Antimicrobial activity and a SAR study of some novel benzimidazole derivatives bearing hydrazone moiety

Article abstract of DOI:10.1016/j.ejmech.2010.04.012

In this study 12 novel benzimidazole compounds bearing hydrazone moiety were synthesized in order to investigate their possible antibacterial and antifungal activity. Structures of the synthesized compounds were elucidated by spectral data. Six different gram-negative and four different gram-positive bacterial strains were used in antibacterial activity tests. Antifungal activity tests were also performed against three different fungal strains. Most of the test compounds found to be significantly effective against Proteus vulgaris,Staphylococcus typhimurium,Klebsiella pneumoniae and Pseudomonas aeruginosa gram-negative bacterial strains. A structureeactivity relationship (SAR) study including some electronic parameters was carried out and a connection between antibacterial activity and electronic properties of the target compounds was determined. Toxicity of the most effective compounds was established by performing BrineeShrimp lethality assay.

Full text of DOI:10.1016/j.ejmech.2010.04.012

European Journal of Medicinal Chemistry 45 (2010) 3293e3298
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antimicrobial activity and a SAR study of some novel benzimidazole
derivatives bearing hydrazone moiety
,
b
b
Yusuf Özkaya , Yagmur Tunalı , Hülya Karaca , Ilhan Is¸ ıkdag
ꢀ a
_
*
ꢀ
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskis¸ehir, Turkey
b Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Anadolu University, 26470 Eskis¸ehir, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study 12 novel benzimidazole compounds bearing hydrazone moiety were synthesized in order to
investigate their possible antibacterial and antifungal activity. Structures of the synthesized compounds
were elucidated by spectral data. Six different gram-negative and four different gram-positive bacterial
strains were used in antibacterial activity tests. Antifungal activity tests were also performed against
three different fungal strains. Most of the test compounds found to be significantly effective against
Proteus vulgaris, Staphylococcus typhimurium, Klebsiella pneumoniae and Pseudomonas aeruginosa gram-
negative bacterial strains. A structureeactivity relationship (SAR) study including some electronic
parameters was carried out and a connection between antibacterial activity and electronic properties of
the target compounds was determined. Toxicity of the most effective compounds was established by
performing BrineeShrimp lethality assay.
Received 10 December 2009
Received in revised form
12 April 2010
Accepted 13 April 2010
Available online 18 April 2010
Keywords:
Benzimidazole
Hydrazone
Antifungal activity
Antimicrobial activity
Structureeactivity relationship
Brine-shrimp lethality assay
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
of researchers toward benzimidazole derivatives has been 5,6-
dimethylbenzimidazole which is a constituent of naturally occur-
Infectious microbial diseases remain pressing problems world-
wide, because resistance to a number of antimicrobial agents (
ring vitamin B12 [16]. Although vitamin B12 is capable of inducing
the growth of bacteria, the benzimidazole component and some of
its derivatives repress the bacterial growth. Due to the structural
similarity to purine, antibacterial ability of benzimidazoles is
explained by their competition with purines resulting in inhibition
of the synthesis of bacterial nucleic acids and proteins [17,18].
In addition to their antibacterial activity, benzimidazole deriv-
atives possess antifungal activity. They can be classified as the most
important group of fungicides with systemic activity and are well
known for their pronounced ability to control a large number of
fungal diseases. Benomyl, thiabendazole and thiophnate methyl are
some main examples of this fungicide class. Owing to their
systemic activity, they can help to control some infectious microbial
diseases. They are also used for the prevention of post-harvest rots
and as soil-drench treatments [9,19].
Hydrazone is another considerable pharmacophore group for
antimicrobial activity. Some widely used antibacterial drugs such as
furacilin, furazolidone and ftivazide are known to contain this
group [20]. In past decades, hydrazones have received much
attention and many studies [21e29] have been reported due to
their chemotherapeutic value in the development of novel anti-
microbial agents.
b
-
lactam antibiotics, macrolides, quinolones, and vancomycin)
among variety of clinically significant species of microorganisms
has become an important global health problem [1]. One way to
battle with this challenge is the conscious usage of the currently
marketed antibiotics; the other is the development of novel anti-
microbial agents [2]. Hence, there will always be a vital need to
discover new chemotherapeutic agents to avert the emergence of
resistance and ideally shorten the duration of therapy.
Benzimidazole is an important pharmacophore and privileged
structure in medicinal chemistry. Literature survey shows that
among the benzimidazole derivatives, 2-substituted ones are found
to be pharmacologically more potent and hence the design and
synthesis of 2-substituted benzimidazoles are the potential area of
research [3,4]. Extensive biochemical and pharmacological studies
have confirmed that its derivatives are effective against various
strains of microorganisms [5e15]. The reason for a special interest
* Corresponding author. Tel.: þ902223350580/3772; fax: þ902223350750.
E-mail address: yozkay@anadolu.edu.tr (Y. Özkay).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.04.012

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Y. Özkay et al. / European Journal of Medicinal Chemistry 45 (2010) 3293e3298
Looking at the antimicrobial importance of benzimidazole and
2.2. Microbiology
hydrazone compounds it was thought that it would be worthwhile
to design, synthesize some new benzimidazole derivatives bearing
hydrazone moiety and screen them for potential antibacterial and
antifungal activities. Furthermore, after extensive literature search,
it was observed that, till date enough efforts have not been made to
combine these two vital moieties as a single molecular scaffold and
to study its antimicrobial activity. Consequently, prompted from
the findings described above, we here reported synthesis and
antimicrobial evaluation of some novel benzimidazole-hydrazone
compounds.
Final products were tested for their in vitro growth inhibitory
activity against human pathogens. Staphylococcus aureus ATCC
25923, Enterococcus faecalis ATCC 29212, Bacillus subtilis and
Listeria monocytogenes were evaluated as gram-positive bacteria.
As gram-negative bacteria; Pseudomonas aeruginosa ATCC 27853,
Klebsiella pneumoniae ATCC 13883, Escherichia coli ATCC 35218, E.
coli ATCC 25922, Salmonella typhimurium NRRL B-4420, and
Proteus vulgaris NRLL B-123 were tested. Candida albicans, andida
tropicalis and andida globrata ATCC 36583 yeasts were also used
in antimicrobial activity tests. Chloramphenicol and ketocanozole
were used as control drugs. The observed antimicrobial data of
the compounds and the reference drugs are given in Tables 2
and 3.
2. Results and discussion
2.1. Chemistry
When compared with the reference drug chloramphenicol,
most of the compounds in the series exhibited considerable anti-
bacterial activity against P. vulgaris and P. aeruginosa (Table 2). The
compounds 4b, 4d, 4e, 4g and the compounds 4ae4e, 4h were
more potent than reference against P. vulgaris and P. aeruginosa,
respectively. MIC value (50
mg/mL) of the other compounds in the
series was equal to reference. Only the compound 4j was found to
be inactive against these two bacterial strains (Table 2).
Present study was undertaken to synthesize some novel benz-
imidazole-hydrazone derivatives and investigate their probable
antibacterial and antifungal effects. Target compounds were
obtained at four steps. First of all, sodium disulfide adduct (1) of 4-
formylbenzoic acid methyl ester was prepared in dilute EtOH.
Secondly, o-phenylenediamine was reacted with the 1 in DMF to
achieve 4-(1H-benzimidazole-2-yl)benzoic acid methyl ester (2)
which was then treated with excess of hydrazine hydrate to obtain
4-(1H-benzimidazole-2-yl)benzoic acid hydrazide (3). At final step,
reaction of the 3 with corresponding 4-substitutedbenzaldehyde
derivative gave the title products (4ae4l). The structures of the
obtained compounds were elucidated by spectral data. Significant
stretching bands in the IR spectra were observed at expected
regions. All of the aromatic and aliphatic protons in the 500 MHz 1H
NMR spectra were also recorded at estimated areas. The mass
spectra (ES-MS) of the compounds showed [M þ 1] peaks, in
agreement with their molecular weight. Elemental analysis results
for C, H and N elements were satisfactory within ꢀ0.4% calculated
values of the compounds. Synthesis procedure of the benzimid-
azole-hydrazone derivatives was outlined in Scheme 1. Some
physicochemical properties and spectral data of the compounds
were given in Table 1.
The compounds 4d and 4fe4h showed significant antibacterial
activity (MIC ¼ 6.25
mg/mL) against S. typhimurium. Higher MIC
value (25 g/mL) than chloramphenicol against this bacterial strain
m
was an introduction of antibacterial inability of the compounds 4b,
4c and 4j. Antibacterial activity of the other five compounds was at
same degree with the reference drug (Table 2).
Some of the compounds exhibited equal antibacterial activity to
chloramphenicol against K. pneumoniae, S. aureus and E. faecalis.
Rest of them was found to be inactive against these bacterial strains.
None of the compounds performed notable growth inhibitory
activity against E. coli 35218, E. coli 25922, L. monocytogenes and B.
subtilis bacterial strains (Tables 2 and 3).
Antifungal activity of the tested benzimidazole-hydrazone
derivatives was observed as insignificant in comparison to their
antibacterial activity. None of the synthesized compounds had
greater activity then reference drug ketaconazole against any of the
fungal strains. However, the compounds 4a, 4d, 4i and the
compounds 4d-f, 4h, 4j, 4l were found to be as active as ketaco-
nazole against C. albicans and C. tropicalis, respectively (Table 3).
A chemical agent is valuable in medicinal field if only it
possesses low toxicity with significant activity. Thus, toxicity of the
compounds 4d, 4e and 4g which have the highest antibacterial
efficacy needs to be revealed. For this purpose Brine-Shrimp (Arte-
mia salina) lethality assay was performed. This assay is regarded as
a useful method for preliminary evaluation of toxicity, and it has
been used for establishing of fungal toxins, plant extract toxicity,
heavy metals, cyanobacteria toxins, pesticides, and cytotoxicity
testing of dental materials [30], natural and synthetic organic
compounds [31]. Moreover, A. salina toxicity test results show
a good correlation with rodent and human acute oral toxicity data.
Likewise, the predictive screening potential of the aquatic inver-
tebrate tests for acute oral toxicity in man, including A. salina
toxicity test, was slightly better than the rat tests for test
compounds [32].
Toxicity test results were analyzed with the LC50 computer
program (Trimmed SpearmaneKarber Method, Version 1.5) so as to
calculate LC50 values and 95% confidence intervals [33]. This
program failed to give LC50 values of the compounds and 95%
confidence intervals because number of dead larvae did not exceed
50% of total larvae. This was a significant result demonstrating that
the tested compounds are non-toxic in the tested concentration
range. Toxicity test results were presented in Table 4.
Scheme 1. Synthesis route of 4-substitutedbenzaldeyde N-[4-(1H-benzimidazol-2-yl)
phenyl]hydrazone derivatives (4ae4l).

Table 1
Some physicochemical properties and spectral data of the compounds 4ae4l.
No
R
M.P. (ꢁC)
280
Yield (%)
81
IR (KBr, cmꢂ1
)
1H NMR (500 MHz, DMSO-d6)
ES-MS
(M þ 1, m/z)
Found (calculated)
C%
H%
N%
4a
eH
3284e3229 (NeH), 1655 (C]O),
1611e1463 (C]N and C]C).
7.10e7.36 (5H, m, H-5,6,300,400,500), 7.50 (2H, m, H-4,7),
7.90e7.96 (4H, m, H-30,50,200,600), 8.14 (2H, d, J ¼ 8.69 Hz, H-20,60),
8.46 (H, s, eN]CHe), 11.76 (H, br, NeH, hydrazone),
12.14 (H, br, NeH, benzimidazole).
7.10e7.21 (4H, m, H-5,6,300,500), 7.49 (2H, m, H-4,7),
7.91e7.96 (4H, m, H-30,50,200,600), 8.14 (2H, d, J ¼ 8.61 Hz,
H-20,60), 8.42 (H, s, eN]CHe), 9.93 (H, s, OeH), 11.71
(H, br, NeH, hydrazone), 12.08 (H, br, NeH, benzimidazole).
2.98 (6H, s, eN(CH3)2), 6.91 (2H, d, J ¼ 8.54, H-300,500),
7.10e7.20 (2H, m, H-5,6), 7.52 (2H, m, H-4,7),
7.90e7.96 (4H, m, H-30,50,200,600), 8.14 (2H, d, J ¼ 8.60 Hz, H-20,60),
8.39 (H, s, eN]CHe), 11.68 (H, br, NeH, hydrazone),
12.08 (H, br, NeH, benzimidazole).
341
357
384
74.10 (73.96)
4.74 (4.73)
4.53 (4.55)
5.52 (5.51)
16.46 (16.51)
4b
4c
eOH
306
186
74
77
3281e3237 (NeH), 1661 (C]O),
1614e1460 (C]N and C]C).
70.78 (70.85)
72.04 (71.84)
15.72 (15.69)
18.26 (18.20)
eN(CH3)2
3287e3242 (NeH), 1659 (C]O),
1608e1459 (C]N and C]C).
4d
4e
4f
eCl
320
305
326
319
289
69
80
73
68
74
3293e3236 (NeH), 1648 (C]O),
1610e1461 (C]N and C]C).
7.10e7.20 (2H, m, H-5,6), 7.50e7.56 (4H, m, H-4,7, 300,500),
7.90e7.98 (4H, m, H-30,50,200,600), 8.14 (2H, d, J ¼ 8.58 Hz, H-20,60),
8.51 (H, s, eN]CHe), 11.65 (H, br, NeH, hydrazone),
12.12 (H, br, NeH, benzimidazole).
376
420
357
355
371
67.29 (67.41)
60.16 (60.10)
70.38 (70.52)
74.56 (74.71)
71.34 (71.29)
4.03 (4.05)
3.61 (3.62)
4.22 (4.24)
5.12 (5.10)
4.90 (4.90)
14.95 (14.89)
13.36 (13.49)
15.63 (15.68)
15.81 (15.86)
15.13 (15.17)
eBr
3271e3234 (NeH), 1657 (C]O),
1612e1464(C]N and C]C).
7.10e7.20 (2H, m, H-5,6), 7.50e7.65 (4H, m, H-4,7, 300,500),
7.90e8.02 (4H, m, H-30,50,200,600),
8.16 (2H, d, J ¼ 8.49 Hz, H-20,60), 8.53 (H, s, eN]CHe),
11.63 (H, br, NeH, hydrazone), 12.11 (H, br, NeH, benzimidazole).
7.10e7.20 (2H, m, H-5,6), 7.39e7.50 (4H, m, H-4,7, 300,500),
7.90e7.95 (4H, m, H-30,50,200,600), 8.16 (2H, d, J ¼ 8.54 Hz, H-20,60),
8.49 (H, s, eN]CHe), 11.63 (H, br, NeH, hydrazone),
12.12 (H, br, NeH, benzimidazole).
2.34 (3H, s, eCH3), 7.10e7.22 (4H, m, H-5,6,300,500),
7.50 (2H, m, H-4,7), 7.89e7.96 (4H, m, H-30,50,200,600),
8.14 (2H, d, J ¼ 8.52 Hz, H-20,60), 8.44 (H, s, eN]CHe),
11.71 (H, br, NeH, hydrazone), 12.05 (H, br, NeH, benzimidazole).
3.88 (3H, s, eOCH3), 7.02 (2H, d, J ¼ 8.47 Hz, H-300,500),
7.10e7.21 (2H, m, H-5,6,), 7.51 (2H, m, H-4,7),
7.89e7.95 (4H, m, H-30,50,200,600),
-F
3286e3239 (NeH), 1658 (C]O),
1609e1460 (C]N and C]C).
4g
4h
eCH3
eOCH3
3290e3230 (NeH), 1659 (C]O),
1611e1462 (C]N and C]C).
3288e3241 (NeH), 1657 (C]O),
1610e1459 (C]N and C]C).
8.11 (2H, d, J ¼ 8.53 Hz, H-20,60), 8.40 (H, s, eN]CHe),
11.64 (H, br, NeH, hydrazone), 12.08 (H, br, NeH, benzimidazole).
7.10e7.20 (2H, m, H-5,6,), 7.52 (2H, m, H-4,7),
7.92e8.09 (4H, m, H-30,50,200,600),
4i
-NO2
335
73
3279e3235 (NeH), 1650 (C]O),
1612e1456 (C]N and C]C).
386
65.45 (65.57)
3.92 (3.90)
18.17 (18.11)
8.15 (2H, d, J ¼ 8.56 Hz, H-20,60),
8.37 (2H, d, J ¼ 8.13 Hz, H-300,500),
8.58 (H, s, eN]CHe),
11.67 (H, br, NeH, hydrazone),
12.09 (H, br, NeH, benzimidazole).
(continued on next page)

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Y. Özkay et al. / European Journal of Medicinal Chemistry 45 (2010) 3293e3298
Table 2
MIC values (mg/mL) of benzimidazole-hydrazone derivatives against gram-negative
bacterial strains.
Compound
A
B
C
D
E
F
4a
4b
4c
4d
4e
4f
4g
4h
4i
25
25
25
25
25
25
25
25
25
25
50
25
12.5
100
100
100
100
50
50
100
50
100
100
200
100
50a
25b
50a
25b
25b
50a
25b
50a
50a
50a
12.5a
25
25
25
25
50
25b
25b
25b
25b
25b
50a
50a
25b
50a
50a
6.25b
12.5a
6.25b
6.25b
6.25b
12.5a
12.5a
25
12.5a
12.5a
25
12.5a
25
25
25
4j
4k
4l
100
50a
50
50
100
50a
50
12.5a
12.5
12.5a
12.5
Ref.
12.5
A: Escherichia coli 35218; B: Escherichia coli 25922; C: Proteus vulgaris; D: Salmonella
thyphimurium; E: Klebsiella pneumoniae; F: Pseudomonas aeruginosa.
Reference: Chloramphenicol.
a
Equal MIC value to reference.
Lower MIC value than reference.
b
2.3. SAR studies
The substitution pattern of the benzimidazole-hydrazone
derivatives was carefully selected to confer different electronic
environment to the molecules. Thus, electron donating groups to
aromatic ring, such as halogens, methyl, methoxy, hydroxyl and
dimethylamine and electron withdrawing groups from aromatic
ring, such as nitro, trifluoromethyl, carboxyl and cyano were chosen
as substituents on the chemical structure of the target compounds.
The compounds 4ie4l, bearing electron withdrawing groups, did
not exhibit stronger antibacterial activity than reference drug
chloramphenicol against any of the bacterial strains. However,
nitro, trifluoromethyl and cyano substituted compounds 4i, 4j and
4l found to be as effective as reference drug against some of the
bacterial strains (Tables
2 and 3). On the other hand, the
compounds 4be4h which were prepared to contain electron
donating groups, showed lower MIC value then chloramphenicol
against at least one bacterial strain. Among the compounds, chloro,
bromo and methyl substituted ones 4d, 4e and 4g possessed wider
antibacterial spectrum than the others (Tables 2 and 3). The
compound 4d was the most active derivative in the series, because
MIC value of it was not only lower than the reference drug against P.
vulgaris, S. thyphimurium and P. aeruginosa but also equal to
Table 3
MIC values (
mg/mL) of benzimidazole-hydrazone derivatives against gram-positive
bacterial and fungal strains.
Compound
A
B
C
D
E
F
G
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
400
400
200
100
200
200
200
200
200
200
400
200
50
25
50
50
12.5a
25
50
25
25
25
25
50
25
12.5
e
12.5a
25
25
25
25
25
25
25
50
25
50
50
50
50
12.5
e
50a
100
100
50
100
100
100
100
100
25
12.5a
12.5a
25
50a
50
50a
100
100
100
100
100
100
100
50
100
100
200
100
e
50a
50a
12.5a
12.5a
25
100
50a
100
50a
200
50a
50a
12.5a
50
100
200
100
e
12.5a
12.5
e
Ref. 1
Ref. 2
e
e
50
25
50
A: Listeria monocytogenes; B: Staphylococcus aureus; C: Enterococcus faecalis; D:
Bacillus subtilis; E: Candida albicans; F: Candida globrata; G: Candida tropicalis.
Reference 1: Chloramphenicol: Reference 2: Ketaconazole.
a
Equal MIC value to reference.

Y. Özkay et al. / European Journal of Medicinal Chemistry 45 (2010) 3293e3298
3297
Table 4
ability causes to decrease on the number of effected strains. Hence,
it can be declared that chloro, bromo and methyl substituents
bearing derivatives are the most suitable compounds for achieving
the best antibacterial spectrum. The reason for this result may be
explained by electron density of the compounds. It has been
reported that electron-donating groups increase the electron
density which makes the compounds effective against microor-
ganisms and enhances the antibacterial activity [28]. However, high
electron density causes more difficult diffusion through the
bacteria cell and substantial activity loss may occur [34]. Thus, for
a compound an optimum electron density is inevitable so as to gain
a significant antibacterial activity. As a result it can be thought that
electron donating ability of the chloro, bromo, and methyl groups
contributes to the compounds 4d, 4e and 4g to reach an optimum
electron density which is important for antibacterial activity.
Brine-shrimp toxicity results of the compounds 4d, 4e, and 4g.
Concentration (
m
g/mL)
Mortalitya
4d
4e
4g
1000
500
250
125
62.5
31.25
15.625
7.8125
3.906
Control
LC50
3
3
3
1
1
1
1
1
1
1
4
3
3
2
2
1
1
1
3
3
2
1
1
1
1
1
1
1
1
1
>1000
mg/mL
>1000
Non-toxic
m
g/mL
>1000 mg/mL
Non-toxic
Toxicity
Non-toxic
a
Ten organisms (Artemia salina) tested for each concentration.
3. Conclusion
The preliminary in vitro antibacterial, antifungal and toxicolog-
ical screening results of novel benzimidazole-hydrazone deriva-
tives reported here have indicated the antimicrobial potent of the
synthesized compounds. Most effective compounds were found to
be non-toxic in A. salina toxicity test. Performed SAR observation
has showed the importance of electronic environment on antimi-
crobial activity. The presence of chloro, bromo and methyl
substituents on the aromatic ring has increased the activity of the
compounds compared to those with other substituents. Findings
from the SAR and toxicity studies have encouraged us to make
some modifications on basic structure of the obtained compounds
to achieve selective, more active and non-toxic derivatives in
ongoing studies. In addition, for further investigations these find-
ings can have a good effect on medicinal chemists to synthesize
similar compounds selectively bearing chloro, bromo and methyl
substituents.
reference against K. pneumoniae, S. aureus and E.s faecalis. Besides,
MIC values of the other derivatives were not lower than the 4d
against any of these bacterial strains (Tables 2 and 3).
In order to clarify relationship between antibacterial activity
and electronic properties of the target compounds some electronic
parameters of the substituents were evaluated. For this purpose,
electronic substituent constant (
effect constant (R) and polarizabilitiy (
s
), field constant (F), resonance
) of the compounds were
a
determined (Table 5). Number of the bacterial strains against which
the test compounds exhibited valuable antibacterial effect (lower
or equal MIC value when compared with reference) were counted
and then probable relationship between number of effected
bacterial strains and electronic substituent constants were sought.
After examining the Table 5, no relationship between antibac-
terial activity and s, F or a constants was established. On the other
hand, a relationship between R values and number of effected
bacterial strains was determined. Order of this constant from the
most electron withdrawing (eCN and eCF3, R ¼ 0.19) substituent to
most electron donating (eN(CH3)2, R ¼ ꢂ0.92) one and number of
effected bacterial strains were concordant at a point. As seen in
Table 5, it is very clear that electron donating abilities of the chloro
(R ¼ ꢂ0.15), bromo (R ¼ ꢂ0.17) and methyl (R ¼ ꢂ0.13) groups,
which are the substituents of the most active compounds 4d, 4e
and 4g, are very close. Besides, the number of significantly inhibited
strains by the 4d, 4e and 4g is higher than the other compounds
(Table 5). This finding indicates that according to chloro, bromo and
methyl substituents, increasing electron withdrawing or donating
4. Materials and methods
4.1. Chemistry
All of the chemicals used in syntheses were obtained from
Merck. Melting points (m.p) of target compounds were measured
in Electrothermal 9001 Digital Melting Point Apparatus and
uncorrected. IR spectra were recorded on Shimadzu, 8400 FTIR
spectrometer as KBr pellets. 1H NMR spectra were recorded on
Bruker UltraShield 500 MHz spectrometer in deutoro dimethyl
sulfoxide. ES-MS data were obtained on Agilent 1100 Series LC/MSD
Trap VL&SL spectrometer. Elemental analyses (C, H, and N) were
determined on a Perkin Elmer analyser.
Table 5
Some electronic constants of compounds 4ae4l and number of inhibited bacterial
strains.
4.1.1. 4-(1H-benzimidazole-2-yl)benzoic acid methyl ester (2)
4-Formylbenzoic acid methyl ester (16.4 g, 100 mmol) and
sodium disulfide (9.5 g, 50 mmol) were dissolved in 100 mL of
aqueous EtOH (80%) and stirred at room temparature for 30 min.
20 mL of EtOH was added and allowed to cool in an ice bath for
a several hours. The precipitate was filtered and dried (yield% 91).
The mixture of this salt (21.44 g, 80 mmol) and o-phenylenedi-
amine (8.64 g, 80 mmol) in DMF (40 mL) were heated at 130 ꢁC for
4 h. The reaction mixture was poured into ice-water and the solid
was filtered, crystallisation of crude product from EtOH gave the 2.
Yield: 63%. m.p. 228 ꢁC. IR (KBr, nmaks cmꢂ1): 1463e1608 (C]C and
C]N), 1737 (C]O), 3284e3231 (NeH).
Compound
R
sa
0.00
ꢂ0.37
ꢂ0.83
0.23
0.23
0.06
ꢂ0.17
ꢂ0.27
0.78
Fa
0.00
Ra
0.00
ab
NBSc
4a
4b
4c
4d
4e
4f
4g
4h
4i
eH
39.95
40.53
44.76
41.83
42.54
39.54
41.72
42.45
41.74
40.81
42.22
41.76
4
2
2
6
5
3
5
4
3
4
0
4
eOH
eN(CH3)2
eCl
eBr
eF
eCH3
eOCH3
eNO2
eCF3
eCOOH
eCN
0.29
0.10
0.41
0.44
0.43
ꢂ0.04
0.26
0.67
0.38
0.33
0.51
ꢂ0.64
ꢂ0.92
ꢂ0.15
ꢂ0.17
ꢂ0.34
ꢂ0.13
ꢂ0.51
0.16
4j
4k
4l
0.54
0.45
0.66
0.19
0.15
0.19
a
Electronic substituent constant taken from Hansch et al. [37].
Calculated using ChemAxon/MarvinSketch computer program.
Number of bacterial strains against which the test compounds exhibited lower
4.1.2. 4-(1H-benzimidazole-2-yl)benzoic acid hydrazide (3)
To a suspansion of the 2 (10.04 g, 40 mmol) in 50 mL EtOH,
15 mL hydrazine hydrate (80%) was added. Reaction mixture was
b
c
or equal MIC value when compared with reference.

3298
Y. Özkay et al. / European Journal of Medicinal Chemistry 45 (2010) 3293e3298
refluxed for 24 h. Then the solvent and excess of hydrazine hydrate
was evaporated and the residue was washed with water, filtered,
dried, and then crystallized from EtOH. Yield % 78. m.p. 304 ꢁC. IR
(KBr, nmaks cmꢂ1): 1458e1604 (C]C and C]N), 1658 (C]O),
3277e3234 (NeH).
water. The flasks were well aerated with the aid of an air pump, and
kept in a water bath at 25e30 ꢁC. The larvae were hatched within
48 h. Ten larvae were transferred with pipetter into each vial
containing test compound and artificial seawater. 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 experiments were performed for each concentration
of compounds.
4.2. General synthesis procedure of 4-Substitutedbenzaldeyde N-[4-
(1H-benzimidazol-2-yl)phenyl] hydrazone derivatives (4ae4l)
Equimolar quantities (2 mmol) of 3 and appropriate 4-sub-
stitutedbenzaldehydes in 25 mL of butanol were refluxed for 3 h
with the presence of catalytic amount of glacial acetic acid. The
resulting solid was filtered and recrystallized from EtOH. Some
physicochemical properties and spectral data of the compounds
were given in Table 1.
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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 concentrations with
MuellereHinton broth and Sabouroud dextrose broth.
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bacteria or yeast growth, a control test was also performed con-
taining inoculated broth supplemented with only DMSO at the
same dilutions used in our experiments and found inactive in
culture medium.
4.3.1. Antimicrobial assay
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well with no growth of microorganism was recorded to represent
the MIC expressed in mg/mL. Each experiment in the antimicrobial
assays was replicated twice in order to define the MIC values.
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levels of the most active compounds (4d, 4e, 4g). Each test
compound was dissolved in DMSO to obtain the stock concentration
of 1000
concentrations (1000e3.906
m
g/ml and then stock solution was diluted to various
g/ml). In order to prevent the toxicity
m
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DMSO was used as a positive control for the toxicityassay. Fresh eggs
of A. salinawere purchased fromthe localpet shop, Eskisehir/Turkey.
The eggs were hatched in a conical flask containing 300 ml artificial
seawater made by dissolving a commercial marine salt in deionised