Antimicrobial activity of a new combination system of benzimidazole and various azoles

Article abstract of DOI:10.1002/ardp.201000172

In the present study a new series of benzimidazole derivatives bearing various (benz)azolylthio moieties were synthesized so as to investigate their antimicrobial activity. Structures of the target compounds (5a-5i) were confirmed by their IR, ¹

Full text of DOI:10.1002/ardp.201000172

264
Arch. Pharm. Chem. Life Sci. 2011, 11, 264–271
Full Paper
Antimicrobial Activity of a New Combination System of
Benzimidazole and Various Azoles
1
2
2
1
¨
˙
˘
˘
Yusuf Ozkay , Yagmur Tunalı , Hu¨lya Karaca , and Ilhan Is¸ıkdag
1 Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Eskis¸ehir, Turkey
2 Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Eskis¸ehir, Turkey
In the present study a new series of benzimidazole derivatives bearing various (benz)azolylthio
moieties were synthesized so as to investigate their antimicrobial activity. Structures of the target
compounds (5a–5i) were confirmed by their IR, 1H-NMR, ES-MS spectral data, and elemental analyses.
The synthesized compounds (5a–5i) exhibited poor activity against bacterial strains. On the other
hand, antifungal activity of the compounds against Candida species was very significant. Brine-Shrimp
lethality assay was performed for determination of toxicity of the compounds. Compounds 5a, 5c, and
5d were evaluated as non-toxic in addition to their attractive antifungal activity. However, the other
compounds (5b, e–i) in the series showed toxicity to different extents.
Keywords: Antimicrobial / (Benz)azolylthio / Benzimidazole / Brine-Shrimp lethality assay / Candida species
Received: June 7, 2010; Revised: August 16, 2010; Accepted: August 20, 2010
DOI 10.1002/ardp.201000172
Introduction
dimethylbenzimidazole, which is a fragment of cyanocobal-
amine (vitamin B12) that is capable of inducing the growth of
bacteria. However, the benzimidazole fragment and some of
its derivatives suppress the bacterial growth owing to struc-
tural similarity to purine. Antibacterial ability of benzimid-
azoles is explained by their competition with purines
resulting in inhibition of the synthesis of bacterial nucleic
acids and proteins [17, 18]. Benzimidazoles exhibit not only
antibacterial activity, but also antifungal activity. They can
be classified as one the most important group of fungicides
with systemic activity and are well-known for their pro-
nounced ability to control a large number of fungal diseases.
Thiabendazole, benomyl, carbendazim, chlorfenazole, cypend-
azole, debacarb, fuberidazole, mecarbinzid, and rabenzazole,
which include the benzimidazole moiety, are the main
examples of this fungicide class [19, 20]. In this group, the
well-known fungicide thiabendazole inhibits fungal micro-
tubular function, resulting in non-disjunction of chromo-
somes at cell division [21, 22].
The development of antimicrobial agents to treat infections
has been one of the most important medical accomplish-
ments of the past century. However, the progresses in
medical care are threatened by a natural occurrence known
as antimicrobial resistance. The increased use of antibacterial
and antifungal drugs in recent years has resulted in the
development of resistance to these agents [1–4] and possible
microbial implications for morbidity, mortality and health
care costs have become a serious fear. Even though, there are
large numbers of antimicrobial drugs available for medical
use, there will always be a vital need to discover new agents
due to antimicrobial resistance [5, 6].
Benzimidazole is an important pharmacophore in medic-
inal chemistry. Recent studies have confirmed that its deriva-
tives are effective against various strains of microorganisms
[7–16]. The basis of
a special interest of researchers
toward benzimidazole derivatives has been 5,6-
In addition to benzimidazole, the other azole moieties
such as imidazole and triazole are also important pharma-
cophores in medicinal chemistry field because of their che-
motherapeutic value. Imidazoles and triazoles constitute two
important classes of antifungal agents which are called with
their names. Treatment of fungal diseases with imidazoles
¨
Correspondence: Yusuf Ozkay, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Anadolu University, 26470, Eskis¸ehir,
Turkey
E-mail: yozkay@anadolu.edu.tr
Fax: þ90 222 335 07 50
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Arch. Pharm. Chem. Life Sci. 2011, 11, 264–271
Benzimidazole Derivatives Bearing Imidazole or Triazole Moieties
265
and triazoles has started with miconazole and ketoconazole
and then fluconazole and itraconazole have been developed.
Thus, imidazoles and triazoles have become the most attrac-
tive groups in the last two decades because they have been the
most successful agents among the antifungal drugs [23, 24].
They act by competitive inhibition of the lanosterol 14a-
demethylase (CYP51), [25] which is a key enzyme in sterol
synthesize a combination system that involves two different
pharmacophores. Thus, in this study, some new benzimida-
zole derivatives bearing imidazole or triazole moieties or
their bioisosters such as benzoxazole, benzothiazole, tetra-
zole and thiadiazole were synthesized in order to investigate
their antibacterial and antifungal activity.
biosynthesis of fungi. Selective inhibition of CYP51 would Results and Discussion
cause depletion of ergosterol and accumulation of lanosterol
and other 14-methyl sterols resulting in the growth inhi-
bition of fungal cells [26]. Unfortunately, imidazoles and
triazoles are fungistatic against yeasts and their broad use
leads to development of resistance showing the urgent need
for new and effective antifungal agents [27].
Chemistry
For synthesis of the target compounds (5a–5i) the reaction
sequences outlined in Scheme 1 were followed. First of all,
sodium bisulfite adduct of 4-acetylaminobenzaldehyde was
prepared in dilute EtOH and reacted with 4,5-dichloro-o-phe-
nylenediamine in DMF to achieve 2-(4-acetylaminophenyl)-1H-
5,6-dichloro-benzimidazole (1). Secondly, N-methylation of 1
Looking at the antimicrobial importance of benzimida-
zole, imidazole and triazole scaffolds, we planned to
Cl
O
O
O
H
N
a
c
N
H
N
H
CH3
CH3
N
H
Cl
1
b
Cl
Cl
O
Cl
Cl
N
N
N
H
H2N
H3C
N
N
2
3
CH3
CH3
d
O
O
Cl
Cl
Cl
Cl
N
N
N
e
(Benz)
azole
N
Cl
N
H
S
H
N
4
CH3
CH3
5a-5i
N
N
N
N
N
N
S
N
N
N
N
N
N
N
(Benz)
azole
:
5b:
5c:
5d:
5a:
5e:
5f:
N
N
N
N
H
N
CH3
CH3
CH3
CH3
Ph
N
N
N
S
5g:
5h:
5i:
N
H
O
Reagents and conditions; a) 1. Na2S2O5, 80% EtOH, r.t., 0.5 h, 2. 4,5-Dichloro-o-phenylenediamine, DMF, 130°C, 4 h;
b) K2CO3, CH3I, acetone, reflux 12 h; c) Concentrated HCl, reflux, 1 h; d) TEA, ClCH2COCl, benzene, ice bath and
then r.t., 1 h; e) Appropriate HS-(benz)azole, K2CO3, acetone, reflux, 12 h.
Scheme 1. Reaction sequence of 2-[4-[2-[(benz)azolylsulfanyl]acetylamino]phenyl]-5,6-dichloro-1-methyl-1H-benzimidazole deriva-
tives (5a–5i).
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Y. Ozkay et al.
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Arch. Pharm. Chem. Life Sci. 2011, 11, 264–271
Table 1. MIC values (mg/mL) of compounds 5a–5i against bacterial strains.
Microbial Strains
Compounds
5a
5b
5c
5d
5e
5f
5g
5h
5i
Reference
A
B
C
D
E
F
G
H
I
12.5ꢂ
400
50ꢂ
200
50
200
400
200
400
400
6.25ꢂꢂ
200
12.5ꢂꢂ
100
50
200
400
200
200
400
12.5ꢂ
200
25ꢂꢂ
100
25
200
400
200
400
400
12.5ꢂ
200
50ꢂ
100
25
200
400
200
400
400
12.5ꢂ
200
50ꢂ
100
25
100
400
200
200
400
12.5ꢂ
200
25ꢂꢂ
100
50
200
400
200
200
400
12.5ꢂ
400
50ꢂ
200
50
400
400
200
400
400
12.5ꢂ
200
25ꢂꢂ
200
50
200
400
100
200
400
12.5ꢂ
200
12.5ꢂꢂ
100
50
200
400
200
200
400
12.5
12.5
50
12.5
12.5
50
50
12.5
12.5
12.5
J
A: Escherichia coli 35218, B: Escherichia coli 25922, C: Proteus vulgaris, D: Salmonella thyphimurium, E: Klebsiella pneumoniae, F: Pseudomonas
aeruginosa, G: Listeria monocytogenes, H: Staphylococcus aureus, I: Enterococcus faecalis, J: Bacillus subtilis. Reference: Chloramphenicol.
ꢂEqual MIC value to reference, ꢂꢂLower MIC value than reference.
with CH3I/K2CO3 in acetone gave 2-(4-acetylaminophenyl)-5,6-
dichloro-1-methyl-1H-benzimidazole (2) which was then deace-
tylated with concentrated HCl to afford 2-(4-aminophenyl)-5,6-
dichloro-1-methyl-1H-benzimidazole (3). Afterwards, 2-[4-(2-
chloroacetylamino)-phenyl)]-5,6-dichloro-1-methyl-1H-benzi-
midazole (4) was prepared in benzene via acetylation reaction
of component 3 and chloroacetyl chloride in the presence of
TEA. Finally, reaction of component 4 and the appropriate
(benz)azolethiol derivative in acetone in the presence
of K2CO3 gave the title products (5a–5i).
Microbiology
Table 1 presents the antibacterial activity of the compounds
(5a–5i) against different strains of bacteria. When compared
with reference drug chloramphenicol, the synthesized com-
pounds (5a–5i) showed no significant antibacterial activity
against Staphylococcus aureus ATCC 25923, Enterococcus faecalis
ATCC 29212, Bacillus subtilis, and Listeria monocytogenes Gram-
positive bacterial strains. On the other hand, antibacterial
ability of the compounds (5a–5i) against Gram-negative
bacteria was more notable. Some of the compounds (5c–5e)
displayed moderate activity against Klebsiella pneumoniae
ATCC 13883. Compound 5b was more active against
Escherichia coli 35218 than the chloramphenicol. Other
compounds (5a, c–i) in the series were found as active as
the reference compound against same bacterial strain.
Compounds 5a, 5d, 5e, and 5g exhibited the same antibacte-
rial activity of the reference against Proteus vulgaris NRLL B-
123. Furthermore, the MIC values (12.5–25 mg/mL) of com-
pounds 5b, 5c, 5f, 5h, and 5i were lower than that of chlor-
amphenicol (50 mg/mL). However, all compounds (5a–5i)
were found as inactive against other Gram-negative bacterial
strains such as Escherichia coli 25922, Pseudomonas aeruginosa
ATCC 27853, and Salmonella thyphimurium NRRL B-4420.
In Table 2 the antifungal activity of the compounds (5a–5i)
against different fungal strains is summarized. In contrast to
their poor antibacterial activity, significant antifungal
activity was displayed by most of the compounds against
Candida yeasts. Especially Candida albicans was more sensitive
to the tested compounds (5a–5i). Most of the compounds (5a–
5f, 5h) in the series exhibited better antifungal activity than
reference drug ketoconazole against C. albicans. Compounds
5g and 5i were found to be as active as the reference
(MIC ¼ 50 mg/mL) against this fungal strain. Only 5g and
5i exhibited lower antifungal activity than ketoconazole
Structure elucidations of the final compounds (5a–5i)
were performed with IR, 1H-NMR, and ES-MS spectroscopic
methods and elemental analysis. Characteristic stretching
–
absorptions of C O groups and N-H bonds were observed
–
at 1671–1681 cmꢀ1 and 3325–3421 cmꢀ1, respectively. The
stretching absorptions at about 1614 and 1451 cmꢀ1 were
–
–
–
assigned to C C and C N bonds, respectively. Disappearance
–
of stretching absorption for S-H bond at about 2550 cmꢀ1
provided evidence for the formation of the target compounds
(5a–5i).
In the 1H-NMR spectra, all of the aromatic and aliphatic
protons were observed at the estimated chemical shifts. The
N-H proton of the acetylamino group gave a singlet signal at
10.47–10.77 ppm. The multiplet belonging to aromatic pro-
tons of 1,4-disubstituted phenyl ring and the signals due to
the protons in positions 4 and 7 of the benzimidazole ring
were found at 7.69–7.82 ppm. The -COCH2 and benzimida-
zole N-CH3 protons singlet signals at 4.52–4.56 ppm and
3.50–3.57 ppm, respectively.
M þ 1 peaks in ES-MS spectra were in agreement with
the calculated molecular weight of the target compounds
(5a–5i). Elemental analysis results for C, H, and N elements
were satisfactory within ꢁ0.4% calculated values of the
compounds.
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Arch. Pharm. Chem. Life Sci. 2011, 11, 264–271
Benzimidazole Derivatives Bearing Imidazole or Triazole Moieties
267
Table 2. MIC values (mg/mL) of compounds 5a–5i against fungal strains.
Microbial Strains
Compounds
5a
5b
5c
5d
5e
5f
5g
5h
5i
Reference
A
B
C
25ꢂꢂ
25ꢂ
50ꢂ
25ꢂꢂ
25ꢂ
50ꢂ
25ꢂꢂ
25ꢂ
50ꢂ
25ꢂꢂ
25ꢂ
50ꢂ
25ꢂꢂ
25ꢂ
50ꢂ
25ꢂꢂ
25ꢂ
50ꢂ
50ꢂ
25ꢂ
100
25ꢂꢂ
25ꢂ
50ꢂ
25ꢂ
50ꢂ
50
25
50
100
A: Candida albicans, B: Candida globrata, C: Candida tropicalis. Reference: Ketaconazole.ꢂEqual MIC value to reference,ꢂꢂLower MIC value
than reference.
against Candida tropicalis. Antifungal activities (MIC ¼ 50 mg/mL)
of the other compounds (5a–f, h) in the series against this
fungal strain were the same of ketoconazole. All of the com-
pounds (5a–5i) indicated the same MIC value (25 mg/mL) of
the reference against Candida globrata.
(Table 3). After evaluating MIC and LC50 values of the com-
pounds (5a–5i) together, the 5a, 5c, and 5d (LC50 > 1000 mg/mL)
were determined as non-toxic. On the other hand, the other
compounds (5b, e–i) showed toxicity to different extents.
Compound 5b (LC50 ¼ 103.11 mg/L) was found to be harmful.
Compound 5e (LC50 ¼ 33.49 mg/L), 5f (LC50 ¼ 51.43 mg/L),
and 5h (LC50 ¼ 71.73 mg/L) were established as toxic.
Compounds 5g and 5i (LC50 < 7.8 mg/L) were designated
as very toxic. The reference drug ketoconazole was also
assayed in toxicity test and it was assessed as non-toxic
(LC50 ¼ 793.70 mg/L). Compounds 5a, c, d, and ketoconazole
found to be as non-toxic, calculation of higher LC50 for the
tested compounds (5a, c, d) than reference was an important
finding which indicates that toxicity level of these three
compounds is weaker than that of the reference.
The successful development of a new drug depends on a
number of criteria that have to be met. For example, in
addition to intrinsic activity, the drug must be able to reach
its target and should not produce toxic effect. Hence, toxicity
of the synthesized compounds (5a–5i), which have significant
antifungal activity, was needed to be investigated. For this
purpose, Brine-Shrimp lethality assay, which has been
regarded as a useful method for preliminary evaluation of
toxicity and used for the establishing of fungal toxins, plant
extract toxicity, heavy metals, cyanobacteria toxins, pesti-
cides, cytotoxicity testing of dental materials [28], natural
and synthetic organic compounds [29], was performed. Such
toxicity test results show a good correlation with rodent and
human acute oral toxicity data. Likewise, the predictive
screening potential of the aquatic invertebrate tests for
acute oral toxicity in man, including Brine-Shrimp toxicity
test, are slightly better than the rat tests for the reference
compounds [30].
Comparison of the results of toxicity, antibacterial and
antifungal activity tests shows that the tested compounds
(5a–5i) have weak antibacterial activity against both Gram-
negative and Gram-positive bacterial strains. On the other
hand, significant inhibitory activity against Candida yeasts
points out the antifungal potential of the compounds.
Compounds 5a, 5c, and 5d are the most noteworthy in the
series because they display antifungal activity at non-toxic
concentrations. Hence, the antifungal activity of the com-
pounds is not due to their general toxicity effect but can be
ascribed to the selectivity of their antifungal properties.
Toxicity test results were analyzed by the LC50 computer
program (Trimmed Spearman-Karber Method, Version 1.5) so
as to calculate LC50 values and 95% confidence intervals [31]
Table 3. Brine-Shrimp toxicity results of compounds 5a–5i.
Compound
LC50 (mg/mL)
Lower 95% limit
Upper 95% limit
Toxicityꢂ
5a
5b
5c
5d
5e
5f
5g
>1000.00
103.11
>1000.00
>1000.00
33.49
51.43
<7.81
71.73
<7.81
793.70
–
44.06
–
–
241.30
–
Non-toxic
Harmful
Non-toxic
Non-toxic
Toxic
Toxic
Very toxic
Toxic
–
–
9.48
23.19
–
13.33
–
566.86
188.88
114.06
–
385.92
–
1111.31
5h
5i
Ketocanazole
Very toxic
Non-toxic
ꢂEvaluated by comparing with MIC values of the compounds against Candida species.
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Y. Ozkay et al.
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Arch. Pharm. Chem. Life Sci. 2011, 11, 264–271
In terms of structure-activity relationships (SARs), the sub-
stitution pattern was explored using various bioisosteric
(benz)azolylthio moieties arising from 1-methyl-1H-imida-
more difficult diffusion through the cell membrane [33]. In
the series, electron density of the non-toxic compounds 5a, c,
d is higher than that of the toxic ones because there is an
electron flow from electron donating methyl group to elec-
tron withdrawing nitrogen atom of the imidazole, triazole or
tetrazole ring. Thus, toxicity loss in the N-methyl substituted
compounds 5a, 5c, and 5d may be due to retardation in
intracellular transport because of high electron density.
zole-2-thiol,
1,2,4-4H-triazole-3-thiol,
1-methyl-1,2,4-4H-
triazole-3-thiol, 1-methyl-1,2,3,4-1H-tetrazole-5-thiol, 1-phe-
nyl-1,2,3,4-1H-tetrazole-5-thiol, 5-methyl-1,2,4-thiadiazole-3-
thiol, 1H-benzimidazole-2-thiol, benzoxazole-2-thiol, and
benzothiazole-2-thiol
in
2-[4-(acetylamino)phenyl]-5,6-
dichloro-1-methyl-1H-benzimidazole main substructure.
Thus, determination of contribution of the various bioiso-
steric (benz)azolylthio moieties to antibacterial and/or anti-
fungal activity and evaluation of SARs were planned.
However, SARs could not be discussed due to poor antibacte-
rial activity of the compounds (5a–5i). Moreover, observation
of very similar antifungal activity displayed by the com-
pounds (5a–5i) indicates that there is no important difference
between contributions of azolylthio moieties to antifungal
activity and makes consideration of the SARs very difficult.
Hence, it can be assumed that antifungal activity of the
compounds (5a–5i) is related to their general structural
characteristics provided by both bioisosteric azolylthio moi-
eties and 2-[4-(acetylamino)phenyl]-5,6-dichloro-1-methyl-1H-
benzimidazole substructure.
Conclusion
Nowadays growing resistance of pathogens against current
antibacterial and antifungal agents is a common and impor-
tant medical problem and research on new compounds
against these pathogens are needed. Hence, we have synthe-
sized some novel benzimidazole-azole derivatives and
screened for their probable antimicrobial activity. Results
of this study have indicated that most of the synthesized
compounds are very effective against Candida species.
Furthermore, compounds 5a, c, d have been recognized as
non-toxic and this has improved their chemotherapeutic
value. It has been observed that non-toxicity of the com-
pounds 5a, 5c, and 5d may be related to their low liphophil-
icity and high electron density. However, there is no
relationship between these physicochemical properties and
antimicrobial potency which can be a result of selective
antifungal effect of the compounds (5a–5i).
Contrary to SARs, a relationship between structural proper-
ties of the compounds (5a–5i) and their toxicological effect is
easily understood because the tested compounds (5a–5i) have
indicated toxicity to different extents, which can be the
result of difference between structural properties. In the
series, the most toxic compounds 5e, 5g, 5h, and 5i contain
1-phenyl-1H-tetrazol-5yl-thio, 1H-benzimidazol-2-ylthio, benz-
oxazol-2-ylthio and benzothiazol-2-ylthio moieties which
enhance lipophilic character of such derivatives. It is known
that there is a relationship between lipophilicity and toxicity.
In general, once the lipophilicity increases, a rise in the
toxicity also occurs. Depending on this respect, it may be
concluded that higher toxic effect of the compounds 5e, 5g–i
than the other derivatives is related to their increased lipo-
philicity. On the other hand, compounds 5b and 5f are not as
Consequently, significant antifungal activity displayed by a
novel combination system of benzimidazole and the other
bioisosteric (benz)azole rings have encouraged us to make
some modifications on basic structure of obtained com-
pounds (5a–5i) to achieve more active derivatives in ongoing
studies. Moreover, we believe that findings of the present
study will have a good impact on medicinal chemists to
synthesize similar compounds which will probably indicate
greater antifungal activity.
lipophilic as the 5e, 5g, 5h, and 5i, but they are the other toxic Materials and Methods
derivatives in the series. Hence, an additional reason to high
lipophilicity, which may be responsible for the toxicity,
should be clarified. If the structures of toxic derivatives (5b
and 5h) are compared with those of the non-toxic derivatives
(5a, 5c, and 5d), the most evident difference consist of pres-
ence of N-methyl groups of the azole rings in 5a, 5c, and 5d. It
has been reported that, in general, the presence of electron-
donating groups, such as methyl, seems to reduce the
toxicity, whereas electron-withdrawing substituents produce
higher toxic effect [32]. This approach has been supported by
the results of our study in which the N-methyl substituted
compounds 5a, c, d displayed no toxicity and can be
explained by the effect of high electron density, which causes
Chemistry
Melting points (m.p.) of the final compounds (5a–5i) were
determined in open capillaries on an Electrothermal 9001
Digital Melting Point Apparatus and are uncorrected. The
purity of the compounds was routinely checked by thin layer
chromatography (TLC) using silica gel 60. IR spectra were
recorded on a Shimadzu 8400 FTIR spectrometer as KBr
pellets. 1H-NMR spectra were recorded on
a Bruker
UltraShield 500 MHz spectrometer in DMSO-d6. MS data were
obtained on an Agilent 1100 Series LC/MSD Trap VL&SL spec-
trometer. Elemental analyses (C, H, and N) were determined
on a Perkin Elmer analyzer.
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Arch. Pharm. Chem. Life Sci. 2011, 11, 264–271
Benzimidazole Derivatives Bearing Imidazole or Triazole Moieties
269
The starting compounds 1 and 2 were synthesized accord-
H, triazole C2-H), 10.47 (s, H, NH-CO). 12.91 (br, H, triazole N-
H). ES-MS (m/z): M þ 1: 434.3. Anal. calcd. for C18H14Cl2N6OS
(433.32): C, 49.89; H, 3.26; N, 19.39. Found: C, 49.96; H, 3.25;
N, 19.42.
ing to previously described methods [9, 34].
2-(4-Aminophenyl)-5,6-dichloro-1-methyl-1H-
benzimidazole (3)
A solution of 2 (20.0g, 59.88 mmol) in conc. aq. HCl (50 mL)
was refluxed for 1 h and then allowed to cool and poured into
ice-water (50 mL). Neutralization with 10% aq. NaOH resulted
in the formation of a solid which was collected by filtration,
washed several times with cold water, and then recrystallized
from ethanol to give 3. Yield 86%. M.p. 2018C. IR (KBr)
5,6-Dichloro-1-methyl-2-[4-[2-[(4-methyl-4H-1,2,4-
triazole-3-yl)sulfanyl]acetylamino]phenyl]-1H-
benzimidazole (5c)
Yield 73%. M.p. 2688C. IR (KBr) nmax (cmꢀ1): 3332 (N-H), 1678
1
(C O), 1610–1451 (C C and C N). H-NMR (500 MHz) (DMSO-
–
–
–
–
–
–
d6) d (ppm): 3.54 (s, 3H, 5,6-dichlorobenzimidazole N-CH3),
3.65 (s, 3H, triazole N-CH3), 4.53 (s, 2H, CO-CH2), 7.73–7.80
(m, 6H, 5,6-dichlorobenzimidazole and 1,4-disubstituted
benzene Ar-H), 8.58 (s, H, triazole C2-H), 10.55 (s, H,
NH-CO). ES-MS (m/z): M þ 1: 448.4. Anal. calcd. for
nmax (cmꢀ1): 3349–3332 (N-H), 1684 (C O), 1607–1451 (C C
–
–
–
–
–
and C N).
–
2-[4-(2-Chloroacetylamino)-phenyl)]-5,6-dichloro-1-
methyl-1H-benzimidazole (4)
C19H16Cl2N6OS (447.35): C, 51.01; H, 3.61; N, 18.79. Found:
Triethylamine (8.5 mL, 60 mmol) was added to a solution of 3
(14.6 g, 50 mmol) in benzene (100 mL). A solution of chloro-
acetyl chloride (4.8 mL, 60 mmol) in benzene (10 mL) was
added dropwise under vigorous stirring to the above mixture
maintaining the temperature below 58C (ice bath). The result-
ing mixture was stirred for 1 h at room temperature and
then evaporated to dryness. The residue was recrystallized
from ethanol to give 4. Yield 73%. M.p. 250–2528C (decomp.).
C, 51.15; H, 3.62; N, 18.65.
5,6-Dichloro-1-methyl-2-[4-[2-[(1-methy-1H-1,2,3,4-
tetrazole-5-yl)sulfanyl]acetylamino]phenyl]-1H-
benzimidazole (5d)
Yield 81%. M.p. 2798C. IR (KBr) nmax (cmꢀ1): 3328 (N-H), 1673
1
(C O), 1610–1466 (C C and C N). H-NMR (500 MHz) (DMSO-
–
–
–
–
–
–
d6) d (ppm): 3.52 (s, 3H, 5,6-dichlorobenzimidazole), 4.01 (s,
3H, tetrazole N-CH3), 4.56 (s, 2H, CO-CH2), 7.73–7.80 (m, 6H,
5,6-dichlorobenzimidazole and 1,4-disubstituted benzene
Ar-H), 10.62 (s, H, NH-CO). ES-MS (m/z): M þ 1: 449.5. Anal.
calcd. for C18H15Cl2N7OS (448.34): C, 48.22; H, 3.37; N, 21.87.
Found: C, 48.11; H, 3.38; N, 21.85.
IR (KBr) nmax (cmꢀ1): 3336 (N-H), 1688 (C O), 1602–1456 (C C
–
–
–
–
–
and C N).
–
General synthesis procedure for target compounds (5a-5i)
A mixture of 4 (737 mg, 2.0 mmol), the appropriate (benz)a-
zolethiol (2.0 mmol), and K2CO3 (276 mg, 2.0 mmol) in
acetone was refluxed for 12 h. The residue was washed with
cold water and recrystallized from ethanol to give 5a–5i.
5,6-Dichloro-[4-[2-[(1-phenyl-1H-1,2,3,4-tetrazole-5-
yl)sulfanyl]acetylamino]phenyl]-1-methyl-2-1H-
benzimidazole (5e)
5,6-Dichloro-1-methyl-2-[4-[2-[(1-methyl-1H-imidazole-2-
yl)sulfanyl]acetylamino]phenyl]-1H-benzimidazole (5a)
Yield 72%. M.p. 2878C. IR (KBr) nmax (cmꢀ1): 3331 (N-H), 1674
1
(C O), 1614–1485 (C C and C N). H-NMR (500 MHz) (DMSO-
–
–
–
–
–
–
Yield 77%. M.p. 2308C. IR (KBr) nmax (cmꢀ1): 3329 (N-H), 1674
d6) d (ppm): 3.50 (s, 3H, 5,6-dichlorobenzimidazole N-CH3),
4.54 (s, 2H, CO-CH2), 7.59–7.64 (m, 3H, tetrazole N-Ph C3,4,5-H),
7.73–7.80 (m, 6H, 5,6-dichlorobenzimidazole and 1,4-disub-
stituted benzene Ar-H), 7.99 (d, 2H, J ¼ 8.6 Hz, tetrazole N-Ph
1
(C O), 1611–1452 (C C and C N). H-NMR (500 MHz) (DMSO-
–
–
–
–
–
–
d6) d (ppm): 2.97 (s, 3H, imidazole N-CH3), 3.57 (s, 3H, 5,6-
dichlorobenzimidazole N-CH3), 4.52 (s, 2H, CO-CH2), 7.71–
7.79 (m, 6H, 5,6-dichlorobenzimidazole and 1,4-disubstituted
benzene Ar-H), 8.01 (d, H, J ¼ 7.3 Hz, imidazole C3-H), 8.17 (d,
H, J ¼ 7.3 Hz, imidazole C2-H), 10.71 (s, H, NH-CO). ES-S (m/z):
M þ 1: 447.5. Anal. calcd. for C20H17Cl2N5OS (446.36): C,
53.82; H, 3.84; N, 16.69. Found: C, 53.94; H, 3.85; N, 16.72.
C
2,6-H), 10.68 (s, H, NH-CO). ES-MS (m/z): M þ 1: 511.4. Anal.
calcd. for C23H17Cl2N7OS (510.41): C, 54.12; H, 3.36; N, 19.21.
Found: C, 54.05; H, 3.34; N, 19.15.
5,6-Dichloro-1-methyl-2-[4-[2-[(5-methyl-1,2,4-
thiadiazole-3-yl)sulfanyl]acetylamino]phenyl]-1H-
benzimidazole (5f)
5,6-Dichloro-1-methyl-2-[4-[2-[(4H-1,2,4-triazole-3-
yl)sulfanyl]acetylamino]phenyl]-1H-benzimidazole (5b)
Yield 64%. M.p. 1788C. IR (KBr) nmax (cmꢀ1): 3421–3325 (N-H),
Yield 78%. M.p. 2218C. IR (KBr) nmax (cmꢀ1): 3335 (N-H), 1672
1
(C O), 1611–1489 (C C and C N). H-NMR (500 MHz) (DMSO-
–
–
–
–
–
–
1671 (C O), 1608–1453 (C C and C N). 1H-NMR (500 MHz)
d6) d (ppm): 2.69 (s, 3H, thiadiazole C2-CH3), 3.52 (s, 3H, 5,6-
dichlorobenzimidazole N-CH3), 4.53 (s, 2H, CO-CH2), 7.74–
7.82 (m, 6H, 5,6-dichlorobenzimidazole and 1,4-disubstituted
benzene Ar-H), 10.62 (s, H, NH-CO). ES-MS (m/z): M þ 1: 465.6.
–
–
–
–
–
–
(DMSO-d6) d (ppm): 3.54 (s, 3H, 5,6-dichlorobenzimidazole
N-CH3), 4.53 (s, 2H, CO-CH2), 7.69–7.77 (m, 6H, 5,6-dichloro-
benzimidazole and 1,4-disubstituted benzene Ar-H), 8.53 (s,
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

¨
Y. Ozkay et al.
270
Arch. Pharm. Chem. Life Sci. 2011, 11, 264–271
Anal. calcd. for C19H15Cl2N5OS2 (464.40): C, 49.14; H, 3.26; N,
15.08. Found: C, 49.05; H, 3.26; N, 15.11.
4420 and Proteus vulgaris NRLL B-123 and yeast as Candida
albicans, C. tropicalis and C. globrata ATCC 36583 (obtained from
Faculty of Medicine Osmangazi University, Eskisehir,
Turkey). Chloramphenicol and ketoconazole were used as
control drugs.
5,6-Dichloro-2-[4-[2-[(1H-benzimidazole-2-
yl)sulfanyl]acetylamino]phenyl]-1-methyl-1H-
benzimidazole (5g)
The synthesized compounds (5a–5i) were tested for their
toxicity by applying Brine-Shrimp lethality assay. Fresh eggs
of Brine-Shrimp (Artemia salina), sold as a fish food, were
purchased from the local pet shop, Eskisehir/Turkey.
Ketoconazole was used in the assay to compare toxicity levels
of the synthesized compounds (5a–5i) with that of reference
antifungal agent.
Yield 82%. M.p. 1978C. IR (KBr) nmax (cmꢀ1): 3408–3346 (N-H),
1677 (C O), 1608–1452 (C C and C N). 1H-NMR (500 MHz)
–
–
–
–
–
–
(DMSO-d6) d (ppm): 3.53 (s, 3H, 5,6-dichlorobenzimidazole
N-CH3), 4.54 (s, 2H, CO-CH2), 7.30–7.33 (m, 2H, benzimidazole
C
3,4-H), 7.72–7.82 (s, 8H, benzimidazole C2,5-H, 5,6-dichloro-
benzimidazole and 1,4-disubstituted benzene Ar-H), 10.77 (s,
H, NH-CO) 12.85 (br, H, benzimidazole N-H). ES-MS (m/z):
M þ 1: 483.5. Anal. calcd. for C23H17Cl2N5OS (482.39): C,
57.27; H, 3.55; N, 14.52. Found: C, 57.21; H, 3.54; N, 14.55.
Antimicrobial assay
Antimicrobial activity assay was performed according to CLSI
reference M7-A7 broth microdilution method as described in
previous study [35]. Twice MIC readings were carried out for
each chemical agent. The compounds (5a–5i) were dissolved
in DMSO for antibacterial and antimycotic assays. Further
dilutions of the compounds (5a–5i) and standard drugs in test
medium were prepared at the required quantities of 800,
400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625 mg/mL con-
centrations with Mueller-Hinton broth and Sabouroud dex-
trose broth. In order to ensure that the solvent per se had no
effect on bacteria or yeast growth, a control test was also
performed containing inoculated broth supplemented with
only DMSO at the same dilutions used in our experiments
and found inactive in culture medium.
5,6-Dichloro-2-[4-[2-[(benzoxazole-2-
yl)sulfanyl]acetylamino]phenyl]-1-methyl-1H-
benzimidazole (5h)
Yield 82%. M.p. 2418C. IR (KBr) nmax (cmꢀ1): 3330 (N-H), 1676
1
(C O), 1604–1454 (C C and C N). H-NMR (500 MHz) (DMSO-
–
–
–
–
–
–
d6) d (ppm): 3.52 (s, 3H, 5,6-dichlorobenzimidazole N-CH3),
4.56 (s, 2H, CO-CH2), 7.35–7.39 (m, 2H, benzoxazole C3,4-H),
7.64–7.69 (m, 2H, benzoxazole C2,5-H), 7.72–7.80 (m, 6H, 5,6-
dichlorobenzimidazole and 1,4-disubstituted benzene Ar-H),
10.68 (s, H, NH-CO). ES-MS (m/z): M þ 1: 484.4. Anal. calcd. for
C
23H16Cl2N4O2S (483.38): C, 57.15; H, 3.34; N, 11.59. Found: C,
57.35; H, 3.33; N, 11.55.
5,6-Dichloro-2-[4-[2-[(Benzothiazole-2-
yl)sulfanyl]acetylamino]phenyl]-1-methyl-1H-
benzimidazole (5i)
Brine-Shrimp lethality assay
Brine-Shrimp toxicity assay was used for determination of
cytotoxicity levels of the synthesized compounds (5a–5i).
Each test compound was dissolved in DMSO to obtain the
stock concentration of 1000 mg/mL and then stock solution
was diluted to various concentrations (1000–7.8125 mg/mL).
In order to prevent the toxicity results from possible false
effects originated from DMSO’s toxicity, stock solutions of
the compounds (5a–5i) were prepared according to suggested
volume range by dissolving 1 mg of test compound in 10 mL
DMSO and completing to 1000 mL with artificial seawater
[29]. Pure DMSO was used as a positive control for the
toxicity assay. The eggs of Brine-Shrimp hatched in a
conical flask containing 300 ml artificial seawater made by
dissolving a commercial marine salt in deionized water. The
flasks were well aerated with the aid of an air pump, and kept
in a water bath at 25–308C. The larvae hatched within 48 h.
Ten larvae were transferred with pipetter into each vial
containing test compound and artificial sea water. A
check count was performed after 24 h of exposure at
room temperature and the number of dead larvae,
exhibiting no internal or external movement during
several seconds of observation, was noted. Three independent
Yield 84%. M.p. 2048C. IR (KBr) nmax (cmꢀ1): 3347 (N-H), 1681
1
(C O), 1609–1452 (C C and C N). H-NMR (500 MHz) (DMSO-
–
–
–
–
–
–
d6) d (ppm): 3.52 (s, 3H, 5,6-dichlorobenzimidazole N-CH3),
4.55 (s, 2H, CO-CH2), 7.25–7.32 (m, 2H, benzothiazole C3,4-H),
7.73–7.81 (m, 6H, 5,6-dichlorobenzimidazole and 1,4-disub-
stituted benzene Ar-H), 7.86 (d, 2H, J ¼ 8.1 Hz benzothiazole
C5-H), 8.05 (d, 2H, J ¼ 8.0 Hz benzothiazole C2-H), 10.68 (s, H,
NH-CO). ES-MS (m/z): M þ 1: 500.5. Anal. calcd. for
C
23H16Cl2N4OS2 (499.44): C, 55.31; H, 3.23; N, 11.22. Found:
C, 55.15; H, 3.24; N, 11.19.
Microbiology
Final products were tested for their in-vitro growth inhibitory
activity against human pathogenic as Gram-positive bacteria;
Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC
29212, Bacillus subtilis and Listeria monocytogenes (obtained from
Faculty of Pharmacy Anadolu University, Eskisehir, Turkey),
as Gram-negative bacteria; Pseudomonas aeruginosa ATCC
27853, Klebsiella pneumoniae ATCC 13883, Escerichia coli ATCC
35218, E. coli ATCC 25922, Salmonella thyphimurium NRRL B-
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2011, 11, 264–271
Benzimidazole Derivatives Bearing Imidazole or Triazole Moieties
271
[17] A. A. Spasov, I. N. Yozhitsa, L. I. Bugaeva, V. A. Anisimova,
Pharm. Chem. J. 1999, 33, 232–243.
experiments were performed for each concentration of
compounds (5a–5i).
[18] F. Arjmand, B. Mohani, S. Ahmad, Eur. J. Med. Chem. 2005, 40,
1103–1110.
The authors have declared no conflict of interest.
[19] Z. A. Kaplancikli, G. Turan-Zitouni, G. Revial, K. Guven, Arch.
Pharm. Res. 2004, 24, 1081–1085.
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ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com