2-Aminopyrimidine based 4-aminoquinoline anti-plasmodial agents. Synthesis, biological activity, structure-activity relationship and mode of action studies

Article abstract of DOI:10.1016/j.ejmech.2012.03.007

2-Aminopyrimidine based 4-aminoquinolines were synthesized using an efficacious protocol. Some of the compounds showed in vitro anti-plasmodial activity against drug-sensitive CQ^S (3D7) and drug-resistant CQ ^R (K1) strains of Plasmodium falciparum in the nM range. In particular, 5-isopropyloxycarbonyl-6-methyl-4-(2-nitrophenyl)-2-[(7- chloroquinolin-4-ylamino)butylamino] pyrimidine depicted the lowest IC ₅₀ (3.6 nM) value (56-fold less than CQ) against CQ^R strain. Structure-activity profile and binding with heme, μ-oxo-heme have been studied. Binding assays with DNA revealed better binding with target parasite type AT rich pUC18 DNA. Most compounds were somewhat cytotoxic, but especially cytostatic. Molecular docking analysis with Pf DHFR allowed identification of stabilizing interactions.

Full text of DOI:10.1016/j.ejmech.2012.03.007

European Journal of Medicinal Chemistry 52 (2012) 82e97
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
2-Aminopyrimidine based 4-aminoquinoline anti-plasmodial agents. Synthesis,
biological activity, structureeactivity relationship and mode of action studies
,
a
b
c
d
e
Kamaljit Singh a , Hardeep Kaur , Kelly Chibale , Jan Balzarini , Susan Little , Prasad V. Bharatam
*
a Organic Synthesis Laboratory, Department of Applied Chemical Sciences and Technology, Guru Nanak Dev University, Amritsar 143005, India
b Department of Chemistry and Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 701, South Africa
c Rega Institute for Medical Research, Katholieke University Leuven, 10 Minderbroedersstraat, B-3000 Leuven, Belgium
d Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK
e Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, S.A.S Nagar 160062, India
a r t i c l e i n f o
a b s t r a c t
Article history:
2-Aminopyrimidine based 4-aminoquinolines were synthesized using an efficacious protocol. Some of
the compounds showed in vitro anti-plasmodial activity against drug-sensitive CQS (3D7) and drug-
resistant CQR (K1) strains of Plasmodium falciparum in the nM range. In particular, 5-
isopropyloxycarbonyl-6-methyl-4-(2-nitrophenyl)-2-[(7-chloroquinolin-4-ylamino)butylamino] pyrimi-
dine depicted the lowest IC50 (3.6 nM) value (56-fold less than CQ) against CQR strain. Structureeactivity
Received 1 February 2012
Received in revised form
2 March 2012
Accepted 2 March 2012
Available online 13 March 2012
profile and binding with heme, m-oxo-heme have been studied. Binding assays with DNA revealed better
binding with target parasite type AT rich pUC18 DNA. Most compounds were somewhat cytotoxic, but
especially cytostatic. Molecular docking analysis with Pf DHFR allowed identification of stabilizing
interactions.
Keywords:
2-Aminopyrimidine
4-Aminoquinolines
DHFR inhibitor
CT DNA interaction
DHPM
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
resistant Plasmodium falciparum, universal chloroquine resistance
[2,3], suspected resistance to artemisinins [4,5], and lack of effec-
Faced with the challenges of drug resistance, poor health
systems, lack of affordable, safe and convenient treatment options,
efficient treatment of malaria, one of the most devastating parasitic
diseases, represents an unmet medical need. Malaria is a major
public health concern in more than 90 countries inhabited by more
than 2.4 billion people e 40% of the world’s population and is
responsible for almost 1 million deaths every year [1]. The majority
of malaria victims in developing countries are pregnant women or
children under the age of five, possessing little or no immunological
protection. The disease is estimated to result in w250 million new
annual infections worldwide. Though the majority of the cases and
approximately 90% of the malaria deaths are found in sub-Saharan
Africa, the disease is now increasing in Asia and Latin America.
Malaria is caused by protozoan parasites of the genus Plasmodium
that infects and destroys red blood cells eventually leading to
death, if untreated. The persistent threat of emergence of multidrug
tive, appropriate and affordable treatment options have given
a new impetus to the research leading to broadening of the range of
therapeutic targets. Thus, creating a new armamentarium of drugs
with promising antimalarial activity coupled with understanding of
their mode of action may lead to the development of a new
generation of treatments both for malaria control and eradication.
The common feature of the drugs based on quinine 1 is the
presence of a quinoline unit, usually a 7-chloroquinoline (chloro-
quine 2 and amodiaquine 3, Chart 1), and are known to cause
parasite death by blocking the polymerization of the toxic heme,
into an insoluble and non-toxic pigment, hemozoin, resulting in
cell lysis and parasite cell autodigestion [6e8]. The mode of action
of 2,4-diaminopyrimidine based drugs, typified by pyrimethamine
4 [9] and the lead compound WR99210 5 [10] is through the
inhibition of P. falciparum Dihydrofolate reductase (Pf DHFR)
enzyme, required for the biosynthesis of tetrahydrofolate involved
in the biotransformation of thymidylate synthase-catalysed deox-
yuridylate to deoxythymidylate (dUMP
/ dTMP), through
a methyl group transfer reaction during DNA biosynthesis [11e16].
In addition, a number of polyamines inhibit ornithine decarbox-
ylase activity in P. falciparum through binding with plasmodial DNA
* Corresponding author. Tel.: þ91 183 2258853; fax: þ91 183 2258819, þ91 183
2258820.
E-mail address: kamaljit19in@yahoo.co.in (K. Singh).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.007

K. Singh et al. / European Journal of Medicinal Chemistry 52 (2012) 82e97
83
OH
N
H
N
OH
N
HN
HN
N
O
Cl
Cl
N
N
Cl
3
1
2
NH2
N
NH2
O
O
Cl
Cl
N
N
H2N
N
Cl
H2N
N
4
5
Chart 1. Antimalarial agents: quinine (1), chloroquine (2), amodiaquine (3), pyrimethamine (4) and WR99210 (5).
[17,18]. Recently, investigations in hybrid antimalarial agents,
combining 4-aminoquinoline with other pharmacophore having
antimalarial activity have been found to be less prone to resistance
to parasite and has thus offered an effective means to overcome the
problem of drug resistance.
a synthetically useful manner [22]. Structures of 6e10 were estab-
lished on the basis of spectral (1H NMR,13C NMR, MS, FT IR) as well as
microanalytical analysis. The yields of the 2-aminopyrimidines 10
are reported in Table 1.
We envisaged that linking 7-chloro-4-aminoquinoline unit,
critical for antimalarial activity through a diversely functionalized
lateral side chain with other antimalarial moiety such as amino-
pyrimidine, might furnish conjugate hybrids [19] capable of
showing useful antimalarial activity. In this communication, we
report synthesis of a set of compounds that possess basic, hydro-
phobic as well as hydrogen bonding substituents, required for
targeting either or both heme as well as DNA, thus providing new
antimalarial agents active against chloroquine resistant strains of
P. falciparum. We have evaluated their anti-plasmodial activity,
cytotoxicity and cytostatic activity, binding studies with DNA and
3. Results and discussion
3.1. In vitro anti-plasmodial activity and structureeactivity
relationships (SARS)
Antiplasmodial activity of pyrimidines linked to CQ as in 10 has
not been described in literature albeit the related dihydropyr-
imidin-2(1H)-ones (DHPMs) have previously been reported [23].
Using the synthetic protocol shown in Scheme 1 allowed consid-
erable diversification around the pyrimidine core for conducting
SAR analysis. The in vitro anti-plasmodial activities of 10aes were
determined in primary and secondary screening against CQS and
CQR strains of P. falciparum. The half maximal inhibitory concen-
tration (IC50) of 10aes are summarised in Table 2 (Fig. 1). Evidently,
the compounds have anti-plasmodial activity in the nM range and
against the CQR strain of P. falciparum, in some cases activity was
found to be even superior to CQ. Systematic variation of the length
as well as nature of the spacer connecting the pharmacophores
discerned useful trends in the anti-plasmodial activity of these
analogs.
heme (monomeric as well as
m-oxo dimeric) using UVevisible,
fluorescence spectrophotometry as well as NMR analysis.
2. Chemistry
Compounds 10aes were synthesized as outlined in Scheme 1, via
a common intermediate 8. 3,4-Dihydropyrimidin-2(1H)-ones 6
were prepared through HCl-catalyzed Biginelli condensation of
appropriate aldehyde (R3CHO), alkylacetoacetate (R2CH2COOR1)
and urea [20]. Dehydrogenation of 6 using pyridinium chlor-
ochromate in DCM furnished pyrimidinones 7 [21]. Refluxing 7 with
POCl3 yielded 8 which upon nucleophilic substitution reaction with
Comparing 10aeg, bearing linear alkyl spacers, revealed an
increase in the anti-plasmodial activity with increase in length of
the spacer up to 4 methylene groups (10ae10c, Table 2). Further
lengthening of the spacer chain length resulted in significant
appropriate 4-amino-7-chloroquinoline
9
gave 10aes in
Fig. 1. Antimalarial activity of compounds 10aes against the CQS and CQR strains of P. falciparum.

84
K. Singh et al. / European Journal of Medicinal Chemistry 52 (2012) 82e97
Fig. 2. Titration of 10h (0e85.7 mM, DMSO:H2O/4:6, v/v) (a) and 10i (0e8.85 mM, DMSO:H2O/4:6, v/v) (b) with heme (2.4 mM, DMSO: H2O/4:6, v/v).
reduction in activity against both the CQR as well as the CQS strains.
Replacing the C-4 phenyl group in 10c by a methyl group to create
10m resulted in nearly 5 times increase in anti-plasmodial activity
(IC50 42.1 nM) against the CQS strain. In fact, compound 10m was
found (Table 2) to be the most active compound of the series,
against the CQS strain. However, 10m exhibited a high resistance
factor (w39) within the series but is 3.5 times less active than CQ,
against the CQR strain. Replacement of the ethyl ester with iso-
propyl ester (10nes), in general showed a decrease in in vitro anti-
plasmodial activity against the CQS strain, although a reverse trend
was observed for the CQR strain, especially in case of 10s, 10p and
10r, where replacing C-4 phenyl by p-nitrophenyl (10s) or o-
nitrophenyl (10p and 10r) groups, in addition to incorporating an
isopropyl ester, resulted in an increase of activity in that order,
rendering 10r as the most potent (IC50 3.6 nM, 56 times more
potent than CQ, Table 2) of all these compounds. These results are in
accordance with the trend observed in case of N, N-bis(7-
chloroquinolin-4-yl)alkane diamines, wherein the alkyl spacer
consisting of four carbon atoms showed optimum potency [24].
The better anti-plasmodial activity of phenyl-substituted
pyrimidine compounds against the CQR strain may be attributed
to optimal fitting of these compounds in the active site of PfDHFR
results in a significant increase in anti-plasmodial activity as well as
resistance against the CQR strain, although it has little effect on
activities against the CQS strain (Table 2). Also, the corresponding o-
, m- or p-nitro derivatives showed considerable variation in anti-
plasmodial activity (Table 2). Comparison of compounds (10n, 10q,
10s) having an identical spacer reveals that the p-NO2 substituted
10s (IC50 175.8 nM) is more active than the o-/m-NO2 substituted
compounds 10q and 10n, respectively, but are less active than the
unsubstituted ethyl ester analog 10b. Further, comparison with the
butyl spacer analog suggests that o-NO2 phenyl derivative 10r is
more potent than the m-NO2 counterpart 10o, as well as its ethyl
ester analog 10c, against the CQR strain. Compound 10r was found
to be the most active compound in this series against the CQR strain
with anti-plasmodial activity (IC50 3.6 nM), 56 times more than CQ
(IC50 201.8 nM) and comparable to artesunate (IC50 2.8 nM)
(Table 2).
Comparing 10j (IC50 28096.7 nM) possessing the branched chain
spacers with identical carbon linear spacer analog 10b (IC50
52.2 nM, Table 2) led to significant decrease in activity. Thus,
compound 10j displayed a 540-fold decrease in activity compared
to 10b, against the CQR strain. Likewise, 10k possessing a branched
C-6 spacer depicted a decrease (IC50 860.4 nM, Table 2) in activity
against linear alkyl (C-7) spacer analog 10d (Table 2), but higher
than 10j. Comparing 10j and 10k, with branched chain spacers, not
only was the anti-plasmodial activity of the latter against both CQS
leading to a favorable conformation for
p-p interaction with the
heme functionality. Moreover, the introduction of a nitro substit-
uent on the phenyl ring at the C-4 position of the pyrimidine core
Fig. 3. Titration of 10r (A) and 10c (B) with monomeric heme at pH 7.5.

K. Singh et al. / European Journal of Medicinal Chemistry 52 (2012) 82e97
85
3.2. Cytotoxicity and antiviral activity
Antiviral activity of the compounds 10aec, 10len, 10p and 10r,
which were active against the CQR strain was also evaluated against
(i) parainfluenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus
B4, Punta Toro virus in vero cell cultures, (ii) herpes simplex virus
ꢀ1 (HSV-1; KOS), herpes simplex virus-2 (HSV-2; G), vaccinia virus,
vesicular stomatitis virus, herpes simplex virus-1 (TKꢀKOS ACVR),
cytomegalovirus, varicella-zoster virus in HEL cell cultures, (iii)
vesicular stomatitis virus, coxsackie virus B4, respiratory syncytial
virus in HeLa cell cultures, (iv) influenza A virus (H1N1 and H3N2)
and influenza B virus in MDCK cell cultures and (v) feline corona
virus (FIPV) and feline herpes virus activity in CRFK cell cultures (SI
Tables S1eS7). Unfortunately, no significant antiviral activity was
noted at subtoxic concentrations. Most compounds were some-
what cytotoxic to the different cell lines (confluent non-
proliferating cultures), but especially cytostatic against prolifer-
ating (Vero/MDCK/CRFK) cell cultures. Chloroquine has been
shown to inhibit HIV through blockade of viral entry via inhibition
of endosomal acidification [25,26]. Compounds 10aec, len, p, r
were also tested against HIV-1 as well as HIV-2 (SI Table S8) in
human T-lymphocyte (CEM) cell cultures. However, none of the
compounds were active at subtoxic concentrations.
Fig. 4. The 300 MHz 1H spectra of 10c upon addition of heme (50 mol % in 40% DMSO-
D2O\ D2SO4 (10
m
l)) [Dd for peak: a ¼ 0.025, b ¼ 0.105, c ¼ 0.052, d ¼ 0.029, e ¼ 0.032,
f ¼ 0.032].
Generally, the compounds exhibit a relatively high cytostatic
activity but displayed a fairly safe selectivity index (except 10m and
10n) (Table 2) in the range of 10.04e638 against MDCK cell
cultures. The most active compound 10r with an IC50 value of
3.6 nM against CQR strain exhibited a highest selectivity index
(SI ¼ 638), 10l with an IC50 value of 160.8 nM against CQR strain
exhibited a high selectivity index (361.9). However, 10m having an
IC50 value of 1659.8 nM was most toxic with a selectivity index of
0.48.
and CQR strains increased, but it also showed an increased resis-
tance factor (Table 2). The replacement of linear chain spacers with
o- and p- linked aryl groups (10h and 10i, respectively), led to much
higher IC50 (Table 2) values compared to former against CQR strain,
while only moderate activity was observed in case of 10i against
CQS strain. These findings could be attributed to the increased steric
bulk (10h more than 10i) affecting the interaction of the iron center
of heme with the compounds. This has been corroborated by per-
forming the titration of monomeric heme with both 10h and 10i.
While the titration of heme (2.4
increasing concentration of 10h (0e85.7
m
M, DMSO:H2O/4:6, v/v) with
3.3. Insight into the mode of action and Fe(III)PPIX-10c/10r
association constants
mM, DMSO:H2O/4:6, v/v)
revealed no change in the absorbance at 402 nm (Fig. 2a), the
addition of 10i, in a similar way showed marked gradual decrease in
absorbance at 402 nm. The decrease in absorbance continued until
The plausible mechanism of in vitro anti-plasmodial action
against CQR strain has been investigated for 10c and 10r, found to be
most potent of the series of the compounds reported herein (IC50
26.1 nM, 10c and IC50 3.6 nM, 10r). These compounds are also
expected to bind to heme [Fe(III)PPIX] (hydroxo or aqua complex of
the concentration of 10i was 8.85
mM (Fig. 2b), representing
1:4 molar ratio of heme and 10i. Hence, the flexibility, chain length
and steric constraints of the spacer linking quinoline moiety and
pyrimidine unit seem to play a role in anti-plasmodial activity of
these derivatives. Although the in vitro activity of 10aec, 10p and
especially 10r (IC50 3.6 nM) was superior to CQ (IC50 201.8 nM)
against the CQR strain, these compounds suffer from high ClogP
values (Table 2), which are suggestive of the fact that these possess
limited aqueous solubility, which in fact is not a serious limitation
in view of recent advancements in formulation methods.
ferriprotoporphyrin IX) in solution and inhibit aggregation to b-
hematin, in much the same way as CQ itself. The higher ClogP
values (Table 2) of these compounds being higher than CQ neces-
sitated the use of aqueous DMSO as solvent. Also in 40% aqueous
DMSO solution, heme is expected to be monomeric, while in purely
aqueous solutions, it exists in aggregated form. Further, it is known
that CQ binds to heme dimer (m-oxo heme) in vitro and can also
Fig. 5. Titration spectra of 10r (A) and 10c (B) with m-oxo heme at pH 5.8.

86
K. Singh et al. / European Journal of Medicinal Chemistry 52 (2012) 82e97
Fig. 6. Absorption spectra of 10c (A) and 10r (B) in the presence of different CT DNA concentration inset show zoom between 280 and 380 nm.
inhibit the formation of
b
-hematin, the in vitro analog of hemozoin
A 1:1 stoichiometry of the most stable complexes of both 10r
and 10c with monomeric heme at pH 7.5 and 5.6 was deduced from
the Job’s plot (SI Figure S3) [33,34]. The absorbance at 402 nm got to
maximum when mole fraction of 10r approached 0.5. This is in
good agreement with the association constant (log K) values cor-
responding to the most stable 1:1:heme: 10r species present in
solution, obtained by fitting the titration data in Hyp Spec - a non-
linear least square fitting programme. The measured log K values
for both 10r and 10c (Table 3) are also in good agreement with the
values reported in literature for CQ [32,35,36]. Thus, the formation
of synthetic Fe(III)PPIX-10r complex, as demonstrated above is
suggestive of inhibition of formation of b-hematin and presumably
results in anti-plasmodial activity of these compounds in a fashion
similar to that of CQ.
Titration of constant concentrations of heme in aqueous NaOH
solution at the physiological pH 5.8 of Plasmodial food vacuole [35]
[27e30]. Therefore the binding assay was also extended to heme
dimer also. The interaction of 10c and 10r with monomeric heme
was followed by spectrophotometric titration at pH 7.4 and 5.6 (the
approximate pH of food vacuole), as described previously [31]. The
spectral changes (Fig. 3A, SI Figure S1), upon addition of increasing
amounts (0e70 mM) of 10r to a solution of heme (2.4 mM, HEPES
buffer pH 7.4) showed substantial decrease of intensity of the Fe(III)
PPIX Soret band at 402 nm with no shift in the absorption
maximum. The longer wavelength Q-bands (494 and 537 nm) of
the metalloporphyrin also decrease in intensity (SI Figure S2) [32].
A sharp isosbestic point was observed at 416 nm (pH 7.4) and at
412 nm (pH 5.6). The association constants for the complexes
formed between monomeric Fe (III) PPIX and 10r at pH 7.5 and 5.6
were calculated from titration data and are presented in Table 3.
The association constant of 10c (Fig. 2A) is found to be greater
(log K 6.018) than 10r (log K 5.078). Decrease in the apparent pH
from 7.5 to 5.6 caused a fairly modest decrease in log K values of
both 10c and 10r, indicating a strong binding to heme even at acidic
pH. The binding of 10c and 10r was also assessed from their 1H
NMR titration with heme. Thus, while 1H NMR spectrum of 10r
depicted considerable broadening of signals under the conditions
(40% DMSO-d6 in D2O) of recording and was inconclusive, the
variation in the chemical shift in signals corresponding to quinoline
unit of 10c were clear (Fig. 4), upon addition of heme.
allowed understanding of the interaction of 10r and 10c with
heme. With addition of increasing amount of 10r to the solution of
-oxo heme, the absorbance of Soret band (362 nm) decreased
m-oxo
m
appreciably with significant red shift (362e405 nm, Fig. 5A). The
association constants and stoichiometry for both 10r and 10c were
determined from titration data and are presented in Table 3.The
calculated association constant of 10r (log K 6.31) is greater than
10c (log K 6.09) and corresponds to 1:1 stoichiometry of the 10r:
m-
oxo heme complex. The comparison of association constants of 10r
Fig. 7. Fluorescence emission spectra of 10r at 380 nm in the presence of increasing concentrations of CT DNA (A) and pUC18 DNA (B).

K. Singh et al. / European Journal of Medicinal Chemistry 52 (2012) 82e97
87
Fig. 8. The interactions of the substrate 10r in the active site cavity of the enzyme Pf DHFR. The hydrogen bonding interactions with the amino acids Ile14, Leu46, Asn108, Ser111
and Try170 are shown with dotted yellow lines.
and 10c for monomeric and
compounds bind strongly with
m
-oxo heme reveals that both
other factors such as degree of accumulation of drug in food
vacuole. Further, 10r showed dose dependent inhibition of b-
m
-oxo heme than monomeric heme
and inhibit hemozoin formation by blocking the growing face of
heme resulting in the observed anti-plasmodial activity. Further, all
hematin formation (SI Figure S4) with an IC50 value lower than
those reported for CQ and quinine, thus demonstrating a better
ability to interact with Fe(III)PPIX. Overall, these compounds
the compounds were also screened for in vitro inhibition of
b-
hematin formation using -hematin inhibitory assay in order to
b
showed strong b-hematin inhibition which seems to be their
further confirm their mechanism of action based on interference
with the heme detoxification process [37,38].
preferred mode of action.
As shown in Table 4, there is a general correlation between anti-
3.4. DNA binding affinity
plasmodial activity and inhibition of
b-hematin formation, but the
same generalisation does not hold for compound 10h, as, in spite of
DNA binding has been considered previously as one of the
possible mechanisms for anti-plasmodial activity and has been
studied using spectrophotometry [39], spectrofluorimetry [40],
DNA melting [41], viscometry [42] etc. Recently, some 4-
aminoquinoline derivatives have been shown to interact with
having 100% b-hematin inhibition(Table 4), it is one of the least
active (IC50 27320.9 nM, CQR and IC50 14828.3 nM, CQS) compounds
of the series. However, it must be noted that anti-plasmodial
activity not only depends upon the b-hematin inhibition but also on
Scheme 1. Synthesis of 10.

Table 1
Synthesis of 2-aminopyrimidines 10.
S. No.
Compounda
Structure
Yield (%)
O
N
O
CH3
1.
10a
89
NH(CH2)2NH
N
CH3
Cl
N
O
N
O
CH3
2.
10b
84
NH(CH2)3NH
N
N
CH3
Cl
O
N
O
CH3
3.
10c
77
NH(CH2)4NH
N
N
CH3
Cl
O
N
O
CH3
4.
10d
67
NH(CH2)7NH
N
N
CH3
Cl
O
N
O
CH3
5.
6.
7.
10e
10f
10g
72
64
65
NH(CH2)8NH
N
N
CH3
Cl
O
N
O
CH3
NH(CH2)10NH
N
CH3
Cl
N
O
N
O
CH3
NH(CH2)12NH
N
CH3
Cl
N

K. Singh et al. / European Journal of Medicinal Chemistry 52 (2012) 82e97
89
Table 1 (continued )
S. No.
Compounda
Structure
Yield (%)
O
N
O
CH3
8.
10hb
53
N
H
N
CH3
NH
N
Cl
O
N
O
CH3
9.
10ib
57
HN
N
N
H
N
CH3
Cl
O
N
O
CH3
CH3
H
N
10.
10j
58
N
CH3
HN
N
Cl
O
N
O
CH3
CH3
H
11.
10k
55
HN
N
N
N
CH3
Cl
Cl
CH3
N
O
N
O
CH3
12.
10l
92
NH(CH2)3NH
N
CH3
CH3
O
N
O
CH3
13.
10m
90
NH(CH2)4NH
N
CH3
Cl
N
O2N
O
CH3
CH3
N
O
14.
10n
71
(continued on next page)
NH(CH2)3NH
N
N
CH3
Cl

90
K. Singh et al. / European Journal of Medicinal Chemistry 52 (2012) 82e97
Table 1 (continued )
S. No.
Compounda
Structure
Yield (%)
O2N
O
CH3
CH3
N
O
15.
10o
78
NH(CH2)4NH
N
N
CH3
Cl
O2N
NH(CH2)2NH
N
O
CH3
CH3
N
O
16.
10p
66
N
CH3
Cl
O2N
O
CH3
CH3
N
O
CH3
17.
10q
73
NH(CH2)3NH
N
Cl
Cl
N
O2N
O
CH3
CH3
N
O
18.
10r
85
NH(CH2)4NH
N
N
CH3
NO2
O
CH3
CH3
19.
10s
82
N
O
NH(CH2)3NH
N
N
CH3
Cl
a
THF, K2CO3, rt.
MeCN, K2CO3, 80 ꢁC.
b
DNA [37] presumably through ionic interactions between phos-
phate groups of DNA and protonated amine sites resulting in
stabilization of the helical configuration of DNA against thermal
denaturation [41,43]. Additionally, interactions between aromatic
nucleuses of the drug with nucleotide bases might also contribute.
Binding of the plasmid DNA with CQ resulted in the elevation of the
thermal melting temperature (Tm) of DNA in addition to other
effects [44e47]. We therefore investigated the binding of the most
active of the series, 10c and 10r toward GC rich Calf Thymus DNA
(CT DNA) as well as AT rich pUC18 DNA through stepwise addition
of small increments of DNA to a solution the compounds at
constant concentration as well as physiological pH (Fig. 6A and B).
A
progressive decrease in the characteristic quinoline ring
absorptions at 320 and 340 nm attributable to the intercalation of
the quinoline into DNA was indicated.
The isosbestic point at 350 nm indicated that the spectra of
limiting systems (i.e. the spectra of free and completely bound
drug) intersect and permitted the selection of a single wavelength
for study of complex formation. The binding constant (log K: 10c
4.67 and 10r 3.39) was calculated from BenesieHildebrand

K. Singh et al. / European Journal of Medicinal Chemistry 52 (2012) 82e97
91
Table 2
In vitro anti-plasmodial activity, cytotoxicity, resistance factor and selectivity index of compounds 10a-s.
Compound
IC50 (nM)
Resistance factore
ClogPf
Cytotoxicity (
m
M)g
MICi
Selectivity indexj
CQS (3D7)a,b
CQR (K1)c,d
CC50
h
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
21.7
247.5
202.3
1134.9
252.2
1776.8
264.8
14828.3
644.4
15888.5
286.7
63.1
139.3
52.2
26.1
6.43
0.210
0.129
0.544
2.047
0.2744
3.22
1.842
9.600
1.7683
3.00
2.5480
39.422
0.385
0.2756
0.5555
0.1173
0.2
0.3518
140.39
0.5418
6.83
6.82
7.35
8.94
9.47
10.53
11.58
8.81
8.80
5.31
8.28
5.64
5.75
7.30
7.30
6.89
7.30
7.30
7.30
5.1
1.4
0.8
0.9
e
0.8
0.8
0.8
e
10.04
15.33
34.41
e
617.7b
516.4
487.7
853.7
27320.9
6186.4b
28096.7
860.4b
160.8
1659.8b
230.5b
192.1
96.0
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
58.2
0.8
0.5
e
20
4
0.8
e
361.89
0.48
2.1
e
42.1
598.7
697.2
172.9
1955.1
18.2
499.9
1.4
5.3
2.2
e
ꢂ0.8
e
22.90
e
229.3
3.6
175.8
201.8
2.8
2.3
e
4.0
e
638
e
10s
2
Artesunate
e
e
e
1.06
e
e
e
a
CQ sensitive strain.
primary screening.
CQ resistant strain.
secondary screening.
calculated by dividing IC50 values of PFK1 by PF3D7.
calculated from Chem draw Ultra 11.0.
determined on Madin Darby canine kidney (MDCK)cells.
b
c
d
e
f
g
h
50% cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan- based MTS assay (reference drugs used: Oseltamivir
carboxylate CC50/MIC >100, Ribavirin CC50/MIC >100, Amantadine CC50/MIC >200 and Rimantadine CC50/MIC >200).
i
minimum compound concentration that causes a microscopically detectable alteration of normal cell morphology.
Selectivity Index (S.I.) is calculated as CC50/IC50 (K1 Strain) ratio.
j
equation [48] and suggested strong interaction with CT DNA.
Comparison of binding constants reveals that 10c binds 20 times
more strongly with CT DNA than 10r.
Groove binding or electrostatic binding of the phosphate back-
bone of DNA gives rise to small changes in thermal denaturation
temperature compared to the intercalation pathway due to stabi-
lization of Watson crick base paired duplex [43,48,49,50]. In the
direction of evaluating strength of interaction of 10c and 10r with
DNA, the thermal behavior (SI Figure S5) of CT DNA in the presence
of drug was evaluated. The thermal denaturation temperature of CT
DNA (60 ꢁC), in the presence of 10c and 10r recorded an increase in
Altering the DNA base composition to see its effect on the drug
binding, we studied the interaction of 10r with GC rich CT DNA by
fluorometric titration. The emission band at 380 nm of 10r (Fig. 7),
decreases in intensity with increasing concentration and eventually
gets completely quenched. The binding constant (log K) calculated
from the fluorescence data is 4.67. Similar titration with AT rich
pUC18 DNA (log K ¼ 5.27) revealed higher affinity of 10r toward AT
rich pUC18 DNA unlike CQ which is known to interact strongly with
GC rich DNA [46]. The strong interaction of the pyrimidine analog
with AT rich pUC18 DNA further suggests that these compounds
might target parasite DNA which has unusually a high AT content
D
Tm of 3 ꢁC and 2.5 ꢁC, respectively (SI Table S9), suggesting
primary groove binding and/or partial intercalative nature of the
interaction.
3.5. Molecular docking analysis
[47] in addition to b-hematin inhibition. The binding of ligand to
Many of the anti-plasmodial agents are known to interact
nucleic acid of DNA (especially AT rich DNA) may possibly induce
conformational changes depending upon the strength and mode of
its interaction with nucleic acids and thus result in increase in the
thermal denaturation temperature, as observed for CQ.
with Pf DHFR for their anti-plasmodial activity. Especially
Table 4
b-hematin inhibitory assay.
Compound
% Inhibitiona
Compound
% Inhibitiona
10a
10b
10c
10d
10e
10f
10g
10h
10i
96
100
100
89
56
e
10l
66
100
79
92
e
Table 3
10m
10n
10o
10p
10q
Binding constant (log K) For 10r and 10c.
Compound
Monomeric heme
m
-oxo heme
CT DNA
log K ꢃ
log K ꢃ
s
log K ꢃ
s
s
89
pH 5.6
pH 7.5
pH 5.8
e
10r
100 (2)b
94
10ra
4.97 ꢃ 0.005
5.284 ꢃ 0.01
4.82
5.075 ꢃ 0.01
6.018 ꢃ 0.008
5.2b
6.31 ꢃ 0.01
6.09 ꢃ 0.02
5.6c
3.39 ꢃ 0.02
4.67 ꢃ 0.04
ND
53
100
35
e
10s
10ca
CQc
18b
324b
CQ
10j
10k
Quininec
Stoichiometry
1:1
1:1
ND
a
a
Calculated from Hyp Spec.
Refer to text Ref. [35].
Refer to text Ref. [36].
The percentage inhibition at 1 mg/ml (highest concentration tested).
b
c
b
c
IC50
(mM).
Refer to text Ref. [37].

92
K. Singh et al. / European Journal of Medicinal Chemistry 52 (2012) 82e97
pyrimethamine (Chart 1) is known to produce anti-plasmodial
effect by binding to Pf DHFR. The best known lead compound for
this inhibitory activity is WR99210 (5). It is worth comparing the
performance of the compounds designed in this work with that of
5, using molecular docking methods. The crystal structure (PDB id:
1J3I, a structure of wild-type Pf DHFR-TS complexed with WR99210,
NADPH, dUMP) was considered for molecular docking analysis.
After appropriately preparing the protein structure of Pf DHFR,
docking of 5 and 10r were carried out. Fig. 8 shows the top scoring
binding pose of 10r in the active site of Pf DHFR. The docking score
for 10r is ꢀ24.5, which is much better than that of the lead
compound 5 (ꢀ20.1). The origin of this improvement in docking
score can be traced to the increased number of stabilizing inter-
actions between 10r and Pf DHFR, in comparison to the interactions
between 5 and Pf DHFR. 10r shows hydrogen bonding interaction
with Tyr170 and Asn108 residues; these two interactions are
common to 10r as well as to 5. In addition, 10r shows hydrogen
bonding interaction with Ser111 and Leu46 residues.
Integrative Medicine (CSIR), Jammu, under electron impact at 70 eV
on a Bruker Daltonics Esquire 3000 spectrometer. Elemental anal-
ysis was performed on FLASH EA 112 (Thermo electron Corpora-
tion) analyzer at Department of Chemistry, Guru Nanak Dev
University, Amritsar and the results are quoted in %. IR recorded on
FTIR Shimadzu 8400 Fourier-transform spectrophotometer in the
range 400e4000 cmꢀ1 using chloroform as medium. Melting
points were determined in open capillaries and are uncorrected.
For monitoring the progress of a reaction and for comparison
purpose, thin layer chromatography (TLC) was performed on pre-
coated aluminum sheets Merck (60F254, 0.2 mm) using an appro-
priate solvent system. The chromatograms were visualized under
UV light. For column chromatography silica gel (60e120 mesh) was
employed and eluents were ethyl acetate/hexane or ethyl acetate/
methanol mixtures.
5.2. General procedure for the synthesis of 2-aminopyrimidines
(10aeg and 10jes)
These additional stabilizing interactions ensure that 10r adopts
a slightly different (and improved) pose in comparison to that of 5.
These improved interactions can be considered as deterministic
factors for the improved anti-plasmodial activity of 10r.
To the stirred solution of 8 (2 mmol) and potassium carbonate
(5 mmol) in dry THF (30 ml), a solution of appropriate 4-
aminoquinoline 9 (1.0 mmol) in dry THF (50 ml) was added. The
reaction mixture was stirred for 48 h at room temperature. The
reaction mixture was filtered and THF was removed under vacuum.
The residue was purified by column chromatography using MeOH/
EtOAc as eluent to obtain corresponding 10, which was recrystal-
lized from DCM/hexane. Using this procedure the following
compounds were isolated.
4. Conclusions
An efficacious transformation for converting readily available 3,
4-dihydropyrimidinones (DHPMs) to 2-aminopyrimidine based 4-
aminoquinolines is presented. The compounds showed anti-plas-
modial activity identical to or superior to CQ. Structureeactivity
relationship has been drawn leading to the identification of at
least one compound 10r as lead compound in this series. The tested
compounds did not depict any antiviral activity and were cytotoxic.
Binding interaction of representative potent compounds with
5.2.1. 5-Ethoxycarbonyl-6-methyl-4-phenyl-2-[(7-chloroquinolin-
4-ylamino)ethylamino] pyrimidine (10a)
White solid. Rf: 0.4 (8% MeOH/EtOAc). Yield: 89%. IR (KBr): nmax
770, 1267, 1709, 2928, 3331 cmꢀ1 1H (300 MHz, CDCl3, 25ꢁC):
d 1.00
heme and
m
-oxo heme using UVevisible and NMR experiments
(t, J ¼ 7.2 Hz, 3H, estereCH3), 2.55 (s, 3H, C6eCH3), 3.47 (m,
J ¼ 4.5 Hz, 2H, CH2), 3.94 (m, 2H, CH2), 4.10 (q, J ¼ 7.2 Hz, 2H,
estereCH2), 5.81 (br, 1H, NH), 6.34 (d, 1H, ArH), 6.44 (br, 1H, NH),
7.26e7.85 (m, 8H, ArH), 8.44 (s, 1H, ArH). 13C NMR (75 MHz, CDCl3,
furnished log K values identical with CQ binding and pointed to 1:1
stoichiometry of the most stable complexes in solution. DNA (both
GC and AT rich) binding affinity using absorption and spectroflu-
orometric data indicated stronger interaction of 10c (20 fold) than
10r with CT DNA, however, 10r showed higher affinity toward AT
rich pUC18 DNA suggesting targeting of parasite AT rich DNA by 10r
25ꢁC):
d 13.5, 23.0, 40.1, 61.3, 98.4, 116.4, 121.3, 124.8, 128.1, 128.3,
128.6, 138.6, 148.8, 149.9, 151.8, 162.1 and 168.3. Anal. Calcd. for
C25H24N5O2Cl: C, 65.00; H, 5.20; N, 15.10; Found: C, 64.92; H, 4.89;
N, 15.32. MS: m/z 462 (Mþþ1).
in addition to b-hematin inhibition as possible mode of action of
these compounds. DNA melting experiments suggested primary
groove binding and/or partial intercalative nature of interaction of
10c and 10r with CT DNA. Thus, the strong electrostatic interaction
of newly designed pyrimidine analogs with AT rich DNA and
blockage of heme polymerisation by complexation of drug with
heme contribute to the observed antimalarial activity in nano
molar range. Further, molecular docking analysis of 10r in the active
site of Pf DHFR indicated superior binding compared to WR99210.
Thus, drug 10r acts on multiple targets (Heme, Enzyme involved in
biosynthesis of DNA (DHFR), parasite DNA) which accounts for its
high anti-plasmodial activity.
5.2.2. 5-Ethoxycarbonyl-6-methyl-4-phenyl-2-[(7-chloroquinolin-
4-ylamino)propylamino]pyrimidine (10b)
White solid. Rf: 0.6 (8% MeOH/EtOAc). Yield: 84%. IR (KBr): nmax
772, 1268, 1707, 2933, 3397 cmꢀ1 1H (300 MHz, CDCl3, 25ꢁC):
d 0.95
(t, J ¼ 7.2 Hz, 3H, estereCH3), 1.98 (q, J ¼ 6.3 Hz, 2H, CH2), 2.50 (s,
3H, C6eCH3), 3.45 (q, J ¼ 6.3 Hz, 2H, CH2), 3.66 (q, J ¼ 6.6 Hz, 2H,
CH2), 4.1 (q, J ¼ 7.2 Hz, 2H, estereCH2), 5.63(s, 1H, NH), 6.39 (d,
J ¼ 5.4 Hz, 1H, ArH), 7.38e7.54 (m, 7H, ArH), 7.90 (d, J ¼ 1.8 Hz,ꢁ1H,
ArH), 8.48 (d, J ¼ 5.4 Hz, 1H, ArH). 13C NMR (75 MHz, CDCl3, 25 C):
d
13.5, 23.0, 38.2, 39.8, 61.1, 98.8, 117.2, 121.0, 125.1, 127.7, 128.4,
129.6, 134.7, 149.0, 149.6, 151.8, 161.6, 167.4 and 168.4. Anal. Calcd.
For C26H26N5O2Cl: C, 65.60; H, 5.46; N, 14.70; Found: C, 65.59; H,
5.40; N, 14.50. MS: m/z 476.1 (Mþ).
5. Experimental
5.1. General
5.2.3. 5-Ethoxycarbonyl-6-methyl-4-phenyl-2-[(7-chloroquinolin-
4-ylamino)butylamino]pyrimidine (10c)
All liquid reagents were dried/purified following recommended
drying agents and/or distilled over 4 Å molecular sieves. THF was
dried (Na- benzophenone ketyl) under nitrogen. 1H NMR
(300 MHz) and 13C (75 MHz) NMR spectra were recorded in CDCl3
on a multinuclear Jeol FT-AL-300 spectrometer with chemical shifts
White solid. Rf: 0.4 (EtOAc). Yield: 77%. IRꢁ(KBr): nmax 772, 1268,
1708, 2986, 3383cmꢀ11H (300 MHz, CDCl3, 25 C):
d
0.94 (t, J ¼ 7.2Hz,
3H, estereCH3), 1.80 (q, J ¼ 6.9 Hz, 4H, CH2), 2.47 (s, 3H, C6eCH3),
3.37 (q, J ¼ 6.3 Hz, 2H, CH2), 3.60 (q, J ¼ 7.2 Hz, 2H, CH2), 4.05 (q,
J ¼ 7.2 Hz, 2H, estereCH2), 5.06 (s, 1H, NH), 5.45 (s, 1H, NH), 6.38 (d,
J ¼ 5.4 Hz, 1H, ArH), 7.3e7.58 (m, 7H, ArH), 7.94 (d, J ¼ 2.1 Hz,ꢁ1H,
ArH), 8.51 (d, J ¼ 5.4 Hz, 1H, ArH). 13C NMR (75 MHz, CDCl3, 25 C):
being reported in parts per million (
methylsilane (TMS,
0.0, 1H NMR) or chloroform (CDCl3,
NMR). Mass spectra were recorded from Indian Institute of
d
) relative to internal tetra-
d
d
77.0, 13
C

K. Singh et al. / European Journal of Medicinal Chemistry 52 (2012) 82e97
93
d
13.5, 22.9, 25.8, 27.4, 40.6, 42.9, 61.1, 99.0, 115.4, 117.0, 120.8, 125.2,
5.2.8. 5-Ethoxycarbonyl-6-methyl-4-phenyl-2-[2-(7-
127.8, 128.2, 128.7, 129.4, 134.8, 139.0, 149.0, 149.6, 151.9, 161.2 and
168.7. Anal. Calcd. For C27H28N5O2Cl: C, 66.18; H, 5.72; N, 14.30;
Found: C, 66.10; H, 5.63; N, 13.95. MS: m/z 490.1 (Mþ).
chloroquinolin-4-ylamino)propylamino]pyrimidine (10j)
White solid. Rf: 0.7 (5% MeOH/EtOAc). Yield: 58%. IR (KBr): nmax
716, 1127, 1700, 2940, 3446 cmꢀ1 1H (300 MHz, CDCl3, 25ꢁC):
d
1.00
(t, J ¼ 7.2 Hz, 3H, estereCH3), 1.46 (d, J ¼ 6.6 Hz, 3H, CH3), 2.54 (s,
3H, C6eCH3), 4.10 (q, J ¼ 6.0 Hz, 2H, estereCH2), 4.69 (m, 1H, CH),
5.35 (br, 1H, NH), 6.39 (d, J ¼ 5.4 Hz, 1H, ArH), 6.60 (br, 1H, NH),
7.50e7.84 (m, 8H, ArH), 8.42 (s, 1H, ArH). 13C NMR (75 MHz, CDCl3,
5.2.4. 5-Ethoxycarbonyl-6-methyl-4-phenyl-2-[(7-chloroquinolin-
4-ylamino)heptylamino]pyrimidine (10d)
White solid. Rf: 0.5 (EtOAc). Yield: 67%. IR (KBr): nmax 769, 1264,
1700, 2930, 3278 cmꢀ1 1H (300 MHz, CDCl3, 25ꢁC):
d
0.95 (t,
25ꢁC):
d 13.1, 19.1, 22.5, 29.2, 45.9, 60.9, 98.1, 120.9, 124.4, 127.7,
J ¼ 7.2 Hz, 3H, estereCH3), 1.44e2.00 (m, 10H, CH2), 2.48 (s, 3H,
C6eCH3), 3.29 (d, J ¼ 5.1 Hz, 2H, CH2), 3.49 (t, J ¼ 6.3 Hz, 2H, CH2),
4.05 (q, J ¼ 7.2 Hz, 2H, estereCH2), 4.93 (br, 1H, NH), 5.29 (br, 1H,
NH), 6.40 (d, J ¼ 5.4 Hz, 1H, ArH), 7.34e7.65 (m, 7H, ArH), 7.96 (d,
J ¼ 1.8 Hz, 1H, ArH), 8.53 (d, J ¼ 5.7 Hz, 1H, ArH). 13C NMR (75 MHz,
127.8, 128.0, 128.1, 128.2, 128.3, 138.2, 149.5, 151.5, 161.1 and
167.9.Anal. Calcd. For C26H26N5O2Cl: C, 65.60; H, 5.46; N, 14.70;
Found: C, 65.39; H, 5.43; N, 14.45. MS: m/z 475.5 (Mþ).
5.2.9. 5-Ethoxycarbonyl-6-methyl-4-phenyl-2-[5-(7-chloroquinolin-
4-ylamino)-2-methylpentylamino] pyrimidine (10k)
CDCl3, 25ꢁC):
d 13.0, 22.5, 26.2, 26.6, 28.4, 28.6, 29.0, 40.7, 42.8, 98.6,
116.6, 120.3, 123.5, 124.8, 127.5, 127.7, 128.4, 128.9, 134.4, 138.8,
148.7, 149.2, 151.6, 160.9 and 168.4. Anal. Calcd. For C30H34N5O2Cl:
C, 67.13; H, 6.39; N, 13.17; Found: C, 67.43; H, 6.05; N, 13.45. MS: m/z
532.1 (Mþ).
Viscous liquid. Rf: 0.6 (8% MeOH/EtOAc). Yield: 55%. IR (KBr):
nmax 716, 1127, 1700, 2940, 3446 cmꢀ1 1H (300 MHz, CDCl3, 25ꢁC):
d
1.03e1.16 (m, 7H, estereCH3 & 2 ꢄ CH2), 1.36 (d, J ¼ 1.5 Hz, 3H,
CH3), 2.58 (s, 3H, C6eCH3), 3.64 (m, 4H, 2 ꢄ CH2), 4.12e4.24 (m, 3H,
CH & estereCH2), 5.24 (br, 1H, NH), 5.64 (br, 1H, NH), 6.48 (s,
J ¼ 5.4 Hz, 1H, ArH), 7.37e7.72 (m, 8H, ArꢁH), 8.07 (s, 1H, ArH), 8.60
5.2.5. 5-Ethoxycarbonyl-6-methyl-4-phenyl-2-[(7-chloroquinolin-
4-ylamino)octylamino]pyrimidine (10e)
(s, 1H, ArH). 13C NMR (75 MHz, CDCl3, 25 C):
d 13.4, 17.8, 17.9, 26.1,
White solid. Rf: 0.6 (EtOAc). Yield: 72%. IR (KBr): nmax 770, 1263,
26.9, 31.5, 31.7, 32.3, 33.1, 41.3, 43.4, 46.8, 49.3, 61.0, 98.9, 115.3,
120.8, 120.9, 125.3, 127.8, 128.1, 128.3, 128.4, 129.4, 135.0, 139.0,
150.0, 151.5 and 168.7. Anal. Calcd. For C29H32N5O2Cl: C, 67.23; H,
6.23; N, 13.52; Found: C, 67.09; H, 6.12; N, 13.36. MS: m/z 518 (Mþ).
1712, 2928, 3269 cmꢀ1 1H (300 MHz, CDCl3, 25ꢁC):
d 0.87 (t,
J ¼ 7.6 Hz, 3H, estereCH3), 1.30 (br, 10H, CH2), 1.52 (d, J ¼ 6.6 Hz, 2H,
CH2), 1.65 (q, J ¼ 6.9 Hz, 2H, CH2), 2.40 (s, 3H, C6eCH3), 3.21 (d,
J ¼ 7.2 Hz, 2H, CH2), 3.41 (t, J ¼ 6.6 Hz, 2H, CH2), 3.97 (q, J ¼ 7.2 Hz,
2H, estereCH2), 5.13 (br, 1H, NH), 5.28 (br, 1H, NH), 6.31 (d,
J ¼ 5.7 Hz, 1H, ArH), 7.18e7. 60 (m, 7H, ArH), 7.86 (d, J ¼ 1.8 Hz,ꢁ1H,
ArH), 8.42 (d, J ¼ 5.1 Hz, 1H, ArH). 13C NMR (75 MHz, CDCl3, 25 C):
5.2.10. 5-Ethoxycarbonyl-6-methyl-4-methyl-2-[(7-chloroquinolin-
4-ylamino)propylamino]pyrimidine (10l)
White solid. Rf: 0.5 (10% MeOH/EtOAc). Yield: 92%. IR (ꢁKBr): nmax
d
13.5, 22.9, 26.7, 27.0, 28.8, 29.1, 29.2, 29.5, 41.2, 43.2, 61.0, 99.0,
801, 1261, 1701, 2930, 3419 cmꢀ1 1H (300 MHz, CDCl3, 25 C):
d 1.38
115.1, 117.0, 120.8, 125.2, 127.9, 128.2, 128.8, 129.3, 134.8, 139.2,
149.0, 149.7, 152.0, 161.2 and 168.9. Anal. Calcd. For C31H36N5O2Cl:
C, 68.19; H, 6.59; N, 12.8; Found: C, 68.03; H, 6.55; N, 12.02. MS: m/z
546.2 (Mþ).
(t, J ¼ 7.2 Hz, 3H, estereCH3), 2.01 (m, 2H, CH2), 2.44 (s, 6H, C6 &
C4eCH3), 3.44 (q, J ¼ 6.0 Hz, 2H, CH2), 3.67 (q, J ¼ 6.3 Hz, 2H, CH2),
4.36 (q, J ¼ 7.2 Hz, 2H, estereCH2), 5.57 (br, 1H, NH), 5.64 (br, 1H,
NH), 6.42 (d, J ¼ 5.4 Hz, 1H, ArH), 7.33 (m, 1H, ArH), 7.73 (d,
J ¼ 9.0 Hz, 1H, ArH), 7.96 (d, J ¼ 2.1 Hz, 1H, ArH), 8.52 (d, J ¼ 5.4 Hz,
5.2.6. 5-Ethoxycarbonyl-6-methyl-4-phenyl-2-[(7-chloroquinolin-
4-ylamino)decylamino]pyrimidine (10f)
1H, ArH). 13C NMR (75 MHz, CDCl3, 25ꢁC):
d 14.2, 23.7, 29.0, 38.3,
40.0, 61.0, 99.0, 121.1, 125.2, 128.8, 134.9, 149.7, 151.9, 161.3 and
167.3. Anal. Calcd. For C21H24N5O2Cl: C, 58.04; H, 5.80; N, 16.90;
Found: C, 57.84; H, 5.93; N, 16.63. MS: m/z 413.5 (Mþ).
Viscous liquid. Rf: 0.3 (1% MeOH/EtOAc). Yield: 64%. IR (KBr): nmax
775, 1256, 1708, 2985, 3325 cmꢀ1 1H (300 MHz, CDCl3, 25ꢁC):
d 0.86
(t, J ¼ 7.2 Hz, 3H, estereCH3),1.25 (br,14H, CH2),1.52 (q, J ¼ 6.9 Hz, 2H,
CH2), 1.67 (q, J ¼ 6.6 Hz, 2H, CH2), 2.41 (s, 3H, C6eCH3), 3.22 (q,
J ¼ 7.2 Hz, 2H, CH2), 3.41 (t, J ¼ 6.6 Hz, 2H, CH2), 3.97 (q, J ¼ 6.9 Hz, 2H,
estereCH2), 5.04 (br,1H, NH), 5.30 (br,1H, NH), 6.32 (d, J ¼ 5.4 Hz,1H,
ArH), 7.19e7.61 (m, 7H, ArH), 7.88 (d, J ¼ 2.1 Hz, 1H, ArH), 8.44 (d,
5.2.11. 5-Ethoxycarbonyl-6-methyl-4-methyl-2-[(7-chloroquinolin-
4-ylamino)butylamino]pyrimidine (10m)
White solid. Rf: 0.6 (10% MeOH/EtOAc). Yield: 90%. IR (KBr): nmax
760, 1257, 1695, 2935, 3431 cmꢀ1 1H (300 MHz, CDCl3, 25ꢁC):
d 1.37
J ¼ 5.7 Hz,1H, ArH).13C NMR (75 MHz, CDCl3, 25ꢁC):
d
13.5,14.5, 26.8,
(t, J ¼ 6.9 Hz, 3H, estereCH3), 1.82 (m, 4H, CH2), 2.41 (s, 6H, C6 &
C4eCH3), 3.40 (q, J ¼ 6.9 Hz, 2H, CH2), 3.57 (q, J ¼ 6.0 Hz, 2H, CH2),
4.35 (q, J ¼ 7.2 Hz, 2H, estereCH2), 5.10 (br, 1H, NH), 5.23 (br, 1H,
NH), 6.42 (d, J ¼ 5.4 Hz, 1H, ArH), 7.34 (m, 1H, ArH), 7.64 (d,
J ¼ 9.0 Hz, 1H, ArH), 7.96 (d, J ¼ 2.1 Hz, 1H, ArH), 8.53 (d, J ¼ 5.1 Hz,
27.1, 28.8, 29.2, 29.3, 29.4, 29.6, 41.3, 43.3, 61.0, 99.0, 115.0, 117.0,
120.8,125.3,127.9,128.2,128.5,129.4,134.9,139.2,149.8,151.6,161.2.
Anal. Calcd. For C33H40N5O2Cl: C, 69.00; H, 6.97; N, 12.20; Found: C,
68.93; H, 6.66; N, 11.82. MS: m/z 574.2 (Mþ).
1H, ArH). 13C NMR (75 MHz, CDCl3, 25ꢁC):
d 9.8, 12.0, 13.6, 26.8, 29.1,
5.2.7. 5-Ethoxycarbonyl-6-methyl-4-phenyl-2-[(7-chloroquinolin-
4-ylamino)dodecylamino]pyrimidine (10g)
47.1, 85.2, 106.9, 111.5, 114.9 and 138.0. Anal. Calcd. For
C22H26N5O2Cl: C, 61.75; H, 6.08; N,16.37; Found: C, 61.43; H, 6.15; N,
15.99. MS: m/z 428.1 (Mþ).
Viscous liquid. Rf: 0.4 (1% MeOH/EtOAc). Yield: 65%. IR (ꢁKBr): nmax
772, 1270, 1700, 2998, 3300 cmꢀ1 1H (300 MHz, CDCl3, 25 C):
d 0.93
(t, J ¼ 7.2 Hz, 3H, estereCH3),1.26 ( s,16H, CH2),1.57 (d, J ¼ 6.9 Hz, 2H,
CH2), 1.71 (d, J ¼ 6.9 Hz, 2H, CH2), 2.48 (s, 3H, C6eCH3), 3.24 (s, 2H,
CH2), 3.45 (q, J ¼ 6.6 Hz, 2H, CH2), 4.04 (q, J ¼ 7.2 Hz, 2H, estereCH2),
5.71 (br, 1H, NH), 6.18 (d, J ¼ 5.4 Hz, 1H, ArH), 6.74 (br, 1H, NH),
7.23e7.40 (m, 7H, ArH), 7.83 (d, J ¼ 6.9 Hz,1H, ArH), 8.23 (s,1H, ArH).
5.2.12. 5-Isopropyloxycarbonyl-6-methyl-4-(3-nitrophenyl)-2-[(7-
chloroquinolin-4-ylamino)propyl amino]pyrimidine (10n)
Yellow solid. Rf: 0.6 (2% MeOH/EtOAc). Yield: 71%. IR (KBr): nmax
752, 1356, 1527, 1700, 2973, 3421 cmꢀ1 1H (300 MHz, CDCl3, 25ꢁC):
d
1.09 (d, J ¼ 6.0 Hz, 6H, 2 ꢄ estereCH3), 2.09 (m, 2H, CH2), 2.52 (s, 3H,
13C NMR (75 MHz, CDCl3, 25ꢁC):
d
13.5, 22.9, 26.8, 27.1, 28.9, 29.2,
C6 eCH3), 3.46 (q, J ¼ 6.0 Hz, 2H, CH2), 3.71 (q, J ¼ 6.6 Hz, 2H, CH2),
5.06 (m, 1H, estereCH), 5.40 (br, 1H, NH), 5.59 (br, 1H, NH), 6.40 (d,
J ¼ 5.4 Hz, 1H, ArH), 7.23e7.28 (m, 2H, ArH), 7.55 (t, J ¼ 7.8 Hz, 1H,
ArH), 7.81 (d, J ¼ 3.3 Hz,1H, ArH), 7.94 (d, J ¼ 2.1 Hz,1H, ArH), 8.26 (d,
J ¼ 1.5 Hz,1H, ArH), 8.42 (t, J ¼ 2.1 Hz,1H, ArH), 8.50 (d, J ¼ 5.4 Hz,1H,
29.3, 29.4, 29.5, 41.3, 43.3, 60.9, 99.0, 115.0, 117.0, 120.7, 125.2, 127.9,
128.1, 128.7, 129.3, 134.8, 139.2, 148.9, 149.7, 151.9, 161.2 and 168.9
Anal. Calcd. For C35H44N5O2Cl: C, 69.82; H, 7.30; N, 11.63; Found: C,
69.53; H, 6.96; N, 11.45. MS: m/z 602 (Mþ).

94
K. Singh et al. / European Journal of Medicinal Chemistry 52 (2012) 82e97
ArH). 13C NMR (75 MHz, CDCl3, 25ꢁC):
69.4, 99.0, 120.8, 123.2, 124.1, 125.1, 128.7, 129.3, 133.9, 149.5 and
161.4. Anal. Calcd. For C27H27N6O4Cl: C, 60.60; H, 5.05; N, 15.70;
Found: C, 60.23; H, 5.02; N, 15.49. MS: m/z 534.5 (Mþ).
d
21.3, 21.4, 28.9, 38.7, 40.3,
25ꢁC):
d
1.05 (s, 6H, 2 ꢄ estereCH3), 2.05 (q, J ¼ 4.2 Hz, 2H, CH2),
2.51 (s, 3H, C6eCH3), 3.46 (t, J ¼ 6 Hz, 2H, CH2), 3.70 (d, J ¼ 6.3 Hz,
2H, CH2), 4.98 (m, 1H, estereCH), 6.39 (d, J ¼ 5.1 Hz, 1H, ArH), 7.63
(d, J ¼ 6.0 Hz, 4H, ArH), 7.90e8.40 (m, 3H, ArH), 8.49 (d, J ¼ 5.1 Hz,
1H, ArH). 13C NMR (75 MHz, CDCl3, 25ꢁC):
d 13.4, 13.5, 20.5, 29.4,
5.2.13. 5-Isopropyloxycarbonyl-6-methyl-4-(3-nitrophenyl)-2-[(7-
chloroquinolin-4-ylamino)butylamino] pyrimidine (10o)
29.7, 31.5, 55.0, 61.6, 61.7, 72.3,111.5,126.5,126.6,128.0,128.3,128.4,
128.9, 129.0, 130.9, 142.6, 158.2 and 166.1. Anal. Calcd. For
C27H27N6O4Cl: C, 60.60; H, 5.05; N, 15.70; Found: C, 60.23; H, 4.97;
N, 15.45. MS: m/z 534.5 (Mþ).
Yellow solid. Rf: 0.6 (5% MeOH/EtOAc). Yield: 78%. IR (KBr): nmax
760, 1357, 1526, 1710, 2935, 3412 cmꢀ1 1H (300 MHz, CDCl3, 25ꢁC):
d
1.2 (d, J ¼ 6.0 Hz, 6H, 2 ꢄ estereCH3),1.84 (m, 4H, CH2), 2.55 (s, 3H,
C6eCH3), 3.37 (m, 2H, CH2), 3.58 (m, 2H, CH2), 5.03 (m, 1H,
estereCH), 5.41 (br, 1H, NH), 5.60 (br, 1H, NH), 6.33 (d, J ¼ 5.4 Hz,
1H, ArH), 7.27e7.30 (m, 2H, ArH), 7.81 (t, J ¼ 1.2 Hz, 1H, ArH), 7.83
(d, J ¼ 1.2 Hz, 1H, ArH), 7.84 (s, 1H, ArH), 7.92 (d, J ¼ 2.1 Hz, 1H, ArH),
5.3. General procedure for the synthesis of 2-aminopyrimidines
(10h & 10i)
To the stirred solution of appropriate 4-aminoquinoline 9 in dry
THF (50 ml) mixture of 8 (in a 1:2 molar ratio) and potassium
carbonate in dry acetonitrile was added. The reaction mixture was
refluxed for 48 h and then filtered. Acetonitrile was removed under
vacuum and the residue was purified by column chromatography
using EtOAc/hexane as eluent to give 10h and 10i which were
recrystallized from DCM/hexane.
8.43 (d, J ¼ 5.7 Hz, 2H, ArH). 13C NMR (75 MHz, CDCl3, 25ꢁC):
d 21.2,
25.7, 28.1, 42.8, 69.2, 98.7, 115.5, 116.7, 121.2, 123.2, 123.9, 125.2,
127.6, 129.0, 133.9, 135.1, 140.5, 147.9, 150.1, 150.7, 161.0 and 167.3.
Anal. Calcd. For C28H29N6O4Cl: C, 61.20; H, 5.28; N, 15.30; Found: C,
60.58; H, 5.48; N, 15.18. MS: m/z 548.1 (Mþ).
5.2.14. 5-Isopropyloxycarbonyl-6-methyl-4-(2-nitrophenyl)-2-[(7-
chloroquinolin-4-ylamino)ethylamino] pyrimidine (10p)
5.3.1. 5-Ethoxycarbonyl-6-methyl-4-phenyl-2-[2-(7-chloroquino-
lin-4-ylamino)phenylamino] pyrimidine (10h)
Yellow solid. Rf: 0.5 (5% MeOH/EtOAc). Yield: 66%. IR (KBr): nmax
760, 1353, 1528, 1709, 2975, 3364 cmꢀ1 1H (300 MHz, CDCl3, 25ꢁC):
Yellow solid. Rf: 0.9 (70% Hexane/EtOAc). Yield: 53%. IR (KBr):
d
0.93 (d, J ¼ 6.3 Hz, 6H, 2 ꢄ estereCH3), 2.67 (s, 3H, C6eCH3), 3.53
nmax 770, 1253, 1717, 2923, 2970, 3192, 3389 cmꢀ1 1H (300 MHz,
(br, 2H, CH2), 3.85 (br, 2H, CH2), 4.98 (m, 1H, estereCH), 5.74 (br, 1H,
NH), 6.45 (d, J ¼ 5.1 Hz,1H, ArH), 7.20e7.60 (m, 6H, ArH), 7.68 (s,1H,
ArH), 8.17 (d, J ¼ 8.1 Hz,1H, ArH), 8.44 (s,1H, ArH).13C NMR (75 MHz,
CDCl3, 25ꢁC):
d
0.99 (t, J ¼ 7.2 Hz, 3H, estereCH3), 2.51 (s, 3H,
C6eCH3), 3.81 (br, 1H, NH), 4.09 (q, J ¼ 7.2 Hz, 2H, estereCH2), 4.13
(br, 1H, NH), 6.81e6.91 (m, 4H, ArH), 7.04 (t, 1H, ArH), 7.42 (t,
J ¼ 1.8 Hz, 4H, ArH), 7.51e7.59 (m, 5H, ArH). 13C NMR (75 MHz,
CDCl3, 25ꢁC):
d 21.2, 40.13, 68.7, 98.9, 124.4, 125.1, 128.7, 129.4, 129.8,
134.7 and 151.9.Anal. Calcd. For C26H27N6O4Cl: C, 59.90; H, 4.80; N,
CDCl3, 25ꢁC):
d 11.5, 20.9, 59.3, 115.4, 117.5, 122.8, 124.1, 126.0, 126.3
16.10; Found: C, 60.04; H, 4.69; N, 15.88. MS: m/z 520.1 (Mþ).
and 127.7.Anal. Calcd. For C29H24N5O2Cl: C, 68.30; H, 4.71; N, 13.70;
Found: C, 68.15; H, 4.47; N, 13.57. MS: m/z 510.5 (Mþ).
5.2.15. 5-Isopropyloxycarbonyl-6-methyl-4-(2-nitrophenyl)-2-[(7-
chloroquinolin-4-ylamino)propyl amino] pyrimidine (10q)
5.3.2. 5-Ethoxycarbonyl-6-methyl-4-phenyl-2-[4-(7-chloroquino-
lin-4-ylamino)phenylamino] pyrimidine (10i)
Yellow solid. Rf: 0.6 (10% MeOH/EtOAc). Yield: 85%. IR (KBr): nmax
757, 1346, 1529, 1705, 2973, 3351 cmꢀ1 (300 MHz, CDCl3, 25ꢁC):
Yellow solid. Rf: 0.4 (50% Hexane/EtOAc). Yield: 57%. IR (KBr):
d
0.90 (s, 6H, 2 ꢄ estereCH3), 2.04 (m, 2H, CH2), 2.57 (s, 3H, C6eCH3),
nmax 768, 1257, 1714, 2929, 3063, 3385 cmꢀ1 1H (300 MHz, CDCl3,
3.19 (br, 2H, CH2), 3.41 (q, J ¼ 5.4 Hz, 2H, CH2), 3.57 (br,1H, NH), 4.87
(m, 1H, estereCH), 6.03 (br, 1H, NH), 6.34 (d, J ¼ 5.7 Hz, 1H, ArH),
7.27e7.64 (m, 5H, ArH), 7.90 (s,1H, ArH), 8.11 (d, J ¼ 7.8 Hzꢁ,1H, ArH),
25ꢁC):
d
1.00 (t, J ¼ 7.2 Hz, 3H, estereCH3), 2.58 (s, 3H, C6eCH3), 4.11
(q, J ¼ 7.2 Hz, 2H, estereCH2), 6.60 (br, 1H, NH), 6.84 (d, J ¼ 5.4 Hz,
1H, ArH), 7.22e7.87 (m, 11H, ArH), 8.02 (d, J ¼ 1.8 Hz, 1H, ArH), 8.52
8.42 (t, J ¼ 5.4 Hz, 1H, ArH). 13C NMR (75 MHz, CDCl3, 25 C):
d
21.1,
(d, J ¼ 2.7 Hz, 1H, ArH). 13C NMR (75 MHz, CDCl3, 25ꢁC):
d 13.5, 29.6,
24.1, 28.5, 38.2, 39.9, 68.5, 98.5,114.3,116.8,121.5,124.3,125.4,126.5,
129.4, 129.7, 133.1, 135.5, 135.6, 149.7, 150.7, 161.0 and 165.6. Anal.
Calcd. For C27H27N6O4Cl: C, 60.60; H, 5.05; N,15.70; Found: C, 60.39;
H, 4.82; N, 15.88. MS: m/z 534.5 (Mþ).
61.3, 76.5, 101.9, 117.5, 117.7, 120.3, 120.4, 121.1, 124.5, 125.9, 128.0,
128.4, 128.5, 128.9, 129.8, 133.8, 135.2, 136.7, 138.5, 148.3, 149.5,
151.8,158.6,165.9,167.3 and 168.4. Anal. Calcd. For C29H24N5O2Cl: C,
68.30; H, 4.71; N, 13.70; Found: C, 67.99; H, 4.92; N, 13.57. MS: m/z
510.5 (Mþ).
5.2.16. 5-Isopropyloxycarbonyl-6-methyl-4-(2-nitrophenyl)-2-[(7-
chloroquinolin-4-ylamino)butylamino] pyrimidine (10r)
6. Materials and methods
Yellow solid. Rf: 0.6 (5% MeOH/EtOAc). Yield: 85%. IR (KBr): nmax
785, 1266, 1346, 1526, 1713, 2932, 3415 cmꢀ1 1H (300 MHz, CDCl3,
6.1. In vitro anti-plasmodial activity assay
25ꢁC):
d
0.89 (d, J ¼ 6.3 Hz, 6H, 2 ꢄ estereCH3), 1.76 (m, 4H, CH2),
2.57 (s, 3H, C6eCH3), 3.13 (br, 1H, NH), 3.30 (m, 2H, CH2), 3.53 (m,
2H, CH2), 4.87 (m, 1H, estereCH), 5.50 (br, 1H, NH), 6.34 (s, 1H, ArH),
7.25e7.58 (m, 4H, ArH), 7.70 (d, J ¼ 8.4 Hz, 1H, ArH), 7.94 (d,
J ¼ 1.5 Hz, 1H, ArH), 8.49 (d, J ¼ 5.4 Hz, 1H, ArH). 13C NMR (75 MHz,
Two clones of P. falciparum are used: (a) 3D7 clone of NF54
which is known to be sensitive to all anti-plasmodials, (b) K1 strain
originating from Thailand that is resistant to chloroquine and
pyrimethamine, but sensitive to mefloquine. The cultures were
naturally asynchronous (65e75% ring stage) and were maintained
in continuous log phase growth in RPMI1640 medium supple-
mented with 5% washed human Aþ erythrocytes, 25 mM Hepes,
32 nM NaHCO3, and AlbuMAXII (lipid-rich bovine serum albumin)
(GIBCO, Grand Island, NY) (CM). All cultures and assays are con-
ducted at 37 ꢁC under an atmosphere of 5% CO2 and 5% O2, with
a balance of N2. Stock drug solutions were prepared in 100% DMSO
(dimethylsulfoxide) at 20 mg/ml. The compound was further
diluted to the appropriate concentration using complete medium
RPMI1640 supplemented with 15 nM cold hypoxanthine and
CDCl3, 25ꢁC):
d 20.8, 20.9, 21.1, 25.6, 29.6, 41.0, 42.8, 42.9, 69.9, 70.1,
98.8, 116.9, 121.5, 124.3, 124.6, 125.3, 127.7, 129.5, 129.7, 130.1, 130.2,
133.3, 135.1, 150.1, 150.2, 150.8, 151.0 and 160.1.Anal. Calcd. For
C28H29N6O4Cl: C, 61.20; H, 5.28; N, 15.30; Found: C, 60.97; H, 4.93;
N, 15.25. MS: m/z 548.1 (Mþ).
5.2.17. 5-Isopropyloxycarbonyl-6-methyl-4-(4-nitrophenyl)-2-[(7-
chloroquinolin-4-ylamino)propyl amino] pyrimidine (10s)
Yellow solid. Rf: 0.9 (1% MeOH/EtOAc). Yield: 82%. IR (KBr): nmax
761, 1271, 1346, 1527, 1710, 2938, 3400 cmꢀ1 1H (300 MHz, CDCl3,

K. Singh et al. / European Journal of Medicinal Chemistry 52 (2012) 82e97
95
AlbuMAXII. Assays were performed in sterile 96-well microtitre
plates; each plate contained 100 l of parasite culture (0.5% para-
increasing concentrations (0e70
m
M) of 10c and 10r. Following
m
each addition, absorbance was recorded at 402 nm. For conducting
the experiment at pH 5.6, solutions of 10c and 10r and Fe (III) PPIX
were prepared in exactly the same manner, except that 2-[N-
morpholino]ethanesulphonate (MES) buffer (pH 5.4) was
substituted for HEPES buffer [31].
sitemia, 2.5% hematocrit). Each compound was tested in triplicate
and parasite growth compared to control and blank (uninfected
erythtocytes) wells. After 24 h of incubation at 37 ꢁC, 3.7 Bq of [3H]
hypoxanthine is added to each well [51]. Cultures were incubated
for a further 24 h before they are harvested onto glass-fiber filter
mats. The radioactivity was counted using a Wallac Microbeta 1450
scintillation counter. The results were recorded as counts per
minute (CPM) per well at each drug concentration, control and
blank wells. Percentage inhibition was calculated from comparison
to blank and control wells, and IC50 values calculated using Graph
Pad Prism 4.0. The preliminary screen uses the 3D7 strain. The
compounds were tested at 6 concentrations (30, 10, 3, 1, 0.3, and
6.3.2. Binding of 10c & 10r with m-oxo dimeric heme
Stock solution (10 mM) of heme was prepared by dissolving
hemin chloride in 0.1 M NaOH. Solution was sonicated for 30 min to
ensure complete dissolution. Heme stock solution was diluted to
60 mM in phosphate buffer (20 mM, pH 5.8). Stock solution of 10c
and 10r (2 mM) were prepared in AR grade DMSO. Titrations were
performed by successive addition of aliquots of stock solution of
0.1
m
g/ml). If the compound did not affect parasite growth at 10
m
g/
g/ml, the
g/ml, the
10c or 10r (0e15
absorbance at 362 nm were recorded. The association constants for
both monomeric &
Hyp Spec [37].
mL) to 60 mM heme solution & changes in the
ml it was classified as inactive, between 10 and 1
compound was designated as partially active, and if <1
m
m
m-oxo dimeric heme were determined by using
compound was classified as active and was further evaluated by
three-fold serial dilutions in a repeat test. For secondary screening
both the 3D7 clone and the K1 line were used. The compound was
diluted three-fold over at 12 different concentrations with an
appropriate starting concentration based on the preliminary
screen. The IC50 is determined by a sigmoidal dose response anal-
ysis using Graph Pad Prism 4.0. For each assay, the IC50 and IC90
values for each parasite line were determined against the known
anti-plasmodials chloroquine and artesunate.
6.3.3. Binding stoichiometry
Binding Stoichiometries of drug with monomeric &
m-oxo
dimeric heme were monitored by UVevisible spectrophotometry
using Job’s method of continuous variation [33,34]. The concen-
tration of drug & heme in solution was kept constant and changes
in absorbance at 402 nm (monomeric)/362 nm (dimeric) were
monitored as a function of the mole fraction.
6.2. Cytotoxicity and antiviral assay
6.3.4. Ferriprotoporphyrin IX biomineralisation inhibition test
(FBIT)
The antiviral assays [except anti-human immunodeficiency
virus (HIV) assays] were based on inhibition of virus-induced
cytopathicity in HEL [herpes simplex virus type 1 (HSV-1), HSV-2
(G), vaccinia virus, and vesicular stomatitis virus], Vero (para-
influenza-3, reovirus-1, Coxsackie B4, and Punta Toro virus), HeLa
(vesicular stomatitis virus, Coxsackie virus B4, and respiratory
syncytial virus) and MDCK (influenza A (H1N1; H3N2) and B virus)
cell cultures. Confluent cell cultures in microtiter 96-well plates
96 well plate containing mixture of 50
chloride dissolved in DMSO, 100 L of 0.5 M sodium acetate buffer
(pH 4.4) and 50 L of different concentrations of drug solution or
50
L of solvent (control), was incubated at 37 ꢁC for 24 h. The plate
was centrifuged at 4500 rpm for 3 min, and supernatant was dis-
carded. The remaining pellet was re-suspended with 200 L of
mL of 0.5 mg\ml of hemin
m
m
m
m
DMSO to eliminate unreacted hemin [38,39]. The plate was
centrifuged again and supernatant similarly discarded. The
were inoculated with 100 cell culture inhibitory dose-50 (CCID50
)
precipitate was dissolved in 150 mL of 0.1 N NaOH & absorbance was
of virus (1 CCID50 being the virus dose to infect 50% of the cell
cultures) in the presence of varying concentrations of the test
compounds. Viral cytopathicity was recorded as soon as it reached
completion in the control virus-infected cell cultures that were not
treated with the test compounds. The anti-HIV activity and anti-
proliferative activity were evaluated against HIV-1 strain IIIB and
HIV-2 strain ROD in human T-lymphocyte CEM cell cultures. Briefly,
virus stocks were titrated in CEM cells and expressed as the 50% cell
culture infective dose (CCID50). CEM cells were suspended in
culture medium at w3 ꢄ 105 cells/ml and infected with HIV at
read at 405 nm. The percentage of inhibition of Ferriprotoporphyrin
IX Biomineralisation was calculated using formula:
Inhibitionð%Þ ¼ 100xf½ðAbs of
controlÞ ꢀ ðAbs of drugÞꢅ=
ðAbs of controlÞg. IC50 values were determined by using Graph pad
Prism 4.0.
6.4. DNA binding studies with 10c and 10r
6.4.1. Preparation of stock solutions
Stock solution of 10c and 10r (2 mM) were prepared in AR grade
methanol. The DNA binding experiments were carried out by
making dilution of the stock with 1:1 buffered methanol.
w100 CCID50. Immediately after viral exposure, 100 mL of the cell
suspension was placed in each well of a flat-bottomed microtiter
plate containing various concentrations of the test compounds.
After a 4-day incubation period at 37 ꢁC, the giant cell formation
was microscopically determined. Compounds were tested in
parallel for cytostatic activity in uninfected CEM cells.
6.4.2. Preparation of CT DNA solution
Stock solution of DNA was prepared by dissolving DNA pellet in
TE buffer (10 mM TriseHCl, 0.1 mM EDTA, pH 7.4). The DNA
concentration was estimated from its absorbance intensity at
260 nm with a known molar absorption coefficient value of
6600 dm3molꢀ1 cmꢀ1. The purity of DNA was established from ratio
of absorbance intensity at 260 nm and at 280 nm. The observed
ratio of 1.8 ensured that DNA was free from protein.
6.3. Heme binding studies
6.3.1. Binding of 10c & 10r with monomeric heme
Hemin stock solution (1.2 mM) was prepared by dissolving
(7.8 mg) hemin chloride in 10 ml AR grade DMSO. Working solu-
tions (2.4
mM) were prepared by mixing 20
mL hemin stock solution
6.4.3. Evaluation of binding constant
with 4 ml DMSO and 1 ml 0.2 M HEPES buffer (pH 7.4) and making
it up to 10 ml with double distilled deionized water. Stock solutions
of 10c and 10r (2 mM) were prepared in AR grade DMSO and were
6.4.3.1. UV-spectrophotometry. The titration experiment was per-
formed by varying the concentration of CT DNA and keeping the
drug concentration constant (30
mM). All the UVevisible spectra
used for titration experiment. Heme (2.4
mM) was titrated with
were recorded after equilibration of solution for 5 min. The binding

96
K. Singh et al. / European Journal of Medicinal Chemistry 52 (2012) 82e97
[5] H. Noedl, D. Socheat, W. Satimal, Artimesinin-resistant malaria in Asia, N. Engl.
J. Med. 361 (2009) 540e541.
[6] R. Ettari, F. Bova, M. Zappala, S. Grasso, N. Micale, Falcipain-2 inhibitors, Med.
Res. Rev. 30 (2010) 136e167.
constants were calculated from absorption at 331 nm by using
BenesieHildebrand equation [48].
[7] M. Foley, L. Tilley, Quinoline antimalarial: mechanisms of action and resis-
tance, Int. J. Parasitol. 27 (1997) 231e240.
[8] J.X. Kelly, M.J. Smilkstein, R. Brun, W. Sergio, A.R. Cooper, K.D. Lane,
A. Janowsky, R.A. Johnson, R.A. Dodean, R. Winter, D.J. Hinrichs, M.K. Riscoe,
Discovery of dual function acridones as a new antimalarial chemotype, Nature
459 (2009) 270e273.
6.4.3.2. Spectrofluorometric titrations. The steady state fluores-
cence experiments were carried out on Varian Cary Eclipse Spec-
trofluorometer at ambient temperature. A slit width of 5 nm was
used with lex ¼ 320 nm and lem ¼ 380 nm. The titration experi-
ment was accomplished by varying the concentration of DNA in
[9] D. Mazier, L. Rénia, G. Snounou, A pre-emptive strike against malaria’s stealthy
hepatic forms, Nat. Rev. Drug Discov. 8 (2009) 855e864.
cuvette (0.3e70
mM) and keeping the compound concentration
constant (30 M). In fluorescence titration experiment working
solutions were prepared by making dilution of DNA stock and drug
stock with methanol.
m
[10] L. Adane, P.V. Bharatam, Modelling and informatics in the analysis of
P. falciparum DHFR enzyme inhibitors, Curr. Med. Chem. 15 (2008)
1522e1569.
[11] R.D. Powell, G.J. Brewer, Effects of pyrimethamine, chlorguanidine and pri-
maquine against exoerythrocytic forms of a strain of chloroquine-resistant
Plasmodium falciparum from Thailand, Am. J. Trop. Med. Hyg. 16 (1967)
693e698.
[12] S. Vangapandu, S. Sachdeva, M. Jain, S. Singh, P. Singh, C.L. Kaul, R. Jain, 8-
Quinolinamines and their pro-drug conjugates as potent blood-
schizontocidal antimalarial agents, Bioorg. Med. Chem. 11 (2003) 4557e4568.
[13] A. Agarwal, K. Srivastava, S.K. Puri, M.S. Chauhan, Synthesis of 4-pyrido-6-
aryl-2-substituted amino pyrimidines as a new class of antimalarial agents,
Bioorg. Med. Chem. 13 (2005) 6226e6232.
6.4.3.3. DNA thermal denaturation. DNA melting experiment were
carried out by monitoring the absorbance of CT DNA (151 mM NP) at
260 nm at various temperature in the presence and absence of drug
in a 5:1 ratio of the DNA and drug with a ramp rate of 0.5ꢁ C/min in
a 40% DMSO/TE buffer (pH 7.4) with 0.5 mM NaCl on a Shimadzu
1601 PC spectrophotometer equipped with a Peltier thermo regu-
lator. The thermal melting temperature was calculated by plotting
dA/dT vs temperature using Microsoft Excel.
[14] J. Wiesner, R. Ortmann, H. Jomaa, M. Schlitzer, New antimalarial drugs,
Angew. Chem. Int. Ed. 42 (2003) 5274e5293.
[15] 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.
6.4.4. Molecular docking method
[16] V.F. Roche, Mechanism based inhibitors of deoxythymidine monophosphate
synthesis as antineoplastic agents, Am. J. Pharm. Educ. 58 (1994) 196e203.
[17] M.R. Hollingdale, P.P. McCann, A. Sjoerdsma, Plasmodium berghei: inhibitors of
ornithine decarboxylase block exoerythrocytic schizogony, Exp. Parasitol. 60
(1985) 111e117.
[18] G. Rastelli, W. Sirawaraporm, P. Sompornpisut, Interaction of pyrimethamine,
cycloguanil, WR99210 and their analogues with Plasmodium falciparum
dihydrofolate reductase: Structural basis of antifolate resistance, Bioorg. Med.
Chem. 8 (2000) 1117e1128.
[19] V. Kouznetsov, A. Gomez-Barrio, Recent developments in the design and
synthesis of hybrid molecules based on aminoquinoline ring and their anti-
plasmodial evaluation, Eur. J. Med. Chem. 44 (2009) 3091e3113.
[20] P. Biginelli, Aldureids of ethylic acetoacetate and ethylic oxaloacetate, Gazz.
Chim. Ital 23 (1893) 360e416.
[21] K. Singh, K. Singh, An efficacious protocol for the oxidation of 3, 4-
dihydropyrimidin-2(1H)-ones using pyridinium chlorochromate as catalyst,
Aust. J. Chem. 61 (2008) 910e913.
[22] K. Singh, K. Singh, B. Wan, S. Franzblau, K. Chibale, J. Balzarini, Facile trans-
formation of 3,4-dihydropyrimidin-2(1H)-ones to pyrimidines in vitro eval-
uation as inhibitor of myobacterium tuberculosis and modulators of cytostatic
activity, Eur. J. Med. Chem. 46 (2011) 2290e2294.
[23] N. October, N.D. Watermeyer, V. Yardley, T.J. Egan, K. Ncokazi, K. Chibale,
Synthesis, antimalarial and cytotoxic evaluation of reversed chloroquines
based on the 3, 4-dihydropyrimidi-2(1H)-one scaffold, Chem. Med. Chem. 3
(2008) 1649e1653.
The molecular docking analysis was carried out using FlexX
molecular docking module [52,53] (FlexX 1.13.5) available in the
SYBYL 7.1 software package from Tripose [54].This docking
approach adopts an incremental construction algorithm for iden-
tifying appropriate pose of the substrate in the active site of the
enzyme. It generates about 30 possible poses of the substrate in the
active site. Most of the 30 poses obtained in this analysis adopted
similar binding mode in the enzyme cavity and hence the top
scoring pose was considered in this analysis. To perform the above
analysis, the crystal structure 1J3I (Wild-type Plasmodium falci-
parum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS)
complexed with WR99210, NADPH, and dUMP) was downloaded
from the protein data bank [55] (PDB) and subjected to protein
preparation and receptor description [56]. The structures of the
substrates were prepared by first building the molecules in 3D and
optimizing the structures using tripos force field. The ionic, tauto-
meric states of substrates were considered while performing
molecular docking analysis. The pose shown in Fig. 7 includes the
neutral state of the substrate in the active site of the enzyme.
[24] J.L. Vennerstrom, W.Y. Ellis, A.L. Ager, S.L. Anderaen Jr., L. Gerena,
W.K. Milhoust, N, N-Bis(7-chloroquinolin-4-yl) alkanediamines with potential
against chloroquine-resistant malaria, J. Med. Chem. 35 (1992) 2129e2134.
[25] F. Romanelli, K.M. Smith, A.D. Hoven, Chloroquine and hydroxychloroquine as
inhibitors of human immunodeficiency virus (HIV-1) activity, Curr. Pharm.
Des 10 (2004) 2643e2648.
[26] A. Savarino, J.R. Boelaert, A. Cassone, G. Majori, R. Cauda, Effects of chloroquine
on viral infections: an old drug against today’s diseases? Lancet Infect. Dis. 3
(2003) 722e727.
Acknowledgments
We thank CSIR [01/(2364)/10/EMR-II], UGC [F. No. 37-188/
2009(SR)] and SAP (DRS-1) UGC, New Delhi and the KU Leuven
(GOA no. 10/14) for providing financial support.
[27] A. Dorn, S.R. Vippagunta, H. Matile, C. Jaquet, J.L. Vennerstrom, R.G. Ridley, An
assessment of drug-haematin binding as a mechanism for inhibition of hae-
matin polymerisation by quinoline antimalarials, Biochem. Pharmacol. 55
(1998) 727e736.
[28] T.J. Egan, Interactions of quinoline antimalarials with hematin in solution,
J. Inorg. Biochem. 100 (2006) 916e926.
Appendix A. Supplementary data
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2012.03.007.
[29] T.J. Egan, K.K. Ncokazi, Quinoline antimalarials decrease the rate of beta-
hematin formation, J. Inorg. Biochem. 99 (2005) 1532e1539.
[30] A.C. Chou, R. Chevli, C.D. Fitch, Ferriprotoporphyrin IX fulfills the criteria for
identification as the chloroquine receptor of malaria parasites, Biochemistry
19 (1980) 1543e1549.
[31] T.J. Egan, W.W. Mavuso, D. Ross, H.M. Marques, Thermodynamic factor
controlling the interaction of quinoline antimalarial drugs with ferriproto-
porphyrin IX, J. Inorg. Biochem. 68 (1997) 137e145.
References
[1] T.N.C. Wells, P.L. Alonso, W.E. Gutteridge, New medicines to improve control
and contribute to the eradication of malaria, Nature 8 (2009) 879e891.
[2] R.E. Martin, R.V. Marchetti, A.I. Cowan, S.M. Howitt, S. Broer, K. Kirk, Chloro-
quine transport via the malaria parasite’s chloroquine resistance transporter,
Science 325 (2009) 1680e1682.
[3] T.E. Wellems, Plasmodium chloroquine resistance and the search for
a replacement antimalarial drug, Science 298 (2002) 124e126.
[4] A.M. Dondrop, F. Nosten, W. Hanpithakpong, S.J. Lec, P. Ringwald, K. Silamut,
M. Imwong, K. Chotivanich, P. Lim, T.A.S. Herdman, S. Yeung, P. Singhasivanon,
N.P. Day, N. Lindegardh, D. Socheat, N.J. White, Artimesinin resistance in
Plasmodium Falciparum malaria, N. Engl. J. Med. 361 (2009) 455e467.
[32] T.J. Egan, R. Hunter, C.H. Kaschula, H.M. Marques, A. Misplon, J. Walden,
Structure-function relationships in aminoquinolines: effect of amino and
chloro groups on quinoline-hematin complex formation, inhibition of
b-hematin formation, and antiplasmodial activity, J. Med. Chem. 43 (2000)
283e291.
[33] K.C. Ingham, Application of jobs method of continuous variation to stoichi-
ometry of protein-ligand complexes, Anal. Biochem. 68 (1975) 660e663.

K. Singh et al. / European Journal of Medicinal Chemistry 52 (2012) 82e97
97
[34] C.Y. Huang, Determination of binding stoichiometry by the continuous vari-
ation method-the job plot, Methods Enzymol. 87 (1982) 509e525.
[35] J.K. Natarajan, J.N. Alumasa, K. Yearick, K.A. Ekoue-Kovi, L.B. Casabianca,
A.C.D. Dios, C. Wolf, P.D. Roepe, 4-N-, 4-S-, and 4-O-Chloroquine analogues:
Influence of side chain length and quinolyl nitrogen pKa on activity vs chlo-
roquine resistant malaria, J. Med. Chem. 51 (2008) 3466e3479.
[36] J.X. Kelly, R. Winter, M. Riscoe, D.H. Peyton, A spectroscopic investigation of
the binding interaction between 4, 5-dihydroxyxanthone and heme, J. Inorg.
Biochem. 86 (2001) 617e625.
[45] Y. Pollack, A.L. Katzen, D.T. Spira, J. Golenser, The genome of Plasmodium
falciparum I: DNA base composition, Nucleic Acids Res. 10 (1982) 539e546.
[46] F. Kwakye-Berko, S.R. Meshnick, Sequence preference of chloroquine binding
to DNA and prevention of Z-DNA formation, Mol. Biochem. Parasitol. 39
(1990) 275e278.
[47] J.M. Woynarowski, M. Krugliak, H. Ginsburg, Pharmacogenomic analyses of
targeting the AT-rich malaria parasite genome with AT-specific alkylating
drugs, Mol. Biochem. Parasitol. 154 (2007) 70e81.
[48] H.A. Benesi, J.H. Hildebrand,
A spectrophotometric investigation of the
[37] F. Rodríguez, I. Rozas, M. Kaiser, R. Brun, B. Nguyen, W.D. Wilson, R.N. García,
C. Dardonville, New Bis (2-aminoimidazoline) and bisguanidine DNA minor
groove binders with potent in vivo antitrypanosomal and antiplasmodial
activity, J. Med. Chem. 51 (2008) 909e923.
[38] E. Deharo, R.N. Garcia, P. Oporto, A. Gimenez, M. Sauvain, V. Jullian,
H. Ginsburg, A non-radiolabelled ferriprotoporphyrin IX biomineralisation
inhibition test for the high throughput screening of antimalarial compounds,
Exp. Parasitol. 100 (2002) 252e256.
[39] F.S. Parker, J.L. Irvin, The interaction of chloroquine with nucleic acids and
nucleoproteins, J. Biol. Chem. 199 (1952) 897e909.
[40] P.H. Bolton, P.A. Mirau, R.H. Shafer, T.L. James, Interaction of the antimalarial
drug fluoroquine with DNA, tRNA, and poly-(A): 19F-NMR chemical-shift and
relaxation, optical absorption, and fluorescence studies, Biopolymers 20
(1981) 435e449.
interaction of iodine with aromatic hydrocarbons, J. Am. Chem. Soc. 71 (1949)
2703e2707.
[49] S.N. Cohen, K.L. Yielding, Spectrophotometric studies of the interaction of
chloroquine with deoxyribonucleic acid, J. Biol. Chem. 240 (1965) 3123e3131.
[50] N.I. Wenzel, N. Chavain, Y. Wang, W. Friebolin, L. Maes, B. Pradines,
M. Lanzer, V. Yardley, R. Brun, C.H. Mende, C. Biot, K.T. Oth, E.D. Charvet,
Antimalarial versus cytotoxic properties of dual drugs derived from 4-
aminoquinolines and Mannich bases: interaction with DNA, J. Med. Chem.
53 (2010) 3214e3226.
[51] R.E. Desjardins, C.J. Canfield, J.D. Haynes, J.D. Chulay, Quantitative assessment
of antimalarial activity in vitro by a semiautomated microdilution technique,
Antimicrob. Agents Chemother. 16 (1979) 710e718.
[52] M. Rarey, B. Kramer, T.J. Lengauer, Multiple automatic base selection: protein-
ligand docking based on incremental construction without manual inter-
vention, J. Comput. Aided Mol. Des 11 (1997) 369e384.
[53] M. Rarey, B. Kramer, T. Lengauer, G. Klebe, A fast flexible docking method
using an incremental construction algorithm, J. Mol. Biol. 261 (1996)
470e489.
[41] J.L. Allison, R.L. O’Brien, F.E. Hahn, DNA: reaction with chloroquine, Science
149 (1965) 1111e1113.
[42] N.B. Kurnick, I.E. Radcliffe, Reaction between DNA and quinacridine and other
antimalarials, J. Lab. Clin. Med. 60 (1962) 669e688.
[43] E.T. Mudasir, D.H. Wahyuni, N. Tjahjono, H. Yoshioka, Inoue, Spectroscopic
studies on the thermodynamic and thermal denaturation of the CT-DNA
binding of methylene blue, Spectrochim. Acta Part A 77 (2010) 528e534.
[44] J. Cheng, R. Zeidan, S. Mishra, A. Liu, S.H. Pun, R.P. Kulkarni, G.S. Jensen,
N.C. Bellocq, M.E. Davis, Structure-function correlation of chloroquine and
analogues as transgene expression enhancers in nonviral gene delivery,
J. Med. Chem. 49 (2006) 6522e6531.
[54] http://www.tripos.com (12.10.11).
[55] J. Yuvaniyama, P. Chitnumsub, S. Kamchonwongpaisan, J. Vanichatanankul,
W. Sirawaraporn, P. Taylor, Insights into antifolate resistance from malarial
DHFR-TS structures, Nat. Struct. Biol. 10 (2003) 357e365.
[56] L. Adane, P.V. Bharatam, V.J. Sharma, A common feature-based 3D-pharma-
cophore model generation and virtual screening: identification of potential
PfDHFR inhibitors, Enz. Inhibit. Med. Chem. 25 (2010) 635e645.