Synthesis, antimicrobial, and cytotoxic activities of novel benzimidazole derivatives bearing cyanopyridine and 4-thiazolidinone motifs

Article abstract of DOI:10.1007/s00044-014-0971-7

A series of 6-(1H-benzo[d]imidazol-2-yl)-2-(2-(3-nitrophenyl)-4- oxothiazolidin-yl)-4-(aryl)nicotinonitriles 5a-l were synthesized and characterized by IR, ¹H NMR, ¹³C NMR, and mass spectrometry techniques. These novel compounds 5a-l were screened for their in vitro antimicrobial activity against different bacterial and fungal strains and in vitro cytotoxicity study (HeLa cell line) using MTT colorimetric assay. The results demonstrated that compounds 5c , 5e, and 5i-k exhibited excellent antibacterial activity, while compounds 5d, 5i, and 5k were found to be the most potent antifungal agents. From the standpoint of SAR studies, it was observed that the presence of electron donating groups remarkably enhanced the antimicrobial activity of newly synthesized compounds. Further, the results of preliminary MTT cytotoxicity studies on HeLa cells suggested that potent antimicrobial activity of 5c-e and 5i-k was accompanied by low cytotoxicity. Springer Science+Business Media 2014.

Full text of DOI:10.1007/s00044-014-0971-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-0971-7
ORIGINAL RESEARCH
Synthesis, antimicrobial, and cytotoxic activities of novel
benzimidazole derivatives bearing cyanopyridine
and 4-thiazolidinone motifs
•
N. C. Desai D. D. Pandya K. A. Bhatt
•
•
•
G. M. Kotadiya Priyanka Desai
Received: 10 October 2013 / Accepted: 17 February 2014
Ó Springer Science+Business Media New York 2014
Abstract A series of 6-(1H-benzo[d]imidazol-2-yl)-2-(2-
(3-nitrophenyl)-4-oxothiazolidin-yl)-4-(aryl)nicotinonitriles
Suree et al., 2007). Therefore, development of new types
of antibacterial agents is a very important task, and much
of the research effort is oriented to the design of new
antibacterial agents with high efficiency (Cui et al., 2005).
It is a well-known fact that more efficacious antibacterial
agents can be designed by joining two or more biologically
active heterocyclic systems together in a single molecular
framework (Xie and Seto, 2007) i.e., benzimidazole, cya-
nopyridine, and thiazolidinone. These synergistic antimi-
crobial combinations have several major advantages,
including the potential to slow down development of drug
resistance, a broader antimicrobial spectrum, and potential
reduction in the dose and undesired side effects of each
drug (Bennett et al., 1979).
1
5a–l were synthesized and characterized by IR, H NMR,
13C NMR, and mass spectrometry techniques. These novel
compounds 5a–l were screened for their in vitro antimi-
crobial activity against different bacterial and fungal strains
and in vitro cytotoxicity study (HeLa cell line) using MTT
colorimetric assay. The results demonstrated that com-
pounds 5c, 5e, and 5i–k exhibited excellent antibacterial
activity, while compounds 5d, 5i, and 5k were found to be
the most potent antifungal agents. From the standpoint of
SAR studies, it was observed that the presence of electron
donating groups remarkably enhanced the antimicrobial
activity of newly synthesized compounds. Further, the
results of preliminary MTT cytotoxicity studies on HeLa
cells suggested that potent antimicrobial activity of 5c–
e and 5i–k was accompanied by low cytotoxicity.
Benzimidazole scaffold is a useful structural motif for
displaying chemical functionality in biologically active
molecules, due to various activities like antihelmintic
(Valdez et al., 2002), antihistaminic (Iemura et al., 1986),
anticancer (Tong et al., 2009), antiviral (Li et al., 2006),
anti-inflammatory (Achar et al., 2010), antiproliferative
Keywords Antimicrobial activity Á Benzimidazole Á
Cyanopyridine Á Cytotoxicity Á MTT assay Á Thiazolidinone
´
(Sann et al., 2006), antioxidant (Kalai et al., 2009), and
anticoagulant (Mederski et al., 2004). This ring system is
also present in numerous antiparasitic and antitumoral
drugs (Boiani and Gonzalez, 2005; Iwao et al., 2004).
Furthermore, pyridine derivatives occupy a pivotal position
in modern heterocyclic chemistry, and consequently, pyr-
idine substructure is one of the most important heterocycles
found in natural products, pharmaceuticals, and functional
materials (Teague, 2008; Movassaghi et al., 2007). Pyri-
dine derivatives containing multi-functional groups such as
streptonigrin, streptonigrone, and lavendamycin are repor-
ted as anticancer drugs, and cerivastatin is reported as
HMGCoA enzyme inhibitor (Bringmann et al., 2004).
Moreover, substituted pyridines are reported as leukotriene
B-4 antagonists (Zhou et al., 2008). On the other hand,
Introduction
The increasing incidences of infection caused by rapid
development of bacterial resistance to most of the known
antibiotics are a serious health problem (Chu et al., 1996;
N. C. Desai (&) Á D. D. Pandya Á K. A. Bhatt Á
G. M. Kotadiya Á Priyanka Desai
Division of Medicinal Chemistry, Department of Chemistry,
UGC NON-SAP & DST-FIST Sponsored Department,
Mahatma Gandhi Campus, Maharaja Krishnakumarsinhji
Bhavnagar University, Bhavnagar 364002, India
e-mail: dnisheeth@rediffmail.com
123

Med Chem Res
cyanopyridone and cyanopyridine derivatives have prom-
ising antimicrobial (Hammam et al., 2000) and anticancer
activities (Abo-Ghalia et al., 2003).
compounds to inhibit cell growth. Structural relevance of
title compounds 5a–l with previously synthesized com-
pounds is shown in Fig. 1.
Numerous thiazolidinone derivatives have shown sig-
nificant bioactivities such as antimicrobial (Gouveia and de
Oliveira, 2009), antidiabetic (Noboyoshi and Heroaki,
2006), anticancer (Wu et al., 2006), anti HIV (Rawal
et al., 2005), etc. Currently, 4-thiazolidinones are consid-
ered as a new class of antidiabetic (insulin-sensitizing)
drug and potent aldose reductase inhibitors. In addition,
4-thiazolidinone nucleus appears frequently in compounds
possessing cardiac and glycemic benefits such as troglit-
azone (Ghazzi et al., 1997). Mode of action of 4-thiazo-
lidinones is a novel inhibition of the bacterial enzyme Mur
B which is precursor acting during the biosynthesis of
peptidoglycan (essential component of the cell wall of
bacteria) (Andres et al., 2000).
Results and discussion
Chemistry
The synthetic strategies adopted to obtain the target com-
pounds are depicted in Scheme 1. Compounds 1-(1H-
benzo[d]imidazol-2-yl)-3-(aryl)prop-2-en-1-ones 2a–l served
as convenient intermediates in the synthesis, which were pre-
pared according to the literature method (Shaharyar et al.,
2010; Ouattara et al., 2011). Compounds 2a–l were treated
with ammonium acetate and malononitrile to achieve deriva-
tives 3a–l, produced via well-known Knoevenagel condensa-
tion reaction. In compounds 3a–l, we observed primary amine
and nitrile groups as well as the disappearance of a, b-unsat-
urated carbonyl group which was confirmed by IR and NMR
spectra. Resultant compounds 3a–l underwent simple con-
densation reaction with 3-nitrobenzaldehyde to provide good
yieldofSchiff bases4a–l whichshowedsuccessfulconversion
of primary amine group into azomethine group. The disap-
pearance of NH2 signals in the FT-IR and 1H NMR supported
the condensation leading to the formation of compounds
4a–l. It is interesting to note that the compounds having azo-
methine structure may exist as E/Z geometrical isomers about
C=N double bond and as cis/trans imine conformers (Galic
et al., 2001; Wyrzykiewicz and Prukah, 1998; Mentese et al.,
2013). According to the literature (Wyrzykiewicz and Prukah,
1998), compounds containing [C=N– bond are present in
higherpercentageindimethyl-d6 sulfoxidesolutionin the form
of geometrical E isomer about [C=N– double bond. The
Z isomer can be stabilized in less polar solvents by an
intramolecular hydrogen bond. In the present study, the
spectral data were obtained in dimethyl-d6 sulfoxide solution,
and no signal belonging to Z isomer was observed. This azo-
methine proton appeared as a singlet atd 8.31–8.42 ppm in the
In this context, our research group had reported the
synthesis of 1-(1-(1H-benzo[d]imidazol-2-yl)ethylidenea-
mino)-6-(arylideneamino)-4-(2-hydroxyphenyl)-2-oxo-1,2-
di-hydropyridine-3,5-dicarbonitriles NCD1–11 (Desai et al.,
2012) containing benzimidazole and 2-pyridone nucleus,
and recently our group also reported the synthesis of N-(5-
(aryl)-2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)-4-(4-oxo-2-
ptolylquinazolin-3(4H)-yl)benzamides NCD1–20 (Desai
et al., 2013) and derived its antimicrobial activity. Consid-
ering this fact, in the present communication, we have
reported the synthesis of 6-(1H-benzo[d]imidazol-2-yl)-2-
(2-(3-nitrophenyl)-4-oxothiazolidin-3-yl)-4-(aryl)nicotino-
nitriles 5a–l by introducing both the cyanopyridine and
4-thiazolidinone nucleus in the second position of benz-
imidazole nucleus. We envision our approach toward the
design and synthesis of novel structurally diverse series of
6-(1H-benzo[d]imidazol-2-yl)-2-(2-(3-nitrophenyl)-4-oxo-
thiazolidin-yl)-4-(aryl)nicotinonitriles 5a–l derivatives
bearing above afore mentioned moieties in single molec-
ular framework in order to investigate their in vitro anti-
microbial activity. Cytotoxicity studies were also
conducted in HeLa cell lines to evaluate the ability of these
Fig. 1 Structural relevance of title compounds 5a–l with previously synthesized compounds NCD1–11 and NCD1–20
123

Med Chem Res
Scheme 1 Synthetic route for the preparation of title compounds
5a–l. Reagents and conditions: (a) EtOH, KOH, stirring, 10 h;
(b) Malononitrile, ammonium acetate, EtOH, reflux, 10 h; (c) 3-
Nitrobenzaldehyde, anhy. ZnCl2, 1,4-dioxane, reflux, 6 h;
(d) SHCH2COOH, 1,4-dioxane, reflux, 12 h
1H NMR spectra and the carbon of resulting compounds res-
onated at d 158.5–158 ppm in the 13C NMR spectra. The
additional support for the formation of derivatives, 4a–l, was
obtained by the appearance of (M?1) ion peaks at corre-
sponding m/z values confirming their molecular masses, and
these compounds gave elemental analysis results consistent
with the proposed structures. In the last step, azomethine
derivatives 4a–l generated finalanalogs5a–l using cyclization
with thioglycolic acid.
reactive methylene group, and methine carbon (asymmetric
carbon) appeared at d 21.4, 33.6, and 71.2 ppm, respectively.
Carbonyl carbon of thiazolidinone ring showed a chemical
shift at d 171.3 ppm, whereas carbon of cyanide group
showed chemical shift value at d 113.8 ppm. The mass
spectrum of 5j showed molecular ion peak at m/z = 532.13
(M?1), which is in agreement with the molecular formula
C29H20N6O3S. Similarly, spectral values for all the com-
pounds and C, H, and N analysis are presented in the
experimental part.
Formation of 6-(1H-benzo[d]imidazol-2-yl)-2-(2-(3-
nitrophenyl)-4-oxothiazolidin-yl)-4-(aryl)nicotinonitriles
5a–l was confirmed on the basis of their IR, H NMR, 13
1
C
Antibacterial activity
NMR spectroscopy, and mass spectrometry. IR spectrum of
compound 5j showed absorption band at 3,440 cm-1 which
was due to N–H stretching. Strong absorption bands at 1,698
and 2,337 cm-1 were observed due to[C=O of thiazolidi-
none ring and cyanide group, respectively. Similarly, strong
stretching vibrations at 3,030 and 1,535 cm-1 were
responsible for C–H bond in reactive methylene group and
N=O bond in –NO2 group, respectively. In addition, the 1H
NMR spectrum of 5j showed a singlet for three protons at d
2.34 ppm due to CH3 group. Two singlets appeared at d 3.95
and 6.44 ppm due to protons of reactive methylene group
and proton attached with asymmetric carbon, respectively.
Aromatic protons displayed multiplets at d 7.22–8.12 ppm.
The singlet signal observed at d 10.25 ppm integrating for
one proton was assigned to –NH group. Moreover, in 13C
NMR spectra of compound 5j, the carbon atoms of methyl,
All the newly synthesized compounds 5a–l were initially
screened for their 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 (Hannan, 2000). The MIC (minimum inhibitory
concentration) values were determined by comparison to
standard ‘‘chloramphenicol’’ as reference drug for evaluat-
ing antibacterial activity which showed MIC of 50 lg/mL
against E. coli, P.aeruginosa, S. aureus, and S. pyogenes.
The results of antibacterial activity are presented in Table 1.
The obtained antimicrobial activity of tested compounds
could be correlated to structural variations. Intermediates
3a–l showed poor antimicrobial activity against all tested
123

Med Chem Res
basis of antibacterial evaluation, compound 5i (3-OCH3-4-
OH) was the most active among the tested derivatives against
E. coli at MIC = 12.5 lg/mL. Compounds 5d (3-OH) and 5l
(4-N(CH3)2) showed good activity at MIC = 100 lg/mL
against all the bacterial strains except S. aureus, whereas
compounds 5c (2-OH) and 5e (4-OH) were found to exhibit
good activity against S. aureus at MIC = 100 lg/mL. When
electron releasing groups (OH, CH3, OCH3) at 4th position
i.e., compounds 5e (4-OH), 5j (4-CH3), and 5k (4-OCH3),
activity increased and all compounds displayed very good
activity at MIC = 50 lg/mL against both E. coli and S. py-
ogenes, while compounds 5e (4-OH) and 5j (4-CH3) also
flaunted very good inhibition at MIC = 50 lg/mL against
P. aeruginosa. Compounds 5c (2-OH)and 5i (3-OCH3-4-OH)
displayed inhibition at MIC = 50 lg/mL against P. aeru-
ginosa and S. aureus, respectively. Similarly, compound 5k
(4-OCH3) showed excellent activity at MIC = 25 lg/mL
against P. aeruginosa and S. aureus, while compound 5j
(4-CH3) exhibited excellent inhibition against S. aureus. In
addition, compound 5i (3-OCH3-4-OH) was found to possess
excellent activity against P. aeruginosa and S. pyogenes at
MIC = 25 lg/mL as compared to the standard chloram-
phenicol.
Table 1 In vitro antimicrobial screening results of compounds 5a–l
Entry -R
Minimum inhibitory concentration (MIC
[lg/mL])
Gram-
positive
bacteria
Gram-
negative
bacteria
Fungi
Sa
Sp
Ec
Pa
Ca
An Ac
5a
5b
5c
5d
5e
5f
2H
1000 1000 500 500 500 500 500
200 200 250 250 500 250 250
-2-Cl
-2-OH
-3-OH
-4-OH
-2-NO2
-3-NO2
-4-NO2
100 62.5 62.5 50
250 100 100 100 100 50 50
100 50 50 50 250 100 100
250 100 100
500 125 250 250 1000 250 250
1000 250 250 500 500 250 250
500 250 250 250 500 250 250
5g
5h
5i
-3-OCH3-4-OH 50
25
50
50
12.5 25
50
25 12.5
5j
-4-CH3
25
25
50
50
50
25
250 100 100
100 50 50
5k
5l
-4-OCH3
-4-N(CH3)2
250 100 100 100 250 100 100
Chloramphenicol
Ketoconazole
50
–
50
–
50
–
50
–
–
–
–
50
50 50
Sa Staphylococcus aureus MTCC 96, Sp Staphylococcus pyogenes
MTCC 442, Ec Escherichia coli MTCC 443, Pa Pseudomonas
aeruginosa MTCC 1688, Ca Candida albicans MTCC 227, An
Aspergillus niger MTCC 282, Ac Aspergillus clavatus MTCC 1323
Antifungal activity
The in vitro antifungal activity of newly synthesized com-
pounds 5a–l was determined using conventional broth
dilution method (Hannan, 2000). In this work, C. albicans,
A. niger, and A. clavatus were used as standard strains to
investigate the activity. The fungal activity of each com-
pound was compared with ketoconazole as a standard drug,
which showed 50 lg/mL MIC against C. albicans, A. niger,
and A. clavatus. 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 ketoconazole (1 U strength)
was used as a positive control. The results of antifungal
activity are presented in Table 1. The results indicated that,
among the tested compounds, 5c (2-OH), 5e (4-OH), 5j
(4-CH3), and 5l (4-N(CH3)2) showed good inhibition against
all the fungal strains, whereas compounds 5d (3-OH) and 5k
(4-OCH3) exhibited very good activity against both
C. albicans and A. niger, respectively. Compound 5i (3-OCH3-
4-OH) showed the highest inhibition against C. albicans at
MIC = 50 lg/mL, while compound 5i (3-OCH3-4-OH)
showed enhanced activity against A. niger at MIC = 25 lg/
mL. Compound 5i (3-OCH3-4-OH) exhibited MIC at 12.5 lg/
mL against A. clavatus which showed the highest activity
among the tested compounds of the series. The activity of the
series of compounds was compared with the standard drug
ketoconazole.
bacterial and fungal strains, among which compounds 3c,
3e, and 3i showed poor activity (MIC = 1 000 lg/mL
against P. aeruginosa and S. pyogenes) and compounds 3d
and 3i displayed poor antifungal activity (MIC = 1 000 lg/
mL against A. clavatus). Precursors 3a–l reacted with
m-nitrobenzaldehyde to generate key scaffolds 4a–l. Further,
these intermediates were tested against different bacterial and
fungal strains and were found to have mild to poor antimi-
crobial activity. Intermediate derivatives 4c, 4e, and 4i
displayed mild activity at MIC = 500 lg/mL against
P. aeruginosa and S. pyogenes, while intermediates 4j and 4k
exhibited mild activity against E. coli and S. aureus at
MIC = 500 lg/mL. Similarly, compounds 4d, 4i, and 4k
exhibited mild activity against A. niger and A. clavatus at
MIC = 500 lg/mL. Now, Schiffbases4a–l weretreatedwith
thioglycolic acid to afford final derivatives 5a–l having
4-thiazolidinone motif, which were found to have broad
spectrum antimicrobial activity. In addition, activity results
revealed that some of the tested compounds showed excellent
inhibitionagainst various testedmicrobialstrains comparedto
the standard drug. From antibacterial activity data, it was
observed that compounds 5c (2-OH), 5e (4-OH), 5i (3-OCH3-
4-OH),5j (4-CH3),and5k (4-OCH3)weremostpotentagainst
all bacterial strains in the range of 12.5–100 lg/mL. On the
123

Med Chem Res
Elemental analysis (% C, H, N) was carried out by a Per-
kin–Elmer 2400 CHN analyzer. IR spectra of all com-
Table 2 Levels of cytotoxicity induced by compounds 5a–l on HeLa
cells
pounds were recorded on
a
Perkin–Elmer FT-IR
Entry
Cytotoxicity IC50 (lM)
HeLa
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.
5a
61.32
74.20
[100
[100
[100
65.12
78.34
58.55
[100
[100
[100
91.70
3.24
5b
5c
5d
5e
5f
5g
General method for the preparation of 1-(1H-
5h
benzo[d]imidazol-2-yl)-3-(aryl)prop-2-en-1-ones (2a–l)
5i
5j
Compounds 1-(1H-benzo[d]imidazol-2-yl)-3-(aryl)prop-2-
en-1-ones 2a–l are prepared according to the literature
method (Shaharyar et al., 2010; Ouattara et al., 2011).
5k
5l
Doxorubicin
General method for the preparation of 2-amino-6-(1H-
benzo[d]imidazol-2-yl)-4-(aryl)nicotinonitriles (3a–l)
IC50 is the concentration required to inhibit 50 % of cell growth
HeLa human cervical cancer cell line
A mixture of an equimolar amount of compound 2a–l and
malononitrile (0.01 mol) was refluxed in ethanol contain-
ing ammonium acetate (0.01 mol) for 10 h. The reaction
mixture was cooled and poured onto crushed ice. The
product obtained was isolated and crystallized from
benzene.
In vitro cytotoxicity studies
In vitro cytotoxic activity of newly synthesized compounds
5a–l was evaluated against human cervical cancer cell line
(HeLa) by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-
tetrazolium bromide (MTT) colorimetric assay (Mosmann,
1983; Alley et al., 1988), which measures the reduction of
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. Cytotoxicity results revealed that the deriva-
tives 5c (2-OH), 5d (3-OH), 5e (4-OH), 5i (3-OCH3-4-
OH), 5j (4-CH3), and 5k (4-OCH3) accounted no toxicity at
concentration of 100 lg/mL (IC50 [ 100 lg/mL), while
other derivatives showed moderate toxicity against HeLa
cell lines. It was confirmed that none of the tested com-
pounds exhibited any significant cytotoxic effects on HeLa
cell line, suggesting that compounds were potential for
their in vivo use as antimicrobial agents.
2-amino-6-(1H-benzo[d]imidazol-2-yl)-4-
phenylnicotinonitrile (3a)
White crystals (MeOH); m.p.: 235 °C; IR (KBr) tmax 3462,
3380, 2922, 2350, 1528, 1565, 1610 cm-1 1H NMR
;
(DMSO-d6, 400 MHz): = 10.25 (s, 1H, –NH), 8.25 (s,
1H, C5–H pyridine ring), 7.70 (s, 2H, Ar–NH2), 7.15–7.80
(m, 9H, Ar–H); 13C NMR (DMSO-d6, 100 MHz):
=
161.9 (C, C–NH2), 158.8 (C, C=N), 154.7 (C, C-2 benz-
imidazole), 113.5 (C, –C:N), 85.8 (C, C–C:N);
LCMS: m/z = 311.12 (M?); Anal. Calcd. for C19H13N5:
C, 73.30; H, 4.21; N, 22.49. Found: C, 73.42; H, 4.34; N,
22.65.
Experimental
2-amino-6-(1H-benzo[d]imidazol-2-yl)-4-(2-
chlorophenyl)nicotinonitrile (3b)
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 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 and 230–400
mesh ASTH for flash chromatography) was applied when
necessary to isolate and purify the reaction products.
Yellowish crystals (MeOH); m.p.: 219 °C, IR (KBr) tmax
1
3460, 3380, 2919, 2347, 1528, 1565, 1615, 724 cm-1; H
NMR (DMSO-d6, 400 MHz): = 10.24 (1H, s, –NH), 8.26
(1H, s, C5–H pyridine ring), 7.72 (2H, s, Ar–NH2),
7.25–7.60 (8H, m, Ar–H); 13C NMR (DMSO-d6,
100 MHz): = 161.9 (C, C–NH2), 158.7 (C, C=N), 154.7
(C, C-2 benzimidazole), 132.0 (C, C–Cl), 113.6 (C,
–C:N), 85.6 (C, C–C:N); LCMS: m/z = 345.08 (M?);
123

Med Chem Res
Anal. Calcd. for C19H12N5Cl: C, 66.00; H, 3.50; N, 20.25.
Found: C, 66.40; H, 4.24; N, 22.59.
(C, C–NH2), 154.7 (C, C–2 benzimidazole), 145.4 (C,
C–NO2), 113.8 (C, –C:N), 85.5 (C, C–C:N); LCMS:
m/z = 356.10 (M?); Anal. Calcd. for C19H12N6O2: C,
64.04; H, 3.39; N, 23.58. Found: C, 64.47; H, 3.34; N,
23.61.
2-amino-6-(1H-benzo[d]imidazol-2-yl)-4-(2-
hydroxyphenyl)nicotinonitrile (3c)
Yellowish crystals (MeOH); m.p.: 219 °C, IR (KBr) tmax
3635, 3435, 3373, 2923, 2340, 1528, 1565, 1598 cm-1; 1H
NMR (DMSO-d6, 400 MHz): = 10.25 (1H, s, –NH), 8.24
(1H, s, C5–H pyridine ring), 7.21–8.40 (8H, m, Ar–H), 7.73
(2H, s, Ar–NH2), 5.34 (1H, s, Ar–OH); 13C NMR (DMSO-
d6, 100 MHz): = 161.9 (C, C–NH2), 155.1 (C, C–OH),
154.7 (C, C-2 benzimidazole), 113.7 (C, –C:N), 85.6 (C,
C–C:N); LCMS: m/z = 327.34 (M?); Anal. Calcd. for
C19H13N5O: C, 69.71; H, 4.00; N, 21.39. Found: C, 69.68;
H, 4.14; N, 21.37.
2-amino-6-(1H-benzo[d]imidazol-2-yl)-4-(3-
nitrophenyl)nicotinonitrile (3g)
Yellow crystals (MeOH); m.p.: 225 °C, IR (KBr) tmax
3436, 3368, 2923, 2342, 1527, 1565, 1591, 1512 cm-1; 1H
NMR (DMSO-d6, 400 MHz): = 10.25 (1H, s, –NH), 8.16
(1H, s, C5–H pyridine ring), 7.22–8.27 (8H, m, Ar–H), 7.74
(2H, s, Ar–NH2); 13C NMR (DMSO-d6, 100 MHz): =
161.9 (C, C–NH2), 154.7 (C, C-2 benzimidazole), 148.4
(C, C–NO2), 113.9 (C, –C:N), 85.5 (C, C–C:N); LCMS:
m/z = 356.10 (M?); Anal. Calcd. for C19H12N6O2: C,
64.04; H, 3.39; N, 23.58. Found: C, 64.47; H, 3.34; N,
23.61.
2-amino-6-(1H-benzo[d]imidazol-2-yl)-4-(3-
hydroxyphenyl)nicotinonitrile (3d)
White crystals (MeOH); m.p.: 245 °C, IR (KBr) tmax 3621,
3440, 3350, 2943, 2350, 1524, 1569, 1587 cm-1; 1H NMR
(DMSO-d6, 400 MHz): = 10.28 (1H, s, –NH), 8.16 (1H,
s, C5–H pyridine ring), 7.22–8.65 (8H, m, Ar–H), 7.73 (2H,
s, Ar–NH2), 5.35 (1H, s, Ar–OH); 13C NMR (DMSO-d6,
100 MHz): = 161.9 (C, C–NH2), 157.5 (C, C–OH), 154.7
(C, C-2 benzimidazole), 113.7 (C, –C:N), 85.6 (C, C–
C:N); LCMS: m/z = 327.11 (M?); Anal. Calcd. for
C19H13N5O: C, 69.71; H, 4.00; N, 21.39. Found: C, 69.70;
H, 4.14; N, 21.41.
2-amino-6-(1H-benzo[d]imidazol-2-yl)-4-(4-
nitrophenyl)nicotinonitrile (3h)
Light yellow crystals (MeOH); m.p.: 225 °C, IR (KBr) tmax
3437, 3370, 2928, 2340, 1525, 1564, 1593, 1516 cm-1; 1H
NMR (DMSO-d6, 400 MHz): = 10.24 (1H, s, –NH), 8.16
(1H, s, C5–H pyridine ring), 7.22–8.32 (8H, m, Ar–H), 7.74
(2H, s, Ar–NH2); 13C NMR (DMSO-d6, 100 MHz): =
161.9 (C, C–NH2), 154.7 (C, C-2 benzimidazole), 148.4 (C,
C–NO2), 113.8 (C, –C:N), 85.6 (C, C–C:N); LCMS: m/
z = 356.10 (M?); Anal. Calcd. for C19H12N6O2: C, 64.04;
H, 3.39; N, 23.58. Found: C, 64.47; H, 3.34; N, 23.61.
2-amino-6-(1H-benzo[d]imidazol-2-yl)-4-(4-
hydroxyphenyl)nicotinonitrile (3e)
2-amino-6-(1H-benzo[d]imidazol-2-yl)-4-(4-hydroxy-3-
Light yellow crystals (MeOH); m.p.: 249 °C, IR (KBr)
tmax 3623, 3430, 3370, 2920, 2342, 1527, 1565,
methoxyphenyl)nicotinonitrile (3i)
1591 cm-1
;
1H NMR (DMSO-d6, 400 MHz): = 10.26
White crystals (CHCl3); m.p.: 231 °C, IR (KBr) tmax 3621,
1
(1H, s, –NH), 8.16 (1H, s, C5–H pyridine ring), 7.73 (2H, s,
Ar–NH2), 6.86–7.62 (8H, m, Ar–H), 5.36 (1H, s, Ar–OH);
13C NMR (DMSO-d6, 100 MHz): = 161.9 (C, C–NH2),
159.0 (C, C–OH), 154.7 (C, C-2 benzimidazole), 113.7 (C,
–C:N), 85.6 (C, C–C:N); LCMS: m/z = 327.11 (M?);
Anal. Calcd. for C19H13N5O: C, 69.71; H-4.00; N, 21.39.
Found: C, 69.68; H, 4.14; N, 21.37.
3436, 3368, 2829, 2923, 2342, 1527, 1565, 1591 cm-1; H
NMR (DMSO-d6, 400 MHz): = 10.25 (1H, s, –NH), 8.16 (1H,
s, C5–H pyridine ring), 6.89–8.16 (7H, m, Ar–H), 7.74 (2H, s,
Ar–NH2), 5.36 (1H, s, Ar–OH), 3.83 (3H, s, Ar–O–CH3–); 13
C
NMR (DMSO-d6, 100 MHz): = 161.9 (C, C–NH2), 154.7 (C,
C-2 benzimidazole), 149.2 (C, C–OH), 148.0 (C, C–OCH3),
113.7 (C, –C:N), 85.6 (C, C–C:N), 56.1 (C, –OCH3);
LCMS: m/z = 357.12 (M?); Anal. Calcd. for C20H15N5O2: C,
67.22; H, 4.23; N, 19.60. Found: C, 67.27; H, 4.27.
2-amino-6-(1H-benzo[d]imidazol-2-yl)-4-(2-
nitrophenyl)nicotinonitrile (3f)
2-amino-6-(1H-benzo[d]imidazol-2-yl)-4-p-
Yellow amorphous; m.p.: 255 °C, IR (KBr) tmax 3439,
3373, 2932, 2345, 1529, 1568, 1595, 1515 cm-1; 1H NMR
(DMSO-d6, 400 MHz): = 10.27 (1H, s, –NH), 8.16 (1H,
s, C5–H pyridine ring), 7.22–8.05 (8H, m, Ar–H), 7.74 (2H,
s, Ar–NH2); 13C NMR (DMSO-d6, 100 MHz): = 161.9
tolylnicotinonitrile (3j)
Brown amorphous; m.p.: 240–243 °C, IR (KBr) tmax 3463,
3370, 2951, 2922, 2348, 1528, 1565, 1610 cm-1; 1H NMR
(DMSO-d6, 400 MHz): = 10.25 (1H, s, –NH), 8.19 (1H,
123

Med Chem Res
s, C5–H pyridine ring), 7.20–7.80 (8H, m, Ar–H), 7.72 (2H,
s, Ar–NH2), 2.34 (3H, s, Ar–CH3); 13C NMR (DMSO-d6,
100 MHz): = 161.9 (C, C–NH2), 154.7 (C, C-2 benz-
imidazole), 132.2 (C, C–CH3), 113.8 (C, –C:N), 85.8 (C,
C–C:N), 21.3 (C, –CH3); LCMS: m/z = 325.13 (M?);
Anal. Calcd. for C20H15N5: C, 73.83; H, 4.65; N, 21.52.
Found: C, 73.42; H, 4.60; N, 21.65.
(DMSO-d6, 400 MHz): = 10.27 (1H, s, –NH), 8.90 (1H,
s, C5–H pyridine ring), 8.35 (1H, s, CH=N), 7.22–8.52
(13H, m, Ar–H); 13C NMR (DMSO-d6, 100 MHz): =
178.7 (C, –N–C–N), 158.8 (C, –C=N), 154.7 (C, C-2
benzimidazole), 148.0 (C, C–NO2), 117.3 (C, –C:N),
98.2 (C, C–C:N); LCMS: m/z = 444.13 (M?); Anal.
Calcd. for C26H16N6O2: C, 70.26; H, 3.63; N, 18.91.
Found: C, 70.30; H, 3.68; N, 19.0.
2-amino-6-(1H-benzo[d]imidazol-2-yl)-4-(4-
methoxyphenyl)nicotinonitrile (3k)
6-(1H-benzo[d]imidazol-2-yl)-4-(2-chlorophenyl)-2-((3-
nitrobenzylidene)amino)nicotino-nitrile (4b)
Pale yellow crystals (MeOH); m.p.: 260–262 °C, IR (KBr)
t
max 3466, 3365, 2922, 2828, 2342, 1530, 1566, 1613 cm-1
;
Yellow crystals (CHCl3); m.p.: 219 °C, IR (KBr) tmax
1H NMR (DMSO-d6, 400 MHz): = 10.23 (1H, s, –NH),
8.15 (1H, s, C5–H pyridine ring), 7.05–7.70 (8H, m, Ar–H),
7.74 (2H, s, Ar–NH2), 3.83 (3H, s, Ar–O–CH3); 13C NMR
(DMSO-d6, 100 MHz): = 161.9 (C, C–NH2), 161.2 (C, C–
OCH3), 154.8 (C, C-2 benzimidazole), 113.8 (C, –C:N),
85.5 (C, C–C:N), 55.7 (C, –OCH3); LCMS: m/z = 341.13
(M?); Anal. Calcd. for C20H15N5O: C, 70.37; H, 4.43; N,
20.52. Found: C, 70.42; H, 4.45; N, 20.56.
3462, 2919, 2347, 1528, 1565, 1615, 1518, 734 cm-1; H
1
NMR (DMSO-d6, 400 MHz): = 10.25 (1H, s, –NH), 8.98
(1H, s, C5–H pyridine ring), 7.22–8.52 (12H, m, Ar–H),
8.36 (1H, s, CH=N); 13C NMR (DMSO-d6, 100 MHz): =
178.6 (C, –N–C–N), 158.7 (C, –C=N), 154.7 (C, C-2
benzimidazole), 148.3 (C, C–NO2), 132.1 (C, C–Cl), 117.4
(C, –C:N), 98.3 (C, C–C:N); LCMS: m/z = 478.09
(M?); Anal. Calcd. for C26H15N6O2Cl: C, 65.21; H, 3.16;
N, 17.55. Found: C, 65.50; H, 3.68; N, 17.46.
2-amino-6-(1H-benzo[d]imidazol-2-yl)-4-(4-
(dimethylamino)phenyl)nicotinonitrile (3l)
6-(1H-benzo[d]imidazol-2-yl)-4-(2-hydroxyphenyl)-2-((3-
nitrobenzylidene)amino)nico-tinonitrile (4c)
Light brown amorphous; m.p.: 240 °C, IR (KBr) tmax
3466, 3365, 2927, 2338, 1537, 1560, 1615 cm-1; 1H NMR
(DMSO-d6, 400 MHz): = 10.27 (1H, s, –NH), 8.16 (1H,
s, C5–H pyridine ring), 6.82–7.61 (8H, m, Ar–H), 7.74 (2H,
s, Ar–NH2), 3.06 (6H, s, Ar–N(CH3)2); 13C NMR (DMSO-
d6, 100 MHz): = 161.9 (C, C–NH2), 155.8 (C, C–
N(CH3)2), 154.8 (C, C-2 benzimidazole), 113.5 (C,
–C:N), 85.5 (C, C–C:N), 41.3 (C, [NCH3); LCMS:
m/z = 354.16 (M?); Anal. Calcd. for C21H18N6: C, 71.17;
H, 5.12; N, 23.71. Found: C, 71.23; H, 5.14; N, 23.70.
Light yellow crystals (MeOH); m.p.: 252 °C, IR (KBr)
tmax 3627, 3459, 2929, 2351, 1523, 1569, 1627,
1525 cm-1 1H NMR (DMSO-d6, 400 MHz): = 10.20
;
(1H, s, –NH), 8.92 (1H, s, C5–H pyridine ring), 7.22–8.52
(12H, m, Ar–H), 8.32 (1H, s, CH=N), 5.33 (1H, s, Ar–OH);
13C NMR (DMSO-d6, 100 MHz): = 178.8 (C, –N–C–N),
158.5 (C, –C=N), 155.2 (C, C–OH), 154.5 (C, C-2 benz-
imidazole), 147.9 (C, C–NO2), 117.1 (C, –C:N), 98.4 (C,
C–C:N); LCMS: m/z = 460.13 (M?); Anal. Calcd. for
C26H16N6O3: C, 67.82; H, 3.50; N, 18.25. Found: C, 67.78;
H, 3.58; N, 18.46.
General method for the preparation of 6-(1H-
benzo[d]imidazol-2-yl)-2-((3-nitrobenzyli-
dene)amino)-4-(aryl)nicotinonitriles (4a–l)
6-(1H-benzo[d]imidazol-2-yl)-4-(3-hydroxyphenyl)-2-((3-
In a round bottom flask, compound (3a–l) (0.01 mol) was
taken in 1,4-dioxane (20 mL), and m-nitrobenzaldehyde
(0.01 mol) was added. To this mixture, a pinch of anhy-
drous zinc chloride was added and refluxed for 6 h and
poured onto crushed ice. The product was filtered and
washed with cold water. The product was dried and re-
crystallized from methanol.
nitrobenzylidene)amino)nicotino-nitrile (4d)
Pale yellow crystals (MeOH); pale yellow crystals; m.p.:
247–249 °C, IR (KBr) tmax 3627, 3449, 2927, 2356,
1527, 1562, 1625, 1524 cm-1 1H NMR (DMSO-d6,
;
400 MHz): = 10.28 (1H, s, –NH), 8.94 (1H, s, C5–H
pyridine ring), 6.91–8.52 (12H, m, Ar–H), 8.36 (1H, s,
CH=N), 5.32 (1H, s, Ar–OH); 13C NMR (DMSO-d6,
100 MHz): = 178.9 (C, –N–C–N), 158.5 (C, –C=N),
157.7 (C, C–OH), 154.7 (C, C-2 benzimidazole), 148.1 (C,
C–NO2), 117.2 (C, –C:N), 98.3 (C, C–C:N); LCMS: m/
z = 460.13 (M?); Anal. Calcd. for C26H16N6O3: C, 67.82;
H, 3.50; N, 18.25. Found: C, 67.78; H, 3.58; N, 18.46.
6-(1H-benzo[d]imidazol-2-yl)-2-((3-
nitrobenzylidene)amino)-4-phenylnicotinonitrile (4a)
Yellowish crystals (MeOH); m.p.: 248 °C, IR (KBr) tmax
3454, 2950, 2346, 1520, 1561, 1621, 1519 cm-1; 1H NMR
123

Med Chem Res
(1H, s, CH=N); 13C NMR (DMSO-d6, 100 MHz):
=
178.8 (C, –N–C–N), 158.7 (C, –C=N), 154.7 (C, C-2
benzimidazole), 148.6 (C, C–NO2), 117.2 (C, –C:N),
98.2 (C, C–C:N); LCMS: m/z = 489.12 (M?); Anal.
Calcd. for C26H15N7O4: C, 63.80; H, 3.09; N, 20.03.
Found: C, 63.78; H, 3.11; N, 20.10.
6-(1H-benzo[d]imidazol-2-yl)-4-(4-hydroxyphenyl)-2-((3-
nitrobenzylidene)amino)nicotino-nitrile (4e)
Light brown crystals (CHCl3); m.p.: 241–243 °C, IR (KBr)
tmax 3638, 3450, 2925, 2349, 1525, 1562, 1629,
1518 cm-1 1H NMR (DMSO-d6, 400 MHz): = 10.29
;
(1H, s, –NH), 8.98 (1H, s, C5–H pyridine ring), 6.86–8.53
(12H, m, Ar–H), 8.31 (1H, s, CH=N), 8.36 (1H, s, Ar–OH);
13C NMR (DMSO-d6, 100 MHz): = 178.9 (C, –N–C–N),
159.5 (C, C–OH), 158.9 (C, –C=N), 154.7 (C, C-2 benz-
imidazole), 148.4 (C, C–NO2), 117.3 (C, –C:N), 98.6 (C,
C–C:N); LCMS: m/z = 460.13 (M?); Anal. Calcd. for
C26H16N6O3: C, 67.82; H, 3.50; N, 18.25. Found: C, 67.85;
H, 3.60; N, 18.45.
6-(1H-benzo[d]imidazol-2-yl)-4-(4-hydroxy-3-
methoxyphenyl)-2-((3-nitrobenzylidene)-
amino)nicotinonitrile (4i)
White crystals (MeOH); m.p.: 259 °C, IR (KBr) tmax 3622,
2839, 3423, 2929, 2351, 1520, 1548, 1628, 1523 cm-1; 1H
NMR (DMSO-d6, 400 MHz): = 10.22 (1H, s, –NH), 8.88
(1H, s, C5–H pyridine ring), 6.90–8.52 (11H, m, Ar–H),
8.41 (1H, s, CH=N) 5.34 (1H, s, Ar–OH), 3.84 (3H, s, Ar–
O–CH3); 13C NMR (DMSO-d6, 100 MHz): = 178.9 (C,
–N–C–N), 158.5 (C, –C=N), 154.4 (C, C-2 benzimidazole),
149.7 (C, C–OH), 148.2 (C, C–NO2), 148.0 (C, C–OCH3),
117.3 (C, –C:N), 98.4 (C, C–C:N), 56.1 (C, –OCH3);
LCMS: m/z = 490.14 (M?); Anal. Calcd. for C27H18N6O4:
C, 66.12; H, 3.70; N, 17.13. Found: C, 66.15; H, 3.78; N,
13.10.
6-(1H-benzo[d]imidazol-2-yl)-2-((3-
nitrobenzyllidene)amino)-4-(2-nitrophenyl)nicotino-
nitrile (4f)
Light yellow crystals (MeOH); m.p.: 240 °C, IR (KBr)
1
tmax 3452, 2936, 2357, 1524, 1559, 1635, 1531 cm-1; H
NMR (DMSO-d6, 400 MHz): = 10.27 (1H, s, –NH), 8.94
(1H, s, C5–H pyridine ring), 7.22–8.52 (12H, m, Ar–H),
8.36 (1H, s, CH=N); 13C NMR (DMSO-d6, 100 MHz): =
178.8 (C, –N–C–N), 158.6 (C, –C=N), 154.7 (C, C-2
benzimidazole), 148.0 (C, C–NO2), 117.1 (C, –C:N), 98.2
(C, C–C:N); LCMS: m/z = 489.12 (M?); Anal. Calcd. for
C26H15N7O4: C, 63.80; H, 3.09; N, 20.03. Found: C, 63.78;
H, 3.11; N, 20.10.
6-(1H-benzo[d]imidazol-2-yl)-2-((3-
nitrobenzylidene)amino)-4-p-tolylnicotinonitrile (4j)
White crystals (MeOH); m.p.: 244 °C, IR (KBr) tmax 3450,
2925, 2349, 2951, 1527, 1553, 1634, 1520 cm-1; 1H NMR
(DMSO-d6, 400 MHz): = 10.29 (1H, s, –NH), 8.94 (1H,
s, C5–H pyridine ring), 7.22–8.52 (12H, m, Ar–H), 8.40
(1H, s, CH=N) 2.35 (3H, s, Ar–CH3); 13C NMR (DMSO-
d6, 100 MHz): = 180.0 (C, –N–C–N), 158.6 (C, –C=N),
154.3 (C, C-2 benzimidazole), 148.5 (C, C–NO2), 132.7
(C, C–CH3), 117.3 (C, –C:N), 98.3 (C, C–C:N), 21.4
(C, –CH3); LCMS: m/z = 460.13 (M?); Anal. Calcd. for
C27H18N6O2: C, 70.73; H, 3.96; N, 18.33. Found: C, 70.85;
H, 3.60; N, 18.40.
6-(1H-benzo[d]imidazol-2-yl)-2-((3-
nitrobenzyllidene)amino)-4-(3-nitrophenyl)nicotino-nitrile
(4g)
Yellowish crystals (MeOH); m.p.: 251 °C, IR (KBr) tmax
3450, 2934, 2356, 1522, 1558, 1631, 1529 cm-1; 1H NMR
(DMSO-d6, 400 MHz): = 10.25 (1H, s, –NH), 8.92 (1H,
s, C5–H pyridine ring), 7.22–8.52 (12H, m, Ar–H), 8.35
(1H, s, CH=N); 13C NMR (DMSO-d6, 100 MHz):
=
178.9 (C, –N–C–N), 158.5 (C, –C=N), 154.7 (C, C-2
benzimidazole), 148.7 (C, C–NO2), 117.1 (C, –C:N),
98.6 (C, C–C:N); LCMS: m/z = 489.12 (M?); Anal.
Calcd. for C26H15N7O4: C, 63.80; H, 3.09; N, 20.03.
Found: C, 63.78; H, 3.11; N, 20.10.
6-(1H-benzo[d]imidazol-2-yl)-4-(4-methoxyphenyl)-
2-((3-nitrobenzylidene)amino)nico-tinonitrile (4k)
Pale white crystals (MeOH); m.p.: 251–253 °C, IR (KBr)
tmax 3450, 2841, 2931, 2330, 1522, 1557, 1631, 1520,
1325 cm-1 1H NMR (DMSO-d6, 400 MHz): = 10.25
;
6-(1H-benzo[d]imidazol-2-yl)-2-((3-
nitrobenzyllidene)amino)-4-(4-nitrophenyl)nicotino-
nitrile (4h)
(1H, s, –NH–), 8.90 (1H, s, C5–H pyridine ring), 7.05–8.52
(12H, m, Ar–H), 8.41 (1H, s, CH=N) 3.84 (3H, s, Ar–O–
CH3); 13C NMR (DMSO-d6, 100 MHz): = 178.6 (C, –N–
C–N), 161.4 (C, C–OCH3), 158.7 (C, –C=N), 154.9 (C, C-2
benzimidazole), 148.2 (C, C–NO2), 117.3 (C, –C:N),
98.0 (C, C–C:N), 55.9 (C, –OCH3); LCMS: m/z =
474.14 (M?); Anal. Calcd. for C27H18N6O3: C, 68.35; H,
3.82; N, 17.71. Found: C, 68.40; H, 3.88; N, 17.76.
Pale yellow crystals (MeOH); m.p.: 231 °C, IR (KBr) tmax
3454, 2932, 2357, 1525, 1559, 1634, 1527 cm-1; 1H NMR
(DMSO-d6, 400 MHz): = 10.26 (1H, s, –NH), 8.95 (1H,
s, C5–H pyridine ring), 7.22–8.52 (12H, m, Ar–H), 8.33
123

Med Chem Res
6-(1H-benzo[d]imidazol-2-yl)-4-(4-dimethylaminophenyl)-
71.2 (C, –S–CH–N), 33.5 (C, –CH2); LCMS: m/z = 522.08
(M?); Anal. Calcd. for C28H17N6O3ClS: C, 60.81; H, 3.10;
N, 15.20. Found: C, 60.90; H, 3.16; N, 15.29.
2-((3-nitrobenzylidene)amino)-nicotinonitrile (4l)
Yellowish crystals (MeOH); m.p.: 248 °C, IR (KBr) tmax
3468, 2927, 2338, 1537, 1560, 1615, 1523 cm-1; 1H NMR
(DMSO-d6, 400 MHz): = 10.25 (1H, s, –NH), 8.93 (1H,
s, C5–H pyridine ring), 6.82–8.54 (12H, m, Ar–H), 8.42
(1H, s, CH=N), 3.06 (6H, s, Ar–N\); 13C NMR (DMSO-
d6, 100 MHz): = 178.1 (C, –N–C–N), 158.6 (C, –C=N),
155.9 (C, C–N(CH3)2), 154.7 (C, C-2 benzimidazole),
148.2 (C, C–NO2), 116.9 (C, –C:N), 98.4 (C, C–C:N),
41.3 (C, [NCH3); LCMS: m/z = 487.18 (M?); Anal.
Calcd. for C28H18N7O2: C, 68.98; H, 4.34; N, 20.11.
Found: C, 69.0; H, 4.38; N, 20.16.
6-(1H-benzo[d]imidazol-2-yl)-4-(2-hydroxyphenyl)-2-(2-
(3-nitrophenyl)-4-oxothiazolidin-3-yl)nicotinonitrile (5c)
Light brown amorphous; m.p.: 352 °C; IR (KBr) tmax
3629, 3434, 3022, 2939, 2340, 1699, 1520, 1618, 1629,
;
1526 cm-1 1H NMR (DMSO-d6, 400 MHz): = 10.26
(1H, s, –NH), 8.75 (1H, s, C5–H pyridine ring), 7.01–8.12
(12H, m, Ar–H), 6.44 (1H, s, S–CH–N group), 5.35 (1H, s,
Ar–OH), 3.95 (2H, s, –CH2 group); 13C NMR (DMSO-d6,
100 MHz): = 171.2 (C, –C=O), 155.1 (C, C–OH), 154.7
(C, C-2 benzimidazole), 147.9 (C, C–NO2), 113.9 (C,
–C:N), 91.5 (C, C–C:N), 71.3 (C, –S–CH–N), 33.2 (C,
–CH2); LCMS: m/z = 534.11 (M?); Anal. Calcd. for
C28H18N6O4S: C, 62.91; H, 3.39; N, 15.75. Found: C,
62.98; H, 3.43; N, 15.85.
General method for the preparation of 6-(1H-
benzo[d]imidazol-2-yl)-2-(2-(3-nitrophenyl)-4-
oxothiazolidin-3-yl)-4-(aryl)nicotinonitriles (5a–l)
Compound (4a–l) (0.01 mol) in an anhydrous 1,4-dioxane
(25 mL) was added to 2-mercaptoacetic acid (0.012 mol).
The mixture was refluxed for 12 h, cooled, and poured into
aqueous saturated solution of sodium bicarbonate to
remove unreacted 2-mercaptoacetic acid. The residue was
filtered, dried, and recrystallized from ethanol (99.5 %).
6-(1H-benzo[d]imidazol-2-yl)-4-(3-hydroxyphenyl)-2-(2-
(3-nitrophenyl)-4-oxothiazolidin-3-yl)nicotinonitrile (5d)
Pale yellow crystals (MeOH); m.p.: 325 °C; IR (KBr) tmax
3630, 3434, 3025, 2937, 2337, 1706, 1521, 1613, 1630,
;
1528, 1327 cm-1 1H NMR (DMSO-d6, 400 MHz): =
6-(1H-benzo[d]imidazol-2-yl)-2-(2-(3-nitrophenyl)-4-
oxothiazolidin-3-yl)-4-phenylnico- tinonitrile (5a)
10.25 (1H, s, –NH), 8.74 (1H, s, C5–H pyridine ring),
6.91–8.12 (12H, m, Ar–H), 6.44 (1H, s, S–CH–N group),
5.36 (1H, s, Ar–OH), 3.94 (2H, s, –CH2 group); 13C NMR
(DMSO-d6, 100 MHz): = 171.2 (C, –C=O), 157.7 (C, C–
OH), 154.3 (C, C-2 benzimidazole), 147.9 (C, C–NO2),
113.7 (C, –C:N), 91.6 (C, C–C:N), 71.3 (C, –S–CH–N),
33.5 (C, –CH2); LCMS: m/z = 534.11 (M?); Anal. Calcd.
for C28H18N6O4S: C, 62.91; H, 3.39; N, 15.75. Found: C,
62.98; H, 3.43; N, 15.85.
Brownish crystals (EtOH); m.p.: 318 °C; IR (KBr) tmax
3421, 3024, 2950, 2346, 1698, 1528, 1619, 1597,
;
1519 cm-1 1H NMR (DMSO-d6, 400 MHz): = 10.29
(1H, s, –NH), 8.73 (1H, s, C5–H pyridine ring), 7.22–8.12
(13H, m, Ar–H), 6.44 (1H, s, S–CH–N group), 3.95 (2H, s,
–CH2 group); 13C NMR (DMSO-d6, 100 MHz): = 171.3
(C, –C=O), 154.6 (C, C-2 benzimidazole), 148.0 (C, C–
NO2), 113.7 (C, –C:N), 91.4 (C, C–C:N), 71.2 (C, –S–
CH–N), 33.6 (C, –CH2); LCMS: m/z = 518.12 (M?);
Anal. Calcd. for C28H18N6O3S: C, 64.85; H, 3.50; N,
16.21. Found: C, 64.77; H, 3.41; N, 16.12.
6-(1H-benzo[d]imidazol-2-yl)-4-(4-hydroxyphenyl)-2-(2-
(3-nitrophenyl)-4-oxothiazolidin-3-yl)nicotinonitrile (5e)
White crystals (EtOH); m.p.: 334 °C; IR (KBr) tmax 3624,
3440, 3029, 2937, 2339, 1710, 1529, 1615, 1632,
;
1531 cm-1 1H NMR (DMSO-d6, 400 MHz): = 10.28
6-(1H-benzo[d]imidazol-2-yl)-4-(2-chlorophenyl)-2-(2-(3-
nitrophenyl)-4-oxothiazolidin-3-yl)nicotinonitrile (5b)
(1H, s, –NH), 8.73 (1H, s, C5–H pyridine ring), 6.86–8.12
(12H, m, Ar–H), 6.45 (1H, s, S–CH–N group), 5.35 (1H, s,
Ar–OH), 3.95 (2H, d, –CH2 group); 13C NMR (DMSO-d6,
100 MHz): = 171.3 (C, –C=O), 157.9 (C, C–OH), 154.4
(C, C-2 benzimidazole), 147.9 (C, C–NO2), 115.5 (C,
–C:N), 91.5 (C, C–C:N), 71.3 (C, –S–CH–N), 33.4 (C,
–CH2); LCMS: m/z = 534.11 (M?); Anal. Calcd. for
C28H18N6O4S: C, 62.91; H, 3.39; N, 15.72. Found: C,
62.98; H, 3.43; N, 15.85.
White crystals (EtOH); m.p.: 327 °C; IR (KBr) tmax 3423,
3034, 2938, 2343, 1703, 1520, 1625, 1600, 1527,
724 cm-1; 1H NMR (DMSO-d6, 400 MHz): = 10.25 (1H,
s, –NH), 8.74 (1H, s, C5–H pyridine ring), 7.22–8.12 (12H,
m, Ar–H), 6.45 (1H, s, S–CH–N group), 3.96 (2H, s, –CH2
group); 13C NMR (DMSO-d6, 100 MHz): = 171.2 (C,
–C=O), 154.7 (C, C-2 benzimidazole), 147.8 (C, C–NO2),
132.2 (C, C–Cl), 113.8 (C, –C:N), 91.4 (C, C–C:N),
123

Med Chem Res
6-(1H-benzo[d]imidazol-2-yl)-4-(2-nitrophenyl)-2-(2-(3-
(11H, m, Ar–H), 6.45 (1H, s, S–CH–N group), 5.35 (1H, s,
Ar–OH), 3.96 (2H, s, –CH2 group), 3.84 (3H, s, Ar–O–
CH3); 13C NMR (DMSO-d6, 100 MHz): = 171.3 (C,
–C=O), 154.8 (C, C-2 benzimidazole), 148.0 (C, C–OCH3),
147.3 (C, C–NO2), 113.9 (C, –C:N), 91.6 (C, C–C:N),
71.1 (C, –S–CH–N), 56.2 (C, –OCH3), 33.5 (C, –CH2);
LCMS: m/z = 564.12 (M?); Anal. Calcd. for
C29H20N6O5S: C, 61.69; H, 3.57; N, 14.89. Found: C,
61.72; H, 3.64; N, 14.33.
nitrophenyl)-4-oxothiazolidin-3-yl)-nicotinonitrile (5f)
Light brown amorphous (EtOH); m.p.: 310 °C; IR (KBr)
tmax 3438, 3031, 2932, 2333, 1711, 1527, 1619, 1635,
1536 cm-1 1H NMR (DMSO-d6, 400 MHz): = 10.25
;
(1H, s, –NH), 8.75 (1H, s, C5–H pyridine ring), 7.22–8.12
(12H, m, Ar–H), 6.44 (1H, s, S–CH–N group), 3.95 (2H, s,
–CH2 group); 13C NMR (DMSO-d6, 100 MHz): = 171.3
(C, –C=O), 154.7 (C, C-2 benzimidazole), 147.8 (C, C–
NO2), 113.8 (C, –C:N), 91.5 (C, C–C:N), 71.1 (C, –S–
CH–N), 33.5 (C, –CH2); LCMS: m/z = 563.10 (M?);
Anal. Calcd. for C28H17N7O5S: C, 59.68; H, 3.04; N,
17.40. Found: C, 59.72; H, 3.14; N, 17.53.
6-(1H-benzo[d]imidazol-2-yl)-2-(2-(3-nitrophenyl)-4-
oxothiazolidin-3-yl)-4-(p-tolyl)-nico-tinonitrile (5j)
White crystals (EtOH); m.p.: 318 °C; IR (KBr) tmax 3440,
3030, 2952, 2930, 2337, 1698, 1529, 1616, 1639,
;
1535 cm-1 1H NMR (DMSO-d6, 400 MHz): = 10.25
6-(1H-benzo[d]imidazol-2-yl)-4-(3-nitrophenyl)-2-(2-(3-
nitrophenyl)-4-oxothiazolidin-3-yl)-nicotinonitrile (5g)
(1H, s, –NH), 8.73 (1H, s, C5–H pyridine ring), 7.22–8.12
(12H, m, Ar–H), 6.44 (1H, s, S–CH–N group), 3.95 (2H, d,
–CH2 group), 2.34 (3H, s, Ar–CH3); 13C NMR (DMSO-d6,
100 MHz): = 171.3 (C, –C=O), 154.7 (C, C-2 benz-
imidazole), 147.8 (C, C–NO2), 132.3 (C, C–CH3), 113.8
(C, –C:N), 91.5 (C, C–C:N), 71.2 (C, –S–CH–N), 33.6
(C, –CH2), 21.4 (C, –CH3); LCMS: m/z = 532.13 (M?);
Anal. Calcd. for C29H20N6O3S: C, 65.40; H, 3.79; N,
15.78. Found: C, 65.48; H, 3.73; N, 15.85.
Yellowish crystals (EtOH); m.p.: 325 °C; IR (KBr) tmax
3438, 3029, 2927, 2330, 1709, 1526, 1616, 1633,
1532 cm-1 1H NMR (DMSO-d6, 400 MHz): = 10.25
;
(1H, s, –NH), 8.73 (1H, s, C5–H pyridine ring), 7.22–8.67
(12H, m, Ar–H), 6.44 (1H, s, S–CH–N group), 3.95 (2H, s,
–CH2 group); 13C NMR (DMSO-d6, 100 MHz): = 171.6
(C, –C=O), 154.5 (C, C-2 benzimidazole), 147.9 (C, C–
NO2), 113.9 (C, –C:N), 91.7 (C, C–C:N), 71.1 (C, –S–
CH–N), 33.7 (C, –CH2); LCMS: m/z = 563.10 (M?);
Anal. Calcd. for C28H17N7O5S: C, 59.68; H, 3.04; N,
17.40. Found: C, 59.72; H, 3.14; N, 17.53.
6-(1H-benzo[d]imidazol-2-yl)-4-(4-methoxyphenyl)-2-(2-
(3-nitrophenyl)-4-oxothiazolidin-3-yl)nicotinonitrile (5k)
6-(1H-benzo[d]imidazol-2-yl)-4-(4-nitrophenyl)-2-(2-(3-
Yellowish crystals (EtOH); crystals (EtOH); m.p.: 348 °C;
IR (KBr) tmax 3441, 3040, 2927, 2843, 2331, 1705, 1533,
1627, 1634, 1531 cm-1; 1H NMR (DMSO-d6, 400 MHz):
= 10.25 (1H, s, –NH), 8.75 (1H, s, C5–H pyridine ring),
7.05–8.12 (12H, m, Ar–H), 6.45 (1H, s, S–CH–N group),
3.95 (2H, s, –CH2 group), 3.84 (3H, s, Ar–O–CH3); 13C
NMR (DMSO-d6, 100 MHz): = 171.3 (C, –C=O), 161.1
(C, C–OCH3), 153.8 (C, C-2 benzimidazole), 147.8 (C, C–
NO2), 113.7 (C, –C:N), 91.6 (C, C–C:N), 71.2 (C, –S–
CH–N), 55.8 (C, –OCH3), 33.4 (C, –CH2); LCMS:
m/z = 548.13 (M?); Anal. Calcd. for C29H20N6O4S: C,
63.49; H, 3.67; N, 15.32. Found: C, 63.56; H, 3.73; N,
15.45.
nitrophenyl)-4-oxothiazolidin-3-yl)-nicotinonitrile (5h)
Yellowish crystals (MeOH); m.p.: 298 °C; IR (KBr) tmax
3434, 3027, 2932, 2331, 1711, 1527, 1615, 1631,
;
1533 cm-1 1H NMR (DMSO-d6, 400 MHz): = 10.25
(1H, s, –NH), 8.47 (1H, s, C5–H pyridine ring), 7.06–8.32
(12H, m, Ar–H), 6.44 (1H, s, S–CH–N group), 3.95 (2H, s,
–CH2 group); 13C NMR (DMSO-d6, 100 MHz): = 171.3
(C, –C=O), 155.1 (C, C-2 benzimidazole), 149.0 (C, C–
NO2), 113.6 (C, –C:N), 94.7 (C, C–C:N), 71.1 (C, –S–
CH–N), 34.5 (C, –CH2); LCMS: m/z = 563.10 (M?);
Anal. Calcd. for C28H17N7O5S: C, 59.68; H, 3.04; N,
17.40. Found: C, 59.72; H, 3.14; N, 17.53.
6-(1H-benzo[d]imidazol-2-yl)-4-(4-dimethylaminophenyl)-
2-(2-(3-nitrophenyl)-4-oxo-thiazolidin-3-yl)
nicotinonitrile (5l)
6-(1H-benzo[d]imidazol-2-yl)-4-(4-hydroxy-3-
methoxyphenyl)-2-(2-(3-nitrophenyl)-4-oxo-thiazolidin-3-
yl)nicotinonitrile (5i)
White crystals (MeOH);m.p.: 271 °C; IR (KBr) tmax 3441,
3033, 2937, 2333, 1700, 1523, 1629, 1630, 1534 cm-1; 1H
NMR (DMSO-d6, 400 MHz): 10.26 (1H, s, –NH), 8.73
(1H, s, C5–H pyridine ring), 6.82–8.12 (12H, m, Ar–H),
6.43 (1H, s, S–CH–N group), 3.94 (2H, s, –CH2 group),
White crystals (EtOH); m.p.: 345 °C; IR (KBr) tmax 3640,
3434, 3021, 2929, 2835, 2329, 1697, 1522, 1619, 1633,
1534 cm-1 1H NMR (DMSO-d6, 400 MHz): = 10.27
;
(1H, s, –NH), 8.76 (1H, s, C5–H pyridine ring), 6.89–8.12
123

Med Chem Res
3.06 (6H, s, Ar–N\); 13C NMR (DMSO-d6, 100 MHz): =
171.3 (C, –C=O), 155.9 (C, C–N(CH3)2), 154.8 (C, C-2
benzimidazole), 147.8 (C, C–NO2), 113.7 (C, –C:N),
91.6 (C, C–C:N), 71.2 (C, –S–CH–N), 41.2 (C,[NCH3),
33.4 (C, –CH2); LCMS: m/z = 561.16 (M?); Anal. Calcd.
for C30H23N7O3S: C, 64.16; H, 4.13; N, 17.46. Found: C,
64.22; H, 4.43; N, 17.49.
Antifungal assay
The newly prepared compounds 5a–l 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 lg/mL MIC against C. albicans, A. niger, and
A. clavatus. 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 ketoconazole (1 U
strength) was used as positive control.
Biological assay
Antibacterial assay
Antibacterial studies of newly synthesized compounds 5a–l
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 col-
lected from Institute of Microbial Technology, Chandigarh.
Primary screening was done first for antibacterial activity
in six sets 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 1000 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 spectrophotomet-
rically. 10 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. DMSO
and sterilized distilled water were used as negative control,
while chloramphenicol antibiotic (1 U strength) was used
as positive control. Standard drug used in the present study
was ‘‘chloramphenicol’’ for evaluating antibacterial activ-
ity which showed 50 lg/mL MIC against E. coli,
P. aeruginosa, S. aureus, and S. pyogenes.
Preliminary cytotoxic activities (MTT assay)
In vitro cytotoxicity activity was measured by means of the
IC50 using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliumbromide] assay method. The IC50
determination was performed according to the National
Committee for Clinical Laboratory Standards (NCCLS)
recommendations. All compounds were dissolved in 0.1 %
DMSO with the stock concentration of 10 g/L and diluted
with medium freshly before drug administration. Cell lines
were seeded into 96-well plates at density of 8,000 cells/
well. After 24 h seeding, each compound dilution was
added in duplicate, and incubation continued at 37 °C in a
humidified atmosphere containing 10 % FBS, 1 % gluta-
mine, and 50 lM/mL gentamicin sulfate in a 5 % CO2 and
95 % air. After 24 h, 20 lL MTT reagent at 5 mg/mL in
PBS (filter sterilized, light protected, and stored at 4 °C)
per well was added , and after 4 h of incubation at 37 °C,
MTT is converted to a blue formazan product by mito-
chondrial succinate dehydrogenase. This product was
eluted from cells by addition of 150 mL of DMSO. The
absorbance at 570 nm was determined by an ELISA using
a ELX800 micro plate spectrophotometer. The IC50 value
was defined as the concentration at which 50 % of the cells
could survive.
Structure activity relationship
The results of the antimicrobial screening were demon-
strated following assumptions about the structural activity
relationship (SAR): substitution pattern of the three biola-
bile benzimidazole, pyridine, and 4-thiazolidinone deriva-
tives was carefully selected to bestow different electronic
environments on the molecules. Antimicrobial activity was
considerably affected by substitution pattern on phenyl ring
123

Med Chem Res
and the most active compounds contained electron donat-
ing substituents. The highest antibacterial activity was
observed when electron donating substituents were present
at ortho or para positions (5c, 5e, 5k) of phenyl ring. In
addition, the highest antifungal activity was observed when
electron donating substituents were placed at meta position
of phenyl ring. In general, ortho- and para-substituted
derivatives showed higher antibacterial activity than meta
substituted, while meta-substituted derivatives exhibited
higher antifungal potency than ortho- and para-substituted
compounds. Compounds 5c and 5e substituted with
hydroxyl group at ortho and para positions, respectively,
showed higher antibacterial activity than the presence of
the same functional group at meta position. In case of
antifungal activity, compound 5d proved to be much more
potent than compounds 5c and 5e. Moreover, on the basis
of activity results, it was observed that introduction of
4-thiazolidinone was also responsible for enhancing anti-
microbial potency, which was confirmed by comparison of
antimicrobial activity of intermediates 3a–l and 4a–l with
final compounds 5a–l. Compounds 5c, 5e, 5–k were found
to be the most potent antibacterial agents, whereas com-
pounds 5d, 5i, and 5k emerged as most effective antifungal
agents indicating that structural requirements are different
for binding of drug to bacterial or fungal targets, respec-
tively (Sortino et al., 2007).
substituents causes a certain change of biological activity.
It may be concluded that the presence of three different
pharmacophore scaffolds enhanced the biological activity.
We have achieved our goal of the present study by
exploring the antimicrobial activity of some new structural
motifs of benzimidazole, cyanopyridine, and 4-thiazolidi-
none with anticipation of generating new leads with potent
activity against various gram-positive, gram-negative bac-
terial, and fungal strains.
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. One of the authors, G M Kotadiya is thankful
to UGC, New Delhi for providing ‘‘Research Fellowships in Science
to Meritorious Students.’’
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