Synthesis and characterization of novel benzimidazole bearing pyrazoline derivatives as potential antimicrobial agents

Article abstract of DOI:10.1007/s00044-013-0756-4

A new series of compounds N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-(aryl)-4, 5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)phenyl)acetamides (5a-u) were synthesized and structures of these compounds were elucidated by spectral (IR, ¹H NMR, ¹³C NMR, and mass spectra) analysis. Antimicrobial activity was measured against Escherichia coli (MTCC 443), Pseudomonas aeruginosa (MTCC 1688), Staphylococcus aureus (MTCC 96), Streptococcus pyogenes (MTCC 442), Candida albicans (MTCC 227), Aspergillus niger (MTCC 282), and Aspergillus clavatus (MTCC 1323) by serial broth dilution method. Evaluation of antimicrobial activity revealed that compounds 5f, 5i, 5q, and 5t were the most active antibacterial, while compounds 5e, 5g, 5h, 5j, 5p, 5r, and 5u were the most potent antifungal agents as compared to standard drugs and thus could be promising new lead molecules.

Full text of DOI:10.1007/s00044-013-0756-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1474–1487
DOI 10.1007/s00044-013-0756-4
ORIGINAL RESEARCH
Synthesis and characterization of novel benzimidazole bearing
pyrazoline derivatives as potential antimicrobial agents
•
•
•
N. C. Desai D. D. Pandya G. M. Kotadiya
Priyanka Desai
Received: 1 May 2013 / Accepted: 21 August 2013 / Published online: 1 September 2013
Ó Springer Science+Business Media New York 2013
Abstract A new series of compounds N-(4-(2-(3-(1H-
benzo[d]imidazol-2-yl)-5-(aryl)-4,5-dihydro-1H-pyrazol-
1-yl)-2-oxoethoxy)phenyl)acetamides (5a–u) were syn-
thesized and structures of these compounds were eluci-
dated by spectral (IR, 1H NMR, 13C NMR, and mass
spectra) analysis. Antimicrobial activity was measured
against Escherichia coli (MTCC 443), Pseudomonas
aeruginosa (MTCC 1688), Staphylococcus aureus (MTCC
96), Streptococcus pyogenes (MTCC 442), Candida albi-
cans (MTCC 227), Aspergillus niger (MTCC 282), and
Aspergillus clavatus (MTCC 1323) by serial broth dilution
method. Evaluation of antimicrobial activity revealed that
compounds 5f, 5i, 5q, and 5t were the most active antibac-
terial, while compounds 5e, 5g, 5h, 5j, 5p, 5r, and 5u were the
most potent antifungal agents as compared to standard drugs
and thus could be promising new lead molecules.
antimicrobial activity through continued broad screen eval-
uation, we have been interested in the effect of structural
variations in title compounds. Here, we have replaced
isoniazide by hydrazide of paracetamol ester compound (4)
at nitrogen of pyrazoline ring. There are no reports of anti-
microbial activity of N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-
5-(aryl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy)
phenyl)acetamides (5a–u). In continuation to this, we have
incorporated the synthesis of new compounds with an
observation of increased antimicrobial activity. Structural
relevance of title compounds (5a–u) with previously syn-
thesized compounds (NCD1–14) and structural similarity of
target compounds (5a–u) with certain commercially
available drugs containing benzimidazole and pyrazole
nucleus are shown in Fig. 1.
Despite numerous attempts to search and develop new
structural prototype as effective antimicrobials, benzimid-
azole derivatives are unique and exhibit a broad spectrum of
biological activities such as antihistaminic, antipyretic, anti-
ulcerative (Scott et al., 2002), antiallergic (Nakano et al.,
2000), antimicrobial (Kus et al., 2009), anticancer (Thim-
megowda et al., 2008), anti-inflammatory (Mader et al.,
2008), antiviral (Vazquez et al., 2001), antiparasitic (Ka-
zimierczuk et al., 2002), and protein kinase inhibitors (Ber-
natowicz et al., 2009). Moreover, benzimidazole derivatives
are structural isosteres of naturally occurring nucleotides,
which allow them to interact easily with the biophores
(Starcevic et al., 2007). The azole group of heterocyclic
compounds possessed significant pharmacokinetic property,
lipophilicity that influence the ability of drug to reach the
target by transmembrane diffusion and showed promising
activity against resistant TB by inhibiting the biosynthesis of
lipids (Andreani et al., 2001; Kini et al., 2009).
Keywords Benzimidazole ꢀ Pyrazoline ꢀ
Antibacterial activity ꢀ Antifungal activity
Introduction
Previously from our laboratory (Desai et al., 2012a), we have
reported (3-(1H-benzo[d]imidazol-2-yl)-5-(aryl)-4,5-dihy-
dro-1H-pyrazol-1-yl)(pyridin-4-yl)methanones (NCD1–14
and these compounds were screened for their antibacterial
and antifungal activity. In an effort to improve the
)
N. C. Desai (&) ꢀ D. D. Pandya ꢀ G. M. Kotadiya ꢀ P. Desai
Division of Medicinal Chemistry, Department of Chemistry,
UGC NON-SAP & DST-FIST Sponsored Department,
Maharaja Krishnakumarsinhji Bhavnagar University,
Mahatma Gandhi Campus, Bhavnagar 364 002, India
e-mail: dnisheeth@rediffmail.com
Pyrazoline derivatives are electron-rich nitrogen het-
erocycles which play an important role in diverse
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Med Chem Res (2014) 23:1474–1487
1475
Fig. 1 Structural relevance of
title compounds (5a–u) with
NCD1–14 and structural
similarity of title compounds
(5a–u) with commercially
available drugs bearing
benzimidazole and pyrazole
nucleus
H3C
H3C
O
CH3
H
N
O
N
S
Cl
N
S
N
N
O
O
N
OCH3
NH
Esomeprazole
Ibipinabant
N
O
H3C
N
O
H
O
O
N
N
N
N
H
H
N
N
R
R
N
N
NCD1-14
5a-u
R
-4-Cl
-3-NO2 500
-4-F 500
-4-NO2 200
MIC (µg/mL)
250
Microorganism
S. aureus
S. pyogenes
E. coli
R
-4-Cl
-3-NO2
-4-F
-4-NO2
MIC (µg/mL)
100
25
100
25
Microorganism
S. aureus
S. pyogenes
E. coli
A. niger
A. niger
biological activities. Considerable attention has been
focused on pyrazolines due to their interesting pharmaco-
logical activities. These compounds have been found to
possess antifungal (Korgaokar et al., 1996), antidepressant
(Prasad et al., 2005), anticonvulsant (Amnerkar and Bhu-
sari, 2010), anti-inflammatory (Udupi et al., 1998), anal-
gesic (Gursoy et al., 2000), antibacterial (Nauduri and
Reddy, 1998), and antitumor (Brzozwskim et al., 2000)
activity and also serve as human acyl-CoA: cholesterol
acyl transferase inhibitors (Sook et al., 2004).
derivatives 1-(1H-benzo[d]imidazol-2-yl)-3-(aryl)prop-2-
en-1-ones (3a–u) were used as precursors for the synthesis
of title compounds (5a–u). This was achieved through the
Claisen–Schmidt condensation of equimolar amounts of
different aldehyde derivatives (2a–u) and compound (1) by
stirring the reactants in aqueous alcoholic solution con-
taining sodium hydroxide at room temperature in accor-
dance with the method described in the literature
(Shaharyar et al., 2010). The cyclocondensation of chal-
cone derivatives (3a–u) with intermediate hydrazide (4) in
acetic acid at 130 °C afforded title compounds (5a–u) in
excellent yields. Here, compounds (5a–u) have pyrazoline
nucleus and were generated through cyclization, migration
of a, b unsaturated double bonds, and diminishing of car-
bonyl group.
Prompted by above mentioned observations and in con-
tinuation of our search for new antimicrobial agents (Desai
et al., 2012b, c), we have focused toward the synthesis of
novel benzimidazole derivatives bearing pyrazoline motifs
and bioevaluation of these derivatives by hybrid approach. In
continuation to this, we have synthesized N-(4-(2-(3-(1H-
benzo[d]imidazol-2-yl)-5-(aryl)-4,5-dihydro-1H-pyrazol-
1-yl)-2-oxoethoxy)phenyl)acetamide derivatives (5a–u) and
screened for their antimicrobial activity. The synthesized
compounds (5a–u) were characterized by IR, 1H NMR,
13C NMR, and mass spectrometry techniques.
Designed series of molecules (5a–u) were characterized
1
by IR, H NMR, 13C NMR, and mass spectrometry tech-
niques before evaluating for in vitro antimicrobial activity.
Two strong absorption bands appeared at 3,388–3,396 and
3,237–3,345 cm-1 in the IR spectra of compounds
(5a–u) corresponding to secondary amine (N–H) group
present in benzimidazole ring and –NHCOCH3- group,
respectively. Compounds (5a–u) showed two C–H stretch-
ing absorption bands at 3,036–3,045 and 2,858–2,866 cm-1
due to aromatic C–H and ethereal C–H (–OCH2–), respec-
tively. Two strong absorption bands were observed at
1,688–1,696 cm-1 and 1,658–1,665 cm-1 due to carbonyl
groups attached to –COCH2 and –NHCOCH3, respectively.
The characteristic signals in 1H NMR of compounds
(5a–u) were of three pyrazoline protons which displayed
Results and discussion
Chemistry
Synthesis of intermediates and target compounds
(5a–u) was accomplished according to the steps illustrated
in Scheme 1. According to Scheme 1, the key chalcone
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Med Chem Res (2014) 23:1474–1487
OCH2CONHNH2
CHO
R
O
H
N
H
N
O
EtOH, NaOH
Stirring, 10 h
+
+
N
N
R
CH3
NHCOCH3
3a-u
2a-u
1
4
CH3COOH
130oC
Reflux, 10 h
O
H3C
N
O
H
O
H
N
N N
For 2a-u, 3a-u and 5a-u, R = -H, 2-OH, -3-OH, -4-OH, -2-Cl, -3-Cl,
-4-Cl, -2-F, -3-F, -4-F, -2-CH3, -3-CH3,
-4-CH3, -3-OCH3, -4-OCH3, 2-NO2,
-3-NO2, -4-NO2, -2-Br, -3-Br, -4-Br,
N
R
5a-u
Scheme 1 Synthetic route for the preparation of title compounds (5a–u)
Antibacterial activity
doublet of doublet. Methine proton (C5–H) of pyrazoline
displayed a signal at d = 6.01–6.12 ppm as a doublet of
doublet with coupling constants in range of 11.11–11.20 and
3.10–3.18 Hz. The two methylene protons (C4–H) attached
with asymmetric carbon (C5–H) displayed two signals; a
doublet of doublet at d = 3.41–3.52 ppm with coupling
constants of 17.46–17.54 and 3.10–3.17 Hz and a doublet
of doublet at d = 3.90–3.96 ppm with coupling constants
of 17.48–17.54 and 11.10–11.18 Hz. In addition, two pro-
tons of methylene group appeared as a singlet at
d = 4.90–4.97 ppm. Three methyl protons of –NHCOCH3
group were observed as a singlet at d = 2.06–2.14 ppm.
Secondary amine present in benzimidazole nucleus showed
characteristic singlet at d = 10.07–10.20 ppm and was
confirmed by the disappearance of peak when exchanged
with D2O. Furthermore, 13C NMR confirmed the proposed
structure by the appearance of signals at d = 172.4–172.9
and 168.2–168.8 ppm due to carbonyl carbon directly
attached to pyrazoline nitrogen and carbonyl group present
in –NHCOCH3 group, respectively. Compounds (5a–u)
showed signals at d = 39.0–39.9 and 67.0–67.4 ppm cor-
responding to carbon of methylene group present in pyraz-
oline nucleus and methylene group carbon linked with
oxygen and carbonyl group, respectively. Methine carbon
showed a chemical shift at d = 60.1–66.6 ppm. Moreover,
the mass spectrum of (5a–u) showed a molecular ion peak
corresponding to molecular formula (5a–u) along with of
other fragment peaks, which supported the structure of
compounds. The details of characterization data are given in
‘‘Experimental’’ section.
All the newly synthesized compounds (5a–u) were initially
evaluated for in vitro antibacterial activity against Gram-
positive bacteria [Staphylococcus aureus (MTCC-96),
Streptococcus pyogenes (MTCC-442)] and Gram-negative
bacteria [Escherichia coli (MTCC-443), Pseudomonas
aeruginosa (MTCC-1688)] using conventional broth dilu-
tion method. The individual minimal inhibitory concentra-
tion (MIC, lg/mL) values of test compounds are listed in
Table 1 along with MIC values of reference compound
ampicillin. From antibacterial activity data (Table 1), it was
observed that compounds 5f (3-Cl), 5i (3-F), 5q (3-NO2), and
5t (3-Br) were the most active antibacterial compounds.
Compounds 5h (2-F), 5j (4-F), and 5r (4-NO2) exhibited
good activity against E. coli. When compounds having
electron-withdrawing groups at 3rd position except fluorine
group, i.e., 5f (3-Cl), 5q (3-NO2), and 5t (3-Br), displayed
very good activity at MIC = 50 lg/mL against E. coli, but
compound having fluorine at 3rd position, i.e., 5i (3-F),
revealedhighest inhibitionat MIC = 25 lg/mL against both
Gram-negative bacterial strains as compared to standard
ampicillin. Further, compounds 5g (4-Cl), 5j (4-F), and 5p
(2-NO2) showed good activity, while compounds 5f (3-Cl),
5q (3-NO2), and 5t (3-Br) exhibited very good activity at
MIC = 50 lg/mL against P. aeruginosa as compared to
standard ampicillin (MIC = 100 lg/mL). In case of
S. aureus, compounds 5e (2-Cl), 5g (4-Cl), 5h (2-F), 5j (4-F),
5p (2-NO2), and 5r (4-NO2) showed very good activity,
while compounds 5f (3-Cl) and 5t (3-Br) displayed excellent
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Med Chem Res (2014) 23:1474–1487
1477
activity at MIC = 50 lg/mL. In addition, compounds 5i
(3-F) and 5q (3-NO2) exhibited highest inhibition at
MIC = 25 lg/mL against S. aureus as compared to standard
ampicillin (MIC = 250 lg/mL). Moreover, compounds 5e
(2-Cl), 5g (4-Cl), 5h (2-F), 5p (2-NO2), and 5r (4-NO2)
revealed good activity, while compound 5j (4-F) displayed
very good activity against S. pyogenes. Furthermore,
hydrogen of phenyl ring in 5a when replaced by chloro, nitro,
and bromo groups at 3rd position, i.e., 5f (3-Cl), 5q (3-NO2),
and 5t (3-Br), increased the activity and compounds exhib-
ited excellent activity against S. pyogenes at MIC = 25
lg/mL. In addition, compound 5i (3-F) showed highest
inhibition at MIC = 12.5 lg/mL against S. pyogenes as
compared to standard ampicillin (MIC = 100 lg/mL).
A. niger, and A. clavatus. Compounds 5e (2-Cl), 5g (4-Cl),
5h (2-F), 5j (4-F), 5p (2-NO2), 5r (4-NO2), and 5u (4-Br)
were most potent against all the fungal strains (Table 2).
Here, electron-withdrawing effects also played a major role
for increasing the antifungal activity. A reverse trend was
observed when substituents at ortho or para positions
disclosed higher potency than meta-substituted derivatives.
Therefore, compounds 5e (2-Cl), 5g (4-Cl), 5p (2-NO2), 5r
(4-NO2), and 5u (4-Br) exhibited very good activity, while
compounds 5h (2-F) and 5j (4-F) displayed excellent
activity at MIC = 50 lg/mL against C. albicans as com-
pared to standard griseofulvin (MIC = 500 lg/mL).
Moreover, compounds 5i (3-F) and 5s (2-Br) showed good
activity against A. niger. In addition, compounds 5g (4-Cl),
5h (2-F), and 5p (2-NO2) showed very good activity
against A. niger, while hydrogen in 5a when replaced by
fluorine, nitro, and bromine at 4th position, i.e., 5j (4-F), 5r
(4-NO2), and 5u (4-Br), enhanced the activity and dis-
played excellent activity at MIC = 25 lg/mL against
Antifungal activity
Compounds (5a–u) were also evaluated for its in vitro
antifungal activity against three fungal strains, C. albicans,
Table 1 Results of antibacterial screening of compounds (5a–u)
Sr. No.
–R
Minimum inhibitory concentration (MIC) for bacteria (lg/mL) SD
Gram-negative
Gram-positive
E. coli
MTCC 443
P. aeruginosa
MTCC 1688
S. aureus
MTCC 96
S. pyogenes
MTCC 442
5a
–H
500 2.64*
250 2.65*
250 1.48
250 1.62**
250 4.65**
50 1.45**
250 1.45**
100 2.02***
25 1.64**
100 1.00**
500 3.64
500 1.45**
1,000
1,000
500 3.04*
500 1.53
250 1.24*
500 3.64*
100 1.90**
50 1.46***
100 1.64**
100 1.86**
25 1.58***
100 2.82**
500 1.45
250 1.86*
1,000
500 4.58*
500 1.54**
250 2.64*
500 1.68**
100 1.73***
25 2.43**
100 2.76***
100 2.49**
12.5 1.00***
50 2.83**
500 2.68*
250 1.49*
500 2.43*
250 2.95*
500 1.12
5b
5c
–2-OH
–3-OH
–4-OH
–2-Cl
500 1.85**
250 1.68*
500 1.85*
250 1.48**
50 1.04***
100 1.63**
250 1.54**
25 1.84***
100 2.62**
500 1.54
250 1.56*
1,000
5d
5e
5f
–3-Cl
5g
–4-Cl
5h
5i
–2-F
–3-F
5j
–4-F
5k
5l
–2-CH3
–3-CH3
–4-CH3
–3-OCH3
–4-OCH3
–2-NO2
–3-NO2
–4-NO2
–2-Br
5m
5n
5o
500 1.68**
1,000
500 2.54*
1,000
500 1.46
1,000
5p
5q
5r
250 1.64**
50 1.02***
100 2.38**
500 2.01*
50 1.38**
500 1.08***
100 2.05
100 1.54**
50 1.56***
250 1.63**
250 2.54*
50 1.03***
250 1.54**
100 1.00
100 2.42**
25 1.45***
100 1.86**
250 1.96***
50 1.46**
250 3.64**
250 1.52*
100 1.83**
25 1.68**
100 1.49*
250 2.43*
25 1.95***
250 1.12**
100 2.06*
5s
5t
–3-Br
5u
Ampicillin
–4-Br
All the values are presented as mean of six experiments (n = 6). All significant differences are considered from control value (0.00). 2 % DMSO
is used as control and its antibacterial activity is nil or zero
SD Standard deviation
*** P \ 0.001 extremely significant; ** P \ 0.01 moderately significant; * P \ 0.05 significant
123

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Med Chem Res (2014) 23:1474–1487
Table 2 Results of antifungal
screening of compounds (5a–u)
Sr. No.
–R
Minimum inhibitory concentration (MIC) for fungi (lg/mL) SD
C. albicans
MTCC 227
A. niger
MTCC 282
A. clavatus
MTCC 1323
5a
–H
500 2.12*
500 1.71
1,000
250 2.67
500 1.43**
500 2.61
500 2.79*
50 1.34***
500 2.82**
50 1.43**
50 1.49**
100 1.49***
25 2.00**
500 4.62*
1,000
500 3.64*
250 1.00
500 2.64*
500 1.16
25 1.48***
100 2.61**
50 1.10**
12.5 1.04**
100 2.54*
25 2.48
5b
–2-OH
–3-OH
–4-OH
–2-Cl
5c
5d
500 1.68*
1001 .93**
250 2.84**
100 1.14**
50 1.64**
250 1.23*
501 .56**
500 3.54*
1,000
5e
5f
–3-Cl
5g
–4-Cl
5h
–2-F
5i
–3-F
5j
–4-F
5k
–2-CH3
–3-CH3
–4-CH3
–3-OCH3
–4-OCH3
–2-NO2
–3-NO2
–4-NO2
–2-Br
250 1.53
500 2.45**
500 2.21**
500 1.29*
1,000
5l
5m
500 2.63*
1,000
500 2.49**
500 1.96
250 1.21*
50 1.43**
500 1.49**
25 1.49***
100 2.00**
250 2.62*
25 2.61*
100 1.10
All the values are presented as
mean of six experiments
(n = 6). All significant
differences are considered from
control value (0.00). 2 %
5n
5o
500 1.49**
100 2.84**
250 1.14**
100 1.64**
250 1.23**
500 1.56**
100 3.54*
500 0.50
5p
50 1.48***
250 1.61**
25 1.10**
50 1.04**
250 2.54*
50 1.48**
100 1.20
5q
DMSO is used as control and its
antifungal activity is nil or zero
5r
5s
SD Standard deviation
5t
–3-Br
*** P \ 0.001 extremely
significant; ** P \ 0.01
moderately significant; *
P \ 0.05 significant
5u
–4-Br
Griseofulvin
A. niger as compared to standard griseofulvin (MIC =
100 lg/mL). Furthermore, compounds 5g (4-Cl), 5p (2-NO2),
5s (2-Br), and 5u (4-Br) exhibited very good activity as well
as compounds 5e (2-Cl), 5j (4-F), and 5r (4-NO2) displayed
excellent activity at MIC = 25 lg/mL against A. clavatus.
Compound 5h (2-F) revealed highest inhibition at MIC =
12.5 lg/mL against A. clavatus as compared to standard
griseofulvin (MIC = 100 lg/mL).
in KBr, frequencies are reported in cm-1. 1H NMR spectra
were run on Varian Gemini 300 MHz and 13C NMR
spectra on Varian Mercury-400 100 MHz in DMSO-d6 as a
solvent and tetramethylsilane (TMS) as an internal stan-
dard. Chemical shifts are reported as units (ppm) values.
Mass spectra were scanned on a Shimadzu LCMS 2010
spectrometer. Anhydrous reactions were carried out in
oven-dried glassware in nitrogen atmosphere.
General synthetic procedure for the preparation of 1-
(1H-benzo[d]imidazol-2-yl)-3-(aryl)-prop-2-en-1-ones
(3a–u)
Experimental
All reactions except those in aqueous media were carried
out by standard techniques for the exclusion of moisture.
Melting points were determined on an electro thermal
melting point apparatus by open capillary method and were
reported uncorrected. TLC on silica gel plates (Merck, 60,
A mixture of compound 1-(1H-benzo[d]imidazol-2-yl)eth-
anone (1) (0.01 mol) and different aromatic aldehydes
(2a–u) (0.01 mol) was stirred in ethanolic sodium hydroxide
for 10 h. The yellow product generated was filtered off,
washed with water, and crystallized from ethanol (99 %).
F254) was used for reaction monitoring. Column chroma-
tography on silica gel (Merck, 70–230 mesh and 230–400
mesh ASTH for flash chromatography) was applied when
necessary to isolate and purify the reaction products. Yields
refer to purified products and were not optimized. Ele-
mental analysis (% C, H, N) was carried out by a Perkin-
Elmer 2400 CHN analyzer. IR spectra of all compounds
were recorded on a Perkin-Elmer FT-IR spectrophotometer
1-(1H-benzo[d]imidazol-2-yl)-3-phenylprop-2-en-1-one
(3a)
Yield: 76 %; m.p.: 176 °C; IR (KBr) mmax/cm-1: 3374 (N–H
stretching, benzimidazole), 3031 (C–H stretching, aromatic
ring), 2910 (C–H stretching, –CH=CH–), 1681 (CO
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Med Chem Res (2014) 23:1474–1487
1479
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-(2-
hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-
oxoethoxy)phenyl)acetamide (5b)
stretching), 1578 (C=N stretching), 1522 (C=C stretching);
1H NMR (300 MHz, DMSO-d6, d, ppm): 6.81 (d, 1H,
J = 14.4 Hz, –COCH=), 7.24 (d, 2H, J = 8.1 Hz, Ar–H),
7.35 (t, 1H, J = 7.8 Hz, Ar–H), 7.44 (t, 2H, J = 7.7 Hz,
Ar–H), 7.58 (d, 2H, J = 8.2 Hz, Ar–H), 7.66 (d, 2H,
J = 7.7 Hz, Ar–H), 7.78 (d, 1H, J = 15.6 Hz, =CH–Ar),
9.96 (s, 1H, –NH D2O exch.); 13C NMR (100 MHz, DMSO-
d6, d, ppm): 121.4 (1C, –CH= attached with C=O), 141.6
(1C, benzimidazole-C2), 145.2 (1C, =CH–Ar), 187.2 (1C,
C=O); LCMS (m/z): 249.09 [(M)?, 78 %]; Anal. Calcd. for
C16H12N2O: C-77.40, H-4.87, N-11.28; Found: C-77.54,
H-4.79, N-11.36 %.
Yield: 68 %; m.p.: 208 °C; IR (KBr) mmax/cm-1: 3432
(O–H stretching), 3392 (N–H stretching, benzimidazole),
3242 (N–H stretching, –NHCOCH3), 3045 (C–H stretching,
aromatic ring), 2856 (C–H stretching, –CH2), 1690 (CO
stretching, –COCH2), 1660 (CO stretching, –NHCOCH3),
1586 (C=N stretching), 1522 (C=C stretching), 1456 (C–H
1
bending, –CH2); H NMR (300 MHz, DMSO-d6, d, ppm):
2.08 (s, 3H, NHCOCH3), 3.44 (dd, 1H, J = 17.48 Hz,
3.10 Hz, C4–H pyrazoline), 3.90 (dd, 1H, J = 17.51 Hz,
11.12 Hz, C4–H pyrazoline), 4.96 (s, 2H, –CH2CO), 5.52 (s,
1H, NHCOCH3), 6.01 (dd, 1H, J = 11.15, 3.12 Hz, C5–H
pyrazoline), 6.90 (t, 2H, J = 7.8 Hz, Ar–H), 7.01 (d, 2H,
J = 8.1 Hz, Ar–H), 7.11 (t, 1H, J = 7.6 Hz, Ar–H), 7.17
(d, 1H, J = 7.6 Hz, Ar–H), 7.26 (d, 2H, J = 8.0 Hz,
Ar–H), 7.52 (d, 2H, J = 8.1 Hz, Ar–H), 7.66 (d, 2H,
J = 8.2 Hz, Ar–H), 9.16 (s, 1H, OH D2O exch.), 10.14 (s,
1H, –NH D2O exch.); 13C NMR (100 MHz, DMSO-d6, d,
ppm): 24.1 (1C, CH3 in NHCOCH3), 39.9 (1C, pyrazoline-
CH2), 60.2 (1C, pyrazoline-CH), 67.2 (1C, CH2 attached
with C=O), 151.4 (1C, benzimidazole-C2), 156.2 (1C,
C–OH), 168.8 (1C, C=O present in NHCOCH3), 172.6 (1C,
C=O attached with pyrazoline ring); LCMS (m/z): 469.13
[(M)?, 79 %]; Anal. Calcd. for C26H23N5O4: C-66.51,
H-4.94, N-14.92; Found: C-66.59, H-4.91, N-14.99 %.
General synthetic procedure for the preparation of N-(4-
(2-(3-(1H-benzo[d]imidazol-2-yl)-5-(aryl)-4,5-dihydro-
1H-pyrazol-1-yl)-2-oxoethoxy)phenyl)acetamides (5a–u)
A mixture of differently substituted benzimidazolyl chal-
cones (3a–u) (0.01 mol) and paracetamol hydrazide com-
pound (4) (0.02 mol) was taken in 20 mL glacial acetic
acid and refluxed at 130 °C for a period of 10 h. The
mixture was concentrated under vacuum and diluted with
ice-cold water. The separated solid is filtered, dried, and
crystallized from chloroform.
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-phenyl-4,5-
dihydro-1H-pyrazol-1-yl)-2-oxoetho-xy)phenyl)acetamide
(5a)
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-(3-
hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-
oxoethoxy)phenyl)acetamide (5c)
Yield: 64 %; m.p.: 188–190 °C; IR (KBr) mmax/cm-1: 3390
(N–H stretching, benzimidazole), 3240 (N–H stretching,
–NHCOCH3), 3042 (C–H stretching, aromatic ring), 2860
(C–H stretching, –CH2), 1690 (CO stretching, –COCH2),
1662 (CO stretching, –NHCOCH3), 1584 (C=N stretching),
Yield: 70 %; m.p.: 190 °C; IR (KBr) mmax/cm-1: 3428
(O–H stretching), 3392 (N–H stretching, benzimidazole),
3240 (N–H stretching, –NHCOCH3), 3036 (C–H stretching,
aromatic ring), 2858 (C–H stretching, –CH2), 1694 (CO
stretching, –COCH2), 1662 (CO stretching, –NHCOCH3),
1584 (C=N stretching), 1524 (C=C stretching), 1457 (C–H
1
1520 (C=C stretching), 1460 (C–H bending, –CH2); H
NMR (300 MHz, DMSO-d6, d, ppm): 2.12 (s, 3H, NHC-
OCH3), 3.41 (dd, 1H, J = 17.52 Hz, 3.13 Hz, C4–H pyraz-
oline), 3.91 (dd, 1H, J = 17.49 Hz, 11.11 Hz, C4–H
pyrazoline), 4.94 (s, 2H, –CH2CO), 5.48 (s, 1H, NHCOCH3),
6.03 (dd, 1H, J = 11.15, 3.12 Hz, C5–H pyrazoline), 6.98 (d,
2H, J = 8.2 Hz, Ar–H), 7.18 (d, 2H, J = 8.2 Hz, Ar–H),
7.27 (t, 1H, J = 7.4 Hz, Ar–H), 7.36 (d, 2H, J = 7.6 Hz,
Ar–H), 7.44 (t, 2H, J = 7.8 Hz, Ar–H), 7.52 (d, 2H,
J = 8.0 Hz, Ar–H), 7.64 (d, 2H, J = 8.1 Hz, Ar–H), 10.12
(s, 1H, –NH D2O exch.); 13C NMR (100 MHz, DMSO-d6, d,
ppm): 24.1 (1C, CH3 in –NHCOCH3), 39.7 (1C, pyrazoline-
CH2), 66.3 (1C, pyrazoline-CH), 67.3 (1C, CH2 attached
with C=O), 151.6 (1C, benzimidazole-C2), 168.7 (1C, C=O
present in NHCOCH3), 172.5 (1C, C=O attached with pyr-
azoline ring); LCMS (m/z): 453.16 [(M)?, 81 %]; Anal.
Calcd. for C26H23N5O3: C-68.86, H-5.11, N-15.44; Found:
C-68.78, H-5.16, N-15.49 %.
1
bending, –CH2); H NMR (300 MHz, DMSO-d6, d, ppm):
2.10 (s, 3H, NHCOCH3), 3.45 (dd, 1H, J = 17.51 Hz,
3.12 Hz, C4–H pyrazoline), 3.92 (dd, 1H, J = 17.51 Hz,
11.10 Hz, C4–H pyrazoline), 4.94 (s, 2H, –CH2CO), 5.54
(s, 1H, NHCOCH3), 6.04 (dd, 1H, J = 11.16, 3.10 Hz,
C5–H pyrazoline), 6.81 (d, 1H, J = 7.8 Hz, Ar–H), 6.91
(d, 1H, J = 7.4 Hz, Ar–H), 7.01 (d, 2H, J = 8.1 Hz, Ar–H),
7.09 (s, 1H, J = 7.6 Hz, Ar–H), 7.18 (d, 2H, J = 8.1 Hz,
Ar–H), 7.28 (t, 1H, J = 7.8 Hz, Ar–H), 7.54 (d, 2H,
J = 8.0 Hz, Ar–H), 7.67 (d, 2H, J = 8.4 Hz, Ar–H), 9.20 (s,
1H, OH D2O exch.), 10.11 (s, 1H, –NH D2O exch.); 13C
NMR (100 MHz, DMSO-d6, d, ppm): 24.2 (1C, CH3 in
NHCOCH3), 39.6 (1C, pyrazoline-CH2), 66.4 (1C, pyrazo-
line-CH), 67.4 (1C, CH2 attached with C=O), 151.6 (1C,
123

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Med Chem Res (2014) 23:1474–1487
benzimidazole-C2), 156.6 (1C, C–OH), 168.6 (1C, C=O
present in NHCOCH3), 172.9 (1C, C=O attached with pyr-
azoline ring); LCMS (m/z): 469.16 [(M)?, 79 %]; Anal.
Calcd. for C26H23N5O4: C-66.51, H-4.94, N-14.92; Found:
C-66.60, H-4.90, N-14.97 %.
(d, 2H, J = 8.2 Hz, Ar–H), 7.32 (d, 1H, J = 7.4 Hz, Ar–
H), 7.42 (t, 1H, J = 7.6 Hz, Ar–H), 7.52 (d, 2H,
J = 8.0 Hz, Ar–H), 7.66 (d, 2H, J = 8.2 Hz, Ar–H), 7.75
(d, 1H, J = 7.8 Hz, Ar–H), 10.14 (s, 1H, –NH D2O exch.);
13C NMR (100 MHz, DMSO-d6, d, ppm): 24.0 (1C, CH3 in
NHCOCH3), 39.2 (1C, pyrazoline-CH2), 61.2 (1C, pyraz-
oline-CH), 67.0 (1C, CH2 attached with C=O), 132.3 (1C,
C–Cl), 151.2 (1C, benzimidazole-C2), 168.5 (1C, C=O
present in NHCOCH3), 172.6 (1C, C=O attached with
pyrazoline ring); LCMS (m/z): 487.16 [(M)?, 79 %]; Anal.
Calcd. for C26H22ClN5O3: C-64.00, H-4.54, N-14.35;
Found: C-64.10, H-4.59, N-14.28 %.
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-(4-
hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-
oxoethoxy)phenyl)acetamide (5d)
Yield: 70 %; m.p.: 190 °C; IR (KBr) mmax/cm-1: 3,434
(O–H stretching), 3,396 (N–H stretching, benzimidazole),
3,244 (N–H stretching, –NHCOCH3), 3042 (C–H stretch-
ing, aromatic ring), 2860 (C–H stretching, –CH2), 1694 (CO
stretching, –COCH2), 1664 (CO stretching, –NHCOCH3),
1588 (C=N stretching), 1526 (C=C stretching), 1458 (C–H
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-(3-
chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-
oxoethoxy)phenyl)acetamide (5f)
1
bending, –CH2); H NMR (300 MHz, DMSO-d6, d, ppm):
Yield: 68 %; m.p.: 198 °C; IR (KBr) mmax/cm-1: 3392
(N–H stretching, benzimidazole), 3240 (N–H stretching,
–NHCOCH3), 3041 (C–H stretching, aromatic ring), 2860
(C–H stretching, –CH2), 1690 (CO stretching, –COCH2),
1662 (CO stretching, –NHCOCH3), 1584 (C=N stretching),
1520 (C=C stretching), 1454 (C–H bending, –CH2), 756
(C–Cl stretching); 1H NMR (300 MHz, DMSO-d6, d,
ppm): 2.10 (s, 3H, NHCOCH3), 3.48 (dd, 1H,
J = 17.51 Hz, 3.16 Hz, C4–H pyrazoline), 3.92 (dd, 1H,
J = 17.52 Hz, 11.18 Hz, C4–H pyrazoline), 4.96 (s, 2H,
–CH2CO), 5.56 (s, 1H, NHCOCH3), 6.14 (dd, 1H,
J = 11.21, 3.14 Hz, C5–H pyrazoline), 6.96 (d, 2H,
J = 8.1 Hz, Ar–H), 7.15 (d, 1H, J = 7.6 Hz, Ar–H), 7.23
(d, 2H, J = 8.2 Hz, Ar–H), 7.32 (d, 1H, J = 7.4 Hz, Ar–
H), 7.44 (t, 1H, J = 7.4 Hz, Ar–H), 7.51 (s, 1H,
J = 7.6 Hz, Ar–H), 7.61 (d, 2H, J = 8.2 Hz, Ar–H), 7.69
(d, 2H, J = 8.4 Hz, Ar–H), 10.18 (s, 1H, –NH D2O exch.);
13C NMR (100 MHz, DMSO-d6, d, ppm): 24.1 (1C, CH3 in
NHCOCH3), 39.3 (1C, pyrazoline-CH2), 65.6 (1C, pyraz-
oline-CH), 67.2 (1C, CH2 attached with C=O), 134.2 (1C,
C–Cl), 151.3 (1C, benzimidazole-C2), 168.4 (1C, C=O
present in NHCOCH3), 172.4 (1C, C=O attached with
pyrazoline ring); LCMS (m/z): 487.15 [(M)?, 81 %]; Anal.
Calcd. for C26H22ClN5O3: C-64.00, H-4.54, N-14.35;
Found: C-64.09, H-4.58, N-14.29 %.
2.12 (s, 3H, NHCOCH3), 3.46 (dd, 1H, J = 17.49 Hz,
3.15 Hz, C4–H pyrazoline), 3.90 (dd, 1H, J = 17.50 Hz,
11.14 Hz, C4–H pyrazoline), 4.96 (s, 2H, –CH2CO), 5.51
(s, 1H, NHCOCH3), 6.06 (dd, 1H, J = 11.18, 3.12 Hz,
C5–H pyrazoline), 6.80 (d, 2H, J = 7.6 Hz, Ar–H), 6.97 (d,
2H, J = 7.6 Hz, Ar–H), 7.12 (d, 2H, J = 7.6 Hz, Ar–H),
7.22 (d, 2H, J = 8.2 Hz, Ar–H), 7.54 (d, 2H, J = 8.0 Hz,
Ar–H), 7.68 (d, 2H, J = 8.2 Hz, Ar–H), 9.18 (s, 1H,
OH D2O exch.), 10.12 (s, 1H, –NH D2O exch.); 13C
NMR (100 MHz, DMSO-d6, d, ppm): 24.1 (1C, CH3 in
NHCOCH3), 39.4 (1C, pyrazoline-CH2), 66.6 (1C, pyrazo-
line-CH), 67.2 (1C, CH2 attached with C=O), 151.4 (1C,
benzimidazole-C2), 155.7 (1C, C–OH), 168.8 (1C, C=O
present in NHCOCH3), 172.7 (1C, C=O attached with pyr-
azoline ring); LCMS (m/z): 469.15 [(M)?, 81 %]; Anal.
Calcd. for C26H23N5O4: C-66.51, H-4.94, N-14.92; Found:
C-66.60, H-4.91, N-14.99 %.
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-(2-
chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-
oxoethoxy)phenyl)acetamide (5e)
Yield: 72 %; m.p.: 222 °C; IR (KBr) mmax/cm-1: 3396
(N–H stretching, benzimidazole), 3244 (N–H stretching,
–NHCOCH3), 3044 (C–H stretching, aromatic ring), 2858
(C–H stretching, –CH2), 1696 (CO stretching, –COCH2),
1664 (CO stretching, –NHCOCH3), 1586 (C=N stretching),
1522 (C=C stretching), 1454 (C–H bending, –CH2), 754
(C–Cl stretching); 1H NMR (300 MHz, DMSO-d6, d,
ppm): 2.09 (s, 3H, NHCOCH3), 3.45 (dd, 1H,
J = 17.51 Hz, 3.16 Hz, C4–H pyrazoline), 3.91 (dd, 1H,
J = 17.48 Hz, 11.15 Hz, C4–H pyrazoline), 4.92 (s, 2H,
–CH2CO), 5.54 (s, 1H, NHCOCH3), 6.08 (dd, 1H,
J = 11.20, 3.12 Hz, C5–H pyrazoline), 6.98 (d, 2H,
J = 8.0 Hz, Ar–H), 7.17 (t, 1H, J = 7.6 Hz, Ar–H), 7.24
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-(4-
chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-
oxoethoxy)phenyl)acetamide (5g)
Yield: 68 %; m.p.: 201 °C; IR (KBr) mmax/cm-1: 3396
(N–H stretching, benzimidazole), 3244 (N–H stretching,
–NHCOCH3), 3044 (C–H stretching, aromatic ring), 2864
(C–H stretching, –CH2), 1694 (CO stretching, –COCH2),
1664 (CO stretching, –NHCOCH3), 1584 (C=N stretching),
1524 (C=C stretching), 1458 (C–H bending, –CH2), 760
123

Med Chem Res (2014) 23:1474–1487
1481
(C–Cl stretching); 1H NMR (300 MHz, DMSO-d6, d, ppm):
2.14 (s, 3H, NHCOCH3), 3.51 (dd, 1H, J = 17.50 Hz,
3.16 Hz, C4–H pyrazoline), 3.91 (dd, 1H, J = 17.48 Hz,
11.16 Hz, C4–H pyrazoline), 4.94 (s, 2H, –CH2CO), 5.54 (s,
1H, NHCOCH3), 6.10 (dd, 1H, J = 11.24, 3.14 Hz, C5–H
pyrazoline), 6.97 (d, 2H, J = 8.2 Hz, Ar–H), 7.18 (d, 2H,
J = 8.0 Hz, Ar–H), 7.41 (d, 2H, J = 7.6 Hz, Ar–H), 7.52
(d, 2H, J = 7.6 Hz, Ar–H), 7.61 (d, 2H, J = 8.1 Hz, Ar–
H), 7.70 (d, 2H, J = 8.4 Hz, Ar–H), 10.20 (s, 1H, –NH D2O
exch.); 13C NMR (100 MHz, DMSO-d6, d, ppm): 24.2 (1C,
CH3 in NHCOCH3), 39.2 (1C, pyrazoline-CH2), 66.2 (1C,
pyrazoline-CH), 67.1 (1C, CH2 attached with C=O), 132.2
(1C, C–Cl), 151.5 (1C, benzimidazole-C2), 168.6 (1C, C=O
present in NHCOCH3), 172.6 (1C, C=O attached with
pyrazoline ring); LCMS (m/z): 487.16 [(M)?, 79 %]; Anal.
Calcd. for C26H22ClN5O3: C-64.00, H-4.54, N-14.35;
Found: C-64.11, H-4.60, N-14.27 %.
–NHCOCH3), 3041 (C–H stretching, aromatic ring), 2862
(C–H stretching, –CH2), 1693 (CO stretching, –COCH2),
1662 (CO stretching, –NHCOCH3), 1582 (C=N stretch-
ing), 1522 (C=C stretching), 1458 (C–H bending, –CH2),
1
1117 (C–F stretching); H NMR (300 MHz, DMSO-d6, d,
ppm): 2.12 (s, 3H, NHCOCH3), 3.47 (dd, 1H,
J = 17.52 Hz, 3.12 Hz, C4–H pyrazoline), 3.92 (dd, 1H,
J = 17.51 Hz, 11.17 Hz, C4–H pyrazoline), 4.93 (s, 2H,
–CH2CO), 5.56 (s, 1H, NHCOCH3), 6.11 (dd, 1H,
J = 11.19, 3.14 Hz, C5–H pyrazoline), 6.84 (s, 1H, Ar–
H), 6.98 (d, 2H, J = 8.1 Hz, Ar–H), 7.06 (d, 1H,
J = 7.6 Hz, Ar–H), 7.11 (d, 1H, J = 7.4 Hz, Ar–H), 7.18
(d, 2H, J = 8.2 Hz, Ar–H), 7.41 (t, 1H, J = 7.6 Hz, Ar–
H), 7.52 (d, 2H, J = 8.0 Hz, Ar–H), 7.67 (d, 2H,
J = 8.4 Hz, Ar–H), 10.16 (s, 1H, –NH D2O exch.); 13C
NMR (100 MHz, DMSO-d6, d, ppm): 24.2 (1C, CH3 in
NHCOCH3), 39.0 (1C, pyrazoline-CH2), 66.1 (1C, pyr-
azoline-CH), 67.2 (1C, CH2 attached with C=O), 151.4
(1C, benzimidazole-C2), 162.7 (1C, C–F), 168.2 (1C, C=O
present in NHCOCH3), 172.6 (1C, C=O attached with
pyrazoline ring); LCMS (m/z): 471.14 [(M)?, 80 %]; Anal.
Calcd. for C26H22FN5O3: C-66.23, H-4.70, N-14.85;
Found: C-66.30, H-4.77, N-14.90 %.
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-(2-fluorophenyl)-
4,5-dihydro-1H-pyrazol-1-yl)-2-
oxoethoxy)phenyl)acetamide (5h)
Yield: 62 %; m.p.: 188 °C; IR (KBr) mmax/cm-1: 3398 (N–H
stretching, benzimidazole), 3245 (N–H stretching, –NHC-
OCH3), 3045 (C–H stretching, aromatic ring), 2866 (C–H
stretching, –CH2), 1696 (CO stretching, –COCH2), 1664
(CO stretching, –NHCOCH3), 1586 (C=N stretching), 1525
(C=C stretching), 1460 (C–H bending, –CH2), 1121 (C–F
stretching); 1H NMR (300 MHz, DMSO-d6, d, ppm): 2.14 (s,
3H, NHCOCH3), 3.48 (dd, 1H, J = 17.54 Hz, 3.14 Hz, C4–
H pyrazoline), 3.94 (dd, 1H, J = 17.49 Hz, 11.16 Hz, C4–H
pyrazoline), 4.94 (s, 2H, –CH2CO), 5.57 (s, 1H, NHCOCH3),
6.12 (dd, 1H, J = 11.19, 3.14 Hz, C5–H pyrazoline), 7.00 (d,
2H, J = 8.1 Hz, Ar–H), 7.16 (t, 1H, J = 7.6 Hz, Ar–H),
7.24 (d, 2H, J = 8.2 Hz, Ar–H), 7.33 (d, 1H, J = 7.6 Hz,
Ar–H), 7.49 (d, 2H, J = 8.0 Hz, Ar–H), 7.57 (d, 1H,
J = 7.4 Hz, Ar–H), 7.63 (t, 1H, J = 7.8 Hz, Ar–H), 7.71 (d,
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-
(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-
oxoethoxy)phenyl)acetamide (5j)
Yield: 60 %; m.p.: 170 °C; IR (KBr) mmax/cm-1: 3396
(N–H stretching, benzimidazole), 3245 (N–H stretching,
–NHCOCH3), 3042 (C–H stretching, aromatic ring), 2862
(C–H stretching, –CH2), 1694 (CO stretching, –COCH2),
1664 (CO stretching, –NHCOCH3), 1584 (C=N stretch-
ing), 1524 (C=C stretching), 1458 (C–H bending, –CH2),
1
1120 (C–F stretching); H NMR (300 MHz, DMSO-d6, d,
ppm): 2.11 (s, 3H, NHCOCH3), 3.48 (dd, 1H,
J = 17.48 Hz, 3.14 Hz, C4–H pyrazoline), 3.96 (dd, 1H,
J = 17.54 Hz, 11.15 Hz, C4–H pyrazoline), 4.94 (s, 2H,
–CH2CO), 5.55 (s, 1H, NHCOCH3), 6.12 (dd, 1H,
J = 11.20, 3.15 Hz, C5–H pyrazoline), 6.97 (d, 2H,
J = 8.1 Hz, Ar–H), 7.16 (d, 2H, J = 7.6 Hz, Ar–H), 7.23
(d, 2H, J = 8.2 Hz, Ar–H), 7.32 (d, 2H, J = 7.4 Hz, Ar–
H), 7.54 (d, 2H, J = 8.0 Hz, Ar–H), 7.66 (d, 2H,
J = 8.3 Hz, Ar–H), 10.12 (s, 1H, –NH D2O exch.); 13C
NMR (100 MHz, DMSO-d6, d, ppm): 24.1 (1C, CH3 in
NHCOCH3), 39.2 (1C, pyrazoline-CH2), 66.0 (1C, pyr-
azoline-CH), 67.0 (1C, CH2 attached with C=O), 151.6
(1C, benzimidazole-C2), 160.8 (1C, C–F), 168.4 (1C,
C=O present in NHCOCH3), 172.5 (1C, C=O attached
with pyrazoline ring); LCMS (m/z): 471.14 [(M)?, 82 %];
Anal. Calcd. for C26H22FN5O3: C-66.23, H-4.70, N-14.85;
Found: C-66.30, H-4.78, N-14.81 %.
2H, J = 8.2 Hz, Ar–H), 10.18 (s, 1H, –NH D2O exch.); 13
C
NMR (100 MHz, DMSO-d6, d, ppm): 24.0 (1C, CH3 in
NHCOCH3), 39.1 (1C, pyrazoline-CH2), 59.4 (1C, pyrazo-
line-CH), 67.0 (1C, CH2 attached with C=O), 151.6 (1C,
benzimidazole-C2), 159.4 (1C, C–F), 168.4 (1C, C=O pres-
ent in NHCOCH3), 172.5 (1C, C=O attached with pyrazoline
ring); LCMS (m/z): 471.18 [(M)?, 82 %]; Anal. Calcd. for
C26H22FN5O3: C-66.23, H-4.70, N-14.85; Found: C-66.29,
H-4.76, N-14.89 %.
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-(3-fluorophenyl)-
4,5-dihydro-1H-pyrazol-1-yl)-2-
oxoethoxy)phenyl)acetamide (5i)
Yield: 60 %; m.p.: 198 °C; IR (KBr) mmax/cm-1: 3395
(N–H stretching, benzimidazole), 3243 (N–H stretching,
123

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Med Chem Res (2014) 23:1474–1487
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-o-tolyl-4,5-
dihydro-1H-pyrazol-1-yl)-2-oxo-ethoxy)phenyl)
acetamide (5k)
66.5 (1C, pyrazoline-CH), 67.2 (1C, CH2 attached with
C=O), 138.3 (1C, C–CH3), 151.6 (1C, benzimidazole-C2),
168.4 (1C, C=O present in NHCOCH3), 172.6 (1C, C=O
attached with pyrazoline ring); LCMS (m/z): 467.17 [(M)?,
83 %]; Anal. Calcd. for C27H25N5O3: C-69.36, H-5.39,
N-14.98; Found: C-69.45, H-5.43, N-14.92 %.
Yield: 64 %; m.p.: 211 °C; IR (KBr) mmax/cm-1: 3390
(N–H stretching, benzimidazole), 3238 (N–H stretching,
–NHCOCH3), 3040 (C–H stretching, aromatic ring), 2931
(C–H stretching, CH3), 2858 (C–H stretching, –CH2), 1688
(CO stretching, –COCH2), 1660 (CO stretching, –NHC-
OCH3), 1581 (C=N stretching), 1521 (C=C stretching),
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-p-tolyl-4,5-
dihydro-1H-pyrazol-1-yl)-2-oxo-ethoxy)phenyl)
acetamide (5m)
1
1452 (C–H bending, –CH2); H NMR (300 MHz, DMSO-
Yield: 61 %; m.p.: 209 °C; IR (KBr) mmax/cm-1: 3391
(N–H stretching, benzimidazole), 3240 (N–H stretching,
–NHCOCH3), 3040 (C–H stretching, aromatic ring),
2934 (C–H stretching, CH3), 2861 (C–H stretching, –CH2),
1692 (CO stretching, –COCH2), 1661 (CO stretching,
–NHCOCH3), 1581 (C=N stretching), 1523 (C=C stretch-
ing), 1457 (C–H bending, –CH2); 1H NMR (300 MHz,
DMSO-d6, d, ppm): 2.06 (s, 3H, NHCOCH3), 2.32 (s, 3H,
CH3), 3.43 (dd, 1H, J = 17.47 Hz, 3.14 Hz, C4–H pyraz-
oline), 3.90 (dd, 1H, J = 17.50 Hz, 11.14 Hz, C4–H pyr-
azoline), 4.91 (s, 2H, –CH2CO), 5.51 (s, 1H, NHCOCH3),
6.07 (dd, 1H, J = 11.12, 3.13 Hz, C5–H pyrazoline), 6.96
(d, 2H, J = 7.9 Hz, Ar–H), 7.09 (d, 2H, J = 7.6 Hz,
Ar–H), 7.16 (d, 2H, J = 7.4 Hz, Ar–H), 7.24 (d, 2H,
J = 8.0 Hz, Ar–H), 7.52 (d, 2H, J = 8.1 Hz, Ar–H), 7.64
(d, 2H, J = 8.2 Hz, Ar–H), 10.06 (s, 1H, –NH D2O exch.);
13C NMR (100 MHz, DMSO-d6, d, ppm): 21.3 (1C, CH3),
24.2 (1C, CH3 in NHCOCH3), 39.4 (1C, pyrazoline-CH2),
66.2 (1C, pyrazoline-CH), 67.1 (1C, CH2 attached with
C=O), 136.4 (1C, C–CH3), 151.4 (1C, benzimidazole-C2),
168.5 (1C, C=O present in NHCOCH3), 172.7 (1C, C=O
attached with pyrazoline ring); LCMS (m/z): 467.19 [(M)?,
80 %]; Anal. Calcd. for C27H25N5O3: C-69.36, H-5.39,
N-14.98; Found: C-69.44, H-5.44, N-14.91 %.
d6, d, ppm): 2.06 (s, 3H, NHCOCH3), 2.36 (s, 3H, CH3),
3.44 (dd, 1H, J = 17.46 Hz, 3.12 Hz, C4–H pyrazoline),
3.91 (dd, 1H, J = 17.51 Hz, 11.12 Hz, C4–H pyrazoline),
4.90 (s, 2H, –CH2CO), 5.51 (s, 1H, NHCOCH3), 6.04 (dd,
1H, J = 11.16, 3.14 Hz, C5–H pyrazoline), 6.91 (d, 1H,
J = 7.6 Hz, Ar–H), 7.00 (d, 2H, J = 8.0 Hz, Ar–H), 7.10
(t, 1H, J = 7.6 Hz, Ar–H), 7.17 (t, 1H, J = 7.4 Hz, Ar–H),
7.24 (d, 2H, J = 8.0 Hz, Ar–H), 7.44 (d, 1H, J = 7.7 Hz,
Ar–H), 7.52 (d, 2H, J = 8.1 Hz, Ar–H), 7.65 (d, 2H,
J = 8.4 Hz, Ar–H), 10.08 (s, 1H, –NH D2O exch.); 13C
NMR (100 MHz, DMSO-d6, d, ppm): 19.2 (1C, CH3), 24.3
(1C, CH3 in NHCOCH3), 39.4 (1C, pyrazoline-CH2), 63.8
(1C, pyrazoline-CH), 67.2 (1C, CH2 attached with C=O),
134.6 (1C, C–CH3), 151.7 (1C, benzimidazole-C2), 168.6
(1C, C=O present in NHCOCH3), 172.6 (1C, C=O attached
with pyrazoline ring); LCMS (m/z): 467.18 [(M)?, 79 %];
Anal. Calcd. for C27H25N5O3: C-69.36, H-5.39, N-14.98;
Found: C-69.45, H-5.44, N-14.93 %.
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-m-tolyl-4,5-
dihydro-1H-pyrazol-1-yl)-2-oxo-ethoxy)phenyl)
acetamide (5l)
Yield: 60 %; m.p.: 232 °C; IR (KBr) mmax/cm-1: 3388
(N–H stretching, benzimidazole), 3237 (N–H stretching,
–NHCOCH3), 3038 (C–H stretching, aromatic ring), 2930
(C–H stretching, CH3), 2858 (C–H stretching, –CH2),
1690 (CO stretching, –COCH2), 1658 (CO stretching,
–NHCOCH3), 1580 (C=N stretching), 1521 (C=C stretch-
ing), 1454 (C–H bending, –CH2); 1H NMR (300 MHz,
DMSO-d6, d, ppm): 2.07 (s, 3H, NHCOCH3), 2.37 (s, 3H,
CH3), 3.44 (dd, 1H, J = 17.46 Hz, 3.12 Hz, C4–H pyraz-
oline), 3.92 (dd, 1H, J = 17.50 Hz, 11.13 Hz, C4–H pyr-
azoline), 4.91 (s, 2H, –CH2CO), 5.52 (s, 1H, NHCOCH3),
6.06 (dd, 1H, J = 11.15, 3.14 Hz, C5–H pyrazoline), 6.98
(d, 2H, J = 8.0 Hz, Ar–H), 7.07 (d, 1H, J = 7.6 Hz,
Ar–H), 7.15 (d, 1H, J = 7.4 Hz, Ar–H), 7.21 (s, 1H,
Ar–H), 7.27 (d, 2H, J = 8.0 Hz, Ar–H), 7.44 (t, 1H,
J = 7.6 Hz, Ar–H), 7.51 (d, 2H, J = 8.2 Hz, Ar–H), 7.67
(d, 2H, J = 8.3 Hz, Ar–H), 10.07 (s, 1H, –NH D2O exch.);
13C NMR (100 MHz, DMSO-d6, d, ppm): 21.7 (1C, CH3),
24.4 (1C, CH3 in NHCOCH3), 39.5 (1C, pyrazoline-CH2),
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-(3-
methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-
oxoethoxy)phenyl)acetamide (5n)
Yield: 64 %; m.p.: 241 °C; IR (KBr) mmax/cm-1: 3388
(N–H stretching, benzimidazole), 3238 (N–H stretching,
–NHCOCH3), 3038 (C–H stretching, aromatic ring), 2861
(C–H stretching, –CH2), 2812 (C–H stretching, OCH3),
1688 (CO stretching, –COCH2), 1660 (CO stretching,
–NHCOCH3), 1580 (C=N stretching), 1521 (C=C stretch-
ing), 1458 (C–H bending, –CH2); 1H NMR (300 MHz,
DMSO-d6, d, ppm): 2.03 (s, 3H, NHCOCH3), 3.42 (dd, 1H,
J = 17.47 Hz, 3.11 Hz, C4–H pyrazoline), 3.81 (s, 3H,
OCH3), 3.92 (dd, 1H, J = 17.49 Hz, 11.12 Hz, C4–H
pyrazoline), 4.91 (s, 2H, –CH2CO), 5.50 (s, 1H, NHC-
OCH3), 6.04 (dd, 1H, J = 11.15, 3.15 Hz, C5–H pyrazo-
line), 6.82 (d, 1H, J = 7.5 Hz, Ar–H), 6.91 (d, 1H,
123

Med Chem Res (2014) 23:1474–1487
1483
J = 7.3 Hz, Ar–H), 7.00 (d, 2H, J = 8.1 Hz, Ar–H), 7.09
(s, 1H, Ar–H), 7.21 (d, 2H, J = 8.1 Hz, Ar–H), 7.31 (t, 1H,
J = 7.4 Hz, Ar–H), 7.51 (d, 2H, J = 8.1 Hz, Ar–H), 7.64
(d, 2H, J = 8.3 Hz, Ar–H), 10.07 (s, 1H, –NH D2O exch.);
13C NMR (100 MHz, DMSO-d6, d, ppm): 24.1 (1C, CH3 in
NHCOCH3), 39.5 (1C, pyrazoline-CH2), 55.7 (1C, OCH3),
66.4 (1C, pyrazoline-CH), 67.3 (1C, CH2 attached with
C=O), 151.5 (1C, benzimidazole-C2), 160.4 (1C, C–
OCH3), 168.4 (1C, C=O present in NHCOCH3), 172.6 (1C,
C=O attached with pyrazoline ring); LCMS (m/z): 483.19
[(M)?, 84 %]; Anal. Calcd. for C27H25N5O4: C-67.07,
H-5.21, N-14.48; Found: C-67.00, H-5.26, N-14.54 %.
J = 17.51 Hz, 3.16 Hz, C4-H pyrazoline), 3.96 (dd, 1H,
J = 17.50 Hz, 11.14 Hz, C4–H pyrazoline), 4.95 (s, 2H,
–CH2CO), 5.58 (s, 1H, NHCOCH3), 6.15 (dd, 1H,
J = 11.17, 3.16 Hz, C5–H pyrazoline), 6.99 (d, 2H,
J = 8.0 Hz, Ar–H), 7.23 (d, 2H, J = 8.1 Hz, Ar–H), 7.52
(d, 2H, J = 7.9 Hz, Ar–H), 7.59 (t, 1H, J = 7.4 Hz, Ar–
H), 7.64 (d, 1H, J = 7.3 Hz, Ar–H), 7. 71 (d, 2H,
J = 8.3 Hz, Ar–H), 7.81 (t, 1H, J = 7.4 Hz, Ar–H), 8.05
(d, 1H, J = 7.6 Hz, Ar–H), 10.14 (s, 1H, –NH D2O exch.);
13C NMR (100 MHz, DMSO-d6, d, ppm): 24.0 (1C, CH3 in
NHCOCH3), 38.5 (1C, pyrazoline-CH2), 61.5 (1C, pyraz-
oline-CH), 67.0 (1C, CH2 attached with C=O), 147.3 (1C,
C–NO2), 151.5 (1C, benzimidazole-C2), 168.4 (1C, C=O
present in NHCOCH3), 172.5 (1C, C=O attached with
pyrazoline ring); LCMS (m/z): 498.17 [(M)?, 81 %]; Anal.
Calcd. for C26H22N6O5: C-62.64, H-4.45, N-16.86; Found:
C-62.71, H-4.49, N-16.80 %.
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-(4-
methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-
oxoethoxy)phenyl)acetamide (5o)
Yield: 66 %; m.p.: 223 °C; IR (KBr) mmax/cm-1: 3390 (N–H
stretching, benzimidazole), 3240 (N–H stretching, –NHC-
OCH3), 3039 (C–H stretching, aromatic ring), 2862 (C–H
stretching, –CH2), 2815 (C–H stretching, OCH3), 1690 (CO
stretching, –COCH2), 1662 (CO stretching, –NHCOCH3),
1581 (C=N stretching), 1522 (C=C stretching), 1459 (C–H
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-
(3-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-
oxoethoxy)phenyl)acetamide (5q)
Yield: 72 %; m.p.: 191 °C; IR (KBr) mmax/cm-1: 3392 (N–H
stretching, benzimidazole), 3242 (N–H stretching, –NHC-
OCH3), 3042 (C–H stretching, aromatic ring), 2862 (C–H
stretching, –CH2), 1692 (CO stretching, –COCH2), 1662
(CO stretching, –NHCOCH3), 1584 (C=N stretching), 1524
(C=C stretching), 1491 (N=O stretching, NO2), 1457 (C–H
1
bending, –CH2); H NMR (300 MHz, DMSO-d6, d, ppm):
2.04 (s, 3H, NHCOCH3), 3.43 (dd, 1H, J = 17.47 Hz,
3.11 Hz, C4–H pyrazoline), 3.84 (s, 3H, OCH3), 3.93 (dd, 1H,
J = 17.48 Hz, 11.11 Hz, C4–H pyrazoline), 4.90 (s, 2H,
–CH2CO), 5.52 (s, 1H, NHCOCH3), 6.03 (dd, 1H, J = 11.14,
3.14 Hz, C5–H pyrazoline), 6.92 (d, 2H, J = 7.4 Hz, Ar–H),
6.99(d, 2H, J = 8.1 Hz, Ar–H), 7.16(d, 2H, J = 7.3 Hz, Ar–
H), 7.23 (d, 2H, J = 8.0 Hz, Ar–H), 7.50 (d, 2H, J = 7.9 Hz,
Ar–H), 7.64 (d, 2H, J = 8.2 Hz, Ar–H), 10.06 (s, 1H, –NH
D2O exch.); 13C NMR (100 MHz, DMSO-d6, d, ppm): 24.2
(1C, CH3 in NHCOCH3), 39.6 (1C, pyrazoline-CH2), 55.8
(1C, OCH3), 66.2 (1C, pyrazoline-CH), 67.1 (1C, CH2
attached with C=O), 151.4 (1C, benzimidazole-C2), 158.7
(1C, C–OCH3), 168.6(1C, C=O present inNHCOCH3), 172.6
(1C, C=Oattachedwithpyrazolinering);LCMS(m/z):483.17
[(M)?, 81 %]; Anal. Calcd. for C27H25N5O4: C-67.07,
H-5.21, N-14.48; Found: C-67.01, H-5.27, N-14.55 %.
1
bending, –CH2); H NMR (300 MHz, DMSO-d6, d, ppm):
2.12 (s, 3H, NHCOCH3), 3.49 (dd, 1H, J = 17.52 Hz,
3.17 Hz, C4–H pyrazoline), 3.96 (dd, 1H, J = 17.52 Hz,
11.15 Hz, C4–H pyrazoline), 4.97 (s, 2H, –CH2CO), 5.60 (s,
1H, NHCOCH3), 6.14 (dd, 1H, J = 11.17, 3.16 Hz, C5–H
pyrazoline), 7.01 (d, 2H, J = 8.01 Hz, Ar–H), 7.24 (d, 2H,
J = 8.1 Hz, Ar–H), 7.54 (d, 2H, J = 8.0 Hz, Ar–H), 7. 69
(d, 2H, J = 8.3 Hz, Ar–H), 7.76 (t, 1H, J = 7.4 Hz, Ar–H),
7.82 (d, 1H, J = 7.6 Hz, Ar–H), 8.01 (d, 1H, J = 7.5 Hz,
Ar–H), 8.18 (s, 1H, Ar–H), 10.16 (s, 1H, –NH D2O exch.);
13C NMR (100 MHz, DMSO-d6, d, ppm): 24.2 (1C, CH3 in
NHCOCH3), 39.5 (1C, pyrazoline-CH2), 65.2 (1C, pyrazo-
line-CH), 67.2 (1C, CH2 attached with C=O), 147.8 (1C, C–
NO2), 151.6 (1C, benzimidazole-C2), 168.6 (1C, C=O
present in NHCOCH3), 172.7 (1C, C=O attached with pyr-
azoline ring); LCMS (m/z): 498.19 [(M)?, 78 %]; Anal.
Calcd. for C26H22N6O5: C-62.64, H-4.45, N-16.86; Found:
C-62.72, H-4.50, N-16.80 %.
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-(2-nitrophenyl)-
4,5-dihydro-1H-pyrazol-1-yl)-2-
oxoethoxy)phenyl)acetamide (5p)
Yield: 70 %; m.p.: 201 °C; IR (KBr) mmax/cm-1: 3394
(N–H stretching, benzimidazole), 3242 (N–H stretching,
–NHCOCH3), 3044 (C–H stretching, aromatic ring), 2864
(C–H stretching, –CH2), 1690 (CO stretching, –COCH2),
1664 (CO stretching, –NHCOCH3), 1584 (C=N stretching),
1524 (C=C stretching), 1492 (N=O stretching, NO2), 1461
(C–H bending, –CH2); 1H NMR (300 MHz, DMSO-d6,
d, ppm): 2.11 (s, 3H, NHCOCH3), 3.48 (dd, 1H,
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-
(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-
oxoethoxy)phenyl)acetamide (5r)
Yield: 68 %; m.p.: 213 °C; IR (KBr) mmax/cm-1: 3394
(N–H stretching, benzimidazole), 3244 (N–H stretching,
123

1484
Med Chem Res (2014) 23:1474–1487
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-(3-
–NHCOCH3), 3044 (C–H stretching, aromatic ring), 2863
(C–H stretching, –CH2), 1692 (CO stretching, –COCH2),
1664 (CO stretching, –NHCOCH3), 1586 (C=N stretching),
1524 (C=C stretching), 1494 (N=O stretching, NO2), 1460
bromophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-
oxoethoxy)phenyl)acetamide (5t)
Yield: 72 %; m.p.: 218 °C; IR (KBr) mmax/cm-1: 3390
(N–H stretching, benzimidazole), 3241 (N–H stretching,
–NHCOCH3), 3040 (C–H stretching, aromatic ring), 2861
(C–H stretching, –CH2), 1692 (CO stretching, –COCH2),
1662 (CO stretching, –NHCOCH3), 1585 (C=N stretching),
1522 (C=C stretching), 1461 (C–H bending, –CH2), 544
(C–Br stretching); 1H NMR (300 MHz, DMSO-d6, d,
ppm): 2.08 (s, 3H, NHCOCH3), 3.44 (dd, 1H,
J = 17.48 Hz, 3.15 Hz, C4–H pyrazoline), 3.94 (dd, 1H,
J = 17.49 Hz, 11.13 Hz, C4–H pyrazoline), 4.95 (s, 2H,
–CH2CO), 5.54 (s, 1H, NHCOCH3), 6.10 (dd, 1H,
J = 11.14, 3.14 Hz, C5–H pyrazoline), 6.97 (d, 2H,
J = 8.0 Hz, Ar–H), 7.18 (d, 2H, J = 8.2 Hz, Ar–H), 7.27
(d, 1H, J = 7.6 Hz, Ar–H), 7.34 (t, 1H, J = 7.5 Hz, Ar–
H), 7.41 (d, 1H, J = 7.4 Hz, Ar–H), 7.48 (s, 1H, Ar–H),
7.54 (d, 2H, J = 7.9 Hz, Ar–H), 7.66 (d, 2H, J = 8.3 Hz,
Ar–H), 10.07 (s, 1H, –NH D2O exch.); 13C NMR
(100 MHz, DMSO-d6, d, ppm): 24.0 (1C, CH3 in NHC-
OCH3), 39.6 (1C, pyrazoline-CH2), 65.4 (1C, pyrazoline-
CH), 67.0 (1C, CH2 attached with C=O), 122.7 (1C, C–Br),
151.3 (1C, benzimidazole-C2), 168.4 (1C, C=O present in
NHCOCH3), 172.5 (1C, C=O attached with pyrazoline
ring); LCMS (m/z): 531.09 [(M)?, 80 %]; Anal. Calcd. for
C26H22BrN5O3: C-58.66, H-4.17, N-13.15; Found:
C-58.59, H-4.21, N-13.19 %.
1
(C–H bending, –CH2); H NMR (300 MHz, DMSO-d6, d,
ppm): 2.13 (s, 3H, NHCOCH3), 3.47 (dd, 1H,
J = 17.51 Hz, 3.16 Hz, C4–H pyrazoline), 3.96 (dd, 1H,
J = 17.51 Hz, 11.16 Hz, C4–H pyrazoline), 4.96 (s, 2H,
–CH2CO), 5.59 (s, 1H, NHCOCH3), 6.15 (dd, 1H,
J = 11.15, 3.18 Hz, C5–H pyrazoline), 7.00 (d, 2H,
J = 8.0 Hz, Ar–H), 7.22 (d, 2H, J = 8.0 Hz, Ar–H), 7.52
(d, 2H, J = 7.9 Hz, Ar–H), 7. 61 (d, 2H, J = 7.6 Hz, Ar–
H), 7.71 (d, 2H, J = 8.2 Hz, Ar–H), 8.22 (d, 2H,
J = 7.6 Hz, Ar–H), 10.15 (s, 1H, –NH D2O exch.); 13C
NMR (100 MHz, DMSO-d6, d, ppm): 24.1 (1C, CH3 in
NHCOCH3), 39.4 (1C, pyrazoline-CH2), 66.2 (1C, pyraz-
oline-CH), 67.1 (1C, CH2 attached with C=O), 145.8 (1C,
C–NO2), 151.4 (1C, benzimidazole-C2), 168.4 (1C, C=O
present in NHCOCH3), 172.5 (1C, C=O attached with
pyrazoline ring); LCMS (m/z): 498.19 [(M)?, 82 %]; Anal.
Calcd. for C26H22N6O5: C-62.64, H-4.45, N-16.86; Found:
C-62.72, H-4.51, N-16.81 %.
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-(2-
bromophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-
oxoethoxy)phenyl)acetamide (5s)
Yield: 74 %; m.p.: 198 °C; IR (KBr) mmax/cm-1: 3392
(N–H stretching, benzimidazole), 3242 (N–H stretching,
–NHCOCH3), 3041 (C–H stretching, aromatic ring), 2862
(C–H stretching, –CH2), 1691 (CO stretching, –COCH2),
1662 (CO stretching, –NHCOCH3), 1584 (C=N stretching),
1524 (C=C stretching), 1462 (C–H bending, –CH2), 541
(C–Br stretching); 1H NMR (300 MHz, DMSO-d6, d,
ppm): 2.10 (s, 3H, NHCOCH3), 3.45 (dd, 1H,
J = 17.49 Hz, 3.15 Hz, C4–H pyrazoline), 3.94 (dd, 1H,
J = 17.49 Hz, 11.13 Hz, C4–H pyrazoline), 4.96 (s, 2H,
–CH2CO), 5.57 (s, 1H, NHCOCH3), 6.12 (dd, 1H,
J = 11.16, 3.15 Hz, C5–H pyrazoline), 6.99 (d, 2H,
J = 8.1 Hz, Ar–H), 7.14 (t, 1H, J = 7.4 Hz, Ar–H), 7.21
(d, 1H, J = 7.6 Hz, Ar–H), 7.26 (d, 2H, J = 8.1 Hz, Ar–
H), 7.35 (t, 1H, J = 7.5 Hz, Ar–H), 7.49 (d, 2H,
J = 7.9 Hz, Ar–H), 7.58 (d, 1H, J = 7.4 Hz, Ar–H), 7.66
(d, 2H, J = 8.4 Hz, Ar–H), 10.09 (s, 1H, –NH D2O exch.);
13C NMR (100 MHz, DMSO-d6, d, ppm): 24.0 (1C, CH3 in
NHCOCH3), 38.8 (1C, pyrazoline-CH2), 62.4 (1C, pyraz-
oline-CH), 67.0 (1C, CH2 attached with C=O), 121.8 (1C,
C–Br), 151.2 (1C, benzimidazole-C2), 168.2 (1C, C=O
present in NHCOCH3), 172.4 (1C, C=O attached with
pyrazoline ring); LCMS (m/z): 531.09 [(M)?, 79 %]; Anal.
Calcd. for C26H22BrN5O3: C-58.66, H-4.17, N-13.15;
Found: C-58.60, H-4.21, N-13.20 %.
N-(4-(2-(3-(1H-benzo[d]imidazol-2-yl)-5-(4-
bromophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-
oxoethoxy)phenyl)acetamide (5u)
Yield: 72 %; m.p.: 209 °C; IR (KBr) mmax/cm-1: 3394
(N–H stretching, benzimidazole), 3245 (N–H stretching,
–NHCOCH3), 3043 (C–H stretching, aromatic ring), 2864
(C–H stretching, –CH2), 1694 (CO stretching, –COCH2),
1665 (CO stretching, –NHCOCH3), 1586 (C=N stretching),
1524 (C=C stretching), 1463 (C–H bending, –CH2), 547
(C–Br stretching); 1H NMR (300 MHz, DMSO-d6, d,
ppm): 2.09 (s, 3H, NHCOCH3), 3.45 (dd, 1H,
J = 17.49 Hz, 3.15 Hz, C4–H pyrazoline), 3.95 (dd, 1H,
J = 17.50 Hz, 11.14 Hz, C4–H pyrazoline), 4.96 (s, 2H,
–CH2CO), 5.54 (s, 1H, NHCOCH3), 6.11 (dd, 1H,
J = 11.15, 3.15 Hz, C5–H pyrazoline), 6.98 (d, 2H,
J = 8.1 Hz, Ar–H), 7.14 (d, 2H, J = 7.7 Hz, Ar–H), 7.22
(d, 2H, J = 8.0 Hz, Ar–H), 7.51 (d, 2H, J = 7.9 Hz, Ar–
H), 7.67 (d, 2H, J = 8.3 Hz, Ar–H), 7.94 (d, 2H,
J = 7.6 Hz, Ar–H), 10.07 (s, 1H, –NH D2O exch.); 13C
NMR (100 MHz, DMSO-d6, d, ppm): 24.1 (1C, CH3 in
NHCOCH3), 39.5 (1C, pyrazoline-CH2), 66.3 (1C, pyraz-
oline-CH), 67.1 (1C, CH2 attached with C=O), 121.2 (1C,
123

Med Chem Res (2014) 23:1474–1487
1485
C–Br), 151.2 (1C, benzimidazole-C2), 168.3 (1C, C=O
present in NHCOCH3), 172.4 (1C, C=O attached with
pyrazoline ring); LCMS (m/z): 531.11 [(M)?, 81 %]; Anal.
Calcd. for C26H22BrN5O3: C-58.66, H-4.17, N-13.15;
Found: C-58.59, H-4.22, N-13.21 %.
antibacterial activity which showed 100, 100, 250, and
100 lg/mL MIC against E. coli, P. aeruginosa, S. aureus,
and S. pyogenes, respectively.
Antifungal bioassay
Antibacterial bioassay
The newly prepared compounds (5a–u) were screened for
their antifungal activity as primary screening in six sets
against C. albicans, A. niger, and A. clavatus at various
concentrations of 1,000, 500, and 250 lg/mL. The primary
active compounds were similarly diluted to obtain 200, 125,
100, 62.5, 50, 25, and 12.5 lg/mL concentrations for sec-
ondary screening to test in a second set of dilution against all
fungi. The fungal activity of each compound was compared
with griseofulvin as a standard drug, which showed 500, 100,
and 100 lg/mL MIC against C. albicans, A. niger, and
A. clavatus, respectively. For fungal growth, in the present
protocol, we have used Sabourauds dextrose broth at 28 °C
in aerobic condition for 48 h. DMSO and sterilized distilled
water were used as negative control while griseofulvin (1 U
strength) was used as positive control.
Antibacterial studies of newly synthesized compounds
(5a–u) were carried out against the representative panel
of Gram-positive [S. aureus (MTCC-96), S. pyogenes
(MTCC-442)] and Gram-negative [E. coli (MTCC-443),
P. aeruginosa (MTCC-1688)] bacteria. All MTCC cultures
were collected from the Institute of Microbial Technology,
Chandigarh. The activity of compounds was determined as
per the National Committee for Clinical Laboratory Stan-
dards (NCCLS) protocol using Mueller–Hinton Broth
(Becton–Dickinson, USA) (Desai et al., 2012d, e). Primary
screening was done first for antibacterial activity in six sets
against E. coli, S. aureus, P. aeruginosa, and S. pyogenes at
different concentrations of 1,000, 500, and 250 lg/mL. The
compounds found to be active in primary screening were
similarly diluted to obtain 200, 125, 100, 62.5, 50, 25, and
12.5 lg/mL concentrations for secondary screening to test
in a second set of dilution against all microorganisms.
Inoculum size for test strain was adjusted to 106 CFU/mL
(Colony Forming Unit per milliliter) by comparing the
turbidity (turbidimetric method). Mueller–Hinton Broth
was used as a nutrient medium to grow and dilute the
compound suspension for test organisms. 2 % DMSO was
used as a diluent/vehicle to obtain the desired concentration
of synthesized compounds and standard drugs to test upon
standard microbial strains. Synthesized compounds were
diluted to 1,000 lg/mL concentration as stock solution.
The control tube containing no antibiotic was immediately
subcultured (before inoculation) by spreading a loopful
evenly over quarter of a plate of medium suitable for the
growth of test organisms. The culture tubes were then
incubated for 24 h at 37 °C and the growth was monitored
visually and spectrophotometrically. 10 lg/mL suspen-
sions were further inoculated on an appropriate media and
growth was noted after 24 and 48 h. The lowest concen-
tration (highest dilution) required to arrest the growth of
bacteria was regarded as minimum inhibitory concentration
(MIC), i.e., the amount of growth from the control tube
before incubation (which represents the original inoculum)
was compared. Solvent had no influence on strain growth.
The result of this was greatly affected by the size of
inoculum. The test mixture contained 106 CFU/mL
organisms. DMSO and sterilized distilled water were used
as negative control while ampicillin antibiotic (1 U
strength) was used as positive control. Standard drug used
in the present study was ‘ampicillin’ for evaluating
The cytotoxic potential of compounds 5e–5j, 5p–5r, 5t,
and 5u was also determined in human cancer cell lines such
as A549, HL-60, and HepG2 according to protocols (Ske-
han et al., 1990). All the tested compounds did not show
significant cytotoxic activity but showed selectivity, in that
they possessed potent antibacterial and antifungal activity
without the cytotoxicity in mammalian cells (Ryu et al.,
2003).
Statistical analysis
Standard deviation was expressed in terms of SD. On the
basis of calculated values by using one-way ANOVA fol-
lowed by independent two sample t test, it was observed
that differences below 0.001 level were considered statis-
tically significant. Compounds (5a–u) were screened for
their antibacterial and antifungal activities in six sets
(n) against bacteria and fungi used in the present protocol.
Structure–activity relationship
The results of the antimicrobial screening of compounds
(5a–u) demonstrated that the substitution pattern of the
hybrid benzimidazole and pyrazoline derivatives was
carefully selected to impart different electronic environ-
ments to the molecules. As seen from activity data, anti-
bacterial activity was considerably affected by substitution
pattern on the phenyl ring and the incorporation of elec-
tron-withdrawing groups was responsible for enhancing the
activity against most of the test microorganisms. The role
of electron-withdrawing group in improving antimicrobial
activity is very well supported by previous studies (Sharma
123

1486
Med Chem Res (2014) 23:1474–1487
Acknowledgments The authors are thankful to the University
Grants Commission, New Delhi and Department of Science &
Technology, New Delhi for financial support under the NON-SAP
and DST-FIST programs, respectively.
et al., 2004, 2009). From antimicrobial activity data, some
analogs of this series were found to have even more
potency than the reference drugs while some of them have
comparable potency. Compounds 5f, 5i, 5q, and 5t with
electron-withdrawing substituents (F, Cl, NO2, and Br) in
the aromatic ring were most active against all test bacterial
strains, while compounds 5e, 5g, 5h, 5j, 5p, 5r, and 5u
were most potent against all the fungal strains. Moreover,
newly synthesized compounds had higher potency against
Gram-positive bacteria as compared to Gram-negative
bacterial strains. In addition, antibacterial activity showed
electron-withdrawing substituents at meta position which
displayed higher activity than substituents at ortho or para
positions. On the other hand, antifungal activity displayed
conflicting results wherein compounds having electron-
withdrawing substituents (Cl, F, NO2, and Br) present at
2nd or 4th positions revealed higher inhibition than com-
pounds having substitution at third position. Clearly, meta
position was a favorable site for high antibacterial, while
ortho or para positions had high antifungal activity indi-
cating that structural requirements are different for binding
of drug to bacterial targets.
References
Amnerkar ND, Bhusari KP (2010) Synthesis, anticonvulsant activity
and 3D-QSAR study of some prop-2-eneamido and 1-acetyl-
pyrazolin derivatives of aminobenzothiazole. Eur J Med Chem
45:149–159
Andreani A, Granaiola M, Leoni A, Morigi R, Ramballdi M (2001)
Synthesis and antitubercular activity of imidazo[2,1-b]thiazoles.
Eur J Med Chem 36:743–746
Bernatowicz AN, Lebska M, Orzeszko A, Kopanska K, Krzywinska
E, Muszynska G, Bretner M (2009) Synthesis of new analogs of
benzotriazole, benzimidazole and phthalimide potential inhibi-
tors of human protein kinase CK2. Bioorg Med Chem
17:1573–1578
Brzozwskim Z, Saczewski F, Gdaniec M (2000) Synthesis, structural
characterization and antitumor activity of novel 2,4-diamino-
1,3,5-triazine derivatives. Eur J Med Chem 35:1053–1064
Desai NC, Pandya DD, Joshi VV, Rajpara KM, Vaghani HV,
Satodiya HM (2012a) Synthesis, characterization and antimicro-
bial screening of hybrid molecules containing benzimidazole-
pyrazole and pyridine nucleus. Med Chem Res
21(12):4463–4472. doi:10.1007/s00044-012-9990-4
Desai NC, Joshi VV, Rajpara KM, Vaghani HV, Satodiya HM
(2012b) Facile synthesis of novel fluorine containing pyrazole
based thiazole derivatives and evaluation of antimicrobial
activity. J Fluor Chem 142:67–78
Desai NC, Rajpara KM, Joshi VV (2012c) Synthesis and character-
ization of some new quinoline based derivatives endowed with
broad spectrum antimicrobial potency. Bioorg Med Chem Lett
15:6871–6875. doi:10.1016/j.bmcl.2012.09.039
Desai NC, Rajpara KM, Joshi VV (2012d) Synthesis of new
quinoline-2-pyrazoline based thiazolinone derivatives as poten-
tial antimicrobial agents. Med Chem Res 22(8):3663–3674.
doi:10.1007/s00044-012-0377-3
Desai NC, Dodiya AM, Shihory NR (2012e) A search of novel
antimicrobial based on benzimidazole and 2-pyridone heterocy-
cles. Med Chem Res 21:2579–2586
Gursoy A, Demirayak S, Capen G, Erol K, Vural K (2000) Synthesis
and preliminary evaluation of new 5-pyrazolinone derivatives as
analgesic agents. Eur J Med Chem 35:359–364
Kazimierczuk Z, Upcroft JA, Upcroft P, Gorska A, Starosciak B,
Laudy A (2002) Synthesis, antiprotozoal and antibacterial
activity of nitro-substituted benzimidazole derivatives. Acta
Biochim Pol 49:185–195
Kini SG, Bhat AR, Bryant B, Williamson JS, Dayan FE (2009)
Synthesis, antitubercular activity and docking study of novel
cyclic azole substituted diphenyl ether derivatives. Eur J Med
Chem 44:492–500
Korgaokar SS, Patil PH, Shah MJ, Parekh HH (1996) Studies on
pyrazolines: preparation and antimicrobial activity of 3-(p-
Conclusion
To conclude, our attempts at exploring benzimidazole-
based pyrazoline derivatives had unexpectedly led to the
identification of a novel chemotype with substantial anti-
microbial activity. Among the newly synthesized com-
pounds (5a–u), analogs 5f (3-Cl), 5i (3-F), 5q (3-NO2), and
5t (3-Br) showed highest inhibition against nearly all the
tested bacteria, while analogs 5e (2-Cl), 5g (4-Cl), 5h
(2-F), 5j (4-F), 5p (2-NO2), 5r (4-NO2), and 5u (4-Br) were
most active against all the fungal strains. Results of anti-
microbial activity clearly demonstrated that the presence of
electron-withdrawing groups/atoms on phenyl ring was
essential for enhancing antimicrobial activity. On the basis
of structure activity relationship, electron-withdrawing
substituents (F, Cl, NO2, and Br) at meta position were
beneficial for antibacterial activity and substituents at 2nd
or 4th positions were crucial for antifungal activity. From
the activity data, compound 5i (3-F) showed highest anti-
bacterial inhibition whereas compound 5 h (2-F) displayed
maximum antifungal inhibition. In addition, the promising
activity of these title compounds could also be attributed to
the incorporation of heterocyclic scaffolds viz., benzimid-
azole and pyrazoline. Thus, suggesting that the compounds
from the present series with electron-withdrawing fluoro,
chloro, nitro, and bromo groups on phenyl ring can serve as
important gateways for the design and development of new
antimicrobial agents with potent activity and minimal
toxicity.
chlorophenylsulphonamidophenyl)-5-aryl-1H/acetyl
lines. Ind J Pharm Sci 58(6):222–225
pyrazo-
Kus C, Sozudonmez F, Altanlar N (2009) Synthesis and antimicrobial
activity of some novel 2-[4-(substituted piperazin-/piperidin-1-
ylcarbonyl)phenyl]-1H-benzimidazole derivatives. Arch Pharm
Chem Life Sci 342:54–60
Mader M, Dios AD, Shih C, Bonjouklian R, Li T, White W, Uralde
BL, Sanchez-Martinez C, Prado MD, Jaramillo C, Diego E,
Cabrejas LMM, Dominguez C, Montero C, Shepherd T, Dally R,
123

Med Chem Res (2014) 23:1474–1487
1487
Toth JE, Chatterjee A, Pleite S, Urgoiti JB, Perez L, Barberis M,
Lorite MJ, Jambrina E, Nevill CR Jr, Lee PA, Schultz RC,
Wolos JA, Li LC, Campbell RM, Anderson BD (2008)
Imidazolyl benzimidazoles and imidazo[4,5-b]pyridines as
potent p38a MAP kinase inhibitors with excellent in vivo
antiinflammatory properties. Bioorg Med Chem Lett 18:179–183
Nakano H, Inoue T, Kawasaki N, Miyataka H, Matsumoto H, Taguchi
T, Inagaki N, Nagai H, Satoh T (2000) Synthesis and biological
activities of novel antiallergic agents with 5-lipoxygenase
inhibiting action. Bioorg Med Chem 8:373–380
Nauduri D, Reddy GBS (1998) Antibacetrials and antimycotics: Part
1: synthesis and activity of 2-pyrazoline derivatives. Chem
Pharm Bull (Tokyo) 46:1254–1260
Prasad YR, Rao AL, Prasoona L, Murali K, Kumar PR (2005)
Synthesis and antidepressant activity of some 1,3,5-triphenyl-2-
pyrazolines and 3-(200-hydroxy naphthalen-100-yl)-1,5-diphenyl-
2-pyrazolines. Bioorg Med Chem Lett 15:5030
Ryu CK, Choi KU, Shim J, You H, Choi IK, Chae MJ (2003)
Synthesis and antifungal activity of 6-arylthio-/6-arylamino-4,7-
dioxobenzothiazoles. Bioorg Med Chem 11:4003–4008
Scott LJ, Dunn CJ, Mallarkey G, Sharpe M (2002) Esomeprazole: a
review of its use in the management of acid-related disorders.
Drugs 62:1503–1538
Shaharyar M, Abdullah MM, Bakht MA, Majeed J (2010) Pyrazoline
bearing benzimidazoles: search for anticancer agent. Eur J Med
Chem 45:114–119
Sharma D, Narasimhan B, Kumar P, Judge V, Narang R, De Clercq E,
Balzarini J (2009) Synthesis, antimicrobial and antiviral evalu-
ation of substituted imidazole derivatives. Eur J Med Chem
44:2347–2353
Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D,
Warren TW, Bokesch H, Kenney S, Boyd MR (1990) New
colorimetric cytotoxicity assay for anticancer-drug screening.
J Natl Cancer Inst 82:1107–1112
Sook JT, Kim KS, An SJ, Cho KH, Lee S, Lee WS (2004) Novel 3,5-
diaryl pyrazolines as human acyl-CoA: cholesterol acyl trans-
ferase inhibitors. Bioorg Med Chem Lett 14:2715–2717
Starcevic K, Kralj M, Ester K, Sabol I, Grace M, Pavelic K, Zambola
GK (2007) Synthesis, antiviral and antitumor activity of
2-substituted-5-amidino-benzimidazoles. Bioorg Med Chem
15:4419–4426
Thimmegowda NR, Swami SN, Kumar CSA, Kumar YCS, Chandr-
appa S, Yip GW, Rangappa KS (2008) Synthesis, characteriza-
tion and evaluation of benzimidazole derivative and its
precursors as inhibitors of MDA-MB-231 human breast cancer
cell proliferation. Bioorg Med Chem Lett 18:432–435
Udupi RH, Kushnoor AS, Bhat AR (1998) Synthesis and biological
evaluation of certain pyrazoline derivative of 2-(6-methoxy-
naphthyl)-propionic acid. Ind J Heterocycl Chem 8:63–66
Vazquez GN, Cedillo R, Campos AH, Yepez L, Luis FH, Valdez J,
´
´
Morales R, Cortes R, Hernandez M, Castillo R (2001) Synthesis
and antiparasitic activity of 2-(trifluoromethyl)benzimidazole
derivatives. Bioorg Med Chem Lett 11:187–190
Sharma P, Rane N, Gurram VK (2004) Synthesis and QSAR studies
of pyrimido[4,5-d]pyrimidine-2,5-dione derivatives as potential
antimicrobial agents. Bioorg Med Chem Lett 14:4185–4190
123