Amino-Substituted Benzamide Derivatives as Promising Antioxidant

Article Cite This: Chem. Res. Toxicol. 2018, 31, 974−984

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Amino-Substituted Benzamide Derivatives as Promising Antioxidant Agents: A Combined Experimental and Computational Study Nataša Perin,† Petra Roškaric,́ † Irena Sovic,́ ‡ Ida Bocě k,† Kristina Starcě vic,́ § Marijana Hranjec,*,† and Robert Vianello*,∥

Chem. Res. Toxicol. 2018.31:974-984. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 09/24/18. For personal use only.

†

Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Marulićev trg 19, 10000 Zagreb, Croatia ‡ Laboratory for Green Chemistry, Division of Physical Chemistry, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia § Department of Animal Husbandry, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatia ∥ Computational Organic Chemistry and Biochemistry Group, Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia S Supporting Information *

ABSTRACT: We prepared a range of N-arylbenzamides with a variable number of methoxy and hydroxy groups, bearing either amino or amino-protonated moieties, and used DPPH and FRAP assays to evaluate their antioxidant capacity. Most of the systems exhibit improved antioxidative properties relative to the reference BHT molecule in both assays. Combining results from both sets of experiments, the most promising antioxidative potential was displayed by the trihydroxy derivative 26, which we propose as a lead compound for a further optimization of the benzamide scaffold. Computational analysis helped in interpreting the observed trends and demonstrated that protonated systems are better antioxidants than their neutral counterparts, while underlying the positive influence of the electron-donating methoxy group on the antioxidant properties, thus confirming the experiments. It also revealed that the introduction of the hydroxy groups shifts the reactivity from both amide and amine groups toward this moiety and additionally enhances antioxidative features. This is particularly evident in 26H•+, which owes its pronounced reactivity to the stabilizing [O•···H−O] hydrogen bonding between the created phenoxyl radical and the two neighboring hydroxy groups. We demonstrated that its antioxidative activities are more favorable than those for analogous trihydroxy derivatives without the N-phenyl group or without the amide moiety, which strongly justifies the employed strategy in utilizing bisphenylamides in designing potent antioxidants.

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INTRODUCTION Antioxidants are organic molecules capable of scavenging free radicals to prevent the cell damage caused by these unstable systems. Reactive oxygen species, continuously produced by oxygen during normal physiological processes, are capable of initiating the oxidative damage of vital biomacromolecules including proteins, lipids, and nucleic acids. Although these compounds have an important role in the normal cell function, their overproduction causes oxidative stress and leads to lowered antioxidative defense. Consequently, oxidative stress is an important factor in the development of some diseases, namely, diabetes, cancer, aging, or different neurodegenerative disorders.1,2 Therefore, the development and application of novel and more efficient natural or synthetic antioxidants is of great importance. In addition, such compounds have also been used as treatments for the prevention of coronary heart disease or stroke. In the last few decades, the search for the novel antioxidants led to the synthesis or isolation of different © 2018 American Chemical Society

organic molecules which showed promising and more effective antioxidant activity in comparison with the standard antioxidants, such as vitamin C (ascorbic acid), β-carotene, lutein, vitamin A, and butylated hydroxyrotoluene (BHT).3,4 Recently, several publications described the antioxidative potential, as well as other biological activities, of a broad range of salicylanilide and benzamide derivatives. Caldarelli and coauthors published the synthesis of N-arylbenzamides developed as novel estrogen receptor agonists.5 Moreover, salicylanilides were shown to be the selective inhibitors of interleukin-12p40 production, which has an important role in inflammatory diseases.6 Also, some salicylanilides (I, Figure 1) revealed a significant antiproliferative activity acting as inhibitors of the epidermal growth factor receptor tyrosine kinase,7,8 while docking simulations aided in revealing the Received: June 29, 2018 Published: August 15, 2018 974

DOI: 10.1021/acs.chemrestox.8b00175 Chem. Res. Toxicol. 2018, 31, 974−984

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Chemical Research in Toxicology

progress of the reactions was routinely checked by a thin layer chromatography (TLC) with Merck silica gel 60F-254 glass plates using dichloromethane/methanol and UV light (254 and 366 nm) for detection. General Method for the Synthesis of Nitro-Substituted Benzamides 7−13. To a solution of the corresponding benzoyl chlorides 1−4 and anilines 5−6 in absolute toluene, triethylamine (TEA) was added in a dropwise fashion. The reaction mixture was refluxed for several hours. After the solution was cooled, it was concentrated or/and the obtained solid was filtered off and recrystallized from the appropriate solvent. 4-Cyano-N-(4-nitrophenyl)benzamide 7. The title compound was synthesized following the general method, from 1 (0.70 g, 4.20 mmol), 5 (0.58 g, 4.20 mmol), and TEA (0.82 mL, 5.88 mmol) in absolute toluene (20 mL) after 24 h and recrystallization from ethanol to obtain 0.61 g (53.5%) of light yellow powder; mp 95−98 °C. 4-Cyano-N-(4-methoxy-2-nitrophenyl)benzamide 8. The title compound was synthesized following the general method, from 1 (0.70 g, 4.20 mmol), 6 (0.70 g, 4.20 mmol), and TEA (0.82 mL, 5.88 mmol) in absolute toluene (20 mL) after 22 h and recrystallization from acetone/MeOH (1:1) to obtain 0.51 g (53.5%) of light yellow powder; mp 190−193 °C. 2-Methoxy-N-(4-nitrophenyl)benzamide 9. The title compound was synthesized following the general method, from 2 (1.00 g, 5.86 mmol), 5 (0.80 g, 5.86 mmol), and TEA (1.62 mL, 11.60 mmol) in absolute toluene (30 mL) after 24 h and recrystallization from MeOH to obtain 1.21 g (61.3%) of white powder; mp 177−181 °C. 2,4-Dimethoxy-N-(4-nitrophenyl)benzamide 10. The title compound was synthesized following the general method, from 3 (0.70 g, 3.49 mmol), 5 (0.48 g, 3.49 mmol), and TEA (0.68 mL, 4.98 mmol) in absolute toluene (20 mL) after 22 h and recrystallization from acetone/MeOH (1:1) to obtain 0.66 g (62.7%) of light yellow powder; mp 187−190 °C. 2,4-Dimethoxy-N-(4-methoxy-2-nitrophenyl)benzamide 11. The title compound was synthesized following the general method, from 3 (1.46 g, 7.35 mmol), 6 (1.23 g, 7.30 mmol), and TEA (1.42 mL, 10.18 mmol) in absolute toluene (30 mL) after 24 h and recrystallization from MeOH to obtain 1.55 g (53.6%) of orange powder; mp 173−178 °C. 3,4,5-Trimethoxy-N-(4-nitrophenyl)benzamide 12. The title compound was synthesized following the general method, from 4 (0.70 g, 3.04 mmol), 5 (0.42 g, 3.04 mmol), and TEA (0.59 mL, 4.26 mmol) in absolute toluene (20 mL) after 22 h and recrystallization from acetone/MeOH (1:1) to obtain 0.60 g (59.5%) of yellow powder; mp 192−195 °C. 3,4,5-Trimethoxy-N-(4-methoxy-2-nitrophenyl)benzamide 13. The title compound was synthesized following the general method, from 4 (1.59 g, 6.89 mmol), 6 (1.16 g, 6.89 mmol), and TEA (1.40 mL, 4.26 mmol) in absolute toluene (30 mL) after 24 h and recrystallization from MeOH to obtain 1.70 g (49.7%) of orange powder; mp 200−204 °C. General Method for the Synthesis of Amino-Substituted Benzamides 14−20. Corresponding nitro-substituted benzamides 7−13 and solution of SnCl2 × 2H2O in MeOH and concentrated HCl were refluxed for 1−3 h. After it was cooled, the reaction mixture was evaporated under vacuum and dissolved in water (50 mL). The resulting solution was treated with 20% NaOH to pH = 14. The resulting product was filtered off and washed with water to obtain amino-substituted benzamides 14−20. N-(4-Aminophenyl)-4-cyanobenzamide 14. The title compound was synthesized following the general method, from 7 (0.30 g, 1.23 mmol), SnCl2 × 2H2O (2.10 g, 9.32 mmol) in concentrated HCl (4.0 mL) and MeOH (4.0 mL) after 1 h to obtain 0.11 g (41.3%) of yellow powder; mp 245−250 °C. N-(2-Amino-4-methoxyphenyl)-4-cyanobenzamide 15. The title compound was synthesized following the general method, from 8 (0.60 g, 2.02 mmol), SnCl2 × 2H2O (3.78 g, 16.76 mmol) in concentrated HCl (7.0 mL) and MeOH (7.0 mL) after 1 h to obtain 0.50 g (92.9.4%) of yellow powder; mp >300 °C.

Figure 1. Previously prepared biologically active benzamide derivatives.

binding modes of the most potent derivatives into this receptor active site. Furthermore, the synthesis and antioxidative activity of various benzamide derivatives were recently demonstrated using electrochemical methods.9 Substituted heterocyclic analogues of salicylanilides have shown promising antimycobacterial activity, thus representing potential antituberculotics.10 Additionally, substituted benzanilides can act as potassium channel activators with some derivatives characterized as potent smooth muscle relaxants.11 Steffen and coauthors investigated the activity of modified salicylanilides as cellpermeable inhibitors of poly(ADP-ribose) glycohydrolase that can modulate both the cell recovery or cell death depending on the level of the exerted DNA damage.12 Novel derivatives of the nitro-substituted salicylic acid were designed and evaluated as potential antimycobacterial, antimicrobial, and antifungal agents (II, Figure 1). The presence of the nitro group in position 4 of the salicylic acid was found to be beneficial for the activity against Mycobacterium tuberculosis.13 Interestingly, some of the salicylanilide derivatives showed a promising antiosteoclastogenic activity due to the revealed inhibition of the osteoclast differentiation and bone resorption (III, Figure 1).14 Combining the demonstrated biological potential of a simple N-arylbenzamide scaffold with the fact that the antioxidant potential of suchlike systems has not been explored enough, we have designed and synthesized novel nitro and amino-substituted derivatives bearing a variable number of the methoxy and hydroxy groups. Amino and amino-protonated derivatives were explored for their antioxidative features using two in vitro assays, while computational analysis at the DFT level with implicit SMD solvation was utilized to predict the structure of the studied systems and rationalize the observed trends in their antioxidative potentials.

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EXPERIMENTAL PROCEDURES

Synthesis. General Methods. Chemicals and solvents used here were purchased from Aldrich and Acros. All solvents were dried using recommended drying agents. Melting points were determined on SMP10 Bibby apparatus. 1H and 13C NMR spectra were acquired on Bruker AV 300 and Bruker AV 600 spectrometers using TMS as an internal standard in DMSO-d6 at 298 K. Elemental analyses for carbon, hydrogen, and nitrogen were performed on a PerkinElmer 2400 analyzer and a PerkinElmer, Series II, CHNS analyzer 2400. The 975


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Chemical Research in Toxicology Table 1. IC50 Values of Investigated Systems for DPPH Free-Radical-Scavenging and FRAP Activitiesa

system

R1

R2

R3

R4

R5

R6

21 22 23 24 25 26 27 28 29 30 32 33 BHT

OH OH H OH OH H H H OCH3 OCH3 H H

H H OH H H OH H H H H OCH3 OCH3

H OCH3 OH H OCH3 OH CN CN H OCH3 OCH3 OCH3

H H OH H H OH H H H H OCH3 OCH3

H H H H H H H NH3+Cl− H H H NH3+Cl−

NH2 NH2 NH2 NH3+Cl− NH3+Cl− NH3+Cl− NH3+Cl− OCH3 NH3+Cl− NH3+Cl− NH3+Cl− OCH3

DPPH IC50 μM 23.80 24.0 16.85 22.25 10.81 12.93 30.73 18.12 30.02 18.4 26.56 30.45 25

± ± ± ± ± ± ± ± ± ± ± ± ±

0.3 1.5 0.1 0.9 3.3 1.9 8.1 0.3 4.1 3.1 1.8 2.1 4.2a

FRAP mmolFe2+/mmolC 2283.67 1530.69 3259.93 2328.80 2174.40 4856.15 1763.47 989.11 2307.42 1905.99 1677.96 1763.47 2089.34

± ± ± ± ± ± ± ± ± ± ± ± ±

62.12 35.87 85.51 118.03 99.85 70.18 32.66 85.02 63.87 87.57 93.20 219.52 55.98

Values are presented as means ± standard deviation. For the DPPH measurements, values lower than those for a standard BHT system indicate more potent antioxidants, while for the FRAP analysis higher values point to a more pronounced antioxidant activity.

a

N-(4-Aminophenyl)-2-methoxybenzamide 16. The title compound was synthesized following the general method, from 10 (0.60 g, 2.21 mmol), SnCl2 × 2H2O (3.48 g, 15.42 mmol) in concentrated HCl (3.0 mL) and MeOH (7.0 mL) after 2 h to obtain 0.23 g (53.3%) of white powder; mp 107−110 °C. N-(4-Aminophenyl)-2,4-dimethoxybenzamide 17. The title compound was synthesized following the general method, from 10 (0.26 g, 0.87 mmol), SnCl2 × 2H2O (1.63 g, 7.28 mmol) in concentrated HCl (3.0 mL) and MeOH (3.0 mL) after 1 h to obtain 0.18 g (77.5%) of yellow powder; mp 179−183 °C. N-(2-Amino-4-methoxyphenyl)-2,4-dimethoxybenzamide 18. The title compound was synthesized following the general method, from 11 (1.20 g, 3.61 mmol), SnCl2 × 2H2O (5.70 g, 25.26 mmol) in concentrated HCl (5.0 mL) and MeOH (10.0 mL) after 3 h to obtain 0.16 g (13.3%) of beige powder; mp 230−233 °C. N-(4-Aminophenyl)-3,4,5-trimethoxybenzamide 19. The title compound was synthesized following the general method, from 12 (0.40 g, 1.20 mmol), SnCl2 × 2H2O (2.25 g, 10.00 mmol) in concentrated HCl (4.3 mL) and MeOH (4.3 mL) after 1 h to obtain 0.17 g (45.5%) of yellow powder; mp 205−209 °C. N-(2-Amino-4-methoxyphenyl)-3,4,5-trimethoxybenzamide 20. The title compound was synthesized following the general method, from 13 (1.20 g, 3.31 mmol), SnCl2 × 2H2O (5.19 g, 23.00 mmol) in concentrated HCl (5.0 mL) and MeOH (10.0 mL) after 2 h to obtain 0.58 g (71.1%) of white powder; mp 196−200 °C. General Method for the Synthesis of Hydroxy-Substituted Benzamides 21−23. A solution of corresponding methoxysubstituted benzamides 16−20 in absolute dichloromethane (DCM) was cooled to −78 °C under argon atmosphere, and BBr3 was added dropwise. The reaction mixture was stirred for 24−48 h at room temperature. After addition of MeOH (10 mL) and water (10 mL), the mixture was stirred for 30 min at room temperature. The obtained recrystallized product was filtered off; MeOH or water layer was extracted with ethyl acetate, and after drying with MgSO4, it was evaporated under vacuum to obtain the powdered product. N-(4-Aminophenyl)-2-hydroxybenzamide 21. The title compound was synthesized following the general method, from 16 (0.20 g, 0.74 mmol), BBr3 (2.2 mL, 2.2 mmol) in absolute DCM (20 mL) to obtain 0.12 g (60.9%) of gray powder; mp 157−161 °C. N-(4-Aminophenyl)-2-hydroxy-4-methoxybenzamide 22. The title compound was synthesized following the general method, from 17 (0.9 g, 1.08 mmol), BBr3 (6.5 mL, 6.5 mmol) in absolute DCM (20 mL) to obtain 0.12 g (42.1%) of white powder; mp 294−296 °C.

N-(4-Aminophenyl)-3,4,5-trihydroxybenzamide 23. The title compound was synthesized following the general method, from 19 (0.41 g, 1.35 mmol), BBr3 (12.1 mL,12.2 mmol) in absolute DCM (20 mL) to obtain 0.22 g (61.2%) of white powder; mp 170−174 °C. General Method for the Synthesis of Amino-Protonated Benzamides 24−26 and 27−33. A stirred suspension of compounds 21−23 and 14−20 in absolute ethanol (5 mL) was saturated with HCl(g). After 24 h of stirring, a small amount of diethyl ether was added, and resulting product was filtered off and washed with diethyl ether to obtain hydrochlorides 24−26 and 27−33. N-(4-Aminophenyl)-2-hydroxybenzamide Hydrochloride 24. The title compound was synthesized following the general method, from 21 (0.06 g, 0.26 mmol) in absolute ethanol (5 mL) to obtain 0.05 g (74.7%) of brown powder; mp 258−260 °C. N-(4-Aminophenyl)-2-hydroxy-4-methoxybenzamide Hydrochloride 25. The title compound was synthesized following the general method, from 22 (0.08 g, 0.32 mmol) in absolute ethanol (5 mL) to obtain 0.06 g (68.1%) of white powder; mp 280−284 °C. N-(4-Aminophenyl)-3,4,5-trihydroxybenzamide Hydrochloride 26. The title compound was synthesized following the general method, from 23 (0.10 g, 0.38 mmol) in absolute ethanol (5 mL) to obtain 0.04 g (68.1%) of gray powder; mp 287−292 °C. N-(4-Aminophenyl)-4-cyanobenzamide Hydrochloride 27. The title compound was synthesized following the general method, from 14 (0.08 g, 3.54 mmol) in absolute ethanol (5 mL) to obtain 0.07 g (74.3%) of light brown powder; mp >300 °C. N-(2-Amino-4-methoxyphenyl)-4-cyanobenzamide Hydrochloride 28. The title compound was synthesized following the general method, from 15 (0.05 g, 1.87 mmol) in absolute ethanol (5 mL) to obtain 0.03 g (51.1%) of gray powder; mp 296−299 °C. N-(4-Aminophenyl)-2-methoxybenzamide Hydrochloride 29. The title compound was synthesized following the general method, from 16 (0.04 g, 0.19 mmol) in absolute ethanol (5 mL) to obtain 0.04 g (75.8%) of beige powder; mp 236−240 °C. N-(4-Aminophenyl)-2,4-dimethoxybenzamide Hydrochloride 30. The title compound was synthesized following the general method, from 17 (0.07 g, 0.28 mmol) in absolute ethanol (5 mL) to obtain 0.04 g (41.9%) of white powder; mp 232−237 °C. N-(2-Amino-4-methoxyphenyl)-2,4-dimethoxybenzamide Hydrochloride 31. The title compound was synthesized following the general method, from 18 (0.03 g, 0.09 mmol) in absolute ethanol (3 mL) to obtain 0.01 g (25.0%) of white powder; mp 183−187 °C. N-(4-Aminophenyl)-3,4,5-trimethoxybenzamide Hydrochloride 32. The title compound was synthesized following the general 976


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Chemical Research in Toxicology Scheme 1. Synthesis of Benzamide Derivatives Investigated Here

method, from 19 (0.07 g, 0.25 mmol) in absolute ethanol (5 mL) to obtain 0.06 g (64.0%) of white powder; mp 277−282 °C. N-(2-Amino-4-methoxyphenyl)-3,4,5-trimethoxybenzamide Hydrochloride 33. The title compound was synthesized following the general method, from 20 (0.04 g, 0.12 mmol) in absolute ethanol (5 mL) to obtain 0.03 g (59.2%) of white powder; mp 200−205 °C. Antioxidative Activity. Determination of the Reducing Activity of the Stable Radical 1,1-Diphenyl-picrylhydrazyl (DPPH). The determination of reducing activity was measured according to the previously reported procedure with modification for use in a 96-well microplate. Briefly, to a solution of DPPH (final concentration 50 μM) in absolute ethanol was added an equal volume of various concentrations of tested compounds dissolved in DMSO. The assay was carried out in a 96-well microtiter plate. Ethanol and DMSO were used as control solutions according to the previously published experimental procedure.44 Determination of Ferric Reducing/Antioxidant Power (FRAP Assay). The FRAP method was performed according to previously reported procedure with minor modifications for an assay on a 96-well microplate.44 All results were then expressed as Fe2+ equivalents (Fe2+ μmol). All tests were done in triplicate, and the results were averaged and presented in Table 1. Computational Details. As a good compromise between accuracy and feasibility, all of the molecular geometries were fully optimized with the density functional theory (DFT) using the B3LYP functional (unrestricted UB3LYP for the radicals), and the 631+G(d) basis set followed by the harmonic frequency calculations as implemented in the Gaussian 09 suite of programs.45 Analysis of different conformations was done to select the most stable structures in each case. The thermal Gibbs free energy corrections were extracted from the corresponding frequency calculations without the application of scaling factors, while the obtained structures were confirmed as true minima by the absence of imaginary vibrational frequencies. The final single-point energies were attained with a highly flexible 6-311++G(2df,2pd) basis set. To account for the solvation effects, we included the SMD polarizable continuum model with all

parameters corresponding to pure ethanol (ε = 24.852), in accordance with presented experiments, giving rise to the B3LYP/6311++G(2df,2pd)//(SMD)/B3LYP/6-31+G(d) model employed here. All reported values correspond to differences in Gibbs free energies obtained at a room temperature of 298 K and a normal pressure of 1 atm. The choice of this computational setup was prompted by its success in modeling mechanisms of various antioxidants46 and in reproducing kinetic and thermodynamic parameters of a variety of organic and enzymatic reactions.47−49 According to the literature, there are multiple mechanisms that relate the antioxidative properties of molecules.15 In this work, we evaluated the two most frequent, and usually thermodynamically most preferred antioxidant mechanisms, namely hydrogen atom transfer (HAT), and single electron transfer (SET) that is commonly followed by proton transfer (SET-PT). All these mechanisms have the same net result (i.e., the formation of corresponding antioxidant radical). HAT is a major mechanism in which the H atom (hydrogen radical) is directly transferred from antioxidant (M) to free radical by the homolytic X−H bond cleavage to break oxidative chain reaction. The capacity of this mechanism is essentially driven by the X−H bond dissociation energy (BDE) calculated here as M−H → M• + H• BDE = G(M•) + G(H•) − G(M−H) The lower BDE value indicates that the stability of the corresponding X−H bond is lower and that it can be easily broken. Therefore, the lower BDE parameter points to a better antioxidant property of the investigated molecule M. Scavenging the free radicals may also be achieved by donating a single electron from a molecule M in the SET-PT process. This mechanism is governed by the adiabatic ionization energy (IE) required to eject an electron from M, calculated here as M → M•+ + e− IE = G(M•+) − G(M) Analogously to BDE, the lower IE value identifies a better antioxidant property of the investigated compound M. 977


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RESULTS AND DISCUSSION Chemistry. All investigated benzamide derivatives were synthesized according to Scheme 1. Nitro-substituted derivatives 7−13 were obtained from the cyano 1 and methoxy 2−4substituted acyl-halogenides and substituted anilines 5−6. Amino-substituted compounds 14−20 were prepared by the reduction of the corresponding nitro precursors with SnCl2 × 2H2O. In order to obtain the corresponding hydroxysubstituted benzamides with an amino moiety 21−23, a removal of the methoxy protecting groups was accomplished with the boron tribromide in absolute dichloromethane at −75 °C. With the dimethoxy derivative 17, only one methoxy group in the ortho position was removed, as confirmed by the 2D NMR. Amino derivatives 14−23 were converted into the aminoprotonated analogues 27−33 with gaseous HCl in absolute ethanol to ensure a better solubility. All compounds were obtained in moderate reaction yields. Structures of 7−26 were confirmed by both 1H and 13C NMR spectroscopies and elemental analysis. Reduction of the nitro group into the amino moiety was monitored by the appearance of the signals related to amino protons in the range 5.5−6.5 ppm in the corresponding 1H NMR spectra. Radical Trapping and Reducing Power of Compounds. To determine the antioxidant capacity of the investigated systems, the reducing activity of the stable 1,1diphenyl-picrylhydrazyl radical (DPPH) and ferric reducing/ antioxidant power (FRAP) parameters were evaluated.15 The former is based on the ability of investigated compounds to donate a hydrogen atom or an electron to DPPH,16−18 and it has been widely used for the measurement of free-radicalscavenging capacity of various systems.19−21 The DPPH radical is a stable organic free radical with the adsorption band at 515−528 nm. It loses this adsorption when accepting an electron or free radical species, which results in a visually noticeable discoloration from purple to yellow. Although this probe is chemically different from the radicals responsible for the autoxidation of real systems, and this approach does not consider kinetic aspects of the radical trapping features, which some authors consider as a limitation of the DPPH method,17,18 we applied this approach in a consistent manner under identical settings for all examined compounds, which is why the obtained results provide a convenient and efficient screening of the radical trapping features of structurally similar systems investigated here. Table 1 displays the obtained results which were collected after 30 min, with the DPPH final concentration being 100 μM. The results are expressed as IC50 values with the exception of 14, 16−18, 20, and 31, which did not exhibit any activity under used assay conditions. All nitrosubstituted derivatives 7−13 as well as 15 and 19 were insoluble in ethanol and were not tested. On the basis of the obtained results, it could be concluded that all measured compounds showed excellent DPPH quenching ability, with few compounds being significantly more active than the standard BHT (IC50 = 25 ± 4.2 μM). The results obtained through DPPH measurements reveal that the most pronounced antioxidative capacity is displayed by the 2-hydroxy-4-methoxy derivative 25 bearing the aminoprotonated group (IC50 = 10.81 ± 3.3 μM). Its analogue with the un-ionized amino moiety 22 showed a lower quenching ability (IC50 = 24.0 ± 1.5 μM), being at the level of the reference BHT. This confirms a persistent trend observed in all

molecules, in which compounds with the amino-protonated group demonstrate a significant improvement in the radicalscavenging activity over the corresponding un-ionized systems, as already noticed in some previous reports.22 Analogously, a trihydroxy-substituted derivative 26 with the amino-protonated group displays an improvement in the scavenging activity in comparison to its neutral derivative 23. Furthermore, the cyano derivative 28, having additional methoxy group at the phenyl ring, was significantly more active (IC50 = 18.12 ± 0.28 μM) in comparison to its analogue 27 (IC50 = 30.73 ± 8.1 μM). Among the methoxy-substituted amino-protonated systems, compound 30 with two methoxy groups showed the highest capacity (IC50 = 18.4 ± 3.1 μM), while the observed positive influence of the methoxy groups did not lead to the enhancement of the quenching ability in the monosubstituted 29. Surprisingly, compound 33 with the additional methoxy group placed at the phenyl ring showed the weakest antioxidative capacity (IC50 = 30.45 ± 2.1 μM) among investigated systems. FRAP assay was used to investigate the reducing power of tested compounds monitored by a change in the absorbance at 593 nm. This method is based on the ability of compounds to reduce the ferric tripyridyl triazine complex (TPTZ) to the ferrous state (Fe2+), which can be observed by an intense blue color.17 As mentioned with the DPPH method, this approach also shows some limitations,18 yet we applied identical conditions for all screened compounds and thus the obtained results are a useful indication of a relative ranking of systems and, together with the DPPH results, provide valuable data on the performance of antioxidants in protecting molecules from oxidative damage. The obtained results reveal that 21, 23, 24, 25, 26, and 29 showed moderate to significant improvement in the reducing power relative to the standard BHT (2089.34 ± 55.98 mmolFe2+/mmolC). The highest and most promising reducing power is found in the trihydroxy-substituted benzamide bearing amino-protonated group 26 (4856.15 ± 70.18 mmolFe2+/mmolC), which is again more active than its un-ionized analogue 23. Similarly, protonated 2-hydroxy-4methoxy derivative 25 also showed an increase in the reducing power compared to neutral 22. In contrast, the cyano derivatives 27 and 28 reveal the opposite trend to that observed in DPPH measurements. Namely, 27 without both the hydroxy and methoxy groups showed an almost 2-fold increase in the reducing power (1763.47 ± 32.66 mmolFe2+/ mmolC). A comparison of the results displayed by aminoprotonated systems reveals that the largest impact on the improvement of the antioxidative capacity is exerted by the increasing number of the hydroxy groups, with the trihydroxy derivative 26 being the most active one. A replacement of one methoxy group with the hydroxy moiety, as in 25, led to a slight improvement of the reducing ability. Surprisingly, the methoxy-substituted benzamide 29 showed higher reducing power (2307.42 ± 63.87 mmolFe2+/mmolC) than all 2hydroxy-4-methoxy- (25), dimethoxy- (30), and trimethoxysubstituted benzamide 32. Also, the introduction of the additional methoxy group in 33 did not improve the reducing power. Taking into account the results from both in vitro assays, the most potent and promising benzamide system is the trihydroxy-substituted derivative with the amino-protonated group 26, which signifies the importance of these two structural elements in promoting the antioxidative activity. Its DPPH radical trapping activity is only slightly improved in 978


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addition, Foti and co-workers demonstrated a linear Evans− Polanyi relationship between BDEs and the kinetic activation energies for the corresponding hydrogen atom transfer for a series of 27 phenols and two unsaturated hydrocarbons,24 and obtained a large proportionality constant of 0.92, which lends credence to the computational approach undertaken here. Before we engage in analyzing these data in detail, let us first discuss some general trends evident in the obtained values. As explained in the Computational Details (see earlier), molecules with lower BDE values display better antioxidant properties through the hydrogen atom transfer mechanism, while lower IP values suggest a better antioxidant through the single electron transfer pathway. In the cationic forms, all molecules are most preferably protonated at the aniline amino moiety, which is an important observation. As expected, cations show lower BDEs than the corresponding un-ionized systems as a rule. This is reasonable, since it becomes easier to abstract a hydrogen atom from a charged, polar and more acidic cationic molecule than it is from a neutral system. In addition, upon the H atom abstraction in the protonated system, the formed cation radical can delocalize both the positive charge and the unpaired electron spin density into the attached aromatic system (Scheme 2), which both contribute to lowering the corresponding BDEs, as already noticed by Liu and Bordwell.22 For example, the reported BDEs for the monocationic Me− NH3+, c-C6H11−NH3+, and Ph−NH3+ in acetonitrile are 114.6, 113.6, and 84.9 kcal mol−1, respectively,22 which emphasizes the important role of the aromatic moiety. Analogously, a neutral molecule is easier to ionize since it is less demanding to strip an electron from a neutral compound M to get a radical cation M•+ than it is from an already positively charged cationic system MH+ to produce a doubly charged radical MH•++. Since both sets of values are highly correlated, in a way that a higher BDE generally yields a higher IE and vice versa (Figure S1), in what follows we will focus our discussion mostly on differences in BDEs unless stated otherwise. This is further justified by the fact that, for each compound, BDE is lower than IE (Table 2), suggesting H atom transfer as the likely predominant antioxidative mechanism in the examined bisphenylamides, in line with previous papers on various phenolic antioxidants23,25−27 or systems with other X−H bond energetic (X = C, N, O, S).28

25, yet 26 exhibits significantly larger potency than all systems examined here as measured with FRAP (Figure 2). Addition-

Figure 2. Most promising antioxidant 26.

ally, as a general conclusion, it was observed that the aminoprotonated derivatives showed much higher antioxidative capacity than their neutral analogues as a rule, in line with previous reports in the literature.22 Computational Analysis. To provide further insight into the structure and properties of the examined systems and to offer rationalization of the measured antioxidant features, we performed a computational analysis using the B3LYP DFT functional in conjunction with the 6-31+G(d) basis set, all immersed in the implicit SMD solvation corresponding to pure ethanol. Since the experimentally considered molecules are structurally very similar and exhibit a relatively narrow span of antioxidant activities, it would be very difficult to interpret and reproduce every single value presented in Table 1 within a reasonable accuracy. Instead, we decided to proceed with a set of model systems M1−M12 (Figure 3), selected to most closely represent the examined set of molecules 21−33, while allowing enough structural and electronic information to offer some general conclusions about studied systems in order to aid in the design of even more potent antioxidants based on the bisphenylamide framework. The calculated bond dissociation energies (BDE) and ionization energies (IE) for both neutral Mn and cationic MnH+ (n = 1−12) are given in Table 2. Both sets of values and the related discussion are based on considering thermodynamic aspects of their reactivities, while neglecting kinetic features.17 Still, in the investigated systems that belong to the same family of compounds, it is reasonable to expect that kinetic properties of either H atom or electron transfer reactions are largely similar along the series and are not predominantly determining the antioxidative activities.23 In

Figure 3. Schematic representation of molecules studied computationally. In the cationic forms, all systems are most preferably protonated at the aniline amino moiety. 979


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Table 2. Bond dissociation energies (BDE) and ionization energies (IE) in ethanol solution calculated at the B3LYP/6-311+ +G(2df,2pd)//(SMD)/B3LYP/6-31+G(d) level of theorya cationic-protonated molecules MnH+

neutral molecules Mn −1

−1

−1

system

BDE (kcal mol )

site of the X−H cleavage

IE (kcal mol )

BDE (kcal mol )

site of the X−H cleavage

IE (kcal mol−1)

M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12

85.3 75.6 75.0 75.4 79.5 81.2 77.5 73.7 71.9 72.7 77.1 67.2

amide N−H amide N−H amide N−H amide N−H amide N−H amide N−H amide N−H C2-phenolic O−H C2-phenolic O−H C2-phenolic O−H amide N−H C4-phenolic O−H

135.7 114.2 115.3 113.1 120.0 125.6 118.7 114.2 115.7 112.6 117.6 113.6

65.8 66.1 65.2 72.8 75.4 71.4 65.7 66.3 64.6 71.2 63.5

aniline N−H aniline N−H aniline N−H aniline N−H aniline N−H aniline N−H aniline N−H aniline N−H aniline N−H aniline N−H C4-phenolic O−H

144.2 145.9 139.9 143.8 150.0 143.4 140.4 145.9 138.8 133.4 134.4

a

The corresponding site of the hydrogen atom abstraction is denoted next to a BDE value.

Scheme 2. Stabilization of the Aniline Radical Cation through Delocalization of the Positive Charge and the Unpaired Electron Spin Density into the Aromatic System

N(amide)−C(aromatic) bond in M1 that changes from 1.414 to 1.342 Å upon hydrogen atom abstraction, as well as in the pronounced localization of the C−C bonds within the N-phenyl ring in the radical. Nevertheless, its BDE(M1) = 85.3 kcal mol−1 is higher than those for all other studied molecules, neutral or cationic. Analogously, its IE(M1) = 135.7 kcal mol−1 is the highest among neutrals, while already being close to IEs calculated for cations. All of this suggests that M1 itself is a very poor antioxidant, yet it will turn out that, with a proper substitution, a much improved antioxidants could be tailored. Still, to put these numbers and values for other molecules in a proper context, we calculated data for the reference BHT molecule as BDE(BHT) = 65.8 kcal mol−1 and IE(BHT) = 123.8 kcal mol−1 in ethanol. The calculated BDE seems to fall in the right range and is comparable to the reported values of 79.9,30 76.9,31 and 72.432 kcal mol−1 in heptane, benzene and toluene, respectively. A significantly lower BDE(BHT) in ethanol calculated here is fully in line with a demonstrated reduction in the O−H BDE values with the polarity of the solvent,23 which follows a trend in the corresponding dielectric constants of ε = 1.9, 2.2, 2.4, and 16.2 for heptane, benzene, toluene, and ethanol, in the same order. This further confirms the poor antioxidant properties of M1 as its BDE and IE values are around 20 and 12 kcal mol−1 less favorable than those for BHT, respectively. Nevertheless, this perspective sets up a distant goal in the design of more potent antioxidants based on the bisphenylamide framework. Addition of the amino group in the para-position (M2) has a significant impact on the calculated data. It reduces BDE and IE values by around 10 and 20 kcal mol−1, respectively, to BDE(M2) = 75.6 and IE(M2) = 114.2 kcal mol−1, thus promoting its antioxidant activity. This underlines the positive effect of the electron-donating substituents that will also be evident in the case of methoxy derivatives later,23 being in line with negative values of their Hammett substituent constants.33 This feature fully agrees with previous reports on the ability of electron-donating groups to reduce BDE values, as, for

Interestingly, regardless of their differences in the hydrogen abstraction site in neutral systems, whether a central amide N− H bond or a hydroxy O−H bond in systems having that functionality, in cations all molecules most preferentially donate a hydrogen atom from the positively charged aniline group. The only exception is M12, which has multiple hydroxy groups, and in which the hydrogen bonding between the newly created phenoxyl radical and the two neighboring − OH groups directs the hydrogen atom abstraction to this site (see later). In this context, electronic effects induced by various substituents in all M1−M11 have a much lower impact on the antioxidant properties of cationic systems than on the thermodynamic parameters of neutral molecules. Specifically, all calculated BDEs span a narrow range between 63.5−75.4 kcal mol−1 for cations, while for neutrals these are between 67.2−85.3 kcal mol−1. It is very interesting to observe that BDE values for neutrals are mostly found to be higher than those calculated for the matching protonated systems, suggesting that un-ionized systems are weaker antioxidants, being in full agreement with experiments here and previous reports in the literature.22 The most significant exception is M12, which turned out to be the most potent neutral antioxidant here, having a BDE value lower than even some of the cations. System M1 is a simple unsubstituted bisphenylamide and will be used as a reference system for M2−M12. It turns out that the N-phenyl moiety is crucial for its radical trapping features. Namely, our calculations show that BDEs for M1 and parent Ph−C(O)−NH2 and CH3−C(O)−NH−Ph are 85.3, 104.0, and 86.8 kcal mol−1, demonstrating the favorable influence of the bonded N-phenyl group, thus justifying the employed strategy. This trend is fully in line with values 97.0, 107.0, and 99.5 kcal mol−1 measured by Bordwell and Ji29 in DMSO, respectively. Thus, the ability of the nitrogen radical in M1 to delocalize the formed spin density into the attached aromatic system is responsible for its lowered BDE. This is nicely evident in the shortening of the corresponding 980


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Chemical Research in Toxicology example, p-dimethylamino group lowers the gas-phase BDE of phenol by 10.1 kcal mol−1, and the corresponding IP value by as much as 37.7 kcal mol−1.21 It is worth noticing that in M2 the most favorable position for the hydrogen atom abstraction remains the amide fragment, as the calculated BDE corresponding to aniline N−H moiety is 78.6 kcal mol−1, thus is 3.0 kcal mol−1 higher. This indicates that in neutral systems the role of the attached amino group is exerted through its electron donating ability that stabilizes the formed amide radical through the resonance effect rather than being the site of the hydrogen atom abstraction. This is evidenced in the reduction of the corresponding C(aromatic)−N(amino) bond from 1.400 to 1.350 Å on going from M2 to M2•, accompanied by the planarization of the amino group in the later. Analogously, the matching N(amide)−C(aromatic) bond is also shortened from 1.419 to 1.345 Å, thus confirming the resonance stabilization of the formed radical. This is a general feature observed in other systems as well, in which a predominant effect leading to enhanced antioxidative properties is the ability of the formed radicals to be stabilized through the extended delocalization that is efficiently assisted through resonance with the attached electron-donating substituents. In terms of BDEs, a simple protonation of M2, as in M2H+, further increases its antioxidant tendency, as the calculated BDE(M2H+) = 65.8 kcal mol−1 is, coincidently, exactly the same as that for the reference BHT, indicating that M2H+ is already a potent antioxidant. As mentioned, in M2H+ the Habstraction occurs on the protonated amino group, while the calculated BDE corresponding to the amide fragment is much higher at 87.9 kcal mol−1. It is worth saying that the homolytic aniline N−H cleavage in the protonated M2H+ is by 12.8 kcal mol−1 more favorable than in neutral M2 (aniline BDE = 78.6 kcal mol−1), which is found in excellent agreement with 14.5 kcal mol−1 reported by Liu and Bordwell for Ph−NH3+ and Ph−NH2 in the gas phase.22 Introduction of the electronwithdrawing cyano group (M3) or the electron-donating methoxy moiety (M4) does not have a huge effect on the calculated data, particularly in neutrals where both substituents do reduce the matching BDEs, but only up to 0.6 kcal mol−1. In cations, the cyano group slightly reduces, while the methoxy group promotes the antioxidant potency to BDE(M4H+) = 65.2 kcal mol−1, again confirming the favorable effect of the electron-donating substituents. This insight rationalizes why, for example, 27 is a poorer antioxidant than both 29 and 30, as well as why the methoxy substitution improves the antioxidant capacity of 25 and 30 relative to 24 and 29, respectively. Moving the free amino group from the para- to ortho-position (M5), as well as substituting the same phenyl ring with the cyano (M6) and methoxy groups (M7) gives molecules with significantly lower activities. This holds consistently for both neutral and cationic systems, as in neutrals the BDEs are increased to 77.5−81.2 kcal mol−1, while in cations to 71.4− 75.4 kcal mol−1. This is in full agreement with experiments, and explains, for instance, why cationic 28 and 33 are among least potent antioxidants. Our calculations reveal that this is likely due to the fact that ortho-amino groups in M5H+−M7H+ and M11H+ are stabilized through the hydrogen bonding with the amide carbonyl. This holds in particular for the closed-shell cationic systems, which then prevents an efficient cleavage of the related N−H bond, in line with previous reports that intramolecular hydrogen bonding, unless being a favorable 5center interaction among vicinal moieties as in M12,21 lowers the reactivity toward free radicals.34,35 Interestingly, even a

large number of the electron-donating methoxy groups, which we demonstrated increase the antioxidant activity, are unable to jointly overcome the unfavorable position of the orthoamino group in 33 (M11), making the latter one of the least potent antioxidants here. Still, systems M5−M7 again confirm a trend in which the electron-accepting cyano group increases, while the electron-donating methoxy group reduces the corresponding BDE value. This fully agrees with N−H BDEs for aniline, 4-CN-aniline, and 4-OMe-aniline in water of 89.1, 91.8, and 87.2 kcal mol−1, respectively, reported by Jonsson and co-workers.36 Systems M8−M10 represent a distinct group of molecules, since in neutrals the most favorable position for the H atom abstraction changes from the amide group to the hydroxy moiety, which turns out to be an important observation. Moreover, these three systems exhibit stronger antioxidant properties than all M1−M11, being in agreement with experiments here. This is because the hydroxy O−H group undergoes a homolytic cleavage easier than the amide N−H,37 thus having a strong emphasis for the design of more potent antioxidants. This observation fully agrees with demonstrated antioxidative features of many phenols and polyhydroxy aromatic compounds reported in the literature.19 In addition, substitution with the − CN and − OMe groups on the phenyl ring bearing the − OH moiety further lowers the BDE values in neutrals to BDE(M9) = 71.9 and BDE(M10) = 72.7 kcal mol−1. These are found in good agreement with those calculated for, for example, sinapic-, caffeic-, and chlorogenic acid in ethanol of 75.0, 75.1, and 75.0 kcal mol −1, respectively.19 As an illustration, our calculations reveal that the homolytic cleavage of the corresponding amide and aniline N−H bonds in M9 is associated with much higher BDE values of 72.6 and 79.4 kcal mol−1, respectively, demonstrating the highest antioxidative potential of the −OH group. Interestingly, although possessing a very reactive phenolic −OH moiety, the H atom abstraction in cationic M8H+−M10H+ again occurs on the protonated amino moiety, however without any noteworthy effect on the calculated BDE and IE values, as evident, for example, in comparison with M2H+− M4H+. Nevertheless, the difference in BDE values between the most favorable cationic −NH3+ site and alternative amide and hydroxy moieties are larger here, being 66.3, 87.7, and 81.7 kcal mol−1 in M9H+, respectively, strongly favoring the former as the prominent radical-scavenging site. Still, even in these cations, one again observes a trend in which the cyano group slightly reduces, while the methoxy group increases the ease of the homolytic cleavage, being a consequence of the positive electron donating features of the latter. A special case is provided by M12 (or 26) which bears three hydroxy groups on the same phenyl ring, while having the amino group on the other ring in a favorable para-position. This system has been experimentally identified as the most potent antioxidant here (Table 1), which is further corroborated by calculations. Namely, the obtained values of BDE(M12) = 67.2 and BDE(M12H+) = 63.5 kcal mol−1 are fully in agreement with a trend in measured data for 23 and 26, respectively. The latter is the lowest BDE here, thus strongly supporting M12H+ as the most potent antioxidant. Relative to the reference BDE(BHT) = 65.8 kcal mol−1, M12H+ exhibits a reduction of 2.3 kcal mol−1. This translates to around 50 times higher antioxidant activity, which is significant and in line with experiments here. Even in the neutral form, the calculated BDE (M12) = 67.2 kcal mol−1 is lower than that for 4 out of 11 examined cations. 981


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Chemical Research in Toxicology Furthermore, M12H+ is the only cationic system where the H atom abstraction occurs on the hydroxy group instead on the amine fragment, for which the calculated BDE assumes 68.0 kcal mol−1, thus is 4.5 kcal mol−1 higher. This is because in M12H•+ the created para-phenoxy radical center forms hydrogen bondings with the neighboring two hydroxy groups at the [O•···H−O] distances of 2.220 and 2.227 Å, which stabilize the system. This structural feature has already been demonstrated as responsible for the enhanced radicalscavenging activity of some natural antioxidants having two or three aromatic hydroxy groups,21 such as gallic acid, for which the calculated BDE is 77.0 kcal mol−1, and is much lower than BDE(phenol) = 82.9 kcal mol−1.19 The rather low BDEs for such catechols are due to the electron-donating character of the second (and third) OH group and to the increase in strength (by several kcal mol−1)37 of the intramolecular hydrogen bonding on going from the catechol to the radical.38 Nevertheless, with BDE = 63.5 kcal mol−1, system M12H+ shows around 10 orders of magnitude an increased antioxidative activity than gallic acid in ethanol, which strongly promotes the investigated systems as a good starting point toward more efficient antioxidants. In addition, a close vicinity of hydroxy moieties is crucial for the stability of the phenoxy radical as, for example, the calculated BDE for 1,2,3-trihydroxybenzene is by 15 kcal mol−1 lower than that of 1,3,5-trihydroxy analogue in toluene.32 Lastly, it is worth mentioning that the calculated BDE(M12H+) = 63.5 kcal mol−1 is lower and more favorable than those obtained for 1,2,3-trihydroxybenzene and 3,4,5-trihydroxybenzamide for which the same computational approach gives 65.9 and 67.9 kcal mol−1, respectively, which offers a further support in utilizing bisphenylamide scaffold for the presented purpose. Still, a certain care must be exerted in designing antioxidants based on polyhydroxy systems, because under certain physiological conditions, involving the presence of transition metal ions such as Fe3+ and Cu2+, and appropriate pH environment, phenoxy radicals can react with oxygen to generate O2•−, H2O2, and a complex combination of semiquinones and quinones39 that may induce lipid peroxidation, DNA damage, and apoptosis.40 Some authors suggest that these side-effects are related only to high doses,39,41,42 but they can play an important role in their selective cytotoxicity toward cancer cells, which contain more copper than do normal cells. Nevertheless, these pro-oxidant activities are a double-edged sword that can occur even in natural antioxidants or various catechols present in our body, and it remains a challenge to find a favorable balance between these two aspects. It is in that context that we are convinced that the results of the present study offer useful guidelines in designing improved molecules and point the attention toward utilizing the N-arylbenzamide scaffold in this direction.

scavenging activity DPPH assay showed that the 2-hydroxy4-methoxy derivative having amino-protonated group 25 exhibits the most potent antioxidative capacity. The highest reducing power evaluated by the FRAP method was demonstrated by the trihydroxy system 26 bearing the amino-protonated group. In general, systems having the protonated amino moiety showed significant improvement in the antioxidative capacity relative to their un-ionized analogues. Also, the presence of the electron-donating methoxy and hydroxy moieties yielded a considerable positive impact on the measured data. Trihydroxy derivative with the −NH3+ group 26 was elucidated as the most active system based on both assays and proposed as a lead compound for further optimization of the investigated scaffold toward more efficient antioxidants. We demonstrated that its antioxidative features are more favorable than those for analogous trihydroxy derivatives without the N-phenyl group or without the amide moiety, which strongly justifies the employed strategy in utilizing bisphenylamides in designing potent antioxidants. For a practical realization, one would also need to consider pro-oxidant features and other toxicological and pharmacological properties of designed compounds,21,41,43 but these aspects are beyond the scope of the current work. Computational analysis helped in interpreting the observed trends in the antioxidative capacity. By investigating homolytic bond dissociation energies and ionization energies for a set of model systems, both in un-ionized neutral and ionized cationic forms, it clearly demonstrated that protonated systems are better antioxidants than their neutral counterparts, and they confirmed the positive influence of the electron-donating methoxy group on the antioxidant parameters. It also showed that, unlike para-amino derivatives, ortho-amino compounds are much less potent antioxidants due to the formation of the hydrogen bonding with the near amide carbonyl, which stabilizes the reactive amino group and prevents an efficient hydrogen atom abstraction. Introduction of the hydroxy groups shifts the reactivity from the amine moiety toward this fragment and promotes the antioxidative properties. This is particularly enhanced in the trihydroxy derivative 26, which was demonstrated by both experiments and computations as the most potent antioxidant studied here. The latter owes its pronounced reactivity to the stabilizing [O•···H−O] hydrogen bonding that the created phenoxyl radical forms with the two neighboring hydroxy groups in 26H•+, the latter also exerting a favorable electron-donating stabilization.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.8b00175. Graphical representation of the correlation between calculated bond dissociation energies (BDE) and ionization energies (IE) for neutral and monoprotonated cationic molecules (Figure S1); structural characterization of the prepared compounds 7−33 together with the corresponding NMR spectra (Figures S2−S54) (PDF)

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CONCLUSIONS Herein we presented the design and synthesis of benzamide derivatives substituted with a variable number of methoxy and hydroxy groups bearing either amino or amino-protonated moieties, which were evaluated for their antioxidative activity. The purpose of this study was to experimentally and computationally assess the impact of a variable number and type of substituents placed on this simple organic scaffold with an already demonstrated biological potential. A large majority of tested compounds displayed a better antioxidative capacity than the standard BHT. Radical-

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (M.H.). 982


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Chemical Research in Toxicology *E-mail: [email protected] (R.V.).

some organic molecules as an antioxidant agents. Pharm. Anal. Acta 5, 1−5. (10) Matyk, J., Waisser, K., Dražková, K., Kuneš, J., Klimešová, V., Palát, K., Jr., and Kaustová, J. (2005) Heterocyclic isosters of antimycobacterial salicylanilides. Farmaco 60, 399−408. (11) Biagi, G., Giorgi, I., Livi, O., Nardi, A., Calderone, V., Martelli, A., Martinotti, E., and LeRoy Salerni, O. (2004) Synthesis and biological activity of novel substituted benzanilides as potassium channel activators. V. Eur. J. Med. Chem. 39, 491−498. (12) Steffen, J. D., Coyle, D. L., Damodaran, K., Beroza, P., and Jacobson, M. K. (2011) Discovery and structure-activity relationships of modified salicylanilides as cell permeable inhibitors of poly(ADPribose) glycohydrolase (PARG). J. Med. Chem. 54, 5403−5413. (13) Paraskevopoulos, G., Krátky, M., Mandíková, J., Trejtnar, F., Stolaríková, J., Pávek, P., Besra, G., and Vinšová, J. (2015) Novel derivatives of nitro-substituted salicylic acids: synthesis, antimicrobial activity and cytotoxicity. Bioorg. Med. Chem. 23, 7292−7301. (14) Chen, C.-L., Liu, F.-L., Lee, C.-C., Chen, T.-C., Ahmed Ali, A. A., Sytwu, H.-K., Chang, D.-M., and Huang, H.-S. (2014) Modified salicylanilide and 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-dione derivatives as novel inhibitors of osteoclast differentiation and bone resorption. J. Med. Chem. 57, 8072−8085. (15) Huang, D., Ou, B., and Prior, R. L. (2005) The chemistry behind antioxidant capacity assays. J. Agric. Food Chem. 53, 1841− 1856. (16) Cheng, Z., Moore, J., and Yu, L. (2006) High-throughput relative DPPH radical scavenging capacity assay. J. Agric. Food Chem. 54, 7429−7436. (17) Foti, M. C. (2015) Use and abuse of the DPPH• radical. J. Agric. Food Chem. 63, 8765−8776. (18) Amorati, R., and Valgimigli, L. (2015) Advantages and limitations of common testing methods for antioxidants. Free Radical Res. 49, 633−649. (19) Chen, Y., Xiao, H., Zheng, J., and Liang, G. (2015) Structurethermodynamics-antioxidant activity relationships of selected natural phenolic acids and derivatives: an experimental and theoretical evaluation. PLoS One 10 (3), e0121276. (20) Xie, J., and Schaich, K. M. (2014) Re-evaluation of the 2,2diphenyl-1-picrylhydrazyl free radical (DPPH) assay for antioxidant activity. J. Agric. Food Chem. 62, 4251−4260. (21) Ingold, K. U., and Pratt, D. A. (2014) Advances in radicaltrapping antioxidant chemistry in the 21st century: a kinetics and mechanisms perspective. Chem. Rev. 114, 9022−9046. (22) Liu, W.-Z., and Bordwell, F. G. (1996) Gas-phase and solutionphase homolytic bond dissociation energies of H−N+ bonds in the conjugate acids of nitrogen bases. J. Org. Chem. 61, 4778−4783. (23) Marteau, C., Nardello-Rataj, V., Favier, D., and Aubry, J. M. (2013) Dual role of phenols as fragrances and antioxidants: mechanism, kinetics and drastic solvent effect. Flavour Fragrance J. 28, 30−38. (24) Foti, M. C., Daquino, C., Mackie, I. D., DiLabio, G. A., and Ingold, K. U. (2008) Reaction of phenols with the 2,2-diphenyl-1picrylhydrazyl radical. Kinetics and DFT calculations applied to determine ArO-H bond dissociation enthalpies and reaction mechanism. J. Org. Chem. 73, 9270−9282. (25) Wright, J. S., Johnson, E. R., and DiLabio, G. A. (2001) Predicting the activity of phenolic antioxidants: theoretical method, analysis of substituent effects, and application to major families of antioxidants. J. Am. Chem. Soc. 123, 1173−1183. (26) Fox, T., and Kollman, P. A. (1996) Calculation of ionization potentials and C−H bond dissociation energies of toluene derivatives. J. Phys. Chem. 100, 2950−2956. (27) Wright, J. S., Carpenter, D. J., McKay, D. J., and Ingold, K. U. J. (1997) Theoretical calculation of substituent effects on the O−H bond strength of phenolic antioxidants related to vitamin E. J. Am. Chem. Soc. 119, 4245−4252. (28) DiLabio, G. A., Pratt, D. A., LoFaro, A. D., and Wright, J. S. (1999) Theoretical study of X−H bond energetics (X = C, N, O, S):

ORCID

Robert Vianello: 0000-0003-1779-4524 Author Contributions

N.P., P.R., I.S., and I.B. performed the synthesis, purification and spectroscopic characterization of prepared compounds. P.R. and K.S. conducted spectroscopic measurements in DPPH and FRAP assays. M.H. performed the SAR study. R.V. performed the computational analysis. K.S., M.H., and R.V. interpreted the results and wrote the manuscript. All authors have given approval to the final version of the manuscript. Funding

This work was supported by the Croatian Science Foundation under the projects IP-2014-09-3386 entitled “Design and synthesis of novel nitrogen-containing heterocyclic fluorophores and fluorescent nanomaterials for pH and metal-ion sensing”, IP-2013-11-5596 entitled “Synthesis and cytostatic evaluations of novel nitrogen heterocycles library”, and IP2016-06-3163 entitled “Dietary lipids, sex and age in pathogenesis of metabolic syndrome”. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS DPPH, 1,1-diphenyl-picrylhydrazyl; FRAP, ferric reducing/ antioxidant power; DNA, deoxyribonucleic acid; DFT, density functional theory; SMD, solvation model density; DMSO, dimethyl sulfoxide

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REFERENCES

(1) Tailor, N., and Sharma, M. (2013) Antioxidant hybrid compounds: a promising therapeutic intervention in oxidative stress induced diseases. Mini-Rev. Med. Chem. 13, 280−297. (2) Pavlin, M., Repič, M., Vianello, R., and Mavri, J. (2016) The chemistry of neurodegeneration: kinetic data and their implications. Mol. Neurobiol. 53, 3400−3415. (3) Nimse, S. B., and Pal, D. (2015) Free radicals, natural antioxidants, and their reaction mechanisms. RSC Adv. 5, 27986− 28006. (4) Augustyniak, A., Bartosz, G., Č ipak, A., Duburs, G., Horáková, Lj., Łuczaj, W., Majekova, M. A., Odysseos, D., Rackova, L., Skrzydlewska, E., Stefek, M., Š trosová, M., Tirzitis, G., Venskutonis, P. R., Viskupicova, J., Vraka, P. S., and Ž arković, N. (2010) Natural and synthetic antioxidants: an updated overview. Free Radical Res. 44, 1216−1262. (5) Caldarelli, A., Minazzi, P., Canonico, P. L., Genazzani, A. A., and Giovenzana, G. B. (2013) N-Arylbenzamides: extremely simple scaffolds for the development of novel estrogen receptor agonists. J. Enzyme Inhib. Med. Chem. 28, 148−152. (6) Brown, M. E., Fitzner, J. N., Stevens, T., Chin, W., Wright, C. D., and Boyce, J. P. (2008) Salicylanilides: selective inhibitors of interleukin-12p40 production. Bioorg. Med. Chem. 16, 8760−8764. (7) Liechti, C., Séquin, U., Bold, G., Furet, P., Meyer, T., and Traxler, P. (2004) Salicylanilides as inhibitors of the protein tyrosine kinase epidermal growth factor receptor. Eur. J. Med. Chem. 39, 11− 26. (8) Zhu, Z.-W., Shi, L., Ruan, X.-M., Yang, Y., Li, H.-Q., Xu, S.-P., and Zhu, H.-L. (2011) Synthesis and antiproliferative activities against Hep-G2 of salicylanide derivatives: potent inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase. J. Enzyme Inhib. Med. Chem. 26, 37−45. (9) Lauriane, N. T. R., Bouali, J., Najih, R., Khouili, M., Hafid, A., and Chtaini, A. (2014) Synthesis and electrochemical evaluation of 983


Article

Chemical Research in Toxicology application to substituent effects, gas phase acidities, and redox potentials. J. Phys. Chem. A 103, 1653−1661. (29) Bordwell, F. G., and Ji, G. Z. (1991) Effects of structural changes on acidities and homolytic bond dissociation energies of the hydrogen-nitrogen bonds in amidines, carboxamides, and thiocarboxamides. J. Am. Chem. Soc. 113, 8398−8401. (30) Lucarini, M., and Pedulli, G. F. (2010) Free radical intermediates in the inhibition of the autoxidation reaction. Chem. Soc. Rev. 39, 2106−2119. (31) Warren, J. J., Tronic, T. A., and Mayer, J. M. (2010) Thermochemistry of proton-coupled electron transfer reagents and its implications. Chem. Rev. 110, 6961−7001. (32) Guitard, R., Nardello-Rataj, V., and Aubry, J.-M. (2016) Theoretical and kinetic tools for selecting effective antioxidants: application to the protection of omega-3 oils with natural and synthetic phenols. Int. J. Mol. Sci. 17, 1220−1245. (33) Hansch, C., Leo, A., and Taft, R. W. (1991) A survey of Hammett substituent constants and resonance and field parameters. Chem. Rev. 91, 165−195. (34) Snelgrove, D. W., Lusztyk, J., Banks, J. T., Mulder, P., and Ingold, K. U. (2001) Kinetic solvent effects on hydrogen-atom abstractions: reliable, quantitative predictions via a single empirical equation. J. Am. Chem. Soc. 123, 469−477. (35) Litwinienko, G., and Ingold, K. U. (2007) Solvent effects on the rates and mechanisms of reaction of phenols with free radicals. Acc. Chem. Res. 40, 222−230. (36) Jonsson, M., Lind, J., Merenyi, G., and Eriksen, T. E. (1995) N−H bond dissociation energies, reduction potentials and pKas of multisubstituted anilines and aniline radical cations. J. Chem. Soc., Perkin Trans. 2 2, 61−65. (37) Benzie, I. F., and Strain, J. J. (1996) The ferric reducing ability of plasma (FRAP) as a measure of ″antioxidant power″: the FRAP assay. Anal. Biochem. 239, 70−76. (38) Lucarini, M., Mugnaini, V., and Pedulli, G. F. (2002) Bond dissociation enthalpies of polyphenols: the importance of cooperative effects. J. Org. Chem. 67, 928−931. (39) Eghbaliferiz, S., and Iranshahi, M. (2016) Prooxidant activity of polyphenols, flavonoids, anthocyanins and carotenoids: updated review of mechanisms and catalyzing metals. Phytother. Res. 30, 1379−1391. (40) Zheng, L. F., Dai, F., Zhou, B., Yang, L., and Liu, Z. L. (2008) Prooxidant activity of hydroxycinnamic acids on DNA damage in the presence of Cu(II) ions: mechanism and structure−activity relationship. Food Chem. Toxicol. 46, 149−156. (41) Rahal, A., Kumar, A., Singh, V., Yadav, B., Tiwari, R., Chakraborty, S., and Dhama, K. (2014) Oxidative stress, prooxidants, and antioxidants: the interplay. BioMed Res. Int. 2014, 761264. (42) Wang, D., Wang, Y., Wan, X., Yang, C. S., and Zhang, J. (2015) Green tea polyphenol (−)-epigallocatechin-3-gallate triggered hepatotoxicity in mice: responses of major antioxidant enzymes and the Nrf2 rescue pathway. Toxicol. Appl. Pharmacol. 283, 65−74. (43) Mulder, P., Korth, H. G., and Ingold, K. U. (2005) Why quantum thermochemical calculations must be used with caution to indicate ″a promising lead antioxidant″. Helv. Chim. Acta 88, 370− 374. (44) Tireli, M., Starčević, K., Martinović, T., Kraljević Pavelić, S., Karminski-Zamola, G., and Hranjec, M. (2017) Antioxidative and antiproliferative activities of novel pyrido[1,2-a]benzimidazoles. Mol. Diversity 21, 201−210. (45) Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., Cheeseman, J. R., Scalmani, G., Barone, V., Mennucci, B., Petersson, G. A., Nakatsuji, H., Caricato, M., Li, X., Hratchian, H. P., Izmaylov, A. F., Bloino, J., Zheng, G. J., Sonnenberg, L., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Vreven, T., Montgomery Jr, J. A., Peralta, J. E., Ogliaro, F., Bearpark, M., Heyd, J. J., Brothers, E., Kudin, K. N., Staroverov, V. N., Kobayashi, R., Normand, J., Raghavachari, K., Rendell, A., Burant, J. C., Iyengar, S. S., Tomasi, J., Cossi, M., Rega, N., Millam, J. M., Klene, M., Knox, J. E., Cross, J. B., Bakken, V.,

Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R. E., Yazyev, O., Austin, A. J., Cammi, R., Pomelli, C., Ochterski, J. W., Martin, R. L., Morokuma, K., Zakrzewski, V. G., Voth, G. A., Salvador, P., Dannenberg, J. J., Dapprich, S., Daniels, A. D., Farkas, Ö ., Foresman, J. B., Ortiz, J. V., Cioslowski, J., and Fox, D. J. Gaussian 09, Revision A.02; Gaussian Inc.: Wallingford, CT, 2009. (46) Leopoldini, M., Russo, N., and Toscano, M. (2011) The molecular basis of working mechanism of natural polyphenolic antioxidants. Food Chem. 125, 288−306. (47) Maršavelski, A., and Vianello, R. (2017) What a difference a methyl group makes: the selectivity of monoamine oxidase B towards histamine and N-methylhistamine. Chem. - Eur. J. 23, 2915−2925. (48) Saftić, D., Vianello, R., and Ž inić, B. (2015) 5-Triazolyluracils and their N1-sulfonyl derivatives: intriguing reactivity differences in the sulfonation of triazole N1′-substituted and N1′-unsubstituted uracil molecules. Eur. J. Org. Chem. 2015 (35), 7695−7704. (49) Gregorić, T., Sedić, M., Grbčić, P., Tomljenović Paravić, A., Kraljević Pavelić, S., Cetina, M., Vianello, R., and Raić-Malić, S. (2017) Novel pyrimidine-2,4-dione-1,2,3-triazole and furo[2,3-d]pyrimidine-2-one-1,2,3-triazole hybrids as potential anti-cancer agents: synthesis, computational and X-ray analysis and biological evaluation. Eur. J. Med. Chem. 125, 1247−1267.

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