Pharmacological and Toxicological Screening of Novel Benzimidazole-Morpholine Derivatives as Dual-Acting Inhibitors

Article abstract of DOI:10.3390/molecules22081374

The aim of this study was to investigate acetylcholinesterase (AChE), monoamine oxidase A (MAO-A), monoamine oxidase B (MAO-B), cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzyme inhibitory, and antimicrobial activities of a new series of 2-(4-s

Full text of DOI:10.3390/molecules22081374

molecules
Article
Pharmacological and Toxicological Screening of
Novel Benzimidazole-Morpholine Derivatives as
Dual-Acting Inhibitors
Nafiz Öncü Can 1,2,
* ,
ID , Ulviye Acar Çevik 2,3, Begüm Nurpelin Sag˘lık 2,3, Yusuf Özkay 2,3
Özlem Atlı 4, Merve Baysal 4, Ümide Demir Özkay 5 and Özgür Devrim Can 5
1
Department of Analytical Chemistry, Faculty of Pharmacy, Anadolu University, Eskis¸ehir 26470, Turkey
Doping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy, Anadolu University,
Eskis¸ehir 26470, Turkey
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eskis¸ehir 26470,
Turkey; uacar@anadolu.edu.tr (U.A.Ç); bnsaglik@anadolu.edu.tr (B.N.S.); yozkay@anadolu.edu.tr (Y.Ö.)
Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University, Eskis¸ehir 26470,
Turkey; oatli@anadolu.edu.tr (Ö.A.); mbaysal@anadolu.edu.tr (M.B.)
2
3
4
5
Department of Pharmacology, Faculty of Pharmacy, Anadolu University, Eskis¸ehir 26470, Turkey;
udemir@anadolu.edu.tr (Ü.D.Ö.); ozgurdt@anadolu.edu.tr (Ö.D.C.)
*
Correspondence: nafizoc@anadolu.edu.tr; Tel.: +90-222-335-0580 (ext. 3770)
Received: 13 July 2017; Accepted: 15 August 2017; Published: 19 August 2017
Abstract: The aim of this study was to investigate acetylcholinesterase (AChE), monoamine oxidase
A (MAO-A), monoamine oxidase B (MAO-B), cyclooxygenase-1 (COX-1) and cyclooxygenase-2
(COX-2) enzyme inhibitory, and antimicrobial activities of a new series of 2-(4-substituted
phenyl)-1-[2-(morpholin-4-yl)ethyl]-1H-benzimidazole derivatives, for their possible use as
multi-action therapeutic agents. Target compounds (n = 15) were synthesized under microwave
irradiation conditions in two steps, and their structures were elucidated by FT-IR, 1H-NMR, 13C-NMR
and high resolution mass spectroscopic analyses. Pharmacological screening studies revealed that
two of the compounds (2b and 2j) have inhibitory potential on both COX-1 and COX-2 enzymes.
In addition, cytotoxic and genotoxic properties of the compounds 2b, 2j and 2m were investigated
via the well-known MTT and Ames tests, which revealed that the mentioned compounds are
non-cytotoxic and non-genotoxic. As a concise conclusion, two novel compounds were characterized
as potential candidates for treatment of frequently encountered inflammatory diseases.
Keywords: benzimidazoles; morpholines; AChE; MAO; COX
1. Introduction
One of the promising advances in drug discovery is the simultaneous combination of two or
more beneficial chemical moieties on the same compound, especially for treatment of a certain
disease. Any pharmaceutical benefit that reduces the demand on polypharmacy, e.g., treatment
while decreasing side effects, eliminating symptoms, or addition of an adjuvant therapeutic activity
is accepted as an ideal starting-point that may lead to the development of novel compounds with
multiple pharmacological effects [1]. There is a consensus that multifactorial disorders can originate
from many sources, so searching for new hybrid compounds with multiple pharmacological profiles
that can be expressed through more than one biochemical pathway would be useful for treating
many kinds of diseases [2,3]. Despite the great challenge in the design and optimization of such
compounds, this strategy possesses clear advantages over drug mixtures or multicomponent drugs
owing to minimization of drug-drug interaction risks [4]. Some available drugs present the capability
Molecules 2017, 22, 1374; doi:10.3390/molecules22081374
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1374
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to modulate more than one bioreceptor, as exemplified by the atypical antipsychotics olanzapine,
risperidone and aripiprazole, which act mainly as dopamine and serotonin receptor antagonists and
have lower affinity to histamine, cholinergic muscarinic and
is another example of dual monoamine oxidase B (MAO-B) and acetylcholine esterase (AChE)
inhibitor [ ] (Figure 1). Despite their great promise in the clinical application, any potential risk
α-adrenergic receptors [5]. Ladostigil
6
of side effects should not be neglected. The dual inhibitors are often characterized by high molecular
weight that may reduce the chance of their drug abilities. Therefore, the safety profiles as well as
pharmacokinetic properties need to be thoroughly considered in the design of dual inhibitors [7].
Figure 1. Chemical structures of some agents with dual inhibitory effect.
Benzimidazole derivatives have a prominent position in medicinal chemistry, which are
always used as one of the essential starting materials for discovery of new therapeutics.
The origin of the special interest towards benzimidazole derivatives has been the 5,6-dimethyl-1-
(
α
-D-ribofuranosyl)-benzimidazole structure, which is a basic part of vitamin B12 [
benzimidazole ring is a structural bioisostere of some of the nucleobases existing in natural nucleotides,
which can interact easily with the biopolymers in living systems [ ]; this feature is often accepted as
8]. Furthermore, the
9
the responsible for its biological importance, either alone or as incorporated into different templates.
It has been reported to show many pharmacological activities including antimicrobial, MAO or
cyclooxygenase (COX) inhibitory, or anticholinesterase [10–18]. Besides, albendazole, mebendazole
and tiabendazole are some examples of antimicrobial agents that carry the benzimidazole ring
(Figure 2). Morpholine is another heterocyclic organic compound, which is also one of the principle
building blocks in organic synthesis; several derivatives of morpholines have received attention in
the past, due to their significant and numerous pharmacological activities such as antiinflammatory,
antidepressant, MAO inhibitor, AChE inhibitor, neuroprotective, antituberculosis, antimalarial and
antiparasitic [19–21]. Moreover, antibacterial agent linezolid, the MAO-A inhibitor moclobemid,
and the antiinflamatory agent emorfazone are some examples of marketed drugs bearing morpholine
moieties (Figure 2).
There are several examples in previously published papers in which the strategy described above
was successfully applied by combining benzimidazole and morpholine pharmacophores on the same
chemical structure; researchers have designed and synthesized many benzimidazole-morpholine

Molecules 2017, 22, 1374
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compounds and investigate their pharmacological activities such as anticholinesterase [22
antimicrobial [24–26], antiinflammatory [27–30] and MAO inhibitory properties [31–39].
,23],
Figure 2. Chemical structures of some drugs containing benzimidazole or morpholine moieties.
The antimicrobial and COX inhibitory potential of benzimidazole and morpholine-based
compounds may be favorable in the design of dual COX inhibitory-antibacterial agents because
inflammation and infection are conditions which are frequently encountered together. A decrease of
fever besides elimination of an infectious microorganism or treatment of inflammation besides removal
of pain are dual outcomes of such agents [40,41]. Besides, the inhibition potency of benzimidazole
and morpholine derivatives against AChE and MAO enzymes may be beneficial in the design
of new anti-Alzheimer’s disease agents. Inhibition of AChE increases neurotransmission in the
cholinergic synapses and temporally decreases the cognitive deficit. AChE also contributes in other
functions related to neuronal development, differentiation, adhesion and β-amyloid protein processing.
Additionally, MAO-B inhibition retards further deterioration of cognitive functions. Thus, discovery
of a dual inhibitor of these enzymes are expected to have potential for the treatment of Alzheimer’s
disease [42].
It is clear that, the benzimidazole-morpholine combination has the potential to serve as
a pharmaceutical source for therapy. With the aim of producing safer and more active hybrid
compounds, the synthesis and pharmacological evaluation of some novel molecules that include
bioactive benzimidazole and morpholine moieties have been presented in this study.
2. Results and Discussion
2.1. Chemistry
Synthesis of the target compounds 2a–2o is outlined in Scheme 1. Initially, 1,2-phenylenediamine
was reacted with various sodium bisulfite adducts of aldehydes under microwave irradiation
conditions to obtain the previously reported 2-(4-substitutedphenyl)-1H-benzimidazoles 1a–1o [43–50].
In the second step, compounds 1a–1o were treated with 2-(morpholin-4-yl)ethyl chloride in the
presence of NaH, and subjected to microwave irradiation again. In the synthesis, various substituents
that may have an impact on biological activity were selected to impart different electronic and
hydrophobic natures to the target compounds.
Structure elucidations of the final compounds 2a–2o were realized by FT-IR, 1H-NMR, 13C-NMR
and HRMS methods. The aromatic C-H and aliphatic C-H stretching bands were observed at
3063–3011 cm−1 and 2986–2945 cm−1, respectively in the IR spectra; C=N stretching bands were
recorded at 1618–1599 cm−1. The out of plane bands, which were assigned to 1,4-disubstituted
benzene, were at 868–818 cm−1. The protons at the positions 3 and 4 of morpholine structure were
recorded at about 2.20 ppm as multiplet, in the 1H-NMR spectra. In addition, the protons of ethylene

Molecules 2017, 22, 1374
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near the morpholine and benzimidazole were observed as a triplet at about 2.50 ppm and 4.40 ppm,
respectively. The protons at the positions 1 and 2 of morpholine were assigned as multiplet at about
3.40 ppm. Aromatic protons are appeared between 7.10 ppm and 7.90 ppm, as expected.
In the 13C-NMR spectra, the signals of the ethylene spacer between the benzimidazole and
morpholine units appeared between 42.03 ppm and 42.36 ppm, and the other carbons of that chain
was recorded between 57.39 ppm and 57.55 ppm. Morpholine carbons had two different peaks at
53.78–53.83 ppm and 66.31–66.50 ppm. The benzimidazole carbon at the position 2 gave a peak between
157.27 ppm and 154.69 ppm, depending on substituent group. The other benzimidazole-carbon peaks
were recorded between 111.01 ppm and 143.30 ppm. The chemical shifts of the substituent groups and
coupling constant of the fluorinated derivatives were consistent with the theoretical values. HRMS
findings were in accordance with the theoretical molecular formula of the compounds 2a–2o.
Scheme 1. Synthesis route to the target compounds 2a–2o.
2.2. Biological Activity Screening
The antimicrobial abilities of the compounds against different human pathogens were evaluated.
Escherichia coli ATCC 35218, Escherichia coli ATCC 25922, Pseudomona aeuroginosa ATCC 27853,
and Klebsiella pneumoniae ATCC 700603 were used as Gram-negative bacteria. As Gram-positive
bacteria Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC29212, and Listeria monocytogenes
ATCC 7644 were tested. Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei
ATCC 6258 and Candida parapsilosis ATCC 22019 yeasts were also used in antimicrobial activity tests.
Chloramphenicol and ketoconazole were used as the reference drugs. Antimicrobial activity results
are given in Table 1.
Table 1 reveals that the microbial strains tested herein are very resistant to compounds
2a–2o; and only Pseudomona aeuroginosa ATCC 27853 was sensitive to some of the compounds.
The compound 2e, which bears an isopropyl substituent, showed equal activity (MIC50 = 250 µg/mL
to the reference drug chloramphenicol against Pseudomona aeuroginosa ATCC 27853. The MIC50
values (MIC50 = 125 g/mL) of compounds 2c and 2m, which bear trifluoromethoxy and chloro
substituents, was 2-fold lower than that of chloramphenicol. The most active compound in
)
µ

Molecules 2017, 22, 1374
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the series was 2-(4-methoxyphenyl)-1-[2-(morpholin-4-yl)ethyl]-1H-benzimidazole (2b), which was
a methoxy-substituted derivative and showed a 16-fold greater activity than the reference drug with
a MIC50 value of 0.0156 µg/mL. These findings show that synthesized compounds do not have
broad antibacterial spectrum and suggest that incorporation of methoxy substituent enhances the
antibacterial activity against Pseudomona aeuroginosa ATCC 27853.
Table 1. Antimicrobial activity results of test compounds 2a–2o (MIC50 values are given in mg/mL).
Compound
A
B
C
D
E
F
G
H
I
J
K
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
Ref-1
Ref-2
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
0.0312
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
0.0156
>1
0.0156
0.125
0.500
0.250
0.500
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
0.0625
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
0.250
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
0.0312
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
0.125
0.500
0.500
>1
0.0039 0.0312 0.0019 0.0625
>1 >1 >1 >1
0.250
A
D
700603;
: Escherichia coli ATCC 35218;
B
: Escherichia coli ATCC 25922;
: Pseudomona aeuroginosa ATCC 27853; : Enterococcus faecalis ATCC 29212;
: Listeria monocytogenes ATCC 7644; : Candida albicans ATCC 90028; I
C
: Staphylococcus aureus ATCC 25923;
: Klebsiella pneumoniae ATCC
: Candida glabrata ATCC 90030;
E
F
G
H
J: Candida krusei ATCC 6258; K: Candida parapsilopsis ATCC 22019; Ref-1: Ketoconazole; Ref-2: Chloramphenicol.
The inhibitory potencies of the synthesized compounds against AChE, MAO-A, MAO-B, COX-1
and COX-2 enzymes were studied according to previously verified protocols [51–54]. In all of the
assays, the compounds were initially tested at two concentrations (10−3 M and 10−4 M); in case of
an inhibition more than 50% for any of the compounds, further studies were performed to determine
the IC50, covering a wider range of concentrations (10−5 M and 10−9 M).
Inhibitory activity results of the synthesized compounds on AChE, MAO-A and MAO-B enzymes
are summarized in Supplementary Table S1; donepezil, moclobemide and selegiline were used as the
reference drugs for AChE, MAO-A and MAO-B inhibition tests, respectively. Relatively low inhibition
against the tested enzymes was observed, when compared to the reference drugs; besides, it can be
underlined that the compounds possessed better inhibitory profile against MAO-B, in comparison to
AChE and MAO-A. Since none of the compounds showed more than 50% inhibition, further assays
were not carried out to determine IC50 values. This indicates that the synthesized compounds have no
potential as drug candidates for AChE- and MAO-related disorders.
The inhibitory potencies of compounds 2a–2o on COX-1 and COX-2 enzymes are given in
Supplementary Table S2; ibuprofen and nimesulide were used as non-selective COX and selective
COX-2 inhibitors in these tests, respectively. The compounds 2a
, 2b, 2f, 2h, 2j and 2m displayed more
than 50% inhibition at the initial concentrations (10−3 M and 10−4 M); consequently, they were assayed
−5
−9
at lower concentrations between 10 M and 10 M, and IC50 values were calculated (Table 2). It has
been determined that compounds 2b and 2j have promising inhibitory activity against both COX-1
and COX-2 enzymes. IC50 values of the compound 2b was 8.096
µM and 8.369 µM versus COX-1 and
COX-2 enzymes, respectively; their IC50 values were also comparable with those of the reference drugs
ibuprofen and nimesulide. Compound 2j possessed 14.18 and 13.09 µM IC50 values on COX-1 and
COX-2, respectively.

Molecules 2017, 22, 1374
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Table 2. Inhibitory potencies of selected test compounds (2a, 2b, 2f, 2h, 2j and 2m) at lower concentrations against COX-1 and COX-2 enzymes.
COX 1 Inhibition % COX 2 Inhibition %
Comp.
−5
−6
−7
−8
−9
−5
−6
−7
−8
−9
10
M
10
M
10
M
10
M
10
M
IC50 µM
10
M
10
M
10
M
10
M
10
M
IC50 µM
2a
2b
2f
2h
2j
2m
Ref-1
Ref-2
47.39
59.37
45.38
46.29
51.41
47.36
74.67
68.39
±
±
±
±
±
±
±
±
1.08 40.12
1.24 42.75
0.99 42.08
1.09 40.88
1.15 44.45
1.16 42.02
1.28 45.08
1.26 41.91
±
±
±
±
±
±
±
±
0.92 35.68
1.02 37.08
0.87 34.23
0.99 35.62
1.07 37.66
0.88 36.65
1.16 28.23
0.98 29.05
±
±
±
±
±
±
±
±
0.97 30.46
0.92 26.77
0.76 32.68
0.81 30.77
0.76 27.46
0.92 26.19
0.91 22.66
0.92 20.37
±
±
±
±
±
±
±
±
0.83 28.81
0.87 19.56
0.78 29.88
0.93 24.23
0.82 23.44
0.79 20.01
0.82 14.76
0.81 18.84
±
±
±
±
±
±
±
±
0.79
0.76
0.68
0.72
0.61
0.62
0.60
0.63
56.08
8.096
43.23
42.97
14.18
21.94
2.435
3.810
43.72
57.85
46.98
47.54
52.54
46.11
69.51
78.69
±
±
±
±
±
±
±
±
1.07 40.32
1.28 43.70
1.13 40.22
1.08 42.60
1.35 43.40
1.13 41.23
1.28 36.38
1.19 48.28
±
±
±
±
±
±
±
±
1.10 38.26
1.12 38.12
1.24 35.62
1.16 31.75
1.27 33.98
1.05 34.48
0.91 29.42
1.09 32.97
±
±
±
±
±
±
±
±
0.92 36.20
0.97 27.45
0.91 31.46
0.99 28.35
1.06 26.85
0.97 28.69
0.84 24.03
0.96 21.30
±
±
±
±
±
±
±
±
0.87 32.44
0.87 21.35
0.84 28.74
0.82 23.04
0.86 20.54
0.83 22.30
0.76 17.08
0.88 14.12
±
±
±
±
±
±
±
±
0.91
0.74
0.63
0.68
0.76
0.52
0.58
0.61
58.66
8.369
44.92
51.19
13.09
22.66
5.327
1.683
Ref-1: Ibuprofen; Ref-2: Nimesulide.

Molecules 2017, 22, 1374
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In terms of structure activity relationships, it is noted that the most active compounds 2b and
2j carry methoxy- and ethoxy- substituents, respectively, at the 4th position of the phenyl on the
benzimidazole ring; in addition, when the nature of the substituents was examined, it can be easily
recognized that only compounds 2b, 2f, 2j and 2m included alkyloxy groups. Besides 2b and 2j,
the compounds 2f and 2m also showed potential activity in initial assays, and were subjected to
further tests. Thus, it may be suggested that the substituents including oxygen atoms may cause more
inhibition than the other substituents; this phenomenon may be explained by the hydrogen accepting
ability of alkyloxy groups.
2.3. Toxicological Studies
It is a well-known fact that a drug candidate should not only possess a beneficial pharmacological
activity, but also has to display a low toxicological profile; from this point of view, the MTT cell viability
and Ames genotoxicity tests were applied. The cytotoxicity results of the most active compounds
(2b
,
2j and 2m) against COX enzymes are presented in Table 3. IC50 values of the compounds 2b
,
2j and 2m against NIH/3T3 was found as 316
µ
M, 316 M and 100 M, respectively. IC50 of the
µ
µ
mentioned compounds against NIH/3T3 is about 5–40 folds higher than their IC50 against COX
enzymes. Thus, it can be stated that the compounds are non-toxic at their effective concentrations
against COX-1 and COX-2.
The Ames assay is a widely used method that use bacteria to test if a compound causes mutations
on the DNA of the test microorganism. More formally, it is a biological assay to assess the mutagenic
potential of a chemical compound. A positive result indicates that the tested chemical is mutagenic
and hence may act as a carcinogen, since cancer is frequently related to mutation. It is a quick and
convenient test to evaluate the carcinogenic potential of a compound [55]. Accordingly, this assay was
performed to investigate the genotoxicity of compounds 2b, 2j and 2m. In Ames MPF assay, more than
25 positive wells were observed with our positive controls, and negative control wells also showed
less than eight positive wells in the presence and absence of S9 with TA98 and TA100; these results are
in compliance with previous studies and the requirements for the validation of the Ames MPF [55].
The results are presented in Table 3.
Table 3. IC50 values of selected test compounds (2b
,
2j and 2m) against NIH3T3 cell lines and results
of Ames MPF test.
REVERTANTS Fold Increase (over Baseline)
Cytotoxicity
NIH3T3 Cells
IC50 (µM)
Concentration
(mg/mL) for
Ames Test
Comp.
2b
TA98
TA100
S9+
S9−
S9+
S9−
0.156
0.3125
0.625
1.25
2.5
0.16
0.32
0.00
0.48
0.80
1.27
0.69
0.75
0.81
0.40
0.40
0.75
0.06
0.52
0.45
0.26
0.26
0.13
1.31 ***
1.37 ***
0.69
0.62
0.25 ***
0.44 *
316
316
100
5
0.156
0.3125
0.625
1.25
2.5
0.25
0.12 *
0.62
0.74
0.12
0.60
0.94
0.68
0.60
0.94
0.15
0.00
0.15
0.38
0.31
0.15
0.80
1.00
0.40
0.67
0.67 ***
1.20
2j
5
0.74 *
0.09 ***
0.156
0.3125
0.625
1.25
2.5
0.46
0.62
0.77
0.46
1.23
0.31
0.95
0.70
0.57
1.08 *
0.82
1.08 *
0.10
0.10
0.88
0.19
0.49
0.29
0.61
0.88
0.47
0.47
0.54
0.61
2m
5
* t-test p value (unpaired, one-sided) < 0.05. *** t-test p value (unpaired, one-sided) < 0.001.

Molecules 2017, 22, 1374
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Compound 2b showed a baseline of 5.77 with TA98 in the absence of S9 and 2.10 in the presence
of S9. None of the tested concentrations reached the mentioned values above the baseline, and also
did not show any significance. Therefore, compound 2b was classified as non-mutagenic against TA98
in the presence/absence of metabolic activation (S9, Figure 3). Compound 2b had a baseline of 5.35
with TA100 in the absence of S9 and 5.14 in the presence of S9. Furthermore, fold inductions over
baseline were less than 1.5 in each concentration level of the compounds, and the results did not show
a dose-response tendency. Therefore, compound 2b was concluded as non-genotoxic against TA100
with/without metabolic activation (Figure 3).
Figure 3. Dose-Response curve of compound 2b against TA98 and TA100 in the presence and absence
of S9 according to Ames MPF test (the thick black line corresponds to mutagenicity threshold).
Compound 2j showed a baseline of 2.70 and 3.89 against TA98 with/without S9, respectively.
Fold inductions over baseline did not reach values more than 1.5 or 2 and, statistically dissimilar
results did not reveal a dose-response tendency. According to these findings, compound 2j accepted as
non-mutagenic against TA98 (Figure 4). Compound 2j was found to show a baseline of 4.37 and 5.00
with/without S9 against TA100. Mentioned fold-increases over the baseline according to the criteria
were not observed with compound 2j, and significant results did not reach these values, showing
a non-monotonic dose-response tendency. Compound 2j was also found to be non-mutagenic against
TA100 in the presence/absence of metabolic activation (Figure 4).
Compound 2m was found to show a baseline of 5.26 and 2.17 in the absence and presence of S9
against TA98, respectively. None of the concentrations reached 1.5 or 2-fold increases over the baseline,
according to the criteria. Significantly different results did not show a dose-response tendency and
they were also lower than the mentioned fold-increases. Compound 2m showed a baseline of 3.43 and
4.94 with/without S9, respectively against TA100. Fold inductions over baseline were less than 1.5 at
each concentration level of the compounds, and there was not any significant difference. Compound
2m was accepted as non-mutagenic against TA98 and TA100 with and without metabolic activation
(Figure 5).

Molecules 2017, 22, 1374
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Figure 4. Dose-Response curve of compound 2j against TA98 and TA100 in the presence and absence
of S9 according to Ames MPF test (the thick black line corresponds to mutagenicity threshold).
Figure 5. Dose-Response curve of compound 2m against TA98 and TA100 in the presence and absence
of S9 according to Ames MPF test (the thick black line corresponds to mutagenicity threshold).

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According to the Ames MPF results, compounds were classified as non-mutagenic. As well as
cytotoxicity results, genotoxicity findings also increased the value of compounds 2b and 2j as possible
COX inhibitors.
2.4. Prediction of ADME and Drug Likeness
Sufficient pharmacological activity and low toxicological profile are not the only prerequisites
for a compound to become a drug candidate; in addition, acceptable pharmacokinetic profile is also
required. Due to importance of such characteristics, in the current study, ADME properties of the
synthesized compounds 2a–2o were also investigated via in-silico routes, and related calculations
were realized by using online Molinspiration software [56]. Drug likeness score (DLS) was calculated
for all of the compounds 2a–2o and reference drugs using Molsoft’s software [57]. The theoretical
calculations of ADME parameters such as molecular weight (MW), logP, topological polar surface
are (tPSA), number of hydrogen donors (nON) and acceptors (nOHNH), volume, and DLS are
presented in Table 4, along with the violations of Lipinski’s rule. Considering Lipinski’s rule of
five, all synthesized compounds have nON smaller than 5, nOHNH smaller than 10 and polar surface
area lesser than 140 Å. Besides, MWs of all of the compounds are in accordance with the value smaller
than 500 g/mol. Synthesized compounds have logP values of less than 5 except for compounds 2e
and 2o. According to theoretical data, the majority of the compounds were found to obey Lipinski’s
rules; only the compounds 2e 2l and 2o showed minor deviation in one parameter. Furthermore,
, 2l
,
high DLSs of 1.12 and 0.88 were calculated for the most active compounds 2b and 2m. Thus, it can
be stated that synthesized compounds have good pharmacokinetics profile, which improves their
biological acceptability.
Table 4. Some physicochemical parameters of test compounds 2a–2o and reference drugs used in
prediction of ADME profiles.
Compound
MW
logP
tPSA
nON
nOHNH Volume
Vio
DLS
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
321.42
337.42
341.84
325.39
349.48
375.39
386.29
378.52
350.47
351.45
332.41
413.52
391.39
353.49
383.50
379.50
268.74
187.29
323.13
531.44
206.28
308.31
3.94
3.54
4.17
3.65
5.00
4.38
4.30
4.34
3.59
3.92
3.24
5.14
4.46
3.92
5.28
4.10
1.69
2.64
0.73
3.77
3.46
2.81
30.30
39.53
30.30
30.30
30.30
30.30
30.30
33.54
33.54
39.53
54.09
39.53
39.53
30.30
30.30
38.78
41.57
3.24
4
5
4
4
4
4
4
5
5
5
5
5
5
4
4
4
4
1
7
8
2
7
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
1
1
308.95
317.94
305.93
297.32
342.34
323.69
310.28
371.90
338.30
334.74
309.25
389.59
332.67
327.08
363.80
367.89
240.70
202.64
249.16
452.47
211.19
248.17
0
0
0
0
1
0
0
0
0
0
0
1
0
0
1
0
0
0
0
1
0
0
0.74
1.12
1.21
1.10
1.15
0.69
0.89
0.61
0.59
1.01
0.63
0.92
0.88
0.92
0.68
1.76
1.36
1.03
0.89
1.32
0.96
−1.40
Donepezil
Moclobemide
Selegiline
Chloramphenicol
Ketoconazole
Ibuprofen
Nimesulide
115.38
69.08
37.30
101.23
MW: Molecular weight; tPSA: Topological polar surface are; nON: Number of hydrogen donors; nOHNH: Number
of hydrogen acceptors; Vio: Violence; DLS: Drug likeness score.

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3. Materials and Methods
3.1. General Information
All of the chemicals used in the study were purchased either from Sigma-Aldrich Corp. (St. Louis,
MO, USA) or Merck KGaA (Darmstadt, Germany), and used without further chemical or biological
purification. Microwave syntheses were realized by using a Monowave 300 high-performance
microwave reactor (Anton-Paar, Graz, Austria). Melting points of the synthesized compounds were
determined by using a MP90 series automatic melting point determination system (Mettler-Toledo,
1
Columbus, OH, USA) and were presented as uncorrected. H- and 13C-NMR spectra were recorded in
DMSO-d6 by a Bruker digital FT-NMR spectrometer (Bruker Bioscience, Billerica, MA, USA) at 500 MHz
and 75 MHz, respectively (splitting patterns in the NMR spectra were designated as follows: s: singlet;
d: doublet; t: triplet; m: multiplet; coupling constants (J) were reported in Hertz). The IR spectra
of the compounds were recorded using an IRAffinity-1S Fourier transform IR (FTIR) spectrometer
and high resolution mass spectrometric studies were performed using a liquid chromatography mass
spectrometry-ion trap-time of flight (LCMS-IT-TOF) instrument, both from Shimadzu (Kyoto, Japan).
Chemical purities of the compounds were checked by classical TLC applications performed on silica
gel 60 F254 (Merck KGaA); LCMS-IT-TOF chromatograms were also used for the same purpose.
The pipetting in the COX-1, COX-2 and AChE assays were performed by using BioTek Precision
XS robotic system (BioTek Instruments, Inc., Winooski, VT, USA). Fluorescence intensities were
measured by BioTek-Synergy H1 multimode microplate reader (BioTek Instruments, Inc). A model
UV-1800 double beam UV-visible spectrophotometer (Shimadzu) was used for spectrophotometric
determinations. Statistical calculations were performed via Prism 5 software from GraphPad (La Jolla,
IL, USA). Water (ultra-pure, with total organic carbon less than 5 ppb and resistivity of at least
18 MOhm·cm−1) was produced in our laboratory using Milli-Q Synthesis A10 system from Millipore
SAS (Molsheim, France) and steam-sterilized before use.
3.2. Chemistry
3.2.1. Microwave-Assisted Synthesis of 2-Substituted-1H-benzimidazole Derivatives 1a–1o
An appropriate aldehyde derivative (0.03 mol), sodium bisulfite (5.7 g, 0.03 mol) and DMF (10 mL)
were added into a 30-mL glass vial of microwave synthesis reactor. The vial was located into the
reactor and heated at 240 ◦C under 10 bars of internal pressure for 5 min; consequently, it was cooled
down to room temperature (RT), 1,2-phenylenediamine (3.24 g, 0.03 mol) was added and the mixture
was subjected to above-mentioned conditions again. After cooling to RT, the mixture was poured
into iced-water, the precipitated product was washed using water, dried at RT and recrystallized
from ethanol.
3.2.2. Microwave-Assisted Synthesis of 2-Substituted-1-[2-(morpholin-4-yl)ethyl]-1H-benzimidazole
Derivatives 2a–2o
The corresponding 2-substituted-1H-benzimidazole derivative 1a–1o (0.0025 mol), NaH (0.072 g,
0.003 mol) and THF (10 mL) were added into a 30-mL glass vial of microwave synthesis reactor.
After addition of 2-(morpholin-4-yl)ethyl chloride (1 mL), the vial was located into the reactor and
heated at 170 ◦C under 10 bars of internal pressure for 30 min. The reaction vial was removed and
cooled to RT, and the mixture was poured into iced-water. The precipitated product was washed using
water, dried at RT and recrystallized from ethanol.
2-(4-Methylphenyl)-1-[2-(morpholin-4-yl)ethyl]-1H-benzimidazole (2a). Yield: 84%. m.p. 117.7–119.1 ◦C.
FTIR (ATR, cm−1): 3017 (aromatic C-H), 2986 (aliphatic C-H), 1610 (C=N), 828 (para-substituted
benzene). 1H-NMR (DMSO-d6, ppm)
δ: 2.19–2.21 (4H, m, morpholine -CH2-), 2.41 (3H, s, -CH3),
2.51 (2H, t, J = 6.40 Hz, -CH2-), 3.37–3.39 (4H, m, morpholine -CH2-), 4.39 (2H, t, J = 6.40 Hz, -CH2-),
7.22–7.27 (2H, m, benzimidazole H5-H6), 7.29 (2H, d, J = 8.00 Hz, phenyl H3,H05,), 7.69–7.71 (2H, m,

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benzimidazole H4,H7), 7.81 (2H, d, J = 8.00 Hz, phenyl H2,H06). 13C-NMR (DMSO-d6, ppm):
δ: 21.40,
42.13, 53.78, 57.43, 66.42, 111.33, 119.51, 122.31, 122.69, 128.30, 129.66, 136.10, 139.69, 143.13, 154.00.
HRMS (m/z): [M + H]+ calcd. for C20H23N3O: 322.1914 found: 322.1901.
2-(4-Methoxyphenyl)-1-[2-(morpholin-4-yl)ethyl]-1H-benzimidazole (2b). Yield: 81%. m.p. 132.7–133.9 ◦C.
FTIR (ATR, cm−1): 3019 (aromatic C-H), 2966 (aliphatic C-H), 1611 (C=N), 847 (para-substituted
1
benzene). H-NMR (DMSO-d6, ppm)
δ: 2.20–2.22 (4H, m, morpholine -CH2-), 2.50 (2H, t, J = 6.50 Hz,
-CH2-), 3.38–3.40 (4H, m, morpholine -CH2-), 3.85 ( 3H, s, -OCH3), 4.38 (2H, t, J = 6.50 Hz, -CH2-),
7.11 (2H, d, J = 8.00 Hz, phenyl H3,H05,), 7.21–7.26 (2H, m, benzimidazole H5-H6), 7.61–7.65 (2H, m,
benzimidazole H4,H7), 7.76 (2H, d, J = 8.00 Hz, phenyl H2,H06). 13C-NMR (DMSO-d6, ppm):
δ: 42.15,
53.79, 55.78, 57.39, 66.44, 111.25, 114.53, 119.38, 122.27, 122.57, 123.32, 131.21, 136.09, 143.12, 153.88,
160.68. HRMS (m/z): [M + H]+ calcd. for C20H23N3O2: 338.1863 found: 338.1844.
2-(4-Chlorophenyl)-1-[2-(morpholin-4-yl)ethyl]-1H-benzimidazole (2c). Yield: 69%. m.p. 133.3–134.0 ◦C.
FTIR (ATR, cm−1): 3055 (aromatic C-H), 2953 (aliphatic C-H), 1602 (C=N), 837 (para-substituted
1
benzene). H-NMR (DMSO-d6, ppm)
δ
: 2.18–2.19 (4H, m, morpholine -CH2-), 2.58 (2H, t, J = 6.50 Hz,
-CH2-), 3.36–3.37 (4H, m, morpholine -CH2-), 4.40 (2H, t, J = 6.50 Hz, -CH2-), 7.23–7.28 (2H, m,
0
benzimidazole H5-H6), 7.62–7.69 (4H, m, benzimidazole H4,H7, phenyl H2,H 6), 7.86–7.88 (2H, phenyl
H3,H05). 13C-NMR (DMSO-d6, ppm)
δ: 42.24, 53.82, 57.47, 66.39, 111.49, 119.70, 122.54, 123.02, 129.20,
130.08, 131.59, 134.92, 136.13, 143.08, 152.82. HRMS (m/z): [M + H]+ calcd. for C19H20N3OCl: 342.1368
found: 342.1348.
◦
2-(4-Fluorophenyl)-1-[2-(morpholin-4-yl)ethyl]-1H-benzimidazole (2d). Yield: 73%. m.p. 91.1–92.0 C.
FTIR (ATR, cm−1): 3055 (aromatic C-H), 2972 (aliphatic C-H), 1605 (C=N), 845 (para-substituted
1
benzene). H-NMR (DMSO-d6, ppm)
δ
: 2.15–2.17 (4H, m, morpholine -CH2-), 2.57 (2H, t, J = 6.50 Hz,
-CH2-), 3.35–3.36 (4H, m, morpholine -CH2-), 4.38 (2H, t, J = 6.50 Hz, -CH2-), 7.23–7.30 (2H, m,
benzimidazole H5-H6), 7.38–7.42 (2H, m, benzimidazole H4,H7), 7.63–7.67 (2H, m, phenyl H2,H06),
7.86–7.88 (2H, phenyl H3,H05). 13C-NMR (DMSO-d6, ppm)
δ
: 42.13, 53.79, 57.43, 66.39, 111.42, 116.15
4
3
(
d, 2JCF = 21.6 Hz), 119.61, 122.45, 122.89, 127.70 (d, JCF = 3.1 Hz), 132.16 (d, JCF = 8.5 Hz), 136.03,
143.03, 153.07, 163.26 (d, 1JCF = 245.1 Hz). HRMS (m/z): [M + H]+ calcd. for C19H20N3OF: 326.1663;
found: 326.1645.
2-(4-Isopropylphenyl)-1-[2-(morpholin-4-yl)ethyl]-1H-benzimidazole (2e). Yield: 76%. m.p. 123.1–124.0 ◦C.
FTIR (ATR, cm−1): 3050 (aromatic C-H), 2967 (aliphatic C-H), 1611 (C=N), 843 (para-substituted
1
benzene). H-NMR (DMSO-d6, ppm) δ: 1.26 (6H, d, J = 7.00 Hz, -CH3), 2.16–2.18 (4H, m, morpholine
-CH2-), 2.51 (2H, t, J = 6.50 Hz, -CH2-), 2.58 (1H, s, -CH-), 3.33–3.35 (4H, m, morpholine -CH2-), 4.41 (2H,
0
t, J = 6.50 Hz, -CH2-), 7.41–7.44 (2H, benzimidazole H5-H6), 7.49 (2H, m, d, J = 8.00 Hz, phenyl H3,H 5),
7.65–7.73 (2H, m, benzimidazole H4,H7), 7.75 (2H, d, J = 8.00 Hz, phenyl H2,H 6). 13C-NMR (DMSO-d6,
0
ppm) δ: 24.22, 33.80, 42.03, 53.78, 57.52, 66.40, 111.31, 119.52, 122.31, 122.69, 127.00, 128.73, 129.80,
136.06, 143.15, 150.44, 154.03.HRMS (m/z): [M + H]+ calcd. for C22H27N3O: 350.2227; found: 350.2199.
2-(4-Benzyloxyphenyl)-1-[2-(morpholin-4-yl)ethyl]-1H-benzimidazole (2f). Yield: 63%. m.p. 160.9–161.9 ◦C.
FTIR (ATR, cm−1): 3051 (aromatic C-H), 2961 (aliphatic -C-H), 1611 (C=N), 831 (para-substituted
benzene), 744 (monosubstituted benzene), 702 (monosubstituted benzene).1H-NMR (DMSO-d6, ppm)
δ
: 2.20 (4H, s, morpholine -CH2-), 2.59 (2H, t, J = 6.50 Hz, -CH2-), 3.38 (4H, s, morpholine -CH2-), 4.38
(2H, t, J = 6.50 Hz, -CH2-), 5.13 (2H, s, -OCH2-), 7.18–7.26 (5H, m, benzyloxy -CH-), 7.35–7.40 (2H,
m, benzimidazole H5-H6), 7.48 (2H, d, J = 8.50 Hz, phenyl H2,H06), 7.61–7.65 (2H, m, benzimidazole
H4,H7), 7.76 (2H, d, J = 8.50 Hz, phenyl H3,H05). 13C-NMR (DMSO-d6, ppm)
δ: 42.13, 53.78, 57.39,
66.43, 66.80, 111.26, 115.40, 119.40, 122.27, 122.57, 123.56, 128.22, 128.39, 128.94, 131.22, 136.08, 137.26,
143.13, 153.85, 159.74. HRMS (m/z): [M + H]+ calcd. for C26H27N3O2: 414.2176; found: 414.2156.
2-(4-Bromophenyl)-1-[2-(morpholin-4-yl)ethyl]-1H-benzimidazole (2g). Yield: 80%. m.p. 166.4–167.3 ◦C.
FTIR (ATR, cm−1): 3053 (aromatic C-H), 2953 (aliphatic C-H), 1608 (C=N), 837 (para-substituted
1
benzene). H-NMR (DMSO-d6, ppm)
δ: 2.18 (4H, s, morpholine -CH2-), 2.58 (2H, t, J = 6.50 Hz, -CH2-),

Molecules 2017, 22, 1374
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3.39 (4H, s, morpholine -CH2-), 4.40 (2H, t, J = 6.50 Hz, -CH2-), 7.25–7.30 (2H, m, benzimidazole H5-H6),
7.66–7.68 (2H, m, phenyl H2,H06), 7.79–7.81 (4H, m, benzimidazole H4,H7, phenyl H3,H05). 13C-NMR
(DMSO-d6, ppm) δ: 42.25, 53.82, 57.48, 66.38, 111.52, 119.69, 122.55, 123.04, 123.66, 130.44, 131.82, 132.13,
136.14, 143.06, 152.89. HRMS (m/z): [M + H]+ calcd. for C19H20N3OBr: 386.0862; found: 386.083.
2-(4-Diethylaminophenyl)-1-[2-(morpholin-4-yl)ethyl]-1H-benzimidazole (2h). Yield: 79%. m.p. 140.2–141.4 ◦C.
FTIR (ATR, cm−1): 3047 (aromatic C-H), 2970 (aliphatic C-H), 1609 (C=N), 818 (para-substituted
1
benzene). H-NMR (DMSO-d6, ppm)
δ
: 1.13 (6H, t, J = 7.00 Hz, -CH3), 2.27–2.29 (4H, m, morpholine
-CH2-), 2.63 (2H, s, -CH2-), 3.41–3.44 (8H, m, morpholine -CH2-, -CH2-), 4.38 (2H, t, J = 6.50 Hz, -CH2-),
0
6.78–6.80 (2H, m, phenyl H2,H 6), 7.20 (2H, s, benzimidazole H5-H6), 7.59–7.63 (4H, m, benzimidazole
H4,H7, phenyl H3,H05). 13C-NMR (DMSO-d6, ppm)
δ: 12.85, 42.23, 44.15, 53.83, 57.44, 66.49, 110.94,
111.39, 116.81, 118.99, 122.02, 122.06, 130.82, 136.25, 143.30, 148.56, 154.69. HRMS (m/z): [M + H]+
calcd. for C23H30N4O: 379.2492; found: 379.2465.
◦
2-(4-Dimethylaminophenyl)-1-[2-(morpholin-4-yl) ethyl]-1H-benzimidazole (2i). Yield: 72%. m.p. 162.0–163.3 C.
FTIR (ATR, cm−1): 3053 (aromatic C-H), 2955 (aliphatic C-H), 1607 (C=N), 824 (para-substituted
1
benzene). H-NMR (DMSO-d6, ppm)
δ: 2.28 (4H, s, morpholine -CH2-), 2.50 (2H, s, -CH2-), 2.99 (6H, s,
-CH3), 3.42–3.44 (4H, m, morpholine -CH2-), 4.38 (2H, t, J = 6.50 Hz, -CH2-), 6.84 (2H, d, J = 8.85 Hz,
phenyl H2,H06), 7.19–7.22 (2H, m, benzimidazole H5-H6), 7.57–7.61 (2H, m, benzimidazole H4,H7),
7.66 ( 2H, d, J = 8.85 Hz, phenyl H3,H05). 13C-NMR (DMSO-d6, ppm)
δ: 40.28, 42.28, 53.83, 57.39,
66.50, 111.01, 112.11, 117.85, 119.06, 122.06, 122.15, 130.53, 136.25, 143.27, 151.36 154.59. HRMS (m/z):
[M + H]+ calcd. for C21H26N4O: 351.2179; found: 351.2172.
◦
2-(4-Ethoxyphenyl)-1-[2-(morpholin-4-yl) ethyl]-1H-benzimidazole (2j). Yield: 76%. m.p. 83.4–85.5 C. FTIR
(ATR, cm−1): 3063 (aromatic C-H), 2972 (aliphatic C-H), 1612 (C=N), 854 (para-substituted benzene).
1H-NMR (DMSO-d6, ppm)
δ: 1.37 (3H, t, J = 6.95 Hz, -CH3), 2.21 (4H, s, morpholine -CH2-), 2.50 (2H,
s, -CH2-), 3.39 (4H, s, morpholine -CH2-), 4.12 (2H, q, J = 6.95 Hz, -CH2-), 4.38 (2H, t, J = 6.50 Hz,
-CH2-), 7.08 (2H, d, J = 8.75 Hz, phenyl H2,H06), 7.21–7.26 (2H, m, benzimidazole H5-H6), 7.61–7.65
(2H, m, benzimidazole H4,H7), 7.75 (2H, d, J = 8.75 Hz, phenyl H3,H05). 13C-NMR (DMSO-d6, ppm)
δ
: 15.05, 42.13, 53.79, 57.39, 63.74 66.44, 111.24, 114.94, 119.37, 122.25, 122.55, 123.17, 131.21, 136.09,
143.13, 153.92, 159.95. HRMS (m/z): [M + H]+ calcd. for C21H25N3O2: 352.2020; found: 352.1985.
2-(4-Cyanophenyl)-1-[2-(morpholin-4-yl)ethyl]-1H-benzimidazole (2k). Yield: 83%. m.p. 134.9–136.2 ◦C.
FTIR (ATR, cm−1): 3059 (aromatic C-H), 2952 (aliphatic C-H), 2226 (C
≡
N), 1612 (C=N), 844
1
(para-substituted benzene). H-NMR (DMSO-d6, ppm)
δ: 2.20 (4H, s, morpholine -CH2-), 2.58 (2H, t,
J = 6.50 Hz, -CH2-), 3.38 (4H, s, morpholine -CH2-), 4.43 (2H, t, J = 6.50 Hz, -CH2-), 7.28–7.33 (2H, m,
benzimidazole H5-H6), 7.70–7.72 (2H, m, phenyl H2,H06), 8.08–8.06 (4H, m, benzimidazole H4,H7,
phenyl H3,H05). 13C-NMR (DMSO-d6, ppm)
δ: 42.36, 53.82, 57.51, 66.34, 111.69, 112.48, 118.98, 119.96,
122.80, 123.45, 130.64, 133.05, 135.77, 136.23, 143.10, 152.27. HRMS (m/z): [M + H]+ calcd. for
C20H20N4O: 333.1710; found: 333.1682.
◦
2-(4-Trifluoromethylphenyl)-1-[2-(morpholin-4-yl)ethyl]-1H-benzimidazole (2l). Yield: 89%. m.p. 124.3–125.2 C.
FTIR (ATR, cm−1): 3052 (aromatic C-H), 2959 (aliphatic C-H), 1618 (C=N), 852 (para-substituted
1
benzene). H-NMR (DMSO-d6, ppm)
δ: 2.15 (4H, s, morpholine -CH2-), 2.57 (2H, t, J = 6.50 Hz, -CH2-),
3.30 (4H, s, morpholine -CH2-), 4.45 (2H, t, J = 6.50 Hz, -CH2-), 7.26–7.33 ( 2H, m, benzimidazole
H5-H6), 7.71 (2H, d, J = 7.50 Hz, phenyl H2,H06), 7.93–7.95 (2H, m, benzimidazole H4,H7), 8.09
(2H, d, J = 7.50 Hz, phenyl H3,H05). 13C-NMR (DMSO-d6, ppm)
δ: 42.28, 53.82, 57.55, 66.31, 111.61,
119.89, 122.69, 123.29, 124.62 (q, 1JCF = 259.2 Hz), 125.98 (q, 3JCF = 3.64 Hz), 130.10 (q, 2JCF = 31.9 Hz),
130.66, 135.33, 136.19, 143.10, 152.49. HRMS (m/z): [M + H]+ calcd. for C20H20N3OF3: 376.1631;
found: 376.1605
◦
2-(4-Trifluoromethoxyphenyl)-1-[2-(morpholin-4-yl)ethyl]-1H-benzimidazole (2m). Yield: 71%. m.p. 122.5–123.6 C.
FTIR (ATR, cm−1): 3055 (aromatic C-H), 2945 (aliphatic C-H), 1614 (C=N), 868 (para-substituted
1
benzene). H-NMR (500 MHz, DMSO-d6, ppm)
δ: 2.12–2.16 (4H, m, morpholine -CH2-), 2.55–2.59 (2H,

Molecules 2017, 22, 1374
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m, -CH2-), 3.30 (4H, s, morpholine -CH2-), 4.29–4.45 (2H, m, -CH2-), 7.22–7.33 (2H, m, benzimidazole
H5-H6), 7.54–7.59 (2H, m, phenyl H2,H06), 7.65–7.61 (4H, m, benzimidazole H4,H7), 7.95–8.00 (2H,
m, phenyl H3,H05). 13C-NMR (DMSO-d6, ppm)
δ
: 42.11, 53.79, 57.53, 66.32, 111.48, 119.73, 120.54
2
(
q, 1jC-F = 255.3 Hz), 121.62, 122.55, 123.05, 130.58, 131.95, 136.06, 143.07, 149.52 (q, jC-F = 1.5 Hz)
152.68. HRMS (m/z): [M + H]+ calcd. for C20H20N3O2F3: 392.1580; found: 392.1557
◦
2-(4-Methylthiophenyl)-1-[2-(morpholin-4-yl)ethyl]-1H-benzimidazole (2n). Yield: 77%. m.p. 153.2–154.1 C.
FTIR (ATR, cm−1): 3041 (aromatic C-H), 2951 (aliphatic C-H), 1599 (C=N), 826 (para-substituted
1
benzene). H-NMR (DMSO-d6, ppm)
δ: 2.22 (4H, s, morpholine -CH2-), 2.50 (3H, s, -SCH3), 2.61 (2H,
t, J = 6.50 Hz, -CH2-), 3.40 (4H, s, morpholine -CH2-), 4.41 (2H, t, J = 6.50 Hz, -CH2-), 7.23–7.26 (2H,
m, benzimidazole H5-H6), 7.43 (2H, d, J = 8.50 Hz, phenyl H2,H06), 7.64–7.66 (2H, m, benzimidazole
H4,H7), 7.78 (2H, d, J = 8.50 Hz, phenyl H3,H05). 13C-NMR (DMSO-d6, ppm)
δ: 14.77, 42.25, 53.82,
57.44, 66.43, 111.35, 119.53, 122.39, 122.77, 125.94, 127.29, 130.13, 136.17, 140.93, 143.14, 153.55. HRMS
(m/z): [M + H]+ calcd. for C20H23N3O2S: 354.1635; found: 354.1593.
◦
2-(1,1’-Biphenyl)-1-[2-(morpholin-4-yl)ethyl]-1H-benzimidazole (2o). Yield: 83%. m.p. 109.4–111.2 C. FTIR
(ATR, cm−1): 3049 (aromatic C-H), 2955 (aliphatic C-H), 1610 (C=N), 845 (para-substituted benzene),
1
746 (monosubstituted benzene), 694 (monosubstituted benzene). H-NMR (DMSO-d6, ppm)
δ: 2.20
(4H, s, morpholine -CH2-), 2.51 (2H, t, J = 6.50 Hz, -CH2-), 3.48 (4H, s, morpholine -CH2-), 4.41 (2H, t,
J = 6.50 Hz, -CH2-), 7.25–7.33 (5H, m), 7.40–7.53 (5H, m), 7.66–7.88 (2H, m), 7.67–7.88 (6H, m). 13C-NMR
(DMSO-d6, ppm) δ: 42.25, 53.82, 57.53, 66.42, 111.38, 119.69, 122.47, 122.88, 127.24, 128.39, 129.51, 130.19,
130.35, 136.23, 139.69, 141.55, 143.29, 153.63. HRMS (m/z): [M + H]+ calcd. for C25H25N3O: 384.2070;
found: 384.2047.
3.3. Antimicrobial Assay
Microbiological studies were performed according to CLSI reference M07-A9 broth microdilution
method for bacterial strains [58] and EUCAST definitive method EDef 7.1 for Candida species [59].
Synthesized compounds were tested for their in vitro growth inhibitory activity against Escherichia coli
ATCC 35218, Escherichia coli ATCC 25922, Staphylococcus aureus, ATCC 25923, Pseudomona aeuroginosa
ATCC 27853, Enterococcus faecalis ATCC29212, Klebsiella pneumoniae ATCC700603 Listeria monocytogenes
ATCC7644, Candida albicans ATCC90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258,
Candida parapsilosis ATCC 22019.
The cultures were obtained from Mueller-Hinton broth (Difco) for the bacterial strains and the
yeasts were main◦tained in Roswell Park Memorial Institute (RPMI) medium, after an overnight
incubation at 37 C for both. The inocula of the test microorganisms were adjusted to match
an equivalent turbidity of a 0.5 McFarland standard, which was determined using a spectrophotometer;
the final inoculum size was determined to be 0.5–2.5
×
105 cfu/mL for antibacterial and antifungal
assays. The tests were carried out for both mediums at pH = 7 and two-fold serial dilutions were
applied. The last well on the microplates, which was containing only the inoculated broth, was kept as
control, and the last well with no growth of microorganism was recorded to represent the minimum
inhibitory concentration (MIC50) in
compounds and reference drugs were firstly dissolved in DMSO, and further dilutions were performed
to the desired concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.6, 7.8, 3.9 and 1.95 g/mL using
Mueller–Hinton broth and RPMI medium. The completed plates were incubated for 24 h, and at
the end of the incubation, resazurin (20 g/mL) was added into each well to control the growth in
µg/mL. For both of the antibacterial and antifungal assays, the test
µ
µ
the wells. Final plates including microorganism strains were incubated for 2 h. MIC50 values were
determined using microplate reader at 590 nm excitation and 560 nm emission wavelengths; MIC50
readings were performed twice for entire compounds. Chloramphenicol and ketoconazole were used
as reference drugs.

Molecules 2017, 22, 1374
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3.4. COX-1 and COX-2 Inhibition Assays
Inhibitory potency of the compounds on COX-1 and COX-2 enzymes was determined by
using fluorimetric COX-1 and COX-2 inhibition screening kits from BioVision (Milpitas, CA, USA).
The experimental protocol as described in the guides provided by the supplier was followed [51,52].
All of the pipetting were performed using a robotized system to increase speed, accuracy and precision.
◦
Fluorescence intensities of the samples were kinetically measured at 25 C for 5–10 min by monitoring
emissions at 587 nm after excitation at 535 nm. Two appropriate points (T1 and T2) in the linear range
of the plot were chosen and the corresponding fluorescence values (RFU1 and RFU2) were obtained.
The slope for all samples including Enzyme Control (EC) was calculated by dividing the net
(RFU2 RFU1) values by the time T (T2 T1). Percentage of relative inhibition was calculated by
using the following equation:
∆RFU
−
∆
−
% Relative inhibition = (Slope of EC − Slope of S/Slope of EC) × 100
where S is the −te3rm representing the tested compound [51
,52]. The initial in vitro assays were
−4
performed at 10 M and 10 M concentrations for all of the test compounds; the ones which showed
inhibition above 50% were assayed by the same protocol within a wider range of concentrations
(10−5 M and 10−9 M) to determine their IC50 against COX-1 and COX-2 enzymes. The IC50
values were calculated using the plots of the enzyme activity against concentrations by applying
regression analyses.
3.5. AChE Inhibition Assay
Inhibition potency of the test compounds against AChE was determined using Ellman’s
method [53]. Enzyme solutions were prepared in gelatin solution (1%, w/v), at the concentration
of 2.5 units/mL. The synthesized compounds, and donepezil, which was used for reference,
were prepared at 10−3 M and 10−4 M concentrations using 2% DMSO solution for initial measurements.
AChE solution (20
(140 L/well, pH 8.0
of the chromogenic reagent 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB, 20
the substrates acetylthiocholine iodide (ATCI, 10 L/well, 75 M) to the enzyme-inhibitor mixture.
µ
L/well) and test solution (20
0.1), and incubated at 25 ◦C for 5 min. The reaction was started by addition
L/well, 10 mM) and
µL/well) were added to phosphate buffer solution
µ
±
µ
µ
µ
The production of the yellow anion was recorded by multimode microplate reader for 10 min at 412 nm.
As a control, an identical solution of the enzyme without the inhibitor was processed. The control
and the inhibitor readings were corrected with blank-readings. All processes were assayed in four
independent determinations.
3.6. MAO Inhibition Assay
Enzyme activity assays were performed according to the procedure reported by Matsumoto et al. [54
]
with slight modifications. In order to prevent the destructive effect of light on MAO enzymes, the assays
were performed in 96-well black plates. Phosphate buffer solution (0.1 M, pH = 7.4) was used for
preparation of stock solutions (5 mg/mL) of both human recombinant MAO-A and MAO-B enzymes.
Enzyme stock solutions were further diluted using assay buffer to achieve a final concentration
of 0.006 mg/mL for MAO-A and 0.015 mg/mL for MAO-B. Kynuramine was dissolved in sterile
deionized water to prepare stock solution (25 mM) and then diluted using assay buffer to get final
concentration of 40
µM for MAO-A enzyme and 20 µM for MAO-B enzyme. Synthesized compounds
and reference drugs, which were moclobemide for MAO-A and selegiline for MAO-B, were diluted to
−3
−4
10 M and 10 M concentrations (100 µL/well) using 2% DMSO. The addition of the test compound
solution was followed by addition of either MAO-A or MAO-B enzyme (50
µ
L/well) solutions.
◦
After 10 min of incubation at 37 C, kynuramine (50
µL/well) was added and enzyme-substrate
reaction was initiated. The plate was incubated for 20 min at 37 ◦C and then reaction was terminated

Molecules 2017, 22, 1374
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by addition of 2 N NaOH (75
µL/well). The fluorimetric reads from the top was performed at 310 nm
excitation and 380 nm emission wavelengths.
3.7. Determination of Cytotoxicity
Cytotoxicity was tested using NIH/3T3 mouse embryonic fibroblast cell line (ATCC® CRL-1658
™,
London, UK). NIH/3T3 cells were incubated according to the supplier’s recommendations. NIH/3T3
cells were seeded as 1
104 cells into each well of 96-well plates. MTT assay was performed
as previously described [60 61]. The compounds were tested between 1 mM and 0.000316 mM
×
,
concentrations (1.0, 0.316, 0.10, 0.0316, 0.01, 0.00316, 0.001, 0.000316 mM). The IC50 values were
determined by plotting a dose-response curve of inhibition % versus tested concentrations of the
compound [62].
3.8. Determination of Genotoxicity
The genotoxicity of selected compounds (2b, 2j, 2m) was determined by Ames assay using
Ames MPF 98/100 mutagenicity assay sample kit (Xenometrix AG, Allschwil, Switzerland) as
previously described elsewhere [63]. Salmonella typhimurium strains, TA98 (frameshift mutations)
and TA100 (base-pair substitutions) were used in the assay. The concentration of the compounds was
between 16 and 5000
in six different concentrations (5.0, 2.5, 1.25, 0.625, 0.3125, 0.156 mg/mL) in DMSO. Mutagenic
potential was determined in the absence or presence of Aroclor -1254 induced male Sprague–Dawley
rat liver microsomal enzyme (S9) mix (Xenometrix AG). Positive controls without S9 mix were
2-nitrofluorene (2.0 g/mL) and 4-nitroquinoline N-oxide (0.1 g/mL), whereas 1.0 g/mL and
2.5 g/mL of 2-aminoanthracene solutions were used as positive controls with S9 against TA98 and
µg/mL, in accordance with the guideline [64]. Compounds were prepared
™
µ
µ
µ
µ
TA100, respectively. Solvent control was prepared with 4% DMSO. The end of an experiment was
determined by the change of the indicator medium colour to yellow, which was originating from the
decrease of pH due to revertant bacteria. Yellow wells were counted as positive, and compared with
the negative control. Fold induction over the negative control and fold induction over the baseline
were calculated (Fold induction over the negative control is accepted as the ratio of the mean number
of positive wells for the dose concentration divided by the mean number of positive wells for the zero
dose (negative) control. Fold induction over the baseline is accepted as the ratio of the mean number
of positive wells for the dose concentration divided by zero dose baseline. The zero-dose baseline is
obtained by adding one standard deviation to the mean number of positive wells of the zero dose
control. If the baseline is less than 1, the value is set to 1 for calculation).
Mutagenicity was determined according to the criteria reported previously [55]. For a baseline
value of
mutagen, and increases
of >3, significant increases between 1.5 and 2.5-fold of the baseline were classified as weak mutagen,
and increases 2.5-fold of the baseline, were classified as mutagen. As a rule, at least two adjacent
≤
3, significant increases between 2 and 3-fold of the baseline were classified as weak
≥
3-fold of the baseline were classified as mutagen. For a baseline value
≥
doses with significant increases or a significant increase at the highest dose level should be observed
for a mutagenic compound. All of the doses were compared according to Student’s t-test at p < 0.05 for
statistical significance. Compounds that did not possessed any of the characteristics mentioned above
were classified as non-mutagenic.
3.9. Prediction of ADME Properties and Drug Likeness
Some physicochemical parameters, which were used to evaluate ADME properties of the
compounds (2a–2o) were calculated via online molecular property and bioactivity score calculation
software of Molinspiration Cheminformatics (ver. 2017, Molinspiration Cheminformatics, Slovensky
Grob, Slovak Republic) [56]. Drug-likeness score of the compounds was assigned by an online
drug-likeness and molecular property prediction software (ver. 2017, Molsoft, San Diego, CA,
USA) [57].

Molecules 2017, 22, 1374
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4. Conclusions
In the present study, synthesis and structural verification of some novel benzimidazole-
morpholine compounds were described, and their biological and pharmaceutical value was evaluated
by applying multiple biological, pharmacological and toxicological tests. Briefly, it can be concluded
that the synthesized compounds did not exceptionally inhibit AChE, MAO-A and MAO-B enzymes.
On the other hand, compounds 2b, 2c, 2e and 2m presented acceptable antibacterial activity especially
against Pseudomona aeuroginosa ATCC 27853. Furthermore, a promising activity was observed for
compounds 2b and 2j against COX-1 and COX-2 enzymes, meanwhile compound 2b was the most
active derivative in both antimicrobial and COX inhibition assays. It is a common knowledge
that microbial infections generally accompanied with fever and inflammation; thus, among the
compounds in the series 2-(4-methoxyphenyl)-1-[2-(morpholin-4-yl)ethyl]-1H-benzimidazole (2b
)
seems to be most powerful candidate for the treatment of a such disease, with dual activity against
Pseudomona aeuroginosa ATCC and COX enzymes.
Supplementary Materials: The following are available online. Table S1: Percentage of inhibition of test
compounds 2a–2o against AChE, MAO-A and MAO-B enzymes, Table S2: Inhibitory potencies of test compounds
2a–2o at higher concentrations against COX-1 and COX-2 enzymes. The 13C-NMR, 1H-NMR, FTIR and HRMS
spectrums of compounds 2a–2o.
Acknowledgments: This study was financially supported by Anadolu University Scientific Research Projects
Commission, Project no. 1105S101 and 1606S567.
Author Contributions: N.Ö.C. and Y.Ö. conceived and designed the experiments; U.A.Ç. and B.N.S. performed
the syntheses; N.Ö.C. and Y.Ö. performed the analytical and spectral studies; Ö.A. and M.B. performed the
cytotoxicity and genotoxicity tests; Ö.D.C. and Ü.D.Ö. conducted the pharmacological parts; N.Ö.C. and Y.Ö.
wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1a–1o and 2a–2o are available from the authors.
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