European Journal of Medicinal Chemistry 122 (2016) 394e407

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European Journal of Medicinal Chemistry

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

Research paper

4-Aminoquinoline derivatives: Synthesis, in vitro and in vivo

antiplasmodial activity against chloroquine-resistant parasites
Shailja Singh a, Drishti Agarwal a, c, Kumkum Sharma a, Manish Sharma a,
Morten A. Nielsen b, Michael Alifrangis b, Ashok K. Singh d, Rinkoo D. Gupta c,
Satish K. Awasthi a, *
a
Chemical Biology Laboratory, Department of Chemistry, University of Delhi, Delhi 110007, India
b
Centre for Medical Parasitology, Institute of International Health, Immunology and Microbiology, University of Copenhagen and Department of Infectious
Diseases, Copenhagen University Hospital, Copenhagen, Denmark
c
Faculty of Life Sciences and Biotechnology, South Asian University, New Delhi 110021, India
d
Department of Zoology, University of Delhi, Delhi 110007, India

a r t i c l e i n f o a b s t r a c t

Article history: Synthetic quinoline derivatives continue to be considered as candidates for new drug discovery if they
Received 14 September 2015 act against CQ-resistant strains of malaria even after the widespread emergence of resistance to CQ. In
Received in revised form this study, we explored the activities of two series of new 4-aminoquinoline derivatives and found them
31 May 2016
to be effective against Plasmodium falciparum under in vitro conditions. Further, we selected four most
Accepted 19 June 2016
Available online 23 June 2016
active derivatives 1m, 1o, 2c and 2j and evaluated their antimalarial potential against Plasmodium berghei
in vivo. These 4-aminoquinolines cured BALB/c mice infected with P. berghei. The ED50 values were
calculated to be 2.062, 2.231, 1.431, 1.623 and 1.18 mg/kg of body weight for each of the compounds 1m,
Keywords:
Antimalarial
1o, 2c, 2j and amodiaquine, respectively. Total doses of 500 mg/kg of body weight were well received.
Amodiaquine The study suggests that these new 4-aminoquinolines should be used for structure activity relationship
Chloroquine to find lead molecules for treating multidrug-resistant Plasmodium falciparum and Plasmodium vivax.
4-Aminoquinoline analogues © 2016 Elsevier Masson SAS. All rights reserved.
Plasmodium falciparum
Plasmodium berghei

1. Introduction avoid an upsurge in malaria attributed morbidity and mortality.

Combination therapy is undoubtedly the best way to treat ma-
Malaria mortality rates have come down by a drastic 47% be- laria and to mitigate the emergence of drug resistance [2]. The
tween 2000 and 2013 globally [1]. The improvements are mainly World Health Organization (WHO), back in 2001, recommended
the result of large scale investments and implementation of the use of artemisinin-based combination therapies (ACTs) in
insecticide treated nets (ITNs), improved diagnostics using rapid endemic areas of Africa and other tropical countries [3]. However,
diagnostics tests and treatment of malaria using the efficacious decreased susceptivity of P. falciparum to artemisinin pose serious
antimalarial drug combinations. However, emergence of drug and concerns [4] and new drugs and drug combinations are highly
insecticide-resistance stand ahead as major obstacles, and attempts needed.
should be directed at overcoming them as soon as possible, so as to Drug candidates based on the 4-aminoquinoline scaffold
continue to be considered as promising candidates for combination
therapy [5]. Till date, no other drug has been able to match the
Abbreviations: ACT, Artemisinin-based combination therapy; RPMI, Roswell antimalarial attributes of chloroquine (CQ). However, widespread
Park Memorial Institute (cell culture medium); HEPES, 4-(2-hydroxyethyl)-1- resistance has rendered it almost useless throughout the world,
piperazine-ethanesulfonic acid; IC50, Median inhibition concentration of growth; particularly against P. falciparum infections [6]. Amodiaquine (AQ),
ED50, Median effective dose; DMSO, Dimethyl sulphoxide; MST, mean survival time; effective against many chloroquine-resistant strains of
SAR, Structure-activity relationship; BW, Body weight; CC50, Cytotoxic concentra-
tion 50; TI, Therapeutic index; TMS, Tetramethylsilane.
P. falciparum, was developed by the US Army-sponsored program to
* Corresponding author. develop alternatives to quinine; its use became widespread both
E-mail addresses: satishpna@gmail.com, skawasthi@chemistry.du.ac.in prophylactically and therapeutically [7]. Soon after, in the 1980s, its
(S.K. Awasthi).

http://dx.doi.org/10.1016/j.ejmech.2016.06.033
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.
 S. Singh et al. / European Journal of Medicinal Chemistry 122 (2016) 394e407 395

use was terminated because of the occurrence of numerous cases of found that there is still some scope for modification of basic scaf-
agranulocytosis, neutropenia and hepatotoxicity in adults taking folds to improve antimalarial activity.
the drug prophylactically [8]. However, later, its therapeutic use In this way newer cost effective compounds with significant
was reiterated and to date, there is no evidence for serious toxicity antimalarial activity can be designed and synthesized. Till now little
associated with amodiaquine therapy [9]. AQ toxicity has been systematic study has been carried out using different electron
explained by the presence of its 4-hydroxyanilino moiety, which is releasing and donating groups on phenyl rings which exhibited the
believed to undergo extensive metabolization to its quinoneimine effect of substituents on the activity of the basic scaffold [14].
variant (Fig. 1), a feature contrasting to chloroquine [10]. Keeping these facts in mind and in the continuation of our ongoing
In this article, we describe the synthesis and antimalarial ac- projects on malaria drug discovery [15e18] and biophysical analysis
tivity of amodiaquine analogues under two series, I and II. These of amodiaquine analogues [19], we have synthesized and charac-
analogues inhibit the formation of toxic metabolites by simple terized two different libraries of small 4-aminoquinoline analogues
oxidation due to absence of hydroxyl group at position 4 of benzene under series I & II as shown in Fig. 2.
unlike amodiaquine. Moreover, these are potent against CQ resis- Since AQ retains antimalarial activity against many CQ-resistant
tant P. falciparum parasites. The compounds were tested: (i) as parasites, several modifications were made to synthesize safer,
blood schizonticides against P. falciparum in vitro; (ii) against P. cost-effective alternative. Other groups have reported the synthesis
berghei infections in mice; (iii) for their in vitro cytotoxicity; (iv) of fluoro amodiaquine (FAQ) [20]. This analogue cannot form toxic
in vivo toxicity; (v) for their binding mode to lactate dehydrogenase metabolites due to absence of hydroxyl group and retains sub-
and dimeric hematin in silico. Apart from an excellent anti- stantial antimalarial activity versus CQ-resistant parasites [21]. In
plasmodial profile, these analogues are extremely cost-effective to order to diversify AQ analogues, in this study, we further report the
synthesize. On the basis of initial data reported in this paper, design and synthesis of newer compounds by introducing different
selected potent analogues may represent new leads for develop- functional groups on phenyl ring to improve antimalarial efficacy
ment of synergistic drug partners in antimalarial combination and lessen toxicity. We assume that these combinations might have
therapy. significant impact on the parasite life cycle by inhibiting hemozoin
formation as well as non-toxic in nature.
We have synthesized 16 analogues (series-I) by direct nucleo-
2. Results and discussion
philic substitution reaction on 4, 7-dichlroquinoline using various
substituted anilines. Further, the introduction of a flexible linear
2.1. Synthesis of 4-aminoquinoline analogues
chain between the two amino functions in CQ and the replacement
of the terminal diethylamino group by different substituents like
Most of the clinical antimalarial drugs including chloroquine
alkyl, phenyl with electron donating and electron withdrawing
(CQ) have lost their utility due to the development of drug resis-
groups were screened and well reported [22]. It is found that 4-
tance. A closely related amodiaquine could be applied as an alter-
aminoquinoline derivatives having 2 or 3 carbon linear chain be-
nate since it retains antimalarial activity against many CQ-resistant
tween two terminal amino groups exhibited promising antima-
parasites [11]. Activity of AQ is linked to a dynamic conformation in
larial activity (Fig. 3) [23]. These modifications were expected to
which the separation between the quinoline nitrogen and the
enhance the lifetime of chloroquine analogues and interesting ac-
diethylamino nitrogen is approximately 8.30 A, which is very much
tivity against CQ resistant strains. Keeping the above facts in mind,
similar to chloroquine [12]. Research studies reveal that intra-
we have synthesized small library of compounds by reductive
molecular hydrogen bond between the hydroxyl and the proton of
amination reaction (series II) in which the length of four carbon
the charged diethylamino function plays a significant role in the
linker chain is similar to that of chloroquine except that one carbon
activity of amodiaquine derivative. However, AQ produces a toxic
is replaced by NH group. Different electron releasing and electron
quinoneimine metabolite (Fig. 1) thus; it has been limited since the
withdrawing groups were placed on o, m or p-positions to get
mid 1980s [13]. According to last WHO’s guidelines for treatment of
maximum inhibitory activity. All new compounds were well char-
malaria, use of AQ is still recommended either in combination with
acterized by NMR and mass spectrometry.
artemisinin derivatives or with sulfadoxine/pyrimethamine. On
Under series-I, we have synthesized 16 compounds (Table 1)
careful examination of the structure of amodiaquine, we have

OH
OH
Metabolic
OH Oxidation HN
CYP2C8 HN
N
HN N
HN
Cl N
Cl N
Quinoneimine
Cl N Formation
Amodiaquine
P 450
GS
Hepatotoxicity OH
OH and
Agranulocytosis
HN
HN
N
NH2

Cl N
Cl N

Fig. 1. Production of a toxic quinoneimine metabolite by amodiaquine.

396 S. Singh et al. / European Journal of Medicinal Chemistry 122 (2016) 394e407

Fig. 2. Series I and II showing reagents and conditions used in synthesis of compounds.

groups on phenyl ring. However, the compound 1e having three

methoxy groups at 3, 4, 5-positions of the phenyl ring showed
significant loss of activity as compared to the compound 1o. This
could be attributed to steric hindrance caused by the presence of
three methoxy groups. Further, compounds 1h, 1i, 1j, 1k, 1l and 1p
exhibit moderate activity, while compound 1c shows least anti-
malarial activity in comparison to the most active compounds in
series I.
In series-II, compound 2c (IC50 95% Confidence
Fig. 3. Basis for the synthesis of 4-aminoquinoline derivatives by reductive amination
intervals ¼ [2.1  105; 5.6  105]g/L) and 2j (IC50 95% Confidence
reaction.
intervals ¼ [1.1  105; 4  105]g/L) were most active. This result
signifies that fluoro and bromo at para position in phenyl ring
using different substituted aromatic anilines. Briefly, equimolar impart significant antimalarial activity. The electron releasing
amount of 4, 7-dichloroquinoline and desired aniline were reflux in groups such as methoxy and methyl at para position on phenyl ring
dry ethanol in basic condition (K2CO3, 1.2 eq.). Compounds were (compounds 2a and 2i, respectively) also enhance antimalarial ac-
purified by crystallization in methanol/methanol þ acetone or tivity while chloro group (compound 2b) has lesser effect on anti-
C2H5OH as solvent. malarial activity. Further, compounds 2d, 2e, 2g and 2h display
All the 11 compounds of series-II (Table 2) were synthesized moderate while compound 2f shows least antimalarial efficacy
using N1-(7-chloroquinolin-4-yl)-ethane-1, 2-diamine (2) as start- with respect to the most active compounds. Interestingly, 3, 5-
ing substrate. In general, commercially available starting material 4, dimethoxy group enhance antimalarial activity in series I while it
7-dichloroquinoline was treated with an excess of aliphatic linear has lesser effect in series II. The reason behind such varying
chain diaminoalkane via a SNAr type of reaction under usual con- behavior of compounds with similar nature of functional groups is
ditions as reported in the literature to afford substituted 4- still elusive. This study clearly demonstrates that there is need of
aminoquinolines with free terminal amino groups in good yield systematic SAR study to establish meaningful correlation among
[24]. This intermediate on reaction with suitable aldehydes yielded various functional groups. Work in this direction is under progress.
desired product in minor to low yield [25].
All the compounds synthesized by the above given procedure 2.3. In vitro cytotoxicity assay
were purified either by crystallization or by column chromatog-
raphy and characterized by different spectroscopic techniques viz. To substantiate the therapeutic value of compounds in series-I
IR, 1H & 13C NMR and MASS. Fig. 2 summarizes the synthesis and series-II, toxicity on mammalian cells was assessed against
strategy behind series-I and II. Huh7 cells; a hepatocyte derived cellular carcinoma cell line
(Tables 1 and 2). However, only compound 2f, containing nitrile
2.2. Efficacies of 4-aminoquinoline analogues against P. falciparum group at para position of phenyl ring appeared to be associated
in vitro with some toxicity (CC50 value of 39.58 mg/mL). All the other
compounds are devoid of any considerable cytotoxicity at the
Two newer series of AQ analogues, series-I (1aep) and series-II highest test concentration (100 mg/mL). Thus, these results further
(2aek) were evaluated in vitro against the erythrocytic stages of support the significance of this study.
P. falciparum (FCR3 strain, a chloroquine, pyrimethamine and
cycloguanil resistant strain). Results in Table 1 show that most of 2.4. In vivo activities of selected derivatives against rodent malaria
the new compounds of series-I exhibited very promising activity
ranges, depending on the functional group on phenyl ring. The We chose 4 compounds that displayed the most potent in vitro
most potent compounds were 1m (IC50 95% Confidence activity (compounds 1m and 1o from series I; compounds 2c and 2j
intervals ¼ [2.9  105; 9.3  105]g/L), which contains fluoro from series II) for evaluation in vivo, against the rodent malaria
group at para position and 1 (IC50 95% Confidence parasite P. berghei (erythrocytic stage of ANKA, a CQ resistant
intervals ¼ [2.2  105; 6.8  105]g/L), with 2, 5-dimethoxy strain). After conducting Peters’ four-day suppressive test, it was
 S. Singh et al. / European Journal of Medicinal Chemistry 122 (2016) 394e407 397

Table 1
List of compounds synthesized using series-I, along with their antiplasmodial activities against P. falciparum FCR3 strain and cytotoxicities against Huh-7 cells.

Compound code R1 IC50 95% confidence intervals (g/L) Mean CC50± SEa (mg/mL)

1a [7.6  104; 3.9  103] >100 mg/mL

1b [4.0  104; 1.3  103] >100 mg/mL

1c [1.9  103; 5.5  103] >100 mg/mL

1d [4.6  104; 1.8  103] >100 mg/mL

1e [6.4  104; 3.2  103] >100 mg/mL

1f [7.6  105; 4.3  104] >100 mg/mL

1g [1.7  104; 6.2  104] >100 mg/mL

1h [9.8  105; 2.7  104] >100 mg/mL

1i [3.6  105; 1.3  104] >100 mg/mL

1j [4.6  105; 1.5  104] >100 mg/mL

1k [5.6  105; 1.9  104] >100 mg/mL

1l [4.5  105; 1.8  104] >100 mg/mL

(continued on next page)

398 S. Singh et al. / European Journal of Medicinal Chemistry 122 (2016) 394e407

Table 1 (continued )

Compound code R1 IC50 95% confidence intervals (g/L) Mean CC50± SEa (mg/mL)
5 5
1m [2.9  10 ; 9.3  10 ] >100 mg/mL

1n [1.6  104; 5.0  104] >100 mg/mL

1o [2.2  105; 6.8  105] >100 mg/mL

1p [6.5  105; 2.1  104] >100 mg/mL

a
Standard error (n ¼ 3).

observed that there was a reduction in the levels of parasitemia in is responsible for the hindrance caused to heme polymerization.
all the test groups, as well as that of the standard drug (AQ) group. The best conformations obtained for the complexes showed that
However, the reverse was the case for the negative control group, as the aromatic rings of all the compounds were parallel to the he-
there was a marked increase in parasitemia level on day 4. The matin ferriprotoporphyrin group (Fig. 6).
in vivo antimalarial activity of the various test compounds is pre-
sented in Fig. 4. 2.6.2. Binding to lactate dehydrogenase (LDH) contributes to the
Results were significant as analyzed by ANOVA (P < 0.05). The antimalarial action
ED50 values were calculated to be 2.062, 2.231, 1.431, 1.623 and Table 5 presents the H-bond energy of the residues involved in
1.18 mg/kg BW for each of the compounds 1m, 1o, 2c, 2j and H-bonds with compounds 1m, 1o, 2c and 2j, docked to PfLDH. Fig. 6
amodiaquine respectively, as indicated in Table 3. The mean sur- shows that these compounds presented the most stable energy
vival time (MST) values of the animals in test groups were deter- conformations in the binding site of NADH. This suggests that they
mined, demonstrating a dose-dependent increase in the number of are NADH competitors. The H-bond energies between the amo-
days the mice survived in various groups survived post-four-day diaquine derivatives and the enzyme were considerably high, as
treatment. MST values of the treated groups were significantly compared with that of NADH H-bond energy (35.3 kcal mol1)
higher than that of control and were comparable to that of the [26]. Furthermore, 2j presented the lowest energy, which was quite
standard drug (Fig. 5). All the results were significant (P < 0.005) as large when compared to NADH. These results imply that all the test
analyzed by Log-rank (Mantel-Cox) test. No significant side-effects compounds are weak inhibitors of PfLDH, as previously reported for
such as weight loss were observed in compound treated groups. CQ [27].
Although PfLDH is not the principal target of CQ, experimental
2.5. Favourable safety profiles in test animals data and modeling studies have explored its weak binding to CQ [28].
The existence of this shared target-binding site suggests that PfLDH
In the LD50 test for determining the therapeutic indices of has an inevitable role to play in drug effectiveness. The docking data
compounds, BALB/c mice died at1000 mg/kg BW of all the com- (Table 5) suggest that the compounds present the most stable en-
pounds and could tolerate 500 mg/kg BW. However, at 800 mg/kg ergetic conformations at the binding site of NADH inside PfLDH. The
BW, half the population of mice died. Therapeutic indices were H-bond energy, residues involved in the H-bonds with NADH, resi-
determined as 387.973, 358.584, 559.05and 492.914 for compounds dues contributing to vanderwaals and other weak interactions and
1m, 1o, 2c and 2j, respectively. the compounds bound to PfLDH (Fig. 7) suggest that these com-
pounds are NADH competitors. Overall, these findings stand in
2.6. In silico studies strong support of these compounds acting as weak inhibitors of
PfLDH, as has been observed in the case of chloroquine [29,30].
2.6.1. Inhibition of hemozoin formation
Whether anti P. falciparum activity of the selected derivatives 3. Conclusion
involves inhibition of hemozoin formation was evaluated by
docking them with dimeric hematin. Docking studies with com- We have successfully designed, synthesized and characterized
pounds 1m, 1o, 2c and 2j showed that the H-bond energies be- two series of new amodiaquine derivatives utilizing readily avail-
tween the antimalarial compounds and the dimeric hematin able and inexpensive chemicals. The present study has led to the
(Table 4) were approximately 6.0 kcal mol1, ascertaining that identification of four antiplasmodial lead compounds: 1m and 1o
hydrophobic interactions were the main contributors for the from series-I; 2c and 2j from series-II. These lead compounds
binding. Thus, all four AQ derivatives, similar to CQ [26], were able display significant activity and low or no toxicity, both in vitro and
to interact with dimeric hematin to form a complex, which in turn in vivo and could be converted to more potent, synergistic
 S. Singh et al. / European Journal of Medicinal Chemistry 122 (2016) 394e407 399

Table 2
List of compounds synthesized using series-II, along with their antiplasmodial activities against P. falciparum FCR3 strain and cytotoxicities against Huh-7 cells.

Compound code R2 IC50 95% confidence interval (g/L) Mean CC50± SEa (mg/mL)

2a [1.3  105; 3.4  105] >100 mg/mL

2b [1.9  103; 3.0  103] >100 mg/mL

2c [2.1  105; 5.6  105] >100 mg/mL

2d [3.0  105; 1.2  104] >100 mg/mL

2e [2.8  105; 1.2  104] >100 mg/mL

2f [1.8  103; 1.1  102] 39.58 mg/mL

2g [5.0  105; 1.9  104] >100 mg/mL

2h [7.2  105; 4.4  104] >100 mg/mL

2i [1.8  105; 6.6  105] >100 mg/mL

2j [1.1  105; 3.4  105] >100 mg/mL

2k [1.4  105; 3.5  105] >100 mg/mL

a
Standard error (n ¼ 3).

antimalarial drug partners to be employed in the combination progress of reactions was monitored by silica gel thin layer chro-
therapy approach. The preliminary screening of antimalarial ac- matography (TLC) plates and visualized under UV. Melting points
tivity should encourage further detail studies aimed at optimizing were determined by using open capillary method and Buchi
their antimalarial activities. Melting Point M 560 melting apparatus and are uncorrected. 1H
NMR and 13C NMR spectral data were recorded on BruckerAvance
4. Experimental procedure spectrometer at 300 MHz and Jeol JNM spectrometer at 400 MHz,
respectively in CDCl3 or CD3OD or DMSO-d6, using TMS as an in-
4.1. General methods ternal standard. The chemical shift values were recorded on d scale
and the coupling constants (J) in hertz. The following abbreviations
Various chemicals and solvents used in this study were pur- were used in reporting spectra: s ¼ singlet, d ¼ doublet, t ¼ triplet,
chased from E. Merk (India) and Sigma-Aldrich chemicals. The q ¼ quartet, m ¼ multiple. IR spectra were obtained on a Perkin
400 S. Singh et al. / European Journal of Medicinal Chemistry 122 (2016) 394e407

Fig. 4. Effects of various compounds and amodiaquine on established P. berghei infections in mice. The experimental hosts were infected on day 0 and treated intraperitoneally with
normal saline; compounds 1m, 1o, 2c, 2j and amodiaquine at 0.1, 1.0, 5, or 10 mg$kg1BW$day1on days 0e3, as described by Ryley and Peters. Data expressed as Mean ± SD of five
mice per condition.

Table 3
Antiplasmodial activity of amodiaquine and derivatives against P. berghei ANKA strain.

Compound code Mean ED50 ± SEa (mg/Kg B. W.) Percentage inhibition (on day 4)

0.1 mg/Kg 1 mg/Kg 5 mg/Kg 10 mg/Kg

1m 2.062 ± 0.433 21.63 32.027 54.19 79.13

1o 2.231 ± 0.502 11.985 21.68 58.073 93.363
2c 1.431 ± 0.353 25.653 45.253 58.79 82.70
2j 1.623 ± 0.381 8.413 36.73 67.597 97.357
Amodiaquine 1.187 ± 0.256 28.023 48.24 67.137 94.07
a
Standard error (n ¼ 3).

Elmer Fourier-transform infrared (FT-IR) Spectrophotometer product was crystallized by using ethanol and the data corresponds
(Spectrum 2000) in potassium bromide disk. ESI-MS spectra were to that reported in the literature [31].
obtained on a Waters micromass LCT Mass spectrometer. Elemental
analysis was done on Elementar GmbH VarioElanalyser. MS and 4.3. General procedure for synthesis of various 7-chloroquinolin-4-
MS/MS data were also recorded automatically on the MALDI-TOF/ yl-phenylamine (1ae1p)
TOF instrument. Spectra were recorded in the reflectron mode
using an Ultraflex III Tof/Tof mass spectrometer (Bruker Daltonics) Compounds were synthesized using 4, 7-dichloroquinoline and
equipped with a 384-sample scout source. The ion acceleration suitable substituted anilines. Briefly, equimolar quantities of ani-
voltage after pulsed extraction was 29,000 V. To record the spectra, lines (1 mmol) and 4, 7-dichloroquinoline (1 mmol) were refluxed
compounds were mixed with an acidic solid matrix such as a- in dry ethanol in basic medium (1.2 mmol K2CO3) for 2e16 h
cyano-4-hydroxy cinnamic acid (CHCA) matrix 10 mg/ml, which depending upon the various substituents. Progress of reaction was
provides high sensitivity and negligible matrix adduction during monitored by thin layer chromatography. After completion of re-
the laser absorption and subjected to laser radiation. The matrix action, reaction mixture was neutralized by 1 N NaOH solution and
was made in 70% acetonitrile and 0.03% TFA. extracted with 3  50 mL of CHCl3, washed with NaHCO3 and brine.
The organic layer was dried over anhydrous Na2SO4. The solvent
4.2. General procedure for the synthesis of N1-(7-chloro-quinolin- was again evaporated under reduced pressure. The ligands were
4-yl)-ethane-1, 2-diamine (2) purified by successive recrystallization [32].

A mixture of 4, 7-dichloroquinoline (1, 5.0 g, 0.025 mol) and 1,2- 4.3.1. Characterization data
ethylene diamine (5.8 g, 0.125 mol) was heated slowly from RT to
120  C and the reaction mixture was stirred at this temperature for Compound 1a: Yield ¼ 79%; mp ¼ 274e278  C; Ms: m/z 323
6 h (Series II). After that the reaction mixture was cooled down to [M þ 1] þ; 1H NMR (300 MHz, CDCl3, ppm): d 8.52 (d, 1H,
room temperature and ice cold water was added to it. The solid thus J ¼ 9.0 Hz, AreH), d 8.34 (d, 1H, J ¼ 6.9 Hz, AreH), d 8.02 (s, 1H,
obtained was filtered and washed with excess water. The crude AreH), d 7.35e7.47 (m, 4H, J ¼ 6.9 Hz, AreH þ NH), d 7.70 (d, 1H,
 S. Singh et al. / European Journal of Medicinal Chemistry 122 (2016) 394e407 401

Fig. 5. Kaplan Meier survival analysis curves of BALB/c mice, administered drugs once daily ip for four consecutive days, as described by Ryley and Peters (5 mice per group). Results
between test and control were significant by P < 0.005 as analyzed by Log-rank test. *Activity in 5 mg/kg BW treated groups is being superimposed by the activity in 10 mg/kg BW
treated groups.

Table 4 J ¼ 7.8 Hz, AreH) 13C NMR (300 MHz, CDCl3 ppm): d 151.97,
Docking energies of the 4-aminoquinoline analogues with dimeric 149.51, 148.83, 136.65, 135.11, 134.52, 134.12, 132.16, 128.96,
hematin. 127.93, 126.29, 125.78, 121.09, 117.42, 101.69, 20.99, 17.68; Anal.
Compound H bond energy (Kcal/mol1) Calcd. for C17H15ClN2: C, 72.35, H, 5.22; N, 9.93. Found: C, 71.68,
1m 6.01
H, 6.20; N, 10.23; IR (KBr, cm1): 3184.15, 2870.07, 1653.93,
1o 5.98 1450.62, 1329.59.
2c 5.81 Compound 1c: Yield ¼ 81%; mp ¼ 298e300  C; Ms: m/z 307
2j 5.13 [M þ 1] þ; 1H NMR (300 MHz, DMSO-d6, ppm): d 8.85 (d, H,
J ¼ 8.4 Hz, AreH), d 8.19 (s, 1 H, AreH), d 8.56 (d, 1H, J ¼ 5.1 Hz,
AreH), d 7.79 (s, 1H, AreH), d 6.83 (d, 1H, J ¼ 6.6 Hz, AreH),
J ¼ 4.8 Hz, AreH), d 7.47 (s, 1H, NH), d 7.41 (s, 1H, AreH), d 7.35 d 7.54e7.64 (m, 2H, AreH), d 5.89 (s, br, 1H, NH); 13C NMR
(1H, AreH), d 6.91 (d, 1H, J ¼ 4.9 Hz, AreH); 13C NMR (300 MHz, (300 MHz, DMSO-d6, ppm): d 155.00, 143.52, 138.99, 138.45,
CDCl3, ppm): d 168.54, 159.40, 150.91, 143.53, 138.57, 130.87, 134.34, 127.85, 127.45, 126.60, 126.5, 126.35, 120.61, 120.36,
129.59, 128.30, 126.76, 122.83, 122.79, 119.65, 118.91, 114.75, 119.17, 117.92, 100.64; Anal. Calcd. for C15H9Cl2FN2: C, 58.95; H,
114.53; Anal. Calcd. for C15H9Cl3N2: C, 54.98, H, 2.78; N, 5.15. 2.66; N, 9.19. Found: C, 60.10; H, 2.32; N, 7.89; IR (KBr, cm1):
Found: C, 55.20; H, 2.52; N, 6.68; IR (KBr, cm1): 3448.63, 3115.37, 3052.15, 2879.07, 2585.09, 1420.19, 1231.56.
2593.15, 1608.33, 1369.54, 1207.25, 1093.78. Compound 1d: Yield ¼ 58%; mp > 310e313  C; Ms: m/z 283
Compound 1b: Yield ¼ 62%; mp ¼ 308e312  C; Ms: m/z 283 [M þ 1] þ; 1H NMR (300 MHz, CD3OD, ppm): d 2.35 (s, 6H, CH3),
[M þ 1]þ; 1H NMR (300 MHz, CDCl3 ppm): d 3.15 (s, 3H, CH3), d 8.85 (d, 1H, J ¼ 9.03 Hz, AreH), d 8.31 (s, 1H, AreH), d 7.67 (d,
d 3.18 (s, 3H, CH3), d 6.33 (d, 1H, J ¼ 5.1 Hz, AreH), d 6.47 (s, 1H, 1H, J ¼ 8.5 Hz, AreH), d 7.50 (d, 1H, J ¼ 7.7 Hz, AreH), d 7.12 (d,
NH), d 8.45 (d, 1H, J ¼ 5.1 Hz, AreH), d 8.01 (d, 1H, J ¼ 1.5 Hz, 1H, J ¼ 3.1 Hz, AreH), d 6.20 (s, 1H, NH), d 6.5 (d, 1H, J ¼ 4.9 Hz,
AreH), d 7.14 (d, 1H, J ¼ 7.5 Hz, AreH),,d 7.86 (d, 1H, J ¼ 9.0 Hz, AreH), d 6.7 (d, 1H, J ¼ 5.4 Hz, AreH); 13C NMR (300 MHz,
AreH), d 7.45 (d, 1H3, J ¼ 3.0 Hz), d 7.14 (2H, AreH), d 7.07 (d, 1H,
402 S. Singh et al. / European Journal of Medicinal Chemistry 122 (2016) 394e407

Fig. 6. Compounds docked in dimeric hematin, illustrating the formation of complexes to inhibit hemozoin polymerization. (A) 1 m, (B) 1o, (C) 2c, (D) 2j.

Table 5
Docking results of the 4-aminoquinoline analogues in the active site of PfLDH.

Compound H bond energy (Kcal/ Residues (H bond Residues (Vander waals/other weak interactions)
mol1) interactions)

1m 6.21 Gly99 Asp53, Ile54, Tyr85, Ala98, Lys118, Ile119, Glu122, Ile123
1o 6.5 Asp53 Gly27, Ser28, Gly29, Met30, Ile31, Val36, Asp53, Ile54, Thr97, Ala98, Gly99, Phe100, Thr101,Ile119,
Glu122
2c 7.85 Val55, Tyr85 Gly99, Glu122, Lys118, Ile123, Gly32, Thr101, Phe52.
2j 5.13 Gly99, Thr97 Gly27, Gly29,Ile31, Ile54,Thr97, Ala98, Gly99, Phe100, Thr101, Val138, Thr139, Asn140

CD3OD, ppm): d 153.02, 151.91, 149.82, 134.60, 133.10, 128.43, N, 9.23; IR (KBr, cm1): 3279.75, 2809.57, 2036.49, 1615.49,
124.56, 123.99, 127.32, 115.21, 110.48, 128.22, 117.24, 115.12, 1337.05, 1239.84, 1164.43.
103.01, 20.20, 12.44; Anal. Calcd. for C17H15ClN2: C, 72.21; H, Compound 1g: Yield ¼ 67%; mp > 298e302  C; Ms: m/z 333
5.35; N, 9.91. Found: C, 70.90; H, 5.63; N, 10.10; IR (KBr, cm1): [M þ 1] þ; 1H NMR (300 MHz, DMSO-d6, ppm): d 8.85 (d,
3180.15, 3004.73, 1540.87, 1231.56, 1056.85. 1H,J ¼ 9.03 Hz, AreH), d 8.31 (s, 1H, AreH), d 7.67 (d, 1H,
Compound 1e: Yield ¼ 85%; mp ¼ 313e317  C; Ms: m/z 345 J ¼ 4.3 Hz, AreH), d 7.50 (d, 1H, J ¼ 5.7 Hz, AreH), d 7.74e7.65 (m,
[M þ 1] þ; 1H NMR (400 MHz, DMSO-d6, ppm): d 3.70 (s, 3H, 4H, J ¼ 25.2 Hz, AreH), d 6.41 (s, 1H, NH); 13C NMR (300 MHz,
OCH3), d 3.78 (s, 6H, OCH3), d 6.06 (s, br, 1H, NH), d 6.81 (s, 2H, DMSO-d6, ppm): d 158.12, 151.45, 149.81, 134.62, 133.02, 128.13,
AreH), d 6.86 (d, 1H, J ¼ 5.1 Hz), d 7.86 (d, 1H, J ¼ 6.7 Hz, AreH), 126.75, 123.93, 127.33, 115.20, 110.47, 128.27, 117.23, 115.24,
d 8.14 (s, 1H, AreH), d 8.48 (d, 1H, J ¼ 5.1 Hz, AreH), d 8.78 (d, 1H, 103.13; Anal. Calcd. for C15H10BrClN2: C, 54.00; H, 3.02; N, 23.95.
J ¼ 6.9 Hz, AreH). 13C NMR (400 MHz, DMSO-d6, ppm): d 155.10, Found: C, 55.12, 2.86; N, 22.41; IR (KBr, cm1): 3185.38, 2880.13,
153.68, 143.37, 139.02, 138.38, 136.63, 132.56, 127.38, 125.94, 2845.73, 1660.02, 1551.02, 1329.89, 1291.91.
119.25, 115.75, 103.21, 100.87, 60.19, 56.14; Anal. Calcd. for Compound 1h: Yield ¼ 87%; mp ¼ 317e319  C; Ms: m/z 273
C18H17ClN2O3: C, 62.28; H, 4.12; N, 8.70. Found: C, 63.24, 3.70, [M þ 1]þ; 1H NMR (400 MHz, DMSO-d6, ppm): d 8.91 (d, 1H,
9.21; IR (KBr, cm1): 3006.19, 2901.43, 1607.65, 1560.24, 1330.02, J ¼ 6.9 Hz, AreH), d 8.54 (d, 1H, J ¼ 5.1 Hz, AreH), d 8.20 (d, 1H,
1242.00. J ¼ 7.3 Hz, AreH), d 7.52 (d, 1H, J ¼ 5.7 Hz, AreH), d 7.40 (t, 1H,
Compound 1f: Yield ¼ 55%; mp ¼ 238e240  C; Ms: m/z 286 J ¼ 18.3 Hz, AreH), d 6.90 (d, 1H, J ¼ 5.1 Hz, AreH), d 6.87 (s, br,
[M þ 1] þ; 1H NMR (300 MHz, DMSO-d6, ppm): d 2.55 (s, 3H, 1H, NH); 13C NMR (400 MHz, DMSO-d6, ppm): d161.23, 154.48,
CH3), d 8.85 (d, 1H, J ¼ 8.01 Hz, AreH), d 8.31 (s, 1H, AreH), d 7.67 143.60, 139.16, 138.45, 131.72, 127.48, 126.31, 121.34, 119.24,
(d, 1H, J ¼ 5.3 Hz, AreH), d 7.50 (d, 1H, J ¼ 7.2 Hz, AreH), d 6.55 (d, 116.12, 114.43, 112.89, 112.12, 100.76; Anal. Calcd. for
2H, J ¼ 4.7, AreH), d 6.44 (s, 1H, NH), d 6.40 (d, 1H, J ¼ 7.1 Hz, C15H10ClFN2: C, 66.70; H, 3.06; N, 10.27. Found: C, 65.82; H, 3.83;
AreH); 13C NMR (300 MHz, DMSO-d6, ppm): d 153.11, 151.59, N, 10.41; IR (KBr, cm1): 3445.09, 3004.97, 1587.72, 1542.37,
149.81, 134.65, 133.01, 128.13, 126.77, 123.92, 127.30, 115.22, 1331.71, 1255.52.
110.74, 128.29, 117.23, 115.12, 103.18, 23.53; Anal. Calcd. for Compound 1i: Yield ¼ 85%; mp ¼ 281e284  C; Ms: m/z 283
C16H12ClFN2: C, 67.02; H, 9.77; N, 8.70. Found: C, 66.80; H, 10.14; [M þ 1]þ; 1H NMR (300 MHz, CDCl3 ppm): d 2.18 (s, 3H, CH3),
 S. Singh et al. / European Journal of Medicinal Chemistry 122 (2016) 394e407 403

Fig. 7. Best conformations of the amodiaquine derivatives in the binding pocket of NADH as generated by the Autodock software. (A) 1m, (B) 1o, (C) 2c, (D) 2j.

d 2.35 (s, 3H, CH3), d 8.29 (d, 1H, J ¼ 5.4 Hz, AreH), d 8.14 (d, 1H, d 155.44, 143.62, 138.0, 138.57, 133.83, 133.26, 132.36, 131.12,
J ¼ 8.7 Hz, AreH), d 7.91 (s, 1H, AreH), d 7.45 (d, 1H, J ¼ 18.9 Hz, 130.41, 129.27, 127.83, 126.40, 19.41,115.70,100.77; Anal. Calcd.
AreH), d 7.06 (s, 1H, AreH), d 7.06e7.09 (d, 3H, AreH þ 1H, NH), (%) for C15H9ClF2N2: C, 61.88; H, 3.22; N, 9.07. Found: C, 60.43, H,
d 6.25 (d, 1H, J ¼ 5.4 Hz, AreH); 13C NMR (300 MHz, CDCl3, ppm): 4.12, N, 9.56; IR (KBr, cm1): 3006.54, 2886.07, 1605.65, 1512.52,
d 150.90, 150.83, 148.48, 137.33, 137.22, 135.99, 132.0, 131.44, 1451.13, 1330.22.
128.14, 127.68, 127.08, 126.03, 122.8, 117.65, 101.47, 20.91, 17.30; Compound 1m: Yield ¼ 87%; mp > 305  C; Ms: m/z 273 [Mþ
Anal. Calcd. for C17H15ClN2: C, 72.24; H, 5.60; N, 9.76. Found: C, 1]þ; 1H NMR (300 MHz, DMSO-d6, ppm): d 4.66 (s, 1H, NH),
72.41; H, 5.28; N, 10.21; IR (KBr, cm1): 348.63, 2593.15, 1578.07, d 8.87 (d, 1H, J ¼ 9.0 Hz), d 8.61 (d, 1H, J ¼ 7.3 Hz), d 8.20 (s, 1H,
1445.69, 1207.25, 1093.78. H8), d 7.84 (d, 1H,H6 J ¼ 8.7 Hz), d 7.34e7.62 (3H, AreH þ 1H,
Compound 1j: Yield ¼ 82%; mp ¼ 166e168  C; Ms: m/z 283 NH), d 7.03 (d, 1H, J ¼ 6.9 Hz, AreH); 13C NMR (300 MHz, CD3OD,
[M þ 1] þ; 1H NMR (300 MHz, DMSO-d6, ppm): d 2.30 (s, 6H, ppm): d 157.25,143.55, 141.38, 141.29, 140.28, 137.51, 130.80,
CH3), d 8.46 (s, 1H, AreH), d 7.83e7.90 (m, 3H, J ¼ 22.5, Are- 129.05, 126.09, 123.91, 120.23, 117.01, 101.51; Anal. Calcd. (%) for
H þ 1H, NH), d 7.71 (d, 2H, J ¼ 8.4 Hz, AreH), d 7.04e7.14 (m, 2H, C15H10ClFN2: C, 66.75; H, 3.06; N, 10.22. Found: C, 66.43; H, 4.22;
AreH), d 6.82 (d, 1H, J ¼ 9.6 Hz, AreH), 13C NMR (300 MHz, N, 10.41; IR (KBr, cm1): 3184.15, 3115.37, 2649.47, 1585.09,
DMSO-d6, ppm): d 158.42, 149.96, 136.98, 135.41, 132.27, 131.83, 1540.87, 1291.49.
131.2, 131.0, 130.31, 129.99, 129.03, 127.34, 126.01, 124.8, 115.5, Compound 1n: Yield ¼ 82%; mp ¼ 314e317  C; Ms: m/z 283
19.72, 13.48; Anal. Calcd. for C17H15ClN2: C, 72.44; H, 5.65; N, [Mþ 1]þ; 1H NMR (300 MHz, CDCl3, ppm): d 2.28 (s, 6H, CH3),
9.96. Found: C, 72.41; H, 5.13; N, 10.20; IR (KBr, cm1): 3279.75, d 8.51 (d, 1H,J ¼ 8.7 Hz, AreH), d 8.16 (d, 1H, J ¼ 6.9 Hz, AreH),
2036.49, 1587.58, 1497.98, 1367.03, 1239.84. d 7.92 (s, 1H, AreH), d 7.54 (s, 1H, AreH), d 7.12 (s, 1H, NH), d 6.73
Compound 1k: Yield ¼ 61%; mp > 281e283  C; Ms: m/z 323 (d, 1H, J ¼ 6.9 Hz, AreH); 13C NMR (300 MHz, CDCl3, ppm):
[M þ 1] þ; 1H NMR (300 MHz, DMSO-d6, ppm): d 8.90 (d, H, d 155.66, 141.44, 139.96, 138.61, 138.33, 136.44, 133.46, 130.59,
J ¼ 9.0 Hz, AreH), d 8.58 (d, 1H,J ¼ 4.0 Hz, AreH), d 8.24 (d, 1H, 127.63, 125.73, 124.54, 122.13, 118.73, 115.31, 99.76, 18.87, 18.48;
J ¼ 3.0 Hz, AreH), d 7.78e7.81 (m, 3H, J ¼ 8.4 Hz, AreH þ 1H, Anal. Calcd. (%) for C17H15ClN2: C, 72.20; H, 5.45; N, 9.82. Found:
NH),d 6.41 (d, 1H, J ¼ 4.0 Hz, AreH); 13C NMR (300 MHz, DMSO- C, 73.12; H, 4.89; N, 8.12; IR (KBr, cm1): 3370.21, 2834.24,
d6, ppm): d 155.31, 144.42, 139.79, 138.92, 136.08, 133.22, 132.63, 1612.59, 1408.51, 1365.66, 1229.94.
130.5, 130.45, 129.96, 128.19, 126.58, 120.15, 116.19.101.72; Anal. Compound 1o:Yield ¼ 75%; mp ¼ 195  C; Ms: m/z 314 [Mþ]; 1H
Calcd. for C15H9Cl3N2: C, 55.77; H, 2.70; N, 8.56. Found: C, 56.20; NMR (300 MHz, DMSO-d6, ppm): d 3.74 (s, 6H, OCH3), d 8.86 (d,
H, 3.12; N, 7.58.; IR (KBr, cm1): 3006.19, 2676.07, 1607.65, 1H,J ¼ 9.0 Hz, AreH), d 8.48 (d, 1H, J ¼ 6.9 Hz, AreH), d 8.21 (s,
1542.56, 1459.16, 1330.02. 1H, AreH), d 7.84 (d, 1H, J ¼ 9.0 Hz, AreH)), d 7.04 (s, 1H, NH),
Compound 1l: Yield ¼ 75%; mp ¼ 210e213  C; Ms: m/z 291 7.07 (s, 1H, AreH), d 6.35 (d, 1H, J ¼ 6.9 Hz, AreH), 13C NMR
[Mþ], 1H NMR (300 MHz, DMSO-d6, ppm): d 9.03 (d, 1H, H2, (300 MHz, DMSO-d6, ppm): d 155.19, 153.45, 148.26, 142.85,
J ¼ 9.0 Hz), d 8.62 (d, 1H, J ¼ 9.0 Hz), d 8.26 (s, 1H, AreH), d 7.86 138.92, 138.25, 127.28, 126.07, 125.19, 119.22, 115.48, 114.39,
(d, 1H,J ¼ 8.7 Hz, AreH), d 7.67e7.53 (q, 2H, J ¼ 22.4 Hz, AreH), 113.9, 113.69, 101.078, 56.08, 55.67; Anal. Calcd. for C17H15N2O2:
d 7.32e7.37 (1H, NH); 13C NMR (300 MHz, DMSO-d6, ppm):
404 S. Singh et al. / European Journal of Medicinal Chemistry 122 (2016) 394e407

C, 64.77; H, 4.90; N, 8.70. Found: C, 65.13; H, 5.20; N, 7.75; IR d 3.61 (d, 2H, CH2, J ¼ 4.5 Hz), d 3.87 (d, 2H, CH2, 3.9 Hz), d 8.51
(KBr, cm1): 3370.21, 3057.92, 1612.59, 1560.07, 1365.66, 1313.57. (br, s, 1 H, NH), d 8.38 (d, 1H, J ¼ 4.5, Hz AreH), d 8.22 (d,
Compound 1p: Yield ¼ 71%; mp ¼ 277e279  C; Ms: m/z 306 J ¼ 7.5 Hz, 1H, AreH), d 7.76 (d, 1H, J ¼ 2.7 Hz, AreH), d 7.44 (d,
[Mþ]; 1H NMR (300 MHz, CDCl3, ppm): d 9.52 (d, 1H, J ¼ 8.7 Hz, 1H, AreH), d 6.84 (d, 2H, AreH þ NH), d 6.59 (d, 1H,J ¼ 4.2 Hz,
AreH), d 9.17 (d, 1H, J ¼ 7.2 Hz,AreH), d 8.93 (s, 1H, AreH), d 8.65 AreH); 13C NMR (400 MHz, CDCl3, ppm): d 168.54, 159.40,
(d, 1H, J ¼ 9.3 Hz, AreH), d 8.56 (s, 1H, AreH), d 8.25 (d, 1H3, 151.85, 149.84, 148.89, 136.67, 130.9, 129.54, 128.50, 128.17,
J ¼ 7.5 Hz, AreH), d 8.07e8.13 (m, 3H,J ¼ 1.5 Hz, AreH þ 1H, NH), 125.30, 121.24, 99.17, 50.23, 46.63, 41.82; Anal. Calcd. for
d 7.75 (d, 1H, AreH); 13C NMR (300 MHz, CDCl3, ppm): d 155.29, C18H17BrClN3: C, 55.33; H, 4.39; N, 10.07. Found: C, 56.35; H,
143.64, 138.96, 138.67, 133.83, 133.26, 132.36, 131.12, 130.41, 4.30; N, 11.20; IR (KBr, cm1): 3236.19, 2903.43, 1617.65, 1566.24,
129.27, 127.83, 126.14, 119.41, 115.61, 101.03; Anal. Calcd. for 1330.02, 1243.00.
C15H9Cl2FN2: C, 58.95; H, 2.66; N, 9.19. Found: C, 57.30; H, 3.32; Compound 2d: Yield ¼ 67%; mp ¼ 148e149  C; Ms: m/z 329.11
N, 10.41; IR (KBr, cm1): 3279.75, 2809.57, 2036.49, 1615.49, [Mþ]; 1H NMR (400 MHz, DMSO-d6, ppm): d 2.62 (s, 2H, CH2),
1539.97, 1337.05. d 3.83 (d, 2H, J ¼ 4.6 Hz, CH2), d 3.83 (d, 2H, J ¼ 3.7 Hz,CH2), d 9.21
(s, 1H, NH), d 8.89 (d, 1H, J ¼ 8.0 Hz, AreH), d 8.34 (s, 1H, NH),
4.4. General procedure for synthesis of various N-benzyl-N0 -(7- d 7.66 (d, 1H, J ¼ 5.3 Hz, AreH), d 7.35 (d, 1H, J ¼ 5.9 Hz, AreH),
chloroquinolin-4-yl)-ethane-1, 2-diamine (2ae2l) d 6.31 (d, 1H, J ¼ 4.2 Hz, AreH), d 6.55 (d, 1H, J ¼ 6.2 Hz, AreH);
13
C NMR (400 MHz, CDCl3, ppm): d 160.71, 158.52, 151.20, 149.23,
In a round bottom flask, added N1-(7-chloroquinolin-4-yl)- 147.34, 144.15, 136.21, 133.22, 131.10, 130.82, 129.20, 121.45,
ethane-1, 2-diamine (13e15 mmol) and aldehydes (10 mmol) un- 112.20, 103.42, 59.10, 48.23, 35.51; Anal. Calcd. for C18H17FClN3:
der nitrogen atmosphere in dry acetonitrile (30e40 mL). Allow the C, 66.55; H, 5.20; N, 12.74. Found: C, 65.83; H, 5.58; N, 13.20. IR
reaction mixture at stirring for 2e4 h at room temperature. Prog- (KBr, cm1): 3162.27, 1620.88, 1467.01, 1381.13, 1292.77.
ress of reaction was monitored by thin layer chromatography. Compound 2e: Yield ¼ 50%; mp ¼ 227e232  C; Ms: m/z 345.08
When the spot of aldehyde and amine get disappeared in TLC, [Mþ]; 1H NMR (400 MHz, CDCl3, ppm): d 2.62 (s, 2H, CH2), d 3.25
15e17 mmol of sodium triacetoxyborohydride in dry CH3CN (5 mL) (d, 2H, J ¼ 3.6,CH2), d 3.80 (d, 2H, J ¼ 4.1,CH2), d 9.31 (s, 1H, NH),
or dry DCM (5 mL) (depending on the aldehydes used) was added d 8.82 (d, 1H, J ¼ 8.1 Hz, AreH), d 8.33 (s, 1H, AreH), d 7.35 (d, 1H,
into the reaction mixture under a nitrogen atmosphere. The reac- J ¼ 5.1 Hz, AreH), d 6.32 (d, 1H, J ¼ 4.2 Hz, AreH), d 6.56 (s, 1H,
tion mixture was left for 18e23 h on constant starring at room J ¼ 3.2 Hz, NH); 13C NMR (400 MHz, CDCl3, ppm): d 151.58,
temperature and progress of reaction was monitored by TLC. Imine 149.10, 147.45, 144.92, 136.18, 134.62, 132.24, 131.10, 130.81,
formation, however, is a reversible reaction and requires long re- 129.22, 121.32, 112.21, 103.14, 48.72, 36.92, 32.51; Anal. Calcd. for
action times and the use of a dehydrating agent such as molecular C18H17Cl2N3: C, 62.44; H, 4.95; N, 11.14. Found: C, 63.24; H, 5.18;
sieves can be used for the reaction to completion. After completion N, 10.15; IR (KBr, cm1): 3143.27, 1667.81, 1462.01, 1392.23,
of the reaction, the reaction mixture was extracted with ether 1207.12.
(3  10 mL). The combined ether extracts were concentrated under Compound 2f: Yield ¼ 88%; mp ¼ 219e221  C; Ms: m/z 336
reduced pressure. The crude product thus obtained was purified by [Mþ]; 1H NMR (400 MHz, DMSO-d6, ppm): d 2.44 (s, 2H, CH2),
column chromatography (silica gel 60e120 mesh) using d 3.53 (d, 2H, J ¼ 4.5 Hz, CH2), d 3.76 (d, 2H, J ¼ 4.8 Hz, CH2),
ethylacetate-hexane as eluent [33,34]. d 8.38 (d, 1H, J ¼ 4.9 Hz, AreH), d 8.22 (d, 1H, J ¼ 6.6, AreH),
d 8.10 (s, 1H, J ¼ 6.6, AreH), d 7.76 (d, 1H, J ¼ 1.5 Hz, AreH),
4.4.1. Characterization data d 7.43e7.41 (m, 2H, J ¼ 8.1, AreH), d 7.17 (d, 1H, J ¼ 1.2, AreH),
d 6.92 (d, 1H, AreH), d 6.33 (s,br, 1H, NH), d 5.28 (s, br, 1H, NH);
13
Compound 2a: Yield ¼ 62%; Brown oily compound; Ms: m/z 341 C NMR (400 MHz, CDCl3, ppm): d160.91, 150.20, 149.49, 142.35,
[Mþ]; 1H NMR (300 MHz, CD3OD, ppm): d 2.62 (s, 2H, CH2), 138.55, 137.26, 137.15, 134.48, 130.19, 122.23, 118.05, 110.1, 106.19,
d 3.65 (d, 2H, J ¼ 3.3 Hz, CH2), d 3.70 (s, 3H, OCH3), d 3.81 (d, 2H, 105.22, 48.77, 30.92, 24.76; Anal. Calcd. (%) for C19H17ClN4: C,
J ¼ 5.2 Hz, CH2), d 8.87 (d, 1H, J ¼ 8.1 Hz, AreH), d 8.24 (s, 1H, 67.75; H, 5.09; N, 16.53. Found: C, 66.89; H, 5.15; N,17.30; IR (KBr,
AreH), d 7.36 (d, 1H, J ¼ 7.3 Hz, AreH), d 6.33 (d, 1H, J ¼ 7.1 Hz, cm1): 3108.27, 1621.24, 1462.01, 1341.20, 1307.12.
AreH), d 6.54e6.88 (m, J ¼ 10.2 Hz, 1H, NH þ 2H, AreH), Compound 2g: Yield ¼ 62%; mp > 133e136  C; Ms: m/z 345
d 7.16e6.94 (m, 3H, J ¼ 20.4,AreH þ1H, NH); 13C NMR (300 MHz, [M þ 1]; 1H NMR (300 MHz, DMSO-d6, ppm): d 3.18 (d, 2H,
CD3OD, ppm): d 158.42, 150.21, 149.59, 143.10, 141.47, 136.18, J ¼ 4.9, CH2), d 3.60 (d, 2H, J ¼ 4.5 Hz, CH2), d 3.18 (s, 2H, CH2),
134.83, 130.52, 129.21, 121.15, 120.82, 119.22, 111.10, 110.29, d 8.39 (d, 1H, J ¼ 4.2 Hz), d 8.33 (s, 1H, NH), d 7.77 (s, 1H, NH),
103.41, 62.40, 58.71, 44.72, 38.14; Anal. Calcd. for C19H20ClN3O: d 6.58 (d, 1H, J ¼ 4.5 Hz, AreH); 13C NMR (400 MHz, CDCl3, ppm):
C, 68.56; H, 5.82; N, 15.49. Found. C: 68.95, H: 5.45, N: 15.15; IR d 168.66, 150.98, 144.60, 138.57, 134.55, 130.10, 129.94, 129.58,
(KBr, cm1): 3327.34, 2626.07, 1815.65, 1563.56, 1445.32, 128.37, 127.86, 126.75, 126.56, 125.97, 124.52, 119.67, 50.73,
1361.03. 46.86, 41.42; Anal. Calcd. for C18H17Cl2N3: C, 62.19; H, 4.98; N,
Compound 2b: Yield ¼ 86%; mp ¼ 158e161  C; Ms: m/z 380 12.34. Found: C, 62.04; H, 5.20; N, 11.74; IR (KBr, cm1): 3125.89,
[Mþ]; 1H NMR (400 MHz, DMSO-d6, ppm): d 3.31 (s, 2H, CH2), 1786.22, 1542.12, 1299.02.11, 1297.79.
d 3.60 (d, 2H, J ¼ 5.4 Hz, CH2), d 3.86 (d, 2H, J ¼ 5.7 Hz, CH2), Compound 2h: Yield ¼ 34%; mp ¼ 210e212  C; Ms: m/z 371; 1H
d 8.39 (d, 1H, J ¼ 5.4 Hz, AreH), d 8.34 (s, 1H, AreH), d 7.75 (t, 1H, NMR (400 MHz, CDCl3, ppm): d 3.20 (s, 2H, CH2), d 3.60 (s, 6 H,
J ¼ 6.9 Hz, AreH), d 7.51 (s, 1H, AreH), d 8.25 (s, 1H, NH), d 8.22 (s, OCH3), d 3.85 (d, 2H, J ¼ 3.2 Hz, CH2), d 4.12 (d, 2H, J ¼ 4.9 Hz,
1H, NH);13C NMR (300 MHz, DMSO-d6, ppm): d149.49, 142.35, CH2), d 8.87 (d, 1H, J ¼ 8.1 Hz, AreH), d 8.22 (s, 1H, AreH), d 7.75
138.35, 137,26, 137.15, 134.48, 130.19, 125.46, 122.23, 118.05, (s, 1H, NH), d 7.33 (d, 1H, J ¼ 7.1 Hz, AreH), d 6.53 (s, 1H, NH),
114.67, 110.18, 106.19, 105.22, 103.89, 55.43, 48.77, 46.30; Anal. d 6.19 (s, 1H, AreH), d 6.09 (d, 1H, J ¼ 5.8 Hz, AreH);13C NMR
Calcd. for C18H16Cl3N3: C, 68.89; H, 7.24; N, 15.14. Found: C, (400 MHz, CDCl3, ppm): d 163.4, 160.2, 151.5, 147.0, 144.7, 135.1,
68.72; H, 7.28; N, 15.21; IR (KBr, cm1): 3316.19, 2453.43, 133.1, 130.8, 130.2, 124.2, 121.9, 112.2, 103.4, 57.1, 48.7, 41.80,
1627.55, 1616.24, 1320.02, 1243.45. 35.52; Anal. Calcd. for C20H22ClN3O2: C, 64.56; H, 6.00; N, 11.30.
Compound 2c: Yield ¼ 71%; mp ¼ 147e150  C; Ms: m/z 390 Found: C, 64.20; H, 5.62; N, 12.14; IR (KBr, cm1): 3336.27,
[Mþ]; 1H NMR (400 MHz, DMSO-d6, ppm): d 2.69 (s, 2H, CH2), 1660.83, 1487.66, 1288.61,1207.12.
 S. Singh et al. / European Journal of Medicinal Chemistry 122 (2016) 394e407 405

Compound 2i: Yield ¼ 55%; mp ¼ 217e220  C; Ms: m/z 326 four-day suppressive test) [36,37]. Rodent malaria parasite Plas-
[Mþ]; 1H NMR (400 MHz, CDCl3, ppm): d 2.39 (s, 3H, CH3), d 3.34 modium berghei ANKA chloroquine resistant strain, obtained from
(s, 2H, J ¼ 3.34, CH2), d 3.65 (s, 2H, J ¼ 3.9, CH2), d 3.96 (t, 3 H, National Institute of Malaria Research (NIMR), Delhi was used for
J ¼ 7.8 Hz, CH2), d 5.51 (s, br, 1H, NH), d 8.30 (s, 1H, J ¼ 8.0 Hz, in vivo studies.
AreH), d 7.97 (d, 1H, J ¼ 1.5 Hz, AreH), d 7.66e7.69 (m, 1H,
J ¼ 9.9 Hz, AreH), d 6.93 (s,br, NH); 13C NMR (400 MHz, CDCl3, 4.6.1. Experimental animals
ppm): d144.69, 142.10, 141.07, 132.11, 131.04, 129.88129.68, Naïve Balb/C mice, males, 25 ± 5g (4e5 week old) free from
129.60, 128.45, 128.27, 120.70, 18.94, 113.90, 55.24, 41.21, 29.67, Eperythrozoon coccoides and Haemobartonella muris, were obtained
21.51; Anal. Calcd. for C19H20ClN3: C, 70.94; H, 6.18; N, 12.00. from the Animal Facility Centre, Department of Zoology, University
Found: C, 68.26; H, 5.58, N, 14.60; IR (KBr, cm1): 3466.57, of Delhi, Delhi, India. They were maintained on a commercial pellet
1710.88, 1432.01, 1324.87,1243.12. diet and housed under appropriate conditions (room temperature
Compound 2j: Yield ¼ 48%; mp ¼ 228e231  C; Ms: m/z 347 at 22 ± 2  C and 50e60% relative humidity, diet with p-amino-
[Mþ]; 1H NMR (400 MHz, CDCl3, ppm): d 3.32 (s, 2H, CH2), d 3.68 benzoic acid content of 45 mg/kg, and water ad libitum.). The study
(d, 2H, J ¼ 4.2 Hz, CH2), d 4.03 (d, 2H, J ¼ 3.9 Hz, CH2), d 8.77 (s, was conducted in accordance with the internationally accepted
1 H, NH), d 8.55 (d, 1H, J ¼ 4.5 Hz, AreH), d 7.95e8.02 (m, 3H, principles for laboratory animal use and care.
AreH), d 7.69 (s, 1 H, AreH), d 7.26 (s, 1 H, NH), 13C NMR
(400 MHz, CDCl3, ppm): d 161.52, 151.90, 149.62, 149.04, 142.04, 4.6.2. Drug solutions
134.91, 133.97, 130.78, 130.23, 128.64, 125.41, 121.04, 99.30, Each compound was made to strength of 2 mg/ml stock solution
59.07, 43.59, 30.89; Anal. Calcd. for C18H16ClF2N3: C, 62.64; H, in 10% DMSO and administered according to desired concentration
4.16; N, 12.08. Found: C, 63.14; H, 4.48; N, 11.20; IR (KBr, cm1): and individual body weight.
3006.54, 2886.07, 1605.65, 1512.52, 1451.13, 1330.22.
Compound 2k: Yield ¼ 41%; mp > 211e213  C; Ms: m/z 371.14 4.6.3. Test procedure
[Mþ]; 1H NMR (400 MHz, CDCl3, ppm): d 2.48 (s, 2H, CH2), d 2.85 On Day 0, heparinized blood was withdrawn from an infected
(d, 2H, J ¼ 7.2 Hz, CH2), d 3.45 (d, 2H, J ¼ 7.7, CH2), d 8.25 (d, 1 H, donor mouse with approximately 25e30% parasitemia, and diluted
J ¼ 6.6 Hz, AreH), d 8.01 (d, 1 H, J ¼ 6.3 Hz, AreH), d 7.64e7.72 in physiological saline to 108 parasitized erythrocytes per ml. An
(m, 5H, AreH þ 1H, NH), d 7.60 (s, 1H, NH), d 6.32 (d, 1H6, aliquot of 0.2 mL (¼2  107 parasitized erythrocytes) of this sus-
J ¼ 7.1 Hz), d 6.53 (d, 1H3, J ¼ 7.5 Hz), d 6.19 (s, 2 H, AreH), d 6.09 pension was injected intraperitoneally (i.p.) into experimental
(s, 1H, AreH). 13C NMR (400 MHz, CDCl3, ppm): d 189.09, 168.49, groups of 5 mice each. One to 3 h post-infection, the experimental
161.63, 150.96, 139.76, 138.51, 132.69, 129.54, 128.52, 127.28, groups were treated with varying doses of each of the test com-
127.10, 126.72, 119.56, 98.94, 60.80, 39.79, 30.90, 24.56; Anal. pounds (0.1, 1, 5, 10 mg/kg BW) by the ip route. Identical doses of
Calcd. (%) for C20H22ClN3O2: C, 61.53; H, 6.30; N, 14.36. Found: C, amodiaquine were administered to the standard drug group and
60.22; H, 5.98; N, 15.65; IR (KBr, cm1): 34548.63, 2673.43 0.2 mL of normal saline to the negative control group. On Days 1 to
1570.07, 1541.61, 1233.59, 1247.30. 3, i.e. 24 h, 48 h and 72 h post-infection, the experimental groups of
mice were treated again with the same dose and by the same route
4.5. In vitro susceptibility assay of P. falciparum as on day 0.24Hrs after the last treatment, on Day 4 (i.e. 96 hrs post-
infection), blood smears from the tail region of mice were made and
Antiplasmodial activity of compounds was determined against stained with Giemsa stain for microscopic study by counting 4
erythrocytic stages of the CQ (100 ng/mL), pyrimethamine (15 nM) fields of approximately 300e500 erythrocytes per slide, to assess
and cycloguanil resistant FCR3 strain of P. falciparum by a modified antimalarial efficacy of the test compound.
[3H]-hypoxanthine incorporation assay [35]. Briefly, all parasites Differences in parasitemia percentage between treated group
were cultured in 96-well microtitre plates and incubated at 37  C animals and untreated animals were analyzed by a one-way
under 2% O2, 5.5% CO2, 92.5% N2 atmosphere in RPMI 1640 (Invi- ANOVA test using IBM SPSS Statistics 16.0 and differences consid-
trogen) with 25 mM HEPES, 25 mM NaHCO3, 200 mM L-glutamine, ered significant if P < 0.05. Further, difference between the mean
50 mg/L gentamycin (Gibco), 5 g/L Albumax II (Life Technologies) value of the control group (taken as 100%) and those of the
and 20 mg/L tritium labeled hypoxanthine (Perkin Elmer) with a experimental groups is calculated and expressed as percent
final reactivity of 0.01 mCi. Parasite cultures were grown and syn- reduction (¼ activity) using the following equation:
chronized to ring stages and used at 1% parasitemia at 2% haema-
tocrit of blood group O erythrocytes. Cultures were exposed during Mean parasitemia treated Activity ¼ 100 e (Mean parasitemia/
48 h of incubation where after the plates were harvested onto a control) x 100
filter plate (Perkin Elmer) using a Filter Mate harvester (Perkin
Elmer). To each well 50 ml of Microscint 20 (Sigma) was added and In untreated control mice mortality was observed approxi-
the plate counted in a MXT top count (Perkin Elmer). Experiments mately one week after infection. Treated mice were observed for a
included untreated controls and serial compound dilutions period of 30 days, and the survival time (in days) was recorded. The
covering a range from 0.05 mg/ml to 2  106 mg/ml, tested in mean survival time was calculated in comparison to untreated
triplicate and in two independent assays. As a control for parasite (Normal saline) and standard drug (amodiaquine) treated groups.
inhibition we used artesunate (IC50 ¼ 0.91 mg/L± 0.17 mg/L). The IC50 Differences in survival time between treated groups and untreated
values were calculated from the dose-response curves using animals were analyzed by Log-rank (Mantel-Cox) test using
GraphPad Prism software. GraphPad Prism 5 and differences considered significant if
P < 0.005. Observations concerning adverse effects due to the
4.6. In vivo efficacy of amodiaquine derivatives against Plasmodium compounds were recorded.
berghei
4.7. Cytotoxicity against Huh-7 cells
Evaluation of the curative potential of the few selected amo-
diaquine derivatives was done using the method described by Ryley Cytotoxicity of the compounds was evaluated in human hepato-
and Peters, 1970 (rodent malaria four-day suppressive test; Peters’ cellular carcinoma cells (Huh-7) using MTT assay [38] and CC50
406 S. Singh et al. / European Journal of Medicinal Chemistry 122 (2016) 394e407

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