Synthesis, spectral studies and biological evaluation of a novel series of 2-substituted-5,6-diarylsubstituted imidazo(2,1-b)-1,3, 4-thiadiazole derivatives as possible anti-tubercular agents

Article abstract of DOI:10.1007/s00044-011-9646-9

A novel series of 18 analogs of 2-substituted-5,6- diarylsubstituted imidazo(2,1-b)-1,3,4-thiadiazole 6a-r have been synthesized by the reaction of 2-amino-5-substituted- 1,3,4-thiadiazoles 5a-d and an appropriately substituted a-bromo-1,2-(p-substituted)diaryl-1-ethanones 4a-e. Structures of these compounds were established by physiochemical, elemental analysis and spectral data. All the title compounds were tested for their in-vitro anti-tubercular activity against Mycobacterium tuberculosis H₃₇Rv using Alamar Blue susceptibility test and the activity expressed as the minimum inhibitory concentration (MIC) in lg/ml. Among synthesized compounds, compound 6h (MIC = 1.25 μg/ml) exhibited excellent anti-tubercular activity with respect to other synthesized compounds and reference drugs. Compounds 6c, 6f, and6g have also displayed an encouraging anti-tubercular activity profile. Further, some title compounds were also assessed for their cytotoxic activity (IC ₅₀) against in a mammalian Vero cell line using MTT assay. The results reveal that these compounds exhibit anti-tubercular activity at non-cytotoxic concentrations. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9646-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1313–1321
DOI 10.1007/s00044-011-9646-9
ORIGINAL RESEARCH
Synthesis, spectral studies and biological evaluation of a novel
series of 2-substituted-5,6-diarylsubstituted imidazo(2,1-b)-1,3,
4-thiadiazole derivatives as possible anti-tubercular agents
•
Mahesh B. Palkar Malleshappa N. Noolvi
•
•
Veeresh S. Maddi Mangala Ghatole
•
Laxmivenkat G. Nargund
Received: 6 January 2011 / Accepted: 12 April 2011 / Published online: 26 April 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A novel series of 18 analogs of 2-substituted-5,6-
diarylsubstituted imidazo(2,1-b)-1,3,4-thiadiazole 6a–r have
been synthesized by the reaction of 2-amino-5-substituted-
1,3,4-thiadiazoles 5a–d and an appropriately substituted
a-bromo-1,2-(p-substituted)diaryl-1-ethanones 4a–e. Struc-
tures of these compounds were established by physiochemi-
cal, elemental analysis and spectral data. All the title
compounds were tested for their in-vitro anti-tubercular
activity against Mycobacterium tuberculosis H37Rv using
Alamar Blue susceptibility test and the activity expressed as
the minimum inhibitory concentration (MIC) in lg/ml.
Among synthesized compounds, compound 6h (MIC =
1.25 lg/ml) exhibited excellent anti-tubercular activity with
respect to other synthesized compounds and reference drugs.
Compounds 6c, 6f, and 6g havealsodisplayed anencouraging
anti-tubercular activity profile. Further, some title compounds
were also assessed for their cytotoxic activity (IC50) against in
a mammalian Vero cell line using MTT assay. The results
reveal that these compounds exhibit anti-tubercular activity at
non-cytotoxic concentrations.
Keywords Anti-tubercular activity ꢀ Cytotoxicity ꢀ
2-Amino-5-substituted-1,3,4-thiadiazoles ꢀ
Imidazo-(2,1-b)-1,3,4-thiadiazoles
Introduction
Tuberculosis (TB) is the oldest documented infectious
disease. It is a chronic necrotizing bacterial infection with
wide variety of manifestations caused by Mycobacterium
tuberculosis, which has plagued humans throughout
recorded and archeological history (Dutt and Stead, 1999).
The introduction of the first line drugs like streptomycin,
para-aminosalicylic acid, isoniazid etc., for treatment some
50 years ago has witnessed in a remarkable decline in TB
cases all over the world. The active TB is currently treated
with a four-drug regimen comprising mainly isoniazid,
rifampicin, pyrazinamide and ethambutol for a period of at
least 6 months (Snider and Roper, 1992; Bass et al., 1994).
Having these powerful weapons in armamentarium, the
battle against TB at least in industrialized countries seemed
to be over during 1970s. But it did not take a long time for
M. tuberculosis to find its way around these compounds,
since the mid of 1980s, the disease has been undergoing a
resurgence driven by variety of changes in social, medical
and economic factors. The long treatment regime can be
difficult to fully complete, fueling the development of
multi-drug resistant (MDR) TB that allowed M. tubercu-
losis to escape the current drug arsenal and created
frightening epidemics in affected countries. In the absence
of an effective therapy, patients with MDRTB continue to
spread the disease, producing new infections with the
M. B. Palkar (&) ꢀ V. S. Maddi
Department of Pharmaceutical Chemistry, K.L.E.S. College
of Pharmacy, Vidyanagar, Hubli 580031, Karnataka, India
e-mail: palkarmahesh4u@rediffmail.com
M. N. Noolvi
Department of Pharmaceutical Chemistry, ASBASJSM College
of Pharmacy, Bela, Ropar 140111, Punjab, India
M. Ghatole
Department of Microbiology, Dr. V. M. Medical College,
Solapur 413 003, Maharastra, India
L. G. Nargund
Department of Pharmaceutical Chemistry, Nargund College
of Pharmacy, Dattatreyanagar, Bangalore 560085,
Karnataka, India
123

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Med Chem Res (2012) 21:1313–1321
resistant strains. Since the MDRTB microbe is more
infectious and virulent than the sensitive M. tuberculosis,
and until new drugs that are effective against MDR strains
are introduced, TB epidemic will grow at an exponential
level (Rattan et al., 1998 and Weil, 1994). The other
alarming fact is that the outbreak of HIV during the 1980s
has exposed the frailties of the current drug armamentar-
ium and has been accelerating the spread of TB all over the
world (Davies, 2000).
activities (Mazzone et al., 1984; Srivastava and Pathak,
1991; Gadad et al., 1999, Terzioglu and Aysel, 2003; Ja-
quith et al., 2010). In addition, the reports of Kolavi et al.,
(2006) and Gadad et al., (2004) on the synthesis, antimi-
crobial and anti-tubercular activity of a series of 2,5,
6-trisubstituted imidazo(2,1-b)-1,3,4-thiadiazoles and 2,6-
disubstituted imidazo(2,1-b)-1,3,4-thiadiazole derivatives
respectively, which have exhibited moderate to excellent
anti-tuberculosis activity, are the driving force for select-
ing imidazo(2,1-b)-1,3,4-thiadiazole nucleus. Although,
recently Gadad et al., (2008) have reported the synthesis
and biological evaluation of 2-trifluoromethyl/sulfona-
mido-5,6-diaryl substituted imidazo(2,1-b)-1,3,4-thiadia-
zole derivatives as cyclooxygenase-2 inhibitors and also
Pyl et al., (1963) have reported earlier the synthesis of
2-methyl-5,6-unsubstituted diaryl imidazo(2,1-b)1,3,4-
thiadiazole derivatives, but there are no reports on anti-
tubercular activity of substituted 5,6-diarylimidazo(2,1-
b)1,3,4-thiadiazoles. Therefore, in view of the above facts
and in continuation of our search on biologically active
heterocyclic systems (Palkar et al., 2010), in this article, we
report the synthesis and spectral studies of novel series of
2-substituted-5,6-diarylsubstituted imidazo(2,1-b)1,3,4-thi-
diazole derivatives (6a–r) and preliminary evaluation of in
vitro anti-tubercular as well as cytotoxic activity.
Today, TB is among the top five causes of global
mortality. One third of the world’s population is currently
infected with M. tuberculosis; 10% of infected cases will
develop clinical disease. According to the World Health
Organization (WHO) facts sheets, TB kills 2 million people
each year. It is estimated that between 2000 and 2020,
nearly 1 billion people will be newly infected; in other
words, someone in the world is newly infected with TB
every second. 200 million people will get sick and 35
million will die from TB if control is not further
strengthened (Maher, 2002; Dye, 2002). No new class of
anti-tuberculosis agents has been developed since the
introduction of rifampin into clinic in 1960s. Therefore,
there is an urgent need for development of innovative anti-
TB agents to effectively combat TB, with improved
properties such as enhanced activity against MDR strains,
reduced toxicity and shortened duration of therapy. From
the mid-1990s, this infectious disease was the focus of
renewed scientific interest. In the last 10 years, the research
on M. tuberculosis has undergone much progress. The
thriving accomplishment of the genome of M. tuberculosis
has offered a promise of a new generation of potent drugs
to combat the emerging epidemic of TB. The emphasis of
mycobacterial research now has shifted from gene hunting
to interpretation of the biology of the whole organism in an
effort to better define which activities is likely to be critical
to survival and thus amenable to the development of new
drugs (Cole et al., 1998; Barry et al., 2000). In this regard,
there have been few additions of some promising new anti-
tuberculosis agents, such as the long acting rifamycins,
fluoroquinolones, oxazolidinones and nitroimidazopyrans
to the existing main-line drugs (Ian, 2001).
Chemistry
Synthesis of 1,2-(p-substituted)diaryl-1-ethanones 3a–e
was carried out by reacting appropriate phenyl acetic acid
1a, b with various substituted aromatic hydrocarbons
2a–d. Subsequently, 3a–e were subjected to bromination
using liquid bromine in chloroform to obtain a-bromo-1,
2-(p-substituted)diaryl-1-ethanones 4a–e as shown in
Scheme 1 (Veeramaneni et al., 2003; Shimokawa et al.,
1979).
The synthesis of 2-substituted-5,6-diarylsubstituted
imidazo(2,1-b)-1,3,4-thiadiazole derivatives 6a–r was
carried out by the condensation of 4a–e with 2-amino-5-
substituted-1,3,4-thiadiazole 5a–d under reflux in dry
ethanol (as depicted in Scheme 2). This reaction proceeds
via intermediate iminothiadiazole I, which spontaneously
undergoes ring closer to II under reflux temperature to
afford the desired fused heterocycles 6a–r with good
yields.
Imidazoles certainly belong among the most important,
significant and abundant five-membered heterocycles,
which are constituents of a variety of natural and synthetic
products. The advent of sulfur drugs and the later discovery
of mesoionic compounds greatly accelerated the rate of
progress in the field of thiadiazoles. The thiadiazole and
imidazole compounds are extensively studied due to their
wide spectrum of bioactivities. Among them the imi-
dazo(2,1-b)-1,3,4-thiadiazole derivatives are pharmaco-
logically important because of their immunostimulant,
anti-inflammatory, analgesic, antifungal, antimicrobial,
antileishmanial, antitumor, anti-tuberculosis and other
The substitution at fifth position of 2-amino-5-substi-
tuted-1,3,4-thiadiazole is crucial in determining the course
of its reaction with substituted a-bromo-1,2-(p-substi-
tuted)diaryl-1-ethanone, such type of reactions have been
studied by deStevens et al., (1993) in case of 2-amino-5-
substituted-1,3,4-thiadiazole derivatives. Pyl et al., (1963)
have described the synthesis 5,6-unsubstituted diaryl
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Med Chem Res (2012) 21:1313–1321
1315
Scheme 1 Synthetic route
of a-bromo-1-(400-
R
R
R
substituted)phenyl-2-(40-
substituted)phenyl-1-ethanones
(4a–e). Reagents and
R1
(a)
(b)
+
R1
R1
conditions: (a) H3PO4,
(CF3CO)2O, 25°C, stir, 1 min;
(b) Br2, CHCl3, 50°C, stir, 0.5 h
Br
O
O
2a, R1= H
COOH
1a, R = H
1b, R =OCH3
3a, R= H, R1= SCH3
3b, R= OCH3, R1= SCH3
3c, R= OCH3, R1= OCH3
3d, R= OCH3, R1= H
3e, R= H, R1= CH3
2b, R1= CH3
2c, R1= OCH3
2d, R1= SCH3
4a, R= H, R1= SCH3
4b, R= OCH3, R1= SCH3
4c, R= OCH3, R1= OCH3
4d, R= OCH3, R1= H
4e, R= H, R1= CH3
Scheme 2 Synthetic route of a
novel series of 2-substituted-
5,6-diarylsubstituted imidazo-
(2,1-b)-1,3,4-thiadiazole (6a–r).
Reagents and conditions:
(a) absolute alcohol, reflux,
6–12 h, P2O5, reflux, 4–9 h,
Na2CO3
R
N N
(a)
+
4a-e
R2
NH2
R1
Br
H
O
S
+
N N
5a, R2= CF3
5b, R2= SO2NH2
5c, R2= 4-F-C6H4
R2
S
NH
5d, R2= 4-OCH3-C6H4
I
- HBr
R
4'
R
R1
5
R1
H
4
N
3
N
4''
- H2O
6
N N
S
7a N
OH
R2
2
N
7
S
R2
1
6a-r
a: R= H, R1= SCH3, R2= CF3
II
k: R= H, R1= SCH3, R2= 4-F-C6H4
b: R= OCH3, R1= SCH3, R2= CF3
c: R= OCH3, R1= OCH3, R2= CF3
d: R= OCH3, R1= H, R2= CF3
e: R= H, R1= CH3, R2= CF3
l: R= OCH3, R1= SCH3, R2= 4-F-C6H4
m: R= OCH3, R1= OCH3, R2= 4-F-C6H4
n: R= OCH3, R1= H, R2= 4-F-C6H4
o: R= H, R1= SCH3, R2= 4-OCH3-C6H4
p: R= OCH3, R1= SCH3, R2= 4-OCH3-C6H4
q: R= OCH3, R1= OCH3, R2= 4-OCH3-C6H4
r: R= H, R1= CH3, R2= 4-OCH3-C6H4
f: R= H, R1= SCH3, R2= SO2NH2
g: R= OCH3, R1= SCH3, R2= SO2NH2
h: R= OCH3, R1= OCH3, R2= SO2NH2
i: R= OCH3, R1= H, R2= SO2NH2
j: R= H, R1= CH3, R2= SO2NH2
imidazo(2,1-b)-1,3,4-thiadiazole derivatives starting with
2-amino-5-substituted-1,3,4-thiadiazole.
of the screening program is to provide a resource
whereby new experimental compounds can be tested for
their capacity to inhibit the growth of virulent M.
tuberculosis.
Biological activity
Cytotoxic activity
Anti-tubercular activity
The cellular conversion of MTT [3-(4,5-dimethylthiazo-2-
yl)-2,5-diphenyl-tetrazolium bromide] into a formazan
product (Mosmann, 1983) was used to evaluate cytotoxic
activity (IC50) of some synthesized compounds (6a–c, f–j,
l, m and o–q) against a mammalian Vero cell line upto
concentrations of 62.5 lg/ml using the Promega Cell Titer
96 non-radioactive cell proliferation assay (Gundersen
et al., 2002). The IC50 values were determined and are
presented in Table 3.
Minimum inhibitory concentration (MIC) values pre-
sented in Table 3 were determined for title compounds
6a–r against M. tuberculosis strain H37Rv using the
Micro plate Alamar Blue assay (MABA) (Franzblau
et al., 1998). This methodology is nontoxic, uses a
thermally stable reagent and shows good correlation with
proportional and BACTEC radiometric methods (Vanitha
and Paramasivan 2004; Reis et al., 2004). The purpose
123

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Med Chem Res (2012) 21:1313–1321
Results and discussion
Synthetic and spectral studies
In general, the IR spectra of these compounds showed
moderately strong bands around 3232–3454, 2921–2969 and
1594–1618 cm-1, characteristic of the SO2NH2, CH3 and
C=N groups, respectively. In the nuclear magnetic resonance
spectra (1H NMR), the signals of the respective protons of the
synthesized compounds were verified on the basis of their
chemical shifts, multiplicities and coupling constants. In 1H-
NMR spectra, the synthesized compounds showed promi-
nent signals for the aromatic protons as multiplets between d
6.77–8.10 ppm. Compounds 6f–j showed a characteristic
singlet between d 8.60–8.82 ppm indicating the presence of
SO2NH2 group. The peaks appeared around d 1.15–1.22,
1.92–2.03 and 3.75–3.88 ppm confirms the presence of CH3,
SCH3 and OCH3 groups, respectively.
We have synthesized a novel series of 2-substituted-5,6-
diarylsubstituted imidazo(2,1-b)-1,3,4-thiadiazole deriva-
tives (6a–r) by reacting 2-amino-5-substituted-1,3,4-thia-
diazole (5a–d) with an appropriately substituted a-bromo-
1,2-(p-substituted)diaryl-1-ethanones (4a–e) as illustrated
in Scheme 2. All the newly synthesized compounds gave
satisfactory analysis for the proposed structures, which
were confirmed on the basis of physicochemical, elemental
analysis and spectral data that are summarized in Tables 1
and 2, respectively.
Table 1 Physico-chemical data of a novel series of 2-substituted-5,6-diarylsubstitued imidazo(2,1-b)-1,3,4-thiadiazole derivatives (6a–r)
R
4'
R1
5
4
N
3
N
4''
6
7a N
R2
2
7
S
1
Compound
Structure
R
Yield (%)
Mp (°C)
Rf valuec
Mol. weight
R1
R2
6a
6b
6c
6d
6e
6f
H
SCH3
SCH3
OCH3
H
CF3
CF3
CF3
CF3
CF3
72.50
60.90
57.35
63.50
46.52
55.30
43.68
39.90
68.91
61.25
49.34
57.26
62.78
44.56
52.89
66.02
59.23
69.62
162–164
142–144
110–112
138–140
178–180
290–292
314–318
298–300
302–304
276–280
210–212
196–200
222–224
216–218
232–234
204–206
184–188
190–194
0.42a
0.54a
0.50a
0.62b
0.52c
0.49c
0.68b
0.60b
0.55b
0.48a
0.53a
0.71b
0.59b
0.43a
0.65b
0.57b
0.62b
0.74b
391.40
421.50
405.40
375.40
359.40
402.50
432.50
416.50
386.45
370.45
417.50
447.55
431.50
401.45
429.60
459.60
443.50
397.50
OCH3
OCH3
OCH3
H
CH3
H
SCH3
SCH3
OCH3
H
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
6g
6h
6i
OCH3
OCH3
OCH3
H
6j
CH3
6k
6l
H
SCH3
SCH3
OCH3
H
4-F-C6H4
4-F-C6H4
4-F-C6H4
4-F-C6H4
OCH3
OCH3
OCH3
H
6m
6n
6o
6p
6q
6r
SCH3
SCH3
OCH3
CH3
4-OCH3-C6H4
4-OCH3-C6H4
4-OCH3-C6H4
4-OCH3-C6H4
OCH3
OCH3
H
All synthesized compounds were purified by flash chromatography using 200–400 mesh silica gel
a
Mobile phase choloroform:hexane (1:9)
b
Mobile phase ethyl acetate:hexane (2:8)
c
Iodine vapors or 10% aqueous potassium permanganate solution was used as visualizing agent
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Med Chem Res (2012) 21:1313–1321
1317
Table 2 Spectral and elemental analysis data of a novel series of 2-substituted-5,6-diarylsubstitued imidazo(2,1-b)-1,3,4-thiadiazole derivatives
(6a–r)
Compound IR (KBr, cm-1
)
1H NMR (DMSO-d6, d, ppm)
Elemental analysis
6f
3440, 3020, 2930, 2855,
1610, 1572, 1463, 1239,
1171, 848
8.82 (s, 2H, SO2NH2), 7.95 (d, J = 8.5 Hz, 2H, aryl-H), Anal. calcd. for C17H14N4O2S3 C, 50.73;
7.79 (d, J = 8.5 Hz, 2H, aryl-H), 7.63-7.74 (m, 3H, aryl- H, 3.51; N, 13.92. Found: C, 50.73; H,
H), 7.42 (d, J = 8.8 Hz, 2H, aryl-H), 1.97 (s, 3H, 400-
SCH3)
3.48; N, 13.97
6g
3454, 3060, 2961, 2873,
1612, 1578, 1469, 1402,
1235, 1162, 881
8.76 (s, 2H, SO2NH2), 8.24 (d, J = 8.3 Hz, 2H, aryl-H), Anal. calcd. for C18H16N4O3S3 C, 49.98;
8.08 (d, J = 8.3 Hz, 2H, aryl-H), 7.57 (d, J = 8.5 Hz, H, 3.73; N, 12.95. Found: C, 50.03; H,
2H, aryl-H), 7.26 (d, J = 8.5 Hz, 2H, aryl-H), 3.84 (s, 3H, 3.72; N, 12.90
40-OCH3), 2.02 (s, 3H, 400-SCH3)
6j
6l
3409, 3115, 2921, 2844,
1618, 1489, 1360, 1243,
1179, 861
8.60 (s, 2H, SO2NH2), 7.52 (d, J = 8.8 Hz, 2H, aryl-H), Anal. Calcd. for C17H14N4O2S2 C,
7.46 (d, J = 8.8 Hz, 2H, aryl-H), 6.94-7.11 (d, 3H, aryl- 55.12; H, 3.81; N, 15.12. Found: C,
H), 6.83 (d, J = 8.0 Hz, 2H, aryl-H), 1.15 (s, 3H, CH3)
55.12; H, 3.77; N, 15.09
3029, 2946, 2844, 1600,
1529, 1435, 1228, 1134,
755
7.91 (d, J = 8.3 Hz, 2H, aryl-H), 7.82 (d, J = 8.3 Hz, 2H, Anal. calcd. for C24H18FN3OS2 C,
aryl-H), 7.69 (d, J = 8.9 Hz, 2H, aryl-H), 7.53 (d,
J = 8.9 Hz, 2H, aryl-H), 6.93 (d, J = 8.0 Hz, 2H, aryl-
H), 6.77 (d, J = 8.0 Hz, 2H, aryl-H), 3.81 (s, 3H, 40-
OCH3), 1.92 (s, 3H, 400-SCH3)
64.41; H, 4.05; N, 9.39. Found: C,
64.40; H, 4.07; N, 9.39
6m
3010, 2949, 2835, 1596,
1461, 1366, 1269, 1114,
771
7.85 (d, J = 8.1 Hz, 2H, aryl-H), 7.76 (d, J = 8.1 Hz, 2H, Anal. calcd. for C24H18FN3O2S C,
aryl-H), 7.49 (d, J = 8.0 Hz, 2H, aryl-H), 7.35 (d,
J = 8.0 Hz, 2H, aryl-H), 7.05 (d, J = 8.6 Hz, 2H, aryl-
H), 6.90 (d, J = 8.6 Hz, 2H, aryl-H), 3.91 (s, 3H, 40-
OCH3), 3.83 (s, 3H, 400-OCH3)
66.81; H, 4.20; N, 9.74. Found: C,
66.85; H, 4.26; N, 9.79
6n
6o
6p
3050, 2938, 2832, 1606,
1502, 1457, 1392, 1255,
1162, 786
8.02 (d, J = 8.9 Hz, 2H, aryl-H), 7.84 (d, J = 8.9 Hz, 2H, Anal. calcd. for C23H16FN3OS C, 68.81;
aryl-H), 7.59 (d, J = 8.3 Hz, 2H, aryl-H), 7.33–7.20 (m, H, 4.02; N, 10.47. Found: C, 68.80; H,
5H, aryl-H), 6.97 (d, J = 8.3 Hz, 2H, aryl-H), 3.75 (s, 3H, 4.06; N, 10.47
40-OCH3)
3035, 2939, 2867, 1603,
1516, 1444, 1287, 1149,
769
8.10 (d, J = 8.7 Hz, 2H, aryl-H), 7.92 (d, J = 8.7 Hz, 2H, Anal. calcd. for C24H19N3OS2 C, 67.11;
aryl-H), 7.65 (d, J = 8.2 Hz, 2H, aryl-H), 7.53–7.38 (m, H, 4.46; N, 9.78. Found: C, 67.10; H,
5H, aryl-H), 7.05 (d, J = 8.2 Hz, 2H, aryl-H), 3.82 (s, 3H, 4.46; N, 9.73
OCH3), 1.93 (s, 3H, 400-SCH3)
3029, 2960, 2840, 1613,
1509, 1488, 1359, 1181,
777
7.94 (d, J = 8.3 Hz, 2H, aryl-H), 7.79 (d, J = 8.3 Hz, 2H, Anal. calcd. for C25H21N3O2S2 C, 65.33;
aryl-H), 7.67 (d, J = 8.5 Hz, 2H, aryl-H), 7.26 (d,
J = 8.5 Hz, 2H, aryl-H), 7.13 (d, J = 8.1 Hz, 2H, aryl-
H), 6.86 (d, J = 8.1 Hz, 2H, aryl-H), 3.87 (s, 3H, 40-
OCH3), 3.80 (s, 3H, OCH3), 1.99 (s, 3H, 400-SCH3)
H, 4.61; N, 9.14. Found: C, 65.35; H,
4.66; N, 9.17
Anti-tubercular activity
compounds having an enhanced anti-tubercular activity
with MIC values ranging from 1.25–5.0 lg/ml. Amongst
them, compounds 6c and 6h (MIC = 1.25 lg/ml) exhib-
ited a significant activity. However, when p-methoxy on
sixth aromatic ring is replaced by p-thiomethyl groups
(compounds 6b, 6g, 6l and 6p), it resulted in compounds
with decreased anti-tubercular activity. Amongst them,
compound 6g (MIC = 2.5 lg/ml) showed a respectable
activity when compared with first line drugs such as iso-
niazid (MIC = 0.25 lg/ml) and gatifloxacin (MIC =
1.0 lg/ml). However, when a methyl group was introduced
on sixth aromatic ring (compounds 6e, 6j and 6r) resulted
in either complete loss of anti-tubercular activity or least
active derivatives. It was also observed that when there is
no substitution on fifth aromatic ring of imidazo(2,1-b)-
1,3,4-thiadiazole nucleus (compounds 6e and 6k), the anti-
tubercular activity was either decreased or lost. Apart
from sulfonamide group at second position of imidazo
All the synthesized compounds (6a–r) were evaluated for
their preliminary in-vitro anti-tubercular activity against M.
tuberculosis strain H37Rv by using MABA method. The
result of anti-tubercular activity is presented in Table 3. All
the synthesized compounds exhibited an interesting activ-
ity profile against the tested mycobacterial strain. The brief
structural activity relationships reveal that the activity is
considerably affected by various substituents at second
position of imidazo(2,1-b)-1,3,4-thiadiazole nucleus. It has
been observed that among the series, the compounds 6f–
j having sulfonamido group at second position of imi-
dazo(2,1-b)-1,3,4-thiadiazole ring exhibited the significant
anti-tubercular activity while other compounds showed
poor to moderate activity. It is interesting to note that when
both fifth and sixth aromatic rings are substituted with p-
methoxy (compounds 6c, 6h, 6m and 6q), it resulted in
123

1318
Med Chem Res (2012) 21:1313–1321
(2,1-b)-1,3,4-thiadiazole nucleus, we have also studied the
influence of trifluoromethyl, p-flurophenyl and p-meth-
oxyphenyl moities on the biological activity, we observed
that such replacement in the core nucleus did not vary the
anti-tubercular activity to a greater extent.
Table 3 The in vitro anti-tubercular and cytotoxic activity data of a
novel series of 2-substituted-5,6-diarylsubstitued imidazo(2,1-b)-
1,3,4-thiadiazole derivatives (6a–r)
R
4'
Cytotoxic activity
R1
5
4
N
3
N
4''
Some compounds 6a–c, f–j, l, m, and o–q were further
examined for toxicity (IC50) in a mammalian Vero cell line
upto 62.5 lg/ml concentrations. After 72 h of exposure,
viability was assessed on the basis of cellular conversion of
MTT into a formazan product using the Promega Cell Titer
96 non-radioactive cell proliferation assay and results are
summarized in Table 3. Among the thirteen derivatives
tested, showed IC50 values ranging from 115.9 to
246.6 lM. All compounds did not show significant activity
against mammalian Vero cell line at concentra-
tions \ 100 lM. Among the test compounds, 2-sulfon-
amido derivatives 6g–i showed inferior toxicity with IC50
values of [200 lM. A comparison of the substitution pat-
tern at second position of imidazo(2,1-b)thiadiazole nucleus
demonstrated that trifluromethyl, p-flurophenyl and
p-methoxyphenyl substituted analogs were more cytotoxic
than the sulfonamido substituted analogs. These results are
important as these compounds with their increased cytoli-
ability are much attractive in the development of new
chemical entities for the treatment of TB. This is primarily
due to the fact that the eradication of TB requires a lengthy
course of treatment, and the need for an agent with a high
margin of safety becomes a primary concern. The IC50 for
the compounds 6h was found to be 246.6 lM against the
cell line tested.
6
7a N
R2
2
7
S
1
(6a-r)
Compound Structure
MICa
IC50 (lM)b
R
R1
R2
6a
H
SCH3 CF3
10.0
5.0
139.6
122.7
147.3
6b
OCH3 SCH3 CF3
OCH3 OCH3 CF3
6c
1.25
6d
OCH3
H
H
CF3
CF3
Resistant NT
Resistant NT
6e
CH3
6f
H
SCH3 SO2NH2
2.5
192.2
6g
OCH3 SCH3 SO2NH2
OCH3 OCH3 SO2NH2
2.5
213.1
246.6
204.5
168.1
6h
1.25
5.0
6i
OCH3
H
H
SO2NH2
SO2NH2
6j
CH3
10.0
6k
H
SCH3 4-F-C6H4
Resistant NT
6l
OCH3 SCH3 4-F-C6H4
OCH3 OCH3 4-F-C6H4
10.0
5.0
115.9
163.4
6m
6n
OCH3
H
H
4-F-C6H4
Resistant NT
6o
SCH3 4-OCH3-C6H4 10.0
134.8
120.3
161.9
6p
OCH3 SCH3 4-OCH3-C6H4 10.0
OCH3 OCH3 4-OCH3-C6H4 5.0
6q
6r
H
CH3
4-OCH3-C6H4 Resistant NT
Conclusion
Isoniazid
Gatifloxacin
0.25
1.0
[450
[550
In this research article, we report the synthesis, spectral
studies, and preliminary biological evaluation of a novel
series of 2-substitued-5,6-diarylsubstituted imidazo(2,1-b)-
1,3,4-thiadiazole derivatives (6a–r) as possible anti-tuber-
cular agents. These fused hetrocyclic compounds were
prepared by the cyclodehydration process between
2-amino-5-substituted-1,3,4-thiadiazole derivatives (5a–
d) and a-bromo-1,2-(p-substituted)diaryl-1-ethanones (4a–
e). The preliminary in-vitro anti-tuberculosis screening of
these title molecules has evidenced that 40-OCH3 and 400-
OCH3 substituted analogs 6c, 6h, 6m, and 6q have
emerged as potential compounds endowed with moderate
to good anti-tuberculosis activity. However, the replace-
ment of sulfonamido by trifluoromethyl, p-flurophenyl and
p-methoxyphenyl group at second position showed
decrease in the anti-tuberculosis activity. Further, some
NT not tested
a
Minimal inhibition concentration is expressed in lg/ml
b
Cytotoxicity is expressed as IC50, is the concentration of com-
pound, which is reduced by 50% of the optical density of treated cells
with respect to untreated cells using the MTT assay
title compounds were also assessed for their cytotoxic
activity (IC50) against in a mammalian Vero cell line using
MTT assay. The results indicated that these compounds
exhibit anti-tubercular activity at non-cytotoxic concen-
trations. The possible improvements in the anti-tubercular
activity can be further achieved by slight modifications in
the ring substituents and/or extensive additional function-
ation warrants further investigations. Our findings will
have impact on medicinal chemists and pharmacists for
123

Med Chem Res (2012) 21:1313–1321
1319
General procedure for the synthesis of a-bromo-1-(400-
substituted)phenyl-2-(40-substituted)phenyl-1-
further investigations in this field in search of potent anti-
tubercular agents.
ethanones (4a–e)
To a constantly stirred solution of 3a–e (0.02 mol) in
chloroform (30 ml) kept at 50°C was added dropwise
bromine (0.022 mol) with stirring. After being stirred at
50°C for 0.5 h, the mixture was washed successively with
aqueous 10% sodium thiosulphate (hypo) solution and
water. The solvent was removed in-vacuo to obtain the
compounds (4a–e) either as oil/ solid mass/crystaline
compounds, which were used in the next step.
Experimental
Chemistry protocols
All research chemicals/ solvents were purchased from
Sigma-Aldrich or Lancaster Co. and used as such for
the reactions. Solvents except LR grade were dried and
purified according to the literature when necessary.
Reactions were monitored by thin layer chromatography
(TLC) on pre-coated silica gel plates from E-Merck Co
and compounds visualized either by exposure to iodine
vapors or dipping in 10% aqueous potassium permanga-
nate solution.
General procedure for the synthesis of 2-substituted-
5,6-diarylsubstituted imidazo(2,1-b)-1,3,4-thiadiazole
(6a–r, Scheme 2)
A mixture of 2-amino-5-substituted-1,3,4-thiadiazole (one of
5a–d, 0.002 mol) and an appropriate a-bromo-1-(400-substi-
tuted)phenyl-2-(40-substituted)phenyl-1-ethanones (one of
4a–e, 0.002 mol) in dry ethanol (150 ml) was heated to reflux
on a water bath for 6–12 h, phosphorous pentoxide
(0.0003 mol) was added and refluxing was continued for
another 4–9 h. The reaction mixture was cooled overnight at
room temperature. Excess of solvent was removed under
reduced pressure and the solid hydrobromide separated was
filtered, washed with cold ethanol and dried. Neutralization of
hydrobromide salts with cold aqueous solution of Na2CO3
yielded the corresponding free bases (6a–r), which were
recrystallized from dry ethanol. Further, the compounds were
purified by flash chromatography using 200–400 mesh silica
gel and eluted either with ethyl acetate:hexane (2:8) or chol-
oroform:hexane (1:9) as mobile phase. The physicochemical,
spectral, and elemental analysis data of the synthesized
compounds are depicted in Tables 1 and 2, respectively.
Melting point of synthesized compounds was deter-
mined in Thermonik melting point apparatus (Thermonik,
Mumbai, India) and is uncorrected. IR spectrum was
recorded on Thermo Nicolet IR200 FT-IR Spectrometer
1
(Madison, WI, USA) by using KBr pellets. The H-NMR
spectra were recorded on Bruker Avance II (400 MHz,
(Bruker, Rheinstetten/ Karlsruhe, Germany)) using CDCl3
or DMSO-d6 as solvent. Chemical shift are given in d ppm
units with respect to TMS. The elemental analysis (CHN)
of the compounds was performed on CHN Elemental
Analyser (Perkin Elmer 2400, Waltham, Massachusetts,
USA). Results of elemental analysis were within 0.3% of
the theoretical values. The purity of the compounds was
examined by TLC on silica gel G plate using choloro-
form:hexane (1:9) or ethyl acetate:hexane (2:8) as mobile
phase and iodine vapors or dipping in 10% aqueous
potassium permanganate solution as visualizing agent. The
compounds were purified by using flash chromatography
SP-01 (Biotage, Sweden) using 200–400 mesh silica gel.
Anti-tubercular activity
General procedure for the synthesis of 1-(400-
substituted)phenyl-2-(40-substituted) phenyl-1-
ethanones (3a–e, Scheme 1)
The anti-tubercular activity of title compounds (6a–r) was
assessed against M. tuberculosis H37Rv (ATTC 27294)
using the Micro plate Alamar Blue assay (MABA, Fran-
zblau et al., 1998). Succinctly, M. tuberculosis H37Rv is
grown in Middlebrook 7H9 broth (7H9 medium) supple-
mented with 0.2% (v/v) glycerol, 10% (v/v) ADC (albu-
min, dextrose, catalase), and 0.05% (v/v) Tween 80. The
bacteria are inoculated in 50 ml of 7H9 medium in 1-l
roller bottles that are placed on a roller bottle apparatus in
an ambient 37°C incubator. When the cells reach an OD600
of 0.150 (equivalent to *1.5 9 107 CFU/ml), they are
diluted 200-fold in 7H9 medium. Further, 200 ll of sterile
deionized water was added to all outer-perimeter wells of
sterile 96 well plates (falcon, 3072: Becton Dickinson,
Lincoln Park, NJ) to minimize evaporation of the medium
To a mixture of phenyl acetic acid/p-substituted phenyl
acetic acid (1a/b, 0.073 mol), substituted aromatic hydro-
carbon (one of 2a–d, 0.088 mol), 88–93% orthophosphoric
acid (0.088 mol) was added, followed by immediate care-
ful addition of trifluoroacetic anhydride (0.0295 mol) with
vigorous stirring at 25°C. The mixture turned into a dark
colored solution with vigorous exothermic reaction. The
reaction mixture was stirred for 1 min at the same tem-
perature and poured into ice-cold water (50 ml) with stir-
ring. Washed with cold hexane (2 9 10 ml) to obtain 3a–
e as solid. The percentage yield of compounds 3a–e were
found to be 76.3, 68.7, 73.7, 66.9, and 74.1%, respectively.
123

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Med Chem Res (2012) 21:1313–1321
biology of M. tuberculosis from complete genome sequence.
Nature 393:537–544
Davies PDO (2000) TB: the global epidemic. J Ind Med Assoc 98:
100–102
deStevens G, Eager M, Tarby C (1993) Steric and electronic effects
controlling the synthesis of bridgehead nitrogen heterocycles.
Heterocycles 35:763–773
Dutt AK, Stead WW (1999) Epidemiology and host factors. In:
Schlossberg D (ed) Tuberculosis and nontuberculous mycobac-
terial infection, 4th edn. W. B. Saunders Company, Philadelphia,
pp 3–16
in the test wells during incubation. The 96 plates received
93.75 ll of the cell suspension (*104 bacteria) in Mid-
dlebrook 7H9 medium (Difco laboratories, Detroit, MI,
USA) and a serial dilution of the compounds 6a–r was
made directly on the plate. The final drug concentrations
tested were 0.01–20.0 lg/ml. Plates were covered and
sealed with parafilm and incubated at 37°C for 5 days.
After this time, 25 ll of a freshly prepared 1:1 mixture of
Alamar Blue (Accumed International, Westlake Ohio)
reagent and 10% tween 80 was added to the plate and
incubated for 24 h. A blue color in the well was interpreted
as no bacterial growth, and a pink color was scored as
growth. The MIC was defined as the lowest drug concen-
tration, which prevented a color change from blue to pink.
Dye C (2002) The TB epidemic 2002–2020 (Abstract). In: Program of
the 4th world congress on tuberculosis, Washington DC, USA,
June 3–5
Franzblau SG, Witzig RS, McLaughlin JC, Torres P, Madico G,
Hernandez A, DegnanMT, Cook MB, Quenzer VK, Ferguson RM,
Gilman RH (1998) Rapid, low-technology MIC determination
with clinical Mycobacterium tuberculosis isolates by using the
microplate Alamar blue assay. J Clin Microbiol 36:362–366
Gadad AK, Karki SS, Rajurkar VG, Bhongade BA (1999) Synthesis
and biological evaluation of 5-formyl-6-arylimidazo(2,1-b)-
1,3,4-thiadiazole-2-N-dimethylaminomethiono) sulfonamides as
antitumor agents. Arzneium-Forsch/Drug Res 49:858–863
Gadad AK, Noolvi MN, Karpoormath RV (2004) Synthesis and
anti-tubercular activity of a series of 2-sulfonamido/trifluoro-
methyl-6-substituted imidazo(2,1-b)-1,3,4-thiadiazole deriva-
tives. Bioorg Med Chem 12:5651–5659
Gadad AK, Palkar MB, Anand K, Noolvi MN, Boreddy TS,
Wagwade J (2008) Synthesis and biological evaluation of
2-trifluoromethyl/sulfonamido-5,6-diaryl substituted imidazo
[2,1-b]-1,3,4-thiadiazoles: a novel class of cyclooxygenase-2
inhibitors. Bioorg Med Chem 16:276–283
Gundersen LL, Nissen-Meyer J, Spilsberg B (2002) Synthesis and
antimycobacterial activity of 6-arylpurines: the requirements for
the N-9 substituent in active antimycobacterial purines. J Med
Chem 45:1383–1386
Cytotoxic activity
The cellular conversion of MTT [3-(4,5-dimethylthiazo-2-
yl)-2,5-diphenyl-tetrazolium bromide] into a formazan
product was used to evaluate cytotoxic activity (IC50)
of some synthesized compounds (6a–c, f–j, l, m and
o–q) against a mammalian Vero cell line upto concentra-
tions of 62.5 lg/ml. After 72 h of exposure, cell viability
was assessed on the basis of cellular conversion of MTT
into a formazan product using the Promega Cell Titer 96
non-radioactive cell proliferation assay (Gundersen et al.,
2002).
Acknowledgments The authors are thankful to Principal, KLES’
College of Pharmacy, Hubli, for providing necessary facilities to
carry out this research work. We sincerely express our gratitude to Dr.
Y. S. Agasimundin, Professor, K.L.E.S’ College of Pharmacy Hubli,
for his encouragement. We are grateful to The Director, SAIF, Punjab
University, for providing elemental and spectral analysis. The authors
sincerely acknowledge AICTE, New-Delhi (India), for financial
support under RPS Scheme (File No. 8023/BOR/RID/RPS-170/2008-
09). Authors also express special thanks to Lupin Pharmaceutical
Industry, Aurangabad, (Maharashtra, India), for providing the gift
sample of INH and Gatifloxacin.
Ian O (2001) Guest commentary: search for new drugs for treatment
of tuberculosis, tuberculosis drug screening program. Antimic-
rob Agents Chemother 45:1943–1946
Jaquith JB, Villeneuve G, Boudreault A, Morris SJ, Durkin J, Gillard
JW, Hewitt K, Marsh HN (2010) Imidazo(2,1-b)-1,3,4-thiadia-
zole sulfonamides. United States Patent US 7,741,349 B2
Kolavi G, Hegde V, Khazi I, Gadad P (2006) Synthesis and
evaluation of anti-tubercular activity of imidazo(2,1-b)-1,3,4-
thiadiazole derivatives. Bioorg Med Chem 14:3069–3080
Maher D, Floyd K, Raviglione M (2002) Strategic framework to
decrease the burden of TB/HIV. WHO document WHO/CDS/
TB/2002. World Health Organization, Geneva, p 296
Mazzone G, Bonina F, Panico AM, Puglisi G, Marchetta G, Amico
RM, Caruso A, Blandino G (1984) Synthesis and preliminary
biological investigation of 2,6-diaryl substituted imidazo(1,2-b)-
1,3-4-thiadiazoles. Farmaco Ed Sci 39:585–589
Mosmann T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays.
J Immunol Methods 65:55–63
Palkar M, Noolvi M, Sankangoud R, Maddi V, Gadad A, Nargund
LVG (2010) Synthesis and antibacterial activity of a novel series
of 2,3-diaryl-substituted-imidazo(2,1-b)-benzothiazole Deriva-
tives. Arch Pharm Chem Life Sci 343:353–359
References
Barry CE, Slayden RA, Sampson AE, Lee RE (2000) Use of
genomics and combinatorial chemistry in the development of
new antimycobacterial drugs. Biochem Pharmacol 59:221–231
Bass JB, Farer LS, Hopewell PC, O0Brien R, Jacobs RF, Ruben F,
Dixie E, Snider J, Thornton G (1994) Treatment of tuberculosis
and tuberculosis infection in adults and children. Am J Respir
Crit Care Med 149:1359–1374
Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D,
Gordon SV, Eiglmeier K, Gas S, Barry CE III, Tekaia F,
Badcock K, Basham D, Brown D, Chillingworth T, Connor R,
Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S,
Hornsby T, Jagels K, Krogh A, McLean J, Moule S, Murphy L,
Oliver K, Osborne J, Quail MA, Rajandream MA, Rogers J,
Rutter S, Seeger K, Skelton J, Squares R, Squares S, Sulston JE,
Taylor K, Whitehead S, Barrell BG (1998) Deciphering the
Pyl T, Waschk F, Beyer H (1963) Synthesis of 1-amino-2-mercapto-
4,5-disubstituted imidazole. Justus Liebigs Ann Chem Bd
663:113–116
Rattan A, Kalia A, Ahmad N (1998) Multidrug-resistant M. tuber-
culosis: molecular perspectives. Emerg Infec Dis 4:195–209
Reis RS, Neves I Jr, Lourenco SLS, Fonseca LS, Lourenco MCS
(2004) Comparison of flow cytometric and alamar blue tests with
123

Med Chem Res (2012) 21:1313–1321
1321
the proportional method for testing susceptibility of Mycobac-
terium tuberculosis to rifampin and isoniazid. J Clin Microbiol
42:2247–2248
b][1,3,4]thiadiazole-5-carbohydrazide. Eur J Med Chem 38:
781–786
Vanitha JD, Paramasivan CN (2004) Evaluation of microplate Alamar
blue assay for drug susceptibility testing of Mycobacterium
avium complex isolates. Diagn Microbiol Infect Dis 49:179–182
Veeramaneni VR, Pal M, Yeleswarapu KR (2003) A high speed
parallel synthesis of 1,2-diaryl-1-ethanones via a clean-chemis-
try C–C bond formation reaction. Tetrahedron 59:3283–3290
Weil DEC (1994) Drug supply: meeting a global need. In: Porter
JDH, McAdam KPWJ (eds) Tuberculosis: back to the future.
Wiley, New York, pp 123–143
Shimokawa N, Nakamura H, Shimakawa K, Minami H, Nishimura H
(1979) Studies on analgesic agents. 1. Preparation of 1, 2-diphenyl-
2-(4-substituted 1-piperazinyl)ethanol derivatives and structure-
activity relationships. J Med Chem 22:58–63
Snider DE Jr, Roper WL (1992) The new tuberculosis (editorial).
New Engl J Med 326:703–705
Srivastava SK, Pathak RB (1991) Synthesis and fungitoxicity of some
1,3,4-thiadiazolo[3,2-a]-imidazoles and 3-(1,3,4-thiadiazol-2-yl)-
4-thiazolidinones. Indian J Chem 30B:620–623
Terzioglu N, Aysel G (2003) Synthesis and anticancer evaluation of
some new hydrazone derivatives of 2,6-dimethylimidazo[2,1-
123