Synthesis, electronic properties, antioxidant and antibacterial activity of some new benzimidazoles

Article abstract of DOI:10.1016/j.bmc.2015.08.029

Two groups of benzimidazole derivatives were synthesized using as precursors 5(6)-substituted 2-mercapto-benzimidazol-thiols and their antioxidant activity was investigated using TBA-MDA test. In the group of 1,3-disubstituted-benzimidazol-2-imines the highest lipid peroxidation inhibition effect 74.04% (IC₅₀ = 141.89 μg/mL) revealed ethyl [3-(2-ethoxy-2-oxoethyl)-2-imino-5-benzoyl-2,3-dihydro-1H-benzimdazol-1-yl]acetate 12 while in the group of 2-substituted-1,3-thiazolo[3,2-a]benzimidazolones the highest inhibition effect showed 2-(4-fluorobenzylidene)-7-(phenylcarbonyl)[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one 17 90.76% (IC₅₀ = 53.70 μg/mL). In order to estimate the capability of the studied benzimidazoles to act as radical scavengers the structure of the most active derivative within the both subseries was optimized at B3LYP/6-311++G?? level and the respective bond dissociation enthalpies were calculated. The appropriate models for the HAT and SET-mechanism of the antioxidant activity were proposed. The antibacterial activity of the compounds was evaluated against two Gram-positive bacteria (Bacillus subtilis ATCC 6633 and Staphylococcus aureus ATCC 6538) and three Gram-negative bacteria (Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027 and Salmonella abony NCTC 6017). 1,3-Diphenylpropyl-5-methyl-1,3-dihydro-2H-benzimidazol-2-imine 14 exhibited significant activity against B. subtilis, S. aureus, S. abony and E. coli (with MIC values of 0.125, 0.016, 0.50 and 0.50 mg/mL, respectively). The group of thiazolobenzimidazolones did not reveal antibacterial activity against the tested strains.

Full text of DOI:10.1016/j.bmc.2015.08.029

Accepted Manuscript
Synthesis, electronic properties, antioxidant and antibacterial activity of some
new benzimidazoles
Anelia Ts. Mavrova, Denitsa Yancheva, Neda Anastassova, Kamelya Anichina,
Jelena Zvezdanovic, Aleksandra Djordjevic, Dejan Markovic, Andrija
Smelcerovic
PII:
S0968-0896(15)30010-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.08.029
BMC 12533
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 June 2015
8 August 2015
25 August 2015
Please cite this article as: Mavrova, A.T., Yancheva, D., Anastassova, N., Anichina, K., Zvezdanovic, J., Djordjevic,
A., Markovic, D., Smelcerovic, A., Synthesis, electronic properties, antioxidant and antibacterial activity of some
new benzimidazoles, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.08.029
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Synthesis, electronic properties, antioxidant and antibacterial activity of some new
benzimidazoles
Anelia Ts. Mavrova a1, Denitsa Yancheva b, Neda Anastassova a, Kamelya Anichina a, Jelena
Zvezdanovic c, Aleksandra Djordjevic d, Dejan Markovic c, Andrija Smelcerovic e
a University of Chemical Technology and Metallurgy, 8 Kliment Ohridski Blvd., 1756 Sofia, Bulgaria
bInstitute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Science, Acad. G Bonchev Str.,
build. 9, 1113 Sofia, Bulgaria
cFaculty of Technology, University of Niš, Bulevar Oslobodjenja 124, 16000 Leskovac, Serbia
dDepartment of Chemistry, Faculty of Science and Mathematics, University of Niš, Višegradska 33, 18000 Niš, Serbia
eDepartment of Chemistry, Faculty of Medicine, University of Nis, Bulevar Dr Zorana Djindjica 81, 18000 Nis, Serbia
Abstract
Two groups of benzimidazole derivatives were synthesized using as precursors 5(6)-
substituted 2-mercapto-benzimidazol-thiols and their antioxidant activity was investigated using
TBA-MDA test.
In the group of 1,3-disubstituted-benzimidazol-2-imines the highest lipid peroxidation
inhibition effect 74.04 % (IC50 = 141.89 g/mL) revealed ethyl [3-(2-ethoxy-2-oxoethyl)-2-imino-5-
benzoyl-2,3-dihydro-1H-benzimdazol-1-yl]acetate 12 while in the group of 2-substituted-1,3-
1
Corresponding author: Anelia Mavrova, Department of Organic Synthesis, University of Chemical
Technology and Metallurgy, 8 Kliment Ohridski Blvd., 1756 Sofia, Bulgaria
e-mail anmav@abv.bg; tel. 0035928163207; fax 003592685488
1

thiazolo[3,2-a]benzimidazolones the highest inhibition effect showed 2-(4-fluorobenzylidene)-7-
(phenylcarbonyl)[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one 17 90.76% (IC50 = 53.70 g/mL). In
order to estimate the capability of the studied benzimidazoles to act as radical scavengers the structure
of the most active derivative within the both subseries was optimized at B3LYP/6-311++G** level and
the respective bond dissociation enthalpies were calculated. The appropriate models for the HAT and
SET-mechanism of the antioxidant activity were proposed.
The antibacterial activity of the compounds was evaluated against two Gram-positive
bacteria (Bacillus subtilis ATCC 6633 and Staphylococcus aureus ATCC 6538) and three Gram-
negative bacteria (Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027 and
Salmonella abony NCTC 6017). 1,3-Diphenylpropyl-5-methyl-1,3-dihydro-2H-benzimidazol-2-
imine 14 exhibited significant activity against B. subtilis, S. aureus, S. abony and E. coli (with MIC
values of 0.125, 0.016, 0.50 and 0.50 mg/mL, respectively). The group of thiazolobenzimidazolones
did not reveal antibacterial activity against the tested strains.
Keywords: 2-imino-benzimidazoles; 1,3-thiazolo[3,2-a]benzimidazolones, lipid peroxidation
inhibition, antibacterial activity, DFT calculations
1. Introduction
Benzimidazole heterocycle system is an important pharmacophore and privileged structure in
the medicinal chemistry. Its derivatives and particularly 2-aminobenzimidazoles exert various
biological activities such as antioxidant,1 antimicrobial,2 anthelmintic,3 anticancer,4 anti-
hypertensives,5 antiviral,6 and antifungal.7 The benzimidazole ring system exists in the structure of
many antiparasitic, anthelmintic, antifungal, antiviral and antitumor drugs. Some of them as
albendazole, omeprazole, lansoprazole, bendamustine etc. have found widespread application in
medicinal practice. Therefore the synthesis of new benzimidazoles and the study of their biological
properties are of pharmacological interest.
2

Many benzimidazoles derivatives were synthesized and proven for their antibacterial
activity.8,9 Benzimidazoles, containing thiadiazole or oxadiazole rings in 1-position were studied for
antibacterial and antifungal activity. All of the reported benzimidazoles showed good activity
towards Gram-positive bacteria and negligible activity towards Gram-negative bacteria.10,11 A series
of 2-substituted-5-nitro-benzimidazole derivatives showed comparable activity to that of ampicilline
against Gram-positive bacteria S. aureus, S. mutans, B. subtilis and to the activity of nalidixic acid
against Gram negative E. coli, S. typhi and P. aeruginosa.12 Furthermore the 2-aminobenzimidazole
dimers are identified as a novel scaffold that possesses antibacterial activity and as such may aid in
the discovery of new antibacterial agents.13 Benzimidazolopeptides as well as head-to-head bis-
benzimidazoles have shown good antimicrobial activity against pathogenic fungal strains and Gram
negative bacterial strains.14, 15
A series of benzimidazoles, containing 1,3,4-thiadiazole or 1,2,4-triazole rings were
synthesized and tested for antioxidant properties by using various in vitro systems.16 Albendazole,
substituted with benzoyl group in 1-position, possesses significant antioxidant activity.17
Thiazolo[3,2-a]benzimidazole derivatives inhibited the oxidation of adrenaline to adrenochrome, and
prevented the formation of the superoxide radical.18 The antioxidant potential of some coumarine-
benzimidazole hybrids as radical scavengers was evaluated.19 2-Aryl-1-arylmethyl-1H-
benzimidazoles can be considered as potential antioxidant and xanthine oxidase inhibitory agents.20
It is known that active oxygen species as superoxide, hydroxyl and peroxyl radicals are
causes of the oxidative stress associated with the pathogenesis of various diseases as Alzheimer's,
Parkinson's, cataracts and DNA damage leading to carcinogenesis. Taking into account that the
antioxidants and enzymes take part of the protective system of the organism it is of interest to
synthesize new benzimidazole derivatives and to study their antioxidant properties.
On the other site the spreading out of various resistant bacterial and fungal strains versus the
antibacterial drugs is a challenge to researchers to create and develop structurally new class of
3

molecules with new mechanisms of action. Thus the generation of new antibacterial agents as well
as antioxidants is of special interest in the field of medical chemistry. Therefore, the aim of the
present study was to synthesize two groups of benzimidazole derivatives using substituted
benzimidazol-2-thiols as starting compounds, 2-imino-2,3-dihydro-benzimidazole and 1,3-
thiazolo[3,2-a]benzimidazolone derivatives, to examine their antibacterial and antioxidant properties
and to perform SAR analysis using DFT calculations.
2. Results and Discussions
2.1. Chemistry
Two groups of some new benzimidazole derivatives were synthesized by use of 2-mercapto-
benzimidazoles as precursors, and their antibacterial and antioxidant properties were investigated.
The nucleophilic substitution of 2-amino-benzimidazoles 7-9 with halogen derivatives was
performed under solid-liquid phase transfer catalysis conditions in dry acetonitrile and resulted in
bis-1,3-disubstituted-2,3-dihydro-benzimidazole 10-14, obtained in good yields (65-86 %). The
second group of compounds was obtained by the reaction of thiazolobenzimidazolone 16 and
substituted arylaldehydes using pyridine as catalyst in good yields. The one-pot synthesis requires
prolonged reaction time, but the yields of the obtained end product are higher.
The
synthesis
of
1,3-thiazolo[3,2-a]benzimidazolones
and
2,3-dihydro-2-
iminobenzimidazoles is illustrated and outlined in Scheme 1.
4

O
N
N
S
O
O
O
H3CO
N
N
19
N
N
S
O
S
20
O
O
O
18
F
O
O
O
O
e
N
N
N
N
f
S
OH
S
F
S
N
N
H
15
16
O
17
O
d
O
N
N
N
OH
O
R
SH
+
R1
Cl
+
S
N
H
1-3
O
R1
1. R = CH3
2. R = C6H5CO
3. R = NO2
17 R1 =p-F
18 R1 = o-F
19 R1 = o-OCH3
O
20 R1 = 1,3-Dioxolyl
a
O
NH
O
R
c
N
N
N
b
R
SO3H
R
NH2
N
H
N
N
H
10 - 11
4. R = CH3
5. R = C6H5CO
6. R = NO2
4 - 6
7- 9
7. R = CH3
8. R = C6H5CO
9. R = NO2
10. R = CH3
11. R = NO2
O
O
O
O
NH
O
O
N
N
N
N
N
NH
O
NH
N
O
O
14
13
12
Scheme 1. Synthesis of the studied benzimidazole compounds. Reagents and conditions: a) KMnO4,
NaOH water solution, refluxing; b) NH4OH, 150 °C in welded ampoule; c) TBAB, dry K2CO3,
halogen derivative, 25 °C, acetonitrile; d) ClCH2COOH, NaOH/ethanol, refluxing 3 h; e)
5

(CH3CO)2O, pyridine medium refluxing 10 min; aromatic aldehydes, cat. Py, ethanol, refluxing 4-6
h.
The starting 4-substituted benzimidazolyl-2-thiols were synthesized by refluxing of 4-
substituted-1,2-diamino-benzenes, carbon disulfide, ethanol and sodium hydroxide according to the
method described in our earlier report.21 The Williamson reaction of the thiols with chloroacetic
acids in ethanol in the presence of excess of sodium hydroxide led to the corresponding thioacetic
o
acids. The cyclocondensation of thioacetic acids with acetic anhydride in pyridine medium at 100 C
resulted to 1,3-thiazolo[3,2-a]benzimidazolones. The compounds 17-20 were synthesized by the use
of two alternative methods. The first one, condensation between thiazolo[3,2-a]benzimidazolones
and aromatic aldehydes performed in ethanol gave the 1,3-thiazolo[3,2-a]benzimidazolones
substituted at the 2-position. By the second method compound 17-20 were synthesized by
Knovenagel condensation and subsequent cyclocondensation of the starting thiol, chloroacetic acid
and the appropriate aromatic aldehydes.
The 5-substituted-2-amino-benzimidazoles 7-9 were synthesized by oxidation of 5-
substituted-2-mercaptobenzimidazoles with water solution of KMnO4 in the presence of sodium
hydroxide to yield benzimidazol-2-yl-sulphonic acids, followed by the treatment of the sulphonic
acids with ammonium hydroxide in welded ampoule for 5 hours at 145-150 °C.22 The introduction
of substituents in 1-th and 3-th position of the 5-substituted 2-aminobenzimidazoles was performed
under solid-liquid phase transfer catalysis conditions in dry acetonitrile using dry potassium
carbonate and different halogen derivatives. Except for compound 12, the synthesis of all other
benzimidazole derivatives is reported here for the first time.
The chemical structures of the compounds were established by elemental analyses, IR and
1H NMR spectra and the results are presented in the Experimental part. The elemental analyses
indicated by the symbols of the elements were within ± 0.4% of theoretical values.
6

2.2. Pharmacology
2.2.1. Antibacterial activity
Antibacterial activity was evaluated against two Gram-positive and three Gram-negative
bacteria. Gram-positive bacteria used were: Bacillus subtilis ATCC 6633 and Staphylococcus aureus
ATCC 6538 while Gram-negative bacteria utilized in the assay were: Escherichia coli ATCC 8739,
Pseudomonas aeruginosa ATCC 9027 and Salmonella abony NCTC 6017. The antibacterial
properties of the studied compounds was determined by using a broth microdilution method in 96
multi-well microtitre plates.
Compounds 10-14 and 17-20 were examined as potential antimicrobial agents, starting from
initial 2 mg/mL concentration for all tested samples. The minimal inhibitory concentrations (MICs)
ranged from 0.016 to 1.0 mg/mL. Only three among the tested samples, i.e. compounds 10, 11 and
14 showed antimicrobial activity (Table 1). Compound 14 was the most active one being effective
against four bacteria, especially against S. aureus with MIC value of 0.016 mg/mL. According to
available data23 compounds 14 and 10 might be considered as antimicrobial agents with strong
activity while compound 11 belongs to the group of agents with moderate activity. The tested
samples were less effective than the antibiotic used as a reference standard. Comparing the
antibacterial activities of the group of 1,3-disubstituted-2-imino-benzimidazoles can be concluded
that the compounds possessing ester groups (12, 13) at positions 1 and 3 do not exhibit antibacterial
activity in contrast to those containing phenylethanone or phenylpropyl groups as substituents. It
could be supposed that the presence of phenylpropyl groups at positions 1 and 3 of 2-imino-
benzimidazol core is responsible for the potent antibacterial activity of the compound 14. This
compound possesses much higher lipophilicity than the other studied 1,3-disubstituted-2-imino-
benzimidazoles as well as the smallest topological surface area (see Table 3). Comparing the
activities of compounds 10 and 11 can be observed that the presence of methyl group at position 5 of
7

2-imino-benzimidazol core is more favorable for antimicrobial activity than the presence of nitro
group. The 2-substituted-1,3-thiazolo[3,2-a]benzimidazolones 17-20 did not show antibacterial
activity, therefore their results were not included in Table 1.
Table 1. Minimal inhibitory concentrations (MIC values given in mg/mL, unless stated otherwise)
of compounds 10-14
Samples
B. subtilis S. aureus
S. abony
n.a.
E. coli
n.a.
P. aeruginosa
0.50
1.00
n.a.
0.50
n.a.
n.a.
n.a.
10
n.a.
n.a.
11
n.a.
n.a.
n.a.
n.a.
12
n.a.
n.a.
n.a.
n.a.
n.a.
13
14
0.125
1.56
n.a.
0.016
0.78
n.a.
0.50
6.25
n.a.
0.50
0.78
n.a.
n.a.
Doxycycline (µg/mL)
DMSO (10%)
12.50
n.a.
2.2.2. Antioxidant activity
Lipid peroxidation (LP), as well as its inhibition in the presence of the synthesized
compounds, was measured by using TBA–MDA test. 24,25
Lipid peroxidation (LP), as well as its inhibition in the presence of the synthesized studied
benzimidazole derivatives, was measured by using TBA–MDA test, the method based on the MDA
(LP secondary product) reaction with TBA to obtain a red coloured complex with maximum
absorption at 530 nm The TBA-MDA complex absorbances in the supernatant read at 530 nm were
used to calculate the inhibition percentage of lipid peroxidation26 by using the equation given in the
experimental section, and the results are given in Table 2. All samples were assayed for LP
8

inhibitory activity at concentrations of 200 µg/mL in the fiinal reaction mixture. Those showing
inhibition greater than 50% at this concentration were tested in a broader concentration range to
allow calculation of IC50 values. The IC50 values represent the concentrations of studied
benzimidazoles in the reaction mixture which induce a 50% LP-inhibition effect after 3h incubation.
Table 2. Lipid peroxidation inhibition effects of studied benzimidazoles 10-14 and 17-20 (example
concentration 200 µg/mL) and IC50 values (given in g/mL)
Comp
No
Lipid Peroxidation
Inhibition Effect
Standard
Lipid Peroxidation
Inhibition Effect
antioxidants
(%)
(%)
IC50 g/mL)
IC50 g/mL)
39.26±3.11
23.43±2.79
74.04±0.90
55.87±3.16
62.15±5.71
90.76±1.04
75.76±6.78
80.35±2.08
60.80±5.35
> 200
> 200
141.89
176.58
144.88
53.70
86.97
74.11
85.51
Trolox
85.61 ± 2.05
91.12 ± 0.36
99.98 ± 2.52
99.90 ± 1.70
7.74
10.47
3.79
10
11
12
13
14
17
18
19
20
Quercetin
Caffeic acid
L-Ascorbic acid
9.12
The obtained results indicated that most of the tested compounds of both groups possessed
good antioxidant properties. The thiazolo[3,2-a]benzimidazolones demonstrated higher activity than
the 1,3-disubstituted-benzimidazol-2-imines. Highest inhibition percentage of lipid peroxidation was
expressed by 2-(4-fluorobenzylidene)-7-(phenylcarbonyl)[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-
one 17 (IC50 = 53.70 g/mL). The next by activity is compound 19 containing a methoxygroup at o-
position of the benzene ring with IC50 value of 74.11 %. The presence of the F-atom in 2-nd position
9

of benzene nucleus as in compound 18 led to decrease of lipid peroxidation inhibition (IC50 = 86.97
g/mL).
In the group of 1,3-disubstituted-2-imino-benzimidazoles compound 12 showed the most
effective LP inhibition (IC50 = 141.89 g/mL).
2.3. SAR
Three different mechanisms could be regarded when concerning how antioxidants deactivate
the free radicals – H-atom abstraction (HAT mechanism, see Eq. 1),27-30 electron transfer (SET
mechanism, see Eq. 2a-2b),30-33 and sequential proton loss electron transfer (SPLET mechanism, see
Eq. 3a-3b)27,34
:
.
.
R-H  R + H
(1)
.
R-H  R+ + e-
(2a)
.
.
R+  R + H+
(2b)
(3a)
(3b)
R-H  R- + H+
.
R-  R + e-
The efficacy of the antioxidant to react via HAT is characterized by the bond dissociation
enthalpy (BDE) – the lower BDE values, the higher the radical scavenging capacity of R-H. In the
SET mechanism, the reactivity is evaluated by ionization potential (IP). A lower IP implies an easier
electron release. The first step of the SPLET mechanism consists in formation of an anion and thus
the proton affinity (PA) of the formed anion is used as a measure for the reactivity. Lower PA values
indicate an easier extraction of the proton. The net result of the three radical scavenging mechanisms
is the same. SET and SPLET mechanisms are favoured in polar environment because the generated
charged species are stabilized by the solvent. Based on calculation of the reaction enthalpies for each
10

of the mechanisms, it is possible to suggest the most probable mechanism of action of a compounds
particular group.27
In order to estimate the capability of the studied iminobenzimidazoles 10-14 to act as radical
scavengers via HAT mechanism the structure of the most active derivative within the series
(compound 12) was optimized at B3LYP/6-311++G** level and the respective bond dissociation
enthalpies were calculated. The most favourable positions for hydrogen atom abstractions are
expected to be the two α-C atoms (site 1 and site 2, Fig. 1) from the side chains and the imino group
N-H (site 3, Fig. 1). It is known that the BDE of the α-C-H bonds in alkylamines decrease with
increasing number of alkyl sibstituents on the N and C atoms.35 In compounds 10-13 the α-C-H
bonds are additionally polarized by the adjacent ester groups.
According to the B3LYP/6-311++G** calculations, the most favorable position for hydrogen
atom abstraction is site 1 (Figure 1).
11

Figure 1. Hydrogen atom abstraction at different sites and corresponding DFT B3LYP/6-311++G**
bond dissociation enthalpies of compound 12 in kJ/mol.
In order to prevent effectively lipid peroxidation the antioxidants should have a lower BDE
value than the free lipid radicals. LOO. radicals typically display a BDE of about 367 kJ/mol, as
reported based on radical kinetics, gas-phase acidity cycles, and photoionization mass spectrometry
measurements.36 However, a lipid BDE value, representative for the chosen level of theory is needed
to enable an accurate comparison. For this purpose the BDE of O-H bond of methanol was
calculated in gas-phase:
12

.
.
BDE(CH3OH) = H(CH3O ) + H(H ) – H(CH3OH) = 413 kJ/mol
Effective chain-breaking antioxidants such as α-tocopherol show substantially lower BDE -
327 kJ/mol according to the calculations in gas-phase the same computational scheme.27
Comparing the BDE value calculated for methanol with those of compound 12, the N-H
bond should be excluded as probable site for HAT reaction. The BDE values related to the formation
of C-centered radicals are lower than those of the N-centered one by 40 kJ/mol. The atomic spin
population analysis show that the formation of C-centered radicals is associated with more efficient
delocalization of the odd electrons through the conjugated system than in the case of the N-centered
one. This leads to better radical stability for the C-centered radicals, and especially those formed by
HAT from site 1. Based on the obtained enthalpies the following hypothetical mechanism could be
suggested:
.
LOH
or LOOH
LO
.
.
or LOO
R2
NH
R2
R2
NH
R2
N
N
N
N
(hydrogen atom
transfer)
R1
R1
Antioxidant mechanism: HAT
Scheme 2. Hypothetical mechanism of antioxidant action of compounds 10-14.
The compounds in the second studied subseries of benzimidazoles 17-20 possess an aromatic
structure without any OH and NH groups. Compounds 17-20 might exist in two diastereoisomeric
forms (Z and E) concerning the relative orientation of the phenyl rings and the thiazolone fragment.
According to the B3LYP/6-311++G** optimization of the structure of the most active derivative
within the series 17, the difference between the ZPVE-corrected total energies of the more stable Z-
isomer and the less stable E-isomer is 16.9 kJ/mol and therefore E-isomer is not expected to be
present in solution or solid state. The NMR data show no evidence of that isomer. The only possible
13

site for H-abstraction is the benzylidene group (Fig. 2), thus feasibility of the HAT mechanism was
estimated based on the respective BDE. As it can be seen in Fig. 2, the calculated value is 421
.
kJ/mol which is higher than those of CH3O , excluding the HAT as possible mechanism of action.
Figure 2. Hydrogen atom abstraction and corresponding DFT B3LYP/6-311++G** bond dissociation
enthalpy of 17 in kJ/mol.
On the other hand, compounds 17-20 might easily donate an electron due to the presence of
their extended conjugated system. They resemble in structure to some recently investigated 2-amino-
5-alkylidenethiazol-4-ones26 and pyrrolopyrimidines37 exhibiting antioxidant action. Similarly to
these cases, a mechanism of stepwise oxidation could be suggested for compounds 17-20 (Scheme
.
3). These compounds could produce radical cations able to scavenge the lipid alkoxyl (LO ), lipid
.
.
peroxyl (LOO ) or hydroxyl ( OH) radicals by initial electron transfer (SET mechanism), then form
an intermediate adduct and terminate the process by a proton transfer (Scheme 3):
14

.
.
LO
or LOO
LO
or LOO
.
H
LO
or LOO
6
O
O
N
.
5
3
4
2
.
1S
S
R1
R1
(electron
transfer)
N
O
O
(LO)
(LO)
LOO
-
.
LOH
or LOOH
LO
or LOO
LOO
LO
or LOO
O
N
O
-
.
H
S
S
R1
R1
(proton
transfer)
N
O
Adduct
O
Antioxidant mechanism: SET
Scheme 3. Hypothetical mechanism of antioxidant action of compounds 17-20.
The structure of the radical cations and adducts formed in the course of the antioxidant action
according to Scheme 3, were further investigated by modeling compound 17 and including a
methoxy radical to represent the lipid. The optimized geometry of the radical cation shows a planar
orientation of the phenyl ring and thiazolobenzimidazolone fragment. The S1-C5 bond is
substantially shortened and the C5-C6 bond is much longer compared to the parent neutral molecule.
The analysis of the spin densities over the atoms indicates that approximately one third of unpaired
electron is localized in the fragment S1-C5-C6 and could be illustrated by the resonance structures
showed on Scheme 4.
15

H
H
O
N
N
O
N
N
S
R
S
R
O
O
H
O
N
N
or
S
R
O
Scheme 4. Resonance structures of the peroxyl scavengers (radical cations) of compounds 17-20.
Thus the attack of the lipid radicals would be expected at S1, C5 and C6. The adducts formed by
addition of methoxy radical at these sites were studied by optimizing the geometry of the relevant
cation products of compound 17. According to the calculated ZPVE-corrected total energies,
addition at C6 seems to be the most favorable. The adduct has tetrahedral structure due to the sp3
hybridization of C6 with two possible stereo configurations – S and R (Fig. 3). In the final step of
the proposed antioxidant reaction, after transferring a proton to the lipid anion, the molecule would
restore its planarity.
A
B
16

Figure 3. Stereo isomers of the methoxy adduct of compound 17 – S configuration at C6 (A) and R
configuration at C6 (B).
In view of future medical application, the promising pharmacological potential of studied
benzimidazoles demonstrated above, should match also favorable pharmacokinetical behavior in
living organisms i.e. sufficient bioavailability and transportation through different membranes to the
site of action, optimal process of metabolization and elimination. In this relation the prediction of
molecular properties such as lipophilicity, molecular size, flexibility and presence of hydrogen-
donor and acceptors could provide useful preliminary information.
Table 3. Calculated molecular properties of compounds 10-14 and 17-20 for assessment of the drug-
likeness
c
e
f
g
h
Compd. milogPa TPSAb Natoms MWd NON NOHNH Nviol. Nrotb.
Voli
No.
( < 10 )
Rule
10
11
12
13
14
17
18
19
20
< 5
2.83
< 500 < 10 < 5
67
113
103
86
29
31
30
23
29
29
29
30
31
383
414
409
319
383
400
400
412
426
5
8
8
7
3
4
4
5
6
1
1
1
1
1
0
0
0
0
0
0
0
0
1
1
1
1
1
6
7
352
2.34
2.02
1.01
5.18
5.30
5.25
5.15
5.03
358
367
293
381
329
329
350
348
10
8
33
8
51
3
51
3
60
4
69
3
17

b
a octanol-water partition coefficient, calculated by the methodology developed by Molinspiration;
c
d
polar surface area; number of nonhydrogen atoms; molecular weight; e number of hydrogen-bond
f
g
acceptors (O and N atoms); number of hydrogen-bond donors (OH and NH groups); Number of
"Rule of five" violations; h number of rotatable bonds; i molecular volume.
Based on the analysis of a large number of drugs, Lipinski and coworkers established an
effective methodology for estimation of potential drug solubility and permeability.38 The required
molecular properties were conveniently set into a "Rule of five": poor absorption or permeation are
more likely to occur when the molecule has molecular weight more than 500, log P over 5, and
contains more than 5 H-bond donors or 10 H-bond acceptors. More than one violation of the rule is
the critical limit for acceptable drug-likeness.
The physico-chemical properties of the studied benzimidazoles, calculated using
Molinspiration tool,39 are shown in Table 3. Data indicate that none of the compounds is above the
critical limit established by the "Rule of five". Molinspiration methodology for calculation of
miLogP implements fragment-based contributions and correlation factors which makes it robust and
widely applicable tool. Compounds 10-13 show miLogP values below 5 which is favorable feature
for their oral bioavalability. The highest lipophilicity among the group of 1,3-disubstituted-2-imino-
benzimidazoles is observed with compound 14 that contains only phenylpropyl substituents without
any polar groups. The derivatives containing thiazolobenzimdazolone moiety 17-20 show
significantly greater lipophilicity with miLogP values slightly above 5. Their higher lipophilicity is
due to their smaller polar surface area which is another useful descriptor of the oral bioavailability40
and drug transport properties.41,42 It is being expressed here as topological surface area (TPSA)
which is a sum of the surface areas occupied by oxygen and nitrogen atoms and the hydrogens
attached to them. TPSA represents the hydrogen bonding capacity of the molecules. Molecules with
TPSA less than 140 Å2 are recognized to have good intestinal absorption, and those with TPSA less
than 60 Å2 show good blood-brain barrier penetration.41,42 All presented benzimidazoles are
18

expected to have good intestinal absorption and most of them – also good blood-brain barrier
penetration.
On the other hand, all compounds containing N-alkyl substituents show higher
conformational flexibility manifested through their greater number of rotatable bonds. The number
of rotatable bonds is an important factor for the efficient binding to receptors and channels as well as
for the oral bioavailability.40 Molecules with more than 10 rotatable bonds tend to show poor oral
bioavailability. All studied compounds, satisfy this criterion and compounds 17-20 have low
conformational flexibility with less than 5 rotatable bonds. The benzimidazole moiety itself has no
rotatable bonds and could be regarded as a rigid linker.
Hydrogen bonding capacity of the drug candidates is also described by the number of H-bond
donors and acceptors. The compounds in the series show 3 to 8 H-bond acceptors, but none (17-20)
or one (10-14) H-bond donor. Molecular volumes of the compounds in the series are less than 400
Å3. Summarizing the physico-chemical properties of studied benzimidazoles, we could conclude that
they obey the "Rule of five" and meet all criteria for good solubility and permeability.
3. Conclusion
Two groups of benzimidazole derivatives were synthesized by use of 2-
mercaptobenzimidazoles as precursors in order to estimate their antioxidant and antibacterial
properties. The results obtained by the screening showed that derivatives of 2-iminobenzimidazoles
10 and 14 possessed strong activity against B. subtilis, S. aureus, S. abony and E. coli whereat their
MIC50 were in the range 0.016 - 0.50 mg/mL. The thiazolo[3,2-a]benzimidazolones demonstrated
higher
activity than the 1,3-disubstituted-benzimidazol-2-imines The 2-substituted
thiazolobenzimidazoles 17 and 19 revealed very strong lipid peroxidation inhibition effects with IC50
values of 53.70 g/mL and 74.11 g/mL, respectively. SAR analysis was performed and some
electronic properties of the compounds were established using B3LYP/6-311++G** calculations. On
the basis of calculated bond dissociation enthalpies, it was suggested that iminobenzimidazoles 10-
19

14 might act as radical scavengers via hydrogen atom abstraction preferably in the α-C atom of the
side chains attached to the iminobenzimidazole rings. Based on the higher BDE for the alkilydene
group in benzimidazoles 17-20, the HAT was excluded as possible mechanism of action of these
compounds. It was suggested that due to their electron-donating properties, compounds 17-20 might
undergo a stepwise oxidation via SET mechanism and produce radical cations able to scavenge the
.
.
.
lipid alkoxyl (LO ), lipid peroxyl (LOO ) or hydroxyl ( OH) radicals.
4. Experimental
4.1. Chemistry
Melting points (mp) were determined on an Electrothermal AZ 9000 3MK4 apparatus and were
uncorrected. IR spectra were recorded on a Bruker spectrophotometer with potassium bromide discs
13
and as well as on aVarian 660-IR, FT-IR Spectrometer. 1H and C NMR spectra were recorded on
a Bruker Avance II+ 250 MHz and a Bruker Avance II+ 600 MHz NMR instrument. The spectra
are referred to the solvent signal. Chemical shifts are expressed in ppm and coupling constants in
1
13
Hz. The precise assignment of the H and C NMR spectra was accomplished by measurement of
2D homonuclear correlation (COSY), DEPT-135 and 2D inverse detected heteronuclear (C–H)
correlations (HMQC and HMBC). The microanalyses for C, H, N and S were performed on Perkin-
Elmer elemental analyzer. The reactions were monitored by thin layer chromatography, which was
performed on Merck pre-coated plates (silica gel. 60 F254, 0.25 mm) and was visualized by
fluorescence quenching under UV light (254 nm).
The starting 4-substituted-1,2-diaminobenzenes were commercial available.
The thiols 1-3 as well as the 2-sulphonic acids and the 2-aminobenzimidazoles were
synthesized according to the procedure described by us earlier21,22
:
4.1.1. 5-Methyl-1H-benzimidazole-2-thiol 1: Yield –71.3 %; Mp >300°C; IR, KBr, (cm-1): νNH
3426; νAr 3126; νSH 2570; δAr 1634, 1618, 1562; δCH3 1372; νC-S 1118; δAr 799; 1H- NMR
20

(DMSO-d6)  (ppm): 13.12 (s, 1H,SH), 10.48 (s, 1H, NH), 7.89 (s, 1H, 9-H, Ar), 7.53 (m, 2H, 6-H,
7-H, J = 8.45), 2.66 (s, 3H, CH3); Analysis: Calc. for C8H8N2S: C, 58.51; H, 4.91; N, 17.06; S,
19.52; Found: 58.48; H, 4.95; N, 17.16; S, 19.60.
6.1.2. (2-Mercapto-1H-benzimidazol-5-yl)(phenyl)methanone 2: Yield – 96,8 %; Mp – 248- 250 °C;
Rf = 0.68, mobile phase: 1H NMR (DMSO-d6)  (ppm): 1.877 (s, 1H, SH), 7.251 (d, J=8.2 Hz, 1H,
7-H), 7.462 (d, J=1.6 Hz, 1H, 4-H), 7.545 (dd, J=8.2 Hz, J=1.6 Hz, 1H, 6-H), 7.558 (t, J=7.7 Hz, 2H,
m-Ph), 7.658 (dt, J = 7.4 Hz, J=1.4 Hz, 1H, p-Ph), 7.700 (dd, J=8.1 Hz, J=1.4 Hz, 2H, o-Ph), 12.75
13
(bs, 1H, NH); C NMR (DMSO-d6)  (ppm): 109.30(7-C), 110.81(4-C), 125.12(6-C), 128.48(m-
Ph), 129.35(o-Ph), 130.51(3a-C), 132.12(p-Ph), 132.92(7a-C), 137.14(5-C),137.99(i-Ph),
170.78(C=N), 195.08(C=O); Analysis: Calc. for C14H10N2OS; C, 66.12; H, 3.96; N, 11.02; O, 6.29;
S, 12.61; Found: C, 66.09; H, 3.91; N, 11.12; O, 6.32; S, 12.64.
4.1.3. 5-Nitro-1H-benzimidazole-2-thiol 3: Yield – 84%; Mp – 245-247°C; Rf - 0.41, mobile phase:
1
CHCl3/ethyl acetate = 6:1. H NMR (DMSO-d6)  (ppm): 7.94(dd, 1H, CH, J = 9.10 Hz); 8.13 (m,
1H, CH); 8.34 (d 1H), 12.32(s, 1H, SH); Analysis: Calculated for C7H5N3O2S: C, 43.07; H, 2.58; N,
21.53; O, 16.39; S, 16.43; Found: C, 43.17; H, 2.53; N, 21.57; O, 16.41; S, 16.42.
4. 2. General procedure for compounds 10-14
To a solution of 5(6)-substituted-2-aminobenzimidazole (0.01 mol) in dry acetonitrile (50
mL) was added anhydrous potassium carbonate (2.7 g, 0.02 mol) and tetrabutylammonium bromide
(TBAB) (0.9 g, 0.003 mol) and 0.02 mol of the halogen organic reagent was dropped by cooling.
The mixture was stirred for 8-10 hours vigorously at 25 ºC and monitored by TLC over the reaction
period. After completing the reaction, the mixture was filtered to separate the solid K2CO3, and the
organic solvent was removed under vacuum.The residue was then crystallized from appropriate
solvent to give the relevant compounds 10-14.
4.2.1.
2-[2-Imino-5-methyl-3-(2-oxo-2-phenylethyl)-2,3-dihydro-1H-benzimdazol-1-yl]-
phenylethanone 10: Yield 69%; Mp. 283-285 °С; Rf - 0.71, mobile phase CHCl3/CH3OH - 8:2; IR,
21

KBr, (cm-1): νNH 3335; νC=O 1689; δNH 1662; 1Н NMR (DMSO-d6)  (ppm): 2.367 (s, 3H, CH3);
5.992 (s, 4H, 2CH2); 7.133 (d, 2H, Ar); 7.630 (t, 3H, Ar), 7.813 (m, 3H, Ar); 8.076 (m, 2H, Ar);
+
8.131 (m, 3H, Ar); 8.834 (m, 2H, NH2 ); 13C NMR (DMSO-d6)  (ppm): 20.98 (C-7); 52.04(C-10,
C-14); 128.18(C-9); 128.18(C-9); 128.51(C-6); 12895(C-8); 133.64 (C-19, C-23, C-25); 127.83 (C-
20, C-22, C-26, C-28); 133.64 (C-21, C-27); 134.10 (C-18, C-24); 151.08(C=N); 191.04 (C-11, C-
15); Analysis: Calc. for C24H21N3O2: C, 75.18; H, 5.52; N, 10.96; O, 8.35; Found: C, 75.20; H,
5.49; N, 10.98; O, 8.39.
4.2.2.
2-[2-Imino-5-nitro-3-(2-oxo-2-phenylethyl)-2,3-dihydro-1H-benzimdazol-1-yl]-1-
phenylethanone 11: Yield 82%, Mp. 246°С, Rf - 0.71, mobile phase CH2Cl2/CH3OH - 8:1; IR, KBr,
1
(cm-1): νNH 3338; νC=O 1679; δNH 1652; δNO2 1335; δAr 751, 691; Н NMR (DMSO): 5.732(s,
4H, 2CH2); 7.445(s, 1H,Ar); 7.636(m, 4H, Ar); 7.752(t, 2H, Ar); 8.072(s, 4H, Ar); 8.119(s, 1H, Ar);
13C NMR (DMSO-d6)  (ppm): 48.04(C-10, C-14); 104.59(C-9); 108.43(C-6); 118.31(C-7);
128.4(C-20, C-24); 129.02(C-26, C-30); 130.00 (C-29, C-27); 134.26(C-22, C-28); 134.35(C-4);
134.38(C-5); 135.34(C-19, C-20), 142.18(C-NO2); 154.35(C=N); 191.67(C=O); 192.78(C=O);
Analysis: Calc. for C23H18N4O4: C, 66.66; H, 4.38; N, 13.52; O, 15.44; Found: C, 66.62; H, 4.36; N,
13.56; O, 15.47.
4.2.3. Ethyl [3-(2-ethoxy-2-oxoethyl)-2-imino-5-benzoyl-2,3-dihydro-1H-benzimdazol-1-yl]acetate
12: IR, KBr, (cm-1): νNH 3457; νC=O 1736; νC=O 1691 δNH 1649; δCH3 1377; νC-O-C1208; δAr
1
746, 713; H NMR (DMSO-d6)  (ppm): 1.191 (t, J=7.1Hz, 6H, CH3), 4.152 (q, J=7.1 Hz, 4H,
OCH2), 4.262 (s, 4H, NCH2), 7.107 (d, J=8.1Hz, 1H, 7-Ar), 7.346(d, J=8.1Hz, 1H, 6-Ar), 7.446 (s,
1H, 4-H), 7.536 (t, J=7.7Hz, 2H, m-Ph), 7.640 (t, J=7.4Hz, 1H, p-Ph), 7.678(dd, J=8.2, 1.2Hz, 2H, o-
Ph); 13C NMR (DMSO-d6)  (ppm): 13.97 (CH3), 47.48 (NCH2), 60.14 (OCH2), 105.77 (7-C),
107.92 (4-Ar), 109.58 (6-Ar), 128.37 (m-Ph), 129.10 (7a-C), 129.30 (o-Ph), 131.95 (p-Ph), 132.25
22

(3a-C), 138.06 (i-Ph), 152.22 (C=N), 167.77 (O-C=O), 171.52 (C=O). Analysis: Calc. for
C22H23N3O5; C, 64.54; H, 5.66; N, 10.26; O, 19.54; Found: C, 64.57; H, 5.61; N, 10.28; O, 19.49.
4.2.4. Ethyl [3-(2-ethoxy-2-oxoethyl)-2-imino-5-methyl-2,3-dihydro-1H-benzimdazol-1-yl]acetate
hydrobromide 13: Yield – 65 %; Mp 297-299 °С, Rf – 0,66; mobile phase CHCl3/ CH3COOC2H5;
IR, KBr, (cm-1): νNH 3342; νC=O 1733; δNH 1666; δCH3 1378; 1Н NMR (DMSO-d6)  (ppm):
1.226 (t, 6H, 2CH3 ), 2.389 (s, 3H, CH3 ), 4.207 (q, 4H, 2O-CH2 ), 5.168 (s, 4H, 2CH2 ), 7.154 (d,
+
13
1H, Ar ), 7.461 (t, 2H, Bz ), 9.039 (bs, 2H, NH2 ); C-NMR(DMSO-d6)  (ppm): 13.53 (C-20),
14.08 (C-23), 21.03 (C-17), 61.85 (C-19, C-22), 110 (C-9), 127.69 (C-6), 133.87 (C-2), 150.65
(C=N), 166.50 (C=O); Analysis: Calc. for C16H21N3O4: C, 60.17; H, 6.63; N, 13.16; O, 20.04;
Found: C, 60.20; H, 6.61; N, 13.14; O, 20.06.
6.2.5. 5-Methyl-1,3-bis(3-phenylpropyl)-1H-benzo[d]imidazol-2(3H)-imine hydrobromide 14:
Yield - 72 %, Mp 223-225 °С, Rf- 0.55, mobile phase CH2Cl2/CH3OH; IR, KBr, (cm-1): νNH-
1
3409; δNH 1676; δCH3 1382; Н NMR (DMSO-d6)  (ppm): 1.987 (t, 4H, 2CH2); 2.399 (s, 3H,
CH3); 2.643 (m, 4H, 2CH2); 3.467 (m, 4H, 2CH2 ); 7.168 (m, 7H, CH); 7.249 (m, 4H, CH); 7.314
+
(s, 1H, CH); 8.738 (s, 2H, NH2 ); Analysis: Calc. for C26H29N3: C, 81.42; H, 7.62; N, 10.96; Found:
C, 81.42; H, 7.62; N, 10.96.
4.3. Synthesis of (5-phenylcarbonyl-1H-benzimidazol-2-ylthio)acetic acid 15: A solution of sodium
hydroxide (0.012 mol) in ethanol (14 mL) and 5(6)-(un)substituted-1H-benzimidazole-2-thiol
(0.0067 mol) was refluxed for 1 h. After cooling, chloroacetic acid (0.0067 mol) was added and the
refluxing was continued for 5 h more. Then the reaction mixture was cooled, poured into water and
acidified with diluted acetic acid. The crystallized product was filtrated, carefully washed with
water and re-crystallized with ethanol; Yield: 85%; Mp 173-175; Rf 0.4 C6H6/CH3OH = 2:1; IR,
KBr, (cm-1): νOH- 3440; νNH 3208; νC=O 1747; νC=O 1649; 1H NMR (DMSO-d6)  (ppm): 3.91
(s, 2H, CH2); 7.58 (dd, 2H, m-Ph, J= 7.15 Hz); 7.70 (m, 1H p-Ph); 7.87 (d 1H, o-Bz, J=8.40 Hz);
7.92 (d, 1H, o-Ph, J=7.38 Hz); 7.94 (m, 1H, o-Bz); 8.04 (d, 1H, m-Bz); 9.82 (bs, 2H, NH, COOH);
23

Analysis: Calc. for C16H12N2O3S; C, 61.53; H, 3.87; N, 8.97; O, 15.37; S, 10.27; Found: , 61.55; H,
3.89; N, 8.95; O, 15.34; S, 10.29.
4.4. Synthesis of 6-(phenylcarbonyl)[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one 16: A solution of
5-phenylcarbonyl-(1H-benzimidazol-2-ylthio)acetic acid (0.104 mol) and acetic anhydride (19.5
mL) in dry pyridine (65 mL) was heated under reflux for 10 min. After completing of the reaction
the solution was cooled and the product was crystallized as pale orange sediment by addition of
water (100 mL) in small portions by stirring. Yield 66%; Mp-147-150; °С, Rf 0.5; mobile phase
-1
;
CH3COOC2H5/C6H6 - 1:4; IR, KBr, (cm ): νC=O 1747; νC=O 1648 νC=O 1648; νC-S 1369, 1293;
1H NMR (DMSO-d6)  (ppm): 4.648 (s, 2H) and 4.652 (s, 2H, CH2), 7.55-7.60 (m, 4H, m-Ph), 7.69-
7.72 (m, 2H, p-Ph+p-Ph), 7.73-7.76 (m, 6H, o-Ph+7-Ar+8-Ar), 7.806 (dd, J=8.4 Hz, J=1.7 Hz, 1H,
7-Ar), 7.894(d, J=1.1 Hz, 1H, 5-Ar), 8.014(d, J=8.2 Hz, 1H, 8-Ar), 8.157(d, J=1.2 Hz, 1H, 5-Ar);
Analysis: Calc. for C16H10N2O2S; C, 65.29; H, 3.42; N, 9.52; O, 10.87; S, 10.89; Found:
C16H10N2O2S; C, 65.31; H, 3.40; N, 9.54; O, 10.88; S, 10.85.
4.5. General procedure for compounds 17-20
A) 0.0105 mol of the relevant aldehyde was added to the solution of 0.01 mol 3a-d in 50 mL
absolute ethanoland 3-4 drops of pyridine. After several hours of reflux, the solution was cooled and
the precipitate obtained was washed with ethanol, followed by double re-crystallization from
ethanol.
B) Mixture of 0.01 mol 1H-benzimidazole-2-thiol, 0.015 mol chloroacetic acid, 0.01 mol aromatic
aldehyde, 2-3 drops piperidine and glacial acetic acid were refluxed for 4 h. After cooling, the
product obtained was re-crystallized with ethanol.
4.5.1. 2-(4-fluorobenzylidene)-6-(phenylcarbonyl)[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one 17:
Yield: 66 /76 %.; Mp 209-210 оС; Rf = 0.3, mobile phase: CH3COOC2H5/C6H6 - 1: 8; IR, KBr, (cm-
1): νC=O 1721; νNO2 1341; δAr 829; 1Н NMR (CDCl3): 8.505 (s, 1H, CH), 8.350 (ds, 1H, CH),
24

8.146 (t, 1H, 1CH), 7.904 (q, 1H, 1CH), 7.849 (d, 2H); 7.783 (d, 1H), 7.672-7.612 (dt, 2H), 7.515
(q, 3H, 3CH), 7.353 (t, 1H, CH); Analysis: Calc. for C23H13FN2O2S: C, 68.99; H, 3.27; F, 4.74; N,
7.00; O, 7.99; S, 8.01; Found: C, 68.95; H, 3.31; F, 4.73; N, 7.04; O, 7.94; S, 8.04;
4.5.2. 2-(2-fluorobenzylidene)-6-(phenylcarbonyl)[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one 18:
Yield - 51/65 %; Mp -232-235°С; Rf 0.64, mobile phase: CH3COOC2H5/C6H6 - 1: 8; IR, KBr, (cm-
1): νC=O 1732; νC=O 1653; νAr 1613; 1550; 1506; δAr 756; 698; 1Н NMR (CDCl3): 8.504 (s, 1H,
CH), 8.077 (m, 1H, CH), 7.917 (d, 1H, 1CH), 7.888 (t, 1H, 1CH), 7.806 (d, 2H); 7.774 (d, 1H),
7.648 (m, 3H), 7.537 (m 3H); Analysis: Calc. for C23H13FN2O2S: C, 68.99; H, 3.27; F, 4.74; N,
7.00; O, 7.99; S, 8.01; Found: C, 68.91; H, 3.27; F, 4.76; N, 7.06; O, 7.96; S, 8.05;
4.5.3. 2-(2-methoxybenzylidene)-6-(phenylcarbonyl)[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one 19
Yield- 62/75 %; Mp - 235-237°C; Rf 0.62, mobile phase: CH3COOC2H5/C6H6 - 1: 8; IR, KBr, (cm-
1): νAr 3031; νCH3 2840; νC=O 1725; νC=O 1654; νAr 1596; 1560; 1487; δCH3 1440; 1362; νC-O-
1
C 1252; 1158; δAr 694, 746; δAr 722; Н NMR (CDCl3): 8.500 (t, 1H, CH, J = 8.52Hz), 8.144(t,
1H, CH), 7,907 (m, 1H), 7.880 (d, 2H, 2CH); 7.769 (d, 1H, CH), 7.607 (m, 5H, 5CH); 7.116 (t, 1H,
CH), 7.008 (d, 1H, CH), 3.958 (s, 3H, CH3); Analysis: Calc. for C24H16N2O3S: C, 69.89; H, 3.91;
N, 6.79; O, 11.64; S, 7.77; Found: C, 69.91; H, 3.89; N, 6.77; O, 11.67; S, 7.74.
4.5.4. 2-(1,3-benzodioxol-5-ylmethylidene)-6-(phenylcarbonyl)-[1,3]thiazolo[3,2-a]benzimidazol-
3(2H)-one 20: Yield: 45 /60 %; Mp - 165-167 °C; Rf 0.60 mobile phase CH3COOC2H5/C6H6 = 1:4;
IR, KBr, (cm-1): νC=O 1717; νC=O 1654 ; νAr 1614; 1580; 1508; δCH2 1440; νC-O-C 1244; 1112;
1H NMR (CDCl3)  (ppm): 5.93 (s, 2H, CH2); 7.24 (dd, 2H, o-Pip, J = 8.22 Hz); 7.36-7.39 (m, 1H,
m-Pip); 7.57 – 7.60 (m, 2H, o-Ph); 7.67 – 7.71 (m, 1H, p-Ph); 7.92 (m, 2H, o-Ph, J = 7.38 Hz);
7.99 (s, 1H, CH=C); 8.20 (d, 1H, o-Bz, J= 8.40 Hz); 8.36 (d, 1H, m-Bz); 8.47 (s, 1H, o-Bz);
Analysis: Calc. for C24H14N2O4S: C, 67.60; H, 3.31; N, 6.57; O, 15.01; S, 7.52; Found: C, 67.62; H,
3.34; N, 6.54; O, 15.03; S, 7.49.
25

4.6. Assay for in vitro antibacterial activity
The in vitro antimicrobial activity of samples 10-14 and 17-20 was tested against a panel of
laboratory control strains belonging to American Type Culture Collection Maryland, USA and
National Collection of Type Cultures, UK. Antimicrobial activity was evaluated against two Gram-
positive bacteria (Bacillus subtilis ATCC 6633 and Staphylococcus aureus ATCC 6538) and three
Gram-negative bacteria (Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027 and
Salmonella abony NCTC 6017).
The minimal inhibitory concentration (MIC) of samples, against tested bacteria was
determined by using a broth microdilution method in 96 multi-well microtitre plates.43 After
overnight cultivation, microbial suspensions were made in Mueller Hinton broth and their turbidity
was standardised to 0.5 McFarland. Dimethyl sulphoxide (10%, v/v aqueous solution) was used to
dissolve and to dilute sample. A serial double dilution of the sample was prepared in 96 well
microtitre plates, using method of Sarker et al.44 The lowest concentration of the sample that
inhibited visible growth was taken as the MIC value. One row was used as a positive control and
contained a broad-spectrum antibiotic (doxycycline in a serial dilution of 200–0.05 μg/ml) to
determine the sensitivity of Gram-negative and Gram-positive bacteria while the other row contained
the solvent as negative control. Tests were carried out in triplicate.
4.7. Lipid peroxidation inhibition
Phospholipids (Phospholipon® 90; PL90) were gifted by PHOSPHOLIPID GMBH, Cologne,
Germany. According to the accompanied declaration the mixture content of PL90 is:
phosphatidylcholine 98%, lyso-phosphatidylcholine 2.1%; fatty acid composition: palmitic acid
12±2%, stearic acid 3±1%, oleic acid 10±3%, linoleic acid 66±5%, linolenic acid 5±2%, peroxide
value max. 1.3. The PL90 were kept in dark to prevent at least the photooxidation process.
26

Thiobarbituric acid (TBA), 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) and
standards of trolox, quercetin, caffeic acid and L-ascorbic acid were obtained from Sigma Aldrich.
4.7.1. TBA-MDA test
The reaction mixture of 0.3 mL contained methanol solution of lipids (PL90) (1·10-2 mol/L) and
methanol solutions of selected synthesized compounds (1 g/L) in 2:1 (v/v) ratio. Lipid peroxidation
was initiated by using 0.2 mL (2.2·10-2 mol/L) aqueous solution of hydrophilic thermal initiator of
lipid peroxidation (AAPH – 2,2′-azobis(2-methylpropionamidine) dihydrochloride) during time
period of 3 h at 40 ºC. One centimeter cube of aqueous trichloroacetic acid (5.5 %), followed by 0.5
mL of TBA (4.2·10-2 mol/L in 5·10-2 mol/L NaOH) and BHT (1·10-3 mol/L) were added in the
reaction mixture immediately after 3 h of initiation. The mixture was incubated for 10 min at 65 ºC
in the dark, and centrifuged for 5 min at 13800 rpm. The TBA-MDA complex absorbances in the
supernatant (the absorbances of control, sample and blank solution monitoring MDA level in the
lipid before LP initiation by AAPH) read at 530 nm were used to calculate the inhibition percentage
of lipid peroxidation.26
Inhibition of Lipid Peroxidation (%) = 100•(Ac-As)/(Ac-Ab)
Ac – the absorbance of control, As – the absorbance of sample and Ab – the absorbance of blank
solution monitoring MDA level in the lipid before LP initiation by AAPH.
All samples were assayed for LP inhibitory activity at concentrations of 200 µg/mL in the fiinal
reaction mixture. Those showing inhibition greater than 50% at this concentration were tested in a
broader concentration range to allow calculation of IC50 values.
4.8. Computational details
All theoretical calculations were performed using the Gaussian 09 package45 of programs.
Geometry and vibrational frequencies of the studied species were performed by analytical gradient
technique without any symmetry constraint. All the results were obtained using the density
27

functional theory (DFT), employing the B3LYP (Becke’s three-parameter non-local exchange46 and
Lee et al. correlation45 potentials).
The geometries of all possible isomers of the studied compounds, radicals, radical cations
were fully optimized by application of the UB3LYP functional in conjunction with the 6-311++G**
basis set. The optimized structures were further characterized by analytical computations of
harmonic vibrational frequencies at the same level. Dissociation enthalpy (BDE), ionization
potential (IP), proton dissociation enthalpy (PDE), proton affinity (PA), and electron transfer
enthalpy (ETE) were calculated according the equations given by Klein et al.27
BDE = H(R.) + H(H.) – H(R-H)
IP = H(R+.) + H(e-) – H(R-H)
.
PDE = H(R.) + H(H+) – H (R+ )
PA = H(R-) + H(H+) – H (R-H)
ETE = H(R.) + H(e-) – H (R-)
The enthalpy of hydrogen atom, H(H) was obtained by the same method and basis set. All
reaction enthalpies were calculated at 298 K. The enthalpies of proton H(H+), and electron, H(e-),
were taken from the literature: 6.197 kJ/mol and 3.145 kJ/mol, respectively.27 Natural bond orbitals
(NBO) analysis47-49 has been performed to characterize the delocalization of electron density within
the molecule.
Acknowledgements: The financial support of this work by the National Science Fund of
Bulgaria (Contracts RNF01/0110), Science Fund / Chemical Technology and Metallurgy University
grant 11252 and the Ministry of Education and Science of the Republic of Serbia (Grant No.172044
and TR34012) is gratefully acknowledged.
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