Synthesis and biological screening of some pyridine derivatives as anti-malarial agents

Article abstract of DOI:10.3109/14756366.2011.575071

Two series of pyridine derivatives were synthesised and evaluated for their in vivo anti-malarial activity against Plasmodium berghei. The anti-malarial activity was determined in vivo by applying 4-day standard suppressive test using chloroquine (CQ)-sen

Full text of DOI:10.3109/14756366.2011.575071

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(1): 69–77
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.575071
RESEARCH ARTICLE
Synthesis and biological screening of some pyridine
derivatives as anti-malarial agents
Adnan A. Bekhit1,2, Ariaya Hymete2, Ashenafi Damtew2,3, Abdel Maaboud I. Mohamed2,4, and
Alaa El-Din A. Bekhit5
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt,
2Department of Pharmaceutical Chemistry, School of Pharmacy, Addis Ababa University, Addis Ababa, Ethiopia,
3Department of Pharmacy, Central University College, Addis Ababa, Ethiopia, 4Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, University of Assiut, Assiut, Egypt, and 5Food Sciences, University of Otago, Dunedin,
New Zealand
Abstract
Two series of pyridine derivatives were synthesised and evaluated for their in vivo anti-malarial activity against
Plasmodium berghei. The anti-malarial activity was determined in vivo by applying 4-day standard suppressive test
using chloroquine (CQ)-sensitive P. berghei ANKA strain–infected mice. Compounds 2a, 2g and 2h showed inhibition
of the parasite multiplication by 90, 91 and 80%, respectively, at a dose level of 50 µmol/kg. Moreover, The most active
compounds (2a, 2g and 2h) were tested in vitro against CQ-resistant Plasmodium falciparum RKL9 strains where
compound 2g showed promising activity with IC50 =0.0402 µM. The compounds were non-toxic at 300 and 100mg/
kg through the oral and parenteral routes, respectively. The docking pose of the most active compounds (2a, 2g and
2h) in the active site of dihydrofolate reductase enzyme revealed several hydrogen and hydrophobic interactions
that contribute to the observed anti-malarial activities.
Keywords: Pyridine derivatives, in vivo, anti-malarial, docking, acute toxicity, dihydrofolate reductase
Introduction
With the continuous resistance to commonly used anti-
malarial drugs is spreading, the search for new effective
anti-malarial drugs is an urgent and a necessity in large
parts of the world to contain and control the disease.
A number of scaffolds, including the pyridine nucleus,
showed anti-malarial activities. us, in an effort to dis-
cover novel anti-malarial agents, new compounds were
designed in the present work by incorporating some
pharmacophoric assemblies to the pyridine nucleus.
e pyridine derivative 4-isonicotinic hydrazide,
which is a leading drug in the treatment of tuberculosis,
is an inhibitor of enoyl-ACP reductase, an important
enzyme in the fatty acid biosynthesis. us, pyridine
analogues may inhibit the biosynthesis of fatty acids that
are fundamental for the survival of P. falciparum in the
host3. Compounds that act on more than one target site
are more liable to be active.
Malaria has infected humans and may have been a
human pathogen for the entire history of the mankind1.
ere are a limited number of drugs that can be used
to treat or prevent malaria, but increased resistance to
these drugs is of concern to health practitioners. is
resistance appears to occur through spontaneous muta-
tions that confer reduced sensitivity to a given drug or
class of drugs. Newer anti-malarials were discovered in
an effort to tackle this problem, but all these drugs are
either expensive or have undesirable side effects.
Even the new generation of anti-malaria drugs appears
to be less effective after a variable length of time due to
the development of resistance in the parasites, especially
the Plasmodium falciparum species. Recently, scientists
reportedthefirstevidenceofresistancetotheworld’smost
effective drug coartem in western Cambodian subjects2.
Address for Correspondence: Dr. Adnan A. Bekhit, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria,
Alexandria 21521, Egypt. Tel.: +20 3 4871317. Fax: +20 3 4873273. E-mail: adnbekhit@hotmail.com, adnbekhit@alexpharmacy.edu.eg
(Received 23 June 2010; revised 07 March 2011; accepted 22 March 2011)
69

70 A.A. Bekhit et al.
Several lead compounds containing a pyridine moi-
ety attached to various cyclic and/or acyclic moieties
have been reported (Figure 1). Pyridine derivatives I,
II4,5, adamantylthiopyridine III6, benzoylpyridinethi-
osemicarbazonesIV7 anddichloropyridinederivativeV8
showed pronounced anti-malarial activity (Figure 1).
Many of the clinically relevant anti-malarial agents
that have a known mechanism of action directly or
indirectly affect pyrimidine metabolism. Drugs target-
ing dihydrofolate reductase (DHFR) or dihydropteroate
synthetase (e.g., pyrimethamine, cycloguanil, sulpho-
namides and sulphones) disrupt folate metabolism,
which is essential for the formation of thymidine9. Also
the lactate dehydrogenase enzyme of P. falciparum has
the ability to rapidly use 3-acetyl pyridine NAD as a
coenzyme in the reaction leading to the formation of
pyruvate from lactate10.
In the present study, various substituents at different
positions on the pyridine nucleus were introduced to
study the effect of such molecular variation on the anti-
malarial activity. In addition to the targeted anti-malarial
activity, docking pose of the most active compounds (2a,
2g and 2h) in the active site of DHFR enzyme and their
acute toxicity profile were obtained.
Splitting patterns were designated as follows: s: singlet; d:
doublet; m: multiplet. Elemental analyses were performed
on Perkin-Elmer 2400 elemental analyzer and were found
within 0.4% of the theoretical values. Follow up of the reac-
tionsandcheckingthepurityofthecompoundswasmadeby
thin layer chromatography on silica gel–pre-coated alumin-
ium sheets (Type 60 GF254; Merck, Darmstadt, Germany)
and the spots were detected by exposure to UV-lamp at
λ=254nm and/or iodine chamber. Parasites were counted
using BIO-PLUS microscope. All the chemicals were from
Sigma Chemical Co. (St. Louis, MO) and the solvents were
from AnalaR BDH (UK) unless otherwise stated.
Synthesis
Nʹ-arylideneisonicotinohydrazide (2a–h)
A mixture of selected aldehyde (10mM), isonicotinic
hydrazide 1 (1.37g, 10mM) and one drop HCl in ethanol
(50mL) was heated under reflux for 6–8h. e reaction
mixture was allowed to cool and the precipitate formed was
filtered, washed with ethanol, dried and crystallized from
ethanol–water mixture (7:3) (Scheme 1, Tables 1 and 2).
4-Aryl-1,2-dihydro-2-oxo-6-phenylpyridine-3-carbonitrile
(4a–e)
A mixture of acetophenone 3 (1.20 g, 10 mM), ethyl
cyanoacetate (1.13 g, 10 mM), the appropriate aldehyde
(10 mM) and ammonium acetate (6.16 g, 80 mM) in etha-
nol (50 mL) was heated under reflux for 6h. e reaction
mixture was cooled and the formed precipitate was fil-
tered, washed with ethanol, then washed successively
with water, dried and crystallized from ethanol–water
mixture (7:4) (Tables 1 and 2).
Material and methods
Melting points were determined in open glass capillar-
ies using an electro thermal BUCHI (B-540) melting point
apparatus and are uncorrected. Infra red (IR) spectra were
recorded on Perkin-Elmer (Postfach, Switzerland) 1430 IR
spectrophotometer (Waltham, MA) using the KBr disc tech-
1
nique. H NMR spectra was recorded on a Bruker Avance
DMX400 FT-NMR spectrometer (Rheinstetten, Germany)
and the chemical shifts are given in δ(ppm) downfield from
tetramethylsilane, which served as an internal standard.
Ethyl 2-cyano-3-(4-(dimethylamino)phenyl)acrylate (5)
is compound was obtained as an unexpected product
applying method used to prepare compounds 4a–e using
4-(dimethylamino)benzaldehyde A. To ensure the struc-
ture, this compound was synthesized by heating under
reflux a mixture of 4-(dimethylamino)benzaldehyde A
(1.49 g, 10 mM), ethyl cyanoacetate (1.13 g, 10 mM) and
ammonium acetate (1.54 g, 20 mL) in ethanol (50 mL) for
5 h. e reaction mixture was concentrated, cooled and
the formed precipitate was filtered, washed with ethanol,
then washed successively with water, dried and crystal-
lized from ethanol–water mixture (4:1) (Tables 1 and 2).
O
HO
O
O
O
H
N
N
N
N
NH
Br
N
O
N
S
N
I
N
II
III
In vivo anti-malarial activity
In vivo anti-malarial activity test of the synthesized com-
pounds was performed using a 4-day standard suppres-
sive test11 with some modifications. is is the most widely
used preliminary test, in which the efficacy of a compound
is assessed by the comparison of blood parasitaemia and
mouse survival times in treated and untreated mice12.
On day 0, the test mice were injected with 0.2ml of
2×107 parasitised erythrocytes, (P. berghei ANKA strain)
intra venously. After 2h, the infected mice were weighed
and randomly divided into 15 groups of six mice each per
cage. Groups 1–13 received the synthesized compounds
N
N
Cl
Cl
N
H
OCF3
N
CF3SO3H
N
CH2CH3
S
NH2
V
IV
Figure 1. Chemical structures of pyridine derivatives having anti-
malarial activity.
Journal of Enzyme Inhibition and Medicinal Chemistry

Pyridine derivatives as anti-malarial agents 71
R
O
O
NH2
N
N
H
N
H
R-CHO
N
N
2
1
R =
F
NO2
OH
F
OH
a
F
OCH3
g
N
f
b
h
c
d
e
Scheme 1. Synthesis of hydrazone derivatives.
at 50 µmol/kg dose levels and served as treatment group13.
Group 14 received the vehicle (7% Tween, 3% ethanol in
water) and served as a negative control. Group 15 received
the standard drug Choloroquine diphosphate (CQ, Sigma-
Aldrich, Deisenhofen, Germany) at the same dose level
(50 µmol/kg) and served as a positive control14.
On days 1–3, animals in the experimental groups were
treated again (with the same dose of the synthesized
compounds and same route daily) as in day 0. On day
4 (i.e. 24 hr after the last dose or 96 hr post-infection),
blood smear from all test animals was prepared using
Giemsa stain. Level of parasitaemia was determined
microscopically by counting 4 fields of approximately 100
erythrocytes per field. e difference between the mean
value for the control group (taken as 100%) and those of
the experimental groups was calculated and expressed as
percent reduction or activity.
Table 1. Physical and analytical data of compounds 2a–h, 4a–e
and 5.
Melting point
Compound
Yield %
81
(°C)
Molecular formula (MW)
C13H11N3O2 (241.08)
C13H10FN3O (243.23)
C13H10FN3O (243.23)
C13H10FN3O (243.23)
C13H10N4O3 (270.08)
C15H16N4O (268.13)
C14H13N3O3 (271.09)
C15H13N3O (251.10)
C18H11FN2O (290.29)
C18H11FN2O (290.29)
C18H11FN2O (290.29)
C18H11N3O3 (317.08)
C20H14N2O (298.11)
C14H16N2O2 (244.28)
2a
2b
2c
2d
2e
2f
245–250
258–259
251–252
260–261
225–227
237–239
228–230
208–210
272–273
281–282
267–268
280–281
271–272
127–128
84
85
82
86
78
2g
2h
4a
4b
4c
4d
4e
5
87
58
84
89
87
89
61
Untreated control mice typically die approximately 1
week after infection15. For treated mice the survival time
(in days) was recorded and the mean survival time was
calculated in comparison with that of the negative con-
trol group16.
76
described by Trager and Jensen12. Parasites were cul-
tured in human AB (+) erythrocytes in RPMI-1640 media
(GIBCOBRL, Paisely, Scotland) supplemented with
25 mM HEPES buffer, 10% human AB (+) serum and 0.2%
sodium bicarbonate (Sigma, St. Louis, MO) and main-
tained at 5% CO2. Cultures containing predominantly
early ring stages were synchronized by addition of 5%
d-sorbitol (Sigma) lysis17 and used for testing. Initial cul-
ture was maintained in small vials with 10% haematocrit,
i.e., 10 mL erythrocytes containing 1.0% ring stage para-
site in 100 µl complete media. e culture volume per
well for the assay was 100 mL. Number of parasites for the
assay was adjusted at 1–1.5% by diluting with fresh human
AB (+) RBC. Assay was done in 96-well microtitre flat-
bottomed tissue culture plates incubated at 37°C for 24 h
in presence of three-fold serial dilutions of compounds
Percentage parasitaemia and suppression were calcu-
lated using the following formulae:
No. of infected RBC
%Parasitaemia =
%Suppression =
×100
No. of total RBC
Parasitaemia in negative control −
Parasitaemia in study group
×100
Parasitaemia in negative control
In vitro anti-malarial assay
P. falciparum strains RKL9 (CQ resistant) was maintained
in a continuous culture using the standard method
© 2012 Informa UK, Ltd.

72 A.A. Bekhit et al.
Table 2. Spectral data of compounds 2a–h, 4a–e and 5.
Compound IR (KBr, cm−1)
1H NMR (DMSO-d6)
2a
3522 (OH), 3207 (NH), 1668 (C=O), 6.91 (d, 2H, J=8.80 Hz, phenyl–C2,6 H), 7.75 (d, 2H, J=8.80 Hz, phenyl–C3,5H), 7.95
1652 (C=N)
(d, 2H, J=5.78 Hz, pyridine–C-βH), 8.37 (s, 1H, CH=N), 8.82 (d, 2H, J=5.78 Hz,
pyridine–C-αH).
2b
2c
2d
3218 (NH), 1669 (C=O), 1654 (C=N)
3220 (NH), 1666 (C=O), 1655 (C=N)
7.21–7.62 (m, 4H, phenyl–H), 7.93 (d, 2H, J=5.78 Hz, pyridine–C-βH), 8.32 (s, 1H,
CH=N), 8.81 (d, 2H, J=5.78 Hz, pyridine–C-αH).
7.01–7.72 (m, 4H, phenyl–H), 7.89 (d, 2H, J=5.78 Hz, pyridine–C-βH), 8.40 (s, 1H,
CH=N), 8.84 (d, 2H, J=5.78 Hz, pyridine–C-αH).
3215 (NH), 1667 (C=O), 1653 (C=N) 7.18 (d, 2H, J=8.80 Hz, phenyl–C3,5H), 7.72(d, 2H, J=8.80 Hz, phenyl–C2,6H), 7.91
(d, 2H, J=5.78 Hz, pyridine–C-βH), 8.39 (s, 1H, CH=N), 8.81 (d, 2H, J=5.78 Hz,
pyridine–C-αH).
2e
2f
3209 (NH), 1670 (C=O), 1656 (C=N),
1522, 1305 (NO2)
7.72–8.11 (m, 3H, phenyl–C4,5,6H), 8.22 (d, 2H, J=5.78 Hz, pyridine–C-βH), 8.39 (s, 1H,
CH=N), 8.85 (d, 2H, J=5.78 Hz, pyridine–C-αH), 8.90 (s, 1H, phenyl–C3H).
3219 (NH), 1664 (C=O), 1656 (C=N) 2.16 (s, 6H, N–CH3)2), 6.52(d, 2H, J=8.80 Hz, phenyl–C3,5H), 6.73(d, 2H, J=8.80 Hz,
phenyl–C2,6H), 7.93 (d, 2H, J=5.78 Hz, pyridine–C-βH), 8.21 (s, 1H, CH=N), 8.82 (d,
2H, J=5.78 Hz, pyridine–C-αH).
2g
3542 (OH), 3227 (NH), 1665 (C=O), 3.81 (s, 3H,−OCH3), 6.90 (d, 1H, J=8.42 Hz, phenyl–C5H), 7.15 (d, 1H, J=8.42 Hz,
1657 (C=N)
phenyl–C6H), 7.41(s, 1H, phenyl–C2H), 8.15 (d, 2H, J=5.78 Hz, pyridine–C-βH), 8.42
(s, 1H, CH=N), 8.91 (d, 2H, J=5.78 Hz, pyridine–C-αH).
2h
4a
4b
4c
4d
4e
3124 (NH), 1662 (C=O), 1651 (C=N) 6.95 (m, 2H, phenyl–CH=CH–), 7.32–7.61 (m, 5H, phenyl–H), 8.23 (d, 1H, CH=N),
8.38 (d, 2H, J=5.78 Hz, pyridine–C-βH), 8.89 (d, 2H, J=5.78 Hz, pyridine–C-αH).
3284 (NH), 2220 (CN), 1654 (C=O)
3278 (NH), 2215 (CN), 1652 (C=O)
3274 (NH), 2217 (CN), 1649 (C=O)
3280 (NH), 2220 (CN), 1656 (C=O)
3279 (NH), 2219 (CN), 1655 (C=O)
7.09 (s, 1H, pyridine–C5H), 7.28–7.65 (m, 9H, phenyl–H), 12.87 (br s, 1H, NH, D2O
exchangeable).
7.13 (s, 1H, pyridine–C5H), 7.19–7.86 (m, 9H, phenyl–H), 12.88 (br s, 1H, NH, D2O
exchangeable).
6.92 (d, 2H, J=8.80 Hz, flourophenyl–C2,6H), 7.11 (s, 1H, pyridine–C5H), 7.36–7.78 (m,
flourophenyl–C3,5 H & phenyl–H), 12.91 (br s, 1H, NH, D2O exchangeable).
7.10 (s, 1H, pyridine–C5H), 7.22–7.94 (m, 8H, nitrophenyl–C4,5,6H & phenyl–H), 8.65
(d, 1H, J=8.64 Hz, nitrophenyl–C3 H), 12.88 (br s, 1H, NH, D2O exchangeable).
6.67 (d, 1H, J=5.22 Hz, Phenyl–CH=CH–),7.12 (s, 1H, pyridine–C5 H), 1.18 (d, 1H,
J=5.22 Hz, Phenyl–CH=CH–), 7.20–8.02 (m, 10H, phenyl–H), 12.89 (br s, 1H, NH, D2O
exchangeable).
5
2214 (CN); 1704 (C=O); 1256, 1123
(C–O–C)
1.24 (t, 3H, CH3), 3.15 (s, 6H,−N(CH3)2), 4.35 (m, 2H, CH2), 6.71 (d, 2H, J=8.75 Hz,
phenyl C2,6H), 7.92 (d, 2H, J=8.75 Hz, phenyl C3,5H), 8.12 (s, 1H,−C=CH).
and CQ diphosphate for their effect on schizont matura-
tion. Test compounds were dissolved in ethanol and fur-
ther diluted with RPMI-1640 medium (the final ethanol
concentration did not exceed 0.5%, which did not affect
parasite growth). CQ diphosphate was dissolved in aque-
ous medium. Test was done in duplicate wells for each
dose of the drugs. Solvent control culture containing the
same concentrations of the solvent as present in the test
wells was done with RPMI-1640 containing 10% AB (+)
serum.
Parasite growth was found to be unaffected by the
solvent concentrations used in the test. Growth of the
parasites from duplicate wells of each concentration was
monitored in Giemsa-stained blood smears by counting
number of schizont per 100 asexual parasites. Percent
schizont maturation inhibition was calculated by the
formula: (1 − Nt/Nc) × 100 where, Nt and Nc represent
the number of schizont in the test and control wells,
respectively.
each, Medical Research Institute, Alexandria University)
according to previously reported methods. e animals
weredividedintogroupsofsixmiceeach. ecompounds
were given orally, suspended in 1% gum acacia, in doses
of 1, 10, 100, 200, 250, 300 mg/kg. e mortality percent-
age in each group was recorded after 24 h18. Additionally
the test compounds were investigated for their parenteral
acute toxicity in groups of mice of six animals each. e
compounds or their vehicle, propylene glycol (control),
were given by intra-peritoneal injection in doses of 10,
25, 50, 75, 100 mg/kg. e percentage survival was fol-
lowed up to 7 days19.
Docking
e co-ordinate from the X-ray crystal structure of
dehydrofolate reductase (DHFR) enzyme used in this
simulation was obtained from the Protein Data Bank
(PDB ID: 1J3I), where the selective DHFR inhibitor
WR99210 is bound to the active site. e ligand mol-
ecules were constructed using the builder module and
were energy minimized. e active site of DHFR was
generated using the MOE-Alpha Site Finder, Molecular
Operating Environment’s (MOE-Dock 2005) module to
Acute toxicity
e oral acute toxicity of the most active compounds
2a, 2g and 2h was investigated using male mice (20 g
Journal of Enzyme Inhibition and Medicinal Chemistry

Pyridine derivatives as anti-malarial agents 73
rationalize the observed biological results in the in vivo
study20. en ligands were docked within this active
site using the MOE-Dock. e lowest energy conforma-
tion was selected and the ligand interactions (hydro-
gen bonding and hydrophobic interaction) with DHFR
were determined.
structure. In contrast, one pot reaction with p-dimeth-
ylaminobenzaldehyde, ethyl cyanoacetate, ammonium
acetate and acetophenone did not produce the target
compound. is may be attributed to the weak electro-
philic property of p-dimethylbenzaldehyde, due to the
electron donating effect of the p-dimethylamino group.
Trials to obtain the target compound 1,2-dihydro-4-
(4-dimethylaminophenyl)-2-oxo-6-phenylpyridine-3-
carbonitrile by preparing the α,β-unsaturated ketone
via condensation of acetophenone and p-dimethyl-
benzaldehyde in the presence of KOH, then cyclisa-
tion by ethyl cyanoacetate and ammonium acetate
went in vain. e results obtained from elemental and
spectral analyses clearly demonstrated that the com-
pound obtained was not the targeted one, instead ethyl
2-cyano-3-(4-(dimethylamino)phenyl)acrylate 5 was
obtained. To ensure the structure of compound 5, this
compound was further synthesized by the reaction of
4-(dimethylamino)benzaldehyde A, ethyl cyanoacetate
and ammonium acetate in ethanol. It is worth men-
tioning that the presence of ester functional group in
Results and discussion
Chemistry
e target compounds were synthesized according to the
steps outlined in Schemes 1 and 2. Condensation of the
appropriate aldehyde derivatives a–h with isonicotinic
hydrazide 1 in ethanol afforded the corresponding hydra-
zones 2a–h. e structures of these compounds were
confirmed by IR spectra that showed lack of the absorp-
tion bands characteristic for NH2 group. e absence of
this primary amine in the IR spectrum and aldehydic
proton in the 1H NMR spectrum confirmed that the target
compounds were formed.
One of the synthetic procedures applied in the
present work (Scheme 2) is regarded to be a multi-
component reaction21,22. Applying one pot reaction, the
selected aldehyde, ethyl cyanoacetate, acetophenone 3
and ammonium acetate in ethanol were heated under
reflux to obtain compound 4a–e. e presence of amidic
carbonyl group in the IR spectrum and the absence of
1
the IR spectra and the number of protons from the H
NMR spectral integration were exactly the same in the
structure of the above unexpected compound and the
targeted compound.
In vivo anti-malarial activity
Table 3 shows the effect of the synthesised compounds
against P. berghei infected mice. Compounds 2a, 2b, 2c,
1
aldehydic proton in the H NMR spectrum showed that
the product obtained is in agreement with the proposed
O
NC
R1CHO/NCCH2COOC2H5
NH
R
CH3COONH4
4a-e
COCH3
CN
O
A/NCCH2COOC2H5
CH3COONH4
O
3
5
CHO
N
NCCH2COOC2H5
CH3COONH4
N
A
R1
=
F
NO2
F
F
a
b
d
e
c
Scheme 2. Synthesis of pyridone derivatives.
© 2012 Informa UK, Ltd.

74 A.A. Bekhit et al.
2d, 2f, 2g, 2h and 4c showed more than 50% suppression
of parasitaemia compared to the control and CQ treated
groups. Compounds 2a, 2g and 2h are the most active
compounds in this study with 90, 91 and 80% suppres-
sion, respectively. e only cyanopyridine derivative that
showed promising activity was 4c. In general, isonicoti-
nohydrazide derivatives were more active than cyano-
pyridine derivatives.
e in vivo biological activities of some of the syn-
thesised isonicotinohydrazide derivatives showed
encouraging results against P. berghei. Substitution of
phenyl ring at the para-position with hydroxyl, flouro
or methoxy group (2a, 2d and 2g) showed remarkable
activity, which might be due the formation of hydrogen
bonding with backbone of the receptor. Compound 2d
showed promising activity. Moreover, compound hav-
ing styryl moiety 2h showed good activity but still less
than 2a and 2g. e presence of dimethylamino group
at the para-position of the phenyl ring might decrease
the activity of compound 2f. When the phenyl ring was
substituted at para-position with hydroxyl group 2a, the
activity was higher than that of compound 2e, which has
nitro group at the para-position of the phenyl ring. is
could be speculated to be due to the large nitro group
that may not be able to interact with the backbone of the
receptor.
interactions with Ile 14, Cys 15, Ala 16, Asp 54, Met 55,
Phe 58 and Pro 113.
Figures 3–5 show the binding interactions of com-
pounds 2a, 2g and 2h to the active site of DHFR, respec-
tively, where they exhibited some similar interactions to
WR99210. Compound 2a displayed hydrogen bond inter-
actions with Phe 116, in addition to hydrophobic interac-
tions with Ala 16, Leu 46, Asp 54, Met 55, Phe 58, Phe 116
and Arg 122. On the other hand, compound 2g showed
hydrogen bond interactions with Phe 116 and Ser 120, in
addition to hydrophobic interactions with Ser 111, Ile 112,
Phe116,Leu119,Ser120andArg122.However,compound
2h displayed hydrogen bond interactions with Ser 111 in
addition to hydrophobic interactions with Gly 44, Val 45,
Leu 46, Met 55, Ser 108, Ser 111, Phe 116 and Leu 119. e
higher activity of compounds 2a and 2g over compound
2h may be attributed to the arene–cation interaction
between hydroxyphenyl group and Arg 122. e hydrogen
bonds profile and their scores of the active compounds 2a,
2g and 2h and standard compound WR99210 are listed in
Table 5. Although all compounds showed hydrogen bond
interactions with the backbone of the enzyme, the residue
of interaction may be different. Both compounds 2a, 2g
interact with Phe 116 with hydrogen donor bond, but com-
pound 2g had additional hydrogen acceptor bond with Ser
120 of excellent score. e standard compound WR99210
showed three hydrogen donor bonds with backbone of
the enzyme, those with Ile 14 and Ile 164 are of weak score
while that with Asp 54 of medium score.
In vitro anti-plasmodial activity
On testing the in vitro anti-plasmodial activity (Table 4),
compounds 2a, 2g and 2h showed better activity than
CQ diphosphate (IC50 = 0.188 µM) against CQ-resistant
(RKL9) strain of parasite. Compound 2g was found to
be the most potent against RKL9 (IC50 = 0.0402 µM). is
may be attributed to the OH group at the para-position,
which is capable of forming hydrogen bonding with the
backbone of the receptor.
Table 3. In vivo anti-malarial activity of compounds 2a–h and
4a–e.
%
%
Compound Parasitaemia Suppression Mean survival time (days)
2a
2b
2c
2d
2e
2f
2g
2h
4a
4b
4c
4d
8
0.8
90
58
61
74
1
11.5 0.6
8.2 1.6
8.6 1.9
9.1 1.2
2.4 0.8
9.22 1.4
9.8 0.5
9.4 1.0
7.4 1.1
6.8 1.8
8.8 1.4
4.2 2.4
7.1 1.5
6.3 0.5
ND
34 1.2
32 1.6
22 0.4
81 2.4
31 1.0
15 0.6
17 1.3
46 3.4
52 2.8
31 3.8
64 1.8
47 2.8
82 1.6
0.0
In vivo acute toxicity test
63
91
80
44
37
63
22
43
0.0
100
Compounds 2a, 2g and 2h were further evaluated for
their oral acute toxicity in male mice using a previously
reported method18,19. e results indicated that the test
compounds proved to be non-toxic and well tolerated by
experimental animals up to 300 mg/kg. Moreover, these
compounds were tested for their toxicity through the
parenteral route. e results revealed that all test com-
pounds were non-toxic up to 100 mg/kg.
4e
Control
Chloroquine
Docking
Molecular docking studies further helps in understand-
ing the various interactions between the ligands and
enzyme active sites in detail. e determination of the 3D
co-crystal structure of DHFR (PDB ID: 1J3I), complexed
with a selective inhibitor, WR99210 (Figure 2), has led
to the development of a model for the topography of the
anti-malarial drugs binding site in DHFR enzyme. is
compound displayed hydrogen bond interactions with
Ile 14, Asp 54 and Ile 164 in addition to hydrophobic
Table 4. In vitro anti-plasmodial activity against chloroquine-
resistant (RKL9) strain of Plasmodium falciparum.
Compound
IC50, µM SD*
0.0422 0.004
0.0402 0.005
0.0660 0.012
0.188 0.003
2a
2g
2h
Chloroquine
*Results of two separate determinations.
Journal of Enzyme Inhibition and Medicinal Chemistry

Pyridine derivatives as anti-malarial agents 75
(57%)
(28%)
(18%)
Figure 2. 3D view from a molecular modelling study, of the minimum-energy structure of the complex of WR99210 docked in DHFRE
(PDB ID: 1J3I). Viewed using Molecular Operating Environment (MOE) module.
ILE_112
PHE_116
(12%)
LEU_119
LEU_46
SER_120
ILE_164
MET_55
PHE_58
Figure 3. 3D view from a molecular modelling study, of the minimum-energy structure of the complex of 2a docked in DHFRE (PDB ID:
1J3I). Viewed using Molecular Operating Environment (MOE) module.
© 2012 Informa UK, Ltd.

76 A.A. Bekhit et al.
SER_120
LEU_119
(14%)
PHE_116
(90%)
ILE_164
ILE_112
PRO_113
PHE_58
LEU_46
MET_55
Figure 4. 3D view from a molecular modelling study, of the minimum-energy structure of the complex of 2g docked in DHFRE (PDB ID:
1J3I). Viewed using Molecular Operating Environment (MOE) module.
PRO_113
ILE_112
(77%)
PHE_116
LEU_46
LEU_119
ILE_164
MET_55
PHE_58
Figure 5. 3D view from a molecular modelling study, of the minimum-energy structure of the complex of 2h docked in DHFRE (PDB ID:
1J3I). Viewed using Molecular Operating Environment (MOE) module.
Journal of Enzyme Inhibition and Medicinal Chemistry

Pyridine derivatives as anti-malarial agents 77
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compounds WR99210, 2a, 2g and 2h.
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Compound
Residue
Ile 14
Type
Score (%) Distance
WR99210
H-don
H-don
H-don
H-don
H-don
H-acc
H-don
28.2
57.1
17.8
72.3
13.6
90.2
76.7
1.98
1.87
2.20
2.17
2.02
2.44
1.67
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2g
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Acknowledgements
e authors wish to thank the Egyptian Fund for
Technical Cooperation with Africa, Ministry of Foreign
Affairs, Egypt, for the financial support that enabled
Prof. Adnan Bekhit and Prof. Abdel Maaboud Mohamed
to be available in School of Pharmacy, Addis Ababa
University.
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Declaration of interest
e authors report no conflicts of interest. e authors
alone are responsible for the content and writing of the
paper.
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