Synthesis of new 4-Aminoquinolines and evaluation of their in vitro activity against chloroquine-sensitive and chloroquine-resistant plasmodium falciparum

Article abstract of DOI:10.1371/journal.pone.0140878

The efficacy of chloroquine, once the drug of choice in the fight against Plasmodium falciparum, is now severely limited due to widespread resistance. Amodiaquine is one of the most potent antimalarial 4-Aminoquinolines known and remains effective against chloroquineresistant parasites, but toxicity issues linked to a quinone-imine metabolite limit its clinical use. In search of new compounds able to retain the antimalarial activity of amodiaquine while circumventing quinone-imine metabolite toxicity, we have synthesized five 4-Aminoquinolines that feature rings lacking hydroxyl groups in the side chain of the molecules and are thus incapable of generating toxic quinone-imines. The new compounds displayed high in vitro potency (low nanomolar IC50), markedly superior to chloroquine and comparable to amodiaquine, against chloroquine-sensitive and chloroquine-resistant strains of P. falciparum, accompanied by low toxicity to L6 rat fibroblasts and MRC5 human lung cells, and metabolic stability comparable or higher than that of amodiaquine. Computational studies indicate a unique mode of binding of compound 4 to heme through the HOMO located on a biphenyl moeity, which may partly explain the high antiplasmodial activity observed for this compound.

Full text of DOI:10.1371/journal.pone.0140878

RESEARCH ARTICLE
Synthesis of New 4-Aminoquinolines and
Evaluation of Their In Vitro Activity against
Chloroquine-Sensitive and Chloroquine-
Resistant Plasmodium falciparum
Chandima S. K. Rajapakse1¤, Maryna Lisai1, Christiane Deregnaucourt2,
Véronique Sinou3, Christine Latour3, Dipankar Roy1, Joseph Schrével2, Roberto
A. Sánchez-Delgado1*
a11111
1 Chemistry Department of Brooklyn College and Ph.D. Program in Chemistry, The Graduate Center of The
City University of New York, New York, New York, United States of America, 2 Unité Molécules de
Communication et Adaptation des Microorganismes, (MCAM, UMR 7245), Sorbonne Universités, Muséum
National d’Histoire Naturelle CNRS, Paris, France, 3 Laboratory of Parasitology, Faculty of Pharmacy, UMR-
MD3, Aix-Marseille University, Marseille, France
¤ Current address: Department of Chemistry, University of Kelaniya, Kelaniya, Sri Lanka
* Rsdelgado@brooklyn.cuny.edu
OPEN ACCESS
Citation: Rajapakse CSK, Lisai M, Deregnaucourt C,
Sinou V, Latour C, Roy D, et al. (2015) Synthesis of
New 4-Aminoquinolines and Evaluation of Their In
Vitro Activity against Chloroquine-Sensitive and
Chloroquine-Resistant Plasmodium falciparum. PLoS
ONE 10(10): e0140878. doi:10.1371/journal.
pone.0140878
Abstract
The efficacy of chloroquine, once the drug of choice in the fight against Plasmodium falcipa-
rum, is now severely limited due to widespread resistance. Amodiaquine is one of the most
potent antimalarial 4-aminoquinolines known and remains effective against chloroquine-
resistant parasites, but toxicity issues linked to a quinone-imine metabolite limit its clinical
use. In search of new compounds able to retain the antimalarial activity of amodiaquine
while circumventing quinone-imine metabolite toxicity, we have synthesized five 4-amino-
quinolines that feature rings lacking hydroxyl groups in the side chain of the molecules and
are thus incapable of generating toxic quinone-imines. The new compounds displayed high
in vitro potency (low nanomolar IC50), markedly superior to chloroquine and comparable to
amodiaquine, against chloroquine-sensitive and chloroquine-resistant strains of P. falcipa-
rum, accompanied by low toxicity to L6 rat fibroblasts and MRC5 human lung cells, and met-
abolic stability comparable or higher than that of amodiaquine. Computational studies
indicate a unique mode of binding of compound 4 to heme through the HOMO located on a
biphenyl moeity, which may partly explain the high antiplasmodial activity observed for this
compound.
Editor: Gabriele Pradel, RWTH Aachen University,
GERMANY
Received: June 16, 2015
Accepted: October 1, 2015
Published: October 16, 2015
Copyright: © 2015 Rajapakse et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: Grant # SC1GM089558 from the National
Institute of General Medical Sciences of the National
Institutes of Health funded the work performed at
Brooklyn College. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Introduction
Malaria continues to be a major global health problem. According to the WHO, an estimated
3.2 billion people are at risk of being infected with Plasmodium and developing disease, and 1.2
billion are at high risk. It is estimated that 198 million cases of malaria occurred globally in
Competing Interests: The authors have declared
that no competing interests exist.
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New 4-Aminoquinoline Antimalarials
2013, leading to 584,000 deaths, mainly in the African Region (90% of all malaria deaths), and
mostly in children under 5 years of age (78% of deaths) [1]. For decades one of the most suc-
cessful and widely used drugs for treating malaria, especially Plasmodium falciparum infection,
was chloroquine (CQ, Fig 1); however, widespread resistance has rendered it essentially useless
in most parts of the world [2]. The 4-aminoquinoline pharmacophore has been the focus of
intense efforts to develop new drugs not susceptible to resistance; for instance, amodiaquine
(AQ, Fig 1), which is structurally related to CQ but contains a p-hydroxyanilino ring in the
side chain of the molecule, is considerably more potent than CQ and remains effective against
most CQ-resistant strains. However, toxicity issues limit its clinical use, in particular the occur-
rence during prolonged treatment or prophylaxis of agranulocytosis and potentially fatal idio-
syncratic hepatotoxicity, linked to the formation of a reactive quinone-imine metabolite [3–7].
Related compounds like tebuquine (Fig 1) and its analogs also display high activity against CQ-
resistant P. falciparum but are equally susceptible to P450-induced oxidation to toxic quinone-
imine metabolites [8]. In order to circumvent this particular type of toxicity, other AQ analogs
unlikely to form quinone-imine intermediates have been considered. They include isoquine
[5,9] and the related GSK369796 [6,9]; amopyroquines [10–12] (Fig 1) and fluoroamodia-
quines [13]; N-tert-butylamino Mannich base derivatives [14,15], and benzoxazines [4], all of
which display high antiplasmodial activity. Other important developments concerning quino-
line antimalarials are the discovery of ferroquine, a highly active and selective organometallic
agent against CQ-resistant P. falciparum [16], and of phenylequine, a compound closely related
to the ones described in this paper, which displays activity comparable to that of ferroquine
against CQ-resistant parasites (Fig 1) [17]. Other aminoquinolines [18–24] and organometallic
CQ derivatives [25–27] with interesting antimalarial properties have been reported in recent
times. In this paper we describe the synthesis and antiplasmodial evaluation of a group of new
4-aminoquinolines 1–5 (Fig 2) structurally related to AQ, but lacking the 4-hydroxyl group in
the side ring. These compounds are highly active in vitro against CQ-sensitive and CQ-resis-
tant P. falciparum and are incapable of generating toxic quinone-imine metabolites.
Results and Discussion
Synthesis and characterization of new aminoquinolines
The new N-benzyl-4-aminoquinolines 1–4, and the reduced N-cyclohexadienylmethyl deriva-
tive 5 (Fig 2) were prepared by condensation of the appropriate amines 6–10 (Fig 2) with
4,7-dichloroquinoline (11) in N-methyl-2-pyrrolidone (NMP) in the presence of K2CO3 and
triethylamine, as exemplified in Fig 3 for compound 1. The precursor amine 6 was obtained
from the reaction of o-cyanobenzylbromide (12) with diethylamine in ethanol to yield o-
(diethylaminomethyl)benzonitrile (13), followed by LiAlH4 reduction of the nitrile group in
diethylether. The other (diethylaminomethyl)benzylamines used in this study (7–9) were pre-
pared by analogous procedures, starting from the corresponding cyanobenzylbromides. Cyclo-
hexadiene 10 was obtained by Birch reduction of 6. All new compounds were characterized by
1H and 13C NMR spectroscopy and high-resolution mass spectrometry (complete data in
Materials and Methods Section); the purity of all samples used in biological tests (> 95%) was
established by elemental analysis and HPLC. It is worth noting that our high yield synthetic
method for 1–5 relies on inexpensive commercially available starting materials.
In vitro evaluation of antiplasmodial activity and cytotoxicity
The activity of compounds 1–5, as well as CQ and AQ, was evaluated in vitro against the CQ-
sensitive 3D7 and CQ-resistant K1 and Dd2 strains of P. falciparum in two independent labo-
ratories designated as A and B. The results of these assays are collected in Table 1, where we
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New 4-Aminoquinoline Antimalarials
Fig 1. Aminoquinoline antimalarial drugs and drug candidates.
doi:10.1371/journal.pone.0140878.g001
note that the IC50 values for CQ toward the Dd2 and K1 from laboratory B were consistently
lower than those from laboratory A. There are precedents for this type of differences in anti-
plasmodial activity as a function of the experimental conditions; more specifically, results of
chemosusceptibility tests have been shown to be affected by the initial parasitemia, hematocrit,
incubation time, time when 3H-hypoxanthine is added, the use of serum substitute, and the gas
mixture [28]. Oxygen tension is a particularly important factor governing CQ activity against
Fig 2. New aminoquinolines (1–5) and precursor amines (6–10) synthesized.
doi:10.1371/journal.pone.0140878.g002
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New 4-Aminoquinoline Antimalarials
Fig 3. Synthetic strategy for compound 1 and other new aminoquinolines.
doi:10.1371/journal.pone.0140878.g003
P. falciparum; it has been shown that high O2 tension (21%) leads to increased efficacy of the
drug compared to lower (10%) O2 tension. Furthermore, the O2 influence was found to be
strain-specific, with particular ocurrence in resistant strains [29]. In accordance with this, for
Dd2 and K1 IC50 values for CQ from laboratory A, using an incubator with a fixed atmosphere
of 5% CO2, 10% O2, 85% N2 for parasite culture, are higher than those from laboratory B,
which employs a candle jar with a ~17% O2, 3% CO2, and 80% N2, whereas IC50 values for 3D7
are similar in both settings.
The new compounds 1–5 display high activity against all the parasites assayed. For the CQ-
sensitive strain 3D7, 1–4 show comparable or slightly higher potency than those measured for
CQ and AQ. More importantly, when tested against the CQ-resistant parasites (K1 and Dd2)
the activity of 1–5 is consistently much higher than that of CQ. Compound 1, which only dif-
fers from the known highly active phenylequine [17] in that it contains a diethylamino instead
of dimethylamino group at the end of the side chain, was about 21 times more potent than CQ
against K1 parasites under conditions A, and close to eigth times more active under conditions
B. Switching to the meta and para isomers 2 and 3, or replacing the arene ring by the corre-
sponding cyclohexadiene in 5 led to a slight decrease of activity under conditions A and a slight
increase under conditions B. On the other hand, the presence of the large biphenyl group in 4
resulted in activity as high as that of 1 under conditions A, and in the highest potency of all
compounds against K1 under conditions B. Compounds 1–5 are also more effective than CQ
against the Dd2 strain, which was found to be CQ-resistant under conditions A but CQ-sensi-
tive under conditions B. The most active compounds are 1 and 4 also in this case, with the lat-
ter again being the most potent under both sets of conditions A and B. Additional
antiplasmodial activity data for selected compounds against CQ-sensitive (F32) and CQ-
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New 4-Aminoquinoline Antimalarials
Table 1. In vitro activities of compounds 1–5, CQ, and AQ against CQ-sensitive and CQ-resistant
strains of P. falciparum.
Antiplasmodial activity, IC50 (nM)
Resistance Indexes
Dd2/3D7 K1/3D7
3D7
Dd2b
K1
A
CQ
AQ
1
17.6 0.71
24.6 3.06
13.5 0.71
18.4 0.66
17.3 0.58
15.2 1.57
-
521.6 95.73
31.6 7.77
21.3 2.52
26.5 5.07
30.5 2.12
20.7 0.20
59.0 1.85
1086.0 163.50
34.3 0.58
52.7 0.99
64.2 6.16
60.3 4.07
53.4 2.03
63.7 1.46
B
29.7
61.7
1.3
1.6
1.4
1.8
1.4
1.4
3.9
3.5
3.5
3.5
2
3
4
5
-
-
CQ
AQ
1
18.0 12.31
11.9 1.00
21.6 3.11
17.1 2.97
16.1 2.44
12.9 0.85
-
35.1 12.08
11.3 0.64
19.4 2.18
17.1 1.72
17.7 3.78
14.4 0.50
-
124.0 48.05
9.60 0.51
14.6 0.36
12.0 1.15
12.4 0.40
11.8 1.00
20.2 6.55
1.9
0.9
0.9
1.0
1.1
1.1
6.9
0.8
0.7
0.7
0.8
0.9
2
3
4
5
-
-
IC50 SD values (nM) determined from independent experiments performed in triplicate in two laboratories
(A, Marseille; B, Paris) under different assay conditions (see main text and Materials and Methods Section
for details). CQ: chloroquine, AQ: amodiaquine, -: not determined.
bNote that Dd2 is CQ-resistant under the culture conditions of laboratory A and CQ-sensitive under the
culture conditions of laboratory B.
doi:10.1371/journal.pone.0140878.t001
resistant (K14 and FcB1) strains are collected in S1 Table (Supporting Information). The trends
observed in those cases are similar to the ones described above, with the new compounds being
comparable or somewhat better than CQ against CQ-sensitive parasites and much more active
than CQ against resistant strains. Compound 4 showed once more the highest potency against
the highly resistant K14 strain under conditions A, with a remarkable IC50 of 7.5 nM.
The low values of the resistance indexes (RI = IC50Dd2/IC503D7 and IC50K1/IC503D7) for
1–4 suggest a low potential for these compounds to develop cross-resistance with CQ [18,23].
It is also important to highlight from the data in Table 1 and S1 Table that the antimalarial
potency of the new 4-aminoquinolines against CQ-resistant strains of P. falciparum is similar
to that of the highly active AQ. As noted above, the clinical use of AQ has been hampered by
its tendency to form toxic quinone-imine metabolites. The molecular design of the compounds
presented here eliminates the potential for this type of toxicity, since all of them lack the
4-hydroxyl group in the ring present in the side chain of each molecular structure and are
therefore incapable of generating quinone-imine intermediates.
In order to further ascertain the possibility of indiscriminate cytotoxicity we also measured
the ability of compounds 1–4 to inhibit normal rat L6 and human MRC5 cell lines by the
method described in the Materials and Methods Section. The data in Table 2 reveal low toxicity
to the mammalian cells, which translates into high selectivity ranges for both CQ-sensitive and
CQ-resistant P. falciparum.
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New 4-Aminoquinoline Antimalarials
Table 2. Cytotoxicity of compounds 1–4 toward mammalian cells.
L6
MRC5
CC50a(μM)
SIb(min-max)
CC50(μM)
SI(min-max)
CQS strains
CQR strains
CQS strains
CQR strains
1
2
3
4
27.7 1.73
20.6 3.92
13.8 1.90
2.8 0.15
923–2052
1505–1884
1140–1720
1639–2147
442–2289
431–1544
459–1565
519–3693
26.4 1.75
20.3 3.40
13.0 2.26
2.6 0.12
880–1955
1435–1796
1086–1639
1562–2046
421–2182
411–1544
438–1491
494–3520
aCC50: concentration of drug inducing 50% of cell growth arrest.
bSI: Selectivity index expressed as (CC50 to each mammalian cell line)/(IC50 to P. falciparum). SI max corresponds to the most sensitive strain of P.
falciparum and SI min toward the less sensitive parasite strain.
doi:10.1371/journal.pone.0140878.t002
In vitro metabolic stability measurements
We also examined the metabolic stability of compounds 1–4, as well as of CQ and AQ, by mea-
suring their intrinsic clearance and half-lives upon incubation with human liver microsomes in
the presence and in the absence of NADPH. The data (Table 3) were compared with values for
the well-characterized positive controls verapamil (CLint 134 mL min-1 mg-1, t1/2 17.2 min) and
warfarin (CLint 0.0 mL min-1 mg-1, t1/2 >240 min), which are considered of low and high meta-
bolic stability, respectively.
In the absence of NADPH no significant metabolism was observed for any of the com-
pounds tested (t1/2 > 240). In the presence of NADPH, CQ displayed the longest half-life of all
the aminoquinolines (133 15.5 min), while AQ showed a short half-life of 5.4 0.42 min.
The t1/2 values for 1–3 are in the range 40–51 min, intermediate between CQ and AQ, while
compound 4, which appears as the most active of the series, shows a t1/2 ~5 min, very similar to
that of AQ. It is known that CQ [30] is rapidly dealkylated via cytochrome P450 enzymes into
the pharmacologically active desethylchloroquine (DECQ), and to a lesser extent, bisdesethyl-
chloroquine, (BDECQ), while AQ is quickly metabolized into active desethylamodiaquine
(DEAQ); elimination of the active metabolites is in turn very slow (in the particular case of the
rapidly metabolized AQ the terminal half-life is ~100 h) [31]. Therefore both CQ and AQ may
be considered pro-drugs, which are bio-activated to DECQ and DEAQ; it is likely that 1–4 are
metabolized by P450 enzymes in a similar manner.
Table 3. Metabolic stability data.
Compd
NADPH-dep. aCLint (μl.min-1.mg-1)
NADPH-dep. bt1/2 (min)
NADPH-free bt1/2 (min)
1
45.6 0.12
56.2 0.55
51.1 3.06
465.9 43.99
17.5 2.03
431 33.4
50.9 0.55
41.1 0.41
45.3 1.36
5.0 0.47
>240
>240
>240
>240
>240
>240
2
3
4
CQ
AQ
133.2 15.52
5.4 0.42
[Compd] = 1 μM. [protein] = 0.3 mg/mL.
aMicrosomal Intrinsic Clearance.
bHalf-life
doi:10.1371/journal.pone.0140878.t003
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New 4-Aminoquinoline Antimalarials
Computational studies
Aminoquinoline drugs exert their antimalarial action by disrupting aggregation of heme into
hemozoin. It is therefore interesting to examine the interactions of new drug candidates with
heme, to try to find correlations with antimalarial potency. The binding of 1–5, CQ, and AQ to
heme was analyzed by use of Autodock 4.2.11 (Details in Materials and Methods Section). For
all the compounds studied the energetically most favorable binding is guided by H-bonding as
well as π-stacking interactions between the aminoquinoline and heme. Although the docking
energies shown in Table 4 are comparable for compounds 1–5 and only marginally stronger
than for CQ, a significant difference in the nature of the interactions among the molecules
studied is that stacking of 4 over the porphyrin takes place through the biphenyl moiety,
whereas for all other systems the stacking happens via the quinoline ring (Fig 4 and S1 Fig in
Supporting Information). This different binding mode of 4 to heme is likely related to the local-
ization of the HOMO over the biphenyl unit, unlike the other four molecules for which the
HOMO is concentrated mostly over the quinoline moeity (Fig 5, and S2 Fig, Supporting Infor-
mation). This structural difference could be in part responsible for the high activity observed
for this compound.
Conclusion
We have synthesized a series of new 4-aminoquinolines designed to display the high antimalar-
ial potency of amodiaquine while avoiding associated toxicity issues caused by quinone-imine
metabolites. This molecular design led to compounds with high in vitro activity, markedly supe-
rior to chloroquine and comparable to amodiaquine, against CQ-sensitive and particularly
against CQ-resistant strains of P. falciparum, accompanied by low toxicity toward L6 rat fibro-
blasts and MRC5 human lung cells. Compounds 1–3 display moderate in vitro metabolic stabil-
ity to human liver microsomes, lower than that of chloroquine but higher than amodiaquine,
while 4 shows a shorter half-life, very similar to that of amodiaquine. The high activity of 4 is
possibly linked with a unique mode of bindig to heme through the biphenyl unit.
Materials and Methods
Synthesis of compounds
General. Solvents (analytical grade, Aldrich) were purified immediately prior to use by
means of an Innovative Technology solvent purification unit. Other reagents (Aldrich) were
used as received. NMR spectra were obtained at 400 MHz for 1H and 75 MHz for 13C using an
AVANCE Bruker 400 instrument; δ values are referred to residual proton or carbon signals in
the deuterated solvents. Elemental analyses were performed by Atlantic Microlab, Norcross,
Georgia. The Mass Spectrometry Service of Hunter College CUNY performed the mass spectral
analyses.
Precursor nitriles 13–16. o-(Diethylaminomethyl)benzonitrile (13): Dropwise addition
of o-cyanobenzylbromide (12) (10.0 g, 50 mmol) in ethanol (30mL) to cold diethylamine (57
mL, 0.55 mol) was followed by 3 h of stirring at room temperature. The solution became
orange and a white precipitate was observed. Aqueous Na2CO3 (20 mL, 0.1 M) was added until
the solution became basic to pH paper. At this time the white precipitate dissolved. Then the
solution was concentrated and extracted into an ether layer. The organic layer was washed with
water 3 times, dried over Na2SO4, and evaporated under vacuum to yield the product as a red-
dish-orange oil. Yield: 8.47 g, 90%. 1H NMR (CDCl3) δ (ppm): 7.54 (m, 2H); 7.47 (td, 1H); 7.22
(td, 2H); 3.69 (s, 2H); 2.49 (q, 4H); 0.98 (t, 6H). ESI-MS (M+H+), 189.1313 (calc), 189.1388
(found).
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New 4-Aminoquinoline Antimalarials
Table 4. Computed docking energies.a
Compd
1
2
3
4
5
CQ
AQ
ΔE Docking (kcal/mol)b
-5.58
-5.11
-5.42
-5.97
-5.31
-4.72
-5.38
aComputed at the B3LYP/6-31G(d) level using the solvation model of Truhlar and co-workers. Computational details are found in the Supporting
Information.
bBis-protonated ligand geometries used for all docking simulations.
doi:10.1371/journal.pone.0140878.t004
m-(Diethylaminomethyl)benzonitrile (14) was prepared by following an analogous proce-
dure, using m-cyanobenzylbromide (6.0 g, 30 mmol) in ethanol (40 mL) and cold diethylamine
(35 mL, 340 mmol) and stirred at room temperature for 3 h. The product was collected as an
orange-colored oil. Yield 4.1 g, 72%. 1H NMR (CDCl3) δ (ppm): 7.7 (s, 1H); 7.61 (d, 1H); 7.56
(d, 1H), 7.45 (t, 1H), 3.26 (s, 2H), 2.55 (q, 4H), 1.05 (t, 6H).
p-(Diethylaminomethyl)benzonitrile (15) was prepared by following an analogous proce-
dure, using p-cyanobenzylbromide (7.2 g, 36 mmol) in THF (25 mL) and cold diethylamine
(40 mL, 403 mmol), stirring for 1.5 h at room temperature. The product was collected as
brown-orange oil. Yield 5.9 g, 86%. 1H NMR (CDCl3) δ (ppm): 7.57 (m, 2H); 7.47 (m, 2H);
3.59 (s, 2H), 2.52 (q, 4H), 1.02 (t, 6H).
4’-((diethylamino)methyl)-[1,1’biphenyl]-4-carbonitrile (16) was prepared by following
an analogous procedure, using 4’-bromomethyl-[1,1’-biphenyl]-4-carbonitrile (10.0 g, 36.7
mmol) in THF (40 mL) and cold diethylamine (40 mL, 403 mmol), stirring for 1.5 h at room
temperature. The product was collected as a colorless oil. Yield 9.4 g, 97%. 1H NMR (CDCl3) δ
(ppm): 7.8 (d, 1H); 7.65 (m, 1H); 7.55 (m, 6H), 3.65 (s, 2H), 2.6 (q, 4H), 1.1 (t, 6H).
Precursor amines 6–10. o-(Diethylaminomethyl)benzylamine (6) o-(Diethylamino-
methyl)benzonitrile (13) (6.0 g, 32 mmol) in anhydrous diethylether (20 mL) was added drop-
wise over a period of 10 min to a suspension of LiAlH4 (2.4 g, 64 mmol) in anhydrous diethyl
ether (50 mL, 0°C, N2 atmosphere). The mixture was allowed to come to room temperature,
stirred for 12 h, cooled in ice, treated dropwise with 20% NaOH (17 mL) with cooling, and
extracted three times with ether (50 mL each). The combined organic phases were dried over
Na2SO4, filtered, and evaporated under vacuum. The product was collected as reddish-orange
colored oil. Yield 6.8 g, 79%. 1H NMR (CDCl3) δ (ppm): 7.17 (m, 4H); 3.75 (s, 2H); 3.50 (s 2H);
2.43 (q, 4H): 1.928 (s, NH2); 0.962 (t, 6H). ESI-MS (M+H+) = 193.1626 (calc), 193.1715
(found).
m-(Diethylaminomethyl)benzylamine (7) was prepared by an analogous procedure using
m-(diethylaminomethyl)benzonitrile (14) (4.1 g, 22 mmol) in anhydrous diethylether (15 mL)
and lithium aluminum hydride (1.7 g, 44 mmol) in anhydrous diethyl ether (40 mL). The
Fig 4. Docked poses of 1 and 4 on heme. Both aminoquinoline molecules were employed in the
diprotonated form. Atom color code: white: H, brown: C, blue: N, red: O, and gold: Fe. Only polar hydrogens
are shown for clarity.
doi:10.1371/journal.pone.0140878.g004
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New 4-Aminoquinoline Antimalarials
Fig 5. HOMO’s of compounds 1 and 4.
doi:10.1371/journal.pone.0140878.g005
product was collected as orange colored oil. Yield 3.4 g, 81%, 1H NMR (CDCl3) δ (ppm): 7.29
(m, 4H); 3.86 (s, 2H); 3.56 (s, 2H), 3.26 (s, 2H), 2.54 (q, 4H), 1.04 (t, 6H).
p-(Diethylaminomethyl)benzylamine (8) was prepared by an analogous procedure using
p-(diethylaminomethyl)benzonitrile (15) (5.9 g, 32 mmol) in anhydrous diethylether (15 mL)
and lithium aluminum hydride (2.4 g, 63 mmol) in anhydrous ether (15 mL) The product was
collected as a pale orange colored oil. Yield 4.1 g, 67%. 1H NMR (CDCl3) δ (ppm): 7.27 (m,
4H); 3.84 (s, 2H); 2.52 (q, 4H), 1.03 (t, 6H).
N-((4’-(Aminomethyl)-[1,1’-biphenyl]-4-yl)methyl)-N,N-diethylamine (9) was prepared
by an analogous procedure using 4’-((diethylamino)methyl)-[1,1’biphenyl]-4-carbonitrile (16)
(9.4 g, 36 mmol) in anhydrous diethylether (25 mL) and lithium aluminum hydride (2.7 g, 71
mmol) in anhydrous ether (25 mL). The product was collected as slightly orange colored oil.
Yield 7.1 g, 78%. 1H NMR (CDCl3) δ (ppm): 7.3 (m, 8H); 3.83 (s, 2H); 3.64 (s, 2H), 2.58 (q,
4H), 1.1 (t, 6H).
N-((2-Aminomethyl)cyclohexa-1,4-dien-1-yl)methyl)-N,N-diethylamine (10) A solution
of o-(diethylaminomethyl)benzylamine (6) (1.5 g, 7.8 mmol) in anhydrous ethanol (24 mL)
was added to liquid ammonia (25 mL) at –78°C. To this mixture was added Li (0.5 g, 10 eq.) in
small portions over two hours. After quenching the reaction with NH4Cl (1.6 g) in H2O (7
mL), the aqueous suspension was extracted with CH2Cl2, and the organic extracts were dried
(Na2SO4) and evaporated under vacuum to give the product as a yellow colored oil. Yield 1.3 g,
89%. 1H NMR (400 MHz, CDCl3) δ 5.72 (m 2H), 3.30 (s, 2H), 3.00 (s, 2H), 2.43 (q, 4H); 2.75
(m, 4H), 2.02 (s, NH2), 1.01 (t, 6H). ESI-MS (M+H+) = 195.1783 (calc), 195.1854 (found).
New 4-aminoquinolines. 7-Chloro-N-(2-((diethylamino)methyl)benzyl)quinolin-
4-amine (1) o-(Diethylaminomethyl)-benzylamine (6) (0.8 g, 4.2 mmol), 4-7-dichloroquino-
line (11) (5.0 g, 25 mmol), K2CO3 (1.0 g, 7.25 mmol), anhydrous triethylamine (5 mL, 36
mmol) and anhydrous NMP (7 mL) were placed in a 25 mL round bottomed flask and heated
under reflux under nitrogen for 15 h. The mixture was allowed to cool to room temperature
before diluting with ethyl acetate. The product was washed 10 times with brine, and then
washed 6 times with a large amount of water to remove NMP. The organic layer was dried over
Na2SO4, filtered, and the solvent was removed under reduced pressure. The product was puri-
fied by column chromatography (ethylacetate: hexane: three drops of Et3N) and collected as a
yellow crystalline product. Yield 0.7 g, 50%. 1H NMR (MeOD), δ (ppm): 8.20 (d, J = 5.6 Hz,
1H), 7.93 (d, J = 9.0 Hz,1 H); 7.68 (d, J = 1.7 Hz, 1H), 7.29 (dd, J = 9.0 Hz, J’ = 1.9 Hz, 1H),
7.28–7.34 (m, 4H), 6.44 (d, J = 5.6 Hz, 1H), 4.59 (s, 2H), 3.57 (s, 2H), 2.48 (q, J = 7.1 Hz, 4H),
0.93 (t, J = 7.1 Hz, 6H), 13C NMR (CDCl3), δ (ppm): 152.2, 150.4, 149.5, 137.5, 137.4, 134.7,
132.2, 130.7, 128.4, 127.9, 127.5, 124.6, 122.6, 118.1, 99.2, 56.4, 47.1, 46.5, 10.5. Anal. Calcd for
C21H24N3Cl: C, 71.27; H, 6.84; N, 11.87. Found: C, 71.19; H, 6.85; N, 11.82. ESI-MS (M+H+) =
354.16588 (calc), 354.17501 (found).
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New 4-Aminoquinoline Antimalarials
7-Chloro-N-(3-((diethylamino)methyl)benzyl)quinolin-4-amine (2) was prepared by an
analogous procedure using m-(diethylaminomethyl)benzylamine (7) (3.4 g, 18 mmol), K2CO3
(3.0 g, 21 mmol), 4-7-dichloroquinoline (11) (20.8 g, 105 mmol), triethylamine (22 mL, 159
mmol) and NMP (25 mL). The resulting product (white powder) was dried under vacuum.
Yield 2.9 g, 47%, 1H NMR (CDCl3), δ (ppm): 8.54 (d, J = 4.8 Hz, 1H); 7.99 (s, 1H); 7.71 (d,
J = 5.0 Hz, 1H), 7.29 (m, 5H), 6.48 (d, J = 4.8 Hz, 1H), 4.54 (s, 2H), 3.59 (s, 2H), 2.54 (q, J = 7.1
Hz, 4H), 1.04 (t, J = 7.1 Hz, 6H). 13C NMR (CDCl3), δ (ppm): 152.1, 149.5, 149.2, 141.1, 137.1,
134.9, 128.8, 128.5, 128.2, 128.1, 120.9, 117.2, 99.7, 57.4, 47.7, 46.8, 11.7. Anal. Calcd for
C21H24N3Cl: C, 71.27; H, 6.84; N, 11.87. Found: C, 71.14; H, 6.95; N, 11.73. ESI-MS (M+H+) =
354.16588 (calc), 354.17610 (found M+H+).
7-Chloro-N-(4-((diethylamino)methyl)benzyl)quinolin-4-amine (3) was prepared by an
analogous procedure using p-(diethylaminomethyl)benzylamine (8) (4.1 g, 21 mmol), K2CO3
(3.5 g, 25.2 mmol), 4-7-dichloroquinoline (11) (20.8 g, 105 mmol), triethylamine (26 mL, 189
mmol) and NMP (30 mL). The resulting product (slightly yellow powder) was dried under vac-
uum. Yield 4.3 g, 57%, 1H NMR (CDCl3), δ (ppm): 8.58 (d, J = 3.9 Hz, 1H); 8.02 (s, 1H); 7.72
(d, J = 4.1 Hz, 1H), 7.30 (m, 4H), 7.41 (d, J = 4.0 Hz, 1H), 6.51 (d, J = 3.9 Hz, 1H), 4.53 (s, 2H),
3.62 (s, 2H), 2.58 (q, J = 7.0 Hz, 4H), 1.10 (t, J = 7.0 Hz, 6H). 13C NMR (CDCl3), δ (ppm):
152.1, 149.5, 149.1, 135.5, 134.9, 128.9, 127.5, 125.5, 120.9, 117.2, 99.7, 57.4, 47.7, 46.8, 11.7.
Anal. Calcd for C21H24N3Cl: C, 71.27; H, 6.84; N, 11.87. Found: C, 71.07; H, 6.92; N, 11.83.
ESI-MS (M+H+) = 354.16588 (calc), 354.17259 (found M+H+).
7-Chloro-N-((4’-((diethylamino)methyl)-[1,1’-biphenyl]-4-yl)methyl)quinolin-4-amine
(4) was prepared by an analogous procedure using N-((4’-(aminomethyl)-[1,1’-biphenyl]-4-yl)
methyl)-N,N-diethylamine (9) (7.8 g, 27.8 mmol), K2CO3 (3.1 g, 22.1 mmol), 4-7-dichloroqui-
noline (11) (14.6 g, 174 mmol), triethylamine (26 mL, 189 mmol), and NMP (30 mL). The
resulting product (light yellow powder) was dried under vacuum. Yield 6.8 g, 56%. 1H NMR
(CDCl3) δ (ppm): 8.49 (d, J = 8.2 Hz, 1H); 7.95 (s, 1H); 7.3 (m, 10H), 6.32 (d, J = 8.2 Hz, 1H),
4.49 (s, 2H), 3.6 (s, 2H), 2.54 (q, J = 7.2 Hz, 4H), 1.09 (t, J = 7.2 Hz, 6H). 13C NMR (CDCl3), δ
(ppm): 152.0, 149.1, 141.9, 134.6, 128.0, 125.3, 120.9, 117.1, 99.5, 57.1, 46.8, 45.8, 11.7. Anal.
Calcd for C27H28N3Cl: C, 75.42; H, 6.56; N, 9.77. Found: C, 75.12; H, 6.76; N, 9.81. ESI-MS
(M+H+) = 430.19718 (calc), 430.20347 (found M+H+).
7-Chloro-N-((2-((diethylamino)methyl)cyclohexa-1,4-dien-1-yl)methyl)quinolin-
4-amine (5) was prepared by an analogous procedure using N-((2-(aminomethyl)cyclohexa-
1,4-dien-1-yl)methyl)-N,N-diethylamine (10) (0.9 g, 4.9 mmol), 4,7-dichloroquinoline (11)
(5.0 g, 25 mmol), K2CO3 (1.0 g, 7.25 mmol), anhydrous triethylamine (5.0 mL, 36 mmol) and
anhydrous NMP (7 mL). The product was isolated as a white crystalline solid. Yield 50%. 1H
NMR (CDCl3): δ 8.45 (d, J = 2.2 Hz, 1H), 7.87 (d, J = 2.2 Hz, 1H), 7.63 (d, J = 8.9 Hz, 1H), 7.25
(dd, J = 8.9 Hz, J’ = 2.1 Hz, 1H), 6.52 (s, NH), 6.36 (d, J = 5.50 Hz, 1H), 5.65 (d, J = 1.24 Hz,
2H), 3.78 (d, J = 4.24 Hz, 2H), 2.98 (s, 2H), 2.80 (m, 4H), 2.50 (q, J = 7.1 Hz, 4H), 0.954 (t,
J = 7.1 Hz, 6H). 13C NMR (CDCl3): δ 152.1, 150.5, 149.3, 134.7, 132.2, 129.8, 128.7, 124.8,
124.5, 123.9, 121.8, 117.7, 99.0, 55.1, 46.8, 45.5, 32.3, 31.7, 11.1. Anal. Calcd for C21H26N3Cl: C,
70.87; H, 7.36; N, 11.81.Found: C, 70.71; H, 7.39; N, 11.70. ESI-MS (M+H+) = 356.1815 (calc),
356.1877 (found).
Biological Assays
Antimalarial Activity Measurements. The following strains of Plasmodium falciparum
were used in this study: F32 (Tanzania), Dd2 (SE Asia), K1 (SE Asia), FcB1 (Colombia), K14
(Cambodia) and 3D7 (Africa). In the routine culture conditions of laboratory A (Marseille),
that is in an incubator with a fixed atmosphere of 5% CO2, 10% O2 and 85% N2 at 37°C, strain
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New 4-Aminoquinoline Antimalarials
3D7 was CQ-sensitive (IC50 < 100 nM), whereas Dd2, K1 and K14 were CQ-resistant (IC50
>
100 nM). In the routine culture conditions of laboratory B (Paris), that is in a candle jar with
an atmosphere of approximately 17% O2, 3% CO2 and 80% N2 at 37°C, 3D7, F32 and Dd2
were CQ-sensitive, whereas K1, K14 and FcB1 were CQ-resistant. Cultures were grown in com-
plete medium consisting of RPMI 1640 (Life Technologies Inc.) supplemented with 11 mM
glucose, 27.5 mM NaHCO3, 100 UI/mL penicillin, 100 μg/mL streptomycin, and 8–10% heat-
inactivated human serum, following the procedure of Trager and Jensen [32]. Parasites at the
ring stage (laboratory A) or asynchronous parasites (laboratory B) were grown in human A
+ or O+ red blood cells at a 1.5% (laboratory A) or 2% (laboratory B) haematocrit and a 0.8–
1% parasitaemia. Synchronization of the parasites was performed by sorbitol treatment [33].
Stock solutions (1 mM) of compounds 1–5 were prepared in DMSO and stored frozen at
-20°C. Further dilutions were in complete culture medium. The complexes were tested for their
inhibitory effect toward P. falciparum intraerythrocytic development. Decreasing concentra-
tions of the compounds and CQ or AQ were established by twofold serial dilutions (maximum
DMSO concentration was 0.5% v/v) and distributed (100 μL/well) in a 96 well microplate;
DMSO was distributed as a control. 100 μL from a culture at a 3% (laboratory A) or 4% (labo-
ratory B) hematocrit in complete medium was added per well. At time zero (laboratory A) or
after 24 h of the incubation (laboratory B), 1.0 μCi (laboratory A) or 0.5 μCi (laboratory B) of
3H-hypoxanthine was added per well. Then the culture proceeded until the parasite cycle was
completed (i.e. to 42 h or 48 h according to the strain). Plates were freeze-thawed and harvested
on filters. Dried filters were moistened in scintillation liquid mixture (OptiScint, Hisafe) and
counted in a 1450 Microbeta counter (Wallac, PerkinElmer). The percentage of growth inhibi-
tion was calculated from the parasite-associated radioactivity. 100% 3H-hypoxanthine incorpo-
ration was determined from a control grown in the absence of drug or test compound.
Antimalarial activity was determined as the concentration of drug inducing 50% of growth
inhibition (IC50) according to Desjardin et al. [34] or by nonlinear regression analysis from the
dose-response relationship as fitted by Riasmart software (Packard).
Cytotoxicity measurements. The rat L-6 and human MRC-5 cell lines were routinely
grown in RPMI 1640 supplemented with 11 mM glucose, 27.5 mM NaHCO3, 100 UI/mL peni-
cillin, 100 μg/mL streptomycin and 10% fetal calf serum (RPMI-FCS), in a 5% CO2 incubator
at 37°C. For cytotoxicity assays, 100 μL per well of a 2×104 cells/mL suspension in RPMI-FCS
were deposited in a 96-well microplate and incubated overnight in culture conditions. Then
decreasing concentrations of the compounds to test were established by twofold serial dilutions
in RPMI-FCS and distributed at a rate of 100 μL per well and the microplate was put back in
the incubator for an additional three (L-6) or five (MRC-5) day period. Then the supernatant
in each well was discarded and replaced by 100 μL of MTT 1 mg/mL in RPMI-FCS. The micro-
plate was re-incubated in culture conditions for three hours, then 100 μL of 10% SDS was
added by well. The cells were left overnight in the incubator then the OD at 540nm was mea-
sured with the Bio-Tek FL600™ microplate reader equipped with the KC4™ software. Cytotoxic
activity was expressed as the concentration of drug inducing 50% of growth arrest (CC50), with
100% growth being determined from cells grown in the absence of test compounds. The selec-
tivity of each compound to P. falciparum was assessed through the selectivity index (SI),
defined as CC50 (to L6 or MRC5 cells) / IC50 (to P. falciparum).
In vitro microsomal metabolic stability measurements. The evaluation of microsomal
stability was performed by Cyprotex US, LLC, Watertown, MA. Samples were analyzed by LC/
MS/MS using an Agilent 6410 mass spectrometer coupled with an Agilent 1200 HPLC and a
CTC PAL chilled autosampler, all controlled by MassHunter software (Agilent). After separa-
tion on an HILIC HPLC colum (Sepax HILIC 3 μM 2.1 x 30 mm) using an acetonitrile-water
gradient system, peaks were analyzed by mass spectrometry (MS) using ESI ionization in
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New 4-Aminoquinoline Antimalarials
MRM mode. The signal was optimized for each compound by ESI positive or negative ioniza-
tion mode. An MS2 scan or an SIM scan was used to optimize the fragmenter voltage and a
product ion analysis was used to identify the best fragment for analysis, and the collision energy
was optimized using a product ion or MRM scan. An ionization ranking was assigned indicat-
ing the compound’s ease of ionization.
Each test compound (1 μM) was incubated in duplicate with human liver microsomes at
37°C. The reaction contains microsomal protein (0.3 mg/mL) in 100 nM potassium phosphate,
2 mM NADPH, 3 mM MgCl2, pH 7.4. A control reaction omitting NADPH was performed for
each compound in order to detect any NADPH-independent degradation. Aliquots were
removed at 0, 10, 20, 40, and 60 min and mixed with an equal volume of ice-cold Stop Solution
(methanol containing haloperidol, diclofenac, or other internal standard). The stopped reac-
tions were further incubated for at least 10 min at -20°C, and an additional volume of water
was added. The samples were centrifuged to remove precipitated protein, and the supernatants
were analyzed by LC/MS/MS to quantify the remaining parent. Data were converted to %
remaining by dividing by the time zero concentration value, and fitted to a first-order decay
model to determine half-life values. Intrinsic clearance values were calculated from the half-life
and protein concentrations by using the equation CLint = ln 2 / t1/2 [microsomal protein].
Computational details
All electronic structure calculations were performed at the B3LYP level of theory in combina-
tion with 6-31G(d) basis set as implemented in the Gaussian09 suite of quantum chemical pro-
grams [35–38]. An ultrafine grid was used for all calculations. The effect of solvation was
modeled using a polarizable continuum model and atomic radii form Truhlar and co-workers
in both water and 1-Octanol continuum [39]. All structures were confirmed to be minima via
frequency calculations in both gas- and solvent-phases. We optimized the neutral, monoproto-
nated, and diprotonated forms of all molecules. Molecular orbital surfaces are generated for gas
phase optimized geometry using an iso-surface value of 0.02. All molecular docking simula-
tions were performed with Autodock4.2.11 [40], with default force field, which includes
parameters for heme Fe, as well as for C/H/N/O/Cl. The structure of monomeric heme used
was from CYP51 (PDB ID: 4H6O; http://www.rcsb.org/pdb/explore/explore.do?structureId=
4H6O), and heme dimers from Pagola et al. [41]. For the ligands, the B3LYP/6-31G(d) opti-
mized geometries of the diprotonated molecules (AQ, CQ, 1–5) were used as starting points
for all docking simulations. All acyclic bonds in the ligands were rotatable for docking calcula-
tions. A grid box with a spacing of 0.375Å, size of 60 x 60 x 60, and centered at the rigid heme
receptor was used to generate atomistic grid by AutoGrid4.2. Molecular docking was per-
formed using Lamarckian genetic algorithm (LGA) with search parameters set for 156 GA
runs, with a population size of 200, a maximum number of 2.5 × 105 energy evaluations, a max-
imum number of 2.7 × 104 generations, a mutation rate of 0.02, and a crossover rate of 0.8.
Other docking parameters were set to default. The docked conformations were clustered into
groups of similar binding modes using a root mean square deviation clustering tolerance of
2.0Å.
Supporting Information
S1 Fig. Docked poses of all molecules studied on a heme receptor. The aminoquinoline mol-
ecules were employed in the diprotonated form. Only polar hydrogens were shown for
improved clarity. Atom color code: white: H, brown: C, blue: N, red: O, and gold: Fe.
(TIF)
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New 4-Aminoquinoline Antimalarials
S2 Fig. HOMO and LUMO plots of all the systems. Computed at the B3LYP/6-31G(d) level
at isosurface value of 0.02.
(TIF)
S1 Table. Additional Antiplasmodial Activity Data from Lab. B.
(DOCX)
S2 Table. B3LYP/6-31GÃ optimized geometry and energy (in Hartrees) of all molecules
studied.
(DOCX)
Acknowledgments
We thank Ms. Victoria Medialdea (Brooklyn College) for assistance in the preparation of the
manuscript.
Author Contributions
Conceived and designed the experiments: CD VS JS RAS-D. Performed the experiments: CR
ML CD VS CL DR. Analyzed the data: CR ML CD VS CL DR JS RAS-D. Wrote the paper: CR
CD VS DR JS RAS-D.
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