European Journal of Medicinal Chemistry 89 (2015) 490e502

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Original article

4-Aminoquinoline-Pyrimidine hybrids: Synthesis, antimalarial

activity, heme binding and docking studies
Deepak Kumar a, Shabana I. Khan b, c, d, Babu L. Tekwani b, c, Prija Ponnan a,
Diwan S. Rawat a, *
a
Department of Chemistry, University of Delhi, Delhi 110007, India
b
National Center for Natural Products Research, University of Mississippi, MS-38677, USA
c
Department of Biomolecular Sciences, University of Mississippi, MS-38677, USA
d
Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia

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 4-aminoquinoline-pyrimidine hybrids has been synthesized and evaluated for their
Received 12 July 2014 antimalarial activity. Several compounds showed promising in vitro antimalarial activity against both CQ-
Received in revised form sensitive and CQ-resistant strains with high selectivity index. All the compounds were found to be non-
18 October 2014
toxic to the mammalian cell lines. Selected compound 7g exhibited significant suppression of para-
Accepted 20 October 2014
Available online 22 October 2014
sitemia in the in vivo assay. The heme binding studies were conducted to determine the mode of action of
these hybrid molecules. These compounds form a stable 1:1 complex with hematin suggesting that heme
may be one of the possible targets of these hybrids. The interaction of these conjugate hybrids was also
Keywords:
Aminoquinoline
investigated by the molecular docking studies in the binding site of PfDHFR. The pharmacokinetic
Antimalarial activity property analysis of best active compounds was also studied using ADMET prediction.
Heme binding studies © 2014 Elsevier Masson SAS. All rights reserved.
Docking studies

1. Introduction administered in combination with a long acting partner drug such

Malaria is an infectious disease caused by protozoan parasites of
the Plasmodium genus and is transmitted to humans by the
infected female Anopheles mosquito [1]. According to a WHO
report, malaria accounted for 207 million cases and an estimated
627,000 deaths worldwide in 2013 [2]. The African sub-continent is
the most badly affected region where almost in every minute one
child gets malaria. Though malaria mortality rates have fallen
during the last decade, mortality figures are still very high for a
disease that is largely preventable and treatable. A major contrib-
utor to the problem is the emergence and spread of resistance to-
wards most drugs in clinical use such as chloroquine, amodiaquine,
pamaquine and mefloquine (Fig. 1). If new drugs are not introduced
onto the market, the spread of malaria could have disastrous con-
sequences. According to the WHO, currently the best available
treatments, particularly for Plasmodium falciparum related un-
complicated and severe malarial infection, are artemisinin combi-
nation therapies (ACTs) where artemisinin or its semi-synthetic
derivative dihydroartemisinin, artemether or artesunate (Fig. 1) is
* Corresponding author.
E-mail address: dsrawat@chemistry.du.ac.in (D.S. Rawat).
as amodiaquine, mefloquine, piperaquine, lumefantrine, sulfadox-
ine and pyrimethamine. Although, artemisinin combination ther-
apy (ACT) is fast acting, well tolerated and is nearly 95% effective in
the treatment of malaria [3], recently cases of resistance to ACTs
have been reported in some south-east Asian countries. The other
limitations of ACTs are low availability of artemisinin, high cost of
production, short biological half-life and reduced pharmacokinetics
[4e10]. All these evidences have intensified the synthetic efforts
towards development of new drugs for the treatment of malaria.
Thus synthesis of new chemical entities for the antimalarial therapy
remains a challenging task for the scientists involved in the malaria
research.
To combat drug resistant problems, various drug discovery ap-
proaches are being engaged and recently the concept of multi-
targeted hybrid drugs has been explored. In this approach, two or
more different target-selective pharmacophores are linked cova-
lently resulting into one molecule [11]. These dual-drug hybrids
have the potential to increase bio-pharmaceutical efficacy, reduce
cost, decrease risk of drugedrug interactions and overcome rapid
development of resistance problems. With these thoughts, various
research groups have synthesized a huge number of hybrid mole-
cules by the combination of chloroquine with other pharmaco-
phores acting on different targets. The most common antimalarial

http://dx.doi.org/10.1016/j.ejmech.2014.10.061
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.
 D. Kumar et al. / European Journal of Medicinal Chemistry 89 (2015) 490e502
Fig. 1. Antimalarial drugs.
agents from these studies include 4-aminoquinoline-isatin conju-
gates [12], quinolizidinyl analogs of chloroquine [13], peroxide-
based trioxaquine derivatives [14e19], ferrocene-chloroquine hy-
brids [20e22], 4-aminoquinoline based tetraoxane [23], 4-
aminoquinoline-thiazolidinone conjugates [24,25],
aminoquinoline-triazine conjugates [26e30], aminoquinoline-
pyrimidine conjugates [31e33], substituted triazines [34e36],
substituted pyrimidines [37e39]. Many of these derivatives have
shown excellent in vitro and in vivo activity against both
chloroquine-sensitive and chloroquine-resistant strains of
P. falciparum and some of these hybrid compounds have also
entered into the clinical trials [11].
Heme and P. falciparum dihydrofolate reductase (PfDHFR) are
among the most important targets for antimalarial drug discovery.
Aminoquinoline based compounds stop hemozoin formation,
while pyrimethamine and cycloguanil exhibit antimalarial activity
due to their ability to inhibit dihydrofolate reductase enzyme. Py-
rimethamine and cycloguanil belong to the triazine and pyrimidine
class of compounds, respectively. Based on these studies, we pro-
posed to incorporate pyrimidine moiety (DHFR inhibitor) in to the
side chain of 4-aminoquinoline (hemozoin inhibitor) in anticipa-
tion that the resultant molecules having two different pharmaco-
phores within one molecule may show potent antimalarial activity
and might be able to prevent the drug resistance to certain extent.
Recently, we synthesized 4-aminoquinoline-pyrimidine hybrids
and studied their antimalarial activity [40]. These conjugates
(Fig. 2) showed potent antimalarial activity (IC50 ¼ 0.005e0.44 mM)
with no cytotoxicity up to 60 mM concentration. Two selected
compounds were also evaluated for their in vivo activity and
showed excellent activity in a mouse model of Plasmodium berghei
with no toxicity (Fig. 2).
Encouraged by these results and as a part of our on-going
research towards the synthesis of novel antimalarial agents
[41e46], we designed a new series of 4-aminoquinoline-pyrimidine
hybrids so as to develop structurally diverse series of compounds in
order to gain structural insight for improved antimalarial activity.
The cyclic secondary amines in our previously reported compounds
491
were replaced by various substituted anilines in order to study the
effect of phenyl ring in place of saturated heterocyclic ring structure
on the antimalarial activity of these compounds (Fig. 2). Thus in the
present investigation we report the synthesis, antimalarial activity
and heme binding studies of a series of novel 4-aminoquinoline-
pyrimidine based molecular hybrids (7e9). Also we investigated
the interaction of these hybrids in the binding site of P. falciparum
dihydrofolate reductase (PfDHFR) protein structures using molec-
ular docking studies.
2. Results and discussion
2.1. Synthesis
Synthesis of aminoquinoline-pyrimidine conjugates was car-
ried out as outlined in Scheme 1. Firstly, N1-(7-chloroquinolin-4-
yl)ethane-1,2-diamine (2a), N1-(7-chloroquinolin-4-yl)propane-
1,3-diamine (2b) and N1-(7-chloroquinolin-4-yl)butane-1,4-
diamine (2c) were synthesized by the reaction of 4,7-
dichloroquinoline (1) with the excess of ethane-1,2-diamine,
propane-1,3-diamine and butane-1,4-diamine, respectively un-
der neat condition at 120  C (Scheme 1) [47]. The 4-
aminoquinolines (2ae2c) with free NH2 group were coupled
with aniline substituted pyrimidines (5ae5g) in the presence of
K2CO3 and using N-methyl pyrrolidone (NMP) as solvent at reflux
condition to give 4-aminoquinoline-pyrimidine conjugates (7e9)
which were purified by column chromatography using MeOH/
CHCl3 as eluent. The substituted pyrimidines were synthesized by
the reaction between commercially available 2,4-
dichloropyrimidine (3) with different substituted anilines
(4ae4g) at 0  C to RT in the presence of triethylamines using
ethanol as a solvent (Scheme 1) [48]. The reaction of pyrimidine
with anilines yielded two regio-isomers 5ae5g and 6ae6g.
Compounds 5ae5g obtained as a major product with very less
amount of compounds 6ae6g (Scheme 1). The major products
5ae5g was separated by column chromatography.
492 D. Kumar et al. / European Journal of Medicinal Chemistry 89 (2015) 490e502

Fig. 2. Design strategy for the synthesis of novel aminoquinoline-pyrimidine conjugates.

Scheme 1. a) Neat, 120e130  C, 4e6 h, 70e90 %; b) 5ae5g, K2CO3, NMP, 140e150  C, 10e12 h, 60e80%; c) Et3N, EtOH, RT, Overnight.

2.2. In vitro and in vivo antimalarial activity
All the twenty one synthesized hybrids were evaluated for their
in vitro antimalarial activity against CQ-sensitive (D6 clone) and
CQ-resistant (W2 clone) strains of P. falciparum using choroquine
and pyrimethamine as reference drugs (Table 1). In order to get
structural insights, two point variations were made in the
aminoquinoline-pyrimidine hybrids (7e9). In the structural motif
of these hybrids the aminoquinoline and pyrimidine rings were
kept common and variation was made in the connecting linker
(C2eC4) while another variation was introduced in the phenyl ring
attached to the 4-position of the pyrimidine ring. Among the series,
compounds having phenyl ring on the pyrimidine nucleus (7a, 8a,
9a) showed antimalarial activity in the range of 0.019e1.509 mM
(D6) and 0.144e1.529 mM (W2) with propylene linked 8a found to
be the most active against both strains. When the phenyl ring was
substituted with halogen groups (7be7d, 8be8d, 9be9d), most of
the compounds showed potent antimalarial activity against both
the strains with the exception of 7b and 9b, in both of which
fluorine is present at the para position of the phenyl ring attached
to pyrimidine ring, and spacer is ethylene and butylene respec-
tively. The analogous compound with a propylene linker (8b)
showed improved antimalarial activity against both the strains.
Compounds 7ce9c and 7de9d with chloro or bromo at para posi-
tion of the phenyl ring showed excellent activity against both the
D6 and W2 strains. Among these, compounds 7d (R ¼ 4-Br, n ¼ 2)
 D. Kumar et al. / European Journal of Medicinal Chemistry 89 (2015) 490e502 493

Table 1 gavage, once daily on days 0, 1 and 2 post infection and monitored
In vitro antimalarial activity and cytotoxicity of 4-aminoquinoline-pyrimidine for apparent signs of toxicity, parasitemia and survival till day 28
hybrids.
post infection (Table 2). The animals were monitored for any signs
Compds P. falciparum P. falciparum Cytotoxicity Resistance of toxicity, suppression in parasitemia and survival until day 28
(D6 clone) (W2 clone) (Vero cells) index (RI) post-infection. As shown in Table 2, no significant in vivo antima-
IC50 S.I. IC50 S.I. IC50 (mM) larial activity was observed for compound 7g. A weak effect was
(mM) (mM) noticed at the highest dose at day 5 (42.12% suppression in para-
7a 1.509 >13.25 1.529 >13.08 NC 1.05 sitemia) but the effect disappeared on day 7 and the mean survival
7b 6.022 >3.32 9.994 >2.0 NC 1.65 time was only 9 days compared to 7.6 days for vehicle treated an-
7c 0.047 335.17 0.046 342.45 15.753 0.978
imals and 26.2 days for chloroquine treated animals.
7d 0.037 >540.54 0.136 >147.05 NC 3.67
7e 0.101 134.71 0.143 95.14 13.606 1.41
7f 0.040 >500.0 0.241 >82.98 NC 6.025
2.3. Mechanistic studies: heme binding studies
7g 0.033 >606.06 0.058 >344.82 NC 1.75
8a 0.019 675.89 0.144 89.18 12.842 7.57
8b 0.028 489.82 0.094 145.90 13.715 3.35 Chloroquine and other quinoline derivatives are believed to
8c 0.047 285.72 0.445 30.17 13.429 9.46 show their antimalarial activity by inhibition of hemozoin forma-
8d 0.049 227.77 0.047 237.46 11.161 0.95 tion within the parasite food vacuole [49]. Hemozoin was originally
8e 0.153 120.76 0.346 53.40 18.477 2.26
8f 0.040 333.37 0.081 164.62 13.335 2.025
considered to be formed by the polymerization of heme [50], but it
8g 0.038 339.57 0.107 120.59 12.904 2.81 has now been demonstrated that it is a crystalline cyclic dimer of
9a 0.044 325.50 0.403 35.53 14.322 9.15 ferriprotoporphyrin IX [51]. It is widely accepted that CQ accumu-
9b 3.196 >6.25 5.831 >3.43 NC 1.82 lates in the plasmodium food vacuole and binds to some form of
9c 0.044 250.63 0.052 212.07 11.028 1.18
parasite heme/hematin, and inhibits hemozoin bio-crystallization
9d 0.047 213.68 0.063 159.41 10.043 1.34
9e 0.138 139.59 0.518 37.19 19.264 3.75 [52e54,59]. This is a non-enzymatic process in which hematin
9f 0.050 222.74 0.067 166.22 11.137 1.34 monomer (heme) released from parasite hemoglobin digestion is
9g 0.044 284.68 0.120 104.38 12.526 2.72 converted into hemozoin, also known as malaria pigment. Hemo-
CQ 0.035 >571.42 0.367 >54.49 NC 10.48 zoin is an insoluble crystal in which adjacent heme dimers are
Pyrimethamine 0.01 e NA e NT
linked via hydrogen bonds between free propionic acids [53].
IC50 the concentration that causes 50% growth inhibition; S.I. Selectivity Cohen et al. were the first to show that CQ forms a complex with
index ¼ (IC50 for cytotoxicity to Vero cells/IC50 for antimalarial activity); R.I.
ferriprotoporphyrin IX (FPIX) in aqueous solution, which was
(Resistance index) ¼ IC50 (W2 strain)/IC50 (D6 strain); NC: No cytotoxicity up to
20 mM; Vero: monkey kidney fibroblasts; NA: Not active up to 19 mM. NT: Not tested. proved by the changes in the UV-spectrum of aqueous hematin in
the presence of drug [55]. Later on several studies explained the
formation of CQ-hematin complex by computational methods as
well as the spectroscopic methods [56e61]. More recently it has
and 8c (R ¼ 4-Cl, n ¼ 3) showed better antimalarial activity against
been determined that CQ forms complexes with both monomeric
CQ-sensitive strain D6 while compounds 7c (R ¼ 4-Cl, n ¼ 2), 9c
and m-oxodimeric FPIX [62,63]. Therefore, we decided to evaluate
(R ¼ 4-Cl, n ¼ 4) and 8d (R ¼ 4-Br, n ¼ 3) and 9d (R ¼ 4-Br, n ¼ 4)
the binding of the most potent compound 7c with heme.
were equally active against CQ-resistant strain W2 (RI ~ 1). Com-
A solution of hematin in 40% DMSO showed a Soret band at
pounds with a methyl group at the para position of the phenyl ring
402 nm, indicating the presence of monomeric heme under the
(7e, 8e, 9e) led to partial decrease in the antimalarial activity
conditions used (0.02 M HEPES buffer, pH 7.4 and 0.02 M MES
against both the strains, while methoxy substitution at 4-position
buffer, pH 5.6). The stepwise addition of small increments of
(7f, 8f, 9f) and 3,5-position (7g, 8g, 9g) of the phenyl ring led to
compound 7c into a constant concentration of monomeric heme
an increase in the antimalarial activity with all the compounds
(5.0 mM) resulted in a substantial decrease in intensity of the Fe(III)
more active against CQ-sensitive strain of P. falciparum (RI > 1).
PPIX Soret band at 402 nm with no shift in the absorption
Most of the compounds showed good values of resistance index (RI)
maximum (Fig. 3). This indicates the association of compounds
(Table 1). The RI is the ratio of the IC50 value against the CQ-
with hematin. Solvent (DMSO) did not affect the binding of com-
resistant strain to the IC50 value against the CQ-sensitive strain.
pound 7c with heme at the pH values used in this experiment. The
The low value of resistance index indicates the activity of a com-
stoichiometry ratio of the most stable complexes of compound 7c
pound regardless of the susceptibility of the parasite strain,
with monomeric heme at pH 7.4 and 5.6 was deduced from the
whereas the large value indicates the loss of activity due to the drug
Job's plot. The absorbance at 402 nm got to maximum when mole
resistance. Therefore, a potent drug molecule should have a smaller
fraction of compound was approximately 0.5. Thus 1:1 ratio was
resistance index. In the present study, resistance index for chloro-
established for the association of compound heme at both the pH
quine was found to be 10.48, whereas tested compounds have RI
values (Fig. 4).
values in the range from 0.97 to 9.46 as shown in Table 1. In fact
some compounds such as 7a, 7c, 8d, 9c, 9d and 9f have resistance
index value near to one which suggests that these molecular hy- Table 2
brids may be active against P. falciparum strains resistant to parent In vivo antimalarial activity of compound 7g in P. berghei-mouse malaria model.
antimalarials chloroquine and pyrimethamine. Cytotoxicity was Treatment Dose (mg/kg  no. % Suppression Mean Toxicity
also determined against Vero cell line (Table 1). All the compounds (PO) of days post-infection) in parasitemiaa survival
showed toxicity at very high concentration as compared to their time
Day 5 Day 7
concentrations (IC50) responsible for their antimalarial activity, as
Vehicle NA  3 e e 7.6 NA
shown by the high selectivity index values in Table 1.
CQ 100  3 100 100 26.2 NC
Compound 7g with significant in vitro antimalarial activity was 7g 11.1  3 1.77 NA 5 NC
selected for further in vivo evaluation. In vivo antimalarial activity 7g 33.3  3 15.14 6.20 8.6 NC
was determined through oral route of administration in a P. berghei- 7g 100  3 42.12 10.43 9 NC
mouse malaria model as described earlier [40]. The compounds a
% suppression in parasitemia is calculated by considering the mean parasitemia
were administered to the P. berghei infected mice, through oral in the vehicle control as 100%.
494 D. Kumar et al. / European Journal of Medicinal Chemistry 89 (2015) 490e502

Fig. 3. A) Titration of compound 7c with monomeric heme at pH 7.4; B) Titration of compound 7c with monomeric heme at pH 5.6.

Fig. 4. Job's plot of monomeric heme complex formation with compound 7c; A) at pH 7.4; B) at pH 5.6; X (mole fraction of compound 7c) ¼ [compd 7c]/[compd 7c]þ[ heme]; A0 is
the absorbance, when x ¼ 1 and A is the absorbance at respective values of x.

As discussed earlier that CQ and its derivatives also bind to heme
dimer (m-oxo-heme). Therefore, the binding of compound 7c was
also studied with m-oxo-dimers of heme at pH 5.8. A solution of
heme in aqueous NaOH showed a peak at 362 nm. Addition of
compound 7c (020 mM) to a solution of m-oxo-dimer (10 mM) in
20 mM phosphate buffer at pH 5.8 resulted in a decrease in in-
tensity of absorbance at 362 nm (Fig. 5), which shows the inter-
action between m-oxo-dimer and compound 7c. The Job's plot
indicated a 1:1 stoichiometry for this complex (Fig. 5).
The association constants for the complexes formed between
monomeric Fe(III)PPIX and compound 7c at pH 7.4 and 5.6 were
calculated by the analysis of titration data and are presented in
Table 3. The association constant for the complexes formed be-
tween monomeric heme and compound 7c at pH 7.4 (log K 5.045)
was comparable to that of the standard antimalarial drug CQ (log K
5.15). Furthermore, decreasing the pH from 7.4 to 5.6 (food vacuole
pH), compound has shown improved binding constant (log K 5.51)
indicating that binding is stronger even at acidic pH of food vacuole.
Interestingly, the compound showed a large value of binding
constant than the standard drug chloroquine at pH 5.6. The asso-
ciation constants for the binding with m-oxo-heme at pH 5.8 (log K
5.123) was found to be even greater than the monomeric heme
complexes. From the data shown in Table 3, it is clear that the
compound 7c binds strongly with monomeric heme (log K 5.51,
determined at digestive vacuole pH 5.6) as well as m-oxo-heme (log
K 5.123, pH 5.8) and the observed results are comparable to the
standard CQ (log K 5.58). Thus the formation of complexes between
soluble hematin and compound 7c suggests the inhibition of for-
mation of b-hematin, a possible mechanism of antimalarial action
of these compounds similarly to that of chloroquine.
2.4. Docking studies
In the present study, the binding mode for novel 4-
aminoquinoline-pyrimidine based molecular hybrids in the active
site of wild type PfDHFR-TS and quadruple mutant PfDHFR-TS
(N51I, C59R, S108 N, I164L) protein structures was explored using
molecular docking studies. For this purpose, the prepared 3D

A B

Fig. 5. A) Titration of compound 7c with m-oxodimeric heme at pH 5.8; B) Job's plot of m-oxodimeric heme complex formation with compound 7c at pH 5.8.
 D. Kumar et al. / European Journal of Medicinal Chemistry 89 (2015) 490e502 495

Table 3 was observed between aromatic ring of Phe58 and pyrimidine as

Binding constants for compound 7c and chloroquine with heme. well as 4-chlorophenyl ring (Fig. 7).
Compd Monomeric Monomeric m-oxo-heme Another compound predicted to have low binding energy
heme log K heme log K log K (pH ¼ 5.8) (58.11 kcal mol1) and high glide score (8.70) was 7g, showing
(pH ¼ 5.6) (pH ¼ 7.4) similar H-bond pattern between linker NH group of compound and
7c 5.510 5.045 5.123 oxygen side chain of Asp54. The p-p interactions between the two
Chloroquine 4.65a 5.15a 5.58a aromatic rings (4-chlorophenyl and pyrimidine) of compounds and
Stoichiometry 1:1 1:1 1:1
aromatic ring of Phe58 of mutant PfDHFR was also observed (Fig. 7).
a
Refer to text Ref. [64]. In wild type PfDHFR, p-p interaction was observed between the
aromatic ring of Phe58 and pyrimidine ring of compound. Another
Hebond interaction was observed between the main chain oxygen
structures of the compounds were docked in the binding pocket of
atom of Ile164 and NH group of 3,5-dimethoxy phenyl ring of
both the wild type PfDHFR-TS and quadruple mutant PfDHFR-TS
compound 7g in wild type PfDHFR (Fig. 6).
(N51I, C59R, S108 N, I164L) structures.
The active aminoquinoline-pyrimidine conjugates interacted
2.5. Prediction of pharmacokinetic properties
with wild and mutant PfDHFR-TS forming H-bond and p-p in-
teractions. Table 4 shows the results of docking for the active
Different pharmacokinetic parameters of compounds in the
compounds. The Glide XP Gscores and glide energies clearly indi-
study, showing good antimalarial activity in malarial parasites were
cate that the most active compounds in the study exhibited sig-
calculated using ADMET predictions by Qikprop v3.5. The most
nificant binding affinities towards the wild (Glide energy
important of these parameters together with its permissible ranges
range 58.11 kcal mol1e44.32 kcal mol1) and quadruple
are listed in Tables 5 and 6. Qikprop results for various parameters
mutant (Glide energy range 56.14 kcal mol1e43.74 kcal mol1)
of Lipinski's rule of 5, a preliminary test of the drug-likeness of the
PfDHFR-TS structures and the energy ranges are comparable to that
compounds is presented in Table 5. An orally active compound
of reference compounds (pyrimethamine, cycloguanil and
should not have more than 2 violations of these rules. In the present
WR99210) and the native substrate of DHFR dihydrofolate (Table 4).
study, all the active test compounds showed Lipinski's rule viola-
Figs. 6 and 7 shows three dimensional binding pose of two
tions less than the maximum permissible value of 2, indicating that
selected active compounds with wild type and mutant PfDHFR-TS,
these active test compounds have good drug likeness properties.
respectively. Compound 9c showed lowest binding energy
The optimum values of descriptors such as number of rotable
(57.14 kcal mol1) and considerable high Glide XP score
bonds (<15) and polar surface area (7e200 Å2) can also have sig-
(8.88 kcal mol1) for wild type PfDHFR and 56.14 and 9.27 for
nificant influence on oral bioavailability of compounds [65]. In the
mutant type PfDHFR. A hydrogen bond interaction was observed
present study, all the test compounds possess a number of rotatable
between NH group of butylene linker of compound 9c and oxygen
bonds <15 and polar surface area falls satisfactorily in the
side chain of Asp54 of both wild and mutant PfDHFR. In addition,
permissible range (7e200 Å). Similarly, the test molecules were
compound 9c forms Hebonding interaction between the NH group
checked for their intestinal absorption or permeation property,
of 4-chlorophenyl ring and the main chain oxygen atom of Ile164 in
which is confirmed by the predicted Caco-2 cells permeability
the wild type PfDHFR (Fig. 6). Further, compound show p-p inter-
(QPPCaco), used as a model for the guteblood barrier [66]. QPPCaco
action between aromatic ring of Phe58 and pyrimidine ring in case
predictions for all the test compounds showed excellent values.
of wild type (Fig. 6), while in mutant type PfDHFR, p-p interaction
Further, QPlogKhsa, the prediction for human serum albumin
binding were calculated and all inhibitors were predicted to lie
Table 4 within the expected range for 95% of known drugs (1.5e1.5). Also,
Glide docking scores (kcal mol1) and docking energies of best active molecules the QikProp descriptor for brain/blood partition coefficient
along with the reference compounds (pyrimethamine, cycloguanil and WR99210) (QPlogBB) and the bloodebrain barrier mimic MDCK cell perme-
and dihydrofolate bound to wild and mutant PfDHFR-TS binding site.
ability (QPPMDCK) showed reliable predictions for all the test
Compounds Docking results Docking results Docking results compounds and the reference compounds. The aqueous solubility
with wild with mutant with PfLDH (QPlogS) parameters for the test compounds were assessed and all
PfDHFR PfDHFR
the compounds were predicted to have QPlogS values in the
XP Glide XP Glide XP Glide permissible range. Furthermore, QPlogHERG descriptor for the
GScore energy GScore energy GScore energy prediction of IC50 value of HERG Kþ channel blockage was predicted
7c 8.45 44.32 8.55 47.95 5.88 45.29 for the test compounds. Compounds 7d, 7f and 7g have been pre-
7d 5.63 48.12 6.43 52.27 5.75 42.84 dicted to possess values for in QPlogHERG in the permissible range
7f 8.71 51.67 7.73 43.19 5.87 44.79
comparable to reference compounds pyrimethamine and cyclo-
7g 8.70 58.11 9.15 53.74 6.35 48.39
8a 8.14 55.20 8.85 51.44 5.70 43.08 guanil (Table 6).
8b 8.54 49.41 8.15 51.55 4.07 42.91
8c 6.89 52.42 6.85 49.99 6.34 44.31 3. Conclusions
8d 6.02 50.35 8.23 47.32 4.49 44.21
8f 8.43 50.30 6.56 48.46 4.62 38.22
8g 8.29 53.83 6.51 48.85 6.28 43.59
In summary, we have reported the synthesis and antimalarial
9a 8.38 48.32 8.04 50.26 3.94 35.84 activity of a series of 4-aminoquinoline-pyrimidine hybrids. The
9c 8.88 57.14 9.27 56.14 6.50 50.54 in vitro evaluation of these hybrids against D6 and W2 strains of
9d 6.77 53.36 9.04 49.05 5.85 49.69 P. falciparum depicted activity in the nanomolar range. Also, these
9f 8.29 51.26 8.61 50.40 5.15 40.07
hybrids exhibited high selectivity indices and low toxicity against
9g 7.23 53.13 6.99 50.72 5.50 40.30
Chloroquine e e e e 6.62 58.32 the tested cell lines. Three compounds (7g, 8a and 8b) exhibited
Dihydrofolate 9.33 64.84 11.00 61.30 e e very potent antimalarial activity (IC50 ¼ 0.019 mMe0.033 mM)
Pyrimethamine 9.04 44.91 9.39 63.55 e e which was comparable to the standard drug chloroquine
Cycloguanil 8.94 38.55 8.95 46.6 e e (IC50 ¼ 0.035 mM) against CQ-sensitive strain, whereas all the
WR99210 4.84 37.03 5.48 34.30 e e
compounds showed better activity than chloroquine and
496 D. Kumar et al. / European Journal of Medicinal Chemistry 89 (2015) 490e502

Fig. 6. 2D and 3D docking pose showing interaction for compounds 9c and 7g in the binding site of wild type PfDHFR-TS (PDB ID: 3QGT).

pyrimethamine against CQ-resistant strain except 7a, 7b, 8c, 9a, 9b
and 9e. Interestingly, the synthesized 4-aminoquinoline-pyrimi-
dine hybrids were more or less equally potent to our previously
reported hybrid molecules [40], however a slight reduction in the
activity was observed on replacing the saturated heterocyclic ring
with a phenyl ring. Some of the compounds showed resistance
index value close to one which indicates that the antimalarial ac-
tivity of these molecular hybrids are comparable between the CQ-
sensitive and CQ-resistant strains of P. falciparum and hence sug-
gesting that the CQ-resistance mechanism has little effect on the
antimalarial activity of these compounds. Further, the binding
capability of compound 7c was evaluated with heme to find out the
probable mode of action of these hybrids. The molecular docking
studies of active compounds from the in vitro studies were per-
formed and all the compounds showed good interaction with the
binding sites of PfDHFR, comparable to the inhibitors and sub-
strates. The calculated ADMET parameters for the test compounds
predicted good pharmacokinetic properties. The promising in vitro
antimalarial activity exhibited by the novel 4-aminoquinoline-py-
rimidine conjugates, docking pattern in the P. falciparum DHFR, also
ADMET properties described in the present study, confirms their
potential for further development as antimalarial lead compounds.

Fig. 7. 2D and 3D docking pose showing interaction for compounds 9c and 7g in the binding site of mutant type PfDHFR-TS (PDB ID: 3QG2).
 D. Kumar et al. / European Journal of Medicinal Chemistry 89 (2015) 490e502 497

Table 5 filtered and washed with excess water. The crude product was
Prediction of Lipinski's ‘Rule of 50 for the active test compounds.a purified by column chromatography using EtOAc/Hexane as eluent
Compound mol_MW donorHB accptHB QPlogPo/w ‘N’ of to afford pure compound 5a.
(<500 amu) (<5) (<10) (<5) violations
(<2) 4.3.1. 2-Chloro-N-phenylpyrimidin-4-amine (5a)
7c 425.31 3 5 4.391 0 Yield 80%; mp 186e187  C; 1H NMR (400 MHz, CDCl3): d 6.59 (d,
7d 469.77 3 5 4.391 0 J ¼ 5.86 Hz, 1H), 7.23 (brs, 1H, NH), 7.25e7.27 (m, 1H), 7.30e7.32 (m,
7f 420.9 3 5 4.003 0
2H), 7.40e7.44 (m, 2H), 8.12 (d, J ¼ 5.86 Hz, 1H).
7g 450.92 3 6.5 4.045 0
8a 404.90 3 5 4.935 0
8b 422.89 3 5 4.727 0 4.3.2. 2-Chloro-N-(4-fluorophenyl)pyrimidin-4-amine (5b)
8c 439.34 3 5 4.95 0 Yield 73%; mp 177e180  C; 1H NMR (400 MHz, CDCl3): d 6.46 (d,
8d 483.79 3 5 5.013 1
J ¼ 5.86 Hz, 1H), 7.09e7.14 (m, 2H), 7.29e7.32 (m, 2H), 7.43 (brs, 1H,
8f 434.92 3 5 4.936 0
8g 464.95 3 6.5 5.084 1
NH), 8.10 (d, J ¼ 5.86 Hz, 1H).
9a 418.92 3 5 4.958 0
9c 453.37 3 5 5.406 1 4.3.3. 2-Chloro-N-(4-chlorophenyl)pyrimidin-4-amine (5c)
9d 497.82 3 5 5.446 1 Yield 75%; mp 198e201  C; 1H NMR (400 MHz, CDCl3): d 6.55 (d,
9f 448.95 3 5 4.758 0
J ¼ 5.86 Hz, 1H), 6.97 (brs, 1H, NH), 7.29 (d, J ¼ 8.05 Hz, 2H), 7.38 (d,
9g 478.98 3 6.5 5.083 1
Chloroquine 319.88 1 4 4.538 0 J ¼ 8.05 Hz, 2H), 8.15 (d, J ¼ 5.86 Hz, 1H).
Pyrimethamine 248.71 4 3 1.809 0
Cycloguanil 253.73 5 3 0.888 0 4.3.4. N-(4-Bromophenyl)-2-chloropyrimidin-4-amine (5d)
a
All values calculated by QikProp v 3.5 and the explanations of the descriptors are Yield 76%; mp 206e209  C; 1H NMR (400 MHz, CDCl3): d 6.55 (d,
given in the text. J ¼ 5.86 Hz, 1H), 6.95 (brs, 1H, NH), 7.23e7.26 (m, 2H), 7.51e7.54 (m,
2H), 8.16 (d, J ¼ 5.86 Hz, 1H).

4. Experimental section 4.3.5. 2-Chloro-N-p-tolylpyrimidin-4-amine (5e)

4.1. General
All the chemicals were purchased from SigmaeAldrich. Solvents
used for the chemical synthesis were of analytical grade and used
without further purification. Thin layer chromatography (Merck
Kiesel 60 F254, 0.2 mm thickness) was used to monitor the progress
of the reactions and the compounds were purified by silica gel
(60e120 mesh) column chromatography. IR spectra were recorded
on a Perkineelmer FT-IR spectrophotometer using KBr pellets or as
a film in chloroform and the values were expressed in cm1.1H NMR
(400 MHz) and 13C NMR (100 MHz) spectra were recorded on Jeol
ECX spectrospin instrument using CDCl3 or DMSO-d6 as solvent and
TMS as internal reference. The chemical shift values were expressed
on d scale and the coupling constant (J) in Hz. Melting points were
recorded on EZ-Melt automated melting point apparatus, Stanford
Research Systems and are uncorrected. Mass data were recorded in
Jeol-Accu TOF JMS-T100LC mass spectrometer.
4.2. Typical procedure for the synthesis of N1-(7-chloroquinolin-4-
yl)ethane-1,2-diamine (2a) and related compounds (2b and 2c)
A mixture of 4,7-dichloroquinoline (1, 5.0 g, 0.025 mol) and 1,2-
ethylene diamine (5.8 g, 0.125 mol) was heated slowly from RT to
120  C and the reaction mixture was stirred at this temperature for
6 h (Scheme 1). After that the reaction mixture was cooled down to
room temperature and ice cold water was added to it. The solid thus
obtained was filtered and washed with excess water. The crude
product was crystallized by using ethanol and the data corresponds
to that reported in the literature [47].
4.3. Typical procedure for the synthesis of 2-chloro-N-
phenylpyrimidin-4-amine (5a) and related compounds (5be5g)
To a solution of 2,4-dichloropyrimidine (3, 2.0 g, 0.013 mol) and
triethylamine (1.63 g, 0.016 mol) in ethanol at 0  C, aniline (4a, 1.2 g,
0.013 mol) was added (Scheme 1). The reaction mixture was stirred
overnight at room temperature. After completion of the reaction as
observed by TLC, excess ethanol was evaporated and the reaction
mixture was diluted with water. The solid thus obtained was
Yield 70%; mp 200e203  C; 1H NMR (400 MHz, CDCl3): d 2.37 (s,
3H, CH3), 6.52 (d, J ¼ 5.86 Hz, 1H), 7.07 (brs, 1H, NH), 7.16 (d,
J ¼ 8.05 Hz, 2H), 7.22 (d, J ¼ 8.05 Hz, 2H), 8.08 (d, J ¼ 5.86 Hz, 1H).
4.3.6. 2-Chloro-N-(4-methoxyphenyl)pyrimidin-4-amine (5f)
Yield 68%; mp 177e180  C; 1H NMR (400 MHz, CDCl3): d 3.83 (s,
3H, OCH3), 6.40 (d, J ¼ 5.86 Hz, 1H), 6.94 (d, J ¼ 8.79 Hz, 2H), 7.21 (d,
J ¼ 8.79 Hz, 2H), 7.28 (brs, 1H, NH), 8.05 (d, J ¼ 5.86 Hz, 1H).
4.3.7. 2-Chloro-N-(3,5-dimethoxyphenyl)pyrimidin-4-amine (5g)
Yield 65%; mp 170e173  C; 1H NMR (400 MHz, CDCl3): d 3.80 (s,
6H, 2OCH3), 6.34 (s, 1H), 6.47 (s, 2H), 6.67 (d, J ¼ 5.86 Hz, 1H), 7.0
(brs, 1H, NH), 8.14 (d, J ¼ 5.86 Hz, 1H).
4.4. Typical procedure for the synthesis of N2-(2-((7-
chloroquinolin-4-yl)amino)ethyl)-N4-phenylpyrimidine-2,4-
diamine (7a) and related compounds (7be7g, 8ae8g and 9ae9g)
To a stirred solution of compound 2a (400 mg, 1.8 mmol) and
compound 5a (372 mg, 1.8 mmol) in N-methyl pyrrolidinone
(NMP), K2CO3 (750 mg, 5.4 mmol) was added. The reaction mixture
was stirred at 140  C for 12 h (Scheme 1). After completion of the
reaction, water was added to the reaction mixture and the product
was extracted with chloroform (3  20 mL). The combined organic
layer was dried over sodium sulphate and excess solvent was
evaporated under reduced pressure. The crude product was puri-
fied by column chromatography using MeOH/CHCl3 as eluent to
afford pure compound 7a in quantitative yield.
4.4.1. N2-(2-(7-Chloroquinolin-4-ylamino)ethyl)-N4-
phenylpyrimidine-2,4-diamine (7a)
Yield 72%; mp 198e199  C; IR (KBr, cm1): 3339, 3241, 3030,
2973, 2921, 1581, 1522, 1498, 1453, 1397, 1248, 1231, 1077, 978, 872,
849, 805; 1H NMR (400 MHz, DMSO-d6): d 3.42e3.44 (m, 2H),
3.56e3.58 (m, 2H), 6.04 (d, J ¼ 5.1 Hz, 1H), 6.56 (brs, 1H), 6.92 (t,
J ¼ 7.3 Hz, 1H), 6.96 (t, J ¼ 5.8 Hz, 1H), 7.23 (t, J ¼ 7.3 Hz, 2H), 7.43 (d,
J ¼ 8.0 Hz, 1H), 7.52 (brs, 1H), 7.70 (d, J ¼ 7.3 Hz, 2H), 7.77 (d,
J ¼ 2.2 Hz, 1H), 7.91 (brs, 1H), 8.17 (d, J ¼ 8.7 Hz, 1H), 8.35 (brs, 1H),
9.18 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 35.83, 39.50, 95.36,
114.06, 116.04, 118.25, 120.54, 120.76, 124.19, 125.28, 130.04, 137.14,
498 D. Kumar et al. / European Journal of Medicinal Chemistry 89 (2015) 490e502

Table 6
Calculated ADMET properties.

Comps PercentHuman QPPCaco nms1 QPlogBB QPPMDCK QPlogKhsa QPlogHERG QPlogS PSA #rotor
OralAbsorption (<25 poor, >500 great)a (3.0e1.2)a (<25 poor >500 great)a (1.5e1.5)a (concern (6.5e0.5) (7.0e200.0)a (0e15)a
(>80%-high, below 5)a
<25% poor)a

7c 100 1926.73 0.236 4022.729 0.382 5.157 4.603 63.606 7

7d 100 1961.069 0.208 4218.681 0.386 4.934 4.483 61.352 7
7f 100 1970.518 0.39 2150.276 0.277 4.803 3.917 69.351 8
7g 100 1760.353 0.467 2085.817 0.289 4.425 3.887 79.658 9
8a 100 1569.034 0.617 1985.673 0.565 6.975 5.93 64.795 8
8b 100 1456.769 0.492 2650.002 0.499 5.888 5.241 64.906 8
8c 100 1460.136 0.448 3419.613 0.563 5.899 5.522 64.942 8
8d 100 1468.714 0.438 3636.597 0.581 5.923 5.607 64.919 8
8f 100 1586.519 0.594 2009.653 0.576 5.958 5.343 72.535 9
8g 100 1570.759 0.751 1988.037 0.576 6.651 6.107 81.529 10
9a 100 1571.443 0.612 1988.98 0.576 6.146 5.388 65.013 9
9c 100 1464.286 0.554 2985.112 0.717 6.14 5.924 65.41 9
9d 100 1481.444 0.523 3217.678 0.731 5.991 5.863 65.429 9
9f 100 1863.611 0.54 2301.046 0.482 5.194 4.58 71.581 10
9g 100 1491.108 0.777 1879.327 0.582 5.929 5.584 79.2 11
CQ 100 1479.516 0.415 2061.643 0.585 6.326 4.49 25.8 8
Pyr 84.346 412.287 0.78 468.849 0.243 4.318 2.978 73.731 4
a
Calculated using QikProp v 3.5. Range/recommended values calculated for 95% known drugs.

145.69, 146.76, 148.62, 157.31, 158.71; ESI-HRMS (m/z) calculated for
C21H19ClN6: 390.1360, found: 391.3322 (MþH)þ, 393.3665
(M þ 2)þ; Anal. calcd. for C21H19ClN6: C, 64.53; H, 4.90; Cl, 9.07; N,
21.50, found: C, 64.68; H, 4.79; Cl, 9.11; N, 21.57.
4.4.2. N2-(2-(7-Chloroquinolin-4-ylamino)ethyl)-N4-(4-
fluorophenyl)pyrimidine-2,4-diamine (7b)
Yield 60%; mp 216e218  C; IR (KBr, cm1): 3452, 3237, 3180,
3059, 2964, 2925, 2855, 1581, 1504, 1458, 1426, 1369, 1332, 1321,
1221, 1151, 1081, 979, 825; 1H NMR (400 MHz, DMSO-d6):
d 3.42e3.43 (m, 2H), 3.55e3.57 (m, 2H), 5.99 (d, J ¼ 5.1 Hz, 1H), 6.55
(brs, 1H), 6.96 (t, J ¼ 5.8 Hz, 1H), 7.06 (d, J ¼ 8.7 Hz, 2H), 7.43 (d,
J ¼ 8.0 Hz, 1H), 7.52 (brs, 1H), 7.69 (brs, 2H), 7.77 (d, J ¼ 2.2 Hz, 1H),
7.91 (brs, 1H), 8.17 (d, J ¼ 8.7 Hz, 1H), 8.35 (brs, 1H), 9.21 (s, 1H); 13C
NMR (100 MHz, DMSO-d6): d 39.58, 43.22, 99.11, 115.40, 115.62,
117.81, 121.44, 121.51, 124.31, 124.52, 127.94, 133.81, 137.25, 149.42,
150.53, 152.34, 156.42, 158.78, 160.96, 162.42; Anal. calcd. for
C21H18ClFN6: C, 61.69; H, 4.44; Cl, 8.67; F, 4.65; N, 20.55, found: C,
61.60; H, 4.62; Cl, 8.74; F, 4.61; N, 20.33.
J ¼ 5.1 Hz, 1H), 6.57 (brs, 1H), 7.01 (t, J ¼ 5.8 Hz, 1H), 7.38 (d,
J ¼ 8.7 Hz, 2H), 7.42e7.44 (m, 1H), 7.69 (d, J ¼ 7.3 Hz, 2H), 7.54 (brs,
1H), 7.76 (d, J ¼ 2.2 Hz, 1H), 7.93 (brs, 1H), 8.18 (d, J ¼ 9.5 Hz, 1H),
8.35 (brs, 1H), 9.32 (s, 1H); Anal. calcd. for C21H18BrClN6: C, 53.69; H,
3.86; Br, 17.01; Cl, 7.55; N, 17.89, found: C, 53.65; H, 3.91; Br, 17.12;
Cl, 7.65; N, 17.94.
4.4.5. N2-(2-(7-Chloroquinolin-4-ylamino)ethyl)-N4-p-
tolylpyrimidine-2,4-diamine (7e)
Yield 70%; mp 216e220  C; IR (KBr, cm1): 3436, 3232, 3033,
2922, 2885, 1580, 1524, 1510, 1379, 1355, 1332, 1257, 131, 975, 904,
852; 1H NMR (400 MHz, DMSO-d6): d 2.20 (s, 3H, CH3), 3.42e3.43
(m, 2H), 3.56e3.57 (m, 2H), 6.0 (d, J ¼ 5.1 Hz, 1H), 6.55 (brs, 1H),
6.91 (t, J ¼ 5.8 Hz, 1H), 7.03 (d, J ¼ 8.0 Hz, 2H), 7.43 (d, J ¼ 8.7 Hz, 1H),
7.55 (d, J ¼ 8.7 Hz, 3H), 7.77 (d, J ¼ 2.2 Hz, 1H), 7.88 (brs, 1H), 8.17 (d,
J ¼ 9.5 Hz, 1H), 8.35 (brs, 1H), 9.07 (s, 1H); ESI-HRMS (m/z) calcu-
lated for C22H21ClN6: 404.1516, found: 405.3329 (MþH)þ, 407.3444
(M þ 2)þ; Anal. calcd. for C22H21ClN6: C, 65.26; H, 5.23; Cl, 8.76; N,
20.76, found: C, 65.33; H, 5.19; Cl, 8.88; N, 20.79.

4.4.3. N4-(4-Chlorophenyl)-N2-(2-(7-chloroquinolin-4-ylamino) 4.4.6. N2-(2-(7-Chloroquinolin-4-ylamino)ethyl)-N4-(4-

ethyl)pyrimidine-2,4-diamine (7c)
Yield 75%; mp 228e232  C; IR (KBr, cm1): 3446, 3227, 3178,
3067, 2965, 2925, 2854, 1609, 1581, 1510, 1489, 1458, 1397, 1371,
1334, 1320, 1288, 1237, 1172, 1148, 1080, 980, 853, 801; 1H NMR
(400 MHz, DMSO-d6): d 3.41e3.44 (m, 2H), 3.56e3.57 (m, 2H),
6.02e6.03 (m, 1H), 6.57 (brs, 1H), 7.02 (t, J ¼ 5.8 Hz, 1H), 7.26 (d,
J ¼ 8.7 Hz, 2H), 7.44 (dd, J ¼ 2.2, 8.7 Hz, 1H), 7.53 (brs, 1H), 7.74e7.75
(m, 2H), 7.77 (d, J ¼ 2.2 Hz, 1H), 7.93 (brs, 1H), 8.18 (d, J ¼ 8.7 Hz, 1H),
8.35 (brs, 1H), 9.33 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 39.48,
42.71, 98.70, 117.39, 120.66, 123.89, 124.10, 124.93, 127.52, 128.39,
133.37, 139.49, 149.01, 150.10, 151.92, 160.39, 161.93; ESI-HRMS (m/
z) calculated for C21H18Cl2N6: 424.0970, found: 425.0135 (MþH)þ,
427.0255 (M þ 2)þ; Anal. calcd. for C21H18Cl2N6: C, 59.30; H, 4.27;
Cl, 16.67; N, 19.76, found: C, 59.25; H, 4.13; Cl, 16.58; N, 19.70.
4.4.4. N4-(4-Bromophenyl)-N2-(2-(7-chloroquinolin-4-ylamino)
ethyl)pyrimidine-2,4-diamine (7d)
Yield 55%; mp 220e224  C; IR (KBr, cm1): 3443, 3178, 3065,
2974, 1580, 1545, 1515, 1488, 1458, 1426, 1394, 1371, 1339, 1319,
1238, 1201, 1148, 1073, 1004, 980, 853, 798; 1H NMR (400 MHz,
DMSO-d6): d 3.42e3.44 (m, 2H), 3.55e3.57 (m, 2H), 6.02 (d,
methoxyphenyl)pyrimidine-2,4 diamine (7f)
Yield 75%; mp 219e222  C; IR (KBr, cm1): 3330, 3238, 3168,
3036, 2924, 1580, 1508, 1451, 1415, 1391, 1360, 1247, 1166, 1043, 924,
805; 1H NMR (400 MHz, DMSO-d6): d 3.40e3.42 (m, 2H), 3.54e3.56
(m, 2H), 3.65 (s, 3H, OCH3), 5.94 (d, J ¼ 5.1 Hz, 1H), 6.55 (brs, 1H),
6.80 (d, J ¼ 8.0 Hz, 2H), 6.86 (t, J ¼ 5.1 Hz, 1H), 7.42 (d, J ¼ 8.7 Hz, 1H),
7.54 (d, J ¼ 8.7 Hz, 2H), 7.76 (d, J ¼ 2.2 Hz, 2H), 7.85 (brs, 1H), 8.17 (d,
J ¼ 8.7 Hz, 1H), 8.34 (brs, 1H), 8.98 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): d 36.66, 39.50, 51.60, 95.23, 110.37, 113.94, 117.99, 120.41,
120.61, 124.08, 129.91, 145.57, 146.64, 148.50, 150.99, 152.25, 157.29,
158.67; Anal. calcd. for C22H21ClN6O: C, 62.78; H, 5.03; Cl, 8.42; N,
19.97; O, 3.80, found: C, 62.84; H, 5.20; Cl, 8.36; N, 18.04; O, 3.92.
4.4.7. N2-(2-(7-Chloroquinolin-4-ylamino)ethyl)-N4-(3,5-
dimethoxyphenyl)pyrimidine-2,4-diamine (7g)
Yield 78%; mp 224e226  C; IR (KBr, cm1): 3406, 3221, 3091,
2937, 1579, 1528, 1492, 1459, 1431, 1331, 1308, 1247, 1224, 1201, 1152,
1081, 1064, 925, 840; 1H NMR (400 MHz, DMSO-d6): d 3.42e3.43
(m, 2H), 3.58 (brs, 2H), 3.66 (s, 6H, 2OCH3), 6.02 (d, J ¼ 5.8 Hz, 1H),
6.08 (s, 1H), 6.56 (brs, 1H), 6.96 (d, J ¼ 2.2 Hz, 3H), 7.42 (d, J ¼ 8.0 Hz,
2H), 7.76 (d, J ¼ 2.2 Hz, 1H), 7.90 (brs, 1H), 8.15 (d, J ¼ 8.7 Hz, 1H),
 D. Kumar et al. / European Journal of Medicinal Chemistry 89 (2015) 490e502
8.33 (brs, 1H), 9.16 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 42.77,
54.97, 93.97, 97.55, 98.70, 117.40, 123.82, 124.10, 127.54, 133.40,
142.14, 149.02, 150.10, 151.96, 160.48, 160.70, 162.0; ESI-HRMS (m/z)
calculated for C23H23ClN6O2: 450.1571, found: 451.1548 (MþH)þ,
453.1848 (M þ 2)þ; Anal. calcd. for C23H23ClN6O2: C, 61.26; H, 5.14;
Cl, 7.86; N, 18.64; O, 7.10, found: C, 61.32; H, 5.23; Cl, 7.80; N, 18.59;
O, 7.15.
4.4.8. N2-(3-(7-Chloroquinolin-4-ylamino)propyl)-N4-
phenylpyrimidine-2,4-diamine (8a)
Yield 70%; mp 190e193  C; IR (KBr, cm1): 3436, 3232, 3038,
2924, 2885, 1580, 1525, 1495, 1436, 1379, 1356, 1332, 1256, 1131,
1079, 975, 904, 879, 853, 792; 1H NMR (400 MHz, DMSO-d6):
d 1.90e1.93 (m, 2H), 3.31e3.33 (m, 2H), 3.38e3.40 (m, 2H), 5.98 (d,
J ¼ 5.8 Hz, 1H), 6.45 (d, J ¼ 5.1 Hz, 1H), 6.85 (brs, 1H), 6.90 (t,
J ¼ 7.3 Hz, 1H), 7.23 (t, J ¼ 8.0 Hz, 2H), 7.34 (t, J ¼ 5.1 Hz, 1H), 7.42
(dd, J ¼ 2.2, 9.5 Hz, 1H), 7.72 (d, J ¼ 8.0 Hz, 2H), 7.76 (d, J ¼ 2.2 Hz,
1H), 7.84 (d, J ¼ 5.1 Hz, 1H), 8.23 (d, J ¼ 8.7 Hz, 1H), 8.34 (d,
J ¼ 5.1 Hz, 1H), 9.13 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 28.76,
39.50, 41.18, 99.55, 118.41, 120.19, 122.34, 124.95, 128.41, 129.48,
134.33, 141.55, 150.0, 150.98, 152,80, 157.0, 161.54, 162.97; Anal.
calcd. for C22H21ClN6: C, 65.26; H, 5.23; Cl, 8.76; N, 20.76, found: C,
65.26; H, 5.23; Cl, 8.76; N, 20.76.
4.4.9. N2-(3-(7-Chloroquinolin-4-ylamino)propyl)-N4-(4-
fluorophenyl)pyrimidine-2,4-diamine (8b)
Yield 65%; mp 192e195  C; IR (KBr, cm1): 3437, 3232, 3042,
2925, 1579, 1525, 1507, 1468, 1445, 1416, 1378, 1235, 1219, 1131, 976,
907, 854, 794; 1H NMR (400 MHz, DMSO-d6): d 1.84e1.89 (m, 2H),
3.27e3.32 (m, 4H), 5.91 (d, J ¼ 5.8 Hz, 1H), 6.41 (d, J ¼ 5.1 Hz, 1H),
6.83 (brs, 1H), 7.02 (t, J ¼ 8.7 Hz, 2H), 7.30 (brs, 1H), 7.37e7.40 (m,
1H), 7.67e7.70 (m, 2H), 7.73 (d, J ¼ 2.2 Hz, 1H), 7.80 (d, J ¼ 5.8 Hz,
1H), 8.20 (d, J ¼ 8.7 Hz, 1H), 8.30 (d, J ¼ 5.8 Hz, 1H), 9.14 (s, 1H); 13C
NMR (100 MHz, DMSO-d6): d 28.76, 39.50, 41.17, 99.57, 115.86,
116.08, 118.42, 121.78, 121.85, 124.94, 124.98, 128.41, 134.37, 137.92,
149.99, 151.02, 152.78, 156.85, 159.22, 161.42, 162.95; ESI-HRMS (m/
z) calculated for C22H20ClFN6: 422.1422, found: 423.1856 (MþH)þ,
425.2231 (M þ 2)þ; Anal. calcd. for C22H20ClFN6: C, 62.48; H, 4.77;
Cl, 8.38; F, 4.49; N, 19.87, found: C, 62.41; H, 4.85; Cl, 8.30; F, 4.53; N,
20.04.
4.4.10. N4-(4-Chlorophenyl)-N2-(3-(7-chloroquinolin-4-ylamino)
propyl)pyrimidine-2,4-diamine (8c)
Yield 72%; mp 181e183  C; IR (KBr, cm1): 3433, 3248, 3087,
2927, 1609, 1582, 1549, 1490, 1332, 1237, 1139, 1085, 981, 902, 875,
853, 790; 1H NMR (400 MHz, DMSO-d6): d 1.90e1.93 (m, 2H),
3.31e3.33 (m, 4H), 5.97 (d, J ¼ 5.1 Hz, 1H), 6.44 (d, J ¼ 5.1 Hz, 1H),
6.91 (brs, 1H), 7.24 (d, J ¼ 8.0 Hz, 2H), 7.34 (brs, 1H), 7.41 (d,
J ¼ 8.0 Hz, 1H), 7.76 (brs, 3H), 7.86 (d, J ¼ 5.1 Hz, 1H), 8.23 (d,
J ¼ 8.7 Hz, 1H), 8.34 (d, J ¼ 4.3 Hz, 1H), 9.28 (s, 1H); 13C NMR
(100 MHz, DMSO-d6): d 28.70, 39.50, 41.13, 99.52, 118.36, 121.42,
124.93, 125.68, 128.36, 129.23, 134.32, 140.53, 149.92, 150.96,
152.70, 161.24, 162.85; Anal. calcd. for C22H20Cl2N6: C, 60.14; H,
4.59; Cl, 16.14; N, 19.13, found: C, 60.26; H, 4.53; Cl, 16.30; N, 19.22.
4.4.11. N4-(4-Bromophenyl)-N2-(3-(7-chloroquinolin-4-ylamino)
propyl)pyrimidine-2,4-diamine (8d)
Yield 50%; mp 203e242  C; IR (KBr, cm1): 3422, 3185, 2927,
1578, 1489, 1394, 1329, 1233, 1136, 1072, 1004, 979, 901, 853, 795;
1
H NMR (400 MHz, DMSO-d6) d: 1H NMR (400 MHz, DMSO-d6) d:
1
H NMR (400 MHz, DMSO-d6): d 1.90e1.93 (m, 2H), 3.31e3.33 (m,
4H), 5.97 (d, J ¼ 5.1 Hz, 1H), 6.44 (d, J ¼ 5.1 Hz, 1H), 6.91 (brs, 1H),
7.24 (d, J ¼ 8.0 Hz, 2H), 7.34 (brs, 1H), 7.41 (d, J ¼ 8.0 Hz, 1H), 7.76
(brs, 3H), 7.86 (d, J ¼ 5.1 Hz, 1H), 8.23 (d, J ¼ 8.7 Hz, 1H), 8.33 (d,
J ¼ 5.8 Hz, 1H), 9.27 (s, 1H); ESI-HRMS (m/z) calculated for
499
C22H20BrClN6: 482.0621, found: 483.1493 (MþH)þ, 485.1466
(M þ 2)þ; Anal. calcd. for C22H20BrClN6: C, 54.62; H, 4.17; Br, 16.52;
Cl, 7.33; N, 17.37, found: C, 54.67; H, 4.29; Br, 16.47; Cl, 7.20; N, 17.31.
4.4.12. N2-(3-(7-Chloroquinolin-4-ylamino)propyl)-N4-p-
tolylpyrimidine-2,4-diamine (8e)
Yield 70%; mp 193e195  C; IR (KBr, cm1): 3432, 3245, 2924,
2861, 1578, 1527, 1510, 1425, 1334, 1238, 1136, 1084, 976, 907, 877,
795; 1H NMR (400 MHz, DMSO-d6): d 1.89e1.93 (m, 2H), 2.18 (s, 3H,
CH3), 3.31e3.34 (m, 4H), 5.94 (d, J ¼ 5.8 Hz, 1H), 6.45 (d, J ¼ 5.1 Hz,
1H), 6.81 (brs, 1H), 7.10 (brs, 2H), 7.34 (t, J ¼ 5.1 Hz, 1H), 7.42 (dd,
J ¼ 2.2, 8.7 Hz, 1H), 7.57 (d, J ¼ 8.0 Hz, 2H), 7.76 (d, J ¼ 2.2 Hz, 1H),
7.80 (d, J ¼ 5.8 Hz, 1H), 8.24 (d, J ¼ 8.7 Hz, 1H), 8.34 (d, J ¼ 5.8 Hz,
1H), 9.02 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 21.24, 28.78,
41.01, 41.18, 99.53, 118.40, 120.30, 124.94, 128.39, 129.87, 131.18,
134.32, 138.93, 149.96, 150.99, 152.76, 156.87, 161.52, 162.98; ESI-
HRMS (m/z) calculated for C23H23ClN6: 418.1673, found: 419.2275
(MþH)þ, 421.2478 (M þ 2)þ; Anal. calcd. for C23H23ClN6: C, 65.94;
H, 5.53; Cl, 8.46; N, 20.06, found: C, 65.88; H, 5.62; Cl, 8.40; N, 20.12.
4.4.13. N2-(3-(7-Chloroquinolin-4-ylamino)propyl)-N4-(4-
methoxyphenyl)pyrimidine-2,4-diamine (8f)
Yield 75%; mp 186e188  C; IR (KBr, cm1): 3436, 3234, 3042,
2930, 2836, 1581, 1526, 1510, 1466, 1442, 1419, 1379, 1333, 1285,
1249, 1131, 1037, 975, 903, 878, 851; 1H NMR (400 MHz, DMSO-d6):
d 1.88e1.92 (m, 2H), 3.29e3.33 (m, 4H), 3.66 (s, 3H, OCH3), 5.89 (d,
J ¼ 5.8 Hz, 1H), 6.44 (d, J ¼ 5.8 Hz, 1H), 6.76 (brs, 1H), 6.81 (d,
J ¼ 8.7 Hz, 2H), 7.32 (brs, 1H), 7.41 (dd, J ¼ 2.2, 8.7 Hz, 1H), 7.57 (d,
J ¼ 9.5 Hz, 2H), 7.75 (d, J ¼ 2.2 Hz, 1H), 7.78 (d, J ¼ 5.8 Hz, 1H), 8.23
(d, J ¼ 8.7 Hz, 1H), 8.33 (d, J ¼ 5.8 Hz, 1H), 8.93 (s, 1H); 13C NMR
(100 MHz, DMSO-d6): d 28.82, 39.50, 41.23, 56.08, 99.62, 114.76,
118.46, 122.15, 125.01, 128.47, 134.35, 134.61, 150.05, 151.03, 152.86,
155.25, 161.61, 163.06; ESI-HRMS (m/z) calculated for C23H23ClN6O:
434.1622, found: 435.1425 (MþH)þ, 437.1761 (M þ 2)þ; Anal. calcd.
for C23H23ClN6O: C, 63.52; H, 5.33; Cl, 8.15; N, 19.32; O, 3.68, found:
C, 63.61; H, 5.28; Cl, 8.22; N, 19.37; O, 3.80.
4.4.14. N2-(3-(7-Chloroquinolin-4-ylamino)propyl)-N4-(3,5-
dimethoxyphenyl)pyrimidine-2,4-diamine (8g)
Yield 70%; mp 191e192  C; IR (KBr, cm1): 3396, 3239, 3129,
3031, 2998, 2963, 1580, 1544, 1480, 1449, 1420, 1374, 1329, 1220,
1207, 1152, 1061, 982, 869; 1H NMR (400 MHz, DMSO-d6):
d 1.91e1.94 (m, 2H), 3.30e3.40 (m, 4H), 3.68 (s, 6H, 2OCH3), 5.97 (d,
J ¼ 5.8 Hz, 1H), 6.09 (brs, 1H), 6.44 (d, J ¼ 5.1 Hz, 1H), 6.90 (brs, 1H),
7.0 (brs, 2H), 7.31 (brs, 1H), 7.41 (dd, J ¼ 2.2, 8.7 Hz, 1H), 7.76 (d,
J ¼ 2.2 Hz, 1H), 7.84 (d, J ¼ 5.1 Hz, 1H), 8.23 (d, J ¼ 8.7 Hz, 1H), 8.34
(d, J ¼ 5.1 Hz, 1H), 9.11 (s, 1H); 13C NMR (100 MHz, DMSO-d6):
d 28.64, 40.99, 41.10, 55.83, 94.51, 98.31, 99.49, 118.34, 124.89,
128.33, 134.23, 143.15, 149.92, 150.93, 152.73, 161.31, 161.48, 162.86;
ESI-HRMS (m/z) calculated for C24H25ClN6O2: 464.1728, found:
465.0541 (MþH)þ, 467.0970 (M þ 2)þ; Anal. calcd. for
C24H25ClN6O2: C, 62.00; H, 5.42; Cl, 7.63; N, 18.08; O, 6.88, found: C,
61.89; H, 5.55; Cl, 7.60; N, 18.17; O, 7.01.
4.4.15. N2-(4-(7-Chloroquinolin-4-ylamino)butyl)-N4-
phenylpyrimidine-2,4-diamine (9a)
Yield 70%; mp 190e192  C; IR (KBr, cm1): 3417, 3238, 3001,
2935, 2863, 1585, 1544, 1497, 1428, 1347, 1234, 1131, 975, 902, 852,
799; 1H NMR (400 MHz, DMSO-d6): d 1.66e1.70 (m, 4H), 3.24e3.31
(m, 4H), 5.96 (d, J ¼ 5.8 Hz, 1H), 6.43 (d, J ¼ 5.1 Hz, 1H), 6.78 (brs,
1H), 6.89 (t, J ¼ 7.3 Hz, 1H), 7.22 (t, J ¼ 5.1 Hz, 2H), 7.29 (t, J ¼ 5.1 Hz,
1H), 7.40 (dd, J ¼ 2.2, 8.7 Hz, 1H), 7.71 (d, J ¼ 8.0 Hz, 2H), 7.75 (d,
J ¼ 1.4 Hz, 1H), 7.81 (d, J ¼ 5.1 Hz, 1H), 8.24 (d, J ¼ 8.7 Hz, 1H), 8.34
(d, J ¼ 5.1 Hz, 1H), 9.10 (s, 1H); 13C NMR (100 MHz, DMSO-d6):
d 25.43, 26.93, 40.46, 42.28, 98.62, 117.46, 119.16, 121.32, 123.94,
500 D. Kumar et al. / European Journal of Medicinal Chemistry 89 (2015) 490e502
124.10, 127.47, 128.52, 133.33, 140.69, 149.10, 150.07, 151.89, 156.17,
160.57, 162.02; ESI-HRMS (m/z) calculated for C23H23ClN6: 418.1673,
found: 419.1407 (MþH)þ, 421.1748 (M þ 2)þ; Anal. calcd. for
C23H23ClN6: C, 65.94; H, 5.53; Cl, 8.46; N, 20.06, found: C, 65.82; H,
5.66; Cl, 8.21; N, 20.10.
4.4.16. N2-(4-(7-Chloroquinolin-4-ylamino)butyl)-N4-(4-
fluorophenyl)pyrimidine-2,4-diamine (9b)
Yield 65%; mp 175e178  C; IR (KBr, cm1): 3448, 3247, 3067,
2933, 2867, 1583, 1507, 1412, 1353, 1219, 1154, 1081, 981, 825, 794;
1
H NMR (400 MHz, DMSO-d6): d 1.64e1.70 (m, 4H), 3.24e3.32 (m,
4H), 5.93 (d, J ¼ 5.8 Hz, 1H), 6.43 (d, J ¼ 5.1 Hz, 1H), 6.81 (brs, 1H),
7.06 (t, J ¼ 8.7 Hz, 2H), 7.33 (t, J ¼ 5.1 Hz, 1H), 7.41 (dd, J ¼ 2.2, 8.7 Hz,
1H), 7.70e7.74 (m, 2H), 7.76 (d, J ¼ 2.2 Hz, 1H), 7.81 (d, J ¼ 5.1 Hz,
1H), 8.25 (d, J ¼ 9.5 Hz, 1H), 8.34 (d, J ¼ 5.1 Hz, 1H), 9.16 (s, 1H); 13C
NMR (100 MHz, DMSO-d6): d 25.42, 26.91, 40.43, 42.29, 98.60,
114.87, 115.09, 117.41, 120.73, 120.81, 123.99, 124.12, 127.29, 133.44,
137.03, 148.88, 150.18, 151.68, 155.85, 158.21, 160.43, 161.97; ESI-
HRMS (m/z) calculated for C23H22ClFN6: 436.1579, found:
437.0507 (MþH)þ, 439.0819 (M þ 2)þ; Anal. calcd. for C23H22ClFN6:
C, 63.23; H, 5.08; Cl, 8.11; F, 4.35; N, 19.24, found: C, 63.28; H, 5.13;
Cl, 8.07; F, 4.29; N, 19.28.
4.4.17. N4-(4-Chlorophenyl)-N2-(4-(7-chloroquinolin-4-ylamino)
butyl)pyrimidine-2,4-diamine (9c)
Yield 70%; mp 229e232  C; IR (KBr, cm1): 3444, 3242, 3066,
3026, 2934, 2869, 1648, 1582, 1549, 1490, 1436, 1342, 1279, 1239,
1132, 1085, 981, 857, 804; 1H NMR (400 MHz, DMSO-d6):
d 1.64e1.70 (m, 4H), 3.24e3.32 (m, 4H), 5.95 (d, J ¼ 5.8 Hz, 1H), 6.42
(d, J ¼ 5.8 Hz, 1H), 6.85 (brs, 1H), 7.26 (d, J ¼ 8.7 Hz, 2H), 7.30 (d,
J ¼ 5.1 Hz, 1H), 7.40 (dd, J ¼ 2.2, 8.7 Hz, 1H), 7.75 (d, J ¼ 2.2 Hz, 2H),
7.77 (brs, 1H), 7.83 (d, J ¼ 5.8 Hz, 1H), 8.24 (d, J ¼ 8.7 Hz, 1H), 8.34 (d,
J ¼ 5.1 Hz, 1H), 9.25 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d: 25.46,
26.93, 40.50, 42.30, 98.61, 117.46, 120.51, 123.95, 124.10, 124.76,
127.46, 128.32, 133.38, 139.69, 149.08, 150.11, 151.85, 156.36, 160.35,
161.98; ESI-HRMS (m/z) calculated for C23H22Cl2N6:452.1283,
found: 453.1317 (MþH)þ, 455.1084 (M þ 2)þ; Anal. calcd. for
C23H22Cl2N6: C, 60.93; H, 4.89; Cl, 15.64; N, 18.54, found: C, 61.06; H,
4.82; Cl, 15.55; N, 18.47.
4.4.18. N4-(4-Bromophenyl)-N2-(4-(7-chloroquinolin-4-ylamino)
butyl)pyrimidine-2,4-diamine (9d)
Yield 50%; mp 240e242  C; IR (KBr, cm1): 3437, 3236, 3064,
2933, 2867, 1653, 1579, 1547, 1491, 1435, 1393, 1342, 1278, 1237,
1132, 980, 857, 802; 1H NMR (400 MHz, DMSO-d6): d 1.64e1.71 (m,
4H), 3.25e3.33 (m, 4H), 5.96 (d, J ¼ 5.8 Hz, 1H), 6.43 (d, J ¼ 5.8 Hz,
1H), 6.85 (brs, 1H), 7.30 (t, J ¼ 5.1 Hz, 1H), 7.38e7.42 (m, 3H), 7.72 (d,
J ¼ 8.7 Hz, 2H), 7.76 (d, J ¼ 2.2 Hz, 1H), 7.84 (d, J ¼ 5.1 Hz, 1H), 8.25
(d, J ¼ 8.7 Hz, 1H), 8.35 (d, J ¼ 5.1 Hz, 1H), 9.27 (s, 1H); 13C NMR
(100 MHz, DMSO-d6): d 25.45, 26.92, 40.49, 42.30, 98.61, 112.63,
117.46, 120.92, 123.96, 124.10, 127.46, 131.21, 133.38, 140.12, 149.07,
150.11, 151.85, 160.33, 161.96; ESI-HRMS (m/z) calculated for
C23H22BrClN6: 496.0778, found: 497.2248 (MþH)þ, 499.2184
(M þ 2)þ; Anal. calcd. for C23H22BrClN6: C, 55.49; H, 4.45; Br, 16.05;
Cl, 7.12; N, 16.88, found: C, 55.54; H, 4.66; Br, 15.97; Cl, 7.05; N,
16.70.
4.4.19. N2-(4-(7-Chloroquinolin-4-ylamino)butyl)-N4-p-
tolylpyrimidine-2,4-diamine (9e)
Yield 70%; mp 202e204  C; IR (KBr, cm1): 3329, 3240, 3164,
3028, 2973, 2923, 1582, 1510, 1451, 1411, 1390, 1359, 1250, 1173, 1147,
1134, 978, 924, 873, 851; 1H NMR (400 MHz, DMSO-d6): d 1.64e1.70
(m, 4H), 2.19 (s, 3H, CH3), 3.25e3.31 (m, 4H), 5.92 (d, J ¼ 5.8 Hz, 1H),
6.43 (d, J ¼ 5.8 Hz, 1H), 6.73 (brs, 1H), 7.02 (d, J ¼ 8.0 Hz, 2H),
7.29e7.32 (m, 1H), 7.41 (dd, J ¼ 2.2, 8.7 Hz, 1H), 7.57 (d, J ¼ 8.7 Hz,
2H), 7.75 (d, J ¼ 2.2 Hz, 1H), 7.78 (d, J ¼ 5.8 Hz, 1H), 8.24 (d,
J ¼ 9.5 Hz, 1H), 8.34 (d, J ¼ 5.1 Hz, 1H), 9.0 (s, 1H); ESI-HRMS (m/z)
calculated for C24H25ClN6: 432.1829, found: 433.1352 (MþH)þ,
435.1620 (M þ 2)þ; Anal. calcd. for C24H25ClN6: C, 66.58; H, 5.82; Cl,
8.19; N, 19.41, found: C, 66.62; H, 5.79; Cl, 8.23; N, 19.54.
4.4.20. N2-(4-(7-Chloroquinolin-4-ylamino)butyl)-N4-(4-
methoxyphenyl)pyrimidine-2,4-diamine (9f)
Yield 70%; mp 200e202  C; IR (KBr, cm1): 3436, 3236, 3067,
3006, 2936, 2869, 1647, 1583, 1557, 1511, 1433, 1349, 1334, 1278,
1235, 1176, 1132, 1085, 1031, 978, 904, 876, 857; 1H NMR (400 MHz,
DMSO-d6): d 1.63e1.70 (m, 4H), 3.24e3.31 (m, 4H), 3.67 (s, 3H,
OCH3), 5.88 (d, J ¼ 5.1 Hz, 1H), 6.43 (d, J ¼ 5.8 Hz, 1H), 6.69 (brs, 1H),
6.82 (d, J ¼ 8.7 Hz, 2H), 7.31 (t, J ¼ 5.1 Hz, 1H), 7.41 (dd, J ¼ 2.2,
8.7 Hz, 1H), 7.57 (d, J ¼ 8.7 Hz, 2H), 7.75e7.77 (m, 2H), 8.25 (d,
J ¼ 8.7 Hz, 1H), 8.34 (d, J ¼ 5.8 Hz, 1H), 8.92 (s, 1H);13C NMR
(100 MHz, DMSO-d6): d 25.45, 26.98, 40.42, 42.32, 55.10, 98.63,
113.77, 117.46, 121.15, 123.97, 124.11, 127.46, 133.38, 133.70, 149.08,
150.12, 151.87, 154.26, 155.85, 160.64, 162.09; ESI-HRMS (m/z)
calculated for C24H25ClN6O: 448.1778, found: 449.3642 (MþH)þ,
451.3801 (M þ 2)þ; Anal. calcd. for C24H25ClN6O: C, 64.21; H, 5.61;
Cl, 7.90; N, 18.72; O, 3.56, found: C, 64.42; H, 5.68; Cl, 7.87; N, 18.66;
O, 3.79.
4.4.21. N2-(4-(7-Chloroquinolin-4-ylamino)butyl)-N4-(3,5-
dimethoxyphenyl)pyrimidine-2,4-diamine (9g)
Yield 70%; mp 224e226  C; IR (KBr, cm1): 3413, 3239, 2946,
2872, 1610, 1586, 1553, 1525, 1479, 1452, 1371, 1329, 1305, 1225,
1204, 1145, 1072, 982, 913, 831; 1H NMR (400 MHz, DMSO-d6):
d 1.64e1.70 (m, 4H), 3.23e3.31 (m, 4H), 3.68 (s, 6H, 2OCH3), 5.94 (d,
J ¼ 5.8 Hz, 1H), 6.07 (t, J ¼ 2.2 Hz, 1H), 6.42 (d, J ¼ 5.8 Hz, 1H), 6.78
(brs, 1H), 6.98 (brs, 2H), 7.27 (brs, 1H), 7.40 (dd, J ¼ 2.2, 8.7 Hz, 1H),
7.75 (d, J ¼ 2.2 Hz, 1H), 7.80 (d, J ¼ 5.1 Hz, 1H), 8.24 (d, J ¼ 8.7 Hz,
1H), 8.33 (d, J ¼ 5.1 Hz, 1H), 9.09 (s, 1H); 13C NMR (100 MHz, DMSO-
d6): d 25.38, 26.90, 40.52, 42.32, 54.99, 93.62, 97.42, 98.65, 117.46,
123.99, 124.10, 127.44, 133.39, 142.33, 149.06, 150.12, 151.87, 156.14,
160.46, 160.63, 162.0; ESI-HRMS (m/z) calculated for C25H27ClN6O2:
478.1884, found: 479.3234 (MþH)þ, 481.3261 (M þ 2)þ; Anal. calcd.
for C25H27ClN6O2: C, 62.69; H, 5.68; Cl, 7.40; N, 17.55; O, 6.68, found:
C, 62.84; H, 5.70; Cl, 7.37; N, 17.82; O, 6.89.
4.5. Assay for in vitro antimalarial activity
The antimalarial activity was determined by measuring plas-
modial LDH activity as described in the literature [67]. A suspension
of red blood cells infected with D6 or W2 strain of P. falciparum
(200 mL, with 2% parasitemia and 2% hematocrit in RPMI 1640
medium supplemented with 10% human serum and 60 mg/mL
amikacin) was added to the wells of a 96-well plate containing
10 mL of serially diluted test samples. The plate was flushed with a
gas mixture of 90% N2, 5% O2, and 5% CO2 and incubated at 37  C, for
72 h in a modular incubation chamber (Billups-Rothenberg, CA).
Parasitic LDH activity was determined according to the procedure
of Makler and Hinrichs [68]. Briefly, 20 mL of the incubation mixture
was mixed with 100 mL of the Malstat™ reagent (Flow Inc., Port-
land, OR) and incubated at room temperature for 30 min. Twenty
microliters of a 1:1 mixture of NBT/PES (Sigma, St. Louis, MO) was
then added and the plate was further incubated in the dark for 1 h.
The reaction was then stopped by the addition of 100 mL of a 5%
acetic acid solution. The plate was read at 650 nm. IC50 values were
computed from the dose response curves. Chloroquine and pyri-
methamine were included as control drugs for comparison. To
determine the selectivity index of antimalarial activity of com-
pounds, in vitro cytotoxicity of these compounds against mamma-
lian cells was also determined. The assay was performed in 96-well
 D. Kumar et al. / European Journal of Medicinal Chemistry 89 (2015) 490e502
tissue culture-treated plates as described earlier [69]. Vero cells
(monkey kidney fibroblasts) were seeded to the wells of 96-well
plate at a density of 25,000 cells/well and incubated for 24 h.
Samples at different concentrations were added and plates were
again incubated for 48 h. The number of viable cells was deter-
mined by Neutral Red assay. The IC50 values were obtained from
dose response curves.
4.6. Computational studies
The molecular structures of all the compounds were drawn
using ChemBioDraw Ultra 12.0 (www.cambridgesoft.com). These
structures were then imported into Maestro implemented in
Schro€dinger, Ligprep module was used to generate energy mini-
mized 3D structures. The possible Lewis structure, tautomers and
ionization states (pH 7.0 ± 2.0) for each of these compounds were
generated and optimized with default settings (Ligprep 2.5,
Schro€dinger, LLC, New York, NY, 2012). Partial atomic charges were
computed using the OPLS_2005 force field. The crystal structures of
wild type PfDHFR-TS (PDB ID: 3QGT; resolution 2.30 Å), quadruple
mutant (N51I þ C59R þ S108N þ I164L) PfDHFR-TS (PDB ID: 3QG2;
resolution: 2.30 Å) complexed with pyrimethamine was extracted
from protein data bank (www.rcsb.org). Protein Preparation
Wizard (Maestro 10.0 Schro €dinger, LLC, New York, NY, 2012) was
used to prepare proteins for docking. Water molecules within 5 Å of
the protein structures were considered. Bond order and formal
charges were assigned and hydrogen atoms were added to the
crystal structure. Further to refine the structure OPLS-2005 force
field parameter was used to remove the steric clashes and the
minimization was terminated when RMSD reached maximum
cutoff value of 0.30 Å.
The location of co-crystalized ligand pyremethamine in both
wild and mutant PfDHFR structures were used to define the centre
of the grid, which was generated using Glide 5.8 (Schro €dinger, LLC,
New York, NY, 2012) with default settings for all parameters. The
grid size was chosen sufficiently large to include all active site
residues involved in substrate binding. The cofactor, NADH in the
PfDHFR-TS wild and mutant structures were also considered as part
of the receptor proteins. All ligand conformers were docked to each
of the receptor grid files (PfDHFR-TS wild and mutant structures)
using Glide extra precision (XP) mode [70]. Default settings were
used for the refinement and scoring.
4.7. In silico ADMET prediction method
The pharmacokinetic profile of the test compounds showing
good antimalarial activity were predicted by using programs Qik-
prop v3.5 (Schro €dinger, Inc., New York, NY, 2012). All the com-
pounds prepared by LigPrep were used for the calculation of
pharmacokinetic properties by QikProp. The program QikProp,
utilizes the method of Jorgensen [71] to compute pharmacokinetic
properties and descriptors such as octanol/water partitioning co-
efficient, aqueous solubility, brain/blood partition coefficient, in-
testinal wall permeability, plasma protein binding and others.
Acknowledgments
D.S.R. thanks Council of Scientific and Industrial Research [No.
02(0049)/12/EMR-II] New Delhi, India for financial support. D.K. is
thankful to CSIR for the award of junior and senior research
fellowship. P.P. is thankful to CSIR for Research Associate. SIK is
thankful to United States Department of Agriculture (USDA), Agri-
cultural Research Service Specific Cooperative Agreement No. 58-
6408-2-0009 for partial support of this work. Mr John Trott is
acknowledged for his excellent technical assistance in biological
501
activity testing at NCNPR. The Authors are also thankful to CIFeU-
SIC, University of Delhi, Delhi for NMR spectral data and RSIC, CDRI,
Lucknow for mass data.
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