Synthesis of novel amides with antiradical capacity from 2-mercaptobenzimidazole and cinnamic acids: Evaluation through donor-acceptor maps and QSAR

Article abstract of DOI:10.1016/j.molstruc.2020.128917

The structures of thioethers I-III and the new amidic compounds 1(a-f)-3(a-f) derived from 2-mercaptobenzimidazole (MBZ) and cinnamic acids were confirmed by NMR and elemental analysis. Antioxidant activity was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH●) radical scavenging assay and 2,2-azinobis (3-ethyl benzothiazoline-6-sulfonic acid) ABTS●+ radical cation decolorization method. Besides, donor-acceptor maps (DAM) and electrophilicity were calculated using DFT/B3LYP method with a 6-311G(d,p) basis set. MBZ, I-III and (1a-3a) compounds showed higher activity in vitro antioxidant assays, confirming with in silico studies that they are the best candidates. The findings found in antiradical activity suggest that these compounds could be promising in the development of new antitumor and antimicrobial agents. QSAR Molecular properties and topological descriptors of the synthesized compounds 1(a-f)-3(a-f) were calculated. The QSAR model indicates that the size and molecular shape are relevant for the antiradical activity for this family of compounds.

Full text of DOI:10.1016/j.molstruc.2020.128917

Journal Pre-proof
Synthesis of novel amides with antiradical capacity from
2-mercaptobenzimidazole and cinnamic acids: Evaluation through
donor-acceptor maps and QSAR
Omar Alejandro Ramos Rodr ´ı guez , Juan Pablo Mojica S a´ nchez ,
Jos e´ Antonio Valc a´ rcel Gami n˜ o , Fernando Obledo Benicio ,
Carlos Eduardo Mac ´ı as Hern a´ ndez ,
Mar ´ı a Teresa Sumaya Mart ´ı nez , Francisco J. Mart ´ı nez Mart ´ı nez ,
´
Zeferino G o´ mez Sandoval , Angel Ramos-Organillo
PII:
S0022-2860(20)31242-4
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2020.128917
MOLSTR 128917
To appear in:
Journal of Molecular Structure
Received date:
Revised date:
Accepted date:
22 May 2020
6 July 2020
15 July 2020
Please cite this article as: Omar Alejandro Ramos Rodr ´ı guez , Juan Pablo Mojica S a´ nchez ,
Jos e´ Antonio Valc a´ rcel Gami n˜ o , Fernando Obledo Benicio , Carlos Eduardo Mac ´ı as Hern a´ ndez ,
Mar ´ı a Teresa Sumaya Mart ´ı nez ,
Francisco J. Mart ´ı nez Mart ´ı nez ,
Zeferino G o´ mez Sandoval ,
´
Angel Ramos-Organillo , Synthesis of novel amides with antiradical capacity from 2-
mercaptobenzimidazole and cinnamic acids: Evaluation through donor-acceptor maps and QSAR,
Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.128917
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2020 Published by Elsevier B.V.

Highlights




Antioxidant activity, DAM, electrophilicity and QSAR of compounds were described.
MBZ and I-III compounds showed high antioxidant activity.
The results of DAM in DMSO phase are consistent of the antioxidant assays.
The size and molecular shape are relevant for the antiradical activity.
1

Synthesis of novel amides with antiradical capacity from 2-
mercaptobenzimidazole and cinnamic acids: Evaluation through donor-
acceptor maps and QSAR
a
b
Omar Alejandro Ramos Rodríguez , Juan Pablo Mojica Sánchez , José Antonio Valcárcel
c
a
a
Gamiño , Fernando Obledo Benicio , Carlos Eduardo Macías Hernández , María Teresa
d
a
a
Sumaya Martínez , Francisco J. Martínez Martínez , Zeferino Gómez Sandoval , and Ángel
a*
Ramos-Organillo
a
Facultad de Ciencias Químicas. Universidad de Colima, Km 9 Carretera Colima-
Coquimatlán, Coquimatlán, Colima. C.P. 28400, México.
b
Tecnológico Nacional de México. Instituto Tecnológico José Mario Molina Pasquel y
Henríquez Campus Tamazula de Gordiano, Carretera Tamazula-Santa Rosa No. 329,
Tamazula de Gordiano, Jalisco. C.P. 49650, México.
c
Centro de Investigación en Dinámica Celular. Universidad Autónoma del Estado de
Morelos, Avenida Universidad No. 1001, Chilapa, Cuernavaca, Morelos. C.P. 62210,
México.
d
Unidad de Tecnología de Alimentos. Universidad Autónoma de Nayarit, Cd. de la Cultura
"
Amado Nervo" Boulevard Tepic-Xalisco s/n, Tepic, Nayarit. C.P. 63190, México.
*
Corresponding author: aaramos@ucol.mx
2

Abstract
The structures of thioethers I-III and the new amidic compounds 1(a-f)-3(a-f)
derived from 2-mercaptobenzimidazole (MBZ) and cinnamic acids were confirmed by
NMR and elemental analysis. Antioxidant activity was evaluated by 1,1-diphenyl-2-
picrylhydrazyl (DPPH●) radical scavenging assay and 2,2-azinobis (3-ethyl
benzothiazoline-6-sulfonic acid) ABTS●+ radical cation decolorization method. Besides,
donor-acceptor maps (DAM) and electrophilicity were calculated using DFT/B3LYP
method with a 6-311G(d,p) basis set. MBZ, I-III and (1a-3a) compounds showed higher
activity in in vitro antioxidant assays, confirming with in silico studies that they are the best
candidates. The findings found in antiradical activity suggest that these compounds could
be promising in the development of new antitumor and antimicrobial agents. QSAR
Molecular properties and topological descriptors of the synthesized compounds 1(a-f)-3(a-
f) were calculated. The QSAR model indicates that the size and molecular shape are
relevant for the antiradical activity for this family of compounds.
Keywords
Donor-Acceptor-Maps; DFT; Antiradical-capacity; 2-mercaptobenzimidazole-and-
●
cinnamic acids-derivates; DPPH ; QSAR.
Introduction
In the search for new drugs for various pathologies, organic chemists have designed
and synthesized heterocyclic compounds. Heterocyclic compounds are relevant in fields of
medicine, biochemistry, environmental chemistry (herbicides and pesticides), coordination,
and organometallic chemistry [1]. This type of compound is used as a nucleus to obtain
3

new functionalized compounds. The 2-mercaptobenzimidazole (MBZ) that has a thiol
group in position-2 that has a tautomeric behavior, where it passes from the thiol to thione
1
13
form and the reverse [2] (Figure 1). In the H and C NMR spectra for the MBZ molecule
1
13
at room temperature show only three signals for H and four signals for C, which would
not happen if the system did not have tautomeric equilibrium, as six signals would be
1
13
expected for the H and seven for the C spectrum [3].
Figure 1. Tautomeric forms of 2-mercaptobenzimidazole (MBZ).
MBZ has pharmacological activity as antimicrobial [4], antihistamine, analgesic,
anticonvulsant, neutropic [5], antioxidant [6, 7], and anti-ulcerative activities. The antiulcer
drugs derived from 2-mercaptobenzimidazole are omeprazole, lansoprazole, rabeprazole,
esomeprazole, which suppress gastric acid secretion by inhibiting the proton pump [8].
MBZ serves as a plant growth regulator and is also used in non-biological
applications [9]. Also, it is widely used as an antioxidant for rubber and plastics [10], and
some derivatives exhibit insecticidal properties [11]. It is also a well-known analytical
reagent for mercury. It has been used for the determination of metallic ions Fe(II), Cu(II),
and Cd(II) in sewage water and industrial wastewater samples [12, 13].
4

Different methods have determined the antioxidant activity of new benzimidazole
derivatives: in vitro at the microsomal level of NADPH-dependent lipid peroxidation in rat
liver, the scavenging of superoxide anion and the stable radical 2,2-diphenyl-1-
picrylhydrazil (DPPH) [14]. It has been found that benzimidazole derivatives present an
excellent antioxidant activity. The compounds 2-styryl-1H-benzimidazole, 2-(2-
chlorostyryl)-1H-benzimidazole, and 2-(2-(1H-benzimidazole-2-yl)vinyl)phenol which
shows an inhibition percentage higher than 90% at nitric oxide radical inhibition test, being
more potent than ascorbic acid (reference antioxidant) [15].
There are different mechanisms by which phenolic antioxidants can scavenge free
radicals: 1) electron transfer (ET), 2) hydrogen atom transfer (HAT), and 3) sequential
proton loss electron transfer (SPLET) [16]. Although it is also true that there are
mechanisms that explain the antioxidant activity and that are not related to free radicals.
There are also non-chemical routes (enzymatic) [17]. In this sense, it is well known that
ionization potential (I) and electron affinity (A) are properties of a system that can measure
its propensity to donate or accept an electron. The best antioxidants have low I value,
because the lower the value, the easier it is too abstract electrons, and vice versa for A to
accept electrons (anti-reducer). It is valid provided that the electron transfer reaction is not
located in the inverted zone of the Marcus parabola. Otherwise, lower values of ionization
energy lead to prolonged reactions [18, 19].
There is now a model that explains the relative search activity and antioxidant
power of the compounds that use these two properties [20]. Quantum chemical density
functional theory (DFT) calculations can be used to obtain ionization potentials, electron
affinities, electron-donation (Rd), and electron-acceptor (Ra) power indices (concerning
the such as fluorine and sodium atoms). A plot of Rd vs. Ra can be used to construct a
5

donator acceptor map (DAM) Figure 2, indicating whether the molecules are good electron
donator or acceptors.
Figure 2. Donator acceptor map (DAM) [21-26].
The DAM is a representation of these properties (Rd vs. Ra), helping to exhibit the
antiradical capacity of any substance and allowing qualitative comparison between
substances [21]. Previous DAM studies by Ana Martínez have included linear polyene-
conjugated molecules, an extensive series of carotenoids, melatonin, and vitamins, and
psittacofulvine and anthocyanins which present a high antiradical capacity [21-26].
In the present study, a new series of amides 1(a-f)-3(a-f) was synthesized using
1
13
Neises & Steglich’s reaction. The chemical structures were confirmed using H-NMR, C-
NMR, IR, and elemental analysis. Besides, the antioxidant activity was calculated to find
new amide derivatives with potential anticancer and antimicrobial activity.
Materials and methods
General experimental procedure
6

The reagents used (2-mercaptobenzimidazole, iodomethane, iodoethane,
iodopropane, cinnamic acid derivatives, DCC, DMAP, silica gel 60 Å) were Sigma-Aldrich
reagent grade and were used without extra purification. The three thioethers I-III, as well
as the 18 synthesized amides 1(a-f)-3(a-f), were characterized by infrared spectroscopy
-1
techniques in a spectral range (4000-600 cm ) using a Varian 3100 FT-IR spectrometer of
1
13
the Excalibur series. H, C NMR, were recorded in CDCl
3
6
or DMSO-d using Bruker
1
13
1
Ultra shield Plus 400 ( H, 400; C, 100.62 MHz). Data for H NMR are reported as
chemical shifts (δ ppm), multiplicity (s= singlet, d= doublet, q= quartet, m= multiple),
1
3
integration, and assignment; data for C NMR are reported as a chemical shift (δ ppm).
Elemental analysis of compounds was performed on the Leco TruSpect Micro (C, H, N).
General procedure for the synthesis of thioethers (I-III)
In a ball flask (6.66 mmol) of 2-mercaptobenzimidazole was placed, 20 mL of
dimethylformamide (DMF) was added as a solvent. It was placed in vigorous agitation at 0
°C. An aqueous solution (2 mL) of NaOH (6.66 mmol) was then added, and agitation
continued at 0 °C for 30 minutes. Alkyl halide (6.66 mmol) was then added and allowed to
react for 24 hours at room temperature (Scheme 1). TLC monitored the progress of the
reaction. After completion of the reaction, the mixture was inactivated with the addition of
1
00 mL of cold water, in which compounds I-III were precipitated and isolated by
filtration and drying. The products were characterized by corresponding spectroscopic data
1
13
(
IR, H, and C NMR).
7

Scheme 1. Reaction to obtain thioethers I-III.
2
-(methylthio)-1H-benzo[d]imidazole (I). White solid; yield 87% (0.95 g); m.p.
-1
1
2
03-206 °C. IR (ⱱ cm ): ⱱ(N-H) 3117; ⱱ(C=N) 1616; ⱱ(C-N) 1346; δ(C-S) 663. H NMR
(
CDCl , 400 MHz): δ (ppm) 7.28-7.23 (1H, m, H-5, H-6); 6.94-6.89 (2H, m, H-7, H-4);
3
1
3
2
3
.52 (1H, s, H-11). C NMR (CDCl , 100.62 MHz): δ (ppm) 151.9 (C2), 139.5 (C8), 139.5
(
C9),
21.7 (C5), 121.7 (C6), 113.7 (C7), 113.7 (C4), 14.2 (C11). E.A C
58.49), H (5.20), N (17.34); Calculated: C (58.10), H (4.91), N (17.06).
-(ethylthio)-1H-benzo[d]imidazole (II). White solid; yield 81% (0.97 g); m.p.
1
8 8 2
H N S (%) Found: C
(
2
-1
1
1
71-174 °C. IR (ⱱ cm ): ⱱ(N-H) 3123; ⱱ(C=N) 1616; ⱱ(C-N) 1344; δ(C-S) 661. H NMR
(
CDCl
3
, 400 MHz): δ (ppm) 7.49-7.12 (4H, m, H-5, H-6, H-4, H-7); 3.27 (1H, c, H-11);
1
3
1
3
.37 (1H, t, H-12). C NMR (CDCl , 100.62 MHz): δ (ppm) 150.5 (C2), 121.8 (C8), 121.7
(
C9), 117.7 (C5), 117.6 (C6), 111.1 (C4), 111.1 (C7), 26.3 (C11), 15.5 (C12). E.A
C
9
H
10
N
2
S (%) Found: C (60.77), H (5.59), N (15.77); Calculated: C (60.64), H (5.65), N
(
15.72).
2
-(propylthio)-1H-benzo[d]imidazole (III). White solid; yield 84% (1.08 g); m.p.
-
1
1
1
64-166 °C. IR (ⱱ cm ): ⱱ(N-H) 3121; ⱱ(C=N) 1620; ⱱ(C-N) 1344; δ(C-S) 673. H NMR
CDCl , 400 MHz): δ (ppm) 7.44 (2H, m, H-4, H-7); 7.13 (2H, m, H-5, H-6); 3.24 (2H, t,
H-18); 1.72 (2H, s, H-19); 0.98 (3H, t, H-20). C NMR (CDCl
47.1 (C2), 138.9 (C9, C8), 123.0 (C6, C5), 115.2 (C4, C7), 38.6 (C18), 21.6 (C19), 13.0
(
3
1
3
3
, 100.62 MHz): δ (ppm)
1
(
(
C20). E.A C10
H
12
N
2
S (%) Found: C (62.67), H (6.39), N (14.79); Calculated: C (62.47), H
6.29), N (14.57).
8

General procedure for the synthesis of amides 1(a-f)-3(a-f)
In a ball flask, I-III (1.83 mmol), (1.83 mmol) of the corresponding trans-cinnamic
acid, and (0.183 mmol) of DMAP were added to 30 mL of dry DCM. These were kept in
agitation at 0°C. Dropwise (1.83 mmol) of DCC previously dissolved in dry DCM was
added. It was left at 0 °C for one hour and then left at room temperature for 24 hours
(
Scheme 2). The progress of the reaction was monitored by TLC. After completion of the
reaction, the mixture was filtered to remove the DCU and then evaporated the solvent at
reduced pressure. Compounds 1(a-f)-3(a-f) were purified by the chromatographic column
of silica gel (60 Å) at elution of 1:9 (ethyl acetate:hexane). The products were characterized
1
13
by corresponding spectroscopic data (IR, H, and C NMR).
9

Scheme 2. Reaction to obtain of the compounds 1(a-f)-3(a-f). (i) hydrocinnamic acid (a);
(
(
ii) trans-cinnamic acid (b), 4-fluoro trans-cinnamic acid (d), 4-chloro trans-cinnamic acid
e), 4-Bromo trans-cinnamic acid (f); (iii) phenylpropiolic acid (c).
1
-(2-(methylthio)-1H-benzo[d]imidazol-1-yl)-3-phenylpropan-1-one
(1a).
-1
Yellow solid; yield 49% (0.35 g); m.p. 43-46 °C. IR (ⱱ cm ): ⱱ(C=O) 1667; ⱱ(C=C) 1625;
1
ⱱ(C=N) 1581; ⱱ(C-N) 1327; δ(C-S) 667. H NMR (CDCl
3
, 400 MHz): δ (ppm) 7.87 (1H,
m, H-7); 7.64 (1H, m, H-4); 7.34 (1H, m, H-5); 7.28 (2H, m, H-16); 7.23 (2H, m, H-15);
1
3
7
.21 (1H, m, H-6); 2.95 (1H, d, H-13); 2.83 (2H, d, H-12); 2.57 (3H, s, H-18). C NMR
CDCl , 100.62 MHz): δ (ppm) 170.3 (C11), 152.7 (C2), 148.3 (C9), 141.3 (C14), 132.1
C8), 128.6 (C16), 127.7 (C15), 125.9 (C17), 123.5 (C6), 122.9 (C5), 119.6 (C4), 119.2
C7), 36.4 (C12), 30.1 (C13), 14.5 (C18). E.A C17 OS (%) Found: C (68.94), H (5.70),
(
(
(
3
16 2
H N
N (9.78); Calculated: C (68.89), H (5.44), N (9.45).
E)-1-(2-(methylthio)-1H-benzo[d]imidazol-1-yl)-3-phenylprop-2-en-1-one (1b).
(
-1
Yellow solid; yield 40% (0.35 g); m.p. 119-121 °C. IR (ⱱ cm ): ⱱ(C=O) 1676; ⱱ(C=C)
1
1
612; ⱱ(C=N) 1574; ⱱ(C-N) 1330; δ(C-S) 669. H NMR (CDCl
3
, 400 MHz): δ (ppm) 7.94
(
1H, d, H-13, J=16 Hz); 7.86 (1H, m, H-4); 7.70-7.64 (2H, m, H-7, H-16, H-17, H-21);
7
2
1
.52-7.41 (2H, m, H-18, H-19, H-20); 7.51 (1H, d, H-14); 7.40-7.31 (2H, m, H-5, H-6);
13
.67 (1H, s, H-11). C NMR (CDCl
3
, 100.62 MHz): δ (ppm) 164.8 (C12), 155.3 (C2),
47.9 (C9), 143.9 (C15), 134.4 (C16), 133.8 (C8), 131.7 (C19), 129.6 (C18, C20), 129.4
(
C17, C21), 124.7 (C6), 123.9 (C5), 119.5 (C14), 118.7 (C4), 114.2 (C7), 15.9 (C11). E.A
OS (%) Found: C (69.25), H (5.12), N (9.52); Calculated: C (69.36), H (4.79), N
17 14 2
C H N
(
9.52).
10

1
-(2-(methylthio)-1H-benzo[d]imidazol-1-yl)-3-phenylprop-2-yn-1-one
(1c).
-1
Yellow solid; yield 65% (0.35 g); m.p. 83-86 °C. IR (ⱱ cm ): ⱱ(C≡C) 2200; ⱱ(C=O) 1668;
1
ⱱ(C=N) 1591; ⱱ(C-N) 1330; δ(C-S) 673. H NMR (CDCl
3
, 400 MHz): δ (ppm) 8.26 (1H,
m, H-7); 7.74 (1H, m, H-4); 7.65 (1H, m, H-6); 7.57 (1H, m, H-5); 7.50 (2H, m, H-15);
1
3
7
.46 (1H, m, H-17); 7.31 (2H, m, H-16); 2.77 (3H, s, H-18). C NMR (CDCl
MHz): δ (ppm) 155.8 (C11), 149.3 (C2), 143.8 (C9), 133.0 (C15), 131.5 (C8), 128.6 (C16),
28.3 (C17), 125.2 (C5), 123.2 (C6), 119.5 (C14), 118.5 (C4), 113.8 (C7), 96.3 (C13), 82.8
C12), 15.6 (C18). E.A C17 OS (%) Found: C (69.84), H (4.46), N (9.67); Calculated:
C (69.84), H (4.14), N (9.58).
E)-3-(4-fluorophenyl)-1-(2-(methylthio)-1H-benzo[d]imidazol-1-yl)prop-2-en-
3
, 100.62
1
(
12 2
H N
(
-
1
1
-one (1d). Yellow solid; yield 56 % (0.32 g); m.p. 138-12 °C. IR (ⱱ cm ): ⱱ(C=O) 1681;
1
ⱱ(C=C) 1622; ⱱ(C=N) 1593; ⱱ(C-N) 1328; δ(C-S) 673. H NMR (CDCl
3
, 400 MHz): δ
(
ppm) 7.96 (1H, d, H-13, J=16 Hz); 7.73 (2H, m, H-15), 7.69 (1H, m, H-4); 7.65 (1H, m,
H-7); 7.35 (2H, m, H-16); 7.25 (2H, d, H-12, J=16 Hz); 7.2 (1H, m, H-5); 7.15 (1H, m, H-
1
3
6
1
1
3
); 2.8 (3H, s, H-18). C NMR (CDCl , 100.62 MHz): δ (ppm) 164.5 (C11), 155.1 (C2),
47.1 (C13), 143.4 (C9), 133.5 (C14), 131.0 (C8), 130.8 (C7), 124.9 (C16), 124.8 (C15),
18.8 (C6), 118.2 (C12), 116.5 (C5), 116.4 (C4), 113.5 (C7), 16.0 (C18). E.A
17 2
C H13FN OS (%) Found: C (65.21), H (4.37), N (9.14); Calculated: C (65.37), H (4.20), N
(
8.97).
(
E)-3-(4-chlorophenyl)-1-(2-(methylthio)-1H-benzo[d]imidazol-1-yl)prop-2-en-
-
1
1
-one (1e). Yellow soli; yield 50 % (0.3 g); m.p. 152-154 °C. IR (ⱱ cm ): ⱱ(C=O) 1685;
1
ⱱ(C=C) 1625; ⱱ(C=N) 1589; ⱱ(C-N) 1329; δ(C-S) 667. H NMR (CDCl
3
, 400 MHz): δ
(
ppm) 7.8 (1H, d, H-13, J=16 Hz); 7.59 (2H, m, H-15), 7.53 (1H, m, H-4); 7.48 (1H, m, H-
7
); 7.32 (2H, m, H-16); 7.23 (1H, m, H-5); 7.18 (2H, d, H-12 J=15.8 Hz); 7.15 (1H, m, H-
11

1
3
6
1
1
3
); 2.66 (3H, s, H-18). C NMR (CDCl , 100.62 MHz): δ (ppm) 164.4 (C11), 155.1 (C2),
46.5 (C13), 143.5 (C9), 137.4 (C14), 133.8 (C8), 132.3 (C17), 129.8 (C16), 129.5 (C15),
24.7 (C6), 123.4 (C5), 118.9 (C12), 118.7 (C4), 113.4 (C7), 15.9 (C18). E.A
17 2
C H13ClN OS (%) Found: C (62.15), H (4.32), N (8.80); Calculated: C (62.10), H (3.99),
N (8.52).
E)-3-(4-bromophenyl)-1-(2-(methylthio)-1H-benzo[d]imidazol-1-yl)prop-2-en-
(
-
1
1
-one (1f). Yellow solid; yield 50 % (0.34 g); m.p. 122-124 °C. IR (ⱱ cm ): ⱱ(C=O) 1672;
1
ⱱ(C=C) 1616; ⱱ(C=N) 1587; ⱱ(C-N) 1336; δ(C-S) 631. H NMR (CDCl
3
, 400 MHz): δ
(
ppm) 7.93 (1H, d, H-13, J=16 Hz); 7.73 (2H, m, H-15), 7.67 (1H, m, H-4); 7.62 (1H, m,
H-7); 7.53 (2H, m, H-16); 7.36 (1H, m, H-5); 7.31 (2H, d, H-12, J=16 Hz); 7.28 (1H, m, H-
1
3
6
1
1
3
); 2.81 (3H, s, H-18). C NMR (CDCl , 100.62 MHz): δ (ppm) 164.4 (C11), 155.4 (C2),
47.0 (C13), 143.3 (C9), 133.5 (C8), 132.9 (C14), 132.5 (C15), 130.0 (C16), 126.0 (C17),
24.7 (C6), 123.7 (C5), 119.0 (C12), 118.6 (C4), 113.4 (C7), 15.9 (C18). E.A
17 2
C H13BrN OS (%) Found: C (54.65), H (3.83), N (7.71); Calculated: C (54.70), H (3.51),
N (7.51).
-(2-(ethylthio)-1H-benzo[d]imidazol-1-yl)-3-phenylpropan-1-one (2a). Yellow
1
-1
solid; yield 62%; m.p. 86-88 °C. IR (ⱱ cm ): ⱱ(C=O); ⱱ(C=C); ⱱ(C=N); ⱱ(C-N); δ(C-S).
1
3
H NMR (CDCl , 400 MHz): δ (ppm) 7.67 (2H, m, H-4, H-7); 7.39-7.22 (5H, m, H-18, H-
1
1
1
1
9, H-20, H-21, H-22, H-5, H-6); 3.39 (1H, c, H-11); 3.35 (1H, t, H-15); 3.21 (1H, t, H-16);
13
3
.50 (1H, t, H-12). C NMR (CDCl , 100.62 MHz): δ (ppm) 171.4 (C13), 155.3 (C2),
44.1 (C17), 139.9 (C9), 132.9 (C8), 128.8 (C19, C21), 128.5 (C18, C22), 126.6 (C20),
24.6 (C6), 123.4 (C5), 118.8 (C4), 113.7 (C7), 39.9 (C15), 30.0 (C16), 27.0 (C11), 13.7
(
(
C12). E.A C18
H
18
N
2
OS (%) Found: C (69.35), H (6.10), N (8.81); Calculated: C (69.65), H
5.85), N (9.02).
12

(
E)-1-(2-(ethylthio)-1H-benzo[d]imidazol-1-yl)-3-phenylprop-2-en-1-one (2b).
-1
Yellow solid; yield 48 % (0.41 g); m.p. 94-96 °C. IR (ⱱ cm ): ⱱ(C=O) 1687; ⱱ(C=C)
1
1
627; ⱱ(C=N) 1597; ⱱ(C-N) 1327; δ(C-S) 644. H NMR (CDCl
3
, 400 MHz): δ (ppm) 7.94
(
1H, d, H-13, J=16 Hz); 7.85 (1H, m, H-4); 7.70-7.63 (5H, m, H-7, H-15, H-16, H-17);
7
1
.53 (1H, d, H-12, J=16 Hz); 7.34-7.27 (2H, m, H-5, 6); 3.29 (1H, c, H-18); 1.39 (1H, t, H-
1
3
3
9). C NMR (CDCl , 100.62 MHz): δ (ppm) 165.1 (C11), 154.3 (C2), 148.1 (C9), 144.2
(
(
C13), 134.7 (C14), 133.8 (C8), 131.7 (C17), 129.6 (C16), 129.4 (C15), 124.8 (C6), 123.9
C5), 120.1 (C12), 118.6 (C4), 114.3 (C7), 26.9 (C18), 14.6 (C19). E.A C18
H
16
N
2
OS (%)
Found: C (70.36), H (5.45), N (8.99); Calculated: C (70.10), H (5.23), N (9.08).
-(2-(ethylthio)-1H-benzo[d]imidazol-1-yl)-3-phenylprop-2-yn-1-one
1
(2c).
-1
Yellow solid; yield 58 % (0.3 g); m.p. 73-75 °C. IR (ⱱ cm ): ⱱ(C≡C) 2117; ⱱ(C=O) 1712;
1
ⱱ(C=N) 1573; ⱱ(C-N) 1342; δ(C-S) 638. H NMR (CDCl
3
, 400 MHz): δ (ppm) 8.29 (1H,
m, H-7); 7.76 (1H, m, H-4); 7.67 (1H, m, H-6); 7.57 (1H, m, H-5); 7.50 (2H, m, H-15);
1
3
7
.37 (1H, m, H-17); 7.31 (2H, m, H-16); 3.40 (2H, c, H-18); 1.52 (3H, t, H-19). C NMR
CDCl , 100.62 MHz): δ (ppm) 154.9 (C11), 149.4 (C2), 143.7 (C9), 133.2 (C8), 133.0
C15), 131.7 (C17), 128.9 (C16), 125.2 (C5), 123.7 (C6), 118.8 (C14), 118.6 (C4), 113.8
C7), 96.2 (C13), 82.6 (C12), 26.9 (C18) 13.8 (C19). E.A C18 OS (%) Found: C
70.85), H (4.92), N (9.40); Calculated: C (70.56), H (4.61), N (9.14).
E)-1-(2-(ethylthio)-1H-benzo[d]imidazol-1-yl)-3-(4-fluorophen-yl)prop-2-en-1-
(
(
(
(
3
14 2
H N
(
-
1
one (2d). Yellow solid; yield 57 % (0.31 g); m.p. 119-121 °C. IR (ⱱ cm ): ⱱ(C=O) 1685;
1
ⱱ(C=C) 1620; ⱱ(C=N) 1597; ⱱ(C-N) 1334; δ(C-S) 637. H NMR (DMSO-d
6
, 400 MHz): δ
(
ppm) 7.95 (1H, d, H13, J=16 Hz), 7.93 (2H, m, H15), 7.68 (1H, m, H4), 7.62 (1H, m, H7),
7
.31 (1H, m, H5), 7.45 (1H, d, H12, J=16 Hz), 7.34 (2H, m, H16), 7.29 (1H, m, H6), 3.28
13
(
6
2H, c, H18), 1.38 (3H, t, H19). C NMR (DMSO-d , 100.62 MHz): δ (ppm) 165.0 (C11),
13

1
1
53.7 (C2), 145.1 (C9), 143.5 (C13), 133.1 (C14), 131.5 (C8), 131.1 (C17), 130.6 (C16),
30.5 (C15), 124.2 (C6), 123.4 (C5), 119.0 (C12), 118.2 (C4), 113.8 (C7), 26.4 (C18), 14.1
(
C19). E.A C18
H (4.63), N (8.58).
E)-3-(4-chlorophenyl)-1-(2-(ethylthio)-1H-benzo[d]imidazol-1-yl)prop-2-en-1-
2
H15FN OS (%) Found: C (66.57), H (4.65), N (8.54); Calculated: C (66.24),
(
-1
one (2e). Yellow solid; yield 56 % (0.32 g); m.p. 125-127 °C. IR (ⱱ cm ): ⱱ(C=O) 1679;
1
ⱱ(C=C) 1616; ⱱ(C=N) 1593; ⱱ(C-N) 1336; δ(C-S) 642. H NMR (DMSO-d
6
, 400 MHz): δ
(
ppm) 7.96 (1H, d, H13, J=16 Hz), 7.93 (2H, m, H15), 7.73 (1H, m, H4), 7.68 (1H, m, H7),
7
.60 (2H, m, H16), 7.55 (1H, d, H12, J=16 Hz), 7.36 (1H, m, H5), 7.33 (1H, m, H6), 3.32
1
3
(
2H, c, H18), 1.43 (3H, t, H19). C NMR (DMSO-d
6
, 100.62 MHz): δ (ppm) 164.5 (C11),
54.0 (C2), 145.9 (C9), 143.5 (C13), 135.9 (C14), 133.1 (C8), 132.9 (C17), 130.7 (C16),
29.2 (C15), 124.3 (C6), 123.4 (C5), 120.0 (C12), 118.2 (C4), 113.8 (C7), 26.4 (C18), 14.1
OS (%) Found: C (63.35), H (4.78), N (8.42); Calculated: C (63.06),
1
1
(
C19). E.A C18
H (4.41), N (8.17).
E)-3-(4-bromophenyl)-1-(2-(ethylthio)-1H-benzo[d]imidazol-1-yl)prop-2-en-1-
H15ClN
2
(
-1
one (2f). Yellow solid; yield 51 % (0.33 g); m.p. 135-137 °C. IR (ⱱ cm ): ⱱ(C=O) 1681;
1
ⱱ(C=C) 1616; ⱱ(C=N) 1583; ⱱ(C-N) 1332; δ(C-S) 632. H NMR (DMSO-d
6
, 400 MHz): δ
(
ppm) 7.92 (1H, d, H13, J=16 Hz), 7.81 (2H, m, H15), 7.70 (1H, m, H4), 7.69 (1H, m, H7),
7
.63 (2H, m, H16), 7.52 (1H, d, H12, J=16 Hz), 7.31 (1H, m, H5), 7.26 (1H, m, H6), 3.29
1
3
(
2H, c, H18), 1.39 (3H, t, H19). C NMR (DMSO-d
6
, 100.62 MHz): δ (ppm) 164.8 (C11),
53.9 (C2), 145.0 (C13), 143.6 (C9), 133.2 (C14), 133.1 (C8), 132.2 (C17), 130.9 (C16),
25.0 (C15), 124.2 (C6), 123.6 (C5), 120.0 (C12), 118.2 (C4), 113.7 (C7), 26.3 (C18), 14.1
OS (%) Found: C (56.11), H (4.25), N (7.47); Calculated: C
55.82), H (3.90), N (7.23).
1
1
(
(
C19). E.A C18H15BrN
2
14

3
-phenyl-1-(2-(propylthio)-1H-benzo[d]imidazol-1-yl)propan-1-one
(3a).
-1
Yellow solid; yield 72% (0.4 g); m.p. 84-86 °C. IR (ⱱ cm ): ⱱ(C=O) 1685; ⱱ(C=N) 1589;
1
ⱱ(C-N) 1334; δ(C-S) 651. H NMR (CDCl
3
, 400 MHz): δ (ppm) 7.69 (1H, m, H-7); 7.67
(
1H, m, H-4); 7.39 (2H, m, H-16); 7.35 (1H, m, H-6); 7.32 (2H, m, H-15); 7.28 (1H, m, H-
5
); 7.23 (1H, m, H-17); 3.42 (1H, d, H-12); 3.35 (2H, t, H-18); 3.22 (1H, d, H-13); 1.88
1
3
(
2H, m, H-19); 1.13 (3H, t, H-20). C NMR (CDCl
3
, 100.62 MHz): δ (ppm) 171.0 (C11),
55.4 (C2), 144.1 (C8), 139.9 (C14), 133.0 (C9), 128.7 (C16), 128.4 (C6), 126.5 (C5),
24.5 (C15), 123.3 (C17), 118.7 (C7), 113.7 (C4), 40.0 (C12), 34.6 (C18), 30.1 (C13), 21.7
OS (%) Found: C (70.12), H (6.48), N (8.78); Calculated:
1
1
(
C19), 13.5 (C20). E.A C19
C (70.34), H (6.21), N (8.63).
E)-3-phenyl-1-(2-(propylthio)-1H-benzo[d]imidazol-1-yl)prop-2-en-1-one (3b).
20 2
H N
(
-1
Yellow solid; yield 60 % (0.3 g); m.p. 76-78 °C. IR (ⱱ cm ): ⱱ(C=O) 1689; ⱱ(C=C) 1624;
1
ⱱ(C=N) 1589; ⱱ(C-N) 1329; δ(C-S) 648. H NMR (DMSO-d
6
, 400 MHz): δ (ppm) 7.93
(
(
(
(
(
(
1H, d, H-13, J=16 Hz); 7.85 (2H, m, H-15); 7.68 (1H, m, H-4); 7.63 (1H, m, H-7); 7.51
2H, m, H-16); 7.50 (1H, m, H-17); 7.48 (1H, d, H-12, J=16 Hz); 7.31 (1H, m, H-5); 7.28
1
3
1H, m, H-6); 3.27 (2H, t, H-18); 1.76 (2H, m, H-19); 1.01 (3H, t, H-20). C NMR
DMSO-d , 100.62 MHz): δ (ppm) 164.4 (C11), 153.7 (C2), 147.2 (C13), 143.3 (C9), 133.8
C14), 133.1 (C8), 131.0 (C17), 129.0 (C15), 128.75 (C16), 124.1 (C5), 123.1 (C6), 119.1
6
C12), 118.1 (C7), 113.5 (C4), 33.6 (C18), 21.6 (C19), 13.0 (C20). E.A C19
H
18
N
2
OS (%)
Found: C (70.57), H (5.85), N (8.50); Calculated: C (70.78), H (5.63), N (8.69).
-phenyl-1-(2-(propylthio)-1H-benzo[d]imidazol-1-yl)prop-2-yn-1-one
3
(3c).
-1
Yellow solid; yield 62 % (0.31 g); m.p. 53-56 °C. IR (ⱱ cm ): ⱱ(C≡C) 2202; ⱱ(C=O)
1
1
664; ⱱ(C=N) 1595; ⱱ(C-N) 1332; δ(C-S) 630. H NMR (DMSO-d
6
, 400 MHz): δ (ppm)
8
.16 (1H, m, H-7); 7.82 (1H, m, H-4); 7.80 (1H, m, H-6); 7.66 (1H, m, H-5); 7.56 (2H, m,
15

H-15); 7.35 (1H, m, H-17); 7.31 (2H, m, H-16); 3.25 (2H, t, H-18); 1.77 (2H, m, H-19);
1
3
1
1
1
6
.02 (3H, t, H-20). C NMR (DMSO-d , 100.62 MHz): δ (ppm) 154.7 (C11), 149.2 (C2),
43.7 (C9), 133.5 (C8), 133.1 (C15), 132.6 (C17), 129.7 (C16), 125.7 (C5), 124.9 (C6),
18.5 (C14), 118.7 (C4), 118.5 (C7), 95.8 (C13), 82.8 (C12), 34.3 (C18), 21.8 (C19), 13.0
(
(
C20). E.A C19
5.03), N (8.74).
E)-3-(4-fluorophenyl)-1-(2-(propylthio)-1H-benzo[d]imidazol-1-yl)prop-2-en-
16 2
H N OS (%) Found: C (70.88), H (5.35), N (8.41); Calculated: C (71.22), H
(
-1
1
-one (3d). Yellow solid; yield 59% (0.31 g); m.p. 96-99 °C. IR (ⱱ cm ): ⱱ(C=O) 1689;
1
ⱱ(C=C) 1622; ⱱ(C=N) 1597; ⱱ(C-N) 1330; δ(C-S) 650. H NMR (DMSO-d
6
, 400 MHz): δ
(
ppm) 7.94 (1H, d, H-13, J=16 Hz); 7.93 (2H, m, H-15); 7.68 (1H, m, H-4); 7.62 (1H, m,
H-7); 7.44 (1H, d, H-12, J=16 Hz); 7.36 (2H, m, H-16); 7.33 (1H, m, H-17); 7.32 (1H, m,
1
3
H-5); 7.29 (1H, m, H-6); 3.26 (2H, t, H-18); 1.76 (2H, m, H-19); 1.01 (3H, t, H-20). C
NMR (DMSO-d , 100.62 MHz): δ (ppm) 164.9 (C11), 154.3 (C2), 146.5 (C13), 143.8
C9), 133.7 (C8), 131.9 (C15), 131.0 (C14), 124.7 (C5), 123.9 (C6), 119.5 (C12), 118.7
C7), 116.7 (C16), 116.5 (C17), 114.1 (C4), 33.9 (C18), 22.0 (C19), 13.6 (C20). E.A
OS (%) Found: C (67.34), H (5.31), N (8.17); Calculated: C (67.04), H (5.03), N
6
(
(
19 2
C H17FN
(
8.23).
(
E)-3-(4-chlorophenyl)-1-(2-(propylthio)-1H-benzo[d]imidazol-1-yl)prop-2-en-
-1
1
-one (3e). Yellow solid; yield 61 % (0.34 g); m.p. 138-140 °C. IR (ⱱ cm ): ⱱ(C=O) 1681;
13
ⱱ(C=C) 1618; ⱱ(C=N) 1587; ⱱ(C-N) 1331; δ(C-S) 644. C NMR (solids): δ (ppm) 164.1
(
(
(
(
C11), 156.8 (C2), 146.4 (C13), 136.0 (C9), 134.3 (C8), 128.3 (C15), 138.4 (C14), 126.0
C5), 122.7 (C6), 121.0 (C12), 116.0 (C7), 129.7 (C16), 131.8 (C17), 113.4 (C4), 34.1
C18), 21.5 (C19), 14.4 (C20). E.A C19
2
H17ClN OS (%) Found: C (64.28), H (5.12), N
8.14); Calculated: C (63.95), H (4.80), N (7.85).
16

(
E)-3-(4-bromophenyl)-1-(2-(propylthio)-1H-benzo[d]imidazol-1-yl)prop-2-en-
-
1
1
-one (3f). Yellow solid; yield 55 % (0.32 g); m.p. 142-145 °C. IR (ⱱ cm ): ⱱ(C=O) 1683;
13
ⱱ(C=C) 1618; ⱱ(C=N) 1583; ⱱ(C-N) 1332; δ(C-S) 632. C NMR (solids): δ (ppm) 164.4
(
(
(
(
C11), 157.0 (C2), 146.5 (C13), 136.4 (C9), 132.1 (C8), 127.2 (C15), 135.8 (C14), 125.5
C5), 123.0 (C6), 121.0 (C12), 116.6 (C7), 126.7 (C16), 130.4 (C17), 113.1 (C4), 34.3
C18), 22.0 (C19), 13.9 (C20). E.A C19
2
H17BrN OS (%) Found: C (57.18), H (4.39), N
7.31); Calculated: C (56.86), H (4.27), N (6.98).
Antioxidant activity
The synthesized compound III was screened for their antioxidant activity by 1,1-
diphenyl-2-picrylhydrazyl (DPPH●) radical scavenging assay and 2,2-azinobis (3-ethyl
●
+
benzothiazoline-6-sulfonic acid) ABTS radical cation decolorization method.
A Power wave XS microplates reader from biotek were used to read the
absorbancies. Micropipettes (Thermofisher 89 finnpippete) of different capacities (25, 200,
1
000 μL) were also used to prepare the required solutions. Eppendorf tubes (1 mL) were
used for sample preparation. All tests were carried out in triplicate. For each run of samples
a standard curve was made using the reference antioxidant in order to calculate the
equivalents per mL of the reference antioxidant [27].
●
DPPH method
●
A solution of DPPH (7.4 mg/100 mL) was prepared and kept in the dark to avoid
photodegradation. 50 μL were taken from a DMSO solution of compound III or the
●
reference antioxidant (trolox) to which 250 μL of DPPH solution was immediately added.
17

The mixture was stirred vigorously with a microvortex and left it to stand at room
temperature for one hour, then the absorbance was measured at 520 nm. The results of anti-
radical activity were expressed as a percentage of inhibition and on μmol trolox/L
equivalents (μmol TE/L) [28-29].
●
+
ABTS method
The radical ABTS was obtained by reacting NH
persulphate (2.45 mM) for 16 hours at 4 ºC in the dark. Once formed, the radical ABTS
was diluted with ethanol to obtain absorbance value of 0.70 (±0.1) at 754 nm. In an
●
+
+
2-
4
ABTS (7mM) with potassium
●
+
●
+
eppendorf tube 490 μL of the diluted solution of free radicals (ABTS ) and 10 μL of the
compounds to be analyzed or vitamin C (reference antioxidant) were added, the mixture
was agitated vigorously and then 200 μL of this mixture were taken and its absorbance was
read at 754 nm in a microplates reader. The results of anti-radical activity were expressed
as a percentage of inhibition and in milligrams ascorbic acid equivalents per liter (mg
AAE/L) [27-28].
Computational details
Geometry optimizations without symmetry restrictions in the gas phase were carried
out for 26 structures within the framework of the density functional theory in the Gaussian
0
9 code [30]. For the exchange, a Becke’s 1988 [31] formalism was used, and for the
correlation, a Perdew and Wang’s 1991 [32] functional was employed. The 6-311G (d, p)
basis set [33] was utilized to describe all electrons, and frequency analysis was performed
in order to verify the stationary points.
18

Once the minima energy geometry of each system has been established, anionic and
cationic states were calculated by adding the negative and the positive charge, respectively,
in the form of single points [34-35].
On the other hand, calculations in solutions were made using the optimized
geometries and utilizing the SMD model [36] under the same level theory, and specifically,
three solvents were considered: water, DMSO, and pentyl ethanoate. In all solvent cases,
single points were effectuated to obtain energies for neutral, anionic, and cationic states. All
calculations were realized in Gaussian 09 [29].
The ionization potential (I) was calculated using the difference between the energy
of cation less the energy of neutral state, and the reduction capacity was estimated from the
electron affinity (A) and calculated how the difference between the energy of neutral
stateless the energy of anion. It is to evaluate the oxidant capacity of the molecules [37].
One tool that allows the measurement of the propensity to donate charge is called
electron-donation power [20], which was defined as (Equation 1).
(
ꢂꢃꢄꢅ)ꢆ
ꢀ
ꢁ
ꢇꢈ(ꢃꢉꢅ)
Equation 1.
Whereas the tool to describe the propensity to accept the charge or called electron-
acceptor power [20], was defined as (Equation 2).
(
ꢃꢄꢂꢅ)ꢆ
ꢊ
ꢁ
ꢇꢈ(ꢃꢉꢅ)
Equation 2.
19

ꢀ
ꢊ
The
and
were calculated for 26 molecules, and as a measure of comparison,
of Na atom and the ꢊof F atom. It is because Na represents a
ꢀ
it was estimated the
suitable electron donor, and F represents an excellent electron acceptor. For any molecule,
it was defined as an electron acceptance index as (Equation 3).
ꢍꢊ
ꢋ ꢁ
ꢌ
ꢊ
ꢍ
ꢎ
Equation 3.
Moreover, an electron donation index was defined as (Equation 4).
ꢍꢀ
ꢋ ꢁ
ꢏ
ꢀ
ꢍ
ꢐꢌ
Equation 4.
If ꢑ =1, the molecule is an excellent electron donor as Na; if ꢑ <1, the molecule is
ꢒ
ꢒ
better electron donor than Na and finally if ꢑ >1 is a poorer electron donor than Na.
ꢒ
Besides, if ꢑ =1, the molecule is a suitable electron acceptor as F; if ꢑ <1 is a poorer
ꢓ
ꢓ
electron acceptor than F and if ꢑ >1 is a better electron acceptor than F.
ꢓ
With the above information, it is possible to correlate the values of ꢋ and ꢋ of the
ꢌ
ꢏ
molecules in the form of graphs called donor-acceptor maps [21-25]. A donor-acceptor map
was created and was compared to the results of the novel amides compounds with known
antioxidants substances: vitamin C, Trolox, Resveratrol, and Quercetin. Additionally, the
calculations of electrophilicity were carried out, and it is defined as infinite differences as
[
38] (Equation 5).
(
ꢃꢄꢅ)ꢆ
ꢍꢁ
ꢔ(ꢃꢉꢅ)
Equation 5.
20

Results and discussion
Synthesis
The synthesis scheme consisted of two stages leading to the formation of a variety
of amides 1(a-f)-3(a-f); the first was the formation of thioethers I-III from 2-
mercaptobenzimidazole with alkyl halides in basic medium with yields of 90% after
precipitation in cold water (Scheme 1). The second stage was the synthesis of the 18 new
amidic compounds 1(a-f)-3(a-f) (Scheme 2), using the N,N’-dicyclohexylcarbodiimide
(
DCC) methodology proposed by Neises & Steglich (1978) [39]. In which the thioethers I-
III were reacted with the different derivatives of cinnamic acid (a-f) in an equimolar
relationship using dry dichloromethane as the solvent, 4-(dimethylamino)pyridine (DMAP)
as catalyst and N,N’-dicyclohexylcarbodiimide (DCC) as an activator of carboxylic acid
towards substitution. Amides 1(a-f)-3(a-f) were obtained with yields of 40-72% after
column chromatography.
Structural elucidation of compounds I-III, 1(a-f)-3(a-f)
1
In H NMR spectra for thioethers I-III, a broad singlet signal resonated at 8.5 ppm
was assigned to the N-H of the imidazole ring. The amplitude of the signal is caused by the
fast exchange of the proton with the other N present in the imidazole ring, caused by the
tautomeric effect, which is faster than the resonance time scale. In spectra of the
compounds (1-3)(b, d, e, f) two doublets were observed between the regions of 7.18-7.51
and 7.80-7.94 ppm due to H12 and H13 of the double bond of the trans-cinnamic acid
21

3
fragment, coupling constant ( J(H12-H13)) was 16 Hz indicating the trans- position of the
double bond.
1
3
In C NMR of compounds 1(a-f)-3(a-f), the signals between 171.1 and 154.7 ppm
were assigned to carbonyl (C11). Where in 1(a-c)-3(a-c) compounds there is an
unprotected phenomenon this is due to the diamagnetic anisotropy present in the double
and triple bond, the effect of this phenomenon can also be seen in the C2 signal where 1a-
3
a derivatives appear at 155.7 ppm, 1b-3b at 155.1 ppm and 1c-3c at 149.3 ppm. (The
NMR spectra are shown in the supplementary material).
Antioxidant activity
Table 1 shows the percentages of scavenging activity and antiradical activity in
●
●+
●
DPPH and ABTS . In the DPPH assay, the MBZ was able to capture 73% of the radical
●
DPPH , also showing better radical entrapment than the Trolox (reference antioxidant),
showing 70%. Thioether I inhibition percentage of 26%, while 1a presented 23%, 1c
trapped only 15%, compound 1f inhibiting 46%, thioether II showed 18%, 2b presented the
lowest percentage with 12%, while 3c showed a low percentage 26%.
Table 1. Antioxidant activity of MBZ and synthesized compounds.
●
●+
DPPH
ABTS
Comp.
MBZ
I
%
%
µmol TE/L
Scavenging
73 ± 0.59
Scavenging mg AAE/L
325 ± 2.3
88 ± 2.68
27 ± 7.80
14 ± 5.54
6 ± 4.44
4 ± 5.24
27 ± 1.00
26 ± 2.05
18 ± 1.20
3 ± 1.86
77 ± 3.6
36 ± 10.7
5 ± 0.6
8 ± 2.89
4 ± 2.08
1 ± 1.53
2 ± 1.15
II
III
1
a
23 ± 1.57
60 ± 4.2
22

1
b
6 ± 2.77
15 ± 0.70
1 ± 1.97
1 ± 1.08
46 ± 1.56
9 ± 0.74
12 ± 1.28
5 ± 4.30
6 ± 1.10
1 ± 3.69
1 ± 2.92
9 ± 4.90
1 ± 2.62
26 ± 2.30
1 ± 0.35
*N/C
**N/F
19 ± 3.6
**N/F
**N/F
184 ± 5.3
**N/F
4 ± 10.6
**N/F
**N/F
**N/F
**N/F
**N/F
**N/F
80 ± 8.7
**N/F
*N/C
5 ± 3.32
4 ± 2.66
3 ± 3.59
3 ± 3.62
5 ± 4.11
8 ± 1.70
6 ± 1.34
5 ± 2.29
3 ± 5.31
2 ± 3.45
11 ± 4.54
5 ± 6.88
3 ± 4.04
5 ± 3.09
2 ± 2.72
*N/C
**N/F
**N/F
**N/F
**N/F
**N/F
2 ± 1.00
**N/F
**N/F
**N/F
**N/F
3 ± 1.53
**N/F
**N/F
**N/F
**N/F
*N/C
1
c
1
d
1
e
1
f
2
2
a
b
2
c
2
d
2
e
2
f
3
3
a
b
3
c
3
d
3
e
3
f
*N/C
*N/C
*N/C
*N/C
Trolox
A. A
70 ± 0.35
---
---
---
---
---
98 ± 0.34
---
*N/C (Not calculated) Insoluble;
*
*N/F (Not found)
Only compounds I, 1a, 1f, and 3c showed significant antiradical activity DPPH● (p
0.05) concerning the target. 2-mercaptobenzimidazole (MBZ) showed significantly
<
elevated antiradical activity (325 µmol ET/L). The compound 1f presented the highest
antiradical activity (184 µmol ET/L) followed by 3c and thioether I, which presented the
23

activity of 80 and 77 µmol ET/L respectively. This difference in antiradical activity
DPPH● between the amide compounds, thioethers (synthetic intermediates) and MBZ (raw
material) may be due to substitution of the two active MBZ sites, on the one hand, the alkyl
substitute in exocyclic sulfur, and the substitute (cinnamic derivative) in N1 to form the
corresponding amides.
To explain the results is necessary to know that two known mechanisms can reduce
the radical DPPH: HAT (Hydrogen Atom Transference) and ET (electron transference)
[
40]. There are differences between the authors, as some define it as an ideal radical for the
ET measurement method, while others classify it for the HAT method. However, it is
necessary to consider the structural characteristics of the antioxidant used [41]. In this assay
of antioxidant activity, the mechanism of HAT can better explain the obtained results.
24

For the transfer mechanism of a hydrogen atom to occur, the dissociation energies
of the N-H and S-H bonds must be considered. Data of the energies of dissociation of the
N-H bond can be calculated much scarcer for aromatic amines [42], it is known that the
energy of this bond is higher than that of the S-H. It explains why the MBZ presented high
antiradical activity compared to thioether derivatives I, II, and III, in which the S-H bond
no longer exists, thus making it impossible to maintain the stability of the newly formed
radical. The Sulfhydryl group is a crucial piece in the stability, either by retaining the
charge or building disulfide bridges, thus neutralizing the free radical [43] (Scheme 3).
Additionally, Figure 3 presents the frontier orbitals of the systems with the highest
antiradical capacity. It is essential to highlight that the areas with the highest energy levels
occupied and the lowest energy levels unoccupied are mostly concentrated on the MBZ
ring except for 2a and 3a compounds in the case of LUMO-type orbital.
25

Scheme 3. Mechanism of the antioxidant activity and stabilization of the MBZ against
●
DPPH .
Figure 3. Frontier molecular orbitals of the compounds that showed higher activity in in
vitro antioxidant assays. From left to right MBZ, I, II, III, 1a, 2a, and 3a.
According to Barbuceanu et al. (2014) [44], the S-alkyl compounds derived from
●
1
,2,4-triazoles showed an inhibitory effect in the antiradical activity DPPH proposing that
the increase of the chain of alkyl substitution in sulfur and with it the increase of
lipophilicity (>Log P) directly affects in the decrease of the antioxidant activity. This effect
can be observed in the tendency of the antiradical activity of the compounds evaluated I
(
26%), II (18%) and III (3%) is that as the S-alkyl chain is lengthened, the antioxidant
activity is considerably reduced.
●
+
In the trial ABTS synthetic intermediates (thioethers) I-III presented the best
radical entrapments, being I the one that presented the highest percentage (27%), followed
by intermediary II with 14% and thioether III with 11%. MBZ presented a very high
antiradical activity (88%) very close to that presented to the reference antioxidant (ascorbic
acid). On the other hand, amide derivatives 1(a-f)-3(a-f) presented low values of radical
entrapment (from 2 to 6%). However, they do not present a significant difference
26

concerning the negative reference. The compounds I and II presented values with
significant differences for the target, with 8 and 4 mg AAE/L (Ascorbic Acid Equivalents),
respectively, MBZ presented the highest antiradical activity (27 mg AAE/L). The data
●
confirm the trend seen in the assay by DPPH , that the presence of the substitute in the
exocyclic sulfur and the lengthening of the chain thus increasing the lipophilicity of the
compound, causes a significant reduction of the antiradical activity.
●
+
The ABTS
method is considered an ET method, although the chemical
characteristics of the antioxidant to be evaluated must be considered since these
characteristics have a direct influence on the most favored mechanism [45]. The mechanism
●
+
can also be influenced by the pH of the ABTS solution, since ethanol has a pH of 7.4,
where an alkaline pH is preferred to the ET mechanism, due to the chemical characteristics
●
+
of ABTS can be adapted to measure the antiradical activity of hydrophilic and lipophilic
compounds in media with different pH [46, 41]. DMSO was the measuring solvent in this
assay due to the insolubility compounds.
Donor Acceptor Map (DAM)
The DAM in different phases (gas, water, DMSO and pentyl ethanoate) were calculated for
MBZ-derived compounds (thioethers and amides). The DAM are shown in Figure 4(a-d).
Water is the dominant solvent in living organisms, DMSO is the solvent utilized for in vitro
●
●+,
tests of antiradical activities with DPPH and ABTS and pentyl ethanoate simulates the
lipid shell of cell membranes.
27

3
.5
2
2
1
0
.5
1
.5
0
0
0.1
0.2
0.3
Ra
0.4
0.5
0.6
(
a)
0
.8
.7
.6
.5
.4
.3
.2
.1
0
0
0
0
0
0
0
0
0
0.2
0.4
0.6
0.8
1
Ra
(
b)
28

0
0
0
0
0
0
0
0
.8
.7
.6
.5
.4
.3
.2
.1
0
0
0.2
0.4
0.6
0.8
1
Ra
(
c)
3
2
.5
2
.5
1
1
0
.5
0
0
0.2
0.4
0.6
0.8
1
Ra
(
d)
Figure 4. Donor Acceptor Maps in gas (a), water (b), DMSO (c) and pentylethanoate (d)
phase for compounds I-III, 1(a-f)-3(a-f) ( ) and MBZ ( ). Antioxidants of reference:
trolox ( ), ascorbic acid ( ), resveratrol ( ) and quercetin ( ).
In the gas-phase, MAD (Figure 4a, Table 2) for the study compounds, as well as the
reference antioxidants (Vitamin C, Trolox, Resveratrol, and Quercetin) included for
comparison. It shows that the evaluated compounds that present unsaturation and triple
29