Antiprotozoal activity of chloroquinoline based chalcones

Article abstract of DOI:10.1016/j.ejmech.2011.02.004

A new series of chloroquinoline based chalcones were synthesized and evaluated for in vitro antiamoebic and antimalarial activities. The results showed that out of fifteen compounds, four were found to be more active against the Entamoeba histolytica; whi

Full text of DOI:10.1016/j.ejmech.2011.02.004

European Journal of Medicinal Chemistry 46 (2011) 1897e1905
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Antiprotozoal activity of chloroquinoline based chalcones
,
Faisal Hayat a, Emma Moseley b, Attar Salahuddin a, Robyn L. Van Zyl b, Amir Azam a
*
a Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
b Pharmacology Division, Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of Witwatersrand, Johannesburg 2193, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of chloroquinoline based chalcones were synthesized and evaluated for in vitro antiamoebic
and antimalarial activities. The results showed that out of fifteen compounds, four were found to be more
active against the Entamoeba histolytica; while one compound was moderatively active compared to the
Received 7 September 2010
Received in revised form
28 January 2011
Accepted 3 February 2011
Available online 5 March 2011
standard drug metronidazole (IC50 ¼ 1.46
chloroquine-sensitive (3D7) strain of P. falciparum indicated relatively low activity when compared to
controls such as chloroquine and quinine (IC50 ¼ 0.0065 M and 0.14 M, respectively). The toxicological
studies of these compounds on human breast cancer MCF-7 cell line showed that all the compounds
were non-toxic at the concentration range of 1.56e50 M.
Ó 2011 Elsevier Masson SAS. All rights reserved.
mM). In contrast, in vitro antimalarial activity against the
m
m
Keywords:
m
2-Chloro-3-formylquinolines
Aromatic ketones
Chalcones
Entamoeba histolytica
Cytotoxicity
Plasmodium falciparum
b
-Haematin
Anti-oxidant
Haemolysis
1. Introduction
threatening diseases such as ulcerative colitis or liver abscess.
Metronidazole (1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole) is
the drug of choice for the treatment of amoebiasis and giardiasis [6]
and is reported to cause several toxic effects such as genotoxicity,
gastric mucus irritation and spermatozoid damage [7,8]. Further-
more, failures in the treatment of several intestinal protozoan
parasites may result from drug resistant by parasites [9,10]
A similar trend has been observed in the treatment of malaria,
a haematoprotozoan parasite of the Plasmodium species, carried by
certain types of the Anopheles mosquito [11]. It is one of the most
prevalent diseases of the developing world, affecting over
a hundred countries and resulting in an estimated 300e500 million
cases annually [12]. In sub-Saharan Africa alone over 90% of
reported malaria cases are attributed to the P. falciparum parasite
[13e15]. Epidemiological models have suggested that P. falciparum
infections outside of Africa, specifically in Southeast Asia, are
expected to be 200% higher than figures originally published by the
World Health Organization, with 76% of the total cases originating
from India [12]. The widespread resistance of the parasite to chlo-
roquine, the backbone of malaria chemotherapy, has highlighted
the fact that the investigation of new chemotherapeutics with
similar mechanisms of action and toxicity profile to that of chlo-
roquine is highly desired. The mechanism of action of chloroquine
lies in the fact that, once haemoglobin has been degraded by
Parasitic protozoa are the causative agents of numerous diseases
and affect an immense proportion of the world’s population [1].
Two such protozoa are Entamoeba histolytica and Plasmodium fal-
ciparum, the causative agents of amoebiasis and malaria, respec-
tively. Amoebiasis continues to be a major problem in developing
countries partly due to a lack of adequate sanitation and health
education [2]. Countries where E. histolytica remains endemic
include India, South Africa and Mexico to name a few [2]. Invasive
amoebiasis is the second most common cause of mortality due to
parasitic infections worldwide [3] killing one in 30 children in
Bangladesh alone [4]. It results primarily in an infection of the
colon, but may also be spread via the haematogenous path to other
organs, especially the liver [5] and is characterized by its high
capacity to destroy host tissues, leading to potentially life-
Abbreviations: mg, microgram; mL, microliter; mM, micromole; mL, milliliter; mg,
milligram; mmol, millimole; nm, nanometer; MTT, 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide; DMSO, dimethyl sulfoxide; DPPH, 2,2-diphenyl-
1-picrylhydrazyl; IC50, the drug concentration at which 50% growth inhibition
occurs.
* Corresponding author. Tel.: þ91 11 2698 1717/3254; fax: þ91 11 2698 0229.
E-mail address: amir_sumbul@yahoo.co.in (A. Azam).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.004

1898
F. Hayat et al. / European Journal of Medicinal Chemistry 46 (2011) 1897e1905
plasmodial cysteine proteases, the toxic haem intermediate cannot
be detoxified into haemozoin, resulting in oxidative stress and
death of the parasite [16]. A therapeutic agent capable of acting in
a similar manner as well as interacting favourably with other
established antimalarials would be invaluable.
were repeated, at least, in triplicate. To determine a possible
mechanism of antimalarial action the inhibition of -haematin
b
formation, as well as free radical scavenging activity were deter-
mined. Drug toxicity was determined by examining the haemolytic
effects of the compounds on healthy erythrocytes. Out of the 14
compounds, one lead compound was selected and combined with
quinine to determine possible drug interactions. The results of the
above are summarized in Table 2 and Fig. 2.
It is well known that chalcones and quinoline incorporating
heterocycles play an important role in medicinal chemistry and
possess various biological activities such as antimicrobial, anti-
inflammatory, analgesic, antimalarial, anticancer, antiviral, anti-
leishmanial and antitubercular [17e22]. According to the literature
iodoquinol is currently used as antiamoebic drug in medical prac-
tice [23] and quinoline based hydrazones have shown antimalarial
and antitubercular activity [24e26]. Many natural products bearing
a quinoline nucleus such as quinidine, quinidinone, and quinine
from the plant Cinchona ledgeriana were also found to be anti-
amoebic [27]. In recent years it is reported that the incorporation of
a quinoline nucleus could alter the course of reaction, as well as the
biological properties of the molecules [28,29].
Since chalcones and quinoline incorporating heterocycles are
biologically active and as a part of our continuous efforts towards
the development of more potent antiprotozoal agents, we herein
report the synthesis, characterization, antiamoebic, antiplasmodial
and cytotoxic properties of a new series of chloroquinoline based
chalcones (4e18) in an attempt to improve upon the efficacy of
current antiamoebic and antimalarial agents.
4. Results and discussion
4.1. Synthesis
The chloroquinoline based chalcones were prepared by one-step
ClaiseneSchmidt condensation [31] of substituted 2-chloro-3-for-
mylquinolines with different aromatic ketones using the reaction
sequence as shown in Scheme 1. For this purpose a series of 2-chloro-
3-formylquinolines (1e3) were prepared from different acetanilides
[32]. The required acetanilides were prepared by direct condensation
of substituted anilines withacetic acidinpresenceofaceticanhydride
[33]. The prepared 2-chloro-3-formylquinolines were condensed
withdifferentcommerciallyavailablearomaticketones inpresence of
aqueous sodium hydroxide (2 mL, 40%) in 50 mL ethanol to yield the
quinolinyl chalcones (4e18) in good yield. All the compounds were
characterized by IR, 1H and 13C NMR and mass spectra. The purity of
the compounds was confirmed by elemental analysis and the data
was found in accordance with ꢀ0.3%.
2. Chemistry
The synthesis of the chloroquinoline based chalcones (4e18)
was performed in a manner as outlined in Scheme 1. The reaction of
2-chloro-3-formylquinolines (1e3) with different commercially
available aromatic ketones in presence of aqueous sodium
hydroxide in ethanol gave the quinolinyl chalcones. All the
compounds are insoluble in water, but soluble in most of the
organic solvents. Melting points were recorded on KSW melting
point apparatus and are uncorrected.
4.2. Antiamoebic activity
Preliminary experiments were carried out to determine the in
vitro antiamoebic activity of all the compounds (4e18) by micro-
dilution method using the HM1:IMSS strain of E. histolytica. The
antiamoebic effect was compared with the most widely used
antiamoebic medication, namely metronidazole which had a 50%
inhibitory concentration (IC50) of 1.46 mM (Table 1). SAR showed
that compounds (4e10) which contained chloro and bromo groups
as substituent’s at C-3 and C-4 position of the phenyl ring; while
compounds (4, 5, 7 and 9) with a methyl group as a substituent at C-
3. Pharmacology
All quinolinyl chalcones (4e18) were screened in vitro against
HM1:IMSS strain of E. histolytica by microdilution method [30]. All
the experiments were carried out in triplicate at each concentration
level and repeated thrice. Cytotoxicity of active compounds has
been studied using the MTT cell viability assay on the breast cancer
MCF-7 cell line. The results of antiamoebic activity and cytotoxicity
are summarized in Table 1. In vitro antimalarial activity was carried
out on the chloroquine-sensitive (3D7) strain of P. falciparum by use
of the [3H]-hypoxanthine-incorporation assay. All experiments
6
and C-7 position of the chloroquinoline ring (excluding
compounds 6, 8 and 10) showed IC50 value in the range of
0.05e7.53 M. Compounds (11e15) besides containing the same
m
substitution of chloro and bromo groups at C-3 and C-4 position of
the phenyl and methyl group at C-6 and C-7 position of the chlor-
oquinoline ring except the compound (13), contain a methyl group
at the
range of 0.06e7.03
a
-b
unsaturated carbonyl position showed IC50 value in the
M. Compounds (16e18) having the same
m
chloroquinoline ring in their structure contain a pyrimidine ring
O
R
R
R
R
CH
H
3
(a)
(b)
NH
O
N
Cl
1
1
H
O
(1-3)
5
8
4
R
R
R
3
6
R
2
7
N
1
Cl
1
(4-18)
Scheme 1. Synthesis of chloroquinoline based chalcone (4e18). Reagent and conditions: (a) DMF/POCl3, 75 ꢁC reflux 17 h, (b) aq. NaOH, C2H5OH, different aromatic ketones, where
R, R1, R2 and R3 are the different substituted groups.

F. Hayat et al. / European Journal of Medicinal Chemistry 46 (2011) 1897e1905
1899
Table 1
In vitro antiamoebic activity of chloroquinoline based chalcones (4e18) against HM1:IMSS strain of E. histolytica and cytotoxicity profile of compounds 5, 10, 11, 15, 17 and
metronidazole.
H
O
4
5
8
R
R
R
3
6
R
2
7
N
1
Cl
1
Compound
R
R1
R2
R3
Antiamoebic activity
Cytotoxicity profile
(MCF-7)
IC50
(m
M) SDa
IC50
(m
M) SDa
Br
Br
Br
4
5
6
H
CH3
H
H
H
7.53
0.14
0.22
0.16
N.D.
N.D.
0.18
N.D.
CH3
H
1.03
>100
H
H
3.45
N.D.
7
CH3
H
H
H
H
H
4.09
3.38
6.91
0.05
0.12
0.23
0.19
0.12
N.D.
N.D.
N.D.
>100
N.D.
N.D.
N.D.
0.14
Br
Br
Cl
8
H
H
9
H
CH3
10
H
H
Cl
Br
11
12
13
14
H
CH3
CH3
CH3
CH3
CH3
0.09
2.79
3.65
7.03
0.22
0.11
0.32
0.11
>100
N.D.
N.D.
N.D.
0.14
N.D.
N.D.
N.D.
Br
Br
Cl
CH3
H
H
H
H
CH3
(continued on next page)

1900
F. Hayat et al. / European Journal of Medicinal Chemistry 46 (2011) 1897e1905
Table 1 (continued ).
Compound
R
R1
R2
R3
Antiamoebic activity
Cytotoxicity profile
(MCF-7)
IC50
(m
M) SDa
IC50 (m
M) SDa
Cl
15
16
17
CH3
CH3
H
H
CH3
0.06
0.14
0.17
0.10
>100
N.D.
0.28
N.D.
0.16
N
N
N
H
H
1.45
CH3
H
0.05
75.7
18
H
H
H
5.30
0.22
0.17
N.D.
N.D.
0.11
Metronidazole
1.46
>100
a
Standard deviation, N.D. ¼ Not determined.
The compounds with bold IC50 values are more active than metronidazole.
instead of the phenyl ring. The compounds (16) and (17) except (18)
having the chloroquinoline ring being substituted by methyl group
at C-6 and C-7 position showed IC50 value in the range of
formation of b-heamatin and scavenge free radicals. The results
have been summarized in Table 2. The hypoxanthine incorporation
assay indicated that, although not comparable to the reference
0.05e5.30
were found to be more active against .E. histolytica than the refer-
ence drug, metronidazole (IC50 ¼ 1.46 M). Out of these five
compounds, four compounds 10 (IC50 0.05 M), 11
(IC50 ¼ 0.09 M), 15 (IC50 ¼ 0.06 M) and 17 (IC50 ¼ 0.05 M)
showed excellent antiamoebic activity being 16e29 times more
m
M. From these 15 compounds, five (5, 10, 11, 15 and 17)
agents chloroquine and quinine (IC50 values ¼ 0.0065
0.14 M, respectively), compounds 10, 16, 17 and 18 (IC50
values ¼ 39.17 M, 31.54 M, 31.31 M and 31.98 M, respectively)
mM and
m
m
m
m
m
m
¼
m
showed the most promising antimalarial activity. Compounds
16e18 were the only compounds to have a pyridine ring substitu-
tions at R3 and a hydrogen group at R2, with either hydrogen or
methyl groups at R and R1. Compound 10, which had the highest
IC50 value out of the 4, has hydrogen groups at R, R1 and R2 with a 4-
chloro phenyl ring at R3. It appears as if the substitution of the
hydrogen group, instead of a methyl group, at R2 positively influ-
ences the activity of compounds 4 and 5 when compared to
compounds 11e13, all of which have a bromophenyl ring. However,
when all methyl groups are replaced with hydrogen groups, as seen
in compound 6, activity diminishes. The positioning of the bromo
m
m
m
active than metronidazole. While compound 5 (IC50 ¼ 1.03
mM) was
moderately active compare to metronidazole.
4.3. Antimalarial activity
A series of chloroquinoline based chalcones (4e18) were
examined for their in vitro antimalarial activity, their interactions
with classic antimalarials, as well as their ability to inhibit the
Table 2
The in vitro antimalarial and haemolytic activity of chloroquinoline based chalcones (4e18) and reference agents along with their effect on
b
-haematin formation.
Compound
Antimalarial activity
IC50 ꢀ S.D. ( M)
Haemolytic activity
Inhibition Of b-haematin formation
m
Maximum inhibition at 100
m
M (%)
% Lysis at 100
mM
IC50 ꢀ S.D. (
m
M)
Maximum inhibition at 100 mM (%)
4
5
6
7
8
9
59.73 ꢀ 1.19
50.11 ꢀ 3.85
>100
74.84 ꢀ 3.39
44.56 ꢀ 1.75
N.D.
1.09 ꢀ 0.55
1.68 ꢀ 0.66
1.38 ꢀ 0.67
1.40 ꢀ 0.96
2.20 ꢀ 0.54
N.D.
>100
>100
>100
>100
>100
N.D.
27.50 ꢀ 4.80
14.88 ꢀ 6.86
16.03 ꢀ 4.81
55.38 ꢀ 19.1
13.55 ꢀ 2.61
N.D.
61.22 ꢀ 13.09
N.D.
10
11
12
13
14
15
16
17
39.17 ꢀ 2.05
47.06 ꢀ 2.76
>100
1.74 ꢀ 0.67
1.04 ꢀ 0.64
1.53 ꢀ 0.34
2.17 ꢀ 0.59
1.44 ꢀ 0.66
1.24 ꢀ 0.78
1.55 ꢀ 1.15
0.47 ꢀ 0.77
1.10 ꢀ 1.04
0.72 ꢀ 0.32
1.22 ꢀ 0.98
>100
>100
>100
>100
>100
>100
>100
>100
19.49 ꢀ 5.35
10.54 ꢀ 0.88
13.30 ꢀ 5.38
9.35 ꢀ 5.76
5.64 ꢀ 3.72
14.42 ꢀ 4.21
11.52 ꢀ 5.20
15.00 ꢀ 0.99
12.29 ꢀ 4.35
51.15 ꢀ 8.1
34.3 ꢀ 8.93
>100
50.44 ꢀ 3.22
>100
8.74 ꢀ 13.66
31.54 ꢀ 3.03
31.31 ꢀ 0.87
31.98 ꢀ 1.27
18
>100
Chloroquine
Quinine
0.0065 ꢀ 0.0002
0.14 ꢀ 0.007
24.97 ꢀ 5.3
65.80 ꢀ 1.77

F. Hayat et al. / European Journal of Medicinal Chemistry 46 (2011) 1897e1905
1901
120
100
80
60
40
20
0
Control
MNZ
5
10
11
15
17
0
1.56
3.14
6.25
12.5
25
50
100
Concentration (µM)
Fig. 1. Percentage of viable cells after 48 h pre-treatment of human breast cancer MCF-7 cells with compounds 5, 10, 11, 15, 17 and metronidazole (MNZ), evaluated by the MTT assay.
group on the phenyl ring appeared not to influence antimalarial
activity. A study conducted against the FCB1 chloroquine-resistant
strain of P. falciparum indicated that quinolinyl chalcones with
a single chloro substitution at position 4 on the phenyl ring dis-
and 2.65%, respectively). The mechanism of action of this class of
compounds remains unclear, when tested against the P. falciparum
cysteine protease, falcipain, the quinolinyl chalcones showed weak
activity that was not correlated with antimalarial activity [34]. In an
examination of the interaction of compound 17 and quinine, an
additive interaction was noted. This is possibly due, to the different
complementary mechanisms of actions of these two compounds.
played no antimalarial activity (>200
substituted at positions 2, 4 and 2, 5, the IC50 values decreased to
19 M and 48.6 M, respectively, whilst di-substitutions at posi-
mM). However, once di-
m
m
tions 3 and 4 diminished the antimalarial activity [34]. The same
study also indicated that the addition of bromo, methoxy or
hydrogen groups to the phenyl ring diminished antimalarial
activity. The above results indicate differences in drug sensitivity
between chloroquine-sensitive and -resistant strains, but also
highlight the significant role that not only the various substitutions,
but also the positioning of the substitutions, play. None of the
4.4. Cytotoxicity profile
Since compounds 5, 10, 11, 15 and 17 were more potent than
metronidazole, they were screened for cytotoxicity against the
human breast cancer MCF-7 cell line. Compounds 5, 10, 11 and
metronidazole inhibited <20% cell growth. Whilst compound 17
compounds were capable of inhibiting the formation of
tin, the synthetic molecule with the same chemical structure as
haemozoin, as effectively as chloroquine or quinine (IC50 ¼ 24.9
and 65.8 M, respectively). Compound 7 exhibited the greatest
activity with 55.38% inhibition at 100 M, however this was not
b-haema-
inhibited cell growth in a dose dependant manner, where 50
mM
inhibited 36% and 100 M, inhibited 66% cell growth (Fig. 1). To
m
mM
ensure the antimalarial activity noted for these compounds were
due to a direct inhibitory effect on the intra-erythrocytic parasite,
all the compounds were screened against healthy red blood cells for
any haemolytic effects (Table 2). None of the compounds showed
any notable haemolytic effects when compared to antimalarials
such as chloroquine or quinine (% haemolysis of 0.72 and 1.22%,
respectively). The greatest haemolytic activity was noted for
compounds 8 and 13 (% haemolysis of 2.2 and 2.17%, respectively).
m
m
well correlated with antimalarial activity. None of the compounds
were capable of scavenging the free electron of DPPHꢂ as effectively
as ascorbic acid (IC50 ¼ 20.10
m
M), compounds 1, 2, 10, 11, 13 and 14
showed some anti-oxidant activity at 80
mM, or a ratio of drug to
DPPHꢂ of 1:1 (% scavenging activity ¼ 7.79, 2.06,3.69, 2.27, 1.43, and
0.78%, respectively) and were comparable to the anti-oxidant
activity of chloroquine and quinine (% scavenging activity ¼ 4.06
5. Conclusion
The 15 chloroquinoline based chalcones (4e18) were synthe-
sized by condensation of substituted 2-chloro-3-formylquinolines
with different aromatic ketones. In vitro antiamoebic and antima-
larial activities were determined against the HM1:IMSS strain of
E. histolytica and 3D7 strain of P. falciparum, respectively. Anti-
amoebic activity indicated that out of the 15 compounds, 5
compounds exhibited more potent activity than the reference drug
1.5
1.0
0.5
0.0
metronidazole (IC50 ¼ 1.46
mM). Antimalarial activity was not as
promising, none of the chloroquinoline based chalcones were
capable of inhibiting parasite growth as effectively as the reference
drugs chloroquine and quinine (IC50 values ¼ 0.0065
0.14 M, respectively). When combined with quinine, compound 17
interacted in a favourable additive manner. Both the inhibition of
-haematin formation and DPPHꢂ free radical scavenging assays
mM and
m
b
indicated that the antimalarial mechanisms of action of the
compounds do not lie in their ability to inhibit haemozoin forma-
tion or act as anti-oxidants. The MTT assay revealed that
compounds 5, 10, 11, 15 and metronidazole were non-toxic,
however compound 17 was found to inhibit 66% cell growth at
0.0
0.5
Quinine
(Relative ratio's)
1.0
1.5
Fig. 2. Isobologram depicting the additive interaction between compound 17 and
quinine.
100 mM. Whilst, the red blood cell toxicity assay showed negligible

1902
F. Hayat et al. / European Journal of Medicinal Chemistry 46 (2011) 1897e1905
amounts of haemolysis when compared to that of chloroquine and
quinine. The results obtained indicate that these compounds show
excellent antiamoebic activity, with 16e29 times the activity of the
standard treatment, metronidazole. No correlation was noted
between the antiamoebic and antimalarial activity with the above
compounds exhibiting only moderate antimalarial activity when
compared to standard treatments. Generally the compounds
proved to be non-toxic. Investigation into derivatives of this class of
compounds is essential, to examine the exact nature of their anti-
malarial activity, as well as the effect of structural changes on
antiamoebal and antiplasmodial activities.
d (ppm): 8.39 (s, 1H, H4 quinoline), 8.22 (s, 1H, phenyl), 8.16 (d, 1H,
J ¼ 15.6 Hz, Hb), 7.98 (d, 1H, J ¼ 7.5 Hz, phenyl), 7.69 (s, 1H, H5
quinoline) 7.66e7.60 [m, 2H, (1H phenyl, H8 quinoline)], 7.57 (d, 1H,
J ¼ 15.6 Hz, Ha), 7.49e7.39 [m, 2H, (1H phenyl, H7 quinoline)], 2.55
(s, 3H, CH3 quinoline); 13C NMR (CDCl3)
d (ppm): 188.8 (C]O),
149.4 (CeCl), 146.5 (C-b), 140.1, 137.9, 136.3, 135.6, 134.0, 132.0,
130.01, 128.1, 127.6, 126.8 (AreC), 122.5 (C-
a), 21.6 (CH3 quinoline);
FAB-MS (m/z): [Mþþ1] 387.25.
6.2.3. (E)-1-(3-Bromophenyl)-3-(2-chloroquinoline-3yl) prop-2-
en-1-one (6)
Yield 78% (Ethyl acetate); mp: 173 ꢁC; Anal. calc. for
C18H11NOClBr: C 59.95, H 3.77, N 3.50%; found: C 59.89, H 3.71, N
3.44%. IR nmax (cmꢃ1): 1659 (C]O), 1583 (C]C); 1H NMR (CDCl3)
6. Experimental protocol
All the required chemicals were purchased from Merck and
Aldrich Chemical Company (USA). 2-Chloro-3-formylquinolines
were synthesized by using the methods reported in literature [32].
Precoated aluminium sheets (silica gel 60 F254, Merck Germany)
were used for thin-layer chromatography (TLC) and spots were
visualized under UV light. Elemental analysis was carried out on
CHNS Elementar (Vario EL-III) and the results were within ꢀ0.3% of
the theoretical values. IR spectra were recorded on PerkineElmer
model 1600 FT-IR RX1 spectrophotometer as KBr discs. 1H NMR and
13C NMR spectrawere 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; 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.
d (ppm): 8.41 (s, 1H, H4 quinoline), 8.24 (s, 1H, phenyl), 8.21 (d, 1H,
J ¼ 15.6 Hz, Hb), 7.97 (d, 1H, J ¼ 7.5 Hz, phenyl), 7.94e7.78 (m, 3H, H5
H7 H8 quinoline), 7.54 (d, 1H, J ¼ 15.6 Hz, Ha), 7.46e7.35 [m, 3H, (2H
phenyl, H6 quinoline)]; 13C NMR (CDCl3)
d (ppm): 188.9 (C]O),
148.9 (CeCl), 145.6 (C-b), 139.0, 137.7, 134.6, 133.4, 129.6, 127.6
(AreC), 126.6 (C-
a
); FAB-MS (m/z): [Mþþ1] 373.11.
6.2.4. (E)-1-(4-Bromophenyl)-3-(2-chloro-6-methylquinoline-3yl)
prop-2-en-1-one (7)
Yield 84% (Ethyl acetate); mp: 276 ꢁC; Anal. calc. for
C19H13NOClBr: C 59.02, H 3.39, N 3.62%; found: C 59.03, H 3.33, N
3.56%. IR nmax (cmꢃ1): 1651 (C]O), 1578 (C]C); 1H NMR (CDCl3)
d
(ppm): 8.41 (s, 1H, H4 quinoline), 8.22 (d, 1H, J ¼ 15.6 Hz, Hb),
8.05e7.91 [m, 3H, (2H phenyl, H8 quinoline)], 7.64 (s,1H, H5 quin-
oline), 7.63e7.52 [m, 3H, (1H phenyl, Ha, H7 quinoline)], 2.56 (s, 3H,
CH3 quinoline); 13C NMR (CDCl3)
d (ppm): 189.4 (C]O), 149.4
(CeCl), 146.4 (C-b),139.5, 137.5, 135.5, 133.1, 128.6, 127.6 (AreC),
122.7 (C-
a
), 22.5 (CH3 quinoline); FAB-MS (m/z): [Mþþ1] 387.39.
6.1. Preparation of 2-chloro-3-formylquinolines (1e3)
6.2.5. (E)-1-(4-Bromophenyl)-3-(2-chloroquinoline-3yl)prop-2-en-
1-one (8)
Substituted 2-chloro-3-formylquinolines were prepared by
a reported method [32].
Yield 88% (Ethyl acetate); mp: 223 ꢁC; Anal. calc. for
C18H11NOClBr: C 58.02, H 2.98, N 3.76%; found: C 58.05, H 2.94, N
3.70%. IR nmax (cmꢃ1): 1651 (C]O), 1586 (C]C); 1H NMR (CDCl3)
6.2. General procedure for the synthesis of chloroquinoline based
chalcones (4e18)
d
(ppm): 8.50 (s, 1H, H4 quinoline), 8.24 (d,1H, J ¼ 15.6 Hz, Hb), 8.05
(d,1H, J ¼ 8.4 Hz, H5 quinoline), 7.95e7.77 [m, 4H, (2H phenyl, H6 H8
quinoline)], 7.69e7.60 [m, 3H, (2H phenyl, H7 quinoline)], 7.55 (d,
A
mixture of substituted 2-chloro-3-formylquinolines
(0.01 mol), the respective aromatic ketones (0.01 mol), and sodium
hydroxide (2 mL, 40% aqueous) in 50 mL ethanol was stirred at
room temperature for 24 h. The resulting precipitate was collected
by filtration, washed with water and recrystallized from ethyl
acetate.
1H, J ¼ 15.6 Hz, H
a d (ppm): 188.86 (C]O), 150.3
); 13C NMR (CDCl3)
(CeCl), 147.9 (C-
b
), 139.9, 136.2, 131.7, 130.1, 129.4, 128.4 (AreC),
125.7 (C-
a
). FAB-MS (m/z): [Mþþ1] 373.44.
6.2.6. (E)-3-(2-Chloro-7-methylquinolin-3-yl)-1-(4-chlorophenyl)
prop-2-en-1-one (9)
6.2.1. (E)-1-(3-Bromophenyl)-3-(2-chloro-7-methylquinoline-3yl)
prop-2-en-1-one (4)
Yield 78% (Ethyl acetate); mp: 158 ꢁC; Anal. calc. for
C19H13NOCl2: C 66.68, H 3.83, N 4.09%; found: C 66.63, H 3.77, N
4.11%. IR nmax (cmꢃ1): 1650 (C]O), 1563 (C]C); 1H NMR (CDCl3)
Yield 88% (Ethyl acetate); mp: 276 ꢁC; Anal. calc. for
C19H13NOClBr: C 59.02, H 3.39, N 3.62%; found: C 59.04, H 3.33, N
3.58%; IR nmax (cmꢃ1): 1652 (C]O), 1579 (C]C); 1H NMR (CDCl3)
d
(ppm): 8.47 (s, 1H, H4 quinoline), 8.19 (d, 1H, J ¼ 15.7 Hz, Hb), 8.06
(d, 1H, J ¼ 7.6 Hz, phenyl), 7.89 (d, 1H, J ¼ 7.5 Hz, phenyl), 7.87e7.76
(m, 2H, H5 H8 quinoline), 7.56 (d, 1H, J ¼ 15.6 Hz, Ha),7.48e7.33 [m,
3H, (2H phenyl, H6 quinoline), 2.55 (s, 3H, CH3 quinoline); 13C NMR
d
(ppm): 8.46 (s,1H, H4 quinoline), 8.24 (s,1H, phenyl), 8.19 (d,1H,
J ¼ 15.6 Hz, Hb), 7.99 (d,1H, J ¼ 7.5 Hz, phenyl), 7.80e7.73 (m, 2H, H5
H8 quinoline), 7.56 (d,1H, J ¼ 15.6 Hz, Ha), 7.46e7.39 [m, 3H, (2H
phenyl, H6 quinoline)], 2.58 (s, 3H, CH3 quinoline); 13C NMR (CDCl3)
(CDCl3) d (ppm): 189.9 (C]O),149.9 (CeCl),147.1 (C-b), 139.2, 138.4,
136.8, 134.6, 132.2, 131.2, 129.7, 128.0, 127.6 (AreC), 124.6 (C-
a),
d
(ppm): 188.3 (C]O), 150.3 (CeCl), 148.2 (C-b), 142.8, 140.4, 139.4,
21.25 (CH3 quinoline); FAB-MS (m/z): [Mþþ1] 343.03.
135.9, 131.6, 130.4, 127.5, 127.1, 126.7 (AreC), 123.0 (C-
a), 22.1 (CH3
quinoline). FAB-MS (m/z): [Mþþ1] 387.34.
6.2.7. (E)-1-(4-Chlorophenyl)-3-(2-chloroquinoline-3yl)prop-2-en-
1-one (10)
6.2.2. (E)-1-(3-Bromophenyl)-3-(2-chloro-6-methylquinolin-3-yl)
prop-2-en-1-one (5)
Yield 86% (Ethyl acetate); mp: 167 ꢁC; Anal. calc. for
C18H11NOCl2: C 65.87, H 3.38, N 4.27%; found: C 65.82, H 3.31, N
4.22%. IR nmax (cmꢃ1): 1662 (C]O),1580 (C]C); 1H NMR (CDCl3)
Yield 74% (Ethyl acetate); mp: 142e144 ꢁC; Anal. calc. for
C19H13NOClBr: C 59.02, H 3.39, N 3.62%; found: C 59.03, H 3.32, N
3.56%. IR nmax (cmꢃ1): 1662 (C]O), 1581 (C]C); 1H NMR (CDCl3)
d
(ppm): 8.50 (s, 1H, H4 quinoline), 8.24 (d, 1H, J ¼ 15.6 Hz, Hb), 8.05
(d,1H, J ¼ 8.2 Hz, H5 quinoline), 8.01e7.77 [m, 4H, (2H phenyl, H6 H8

F. Hayat et al. / European Journal of Medicinal Chemistry 46 (2011) 1897e1905
1903
quinoline), 7.65e7.60 [m, 3H, (2H phenyl, H7 quinoline)], 7.56 (s,1H,
phenyl), 7.82 (d, 1H, J ¼ 7.5 Hz, H8 quinoline), 7.74 (s, 1H, CHb]
CMe), 7.63 (s, 1H, H5 quinoline) 7.48e7.32 [m, 3H, (2H phenyl, H7
J ¼ 15.6 Hz, Ha); 13C NMR (CDCl3)
d (ppm): 188.6 (C]O), 150.6
(CeCl), 147.7 (C-
126.3 (AreC), 125.2 (C-
b
), 139.4, 136.2, 135.2, 131.2, 130.0, 128.3, 127.5,
quinoline)], 2.56 (s, 3H, CH3 quinoline), 2.20 (s, 3H, CHb]CMe); 13
C
a
); FAB-MS (m/z): [Mþþ1] 329.24.
NMR (CDCl3)
d
(ppm): 196.3 (C]O), 148.7 (CeCl), 145.9 (C-
b
), 138.5,
) 21.3,
137.4, 136.3, 134.1, 133.4, 132.2, 129.1, 127.6 (AreC), 124.3 (C-
a
6.2.8. (E)-1-(3-Bromophenyl)- 3-(2-chloro-7-methylquinoline-
3yl)-2-methylprop-2-en-1-one (11)
(CH3 quinoline), 14.5 [CH3 (CHb]CMe)]; FAB-MS (m/z): [Mþþ1]
357.41.
Yield 82% (Ethyl acetate); mp: 275 ꢁC; Anal. calc. for
C20H15NOClBr: C 59.95, H 3.77, N 3.50%; found: C 59.89, H 3.73, N
3.46%; IR nmax (cmꢃ1): 1657 (C]O), 1580 (C]C); 1H NMR (CDCl3)
6.2.13. (E)-3-(2-Chloro-6-methylquinoline-3yl)-1-(pyridine-2yl)
prop-2-en-1-one (16)
d
(ppm): 8.11 (s, 1H, H4 quinoline), 8.01 (s, 1H, phenyl), 7.79e7.74
Yield 78% (Ethyl acetate); mp: 188 ꢁC; Anal. calc. for
C18H13N2OCl: C 70.02, H 4.24, N 9.07%; found: C 70.03, H 4.19, N
9.02%. IR nmax (cmꢃ1): 1651 (C]O), 1560 (C]C); 1H NMR (CDCl3)
[m, 3H, (1H phenyl, H5 H8 quinoline)], 7.72 (s, 1H, Hb CHb]CMe),
,
7.44e7.36 [m, 3H, (2H phenyl, H6 quinoline)], 2.57 (s, 3H, CH3
quinoline), 2.20 (s, 3H, CHb]CMe); 13C NMR (CDCl3)
d
(ppm): 196.8
d
(ppm): 8.78 (d, 1H, J ¼ 4.2 Hz, pyridine), 8.58 (s,1H, H4 quinoline),
(C]O), 149.2 (CeCl), 147.2 (C-
b), 141.9, 139.2, 138.7, 138.0, 137.1,
8.46 (d,1H, J ¼ 15.9 Hz, Hb), 8.39 (d,1H, J ¼ 2.4 Hz, pyridine), 8.26 (d,
1H, J ¼ 7.8 Hz, H8 quinoline), 7.95e7.90 [m, 2H, (1H pyridine, H5
quinoline)] 7.66e7.59 [m, 2H, (1H pyridine, H7 quinoline)], 7.56 (d,
1H, J ¼ 15.6 Hz, Ha), 2.56 (s, 3H, CH3 quinoline); 13C NMR (CDCl3)
129.8, 129.7, 128.0, 127.3, 126.9 (AreC), 124.5 (C-a), 21.9 (CH3
quinoline), 14.1 [CH3 (CHb]CMe)]; FAB-MS (m/z): [Mþþ1] 401.22.
6.2.9. (E)-1-(3-Bromophenyl)-3-(2-chloro-6-methylquinolin-3-yl)-
2-methylprop-2-en-1-one (12)
d
(ppm): 188.5 (C]O), 149.7 (CeCl), 148.8 (C-
b), 146.5, 139.1, 137.6,
135.6, 133.8, 128.0, 127.1, 126.8 (AreC), 124.5 (C-
a
), 21.5 (CH3
Yield 84% (Ethyl acetate); mp: 181e183 ꢁC; Anal. calc. for
C20H15NOClBr: C 59.95, H 3.77, N 3.50%; found: C 59.87, H 3.71, N
3.44%; IR nmax (cmꢃ1): 1645 (C]O), 1577 (C]C); 1H NMR (CDCl3)
quinoline). FAB-MS (m/z): [Mþþ1] 309.37.
6.2.14. (E)-3-(2-Chloro-7-methylquinoline-3yl)-1-(pyridine-2yl)
prop-2-en-1-one (17)
d
(ppm): 8.17 (s, 1H, H4 quinoline), 8.02 (s, 1H, phenyl), 7.94 (d, 1H,
J ¼ 8.3 Hz, phenyl), 7.81 (d, 1H, J ¼ 7.5 Hz, H8 quinoline), 7.73 (s, 1H,
Hb CHb]CMe),7.63 (s, 1H, H5 quinoline), 7.42e7.37 (m, 2H, phenyl),
7.33 (d,1H, J ¼ 7.8 Hz, H7 quinoline), 2.55 (s, 3H, CH3 quinoline), 2.19
Yield 88% (Ethyl acetate); mp: 235 ꢁC; Anal. calc. for
C18H13N2OCl: C 70.02, H 4.24, N 9.07%; found: C 70.05, H 4.18, N
9.03%; IR nmax (cmꢃ1): 1658 (C]O), 1578 (C]C); 1H NMR (CDCl3)
(s, 3H, CH3 CHb]CMe); 13C NMR (CDCl3)
d
(ppm): 196.3 (C]O),
d
(ppm): 8.76 (d, 1H, J ¼ 4.2 Hz, pyridine), 8.63 (s,1H, H4 quinoline),
148.0 (CeCl),147.2 (C-
b), 141.3,138.7,137.6,135.5, 133.6,132.3, 129.1,
8.40 (d,1H, J ¼ 15.9 Hz, Hb), 8.38 (d,1H, J ¼ 2.3 Hz, pyridine), 8.26 (d,
1H, J ¼ 7.5 Hz, H5 quinoline), 7.95e7.81 [m, 2H (1H pyridine, H8
quinoline)], 7.67e7.57 [m, 2H ( 1H pyridine, H6 quinoline)], 7.54 (d,
1H, J ¼ 15.9 Hz, Ha), 2.56 (s, 3H, CH3 quinoline); 13C NMR (CDCl3)
128.1, 127.6 (AreC), 126.4 (C-a), 21.3 (CH3 quinoline), 14.5 [CH3
(CHb ¼ CMe)]; FAB-MS (m/z): [Mþþ1] 401.32.
6.2.10. (E)-1-(3-Bromophenyl)-3-(2-chloroquinoline-3yl)-2-
methylprop-2-en-1-one (13)
d
(ppm): 188.4 (C]O), 150.3 (CeCl), 148.4 (C-
b
), 142.7, 140.4, 139.9,
), 22.4 (CH3
137.6, 129.1, 127.2, 127.9, 125.3 (AreC), 124.2 (C-
a
Yield 89% (Ethyl acetate); mp: 217 ꢁC; Anal. calc. for
C19H13NOClBr: C 59.02, H 3.39, N 3.62%; found: C 59.03, H 3.32, N
3.58%. IR nmax (cmꢃ1): 1644 (C]O), 1579 (C]C); 1H NMR (CDCl3)
quinoline); FAB-MS (m/z): [Mþþ1] 309.41.
6.2.15. (E)-3-(2-Chloroquinoline-3yl)-1 (pyridine-2yl)prop-2-en-1-
one (18)
d
(ppm): 8.17 (s, 1H, H4 quinoline), 8.05 (s, 1H, phenyl), 7.89 (d, 1H,
J ¼ 8.4 Hz, phenyl), 7.82 (d, 1H, J ¼ 7.8 Hz, H8 quinoline), 7.79e7.73
(m,2H, H5 H6 quinoline), 7.71(s, 1H, CHb ¼ CMe), 7.64e7.59 (1H, t,
J ¼ 7.2 Hz, H7 quinoline), 7.42e7.33 (m, 2H, phenyl), 2.21 (s, 3H,
Yield 89% (Ethyl acetate); mp: 248 ꢁC; Anal. calc. for
C17H11N2OCl: C 69.28, H 3.76, N 9.50%; found: C 69.24, H 3.72, N
9.47%. IR nmax (cmꢃ1): 1653 (C]O), 1566 (C]C); 1H NMR (CDCl3)
CHb]CMe); 13C NMR (CDCl3)
d
(ppm): 196.5 (C]O), 149.5
d
(ppm): 8.78 (d, 1H, J ¼ 4.2 Hz, pyridine), 8.67 (s, 1H, H4 quinoline),
(CeCl), 147.3 (C-
b
), 139.2, 138.5, 136.1, 135.4, 132.4, 128.6, 127.5
8.42 (d,1H, J ¼ 15.9 Hz, Hb), 8.35 (d,1H, J ¼ 2.4 Hz, pyridine), 8.26 (d,
1H, J ¼ 7.5 Hz, H5 quinoline), 8.05e7.76 [m, 3H, (1H pyridine, H6 H8
quinoline)], 7.63e7.58 [m, 2H, (1H pyridine, H7 quinoline)], 7.55 (d,
(AreC), 124.2 (C-
a
), 14.3[CH3 (CHb]CMe)]. FAB-MS (m/z): [Mþþ1]
387.16.
1H, J ¼ 15.9 Hz, Ha), 13C NMR (CDCl3)
d (ppm): 188.6 (C]O), 150.9
6.2.11. (E)-3-(2-Chloro-7-methylquinoline-3yl)-1-(3-chlorophenyl)-
2-methylprop-2-en-1-one (14)
(CeCl), 148.1 (C-b), 139.9, 137.3, 128.2, 127.8, 125.3 (AreC), 124.4
(C-
a
); FAB-MS (m/z): [Mþþ1] 295.13.
Yield 84% (Ethyl acetate); mp: 236 ꢁC; Anal. calc. for
C20H15NOCl2: C 67.43, H 4.24, N 3.93%; found: C 67.38, H 4.18, N
3.86%. IR nmax (cmꢃ1): 1655 (C]O), 1576 (C]C); 1H NMR (CDCl3)
6.3. In vitro antiamoebic assay
d
(ppm): 8.12 (s, 1H, H4 quinoline), 7.86 (s, 1H, phenyl), 7.81 (s, 1H,
All the compounds (4e18) were screened in vitro for anti-
amoebic activity against HM1:IMSS strain of E. histolytica by
microdilution method [30]. E. histolytica trophozoites were
cultured in a 96-well microtiter plate suspended in Diamond TYIS-
33 growth medium [35]. The test compounds (1 mg) were dis-
H8 quinoline), 7.77e7.74 [m, 2H, (1H phenyl, H5 quinoline)], 7.73 (s,
1H, CHb]CMe), 7.47e7.33 [m, 3H, (2H phenyl, H6 quinoline)] 2.55
(s, 3H, CH3 quinoline), 2.20 (s, 3H, CHb]CMe); 13C NMR (CDCl3)
d
(ppm): 197.0 (C]O), 149.8 (CeCl), 145.0 (C-
b
), 139.1, 137.1, 134.6,
132.1, 129.8, 127.6 (AreC), 124.5 (C-
a
), 22.0 (CH3 quinoline), 14.2
solved in DMSO (40 mL, concentration at which no inhibition of
[CH3 (CHb]CMe)]; FAB-MS (m/z): [Mþþ1] 357.08.
amoeba growth occurred) [27,36]. The stock solutions (1 mg/mL) of
the compounds were freshly prepared and twofold serial dilutions
were made in the wells of a 96-well microtiter plate. The following
controls were included in each plate: 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 and repeated thrice.
The amoeba suspension was prepared from a confluent culture by
6.2.12. (E)-3-(2-Chloro-6-methylquinolin-3-yl)-1-(3-chlorophenyl)-
2-methylprop-2-en-1-one (15)
Yield 88% (Ethyl acetate); mp: 178 ꢁC Anal. calc. for C20H15NOCl2:
C 67.43, H 4.24, N 3.93%; found: C 67.39 H 4.19, N 3.88%. IR nmax
(cmꢃ1): 1651 (C]O), 1578 (C]C); 1H NMR (CDCl3)
d
(ppm): 8.16 (s,
1H, H4 quinoline), 8.07 (s, 1H, phenyl), 7.94 (d, 1H, J ¼ 8.4 Hz,

1904
F. Hayat et al. / European Journal of Medicinal Chemistry 46 (2011) 1897e1905
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 the flask. The number of amoeba/mL was estimated with
a haemocytometer, using the Trypan blue exclusion assay to
confirm viability. The suspension was diluted to 105 organism per
to construct an isobologram from which the type of interaction
could be determined [40,41]. Each experiment was repeated, at
least, in triplicate.
6.4. Inhibition of b-haemozoin formation assay
mL in fresh medium and 170 mL of this suspension was added to the
test and control wells in the plate such that an inoculum of 1.7 ꢄ 104
organisms/well was achieved to ensure confluency but no excessive
growth in control wells. Plates were sealed and gassed for 10 min
with nitrogen before incubation at 37 ꢁC for 72 h. After incubation,
the growth of amoeba in the plate was checked with a low power
microscope. The culture medium was removed by inverting the
plate and shaking gently. The plate was then immediately washed
with prewarmed (37 ꢁC) 0.9% (w/v) sodium chloride solution. This
procedure was completed as quickly as possible to ensure the plate
did not cool, in order to prevent the detachment of amoebae. The
plate was allowed to dry at room temperature and the amoebae
were fixed with chilled (ꢃ20 ꢁC) 100% methanol and when dried,
stained with 0.5% aqueous eosin for 15 min. The stained plate was
washed 3 times with distilled water and allowed to dry before
To determine whether the compounds had a similar mechanism
of action to that of chloroquine, the following were combined in
a 96-well microtiter plate: 12.5
a 1 mg/mL haemin (Sigma) solubilised in DMSO, 25
finally 50 l of a 0.5M acetate buffer. The acetate buffer was utilized
to simulate the acidic conditions (pH 4.7) of the parasitic food
vacuole. The plates were then incubated for 24 h and 100 L of the
mL of the test compound, 12.5
mL of
mL H2O and
m
m
solution removed and the same volume substituted with DMSO,
the plates were then centrifuged at 1500 g for 10 min. This was
repeated 3 times to remove any unreacted haemin. Following this,
100
dissolve the
and the absorbance read at 405 nm [42]. From the data obtained the
mL was removed and substituted with a 2M NaOH solution to
b
-haematin crystals. The solution was diluted twofold
concentration at which b-haematin formation was inhibited by 50%
(IC50 value) was determined by use of EnzfitterÒ software. Classic
antimalarials, such as quinine and chloroquine, were used as
positive control. Each experiment was repeated, at least, in
triplicate.
200 mL 0.1 N sodium hydroxide was added to each well to dissolve
the protein and release the dye. The optical density of the resulting
solution was determined at 490 nm with a microplate reader. The %
inhibition of amoebal growth was calculated taking into account
the controls and then plotted against the logarithm of the
compound concentration. Linear regression analysis was used to
determine the best fitting line from which the IC50 value was found
(Table 1).
6.5. DPPHꢂ assay
The 2,2-diphenyl-1-picrylhydrazyl (DPPHꢂ) assay was used to
measure the ability of the compounds to scavenge the stable free
6.3.1. In vitro antimalarial assay
radical of DPPHꢂ in comparison to ascorbic acid. DPPHꢂ (100
mL)
Antimalarial activity of the compounds, against the chloro-
quine-sensitive (3D7) strain of P. falciparum, was performed using
the [3H]-hypoxanthine-incorporation assay [37]. The 3D7 strain
was continuously maintained in vitro in supplemented RPMI-1640
culture media and at a haematocrit of 5%. The culture was incu-
bated at 37 ꢁC in a gaseous atmosphere of 5% CO2, 3% O2, 92% N2 [38]
dissolved in HPLC grade methanol, was added along with 25
mL of
the compounds to a 96-well microtiter plate. Controls included
a drug-free DPPHꢂ control and a background control consisting of
MeOH and DMSO. The plate was then incubated in the dark at room
temperature for 30 min before the absorbance read at 540 nm. The
DPPHꢂ scavenging activity of the compounds were expressed as the
percentage decrease in the absorbance compared with the drug-
free DPPHꢂ control. Each experiment was triplicated.
and synchronized at the ring stage with 5%
being adjusted to a final parasitemia of 0.5% and haematocrit of 1%.
This suspension (200 L) was added to each well of the 96-well
D-sorbitol [39] before
m
plate with the exception of some wells which received non-para-
sitized red blood cells. Stock solutions of the test compounds were
made up in DMSO and serially diluted in a 96-well microtiter plate
[37]. The microtiter plate was then incubated for 24 h. Following
6.6. Cytotoxicity assays
6.6.1. MTT assay
the incubation period, 25
m
L of the radiolabeled [3H]-hypoxanthine
Ci/well was added to
MCF-7 cells were cultured and maintained as a monolayer in
Dulbecco’s modified Eagle’s medium (DMEM, Sigma) supple-
mented with 10% of fetal calf serum (Sigma) and antibiotics
isotope (Amersham) at a concentration of 0.5
m
each well. The microtiter plate was then incubated for a further
24 h. The parasitic DNA was harvested onto glass fibre filter mats by
use of a TitertekÔ semi-automatic cell harvester. The mats were
then transferred to sample bags containing scintillation fluid
(100 IU/mL of penicillin and 100 mg/mL of streptomycin, Sigma).
All cells were cultured at 37 ꢁC in a 100% humidity atmosphere
and 5% CO2 [43]. Exponentially growing viable cells were plated
at 1.2ꢄ104 cells per well into 96-well plates and incubated for
48 h before the addition of the compounds/metronidazole. 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. After 48 h of incubation at 37 ꢁC,
(PerkineElmer) and the b-radioactivity counted on the Wallac 1205
Betaplate scintillation counter. The counts per minute (cpm) were
generated and the parasite survival rate calculated. The concen-
tration required to inhibit parasite growth by 50% (IC50 value) was
determined from log sigmoid dose-response curves using the
EnzfitterÒ software. Chloroquine and quinine were used as refer-
ence agents. Each experiment was repeated, at least, in triplicate.
the medium was removed and 25 mL of MTT (5 mg/mL) in serum
free medium was added to each well. The plates were incubated
6.3.2. Combination study
at 37 ꢁC for 4 h. At the end of the incubation period, the medium
The antagonistic, synergistic or additive effect of the compound
exhibiting the most promising antimalarial activity and quinine
were determined by using the tritiated hypoxanthine incorporation
assay and constructing isobolograms. This involved preparing
various ratios of the two drugs (9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8
and 1:9) from which serial dilutions were prepared and from each
combination, an IC50 value was obtained. The IC50 values were used
was removed and 100 mL DMSO added to all wells. The metab-
olized MTT product dissolved in DMSO was quantified by reading
the absorbance at 570 nm with a reference wavelength of 655 nm
in an ELISA plate reader (Labsystems Multiskan RC, Helsinki,
Finland). All assays were performed in triplicate. Percent viability
was defined as the relative absorbance of treated versus
untreated control cells.

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This work was supported by Council of Scientific and Industrial
Research (grant # 01(2278)/08/EMR-II New Delhi, India) and the
Medical Faculty Research Endowment Fund of the University of
the Witwatersrand, South Africa. We would like to acknowledge the
financial support for F. Hayat from the University Grant Commission
(UGC), India and E. Moseley from the National Research Fund (South
Africa), Belgium Technical Cooperation and the University of the
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