4-Aminoquinoline-Pyrimidine hybrids: Synthesis, antimalarial activity, heme binding and docking studies

Article abstract of DOI:10.1016/j.ejmech.2014.10.061

A series of novel 4-aminoquinoline-pyrimidine hybrids has been synthesized and evaluated for their antimalarial activity. Several compounds showed promising in vitro antimalarial activity against both CQ-sensitive and CQ-resistant strains with high select

Full text of DOI:10.1016/j.ejmech.2014.10.061

European Journal of Medicinal Chemistry 89 (2015) 490e502
Contents lists available at ScienceDirect
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
,
c
,
,
Deepak Kumar a, Shabana I. Khan b 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
antimalarial activity. Several compounds showed promising in vitro antimalarial activity against both CQ-
sensitive and CQ-resistant strains with high selectivity index. All the compounds were found to be non-
toxic to the mammalian cell lines. Selected compound 7g exhibited significant suppression of para-
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
investigated by the molecular docking studies in the binding site of PfDHFR. The pharmacokinetic
property analysis of best active compounds was also studied using ADMET prediction.
Received 12 July 2014
Received in revised form
18 October 2014
Accepted 20 October 2014
Available online 22 October 2014
Keywords:
Aminoquinoline
Antimalarial activity
Heme binding studies
Docking studies
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
administered in combination with a long acting partner drug such
as amodiaquine, mefloquine, piperaquine, lumefantrine, sulfadox-
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
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
* Corresponding author.
E-mail address: dsrawat@chemistry.du.ac.in (D.S. Rawat).
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
491
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-
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.
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].
2. Results and discussion
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
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
(Fig. 2) showed potent antimalarial activity (IC50 ¼ 0.005e0.44
mM)
with no cytotoxicity up to 60 M concentration. Two selected
m
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
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
9a) showed antimalarial activity in the range of 0.019e1.509
mM
(D6) and 0.144e1.529 M (W2) with propylene linked 8a found to
m
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,
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
for apparent signs of toxicity, parasitemia and survival till day 28
post infection (Table 2). The animals were monitored for any signs
of toxicity, suppression in parasitemia and survival until day 28
post-infection. As shown in Table 2, no significant in vivo antima-
larial activity was observed for compound 7g. A weak effect was
noticed at the highest dose at day 5 (42.12% suppression in para-
sitemia) but the effect disappeared on day 7 and the mean survival
time was only 9 days compared to 7.6 days for vehicle treated an-
imals and 26.2 days for chloroquine treated animals.
In vitro antimalarial activity and cytotoxicity of 4-aminoquinoline-pyrimidine
hybrids.
Compds
P. falciparum
(D6 clone)
P. falciparum
(W2 clone)
Cytotoxicity Resistance
(Vero cells) index (RI)
IC50
M)
S.I.
IC50
M)
S.I.
IC50 (mM)
(
m
(
m
7a
7b
7c
7d
7e
7f
7g
8a
8b
8c
8d
8e
8f
8g
9a
9b
9c
9d
9e
9f
1.509 >13.25
6.022 >3.32
0.047 335.17
0.037 >540.54 0.136 >147.05 NC
0.101 134.71
1.529 >13.08
9.994 >2.0
0.046 342.45
NC
NC
15.753
1.05
1.65
0.978
3.67
1.41
6.025
1.75
7.57
3.35
9.46
0.95
2.26
2.025
2.81
9.15
1.82
1.18
1.34
3.75
1.34
2.72
10.48
0.143 95.14
0.241 >82.98
13.606
NC
0.040 >500.0
0.033 >606.06 0.058 >344.82 NC
2.3. Mechanistic studies: heme binding studies
0.019 675.89
0.028 489.82
0.047 285.72
0.049 227.77
0.153 120.76
0.040 333.37
0.038 339.57
0.044 325.50
3.196 >6.25
0.044 250.63
0.047 213.68
0.138 139.59
0.050 222.74
0.044 284.68
0.144 89.18
0.094 145.90
0.445 30.17
0.047 237.46
0.346 53.40
0.081 164.62
0.107 120.59
0.403 35.53
5.831 >3.43
0.052 212.07
0.063 159.41
0.518 37.19
0.067 166.22
0.120 104.38
12.842
13.715
13.429
11.161
18.477
13.335
12.904
14.322
NC
11.028
10.043
19.264
11.137
12.526
NC
Chloroquine and other quinoline derivatives are believed to
show their antimalarial activity by inhibition of hemozoin forma-
tion within the parasite food vacuole [49]. Hemozoin was originally
considered to be formed by the polymerization of heme [50], but it
has now been demonstrated that it is a crystalline cyclic dimer of
ferriprotoporphyrin IX [51]. It is widely accepted that CQ accumu-
lates in the plasmodium food vacuole and binds to some form of
parasite heme/hematin, and inhibits hemozoin bio-crystallization
[52e54,59]. This is a non-enzymatic process in which hematin
monomer (heme) released from parasite hemoglobin digestion is
converted into hemozoin, also known as malaria pigment. Hemo-
zoin is an insoluble crystal in which adjacent heme dimers are
linked via hydrogen bonds between free propionic acids [53].
Cohen et al. were the first to show that CQ forms a complex with
ferriprotoporphyrin IX (FPIX) in aqueous solution, which was
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
been determined that CQ forms complexes with both monomeric
9g
CQ
0.035 >571.42 0.367 >54.49
NA
Pyrimethamine 0.01
e
e
NT
IC50 the concentration that causes 50% growth inhibition; S.I. Selectivity
index ¼ (IC50 for cytotoxicity to Vero cells/IC50 for antimalarial activity); R.I.
(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.
and 8c (R ¼ 4-Cl, n ¼ 3) showed better antimalarial activity against
CQ-sensitive strain D6 while compounds 7c (R ¼ 4-Cl, n ¼ 2), 9c
(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)
were equally active against CQ-resistant strain W2 (RI ~ 1). Com-
pounds with a methyl group at the para position of the phenyl ring
(7e, 8e, 9e) led to partial decrease in the antimalarial activity
against both the strains, while methoxy substitution at 4-position
(7f, 8f, 9f) and 3,5-position (7g, 8g, 9g) of the phenyl ring led to
an increase in the antimalarial activity with all the compounds
more active against CQ-sensitive strain of P. falciparum (RI > 1).
Most of the compounds showed good values of resistance index (RI)
(Table 1). The RI is the ratio of the IC50 value against the CQ-
resistant strain to the IC50 value against the CQ-sensitive strain.
The low value of resistance index indicates the activity of a com-
pound regardless of the susceptibility of the parasite strain,
whereas the large value indicates the loss of activity due to the drug
resistance. Therefore, a potent drug molecule should have a smaller
resistance index. In the present study, resistance index for chloro-
quine was found to be 10.48, whereas tested compounds have RI
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-
brids may be active against P. falciparum strains resistant to parent
antimalarials chloroquine and pyrimethamine. Cytotoxicity was
also determined against Vero cell line (Table 1). All the compounds
showed toxicity at very high concentration as compared to their
concentrations (IC50) responsible for their antimalarial activity, as
shown by the high selectivity index values in Table 1.
and m-oxodimeric FPIX [62,63]. Therefore, we decided to evaluate
the binding of the most potent compound 7c with heme.
A solution of hematin in 40% DMSO showed a Soret band at
402 nm, indicating the presence of monomeric heme under the
conditions used (0.02 M HEPES buffer, pH 7.4 and 0.02 M MES
buffer, pH 5.6). The stepwise addition of small increments of
compound 7c into a constant concentration of monomeric heme
(5.0 mM) resulted in a substantial decrease in intensity of the Fe(III)
PPIX Soret band at 402 nm with no shift in the absorption
maximum (Fig. 3). This indicates the association of compounds
with hematin. Solvent (DMSO) did not affect the binding of com-
pound 7c with heme at the pH values used in this experiment. The
stoichiometry ratio of the most stable complexes of compound 7c
with monomeric heme at pH 7.4 and 5.6 was deduced from the
Job's plot. The absorbance at 402 nm got to maximum when mole
fraction of compound was approximately 0.5. Thus 1:1 ratio was
established for the association of compound heme at both the pH
values (Fig. 4).
Table 2
In vivo antimalarial activity of compound 7g in P. berghei-mouse malaria model.
Treatment
(PO)
Dose (mg/kg ꢁ no.
% Suppression
in parasitemiaa
Mean
survival
time
Toxicity
of days post-infection)
Day 5
Day 7
Vehicle
CQ
7g
7g
7g
NA ꢁ 3
e
e
7.6
26.2
5
8.6
9
NA
NC
NC
NC
NC
100 ꢁ 3
11.1 ꢁ 3
33.3 ꢁ 3
100 ꢁ 3
100
100
NA
6.20
10.43
Compound 7g with significant in vitro antimalarial activity was
selected for further in vivo evaluation. In vivo antimalarial activity
was determined through oral route of administration in a P. berghei-
mouse malaria model as described earlier [40]. The compounds
were administered to the P. berghei infected mice, through oral
1.77
15.14
42.12
a
% suppression in parasitemia is calculated by considering the mean parasitemia
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 ( -oxo-heme). Therefore, the binding of compound 7c was
also studied with -oxo-dimers of heme at pH 5.8. A solution of
heme in aqueous NaOH showed a peak at 362 nm. Addition of
M) to a solution of -oxo-dimer (10 M) in
constant than the standard drug chloroquine at pH 5.6. The asso-
ciation constants for the binding with -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,
m
m
m
compound 7c (0ꢂ20
m
m
m
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-
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-
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
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
well as 4-chlorophenyl ring (Fig. 7).
Binding constants for compound 7c and chloroquine with heme.
Another compound predicted to have low binding energy
(ꢂ58.11 kcal molꢂ1) and high glide score (ꢂ8.70) was 7g, showing
similar H-bond pattern between linker NH group of compound and
Compd
Monomeric
heme log K
(pH ¼ 5.6)
Monomeric
heme log K
(pH ¼ 7.4)
m-oxo-heme
log K (pH ¼ 5.8)
7c
5.510
4.65a
1:1
5.045
5.15a
1:1
5.123
5.58a
1:1
oxygen side chain of Asp54. The p-p interactions between the two
aromatic rings (4-chlorophenyl and pyrimidine) of compounds and
aromatic ring of Phe58 of mutant PfDHFR was also observed (Fig. 7).
Chloroquine
Stoichiometry
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
atom of Ile164 and NH group of 3,5-dimethoxy phenyl ring of
compound 7g in wild type PfDHFR (Fig. 6).
structures of the compounds were docked in the binding pocket of
both the wild type PfDHFR-TS and quadruple mutant PfDHFR-TS
(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
compounds. The Glide XP Gscores and glide energies clearly indi-
cate that the most active compounds in the study exhibited sig-
nificant binding affinities towards the wild (Glide energy
range ꢂ58.11 kcal molꢂ1eꢂ44.32 kcal molꢂ1) and quadruple
Different pharmacokinetic parameters of compounds in the
study, showing good antimalarial activity in malarial parasites were
calculated using ADMET predictions by Qikprop v3.5. The most
important of these parameters together with its permissible ranges
are listed in Tables 5 and 6. Qikprop results for various parameters
of Lipinski's rule of 5, a preliminary test of the drug-likeness of the
compounds is presented in Table 5. An orally active compound
should not have more than 2 violations of these rules. In the present
study, all the active test compounds showed Lipinski's rule viola-
tions less than the maximum permissible value of 2, indicating that
these active test compounds have good drug likeness properties.
The optimum values of descriptors such as number of rotable
bonds (<15) and polar surface area (7e200 Å2) can also have sig-
nificant influence on oral bioavailability of compounds [65]. In the
present study, all the test compounds possess a number of rotatable
bonds <15 and polar surface area falls satisfactorily in the
permissible range (7e200 Å). Similarly, the test molecules were
checked for their intestinal absorption or permeation property,
which is confirmed by the predicted Caco-2 cells permeability
(QPPCaco), used as a model for the guteblood barrier [66]. QPPCaco
predictions for all the test compounds showed excellent values.
Further, QPlogKhsa, the prediction for human serum albumin
binding were calculated and all inhibitors were predicted to lie
within the expected range for 95% of known drugs (ꢂ1.5e1.5). Also,
the QikProp descriptor for brain/blood partition coefficient
(QPlogBB) and the bloodebrain barrier mimic MDCK cell perme-
ability (QPPMDCK) showed reliable predictions for all the test
compounds and the reference compounds. The aqueous solubility
(QPlogS) parameters for the test compounds were assessed and all
the compounds were predicted to have QPlogS values in the
permissible range. Furthermore, QPlogHERG descriptor for the
prediction of IC50 value of HERG Kþ channel blockage was predicted
for the test compounds. Compounds 7d, 7f and 7g have been pre-
dicted to possess values for in QPlogHERG in the permissible range
comparable to reference compounds pyrimethamine and cyclo-
guanil (Table 6).
mutant (Glide energy range ꢂ56.14 kcal molꢂ1eꢂ43.74 kcal molꢂ1
)
PfDHFR-TS structures and the energy ranges are comparable to that
of reference compounds (pyrimethamine, cycloguanil and
WR99210) and the native substrate of DHFR dihydrofolate (Table 4).
Figs. 6 and 7 shows three dimensional binding pose of two
selected active compounds with wild type and mutant PfDHFR-TS,
respectively. Compound 9c showed lowest binding energy
(ꢂ57.14 kcal molꢂ1
) and considerable high Glide XP score
(ꢂ8.88 kcal molꢂ1) for wild type PfDHFR and ꢂ56.14 and ꢂ9.27 for
mutant type PfDHFR. A hydrogen bond interaction was observed
between NH group of butylene linker of compound 9c and oxygen
side chain of Asp54 of both wild and mutant PfDHFR. In addition,
compound 9c forms Hebonding interaction between the NH group
of 4-chlorophenyl ring and the main chain oxygen atom of Ile164 in
the wild type PfDHFR (Fig. 6). Further, compound show
p-p inter-
action between aromatic ring of Phe58 and pyrimidine ring in case
of wild type (Fig. 6), while in mutant type PfDHFR, p-p interaction
Table 4
Glide docking scores (kcal molꢂ1) and docking energies of best active molecules
along with the reference compounds (pyrimethamine, cycloguanil and WR99210)
and dihydrofolate bound to wild and mutant PfDHFR-TS binding site.
Compounds
Docking results
with wild
PfDHFR
Docking results
with mutant
PfDHFR
Docking results
with PfLDH
XP
Glide
XP
Glide
XP
Glide
GScore
energy
GScore
energy
GScore
energy
7c
7d
7f
7g
8a
8b
8c
8d
8f
8g
9a
9c
9d
9f
9g
ꢂ8.45
ꢂ5.63
ꢂ8.71
ꢂ8.70
ꢂ8.14
ꢂ8.54
ꢂ6.89
ꢂ6.02
ꢂ8.43
ꢂ8.29
ꢂ8.38
ꢂ8.88
ꢂ6.77
ꢂ8.29
ꢂ7.23
e
ꢂ44.32
ꢂ48.12
ꢂ51.67
ꢂ58.11
ꢂ55.20
ꢂ49.41
ꢂ52.42
ꢂ50.35
ꢂ50.30
ꢂ53.83
ꢂ48.32
ꢂ57.14
ꢂ53.36
ꢂ51.26
ꢂ53.13
e
ꢂ8.55
ꢂ6.43
ꢂ7.73
ꢂ9.15
ꢂ8.85
ꢂ8.15
ꢂ6.85
ꢂ8.23
ꢂ6.56
ꢂ6.51
ꢂ8.04
ꢂ9.27
ꢂ9.04
ꢂ8.61
ꢂ6.99
e
ꢂ47.95
ꢂ52.27
ꢂ43.19
ꢂ53.74
ꢂ51.44
ꢂ51.55
ꢂ49.99
ꢂ47.32
ꢂ48.46
ꢂ48.85
ꢂ50.26
ꢂ56.14
ꢂ49.05
ꢂ50.40
ꢂ50.72
e
ꢂ5.88
ꢂ5.75
ꢂ5.87
ꢂ6.35
ꢂ5.70
ꢂ4.07
ꢂ6.34
ꢂ4.49
ꢂ4.62
ꢂ6.28
ꢂ3.94
ꢂ6.50
ꢂ5.85
ꢂ5.15
ꢂ5.50
ꢂ6.62
e
ꢂ45.29
ꢂ42.84
ꢂ44.79
ꢂ48.39
ꢂ43.08
ꢂ42.91
ꢂ44.31
ꢂ44.21
ꢂ38.22
ꢂ43.59
ꢂ35.84
ꢂ50.54
ꢂ49.69
ꢂ40.07
ꢂ40.30
ꢂ58.32
e
3. Conclusions
In summary, we have reported the synthesis and antimalarial
activity of a series of 4-aminoquinoline-pyrimidine hybrids. The
in vitro evaluation of these hybrids against D6 and W2 strains of
P. falciparum depicted activity in the nanomolar range. Also, these
hybrids exhibited high selectivity indices and low toxicity against
the tested cell lines. Three compounds (7g, 8a and 8b) exhibited
Chloroquine
Dihydrofolate
Pyrimethamine
Cycloguanil
WR99210
very potent antimalarial activity (IC50 ¼ 0.019
which was comparable to the standard drug chloroquine
(IC50 ¼ 0.035 M) against CQ-sensitive strain, whereas all the
compounds showed better activity than chloroquine and
mMe0.033 mM)
ꢂ9.33
ꢂ9.04
ꢂ8.94
ꢂ4.84
ꢂ64.84
ꢂ44.91
ꢂ38.55
ꢂ37.03
ꢂ11.00
ꢂ9.39
ꢂ8.95
ꢂ5.48
ꢂ61.30
ꢂ63.55
ꢂ46.6
e
e
e
e
m
ꢂ34.30
e
e

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
purified by column chromatography using EtOAc/Hexane as eluent
to afford pure compound 5a.
Prediction of Lipinski's ‘Rule of 50 for the active test compounds.a
Compound
mol_MW
donorHB accptHB QPlogPo/w ‘N’ of
(<500 amu) (<5)
(<10)
(<5)
violations
(<2)
4.3.1. 2-Chloro-N-phenylpyrimidin-4-amine (5a)
Yield 80%; mp 186e187 ꢀC; 1H NMR (400 MHz, CDCl3):
d 6.59 (d,
7c
7d
7f
7g
8a
8b
8c
8d
8f
8g
9a
9c
9d
9f
9g
425.31
469.77
420.9
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
1
4
5
5
5
5
6.5
5
5
5
5
5
6.5
5
5
5
5
6.5
4
3
4.391
4.391
4.003
4.045
4.935
4.727
4.95
5.013
4.936
5.084
4.958
5.406
5.446
4.758
5.083
4.538
1.809
0.888
0
0
0
0
0
0
0
1
0
1
0
1
1
0
1
0
0
0
J ¼ 5.86 Hz, 1H), 7.23 (brs, 1H, NH), 7.25e7.27 (m, 1H), 7.30e7.32 (m,
2H), 7.40e7.44 (m, 2H), 8.12 (d, J ¼ 5.86 Hz, 1H).
450.92
404.90
422.89
439.34
483.79
434.92
464.95
418.92
453.37
497.82
448.95
478.98
319.88
4.3.2. 2-Chloro-N-(4-fluorophenyl)pyrimidin-4-amine (5b)
Yield 73%; mp 177e180 ꢀC; 1H NMR (400 MHz, CDCl3):
d 6.46 (d,
J ¼ 5.86 Hz, 1H), 7.09e7.14 (m, 2H), 7.29e7.32 (m, 2H), 7.43 (brs, 1H,
NH), 8.10 (d, J ¼ 5.86 Hz, 1H).
4.3.3. 2-Chloro-N-(4-chlorophenyl)pyrimidin-4-amine (5c)
Yield 75%; mp 198e201 ꢀC; 1H NMR (400 MHz, CDCl3):
d 6.55 (d,
J ¼ 5.86 Hz, 1H), 6.97 (brs, 1H, NH), 7.29 (d, J ¼ 8.05 Hz, 2H), 7.38 (d,
J ¼ 8.05 Hz, 2H), 8.15 (d, J ¼ 5.86 Hz, 1H).
Chloroquine
Pyrimethamine 248.71
Cycloguanil
253.73
3
4.3.4. N-(4-Bromophenyl)-2-chloropyrimidin-4-amine (5d)
Yield 76%; mp 206e209 ꢀC; 1H NMR (400 MHz, CDCl3):
d 6.55 (d,
a
All values calculated by QikProp v 3.5 and the explanations of the descriptors are
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)
Yield 70%; mp 200e203 ꢀC; 1H NMR (400 MHz, CDCl3):
d 2.37 (s,
4.1. General
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).
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 cmꢂ1.1H 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
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-
on
d scale and the coupling constant (J) in Hz. Melting points were
chloroquinolin-4-yl)amino)ethyl)-N4-phenylpyrimidine-2,4-
diamine (7a) and related compounds (7be7g, 8ae8g and 9ae9g)
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.
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.
4.2. 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].
4.4.1. N2-(2-(7-Chloroquinolin-4-ylamino)ethyl)-N4-
phenylpyrimidine-2,4-diamine (7a)
4.3. Typical procedure for the synthesis of 2-chloro-N-
phenylpyrimidin-4-amine (5a) and related compounds (5be5g)
Yield 72%; mp 198e199 ꢀC; IR (KBr, cmꢂ1): 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),
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
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
OralAbsorption
(>80%-high,
QPPCaco nmsꢂ1
QPlogBB
QPPMDCK
QPlogKhsa
QPlogHERG
(concern
QPlogS
(ꢂ6.5e0.5)
PSA
#rotor
(<25 poor, >500 great)a
(ꢂ3.0e1.2)a
(<25 poor >500 great)a
(ꢂ1.5e1.5)a
(7.0e200.0)a
(0e15)a
below ꢂ5)a
<25% poor)a
7c
7d
7f
7g
8a
8b
8c
8d
8f
8g
9a
9c
9d
9f
9g
CQ
Pyr
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
84.346
1926.73
ꢂ0.236
ꢂ0.208
ꢂ0.39
4022.729
4218.681
2150.276
2085.817
1985.673
2650.002
3419.613
3636.597
2009.653
1988.037
1988.98
2985.112
3217.678
2301.046
1879.327
2061.643
468.849
0.382
0.386
0.277
0.289
0.565
0.499
0.563
0.581
0.576
0.576
0.576
0.717
0.731
0.482
0.582
0.585
ꢂ0.243
ꢂ5.157
ꢂ4.934
ꢂ4.803
ꢂ4.425
ꢂ6.975
ꢂ5.888
ꢂ5.899
ꢂ5.923
ꢂ5.958
ꢂ6.651
ꢂ6.146
ꢂ6.14
ꢂ4.603
ꢂ4.483
ꢂ3.917
ꢂ3.887
ꢂ5.93
63.606
61.352
69.351
79.658
64.795
64.906
64.942
64.919
72.535
81.529
65.013
65.41
65.429
71.581
79.2
25.8
73.731
7
7
8
9
8
8
8
8
9
1961.069
1970.518
1760.353
1569.034
1456.769
1460.136
1468.714
1586.519
1570.759
1571.443
1464.286
1481.444
1863.611
1491.108
1479.516
412.287
ꢂ0.467
ꢂ0.617
ꢂ0.492
ꢂ0.448
ꢂ0.438
ꢂ0.594
ꢂ0.751
ꢂ0.612
ꢂ0.554
ꢂ0.523
ꢂ0.54
ꢂ5.241
ꢂ5.522
ꢂ5.607
ꢂ5.343
ꢂ6.107
ꢂ5.388
ꢂ5.924
ꢂ5.863
ꢂ4.58
10
9
9
ꢂ5.991
ꢂ5.194
ꢂ5.929
ꢂ6.326
ꢂ4.318
9
10
11
8
ꢂ0.777
0.415
ꢂ0.78
ꢂ5.584
ꢂ4.49
ꢂ2.978
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.
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.
4.4.2. N2-(2-(7-Chloroquinolin-4-ylamino)ethyl)-N4-(4-
fluorophenyl)pyrimidine-2,4-diamine (7b)
Yield 60%; mp 216e218 ꢀC; IR (KBr, cmꢂ1): 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):
4.4.5. N2-(2-(7-Chloroquinolin-4-ylamino)ethyl)-N4-p-
tolylpyrimidine-2,4-diamine (7e)
Yield 70%; mp 216e220 ꢀC; IR (KBr, cmꢂ1): 3436, 3232, 3033,
2922, 2885, 1580, 1524, 1510, 1379, 1355, 1332, 1257, 131, 975, 904,
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),
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.
7.91 (brs, 1H), 8.17 (d, J ¼ 8.7 Hz, 1H), 8.35 (brs, 1H), 9.21 (s, 1H); 13
C
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
C
21H18ClFN6: 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.
4.4.3. N4-(4-Chlorophenyl)-N2-(2-(7-chloroquinolin-4-ylamino)
ethyl)pyrimidine-2,4-diamine (7c)
4.4.6. N2-(2-(7-Chloroquinolin-4-ylamino)ethyl)-N4-(4-
methoxyphenyl)pyrimidine-2,4 diamine (7f)
Yield 75%; mp 228e232 ꢀC; IR (KBr, cmꢂ1): 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
Yield 75%; mp 219e222 ꢀC; IR (KBr, cmꢂ1): 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
(400 MHz, DMSO-d6):
d
3.41e3.44 (m, 2H), 3.56e3.57 (m, 2H),
(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,
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,
DMSO-d6): d 36.66, 39.50, 51.60, 95.23, 110.37, 113.94,117.99, 120.41,
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.
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)
4.4.4. N4-(4-Bromophenyl)-N2-(2-(7-chloroquinolin-4-ylamino)
ethyl)pyrimidine-2,4-diamine (7d)
Yield 78%; mp 224e226 ꢀC; IR (KBr, cmꢂ1): 3406, 3221, 3091,
2937,1579,1528,1492,1459,1431,1331,1308,1247,1224,1201,1152,
Yield 55%; mp 220e224 ꢀC; IR (KBr, cmꢂ1): 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,
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),
DMSO-d6):
d 3.42e3.44 (m, 2H), 3.55e3.57 (m, 2H), 6.02 (d,

D. Kumar et al. / European Journal of Medicinal Chemistry 89 (2015) 490e502
499
8.33 (brs, 1H), 9.16 (s, 1H); 13C NMR (100 MHz, DMSO-d6):
d
42.77,
C
22H20BrClN6: 482.0621, found: 483.1493 (MþH)þ, 485.1466
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.
(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, cmꢂ1): 3432, 3245, 2924,
2861, 1578, 1527, 1510, 1425, 1334, 1238, 1136, 1084, 976, 907, 877,
4.4.8. N2-(3-(7-Chloroquinolin-4-ylamino)propyl)-N4-
phenylpyrimidine-2,4-diamine (8a)
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,
Yield 70%; mp 190e193 ꢀC; IR (KBr, cmꢂ1): 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,
1H), 9.02 (s, 1H); 13C NMR (100 MHz, DMSO-d6):
d 21.24, 28.78,
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,
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.
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.
4.4.13. N2-(3-(7-Chloroquinolin-4-ylamino)propyl)-N4-(4-
methoxyphenyl)pyrimidine-2,4-diamine (8f)
Yield 75%; mp 186e188 ꢀC; IR (KBr, cmꢂ1): 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):
4.4.9. N2-(3-(7-Chloroquinolin-4-ylamino)propyl)-N4-(4-
fluorophenyl)pyrimidine-2,4-diamine (8b)
d
1.88e1.92 (m, 2H), 3.29e3.33 (m, 4H), 3.66 (s, 3H, OCH3), 5.89 (d,
Yield 65%; mp 192e195 ꢀC; IR (KBr, cmꢂ1): 3437, 3232, 3042,
2925, 1579, 1525, 1507, 1468, 1445, 1416, 1378, 1235, 1219, 1131, 976,
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
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,
(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.
1H), 8.20 (d, J ¼ 8.7 Hz, 1H), 8.30 (d, J ¼ 5.8 Hz, 1H), 9.14 (s, 1H); 13
C
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.
4.4.14. N2-(3-(7-Chloroquinolin-4-ylamino)propyl)-N4-(3,5-
dimethoxyphenyl)pyrimidine-2,4-diamine (8g)
Yield 70%; mp 191e192 ꢀC; IR (KBr, cmꢂ1): 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):
4.4.10. N4-(4-Chlorophenyl)-N2-(3-(7-chloroquinolin-4-ylamino)
propyl)pyrimidine-2,4-diamine (8c)
d
1.91e1.94 (m, 2H), 3.30e3.40 (m, 4H), 3.68 (s, 6H, 2OCH3), 5.97 (d,
Yield 72%; mp 181e183 ꢀC; IR (KBr, cmꢂ1): 3433, 3248, 3087,
2927, 1609, 1582, 1549, 1490, 1332, 1237, 1139, 1085, 981, 902, 875,
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):
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
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:
(100 MHz, DMSO-d6):
d
28.70, 39.50, 41.13, 99.52, 118.36, 121.42,
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,
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.
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)
4.4.11. N4-(4-Bromophenyl)-N2-(3-(7-chloroquinolin-4-ylamino)
propyl)pyrimidine-2,4-diamine (8d)
Yield 70%; mp 190e192 ꢀC; IR (KBr, cmꢂ1): 3417, 3238, 3001,
2935, 2863, 1585, 1544, 1497, 1428, 1347, 1234, 1131, 975, 902, 852,
Yield 50%; mp 203e242 ꢀC; IR (KBr, cmꢂ1): 3422, 3185, 2927,
1578, 1489, 1394, 1329, 1233, 1136, 1072, 1004, 979, 901, 853, 795;
799; 1H NMR (400 MHz, DMSO-d6):
d 1.66e1.70 (m, 4H), 3.24e3.31
1H NMR (400 MHz, DMSO-d6)
d
d
:
1H NMR (400 MHz, DMSO-d6)
1.90e1.93 (m, 2H), 3.31e3.33 (m,
d:
(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):
1H NMR (400 MHz, DMSO-d6):
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
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
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.
C
23H23ClN6: 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)
4.4.20. N2-(4-(7-Chloroquinolin-4-ylamino)butyl)-N4-(4-
methoxyphenyl)pyrimidine-2,4-diamine (9f)
Yield 65%; mp 175e178 ꢀC; IR (KBr, cmꢂ1): 3448, 3247, 3067,
2933, 2867, 1583, 1507, 1412, 1353, 1219, 1154, 1081, 981, 825, 794;
Yield 70%; mp 200e202 ꢀC; IR (KBr, cmꢂ1): 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,
1H 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,
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
1H), 8.25 (d, J ¼ 9.5 Hz, 1H), 8.34 (d, J ¼ 5.1 Hz, 1H), 9.16 (s, 1H); 13
C
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-
(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.
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)
4.4.21. N2-(4-(7-Chloroquinolin-4-ylamino)butyl)-N4-(3,5-
dimethoxyphenyl)pyrimidine-2,4-diamine (9g)
Yield 70%; mp 229e232 ꢀC; IR (KBr, cmꢂ1): 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):
Yield 70%; mp 224e226 ꢀC; IR (KBr, cmꢂ1): 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), 3.24e3.32 (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,
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-
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,
d6):
d 25.38, 26.90, 40.52, 42.32, 54.99, 93.62, 97.42, 98.65, 117.46,
161.98; ESI-HRMS (m/z) calculated for
C
23H22Cl2N6:452.1283,
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.
found: 453.1317 (MþH)þ, 455.1084 (M þ 2)þ; Anal. calcd. for
C
23H22Cl2N6: 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)
4.5. Assay for in vitro antimalarial activity
Yield 50%; mp 240e242 ꢀC; IR (KBr, cmꢂ1): 3437, 3236, 3064,
2933, 2867, 1653, 1579, 1547, 1491, 1435, 1393, 1342, 1278, 1237,
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
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
(200
medium supplemented with 10% human serum and 60
amikacin) was added to the wells of a 96-well plate containing
mL, with 2% parasitemia and 2% hematocrit in RPMI 1640
mg/mL
(100 MHz, DMSO-d6):
d
25.45, 26.92, 40.49, 42.30, 98.61, 112.63,
10 mL of serially diluted test samples. The plate was flushed with a
117.46, 120.92, 123.96, 124.10, 127.46, 131.21, 133.38, 140.12, 149.07,
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
150.11, 151.85, 160.33, 161.96; ESI-HRMS (m/z) calculated for
C
23H22BrClN6: 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.
of Makler and Hinrichs [68]. Briefly, 20
mL of the incubation mixture
was mixed with 100 L of the Malstat™ reagent (Flow Inc., Port-
m
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.
4.4.19. N2-(4-(7-Chloroquinolin-4-ylamino)butyl)-N4-p-
tolylpyrimidine-2,4-diamine (9e)
The reaction was then stopped by the addition of 100 mL of a 5%
Yield 70%; mp 202e204 ꢀC; IR (KBr, cmꢂ1): 3329, 3240, 3164,
3028, 2973, 2923,1582,1510,1451,1411,1390,1359,1250,1173,1147,
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
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,

D. Kumar et al. / European Journal of Medicinal Chemistry 89 (2015) 490e502
501
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.
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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