Quinoline-pyrimidine hybrids: Synthesis, antiplasmodial activity, SAR, and mode of action studies

Article abstract of DOI:10.1021/jm4014778

For the treatment of malaria which affects nearly 200 million people each year and the continued exacerbation by the emergence of drug resistance to most of the available antimalarials, the "covalent bitherapy" suggests hybrid molecules to be the next-generation antimalarial drugs. In this investigation, new hybrids of 4-aminoquinoline and pyrimidine moieties that show antiplasmodial activity in the nM range against chloroquine-resistant as well as chloroquine-sensitive strains of Plasmodium falciparum have been prepared. Cytotoxicity evaluation and mode of action of most potent hybrid molecule have been conducted.

Full text of DOI:10.1021/jm4014778

Article
pubs.acs.org/jmc
Quinoline−Pyrimidine Hybrids: Synthesis, Antiplasmodial Activity,
SAR, and Mode of Action Studies
Kamaljit Singh,* Hardeep Kaur, Peter Smith, Carmen de Kock, Kelly Chibale, and Jan Balzarini
,
†
†
‡
‡
§
∥
†
Department of Chemistry, UGC Centre of Advance Study-I, Guru Nanak Dev University, Amritsar, Punjab 143005, India
‡
Division of Pharmacology, Department of Medicine, University of Cape Town, Observatory 7925, South Africa
§
Department of Chemistry, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 701,
South Africa
∥
Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
*
S Supporting Information
ABSTRACT: For the treatment of malaria which affects
nearly 200 million people each year and the continued
exacerbation by the emergence of drug resistance to most of
the available antimalarials, the “covalent bitherapy” suggests
hybrid molecules to be the next-generation antimalarial drugs.
In this investigation, new hybrids of 4-aminoquinoline and
pyrimidine moieties that show antiplasmodial activity in the
nM range against chloroquine-resistant as well as chloroquine-
sensitive strains of Plasmodium falciparum have been prepared. Cytotoxicity evaluation and mode of action of most potent hybrid
molecule have been conducted.
INTRODUCTION
triggered a new strategy in drug design which involves linking
■
1
6,17
1
,2
two drugs with intrinsic activity into a single agent.
In most
Malaria is one of the major infectious diseases along with
tuberculosis and HIV/AIDS which infects over 200 million
people each year and results in over 1 million deaths. Malaria is
endemic in more than 100 countries throughout the tropics and
in some temperate regions. According to the latest WHO
estimates, malaria accounted for 219 million illnesses and an
estimated 660000 deaths worldwide in 2010; about 90% of all
malaria deaths occur in Africa and were due to Plasmodium
such hybrids, two pharmacophores have independent modes of
action against different targets that make the emergence of drug
resistance less likely. Recently, the fast-acting artemisinin has
been combined with the slow-acting quinine into a hybrid drug
1
8
4
for malaria. Drugs based on fully synthetic peroxidic
19
molecules, trioxaquines represented by 5 (DU1301) and 6
PA1103/SAR116242), constitute other examples of the
(
3
,4
synthetic hybrid molecules containing a 1,2,4-trioxane motif
linked to a 4-aminoquinoline unit and have shown promising
activity against early erythrocytic stages of P. falciparum as
falciparum infections of young children. The traditional drugs
such as chloroquine (1, CQ), pamaquine 2, and mefloquine 3
Figure 1), which were once significantly active and affordable,
(
R
tested against both CQ-resistant (CQ ) and CQ-sensitive
lost significance owing to the emergence of parasite strains that
are resistant to such drugs. The drug resistance of CQ is
strongly linked to mutations in the gene that give rise to the
protein, PfCRT (P. falciparum chloroquine resistance trans-
porter), located in the parasite’s digestive vacuole (DV)
S
20,21
(
CQ ) strains.
In addition to alkylation of heme by
trioxaquine, the aminoquinoline partner of 5 and 6 promotes
heme stacking and prevents polymerization of heme into the
nontoxic hemazoin and thus act at different stages of the
parasite’s life cycle through a dual mode of action.
5
−7
membrane.
Consequently, excessive export of CQ from its
The hybrid antimalarials featuring quinoline as one partner
are represented (A−L, Figure 2) by quinoline−cinnamic acid
24
site of action and decrease in the accumulation of the drug in
8,9
the DV eventually results in loss of antimalarial activity. Small
molecules structurally related to the 4-aminoquinoline motif are
known to exert antimalarial action through prevention of
polymerization of toxic heme, leading to its accumulation which
2
2
23
A, quinoline−ferrocenophane B, quinoline−rhodanine C,
25
26
quinoline−primaquine D, quinoline−triazine E, quinoline−
27
28
isatin F, quinoline−clotrimazole G, quinoline-γ-hydroxy-γ-
29
30
10
lactam H, quinoline−furoxan I, quinoline−hydroxypyri-
results in parasite death. Heme targeting, molecules that act
on several other targets such as phospholipids, tyrosine
31 32−34
done J, quinoline−pyrimidines K,
and quinoline−
3
5,36
1
1
12
pyrimidine carboxylates L,
etc., that possess promising
kinase, DNA via intercalation, hemoglobin degrading
R
1
3,14
13−15
activities against CQ strains and hit different targets. Spurred
proteases,
and phospholipases,
have shown a useful
level of antimalarial action.
To tackle the development of resistance to the antimalarial
drugs, the concept of hybrid drugs (covalent bitherapy) has
Received: September 21, 2013
©
XXXX American Chemical Society
A
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Figure 1. Quinoline antimalarials.
Figure 2. Representative quinoline hybrid antimalarial compounds A−L.
by the therapeutic advantage of the hybrid antimalarials,
recently we reported on the structure−activity relationship,
cytotoxicity, and mode of action studies of some quinoline−
uracil, lacking a carboxylate at the 5-position. Moreover, side
chain diversity was also explored by varying the nature and
length of the linker between the two “N” atoms as well as the
substitution pattern and basicity of the distal amino group.
3
5,36
pyrimidine carboxylate hybrids L (Figure 2).
It was found
1
that the activity of these hybrids was dependent upon the
length of the linker (aromatic/aliphatic) connecting the two
pharmacophores as well as the substituents on the pyrimidine
motif. However, these hybrids displayed quite a high toxicity
Furthermore, binding experiments ( H NMR, FTIR, and
LCMS) were performed with the heme (monomeric and μ-
oxo) as well as DNA (CT-DNA and pUC18 DNA) to explore
possible mode of antimalarial action of these hybrids.
3
5,36
and ClogP values.
In this investigation, we report the
CHEMISTRY
synthesis, evaluation of in vitro antiplasmodial activity,
physicochemical properties such as acid dissociation constant
■
The quinoline−pyrimidine hybrids were synthesized in an
economical way using an expedient approach that entails
linking the 2,4-dichloropyrimidine 8 unit with appropriate
derivatives of 4-amino-7-chloroquinoline 9 or morpholine 10 as
(
pK ), aqueous solubility and distribution coefficient (log D),
a
and mechanism of action studies on new hybrids comprising of
-aminoquinoline unit linked to pyrimidine motifs derived from
4
B
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Scheme 1. Synthesis of 4-Aminoquinoline−Pyrimidine Hybrids
outlined in Scheme 1. The reaction of uracil 7 with refluxing
RESULTS AND DISCUSSION
■
POCl under solvent-free conditions readily furnished 8 in 98%
3
Antiplasmodial Activity and Structure−Activity Rela-
tionships (SARs). The in vitro antiplasmodial activity of the
yield. Treatment of 4,7-dichloroquinoline with an excess
amount of appropriate diaminoalkane or aminoalcohol under
inert atmosphere conditions furnished corresponding N-(7-
chloro-4-quinolyl)diaminoalkane 9 in quantitative yield, as
S
hybrids 11−15 was evaluated against the D10 (CQ ) as well as
R
Dd2 (CQ ) strains of P. falciparum using CQ, artesunate
38
(
ASN), and MMV390048 as positive controls. Among the 18
3
7
compounds tested, five compounds displayed IC values in the
described. The nucleophilic substitution reaction of 8 with
appropriate 9 or morpholine 10 in the presence of a base in
THF afforded 4-chloro-substituted hybrids 11a−h in 58−97%
yield and 2-chloro-substituted structural isomers 12a−c in 25−
50
range of 22−70 nM against D10 strain. One of the compounds
bearing the 2-morphonylpyrimidine core (13) was devoid of
significant activity, and the remaining compounds had IC50
values ranging between 113 and 4310 nM (Table 1) as
described below.
3
0% yields (Table 1), after column chromatographic
separation. Finally, substitution of a second chloro substituent
of 11b or 12b with different heterocyclic amines in acetonitrile
gave another set of hybrids 14 (89−98%) and 15 (94−98%),
respectively (Table 1). The structures of all the compounds
Structure−activity relationship studies indicate that increas-
ing the length of the methylene spacer from −(CH ) − to
2
2
−
(CH ) − in 11a−g and 12a−c has a significant effect on the
2 12
antiplasmodial activity. Among the C-2 quinolinyl hybrids
S
R
1
13
11a−g, the antiplasmodial activity against both CQ and CQ
were established on the basis of spectral ( H NMR, C NMR,
LCMS, FTIR) as well as microanalytical data (vide
experimental). Further, heteronuclear multiple quantum
correlation (HSQC) and heteronuclear multiple bond
correlation (HMBC) NMR experiments were recorded for
the isomeric 11b/12b and 14a/15a pairs (Supporting
Information, Section S1) to corroborate the structures of
these compounds. To unequivocally establish the structures of
the isomeric compounds 14a,b and 15a,b, the structure of 14a
was additionally confirmed by determing the single crystal X-
ray structure (Supporting Information, Table S1). The Ortep
diagram is given in Figure 3. The structures of 15a as well as
the pair 14b/15b were established by analogy with the
spectroscopic data of 14a.
increases only up to a −(CH ) − spacer (11a−c, Table 1),
2
4
further chain lengthening resulted in reduction of the activity
S
against the CQ strain. These results are in accordance with the
trend observed in case of N,N-bis-(7-chloroquinolin-4-yl)alkane
diamines, wherein the alkyl spacer consisting of four carbon
39
atoms showed optimum potency. On the other hand, the
antiplasmodial activity against the CQS strain of the C-4
quinolinyl-substituted hybrids 12a−c, bearing −(CH ) −,
2
2
−
(CH ) −, and −(CH ) − alkane spacers, respectively, is
2
3
2 8
maximized in 12b, which has three methylene groups. Further,
the C-2 and C-4 quinolinyl structural isomers 11b and 12b,
respectively, bearing a three carbon spacer, displayed (Table 1)
comparable (IC50 244 nM, 11b; IC50 270 nM, 12b) activities
S
R
against CQ (D10) strain; although against the CQ (Dd2)
strain, 12b was nearly 2.3-fold more active. The above trend of
C
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S
R
Table 1. In Vitro Antimalarial Activity of Compounds 11−15 against P. falciparum (CQ ) D10 Strain and (CQ ) Dd2 Strain for
n = 3 (n = Number of Replicates)
D
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Table 1. continued
a
b
c
d
CQ sensitive strain. Data represents the mean of three independent experiments. CQ resistant strain. Calculated from Chem Draw Ultra 11.0
e
f
plus. Required to cause a microscopically detectable alteration of the normal cell morphology. Determined on HeLA cell cultures (reference drugs
used: DS-5000 (μg/mL)/MIC >100, and (S)-DHPA/MIC (μM) >250). Determined on Crandell−Rees feline kidney cells (CRFK) cells. 50%
cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan-based MTS assay (reference drugs used: HHA
g
h
i
CC /MIC >100, UDA CC /MIC 63.2 and, Ganciclovir CC /MIC >100) Selectivity Index (SI) is calculated as CC /IC (Dd2 strain) ratio.
50
50
50
50
50
j
IC50 (50% inhibitory concentration for cell proliferation). nd = not determined.
activity for the structural isomers was not observed when the
chloro substituent of 11b and 12b was replaced by either
morpholine or piperidine to create 14a,b and 15a,b,
respectively. Thus, while 14a bearing a morpholino group at
the C-4 position of the pyrimidine was more effective against
the D10 strain, 14b bearing a piperidine group at the same
position was active against both Dd2 (IC50 43 nM) as well as
D10 (IC 22.6 nM) strains. Both 15a and 15b showed almost
D10 strain. Replacing the more basic diaminoalkyl linker of 11b
with a relatively less basic alkoxy amino linker in 11h resulted in
a decrease in the antiplasmodial activity of 11h, which may be
attributed to the reduced accumulation of 11h in the DV of the
40
parasite in analogy to the work reported in the literature and
supporting the notion that the basicity of an alkyl chain linker
plays a crucial role in determining the activity of quinoline-
50
37
similar activity (IC50 70 nM, 15a; IC50 65.5 nM, 15b) against
based antimalarials.
E
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quinoline group decreased from 8.21 to 8.12 and the pK 7.10,
a1
corresponding to the linker NH in 11b, was absent for 11h.
This supports the observed trend in the antiplasmodial activity
of 11b and 11h (Table 1). Among the compounds 12a−c, the
pK value of the less active 12a (IC 1430 ± 179) with a two
a
50
carbon chain was considerably lower than the more active 12b
IC50 270 ± 5.1), the three carbon counterpart. The pK value
(
a
increased marginally when the chain length of the linker
increased to eight carbon analogue 12c. Hence, the length and
nature of the spacer linking a quinoline and pyrimidine moieties
appears to play a crucial role in determining the antiplasmodial
activity of 11−15.
Physicochemical parameters such as aqueous solubility (SW)
and lipophilicity are important determinants for drug
absorption as well as pharmacokinetic profiles. A good balance
of the lipophilic−hydrophilic properties has been reported to
influence the way a drug molecule passes through biological
membranes and barriers to eventually enter the systemic
circulation. Thus, optimal solubility to both water and octanol
is a prerequisite for drugs intended to be administered
Figure 3. X-ray structure and crystallographic numbering of 14a
(
CCDC number 974702).
41−43
orally.
The aqueous solubility and the distribution coefficient (log
D), a pH dependent version of the partition coefficient (log P)
of all compounds, is compiled in Table 2. The solubility in
The pK of the titratable nitrogens were measured for the
a
series of compounds 11−15. The pK data (Table 2) shows
a
that compounds 11a−11g in which the chain length of the
linker increases from 2 to 12 carbon atoms, the first three
members 11a−11c recorded an increasing trend in basicity,
while increasing the chain length beyond four carbons up to 10
carbons (11d−11g) recorded a decreasing trend in the
octanol (SOC) was calculated from the experimental S and log
W
D data using the equation: log SOC = log D + log S . The
W
aqueous solubility (S
octanol/buffer mixture at the cytosolic pH 7.4 of the parasite
was determined using HPLC (Supporting Information, Section
) in PBS buffer and log D in an n-
W
44
experimentally determined pK values. The more interesting
a
45
comparison was evident in the members 11b and 11h, in which
the more basic aminoalkyl linker of 11b was replaced with less
S1). The data in Table 2 shows that all the compounds
exhibit appreciable SW in μM range, which decreases as the
length of spacer increases from 2 to 12 carbon atoms. Further,
basic alkoxy amino linker (11h), the pK corresponding to the
a
Table 2. Acid Dissociation Constants (pK ), Aqueous Solubility (S ), Distribution Coefficients (log D), and Solubility in
a
w
Octanol (SOC) of Compounds 11−15
a
pKa
b
b
e
e
f
compd
pKa1
pKa2
Mw (g/mol)
SW (μM)
log D
SOC (μM)
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1a
1b
1c
1d
1e
1f
7.01
7.10
7.45
6.82
6.74
5.97
nd
8.10
8.21
8.64
8.11
8.00
7.85
nd
334.2
348.2
362.2
404.3
418.3
446.4
474.4
349.2
334.2
348.2
418.3
199.6
398.1
396.9
397.9
382.9
398.1
396.9
319.8
0.90 ± 0.07
0.40 ± 0.05
0.70 ± 0.09
0.13 ± 0.04
0.09 ± 0.03
0.07 ± 0.01
0.05 ± 0.01
0.20 ± 0.06
0.68 ± 0.08
0.60 ± 0.02
0.08 ± 0.01
9.00 ± 0.36
0.53 ± 0.12
0.25 ± 0.09
0.60 ± 0.06
0.22 ± 0.09
0.55 ± 0.12
0.32 ± 0.07
nd
0.501 ± 0.021
0.657 ± 0.081
0.742 ± 0.016
1.162 ± 0.017
1.510 ± 0.143
1.989 ± 0.261
2.590 ± 0.109
1.040 ± 0.270
0.489 ± 0.034
0.229 ± 0.059
1.480 ± 0.181
0.130 ± 0.026
0.257 ± 0.068
0.845 ± 0.111
0.102 ± 0.018
0.756 ± 0.094
0.363 ± 0.083
0.801 ± 0.053
nd
2.85
1.81
3.86
1.92
2.91
6.82
19.40
2.19
2.09
1.01
2.41
12.14
0.95
1.74
0.75
1.25
1.27
2.02
nd
1g
1h
2a
2b
2c
3
na
8.12
8.20
8.62
8.43
na
5.85
7.22
7.29
na
4a
4b
5.20
7.18
5.53
6.80
7.30
7.35
7.95
8.33
9.17
8.08
8.70
8.63
10.98
c
4c
4d
5a
5b
d
, CQ
8.30
a
b
The pK data represents mean of the three determinations obtained at 298.1 K. pK represent the pK of the secondary nitrogen atom bonded to
a
a1
a
c
the pyrimidine core and pK represents the pK of the quinoline N. pK corresponding to the secondary NH of piperazine, bonded to pyrimidine
core is found to be 6.85. pK represent the pK of the side chain tertiary nitrogen. Experimental data represent the mean ± SD of three
independent measurements. Solubility in octanol (S ) at pH 7.4 was calculated from experimental aqueous solubility (S ) and distribution
a2
a
a3
d
e
a1
a
f
OC
W
coefficient log D (n-octanol/PBS buffer) at pH 7.4 using log SOC = log D + log S . nd = not determined. na = not applicable.
w
F
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Figure 4. Titration of monomeric heme (2.4 μM) (A) at pH 7.4 (0.02 M HEPES buffer in aqueous DMSO solution) and (B) pH 5.6 (0.02 M MES
buffer in aqueous DMSO solution) with increasing concentration of 14b (0−69 μM in DMSO). (Inset: plot of A402 nm vs conc of 14b).
S
R
compared to the compounds with long chain (7−12 carbon
atoms) diamino linkers 11d−11g and the aminoalkoxy linker in
range against both the CQ and CQ strains of P. falciparum.
The most active compound of this series 14b (43 nM)
displayed activity greater than CQ against the Dd2 strain, which
also approaches that of artesunate (31.2 nM). Further, the SAR
study revealed that the alterations in length and nature of
methylene spacer as well as incorporation of a variety of
heterocyclic rings on the pyrimidine motif have considerable
effect on antiplasmodial activity.
1
1h, the most active compound 14b of the series depicted
higher aqueous solubility (Table 2).
Further, we evaluated the trends in the antiplasmodial
activity upon replacing the chloro substituent of the
representative hybrids 11b and 12b with cyclic amines
(
morpholine, piperidine, piperazine, and pyrrolidine) of varying
basicity to generate compounds 14a−d and the structural
isomers 15a−b, respectively (Scheme 1 and Table 1). Overall,
it was found that all compounds bearing cyclic amines 14a−d
and 15a−b were more active than their precursor Cl-
substituted analogues 11b and 12b against both D10 and
Dd2 strains. In the series of the compounds 14a−d, the
antiplasmodial activity increased in the order 14b > 14d > 14a
Cytotoxicity and Antiviral Activity. The cytotoxicity
(CC50 values, Table 1) of all the synthesized hybrids 11−15
was determined against various (HeLa, Vero, CRFK, Hel, and
MDCK) mammalian cell cultures (Supporting Information,
Tables S2−S7). These studies reveal that most of the
compounds are noncytotoxic at submicromolar concentrations
against any of the tested cell cultures. Further, the ratio of the
cytotoxicity (CC50 in μM) and in vitro antiplasmodial activity
(IC50 in nM for Dd2 strain) enabled the determination of a
selectivity index (SI) for these compounds. Nine compounds
displayed a fairly good selectivity index (SI) ranging from 209
to 632 (Table 1), whereas two compounds, 12c and 14c,
exhibited only moderate SI values (9 and 36). All the hybrids of
the series displayed structure-dependent SI values which are
greater than 209, except 12c and 14c.
>
14c against D10 strain (Table 1), while 15a displayed activity
similar to 15b. Unlike 11b and 11h, the observed trend of
antiplasmodial activity in 14a−14d does not follow the trend of
their pKa (Table 2) values but is consistent with the
lipophilicity trend [ClogP: 14b (6.15) > 14d (5.56) > 14a
(
4.61) > 14c (3.73)] as shown in Table 1 as well as log D [14b
(
0.845) > 14d (0.756) > 14a (0.257) > 14c (0.102)] values
shown in Table 2. The more basic compound 14c recorded an
additional pKa (pK , Table 2) value corresponding to the
additional nitrogen of piperazine. Similarly, the antiplasmodial
activities of 15a and 15b are also in the order of their ClogP
CQ shows inhibitory effects against several viruses, including
human immunodeficiency virus type 1 and hepatitis B virus. It
is a weak base that increases the pH of acidic vesicles. When
added extracellularly, the nonprotonated portion of CQ enters
a3
[15b (6.15) > 15a (4.61)] as well as log D [15b (0.801) >
46
15a(0.363)] values (Table 2). Thus, the compound 14b
the cell, where it gets protonated and concentrated in acidic,
low-pH organelles such as endosomes, Golgi vesicles, and
exhibited the highest antiplasmodial activity (IC50 43 nM
against Dd2 strain, respectively) within the series, which is 3.2-
fold higher than the standard chloroquine against Dd2 strain. It
is important to recall that in addition to basicity and
lipophilicity, other factors such as binding with heme also
contribute to the antiplasmodial activity. Thus, for the
structural isomeric 4-aminoquinoline hybrids 14a/15a and
47,48
lysosomes.
CQ can affect virus infection in many ways, and
the antiviral effect depends in part on the efficiency of
46−50
endosome-mediated virus entry.
Thus, we determined in
vitro antiviral activities of 11−15 against (i) herpes simplex
virus-1 (HSV-1; KOS), herpes simplex virus-2 (HSV-2; G),
vaccinia virus, vesicular stomatitis virus, herpes simplex virus-1
R
14b/15b, which exhibit identical ClogP values (Table 1),
(KOS TK-ACV ) in HEL cell cultures, (ii) parainfluenza-3
variation in antimalarial activity may in part be attributed to the
way these compounds bind to heme or other drug targets.
Further, the compound 13 lacking a 7-chloroquinolinyl group is
devoid of any antiplasmodial activity further points to the
involvement of the quinoline moiety for the observed
antiplasmodial activity of hybrids 11, 12, 14, and 15.
virus, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro
virus in Vero cell cultures, (iii) influenza A virus (H1N1 and
H3N2) and influenza B virus in MDCK cell cultures, (iv)
vesicular stomatitis virus, Coxsackie virus B4, respiratory
syncytial virus in HeLa cell cultures, (v) cytomegalovirus
using AD-169 and Davis strain in HEL cell cultures, (vi)
varicella-zoster virus (VZV) in HEL cell cultures, (Supporting
Information, Tables S2−S7), and (vii) feline corona virus
Thus, these hybrids which have molecular weights below 500
(Table 2) exhibit antiplasmodial activities in the nanomolar
G
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Table 3. Binding Constant (log K) for 14b and CQ
monomeric heme log K ± σ
μ-oxo heme log K ± σ
compd
4b
pH 5.6
pH 7.4
pH 5.8
CT DNA log K
pUC18 DNA log K
1
4.5909 ± 0.0161
4.65 ± 0.052
4.6344 ± 0.3050
5.15 ± 0.176
5.8427 ± 0.1044
5.58 ± 0.006
1:1
3.64
nd
3.99
nd
CQ
stoichiometry
1:1
nd
(
(
FIPV) and feline herpes virus activity in CRFK cell cultures
Supporting Information, Table S8). No significant antiviral
activity was noted at subtoxic concentrations, pointing to some
degree of selectivity of the antiplasmodial agents.
Mode of Action Studies. Heme Binding Studies. CQ and
related 4-aminoquinoline antimalarials bind to FPIX via π−π
stacking interaction of the quinoline ring with the porphyrin
ring, thus preventing the sequestration of toxic heme to
1
0
hemozoin. One of the possible mechanisms of antimalarial
action of quinoline−pyrimidine hybrids is through inhibition of
hemozoin formation. Therefore, we decided to evaluate the
binding of the most potent compound 14b of the series with
heme. The stepwise addition of small increments of 14b (0−69
μM, DMSO) into a constant concentration of monomeric
heme (2.4 μM) in 0.02 M HEPES buffer in aqueous DMSO at
pH 7.4 and at the plasmodial food vacuole acidic pH 5.6 (0.02
M MES buffer in aqueous DMSO) resulted in a substantial
decrease in intensity of the Fe(III) PPIX Soret band at 402 nm
with no shift in the absorption maximum (Figure 4). Solvent
1
Figure 5. H NMR spectral changes observed for 14b after addition of
increasing amounts of heme: (A) 0 mol %, (B) 10 mol, and (C) 30
mol in 40% DMSO:D
.052, c = 0.015, d = 0.002, e = 0.026, f = 0.015, g = 0.010, h = 0.091, i
0.063, j = 0.023, k = 0.008, l = 0.071, m = 0.013 ppm].
O/D
SO (10 μL) [Δδ for peak: a = 0.021, b =
2 4
2
0
=
(
DMSO) does not affect the binding of 14b with heme at the
3
5
pH values used in this experiment. A 1:1 stoichiometry of the
most stable complex of 14b with monomeric heme at pH 7.5
and 5.6 was established from the Job’s plot (Supporting
Information, Figure S1). The association constants (Table 3)
were calculated by analyzing the titration curves obtained at pH
procedure. The stepwise addition of compound 14b (0.3−16
μM) to a solution of μ-oxo dimer (10 μM) in 20 mM
phosphate buffer at pH 5.8 resulted in a decrease in intensity of
absorbance at 362 nm (Supporting Information, Figure S2).
The Job’s plot indicated a 1:1 stoichiometry for the most stable
μ-oxo:14b complex (Supporting Information, Figure S2). The
association constants calculated using HypSpec suggests that
the binding of 14b with μ-oxoheme (log K 5.84) is stronger
than the monomeric heme (log K 4.63) and also is comparable
to the standard CQ (log K 5.58). Thus, compound 14b can be
proposed to inhibit hemozoin formation by blocking the
growing face of heme, which could be correlated with the
observed antiplasmodial activity.
51
7
.4 using HypSpec, a nonlinear least-squares fitting program.
The titration of CQ with heme were also performed under
identical conditions, and the binding constants were calculated
for comparison purposes. Table 3 shows that the association
constants for the complexes formed between monomeric heme
and 14b (log K 4.63) is less than those of the standard
antimalarial drug CQ (log K 5.15). Furthermore, the decrease
of apparent pH from 7.4 to 5.6 (Table 3) has little effect on the
binding constants, indicating that binding is stronger even at
acidic pH.
To further provide evidence for the inhibition of β-hematin
formation by binding of 14b with heme, polymerization of
5
3
The binding of 14b with monomeric heme was further
heme to β-hematin was conducted at 60 °C in 4.5 M acetate
1
supported by the H NMR titration. The titration experiment
was performed by recording the H NMR spectra of 14b in
4
buffer and the FT-IR of β-hematin shows bands at 1662 and
1
−1
1209 cm , characteristic of the iron carboxylate bonds. The
0% DMSO:D O/D SO (10 μL) after addition of heme (10
disappearance of Fe-carboxylate bands upon binding to
quinoline antimalarials has been established as proof-of-
2
2
4
and 30 mol %) dissolved in DMSO. Shift in both the aromatic
as well as aliphatic proton signals of 14b (Figure 5) was noted
upon addition of 10 mol % of heme to the solution, indicating
binding of 14b with heme. However, further addition of heme
led to broadening of the peaks which may be due to the
paramagnetic effect of Fe(III) of the ferriprotoporphyrin(IX).
Further, the mass spectral analysis of an equimolar (5 μmol)
solution of hemin chloride and 14b depicted an intense
molecular ion peak at 1012.2993 Da (Figure 6A), correspond-
ing to the molecular formula C H ClFeN O and further
54
concept to ascertain inhibition of β-hematin formation. The
FT-IR spectra of the precipitate obtained upon incubation of
heme with 1.2 equiv of 14b using the literature-reported
54
procedure showed disappearance of peaks at 1662 and 1209
−1
cm assigned to β-hematin, indicating binding of heme with
14b (Supporting Information, Figure S3). Further, the IR
spectrum of the adduct of 14b with heme is also significantly
different from that of 14b and heme itself (Supporting
Information, Figures S4−S5).
DNA Binding Studies. 4-Aminoquinoline-based antimalarials
such as 1 (CQ) interact with DNA in vitro through ionic
interactions between the protonated nitrogen of the drug and
the anionic phosphate groups of DNA in addition to the
interactions between the aromatic nuclei of the drug with
5
5
57
10
4
corroborating the formation of 1:1 complex. Thus, on the basis
52
of this data as well as the literature precedence, we propose
that 14b interacts with the iron atom of heme through the
quinoline nitrogen as shown in Figure 6B.
The binding of the most potent compound 14b was also
studied with μ-oxo dimers of heme at pH 5.8 using a standard
5
5,56
nucleotide bases,
thereby stabilizing the helical config-
H
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Journal of Medicinal Chemistry
Article
Figure 6. (A) The solution phase mass spectra of 14b (5 μmol) upon addition of monomeric heme (5 μmol) in 40% aq DMSO solution (inset
shows zoom between 1008 and 1024 Da); (B) proposed binding of heme with 14b.
Figure 7. Fluorescence emission spectra (λex = 330 nm, λem = 376 nm) of 14b (17.1 μM) in buffered CH OH upon addition of increasing
3
concentrations of (A) CT DNA (0.5−251 μM) and (B) pUC18 DNA (0.02−11.4 μM).
uration against thermal denaturation. Further, the buffering
activity of 1 could additionally improve gene transfection
efficiency by facilitating the DNA release from the endocytic
pathways or by inhibiting lysosomal enzyme degradation.
Thus, the DNA binding ability of the new quinoline−
pyrimidine hybrids was studied.
constant estimated using HypSpec revealed that 14b had a
higher affinity for AT rich pUC18DNA.
57
CONCLUSIONS
■
In summary, we have reported the synthesis and systematic
evaluation of structure−activity relationships of series of potent
quinoline−pyrimidine hybrids. The in vitro evaluation of these
hybrids against Dd2 and D10 strains of P. falciparum depicted
activity in the nanomolar range. Among others, the compound
14b exhibited the lowest IC value within the series against
The addition of small increments of CT-DNA (0.5−251
μM) to the buffered methanolic solution of 14b (17.1 μM)
showed remarkable shifts in λmax as well as in the absorption
intensity. The characteristic quinoline ring absorption at 331
nm (π→π*) showed a hypochromic shift by 23% (Supporting
Information, Figure S6) with a red-shift of ∼14 nm, suggesting
50
S
R
both the CQ and CQ strains of P. falciparum. Also, these
hybrids exhibited high selectivity indices and low toxicity
against the tested cell lines. Further, the binding capability of
14b was evaluated with both heme and DNA to shed light on
the mode of action of this class of hybrids.
58
an intercalative binding of 14b with DNA base pairs, which
should also stabilize DNA and show an increment in the
59
thermal melting temperature (Tm). The melting temperature
of CT DNA under our experimental conditions was 59.5 °C
and it increases to 67.8 °C upon addition of 14b, indicating the
stabilization of the DNA duplex structure (Supporting
Information, Table S9). Further, the derivative melting curve
presented in Supporting Information, Figure S7 shows that the
Tm of CT DNA upon addition of 14b is comparable to that
observed for the CQ.
EXPERIMENTAL PROCEDURES
General. All liquid reagents were dried/purified following
recommended drying agents and/or distilled over 4 Å molecular
■
1
sieves. THF was dried (Na-benzophenoneketyl) under nitrogen. H
NMR (300 and 400 MHz), 13C (75 MHz and 100 MHz), HSQC, and
HMBC NMR spectra were recorded in CDCl and DMSO-d on a
3
6
multinuclear Jeol FT-AL-300 spectrometer and BrukerAvance II 400
spectrometer with chemical shifts being reported in parts per million
Due to the fact that the malaria parasite exhibit unusually a
60
1
higher AT content compared to human DNA, the binding
affinities of 14b toward two DNA types having different
nucleotide base composition were investigated using fluo-
rescence spectrophotometery to allow the comparison of drug
binding affinity specifically to GC vs AT-rich DNA. Addition of
DNA (CT DNA and pUC18 DNA) to the buffered methanolic
solution of 14b (17.1 μM) induced a monotonous decrease of
fluorescence intensity (Figure 7). Comparison of association
(δ) relative to internal tetramethylsilane (TMS, δ 0.0, H NMR) or
13
chloroform (CDCl , δ 77.0, C NMR). Mass spectra were recorded
3
on a Bruker LC-MS MICROTOF II spectrometer. Elemental analysis
was performed on FLASH EA 112 (Thermo Electron Corporation)
analyzer, and the results are quoted in %. IR spectra were recorded on
Perkin-Elmer FTIR-C92035 Fourier transform spectrometer in the
−1
range 400−4000 cm using KBr pellets. For monitoring the progress
of a reaction and for comparison purpose, thin layer chromatography
(TLC) was performed on precoated aluminum sheets of Merck
I
dx.doi.org/10.1021/jm4014778 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(
60F254, 0.2 mm) using an appropriate solvent system. The
J = 5.4 Hz, 1H, ArH). 13C NMR (100 MHz, DMSO-d , 25 °C): δ 26.9,
6
chromatograms were visualized under UV light. For column
chromatography silica gel (60−120 mesh) was employed, and eluents
were ethyl acetate/hexane or ethyl acetate/methanol mixtures. The pH
measurements were performed with the Equip-Tronics digital pH
meter model EQ 610. The HPLC system consisted of a Waters 2489
HPLC separations module equipped with Waters 2489 photodiode
array detector, Waters 515 HPLC pump, and Empower software
37.8, 40.1, 98.5, 104.9, 117.2, 124.4, 126.4, 133.9, 147.7, 150.6, 154.9,
159.9, and 163.4. Anal. Calcd for C H N Cl : C, 55.10; H, 4.31; N,
16
15
5
2
+
20.11. Found: C, 54.99; H, 4.38; N, 20.01. MS: m/z 348 (M ).
4-Chloro-2-[(7-chloroquinolin-4-ylamino)butylamino]pyrimidine
(11c). Chromatographic eluent: MeOH/EtOAc (1:99 v/v). White
−1 1
solid. Yield: 56%. IR (KBr): νmax 1581, 2928, 3253 cm . H (300
MHz, DMSO-d , 25 °C): δ 1.61 (m, 4H, CH ), 3.25 (m, 2H, CH ),
6
2
2
(
Waters Corporation, Milford, MA, USA) for system control, data
6.44 (m, 1H, ArH), 6.60 (m, 1H, ArH), 7.30 (br, 1H, NH), 7.41 (m,
1H, ArH), 7.62 (br, 1H, NH), 7.75 (m, 1H, ArH), 8.19 (m, 1H, ArH),
collection, and processing. Liquid chromatography was carried out
using a Symmetry (4.6 mm × 75 mm), 3.5 μm C-18 reversed-phase
column. The mobile phase consisted of premixed HPLC grade
methanol and water in the varying ratio and degassed prior to
operating under isocratic conditions at a flow rate of 0.4 mL/min with
8.34 (m, 1H, ArH). 13C NMR (75 MHz, DMSO-d
, 25 °C): δ 29.5,
99.2, 109.8, 117.8, 124.6, 124.8, 127.3, 134.2, 148.7, 151.0, 151.7,
160.8, and 162.9. Anal. Calcd for C17 Cl : C, 56.36; H, 4.73; N,
6
H
N
17
5
2
+
19.23. Found: C, 56.42; H, 4.69; N, 19.05. MS: m/z 362 (M ).
4-Chloro-2-[(7-chloroquinolin-4-ylamino)heptylamino]-
pyrimidine (11d). Chromatographic eluent: EtOAc. Yellow solid.
20 μL standard sample injections. A calibration plot of the peak area
versus compound concentration for each compound showed excellent
−1 1
2
Yield: 65%. IR (KBr): 1612, 2930, 3415 cm . H (400 MHz, DMSO-
d , 25 °C): δ 1.33 (m, 6H, CH ), 1.49 (m, 2H, CH ), 1.67 (m, 2H,
linearity (0.99 < r < 1) over the concentration range (0−10 μM)
employed for the assays. The HPLC data was recorded at the
6
2
2
CH ), 3.24 (m, 4H, CH ), 6.47 (d, J = 5.5 Hz, 1H, ArH),6.85 (d, J =
absorption maximum (330 nm) for compounds 11−15 and 250 nm,
2
2
6
.5 Hz, 1H, ArH), 7.39 (m, 1H, ArH), 7.85 (d, J = 4.6 Hz, 1H,
the wavelength of maximum absorption (λ ) for 13. The retention
max
ArH),7.95 (m, 1H, ArH), 8.52 (d, J = 6.3 Hz, 1H, ArH),8.72 (d, J =
time (t ) is expressed in minutes (min). The purity of compounds
R
1
3
11−15 was evaluated by C,H,N analysis (FLASH EA 112 (Thermo
9.0 Hz, 1H, ArH),8.82 (d, J = 3.6 Hz, 1H, Ar), 9.66 (br, 1H, NH).
NMR (100 MHz, DMSO-d , 25 °C): δ 26.2, 27.7, 28.3, 43.0, 98.7,
100.1, 104.9, 115.3, 118.9, 125.8, 126.6, 137.8, 138.4, 142.1, 151.4,
154.9, 159.7, 163.3, and 164.3. Anal. Calcd for C20 Cl : C, 59.41;
C
Electron Corporation) analyzer) and HPLC methods (Supporting
Information, Table S10). The purities of all the final compounds were
confirmed to be ≥95% by combustion and HPLC analytical methods.
The steady state fluorescence experiments were carried out on
Perkin-Elmer LS55 fluorescence spectrometer at ambient temperature.
UV−visible spectral studies were conducted on Shimadzu 1601 PC
spectrophotometer with a quartz cuvette (path length, 1 cm). The
absorption spectra have been recorded between 1100 and 200 nm.
The cell holder of the spectrophotometer was thermostatted at 25 °C
for consistency in the recordings.
6
H
23
N
5
2
H, 5.73; N, 17.32. Found: C, 59.46; H, 5.79; N, 17.33. MS: m/z 404
+
(M ).
4-Chloro-2-[(7-chloroquinolin-4-ylamino)octylamino]pyrimidine
11e). Chromatographic eluent: EtOAc. Yellow solid. Yield: 65%. IR
(
−1 1
(KBr): νmax 1576, 2930, 3292 cm . H (400 MHz, DMSO-d , 25 °C):
6
δ 1.34 (m, 8H, CH ), 1.48 (m, 2H, CH ), 1.64 (m, 2H, CH ), 3.18
2
2
2
(
m, 2H, CH ), 3.23 (m, 2H, CH ), 6.42 (m, 2H, ArH), 7.28 (br, 1H,
2
2
NH), 7.43 (dd, J1,2 = 8.9 Hz, J1,3 = 2.2 Hz, 1H, ArH), 7.78 (d, J = 2.2
General Procedure for the Synthesis of 2,4-Dichloropyr-
imidine (8). Uracil (5 g, 0.04 mol) was suspended in phosphorus
Hz, 1H, ArH), 7.86 (m, 2H, ArH and NH), 8.27 (d, J = 9.0 Hz, 1H,
ArH), 8.38 (d, J = 5.4 Hz, 1H, Ar). 13C NMR (100 MHz, DMSO-d
,
6
oxychloride (POCl ) (25 mL) and heated at 105 °C for 6 h. Excess
3
POCl was removed under reduced pressure, and last traces were
25 °C): δ 26.6, 28.1, 29.0, 42.6, 48.9, 98.9, 105.3, 117.7, 124.4, 127.5,
134.0, 149.2, 150.6, 152.2, 155.5, 160.3, and 163.7. Anal. Calcd for
3
removed through azeotropic distillation with dry benzene (2 × 20
mL). The residue was then further purified by column chromatog-
raphy (60−120 mesh silica) using hexane/EtOAc (92:8 v/v) as eluent
C H N Cl : C, 60.29; H, 6.02; N, 16.74. Found: C, 60.14; H, 6.43;
21 25 5 2
N, 16.83. MS: m/z 418 (M ).
+
1
to furnish 8 as white crystalline solid. Yield: 98%. H (300 MHz,
4-Chloro-2-[(7-chloroquinolin-4-ylamino)decylamino]pyrimidine
(
11f). Chromatographic eluent: hexane/EtOAc (85:15 v/v). Yellow
CDCl , 25 °C): δ 7.33 (d, J = 5.1 Hz, 1H, ArH), 8.52 (d, J = 5.1 Hz,
3
−1 1
13
solid. Yield: 60%. IR (KBr): νmax 1599, 2919, 3260 cm . H (400
1
H, ArH). C NMR (75 MHz, CDCl , 25 °C): δ 120.3, 160.0, 161.07,
3
MHz, DMSO-d , 25 °C): δ 1.27 (m, 12H, CH ), 1.54 (m, 4H, CH ),
and 162.6. Anal. Calcd for C H N Cl : C, 32.25; H, 1.35; N, 18.80.
Found: C, 32.09; H, 1.27; N, 18.85. MS: m/z 147.9 (M ).
6
2
2
4
2
2
2
+
3
.15 (m, 4H, CH ), 6.48 (m, 2H, ArH), 6.66 (dd, J = 5.6 Hz, 1H,
2
ArH), 7.55 (d, J1,2 = 8.8 Hz, 1H, ArH), 7.71 (br, 1H, NH), 7.86 (s, 1H,
General Procedure for the Synthesis of 11−13. The solution
of appropriate aminoquinoline 9 or morpholine (0.021 mol) in dry
THF (5 mL) was added to the stirred solution of 8 (0.018 mol) and
potassium carbonate (0.09 mol) in dry THF (30 mL). The reaction
mixture was stirred at room temperature for 48 h, and upon
completion (TLC), the reaction mixture was filtered and the filtrate
was concentrated under vacuum. The residue was purified by column
chromatography using MeOH/EtOAc (11 and 12) or hexane/EtOAc
ArH), 8.42 (d, J = 9.0 Hz, 1H, ArH), 8.46 (d, J = 5.6 Hz, 1H, ArH).
13
C NMR (100 MHz, DMSO-d , 25 °C): δ 21.0, 26.3, 42.5, 98.5,
6
100.3, 104.9, 117.0, 124.4, 125.7, 134.2, 151.0, 155.1, 157.6, 159.9,
163.3, and 172.0. Anal. Calcd for C23 Cl : C, 61.88; H, 6.55; N,
H
N
29
5
2
+
15.69. Found: C, 61.96; H, 6.37; N, 15.63. MS: m/z 446 (M ).
4-Chloro-2-[(7-chloroquinolin-4-ylamino)dodecylamino]-
pyrimidine (11g). Chromatographic eluent: hexane/EtOAc (82:18 v/
−1 1
v). Yellow solid. Yield: 58%. IR (KBr): νmax 1596, 2920, 3259 cm . H
400 MHz, DMSO-d , 25 °C): δ 1.32 (m, 16H, CH ), 1.53 (m, 4H,
(13) as eluent. Using this procedure, the following compounds were
(
isolated.
6
2
CH ), 3.27 (m, 4H, CH ), 6.49 (m, 2H, ArH), 6.68 (d, J = 5.1 Hz, 1H,
4
-Chloro-2-[(7-chloroquinolin-4-ylamino)ethylamino]pyrimidine
2
2
(
11a). Chromatographic eluent: MeOH/EtOAc (2:98 v/v). White
ArH), 7.43 (br, 1H, NH), 7.50 (dd, J1,2 = 8.9 Hz, J1,3 = 2.2 Hz, 1H,
−1 1
solid. Yield: 58%. IR (KBr): νmax 1579, 3055, 3275 cm . H (400
MHz, DMSO-d , 25 °C): δ 3.17 (m, 4H, CH ), 6.46 (d, J = 6 Hz, 1H,
ArH), 6.75 (d, J = 5.6 Hz, 1H, ArH), 7.47 (m, 2H, NH and ArH), 7.80
ArH), 7.72 (br, 1H, NH), 7.83 (s, 1H, ArH), 8.34 (d, J = 9.0 Hz, 1H,
ArH), 8.44 (d, J = 5.5 Hz, 1H, ArH). 13C NMR (100 MHz, DMSO-d
,
6
6
2
25 °C): δ 26.3, 28.3, 28.6, 28.9, 42.3, 104.9, 117.3, 124.0, 133.4, 155.2,
(
(
s, 1H, ArH), 7.92 (d, J = 5.6 Hz, 1H, ArH), 8.11 (br, 1H, NH), 8.20
m, 1H, ArH), 8.41 (d, J = 5.2 Hz, 1H, ArH). C NMR (75 MHz,
159.9, and 163.3. Anal. Calcd for C25 Cl : C, 63.28; H, 7.01; N,
H
N
33
5
2
13
+
14.76. Found: C, 63.35; H, 7.07; N, 14.81. MS: m/z 474 (M ).
4-Chloro-2-[(7-chloroquinolin-4-ylamino)propoxy]pyrimidine
(11h). Chromatographic eluent: EtOAc. White solid. Yield: 97%. IR
CDCl , 25 °C): δ 22.4, 37.7, 104.8, 154.7, 163.4, and 169.6. Anal.
3
Calcd for C H N Cl : C, 53.91; H, 3.92; N, 20.96. Found: C, 53.75;
15
13
5
2
+
−1 1
H, 3.86; N, 20.91. MS: m/z 334 (M ).
-Chloro-2-[(7-chloroquinolin-4-ylamino)propylamino]-
pyrimidine (11b). Chromatographic eluent: MeOH/EtOAc (2:98 v/
(KBr): νmax 1576, 2962, 3215 cm . H (400 MHz, DMSO-d
δ 2.19 (quin, J = 5.1 Hz, 2H, CH ), 3.49 (q, J = 6.6 Hz, 2H, CH
2
6
, 25 °C):
4
2
), 4.51
(t, J = 6.3 Hz, 2H, CH ), 6.57 (d, J = 5.5 Hz, 1H, ArH), 7.06 (d, J = 5.7
2
38
−1 1
v). White solid. Yield: 65%. IR (KBr): νmax 1596, 30, 3111 cm . H
300 MHz, DMSO-d , 25 °C): δ 2.00 (m, 2H, CH ), 3.47 (m, 4H,
Hz, 1H, ArH), 7.43 (br, 1H, NH), 7.51 (dd, J1,2 = 9 Hz, J1,3 = 2.2
Hz,1H, ArH), 7.84 (d, J = 2.4 Hz, 1H, ArH), 8.32 (d, J = 9.0 Hz, 1H,
ArH), 8.45 (d, J = 5.4 Hz, 1H, ArH), 8.52 (d, J = 5.8 Hz, 1H, ArH).
(
6
2
CH ), 6.44 (m, 2H, ArH), 7.05 (br, 1H, NH), 7.35 (m, 1H, ArH), 7.61
2
13
(br, 1H, NH), 7.85 (d, J = 1.8, 2H, ArH), 8.02 (m, 1H, ArH), 8.42 (d,
C NMR (100 MHz, DMSO-d , 25 °C): δ 27.2, 65.8, 99.2, 108.0,
6
J
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Journal of Medicinal Chemistry
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1
24.6, 127.9, and 160.3. Anal. Calcd for C H N Cl O: C, 55.03; H,
MHz, CDCl , 25 °C): δ 24.9, 25.7, 28.5, 38.7, 41.0, 44.8, 98.9, 117.1,
16
14
5
2
3
+
4
−
.04; N, 16.04. Found: C, 55.13; H, 4.18; N, 16.21. MS: m/z 348 (M
1).
-Chloro-4-[(7-chloroquinolin-4-ylamino)ethylamino] pyrimidine
12a). Chromatographic eluent: EtOAc. White solid. Yield: 25%. IR
121.0, 125.3, 128.4, 135.0, 148.8, 149.6, 151.7, 156.1, 161.6, and 162.8.
Anal. Calcd for C H N Cl O: C, 60.22; H, 5.81; N, 21.07. Found: C,
2
0
23
6
+
2
60.36; H, 5.92; N, 21.14. MS: m/z 397 (M ).
(
4-Piprazinyl-2-[(7-chloroquinolin-4-ylamino)propylamino]-
−1 1
(KBr): νmax, 1583, 3064, 3252 cm . H (400 MHz, DMSO-d , 25
pyrimidine (14c). White solid. Yield: 97%. IR (KBr): ν 1567, 3088,
6
max
−1 1
°
7
8
C): δ 3.34 (m, 4H, CH ), 6.68 (m, 2H, ArH), 7.42 (m, 1H, ArH),
3403 cm . H (400 MHz, DMSO-d , 25 °C): δ 1.27 (m, 2H, CH ),
2
6
2
.77 (s, 1H, ArH), 7.88 (d, J = 5.6 Hz, 1H, ArH), 8.00 (br, 1H, NH),
3.33 (m, 2H, CH ), 3.50 (m, 4H, CH ), 3.66 (m, 4H, ArH), 5.78 (d, J
2 2
.17 (m, 2H, ArH), 8.39 (d, J = 4.2 Hz, 1H, ArH). 13C NMR (75
= 5.7 Hz, 1H, ArH), 6.42 (d, J = 5.5 Hz, 1H, ArH), 6.85 (br, 1H,
ArH), 7.24 (br, 1H, NH), 7.34 (dd, J1,2 = 9 Hz, J1,3 = 2.1 Hz,1H, ArH),
7.74 (m, 2H, ArH), 7.75 (m, 1H, ArH), 8.21 (d, J = 9 Hz, 1H, ArH),
MHz, CDCl , 25 °C): δ 29.5, 99.2, 109.8, 117.7, 124.6, 127.3, 134.2,
3
148.7, 151.0, 160.7, and 162.8. Anal. Calcd for C H N Cl : C, 53.91;
15 13 5 2
8.38 (d, J = 5.4 Hz, 1H, ArH). 13C NMR (75 MHz, CDCl , 25 °C): δ
H, 3.92; N, 20.96. Found: C, 53.95; H, 4.07; N, 20.80. MS: m/z 334
3
+
(
M ).
-Chloro-4-[(7-chloroquinolin-4-ylamino)propylamino]-
pyrimidine (12b). Chromatographic eluent: EtOAc. White solid.
25.4, 33.2, 39.6, 98.7, 123.4, 117.1, 124.2, 125.3, and 161.2. Anal. Calcd
for C H N Cl: C, 60.37; H, 6.08; N, 24.64. Found: C, 60.49; H,
2
2
0
24
7
+
5.95; N, 24.53. MS: m/z 398 (M + 1).
−1 1
Yield: 25%. IR (KBr): νmax1670, 3111, 3201 cm . H (300 MHz,
CDCl , 25 °C): δ 2.01 (m, 2H, CH ), 3.44 (m, 2H, CH ), 3.60 (m,
4-Pyrolidinyl-2-[(7-chloroquinolin-4-ylamino)propylamino]-
pyrimidine (14d). Yellow solid. Yield: 89%. IR (KBr): ν 1582, 2957,
3
2
2
max
−
1 1
2
1
1
H, CH ), 6.26 (br, 1H, NH), 6.57 (d, J = 5.7 Hz, 2H, ArH), 6.72 (br,
H, NH), 7.36 (m, 1H, ArH), 7.92 (m, 2H, ArH), 8.15 (d, J = 5.1 Hz,
H, ArH), 8.40 (d, J = 5.7 Hz, 1H, ArH). C NMR (100 MHz,
3336 cm . H (400 MHz, CDCl , 25 °C): δ 1.74 (m, 2H, CH ), 2.04
2
3
2
(m, 4H, CH
), 3.33 (m, 2H, CH
), 3.45 (m, 6H, CH
2
), 5.52 (br, 1H,
2
2
13
NH), 5.65 (m, 1H, ArH), 5.98 (br, 1H, NH), 6.39 (d, J = 5.4 Hz, 1H,
ArH), 7.29 (dd, J1,2 = 9 Hz, J1,3 = 1.9 Hz, 1H, ArH), 7.61 (m, 1H,
ArH), 7.83 (m, 1H, ArH), 7.93 (m, 1H, ArH), 8.49 (d, J = 5.3 Hz, 1H,
DMSO-d , 25 °C): δ 27.2, 38.5, 39.3, 98.6, 108.7, 117.3, 117.4, 124.0,
1
for C H N Cl : C, 55.10; H, 4.31; N, 20.11. Found: C, 55.24; H,
6
27.4, 133.3, 148.9, 149.9, 150.0, 151.8, 159.9, and 162.3. Anal. Calcd
ArH). 13C NMR (75 MHz, CDCl
, 25 °C): δ 23.2, 39.1, 28.4, 29.6,
16
15
5
2
3
+
4
.35; N, 20.08. MS: m/z 348 (M ).
-Chloro-4-[(7-chloroquinolin-4-ylamino)octylamino]pyrimidine
12c). Chromatographic eluent: EtOAc. Yellow solid. Yield: 30%. IR
36.7, 38.6, 41.1, 93.5, 98.8, 117.1, 121.2, 125.1,128.3, 134.8, 148.8,
149.8, 151.6, 155.6, 160.1, 162.7, and 170.5. Anal. Calcd for
C H N Cl: C, 62.74; H, 6.05; N, 21.95. Found: C, 62.64; H, 6.23;
2
(
2
0
23
6
−1
1
+
(
KBr): νmax 1579, 2953, 3250 cm . H (400 MHz, DMSO-d , 25 °C):
N, 21.76. MS: m/z 383 (M +1).
6
δ 1.36 (m, 8H, CH ), 1.49 (m, 2H, CH ), 1.65 (m, 2H, CH ), 3.24
2-Morpholinyl-4-[(7-chloroquinolin-4-ylamino)propylamino]-
2
2
2
(
m, 4H, CH ), 6.44 (m, 2H, ArH), 6.63 (dd, J1,2 = 9.0 Hz, J1,3 = 2.2
pyrimidine (15a). White solid. Yield: 98%. IR (KBr): νmax 1586, 2955,
2
−
1 1
Hz, 1H, ArH), 7.29 (br, 1H, NH), 7.43 (d, J = 6.7 Hz, 1H, ArH), 7.67
br, 1H, NH), 7.77 (d, J = 2.2 Hz, 1H, ArH), 8.27 (d, J = 9.0 Hz, 1H,
3245 cm . H (400 MHz, CDCl
(m, 2H, CH ), 3.57 (m, 6H, CH
3
, 25 °C): δ 1.94 (m, 2H, CH
), 3.42
2
(
2
2
), 3.70 (m, 4H, CH ), 4.97 (br, 1H,
2
13
ArH), 8.39 (d, J = 5.4 Hz, 1H, Ar). C NMR (100 MHz, CDCl , 25
NH), 5.91 (d, J = 6.0 Hz, 1H, ArH), 6.40 (d, J = 5.4 Hz, 1H, ArH),
7.34 (dd, J1,2 = 8.7 Hz, J1,3 = 2.1 Hz,1H, ArH), 7.81 (d, J = 9 Hz, 1H,
3
°
1
C): δ 26.6, 28.7, 29.0, 29.1, 29.3, 41.4, 43.2, 98.9, 109.7, 121.0, 125.3,
51.3, 159.0, and 163.6. Anal. Calcd for C H N Cl : C, 60.29; H,
1
3
ArH), 7.95 (m, 2H, ArH), 8.49 (d, J = 5.4 Hz, 1H, ArH). C NMR
(100 MHz, CDCl , 25 °C): δ 28.9, 38.3, 39.7, 44.0, 46.4, 66.5, 68.0,
93.8, 98.7, 117.6, 121.7, 125.0, 128.5, 134.7, 149.2, 150.1, 151.9, 156.3,
162.3, and 162.7. Anal. Calcd for C20 Cl O: C, 60.22; H, 5.81; N,
21
25
5
2
6.02; N, 16.74. Found: C, 60.42; H, 6.12; N, 16.49. MS: m/z 418
3
+
(
M ).
-Chloro-2-morpholinopyrimidine (13). Chromatographic eluent:
hexane/EtOAc (90:10 v/v). White solid. Yield: 98%. IR (KBr): ν
4
H N
23 6
+
21.07. Found: C, 60.12; H, 5.59; N, 21.26. MS: m/z 398 (M ).
max
−
1 1
1
589, 2972, 3091 cm . H (300 MHz, CDCl , 25 °C): δ 3.64 (m, 4H,
2-Piperidinyl-4-[(7-chloroquinolin-4-ylamino)propylamino]-
3
13
pyrimidine (15b). Yellow solid. Yield: 94%. IR (KBr): ν 1583, 2976,
CH ), 3.76 (m, 4H, CH ), 6.37 (m, 1H, ArH), 8.06 (m, 1H, ArH).
NMR (75 MHz, CDCl , 25 °C): δ 29.6, 44.2, 66.3, 101.0, and 157.5.
Anal. Calcd for C H N OCl: C, 48.13; H, 5.05; N, 21.05. Found: C,
4
C
max
2
2
−
1 1
3
(
310 cm . H (400 MHz, CDCl , 25 °C): δ 1.6 (m, 4H, CH ), 1.93
3
2
3
m, 2H, CH ), 3.31 (m, 2H, CH ), 3.44 (m, 2H, CH ), 3.54 (m, 4H,
2 2 2
8
10
3
+
CH ), 3.70 (m, 2H, CH ), 5.64 (br, 1H, NH), 5.93 (dd, J = 17.2 Hz,
8.07; H, 5.19; N, 21.17. MS: m/z 200.0 (M +1).
2
2
1,2
J
=
6
.
2
H
z
,
1
H
,
A
r
H
)
,
6
.
3
8
(
d
,
J
=
5
.
5
H
z
,
1
H
,
A
r
H
)
,
7
.
3
3
(
d
d
,
J
=
General Procedure for the Synthesis of 14−15. To the stirred
1
,
3
1
,
2
8
1
2
1
.8 Hz, J1,3 = 2 Hz, 1H, ArH), 7.86 (m,2H, ArH), 7.91 (d, J = 2.4 Hz,
solution of compound 11b or 12b (0.055 mol) and potassium
carbonate (0.25 mol) in dry acetonitrile (20 mL), an appropriate
amine (morpholine/piperidine/piperazine/pyrrolidine) (0.018 mol)
was added. The reaction mixture was refluxed for 2 h, and upon
completion (TLC), the crude product was filtered and recrystallized
from DCM/hexane. The following compounds were isolated.
13
H, ArH), 8.47 (m, 1H, ArH). C NMR (75 MHz, CDCl , 25 °C): δ
3
4.9, 25.7, 28.5, 38.7, 41.0, 44.8, 98.9, 117.1, 121.0, 125.3, 128.4, 135.0,
48.8, 149.6, 151.7, 156.1, 161.6, and 162.8. Anal. Calcd for
C H N Cl O: C, 60.22; H, 5.81; N, 21.07. Found: C, 60.03; H,
20 23
6
+
6
.08; N, 21.38. MS: m/z 397 (M ).
In Vitro Antimalarial Activity Assay. The test samples were
4
-Morpholinyl-2-[(7-chloroquinolin-4-ylamino)propylamino]-
pyrimidine (14a). White solid. Yield: 97%. IR (KBr): ν 1586, 2962,
3
tested in triplicate on one or two separate occasions against
max
−1
1
S
R
376 cm . H (400 MHz, CDCl , 25 °C): δ 2.07 (m, 2H, CH ), 3.45
chloroquine sensitive (CQ ) D10 and chloroquine-resistant (CQ )
Dd2 strains of P. falciparum. Continuous in vitro cultures of asexual
erythrocyte stages of P. falciparum were maintained using a modified
3
2
(
m, 2H, CH ), 3.56 (m, 2H, CH ), 3.70 (m, 8H, CH ), 4.78 (br, 1H,
2 2 2
NH), 5.13 (br, 1H, NH), 5.71(d, J = 5.7 Hz, 1H, ArH), 6.40 (d, J = 5.4
Hz, 1H, ArH), 7.34 (dd, J1,2 = 8.9 Hz, J1,3 = 2 Hz,1H, ArH), 7.54 (d, J
6
1
method of Trager and Jensen. Quantitative assessment of
=
8.7 Hz, 1H, ArH), 7.89 (d, J = 5.4 Hz, 1H, ArH), 7.96 (d, J = 2.1 Hz,
antiplasmodial activity in vitro was determined via the parasite lactate
H, ArH), 8.51 (d, J = 5.1 Hz, 1H, ArH). 13C NMR (75 MHz, CDCl3,
5 °C): δ 28.5, 29.7, 38.7, 41.1, 44.3, 66.8, 98.9, 117.1, 121.1, 125.5,
28.4, 128.6, 129.7, 135.0, 148.8, 149.8, 151.7, 156.0, 161.7, and 162.8.
62
1
2
1
dehydrogenase assay using a modified method described by Makler.
The test samples were prepared to a 20 mg/mL stock solution in
100% DMSO. Stock solutions were stored at −20 °C. Further
dilutions were prepared on the day of the experiment. Chloroquine
diphosphate (CQ) (Sigma), artesunate (Sigma), and an in-house
control MMV390048 were used as the reference drugs in all
experiments. A full dose−response was performed for all compounds
to determine the concentration inhibiting 50% of parasite growth
(IC50 value). Test samples were tested at a starting concentration of 10
μg/mL, which was then serially diluted 2-fold in complete medium to
give 10 concentrations, with the lowest concentration being 0.02 μg/
mL. The same dilution technique was used for all samples. Reference
drugs were tested at a starting concentration of 1000 ng/mL. Several
Anal. Calcd for C H N Cl O: C, 60.22; H, 5.81; N, 21.07. Found: C,
20
23
6
+
6
0.18; H, 5.98; N, 21.24. MS: m/z 398 (M ).
-Piperidinyl-2-[(7-chloroquinolin-4-ylamino)propylamino]-
pyrimidine (14b). Yellow solid. Yield: 98%. IR (KBr): ν 1580, 2923,
4
max
−1 1
3
401 cm . H (400 MHz, CDCl , 25 °C): δ 1.51 (m, 4H, CH ), 2.05
3
2
(
m, 4H, CH ), 3.43 (m, 2H, CH ), 3.55 (m, 2H, CH ), 3.70 (m, 4H,
2 2 2
CH ),4.91 (br, 1H, NH), 5.40 (br, 1H, NH), 5.63 (d, J = 5.7 Hz, 1H,
2
ArH), 6.38 (d, J = 5.4 Hz, 1H, ArH), 7.30 (dd, J1,2 = 8.8 Hz, J1,3 = 1.9
Hz,1H, ArH), 7.56 (d, J = 8.9 Hz, 1H, ArH), 7.86 (d, J = 5.7 Hz, 1H,
ArH), 7.92 (s, 1H, ArH), 8.49 (d, J = 5.3 Hz, 1H, ArH). 1 C NMR (75
3
K
dx.doi.org/10.1021/jm4014778 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
compounds were tested at a starting concentration of 1000 ng/mL.
The highest concentration of solvent to which the parasites were
exposed to had no measurable effect on the parasite viability (data not
shown). The IC50 values were obtained using a nonlinear dose−
response curve fitting analysis via Graph Pad Prism v.4.0 software.
Cytotoxicity and Antiviral Activity Assays. Cytotoxicity was
determined by exposing different concentrations of the samples to
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AUTHOR INFORMATION
Corresponding Author
Phone: +91 183 2258853; +91 183 2258802-09 ext 3508. Fax:
91 183 2258819/20. E-mail: kamaljit19in@yahoo.co.in.
■
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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(
16) Muregi, F. W.; Ishih, A. Next-generation antimalarial drugs:
hybrid molecules as a new strategy in drug design. Drug Dev. Res. 2010,
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K.S. gratefully acknowledges financial assistance from CSIR,
New Delhi (project 01(2364)/10/EMR-II), and UGC, New
Delhi, for Special Assistance Programme (SAP). Thanks are
due to the Sophisticated Instrumentation Centre, IIT Indore,
for single crystal X-ray analysis. H.K. thanks CSIR, New Delhi
F. no. 09/254 (0195)/2009-EMR-I) for a senior research
fellowship. J.B. thanks KU Leuven for financial support (GOA
0/14).The South African Research Chairs Initiative of the
7
(
2
(
(
novel artemisinin−quinine hybrid with potent antimalarial activity.
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1
Department of Science and Technology, the South African
Medical Research Council, and the University of Cape Town
are gratefully acknowledged for financial support (K.C.).
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ABBREVIATIONS USED
■
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PfCRT, Plasmodium falciparum chloroquine resistance trans-
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ASN, artesunate; FPIX, ferriprotoporphyrin IX; HSQC,
heteronuclear multiple quantum correlation; HMBC, hetero-
nuclear multiple bond correlation
1
(
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