Mannich base derivatives of 1,3,4-oxadiazole: Synthesis and screening against Entamoeba histolytica

Article abstract of DOI:10.1007/s00044-012-0108-9

Mannich base derivatives of 5-(pyridine-4-yl)-1,3,4-oxadiazole-2-(3H)- thione with substituted piperazine were synthesized and characterized. In vitro antiamoebic activity was performed against HM1: IMSS strain of Entamoeba histolytica and the cytotoxicit

Full text of DOI:10.1007/s00044-012-0108-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0108-9
ORIGINAL RESEARCH
Mannich base derivatives of 1,3,4-oxadiazole: synthesis
and screening against Entamoeba histolytica
•
Shadab Miyan Siddiqui Attar Salahuddin
•
Amir Azam
Received: 3 December 2011 / Accepted: 22 May 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Mannich base derivatives of 5-(pyridine-4-yl)-
1,3,4-oxadiazole-2-(3H)-thione with substituted piperazine
were synthesized and characterized. In vitro antiamoebic
activity was performed against HM1: IMSS strain of
Entamoeba histolytica and the cytotoxicity of the com-
pounds having IC50 value less than metronidazole was
assessed by MTT assay on human breast cancer MCF-7
cell line. The results revealed that compounds 2, 3 and 5
were found to be better inhibitors of E. histolytica than the
reference drug metronidazole and found low cytotoxic in
the concentration range of 2.5–250 lM. This study sug-
gests the possibility of developing 1,3,4-oxadiazole ana-
logues as potential drug candidates.
2009). Amoebiasis is emerging parasitic complication in
the human immunodeficiency virus (HIV)-infected patients
(Watanabe et al., 2011; Wu et al., 2008) and is responsible
for approximately 100,000 fatalities annually, making it the
second leading cause of death due to parasitic disease
(Ralston and Petri, 2011).
The treatment for amoebiasis is the administration of
nitroimidazoles such as metronidazole and tinidazole
(Fig. 1) but long-term uses of these medicaments produce
several side effects in patients (Ordaz-Pichardo et al.,
2005). Moreover, resistance of E. histolytica to metroni-
dazole has been reported (Becker et al., 2011) and the
treatment failure may emerge as major public health issue.
Therefore, new effective antiamoebic agents are required.
Some compounds having oxadiazole ring have been
reported to exhibit remarkable antiamoebic activities. For
instance, BTI 2286E ( )-E-3-(4-methylsulphinylstyryl)-
1,2,4-oxadiazole has been found to show potent amoebicidal
activity in a single-dose treatment against E. histolytica
infection in the liver of golden hamsters and the caeca of mice,
hamsters and rats (Bhopale et al., 1993). Apart from this,
5-(pyridine-4-yl)-1,3,4-oxadiazole-2-(3H)-thione, a promi-
nent heterocyclic scaffold, also have been found to be
endowed with antiamoebic activity (Siddiqui et al., 2012a).
Pyridine and piperazine rings are important components of
several anti-amoebic compounds (Hayat et al., 2010; Bhat
et al., 2009; Singh et al., 2006). Some N-phenyl piperazine
containingthiocarbamoyl pyrazolines (Abid and Azam, 2005;
Budakoti et al., 2007) and their metal complexes (Husain
etal., 2008) havebeenreportedto showexcellentantiamoebic
activities. Considering these perspectives, we decided to
design compounds derived from active core of 5-(pyridine-4-
yl)-1,3,4-oxadiazole-2-(3H)-thione containing substituted
piperazine (Fig. 2). The final compounds were considered to
be synthetic equivalents of thiosemicarbazone fragment
Keywords 1,3,4-Oxadiazole Á Mannich base Á
Antiamoebic activity Á E. histolytica Á MTT assay
Introduction
During the last few decades, the number of life threatening
infections caused by pathogenic protozoa has reached an
alarming level in the hospitals and community. Amoebia-
sis, a contagious disease of the human gastrointestinal tract
caused by parasitic protozoa Entamoeba histolytica, is one
of them (Tengku and Norhayati, 2011; Watanabe et al.,
2011). In general, the infection occurs by the ingestion of
cysts of E. histolytica in contaminated food and water, but
it has been reported that the parasite can be transmitted by
homosexual as well as heterosexual activity (Salit et al.,
S. M. Siddiqui Á A. Salahuddin Á A. Azam (&)
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar,
New Delhi 110025, India
e-mail: amir_sumbul@yahoo.co.in
123

Med Chem Res
O
NH
N
N
N
S
(a)
NH2
NO2
N
H
O
N
O
NO2
( 1 )
N
N
N
(b)
S
OH
Metronidazole
R
N
O
N
Tinidazole
N
O
N
S
Fig. 1 Standard drugs for amoebiasis treatment
N
(2-11)
embedded within the 1,2,4-triazole ring (Siddiqui et al.,
2012b). Combination of these active components within a
single molecular framework was expected to show remark-
able antiamoebic activity. Although some of the Mannich
base derivatives of 5-(pyridine-4-yl)-1,3,4-oxadiazole-2-
(3H)-thione were synthesized and evaluated for antimicrobial
and antimycobacterialactivities (Bayrak etal., 2009; Mamolo
et al., 2005; Oza and Patel, 2010; Singh et al., 1986), it was
important to evaluate these compounds against amoebiasis. In
this view a series of Mannich base derivatives of 5-(pyridine-
4-yl)-1,3,4-oxadiazole-2-(3H)-thione incorporating pipera-
zine ring (2–11) was synthesized and evaluated in vitro
against HM1: IMSS strain of E. histolytica.
Scheme 1 Synthesis of Mannich base derivatives of 5-(pyridine-4-
yl)-1,3,4-oxadiazole-2-(3H) thione (2–11). Reagents and conditions:
a CS2, KOH/H?, reflux 10 h b different substituted piperazines, 37 %
HCHO, EtOH, rt, 2 h
reaction. Mannich reaction is a three-component condensation
reaction involving active hydrogen containing compound,
formaldehyde and a primary or secondary amine and has
considerable importance for the synthesis and modification of
biologically active compounds (Reddy et al., 2008). The
structureofallthedesiredcompounds(2–11) waselucidatedby
spectral studies and their purity was confirmed by elemental
analyses.
Antiamoebic activity
Results and discussion
Preliminary experiments were carried out to determine the
in vitro antiamoebic activity of all the compounds (2–11)
by microdilution method using HM1: IMSS strain of
E. histolytica (Wright et al., 1988). All the experiments
were carried out in triplicate at each concentration level
and repeated thrice. The results are summarized in Table 1.
The data are present in terms of percent growth inhibition
relative to untreated controls, and plotted as probit values
as a function of drug concentration. The antiamoebic effect
Chemistry
Synthesis of Mannich base derivatives of 5-(pyridine-4-yl)-
1,3,4-oxadiazole-2-(3H)-thione (2–11) is out lined in
Scheme 1. The precursor 5-(pyridine-4-yl)-1,3,4-oxadiazole-
2-(3H)-thione (1) was prepared by the reported method (Reid
and Heindel, 1976). The NH proton at position-3 of 5-(pyri-
dine-4-yl)-1,3,4-oxadiazole-2-(3H)-thione (1) is adequately
acidic to obtain the target compounds (2–11) by Mannich
N
NH
Variation in substituted piperazines
S
O
Cl
N
N
N
IC50 = 0.55 µM
Active core moiety
Pyridyl-oxadiazole scaffold
R
N
S
N
Structural
Modification
N
N
N
NH
N
N
S
N
N
Shift in Piperazine position
and introduction of O in
thiosemicarbazone fragment
S
O
IC50 = 1.2 µM
Piperazine TSC
N
Ar
1, 3, 4-oxadiazole-2(3H)-thiones as
embeded thiosemicarbazone equivalents
IC50 = 0.28-10.8 µM
TSC-embeded triazoles
Fig. 2 Structural modification and rational designing
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Med Chem Res
Table 1 In vitro antiamoebic activity of Mannich base derivatives of 5-(pyridine-4-yl)-1,3,4-oxadiazole-2-(3H)-thione (2–11) against HM1:
IMSS strain of E. histolytica and cytotoxicity profile of compounds 2, 3, 5 and metronidazole
R
N
N
N
N
S
O
N
(2-11)
(2–11)
Compound no.
R
Antiamoebic activity
Cytotoxicity profile
IC50 (lM)
SDa
IC50 (lM)
SDa
0.24
2
3
0.245
1.06
0.009
[250
[250
F
0.015
0.018
0.27
O
4
3.47
N.D.
N.D.
5
6
C2H5
CH3
0.327
5.11
0.005
0.001
[250
N.D.
0.21
N.D.
Cl
7
2.57
0.007
N.D.
N.D.
8
9
3.40
2.82
0.013
0.014
N.D.
N.D.
N.D.
N.D.
N
O
O
10
3.62
0.006
N.D.
N.D.
123

Med Chem Res
Table 1 continued
Compound no.
R
Antiamoebic activity
Cytotoxicity profile
IC50 (lM)
SDa
IC50 (lM)
SDa
CF3
11
2.87
0.013
N.D.
N.D.
1.81
0.011
[250
0.89
N
N
NO2
HO
N.D. not done
a
Standard deviation
was compared with the most widely used antiamoebic
medication metronidazole which had a 50 % inhibitory
concentration (IC50) 1.81 lM in our experiments. The
results revealed that the oxadiazole derivatives (2–11)
(Fig. 3) showed IC50 values in the range 0.25–5.11 lM.
Among all the screened compounds (2–11), compounds 2,
3, and 5 exhibited IC50 values less than the standard drug
metronidazole, while the remaining compounds showed
IC50 values more than metronidazole and were considered
to be inactive. Structure activity relationship (SAR) showed
that the maximum activity was shown by compound 2
(IC50 = 0.245 lM), having 4-fluorophenyl group attached
with piperazine ring. The replacement of the 4-fluorophenyl
group with ethyl group (5, IC50 =0.327 lM)) did not reduce
the antiamoebic activity significantly, but the replacement
of the 4-fluoro phenyl group with 2-methoxyphenyl group
(3, IC50 = 1.06 lM) drastically reduced the antiamoebic
activity. The biological data showed that the antiamoebic
activity is dependent on substituent (R).
Cytotoxicity profile
The compounds (2, 3 and 5) having less IC50 value than
metronidazole were screened against human breast cancer
MCF-7 cell line. A sub-confluent population of MCF-7
cells was treated with increasing concentrations of com-
pounds and the number of viable cells was measured after
48 h by MTT cell viability assay based on mitochondrial
reduction of the yellow MTT tetrazolium dye to a highly
coloured blue formazone product. This assay usually shows
high correlation with number of living cells and cell pro-
liferation. The % cell viability shown by the compounds
(2, 3, 5 and metronidazole) at concentration range of
2.5–500 lM is given in Fig. 3. These results showed that
all the compounds and metronidazole were low cytotoxic
in the concentration range of 2.5–250 lM.
MTT Assay
100
Control
2
80
Experimental
3
60
40
20
0
5
Synthesis
MNZ
Melting points (mp) were recorded on a KSW apparatus
and are uncorrected. Elemental analysis was carried out on
CHNS Elemental Analyzer vario MICRO, Elementar
Analysensysteme GmbH, Germany. IR spectra were
recorded on Perkine-Elmer model 1600 FT-IR RX1 spec-
1
Concentration (µM)
trophotometer as KBr discs. The H NMR spectra were
recorded on Bruker Spectrospin DPX 300 MHz spec-
trometer using DMSO and CDCl3 as a solvent and tri-
methylsilane (TMS) as an internal standard. Chemical shift
Fig. 3 Percentage of viable cells after 48 h pre-treatment of human
breast cancer MCF-7 cells with compounds 2, 3, 5, and metronidazole
(MNZ), evaluated by the MTT assay
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Med Chem Res
3-[(4-Ethylpiperazin-1-yl)methyl]-5-pyridin-4-yl-
values are given in part per million (ppm) with respect to
TMS. Mass spectra were recorded by GC–MS (Perkin-
Elmer Clarus 500 GC).
1,3,4-oxadiazole-2(3H)-thione (5) Yield: 66 %; mp.160 °C;
Anal. calc. for C14H19N5OS: C 55.06, H 6.27, N 22.93, S
10.50 %; found: C 55.01, H 6.23, N 22.89, S 10.54 %; IR
tmax (cm-1): 2910 (CH3), 2849 (CH2), 1651, 1437 (C=N),
1355 (C=S), 1243 (C–O–C); 1H NMR (CDCl3) d (ppm): 1H
NMR (CDCl3) d (ppm): 8.71 (d, 2H, J = 3.0 Hz, Ar–H),
8.65 (d, 2H, J = 3.0 Hz, Ar–H), 7.76–7.62 (m, 4H, Ar–H),
5.14 (s, 2H, N–CH2–N), 3.55–2.96 (m, 10H, piperazine and
CH2) 1.42–1.37 (t, 3H, CH3); MS (EI, 70 eV): m/
z = 305.39 [M?].
Preparation of 5-(pyridine-4-yl)-1,3,4-oxadiazole-
2-(3H)-thione (1)
It was prepared by the reported method (Reid and Heindel,
1976).
General procedure for the synthesis of Mannich
bases of 5-(pyridine-4-yl)-1,3,4-oxadiazole-2-(3H)-
thione (2–11)
3-[(4-Methylpiperazin-1-yl)methyl]-5-(pyridin-4-yl)-
1,3,4-oxadiazole-2(3H)-thione (6) Yield: 65 %; m.p.:
240 °C; Anal. calc. for C13H17N5OS: C 53.59, H 5.88, N
24.04, S 11.00 %; found: C 53.63, H 5.91, N 24.01, S
11.04 %; IR tmax(cm-1): 2930 (CH3), 2852 (CH2), 1658,
1449 (C=N), 1365 (C=S),1263 (C–O–C); 1H NMR
(DMSO-d6) d (ppm): 8.67 (d, 2H, J = 6.0 Hz, Ar–H), 7.68
(d, 2H, J = 6.0 Hz, Ar–H), 5.19 (s, 2H, N–CH2–N),
3.28–2.80 (m, 8H, piperazine), 2.37 (s, 3H, CH3); MS (EI,
70 eV): m/z = 291.37 [M?].
A mixture of 5-(pyridine-4-yl)-1,3,4-oxadiazole-2 (3H)-
thione (0.01 mol), substituted piperazine (0.01 mol) and
37 % formaldehyde solution (1.5 mL), in ethanol (15 mL),
was stirred at room temperature for 2 h and allowed to stand
overnight. The precipitated crude product was filtered,
washed with water, dried, and recrystallized from ethanol.
3-{[4-(4-Fluorophenyl)piperazin-1-yl]methyl}-5-
(pyridin-4-yl)-1,3,4-oxadiazole-2(3H)-thione
(2) Yield:
3-{[4-(3-Chlorophenyl)piperazin-1-yl]methyl}-5-
(pyridin-4-yl)-1,3,4-oxadiazole-2(3H)-thione
68 %; m.p.: 172 °C; Anal. calc. for C18H18FN5OS: C
58.21, H 4.88, N 18.86, S 8.63 %; found: C 58.19, H 4.91,
N 18.83, S 8.59 %; IR tmax (cm-1): 3052 (Ar–CH), 2852
(CH2), 1612, 1446 (C=N), 1358 (C=S), 1239 (C–O–C); 1H
NMR (CDCl3) d (ppm): 8.82 (d, 2H, J = 3.0 Hz, Ar–H),
7.79 (d, 2H, J = 6.0 Hz, Ar–H), 6.98–6.82 (m, 4H, Ar–H),
5.17 (s, 2H, N–CH2–N), 3.14–3.00 (m, 8H, piperazine);
MS (EI, 70 eV): m/z = 371.43 [M?].
(7) Yield:
62 %; m.p.: 166 °C; Anal. calc. for C18H18ClN5OS: C
55.74, H 4.68, N 18.06, S 8.27 %; found: C 55.71, H 4.63,
N 18.09, S 8.31 %; IR tmax (cm-1): 3063 (Ar–CH), 2876
(CH2), 1636, 1458 (C=N), 1352 (C=S), 1263 (C–O–C); 1H
NMR (DMSO-d6) d (ppm): 8.78 (d, 2H, J = 6.0 Hz, Ar–
H), 7.78 (d, 2H, J = 6.0 Hz, Ar–H), 7.13–6.90 (m, 4H, Ar–
H), 5.13 (s, 2H, N–CH2–N), 3.27–2.92 (m, 8H, piperazine);
MS (EI, 70 eV): m/z = 387.88 [M?].
3-{[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-5-
(pyridin-4-yl)-1,3,4-oxadiazole-2(3H)-thione
(3) Yield:
3-[(4-Benzylpiperazin-1-yl)methyl]-5-(pyridin-4-yl)-
72 %; m.p.: 110 °C; Anal. calc. for C19H21N5O2S: C 59.51,
H 5.52, N 18.26, S 8.36 %; found: C 59.55, H 5.50, N
18.23, S 8.36 %; IR tmax (cm-1): 3032 (Ar–CH), 2941
(CH3), 2827 (CH2), 1618, 1439 (C=N), 1352 (C=S), 1233
(C–O–C); 1H NMR (CDCl3) d (ppm): 8.82 (d, 2H,
J = 6.0 Hz, Ar–H), 7.78 (d, 2H, J = 6.0 Hz, Ar–H),
7.00–6.82 (m, 4H, Ar–H), 5.18 (s, 2H, N–CH2–N), 3.82 (s,
3H, OCH3), 3.17–2.91 (m, 8H, piperazine); MS (EI,
70 eV): m/z = 383.46 [M?].
1,3,4-oxadiazole-2(3H)-thione (8) Yield: 58 %; mp:
180 °C; Anal. calc. for C19H21N5OS: C 62.10, H 5.76, N
19.06, S 8.73 %; found: C 62.14, H 5.73, N 19.07, S
8.75 %; IR tmax (cm-1): 3046 (Ar–CH), 2851 (CH2), 1608,
1440 (C=N), 1359 (C=S), 1234 (C–O–C); 1H NMR
(DMSO-d6) d (ppm): 8.69 (d, 2H, J = 3.0 Hz, Ar–H), 7.70
(d, 2H, J = 3.0 Hz, Ar–H), 7.22–7.18 (m, 5H, Ar–H), 4.97
(s, 2H, N–CH2–N), 3.59–2.99 (m, 10H, CH2Ph and
piperazine); MS (EI, 70 eV): m/z = 367.46 [M?].
3-[(4-Phenylpiperazin-1-yl)methyl]-5-(pyridin-4-yl)-
5-(Pyridin-4-yl)-3-{[4-(pyridin-2-yl)piperazin-1-yl]
1,3,4-oxadiazole-2(3H)-thione (4) Yield: 67 %; mp:
188 °C; Anal. calc. for C18H19N5OS: C 61.17, H 5.42, N
19.81, S 9.07 %; found: C 61.19, H 5.38, N 19.80, S
9.03 %; IR tmax (cm-1): 3042 (Ar–CH), 2854 (CH2), 1609,
1436 (C=N), 1354 (C=S), 1243 (C–O–C). 1H NMR
(CDCl3) d (ppm): 8.82–7.25 (m, 6H, Ar–H), 6.90 (d, 2H,
J = 6.0 Hz, Ar–H), 5.17 (s, 2H, N–CH2–N), 3.20–3.02 (m,
8H, piperazine); MS (EI, 70 eV): m/z = 353.44 [M?].
methyl}-1,3,4-oxadiazole-2(3H)-thione (9) Yield: 61 %;
m.p.: 154 °C; Anal. calc. for C17H18N6OS : C 57.61, H
5.12, N 23.17, S 9.05 %; found: C 57.66, H 5.15, N 23.13,
S 9.04 %; IR tmax (cm-1): 3051 (Ar–CH), 2823 (CH2),
1648, 1430 (C=N), 1363 (C=S), 1248 (C–O–C);1H NMR
(DMSO-d6) d (ppm): 8.57 (d, 2H, J = 6.0 Hz, Ar–H),
8.05–8.01 (m, 2H, Ar–H), 7.59 (d, 2H, J = 6.0 Hz, Ar–H),
123

Med Chem Res
7.52–7.39 (m, 2H, Ar–H), 4.05 (s, 2H, N–CH2–N),
3.58–3.06 (m, 8H, piperazine); MS (EI, 70 eV):
m/z = 354.42 [M?].
wells in the plate so that the wells were completely filled
(total volume, 340 lL). An inoculum of 1.7 9 104 organ-
isms/well was chosen so that confluent, but not excessive
growth, took place in control wells. Plate was sealed and
gassed for 10 min with nitrogen before incubation at 37 °C
for 72 h. After incubation, the growth of amoeba in the
plate was checked with a low power microscope. The
culture medium was removed by inverting the plate and
shaking gently. Plate was then immediately washed with
sodium chloride solution (0.9 %) at 37 °C. This procedure
was completed quickly and the plate was not allowed to
cool to prevent the detachment of amoeba. The plate was
allowed to dry at room temperature and the amoebae were
fixed with methanol and when dried, stained with (0.5 %)
aqueous eosin for 15 min. The stained plate was washed
once with tap water, then twice with distilled water and
then allowed to dry. A 200 lL portion of 0.1 N sodium
hydroxide solution was added to each well to dissolve the
protein and release the dye. The optical density of the
resulting solution in each well was determined at 490 nm
with a microplate reader. The % inhibition of amoebal
growth was calculated from the optical densities of the
control and test wells and plotted against the logarithm of
the dose of the drug tested. Linear regression analysis was
used to determine the best fitting line from which the IC50
value was found. The IC50 values are reported in Table (1).
3-((4-((Benzo[d][1,3]dioxol-5-yl)methyl)piperazine-1-
yl)methyl)-5(pyridine-4-yl)-1,3,4-oxadiazole-2(3H)-
thione (10) Yield: 64 %; mp: 146 °C; Anal. calc. for
C20H21N5O3S: C 58.38, H 5.14, N 17.02, S 7.79 %; found:
C 58.41, H 5.11, N 17.06, S 7.81 %; IR tmax (cm-1): 3024
(Ar–CH), 2878 (CH2), 1608, 1438 (C=N), 1363 (C=S),
1248 (C–O–C); 1H NMR (DMSO-d6) d (ppm): 8.67 (d, 2H,
J = 6.0 Hz, Ar–H), 7.69 (d, 2H, J = 6.0 Hz, Ar–H),
6.86–6.72 (m, 3H, Ar–H), 6.03–5.98 (m, 4H, N–CH2–N
and O–CH2–O), 3.45–2.90 (m, 4H, piperazine), 2.44–2.34
(m, 4H, piperazine); MS (EI, 70 eV): m/z = 411.47 [M?].
3-(2-(Trifluoromethyl)benzyl)-5-(pyridine-4-yl)-
1,3,4-oxadiazole-2(3H)-thione (11) Yield: 62 %; mp:
152 °C; Anal. calc. for C20H20N5F3OS: C 55.16, H 4.63, N
16.08, S 7.36 %; found: C 55.12, H 4.67, N 16.12, S
7.39 %; IR tmax (cm-1): 3030 (Ar–CH), 2832 (CH2), 1649,
1433 (C=N), 1365 (C=S), 1249 (C–O–C); 1H NMR
(CDCl3) d (ppm): 8.69 (d, 2H, J = 6.0 Hz, Ar–H), 7.65 (d,
2H, J = 6.0 Hz, Ar–H), 7.25–7.20 (t, 1H, Ar–H), 7.02–6.94
(m, 3H, Ar–H), 5.05 (s, 2H, N–CH2–N), 3.14–2.50 (m, 8H,
piperazine); MS (EI, 70 eV): m/z = 435.46 [M?].
In vitro antiamoebic assay
MTT assay
All the title compounds were screened for in vitro anti-
amoebic activity against HM1: IMSS strain of E. histoly-
tica by microdilution method (Wright et al., 1988).
Entamoeba histolytica trophozoites were cultured in cul-
ture tubes using Diamond TYIS-33 growth medium. The
test compounds (1 mg) were dissolved in DMSO (40 mL,
level at which no inhibition of amoeba occurs) (Gillin
et al., 1982). The stock solutions of the compounds were
prepared freshly before use at a concentration of 1 mg/mL.
Two-fold serial dilutions were made in the wells of 96-well
microtiter plate (costar). Each test includes metronidazole
as a standard amoebicidal drug, control wells (culture
medium plus amoebae) and a blank (culture medium only).
All the experiments were carried out in triplicate at each
concentration level and repeated thrice. The amoeba sus-
pension was prepared from a confluent culture by pouring
off the medium at 37 °C and adding 5 mL of fresh med-
ium, chilling the culture tube on ice to detach the organ-
isms from the side of flask. The number of amoeba/ml was
estimated with the help of a haemocytometer, using trypan
blue exclusion to confirm the viability. The suspension was
diluted to 105 organism/mL by adding fresh medium and
170 lL of this suspension was added to the test and control
The human breast cancer MCF-7 cells were obtained from
NCCS (Pune, India). The cells were cultured in DMEM
(Sigma) with 10 % foetal bovine serum and 1 % penicillin/
streptomycin/neomycin. The effect of compounds 2, 3, 5
and the standard drug metronidazole on cell proliferation
was measured using an MTT-based assay (Mosmann,
1983). In brief, the cells (10,000 well-1) were incubated in
triplicate in a 96-well plate in the presence of various
concentrations of compounds 2, 3, 5 as well as metroni-
dazole or vehicle (DMSO) alone in a final volume of
200 lL at 37 °C and 5 % CO2 in and humidified chamber
for 48 h. At the end of this time period, 20 lL of MTT
solution (5 mg/mL in PBS) was added to each well, and the
cells were incubated at 37 °C in a humidified chamber for
4 h. After 4 h, the supernatant was removed from each
well. The coloured formazan crystal produced from MTT
was dissolved in 200 lL of DMSO, and then the absor-
bance (A) value was measured at 570 nm by a multiscanner
autoreader. The following formula was used for the cal-
culation of the percentage of cell viability (CV): CV
(%) = (A of the experimental samples/A of the control)
9 100.
123

Med Chem Res
Ordaz-Pichardo C, Shibayama M, Villa-Trevino S, Arriaga-Alba M,
Angeles E, de la Garza M (2005) Antiamoebic and toxicity
studies of a carbamic acid derivative and its therapeutic effect in
a hamster model of hepatic amoebiasis. Antimicrob Agents
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Oza KK, Patel HS (2010) Antimicrobial activity of novel 3-substi-
tuted 5-(pyridine-4-yl)-3H-1,3,4-oxadiazole-2-thione deriva-
tives. Bul Chem Commun 42:103–106
Conclusion
It was concluded that the compounds 2, 3 and 5 showed
better antiamoebic activity than the reference drug metro-
nidazole and found low cytotoxic against human breast
cancer MCF-7 cell line. This study suggests the possibility
of developing 1,3,4-oxadiazole analogues as potential drug
candidates.
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