Synthesis of some 1,3,4-thiadiazole derivatives as inhibitors of Entamoeba histolytica

Article abstract of DOI:10.1007/s00044-012-0107-x

In the quest for potent anti-amoebic agents, some 1,3,4-thiadiazole derivatives were synthesized and characterized by spectral data. The purity of the compounds was confirmed by elemental analysis. All the compounds were screened in vitro against HM1:IMSS strain of Entamoeba histolytica by microdilution method. The results revealed that compounds 1 (IC₅₀ = 0.670 μM), 3 (IC₅₀ = 1.60 μM) and 8 (IC₅₀ = 0.522 μM) had much better anti-amoebic activity than the reference drug metronidazole (IC₅₀ = 1.80 μM). Further, cytotoxicity of the compounds having IC₅₀ value less than metronidazole was assessed by MTT assay on human breast cancer MCF-7 cell line and all the compounds were found low cytotoxic in the concentration range of 2.5-250 μM. Preliminary results indicate that these three compounds (1, 3 and 8) may be subjected to further investigations and it may be hoped that the present study will stimulate efforts towards the development of novel effective anti-amoebic agents.

Full text of DOI:10.1007/s00044-012-0107-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0107-x
ORIGINAL RESEARCH
Synthesis of some 1,3,4-thiadiazole derivatives as inhibitors
of Entamoeba histolytica
•
Shadab Miyan Siddiqui Attar Salahuddin
•
Amir Azam
Received: 28 November 2011 / Accepted: 22 May 2012
Ó Springer Science+Business Media, LLC 2012
Abstract In the quest for potent anti-amoebic agents,
some 1,3,4-thiadiazole derivatives were synthesized and
characterized by spectral data. The purity of the com-
pounds was confirmed by elemental analysis. All the
compounds were screened in vitro against HM1:IMSS
strain of Entamoeba histolytica by microdilution method.
The results revealed that compounds 1 (IC50 = 0.670 lM),
3 (IC50 = 1.60 lM) and 8 (IC50 = 0.522 lM) had much
better anti-amoebic activity than the reference drug met-
ronidazole (IC50 = 1.80 lM). Further, cytotoxicity of the
compounds having IC50 value less than metronidazole was
assessed by MTT assay on human breast cancer MCF-7
cell line and all the compounds were found low cytotoxic
in the concentration range of 2.5–250 lM. Preliminary
results indicate that these three compounds (1, 3 and 8)
may be subjected to further investigations and it may be
hoped that the present study will stimulate efforts towards
the development of novel effective anti-amoebic agents.
gastrointestinal tract, caused by parasitic protozoa
Entamoeba histolytica (Ralston and Petri, 2011). It has
been reported that E. histolytica causes up to 100,000
deaths worldwide annually (Tengku and Norhayati, 2011);
therefore, virulence of this obligate parasite is a challenge
for human health research. Amoebiasis is increasing among
men who have sex with men due to oral-anal sexual contact
(Park et al., 2007). Further, co-infection of amoeba and
HIV-1 is an emerging problem in developed East Asian
countries (Watanabe et al., 2011). Modern chemotherapy
offers a range of anti-amoebic agents, but there is not an
ideal treatment for amoebiasis because many of the avail-
able drugs elicit serious adverse reactions (Astelbauer and
Walochnik, 2011). Moreover, relapses of intestinal and
hepatic amoebiasis have been reported (Hwang et al.,
2011). Therefore, to ensure successful treatment, novel
efficacious anti-amoebic agents are required.
Sulphur-nitrogen heterocycles are important compounds
due to their significant and versatile biological activity
(Rakitin, 2009), and therefore these compounds have
retained the interest of the researchers. Among these, 1,3,
4-thiadiazoles represent one of the most promising class of
heterocycles in drug discovery. Acetazolamide, desagly-
buzole and furidiazine are commercially available 1,3,
4-thiadiazole drugs (Ali and Kazak, 2010). Thiadiazole
nucleus exhibits multifaceted biological activities (Clerici,
2002) possibly due to the presence of –N=C=S moiety
(Serban et al., 2010). The antiparasitic activity of 1,3,4-thi-
adiazoles is well documented. Several derivatives of 1,3,
4-thiadiazole have been found active against parasitic dis-
eases like leishmaniasis and trypanosomiasis (Carvalho
et al., 2004; Fardmoghadam et al., 2008; Poorrajab et al.,
2009a). Nitroheteroaryl-1,3,4-thiadiazole-based compounds
have been reported to exhibit potent anti-leishmanial activity
(Fardmoghadam et al., 2008) as well as anti-trypanosomal
Keywords 1,3,4-Thiadiazole ꢀ Anti-amoebic activity ꢀ
HM1:IMSS ꢀ E. histolytica ꢀ MTT assay
Introduction
Despite the significant progress in their treatment and
prevention, infectious diseases remain the major cause of
death and debility and are responsible for worsening the
living condition of millions of people around the world.
Amoebiasis is a life threatening infection of the human
S. M. Siddiqui ꢀ A. Salahuddin ꢀ A. Azam (&)
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar,
New Delhi 110025, India
e-mail: dr.amir.azam@gmail.com; amir_sumbul@yahoo.co.in
123

Med Chem Res
O
N
N
H
H
( d )
( a - c )
N
N
R
N
N
R
S
H
H
S
( 1 - 8 )
( TSC-1 - TSC-8 )
S
H
N N
S
O
( a - b)
( e )
R
N
( f )
N
SK
SH
R
H
R
OH
O
( 9 )
N N
S
( g )
R
NH2
( 10 )
Scheme 1 Synthesis of 1,3,4-thiadiazole derivatives (1–10).
Reagents and conditions: a CH3OH/H2SO4, reflux 6 h; b N2H4ꢀ
H2O/C2H5OH, reflux 6 h; c C6H5NCS, C2H5OH, reflux 1 h; d Conc.
H2SO4, rt, 30 min; e CS2, KOH/C2H5OH, rt, 10 h; f Conc. H2SO4, rt,
24 h; g NH2NHCSNH2, Conc. H2SO4, reflux, 7 h
activity (Poorrajab et al., 2009b). Like amoebiasis, both of
these are protozoal diseases; therefore, we expected that the
1,3,4-thiadiazole derivatives might be effective against
amoebiasis. Furthermore, it is a well-known fact that the
thiadiazole system is a cyclic analogue of thiosemicarbazide
having toxophoric N=C=S linkage (Siddiqui et al., 2011).
Our previous studies have proved that thiosemicarbazides
are useful building blocks for the development of anti-
amoebic compounds (Singh et al., 2006; Abid et al., 2009).
Moreover, in our recent studies, 1,2,3-thiadiazole deriva-
tives have emerged as potential anti-amoebic agents (Hayat
et al., 2010). Keeping all these observations in mind, some
1,3,4-thiadiazolederivatives were synthesized with an aim to
achieve the compounds having better anti-amoebic activity
and lesser cytotoxicity than the standard drug metronidazole.
required phenylthiosemicarbazides (TSC-1–TSC-8) were
prepared by addition reaction between acid hydrazides and
phenylisothiocyanate according to the reported method (Iqbal
et al., 1996) with suitable modifications. It is well known that
isothiocyanates are sensitive to water; therefore, the reaction
medium was anhydrous ethanol instead of aqueous ethanol
reported by Iqbal et al. Different acid hydrazides, being the key
intermediates for the synthesis of 2,5-disubstituted-1,3,4-thia-
diazole derivatives (1–8) and 5-phenyl-1,3,4-thiadiazole-
2-thiol (9), were obtained from the corresponding acids through
esterification followed by hydrazination (Furniss et al.,1989).
Synthesis of 5-phenyl-1,3,4-thiadiazole-2-thiol (9) involved
four steps: (i) esterification of benzoic acid with methanol; (ii)
subsequent hydrazination; (iii) salt formation; and (iv) dehyd-
rative cyclization in acidic medium. Benzoic acid hydrazide,
obtained from benzoic acid via esterification followed by
hydrazination, was treated with carbon disulphide in ethanolic
potassium hydroxide by means of the method of Reid and
Heindel (1976) to afford potassium salt of N0-benzoyl-hydra-
zinecarbodithioic acid, which underwent cyclization in acidic
medium to furnish 5-phenyl-1,3,4-thiadiazole-2-thiol (9). On
the other hand, 5-phenyl-1,3,4-thiadiazol-2-amine (10) was
prepared by a single-step reaction based on the method of
Chubb and Nissenbaum (1959). The intermediate compounds
phenylthiosemicarbazides (TSC-1–TSC-8) were character-
ized by FT-IR analysis (Table 1), while the structures of all the
Results and discussion
Chemistry
The strategy adopted for the synthesis of the target com-
pounds is depicted in Scheme 1.
2,5-Disubstituted-1,3,4-thiadiazole derivatives (1–8) were
synthesized by acid-catalysed intramolecular dehydrative
cyclization of corresponding phenylthiosemicarbazides. The
123

Med Chem Res
Table 1 FT-IR spectral data of intermediate phenylthiosemicarbazides (TSC-1–TSC-8) and title compounds (1–10)
O
H
N
H
N
N
N
R
R
N
H
S
S
R
(TSC-1-TSC-8)
(1-8)
SH
S
NH2
S
R
N
N
N
N
(9)
(10)
Compound code
mmax (cm-1
)
NH
C=O
C=S
C=N N–N C–S-C (thiadiazole ring)
SH
NH2
TSC-1
3,406; 3,256
3,409; 3,260
3,407; 3,260
3,403; 3,260
3,405; 3,259
3,406; 3,257
3,409; 3,256
3,406; 3,259
3,270
1,666
1,305
–
–
–
–
–
TSC-2
1,669
1,306
–
–
–
–
–
TSC-3
1,663
1,302
–
–
–
–
–
TSC-4
1,669
1,305
–
–
–
–
–
TSC-5
1,666
1,305
–
–
–
–
–
TSC-6
1,666
1,305
–
–
–
–
–
TSC-7
1,666
1,305
–
–
–
–
–
TSC-8
1,662
1,305
–
–
–
–
–
1
2
3
4
5
6
7
8
9
10
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1,601
1,603
1,604
1,600
1,621
1,622
1,612
1,611
1,601
1,595
1,032
1,028
1,031
1,031
1,027
1,028
1,028
1,030
1,032
1,029
701
699
704
705
691
692
698
697
702
699
–
–
3,253
–
–
3,266
–
–
3,257
–
–
3,258
–
–
3,261
–
–
3,253
–
–
3,251
–
–
–
2,338
–
–
–
3,436
final compounds (1–10) were assigned on the basis of FT-IR,
1H NMR and 13C NMR and mass spectral data, which were in
full agreement with the proposed structures.
The biological behaviour revealed that compound 1 (IC50 =
0.670 lM), 3 (IC50 = 1.60 lM) and 8 (IC50 = 0.522 lM)
were more potent inhibitors of E. histolytica than the refer-
ence drug metronidazole (IC50 = 1.80 lM). However,
compound (8), having methyl group at the C-5 position of
thiadiazole ring, exhibited an excellent activity in terms of
IC50 value and was the most active compound in the series.
Having replaced this methyl group with the 4-nitrophenyl
did not effectively reduce anti-amoebic activity, but the
replacement of methyl group with phenyl group reduced
anti-amoebic activity drastically. Further, to ensure the role
of different substituents on the anti-amoebic activity, we
evaluated the anti-amoebic activity of 5-phenyl-1,3,4-thia-
diazole-2-thiol (9) and 5-phenyl-1,3,4-thiadiazol-2-amine
(10), which had thiol (SH) & primary amino (NH2) group at
C-2 position of 1,3,4-thiadiazole ring (Fig. 2). They exhib-
ited IC50 values more than that of the reference drug,
Anti-amoebic activity
All the 1,3,4-thiadiazole derivatives were screened against
HM1:IMSS strain of E. histolytica by microdilution
method (Wright et al., 1988). All the experiments were
carried out in triplicate at each concentration level and
repeated thrice. The anti-amoebic effect was compared
with the most widely used medication metronidazole,
which had a 50 % inhibitory concentration (IC50) of
1.80 lM in our experiments. The results are given in
Table 2. Among all 2,5-disubstituted-1,3,4-thiadiazole
derivatives (1–8), we studied the effects of different sub-
stituents (R) on C-5 position of thiadiazole ring (Fig. 1).
123

Med Chem Res
Table 2 In vitro anti-amoebic activity of 1,3,4-thiadiazole derivatives against HM1:IMSS strain of E. histolytica and their cytotoxicity profile
2
1
NH2
3
1
S
N
SH
4
2
S
3
N
R
5
4
5
N
R
3
N
5
NH
N
2
N
S
4
1
R
(1-8)
(9)
(10)
Compound number
R
Antiamoebic activity
Cytotoxicity profile
IC50 (lM)
SD
IC50 (lM)
SD
1
0.670
0.007
[250
0.024
NO2
2
4.43
0.011
ND
ND
Cl
3
4
1.60
0.009
0.007
[250
0.027
ND
5.49
ND
F
5
6
7
3.50
4.64
5.55
0.013
0.018
0.011
ND
ND
ND
ND
ND
ND
SH
OH
NH2
8
9
CH3
0.522
0.008
0.016
[250
ND
0.021
ND
5.14
123

Med Chem Res
Table 2 continued
Compound number
R
Antiamoebic activity
Cytotoxicity profile
IC50 (lM)
SD
IC50 (lM)
SD
10
5.46
0.017
ND
ND
Metronidazole
1.80
0.029
[250
0.022
SD standard deviation, ND not done
Cytotoxicity profile
Fig. 1 Structure of 2,5-
disubstituted-1,3,4-thiadiazole
derivatives (1–8) where
R = different substituents given
in Table 2
3
4
N
S
2
N
Cytotoxicity of the compounds having IC50 values less than
metronidazole was assessed by MTT assay on human
breast cancer MCF-7 cell line. A confluent population of
MCF-7 cells was treated with increasing concentrations of
compounds and the number of viable cells was measured
after 48 h by MTT cell viability assay based on mito-
chondrial reduction of the yellow MTT tetrazolium dye to a
highly coloured blue formazan crystals. This assay usually
shows high correlation with the number of living cells and
cell proliferation. The concentration range for all the
compounds (1, 3, 8 and MNZ) is mentioned in Fig. 4,
which illustrates that all the three compounds were low
cytotoxic in the concentration range of 2.5–250 lM.
NH
5
R
1
2
1
NH2
3
1
S
SH
2
S
R
5
4
R
N
5
3
N
4
N
N
(9)
(10)
Fig. 2 Structure of 5-phenyl-1,3,4-thiadiazole-2-thiol (9) and 5-phe-
nyl-1,3,4-thiadiazol-2- amine (10)
Experimental protocol
metronidazole (MNZ), and therefore were considered to be
inactive. These results indicated that the anti-amoebic activity
was influenced by the type of substituent (R) present at the C-5
position of thiadiazole ring as well as the left side chain attached
on the C-2 position of thiadiazole ring. In our previous paper
(Hayat et al., 2010), we described the synthesis and in vitro
anti-amoebic activity of 1,2,3-thiadiazole derivatives. Among
those derivatives, the compounds having 4-chloro phenyl
(1, IC50 = 0.24 lM), 4-bromo phenyl (2, IC50 = 0.39 lM)
and furan ring (3, IC50 = 0.23 lM) substituted at C-4 position
of 1,2,3-thiadiazole rings were found active against E. histoly-
tica (Fig. 3). On the other hand, in the present study, only
three compounds: 5-(4-nitrophenyl)-N-phenyl-1,3,4-thiadiazol-
2-amine (1, IC50 = 0.670 lM), N, 5-diphenyl-1,3,4-thiadiazol-
2-amine (3, IC50 = 1.60 lM) and 5-methyl-N-phenyl-1,3,
4-thiadiazol-2-amine (8, IC50 = 0.522 lM) were found more
active than the metronidazole yet their IC50 values were higher
than that of the active compounds of 1,2,3-thiadiazole series
(Hayat et al., 2010). These differences in their IC50 values may
be due to the presence of different side chains. However, the
mechanism of the biological activity needs further investiga-
tions, which are in progress.
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
trophotometer as KBr discs. The H NMR and 13C NMR
spectra were recorded on Bruker Spectrospin DPX
300 MHz and Bruker Spectrospin DPX 75 MHz spec-
trometer, respectively, using DMSO-d6 as a solvent and
trimethylsilane (TMS) as an internal standard. Chemical
shift values are given in parts per million (ppm) with
respect to TMS. Mass spectra were recorded by GC–MS
(Perkin-Elmer Clarus 500 GC).
General procedure for the preparation of 2,5-disubstituted-
1,3,4-thiadiazole derivatives (1–8)
A mixture of the corresponding phenylthiosemicarbazides
(0.01 mol) and conc.H2SO4 (10 mL) was stirred at room
temperature for 30 min. The reaction content was poured
over crushed ice. The solid obtained was washed with
123

Med Chem Res
Fig. 3 Structure of 1,2,3-
thiadiazole derivatives (Hayat
et al., 2010)
2
1
S
N
3
N
5
4
R
Br
Cl
Where R =
O
( 1 )
( 2 )
( 3 )
8.03–8.01 (m, 2H, Ar–H), 7.79–7.66 (m, 8H, Ar–H); 13C
NMR (DMSO-d6) d (ppm): 164.02 (thiadiazole C-2),
158.03 (thiadiazole C-5), 141.42, 140.85, 130.47, 130.27,
129.41, 126.91, 126.79, 116.67 (Ar–C); MS (EI, 70 eV):
m/z = 253.32 [M?].
MTT Assay
100
80
60
40
20
0
Control
1
3
8
MNZ
5-(4-Fluorophenyl)-N-phenyl-1,3,4-thiadiazol-2-amine (4):
Yield: 73 %; mp: 200 °C; Anal. calc. for C14H10FN3S: C
61.98, H 3.72, N 15.49, S 11.82 %; found: C 61.93, H 3.77, N
1
15.53, S 11.79 %; H NMR (DMSO-d6) d (ppm) : 10.63 (s,
1H, NH), 8.09–7.89 (m, 2H, Ar–H), 7.82–7.51 (m, 5H,
Ar–H), 7.44–7.29 (t, 2H, Ar–H); 13C NMR (DMSO-d6) d
(ppm): 164.85 (thiadiazole C-2), 157.26 (thiadiazole C-5),
141.82, 141.17, 136.70, 130.90, 129.58, 129.47, 127.13,
116.97 (Ar–C); MS (EI, 70 eV): m/z = 271.31 [M?].
Concentration (µM)
Fig. 4 Percentage of viable cells after 48 h pre-treatment of human
breast cancer MCF-7 cells with compounds 1, 3, 8, and metronidazole
(MNZ) evaluated by the MTT assay
2-(5-Anilino-1,3,4-thiadiazol-2-yl)benzenethiol (5): Yield:
62 %, m.p. 212 °C; Anal. calc. for C14H11N3S2: C 58.92, H
3.89, N 14.72, S 22.47 %; found: C 58.96, H 3.83, N 14.75, S
1
sodium carbonate solution followed by water and finally
recrystallized from ethanol.
22.49 %; H NMR (DMSO-d6) d (ppm): 10.65 (s, 1H, SH),
10.44 (s, 1H, NH), 8.10–7.00 (m, 9H, Ar–H); 13C NMR
(DMSO-d6) d (ppm): 165.50 (thiadiazole C-2), 157.72 (thia-
diazole C-5), 146.32, 146.05, 145.74, 139.18, 129.20, 128.03,
125.98, 117.13 (Ar–C); MS (EI, 70 eV): m/z = 285.38 [M?].
2-(5-Anilino-1,3,4-thiadiazole-2-yl) phenol (6): Yield:
60 %; m.p. 204 °C; Anal. calc. for C14H11N3OS: C 62.43,
H 4.12, N 15.60, S 11.91 %; found: C 62.41, H 4.11, N 15.64,
S 11.94 %; 1H NMR (DMSO-d6) d (ppm) : 9.84 (s, 1H, NH),
8.25 (s, 1H, OH), 7.59–7.52 (m, 5H, Ar–H), 7.32–7.27 (m, 2H,
Ar–H), 6.96–6.91 (m, 2H, Ar–H); 13C NMR (DMSO-d6) d
(ppm): 165.78 (thiadiazole C-2), 158.00 (thiadiazole C-5),
147.10, 141.77, 137.43, 130.34, 129.78, 129.55, 128.48, 117.52
(Ar–C); MS (EI, 70 eV): m/z = 269.32 [M?].
5-(4-Nitrophenyl)-N-phenyl-1,3,4-thiadiazol-2-amine (1):
Yield: 52 %; mp: 220 °C; Anal. calc. for C14H10N4O2S: C
56.37, H 3.38, N 18.78, S 10.75 %; found: C 56.41, H 3.34, N
1
18.77, S 10.71 %; H NMR (DMSO-d6) d (ppm): 10.87 (s,
1H, NH), 8.34 (d, 2H, J = 9.0 Hz, Ar–H), 8.13 (d, 2H,
J = 9.0 Hz, Ar–H), 7.61 (m, 5H, Ar–H); 13C NMR (DMSO-
d6) d (ppm): 165.75 (thiadiazole C-2), 156.06 (thiadiazole
C-5), 148.34, 143.06, 140.60, 136.55, 128.19, 127.14, 124.99,
117.15 (Ar–C); MS (EI, 70 eV): m/z = 298.31 [M?].
5-(4-Chlorophenyl)-N-phenyl-1,3,4-thiadiazol-2-amine (2):
Yield: 78 %; mp: 214 °C; Anal. calc. for C14H10ClN3S: C
58.43, H 3.50, N 14.60, S 11.14 %; found: C 58.39, H 3.53, N
1
14.64, S 11.09 %; H NMR (DMSO-d6) d (ppm): 10.68 (s,
5-(2-Aminophenyl)-N-phenyl-1,3,4-thiadiazol-2-amine (7):
Yield: 58 %; m.p. 220 °C, Anal.calc. for C14H12N4S: C
62.66, H 4.51, N 20.88, S 11.95 %, found: C 62.69, H 4.47, N
1H, NH), 7.87–7.56 (m, 9H, Ar–H); 13C NMR (DMSO-d6) d
(ppm): 164.61 (thiadiazole C-2), 157.20 (thiadiazole C-5),
141.67, 141.20, 135.24, 131.61, 129.78, 128.86, 127.16,
117.09 (Ar–C); MS (EI, 70 eV): m/z = 287.76 [M?].
1
20.90, S 11.92 %; H NMR (DMSO-d6) d (ppm): 12.98 (s,
1H, NH), 7.93–7.22 (m, 9H, Ar–H), 7.04 (s, 2H, NH2); 13C
NMR (DMSO-d6) d (ppm): 164.07 (thiadiazole C-2), 158.10
(thiadiazole C-5), 141.27, 140.96, 130.50, 130.27, 129.43,
126.93, 126.83, 116.77 (Ar–C); MS (EI, 70 eV): m/
z = 268.33 [M?].
N, 5-Diphenyl-1,3,4-thiadiazol-2-amine (3): Yield:
71 %; mp: 178 °C; Anal. calc. for C14 H11N3S: C 66.38, H
4.38, N 16.59, 12.66 %; found: C 66.33, H 4.41, N 16.63, S
12.63 %; 1H NMR (DMSO-d6) d (ppm): 10.84 (s, 1H, NH),
123

Med Chem Res
5-Methyl-N-phenyl-1,3,4-thiadiazol-2-amine (8): Yield:
66 %; m.p. 264 °C; Anal. calc. for C9H9N3S: C 56.52, H
4.74, N 21.97, S 16.77 %; found: C 56.49, H 4.77, N 21.99,
by microdilution method (Wright et al., 1988). E. histolytica
trophozoites were cultured in culture tubes using Diamond
TYIS-33 growth medium. The test compounds (1 mg)
were dissolved in DMSO (40 mL, level at which no inhi-
bition 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. Twofold 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 suspension was prepared from
a confluent culture by pouring off the medium at 37 °C and
adding 5 mL of fresh medium, chilling the culture tube on
ice to detach the organisms 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 wells in the plate so that
the wells were completely filled (total volume, 340 lL).
An inoculum of 1.7 9 104 organisms/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 incu-
bation, 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 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 it was 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
using 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 2.
1
S 16.81 %; H NMR (DMSO-d6) d (ppm): 10.37 (s, 1H,
NH), 7.58–7.50 (m, 5H, Ar–H), 2.56 (s, 3H, CH3); 13C
NMR (DMSO-d6) d (ppm): 164.63 (thiadiazole C-2),
155.76 (thiadiazole C-5), 142.04, 141.14, 127.05, 116.69
(Ar–C), 15.60 (CH3). MS (EI, 70 eV): m/z = 191.25 [M?].
General procedure for the preparation of 5-phenyl-1,3,4-
thiadiazole-2-thiol (9)
Potassium salt of N0-benzoyl-hydrazinecarbodithioic acid
(0.05 mol) was added portionwise to 98 % H2SO4 (15 mL)
and the resulting clear solution was stirred at room tem-
perature for 4 h. The mixture was cautiously added to
crushed ice, stirred for 20 h, refrigerated for 2 h and the
separated precipitates were filtered, washed with water,
dried and finally recrystallized from ethanol to furnish the
desired compound (9).
5-Phenyl-1,3,4-thiadiazole-2-thiol (9): Yield: 80 %; mp:
198 °C; Anal. calc. for C8H6N2S2: C 49.46, H 3.11, N
14.42, S 33.01 %; found: C 49.50, H 3.07, N 14.40, S
33.08 %; 1H NMR (DMSO-d6) d (ppm): 14.74 (s, 1H, SH),
8.19–7.60 (m, 5H, Ar–H); 13C NMR (DMSO-d6) d (ppm):
187.22 (C–SH), 160.55 (C=N), 135.27, 128.66, 127.35,
125.66 (Ar–C); MS (EI, 70 eV): m/z = 194.27 [M?].
General procedure for the preparation of 5-phenyl-1,3,
4-thiadiazol-2-amine (10)
A mixture of carboxylic acid (0.15 mol) and thiosemicar-
bazide (0.125 mol) was stirred in 16 mL of concentrated
sulphuric acid in a round bottomed flask at 80–90 °C for
7 h under reflux. After the reaction was complete, the
reaction mixture was allowed to cool and was poured over
crushed ice. The mixture was basified using concentrated
ammonium hydroxide solution. On addition of the base, the
thiadiazole product precipitated was filtered and recrys-
tallized from aqueous ethanol (10–15 %).
5-Phenyl-1,3,4-thiadiazol-2-amine (10): Yield: 66 %;
mp: 224 °C; Anal. calc. for C8H7N3S: C 54.22, H 3.98, N
23.71, S 18.09 %; found: C 54.24, H 3.95, N 23.75, S
18.11 %; 1H NMR (DMSO-d6) d (ppm): 7.82–7.80 (m, 2H,
Ar–H), 7.39–7.34 (m, 2H, Ar–H), 7.27–7.23 (t, 1H, Ar–H),
7.11 (s, 2H, NH2); 13C NMR (DMSO-d6) d (ppm): 168.33,
149.93 (thiadiazole ring C), 134.98, 128.56, 127.27, 125.61
(Ar–C); MS (EI, 70 eV): m/z = 177.22 [M?].
MTT assay
In vitro anti-amoebic assay
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/
All the title compounds were screened for in vitro anti-
amoebic activity against HM1:IMSS strain of E. histolytica
123

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