Synthesis and biological evaluation of benzhydryl-based antiplasmodial agents possessing Plasmodium falciparum chloroquine resistance transporter (PfCRT) inhibitory activity

Article abstract of DOI:10.1016/j.ejmech.2021.113227

Due to the surge in resistance to common therapies, malaria remains a significant concern to human health worldwide. In chloroquine (CQ)-resistant (CQ-R) strains of Plasmodium falciparum, CQ and related drugs are effluxed from the parasite's digestive vacuole (DV). This process is mediated by mutant isoforms of a protein called CQ resistance transporter (PfCRT). CQ-R strains can be partially re-sensitized to CQ by verapamil (VP), primaquine (PQ) and other compounds, and this has been shown to be due to the ability of these molecules to inhibit drug transport via PfCRT. We have previously developed a series of clotrimazole (CLT)-based antimalarial agents that possess inhibitory activity against PfCRT (4a,b). In our endeavor to develop novel PfCRT inhibitors, and to perform a structure-activity relationship analysis, we synthesized a new library of analogues. When the benzhydryl system was linked to a 4-aminoquinoline group (5a-f) the resulting compounds exhibited good cytotoxicity against both CQ-R and CQ-S strains of P. falciparum. The most potent inhibitory activity against the PfCRT-mediated transport of CQ was obtained with compound 5k. When compared to the reference compound, benzhydryl analogues of PQ (5i,j) showed a similar activity against blood-stage parasites, and a stronger in vitro potency against liver-stage parasites. Unfortunately, in the in vivo transmission blocking assays, 5i,j were inactive against gametocytes.

Full text of DOI:10.1016/j.ejmech.2021.113227

European Journal of Medicinal Chemistry 215 (2021) 113227
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Synthesis and biological evaluation of benzhydryl-based
antiplasmodial agents possessing Plasmodium falciparum chloroquine
resistance transporter (PfCRT) inhibitory activity
,
,
,
, b
Nicola Relitti a b, Stefano Federico a b, Luca Pozzetti a b, Stefania Butini a
,
Stefania Lamponi a b, Donatella Taramelli c b, Sarah D’Alessandro d b, Rowena E. Martin e,
,
,
,
Sarah H. Shafik e, Robert L. Summers e, Simone K. Babij e, Annette Habluetzel f
,
,
b
Sofia Tapanelli f b, Reto Caldelari g, Sandra Gemma a *, Giuseppe Campiani a
,
,
b
,
, b
a Department of Biotechnology, Chemistry and Pharmacy (DoE 2018-2022), University of Siena, Via Aldo Moro 2, 53100 Siena, Italy
b Centro Interuniversitario di Ricerche Sulla Malaria (CIRM), University of Milan, Milano, Italy
c Department of Pharmacological and Biomolecular Sciences, University of Milan, Via Pascal 36, 20133, Milan, Italy
d Department of Biomedical, Surgical and Dental Sciences, University of Milan, Via Pascal 36, 20133, Milan, Italy
e Research School of Biology, Australian National University, Canberra, ACT, 2600, Australia
f School of Pharmacy, University of Camerino, Piazza Cavour 19F, 62032, Camerino, Italy
g Institute of Cell Biology, University of Bern, Baltzerstrasse 4, 3012, Bern, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Due to the surge in resistance to common therapies, malaria remains a significant concern to human
health worldwide. In chloroquine (CQ)-resistant (CQ-R) strains of Plasmodium falciparum, CQ and related
drugs are effluxed from the parasite’s digestive vacuole (DV). This process is mediated by mutant iso-
forms of a protein called CQ resistance transporter (PfCRT). CQ-R strains can be partially re-sensitized to
CQ by verapamil (VP), primaquine (PQ) and other compounds, and this has been shown to be due to the
ability of these molecules to inhibit drug transport via PfCRT. We have previously developed a series of
clotrimazole (CLT)-based antimalarial agents that possess inhibitory activity against PfCRT (4a,b). In our
endeavor to develop novel PfCRT inhibitors, and to perform a structure-activity relationship analysis, we
synthesized a new library of analogues. When the benzhydryl system was linked to a 4-aminoquinoline
group (5a-f) the resulting compounds exhibited good cytotoxicity against both CQ-R and CQ-S strains of
P. falciparum. The most potent inhibitory activity against the PfCRT-mediated transport of CQ was ob-
tained with compound 5k. When compared to the reference compound, benzhydryl analogues of PQ
(5i,j) showed a similar activity against blood-stage parasites, and a stronger in vitro potency against liver-
stage parasites. Unfortunately, in the in vivo transmission blocking assays, 5i,j were inactive against
gametocytes.
Received 16 December 2020
Received in revised form
21 January 2021
Accepted 21 January 2021
Available online 6 February 2021
Keywords:
Malaria
Plasmodium falciparum
Chloroquine resistance
PfCRT
Chemosensitization
Liver-stage antimalarial
Xenopus oocytes
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
efficacy together with a fast action and thus reduced potential for the
development of resistant parasite strains. However, in recent years
Despite recent progress in the fight against malaria, it remains a
serious problem worldwide with more than 405.000 deaths in 2018
[1]. The current first-line treatments for malaria are the artemisinin-
based combination therapies (ACTs), which are characterized by high
some regions of Asia (the Greater Mekong Subregion) have reported
a lower vulnerability of the malaria parasite to ACTs [2]. Given the
importance of the ACTs for the treatment of drug-resistant malaria, it
is necessary to search for new and affordable antiplasmodial drugs
that are active against multiple stages of the Plasmodium life cycle
and/or that are able to rescue older antimalarial drugs. Chloroquine
(CQ,1, Fig. 1) was one of the first safe and effective antimalarials to be
widely employed. It is characterized by potent and selective cyto-
toxic action against Plasmodium falciparum, together with a good
* Corresponding author. Department of Biotechnology, Chemistry and Pharmacy
(DoE 2018-2022), University of Siena, Via Aldo Moro 2, 53100, Siena, Italy.
E-mail address: gemma@unisi.it (S. Gemma).
https://doi.org/10.1016/j.ejmech.2021.113227
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

N. Relitti, S. Federico, L. Pozzetti et al.
European Journal of Medicinal Chemistry 215 (2021) 113227
Abbreviations
hpi
hours post infection
i.p
intraperitoneal
ACTs
CP
CQ
CQ-R
CQ-S
VP
artemisinin-based combination therapies
chlorpheniramine
chloroquine
CQ-resistant
chloroquine-sensitive
verapamil
i.v
intravenous
microwaves
MW
PetEt
PfCRT
PQ
petroleum ether
P. falciparum chloroquine-resistance transporter
primaquine
CLT
clotrimazole
DCE
DCM
DFA
DMF
DMSO
DV
dichloroethane
TBAF
tetrabutylammonium fluoride
dichloromethane
direct feeding assay
dimethylformamide
dimethyl sulfoxide
digestive vacuole
green fluorescent protein
TBDPSCl tert-butyldiphenylchlorosilane
TEA
TFA
THF
TZP
SAR
triethylamine
trifluoroacetic acid
tetrahydrofuran
triazolopyrimidine
structure-activity relationship
GFP
no intrinsic antiplasmodial activity against the intraerythrocytic
stage of Plasmodium [15]. Their mode of action arises from the in-
hibition of CQ transport via the mutant isoforms of PfCRT
[14,16e18], thus reinstating the ability of CQ to accumulate inside
the DV by a weak-base trapping mechanism [19]. Aside from its
roles in the phenomenon of multidrug resistance [6], PfCRT is
essential for the survival of the parasite [20]. Its natural function
was recently revealed to be the efflux of large host-derived peptides
(4e11 residues in length) from the DV, with PfCRT3D7 translocating
a broader range of peptides and peptide mimics, and at a higher
capacity, than almost all of the mutant isoforms (including
PfCRTDd2) [21].
We have been pursuing the design and development of new
antiplasmodial drugs endowed with various modes of actions
[22,23]. These included clotrimazole (CLT)-based antiplasmodials
agents as a novel scaffold able to interfere with heme metabolism
inside the DV, as well as a new class of CQ-CLT-hybrid compounds
[24e26]. Benzhydryl-derivatives belonging to the latter class,
typified by compounds 4a,b (Fig. 1), showed an excellent anti-
plasmodial activity profile against both CQ-S and CQ-R strains of
P. falciparum in vitro and in vivo. These molecules share all or most
of the following features: a weak basic heme-complexing group, a
quinoline moiety (to provide the antiplasmodial effect), and a
benzhydryl system presenting a basic group (to enable accumula-
tion of the compound inside the DV via weak-base trapping). Most
of these molecules showed good antiplasmodial potency against
the CQ-R strains, which led us to explore the mechanisms involved
in their activity. It was observed that both 4a and 4b were able to
inhibit CQ transport via PfCRTDd2 in the Xenopus oocyte expression
system [24]. Encouraged by this discovery and the growing interest
in PfCRT as a promising drug target [21,27e29], we investigated a
series of new analogues of 4 in order to improve their activity
against PfCRTDd2 and to explore the structural features required for
the inhibition of PfCRTDd2-mediated CQ transport, whilst main-
taining the intrinsic antiplasmodial properties. Here, we designed
and tested analogues 5a-k, and 6c,h,i (Table 1) to evaluate the effect
of substitutions at (1) the benzhydryl moiety and (2) the quinoline
ring on activity against PfCRTDd2-mediated CQ transport as well as
on antiplasmodial activity. In addition, compounds 5l,m were
evaluated in an attempt to improve the inhibitory activity against
Fig. 1. Structure of chloroquine (1), verapamil (2), primaquine (3) and 4a,b.
safety profile and low-cost of the treatment regimen. However, after
decades of use, a dramatic spread of CQ-resistant (CQ-R) strains of
malaria parasites occurred [3]. The resistance of P. falciparum to CQ is
related to an increased capacity of the parasite to expel the drug from
the digestive vacuole (DV), which is its site of action. CQ-R parasites
accumulate much less CQ in their DV than CQ-sensitive (CQ-S) par-
asites do [4,5], and the presence of a resistance-reverser (e.g., the
calcium channel blocker verapamil, VP, 1, Fig. 1) increases the accu-
mulation of CQ in CQ-R parasites. The mechanism behind the CQ-
efflux from the DV of P. falciparum has been established [6]. CQ is
transported out of the DV by a mutant isoform of the P. falciparum
chloroquine-resistance transporter (PfCRT) [7e10], a protein that
localizes to the membrane of this organelle [11,12].
A cryo-electron microscopy structure of PfCRT (49 kDa) com-
plexed with a recombinant PfCRT-specific antigen-binding frag-
ment (50 kDa) has recently been published [7]. PfCRT consists of
424 amino acids, with ten transmembrane domains that cross the
DV membrane. Expression of PfCRT at the surface of Xenopus oo-
cytes has revealed that the isoforms of the protein associated with
CQ resistance, such as the ‘Dd2’ variant (PfCRTDd2), mediate CQ
transport [7,8]. By contrast, wild-type PfCRT (PfCRT3D7) lacks sig-
nificant CQ transport activity. From in vitro assays, it has been
observed that the sensitivity of CQ-R P. falciparum strains to CQ can
be partially restored by VP (2, Fig. 1) as well as by various other
compounds, such as the liver-stage antimalarial primaquine (PQ, 3
Fig. 1) [5,13,14]. These CQ resistance-reversers do not usually affect
the accumulation of CQ in the DV of CQ-S strains, and exert little or
PfCRTDd2
.
2. Chemistry
Compounds 5a-k, 5m were synthesized as described in
Schemes 1e4, while the synthesis of 5l and 6a-i is reported in the
2

Table 1
Antiplasmodial activity of compounds 5a-l, 6c,h,i and of reference compounds CQ (1), PQ (3) and 4a,b.
Compound
1 (CQ)
3 (PQ)
R1
-
-
R2
-
-
R3
-
-
X
-
-
IC50
(
m
M) D10
IC50 (mM) W2
0.022
5.7
0.280
4.4
4a[24]
m-Cl
H
0.025
0.033
4b[24]
5a
p-Cl
p-Cl
H
H
H
H
H
H
H
0.062
0.065
0.084
0.019
0.023
0.040
0.179
0.058
0.069
0.088
0.027
0.022
0.075
0.475
5b
m-Cl
5c
p-Cl
5d
m-Cl
5e
m-OMe
m-OH
5f
5g
5h
5i
p-Cl
p-Cl
m-Cl
H
OH
H
3.70
2.00
2.80
2.70
2.00
2.30
5j
m-Cl
m-Cl
H
H
4.50
3.70
3.70
0.92
5k
5l
-
-
-
-
0.595
0.460
6c
p-Cl
-OH
H
>15
>15
6h
6i
p-Cl
p-Cl
¼O
-
>15
>15
8.6
11
-OH
H
3

N. Relitti, S. Federico, L. Pozzetti et al.
European Journal of Medicinal Chemistry 215 (2021) 113227
Supporting Information file (Schemes S1-5). For the synthesis of
compounds 5a-f and 5i,j an approach that parallels our previously
described synthetic pathway was followed (Scheme 1). Starting
from the appropriate benzhydrols 6a-d, the corresponding benz-
hydryl chlorides were obtained by treatment with SOCl2 in dry
DCM. These chlorides were used for the alkylation of the 7-chloro-
4-aminoquinoline obtaining compounds 5a-d. For the synthesis of
compounds 5e,f, benzhydrols intermediates 11 and 12 were pre-
pared starting from aldehydes 8 and 9. For the synthesis of inter-
mediate 10, the free OH group of 8 was protected to the
corresponding silyl-derivative. Aldehydes 9 and 10 were then
subjected to a lithium halogen exchange reaction to afford com-
pounds 11 and 12, respectively. Deprotection of the cyclic ketal of
both compounds released the free aldehyde that was then sub-
mitted to a reductive amination protocol to afford the tertiary
amines 13 and 14. Starting from these latter compounds, the sub-
sequent steps, namely chlorination to 15, 16, and reaction with 7-
chloro-4-aminoquinolines, afforded protected derivatives 17 and
18. Their deprotection afforded the final compounds 5e,f. Following
Scheme 2. Reagents and Conditions: a) Methylmagnesium bromide, [1,2-
bis(diphenylphosphino)ethane]dichloronickel (II), Et2O, 0e25 ꢀC, 12 h; b) tert-butyl
iodide, I2, FeCl2, TFA, DMSO, 80 ꢀC, 6 h; c) 4-bhlorophenylmagnesium bromide, THF,
0e25 ꢀC, 6 h; d) MnO2, CHCl3, 25 ꢀC, 3 h; e) 2-(4-bromophenyl)-1,3-dioxolane, n-BuLi,
THF, ꢁ70 ꢀC, 2 h; f) acetone, H2O, 140 W, 100 ꢀC, 30 min; g) 1-Boc-piperazine,
NaBH3CN, EtOH, AcOH, 25 ꢀC, 12 h; h) TFA, DCM, 25 ꢀC, 1.5 h; i) H3PO2, I2, AcOH, H2O,
60 ꢀC, 12 h.
a
similar reaction pathway, alkylation of 8-amino-6-
reaction with 4-chlorophenylmagnesium bromide obtaining an
alcohol intermediate that was oxidized to the ketone 24 with
MnO2. This latter compound was submitted to a reaction with n-
BuLi and 2-(4-bromophenyl)-1,3-dioxolane affording compound
25. The dioxolane group was converted to aldehyde through a
treatment with a mixture of water/acetone under microwave
irradiation [33]. The obtained aldehyde was used in the following
reductive amination reaction with 1-Boc-piperazine giving com-
pound 26. From this intermediate, the two final compounds 5g,h
were obtained after Boc deprotection and a dehydroxylation pro-
cedure involving H3PO2 and I2 (5h) [34].
For the synthesis of 5k (Scheme 3), aminoguanidine 27 was
cyclized to triazole 28 by treatment with AcOH [35], then this
compound was converted to the 2-methyl-[1,2,4]triazolo [1,5-a]
pyrimidine-5,7-diol by treatment with diethyl malonate in the
presence of NaOEt [36], afterward the resulting intermediate was
converted to the dichloro-derivative 29 through a reaction con-
ducted in POCl3 [37,38]. Intermediate 29 was then submitted to an
aromatic substitution with the benzhydrylamine 30 and subse-
quently treated with a methanolic solution of NaOMe under MW
irradiation, to afford compound 31. After deprotection of the Boc-
group, the final compound 5k was isolated. Amine 30 was in turn
prepared by treatment of ketone 32 (obtained as previously re-
ported [24,25]) with hydroxylamine and successive reduction of
the oxime with Zn in acidic medium.
methoxyquinoline with benzhydryl chlorides 19 and 20 (synthe-
sized as reported [25,30]) afforded 21 and 5j, respectively. Starting
from 21, final compound 5h was obtained by acid-promoted N-Boc
deprotection.
In Scheme 2 the synthesis of the final compounds 5g,h is re-
ported. 4,7-Dichloroquinoline (22) was converted to 7-chloro-4-
methylquinoline by treatment with methylmagnesium bromide,
in the presence of [1,2-bis(diphenylphosphino)ethane]dichlor-
onickel (II) as catalyst [31]. The methyl group was oxidized to the
corresponding aldehyde through a reaction carried out in the
presence of tert-butyl iodide, DMSO, AcOH and FeCl2 obtaining
compound 23 [32]. This intermediate was used in the next Grignard
Scheme 1. Reagents and Conditions: a) SOCl2, DCM, 50 ꢀC, 2 h; b) 4-amino-7-
chloroquinoline CH3CN, TEA, 80 ꢀC, 12 h; c) TBDPSCl, imidazole, THF, 0e25 ꢀC, 12 h;
d) 2-(4-bromophenyl)-1,3-dioxolane, n-BuLi, THF, ꢁ70 ꢀC, 2 h; e) HCl, 1 N, THF, 4 h,
25 ꢀC; f) 1-Boc-piperazine, NaBH3CN, 1% AcOH in EtOH, 12 h, 25 ꢀC; g) 1 N HCl in
MeOH, 40 ꢀC, 30 min; h) TBAF, THF, 0e25 ꢀC, 12 h; i) 8-amino-6-methoxy quinoline,
CH3CN, TEA, 80 ꢀC, 12 h.
Scheme 3. Reagents and Conditions: a) AcOH, toluene, 110 ꢀC, 12 h; b) diethyl mal-
onate, EtONa, EtOH, 0e80 ꢀC, 12 h; c) POCl3, 110 ꢀC, 12 h; d) EtOH, 25 ꢀC, 12 h; e)
NaOMe, MeOH, 100 W, 85 ꢀC, 30 min; f) 1 N HCl in MeOH, 40 ꢀC, 30 min; g)
NH2OH$HCl, BaCO3, MeOH, 65 ꢀC, 12 h; h) Zn, AcOH, NH4Cl, 50 ꢀC, 12 h.
4

N. Relitti, S. Federico, L. Pozzetti et al.
European Journal of Medicinal Chemistry 215 (2021) 113227
The synthesis of the dimeric derivative 5m is reported in
Scheme 4. Alcohol 14 was oxidized with MnO2 to the corresponding
ketone, and upon deprotection of the silyl group, intermediate 33
was obtained. Compound 33 was then reacted with ethylene glycol
dimesylate in the presence of sodium hydride. The monoalkylated
derivative 34 was the major product of the reaction, while only
traces of 35 were collected from the reaction mixture. The reaction
between 34 and another equivalent of 33 afforded the bis-alky-
lated-derivative 35 in reasonable yield. Compound 5m was finally
obtained from 35 through chlorination, reaction with 7-chloro-4-
aminoquinoline and N-Boc deprotection.
values of 40 and 75 nM against the CQ-S and CQ-R strains,
respectively. Since the phenolic analog 5f showed a marked
decrease in antiplasmodial activity, especially against the CQ-R
strain (IC50 of 475 nM), coupled with a moderate cross-resistance
to CQ, its activity against PfCRTDd2 was not evaluated.
To confirm the importance of the 4-aminoquinoline moiety on
biological activity, a further set of compounds (5g,h and 6c,h,i) was
designed, synthesized, and tested for inhibitory activity against
PfCRTDd2-mediated transport and P. falciparum growth. Compounds
6c,h,i lack the 4-aminoquinoline moiety, whereas compounds 5g,h
lack the exocyclic nitrogen e which affects both the distance of the
endocyclic quinoline nitrogen from the benzhydryl system and the
basicity of the resulting quinoline. All of these compounds exhibi-
ted reduced activity against PfCRTDd2 (relative to 4a,b) and a sub-
stantial decrease in antiplasmodial activity against both the CQ-R
and CQ-S strains. Compounds 6c,h,i were the weakest inhibitors of
PfCRTDd2-mediated CQ transport (inhibition of only 22e43% when
3. Results and structure-activity relationship (SAR) analysis
All synthesized compounds were evaluated for their in vitro
antiplasmodial activity against the CQ-S strain D10 (which carries
PfCRT3D7
) and the CQ-R resistant strain W2 (which carries
present at 100
4-aminoquinoline moiety and of its basicity to get a potent anti-
plasmodial activity and for inhibitory activity against PfCRTDd2
mM). These results confirmed the importance of the
PfCRTDd2) and for their ability to inhibit the PfCRTDd2-mediated
transport of CQ (Table 1 and Fig. 2). P. falciparum viability was
assessed using the activity of the parasite lactate hydrogenase as a
marker, and inhibitory activity against PfCRTDd2 was assessed in the
Xenopus oocyte system. A subset of 15 compounds were screened
for the ability to inhibit the transport of [3H]CQ via PfCRTDd2 when
.
Dimers of 4a were also synthesized in an attempt to increase the
inhibitory activity against PfCRTDd2 [29]. Two different approaches
were used to obtain those compounds: (i) the piperazine moiety
was used as a dimerizing unit to produce compound 5l; (ii) an ethyl
glycol linker was used to connect two piperazine moieties, yielding
compound 5m. However, when compared with 4a, both 5l and 5m
exhibited reduced inhibitory activities against PfCRTDd2 that could
be ascribed to a non-correct orientation of the quinoline/proto-
natable chain moieties in the transporter induced by the linkers.
Interestingly, the derivatives bearing the 6-methoxy-8-
aminoquinoline system characteristic of primaquine (compounds
5i,j) retained appreciable activity against PfCRTDd2 (~65% inhibition
present at 100
mM in the extracellular solution. Treatments con-
taining 100
m
M of a known inhibitor of PfCRTDd2 e VP, chlor-
pheniramine (CP), or PQ e were included for comparison, and non-
expressing oocytes were used as a negative control. In all cases, the
lack of an effect in the non-expressing oocytes indicated that none
of the compounds affect the simple diffusion of [3H]CQ into the
oocytes (Fig. 2A). Beginning with the two reference compounds
4a,b, we explored small modifications to their chemical structures
to understand the key substitutions of our molecules necessary for
the inhibition of PfCRTDd2. First, we verified the importance of the
piperazine nitrogen atoms by isosteric replacement. Removal of the
distal nitrogen gave rise to the isosteric piperidines 5a,b or pyrro-
lidines 5c,d through ring contraction. In all four cases, a reduction
in the ability to inhibit PfCRTDd2 was observed, but this effect was
more evident with the piperidines (5a,b). Regarding the anti-
plasmodial activity, only modest changes in the IC50 values against
CQ-S and CQ-R strains were observed, with compounds 5c,d
exhibiting the lowest IC50 values (27 and 22 nM, respectively)
against the CQ-R strain. Comparisons between 5a,b and 5c,d
revealed that the position of the chloride on the benzene ring did
not affect inhibitory activity against PfCRTDd2. The replacement of
the m-Cl with a m-OMe yielded compound 5e, which inhibited
PfCRTDd2 to a similar extent as compounds 4a,b and produced IC50
at 100 mM), whilst also exhibiting poor antiplasmodial activity, which
is slightly higher than PQ. The final modification to the quinoline
group was the substitution of the 6-methoxy-8-aminoquinoline
moiety with a bioisosteric heterocycle (5k). PQ and its metabolic
derivatives have an important role in the toxicity associated with this
drug. Indeed, PQ and several of its metabolites, the most represen-
tative of which is 5-hydroxyprimaquine, induce the formation of
methemoglobinemia at a rate and extension higher than usual,
leading to the occurrence of hemolytic anemia [39]. Based on these
observations, we replaced the quinoline group of 5i,j with a tri-
azolopyrimidine (TZP) as a bioisostere of the quinoline moiety of CQ
[36,40e43]. Relative to compounds 5i,j, 5k was considerably more
potent against the CQ-R strain, with a >10-fold reduction in the IC50
(920 nM), but showed a similar level of activity against the CQ-S
strain (3.70
this study, 5k emerged as the most potent inhibitor of CQ transport
via PfCRTDd2 (~98% inhibition at 100
M and an IC50 of 12.5 M).
mM). Furthermore, amongst the compounds tested in
m
m
The poor antiplasmodial activity of the primaquine derivatives
5i,j against asexual blood-stage P. falciparum parasites can be
readily explained by the mode of action of PQ and its derivative,
which target exoerythrocytic forms of the parasites such as ga-
metocytes and hepatic schizonts via a P450-dependent bio-
activation mechanism [44]. On the other hand, metabolic and/or
non-enzymatic formation of PQ hydroxylated metabolites also
play an important role in the toxicity of this drug [45]. Based on the
above datasets, we sought to measure the activity of our PQ ana-
logues against the exoerythrocytic forms of Plasmodium. A pre-
liminary evaluation of the cytotoxicity of compounds 5i-k revealed
that 5i,j exhibited a moderate level of cytotoxicity in the NIH3T3
cell line (TC50 ¼ 19 and 28
m
M, respectively), whereas compound 5k
Scheme 4. Reagents and Conditions: a) MnO2, DCE, 80 ꢀC, 6 h; b) TBAF, THF, 2 h,
25 ꢀC; c) ethylene glycol dimesylate, NaH, DMF, 2 h, 80 ꢀC; d) 33, NaH, DMF, 2 h, 80 ꢀC;
e) NaBH4, THF, 12 h, 25 ꢀC; f) SOCl2, 3 h, 80 ꢀC; g) 4-amino-7-chloroquinoline, TEA,
CH3CN, 12 h, 80 ꢀC; h) 1 N HCl in MeOH, 40 ꢀC, 30 min.
produced a TC50 value lower than 3.5
mM. Hence, only compounds
5i,j were progressed to assays with exoerythrocytic forms of
Plasmodium.
5

N. Relitti, S. Federico, L. Pozzetti et al.
European Journal of Medicinal Chemistry 215 (2021) 113227
Fig. 2. Effect of compounds 5a-m and 6c,h,i on the PfCRTDd2-mediated transport of CQ into Xenopus oocytes. (A) The uptake of [3H]CQ into non-expressing oocytes (white bars) and
oocytes expressing PfCRTDd2 (grey bars) was measured in the absence or presence of 100
M of the test compound. The reference drugs were VP, CP, and PQ, all of which partially
m
reverse CQ resistance in vitro by inhibiting the transport of CQ via PfCRTDd2 [14,17]. The component of transport attributable to PfCRTDd2 (i.e. PfCRT-mediated transport) was
calculated by subtracting the level of uptake measured in non-expressing oocytes from that measured in oocytes expressing PfCRTDd2 (see Fig. S1). Within each experiment,
measurements were made from 10 oocytes per treatment and uptake was expressed relative to that measured in the PfCRTDd2-expressing oocytes under control conditions. The
data are the mean of n ¼ 3e11 independent experiments (each yielding similar results and overlaid as individual data points) and are shown s.e.m. The grey asterisks denote a
significant difference (***P < 0.001, **P < 0.01, and *P < 0.005; one-way ANOVA) in [3H]CQ accumulation between the control PfCRTDd2 treatment and that measured in the
presence of a test compound. Note that non-expressing oocytes take up [3H]CQ to a low level via simple diffusion of the neutral species of the drug; this represents the “background”
level of CQ accumulation in oocytes [7]. The concentration-dependence of the effects of 5i (B), 5j (C), 5k (D), and 5l (E) on [3H]CQ uptake into oocytes expressing PfCRTDd2 (see
Fig. S1). PfCRTDd2-mediated transport (grey circles) was calculated by subtracting the level of uptake measured in non-expressing oocytes from that measured in oocytes expressing
PfCRTDd2. The IC50s derived from these data were obtained by a least-squares fit of the equation Y ¼ Ymin þ [(Ymax ꢁ Ymin)/(1 þ ([inhibitor]/IC50)C], where Y is the component of CQ
uptake attributable to PfCRTDd2, Ymin and Ymax are the minimum and maximum values of Y, and C is a constant. Uptake is shown as the mean s.e.m. from four (D and E) or six (B
and C) independent experiments, within which measurements were made from 10 oocytes per treatment. Where not shown, error bars fall within the symbols.
3.1. In vitro evaluation of 5i and 5j against liver-stage parasites
dose-response effect on the number of parasites. PQ was the least
active (8 M) to reduce the parasite numbers to about 50%. Com-
pound 5i reached this level at doses as low as 0.5 M and com-
pound 5j even lower at 0.125 M. Not only the number of parasites
was reduced but also their size. Whereas PQ reduced the average
parasite size to about half at 2 M, compound 5i showed a 50%
m
Since PQ is the most important commercially available drug
against the liver-stage of malaria, the PQ derivatives 5i and 5j were
also tested against the liver-stage of P. berghei. Infected HepG2 cells
were treated from 2 hpi onwards with different doses of PQ, 5i or 5j.
At 48 hpi, parasite number (Fig. 3A) and size (Fig. 3B) were evalu-
ated by automated microscopy. All the three drugs tested showed a
m
m
m
reduction at 125 nM, and compound 5j at 62.5 nM. These data
demonstrate that the two PQ derivatives, compared with PQ, are
6

N. Relitti, S. Federico, L. Pozzetti et al.
European Journal of Medicinal Chemistry 215 (2021) 113227
Fig. 3. Effects of compounds 5i and 5j on liver-stage growth and development compared to primaquine. HepG2 cells infected with mCherry expressing Plasmodium berghei
sporozoites were exposed to different drug concentrations from 2 hpi. At 48 h the cells were fixed, and parasite size and count were evaluated by automated microscopy (INCell
analyzer 2000, GE healthcare). The dotted red line indicates the host cell growth inhibiting concentration. DMSO was used as solvent control at the highest concentration used (e.g.
for 5i, same as for 8 mM concentration). In A) mean parasite numbers (6 replicates) with SD are shown for the individual compound concentrations. In B) median parasite sizes with
interquartile range are shown for each concentration. The number of parasite size measurements were limited to 100 parasites for illustrative purposes. Statistical evaluations (One-
way ANOVA with Dunnett’s multiple comparisons) were done using Prism 8 (GraphPad) (ns: not significant, **: P < 0.01,****: P < 0.0001).
more potent inhibitors against liver-stage parasites of P. berghei.
3.2. In vivo evaluation of 5i and 5j as transmission blocking
compounds
The effects of the PQ analogues 5i and 5j on transmissible stages
of Plasmodium were assessed with the murine malaria parasite
P. berghei (ANKA strain GFP-con) using BALB/c mice and Anopheles
stephensi mosquitoes. Neither 5i nor 5j showed any impact on
transmissible stages. Mosquitoes fed on gametocytaemic mice,
previously treated with 5i or 5j at 10 mmol/kg resulted in being
highly infected (Fig. 4). Mean oocyst numbers amounted to 454 in
the 5i group and 491 in 5j treated mosquitoes. For comparison, a
similarly high number of oocysts (357) was counted in solvent
control mosquitoes whereas in the positive reference group, in
which mice were treated with PQ at the same dosage (10 mmol/kg),
Fig. 4. Effects of 5i and 5j on transmissible stages of P. berghei in vivo. Gametocytaemic
mice were treated with 5i and 5j at 10 mol/kg twice (3 days after mouse infection and
1.5 h before mosquito feeds). PQ (10 mol/kg) served as positive reference and PBS as
solvent control. For each treatment 3 mouse - mosquito cage replicates were per-
formed. Oocyst numbers were assessed on 20e30 mosquitoes (minimum 8) of each
replicate cage by gut dissection on day 8 after infection. Columns represent geometric
mean numbers of oocysts from positive mosquitoes of 3 cages per treatment. Vertical
bars depict the SD. Mean oocyst numbers of the 5i and 5j treatments were not
different from solvent control replicates (2-tailed t-test: 5i vs control: p ¼ 0.2024; 5j
versus control: p ¼ 0.2118).
the mean oocyst number was markedly decreased (17). Since
experimental mice have received 2 treatments, the first on day 3 of
infection at the moment of gametocyte maturation and the second
on day 4 just 1.5 h before mosquito feeding, it can be concluded that
5i and 5j did not interfere neither with gametocytes nor with the
development of early sporogonic stages in the mosquito midgut.
m
m
4. Conclusions
following newly developed synthetic schemes. The SAR analysis
revealed that the presence of the 4-aminoquinoline moiety is
crucial for retaining strong potency against both CQ-S and CQ-R
strains of P. falciparum (5a-f). N-methylenpyrrolidine as a basic
substituent yielded the most potent activity against the
In this work we describe the discovery of a new library of
antimalarial compounds endowed with an inhibitory activity
against the Dd2 isoform of PfCRT. The synthesis of compounds
5a,b,i-l was performed by applying procedures previously
described by us, while the other final products were obtained
7

N. Relitti, S. Federico, L. Pozzetti et al.
European Journal of Medicinal Chemistry 215 (2021) 113227
P. falciparum strains. No particular differences in potency were
highlighted by p- or m-substitution on the aromatic moiety. In this
context, a chloride or a methoxy group generated the compounds
with the highest activities. Among these compounds, 5d and 5e
exhibited potent inhibition of CQ transport via PfCRTDd2. However,
within all of the synthesized products, 5k was the most potent
against PfCRTDd2, and this property correlates with the marked
increase in its cytotoxicity against the CQ-R P. falciparum strain
glassware and anhydrous solvents.
5.2. Chemistry
5.2.1. 1-(4-(Chloro(4-chlorophenyl)methyl)benzyl)piperidine (7a)
To a solution of 6a (0.20 g, 0.63 mmol) in dry DCM (10 mL) SOCl2
(274 m
L, 3.78 mmol) was added and the reaction was stirred at 45 ꢀC
for 1 h. The solvent was evaporated and crude 7a (0.21 g, quanti-
tative yield) was used in the next step without further purifications.
(IC50 ¼ 0.92
m
M) versus its activity against the CQ-S strain
(IC50 ¼ 3.70
m
M). Compounds 5l and 5m, which are dimers of
1H NMR: (300 MHz, CDCl3)
d
7.66 (d, 2H, J ¼ 7.9 Hz), 7.43 (d, 2H,
compound 4a, were also synthesized in an attempt to increase the
inhibitory activity against PfCRT. However, when compared to 4a,b,
both 5l and 5m exhibited reduced inhibitory and cytotoxicity
profiles. Compounds 5i and 5j, linked to an 8-aminoquinoline
moiety, showed a similar activity to their parent compound (PQ)
against blood-stage parasites. Interestingly, in the in vitro assays,
these compounds were up to 64 times more active against the liver-
stage than PQ. These findings will be the subject of further studies
aimed at defining their mode of action. These compounds showed
an inhibitory activity against PfCRT in line with the other analogues
described in this work. From the cytotoxicity assays against murine
fibroblasts, compounds 5i and 5j showed a safe profile. Given that
PQ has a cytotoxic action against the sexual stages of malaria, and
that 5i and 5j were more active than PQ against the liver-stage
parasites, we investigated their activity in an in vivo transmission
blocking assay. Unfortunately, they did not interfere with the
development of either gametocytes or the early sporogonic stages
in the mosquito midgut. We believe that this work may pave the
way for the development of potent antimalarial agents, active
against liver- and blood-stage CQ-S and CQ-R parasites, that are also
potent blockers of PfCRT. Additional optimization of these com-
pounds may be useful to identify potential lead compounds for
further biological investigation.
J ¼ 7.9 Hz), 7.29e7.14 (m, 4H), 6.06 (s, 1H), 4.11 (s, 2H), 3.41 (d, 2H,
J ¼ 11 Hz) 2.64e2.53 (m, 2H), 2.34e2.25 (m, 2H), 1.88e1.77 (m, 2H),
1.37e1.32 (m, 2H). ESI MS m/z: [M þ H]þ 334.
5.2.2. 1-(4-(Chloro(3-chlorophenyl)methyl)benzyl)piperidine (7b)
Starting from 6b (0.18 g, 0.60 mmol) the title compound was
prepared following the procedure of 6a. Crude was used in the next
reaction without any further purification. 7b (0.20 g, quantitative
yield) was obtained as a brown oil. 1H NMR: (300 MHz, CDCl3)
d
7.68e7.65 (m, 2H), 7.38e7.09 (m, 6H), 6.00 (s, 1H), 4.15 (s, 2H),
3.35 (br, 2H), 2.72e2.67 (m, 2H), 2.18e2.12 (m, 2H), 1.77e172 (m,
2H), 1.36e1.31 (m, 2H). ESI MS m/z: [M þ H]þ 334.
5.2.3. 1-(4-(Chloro(4-chlorophenyl)methyl)phenethyl)pyrrolidine
(7c)
Starting from 6c (0.18 g, 0.60 mmol) the title compound was
prepared following the procedure of 7a. Crude was used in the next
reaction without any further purification. 7c (0.20 g, quantitative
yield) was obtained as a brown oil. 1H NMR (300 MHz, CDCl3)
d
7.38e7.29 (m, 6H), 7.28e7.22 (m, 2H), 6.05 (s, 1H), 3.81 (br, 2H),
3.24 (s, 4H), 2.78 (br, 2H), 2.24 (br, 2H), 2.06 (br, 2H). MS (ESI) m/z
334 [MþH]þ.
5.2.4. 1-(4-(Chloro(3-chlorophenyl)methyl)phenethyl)pyrrolidine
(7d)
5. Experimental section
Starting from 6d (0.18 g, 0.60 mmol) the title compound was
prepared following the procedure of 7a. Crude was used in the next
reaction without any further purification. 7d (0.19 g, quantitative
yield) was obtained as a brown oil. 1H NMR (300 MHz, CDCl3)
5.1. General information
Unless otherwise specified, materials were purchased from
commercial suppliers and used without further purification. Re-
action progress was monitored by TLC using silica gel 60 F254
(0.040e0.063 mm) with detection by UV. Silica gel 60
(0.040e0.063 mm) was used for column chromatography. 1H NMR
and 13C NMR spectra were recorded on a Varian 300 MHz spec-
trometer or a Bruker 400 MHz spectrometer by using the residual
signal of the deuterated solvent as internal standard. Splitting
patterns are described as singlet (s), doublet (d), triplet (t), quartet
d
7.44e6.98 (m, 8H), 5.99 (s,1H), 3.73 (br, 2H), 3.21 (br, 4H), 2.81 (br,
2H), 2.26e1.84 (m, 4H). MS (ESI) m/z 334 [MþH]þ.
5.2.5. 7-Chloro-N-((4-chlorophenyl)(4-(piperidin-1-ylmethyl)
phenyl)methyl)quinolin-4-amine (5a)
To a solution of 7a (0.84 g, 2.52 mmol) in MeCN (5 mL) TEA
(1.05 mL, 7.56 mmol) and 4-Amino-7-chloroquinoline (0.54 g,
3.024 mmol) were added. The reaction was stirred at 80 ꢀC for 12 h,
then the solvent was evaporated, water was added, and the mixture
was extracted with DCM. The crude material was purified by
chromatography on silica gel (2% of MeOH in DCM) obtaining 5a
(q) and broad (br); the values of chemical shifts (d) are given in ppm
and coupling constants (J) in Hertz (Hz). HPLC analysis were per-
formed with a Shimadzu Prominence apparatus equipped with a
scanning absorbance UV-VIS detector (Diode Array SPD-M20A) also
equipped with a thermostatic chamber or with Agilent 1100 Series
equipped with UV-VIS detector or LaPrep P130 apparatus using a
P311 UV detector. Purity of final products (>95%) was determined
(0.406 g, 34%) as a white solid. 1H NMR: (300 MHz, CDCl3)
d 8.40 (d,
1H, J ¼ 5.3 Hz), 7.95 (d, 1H, J ¼ 2.1 Hz), 7.74 (d, 1H, J ¼ 9.1 Hz),
7.36e7.23 (m, 8H), 6.22 (d, 1H, J ¼ 5.6 Hz), 5.69 (d, 1H, J ¼ 4.1 Hz),
5.50 (d, 1H, J ¼ 4.4 Hz), 3.44 (s, 2H), 2.36 (br, 4H), 1.60e1.52 (m, 4H),
by analytical HPLC Merk Purospher® STAR RP-18e (5
m
m) LiChro-
1.44e1.42 (m, 2H); 13C NMR (75 MHz, CDCl3)
d 152.0, 149.0, 148.2,
CART® 250-4 column; detection at 254 nm; flow rate ¼ 1.0 mL/
min; mobile phase A, 0.01% TFA (v/v) in water; mobile B, MeCN;
gradient, 90/10e10/90 A/B in 20 min. The gradient was optimized
based on the compound polarity. Unless otherwise indicated,
retention times (RT) refer to the above-described conditions. ESI-
MS spectra were performed by an Agilent 1100 Series LC/MSD
spectrometer. Melting points were detected by a BÜCHI melting
point B-450 and reported as ꢀC. The yields are referred to purified
products and are not optimized. All moisture-sensitive reactions
were performed under argon atmosphere using oven-dried
139.3,135.0,133.7,130.0,129.2,129.0,128.6,127.3,125.7,120.8,117.1,
110.0, 101.3, 63.3, 61.5, 54.5, 25.8, 24.3; HRMS-ESI m/z: calcd for
C
28H28Cl2Nþ3 [MþH]þ: 476.1655; found: 476.1651; ESI MS m/z: 476
[M þH]þ m.p.: 200e202 ꢀC; HPCL RT: 11.2 min.
5.2.6. 7-Chloro-N-((3-chlorophenyl)(4-(piperidin-1-ylmethyl)
phenyl)methyl)quinolin-4-amine (5b)
Starting from 7c (0.12 g, 0.36 mmol) the title compound was
prepared following the procedure of 5a. The crude residue was
purified by flash column chromatography on alumina (5% MeOH in
8

N. Relitti, S. Federico, L. Pozzetti et al.
European Journal of Medicinal Chemistry 215 (2021) 113227
DCM) obtaining 5c (0.50 g, 29%) as a white solid. 1H NMR:
5.2.11. (4-(1,3-Dioxolan-2-yl)phenyl)(3-((tert-butyldiphenylsilyl)
oxy)phenyl)methanol (12)
(300 MHz, CDCl3)
d
8.44 (d, 1H, J ¼ 5.3 Hz), 7.98 (d, 1H, J ¼ 2.3 Hz),
7.73 (d, 1H, J ¼ 8.8 Hz) 7.40e7.25 (m, 8H), 6.24 (d, 1H, J ¼ 5.3 Hz),
5.68 (d, 1H, J ¼ 4.4 Hz), 5.42 (d, 1H, J ¼ 4.1 Hz), 3.46 (s, 2H), 2.38 (br,
4H), 1.61e1.54 (m, 4H),1.47e1.44 (m, 2H); 13C NMR (75 MHz, CDCl3)
Starting from 10 (2.2 g, 6.0 mmol) and 2-(4-bromophenyl)-1,3-
dioxolane (1.3 g, 5.4 mmol) the title compound was prepared
following the procedure of 25. The crude residue was purified by
flash column chromatography on silica gel (25% EtOAc in PetEt)
obtaining 12 (1.5 g, 54%) as colorless oil. 1H NMR: (300 MHz, CDCl3)
d
151.9, 149.0, 148.2, 142.8, 139.2, 138.4, 135.0 (2C), 130.4, 130.2,
129.0, 128.2, 127.4, 127.3, 125.7, 125.3, 120.9, 117.1, 101.3, 63.1, 61.7,
54.5, 25.7, 24.1; HRMS-ESI m/z: calcd for C28H28Cl2Nþ3 [MþH]þ:
476.1655; found: 476.1660; ESI MS m/z: 476 [M þ H]þ; Melting
point 204e206 ꢀC; HPLC RT: 11.0 min.
d
7.67 (d, J ¼ 6.7 Hz, 4H), 7.49e7.30 (m, 8H), 7.19 (d, J ¼ 8.1 Hz, 2H),
7.02 (t, J ¼ 7.9 Hz, 1H), 6.82 (d, J ¼ 7.6 Hz, 1H), 6.77 (s, 1H), 6.64 (d,
J ¼ 9.5 Hz, 1H), 5.79 (s, 1H), 5.62 (s, 1H), 4.16e3.97 (m, 4H), 1.08 (s,
9H); MS (ESI) m/z: 533 [M þ Na]þ.
5.2.7. 7-Chloro-N-((4-chlorophenyl)(4-(2-(pyrrolidin-1-yl)ethyl)
phenyl)methyl)quinolin-4-amine (5c)
Starting from 7c (0.20 g, 0.6 mmol) the title compound was
prepared following the procedure of 5a. The crude residue was
purified by flash column chromatography on alumina (5% MeOH in
DCM) obtaining 5c (0.05 g, 18%) as a pale yellow solid. 1H NMR
5.2.12. tert-Butyl 4-(4-(hydroxy(3-methoxyphenyl)methyl)benzyl)
piperazine-1-carboxylate (13)
Step e: To a solution of 11 (0.40 g, 1.4 mmol) in THF (8 mL) a 1 N
solution of HCl was added (4 mL). Mixture was stirred at 25 ꢀC for
4 h; NaHCO3 was added and THF was evaporated. Residue was
extracted with EtOAc and the combined organic layers were dried
over Na2SO4 and concentrated in vacuo; the crude residue was used
in the next reaction without any further purification. Compound 4-
(hydroxy(3-methoxyphenyl)methyl)benzaldehyde (0.38 g, quantita-
tive yield) was obtained as a colorless oil. 1H NMR: (300 MHz,
(300 MHz, CDCl3)
d
8.41 (d, J ¼ 5.2 Hz, 1H), 7.96 (s, 1H), 7.71 (d,
J ¼ 8.9 Hz, 1H), 7.42e7.12 (m, 9H), 6.21 (d, J ¼ 5.2 Hz, 1H), 5.67 (d,
J ¼ 3.8 Hz, 1H), 5.42 (d, J ¼ 3.1 Hz,1H), 2.93e2.76 (m, 2H), 2.76e2.62
(m, 2H), 2.57 (br, 4H), 1.80 (br, 4H); 13C NMR (75 MHz, CDCl3)
d
151.9, 149.0, 148.2, 140.3, 139.3, 138.5, 135.0, 133.7, 129.5, 129.2,
128.6, 127.6, 125.7, 121.0, 117.1, 101.4, 61.4, 57.9, 54.1, 35.0, 23.5; MS
(ESI) m/z 476 [MþH]þ; HRMS-ESI m/z: calcd for C28H28Cl2N3þ
[MþH]þ: 476.1655; found: 476.1663; HPLC RT: 11.4 min.
CDCl3)
d
9.91 (s, 1H), 7.80 (d, J ¼ 6.7 Hz, 2H), 7.54 (d, J ¼ 8.1 Hz, 2H),
7.24 (t, J ¼ 7.3 Hz, 1H), 6.95e6.86 (m, 2H), 6.80 (d, J ¼ 7.5 Hz, 1H),
5.81 (s, 1H), 3.75 (s, 3H), 3.06 (br, 1H); MS (ESI) m/z: 241 [M ꢁ H]-.
Step f: Compound 13 was obtained starting from the above-
obtained compound (0.35 g, 1.4 mmol) following the procedure
used for 26g. Crude compound was purified by chromatography on
silica gel (40% PetEt in EtOAc) obtaining 13 (0.578 g, 89%) as a pale
5.2.8. 7-Chloro-N-((3-chlorophenyl)(4-(2-(pyrrolidin-1-yl)ethyl)
phenyl)methyl)quinolin-4-amine (5d)
Starting from 7d (0.191 g, 0.6 mmol) the title compound was
prepared following the procedure of 5a. The crude residue was
purified by flash column chromatography on alumina (5% MeOH in
DCM) obtaining 5d (0.117 g, 43%) as a pale-yellow oil. 1H NMR
yellow oil. 1H NMR: (300 MHz, CDCl3)
d 7.35e7.11 (m, 5H), 6.91 (d,
J ¼ 8.9 Hz, 2H), 6.74 (s, 1H), 5.68 (s, 1H), 3.70 (s, 3H), 3.40 (s, 2H),
3.32 (br, 4H), 2.28 (br, 4H), 1.42 (s, 9H); MS (ESI) m/z: 413 [M þ H]þ.
(400 MHz, CDCl3)
d
8.39 (d, J ¼ 5.1 Hz, 1H), 7.96 (s, 1H), 7.70 (d,
J ¼ 8.9 Hz, 1H), 7.44e7.29 (m, 2H), 7.29e7.04 (m, 7H), 6.21 (d,
J ¼ 5.2 Hz, 1H), 5.65 (d, J ¼ 3.9 Hz, 1H), 5.42 (d, J ¼ 3.6 Hz, 1H),
2.87e2.74 (m, J ¼ 8.9 Hz, 2H), 2.74e2.63 (m, 2H), 2.56 (br, 4H), 1.78
(br, 4H); 13C NMR (75 MHz, CDCl3)
d 151.4, 148.6, 148.5, 142.7, 139.0,
5.2.13. tert-Butyl (4-(4-((3-((tert-butyldiphenylsilyl)oxy)
phenyl)(hydroxy)methyl)benzyl)piperazine-1-carboxylate (14)
Starting from 12 (1.5 g, 2.9 mmol) the title compound was
prepared following the procedure of 13. The crude residue was
purified by flash column chromatography on silica gel (25% EtOAc
in PetEt) obtaining 14 (1.5 g, 70%) as colorless oil. 1H NMR:
137.6, 135.2, 135.0, 130.4, 129.5, 128.4, 128.2, 128.1, 127.4, 125.8,
125.5, 121.5, 117.4, 101.2, 61.5, 57.0, 53.9, 32.8, 23.4; MS (ESI) m/z 476
[MþH]þ; HRMS-ESI m/z: calcd for C28H28Cl2Nþ3 [MþH]þ: 476.1655;
found: 476.1660; HPLC RT: 11.1 min.
(300 MHz, CDCl3) d 7.72e7.62 (m, 4H), 7.47e7.28 (m, 6H), 7.24e7.10
5.2.9. 3-((tert-Butyldiphenylsilyl)oxy)benzaldehyde (10)
(m, 4H), 7.04 (t, J ¼ 7.9 Hz, 1H), 6.85 (d, J ¼ 7.2 Hz, 1H), 6.79 (s, 1H),
6.66 (d, J ¼ 8.1 Hz, 1H), 5.61 (s, 1H), 3.46 (s, 2H), 3.41 (br, 4H), 2.36
(br, 4H), 1.45 (s, 9H), 1.08 (s, 9H); MS (ESI) m/z: 637 [M þ H]þ.
To a solution of 8 (2.0 g, 16.4 mmol) in dry THF (100 mL) cooled
at 0 ꢀC, imidazole (1.3 g, 19.7 mmol) and TBDPSCl (5.1 mL,
19.7 mmol) were added. Reaction was stirred at 25 ꢀC for 12 h, then
water was added, solvent was evaporated, and the residue was
extracted with DCM. The combined organic layers were dried over
Na2SO4 and concentrated in vacuo. The crude material was purified
by chromatography on silica gel (5% EtOAc in PetEt) obtaining 10
5.2.14. tert-Butyl 4-(4-(((7-chloroquinolin-4-yl)amino)(3-
methoxyphenyl)methyl)benzyl)piperaz-ine-1-carboxylate (17)
Step e: Starting from 13 (0.124 g, 0.3 mmol), 15 was prepared
following the procedure used for compound 7a. 15 (0.10 g, 76%) was
obtained as a pale yellow oil and was used in the next step without
(5.3 g, 90%) as colorless oil. 1H NMR: (300 MHz, CDCl3)
d 9.82 (s, 1H),
7.76e7.66 (m, 4H), 7.47e7.34 (m, 7H), 7.30e7.27 (m, 1H), 7.24e7.18
(m, 1H), 6.99e6.91 (m, 1H), 1.12 (s, 9H); MS (ESI) m/z: 361 [M þ H]þ.
any further purification. 1H NMR: (300 MHz, CDCl3)
d 7.41e7.28 (m,
5.2.10. (4-(1,3-Dioxolan-2-yl)phenyl)(3-methoxyphenyl)methanol
(11)
4H), 7.26e7.14 (m, 1H), 7.05e6.96 (m, 2H), 6.84 (d, J ¼ 8.2 Hz, 1H),
6.09 (s, 1H), 3.80 (s, 3H), 3.49 (s, 2H), 3.43 (br, 4H), 2.38 (br, 4H),1.46
(s, 9H); MS (ESI) m/z: 431 [M þ H]þ.
Starting from 9 (585 mL, 4.8 mmol) and 2-(4-bromophenyl)-1,3-
dioxolane (1.0 g, 4.4 mmol) the title compound was prepared
following the procedure of 25. The crude residue was purified by
flash column chromatography on silica gel (25% EtOAc in PetEt)
obtaining 11 (0.505 g, 44%) as colorless oil. 1H NMR: (300 MHz,
Starting from 15 (0.10 g, 0.2 mmol), the title compound 17
(0.034 g, 26%) was obtained following the procedure used for
compound 5a. 17 was obtained as a pale-yellow oil. 1H NMR:
(300 MHz, CDCl3)
d
8.38 (d, J ¼ 5.3 Hz, 1H), 7.93 (d, J ¼ 2.1 Hz, 1H),
Acetone)
d
7.50e7.34 (m, Hz, 4H), 7.20 (t, J ¼ 7.9 Hz, 1H), 7.03 (s, 1H),
7.73 (d, J ¼ 9.0 Hz, 1H), 7.37e7.19 (m, 6H), 6.93 (d, J ¼ 7.7 Hz, 1H),
6.90e6.87 (m, 1H), 6.84e6.78 (m, 1H), 6.25 (d, J ¼ 5.4 Hz, 1H), 5.67
(d, J ¼ 4.4 Hz, 1H), 5.55 (d, J ¼ 4.5 Hz, 1H), 3.73 (s, 3H), 3.47 (s, 2H),
3.40 (br, 4H), 2.36 (br, 4H), 1.43 (s, 9H); MS (ESI) m/z: 573 [M þ H]þ.
6.96 (d, J ¼ 6.3 Hz, 1H), 6.77 (dd, J ¼ 8.2, 2.6 Hz, 1H), 5.81 (d,
J ¼ 3.9 Hz, 1H), 5.70 (s, 1H), 4.85 (d, J ¼ 3.9 Hz, 1H), 4.18e3.87 (m,
4H), 3.76 (s, 3H); MS (ESI) m/z: 287 [M þ H]þ.
9

N. Relitti, S. Federico, L. Pozzetti et al.
European Journal of Medicinal Chemistry 215 (2021) 113227
5.2.15. tert-Butyl 4-(4-((3-((tert-butyldiphenylsilyl)oxy)phenyl)((7-
chloroquinolin-4-yl)amino)methyl)benzyl)piperazine-1-carboxylate
(18)
Starting from 14 (0.088 g, 0.1 mmol) the title compound was
prepared following the procedure of 5a. The crude residue was
purified by flash column chromatography on silica gel (2% MeOH in
DCM) obtaining 18 (0.035 g, 34%) as pale-yellow oil. 1H NMR:
chromatography on silica gel (50% EtOAc in PetEt), and it was ob-
tained as a pale-yellow oil (0.05 g, 30%). 1H NMR: (300 MHz, CDCl3)
d
8.54 (d, J ¼ 4.2 Hz, 1H), 7.94 (d, J ¼ 8.3 Hz, 1H), 7.41 (s, 1H),
7.36e7.22 (m, 8H), 6.73 (d, J ¼ 4.8 Hz, 1H), 6.38 (d, J ¼ 2.3 Hz, 1H),
6.10 (d, J ¼ 2.3 Hz, 1H), 5.59 (d, J ¼ 4.9 Hz, 1H), 3.77 (s, 3H), 3.48 (s,
2H), 3.43 (br, 4H), 2.38 (br, 4H), 1.45 (s, 9H); MS (ESI) m/z: 573 [M þ
H]þ.
(300 MHz, CDCl3):
d
8.36 (d, J ¼ 5.1 Hz, 1H), 7.97 (s, 1H), 7.69e7.60
(m, 2H), 7.57e7.50 (m, 2H), 7.45e7.09 (m, 11H), 7.00 (d, J ¼ 7.0 Hz,
2H), 6.84 (d, J ¼ 7.9 Hz, 2H), 6.67 (s, 1H), 6.10 (d, J ¼ 5.4 Hz, 1H), 5.44
(d, J ¼ 4.0 Hz, 1H), 5.08 (d, J ¼ 4.0 Hz, 1H), 3.46 (s, 2H), 3.43 (br, 4H),
2.36 (br, 4H), 1.46 (s, 9H), 1.04 (s, 9H); MS (ESI) m/z: 798 [M þ H]þ.
5.2.19. N-((3-Chlorophenyl)(4-(piperazin-1-ylmethyl)phenyl)
methyl)-6-methoxyquinolin-8-amine (5i)
Compound 5i was obtained starting from 21 (0.017 g,
0.03 mmol) following the procedure described before to obtain
compound 5e. Crude 5i was purified by chromatography on silica
gel (3% MeOH in DCM) and was obtained as a pale yellow oil
5.2.16. 7-Chloro-N-((3-methoxyphenyl)(4-(piperazin-1-ylmethyl)
phenyl)methyl)quinolin-4-amine (5e)
17 (0.085 g, 0.2 mmol) was dissolved in MeOH and a 1 M solu-
tion of HCl in MeOH was slowly added. The mixture was evaporated
at 45 ꢀC under vacuum. This procedure was repeated three times.
5e was obtained as a colorless oil (0.07 g, quantitative yield). 1H
(0.012 g, 86%). 1H NMR: (300 MHz, CDCl3)
d
8.54 (d, J ¼ 4.1 Hz, 1H),
7.94 (d, J ¼ 8.3 Hz, 1H), 7.41 (s, 1H), 7.39e7.16 (m, 8H), 6.73 (d,
J ¼ 4.8 Hz, 1H), 6.38 (d, J ¼ 2.1 Hz, 1H), 6.11 (d, J ¼ 2.0 Hz, 1H), 5.59
(d, J ¼ 4.8 Hz, 1H), 3.80 (s, 3H), 3.46 (s, 2H), 2.88 (br, 4H), 2.41 (br,
4H); 13C NMR: (75 MHz, CDCl3)
d 159.3, 144.9, 144.8, 144.7, 140.9,
NMR: (300 MHz, CDCl3)
d
8.43 (d, J ¼ 5.3 Hz, 1H), 7.98 (d, J ¼ 2.0 Hz,
137.9, 135.6, 135.0, 134.9, 130.3, 129.9, 129.8 (2C), 127.8. 127.7, 125.8,
122.2, 99.0, 93.2, 63.5, 62.3, 55.4, 54.6, 46.2; MS (ESI) m/z: 473 [M þ
H]þ. HPLC (gradient, 95/5e55/45 A/B in 12 min) RT: 9.5 min.
1H), 7.71 (d, J ¼ 9.0 Hz, 2H), 7.43e7.26 (m, 5H), 7.01e6.79 (m, 3H),
6.27 (d, J ¼ 5.4 Hz, 1H), 5.68 (d, J ¼ 4.3 Hz, 1H), 5.40 (d, J ¼ 4.3 Hz,
1H), 3.77 (s, 3H), 3.48 (s, 2H), 2.88 (br, 4H), 2.41 (br, 4H); 13C NMR:
(75 MHz, CDCl3)
d
159.1, 151.0, 148.0, 147.4, 141.6, 138.7, 137.1, 133.9,
5.2.20. N-((3-Chlorophenyl)(4-(pyrrolidin-1-ylmethyl)phenyl)
methyl)-6-methoxyquinolin-8-amine (5j)
Starting from 19 (0.10 g, 0.3 mmol) and SI19 (0.065 g, 0.4 mmol)
the title compound was prepared following the procedure of 21. The
crude material was purified by flash chromatography on silica gel (2%
MeOH in DCM) to give 5j as a pale yellow oil (0.091 g, 50%). 1H NMR
129.1, 128.8, 127.9, 126.3, 124.5, 120.0, 118.5, 116.1, 112.4, 111.7, 100.2,
62.1, 61.0, 54.2, 53.3, 44.9; MS (ESI) m/z: 473 [M þ H]þ; HRMS-ESI
m/z: calcd for C28H30ClN4Oþ [MþH]þ: 473.2103; found: 473.2110;
HPLC RT: 8.9 min.
5.2.17. 3-(((7-Chloroquinolin-4-yl)amino)(4-(piperazin-1-
ylmethyl)phenyl)methyl)phenol (5f)
(300 MHz, CDCl3)
d
8.54 (d, J ¼ 4.2 Hz, 1H), 7.94 (d, J ¼ 8.3 Hz, 1H),
7.51e7.13 (m, 8H), 6.73 (d, J ¼ 4.8 Hz, 1H), 6.38 (s, 1H), 6.11 (s, 1H),
Step g: To a solution of 18 (0.035 g, 0.04 mmol) in dry THF,
cooled to 0 ꢀC (5 mL) a 1 M solution of TBAF in THF (130 L, 0.1 mmol)
was added. Reaction was allowed to reach 25 ꢀC and stirred for 2 h.
Water was added, THF was evaporated and residue was extracted
with DCM and the combined organic layers were dried over Na2SO4
and concentrated in vacuo. Crude was purified by flash chroma-
tography on silica gel (MeOH 3% in DCM). Compound tert-butyl 4-
(4-(((7-chloroquinolin-4-yl)amino)(3-hydroxyphenyl)methyl)benzyl)
piperazine-1-carboylate (0.02 g, 81%) was obtained as pale yellow
5.59 (d, J ¼ 4.9 Hz,1H), 3.80 (s, 3H), 3.60 (s, 2H), 2.51 (br, 4H), 1.78 (br,
5H); 13C NMR (75 MHz, CDCl3)
d 159.3, 144.9, 144.8, 144.8, 140.7,
139.2, 137.5, 135.6, 135.0, 134.9, 130.3, 129.8, 129.7, 127.8, 127.7, 125.8,
122.2, 99.0, 93.2, 62.3, 60.6, 55.4, 54.5, 23.7; ESI MS m/z: 458 [M þ
H]þ; HRMS-ESI m/z: calcd for C28H29ClN3Oþ [MþH]þ: 458,1994;
found: 458.1998; HPLC RT: 14.6 min.
5.2.21. 7-Chloroquinoline-4-carbaldehyde (23)
To a solution of 4,7-dichloroquinoline (1.5 g, 7.6 mmol) in dry
oil. 1H NMR: (300 MHz, CDCl3)
d
8.20 (d, J ¼ 5.5 Hz, 1H), 7.92 (d,
Et2O,
[1,2-bis(diphenylphosphino)-ethane]dichloronickel
(II)
J ¼ 2.0 Hz,1H), 7.54 (d, J ¼ 9.0 Hz,1H), 7.28 (s, 4H), 7.26 (d, J ¼ 0.6 Hz,
1H), 7.25 (d, J ¼ 2.0 Hz, 1H), 7.20 (t, J ¼ 7.8 Hz, 1H), 6.90 (s, 1H), 6.82
(d, J ¼ 8.0 Hz,1H), 6.74 (d, J ¼ 7.8 Hz,1H), 6.15 (d, J ¼ 5.6 Hz,1H), 5.60
(d, J ¼ 4.3 Hz, 1H), 5.48 (d, J ¼ 4.5 Hz, 1H), 3.48 (s, 2H), 3.42 (br, 4H),
2.38 (br, 4H), 1.45 (s, 9H); MS (ESI) m/z: 559 [M þ H]þ.
(0.04 g, 0.07 mmol) was added. Suspension was cooled to 0 ꢀC and a
3.0 M solution of methylmagnesium bromide (2.6 mL, 7.6 mmol) in
Et2O was added. Reaction was stirred at 25 ꢀC for 12 h. A saturated
solution of NH4Cl was added to the reaction and mixture was
extracted with Et2O. The combined organic layers were dried over
Na2SO4 and concentrated in vacuo. The crude material was purified
by chromatography on silica gel (5% of EtOAc in PetEt) obtaining 7-
chloro-4-methylquinoline (1.9 g, 70%) as a low melting point white
Step h: Starting from the above obtained compound (0.02 g,
0.04 mmol), the title compound was prepared following the pro-
cedure of 5e. 5f was obtained as a colorless oil (0.017 g, quantitative
yield). 1H NMR: (300 MHz, CDCl3)
d
8.26 (d, J ¼ 5.4 Hz, 1H), 7.92 (d,
solid. 1H NMR (300 MHz, CDCl3)
d
8.77 (d, J ¼ 4.4 Hz, 1H), 8.10 (s,
J ¼ 2.1 Hz, 1H), 7.59 (d, J ¼ 9.1 Hz, 1H), 7.33e7.17 (m, 6H), 6.90e6.69
(m, 3H), 6.19 (d, J ¼ 5.6 Hz, 1H), 5.63 (d, J ¼ 4.4 Hz, 1H), 5.44 (d,
J ¼ 4.5 Hz, 1H), 3.44 (s, 2H), 2.84 (br, 4H), 2.38 (br, 4H); 13C NMR:
1H), 7.93 (d, J ¼ 8.9 Hz, 1H), 7.52 (d, J ¼ 8.9 Hz, 1H), 7.23 (d,
J ¼ 4.5 Hz, 1H), 2.70 (s, 3H); MS (ESI) m/z 178 [MþH]þ.
To a solution of 7-chloro-4-methylquinoline (0.50 g, 2.8 mmol) in
(75 MHz, CDCl3)
d
158.1, 151.5, 148.9, 148.3, 142.1, 140.1, 137.9, 135.3,
DMSO (15 mL) TFA (274 mL, 3.6 mmol), tert-butyliodide (342 mL,
130.6, 130.2, 128.2, 127.7, 125.9, 121.1, 119.0, 117.0, 115.8, 114.1, 101.3,
2.9 mmol), iodine (0.75 g, 3.0 mmol) and FeCl2 (0.13 g, 0.7 mmol)
were added in this order and mixture was heated at 80 ꢀC for 6 h.
Thereafter a saturated solution of Na2S2O3 and a saturated solution
of NaHCO3 were added. Mixture was extracted with EtOAc and the
combined organic layers were washed with brine, dried over
Na2SO4 and concentrated in vacuo. The crude material was purified
by chromatography on silica gel (20% of EtOAc in PetEt) obtaining
63.2, 61.9, 54.1, 45.8; MS (ESI) m/z: 459 [M þ H]þ. HPLC RT: 7.3 min.
5.2.18. tert-Butyl 4-(4-((3-chlorophenyl)((6-methoxyquinolin-8-yl)
amino)methyl)benzyl)piperazine-1-carboxylate (21)
To a solution of 20 (0.104 g, 0.2 mmol) in dry CH3CN (5 mL)
DIPEA (110 mL, 0.6 mmol) and SI19 (0.05 g, 0.3 mmol) were added.
The resulting solution was stirred at 80 ꢀC for 12 h, then water was
added, solvent was evaporated, and residue was extracted with
DCM. The combined organic layers were dried over Na2SO4 and
concentrated in vacuo. Crude compound 21 was purified by
23 (0.468 g, 87%) as a white solid. 1H NMR (300 MHz, CDCl3)
d 10.45
(s, 1H), 9.21 (d, J ¼ 4.2 Hz, 1H), 9.01 (d, J ¼ 9.1 Hz, 1H), 8.22 (d,
J ¼ 2.1 Hz, 1H), 7.79 (d, J ¼ 4.2 Hz, 1H), 7.68 (dd, J ¼ 9.1, 2.1 Hz, 1H);
MS (ESI) m/z 192 [MþH]þ.
10

N. Relitti, S. Federico, L. Pozzetti et al.
European Journal of Medicinal Chemistry 215 (2021) 113227
5.2.22. (4-Chlorophenyl)(7-chloroquinolin-4-yl)methanone (24)
Step c: To a solution of 23 (1.0 g, 5.2 mmol) in dry THF (5 mL)
cooled at 0 ꢀC, a 1 M solution of 4-chlorophenylmagnesium bro-
mide (5.7 mL, 5.7 mmol) in Et2O was added. Reaction was stirred at
25 ꢀC for 6 h. The reaction was quenched with a saturated solution
of NH4Cl, THF was evaporated and residue was extracted with
EtOAc. The combined organic layers were dried over Na2SO4 and
concentrated in vacuo. The crude material was purified by chro-
matography on silica gel (33% of EtOAc in PetEt) obtaining (4-
7.98 (d, J ¼ 2.1 Hz, 1H), 7.39e7.08 (m, 9H), 6.75 (d, J ¼ 4.6 Hz, 1H),
4.44 (br,1H), 3.49 (s, 2H), 3.39 (br, 4H), 2.36 (br, 4H),1.42 (s, 9H); MS
(ESI) m/z 600 [MþNa]þ.
5.2.25. 7-Chloro-4-((4-chlorophenyl)(4-(piperazin-1-ylmethyl)
phenyl)methyl)quinoline (5g)
Compound 26 (0.025 g, 0.04 mmol) was dissolved in AcOH
(5 mL) then a solution of H3PO4 (123 mL, 0.19 mmol) 50% in water
and a catalytic amount of iodine were added. Reaction was stirred
at 60 ꢀC for 12 h and water was added. AcOH was evaporated under
reduced pressure; residue was neutralized with NaHCO3 and
extracted with DCM. The combined organic layers were dried over
Na2SO4 and concentrated in vacuo. The crude material was purified
by chromatography on alumina (5% of MeOH in DCM) obtaining 28
(0.015 g, 80%) as amorphous white solid. 1H NMR (400 MHz, CDCl3)
chlorophenyl)(7-chloroquinolin-4-yl)methanol (1.3 g, 82%) as
a
white solid. 1H NMR (300 MHz, CDCl3)
d
8.84 (d, J ¼ 4.3 Hz,1H), 8.04
(s, 1H), 7.82 (d, J ¼ 9.0 Hz, 1H), 7.63 (d, J ¼ 4.4 Hz, 1H), 7.39 (d,
J ¼ 9.0 Hz, 1H), 7.33e7.21 (m, 4H), 6.40 (s, 1H); MS (ESI) m/z 304
[MþH]þ.
Step d: To a solution of the above obtained compound (1.3 g,
4.3 mmol) in CHCl3 (100 mL) MnO2 (1.5 g, 17.1 mmol) was added.
Suspension was stirred for 3 h at 25 ꢀC and reaction was filtered
through paper and solvent evaporated. Crude was purified by flash
chromatography on silica gel (20% of EtOAc in PetEt) obtaining 24
d
8.78 (d, J ¼ 4.5 Hz, 1H), 8.10 (d, J ¼ 1.9 Hz, 1H), 7.81 (d, J ¼ 9.0 Hz,
1H), 7.37 (dd, J ¼ 9.0, 2.0 Hz, 1H), 7.33e7.19 (m, 5H), 6.98 (dd, J ¼ 8.1,
3.7 Hz, 3H), 6.81 (d, J ¼ 4.5 Hz, 1H), 6.10 (s, 1H), 3.45 (s, 2H), 2.86 (br,
4H), 2.39 (br, 4H); 13C NMR (75 MHz, CDCl3)
d 151.3, 149.1, 149.0,
(1.2 g, 92%) as a white solid. 1H NMR (300 MHz, CDCl3)
d
9.03 (d,
140.4, 140.2, 136.3, 135.0, 133.0, 130.7, 129.5, 129.4, 129.2, 129.0,
127.8, 125.5, 122.1, 62.3, 52.9, 51.8, 50.9, 44.2; HRMS-ESI m/z: calcd
for C27H26Cl2Nþ3 [MþH]þ: 462.1498; found: 462.1504; MS (ESI) m/z
462 [MþH]þ. HPLC RT: 10.2 min.
J ¼ 4.3 Hz, 1H), 8.21 (d, J ¼ 2.1 Hz, 1H), 7.82 (s, 1H), 7.81e7.72 (m,
2H), 7.52 (d, J ¼ 2.2 Hz, 1H), 7.51e7.43 (m, 2H), 7.38 (d, J ¼ 4.3 Hz,
1H). MS (ESI) m/z 302 [MþH]þ.
5.2.23. (4-(1,3-Dioxolan-2-yl)phenyl)(4-chlorophenyl)(7-
chloroquinolin-4-yl)methanol (25)
5.2.26. (4-Chlorophenyl)(7-chloroquinolin-4-yl)(4-(piperazin-1-
ylmethyl)phenyl)methanol (5h)
To a solution of 2-(4-bromophenyl)-1,3-dioxolane (0.186 g,
To a solution of 26 (0.035 g, 0.06) in dry DCM (3 mL) TFA (250 mL)
0.8 mmol) in dry THF (10 mL) cooled at ꢁ70 ꢀC a solution of n-BuLi
was added and mixture was stirred at 1.5 h at 25 ꢀC. Reaction was
quenched with NaHCO3 and extracted with DCM. The combined
organic layers were dried over Na2SO4 and concentrated in vacuo.
The crude material was purified by chromatography on alumina (2%
of MeOH in DCM) obtaining 5h (0.02 g, 68%) as amorphous white
(485
m
L, 1.0 mmol) was added. Mixture was stirred at ꢁ70 ꢀC for
30 min, then a solution of 24 (0.27 g, 0.9 mmol) in dry THF (5 mL)
was added, and reaction was stirred for another 1.5 h at ꢁ70 ꢀC.
Thereafter, the reaction was allowed to reach 25 ꢀC, water was
added and THF was evaporated. Residue was extracted with EtOAc
and the combined organic layers were washed with brine, dried
over Na2SO4 and concentrated in vacuo. The crude material was
purified by chromatography on silica gel (20% of EtOAc in PetEt)
obtaining 25 (0.287 g, 78%) as amorphous white solid. 1H NMR
solid. 1H NMR (400 MHz, CDCl3)
d
8.64 (d, J ¼ 4.6 Hz, 1H), 8.15e7.88
(m, 2H), 7.42e7.03 (m, 9H), 6.77 (d, J ¼ 4.6 Hz, 1H), 3.43 (s, 2H), 2.73
(br, 4H), 2.33 (br, 4H); 13C NMR (75 MHz, CDCl3)
151.0,150.5,150.0,
d
144.1, 143.8, 137.3, 134.8,133.8, 129.5,129.1, 128.9, 128.5, 127.5, 127.1,
124.7, 121.8, 82.1, 62.5, 52.0 (2C), 44.7; HRMS-ESI m/z: calcd for
(300 MHz, CDCl3)
d
8.57 (d, J ¼ 4.5 Hz, 1H), 8.11e7.84 (m, 2H), 7.44
C
27H26Cl2N3Oþ [MþH]þ: 478.1447; found: 478.1455; MS (ESI) m/z
(d, J ¼ 8.0 Hz, 2H), 7.37e7.02 (m, 7H), 6.73 (d, J ¼ 4.5 Hz, 1H), 5.77 (s,
478 [MþH]þ. HPLC RT 9.6 min.
1H), 4.31e3.79 (m, 5H); MS (ESI) m/z 452 [MþH]þ.
5.2.27. 3-Methyl-1H-1,2,4-triazol-5-amine (28)
5.2.24. tert-Butyl 4-(4-((4-chlorophenyl)(7-chloroquinolin-4-
yl)(hydroxy)methyl)benzyl)piperazine-1-carboxylate (26)
To aminoguanidine bicarbonate (2.0 g, 14.7 mmol) AcOH (2 mL)
was added. Mixture was stirred at 25 ꢀC until effervescence had
completely vanished, then toluene (100 mL) was added, reaction
was linked to a dean-stark system and heated at 110 ꢀC for 12 h.
Solvent was evaporated and residue was purified by chromatog-
raphy on silica gel (11% MeOH, 1.1% NH4OH in DCM). 28 (1.2, 86%)
Step f: Compound 25 (0.05 g, 0.1 mmol) was dissolved in water
(2 mL) and the minimum amount of acetone necessary to have a
limpid solution was added. Mixture was submitted under MW
irradiation at 140 W, 100 ꢀC for 30 min. Mixture was extracted with
EtOAc and the combined organic layers were dried over Na2SO4 and
concentrated in vacuo. Residue 4-((4-chlorophenyl)(7-chloroqu
inolin-4-yl)(hydroxy)methyl)benzaldehyde (quantitative yield) was
used in the next reaction without any further purification. 1H NMR
was obtained as a white solid. 1H NMR: (300 MHz, DMSO)
d 5.47 (br,
2H), 2.03 (s, 3H), 1.85 (s, 1H); ESI MS m/z: 99 [M þ H]þ; m.p.
143e145 ꢀC.
(300 MHz, CDCl3)
d
10.02 (s, 1H), 8.72 (d, J ¼ 4.6 Hz, 1H), 8.08 (s, 1H),
5.2.28. 5,7-Dichloro-2-methyl-[1,2,4]triazolo[1,5-a]pyrimidine (29)
To 20 mL of dry EtOH Na0 (0.32 g, 13.9 mmol) was added at 0 ꢀC;
after complete Na solubilization compound 28 (1.1 g, 11.6 mmol)
and diethyl malonate (2 mL, 13.2 mmol) were added. The reaction
was heated at 80 ꢀC for 12 h. A white precipitate was obtained and
collected, then it was washed with EtOH, solubilized in water, and
precipitated again with HCl 37%. The white solid obtained was
collected, obtaining 2-methyl-[1,2,4]triazolo[1,5-a]pyrimidine-5,7-
7.95 (d, J ¼ 9.2 Hz, 1H), 7.87 (d, J ¼ 8.0 Hz, 2H), 7.45 (d, J ¼ 8.0 Hz,
2H), 7.35 (d, J ¼ 8.4 Hz, 2H), 7.33e7.23 (m, 1H), 7.19 (d, J ¼ 8.4 Hz,
2H), 6.76 (d, J ¼ 4.4 Hz, 1H); MS (ESI) m/z 408 [MþH]þ.
Step g: The above obtained compound (0.05 g, 0.12 mmol) was
dissolved in a 1% solution of AcOH in EtOH (5 mL) and 1-Boc-
piperazine (0.022 g, 0.1 mmol) and NaBH3CN (0.015 g, 0.3 mmol)
were added. Reaction was stirred at 25 ꢀC for 12 h, then a saturated
solution of NaHCO3 was added, and EtOH was evaporated. Residue
was extracted with DCM and the combined organic layers were
dried over Na2SO4 and concentrated in vacuo. The crude material
was purified by chromatography on silica gel (1% of MeOH in DCM)
obtaining 26 (0.06 g, 87%) as amorphous white solid. 1H NMR
diol (1.2 g, 62%). 1H NMR: (300 MHz, DMSO)
d 12.24 (br, 2H), 5.04
(s, 1H), 2.33 (s, 3H); ESI MS m/z: 167 [M þ H]þ; m.p. decomposition
at 220 ꢀC.
The above obtained compound ((1.2 g, 7.22 mmol) was sus-
pended in POCl3 (20 mL) and stirred at 110 ꢀC for 12 h POCl3 was
evaporated and the residue was neutralized with a saturated
(300 MHz, CDCl3)
d
8.59 (d, J ¼ 4.6 Hz, 1H), 8.05 (d, J ¼ 9.3 Hz, 1H),
11

N. Relitti, S. Federico, L. Pozzetti et al.
European Journal of Medicinal Chemistry 215 (2021) 113227
solution of NaHCO3, and extracted with DCM. The combined
organic layers were dried over Na2SO4 and concentrated in vacuo.
Crude compound was purified by chromatography on silica gel (2%
MeOH in DCM) obtaining 29 (0.883 g, 60%) as a white solid. 1H
5.2.32. tert-Butyl 4-(4-(3-hydroxybenzoyl)benzyl)piperazine-1-
carboxylate (33)
Step a: 14 (1.0 g, 1.6 mmol) was dissolved in DCE (50 mL) and
85% MnO2 (0.655 g, 6.40 mmol) was added. The reaction was stirred
at 80 ꢀC for 6 h then it was filtered through paper and the solvent
was evaporated. tert-Butyl 4-(4-(3-((tert-butyldiphenylsilyl)-oxy)-
benzoyl)benzyl)piperazine-1-carboxylate (0.725 g, 70%) was ob-
tained as a pale yellow oil and it was used in the next step without
NMR: (300 MHz, CDCl3) d 7.17 (s, 1H), 2.67 (s, 3H); MS (ESI) m/z: 202
[M þ H]þ; m.p. 222e224 ꢀC.
5.2.29. tert-Butyl 4-(4-(amino(3-chlorophenyl)methyl)benzyl)
piperazine-1-carboxylate (30)
further purifications. 1H NMR: (300 MHz, CDCl3)
d 7.75e7.63 (m,
3H), 7.58 (d, J ¼ 8.0 Hz, 2H), 7.47e7.24 (m, 10H), 7.22e7.12 (m, 2H),
6.99e6.91 (m, 1H), 3.55 (s, 2H), 3.45 (br, 4H), 2.40 (br, 4H), 1.46 (s,
9H), 1.10 (s, 9H); MS (ESI) m/z: 635 [M þ H]þ.
Step g: To a solution of 32 (0.44 g, 1.1 mmol) in EtOH (20 mL)
BaCO3 (0.481 g, 2.4 mmol) and NH2OH$HCl (0.157 g, 2.4 mmol)
were added. Reaction was stirred at 65 ꢀC for 12 h, then was filtered
through celite and solvent was evaporated. (E,Z)-tert-Butyl 4-(4-((3-
chlorophenyl)(hydroxyimino)-methyl)benzyl)piperazine-1-
carboxylate (0.46 g, quantitative yield) was obtained as an amor-
phous white solid and was used in the next step without any
Step b: Compound 33 was obtained from the above compound
(0.084 g, 0.1 mmol) following the procedure described before for
compound 5f step g. Crude was purified by chromatography on
silica gel (3% MeOH, in DCM) obtaining 33 (0.05 g, 97%) as a white
solid. 1H NMR: (300 MHz, CDCl3)
J ¼ 8.1 Hz, 2H), 7.30e7.19 (m, 3H), 7.05 (d, J ¼ 7.7 Hz, 1H), 3.54 (s,
2H), 3.42 (br, 4H), 2.40 (br, 4H), 1.43 (s, 9H); MS (ESI) m/z: [M þ H]þ
397; m.p. 157e159 ꢀC.
d
7.70 (d, J ¼ 8.0 Hz, 2H), 7.37 (d,
further purification. 1H NMR: (400 MHz, CDCl3)
d
8.34 (br, 1H),
7.51e7.19 (m, 8H), 3.52 (br, 6H), 2.31 (br, 4H), 1.43 (s, 9H); MS (ESI)
m/z: 430 [M þ H]þ.
Stepe h: To a solution of the above-obtained compound (0.46 g,
1.1 mmol) in AcOH (50 mL) NH4Cl (1.2 g, 22.3 mmol) and Zn0 (2.7 g,
41.9 mmol) were added. Reaction was stirred at 50 ꢀC for 12 h then
was filtered through celite, solvent was evaporated, and residue
neutralized with NaHCO3. Mixture was extracted with DCM, and
the combined organic layers were dried over Na2SO4 and concen-
trated in vacuo. Crude 30 (0.30 g, 70%) obtained as a pale-yellow oil,
was used in the next step without any further purification. 1H NMR:
5.2.33. tert-Butyl 4-(4-(3-(2-((methylsulfonyl)oxy)ethoxy)benzoyl)
benzyl)piperazine-1-carboxylate (34)
To a solution of 33 (0.10 g, 0.3 mmol) in dry DMF (5 mL) NaH
(0.012 g, 0.5 mmol) was added. The reaction was heated at 80 ꢀC
and a solution of ethylene glycol dimesylate (0.065 g, 0.3 mmol) in
dry DMF (2 mL) was added. The mixture was stirred at 80 ꢀC for 2 h,
then DMF was evaporated and crude was treated with water. The
residue was extracted with DCM and the combined organic layers
were washed with brine, dried over Na2SO4 and concentrated in
vacuo. Crude compound was purified by chromatography on silica
gel (2% MeOH in DCM). 34 (0.041 g, 27%) was obtained as a pale-
(400 MHz, CDCl3) d 7.36 (s, 1H), 7.30e7.12 (m, 7H), 5.12 (s, 1H), 3.43
(s, 2H), 3.38 (br, 4H), 2.33 (br, 4H),1.83 (br, 2H),1.42 (s, 9H); MS (ESI)
m/z: 416 [M þ H]þ.
yellow oil. 1H NMR: (400 MHz, CDCl3)
d
7.74 (d, J ¼ 8.1 Hz, 2H),
5.2.30. tert-Butyl 4-(4-(((5-chloro-2-methyl-[1,2,4]triazolo[1,5-a]
pyrimidin-7-yl)amino)(3-chlorophenyl)methyl)benzyl)piperazine-
1-carboxylate (31)
7.43 (d, J ¼ 8.0 Hz, 2H), 7.36 (t, J ¼ 9.6 Hz, 3H), 7.12 (d, J ¼ 7.2 Hz,1H),
4.57 (t, J ¼ 4.0 Hz, 2H), 4.29 (t, J ¼ 4.0 Hz, 2H), 3.56 (s, 2H), 3.42 (br,
4H), 3.05 (s, 3H), 2.39 (br, 4H), 1.44 (s, 9H); MS (ESI) m/z: 519 [M þ
H]þ.
Compound 29 (0.07 g, 0.4 mmol) and 30 (0.216 g, 0.5 mmol)
were dissolved in EtOH (5 mL) and the reaction was stirred at 25 ꢀC
for 12 h. The solvent was evaporated, and the crude was purified by
chromatography on silica gel (3% MeOH in DCM) obtaining 31
5.2.34. Di-tert-butyl 4,4’-(((((ethane-1,2-diylbis(oxy))bis(3,1-
phenylene))bis(hydroxymethylene))-bis(4,1-phenylene))
bis(methylene))bis(piperazine-1-carboxylate) (35)
(0.20 g, 66%). 1H NMR: (400 MHz, CDCl3)
d
7.44e7.17 (m, 8H), 6.59
(d, J ¼ 5.9 Hz, 1H), 5.90 (s, 1H), 5.72 (d, J ¼ 6.0 Hz, 1H), 3.48 (s, 2H),
3.42 (br, 4H), 2.52 (s, 3H), 2.37 (br, 4H), 1.43 (s, 9H); MS (ESI) m/z:
582 [M þ H]þ.
Step d: To a solution of 33 (0.031 g, 0.1 mmol) in dry DMF (2 mL)
NaH (0.003 g, 0.1 mmol) was added. The mixture was stirred at
80 ꢀC and 34 (0.034 g, 0.1 mmol) was added; the reaction was
stirred at 80 ꢀC for 2 h. Then DMF was evaporated, water was added
to the residue and mixture was extracted with DCM. The combined
organic layers were washed with brine, dried over Na2SO4 and
concentrated in vacuo. Crude compound was purified by chroma-
tography on silica gel (2% MeOH in DCM) obtaining di-tert-butyl
4,4’-(((3,3’-(ethane-1,2-diylbis(oxy))bis(benzoyl))bis(4,1-phenylene))
bis(methylene))bis(piperazine-1-carboxylate (0.038 g, 71%) as a pale
5.2.31. N-((3-Chlorophenyl)(4-(piperazin-1-ylmethyl)phenyl)
methyl)-5-methoxy-2-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-
amine (5k)
To dry MeOH (2 mL) placed in a microwave tube, and cooled at
0
ꢀC, metallic sodium (0.015 g, 0.70 mmol) was added. After com-
plete dissolution of sodium, compound 31 (0.06 g, 0.10 mmol) was
added and the mixture was heated to 85 ꢀC under microwave
irradiation 80 W for 30 min. Water was slowly added and the
mixture was extracted with DCM; the combined organic layers
were dried over Na2SO4 and concentrated in vacuo. Crude 5k was
purified by chromatography on alumina (MeOH 5% in DCM) and
was obtained as a colorless oil (0.025 g, 54%). 1H NMR: (400 MHz,
yellow oil. 1H NMR: (300 MHz, CDCl3)
d
7.76 (d, J ¼ 7.9 Hz, 4H),
7.50e7.31 (m, 9H), 7.23e7.09 (m, 3H), 4.39 (s, 4H), 3.58 (s, 4H), 3.44
(br, 8H), 2.41 (br, 8H), 1.45 (s, 18H); MS (ESI) m/z: 819 [M þ H]þ.
Step e: The above obtained compound (0.098 g, 0.13 mmol) was
dissolved in THF (5 mL), cooled at 0 ꢀC, then NaBH4 (0.012 g,
0.33 mmol) was added. The reaction was stirred at 25 ꢀC for 12 h
then water was added, and the mixture was extracted with EtOAc.
The combined organic layers were dried over Na2SO4 and
concentrated in vacuo. 35 (0.10 g, quantitative yield) was obtained
as a colorless oil and used in the next step without any further
CDCl3)
3.91 (s, 3H), 3.45 (s, 2H), 2.86 (br, 4H), 2.47 (s, 3H), 2.35 (br, 4H); 13
NMR: (75 MHz, CDCl3) 167.4,164.2,155.5,146.8,141.8,139.2,138.0,
d 7.48e7.10 (m, 8H), 6.32 (s, 1H), 5.62 (s, 1H), 5.28 (s, 1H),
C
d
135.4, 130.7, 130.2 (2C), 128.8,127.5 (2C), 125.4, 63.2, 60.9, 54.4, 46.1,
purification. 1H NMR (400 MHz, CDCl3)
d 7.42e7.13 (m, 10H),
15.1; MS (ESI) m/z: 500 [M þ Na]þ; HRMS-ESI m/z: calcd for
7.02e6.87 (m, 4H), 6.79 (dd, J ¼ 8.3, 2.6 Hz, 2H), 5.74 (s, 2H), 4.24 (s,
4H), 3.43 (s, 4H), 3.35 (t, J ¼ 5.0 Hz, 8H), 2.82 (s, 2H), 2.31 (t,
J ¼ 5.0 Hz, 8H), 1.42 (s, 18H); MS (ESI) m/z: 823 [M þ H]þ.
C
25H29ClN7Oþ [MþH]þ: 478.2117; found: 478.2121; HPLC RT:
9.2 min.
12

N. Relitti, S. Federico, L. Pozzetti et al.
European Journal of Medicinal Chemistry 215 (2021) 113227
5.2.35. N,N’-(((Ethane-1,2-diylbis(oxy))bis(3,1-phenylene))bis((4-
(piperazin-1-ylmethyl)phenyl)-methylene))bis(7-chloroquinolin-4-
amine) (5m)
Step f: Compound di-tert-butyl 4,4’-(((((ethane-1,2-diylbis(oxy))
bis(3,1-phenylene))-bis(chloro-methylene))bis(4,1-phenylene))bis(-
methylene))bis(piperazine-1-carboxylate) (0.103 g, quantitative
yield) was obtained as a brown oil, starting from 35 and following
the procedure used before to obtain compound 6a. It was used in
the next step without any further purification. 1H NMR: (300 MHz,
were stored in the dark at 16e18 ꢀC in OR2þ buffer (82.5 mM NaCl,
2.5 mM KCl, 1 mM MgCl2, 1 mM Na2HPO4, 5 mM HEPES, 1 mM
CaCl2, and 50
m
g/mL gentamicin; pH 7.8), which was replaced daily.
The uptake of [3H]CQ (0.25
mM; 20 Ci/mmol; American Radio-
labeled Chemicals) was measured 3e4 days post-injection using a
MicroBeta2 microplate liquid scintillation analyzer (PerkinElmer)
and a protocol described in detail previously [28]. Briefly, the [3H]
CQ influx assays were performed over 1.5 h at 27.5 ꢀC and in a
medium that contained 96 mM NaCl, 2 mM KCl, 2 mM MgCl2,
CDCl3)
d
7.41e7.18 (m, 10H), 7.09e6.96 (m, 4H), 6.86 (dd, J ¼ 7.9,
1.8 mM CaCl2, 10 mM MES, 10 mM Tris base (pH 5.5), and 15 mM
2.1 Hz, 2H), 6.07 (s, 2H), 4.29 (s, 4H), 3.47 (s, 4H), 3.41 (br, 8H), 2.37
(br, 8H), 1.46 (s, 18H); MS (ESI) m/z: 859 [M þ H]þ.
unlabeled CQ. In all cases, at least three independent experiments
were performed (on oocytes from different frogs), and in each
experiment measurements were made from 10 oocytes per treat-
ment. All errors cited in the text and shown in the figures represent
the s.e.m. Statistical comparisons were made using one-way
ANOVAs in conjunction with Tukey’s multiple comparisons test.
Step g: Compound di-tert-butyl 4,4’-(((((ethane-1,2-diylbis(oxy))
bis(3,1-phenylene))bis(((7-chloroquinolin-4-yl)amino)methylene))
bis(4,1-phenylene))bis(methylene))bis(piperazine-1-carboxylate) was
obtained starting from the above-obtained compound (0.075 g,
0.1 mmol) following the procedure used for compound 5a. Crude
was purified by chromatography obtaining the desired compound
as a pale yellow oil (0.017 g, 17%). 1H NMR: (300 MHz, CDCl3)
5.4. Transmission blocking assay
d
8.48e8.38 (m, 2H), 7.97 (s, 2H), 7.72 (d, J ¼ 9.1 Hz, 2H), 7.40e7.24
5.4.1. Murine malaria model
(m, 12H), 7.04e6.90 (m, 4H), 6.84 (d, J ¼ 8.1 Hz, 2H), 6.25 (d,
J ¼ 5.4 Hz, 2H), 5.67 (s, 2H), 5.49 (s, 2H), 4.23 (s, 4H), 3.49 (s, 4H),
3.42 (br, 8H), 2.38 (br, 8H), 1.45 (s, 18H); MS (ESI) m/z: 572
Plasmodium berghei ANKA GFP-con strain, expressing a green
fluorescent protein (GFP) at all life cycle stages was employed in the
in vivo Direct Feeding Assay (DFA). BALB/c mice were used as
vertebrate hosts and An. stephensi mosquitoes as experimental
vectors.
[M þ 2H]2þ
.
Step h: Compound 5m was obtained starting from the above-
obtained compound (0.017 g, 0.02 mmol) following the same pro-
cedure used to obtain 5e. Crude was purified by HPLC equipped
with a UV-VIS detector (Diode Array SPD-M20A) and the column
used was Chromolith® Semiprep RP-18e (100-10 mm). All runs
were performed in gradient mode (flow rate ¼ 2.5 mL/min;
gradient, 80/20e5/95 A/B in 10 min; isocratic 5/95 A/B 5 min) at
25 ꢀC with detection at 254 nM. Sample solutions were prepared
(approximately 2 mg/mL) by dissolving the analytes in the eluent
5.4.2. Mice
Eight to ten-week-old BALB/c mice, weighing 18e25 g were
used in this study. The mice were reared in the animal house (24 ꢀC,
14 h light/10 h dark cycle and 70% relative humidity) of the Uni-
versity of Camerino and were fed ad-libitum with standard mouse
food (Mucedola s.r.l., Milano, Italy) and tap water.
and filtering through 0.45
mM Acrodisc filters. The injection volume
5.4.3. Mosquitoes
was 50 L and RT of 5e was 5.94 min 5e (0.006 g, 50%) was obtained
as a pale-yellow oil.
m
Anopheles stephensi mosquitoes were maintained at a temper-
ature of 30 C, 12 h light/12 h dark cycle and 75e85% relative hu-
ꢀ
1H NMR: (300 MHz, CDCl3)
d
8.41 (d, J ¼ 3.1 Hz, 2H), 8.09 (d,
midity in the insectary of the University of Camerino. Larvae were
raised in the same facility in trays filled with well water and fed
with grinded mouse food. Four to five-day old female mosquitoes
were used in the experimentations. Mosquito cages were trans-
ferred to a 19 ꢀC chamber 24 h prior to the experimental blood-
meals to allow sporogonic development to be completed which
in the case of P. berghei requires a temperature between 18 and
20 ꢀC.
J ¼ 5.1 Hz, 1H), 7.96 (d, J ¼ 1.8 Hz, 1H), 7.71 (d, J ¼ 9.0 Hz, 2H),
7.43e7.22 (m, 14H), 7.04e6.71 (m, 6H), 6.25 (d, J ¼ 5.3 Hz, 2H), 5.66
(s, 2H), 5.45 (s, 2H), 4.23 (s, 4H), 3.47 (s, 4H), 2.87 (br, 8H), 2.41 (br,
8H); MS (ESI) m/z: 472 [M þ 2H]2þ
.
5.3. Expression of PfCRT in Xenopus oocytes and measurements of
CQ transport
Ethical approval of the work performed with the Xenopus laevis
frogs was obtained from the Australian National University Animal
Experimentation Ethics Committee (Animal Ethics Protocol Num-
ber A2013/13) in accordance with the Australian Code of Practice
for the Care and Use of Animals for Scientific Purposes. Sections of
ovary were surgically removed from adult female frogs (purchased
from NASCO) using a procedure outlined in detail elsewhere [46].
Single oocytes were obtained by incubating the ovary sections with
collagenase in accordance with a protocol described previously [46]
and with the following minor modification: the ovary tissue
(weighing 7e14 g) was transferred to a 100 mL Erlenmeyer flask
and incubated in the dark in OR2ꢁ buffer supplemented with 0.5 M
Na2HPO4 (Sigma-Aldrich), bovine serum albumin (250 mg/mL,
5.5. Direct Feeding Assay (DFA)
The transmission blocking activity of the compounds was
evaluated in a double in vivo assay including both, the mouse, and
the mosquito host. The effects of the compounds on gametocytes in
the mouse and on early sporogonic development to oocyst for-
mation in mosquitoes was tested as described in detail before [47].
Experimental mice received a standardized i.p. infection with
107 P. berghei GFP-con infected red blood cells. In the morning of
day 3 after mouse infection parasitemia and gametocytemia was
determined on Giemsa stained thin films. Mice showing a para-
sitemia of about 5% (3e10%) with the presence of micro- and
macrogametocytes were selected for the DFA and allocated
randomly to the four treatment groups (3 mice per group), namely
PQ derivative 5i and 5j, PBS solvent control and PQ as positive
reference. In the afternoon of day 3 post infection, i.e. at the time
point of P. berghei gametocyte maturation, a first treatment was
carried out by administering (i.p.) compounds 5i, 5j and PQ at a
Sigma-Aldrich), 5e10
mg/mL of collagenase (of which 60% was
collagenase A and 40% collagenase D; Roche), and 40e100
mg/mL of
soybean trypsin inhibitor (type II-S; Sigma-Aldrich). Healthy, de-
folliculated oocytes of a suitable size and age (developmental
stages V and VI) were selected for microinjection. cRNA encoding
PfCRTDd2 was transcribed in vitro and microinjected (20 ng per
oocyte) into the oocytes as outlined elsewhere [28,46]. The oocytes
dosage of 10
mmol/kg to the treatment mice and giving plain PBS to
controls. On day four post-infection, the treatment was repeated
13

N. Relitti, S. Federico, L. Pozzetti et al.
European Journal of Medicinal Chemistry 215 (2021) 113227
1.5 h before the exposure of mice to mosquitoes. Experimental
animals were anaesthetized and kept on top of cages containing
approximately 150 female A. stephensi mosquitoes for 1 h. Unfed
females were removed the following day. On day 8 after blood-
meal, 20e30 mosquitoes (minimum 8) from each cage were
dissected and oocyst densities assessed by counting the number of
fluorescent oocysts on mosquito mid-guts at the fluorescent mi-
croscope (Zeiss Axio observer Z1, 60e100 x magnification) (Fig. S2).
Experimental treatments were affected in triplicates, using for each
treatment or control group 3 mice for the infection of 3 mosquito
cages. For each mosquito cage, the geometric mean number of
oocysts and the standard deviation was assessed on positive
mosquitoes and the arithmetic mean calculated for each treatment
using the geometric means of the 3 replicates. Differences in mean
oocyst numbers between each of the 2 PQ derivative treatment
groups and the solvent control were evaluated by t-test (two-
tailed).
First, the following solutions were prepared in order to deter-
mine the percentage of viable cells:
1. Neutral Red (NR) stock solution: 0.33 g NR dye powder in 100 ml
sterile H2O
2. NR medium: 1.0 mL NR stock solution þ99.0 routine culture
ꢀ
medium pre-warmed to 37 C
3. NR desorb solution: 1% glacial acetic acid solution þ50%
ethanol þ49% H2O
At the end of incubation, the routine culture medium was
removed from each plate and the cells were carefully rinsed with
1 ml pre-warmed D-PBS 0.1 M. Plates were then gently blotted with
paper towels.1.0 ml NR medium was added to each dish and further
incubated at 37 ꢀC, 95% humidity, 5.0% CO2 for 3 h. The cells were
checked during incubation for NR crystal formation. After incuba-
tion, the NR medium was removed, and the cells were carefully
rinsed with 1 mL pre-warmed D-PBS 0.1 M. PBS was decanted and
blotted from the dishes and exactly 1 mL NR desorb solution was
added to each sample. Plates were placed on a shaker for
20e45 min to extract NR from the cells and form a homogeneous
solution. During this step the samples were covered to protect them
from light. Five minutes after removal from the shaker, absorbance
was read at 540 nm with a UV/visible spectrophotometer (Varian
Cary 1 E).
5.6. Liver-stage analysis
6e12 weeks of age BALB/cJRj (Janvier-laboratories) mice were
infected by i.p. injection of PbmCherryhsp70 blood stabilities [48]. At
around 2% parasitemia (~day 3) blood was passaged i.v. into naïve
mice. When these mice reached parasitemia of about 5% (around
day 3) thin blood smears were stained using Wright stain and
gametocyte development was examined. Mice necessary to infect
mosquitos were anesthetized with a mixture of 50 mg/kg Ketamine
and 10 mg/kg.
Author contributions
Xylazine in PBS by i.p., route. After reaching deep anesthesia
they were placed on a mosquito cage for 30 min where mosquitos
(female Anopheles stephensi) could take a blood meal. Mosquitos
were kept at 20.5 ꢀC and 80% humidity (12 h/12 h light-dark cycle).
Between 18- and 26-days post feed, salivary glands of Plasmodium
berghei infected mosquitos were dissected and used to infect
HepG2 cells.
Confluent HepG2 cell cultures (60000 cells/96 well seeded the
day before) were infected with 200000 P. berghei sporozoites each
(PbmCherryhsp70) for 2 h. In total 6 replicates with 3 wells each were
infected. The cells of each replicate were detached by the use of
accutase (Innovative Cell Technologies) pooled and distributed to 3
times 8 wells containing either primaquine, 5i or 5j, each at
different concentrations plus control (DMSO same concentration as
in highest drug concentration). Media were changed at 24 hpi, and
the cells were fixed with 4% PFA/PBS at 48 hpi. Host cell growth was
monitored by phase contrast microscopy to evaluate growth
inhibiting drug concentrations. Parasite numbers and sizes were
determined by automated microscopy (IN Cell analyzer, GE life
sciences) monitoring the mCherry signal of PbmCherryhsp70 para-
sites [49]. Statistical evaluations (One-way ANOVA with Dunnett’s
multiple comparisons) were done using Prism 8 (GraphPad).
The manuscript was written through contribution of all authors.
All authors have given approval to the final version of the
manuscript.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgement
Authors thank MIUR Progetto Dipartimento di Eccellenza DBCF-
UNISI 2018e2022. Financial contribution from MIUR is kindly
acknowledged (Project PRIN 20154JRJPP).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113227.
References
5.7. Cytotoxicity assay on NIH3T3 cell line
[1] World Health Organization, World Malaria Report 2019, Geneva, 2019.
[2] O. Müller, G.Y. Lu, L. von Seidlein, Geographic expansion of artemisinin
resistance, J. Trav. Med. 26 (2019), https://doi.org/10.1093/jtm/taz030.
[3] J.-F. Trape, G. Pison, A. Spiegel, C. Enel, C. Rogier, Combating malaria in africa,
Trends Parasitol. 18 (2002) 224e230, https://doi.org/10.1016/S1471-4922(02)
02249-3.
Cells (5 ꢂ 104) suspended in 1 mL of complete medium were
seeded in each well of a 24 well round multidish and incubated at
37 ꢀC in an atmosphere of 5% CO2. After 24 h of culture, the culture
medium was discharged and test samples, solubilized in DMSO,
were added to each well at different concentration values. For the
evaluation of cytotoxicity, three experiments repeated in six rep-
licates were performed and all compounds were tested at
[4] S. Pulcini, H.M. Staines, A.H. Lee, S.H. Shafik, G. Bouyer, C.M. Moore, D.A. Daley,
ꢀ
M.J. Hoke, L.M. Altenhofen, H.J. Painter, J. Mu, D.J.P. Ferguson, M. Llinas,
R.E. Martin, D.A. Fidock, R.A. Cooper, S. Krishna, Mutations in the Plasmodium
falciparum chloroquine resistance transporter, PfCRT, enlarge the parasite’s
food vacuole and alter drug sensitivities, Sci. Rep. 5 (2015) 14552, https://
doi.org/10.1038/srep14552.
increasing concentrations ranging from 8 to 160 mM. The samples
[5] S.K. Martin, A.M. Oduola, W.K. Milhous, Reversal of chloroquine resistance in
Plasmodium falciparum by verapamil, Science 235 (1987) 899e901, https://
doi.org/10.1126/science.3544220, 80-.
[6] R.E. Martin, S.H. Shafik, S.N. Richards, Mechanisms of resistance to the partner
drugs of artemisinin in the malaria parasite, Curr. Opin. Pharmacol. 42 (2018)
71e80, https://doi.org/10.1016/j.coph.2018.07.010.
were set up in six replicates for each tested concentration. Com-
plete medium was used as negative control. Cell viability and
proliferation was evaluated by Neutral Red uptake after 24 h of
incubation with NIH3T3 as follows.
14

N. Relitti, S. Federico, L. Pozzetti et al.
European Journal of Medicinal Chemistry 215 (2021) 113227
€
[7] R.E. Martin, R. V Marchetti, A.I. Cowan, S.M. Howitt, S. Broer, K. Kirk, Chloro-
quine transport via the malaria parasite’s chloroquine resistance transporter,
Science 325 (2009) 1680e1682, https://doi.org/10.1126/science.1175667, 80-.
[8] R.L. Summers, A. Dave, T.J. Dolstra, S. Bellanca, R. V Marchetti, M.N. Nash,
S.N. Richards, V. Goh, R.L. Schenk, W.D. Stein, K. Kirk, C.P. Sanchez, M. Lanzer,
R.E. Martin, Diverse mutational pathways converge on saturable chloroquine
transport via the malaria parasite’s chloroquine resistance transporter, Proc.
Natl. Acad. Sci. Unit. States Am. 111 (2014) E1759, https://doi.org/10.1073/
pnas.1322965111. LP-E1767.
[25] S. Gemma, G. Campiani, S. Butini, G. Kukreja, S.S. Coccone, B.P. Joshi,
M. Persico, V. Nacci, I. Fiorini, E. Novellino, E. Fattorusso, O. Taglialatela-Scafati,
L. Savini, D. Taramelli, N. Basilico, S. Parapini, G. Morace, V. Yardley, S. Croft,
M. Coletta, S. Marini, C. Fattorusso, Clotrimazole scaffold as an innovative
pharmacophore towards potent antimalarial agents: design, synthesis, and
biological and structureeactivity relationship studies, J. Med. Chem. 51 (2008)
1278e1294, https://doi.org/10.1021/jm701247k.
[26] S. Gemma, G. Campiani, S. Butini, G. Kukreja, B.P. Joshi, M. Persico,
B. Catalanotti, E. Novellino, E. Fattorusso, V. Nacci, L. Savini, D. Taramelli,
N. Basilico, G. Morace, V. Yardley, C. Fattorusso, Design and synthesis of potent
antimalarial agents based on clotrimazole Scaffold: exploring an innovative
pharmacophore, J. Med. Chem. 50 (2007) 595e598, https://doi.org/10.1021/
jm061429p.
[9] A.M. Lehane, R. Hayward, K.J. Saliba, K. Kirk,
A verapamil-sensitive
chloroquine-associated Hþ leak from the digestive vacuole in chloroquine-
resistant malaria parasites, J. Cell Sci. 121 (2008) 1624, https://doi.org/
10.1242/jcs.016758. LP e 1632.
[10] A.M. Lehane, K. Kirk, Chloroquine resistance-conferring mutations in pfcrt
give rise to a chloroquine-associated Hþ leak from the malaria parasite’s
digestive vacuole, Antimicrob. Agents Chemother. 52 (2008) 4374, https://
doi.org/10.1128/AAC.00666-08. LP e 4380.
[27] J.L. Small-Saunders, L.M. Hagenah, D.A. Fidock, Turning the tide: targeting
PfCRT to combat drug-resistant P. falciparum? Nat. Rev. Microbiol. 18 (2020)
261e262, https://doi.org/10.1038/s41579-020-0353-8.
[28] S.N. Richards, M.N. Nash, E.S. Baker, M.W. Webster, A.M. Lehane, S.H. Shafik,
R.E. Martin, Molecular mechanisms for drug hypersensitivity induced by the
malaria parasite’s chloroquine resistance transporter, PLoS Pathog. 12 (2016),
e1005725, https://doi.org/10.1371/journal.ppat.1005725.
[29] C.A. Hrycyna, R.L. Summers, A.M. Lehane, M.M. Pires, H. Namanja, K. Bohn,
J. Kuriakose, M. Ferdig, P.P. Henrich, D.A. Fidock, K. Kirk, J. Chmielewski,
R.E. Martin, Quinine dimers are potent inhibitors of the plasmodium falcip-
arum chloroquine resistance transporter and are active against quinoline-
resistant P. Falciparum, ACS Chem. Biol. 9 (2014) 722e730, https://doi.org/
10.1021/cb4008953.
[30] S. Gemma, G. Kukreja, G. Campiani, S. Butini, M. Bernetti, B.P. Joshi, L. Savini,
N. Basilico, D. Taramelli, V. Yardley, A. Bertamino, E. Novellino, M. Persico,
B. Catalanotti, C. Fattorusso, Development of piperazine-tethered hetero-
dimers as potent antimalarials against chloroquine-resistant P. falciparum
strains. Synthesis and molecular modeling, Bioorg. Med. Chem. Lett 17 (2007)
3535e3539, https://doi.org/10.1016/j.bmcl.2007.04.077.
[11] D.A. Fidock, T. Nomura, A.K. Talley, R.A. Cooper, S.M. Dzekunov, M.T. Ferdig,
ꢀ
L.M.B. Ursos, A. bir Singh Sidhu, B. Naude, K.W. Deitsch, X. Su, J.C. Wootton,
P.D. Roepe, T.E. Wellems, Mutations in the P. Falciparum digestive vacuole
transmembrane protein PfCRT and evidence for their role in chloroquine
resistance, Mol. Cell.
2765(05)00077-8.
6 (2000) 861e871, https://doi.org/10.1016/S1097-
[12] R.A. Cooper, M.T. Ferdig, X.-Z. Su, L.M.B. Ursos, J. Mu, T. Nomura, H. Fujioka,
D.A. Fidock, P.D. Roepe, T.E. Wellems, Alternative mutations at position 76 of
the vacuolar transmembrane protein PfCRT are associated with chloroquine
resistance and unique stereospecific quinine and quinidine responses in
plasmodium falciparum, Mol. Pharmacol. 61 (2002) 35, https://doi.org/
10.1124/mol.61.1.35. LP e 42.
[13] P.G. Bray, S. Deed, E. Fox, M. Kalkanidis, M. Mungthin, L.W. Deady, L. Tilley,
Primaquine synergises the activity of chloroquine against chloroquine-
resistant P. falciparum, Biochem. Pharmacol. 70 (2005) 1158e1166, https://
doi.org/10.1016/j.bcp.2005.07.021.
[14] K.J. Deane, R.L. Summers, A.M. Lehane, R.E. Martin, R.A. Barrow, Chlorphen-
iramine analogues reverse chloroquine resistance in plasmodium falciparum
by inhibiting PfCRT, ACS Med. Chem. Lett. 5 (2014) 576e581, https://doi.org/
10.1021/ml5000228.
[15] P.G. Bray, S.A. Ward, A comparison of the phenomenology and genetics of
multidrug resistance in cancer cells and quinoline resistance in Plasmodium
falciparum, Pharmacol. Ther. 77 (1998) 1e28, https://doi.org/10.1016/S0163-
7258(97)00083-1.
[31] E.D. Thorsett, F.R. Stermitz, Alkylation of haloquinolines by grignard reagents
with nickel-phosphine complex catalysts, J. Heterocycl. Chem. 10 (1973)
243e244, https://doi.org/10.1002/jhet.5570100224.
[32] E. Vismara, F. Fontana, F. Minisci, A new method for the oxidation of methyl to
formyl groups in heteroaromatic derivatives, Gazz. Chim. Ital. 117 (1987)
135e136.
[33] A. Procopio, M. Gaspari, M. Nardi, M. Oliverio, A. Tagarelli, G. Sindona, Simple
and efficient MW-assisted cleavage of acetals and ketals in pure water, Tet-
rahedron
Lett.
48
(2007)
8623e8627,
https://doi.org/10.1016/
[16] R.E. Martin, A.S. Butterworth, D.L. Gardiner, K. Kirk, J.S. McCarthy, T.S. Skinner-
Adams, Saquinavir inhibits the malaria parasite’s chloroquine resistance
transporter, Antimicrob. Agents Chemother. 56 (2012) 2283e2289, https://
doi.org/10.1128/AAC.00166-12.
[17] S. Bellanca, R.L. Summers, M. Meyrath, A. Dave, M.N. Nash, M. Dittmer,
C.P. Sanchez, W.D. Stein, R.E. Martin, M. Lanzer, Multiple drugs compete for
transport via the Plasmodium falciparum chloroquine resistance transporter
at distinct but interdependent sites, J. Biol. Chem. 289 (2014) 36336e36351,
https://doi.org/10.1074/jbc.M114.614206.
[18] A.M. Lehane, K. Kirk, Efflux of a range of antimalarial drugs and ‘chloroquine
resistance reversers’ from the digestive vacuole in malaria parasites with
mutant PfCRT, Mol. Microbiol. 77 (2010) 1039e1051, https://doi.org/10.1111/
j.1365-2958.2010.07272.x.
j.tetlet.2007.10.038.
[34] P.E. Gordon, A.J. Fry, Hypophosphorous acideiodine: a novel reducing system.
Part 2: reduction of benzhydrols to diarylmethylene derivatives, Tetrahedron
Lett. 42 (2001) 831e833, https://doi.org/10.1016/S0040-4039(00)02159-6.
[35] N. Boechat, L.C.S. Pinheiro, O.A. Santos-Filho, I.C. Silva, Design and synthesis of
new N-(5-Trifluoromethyl)-1H-1,2,4-triazol-3-yl benzenesulfonamides as
possible antimalarial prototypes, Molecules 16 (2011) 8083e8097, https://
doi.org/10.3390/molecules16098083.
[36] N. Boechat, L.C.S. Pinheiro, T.S. Silva, A.C.C. Aguiar, A.S. Carvalho, M.M. Bastos,
C.C.P. Costa, S. Pinheiro, A.C. Pinto, J.S. Mendonça, K.D.B. Dutra, A.L. Valverde,
O.A. Santos-Filho, I.P. Ceravolo, A.U. Krettli, New trifluoromethyl tri-
azolopyrimidines as Anti-Plasmodium falciparum agents, Molecules 17 (2012)
8285e8302, https://doi.org/10.3390/molecules17078285.
[19] D.A. van Schalkwyk, T.J. Egan, Quinoline-resistance reversing agents for the
[37] Y. Makisumi, Synthesis of potential anticancer agents. IV. 5, 7-disubstituted s-
triazolo [2, 3-a] pyrimidines, Chem. Pharm. Bull. (Tokyo) 9 (1961) 801e808,
https://doi.org/10.1248/cpb.9.801.
malaria parasite Plasmodium falciparum, Drug Resist. Updates
9 (2006)
211e226, https://doi.org/10.1016/j.drup.2006.09.002.
[20] R.E. Martin, The transportome of the malaria parasite, Biol. Rev. 95 (2020)
[38] Y. Makisumi, Studies on azaindolizine compounds. XIV. Transetherification in
s-triazolo [1, 5-a] pyrimidines, Chem. Pharm. Bull. (Tokyo) 11 (1963)
845e850, https://doi.org/10.1248/cpb.11.845.
305e332, https://doi.org/10.1111/brv.12565.
ꢀ
[21] S.H. Shafik, S.A. Cobbold, K. Barkat, S.N. Richards, N.S. Lancaster, M. Llinas,
S.J. Hogg, R.L. Summers, M.J. McConville, R.E. Martin, The natural function of
the malaria parasite’s chloroquine resistance transporter, Nat. Commun. 11
(2020) 3922, https://doi.org/10.1038/s41467-020-17781-6.
[39] N. Vale, R. Moreira, P. Gomes, Primaquine revisited six decades after its dis-
covery, Eur. J. Med. Chem. 44 (2009) 937e953, https://doi.org/10.1016/
j.ejmech.2008.08.011.
[22] S. D’Alessandro, G. Alfano, L. Di Cerbo, S. Brogi, G. Chemi, N. Relitti, M. Brindisi,
S. Lamponi, E. Novellino, G. Campiani, S. Gemma, N. Basilico, D. Taramelli,
M.C. Baratto, R. Pogni, S. Butini, Bridged bicyclic 2,3-dioxabicyclo[3.3.1]non-
anes as antiplasmodial agents: synthesis, structure-activity relationships and
studies on their biomimetic reaction with Fe(II), Bioorg. Chem. 89 (2019)
103020, https://doi.org/10.1016/j.bioorg.2019.103020.
[23] A. Vallone, S. D’Alessandro, S. Brogi, M. Brindisi, G. Chemi, G. Alfano,
S. Lamponi, S.G. Lee, J.M. Jez, K.J.M. Koolen, K.J. Dechering, S. Saponara, F. Fusi,
B. Gorelli, D. Taramelli, S. Parapini, R. Caldelari, G. Campiani, S. Gemma,
S. Butini, Antimalarial agents against both sexual and asexual parasites stages:
structure-activity relationships and biological studies of the Malaria Box
[40] E.R. Da Silva, N. Boechat, L.C.S. Pinheiro, M.M. Bastos, C.C.P. Costa,
J.C. Bartholomeu, T.H. Da Costa, Novel selective inhibitor of leishmania
(leishmania) amazonensis arginase, Chem. Biol. Drug Des. 86 (2015) 969e978,
https://doi.org/10.1111/cbdd.12566.
[41] R. Gujjar, F. El Mazouni, K.L. White, J. White, S. Creason, D.M. Shackleford,
X. Deng, W.N. Charman, I. Bathurst, J. Burrows, D.M. Floyd, D. Matthews,
F.S. Buckner, S.A. Charman, M.A. Phillips, P.K. Rathod, Lead optimization of aryl
and aralkyl amine-based triazolopyrimidine inhibitors of plasmodium fal-
ciparum dihydroorotate dehydrogenase with antimalarial activity in mice,
J. Med. Chem. 54 (2011) 3935e3949, https://doi.org/10.1021/jm200265b.
[42] J.M. Coteron, M. Marco, J. Esquivias, X. Deng, K.L. White, J. White, M. Koltun,
F. El Mazouni, S. Kokkonda, K. Katneni, R. Bhamidipati, D.M. Shackleford,
compound
1-[5-(4-bromo-2-chlorophenyl)furan-2-yl]-N-[(piperidin-4-yl)
ꢀ
methyl]methanamine (MMV019918) and analog, Eur. J. Med. Chem. 150
(2018) 698e718, https://doi.org/10.1016/j.ejmech.2018.03.024.
I. Angulo-Barturen, S.B. Ferrer, M.B. Jimenez-Díaz, F.J. Gamo, E.J. Goldsmith,
W.N. Charman, I. Bathurst, D. Floyd, D. Matthews, J.N. Burrows, P.K. Rathod,
S.A. Charman, M.A. Phillips, Structure-guided lead optimization of
triazolopyrimidine-ring substituents identifies potent plasmodium falciparum
dihydroorotate dehydrogenase inhibitors with clinical candidate potential,
J. Med. Chem. 54 (2011) 5540e5561, https://doi.org/10.1021/jm200592f.
[43] R. Gujjar, A. Marwaha, F. El Mazouni, J. White, K.L. White, S. Creason,
D.M. Shackleford, J. Baldwin, W.N. Charman, F.S. Buckner, S. Charman,
[24] S. Gemma, C. Camodeca, S. Sanna Coccone, B.P. Joshi, M. Bernetti, V. Moretti,
S. Brogi, M.C. Bonache de Marcos, L. Savini, D. Taramelli, N. Basilico,
S. Parapini, M. Rottmann, R. Brun, S. Lamponi, S. Caccia, G. Guiso,
R.L. Summers, R.E. Martin, S. Saponara, B. Gorelli, E. Novellino, G. Campiani,
S. Butini, Optimization of 4-aminoquinoline/clotrimazole-based hybrid anti-
malarials: further structureeactivity relationships, in vivo studies, and pre-
liminary toxicity profiling, J. Med. Chem. 55 (2012) 6948e6967, https://
doi.org/10.1021/jm300802s.
P.K. Rathod, M.A. Phillips, Identification of
a
metabolically stable
triazolopyrimidine-based dihydroorotate dehydrogenase inhibitor with
15

N. Relitti, S. Federico, L. Pozzetti et al.
European Journal of Medicinal Chemistry 215 (2021) 113227
antimalarial activity in mice, J. Med. Chem. 52 (2009) 1864e1872, https://
doi.org/10.1021/jm801343r.
J. Infect. Dis. 213 (2016) 800e810, https://doi.org/10.1093/infdis/jiv509.
[47] L. Lucantoni, R.S. Yerbanga, G. Lupidi, L. Pasqualini, F. Esposito, A. Habluetzel,
Transmission blocking activity of a standardized neem (Azadirachta indica)
seed extract on the rodent malaria parasite Plasmodium berghei in its vector
Anopheles stephensi, Malar. J. 9 (2010) 66, https://doi.org/10.1186/1475-
2875-9-66.
[48] P.-C. Burda, M.A. Roelli, M. Schaffner, S.M. Khan, C.J. Janse, V.T. Heussler,
A plasmodium phospholipase is involved in disruption of the liver stage
parasitophorous vacuole membrane, PLoS Pathog. 11 (2015), e1004760,
https://doi.org/10.1371/journal.ppat.1004760.
[49] P. Pino, R. Caldelari, B. Mukherjee, J. Vahokoski, N. Klages, B. Maco, C.R. Collins,
M.J. Blackman, I. Kursula, V. Heussler, M. Brochet, D. Soldati-Favre,
A multistage antimalarial targets the plasmepsins IX and X essential for in-
vasion and egress, Science 80e (358) (2017) 522, https://doi.org/10.1126/
science.aaf8675. LP e 528.
[44] G. Camarda, P. Jirawatcharadech, R.S. Priestley, A. Saif, S. March, M.H.L. Wong,
S. Leung, A.B. Miller, D.A. Baker, P. Alano, M.J.I. Paine, S.N. Bhatia, P.M. O’Neill,
S.A. Ward, G.A. Biagini, Antimalarial activity of primaquine operates via a two-
step biochemical relay, Nat. Commun. 10 (2019) 3226, https://doi.org/
10.1038/s41467-019-11239-0.
[45] P.S. Fasinu, N.P.D. Nanayakkara, Y.-H. Wang, N.D. Chaurasiya, H.M.B. Herath,
J.D. McChesney, B. Avula, I. Khan, B.L. Tekwani, L.A. Walker, Formation pri-
maquine-5,6-orthoquinone, the putative active and toxic metabolite of pri-
maquine via direct oxidation in human erythrocytes, Malar. J. 18 (2019) 30,
https://doi.org/10.1186/s12936-019-2658-5.
[46] D.A. van Schalkwyk, M.N. Nash, S.H. Shafik, R.L. Summers, A.M. Lehane,
P.J. Smith, R.E. Martin, Verapamil-sensitive transport of quinacrine and
methylene blue via the plasmodium falciparum chloroquine resistance
transporter reduces the parasite’s susceptibility to these tricyclic drugs,
16