Synthesis and biological evaluation of new imidazo[2,1-b][1,3,4]thiadiazole-benzimidazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2015.03.024

In this report, we describe the synthesis and biological evaluation of a new series of 2-(imidazo[2,1-b][1,3,4]thiadiazol-5-yl)-1H-benzimidazole derivatives (5a-ac). The molecules were analyzed by ¹H NMR, ¹³C NMR, mass spectral and elemental data. The structure of one of the pre-final compounds, 6-(4-methoxyphenyl)-2-(4-methylphenyl)imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde (4d) and that of a target compound, 2-[2-methyl-6-(4-methyl phenyl) imidazo[2,1-b][1,3,4]thiadiazol-5-yl]-1H-benzimidazole (5aa) were confirmed by single crystal XRD studies. All the target compounds were screened for in vitro anti-tuberculosis activity against Mycobacterium tuberculosis H37Rv strain. Seven (5c, 5d, 5l, 5p, 5r, 5z and 5aa) out of twenty nine compounds showed potent anti-tubercular activity with a MIC of 3.125 μg/mL. A p-substituted phenyl group (p-tolyl or p-chlorophenyl) in the imidazo[2,1-b][1,3,4]thiadiazole ring and/or a chloro group in the benzimidazole ring enhance anti-tuberculosis activity whereas a nitro group in the benzimidazole ring reduces the activity. In the antibacterial screening, compounds 5i, 5w and 5ac showed promising activity against the tested bacterial strains. Further, antifungal and antioxidant activities of these molecules were also investigated. In the cytotoxicity study, the active antitubercular compounds exhibited very low toxicity against a normal cell line.

Full text of DOI:10.1016/j.ejmech.2015.03.024

European Journal of Medicinal Chemistry 95 (2015) 49e63
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of new imidazo[2,1-b][1,3,4]
thiadiazole-benzimidazole derivatives
,
*
Jurupula Ramprasad a, Nagabhushana Nayak a, Udayakumar Dalimba a
,
Perumal Yogeeswari b, Dharmarajan Sriram b, S.K. Peethambar c, Rajeshwara Achur c,
H. S. Santosh Kumar d
a Organic Chemistry Laboratory, Department of Chemistry, National Institute of Technology Karnataka, Surathkal, Srinivasanagar, Mangalore 575025, India
b Medicinal Chemistry and Drug Discovery Research Laboratory, Pharmacy Group, Birla Institute of Technology and Science-Pilani, Hyderabad Campus,
Jawahar Nagar, Telangana 500078, India
c Department of Biochemistry, Kuvempu University, Jnanasahyadri, Shankaraghatta 577451, India
d Department of Bioinformatics, Kuvempu University, Jnanasahyadri, Shankaraghatta 577451, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In this report, we describe the synthesis and biological evaluation of a new series of 2-(imidazo[2,1-b]
[1,3,4]thiadiazol-5-yl)-1H-benzimidazole derivatives (5a-ac). The molecules were analyzed by 1H NMR,
13C NMR, mass spectral and elemental data. The structure of one of the pre-final compounds, 6-(4-
methoxyphenyl)-2-(4-methylphenyl)imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde (4d) and that of a
target compound, 2-[2-methyl-6-(4-methyl phenyl) imidazo[2,1-b][1,3,4]thiadiazol-5-yl]-1H-benzimid-
azole (5aa) were confirmed by single crystal XRD studies. All the target compounds were screened for
in vitro anti-tuberculosis activity against Mycobacterium tuberculosis H37Rv strain. Seven (5c, 5d, 5l, 5p,
5r, 5z and 5aa) out of twenty nine compounds showed potent anti-tubercular activity with a MIC of
Received 11 November 2014
Received in revised form
11 March 2015
Accepted 12 March 2015
Available online 14 March 2015
Keywords:
Imidazo[2,1-b][1,3,4]thiadiazole
Benzimidazole
3.125 mg/mL. A p-substituted phenyl group (p-tolyl or p-chlorophenyl) in the imidazo[2,1-b][1,3,4]thia-
diazole ring and/or a chloro group in the benzimidazole ring enhance anti-tuberculosis activity whereas
a nitro group in the benzimidazole ring reduces the activity. In the antibacterial screening, compounds 5i,
5w and 5ac showed promising activity against the tested bacterial strains. Further, antifungal and
antioxidant activities of these molecules were also investigated. In the cytotoxicity study, the active
antitubercular compounds exhibited very low toxicity against a normal cell line.
Mycobacterium tuberculosis
Antibacterial agent
Antioxidant activity
Cytotoxicity
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
in the body. However, if immune system disorders cause abnor-
mally low resistance, such as in people with HIV, TB bacteria can
Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb).
It is an instant disease and is the most single infectious killer dis-
ease after HIV/AIDS. According to the report released from the
world health organization (WHO) in 2013 [1], 8.6 million people fell
sick with TB and 1.3 million died from TB. Over 95% of TB deaths
occur in low and middle income countries. In 2012, an estimated
5,30,000 children became ill with TB and 74,000 HIV negative
children died of TB. In most of the cases, TB does not show any
serious symptoms because the bacteria can live in an inactive form
become active. Some of the available anti-tuberculosis drugs such
as isoniazid and rifampin have a vital effect in the treatment of TB
over past decades. However, the global dominance of the exten-
sively drug-resistant tuberculosis (XDRTB) and multidrug resistant
tuberculosis (MDRTB) made these traditional anti-TB agents with
limited efficacy. Many active molecules were developed in the last
four decades to treat multidrug resistant tuberculosis. But only one
molecule, Bedaquiline, was approved by U.S. Food and Drug
Administration [2]. Thus there is an emergent need to develop
more effective drugs to treat TB. In this direction, a variety of het-
erocyclic compounds are being synthesized to check the possible
potency of the molecules to act against TB [3e6].
* Corresponding author.
The imidazo[2,1-b][1,3,4]thiadiazole moiety is an important
class of heterocyclic compounds [7] in medicinal chemistry
E-mail
addresses:
udayaravi80@gmail.com,
udayakumar@nitk.ac.in
(U. Dalimba).
http://dx.doi.org/10.1016/j.ejmech.2015.03.024
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

50
J. Ramprasad et al. / European Journal of Medicinal Chemistry 95 (2015) 49e63
research. There are several reports available in the literature
describing the imidazo[2,1-b][1,3,4]thiadiazole derivatives for their
various biological activities. The most relevant and recent studies
revealed that these molecules exhibit antimicrobial [8e10], anti-
inflammatory [11], anticonvulsant [12,13], antituberculosis
[14e16], anticancer [17e19] and anti hyperlipidemic [20] activities.
In fact, 1,3,4-thiadiazole core is found in several marketed drugs
such as Acetazolamide (I) and Methazolamide (II) (which are car-
bonic anhydrase inhibitors for treatment of glaucoma), and sulfa-
methizole (III), Cefazedone (IV), Cefazolin (V), Ceftezole (VI) (which
are used as antibacterial drugs) (Fig. 1). A few imidazo[2,1-b][1,3,4]
thiadiazole derivatives carrying other active heterocyclic pharma-
cophores particularly at position-5 (XI, XII) have been found to
possess good activity against Mtb H37Rv strain [21e23]. For
instance, Alegaon et al. reported the synthesis and antitubercular
evaluation of a series of imidazo[2,1-b][1,3,4]thiadiazoles carrying
different heterocyclic moieties at position-5 (XIII) [21]. On the other
hand, benzimidazole and its derivatives are finding great impor-
tance in medicinal chemistry research due to their important bio-
logical actions as well as their synthetic applications [24].
Benzimidazole is a core structural moiety found in some of the
important drugs like albendazole (VII), mebendazole (VIII), thia-
bendazole (IX), rabeprazole (X) etc (Fig. 1). A few recent reports
demonstrated the promising antitubercular activity of benzimid-
azole derivatives. For example, a series of pyrido[1,2-a]benzimid-
azole based molecules (XIV) exhibit excellent bactericidal activity,
2. Results and discussion
2.1. Chemistry
The twenty nine new 2-subsituted-6-arylimidazo[2,1-b][1,3,4]
thiadiazole derivatives (5a-ac) were synthesized according to the
synthetic route presented in Scheme 1. One of the key in-
termediates, 5-methyl-1,3,4-thiadiazol-2-amine (2a) was synthe-
sized by treating acetyl chloride with thiosemicarbazide using the
reported procedure [28] with a modification of the work up
method to get a better yield (80% as against the reported yield of
23%) of the product (without column chromatography purifica-
tion). Other intermediate compounds, 5-aromatic-1,3,4-
thiadiazole-2-amines (2b-f) were synthesized by treating the cor-
responding aromatic acid with thiosemicarbazide in the presence
of phosphorous oxychloride [29]. The 2-subsituted-6-arylimidazo
[2,1-b][1,3,4]thiadiazole derivatives (3aem) were synthesized by
the reaction between 5-substituted-1,3,4-thiadiazol-2-amine (2a-
f) and the corresponding substituted
a-halo aryl ketone under
heating conditions. The formation of compounds 3a-m was
confirmed by 1H NMR, 13C NMR and mass spectral analysis. For
instance, the 1H NMR spectrum of compound 3b showed a singlet
with one proton at
imidazole ring proton (C5eH). Further, the spectrum displayed
singlets at 2.38 and 2.71 ppm representing the methyl protons on
d 7.92 ppm, which indicates the presence of
d
phenyl and 1,3,4-thiadiazole rings respectively. Also, its mass
spectrum showed a molecular ion peak at m/z 230.0, which cor-
responds to Mþ1 peak of the molecule.
with several of them having MICMABA values lower than 1 mg/mL
[25]. Further, Stanley et al. identified a novel benzimidazole com-
pound, N-(2,4-dichlorobenzyl)-1-propyl-1H-benzo[d]imidazol-5-
amine (XV), which targets mycobacterial membrane protein large
3 (MmpL3) [26]. Also, a library of novel trisubstituted benzimid-
azoles were developed through rational drug design by Kumar et al.
and some of these compounds (XVI, XVII) exhibited promising MIC
In the next step, imidazo[2,1-b][1,3,4]thiadiazoles (3aem) were
subjected to VilsmeiereHaack formylation reaction to afford 2-
substituted-6-arylimidazo[2,1-b][1,3,4]thiadiazole-5-
carbaldehydes (4aem), the structures of which were confirmed by
spectral analysis. In the 1H NMR spectrum of 4b, the singlet peak
due to imidazole ring proton (C5eH) disappeared whereas a new
values in the range of 0.5e6.1 mg/mL against Mtb H37Rv strain [27].
Thus, in view of the promising bactericidal activity exhibited by the
imidazo[2,1-b][1,3,4]thiadiazole and benzimidazole systems
(Fig. 2), we envisaged to amalgamate these two structural units in a
single molecular frame and to explore the effects of this structural
amalgamation towards their antitubercular activity. Hence we
synthesized a series of imidazo[2,1-b][1,3,4]thiadiazole containing
benzimidazole derivatives and evaluated their antitubercular,
antibacterial, antifungal and antioxidant activities.
singlet appeared at d 10 ppm confirming the presence of aldehyde
(-CHO) group. Also, its mass spectrum showed a molecular ion peak
at m/z 257.9, which corresponds to Mþ1 peak of the molecule. The
1H NMR spectrum of 4d shows two singlets at 2.5 and 3.9 ppm
which indicates the presence of methyl and methoxy groups in the
molecule respectively, along with other characteristic signals. Sin-
gle crystals of 4d were analyzed by single crystal X-ray diffrac-
tometer to confirm its three dimensional structure. Finally, the
Fig. 1. Representative thiadiazole and benzimidazole based drug molecules (IeX).

J. Ramprasad et al. / European Journal of Medicinal Chemistry 95 (2015) 49e63
51
Fig. 2. Imidazo [2,1-b][1,3,4]thiadiazole and benzimidazole based antitubercular agents.
Scheme 1. Synthesis of imidazo[2,1-b][1,3,4]thiadiazole-benzimidazole derivatives. Reaction condition a: Acetyl chloride, 0 ꢀC - RT, 4 h; b: Substituted aromatic acid, POCl3, 75 ꢀC,
30 min; c: Phenacyl bromide, ethanol, 80e85 ꢀC, 24 h; d: DMF, POCl3, 60 ꢀC, 6 h; e: sodium meta bisulfite, DMF, 120 ꢀC, 3 h.
target compounds, 2-(2-substituted-6-phenylimidazo[2,1-b][1,3,4]
thiadiazol-5-yl)-1H-benzimidazoles (5a-ac), were synthesized by
treating compounds 4aem with different substituted o-phenyl-
enediamines in the presence sodium meta bisulfite (Na2S2O5) un-
der heating conditions using DMF as the solvent [30,31]. The
structural features and the physical constant of compounds 5a-ac
are given in Table 1. The chemical structure of the target com-
pounds was confirmed by spectral analysis and elemental data. The
aromatic (phenyl) protons. Also, its mass spectrum showed a mo-
lecular ion peak at m/z 346.0, which corresponds to Mþ1 peak of
the molecule and is in agreement with its molecular formula
C19H15N5S. The three dimensional structure of 5aa was evidenced
by single crystal X-ray diffraction study. The spectral and elemental
analysis data of all target compounds are given in the experimental
part.
1H NMR spectrum of 5aa showed a broad singlet at
to NH proton of the benzimidazole ring. Further, singlets at
and 2.3 ppm indicate the methyl protons of the 1,3,4-thiadiazole
and phenyl rings respectively. In addition, the spectrum displayed
two doublets at 7.18 and 7.8 ppm, multiplet in the region of
7.65 ppm and double of doublet in the region of 7.26 ppm due to
d
12.8 ppm due
2.9
2.2. Single crystal X-ray crystallography studies
d
d
Single crystals of 4d and 5aa were grown from a solvent mixture
of methanol and chloroform (1:1) by the slow evaporation of the
solvent mixture at room temperature. The compounds (4d and 5aa)
crystallized in the monoclinic system with P 21/n space group. The
d
d
d

52
J. Ramprasad et al. / European Journal of Medicinal Chemistry 95 (2015) 49e63
Table 1
nitrobenzimidazole substitution also have shown moderate activity
when a methyl group is replaced with a p-substituted phenyl
group. For instance, compounds 5e and 5j showed improved ac-
Physical properties and structural features of target compounds (5a-5ac).
Entry
Product
R1
R2
R3
Yield (%)
m.p (ꢀC)
tivity (MIC ¼ 6.25
m
g/mL) when compared to the activity of com-
1
2
3
4
5
6
7
8
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
4-CH3$C6H4
4-CH3$C6H4
4-CH3$C6H4
4-Cl$C6H4
4-Cl$C6H4
4-Cl$C6H4
2-Cl$C6H4
C6H5
4-CH3$C6H4
4-CH3$C6H4
4-Cl$C6H4
4-Cl$C6H4
4-F$C6H4
4-F$C6H4
4-F$C6H4
4-CH3$C6H4
4-CH3$C6H4
4-CH3$C6H4
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OCH3
OCH3
OCH3
CH3
CH3
CH3
OCH3
OCH3
F
H
NO2
Cl
H
NO2
Cl
NO2
NO2
H
NO2
H
H
H
NO2
Cl
H
NO2
Cl
H
NO2
H
NO2
Cl
78
73
76
74
72
76
78
74
77
74
72
79
77
78
74
78
76
79
82
86
80
82
79
86
87
78
88
84
82
247e248
243e244
218e219
269e270
226e227
259e260
235e236
259e260
243e244
266e267
264e265
258e259
236e237
211e212
201e202
222e223
208e209
268e269
179e180
189e190
186e187
192e193
170e171
179e180
181e182
180e181
173e174
202e203
193e194
pounds 5v and 5ab (MIC ¼ 50
mg/mL) respectively. This information
on structureeactivity relationship explored in the present study
could be helpful in further structural modification and develop-
ment of new imidazo[2,1-b][1,3,4]thiadiazole-benzimidazole hy-
brids as potent antitubercular agents.
9
2.4. Antibacterial activity
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
F
OCH3
F
OCH3
OCH3
OCH3
Cl
Cl
Cl
OCH3
OCH3
F
The in vitro antibacterial activity of synthesized compounds 5a-
5ac were determined using Muller Hinton Agar method [32,33].
These compounds were screened against Staphylococcus aureus,
Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi. The
compounds were dissolved in DMSO and appropriate dilutions
were made (1 mg/mL and 0.5 mg/mL). Streptomycin was taken as
the standard drug for the screening. The result showed that among
the tested compounds, 5i exhibit good activity against all the tested
bacterial strains at concentrations of 1 and 0.5 mg/mL, whereas
compounds 5w and 5ac are moderately active against all these
microbial strains. The results of the antibacterial screening are
summarized in Table 3, where in, the figures represent the zone of
inhibition.
5s
5t
5u
5v
5w
5x
5y
5z
5aa
5ab
5ac
F
F
Cl
Cl
H
NO2
Cl
H
NO2
Cl
Cl
CH3
CH3
CH3
CH3
CH3
2.5. Antifungal activity
The synthesized compounds 5a-5ac were screened also for their
antifungal activity against Aspergillus flavus, Chrysosporium kerati-
nophilum and Candida albicans. These compounds were dissolved in
DMSO and antifungal activity was determined using the well plate
method [34,35] at concentrations of 1 and 0.5 mg/mL and the re-
sults are tabulated in Table 4. Fluconazole was taken as the standard
drug. It is evident from Table 4 that none of the compounds show
any significant activity against the tested fungal strains. Only two
compounds 5w and 5x, both containing a 4-chlorophenyl substi-
tution on the imidazo[2,1-b][1,3,4]thiadiazole ring and a chloro
substitution on the benzimidazole ring, exhibited moderate activ-
ity. These in vitro screening results show that the new imidazo[2, 1-
b][1,3,4]thiadiazole-benzimidazole hybrids (5a-5ac) are very se-
lective against M. tuberculosis H37Rv strain, 19 compounds of the
crystal structure of the compounds is shown in Fig. 3. The crystal
data and measurement details for compounds 4d and 5aa are given
in Table 2.
2.3. In vitro antimycobacterial activity
The synthesized imidazo[2,1-b][1,3,4]thiadiazole-benzimid-
azole hybrids (5a-5ac) were screened against M. tuberculosis H37Rv
(ATCC27294) by agar dilution method to evaluate their anti-
mycobacterial activity in terms of minimum inhibitory concentra-
tion (MIC) values. The MIC is defined as the minimum
concentration of a compound required to completely inhibit the
series exhibited MIC of 12.5 mg/mL or less.
bacterial growth. The MIC values in
those of standard drugs for comparison are presented in Fig. 4,
which shows that the values are in the range 3.125e50.0 g/mL. It is
mg/mL of 5a-5ac along with
2.6. Antioxidant studies: DPPH radical scavenging assay
m
evident that among the twenty nine compounds, seven compounds
namely 5c, 5d, 5l, 5p, 5r, 5z and 5aa showed potent anti-tubercular
The free radical scavenging activity of test samples 5aeac was
measured using the DPPH method [36]. Butylated hydroxytoluene
(BHT) was used as the standard. Among the tested compounds, 5r
and 5z showed significant DPPH scavenging activity (>65%) at
activity with MIC of 3.125
mg/mL and are equipotent with the
standard Ethambutol. Four other compounds (5e, 5j, 5s and 5u)
showed promising activity against M. tuberculosis with MIC of
6.25 mg/mL.
100 mg concentration (Fig. 5).
The nature of the substituents on the imidazo[2,1-b][1,3,4]
thiadiazole (R1/R2) and benzimidazole (R3) rings was found to affect
the activity of these compounds. The presence of a nitro group on
the benzimidazole ring substantially decreased the anti-TB activity
as can be seen in case of compounds 5g, 5h, 5v, 5y and 5ab which
2.7. In vitro cytotoxicity studies
The in vitro cytotoxicity of the active compounds (MIC ꢁ6.25
mg/
mL against M. tuberculosis) were evaluated by MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay
[37] against normal Vero cell line. The graphical representation of
the cell growth inhibition of the compounds at a concentration of
showed an MIC value of 50
mg/mL. Whereas compounds with
unsubstituted or chlorosubstituted benzimidazole rings showed
excellent to moderate activity. Though two compounds (5z and
5aa) with a methyl substitution at position-2 of the imidazo[2,1-b]
[1,3,4]thiadiazole ring exhibited significant activity, replacing the
methyl group with a p-substituted phenyl group (p-tolyl or p-
chlorophenyl) was found to be effective in enhancing anti-TB ac-
tivity. This is evident from the fact that compounds with a
62.5 mg/mL is shown in Fig. 6. Some of the most potent antituber-
cular compounds 5r, 5s and 5aa exhibited very low toxicity of 7.4,
7.1 and 5.03% respectively. Also, none of the other active com-
pounds are toxic to the normal cells thus proving the lack of general
cellular toxicity.

J. Ramprasad et al. / European Journal of Medicinal Chemistry 95 (2015) 49e63
53
Fig. 3. ORTEP diagram showing the X-ray crystal structure of compounds a) 4d and b) 5aa.
3. Conclusions
spectroscopic techniques and elemental analysis. The compounds
were screened for their anti-tubercular activity against M. tuber-
culosis H37Rv by Agar dilution method. Among the tested com-
pounds, seven compounds (5c, 5d, 5l, 5p, 5r, 5z and 5aa) exhibited
significant activity against the growth of M. tuberculosis with MIC of
A series of twenty nine 2-(imidazo [2,1-b][1,3,4]thiadiazol-5-yl)-
1H-benzimidazole derivatives were designed and synthesized.
These compounds were characterized by 1H NMR, 13C NMR, mass
3.125 mg/mL and a moderate activity with MIC of 6.25 mg/mL was
observed for compounds 5u, 5e, 5s and 5j. The structureeactivity
relationship reveals that various substituents on the imidazo[2,1-b]
[1,3,4]thiadiazole and the benzimidazole rings affect significantly
the anti-TB activity of these compounds. Compounds with a chloro
substituted or unsubstituted benzimidazole ring exhibit better ac-
tivity when compared with the activity of nitro substituted de-
Table 2
Crystal data and measurement details for compounds 4d and 5aa.
Parameters
Crystal data of 4d
Crystal data of 5aa
Empirical formula
Formula weight
Crystal system
C19H15N3O2S
349.41
Monoclinic
0.5 mm ꢂ 0.24 mm
ꢂ 0.14 mm
P 21/n
7.1761(7)
19.468(17)
12.022(11)
1611.2(3)
90, 106.408(6), 90
4
C19H15N5S$H2O
363.45
Monoclinic
0.42 mm ꢂ 0.22 mm
ꢂ 0.10 mm
P 21/n
7.1846(5)
22.9252(13)
11.0542(6)
1820.72(19)
90, 90.087(3), 90
4
rivatives. Further,
a phenyl group with a p-methyl/p-chloro
Crystal dimension
substituent at position-2 of the imidazo[2,1-b][1,3,4]thiadiazole
ring enhanced the activity. Hence, these compounds with prom-
ising anti-TB activity could serve as promising lead molecules for
further generation of potent antitubercular agents. Further, these
compounds were screened also for their antibacterial, antifungal
and antioxidant activity. Among the tested compounds, 5i showed
good activity and compounds 5w and 5ac showed moderate ac-
tivity against the tested bacterial strains. Compounds 5w and 5x
showed moderate activity against the fungal strains. The antioxi-
dant property of compound 5z is comparable with that of the
standard BHT. In conclusion, the new imidazo[2,1-b][1,3,4]thia-
diazole-benzimidazole hybrids (5a-5ac) show remarkable selec-
tivity against M. tuberculosis H37Rv strain and interestingly, most
Space group
a (Å)
b (Å)
c (Å)
Volume (Å3)
Angle
Z
a, b, g
F000
728
0.219
296k
0.71073
760
0.196
296k
0.71073
m
(mmꢃ1
)
Temperature (T)
Radiation wavelength (Å)
Radiation type
Radiation source
CCDC number
Mo K
Mo
a
Mo K
a
Mo
1,032,193
1,032,180

54
J. Ramprasad et al. / European Journal of Medicinal Chemistry 95 (2015) 49e63
Fig. 4. Antitubercular activity of 5a-5ac against Mycobacterium tuberculosis H37RV (INH: Isoniazid; EMB: Ethambutol; PZA: Pyrazinamide).
Table 3
Antibacterial activity of target compounds (5a-5ac) against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi.
Compound
Escherichia coli
Staphylococcus aureus
Pseudomonas aeruginosa
Salmonella typhi
Cocn. in mg/ml
1
0.5
1
0.5
1
0.5
1
0.5
Control
SM
5a
0
0
0
0
18.6 0.2
e
14.3 0.1
e
16.8 0.2
e
12.5 0.2
e
16.7 0.2
e
13.6 0.2
e
17.2 0.2
e
15.7 0.2
e
5b
5c
5d
5e
e
e
e
e
e
e
e
e
04 0.1
05 0.2
e
02 0.2
03 0.1
e
06 0.1
04 0.1
e
03 0.2
01 0.1
e
07 0.1
04 0.2
e
03 0.2
01 0.1
e
3
0.2
01 0.1
03 0.5
e
05 0.4
e
5f
5g
04 0.1
e
02 0.4
e
03 0.1
e
03 0.1
e
04 0.1
e
03 0.1
e
03 0.1
e
02 0.2
e
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
04 0.4
13 0.5
04 0.5
03 0.2
05 0.1
e
03 0.3
09 0.5
02 0.1
01 0.1
03 0.2
e
04 0.2
14 0.1
04 0.1
03 0.1
07 0.1
e
02 0.1
11 0.2
02 0.2
02 0.2
03 0.2
e
05 0.1
13 0.1
03 0.2
02 0.2
08 0.1
e
03 0.2
10 0.2
02 0.2
01 0.2
05 0.2
e
06 0.1
15 0.3
04 0.1
03 0.2
03 0.3
e
03 0.2
13 0.2
01 0.2
01 0.1
01 0.1
e
05 0.3
05 0.2
04 0.2
e
03 0.4
01 0.1
02 0.2
e
05 0.1
04 0.1
03 0.1
e
03 0.2
01 0.1
02 0.1
e
06 0.1
02 0.1
06 0.1
e
03 0.2
01 0.1
03 0.2
e
04 0.1
03 0.5
04 0.2
e
03 0.2
0
02 0.2
e
04 0.1
e
02 0.2
e
05 0.2
e
03 0.1
e
03 0.1
e
02 0.1
e
e
e
5s
e
e
5t
e
e
e
e
e
e
e
e
5u
5v
5w
5x
5y
5z
5aa
5ab
5acc
04 0.1
04 0.3
11 0.2
04 0.2
06 0.1
04 0.1
05 0.1
e
03 0.1
02 0.4
08 0.2
03 0.1
04 0.2
02 0.4
02 0.2
e
02 0.1
03 0.1
09 0.4
04 0.2
09 0.1
03 0.2
05 0.2
e
01 0.2
02 0.2
06 0.6
03 0.3
07 0.2
02 0.1
03 0.1
e
03 0.2
01 0.1
13 0.1
05 0.2
08 0.1
04 0.1
03 0.1
e
02 0.3
03 0.2
10 0.2
03 0.1
05 0.2
01 0.1
02 0.1
e
02 0.1
02 0.1
10 0.1
03 0.1
06 0.3
04 0.2
06 0.3
e
01 0.5
01 0.2
07 0.2
01 0.1
04 0.5
02 0.2
e
e
08 0.1
05 0.2
10 0.1
07 0.2
09 0.1
06 0.2
08 0.1
07 0.2
SM: Streptomycin (anti-bacterial standard); e: inhibition not detected; control: dimethylsulfoxide.
derivatives (19 compounds) of the series exhibited MIC of 12.5
m
g/
Ltd. All the solvents were distilled and dried before usage. The
progress of the reaction was monitored by TLC using pre coated
aluminum sheets with 60 F254 silica gel (Merck KGaA). Melting
point of the synthesized compounds was recorded by a Stuart SMP3
melting point apparatus. Infrared spectra of the compounds were
recorded on a Jasco FTIR 4200 spectrometer. 1H NMR spectra of the
intermediates and final compounds were recorded using Bruker
mL or less. Further, the cytotoxicity study revealed that none of the
active molecules are toxic to the Vero cell line thus proving the lack
of general cellular toxicity.
4. Experimental
400 MHz NMR spectrometers using TMS as internal standard. 13
C
4.1. Materials and instruments
NMR spectra of the compounds were recorded using a Bruker
100 MHz NMR spectrometer. Elemental analysis was done using a
Thermo electron corporation EA-112 series C, H, N, S analyzer. Mass
The required chemicals and solvents were procured from Sigma
Aldrich (Germany), Merck (India) and Spectrochem Chemicals Pvt.

J. Ramprasad et al. / European Journal of Medicinal Chemistry 95 (2015) 49e63
55
Table 4
Antifungal activity of target compounds (5a-5ac) against Aspergillus flavus, Chrys-
osporium keratinophilum and Candida albicans.
Compound
Aspergillus flavus
Chrysosporium
keratinophilum
Candida albicans
Conc. in mg/ml
Control
FZ
1
0
0.5
1
0
0.5
1
0
0.5
14 0.2 10 0.1 16 0.2 14 0.2 23 0.2 20 0.2
5a
e
e
e
e
e
e
5b
5c
5d
5e
e
e
e
e
e
e
03 0.1 02 0.1 04 0.2 02 0.1 03 0.1 02 0.1
05 0.1 03 0.1 05 0.1 02 0.1 04 0.2 02 0.1
e
e
e
e
e
e
5f
03 0.2 02 0.1 03 0.1 02 0.1 04 0.1 03 0.1
5g
5h
5i
5j
5k
5l
e
e
e
e
e
e
03 0.2 01 0.1 04 0.2 02 0.2 03 0.1
06 0.2 04 0.1 05 0.2 03 0.2 06 0.1 04 0.2
05 0.1 02 0.1 04 0.2 02 0.1 05 0.1 02 0.2
e
Fig. 6. Growth inhibition activity of active compounds (at a concentration of 62.5 mg/
mL) against VERO cell line.
e
e
e
e
e
e
04 0.1 02 0.1 04 0.1 03 0.2 03 0.2 01 0.3
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
5x
5y
5z
5aa
5ab
5ac
e
e
e
e
e
e
05 0.1 03 0.2 04 0.1 02 0.2 05 0.1 02 0.1
04 0.2 02 0.2 04 0.1 01 0.1 05 0.1 02 0.2
4.2. Synthesis
e
e
e
e
e
e
e
e
e
e
e
e
4.2.1. Synthesis of 5-amino-2-methyl-1, 3, 4-thiadiazole (2a)
05 0.1 02 0.2 03 0.2 01 0.1 06 0.1 04 0.2
e
To the thiosemicarbazide (15.0 g, 164.58 mmol), acetyl chloride
(28.54 mL, 329.16 mmol) was added slowly and the mixture was
stirred for 4 h at RT. To the reaction mixture, ice cold water was
added and the solid obtained was filtered off. The solid was then
added to ice cold water. To this slurry, a solution of 50% NaOH was
added till the pH of the solution becomes basic. The solid obtained
was filtered off and dried under vacuum to get the compound (2a)
as white solid. Yield: 14.4 g, 76%; m.p: 269e270 ꢀC; FTIR (ATR,
cmꢃ1): 3288, 3105, 2925, 1614, 689; 1H NMR (400 MHz, DMSO-d6)
e
e
e
e
e
02 0.1 01 0.1 03 0.1 02 0.2 02 0.1 01 0.2
03 0.1 02 0.2 04 0.1 03 0.2 02 0.1 01 0.2
e
e
e
e
e
e
07 0.1 05 0.2 08 0.2 06 0.1 09 0.2 07 0.2
06 0.1 05 0.1 07 0.2 06 0.1 06 0.1 04 0.1
e
e
e
e
e
e
03 0.1 01 0.2 05 0.1 03 0.2 04 0.1 02 0.2
e
e
e
e
e
e
e
e
e
e
e
e
e
03 0.1 01 0.2 02 0.1
04 0.1 01 0.2
d
(ppm): 2.18 (s, 3H, CH3), 13.05 (br s, 2H, NH2); 13C NMR (100 MHz,
DMSO-d6) (ppm): 10.9, 148.8, 165.7; ESI-MS (m/z) 116.02
FZ: Flucanazole (anti-fungal standard); e: inhibition not detected; control:
dimethylsulfoxide.
d
(M þ H) þ; calculated for C3H5N3S; C, 31.9; H, 4.38; N, 36.49; S,
27.84. Found: C, 31.89; H, 4.41; N, 36.42; S, 27.44.
spectra were recorded using a Waters micro mass Q-Tofmicro
spectrometer with an ESI source. X-ray intensity data for com-
pounds 4d and 5aa was collected at room temperature using a
Bruker smart Apex Duo single crystaleray diffractometer equipped
with dual system (compact copper micro-porous source plus mo-
lybdenum scaled tube source) CCD detector. Monochromatic Mo-
4.2.2. General procedure for the synthesis of 5-subsituted-1,3,4-
thiadiazole-2-yl amine (2b-f)
4.2.2.1. 5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-amine
(2b). A
mixture of 4-fluoro benzoic acid (5.0 g, 35.70 mmol), thio-
semicarbazide (3.25 g, 35.70 mmol) and POCl3 (9.25 ml) was heated
to 75 ꢀC and maintained same temperature for 30 min under
Ka
radiation (
l
¼ 0.71073 Å) was used for the measurement.
Fig. 5. Antioxidant activity of compounds 5a-ac.

56
J. Ramprasad et al. / European Journal of Medicinal Chemistry 95 (2015) 49e63
stirring. The reaction mixture cooled to room temperature, water
(55 ml) was added and refluxed for 4 h. After cooling, the mixture
was basified with 50% NaOH to pH 8 by the drop wise addition
under stirring. The obtained solid was filtered and recrystallized
from ethanol to give the target compound 2b as a colorless solid.
Yield: 5.69 g, 82%; m.p: 230e231 ꢀC; FTIR (ATR, cmꢃ1): 3346, 3251,
bromide (4.032 g, 17.39 mmol) was refluxed in dry ethanol (20 mL)
for 24 h. The excess of solvent was removed under reduced pres-
sure and the solid hydrobromide salt was suspended in water, and
neutralized by the aqueous sodium carbonate solution to get free
base. It was then filtered, washed with water, dried, and recrys-
tallized from ethanol to get compound 3a as light yellow solid.
Yield: 3.4 g, 80%; m.p: 184e185 ꢀC; FTIR (ATR, cmꢃ1): FTIR (ATR,
cmꢃ1): 3066, 2938, 1592, 1504, 1466, 843, 741, 682; 1H NMR
2934, 1633, 1063, 683; 1H NMR (400 MHz, DMSO-d6)
d (ppm):
7.29e7.33 (t, J ¼ 8.77 Hz, 2H, AreH), 7.41 (s, 2H), 7.79e7.83 (dd,
J ¼ 5.46 Hz, 8.59 Hz, 2H, AreH); 13C NMR (100 MHz, DMSO-d6)
(400 MHz, CDCl3)
AreH),7.74 (dd, J ¼ 1.8, 6.6 Hz, 2H, AreH), 7.94 (s, 1H, H-5 imid-
azole), 13C NMR (100 MHz, CDCl3)
(ppm): 17.86, 102.29, 126.24,
d
(ppm): 2.72 (s, 3H), 7.37 (dd, J ¼ 2.0, 6.8 Hz, 2H,
d
(ppm): 118.13, 129.43, 130.21, 154.32, 157.28, 168.10; ESI-MS: m/z
196.4 [MþH] þ; calculated for C8H6FN3S; C, 49.22; H, 3.10; N, 21.52;
d
S, 16.43. Found: C, 49.20; H, 3.11; N, 21.52; S, 16.44.
128.87, 132.48, 1 33.09, 144.93, 145.86, 159.59; ESI-MS (m/z) 249.9
(M þ H)þ; calculated for C11H8ClN3S; C, 52.91; H, 3.23; N, 16.83; S,
12.84. Found: C, 52.98; H, 3.28; N, 16.92; S, 12.54.
4.2.2.2. 5-(4-Methylphenyl)-1,3,4-thiadiazol-2-amine
(2c).
Compound 2c was synthesized by following the above procedure
with 4-methyl benzoic acid (5.0 g, 36.76 mmol), thiosemicarbazide
(3.35 g, 36.76 mmol) and POCl3 (9.25 ml). White solid; yield: 6.31 g,
90%; m.p: 213e214 ꢀC; FTIR (ATR, cmꢃ1): 3278, 3103, 2955, 1611,
Compounds 3b-m were synthesized by following the above
procedure by reacting 2 g of 5-subsituted-1,3,4-thiadiazole-2-yl
amine with same equivalent of corresponding phenacyl bromide
derivative. The structural characterization data for compounds 3b-
m are given below.
690; 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 2.33 (s, 3H), 7.25e7.27
(d, J ¼ 8.00 Hz, 2H, AreH), 7.36 (s, 2H), 7.63e7.65 (d, J ¼ 8.12 Hz, 2H,
AreH); 13C NMR (100 MHz, DMSO-d6)
(ppm): 21.35, 126.72,
d
4.2.3.2. 2-Methyl-6-(4-methylphenyl)imidazo[2,1-b][1,3,4]thiadia-
zole (3b). Yellow solid, yield: 3.26 g, 82.0%; m.p: 211e212 ꢀC; FTIR
(ATR, cmꢃ1): 3067, 2924, 1592, 1504, 1477, 842, 668; 1H NMR
128.80, 130.11, 139.76, 156.95, 168.65; ESI-MS: m/z 192.2 [MþH]þ.
calculated for C9H9N3S; C, 56.22; H, 4.74; N, 21.97; S, 16.77. Found:
C, 56.20; H, 4.74; N, 21.90; S, 16.74.
(400 MHz, CDCl3):
d
2.37(s, CH3), 2.71 (s, CH3), 7.21 (d, J ¼ 7.9 Hz, 2H,
AreH),7.70 (d, J ¼ 7.9 Hz, 2H, AreH) 7.92 (s, H-5 imidazole, 1H), 13C
4.2.2.3. 5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-amine
(2d). The
NMR (100 MHz, CDCl3) d (ppm): 17.82, 21.26, 108.76, 124.94, 129.41,
above procedure was followed for 4-chloro benzoic acid (5.0 g,
32.05 mmol), thiosemicarbazide (2.92 g, 32.05 mmol) and POCl3
(9.25 ml) to afford compound 2d as White solid. Yield: 5.20 g, 77%;
m.p: 210e211 ꢀC; FTIR (ATR, cmꢃ1): 3258, 3093, 1633, 751, 684; 1H
131.14, 137.27, 145.53, 146.17, 159.14; ESI-MS m/z 230.0 [MþH]þ;
calculated for C12H11N3S; C, 62.86; H, 4.84; N,18.33; S, 13.98. Found:
C, 62.98; H, 4.78; N, 18.40; S, 14.02.
NMR (400 MHz, DMSO-d6)
d
(ppm): 7.47 (s, 2H), 7.52e7.54 (d,
4.2.3.3. 6-(4-Methoxyphenyl)-2-methylimidazo[2,1-b][1,3,4]thiadia-
zole (3c). Yellow solid, yield: 3.44 g, 81%; m.p:192e193 ꢀC; FTIR
(ATR, cmꢃ1): 3043, 2935, 1540, 1462, 1239, 1023, 830, 670; 1H NMR
J ¼ 6.55 Hz, 2H, AreH), 7.76e7.78 (d, J ¼ 6.24 Hz, 2H, AreH); 13C
NMR (100 MHz, DMSO-d6)
d (ppm):128.38, 129.63, 130.34, 134.43,
155.59, 168.30; ESI-MS: m/z 212.6 [MþH]þ. calculated for
C8H6ClN3S; C, 45.39; H, 2.86; N, 19.85; S, 15.15. Found: C, 45.30; H,
2.88; N, 19.90; S, 15.14.
(400 MHz, CDCl3)
(m, 2H, ArH),7.73 (d, J ¼ 8.8 Hz, 2H, ArH),7.871 (s, H-5 imidazole,
1H), 13C NMR (100 MHz, CDCl3)
(ppm): 17.52, 54.60, 109.77,
d (ppm): 2.70 (s, CH3), 3.84 (s, OCH3), 6.95e6.97
d
125.65, 128.42, 131.15, 132.78, 145.23, 146.20, 159.40; ESI-MS m/z
245.9 [MþH]þ; calculated for C12H11N3OS; C, 58.76; H, 4.52; N,
17.13; S, 13.07. Found: C, 58.86; H, 4.60; N, 17.12; S, 13.14.
4.2.2.4. 5-(2-Chlorophenyl)-1,
3,
4-thiadiazol-2-amine
(2e).
Compound 2e was synthesized by following the above procedure
for the 2-chloro benzoic acid (5.0 g, 32.05 mmol), thio-
semicarbazide (2.92 g, 32.05 mmol) and POCl3 (9.25 ml). Yield:
5.41 g, 80% (white solid); m.p: 220e221 ꢀC; FTIR (ATR, cmꢃ1): 3269,
4.2.3.4. 6-(4-Methoxyphenyl)-2-(4-methylphenyl)imidazo[2,1-b]
[1,3,4]thiadiazole (3d). Yellow solid, yield: 2.62 g, 78.0%;
m.p:177e178 ꢀC; FTIR (ATR, cmꢃ1): 3042, 2934, 1540, 1461, 1234,
3098, 1636, 748, 694; 1H NMR (400 MHz, DMSO-d6)
d
(ppm):
7.97e8.01 (m, 2H, AreH), 7.57e7.60 (m, 1H, AreH), 7.44e7.47 (m,
1H, AreH), 7.42 (s, 2H); 13C NMR (100 MHz, DMSO-d6)
(ppm):
1023, 830, 671; 1H NMR (400 MHz, CDCl3)
d (ppm): 2.43 (s, 3H,
d
CH3), 3.85 (s, 3H, OCH3), 6.96 (d, J ¼ 8.8 Hz, 2H, ArH),7.29 (d,
126.50, 128.45, 129.32, 130.21, 132.01, 136.81, 155.70, 168.41; ESI-
MS: m/z 212.10 [MþH] þ. calculated for C8H6ClN3S; C, 45.39; H,
2.86; N, 19.85; S, 15.15. Found: C, 45.31; H, 2.89; N, 19.85; S, 15.16.
J ¼ 22.82 Hz, 2H, AreH), 7.75e7.78 (m, 4H, AreH), 7.94 (s,
imidazole-H, 1H); 13C NMR (100 MHz, CDCl3)
d (ppm): 21.57, 55.34,
108.35, 114.17, 126.37, 126.61, 126.72, 127.60, 129.95, 142.18, 144.97,
146.39, 159.26, 161.28; ESI-MS m/z 322.0 [MþH]þ; calculated for
4.2.2.5. 5-Phenyl-1, 3, 4-thiadiazol-2-amine (2f). Compound 2f was
synthesized by following the above procedure for benzoic acid
(5.0 g, 40.32 mmol), thiosemicarbazide (3.67 g, 40.321 mmol) and
POCl3 (9.3 ml). Yield: 5.56 g, 78.0% (white solid); m.p:224e225 ꢀC;
FTIR (ATR, cmꢃ1): FTIR (ATR, cmꢃ1): 3288, 3100, 1611, 690; 1H NMR
C18H15N3OS; C, 67.27; H, 4.70; N, 13.07; S, 9.98. Found: C, 67.15; H,
4.75; N, 13.10; S, 10.01.
4.2.3.5. 6-(4-Chlorophenyl)-2-p-tolylimidazo[2,1-b][1,3,4]thiadiazole
(3e). Off white solid, yield: 2.72 g, 80%; m.p: 234e235 ꢀC; FTIR
(ATR, cmꢃ1): 3068, 2924, 1592, 1511, 1474, 840, 729, 670; 1H NMR
(400 MHz, DMSO-d6)
d (ppm): 7.40 (s, 2H), 7.43e7.49 (dd,
J ¼ 6.38 Hz, 14.00 Hz, 2H, AreH), 7.74e7.76 (d, J ¼ 6.94 Hz, 2H,
(400 MHz, CDCl3) d (ppm): 2.45 (s, CH3), 7.38 (m, 2H, AreH), 7.74
AreH); 13C NMR (100 MHz, DMSO-d6)
d
(ppm): 126.71, 128.70,
(dd, J ¼ 2.0, 6.8 Hz, 2H, AreH), 7.96 (s, 1H, imidazole-H); 13C NMR
129.98, 138.56, 156.94, 168.45; ESI-MS: m/z 178.3 [MþH]þ. calcu-
lated for C8H7N3S; C,54.22; H, 3.98; N, 23.71; S, 18.09. Found: C,
54.21; H, 3.90; N, 23.80; S, 18.10.
(100 MHz, CDCl3) d (ppm): 17.82, 102.30, 126.30, 128.78, 128.87,
129.21, 132.48, 133.10, 141.21, 144.89, 158.54; ESI-MS m/z 326.0
[MþH]þ; calculated for C17H12ClN3S; C, 62.67; H, 3.71; N, 12.90; S,
9.84. Found: C, 62.65; H, 3.75; N, 12.85; S, 9.86.
4.2.3. General procedure for the synthesis of 2-subsituted-6-
arylimidazo[2,1-b][1,3,4] thiadiazoles (3a-m)
4.2.3.1. 6-(4-Chlorophenyl)-2-methyl imidazo[2,1-b][1,3,4]thiadia-
zole (3a). A mixture of 2a (2 g, 17.39 mmol) and 4-chloro phenacyl
4.2.3.6. 6-(4-Fluorophenyl)-2-p-tolylimidazo[2,1-b][1,3,4]thiadiazole
(3f). White solid, yield: 2.55 g, 79.0%; m.p: 228e230 ꢀC; FTIR (ATR,
cmꢃ1): 3067, 2924, 1539, 1464, 1062, 827, 671; 1H NMR (400 MHz,

J. Ramprasad et al. / European Journal of Medicinal Chemistry 95 (2015) 49e63
57
CDCl3)
d
(ppm): 2.42 (s, 3H, CH3),7.431 (d, J ¼ 8.4 Hz, 2H, AreH),
3.56; N, 12.26; S, 9.40.
7.58e7.60 (m, 2H, AreH), 7.95 (d, J ¼ 7.9 Hz, AreH), 8.12e8.15 (m,
2H, AreH),7.98 (s, 1H, imidazole-H); 13C NMR (100 MHz, CDCl3)
4.2.3.12. 6-(4-Methoxyphenyl)-2-phenylimidazo[2,1-b][1,3,4]thia-
diazole (3l). White solid, yield: 2.84 g, 82.0%; m.p: 219e220 ꢀC;
FTIR (ATR, cmꢃ1): 3066, 2924, 1593, 1503, 1465, 1244, 1028, 840,
d
(ppm): 21.57, 109.02, 115.61, 115.82, 126.72, 126.79, 126.87, 127.42,
130.02, 142.46, 142.49, 145.20, 161.25; ESI-MS m/z 310.1 [MþH]þ;
calculated for C17H12FN3S; C, 66.00; H, 3.91; N, 13.58; S, 10.36.
Found: C, 66.12; H, 3.95; N, 13.54; S, 10.41.
668; 1H NMR (400 MHz, CDCl3)
d (ppm): 3.79 (s, 3H, OCH3), 7.02 (d,
J ¼ 8.3 Hz, 2H, AreH), 7.32e7.42 (m, 3H, AreH), 7.43e7.49 (m, 2H),
7.61 (d, J ¼ 8.8 Hz, 2H) 7.87 (s,1H, imidazole-H); 13C NMR (100 MHz,
4.2.3.7. 2-(4-Fluorophenyl)-6-(4-methoxyphenyl)imidazo[2,1-b]
[1,3,4]thiadiazole (3g). Light yellow solid, yield: 2.47 g, 75%; m.p:
203e204 ꢀC; FTIR (ATR, cmꢃ1): 3063, 2935, 1539, 1463, 1243, 1066,
CDCl3) d (ppm): 54.60, 108.27, 124.65, 128.22, 128.84, 129.10, 130.61,
131.21, 131.75, 132.78, 142.55, 150.15, 158.40; ESI-MS (m/z) 308.10
(M þ H)þ; calculated for C17H13N3OS; C, 66.43; H, 4.26; N, 13.67; S,
10.43. Found: C, 66.45; H, 4.24; N, 13.66; S, 10.42.
1022, 828, 671; 1H NMR (400 MHz, CDCl3)
d (ppm): 3.81 (s, 3H,
OCH3), 7.22e7.30 (m, 4H, ArH), 7.64e7.68 (m, 2H, ArH), 7.85 (d,
J ¼ 7.4 Hz, 2H, AreH), 7.92 (s, imidazole-H); 13C NMR (100 MHz,
4.2.3.13. 6-(4-Fluorophenyl)-2-methylimidazo[2,1-b][1,3,4]thiadia-
zole (3m). Light yellow solid, yield: 2.56 g, 79.0%; m.p:179e180 ꢀC;
FTIR (ATR, cmꢃ1): 3068, 2926, 1597, 1538, 1470, 1064, 834, 670; 1H
CDCl3) d (ppm): 54.78, 109.77, 115.61, 125.65, 126.38, 128.45, 130.97,
131.15, 132.80, 145.33, 146.21, 157.28, 168.10; ESI-MS m/z 326.10
[MþH]þ; calculated for C17H12FN3OS; C, 62.76; H, 3.72; N, 12.91; S,
9.86. Found: C, 62.66; H, 3.75; N, 12.94; S, 9.81.
NMR (400 MHz, CDCl3)
AreH),7.741 (d, J ¼ 7.6 Hz, 2H, AreH), 7.96 (s, 1H, H-5 imidazole);
13C NMR (100 MHz, CDCl3)
(ppm): 17.86, 102.29, 126.24, 128.87,
d (ppm): 2.73 (s, 3H),7.38 (m, 2H,
d
4.2.3.8. 2-(4-Chlorophenyl)-6-p-tolylimidazo[2,1-b][1,3,4]thiadiazole
(3h). Yellow solid, yield: 2.40 g,78.2%; m.p: 239e240 ꢀC; FTIR (ATR,
cmꢃ1): 3087, 2919, 1593, 1513, 1460, 828, 761, 668; 1H NMR
132.48, 133.09, 144.93, 145.86, 159.59; ESI-MS (m/z) 234.1
(M þ H)þ; calculated for C11H8FN3S; C, 56.64; H, 3.46; N, 18.01; S,
13.75. Found: C, 56.66; H, 3.44; N, 18.06; S, 13.72.
(400 MHz, CDCl3)
AreH), 7.30 (m, 2H, AreH), 7.70e7.66 (m, 4H, AreH), 7.94 (s,
imidazole-H, 1H); 13C NMR (100 MHz, CDCl3)
(ppm): 21.26,
108.78, 124.89, 129.42, 128.89, 130.56, 131.14, 136.61, 137.27, 138.42,
145.54, 146.27, 160.21; ESI-MS (m/z) 326.04 (M þ H)þ; calculated
for C17H12ClN3S; C, 62.67; H, 3.71; N, 12.90; S, 9.84. Found: C, 62.66;
H, 3.73; N, 12.84; S, 9.83.
d
(ppm): 2.34 (s, 3H, CH3), 7.21 (d, J ¼ 7.6 Hz, 2H,
4.2.4. General procedure for the preparation of 2-subsituted-6-
arylimidazo [2,1-b][1,3,4]-thiadiazole-5-carbaldehydes (4 a-m)
4.2.4.1. 6-(4-Chlorophenyl)-2-methylimidazo [2,1-b][1,3,4]thiadia-
zole-5-carbaldehyde(4a). Vilsmeier-Haack salt was synthesized by
adding POCl3 (1.49 mL, 16.06 mmol) drop-wise to a dry RB con-
taining DMF (1.23 mL, 16.06 mmol) maintaining temperature at
0e5 ꢀC under N2 atmosphere. Later, a solution of 3a (2.0 g,
8.032 mmol) in 20 mL of DMF was added to the resulting complex
at a stretch. The resulting solution was stirred at 60 ꢀC for about 6 h.
The container was cooled to room temperature and quenched to ice
cold water while stirring. The solid obtained was filtered, washed
with excess of water and then purified by column chromatographic
technique using ethyl acetate and hexane (3:7) system to give
compound 4a as white solid. Yield:1.9 g, 85%, m.p: 174e175 ꢀC; FTIR
(ATR, cmꢃ1): 3066, 2924, 1679, 1592, 1504, 1479, 843, 728, 671; 1H
d
4.2.3.9. 2-(4-Chlorophenyl)-6-(4-methoxyphenyl)imidazo[2,1-b]
[1,3,4]thiadiazole (3i). Yellow solid, yield: 2.54 g,79.0%; m.p:
159e160 ꢀC; FTIR (ATR, cmꢃ1): 3067, 2934, 1539, 1462, 1239, 1023,
828, 758, 671; 1H NMR (400 MHz, CDCl3)
d (ppm): 3.81 (s, 3H, CH3),
7.23e7.33 (m, 4H, AreH), 7.66e7.75 (m, 2H, AreH),7.96 (d,
J ¼ 7.9 Hz, 2H, AreH),7.93 (s, imidazole-H); 13C NMR (100 MHz,
CDCl3) d (ppm): 54.80, 108.77, 125.75, 128.42, 128.94, 129.10, 130.61,
131.85, 132.78, 136.56, 143.65, 150.15, 159.40; ESI-MS (m/z) 342.0
(M þ H)þ; calculated for C17H12ClN3OS; C, 59.73; H, 3.54; N, 12.29;
S, 9.38. Found: C, 59.76; H, 3.54; N, 12.32; S, 9.33.
NMR (400 MHz, CDCl3)
6.6 Hz, 2H, AreH),7.82 (d, J ¼ 8.3 Hz, 2H, AreH),10.35 (s, CHO, 1H),
13CNMR (100 MHz, CDCl3)
(ppm): 18.09, 124.04, 129.09, 130.28,
d
(ppm): 2.85 (3H, CH3), 7.48 (dd, J ¼ 1.7,
d
130.76, 136.05, 151.20, 154.51, 162.28, 177.18; ESI-MS m/z 277.9
[MþH]þ;. calculated for C12H8ClN3OS; C, 51.90; H, 2.90; N, 15.13; S,
11.55. Found: C, 51.86; H, 2.88; N, 15.15; S, 11.44.
Compounds 4b-m were synthesized by following the above
procedure and structural characterization data for the compounds
are given below.
4.2.3.10. 2-(4-Chlorophenyl)-6-(4-fluorophenyl)imidazo[2,1-b][1,3,4]
thiadiazole (3j). White solid, yield: 2.52 g, 81.2%; m.p: 225e226 ꢀC;
FTIR (ATR, cmꢃ1): 3066, 2925, 1594, 1503, 1474, 1087, 839, 729, 670;
1H NMR (400 MHz, CDCl3)
d
(ppm): 7.45 (d, J ¼ 8.3 Hz, 2H, AreH),
7.66e7.70 (m, 4H, AreH), 8.09 (d, J ¼ 8.0 Hz, 2H, AreH), 7.96 (1H, s,
imidazole-H); 13C NMR (100 MHz, CDCl3)
(ppm): 108.26, 115.34,
d
118.21, 128.56, 130.10, 131.21, 137.21, 149.45, 154.33, 161.45, 162.44,
164.21; ESI-MS (m/z) 330.0 (M þ H)þ; calculated for C16H9ClFN3S;
C, 58.27; H, 2.75; N, 12.74; S, 9.72. Found: C, 58.26; H, 2.76; N, 12.72;
S, 9.73.
4.2.4.2. 2-Methyl-6-(4-methylphenyl)imidazo[2,1-b][1,3,4]thiadia-
zole-5-carbaldehyde (4b). Light yellow solid, yield: 1.84 g, 82.2%;
m.p:145e146 ꢀC; FTIR (ATR, cmꢃ1): 3068, 2924, 1678, 1592, 1504,
1478, 842, 670; 1H NMR (400 MHz, CDCl3)
d
(ppm): 2.48(s, CH3),
2.84 (s, CH3), 7.28 (d, J ¼ 7.9 Hz, 2H, AreH),7.71 (d, J ¼ 7.9 Hz, 2H,
AreH) 10.00 (s, CHO, 1H),13C NMR (100 MHz, CDCl3)
(ppm): 18.08,
4.2.3.11. 2-(2-Chlorophenyl) -6- (4-methoxyphenyl)imidazo[2,1-b]
[1,3,4]thiadiazole (3k). Off white solid, yield: 2.30 g, 75.0%; m.p:
213e214 ꢀC; FTIR (ATR, cmꢃ1): 3068, 2921, 1594, 1503, 1476, 1245,
d
21.41, 123.98, 129.06, 129.44, 129.63, 140.10, 151.45, 156.76, 161.85,
177.61; ESI-MS: m/z 257.9 [MþH]þ; calculated for C13H11N3OS; C,
60.68; H, 4.31; N, 16.33; S, 12.46. Found: C, 60.66; H, 4.31; N, 16.35;
S, 12.44.
1024, 840, 752, 670; 1H NMR (400 MHz, CDCl3)
d (ppm): 3.81 (s, 3H,
OCH3), 7.02 (d, J ¼ 8.3 Hz, 2H, AreH),7.62 (d, J ¼ 6.6 Hz, 1H, AreH),
7.67 (s, 1H, AreH), 7.71 (d, J ¼ 7.5 Hz, 1H, AreH),7.84 (d, J ¼ 8.7 Hz,
1H, AreH), 8.67 (d, J ¼ 8.3 Hz, 2H, AreH), 7.97 (s, 1H, imidazole-H);
13C NMR (100 MHz, CDCl3)
d
(ppm): 54.80, 108.77, 115.21, 125.75,
4.2.4.3. 6-(4-Methoxyphenyl)-2-methylimidazo[2,1-b][1,3,4]thiadia-
zole-5-carbaldehyde (4c). Off white solid, yield: 1.89 g, 85%;
m.p:161e162 ꢀC; FTIR (ATR, cmꢃ1): 3044, 2935, 1679, 1540, 1462,
128.42, 128.94, 129.10, 130.61, 131.85, 132.51, 132.82, 136.46, 143.65,
150.25, 160.40; ESI-MS (m/z) 342.0 (M þ H)þ; calculated for
C
17H13N3OS; C, 59.73; H, 3.54; N, 12.29; S, 9.38. Found: C, 59.69; H,
1238, 1024, 830, 671; 1H NMR (400 MHz, CDCl3)
d (ppm): 2.84 (s,

58
J. Ramprasad et al. / European Journal of Medicinal Chemistry 95 (2015) 49e63
3H, CH3), 3.88 (s, 3H, OCH3), 7.02e7.04 (m, 2H, AreH),7.7e7.8 (m,
2H, AreH), 9.99 (s, 1H, CHO); 13C NMR (100 MHz, CDCl3)
(ppm):
Found: C, 61.25; H, 3.42; N, 11.91; S, 9.16.
d
17.82, 55.60, 109.97, 125.95, 128.62, 131.25, 133.1, 145.83, 146.60,
159.70, 177.41; ESI-MS: m/z 274.0 [MþH]þ; calculated for
4.2.4.9. 2-(4-Chlorophenyl)-6-(4-methoxyphenyl)imidazo[2,1-b]
[1,3,4]thiadiazole-5-carbaldehyde (4i). White solid, yield:1.71 g,
79.2%; m.p: 196e197 ꢀC; FTIR (ATR, cmꢃ1): 3043, 2935, 1679, 1539,
C
13H11N3O2S; C, 57.13; H, 4.06; N, 15.37; S, 11.73. Found: C, 57.16; H,
4.01; N, 15.35; S, 11.74.
1462, 1239, 1023, 828, 758, 671; 1H NMR (400 MHz, CDCl3)
d (ppm):
3.821 (s, 3H, CH3), 7.25e7.34 (m, 4H, AreH), 7.68e7.78 (m, 2H,
4.2.4.4. 6-(4-Methoxyphenyl)-2-(4-methylphenyl)imidazo[2,1-b]
[1,3,4]thiadiazole-5- carbaldehyde (4d). Light yellow solid, yield:
1.73 g, 80%; m.p: 195e196 ꢀC; FTIR (ATR, cmꢃ1): 3043, 2935, 1679,
1540, 1462, 1238, 1024, 830, 671; 1H NMR (400 MHz, CDCl3)
AreH), 7.98 (d, J ¼ 7.4 Hz, 2H, AreH), 10.22 (s, 1H, CHO); 13C NMR
(100 MHz, CDCl3)
d (ppm): 54.84, 116.25, 126.15, 128.52, 128.84,
129.25, 130.81, 131.95, 133.81, 136.66, 143.15, 150.25, 160.21, 177.21;
ESI-MS (m/z) 370.1 (M þ H)þ; calculated for C18H12ClN3O2S; C,
58.46; H, 3.27; N, 11.36; S, 8.67. Found: C, 58.45; H, 3.25; N, 11.35; S,
8.66.
d
(ppm): 2.45 (s, 3H, CH3), 3.89 (s, 3H, OCH3), 7.03e7.05 (m, 2H,
AreH), 7.33 (d, J ¼ 7.9 Hz, 2H, AreH),7.85e7.91 (m, 4H, AreH), 10.10
(s, 1H, CHO), 13C NMR (100 MHz, CDCl3)
(ppm): 21.65, 55.44,
d
114.28, 123.71, 124.91, 126.82, 127.14, 130.06, 130.55, 143.06, 150.45,
156.02, 161.10, 163.85, 177.44; ESI-MS: m/z [MþH]þ 350.0; calcu-
lated for C19H15N3O2S; C, 65.31; H, 4.33; N, 12.03; S, 9.18. Found: C,
65.28; H, 4.32; N, 12.05; S, 9.20.
4.2.4.10. 2-(4-Chlorophenyl)-6-(4-fluorophenyl)imidazo[2,1-b][1,3,4]
thiadiazole-5-carbaldehyde (4j). Off white solid, yield: 1.66 g, 76.6%;
m.p: 189e190 ꢀC; FTIR (ATR, cmꢃ1): 3068, 2921, 1676, 1594, 1503,
1476, 1088, 839, 729, 668; 1H NMR (400 MHz, CDCl3)
d (ppm): 7.48
4.2.4.5. 6-(4-Chlorophenyl)-2-p-tolylimidazo[2,1-b][1,3,4]thiadia-
zole-5-carbaldehyde (4e). Light brown solid, yield: 1.69 g, 78%; m.p:
209e210 ꢀC; FTIR (ATR, cmꢃ1): 3068, 2924, 1679, 1592, 1504, 1476,
(d, J ¼ 8.6 Hz, 2H, AreH), 7.72e7.68 (m, 4H, AreH), 8.12 (d,
J ¼ 8.3 Hz, 2H, AreH), 10.20 (s, 1H, CHO); 13C NMR (100 MHz, CDCl3)
d
(ppm):115.84,116.06, 128.39, 129.80,131.00,131.09,138.74, 150.15,
840, 729, 669; 1H NMR (400 MHz, CDCl3)
d (ppm): 2.48 (s, 3H,
154.73, 162.67, 162.82, 165.17, 177.18; ESI-MS (m/z) 358.1 (M þ H)þ;
calculated for C17H9ClFN3OS; C, 57.07; H, 2.54; N, 11.74; S, 8.96.
Found: C, 57.15; H, 2.52; N, 11.71; S, 8.96.
CH3),7.39 (m, 2H, AreH), 7.05e7.08 (m, 2H, AreH), 7.50 (d,
J ¼ 7.5 Hz, 2H, AreH),7.75 (d, J ¼ 8.72 Hz 2H, AreH),10.18 (s,
1H,CHO); 13C NMR (100 MHz, CDCl3)
d (ppm): 17.86, 115.21, 116.21,
127.30, 128.18, 128.97, 129.21, 131.15, 132.68, 134.10, 142.31, 145.39,
159.54, 162.51, 177.83; ESI-MS m/z 354.0 [MþH]þ; calculated for
4.2.4.11. 2-(2-Chlorophenyl)-6-(4-methoxyphenyl)imidazo[2,1-b]
[1,3,4]thiadiazole-5-carbaldehyde (4k). Off white solid, yield: 1.69 g,
78.2%; m.p: 178e180 ꢀC; FTIR (ATR, cmꢃ1): 3068, 2921, 1676, 1594,
1503, 1244, 1028, 1476, 839, 752, 671; 1H NMR (400 MHz, CDCl3)
C
18H12ClN3OS; C, 61.10; H, 3.42; N, 11.88; S, 9.06. Found: C, 61.12; H,
3.42; N, 11.90; S, 9.09.
4.2.4.6. 6-(4-Fluorophenyl)-2-p-tolylimidazo[2,1-b][1,3,4]thiadia-
zole-5-carbaldehyde (4f). White solid, yield: 1.78 g, 82.0%; m.p:
193e194 ꢀC; FTIR (ATR, cmꢃ1): 3066, 2925, 1678, 1539, 1462, 1061,
d
(ppm):3.81 (s, 3H, OCH3),7.026 (d, J ¼ 8.7 Hz, 2H, AreH), 7.623 (d,
J ¼ 6.6 Hz, 1H, AreH),7.67 (s, 1H, AreH) 7.72 (d, J ¼ 8.4 Hz, 1H,
AreH),7.84 (d, J ¼ 8.5 Hz, 1H, AreH), 8.70 (d, J ¼ 8.3 Hz, 2H, AreH),
827, 671; 1H NMR (400 MHz, CDCl3)
d (ppm): 2.46 (s, 3H, CH3), 7.53
10.28 (s, 1H, CHO); 13C NMR (100 MHz, CDCl3)
d (ppm): 54.80,
(m, 2H, AreH), 7.59 (d, J ¼ 8.4 Hz, 2H, ArH), 7.98 (d, J ¼ 7.8 Hz, 2H,
115.21, 116.81, 126.35, 128.42, 128.94, 129.10, 130.81, 131.85, 132.51,
133.12, 137.16, 143.85, 151.25, 162.40, 177.38; ESI-MS (m/z) 370.1
(M þ H)þ; calculated for C18H12ClN3O2S; C, 58.46; H, 3.27; N, 11.36;
S, 8.67. Found: C, 58.44; H, 3.30; N, 11.41; S, 8.68.
ArH), 8.14e8.17 (m, 2H, ArH), 10.25 (s, CHO); 13C NMR (100 MHz,
CDCl3)
d (ppm): 21.64, 115.76, 115.98, 126.70, 127.15, 130.11, 130.97,
131.05, 143.26, 150.11, 154.25, 162.61, 164.34, 165.10, 177.23; ESI-MS
m/z 338.1 [MþH]þ; calculated for C18H12FN3OS; C, 64.08; H, 3.59;
N, 12.46; S, 9.50. Found: C, 64.12; H, 3.52; N, 12.45; S, 9.56.
4.2.4.12. 6-(4-Methoxyphenyl)-2-phenylimidazo[2,1-b][1,3,4]thia-
diazole-5-carbaldehyde (4l). White solid, yield: 1.63 g, 75.0%; m.p:
179e180 ꢀC; FTIR (ATR, cmꢃ1): 3067, 2925, 1678, 1594, 1503, 1464,
4.2.4.7. 2-(4-Fluorophenyl)-6-(4-methoxyphenyl)
imidazo[2,1-b]
[1,3,4]thiadiazole-5-carbaldehyde (4g). Light yellow solid, yield:
1.78 g, 82.0%; m.p: 189e190 ꢀC; FTIR (ATR, cmꢃ1): 3043, 2935, 1677,
1539, 1463, 1244, 1062, 1023, 828, 671; 1H NMR (400 MHz, CDCl3)
1244,1028, 1476, 839, 668; 1H NMR (400 MHz, CDCl3)
d (ppm): 3.81
(s, 3H, OCH3), 7.10 (d, J ¼ 8.3 Hz, 2H, AreH), 7.32e7.42 (m, 3H,
d
(ppm): 3.82 (s, 3H, OCH3), 7.26e7.30 (m, 4H, ArH), 7.66e7.70 (m,
AreH), 7.45e7.52 (m, 2H, ArH), 7.63 (d, J ¼ 8.8 Hz, 2H),10.18 (s, 1H,
2H, ArH), 7.87 (d, J ¼ 7.9 Hz, 2H, AreH), 10.21 (s, 1H, CHO); 13C NMR
CHO); 13CNMR (100 MHz, CDCl3) (ppm): 13C NMR (100 MHz,
d
(100 MHz, CDCl3)
d (ppm): 54.88, 115.71, 116.21, 125.95, 126.38,
CDCl3)
d (ppm): 54.60, 116.21, 125.15, 127.32, 128.94, 129.10, 130.81,
129.45, 130.97, 131.75, 132.98, 145.33, 146.31, 157.28, 162.61, 168.10,
177.34; ESI-MS m/z 354.0 [MþH]þ; calculated for C18H12FN3O2S; C,
61.18; H, 3.42; N, 11.89; S, 9.07. Found: C, 61.22; H, 3.39; N, 11.89; S,
9.06.
131.31, 131.95, 133.16, 142.55, 150.15, 160.10, 177.31; ESI-MS (m/z)
336.10 (M þ H)þ; calculated for C18H13N3O2S; C, 64.46; H, 3.91; N,
12.53; S, 9.56. Found: C, 64.50; H, 3.90; N, 12.46; S, 9.58.
4.2.4.8. 2-(4-Chlorophenyl)-6-p-tolylimidazo[2,1-b][1,3,4]thiadia-
zole-5-carbaldehyde (4h). Light yellow solid, yield: 1.69 g, 78.0%;
m.p: 207e208 ꢀC; FTIR (ATR, cmꢃ1): 3087, 2918, 1678, 1592, 1512,
4.2.4.13. 6-(4-Fluorophenyl)-2-methylimidazo[2,1-b][1,3,4]thiadia-
zole-5-carbaldehyde (4m). Light yellow solid, yield: 1.81 g, 81.1%;
m.p:169e170 ꢀC; FTIR (ATR, cmꢃ1): 3068, 2926, 1678, 1597, 1538,
1459, 828, 760, 668; 1H NMR (400 MHz, CDCl3)
d
(ppm): 2.35 (s, 3H,
CH3), 7.21 (m, 2H, AreH), 7.32 (m, 2H, AreH), 7.68e7.72 (m, 4H,
AreH), 10.10 (s, 1H, CHO); 13C NMR (100 MHz, CDCl3)
(ppm):
1470,1064, 834, 670; 1H NMR (400 MHz, CDCl3)
d
(ppm): 2.76 (s, 3H,
CH3), 7.40 (m, 2H, AreH),7.761 (d, J ¼ 8.6 Hz, 2H, AreH),10.25 (s,
CHO, 1H); 13C NMR (100 MHz, DMSO-d6)
(ppm): 17.82, 115.62,
d
d
21.36, 116.26, 124.89, 128.62, 128.99, 130.76, 131.24, 136.71, 137.27,
138.62, 145.74, 147.27, 160.21, 177.21; ESI-MS (m/z) 354.0 (M þ H)þ;
calculated for C18H12ClN3OS; C, 61.10; H, 3.42; N, 11.88; S, 9.06.
116.09, 128.32, 130.17, 130.94, 146.20, 159.23, 163.10, 177.21; ESI-MS
m/z 262.0 [MþH]þ; calculated for C12H8FN3OS; C, 55.16; H, 3.09; N,
16.08; S, 12.27. Found: C, 55.20; H, 3.10; N, 16.06; S, 12.21.

J. Ramprasad et al. / European Journal of Medicinal Chemistry 95 (2015) 49e63
59
4.2.5. General procedure and spectral data for the synthesis of
target molecules 5a-ac
2H, AreH),7.30 (m, 2H, AreH), 7.70e7.68 (m, 4H, AreH), 7.908 (d,
J ¼ 7.6 Hz, 2H, AreH), 8.06 (d, J ¼ 8.0 Hz, 2H, AreH), 12.88 (1H, br,
4.2.5.1. 2-(6-(4-Methoxy phenyl)-2-p-tolyl imidazo[2,1-b][1,3,4]
thiadiazol-5-yl)-1H-benzo[d]imidazole (5a). To a stirred solution of
o-phenylenediamine (0.046 g, 0.429 mmol) in DMF (3 mL) under
nitrogen atmosphere, compound 4d (0.15 g, 0.0429 mmol) was
added and stirred for 5 min after that sodium met bisulfite (0.122 g,
0.644 mmol) was added. The reaction mixture was then heated to
120 ꢀC and maintained same temperature for 3 h. Reaction was
monitored with TLC and once starting material was completely
consumed the solvent was removed by under vacuum. The solid
obtained was dissolved in ethyl acetate (10 mL) and washed with
water (10 mL). The aqueous layer was extracted with ethyl acetate
(3 ꢂ 10 mL). The organic extracts were combined, dried over
anhydrous Na2SO4, filtered, and concentrated in vacuum. The crude
product was purified by flash column chromatography eluting with
methanol/chloroform (1:9) to give 5a as Off white solid. FTIR (ATR,
cmꢃ1): 3352, 3077, 2938, 1603, 1563, 1466, 1244, 1026, 827, 691; 1H
NH), 13C NMR (100 MHz, DMSO-d6)
d (ppm): 21.33, 115.11, 123.06,
127.96, 128.70, 129.29, 129.34, 130.12, 131.04, 137.35, 138.11, 141.78,
145.66, 146.02, 161.44; ESI-MS (m/z) 442.0 (M þ H)þ; calculated for
C24H16ClN5S; C, 65.23; H, 3.65; N, 15.85; S,7.26. Found: C, 65.25; H,
3.67; N, 15.83; S, 7.24.
4.2.5.5. 2-(2-(4-Chlorophenyl)-6-p-tolylimidazo[2,1-b][1,3,4]thiadia-
zol-5-yl)-5-nitro-1H-benzo[d]imidazole (5e). Yellow solid. FTIR
(ATR, cmꢃ1): 3348, 3072, 2934, 1596, 1517, 1462, 827, 754, 682; 1H
NMR (DMSO-d6, 400 MHz)
d (ppm): 2.34 (3H, s, CH3), 7.21 (m, 2H,
ArH), 7.30 (m, 2H, ArH), 7.70e7.66 (m, 4H, ArH),8.18 (1H, d,
J ¼ 8.5 Hz), 8.26 (d, J ¼ 7.6 Hz, 1H, ArH), 8.55 (s, 1H, ArH), 12.91 (s,
1H, NH); 13C NMR (100 MHz, DMSO-d6): 21.33, 110.5, 116.2, 118.4,
128.70, 129.29, 130.34, 130.72, 131.04, 137.35, 138.11, 139.8, 141.78,
142.8, 145.86, 145.2, 146.12, 161.54; ESI-MS (m/z) 487.0 (M þ H)þ;
calculated for C24H15ClN6O2S; C, 59.20; H, 3.10; N, 17.26; S,6.59.
Found: C, 69.10; H, 3.10; N, 17.20; S, 6.48.
NMR (400 MHz, DMSO-d6)
d (ppm): 2.41 (s, 3H, CH3), 3.81 (s, 3H,
OCH3), 7.30 (dd, J ¼ 3.0, 6.1 Hz, 2H, AreH), 7.43 (d, J ¼ 8.3 Hz, 2H,
AreH), 7.48 (d, J ¼ 8.5 Hz, 2H, AreH), 7.70e7.72 (m, 2H, AreH), 7.96
(d, J ¼ 8.3 Hz, 2H, AreH), 8.12 (d, J ¼ 8.6 Hz, 2H, AreH), 11.6e14.2 (s,
4.2.5.6. 5-Chloro-2-(2-(4-chlorophenyl)-6-p-tolylimidazo[2,1-b]
[1,3,4]thiadiazol-5-yl)-1H-benzo[d]imidazole (5f). Green solid. FTIR
(ATR, cmꢃ1): 3351, 3084, 2936, 1598, 1463, 827, 756, 693; 1H NMR
1H, NH); 13C NMR (100 MHz, DMSO-d6)
d (ppm): 21.60, 55.52,
115.70, 123.21, 127.02, 128.80, 129.86, 132.77, 133.26, 143.23, 144.43,
(DMSO-d6, 400 MHz) d (ppm): 2.34 (s, 3H, CH3), 7.21 (m, 2H, 4H),
145.83, 160.7, 163.21; ESI-MS (m/z) 438.1 (M þ H)þ; calculated for
7.27 (d, J ¼ 8.5 Hz, 1H, AreH), 7.30 (m, 2H, AreH), 7.64 (d, J ¼ 7.4 Hz,
C
25H19N5OS; C, 68.63; H, 4.38; N, 16.01; S, 7.33. Found: C, 68.54; H,
1H, AreH), 7.66e7.70 (m, 4H, AreH), 7.72 (s, 1H, AreH),12.91 (s, 1H,
4.40; N, 16.10; S, 7.32.
NH); 13C NMR (100 MHz, DMSO-d6)
d (ppm): 21.33, 115.8, 116.2,
Compounds 5b-ac were synthesized by following the above
procedure by reacting 0.15 g of imidazo[2,1-b][1,3,4]thiadiazole-5-
carbaldehyde (4a-m) with same equivalent of corresponding
substituted o-phenylenediamine.
124.1, 128.70, 129.20,129.29,130.34, 130.72, 131.04, 137.35, 138.11,
140.3, 141.78, 145.86, 145.2, 146.12, 161.54;ESI-MS (m/z) 476.0
(M þ H)þ; calculated for C24H15Cl2N5S; C, 60.51; H, 3.17; N, 14.88;
S,6.73. Found: C, 60.55; H, 3.18; N, 14.90; S, 6.70.
4.2.5.2. 2-(6-(4-Methoxyphenyl)-2-p-tolylimidazo[2,1-b][1,3,4]thia-
diazol-5-yl)-5-nitro -1H-benzo[d]imidazole(5b). Yellow solid. FTIR
(ATR, cmꢃ1): 3348, 3073, 2934, 1604, 1517, 1457, 1244, 1024, 836,
4.2.5.7. 2-(2-(2-Chlorophenyl)-6-(4-methoxyphenyl)imidazo[2,1-b]
[1,3,4]thiadiazol-5-yl)-5-nitro-1H-benzo[d]imidazole
(5g). Light
yellow solid. FTIR (ATR, cmꢃ1): 3351, 3068, 2921, 1594, 1517, 1503,
681; 1H NMR (DMSO-d6, 400 MHz)
d
(ppm): 2.42 (s, 3H, CH3), 3.81
1463, 1244, 1028, 838, 752, 681; 1H NMR (DMSO-d6, 400 MHz)
(s, 3H, OCH3), 7.01 (d, J ¼ 8.5 Hz, 2H, AreH), 7.45 (2H, d, J ¼ 7.8 Hz,
ArH), 7.86 (d, J ¼ 8.9 Hz, 1H, AreH),8.00e8.20 (m, 4H, AreH), 8.612
(d, J ¼ 1.8 Hz, 1H, AreH), 13.10e13.29 (s, 1H, NH); 13C NMR
d
(ppm): 3.81 (s, 3H, OCH3), 7.02 (d, J ¼ 8.3 Hz, 2H, AreH), 7.62 (d,
J ¼ 6.6 Hz, 1H, AreH), 7.67 (s, 1H, AreH), 7.70 (d, J ¼ 7.5 Hz, 1H,
AreH),7.84 (d, J ¼ 8.7 Hz, 1H, AreH), 8.07 (d, J ¼ 8.3 Hz, 2H, AreH),
8.18 (d, J ¼ 8.4 Hz, 1H, AreH), 8.268 (d, J ¼ 7.5 Hz, 1H, AreH), 8.589
(100 MHz, DMSO-d6)
d (ppm): 21.69, 55.82, 114.24, 115.12, 123.39,
126.51, 127.19, 127.28, 127.46, 130.74, 142.90, 143.08, 143.63, 146.90,
(s, 1H, AreH); 13C NMR (100 MHz, DMSO-d6)
d (ppm): 159.72, 18.17,
146.09, 159.72, 162.54; ESI-MS (m/z) 483.1 (M þ H)þ; calculated for
55.82, 114.24, 115.12,127.0, 129.0, 130.6, 132.5, 131.6, 134.02, 142.90,
143.08, 143.63, 146.90, 146.09, 159.72, 159.72; ESI-MS (m/z) 503.0
(M þ H)þ; calculated for C24H15ClN6O3S; C, 57.32; H, 3.01; N, 16.71;
S,6.38. Found: C, 57.34; H, 3.08; N, 16.75; S, 6.35.
C
25H18N6O3S; C, 62.23; H, 3.76; N, 17.42; S, 6.65. Found: C, 62.25; H,
3.78; N, 17.40; S, 6.67.
4.2.5.3. 5-Chloro-2-(6-(4-methoxyphenyl)-2-p-tolylimidazo[2,1-b]
[1,3,4]thiadiazol-5-yl)-1H-benzo[d]imidazole(5c). Green solid. FTIR
(ATR, cmꢃ1): 3353, 3044, 2935, 1540, 1462, 1238, 1024, 828, 751,
4.2.5.8. 2-(6-(4-Methoxyphenyl)-2-phenylimidazo[2,1-b][1,3,4]thia-
diazol-5-yl)-5-nitro-1H-benzo[d]imidazole (5h). Light yellow solid.
FTIR (ATR, cmꢃ1): 3351, 3078, 2935, 1563, 1519, 1466, 1248, 1028,
691; 1H NMR (DMSO-d6, 400 MHz)
d (ppm): 2.41 (s, 3H, CH3), 3.78
(s, 3H, OCH3), 6.98 (d, J ¼ 8.8 Hz, 2H, AreH), 7.30 (dd, J ¼ 1.7, 8.3 Hz,
1H, AreH), 7.43 (d, J ¼ 8.3 Hz, 2H, AreH), 7.71 (d, J ¼ 8.8 Hz, 1H,
AreH),7.76 (s, 1H, AreH), 7.96 (d, J ¼ 8.3 Hz, 2H, AreH), 8.01 (d,
J ¼ 8.8 Hz, 2H, AreH), 12.4e13.6 (s, NH, 1H); 13C NMR (100 MHz,
DMSO-d6): 21.59, 55.62, 114.14, 114.22, 123.19, 126.31, 127.09,
127.28, 127.46, 129.54, 130.54, 143.08, 143.53, 145.60, 146.09, 159.82,
162.64; ESI-MS (m/z) 472.0 (M þ H)þ; calculated for C25H18ClN5OS;
C, 63.62; H, 3.84; N, 14.84; S,6.79. Found: C, 63.52; H, 3.82; N, 14.86;
S, 6.74.
833, 683; 1H NMR (DMSO-d6, 400 MHz)
d (ppm): 3.78 (s, 3H, OCH3),
7.104 (d, J ¼ 8.8 Hz, 2H, AreH), 7.34e7.45 (m, 3H, AreH), 7.58e7.60
(m, 2H, AreH), 7.913 (d, J ¼ 8.4 Hz, 2H, AreH), 8.18 (d, J ¼ 8.5 Hz, 1H,
AreH), 8.26 (d, J ¼ 7.6 Hz, 1H, AreH),8.55 (s, 1H, AreH),12.91 (s, 1H,
NH); 13C NMR (100 MHz, CDCl3)
d (ppm): 55.59, 114.62, 115.44,
115.65, 123.18, 126.37, 129.43, 126.91, 132.45, 144.12, 142.70, 146.20,
160.74, 162.32; ESI-MS (m/z) 469.1 (M þ H)þ; calculated for
C14H16N6O3S; C, 61.53; H, 3.44; N, 17.94; S,6.84. Found: C, 61.60; H,
3.45; N, 17.96; S, 6.85.
4.2.5.4. 2-(2-(4-Chlorophenyl)-6-p-tolylimidazo[2,1-b][1,3,4]thiadia-
zol-5-yl)-1H-benzo[d]imidazole (5d). Light yellow solid. FTIR (ATR,
cmꢃ1): 3353, 3072, 2936, 1596, 1518, 1459, 828, 756, 693; 1H NMR
4.2.5.9. 2-[6-(4-Fluorophenyl)-2-(4-methylphenyl)imidazo[2,1-b]
[1,3,4]thiadiazol-5-yl]-1H-benzimidazole (5i). Yellow solid. FTIR
(ATR, cmꢃ1): 3353, 3077, 2934, 1602, 1538, 1464, 1061, 827, 688; 1H
(DMSO-d6, 400 MHz)
d
(ppm): 2.34 (s, 3H, CH3),7.21 (d, J ¼ 7.6 Hz,
NMR (DMSO-d6, 400 MHz) d (ppm): 2.50 (s, 3H, CH3), 7.23e7.33 (m,

60
J. Ramprasad et al. / European Journal of Medicinal Chemistry 95 (2015) 49e63
4H, AreH), 7.431 (d, J ¼ 8.4 Hz, 2H, AreH), 7.66e7.76 (m, 2H,
AreH),7.96 (d, J ¼ 7.9 Hz,2H,AreH),8.12e8.15 (m, 2H, AreH), 12.82
127.63, 129.37, 129.21, 130.32, 134.31, 142.98, 145.70, 145.66, 159.80,
161.18, 165.20; ESI-MS (m/z) 487.1 (M þ H) þ; calculated for
(s, 1H, NH); 13C NMR (100 MHz, DMSO-d6)
d
(ppm): 21.59, 115.70,
C24H15FN6O3S; C, 59.25; H, 3.11; N, 17.28; S, 6.59. Found: C, 59.22; H,
123.55, 127.05, 127.50, 130.12, 130.47, 141.81, 143.13, 145.58, 161.25,
3.12; N, 17.26; S, 6.62.
162.85, 163.69; ESI-MS (m/z) 426.0 (M þ H)þ; calculated for
C
24H16FN5S; C, 67.75; H, 3.79; N, 16.46; S,7.54. Found: C, 67.70; H,
4.2.5.15. 5-Chloro-2-(2-(4-fluorophenyl)-6-(4-methoxy phenyl) imi-
dazo [2,1-b][1,3,4] thiadiazol-5-yl)-1H-benzo[d]imidazole (5o).
Light brown solid. FTIR (ATR, cmꢃ1): 3351, 3078, 2937, 1601, 1538,
1463, 1068, 1024, 1028, 751, 686; 1H NMR (DMSO-d6, 400 MHz)
3.81; N, 16.50; S, 7.52.
4.2.5.10. 2-(6-(4-Fluorophenyl)-2-p-tolylimidazo[2,1-b][1,3,4]thia-
diazol-5-yl)-5-nitro-1H-benzo[d]imidazole (5j). Light yellow solid.
FTIR (ATR, cmꢃ1): 3351, 3068, 2926, 1597, 1538, 1475, 1064, 834,
d
(ppm): 3.84 (s, 3H, OCH3), 6.99 (d, J ¼ 8.4 Hz, 2H, AreH), 7.30 (dd,
J ¼ 1.7, 8.3 Hz, 1H, AreH), 7.43 (m, 2H, AreH),7.71 (d, J ¼ 8.8 Hz, 1H,
AreH),7.76 (s, 1H, AreH), 7.96 (d, J ¼ 8.3 Hz, 2H, AreH), 8.01 (m, 2H,
AreH), 12.4e13.6 (s, 1H, br); 13C NMR (100 MHz, DMSO-d6): 55.42,
114.24, 114.32, 123.29, 126.61, 127.19, 127.56, 129.94, 131.44, 143.08,
143.53, 145.70, 146.09, 159.92, 162.64, 165.20; ESI-MS (m/z) 476.1
(M þ H)þ; calculated for C24H15ClFN5OS; C, 60.57; H, 3.18; N, 14.72;
S,6.74. Found: C, 60.48; H, 3.20; N, 14.68; S, 3.20.
687; 1H NMR (DMSO-d6, 400 MHz)
d (ppm): 2.42 (s, 3H, CH3), 7.29
(t, J ¼ 17.5 Hz, 2H, AreH), 7.39e7.46 (m, 2H, AreH), 7.83e07.91 (m,
2H, AreH), 8.301 (d, J ¼ 10.1 Hz, 2H, AreH), 8.20 (m, 2H, AreH), 8.60
(s, 1H, AreH), 13.29 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6)
d
(ppm): 21.62, 114.58, 115.44, 115.65, 126.91, 127.02, 127.63, 130.21,
130.53, 130.95, 143.31, 161.45, 163.90; ESI-MS (m/z) 468.9 (MꢃH)þ;
calculated for C24H15FN6O2S; C, 61.27; H, 3.21; N, 17.86; S,6.82.
Found: C, 61.25; H, 3.23; N, 17.82; S, 6.80.
4.2.5.16. 2-(6-(4-Chlorophenyl)-2-p-tolylimidazo[2,1-b][1,3,4]thia-
diazol-5-yl)-1H-benzo [d] imidazole (5p). Light yellow solid. FTIR
(ATR, cmꢃ1): 3348, 3068, 2924, 1592, 1534, 1476, 840, 727, 689; 1H
4.2.5.11. 2-[2-(4-Chlorophenyl)-6-(4-methoxyphenyl)imidazo[2,1-b]
[1,3,4]thiadiazol-5-yl]-1H-benzimidazole(5k). Light yellow solid.
FTIR (ATR, cmꢃ1): 3350, 3077, 2937, 1608, 1538, 1437, 1028, 826, 751,
NMR (400 MHz, DMSO-d6) d(ppm): 2.41 (s, 3H, CH3), 7.30 (dd,
J ¼ 3.0, 6.1 Hz, 2H, AreH), 7.40 (d, J ¼ 8.3 Hz, 2H, AreH),7.48 (d,
J ¼ 8.7 Hz, 2H, AreH), 7.70e7.72 (m, 2H, AreH), 7.96 (d, J ¼ 8.3 Hz,
2H, AreH), 8.10 (d, J ¼ 8.3 Hz, 2H, AreH), 11.6e14.2 (s, 1H, NH); 13C
689; 1H NMR (DMSO-d6, 400 MHz)
d (ppm): 3.783 (s, 3H,
CH3),6.983 (d, J ¼ 8.8 Hz, 2H, AreH), 7.2e7.4 (m, 2H, AreH), 7.694
(d, J ¼ 8.8 Hz, 4H, AreH), 8.02e8.10 (m, 4H, AreH), 12.77 (s, NH,
NMR (100 MHz, DMSO-d6) d(ppm): 21.60, 115.60, 123.21, 127.02,
1H); 13C NMR (100 MHz, DMSO-d6)
d
(ppm): 55.63, 114.8, 126.35,
127.55, 128.80, 129.76, 130.57, 132.77, 133.26, 143.23, 144.43, 145.83,
163.21; ESI-MS (m/z) 442.0 (M þ H)þ; calculated for C24H16ClN5S;
C, 65.23; H, 3.65; N, 18.44; S, 7.26. Found: C, 65.18; H, 3.68; N, 18.46;
S, 7.28.
123.06, 128.73, 129.27, 129.41, 130.12, 137.30,141.78, 145.50, 145.66,
159.80, 161.18; ESI-MS (m/z) 458.0 (M þ H)þ; calculated for
C
24H16ClN5S; C, 62.95; H, 3.52; N, 15.29; S,7.00. Found: C, 62.90; H,
3.53; N, 15.50; S, 6.90.
4.2.5.17. 2-(6-(4-Chlorophenyl)-2-p-tolylimidazo[2,1-b][1,3,4]thia-
diazol-5-yl)-5-nitro-1H-benzo[d]imidazole (5q). Yellow solid. FTIR
(ATR, cmꢃ1): 3351, 3068, 2924, 1592, 1534, 1518,1476, 837, 748, 686;
4.2.5.12. 2-[2-(4-Chlorophenyl)-6-(4-fluorophenyl)imidazo[2,1-b]
[1,3,4]thiadiazol-5-yl]-1H-benzimidazole (5l). Light yellow solid.
FTIR (ATR, cmꢃ1): 3351, 3068, 2936, 1616, 1537, 1467, 1068, 837, 748,
1H NMR (400 MHz, DMSO-d6)
d (ppm): 2.41 (s, 3H, CH3),7.43 (d,
693; 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 7.24e7.29 (m, 4H,
AreH), 7.70 (d, J ¼ 8.4 Hz, 4H, AreH), 8.10e8.17 (m, 4H, AreH),
12,83 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6)
(ppm): 115.50,
J ¼ 7.9 Hz, 2H, AreH), 7.49 (d, J ¼ 8.3 Hz, 2H, AreH), 7.72 (d,
J ¼ 7.5 Hz, 1H, AreH), 7.98 (d, J ¼ 7.9 Hz, 2H, AreH), 8.10 (d,
J ¼ 8.4 Hz, 2H, AreH), 8.26 (d, J ¼ 7.5 Hz, 1H, AreH), 8.58 (s, 1H,
AreH), 12.6e14.2 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6)
d
115.72, 123.12, 128.63, 129.33, 130.10, 130.20, 130.28, 137.43, 144.80,
145.71, 161.30, 161.68, 163.78; ESI-MS (m/z) 446.0 (M þ H)þ;
calculated for C23H13ClFN5S; C, 61.95; H, 2.94; N, 15.71; S,7.19.
Found: C, 61.90; H, 2.96; N, 15.65; S, 7.20.
d
(ppm): 21.39, 112.64, 113.32, 123.19, 126.31, 127.29, 127.38, 127.86,
130.44, 134.31, 142.6, 143.18, 143.33, 145.30, 146.29, 160.32; ESI-MS
(m/z) 487.0 (M þ H)þ; calculated for C24H15ClN6O2S; C, 59.20; H,
3.10; N, 17.26; S, 6.59. Found: C, 59.22; H, 3.12; N, 17.26; S, 6.60.
4.2.5.13. 2-(2-(4-Fluorophenyl)-6- (4-methoxyphenyl) imidazo [2,1-
b][1,3,4] thiadiazol-5-yl)-1H-benzo[d] imidazole (5m). Off white
solid. FTIR (ATR, cmꢃ1): 3349, 3073, 2937, 1611, 1540, 1467, 1244,
4.2.5.18. 5-Chloro-2-(6-(4-chlorophenyl)-2-p-tolylimidazo[2,1-b]
[1,3,4]thiadiazol-5-yl)-1H-benzo[d]imidazole (5r). Light yellow
solid. FTIR (ATR, cmꢃ1): 3351, 3073, 2935, 1598, 1540, 1464, 827,
1061, 1028, 827, 686; 1H NMR (DMSO-d6, 400 MHz)
d (ppm): 3.79
(s, 3H, OCH3),6.97 (d, J ¼ 8.4 Hz, 2H, AreH), 7.3e7.5 (m, 2H, AreH),
693; 1H NMR (DMSO-d6, 400 MHz)
d (ppm): 2.416 (s, 3H, CH3),7.31
7.69e7.72 (m, 4H, AreH), 8.04e8.08 (m, 4H, AreH), 12.76 (s, 1H,
(d, J ¼ 8.4 Hz, 1H, AreH), 7.43 (d, J ¼ 7.9 Hz, 2H, AreH), 7.49 (d,
J ¼ 8.3 Hz, 2H, AreH), 7.72 (d, J ¼ 7.9 Hz, 1H, AreH), 7.77 (s, 1H,
AreH), 7.98 (d, J ¼ 7.9 Hz, 2H, AreH), 8.10 (d, J ¼ 8.4 Hz, 2H, AreH),
NH); 13C NMR (100 MHz, DMSO-d6)
d (ppm): 55.73, 114.7, 126.45,
123.16, 127.63, 129.37, 129.21, 130.32, 142.98, 145.70,145.66, 159.80,
161.18, 165.20; ESI-MS (m/z) 442.1 (M þ H)þ; calculated for
12.6e13.1 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6)
d (ppm):
C
24H16CFN5S; C, 65.29; H, 3.65; N, 15.86; S,7.26. Found: C, 64.90; H,
21.49, 112.14, 113.12, 123.09, 126.21, 127.19, 127.38, 127.56, 129.44,
130.44, 134.31, 143.08, 143.43, 145.50, 146.19, 160.82; ESI-MS (m/z)
476.0 (M þ H)þ; calculated for C24H15ClN6O2S; C, 59.20; H, 3.10; N,
17.26; S, 6.59. Found: C, 59.22; H, 3.12; N, 17.26; S, 6.60.
3.68; N, 15.60; S, 7.20.
4.2.5.14. 2-(2-(4-Fluorophenyl)-6-(4-methoxyphenyl)imidazo[2,1-b]
[1,3,4]thiadiazol-5-yl)-5-nitro-1H-benzo[d]imidazole
Yellow solid. FTIR (ATR, cmꢃ1): 3350, 3077, 2936, 1603, 1518, 1464,
1244, 1071, 1028, 831, 691; 1H NMR (400 MHz, DMSO-d6)
(ppm):
3.81 (s, 3H, OCH3),7.02 (d, J ¼ 8.8 Hz, 2H, AreH),7.46 (d, J ¼ 7.4 Hz,
2H, AreH), 7.86 (d, J ¼ 8.8 Hz, 2H, AreH), 8.00e8.20 (m, 4H, AreH),
8.61 (s, 1H, AreH), 13.10e13.293 (s, 1H, NH); 13C NMR (100 MHz,
(5n).
4.2.5.19. 2-(6-(4-Methoxyphenyl)-2-methylimidazo[2,1-b][1,3,4]
thiadiazol-5-yl)-1H-benzo [d]imidazole (5s). White solid. FTIR (ATR,
cmꢃ1): 3273, 3071, 2965, 1592, 1494, 1437, 1248, 1028, 835, 691; 1H
d
NMR (DMSO-d6, 400 MHz)
d (ppm): 2.78 (s, 3H, CH3), 3.75 (s, 3H,
OCH3), 6.94 (d, J ¼ 8.8 Hz, 2H, AreH),7.25 (m, 2H, AreH),7.58 (d,
DMSO-d6)
d (ppm): 55.73, 114.7, 115.32, 123.19, 126.45, 123.16,
J ¼ 7 Hz, 1H, AreH), 7.70 (d, J ¼ 7.4 Hz, 1H, AreH), 7.91 (d, J ¼ 8.8 Hz,

J. Ramprasad et al. / European Journal of Medicinal Chemistry 95 (2015) 49e63
61
2H, AreH),12.73 (s, 1H, NH) 13C NMR (100 MHz, DMSO-d6): 18.07,
3.31; N, 19.14; S, 8.76. Found: C, 59.06; H, 3.33; N, 19.12; S, 8.74.
55.60, 114.19, 114.33, 119.69, 126.56, 129.10, 142.23, 144.99, 146.12,
159.61, 161.93; ESI-MS (m/z) 362.0 (M þ H)þ; calculated for
4.2.5.25. 2-(6-(4-Chlorophenyl)-2-methylimidazo[2,1-b][1,3,4]thia-
diazol-5-yl)-5-nitro-1H-benzo[d]imidazole (5y). Yellow solid. FTIR
(ATR, cmꢃ1): 3278, 3070, 2968, 1591, 1496, 1434, 834, 758, 689; 1H
C
19H15N5OS; C, 63.14; H, 4.18; N, 19.38; S,8.87. Found: C, 63.10; H,
4.20; N, 19.40; S, 8.85.
NMR (DMSO-d6, 400 MHz)
d (ppm): 2.83 (s, 3H, CH3),7.68 (dd,
4.2.5.20. 2-(6-(4-Methoxyphenyl)-2-methylimidazo[2,1-b][1,3,4]
J ¼ 1.9, 7.6 Hz, 2H, AreH),7.70 (d, J ¼ 7.5 Hz, 1H, AreH), 8.08 (dd,
thiadiazol-5-yl)-5-nitro-1H-benzo[d]imidazole(5t). Yellow
FTIR (ATR, cmꢃ1): 3273, 3068, 2965, 1604, 1518, 1497, 1436, 1246,
1024, 836, 687; 1H NMR (DMSO-d6, 400 MHz)
(ppm): 2.78 (s, 3H,
solid.
J ¼ 1.8, 8.8 Hz, 2H, AreH), 8.26 (d, J ¼ 7.6 Hz, 1H, AreH), 8.58 (s, 1H,
AreH), 12.85 (s, 1H, NH); 13C NMR (100 MHz, CDCl3)
d (ppm): 18.16,
d
115.96, 123.42, 127.58, 129.21, 121.93, 130.83, 132.89, 133.27, 145.14,
CH3), 3.78 (s, 3H, OCH3),7.913 (d, J ¼ 8.8 Hz, 2H, AreH),7.58e7.60
(m, 2H, AreH), 8.18 (d, J ¼ 8.5 Hz, 1H, AreH), 8.26 (d, J ¼ 7.6 Hz, 1H,
AreH), 8.55 (s, 1H, AreH),12.91 (s, 1H, NH); 13C NMR (100 MHz,
146.28, 162.96; ESI-MS (m/z) 411.0 (M þ H)þ; calculated for
C18H11ClN6O2S; C, 52.62; H, 2.70; N, 20.46; S, 7.80. Found: C, 52.55;
H, 2.71; N, 20.48; S, 7.82.
CDCl3)
d (ppm): 18.22, 55.60, 114.52, 123.08, 127.37, 129.43, 132.35,
144.12, 142.8, 146.20, 160.7, 162.42; ESI-MS (m/z) 407.0 (M þ H)þ;
calculated for C19H14N6O3S; C, 56.15; H, 3.47; N, 20.68; S,7.89.
Found: C, 56.10; H, 3.45; N, 20.70; S, 7.85.
4.2.5.26. 5-Chloro-2-(6-(4-chlorophenyl)-2-methylimidazo[2,1-b]
[1,3,4]thiadiazol-5-yl)-1H-benzo[d]imidazole(5z). Light green solid.
FTIR (ATR, cmꢃ1): 3275, 3069, 2965, 1592, 1497, 837, 753, 690; 1H
4.2.5.21. 2-(6-(4-Fluorophenyl)-2-methylimidazo[2,1-b][1,3,4]thia-
diazol-5-yl)-1H-benzo [d] imidazole (5u). White solid. FTIR (ATR,
cmꢃ1): 3273, 3074, 2964, 1597, 1538, 1437, 1062, 834, 687; 1H NMR
NMR (DMSO-d6, 400 MHz)
d (ppm): 2.81 (s, 3H, OCH3), 7.29 (dd,
J ¼ 8.7, 1.7 Hz, 1H, ArH), 7.46 (d, J ¼ 8.3 Hz, 2H, ArH), 7.67 (d,
J ¼ 8.3 Hz, 1H, ArH), 7.73 (s, 1H, ArH), 8.00 (d, J ¼ 8.3 Hz, 2H, AreH);
13C NMR (100 MHz, DMSO-d6)
d (ppm): 18.15, 114.96, 123.32,
(DMSO-d6, 400 MHz)
d (ppm): 2.804 (s, 3H, CH3),7.22 (d, 2H, ArH,
J ¼ 8.8 Hz), 7.26e7.28 (m, 2H, AreH), 7.67(dd, J ¼ 5.6, 3.2 Hz, 2H,
127.38, 128.77, 129.20, 129.73, 130.89, 132.79, 133.17, 143.14, 144.18,
AreH), 8.04 (dd, J ¼ 8.4, 6.0 Hz, 2H, AreH), 12.82 (s, 1H, NH); 13C
146.87, 162.94; ESI-MS (m/z) 400.0 (M þ H)þ; calculated for
NMR (100 MHz, DMSO-d6)
d (ppm): 18.10, 115.09, 115.52, 115.74,
C18H11Cl2N5S; C, 54.01; H, 2.77; N, 17.50; S, 8.01. Found: C, 54.02; H,
122.99, 129.87, 129.95, 130.52, 130.55, 141.87, 144.04, 146.42, 161.17,
163.61; ESI-MS (m/z) 350.08 (M þ H)þ; calculated for C18H12FN5S;
C, 61.88; H, 3.46; N, 20.04; S, 9.18. Found: C, 61.76; H, 3.48; N, 21.06;
S, 9.16.
2.80; N, 17.52; S, 8.04.
4.2.5.27. 2-(2-Methyl-6-p-tolylimidazo[2,1-b][1,3,4]thiadiazol-5-yl)-
1H-benzo[d]imidazole (5aa). Off white solid. FTIR (ATR, cmꢃ1):
3273, 3069, 2964, 1590, 1497, 1437, 838, 751, 691; 1H NMR
4.2.5.22. 2-(6-(4-Fluorophenyl)-2-methyl imidazo [2,1-b][1,3,4]thia-
diazol-5-yl)-5-nitro-1H-benzo[d]imidazole (5v). Yellow solid. FTIR
(ATR, cmꢃ1): 3347, 3077, 2935, 1603, 1518, 1464, 1092, 831, 690; 1H
(400 MHz, DMSO-d6) d(ppm): 2.30 (s, 3H, CH3), 2.89 (s, 3H, CH3),
7.18 (d, J ¼ 8.4 Hz, 2H, AreH),7.26 (dd, J ¼ 3.1, 6.2 Hz, 2H, AreH),7.65
(m, 2H, AreH),7.80 (d, J ¼ 8.4 Hz, 2H, AreH),12.76 (s, 1H, NH); 13C
NMR (DMSO-d6, 400 MHz)
d (ppm): 2.82 (s, 3H, CH3),7.68 (dd,
NMR (100 MHz, DMSO-d6)
d (ppm): 18.07, 21.29, 114.82, 122.88,
J ¼ 2.0, 6.8 Hz, 2H, AreH), 7.70 (d, J ¼ 7.6 Hz, 1H, AreH), 8.06 (dd,
127.67, 129.33, 131.25, 137.78, 142.09, 145.09, 146.22, 162.15; ESI-MS
(m/z) 346.0 (M þ H)þ; calculated for C19H15N5S; C, 66.07; H, 4.38;
N, 20.27; S, 9.28. Found: C, 66.12; H, 4.40; N, 20.25; S, 9.25.
J ¼ 3.2, 5.6 Hz, 2H, AreH),8.26 (d, J ¼ 7.6 Hz, 1H, AreH), 8.58 (s, 1H,
AreH), 12.89 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6)
d (ppm):
18.15, 115.19, 123.19, 129.87, 129.97, 130.52, 138.55, 141.77, 144.04,
146.82, 161.27, 163.61; ESI-MS (m/z) 395.1 (M þ H)þ; calculated for
C
18H11FN6O2S; C, 54.82; H, 2.81; N, 21.31; S,8.13. Found: C, 54.80; H,
4.2.5.28. 2-(2-Methyl-6-p-tolyl imidazo [2, 1-b][1,3,4] thiadiazol-5-
yl)-5- nitro- 1H-benzo[d ]imidazole (5ab). Yellow solid. FTIR (ATR,
cmꢃ1): 3271, 3068, 2924, 1598, 1518, 1438, 842, 688; 1H NMR
2.83; N, 21.32; S, 8.11.
4.2.5.23. 5-Chloro-2-(6-(4-fluorophenyl)-2-methylimidazo[2,1-b]
(DMSO-d6, 400 MHz)
d (ppm): 2.30 (s, 3H, CH3), 2.78 (s, 3H, CH3),
[1,3,4]thiadiazol-5-yl)-1H-benzo[d]imidazole
cmꢃ1): 3352, 3071, 2937, 1603, 1478, 1437, 1071, 837, 752, 690; Light
green solid. 1H NMR (DMSO-d6, 400 MHz)
(ppm): 2.80 (s, 3H,
(5w). FTIR
(ATR,
7.26 (dd, J ¼ 3.1, 6.2 Hz, 1H, AreH), 7.29 (d, J ¼ 8.7 Hz, 1H, AreH),
7.65 (m, 2H, AreH), 8.12 (d, J ¼ 7.4 Hz, 1H, AreH), 8.26 (d, J ¼ 8.4 Hz,
1H, AreH), 8.68 (s, 1H, AreH), 12.90 (s, 1H, NH); 13C NMR (100 MHz,
d
CH3), 7.28 (dd, J ¼ 1.8, 8.8 Hz, 1H, AreH), 7.45 (d, J ¼ 8.3 Hz, 2H,
AreH), 7.64 (d, J ¼ 8.7 Hz, 1H, AreH), 7.72 (s, 1H, AreH), 8.09 (d,
J ¼ 8.0 Hz, 2H, AreH), 12.83 (s, 1H, NH); 13C NMR (100 MHz, CDCl3)
DMSO-d6)
d (ppm): 18.12, 21.30, 114.62, 122.98, 127.27, 129.43,
131.35, 137.78, 145.12, 142.8, 146.30, 162.32; ESI-MS (m/z) 391.08
(M þ H)þ; calculated for C19H14N6O2S; C, 58.45; H, 3.61; N, 21.53; S,
8.21. Found: C, 58.29; H, 3.62; N, 21.55; S, 8.23.
d
(ppm): 18.25, 115.0, 115.7, 123.62, 127.48, 129.81, 121.63, 131.29,
132.89, 133.27, 138.90, 142.1, 143.44, 144.28, 158.9, 162.97; ESI-MS
(m/z) 383.9 (M þ H) þ; calculated for C18H11ClFN5S; C, 56.33; H,
2.89; N, 18.25; S, 8.35. Found: C, 56.31; H, 2.90; N, 18.19; S, 8.31.
4.2.5.29. 5-Chloro-2-(2-methyl-6-p-tolylimidazo[2,1-b][1,3,4]thia-
diazol-5-yl)-1H-benzo[d] imidazole (5ac). Light green solid. FTIR
(ATR, cmꢃ1): 3273, 3070, 2965, 1590, 1498, 1435, 835, 751, 691; 1H
4.2.5.24. 2-(6-(4-Chlorophenyl)-2-methylimidazo[2,1-b][1,3,4]thia-
diazol-5-yl)-1H-benzo[d] imidazole (5x). Off white solid. FTIR (ATR,
cmꢃ1): 3272, 3070, 2965, 1590, 1495, 1436, 835, 751, 690; 1H NMR
NMR (DMSO-d6, 400 MHz) d(ppm): 2.30 (s, 3H, CH3), 2.79 (s, 3H,
CH3), 7.01 (d, J ¼ 8.8 Hz, 2H, AreH), 7.49 (d, J ¼ 8.3 Hz, 2H, AreH),
8.10 (d, J ¼ 8.4 Hz, 1H, AreH), 8.26 (d, J ¼ 7.6 Hz, 1H, AreH), 8.58 (s,
1H, AreH), 12.94 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6)
(DMSO-d6, 400 MHz)
d
(ppm): 2.81 (s, 3H, CH3), 7.23 (d, J ¼ 8.8 Hz,
2H, AreH), 7.28e7.30 (m, 2H, AreH), 7.69 (dd, J ¼ 1.8, 8.8 Hz, 2H,
AreH), 8.06 (dd, J ¼ 6.0, 8.4 Hz, 2H, AreH), 12.83 (s, 1H, NH); 13C
d
(ppm): 18.16, 21.35, 114.98, 122.42, 127.48, 129.61, 121.93, 130.89,
NMR (100 MHz, CDCl3)
d
(ppm): 18.20, 115.09, 123.29, 129.89,
132.79, 133.17, 143.14, 144.18,151.2, 152.3, 162.94; ESI-MS (m/z)
380.0 (M þ H)þ; calculated for C19H14ClN5S; C, 66.07; H, 3.71; N,
18.44; S, 8.44. Found: C, 66.06; H, 3.70; N, 18.46; S, 8.40.
129.95, 130.53, 130.75, 134.2, 141.77, 144.14, 146.32, 161.13; ESI-MS
(m/z) 366.08 (M þ H)þ; calculated for C18H12ClN5S; C, 59.09; H,

62
J. Ramprasad et al. / European Journal of Medicinal Chemistry 95 (2015) 49e63
4.3. Antitubercular studies
radical solution and each test tube was made up to final volume of
4 ml. BHA was used as a reference standard and dissolved in
methanol to get the same concentration as that of synthesized
compounds. Each mixture was vortexed for a few seconds and
allowed to stand in the dark for 10 min at ambient temperature. The
absorbance of each reaction mixture was measured at 517 nm
against a blank of methanol using a UVevisible spectrometer
(Shimadzu UV-1800, Japan). The level of DPPH radical scanning
activity was calculated as:
Two-fold serial dilutions of each test compound/drug were
prepared and incorporated into Middlebrook 7H11 agar medium
with oleic acid, albumin, dextrose, and catalase (OADC) growth
supplement to get final concentrations of 50, 25, 12.5, 6.25, 3.13,
1.56 and 0.78 mg/mL. Inoculum of MTB H37Rv ATCC 27294/XDR-TB
was prepared from fresh Middlebrook 7H11 agar slants with OADC
(Difco) growth supplement adjusted to 1 mg/mL (wet weight) in
Absorbance of the control ꢃ Absorbance of the test sample
%Scavenging Activity ¼
ꢂ 100
Absorbance of the control
Tween 80 (0.05%) saline diluted to 10ꢃ2 to give a concentration of
~107 cfu/mL. Five microliters of this bacterial suspension was
spotted onto 7H11 agar tubes containing different concentrations
of the drug as discussed above. The tubes were incubated at 37 ꢀC,
and final readings (as MIC in mg/mL) were determined after 28
days. This method is similar to that recommended by the National
Committee for Clinical Laboratory Standards for the determination
of MIC in triplicate.
4.7. In vitro cytotoxicity studies
Vero (African green monkey kidney) cell line was procured from
National Centre for Cell Sciences (NCCS), Pune, India. Stock cells
were cultured in MEM supplemented with 10% inactivated Fetal
Bovine Serum (FBS), penicillin (100 IU/ml), streptomycin (100
mg/
ml) and amphotericin B (5 g/ml) in an humidified atmosphere of
m
5% CO2 at 37 ꢀC until confluent. The cells were dissociated with
TPVG solution (0.2% trypsin, 0.02% EDTA, 0.05% glucose in PBS). The
stock cultures were grown in 25 cm2 culture flasks and all experi-
ments were carried out in 96 microtitre plates (Tarsons India Pvt.
Ltd., Kolkata, India). For cytotoxicity studies, each weighed test
drugs were separately dissolved in distilled DMSO and volume was
made up with MEM supplemented with 2% inactivated FBS to
obtain a stock solution of 1 mg/ml concentration and sterilized by
filtration. Serial two fold dilutions were prepared from this for
carrying out MTT assay. The monolayer cell culture was trypsinized
and the cell count was adjusted to 1.0 ꢂ 105 cells/ml using MEM
containing 10% FBS. To each well of the 96 well microtitre plate,
0.1 ml of the diluted cell suspension (approximately 10,000 cells)
was added. After 24 h, when a partial monolayer was formed, the
supernatant was flicked off, washed the monolayer once with
4.4. Antibacterial studies
All bacterial strains were maintained on nutrient agar medium
at 37 ꢀC. The cultures were inoculated in fresh 10 mL Nutrient Broth
to yield an initial suspension of approximately 10e100 cfu/mL. All
broths were then incubated statically at 37 ꢀC for 18e24 h. Sus-
ceptibility of the test organism to the organic compound was
determined by well plate technique. The bacterial suspensions
were serially diluted in saline and 0.1 ml from the appropriate
dilution was spread on nutrient agar. The wells were dug in each
petri plate by sterilized cork borer. The compounds were dissolved
in DMSO and appropriate dilutions were made (1 mg/mL and
0.5 mg/mL). Each experiment was carried out in triplicate. The
same procedure was repeated for all microorganisms. After the
inoculation of organism and compound, the petri plates were
incubated for 18 h at 37 ꢀC. The diameter of zone of inhibition was
measured and the values for DMSO solvent were subtracted to get
the actual values.
medium and 100 mL of different test concentrations of test drugs
were added on to the partial monolayer in microtitre plates. The
plates were then incubated at 37 ꢀC for 3 days in 5% CO2 atmo-
sphere, and microscopic examination was carried out and obser-
vations were noted every 24 h interval. After 72 h, the drug
solutions in the wells were discarded and 50 ml of MTT in PBS was
added to each well. The plates were gently shaken and incubated
4.5. Antifungal studies
for 3 h at 37 ꢀC in 5% CO2 atmosphere. The supernatant was
removed and 100 mL of propanol was added and the plates were
gently shaken to solubilize the formed formazan. The absorbance
was measured using a microplate reader at a wavelength of 540 nm.
The compounds were dissolved in DMSO and antimicrobial ac-
tivity was determined at concentration of 1 and 0.5 mg/mL. The
required amounts of each fungal strain were removed from the
stock and suspended in 5 mL of distilled water with 2 drops of
Tween 80. This suspension was uniformly spread on petri plates
containing Potato dextrose agar media using sterile swabs. After
adding the test samples into the wells, the plates were incubated at
25 ꢀC for 3 days. The plates were then examined for the presence of
zones of inhibition and the results were recorded.
ꢀ
MeanODof individualtest group
% GrowthInhibition ¼ 100ꢃ
MeanODof controlgroup
ꢁ
ꢂ100
4.6. Antioxidant studies
Acknowledgments
Free radical-scavenging capacities of the compounds 5aeac
were determined using the stable 2,2-diphenyl-1-picrylhydrazyl
Authors are thankful to NITK, Surathkal for providing the
research facilities and to Dr. Reddy's institute of life sciences, HCU
campus for providing analytical facilities.
radical (DPPH). An aliquot of 100
mg concentrations of synthesized
compounds in methanol was added to 3 ml of 0.004% w/v DPPH

J. Ramprasad et al. / European Journal of Medicinal Chemistry 95 (2015) 49e63
63
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.03.024.
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