Primaquine-pyrimidine hybrids: Synthesis and dual-stage antiplasmodial activity

Article abstract of DOI:10.1016/j.ejmech.2015.06.045

Abstract A series of novel pyrimidine-primaquine hybrids were synthesized and their effectiveness against the blood and liver stages of malaria parasites was evaluated. The hybrids displayed enhanced liver stage in vitro activity against P. berghei liver

Full text of DOI:10.1016/j.ejmech.2015.06.045

European Journal of Medicinal Chemistry 101 (2015) 266e273
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Primaquineepyrimidine hybrids: Synthesis and dual-stage
antiplasmodial activity
Hardeep Kaur a, Marta Machado d, Carmen de Kock b, Peter Smith b, Kelly Chibale c,
d
a, *
^
Miguel Prudencio , Kamaljit Singh
a Department of Chemistry, UGC-Centre for Advanced Studies-II, Guru Nanak Dev University, Amritsar 143005, India
b Division of Pharmacology, Department of Medicine, University of Cape Town, Observatory 7925, South Africa
c Department of Chemistry, South African Medical Research Council Drug Discovery and Development Research Unit, Institute of Infectious Disease and
Molecular Medicine, University of Cape Town, Rondebosch 701, South Africa
d Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel pyrimidineeprimaquine hybrids were synthesized and their effectiveness against the
blood and liver stages of malaria parasites was evaluated. The hybrids displayed enhanced liver stage
in vitro activity against P. berghei liver stage infection.
Received 21 March 2015
Received in revised form
21 June 2015
Accepted 22 June 2015
Available online 29 June 2015
© 2015 Elsevier Masson SAS. All rights reserved.
Keywords:
Pyrimidine
Primaquine
Hybrid drugs
Antiplasmodial agents
Liver stage activity
Blood stage activity
1. Introduction
sporozoites into their mammalian hosts during the bite of an
infected female Anopheles mosquito. Once in circulation, sporozo-
The magnitude of the devastation caused by malaria disease is
significant, since as many as 250 million clinical cases (approx. 40
percent of human population) and a mortality of 584,000 people
are reported annually [1].
ites make their way to the liver, where they invade hepatocytes.
During the exo-erythrocytic stage of their life cycle, sporozoites
develop and multiply asymptomatically to produce tens of thou-
sands of merozoites. Merozoites are eventually released into the
blood stream, where they cyclically invade, multiply and rupture
red blood cells, causing the symptoms of malaria. During this
erythrocytic stage of their life cycle, some parasites develop into
sexual forms called gametocytes that can be taken up by a mosquito
upon a subsequent blood meal. The sexual phase of Plasmodium
reproduction takes place in the mosquito's midgut and culminates
in the production of thousands of sporozoites, which migrate to the
mosquito's salivary glands, completing the cycle. In the arma-
mentarium of antimalarial drugs, a major thrust is in the devel-
opment of drugs that are efficacious against asexual blood
(erythrocytic) stages of P. falciparum, which are responsible for
illness but represent only a small proportion of the complex life-
cycle of the parasite. In fact, the first obligatory phase of infection
of a mammalian host occurs in the liver, where the parasite
Plasmodium falciparum is responsible for most malaria-
associated mortality worldwide, and it is by far the predominant
species in tropical and sub-tropical countries. Other species that
cause malaria in humans are Plasmoduim vivax, P. ovale, P. malariae
and Plasmodium knowlesi. P. vivax is less lethal than P. falciparum
but, like P. ovale, can remain dormant in hepatic cells for many years
and cause malaria recurrence/relapse. Similarly, P. knowlesi causes
malaria in primates, but of late there are reports of malaria in-
fections in humans also due to this species [2,3] The life cycle of
malaria parasites [4] is initiated with the injection of Plasmodium
* Corresponding author.
E-mail address: kamaljit.chem@gndu.ac.in (K. Singh).
http://dx.doi.org/10.1016/j.ejmech.2015.06.045
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

H. Kaur et al. / European Journal of Medicinal Chemistry 101 (2015) 266e273
267
asymptomatically infects and develops inside hepatocytes [4].
Despite constituting a privileged target for prophylactic interven-
tion, the liver stage of infection is largely understudied and effec-
tive drugs against Plasmodium liver stages are very scarce [5]. This is
particularly relevant in the case of infections by P. vivax and P. ovale,
as these parasite species can form hypnozoites that remain
dormant in liver cells for extended periods may lead to malaria
relapses malaria.
The treatment regimen for malaria infection is complicated by
the emergence of drug resistant parasite strains and resistance to
the classical 4-aminoquinoline based chloroquine (CQ) 1, a one-
time effective blood stage schizonticidal drug [6,7]. The only
highly effective and fast acting treatment effective against all spe-
cies capable of causing human malaria resistant to other agents is
represented by artemisinin (ART) and its derivatives that act on the
asexual blood stages of malaria parasites [8]. They are generally
implicated in the treatment of uncomplicated P. falciparum malaria
as components of ART-based combination therapies (ACTs) in
which the fast-acting ART is combined with another antimalarial
agent with a long terminal half-life [9,10].
On the other hand, the 8-aminoquinoline based primaquine (PQ,
Chart 1) 2 is the only drug that is used as a prophylactic agent
against the clinically silent liver stage schizonts, and can eliminate
hypnozoites, thus avoiding relapse of P. vivax infections [11]. PQ also
acts against gametocytes produced during the life cycle sexual
stages of the parasite and this activity disrupts the transmission of
infection to mosquitoes [11]. Thus, PQ is frequently administered in
combination with a blood-stage schizonticidal drug to achieve a
radical cure of infections with P. vivax or P. ovale and to prevent
relapses due to the development of subsequent blood-stage in-
fections from hypnozoites [12,13]. However, unfortunately, the
reliability of PQ has been questioned due to its possible side effects,
which include methemoglobinemia and hemolytic anemia in
glucose-6-phosphate dehydrogenase-deficient patients [14]. Be-
sides, there are indications of the emergence of resistance to PQ
[15].
Thus, in the struggle to fight malaria, new strategies are needed
that provide cure for all Plasmodium species that infect human red
blood cells, as well as replicative and dormant liver forms.
Hybrid drugs are purported to be the drugs of the future [16,17].
While a vast number of hybrids of chloroquine have been synthe-
sized and evaluated [18e22], there are very few instances of eval-
uation of hybrids of PQ [23e26], for example, the ability of the
primaquine-chloroquine hybrid 3 to inhibit (in vitro and in vivo)
the liver and blood stages of mammalian Plasmodium infection has
been demonstrated. Similarly, hybrids comprising of PQ and ART
are able to inhibit both blood as well as liver stages of the parasite
life cycle [23].
The enzyme dihydrofolate reductase (DHFR) has received
considerable attention as a putative target of pyrimethamine 4 and
other antifolate prophylaxis agents against P. falciparum infection.
DHFR is implicated in the reduction of dihydrofolate to tetrahy-
drofolate (Fig. 1), which upon methylation results in the formation
of N5,N10-methylene tetrahydrofolate coenzyme. The latter acts as a
single carbon unit transfer agent to convert deoxyuridine mono-
phosphate (dUMP) to deoxythymidine monophosphate (dTMP) in
the presence of thymidylate synthase (TS) and is indirectly impli-
cated in the metabolism of amino acids and purine nucleotides [27].
Overall, inhibition of DHFR prevents biosynthesis of DNA leading to
death of the parasite. We have previously reported various types of
hybrids containing pyrimidines and chloroquine pharmacophores,
linked covalently through a variety of basic chains [28]. We have
also evaluated the binding interactions and stoichiometry of
binding with heme and m-oxo heme. In addition these mechanistic
studies have also revealed that these hybrids showed tendency to
bind with AT rich pUC18 DNA and GC rich CT DNA.
Fig. 1. Mode of action of dihydrofolate reductase (DHFR) in the biotransformation of dUMP to dTMP.

268
H. Kaur et al. / European Journal of Medicinal Chemistry 101 (2015) 266e273
Fig. 2. Design strategy of “pyrimidine-primaquine” hybrids.
Fig. 3. Final screening of selected hybrid against liver stages. Activity (Infection scale, bars) and toxicity to hepatoma cells (cell confluency scale, circles).
Chart 1. Antiplasmodial agents (chloroquine 1, primaquine 2, primaquine-chloroquine hybrid 3, pyrimethamine 4).
We hypothesized that pyrimidines, when combined with pri-
substituents when incorporated on hybrids exert significant anti-
maquine, will result in hybrids, which might display superior
antimalarial activity. In this work, we exploit a dual-stage hybrid
approach, wherein a pyrimidine pharmacophore has been cova-
lently linked with PQ to obtain “pyrimidine-primaquine” hybrids
(Fig. 2) for the first time. The choice of substituents on the hybrids
was guided by our earlier investigations [28] wherein such
malarial action as well as impart adequate solubility.
2. Chemistry
The synthesis of target PQ-pyrimidine hybrids 8 is outlined in
Scheme 1. The precursor 3,4-dihydropyrimidin-2(1H)-one 5 was

H. Kaur et al. / European Journal of Medicinal Chemistry 101 (2015) 266e273
269
Scheme 1. (a) EtOH, HCl, rt; (b) 60% nitric acid, 0 ꢀC; (c) POCl3, 105 ꢀC, 45 min; (d) NEt3, DMF, rt. Atoms in 8 are numbered to facilitate assignment of 13C NMR signals (Table 2).
prepared through NH4Cl [29]/polyphosphate ester (PPE) [30]/HCl
[31] catalyzed three-component Biginelli condensation of an alkyl/
aryl acetoacetate, urea and appropriate aldehyde in C2H5OH/THF.
Subsequent oxidation of 5 using 60% nitric acid [32] readily fur-
nished pyrimidin-2(1H)-ones 6, which upon chlorination with
POCl3 yielded 2-chloropyrimidin-2(1H)-ones 7. The condensation
of 7 with the commercially available primaquine biphosphate 2 in
the presence of triethylamine in anhydrous DMF under an atmo-
sphere of nitrogen at room temperature provided the target hybrids
8aej in excellent yields (Table 1). Structures of all the hybrids and
intermediates were unambiguously established on the basis of
spectral (1H NMR, 13C NMR, HRMS, FT IR) as well as microanalytical
analysis.
CQ-sensitive P. falciparum NF54 strain (Table 1) in both assays.
Atovaquone (ATQ) was used a positive control in liver stage testing
whereas CQ and artesunate (ASN) were additional controls in the
blood stage assays.
The hybrids 8aej displayed blood stage activity against
P. falciparum with IC50 values in the range 1.93e6.46
mM. The
analysis of the blood stage activity data of hybrids 8aej revealed
that none of the hybrids exhibited better activity than the standard
drugs (ASN, CQ and PQ), although the activity of hybrid 8j was only
2.1 fold less than PQ. These results suggested that the conjugation
of PQ with pyrimidine-5-carboxylate motif did not result in sig-
nificant blood stage activity. The observed lack of blood stage ac-
tivity of these hybrids may be attributed to the lack of aliphatic
amine linkers in analogy to the work reported in the literature
[24e26].
3. Results and discussion
Furthermore, the nature of the substitution pattern on the py-
rimidine pharmacophore has a significant effect on the in vitro anti-
All the synthesized hybrids were tested against liver stages of
the rodent parasite P. berghei and in vitro against blood stages of the
plasmodial activity of hybrids 8. The presence of
a
phenyl
Table 1
In vitro antiplasmodial activity, CC50 values and selectivity indices of hybrids 8aej and control drugs.
Hybrid
Yield (%)a
IC50
(m
M)
SI
Blood stage Pf NF54 strainb,c
Liver staged
CC50 (m
M)e
8a
8b
8c
8d
8e
8f
8g
8h
8i
90
92
94
92
95
91
90
85
88
91
e
6.462
4.803
4.320
2.000
3.778
3.431
3.278
2.791
2.671
1.934
0.892
nd
1.261 0.310
nd
nd
nd
1.857 0.302
nd
1.571 0.248
nd
1.500 0.594
nd
8.428 3.389
0.0018 0.0003
>100
e
>79.3
e
e
e
e
e
61
e
32.6
e
>100
e
>63.6
e
>100
e
>66.6
e
8j
PQ
ATQ
CQ
ASN
e
e
e
e
e
e
0.0125
0.0044
e
e
e
nd ¼ not determined.
a
Isolated after column chromatography.
Mean of three observation.
Determined against CQ-sensitive strain.
Determined against P. berghei.
b
c
d
e
Toxicity determined against Huh 7 cell line.

270
H. Kaur et al. / European Journal of Medicinal Chemistry 101 (2015) 266e273
substituent at C-4 of the pyrimidine core (8a-b) was detrimental to
blood stage activity, whereas the presence of a p-nitrophenyl group
at the C-4 position (8j) resulted in the most active hybrid of the
on a Bruker Avance II 400 spectrometer with chemical shifts being
reported in parts per million ( ) relative to internal tetramethylsi-
lane (TMS,
0.0, 1H NMR) or chloroform (CDCl3, 77.0, 13C NMR).
d
d
d
series with an IC50 value of 1.93
m
M. A comparison of the hybrids
Mass spectra were recorded at Department of Chemistry, Guru
Nanak Dev University, Amritsar on a Bruker LC-MS MICROTOF II
spectrometer. Elemental analysis was performed on FLASH EA 112
(Thermo electron Corporation) analyzer at Department of Chem-
istry, Guru Nanak Dev University, Amritsar and the results are
quoted in %. IR spectra were recorded on Perkin Elmer FTIR-C92035
Fourier transform spectrometer in the range 400e4000 cmꢁ1 using
KBr pellets. For monitoring the progress of a reaction and for
comparison purpose, thin layer chromatography (TLC) was per-
formed on pre-coated aluminum sheets of Merck (60F254, 0.2 mm)
using an appropriate solvent system. The chromatograms were
visualized under UV light. For column chromatography silica gel
(60e120 mesh) was employed and eluents were ethyl acetate/
hexane mixtures.
8hej (o/m/p-nitro), 8f (p-F), and 8g (p-OCH3) revealed that nitro
substitution resulted in slightly improved blood stage activity.
Switching the substituents (R2 and R3) in the isomeric hybrids 8a
and 8c led to 8c [IC50 ¼ 4.320
against N54 strain of P. falciparum than 8a (IC50 ¼ 6.462
variation of the C-5 ester groups among the hybrids resulted in the
m
M], which was 1.5 fold more active
m
M). The
i-propyl ester containing 8b (IC50 ¼ 4.803
mM) to be more active
than 8a.
Varying the substituents at C-6 position of the pyrimidine core
suggested that 8e bearing a methyl group at the C-6 position was
marginally more active (8e: IC50
¼
3.778 mM) than 8c
(IC50 ¼ 4.320
m
M) bearing a phenyl group at the same position.
Thus, this brief SAR analysis suggested that the presence of phenyl
substituents at C-4 or C-6 positions on the pyrimidine core led to
lowering of blood stage antiplasmodial activity.
5.2. General procedure for synthesis of 8
The potential of hybrids 8aej to inhibit the liver stages of Plas-
modium infection was assessed on an infection system that uses a
human heptoma cell line (Huh 7) and the rodent parasite P. berghei.
In an initial screening we determined the effect of all the hybrids at
To the stirred solution of primaquine biphosphate 2 (0.87 mmol)
and triethylamine (2.6 mmol) in dry DMF (10 ml) nitrogen atmo-
sphere, a solution of appropriate 2-chloropyrimidine 7 (0.87 mmol)
in dry DMF (5 ml) was added. The reaction mixture was strirred at
room temerature for 48 h under nitrogen atmosphere. The reaction
mixture was concenterated under vacuum and the residue was
purified by column chromatography using hexane/ethyl acetate as
eluent to obtain corresponding 8, which was then recrystallized
from DCM/hexane to obtain yellow solid.
1 mM, 5 mM and 10 mM on infection and cell confluence. None of the
hybrid 8aej significantly affected Huh7 cell proliferation (Table 1)
indicating that the hybrids are non-toxic antimalarial agents.
Moreover, these hybrids depicted high selectivity index (Table 1).
An inspection of the data suggested that hybrids 8a, 8e, 8g and 8i
led to the strongest decrease infection at the lowest tested con-
centration of 1
mM. Thus, we selected these hybrids for a dose
response study and determined their IC50 values for the inhibition
of hepatic P. berghei infection (Fig. 3). The comparison of IC50 values
of hybrid 8 and standard drugs presented in Table 1 showed that
the hybrids 8aej displayed superior activity relative to PQ but lower
5.2.1. Ethyl 2-(4-(6-methoxyquinolin-8-ylamino)pentylamino)-6-
methyl-4-phenylpyrimidin-5-carboxylate (8a)
Yellow solid. Yield: 90%. m.p: 65 ꢀC. IR (KBr): nmax 1266, 1565,
1710, 2928, 3058, 3385 cmꢁ1 1H NMR (400 MHz, CDCl3, 25 ꢀC):
.
than ATQ. Among all the hybrids tested, hybrid 8a (IC50 ¼ 1.261
showed the highest activity against the liver stage of infection,
which is 6.7 fold greater than PQ (IC50 ¼ 8.428 M) albeit its blood
stage activity is weaker. On the other hand, hybrid 8i has good
blood stage activity (IC50 ¼ 2.671 M), although lower than that of
PQ (IC50 ¼ 0.890 M), along with excellent liver stage activity
(IC50 ¼ 1.5
m
M)
d
0.91 (d, J ¼ 6.2 Hz, 3H, CHCH3), 1.29 (t, J ¼ 6.6 Hz, 3H, ester-CH3),
1.57e1.68 (m, 4H, 2 ꢂ CH2), 2.44 (s, 3H, C6eCH3), 3.48e3.50 (m, 2H,
CH2), 3.64e3.67 (m, 1H, CHCH3), 3.85 (s, 3H, OCH3), 4.01 (q,
J ¼ 6.6 Hz, 2H, ester-CH2), 5.32 (br, 1H, NH), 5.99 (br, 1H, NH),
6.23e6.28 (m, 2H, ArH), 7.20e7.37 (m, 4H, ArH), 7.49 (s, 2H, ArH),
7.87 (d, J ¼ 8.7 Hz, 1H, ArH), 8.50 (s, 1H, ArH). 13C NMR (125 MHz,
m
m
m
m
M) and is non-toxic (CC50 ¼ > 100). Our preliminary
CDCl3, 25 ꢀC):
d 13.4, 20.5, 20.9, 26.3, 33.9, 41.1, 47.7, 55.1, 60.9, 96.7,
liver stage activity studies revealed no in vivo activity for 8i. How-
ever, 8i may serve as good starting point for further structural
optimization. Overall, it is concluded that the hybridization of PQ
with pyrimidine-5-carboxylate enhances the inherent liver stage
activity of the PQ motif.
121.7, 127.9, 128.1, 129.3, 134.7, 139.2, 144.2, 159.4, 170.3. Anal. Calcd.
For C29H33N5O3: C, 69.72; H, 6.66; N,14.02; Found: C, 69.81; H, 6.73;
N, 14.09. HRMS: m/z 500.2592 [Mþ þ 1].
5.2.2. Isopropyl 2-(4-(6-methoxyquinolin-8-ylamino)
pentylamino)-6-methyl-4-phenylpyrimidin-5-carboxylate (8b)
4. Conclusions
Yellow solid. Yield: 92%. m.p: 72 ꢀC. IR (KBr): nmax 1266, 1561,
1710, 2924, 3058, 3388 cmꢁ1 1H NMR (400 MHz, CDCl3, 25 ꢀC):
.
The synthesis and antiplasmodial activity of novel class of hy-
brids combining pyrimidine-5-carboxylate motif with primaquine
has been described. The synthesized hybrids exhibit enhanced liver
stage activities in comparison to primaquine. One of the hybrids, 8i,
has good blood stage activity as well as excellent liver stage activity
and may serve as good starting point for the development of po-
tential antiplasmodial agents targeting both blood and liver stage
infections.
d
0.99 (d, J ¼ 6.2 Hz, 6H, 2 ꢂ ester-CH3), 1.31 (d, J ¼ 6.4 Hz, 3H, CH3),
1.70e1.81 (m, 4H, 2 ꢂ CH2), 2.45 (s, 3H, C6eCH3), 3.48e3.55 (m, 2H,
CH2), 3.66e3.68 (m, 1H, CH), 3.88 (s, 3H, OCH3), 4.92e4.96 (m, 1H,
ester-CH), 5.43 (br, 1H, NH), 6.02 (br, 1H, NH), 6.29 (d, J ¼ 2.4 Hz, 1H,
ArH), 6.33 (d, J ¼ 2.4 Hz, 1H, ArH), 7.30 (dd, J ¼ 4.2, 8.2 Hz, 1H, ArH),
7.36e7.41 (m, 3H, ArH), 7.53 (s, 2H, ArH), 7.92 (dd, J ¼ 1.6, 8.2 Hz, 1H,
ArH), 8.52 (dd, J ¼ 1.6, 4.3 Hz, 1H, ArH). 13C NMR (125 MHz, CDCl3,
25 ꢀC):
d 20.6, 21.3, 26.4, 34.0, 41.3, 47.8, 55.2, 68.7, 91.6, 96.7, 115.7,
121.8, 128.0, 128.2,129.3, 129.9, 134.8,135.3,139.1,144.2,145.0, 159.4,
161.1, 168.3. Anal. Calcd. For C30H35N5O3: C, 70.15; H, 6.87; N, 13.63;
Found: C, 69.99; H, 6.92; N, 13.560. HRMS: m/z 514.2728 [Mþ þ 1].
5. Experimental
5.1. General
5.2.3. Ethyl 2-(4-(6-methoxyquinolin-8-ylamino)pentylamino)-4-
methyl-6-phenylpyrimidin-5-carboxylate (8c)
All liquid reagents were dried/purified following recommended
drying agents and/or distilled over 4 Å molecular sieves. 1H NMR
(400 MHz) and 13C (125 MHz) NMR spectra were recorded in CDCl3
Yellow solid. Yield: 94%. m.p: 68 ꢀC. IR (KBr): nmax 1266, 1560,
1712, 2926, 3057, 3388 cmꢁ1 1H NMR (400 MHz, CDCl3, 25 ꢀC):
.

H. Kaur et al. / European Journal of Medicinal Chemistry 101 (2015) 266e273
271
d
0.87 (t, J ¼ 7.2 Hz, 3H, ester-CH3), 1.21 (d, J ¼ 6.3 Hz, 3H, CH3),
(m, 2H, CH2), 3.65e3.66 (m, 1H, CH), 3.83 (s, 3H, OCH3), 3.88 (s, 3H,
OCH3), 4.10 (q, J ¼ 7.1 Hz, 2H, CH2), 5.37 (br, 1H, NH), 6.04 (br, 1H,
NH), 6.29 (d, J ¼ 2.4 Hz, 1H, ArH), 6.33 (d, J ¼ 2.4 Hz, 1H, ArH),
6.88e6.92 (m, 2H, ArH), 7.28 (dd, J ¼ 4.2, 8.2 Hz, 1H, ArH), 7.53 (d,
J ¼ 8.0 Hz, 2H, ArH), 7.90 (dd, J ¼ 1.6, 8.2 Hz, 1H, ArH), 8.52 (dd,
1.63e1.75 (m, 4H, 2 ꢂ CH2), 2.40 (s, 3H, C4eCH3), 3.42e3.49 (m, 2H,
CH2), 3.59e3.61 (m, 1H, CH), 3.80 (s, 3H, OCH3), 3.97 (q, J ¼ 7.1 Hz,
2H, ester-CH2), 5.42 (br, 1H, NH), 6.00 (br, 1H, NH), 6.22 (d,
J ¼ 2.4 Hz, 1H, ArH), 6.26 (d, J ¼ 2.4 Hz, 1H, ArH), 7.23 (dd, J ¼ 4.2,
8.2 Hz, 1H, ArH), 7.29e7.34 (m, 3H, ArH), 7.45e7.50 (m, 2H, ArH),
7.85 (dd, J ¼ 1.6, 2.8 Hz, 1H, ArH), 8.45 (dd, J ¼ 1.6, 4.2 Hz, 1H, ArH).
J ¼ 1.5, 4.1 Hz, 1H, ArH). 13C NMR (125 MHz, CDCl3, 25 ꢀC):
d 13.7,
20.6, 22.8, 26.4, 34.0, 41.3, 47.8, 55.2, 55.3, 61.0, 91.6, 96.7, 113.6,
114.8, 121.8, 129.6, 129.9, 131.4, 134.7, 135.3, 144.3, 145.0, 159.4,
160.8, 161.1, 165.1, 169.3. Anal. Calcd. For C30H35N5O4: C, 68.03; H,
6.66; N, 13.22; Found: C, 67.89; H, 6.74; N, 13.05. HRMS: m/z
530.2678 [Mþ þ 1].
13C NMR (125 MHz, CDCl3, 25 ꢀC):
d 13.5, 20.6, 26.3, 34.0, 41.3, 47.8,
55.2, 61.0, 91.6, 96.8, 115.1, 121.8, 127.9, 128.2, 129.4, 129.9, 134.9,
135.2, 139.1, 144.2, 144.9, 159.4, 166.2, 168.8. Anal. Calcd. For
C
29H33N5O3: C, 69.72; H, 6.66; N, 14.02; Found: C, 69.65; H, 6.78; N,
13.94. HRMS: m/z 500.2583 [Mþ þ 1].
5.2.8. Isopropyl 2-(4-(6-methoxyquinolin-8-ylamino)
pentylamino)-6-methyl-4-(2-nitrophenyl)pyrimidin-5-carboxylate
(8h)
5.2.4. Methyl 2-(4-(6-methoxyquinolin-8-ylamino)pentylamino)-
4,6-dimethylpyrimidin-5-carboxylate (8d)
Yellow solid. Yield: 92%. m.p: 44 ꢀC. IR (KBr): nmax 1266, 1566,
Yellow solid. Yield: 85%. m.p: 84 ꢀC. IR (KBr): nmax 1266, 1385,
1711, 2928, 3083, 3388 cmꢁ1 1H NMR (400 MHz, CDCl3, 25 ꢀC):
.
d
1524, 1566, 1712, 2929, 3082, 3388 cmꢁ1. 1H NMR (400 MHz, CDCl3,
1.28 (d, J ¼ 6.0 Hz, 3H, CH3),1.74e1.72 (m, 4H, 2 ꢂ CH2), 2.38 (s, 6H,
25 ꢀC):
d
0.90 (d, J ¼ 6.4 Hz, 6H, 2 ꢂ ester-CH3), 1.30 (d, J ¼ 5.3 Hz,
C4 & C6eCH3), 3.44e3.49 (m, 2H, CH2), 3.64e3.67 (m, 1H, CH),
3.83e3.89 (m, 6H, OCH3 & ester CH3), 5.32 (br, 1H, NH), 6.00 (br, 1H,
NH), 6.26 (s, 1H, ArH), 6.31 (d, J ¼ 2.1 Hz, 1H, ArH), 7.24e7.29 (m, 1H,
3H, CH3), 1.67e1.74 (m, 4H, 2 ꢂ CH2), 2.58 (s, 3H, C6eCH3),
3.36e3.56 (m, 2H, CH2), 3.58e3.65 (m, 1H, CH), 3.88 (s, 3H, OCH3),
4.84e4.86 (m, 1H, ester-CH), 6.00 (br, 1H, NH), 6.28 (s, 1H, ArH),
6.33 (d, J ¼ 2.3 Hz, 1H, ArH), 7.27e7.32 (m, 1H, ArH), 7.44 (br, 1H,
NH), 7.52e7.62 (m, 2H, ArH), 7.93 (d, J ¼ 7.4 Hz, 1H, ArH), 8.04 (t,
J ¼ 7.0 Hz, 1H, ArH), 8.14 (d, J ¼ 7.7 Hz, 1H, ArH), 8.52 (dd, J ¼ 1.6,
ArH), 7.89 (d, J ¼ 8.1 Hz, 1H, ArH), 8.50 (d, J ¼ 4.2 Hz, 1H, ArH). 13
C
NMR (125 MHz, CDCl3, 25 ꢀC):
d 20.8, 23.3, 25.5, 34.7, 42.4, 47.3,
55.9, 63.5, 96.2, 99.3, 119.9, 121.7, 124.4, 132.1, 135.7, 145.3, 147.1,
160.5,168.2. Anal. Calcd. For C23H29N5O3: C, 65.23; H, 6.90; N,16.54;
Found: C, 65.31; H, 6.94; N, 16.46. HRMS: m/z 424.2335 [Mþ þ 1].
4.2 Hz,1H, ArH). 13C NMR (125 MHz, CDCl3, 25 ꢀC):
d 20.6, 21.2, 26.2,
33.9, 41.5, 47.8, 55.2, 62.1, 91.6, 95.8, 119.3, 121.8, 124.3, 129.0, 148.1,
154.0, 161.0, 166.3, 179.7, 184.3, 189.4, 190.9. Anal. Calcd. For
5.2.5. Ethyl 2-(4-(6-methoxyquinolin-8-ylamino)pentylamino)-
4,6-dimethylpyrimidin-5-carboxylate (8e)
C30H34N6O5: C, 64.50; H, 6.13; N, 15.04; Found: C, 64.59; H, 6.26; N,
15.09. HRMS: m/z 559.2583 [Mþ þ 1].
Yellow solid. Yield: 95%. m.p: 47 ꢀC. IR (KBr): nmax 1266, 1520,
1561, 1710, 2924, 3058, 3388 cmꢁ1
.
1H NMR (400 MHz, CDCl3,
5.2.9. Isopropyl 2-(4-(6-methoxyquinolin-8-ylamino)
pentylamino)-6-methyl-4-(3-nitrophenyl)pyrimidin-5-carboxylate
(8i)
25 ꢀC):
d
0.78 (d, J ¼ 5.7 Hz, 3H, CH3), 1.29 (t, J ¼ 7.2 Hz, 3H, ester-
CH3), 1.66e1.70 (m, 4H, 2 ꢂ CH2), 2.33 (s, 6H, C4 & C6eCH3), 3.41 (t,
J ¼ 6.0 Hz, 2H, CH2), 3.57e3.61 (m, 1H, CH), 3.81 (s, 3H, OCH3), 4.26
(q, J ¼ 7.2 Hz, 2H, ester-CH2), 5.18 (br, 1H, NH), 5.95 (br, 1H, NH),
6.21 (s, 1H, ArH), 6.25 (d, J ¼ 2.4 Hz, 1H, ArH), 7.23 (t, J ¼ 4.2 Hz, 1H,
ArH), 7.85 (d, J ¼ 8.4 Hz, 1H, ArH), 8.45 (dd, J ¼ 1.5 Hz, 4.2 Hz, 1H,
Yellow solid. Yield: 88%. m.p: 67 ꢀC. IR (KBr): nmax 1266, 1385,
1524, 1566, 1712, 2929, 3082, 3388 cmꢁ1. 1H NMR (400 MHz, CDCl3,
25 ꢀC):
d
1.08 (d, J ¼ 6.1 Hz, 6H, 2 ꢂ ester-CH3),1.30 (d, J ¼ 6.2 Hz, 3H,
CH3), 1.66e1.79 (m, 4H, 2 ꢂ CH2), 2.49 (s, 3H, C6eCH3), 3.49e3.53
(m, 2H, CH2), 3.65e3.66 (m, 1H, CH), 3.88 (s, 3H, OCH3), 5.01e5.20
(m, 1H, ester-CH), 5.45 (br, 1H, NH), 6.10 (br, 1H, NH), 6.28 (d,
J ¼ 2.4 Hz, 1H, ArH), 6.33 (d, J ¼ 2.4 Hz, 1H, ArH), 7.29e7.32 (m, 1H,
ArH), 7.56 (t, J ¼ 7.8 Hz, 1H, ArH), 7.80e7.91 (m, 2H, ArH), 8.28 (d,
J ¼ 7.3 Hz, 1H, ArH), 8.41 (s, 1H, ArH), 8.52 (s, 1H, ArH). 13C NMR
ArH). 13C NMR (125 MHz, CDCl3, 25 ꢀC):
d 14.1, 21.4, 22.5, 25.3, 34.9,
45.2, 46.9, 55.7, 60.9, 96.3, 99.7, 121.0, 125.4, 132.3, 134.8, 144.9,
146.1, 160.3, 166.4. Anal. Calcd. For C24H31N5O3: C, 65.88; H, 7.14; N,
16.01; Found: C, 65.80; H, 6.99; N, 16.11. HRMS: m/z 438.2498
[Mþ þ 1].
(125 MHz, CDCl3, 25 ꢀC):
d 20.6, 21.4, 23.3, 26.3, 34.0, 41.3, 47.8, 55.2,
5.2.6. Ethyl 4-(4-fluorophenyl)-2-(4-(6-methoxyquinolin-8-
ylamino)pentylamino)-6-methylpyrimidin-5-carboxylate (8f)
Yellow solid. Yield: 91%. m.p: 60 ꢀC. IR (KBr): nmax 1266, 1564,
69.1, 91.7, 96.8, 121.8, 123.3, 123.9, 129.2, 129.9, 134.1, 134.8, 140.8,
148.2, 159.5, 161.1, 167.1. Anal. Calcd. For C30H34N6O5: C, 64.50; H,
6.13; N, 15.04; Found: C, 64.62; H, 6.28; N, 15.17. HRMS: m/z
559.2599 [Mþ þ 1].
1710, 2920, 3084, 3388 cmꢁ1 1H NMR (400 MHz, CDCl3, 25 ꢀC):
.
d
1.01 (t, J ¼ 6.9 Hz, 3H, ester-CH3), 1.31 (d, J ¼ 6.3 Hz, 3H, CH3),
1.77e1.81 (m, 4H, 2 ꢂ CH2), 2.47 (s, 3H, C6eCH3), 3.52e3.55 (m, 2H,
CH2), 3.65e3.67 (m, 1H, CH), 3.88 (s, 3H, OCH3), 4.04 (q, J ¼ 7.2 Hz,
2H, CH2), 5.30 (br, 1H, NH), 6.10 (br, 1H, NH), 6.31 (s, 1H, ArH), 6.35
(d, J ¼ 2.4 Hz,1H, ArH), 7.07 (t, J ¼ 7.1 Hz, 2H, ArH), 7.29e7.40 (m,1H,
ArH), 7.53 (m, 1H, ArH), 7.95 (m, 2H, ArH), 8.52 (dd, J ¼ 1.5, 3.9 Hz,
5.2.10. Isopropyl 2-(4-(6-methoxyquinolin-8-ylamino)
pentylamino)-6-methyl-4-(4-nitrophenyl)pyrimidin-5-carboxylate
(8j)
Yellow solid. Yield: 91%. m.p: 90 ꢀC. IR (KBr): nmax 1266, 1385,
1524, 1566, 1712, 2929, 3082, 3388 cmꢁ1. 1H NMR (400 MHz, CDCl3,
1H, ArH). 13C NMR (125 MHz, CDCl3, 25 ꢀC):
d
13.7, 20.7, 21.2, 26.5,
25 ꢀC):
d
1.03 (d, J ¼ 6.2 Hz, 6H, 2 ꢂ ester-CH3),1.27 (d, J ¼ 6.2 Hz, 3H,
34.2, 41.3, 48.0, 55.4, 61.1, 96.9,122.0,128.1,128.4,129.5,130.1,134.9,
139.4, 144.5, 159.6, 170.6. Anal. Calcd. For C29H32FN5O3: C, 67.29; H,
6.23; N, 13.53; Found: C, 67.21; H, 6.37; N, 13.61. HRMS: m/z
518.2582 [Mþ þ 1].
CH3), 1.69e1.81 (m, 4H, 2 ꢂ CH2), 2.48 (s, 3H, C6eCH3), 3.50e3.52
(m, 2H, CH2), 3.65e3.67 (m, 1H, CH), 3.88 (s, 3H, OCH3), 4.95e4.97
(m, 1H, ester-CH), 5.48 (br, 1H, NH), 6.02 (br, 1H, NH), 6.28 (s, 1H,
ArH), 6.34 (d, J ¼ 2.4 Hz, 1H, ArH), 7.30 (dd, J ¼ 4.2, 8.2 Hz, 1H, ArH),
7.66 (d, J ¼ 8.6 Hz, 2H, ArH), 7.92 (dd, J ¼ 1.4, 8.2 Hz, 1H, ArH), 8.22
(d, J ¼ 7.7 Hz, 2H, ArH), 8.52 (dd, J ¼ 1.5, 4.2 Hz, 1H, ArH). 13C NMR
5.2.7. Ethyl 2-(4-(6-methoxyquinolin-8-ylamino)pentylamino)-6-
methyl-4-(4-methoxyphenyl)pyrimidin-5-carboxylate (8g)
Yellow solid. Yield: 90%. m.p: 45 ꢀC IR (KBr): nmax 1268, 1524,
(125 MHz, CDCl3, 25 ꢀC):
d 20.6, 21.0, 22.7, 26.3, 33.9, 41.3, 47.7, 55.2,
69.0, 91.6, 96.8, 115.4, 121.8, 123.4, 129.1, 129.9, 134.8, 135.3, 144.3,
144.9, 145.5, 148.1, 159.4, 161.1, 163.9, 167.4. Anal. Calcd. For
1566, 1710, 2929, 3081, 3387 cmꢁ1 1H NMR (400 MHz, CDCl3,
.
25 ꢀC):
CH3), 1.70e1.81 (m, 4H, 2 ꢂ CH2), 2.44 (s, 3H, C6eCH3), 3.48e3.55
d
1.06 (t, J ¼ 7.1 Hz, 3H, ester-CH3), 1.29 (d, J ¼ 6.3 Hz, 3H,
C30H34N6O5: C, 64.50; H, 6.13; N, 15.04; Found: C, 64.44; H, 6.07; N,
15.20. HRMS: m/z 559.2593 [Mþ þ 1].

272
H. Kaur et al. / European Journal of Medicinal Chemistry 101 (2015) 266e273
Table 2
Assignment (partial) of signals in13C NMR spectra of 8aej.
Compound
Partial13C NMR assignment (
d
/ppm)
6
C-6 Me
C-5 ester
5
a
b
c
d
e
C-60 OMe
C-4 methyl or substituent on C-4 aromatic ring
CO
8a
8b
8c
8d
8e
8f
8g
8h
8i
20.9
21.3
20.6
23.3
22.5
21.2
22.8
21.2
23.3
22.7
13.4, 60.9
21.3, 68.7
13.5, 61.0
63.5
14.1, 60.9
13.7, 61.3
13.7, 61.0
21.2, 62.1
21.4, 69.1
21.0, 69.0
96.7
96.7
96.8
96.2
96.3
96.9
96.7
95.8
96.8
96.8
159.4
41.1
41.3
41.3
42.4
45.2
41.3
41.3
41.5
41.3
41.3
26.3
26.4
26.3
25.5
25.3
26.5
26.4
26.2
26.3
26.3
33.9
34.0
34.0
34.7
34.9
34.2
34.0
33.9
34.0
33.9
47.7
47.8
47.8
47.3
46.9
48.0
47.8
47.8
47.8
47.7
20.5
20.6
20.6
20.8
21.4
20.7
20.6
20.6
20.6
20.6
55.1
55.2
55.2
55.9
55.7
55.4
55.2
55.2
55.2
55.2
e
170.3
168.3
168.8
168.2
166.4
170.6
169.3
166.3
167.1
167.4
159.4
159.4
160.5
160.3
159.6
159.4
161.0
159.5
159.4
e
26.3
20.8
21.4
e
55.3
148.1
148.2
148.1
8j
Assignment of the key signals in the 13C NMR spectra of 8aej is
presented in Table 2.
measured 48 h after infection by a bioluminescence assay (Bio-
tium). The effect of the compounds on the viability of Huh-7 cells
was assessed by the Alamar Blue assay (Invitrogen, U.K.) using the
manufacturer's protocol.
6. In vitro blood stage activity assay
The test samples were tested in triplicate on one or two
separate occasions against chloroquine sensitive (CQS) NF54 strain
of P. falciparum. Continuous in vitro cultures of asexual erythrocyte
stages of P. falciparum were maintained using a modified method
of Trager and Jensen [33]. Quantitative assessment of anti-
plasmodial activity in vitro was determined via the parasite lactate
dehydrogenase assay using a modified method described by
Makler [34].
The test samples were prepared to a 20 mg/ml stock solution in
100% DMSO. Samples were tested as a suspension. Stock solutions
were stored at ꢁ20 ꢀC. Further dilutions were prepared on the day
of the experiment. Chloroquine (CQ) and artesunate were used as
the reference drugs in all experiments. A full doseeresponse was
performed for all compounds to determine the concentration
inhibiting 50% of parasite growth (IC50evalue). Test samples were
8. In vivo liver stage activity assay
C57Bl/6J mice were infected by intravenous inoculation of
5 ꢂ 103 luciferase-expressing P. berghei ANKA sporozoites freshly
dissected from the salivary glands of infected A. stephensi mosqui-
toes. Compound 8i was dissolved in DMSO (stock solution) and a
50 mg/kg bw suspension of the compound in sunflower oil was
administered at by oral gavage injection 8 h prior to infection, at the
time of infection, and 24 h after infection. An equivalent amount of
drug vehicle was injected in control mice.
Determination of liver parasite loads in vivo was carried out as
previously described [36]. Briefly, livers were collected and ho-
mogenized in a denaturing solution (4 M guanidine thiocyanate;
25 mM sodium citrate [pH 7], 0.5% sarcosyl, and 0.7% b-mercap-
toethanol in diethyl pyrocarbonate-treated water) 47 h after
sporozoite injection. Total RNA was extracted using the Qiagen
RNeasy Mini kit according to the manufacturer's instructions. RNA
for infection measurements was converted into cDNA by using the
Nzytech kit according to the manufacturer's protocol. The quanti-
tative real-time PCRs (qRT-PCRs) used the Applied Biosystems Po-
wer SYBR green PCR master mix and were performed according to
the manufacturer's instructions on an ABI Prism 7500 Fast system
(Applied Biosystems). Amplification reactions were carried out in a
tested at a starting concentration of 100
serially diluted 2-fold in complete medium to give 10 concentra-
tions; with the lowest concentration being 0.2 g/ml. The same
mg/ml, which was then
m
dilution technique was used for all samples. Reference drugs were
tested at a starting concentration of 1000 ng/ml. Active samples
were tested at a starting concentration of 1000 ng/ml. The highest
concentration of solvent to which the parasites were exposed to
had no measurable effect on the parasite viability (data not shown).
The IC50-values were obtained using a non-linear dose response
curve fitting analysis via Graph Pad Prism v.4.0 software.
total reaction volume of 20 ml, using 1 mg cDNA and employing P.
berghei ANKA 18S rRNA gene- or housekeeping gene-specific
primers. Relative amounts of P. berghei ANKA mRNA were calcu-
lated against the amount of the hypoxanthine guanine phosphor-
ibosyltransferase (HPRT) housekeeping gene. Primer sequences
specific to each gene were as follows: for the P. berghei ANKA 18S
rRNA gene, 50-AAG CAT TAA ATA AAG CGA ATA CAT CCT TAC-30 and
50-GGA GAT TGG TTT TGA CGT TTA TGT G-3’; for the mouse HPRT
gene, 50-TGC TCG AGA TGT GAT GAA GG-30 and 50-TCC CCT GTT GAC
TGG TCA TT-3’; and for the human HPRT gene, 50-TGC TCG AGA TGT
GAT GAA GG-30 and 50-TCC CCT GTT GAC TGG TCA TT-3’.
7. In vitro liver stage activity assay
Inhibition of liver-stage infection by test compounds was
determined by measuring the luminescence intensity in Huh-7
cells infected with a firefly luciferase-expressing P. berghei line, as
previously described [35]. Briefly, Huh-7 cells, a human hepatoma
cell line, were cultured in 1640 RPMI medium supplemented with
10% v/v fetal bovine serum, 1% v/v nonessential amino acids, 1% v/v
penicillin/streptomycin, 1% v/v glutamine, and 10 mM 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7, and
maintained at 37 ꢀC with 5% CO2. For infection assays, Huh-7 cells
(1.0 ꢂ 104 per well) were seeded in 96-well plates the day before
drug treatment and infection. The medium was replaced by me-
dium containing the appropriate concentration of each compound
approximately 1 h prior to infection with sporozoites freshly ob-
tained through disruption of salivary glands of infected female
Anopheles stephensi mosquitoes. Sporozoite addition was followed
by centrifugation at 1700 g for 5 min. Parasite infection load was
Acknowledgments
We gratefully acknowledge financial assistance from CSIR, New
~
^
Delhi and Fundaçao para a Ciencia e Tecnologia, Portugal (grant
PTDC/SAU-MIC/117060/2010 to MP). H.K. thanks CSIR, New Delhi
for senior research fellowship (F. No. 09/254 (0195)/2009-EMR-I).
The University of Cape Town, South African Medical Research
Council, and South African Research Chairs Initiative of the
Department of Science and Technology, administered through the

H. Kaur et al. / European Journal of Medicinal Chemistry 101 (2015) 266e273
273
chloroquine-bridged ferrocenophane analogues of ferroquine may obviate
malaria drug resistance mechanisms, J. Med. Chem. 56 (2013) 1596e1613.
South African National Research Foundation are gratefully
acknowledged for support (K.C).
[21] Nisha, J. Gut, P.J. Rosenthal, V. Kumar,
b-amino-alcohol tethered 4-
aminoquinoline-isatin conjugates: synthesis and antimalarial evaluation,
Eur. J. Med. Chem. 84 (2014) 566e573.
[22] A. Mishra, H. Batchu, K. Srivastava, P. Singh, P.K. Shukla, S. Batra, Synthesis and
evaluation of new diaryl ether and quinoline hybrids as potential anti-
plasmodial and antimicrobial agents, Bioorg. Med. Chem. Lett. 24 (2014)
1719e1723.
[23] R. Capela, G.G. Cabal, P.J. Rosenthal, J. Gut, M.M. Mota, R. Moreira, F. Lopes,
M. Prudencio, Design and evaluation of primaquine-artemisinin hybrids as a
multistage antimalarial strategy”, Antimicrob. Agents Chemother. 55 (2011)
4698e4706.
[24] D. Miranda, R. Capela, I.S. Albuquerque, P. Meireles, I. Paiva, F. Nogueira,
R. Amewu, J. Gut, P.J. Rosenthal, R. Oliveira, M.M. Mota, R. Moreira, F. Marti,
M. Prudencio, P.M. O‟Neill, F. Lopes, Novel endoperoxide-based transmission-
blocking antimalarials with liver- and blood-schizontocidal activities, ACS
Med. Chem. Lett. 5 (2014) 108e112.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.06.045.
References
[1] World Malaria Report, World Health Organization, Geneva, 2012. Available
from,
http://www.who.int/iris/bitstream/10665/78945/1/9789241564533_
eng.pdf.
[2] C. Daneshvar, Clinical and laboratory features of human Plasmodium knowlesi
infection, Clin. Infect. Dis. 49 (2009) 852e860.
[3] M.D.S. Sabbatani, M.D. Sirio Fiorino, M.D. Roberto Manfredi, The emerging of
the fifth malaria parasite(Plasmodium knowlesi). A public health concern?
Braz. J. Infect. Dis. 14 (2010) 1e13.
[25] J. Matos, N. Vale, M.S. Collins, J. Gut, P.J. Rosenthal, M.T. Cushion, R. Moreiraf,
P. Gomes, PRIMACENES: novel non-cytotoxic primaquine-ferrocene conju-
gates with anti-Pneumocystis carinii activity, Med. Chem. Commun. 1 (2010)
199e201.
[26] B. Perez, C. Teixeira, I.S. Albuquerque, J. Gut, P.J. Rosenthal, M. Prudencio,
P. Gomes, PRIMACINS, N-cinnamoyl-primaquine conjugates, with improved
liver-stage antimalarial activity, Med. Chem. Commun. 3 (2012) 1170e1172.
[27] (a) C.E. Garrett, J.A. Coderre, T.D. Meek, E.P. Garvey, S.M. Beverly, D.V. Santi,
A bifunctional thymidylate synthetase-dihydrofolate reductase in protozoa,
Mol. Biochem. Parasitol. 11 (1984) 257e265;
^
[4] M. Prudencio, A. Rodriguez, M.M. Mota, The silent path to thousands of
merozoites: the Plasmodium liver stage, Nat. Rev. Microbiol.
4 (2006)
849e856.
^
[5] T. Rodrigues, M. Prudencio, R. Moreira, M.M. Mota, F. Lopes, Targeting the liver
stage of malaria parasites:
a yet unmet goal, J. Med. Chem. 55 (2012)
995e1012.
[6] P. Winstanley, S. Ward, Malaria chemotherapy, Adv. Parasitol. 61 (2006)
47e76.
[7] T.J. Egan, Quinoline antimalarials, Expert Opin. Ther. Pat. 11 (2001) 185e209.
[8] World Health Organization, Guidelines for the Treatment of Malaria, second
ed., WHO Press, Geneva, 2010.
(b) V.F. Roche, Mechanism based inhibitors of deoxythymidine mono-
phosphate synthesis as antineoplastic agents, Am. J. Pharm. Educ. 58 (1994)
196e203.
[28] (a) K. Singh, H. Kaur, K. Chibale, J. Balzarini, S. Little, P.V. Bharatam, 2-
Aminopyrimidine based 4-aminoquinoline anti-plasmodial agents. Synthe-
sis, biological activity, structure-activity relationship and mode of action
studies, Eur. J. Med. Chem. 52 (2012) 82e97;
[9] World Health Organization, Global Report on Antimalarial Drug Efficacy and
Drug Resistance: 2000ꢁ2010, WHO Press, Geneva, 2010.
[10] R.N. Price, Artemisinin drugs: novel antimalarial agents, Exp. Opin. Invest.
Drugs 9 (2000) 1815e1827.
(b) K. Singh, H. Kaur, K. Chibale, J. Balzarini, Synthesis of 4-
aminoquinolineepyrimidine hybrids as potent antimalarials and their mode
of action studies, Eur. J. Med. Chem. 66 (2013) 314e323;
(c) K. Singh, H. Kaur, P. Smith, C. de Kock, K. Chibale, J. Balzarini, Quinoli-
neepyrimidine hybrids: synthesis, antiplasmodial activity, SAR and mode of
action studies, J. Med. Chem. 57 (2014) 435e448.
[11] L. Tilley, P. Loria, M. Foley, Chloroquine and other quinoline antimalarials, in:
P.J. Rosenthal (Ed.), Antimalarial Chemotherapy: Mechanisms of Action,
Resistance, and New Directions in Drug Discovery, Humana Press, Totowa, NJ,
2001.
[12] Pasaribu, W. Chokejindachai, C. Sirivichayakul, N. Tanomsing, I. Chavez,
E. Tjitra, S. Pasaribu, M. Imwong, N. White, J.A.M. Dondorp, A randomized
comparison of dihydroartemisinin-piperaquine and artesunate-amodiaquine
combined with primaquine for radical treatment of vivax malaria in Suma-
tera, Indonesia, J. Infect. Dis. 208 (2013) 1906e1913.
[13] S. Pukrittayakamee, J. Tarning, P. Jittamala, P. Charunwatthana, S. Lawpoolsri,
S.J. Lee, W. Hanpithakpong, B. Hanboonkunupakarn, N.P.J. Day, E.A. Ashley,
N.J. White, Pharmacokinetic interactions between primaquine and chloro-
quine, Antimicrob. Agents Chemother. 58 (2014) 3354e3359.
[14] G.D. Shanks, A.J. Oloo, G.M. Aleman, C. Ohrt, F.W. Klotz, D. Braitman, J. Horton,
R. Brueckner, A new primaquine analogue, tafenoquine (WR 238605), for
prophylaxis against Plasmodium falciparum malaria, Clin. Infect. Dis. 33 (2001)
1968e1974.
[29] A. Shaabani, A. Bazgir, F. Teimouri, Ammonium chloride-catalyzed one-pot
synthesis of 3,4-dihydropyrimidin-2-(1H)-ones under solvent-free conditions,
Tetrahedron Lett. 44 (2003) 857e859.
[30] F.S. Falsone, C.O. Kappe, The Biginelli dihydropyrimidone synthesis, Arkivoc ii
(2001) 122e134.
[31] P. Biginelli, Aldureids of ethylic acetoacetate and ethylic oxaloacetate, Gazz.
Chim. Ital. 23 (1893) 360e413.
[32] A. Puchala, F. Belaj, J. Bergman, C.O. Kappe, On the reaction of 3,4- dihy-
dropyrimidones with nitric acid. Preparation and X-ray structure analysis of a
stable nitrolic acid, J. Heterocycl. Chem. 38 (2001) 1345e1352.
[33] W. Trager, J.B. Jensen, Human malaria parasite in continuous culture, Science
193 (1976) 673e675.
[15] W.E. Collins, G.M. Jeffery, Primaquine resistance in Plasmoduim vivax, Am. J.
Trop. Med. Hyg. 55 (1996) 243e249.
[34] M.T. Makler, J.M. Ries, J.A. Williams, J.E. Bancroft, R.C. Piper, B.L. Gibbins,
D.J. Hinrichs, Parasite lactate dehydrogenase as an assay for Plasmodium fal-
ciparum drug sensitivity, Am. J. Trop. Med. Hyg. 48 (1993) 739e741.
[16] F.W. Muregi, A. Ishih, Next-generation antimalarial drugs: hybrid molecules
as a new strategy in drug design, Drug Dev. Res. 71 (2010) 20e32.
[17] S.S. Chauhan, M. Sharma, P.M.S. Chauhan, Trioxaquines: hybrid molecules for
the treatment of malaria, Drug News Perspect. 23 (2010) 632e646.
[18] F. Cosledan, L. Fraisse, A. Pellet, F. Guillou, B. Mordmüller, P.G. Kremsner,
A. Moreno, D. Mazier, J.-P. Maffrand, B. Meunier, Selection of a trioxaquine as
an antimalarial drug candidate, Proc. Natl. Acad. Sci. U. S. A. 105 (2008)
17579e17584.
^
[35] I.H. Ploemen, M. Prudencio, B.G. Douradinha, J. Ramesar, J. Fonager, G.J. van
Gemert, A.J. Luty, C.C. Hermsen, R.W. Sauerwein, F.G. Baptista, M.M. Mota,
A.P. Waters, I. Que, C.W. Lowik, S.M. Khan, C.J. Janse, B.M. Franke-Fayard,
Visualisation and quantitative analysis of the rodent malaria liver stage by real
time imaging, PLoS ONE 4 (2009) e7881.
^
[36] M. Prudencio, C.D. Rodrigues, M. Hannus, C. Martin, E. Real, L.A. Gonçalves,
€
C. Carret, R. Dorkin, I. Rohl, K. Jahn-Hoffmann, A.J.F. Luty, R. Sauerwein,
[19] B.C. Perez, C. Teixeira, I.S. Albuquerque, J. Gut, P.J. Rosenthal, J.R.B. Gomes,
C. Echeverri, J.M.M. Motaet, Kinome-wide RNAi screen implicates at least 5
host hepatocyte kinases in Plasmodium sporozoite infection, PLoS Pathog. 4
(2008) e1000201.
^
M. Prudencio, P. Gomes, N-Cinnamoylated chloroquine analogues as dual-
stage antimalarial leads, J. Med. Chem. 56 (2013) 556e567.
[20] P.F. Salas, C. Herrmann, J.F. Cawthray, C. Nimphius, A. Kenkel, J. Chen, C. de
Kock, P.J. Smith, B.O. Patrick, M.J. Adam, C. Orvig, Structural characteristics of
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