Synthesis, antibacterial and antitubercular activities of benzimidazole bearing substituted 2-pyridone motifs

Article abstract of DOI:10.1016/j.ejmech.2014.06.004

A series of benzimidazole bearing 2-pyridones 5a-r were synthesized and evaluated for their in vitro antibacterial and antitubercular activity. Further, all compounds were examined for their cytotoxic study on VERO cell line and characterized by well-known spectral techniques. It was observed that the compounds 5h, 5i, 5k, 5q and 5r were found to possess significant broad spectrum antibacterial activity (12.5-100 μg/mL of MIC), while compounds 5g-5i, 5k and 5l proved to be the most potent antitubercular activity in range of 2.76-20.4 μM of MIC at low level of cytotoxicity, indicating good selectivity. From SAR studies, lipophilic profile of compounds was remarkably vital for antibacterial activity, while MIC values of antitubercular activity could not be directly correlated with lipophilicity.

Full text of DOI:10.1016/j.ejmech.2014.06.004

Accepted Manuscript
Synthesis, antibacterial and antitubercular activities of benzimidazole bearing
substituted 2-pyridone motifs
N.C. Desai , N.R. Shihory , G.M. Kotadiya , Priyanka Desai
PII:
S0223-5234(14)00522-4
10.1016/j.ejmech.2014.06.004
EJMECH 7046
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 February 2014
Revised Date: 5 April 2014
Accepted Date: 3 June 2014
Please cite this article as: N. Desai, N. Shihory, G. Kotadiya, P. Desai, Synthesis, antibacterial and
antitubercular activities of benzimidazole bearing substituted 2-pyridone motifs, European Journal of
Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.06.004.
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A series of benzimidazole bearing substituted 2-pyridones were synthesized.
All the compounds were subjected for their in vitro biological screening.
Some of the tested compounds exhibited higher potency compared to the standard.
None of the tested compounds exhibited significant cytotoxic activity.
Lipophilicity plays an important role in antibacterial effect of tested compounds.

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Synthesis, antibacterial and antitubercular activities of benzimidazole bearing
substituted 2-pyridone motifs
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N C Desai*, N R Shihory, G M Kotadiya, Priyanka Desai
Division of Medicinal Chemistry, Department of Chemistry
(UGC NON-SAP & DST-FIST Sponsored),
Mahatma Gandhi Campus, Maharaja Krishnakumarsinhji Bhavnagar University,
Bhavnagar 364 002, India
E-mail: dnisheeth@rediffmail.com,
Telefax: +91 278 2439852
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Abstract
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A series of benzimidazole bearing 2-pyridones 5a-r were synthesized and evaluated for
their in vitro antibacterial and antitubercular activity. Further, all compounds were examined for
their cytotoxic study on VERO cell line and characterized by well-known spectral techniques. It
was observed that the compounds 5h, 5i, 5k, 5q and 5r were found to possess significant broad
spectrum antibacterial activity (12.5-100 µg/mL of MIC), while compounds 5g-5i, 5k and 5l
proved to be the most potent antitubercular activity in range of 2.76-20.4 µM of MIC at low level
of cytotoxicity, indicating good selectivity. From SAR studies, lipophilic profile of compounds
was remarkably vital for antibacterial activity, while MIC values of antitubercular could not be
directly correlate with lipophilicity.
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Keywords: Antibacterial activity; Antitubercular activity; Benzimidazole; Cytotoxicity; MABA
assay; 2-Pyridone
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1. Introduction
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The current first-line tuberculosis drug treatment is more than 40 years old and consists
primarily of rifampicin and isoniazid. These antibiotics are drug-susceptible and require longer
time and large number of doses, which are multi-drug resistant (MDR) and extensively drug
resistant (XDR) to tuberculosis strains [1-3]. However, the rapid increase of multi-drug-resistant
tuberculosis (MDR-TB) (resistant to at least isoniazid and rifampicin) and extensively drug-
resistant tuberculosis (XDR-TB) (resistant to isoniazid, rifampicin in addition to
fluoroquinolone, kanamycin, amikacin or capreomycin among second line anti-TB drugs) has led
to an urgent need for the identification of new drug targets and the growth of novel anti-TB
drugs. Furthermore, The most commonly encountered antibiotic-resistant bacteria, methicillin-
resistant Staphylococcus aureus (MRSA), has a major impact on infections in both hospitals and
community settings [4,5]. Unfortunately, as antibiotic resistant organisms have become more
commonplace, the pipeline for the discovery of new antimicrobial agents has decreased [6].
Thus, there is a pressing need for new antimicrobial agents that are capable of treating resistant
bacterial strains.
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Moreover, dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction
of dihydrofolate to tetrahydrofolate that is essential for DNA synthesis. Inhibition of its activity
leads to arrest of DNA synthesis and hence cell death [7]. Mycobacterium tuberculosis DHFR is
an attractive novel drug target for developing anti-tuberculosis drugs. To overcome this problem,
we have synthesized a series of benzimidazole bearing 2-pyridones by replacing the pyrazole
motif in our previously synthesized compounds NCD1-20 [8] and screened them for their
antibacterial property. In this attempt, we got excellent antibacterial results. Antibacterial studies
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impelled us to inspect 5a-r for their in vitro antitubercular activity as well. Structural relevance
of title compounds 5a-r with previously synthesized compounds is shown in Figure 1.
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Benzimidazole nucleus is a the key building block for numerous compounds that play
beneficial roles in the functioning of biologically important molecules [9] and are remarkably
effective both with respect to their inhibitory activity and their favorable selectivity ratio [10-12].
Benzimidazoles are considered a promising class of bioactive heterocyclic compounds
encompassing a diverse range of biological activities such as antiulcer [13], antihelminthic [14],
antihypertensive [15], anticoagulant [16], anti-inflammatory [17], antimicrobial [18-20] and
antiparasitic [21]. The azole group of heterocyclic compounds possesses significant
pharmacokinetic profile and lipophilicity that influence the ability of drug to reach the target by
transmembrane diffusion and along with promising activity against resistant TB by inhibiting the
biosynthesis of lipids [22,23]. Further, 2-pyridones represent a unique class of pharmacophores,
which are observed in various therapeutic agents [24]. In recent years, 2-pyridones have
assimilated much importance as these compounds exhibit several biological activities such as
antitumoral [25], antimalarial [26], analgesic [27] and anti-HIV [28] properties. Moreover, 2-
pyridones are a class of recently discovered potent antibacterial agents that are of particular
interest due to their in vitro and in vivo antibacterial potencies against the bacterial type II DNA
topoisomerases, which include two highly homologous enzymes-DNA gyrase and topoisomerase
IV [29,30]. Moreover, among the pharmacokinetic properties, a low and highly variable
bioavailability is indeed the main reason for stopping further development of the drug [31].
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Motivated by the above findings and from our previous work [32,33], the main aim of the
work is to obtain more active antibacterial and antitubercular agents with plausible novel
mechanisms of action. It was thought worthwhile to synthesize some new benzimidazole bearing
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2-pyridone derivatives comprising of the above aforementioned moieties in single molecular
framework in order to investigate their in vitro antibacterial and antitubercular activity. In
continuation to this, in our present communication, we have synthesized benzimidazole bearing
2-pyridones 5a-r and evaluated for their in vitro antibacterial and antitubercular activity. In
addition, cytotoxicity studies were also conducted in VERO cell lines to evaluate the ability of
these compounds to inhibit the cell growth. Most active compounds 5h, 5q and 5r were also
screened against MRSA strain.
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{Insert Figure 1 here}
2. Results and discussion
2.1. Chemistry
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We have synthesized new analogues in which 2-pyridone motif is connected to the
benzimidazole system. The synthetic strategies adopted for the synthesis of target benzimidazole
bearing 2-pyridone derivatives 5a-r are depicted in Schemes 1 and 2. In Scheme 1, condensation
of 1-(1H-benzo[d]imidazol-2-yl)ethanone 1 with equimolar quantity of cyanoacetic acid
hydrazide in refluxing 1,4-dioxane afforded a single product, that was identified as N'-(1-(1H-
benzo[d]imidazol-2-yl)ethylidene)-2-cyanoacetohydrazide 2. The elemental analysis and spectral
data were in accordance with the proposed N'-(1-(1H-benzo[d]imidazol-2-yl)ethylidene)-2-
cyanoacetohydrazide 2 structure. The IR spectrum of 2 showed strong absorption bands at 2248
and 1681 cm-1 were due to cyanide and carbonyl group, respectively. Its 1H NMR spectrum apart
from the expected aromatic signals, showed two new singlets at δ 3.38 and 8.92 ppm assigned to
the reactive methylene protons and proton of secondary amine attached with carbonyl group
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respectively. The C NMR spectrum displayed nine carbon signals, the most important signals
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appeared at δ 13.4, 27.3, 125.8, 171.3 ppm characteristic of methyl, methylene, cyanide and
carbonyl carbons, respectively. The mass spectrum revealed a molecular ion peak at m/z =
241.11 (M + 1), in agreement with its proposed structure. The presence of the reactive methylene
group in the hydrazide 2 makes it a versatile precursor for the Michael type condensation with
Knoevenagel product p-nitrobenzaldehyde and malononitrile compound 3 in presence of
catalytic amount of piperidine utilizing ethanol (95%) as a solvent furnished 2-pyridone
derivative identified as 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-amino-4-(4-
nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile 4. Structure of the latter product was
confirmed by IR spectra which showed characteristic absorption bands at 1688 and 3446 cm-1for
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1
conjugated >C=O and primary amine group respectively. Their H NMR spectra displayed a
broad singlet at
δ 8.78 ppm for primary amine protons, besides the disappearance of reactive
methylene group and secondary amine singlets due to its involvement in cyclization.
Condensation of 2-pyridone derivative 4 with appropriate aromatic aldehydes in boiling ethanol
afforded the respective targeted benzimidazole bearing 2-pyridones acknowledged as 1-((1-(1H-
benzo[d]imidazol-2-yl)ethylidene)amino)-6-((arylbenzylidene)amino)-4-(4-nitrophenyl)-2-oxo-
1,2-dihydropyridine-3,5-dicarbonitriles 5a-r. The structures of the final analogues 5a-r were
established by IR spectra which showed disappearance of -NH2 band in compound 4. A strong
absorption band appeared between 1679-1688 cm-1 was assigned to conjugated >C=O group.
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Their H NMR spectra revealed a singlet of methine proton at
δ 9.40-9.57 ppm involved in
azomethine formation, along with the vanishing of primary amine singlet. The 13C NMR
spectrum of compound 5l, displayed, besides the expected methyl and aromatic signals, three
characteristic signals at
δ 115.9, 160.1 and 163.8 ppm due to the carbons of CN, C=O and CH=N
respectively. The mass spectrum of 5l showed molecular ion peak at m/z = 540.17 (M + 1), in
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agreement with its proposed structure. Similarly, the spectral values for all the compounds and
C, H, N analysis are presented in the experimental part.
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{Insert Scheme 1 here}
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A plausible mechanistic pathway for the formation of compounds 5a-r is suggested in
Scheme 2. Firstly, hydrazone (A) underwent Michael addition with Knoevenagel product (B)
and furnished the intermediate (C), which further experienced intramolecular nucleophilic attack
on cyanide carbon followed by annulation to yield intermediate (D). The intermediate (D)
transformed to compound (E) by intramolecular electron transfer to nitrogen atom. In the last
step, intermediate (E) was transformed to targeted compounds by intermolecular nucleophilic
attack on carbonyl carbon of different aromatic aldehydes.
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{Insert Scheme 2 here}
2.2. Biological Evaluation
2.2.1. Antibacterial activity
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The intermediates 2, 4 and target compounds 5a-r were investigated for their in vitro
antibacterial activity against two gram-positive and two gram-negative bacteria and the yeast like
pathogenic fungus Candida albicans using conventional broth-dilution method [34,35].
Minimum inhibitory concentration (MIC) is defined as the concentration of the compound
required to obtain complete inhibition of bacterial growth. MICs of the synthesized compounds
were compared with ciprofloxacin and chloramphenicol and the results were depicted in Table 1.
The antibacterial activity assessed (Table 1) for analogues 2, 4 and 5a-r against several strains
flaunted good activity. From the bioassay, it was observed that the final analogues 5a-r with the
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substitutions of phenyl ring were most active against all the pathogenic strains studied than the
intermediate hydrazone 2 and 2-pyridone derivative 4. Intermediate 2 showed very poor
antibacterial activity against all tested bacterial strains and it was observed that compound 2 with
low lipophilicity (Clog P = 0.7220) displayed very low activity as compared to intermediate 4
and final analogues 5a-r. Precursor 2 reacted with Knoevenagel compound 3 to generate
intermediate 4 which was tested against different bacterial strains, and it showed more inhibitory
efficacy than compound 2 due to high lipophilicity (Clog P = 1.4772) and less activity compared
to all final analogues. Therefore, it was observed that formation of 2-pyridone nucleus was
responsible for enhancing antibacterial potency. Now, intermediate 4 was treated with different
aromatic aldehydes to afford targeted compounds 5a-r which were found to have broad spectrum
antimicrobial efficacy. This could be correlated to structural variations and different aromatic
substitutions in phenyl ring.
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Good bioavailability can be achieved by an appropriate balance between solubility and
partitioning properties. Membrane permeability and bioavailability is always associated with
some basic molecular descriptor such as Clog P (lipophilicity). Thus, prediction of
bioavailability-related properties, such as lipophilicity is important before actual synthesis, in
order to reduce enormous wastage of expensive chemicals and precious time. The computed
Clog P values (P is the partition coefficient of the molecule in the water/octanol system) are
shown in Table 1 and 3. The ChemBioDraw Ultra, version 12.0, software by Cambridge Soft is
a program used to predict lipophilicity of compounds.
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From the bioassay, final analogues with electron withdrawing halogen substitution were
found to be more active than the remaining final analogues against all the bacterial strains.
Among all the final active analogues, compound 5q (3-Br, Clog P = 3.9272) endowed with
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bromine exerted highest inhibition against all the bacterial strains and showed highest efficacy
against S. aureus at 12.5 µg/mL of MIC as compared to standards ciprofloxacin (50 µg/mL, Clog
P = -0.7252) and chloramphenicol (50 µg/mL, Clog P = 1.293). In addition, compound 5q (Clog
P = 3.9272) with higher lipophilicity flaunted potential inhibitory action against E. coli and S.
pyogenes at MIC of 25 µg/mL. Moreover, the above mentioned derivative 5q displayed excellent
inhibition against P. aeruginosa at 25 µg/mL of MIC. Furthermore, among the second line active
compounds, 5h (3-Cl, Clog P = 3.7772) and 5r (4-Br, Clog P = 3.9272) indicated MIC of 50
µg/mL, in which more lipophilic compound 5r showed better results against both E. coli and S.
pyogenes. In addition, the above mentioned derivatives 5h and 5r were also found to
demonstrate excellent efficacy against S. aureus at 25 µg/mL of MIC and analogue 5r also
demonstrated MIC of 50 µg/mL against P. aeruginosa. Derivatives 5e (3-F, Clog P = 3.2072)
with fluoro, 5g (2-Cl, Clog P = 3.7772) with chloro and 5k (3-CH3, Clog P = 3.5632) with
methyl group to the aromatic nucleus bequeathed appreciable potency towards E. coli at 100
µg/mL of MIC, in which compound 5g showing highest lipophilicity gave better results.
Compound 5h (3-Cl, Clog P = 3.7772) with electron withdrawing chlorine substituent indicated
diminished activity at 62.5 µg/mL of MIC against P. aeruginosa. Another analogue 5i (4-Cl,
Clog P = 3.7772) with chlorine and 5k (3-CH3, Clog P = 3.5632) endowed with electron
donating methyl group exerted MIC of 100 µg/mL against P. aeruginosa as compared to
standard ciprofloxacin (25 µg/mL, Clog P = -0.7252) and chloramphenicol (50 µg/mL, Clog P =
1.293) antibiotics. Derivative 5g (2-Cl, Clog P = 3.7772) displayed more potency against S.
aureus than E. coli at MIC of 50 µg/mL and diminished activity at MIC of 62.5 µg/mL against S.
pyogenes. Final derivatives 5c (3-OH, Clog P = 3.2982), 5e (3-F, Clog P = 3.2072), 5k (3-CH3,
Clog P = 3.5632) and 5m (3-OCH3, Clog P = 3.4082) displayed remarkable inhibitory effects at
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MIC of 100 µg/mL against S. aureus. Among them compound 5k contemplated same action
against S. pyogenes. In addition, compound 5i (4-Cl, Clog P = 3.7772) exhibited activity at MIC
of 50 and 62.5 µg/mL against S. pyogenes and S. aureus respectively. Moreover, compound 5e
possessing fluorine atom at 3rd position of phenyl ring increased the potency and showed
inhibition at 62.5 µg/mL of MIC against S. pyogenes. Furthermore, the inhibitory activity of all
the synthesized compounds against C. albicans was rather lower than their antibacterial activity,
only compound 5q (3-Br, MIC = 250 µg/mL) exhibited moderate activity as compared to
standard ketoconazole.
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{Insert Table 1 here}
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Furthermore, the most active compounds 5h, 5q and 5r against S. aureus and S. pyogenes
(MIC = 12.5-50 µg/mL) were also tested against methicillin-resistant S. aureus (MRSA isolate
ATCC 43300) and results are given in Table 2. Compound 5r exhibited more potent activity
than the standard drugs against methicillin-resistant S. aureus. Compound 5r, with MIC value of
6.25 µg/mL against MRSA showed four-fold more potency than ciprofloxacin (MIC = 25
µg/mL) and eight-fold more activity than chloramphenicol (MIC = 50 µg/mL). In addition,
compound 5q endowed with bromo group showed two-fold more inhibition at MIC value of 12.5
µg/mL than ciprofloxacin and four-fold higher potency than chloramphenicol against MRSA.
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{Insert Table 2 here}
2.2.2. Antitubercular activity
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The encouraging results from the antibacterial studies impelled us to go for the screening
of title compounds for their in vitro antitubercular activity. Intermediates (2 and 4) and all the
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final compounds along with the standard drug for comparison were firstly evaluated for their
activity against the Mycobacterium tuberculosis H37Rv strain in Middlebrook 7H12 medium
using Microplate Alamar Blue Assay (MABA) MIC method [36]. The drug in clinical use,
isoniazid was used as a reference drug. The results of actual MICs of tested compounds were
reported in Table 3. It was observed that final analogues 5a-r displayed superior anti-tubercular
activity compared to both the intermediates (2 and 4). Among the eighteen synthesized
compounds, compounds 5g-5i, 5k and 5l endowed with inductively electron withdrawing
chlorine and electron donating methyl groups afforded maximum MICs ranging from 0.85 to
6.28 µg/mL against Mycobacterium tuberculosis. For final derivatives, introduction of chlorine
substituent in 5g-5i was tolerated, 3-chloro yielding the best activity (3-Cl > 4-Cl > 2-Cl).
Furthermore, among the second line active compounds, 5k and 5l brandished MICs in range of
2.07-6.28 µg/mL, in which compound 5k having methyl in meta position showed better result at
MIC of 2.07 µg/mL whereas compound 5l displayed inhibition at MIC of 6.28 µg/mL.
Compound 5h (3-Cl) possessed highest inhibition at MIC of 0.85 µg/mL amongst all the tested
derivatives as compared to standard isoniazid, while their ortho and para derivatives 5i (4-Cl)
and 5g (2-Cl) showed MICs of 1.79 and 2.65 µg/mL respectively. Here, we have discussed and
compared antitubercular activity based on first line drug Isoniazid (0.24 µg/mL, Clog P = -
0.668). From the antitubercular activity results, it may be concluded that final derivatives 5a-o
were mixdly active and no specific relationship was observed in case of Clog P or lipophilicity
verses antitubercular activity profiles.
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2.2.3. Cytotoxicity study
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After having identified numerous active antibacterial and antimycobacterial
benzimidazole bearing 2-pyridones, the next step was to examine the toxicity of the drug
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contenders. In vitro cytotoxicity of compounds 5a-r was determined against mammalian VERO
cell lines [37]. After 72 h of exposure, viability was considered on the basis of cellular
conversion of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium and phenylmethasulfazone] (100:20) into formazan product using Promega Cell Titer
96 non-radioactive cell proliferation assay. The cytotoxicity results presented in Table 3 are
expressed as the concentration inhibiting 50% of the cell growth IC50. The compounds exhibited
moderate to low level of cytotoxicity with the IC50 values in the range of 13.73 - >39.42 µg/mL.
It was observed that none of the tested compounds exhibited any significant cytotoxic effects on
VERO cells, suggesting a great potential for their in vivo use as antibacterial and anti-tubercular
agents. As for activity against VERO cell lines, the highest cytotoxic activity was flaunted by
compound 5d which showed percentage viability IC50 at 13.73 µg/mL, whereas, the second line
cytotoxic activity was displayed by compounds 5a, 5c and 5p which showed the percentage
viability IC50 at 17.61, 17.00 and 16.05 µg/mL respectively. Moreover, final analogues 5g-5i, 5k,
5l, 5q and 5r showed no toxicity for the percentage viability IC50 > 39.42 µg/mL.
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{Insert Table 3 here}
2.3. Structure-activity relationship
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The results of antibacterial and antitubercular screening demonstrated the following
assumptions about the structural activity relationship (SAR): in the present study, we
investigated the effects of the substitution pattern of the hybrid benzimidazole and 2-pyridone
derivatives were carefully selected to impart different electronic environment on the molecules.
By comparing the antibacterial activity of the synthesized compounds, it was found that the
tested compounds were more effective against Gram-positive bacteria. In addition, the
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antibacterial activity was considerably affected by substitution pattern on the phenyl ring and
lipophilic profile of the compounds. It is believed that the strong lipophilic character of the
molecule plays an essential role in producing antibacterial effect. These properties are seen as an
important parameter related to membrane permeation in biological system. Many processes of
drug disposition depend on the capability to cross membranes and hence there is a high
correlation with measures of lipophilicity. Lipophilicity plays a major role in determining where
drugs are distributed within the body after adsorption and as a consequence how rapidly they are
metabolized and excreted. In this context, the presence of lipophilic moiety would be important
for such activity. In case of antibacterial activity, some analogues of this series were found to
have even more potency than the standard drug ‘chloramphenicol’ while some of them exhibited
comparable potency. Compounds 5h, 5i, 5k, 5q and 5r bearing Cl, Br, CH3 groups were found to
be most potent antibacterial agents due to its high lipophilicity. Among them, compound 5q
endowed with inductively electron withdrawing bromine at meta position emerged as the most
effective antibacterial agent at MIC of 12.5 µg/mL. The MIC values of these novel compounds
confirmed that the presence of bromine, chlorine and methyl substituent at meta position gave
rise to better pharmacological potency rather than the same substituent present at ortho or para
positions. Whereas, the opposite trend was observed for antitubercular activity results and there
was no specific relationship observed between MIC profiles versus lipophilicity. Among the
eighteen synthesized compounds, 5g-5i, 5k and 5l endowed with inductively electron
withdrawing chlorine and electron donating methyl group illustrated maximum MICs in the
range of 0.85 to 6.28 µg/mL against Mycobacterium tuberculosis. Out of them, 3-chloro yielded
the best activity at MIC of 0.85 µg/mL as compared to first line drug isoniazid (0.24 µg/mL).
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3. Conclusion
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The focal point of the current work was on the development of new bioactive based on
benzimidazole clubbed 2-pyridone compact system with the hope of generating new bioactive
chemical entities that could be useful as potent antibacterial and antitubercular agents. Many of
the synthesized motifs (5h, 5i, 5k, 5q and 5r), possessing atom/group such as bromo, chloro and
methyl at meta or para positions were identified as the most compelling antibacterial agents.
Albeit, it was observed that the strong lipophilic character of the molecule plays an imperative
role in producing antibacterial effect. Compounds 5g-5i, 5k and 5l came out as the most
promising antitubercular agents. In addition, the relationship between activity profiles and
lipophilicity of the newer analogues was also discussed in which higher lipophilic compounds
showed higher bioactivities, while, in case of antitubercular activity, there was no specific
relationship observed between MIC profiles versus lipophilicity. Moreover, the potent
antibacterial and antitubercular activity of most active compounds 5g-5i, 5k, 5l, 5q and 5r were
accompanied with relatively low level of cytotoxicity. Consequently, the degree of activity and
the encouraging physicochemical parameters displayed by a novel innovative structural
combination system of benzimidazole and 2-oxopyridine rings make such compounds a
privileged structure to achieve more active derivatives in ongoing studies. Synthesized
compounds were screened against C. albicans, but it was our observation that none of the
compounds exhibited significant activity against this fungi.
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4. Experimental
4.1. General Methods
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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
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apparatus and were reported uncorrected. TLC on silica gel plates (Merck, 60, F254) was used for
purity checking and reaction monitoring. Column chromatography 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
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spectrophotometer in KBr. H NMR and spectra were recorded on Varian Gemini 400 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 spectrometer. Anhydrous reactions were carried out in oven-dried glassware in
nitrogen atmosphere.
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4.2. General procedure for the synthesis of N'-(1-(1H-benzo[d]imidazol-2-yl)ethylidene)-2-
cyanoacetohydrazide (2)
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A mixture of equimolar amount of acetyl benzimidazole compound 1 (0.01 mol) and cyanoacetic
acid hydrazide (0.01 mol) in 1,4-dioxane (50 mL) was refluxed for 3 h. The reaction mixture was
concentrated by evaporating to dryness under reduced pressure and then cooled down to room
temperature. The separated crystals were filtered, air dried and recrystallized from absolute
alcohol. Yield 70%; mp 165 °C. IR (νmax, cm-1, KBr): 3371 (-NH, benzimidazole), 3278 (-NH, -
CONH-), 3028 (C-H, aromatic), 2917 (C-H, CH3), 2248 (CN), 1681 (CO), 1641 (C=C), 1532
(C=N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 1.88 (s, 3H, CH3), 3.38 (s, 2H, -CH2CN), 7.20
(d, 2H, J = 8.0 Hz, Ar-H), 7.61 (d, 2H, J = 8.2 Hz, Ar-H), 8.92 (s, 1H, -NHCO- D2O exch.),
13
10.32 (s, 1H, -NH benzimidazole D2O exch.). C NMR (100 MHz, DMSO-d6, δ, ppm): 171.3,
155.7, 151.6, 134.9, 125.8, 123.2, 115.1, 27.3, 13.4. LCMS (ESI): M/Z = 241.11 [M+]. Anal.
Calcd. for C12H11N5O: C, 59.74; H, 4.60; N, 29.03. Found: C, 59.69; H, 4.55; N, 29.09.
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4.3. General procedure for the synthesis of 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)-
amino)-6-amino-4-(4-nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (4)
318
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323
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326
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A mixture containing compound 2 (0.01 mol) and Knoevenagel compound 2-(4-
nitrobenzylidene)malononitrile 3 (0.01 mol) in ethanol (50 mL) was refluxed for 3 h using 2
drops of piperidine as catalyst. The excess of solvent was distilled out and the mixture was then
cooled down to room temperature. The crystals formed were filtered, air dried and recrystallized
from aqueous DMF. Yield 71%; mp 208-210 °C. IR (νmax, cm-1, KBr): 3446 (NH2), 3376 (-NH,
benzimidazole), 3030 (C-H, aromatic), 2921 (C-H, CH3), 2232 (CN), 1688 (CO), 1642 (C=C),
1
1536 (C=N), 1516, 1332 (N=O, Ar-NO2). H NMR (400 MHz, DMSO-d6, δ, ppm): 1.91 (s, 3H,
CH3), 7.22 (d, 2H, J = 8.0 Hz, Ar-H), 7.44 (d, 2H, J = 7.6 Hz, Ar-H), 7.62 (d, 2H, J = 8.4 Hz,
Ar-H), 8.21 (d, 2H, J = 7.7 Hz, Ar-H), 8.78 (s, 2H, NH2 D2O exch.), 10.36 (s, 1H, -NH
benzimidazole D2O exch.). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 169.5, 160.2, 159.4, 155.8,
151.7, 147.2, 138.7, 134.9, 130.2, 123.1, 123.8, 115.1, 115.5, 115.9, 76.6, 13.8. LCMS (ESI):
M/Z = 438.11 [M+]. Anal. Calcd. for C22H14N8O3: C, 60.27; H, 3.22; N, 25.56. Found: C, 60.34;
H, 3.25; N, 25.51.
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332
333
4.4. General procedure for the synthesis of 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-
6-((arylbenzylidene)amino)-4-(4-nitrophenyl)-2-oxo-1,2-dihydro-pyridine-3,5-dicarbonitriles
(5a-r)
334
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336
Compound 4 (0.01 mol), different substituted aromatic aldehydes (0.01 mol) and ethanol (50
mL) were taken in a round bottom flask and refluxed for 5 h. After 5 h, the reaction mass was
poured onto crushed ice and separated solid was filtered, dried and recrystallized from DMSO.
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4.4.1.
1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-(benzylideneamino)-4-(4-nitro-
phenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5a)
339
340
341
342
343
344
345
346
347
348
Yield 66%; mp 221 °C. IR (νmax, cm-1, KBr): 3380 (-NH, benzimidazole), 3034 (C-H, aromatic),
2992 (C-H, CH=N), 2924 (C-H, CH3), 2228 (CN), 1682 (CO), 1641 (C=C), 1532 (C=N), 1520,
1331 (N=O, Ar-NO2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 1.90 (s, 3H, CH3), 7.20 (d, 2H, J
= 8.1 Hz, Ar-H), 7.41 (d, 2H, J = 7.5 Hz, Ar-H), 7.47 (d, 2H, J = 7.6 Hz, Ar-H), 7.54 (t, 1H, J =
7.4 Hz, Ar-H), 7.63 (d, 2H, J = 8.3 Hz, Ar-H), 7.84 (d, 2H, J = 7.7 Hz, Ar-H), 8.24 (d, 2H, J =
7.6 Hz, Ar-H), 9.51 (s, 1H, CH=N), 10.41 (s, 1H, -NH benzimidazole D2O exch.). 13C NMR
(100 MHz, DMSO-d6, δ, ppm): 169.5, 163.8, 160.1, 155.7, 153.3, 151.6, 147.1, 138.6, 134.8,
133.8, 131.2, 130.1, 129.4, 128.9, 123.2, 123.8, 115.0, 115.5, 115.9, 114.7, 13.5. LCMS (ESI):
M/Z = 526.14 [M+]. Anal. Calcd. for C29H18N8O3: C, 66.16; H, 3.45; N, 21.28. Found: C, 66.22;
H, 3.49; N, 21.21.
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350
4.4.2. 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-((2-hydroxybenzylidene)amino)-4-
(4-nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5b)
351
352
353
354
355
356
357
358
Yield 69%; mp 242 °C. IR (νmax, cm-1, KBr): 3441 (OH), 3382 (-NH, benzimidazole), 3031 (C-H,
aromatic), 2990 (C-H, CH=N), 2924 (C-H, CH3), 2225 (CN), 1681 (CO), 1637 (C=C), 1531
(C=N), 1522, 1330 (N=O, Ar-NO2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 1.92 (s, 3H, CH3),
7.01 (d, 1H, J = 7.5 Hz, Ar-H), 7.11 (t, 1H, J = 7.6 Hz, Ar-H), 7.21 (d, 2H, J = 8.2 Hz, Ar-H),
7.44 (d, 2H, J = 7.6 Hz, Ar-H), 7.53 (t, 1H, J = 7.4 Hz, Ar-H), 7.62 (d, 2H, J = 8.4 Hz, Ar-H),
7.71 (d, 1H, J = 7.6 Hz, Ar-H), 8.21 (d, 2H, J = 7.6 Hz, Ar-H), 8.74 (s, 1H, OH D2O exch.), 9.54
13
(s, 1H, CH=N), 10.42 (s, 1H, -NH benzimidazole D2O exch.). C NMR (100 MHz, DMSO-d6,
δ, ppm): 169.6, 163.7, 160.2, 155.6, 153.1, 151.5, 147.0, 138.5, 134.8, 134.1, 133.5, 130.1,
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127.9, 127.4, 123.8, 123.1, 115.9, 115.5, 115.0, 115.9, 114.8, 59.4, 13.7, 13.2. LCMS (ESI): M/Z
= 542.16 [M+]. Anal. Calcd. for C29H18N8O4: C, 64.20; H, 3.34; N, 20.65. Found: C, 64.28; H,
3.39; N, 20.71.
362
363
4.4.3. 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-((3-hydroxybenzylidene)amino)-4-
(4-nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5c)
364
365
366
367
368
369
370
371
372
373
Yield 68%; mp 256 °C. IR (νmax, cm-1, KBr): 3440 (OH), 3384 (-NH, benzimidazole), 3034 (C-H,
aromatic), 2992 (C-H, CH=N), 2927 (C-H, CH3), 2223 (CN), 1684 (CO), 1639 (C=C), 1534
(C=N), 1524, 1334 (N=O, Ar-NO2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 1.90 (s, 3H, CH3),
7.02 (d, 1H, J = 7.6 Hz, Ar-H), 7.18 (d, 2H, J = 8.0 Hz, Ar-H), 7.27 (t, 1H, J = 7.6 Hz, Ar-H),
7.39 (d, 1H, J = 7.4 Hz, Ar-H), 7.46 (d, 2H, J = 7.6 Hz, Ar-H), 7.53 (s, 1H, Ar-H), 7.63 (d, 2H, J
= 8.4 Hz, Ar-H), 8.22 (d, 2H, J = 7.6 Hz, Ar-H), 8.71 (s, 1H, OH D2O exch.), 9.55 (s, 1H,
CH=N), 10.44 (s, 1H, -NH benzimidazole D2O exch.). 13C NMR (100 MHz, DMSO-d6, δ, ppm):
169.4, 163.8, 160.1, 158.6, 155.5, 153.2, 151.5, 147.1, 138.7, 135.2, 134.9, 130.5, 130.1, 123.9,
123.1, 121.8, 118.2, 115.9, 115.6, 115.0, 114.8, 114.3, 13.6. LCMS (ESI): M/Z = 542.14 [M+].
Anal. Calcd. for C29H18N8O4: C, 64.20; H, 3.34; N, 20.65. Found: C, 64.29; H, 3.38; N, 20.72.
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375
4.4.4. 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-((4-hydroxybenzylidene)amino)-4-
(4-nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5d)
376
377
378
379
380
Yield 63%; mp 236 °C. IR (νmax, cm-1, KBr): 3444 (OH), 3386 (-NH, benzimidazole), 3036 (C-H,
aromatic), 2992 (C-H, CH=N), 2928 (C-H, CH3), 2224 (CN), 1685 (CO), 1637 (C=C), 1531
(C=N), 1522, 1332 (N=O, Ar-NO2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 1.89 (s, 3H, CH3),
6.88 (d, 2H, J = 7.8 Hz, Ar-H), 7.20 (d, 2H, J = 7.9 Hz, Ar-H), 7.44 (d, 2H, J = 7.5 Hz, Ar-H),
7.62 (d, 2H, J = 8.3 Hz, Ar-H), 7.82 (d, 2H, J = 7.4 Hz, Ar-H), 8.24 (d, 2H, J = 7.5 Hz, Ar-H),
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8.70 (s, 1H, OH D2O exch.), 9.57 (s, 1H, CH=N), 10.44 (s, 1H, -NH benzimidazole D2O exch.).
13C NMR (100 MHz, DMSO-d6, δ, ppm): 169.5, 163.9, 160.8, 160.0, 155.6, 153.1, 151.6, 147.2,
138.5, 134.8, 130.7, 130.1, 126.2, 123.8, 123.0, 116.1, 115.9, 115.4, 115.0, 114.8, 13.7. LCMS
(ESI): M/Z = 542.15 [M+]. Anal. Calcd. for C29H18N8O4: C, 64.20; H, 3.34; N, 20.65. Found: C,
64.30; H, 3.38; N, 20.73.
386
387
4.4.5. 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-((3-fluorobenzylidene)amino)-4-(4-
nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5e)
388
389
390
391
392
393
394
395
396
397
Yield 62%; mp 192 °C. IR (νmax, cm-1, KBr): 3388 (-NH, benzimidazole), 3038 (C-H, aromatic),
2994 (C-H, CH=N), 2930 (C-H, CH3), 2226 (CN), 1688 (CO), 1638 (C=C), 1532 (C=N), 1524,
1
1334 (N=O, Ar-NO2), 1122 (C-F). H NMR (400 MHz, DMSO-d6, δ, ppm): 1.87 (s, 3H, CH3),
7.21 (d, 2H, J = 8.0 Hz, Ar-H), 7.37 (d, 1H, J = 7.4 Hz, Ar-H), 7.45 (d, 2H, J = 7.4 Hz, Ar-H),
7.58 (d, 2H, J = 8.3 Hz, Ar-H), 7.65 (d, 1H, J = 7.5 Hz, Ar-H), 7.71 (t, 1H, J = 7.6 Hz, Ar-H),
7.84 (d, 1H, J = 7.4 Hz, Ar-H), 8.22 (d, 2H, J = 7.5 Hz, Ar-H), 9.54 (s, 1H, CH=N), 10.41 (s, 1H,
13
-NH benzimidazole D2O exch.). C NMR (100 MHz, DMSO-d6, δ, ppm): 169.3, 163.8, 163.1,
160.1, 155.7, 153.1, 151.5, 147.1, 138.5, 135.2, 134.9, 130.5, 130.1, 124.8, 123.9, 123.2, 117.7,
115.9, 115.5, 115.0, 114.8, 114.1, 13.7. LCMS (ESI): M/Z = 544.12 [M+]. Anal. Calcd. for
C29H17FN8O3 : C, 63.97; H, 3.15; N, 20.58. Found: C, 63.90; H, 3.19; N, 20.52.
398
399
4.4.6. 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-((4-fluorobenzylidene)amino)-4-(4-
nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5f)
400
401
402
Yield 64%; mp 208 °C. IR (νmax, cm-1, KBr): 3386 (-NH, benzimidazole), 3037 (C-H, aromatic),
2993 (C-H, CH=N), 2931 (C-H, CH3), 2227 (CN), 1686 (CO), 1638 (C=C), 1531 (C=N), 1522,
1
1332 (N=O, Ar-NO2), 1125 (C-F). H NMR (400 MHz, DMSO-d6, δ, ppm): 1.88 (s, 3H, CH3),
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7.22 (d, 2H, J = 8.1 Hz, Ar-H), 7.37 (d, 2H, J = 7.6 Hz, Ar-H), 7.44 (d, 2H, J = 7.5 Hz, Ar-H),
7.62 (d, 2H, J = 8.4 Hz, Ar-H), 7.83 (d, 2H, J = 7.7 Hz, Ar-H), 8.23 (d, 2H, J = 7.5 Hz, Ar-H),
9.51 (s, 1H, CH=N), 10.45 (s, 1H, -NH benzimidazole D2O exch.). 13C NMR (100 MHz, DMSO-
d6, δ, ppm): 169.5, 165.3, 163.7, 160.1, 155.7, 153.3, 151.7, 147.2, 138.7, 134.8, 130.9, 130.2,
129.2, 123.8, 123.1, 115.9, 115.6, 115.2, 114.9, 114.2, 13.5. LCMS (ESI): M/Z = 544.12 [M+].
Anal. Calcd. for C29H17FN8O3 : C, 63.97; H, 3.15; N, 20.58. Found: C, 63.91; H, 3.20; N, 20.51.
409
410
4.4.7. 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-((2-chlorobenzylidene)amino)-4-(4-
nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5g)
411
412
413
414
415
416
417
418
419
420
Yield 67%; mp 223 °C. IR (νmax, cm-1, KBr): 3384 (-NH, benzimidazole), 3034 (C-H, aromatic),
2991 (C-H, CH=N), 2929 (C-H, CH3), 2226 (CN), 1684 (CO), 1634 (C=C), 1530 (C=N), 1521,
1
1331 (N=O, Ar-NO2), 756 (C-Cl). H NMR (400 MHz, DMSO-d6, δ, ppm): 1.92 (s, 3H, CH3),
7.19 (d, 2H, J = 8.2 Hz, Ar-H), 7.38 (t, 1H, J = 7.4 Hz, Ar-H), 7.44 (d, 2H, J = 7.6 Hz, Ar-H),
7.50 (t, 1H, J = 7.5 Hz, Ar-H), 7.57 (d, 1H, J = 7.5 Hz, Ar-H), 7.68 (d, 2H, J = 8.4 Hz, Ar-H),
7.81 (d, 1H, J = 7.6 Hz, Ar-H), 8.22 (d, 2H, J = 7.5 Hz, Ar-H), 9.48 (s, 1H, CH=N), 10.42 (s, 1H,
13
-NH benzimidazole D2O exch.). C NMR (100 MHz, DMSO-d6, δ, ppm): 169.4, 163.6, 160.1,
155.7, 153.3, 151.6, 147.2, 138.7, 134.8, 133.9, 133.3, 132.5, 130.6, 130.1, 127.3, 126.8, 123.8,
123.2, 115.9, 115.5, 115.0, 114.9, 13.6. LCMS (ESI): M/Z = 561.12 [M+]. Anal. Calcd. for
C29H17ClN8O3: C, 62.09; H, 3.05; N, 19.98. Found: C, 62.19; H, 3.09; N, 19.92.
421
422
4.4.8. 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-((3-chlorobenzylidene)amino)-4-(4-
nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5h)
423
424
Yield 64%; mp 199 °C. IR (νmax, cm-1, KBr): 3384 (-NH, benzimidazole), 3035 (C-H, aromatic),
2992 (C-H, CH=N), 2930 (C-H, CH3), 2228 (CN), 1682 (CO), 1632 (C=C), 1528 (C=N), 1520,
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426
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1330 (N=O, Ar-NO2), 758 (C-Cl). H NMR (400 MHz, DMSO-d6, δ, ppm): 1.90 (s, 3H, CH3),
7.21 (d, 2H, J = 8.1 Hz, Ar-H), 7.41 (d, 2H, J = 7.4 Hz, Ar-H), 7.47 (t, 1H, J = 7.4 Hz, Ar-H),
7.58 (d, 1H, J = 7.5 Hz, Ar-H), 7.69 (d, 2H, J = 8.3 Hz, Ar-H), 7.74 (d, 1H, J = 7.5 Hz, Ar-H),
7.92 (d, 1H, J = 7.6 Hz, Ar-H), 8.24 (d, 2H, J = 7.7 Hz, Ar-H), 9.44 (s, 1H, CH=N), 10.43 (s, 1H,
13
-NH benzimidazole D2O exch.). C NMR (100 MHz, DMSO-d6, δ, ppm): 169.5, 163.8, 160.2,
155.6, 153.4, 151.4, 147.1 138.6, 135.1, 134.9, 134.3, 131.0, 130.6, 130.0, 127.7, 127.2, 123.9,
123.1, 115.8, 115.5, 115.0, 114.7, 13.5. LCMS (ESI): M/Z = 561.11 [M+]. Anal. Calcd. for
C29H17ClN8O3: C, 62.09; H, 3.05; N, 19.98. Found: C, 62.18; H, 3.10; N, 19.93.
433
434
4.4.9. 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-((4-chlorobenzylidene)amino)-4-(4-
nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5i)
435
436
437
438
439
440
441
442
443
Yield 67%; mp 229 °C. IR (νmax, cm-1, KBr): 3386 (-NH, benzimidazole), 3036 (C-H, aromatic),
2994 (C-H, CH=N), 2932 (C-H, CH3), 2229 (CN), 1682 (CO), 1634 (C=C), 1528 (C=N), 1522,
1331 (N=O, Ar-NO2), 760 (C-Cl). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 1.88 (s, 3H, CH3),
7.22 (d, 2H, J = 8.2 Hz, Ar-H), 7.44 (d, 2H, J = 7.5 Hz, Ar-H), 7.57 (d, 2H, J = 7.6 Hz, Ar-H),
7.68 (d, 2H, J = 8.2 Hz, Ar-H), 7.79 (d, 2H, J = 7.6 Hz, Ar-H), 8.22 (d, 2H, J = 7.7 Hz, Ar-H),
9.49 (s, 1H, CH=N), 10.44 (s, 1H, -NH benzimidazole D2O exch.). 13C NMR (100 MHz, DMSO-
d6, δ, ppm): 169.6, 163.7, 160.1, 155.5, 153.2, 151.5, 147.0, 138.5, 136.7, 134.8, 131.9, 130.7,
130.0, 128.9, 123.8, 123.2, 115.9, 115.6, 115.2, 114.8, 13.7. LCMS (ESI): M/Z = 561.11 [M+].
Anal. Calcd. for C29H17ClN8O3: C, 62.09; H, 3.05; N, 19.98. Found: C, 62.19; H, 3.09; N, 19.92.
444
445
4.4.10. 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-((2-methylbenzylidene)amino)-4-
(4-nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5j)
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447
448
449
450
451
452
453
454
455
Yield 63%; mp 244 °C. IR (νmax, cm-1, KBr): 3381 (-NH, benzimidazole), 3032 (C-H, aromatic),
2990 (C-H, CH=N), 2928 (C-H, CH3), 2224 (CN), 1679 (CO), 1630 (C=C), 1523 (C=N), 1520,
1
1328 (N=O, Ar-NO2). H NMR (400 MHz, DMSO-d6, δ, ppm): 1.91 (s, 3H, CH3), 2.51 (s, 3H,
CH3), 7.17 (d, 2H, J = 8.1 Hz, Ar-H), 7.24 (t, 1H, J = 7.4 Hz, Ar-H), 7.30 (d, 1H, J = 7.5 Hz, Ar-
H), 7.38 (t, 1H, J = 7.6 Hz, Ar-H), 7.46 (d, 2H, J = 7.9 Hz, Ar-H), 7.67 (d, 2H, J = 8.3 Hz, Ar-
H), 7.74 (d, 1H, J = 7.4 Hz, Ar-H), 8.23 (d, 2H, J = 7.6 Hz, Ar-H), 9.43 (s, 1H, CH=N), 10.45 (s,
1H, -NH benzimidazole D2O exch.). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 169.5, 163.6,
160.1, 155.7, 153.3, 151.6, 147.1, 138.6, 135.3, 134.9, 131.2, 130.8, 130.1, 129.1, 126.6, 125.9,
123.9, 123.1, 115.9, 115.5, 115.0, 114.7, 18.8, 13.7. LCMS (ESI): M/Z = 540.18 [M+]. Anal.
Calcd. for C30H20N8O3: C, 66.66; H, 3.73; N, 20.73. Found: C, 66.60; H, 3.77; N, 20.78.
456
457
4.4.11. 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-((3-methylbenzylidene)amino)-4-
(4-nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5k)
458
459
460
461
462
463
464
465
466
467
Yield 64%; mp 218 °C. IR (νmax, cm-1, KBr): 3380 (-NH, benzimidazole), 3031 (C-H, aromatic),
2990 (C-H, CH=N), 2927 (C-H, CH3), 2222 (CN), 1678 (CO), 1630 (C=C), 1523 (C=N), 1521,
1
1328 (N=O, Ar-NO2). H NMR (400 MHz, DMSO-d6, δ, ppm): 1.88 (s, 3H, CH3), 2.36 (s, 3H,
CH3), 7.18 (d, 2H, J = 8.2 Hz, Ar-H), 7.29 (d, 1H, J = 7.5 Hz, Ar-H), 7.39 (t, 1H, J = 7.6 Hz, Ar-
H), 7.45 (d, 2H, J = 7.5 Hz, Ar-H), 7.61 (d, 1H, J = 7.4 Hz, Ar-H), 7.67 (d, 2H, J = 8.4 Hz, Ar-
H), 7.76 (s, 1H, Ar-H), 8.21 (d, 2H, J = 7.4 Hz, Ar-H), 9.41 (s, 1H, CH=N), 10.44 (s, 1H, -NH
benzimidazole D2O exch.). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 169.4, 163.8, 160.0, 155.6,
153.2, 151.7, 147.2, 138.8, 138.2, 134.8, 133.7, 131.4, 130.2, 129.5, 128.8, 126.3, 123.9, 123.2,
115.9, 115.5, 115.0, 114.7, 21.4, 13.6. LCMS (ESI): M/Z = 540.17 [M+]. Anal. Calcd. for
C30H20N8O3: C, 66.66; H, 3.73; N, 20.73. Found: C, 66.58; H, 3.78; N, 20.77.
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4.4.12. 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-((4-methylbenzylidene)amino)-4-
(4-nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5l)
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Yield 62%; mp 247 °C. IR (νmax, cm-1, KBr): 3381 (-NH, benzimidazole), 3030 (C-H, aromatic),
2990 (C-H, CH=N), 2928 (C-H, CH3), 2224 (CN), 1680 (CO), 1631 (C=C), 1524 (C=N), 1520,
1
1326 (N=O, Ar-NO2). H NMR (400 MHz, DMSO-d6, δ, ppm): 1.87 (s, 3H, CH3), 2.37 (s, 3H,
Ar-CH3), 7.19 (d, 2H, J = 8.1 Hz, Ar-H), 7.29 (d, 2H, J = 7.7 Hz, Ar-H), 7.44 (d, 2H, J = 7.5 Hz,
Ar-H), 7.67 (d, 2H, J = 8.3 Hz, Ar-H), 7.74 (d, 2H, J = 7.4 Hz, Ar-H), 8.24 (d, 2H, J = 7.6 Hz,
Ar-H), 9.44 (s, 1H, CH=N), 10.46 (s, 1H, -NH benzimidazole D2O exch.). 13C NMR (100 MHz,
DMSO-d6, δ, ppm): 169.5, 163.8, 160.1, 155.7, 153.1, 151.6, 147.0, 140.7, 138.5, 134.8, 130.7,
130.1, 129.7, 129.2, 123.8, 123.1, 115.9, 115.5, 115.1, 114.7, 21.5, 13.7. LCMS (ESI): M/Z =
540.17 [M+]. Anal. Calcd. for C30H20N8O3: C, 66.66; H, 3.73; N, 20.73. Found: C, 66.58; H,
3.78; N, 20.78.
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4.4.13. 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-((3-methoxybenzylidene)- amino)-
4-(4-nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5m)
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Yield 66%; mp 201 °C. IR (νmax, cm-1, KBr): 3380 (-NH, benzimidazole), 3027 (C-H, aromatic),
2988 (C-H, CH=N), 2928 (C-H, CH3), 2851 (C-H, OCH3), 2221 (CN), 1678 (CO), 1630 (C=C),
1
1522 (C=N), 1518, 1322 (N=O, Ar-NO2). H NMR (400 MHz, DMSO-d6, δ, ppm): 1.87 (s, 3H,
CH3), 3.86 (s, 3H, OCH3), 7.07 (d, 1H, J = 7.5 Hz, Ar-H), 7.18 (d, 2H, J = 8.1 Hz, Ar-H), 7.32 (t,
1H, J = 7.6 Hz, Ar-H), 7.39 (d, 1H, J = 7.4 Hz, Ar-H), 7.45 (d, 2H, J = 7.6 Hz, Ar-H), 7.54 (s,
1H, Ar-H), 7.66 (d, 2H, J = 8.4 Hz, Ar-H), 8.21 (d, 2H, J = 7.5 Hz, Ar-H), 9.41 (s, 1H, CH=N),
13
10.42 (s, 1H, -NH benzimidazole D2O exch.). C NMR (100 MHz, DMSO-d6, δ, ppm): 169.5,
163.7, 160.8, 160.1, 155.7, 153.1, 151.5, 147.1, 138.7, 134.9, 134.3, 130.1, 129.7, 123.9, 123.2,
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121.6, 116.7, 115.8, 115.4, 115.0, 114.7, 111.3, 55.6, 13.6. LCMS (ESI): M/Z = 556.15 [M+].
Anal. Calcd. for C30H20N8O4: C, 64.74; H, 3.62; N, 20.13. Found: C, 64.68; H, 3.67; N, 20.18.
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4.4.14. 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-((4-methoxybenzylidene)- amino)-
4-(4-nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5n)
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Yield 67%; mp 211 °C. IR (νmax, cm-1, KBr): 3381 (-NH, benzimidazole), 3028 (C-H, aromatic),
2990 (C-H, CH=N), 2929 (C-H, CH3), 2854 (C-H, OCH3), 2223 (CN), 1679 (CO), 1632 (C=C),
1
1524 (C=N), 1520, 1323 (N=O, Ar-NO2). H NMR (400 MHz, DMSO-d6, δ, ppm): 1.86 (s, 3H,
CH3), 3.87 (s, 3H, OCH3), 7.07 (d, 2H, J = 7.5 Hz, Ar-H), 7.19 (d, 2H, J = 8.0 Hz, Ar-H), 7.41
(d, 2H, J = 7.4 Hz, Ar-H), 7.67 (d, 2H, J = 8.4 Hz, Ar-H), 7.84 (d, 2H, J = 7.3 Hz, Ar-H), 8.20
(d, 2H, J = 7.5 Hz, Ar-H), 9.40 (s, 1H, CH=N), 10.42 (s, 1H, -NH benzimidazole D2O exch.).
13C NMR (100 MHz, DMSO-d6, δ, ppm): 169.4, 163.9, 162.8, 160.0, 155.7, 153.2, 151.7, 147.2,
138.6, 134.9, 130.8, 130.3, 126.1, 123.8, 123.1, 115.9, 115.5, 115.0, 114.8, 114.3, 55.9, 13.7.
LCMS (ESI): M/Z = 556.14 [M+]. Anal. Calcd. for C30H20N8O4: C, 64.74; H, 3.62; N, 20.13.
Found: C, 64.68; H, 3.68; N, 20.19.
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4.4.15. 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-((3-nitrobenzylidene)amino)-4-(4-
nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5o)
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Yield 69%; mp 251 °C. IR (νmax, cm-1, KBr): 3385 (-NH, benzimidazole), 3031 (C-H, aromatic),
2994 (C-H, CH=N), 2932 (C-H, CH3), 2226 (CN), 1684 (CO), 1634 (C=C), 1527 (C=N), 1524,
1326 (N=O, Ar-NO2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 1.90 (s, 3H, CH3), 7.21 (d, 2H, J
= 8.0 Hz, Ar-H), 7.42 (d, 2H, J = 7.5 Hz, Ar-H), 7.69 (d, 2H, J = 8.3 Hz, Ar-H), 7.80 (t, 1H, J =
7.5 Hz, Ar-H), 8.14 (d, 1H, J = 7.5 Hz, Ar-H), 8.20 (d, 2H, J = 7.6 Hz, Ar-H), 8.27 (d, 1H, J =
7.7 Hz, Ar-H), 8.51 (s, 1H, Ar-H), 9.44 (s, 1H, CH=N), 10.48 (s, 1H, -NH benzimidazole D2O
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exch.). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 169.4, 163.6, 160.1, 155.7, 153.2, 151.6, 148.1,
147.0, 138.5, 135.4, 134.8, 134.4, 130.2, 129.6, 126.3, 123.8, 123.2, 121.7, 115.8, 115.5, 115.0,
114.8, 13.7. LCMS (ESI): M/Z = 571.12 [M+]. Anal. Calcd. for C29H17N9O5: C, 60.95; H, 3.00;
N, 22.06. Found: C, 60.89; H, 3.04; N, 22.11.
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4.4.16. 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-((4-nitrobenzylidene)amino)-4-(4-
nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5p)
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Yield 66%; mp 261 °C. IR (νmax, cm-1, KBr): 3387 (-NH, benzimidazole), 3034 (C-H, aromatic),
2996 (C-H, CH=N), 2934 (C-H, CH3), 2229 (CN), 1688 (CO), 1637 (C=C), 1529 (C=N), 1526,
1327 (N=O, Ar-NO2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 1.90 (s, 3H, CH3), 7.21 (d, 2H, J
= 8.0 Hz, Ar-H), 7.42 (d, 2H, J = 7.5 Hz, Ar-H), 7.69 (d, 2H, J = 8.3 Hz, Ar-H), 7.80 (t, 1H, J =
7.5 Hz, Ar-H), 8.14 (d, 1H, J = 7.5 Hz, Ar-H), 8.20 (d, 2H, J = 7.6 Hz, Ar-H), 8.27 (d, 1H, J =
7.7 Hz, Ar-H), 8.51 (s, 1H, Ar-H), 9.44 (s, 1H, CH=N), 10.48 (s, 1H, -NH benzimidazole D2O
exch.). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 169.6, 163.8, 160.1, 155.7, 153.1, 151.7, 150.4,
147.2, 139.9, 138.6, 134.9, 130.1, 127.8, 124.1, 123.9, 123.3, 115.9, 115.5, 115.0, 114.7, 13.5.
LCMS (ESI): M/Z = 571.14 [M+]. Anal. Calcd. for C29H17N9O5: C, 60.95; H, 3.00; N, 22.06.
Found: C, 60.87; H, 3.06; N, 22.12.
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4.4.17. 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-((3-bromobenzylidene)amino)-4-
(4-nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5q)
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531
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Yield 70%; mp 193 °C. IR (νmax, cm-1, KBr): 3386 (-NH, benzimidazole), 3033 (C-H, aromatic),
2992 (C-H, CH=N), 2931 (C-H, CH3), 2224 (CN), 1684 (CO), 1635 (C=C), 1525 (C=N), 1526,
1
1324 (N=O, Ar-NO2), 543 (C-Br). H NMR (400 MHz, DMSO-d6, δ, ppm): 1.89 (s, 3H, CH3),
7.21 (d, 2H, J = 8.0 Hz, Ar-H), 7.39 (t, 1H, J = 7.5 Hz, Ar-H), 7.45 (d, 2H, J = 7.4 Hz, Ar-H),
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7.57 (d, 1H, J = 7.5 Hz, Ar-H), 7.67 (d, 2H, J = 7.6 Hz, Ar-H), 7.79 (d, 1H, J = 7.4 Hz, Ar-H),
7.85 (s, 1H, Ar-H), 8.21 (d, 2H, J = 7.6 Hz, Ar-H), 9.49 (s, 1H, CH=N), 10.43 (s, 1H, -NH
benzimidazole D2O exch.). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 169.5, 163.7, 160.2, 155.7,
153.3, 151.6, 147.1, 138.9, 135.8, 134.7, 133.7, 132.8, 130.2, 129.8, 128.3, 123.9, 123.5, 123.0,
115.8, 115.5, 115.1, 114.8, 13.7. LCMS (ESI): M/Z = 604.08 [M+]. Anal. Calcd. for
C29H17BrN8O3: C, 57.53; H, 2.83; N, 18.51. Found: C, 57.60; H, 2.87; N, 18.57.
540
541
4.4.18. 1-((1-(1H-benzo[d]imidazol-2-yl)ethylidene)amino)-6-((4-bromobenzylidene)amino)-4-
(4-nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5r)
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549
550
Yield 71%; mp 223 °C. IR (νmax, cm-1, KBr): 3387 (-NH, benzimidazole), 3034 (C-H, aromatic),
2994 (C-H, CH=N), 2932 (C-H, CH3), 2225 (CN), 1686 (CO), 1636 (C=C), 1526 (C=N), 1527,
1
1325 (N=O, Ar-NO2), 546 (C-Br). H NMR (400 MHz, DMSO-d6, δ, ppm): 1.88 (s, 3H, CH3),
7.20 (d, 2H, J = 8.0 Hz, Ar-H), 7.42 (d, 2H, J = 7.5 Hz, Ar-H), 7.58 (d, 2H, J = 7.7 Hz, Ar-H),
7.68 (d, 2H, J = 7.6 Hz, Ar-H), 7.75 (d, 2H, J = 7.6 Hz, Ar-H), 8.20 (d, 2H, J = 7.6 Hz, Ar-H),
9.46 (s, 1H, CH=N), 10.45 (s, 1H, -NH benzimidazole D2O exch.). 13C NMR (100 MHz, DMSO-
d6, δ, ppm): 169.5, 163.7, 160.2, 155.5, 153.2, 151.6, 147.1, 138.7, 134.8, 132.6, 131.8, 130.2,
128.6, 125.5, 123.8, 123.0, 115.9, 115.4, 115.0, 114.8, 13.7. LCMS (ESI): M/Z = 604.07 [M+].
Anal. Calcd. for C29H17BrN8O3: C, 57.53; H, 2.83; N, 18.51. Found: C, 57.60; H, 2.87; N, 18.58.
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4.5. Biological assay
4.5.1. Antibacterial assay
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Antibacterial studies of newly synthesized compounds 5a-r were carried out against the
representative panel of bacteria such as Staphylococcus aureus MTCC-96, Streptococcus
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pyogenes MTCC-442, Escherichia coli MTCC-443, Pseudomonas aeruginosa MTCC-1688 and
methicillin-resistant S. aureus (MRSA isolate ATCC 43300). Antifungal activity was carried out
against the yeast-like pathogenic fungus Candida albicans MTCC 227. All MTCC and ATCC
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 1000, 500, 250 ꢀg/mL. The compounds found to be
active in primary screening were similarly diluted to obtain 200, 125, 100, 62.5, 50, 25, 12.5 and
6.25 ꢀg/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 1000 ꢀg/mL concentration, as stock solution. The control tube containing no
antibiotic was immediately subcultured [before inoculation] by spreading a loopful evenly over a
quarter of plate in a suitable medium for the growth of test organisms. The culture tubes were
then incubated for 24 h at 37 °C and the growth was monitored visually and
spectrophotometrically. Ten ꢀg/mL suspensions were further inoculated on an appropriate media
and growth was noted after 24 h and 48 h. The lowest concentration (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
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inoculum) was compared. Solvent had no influence on strain growth. The result of this was
greatly affected by the size of the inoculums. The test 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 drugs used in the present
study were ‘ciprofloxacin’ and ‘chloramphenicol’ for evaluating antibacterial activity.
583
4.5.2. Antitubercular assay
584
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Antitubercular activity was determined using the modified radiometric 7H12 broth
(BACTEC 12B system) in which stock solutions as test compounds were prepared in
dimethylsulfoxide (DMSO) at a concentration of 12.8 mM and the final test concentrations
ranged from 39.42 to 0.15 µg/mL. Controls received 50 µL DMSO. INH was included as a
positive drug control. INH was solubilized and diluted in DMSO and added to BACTEC-12
broth to achieve a range of concentration for determination of minimum inhibitory concentration
(MIC, lowest concentration inhibiting ≥ 90% of the inoculums, MIC value of INH is 0.24 µg/mL
at 95% inhibition of H37Rv strain). M. tuberculosis H37Rv strain (ATCC 27294) was cultured at
37 ˚C in 100 ml of Middlebrook 7H9 broth (Difco, Detroit, Mich.) supplemented with 0.2%
(vol/vol) glycerol, 10% (vol/vol) OADC (oleic acid, albumin, dextrose, catalase) and 0.05%
(vol/vol) Tween 80. The complete medium was referred to as 7H9GC-Tween. Cultures were
incubated in 500-ml nephelometer flasks on a rotary shaker at 150 rpm and 37 °C until they
reached an optical density of 0.4 to 0.5 at 550 nm. Bacteria were washed and suspended in 20 ml
of phosphate-buffered saline and passed through an 8-mm pore size filter to eliminate clumps.
The filtrates were aliquoted, stored at 280 °C and used within 30 days. Cultures were prepared
and an appropriate dilution performed such that a BACTEC-12B vial inoculated with 0.1 mL
would reach a growth index (GI) of 999 in five days. Antimicrobial susceptibility testing was
27