Amino-Substituted Benzamide Derivatives as Promising Antioxidant Agents: A Combined Experimental and Computational Study

Article abstract of DOI:10.1021/acs.chemrestox.8b00175

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.

Full text of DOI:10.1021/acs.chemrestox.8b00175

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Article
Amino substituted benzamide derivatives as promising antioxidant
agents: A combined experimental and computational study
Nataša Perin, Petra Roškari#, Irena Sovi#, Ida Bo#ek,
Kristina Star#evi#, Marijana Hranjec, and Robert Vianello
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00175 • Publication Date (Web): 15 Aug 2018
Downloaded from http://pubs.acs.org on August 15, 2018
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Amino substituted benzamide derivatives as promising
antioxidant agents: A combined experimental and
computational study
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Nataša Perin,a Petra Roškarić,a Irena Sović,b Ida Boček,a Kristina Starčević,c Marijana Hranjec*a and
Robert Vianello*d
a Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb,
Marulićev trg 19, 10000 Zagreb, Croatia. E-mail: mhranjec@fkit.hr
b Laboratory for Green Chemistry, Division of Physical Chemistry, Ruđer Bošković Institute, Bijenička cesta
54, HR 10000 Zagreb, Croatia
c Department of Animal Husbandry, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55,
HR-10000 Zagreb, Croatia
d Computational Organic Chemistry and Biochemistry Group, Division of Organic Chemistry and
Biochemistry, Ruđer Bošković Institute, Bijenička cesta 54, HR 10000 Zagreb, Croatia. E-mail:
robert.vianello@irb.hr
Keywords: amines, benzamides, computational analysis, homolytic bond dissociation energy, antioxidant
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TABLE OF CONTENTS
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enhancing antioxidative activity
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R2
R3
R4
R1
O
NH3 Cl-
+
OH
R5
H
N
R6
DPPH (IC50) 12.93 ± 1.9 µM
+
FRAP (mmolFe2 /mmolc) 4856.15 ± 70.18
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ABSTRACT
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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 towards 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 favourable 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
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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 the 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 over-production causes oxidative stress leading to the lowered antioxidative defence.
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 a coronary heart disease or stroke. In the
last few decades, the search for the novel antioxidants led to the synthesis or isolation of different 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
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Recently, several publications described the antioxidative potential, as well as other biological activities, of
a broad range of salicylanilide and benzamide derivatives. Caldarelli and co-authors 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 the
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 binding modes of the most potent derivatives into this receptor active site.
Furthermore, the synthesis and antioxidative activity of various benzamide derivatives was recently
demonstrated using electrochemical methods.9 Substituted heterocyclic analogues of salicylanilides have
shown promising antimycobacterial activity, thus representing potential antituberculotics.10
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Additionally, substituted benzanilides can act as potassium channel activators with some derivatives
characterized as potent smooth muscle relaxants.11 Steffen and co-authors investigated the activity of
modified salicylanilides as cell permeable 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
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R1
R2
R3
OH
OH
R4
H
R1
H
N
N
R2
O
O
R5
R1 = H, Br
R2 = H, CH3
R3 = H, I, Br
R1 = I, Br, Cl, F, CF3, NO2, diCl
R2 = o-, m- or p-NO2
II
R4 = H, F, Cl, Br
R5 = H, F, Cl, Br, CH3, OCH3, CH(CH3)2
I
R3
R1
O
F
H
N
O
R2
R1 = H, OCH3
R2 = F, Cl, I
R3 = CH3, OCH3, fluorbenzyl
III
Figure 1. Previously prepared biologically active benzamide derivatives.
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
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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 Perkin-Elmer 2400 analyzer and a Perkin-Elmer, Series II, CHNS analyzer
2400. The 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 cooling, the solution was
concentrated or/and the obtained solid was filtered off and recrystallized from the appropriate solvent.
4-cyano-N-(4-nitrophenyl)benzamide 7
Following 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 recrystalization from ethanol to obtain 0.61 g (53.5%) of
light yellow powder; mp 95–98 °C.
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4-cyano-N-(4-methoxy-2-nitrophenyl)benzamide 8
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Following 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 recrystalization from acetone/MeOH (1:1) to obtain 0.51 g
(53.5%) of light yellow powder; mp 190–193 °C.
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2-methoxy-N-(4-nitrophenyl)benzamide 9
Following 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 recrystalization from MeOH to obtain 1.21 g (61.3%) of
white powder; mp 177–181 °C.
2,4-dimethoxy-N-(4-nitrophenyl)benzamide 10
Following 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 recrystalization 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
Following 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 recrystalization 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
Following 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 recrystalization 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
Following 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 recrystalization from MeOH to obtain 1.70 g (49.7%) of
orange powder; mp 200–204 °C.
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General method for the synthesis of amino substituted benzamides 14–20. Corresponding nitro
substituted benzamides 7–13 and solution of SnCl2 x 2H2O in MeOH and concentrated HCl were refluxed for
1–3 hours. After cooling, the reaction mixture was evaporated under vacuum and dissolved in water (50 ml).
The resulting solution was treated with 20% NaOH to pH = 14. Resulting product was filtered off and
washed with water to obtain amino substituted benzamides 14–20.
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N-(4-aminophenyl)-4-cyanobenzamide 14
Following general method, from 7 (0.30 g, 1.23 mmol), SnCl2×2H2O (2.10 g, 9.32 mmol) in concentratred
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
Following general method, from 8 (0.60 g, 2.02 mmol), SnCl2×2H2O (3.78 g, 16.76 mmol) in concentratred
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.
N-(4-aminophenyl)-2-methoxybenzamide 16
Following general method, from 10 (0.60 g, 2.21 mmol), SnCl2×2H2O (3.48 g, 15.42 mmol) in concentratred
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
Following 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
Following general method, from 11 (1.20 g, 3.61 mmol), SnCl2×2H2O (5.70 g, 25.26 mmol) in concentratred
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
Following general method, from 12 (0.40 g, 1.20 mmol), SnCl2×2H2O (2.25 g, 10.00 mmol) in concentratred
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.
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N-(2-amino-4-methoxyphenyl)-3,4,5-trimethoxybenzamide 20
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Following general method, from 13 (1.20 g, 3.31 mmol), SnCl2×2H2O (5.19 g, 23.00 mmol) in concentratred
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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.
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General method for the synthesis of hydroxy substituted benzamides 21–23. A solution of
corresponding methoxy substituted benzamides 16–20 in absolute dichlormethane (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. Obtained product was filtered of recrystallized from MeOH or water layer was extracted
with ethyl acetate and after drying with MgSO4, evaporated under vacuum to obtain powdered product.
N-(4-aminophenyl)-2-hydroxybenzamide 21
Following 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 grey powder; mp 157–161 °C.
N-(4-aminophenyl)-2-hydroxy-4-methoxybenzamide 22
Following 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-trihydroxy benzamide 23
Following 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
hours of stirring small amount of diethylether was added, resulting product was filtered off and washed with
diethylether to obtain hydrochlorides 24–26 and 27–33.
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N-(4-aminophenyl)-2-hydroxybenzamide hydrochloride 24
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Following 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.
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N-(4-aminophenyl)-2-hydroxy-4-methoxybenzamide hydrochloride 25
Following 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
Following general method, from 23 (0.10 g, 0.38 mmol) in absolute ethanol (5 ml) to obtain 0.04 g (68.1%)
of grey powder; mp 287–292 °C.
N-(4-aminophenyl)-4-cyanobenzamide hydrochloride 27
Following 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
Following general method, from 15 (0.05 g, 1.87 mmol) in absolute ethanol (5 ml) to obtain 0.03 g (51.1%)
of grey powder; mp 296–299 °C.
N-(4-aminophenyl)-2-methoxybenzamide hydrochloride 29
Following 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
Following 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
Following 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.
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N-(4-aminophenyl)-3,4,5-trimethoxybenzamide hydrochloride 32
Following general 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.
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N-(2-amino-4-methoxyphenyl)-3,4,5-trimethoxybenzamide hydrochloride 33
Following 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 the use in a 96-well microplate. Briefly, to solution of DPPH (final concentration 50 µM) in
absolute ethanol was added 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 6–31+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
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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/6–
311++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 modelling mechanisms of various
antioxidants,46 and in reproducing kinetic and thermodynamic parameters of a variety of organic and
enzymatic reactions.47–49
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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 transfers 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)
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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.
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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.
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 2–4 substituted 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 hydroxy substituted
benzamides with 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.
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R2
R2
R2
R5
R3
R4
R1
R3
R4
R1
O
R3
R4
R1
O
SnCl2 x 2H2O
MeOH/HCl
Et3N
R5
R5
+
H2N
R6
H
H
Toluene abs.
N
N
COCl
5 R5=H; R6=NO2
6 R5=NO2; R6=OCH3
1 R3=CN; R1=R2=R4=H
R6
R6
2 R1=OCH3; R2=R3=R4=H
3 R1=R3=OCH3; R2=R4=H
4 R2=R3=R4=OCH3; R2=H
7 R3=CN; R1=R2=R4=R5=H; R6=NO2
14 R3=CN; R1=R2=R4=R5=H; R6=NH2
8 R3=CN; R1=R2=R4=H; R5=NO2; R6=OCH3
9 R1=OCH3; R2=R3=R4=R5=H; R6=NO2
10 R1=R3=OCH3; R2=R4=R5=H; R6=NO2
11 R1=R3=OCH3; R2=R4=H; R5=NO2; R6=OCH3
12 R2=R3=R4=OCH3; R1=R5=H; R6=NO2
13 R2=R3=R4=OCH3; R1=H; R5=NO2; R6=OCH3
15 R3=CN; R1=R2=R4=H; R5=NH2; R6=OCH3
16 R1=OCH3; R2=R3=R4=R5=H; R6=NH2
17 R1=R3=OCH3; R2=R4=R5=H; R6=NH2
18 R1=R3=OCH3; R2=R4=H; R5=NH2; R6=OCH3
19 R2=R3=R4=OCH3; R1=R5=H; R6=NH2
20 R2=R3=R4=OCH3; R1=H; R5=NH2; R6=OCH3
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R2
R2
HCl(g)
EtOHabs.
R3
R4
R1
CH2Cl2abs.
BBr3
R3
R4
R1
O
R5
R5
H
H
N
N
HCl(g)
EtOHabs.
O
R6
R6
21 R1=OH; R2=R3=R4=R5=H; R6=NH2
22 R1=OH; R3=OCH3; R2=R4=R5=H; R6=NH2
23 R2=R3=R4=OH; R1=R5=H; R6=NH2
24 R1=OH; R2=R3=R4=R5=H; R6=NH3+Cl-
25 R1=OH; R3=OCH3; R2=R4=R5=H; R6=NH3+Cl-
26 R2=R3=R4=OH; R1=R5=H; R6=NH3+Cl-
R2
R3
R1
O
R5
H
N
R4
R6
27 R3=CN; R1=R2=R4=R5=H; R6=NH3+Cl-
28 R3=CN; R1=R2=R4=H; R5=NH3+Cl-; R6=OCH3
29 R1=OCH3; R2=R3=R4=R5=H; R6=NH3+Cl-
30 R1=R3=OCH3; R2=R4=R5=H; R6=NH3+Cl-
31 R1=R3=OCH3; R2=R4=H; R5=NH3+Cl-; R6=OCH3
32 R2=R3=R4=OCH3; R1=R5=H; R6=NH3+Cl-
33 R2=R3=R4=OCH3; R1=H; R5=NH3+Cl-; R6=OCH3
Scheme 1. Synthesis of benzamide derivatives investigated here.
Amino derivatives 14–23 were converted into the amino protonated 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 H and 13C NMR spectroscopies and elemental analysis.
1
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 1,1-diphenyl-picrylhydrazyl radical (DPPH) and
ferric reducing/antioxidant power (FRAP) parameters were evaluated.15 The former is based on the ability of
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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 radical scavenging 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 minutes, 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 nitro substituted derivatives 7–13 as well as 15 and 19
were insoluble in ethanol and were not tested. Based on 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).
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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 amino protonated group (IC50 = 10.81 ±
3.3 µM). Its analogue with the unionized 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 radical
scavenging activity over the corresponding unionized 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
=
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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.
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Table 1. IC50 values of investigated systems for DPPH free radical scavenging and FRAP activities.a
DPPH
FRAP
System
R1
R2
R3
R4
R5
R6
IC50 µM
mmolFe2+/mmolC
OH
OH
H
H
H
H
H
NH2
23.80 ± 0.3
24.0 ± 1.5
2283.67 ± 62.12
1530.69 ± 35.87
21
22
H
OH
H
OCH3
OH
H
OH
H
NH2
H
OH
OH
H
H
H
H
H
H
NH2
16.85 ± 0.1
22.25 ± 0.9
10.81 ± 3.3
12.93 ± 1.9
30.73 ± 8.1
18.12 ± 0.3
30.02 ± 4.1
18.4 ± 3.1
26.56 ± 1.8
30.45 ± 2.1
25 ± 4.2a
3259.93 ± 85.51
2328.80 ± 118.03
2174.40 ± 99.85
4856.15 ± 70.18
1763.47 ± 32.66
989.11 ± 85.02
2307.42 ± 63.87
1905.99 ± 87.57
1677.96 ± 93.20
1763.47 ± 219.52
2089.34 ± 55.98
23
24
+
H
NH3 Cl–
+
H
OCH3
OH
H
NH3 Cl–
25
+
OH
H
OH
H
NH3 Cl–
26
+
H
CN
NH3 Cl–
27
+
H
H
CN
H
NH3 Cl–
OCH3
28
+
OCH3
OCH3
H
H
H
H
H
H
H
NH3 Cl–
29
+
H
OCH3
OCH3
OCH3
H
NH3 Cl–
30
+
OCH3
OCH3
OCH3
OCH3
NH3 Cl–
32
+
H
NH3 Cl–
OCH3
33
BHT
a
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.
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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, thus the obtained results are 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 unionized analogue 23. Similarly, protonated 2-hydroxy-4-methoxy 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 twofold increase in the reducing power (1763.47 ± 32.66
mmolFe2+/mmolC). A comparison of the results displayed by amino protonated 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 2-hydroxy-4-methoxy- (25), dimethoxy- (30) and trimethoxy-substituted benzamide 32. Also, the
introduction of the additional methoxy group in 33 did not improve the reducing power.
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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
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only slightly improved in 25, yet 26 exhibits significantly larger potency than all systems examined here as
measured with FRAP. Additionally, as a general conclusion, it was observed that the amino protonated
derivatives showed much higher antioxidative capacity than their neutral analogues as a rule, in line with
previous reports in the literature.22
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Figure 2. The most promising antioxidant 26.
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.
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M1
M2 (R = H)
M5 (R = H)
M6 (R = CN)
M7 (R = OMe)
M8 (R = H)
M9 (R = CN)
M10 (R = OMe)
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21
22
M3 (R = CN)
M4 (R = OMe)
M11 (33)
M12 (26)
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26
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45
46
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48
49
50
51
52
53
54
55
56
57
58
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Figure 3. Schematic representation of molecules studied computationally. In the cationic forms, all systems
are most preferably protonated at the aniline amino moiety.
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 addition, Foti and co-workers demonstrated a
liner 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 later), molecules with lower BDE values
display better antioxidant properties through the hydrogen atom transfer mechanism, while lower IP values
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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 unionized 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
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+
+
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, neutral molecules are 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 1), 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
Scheme 2. Stabilization of the aniline radical cation through delocalization of the positive charge and the
unpaired electron spin density into the aromatic system.
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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 theory. The corresponding site of the
hydrogen atom abstraction is denoted next to a BDE value.
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16
17
18
19
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21
22
23
24
25
26
27
28
29
30
31
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33
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neutral molecules Mn
cationic protonated molecules MnH+
system
BDE (kcal
site of the X–H
IE (kcal
BDE (kcal
site of the X–H
IE (kcal
M1
M2
M3
M4
M5
M6
M7
M8
M9
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
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
amide N–H
amide N–H
C2-phenolic O–H
C2-phenolic O–H
C2-phenolic O–H
amide N–H
M10
M11
M12
C4-phenolic O–H
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 neighbouring –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 unionized systems are weaker antioxidants, being in full agreement with experiments here
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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.
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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 favourable 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 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 favourable than
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those for BHT, respectively. Nevertheless, this perspective sets up a distant goal in the design of more potent
antioxidants based on the bisphenylamide framework.
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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 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 favourable 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
with 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 H-abstraction occurs
on the protonated amino group, while the calculated BDE corresponding to the amide fragment is much
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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 favourable than in neutral M2 (aniline BDE = 78.6 kcal mol–1), which is found in
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excellent agreement with 14.5 kcal mol–1 reported by Liu and Bordwell for Ph–NH3 and Ph–NH2 in the gas-
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phase.22 Introduction of the electron-withdrawing 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 favourable 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 favourable 5-center interaction among vicinal moieties as in M12,21 lowers the
reactivity towards 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
unfavourable position of the ortho-amino 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
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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
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Systems M8–M10 represent a distinct group of molecules, since in neutrals the most favourable 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
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values between the most favourable 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 favouring 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
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ring in a favourable 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 four out of eleven examined cations. 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 centre forms hydrogen bondings with the neighbouring 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 radical scavenging 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 towards 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 favourable than
those obtained for 1,2,3-trihydroxybenzene and 3,4,5-trihydroxybenzamide for which the same
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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.
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Still, a certain care must be exerted in designing antioxidants based on polyhydroxy systems, since 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 while 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 favourable
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 towards utilizing the N-
arylbenzamide scaffold in this direction.
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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 scavenging activity DPPH assay showed that the 2-hydroxy-4-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.
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In general, systems having the protonated amino moiety showed significant improvement in the
antioxidative capacity relative to their unionized analogues. Also, the presence of the electron-donating
methoxy and hydroxy moieties yielded a considerable positive impact on the measured data. Trihydroxy
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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 towards more efficient
antioxidants. We demonstrated that its antioxidative features are more favourable 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
unionized neutral and ionized cationic forms, it clearly demonstrated that protonated systems are better
antioxidants than their neutral counterparts, and 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 towards 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 neighbouring hydroxy groups in 26H•+, the latter also
exerting a favourable electron-donating stabilization.
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ASSOCIATED CONTENT
Supporting Information
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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).
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AUTHOR INFORMATION
Corresponding Authors
*Email: mhranjec@fkit.hr (M.H.)
*Email: robert.vianello@irb.hr (R.V.)
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 IP-2016-06-3163 entitled "Dietary lipids, sex and age in pathogenesis of
metabolic syndrome".
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