant benzoquinazoline or quinazoline derivatives, which could be considered for developing useful templates for novel potent antioxidant compounds in the future.²³⁻²⁶ The grafting degree of hydrazine-thiosemicarbazone groups in the 2-[phenylhydrazine (or hydrazine) thiosemicarbazone]-chitosan complex improved their free radical scavenging activity compared to chitosan, and their polymerization on chitosan increased their antioxidant activity obviously with an IC₅₀ range of 0.263–0.531 mg/mL in comparison to ascorbic acid.¹⁹

Targets 1–13 (Scheme 1) were synthesized and characterized in our previous work.²⁷

Scheme 1. Synthesis Routes for Targets 1–13.

The DPPH steady radical could be attributed to the transfer of hydrogen or an electron to a stable compound. The DPPH methanolic solution demonstrated strong absorbance at 517 nm due to single electrons (odd electrons) or pairing electrons.²⁸⁻³⁰ When the solution was mixed with suitable reducing agents (antioxidants), the color disappeared. Reduction in color is stoichiometrically increased with the number of electrons, and the decrease in the absorbance measurement could be directly compared with BHA. The DPPH radical scavenging ability of hydrazinobenzoic acid derivatives 1–13 was evaluated and compared with BHA as a standard antioxidant. A graphical representation of the antioxidant activity in parameters of % inhibition versus the concentration of compounds 1–13 is shown in Figure 1. The results indicate that most of the products possess high radical scavenging activity comparable to BHA (Figure 1). It is worth mentioning that compounds 3 and 5–9 exhibited a potent DPPH radical scavenging activity at a lower concentration (20 μg/mL) in relation to the other substituted compounds and the standard BHA at a concentration of 40 μg/mL. Their radical scavenging activities varied from 50.27 ± 0.15 to 72.29 ± 0.24%. Parent compound 1 exhibited a moderate DPPH radical scavenging ability of 41.48 ± 0.23%, while 11 and 12 showed the lowest abilities of 17.21 ± 0.16 and 18.52 ± 0.14%, respectively. Various substitutions (isothiocyanate and benzylidene) on the structure of 1 resulted in an increase in the radical scavenging activity, particularly for compounds 3 and 5–9 that showed intrinsically the highest activity at a very low concentration (20 μg/mL).

Figure 1.

DPPH radical scavenging activity of targets 1–13; data are mean ± standard deviation of triplicate experiments.

2.2. ABTS Radical Quenching Activity

Further examination of the antioxidant potency of targets 1–13 was performed by evaluation of their ABTS free radical scavenging ability, which is considered a commonly in vitro used method as well. The results presented in Figure 2 support the DPPH scavenging activity illustrated in Figure 1 and show that derivatives 7–10 possessed more superior scavenging capabilities compared to BHA activity. Except for compounds 11–13, data illustrated in Figure 2 clearly indicate that all substituted derivatives showed an excellent ABTS radical scavenging activity. The hydroxyl-methoxy derivative (3) exhibited the highest ABTS radical quenching activity (85.19 ± 0.17%), mostly equal to that of BHA (85.20 ± 0.33) at the same concentration (20 μg/mL). Compounds 2 and 5–10 revealed potent ABTS radical scavenging ability in relation to BHA. Their radical scavenging effects ranged from 80.60 ± 0.15 for compound 6 to 84.34 ± 0.10% for 7 at the same concentration. Compound 1 demonstrated good antioxidant activity (74.52 ± 0.11%), while the substitution in compounds 11 and 13 resulted in moderate activities of 34.77 ± 0.15 and 46.55 ± 0.16%, respectively. The lowest ABTS radical scavenging ability was recorded by derivative 12 (18.51 ± 0.15%).

Figure 2.

ABTS radical scavenging ability of 1–13; data are mean ± standard deviation of triplicate experiments.

The DPPH scavenging effect of tested 1–13 was found to be in the descending order of BHA, 10, 9, 8, 7, 3, 2, 5, 6, 1, 4. On the other hand, the percentage values of ABTS scavenging activities of the same group were ordered in the descending manner of BHA, 3, 7, 8, 9, 10, 2, 5, 6, 1, 4. In our previous work, we reported that the radical scavenging activity of triazoloquinazolines²² depended mostly on the hydrazine and isothiocyanate units, which exerted their abilities as a better hydrogen supply or electron-donating group. The present outcomes affirmed the previous findings that 5–10 were the most active. The presence of hydrazine and isothiocyanate functional groups may result in maximum scavenging capabilities via their rules to stabilize the free radical form after electron donation or hydrogen supply.

2.3. FRAP

Iron plays a vital role in various normal neurological functions of living organisms; however, it can contribute to electron transfer reactions (redox reaction) with several substrates, making it an essential inducer for reactive species in humans.³¹ In view of this fact, the FRAP method was employed to estimate the capability of the electron donation of hydrazinobenzoic acid derivatives 1–13. The FRAP of a compound is a valuable indicator of its significant antioxidant ability. As shown in Figure 3, the majority of the examined substituents on the compound 1 structure led to a positive response to ferric reducing power capacity. Compound 1 showed a ferric reducing power capacity of 2864 ± 32.56 μmol Trolox/100 g, which is sharply increased to 4120 ± 20.53, 4080 ± 14.57, 4075 ± 11.06, and 4059 ± 33.42 μmol Trolox/100 g for compounds 7, 2, 3, and 5, respectively. An appropriate increase in FRAP by the substitution occurred in compounds 6, 8, 10, and 9 to achieve 3810 ± 10.50, 3733 ± 17.00, 3345 ± 20.52, and 3055 ± 21.01 μmol Trolox/100 g, respectively, at the same concentration (40 μg/mL). On the contrary, the substitutions in compounds 11, 12, and 13 caused a pronounced reduction in the ferric reducing power ability to 1047 ± 8.62, 946 ± 15.52, and 1263 ± 11.60 μmol Trolox/100 g, respectively, at 40 μg/mL. The FRAP of the targets was found in the order of 7 > 2 > 3 > 5 > 6 > 8 > 10 > 9 > 11 > 12 > 13.

Figure 3.

FRAP activity of targets 1–13; data are mean ± standard deviation of triplicate experiments.

2.4. Reducing Power Ability

In this method, the change of color from yellow to various green and blue shades relies on the reducing power of each substance at a concentration of 40 μg/mL. The antioxidant effect of 1–13 causes reduction of ferric (Fe³⁺) in the ferricyanide complex into the ferrous (Fe²⁺) form. Therefore, measuring the formation of Perls Prussian blue at 700 nm can control the concentration of Fe²⁺.³¹ The reducing power ability of targets 1–13 varied in high-to-moderate activity in comparison with BHA (Figure 4). Compound 1 demonstrated a high reducing power capability of 1.06 ± 0.03 at an absorbance of 700 nm. Substitutions on compound 1 resulted in a slight increase in the reducing power capability as observed by compounds 3, 2, and 5 (1.23 ± 0.04, 1.17 ± 0.02, and 1.10 ± 0.04, respectively), relative to BHA (1.35 ± 0.06) at the same concentration (40 μg/mL). Compounds 6–10 exhibited moderate reducing power activity ranging from 0.86 ± 0.01 to 1.02 ± 0.02. On the other hand, compounds 11, 12, and 13 recorded the lowest reducing power ability among all investigated 4-hydrazinobenzoic acid derivatives and showed the lowest antioxidant activity in all of the abovementioned assays.

Figure 4.

Reducing power ability of targets 1–13; data are mean ± standard deviation of triplicate experiments.

The strong correlations between the antioxidant ability assayed by ABTS, DPPH, FRAP, and reducing power capacity methods and compounds 1–13 (Figures 1–4) indicate that the hydrazine, isothiocyanate, and hydrazone units largely contribute to the antioxidant activities of 1–13. The presence of an anhydride moiety in 11–13 does not offer advantages in the antioxidant profile. The results are in accordance with several studies, indicating that hydrazine and isothiocyanate core units are major antioxidant constituents in heterocyclic compounds.

2.5. DFT Study

2.5.1. Geometry Optimization and Reactivity Descriptors

The physicochemical descriptors like BDE, PDE, PA, IP, ETE, η, S, and μ (Table S1) were employed to visualize the existing relation between 1–13 structures and chemical reactivity. Accurate data for structural and electronic features of antioxidants are of great significance in describing their scavenging behavior. Thus, various initial geometries of the target compounds were selected to undergo full optimization using the B3LYP/6-311G(d,p) level of theory. In the present work, full optimization of compounds 1, 2–4, 5–10, and 11–13 was considered (Figure S1), and the orientation possibilities as H₁ (aniline–Ar–NH), H₂ (hydrazine–N–NH or hydrazinecarbothioamide N–NH–CS–NH), and H₃ (phenol Ar–OH) were chosen.

Furthermore, in the presence of a weak external electric field, the electronic cloud system can be distorted from its normal shape.³² Different factors like compound complexity, molecular size, and the number of electrons can influence the polarizability parameter. The mean polarizability and dipole moment values are listed in Table 2. The tendency of charge distribution is accompanied by molecule polarizability. In our study, most of the target compounds showed higher polarizability values than BHA; however, it points out that the polarizability of 11 (characterized by two carboxylic groups) was influenced by the complexity system and the number of electrons; hence, it is considered the highest polar compound among this series.

Table 2. HOMO and LUMO Energies, Mean Polarizability, and Dipole Moment of Compounds 1–13 at the B3LYP/6-311G(d,p) Level of Theory.

	EHOMO (eV)		ELUMO (eV)		ΔEHOMO–LUMO (eV)		dipole moment (debye)		polarizability (α) (au)
cps	MeOH	gas	MeOH	gas	MeOH	gas	MeOH	gas	MeOH	gas
1	–5.89	–5.859	–1.18	–0.933	4.71	4.925	6.535	5.1315	134.839	101.13
2	–5.701	–5.622	–1.647	–1.584	4.053	4.037	7.976	6.3430	289.455	218.926
3	–5.669	–6.143	–1.7	–1.729	3.969	4.414	9.339	7.3883	296.714	225.846
4	–5.892	–5.964	–2.675	–2.764	3.217	3.2	9.692	7.4601	299.938	225.050
5	–6.229	–5.971	–1.404	–1.469	4.825	4.503	3.809	2.7875	203.252	155.961
6	–6.219	–5.934	–1.405	–1.455	4.814	4.478	4.05	3.0199	219.315	168.513
7	–6.191	–5.999	–1.459	–1.488	4.732	4.511	5.227	2.7013	230.721	180.334
8	–6.167	–6.017	–1.367	–1.134	4.8	4.884	10.687	7.7614	244.068	190.842
9	–6.195	–5.952	–1.446	–1.448	4.749	4.503	4.729	3.0260	286.6	219.901
10	–6.181	–5.866	–1.444	–1.445	4.737	4.42	4.954	2.9235	298.64	227.654
11	–6.412	–6.622	–3.46	–3.806	2.952	2.816	3.451	2.0656	426.991	329.818
12	–6.347	–6.329	–2.611	–2.719	3.736	3.61	7.749	5.8543	250.778	190.286
13	–6.411	–6.509	–2.878	–3.097	3.533	3.412	4.273	3.0994	287.514	219.863
BHA	–5.627	–5.546	–0.371	–0.228	5.256	5.318	3.156	2.5045	172.023	131.446

2.5.2. Frontier Molecular Orbitals and Reactivity Descriptors

The molecular orbital (MO) analysis of E_HOMO and E_LUMO is correlated to the chemical reactivity and stability of the compounds, in which molecules can be polarized effectively when small gap energy (ΔE_HOMO–LUMO) occurs between HOMO and LUMO. This is attributed to the transfer of electronic charges from donor to withdrawal groups. However, when the ΔE_HOMO–LUMO gaps of molecules are very large, the polarization of molecules appears to be less effective and reactive than that of the former (with a smaller gap). As illustrated in Table 2, the results of calculated gaps for 1–13 are in ranges of 2.952–4.825 and 2.816–4.925 eV in methanol and gas phases, respectively, compared to those of BHA (5.20 and 5.32 eV). Figure 5 shows the electron density distribution of HOMO in the gas phase for compounds 1–4 and 11–13, which mainly occurred on (Ar–NH) and (NH–CS–NH–NH−) moieties as the electron transfer zones, while in 5–10, it distributed mostly on the (NH–CS–NH–NH₂) group. LUMO spatial distributions of 1–4 and 11–13 are located on the carboxylic, phenyl hydroxyl, and isoindoline-1,3-dionyl moieties (the electron acceptor zones); however, they are mostly located on −NH–NH– in 5–10. The −N–NH, −N–NH–CS–NH–, and Ar–OH groups could be the most plausible reaction sites for the free radical attack. Molecules with lower frontier molecular orbital gaps are more polarized.³³ Theoretically, the HOMO energy is a valid indicator for scavenging activity rather than LUMO energy.³⁴ All values of global reactivity descriptors are presented in Tables 1 and 2 and reported in the eV unit. The chemical hardness (η) is correlated to the chemical potential with the resistance of change in the number of electrons (molecule stability), whereas S provides an insight into the capacity of an atom to receive an electron (molecule reactivity). In comparison with the calculated S of BHA (0.381 and 0.376 in the methanol and gas phases, respectively), compounds 1, 3, and 5–10 showed S values ranging between 0.405 and 0.453 in the gas phase, making them more chemically active and easier to donate electrons or H supply. However, the calculated η values (2.207–2.463) in the gas phase reflect their good stabilities in relation to BHA (2.659). The S parameter demonstrates that compounds 1, 3, and 5–10 are more preferable in the charge-transfer mechanism. For understanding the charge-transfer reaction, the χ and μ parameters were applied to measure the electron escaping tendency. In accordance with the obtained outcomes, compounds 1–3 and 5–10 showed the lowest χ values (3.396–3.936) in comparison to 4 and 11–13 (4.364–5.214) in the gas phase, indicating their good antioxidant abilities via their ability to donate electrons. Generally, all μ, χ, S, and η descriptor values suggest that compounds 1, 3, and 5–10 were characterized as more promising targets for electron scavenging reactions, which is in agreement with the experimental assay results.

Figure 5.

HOMO and LUMO molecular orbital plots for 1–13 in the gas phase.

Table 1. Global Reactivity Descriptors of Compounds 1–13 at the B3LYP/6-311G(d,p) Level of Theory.

	η		S		ω		μ		χ
formula	= (EHOMO – ELUMO)/2		= 1/η		μ2/2η		= (EHOMO + ELUMO)/2		= −μ
cps	MeOH	gas	MeOH	gas	MeOH	gas	MeOH	gas	MeOH	gas
1	2.355	2.463	0.425	0.406	2.653	2.342	–3.535	–3.396	3.535	3.396
2	2.027	2.019	0.493	0.495	3.33	3.215	–3.674	–3.603	3.674	3.603
3	1.984	2.207	0.504	0.453	3.421	3.51	–3.685	–3.936	3.685	3.936
4	1.609	1.6	0.622	0.625	5.704	5.952	–4.284	–4.364	4.284	4.364
5	2.412	2.251	0.415	0.444	3.019	3.073	–3.817	–3.72	3.817	3.72
6	2.407	2.239	0.415	0.447	3.019	3.048	–3.812	–3.694	3.812	3.694
7	2.366	2.256	0.423	0.443	3.091	3.106	–3.825	–3.743	3.825	3.743
8	2.4	2.442	0.417	0.41	2.956	2.618	–3.767	–3.576	3.767	3.576
9	2.375	2.252	0.421	0.444	3.073	3.04	–3.82	–3.7	3.82	3.7
10	2.368	2.21	0.422	0.452	3.068	3.023	–3.812	–3.656	3.812	3.656
11	1.476	1.408	0.678	0.71	8.254	9.655	–4.936	–5.214	4.936	5.214
12	1.868	1.805	0.535	0.554	5.371	5.668	–4.479	–4.524	4.479	4.524
13	1.766	1.706	0.566	0.586	6.105	6.761	–4.644	–4.803	4.644	4.803
BHA	2.628	2.659	0.381	0.376	1.711	1.568	–2.999	–2.887	2.999	2.887

Molecular electrostatic potential (MEP) is a crucial descriptor used to validate the evidence parts of the molecular reactivity system. The MEP three-dimensional surface provides us with information about the position, shape, positive/negative size, and neutral electrostatic potentials.³² Such information highlighted the structural relationship and molecular reactivity toward electrophilic and nucleophilic attacks. In Figure 6, the maximum negative electronic potential (in red color) is the favored site for the electrophilic attack, while the positive electrostatic potential (in blue color) is attracted by radical charged molecules. The high electrostatic potential regions (Figure 6) are observed in the vicinity of the oxygen of the carboxylic group in 1–13 and in the sulfur of the thiourea group in 5–10, while a low electropositive potential existed on the H₁ of (Ar–NH) of all molecules, H₂ of hydrazine (Ar–NH–NH) in molecules of 1 and 5–10, Ar–OH in 2–4 and (R–NHCSNH−) in 5–10. By comparing the blue color code values, +0.104 au in 4, +0.0844 in 2, +0.0844 in 3, +0.08.371 in 8, and +0.08153 in 7 suggest that Ar–NH in 7 and 8 and Ar–OH in 2–4 are the most favored nucleophilic attacking sites. Moreover, in 5–10, the codes ranging between +0.08371 and +0.07394 indicate their favored site for nucleophilic interactions.

Figure 6.

Molecular electrostatic potential of 1–13.

In addition, the H atoms of the hydrazine group are appropriate sites for nucleophiles, whereas the oxygen atom of carboxylic acid is favored for electrophiles.

Free radicals interact with organic molecules by one or more of the following three fundamental mechanisms: HAT, SETPT, and SPLET (Table S2).³² The resulting parameters (BDE, IP, PDE, and PA) shown in Table 3 indicate which radical scavenging mechanism is favored over another and the detailed HAT, SETPT, and SPLET mechanisms are reported.³² To describe the HAT mechanism, BDE is the best consistent thermodynamic parameter. This mechanism includes the transformation of the H atom from the NH– or OH– of the antioxidant compounds to the free radical (Scheme S1). The lowest BDE is the most readily available antioxidant in the highest quantity. As illustrated in Table 3, the results revealed that the N–H and O–H of compounds 3 and 5–10 have the least BDE values and demonstrated the highest radical scavenging properties, which is in agreement with the experimental results. In the present study, BDEs were calculated in the methanol and gas phases.³⁵ The obtained results revealed that the BDE values in methanol are in a range of 4.83–29.79 kcal/mol, which are higher than those in the gas phase for 5–10. However, the lowest BDE values in the gas phase are 80.137, 70.67, 67.597, 67.071, 60.863, 67.532, and 66.296 kcal/mol for 1, 3, 5–7, 9, and 10, respectively (Table 3), with regard to BHA (69.77 kcal/mol). Surprisingly, the N–H bonds of 1, 5, 6, 7, 9, and 10 are still the easiest dissociation bonds in methanol solvent. Based on the calculated BDE values, compounds 3 and 5–10 are considered the most potential antioxidants. The IP and PDE (Table 3) are the most important parameters for the SETPT mechanism.³² In general, lower IPs are more subject to ionization and increase the electron transfer rate between free radicals and antioxidants. By comparison, the IP values of the target compounds were ordered as 3 < 2 < BHA < 4 < 1 < 8 < 7 < 10 < 9 < 6 < 5 < 12 < 13 < 11. The IP values of the investigated compounds 1 and 4–13 are larger than the PI value of BHA except for 2 and 3. Thus, compounds 2 and 3 are the easiest to transfer electrons and possess one of the highest antioxidant activities. Moreover, compounds 5, 6, 9, 10 and 11 revealed the lowest calculated PDEs of approximately 150–260 kcal/mol. In accordance with these results, compounds 5–7, 9, and 10 should be the most potental antioxidants.³²

Table 3. Calculated BDE, IP, PDE, and PA for 1–13 at the B3LYP/6-311G(d,p) Level of Theory (in kcal/mol).

		BDE
comp.	H position	MeOH	gas	PDE	IP	PA	ABTS%
1	Ar–NH	86.65	80.137	266.321	136.189	188.479	74.52 ± 0.11
	R–NH	86.338	81.513	274.785		319.852
2	Ar–NH	84.649	79.825	270.859	129.65	306.949	82.28 ± 0.20
	Ar–OH	85.763	80.939	271.973		295.383
3	Ar–NH	85.307	72.514	271.775	129.392	307.6	85.19 ± 0.17
	Ar–OH	84.136	70.67	270.604		303.835
4	Ar–NH	84.997	72.061	265.59	135.268	303.998	69.23 ± 0.15
	Ar–OH	91.062	75.365	271.654		283.123
5	Ar–NH	84.472	67.597	258.693	141.64	305.743	81.54 ± 0.20
	R–NH2	89.542	72.672	263.762		307.873
	R–NH3	102.028	91.604	276.248		302.757
6	Ar–NH	84.223	67.529	258.77	141.314	305.123	80.60 ± 0.15
	R–NH2	89.324	72.693	263.871		307.823
	R–NH3	102.32	91.267	276.867		310.686
7	Ar–NH	86.013	67.071	261.054	140.82	304.845	84.34 ± 0.10
	R–NH2	92.574	71.943	267.615		305.255
	R–NH3	101.349	92.388	276.39		309.637
8	Ar–NH	89.319	74.163	265.171	140.009	309.197	82.49 ± 0.12
	R–NH2	90.656	60.863	266.508		299.765
	R–NH3	110.757	91.335	286.609		308.775
9	Ar–NH	85.036	67.532	259.785	141.112	304.476	83.41 ± 0.21
	R–NH2	91.311	73.181	266.06		305.667
	R–NH3	102.635	86.156	277.384		309.372
10	Ar–NH	85.445	66.296	260.46	140.845	304.271	83.22 ± 0.20
	R–NH2	91.202	71.583	266.218		305.868
	R–NH3	101.797	90.582	276.813		302.004
11	Ar–NH	89.691	77.423	150.281	255.271	301.252	34.77 ± 0.15
12	Ar–NH	89.716	77.505	259.517	146.06	304.715	18.51 ± 0.17
13	Ar–NH	89.824	77.467	258.044	147.64	303.076	46.55 ± 0.16
BHA	Ar–OH	82.929	69.77	266.122	132.667	309.53	85.18 ± 0.33

The SPLET mechanism, as shown in Table S2, initiates with the dissociation of the acidic moiety, which can be characterized by the proton affinity (suggested as the possible path to trap the radical in the polar environment); this is followed by an electron transfer to the free radical, at a cost of the electron transfer energy.³² The PA parameter is employed in this regard for elucidation purposes. Thus, lower PA has higher antioxidant capacity. As can be seen from Table 3, the PA values of O–H bonds are lower than those of N–H bonds. For instance, the O–H bonds have the lowest PA values in the methanol phase for 2–4 (295.383, 303.835, and 283.123 kcal/mol, respectively), whereas the N–H bonds have PA values of 305.743, 305.123, 304.845, 304.476, and 304.271 kcal/mol for 5–7, 9, and 10, respectively. Thus, the N–H bonds are also favored for the SPLET mechanism. This variant in activity is mostly associated with NH/OH group numbers that can be transferred to the DPPH free radical. Thus, an increase in hydrogen atoms is accompanied by an increase in scavenging activities. The findings suggested that the studied compounds might demonstrate their antioxidant activity via the three abovementioned antioxidant mechanisms. Due to the polarizability of 1–13, their electron scavenging reactions were obviously influenced by the polar environment, and polarizability could positively enhance the contribution of the SETPT mechanism followed by the SPLET mechanism. An agreement was noticed between some calculated values and the experimental results.

3. Conclusions

Inspired by the antioxidant properties of molecules bearing isothiocyanate and hydrazine core units, targets 1–13 were evaluated for their antioxidant activities. The results revealed that all investigated compounds showed antioxidant activity, particularly compounds 5–10 exhibited the highest scavenging effects. The radical scavenging activity of 1–13 was evaluated theoretically at the B3LYP/6-311++G(d,p) level of theory. The computed global reactivity descriptors suggested that molecules 1 and 5–10 were more favorable in the electron scavenging reactions. A good correlation was found between the experimental and calculated data. Compounds 3 and 5–10 had the least BDE values and demonstrated the highest radical scavenging properties. The calculated chemical descriptor values (μ, χ, S, and η) suggested 1, 3, and 5–10 as more promising targets for electron scavenging reactions. In addition, the HOMO and LUMO energies of targets 1–13 were calculated using the above DFT set to determine the properties of the target compounds and facilitate their antioxidant mechanisms.

4. Materials and Methods

4.1. Antioxidant Activity Investigation Assays

The investigated compounds 1–13 were described and characterized in our previous work.²⁷ In accordance with the procedures of Brand-Williams, Oyaizu, Benzie, and Re et al.,³⁶⁻³⁹ the DPPH, reducing power capability, FRAP, and ABTS radical scavenging assays were performed. The detailed methodologies were reported in our previous papers.⁴⁰⁻⁴²

4.2. DFT Study and Antioxidant Computation Descriptors

By employing the Gaussian 09 program package, all calculations were enforced.²⁰ Compounds structures, radicals, and ions were optimized by DFT, combined with the B3LYP/6-311G(d,p) set.^32,43 Assessment of free radical scavenging mechanisms for hydrazinobenzoic acids 1–13 was accomplished by the computation of various antioxidant descriptors, as presented in Tables S1 and S2.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c04772.

• Initial inputs for geometry optimizations of 1–13 and BHA (Figure S1); chemical reactivity descriptors (Table S1); antioxidant reaction mechanism and computation of antioxidant descriptors (Table S2); and example of the expected reaction mechanism between the DPPH free radical and compounds 5–10 (Scheme S1) (PDF)

The authors declare no competing financial interest.