Synthesis, antimicrobial and cytotoxic activity of 2-azetidinone derivatives of pyridyl benzimidazoles

Article abstract of DOI:10.1007/s00044-013-0775-1

In the present study, a series of novel 6-(1H-benzo[d]imidazol-2-yl)-2-(3- chloro-2-oxo-4-phenylazetidin-1-yl)-4-(aryl)nicotinonitriles 6a-o were efficiently synthesized and evaluated for their in vitro antibacterial activity against Gram-positive (Staphylococcus aureus and Streptococcus pyogenes), Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria and fungal (Candida albicans, Aspergillus niger and Aspergillus clavatus) strains. The results of antimicrobial study revealed that compounds 6b, 6c, 6d, 6h and 6i exhibited substantial antibacterial activity while compounds 6c and 6h emerged as the most potent antifungal agents compared to the standard drugs chloramphenicol and ketoconazole, respectively. From the standpoint of SAR studies, it was observed that the presence of electron-withdrawing groups remarkably enhances the antibacterial activity of newly synthesized compounds. Further, the results of preliminary MTT cytotoxicity studies on HeLa cells suggested that potent antimicrobial activity of 6b, 6c, 6d, 6h and 6i is accompanied by low level of cytotoxic concentrations. All the newly synthesized analogues were characterized by IR, ¹H NMR, ¹³C NMR and mass spectral data. Springer Science+Business Media 2013.

Full text of DOI:10.1007/s00044-013-0775-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1725–1741
DOI 10.1007/s00044-013-0775-1
ORIGINAL RESEARCH
Synthesis, antimicrobial and cytotoxic activity of 2-azetidinone
derivatives of pyridyl benzimidazoles
•
•
•
N. C. Desai D. D. Pandya G. M. Kotadiya
Priyanka Desai
Received: 5 June 2013 / Accepted: 28 August 2013 / Published online: 12 September 2013
Ó Springer Science+Business Media New York 2013
Abstract In the present study, a series of novel 6-(1H-
benzo[d]imidazol-2-yl)-2-(3-chloro-2-oxo-4-phenylazeti-
din-1-yl)-4-(aryl)nicotinonitriles 6a–o were efficiently
synthesized and evaluated for their in vitro antibacterial
activity against Gram-positive (Staphylococcus aureus
and Streptococcus pyogenes), Gram-negative (Esche-
richia coli and Pseudomonas aeruginosa) bacteria and
fungal (Candida albicans, Aspergillus niger and Asper-
gillus clavatus) strains. The results of antimicrobial study
revealed that compounds 6b, 6c, 6d, 6h and 6i exhibited
substantial antibacterial activity while compounds 6c and
6h emerged as the most potent antifungal agents com-
pared to the standard drugs chloramphenicol and keto-
conazole, respectively. From the standpoint of SAR
studies, it was observed that the presence of electron-
withdrawing groups remarkably enhances the antibacte-
rial activity of newly synthesized compounds. Further,
the results of preliminary MTT cytotoxicity studies on
HeLa cells suggested that potent antimicrobial activity of
6b, 6c, 6d, 6h and 6i is accompanied by low level of
cytotoxic concentrations. All the newly synthesized
Introduction
The treatment of bacterial infections still remains an
important and challenging therapeutic problem because of
factors that include emerging infectious diseases and the
increasing number of multidrug-resistant microbial patho-
gens. Since the design of new compounds to deal with
resistant bacteria and fungi has become one of the most
important areas of antibacterial and antifungal research
today, resistance of pathogenic bacteria and fungi toward
available antimicrobial drug is rapidly becoming a major
problem worldwide. So the discovery of novel and potent
antibacterial as well as antifungal agent is more demanding
and challenging for chemists and pharmacists nowadays
(Grare et al., 2007).
The benzimidazole nucleuses has been an important
pharmacophore and privileged structure in medicinal
chemistry and are remarkably effective compounds both
with respect to their inhibitory activity and their favourable
selectivity ratio (Ansari and Lal, 2009; Kazimierczuk et al.,
2002). The azole group of heterocyclic compounds pos-
sessed 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). Benzimidazoles
are considered as a promising class of bioactive heterocy-
clic compounds encompassing a diverse range of biological
activities such as antioxidant (Kus et al., 2008), antiviral
(Chien et al., 2004), antihypertensive (Kohara et al., 1996),
analgesic (Elmer et al., 1995), anti-allergic (Richards et al.,
2004), anticoagulant (Mederski et al., 2004), anti-inflam-
matory (Mader et al., 2008), antimicrobial (Arjmand et al.,
2005), antihelminthic (Haugwitz et al., 1982) and anti-
parasitic (Alp et al., 2009).
1
analogues were characterized by IR, H NMR, 13C NMR
and mass spectral data.
Keywords 2-Azetidinone ꢀ Cyanopyridine ꢀ
Benzimidazole ꢀ Antimicrobial activity ꢀ Cytotoxicity
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
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Med Chem Res (2014) 23:1725–1741
The synthesis of the pyridine-containing heterocyclic
target of contemporary organic synthesis (Buttero et al.,
2003). Recently, 2-azetidinones are associated with various
other biological activities such as antifungal (Halve et al.,
2007), antitubercular (Kagthara et al., 2000), antitumor
(Chen et al., 2008), anti-inflammatory (Gurupadayya et al.,
2008), anticonvulsant (Rajasekaran and Murugesan, 2005)
and analgesic (Rawls et al., 2010). Structural relevance of
title compounds (6a–o) with some commercially available
drugs containing 2-azetidinone, benzimidazole and pyridine
nucleus are presented in Fig. 1.
systems occupies an important place in the realm of syn-
thetic organic chemistry, due to their therapeutic and
pharmacological properties (Henry, 2004; Bagley et al.,
2005; Gilchrist, 2001). In many enzymes of living organ-
isms, it is the prosthetic pyridine nucleotide (NADP) that is
involved in various oxidation–reduction processes. Other
evidence of potent activity of pyridine in biological sys-
tems is its presence in important vitamins such as niacin
and pyridoxine (vitamin B6). The pyridine nucleus is an
integral part of anticancer and anti-inflammatory agents
(Son et al., 2008; Amr and Abdulla, 2006) too. In addition,
cyanopyridone and cyanopyridine derivatives have shown
to possess promising antimicrobial (Hammam et al., 2000)
and anticancer activity (Abo-Ghalia et al., 2003). The
derivatives which contain pyridine nucleus such as strep-
tonigrin, streptonigrone and lavendamycin are reported as
anticancer drugs and cerivastatin is reported as HMG-CoA
enzyme inhibitor (Bringmann et al., 2004). Moreover,
substituted pyridines are reported as leukotriene B-4
antagonists (Taylor et al., 1991).
Prompted by the above-mentioned observations and in
continuation of our search for new antimicrobial agents
(Desai et al., 2012a, b, 2013), we herein report the syn-
thesis of some novel 2-azetidinone derivatives that com-
prises the above-mentioned moieties in single molecular
framework in order to investigate their in vitro antimicro-
bial activity. In addition, cytotoxicity studies were also
conducted in HeLa cell lines to evaluate the ability of these
compounds to inhibit the cell growth. Keeping this in mind,
we designed and synthesized 6-(1H-benzo[d]imidazol-2-
yl)-2-(3-chloro-2-oxo-4-phenylazetidin-1-yl)-4-(aryl)nic-
otinonitriles 6a–o with fascinating structural features.
On the other side, 2-azetidinones commonly known as
b-lactams are still the most prescribed chemotherapeutic
agents used in medicine to treat bacterial infections and
microbial diseases (Georg, 1993). Further, 2-azetidinone
molecules operate by forming a covalent adduct with
membrane-bound bacterial transpeptidases, which are also
known as penicillin-binding proteins (PBPs), involved in the
biosynthesis of cell walls (Knowles, 1985; Walsh, 2000). On
the basis of this mechanism, inhibitors prevent the con-
struction of cell wall and eventually lead to cell lysis and
death. Moreover, due to their b-lactamase inhibitory action,
2-azetidinone-based heterocycles represent an attractive
Results and discussion
Chemistry
The synthetic route for the preparation of 6-(1H-benzo[d]imi-
dazol-2-yl)-2-(3-chloro-2-oxo-4-phenylazetidin-1-yl)-4-(aryl)
nicotinonitriles 6a–o is summarized in Scheme 1. The chal-
cone derivatives 1-(1H-benzo[d]imidazol-2-yl)-3-(aryl)prop-
2-en-1-ones 3a–o were used as precursors for the synthesis of
Fig. 1 Structural relevance of
title compounds 6a–o with some
commercially available drugs
containing 2-azetidinone,
benzimidazole and pyridine
nucleus
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Med Chem Res (2014) 23:1725–1741
1727
Scheme 1 Synthetic route for the preparation of title compounds 6a–o.
Reagents and conditions: (a) EtOH, KOH, stirring, 10 h; (b) Malono-
nitrile, ammonium acetate, EtOH, reflux, 10 h; (c) Benzaldehyde, anhy.
ZnCl2, 1,4-dioxane, reflux, 6 h; (d) ClCOCH2Cl, Et3N, 1,4-dioxane,
reflux, 6 h
title compounds 6a–o and their synthesis was achieved using
previously published method (Shaharyar et al., 2010). The
cyclocondensation of chalcone derivatives 3a–o with malon-
onitrile in the presence of ammonium acetate afforded the
corresponding pyridyl benzimidazole derivatives 4a–o, which
on further reaction with benzaldehyde in the presence of
anhydrous zinc chloride resulted in the formation of interme-
diate compounds 5a–o. IR spectra of compounds 4a-o showed
the characteristic –C:N and –C–NH2 stretching absorption
bands in the region of 2,212–2,222 cm-1 and
3,468–3,480 cm-1, respectively. Whilethe 1H NMR spectraof
4a–o displayed characteristic singlets for a methine proton of
pyridine ring around at d8.18–8.26 ppm, two protons of amino
group showed around at d 8.78–8.84 ppm and a secondary
amine proton of benzimidazole ring displayed around at
d 10.16–10.24 ppm. Furthermore, 13C NMR spectra of 4a–o
demonstrated characteristic signals around at d 110.8–111.5,
113.5–115.6 and 161.6–162.4 ppm corresponding to methine,
C–CN and C–NH2 carbon, respectively. For compounds 5a–o,
IR spectra proved useful in tracing the disappearance of the
–C:N stretching absorption of the parent compounds 4a–o.
electrocyclisation reaction with chloroacetyl chloride and tri-
ethylamine in 1,4-dioxane which afforded the target com-
pounds 6-(1H-benzo[d]imidazol-2-yl)-2-(3-chloro-2-oxo-4-
phenylazetidin-1-yl)-4-(aryl)nicotinonitriles 6a–o. IR spectra
of the title compounds 6a–o showed strong absorption band in
the region of 1,682–1,692 cm-1 due to the C=O group of
2-azetidinone. 1H NMR spectra of compounds 6a–o showed a
doublet at around d 5.04–5.12 and 5.44–5.51 ppm equivalent
to one proton due to N–CH (C4–H) and CH–Cl (C3–H) of
2-azetidinone ring, respectively. Further, 13C NMR spectra
displayed signals in the range of d 162.0–162.4 and
62.0–62.4 ppm assignable to [C=O and C–Cl carbon of
2-azetidinone ring, respectively. In addition, mass spectra for
the title compounds 6a–o were in full agreement with their
proposed structures.
Antibacterial activity
All the newly synthesized compounds 6a–o were evaluated
for their in vitro antibacterial activity against Staphylococcus
aureus and Streptococcus pyogenes (Gram-positive) and
Escherichia coli and Pseudomonas aeruginosa (Gram-neg-
ative) bacteria by conventional broth-dilution method
(Hannan, 2000) using chloramphenicol as control drug. The
results of the antibacterial studies are presented in Table 1.
The obtained antimicrobial activity of tested compounds
could be correlated to structural variations. Intermediates
4a–o showed poor antimicrobial activity against all tested
bacterial and fungal strains, amongst which compounds 4c,
1
While, H NMR spectra revealed the appearance of singlet
peak in the range of d 8.60–8.66 ppm corresponding to one
proton of –CH=N– group (Schiff base). 13C NMR confirmed
the proposed structures 5a–o with the appearance of a signal
aroundat d158.4–158.9 ppm due to alkenecarbon of –CH=N–
group (Schiff base). The mass spectra for the compounds 4a–o
and 5a–o were consistentwith theproposed structures. Further,
initially formed Schiff bases 5a–o underwent an
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Med Chem Res (2014) 23:1725–1741
Table 1 Antimicrobial
screening results of compounds
6a–o
Entry
R
Minimum inhibitory concentration (MIC) (lg/mL)
Gram-positive bacteria Gram-negative bacteria
Fungi
Ca
Sa
Sp
Ec
Pa
An
250
Ac
6a
–H
250
100
25
500
100
50
250
100
50
500
500
100
50
500
100
50
6b
-2-Cl
250
25
100
25
6c
-3-Cl
6d
-4-Cl
12.5
25
12.5
12.5
100
500
250
500
50
100
500
500
500
50
100
500
250
250
50
6e
-2-OH
-3-OH
-4-OH
-3-NO2
-4-NO2
-2-CH3
-3-CH3
-4-CH3
-3-OCH3
-4-OCH3
-2-OH-4-Cl
500
500
250
25
500
500
250
50
500
500
250
50
500
500
250
50
6f
6g
6h
6i
12.5
25
25
12.5
100
500
500
500
500
1000
250
–
100
1000
250
500
500
500
250
–
100
500
500
500
250
500
250
–
6j
500
250
250
500
500
100
50
500
250
500
500
250
250
50
250
500
100
250
100
500
50
1000
500
500
500
250
200
50
Sa: Staphylococcus aureus,
Sp: Staphylococcus pyogenes,
Ec: Escherichia coli,
Pa: Pseudomonas aeruginosa,
Ca: Candida albicans,
An: Aspergillus niger,
6k
6l
6m
6n
6o
Ac: Aspergillus clavatus
a
Chloramphenicol
Standarda
Standardb
–
–
–
–
50
50
50
b
Ketoconazole
4d, 4h and 4i showed poor activity (MIC C 1,000 lg/mL
against S. aureus, P. aeruginosa and S. pyogenes) and
compounds 4c and 4h displayed very poor antifungal activity
(MIC C 1,000 lg/mL against all the fungal strains). Pre-
cursors 4a–o reacted with benzaldehyde to generate key
scaffolds 5a–o. Further, these intermediates were tested
against different bacterial and fungal strains. Intermediate
derivatives 5b, 5c, 5d, 5h and 5i displayed mild activity at
MIC = 1,000 lg/mL against P. aeruginosa and S. pyoge-
nes, while intermediates 5c and 5h exhibited poor activity
against all the fungal strains. Now, Schiff bases 5a–o were
treated with chloroacetyl chloride to afford targeted com-
pounds 6a–o having 2-azetidinone motif, which were found
to have broad-spectrum antimicrobial activity. Therefore, it
was observed that formation of 2-azetidinone nucleus was
responsible for enhancing antimicrobial potency. It was
observed that compounds 6b (2-Cl), 6c (3-Cl), 6d (4-Cl), 6h
(3-NO2) and 6i (4-NO2)were most active compounds. On the
basis of antibacterial evaluation, compounds 6b (2-Cl), 6l
(4-CH3) and 6n (4-OCH3) showed good activity at
MIC = 100 lg/mL, whereas compounds 6c (3-Cl) and 6h
(3-NO2) were found to show very good activity against
E. coli at MIC = 50 lg/mL. When chloro and nitro groups
were migrated from 3rd to 4th position i.e. 6d (4-Cl) and 6i
(4-NO2), activity increased and compound 6i (4-NO2)
displayed excellent activity at MIC = 25 lg/mL,
while compound 6d (4-Cl) observed the highest inhibition at
MIC = 12.5 lg/mL against E. coli as compared to
standard chloramphenicol (MIC = 50 lg/mL). Compound
6h (3-NO2) possessed very good activity at MIC = 50
lg/mL whereas compound 6c (3-Cl) possessed excellent
activity against P. aeruginosa at MIC = 25 lg/mL. When
we replaced hydrogen of phenyl ring in 6a by chloro and
nitro groups at 4th position we got compounds 6d (4-Cl) and
6i (4-NO2) which underwent enhancement in activity and
exhibited the highest inhibition against P. aeruginosa at
MIC = 12.5 lg/mL as compared to chloramphenicol
(MIC = 50 lg/mL). Compounds 6b (2-Cl) and 6o (2-OH-4-
Cl) were found to exhibit activity at MIC = 100 lg/mL,
while compounds 6c (3-Cl) and 6h (3-NO2) showed excel-
lent activity at MIC = 25 lg/mL. In addition, the above-
mentioned two groups were migrated from 3rd to 4th posi-
tion i.e. 6d (4-Cl) and 6i (4-NO2), enhanced activity and
showed the highest inhibition at MIC = 12.5 lg/mL against
S. aureus as compared to chloramphenicol (MIC = 50
lg/mL). Compound 6b (2-Cl) showed good activity, while
compounds 6c (3-Cl) and 6h (3-NO2) observed very good
activity (MIC = 50 lg/mL). Moreover, Compounds 6d
(4-Cl) and 6i (4-NO2) exhibited the highest inhibition at
MIC = 25 lg/mL against S. pyogenes as compared to
standard drug (MIC = 50 lg/mL).
Antifungal activity
All the newly synthesized compounds 6a–o were also
evaluated for their in vitro antifungal activity against
Candida albicans, Aspergillus niger and Aspergillus clav-
atus ketoconazole as standard drug. The results of the
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Med Chem Res (2014) 23:1725–1741
1729
antifungal studies are presented in Table 1. Antifungal
activity showed that compounds 6b (2-Cl), 6d (4-Cl) and 6i
(4-NO2) were found to have good activity at
MIC = 100 lg/mL against all the three fungal strains;
when we replaced chloro and nitro groups from 2nd or 4th
to 3rd position i.e. 6c (3-Cl) and 6h (3-NO2), activity
increased and compounds exhibited the highest inhibition
against both C. albicans and A. clavatus at MIC = 50
lg/mL as compared to standard ketoconazole (MIC = 50
lg/mL). Moreover, in case of A. niger, compound 6h
(3-NO2) revealed very good activity at MIC = 50 lg/mL
and compound 6c (3-Cl) showed excellent activity at
MIC = 25 lg/mL as compared to standard ketoconazole
(MIC = 50 lg/mL).
dryness under reduced pressure and then poured into
250 mL ice-cold water. The product obtained was isolated
and crystallized from benzene.
2-Amino-6-(1H-benzo[d]imidazol-2-yl)-4-
phenylnicotinonitrile (4a)
Yield: 79 %; m.p.: 242 °C; IR (mmax, cm-1, KBr): 3,470
(NH2), 3,312 (–NH) 3,058 (C–H, aromatic), 2,220 (CN),
1
1,588 (C=N), 1,518 (C=C); H NMR (300 MHz, DMSO-
d6, d, ppm): 7.27 (d, 2H, J = 8.4 Hz, Ar–H), 7.37 (t, 1H,
J = 8.1 Hz, Ar–H), 7.42 (t, 2H, J = 7.8 Hz, Ar–H), 7.54
(d, 2H, J = 7.8 Hz, Ar–H), 7.68 (d, 2H, J = 8.4 Hz,
Ar–H), 8.22 (s, 1H, C5–H pyridine), 8.84 (s, 2H, NH2 D2O
exch.), 10.18 (s, 1H, –NH D2O exch.); 13C NMR
(100 MHz, DMSO-d6, d, ppm): 161.9, 158.9, 154.8, 154.2,
141.8, 139.2, 129.9, 129.4, 127.4, 123.2, 115.1, 113.9,
111.2, 85.7; LCMS (M/Z): 311.11 [M?]; Anal. Calcd. for
C19H13N5: C, 73.30; H, 4.21; N, 22.49; Found: C, 73.31;
H, 4.22; N, 22.48 %.
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 electrothermal
melting point apparatus and were reported uncorrected.
TLC on silica gel plates (Merck, 60, F254) was used for
purity checking and reaction monitoring. Column chro-
matography 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. Elemental 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 in KBr. 1H NMR spectra were recorded
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 standard. Mass
spectra were scanned on a Shimadzu LC–MS 2010 spec-
trometer. Anhydrous reactions were carried out in oven-
dried glassware in nitrogen atmosphere.
2-Amino-6-(1H-benzo[d]imidazol-2-yl)-4-(2-
chlorophenyl)nicotinonitrile (4b)
Yield: 76 %; m.p.: 222 °C; IR (mmax, cm-1, KBr): 3,472
(NH2), 3,318 (–NH) 3,060 (C–H, aromatic), 2,218 (CN),
1,590 (C=N), 1,512 (C=C), 743 (C–Cl); 1H NMR
(300 MHz, DMSO-d6, d, ppm): 7.22 (d, 2H, J = 8.3 Hz,
Ar–H), 7.32 (t, 1H, J = 7.8 Hz, Ar–H), 7.44 (t, 1H,
J = 7.8 Hz, Ar–H), 7.56 (d, 1H, J = 8.0 Hz, Ar–H), 7.67
(d, 2H, J = 8.2 Hz, Ar–H), 7.80 (d, 1H, J = 8.0 Hz,
Ar–H), 8.24 (s, 1H, C5–H pyridine), 8.82 (s, 2H, NH2 D2O
exch.), 10.16 (s, 1H, –NH D2O exch.); 13C NMR
(100 MHz, DMSO-d6, d, ppm): 161.8, 158.9, 156.7, 154.7,
141.9, 138.7, 132.2, 130.6, 129.4, 128.8, 127.4, 123.1,
115.2, 113.7, 111.2, 85.7; LCMS (M/Z): 345.08 [M?];
Anal. Calcd. for C19H12ClN5: C, 66.00; H, 3.50; N, 20.25;
Found: C, 66.04; H, 3.51; N, 20.27 %.
Synthesis of 1-(1H-benzo[d]imidazol-2-yl)-3-
(aryl)prop-2-en-1-ones (3a–o)
2-Amino-6-(1H-benzo[d]imidazol-2-yl)-4-(3-
chlorophenyl)nicotinonitrile (4c)
Synthesis of 1-(1H-benzo[d]imidazol-2-yl)-3-(aryl)prop-2-
en-1-ones 3a–o was achieved by reported method (Sha-
haryar et al., 2010).
Yield: 80 %; m.p.: 238 °C; IR (mmax, cm-1, KBr): 3,470
(NH2), 3,320 (–NH) 3,060 (C–H, aromatic), 2,220 (CN),
1,586 (C=N), 1,510 (C=C), 741 (C–Cl); 1H NMR
(300 MHz, DMSO-d6, d, ppm): 7.26 (d, 2H, J = 8.1 Hz,
Ar–H), 7.37 (d, 1H, J = 7.8 Hz, Ar–H), 7.44 (t, 1H,
J = 7.9 Hz, Ar–H), 7.55 (d, 1H, J = 8.0 Hz, Ar–H), 7.70
(d, 2H, J = 8.2 Hz, Ar–H), 8.06 (s, 1H, Ar–H), 8.26 (s, 1H,
C5–H pyridine), 8.80 (s, 2H, NH2 D2O exch.), 10.20 (s, 1H,
–NH D2O exch.); 13C NMR (100 MHz, DMSO-d6, d,
ppm): 161.9, 158.6, 154.9, 154.3, 141.7, 139.5, 134.8,
129.9, 129.3, 127.1, 123.0, 115.1, 113.8, 111.4, 85.7;
General synthetic procedure for the preparation
of 2-amino-6-(1H-benzo[d]imidazol-2-yl)-4-
(aryl)nicotinonitriles (4a–o)
A mixture of an equimolar amount of compounds 3a–o and
malononitrile (0.01 mol) was refluxed in ethanol (99 %)
containing ammonium acetate (0.01 mol) for 10 h. The
reaction mixture was then concentrated by evaporate to
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Med Chem Res (2014) 23:1725–1741
LCMS (M/Z): 345.08 [M?]; Anal. Calcd. for C19H12ClN5:
C, 66.00; H, 3.50; N, 20.25; Found: C, 66.02; H, 3.50;
N, 20.26 %.
115.2, 113.7, 111.2, 85.8; LCMS (M/Z): 327.12 [M?]; Anal.
Calcd. for C19H13N5O: C, 69.71; H, 4.00; N, 21.39; Found:
C, 69.70; H, 4.03; N, 21.40 %.
2-Amino-6-(1H-benzo[d]imidazol-2-yl)-4-(4-
2-Amino-6-(1H-benzo[d]imidazol-2-yl)-4-(4-
chlorophenyl)nicotinonitrile (4d)
hydroxyphenyl)nicotinonitrile (4g)
Yield: 75 %; m.p.: 210–212 °C; IR (mmax, cm-1, KBr):
3,473 (NH2), 3,315 (–NH), 3,056 (C–H, aromatic), 2,218
Yield: 72 %; m.p.: 224 °C; IR (mmax, cm-1, KBr): 3,471
(NH2), 3,415 (OH), 3,318 (–NH), 3,060 (C–H, aromatic),
2,218 (CN), 1,586 (C=N), 1,514 (C=C); 1H NMR
(300 MHz, DMSO-d6, d, ppm): 6.88 (d, 2H, J = 7.4 Hz,
Ar–H), 7.24 (d, 2H, J = 8.0 Hz, Ar–H), 7.66 (d, 2H,
J = 8.0 Hz, Ar–H), 7.78 (d, 2H, J = 7.6 Hz, Ar–H), 8.24
(s, 1H, C5–H pyridine), 8.82 (s, 2H, NH2 D2O exch.), 9.22
(s, 1H, OH D2O exch.), 10.20 (s, 1H, –NH D2O exch.);
13C NMR (100 MHz, DMSO-d6, d, ppm): 161.8, 159.0,
158.6, 154.8, 154.1, 141.7, 131.4, 130.1, 123.1, 116.5,
115.0, 113.6, 111.0, 85.8; LCMS (M/Z): 327.12 [M?];
Anal. Calcd. for C19H13N5O: C, 69.71; H, 4.00; N, 21.39;
Found: C, 69.72; H, 4.01; N, 21.40 %.
1
(CN), 1,590 (C=N), 1,511 (C=C), 744 (C–Cl); H NMR
(300 MHz, DMSO-d6, d, ppm): 7.27 (d, 2H, J = 8.0 Hz,
Ar–H), 7.57 (d, 2H, J = 7.9 Hz, Ar–H), 7.69 (d, 2H,
J = 8.1 Hz, Ar–H), 7.84 (d, 2H, J = 7.8 Hz, Ar–H), 8.24
(s, 1H, C5–H pyridine), 8.82 (s, 2H, NH2 D2O exch.), 10.18
(s, 1H, –NH D2O exch.); 13C NMR (100 MHz, DMSO-d6,
d, ppm): 161.7, 158.6, 154.8, 154.1, 141.8, 137.1, 134.8,
129.7, 128.8, 123.1, 115.2, 113.7, 111.0, 85.7; LCMS
(M/Z): 345.08 [M?]; Anal. Calcd. for C19H12ClN5: C, 66.00;
H, 3.50; N, 20.25; Found: C, 66.04; H, 3.52; N, 20.24 %.
2-Amino-6-(1H-benzo[d]imidazol-2-yl)-4-(2-
hydroxyphenyl)nicotinonitrile (4e)
2-Amino-6-(1H-benzo[d]imidazol-2-yl)-4-(3-
nitrophenyl)nicotinonitrile (4h)
Yield: 74 %; m.p.: 232–234 °C; IR (mmax, cm-1, KBr):
3,468 (NH2), 3,416 (OH), 3,314 (–NH), 3,058 (C–H, aro-
Yield: 82 %; m.p.: 218 °C; IR (mmax, cm-1, KBr): 3,476
(NH2), 3,320 (–NH), 3,061 (C–H, aromatic), 2,218 (CN),
1,586 (C=N), 1,514 (C=C), 1,486 (NO2); 1H NMR
(300 MHz, DMSO-d6, d, ppm): 7.20 (d, 2H, J = 8.0 Hz,
Ar–H), 7.65 (d, 2H, J = 8.2 Hz, Ar–H), 7.76 (t, 1H,
J = 7.8 Hz, Ar–H), 7.86 (d, 1H, J = 7.8 Hz, Ar–H), 7.98
(d, 1H, J = 7.8 Hz, Ar–H), 8.10 (s, 1H, Ar–H), 8.22 (s, 1H,
C5–H pyridine), 8.84 (s, 2H, NH2 D2O exch.), 10.22 (s, 1H,
–NH D2O exch.); 13C NMR (100 MHz, DMSO-d6, d,
ppm): 161.7, 158.7, 154.7, 154.1, 148.5, 141.6, 138.9,
133.6, 130.1, 124.5, 123.0, 122.8, 115.1, 113.6, 111.0,
85.7; LCMS (M/Z): 356.12 [M?]; Anal. Calcd. for
C19H12N6O2: C, 64.04; H, 3.39; N, 23.58; Found: C, 64.01;
H, 3.37; N, 23.60 %.
1
matic), 2,214 (CN), 1,585 (C=N), 1,512 (C=C); H NMR
(300 MHz, DMSO-d6, d, ppm): 7.05 (d, 1H, J = 7.6 Hz,
Ar–H), 7.16 (t, 1H, J = 7.8 Hz, Ar–H), 7.27 (d, 2H,
J = 8.0 Hz, Ar–H), 7.37 (t, 1H, J = 7.8 Hz, Ar–H), 7.66
(d, 2H, J = 8.1 Hz, Ar–H), 7.76 (d, 1H, J = 7.6 Hz,
Ar–H), 8.21 (s, 1H, C5–H pyridine), 8.78 (s, 2H, NH2 D2O
exch.), 9.21 (s, 1H, OH D2O exch.), 10.16 (s, 1H, –NH
D2O exch.); 13C NMR (100 MHz, DMSO-d6, d, ppm):
161.9, 158.7, 156.8, 155.1, 154.8, 141.7, 130.8, 130.1,
128.7, 123.1, 121.8, 117.9, 115.2, 113.6, 111.2, 85.8;
LCMS (M/Z): 327.12 [M?]; Anal. Calcd. for C19H13N5O:
C, 69.71; H, 4.00; N, 21.39; Found: C, 69.74; H, 4.02;
N, 21.41 %.
2-Amino-6-(1H-benzo[d]imidazol-2-yl)-4-(3-
2-Amino-6-(1H-benzo[d]imidazol-2-yl)-4-(4-
hydroxyphenyl)nicotinonitrile (4f)
nitrophenyl)nicotinonitrile (4i)
Yield: 75 %; m.p.: 218 °C; IR (mmax, cm-1, KBr): 3,470
(NH2), 3,417 (OH), 3,316 (–NH), 3,054 (C–H, aromatic),
2,215 (CN), 1,588 (C=N), 1,510 (C=C); 1H NMR
(300 MHz, DMSO-d6, d, ppm): 6.97 (d, 1H, J = 7.4 Hz,
Ar–H), 7.10 (d, 1H, J = 7.6 Hz, Ar–H), 7.22 (d, 2H,
J = 8.0 Hz, Ar–H), 7.30 (s, 1H, Ar–H), 7.44 (t, 1H,
J = 7.8 Hz, Ar–H), 7.67 (d, 2H, J = 8.0 Hz, Ar–H), 8.20
(s, 1H, C5–H pyridine), 8.80 (s, 2H, NH2 D2O exch.), 9.20
(s, 1H, OH D2O exch.), 10.18 (s, 1H, –NH D2O exch.);
13C NMR (100 MHz, DMSO-d6, d, ppm): 161.8, 158.5, 157.6,
154.8, 154.1, 141.8, 139.4, 130.7, 123.1, 120.1, 116.5, 115.7,
Yield: 80 %; m.p.: 228 °C; IR (mmax, cm-1, KBr): 3,480
(NH2), 3,318 (–NH), 3,064 (C–H, aromatic), 2,220 (CN),
1,590 (C=N), 1,520 (C=C), 1,490 (NO2); 1H NMR
(300 MHz, DMSO-d6, d, ppm): 7.22 (d, 2H, J = 8.1 Hz,
Ar–H), 7.66 (d, 2H, J = 8.2 Hz, Ar–H), 7.86 (d, 2H,
J = 7.8 Hz, Ar–H), 8.12 (d, 2H, J = 7.7 Hz, Ar–H), 8.21
(s, 1H, C5–H pyridine), 8.84 (s, 2H, NH2 D2O exch.), 10.20
(s, 1H, –NH D2O exch.); 13C NMR (100 MHz, DMSO-d6,
d, ppm): 161.6, 158.8, 154.9, 154.1, 148.6, 145.2, 141.6,
128.4, 124.5, 123.1, 115.2, 113.6, 111.2, 85.8; LCMS
123

Med Chem Res (2014) 23:1725–1741
1731
(M/Z): 356.12 [M?]; Anal. Calcd. for C19H12N6O2:
C, 64.04; H, 3.39; N, 23.58; Found: C, 64.02; H, 3.40;
N, 23.61 %.
111.1, 85.8, 21.2; LCMS (M/Z): 325.12 [M?]; Anal. Calcd.
for C20H15N5: C, 73.83; H, 4.65; N, 21.52; Found:
C, 73.86; H, 4.66; N, 21.54 %.
2-Amino-6-(1H-benzo[d]imidazol-2-yl)-4-o-
2-Amino-6-(1H-benzo[d]imidazol-2-yl)-4-(3-
tolylnicotinonitrile (4j)
methoxyphenyl)nicotinonitrile (4m)
Yield: 72 %; m.p.: 212 °C; IR (mmax, cm-1, KBr): 3,468
(NH2), 3,316 (–NH), 3,058, 2,930 (C–H, aromatic, CH3),
2,220 (CN), 1,588 (C=N), 1,518 (C=C); 1H NMR
(300 MHz, DMSO-d6, d, ppm): 2.34 (s, 3H, CH3), 7.17 (d,
2H, J = 8.0 Hz, Ar–H), 7.27 (d, 1H, J = 7.6 Hz, Ar–H),
7.36 (t, 1H, J = 7.4 Hz, Ar–H), 7.44 (t, 1H, J = 7.8 Hz,
Ar–H), 7.66 (d, 2H, J = 8.2 Hz, Ar–H), 7.77 (d, 1H,
J = 7.8 Hz, Ar–H), 8.20 (s, 1H, C5–H pyridine), 8.78
(s, 2H, NH2 D2O exch.), 10.18 (s, 1H, –NH D2O exch.);
13C NMR (100 MHz, DMSO-d6, d, ppm): 161.7, 158.7,
156.7, 154.8, 141.6, 135.8, 135.1, 129.7, 129.1, 126.3,
125.1, 123.2, 115.1, 113.7, 111.0, 85.7, 18.9; LCMS (M/Z):
325.12 [M?]; Anal. Calcd. for C20H15N5: C, 73.83;
H, 4.65; N, 21.52; Found: C, 73.85; H, 4.66; N, 21.54 %.
Yield: 76 %; m.p.: 196 °C; IR (mmax, cm-1, KBr): 3,466
(NH2), 3,318 (–NH), 3,054, 2,815 (C–H, aromatic, OCH3),
2,216 (CN), 1,586 (C=N), 1,512 (C=C); 1H NMR
(300 MHz, DMSO-d6, d, ppm): 3.87 (s, 3H, OCH3), 7.01
(d, 1H, J = 7.6 Hz, Ar–H), 7.12 (d, 1H, J = 7.6 Hz,
Ar–H), 7.20 (d, 2H, J = 8.1 Hz, Ar–H), 7.34 (s, 1H,
J = 7.6 Hz, Ar–H), 7.46 (t, 1H, J = 7.4 Hz, Ar–H), 7.67
(d, 2H, J = 8.2 Hz, Ar–H), 8.20 (s, 1H, C5–H pyridine),
8.78 (s, 2H, NH2 D2O exch.), 10.18 (s, 1H, –NH D2O
exch.); 13C NMR (100 MHz, DMSO-d6, d, ppm): 161.8,
161.0, 158.7, 154.9, 154.2, 141.7, 139.0, 130.2, 123.0,
119.7, 115.2, 114.9, 113.7, 113.2, 111.0, 85.9, 55.9; LCMS
(M/Z): 341.12 [M?]; Anal. Calcd. for C20H15N5O:
C, 70.37; H, 4.43; N, 20.52; Found: C, 70.39; H, 4.45;
N, 20.54 %.
2-Amino-6-(1H-benzo[d]imidazol-2-yl)-4-m-
tolylnicotinonitrile (4k)
2-Amino-6-(1H-benzo[d]imidazol-2-yl)-4-(4-
methoxyphenyl)nicotinonitrile (4n)
Yield: 74 %; m.p.: 201 °C; IR (mmax, cm-1, KBr): 3,472
(NH2), 3,318 (–NH), 3,059, 2,931 (C–H, aromatic, CH3),
2,218 (CN), 1,586 (C=N), 1,514 (C=C); 1H NMR
(300 MHz, DMSO-d6, d, ppm): 2.36 (s, 3H, CH3), 7.18 (d,
2H, J = 8.0 Hz, Ar–H), 7.26 (d, 1H, J = 7.8 Hz, Ar–H),
7.38 (d, 1H, J = 7.6 Hz, Ar–H), 7.44 (t, 1H, J = 7.8 Hz,
Ar–H), 7.67 (d, 2H, J = 8.1 Hz, Ar–H), 7.84 (s, 1H,
Ar–H), 8.22 (s, 1H, C5–H pyridine), 8.80 (s, 2H, NH2 D2O
exch.), 10.20 (s, 1H, –NH D2O exch.); 13C NMR
(100 MHz, DMSO-d6, d, ppm): 161.8, 158.7, 154.7, 154.1,
141.8, 138.9, 137.9, 129.8, 129.1, 128.0, 124.5, 123.0,
115.1, 113.6, 111.1, 85.6, 21.7; LCMS (M/Z): 325.12
[M?]; Anal. Calcd. for C20H15N5: C, 73.83; H, 4.65;
N, 21.52; Found: C, 73.80; H, 4.64; N, 21.55 %.
Yield: 72 %; m.p.: 190 °C; IR (mmax, cm-1, KBr): 3,471
(NH2), 3,316 (–NH), 3,061, 2,817 (C–H, aromatic, OCH3),
2,212 (CN), 1,586 (C=N), 1,515 (C=C); 1H NMR
(300 MHz, DMSO-d6, d, ppm): 3.85 (s, 3H, OCH3), 7.11
(d, 2H, J = 7.6 Hz, Ar–H), 7.20 (d, 2H, J = 8.1 Hz,
Ar–H), 7.65 (d, 2H, J = 8.2 Hz, Ar–H), 7.76 (d, 2H,
J = 7.4 Hz, Ar–H), 8.18 (s, 1H, C5–H pyridine), 8.80
(s, 2H, NH2 D2O exch.), 10.20 (s, 1H, –NH D2O exch.);
13C NMR (100 MHz, DMSO-d6, d, ppm): 161.7, 161.1,
158.7, 154.7, 154.1, 141.8, 131.4, 129.7, 123.0, 115.2,
114.9, 113.7, 111.0, 85.6, 55.7; LCMS (M/Z): 341.12
[M?]; Anal. Calcd. for C20H15N5O: C, 70.37; H, 4.43;
N, 20.52; Found: C, 70.40; H, 4.44; N, 20.55 %.
2-Amino-6-(1H-benzo[d]imidazol-2-yl)-4-p-
2-Amino-6-(1H-benzo[d]imidazol-2-yl)-4-(4-chloro-2-
tolylnicotinonitrile (4l)
hydroxyphenyl)nicotinonitrile (4o)
Yield: 76 %; m.p.: 221 °C; IR (mmax, cm-1, KBr): 3,470
(NH2), 3,320 (–NH), 3,054, 2,927 (C–H, aromatic, CH3),
2,222 (CN), 1,588 (C=N), 1,516 (C=C); 1H NMR
(300 MHz, DMSO-d6, d, ppm): 2.34 (s, 3H, CH3), 7.20 (d,
2H, J = 8.0 Hz, Ar–H), 7.34 (d, 2H, J = 7.8 Hz, Ar–H),
7.44 (d, 2H, J = 7.6 Hz, Ar–H), 7.66 (d, 2H, J = 8.2 Hz,
Ar–H), 8.20 (s, 1H, C5–H pyridine), 8.80 (s, 2H, NH2 D2O
exch.), 10.22 (s, 1H, –NH D2O exch.); 13C NMR
(100 MHz, DMSO-d6, d, ppm): 161.7, 158.8, 154.8, 154.2,
141.9, 136.0, 132.3, 129.6, 127.3, 123.0, 115.2, 113.7,
Yield: 81 %; m.p.: 216 °C; IR (mmax, cm-1, KBr): 3,476
(NH2), 3,421 (OH), 3,315 (–NH), 3,060 (C–H, aromatic),
2,220 (CN), 1,588 (C=N), 1,513 (C=C), 748 (C–Cl);
1H NMR (300 MHz, DMSO-d6, d, ppm): 7.06 (d, 1H,
J = 7.6 Hz, Ar–H), 7.14 (s, 1H, Ar–H), 7.22 (d, 2H,
J = 8.2 Hz, Ar–H), 7.57 (d, 1H, J = 7.6 Hz, Ar–H), 7.67
(d, 2H, J = 8.4 Hz, Ar–H), 8.20 (s, 1H, C5–H pyridine),
8.82 (s, 2H, NH2 D2O exch.), 9.24 (s, 1H, OH D2O exch.),
10.24 (s, 1H, –NH D2O exch.); 13C NMR (100 MHz,
DMSO-d6, d, ppm): 161.9, 158.8, 156.9, 156.3, 154.7,
123

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Med Chem Res (2014) 23:1725–1741
6-(1H-Benzo[d]imidazol-2-yl)-2-(benzylideneamino)-4-(3-
141.6, 137.4, 126.7, 123.1, 121.9, 117.3, 115.2, 113.6,
111.2, 85.6; LCMS (M/Z): 361.10 [M?]; Anal. Calcd. for
C19H12ClN5O: C, 63.08; H, 3.34; N, 19.36; Found:
C, 63.10; H, 3.36; N, 19.38 %.
chlorophenyl)nicotinonitrile (5c)
Yield: 70 %; m.p.: 194 °C; IR (mmax, cm-1, KBr): 3,318
(–NH), 3,078 (=C–H), 3,044 (C–H, aromatic), 2,218 (CN),
1,595 (C=N), 1,522 (C=C), 745 (C–Cl); 1H NMR
(300 MHz, DMSO-d6, d, ppm): 7.20 (d, 2H, J = 8.2 Hz,
Ar–H), 7.32 (d, 1H, J = 7.8 Hz, Ar–H), 7.39 (t, 1H,
J = 7.6 Hz, Ar–H), 7.47 (d, 1H, J = 7.6 Hz, Ar–H), 7.54
(t, 1H, J = 8.2 Hz, Ar–H), 7.62 (t, 2H, J = 8.1 Hz, Ar–H),
7.71 (d, 2H, J = 8.4 Hz, Ar–H), 7.84 (d, 2H, J = 8.2 Hz,
Ar–H), 8.04 (s, 1H, Ar–H), 8.62 (s, 1H, CH=N), 9.01
(s, 1H, C5–H pyridine), 10.60 (s, 1H, –NH D2O exch.);
13C NMR (100 MHz, DMSO-d6, d, ppm): 178.6, 160.8,
158.5, 155.7, 154.7, 141.5, 139.2, 136.4, 134.8, 131.0,
129.9, 129.5, 129.0, 128.8, 127.2, 125.4, 123.1, 120.4,
117.1, 115.2, 98.0; LCMS (M/Z): 433.12 [M?]; Anal.
Calcd. for C26H16N5Cl: C, 71.97; H, 3.72; N, 16.14; Found:
C, 71.94; H, 3.71; N, 16.17 %.
General synthetic procedure for the preparation of 6-
(1H-benzo[d]imidazol-2-yl)-2-(benzylideneamino)-4-
(aryl)nicotinonitriles (5a–o)
In a round-bottom flask, compound (4a–o) (0.01 mol) was
taken in 1,4-dioxane (20 mL) and benzaldehyde (0.01 mol)
was added. To this mixture, a pinch of anhydrous zinc
chloride was added and refluxed for 6 h and poured onto
crushed ice. The product was filtered, washed with cold
water and recrystallized from methanol.
6-(1H-Benzo[d]imidazol-2-yl)-2-(benzylideneamino)-4-
phenylnicotinonitrile (5a)
Yield:66 %;m.p.:190 °C;IR(mmax,cm-1, KBr):3,318(–NH),
3,078 (=C–H), 3,042 (C–H, aromatic), 2,220 (CN), 1,592
(C=N), 1,518 (C=C); 1H NMR (300 MHz, DMSO-d6, d, ppm):
7.20 (d, 2H, J = 8.0 Hz, Ar–H), 7.40 (t, 1H, J = 7.8 Hz, Ar
–H), 7.50 (t, 2H, J = 7.6 Hz, Ar–H), 7.57 (d, 2H, J = 7.8 Hz,
Ar–H), 7.63 (t, 1H, J = 8.2 Hz, Ar–H), 7.69 (d, 2H,
J = 8.2 Hz, Ar–H), 7.77 (t, 2H, J = 8.1 Hz, Ar–H), 7.86 (d,
2H, J = 8.2 Hz, Ar–H), 8.62 (s, 1H, CH=N), 8.98 (s, 1H, C5–H
pyridine), 10.56 (s, 1H, –NH D2O exch.); 13C NMR (100 MHz,
DMSO-d6, d, ppm): 178.8, 160.8, 158.6, 155.7, 154.7, 141.7,
139.0, 136.4, 131.0, 129.9, 129.5, 129.0, 128.7, 127.5, 123.1,
120.1, 117.0, 115.2, 98.2; LCMS (M/Z): 399.12 [M?]; Anal.
Calcd. for C26H17N5: C, 78.18; H, 4.29; N, 17.53; Found:
C, 78.20; H, 4, 27; N, 17.55 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(benzylideneamino)-4-(4-
chlorophenyl)nicotinonitrile (5d)
Yield: 68 %; m.p.: 202 °C; IR (mmax, cm-1, KBr): 3,324
(–NH), 3,084 (=C–H), 3,046 (C–H, aromatic), 2,221 (CN),
1,598 (C=N), 1,520 (C=C), 752 (C–Cl); 1H NMR
(300 MHz, DMSO-d6, d, ppm): 7.16 (d, 2H, J = 8.1 Hz,
Ar–H), 7.48 (t, 1H, J = 8.0 Hz, Ar–H), 7.54 (t, 2H,
J = 8.2 Hz, Ar–H), 7.61 (d, 2H, J = 7.6 Hz, Ar–H), 7.70
(d, 2H, J = 8.4 Hz, Ar–H), 7.78 (d, 2H, J = 7.6 Hz,
Ar–H), 7.87 (d, 2H, J = 8.0 Hz, Ar–H), 8.60 (s, 1H,
CH=N), 9.03 (s, 1H, C5–H pyridine), 10.62 (s, 1H, –NH
D2O exch.); 13C NMR (100 MHz, DMSO-d6, d, ppm):
178.8, 160.7, 158.6, 155.7, 154.6, 141.7, 137.2, 136.3,
134.8, 131.1, 129.8, 129.3, 128.7, 128.1, 123.2, 120.2,
117.1, 115.1, 98.0; LCMS (M/Z): 433.12 [M?]; Anal.
Calcd. for C26H16N5Cl: C, 71.97; H, 3.72; N, 16.14; Found:
C, 71.99; H, 3.75; N, 16.17 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(benzylideneamino)-4-(2-
chlorophenyl)nicotinonitrile (5b)
Yield: 69 %; m.p.: 198 °C; IR (mmax, cm-1, KBr): 3,321
(–NH), 3,081 (=C–H), 3,045 (C–H, aromatic), 2,219 (CN),
1,595 (C=N), 1,520 (C=C), 748 (C–Cl); 1H NMR
(300 MHz, DMSO-d6, d, ppm): 7.18 (d, 2H, J = 8.0 Hz,
Ar–H), 7.34 (t, 1H, J = 7.8 Hz, Ar–H), 7.42 (t, 1H,
J = 7.8 Hz, Ar–H), 7.50 (t, 1H, J = 8.0 Hz, Ar–H), 7.58
(t, 2H, J = 8.2 Hz, Ar–H), 7.64 (d, 1H, J = 8.0 Hz,
Ar–H), 7.71 (d, 2H, J = 8.2 Hz, Ar–H), 7.78 (d, 1H,
J = 8.2 Hz, Ar–H), 7.87 (d, 2H, J = 8.2 Hz, Ar–H), 8.64
(s, 1H, CH=N), 8.98 (s, 1H, C5–H pyridine), 10.58 (s, 1H,
–NH D2O exch.); 13C NMR (100 MHz, DMSO-d6, d, ppm):
178.7, 160.9, 158.5, 155.7, 154.6, 141.7, 138.9, 136.4, 132.0,
131.1, 130.7, 129.8, 129.3, 128.8, 128.2, 127.4, 123.1, 120.2,
117.1, 115.0, 98.2; LCMS (M/Z): 433.12 [M?]; Anal. Calcd.
for C26H16N5Cl: C, 71.97; H, 3.72; N, 16.14; Found:
C, 71.93; H, 3.74; N, 16.15 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(benzylideneamino)-4-(2-
hydroxyphenyl)nicotinonitrile (5e)
Yield: 66 %; m.p.: 192 °C; IR (mmax, cm-1, KBr): 3,421
(OH), 3,317 (–NH), 3,081 (=C–H), 3,042 (C–H, aromatic),
2,218 (CN), 1,595 (C=N), 1,520 (C=C); 1H NMR
(300 MHz, DMSO-d6, d, ppm):7.04 (d, 1H, J = 7.8 Hz,
Ar–H), 7.12 (t, 1H, J = 7.4 Hz, Ar–H), 7.20 (d, 2H,
J = 8.0 Hz, Ar–H), 7.34 (t, 1H, J = 7.8 Hz, Ar–H), 7.44
(t, 1H, J = 8.0 Hz, Ar–H), 7.54 (t, 2H, J = 8.2 Hz, Ar–H),
7.64 (d, 2H, J = 8.4 Hz, Ar–H), 7.72 (d, 1H, J = 7.8 Hz,
Ar–H), 7.85 (d, 2H, J = 8.0 Hz, Ar–H), 8.62 (s, 1H,
CH=N), 8.98 (s, 1H, C5–H pyridine), 9.38 (s, 1H, OH D2O
exch.), 10.60 (s, 1H, –NH D2O exch.); 13C NMR
123

Med Chem Res (2014) 23:1725–1741
1733
(100 MHz, DMSO-d6, d, ppm): 178.6, 160.6, 158.7, 155.7,
155.0, 154.6, 141.6, 136.2, 131.1, 130.8, 130.1, 129.2,
128.8, 128.3, 123.1, 121.8, 120.2, 117.9, 117.2, 115.1,
98.1; LCMS (M/Z): 415.15 [M?]; Anal. Calcd. for
C26H17N5O: C, 75.17; H, 4.12; N, 16.86; Found: C, 75.14;
H, 4.11; N, 16.89 %.
(300 MHz, DMSO-d6, d, ppm): 7.20 (d, 2H, J = 8.2 Hz,
Ar–H), 7.44 (t, 1H, J = 8.0 Hz, Ar–H), 7.54 (t, 2H,
J = 8.0 Hz, Ar–H), 7.67 (d, 2H, J = 8.2 Hz, Ar–H), 7.78
(t, 1H, J = 7.6 Hz, Ar–H), 7.87 (d, 2H, J = 8.0 Hz,
Ar–H), 7.94 (d, 1H, J = 7.6 Hz, Ar–H), 8.07 (d, 1H,
J = 7.4 Hz, Ar–H), 8.24 (s, 1H, Ar–H), 8.64 (s, 1H,
CH=N), 9.06 (s, 1H, C5–H pyridine), 10.64 (s, 1H, –NH
D2O exch.); 13C NMR (100 MHz, DMSO-d6, d, ppm):
178.8, 160.7, 158.7, 155.7, 154.7, 148.3, 141.7, 138.9,
136.4, 133.6, 131.1, 130.1, 129.3, 128.7, 124.5, 123.2,
122.8, 120.0, 117.0, 115.1, 98.0; LCMS (M/Z): 444.15
[M?]; Anal. Calcd. for C26H16N6O2: C, 70.26; H, 3.63;
N, 18.91; Found: C, 70.29; H, 3.62; N, 18.90 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(benzylideneamino)-4-(3-
hydroxyphenyl)nicotinonitrile (5f)
Yield: 80 %; m.p.: 191 °C; IR (mmax, cm-1, KBr): 3,418
(OH), 3,320 (–NH), 3,080 (=C–H), 3,045 (C–H, aromatic),
2,219 (CN), 1,596 (C=N), 1,524 (C=C); 1H NMR
(300 MHz, DMSO-d6, d, ppm):6.97 (d, 1H, J = 7.4 Hz,
Ar–H), 7.11 (d, 1H, J = 7.6 Hz, Ar–H), 7.22 (d, 2H,
J = 8.1 Hz, Ar–H), 7.32 (s, 1H, J = 7.8 Hz, Ar–H), 7.44
(t, 1H, J = 7.8 Hz, Ar–H), 7.51 (t, 1H, J = 8.0 Hz, Ar–H),
7.61 (t, 2H, J = 8.2 Hz, Ar–H), 7.69 (d, 2H, J = 8.4 Hz,
Ar–H), 7.84 (d, 2H, J = 8.1 Hz, Ar–H), 8.64 (s, 1H,
CH=N), 8.97 (s, 1H, C5–H pyridine), 9.40 (s, 1H, OH D2O
exch.), 10.64 (s, 1H, –NH D2O exch.); 13C NMR
(100 MHz, DMSO-d6, d, ppm): 178.7, 160.8, 158.7, 157.6,
155.6, 154.7, 141.7, 139.3, 136.3, 131.0, 130.7, 129.2,
128.9, 123.1, 120.7, 120.1, 117.2, 116.5, 115.7, 115.3,
98.2; LCMS (M/Z): 415.15 [M?]; Anal. Calcd. for
C26H17N5O: C, 75.17; H, 4.12; N, 16.86; Found: C, 75.15;
H, 4.13; N, 16.88 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(benzylideneamino)-4-(4-
nitrophenyl)nicotinonitrile (5i)
Yield: 72 %; m.p.: 214 °C; IR (mmax, cm-1, KBr): 3,325
(–NH), 3,084 (=C–H), 3,044 (C–H, aromatic), 2,221 (CN),
1,598 (C=N), 1,520 (C=C), 1,490 (NO2); 1H NMR
(300 MHz, DMSO-d6, d, ppm): 7.22 (d, 2H, J = 8.1 Hz,
Ar–H), 7.45 (t, 1H, J = 8.0 Hz, Ar–H), 7.54 (t, 2H,
J = 8.0 Hz, Ar–H), 7.68 (d, 2H, J = 8.4 Hz, Ar–H), 7.78
(d, 2H, J = 7.6 Hz, Ar–H), 7.88 (d, 2H, J = 8.0 Hz,
Ar–H), 8.24 (d, 2H, J = 7.8 Hz, Ar–H), 8.66 (s, 1H,
CH=N), 9.04 (s, 1H, C5–H pyridine), 10.65 (s, 1H, –NH
D2O exch.); 13C NMR (100 MHz, DMSO-d6, d, ppm):
178.7, 160.7, 158.6, 155.6, 154.7, 148.4, 145.1, 141.7,
136.4, 131.0, 129.4, 128.7, 128.1, 124.3, 123.2, 120.2,
117.1, 115.1, 98.0; LCMS (M/Z): 444.14 [M?]; Anal.
Calcd. for C26H16N6O2: C, 70.26; H, 3.63; N, 18.91;
Found: C, 70.30; H, 3.64; N, 18.93 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(benzylideneamino)-4-(4-
hydroxyphenyl)nicotinonitrile (5g)
Yield: 68 %; m.p.: 199 °C; IR (mmax, cm-1, KBr): 3,422
(OH), 3,322 (–NH), 3,081 (=C–H), 3,040 (C–H, aromatic),
2,216 (CN), 1,595 (C=N), 1518s (C=C); 1H NMR
(300 MHz, DMSO-d6, d, ppm):6.88 (d, 2H, J = 7.6 Hz,
Ar–H), 7.18 (d, 2H, J = 8.0 Hz, Ar–H), 7.46 (t, 1H,
J = 8.0 Hz, Ar–H), 7.57 (t, 2H, J = 8.0 Hz, Ar–H), 7.68
(d, 2H, J = 8.2 Hz, Ar–H), 7.77 (d, 2H, J = 7.6 Hz,
Ar–H), 7.87 (d, 2H, J = 8.2 Hz, Ar–H), 8.63 (s, 1H,
CH=N), 8.99 (s, 1H, C5–H pyridine), 9.38 (s, 1H, OH D2O
exch.), 10.62 (s, 1H, –NH D2O exch.); 13C NMR
(100 MHz, DMSO-d6, d, ppm): 178.7, 160.7, 159.0, 158.8,
155.6, 154.7, 141.6, 136.3, 131.7, 131.0, 130.1, 129.3,
128.7, 123.1, 120.2, 117.0, 116.5, 115.2, 98.0; LCMS
(M/Z): 415.15 [M?]; Anal. Calcd. for C26H17N5O: C, 75.17;
H, 4.12; N, 16.86; Found: C, 75.19; H, 4.11; N, 16.89 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(benzylideneamino)-4-o-
tolylnicotinonitrile (5j)
Yield: 64 %; m.p.: 194 °C; IR (mmax, cm-1, KBr): 3,320
(–NH), 3,075 (=C–H), 3,040, 2,928 (C–H, aromatic, CH3),
2,214 (CN), 1,588 (C=N), 1,514 (C=C); 1H NMR
(300 MHz, DMSO-d6, d, ppm): 2.34 (s, 3H, CH3), 7.18 (d,
2H, J = 8.0 Hz, Ar–H), 7.27 (t, 1H, J = 7.6 Hz, Ar–H),
7.35 (d, 1H, J = 7.4 Hz, Ar–H), 7.42 (t, 1H, J = 7.6 Hz,
Ar–H), 7.48 (t, 1H, J = 8.0 Hz, Ar–H), 7.56 (t, 2H,
J = 8.0 Hz, Ar–H), 7.66 (d, 2H, J = 8.2 Hz, Ar–H), 7.78
(d, 1H, J = 7.6 Hz, Ar–H), 7.88 (d, 2H, J = 8.1 Hz,
Ar–H), 8.62 (s, 1H, CH=N), 8.97 (s, 1H, C5–H pyridine),
10.61 (s, 1H, –NH D2O exch.); 13C NMR (100 MHz,
DMSO-d6, d, ppm): 178.6, 160.7, 158.6, 155.5, 154.7,
141.6, 136.5, 135.7, 135.1, 131.0, 129.9, 129.5, 129.1, 128.8,
126.2, 125.1, 123.0, 120.2, 117.1, 115.0, 98.2, 18.8; LCMS
(M/Z): 413.14 [M?]; Anal. Calcd. for C27H19N5: C, 78.43;
H, 4.63; N, 16.94; Found: C, 78.46; H, 4.64; N, 16.95 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(benzylideneamino)-4-(3-
nitrophenyl)nicotinonitrile (5h)
Yield: 70 %; m.p.: 205 °C; IR (mmax, cm-1, KBr): 3,324
(–NH), 3,081 (=C–H), 3,040 (C–H, aromatic), 2,216 (CN),
1,595 (C=N), 1,518 (C=C), 1,492 (NO2); 1H NMR
123

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Med Chem Res (2014) 23:1725–1741
6-(1H-Benzo[d]imidazol-2-yl)-2-(benzylideneamino)-4-m-
DMSO-d6, d, ppm): 178.6, 161.1, 160.8, 158.6, 155.7,
154.7, 141.8, 139.0, 136.3, 131.1, 130.2, 129.2, 128.8, 123.1,
120.1, 119.7, 117.0, 115.2, 114.8, 113.2, 98.2, 55.7; LCMS
(M/Z): 429.15 [M?]; Anal. Calcd. for C27H19N5O: C, 75.51;
H, 4.46; N, 16.31; Found: C, 75.54; H, 4.45; N, 16.34 %.
tolylnicotinonitrile (5k)
Yield: 65 %; m.p.: 201 °C; IR (mmax, cm-1, KBr): 3,316
(–NH), 3,074 (=C–H), 3,042, 2,930 (C–H, aromatic, CH3),
2,216 (CN), 1,586 (C=N), 1,515 (C=C); 1H NMR
(300 MHz, DMSO-d6, d, ppm): 2.32 (s, 3H, CH3), 7.11 (d,
1H, J = 7.6 Hz, Ar–H), 7.20 (d, 2H, J = 8.0 Hz, Ar–H),
7.32 (d, 1H, J = 7.4 Hz, Ar–H), 7.40 (t, 1H, J = 7.6 Hz,
Ar–H), 7.48 (t, 1H, J = 8.0 Hz, Ar–H), 7.57 (t, 2H,
J = 8.1 Hz, Ar–H), 7.67 (d, 2H, J = 8.2 Hz, Ar–H), 7.80
(s, 1H, Ar–H), 7.87 (d, 2H, J = 8.1 Hz, Ar–H), 8.60 (s, 1H,
CH=N), 8.96 (s, 1H, C5–H pyridine), 10.60 (s, 1H, –NH
D2O exch.); 13C NMR (100 MHz, DMSO-d6, d, ppm):
178.7, 160.5, 158.6, 155.7, 154.7, 141.8, 138.9, 137.7,
136.5, 131.2, 129.9, 129.5, 129.1, 128.8, 128.2, 124.5,
123.2, 120.0, 117.1, 115.2, 98.2, 21.8; LCMS (M/Z):
413.16 [M?]; Anal. Calcd. for C27H19N5: C, 78.43;
H, 4.63; N, 16.94; Found: C, 78.40; H, 4.65; N, 16.92 %.
6-(1H-benzo[d]imidazol-2-yl)-2-(benzylideneamino)-4-(4-
methoxyphenyl)nicotinonitrile (5n)
Yield: 69 %; m.p.: 182 °C; IR (mmax, cm-1, KBr): 3,314
(–NH), 3,075 (=C–H), 3,040, 2,822 (C–H, aromatic,
1
OCH3), 2,212 (CN), 1,587 (C=N), 1,515 (C=C); H NMR
(300 MHz, DMSO-d6, d, ppm): 3.86 (s, 3H, OCH3), 7.08
(d, 2H, J = 7.6 Hz, Ar–H), 7.20 (d, 2H, J = 8.1 Hz,
Ar–H), 7.48 (t, 1H, J = 8.0 Hz, Ar–H), 7.56 (t, 2H,
J = 8.1 Hz, Ar–H), 7.67 (d, 2H, J = 8.2 Hz, Ar–H), 7.76
(d, 2H, J = 7.6 Hz, Ar–H), 7.87 (d, 2H, J = 8.1 Hz,
Ar–H), 8.62 (s, 1H, CH=N), 8.97 (s, 1H, C5–H pyridine),
10.61 (s, 1H, –NH D2O exch.); 13C NMR (100 MHz,
DMSO-d6, d, ppm): 178.7, 161.2, 160.8, 158.7, 155.7,
154.8, 141.8, 136.4, 131.7, 131.1, 129.8, 129.2, 128.8,
123.2, 120.1, 117.1, 115.0, 114.9, 98.1, 55.9; LCMS (M/Z):
429.14 [M?]; Anal. Calcd. for C27H19N5O: C, 75.51;
H, 4.46; N, 16.31; Found: C, 75.55; H, 4.48; N, 16.33 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(benzylideneamino)-4-p-
tolylnicotinonitrile (5l)
Yield: 63 %; m.p.: 190 °C; IR (mmax, cm-1, KBr): 3,318
(–NH), 3,075 (=C–H), 3,042, 2,929 (C–H, aromatic, CH3),
2,218 (CN), 1,584 (C=N), 1,518 (C=C); 1H NMR
(300 MHz, DMSO-d6, d, ppm): 2.30 (s, 3H, CH3), 7.18 (d,
2H, J = 8.0 Hz, Ar–H), 7.30 (d, 2H, J = 7.6 Hz, Ar–H),
7.38 (d, 2H, J = 7.6 Hz, Ar–H), 7.47 (t, 1H, J = 8.0 Hz,
Ar–H), 7.56 (t, 2H, J = 8.1 Hz, Ar–H), 7.67 (d, 2H,
J = 8.4 Hz, Ar–H), 7.88 (d, 2H, J = 8.1 Hz, Ar–H), 8.61
(s, 1H, CH=N), 8.98 (s, 1H, C5–H pyridine), 10.62 (s, 1H,
–NH D2O exch.); 13C NMR (100 MHz, DMSO-d6, d,
ppm): 178.7, 160.8, 158.6, 155.7, 154.6, 141.8, 136.9,
136.3, 131.1, 129.7, 129.2, 128.8, 127.1, 123.2, 120.2,
117.1, 115.0, 98.2, 21.2; LCMS (M/Z): 413.15 [M?]; Anal.
Calcd. for C27H19N5: C, 78.43; H, 4.63; N, 16.94; Found:
C, 78.41; H, 4.62; N, 16.96 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(benzylideneamino)-4-(4-
chloro-2-hydroxyphenyl)-nicotinonitrile (5o)
Yield: 69 %; m.p.: 207 °C; IR (mmax, cm-1, KBr):
3,423(OH), 3,320 (–NH), 3,080 (=C–H), 3,046 (C–H,
aromatic), 2,221 (CN), 1,591 (C=N), 1,520 (C=C), 751
1
(C–Cl); H NMR (300 MHz, DMSO-d6, d, ppm): 7.06 (d,
1H, J = 7.6 Hz, Ar–H), 7.12 (d, 1H, J = 7.4 Hz, Ar–H),
7.20 (d, 2H, J = 8.1 Hz, Ar–H), 7.48 (t, 1H, J = 8.0 Hz,
Ar–H), 7.56 (t, 2H, J = 8.1 Hz, Ar–H), 7.61 (d, 1H,
J = 7.6 Hz, Ar–H), 7.69 (d, 2H, J = 8.4 Hz, Ar–H), 7.86
(d, 2H, J = 8.2 Hz, Ar–H), 8.62 (s, 1H, CH=N), 8.98 (s,
1H, C5–H pyridine), 9.40 (s, 1H, OH D2O exch.), 10.62 (s,
1H, –NH D2O exch.); 13C NMR (100 MHz, DMSO-d6, d,
ppm): 178.6, 160.8, 158.7, 156.5, 155.6, 154.6, 141.7,
137.4, 136.2, 131.1, 130.2, 129.8, 128.8, 126.7, 123.0,
121.9, 120.2, 117.7, 117.1, 115.2, 98.1, 55.9; LCMS (M/Z):
449.12 [M?]; Anal. Calcd. for C26H16N5ClO: C, 69.41;
H, 3.58; N, 15.57; Found: C, 69.45; H, 3.57; N, 15.57 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(benzylideneamino)-4-(3-
methoxyphenyl)nicotinonitrile (5m)
Yield: 66 %; m.p.: 190 °C; IR (mmax, cm-1, KBr): 3,315
(–NH), 3,074 (=C–H), 3,038, 2,824 (C–H, aromatic,
1
OCH3), 2,214 (CN), 1,586 (C=N), 1,517 (C=C); H NMR
(300 MHz, DMSO-d6, d, ppm): 3.85 (s, 3H, OCH3), 6.97
(d, 1H, J = 7.6 Hz, Ar–H), 7.10 (d, 1H, J = 7.6 Hz,
Ar–H), 7.18 (d, 2H, J = 8.1 Hz, Ar–H), 7.37 (s, 1H,
Ar–H), 7.44 (t, 1H, J = 7.4 Hz, Ar–H), 7.51 (t, 1H,
J = 8.0 Hz, Ar–H), 7.57 (t, 2H, J = 8.1 Hz, Ar–H), 7.67
(d, 2H, J = 8.4 Hz, Ar–H), 7.86 (d, 2H, J = 8.1 Hz,
Ar–H), 8.60 (s, 1H, CH=N), 8.97 (s, 1H, C5–H pyridine),
10.60 (s, 1H, –NH D2O exch.); 13C NMR (100 MHz,
General synthetic procedure for the preparation of 6-
(1H-benzo[d]imidazol-2-yl)-2-(3-chloro-2-oxo-4-
phenylazetidin-1-yl)-4-(aryl)nicotinonitriles (6a–o)
To a stirred solution of compound (5a–o) (0.05 mol) and
triethylamine (0.01 mol) in 1,4-dioxane, chloroacetyl
chloride (0.01 mol) was added dropwise at 0–5 °C with
stirring. The reaction mixture was stirred for about 1 h and
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1735
the precipitated amine hydrochloride was filtered off. The
filtrate was refluxed for about 8 h and excess of solvent
was evaporated under reduced pressure. The residue was
poured onto crushed ice. The solid obtained was filtered,
washed with water and crystallized from methanol.
(300 MHz, DMSO-d6, d, ppm): 5.08 (d, 1H, J = 7.1 Hz,
C4–H azetidinone), 5.49 (d, 1H, J = 6.8 Hz, C3–H aze-
tidinone), 7.16 (d, 2H, J = 8.0 Hz, Ar–H), 7.27 (t, 1H,
J = 7.9 Hz, Ar–H), 7.36 (d, 2H, J = 7.8 Hz, Ar–H), 7.40
(d, 1H, J = 7.6 Hz, Ar–H), 7.46 (t, 2H, J = 7.8 Hz,
Ar–H), 7.52 (t, 1H, J = 7.6 Hz, Ar–H), 7.57 (d, 1H,
J = 7.4 Hz, Ar–H), 7.67 (d, 2H, J = 8.4 Hz, Ar–H), 7.97
(s, 1H, Ar–H), 8.92 (s, 1H, C5–H pyridine), 10.50 (s, 1H,
–NH D2O exch.); 13C NMR (100 MHz, DMSO-d6, d,
ppm): 162.1, 157.3, 154.9, 154.4, 151.3, 143.6, 141.6,
139.5, 134.7, 129.8, 129.2, 128.7, 127.3, 126.9, 126.3, 125.6,
123.2, 117.8, 115.1, 113.7, 91.6, 67.7, 62.1; LCMS (M/Z):
509.08 [M?]; Anal. Calcd. for C28H17N5Cl2O: C, 65.89;
H, 3.36; N, 13.72; Found: C, 65.91; H, 3.35; N, 13.73 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(3-chloro-2-oxo-4-
phenylazetidin-1-yl)-4-phenyl-nicotinonitrile (6a)
Yield: 58 %; m.p.: 262 °C; IR (mmax, cm-1, KBr): 3,324
(–NH), 3,051, (C–H, aromatic), 2,228 (CN), 1,684 (CO),
1,594 (C=N), 1,524 (C=C), 751 (C–Cl); 1H NMR (300 MHz,
DMSO-d6, d, ppm): 5.12 (d, 1H, J = 7.1 Hz, C4–H azetid-
inone), 5.48 (d, 1H, J = 6.6 Hz, C3–H azetidinone), 7.18 (d,
2H, J = 8.2 Hz, Ar–H), 7.27 (t, 1H, J = 8.0 Hz, Ar–H),
7.34 (d, 2H, J = 7.8 Hz, Ar–H), 7.40 (t, 2H, J = 7.9 Hz,
Ar–H), 7.46 (t, 1H, J = 7.6 Hz, Ar–H), 7.52 (t, 2H,
J = 8.0 Hz, Ar–H), 7.59 (d, 2H, J = 7.6 Hz, Ar–H), 7.69
(d, 2H, J = 8.4 Hz, Ar–H), 8.94 (s, 1H, C5–H pyridine),
10.48 (s, 1H, –NH D2O exch.); 13C NMR (100 MHz,
DMSO-d6, d, ppm): 162.2, 157.3, 154.9, 154.4, 151.2, 143.6,
141.7, 139.0, 129.7, 129.1, 128.7, 127.5, 126.8, 126.1, 123.0,
117.7, 115.2, 113.7, 91.6, 67.7, 62.1; LCMS (M/Z): 475.12
[M?]; Anal. Calcd. for C28H18N5ClO: C, 70.66; H, 3.81; N,
14.72; Found: C, 70.70; H, 3.81; N, 14.74 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(3-chloro-2-oxo-4-
phenylazetidin-1-yl)-4-(4-chlorophenyl)nicotinonitrile (6d)
Yield: 61 %; m.p.: 251 °C; IR (mmax, cm-1, KBr): 3,329
(–NH), 3,053, (C–H, aromatic), 2,230 (CN), 1,690 (CO),
1,598 (C=N), 1,528 (C=C), 767 (C–Cl); 1H NMR
(300 MHz, DMSO-d6, d, ppm): 5.10 (d, 1H, J = 6.9 Hz,
C4–H azetidinone), 5.47 (d, 1H, J = 6.6 Hz, C3–H aze-
tidinone), 7.20 (d, 2H, J = 8.1 Hz, Ar–H), 7.27 (t, 1H,
J = 7.9 Hz, Ar–H), 7.37 (d, 2H, J = 7.8 Hz, Ar–H), 7.42
(t, 2H, J = 7.4 Hz, Ar–H), 7.57 (d, 2H, J = 7.8 Hz,
Ar–H), 7.68 (d, 2H, J = 8.4 Hz, Ar–H), 7.77 (d, 2H,
J = 7.4 Hz, Ar–H), 8.94 (s, 1H, C5–H pyridine), 10.48 (s,
1H, –NH D2O exch.); 13C NMR (100 MHz, DMSO-d6, d,
ppm): 162.3, 157.4, 154.9, 154.3, 151.3, 143.6, 141.8,
137.1, 134.7, 129.4, 128.8, 128.2, 126.9, 126.1, 123.1,
117.6, 115.2, 113.7, 91.8, 67.5, 62.2; LCMS (M/Z): 509.12
[M?]; Anal. Calcd. for C28H17N5Cl2O: C, 65.89; H, 3.36;
N, 13.72; Found: C, 65.92; H, 3.37; N, 13.74 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(3-chloro-2-oxo-4-
phenylazetidin-1-yl)-4-(2-chlorophenyl)nicotinonitrile (6b)
Yield: 62 %; m.p.: 243 °C; IR (mmax, cm-1, KBr): 3,327
(–NH), 3,054, (C–H, aromatic), 2,229 (CN), 1,689 (CO),
1,597 (C=N), 1,527 (C=C), 766 (C–Cl); 1H NMR (300 MHz,
DMSO-d6, d, ppm): 5.10 (d, 1H, J = 7.2 Hz, C4–H azetid-
inone), 5.47 (d, 1H, J = 6.7 Hz, C3–H azetidinone), 7.16 (d,
2H, J = 8.1 Hz, Ar–H), 7.25 (t, 1H, J = 7.9 Hz, Ar–H),
7.34 (d, 2H, J = 7.8 Hz, Ar–H), 7.41 (t, 1H, J = 7.6 Hz,
Ar–H), 7.47 (t, 1H, J = 7.6 Hz, Ar–H), 7.52 (t, 2H,
J = 8.0 Hz, Ar–H), 7.61 (d, 1H, J = 7.6 Hz, Ar–H), 7.70 (d,
2H, J = 8.4 Hz, Ar–H), 7.78 (d, 1H, J = 7.6 Hz, Ar–H),
8.96 (s, 1H, C5–H pyridine), 10.48 (s, 1H, –NH D2O exch.);
13C NMR (100 MHz, DMSO-d6, d, ppm): 162.1, 157.7,
157.1, 154.8, 151.1, 143.6, 141.8, 138.8, 132.1, 130.6, 129.4,
128.9, 128.3, 127.4, 126.9, 126.3, 123.1, 117.7, 115.1, 113.5,
91.6, 67.6, 62.1; LCMS (M/Z): 509.10 [M?]; Anal. Calcd. for
C28H17N5Cl2O: C, 65.89; H, 3.36; N, 13.72; Found:
C, 65.92; H, 3.36; N, 13.74 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(3-chloro-2-oxo-4-
phenylazetidin-1-yl)-4-(2-hydroxyphenyl)nicotinonitrile
(6e)
Yield: 57 %; m.p.: 221 °C; IR (mmax, cm-1, KBr): 3,424
(OH), 3,325 (–NH), 3,049 (C–H, aromatic), 2,226 (CN),
1,685 (CO), 1,596 (C=N), 1,524 (C=C), 754 (C–Cl);
1H NMR (300 MHz, DMSO-d6, d, ppm): 5.11 (d, 1H,
J = 7.0 Hz, C4–H azetidinone), 5.46 (d, 1H, J = 6.8 Hz,
C3–H azetidinone), 7.05 (d, 1H, J = 7.6 Hz, Ar–H), 7.12
(t, 1H, J = 7.4 Hz, Ar–H), 7.22 (d, 2H, J = 8.2 Hz,
Ar–H), 7.27 (t, 1H, J = 7.8 Hz, Ar–H), 7.34 (t, 1H,
J = 7.9 Hz, Ar–H), 7.40 (d, 2H, J = 8.0 Hz, Ar–H), 7.47
(t, 2H, J = 7.8 Hz, Ar–H), 7.67 (d, 2H, J = 8.4 Hz,
Ar–H), 7.74 (d, 1H, J = 7.4 Hz, Ar–H), 8.97 (s, 1H, C5–H
pyridine), 9.47 (s, 1H, OH D2O exch.), 10.45 (s, 1H, –NH
D2O exch.); 13C NMR (100 MHz, DMSO-d6, d, ppm):
162.1, 157.8, 157.2, 155.1, 154.6, 151.3, 143.7, 141.7,
6-(1H-Benzo[d]imidazol-2-yl)-2-(3-chloro-2-oxo-4-
phenylazetidin-1-yl)-4-(3-chlorophenyl)nicotinonitrile (6c)
Yield: 59 %; m.p.: 232 °C; IR (mmax, cm-1, KBr): 3,325
(–NH), 3,051, (C–H, aromatic), 2,228 (CN), 1,688 (CO),
1,597 (C=N), 1,526 (C=C), 764 (C–Cl); 1H NMR
123

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Med Chem Res (2014) 23:1725–1741
130.9, 130.4, 128.8, 128.1, 126.9, 126.3, 123.1, 121.9,
117.9, 117.5, 115.0, 113.6, 91.5, 67.5, 62.2; LCMS (M/Z):
491.12 [M?]; Anal. Calcd. for C28H18N5ClO2: C, 68.36;
H, 3.69; N, 14.24; Found: C, 68.39; H, 3.68; N, 14.25 %.
1,598 (C=N), 1,528 (C=C), 1,490 (NO2), 754 (C–Cl);
1H NMR (300 MHz, DMSO-d6, d, ppm): 5.12 (d, 1H,
J = 6.9 Hz, C4–H azetidinone), 5.49 (d, 1H, J = 6.6 Hz,
C3–H azetidinone), 7.22 (d, 2H, J = 8.2 Hz, Ar–H), 7.28
(t, 1H, J = 8.0 Hz, Ar–H), 7.34 (d, 2H, J = 7.9 Hz, Ar–H),
7.44 (t, 2H, J = 7.8 Hz, Ar–H), 7.68 (d, 2H, J = 8.4 Hz,
Ar–H), 7.78 (t, 1H, J = 7.6 Hz, Ar–H), 7.89 (d, 1H,
J = 7.6 Hz, Ar–H), 8.01 (d, 1H, J = 7.4 Hz, Ar–H), 8.11 (s,
1H, Ar–H), 9.02 (s, 1H, C5–H pyridine), 10.54 (s, 1H, –NH
D2O exch.); 13C NMR (100 MHz, DMSO-d6, d, ppm):
162.2, 157.4, 154.9, 154.2, 151.3, 148.5, 143.7, 141.8, 138.9,
133.6, 130.1, 128.6, 126.9, 126.4, 124.4, 123.1, 122.8, 117.8,
115.1, 113.7, 91.4, 67.8, 62.2; LCMS (M/Z): 520.12 [M?];
Anal. Calcd. for C28H17N6ClO3: C, 64.56; H, 3.29; N, 16.13;
Found: C, 64.60; H, 3.28; N, 16.16 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(3-chloro-2-oxo-4-
phenylazetidin-1-yl)-4-(3-hydroxyphenyl)nicotinonitrile
(6f)
Yield:58 %;m.p.: 224 °C;IR(mmax, cm-1, KBr): 3,422 (OH),
3,324 (–NH), 3,051 (C–H, aromatic), 2,225 (CN), 1,688 (CO),
1,597 (C=N), 1,523 (C=C), 750 (C–Cl); 1H NMR (300 MHz,
DMSO-d6, d, ppm): 5.07 (d, 1H, J = 7.2 Hz, C4–H azetidi-
none), 5.44 (d, 1H, J = 6.9 Hz, C3–H azetidinone), 6.97 (d,
1H, J = 7.6 Hz, Ar–H), 7.11(d, 1H, J = 7.6 Hz, Ar–H), 7.18
(d, 2H, J = 8.0 Hz, Ar–H), 7.27 (t, 1H, J = 8.0 Hz, Ar–H),
7.34 (d, 2H, J = 7.8 Hz, Ar–H), 7.39 (s, 1H, Ar–H), 7.44
(t, 1H, J = 7.4 Hz, Ar–H), 7.49 (t, 2H, J = 7.8 Hz, Ar–H),
7.68 (d, 2H, J = 8.4 Hz, Ar–H), 8.98 (s, 1H, C5–H pyridine),
9.48 (s, 1H, OH D2O exch.), 10.47 (s, 1H, –NH D2O exch.);
13C NMR (100 MHz, DMSO-d6, d, ppm): 162.2, 157.7,
157.2, 154.8, 154.1, 151.1, 143.6, 141.6, 139.3, 130.5, 128.8,
126.9, 126.4, 123.2, 120.1, 117.7, 116.4, 115.8, 115.2, 113.7,
91.7, 67.7, 62.1; LCMS (M/Z): 491.10 [M?]; Anal. Calcd. for
C28H18N5ClO2: C, 68.36; H, 3.69; N, 14.24; Found: C, 68.40;
H, 3.70; N, 14.25 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(3-chloro-2-oxo-4-
phenylazetidin-1-yl)-4-(4-nitrophenyl)nicotinonitrile (6i)
Yield: 61 %; m.p.: 253 °C; IR (mmax, cm-1, KBr): 3,331
(–NH), 3,054 (C–H, aromatic), 2,228 (CN), 1,688 (CO),
1,596 (C=N), 1,526 (C=C), 1,488 (NO2), 755 (C–Cl);
1H NMR (300 MHz, DMSO-d6, d, ppm): 5.14 (d, 1H,
J = 7.1 Hz, C4–H azetidinone), 5.51 (d, 1H, J = 6.8 Hz,
C3–H azetidinone), 7.22 (d, 2H, J = 8.2 Hz, Ar–H), 7.27 (t,
1H, J = 7.9 Hz, Ar–H), 7.36 (d, 2H, J = 7.9 Hz, Ar–H),
7.44 (t, 2H, J = 7.8 Hz, Ar–H), 7.70 (d, 2H, J = 8.4 Hz,
Ar–H), 7.82 (d, 2H, J = 7.6 Hz, Ar–H), 8.12 (d, 2H,
J = 7.8 Hz, Ar–H), 9.01 (s, 1H, C5–H pyridine), 10.52 (s,
1H, –NH D2O exch.); 13C NMR (100 MHz, DMSO-d6, d,
ppm): 162.1, 157.4, 154.9, 154.1, 151.4, 148.3, 145.2, 143.6,
141.7, 128.7, 128.1, 126.9, 126.3, 124.4, 123.1, 124.4, 117.1,
115.2, 113.8, 91.6, 67.8, 62.1; LCMS (M/Z): 520.10 [M?];
Anal. Calcd. for C28H17N6ClO3: C, 64.56; H, 3.29; N, 16.13;
Found: C, 64.58; H, 3.30; N, 16.14 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(3-chloro-2-oxo-4-
phenylazetidin-1-yl)-4-(4-hydroxyphenyl)nicotinonitrile
(6g)
Yield:60 %;m.p.: 198 °C;IR(mmax, cm-1, KBr): 3,426 (OH),
3,321 (–NH), 3,051 (C–H, aromatic), 2,227 (CN), 1,687 (CO),
1,595 (C=N), 1,522 (C=C), 752 (C–Cl); 1H NMR (300 MHz,
DMSO-d6, d, ppm): 5.06 (d, 1H, J = 7.1 Hz, C4–H azetidi-
none), 5.45 (d, 1H, J = 6.7 Hz, C3–H azetidinone), 6.90 (d,
2H, J = 7.6 Hz, Ar–H), 7.18(d, 2H, J = 8.0 Hz, Ar–H), 7.27
(t, 1H, J = 8.0 Hz, Ar–H), 7.35 (d, 2H, J = 7.8 Hz, Ar–H),
7.44 (t, 2H, J = 7.8 Hz, Ar–H), 7.66 (d, 2H, J = 8.2 Hz,
Ar–H), 7.75 (d, 2H, J = 7.6 Hz, Ar–H), 8.96 (s, 1H, C5–H
pyridine), 9.45(s, 1H, OHD2O exch.), 10.47 (s, 1H, –NH D2O
exch.); 13C NMR (100 MHz, DMSO-d6, d, ppm): 162.1,
159.0, 157.2, 154.9, 154.3, 151.3, 143.6, 141.7, 131.7, 130.2,
128.5, 126.8, 126.2, 123.2, 117.7, 116.5, 115.2, 113.8, 91.9,
67.7, 62.0; LCMS (M/Z): 491.11 [M?]; Anal. Calcd. for
C28H18N5ClO2: C, 68.36; H, 3.69; N, 14.24; Found: C, 68.48;
H, 3.71; N, 14.26 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(3-chloro-2-oxo-4-
phenylazetidin-1-yl)-4-o-tolyl-nicotinonitrile (6j)
Yield: 57 %; m.p.: 216 °C; IR (mmax, cm-1, KBr): 3,321
(–NH), 3,046, 2,927 (C–H, aromatic, CH3), 2,224 (CN),
1,684 (CO), 1,591 (C=N), 1,522 (C=C), 748 (C–Cl);
1H NMR (300 MHz, DMSO-d6, d, ppm): 2.34 (s, 3H, CH3),
5.07 (d, 1H, J = 7.0 Hz, C4–H azetidinone), 5.47 (d, 1H,
J = 6.7 Hz, C3–H azetidinone), 7.16 (d, 2H, J = 8.0 Hz,
Ar–H), 7.27 (t, 1H, J = 7.9 Hz, Ar–H), 7.34 (d, 2H,
J = 7.9 Hz, Ar–H), 7.39 (t, 1H, J = 7.6 Hz, Ar–H), 7.47 (d,
1H, J = 7.6 Hz, Ar–H), 7.54 (t, 1H, J = 7.6 Hz,
Ar–H), 7.61 (t, 2H, J = 7.8 Hz, Ar–H), 7.67 (d, 2H,
J = 8.2 Hz, Ar–H), 7.78 (d, 1H, J = 7.6 Hz, Ar–H), 8.92
(s, 1H, C5–H pyridine), 10.47 (s, 1H, –NH D2O exch.);
13C NMR (100 MHz, DMSO-d6, d, ppm): 162.2, 157.8,
157.3, 154.8, 151.2, 143.6, 141.6, 135.8, 135.1, 129.5, 129.1,
6-(1H-Benzo[d]imidazol-2-yl)-2-(3-chloro-2-oxo-4-
phenylazetidin-1-yl)-4-(3-nitrophenyl)nicotinonitrile (6h)
Yield: 62 %; m.p.: 241 °C; IR (mmax, cm-1, KBr): 3,330
(–NH), 3,056, (C–H, aromatic), 2,230 (CN), 1,690 (CO),
123

Med Chem Res (2014) 23:1725–1741
1737
128.4, 126.9, 126.5, 126.1, 125.1, 123.0, 117.7, 115.1, 113.8,
91.6, 67.7, 62.1, 18.8; LCMS (M/Z): 489.12 [M?]; Anal.
Calcd. for C29H20N5ClO: C, 71.09; H, 4.11;
N, 14.29; Found: C, 71.14; H, 4.10; N, 14.27 %.
1,682 (CO), 1,590 (C=N), 1,520 (C=C), 746 (C–Cl);
1H NMR (300 MHz, DMSO-d6, d, ppm): 3.87 (s, 3H,
OCH3), 5.04 (d, 1H, J = 7.1 Hz, C4–H azetidinone), 5.45
(d, 1H, J = 6.8 Hz, C3–H azetidinone), 6.98 (d, 1H,
J = 7.4 Hz, Ar–H), 7.10 (d, 1H, J = 7.6 Hz, Ar–H), 7.17
(d, 2H, J = 8.0 Hz, Ar–H), 7.26 (t, 1H, J = 7.8 Hz,
Ar–H), 7.34 (d, 2H, J = 8.0 Hz, Ar–H), 7.39 (s, 1H,
Ar–H), 7.47 (t, 1H, J = 7.6 Hz, Ar–H), 7.54 (t, 2H,
J = 7.8 Hz, Ar–H), 7.67 (d, 2H, J = 8.4 Hz, Ar–H), 8.90
(s, 1H, C5–H pyridine), 10.47 (s, 1H, –NH D2O exch.);
13C NMR (100 MHz, DMSO-d6, d, ppm): 162.3, 161.1,
157.4, 154.9, 154.3, 151.2, 143.7, 141.6, 139.1, 130.3,
128.5, 126.9, 126.4, 123.1, 117.7, 115.1, 114.9, 113.9,
113.2, 91.7, 67.9, 62.2, 55.9; LCMS (M/Z): 505.14 [M?];
Anal. Calcd. for C29H20N5ClO2: C, 68.84; H, 3.98;
N, 13.84; Found: C, 68.88; H, 3.99; N, 13.87 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(3-chloro-2-oxo-4-
phenylazetidin-1-yl)-4-m-tolyl-nicotinonitrile (6k)
Yield: 58 %; m.p.: 237 °C; IR (mmax, cm-1, KBr): 3,321
(–NH), 3,044, 2,930 (C–H, aromatic, CH3), 2,221 (CN),
1,685 (CO), 1,590 (C=N), 1,520 (C=C), 748 (C–Cl);
1H NMR (300 MHz, DMSO-d6, d, ppm): 2.32 (s, 3H, CH3),
5.06 (d, 1H, J = 7.0 Hz, C4–H azetidinone), 5.47 (d, 1H,
J = 6.7 Hz, C3–H azetidinone), 7.12 (d, 1H, J = 7.6 Hz,
Ar–H), 7.18 (d, 2H, J = 8.0 Hz, Ar–H), 7.26 (t, 1H,
J = 7.8 Hz, Ar–H), 7.34 (d, 2H, J = 8.0 Hz, Ar–H), 7.39
(d, 1H, J = 7.6 Hz, Ar–H), 7.47 (t, 1H, J = 7.6 Hz, Ar–H),
7.54 (t, 2H, J = 7.8 Hz, Ar–H), 7.69 (d, 2H, J = 8.4 Hz,
Ar–H), 7.80 (s, 1H, Ar–H), 8.94 (s, 1H, C5–H pyridine),
10.48 (s, 1H, –NH D2O exch.); 13C NMR (100 MHz,
DMSO-d6, d, ppm): 162.3, 157.3, 154.8, 154.2, 151.1, 143.5,
141.7, 138.8, 137.9, 129.8, 129.2, 128.7, 128.0, 126.9, 126.3,
124.5, 123.1, 117.7, 115.2, 113.8, 91.6, 67.8, 62.1, 21.8;
LCMS (M/Z): 489.11 [M?]; Anal. Calcd. for C29H20N5ClO:
C, 71.09; H, 4.11; N, 14.29; Found: C, 71.12; H, 4.10;
N, 14.28 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(3-chloro-2-oxo-4-
phenylazetidin-1-yl)-4-(4-methoxyphenyl)nicotinonitrile
(6n)
Yield: 59 %; m.p.: 216–218 °C; IR (mmax, cm-1, KBr):
3,321 (–NH), 3,041, 2,820 (C–H, aromatic, OCH3), 2,225
(CN), 1,686 (CO), 1,588 (C=N), 1,522 (C=C), 748 (C–Cl);
1H NMR (300 MHz, DMSO-d6, d, ppm): 3.86 (s, 3H,
OCH3), 5.04 (d, 1H, J = 7.0 Hz, C4–H azetidinone), 5.46
(d, 1H, J = 6.8 Hz, C3–H azetidinone), 7.08 (d, 2H,
J = 7.6 Hz, Ar–H), 7.17 (d, 2H, J = 8.0 Hz, Ar–H), 7.25
(t, 1H, J = 7.8 Hz, Ar–H), 7.35 (d, 2H, J = 8.0 Hz,
Ar–H), 7.47 (t, 2H, J = 7.8 Hz, Ar–H), 7.66 (d, 2H,
J = 8.2 Hz, Ar–H), 7.77 (d, 2H, J = 7.6 Hz, Ar–H), 8.90
(s, 1H, C5–H pyridine), 10.45 (s, 1H, –NH D2O exch.);
13C NMR (100 MHz, DMSO-d6, d, ppm): 162.1, 161.2,
157.4, 154.8, 154.2, 151.1, 143.5, 141.8, 131.4, 129.7,
128.5, 126.8, 126.2, 123.2, 117.8, 115.3, 114.9, 113.9,
91.5, 67.6, 62.1, 55.7; LCMS (M/Z): 505.14 [M?]; Anal.
Calcd. for C29H20N5ClO2: C, 68.84; H, 3.98; N, 13.84;
Found: C, 68.88; H, 3.99; N, 13.86 %.
6-(1H-Benzo[d]imidazol-2-yl)-2-(3-chloro-2-oxo-4-
phenylazetidin-1-yl)-4-p-tolyl-nicotinonitrile (6l)
Yield: 60 %; m.p.: 224 °C; IR (mmax, cm-1, KBr): 3,322
(–NH), 3,045, 2,928 (C–H, aromatic, CH3), 2,220 (CN),
1,686 (CO), 1,588 (C=N), 1,522 (C=C), 744 (C–Cl);
1H NMR (300 MHz, DMSO-d6, d, ppm): 2.36 (s, 3H, CH3),
5.08 (d, 1H, J = 7.2 Hz, C4–H azetidinone), 5.46 (d, 1H,
J = 6.7 Hz, C3–H azetidinone), 7.18 (d, 2H, J = 8.0 Hz,
Ar–H), 7.25 (t, 1H, J = 7.8 Hz, Ar–H), 7.33 (d, 2H,
J = 8.0 Hz, Ar–H), 7.39 (t, 2H, J = 7.6 Hz, Ar–H), 7.45 (d,
2H, J = 7.6 Hz, Ar–H), 7.52 (t, 2H, J = 7.8 Hz, Ar–H),
7.67 (d, 2H, J = 8.4 Hz, Ar–H), 8.91 (s, 1H, C5–H pyri-
dine), 10.46 (s, 1H, –NH D2O exch.); 13C NMR (100 MHz,
DMSO-d6, d, ppm): 162.2, 157.3, 154.9, 154.1, 151.2, 143.5,
141.6, 136.1, 132.3, 129.5, 128.6, 127.4, 126.7, 126.1, 123.1,
117.5, 115.2, 113.6, 91.5, 67.8, 62.0, 21.1; LCMS (M/Z):
489.14 [M?]; Anal. Calcd. for C29H20N5ClO: C, 71.09;
H, 4.11; N, 14.29; Found: C, 71.13; H, 4.11; N, 14.30 %.
6-(1H-Benzo[d]imidazol-2-yl)-4-(4-chloro-2-
hydroxyphenyl)-2-(3-chloro-2-oxo-4-phenylazetidin-1-
yl)nicotinonitrile (6o)
Yield: 62 %; m.p.: 248 °C; IR (mmax, cm-1, KBr): 3,428
(OH), 3,324 (–NH), 3,053 (C–H, aromatic), 2,230 (CN),
1,692 (CO), 1,596 (C=N), 1,528 (C=C), 767 (C–Cl);
1H NMR (300 MHz, DMSO-d6, d, ppm): 5.08 (d, 1H,
J = 7.1 Hz, C4–H azetidinone), 5.48 (d, 1H, J = 6.7 Hz,
C3–H azetidinone), 7.11 (d, 1H, J = 7.6 Hz, Ar–H), 7.16
(d, 2H, J = 8.0 Hz, Ar–H), 7.22 (d, 2H, J = 8.0 Hz,
Ar–H), 7.28 (t, 1H, J = 8.0 Hz, Ar–H), 7.36 (d, 2H,
J = 7.9 Hz, Ar–H), 7.45 (t, 2H, J = 8.2 Hz, Ar–H), 7.57
6-(1H-Benzo[d]imidazol-2-yl)-2-(3-chloro-2-oxo-4-
phenylazetidin-1-yl)-4-(3-methoxyphenyl)nicotinonitrile
(6m)
Yield: 60 %; m.p.: 202 °C; IR (mmax, cm-1, KBr): 3,320
(–NH), 3,044, 2,820 (C–H, aromatic, OCH3), 2,224 (CN),
123

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Med Chem Res (2014) 23:1725–1741
(d, 1H, J = 7.6 Hz, Ar–H), 7.67 (d, 2H, J = 8.4 Hz,
Ar–H), 8.96 (s, 1H, C5–H pyridine), 9.47 (s, 1H, OH D2O
exch.), 10.49 (s, 1H, –NH D2O exch.); 13C NMR
(100 MHz, DMSO-d6, d, ppm): 162.3, 157.9, 157.3, 156.4,
154.8, 151.3, 143.7, 141.8, 137.4, 130.3, 128.5, 126.9,
126.5, 126.0, 123.2, 121.8, 117.8, 117.1, 115.3, 113.6,
91.7, 67.8, 62.2; LCMS (M/Z): 525.12 [M?]; Anal. Calcd.
for C28H17N5Cl2O2: C, 63.89; H, 3.26; N, 13.30; Found:
C, 63.84; H, 3.24; N, 13.32 %.
mixture should contain 106 CFU/mL organisms. DMSO
and sterilized distilled water were used as negative control
while chloramphenicol (1 U strength) was used as positive
control. Standard drug used in the present study was
‘chloramphenicol’ for evaluating antibacterial activity
which showed 50, 50, 50 and 50 lg/mL MIC against
E. coli, P. aeruginosa, S. aureus and S. pyogenes,
respectively.
Antifungal assay
Antibacterial assay
The newly prepared compounds 6a–o 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
secondary screening to test in a second set of dilution
against all fungi. The fungal activity of each compound
was compared with ketoconazole as a standard drug, which
showed 50, 50 and 50 lg/mL MIC against C. albicans,
A. niger and A. clavatus, respectively. For fungal growth,
in the present protocol, we have used Sabouraud’s dextrose
broth at 28 °C in aerobic condition for 48 h. DMSO and
sterilized distilled water were used as negative control
while ketoconazole (1 U strength) was used as positive
control.
Antibacterial studies of newly synthesized compounds
6a–o were carried out against the representative panel of
Gram-positive [Staphylococcus aureus (MTCC-96) and
Streptococcus pyogenes (MTCC-442)] and Gram-negative
[Escherichia coli (MTCC-443) and and Pseudomonas
aeruginosa (MTCC-1688)] bacteria. All MTCC cultures
were collected from Institute of Microbial Technology,
Chandigarh. The activity of compounds was determined as
per National Committee for Clinical Laboratory Standards
(NCCLS) protocol using Mueller–Hinton Broth (Becton–
Dickinson, USA). 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 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 con-
taining no antibiotic was immediately subcultured [before
inoculation] by spreading a loopful evenly over quarter of
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 spectrophoto-
metrically. Ten lg/mL suspensions were further inoculated
on an appropriate media and growth was noted after 24 and
48 h. The lowest concentration (the 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 the inoculum. The test
Structure–activity relationship
From the results of the antimicrobial activity of the newly
synthesized compounds 6a–o, the following structure–
activity relationships may be derived: in general, com-
pounds 6a–o showed improved antibacterial activity com-
pared to the antifungal activity and deduced patterns of
their antimicrobial activity are in the following order:
antibacterial [ antifungal. Moreover, antibacterial activity
was considerably affected by substitution pattern on the
phenyl ring and the most active compounds contain elec-
tron-withdrawing substituents at ortho, meta and para
positions of the phenyl ring (p [ m [ o). The compounds
6d and 6i, substituted with chloro and nitro group,
respectively, at para position showed the highest antibac-
terial activity. On the other hand, the presence of the same
functional groups at meta position resulted in slight
decrease in the antimicrobial activity (6c and 6h) but still
produced potent inhibitory action compared to the standard
drug chloramphenicol. While, substituting the phenyl ring
with chloro group at ortho position resulted in a noticeable
decrease in the antimicrobial activity of compound 6b.
Furthermore, compounds having electron-withdrawing
substituents (Cl and NO2) at meta position possessed the
highest antifungal activity compared to ortho- or para-
123

Med Chem Res (2014) 23:1725–1741
1739
substituted compounds. The compound 6c having chloro
substituent at meta position showed the most potent anti-
fungal activity. In contrast, the presence of electron-
releasing groups on the phenyl ring witnessed a substantial
decrease in antimicrobial activity for compounds 6a, 6e–6g
and 6j–6o. The role of electron-withdrawing groups in
improving antimicrobial activity is very well supported by
previous studies (Sharma et al., 2004, 2009). Moreover, on
the basis of activity results, it was observed that the
introduction of 2-azetidinone was also responsible for
enhancing antimicrobial potency, which was confirmed by
the comparison of antimicrobial activity of intermediates
4a–o and 5a–o with final compounds 6a–o. Compounds
6b, 6c, 6d, 6h and 6i were found to be most potent anti-
bacterial agents, whereas compounds 6c and 6h emerged as
most effective antifungal agents indicating that the struc-
tural requirements are different for binding of drug to
bacterial or fungal targets, respectively (Sortino et al.,
2007).
accounted no toxicity at concentration of 100 lM
(IC50 [ 100 lM), while other derivatives displayed mod-
erate toxicity against HeLa cell lines. It was confirmed that
none of the tested compounds exhibited any significant
cytotoxic effects on HeLa cell lines, suggesting that com-
pounds were potential for their in vivo use as antimicrobial
agents.
Conclusion
On the basis of the biological activity results, we may
conclude that the synthesis and screening of antimicrobial
activity of some new 2-azetidinone derivatives of pyridyl
benzimidazoles with the hope of discovering new bioactive
molecules that could be useful as potent antimicrobial
agents. The focus has been given for the synthesis of
structural hybrids comprising basically benzimidazole
moiety attached to pyridyl and 2-azetidinones. All the
synthesized compounds were characterized by IR,
1H NMR, 13C NMR and mass spectrometry techniques.
The final compounds 6a–o showed antimicrobial efficacy
considerably greater than the intermediate compounds
4a–o and 5a–o. From the activity data, it was observed that
the generation of 2-azetidinone nucleus was accountable
for enhancing antimicrobial potency. As discussed from the
SAR and toxicity studies, we were encouraged to make
modifications in electronic diversity on the basic structure
of final compounds 6a–o for the generation of non-toxic
antimicrobial agents. Moreover, the presence of different
substituents cause a certain change of activity. In addition,
it may also be concluded that the promising activities of
these title compounds could also be attributed to the
combination of three different heterocyclic scaffolds viz.,
benzimidazole, cyanopyridine and 2-azetidinone. Many of
the synthesized motifs (6b, 6c, 6d, 6h and 6i) possessing
electron-withdrawing groups such as chloro and nitro were
identified as most potent antimicrobial agents compared to
the standard drugs with relatively low level of cytotoxicity.
The results described here merit further investigations in
our laboratories using a forward chemical approach for
finding lead molecules as antimicrobial agents.
In vitro cytotoxicity assay
In vitro cytotoxic activity of newly synthesized compounds
(6a–o) was evaluated against human cervical cancer cell
line (HeLa) by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-
nyltetrazolium bromide (MTT) colorimetric assay (Mos-
mann, 1983), which measures the reduction of the
tetrazolium bromide salt into a formazan dye by mito-
chondrial dehydrogenases in treated versus untreated cells.
The IC50 values obtained for these compounds are shown
in Table 2. IC50 values were determined in triplicate at 24,
48 and 72 h of drug incubation. In vitro cytotoxic activity
results revealed that the derivatives 6b, 6c, 6d, 6h and 6i
Table 2 Levels of cytotoxicity
induced by compounds 6a–o on
HeLa cells
Entry
Cytotoxicity
a
(IC50
)
HeLa
6a
6b
6c
6d
6e
6f
81
[100
[100
[100
78
86.50
67.40
[100
[100
66
Acknowledgments 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.
6g
6h
6i
6j
6k
6l
78.20
74.50
64
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