Structure-activity relationship of new antimalarial 1-aryl-3-susbtituted propanol derivatives: Synthesis, preliminary toxicity profiling, parasite life cycle stage studies, target exploration, and targeted delivery

Article abstract of DOI:10.1016/j.ejmech.2018.04.038

Design, synthesis, structure-activity relationship, cytotoxicity studies, in silico drug-likeness, genotoxicity screening, and in vivo studies of new 1-aryl-3-substituted propanol derivatives led to the identification of nine compounds with promising in vitro (55, 56, 61, 64, 66, and 70–73) and in vivo (66 and 72) antimalarial profiles against Plasmodium falciparum and Plasmodium berghei. Compounds 55, 56, 61, 64, 66 and 70–73 exhibited potent antiplasmodial activity against chloroquine-resistant strain FCR-3 (IC₅₀s < 0.28 μM), and compounds 55, 56, 64, 70, 71, and 72 showed potent biological activity in chloroquine-sensitive and multidrug-resistant strains (IC₅₀s < 0.7 μM for 3D7, D6, FCR-3 and C235). All of these compounds share appropriate drug-likeness profiles and adequate selectivity indexes (77 < SI < 184) as well as lack genotoxicity. In vivo efficacy tests in a mouse model showed compounds 66 and 72 to be promising candidates as they exhibited significant parasitemia reductions of 96.4% and 80.4%, respectively. Additional studies such as liver stage and sporogony inhibition, target exploration of heat shock protein 90 of P. falciparum, targeted delivery by immunoliposomes, and enantiomer characterization were performed and strongly reinforce the hypothesis of 1-aryl-3-substituted propanol derivatives as promising antimalarial compounds.

Full text of DOI:10.1016/j.ejmech.2018.04.038

Accepted Manuscript
Structure-activity relationship of new antimalarial 1-aryl-3-susbtituted propanol
derivatives: Synthesis, preliminary toxicity profiling, parasite life cycle stage studies,
target exploration, and targeted delivery
Miguel Quiliano, Adriana Pabón, Ernest Moles, Leonardo Bonilla-Ramirez, Isabelle
Fabing, Kim Y. Fong, Diego A. Nieto-Aco, David W. Wright, Juan C. Pizarro, Ariane
Vettorazzi, Adela López de Cerain, Eric Deharo, Xavier Fernández-Busquets,
Giovanny Garavito, Ignacio Aldana, Silvia Galiano
PII:
S0223-5234(18)30372-6
10.1016/j.ejmech.2018.04.038
EJMECH 10387
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 February 2018
Revised Date: 16 April 2018
Accepted Date: 18 April 2018
Please cite this article as: M. Quiliano, A. Pabón, E. Moles, L. Bonilla-Ramirez, I. Fabing, K.Y. Fong,
D.A. Nieto-Aco, D.W. Wright, J.C. Pizarro, A. Vettorazzi, A. López de Cerain, E. Deharo, X. Fernández-
Busquets, G. Garavito, I. Aldana, S. Galiano, Structure-activity relationship of new antimalarial 1-aryl-3-
susbtituted propanol derivatives: Synthesis, preliminary toxicity profiling, parasite life cycle stage studies,
target exploration, and targeted delivery, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.04.038.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Structure-activity relationship of new
antimalarial 1-aryl-3-susbtituted propanol
derivatives: Synthesis, preliminary toxicity
profiling, parasite life cycle stage studies, target
exploration, and targeted delivery
a,b
c
d,e,f
c
Miguel Quiliano , Adriana Pabón , Ernest Moles , Leonardo Bonilla-Ramirez ,
g
h
a,b,1
h
Isabelle Fabing , Kim Y. Fong , Diego A. Nieto-Aco , David W. Wright , Juan C.
i,j
k
k
l
Pizarro , Ariane Vettorazzi , Adela López de Cerain , Eric Deharo , Xavier Fernández-
d,e,f
m
a,b
a,b
Busquets , Giovanny Garavito , Ignacio Aldana , Silvia Galiano (*)
a
Universidad de Navarra, Instituto de Salud Tropical (ISTUN), Campus Universitario,
3
1008 Pamplona, Spain
b
Universidad de Navarra, Facultad de Farmacia y Nutrición, Departamento de Química
Orgánica y Farmacéutica, Campus Universitario, 31008 Pamplona, Spain
c
Grupo Malaria, Universidad de Antioquía, Medellín, Colombia
d
Nanomalaria Group, Institute for Bioengineering of Catalonia (IBEC), Baldiri Reixac
1
0-12, 08028 Barcelona, Spain
1

ACCEPTED MANUSCRIPT
e
Barcelona Institute for Global Health (ISGlobal), Barcelona Center for International
Health Research (CRESIB, Hospital Clínic-Universitat de Barcelona), Rosselló 149-
53, 08036 Barcelona, Spain
1
f
Nanoscience and Nanotechnology Institute (IN2UB), University of Barcelona, Martí i
Franquès 1, 08028 Barcelona, Spain
g
Laboratoire de Synthese et Physicochimie de Molécules d’Intéret Biologique
SPCMIB-UMR5068, CNRS - Université Paul Sabatier, 118, route de Narbonne, 31062,
Toulouse Cedex 09, France
h
Department of Chemistry, Vanderbilt University, Station B 351822, Nashville, TN
3
7235, USA
i
Department of Tropical Medicine, School of Public Health and Tropical Medicine,
Tulane University
j
Vector-Borne Infectious Diseases Research Center, Tulane University
k
Universidad de Navarra, Facultad de Farmacia y Nutrición, Department of
Pharmacology and Toxicology, Campus Universitario, 31008 Pamplona, Spain
l
UMR 152 PHARMA-DEV, Université Toulouse, IRD, UPS, 31062, Toulouse, France
m
Universidad Nacional de Colombia, Sede Bogotá, Facultad de Ciencias,
Departamento de Farmacia (DFUNC), Grupo de investigación FaMeTra (Farmacología
de la Medicina tradicional y popular), Carrera 30 45-03, Bogotá D.C., Colombia
2

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*
Corresponding author. Universidad de Navarra, Facultad de Farmacia y Nutrición,
Department of Organic and Pharmaceutical Chemistry, Institute of Tropical Health
ISTUN), Campus Universitario, 31008 Pamplona, Spain. Tel.: +34 948 425600
(
extension #803329. E-mail address: silvia.galiano@gmail.com (S. Galiano).
1
Present address: Laboratorio de investigación en Biopolímeros y Metalofármacos,
Facultad de Ciencias, Universidad Nacional de Ingeniería, Av. Túpac Amaru 210, Lima
2
5, Peru
ABSTRACT
Design, synthesis, structure-activity relationship, cytotoxicity studies, in silico drug-
likeness, genotoxicity screening, and in vivo studies of new 1-aryl-3-substituted
propanol derivatives led to the identification of nine compounds with promising in vitro
(55, 56, 61, 64, 66, and 70-73) and in vivo (66 and 72) antimalarial profiles against
Plasmodium falciparum and Plasmodium berghei. Compounds 55, 56, 61, 64, 66 and
7
0-73 exhibited potent antiplasmodial activity against chloroquine-resistant strain FCR-
(IC s < 0.28 µM), and compounds 55, 56, 64, 70, 71, and 72 showed potent
3
5
0
biological activity in chloroquine-sensitive and multidrug-resistant strains (IC s < 0.7
5
0
µM for 3D7, D6, FCR-3 and C235). All of these compounds share appropriate drug-
likeness profiles and adequate selectivity indexes (77 < SI < 184) as well as lack
genotoxicity. In vivo efficacy tests in a mouse model showed compounds 66 and 72 to
be promising candidates as they exhibited significant parasitemia reductions of 96.4%
and 80.4%, respectively. Additional studies such as liver stage and sporogony
inhibition, target exploration of heat shock protein 90 of P. falciparum, targeted
delivery by immunoliposomes, and enantiomer characterization were performed and
3

ACCEPTED MANUSCRIPT
strongly reinforce the hypothesis of 1-aryl-3-substituted propanol derivatives as
promising antimalarial compounds.
Keywords: Antiplasmodial, Antimalarial, Arylamino alcohol, Multi-stage activity,
Hsp90, Enantiomer separation
1
. Introduction
Malaria continues to be one of the major tropical diseases affecting the world.
According to the latest World Malaria Report produced by the WHO, nearly half of the
world's population distributed in 91 countries are at risk of being infected. In 2016
alone, 216 million cases and 445,000 deaths were reported globally, mostly in small
African children [1]. The disease is caused by protozoan parasites of the genus
Plasmodium, with the deadliest being Plasmodium falciparum (P. falciparum).
Plasmodium-infected female Anopheles mosquitoes bite humans initiating the infection.
As a result, the asymptomatic liver stage begins, and newly formed merozoites are
released into the bloodstream, where they invade red blood cells, and cause clinical
symptoms. Parasite transmission to the mosquito vector occurs when the mosquito
ingests circulating gametocytes, and the sexual stage of the parasite`s life cycle is then
started [2]. Artemisinin-based combination therapies (ACTs) are currently used as the
first-line treatment for this disease [1-3]; however, patients with resistance to
artemisinin and its derivatives are routinely identified in Southeast Asia (e.g., along the
Cambodia-Thailand border) [3, 4]. Consequently, there is an urgent need for the
continued development and innovation of new antimalarial chemical entities targeting
the liver stage, sexual forms, and new therapeutic pathways that is being addressed by
the academic, industrial and non-profit sectors [5, 6]. Due to the high cost, high rate of
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failure and considerable time needed to develop new antimalarial drugs, the study of
classic antimalarial drugs or drug repurposing is a promising approach [7, 8].
Present in classic arylamino alcohols (CAA) such as quinine, mefloquine,
lumefantrine, and halofantrine, the arylamino alcohol pharmacophore (ß-amino or γ-
amino alcohol moiety) is part of the antimalarial arsenal. Mandatory structural features
for antiplasmodial activity include the presence of aromatic (AR) and amino alcohol
portions linked by a carbon chain of two or three atoms in length [9] (Figure 1).
HO
AMINO ALCOHOL
PHARMACOPHORE
N
Cl
HO
N
H
Cl
HO
HO
N
NRR'
3
H CO
N
CF
3
AR
β
N
CF3
Cl
Quinine
Mefloquine
Lumefantrine
AMINO ALCOHOL
PHARMACOPHORE
Previous hit in APD
Selectivity index (VERO cells)= 37
No genotoxic
HO
N
F
OH
HO
NRR'
Cl
3
F C
γ
3
F C
AR
HN
Cl
8*
N
D6 IC50 = 0.11 µM
FCR-3 IC50 = 0.15 µM
C235 IC50 = 0.13 µM
NO
2
Halofantrine
Excellent parasitemia reduction in vivo (98%)
Figure 1. Arylamino alcohol pharmacophores (ß-amino or γ-amino alcohol moiety).
Classic arylamino alcohols, APD 8*, and their respective pharmacophores in colors red
and blue. Compound 8* was reported as hit in previous studies [10]
In the last fourteen years, research groups were attracted to the study of aryl amino
alcohols due to their high antimalarial activity and ADMET profile. Among them, ß-
amino alcohols have been more widely studied in comparison to those with a γ-amino
5

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alcohol moiety. Such studies were listed in our previous publication [10]. In the last
months, two new studies explored and identified new amino alcohols as selective
inhibitors of the heat shock protein 90 (Hsp90) of P. falciparum [11] and the hemozoin
formation pathway [12].
Our group has been exploring the structure-activity relationship (SAR) of γ-amino
alcohols, more specifically, 1-aryl-3-substituted propanol derivatives (APD), during the
last decade [10, 13-15]. Recently, we reported the synthesis, antimalarial evaluation,
toxicological studies, and target exploration of thirteen new APD [10]. In the study, the
new APD exhibited promising antiplasmodial activity against drug-sensitive (D6 0.11
µM ≤ IC ≤ 0.49 µM) and multidrug-resistant (FCR-3 0.40 µM ≤ IC ≤ 14 µM and
5
0
50
C235 0.13 µM ≤ IC ≤ 1.05 µM) strains of P. falciparum (blood stage). Adequate in
5
0
silico drug-likeness and the absence of genotoxicity were essential factors to explore the
scope of this chemotype series. Among them, compound 8* (Figure 1) displayed an
excellent parasitemia reduction (98 ± 1%) and complete curing of all the treated mice
infected with Plasmodium berghei (P. berghei). However, its selectivity index (SI = 37)
remain a priority problem to overcome as well as its unexplored multistage activity.
Two important therapeutic targets in the fight against malaria, i.e., the plasmepsin II
enzyme of P. falciparum and hemozoin inhibition pathway, were explored to establish a
mechanism of action, but the targets were discarded; APD may provide an alternative
antimalarial mechanism to others arylamino alcohols referenced in the literature.
Although our recent results represent a significant advance within APD (the γ-amino
alcohol field), more studies are needed to expand, and optimize the chemical series.
In this manuscript, we explored and addressed new assays to reinforce the hypothesis
of APD as promising antimalarial compounds. The new exploration and development of
APD were performed by: (1) expanding SAR studies using physicochemical, electronic,
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bulky and bioisosteric replacements at aromatic regions Ar and Ar (Figure 2), (2)
1
2
generating additional derivatives with high potency against both chloroquine-sensitive
and multidrug resistant strains of P. falciparum (blood stage), (3) reducing the
cytotoxicity or improving the selectivity index of the most active compounds, (4)
improving or maintaining high values of parasitemia reduction in the P. berghei mouse
model as was exhibited by compound 8* previously, (5) evaluating multistage activity
(
liver stage activity and sporogony inhibition), (6) exploring a new biological target in
P. falciparum, (7) testing new strategies to deliver APD into parasitized red blood cells
pRBCs) as an alternative to oral administration, and (8) exploring antiplasmodial and
(
cytotoxicity profiles on racemic mixtures and individual enantiomers on APD as
strategy for future optimizations.
Bioisosteric replacement of the substituents:
Exploration of optimum substituents:
Vital for antiplasmodial activity
CN
CN
CN
Cl
Br
3
F C
Exploration of bulky substituens:
HO
H CO
Cl
3
Br
New
New
e.g.
<
<
OH
-
-
Physicochemical properties
Electronic behavior
H
3
CO
3
F C
Amine
Ar
2
Ar
1
Addressing efficacy
Addressing efficacy and safety
Previous
Previous
Exploration of substituents:
F
Exploration of substituents:
Reduction of chain length decreases
antiplasmodial activity
F
CF
3
NO
2
F
3
F C
S
Figure 2. General SAR landscape for APD: Previous and new substitutions in Ar and
1
Ar regions.
2
2
. Results and discussion
2
.1. Design criteria for the new APD
The design criteria of the new APD were based on the consensus pharmacophore model
proposed by us previously [10]. The model was obtained taking into account the most
7

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active APD reported by our group in which different chemical scaffolds share common
structural features, and therefore, a common SAR landscape. The design criteria can be
broken down as follows:
2
.1.1. Exploration of the Ar region using 4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridine
1
as a common scaffold
A common substituent in the Ar region for APD is an aromatic fragment attached to a
1
fluorine atom. Indeed, our most active compounds previously reported for APD
presented 4-fluorophenyl or 4-fluoronaphthyl groups in the Ar region [10, 13].
1
However, the use of alternative substituents remains unexplored for APD. As was
shown in Figure 2, the exploration of an optimum substituent was proposed, considering
substituents with electron-donating (OCH , OH, OCH O) and electron-withdrawing (F,
3
2
Cl, Br, COOCH , CN) behaviors, and a lipophilicity contribution (hydrophobic and
3
hydrophilic) to the entire molecule. According to recent in vitro studies performed by
our group [10], two of the five APD that resulted with FCR-3 IC values less than 0.7
5
0
µM and adequate selectivity index (SI) (37 ≤ SI ≤ 244) presented in their structure the
scaffold 4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridine. In fact, the scaffold itself
showed moderate antiplasmodial activity (FCR-3 IC = 8.2 µM). Thus, based on its
5
0
versatile antiplasmodial activity and easy commercial availability, we decided to
explore the Ar1 region using 4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridine as a
common scaffold.
2
.1.2. Exploring the versatility of the antiplasmodial scaffold 1-[4-nitro-2-
trifluoromethyl)phenyl]piperidin-4-amine
Another scaffold present in two potent compounds recently studied by us (FCR-3 IC ≤
(
5
0
0
.5 µM, 37 ≤ SI ≤ 63) is 1-[4-nitro-2-(trifluoromethyl)phenyl]piperidin-4-amine. These
compounds showed marked in vivo parasitemia reductions (87-98%), and the scaffold
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itself exhibited moderate antiplasmodial activity against the FCR-3 strain (IC = 4.3
5
0
µM) [10]. However, its slow and tedious synthetic route precludes an easy and fast
exploration. Taking the above into account and based on the results obtained in section
2
.1.1, the exploration of new analogues was performed using the most active and
interesting substituents in the Ar region.
1
2
.1.3. Exploring old and new groups in the Ar region
2
In this section, the SAR information obtained in section 2.1.1 was used to explore the
classical groups commonly used at the ortho (CF ) or para (NO and F) position on the
3
2
phenyl ring of the Ar region. Additionally, based on the potent antiplasmodial
2
compounds reported by numerous academic groups in which the diphenylmethyl group
was used [16-18], the new scaffold was incorporated in the structure of the APD and its
versatility was explored.
2
.1.4. Decoding the substitutions in the Ar region and their influence on the
2
antiplasmodial activity.
One of the hypotheses used to design new APD was the mandatory presence of a
hydrophobic group such as CF at the ortho position on the phenyl ring in the Ar region
3
2
[
10]. However, not only presented the group that physicochemical property but also
exhibited an electron-withdrawing behavior. In fact, in our last publications, the
predilect substituents used on the phenyl ring in the Ar region were both electron-
2
withdrawing groups (CF and NO ) [10, 13, 14]. There is only one antecedent where an
3
2
electron-donating group such as OH was used by us, obtaining a marked decrease in the
antiplasmodial activity [14]. This observation led us to ask: 1) is the presence of an
electron-withdrawing or hydrophobic group responsible for maintaining the
9

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antiplasmodial activity? and 2) how much could this group contribute to or affect the
antimalarial activity compared to aromatic derivatives in region Ar ? To explore these
1
questions, the OCH group was chosen due to its electron-donating behavior, less
3
lipophilic contribution, structural similarity to the OH group, bulkiness, and synthetic
availability. To more effectively compare the analogues, 4-fluorophenyl, 4-
fluoronaphthyl and 4-methoxyphenyl groups were used in the Ar region. Additionally,
1
5
*, 6*, and 7* from our in-house library were tested to establish a clear SAR
(contributing long and bulky groups such as phenyl, biphenyl, and 2-naphthyl) [14, 19].
Piperazine and tetrahydropyridine groups were used as amines due to their synthetic
accessibility.
2
.1.5. Reducing the cytotoxicity of compound 8* using bioisostere and non-bioisostere
groups
As previously reported by us, the APD 8* was used as a reference or starter compound
for bioisosteric replacements. Because of its high antiplasmodial activity against drug-
sensitive (D6 ≤ 0.19 µM) and multidrug-resistant (FCR-3 ≤ 0.19 µM and C235 ≤ 0.28
µM) strains of P. falciparum, excellent parasitemia reduction (98%, in vivo) and
complete cure of all the treated mice, compound 8* was considered a promising
compound [10]. However, the presence of a nitro group in its structure represents a
structural alert always associated with toxicity [20]. Additionally, based on our previous
data and pharmacophore model proposed for APD, the presence of a hydrophobic group
at the ortho position on the phenyl ring of Ar should ensure the antiplasmodial activity
2
[
10, 13]; however, no complementary studies have yet been performed to modulate this
structural requirement. Therefore, bioisostere groups on the new APD can be used to 1)
maintain high antiplasmodial and antimalarial activity while at the same time improving
10

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the toxicity and thus avoiding future complications related to the presence of the nitro
group; 2) modulate the presence of a hydrophobic group at the ortho position on the
phenyl ring as mandatory to the antiplasmodial and antimalarial activity. The
bioisosteric replacement was performed using Spark™ software [21] containing 536184
commercial fragments from the Zinc database (drug-like) [22]. The results of the
Spark™ search showed 1000 possible hits ranked according to their internal scores. The
first hit reported was the starter compound 8* itself, which served as the internal control
(Table 1).
Table 1. Selected four hits from Spark experiment. Bioisosteres are ranked according to
its internal scores.
OH
F
NO2
N
N
H
F3C
Ar region to be modified
2
Starter compound
a
b
c
f
Results
BIF%
Score
Field score
Shape
score
ALOGPs 2D SIM
d
e
2.1
Hit 1
Hit 14
Hit 128
Hit 221
97
86
75
72
0.992
0.969
0.945
0.938
0.993
0.956
0.932
0.931
0.992
0.983
0.958
0.946
4.39
4.60
4.91
4.92
1
0.752
0.554
0.554
a
b
BIF (Bio-Isostere Factor) indicates favorable bioisosteres at geometry, electronic and surface properties; Score,
c
combination of Field and Shape score; Field score, similarity of the result and the starter molecule using only fields
excluding shape data; Shape score, similarity of the result and the starter molecule using shapes and excluding all
electrostatics data; ALOGPs 2.1 (LogP), logarithm of compound partition coefficient between n-octanol and water;
D SIM, structural similarity of the result and starter molecule, it is used to indicate novelty.
d
e
f
2
To make more feasible the exploration of the bioisostere groups in the Ar region, filters
2
2
such as molecular weight (≤ 500 Da), TPSA (≤ 140 Å ), LogP (ALOGPs 2.1 ≤ 5), and
11

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the presence of a nitro group in the final structure were applied to reduce the numbers of
hits. Thus, 514 hits were finally obtained and analyzed. Halogen atoms (Br and Cl) and
a carbonitrile group at the ortho and para positions, respectively, on the phenyl ring of
Ar were found to be the most promising hits to be synthesized based on their ability to
2
mimic the original moiety (BIF%), their similar electrostatic properties (field score),
their similar surface properties (shape score) and their novelty (2D SIM or 2D Tanimoto
similarity) (Table 1 and Figure 3).
Figure 3. Comparison of the starter compound (8*) and the 4 selected hits showing the
three-dimensional structure and field points (Hit 1, Hit 14, Hit128, and Hit 221). The
four field types are represented as follows: red (electrophilic), blue (nucleophilic),
orange (hydrophobic), and yellow (van der Waals attractive forces). The size of the field
or spheres indicates more energetically favorable interactions can be made.
Therefore, hits 14, 128, and 221 were selected to be synthesized and tested against
strains of P. falciparum. Of the hit population, the selected hits were ranked among the
top 250 of 1000 (without filters) and among the top 70 of 514 (with filters). Finally, a
substitution of the NO group by a NH group (non-bioisostere group) was performed to
2
2
clarify the role of the substituents at the para position on the phenyl ring in the Ar2
region, a modification in the structure never explored before in APD. In a
12

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complementary way, the synthetic viability was an important criterion used to select the
final compounds because the implementation of a new synthetic pathway was not
required, consequently obtaining fast results and reducing costs.
2
.2. Chemistry
The new APD (37-73) were synthesized according to the previous reported
procedures [10, 13, 14] outlined in Schemes 1 and 2. The starting arylamines 1-
diphenylmethyl)piperazine, 4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridine, 1-(4-
fluorophenyl)piperazine, 1-(2-nitrophenyl)piperazine, 1-(4-nitrophenyl)piperazine, 1-(2-
trifluoromethylphenyl)piperazine, 1-(2-methoxyphenyl)piperazine, and 4-(2-
(
methoxyphenyl)-1,2,3,6-tetrahydropyridine (IVa-g) were commercially available. Non-
commercially available arylamines (IIIa-d) were synthesized using 4-(N-Boc-
amino)piperidine and the corresponding fluorinated derivatives (Ia-d) such as 2-fluoro-
5
-nitrobenzotrifluoride, 3-bromo-4-fluorobenzonitrile, 3-chloro-4-fluorobenzonitrile,
and 4-fluoro-3-(trifluoromethyl)benzonitrile by a nucleophilic aromatic substitution
reaction (S Ar) via Meisenheimer complex formation and the later removal of the Boc-
N
group with hydrochloric acid (HCl) and acetic acid (AcOH) (Scheme 1.ii). All methyl
ketone compounds (V) were commercially available. The ketone intermediates (β-
amino ketones) (1-36) were prepared using the Mannich reaction (Scheme 1.iii) that
involved the condensation of the corresponding methyl ketone (V) with different aryl
amines (IIIa-d and IVa-g). The final hydroxyl derivatives (γ-amino alcohols) (37-72)
were synthesized by the reduction of the corresponding carbonyl group with sodium
borohydride (NaBH ) in methanol (MeOH) (Scheme 1.iv). The final hydroxyl
4
compound 73 was obtained by the reduction of the aromatic nitro group to an amine
13

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with hydrazine monohydrate and Raney nickel as catalyst in the presence of MeOH
using as an intermediate the previously published compound VI (Scheme 2.i) [10].
R
1
/R
2
: Cl/CN; Br/CN; CF
H/F; CH O/H; CF
3
/CN; CF
3
/NO
; NO
2
R
2
3
3
/H; H/CF
3
2 2
/H; H/NO
NH
BOC
N
+
F
H
R
Amine:
N
N
1
N
Ia-d
F
F
Cl
i
Ar:
F
Cl
Br
F
R
2
OCH
3
N
BOC
R
1
F
N
H
O
HO
H
3
CO
3
H CO
IIa-d
OCH
3
O
O
ii
NC
R
O
H
R
2
2
R
2
H
n
O
N
OH
N
N
R
1
R
1
R
1
Ar
N
H
Ar
N
H
H
2
N
iii
iv
IIIa-d
12-15, 34-36
48-51, 70-72
O
OH
O
H
HN
Ar
N
Ar
N
O
H
N
n
N
N
Ar
iii
iv
V
IVa
20-27
56-63
O
H
R
2
R
2
R
2
H
O
OH
n
Amine
Ar
Amine
Ar
Amine
R
1
iii
R
1
iv
R
1
IVb-g
1-11, 16-19, 28-33
3
7-47, 52-55, 64-69
Reagents and conditions: (i) CH CN, K CO , 12 h, reflux; (ii) AcOH / HCl (1:1), rt., 2 h, NaOH 2M;
3
2
3
o
o
(
iii) 1,4-dioxane, microwaves, 5 min, 100-110 C, 20 PSI, 150 W; (iv) MeOH, NaBH , 0 C, 2 h
4
Scheme 1. Synthesis of arylamino alcohols 37-72
14

ACCEPTED MANUSCRIPT
NO2
NH
2
OH
N
OH
N
CF3
CF
3
N
N
H
H
F
F
i
VI
73
o
Reagents and conditions: (i) MeOH, 0 C, Raney nickel, hydrazine monohydrate, 3 h
Scheme 2. Synthesis of arylamino alcohol 73
2
.3. In vitro antiplasmodial activity (3D7 and FCR-3 strain) and cytotoxicity studies.
The activities of thirty-seven new synthesized hydroxyl analogues (37-73) were
determined against the 3D7 chloroquine-sensitive strain and FCR-3 multidrug-resistant
strain of P. falciparum (Tables 2-7). Chloroquine was used as a reference drug in all
experiments for comparison (3D7 IC50 = 0.02, FCR-3 IC50 = 0.12 µM). Their
cytotoxicity was determined against HepG-2 cells.
2
.3.1. Exploration of Ar region using 4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridine as
1
a common scaffold
The antiplasmodial activities of eleven newly synthesized hydroxyl analogues (37-47)
are shown in Table 2. Although none of the compounds showed submicromolar activity
against the strains (IC < 1 µM), compounds 40-43 exhibited similar antiplasmodial
5
0
activities against the 3D7 (2.27 µM ≤ IC ≤ 5.42 µM) and FCR-3 strains (2.01 µM ≤
5
0
IC50 ≤ 7.06 µM). On the contrary, compounds 37-39 and 45-47 showed significant
differences in potency between strains (e.g., 39 3D7 IC = 17.38 µM vs. FCR-3 IC =
5
0
50
1
.70). Overall, compounds 40, 41, 42, and 43 were the most active compounds against
the 3D7 strain (2.27 µM ≤ IC ≤ 5.42 µM), while for the FCR-3 strain, compounds 37-
5
0
3
9, 41-43, and 47 were the most active (1.13 µM ≤ IC ≤ 2.88 µM). In both cases,
50
moderate antiplasmodial activity was exhibited (1 µM ≤ IC ≤ 10 µM). An analysis of
5
0
the results obtained shows that the different substitutions on the phenyl ring of the Ar1
15

ACCEPTED MANUSCRIPT
region did not contribute to the increase of the antiplasmodial activity in comparison
with the previous analogues synthesized, where IC values of 0.66 and 0.40 µM were
50
reported for 4-fluorophenyl and 4-fluoronaphthyl, respectively [10]. Additionally, it
must be highlighted that the presence of halogen atoms, such as Br, Cl, and F, increases
the antiplasmodial activity (F > Cl > Br) and contributes to maintaining the potency in
both strains.
Table 2. In vitro antimalarial activity against chloroquine sensitive strain (3D7) and
multidrug-resistant strain (FCR-3) of P. falciparum, and HepG-2 cytotoxicity of
compounds 37-47
OH
Ar1
N
F
IC50 (µM)a
P. falciparum
CC50(µM)b
HepG-2d
Compound
Ar
1
SIc
3
D7
FCR-3
3
7
8
8.48 ± 1.40
1.33 ± 0.20
1.13 ± 0.50
82.43 ± 4.00
62
NC
3
O
10.46 ± 3.40
509.30 ± 19.80
451
OCH3
O
39
40
41
42
17.38 ± 4.70
2.27 ± 0.20
2.79 ± 0.90
3.57 ± 0.50
1.70 ± 0.20
7.06 ± 1.70
2.01± 0.60
2.29 ± 0.20
164.88 ± 23.10
54.08 ± 4.60
97
8
O
Cl
F
F
96.97 ± 16.20
109.81± 15.40
48
48
F
Cl
Br
43
44
45
5.42 ± 1.30
10.64 ± 1.40
18.25 ± 5.80
2.68 ± 0.50
9.04 ± 0.80
11.54 ± 0.10
108.58 ± 2.40
121.15 ± 9.10
151.94 ± 4.30
41
13
13
3
H CO
OCH3
4
6
7
24.09 ± 1.40
9.48 ± 1.00
91.36 ± 3.00
10
H CO
3
4
22.17 ± 2.10
0.02
2.88 ± 0.10
0.12
253.23 ± 10.90
137.49
88
HO
CQe
-
1146
a
IC50: Concentration that produces 50% of the inhibitory effect.
16

ACCEPTED MANUSCRIPT
b
c
CC50: Concentration that produces 50% of cytotoxicity.
SI: Selectivity index = CC50 HepG-2 cells/ FCR-3 IC50 P. falciparum.
HepG-2: Human hepatocellular carcinoma cells.
CQ: chloroquine.
d
e
2
.3.2. Exploring the versatility of the antiplasmodial scaffold 1-[4-nitro-2-
(trifluoromethyl)phenyl]piperidin-4-amine
Based on the SAR information obtained in the exploration of the Ar region (section
1
2
.3.1), the introduction of F, Cl, and CN substituents was chosen to explore new
analogues. The antiplasmodial activities of four newly synthesized hydroxyl analogues
48-51) are shown in Table 3. Compounds 48 and 49 containing halogen atoms (Cl and
(
F) were found to be the most active compounds in this series showing marked
antiplasmodial activity in both strains, and almost equaling the antiplasmodial activity
previously shown by the synthesized compound 1* (FCR-3 IC = 0.48 µM) [10]. No
5
0
substitution (50) or introduction of a carbonitrile group on the phenyl group (51) led to a
drop in potency.
Table 3. In vitro antimalarial activity against chloroquine sensitive strain (3D7), and
multidrug-resistant strain (FCR-3) of P. falciparum, and HepG-2 cytotoxicity of
compounds 48-51
H
HO
Ar1
N
CF3
N
NO2
a
IC50 (µM)
P. falciparum
D7 FCR-3
CC50 (µM)b
c
Compound
Ar
1
SI
d
3
HepG-2
Cl
4
4
5
5
8
9
0
1
0.71 ± 0.20
0.67 ± 0.10
4.22 ± 2.90
4.42 ± 0.20
0.51 ± 0.02
0.53 ± 0.08
2.02 ± 0.20
1.80 ± 0.50
4.61 ± 0.04
13.33 ± 1.70
23.0 ± 2.80
27.63 ± 0.20
9
F
F
25
11
15
F
NC
17

ACCEPTED MANUSCRIPT
*
f
1
ND
0.48 ± 0.04
10.40 ± 1.60
22
F
CQe
-
0.02
0.12
137.49
1146
a
b
c
IC50: Concentration that produces 50% of the inhibitory effect.
CC50: Concentration that produces 50% of cytotoxicity.
SI: Selectivity index = CC50 HepG-2 cells/ FCR-3 IC50 P. falciparum.
d
HepG-2: Human hepatocellular carcinoma cells
CQ: chloroquine.
e
f
ND: Not determined.
*
Previously synthesized by our group [10]
2
.3.3. Exploring old and new groups in the Ar region
2
To expand the landscape in the Ar region, different substitutions on the phenyl ring
2
attached to a piperazine were carried out. In this series, 4-fluoronaphthyl was introduced
in the Ar region according to the SAR shown in section 2.3.1. The activities of five
1
newly synthesized hydroxyl analogues (52-56) are shown in Table 4. Compounds 55
and 56 were the most potent APD, with comparable submicromolar values for both
strains. In contrast, the rest of the compounds within this series displayed significant
differences between the two strains, with compounds 52 and 53 achieving
submicromolar values for the 3D7 strain. In this new exploration, the complete SAR
data are revealed for electron-withdrawing substituents at the ortho and para positions
on the phenyl ring such as CF , NO , and F. These groups had been previously tested by
3
2
our group in different positions and combinations (e.g., the reference compounds 2*-4*)
10, 13], but the exact role of each one had not been clarified (e.g., electrostatic, steric,
lipophilic). The removal of the CF group at the ortho position maintaining the NO
[
3
2
group at the para position led to a decrease in antiplasmodial activity (54 FCR-3 IC =
5
0
1
.71 µM vs. 4* FCR-3 IC50 = 0.5 µM). In contrast, an improvement of the
antiplasmodial efficacy was achieved with the corresponding o-CF analogue in which
3
the NO group had been removed (55 FCR-3 IC = 0.15 µM vs. 4* FCR-3 IC = 0.5
2
50
50
µM). This last improvement is also observed in the case of the NO group at the ortho
2
position and the CF group removed from the para position (53 FCR-3 IC = 2.62 µM
3
50
18

ACCEPTED MANUSCRIPT
vs. 3* FCR-3 IC = 5.0 µM). Additionally, when a CF group is substituted at the para
5
0
3
position for a NO or F group, the activity decreases. Therefore, our data reinforces our
2
previous 3D pharmacophore model, in which the presence of a hydrophobic group is
necessary to obtain potent antimalarial compounds against chloroquine-resistant strain
FCR-3. It must be noticed that bulky groups (e.g., NO ) at the ortho position retained
2
antiplasmodial activity in chloroquine-sensible strain 3D7 even if they are not
hydrophobic. However, the electronic role of the substituents at the ortho and para
positions remains unsolved. On the other hand,
diphenylmethyl)piperazine was incorporated in the structure of APD, and its versatility
was explored in the following section.
a
novel scaffold 1-
(
Table 4. In vitro antimalarial activity against chloroquine sensitive strain (3D7) and
multidrug-resistant strain (FCR-3) of P. falciparum, and HepG-2 cytotoxicity of
compounds 52-56
OH
N
N
F
Ar2
IC50 (µM)a
P. falciparum
CC50 (µM)b
HepG-2d
Compound
Ar
2
SIc
3
D7
FCR-3
52
53
54
0.43 ± 0.10
0.92 ± 0.30
4.74 ± 0.40
4.05 ± 1.40
2.62 ± 0.30
1.71 ± 0.60
29.38 ± 4.30
19.86 ± 0.40
12.47 ± 1.10
7
8
7
F
2
O N
NO
2
5
5
2
5
6
*
0.16 ± 0.06
0.62 ± 0.02
0.15 ± 0.03
0.33 ± 0.04
27.52 ± 0.60
19.90 ± 2.70
183
60
F
3
C
NDf
ND
0.93 ± 0.70
5 ± 0.08
ND
ND
-
-
CF
3
3
4
*
*
O2N
CF3
ND
0.5 ± 0.02
0.12
ND
-
F
3
C
NO
2
CQe
-
0.02
137.49
1146
19

ACCEPTED MANUSCRIPT
a
b
c
IC50: Concentration that produces 50% of the inhibitory effect.
CC50: Concentration that produces 50% of cytotoxicity.
SI: Selectivity index = CC50 HepG-2 cells/ FCR-3 IC50 P. falciparum.
HepG-2: Human hepatocellular carcinoma cells
CQ: chloroquine.
d
e
f
ND: Not determined.
*
Previously synthesized by our group [10, 13]
2
.3.3.1. Exploration of the 1-(diphenylmethyl)piperazine scaffold
Encouraged by the potent activity of compound 56 bearing
a
1-
(diphenylmethyl)piperazine scaffold against both strains (3D7 IC50 = 0.62 µM and
FCR-3 IC = 0.33 µM), we decided to expand this chemical series using the most
5
0
active variations used in the Ar region. The antiplasmodial activities of seven newly
1
synthesized hydroxyl analogues (57-63) are shown in Table 5. Compounds containing
fluorine atoms were the most active (57 and 61). Compounds 58-60, 62, and 63 showed
moderate to low antiplasmodial activity (1 µM ≤ IC s ≤ 52 µM). From a structural
5
0
point of view, the new scaffold meets the requirement of having a hydrophobic portion
in the Ar region, which is important for the antiplasmodial activity [10]. The use of the
2
substituents 4-fluoronaphthyl and 3,4-fluorophenyl in the Ar region showed almost the
1
same IC values for both strains (56 vs. 61; IC s ≤ 0.62 µM). Interestingly, the same
5
0
50
substituents reported a similar modulation of the antiplasmodial activity using the 1-[4-
nitro-2-(trifluoromethyl)phenyl]piperidin-4-amine scaffold (49 vs. 8*; IC s ≤ 0.67
5
0
µM). In contrast, the substituents 4-fluoronaphthyl and 3,4-fluorophenyl with the 4-(4-
fluorophenyl)-1,2,3,6-tetrahydropyridine as a common scaffold did not show this
pattern (a scaffold without a hydrophobic group at ortho position in the Ar region).
2
Thus, 3,4-fluorophenyl can be considered a good alternative to classical groups such as
4
-fluoronaphthyl and 4-fluorophenyl when chemical scaffolds containing a hydrophobic
group in the Ar region are used.
2
20

ACCEPTED MANUSCRIPT
Table 5. In vitro antimalarial activity against chloroquine sensitive strain (3D7) and
multidrug-resistant strain (FCR-3) of P. falciparum, and HepG-2 cytotoxicity of
compounds 57-63
OH
N
Ar1
N
IC50 (µM)a
CC50 (µM)b
Compound
Ar
1
P. falciparum
SIc
3
D7
FCR-3
HepG-2d
57
58
59
60
1.08 ± 0.04
52.22 ± 6.80
24.85 ± 4.90
5.11 ± 0.30
0.62 ± 0.20
252 ± 20.60
406
114
118
194
F
3.09 ± 0.20
3.93 ± 1.10
1.39 ± 0.40
351.40 ± 27.70
462.43 ± 2.20
269.16 ± 2.10
Br
Cl
Cl
F
F
6
1
2
0.38 ± 0.07
1.66 ± 0.04
0.37 ± 0.02
1.44 ± 0.20
54.27 ± 6.90
36.10 ± 4.80
147
25
F
O
6
O
O
6
3
1.69 ± 0.06
0.02
1.46 ± 0.20
0.12
83.96 ± 5.30
137.49
58
OCH
3
CQe
-
1146
a
b
c
IC50: Concentration that produces 50% of the inhibitory effect.
CC50: Concentration that produces 50% of cytotoxicity.
SI: Selectivity index = CC50 HepG-2 cells/ FCR-3 IC50 P. falciparum.
d
HepG-2: Human hepatocellular carcinoma cells
CQ: chloroquine.
e
2
.3.4. Decoding the substitutions in the Ar region and their influence on the
2
antiplasmodial activity
The activities of six newly synthesized hydroxyl analogues (64-69) are shown in
Table 6. Substitutions in the Ar region on the phenyl ring at the ortho position by CF
2
3
21

ACCEPTED MANUSCRIPT
and OCH groups while maintaining the 4-fluorophenyl group in the Ar region and
3
1
piperazine as the amine showed potent to moderate activity against both strains (66 and
6
8, 0.19 µM ≤ IC s ≤ 7.31 µM). Meanwhile, when a 4-fluoronaphthyl group replaces
50
4
-fluorophenyl on the left hand side of these derivatives, both compounds exhibited
potent activity (55 and 64; 3D7 and FCR-3 IC s ≤ 0.22 µM). Therefore, the SAR
5
0
analysis revealed that the essential structural requirement for antiplasmodial activity at
the ortho position was the presence of groups with a hydrophobic surface and a bulky
nature (e.g., CF , OCH ), regardless of the electronic nature of the substituents
3
3
(electron-withdrawing or electron-donating). For example, compound 53 with a bulky
NO group but no hydrophobic surface led to a decrease in the antiplasmodial activity in
2
comparison with compounds 55 and 64 (Figure 4).
Figure 4. Electrostatic and hydrophobic surfaces for compounds 53, 55, and 64. (a)
Negative (cyan) and positive (red) electrostatic surface; (b) hydrophobic surface
(orange). The change of substituents at ortho position on the phenyl ring in the Ar2
22

ACCEPTED MANUSCRIPT
region introduce negative charges but also withdraws or donates electrons from the ring
modifying the positive charge of aromatic hydrogens
As a second approach, the comparison of compounds 66, 69, 5*, 6*, and 7* clearly
shows that the presence of the OCH group itself in the Ar region did not guarantee
3
2
potent antimalarial activity. By contrast, the absence of a hydrophobic surface or bulky
substituent at the ortho position in cases where 4-fluorophenyl and 4-fluoronaphthyl
were present in the Ar region was partially tolerated, reaching moderate to potent
1
antimalarial activity against at least one strain of P. falciparum (e.g., compounds 52, 53,
2
*). Finally, the comparison of piperazine and tetrahydropyridine groups as the central
amine (64 vs. 65 and 66 vs. 67) indicates that piperazine analogues are more active than
tetrahydropyridine analogues against FCR-3 strain.
Table 6. In vitro antimalarial activity against chloroquine sensitive strain (3D7) and
multidrug-resistant strain (FCR-3) of P. falciparum, and HepG-2 cytotoxicity of
compounds 64-69
OH
R1
Ar1
Amine
a
IC50 (µM)
b
CC50 (µM)
c
Compound
Ar
1
Amine
R
1
P. falciparum
SI
d
3
D7
FCR-3
HepG-2
6
6
4
5
N
N
N
OCH
3
3
0.07 ± 1.60
0.10 ± 1.90
0.22 ± 0.02
30.37 ± 5.40
17.69 ± 3.10
138
14
F
F
OCH
1.30 ± 0.20
6
6
6
6
7
8
N
N
N
N
N
OCH
3
3
7.31 ± 0.02
2.82 ± 9.10
1.01 ± 0.10
0.19 ± 0.10
1.32 ± 0.02
1.69 ± 0.40
19.54 ± 0.02
67.03 ± 9.10
1.35 ± 0.18
103
51
1
F
OCH
F
F
CF
3
6
5
9
*
N
N
N
N
OCH
3
3
10.53 ± 1.20
13.90 ± 0.90
7.98 ± 1.20
370.83 ± 33.30
> 612
46
3
H CO
OCH
15.66 ± 1.20
> 39
23

ACCEPTED MANUSCRIPT
6
7
*
*
N
N
N
OCH
3
7.25 ± 2.80
2.78 ± 0.02
24.44 ± 4.30
9
N
OCH
3
4.08 ± 1.60
2.63 ± 0.10
95.01 ± 4.20
36
CQe
-
-
-
0.02
0.12
137.49
1146
a
b
c
IC50: Concentration that produces 50% of the inhibitory effect.
CC50: Concentration that produces 50% of cytotoxicity.
SI: Selectivity index = CC50 HepG-2 cells/ FCR-3 IC50 P. falciparum.
d
HepG-2: Human hepatocellular carcinoma cells.
CQ: chloroquine.
e
*
Previously synthesized by our group [14, 19]
2
.3.5. Reducing the cytotoxicity of compound 8* using bioisostere and non-bioisostere
groups
Based on the detailed criteria used to design new analogues of compound 8* (see
section 2.1.5.), the activities of four newly synthesized hydroxyl analogues (70-73) are
shown in Table 7. Compounds 70-73 exhibited IC values below 0.1 µM against the
5
0
FCR-3 strain and IC values below 0.22 µM against the 3D7 strain. As was expected
5
0
for compounds 70-72, the use of the bioisosteric replacements generated analogues with
similar antimalarial potency against P. falciparum, while the cytotoxicity of HepG-2
was attenuated (for a more in-depth discussion of the cytotoxicity, see section 2.3.6.).
Interestingly, hydrophobic groups of different sizes (Cl < Br < CF ) at the ortho position
3
were well tolerated. Although compound 73 had an electron-donating group (NH ) at
2
the para position on the phenyl ring in the Ar region, the antimalarial potency of the
2
molecule was maintained against both strains (IC50 ≤ 0.14 µM). This observation
modifies our previous hypothesis in which only electron-withdrawing groups at the para
position contributed to the activity [10]. In fact, the para position can be used to
introduce groups that help to improve the water solubility in future optimization
campaigns.
Table 7. In vitro antimalarial activity against chloroquine sensitive strain (3D7) and
multidrug-resistant strain (FCR-3) of P. falciparum, and HepG-2 cytotoxicity of
compounds 70-73
24

ACCEPTED MANUSCRIPT
H
HO
N
R1
N
R2
F
a
IC50 (µM)
b
CC50 (µM)
c
Compound
R
1
R
2
P. falciparum
FCR-3
SI
d
3
D7
HepG-2
7
7
7
7
8
CQ
0
1
2
3
Br
Cl
CN
CN
CN
0.22 ± 0.10
0.15 ± 0.01
0.13 ± 0.01
0.14 ± 0.06
0.07 ± 0.00
0.09 ± 0.06
0.08 ± 0.03
0.08 ± 0.00
0.15 ± 0.01
0.12
9.87 ± 2.70
7.95 ± 1.20
6.22 ± 0.60
10.95 ± 0.60
5.70 ± 1.80
137.49
141
88
78
137
38
1146
CF
CF
CF
-
3
3
3
NH
NO
-
2
f
*
2
ND
0.02
e
a
b
c
IC50: Concentration that produces 50% of the inhibitory effect.
CC50: Concentration that produces 50% of cytotoxicity.
SI: Selectivity index = CC50 HepG-2 cells/ FCR-3 IC50 P. falciparum.
d
HepG-2: Human hepatocellular carcinoma cells
CQ: Chloroquine.
e
f
ND: Not determined.
*
Previously synthesized by our group [10]
2
.3.6. General cytotoxicity
The cytotoxicities of the thirty-seven newly synthesized hydroxyl analogues (38-73)
were determined against HepG2-A16 cells using the MTT assay [23] (Tables 2-7). An
exploration of the Ar region showed that compounds with substituents on the phenyl
1
ring containing halogen atoms such as F, Cl, or Br (41-43) exhibited a moderate
selectivity index (41 ≤ SI ≤ 48), except for compound 40 (low SI = 8). Meanwhile,
substituents such as CN, COOCH , -OCH O-, and OH resulted in a moderate to high
3
2
selectivity index (62 ≤ SI ≤ 451). Unsubstituted phenyl ring and OCH groups resulted
3
in a significant drop in SI (44, 45, and 46). The use of the different Ar substituents with
1
the 1-[4-nitro-2-(trifluoromethyl)phenyl]piperidin-4-amine scaffold did not improve on
the SI of the previous analogue compound 1* (SI = 22). Compound 49 was the only
analogue with a similar SI (49 SI = 25). Therefore, the 3,4-difluoro substitution on the
phenyl ring of the Ar region can be considered an alternative variation because of the
1
moderate SI presented in APD. On the other hand, the cytotoxicity exploration of the
Ar2 region showed that compounds 55 and 56, bearing trifluoromethyl and
25

ACCEPTED MANUSCRIPT
diphenylmethyl groups, respectively, exhibited high to moderate SI (183 ≥ SI ≥ 60). In
fact, the high selectivity of the diphenylmethyl scaffold is reinforced by compounds 57-
6
3, which exhibited moderate to high SI (25 ≥ SI ≥ 406), even though variations in the
Ar region were responsible for the low selectivity in other series (40 vs. 48 vs. 60).
1
High selectivity was also observed for compound 64 (OCH ; SI ≥ 100), which is a
3
structural analogue of compound 55 (CF ). A comparison between the piperazine and
3
tetrahydropyridine groups showed that analogues with piperazine as the central amine
are less cytotoxic than tetrahydropyridine (64 vs. 65 and 66 vs. 67). The use of
bioisostere and non-bioisostere groups allowed us to obtain new analogues of
compound 8* with a marked decrease in cytotoxicity (70-73). Taking as a starting point
the SI of compound 8* (SI = 38), high values of selectivity were reached for analogues
7
0-73 (78 ≤ SI ≤ 141). This increase in the SI values can be explained by the absence of
the nitro group and its well-documented mutagenic and carcinogenic potential [20]. The
most remarkable result to emerge from the bioisosteric replacement is the feasibility of
obtaining a high selectivity using the aminopiperidine scaffold (SI ≥ 78). In fact, in our
previous studies, because of its high antiplasmodial activity, the aminopiperidine
scaffold was considered an attractive fragment, but its selectivity was always of concern
[
10, 14].
2
.4. In vitro antiplasmodial activity (D6 and C235 strains)
As part of our antimalarial drug discovery pipeline, new APD must exhibit
remarkable potency against both chloroquine-sensitive and multidrug-resistant strains of
P. falciparum. More specifically, the objective is to explore and establish a broad
spectrum of antiplasmodial efficacy for new APD against mefloquine-resistant strains.
Mefloquine, a classical arylamino alcohol, is a molecule widely used by the WHO in
26

ACCEPTED MANUSCRIPT
chemoprotection campaigns [1, 2]. Therefore, nine compounds with marked
antiplasmodial activities (FCR-3 IC ≤ 1.32 µM) were independently evaluated and
5
0
validated in two laboratories using two different strains of P. falciparum. The newly
synthesized APD were tested against the D6 chloroquine-sensitive (but naturally less
susceptible to mefloquine) and C235 multidrug-resistant strains (resistant to
mefloquine, chloroquine, and pyrimethamine) (Table 8). All the selected compounds
except 66 and 67 exhibited potent antimalarial activity against both strains (D6 IC ≤
5
0
0
.28 µM, C235 IC ≤ 0.41 µM). Compounds 55 and 72 were found to be the most
50
active compounds, with IC values in the range of 0.08-0.14 µM for the D6 and C235
5
0
strains. Based on our results and previous IC data against the D6 and C235 strains
5
0
[
10], it is clear that, in general, the substituent 4-fluoronaphthyl was present in the Ar1
region in the most active compounds. Such a characteristic can be observed more
clearly if the analogues using 4-fluoronaphthyl and 4-fluorophenyl are compared (64 vs.
6
6 and 65 vs. 67). Our results reinforce the idea that APD have remarkable potency
against chloroquine-sensitive (3D7 and D6) and multidrug-resistant (FCR-3 and C235)
strains of P. falciparum.
Table 8. In vitro antimalarial activity against chloroquine sensitive strain (D6) and
multidrug-resistant strain (C235) of P. falciparum
a
IC50 (µM)
P. falciparum
Compound
Structure
D6
C235
OH
N
5
5
5
6
0.10 ± 0.01
0.14 ± 0.01
N
F
F
F3C
OH
N
N
0.10 ± 0.02
0.17 ± 0.01
27

ACCEPTED MANUSCRIPT
OH
N
OCH3
6
6
4
5
0.28 ± 0.03
0.41 ± 0.00
0.23 ± 0.00
N
F
OH
N
OCH3
0.15 ± 0.01
F
OH
OH
N
N
OCH3
OCH3
6
6
6
7
6.32 ± 0.33
3.73 ± 0.43
7.26 ± 0.17
3.69 ± 0.17
N
F
F
OH
CN
F
N
N
N
0.12 ± 0.00
0.12 ± 0.00
0.15 ± 0.01
0.17 ± 0.02
7
7
7
0
1
2
N
H
Br
Cl
OH
OH
CN
F
N
H
CN
F
0.08 ± 0.00
0.01 ± 0.00
0.12 ± 0.02
0.05 ± 0.00
N
H
3
F C
CQ
a
IC : Concentration that produces 50% of the inhibitory effect.
5
0
2
.5. In silico physicochemical properties and druglikeness evaluation
An in silico study was performed for the prediction of the druglikeness profile of
synthesized APD (Table 9). Used as a good descriptor of the drug absorbance in the
intestines, blood-brain barrier crossing, and Caco-2 monolayer penetration the
topological polar surface area (TPSA) was calculated [24], and the theoretical
percentage of human intestinal absorption (%ABS) was obtained according to the
equation %ABS = 109 – 0.345 x TPSA [25]. Additionally, the number of rotatable
bonds (n-ROTB) were calculated, and the druglikeness profile according to Lipinski’s
rule of five [26] and Veber’s rule [27] was evaluated for APD. From these parameters,
all compounds exhibited a %ABS ranging between 80 to 100%, except for compound
28