Novel benzimidazole-oxadiazole hybrid molecules as promising antimicrobial agents

Article abstract of DOI:10.1039/c5ra23282a

In the present study, we describe the design and expeditious synthesis of novel 2-aryl-5-(3-aryl-[1,2,4]-oxadiazol-5-yl)-1-methyl-1H-benzo[d]imidazole hybrid molecules as promising antimicrobial agents. The core moiety 2-aryl-ethyl-1H-benzo[d]imidazole-5-

Full text of DOI:10.1039/c5ra23282a

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Novel benzimidazole-oxadiazole hybrid molecules as promising antimicrobial agents
Shruthi Na, Boja Poojarya*, Vasantha Kumar a, Mohammed Mumtaz Hussainb, Vaishali M Rai c,
Vinitha R Paic, Mahima Bhata, B C Revanasiddappad
aDepartment of Chemistry, Mangalore University, Mangalagangothriꢀ574199, Karnataka, India.
bDepartment of Pharmaceutical Chemistry, S.J.M. College of Pharmacy, Chitradurgaꢀ575501,
Karnataka, India.
cDepartment of Bioꢀchemistry, Yenepoya University, Deralakattaꢀ575 018, Mangalore,
Karnataka, India
dDepartment of Pharmacology, N.G.S.M. Institute of Pharmaceutical Sciences, Paneer,
Deralakatta, Mangalore ꢀ 575 018, Karnataka, India.
*Corresponding Author: Boja Poojary, Department of Chemistry, Mangalore University,
Mangalagangothriꢀ574199, Karnataka, India Tel./fax:+91ꢀ824ꢀ2287262/2287367
Eꢀmail: bojapoojary@gmail.com
Abstract
In the present study, we describe the design and expeditious synthesis of novel 2ꢀarylꢀ5ꢀ
(3ꢀarylꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ1ꢀmethylꢀ1Hꢀbenzo[d]imidazole hybrid molecules as promising
antimicrobial agents. The core moiety 2ꢀarylꢀethylꢀ1Hꢀbenzo[d]imidazoleꢀ5ꢀcarboxylate was
efficiently synthesized by rapid ‘one pot’ nitro reductive cyclization reaction using sodium
dithionite as reagent. All the compounds were screened for their antimicrobial activities and the
active compounds were screened for their antiꢀtubercular activity against Mycobacterium
tuberculosis H37Rv strain by Microplate Almer Blue Assay method. Compounds 8k, 8n, 8p and
8r exhibited potent antitubercular activity with MIC 1.6 ꢁg/mL which is two times more potent
than the standard drugs Pyrazinamide and Ciprofloxacin and four fold superior than
Streptomycin and Isoniazid. Further, potent compounds were tested for its preliminary toxicity
by hemolytic assay, where the compounds remain non toxic even at higher concentration and
showed good selectivity index. The investigation of cytotoxicity against normal embryonic
kidney cell line, HEK 293 by MTT assay showed IC50 value more than 355 ꢁg/mL for all the

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tested potent compounds. From the screening study 8k, 8n, 8p and 8n emerged as strong
candidates with potent antimicrobial activities and good ADME parameters. Attempts were also
made to establish the structural activity relationships among the tested compounds.
Keywords
Benzimidazole; [1,2,4]ꢀOxadiazole; ‘Oneꢀpot’ nitroreductive cyclisation; Antimicrobial;
Antitubercular, Hemolytic
1. Introduction
Antibiotics are the most important weapons to fight against microbial infections and have
enormously benefited the healthꢀrelated quality of human life since their introduction. However,
over the past few decades, these health benefits are under threat as many commonly used
antibiotics becoming less sensitive, effective against certain illnesses, not only because of toxic
reactions, but also due to the emergence of drugꢀresistant bacteria. After AIDS, tuberculosis (TB)
caused by Mycobacterium tuberculosis, is the world's second most serious and fatal chronic
infections disease.1 People with weak immune systems, for example, having HIV positive,
diabetes,2 cancer3 and transplant4 are more susceptible to TB infection. The unique structure and
chemical composition of the mycobacterial cell wall prevents successful entry of drugs which
renders most antibiotics ineffective making TB treatment very difficult. Furthermore, this
problem has become more critical with the emergence of multiꢀdrug resistant (MDR) and
extensivelyꢀdrug resistant (XDR) strains of M. tuberculosis against both the first line as well as
second line drugs.5ꢀ7 But, no such new effective TB drugs have been launched in the market for
the past few decades. It is also necessary that effective therapeutic agents should possess good
safety profile i.e. good selectivity index and less toxicity. But there are many reports found in the
literature where most potent compounds are not effective because of its toxicity. And also,
potency and selectivity are extremely sensitive to minor changes in the chemical structure of the
drug. 8ꢀ12 Hence, there is an urgent requirement of developing new antimycobacterial drugs with
a unique structure, effective mode of action with reduced toxicity and good selectivity than the
existing drugs.
Benzimidazole nucleus is considered as ‘Master Key Core’ in many compounds acting at
different targets to bring out varied pharmacological properties. The benzimidazole chemistry
has gained prominent importance with a discovery of Nꢀribosylꢀdimethyl benzimidazole which

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serves as an axial ligand for cobalt in vitamin B12.13 In the synthesis of benzimidazoles reduction
of nitro group followed by cyclization is main step which conventionally involve two steps. One
pot reactions gaining significant importance to conduct synthesis in a more sustainable fashion
with an aim of minimizing reagents and solvents use, as well as to reduce the isolation steps.14
Benzimidazole and its derivatives represent one of the highly important biologically active class
of compounds possessing various biological activities including antiviral,15 anthelmintic,16
anticancer,17,18 antihistamine,19 antihypertensive,20 antineoplastic,21 antiprotozoal,22 antiꢀhepatitis
B virus agents.23 Only few reports are available for antitubercular activity of benzimidazoles.24ꢀ27
These developments sparked active research to explore the nucleus with varied activities.
Optimization of substituents around the benzimidazole nucleus has resulted in many drugs like
Carbendazim (antifungal agent), Albendazole, Mebendazole (antiꢀhelmintic agent), Pimozide
(antiꢀpsychotic drug), Omeprazole, Pantoprazole (proton pump inhibitors) and many lead
compounds are used in wide therapeutic areas.
Moreover, most of the drugs have 2nd and 5th position substitutions at benzimidazole core.
Substitutions at these two positions of benzimidazole moiety were identified as essential for
receptor activation and potency of receptor binding.28 On the other hand, [1,2,4]ꢀoxadiazole is
another privileged scaffold finding great importance in medicinal chemistry research due to their
synthetic applications such as anticonvulsant,29 analgesic,30 antiꢀinflammatory,31 antitumor.32
Also, [1,2,4]ꢀoxadiazoles are reported for their selective inhibition of receptors such as
35
tyrosinase inhibitors,33 muscarinic,34 histamineꢀH3 and Benzodiazepines.36 In addition,
significant number of drugs and drug candidates in clinical development are halogenated.
Insertion of halogen atoms on hit or lead compounds was predominantly causes development in
their steric effects by increasing the ability of bulk atoms to occupy the binding site of molecular
targets and also to increase the polar hydrophobicity.37
Based on the biological profiles of the benzimidazoles and [1,2,4]ꢀoxadiazole and also in
continual part of our research work on synthesis of benzimidazoles by ‘one pot’ nitro reductive
cyclization method,38, 39 we report the synthesis of a series of novel benzimidazole with [1,2,4]ꢀ
oxadiazole in single molecular frame and evaluated them for antibacterial, antifungal and
antimycobacterial activities. (Figure. 1)
[Insert Figure. 1]

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2. Results and discussion
2.1. Chemistry
The twenty new compounds were synthesized by a series of reactions as depicted in the
Scheme 1, 2 and 3. The intermediates N1ꢀhydroxyamidines (2a-2e) were synthesized by treating
various aromatic/heterocyclic nitriles (1a-1e) with hydroxylamine hydrochloride in ethanol using
sodium bicarbonate. The intermediate ethyl 4ꢀchloroꢀ3ꢀnitro benzoate 4 was synthesized by
esterification of commercially available 4ꢀchloroꢀ3ꢀnitro benzoic acid 3 according to reported
literature procedure.40 The compound ethyl 3ꢀnitroꢀ4ꢀ(methylamino)ꢀbenzoate 5 was obtained by
the nucleophilic displacement reaction of compound 4 with methylamine in the presence of
triethylamine as a base in THF solvent. The core nucleus ethyl 2ꢀarylꢀ1ꢀmethylꢀ1Hꢀ
benzo[d]imidazoleꢀ5ꢀcarboxylate (6a-6d) was prepared by ‘oneꢀpot’ nitro reductive cyclization
of 5 with various halo substituted aromatic aldehydes using sodium dithionite reagent under
0
dimethyl sulphoxide medium at 90 C. This ‘one pot’ reductive synthesis of 6a-6d gave good
yield (more than 90%) with high purity in very short reaction time as compared to conventional
stepwise cyclization methods. Usually, in stepwise method, first step involves reduction of nitro
group to an amine by hydrogenation or reduction reaction, followed by cyclization which
requires strong acidic or oxidizing conditions. But, this method involve several drawbacks like,
harsh reaction conditions, use of expensive reagents, isolation of intermediates, tedious work up
procedures, long reaction path and low yield. Hence, compared to stepwise method, ‘one
pot’reductive cyclization is simple, convenient, cheap and high yielding method which is much
more superior over stepwise cyclization method.38 The compound 2ꢀarylꢀ1ꢀmethylꢀ1Hꢀ
benzo[d]imidazoleꢀ5ꢀcarboxylic acid 7 was obtained by ester hydrolysis of ‘one pot’
intermediate 6a-6d by using NaOH in ethanol medium. Finally, intermediate N1ꢀhydroxyamidine
(2a-2e) undergo Oꢀacylation with carboxylic acid group of intermediate 6a-6d followed by
intramolecular cyclodehydration in the presence of coupling agent, TBTU and a base Nꢀmethyl
morpoline in dimethylformamide medium afforded 2ꢀarylꢀ5ꢀ(3ꢀarylꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ1ꢀ
methylꢀ1Hꢀbenzo[d]imidazoles (8a-8t). Physical properties and structural features of target
compounds 8a-8t are presented Table 1.
[Insert Scheme 1, 2 and 3]
[Insert Table 1]

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1
13
The newly synthesized compounds were characterized by FT (ATR) IR, H NMR, C
NMR, ESI MS and elemental analyses. The formation of intermediate 2a-2e from corresponding
aromatic nitriles was confirmed by the appearance of characteristic stretching vibrations for NH
and OH groups. For the compound 2e, NH and OH stretching vibrations were observed at 3342
and 3197cmꢀ1. The characteristic stretching vibrations for SO2 group was observed at1382 cmꢀ1
(asym) and 1123 cmꢀ1 (sym). And also, in its 1H NMR spectrum, free NH2 signal was appeared at
δ 5.45 ppm and distinct singlet for OH was appeared at δ 8.93 ppm. Further, a singlet at δ 2.71
ppm indicated the presence of three protons of methylsulfonyl group. Two distinct doublets were
observed at δ 7.47 and δ 7.60 ppm for four protons of methylsulfonyl phenyl ring, each
integrating for two protons with coupling constants J = 8.4 Hz and 8.0 Hz respectively.
The formation of ‘one pot’ reductive cyclized core nucleus 6a-6d was confirmed by the
absence of characteristic NH stretching at 3350cmꢀ1 of the previous step methylamine compound
1
5. Further, by considering prototype 6a, its H NMR showed three protons of 2ꢀchloroꢀ6ꢀflouro
phenyl ring as a triplet at δ 7.52 ppm (coupling constant J = 8.4 Hz) and a multiplet in the region
δ 7.42ꢀ7.46 ppm which clearly indicated the cyclization. In addition, the spectrum displayed
doublet centered at δ 7.62 ppm, doublet of doublet in the region of δ 7.71ꢀ7.81 ppm and a singlet
at δ 8.34 ppm due to three aromatic protons of benzimidazole ring. The –CH3 and –CH2 protons
of ester group were appeared as a triplet at δ 1.36 ppm and a quartet in the region δ 4.33ꢀ4.39
ppm. In its spectrum the disappearance of a broad –NH peak in the downfield region strongly
confirmed the formation of the compound 6a. Its formation was also evidenced by ESI MS
spectral data which showed (M++H) peak at m/e value 333.1. In the next step, FT(ATR) IR
spectrum of compound 7a showed the characteristic OH vibration at 3220cmꢀ1 which was absent
in corresponding previous ester 6a. The shifting of carbonyl stretching frequency to a lower
region of 1693cmꢀ1 from 1735cmꢀ1 gave strong evidence for the conversion of ester group to
acid. Further, its 1H NMR spectrum displayed broad OH signal at δ 12.82 ppm and also absence
of signals due to ester protons in the shielded region strongly supported the conversion of ester
6a to acid 7a. In its ESI MS spectrum, presence of deprotonated molecular ion peak at m/e value
303.0 further confirmed its formation.
Formation of new target compounds 8a-8t also characterized by spectral data and
elemental analyses. In the FT (ATR) IR spectrum, absence of characteristic OH/NH vibrations
strongly evidenced the formation of 8a-8t, which was found in the intermediate 2a-2e. Aromatic

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protons showed stretching vibrations around 3018ꢀ3080 cmꢀ1. For the prototype compound 8a,
CꢀF and CꢀCl stretching bands were observed at 1165cmꢀ1 and 764 cmꢀ1 respectively. Further,
1
in H NMR of 8a showed a characteristic singlet for NꢀCH3 group at δ 3.67 ppm. Aromatic
protons of 2ꢀchloroꢀ6ꢀfluoro ring resonated as a multiplet in the region δ 7.42ꢀ7.46 ppm. The
proton at C7 of benzimidazole appeared as a doublet at δ 7.34 ppm with coupling constant J = 7.6
Hz, C6 as doublet of doublet in the region δ 8.17ꢀ8.19 ppm (J = 8.4 and 1.2 Hz) and for C4 as a
13
broad singlet at 8.65 ppm. In its C NMR spectrum, signal due to carbonyl group was absent.
NꢀCH3 carbon of benzimidazole displayed signal in the region δ 30ꢀ34 ppm, along with all
aromatic carbons in the region δ 110ꢀ160 ppm. Oxadiazole ring carbons showed signals in the
down field region δ 162ꢀ176 ppm, which provide an additional support for the formation of
compound. Further, compound 8a displayed protonated peak at m/z value 439.2 along with its
isotopic peaks at m/z value 441.2 and 443.2 in its ESI MS spectrum which is in accordance with
its molecular formula weight evidences the formation of target molecule. The spectral and
elemental analysis data of all target compounds are presented in experimental section.
2.2. Biological screening
2.2.1. Antibacterial activity
To investigate the antimicrobial potency, all the newly synthesized compounds 8a-8t
were screened for their inꢀvitro antibacterial activity against two representative Gram positive
bacterial species S. aureus, E. faecalis; two Gram negative bacterial strains E. coli, K. pneumonia
by broth dilution method41 using Ciprofloxacin as reference drug in terms of minimum inhibitory
concentration (MIC). Results are summerised in Table 2. From the obtained results, it has been
noticed that the tested new compounds were exhibited moderate to good inhibition in the range
of 1.6 ꢁg/mL to 50 ꢁg/mL. The compounds 8a and 8k showed highest inhibition among the
compounds in the series. Compound 8a with 2ꢀchloroꢀ6ꢀfluorophenyl substitution at 2nd position
of benzimidazole and pꢀchlorophenyl substitution at 3rd position of oxadiazole ring at other
extreme end showed MIC value of 1.6 ꢁg/mL against E. faecalis, 3.125 ꢁg/mL against S. aureus,
6.25 ꢁg/mL against K. pneumonia and E. coli. Compound 8k with 3ꢀchloroꢀ2ꢀfluorophenyl
substitution at 2nd position of benzimidazole and pꢀchlorophenyl substitution at oxadiazole ring
exhibited good inhibition with MIC value of 1.6 ꢁg/mL against both Gram positive bacterial
strains, which is lower than the standard drug Ciprofloxacin (MIC: 2.0 ꢁg/mL) against S.aureus,

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6.25 ꢁg/mL against E. coli and 12.5 ꢁg/mL against K. pneumonia. Surprisingly, both compounds
8a and 8k possess chlorofluorophenyl substitutions at benzimidazole ring. The higher activity
could be attributed to the presence of chlorofluoro groups; especially the presence of highly
active small atom ‘fluorine’ may impart the potentiality. Among the other 3ꢀchloroꢀ2ꢀfluoro
derivatives, 8e with 4ꢀmethylsulfonylphenyl at oxadiazole ring displayed good inhibition with
MIC value 3.125 ꢁg/mL against S. aureus and E. faecalis, but exhibited moderate activity
against Gram negative bacterial strains. While other compounds 8b, 8c and 8d did not exert good
inhibition against all bacterial strains except compound 8c with MIC value 6.25ꢁg/mL against E.
coli. Among the 2,4ꢀdichlorophenyl derivatives, compound 8f showed good inhibition against E.
faecalis with MIC value of 3.125 ꢁg/mL and 6.25 ꢁg/mL against S. aureus and also compound
8j having methyl sulfonyl group showed moderate inhibition against both Gram positive
bacterial strains with MIC value of 6.25 ꢁg/mL. Whereas, rest of the compounds did not show
any pronounced inhibition. From the results of 3ꢀchloroꢀ2ꢀfluoro derivatives, compound 8n with
3ꢀchloro benzyl group at oxadiazole ring showed good inhibition against S. aureus with MIC
value 3.125 ꢁg/mL and against E. faecalis with MIC 6.25 ꢁg/mL. Among the compounds 8p, 8q,
8r, 8s and 8t, all the compounds displayed good inhibition against Gram positive bacterial
strains, except compound 8t with MIC≥ 12.5 ꢁg/mL. Against K. pneumonia and E. coli, did not
show any considerable activity. The high activity of the derivatives might be due to the presence
of dihalo substitution at phenyl ring beside benzimidazole moiety.
2.2.2. Antifungal activity
Inꢀvitro antifungal activity of the synthesized compounds 8a-8t were assessed against
two representative fungal pathogens C. albicans and A. fumigates by broth dilution method using
Flucanazole as a standard drug. Results are presented in Table 2. Most of the compounds
showed excellent inhibition with MIC value less than the standard drug Flucanazole (MIC: 8.0
ꢁg/mL) against fungal pathogen. A. fumigates, except compounds 8c, 8d, 8g and 8j.
Interestingly, compounds 8k, 8m and 8n with 3ꢀchloroꢀ2ꢀfluoro phenyl substitutions at 2nd
position of benzimidazole and chloro substitution at oxadiazole end showed minimum MIC
value (1.6ꢁg/mL) with highest inhibition among the compounds in the series against A.
fumigates. Against C. albicans, compound 8a showed highest inhibition with MIC value of 1.6
ꢁg/mL and compounds 8b, 8c, 8d, 8e, 8f, 8h, 8i, 8j, 8k, 8l, 8m, 8n, 8o, 8p and 8r showed more

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inhibition than standard drug. Rest of the derivatives showed moderate activity. Presence of
chlorofluoro substitution at benzimidazole end enhanced the activity.
[Insert Table 2]
2.2.3. Antimycobacterial study
Based on encouraging results from the antibacterial screening, the selected title
compounds 8a, 8e, 8f, 8j, 8p, 8n, 8p, 8q, 8r and 8s which showed good antibacterial activity
were further screened for their inꢀvitro antimycobacterial activity against Mycobacterium
tuberculosis H37Rv using Pyrazinamide, Streptomycin, Ciprofloxacin and Isoniazid as standards
by Microplate Almer Blue Assay (MABA) method.42 The corresponding results are summerised
in Table 3. As evident from the table, all the tested compounds exhibited moderate to excellent
antiꢀtubercular activity with MIC ranging from 1.6ꢀ25.0 ꢁg/mL. It is interesting to note that most
of the tested compounds showed either enhanced activity or activity in line with the reference
drugs used. Surprisingly, compounds 8k, 8n, 8p and 8r showed excellent inꢀvitro activity against
M. tuberculosis H37Rv with MIC value of 1.6 ꢁg/mL which was approximately one fold times
lower than reference drugs Pyrazinamide and Ciprofloxacin (MIC: 3.12 ꢁg/mL) and three fold
time lower than Streptomycin and Isoniazid (MIC: 6.25 ꢁg/mL). Compounds 8k and 8n contain
3ꢀchloroꢀ2ꢀfluoro phenyl substitution at 2nd position of benzimidazole and chloro substitution at
other extreme end aryl group attached to 5th position of oxadiazole ring. Whereas, compounds 8p
and 8r carries 2,3ꢀdichloro phenyl substitution at phenyl ring attached to benzimidazole moiety
at 2nd position and, parachloro and orthochlorophenyl substitution respectively at oxadiazole ring
end. And also, compounds 8a, 8f and 8s showed potent activity with MIC value of 3.12 ꢁg/mL,
which was equipotent with standard drugs Pyrazinamide and Ciprofloxacin (MIC: 3.12 ꢁg/mL)
and in about one fold times more active than Streptomycin and Isoniazid (MIC: 6.25 ꢁg/mL).
However all these highly active compounds possess 3ꢀchloroꢀ2ꢀfluoro and 2,3ꢀdichlorophenyl
substitutions at benzimidazole end and mono haloaryl substitution at 5th position of oxadiazole
moiety. It has been well reported that lipophilicity of compound plays a major role to study the
biological activity of compound.43 These properties are found to be an important parameter
which is related to the membrane permeation in biological system. In addition, halogen
substituted molecules have a prominent place in modern medicinal chemistry and they will

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improve the lipophilic nature of the compound.44 The better activity of the compounds could be
due to the presence of 2,3ꢀdihalophenyl substitutions at 2nd position of the benzimidazole ring
which increases the lipophilic nature of the compound, thereby making the molecule to be more
cell permeable. However, isosteric displacement of 4ꢀchloro phenyl group in 8p with 3ꢀpyridinyl
group in compound 8q at phenyl ring attached to 3rd position of oxadiazole ring showed
depletion in the activity with MIC value of 12.5 ꢁg/mL. Similarly, introduction of methyl
sulfonyl group (8j) in place of 4ꢀchloro substitution (8f) showed significant loss in potential
against M. tuberculosis M37Rv with MIC value 25 ꢁg/mL. Further, when 4ꢀchloro group of 8a
was replaced with methylsulphonyl group, activity against M. tuberculosis M37Rv was
drastically reduced. In addition, it could also be observed that all the 4ꢀchloro phenyl substituted
compounds 8a, 8f, 8k and 8p at 3rd position of oxadiazole ring, with 2ꢀchloroꢀ6ꢀfluoro, 2,4,ꢀ
dichloro, 3ꢀchloroꢀ2ꢀfluoro, 2,3ꢀdichloro phenyl substitutions at 2nd position of benzimidazole
showed potent inꢀvitro activity against M. tuberculosis M37Rv (MIC ≤ 3.12 ꢁg/mL). And also,
could be noted that 2,3ꢀdichloro (8p) and 3ꢀchloroꢀ2ꢀfluoro (8k) derivatives showed higher
activity than 2ꢀchloroꢀ6ꢀfluoro (8a) and 2,4ꢀdichloro (8f) derivatives.
[Insert Table 3]
2.2.4. Hemolytic activity
The preliminary toxicity of selected compounds was investigated against human
erythrocytes (hRBCs) 45 and results are displayed in Table 2. The concentration corresponding to
50% cell lysis was expressed as their HC50 values. The results indicated that the compounds
being good antitubercular showed significant lysis only at higher concentration in the range of
93ꢀ874 ꢁg/mL. The most potent compounds 8k and 8r remain nontoxic even at very high HC50
value of 874 and 369 ꢁg/mL respectively. Compounds 8n and 8p are also found to be non toxic
at higher concentration (HC50 value 93.69 and 129.46 ꢁg/mL). The selectivity ratio (HC50/MIC)
was also calculated for selected compounds. All the tested compounds showed good selectivity
ratio (HC50/MIC) ≥ 58. The compound 8k with 3ꢀchloroꢀ2ꢀfluoro substitution emerged as most
effective compound, being 546 fold selective toward M. tuberculosis M37Rv and compound 8r
found to be 230 fold selective. The selectivity for these compounds higher than the standard drug

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Streptomycin (Selectivity index: 162). The high selectivity of compounds clearly implies the
suitability of these compounds for drug development as new antiꢀTB candidates.
2.2.5. Cytotoxicity test
In order to further investigate these compounds as selective antimicrobial agents, toxicity
test was performed against human normal embryonic kidney cell line, HEK 293 using MTT
assay 46 with most potent compounds 8k, 8n, 8p and 8r. The dose dependent cytotoxic effect
was obtained with increase in the concentration of the selected compounds as shown in Figure 2
and IC50 concentrations are given in Table 4. As we can observe from the results, the IC50 value
for the compound 8r was found to be 355.42 ꢁg/mL and the remaining compounds 8k, 8n and
8p showed IC50 value more than 500 ꢁg/mL, which is many fold higher concentration than the
MIC for antitubercular activity. In order to compare the effectiveness of the compounds, the
toxicity test was performed with standard drug Mitomycin C which showed IC50 value less than
100 ꢁg/mL. It clearly supports that, these compounds are non toxic even at very high
concentration.
[insert Table 4 and Figure 2]
2.2.6. Inꢀsilico physicoꢀchemical properties
The assessment of druglikelyness nature of active compounds in pharmaceutical research
plays an important role in lead generation and lead optimization. Lipinski’s “rule of five” helps
to develop bioactive drug candidate.47,48 For a bioactive molecule to be a therapeutic agent, it
should satisfy Lipinski’s Rule of Five along with high oral bioavailability, which is decided by
ADME (Absorption, Distribution, Metabolism and Elimination) profile. According to this rule,
active compound should have molecular weight ≤500, partition coefficient (logP) should be ≤5,
and number of hydrogen bond donors should be ≤5 and number of hydrogen bond acceptors ≤10.
Along with this rule, number of rotatable bonds in a molecule should be ≤10 and Tropical polar
surface area (TPSA) should be ≤ 140 A0 for an effective orally bioavailable compound. TPSA is
the sum of surfaces of polar atoms such as oxygen, nitrogen and attached hydrogen and number
of rotatable bonds is a measure of conformational flexibility of the molecule in turn decides the

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binding of receptors. The percentage absorption was calculated by the equation %ABS = [109–
(0.345) TPSA].
In our present study, the physicoꢀchemical descriptors of the potent compounds 8a, 8f,
8k, 8n, 8p, 8r and 8s were assessed to determine its ADME parameters by using Molinispiration
online property calculation toolkit and the results are summarized in Table 5. From the table we
can observe that, all the compounds showed excellent passive oral absorption with percentage
absorption ˃ 90. All the target compounds satisfy all the parameters except the log p value,
which is slightly higher than limiting value for all the compounds. But, violation of any one of
49
the rule does not affect the bioavailability of the molecule and druglikeness, if the percentage
of absorption is good. TPSA was found to be 56.75 A0 for all the compounds and numbers of
rotatable bonds were less than 10 for all the compounds which decide molecular flexibility.
[Insert Table 5]
3. Conclusion
In conclusion, this work is focused on the development of novel potent compounds based
on benzimidazoleꢀoxadiazole compact system. A series of twenty novel compounds of
benzimidazoles with [1,2,4]ꢀoxadiazoles were designed and efficiently synthesized with good
yield by employing rapid ‘one pot’ nitro reductive cyclization. Compounds 8a and 8k are
emerged as promising antibacterial agents against all the bacterial and fungal pathogens and are
found to be superior over Ciprofloxacin and Flucanazole with good ADME parameters.
Compounds 8k and 8n with 3ꢀchloroꢀ2ꢀfluoro aryl ring at 2nd position of benzimidazole and 4ꢀ
chlorophenyl (8k) and 3ꢀchorobenzyl (8n) substitution at oxadiazole ring emerged as excellent
antitubercular agents with two fold more active than Pyrazinamide and Ciprofloxacin, four times
than Streptomycin and Isoniazid. And also, compounds 8p and 8r with 2,3,ꢀdichloro phenyl
substitutions at benzimidazole end and 4ꢀchloro (8p) and 2ꢀchlorophenyl (8r) substitutions at
oxadiazole phenyl ring are found to be around two times higher active than Pyrazinamide and
Ciprofloxacin, four fold than Streptomycin and Isoniazid against M. tuberculosis M37Rv which
were emerged as potent antiꢀtuberculosis agents along with good drug like properties. The
preliminary toxicity test of potent compounds 8a, 8k, 8n, 8p, 8r and 8s against human
erythrocytes showed less toxicity and good selectivity index. Compound 8k showed highest

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selectivity index of 546 fold. Further investigation of cytotoxicity of potent compounds against
human embryonic kidney cell line HEK 293, displayed nonꢀtoxicity of these compounds.
Structureꢀ activity relationship revealed that presence of dihalo substitution at the 2nd position
aryl ring of benzimidazole moiety as well as halogen substitution at the phenyl ring of [1,2,4]ꢀ
oxadiazole are crucial for pronounced activity and significantly enhances antibacterial,
antifungal and antimycobacterial activities. Further detailed structural modifications at 1st, 2nd
and 5th position of benzimidazole moiety with different heterocyclic rings are underway to get
better analogs with improved efficacy.
4. Experimental section
4.1. Materials and methods
4.1.1. Preparation of N1ꢀhydroxy amidines (2)
Sodium bicarbonate (70 mmol, 5.88 g) was added in portions to a solution of
hydroxylamine hydrochloride (70 mmol, 4.79 g) in 25 ml of water. A solution of substituted
acetonitrile (35 mmol) in 40 ml of ethanol was then added, and the mixture stirred under reflux
for 8 h. The precipitate formed was filtered off and recrystallized from ethanol.
4.1.1.1. 4ꢀChloroꢀN’ꢀHydroxybenzamidine (2a)
White crystalline solid; FT IR (ATR, ν max, cmꢀ1): 3348 (NꢀH), 3207 (OH), 3030 (ArꢀH), 2979,
2928 (CꢀH), 1652 (C=N); 1H NMR (400 MHz, DMSOꢀd6, δ ppm): 8.98 (s, OH), 7.78 (d, 2H, pꢀ
Cl phenyl ring, J = 8.8 Hz), 7.50 (d, 2H, pꢀCl phenyl ring, J = 8.4 Hz), 5.45 (s, 2H, NH2),; ESIꢀ
MS: 171.1 (M++H), 173.1[(M+H)+2]+; Anal. Calcd. for C7H7ClN2O: C, 49.28; H, 4.14; N,
16.42. Found: C, 49.27; H, 4.15; N, 16.41.
4.1.1.2. N’ꢀHydroxynicotinamidine (2b)
Off white solid; FT IR(ATR, ν max, cmꢀ1): 3350 (NꢀH), 3200 (OH), 3020 (ArꢀH), 2989, 2933
(CꢀH), 1650(C=N); 1H NMR (400 MHz, DMSOꢀd6, δ ppm): 5.49 (s, 2H, NH2), 7.58 (d, 1H, Arꢀ
H, J = 8.4 Hz), 8.03 (s, 1H, ArꢀH), 8.16ꢀ8.20 (m, 2H, ArꢀH), 8.98 (s, OH); ESIꢀMS: 138.14
(M++H); Anal. Calcd. for C6H7N3O: C, 52.55; H, 5.14; N, 30.64. Found: C, 52.54; H, 5.15; N,
30.65.

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4.1.1.3. 2ꢀChloroꢀN’ꢀHydroxybenzamidine (2c)
White crystalline solid; FT IR(ATR, ν max, cmꢀ1): 3360 (NꢀH), 3220 (OH), 3025 (ArꢀH), 2980,
2930 (CꢀH), 1649 (C=N); 1H NMR (400 MHz, DMSOꢀd6, δ ppm): 5.49 (s, 2H, NH2), 7.26ꢀ7.37
(m, 2H, ArꢀH), 7.42 (m, 1H, ArꢀH), 7.52 (dd, 1H, ArꢀH, J = 7.5 and 1.4 Hz), 8.98 (s, OH); ESIꢀ
ESIꢀMS: 171.6 (M+H)+, 173.3 [(M+H)+2]+; Anal. Calcd. for C7H7ClN2O: C, 49.28; H, 4.14; N,
16.42. Found: C, 49.27; H, 4.15; N, 16.43.
4.1.1.4. 3ꢀChlorophenylꢀN’ꢀHydroxyacetamidine (2d)
White crystalline solid; FT IR(ATR, ν max, cmꢀ1): 3366 (NꢀH), 3204 (OH), 3015 (ArꢀH), 2985,
1
2937 (CꢀH), 1649 (C=N); H NMR (400 MHz, DMSOꢀd6, δ ppm): 3.32 (S, 2H, benzylꢀCH2),
7.93 (d, 1H, ArꢀH, J = 8.8 Hz), 8.08 (t, 1H, ArꢀH, J = 8.4 Hz), 8.26 (d, 1H, ArꢀH, J = 8.4 Hz),
8.48 (s, 1H, ArꢀH); ESIꢀMS: 185.06 (M++H), 187.4[(M+H)+2]+; Anal. Calcd. for C8H9ClN2O:
C, 52.04; H, 4.91; N, 15.17. Found: C, 52.05; H, 4.90; N, 15.18.
4.1.1.5. N’ꢀHydroxyꢀ4ꢀ(methylsulfonyl)benzamidine (2e)
Light brown crystals; FT IR(ATR, ν max, cmꢀ1): 3414 (OH), 3263 (NH), 3081 (ArꢀH), 2968 (Cꢀ
1
H), 1653 (C=N), 1379 (asym SO2), 1083 (sym SO2); H NMR (400 MHz, DMSOꢀd6, δ ppm):
2.71 (s,2H, ꢀCH3), 5.45 (s, 2H, NH2), 7.47 (d, 2H, ArꢀH, J = 8.4 Hz), 7.60 (d, 2H, ArꢀH, J = 8
Hz), 8.93 (s, OH); ESIꢀMS: 214.5 (M++H), 215.1[(M+H)+1]+; Anal. calcd. for C8H10N2O3S: C,
44.85; H, 4.70; N, 13.08; S, 14.97. Found: C, 44.86; H, 4.71; N, 13.09; S, 14.98.
4.1.2. Preparation of ethyl 4ꢀchloroꢀ3ꢀnitrobenzoate (4)
A mixture of ethyl 4ꢀchloroꢀ3ꢀnitrobenzoic acid (10 mmol) in 30ml of ethanol and 12 ml
of Conc. hydrochloric acid were refluxed for 10 h. After completion of the reaction, solvent was
removed under reduced pressure and the residue was taken up in ether (100 ml). The solution
was extracted with saturated aqueous sodium bicarbonate solution (2x100 ml) and water (2x100
ml). Reaction progress was confirmed by Dichloromethaneꢀmethanol mixture (9:1).
Yellow solid; FT IR (ATR, ν max, cmꢀ1): 3097 (ArꢀH), 2976, 2941 (CꢀH), 1535 (NO2 asym),
1354 (NO2 sym); 1H NMR (400 MHz, DMSOꢀd6, δ ppm): 1.34 (t, 3H, ꢀCH3, J = 7.2 Hz), 4.34ꢀ
4.39 ( q, 2H, ester CH2), 7.94 (d, 1H, ArꢀH), 8.18ꢀ8.21(dd, 1H, ArꢀH, J = 8.4 Hz and J = 2 Hz),

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+
8.51 (s, 1H, ArꢀH); ESIꢀMS: 232.0 [(M+H)+1] , 234.1 [(M+H)+3]+; Anal. Calcd. for
C9H8ClNO4: C, 47.08; H, 3.51; N, 15.44. Found: C, 47.07; H, 3.50; N, 15.43.
4.1.3. Preparation of ethyl 4ꢀ(methylamino)ꢀ3ꢀnitrobenzoate (5)
Ethyl 4ꢀchloroꢀ3ꢀnitrobenzoate (4) (10 mmol, 4.5g) in 25ml of THF was taken. To this
compound methylamine (88 mmol, 2.7g) and triethylamine (44 mmol, 4.4g) was added. Reaction
mixture was kept for stirring overnight with heating. After completion of the reaction, reaction
mixture was quenched in to ice water. Pure product obtained was obtained which was collected
by filtration.
Light yellow solid; FT IR(ATR, ν max, cmꢀ1): 3369 (NꢀH), 3037 (ArꢀH), 2926, 2883 (CꢀH),
1535 (NO2 asym), 1354 (NO2 sym); 1H NMR (400 MHz, DMSOꢀd6, δ ppm): 1.36 (t, 3H, ꢀCH3,
J = 7.2 Hz), 3.01 (s, 3H, ꢀCH3), 4.34ꢀ4.39 ( q, 2H, ester CH2), 6.75 (d, 1H, ArꢀH, J = 8.8 Hz)),
7.59ꢀ7.72 (m, 1H, ArꢀH), 8.11 (s, 1H, ArꢀH), 12.41 (s, NH); ESIꢀMS: 224.5 (M+ꢀH); Anal.
Calcd. for C10H12N2O4: C, 53.57; H, 5.39; N, 12.49. Found: C, 53.58; H, 5.39; N, 12.48.
4.1.4. General Procedure for the Synthesis of ethyl 2ꢀarylꢀ1ꢀmethylꢀ1Hꢀbenzo[d]imidazoleꢀ5ꢀ
carboxylate (6)
Sodium dithionite (40 mmol, 6.98 g) was added to a stirred solution of ethyl 4ꢀ
(methylamino)ꢀ3ꢀnitrobenzoate (5) (13 mmol, 2.12g) and substituted benzaldehyde (13 mmol) in
O
DMSO (20 ml). The reaction mixture was stirred at 90 C for 3 h. After the completion of
reaction (monitored by TLC ethyl acetate: hexane ; 3: 7, v/v), it was poured into crushed ice. The
solid separated was filtered off, washed with water, and dried. Collected product was
recrystallized from dimethylformamide.
4.1.4.1. Ethyl 2ꢀ(2ꢀchloroꢀ6ꢀflourophenyl)ꢀ1ꢀmethylꢀ1Hꢀbenzo[d]imidazoleꢀ5ꢀcarboxylate (6a)
White powder; FT IR(ATR, ν max, cmꢀ1): 3061 (ArꢀH), 2972, 2926 (CꢀH), 1699 (C=O), 1620
(C=N), 1284 (CꢀF), 896 (CꢀCl); 1H NMR (400 MHz, DMSOꢀd6, δ ppm): 1.36 (t, 3H, J = 7.2 Hz,
ꢀCH3), 3.70 (s, 3H, ꢀCH3), 4.33–4.39 (q, 2H, ꢀCH2ꢀCH3, J = 14.4 and 6.8 Hz), 7.52 (t, 1H, ArꢀH,
J = 8.4 Hz), 7.62 (d,1H, ArꢀH, J = 7.2 Hz), 7.71–7.77 (m, 1H, ArꢀH), 7.80 (d, 1H, ArꢀH, J = 8.4
Hz), 7.98ꢀ8.01 (dd, 1H, ArꢀH, J = 8.8 and 1.6 Hz), 8.34 (s, 1H, ArꢀH); ESIꢀMS (m/z): 333.1

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(M++H), 335.1 [(M+H)+2]+; Anal. calcd. for C17H14ClFN2O2: C, 61.36; H, 4.24; N, 8.42. Found:
C, 61.32; H, 4.25; N, 8.46.
4.1.4.2. Ethyl 2ꢀ(2,4ꢀdichlorophenyl)ꢀ1ꢀmethylꢀ1Hꢀbenzo[d]imidazoleꢀ5ꢀcarboxylate (6b)
White amorphous solid; FT IR (ATR, ν max, cmꢀ1): 3040 (ArꢀH), 2983, 2932 (CꢀH), 1692
1
(C=O), 1614 (C=N), 1538 (C=C), 890 (CꢀCl); H NMR (400 MHz, DMSOꢀd6, δ ppm): 1.38 (t,
3H, J = 7.2 Hz, ꢀCH3), 3.73 (s, 3H, ꢀCH3), 4.34–4.40 (q, 2H, ꢀCH2ꢀCH3, J = 14.4 and 6.8 Hz),
7.58 (s, 1H, ArꢀH), 7.66 (d,1H, ArꢀH, J = 7.2 Hz), 7.98ꢀ8.01 (dd, 1H, ArꢀH, J = 8.8 and 1.6 Hz),
8.09 (d, 2H, ArꢀH, J = 8.8 Hz), 8.37 (s, 1H, ArꢀH); ESIꢀMS (m/z): 349.1 (M++H),
351.6[(M+H)+2]+, 353.4 [(M+H)+4]+; Anal. calcd. for C17H14Cl2N2O2: C, 58.47; H, 4.04; N,
8.02. Found: C, 58.48; H, 4.07; N, 8.02.
4.1.4.3. Ethyl 2ꢀ(3ꢀchloroꢀ2ꢀfluorophenyl)ꢀ1ꢀmethylꢀ1Hꢀbenzo[d]imidazoleꢀ5ꢀcarboxylate (6c)
White amorphous solid; FT IR(ATR, ν max, cmꢀ1): 3045 (ArꢀH), 2981, 2931 (CꢀH), 1699 (C=O),
1609 (C=N), 1539 (C=C), 1272 (CꢀF), 890 (CꢀCl); 1H NMR (400 MHz, DMSOꢀd6, δ ppm): 1.36
(t, 3H, 7.2 Hz, ꢀCH3), 3.73 (s, 3H, ꢀCH3), 4.32–4.39 (q, 2H, ꢀCH2ꢀCH3, J = 14.4 and 6.8 Hz),
7.42ꢀ7.47 (m, 3H, 3ꢀClꢀ2ꢀF phenyl ring), 7.69 (d,1H, ArꢀH, J = 7.2 Hz), 7.99ꢀ8.02 (dd, 1H, Arꢀ
H, J = 8.8 and 1.6 Hz), 8.38 (s, 1H, ArꢀH); ESIꢀMS (m/z): 333.1 (M++H), 335.7 [(M+H)+2]+;
Anal. calcd. for C17H14ClFN2O2: C, 61.36; H, 4.24; N, 8.42. Found: C, 61.38; H, 4.22; N, 8.43.
4.1.4.4. Ethyl 2ꢀ(2,3ꢀdichlorophenyl)ꢀ1ꢀmethylꢀ1Hꢀbenzo[d]imidazoleꢀ5ꢀcarboxylate (6d)
White crystalline solid; FT IR (ATR, ν max, cmꢀ1): 3049 (ArꢀH), 2990, 2939 (CꢀH), 1700(C=O),
1
1623 (C=N), 1539 (C=C), 889 (CꢀCl); H NMR (400 MHz, DMSOꢀd6, δ ppm): 1.38 (t, 3H, 7.2
Hz, ꢀCH3), 3.75 (s, 3H, ꢀCH3), 4.31–4.39 (q, 2H, ꢀCH2ꢀCH3, J = 14.4 and 6.8 Hz), 7.60 (d,1H,
ArꢀH, J = 7.2 Hz), 7.68 (d, 1H, ArꢀH, J = 8.4 Hz), 7.83 (t, 1H, ArꢀH, J = 8.4 Hz), 7.99ꢀ8.02 (dd,
1H, ArꢀH, J = 8.8 and 1.6 Hz), 8.09 (d, 1H, ArꢀH, J = 8.8 Hz), 8.38 (s, 1H, ArꢀH); ESIꢀMS
(m/z): 350.1 (M++H), 352.6 [(M+H)+2]+, 354.2 [(M+H)+4]+; Anal. calcd. for C17H14Cl2N2O2:
C, 58.47; H, 4.04; N, 8.02. Found: C, 58.45; H, 4.05; N, 8.04.
4.1.5. Procedure for the preparation of 2ꢀarylꢀ1ꢀmethylꢀ1Hꢀbenzo[d]imidazoleꢀ5ꢀcarboxylic
acid (7)

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To the solution of ethyl 2ꢀarylꢀ1ꢀmethylꢀ1Hꢀbenzo[d]imidazoleꢀ5ꢀcarboxylate (6) (9
mmol, 3g) in 20 mL of ethanol, sodium hydroxide in dissolved in minimum quantity of water
0
was added slowly with stirring. After complete addition, reaction mixture was refluxed at 80 C
for 5 h. After completion of the reaction, reaction mixture was quenched into ice water. The pure
product was collected by filtration.
4.1.5.1. 2ꢀ(2ꢀchloroꢀ6ꢀfluorophenyl)ꢀ1ꢀmethylꢀ1Hꢀbenzo[d]imidazoleꢀ5ꢀcarboxylic acid (7a)
Creamy white powder; FT IR(ATR, ν max, cmꢀ1): 3196 (OH), 3030 (ArꢀH), 2970, 2924 (CꢀH),
1689 (C=O), 1618 (C=N), 1566 (C=C), 1033 (CꢀF), 752 (CꢀCl); 1H NMR (400 MHz, DMSOꢀd6,
δ ppm): 3.69 (s, 1H, ꢀCH3), 7.52 (t,1H, ArꢀH, J = 8.8 Hz), 7.61 (d, 1H, ArꢀH, J = 8 Hz), 7.71ꢀ
7.78 (m, 2H, ArꢀH), 7.98 (d, 1H, ArꢀH, J = 8.4 Hz), 8.30 (s, 1H, ArꢀH), 12.82 (bs, OH, 1H); ESIꢀ
MS (m/z): 305.0 (M++H), 307.1 [(M+H)+2]+. Anal. calcd. for C15H10ClFN2O2: C, 59.13; H,
3.31; N, 9.19. Found: C, 59.16; H, 3.32; N, 9.22.
4.1.5.2. 2ꢀ(2,4ꢀdichlorophenyl)ꢀ1ꢀmethylꢀ1Hꢀbenzo[d]imidazoleꢀ5ꢀcarboxylic acid (7b)
White amorphous solid; FT IR(ATR, ν max, cmꢀ1): 3240 (OH), 2984, 2939 (CꢀH), 1684 (C=O),
1
1616 (C=N), 1528 (C=C), 895 (CꢀCl); H NMR (400 MHz, DMSOꢀd6, d ppm): 3.73 (s, 3H, ꢀ
CH3), 7.59 (s, 1H, ArꢀH), 7.66 (d,1H, ArꢀH, J = 7.2 Hz), 7.99ꢀ8.02 (dd, 1H, ArꢀH, J = 8.8 and
1.6 Hz), 8.09 (d, 2H, ArꢀH, J = 8.8 Hz), 8.37 (s, 1H, ArꢀH), 12.80 (bs, OH, 1H); ESIꢀMS (m/z):
320.1 (M+ꢀH), 322.4 (M+H)+; Anal. calcd. for C15H10Cl2N2O2: C, 56.10; H, 3.14; N, 8.72.
Found: C, 56.08; H, 3.17; N, 8.75.
4.1.5.3. 2ꢀ(3ꢀchloroꢀ2ꢀfluorophenyl)ꢀ1ꢀmethylꢀ1Hꢀbenzo[d]imidazoleꢀ5ꢀcarboxylic acid (7c)
Light cream solid; FT IR(ATR, ν max, cmꢀ1): 3194 (OH), 2981, 2931 (CꢀH), 1689 (C=O), 1599
1
(C=N), 1539 (C=C), 1279(CꢀF), 892 (CꢀCl); H NMR (400 MHz, DMSOꢀd6, δ ppm): 3.73 (s,
3H, ꢀCH3), 7.43ꢀ7.48 (m, 3H, 3ꢀClꢀ2ꢀF phenyl ring), 7.69 (d,1H, ArꢀH, J = 7.2 Hz), 7.99ꢀ8.02
(dd, 1H, ArꢀH, J = 8.8 and 1.6 Hz), 8.38 (s, 1H, ArꢀH), 12.73 (bs, OH, 1H); ESIꢀMS (m/z):
305.7 (M++H), 307.2 [(M+H)+2]+; Anal. calcd. for C15H10ClFN2O2: C, 59.13; H, 3.31; N, 9.19.
Found: C, 59.10; H, 3.34; N, 9.18.
4.1.5.4. 2ꢀ(2,3ꢀdichlorophenyl)ꢀ1ꢀmethylꢀ1Hꢀbenzo[d]imidazoleꢀ5ꢀcarboxylic acid (7d)

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White amorphous sold; FT IR(ATR, ν max, cmꢀ1): 3149 (OH), 2990, 2939 (CꢀH), 1690(C=O),
1
1603 (C=N), 1549 (C=C), 886 (CꢀCl); H NMR (400 MHz, DMSOꢀd6, δ ppm): 3.75 (s, 3H, ꢀ
CH3), 7.60 (d,1H, ArꢀH, J = 7.2 Hz), 7.68 (d, 1H, ArꢀH, J = 8.4 Hz), 7.83 (t, 1H, ArꢀH, J = 8.4
Hz), 7.99ꢀ8.02 (dd, 1H, ArꢀH, J = 8.8 and 1.6 Hz), 8.09 (d, 1H, ArꢀH, J = 8.8 Hz), 8.38 (s, 1H,
ArꢀH), 12.43 (bs, OH, 1H); ESIꢀMS (m/z): 322.1 (M++H), 324.3 [(M+H)+2]+, 326.3
[(M+H)+4]+; Anal. calcd. for C15H10Cl2N2O2: C, 56.10; H, 3.14; N, 8.72. Found: C, 56.14; H,
3.15; N, 8.74.
4.1.6. General procedure for the preparation of 2ꢀarylꢀ5ꢀ(3ꢀ(4ꢀchlorophenyl)ꢀ[1,2,4]ꢀoxadiazolꢀ
5ꢀyl)ꢀ1ꢀmethylꢀ1Hꢀbenzo[d]imidazole (8a-t)
A mixture of 2ꢀarylꢀ1ꢀmethylꢀ1Hꢀbenzo[d]imidazoleꢀ5ꢀcarboxylic acid (7)(10mmol,
2.84g), TBTU (12.5mmol, 4g) and NMM (15mmol, 1.51g) in DMF solvent were kept for stirring
under cold condition. After one hour of stirring, N1ꢀhydroxyamidine (2) was added to reaction
mixture in portionwise and allowed to stir overnight at room temperature. After completion of
the reaction, the separated solid was collected by filtration and washed well with water. Obtained
product was dried well.
4.1.6.1.
2ꢀ(2ꢀChloroꢀ6ꢀfluorophenyl)ꢀ5ꢀ(3ꢀ(4ꢀchlorophenyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ1ꢀmethylꢀ
1Hꢀbenzo[d]imidazole (8a)
Pale yellow solid; FT IR(ATR, ν max, cmꢀ1): 3018 (ArꢀH), 2976,2945 (CꢀH), 1618 (C=N), 1596
(C=C), 1165 (CꢀF), 754(CꢀCl); 1H NMR (400 MHz, DMSO, δ ppm): 3.67 (s, 3H, ꢀCH3), 7.34 (d,
2H, ArꢀH, J = 7.6 Hz), 7.42ꢀ7.46 (m, 3H, 2ꢀClꢀ6ꢀF phenyl ring), 7.52 (d, 2H, pꢀCl phenyl ring, J
= 8.4 Hz), 8.08 (d, 2H, pꢀCl phenyl ring, J = 8.8 Hz), 8.17ꢀ8.19 ( dd, 1H, , J = 8.4 Hz and 1.2
Hz), 8.65 (s, 1H, benzimidazoleꢀH); 13C NMR (125 MHz, DMSO, δ ppm):30.72 (ꢀCH3), 110.56,
114.60, 118.63, 121.17, 123.31, 125.78, 128.85, 132.63, 132.70, 137.18, 138.44, 143.06, 160.43,
162.44 (ꢀC=N), 168.13 (ꢀC=N), 176.53(ꢀC=N); ESIꢀMS: 439.2 (M++H), 441.2 [(M+H)+2]+,
443.2 [(M+H)+4]+; Anal. calcd. For C22H13Cl2FN4O: C, 60.15; H, 2.98; N, 12.75. Found: C,
60.18; H, 2.99; N, 12.79.
4.1.6.2. 2ꢀ(2ꢀChloroꢀ6ꢀfluorophenyl)ꢀ5ꢀ(3ꢀ(pyridinꢀ3ꢀyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ 1ꢀmethylꢀ1Hꢀ
benzo[d]imidazole (8b)

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Yellow solid; FT IR(ATR, ν max, cmꢀ1): 3028 (ArꢀH), 2996, 2935 (CꢀH), 1608 (C=N), 1596
1
(C=C), 1145 (CꢀF), 784 (CꢀCl); H NMR (400 MHz, DMSO, δ ppm): 3.69 (s, 3H, ꢀCH3), 7.43ꢀ
7.47 (m, 3H, 2ꢀClꢀ6ꢀF phenyl ring), 7.54 (d, 1H, ArꢀH, J = 8.4 Hz),7.58 (d, 1H, ArꢀH, J = 8.4
Hz), 8.08 (s, 1H, ArꢀH), 8.16ꢀ8.20 (m, 2H, ArꢀH), 8.24ꢀ8.27 (dd, 1H, benzimidazoleꢀH, J = 8.4
13
Hz and 1.2 Hz), 8.69 (s, 1H, benzimidazoleꢀH); C NMR (125 MHz, DMSO, δ ppm): 31.72 (ꢀ
CH3), 111.50, 114.65, 119.64, 120.17, 122.61, 124.88, 129.87, 131.64, 132.75, 136.18, 139.44,
144.06, 148.37, 159.43, 161.47 (ꢀC=N), 169.13 (ꢀC=N), 177.53 (ꢀC=N); ESIꢀMS: 407.3 (M++H),
409.3 [(M+H)+2]+; Anal. calcd. for C21H13ClFN5O: C, 62.15; H, 3.23; N, 17.26. Found: C,
62.16; H, 3.26; N, 17.28.
4.1.6.3. 2ꢀ(2ꢀChloroꢀ6ꢀfluorophenyl)ꢀ5ꢀ(3ꢀ(2ꢀchlorophenyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ 1ꢀmethylꢀ
1Hꢀbenzo[d]imidazole (8c)
White solid; FT IR(ATR, ν max, cmꢀ1): 3054 (ArꢀH), 2986, 2935 (CꢀH), 1618 (C=N), 1586
1
(C=C), 1148 (CꢀF), 786 (CꢀCl); H NMR (400 MHz, DMSO, δ ppm): 3.64 (s, 3H, ꢀCH3), 7.34ꢀ
7.38 (m, 3H, ArꢀH, J = 8 Hz), 7.49 (d, 1H, ArꢀH, J = 8.4 Hz), 7.53ꢀ7.56 (m, 4H, ArꢀH), 8.20ꢀ
13
8.22 ( dd, 1H, benzimidazoleꢀH , J = 8.8 Hz and 1.6 Hz), 8.60 (s, 1H, benzimidazoleꢀH); C
NMR (125 MHz, DMSO, δ ppm):32.32 (ꢀCH3), 113.55, 115.65, 120.64, 122.17, 124.65, 126.88,
127.33, 129.87, 132.64, 133.75, 136.58, 138.49, 144.06, 157.43, 160.47(ꢀC=N), 166.13 (ꢀC=N),
177.53 (ꢀC=N); ESIꢀMS: 440.2 (M++H), 442.4 [(M+H)+2]+, 444.7 [(M+H)+4]+; Anal. calcd. for
C22H13Cl2FN4O: C, 60.15; H, 2.98; N, 12.75. Found: C, 60.17; H, 2.96; N, 12.78.
4.1.6.4. 2ꢀ(2ꢀChloroꢀ6ꢀfluorophenyl)ꢀ5ꢀ(3ꢀ(3ꢀchlorobenzyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ 1ꢀmethylꢀ
1Hꢀbenzo[d]imidazole (8d)
White amorphous solid; FT IR(ATR, ν max, cmꢀ1): 3069 (ArꢀH), 2976, 2929 (CꢀH), 1602(C=N),
1576 (C=C), 1188 (CꢀF), 796 (CꢀCl); 1H NMR (400 MHz, DMSO, δ ppm): 1H NMR (400 MHz,
DMSO, δ ppm): 3.30 (S, 2H, benzylꢀCH2), 3.71 (s,3H, imidazoleꢀCH3), 7.47ꢀ7.54 (m, 3H, 2ꢀClꢀ
6ꢀF phenyl ringꢀH, 7.59 (d, 1H, ArꢀH, J = 8 Hz), 7.70ꢀ7.73 (m, 1H, ArꢀH, benzimidazoleꢀH),
7.76 (d, 1H, ArꢀH, J = 8.4 Hz, benzimidazoleꢀH), 7.90 (d, 1H, ArꢀH, J = 8.8 Hz), 8.08 (t, 1H, Arꢀ
H, J = 8.4 Hz), 8.43 (s, 1H, ArꢀH), 8.57 (s, 1H, benzimidazoleꢀH); 13C NMR (125 MHz, DMSO,

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δ ppm): 30.99 (ꢀCH3), 36.46 (CH2), 110.79, 121.59, 124.24, 125.97, 126.64, 127.47, 127.82,
128.56, 128.52, 130.16, 146.92, 157.59, 163.92 (ꢀC=N), 169.53 (ꢀC=N), 175.86 (ꢀC=N); ESIꢀ
MS: 453.2 (M++H), 455.2 [(M+H)+2]+, 457.2 [(M+H)+4]+; Anal. calcd. for C23H15Cl2FN4O: C,
60.94; H, 3.34; N, 12.36. Found: C, 60.95; H, 3.35; N, 12.38.
4.1.6.5. 2ꢀ(2ꢀChloroꢀ6ꢀfluorophenyl)ꢀ5ꢀ(3ꢀ(4ꢀ(methylsulfonyl)phenyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ1ꢀ
methylꢀ1Hꢀbenzo[d]imidazole (8e)
White amorphous solid; FT IR(ATR, ν max, cmꢀ1): 3099 (ArꢀH), 2996, 2939 (CꢀH), 1613(C=N),
1
1571 (C=C), 1388 (asym SO2), 1185 (CꢀF), 1129 (sym SO2), 798 (CꢀCl); H NMR (400 MHz,
DMSO, δ ppm): 3.24 (s, 3H, CH3), 3.63 (s, 3H, CH3), 7.66ꢀ7.75 (m, 5H, ArꢀH), 7.96 (d, 2H, Arꢀ
H, J = 8.8 Hz), 8.12 (d, 2H, ArꢀH, J = 8.4 Hz), 8.57 (s, 1H, ArꢀH); 13C NMR (125 MHz, DMSO,
δ ppm):31.72 (ꢀCH3), 111.56, 116.60, 119.63, 120.17, 126.73, 129.75, 133.63, 134.70, 136.62,
137.11, 139.49, 148.06, 161.43, 163.44 (ꢀC=N), 169.13 (ꢀC=N), 176.53 (ꢀC=N); ESIꢀMS: 483.2
(M++H), 484.2 [(M+H)+2]+; Anal. calcd. for C23H16ClFN4O3S: C, 57.20; H, 3.34; N, 11.60; S,
6.64. Found: C, 57.21; H, 3.37; N, 11.63; S, 6.63.
4.1.6.6.
2ꢀ(2,4ꢀDichlorophenyl)ꢀ5ꢀ(3ꢀ(4ꢀchlorophenyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ1ꢀmethylꢀ1Hꢀ
benzo[d]imidazole (8f)
Light yellow solid; FT IR (ATR, ν max, cmꢀ1): 3077 (ArꢀH), 2992, 2930 (CꢀH), 1623(C=N),
1
1571 (C=C), 778 (CꢀCl); H NMR (400 MHz, DMSO, δ ppm): 3.68 (s, 3H, ꢀCH3), 7.39 (d, 1H,
benzimidazoleꢀH, J = 8 Hz), 7.43 (d, 2H, 4ꢀchlorophenyl, J = 8.4 Hz), 7.50 (d, 2H, 4ꢀ
chlorophenyl, J = 8.4 Hz), 7.53 (s, 1H, 2,4ꢀdichlorophenyl), 8.08 (d, 2H, 2,4ꢀdichlorophenyl, J =
8.8 Hz), 8.16ꢀ8.19 (dd, 1H, benzimidazoleꢀH, J = 8.4 Hz and 1.6 Hz), 8.62 (s, 1H,
benzimidazoleꢀH); 13C NMR (125 MHz, DMSO, δ ppm): 31.20 (ꢀCH3), 111.55, 119.75, 121.97,
123.36, 125.62, 127.74, 128.86, 129.15, 129.88, 133.18, 137.22, 152.60 (ꢀC=N), 168.16 (ꢀC=N),
176.51 (ꢀC=N); ESIꢀMS: 455.1 (M++H), 457.1 [(M+H)+2]+, 459.1 [(M+H)+4]+, 461.0
[(M+H)+6]+; Anal. calcd. for C23H15Cl2FN4O: C, 57.98; H, 2.88; N, 12.29. Found: C, 57.96; H,
2.86; N, 12.30.

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4.1.6.7.
2ꢀ(2,4ꢀDichlorophenyl)ꢀ5ꢀ(3ꢀ(pyridinꢀ3ꢀyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ1ꢀmethylꢀ1Hꢀ
benzo[d]imidazole (8g)
Light brown solid; FT IR(ATR, ν max, cmꢀ1): 3057 (ArꢀH), 2994, 2932 (CꢀH), 1629 (C=N),
1
1576 (C=C), 778 (CꢀCl); H NMR (400 MHz, DMSO, δ ppm):3.68 (s, 3H, ꢀCH3), 7.40 (d, 2H,
ArꢀH, J = 8.4 Hz), 7.56 (s, 1H, ArꢀH), 7.59 (d, 1H, ArꢀH, J = 8 Hz), 7.63 (d, 1H, ArꢀH, J = 7.6
Hz), 8.08ꢀ8.12 (m, 3H, ArꢀH), 8.17ꢀ8.20 (dd, 1H, benzimidazoleꢀH, J = 8.4 Hz and 1.2 Hz), 8.64
13
(s, 1H, benzimidazoleꢀH); C NMR (125 MHz, DMSO, δ ppm): 30.20 (ꢀCH3), 109.55, 116.57,
118.24, 121.98, 122.36, 124.62, 125.41, 127.74, 128.36, 129.45, 129.88, 130.31, 133.18, 137.22,
138.20, 154.64 (ꢀC=N), 169.16 (ꢀC=N), 175.59 (ꢀC=N); ESIꢀMS: 455.1 (M++H), 457.5
[(M+H)+2]+; Anal. calcd. for C21H13Cl2N5O: C, 59.73; H, 3.10; N, 16.79. Found: C, 59.75; H,
3.11; N, 16.82.
4.1.6.8.
2ꢀ(2,4ꢀDichlorophenyl)ꢀ5ꢀ(3ꢀ(2ꢀchlorophenyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ 1ꢀmethylꢀ1Hꢀ
benzo[d]imidazole (8h)
White amorphous solid; FT IR(ATR, ν max, cmꢀ1): 3057 (ArꢀH), 2994, 2932 (CꢀH), 1629 (C=N),
1
1576 (C=C), 778 (CꢀCl); H NMR (400 MHz, DMSO, δ ppm): 3.64 (s, 3H, ꢀCH3), 7.34 (d, 2H,
ArꢀH, J = 8.4 Hz), 7.52ꢀ7.55 (m, 4H, ArꢀH), 7.77 (d, 1H, benzimidazoleꢀH, J = 8 Hz), 7.87 (s,
1H, ArꢀH), 8.22ꢀ8.25 ( dd, 1H, , J = 8.4 Hz and 1.2 Hz), 8.65 (s, 1H, benzimidazoleꢀH); 13C
NMR (125 MHz, DMSO, δ ppm): 30.29 (ꢀCH3), 110.59, 118.45, 120.94, 124.36, 125.82, 127.76,
128.80, 129.85, 129.99, 133.18, 135.32, 136.22, 137.46, 140.39, 142.36, 153.60 (ꢀC=N), 166.76
(ꢀC=N), 174.71 (ꢀC=N); ESIꢀMS: 455.1 (M++H), 457.1 [(M+H)+2]+, 459.5 [(M+H)+4]+, 461.0
[(M+H)+6]+; Anal. calcd. for C22H13Cl3N4O: C, 57.98; H, 2.88; N, 12.29. Found: C, 57.99; H,
2.89; N, 12.30.
4.1.6.9.
2ꢀ(2,4ꢀDichlorophenyl)ꢀ5ꢀ(3ꢀ(3ꢀchlorobenzyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ 1ꢀmethylꢀ1Hꢀ
benzo[d]imidazole (8i)
White solid; FT IR(ATR, ν max, cmꢀ1): 3059 (ArꢀH), 2973, 2928 (CꢀH), 1629 (C=N), 1570
(C=C), 783 (CꢀCl); 1H NMR (400 MHz, DMSO, δ ppm): 3.32 (S, 2H, benzylꢀCH2), 3.75 (s, 3H,
imidazoleꢀCH3), 7.47 (d, 2H, 2,4ꢀdichlorophenylꢀH, J = 8.4 Hz), 7.59 (s, 1H, 2,4ꢀ
dichlorophenylꢀH), 7.76 (d, 1H, benzimidazoleꢀH, J = 8.4 Hz,), 7.80ꢀ7.83 (m, 1H, ArꢀH,
benzimidazoleꢀH), 7.90 (d, 1H, ArꢀH, J = 8.8 Hz), 8.08 (t, 1H, ArꢀH, J = 8.4 Hz), 8.16 (d, 1H,

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ArꢀH, J = 8.4 Hz), 8.43 (s, 1H, ArꢀH), 8.57 (s, 1H, benzimidazoleꢀH); 13C NMR (125 MHz,
DMSO, δ ppm): 30.82 (ꢀCH3), 35.39 (ꢀCH2), 111.59, 119.45, 121.94, 124.48, 126.82, 128.76,
129.80, 129.85, 130.99, 132.46, 133.28, 134.18, 136.45, 138.43, 142.40, 144.20, 153.68 (ꢀC=N),
166.77 (ꢀC=N), 174.77 (ꢀC=N); ESIꢀMS: 471.4 (M++H), 473.1 [(M+H)+2]+, 475.4 [(M+H)+4]+,
477.0 [(M+H)+6]+; Anal. calcd. for C23H15Cl3N4O: C, 58.81; H, 3.22; N, 11.93. Found: C, 58.83;
H, 3.23; N, 11.95.
4.1.6.1.0.
2ꢀ(2,4ꢀDichlorophenyl)ꢀ5ꢀ(3ꢀ(4ꢀ(methylsulfonyl)phenyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ1ꢀ
methylꢀ1Hꢀbenzo[d]imidazole (8j)
Light cream solid; FT IR(ATR, ν max, cmꢀ1): 3081(ArꢀH), 2971,2935 (CꢀH), 1382 (asym SO2),
1
1123 (sym SO2); H NMR (400 MHz, DMSO, δ ppm): 3.20 (s, 3H, CH3), 3.68 (s, 3H, CH3),
7.66ꢀ7.75 (m, 5H, ArꢀH), 7.91 (d, 2H, ArꢀH, J = 8.8 Hz), 8.09 (d, 2H, ArꢀH, J = 8.4 Hz), 8.57 (s,
1H, ArꢀH). 13C NMR (125 MHz, DMSO, δ ppm): 30.91(CH3), 43.60 (CH2), 110.69, 121.45,
123.49, 124.14, 127.06, 127.86, 128.22, 129.42, 129.65, 133.72, 134.25, 136.00, 138.60, 139.25,
141.90, 143.18, 151.67 (ꢀC=N), 157.31(ꢀC=N), 163.95 (ꢀC=N); ESIꢀMS: 513.2 (M++H), 515.2
[(M+H)+2]+, 417.1 [(M+H)+4]+; Anal. calcd. for C23H16Cl2N4O3S: C, 55.32; H, 3.23; N, 11.22;
S, 6.42. Found: C, 55.33; H, 3.24; N, 11.24; S, 6.43.
4.1.6.1.1. 2ꢀ(3ꢀChloroꢀ2ꢀfluorophenyl)ꢀ5ꢀ(3ꢀ(4ꢀchlorophenyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ1ꢀmethylꢀ
1Hꢀbenzo[d]imidazole (8k)
White crystalline solid; FT IR(ATR, ν max, cmꢀ1): 3034 (ArꢀH), 2970.2924 (CꢀH), 1618 (C=N),
1
1566 (C=C), 1133 (CꢀF), 782 (CꢀCl); H NMR (400 MHz, DMSO, δ ppm): 3.64 (s, 3H, ꢀCH3),
7.35 (d, 2H, ArꢀH, J = 7.6 Hz), 7.41ꢀ7.45 (m, 3H, 3ꢀClꢀ2ꢀF phenyl ring), 7.55 (d, 1H, pꢀCl
phenyl ring, J = 8.4 Hz), 8.08 (d, 2H, pꢀCl phenyl ring, J = 8.8 Hz), 8.18ꢀ8.20 ( dd, 1H, , J = 8.4
Hz and 1.2 Hz), 8.65 (s, 1H, benzimidazoleꢀH); 13C NMR (125 MHz, DMSO, δ ppm): 30.47,
111.64, 118.74, 119.86, 122.61, 123.48, 124.40, 126.97, 128.57, 131.56, 132.26, 138.05, 142.09,
143.04, 155.89 (ꢀC=N), 156.73 (ꢀC=N), 162.86 (ꢀC=N); ESIꢀMS: 440.3 (M++H), 441.2
[(M+H)+2]+, 443.2 [(M+H)+4]+; Anal. calcd. for C22H13Cl2FN4O: C, 60.15; H, 2.98; N, 12.75.
Found: C, 60.17; H, 2.97; N, 12.78.

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4.1.6.1.2. 2ꢀ(3ꢀChloroꢀ2ꢀfluorophenyl)ꢀ5ꢀ(3ꢀ(pyridinꢀ3ꢀyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ1ꢀmethylꢀ1Hꢀ
benzo[d]imidazole (8l)
Light cream solid; FT IR(ATR, ν max, cmꢀ1): 3039 (ArꢀH), 2966, 2935 (CꢀH), 1608 (C=N), 1596
1
(C=C), 1145 (CꢀF), 784 (CꢀCl); H NMR (400 MHz, DMSO, δ ppm): 3.69 (s, 3H, ꢀCH3), 7.42ꢀ
7.46 (m, 3H, 3ꢀClꢀ2ꢀF phenyl ring), 7.55 (d, 1H, ArꢀH, J = 8.4 Hz),7.62 (d, 1H, ArꢀH, J = 8.4
Hz), 8.08 (s, 1H, ArꢀH), 8.16ꢀ8.20 (m, 2H, , ArꢀH), 8.70 (s, 1H, benzimidazoleꢀH); 13C NMR
(125 MHz, DMSO, δ ppm): 30.87 (CH3), 112.63, 117.72, 118.86, 123.61, 124.48, 124.98,
126.97, 128.47, 129.53, 130.56, 132.66, 134.72, 138.55, 141.49, 143.04, 144.25, 155.89(ꢀC=N),
156.73 (ꢀC=N), 162.86 (ꢀC=N); ESIꢀMS: 407.3 (M++H), 409.3 [(M+H)+2]+; Anal. calcd. for
C21H13ClFN5O: C, 62.15; H, 3.23; N, 17.26. Found: C, 62.17; H, 3.22; N, 17.29.
4.1.6.1.3. 2ꢀ(3ꢀChloroꢀ2ꢀfluorophenyl)ꢀ5ꢀ(3ꢀ(2ꢀchlorophenyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ1ꢀmethylꢀ
1Hꢀbenzo[d]imidazole (8m)
Creamy white solid; FT IR(ATR, ν max, cmꢀ1): 3059 (ArꢀH), 2986, 2932 (CꢀH), 1628 (C=N),
1
1583 (C=C), 1149 (CꢀF), 787 (CꢀCl); H NMR (400 MHz, DMSO, δ ppm): 3.64 (s, 3H, ꢀCH3),
7.35ꢀ7.39 (m, 3H, 3ꢀClꢀ2ꢀF phenyl ring, J = 8 Hz), 7.50 (d, 1H, ArꢀH, J = 8.4 Hz), 7.53ꢀ7.56 (m,
4H, ArꢀH), 8.21ꢀ8.23 ( dd, 1H, , J = 8.8 Hz and 1.6 Hz), 8.65 (s, 1H, benzimidazoleꢀH); 1H NMR
(400 MHz, DMSO, δ ppm):3.80 (s, 3H,ꢀCH3), 7.19ꢀ7.87 (m, 9H, ArꢀH), 8.71 (s, ArꢀH); 13C
NMR (125 MHz, DMSO, δ ppm): 31.27 (CH3), 110.84, 119.44, 119.56, 121.67, 123.46, 124.40,
125.93, 127.03, 129.57, 131.22, 132.16, 132.26, 132.88, 139.05, 142.09, 154.04, 155.89 (ꢀC=N),
156.03 (ꢀC=N), 163.81 (ꢀC=N); ESIꢀMS: 440.6 (M++H), 442.4 [(M+H)+2]+, 444.7 [(M+H)+4]+;
Anal. calcd. for C22H13Cl2FN4O: C, 60.15; H, 2.98; N, 12.75. Found: C, 60.17; H, 2.97; N, 12.76.
4.1.6.1.4. 2ꢀ(3ꢀChloroꢀ2ꢀfluorophenyl)ꢀ5ꢀ(3ꢀ(3ꢀchlorobenzyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ1ꢀmethylꢀ
1Hꢀbenzo[d]imidazole (8n)
White amorphous solid; FT IR(ATR, ν max, cmꢀ1): 3059 (ArꢀH), 2973, 2925 (CꢀH), 1602 (C=N),
1586 (C=C), 1184 (CꢀF), 798 (CꢀCl); 1H NMR (400 MHz, DMSO, δ ppm): 3.33 (S, 2H, benzylꢀ
CH2), 3.72 (s,3H, imidazoleꢀCH3), 7.48ꢀ7.55 (m, 3H, 3ꢀClꢀ2ꢀF phenyl ringꢀH), 7.60 (d, 1H, Arꢀ
H, J = 8 Hz), 7.70ꢀ7.73 (m, 1H, ArꢀH, benzimidazoleꢀH), 7.78 (d, 1H, ArꢀH, J = 8.4 Hz,
benzimidazoleꢀH), 7.90 (d, 1H, ArꢀH, J = 8.8 Hz), 8.08 (t, 1H, ArꢀH, J = 8.4 Hz), 8.43 (s, 1H,
ArꢀH), 8.61 (s, 1H, benzimidazoleꢀH); 13C NMR (125 MHz, DMSO, δ ppm): 31.77 (CH3), 42.42

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(CH2), 110.84, 118.64, 119.45, 120.52, 122.61, 124.46, 125.40, 126.93, 127.03, 130.57, 131.82,
132.16, 132.86, 132.98, 139.65, 142.59, 154.34, 155.89 (ꢀC=N), 157.03 (ꢀC=N), 162.81 (ꢀC=N);
ESIꢀMS: 454.4 (M++H), 455.2 [(M+H)+2]+, 457.2 [(M+H)+4]+; Anal. calcd. for
C23H15Cl2FN4O: C, 60.91; H, 3.34; N, 12.36. Found: C, 60.93; H, 3.36; N, 12.33
4.1.6.1.5. 2ꢀ(3ꢀChloroꢀ2ꢀfluorophenyl)ꢀ5ꢀ(3ꢀ(4ꢀ(methylsulfonyl)phenyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ
1ꢀmethylꢀ1Hꢀbenzo[d]imidazole (8o)
FT IR(ATR, ν max, cmꢀ1): 3029 (ArꢀH), 2986, 2929 (CꢀH), 1618 (C=N), 1561 (C=C), 1386
(asym SO2), 1188 (CꢀF), 1128 (sym SO2), 794 (CꢀCl); 1H NMR (400 MHz, DMSO, δ ppm): 3.26
(s, 3H, CH3), 3.60 (s, 3H, CH3), 7.66ꢀ7.75 (m, 5H, ArꢀH), 7.96 (d, 2H, ArꢀH, J = 8.8 Hz), 8.12
(d, 2H, ArꢀH, J = 8.4 Hz), 8.57 (s, 1H, ArꢀH); 13C NMR (125 MHz, DMSO, δ ppm): 30.73
(CH3), 44.49 (CH3), 112.84, 118.68, 120.59, 122.51, 124.49, 125.70, 126.93, 128.03, 130.27,
131.87, 132.06, 132.86, 134.98, 139.62, 143.51, 154.34, 155.89 (ꢀC=N), 159.03 (ꢀC=N), 161.48
(ꢀC=N); ESIꢀMS: 483.2 (M++H), 484.2 [(M+H)+2]+; Anal. calcd. for C23H16ClFN4O3S: C,
57.20; H, 3.34; N, 11.60; S, 6.64. Found: C, 57.21; H, 3.35; N, 11.61; S, 6.64.
4.1.6.1.6.
2ꢀ(2,3ꢀDichlorophenyl)ꢀ5ꢀ(3ꢀ(4ꢀchlorophenyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ1ꢀmethylꢀ1Hꢀ
benzo[d]imidazole (8p)
Light yellow solid; FT IR(ATR, ν max, cmꢀ1): 3077 (ArꢀH), 2992, 2930 (CꢀH), 1623 (C=N),
1
1571 (C=C), 778 (CꢀCl); H NMR (400 MHz, DMSO, δ ppm):3.68 (s, 3H, ꢀCH3), 7.42 (d, 1H,
benzimidazoleꢀH, J = 8 Hz), 7.45 (d, 2H, 4ꢀchlorophenyl, J = 8.4 Hz), 7.50 (d, 2H, 4ꢀ
chlorophenyl, J = 8.4 Hz), 7.68 (d, 1H, 2,3ꢀdichlorophenyl, J = 8.4 Hz), 7.83 (t, 1H, 2,3ꢀ
dichlorophenyl, J = 8.4 Hz), 8.09 (d, 1H, 2,3ꢀdichlorophenyl, J = 8.8 Hz), 8.15ꢀ8.18 (dd, 1H,
benzimidazoleꢀH, J = 8.4 Hz and 1.6 Hz), 8.66 (s, 1H, benzimidazoleꢀH); 13C NMR (125 MHz,
DMSO, δ ppm): 31.30 (ꢀCH3), 112.55, 119.75, 121.77, 123.36, 125.61, 127.84, 128.86, 129.15,
129.88, 133.18, 137.22, 154.60 (ꢀC=N), 169.16 (ꢀC=N), 176.81 (ꢀC=N); ESIꢀMS: 455.5 (M++H),
457.1 [(M+H)+2]+, 459.1 [(M+H)+4]+, 461.0 [(M+H)+6]+; Anal. calcd. for C23H15Cl2FN4O: C,
57.98; H, 2.88; N, 12.29. Found: C, 57.97; H, 2.89; N, 12.31.
4.1.6.1.7.
2ꢀ(2,3ꢀDichlorophenyl)ꢀ5ꢀ(3ꢀ(pyridinꢀ3ꢀyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ1ꢀmethylꢀ1Hꢀ
benzo[d]imidazole (8q)

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White solid; FT IR(ATR, ν max, cmꢀ1): 3055 (ArꢀH), 2993, 2927 (CꢀH), 1629 (C=N), 1579
1
(C=C), 773 (CꢀCl); H NMR (400 MHz, DMSO, δ ppm):3.69 (s, 3H, ꢀCH3), 7.40 (d, 1H, 2,3ꢀ
dichlorophenyl, J = 8.4 Hz), 7.50 (t, 1H, 2,3ꢀdichlorophenyl, J = 8.4 Hz), 7.55 (d, 1H, 2,3ꢀ
dichlorophenyl, J = 8.4 Hz), 7.59 (d, 1H, ArꢀH, J = 8 Hz), 7.63 (d, 1H, ArꢀH, J = 7.6 Hz), 8.08ꢀ
8.12 (m, 3H, ArꢀH), 8.17ꢀ8.20 (dd, 1H, benzimidazoleꢀH, J = 8.4 Hz and 1.2 Hz), 8.64 (s, 1H,
benzimidazoleꢀH); 13C NMR (125 MHz, DMSO, δ ppm): 30.20 (ꢀCH3), 109.99, 116.57, 118.24,
121.98, 122.36, 124.62, 125.41, 127.74, 128.36, 129.45, 129.88, 130.31, 133.18, 137.22, 138.20,
154.64 (ꢀC=N), 169.16 (ꢀC=N), 175.59 (ꢀC=N); ESIꢀMS: 455.1 (M++H), 457.5 [(M+H)+2]+;
Anal. calcd. for C21H13Cl2N5O: C, 59.73; H, 3.10; N, 16.79. Found: C, 59.72; H, 3.09; N, 16.80.
4.1.6.1.8.
2ꢀ(2,3ꢀDichlorophenyl)ꢀ5ꢀ(3ꢀ(2ꢀchlorophenyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ1ꢀmethylꢀ1Hꢀ
benzo[d]imidazole (8r)
White solid; FT IR(ATR, ν max, cmꢀ1): 3057 (ArꢀH), 2993, 2930 (CꢀH), 1621 (C=N), 1572
1
(C=C), 779 (CꢀCl); H NMR (400 MHz, DMSO, δ ppm): 3.64 (s, 3H, ꢀCH3), 7.34 (d, 1H, 2,3ꢀ
dichlorophenyl, J = 8.4 Hz), 7.52ꢀ7.55 (m, 4H, ArꢀH), 7.77 (d, 1H, benzimidazoleꢀH, J = 8 Hz),
7.87 (t, 1H, 2,3ꢀdichlorophenyl, J = 8.4 Hz), 8.04 (d, 1H, 2,3ꢀdichloro phenylꢀH, J = 8.6 Hz),
8.22ꢀ8.25 ( dd, 1H, , J = 8.4 Hz and 1.2 Hz), 8.65 (s, 1H, benzimidazoleꢀH); 13C NMR (125
MHz, DMSO, δ ppm): 30.56 (ꢀCH3), 111.59, 117.45, 120.94, 124.36, 125.82, 126.76, 128.80,
129.85, 129.99, 133.18, 135.32, 136.22, 137.46, 140.39, 142.36, 153.70 (ꢀC=N), 166.76 (ꢀC=N),
134.71 (ꢀC=N); ESIꢀMS: 455.1 (M++H), 457.1 [(M+H)+2]+, 459.5 [(M+H)+4]+, 461.0
[(M+H)+6]+; Anal. calcd. for C22H13Cl3N4O: C, 57.98; H, 2.88; N, 12.29. Found: C, 57.99; H,
2.89; N, 12.28.
4.1.6.1.9. 2ꢀ(2,3ꢀDichlorophenyl)ꢀ5ꢀ(3ꢀ(3ꢀchlorobenzyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ 1ꢀmethylꢀ1Hꢀ
benzo[d]imidazole (8s)
White solid; FT IR (ATR, ν max, cmꢀ1): 3059 (ArꢀH), 2973, 2928 (CꢀH), 1629 (C=N), 1575
(C=C), 814 (CꢀCl); 1H NMR (400 MHz, DMSO, δ ppm): 3.32 (S, 2H, benzylꢀCH2), 3.75 (s, 3H,
ꢀCH3), 7.47 (d, 1H, dichlorophenyl, J = 8 Hz), 7.59 (t, 1H, 2,3ꢀdichlorophenyl, J = 8 Hz), 7.76
(d, 1H, benzimidazoleꢀH, J = 8.4 Hz), 7.80 (d, 1H, dichlorophenyl , J = 8 Hz), 7.85ꢀ7.88 (m,
1H, ArꢀH, benzimidazoleꢀH), 7.96 (d, 1H, ArꢀH, J = 8.8 Hz), 8.08 (t, 1H, ArꢀH, J = 8.4 Hz),
8.16 (d, 1H, ArꢀH, J = 8.4 Hz), 8.43 (s, 1H, ArꢀH), 8.57 (s, 1H, benzimidazoleꢀH); 13C NMR

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(125 MHz, DMSO, δ ppm): 32.82 (ꢀCH3), 39.39 (ꢀCH2), 111.59, 119.45, 121.44, 124.48, 126.72,
129.76, 129.80, 129.85, 131.99, 132.46, 133.28, 134.18, 137.45, 138.43, 142.46, 144.54, 153.69
(ꢀC=N), 166.77 (ꢀC=N), 175.77 (ꢀC=N); ESIꢀMS: 471.4 473.1 [(M+H)+2]+, 475.4 [(M+H)+4]+,
477.0 [(M+H)+6]+; Anal. calcd. for C23H15Cl3N4O: C, 58.81; H, 3.22; N, 11.93. Found: C,
58.83; H, 3.24; N, 11.92.
4.1.6.2.0. 2ꢀ(2,3ꢀDichlorophenyl)ꢀ5ꢀ(3ꢀ(4ꢀ(methylsulfonyl)phenyl)ꢀ[1,2,4]ꢀoxadiazolꢀ5ꢀyl)ꢀ 1ꢀ
methylꢀ1Hꢀbenzo[d]imidazole (8t)
Light orange solid; FT IR (ATR, ν max, cmꢀ1): 3071(ArꢀH), 2971,2935 (CꢀH), 1386 (asym SO2),
1
1113 (sym SO2), 803 (CꢀCl); H NMR (400 MHz, DMSO, δ ppm): 3.24 (s, 3H, ꢀCH3), 3.69 (s,
3H, ꢀCH3), 7.68ꢀ7.77 (m, 5H, ArꢀH), 7.98 (d, 2H, ArꢀH, J = 8.8 Hz), 8.18 (d, 2H, ArꢀH, J = 8.4
Hz), 8.60 (s, 1H, ArꢀH). 13C NMR (125 MHz, DMSO, δ ppm): 30.95(CH3), 43.63 (CH2), 111.69,
122.45, 123.49, 124.14, 127.06, 127.86, 128.22, 129.45, 129.64, 133.71, 134.25, 136.04, 138.67,
139.25, 141.90, 144.18, 152.67 (ꢀC=N), 158.31(ꢀC=N), 164.95 (ꢀC=N); ESIꢀMS: 513.1 (M++H),
515.5 [(M+H)+2]+, 417.4 [(M+H)+4]+; Anal. calcd. for C23H16Cl2N4O3S: C, 55.32; H, 3.23; N,
11.22; S, 6.42. Found: C, 55.34; H, 3.25; N, 11.24; S, 6.44.
4.2. Antimicrobial activity
The MIC of the samples in DMSO was determined by using different concentrations of
compounds in Brain Heart Infusion Broth for bacteria and fungi by macro dilution method. The
lowest concentration of the sample in DMSO inhibiting the visible growth of microorganisms
was considered as MIC. Here broth dilution test is used to determine Minimum Inhibitory
Concentration (MIC) of the samples 8a-8t. Freshly prepared nutrient broth was used as diluents.
The 24 h old culture of the test bacteria S. aureus, E. fecalis, E. coli and K. pneumoniae and the
test fungi C. albicans and A. fumigatus were diluted 100 folds in nutrient broth (NB) (100 µl
bacterial cultures in10 ml NB). The stock solution of the synthesized compounds with
concentration 1mg/1mL was prepared in DMSO. Different concentrations of the test samples
(0.2, 0.4, 0.8, 1.6, 3.125, 6.25, 12.5, 25, 50 and 100 µg/ml) were added to the test tubes
containing the bacterial and fungal cultures. All the tubes were incubated at 37oC for 24 h for
bacteria and at 28oC for 48 h for fungi. The tubes were examined for visible turbidity and using
NB as a control. Control without test samples and with solvent (DMSO) was assayed

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simultaneously. The lowest concentration that inhibited visible growth of the tested organisms
was recorded as MIC.
4.3. Inꢀvitro antitubercular activity
Antitubercular activity was evaluated against Mycobacterium tuberculosis H37Rv by
Microplate Almer Blue Assay (MABA). In the presence of cellular metabolism, the colour of
Alamar Blue (ALB) changes from a fully oxidized, non fluorescent blue to a fully reduced,
fluorescent pink colour. This methodology is nonꢀtoxic, uses a thermally stable reagent and
shows good correlation with BACTEC radiometric method. Middlebrook 7H9 broth base with
Middlebrook OADC growth supplement used as medium. 200µl of sterile deionzed water was
added to all outer perimeter wells of sterile 96 wells plate to minimize evaporation of medium in
the test wells during incubation. The 96 wells plate received 100 µl of the Middlebrook 7H9
broth. 10 mg of each compound was dissolved in 10.0 mL of DMSO separately and was further
diluted with DMSO to make 100 µg/mL and 10 µg/mL stock solution. Using the above stock
solution, serial dilutions of 0.2 µg/mL, 0.8 µg/mL, 1.6 µg/mL, 3.12 µg/mL, 6.25 µg/mL, 12.5
µg/mL, 25 µg/mL, 50 µg/mL and 100 µg/mL were made with DMSO directly on the plate.
Further the positive control was prepared by inoculating the test organism with medium
containing standard drugs and a negative control was prepared by inoculating test organism with
medium containing only DMSO. 50 ꢁL of DMSO used as control. Medium without test
compound/DMSO was also inoculated with the test organism to check whether the media will
support the growth of the tubercle bacilli or not, and observed that no mycobacteria growth. The
above said wells were inoculated with M. tuberculosis H37Rv Strain. Plates were covered and
sealed with parafilm and incubated at 37ºC for five days. After this time, 25µl of freshly
prepared 1:1 mixture of Almar Blue reagent and 10% tween 80 was added to the plate and
incubated for 24 hrs. A blue color in the well was interpreted as no bacterial growth, and pink
color was scored as growth.
4.4. Hemolytic assay
Human blood was collected in EDTA (2mg/ml) containing vacutainer. Plasma and buffy
coat was removed from the EDTAꢀblood by centrifugation at 800 X g for 10 mins. Later, the
erythrocytes were washed three times by using normal saline (0.9%) and resuspended in saline to

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5% erythrocytes suspension. The cells were incubated for 1h at 37 °C in the presence of test
compounds. After the incubation, the samples were centrifuged at 800 X g for 10 min and the
absorbance was read using UV spectrophotometer. 2% Triton Xꢀ100 (Sigmaꢀ Aldrich, St. Louis,
USA) served as positive control. Phosphate buffered saline served as negative control. The
absorbance of haemoglobin release was measured at 540 nm and is expressed as % of Triton Xꢀ
100 induced hemolysis. The result was calculated by using the formula,
% Hemolysis =[(Absorbance of sample) – (Absorbance of blank)/ Highest absorbance of positive
control] x 100
Each concentration had triplicate values. HC50 was determined for each triplicate OD and
average of HC50 was plotted against concentration fitted with a sigmoid plot. From the curve
concentration corresponding to 50% hemolysis was determined.
4.4. Inꢀvitro cytotoxic activity
Human embryonic kidney cell line HEK 293 was procured from NCCS, Pune. The
cytotoxicity effects of 8k, 8n, 8p and 8r were studied by MTT assay. The cells were cultured
using Minimum Essential Medium Eagle (MEM) medium supplemented with 10 % fetal bovine
serum (FBS), penicillin (100 U/mL) and streptomycin (100 µg/mL) and grown in 25 cm2 tissue
culture flasks at 37°C with 5% CO2 in incubator (Galaxy 170S, New Brunswick). After attaining
70 to 80 % confluence, cells were trypsinized and seeded to 96 well plates in 100 ꢁL of MEM
(5,000 cells/well). After 24 h different concentrations of drugs were dissolved in DMSO and
added to 96 well plates and incubated for 24 h at standard condition. Untreated cells were
representing as a control group. Four replicates were set up for each test group. After completion
of incubation time, 20 µL of MTT dye (5 mg/mL in PBS) was added to all wells; sealed the plate
with aluminium foil and incubate for 4 h in incubator. After 4 h, 100 µL of dimethyl sulfoxide
was added to all the wells and mixed by uniform shaking. Using multiꢀplate reader
(LisaScan®EM) the absorbance was recorded at 570 nm. The percentage viable cell was
calculated using following formula:
Percentage of cell viability = (Test sampleꢀblank / Controlꢀblank) x 100
Each concentration had triplicate values and the average value was plotted against concentration
by fitting with a sigmoid plot. From the curve concentration corresponding to 50% cell viability
was noted.

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Acknowledgements
The authors are grateful to UGCꢀSAP and DSTꢀPURSE for providing financial
assistance. We are thankful to USIC Mangalore University and SAIF cochin for providing
spectral analyses. We also gratefully acknowledge The Director and Mr. Vishwanatha U, SDM
Centre for Research in Ayurveda, Udupi for their help in carrying out cytotoxicity study.
Supplementary data
Supplementary data contain scanned spectra of compounds and dose response curve of
Mitomycin C against normal cell line.
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