Synthesis, β-hematin inhibition studies and antimalarial evaluation of new dehydroxy isoquine derivatives against Plasmodium berghei: A promising antimalarial agent

Article abstract of DOI:10.1016/j.ejmech.2017.10.051

Many people are affected by Malaria around the world, and the parasite is developing resistance against available drugs. Currently, isoquine and N-tert-butyl isoquine are some of the most promising antimalarial candidates that have already reached Phase I

Full text of DOI:10.1016/j.ejmech.2017.10.051

Accepted Manuscript
Synthesis, β-hematin inhibition studies and antimalarial evaluation of new dehydroxy
isoquine derivatives against Plasmodium berghei: A promising antimalarial agent
Edgar A. Valverde, Angel H. Romero, María E. Acosta, Neira Gamboa, Genesis
Henriques, Juan R. Rodrigues, Carlos Ciangherotti, Simón E. López
PII:
S0223-5234(17)30853-X
10.1016/j.ejmech.2017.10.051
EJMECH 9843
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 August 2017
Revised Date: 14 October 2017
Accepted Date: 16 October 2017
Please cite this article as: E.A. Valverde, A.H. Romero, Marí.E. Acosta, N. Gamboa, G. Henriques, J.R.
Rodrigues, C. Ciangherotti, Simó.E. López, Synthesis, β-hematin inhibition studies and antimalarial
evaluation of new dehydroxy isoquine derivatives against Plasmodium berghei: A promising antimalarial
agent, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.10.051.
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ACCEPTED MANUSCRIPT

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1
ACCEPTED MANUSCRIPT
Synthesis, β-hematin inhibition studies and antimalarial evaluation of new
dehydroxy isoquine derivatives against Plasmodium berghei: A promising
antimalarial agent
Edgar A. Valverde,a Angel H. Romeroa*, María E. Acosta,b Neira Gamboa,b Genesis
Henriques,b Juan R. Rodrigues,b Carlos Ciangherotti,b Simón E. Lópeza*
aLaboratorio de Química Medicinal y Heterociclos, Departamento de Química, Universidad Simón Bolívar,
Valle de Sartenejas, Baruta, Caracas 1080-A, Apartado 89000, Venezuela.
bUnidad de Bioquímica, Facultad de Farmacia, Universidad Central de Venezuela, Aptdo. 47206, Los
Chaguaramos, 1041-A Caracas, Venezuela.
a*Corresponding authors: E-mail: angel.ucv.usb@gmail.com (AH Romero); slopez@usb.ve (SE López)
ABSTRACT: Many people are affected by Malaria around the world, and the parasite is developing resistance
against available drugs. Currently, isoquine and N-tert-butyl isoquine are some of the most promising
antimalarial candidates that have already reached Phase I and II clinical trials, respectively. Nevertheless,
pharmacodynamic studies have demonstrated that isoquine is highly sensitive to form O-glucuronide metabolite,
which may affect its accumulation in tissues. To avoid the O-glucuronide formation and its negative influence in
the accumulation process, a series of novel five dehydroxy isoquine derivatives were designed and prepared
herein as potential antimalarial agents. By a simple three-step procedure, five dehydroxy isoquines were
prepared and subsequently examined on the inhibition of haemozoin formation, the main target of the 4-
aminoquinolines. Four derivatives displayed significant inhibitory activities at low IC50 values from 1.66 to 1.86
µM comparable to CQ. On the basis of the results, these four compounds were subsequently tested against
Plasmodium berghei ANKA model in mice, showing to be as active as CQ with significant curative responses
and parasitemia suppresion in mice infected. On the other hand, these four compounds showed an acceptable
non specific cytotoxicity on murine peritoneal macrophague and human erythrocyte cells. Thus, the presented
data indicate that the dehydroxy isoquines 4b, 4c and 4e constitute promising cost-effective leads for the
development of new antiplasmodial targeted at blood-stage malaria parasites.
Keywords: Malaria, Isoquine, 4-aminoquinoline, antimalarial activity, Plasmodium berghei, Lipinski rule.
1. Introduction
Malaria is one of the largest tropical diseases caused by parasites of the genus
Plasmodium spp., which are transmitted to humans via the bites of an infected female
Anopheles mosquito. Plasmodium falciparum is the most virulent species in human
malaria parasite, responsible for about 1 million deaths every year [1-2]. According to
the World Health Organization (WHO) 2015 report, about 3.2 billion people are at risk
of malaria, particularly in poor or developing countries, registering 250 million clinical

2
cases of malaria and more
and pregnant women [3].
thanCE400,000 deaths annually, especially among children
AC PTED MANUSCRIPT
OH
N
N
HN
N
HN
N
Cl
Cl
1
2
N
N
H
HN
N
OH
HN
OH
Cl
Cl
N
4
3
N
R1
R2
OH
N
HN
N
HN
N
Cl
Cl
Cl
5
6
Figure 1. Structure of most important 4-aminoquinolines antimalarial derivatives 1-6.
Classically, chloroquine (CQ) 1 has been used widely as a standard antimalarial drug
for more than 60 years due to many advantage such as excellent availability, low
toxicity, effectiveness and facile preparation [1-4]. However, its efficacy has been
compromised by the increasing resistant P. falciparum in worldwide [5]. As a
consequence of the emergence of chloroquine resistance, alternative 4-aminoquinoline
such as amodiaquine (AQ) 2, isoquine (IQ) 3 and tebuquine (TBQ) 5 were subsequently
designed and prepared (see structures in Figure 1) [5a,6]. In particular, AQ is effective
against many chloroquine resistant strain of P. falciparum, although its clinical use has
been restricted by the hepatotoxic effect and agranulocytosis found in the treated patients
[7]. The AQ toxicities are mainly associated to the formation “in vivo” of a
quinoneimine reactive intermediate from oxidation of the phenolic ring. The oxidation of
AQ is facilitated by the para position of hydroxyl group on the aniline ring. Although
structure activity relationship (SAR) studies have demonstrated that the phenolic group
plays an important role in the antimalarial activity of AQ [8-14]. To prevent oxidation to
toxic metabolites in in vivo models, isoquine (IQ), that posses the hydroxyl group placed
at meta-position of aniline ring, was designed and evaluated as an alternative
antimalarial agent [6a,15]. IQ and derivatives have exhibited excellent in vitro and in

3
vivo antimalarial results, with good bioavailabilities. InIPcontrast to AQ, IQ can be
ACCEPTED MANUSCR T
excreted primarily by the formation of its O-glucuronide derivative (metabolism Phase
II), which has been found in bile and urine samples of treated host. This last may affect
the accumulation of drug in tissues. Currently, isoquine and N-tert-butyl isoquine 4 are
in phase I and II clinical trials [16a-c].
Other group of novel 4-aminoquinolines have been demonstrated excellent
antimalarial activity against resistant P. falciparum strains [16d-i]. Recently, as part of a
preliminary investigation to the present report, we designed and synthesized a series of
new dehydroxy isotebuquine derivatives 6 to prevent Phase II-glucuronidation and
clarify the relevance of the hydroxy group on the antimalarial response of isotebuquine
derivatives. These dehydroxy isotebuquines showed excellent in vitro and in vivo
antimalarial results comparable to standard CQ [17]. Then, in order to complement the
mentioned investigation [17], we propused herein the design, synthesis and antimalarial
evaluation of new low-cost dehydroxy isoquines 4a-e (see Scheme 1) as potential
antimalarial targeted at blood-stage malarie parasite. Unable to produce the O-
glucuronide, these new dehydroxy isoquines may exhibit similar or better antimalarial
activity than isoquine derivatives.
2. Results and discussion
A series of novel dehydroxy isoquines were designed herein as an alternative isoquine
analogue incapable of producing O-glucuronide metabolites. This type of metabolite
derived of hydroxylated compounds can affect the accumulation of the drug inside host.
It is important to mention that the absence of hydroxyl substitution in isoquine
derivatives may have profound effect on physicochemical properties such as lipid
solubility, water solubility, pKa and hydrogen bondings. The hydroxyl substitution has
demonstrated to play an important role on the antimalarial efficacy of AQ, where the
replacement of hydroxyl group by either fluorine or chlorine in AQ led a decrease in the
antimalarial response against Plasmodium falciparum strains [8,15]. The relevance of
the hydroxyl substitution in the antimalarial activity of isoquine derivatives has not been
investigated previously, which motivated the design, synthesis and antimalarial
evaluation of the new dehydroxy isoquine derivatives 4a-e.

4
2.1. Chemistry
ACCEPTED MANUSCRIPT
New dehydroxy isoquine derivatives 4a-e were synthesized employing a
straightforward and efficient protocol reported recently by our group [17], which is
depicted in Scheme 1. The synthetic sequence involves a facile three-step procedure
from low cost starting materials. Initially, 4-nitrobenzyl chloride 1 was reacted with
different secondary amines by a nucleophilic sustitution (SN2) to lead the corresponding
benzylamines 2a-e. These resulting derivatives 2a-e were subjected to a subsequent
reduction reaction using tin in hydrochloric acid, affording the desired anilines 3a-e in
good yields. Stage 3 of the sequence involves the heteroaromatic nucleophilic
substitution (SNAr) reaction of 4,7-dichloroquinolines at position 4 with the synthesized
anilines 3a-e under refluxing ethanol. The reaction requires a catalytic amount of
hydrochloric acid (HCl) to give the corresponding substituted 4-aminoquinolines 4a-e
in good 70-80% yields (see Table 1). Particularly, the synthesis of tert-butyl quinoline 4
required at least 0.4 eq. of HCl to complete the reaction. In this synthetic sequence, only
the anilines derivatives 3a-e and dehydroxy isoquines 4a-e were well purified and
characterized.
X
Cl
Cl
X
HN
X=
N
N
O
a
b
c
N
N
NH
Cl
N
NO2
2a-e
NO2
NH2
4a-e
3a-e
1
Scheme 1. Synthesis of novel dehydroxy isoquine derivatives 4a-e. Conditions: (a)
dialkylamine- or alkylamine (5 eq.), toluene, 90 ºC, 5 h; (b) Sn, HCl, 70ºC, 2 h. (c) 4,7-
dichloroquinoline (1.2 eq.), ethanol, HCl (cat.), reflux, 6 h.
Table 1. Yields for the last step of the synthesis of dehydroxy isoquine derivatives 4a-e.
Compounds
3a
Xa
Yield (%)
=
N
79
3b
3c
N
N
N
80
81
3d
3e
83
O
79b
NH
aThe molecular structure of 4a-e are shown in Scheme 1 and Supplementary material.
bThe tert-butyl substituted quinoline 4e was obtained as the monohydrochloride salt.

5
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2.2. Biological evaluations
In vitro studies on the haem polymerization. Initially, the ability of the five
synthesized dehydroxy isoquine 4a-e to inhibit β-hematin formation was assessed by
previously reported methods [17-18]. The percentage of inhibition of haem
polymerization (% IHP) and IC50 values for each compound are listed in Table 2. The β-
hematin was formed in vitro from hemin under acidic and low oxygen conditions,
simulating the natural conditions found in the food vacuole of the parasite. It is well
known that the inhibition of the β-hematin formation is the main mode of action of
diverse 4-amino-7-chloroquinolines such as CQ, AQ, IQ and TBQ [18-23]. The
formation of the β-hematin is a Plasmodium-specific defense mechanism in which the
toxic ferriprotoporphyrin IX (FPIX) is converted into an insoluble non-toxic crystalline
polymer [24-25]. It is not completely clear how 4-amino-7-chloroquinoline agents
inhibit the β-hematin formation in vivo; however, several evidences have demonstrated
that CQ and other 4-aminoquinolines interfere with the haem polymerization through a
π-π stacking interaction between the porphyrin ring of the hematin and the quinoline
ring, which facilitate an effective accumulation of the heme/drug complex within the
erythrocyte leading the parasite death by oxidative stress [26-30]. On these basis, the
inhibition of the β-hematin represents the main mode of action of our designed
dehydroxy isoquine 4a-e as antimalarial compounds.
In general, four of the five synthesized quinolines showed an excellent capability to
block the haem polymerization (IHP), exhibiting relatively low IC50 values from 1.66 to
1.86 µM (see Table 2). It should be noted that these four derivatives presented IC50
values against the IHP comparable to that obtained for standard chloroquine (1.48 µM).
Only derivative 4d bearing a morpholine group showed a marginal inhibition of the
haem polymerization with a minimum percentage of 5.17% at 50 µM. In addition, it is
important to mention that the capacity of the tested dehydroxy isoquine derivatives to
inhibit the haem polymerization is comparable to those obtained for diverse
hydroxylated 4-amino-7-chloroquinolines such as AQ, IQ or TBQ [18-20], which
reflects that this inhibition process is not affected by the absence of the hydroxyl
substitution on the aniline ring of the 4-amino7-chloroquinolines. On the other hand, the
inhibitory activity found for the dehydroxy isoquines are completely expected because
these derivatives keep some important pharmacophore such as 7-chloroquinoline

6
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scaffold, the terminal amine moiety and nonpolar linker between these two mentioned
fragments.
Table 2. Results of inhibition of haem polymerization (IHP) and toxicity evaluations for
compounds 4a-e.
% IHPa,b (± SD) IC50c (µM) ± SD LD50d (µM) ± SD LyticC50e (µM) ± SD
Entries
98.21 ± 1.52
97.98 ± 1.79
98.11 ± 2.14
5.17 ± 1.05
98.56 ± 2.13
98.57 ± 2.01
1.78 ± 0.13
1.86 ± 0.22
1.81 ± 0.23
>50.0
1.66 ± 0.11
1.48 ± 0.14
42.4 ± 2.3
52.3 ± 2.8
63.2 ± 3.4
36.2 ± 3.1
47.1 ± 2.5
134.5 ± 1.7
2924.1 ± 13.3
1405.7 ± 23.4
959.2 ± 24.8
591.3 ± 13.7
1485.4 ± 9.8
> 1500.0
4a
4b
4c
4d
4ef
CQg
aSD: Standard desviation; b %IHP: Percentage of inhibition of haem polymerization at 50 µM; c Inhibitory doses for the inhibition
of haem polymerization.dLethal dose of the tested compounds against murine peritoneal macrophague cells.e LyticC50, half-lytic
concentration on red blood cells of the tested compounds. f Monohydrochloride quinoline; gPositive control: CQ (chloroquine).
In vitro toxicity on murine peritoneal macrophage and human red blood cells
(RBCs). Initially, toxicity of the synthesized derivatives was determined using
peritoneal macrophages derived from murine model. Macrophages are resident cells
found in almost all tissues of the mammal bodies, where they assume specific
phenotypes and develop distinct functions. Tissue macrophages are considered as
immune sentinels because of their strategic localization and their ability to initiate and
modulate immune responses during pathogenic infection or tissue injury and to
contribute to the maintenance of tissue homeostasis [31]. LD50 was used as parameter to
evaluate the toxicity against murine peritoneal macrophages. In general, four of the five
tested dehydroxy isoquine derivatives showed an acceptable toxicity toward murine
peritoneal macrophages with LD50 values ranging from 40 to 63 µM [32]. In particular,
the derivatives 4b (52.3 µM) and 4c (63.2 µM) exhibited the lowest toxicity ranges
against peritoneal macrophagues, while the compound 4d (36.2 µM) was relatively the
most toxic among the tested quinolines. All synthesized compounds were relatively
more toxic than CQ against murine peritoneal macrophagues.
Secondly, cytotoxicity effect of these five derivatives was evaluated on human red
blood cells (RBCs). RBCs are crucially important because of these compounds act on
the blood-stage malaria parasites. To evaluate the in vitro toxicological effect of the
most active dehydroxyl isoquine derivative, we used a model based on the lysis of
RBCs, measuring the haemoglobin released in the supernatant fraction [33]. The release
of haemoglobin by an equal number of RBCs by hypotonic lysis in 0.05 volumes of

7
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water was used as a 100% positive control, while RBCs treated with saline solution
served as negative controls. Their toxicity results were expressed as the concentration at
which half of the RBCs lysed (LyticCC50). Generally speaking, all tested derivatives 4a-
e showed a significant cytotoxicity against human RBCs with high LyticC50 values from
591 to 2924 µM, indicating that the action of these compounds has a negligible effect on
the viability of the RBCs in in vivo models. This result is very promising because our
dehydroxy isoquines act on the blood stage of parasite. Similarly to toxicity against
macrophages, the compound 4d exhibited the highest toxicity among the tested
quinolines on RBCs. On the basis of inhibition of haem polymerization and toxicity
results presented in table 2, the compounds 4a, 4b, 4c and 4e were selected for further
studies in the mouse model of malaria.
In vivo antimalarial effect of the dehydroxy isoquines. In order to know the
therapeutic efficacy of the synthesized dehydroxy isoquines the best compounds 4a, 4b,
4c and 4e were evaluated in a standard “4-day test” in mice infected intravenously with
murine pathogen Plasmodium berghei ANKA, a chloroquine-susceptible strain. Mice
were treated with the compound (CQ or 4a-e, ip once daily) for four consecutive days
(days 0-4 post-infection). Then, parasitemias in peripheral blood were measured at day
fourth post-infection and the survival days were monitored for the treated mice. These
results on treated mice were compared with control mice receiving saline (untreated
mice) (see Table 3). In general, dehydroxy isoquines 4a, 4b, 4d and 4e have the ability
to increase significantly the survival time in more than 20 days post-infection, results
comparable to those obtained for CQ drug. It is important to emphasize that the
untreated mice died between days 6 and 7 post-infection, demonstrating the significant
in vivo efficacy of the four tested compounds. On other hand, the treated mice presented
a remarkable suppression of parasitemia with percentages from 2 to 4 % post-infection,
which are lower than those found for untreated mice of 63% post-infection. Thus, the
significant survival time of infected mice is associated to a curative response of the
compounds on the parasitemia (see Table 3). Finally, the satisfactory correlation
between the antimalarial in vivo and inhibition of β-hematin formation results indicates
that the significant antimalarial efficacy of our designed dihydroxy isoquines may be
associated mainly to the inhibition of haem polymerization within the erythrocytes.

8
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Table 3. The effect of dehydroxy isoquine derivatives on P.berghei infected mice.
Entries
Post-inf. days of survival (± SD)a
24.90 (2.95)
%P (± SD)b,c
3.80(1.20)
4a
27.70 (1.80)
28.20 (1.50)
25.80 (2.05)
29.40 (0.40)
2.50 (0.75)
2.20 (0.86)
4.00 (0.25)
0.60 (0.08)
65.20 (2.58)
4b
4c
4e
CQd,e
Untreated control
6.80 (0.70)
aResults are expressed as the media±standard desviation (SD). n=6 (number of treated mice); Standard desviation; b%P:
c
d
Percentage of parasitemia; P<0,05 comparing to control treated group; Positive control treated with cholorquine (25 mg kg-1);
eP<’0,01 comparing to control treated group.
In general, the nature of the alkylamine moiety seems to have an important influence
on the effectiveness of these classes of derivatives. Particularly, the pyrrolidine and
piperidine derivatives exhibited the best values of inhibition of haem polymerization,
parasitemia and survival days for infected mice with P. berghei among the compounds
tested, while the morpholine substituted quinoline was discarded as potential
antimalarial agent due to its poor inhibitory activity on the haem polymerization. The
effect of the alkylamine group on the antimalarial activity of the dehydroxy isoquines is
in good agreement with those reported for amodiaquine and isoquine derivatives [6].
The particular negative effect of morpholine moiety was also found for the dehydroxy
isotebuquine reported recently by our group [17]. Curiously, the less toxic compounds
4b, 4c and CQ against macrophague cells exhibited the best in vivo antimalarial effect
(compared Tables 2 and 3). Therefore, the new dehydroxy isoquines 4b and 4c
represent promising antimalarial candidates for further studies with a significant
curative antimalarial response and low toxicological effect.
The significant antimalarial activity found for the dehydroxy isoquine herein and
those previously reported for the dehydroxy isotebuquine derivatives [17], make us to
conclude that the hydroxyl group is not essential to lead a significant antimalarial
activity for the studied isoquine and isotebuquine compounds. In addition, the inclusion
of hydroxyl group in isoquine derivatives contributes to its fast elimination in an in vivo
model by excretion as glucuronide, which affect its effective concentration in animal
tissues. This fast elimination mechanism is not possible in our designed structures;
although additional pharmacodynamics, pharmacokinetic and metabolism assays are
necessary for our derivative to know the real positive effect of the absence of the
hydroxyl group. On the other hand, no appreciable differences in antimalarial activities
between the dehydroxy isoquine 4a-e and reported dehydroxy isotebuquine17 were

9
observed, by which the inclusion of additional phenyl ring on the aniline ring of
ACCEPTED MANUSCRIPT
dehydroxy isoquine seem to have a negligible effect in the biological response of these
interesting promising 4-aminoquinolines.
2.3. Molecular Docking studies
In order to know if the action of dehydroxy isoquines may affect other stages of the
erythrocytic cycle of the parasite, a molecular docking study was performed on a
protease involved in hemoglobin degradation such as the plasmepsins of Plasmodium
falciparum. In general, there are several proteases involved in the hemoglobin
degradation pathway such as plasmepsins (Plm) I, II and IV and HAP (a histo-aspartic
protease). These proteases are located in the acidic food vacuole of the parasite and are
essential for the survival and reproduction of the most virulent malaria parasite,
Plasmodium falciparum [34a]. In particular, the plasmepsins are aspartic proteases with
the catalytic aspartic residues situated in an active site cleft formed by two domains.
The catalytic mechanism of peptide bond cleavage in aspartic proteases is generally
considered to involve the activation of a water molecule by one of the aspartates
accompanied by nucleophilic attack of the water/hydroxide on the carbonyl carbon of
the substrate [34b]. Thus, taken into account the well known good acumulation of 4-
aminoquinolines in the acidic vacuole of the parasite, these type of proteases emerge as
highly promising drug targets for theraphies against malaria [35]. In this sense,
molecular docking of our five derivatives were carried out on Plm I and Plm II
enzymes, deposited in the Protein Data Bank website under codes 1LEE and 1LF2,
respectively. Molecular docking calculations were performed using the AMBER force
field, applied to the complete structure of both enzymes. The molecular docking results
of the tested compounds 4a-e, CQ and references drugs (R36 and R37) 1-5 are shown in
Table 4 and Figure 2.
The molecular docking results showed clearly that our dehydroxy isoquines exhibited
weaker affinity towards both Plm I and Plm II enzymes than the reference drugs, by
which seem, at least theoretically, that our dehydroxy isoquines are should not be good
inhibitors of these enzymes. On the other hand, a detailed analysis of ligand-enzyme
complex showed that reference drugs R36 and R37 are more extended along active site
than our dehydroxy isoquines (see Figure 2), which permits to facilitate the stabilization
of the ligand-enzyme complex and explain the remarkable binding affinity found for
R36 and R37 compared to our derivatives. Despite the exclusive theoretical nature of
this study, it is possible to conclude that a good inhibitor of the Plm I and Plm II

10
ACCEolPTED MANU R T
enzymes is represented by a m ecule containingSClongIP, flexible and coordinative
chains (see structure of R36 and R37). Then, based on molecular features, this mode of
action is practically discared for our synthesized compounds, and the significant
antimalarial response of our synthesized compounds is associated mainly to the
inhibition of haem polymerization. Ligand-enzyme interactions into active site of the
Plm I and PlmII enzymes are shown clearly in Supplementary material.
Table 4 shows the hydrogen bonding interactions exhibited by the five derivatives
with essential residues of active sites. In addition, all derivatives exhibited two π-π
interactions between quinoline ring of 4a-e with the phenyl ring of Tyr-77 and Phe-111
residues in the Plm I enzyme.
Figure 2. Representation of ligand-enzyme complex for the dehydroxy isoquines 4a-e
in the active site of the Plm I enzyme.

11
Table 4. Docking statistics
A
on
C
th
C
e
E
P
P
lmTE
I
D
an
M
d P
A
lmNUII
S
en
C
z
R
ymIP
e
T
s for the derivatives 4a-e.
Molecules
Plm I
Plm II
E (kcal/mol)
-10.751
H-interactionsc
No interactions
E(kcal/mol) H-interactionsd
-10.342
Asp-214 (3.00Aº) and Thr-
217 (2.75Aº)
Asp-34 (2.88Aº), Gly-36
(2.78Aº), Tyr-192 (3.00 Aº)
Ser-79 (2.53Aº)
4a
4b
Ser-79 (2.94 Aº)
-11.855
-11.485
Ser-79 (2.81 Aº)
Try-192 (2.53 Aº)
-10.674
-10.163
4c
4d
-11.287
-11.034
Ser-218 (2.34 Aº), Thr-217
(2.62 Aº)
-10.888
Asp-214 (3.00 Aº)
-10.996
Ser-79 (2.98 Aº), Gly-36
(3.00 Aº)
4e
-10.125
-14.137
Ser-79 (2.98 Aº)
-9.781
Ser-79 (2.52 Aº)
----------
CQ
R36a
Ser-79 (2.67 Aº), Ser- -----------
218 (2.64 Aº), Ala-219
(2.88 Aº).
R37b
---------
-------------
-13.452
Ser-79 (2.23 A º), Gly-216
(2.31 Aº), Thr-217 (2.03 Aº)
aReference drug for the Plm I enzyme (PDB code: 1LEE). Name: 4-amino-N-{4-[2-(2,6-dimethyl-phenoxy)-acetylamino]- 3-
hydroxy-1-isobutyl-5-phenyl-pentyl}-benzamide.
bReference drug for the Plm II enzyme (PDB code: 1LF2). Name: 3-amino-N-{4-[2-(2,6-dimethyl-phenoxy)-acetylamino]- 3-
hydroxy-1-isobutyl-5-phenyl-pentyl}-benzamide.
cH-interactions (in Aº) in Plm I is through the NH of aniline ring. 4d interact through oxygen of morpholine ring.
dH-interactions (in Aº) in Plm II is through the NH of aniline ring and the nitrogen of the dialkylamine moiety.
As last topic, in order to identify quinolines orally active, the five Lipinski´s
parameters such as partition coefficient (LogP), molacular weight (MW), number of H-
donors (H-D) and H-acceptors (H-A), were calculated for the tested dehydroxy
isoquines 4a-e (see Table 5). The rule of five is an important criteria for the design of
new orally active drug candidate. By applying the rule of five (RO5) and its variants,36
all our derivatives showed good drug-like properties, except to their Log P whose
values are near to the upper limit of lipophilicity. This fact is very important due to that
the absence of hydroxy moiety could compromise the bioavailability of these new
derivatives; however still it is required to make additional studies of bioavailability on
the most promising compounds.
Table 5. Calculation of Lipinski´s parameters for the dehydroxy isoquines 4a-e and CQ.a
Compounds Log P4.5 Log P7.4
MW
nON
nOHNH
nViol
<5
<5
<500
<10
3
3
3
4
<5
1
1
1
1
1
2
3
4
5
6
4.77
4.82
4.40
3.68
4.77
3.73
4.76
4.87
4.42
3.42
4.66
3.68
339.86
351.87
337.85
353.85
375.13
319.18
0
0
0
0
0
0
4a
4b
4c
4d
4eb
CQ
3
3
2
1
anViol, number of violations; MW, molecular weight; nON, number of hydrogen bond acceptors; nOHNH, number of hydrogen
bond donors. b Monohydrochloride quinoline.

12
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3. Conclusion
In summary, novel dehydroxyl-isoquines 4b, 4c and 4e emerged as excellent
candidates for further studies with significant activities on the inhibition of the haem
polymerization, satisfactory in vivo results and acceptable toxicity ranges. The
dehydroxy isoquines 4a-e were prepared in good yields using a simple three step
synthetic sequence from low-cost starting materials. The good correlation between the
antimalarial data and inhibition of the haem polymerization suggests that the inhibition
of the β-hematin is the main mechanism of action of these novel dehydroxyl isoquines.
A structure-activity relationship indicates that the 3-hydroxyl substitution on the
isoquine compound is not necessary to lead a potent antimalarial effect such as was
observed in our dehydroxyl isotebuquines. However, further in vitro and in vivo assays
on resistant strain of Plasmodium falciparum are required to evaluate the real potential
of the most active derivatives 4b, 4c and 4e.
4. Experimental section
4.1. Chemistry
All reagents and starting materials were purchased from commercial sources and used
without further purification; solvents were anhydrous HPLC grade. Melting points are
recorded with a micro melting point apparatus und uncorrected. 1H NMR and 13C NMR
spectra are recorded at JEOL Eclipse Plus 400 or JEOL Eclipse Plus 270. Multiplicity is
indicated as follows: s (singlet), d (doublet), t (triple), m (multiplet), dd (doublet of
doublets), br s (broad singlet); chemical shifts were measured in parts per million (δ)
and coupling constant (J) are given in Hz. Proton chemical shifts were given in relative
to tetramethylsilane (δ 0.00 ppm) in CDCl3 or CD3OD solvents. Carbon chemical shifts
are internally referenced to the deuterated solvent signals in CDCl3 (δ 77.00 ppm) or
CD3OD (δ 48.67 ppm). IR spectra were recorded on NICOLET Magna IR 760 using
KBr tablets. Elemental analyses of the synthesized compounds were performed using a
Perkin Elmer 2400 CHN analyser: results fell in the range of 0.4% of the required
theoretical values.
4.1.1. General procedure for the preparation of 4-(N,N-dialkylamine-1-
methyl)aniline 3a-e: These derivatives were prepared following a similar procedure
reported in literature [17]. To a solution of 1-(chloromethyl)-4-nitrobenzene 1 (2 mmol, 1
equiv.) in toluene was added slowly the corresponding dialkylamine or tert-butylamine

13
ACCixEP D MAN CR T
(10 mmol, 5 equiv.). The m tureTEwas stirredUSunderIPreflux for 4-5 hours and
monitored by TLC using solvent mixture Hex/AcOEt (7:3). The reaction mixture was
then cooled to room temperature, the solvent and the unreacted alkyl(dialkyl)amine was
evaporated under reduced pressure to give
a
yellow-white solid of 1-
1
(alkyl(dialkyl)amine-methyl)-4-nitrobenzenes 2a-e (see H-NMR and 13C-NMR data in
supplementary data from page 8 to page 23), which was pure enough to proceed with the
oxidation step. These 1-(dialkylaminemethyl)-4-nitrobenzene 2a-e (2 mmol, 1 equiv.)
were treated with a solution of hydrochloric acid (20 mmol) and tin powder (6 mmol, 3
equiv.) under reflux for 2 hours. The reaction mixture was then cooled to room
temperature and neutralized carefully with sodium hydroxide solution (aq. 10%) and
extracted with dichloromethane (3x20 mL). The organic lawyer was washed with
distilled water, dried using anh. MgSO4, filtered and the solvent evaporated under
reduced pressure to give the aniline product, which was purified by flash chromatography
column using as eluent dichloromethane: methanol (9/1) to give a yellow-orange
compound 3a-e.
4.1.1.1. 4-(diethylamino)methyl-aniline 3a. Orange oil, yield 72%. FT-IR (KBr, ν cm-
1
1): 3354 (st. N-H), 3158 (st. N-H), 2934-2890 (st. C-H), 1608-1522 (st. C=C, Ar). H-
NMR (400 MHz, CDCl3): δ 7.09 (dd, 2H, J=8.40; J=2.52); 6.62 (dd, 2H, J=8.20;
J=1.80); 3.61 (s, 2H, NH2); 3.46 (s, 2H, CH2-N); 2.49 (q, 4H, CH2-N); 1.03 (t, 6H,
CH3). 13C-NMR (100 MHz, CDCl3): δ 145.2; 130.3 (2C); 129.2; 115.0 (2C); 56.8 (CH2-
N); 46.4 (2C, CH2-N); 11.6 (2C, CH3). DEPT-135 (100 MHz, CDCl3): 130.3 (2C);
115.0 (2C); 56.8 (1C, CH2-N); 46.4 (2C, CH2-N); 11.6 (2C, CH3). Anal. Calc. for
C11H18N2: C, 74.11; H, 10.18; N, 15.71. Found: C, 74.03; H, 10.12; N, 15.58.
4.1.1.2. 4-(piperidino-1-ylmethyl)-aniline 3b. Orange oil, yield 78%. FT-IR (KBr, ν
cm-1): 3332 (st. N-H), 3142 (st. N-H), 2934-2890 (st. C-H), 1601-1526 (st. C=C, Ar).
1H-NMR (400 MHz, CDCl3): δ 7.07 (dd, 2H, J=8.44; J=2.56); 6.62 (dd, 2H, J=8.30;
J=1.84); 3.60 (s, 2H, NH2); 3.39 (s, 2H, CH2-N); 2.37 (s, 4H, CH2-N); 1.56 (m, 4H,
CH2); 1.40 (m, 2H, CH2). 13C-NMR (100 MHz, CDCl3): δ 145.8; 130.8 (2C); 128.2;
114.9 (2C); 63.2 (CH2-N); 54.1 (2C, N-CH2); 25.7 (2C, CH2); 24.3 (1C, CH2). DEPT-
135 (100 MHz, CDCl3): 130.8 (2C); 114.9 (2C); 63.2 (CH2-N); 54.1 (2C, N-CH2); 25.7
(2C, CH2); 24.3 (2C, CH2). Anal. Calc. for C12H18N2: C, 75.74; H, 9.53; N, 14.72.
Found: C, 75.68; H, 9.48; N. 14.63.
4.1.1.3. 4-(pyrrolidino-1-ylmethyl)-aniline 3c. Orange oil, yield 81%. FT-IR (KBr, ν
cm-1): 3351 (st. N-H), 3167 (st. N-H), 2934-2890 (st. C-H), 1599-1529 (st. C=C, Ar).

14
1H-NMR (400 MHz, CDCl3
): δ 7.10 (d, 2H, J=8.08); 6.6IP3 (dd, 2H, J=8.20; J=2.60);
ACCEPTED MANUSCR T
3.57 (s, 2H, NH2); 3.51 (s, 2H, CH2-N); 2.50 (s, 4H, N-CH2); 1.75 (m, 4H, CH2).
DEPT-135 (100 MHz, CDCl3): 129.8 (2C); 115.4 (2C); 68.2 (CH2-N); 46.6 (2C, N-
CH2); 20.5 (2C, CH2). Anal. Calc. for C11H16N2: C, 74.96; H, 9.15; N, 15.89. Found: C,
74.83; H, 9.06; N, 15.80.
4.1.1.4. 4-(morpholinomethyl)-aniline 3d. Yellow solid, 84%, m.p.: 82-84°C. FT-IR
(KBr, ν cm-1): 3359 (st. N-H), 3175 (st. N-H), 2934-2890 (st. C-H), 1603-1524 (st.
1
C=C, Ar). ). H-NMR (400 MHz, CDCl3): δ 7.07 (dd, 2H, J=8.44; J=2.56); 6.61 (dd,
2H, J=8.44; J=2.54); 3.70 (t, 4H, CH2-O); 3.62 (s, 2H, NH2); 3.37 (s, 2H, CH2-N); 2.40
13
(s, 4H, CH2-N). C-NMR (100 MHz, CDCl3): δ 145.6; 130.5 (2C); 127.5; 115.0 (2C);
67.1 (CH2-N); 63.1 (2C, N-CH2); 53.6 (2C, CH2). Anal. Calc. for C11H16N2O: C, 68.72;
H, 8.39; N, 14.57. Found: C, 68.61; H, 8.28; N, 14.45.
4.1.1.5. 4-(tert-butylamino)methyl-aniline 3e. Yellow oil, yield, 87%. FT-IR (KBr, ν
cm-1): 3354 (st. N-H), 3150 (st. N-H), 2934-2890 (st. C-H), 1602-1524 (st. C=C, Ar).
1H-NMR (400 MHz, CDCl3): δ 7.10 (d, 2H, J=8.04); 6.63 (dd, 2H, J=8.20; J=1.84);
3.60 (s, 2H, CH2); 1.11 (s, 9H, CH3). 13C-NMR (100 MHz, CDCl3): 145.2; 131.4 (2C);
129.5; 115.3 (2C); 50.7 (CH2-N); 46.8; 29.2 (3C, CH3). Anal. Calc. for C11H18N2: C,
74.11; H, 10.18; N, 15.71. Found: C, 74.07; H, 10.02; N, 15.59.
4.1.2. General procedure for the preparation of 4-amino-quinolines 4a-e: These
derivatives were prepared following a similar procedure reported by our group [17]. To
a solution of 4,7-dichloroquinoline (1 mmol, 1.2 equiv.) in ethanol (20 mL) was added
concentrated hydrochloric acid (catalytic amount from one to two drops) and stirred by
4 minutes. To this mixture was added the corresponding aniline 3a-e (1 equiv.) and
stirred under reflux for 4 hours. During the reaction a yellow precipitate was observed,
the mixture was then cooled to room temperature and the precipitated solid filtered,
washed with ethanol and dried under reduced pressure to yield the desired quinolines
4a-e.
4.1.2.1. 7-chloro-N-(4-((diethylamino)methyl)phenyl)quinolin-4-amine 4a. Yellow
solid, yield 79%, m.p.: 198-200°C. FT-IR (KBr, ν cm-1): 3165-3090 (st. N-H), 2967-
1
2933 (st. C-H), 1614-1590-1534-1511 (st. C=C, Ar). H-NMR (400 MHz, CD3OD): δ
8.28 (d, 1H, J=5.48); 8.24 (d, 1H, J=9.0); 7.82 (d, 1H, J=1.64); 7.46 (d, 1H, J=8.96,
J=1.72); 7.37 (d, 2H, J=8.2); 7.30 (d, 2H, J=8.2); 6.88 (d, 1H, J=5.48); 4.89 (s, 1H,
NH); 3.30 (s, 2H); 2.54 (q, 4H, CH2); 1.08 (t, 6H, CH3). 13C-NMR (100 MHz, CD3OD):

15
δ 152.4; 151.1; 150.2; 140.2; 136.7; 136.1; 131.8 (2C); 127.8; 126.7; 124.6; 124.4
ACCEPTED MANUSCRIPT
(2C); 119.5; 102.6; 58.8 (CH2-N); 47.8 (2C,N-CH2); 11.4 (2C, CH3). Anal. Calc. for
C20H22ClN3: C,70.68; H, 6.52; N, 12.36. Found: C, 70.71; H, 6.37; N, 12.14.
4.1.2.2. 7-chloro-N-(4-(piperidino-1-ylmethyl)phenyl)quinolin-4-amine 4b. Yellow
solid, yield 80%, m.p.:229-231°C. FT-IR (KBr, ν cm-1): 3156-3093 (st. N-H), 2967-
2925 (st. C-H), 1611-1590-1530 (st. C=C, Ar). 1H-NMR (400 MHz, CD3OD): δ 8.36 (d,
1H, J=4.56); 8.27 (d, 1H, J=8.8); 7.85 (s, 1H); 7.48 (d, 1H, J=8.8); 7.41 (d, 2H, J=7.0);
7.33 (d, 2H, J=7.3); 6.93 (d, 1H, J=4.6); 4.61 (s, 1H, NH); 3.58 (s, 2H, CH2-N); 2.52 (q,
13
4H, N-CH2); 1.64 (s, 4H, CH2); 1.49 (m, 2H, CH2). C-NMR (100 MHz, CD3OD): δ
152.4; 151.1; 150.2; 140.7; 136.8; 135.1; 132.4 (2C); 127.8; 126.7; 124.7; 124.3 (2C);
119.6; 102.8; 63.8 (CH2-N); 55.0 (2C, N-CH2); 26.2 (2C, CH2); 24.9 (1C, CH2). Anal.
Calc. for C20H22ClN3: C,70.68; H, 6.52; N, 12.36. Found: C, 70.71; H, 6.37; N, 12.14.
Anal. Calc. for C21H22ClN3: C, 71.68; H, 6.30; N, 11.94. Found: C, 71.59; H, 6.09; N,
11.42.
4.1.2.3. 7-chloro-N-(4-(pyrrolidino-1-ylmethyl)phenyl)quinolin-4-amine 4c. Yellow
solid, yield 81%, m.p.: 196-198°C. FT-IR (KBr, ν cm-1): 3160-3096 (st. N-H), 2964-
2930 (st. C-H), 1612-1575-1532-1512 (st. C=C, Ar). 1H-NMR (400 MHz, CD3OD): δ
8.33 (d, 1H, J=4.56); 8.25 (d, 1H, J=8.8); 7.83 (s, 1H); 7.45 (d, 1H, J=8.8); 7.39 (d, 2H,
J=7.0); 7.31 (d, 2H, J=7.3); 6.89 (d, 1H, J=4.6); 4.61 (s, 1H, NH); 3.63 (s, 2H, CH2-N);
2.56 (q, 4H, N-CH2); 1.81 (m, 2H, CH2). 13C-NMR (100 MHz, CD3OD): δ 152.4;
151.1; 150.2; 140.4; 136.8; 136.0; 131.7 (2C); 127.9; 126.7; 124.7; 124.4 (2C); 119.6;
102.7; 60.9 (CH2-N); 54.9 (2C, N-CH2); 24.1 (2C, CH2). Anal. Calc. for C20H20ClN3: C,
71.10; H, 5.97; N, 12.44. Found: C, 71.25; H, 6.02; N, 11.77.
4.1.2.4. 7-chloro-N-(4-(morpholinomethyl)phenyl)quinolin-4-amine 4d. Yellow
solid, yield 83%, m.p.: 219-221°C. FT-IR (KBr, ν cm-1): 3160-3080 (st. N-H), 2953-
2926 (st. C-H), 1613-1593-1535-1511 (st. C=C, Ar). 1H-NMR (400 MHz, CD3OD): δ
8.36 (d, 1H, J=5.6); 8.29 (d, 1H, J=9.1); 7.87 (d, 1H, J=2.1); 7.52 (dd, 1H, J=9.0;
J=2.1); 7.42 (d, 2H, J=8.5); 7.33 (d, 2H, J=8.5); 6.91 (d, 1H, J=5.6); 4.6 (s, 1H); 3.71 (t,
4H, CH2-O); 3.57 (s, 2H, CH2-N); 2.52 (t, 4H, N-CH2). 13C-NMR (100 MHz, CD3OD):
δ 151.9; 151.6; 149.6; 140.3; 137.1; 134.9; 132.1 (2C); 127.3; 126.9; 124.8; 124.6 (2C);
119.4; 102.6; 67.6 (2C, CH2-O); 63.7 (1C, N-CH2); 52.4 (2C, CH2-N). Anal. Calc. for
C20H20ClN3O.(0,06.CH3OH): C, 66.06; H, 6.11; N, 11.17. Found: C, 66.05; H, 5.39; N,
10.81.

16
ACCEPTED MANUSCRIPT
N-(4-((tert-butylamino)methyl)phenyl)-7-chloroquinolin-4-amine
4.1.2.5.
hydrochloride 4e. In this case, the resulting compound was not treated with alcaline
solution, and the product was recrystalizated directly to give the monohidrochloride salt.
Yellow solid, yield 79%, m.p.: 220-222 °C. FT-IR (KBr, ν cm-1): 3284 (st. N-H), 2960
(st. C-H), 1613-1575-1528-1514 (st. C=C, Ar). 1H-NMR (400 MHz, CD3OD): δ 9.06 (s,
1H, N-H); 8.43 (s, 1H); 8.42 (d, 1H, J=2.92); 7.88 (d, 1H, J=2.20); 7.55 (d, 1H, J=8.80;
J=2.20); 7.39 (d, 2H, J=8.44); 7.27 (d, 2H, J=8.44); 6.83 (d, 1H, J=5.52 ); 3.66 (s, 2H,
CH2-N); 1.11 (s, 9H, CH3). 13C-NMR (100 MHz, CD3OD): δ 152.4; 151.3; 150.2;
140.1; 138.0; 136.7; 131.0 (2C); 127.8; 126.7; 124.8; 124.7 (2C); 119.6; 102.5; 52.5
(CH2-N); 48.2; 28.6 (3C, CH3). Anal. Calc. for C20H23Cl2N3: C, 63.83; H, 6.16; N,
11.17. Found: C, 64.35; H, 6.28; N, 10.73.
4.2. Biological assays
4.2.1. Inhibition of haemozoin formation
The haemozoin formation assay was performed according to the literature [31]. Briefly,
a solution of hemin chloride (50 µL, 4 mM), dissolved in DMSO (5.2 mg.mL–1), was
distributed in 96-well micro plates. Different concentrations (50-5 µM) of the quinoline
compounds 4a-e dissolved in DMSO, were added in triplicate in test wells (50 µL).
Controls contained either water (50 µL) or DMSO (50 µL). β-Hematin formation was
initiated by the addition Acetate buffer (100 µL 0.2 M, pH 4.4). Plates were incubated at
37 °C for 48 h to allow completion of the reaction and centrifuged (4000 rpm × 15 min,
IEC-CENTRA, MP4R). After discarding the supernatant, the pellet was washed twice
with DMSO (200 µL) and finally, dissolved in NaOH (200 µL, 0.2 N). The solubilized
aggregates were further diluted 1:2 with NaOH (0.1 N) and absorbances recorded at 405
nm (Microplate Reader, BIORAD-550). The results were expressed as a percentage of
inhibition of haemozin formation (% IHF). CQ was used as positive control.
4.2.2. Parasite, experimental host and strain maintenance
Male Balb-C mice, weighing 18-22 g were maintained on a commercial pellet diet and
housed under conditions approved by Ethics Committee. P. berghei (ANKA strain), a
rodent malaria parasite, was used for infection. Mice were infected by i.p. injection with
1×106 infected erythrocytes diluted in phosphate buffered saline solution (PBS, 10 mM,
pH 7.4, 0.1 mL). Parasitemia was monitored by microscopic examination of Giemsa
stained smears [37].

17
ACCEPTED MANUSCRIPT
4.2.3. Four-days suppressive test
Balb-C mice (18-22 g) were infected i.v. (using caudal vein) with 106 infected RBC
with P. berghei (n=6). Two hours after infection, treatment began with the best
compounds tested in the in vitro assays. These quinolines 4a, 4b, 4c and 4e were
dissolved in DMSO (0.1 M), diluted with Saline-Tween 20 solution (2%). Each
compound (20 mg.kg–1) was administered once by i.p. for 4 days. At day 4, the
parasitemia was counted by examination of Giemsa stained smears. Chloroquine (25
mg.kg–1) was used as a positive control. The survival time beyond the control group
(saline treated) was recorded. The results were expressed as percentage of parasitemia
(% of parasitemia) and survival days of each compound treated-group over the control
(saline treated group) [38].
4.2.4. Murine peritonial macrophages growth inhibition assay.
Murine peritoneal macrophages were were plated in 96-well plates at 5x104 cell per
well in 150 mL culture medium composed of DMEM supplemented with 5 mM L-
glutamine, 10% (v/v) fetal bovine serum and antibiotics (penicillin/streptopmycin/
amphotericin B). After an overnight incubation in a 5% CO2 humidified incubator,
different concentrations of the tested dehydroxy isoquines 4a, 4b, 4c and 4e were added
to the cultures (duplicate wells; 200 mL final culture volume) and incubation continued
for an additional 48 h. Cell viability in individual wells was assessed by using the
colorimetric MTT reduction assay and expressed as percentage of MTT reduction
relative to control wells containing untreated cell and used to derive LD50 values from
dose response plots of % cell viability vs. log (compound concentration) using the non-
linear regression function of GraphPad Prism v.5.02 [39].
4.2.5. In vitro toxicity on human red blood cells (RBCs)
To evaluate the in vitro toxicological effect of the dehydroxyl isoquine derivative 4a-e,
we used a model based on the lysis of red blood cells (RBCs), measuring the
haemoglobin released in the supernatant fraction [33]. The haemoglobin released is
measured using spectrophotometer at 550 nm. RBCs in 50% Alsever’s solution were
centrifuged at 800g for 10 min and then washed three times with saline solution to
obtain RBCs in 100%. The synthesized compound (5-5000 µM) was incubated with a
2% final suspension of RBCs at 37°C for 45 min. The release of haemoglobin by an
equal number of RBCs by hypotonic lysis in 0.05 volumes of water was used as a 100%

18
positive control, while RBCs treated with saline solution rved as negative controls.
ACCEPTED MANUSCRIPseT
Results were expressed as the concentration at which half of the RBCs lysed (LyticC50).
4.2.6. Molecular docking studies
Five dehydroxylisoquines 4a-e were taken from our previous investi-gations as
potential inhibitors of the Plasmepsin I and II enzymes. Each quinoline structures were
built using ArgusLab v4.0.1 [40] and their respectives geometry was optimized using
semiempiricalmethods: PM3 [41a] and AM1 [41b]. In the optimization geometry
calculations of 4a-e, the requested convergence on density matrix was 10-12 and the
maximum gradient component was 0.000084. Each optimized quinoline was exported
to Protein document for the corresponding molecular docking. The X-ray
crystallographic structure of the P. falciparum enzymes: Plm I (PDB Code: 1LEE) and
Plm II (PDB Code: 1LF2) (see Tables 4 and Figure 2). Addition of hydrogens atoms to
the tested proteins was performed. Energy minimization of the proteins was performed
with the AMBER force field by using conjugate gradient method with a RMA gradient
of 0.01 kcal/A◦mol on Swiss-PdbViewer v4.0.1 [42] software. Binding site definition
was determined by comparison with reported interactions for the respective co-
crystallized ligand. The prepared protein was exported to ArgusLab v.4.0.1 program
package and save as Agl document.4.2. Molecular docking of the 102 diastereomers
over the three selected proteins was performed using ArgusLab (v4.0.1) package
program under Windows 7.0 enviroment, using AMBER force field. The protein and
ligand molecules were prepared as described above. The docking experiment on the
tested enzymes was carried out between the optimized ligand into the binding site
through their respective grid map dimensions and with a grid point spacing of 0.375 A◦.
Flexible ligand model was used in the docking and subsequent optimization scheme. As
a test of docking accuracy and for docking energy comparison, co-crystallized ligands
were re-docked into the protein structures (see Tables 4 and Figure 2). Different
orientations of the ligands were scanned and ranked from their energy scores.
Reproducibility of the calculated affinity energy and the minimum energy pose were
evaluated through 10 replicates for each ligand [43]. Affinity energy is reported as mean
of the 10 replicates. The lowest energy (strongest docking) poses for each ligand in each
protein target are summarized in Tables 4.

19
4.2.6. Data analysis
ACCEPTED MANUSCRIPT
Data were statistically analyzed using one-way ANOVA and t-tests for specific group
comparisons; assuming 95% of confidence according to GraphPad Prism 3.02 [44].
Acknowledgements
Authors thanks to Decanato de Investigacion y Desarrollo (DID, Universidad Simón
Bolívar) for its finacial support as well as to Lic. Noelani Ciguela for NMR spectras.
EA. Valverde and A.H. Romero thank to Decanato de Estudios de Postgrado
(Universidad Simón Bolívar) for Doctor Fellowship programs.
Supporting Information
Information on compound synthesis and characterization data, and protocols and results
for various antimalarial analyses is listed in this file. This material is available at
http://sciencedirect.com.
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Highlights
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Novel dehydroxy isoquines were synthesized and evaluated as antimalarial agents.
Derivatives 4b and 4c exhibited high potent activity against Plasmodium berghei.
4a, 4b, 4c and 4e inhibited the β-hematin formation at low sub-micromolar ranges.
The prepared dehydroxy isoquines exhibited acceptable in vitro cytotoxicities.