Synthesis, characterization and antiamoebic activity of some hydrazone and azole derivatives bearing pyridyl moiety as a promising heterocyclic scaffold

Article abstract of DOI:10.1016/j.ejmech.2012.01.030

In an effort to develop effective antiamoebic agents, some hydrazones and azoles containing pyridyl moiety were synthesized and screened for in vitro antiamoebic activity against HM1:IMSS strain of Entamoeba histolytica. Among all the compounds, only five compounds (1, 3, 5, 9 and 11) were found to be better inhibitors of growth of E. histolytica than the reference drug metronidazole. The cytotoxic studies of these compounds on human breast cancer MCF-7 cell line revealed that all the compounds were low-cytotoxic in the concentration range of 2.5-250 μM.

Full text of DOI:10.1016/j.ejmech.2012.01.030

European Journal of Medicinal Chemistry 49 (2012) 411e416
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, characterization and antiamoebic activity of some hydrazone and azole
derivatives bearing pyridyl moiety as a promising heterocyclic scaffold
Shadab Miyan Siddiqui, Attar Salahuddin, Amir Azam*
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 September 2011
Received in revised form
In an effort to develop effective antiamoebic agents, some hydrazones and azoles containing pyridyl
moiety were synthesized and screened for in vitro antiamoebic activity against HM1:IMSS strain of
Entamoeba histolytica. Among all the compounds, only five compounds (1, 3, 5, 9 and 11) were found to
be better inhibitors of growth of E. histolytica than the reference drug metronidazole. The cytotoxic
studies of these compounds on human breast cancer MCF-7 cell line revealed that all the compounds
27 December 2011
Accepted 14 January 2012
Available online 21 January 2012
were low-cytotoxic in the concentration range of 2.5e250 mM.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Hydrazone
Azole
Antiamoebic activity
MTT assay
1
. Introduction
The critical role played by small heterocyclic molecules in drug
design cannot be denied. These molecules act as highly function-
alized scaffolds. In this context, pyridyl ring, a prominent scaffold
present in plenty of bioactive molecules, has played a vital role in
the development of different medicinal agents [10]. On the other
hand, the interesting and versatile biological activities of hydra-
zones [11], established them as important pharmacophore. Azoles,
especially the bioisosteric 1, 3, 4-oxadiazoles, 1, 2, 4-triazoles, and 1,
3, 4-thiadiazoles, are important molecules known to interact with
particular receptors on enzyme active sites [12]. Moreover, in our
earlier reports, we have reported the synthesis of different azole
and hydrazone derivatives and it was found that the combination of
these derivatives with pyridyl ring exhibited antiamoebic activities
[13,14]. Encouraged by these reports, some hydrazone and azole
derivatives bearing a pyridyl moiety were synthesized with an aim
to achieve the compounds having better antiamoebic activity and
lesser cytotoxicity than the standard drug metronidazole.
Several species of protozoa have been associated with acute and
chronic diarrhoea in HIV-infected patients. Entamoeba histolytica,
the causative agent of amoebiasis, is one of them [1]. This patho-
genic parasite is well known for its high potential for invading and
destroying human tissue, leading to diseases like hemorrhagic
colitis and extra intestinal abscess [2]. It produces considerable
amounts of cysteine proteinases (CPs) and this class of enzymes is
instrumental for E. histolytica-induced pathology [3]. Infection of
this parasite occurs in a number of countries of the world, resulting
5
0 million cases of invasive disease and up to 110,000 deaths per
year [4]. Since their introduction, nitroimidazoles are one of most
significant weapons in fighting against protozoal infections. Over
the past few decades, these health benefits are under threat as
many of them have become less effective due to their toxic nature.
Literature survey reveals that cross resistance exists among nitro-
imidazoles [5]. Metronidazole, the most widely used medication
against amoebiasis, is potentially carcinogenic to humans because
it is genotoxic to human cells [6]. Several serious side effects such as
seizures, ataxia, peripheral neuropathy, transient myopia, gastric
mucus irritation, spermatozoid damage and haematuria are asso-
ciated with metronidazole [7e9]. Therefore, it is essential to
investigate new compounds for the development of novel drugs
with better amoebicidal activity and lesser toxicity for the host.
2. Chemistry
The synthetic pathway leading to the title compounds is given in
Scheme 1. Isonicotinyl hydrazones (1e8) were synthesized by
a single step reaction of isonicotinic hydrazide (isoniazid) with
appropriate aryl aldehydes, in presence of catalytic amount of
glacial acetic acid [15]. 5-(Pyridine-4-yl)-1, 3, 4-oxadiazole-2-(3H)-
thione (9), was afforded by cyclization of isonicotinic acid hydrazide
using carbon disulfide in presence of KOH [16]. N-Phenyl-2-
*
Corresponding author. Tel.: þ91 11 26981717/3254; fax: þ91 11 26980229.
E-mail address: amir_sumbul@yahoo.co.in (A. Azam).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.030

412
S.M. Siddiqui et al. / European Journal of Medicinal Chemistry 49 (2012) 411e416
O
N
Ar
(
N
H
(
a)
N
N
1 - 8 )
H
N
N
O
(b)
S
NH
O
N
NH2
(
9 )
O
H
N
(c)
NH
N
N
H
S
(
10 )
(d)
(e)
N
N
N
N
SH
NH
N
S
N
N
(
11)
( 12 )
þ
2 6 5
OH, reflux 1 h, (b) CS , KOH/H , reflux 10 h, (c) C H NCS,
Scheme 1. Synthesis of hydrazone (1e8) and azole (9, 11, 12) derivatives. Reagents and conditions: (a) AreCHO/C
OH, reflux 1 h, (d) 2 N NaOH, reflux 2 h, (e) H SO , rt, 1 h.
2
H
5
C
H
2 5
2
4
(
pyridine-4-yl carbonyl) hydrazinecarbothiamide (10), was ob-
Additionally, in 13C NMR spectra the appearance of characteristic
signals for C]O and C]N in the range of 161.19e162.03 ppm and
140.08e141.10 ppm respectively confirmed the formation of the
compounds 1e8. The IR spectrum of 5-(pyridine-4-yl)-1, 3, 4-
oxadiazole-2-(3H)-thione (9), showed NeH absorption band at
tained by the reaction of isoniazid with phenylisothiocyanate [17].
Compound 10 was used as the key intermediate for the synthesis of
compounds 11 and 12. The cyclization of compound 10 in the
presence of 2 N NaOH afforded 4-phenyl-5-pyridin-4-yl-4H-1, 2, 4-
triazole-3-thiol (11) [18]. On the other hand, the acid catalyzed
intramolecular dehydrative cyclization of compound 10 furnished
N-phenyl-5-pyridin-4-yl-1, 3, 4-thiadiazol-2-amine (12) [17].
ꢀ
1
ꢀ1
3140 cm while C]S absorption band at 1312 cm . Moreover, the
formation of oxadiazole ring was supported by the presence of
characteristic C]N and CeOeC absorption bands of oxadiazole ring
ꢀ1
1
at 1586 and 1244 cm respectively. In the H NMR spectrum of
3
. Pharmacology
compound 9, all aromatic protons appeared in the expected region,
while the NH proton resonated as a singlet at
d 6.20 ppm. The
13
All the title compounds were screened in vitro against
structure of the compound 9 was further established by C NMR
spectrum, which showed characteristic signals of oxadiazole ring at
177.87 and 158.80 ppm. The infrared spectrum of compound 10
HM1:IMSS strain of E. histolytica by microdilution method [19]. All
the experiments were carried out in triplicate at each concentration
level and repeated thrice. Cytotoxicity of the compounds having
IC50 value less than metronidazole was assessed by MTT assay on
human breast cancer MCF-7 cell line. The results of antiamoebic
activity and cytotoxicity are summarized in Table 1.
ꢀ
1
exhibited characteristic absorption bands at 1305 cm
and
due to the C]S and C]O groups. In the 1H NMR
spectrum of compound 10, NH signals were observed at 9.75, 9.85
ꢀ
1
1666 cm
13
and 10.84 ppm while C NMR spectra showed characteristic
signals of C]S and C]O at 181.24 ppm and 164.56 ppm respec-
tively. When compound 10 was converted in compound 11, NH
peaks disappeared and a new signal due to eSH group was
4
. Results and discussion
1
4.1. Synthesis
observed at 14.42 ppm in the H NMR spectrum of compound 11. IR
spectrum of compound 12, exhibited absorption bands at 3261,
ꢀ
1
All the synthesized compounds gave satisfactory analyses for
1622, 1028 and 692 cm
due to NH, C]N, NeN, and CeSeC
respectively. In the H NMR spectrum of compound 12, NH
proton appeared as a singlet at 10.91 ppm and all the aromatic
1
the proposed structures, which were confirmed on the basis of
their spectral data. The IR spectra of compounds 1e8 exhibited
d
ꢀ
1
characteristic absorption bands at 1648e1676 cm
and
162e3390 cm due to C]O and NH stretching respectively. Iso-
nicotinyl hydrazones exhibited a characteristic absorption band
protons appeared in the expected region. Further, appearance of
characteristic signals of thiadiazole ring at 164.45 ppm and
157.26 ppm in C NMR spectrum was the significant parameter
considered for the confirmation of the structure of compound 12.
ꢀ
1
3
13
ꢀ1
due to azomethine group in the region of 1539e1594 cm
,
providing confirmatory evidence for the condensation reaction
between aldehyde group and hydrazide. The structures of
compounds 1e8 were further confirmed by H NMR spectral data. A
4.2. Antiamoebic activity
1
singlet in the range of 8.41e8.68 ppm due to (CH]N) proton dis-
played the successful formation of the desired products.
In order to explore the potential of the hydrazones (1e8) and
azoles (9, 11 & 12) derivatives as possible scaffold for E. histolytica

S.M. Siddiqui et al. / European Journal of Medicinal Chemistry 49 (2012) 411e416
413
Table 1
inhibitors were evaluated against HM1:IMSS strain of E. histolytica.
The antiamoebic effect was compared with the most widely used
antiamoebic medication metronidazole which had a 50% inhibitory
In vitro antiamoebic activity of hydrazone (1e8) and azole (9, 11 & 12) derivatives
against HM1:IMSS strain of E. histolytica and cytotoxicity profile.
O
concentration (IC50) of 1.81 mM in our experiments. All the
N
Ar
compounds shared a common feature: the presence of pyridyl ring,
but they were found differ in their corresponding antiamoebic
activity. In terms of structure activity relationship (SAR), it was
concluded that among all the screened hydrazones (1e8), none of
the derivatives without a para-position substituent displayed
antiamoebic activity. However, compound 5 having methyl group
at para-position of phenyl ring, exhibited most promising anti-
N
H
N
(
1-8)
H
N
N
N
N
N
SH
S
O
amoebic activity (IC50 ¼ 0.131
IC50 ¼ 0.37 M) and methoxy (3, IC50 ¼ 0.39
position of phenyl ring exerts significant inhibitory activity,
whereas the introduction of dimethylamino (7, IC50 ¼ 5.55 M) &
isopropyl (8, IC50 ¼ 3.74 M) group at the same position did not
mM). Incorporation of nitro (1,
N
m
m
M) group at para-
N
(
9)
m
N N
S
(11)
m
exhibit antiamoebic activity. In terms of SAR, it can be concluded
that pyridyl, azomethine group (CH]N) as well as the substituents
played the crucial role in activity against HM1:IMSS strain of
E. histolytica. In fact, the entire molecular-skeleton of the particular
compound is concerned with its IC50 value and therefore, respon-
sible for its antiamoebic activity. Apart from this, among azole
derivatives (9, 11, & 12), combination of 1, 2, 4-triazole ring with
NH
N
(
12)
Compound No. Ar
Antiamoebic
activity
Cytotoxicity
profile
M) SDa
M) SDa
IC50 (m
pyridyl ring i.e. compound 11 (IC50 ¼ 0.16
mM) has an excellent
IC50
(
m
impact on antiamoebic activity against E. histolytica. It is interesting
to note that the replacement of 1, 2, 4-triazole ring with 1, 3, 4-
oxadiazole ring (9) reduced the antiamoebic activity
1
0.37
0.005 >250
0.9
NO2
(IC50 ¼ 0.558
thiadiazole derivative (12, IC50 ¼ 4.43
more than that of the standard drug metronidazole (IC50 ¼ 1.81
and therefore it was considered to be inactive against E. histolytica.
m
M). Further, the results revealed that 1, 3, 4-
M) showed IC50 value
M)
m
m
2
3
3.84
0.39
0.006 N.D.
N.D.
1.2
4.3. Cytotoxicity profile
0.001 >250
Cytotoxicity of compounds (1, 3, 5, 9 & 11) was assessed by MTT
OCH3
Cl
assay on human breast cancer MCF-7 cell line. A confluent pop-
ulation of MCF-7 cells was treated with increasing concentrations
of compounds and the number of viable cells was measured after
4
8 h by MTTcell viability assay based on mitochondrial reduction of
4
5
12.11
0.13
0.017 N.D.
N.D.
0.45
the yellow MTT tetrazolium dye to a highly coloured blue formazan
product. This assay usually shows high correlation with number of
living cells and cell proliferation. The concentration range for all the
compounds (1, 3, 5, 9, 11 & MNZ) is mentioned in Fig. 1, which
illustrates that all the active compounds and metronidazole were
0.010 >250
CH3
low-cytotoxic in the concentration range of 2.5e250 mM, while
OH
6
4.14
0.004 N.D.
N.D.
7
8
5.55
3.74
0.006 N.D.
0.007 N.D.
N.D.
N.D.
N
9
1
e
e
0.558
0.160
0.008 >250
0.007 >250
1.1
0.55
1
2
1
e
4.43
1.81
0.011 N.D.
0.011 >250
N.D.
0.89
Fig. 1. Percentage of viable cells after 48 h pre-treatment of human breast cancer MCF-
cells with compounds 1, 3, 5, 9, 11 and metronidazole (MNZ), evaluated by the MTT
assay.
Metronidazole
7
a
Standard deviation, N.D. ¼ Not Done.

414
S.M. Siddiqui et al. / European Journal of Medicinal Chemistry 49 (2012) 411e416
they showed cytotoxic effects above 250
m
M. Therefore, it can be
(m, 2H, AreH), 7.68 (d, 2H, J ¼ 6.0 Hz, AreH), 7.00 (d, 2H, J ¼ 6.0 Hz,
13
concluded that the cytotoxicity of all the compounds (1, 3, 5, 9 & 11)
was found to be concentration-dependent and all the screened
compounds were low-cytotoxic against the human breast cancer
3 6
AreH), 3.78 (s, 3H, OCH ); C NMR (DMSO-d ) d (ppm): 161.52 (C]
O), 141.10 (CH]N), 150.68, 149.78, 142.51, 129.41, 126.97, 123.73,
þ
122.04 (AreC), 55.74 (OCH
100%), 256.09 (M þ 1, 15.1%).
3
); MS (EI, 70 eV): m/z ¼ 255.27 (M ,
þ
MCF-7 cell line in the concentration range of 2.5e250
. Conclusion
Taking all the biological data in consideration, it can be
mM.
0
5
6.1.4. N -(3-Chlorobenzylidene) isonicotinohydrazide (4)
ꢁ
Yield: 94%; mp: 216 C; Anal. calc. for C13
H10ClN
3
O: C 60.12, H
n
ꢀ
1
3.88, N 16.18%; found: C 60.15, H 3.87, N 16.20%; IR
max (cm ):
3168 (NH), 1673 (CO), 1539 (C]N); H NMR (DMSO-d (ppm):
12.13 (s, 1H, NH), 8.45 (s, 1H, CH]N), 8.78e8.76 (m, 2H, AreH),
1
concluded that among all the screened compounds only five
compounds (1, 3, 5, 9 and 11) were found to be better inhibitors of
growth of E. histolytica than the reference drug metronidazole and
all five of them were low-cytotoxic against the human breast cancer
MCF-7 cell line in the concentration range of 2.5e250
hoped that these studies will stimulate further efforts towards the
development of novel effective antiamoebic agents.
6
) d
13
6
7.90e7.47 (m, 6H, AreH); C NMR (DMSO-d ) d (ppm): 161.86 (C]
O), 140.30 (CH]N), 150.40, 147.35, 136.30, 133.77, 130.79, 130.05,
þ
m
M. It is
126.56, 126.02, 121.64 (AreC); MS (EI, 70 eV): m/z ¼ 259.69 (M ,
þ
þ
100%), 261.05 (M þ 2, 31.9%), 262.05 (M þ 3, 4.6%).
0
6
.1.5. N -(4-Methylbenzylidene) isonicotinohydrazide (5)
ꢁ
6
. Experimental protocol
Yield: 92%; mp: 204 C; Anal. calc. for C14
.48, N 17.56%; found: C 70.23, H 5.52, N 17.57%; IR
3252 (NH), 1650 (CO), 1545 (C]N); H NMR (DMSO-d
13
H N
3
O: C 70.28, H
ꢀ
1
5
n
max (cm ):
(ppm):
1
Melting points (mp) were recorded on a KSW apparatus and are
6
)
d
uncorrected. Elemental analysis was carried out on CHNS
Elemental Analyzer vario MICRO, Elementar Analysensysteme
11.99 (s, 1H, NH), 8.42 (s, 1H, CH]N), 8.77e8.73 (m, 2H, AreH), 7.81
(d, 2H, J ¼ 6.0 Hz, AreH), 7.62 (d, 2H, J ¼ 9.0 Hz, AreH), 7.25 (d, 2H,
13
GmbH, Germany. IR spectra were recorded on PerkineElmer model
J ¼ 9.0 Hz, AreH), 2.27 (s, 3H, CH
3 6
); C NMR (DMSO-d ) d (ppm):
1
1
600 FT-IR RX1 spectrophotometer as KBr discs. The H NMR and
162.02 (C]O), 140.75 (CH]N), 150.76, 149.54, 140.98, 131.76,
129.95, 127.73, 122.01 (AreC), 21.51 (CH ); MS (EI, 70 eV): m/
1
3
C NMR spectra were recorded on Bruker Spectrospin DPX
00 MHz and Bruker Spectrospin DPX 75 MHz spectrometer
3
þ
þ
3
z ¼ 239.27 (M , 100%), 240.21 (M þ 1, 15.2%).
6
respectively using DMSO-d as a solvent and trimethylsilane (TMS)
0
as an internal standard. Chemical shift values are given in part per
million (ppm) with respect to TMS. Mass spectra were recorded by
GCeMS (PerkineElmer Clarus 500 GC).
6.1.6. N -(2-Hydroxybenzylidene) isonicotinohydrazide (6)
ꢁ
Yield: 82%; mp: 264 C; Anal. calc. for C13
H
11
N
n
3
O
2
: C 64.72, H
ꢀ
1
4.60, N 17.42%; found: C 64.77, H 4.62, N 17.41%; IR
max (cm ): 3162
(ppm): 12.28 (s,
1H, NH),11.08 (s,1H, OH), 8.68 (s,1H, CH]N), 8.78 (d, 2H, J ¼ 6.0 Hz,
1
(
6
NH), 1673 (CO), 1558 (C]N); H NMR (DMSO-d ) d
6.1. General procedure for preparation of isonicotinyl hydrazones
1
3
(1e8)
AreH), 7.84 (d, 2H, J ¼ 6.0 Hz, AreH), 7.64e6.79 (m, 4H, AreH);
NMR (DMSO-d (ppm): 161.39 (C]O), 140.08 (CH]N), 150.57,
157.57, 149.08, 131.81, 129.31, 121.64, 119.49, 118.73, 116.52 (AreC);
C
6
) d
A mixture of isoniazid (0.01 mol) and aromatic aldehyde
0.01 mol) in ethanol was allowed to reflux for 1 h. After comple-
þ
þ
(
MS (EI, 70 eV): m/z ¼ 241.24 (M , 100%), 242.19 (M þ 1, 14.1%).
tion of reaction, the solution was cooled to room temperature and
the resulting precipitates were collected by filtration to provide the
corresponding hydrazones.
0
6.1.7. N -(4-Dimethylaminobenzylidene) isonicotinohydrazide (7)
ꢁ
Yield: 95%; mp: 106 C; Anal. calc. for C15
H
16
N
n
4
O: C 67.15, H 6.01,
max (cm ): 3384
ꢀ1
N 20.88%; found: C 67.19, H 6.07, N 20.81%; IR
(NH), 1662 (CO), 1592 (C]N); H NMR (DMSO-d
0
1
6
.1.1. N -(4-Nitrobenzylidene) isonicotinohydrazide (1)
6
)
d
(ppm): 11.82 (s,
ꢁ
Yield: 94%; mp: 256 C; Anal. calc. for C13
N 20.73%; found: C 57.82, H 3.76, N 20.69%; IR
660 (CO), 1594 (C]N); H NMR (DMSO-d
H
n
10
N
4
O
3
: C 57.78, H 3.73,
1H, NH), 8.33 (s, 1H, CH]N), 8.78e8.76 (m, 2H, AreH), 7.83e7.81
ꢀ
1
max (cm ): 3242 (NH),
(m, 2H, AreH), 7.57 (d, 2H, J ¼ 9.0 Hz, AreH), 6.76 (d, 2H,
1
13
1
6
)
d
(ppm): 12.36 (s, 1H,
J ¼ 9.0 Hz, AreH), 2.98 (s, 6H, CH
3
); C NMR (DMSO-d
6
)
d
(ppm):
NH), 8.54 (s, 1H, CH]N), 8.80 (d, 2H, J ¼ 6.0 Hz, AreH), 8.29 (d, 2H,
161.19 (C]O),140.90 (CH]N), 150.35,151.79, 149.97, 128.78, 121.58,
121.22, 111.85 (AreC); MS (EI, 70 eV): m/z ¼ 268.31 (M , 100%),
þ
J ¼ 9.0 Hz, AreH), 7.99 (d, 2H, J ¼ 9.0 Hz, AreH), 7.83 (d, 2H, J ¼ 6.0 Hz,
AreH); 13C NMR (DMSO-d
d
(ppm): 162.03 (C]O),140.30 (CH]N),
269.14 (M þ 1, 15.2%).
þ
6
)
1
50.44, 149.70, 148.06, 146.53, 128.26, 124.13, 121.62 (AreC); MS (EI,
þ
þ
0
70 eV): m/z ¼ 270.24 (M , 100.0%), 271.08 (M þ 1, 13.8%).
6.1.8. N -(4-Isopropyl benzylidene) isonicotinohydrazide (8)
ꢁ
Yield: 84%; mp:210 C; Anal. calc. for C16
H
n
17
N
3
O: C 71.89, H 6.41, N
0
ꢀ1
6
.1.2. N -Benzylidene isonicotinohydrazide (2)
15.72%; found: C 71.87, H 6.44, N 15.73%; IR
1662 (CO), 1575 (C]N); H NMR (DMSO-d
max (cm ): 3390 (NH),
d
ꢁ
1
Yield: 96%; mp: 198 C; Anal. calc. for C13
H
11
N
3
O: C 69.32, H 4.92,
n
6
)
(ppm): 11.97 (s, 1H,
ꢀ
1
N 18.66%; found: C 69.34, H 4.89, N 18.62%; IR
max (cm ): 3186
(ppm): 12.02 (s,
H, NH), 8.48 (s, 1H, CH]N), 8.77 (d, 2H, J ¼ 3.0 Hz, AreH), 7.82 (d,
H, J ¼ 6.0 Hz, AreH), 7.73 (d, 2H, J ¼ 3.0 Hz, AreH), 7.45e7.16 (m,
H, AreH); 13C NMR (DMSO-d
(ppm): 161.76 (C]O), 140.51
NH), 8.44 (s, 1H, CH]N), 8.76 (d, 2H, J ¼ 3.0 Hz, AreH), 7.81 (d, 2H,
1
(
6
NH), 1676 (CO),1551 (C]N); H NMR (DMSO-d ) d
J ¼ 6.0 Hz, AreH), 7.66 (d, 2H, J ¼ 6.0 Hz, AreH), 7.32 (d, 2H, J ¼ 6.0 Hz,
1
3
1
2
3
AreH), 2.95e2.86 (m, 1H, CH), 1.19 (d, 6H, J ¼ 9.0 Hz, CH
3
); C NMR
(DMSO-d
6
)
d
(ppm): 161.63 (C]O), 140.58 (CH]N), 150.68, 150.39,
), 23.69 (CH );
MS (EI, 70 eV): m/z ¼ 267.32 (M , 100%), 268.14 (M þ 1, 16.9%).
6
)
d
149.15,131.78,127.47,126.92,121.62 (AreC), 33.49 (CH
2
3
þ
þ
(
CH]N),150.41,149.12,134.08,130.47,128.95,127.37,121.65 (AreC);
þ
þ
MS (EI, 70 eV): m/z ¼ 225.24 (M 100%), 226.09 (M þ 1, 15.2%).
6.2. General procedure for preparation of compound 5-(pyridine-4-
0
6
.1.3. N -(4-Methoxybenzylidene) isonicotinohydrazide (3)
yl)-1, 3, 4-oxadiazole-2-(3H)-thione (9)
ꢁ
Yield: 96%; mp: 170 C; Anal. calc. for C14
.13, N 16.46%; found: C 65.89, H 5.11, N 16.45%; IR
211 (NH), 1648 (CO), 1541 (C]N); H NMR (DMSO-d (ppm):
H
13
N
3
O
2
: C 65.87, H
ꢀ
1
5
3
n
max (cm ):
A
mixture of isonicotinic hydrazide (0.1 mol), potassium
1
6
)
d
hydroxide (0.1 mol) and carbon disulfide (0.15), in ethanol (200 mL)
was heated under reflux with constant stirring for 10 h and the
11.94 (s, 1H, NH), 8.41 (s, 1H, CH]N), 8.75 (s, 2H, AreH), 7.82e7.81

S.M. Siddiqui et al. / European Journal of Medicinal Chemistry 49 (2012) 411e416
415
solvent was distilled in vacuo. The obtained residue was dissolved
in water (100 mL), filtered, and the filtrate was acidified with
hydrochloric acid. The precipitated crude product was filtered,
washed with water, dried, and crystallized from methanol.
(DMSO-d
7.67e7.36 (m, 5H, AreH); C NMR (DMSO-d
157.26 (thiadiazole ring C), 141.82, 141.17, 136.70, 135.32, 129.58, 127.13,
117.01; MS (EI, 70 eV): m/z ¼ 254.31 (M ,100%), 255.07 (M þ 1, 14.2%).
6
) d (ppm): 10.91 (s, 1H, NH), 8.85e7.98 (m, 4H, AreH),
13
6
) d (ppm): 164.45,
þ
þ
6
.2.1. 5-(Pyridine-4-yl)-1, 3, 4-oxadiazole-2-(3H)-thione(9)
6.6. In vitro antiamoebic assay
ꢁ
Yield 84%; mp: 234 C; Anal. calc. for C
7
H
5
N
3
OS: C 46.92, H 2.81,
N 23.45, S 17.89%; found: C 46.87, H 2.83, N 23.41, S 17.93%; IR
n
max
H
All the synthesized compounds were screened in vitro for anti-
amoebic activity against HM1:IMSS strain of E. histolytica by
microdilution method. E. histolytica trophozoites were cultured in
culture tubes by 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) [20,21]. 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 suspension was prepared from
ꢀ1
1
(
cm ): 3140 (NH), 1586 (C]N), 1312 (C]S), 1244 (CeOeC);
NMR (DMSO-d6)
d (ppm): 8.76e8.75 (m, 2H, AreH), 7.76e7.75 (m,
13
2
H, AreH), 6.20 (s, 1H, NH); C NMR (DMSO-d6)
d (ppm): 177.87,
1
58.80 (oxadiazole ring C), 150.86, 129.81, 119.65 (pyridine ring C);
þ
þ
MS (EI, 70 eV): m/z ¼ 179.19 (M , 100%), 180.02 (M þ 1, 8.1%).
6.3. General procedure for preparation of N-phenyl-2-(pyridine-4-
yl carbonyl) hydrazinecarbothiamide (10)
A mixture of isoniazid (0.01 mol) and phenylisothiocyanate
(
0.01 mol) in ethanol (20 ml) was allowed to reflux for 1 h. On
cooling, the colourless solid was obtained, which was filtered,
washed with cold ethanol, and dried to yield compound 10.
ꢁ
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
6
.3.1. N-Phenyl-2-(pyridine-4-ylcarbonyl)hydrazinecarbothiamide(10)
organisms from the side of flask. The number of amoeba/ml was
ꢁ
Yield: 92%; mp: 120 C; Anal. calc. forC13
H N
12 4
OS:C 57.34, H 4.44,
estimated with the help of a haemocytometer, using trypan blue
5
N 20.57, S 11.77%; found: C 57.38, H 4.47, N 20.51, S 11.74%; IR
n
max
exclusion to confirm the viability. The suspension was diluted to 10
ꢀ
1
1
(
d
(
5
cm ): 3411, 3258 (NH),1666 (CONH),1305 (C]S); H NMR (DMSO-
(ppm): 10.84 (s, 1H, NH), 9.85 (s, 1H, NH), 9.75 (s, 1H, NH), 8.75
d, 2H, J ¼ 3.0 Hz, AreH), 7.83 (d, 2H, J ¼ 6.0 Hz, AreH), 7.84e7.12 (m,
H, AreH); 13C NMR (DMSO-d
(ppm): 181.24 (C]S), 164.56 (C]
O), 150.81, 139.65, 139.46, 128.88, 128.12, 126.00, 121.74 (AreC); MS
organism/mL by adding fresh medium and 170 mL of this suspen-
sion was added to the test and control wells in the plate so that the
6
)
d
wells were completely filled (total volume, 340 mL). An inoculum
4
6
)
d
of 1.7 ꢂ 10 organisms/well was chosen so that confluent, but not
excessive growth, took place in control wells. Plate was sealed and
þ
þ
ꢁ
(
EI, 70 eV): m/z ¼ 272.32 (M , 100%), 273.08 (M þ 1, 14.1%).
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
6
4
.4. General procedure for preparation of 4-phenyl-5-pyridin-4-yl-
H-1, 2, 4-triazole-3-thiol (11)
ꢁ
washed with sodium chloride solution (0.9%) at 37 C. This proce-
A solution of N-phenyl-2-(pyridine-4-ylcarbonyl) hydrazine-
dure was completed quickly and the plate was not allowed to cool
in order 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
carbothiamide (10) (0.01 mol) in 2 N NaOH was allowed to reflux
for 2 h. The resulting solution was cooled to room temperature and
acidified to pH 3e4 with 37% hydrochloric acid. The precipitate
formed was filtered and washed with distilled water, dried and
crystallized to furnish the compound 11.
twice with distilled water and then allowed to dry. A 200 mL 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
6
.4.1. 4-Phenyl-5-pyridin-4-yl-4H-1, 2, 4-triazole-3-thiol (11)
ꢁ
Yield: 84%; mp: 200 C; Anal. calc. for C13
10
H N
4
S: C 61.40, H 3.96,
N 22.03, S 12.61%; found: C 61.37, H 3.91, N 22.07, S 12.67%; IR n
max
ꢀ1
1
(
cm ): 2489 (SH), 1600, 1490 (C]N); H NMR (DMSO-d
6
) d (ppm):
14.42 (s, 1H, SH), 8.98e8.75 (m, 2H, AreH), 7.69e7.40 (m, 7H,
13
AreH); C NMR (DMSO-d
6
) d (ppm): 169.19, 148.47 (triazole ring
C), 150.09, 134.21, 133.32, 129.83, 129.57, 128.70, 122.01 (AreC); MS
line from which the IC50 value was found. The IC50 values in
reported in Table 1.
mM are
þ
þ
(
EI, 70 eV): m/z ¼ 254.31 (M , 100%), 255.07 (M þ 1, 14.2%).
6.5. General procedure for preparation of N-phenyl-5-pyridin-4-yl-
6.7. MTT assay
1, 3, 4-thiadiazol-2-amine (12)
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 1, 3, 5, 9, 11 & the standard drug metro-
nidazole on cell proliferation was measured by using an MTT-based
assay [22]. Briefly, the cells (10,000/well) were incubated in tripli-
cate in a 96-well plate in the presence of various concentrations of
A mixture of 0.001 mol of N-phenyl-2-(pyridine-4-yl carbonyl)
hydrazinecarbothiamide (10) and concentrated H SO (1 mL) was
stirred at room temperature for 1 h. Then the reaction mixture was
poured over crushed ice. The precipitated solid was washed with
sodium carbonate solution followed by water and recrystallized
from ethanol.
2
4
compounds 1, 3, 5, 9, 11 as well as metronidazole or vehicle (DMSO)
ꢁ
6
.5.1. N-Phenyl-5-pyridin-4-yl-1, 3, 4-thiadiazol-2-amine (12)
alone in a final volume of 200
m
L at 37 C and 5% CO
2
in and
ꢁ
10 4
Yield: 32%; mp: 238 C; Anal. calc. for C13H N S: C 61.40, H 3.96, N
humidified chamber for 48 h. At the end of this time period, 20 mL
2
2.03, S 12.61%; found: C 61.44, H 3.93, N 22.07, S 12.66%. IR
n
max
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
ꢀ1
1
ꢁ
(cm ): 3261 (NH), 1622 (C]N), 1028 (NeN), 692(CeSeC); H NMR

416
S.M. Siddiqui et al. / European Journal of Medicinal Chemistry 49 (2012) 411e416
4
h, the supernatant was removed from each well. The coloured
(c) G.R. Luedtke, K. Schinzel, X. Tan, R.W. Tester, I. Nashashibi, Y. Xu, S. Dugar,
D.E. Levy, J. Jung, Bioorg. Med. Chem. Lett. 20 (2010) 2556e2559;
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formazan crystal produced from MTT was dissolved in 200 L of
m
(
DMSO, and then the absorbance (A) value was measured at 570 nm
by a multiscanner autoreader. The following formula was used for
the calculation of the percentage of cell viability (CV): CV (%) ¼ (A of
the experimental samples/A of the control) ꢂ 100.
(
4
(
e) P.M. Dewang, Dae-Kee Kim, Bioorg. Med. Chem. Lett. 20 (2010)
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