Analgesic, anti-inflammatory, and antimicrobial activities of novel isoxazole/pyrimidine/pyrazole substituted benzimidazole analogs

Article abstract of DOI:10.1007/s00044-017-2000-0

From o-phenylenediamine and p-amino benzoic acid, several new 3-methylisoxazol-5(4H)-one/2-hydroxy/mercapto-6-methylpyrimidin-4(5H)-one/3-methyl-1-substituted-1H-pyrazol-5(4H)-one substituted benzimidazole derivatives 5–16 were synthesized through multi step synthesis based on hybrid approach. All test compounds were screened for its analgesic, anti-inflammatory, and in vitro antimicrobial activity by tail flick method, carrageenan induced foot paw edema method and agar streak dilution method, respectively. Most active compounds were examined for its ulcerogenicity by pylorus ligation method. The relationship between chemical structure and biological activities of the test compounds were discussed. Among various tested compounds, 4-(2-(4-(1H-benzimidazol-2-yl)phenyl)hydrazono)-1-(4-chlorophenyl)-3-methyl-1H-pyrazol-5(4H)-one 10 was found to be most potent compound. This compound showed 72% analgesic activity (20 mg/kg; second hour) and 67% protection in paw edema test (20 mg/kg; second hour) which are comparable with that of standard Diclofenac [69% analgesic activity (20 mg/kg; second hour) and 65% protection in paw edema test (20 mg/kg; second hour)]. In addition, compound 10 exhibited least ulcer index which is about 1/3 of the ulcer index of reference standard and showed good antibacterial and moderate antifungal activity.

Full text of DOI:10.1007/s00044-017-2000-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2017) 26:3026–3037
DOI 10.1007/s00044-017-2000-0
ORIGINAL RESEARCH
Analgesic, anti-inflammatory, and antimicrobial activities of novel
isoxazole/pyrimidine/pyrazole substituted benzimidazole analogs
Krishna Veni Chikkula1 Raja Sundararajan2
●
Received: 23 April 2017 / Accepted: 20 July 2017 / Published online: 9 August 2017
© Springer Science+Business Media, LLC 2017
Abstract From o-phenylenediamine and p-amino benzoic
acid, several new 3-methylisoxazol-5(4H)-one/2-hydroxy/
mercapto-6-methylpyrimidin-4(5H)-one/3-methyl-1-sub-
stituted-1H-pyrazol-5(4H)-one substituted benzimidazole
derivatives 5–16 were synthesized through multi step
synthesis based on hybrid approach. All test compounds
were screened for its analgesic, anti-inflammatory, and
in vitro antimicrobial activity by tail flick method, carra-
geenan induced foot paw edema method and agar streak
dilution method, respectively. Most active compounds were
examined for its ulcerogenicity by pylorus ligation method.
The relationship between chemical structure and biological
activities of the test compounds were discussed. Among
various tested compounds, 4-(2-(4-(1H-benzimidazol-2-yl)
phenyl)hydrazono)-1-(4-chlorophenyl)-3-methyl-1H-pyrazol-
5(4H)-one 10 was found to be most potent compound. This
compound showed 72% analgesic activity (20 mg/kg; second
hour) and 67% protection in paw edema test (20 mg/kg;
second hour) which are comparable with that of standard
Diclofenac [69% analgesic activity (20 mg/kg; second hour)
and 65% protection in paw edema test (20 mg/kg; second
hour)]. In addition, compound 10 exhibited least ulcer index
which is about 1/3 of the ulcer index of reference standard
and showed good antibacterial and moderate antifungal
activity.
●
●
●
Keywords Benzimidazole Isoxazole Pyrimidine
●
●
Analgesic Anti-inflammatory Antimicrobial
Introduction
Non steroidal anti-inflammatory drugs (NSAIDs) are among
the most commonly prescribed drugs worldwide. They are
also consumed on non-prescription basis as well due to their
“over the counter” availability. Conversely, their intake is
normally associated with gastrointestinal (GI) side effects,
representing an important medical and socioeconomic pro-
blem (Abdel Tawab et al. 2009). Long term use of NSAIDs
is associated with side effects like hepatotoxicity, platelet
dysfunction, and bleeding (Feldman et al. 2000; Trepanier
2002; Bergh and Budsberg 2005). But the major and
potential side effect with chronic use of NSAIDs is GI
ulcerations (Dannhardt and Kiefer 2001). These drugs can
affect all segments of the GI tract and be responsible for
ulceration, dyspepsia, or gastric bleeding. In a few cases,
they can cause severe toxicity requiring hospital admission
and aggressive management (Dhikav et al. 2003)
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-2000-0) contains supplementary material,
which is available to authorized users.
In the field of pharmaceutical and medicinal chemistry,
benzimidazole and its derivatives are found to be trendy
structures employed for discovery of drugs within the vast
range of heterocycles. In drug discovery, the unique struc-
tural features of benzimidazole and a wide range of biolo-
gical activities of its derivatives made it privileged structure
(Immadisetty et al. 2014). Recently, benzimidazole scaffold
has emerged as a pharmacophore of choice for designing
* Raja Sundararajan
sraja61@gmail.com
1
Sarojini Naidu Vanita Pharmacy Maha Vidyalaya, Secunderabad,
Telangana 500017, India
2
Department of Pharmaceutical Chemistry, GITAM Institute of
Pharmacy, GITAM University, Gandhi Nagar, Rushikonda,
Visakhapatnam, Andhra Pradesh 530 045, India

Med Chem Res (2017) 26:3026–3037
3027
Fig. 1 Hybrid approach design of isoxazole/pyrimidine/pyrazole substituted benzimidazole analogs (5–16)
analgesic and anti-inflammatory agents acting on different
clinically approved targets for pain and inflammation
(Monika et al. 2014; Monika et al. 2015). In addition ben-
zimidazole is a well established class of H+/K+ATPase
inhibitors, therapeutically useful in the treatment of acute
and chronic ulcer conditions (Anderson and Carlsson 2005;
Avinash et al. 2008). Hydrazones which is considered to be
a advantaged structure, has been used as the foundation for
designing new analgesic and anti-inflammatory compounds,
because the hydrazone subunit is a key pharmacophore for
binding to and inhibiting cyclooxygenases (Thais et al.
2014; Chelucci et al. 2014). Azole containing compounds
exhibited variety of biological activities and some of them
are in clinical practice as antimicrobial agents and NSAIDs
(Theivendren et al. 2014; Javed et al. 2016). Some pyrazole
(Penning et al. 1997; Panneer Selvam et al. 2011; Surendra
et al. 2016) and isoxazole (Govindaraj et al. 2013) deriva-
tives are extensively studied for its anti-inflammatory and
antimicrobial potency. More over pyrimidines are an
important class of heterocyclic compound and many pyr-
imidine derivatives are reported to have broad spectrum of
biological activities (Mona 2012; Bhimanna and Hesham
2016).
developing effective NSAID agents with less GI toxicity by
a hybrid approach using the benzimidazole scaffold (Fig. 1).
In the present study, we have placed the substituted iso-
xazole/pyrimidine/pyrazole moiety at the C-2 position of
benzimidazole ring as hydrazones and studied their
analgesic, anti-inflammatory, ulcerogenicity, and anti-
microbial activities.
Results and discussion
Chemistry
The title compounds 4–16 were synthesized as per the
protocol shown in Scheme 1. By substituting different
groups at iminie nitrogen, a sequence of novel 1-(4-(1H-
benzimidazol-2-yl)phenyl)hydrazone analogs were synthe-
sized in the present study. From o-phenylenediamine and p-
amino benzoic acid, a sequence of novel heterocyclic sub-
stituted
1-(4-(1H-benzimidazol-2-yl)phenyl)hydrazono
analogs 4–16 were synthesized by a multistep synthesis.
Primarily o-phenylenediamine 1 was treated with p-amino
benzoic acid 2 in presence of polyphosphoric acid (PPA) to
obtain 4-(1H-benzimidazol-2-yl)benzenamine 3 by cycli-
sation reaction. In the second step, obtained amino deriva-
tive 3 was diazotized using sodium nitrite and hydrochloric
acid. Subsequently the diazotized salt was treated with
ethylacetoacetate to produce ethyl 2-(2-(4-(1H-benzimida-
Hybrid approach is an approach where two or more
pharmacophores are merged into a single molecule.
Therefore, a single molecule containing more than one
pharmacophore, each pharmacophores may be addressing
the active site of targets and offer the possibility to selec-
tivity, further it can also reduce unwanted side effects. The
present work is an extension of our ongoing efforts towards
zol-2-yl)phenyl)hydrazono)-3-oxobutanoate
4
through
intramolecular rearrangement reaction. In the final step, the

3028
Med Chem Res (2017) 26:3026–3037
Scheme 1 Synthesis of novel isoxazole/pyrimidine/pyrazole substituted benzimidazole analogs (5–16)
obtained keto ester 4 undergoes dehydrative cyclisation
with various amine derivatives such as hydroxylamine
hydrochloride, urea/thiourea, and different hydrazine
hydrochloride analogs and produces corresponding iso-
xazole 5, pyrimidine 6–7, and pyrazole derivatives 8–16,
respectively. To optimize the reactions for purity and
completion, thin layer chromatography (TLC) was per-
formed throughout the reactions.
Assigned structures of the test compound were confirmed
using various spectral studies (infrared (IR), nuclear mag-
netic resonance (NMR), mass spectra, and elemental ana-
lysis). Presence of particular groups was identified from IR
spectra by means of some characteristic absorption peaks.
Absence of a broad absorption peak between 3000 and
3500 cm−1 corresponds to COOH and presence of absorp-
tion peak at 3357 cm−1 in IR due to presence of NH
stretching, confirms the formation of 4-(1H-benzimidazol-
2-yl)benzenamine 3. It is further supported by appearance
of singlet in its 1H-NMR spectra at δ 5.78 and 4.30 ppm for
one and two proton which might be assigned to NH and
NH2 proton, respectively. The formations of keto ester 4
was confirmed from the appearance of sharp peak at 1703
cm−1 in IR corresponds to C=O stretching and appearance
of three singlet peaks at δ 6.47, 5.83, and 2.22 ppm for one,
one, and three protons which might be assigned to NH of
hydrazone, NH of benzimidazole, and CH3 proton,
respectively. This is further confirmed from NMR spectro-
scopy by the appearance of quartet peak at δ 4.49–4.71 ppm
for two protons, which might be assigned to CH2 of C2H5
and triplet peak at δ 1.71–1.80 ppm for three protons which
might be assigned to CH3 of C2H5. The formation of novel
heterocyclic substituted 1-(4-(1H-benzimidazol-2-yl)phe-
nyl)hydrazone analogs 5–16 from ethyl 2-(2-(4-(1H-benzi-
midazol-2-yl)phenyl)hydrazono)-3-oxobutanoate 4 can be
recognized from the disappearance of quartet and triplet
1
peaks in its H-NMR spectra corresponds to the CH2 and
CH3 of C2H5. The formation of compounds 5, 6, and 7 are
confirmed by the appearance of singlet peak in its 1H-NMR
spectra at δ 2.28, 2.62 and 2.25 ppm corresponds to the
three protons of CH3, one proton of OH, and one proton of

Med Chem Res (2017) 26:3026–3037
3029
SH, respectively. Appearance of singlet for one proton of
pyrazole NH at δ 6.65 ppm confirms the formation of
compound 8. Multiplet for 13 protons observed at δ
6.86–8.30 ppm confirms the structure of compound 9.
Appearance of sharp peak at 858 and 879 cm−1, in IR
corresponds to chlorine confirms the presence of chlorine in
compounds 10 and 13, respectively. Similarly, appearance
of sharp peak at 1085 and 1065 cm−1, in IR corresponds to
fluorine confirms the presence of fluorine in compounds 11
and 14, respectively. Appearance of singlet for three protons
of methoxy group at δ 3.59 ppm confirms the formation of
compound 12. Meanwhile, appearance of singlet for two
protons of amino group at δ 2.88 ppm confirms the forma-
tion of compound 15. The formation of compound 16 was
confirmed by appearance of multiplet at δ 7.38–8.59 ppm
for 12 protons. The structure of title compounds 5–16 were
further confirmed by appearance of various other peaks in
NMR spectroscopy corresponds to assigned structure. Mass
spectrum further confirmed their molecular weight and
purity.
unsubstituted derivative). Moreover, no change in activity
was observed when N-1 hydrogen of pyrazole was sub-
stituted with thioamide or isonicotinoyl group.
Anti-inflammatory activity
To study the anti-inflammatory activity of test compounds
3–16 Carrageenan induced paw edema test was performed
using Wistar rats and the obtained results are summarized in
Table 2. Entire synthesized benzimidazole analogs showed
anti-inflammatory activity in similar way like analgesic
activity. From the study the most potent compounds were
found to be 4-(2-(4-(1H-benzimidazol-2-yl)phenyl)hydra-
zono)-1-(4-chlorophenyl)-3-methyl-1H-pyrazol-5(4H)-one 10,
4-(2-(4-(1H-benzimidazol-2-yl)phenyl)hydrazono)-1-(4-fluor-
ophenyl)-3-methyl-1H-pyrazol-5(4H)-one 11, and 4-(2-(4-
(1H-benzimidazol-2-yl)phenyl)hydrazono)-1-(4-chlorophe-
nyl)-3-methyl-1H-pyrazol-5(4H)-one 12. Among fourteen
tested analogs, -(1H-benzimidazol-2-yl)benzenamine 3 and
ethyl 2-(2-(4-(1H-benzimidazol-2-yl)phenyl)hydrazono)-3-
oxobutanoate 4 exhibited worst anti-inflammatory activity.
Biological activities
Ulcerogenicity
Analgesic activity
Due to poor analgesic and anti-inflammatory activity,
compounds 3–7 and 15–16 were not tested for its ulcer-
ogenicity; whereas rest of compounds 8–14 which exhibited
good analgesic and anti-inflammatory activity were exam-
ined for its ulcerogenicity and the results are summarized in
Table 2. Compared to standard Diclofenac entire tested
compounds exhibited less ulcer index. Ulcer index of test
compounds revealed that para substituted phenyl ring pos-
sessing pyrazole derivatives 10–12 exhibited negligible
ulcer index, whereas shifting of position from para to meta
leads to slight increases in ulcer index. Over other test
compounds, compounds 13–14 possessing m-chloro and
m-fluoro group exhibited higher ulcer index. Compared to
the reference drug Diclofenac (1.68 0.52) the test com-
pounds exhibited 32–64% of the ulcer index. Compound 4-
(2-(4-(1H-benzimidazol-2-yl)phenyl)hydrazono)-1-(4-
chlorophenyl)-3-methyl-1H-pyrazol-5(4H)-one 10 ex-
hibited least ulcer index (0.53 0.29) among the tested
compounds which is about one-third of the ulcer index of
reference standard. Out of entire tested compounds, 4-(2-(4-
(1H-benzimidazol-2-yl)phenyl)hydrazono)-1-(3-fluorophen-
yl)-3-methyl-1H-pyrazol-5(4H)-one 14 was found to
possess highest ulcer index (1.08 0.45) which is about
64% of the ulcer index of Diclofenac.
Using Wistar albino mice, novel synthesized compounds
3–16 were subjected to analgesic activity test by tail flick
technique and the obtained results are presented effectively
in Table 1. Graded dose response and significant activity
was observed for almost entire test compounds. The
analgesic activity of novel benzimidazole derivatives 3–16
were varied with reaction time. In general test compounds,
at 30 min of reaction time displayed moderate analgesic
activity; at first hour the activity increased, further at second
hour it reached to peak level and at third hour the activity
decreased. The amino derivative 3 and keto ester analog 4
exhibited weaker analgesic activity. When a keto ester 4
was cyclised to isoxazole derivative 5, slight increase in
activity was observed. To increase the activity further,
isoxazole nucleus was replaced with pyrimidine analogs
6–7 which results in slight increased analgesic activity;
whereas replacement with pyrazole nucleus 8–16 greatly
increased the activity. Within pyrazole derivatives, the
activity varies with substitution present on N-1 of pyrazole.
Substitution of N-1 hydrogen of pyrazole with phenyl ring
results in significant increased activity. Further introduction
of substituent such as chloro, fluoro, and methoxy group at
para position of phenyl ring produced very good analgesic
activity. These para substituted derivative 10–12 exhibited
superior or equipotent analgesic activity when compared
against standard Diclofenac. Whereas, introduction of sub-
stituent such as chloro, and fluoro, at meta position of
phenyl ring results in retaining of activity (almost similar to
Antimicrobial activity
In vitro antibacterial and antifungal activity of entire syn-
thesized compounds were screened by agar streak dilution

3030
Med Chem Res (2017) 26:3026–3037
Table 1 Percent analgesic
activity of the synthesized
compounds 3–16 (Tail flick
method)
Compound
Dose (mg/kg)
Percent analgesic activity
30 min
1 h
2 h
3 h
3
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
NA
10
20
04 1.50*
05 2.13*
08 1.72**
08 1.17*
15 0.51*
20 0.92*
18 1.08NS
22 1.25***
19 0.72*
24 1.44*
25 1.80*
31 1.56**
28 1.32**
33 1.58*
37 0.71**
47 1.46*
36 1.73***
45 1.03*
35 0.96*
44 0.51**
24 1.28*
31 1.18*
24 1.41**
30 1.09*
24 1.14**
31 1.51NS
25 1.24*
30 1.30*
03 0.56
05 1.41**
05 1.15NS
11 0.58*
12 1.41*
21 1.91***
27 1.24*
23 1.36**
28 1.17*
25 0.47*
31 0.91**
30 1.19*
44 1.65*
34 1.81**
47 0.52*
48 0.71***
60 1.28**
47 1.56**
58 1.25*
45 1.81***
58 1.93*
33 1.62**
43 0.47*
30 1.18*
41 1.61***
29 1.30*
42 1.02**
29 0.75*
43 1.87*
05 1.36
05 1.38*
07 1.27**
12 1.14*
13 0.52NS
24 1.42*
31 0.47*
25 1.80*
34 1.55*
26 1.54**
35 0.47*
33 0.71*
50 1.97**
36 0.91*
54 1.14***
55 2.15**
72 1.71**
53 1.87*
70 1.69***
52 1.57*
68 2.17**
34 1.54*
49 0.73**
32 1.28*
46 1.70*
33 1.52*
49 0.81*
31 1.47**
48 0.53NS
06 1.82
03 1.35*
03 0.49*
05 1.83*
07 2.24**
13 1.38**
17 1.69*
15 0.74*
20 0.81**
17 1.94*
21 0.64*
20 2.05***
28 1.86*
25 1.82*
30 0.92**
34 0.53*
41 1.42*
35 1.27*
41 0.64**
33 1.02*
40 0.76*
24 0.62**
27 2.06*
21 1.38*
26 1.33**
22 0.79NS
27 1.25*
21 1.48*
29 1.84***
04 1.13
4
5
6
7
8
9
10
11
12
13
14
15
16
Control
Diclofenac
36 0.66**
45 0.84*
45 1.18***
58 1.26**
53 0.69**
69 0.43*
34 1.94*
42 2.05***
Each value represents the mean SEM (n = 6)
NA not applicable, NS non-significant
Significance levels *p < 0.5, **p < 0.01, ***p < 0.001 as compared with the respective control
method. In order to control the sensitivity of the test
organisms minimum inhibitory concentrations (MICs) of
Ciprofloxacin and Ketoconazole were determined simulta-
neously. MICs of synthesized compounds and standard
drugs were summarized in Table 3. Test compounds
exhibited varying degree of antimicrobial activity. From
antimicrobial study it was found that, in general entire test
compounds exhibited mild to good anti-bacterial activity
and weak to moderate anti-fungal activity. Out of several
tested compounds, compounds 10, 11, 13, and 14 exhibited
very good antimicrobial activity; whereas compounds 5–7
displayed moderate antimicrobial activity. Rest of
compounds 3, 4, 8, 9, 12, 15, and 16 showed weaker anti-
microbial activity. Compound 4-(2-(4-(1H-benzimidazol-2-
yl)phenyl)hydrazono)-1-(4-chlorophenyl)-3-methyl-1H-pyr-
azol-5(4H)-one 10, displayed equal activity like standard
Ciprofloxacin against S. aureus (MIC: 7.81 µg/ml), S. epi-
dermidis (MIC: 1.95 µg/ml), and E. coli (MIC: 7.81 µg/ml).
Similarly against M. luteus (MIC: 3.9 µg/ml) and E. coli
(MIC: 7.81 µg/ml), compound 4-(2-(4-(1H-benzimidazol-2-
yl)phenyl)hydrazono)-1-(3-chlorophenyl)-3-methyl-1H-pyr-
azol-5(4H)-one 13, showed equipotent activity with
Ciprofloxacin.

Med Chem Res (2017) 26:3026–3037
3031
Table 2 Percent anti-
inflammatory activity
Compound Dose (mg/kg) Percent protection
Ulcer index
(Carrageenan induced paw
oedema test in rats) and ulcer
index of the synthesized
30 min
1 h
2 h
3 h
3
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
NA
10
20
07 0.42*
10 0.79*
10 1.57**
11 0.83*
16 0.84*
21 2.16**
18 0.43*
23 1.91*
19 1.05**
24 0.68*
21 0.96*
27 0.79*
26 1.45**
32 2.17*
35 1.04*
43 1.32*
08 0.59NS
12 0.96*
11 0.82*
13 0.49**
19 1.12*
25 1.02*
20 0.61NS
26 0.46*
22 2.19*
26 0.90**
23 1.35**
30 2.34*
30 0.57*
39 0.47**
10 1.91**
13 0.87*
12 0.40NS
15 1.25*
24 1.35*
31 1.47*
27 1.16**
34 2.14*
28 0.96*
36 1.01*
31 1.16*
38 1.54**
05 0.86*
07 1.24*
07 1.09**
10 0.98*
15 1.57*
19 0.83***
16 2.09*
20 1.95*
16 1.97*
22 1.83*
20 0.75*
26 1.38***
ND
compounds 3–16
4
ND
5
ND
6
ND
7
ND
8
0.95 0.23
0.82 0.34
0.53 0.29
0.59 0.14
0.66 0.21
1.04 0.17
1.08 0.45
ND
9
37 2.34*** 26 0.72*
50 0.92*
30 1.03**
31 1.72*
10
11
12
13
14
15
16
38 0.72*** 47 1.13*
56 1.37*
33 1.27*** 37 1.84*
67 1.74*** 42 2.30*
45 1.19*
66 0.51*
44 1.59**
65 0.63*
36 2.42*
47 1.18*
36 1.59*
47 1.21*
31 0.78*
40 1.40**
30 1.28*
39 1.60*
23 0.65**
28 2.11*
22 0.77*
26 1.62*
42 0.62*
31 0.54*
40 1.03**
25 1.01*
30 1.80*
24 2.36*
29 0.77**
20 0.31*
54 0.64**
35 1.01*
54 2.18*
28 0.83*
38 1.95**
26 1.21*
36 1.59*
22 2.06NS
31 0.59*** 19 1.42*
25 1.48*** 32 0.70*
38 1.82*
30 2.53*
37 1.35*
05 1.64
22 1.30*
17 0.78NS
22 1.41**
04 0.89
19 2.60*
24 0.54NS
05 0.93
22 1.23**
30 1.78*
06 1.25
ND
Control
0.12 0.07
1.68 0.52
Diclofenac
32 1.89*
40 0.35*
36 2.13*** 44 0.37**
53 0.68*
30 1.26*
65 1.27*** 42 1.61**
Each value represents the mean SEM (n = 6)
NA not applicable, ND not determined, NS non-significant
Significance levels: *p < 0.5, **p < 0.01, ***p < 0.001 as compared with the respective control
The potent antibacterial activity exhibited by compounds
10, 11, 13, and 14 might be due to the presence of electron
withdrawing substituent like chloro, and fluoro group in the
phenyl ring attached to pyrazole nucleus. In general, elec-
tron withdrawing group substitiuted pyrazole analogs 10–11
and 13–14 exhibited better antimicrobial activity than iso-
xazole 5, and pyrimidine 6–7 derivatives. The chemical
structure and antimicrobial activity relationship of the syn-
thesized compounds revealed that the compounds having
electron withdrawing moiety 10–11 and 13–14 exhibited
better activity than compounds has electron releasing
moieties 12. Among tested compounds, 4-(2-(4-(1H-
benzimidazol-2-yl)phenyl)hydrazono)-1-(4-chlorophenyl)-3-
methyl-1H-pyrazol-5(4H)-one 10 was found to be potent
antibacterial agent. Unlike analgesic and anti-inflammatory
activity, here nature of substituent played a major role than
position of the substituent.
Experimental
General
The chemicals and reagents used were obtained from var-
ious chemical units Qualigens, E. Merck India Ltd., CDH,

3032
Med Chem Res (2017) 26:3026–3037
Table 3 MIC (Minimum inhibitory concentration in µg/ml) of synthesized compounds 3–16
Compound Antibacterial activity
Antifungal activity
B. cereus S. aureus M. luteus S. epidermidis K. pneumoniae P. aeruginosa E. coli A. fumigatus A. niger
3
>125
>125
62.5
62.5
62.5
125
>125
>125
31.25
62.5
>125
>125
15.62
31.25
31.25
62.5
125
>125
>125
15.62
31.25
15.62
31.25
31.25
1.95
3.9
>125
>125
7.81
15.62
15.62
31.25
31.25
3.9
>125
>125
15.62
31.25
15.62
62.5
62.5
7.81
7.81
>125
7.81
7.81
62.5
125
>125
>125
31.25
62.5
62.5
125
>125
>125
62.5
62.5
62.5
125
>125
>125
31.25
62.5
4
5
6
7
31.25
62.5
62.5
8
62.5
9
125
62.5
125
62.5
15.62
31.25
>125
15.62
31.25
125
125
10
15.62
15.62
125
7.81
7.81
7.81
>125
3.9
7.81
15.62
125
15.62
15.62
>125
15.62
31.25
125
11
15.62
125
7.81
125
12
62.5
3.9
13
15.62
31.25
125
15.62
15.62
62.5
3.9
7.81
15.62
125
14
7.81
125
7.81
62.5
62.5
1.95
7.81
31.25
62.5
1.95
15
16
Standarda
125
125
125
125
125
125
7.81
7.81
3.9
3.9
7.81
3.9
7.81
a
Ciprofloxacin used as a reference standard for bacteria and Ketoconazole used as a reference standard for fungi
and SD Fine Chem. These solvents used were of LR grade
and purified before their use. The silica gel G used for
analytical chromatography (TLC) was obtained from E.
Merck India Ltd. All the melting points were taken in open
glass capillary and are uncorrected. 1H-nuclear magnetic
resonance (NMR) and 13C-NMR spectra were recorded at
500 and 125 MHz respectively on Bruker Avance-500
NMR spectrometer in CDCl3 using tetramethylsilane
(TMS) for 1H-NMR and CDCl3 for 13C-NMR as an internal
standard. The chemical shifts are reported in ppm scale.
Mass spectra were obtained on a JEOL-SX-102 instrument
using electron impact ionization. All the infrared (IR)
spectra were recorded in KBr pellets on a Jasco FT-IR
410 spectrometer. Elemental analyzes were performed on a
Perkine Elmer model 2400 C analyzer and were within
0.4% of the theoretical values.
MHz) δ ppm: 155.2 (C-2), 148.5 (C′-1), 139.6 (C-8 and C-
9), 130.3 (C′-3 and C′-5), 124.7 (C-5 and C-6), 122.2 (C′-4),
119.5 (C′-2 and C′-6), 118.1 (C-4 and C-7). EI-MS m/z: 209
(M+). Anal. calcd for C13H11N3: C, 74.62; H, 5.30; N,
20.08. Found: C, 74.86; H, 5.28; N, 20.01.
Synthesis of ethyl 2-(2-(4-(1H-benzimidazol-2-yl)phenyl)
hydrazono)-3-oxobutanoate (4) 4-(1H-Benzimidazol-2-yl)
benzenamine 3 (2.09 g; 0.01 mol) was dissolved in a mix-
ture of concentrated hydrochloric acid (5 ml) and water
(5 ml). The solution is cooled to 5 °C by immersing the flask
in a mixture of ice and water. The solution of powdered
sodium nitrite (1.38 g; 0.02 mol) dissolved in water (5 ml)
was drop wise added to the solution of 4-(1H-benzimidazol-
2-yl)benzenamine hydrochloride with stirring. The stirring
was continued for 30 min after complete addition of sodium
nitrite. The obtained diazonium salt was added to a solution
of ethylacetoacetate (1.30 g; 0.01 mol) in ethanol (10 ml)
with stirring. Then the reaction mixture was stirred for 2 h
magnetically at room temperature and kept aside at room
temperature for further 2 h. The solid product 4, so formed,
was collected by filtration and recrystallized from ethanol.
Yield = 72 %, m.p. 183–186 °C. IR (KBr) cm−1: 3392
(NH), 3008 (Ar–CH), 2919 (CH3–CH), 1703 (C=O), 1608
Synthesis of 4-(1H-benzimidazol-2-yl)benzenamine (3) A
mixture of o-phenylenediamine 1 (1.08 g, 0.01 mol) and p-
aminobenzoic acid 2 (1.37 g, 0.01 mol) were stirred in a
syrupy o-phosphoric acid (15 ml) at 200 °C for 2 h. The
reaction mixture was cooled and poured on crushed ice. The
bulky white precipitate obtained was stirred in cold water
(150 ml) and sodium hydroxide solution (5 M) was added
until the pH 7. The resulting solid obtained 3 was filtered
and recrystallized from methanol. Yield = 70%, m.p.
246–248 °C. IR (KBr) cm−1: 3357 (NH), 2985 (Ar–CH),
1
(C=N), 1559 (C=C); 1096 (C–O–C). H-NMR (CDCl3,
500 MHz) δ ppm: 6.89–7.97 (m, 8H, Ar–CH), 6.47 (s, 1H,
NH of hydrazone), 5.83 (s, 1H, NH of benzimidazole),
4.49–4.71 (t, 2H, CH2), 2.22 (s, 3H, CH3), 1.71–1.80 (t, 3H,
CH3). 13C-NMR (CDCl3, 500 MHz) δ ppm: 197.8 (C=O),
160.4 (COO), 154.7 (C-2), 149.3 (C′-1), 136.8 (C-8 and
1
1675 (C=N), 1586 (C=C). H-NMR (CDCl3, 500 MHz) δ
ppm: 7.09–8.24 (m, 8H, Ar–CH), 5.78 (s, 1H, NH of
benzimidazole), 4.30 (s, 2H, NH2). 13C-NMR (CDCl3, 500

Med Chem Res (2017) 26:3026–3037
3033
C-9), 135.0 (C=N), 128.5 (C′-3 and C′-5), 126.4 (C-5 and
C-6), 121.9 (C′-4), 118.1 (C′-2 & C’-6), 117.4 (C-4 and C-
7), 64.5 (CH2CH3), 25.7 (COCH3), 20.9 (CH2CH3). EI-MS
m/z: 350 (M+). Anal. calcd for C19H18N4O3: C, 65.13; H,
5.18; N, 15.99. Found: C, 64.91; H, 5.20; N, 16.04.
5-(2-(4-(1H-Benzimidazol-2-yl)phenyl)hydrazono)-2-mer-
capto-6-methylpyrimidin-4(5H)-one (7) Yield = 80 %, m.
p. 109–112 °C. IR (KBr) cm−1: 3389 (NH), 3073 (Ar–CH),
2914 (CH3–CH), 2514 (SH), 1734 (C=O), 1670 (C=N),
1644 (C=C). 1H-NMR (CDCl3, 500 MHz)
δ ppm:
7.02–8.11 (m, 8H, Ar–CH), 6.49 (s, 1H, NH of hydrazone),
5.37 (s, 1H, NH of benzimidazole), 2.25 (s, 1H, SH), 1.28
(s, 3H, CH3). 13C-NMR (CDCl3, 500 MHz) δ ppm: 195.6
(C=O), 171.9 (C″-2), 165.4 (C″-6), 158.1 (C″-5), 155.6 (C-
2), 148.0 (C′-1), 139.1 (C-8 and C-9), 133.8 (C′-3 and C′-5),
125.2 (C-5 and C-6), 120.5 (C′-4), 119.9 (C′-2 and C′-6),
118.5 (C-4 and C-7), 19.4 (CH3). EI-MS m/z: 362 (M+).
Anal. calcd for C18H14N6OS: C, 59.65; H, 3.89; N, 23.19.
Found: C, 59.81; H, 3.88; N, 23.10.
Synthesis of 4-(2-(4-(1H-benzimidazol-2-yl)phenyl)hydra-
zono)-3-methylisoxazol-5(4H)-one (5) A mixture of
ethyl 2-(2-(4-(1H-benzimidazol-2-yl)phenyl)hydrazono)-3-
oxobutanoate 4 (3.50 g; 0.01 mol) and hydroxylamine
hydrochloride (1.04 g; 0.015 mol) in ethanol (20 ml) was
refluxed for 8 h in water bath. After removal of ethanol in
vacuum, the oil obtained was poured into ice cold water and
stirred well. The product obtained 5 was filtered, washed
with water twice, dried, and recrystallized using ethanol.
Yield = 75%, m.p. 120–122 °C. IR (KBr) cm−1: 3277
(NH), 3016 (Ar–CH), 2950 (CH3–CH), 1738 (C=O), 1666
Synthesis of 4-(2-(4-(1H-benzimidazol-2-yl)phenyl)hydrazi-
nyl)-3-methyl-1-substituted-1H-pyrazol-5(4H)-one (8-12)
A mixture of ethyl 2-(2-(4-(1H-benzimidazol-2-yl)phenyl)
hydrazono)-3-oxobutanoate 4 (3.50 g; 0.01 mol) and var-
ious hydrazine hydrochloride (0.015 mol) in ethanol (20 ml)
was refluxed for 12 h in water bath. After removal of
ethanol in vacuum, the oil obtained was poured into ice cold
water and stirred well. The product separated 8–12 was
filtered, dried and recrystallized.
1
(C=N), 1647 (C=C). H-NMR (CDCl3, 500 MHz) δ ppm:
7.05–7.84 (m, 8H, Ar–CH), 6.53 (s, 1H, NH of hydrazone),
5.30 (s, 1H, NH of benzimidazole), 2.28 (s, 3H, CH3). 13C-
NMR (CDCl3, 500 MHz) δ ppm: 165.6 (C″=O), 163.1 (C
″-3), 160.8 (C″-4), 157.3 (C-2), 149.1 (C′-1), 138.2 (C-8 and
C-9), 131.6 (C′-3 and C′-5), 126.9 (C-5 and C-6), 123.5 (C
′-4), 120.4 (C′-2 and C′-6), 118.6 (C-4 & C-7), 22.7 (CH3).
EI-MS m/z: 319 (M+). Anal. calcd for C17H13N5O2: C,
63.94; H, 4.10; N, 21.93. Found: C, 63.71; H, 4.11; N,
21.99.
4-(2-(4-(1H-Benzimidazol-2-yl)phenyl)hydrazono)-3-
methyl-1H-pyrazol-5(4H)-one (8) Yield = 73%, m.p.
188–190 °C. IR (KBr) cm−1: 3389 and 3281 (NH), 3057
(Ar–CH), 2917 (CH3–CH), 1735 (C=O), 1675 (C=N),
Synthesis of 5-(2-(4-(1H-benzimidazol-2-yl)phenyl)hydra-
zinyl)-2-substituted-6-methylpyrimidin-4(5H)-one (6–7)
A mixture of ethyl 2-(2-(4-(1H-benzimidazol-2-yl)phenyl)
hydrazono)-3-oxobutanoate 4 (3.50 g; 0.01 mol), urea/
thiourea (0.015 mol) and potassium carbonate (0.2 g) in
ethanol (20 ml) was refluxed for 10 h on a water bath. On
cooling the solid separated out was filtered. The residue was
dissolved in hot water and filtered when hot. The filtrate
was neutralized with acetic acid and the solid precipitated
out 6–7 was filtered and recrystallized from ethanol.
1614 (C=C). 1H-NMR (CDCl3, 500 MHz)
δ ppm:
7.22–8.54 (m, 8H, Ar–CH), 6.91 (s, 1H, NH of hydrazone),
6.65 (s, 1H, NH), 5.87 (s, 1H, NH of benzimidazole), 1.59
(s, 3H, CH3). 13C-NMR (CDCl3, 500 MHz) δ ppm: 163.2
(C=O), 153.7 (C-2), 150.5 (C″-3), 146.9 (C′-1), 138.6 (C-8
and C-9), 137.1 (C″-4), 131.3 (C′-3 and C′-5), 125.0 (C-5
and C-6), 123.6 (C′-4), 119.5 (C′-2 and C′-6), 114.8 (C-4
and C-7), 24.1 (CH3). EI-MS m/z: 318 (M+). Anal. calcd for
C17H14N6O: C, 64.14; H, 4.43; N, 26.40. Found: C, 63.93;
H, 4.42; N, 26.49.
5-(2-(4-(1H-Benzimidazol-2-yl)phenyl)hydrazono)-2-
hydroxy-6-methylpyrimidin-4(5H)-one (6) Yield = 78%,
m.p. 176–178 °C. IR (KBr) cm−1: 3515 (OH), 3312 (NH),
3042 (Ar–CH), 2938 (CH3–CH), 1752 (C=O), 1642
4-(2-(4-(1H-Benzimidazol-2-yl)phenyl)hydrazono)-3-methyl-
1-phenyl-1H-pyrazol-5(4H)-one (9) Yield = 79%, m.p.
152–154 °C. IR (KBr) cm−1: 3366 and 3290 (NH), 3146
(Ar–CH), 2908 (CH3–CH), 1691 (C=O), 1623 (C=N),
1
(C=N), 1603 (C=C). H-NMR (CDCl3, 500 MHz) δ ppm:
7.17–8.19 (m, 8H, Ar–CH), 6.74 (s, 1H, NH of hydrazone),
5.19 (s, 1H, NH of benzimidazole), 2.62 (s, 1H, OH), 1.70
(s, 3H, CH3). 13C-NMR (CDCl3, 500 MHz) δ ppm: 190.5
(C=O), 168.7 (C″-6), 166.1 (C″-2), 157.4 (C″-5), 154.9 (C-2),
150.2 (C′-1), 138.4 (C-8 and C-9), 132.0 (C′-3 and C′-5),
125.3 (C-5 and C-6), 121.6 (C′-4), 119.7 (C′-2 and C′-6),
116.8 (C-4 and C-7), 22.3 (CH3). EI-MS m/z: 346 (M+).
Anal. calcd for C18H14N6O2: C, 62.42; H, 4.07; N, 24.27.
Found: C, 62.60; H, 4.05; N, 24.19.
1559 (C=C). 1H-NMR (CDCl3, 500 MHz)
δ ppm:
6.86–8.30 (m, 13H, Ar–CH), 6.48 (s, 1H, NH of hydra-
zone), 5.39 (s, 1H, NH of benzimidazole), 1.42 (s, 3H,
CH3). 13C-NMR (CDCl3, 500 MHz) δ ppm: 160.5 (C=O),
152.9 (C-2), 150.1 (C″-3), 148.6 (C′-1), 136.2 (C-8 and C-
9), 134.7 (C″′-1), 133.0 (C″′-3 and C″′-5), 128.4 (C″-4),
126.6 (C′-3 and C′-5), 125.1 (C″′-4), 123.8 (C-5 and C-6),
122.3 (C″′-2 & C″′-6), 121.9 (C′-4), 118.0 (C′-2 and C′-6),
115.7 (C-4 and C-7), 21.2 (CH3). EI-MS m/z: 394 (M+).

3034
Med Chem Res (2017) 26:3026–3037
Anal. calcd for C23H18N6O: C, 70.04; H, 4.60; N, 21.31.
Found: C, 70.29; H, 4.61; N, 21.25.
4-(2-(4-(1H-Benzimidazol-2-yl)phenyl)hydrazono)-1-(3-
chlorophenyl)-3-methyl-1H-pyrazol-5(4H)-one (13) Yield
= 75%, m.p. 129–131 °C. IR (KBr) cm−1: 3386 (NH), 3064
(Ar–CH), 2985 (CH3–CH), 1741 (C=O), 1622 (C=N),
1
4-(2-(4-(1H-Benzimidazol-2-yl)phenyl)hydrazono)-1-(4-
1605 (C=C), 879 (C–Cl). H-NMR (CDCl3, 500 MHz) δ
chlorophenyl)-3-methyl-1H-pyrazol-5(4H)-one
(10)
ppm: 6.85–8.14 (m, 12H, Ar–CH), 6.76 (s, 1H, NH of
hydrazone), 5.39 (s, 1H, NH of benzimidazole), 2.01 (s, 3H,
CH3). 13C-NMR (CDCl3, 500 MHz) δ ppm: 160.5 (C=O),
154.2 (C-2), 152.0 (C″-3), 147.8 (C′-1), 137.6 (C″′-1), 137.1
(C-8 and C-9), 133.9 (C″′-3), 130.2 (C″′-5), 128.5 (C″-4),
127.4 (C′-3 and C′-5), 126.0 (C″′-4), 125.8 (C-5 and C-6),
120.7 (C″′-2), 119.3 (C′-4), 118.6 (C″′-6), 117.1 (C′-2 and C
′-6), 110.5 (C-4 and C-7), 18.4 (CH3). EI-MS m/z: 430
(M+2), 428 (M+). Anal. calcd for C23H17ClN6O: C, 64.41;
H, 4.00; N, 19.60. Found: C, 64.58; H, 3.99; N, 19.52.
Yield = 82%, m.p. 197–200 °C. IR (KBr) cm−1: 3319
(NH), 3001 (Ar–CH), 2942 (CH3–CH), 1728 (C=O), 1645
1
(C=N), 1611 (C=C), 858 (C–Cl). H-NMR (CDCl3, 500
MHz) δ ppm: 7.14–8.26 (m, 12H, Ar–CH), 6.89 (s, 1H, NH
of hydrazone), 5.09 (s, 1H, NH of benzimidazole), 1.70 (s,
3H, CH3). 13C-NMR (CDCl3, 500 MHz) δ ppm: 155.3
(C=O), 152.7 (C-2), 151.4 (C″-3), 148.0 (C′-1), 138.9 (C-8
and C-9), 135.1 (C″′-1), 134.5 (C″′-4), 131.8 (C″′-3 and C″
′-5), 129.2 (C″-4), 125.0 (C′-3 and C′-5), 124.6 (C-5 and C-
6), 121.3 (C″′-2 and C″′-6), 120.5 (C′-4), 117.8 (C′-2 and C
′-6), 112.3 (C-4 and C-7), 17.6 (CH3). EI-MS m/z: 430
(M+2), 428 (M+). Anal. calcd for C23H17ClN6O: C, 64.41;
H, 4.00; N, 19.60. Found: C, 64.62; H, 4.02; N, 19.54.
4-(2-(4-(1H-Benzimidazol-2-yl)phenyl)hydrazono)-1-(3-
fluorophenyl)-3-methyl-1H-pyrazol-5(4H)-one (14) Yield
= 71%, m.p. 116–118 °C. IR (KBr) cm−1: 3285 (NH), 3067
(Ar–CH), 2970 (CH3–CH), 1744 (C=O), 1636 (C=N),
1
1608 (C=C), 1065 (C–F). H-NMR (CDCl3, 500 MHz) δ
4-(2-(4-(1H-Benzimidazol-2-yl)phenyl)hydrazono)-1-(4-
fluorophenyl)-3-methyl-1H-pyrazol-5(4H)-one (11) Yield
= 77%, m.p. 103–104 °C. IR (KBr) cm−1: 3335 (NH), 3042
(Ar–CH), 2959 (CH3–CH), 1734 (C=O), 1640 (C=N),
ppm: 7.35–8.19 (m, 12H, Ar–CH), 7.16 (s, 1H, NH of
hydrazone), 5.28 (s, 1H, NH of benzimidazole), 2.20 (s, 3H,
CH3). 13C-NMR (CDCl3, 500 MHz) δ ppm: 7.35–8.19 (m,
12H, Ar–CH), 7.16 (s, 1H, NH of hydrazone), 5.28 (s, 1H,
NH of benzimidazole), 2.20 (s, 3H, CH3). 13C-NMR
(CDCl3, 500 MHz) δ ppm:166.7 (C″′-3), 153.4 (C=O),
152.0 (C-2), 151.9 (C″-3), 149.6 (C′-1), 140.5 (C″′-1), 139.1
(C-8 and C-9), 132.8(C″′-5), 126.3 (C″-4), 125.2 (C′-3 and
C′-5), 124.0 (C-5 and C-6), 122.8 (C′-4), 114.6 (C″′-6),
113.1 (C′-2 and C′-6), 112.5 (C-4 and C-7), 109.8 (C″′-4),
109.0 (C″′-2), 23.4CH3). EI-MS m/z: 412 (M+). Anal. calcd
for C23H17FN6O: C, 66.98; H, 4.15; N, 20.38. Found: C,
66.75; H, 4.17; N, 20.46.
1
1628 (C=C), 1085 (C–F). H-NMR (CDCl3, 500 MHz) δ
ppm: 7.03–8.10 (m, 12H, Ar–CH), 6.55 (s, 1H, NH of
hydrazone), 5.28 (s, 1H, NH of benzimidazole), 2.03 (s, 3H,
CH3). 13C-NMR (CDCl3, 500 MHz) δ ppm: 161.8 (C″′-4),
154.0 (C=O), 151.3 (C-2), 150.9 (C″-3), 149.2 (C′-1),
140.6 (C-8 and C-9), 133.5 (C″′-1), 128.1 (C″-4), 124.9 (C
′-3 and C′-5), 120.5 (C″′-2 and C″′-6), 119.7 (C-5 and C-6),
119.2 (C′-4), 118.0 (C′-2 and C′-6), 114.4 (C″′-3 and C″′-5),
111.7 (C-4 and C-7), 20.3 (CH3). EI-MS m/z: 412 (M+).
Anal. calcd for C23H17FN6O: C, 66.98; H, 4.15; N, 20.38.
Found: C, 67.20; H, 4.13; N, 20.31.
4-(2-(4-(1H-Benzimidazol-2-yl)phenyl)hydrazono)-3-methyl-
5-oxo-4,5-dihydropyrazole-1-carbothioamide (15) Yield
= 71%, m.p. 141–143 °C. IR (KBr) cm−1: 3317 (NH), 3002
(Ar–CH), 2941 (CH3–CH), 1743 (C=O), 1642 (C=N),
4-(2-(4-(1H-Benzimidazol-2-yl)phenyl)hydrazono)-1-(4-
methoxyphenyl)-3-methyl-1H-pyrazol-5(4H)-one
(12)
Yield = 72%, m.p. 145–147 °C. IR (KBr) cm−1: 3359
(NH), 3070 (Ar–CH), 2953 (CH3–CH), 1747 (C=O), 1633
1610 (C=C). 1H-NMR (CDCl3, 500 MHz)
δ ppm:
7.11–8.20 (m, 8H, Ar–CH), 6.72 (s, 1H, NH of hydrazone),
5.39 (s, 1H, NH of benzimidazole), 2.88 (s, 2H, NH2), 1.13
(s, 3H, CH3). 13C-NMR (CDCl3, 500 MHz) δ ppm: 180.6
(C=S), 160.5 (C=O), 156.1 (C-2), 149.3 (C″-3), 147.2 (C
′-1), 140.5 (C-8 and C-9), 138.7 (C″-4), 130.1 (C′-3 and C
′-5), 125.8 (C-5 and C-6), 124.4 (C′-4), 115.6 (C′-2 and C
′-6), 113.0 (C-4 and C-7), 22.3 (CH3). EI-MS m/z: 377
(M+). Anal. calcd for C18H15N7OS: C, 57.28; H, 4.01; N,
25.98. Found: C, 57.47; H, 4.02; N, 26.05.
1
(C=N), 1608 (C=C), 1046 (C–O–C). H-NMR (CDCl3,
500 MHz) δ ppm: 6.91–8.05 (m, 12H, Ar–CH), 6.62 (s, 1H,
NH of hydrazone), 5.34 (s, 1H, NH of benzimidazole), 3.59
(s, 3H, OCH3), 1.96 (s, 3H, CH3). 13C-NMR (CDCl3, 500
MHz) δ ppm: 159.1 (C=O), 157.5 (C″′-4), 153.2 (C-2),
150.9 (C″-3), 149.4 (C′-1), 136.5 (C-8 and C-9), 134.7 (C
″-4), 134.0 (C″′-1), 128.3 (C′-3 and C′-5), 126.8 (C-5 and C-
6), 124.6 (C″′-2 and C″′-6), 119.3 (C′-4), 118.5 (C′-2 and C
′-6), 115.8 (C-4 and C-7), 113.6 (C″′-3 and C″′-5), 52.4
(OCH3), 24.0 (CH3). EI-MS m/z: 424 (M+). Anal. calcd for
C24H20N6O2: C, 67.91; H, 4.75; N, 19.80. Found: C, 67.68;
H, 4.77; N, 19.87.
4-(2-(4-(1H-Benzimidazol-2-yl)phenyl)hydrazono)-1-isonico-
tinoyl-3-methyl-1H-pyrazol-5(4H)-one (16) Yield = 76%,
m.p. 162–165 °C. IR (KBr) cm−1: 3320 (NH), 3074

Med Chem Res (2017) 26:3026–3037
3035
(Ar–CH), 2899 (CH3–CH), 1744 (C=O), 1659 (C=N), 1612
(C=C). H-NMR (CDCl3, 500 MHz) δ ppm: 7.38–8.59 (m,
standard drug for comparison. The test compounds were
administered at two dose levels of 10 and 20 mg/kg. The
paw volumes were measured using the mercury displace-
ment technique with the help of plethysmograph immedi-
ately before and 30 min, 1, 2, and 3 h after carrageenan
injection. The percent inhibition of paw oedema was cal-
culated according to the following formula, percent inhibi-
tion I = 100[1−(a−x)/(b−y)] where x is the mean paw
volume of rats before the administration of carrageenan and
test compounds or reference compound (test group), a is the
mean paw volume of rats after the administration of carra-
geenan in the test group (drug treated), b is the mean paw
volume of rats after the administration of carrageenan in the
control group, y is the mean paw volume of rats before the
administration of carrageenan in the control group. All the
percent inhibition results are shown in Table 2.
1
12H, Ar–CH), 7.06 (s, 1H, NH of hydrazone), 5.21 (s, 1H,
NH of benzimidazole), 1.98 (s, 3H, CH3). 13C-NMR (CDCl3,
500 MHz) δ ppm: 170.8 (C=O), 162.5 (C=O), 155.3 (C-2),
153.0 (C″′-2 and C″′-6), 150.9 (C″-3), 148.6 (C′-1), 142.1 (C″
′-4), 138.4 (C-8 and C-9), 137.5 (C″-4), 133.2 (C′-3 and C′-5),
125.4 (C-5 and C-6), 123.9 (C″′-3 and C″′-5), 123.0 (C′-4),
115.8 (C′-2 and C′-6), 112.5 (C-4 and C-7), 19.3 (CH3). EI-
MS m/z: 423 (M+). Anal. calcd for C23H17N7O2: C, 65.24; H,
4.05; N, 23.16. Found: C, 65.46; H, 4.03; N, 23.22.
Biological activities
Pharmacology
The synthesized compounds were evaluated for analgesic,
anti-inflammatory, and ulcerogenic activities. One-way
analysis of variance (ANOVA) was performed to certain
the significance of all the exhibited activities. The test
compounds and the standard drugs were administered in the
form of a suspension (1% carboxy methyl cellulose as a
vehicle) by oral route of administration for analgesic and
anti-inflammatory but for ulcerogenicity studies by intra-
peritoneally as suspension in 10% v/v Tween-80. Each
group consisted of six animals. The animals were main-
tained in colony cages at 25 2 °C, relative humidity of
45–55%, under a 12 h light and dark cycle; were fed stan-
dard animal feed (Olfert et al. 1993). All the animals were
acclimatized for a week before use.
Ulcerogenicity
Ulceration in rats was induced as reported method (Goyal
et al. 1985). Albino rats of Wistar strain weighing 150–200
g of either sex were divided into various groups each of six
animals. Control group of animals were administered only
with 10% v/v Tween-80 suspension intraperitoneally. One
group was administered with Aspirin intraperitoneally in a
dose of 200 mg/kg once daily for 3 days. Diclofenac was
also administered as standard drug at 20 mg/kg once daily
for 3 days to another group of animals in the same route.
The remaining group of animals was administered with test
compounds intraperitoneally in a dose of 20 mg/kg. On
fourth day, pylorus was ligated as per previous reported
method (Shay et al. 1945). Animals were fasted for 36 h
before the pylorus ligation procedure. Four hours after the
ligation, animals were sacrificed. The stomach was removed
and opened along with the greater curvature. Ulcer index
was determined by earlier reported method (Ganguly and
Bhatnagar 1973) and presented in Table 2.
Analgesic activity
The analgesic activity was performed by tail flick technique
using Wistar albino mice (25–35 g) of either sex selected by
random sampling technique (Kulkarni 1980 and D’Amour and
Smith 1941). Diclofenac sodium at a dose level of 10 and 20
mg/kg was administered orally as reference drug for com-
parison. The test compounds at two dose levels i.e. 10 and 20
mg/kg were administered orally. The reaction times were
recorded immediately before and 30 min, 1, 2, and 3 h after
the treatment and cut-off time was 10 s. The percent analgesic
activity (PAA) was calculated by the following formula. PAA
= [T2−T1/10−T1] × 100; where T1 is the reaction time (s)
before treatment, and T2 is the reaction time (s) after treatment.
The observed results are presented in Table 1.
Antimicrobial activity
In this study, all the synthesized compounds were screened
for antimicrobial activity by agar streak dilution method.
The antibacterial activity of the compounds were evaluated
against four Gram-positive bacteria Bacillus cereus ATCC
11778, Staphylococcus aureus ATCC 9144, Micrococcus
luteus ATCC 4698 and Staphylococcus epidermidis ATCC
155, and three Gram-negative bacteria Klebsiella pneumo-
niae ATCC 11298, Pseudomonas aeruginosa ATCC 2853,
and Escherichia coli ATCC 25922. The antifungal activities
of the synthesized compounds were evaluated against two
fungi Aspergillus fumigatus ATCC 46645 and Aspergillus
niger ATCC 9029. Bacterial strains were cultured over
night at 37 °C in Mueller–Hinton broth and the yeast was
Anti-inflammatory activity
Anti-inflammatory activity was evaluated by carrageenan
induced paw oedema test in rats (Winter et al. 1962).
Diclofenac sodium 10 and 20 mg/kg was administered as

3036
Med Chem Res (2017) 26:3026–3037
cultured overnight at 30 °C in YEPDE agar for antibacterial
and antifungal activity tests. Test strains were suspended in
nutrient agar to give a final density of 5 × 10−5 cfu/ml.
inflammatory activity with that of standard Diclofenac
with negligible ulcer index and displayed good antibacterial
and moderate antifungal activity. Consequently, this deri-
vative could be developed as a new class of analgesic, anti-
inflammatory and antimicrobial agents. Nevertheless, fur-
ther structural modification is designed to enhance these
activities with the low ulcerogenic index.
Minimum inhibitory concentration (MIC)
MIC of the compound was determined by agar streak
dilution method (Hawkey and Lewis 1994). A stock solu-
tion of the synthesized compound (100 µg/ml) in dimethyl
formamide was prepared and graded quantities of the test
compounds were incorporated in specified quantity of
molten sterile agar (nutrient agar for antibacterial activity
and Sabouraud’s dextrose agar medium for antifungal
activity). A specified quantity of the medium (40–50 °C)
containing the compound was poured into a petridish to
give a depth of 3–4 mm and allowed to solidify. Suspension
of the micro-organism were prepared to contain approxi-
mately 5 × 10−5 cfu/ml and applied to plates with serially
diluted compounds in dimethyl formamide to be tested and
incubated at 37 °C for 24 and 48 h for bacteria and fungi
respectively. The MIC was considered to be the lowest
concentration of the test substance exhibiting no visible
growth of bacteria or fungi on the plate. The observed MIC
is presented in Table 3.
Acknowledgements The authors gratefully acknowledge the man-
agement of Sarojini Naidu Vanita Pharmacy Maha Vidyalaya,
Secunderabad, for providing infrastructure facilities to carry out this
research work.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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