Synthesis and evaluation of 7-chloro-4-(piperazin-1-yl)quinoline- sulfonamide as hybrid antiprotozoal agents

Article abstract of DOI:10.1016/j.bmc.2013.03.052

A new series of 4-aminochloroquinoline based sulfonamides were synthesized and evaluated for antiamoebic and antimalarial activities. Out of the eleven compounds evaluated (F1-F11), two of them (F3 and F10) showed good activity against Entamoeba histolyti

Full text of DOI:10.1016/j.bmc.2013.03.052

Accepted Manuscript
Synthesis and evaluation of 7-chloro-4-(piperazin-1-yl)quinoline-sulfonamide
as hybrid antiprotozoal agents
Attar Salahuddin, Afreen Inam, Robyn L. van Zyl, Donovan C. Heslop, Chien-
Teng Chen, Fernando Avecilla, Subhash M. Agarwal, Amir Azam
PII:
S0968-0896(13)00274-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.03.052
BMC 10709
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
31 January 2013
16 March 2013
19 March 2013
Please cite this article as: Salahuddin, A., Inam, A., van Zyl, R.L., Heslop, D.C., Chen, C-T., Avecilla, F., Agarwal,
S.M., Azam, A., Synthesis and evaluation of 7-chloro-4-(piperazin-1-yl)quinoline-sulfonamide as hybrid
antiprotozoal agents, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.03.052
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Graphical Abstract
Synthesis and evaluation of 7-chloro-4-
(piperazin-1-yl)quinoline-sulfonamide as
hybrid antiprotozoal agents
Leave this area blank for abstract info.
Attar Salahuddina, Afreen Inama, Robyn L. van Zylb, Donovan C. Heslopb, Chien-Teng Chenb, Fernando
Avecillac, Subhash M. Agarwald,* and Amir Azama,*
aDepartment of Chemistry, Jamia Millia Islamia, New Delhi-110025, India
bPharmacology Division, Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University
of Witwatersrand, Johannesburg 2193, South Africa
cDepartamento de Química Fundamental, Universidade da Coruña, Campus da Zapateira s/n, 15071, A
Coruña, Spain
dBioinformatics Division, Institute of Cytology and Preventive Oncology (ICMR), I-7, Sector-39, Noida-201301,
Uttar Pradesh, India
Hybrid antimalarials
P. falciparum (CQ-resistant FCR-3 strain)
IC50 = 1.16 M
Piperazine (linker)
Increases hydrophilicity
Sulphonamide
(DHPS-Inhibition in
folate biosynthesis)
(F8)
(F)
7-Chloroquinoline
(Accumulation in
haem binding)

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Synthesis and evaluation of 7-chloro-4-(piperazin-1-yl)quinoline-sulfonamide as
hybrid antiprotozoal agents
Attar Salahuddina, Afreen Inama, Robyn L. van Zylb, Donovan C. Heslopb, Chien-Teng Chenb, Fernando
Avecillac, Subhash M. Agarwald,* and Amir Azama,*
aDepartment of Chemistry, Jamia Millia Islamia,New Delhi-110025, India
bPharmacology Division, Department of Pharmacy and Pharmacology, Faculty of Health Sciences,University of Witwatersrand, Johannesburg 2193, South
Africa
c Departamento de Química Fundamental, Universidade da Coruña, Campus da Zapateira s/n, 15071, A Coruña, Spain
dBioinformatics Division, Institute of Cytology and Preventive Oncology (ICMR) I-7, Sector-39, Noida -201301, Uttar Pradesh, India
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A new series of 4-aminochloroquinoline based sulfonamides were synthesized and evaluated for
antiamoebic and antimalarial activities. Out of the eleven compounds evaluated (F1-11), two of
them (F3 and F10) showed good activity against Entamoeba histolytica (IC50 < 5µM). Three of
the compounds (F5, F7 and F8) also displayed antimalarial activity against the chloroquine-
resistant (FCR-3) strain of Plasmodium falciparum with IC50 values of 2 µM. Compound F7,
whose crystal structure was also determined, inhibited β–haematin formation more potently than
quinine. To further understand the action of hybrid molecules F7 and F8, molecular docking was
carried out against the homology model of P. falciparum enzyme dihydropteroate synthase
(PfDHPS). The complexes showed that the inhibitors place themselves nicely into the active site
of the enzyme and exhibit interaction energy which is in accordance with our activity profile
data. Application of Lipinski “rule of five” on all the compounds (F1-F11) suggested high drug
likeness of F7 and F8, similar to quinine.
Available online
Keywords:
Antiprotozoal agents
Sulfonamides
E. histolytica
P. falciparum
Cytotoxicity
Homology modeling
Docking
2009 Elsevier Ltd. All rights reserved.
1. Introduction
The protozoan parasites have now become resistant to some of
the more effective antiprotozoal drugs, thereby pressurizing the
Diseases caused by protozoal organisms are responsible for
considerable mortality and morbidity, affecting more than 500
million people in the world.1 Two such protozoan diseases are
malaria caused by Plasmodium falciparum and amoebiasis
caused by Entamoeba histolytica.2,3 Malaria alone affects nearly
40% of the global population, while amoebiasis results in 50
million cases of invasive disease and up to a million fatalities per
year.3 Chemotherapy remains the mainstay as the control strategy
for both these diseases. 7-Chloroquinolines and nitroimidazoles
as core moieties are active against malaria and amoebiasis,
respectively. Chloroquines, amodiaquine and ferroquine are the
standard drugs that bear 4-aminochloroquinoline core in their
structures while metronidazole, ornidazole and tinidazole are a
class of nitroimidazole ring bearing drugs.
control measures in place to treat patients infected with malaria
and amoebiasis. This scenario has necessitated the search for
novel drugs to contribute to the global chemotherapeutic
regimens.4 Presently, the most promising and so far successful
strategy in fighting malaria is the artemisinin combination
chemotherapy (ACT), in which an artemisinin derivative is used
together with conventional antimalarial drug to improve efficacy
and to delay the onset of resistance.5 A recent rational approach
of antimalarial drug design characterized as „„covalent
bitherapy‟‟ involves linking two molecules with individual
intrinsic activity into a single agent, thus packaging dual activity
into a single hybrid molecule.6,7 Current research in this field
seems to support hybrid molecules as the next-generation
antimalarial drugs, for example Trioxaferroquines.6-8 Many
conjugates of the available drugs for malaria have been reported
since the establishment of the concept of covalent bitherapy.9 In
many cases the conjugates have displayed more potency towards
both drug resistant and non-resistant strains.
* Corresponding authors. Fax: +91 11 26980229.
E-mail
addresses:
amir_sumbul@yahoo.co.in
(A.A),
smagarwal@yahoo.com (S.M.A)

The two enzymes DHPS (dihydropteroate synthetase) and
DHFR (dihydrofolate reductase) present within folate
biosynthetic pathway are ideal targets for antimicrobial therapy
as folate is necessary for the cell to synthesize nucleic acids and
in its absence cells is unable to divide. Sulfonamides are well
known competitive inhibitors of the enzyme DHPS10 which
catalyses the conversion of PABA (para-aminobenzoic acid) to
dihydropteroate, a key step in folate synthesis.11 Sulfadoxine is
an ultra-long-lasting sulfonamide often used in combination with
pyrimethamine to treat or prevent malaria.12
showed common peaks of the core 7-chloro-4-(piperazin-1-
yl)quinoline. The piperazine ring carbons showed peak around 45
and 51 ppm. Quinoline ring moiety showed peaks at 109, 121,
126 (for pyridine ring), the fused benzene part showed peaks
around 120-160 ppm. The quaternary carbon attached to the
piperazine showed at 135 ppm. The different aromatic sulfonyl
derivatives showed their respective peaks in the aromatic region
around 120 – 160 ppm.
R
Therefore, using this recent strategy of linking two scaffolds
in a single molecule, in the present study a series of novel
chloroquinoline based sulfonamide hybrids were designed and
evaluated for antiprotozoal activity. The design of the new hybrid
molecules is illustrated (Fig. 1), whereby
a
4-
aminochloroquinoline was joined via a piperazine linker to a
sulfonamide. It is proposed that 4-aminochloroquinoline will
promote haem binding, piperazine will act as the linker and the
sulfonamide group will act as a DHPS inhibitor.10, 13
Scheme 1. Reagents and conditions: (a) Piperazine, EtOH, reflux
12 h (77 %); b) R-Sulfonyl chlorides, Et3N, DCM, 0°C – rt (85-
95%).
2.2. Single Crystal Structure of F7
7-Chloro-4-[4-(propylsulfonyl)piperazin-1-yl]quinoline,
F7,
crystallizes from 30% dichloromethane/hexane solution as a
colorless prism (crystal dimensions 0.22 x 0.21 x 0.20). Fig. 2
shows an ORTEP representation of F7. Hydrogen bonds were not
found in the structure. Crystal data and details of the data
collection and refinement for the compound F7 are mentioned in
Table 1.
R
Figure 1. Rational design of hybrid antimalarials
2. Results and Discussion
2.1. Chemistry
Figure 2. ORTEP plot for the compound F7. All the non-
hydrogen atoms are presented by their 30% probability
ellipsoids.
The chloro-4-piperazin-1-yl-quinoline (2) was synthesized by
aromatic nucleophilic substitution of piperazine on the
commercially available 4,7-dichloroquinoline (1) under refluxing
conditions in good yield. The sulfonamide formation of the final
compounds (F1 to F11) was achieved by reacting 7-chloro-4-
piperazin-1-yl-quinoline (2) with different sulfonyl chlorides
using triethylamine as a base and dichloromethane as a solvent at
0 °C to room temperature (Scheme 1). All the products were
soluble in polar solvents and recrystallization was done in
dichloromethane hexane system and the compounds are stable in
solid states at room temperature. Melting points were recorded on
KSW melting point apparatus and are uncorrected. The 1H NMR
showed common signals of core 7-chloro-4-(piperazin-1-
yl)quinoline present in all the final compounds. The C2-H is
more deshielded than the C3-H due to electron withdrawing
nature of the pyridine nitrogen and the electron donating capacity
of piperazinyl group on the C4, and therefore appeared around δ
8.7 and 6.8, respectively. The C5-H and C6-H appeared as
separate doublets around δ 7.36 and 7.76, respectively. The C8-H
appeared as a singlet in all the compounds around δ 8.02. All the
aromatic and aliphatic substituents in the sulfonamide group
appeared as expected. The 13C spectra of all the compounds
Table 1. Crystal data and structure refinement for F7
Compound
F7
C16H20ClN3O2S
353.86
Formula
Formula weight
T, K
100(2)
Wavelength, Å
Crystal system
Space group
a/Å
b/Å
c/Å
α/º
β/º
0.71073
Triclinic
P1
9.4587(8)
9.9170(9)
10.2481(9)
90.922(7)
92.776(6)
118.318(6)
844.42(13)
2
γ/º
V/Å3
Z
F000
372
1.392
0.362
Dcalc/g cm-3
/mm-1
/ (º)
1.99 to 24.71

MTT cell viability assay on the human colon adenocarcinoma
(HT29) cell line.16 In vitro antimalarial activity was carried out
on the chloroquine-resistant (FCR-3) strain of P. falciparum by
use of the [3H]-hypoxanthine-incorporation assay.17 To
determine a possible mechanism of antimalarial action the
inhibition of β-haematin formation was assessed.18 Drug toxicity
was determined by examining the haemolytic effects of the
compounds on healthy erythrocytes.19 The results of the
experiments are summarized in Table 3.
Rint
0.0662
0.22 x 0.21 x 0.20
1.049
Crystal size/ mm3
Goodness-of-fit on F2
a
R1
0.0401
0.1180
0.533 and -0.392
wR2 (all data) b
Largest differences peak and hole
(eÅ-3)
aR1 = Fo - Fc/Fo.
bwR2 = [w(Fo2 -Fc2)2]/[w(Fo )]1/2
4
2.3.1 Antiamoebic activity
In the crystal packing, a weak - stacking could account
between quinoline rings [C(1)-C(2)-C(3)-C(4)-C(5)-N(3)-C(6)-
C(7)-C(8)-C(9)] of an inversion-related molecules (mean
separation between the quinoline rings ca 4.699 Å),14 see Fig. 3.
The benzene and pyridine quinoline rings present a distortion
with respect to the planarity and they are inclined at a dihedral
angle of 4.95(16)º with respect to each other. This planarity loss
is related to the crystal packing forces increasing the stress of its
structure. Table 2 contains selected bond lengths and angles for
compound F7.
Preliminary experiments were carried out to determine the in
vitro antiamoebic activity of all the compounds (F1-F11) by
microdilution 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 μM
(Table 3). The structure activity relationship (SAR) showed that
compounds (F1-F11) which contained aliphatic substituents in
the benzene ring of the sulfonamide group increased with the
increasing hydrophobicity, hence F3 (IC50: 2.86 μM) with the
most bulky and hydrophobic t-butyl group showed highest
antiamoebic activity, while F10 (IC50: 4.54 μM) with a p-tolyl
sulfonamide group showed considerable activity (Fig. 4). The
compounds F4 (IC50: 8.23 μM) and F8 (IC50: 9.61 μM) with a
para substitution in the benzene ring of the sulfonamide group
have comparatively less IC50 values than the other substituted
compounds (Fig. 4). Aliphatic sulfonamide F7 (IC50: >100 μM)
and F11 (IC50: >100 μM) were found to be the least active
compounds in the series. Therefore it was concluded that for
display of antiamoebic activity, an aromatic sulfonamide with
aliphatic/hydrophobic substitutions at para position is required in
the final structure of 7-chloro-4-piperazin-1-yl-quinoline
sulfonamides. Collectively only one compound F3 (IC50: 2.86
μM) was found to be nearly as active to the standard drug
metronidazole (IC50: 1.46 μM).
Figure 3. Crystal packing of F7. Hydrogen atoms are omitted for
clarity. π-π stacking between benzene and pyridine quinoline
groups are presented in dashed lines.
Relative antiamoebic activity
IC50
SD
Table 2. Bond lengths [Å] and angles [°] for F7
Compound F7
10
8
Bond lengths (Å)
6
S(1)-O(1)
S(1)-O(2)
S(1)-N(2)
S(1)-C(14)
1.4330(18)
1.4276(19)
1.633(2)
C(1)-N(3)
N(3)-C(5)
C(4)-C(5)
1.319(3)
1.375(3)
1.412(4)
value
50
4
IC
2
0
F3
F4
F8
Compound Code
F10
MNZ
1.778(3)
Bond Angles (º)
Figure 4. Relative antiamoebic activities
N(3)-C(5)-C(4)
C(6)-C(5)-C(4)
C(5)-C(4)-C(3)
C(5)-C(4)-C(9)
123.4(2)
119.4(2)
118.0(2)
118.9(2)
O(2)-S(1)-O(1) 118.98(11)
O(1)-S(1)-N(2) 106.90(11)
O(2)-S(1)-N(2) 107.30(11)
O(2)-S(1)-C(14) 108.65(13)
O(1)-S(1)-C(14) 107.63(12)
N(2)-S(1)-C(14) 106.78(12)
2.3.2 Antimalarial, haemolytic and β–haematin inhibitory
activity
The series of chloroquinolinyl piperazine sulfonamides (F1-F11)
were examined for their in vitro antimalarial activity, haemolytic
properties and to elucidate a possible mechanism of action, their
ability to inhibit the formation of β-haematin was also assessed
(Table 3). In contrast to the activity observed against Entamoeba,
where compounds F3 and F10 were the most active, compounds
F8, F7 and F5 were the most active against Plasmodium
falciparum with IC50 values less than 2 μM. None of the
compounds caused significant haemolysis of the uninfected
human erythrocytes, thus, indicating that the compounds entered
the parasite and directly inhibited parasite growth, rather than
2.3. Pharmacology
All chloroquinolinyl sulfonamides (F1-F11) were screened in
vitro against HM1: IMSS strain of E. histolytica by the
microdilution method.15 All the experiments were carried out in
triplicate at each concentration level and repeated thrice.
Cytotoxicity of active compounds has been studied using the

interfering with the red blood cell membrane integrity. The N-
acetylaniline substitution in compound F8 (4-{[4-(7-
chloroquinolin-4-yl)piperazin-1-yl]sulfonyl}-N-acetylaniline)
was the most active in the series, although activity was at least 7
fold less than the standard antimalarial agent, quinine. The
decrease in inhibitory activity can be attributed to the
sulfonamide/sulfadoxine group, which against this Plasmodium
strain has an IC50 value of greater than 100 µM. Thus, it does not
contribute to the overall antimalarial activity of the new complex.
Although the antimalarial activity of the compounds did not
correlate with their ability to inhibit β-haematin formation,
several structures did display interesting properties to be used as
lead compounds to improve upon the antimalarial efficacy.
Namely, the ability of a compound F8 to inhibit β-haematin
formation (IC50 = 8.84µM) as potently as chloroquine, a known
inhibitor of β-haematin formation (IC50 value: 8.63 ± 2.13 µM),
which could indicate one possible mechanism of action of this
compound. In contrast, although the nitrophenyl group on the
benzene ring of compound F5 retained the compounds activity
against the intra-erythrocytic parasite, its ability to inhibit β-
haematin formation was not comparable to that of chloroquine or
quinine (Table 3). The aliphatic propyl group substituted on the
benzene ring (compound F7) not only increased the antimalarial
activity when compared to the methyl substitution (compound
F11), but it also increased the ability of the compound to inhibit
β-haematin formation.
2.3.3 Cytotoxicity profile
Overall, the compounds inhibited approximately 30% cell
viability at 100 µM, with compound F8 being the least toxic
(Table 3). In contrast, compound F9 was the most inhibitory
against the colon adenocarcinoma by inhibiting 45% cell growth,
in comparison to 52% by camptothecin (CTT), a cytotoxic
quinoline alkaloid which inhibits the DNA enzyme,
topoisomerase I.20 The two most active compounds against
amoebiasis, compounds F3 and F10, had a safety index of 24 and
14, respectively. The three most active compounds against P.
falciparum, compounds F8, F7 and F5, were relatively non-toxic
to the colon adenocarcinoma cells, where overall 17% of cells
were inhibited at 100 µM. The safety index of these three
compounds was 84, 39 and 44, respectively.
Table 3. Biological activity results
Compd
R-substitution
Antiamoebic
activity
(HM1:IMSS)
IC50 ± S.D.
(M)
Antimalarial
activity
(FCR-3)
IC50 ± S.D.
(M)
Haemolytic
activity
Inhibition of
β-haematin
formation
IC50 ± S.D.
(µM)
Cytotoxicity
(HT29)
% Lysis ±
S.D.
at 100 µM
% Cell viability
± S.D. at 100
M
Cl
Cl
F1
2.55 ± 0.38
2.06 ± 0.46
47.22 ± 3.67
30.2 ± 0.23
63.39 ± 9.57
F2
F3
F4
3.06 ± 0.59
6.09 ± 0.30
3.30 ± 0.31
0.97 ± 0.01
1.36 ± 0.32
6.03 ± 0.11
71.17 ± 5.60
72.40 ± 1.93
9.04 ± 1.90
23.4 ± 0.02
2.86 ± 0.03
8.23 ± 0.012
59.76 ± 2.76
67.69 ± 7.74
68.11 ± 19.15
O
F5
1.94 ± 0.37
0.01 ± 0.01
1.40 ± 0.18
63.54 ± 1.82
34.77 ± 0.66
>100 ± 0.9
86.11 ± 14.12
NO2
Cl
F6
F7
3.27 ± 0.23
25.7 ± 0.1
61.27 ±12.58
67.07 ± 12.01
>100 ± 0.91
1.72 ± 0.22
1.16 ± 0.36
0.82 ± 0.20
0.83 ± 0.12
1.79 ± 0.22
8.84 ± 1.27
H
N
F8
9.61 ± 0.02
96.84 ± 11.20
O
F9
4.02 ± 1.00
4.22 ± 0.62
0.01 ± 0.01
0.62 ± 0.13
18.09 ± 4.05
32.8 ± 0.26
55.15 ± 17.42
64.45 ± 12.66
F10
8.92 ± 2.39
4.54 ± 0.1
F11
(MNZ)
Qu.
CH3
>100 ± 0.21
1.45 ± 0.06
N.D.
8.54 ± 1.49
N.D.
0.01 ± 0.01
N.D.
13.54 ± 1.73
N.D.
72.96 ± 14.55
N.D.
Metronidazole
Quinine
0.17 ± 0.03
2.14 ± 0.34
22.08 ± 3.10
N.D.

CTT
Camptothecin
N.D.
N.D.
N.D.
N.D.
48.33 ± 4.18
2.4. Measuring drug-likeness
Good bioavailability can be achieved with a balance between
solubility and partitioning properties. Thus in order to gain
insight into the drug likeness of our compounds in comparison
to standard drugs we subjected the compounds F1-F11 for the
prediction of lipophilicity and Lipinski‟s “Rule of Five”.21
High oral bioavailability is an important factor for the
development of bioactive molecules as therapeutic agents.
Good intestinal absorption, reduced molecular flexibility
(measured by the number of rotatable bonds), low polar
surface area (PSA) or total hydrogen bond count (sum of
donors, HBDs and acceptors, HBAs), are important predictors
of good oral bioavailability.22 Molecular properties such as
membrane permeability and bioavailability are associated with
log P (partition coefficient), molecular weight (MW), or
hydrogen bond acceptors and donors count in a molecule.23
The rule states that most molecules with good membrane
permeability have log P <5, molecular weight <500, number of
hydrogen bond acceptors <10, and number of hydrogen bond
donors <5. This rule is widely used as a filter for drug-like
properties. An analysis of small drug-like molecules suggests a
filter of log D > 0 and <3, which enhances the probability of a
compound to exhibit good intestinal permeability. A poor
permeation or absorption is more likely when there are more
than 5 H-bond donors and 10 H-bond acceptors. All the
compounds (F1-F11), quinine and chloroquine have obeyed
the “Rule of Five” with logP values <5 and HBA ≤5. All the
compounds (F1-F11) under investigation possess hydrogen
bond donor‟s ≤1 and a considerable number of hydrogen bond
acceptors (≤5) as shown in Table 4. The most active
compounds against P. falciparum F7 and F8 have peculiar
properties with log P values nearly equal to that of quinine. F7
has a PSA near to that of quinine, while F8 has distinctly large
PSA value.
Figure 5. Graph showing correlation between log P and IC50
values for P. falciparum growth inhibition by F1-F11,
chloroquine and quinine. The compounds whose log P values
are close to the linear line and have lower IC50 values are the
most active. The most active compounds are encircled.
2.5. Comparative modeling of PfDHP
As we have designed hybrid molecules capable of exhibiting
dual activity i.e. β-haematin and DHPS inhibition, we also
generated a homology model of PfDHPS so as to analyze the
interactions of the quinoline based sulfonamides with the
enzyme. To construct the 3D model of PfDHPS, a BLAST
search was performed against the Protein Data Bank (PDB) 24
for template identification. BLAST analysis revealed that
Bacillus anthracis DHPS (PDB code 1TX0) shares 35%
identity and 49% similarity, the highest with PfDHPS and
therefore it was selected as the template for model
construction. The sequence alignment of the 1TX0 with
PfDHPS sequence that was used for model construction is
shown in Fig. 6. Homology modeling was then carried out
through Modeller 9v9.25 The constructed model was then
subjected to loop refinement followed by energy minimization.
The stereo chemical quality of the final predicted structure was
assessed using PROCHECK26, which showed that 84.1% of the
residues were in the „most favored region‟ and 15.4% in the
combined „allowed region‟ and one residue (Asp110) was
found in the disallowed region. Also, it is established that the
score for G-factors should be above −0.50 for a reliable
model.27 We observed that the G-factor scores of the model
was -0.22 for dihedral bonds, 0.06 for covalent bonds and
−0.09 overall. The distribution of the main chain bond lengths
and bond angles was 99.9% and 93.0% within limits. The
quality of the structure is further evident by the fact that
according to VERIFY3D28, 82.33% of the residues have a
score of greater than 0.2, which indicates a good quality model.
Table 4. Lipinski tools for measuring drug likeness
No of No of
HBAs* HBDs*
No.
Mol.wt LogP Log D
PSA
F1
F2
F3
F4
F5
F6
F7
F8
F9
456.773 4.73
437.942 4.52
443.989 5.07
417.909 3.37
432.881 3.47
422.328 4.13
353.867 2.44
444.934 2.76
387.883 3.53
4.54
4.32
4.88
3.18
3.28
3.94
2.25
2.57
3.34
3.85
1.22
0.88
0.86
4
4
4
5
5
4
4
5
4
4
4
3
4
0
0
0
0
0
0
0
1
0
0
0
1
1
53.51
53.51
53.51
62.74/
99.33
53.51
53.51
82.61
53.51
53.51
53.51
28.16
45.59
F10 401.910 4.04
F11 325.814 1.41
CHQ 325.814 3.93
QU 324.416 2.51
*HBA- hydrogen bond acceptor, HBD-Hydrogen bond donor,
PSA-polar surface area obtained by Marvin Sketch 5.1
A graph of IC50 versus the logP values clearly indicates that F7
and F8 have high drug-likeness similar to chloroquine and
quinine. F7, F8 and standard drugs used have high proximity
therefore lie near the Y-axis with low IC50 and logP values
(circled) (Fig. 5)

PfDHPS 1
1TX0
PfDHPS 61
1TX0 61
EKTNIVGILNVNYDSFSDGGIFVEPKRAVQRMFEMINEGASVIDIGGESSAPFVIPNPKI 60
EKT I+GILNV DSFSDGG + E AV+ EM +EGA +IDIGGES+ P
EKTLIMGILNVTPDSFSDGGSYNEVDAAVRHAKEMRDEGAHIIDIGGESTRPGFAKVSVE 60
1
SERDLVVPVLQLFQKEWNDIKNKIVKCDAKPIISIDTINYNVFKECVDNDLVDILNDISA 120
E
VVP++Q
KE
K
ISIDT
V K+ ++
I+NDI
EEIKRVVPMIQAVSKE------------VKLPISIDTYKAEVAKQAIEAG-AHIINDIWG 107
PfDHPS 121 CTNNPEIIKLLKKKNKFYSVVLMHKRGNPHTMDKLTNYDNLVYDIKNYLEQRLNFLVLNG 180
P+I ++ ++LMH R N NY NL+ D+
108 AKAEPKIAEVAAHYD--VPIILMHNRDN-------MNYRNLMADMIADLYDSIKIAKDAG 158
+
L
+
G
1TX0
PfDHPS 181 IPRYRILFDIGLGFAKKHDQSIKLLQNIHVYD--EYPLFIGYSRKRFIAHCMN
I+ D G+GFAK +Q+++ ++N+ YP+ +G SRK FI H ++
159 VRDENIILDPGIGFAKTPEQNLEAMRNLEQLNVLGYPVLLGTSRKSFIGHVLD
231
211
+
+
1TX0
Figure 6. Sequence alignment of PfDHPS with the template (Pdb id: 1TX0)
Also we analyzed the model using ProSA-web29 which
measures the z-score (indicating overall model quality) and
deviation of the total energy of the structure with respect to
energy distribution derived from random conformations. The
overall ProSA z-score evaluated for the model developed in
this study was -7.29, which is comparable to X-ray crystallized
template structure of 1TX0 (z-score = -7.71). Further, the root-
mean-square deviation (RMSD) between the backbone atoms
of the template and the homology model was observed to be
0.734 Å indicating reasonably good structural parameters of
the predicted structure (Fig. 7).
with F8 reveals that the residues Glu 48, Ala 51, Pro 52, Phe
53, Lys 196, His 198, Gly 219, Arg 222 and Lys 223 are
involved in hydrophobic interaction, while Gly 193 and Arg
224 forms hydrogen bond with N3 and O3 of F8, respectively
(Fig. 8a). Previously too, P. falciparum DHPS homology
modeling followed by molecular docking has demonstrated
that sulfadoxine docks into the same active site having similar
active site forming residues in interaction with the inhibitor.32
Further, we observe that F7 also interacts with similar residues
(Ala 51, Pro 52, Phe 53, Gly 193, Lys 196, Lys 197, His 193
and Phe 225) that contribute towards a hydrophobic interaction
and exhibits hydrogen bond with Arg 224 (Fig. 8b). Thus the
docking studies are in concurrence with previous studies and
provide additional evidence that these compounds may inhibit
the growth of P. falciparum by interacting with PfDHPS.
Figure 7. Structural superimposition of Cα trace of PfDHPS
model (represented in blue color) with known crystal structure
(represented in green color). The root-mean-square deviation
(RMSD) between the template and the homology model was
0.734 Å
2.6. Molecular docking
After the homology model was constructed, the active site
information was obtained by superimposing 3-D structure of
the PfDHPS with that of E. coli DHPS (Pdb id: 1AJO), as it
contains sulfonamide bound in the active site. Thereafter,
docking in the binding pocket of the modeled protein was
undertaken using Autodock 4.230 and the interacting residues
with two most promising P. falciparum inhibitors (F7 and F8)
were determined. It was noted that Autodock binding energy of
F7 and F8 was -7.37 and -7.89 with PfDHPS respectively,
which is in accordance with our activity profile data that
indicated F8 to be the most active inhibitor of Plasmodium
growth, followed by F7. Ligplot31 analysis of docked complex

4.1.1 7-Chloro-4-piperazin-1-yl-quinoline (2).
To a stirred solution of 4,7-dichloroquinoline (1) (10 g, 50.49
mmol) in 150 mL ethanol was added piperazine (30.44 g,
353.44 mmol), the resulting solution was then refluxed for 12
h. On reaction completion (TLC) the reaction was then
concentrated under vacuum to give a crude solid mixture
which was taken up in 200 mL DCM and washed with
saturated sodium bicarbonate solution until no piperazine was
seen in the organic layer (TLC). The combined organic
extracts were dried over anhydrous sodium sulfate and
concentrated to give a crude product which was purified by
recrystallization in 30% DCM: Hexane as a white powder (9.7
g, 77.6%) of compound 2.: Rf = 0.25 (10%MeOH/CHCl3) mp:
118-120 °C; Anal. calc. for C13H14ClN3 : C 63.03, H 5.70 N
16.96 % found: C 63.23, H 5.61, N 17.02 %; IR νmax (cm-1):
1
1652 (C=O), 1579 (C=C); H NMR (CDCl3) (ppm): 3.14-
3.21(m, 8H), 3.33 (s, 1H), 6.83 (d, 1H J=5.1Hz), 7.42 (dd, 1H,
J=2.1Hz and 9Hz), 7.97 (t, 1H, J=9Hz) 8.06 (dd, 1H, J= 2.1Hz
and 9Hz), 8.72 (d, 1H, J=5.1 Hz); FAB-MS (m/z): [M++1] 248
4.1.2
7-Chloro-4-(4-Alkyl/Aryl
sulfonyl-piperazin-1-yl)-
quinoline (F1 to F11).
To a stirred solution of 7-chloro-4-piperazin-1-yl-quinoline (2)
(0.25 g, 1 mmol) in 8-10 mL of DCM at 0 °C was added
triethylamine (0.12 mL, 1.26 mmol). Different aryl sulfonyl
chlorides (1 mmol) were added dropwise / portion wise and the
resulting solution was stirred further for 15 min at 0 °C and
then stirred at room temperature for different time intervals.
On reaction completion (TLC), the reaction mix was diluted
with 20 mL water and 20 mL DCM and partitioned in
separating funnel, where the organic layer was washed with
water (3x 20 mL) before being separated and dried over
Figure 8. Schematic 2D representation of interactions of (a) F8
and (b) F7. Hydrogen bonds are shown with green dashed
lines, and hydrophobic contacts by red arcs with radiating
lines.
sodium sulfate to yield compound
3 in which was
recrystallized in DCM:Hexane to yield pure products in 85-
95% yields.
3. Conclusions
The present study indicates that the complex consisting of 4-
aminochloroquinoline and sulfadoxine linked via piperazine is
a viable combination as it acts against both Entamoeba
histolytica and Plasmodium falciparum. Overall, the
4.1.3 7-Chloro-4-{4-[(2,5-dichlorophenyl)sulfonyl] piperazin-
1-yl} quinoline (F1)
Yield 95%; (DCM/Hexane); Rf = 0.65 (5%MeOH/CHCl3) mp:
165-167°C; Anal. calc. for C19H16Cl3N3O2S : C 49.96, H 3.53,
N 9.20, S 7.02% found: C 49.7, H 3.32, N 9.25, S 7.2%. IR
max (cm-1): 1569.29, 1157.53, 1034.73, 817.46, 575.83,
671.67. 1H NMR (CDCl3) (ppm): 3.254 (t,4H, J=3.3Hz),
3.613 (t, 4H, J=3.3Hz), 6.849 (d, 1H, J=4.8Hz), 7.424 (d, 1H,
J=9Hz), 7.509 (s, 2H), 7.863 (d, 1H, J=8.7Hz), 8.072 (d, 2H,
J=15.6), 8.743 (d, 1H, J=4.8Hz). 13CNMR  (ppm): 45.76,
51.95, 109.50, 121.67, 124.51, 126.64, 129.04, 130.46, 131.80,
133.31, 133.35, 135.14, 137.39, 150.05, 151.90, 156.06. FAB-
MS (m/z): [M++1] 456.25
compounds
F3
(7-chloro-4-{4-[(4-t-
butylphenyl)sulfonyl]piperazin-1-yl}quinoline) and F8 ((4-{[4-
(7-chloroquinolin-4-yl)piperazin-1-yl]sulfonyl}-N-
acetylaniline)) which possess favourable structural features
need to be further developed to increase antiamoebiasis and
antiplasmodial activity respectively.
4. Experimental Section
4.1. Chemistry
All the required chemicals were purchased from Merck and
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 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 Bruker FT-
IR spectrophotometer under neat condition on ZnSe Crystal.
1H NMR and 13C NMR spectra were recorded on Bruker
Spectrospin DPX 300 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.
4.1.4
7-Chloro-4-[4-(2-naphthylsulfonyl)piperazin-1-
yl]quinoline (F2)
Yield: 93%,(DCM/Hexane): Rf = 0.6 (5%MeOH/CHCl3) m.p.:
179-182°C; Anal. Calc. for C23H20ClN3O2S: C 63.08, H 4.60,
N 9.59, S 7.32%; found: C 62.89, H 4.71, N 9.44, S 7.3%. IR
max (cm-1): 1569.36, 1160.65, 1069.27, 818.68, 571.09,
1
606.33, 646.23; H NMR (CDCl3) (ppm): 3.29 (t, 4H, J =
4.2Hz), 3.38 (t, 4H, J=4.2Hz), 6.851 (d, 1H, J=4.8Hz), 7.31-
7.35 (dd, 1H, J = 2.1 & 9Hz), 7.64-7.73 (m, 3H), 7.81-7.85
(dd, 1H, J=1.8 & 8.7Hz), 7.96-8.09 (m, 4H), 8.42 (s, 1H), 8.72
(d, 1H, J=4.8Hz); 13C NMR (CDCl3) (ppm): 46.03, 51.46,
109.36, 121.52, 122.83, 124.53, 126.46, 127.73, 127.96,
128.83, 129.05, 129.18, 129.21, 129.46, 132.15, 132.36,
134.95, 135.04, 149.82, 151.78, 156.00; FAB-MS (m/z):
[M++1] 438.

4.1.5 7-Chloro-4-{4-[(4-t-butylphenyl)sulfonyl]piperazin-1-
yl}quinoline (F3)
54.39, H 5.73, N 11.66, S 9.11 %; IR max (cm-1): 1579.55,
1258.12, 1373.47, 1152.19,, 1492.81, 826.67, 608.37; 1H NMR
(CDCl3) (ppm): 1.11 (t, 3H, J=14.7Hz), 1.927 (m, 2H), 2.99
(t, 2H, J=15.6Hz), 3.277(t, 4H, J=9Hz), 3.5 (t, 4H, J=9Hz),
6.86 (d, 1H, J=4.8Hz), 7.42-7.46 (dd, 1H, J=11.8 & 9Hz), 7.95
(d, 1H, J=9Hz), 8.053 (d, 1H, J=1.5Hz), 8.749 (d, 1H,
J=5.1Hz); 13C NMR (CDCl3) (ppm): 13.10, 16.78, 45.64,
51.13, 51.98, 109.46, 121.65, 124.54, 126.52, 128.96, 135.00,
150.00, 151.89, 156.89, 156.07; FAB-MS (m/z): [M++1] 354.2
Yield: 91%, (DCM/Hexane); Rf = 0.7 (5%MeOH/CHCl3)m.p.:
170-173°C; Anal. Calc. for C23H26ClN3O2S: C 62.22, H 5.90,
N 9.46, S 7.22%; found: C 62.3, H 5.72, N 9.56, S 7.13%. IR
max (cm-1): 1569.33, 1163.35, 832.21, 578.93, 611.72,
1
2962.37, 942.08; H NMR (CDCl3) (ppm): 1.385 (s, 9H),
3.315 (d, 8H, J=4.5Hz), 6.853 (d, 1H, J=5.1Hz), 7.362-7.398
(d, 1H, J=1.8 & 8.85Hz), 7.612 (d,2H, J=8.4Hz), 7.772 (m,
3H), 8.031 (d, 1H, J=1.8Hz), 8.720 (s, 1H); 13C NMR (CDCl3)
(ppm): 31.10, 35.26, 45.79, 45.98, 51.57, 109.45, 121.62,
124.64, 126.30, 126.55, 127.73, 128.98, 132.42, 135.11,
149.98, 151.93, 156.13, 157.02. FAB-MS (m/z): [M++1]
444.39
4.1.10 4-{[4-(7-Chloroquinolin-4-yl)piperazin-1-yl]sulfonyl}-
N-acetylaniline (F8)
Yield: 82.4%, (White solid, DCM/Hexane), Rf = 0.75
(5%MeOH/CHCl3) m.p.: 265-268°C; Anal. calc. for
C21H21ClN4O3S: C 56.69, H 4.76, N 12.59, S 7.21%; found: C
59.87, H 4.71, N 12.44, S 7.12%; IR max (cm-1): 3186.04,
1669.62, 13340.60, 1158,41, 1262.29, 829.51, 608.82; 1H
NMR (CDCl3) (ppm): 2.263 (s, 3H), 3.300 (s, 8H), 6.862 (d,
1H, J=4.8Hz), 7.391-7.49 (dd, 1H, J=2.1 & 9Hz), 7.776-7.824
(m, 5H), 8.042 (d, 1H, J=1.8Hz), 8.199 (s, 1H), 8.747 (d, 1H,
J=4.8Hz); 13C NMR (CDCl3) (ppm): 24.33, 45.88, 51.28,
53.36, 108.82, 109.48, 119.53, 121.71, 124.69, 126.61, 128.98,
129.03, 135.15, 150.05, 151.96, 156.11; FAB-MS (m/z):
[M++1] 445.2
4.1.6 7-Chloro-4-{4-[(4-methoxyphenyl)sulfonyl]piperazin-1-
yl}quinoline (F4)
Yield: 90%, (Dull white solid, DCM/Hexane), Rf = 0.6
(5%MeOH/CHCl3) m.p.: 220-223°C; Anal. calc. for
C20H20ClN3O3S: C 57.48, H 4.82, N 10.05, S 7.67%; found: C
57.32, H 4.94, N 10.02, S 7.37%; IR max (cm-1): 1571.59,
1189.23, 1156.81, 1063.59, 823.62, 550.33, 653.20, 608.15,
1
1253.95; H NMR (CDCl3) (ppm): 3.288 (s, 8H), 3.194 (s,
3H), 6.843 (d, 1H, J=1.6Hz), 7.073 (d, 2H, J=8.4Hz), 7.375 (d,
1H, J=9Hz), 7.75-7.79 (m, 3H), 8.029 (s, 1H), 8.73 (d, 1H,
J=4.8Hz); 13C NMR (CDCl3) (ppm): 46.05, 51.51, 55.72,
109.47, 114.47, 121.67, 124.65, 126.53, 126.84, 129.04,
130.00, 135.09, 150.05, 151.98, 156.09, 163.33. FAB-MS
(m/z): [M++1] 418.12
4.1.11
7-Chloro-4-[4-(phenylsulfonyl)piperazin-1-
yl]quinoline (F9)
Yield: 85.6%, (Dark brown solid, DCM/Hexane), Rf = 0.6
(5%MeOH/CHCl3) m.p.: 148-151°C; Anal. calc. for
C19H18ClN3O2S: C 58.83, H 4.68, N 10.83, S 8.27%; found: C
58.8, H 4.62, N 10.68, S 8.21%. IR max (cm-1): 1337.20,
4.1.7
yl}quinoline (F5)
7-Chloro-4-{4-[(2-nitrophenyl)sulfonyl]piperazin-1-
1
1158.52, 1237.26, 813.19, 733.44, 606.73; H NMR (CDCl3)
(ppm): 3.32-3.34 (m, 4H), 3.41-3.42 (m, 4H), 6.90 (d, 1H,
J=5.4Hz), 7.39-7.43 (dd, 1H, J=2.1 & 8.85Hz), 7.55-7.78 (m,
3H), 7.76 (d, 1H, J=9Hz), 7.75-7.85 (m, 2H), 8.12 (d, 1H,
J=2.1Hz), 8.73 (d, 1H, J=5.4Hz; 13C NMR (CDCl3) (ppm):
45.92, 45.98, 51.54, 108.98, 120.96, 124.93, 125.97, 127.03,
127.81, 128.31, 133.36, 135.36, 136.33, 150.13, 157.24. FAB-
MS (m/z): [M++1] 388.16
Yield: 94%,(Off white solid, DCM/Hexane), Rf = 0.5
(5%MeOH/CHCl3)
C19H17ClN4O4S: C 52.72, H 3.96, N 12.94, S 7.41%; found: C
52.63, H 3.77, N 12.91, S 7.33%. IR max (cm-1): 1546.68,
1165.92, 1012.97, 814.70, 569.42, 696.93, 611.15, 859.81,
m.p.: 152-155°C; Anal. calc. for
1
1546.63, 1350.76; H NMR (CDCl3) (ppm): 3.282 (t, 4H,
J=4.5Hz), 3.615 (t, 4H, J=4.8Hz), 6.851 (d, 1H, J=5.1Hz),
7.40-7.43 (dd, 1H, J=1.8 & 9Hz), 7.65-7.70 (m, 1H), 7.72-7.80
(m, 2H), 7.85 (d, 1H, J=9Hz), 8.03-8.06 (dd, 2H, J = 1.8 &
6.6Hz), 8.74 (d, 1H, J=5.1Hz); 13C NMR (CDCl3) (ppm):
45.95, 51.84, 109.60, 121.73, 124.30, 124.60, 126.69, 129.09,
130.98, 131.09, 131.78, 134.12, 135.17, 148.44, 150.10,
152.00, 156.07. FAB-MS (m/z): [M++1] 433.3
4.1.12 7-Chloro-4-{4-[(4-methylphenyl)sulfonyl]piperazin-1-
yl}quinoline (F10)
yield: 80.2%, (Light brown solid, DCM/Hexane) Rf = 0.7
(5%MeOH/CHCl3) m.p.: 172-175°C; Anal. calc. for
C20H20ClN3O2S: C 59.77, H 5.02, N 10.45, S 7.98%; found: C
59.68, H 5.18, N 10.6, S 8.0%. IR max (cm-1): 1570.29,
1
1011.62, 1336.34, 1157.93, 867.99, 812.82, 610.28; H NMR
4.1.8 7-Chloro-4-{4-[(4-chlorophenyl)sulfonyl]piperazin-1-
yl}quinoline (F6)
(CDCl3) (ppm): 2.485 (s,3H), 3.296 (s, 8H), 6.843 (d, 1H,
J=5.1Hz), 7.36-7.42 (m, 3H), 7.71-7.77 (m, 3H), 8.037 (d, 1H,
J=1.8Hz), 8.731 (d, 1H, J=6.9Hz); 13C NMR (CDCl3) (ppm):
21.55, 45.95, 51.44, 109.36, 121.55, 124.57, 126.44, 127.79,
128.88, 129.85, 132.29, 135.03, 144.05, 149.88, 151.81,
156.04; FAB-MS (m/z): [M++1] 402.1
Yield: 93.3%, (White solid, DCM/Hexane), Rf = 0.65
(5%MeOH/CHCl3) m.p.: 164-167°C; Anal. calc. for
C19H17Cl2N3O2S: C 54.04, H 4.06, N 9.95, S 7.59%; found: C
54.22, H 4.3, N 9.52, S 7.43%; IR max (cm-1): 1571.66,
1
1340.79, 1162.67, 607.60,759.46, 815.34, 1259.38; H NMR
(CDCl3) (ppm): 3.299 (m, 8H), 6.843 (d, 1H, J=4.8Hz),
7.396-7.359 (dd, 1H, J=2.1 & 9Hz), 7.588 (d, 2H, J=8.7Hz),
7.79-7.74 (m, 3H), 8.301 (d, 1H, J=1.8Hz), 8.726 (s, 1H); 13C
NMR (CDCl3) (ppm): 45.95, 57.42, 109.46, 121.58, 124.48,
126.55, 129.02, 129.15, 129.61, 133.91, 135.08, 150.00,
151.92, 155.88; FAB-MS (m/z): [M++1] 423.4
4.1.13
yl]quinoline (F11)
7-Chloro-4-[4-(methylsulfonyl)piperazin-1-
Yield: 91.3%, (Pale yellow solid, DCM/Hexane), Rf = 0.45
(5%MeOH/CHCl3)mp: 197-200 °C Anal. calc. for
C14H16ClN3O2S: C 51.61, H 4.95, N 12.90, S 9.84%; found: C
51.39 H 4.9, N 12.88, S 9.81%. IR max (cm-1): 1592.25,
4.1.9
(7-Chloro-4-[4-(propylsulfonyl)piperazin-1-
1
1332.69, 1155.43, 815.32, 609.39; H NMR (CDCl3) (ppm):
yl]quinoline (F7)
2.924 (s, 3H), 3.88 (t, 4H, J=9Hz), 3.554 (t, 4H, J=9Hz), 6.69
(d, 1H, J=5.1Hz), 7.46-7.50 (dd, 1H, J=2.1 & 9Hz), 7.931 (d,
1H, J=9Hz), 8.09 (s, 1H), 8.776 (d, 1H, J=5.1Hz); 13C NMR
(CDCl3) (ppm): 34.82, 45.77, 51.71, 109.41, 121.52, 124.61,
Yield: 94.8%, (Pale yellow solid, DCM/Hexane), Rf = 0.55
(5%MeOH/CHCl3) m.p.: 118-121°C; Anal. calc. for
C16H20ClN3O2S: C 54.31, H 5.70, N 11.87, S 9.06%; found: C

126.79, 128.6, 135.51, 149.47, 151.46, 156.39; FAB-MS
(m/z): [M++1] 326.41
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 Titertek™ semi-automatic cell harvester. The
mats were then transferred to sample bags containing
scintillation fluid (Wallac®) and the β-radioactivity counted on
the Wallac® 1205 Betaplate scintillation counter. The counts
per minute (cpm) were generated and the % parasite growth
calculated. The concentration required to inhibit parasite
growth by 50% (IC50 value) was determined from log sigmoid
dose response curves using the GraphPad Prism® 5.0 software.
Quinine a clinically used anti-malarial agent was used as the
positive control. Each experiment was repeated, at least, in
triplicate.
4.2. In vitro antiamoebic assay
All the compounds (F1- F11) were screened in vitro for
antiamoebic activity against HM1: IMSS strain of E.
histolytica by
a
microdilution method.15 E. histolytica
trophozoites were cultured in a 96-well microtiter plate
suspended in Diamond TYIS-33 growth medium.33 The test
compounds (1 mg) were dissolved in DMSO (40 L,
concentration at which no inhibition of amoeba growth
occurred).34,35 The stock solutions (1 mg/mL) of the
compounds were freshly prepared and two-fold 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
4.4. Inhibition of β-haematin formation assay
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: 25 μL of the test compound, 25
μL of a 1 mg/mL haemin (Sigma) solubilised in DMSO, 50 μL
H2O and finally 100 μl 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 μL of the 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 μL was removed and
substituted with a 2M NaOH solution to dissolve the β-
haematin crystals. The solution was diluted twofold and the
absorbance read at 405 nm.17 From the data obtained the
concentration at which β-haematin formation was inhibited by
50% (IC50 value) was determined using GraphPad Prism® 5.0
software. Each experiment was repeated at least in triplicate.
and repeated thrice. The amoeba suspension was prepared from
o
a confluent culture by pouring off the medium at 37 C and
adding 5 mL of fresh medium, chilling the culture tube on ice
to detach the organisms from the side of 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 mL in fresh
medium and 170 L of this suspension was added to the test
and control wells in the plate such that an inoculum of 1.7 
104 organisms/well was achieved to ensure confluency, but no
excessive growth in the control wells. Plates were sealed and
o
gassed for 10 minutes 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
o
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 (-20 oC) 100%
methanol and then dried, stained with 0.5% aqueous eosin for
15 minutes. The stained plate was washed three times with
distilled water and allowed to dry before 200 L 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.
4.5. MTT cell viability assay
A human colon adenocarcinoma (HT29) cell line was cultured
and maintained as a monolayer in Dulbecco‟s modified Eagle‟s
medium (DMEM, Sigma) supplemented with 10% fetal calf
serum (Sigma) and antibiotics (100 IU/mL of penicillin and
100 μg/mL of streptomycin, Sigma). All cells were cultured at
37 °C in a humid atmosphere and 5% CO2.18 Exponentially
growing viable cells were plated at 0.15 x 106 cells per well
into 96-well plates and incubated along with the test and
control compounds. Stock solutions of compounds were
initially dissolved in DMSO and all compounds screened at
100 µM, where the final 1% DMSO in the well did not
adversely affect cell growth. The growth-inhibitory effects of
the compounds and positive control camptothecin were
measured using the tetrazolium MTT assay. After 46 h of
4.3. In vitro antimalarial assay
o
incubation at 37 C, the medium was removed and 20 µL of
Antimalarial activity of the compounds, against the
chloroquine-resistant (FCR-3) strain of P. falciparum, was
performed using the [3H]-hypoxanthine incorporation assay.16
The FCR-3 strain was continuously maintained in vitro in
supplemented RPMI-1640 culture media and at a haematocrit
of 5%. The culture was incubated at 37 ºC in a gaseous
atmosphere of 5% CO2, 3% O2, 92% N2 and synchronized at
the ring stage with 5% D-sorbitol before being adjusted to a
final parasitemia of 0.5% and haematocrit of 1%.17 This
suspension (200 µL) was added to each well of the 96-well
plate with the exception of four wells which received non-
parasitized red blood cells. Stock solutions of the test
compounds were made up in DMSO and serially diluted in
culture medium in a 96-well microtiter plate.16 The microtiter
plate was then incubated for 24 h. Following the incubation
period, 25 μL of the radiolabeled [3H]-hypoxanthine isotope
(Amersham) at a concentration of 1.85 μCi/well was added to
MTT (5 mg/mL in phosphate buffered saline (pH 7.4)) was
added to each well. The plates were incubated at 37 C for 2 h.
o
At the end of the incubation period, the medium was removed
and 150 µL DMSO added to all wells. The metabolised MTT
product dissolved in DMSO was quantified by reading the
absorbance at 540 nm with a reference wavelength of 690 nm.
All assays were performed at least in triplicate. Percent cell
viability of the treated cells was calculated as a percentage of
the untreated control cells taking into account the reference
wavelength and cell free controls.
4.6. Red blood cell toxicity assay
The haemolytic activities of the compounds were evaluated in
comparison to the standard antimalarial agent, quinine.19
A
suspension of fresh human red blood cells was adjusted to a
1% haematocrit in culture media and plated together with each

38
test compound (100 µM). This suspension was incubated for
48 h at 37 oC before the absorbance was read at 414 nm. The %
haemolysis was calculated using a 2.0% (v/v) Triton X100
solution as the 100% haemolytic control. These results were
used to generate a log sigmoid dose-response curve to calculate
IC50 values and the mean ± s.d. were calculated from at least
triplicate values.
minimization in Swiss PDB viewer.
The goodness of the
predicted PfDHPS model was then assessed using
PROCHECK and VERIFY3D. Further, in order to assess the
reliability of the modelled structure of PfDHPS, we calculated
the root mean square deviation (RMSD) by superimposing it
on the known template structure.
4.9. Molecular docking
4.7. X-ray Crystal Structure determination
To determine the key residues that interact with the
sulfonamide active site of the PfDHPS, the biomolecular
interactions between the inhibitors (F7 and F8) and modeled
PfDHPS was analyzed using Autodock 4.2.30 Active site was
obtained by structural superimposition of modeled PfDHPS
with that of E. coli DHPS (Pdb id: 1AJO). The crystal structure
1AJO was used for structural imposition as it contains
sulfonamide bound in the active site. Docking simulations
were performed using Lamarckian Genetic algorithm (LGA).
The grid maps representing the ligand were calculated with
Autogrid. The dimensions of the grid were 60 x 60 x 60 grid
points with a spacing of 0.375 Å between the grid points and
centered on the ligand (41.92, 8.09 and 1.876 coordinates). In
the present study docking was performed by creating an initial
population of 150 individuals, maximum number of evaluation
2500000, maximum number of generations 27000, rate of gene
mutation 0.02, cross-over rate 0.8 and the remaining
parameters were set as default. 10 docking conformations
(poses) were generated and the best docked conformation was
selected based on the Autodock binding energy, for further
analysis. Finally, Ligplot31 was used to map the hydrogen and
hydrophobic interaction of the docked inhibitor to the modeled
structure.
Three-dimensional X-ray data for F7 was collected on a Bruker
SMART Apex CCD diffractometer at 100(2) K, using a
graphite monochromator and Mo-K radiation ( = 0.71073
Å) by the -ω scan method. Reflections were measured from a
hemisphere of data collected of frames each covering 0.3
degrees in ω. Of the 20403 reflections measured in F7, all of
which were corrected for Lorentz and polarization effects, and
for absorption by semi-empirical methods based on symmetry-
equivalent and repeated reflections, 2244 independent
reflections exceeded the significance level F/(F) > 4.0.
Complex scattering factors were taken from the program
package SHELXTL36 The structures were solved by direct
methods and refined by full-matrix least-squares methods on
F2. The non-hydrogen atoms were refined with anisotropic
thermal parameters in all cases. The hydrogen atoms were
located in difference Fourier map and freely refined. A final
difference Fourier map showed no residual density outside:
0.533 and -0.392 e.Å-3 for F7. A weighting scheme w =
1/[σ2(Fo2) + (0.061900P)2 + 0.497700P] for F7, where P =
(|Fo|2 + 2|Fc|2)/3, were used in the latter stages of refinement.
CCDC 911483 contains the supplementary crystallographic
data for the structure reported in this paper. These data can be
Acknowledgments
obtained
free
of
charge
via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: (+44) 1223 336 033; or e-mail:
deposit@ccdc.cam.ac.uk.
This work was supported by Council of Scientific and
Industrial Research (Grant # 01(2278)/08/EMR-II New Delhi,
India) and the Faculty of Health Sciences of the University of
the Witwatersrand, South Africa.
4.8. Homology modelling of Plasmodium falciparum
dihydroperoate synthase (PfDHPS)
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