Synthesis, characterization, antiamoebic activity and cytotoxicity of novel 2-(quinolin-8-yloxy) acetohydrazones and their cyclized products (1,2,3-thiadiazole and 1,2,3-selenadiazole derivatives)

Article abstract of DOI:10.1016/j.ejmech.2010.09.066

A series of 1,2,3-thiadiazole and 1,2,3-selenadiazole derivatives were synthesized by the cyclization of novel 2-(quinolin-8-yloxy) acetohydrazones. In vitro antiamoebic activity was performed against HM1: IMSS strain of Entamoeba histolytica. The results showed that all the 2-(quinolin-8-yloxy) acetohydrazones were more active than their cyclized products (1,2,3-thiadiazole and 1,2,3-selenadiazole derivatives). SAR showed that the compounds having quinoline ring and hydrazone linkage with free N-H group are responsible for higher antiamoebic activity. The cytotoxic studies of these compounds on human breast cancer MCF-7 cell line showed that all the compounds were nontoxic at the concentration range of 1.56-50 μM.

Full text of DOI:10.1016/j.ejmech.2010.09.066

European Journal of Medicinal Chemistry 45 (2010) 6127e6134
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, characterization, antiamoebic activity and cytotoxicity of novel
2-(quinolin-8-yloxy) acetohydrazones and their cyclized products
(1,2,3-thiadiazole and 1,2,3-selenadiazole derivatives)
,
Faisal Hayat a, Attar Salahuddin a, Jamil Zargan b, Amir Azam a
*
a Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110 025, India
b Department of Toxicology, Jamia Hamdard, Hamdard Nagar, New Delhi 110 062, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1,2,3-thiadiazole and 1,2,3-selenadiazole derivatives were synthesized by the cyclization of
novel 2-(quinolin-8-yloxy) acetohydrazones. In vitro antiamoebic activity was performed against HM1:
IMSS strain of Entamoeba histolytica. The results showed that all the 2-(quinolin-8-yloxy) acetohy-
drazones were more active than their cyclized products (1,2,3-thiadiazole and 1,2,3-selenadiazole
derivatives). SAR showed that the compounds having quinoline ring and hydrazone linkage with free
NeH group are responsible for higher antiamoebic activity. The cytotoxic studies of these compounds
on human breast cancer MCF-7 cell line showed that all the compounds were nontoxic at the
Received 17 March 2010
Received in revised form
2 September 2010
Accepted 22 September 2010
Available online 7 October 2010
Keywords:
2-(Quinolin-8-yloxy)acetohydrazones
1,2,3-Thiadiazoles
concentration range of 1.56e50 mM.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1,2,3-Selenadiazoles
Entamoeba histolytica
MTT assay
1. Introduction
It is well known that nitrogen, oxygen, sulfur and selenium
containing heterocycles possess different pharmacophoric proper-
ties. Among these type of molecules, quinolinyl hydrazones, thia-
diazoles and selenadiazoles have shown various important
biological activities such as antimalarial, antitubercular, antitumor,
antibacterial, anticancer, CNS depressant, anticonvulsant, mollus-
cicidal, analgesic, anti inflammatory and anti HIV [8e21]. In our
earlier studies, we have reported the synthesis and antiamoebic
activity of different heterocyclic compounds such as thio-
semicarbazones, pyrazolines and dioxazoles which showed very
promising results [22e25].
Considering the fact that nearly all classes of heterocyclic
compounds are biologically active and as a part of our continuous
efforts towards the development of more potent amoebicidal
agents, we herein report the synthesis, characterization, anti-
amoebic activity and cytotoxicity of novel 2-(quinolin-8-yloxy)
acetohydrazones 2e7 and their cyclized products, 1,2,3-thiadiazole
and 1,2,3-selenadiazole derivatives 8e19.
Amoebiasis is the second leading cause of death from parasitic
disease worldwide [1]. Entamoeba histolytica, the causative organism
of amoebiasis, is a protozoan parasite which affects two main organ
systems in human body: the gastrointestinal tract and the liver.
Gastrointestinal involvement occurs as a result of ingestion of the
cysts of the parasite from food or water contaminated with faeces.
The cysts are digested in the intestinal lumen releasing trophozoites
[2]. Occasionally these trophozoites penetrate through to the portal
circulation, reaching the liver and brain and result in the formation
of abscesses which could be life threatening [3]. Metronidazole is
known to be a highly effective amoebicide and is considered to be
the drug of choice for the treatment of amoebiasis, but recent studies
have shown that this drug has several toxic effects such as geno-
toxicity, gastric mucus irritation, and spermatozoid damage [4,5].
Furthermore, failures in the treatment of several intestinal proto-
zoan parasites may result from drug resistant to parasites [6,7].
Therefore, it is especially necessary to search constantly for new
molecules for the treatment of amoebiasis, and to diminish and/or
eliminate the use of metronidazole and other toxic drugs.
2. Chemistry
The synthesis of 1,2,3-thiadiazole and 1,2,3-selenadiazole deriv-
atives 8e19 was performed by the cyclization of corresponding
* Corresponding author. Tel.: þ91 11 2698 1717/325 4; fax: þ91 11 2698 0229.
E-mail address: amir_sumbul@yahoo.co.in (A. Azam).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.066

6128
F. Hayat et al. / European Journal of Medicinal Chemistry 45 (2010) 6127e6134
2-(quinolin-8-yloxy) acetohydrazones 2e7 on treatment with sele-
nium dioxide/acetic acid and thionyl chloride in a manner as out-
linedin Scheme 1. The probable mechanism for cyclization is well
known [26]. All the compounds were insoluble in water but soluble
in most of the organic solvents. Melting points were recorded on
KSW melting point apparatus and are uncorrected. All the
compounds 2e19 were characterized by IR, 1H NMR, 13C NMR and
Mass spectra. The purity of the compounds was confirmed by
elemental analysis and data was found in accordance with ꢀ0.3%.
was confirmed by elemental analysis and data was found in
accordance with ꢀ0.3%.
Characteristic IR spectra showed significant bands for
the formation of 2-(quinolin-8-yloxy) acetohydrazone derivatives
2e7 where the appearance of two characteristic bands at
3137e3268 cmꢁ1 and 1668e1691 cmꢁ1 were assigned to the NeH
and C]O stretching and the band at 1621e1646 cmꢁ1 due to (C]
N) suggested the condensation of different aromatic ketones
with 2-(quinolin-8-yloxy) acetohydrazide 1. The symmetric and
asymmetric CeH stretching modes of methyl group appeared as
a shoulder just below 3000 cmꢁ1 in all the 2-(quinolin-8-yloxy)
acetohydrazone derivatives 2e7, which was found absent in case
of compounds 8e19, revealed their formation. The appearance of
bands at 1538e1595 cmꢁ1 (C]C) and 1447e1474 cmꢁ1 (N]N)
also showed the formation of 1,2,3-thiadiazole and 1,2,3-selena-
diazole rings in all the compounds 8e19. The structures of the
compounds 2e7 and 8e19 were further confirmed by 1H NMR
and 13C NMR. The NMR spectra of all the compounds were
recorded in CDCl3 and that also favors the proposed structures. In
all the compounds 2e7, (NeH) protons appeared as singlet at
3. Pharmacology
All the synthesized compounds 2e19 were screened in vitro
against HM1: IMSS strain of E. histolytica by microdilution method
[27]. All the experiments were carried out in triplicate at each
concentration level and repeated thrice. Cytotoxicity of compounds
2e7, 9, 10, 12, 16, 17 and metronidazole were studied by MTT assay
on human breast cancer MCF-7 cell line. The results of biological
activity and cytotoxicity are summarized in Tables 1 and 2.
d
9.94e11.8 ppm and the appearance of two doublets in the range
4. Results and discussion
of 8.90e8.96 ppm and 8.21e8.32 ppm corresponds to the C-2
and C-4 protons of quinoline moiety. Further, the protons of
methyl group of substituted aromatic ketones appeared at
4.1. Synthesis
d
2.40e2.48 ppm (Fig. 1). The structures of 2e7 were further
The 1,2,3-thiadiazole and 1,2,3-selenadiazole derivatives 8e19
were prepared by the cyclization of novel 2-(quinolin-8-yloxy)
acetohydrazones 2e7 as shown in Scheme 1. The starting material,
2-(quinolin-8-yloxy) acetohydrazide 1 was synthesized according
to a known method [28]. The key intermediates, 2-(quinolin-8-
yloxy) acetohydrazone derivatives 2e7 were synthesized by the
condensation of 8-quinolinoxyacetic acid hydrazide with different
aromatic ketones. The cyclization of corresponding 2-(quinolin-8-
yloxy) acetohydrazone derivatives 2e7 on treatment with thionyl
chloride gave 1,2,3-thiadiazoles 8e13, whereas the same 2-(qui-
nolin-8-yloxy) acetohydrazone derivatives 2e7 when treated with
selenium dioxide/acetic acid gave the compounds 1,2,3-selena-
diazoles 14e19. All the compounds 2e19 were characterized by IR,
1H NMR, 13C NMR and Mass spectra. The purity of the compounds
supported by 13C NMR spectra. The disappearance of ketonic
carbon (C]O) signal at 191 ppm confirmed the formation of 2-
(quinolin-8-yloxy) acetohydrazone derivatives 2e7. The forma-
tion of 1,2,3-thiadiazole and 1,2,3-selenadiazole rings was also
supported by the absence of signals at 2.44 and 10.9 ppm due to
eCH3 and (eN]NHe) respectively in all the compounds 8e19.
The singlets at 8.51e8.67 and 9.27e9.47 ppm, which arised due to
(C]CeH) group present at C-50 of the 1,2,3-thiadiazole and 1,2,3-
selenadiazole rings (Fig. 2), confirmed the cyclization of 2-(qui-
nolin-8-yloxy) acetohydrazons 2e7 into 1,2,3-thiadiazole 8e13
and 1,2,3-selenadiazole rings 14e19. In addition, the signals for
aromatic region appeared in the range of 6.67e7.99 ppm for all
the compounds.
(a)
(b)
N
N
N
(1)
OH
O
O
NH
2
O
NH
CH3
O
O
(c)
N
(d)
N
S
R
R
N
(8-13)
O
N
R
N
O
NH
(e)
N
Se
CH3
(2-7)
(14-19)
Scheme 1. General method for the synthesis of 2-(quinolin-8-yloxy) acetohydrazone (2e7), 1,2,3-thiadiazole (8e13) and 1,2,3-selenadiazole (14e19) derivatives. Reagent and
conditions: (a) Ethyl chloroacetate, Acetone, K2CO3, Reflux (b) NH2NH2$2H2O, C2H5OH, reflux 3 h (c) RCOCH3, C2H5OH, reflux 4 h (d) 0e5 ꢂC, SOCl2, Stirring at room temp. 24 h. (e)
SeO2, Glacial acetic acid, (R ¼ Aryl group of different ketones).

F. Hayat et al. / European Journal of Medicinal Chemistry 45 (2010) 6127e6134
6129
Table 1
2-(Quinolin-8-yloxy) acetohydrazones (2e7), their in vitro antiamoebic activity against HM1: IMSS strain of E. histolytica and cytotoxicity profile.
N
O
N
R
O
NH
CH
3
(2-7)
.
Compound
R
Antiamoebic activity
Cytotoxicity profile
IC50 (
m
M)
SDa
IC50 (
m
M)
SDa
2
0.03
0.12
>100
0.11
3
4
5
0.04
0.04
0.05
0.08
0.05
0.06
>100
>100
>100
0.24
Br
Cl
0.18
0.14
N
6
0.05
0.08
1.80
0.09
0.16
0.13
>100
>100
>100
0.11
0.21
0.16
O
S
7
Metronidazole
The compound with bold font IC50 values are more active than metronidazole.
a
Standard deviation, N.D. ¼ Not Done.
The structures of the compounds 8e19 were further supported
by 13C NMR spectra. The appearance of characteristic signals for C-
40 and C-50 carbon atoms in the range of 161.1e162.8 ppm and
133.2e134.8 ppm clearly favored the formation of 1,2,3-thiadiazole
and 1,2,3-selenadiazole rings in all the compounds 8e19. The
signals due to the aromatic carbons resonate at their usual posi-
tions and the values are given in the Experimental section.
microdilution method. E. histolytica trophozoites were cultured
in TYIS-33 growth medium in 96-well microtitre plate. The test
compounds (1 mg) were dissolved in DMSO (40 ml). The maximum
concentrations of DMSO in the test did not exceed 0.25%, at which
level no inhibition of amoebal growth occurred. The stock solutions
of the compounds were prepared freshly before use at a concen-
tration of 1 mg/ml. The IC50 values in mM are given in Tables 1 and
2. The results were estimated as the percentage of growth inhibi-
tion compared with the untreated controls and plotted as probit
values as a function of the drug concentration. The IC50 and
95% confidence limits were interpolated in the corresponding
doseeresponse curve. Metronidazole was used as the reference
4.2. Antiamoebic activity
All the compounds 2e19 were screened in vitro for antiamoebic
activity against HM1: IMSS strain of E. histolytica by the

6130
F. Hayat et al. / European Journal of Medicinal Chemistry 45 (2010) 6127e6134
Table 2 (continued)
Table 2
1,2,3-Thiadiazoles (8e13) and 1,2,3-selenadiazoles (14e19), their in vitro anti-
amoebic activity against HM1: IMSS strain of E. histolytica and cytotoxicity profile of
compounds 9, 10, 12, 16, 17 and metronidazole.
Compound
R
X
Antiamoebic
activity
Cytotoxicity
profile
IC50 (m m
M) SDa IC50 ( M) SDa
N
17
Se 0.46
0.05 86.62
N.D
N
X= S (8-13)
X= Se (14-19)
N
X
R
18
Se 5.72
Se 5.28
1.80
0.04 N.D
0.02 N.D
0.13 >100
N.D
N.D
0.16
Compound
R
X
S
Antiamoebic
activity
Cytotoxicity
profile
O
S
IC50 (
7.46
m m
M) SDa IC50 ( M) SDa
19
8
0.18 N.D
N.D
Metronidazole
9
S
S
0.24
0.39
0.05 68.11
0.18
The compound with bold font IC50 values are more active than metronidazole.
a
Standard deviation, N.D. ¼ Not Done.
Br
Cl
drug which had a 50% inhibitory concentration of 1.80
experiments. The 2-(quinolin-8-yloxy) acetohydrazone derivatives
2e7 showed an IC50 value in the range 0.03e0.08 M. However, the
cyclized compounds 8e19 showed decreased antiamoebic
activity in the range of 0.23e7.46 M. Among all the 1,2,3-thia-
diazole and 1,2,3-selenadiazole derivatives 8e19, the compounds
having 4-chloro phenyl (9, IC50 ¼ 0.24 M), 4-bromo phenyl (10,
IC50 0.39 M; 16, IC50 0.58 M), furan ring (12,
IC50 ¼ 0.23 M) and thiophene ring (17, IC50 ¼ 0.46 M)
mM in our
10
0.08 >100
0.29
N.D
m
a
m
m
¼
m
¼
m
11
12
S
5.46
0.04 N.D
N
m
m
substituted at C-4 position of 1,2,3-thia and selenadiazole rings
were distinctly more active. Compounds 9 and 12 are the most
active among the azole series 8e19. In terms of structural activity
relationship it was concluded that among the whole series of
compounds 2e19, all the 2-(quinolin-8-yloxy) acetohydrazone
derivatives 2e7 showed excellent antiamoebic activity compared to
the cyclized products 8e19. This may be due to the presence of
quinoline nucleus and hydrazone linkage [eNHeN]C(CH3)eR]
which resembles Idoquinol, currently used as an antiamoebic drug
[29] and quinoline based hydrazones have shown antimalarial and
antitubercular activity [19e21]. Many natural product bearing
quinoline nucleus such as quinidine, quinidinone, and quinine
from the plant Cinchona ledgeriana were also found to be anti-
amoebic [30].
S
S
0.23
0.13 95.07
0.27
O
S
13
14
2.25
0.06 66.90
0.25
N.D
The considerable loss of antiamoebic activity displayed in the
1,2,3-thiadiazoles and 1,2,3-selenadiazoles is attributed to the
loss of active quinoline nucleus in the conversion of hydrazone
linkage bearing a free NeH group to a ring constrained structures
Se 4.56
0.07 N.D
4
5
9
15
16
Se 2.95
0.04 N.D
N.D
3
2
6
7
10
N
8
Br
Cl
1
O
Se 0.58
0.11 57.05
0.13
N
R
O
NH
CH
3
Fig. 1. Structure of 2(quinolin-8-yloxy) acetohydrazones (2e7).

F. Hayat et al. / European Journal of Medicinal Chemistry 45 (2010) 6127e6134
6131
5. Conclusion
2'
N
1'
X
This research involves the synthesis of 2-(quinolin-8-yloxy)
acetohydrazone, 1, 2, 3-thiadiazole and 1,2,3-selenadiazole deriva-
tives 2e19. The antiamoebic activity of all these compounds was
examined using HM1: IMSS strain of E. histolytica and the results
showed that the compounds 2e7 having quinoline nucleus with
a hydrazone linkage [eNHeN]C(CH3)eR] bearing a free NeH
group exhibited higher antiamoebic activity as compared with
derived cyclized products 8e19. SAR studies showed that the
considerable loss of antiamoebic activity displayed in the 1,2,3-
thiadiazole and 1,2,3-selenadiazole derivatives 8e19 is attributed
to the loss of 2-(quinolin-8-yloxy) acetohydrazones moiety. The
MTT assay results showed that all the compounds were nontoxic.
Only compounds 9 and 16 showed moderate cytotoxicity against
the human breast cancer MCF-7 cell line at a concentration of
3'
N
4'
5'
R
X = S, Se
Fig. 2. Structure of 1,2,3-thiadiazole and 1,2,3-selenadiazole derivatives (8e19).
8e19. All the biologically active hydrazones possess a free NeH
group in their structure [31]. Therefore it can be concluded that
a quinoline nucleus with a hydrazone linkage [eNHeN]C(CH3)e
R] bearing a free NeH group is responsible for higher anti-
amoebic activity.
100 mM.
6. Experimental protocol
All the chemicals were purchased from Aldrich Chemical
Company (USA). Precoated aluminium sheets (silica gel 60 F254,
Merck Germany) were used for thin-layer chromatography (TLC)
and spots were visualized under UV light. Elemental analyses were
performed on Heraeus Vario EL III analyzer the results were within
ꢀ0.3% of the theoretical values. IR spectra on KBr disks were
recorded on a Perkin Elmer model 1620 FT-IR spectrophotometer.
1H NMR and 13C NMR spectra were recorded on Bruker Spectrospin
DPX 300 MHz and Bruker Spectrospin DPX 75 MHz spectrometer
respectively using CDCl3 as a solvent and trimethylsilane (TMS) as
an internal standard. Splitting patterns are designated as follows; s,
singlet; d, doublet; t, triplet; m, multiplet. Chemical shift values are
given in ppm. The FAB mass spectra of the compounds were
recorded on JEOL SX 102/DA-6000 mass spectrometer using Argon/
Xenon 6 KV, 10 mA as the FAB gas and m-nitrobenzyl alcohol (NBA)
was used as the matrix.
4.3. Cytotoxicity profile
To examine the effect of antiamoebic compounds 2e7, 9, 10,
12, 16, 17 and metronidazole on cell proliferation, we studied
their cytotoxicity on human breast cancer MCF-7 cell line. A
subconfluent 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 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 proliferation. The concentration range for all the compounds
was 1.56e100
metronidazole exhibited 100% viability at the concentration
range of 1.56e100 M whereas the compound 7 showed 88%
viability at 100 M. These results mean that all the compounds
mM. Fig. 3 depicts the compounds 2e6 and
m
m
6.1. Preparation of 2-(quinolin-8-yloxy) acetohydrazide (1)
2e7 and metronidazole were nontoxic against the human breast
cancer MCF-7 cell line. Compounds 9, 10, 12, 17 and metronida-
zole exhibited >80% viability (Fig. 4); compound 16 showed 66%
2-(Quinolin-8-yloxy) acetohydrazide was prepared by a repor-
ted method [27].
viability at the concentration range of 1.56e50
m
M (Fig. 4). On
only
increasing the concentration range up to 100
m
M
compounds 9 and 16 (Viability 68% and 57%) showed moderate
cytotoxicity against the breast cancer MCF-7 cell line. The cyto-
toxicity IC50 values along with the standard deviation values of
compounds 2e7, 9, 10, 12, 16, 17 and metronidazole are given in
Tables 1 and 2.
6.2. Synthesis of 2-(quinolin-8-yloxy) acetohydrazones (2e7):
general procedure
A mixture of 2-(quinolin-8-yloxy) acetohydrazide 2 (10 mmol)
and appropriate aromatic ketone (10 mmol) in absolute ethanol
102
100
98
96
94
92
90
88
86
84
82
Control
MNZ
2
3
4
5
6
7
0
1.65
3.14
6.25
Concentration (µM)
12.5
25
50
100
Fig. 3. Percentage of viable cells after 48 h pre-treatment of human breast cancer MCF-7 cells with compounds 2, 3, 4, 5, 6, 7 and metronidazole (MNZ), evaluated by MTT assay.

6132
F. Hayat et al. / European Journal of Medicinal Chemistry 45 (2010) 6127e6134
120
100
80
60
40
20
0
Control
MNZ
9
10
12
16
17
0
1.56
3.14
6.25
12.5
25
50
100
Concentration (µM)
Fig. 4. Percentage of viable cells after 48 h pre-treatment of human breast cancer MCF-7 cells with compounds 9, 10, 12, 16, 17 and metronidazole (MNZ), evaluated by MTT assay.
(50 ml) was refluxed for 4 h with continuous stirring. After cooling,
the solid was filtered and recrystallized from appropriate solvent.
(NH), 3097 (Ar-H), 2921, 2844 (CeH),1686 (C]O),1624 (C]N),1579
(C]C); 1H NMR (CDCl3)
(ppm): 11.08 (s,1H, NH), 8.96 (d, 1H,
d
J ¼ 4.1 Hz, quinoline ring C-2), 8.32 (d, 1H, J ¼ 8.1 Hz, quinoline ring
6.2.1. N0-[(1E)-1-Phenylethylidene]-2-(quinolin-8-yloxy)
acetohydrazone (2)
C-4), 8.24e7.25 (m, 8H, quinoline and pyridine ring), 4.92 (s,2H,
CH2), 2.40(s, 3H, CH3); 13C NMR (CDCl3)
d (ppm): 165.32 (C]O),
Yield 82%; m.p. 189 ꢂC; Anal. calc. for C19H17N3O2: C 71.46, H 5.37,
N 13.16%; found: C 71.38, H 5.29, N 13.11%; IR nmax (cmꢁ1): 3249
(NH), 3023 (Ar-H), 2912, 2827 (CeH),1668 (C]O),1626 (C]N),1545
(149.79, 148.44, 136.31, 129.64, 126.84, 122.14, 121.55 quinoline and
pyridine ring), 70.46 (CH2), 11.85 (methyl); FAB-MS (m/z): 320 (Mþ,
100%), 321 (Mþ þ 1, 82%).
(C]C); 1H NMR (CDCl3)
d (ppm): 9.94 (s, 1H, NH), 8.95 (d, 1H,
J ¼ 4.1 Hz, quinoline ring C-2), 8.24 (d, 1H, J ¼ 8.3 Hz, quinoline ring
6.2.5. N΄-[(1E)-1-(Furan-2-yl) ethylidene]-2-(quinolin-8-yloxy)
acetohydrazone (6)
C-4), 7.64e6.96 (m, 9H, quinoline and phenyl ring), 4.95 (s, 2H, CH2),
2.44 (s, 3H, CH3); 13C NMR (CDCl3)
d
(ppm): 165.66 (C]O), (149.51,
Yield 78%; m.p: 176 ꢂC; Anal. calc. for C17H15N3O3: C 66.01, H 4.89,
N 13.58%; found: C 66.04, H 4.72, N 13.46%; IR nmax(cmꢁ1): 3268
(NH), 3107 (Ar-H), 2972, 2851 (CeH),1675 (C]O), 1646 (C]N), 1542
147.32, 136.35, 135.12, 127.23, 122.54, 121.55 quinoline and phenyl
ring), 70.56 (CH2), 11.87 (methyl); FAB-MS (m/z): 319 (Mþ, 100%),
320 (Mþ þ 1, 65%), 319 (Mþ ꢁ CH3, 28%).
(C]C); 1H NMR (CDCl3)
d (ppm): 10.8 (s,1H, NH), 8.90 (d, 1H,
J ¼ 4.2 Hz, quinoline ring C-2), 8.22 (d, 1H, J ¼ 8.4 Hz, quinoline ring
6.2.2. N0-[(1E)-1-(4-bromophenyl) ethylidene]-2-(quinolin-8-
yloxy) acetohydrazone (3)
C-4), 7.53e6.42 (m, 7H, quinoline and furan ring), 4.94 (s, 2H, CH2),
2.48 (s, 3H, CH3); 13C NMR (CDCl3)
d (ppm): 164.77 (C]O), (149.64,
Yield 76%; m.p: 189 ꢂC; Anal. calc. for C19H16N3O2Br: C 56.27, H
3.67, N 10.94%; found: C 56.19, H 3.62, N 10.88%; IR nmax (cmꢁ1): 3165
(NH), 3065 (Ar-H), 2987, 2834 (CeH),1672 (C]O),1628 (C]N),1546
145.88,136.36,135.12,129.61,122.17 quinoline and furan ring), 70.40
(CH2), 12.86 (methyl); FAB-MS (m/z): 309 (Mþ, 100%), 310 (Mþ þ 1,
68%), 294 (Mþ ꢁ CH3, 28%).
(C]C); 1H NMR (CDCl3)
d (ppm): 10.5 (s, 1H, NH), 8.94 (d, 1H,
J ¼ 4.2 Hz, quinoline ring C-2), 8.22 (d, 1H, J ¼ 8.3 Hz, quinoline ring
6.2.6. 2-(Quinolin-8-yloxy) N0-[(1E)-1-(thiophen-2-yl) ethylidene]-
acetohydrazone (7)
C-4), 7.66e6.97 (m, 8H, quinoline and phenyl ring), 4.94(s, 2H, CH2),
2.42 (s, 3H, CH3); 13C NMR (CDCl3)
d
(ppm): 165.23 (C]O), (149.21,
Yield 81%; m.p: 164 ꢂC; Anal. calc. for C17H15N3O2S: C 61.72, H
4.21, N 13.50%; found: C 61.64, H 4.17, N 13.41%; IR nmax(cmꢁ1): 3248
(NH), 3062 (Ar-H), 2972, 2863 (C-H),1691 (C]O),1626 (C]N),1552
147.32, 136.86, 135.23, 126.83, 121.93 quinoline and phenyl ring),
70.12 (CH2), 11.93 (methyl); FAB-MS (m/z): 398 (Mþ, 100%), 399
(Mþ þ 1, 74%).
(C]C); 1H NMR (CDCl3)
d (ppm): 10.8 (s, 1H, NH), 8.90 (d, 1H,
J ¼ 4.2 Hz, quinoline ring C-2), 8.22 (d, 1H, J ¼ 8.1 Hz, quinoline ring
6.2.3. N΄-[(1E)-1-(4-chlorophenyl) ethylidene]-2-(quinolin-8-
yloxy) acetohydrazone (4)
C-4), 7.54e7.01 (m, 7H, quinoline and thiophene ring), 4.98 (s, 2H,
CH2), 2.44 (s, 3H, CH3); 13C NMR (CDCl3)
d (ppm): 165.93 (C]O),
Yield 78%; m.p: 171 ꢂC; Anal. calc. for C19H16N3O2Cl: C 63.63, H
4.15, N 12.37%; found: C 63.57, H 4.09, N 12.31%; IR nmax (cmꢁ1): 3246
(NH), 3035 (Ar-H), 2954, 2842 (CeH),1682 (C]O),1621 (C]N),1535
(149.28, 148.72, 134.86, 132.23, 127.83, 121.93 quinoline and thio-
phene ring), 70.42 (CH2), 12.03 (methyl); FAB-MS (m/z): 325 (Mþ,
100%), 326 (Mþ þ 1, 42%), 319 (Mþ ꢁ CH3, 21%).
(C]C); 1H NMR (CDCl3)
d (ppm): 10.3 (s,1H, NH), 8.92 (d, 1H,
J ¼ 4.3 Hz, quinoline ring C-2), 8.21(d, 1H, J ¼ 8.4 Hz, quinoline ring
6.3. Synthesis of 1,2,3-thiadiazole derivatives (8e13): general
procedure
C-4), 7.63e6.97 (m, 8H, quinoline and phenyl ring), 4.93 (s, 2H, CH2),
2.45 (s, 3H, CH3); 13C NMR (CDCl3)
d (ppm): 164.98 (C]O), (149.86,
148.68, 135.43, 128.68, 125.12, 122.13 quinoline and phenyl ring),
70.58 (CH2), 12.16 (methyl); FAB-MS (m/z): 353 (Mþ, 100%), 354
(Mþ þ 1, 5%), 338 (Mþ ꢁ CH3, 43%).
All the 1,2,3-thiadiazole derivatives were synthesized by the
cyclization of 2-(quinolin-8-yloxy) acetohydrazones 2e7 (5 mmol)
in an excess amount of thionyl chloride (10 ml) at 0e5 ꢂC for 30 min
and then stirred at room temperature overnight until no more
hydrogen chloride was produced. The remaining thionyl chloride
was evaporated under vacuum to afford the crude title compounds
8e13, which were further purified by column chromatography
using 7:3 hexane: ethylacetate as eluent [32].
6.2.4. N΄-[(1E)-1-(Pyridin-2-yl) ethylidene]-2-(quinolin-8-yloxy)
acetohydrazone (5)
Yield 87%; m.p: 188 ꢂC; Anal. calc. for C18H16N4O2: C 67.49, H
5.03, N 17.49%; found: C 67.36, H 5.07, N 17.38%; IR nmax (cmꢁ1): 3137

F. Hayat et al. / European Journal of Medicinal Chemistry 45 (2010) 6127e6134
6133
6.3.1. 4-Phenyl-[1,2,3]-thiadiazole (8)
poured into ice water and extracted with chloroform. The
combined organic layers were washed with water and dried over
anhydrous sodium sulphate. The solvent was removed under
vacuum to afford the crude title compounds 14e19, which were
further purified by column chromatography using 7:3 hexane:-
ethylacetate as eluent [32].
Yield: 69%; mp: 162 ꢂC; Anal. calc. for C8H6N2S: C 59.23, H 3.73,
N 17.27%; found: C 59.12, H 3.67, N 17.19%; IR nmax (cmꢁ1): 3018
(AreH), 1550 (C]C), 1447(N]N); 1H NMR (CDCl3)
d
(ppm): 8.67 (s,
1H, thiadiazole), 7.68e7.65 (m, 2H, Ar-H), 7.45e7.28 (m, 3H, Ar-H);
13C NMR (CDCl3) (ppm): 161.5 (C-40, thiadiazole), 134.7 (C-50,
d
thiadiazole), 130.5, 129.2 128.6, 126.6 (Ar-C); FAB-MS (m/z): 162
(Mþ, 100%), 163 (Mþ þ 1, 37%).
6.4.1. 4-Phenyl-[1,2,3]-selenadiazole (14)
Yield: 66%; mp: 198 ꢂC; Anal. calc. for C8H6N2Se: C 45.95, H 2.89,
6.3.2. 4-(4-Bromophenyl)-[1,2,3]-thiadiazole (9)
N 13.40%; found: C 45.88, H 2.82, N 13.29%; IR nmax (cmꢁ1): 3090
Yield: 55%; mp: 123 ꢂC; Anal. calc. for C8H5N2SBr: C 39.85, H
(AreH), 1542 (C]C), 1474 (N]N); 1H NMR (CDCl3)
d
(ppm): 9.27 (s,
2.09, N 11.62%; found: C 39.74, H 2.16, N 11.57%; IR nmax (cmꢁ1):
1H, selenadiazole), 7.57e7.54 (m, 2H, Ar-H), 7.38e7.34 (m, 3H,
3087 (AreH), 1542 (C]C), 1454 (N]N); 1H NMR (CDCl3)
d
(ppm):
Ar-H); 13C NMR (CDCl3) (ppm): 163.6 (C-40, selenadiazole), 134.8
d
8.54 (s, 1H, thiadiazole), 7.66e7.63 (m, 2H, Ar-H), 7.41e7.39 (m, 2H,
(C-50, selenadiazole), 130.6, 129.2, 129.1, 128.3, 126.6 (Ar-C); FAB-MS
Ar-H); 13C NMR (CDCl3)
d
(ppm): 162.5 (C-40, thiadiazole), 133.2
(m/z): 209 (Mþ, 100%), 210 (Mþ þ 1, 18%).
(C-50, thiadiazole), 130.4, 129.7, 128.2, 126.5 (Ar-C); FAB-MS (m/z):
241(Mþ, 100%), 242(Mþ þ 1, 22%), 161 (Mþ ꢁ Br, 5%).
6.4.2. 4-(4-Bromophenyl)-[1,2,3]-selenadiazole (15)
Yield: 56%; mp: 177 ꢂC; Anal. calc. for C8H5N2BrSe: C 33.36, H
6.3.3. 4-(4-Chlorophenyl)-[1,2,3]-thiadiazole (10)
1.75, N 9.73%; found: C 33.24, H 1.66, N 9.66%; IR nmax (cmꢁ1): 3068
Yield: 62%; mp: 113 ꢂC; Anal. calc. for C8H5N2SCl: C 48.86, H 2.56,
(AreH), 1558 (C]C), 1474 (N]N); 1H NMR (CDCl3)
d
(ppm): 9.36 (s,
N14.24%; found: C 48.77, H 2.49, N 14.18%; IR nmax (cmꢁ1): 3086
1H, selenadiazole), 7.78e7.69 (m, 2H, Ar-H), 7.45e7.24 (m, 2H,
(AreH), 1551 (C]C), 1457 (N]N); 1H NMR (CDCl3)
d
(ppm): 8.56
(s,1H, thiadiazole), 7.79e7.81 (m, 2H, Ar-H), 7.61e7.48 (m, 2H, Ar-H);
13C NMR (CDCl3) (ppm): 162.6 (C-40, thiadiazole), 134.2 (C-50,
Ar-H); 13C NMR (CDCl3) (ppm): 162.8 (C-40, selenadiazole), 134.3
d
(C-50, selenadiazole), 130.5, 129.7, 128.3, 126.3 (Ar-C); FAB-MS (m/z):
288 (Mþ, 100%), 289 (Mþþ1, 31%).
d
thiadiazole),130.2,129.6 128.7,127.1126.6 (Ar-C); FAB-MS (m/z): 196
(Mþ, 100%), 197 (Mþ þ 1, 24%),198 (Mþ þ 2, 32%), 192 (Mþ ꢁ 4, 98%).
6.4.3. 4-(4-Chlorophenyl)-[1,2,3]-selenadiazole (16)
Yield: 62%; mp: 154 ꢂC; Anal. calc. for C8H5N2ClSe: C 39.45, H
6.3.4. 2-(1,2,3-Thiadiazole-4-yl)pyridine (11)
2.07, N 11.50%; found: C 39.38, H 2.09, N 11.43%; IR nmax (cmꢁ1):
Yield: 38%; mp: 163 ꢂC; Anal. calc. for C7H5N3S: C 51.52, H 3.09, N
3079 (AreH), 1595 (C]C), 1452 (N]N); 1H NMR (CDCl3)
d
(ppm):
25.75%; found: C 51.41, H 3.12, N 25.67%; IR nmax (cmꢁ1): 3011
9.47 (s, 1H, selenadiazole), 8.02 (d, 2H, J ¼ 8.4 Hz, Ar-H), 7.51e7.48
(AreH), 1544 (C]C), 1471 (N]N); 1H NMR (CDCl3)
d
(ppm): 8.52 (s,
(m, 2H, Ar-H); 13C NMR (CDCl3) (ppm): 162.4 (C-40, selenadiazole),
d
1H, thiadiazole), 8.03 (d, 1H, J ¼ 7.1 Hz, Ar-H), 7.95 (d, 1H, J ¼ 7.1 Hz,
133.7 (C-50, selenadiazole), 130.9, 129.5, 129.4, 128.6, 126.3 (Ar-C);
Ar-H), 7.59e6.31 (m, 2H, Ar-H); 13C NMR (CDCl3)
d
(ppm): 162.6 (C-
FAB-MS (m/z): 243 (Mþ, 100%), 244 (Mþ þ 1, 48%).
40, thiadiazole), 134.3 (C-50, thiadiazole), 128.6, 124.7, 123.7, 126.8
(Ar-C); FAB-MS (m/z): 163 (Mþ, 100%), 164 (Mþ þ 1, 28%).
6.4.4. 2-(1,2,3-Selenadiazole-4-yl)pyridine (17)
Yield: 43%; mp: 186 ꢂC; Anal. calc. for C7H5N3Se: C 40.02, H 2.40,
N 20.00%; found: C 40.05, H 2.34, N 20.09%; IR nmax (cmꢁ1): 3088
6.3.5. 4-(Furan-2-yl)-[1,2,3]-thiadiazole (12)
Yield: 35%; mp: 178 ꢂC; Anal. calc. for C6H4N2OS: C 47.36, H 2.65,
(AreH), 1565 (C]C), 1466 (N]N); 1H NMR (CDCl3)
d (ppm): 9.46 (s,
N 18.41%; found: C 47.28, H 2.57, N 18.29%; IR nmax (cmꢁ1): 3075
1H, selenadiazole), 8.04 (d, 1H, J ¼ 7.1 Hz, Ar-H), 7.95 (d, 1H,
(AreH), 1546 (C]C), 1456 (N]N); 1H NMR (CDCl3)
d
(ppm): 8.58 (s,
J ¼ 7.1 Hz, Ar-H), 7.54e7.33 (m, 2H, Ar-H); 13C NMR (CDCl3)
d (ppm):
1H, thiadiazole), 7.26e6.13 (m, 2H, Ar-H), 6.37 (d, 1H, J ¼ 3.3 Hz,
161.1 (C-40, selenadiazole), 134.2 (C-50, selenadiazole), 129.4, 128.6,
Ar-H); 13C NMR (CDCl3)
d
(ppm): 162.2 (C-40, thiadiazole), 133þ.6
127.7, 126.1 (Ar-C); FAB-MS (m/z): 210 (Mþ, 100%), 211 (Mþ þ 1, 42%).
(C-50, thiadiazole),121.6, 117.6,109.4 (Ar-C); FAB-MS (m/z): 152 (M ,
100%), 153 (Mþ þ 1, 46%).
6.4.5. 4-(Furan-2-yl)-[1,2,3]-selenadiazole (18)
Yield: 28%; mp: 177 ꢂC; Anal. calc. for C6H4N2OSe: C 36.20, H
6.3.6. 4-(Thiophen-2-yl)-[1,2,3]-thiadiazole (13)
2.03, N 14.07%; found: C 36.13, H 2.11, N 14.05%; IR nmax (cmꢁ1):
Yield: 43%; mp: 189 ꢂC; Anal. calc. for C6H4N2S2: C 42.83, H 2.40,
3078 (AreH), 1538 (C]C), 1462(N]N); 1H NMR (CDCl3)
d
(ppm):
N 16.65%; found: C 42.74, H 2.29, N 16.52%; IR nmax (cmꢁ1): 3063
9.32 (s, 1H, selenadiazole), 7.54e6.58 (m, 3H, Ar-H); 13C NMR
(AreH), 1548 (C]C), 1471 (N]N); 1H NMR (CDCl3)
d
(ppm): 8.51 (s,
(CDCl3) d
(ppm): 162.8 (C-40, selenadiazole), 133.8 (C-50, selena-
1H, thiadiazole), 7.66 (d, 1H, J ¼ 4.2 Hz, Ar-H), 7.42 (d, 1H, J ¼ 4.8 Hz,
diazole), 129.8, 128.4, 126.1 (Ar-C); FAB-MS (m/z): 199 (Mþ, 100%),
200 (Mþ þ 1, 37%).
Ar-H), 7.12 (t, 1H, J ¼ 3.6 Hz, Ar-H); 13C NMR (CDCl3)
d (ppm): 161.8
(C-40, thiadiazole), 134.8 (C-50, thiadiazole), 131.4, 129.3, 128.9, 126.4,
117.4 (Ar-C); FAB-MS (m/z): 168 (Mþ, 100%), 169 (Mþ þ 1, 22%).
6.4.6. 4-(Thiophen-2-yl)-[1,2,3]-selenadiazole (19)
Yield: 28%; mp: 164 ꢂC; Anal. calc. for C6H4N2SSe: C 33.50, H
1.87, N 13.02%; found: C 33.48, H 1.76, N 13.07%; IR nmax (cmꢁ1):
6.4. Synthesis of 1,2,3-selenadiazole derivatives (14e19): general
procedure
3090 (AreH), 1542 (C]C), 1474 (N]N); 1H NMR (CDCl3)
d (ppm):
9.30 (s, 1H, selenadiazole), 7.41 (d, 1H, J ¼ 4.8 Hz, Ar-H), 7.16 (t, 2H,
All the 1,2,3-selenadiazole derivatives were synthesized by
dissolving of respective 2-(quinolin-8-yloxy) acetohydrazones 2e7
(5 mmol) in glacial acetic acid (15 ml) at room temperature and the
resulting mixture was treated with SeO2 powder (6.5 mmol). The
mixture was kept under vigorous stirring. After 2e3 min the color
of the mixture becomes red. Monitoring of the reaction by TLC
(eluent: 7:3 hexane:ethyl acetate) showed that the reaction was
complete in 10e24 h. The mixture was filtered and the filtrates
J ¼ 5.1 Hz, Ar-H); 13C NMR (CDCl3)
d
(ppm): 162.5 (C-40, selena-
diazole), 133.3 (C-50, selenadiazole), 128.3, 127.5, 124.9, 104.5 (Ar-C).
FAB-MS (m/z): 215 (Mþ, 100%), 216 (Mþ þ 1, 29%).
6.5. In vitro antiamoebic assay
All the compounds (2e19) were screened in vitro for antiamoebic
activity against HM1: IMSS strain of E. histolytica by microdilution

6134
F. Hayat et al. / European Journal of Medicinal Chemistry 45 (2010) 6127e6134
method [27]. E. histolytica trophozoites were cultured in culture
tubes by using Diamond TYIS-33 growth medium [33]. The test
compounds (1 mg) were dissolved in DMSO (40 ml, level at which no
was defined as the relative absorbance of treated versus untreated
control cells. Plates were analyzed in an ELISA plate reader (Lab-
systems Multiskan RC, Helsinki, Finland) at 570 nm with a reference
wavelength of 655 nm.
inhibition of amoeba occurs) [30,34]. 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
a confluent culture by pouring off the medium at 37 ꢂC and adding
5 ml of fresh medium, chilling the culture tube on ice to detach the
organisms from the side of the 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
Acknowledgments
This work was supported by Council of Scientific and Industrial
Research (grant # 01(2278)/08/EMR-II New Delhi, India). One of us
(F.H.) is thankful to the University Grant Commission (UGC), India
for the financial support.
References
[1] S.L. Stanley Jr., Lancet 361 (2003) 1025e1034.
[2] D.C. Ng, S.Y. Kwok, Y. Cheng, C.C. Chung, M.K. Li, Hong Kong Med. J. 12 (2006)
71e73.
[3] M. Espinosa-Cantellano, A. Martinez-Palmo, Clin. Microbiol. Rev. 13 (2000)
318e331.
organism/ml by adding fresh medium and 170
was added to the test and control wells in the plate so that the wells
were completely filled (total volume, 340 l). An inoculum of
ml of this suspension
m
1.7 ꢃ 104 organisms/well was chosen so that confluent, but not
excessive growth, took place in control wells. Plates were sealed and
gassed for 10 min with nitrogen before incubation at 37 ꢂC 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 ꢂC. This proce-
dure was completed quickly and the plate was not allowed to cool in
order to prevent the detachment of amoebae. 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
[4] F.A. el-Nahas, M.I. el-Ashmawy, Basic Clin. Pharmacol. Toxicol. 94 (2004)
226e231.
[5] V. Purohit, K.A. Basu, Chem. Res. Toxicol. 13 (2000) 673e692.
[6] P. Abboud, V. Lemee, G. Gargala, P. Brasseur, J.J. Ballet, F. Borsa-Lebas, F. Caron,
L. Favennec, Clin. Infect. Dis. 32 (2001) 1792e1794.
[7] W. Petri, Trends Parasitol. 19 (2003) 523e526.
[8] D.A. Ibrahim, Eur. J. Med. Chem. 44 (2009) 2776e2781.
[9] S. Talath, A.K. Gadad, Eur. J. Med. Chem. 41 (2006) 918e924.
ꢀ
[10] W. Rzeski, J. Matysiak, M. Kandefer-Szerszen, Bioorg. Med. Chem. 15 (2007)
3201e3207.
[11] V. Jatav, P. Mishra, S. Kashaw, J.P. Stables, Eur. J. Med. Chem. 43 (2008)
1945e1954.
[12] A.A. Fadda, E. Abdel-Latif, R.E. El-Mekawy, Eur. J. Med. Chem. 44 (2009)
1250e1256.
[13] H.N. Hafez, M.I. Hegab, I.S. Ahmed-Farag, A.B.A. El-Gazzar, Bioorg. Med. Chem.
Lett. 18 (2008) 4538e4543.
[14] R.S. Lamani, N.S. Shetty, R.R. Kamble, I.A.M. Khazi, Eur. J. Med. Chem. 44 (2009)
2828e2833.
[15] P. Arsenyan, K. Rubina, I. Shestakova, I. Domracheva, Eur. J. Med. Chem. 42
(2007) 635e640.
[16] P. Zhan, X. Liu, Z. Fang, C. Pannecouque, E.D. Clercq, Bioorg. Med. Chem. 17
(2009) 6374e6379.
[17] A.V. Karnik, A.M. Kulkarni, N.J. Malviya, B.R. Mourya, B.L. Jadhav, Eur. J. Med.
Chem. 43 (2008) 2615e2617.
[18] A.R. Jalilian, S. Sattari, M. Bineshmarvasti, M. Daneshtalab, A. Shafiee, Farm-
acognosia 58 (2003) 63e68.
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 line
[19] S. Gemma, G. Kukreja, C. Fattorusso, M. Persico, M. Romano, M. Altarelli,
L. Savini, G. Campiani, E. Fattorusso, N. Basilico, Bioorg. Med. Chem. Lett. 16
(2006) 5384e5388.
from which the IC50 value was found. The IC50 values in
reported in Tables 1 and 2.
mM are
[20] L. Savini, L. Chiasserini, A. Gaeta, C. Pellerano, Bioorg. Med. Chem. 10 (2002)
2193e2198.
[21] A. Nayyar, A. Malde, E. Coutinho, R. Jain, Bioorg. Med. Chem. 14 (2006)
7302e7310.
[22] N. Bharti, K. Husain, M.T.G. Garza, D.E. Cruz-Vega, J. Castro-Garza, B.D. Mata-
Cardenas, F. Naqvi, A. Azam, Bioorg. Med. Chem. Lett. 12 (2002) 3475e3478.
[23] M. Abid, A. Azam, Eur. J. Med. Chem. 40 (2005) 935e942.
[24] I. Irfan, N. Sawangjaroen, A.R. Bhat, A. Azam, Eur. J. Med. Chem. 45 (2010)
1648e1653.
6.6. MTT assay
MCF-7 cells were cultured and maintained as monolayer in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% of fetal calf serum (FCS), antibiotics: 100 IU/ml of penicillin and
100 m
g/ml of streptomycin. All cells were cultured in flasks at 37 ꢂC
in the 100% humidity atmosphere and 5% of CO2 [35]. Only viable
cells were used in the assay. Exponentially growing cells were
plated at 1.2 ꢃ 104 cells per well into 96-well plates and incubated
for 48 h before the addition of drugs. Stock solutions of compounds
were initially dissolved in 20% (v/v) DMSO and further diluted with
fresh complete medium. The growth-inhibitory effects of the
compounds were measured using standard tetrazolium MTT assay.
[25] P.F. Iqbal, H. Parveen, A.R. Bhat, F. Hayat, A. Azam, Eur. J. Med. Chem. 44 (2009)
4747e4751.
[26] C.D. Hurd, R.I. Mori, J. Am. Chem. Soc. 77 (1955) 5359.
[27] C.W. Wright, M.J. O’Neill, J.D. Phillipson, D.C. Warhurst, Antimicrob. Agents
Chemother. 32 (1988) 1725e1729.
[28] M. Ahmed, R. Sharma, D.P. Nagda, J.L. Jat, G.L. Talesara, Arkivoc xi (2006)
66e75.
[29] S. Singh, N. Bharti, P.P. Mohapatra, Chem. Rev. 109 (2009) 1900e1947.
[30] A.T. Keene, A. Harris, J.D. Phillipson, D.C. Warhurst, Planta Med. 52 (1986)
278e284.
After 48 h of incubation at 37 ꢂC, the medium was removed and 5
ml
[31] S. Rollas, G. Küçükgüzel, Molecules 12 (2007) 1910e1939.
[32] M. Al-Smadi, S. Ratrout, Molecules 9 (2004) 957e967.
[33] L.S. Diamond, D.R. Harlow, C.C.R. Cunnick, Trans. R. Soc. Trop. Med. Hyg. 72
(1978) 431e432.
of MTT reagent (5 mg/ml) in serum free medium was added to each
well. The plates were incubated at 37 ꢂC for 4 h. At the end of the
incubation period, the medium was removed and pure DMSO
(100 ml) was added to each well. The metabolized MTT product
dissolved in DMSO was quantified by reading the absorbance at
[34] F.D. Gillin, D.S. Reiner, M. Suffness, Antimicrob. Agents Chemother. 22 (1982)
342e345.
ꢁ
[35] F. Gümüs, Ö Algül, G. Eren, H. Eroglu, N. Diril, S. Gür, A.Ö zkul, Eur. J. Med.
570 nm. All assays were performed in triplicate. Percent viability
Chem. 38 (2003) 473e480.