Computational modeling, synthesis, and antioxidant potential of novel phenylcarbamoylbenzoic acid analogs in combating oxidative stress

Article abstract of DOI:10.1002/ardp.201200183

A new series of phenylcarbamoylbenzoic acid analogs were designed, synthesized, and evaluated for their in vitro antioxidant activity, aiming to find a lead for the treatment of oxidative stress. The target compounds were prepared by substituting the core

Full text of DOI:10.1002/ardp.201200183

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
1
Full Paper
Computational Modeling, Synthesis, and Antioxidant
Potential of Novel Phenylcarbamoylbenzoic Acid Analogs
in Combating Oxidative Stress
Waleed A. Bayoumi1,3, Magda A. Elsayed1,4, Hany N. Baraka2, and Laila Abou-zeid1
1 Faculty of Pharmacy, Department of Pharmaceutical Organic Chemistry, University of Mansoura,
Mansoura, Egypt
2 Faculty of Pharmacy, Department of Pharmacognosy, University of Mansoura, Mansoura, Egypt
3 Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Delta University for Science and
Technology, Mansoura, Gamassa, Egypt
4 Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Taibah University, Madinah, Saudi Arabia
A new series of phenylcarbamoylbenzoic acid analogs were designed, synthesized, and evaluated for
their in vitro antioxidant activity, aiming to find a lead for the treatment of oxidative stress. The target
compounds were prepared by substituting the core phenylcarbamoylbenzoic acid moiety with certain
functionalities via simple and efficient synthetic strategies. A molecular modeling study was
performed to evaluate the recognition of target compounds at the 3MNG binding pocket. The
docking data revealed that compounds 8c and 9a selectively bind to the crucial amino acid
residues in the active site of 3MNG. The in vitro antioxidant activity was determined by ABTS
antioxidant assay. Compounds 8c, 9a, and 9b showed high antioxidant activity and are thus
promising as potent antioxidant leads.
Keywords: Antioxidant activity / Molecular modeling / Phenylcarbamoylbenzoic acid / Synthesis
Received: May 1, 2012; Revised: July 4, 2012; Accepted: July 11, 2012
DOI 10.1002/ardp.201200183
Introduction
and redox signaling. They use a conserved residue, Cys47,
to reduce peroxide substrates. The peroxiredoxins (Prxs) have
a remarkably high catalytic efficiency, which makes them a
dominant player in cell-wide peroxide reduction [10, 11].
Due to their wide spectrum of reported biological activity
[12] and the facile and efficient synthesis methods, carba-
moylbenzoic acid derivatives were selected for this study. The
carbamoylbenzoic acid moiety was hybridized with different
pharmacophoric moieties of potential antioxidant activity,
for example, hydrazones [13], Schiff’s bases [14], semicarba-
zone [15], thiosemicarbazone [16], acid hydrazide [17], and
chalcone analogs [18–20]. In addition, cyclized heterocyclic
moieties were synthesized for comparison with their pre-
cursors: chalcone analogs, including cyano-pyridones,
amino-cyano-pyridines, and pyrazolines [21].
Growing evidence suggests that the free radical-reactive
nitrogen and oxygen species (RNOS) are involved in the
damage of biomolecules, contributing to the etiology of
several human diseases [1–3]. Antioxidants either of natural
or synthetic origin are molecules capable of neutralizing free
radicals by acting at different levels: prevention, intercep-
tion, and repair [4–6]. Hence antioxidants have significant
importance in human health. Thus, the demand for anti-
oxidants has stimulated the search for new compounds of
potential use in this field [7–9].
At the molecular level, the human antioxidant ‘‘per-
oxiredoxin enzyme family’’ is responsible for protection
The phenylcarbamoylbenzoic analogs were evaluated for
their potential in vitro antioxidant activity, which indicates
the presence of potentially active leads, namely, the 8c, 9a,
and 9b analogs. The computational modeling data indicated
that the highly effective analogs have special structural
Correspondence: Waleed A. Bayoumi, Faculty of Pharmacy, Department
of Pharmaceutical Organic Chemistry, University of Mansoura, Mansoura
35516, Egypt.
E-mail: waleedbayoumi@mans.edu.eg
Fax: þ2 050 2247496
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W. A. Bayoumi et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
features. This makes them promising to be considered as
novel antioxidant scavengers for use in the treatment of
oxidative stress-related diseases.
solvent and a catalytic amount of acetic acid was utilized. In
addition, reaction of benzoic acid hydrazide with 1 afforded
benzoyl hydrazone 4 in good yield (Scheme 1).
The chalcone analogs 5a–c were used as precursors for
several cyclization reactions to prepare different potential
heterocyclic moieties. The synthesis of the chalcone analogs
5a–c was achieved through Claisen–Schmidt condensation
reaction (crossed aldol condensation) between 1 and the
appropriate aromatic aldehyde. The reaction was base-
catalyzed by alcoholic KOH and the yields were found to
be reasonable (Scheme 1).
Results and discussion
Chemistry
The synthetic methods for the preparation of the target
compounds are illustrated in Schemes 1–3. The ketonic
derivative 1 [22] was selected as starting material. It was
prepared in good yield via a nucleophilic amide formation
reaction, in which 4-aminoacetophenone was acylated with
phthalic acid anhydride in the presence of ethyl acetate as
solvent. The hydrazine derivative 2a [6] and its phenyl analog
2b were prepared by Schiff’s base condensation reaction of 1
with hydrazine hydrate and phenyl hydrazine, respectively
(Scheme 1).
Construction of two pyridine ring moieties (Scheme 2),
namely cyano-pyridones 6a–c and amino-cyano-pyridines
7a–c, was achieved by two methods. The one-pot multicom-
ponent reaction [23, 24] (method 1) was performed by reflux-
ing a mixture of 1, the appropriate aromatic aldehyde,
ammonium acetate, and ethyl cyanoacetate for the cyano-
pyridinone ring derivatives 6a–c or malononitrile for the
amino-cyano-pyridine ring derivatives 7a–c. Moreover, 6a–
c, and 7a–c were synthesized through a conventional step-
wise reaction [25, 26] (method 2), starting from the inter-
mediate chalcone analogs 5a–c via condensation with
ammonium acetate and ethyl cyanoacetate for 6a–c or malo-
nonitrile for 7a–c. The mechanism of 6a–c formation was
thought to proceed under Michael condensation [27], in
Condensation of 1 with semicarbazide hydrochloride in
the presence of sodium acetate afforded the corresponding
semicarbazone derivative 3a, whereas condensation of 1 with
thiosemicarbazide afforded the corresponding thiosemi-
carbazone derivative 3b. In both cases, ethanol was used as
Scheme 1. Synthesis of compounds 2–5. Reagents and conditions:
(i) NH2NH2/PhNHNH2, C2H5OH, reflux, 5–6 h; (ii) NH2NHCONH2.HCl,
CH3COONa/NH2NHCSNH2, C2H5OH, CH3COOH, reflux, 5 h; (iii)
PhCONHNH2, CH3COOH, reflux, 8 h; (iv) ArCHO, ethanolic KOH,
stirring, rt, 6 h.
Scheme 2. Synthesis of compounds 6a–c and 7a–c. Reagents
and conditions: (i) ArCHO, CH3COONH4, C2H5OH, reflux, 6 h;
(ii) CH3COONH4, C2H5OH, reflux, 6 h; (iii) ArCHO, CH2(CN)2,
CH3COONH4, n-butanol, reflux, 8 h; (iv) CH2(CN)2, CH3COONH4,
n-butanol, reflux, 8 h.
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Phenylcarbamoylbenzoic Acid Analogs against Oxidative Stress
3
component reaction (method 1) were slightly higher than
those of the conventional method for 6a–c, whereas there
was no significant difference between both methods for 7a–c.
The pyrazoline derivatives 8a–c and phenylpyrazoline
derivatives 9a–c were prepared via cyclocondensation of
5a–c with hydrazine and phenylhydrazine, respectively
(Scheme 3).
All target compounds were characterized by physical ana-
lytical and spectral data and were found to be in agreement
with calculated and predicted values.
Molecular docking
Molecular modeling studies were performed in an attempt
to gain insights into the molecular structures of the active
compounds 8c, 9a, 9b, and the inactive compound 7a,
in comparison to the competitive antioxidant inhibitor,
1,2-dithiane-4,5-diol (DTT) [10].
The synthesized target compounds were comparatively
evaluated in terms of their binding mode to the human
antioxidant enzyme (3MNG) pocket (Fig. 1). The investigated
compounds were subjected to a molecular modeling study
to evaluate their recognition profile at the human 3MNG
binding pocket, in comparison to the competitive anti-
oxidant inhibitor DTT. At the 3MNG pocket, DTT formed
two bifurcated H-bonds with the key residues Gly46
and Cys47; the latter is an essential residue for binding
and biological activity. Besides, an additional H-bond was
formed with the catalytic residue Thr44 [11] (Fig. 1).
Molecular docking was performed for the proposed com-
pound 8c to evaluate its recognition profiles at the 3MNG
binding pocket. Analysis of the docking data revealed that
the hydroxyl group of the carboxylic moiety contributed a
stable H-bond with the key pocket residue Thr147. Besides,
Scheme 3. Synthesis of compounds 8a–c and 9a–c. Reagents and
conditions: (i) NH2NH2, C2H5OH, reflux, 3 h; (ii) PhNHNH2, C2H5OH,
reflux, 6 h.
which ethyl cyanoacetate readily condenses with the
a,b-unsaturated carbonyl compounds 5a–c, giving rise to
the corresponding 6a–c. The assignment of the structures
of 6a–c was based on the more predominant keto form [28],
whereas the synthesis of 7a–c was reported to proceed via
initial formation of arylidenemalononitrile [29] followed
by reaction with the a-hydrogen in the ketonic moiety.
It was observed that the reaction yields of the one-pot multi-
Figure 1. Putative hydrogen bonds between the competitive inhibitor ligand DTT and the conserved key triad residues, Thr44, Gly46, and
Cys47, in the active site of the human antioxidant enzyme 3MNG.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
the terminal methoxy oxygen atom formed an H-bond with
the catalytic residue Cys72 of the 3MNG pocket. Furthermore,
the N1 and N2 of the pyrazoline ring formed two H-bonds with
Thr44 and Ser48; the latter is a non-conserved key residue,
but plays a crucial role in the recognition of the tested
compounds (Fig. 2).
changes in the size and shape of the ligand-binding crevice,
accompanied by a high degree of RMS deviations (Fig. 4).
However, the cyano group and the terminal phenyl group
protruded out into the solvent cavity, away from the con-
served residues of this cavity, resulting in an entropy penalty
due to the created unfavorable grab detected by the RMS
deviations. The overall binding recognition was formed with
the main conserved residue: Thr44. The low binding affinity
eradicated the antioxidant activity.
The molecular docking study indicated that the amide
oxygen of compound 9a is professionally recognized by
the key amino acid Arg127, while the N2 of pyrazoline
showed proper binding to Phe43. Figure 3 shows that the
phenylpyrazole ring aligns at the narrow tubule cavity
enriched with the conserved residues, allowing proper hydro-
phobic interaction with Cys47 and Thr44 from the upside of
the cavity, and at the same time faces the three aromatic
residues Phe120, Phe43, and Trp84.
Antioxidant screening
All the newly synthesized compounds 1–9 were evaluated for
their potential in vitro antioxidant activity by qualitative
screening using L-ascorbic acid as a positive control according
to the ABTS antioxidant assay method [30, 31]. ABTS is the
abbreviation of 2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic
acid) diammonium salt. The ABTS test is a radical cation
Switching the pyrazoline ring by introducing the amino-
cyano-pyridine ring of compound 7a induced conformational
Figure 2. Binding mode for 8c docked and minimized in the 3MNG binding pocket, showing the conserved residue Thr44 and the non-
conserved residues Ser48, Cys72, and Thr147 involved in its recognition.
Figure 3. Binding mode for 9a docked and minimized in the 3MNG binding pocket, showing the hydrogen bonding interaction with the active
site residues, Phe43, Trp84, and Arg127. Lipophilic interaction stabilized with Cys47 and Thr44.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
Phenylcarbamoylbenzoic Acid Analogs against Oxidative Stress
5
Figure 4. Binding mode for 7a docked and minimized in the 3MNG binding pocket, showing the hydrogen bonding interaction with the active
site residue, Thr44.
decolorization test and is also a spectrophotometric method Conclusions
widely used for the assessment of the antioxidant activity of
various substances. It is applicable to both lipophilic and
hydrophilic compounds. The radical cation (ABTS.þ) was
generated by oxidation of ABTS with potassium persulfate.
The absorbance is bleached by antioxidants because of their
capacity to reduce the preformed radical. The % inhibition of
the absorbance was calculated to indicate the inhibition
of superoxide production and to measure the antioxidant
activity.
The molecular modeling study revealed that the carbamoyl-
benzoic acid ring system is an essential backbone for com-
petitive inhibition of the human antioxidant 3MNG as it
carries the recognition feature with the key amino acid
residues of the active tubule cavity: Cys47 and Thr44.
Replacement of the pyrazoline ring by the amino-cyano-
pyridine ring leads to a decrease in the degree of recognition
at the binding pocket as in the case of compound 7a.
The carbamoylbenzoic acid analogs represent a unique
class of organic compounds, characterized by efficient and
facile methods of preparation. Hybridization with other
pharmacophores results in alteration of their biological
activity. This class of compounds can serve as a scaffold for
the modulation of biological activity.
The results of the antioxidant screening (scavenger
activity) are listed in Table 1. It was found that compounds
8a–c, 9a, and 9b, in which the carbamoylbenzoic acid core is
hybridized with monoaryl-substituted pyrazoline or diaryl-
substituted pyrazoline moieties, showed the highest anti-
oxidant activity, comparable to the standard reference
ascorbic acid. The antioxidant activities of the rest of the
compounds ranged from mild to low activity.
Compounds 8a–c, 9a, and 9b, in which the carbamoyl-
benzoic acid core is hybridized with monoaryl-substituted
Table 1. Results of the ABTS antioxidant assay.
Tested
compound
Absorbance
of sample
(mean)
% Inhibition of
superoxide
production
Tested
compound
Absorbance of
sample (mean)
% Inhibition of
superoxide
production
Ascorbic acid
1
0.051
0.473
0.419
0.231
0.433
0.114
0.266
0.335
0.290
0.466
0.459
90.04%
7.60%
6b
6c
7a
7b
7c
8a
8b
8c
9a
9b
9c
0.436
0.469
0.504
0.472
0.239
0.063
0.091
0.054
0.051
0.053
0.335
14.82%
8.39%
1.65%
2a
2b
3a
3b
4
5a
5b
5c
6a
18.14%
54.92%
15.41%
77.65%
48.09%
34.53%
43.41%
8.97%
7.90%
53.26%
87.70%
82.14%
89.46%
90.04%
89.65%
34.53%
10.43%
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W. A. Bayoumi et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
65%. Analysis for C17H16N4O3S (356.40), Calcd.: C, 57.29; H, 4.52;
1
N, 15.72. Found: C, 57.461; H, 4.31; N, 15.33. H NMR (DMSO-d6):
pyrazoline or diaryl-substituted pyrazoline moieties,
showed the highest antioxidant activity, comparable to the
standard reference ascorbic acid. The antioxidant activities of
the remaining compounds ranged from mild to low activity.
d 2.49 (s, 3H, CH3), d 6.75 (s, 2H, NH2, D2O exchangeable), d 7.55–
–
8.20 (m, 8H, Ar–H), d 9.15 (s, 1H, N–NH, D O exchangeable),
–
d 10.19 (s, 1H, CONH, D2O exchangeable), d 11.00 (s, 1H, COOH,
2
D2O exchangeable). MS m/z (%): 356 (5.45).
Experimental
2-((4-(1-(2-Benzoylhydrazono)ethyl)phenyl)carbamoyl)-
benzoic acid (4)
Chemistry
A mixture of 1 (2.83 g, 10 mmol) and benzoic acid hydrazide
(1.36 g, 10 mmol) in glacial acetic acid (20 mL) was heated under
reflux for 8 h, allowed to cool to room temperature and then
poured over ice-water. The separated solid was collected by filtra-
tion, dried, and recrystallized from glacial acetic acid to give the
pure products. White crystals, mp 187–1898C, yield 78%. Analysis
for C23H19N3O4 (401.41), Calcd.: C, 68.82; H, 4.77; N, 10.47. Found: C,
69.01; H, 4.55; N, 10.27. 1H NMR (DMSO-d6): d 2.53 (s, 3H, CH3), d 7.45–
Melting points were determined on a Fisher–Johns melting point
apparatus and were uncorrected. 1H NMR spectra were recorded
on a Bruker Ac 250 FT-NMR spectrometer (250 MHz) in DMSO-d6
using TMS as internal standard; chemical shifts in ppm were
expressed in d units. MS analyses were performed on a JEOL JMS-
600H spectrometer. Reaction times and purity of compounds
were determined and checked using TLC on silica gel plates 60
F245 from E. Merck, and the spots were visualized by UV light
(366, 245 nm). Compounds 1 [22] and 2a [6] were prepared
according to the reported procedures.
–
8.23 (m, 13H, Ar–H), d 9.96 (s, 1H, N–NH, D O exchangeable),
–
d 10.81 (s, 1H, CONH, D2O exchangeable), d 11.00 (s, 1H, COOH,
2
D2O exchangeable). MS m/z (%): 400 (3.69).
2-((4-(1-(2-Phenylhydrazono)ethyl)phenyl)carbamoyl)-
benzoic acid (2b)
2-(4-(Un)substituted cinnamoylphenylcarbamoyl)benzoic
acid (5a–c)
A mixture of 1 (2.83 g, 10 mmol) and phenyl hydrazine (2.16 g,
20 mmol) in absolute ethanol (30 mL) was stirred under reflux for
6 h. The product formed after cooling was collected by filtration,
dried, and recrystallized from isopropyl alcohol to afford the pure
product. Yellow crystals, mp 195–1978C, yield 73%. Analysis
for C22H19N3O3 (373.40), Calcd.: C, 70.76; H, 5.13; N, 11.25.
Found: C, 70.51; H, 5.05; N, 11.37. 1H NMR (DMSO-d6): d 2.37
A mixture of 1 (2.83 g, 10 mmol) and the appropriate aldehyde
(10 mmol) in 5% ethanolic sodium hydroxide (40 mL) was
stirred at room temperature for 5 h. The reaction mixture was
poured onto ice-cold water and neutralized with hydrochloric
acid solution (10%). The formed precipitate was filtered, dried,
and recrystallized from the proper solvent to give the pure
products.
–
(s, 3H, CH3), d 7.12–8.20 (m, 13H, Ar–H), d 9.15 (s, 1H, N–NH,
–
D2O exchangeable), d 10.02 (s, 1H, CONH, D2O exchangeable),
d 11.00 (s, 1H, COOH, D2O exchangeable). MS m/z (%): 373 (15.35).
2-(4-Cinnamoylphenylcarbamoyl)benzoic acid (5a)
Yellow crystals (ethanol), mp 140–1428C, yield 68%. Analysis
for C23H17NO4 (371.39), Calcd.: C, 74.38; H, 4.61; N, 3.77. Found:
C, 74.40; H, 4.65; N, 3.79. 1H NMR (DMSO-d6): d (7.22–8.20 (m, 15H,
Ar–H and vinyl-H), d 9.85 (s, 1H, CONH, D2O exchangeable), 11.31
(s, 1H, COOH, D2O exchangeable). MS m/z (%): 371 (9.2).
2-((4-(1-(2-Carbamoylhydrazono)ethyl)phenyl)carbamoyl)-
benzoic acid (3a)
A mixture of 1 (2.83 g, 10 mmol), semicarbazide hydrochloride
(1.11 g, 10 mmol), and anhydrous sodium acetate (0.82 g,
10 mmol) in absolute ethanol (20 mL) was heated under reflux
for 5 h in the presence of a catalytic amount of glacial acetic acid
(1 mL). The reaction mixture was concentrated to half its volume
and allowed to cool to room temperature. The formed crystalline
precipitate was collected by filtration, dried, and recrystallized
from ethyl acetate to afford a crystalline product. White crystals,
mp 251–2538C, yield 60%. Analysis for C17H16N4O4 (340.33), Calcd.:
C, 59.99; H, 4.74; N, 16.46. Found: C, 60.21; H, 4.51; N, 16.59. 1H NMR
(DMSO-d6): d 2.32 (s, 3H, CH3), d 6.64 (s, 2H, NH2, D2O exchangeable),
2-(4-(3-(4-Chlorophenyl)acryloyl)phenylcarbamoyl)-
benzoic acid (5b)
Yellow crystals (ethanol), mp 170–1728C, yield 73%. Analysis
for C23H16ClNO4 (405.83), Calcd.: C, 68.07; H, 3.97; N, 3.45.
1
Found: C, 68.09; H, 3.10; N, 3.49. H NMR (DMSO-d6): d 7.30–8.20
(m, 14H, Ar–H and vinyl-H), d 9.84 (s, 1H, CONH, D2O exchangeable),
11.20 (s, 1H, COOH, D2O exchangeable). MS m/z (%): 405 (100).
–
d 7.56–8.26 (m, 8H, Ar–H), d 9.15 (s, 1H, N–NH, D O exchangeable),
2-(4-(3-(4-Methoxyphenyl)acryloyl)phenylcarbamoyl)-
benzoic acid (5c)
Yellow crystals (methanol), mp 184–1858C, yield 65%. Analysis
for C24H19NO5 (401.41), Calcd.: C, 71.81; H, 4.77; N, 3.49. Found: C,
71.86; H, 4.79; N, 3.51. 1H NMR (DMSO): d 3.79 (s, 3H, OCH3), d 6.90–8.22
(m, 14H, Ar–H and vinyl-H), d 9.85 (s, 1H, CONH, D2O exchangeable),
11.31 (s, 1H, COOH, D2O exchangeable). MS m/z (%): 401 (100).
–
d 10.17 (s, 1H, CONH, D2O exchangeable), d 11.30 (s, 1H, COOH,
2
D2O exchangeable). MS m/z (%): 340 (17.62).
2-((4-(1-(2-Carbamothioylhydrazono)ethyl)phenyl)-
carbamoyl)benzoic acid (3b)
A mixture of 1 (2.83 g, 10 mmol) and thiosemicarbazide (0.91 g,
10 mmol) in a mixture of absolute ethanol (20 mL) and glacial
acetic acid (1 mL) was heated under reflux for 5 h. The reaction
mixture was concentrated to half of its volume and allowed to cool
to room temperature. The formed crystalline precipitate was col-
lected by filtration, dried, and recrystallized from ethyl acetate to
afford a crystalline product. Yellow crystals, mp 211–2138C, yield
2-((4-(5-Cyano-6-oxo-4-(un)substituted phenyl-1,6-
dihydropyridin-2-yl)phenyl)carbamoyl)benzoic acid (6a–c)
Method 1: A mixture of 1 (2.83 g, 10 mmol), ethyl cyanoacetate
(1.13 g, 10 mmol), the appropriate aldehyde (10 mmol), and
ammonium acetate (6.16 g, 80 mmol) in absolute ethanol
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
Phenylcarbamoylbenzoic Acid Analogs against Oxidative Stress
7
(50 mL) was refluxed for 6 h. The reaction mixture was allowed
to cool to room temperature; the separated solid was collected by
filtration, washed several times with warm ethanol, dried, and
recrystallized from ethanol/DMF (70:30) to afford the pure
products.
Calcd.: C, 71.88; H, 4.18; N, 12.90. Found: C, 71.90; H, 4.22; N, 12.95.
1H NMR (DMSO-d6): d 6.23 (s, 2H, NH2, D2O exchangeable), d 7.40–
8.22 (m, 14H, Ar–H), d 9.88 (s, 1H, CONH, D2O exchangeable), 10.91
(s, 1H, COOHNH, D2O exchangeable), MS m/z (%): 434 (48.88).
Method 2: A mixture of the appropriate 5a–c (10 mmol), ethyl
cyanoacetate (1.13 g, 10 mmol), and ammonium acetate (6.16 g,
80 mmol) in absolute ethanol (50 mL) was refluxed for 6 h. The
reaction mixture was allowed to cool to room temperature. The
separated solid was then worked up as in method 1.
2-(4-(6-Amino-4-(4-chlorophenyl)-5-cyanopyridin-2-yl)-
phenylcarbamoyl)benzoic acid (7b)
Yellow crystals (n-butanol), mp 210–2128C, yield 63% (method 1),
59% (method 2). Analysis for C26H17ClN4O3 (468.89), Calcd.: C,
66.60; H, 3.65; N, 11.95. Found: C, 66.64; H, 3.67; N, 11.94. 1H NMR
(DMSO-d6): d 6.50 (s, 2H, NH2, D2O exchangeable), d 7.50–8.20
(m, 13H, Ar–H), d 9.85 (s, 1H, CONH, D2O exchangeable), 11.10
(s, 1H, COOHNH, D2O exchangeable), MS m/z (%): 468 (13.89).
2-((4-(5-Cyano-6-oxo-4-phenyl-1,6-dihydropyridin-2-yl)-
phenyl)carbamoyl)benzoic acid (6a)
Orange crystals, mp 256–2588C, yield 63% (method 1), 55% (method
2). Analysis for C26H17N3O4 (435.43), Calcd.: C, 71.72; H, 3.94; N, 9.65.
Found: C, 71.87; H, 3.68; N, 9.91. 1H NMR (DMSO-d6): d 6.51–8.19
(m, 14H, Ar–H), d 9.91 (s, 1H, CONH, D2O exchangeable), d 11.00
(s, 1H, COOH, D2O exchangeable), NH of the pyridine ring seemed to
be exchanged by the solvent. MS m/z (%): 435 (12.34).
2-(4-(6-Amino-5-cyano-4-(4-methoxyphenyl)pyridin-2-yl)-
phenylcarbamoyl)benzoic acid (7c)
Yellowish brown crystals (ethanol), mp 137–1398C, yield 60%
(method 1), 57% (method 2). Analysis for C27H20ClN4O4 (464.47),
Calcd.: C, 69.82; H, 4.34; N, 12.06. Found: C, 69.85; H, 4.36;
N, 12.09. 1H NMR (DMSO-d6): d 3.80 (s, 3H, CH3), d 6.32 (s, 2H,
NH2, D2O exchangeable), d 7.40–8.22 (m, 14H, Ar–H), d 9.88
(s, 1H, CONH, D2O exchangeable), 10.91 (s, 1H, COOHNH,
D2O exchangeable), MS m/z (%): 464 (24.67).
2-((4-(4-(4-Chlorophenyl)-5-cyano-6-oxo-1,6-
dihydropyridin-2-yl)phenyl)carbamoyl)benzoic acid (6b)
Orange crystals, mp 273–2758C, yield 68% (method 1), 60%
(method 2). Analysis for C26H16ClN3O4 (469.88), Calcd.: C, 66.46;
H, 3.43; N, 8.94. Found: C, 66.70; H, 3.54; N, 9.19. 1H NMR (DMSO-d6):
d 6.50–8.22 (m, 13H, Ar–H), d 9.90 (s, 1H, CONH, D2O exchangeable),
d 11.20 (s, 1H, COOH, D2O exchangeable), NH of the pyridine ring
seemed to be exchanged by the solvent. MS m/z (%): 469 (17.33).
2-(4-((Un)substituted)-4,5-dihydro-1H-pyrazol-3-yl)-
phenylcarbamoyl)benzoic acid (8a–c and 9a–c)
A mixture of 5a–c (3 mmol) and hydrazine hydrate 98% (0.32 g,
3 mmol) or phenyl hydrazine (0.14 g, 3 mmol) in absolute etha-
nol (30 mL) was refluxed for 3–6 h. Upon cooling, the obtained
product was filtered off and recrystallized from the proper sol-
vent to obtain the title products.
2-((4-(5-Cyano-4-(4-methoxyphenyl)-6-oxo-1,6-
dihydropyridin-2-yl)phenyl)carbamoyl)benzoic acid (6c)
Orange crystals, mp 213–2158C, yield 65% (method 1), 57% (method
2). Analysis for C27H19N3O5 (465.46), Calcd.: C, 69.67; H, 4.11; N, 9.03.
Found: C, 69.78; H, 4.24; N, 9.15. 1H NMR (DMSO-d6): d 3.80 (s, 3H,
2-(4-(5-Phenyl-4,5-dihydro-1H-pyrazol-3-yl)-
phenylcarbamoyl)benzoic acid (8a)
OCH3), d 7.51–8.18 (m, 13H, Ar–H),
d 9.90 (s, 1H, CONH,
White crystals (ethanol), mp 245–2478C, yield 60%. Analysis
for C23H19N3O3 (385.42), Calcd.: C, 71.67; H, 4.96; N, 10.90.
Found: C, 71.69; H, 4.99; N, 10.94. 1H NMR (DMSO-d6): d 2.64–
2.83 (m, 2H, pyraz-CH2), d 4.91 (t, 1H, pyraz-CH), d 6.35 (s, 1H,
NH of pyraz.), d 7.23–8.14 (m, 13H, Ar–H), d 9.90 (s, 1H, CONH,
D2O exchangeable with), d 11.10 (s, 1H, COOH, D2O exchangeable).
MS m/z (%): 385 (28.9).
D2O exchangeable), d 11.00 (s, 1H, COOH, D2O exchangeable),
NH of the pyridine ring seemed to be exchanged by the solvent.
MS m/z (%): 465 (11.89).
2-(4-(6-Amino-5-cyano-4-(un)substituted phenylpyridin-2-yl)-
phenylcarbamoyl)benzoic acid (7a–c)
Method 1: A mixture of 5a–c (10 mmol), malononitrile (0.66 g,
10 mmol), and ammonium acetate (8.16 g, 80 mmol) in n-buta-
nol (30 mL) was heated under reflux for 8 h. The reaction mix-
ture was concentrated in vacuo. On cooling, the precipitated solid
was filtered, dried, and recrystallized from the proper solvent to
obtain the desired products.
Method 2: A mixture of 1 (2.83 g, 10 mmol), malononitrile
(0.66 g, 10 mmol), the appropriate aldehyde (10 mmol), and
ammonium acetate (8.16 g, 80 mmol) in n-butanol (50 mL)
was heated under reflux for 6 h. The solid formed upon concen-
trating and cooling was filtered off, washed with water, and
recrystallized from the appropriate solvent to obtain the desired
products.
2-(4-(5-(4-Chlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)-
phenylcarbamoyl)benzoic acid (8b)
White crystals (methanol), mp 258–2608C, yield 65%. Analysis
for C23H18ClN3O3 (419.86), Calcd.: C, 65.79; H, 4.32; N, 10.01.
Found: C, 65.81; H, 4.35; N, 10.05. 1H NMR (DMSO-d6): d 2.62–
2.83 (m, 2H, pyraz-CH2), d 4.87 (t, 1H, pyraz-CH), d 6.35 (s, 1H,
NH of pyraz.), d 7.24–8.27 (m, 12H, Ar–H), d 10.11 (s, 1H, CONH,
D2O exchangeable with), d 11.10 (s, 1H, COOH, D2O exchangeable).
MS m/z (%): 419 (45.23).
2-(4-(5-(4-Methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)-
phenylcarbamoyl)benzoic acid (8c)
White crystals (ethanol), mp 275–2778C, yield 62%. Analysis
for C24H21N3O4 (415.44), Calcd.: C, 69.39; H, 5.10; N, 10.11.
Found: C, 69.44; H, 5.13; N, 10.19. 1H NMR (DMSO-d6): d 2.50–
2.68 (m, 2H, pyraz-CH2), d 4.51 (t, 1H, pyraz-CH), d 6.12 (s, 1H,
2-(4-(6-Amino-5-cyano-4-phenylpyridin-2-yl)-
phenylcarbamoyl)benzoic acid (7a)
Yellowish white crystals (n-butanol), mp 238–2408C, yield 52%
(method 1), 53% (method 2). Analysis for C26H18N4O3 (434.45),
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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8
W. A. Bayoumi et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
˚
cut-off 8 A and the Cartesian minimization RMS gradient
NH of pyraz.), d 6.99–8.31 (m, 12H, Ar–H), d 10.89 (s, 1H, CONH,
D2O exchangeable with), d 11.10 (s, 1H, COOH, D2O exchangeable).
MS m/z (%): 415 (36.6).
0.001 kcal molꢀ1
ations where the root mean square (RMS) tolerance was fixed to
ꢀ1. The iteration limit was set to 1000 iter-
˚
A
0.1 kcal molꢀ1 ꢀ1. The conformers with the lowest energy were
A
˚
selected as the global minimal for further modeling studies.
2-(4-(1,5-Diphenyl-4,5-dihydro-1H-pyrazol-3-yl)-
phenylcarbamoyl)benzoic acid (9a)
Docking
Yellowish white crystals (ethyl acetate), mp 188–1908C, yield 58%.
Analysis for C29H23N3O3 (461.51), Calcd.: C, 75.47; H, 5.02; N, 9.10.
All the novel benzoic acid analogs were docked against the lead
competitive inhibitor ligand DTT at the crystal enzyme structure
of the human antioxidant enzyme ‘‘3MNG’’.
1
Found: C, 75.43; H, 5.05; N, 9.14. H NMR (DMSO-d6): d 2.51–2.79
(m, 2H, pyraz-CH2), d 4.45 (t, 1H, pyraz-CH), d 7.08–8.15 (m, 18H,
Ar–H), d 10.15 (s, 1H, CONH, D2O exchangeable with), d 11.10 (s, 1H,
COOH, D2O exchangeable). MS m/z (%): 461 (65.6%).
ABTS antioxidant assay
Preparation of reagents
2-(4-(5-(4-Chlorophenyl)-1-phenyl-4,5-dihydro-1H-
pyrazol-3-yl)phenylcarbamoyl)benzoic acid (9b)
ABTS solution was prepared as 0.1 g/100 mL. MnO2 solution
(25 mg/mL) was used instead of potassium persulfate. All
reagents were prepared in phosphate buffer (pH 7, 0.1 M). The
two reagents ABTS/MnO2 (2:3) were mixed and centrifuged. The
supernatant was obtained as green-blue solution (ABTS.þ radical
solution). This color remained stable for more than 1 h. The
absorbance was adjusted at about 0.2 at 734 nm. L–Ascorbic acid
solution was prepared as 2% solution: 1 g/50 mL distilled water.
Each test sample was used at a concentration of 0.01 mg/mL in
methanol/phosphate buffer (1:1).
Yellowish white crystals (ethyl acetate), mp 218–2208C, yield 70%.
Analysis for C29H22ClN3O3 (495.96), Calcd.: C, 70.23; H, 4.47; N, 8.47.
1
Found: C, 70.26; H, 4.50; N, 8.49. H NMR (DMSO-d6): d 2.70–2.92
(m, 2H, pyraz-CH2), d 4.92 (t, 1H, pyraz-CH), d 7.12–8.23 (m, 17H,
Ar–H), d 9.95 (s, 1H, CONH, D2O exchangeable with), d 11.10 (s, 1H,
COOH, D2O exchangeable). MS m/z (%): 495 (27.85).
2-(4-(5-(4-Methoxyphenyl)-1-phenyl-4,5-dihydro-1H-
pyrazol-3-yl)phenylcarbamoyl)benzoic acid (9c)
Starting assay
Yellowish white crystals (methanol), mp 212–2148C, yield 63%.
Analysis for C30H25N3O4 (491.54), Calcd.: C, 73.30; H, 5.13; N, 8.55.
Found: C, 73.26; H, 5.14; N, 8.49. 1H NMR (DMSO-d6): d 2.67–2.89
(m, 2H, pyraz-CH2), d 3.79 (s, 3H, OCH3), d 4.81 (t, 1H, pyraz-CH),
d 6.91–8.20 (m, 17H, Ar–H), d 9.27 (s, 1H, CONH, D2O exchangeable
with), d 11.10 (s, 1H, COOH, D2O exchangeable). MS m/z (%): 491 (29.3).
Of ABTS/MnO2, 900 mL was added to a spectrophotometer cuvette
(SPEKOL 11), and the absorbance was measured at 734 nm
against a blank made of methanol/phosphate buffer (1:1). Of
the ABTS/MnO2 mixture, 900 mL was added to 100 mL standard
L-ascorbic acid, and the absorbance was measured against the
blank: methanol/phosphate buffer (1:1) þ 100 mL of L-ascorbic
acid. Nine hundred microliter of the ABTS/MnO2 mixture was
added to 100 mL of sample, and the absorbance was measured
against the blank: methanol/phosphate buffer (1:1) þ 100 mL of
sample. The % inhibition of absorbance for each of the tested
compounds was calculated from the equation: % Inhibition ¼
Molecular modeling and docking methods
The molecular modeling study was conducted with the
Hyperchem 6.03 package from Hypercube and the Molegro
Molecular Viewer programs running on a personal computer
[32, 33], to illustrate the ligand receptor interaction and define
their degree of complementarity.
(Abscontrol ꢀ Abstest)/Abscontrol
.
The authors have declared no conflict of interest.
Enzyme structure
The starting coordinate of the human antioxidant enzyme in
complex with the competitive inhibitor DTT (code ID 3MNG) was
obtained from the Protein Data Bank of Brookhaven National
Laboratory (Fig. 1).
References
[1] H. Adibi, A. Rashidi, M. M. Khodaei, A. Alizadeh, M. B.
Majnooni, N. Pakravan, R. Abiri, D. Nematollahi, Chem.
Pharm. Bull. 2011, 59, 1149–1152.
Molecular structures of the synthesized benzoic acid
analogs
The benzoic acid analogs were constructed from fragment libraries
in the Hyperchem program, followed by energy minimization
using the ‘‘Amber force field’’. The partial atomic charges for each
analog were assigned with the semi-empirical mechanical calcu-
lation method ‘‘AM1’’ implemented in Hyperchem 6.03.
[2] R. Bai, F. Ma, D. Liang, X. Zhao, J. Chem. Ecol. 2009, 35, 488–494.
[3] S. M. Salga, H. M. Ali, M. A. Abdullah, S. I. Abdelwahab, L. K.
Wai, M. J. Buckle, S. D. Sukumaran, A. H. Hadi, Molecules
2011, 7, 9316–9330.
[4] K. M. Khan, M. Ali, A. Wadood, Zaheer-ul-Haq, M. Khan,
M. A. Lodhi, S. Perveen, M. I. Choudhary, W. Voelter, J. Mol.
Graphics Modell. 2011, 30, 153–156.
[5] M. K. Lehtinen, A. Bonni, Curr. Mol. Med. 2006, 6, 871–881.
Conformational search and the derived structural
global minima
The partially charged and energy-minimized structures of the
tested compounds were subjected to conformational search
using the conformational search module implemented in
MOE. Conformational search was performed using the energy
[6] W. A. Bayoumi, M. A. Elsayed, Med. Chem. Res. 2012, 21, 1633–
1640.
[7] I. Mitra, A. Saha, K. Roy, Sci. Pharm. 2011, 79, 31–57.
[8] I. Mitra, A. Saha, A. K. Roy, Chem. Biol. Drug Des. 2009, 73, 526–
536.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
Phenylcarbamoylbenzoic Acid Analogs against Oxidative Stress
9
[9] K. V. Balakin, Y. A. Ivanenkov, A. V. Skorenko, Y. V. Nikolsky,
N. P. Savchuk, A. A. Ivashchenko, J. Biomol. Screening 2004, 9,
22–31.
[21] D. M. Martin, B. G. Torres, P. R. Spohr, P. Machado, H. G.
Bonacorso, N. Zanatta, M. A. Martins, T. Emanuelli, Basic
Clin. Pharmacol. Toxicol. 2009, 104, 107–112.
[10] A. Hall, D. Parsonage, L. B. Poole, P. A. Karplus, J. Mol. Biol.
2010, 10, 194–209.
[22] A. V. Dolzhenko, V. O. Koz’minykh, N. V. Kolotova, N. Z.
Burdi, V. P. Kotegov, G. N. Novoselova, B. Y. Syropyatov,
Pharm. Chem. J. 2003, 37, 342–344.
[11] J. P. Declercq, C. Evrard, A. Clippe, D. V. Stricht, A. Bernard,
B. Knoops, J. Mol. Biol. 2001, 311, 751–759.
[23] M. A. Moustafa, H. M. Eisa, A. A. El-Emam, M. M. El-Kerdawy,
J. Pharm. Belg. 1987, 42, 38–43.
[12] V. O. Koz’minykh, Pharm. Chem. J. 2006, 40, 8–17.
[24] N. Latif, N. Mishriky, N. Grigis, Ind. J. Chem. 1981, 20, 147–
149.
[13] Q. M. Angelo, K. B. George, S. G. Cezar, G. O. Ricardo,
A. Antonio, H.-L. Marcelo, Biochim. Biophys. Acta 2003, 1620,
15–24.
[25] J. B. Bardham, J. Chem. Soc. 1929, 2223–2224.
[14] A. Chen, T. Taguchi, S. Aoyama, M. Sugiura, M. Haruna,
M. Wang, I. Miwa, Free Radical Biol. Med. 2003, 35, 1392–1403.
[26] S. Kambe, K. Saito, A. Sakurai, H. Midorikawa, Synthesis 1980,
366–367.
[27] C. Barat, Ind. J. Chem. Soc. 1930, 7, 321–324.
[15] S. Manmohan, P. Arindam, P. S. Hemendra, K. D. Sushil, K. S.
Rajendra, Int. J. Pharm. Sci. Drug Res. 2011, 3, 150–154.
[28] A. R. Katritzky, R. A. Jones, J. Chem. Soc. 1960, 2947–2953.
[16] S. Ghosh, A. K. Misra, G. Bhatia, M. M. Khan, A. K. Khanna,
Bioorg. Med. Chem. Lett. 2009, 19, 386–389.
[29] K. M. Ghoneim, M. M. Badran, M. A. Shaaban, S. El-Meligi,
Egypt. J. Pharm. Sci. 1988, 29, 553–561.
[17] G. Gu¨rko¨k, T. Coban, S. Suzen, J. Inhib. Med. Chem. 2009, 24,
506–515.
[30] N. Re, A. Pellegrini, A. Proteggente, M. Y. Pannala, C. R.
Evans, Free Radical Biol. Med. 1999, 29, 1231–1237.
[18] P. M. Sivakumar, P. K. Prabhakar, M. Doble, Med. Chem. Res.
2011, 20, 482–492.
[31] N. J. Miller, C. Rice-Evans, Free Radical Res. 1997, 26, 195–
199.
[19] R. N. Gacche, N. A. Dhole, S. G. Kamble, B. P. Bandgar,
J. Enzyme Inhib. Med. Chem. 2008, 23, 28–31.
[32] HyperChem: Molecular Modeling System Hypercube, Inc.
Release 6.03, Florida, USA, 1997.
[20] C. B. Patil, S. K. Mahajan, S. A. Katti, J. Pharm. Sci. Res. 2009, 1,
11–22.
[33] Molegro Molecular Viewer (MMV), 2008. 1.0, Molegro,
Denmark.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com