Novel molecule combinations and corresponding hybrids targeting artemisinin-resistant Plasmodium falciparum parasites

Article abstract of DOI:10.1016/j.bmcl.2021.127884

Malaria is still considered as the major parasitic disease and the development of artemisinin resistance does not improve this alarming situation. Based on the recent identification of relevant malaria targets in the artemisinin resistance context, novel

Full text of DOI:10.1016/j.bmcl.2021.127884

Bioorg. Med. Chem. Lett. 39 (2021) 127884
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel molecule combinations and corresponding hybrids targeting
artemisinin-resistant Plasmodium falciparum parasites
a
,
b
,
c
a
,
b
,
c
a
a
Manel Ouji
, Michel Nguyen
, Romain Musti `e re , Tony Jimenez ,
a,
b
,
c
a,
b
,
c,
*
a,*
Jean-Michel Augereau
, Françoise Benoit-Vical
, C ´e line Deraeve
a
LCC-CNRS, Laboratoire de Chimie de Coordination, Universit ´e de Toulouse, CNRS, UPS, Toulouse, France
Institut de Pharmacologie et de Biologie Structurale, IPBS, Universit ´e de Toulouse, CNRS, UPS, France
New Antimalarial Molecules and Pharmacological Approaches, ERL Inserm UMR 1289, Toulouse, France
b
c
A R T I C L E I N F O
A B S T R A C T
Keyword:
Malaria is still considered as the major parasitic disease and the development of artemisinin resistance does not
improve this alarming situation. Based on the recent identification of relevant malaria targets in the artemisinin
resistance context, novel drug combinations were evaluated against artemisinin-sensitive and artemisinin-
resistant Plasmodium falciparum parasites. Corresponding hybrid molecules were also synthesized and evalu-
ated for comparison with combinations and individual pharmacophores (e.g. atovaquone, mefloquine or tri-
closan). Combinations and hybrids showed remarkable antimalarial activity (IC50 = 0.6 to 1.1 nM for the best
compounds), strong selectivity, and didn’t present any cross-resistance with artemisinin. Moreover, the combi-
nation triclosan + atovaquone showed high activity against artemisinin-resistant parasites at the quiescent stage
but the corresponding hybrid lost this pharmacological property. This result is essential since only few molecules
active against quiescent artemisinin-resistant parasites are reported. Our promising results highlight the potential
of these combinations and paves the way for pharmacomodulation work on the best hybrids.
Antimalarial drug
Plasmodium falciparum
Artemisinin resistance
Quiescence
Drug combination
Hybrid molecule
Malaria is one of the leading causes of mortality by infectious dis-
eases, killing >400 000 people each year, mainly in tropical and sub-
tropical regions.1 The disease is caused by the Plasmodium parasite,
transmitted through the bite of an infected mosquito of the genus
Anopheles. A notable decrease of malaria mortality has been observed for
the 20 last years due in part to the introduction of artemisinin and its
derivatives (ARTs) in the antimalarial therapeutic arsenal. To reduce the
risk of drug resistance, ARTs are used in combination with one or two
other antimalarial drugs having different modes of action and pharma-
cokinetic properties. These Artemisinin-based Combination Therapies
state of quiescence is characterized by a drastically lowered metabolism
1
3,14
that allows parasites to limit ART-induced cellular damages.
How-
ever, quiescent parasites still possess a maintained mitochondrial ac-
tivity and an implemented fatty acid synthesis type II (FAS-II) pathway
in the apicoplast that enable parasites to restart their cell cycle after drug
elimination.1
0,13,14
Interestingly, the antimalarial drug atovaquone
complex of mitochondrial electron
transport chain, was reported to kill dihydroartemisinin-induced
(ATQ, Table 1), which targets the bc
1
1
5
quiescent parasites.1
6,17
Similarly, triclosan (TCS) and haloxyfop,
which inhibit the FabI and acetyl-CoA carboxylase of the Plasmodium
(
ACTs) have been recommended by the WHO, since 2001, as first-line
FAS-II pathway respectively, delayed the recrudescence of quiescent
2
13
treatments of uncomplicated falciparum malaria worldwide. However,
parasites after dihydroartemisinin treatment. Simultaneously target-
the efficacy of the ACTs rapidly declined in South-East Asia, due to the
ing the mitochondrion with ATQ and the apicoplast with TCS would thus
be of valuable interest to face the issue of ART-resistance. Compound
GW844520 reported, like ATQ, as an inhibitor of the bc1 complex of the
mitochondrial electron transport chain,18 was also selected to be studied
in the context of ART-resistance. Finally, the antimalarial drug meflo-
3
–7
emergence of P. falciparum resistance to ARTs
but also to partner
8
,9
drugs, and is now threatening malaria eradication. Targeting ARTs-
resistant parasites is thus an urgent concern.
ART-resistance is based on an original mechanism relying on a
parasite quiescence state induced by ARTs exposure.1
0–12
Indeed, this
quine (MQ) was picked out as a relevant compound for drug
*
Corresponding authors at: LCC-CNRS, Laboratoire de Chimie de Coordination, Universit ´e de Toulouse, CNRS, UPS, Toulouse, France (F. Benoit-Vical and C.
Deraeve).
E-mail addresses: francoise.vical@inserm.fr (F. Benoit-Vical), celine.deraeve@lcc-toulouse.fr (C. Deraeve).
https://doi.org/10.1016/j.bmcl.2021.127884
Received 18 December 2020; Received in revised form 5 February 2021; Accepted 13 February 2021
Available online 24 February 2021
0
960-894X/© 2021 Elsevier Ltd. All rights reserved.

M. Ouji et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127884
combinations studies because it is one of the only two partner drugs used
in ACTs to be reported as active on quiescent ART-resistant parasites.17
We report here the evaluation of different combinations of two of these
compounds, each targeting one essential pathway for quiescence sur-
vival: ATQ + TCS, ATQ + MQ, GW844520 + TCS. This evaluation was
performed first on proliferating parasites then in an artemisinin resis-
tance context on dihydroartemisinin (DHA)-induced quiescent state,
IC50 value of 0.9 nM, corresponding to a more potent anti-proliferative
effect than the two compounds alone. This combination appeared as the
best one tested. The combination ATQ + TCS also had a good anti-
plasmodial activity but its IC50 value (1.8 nM) close to the ATQ one
suggests that the result is mainly due to ATQ activity. The same
reasoning can be made for the combination GW844520 + TCS. The low
efficacy of TCS, alone and in combination, can be explained by the fact
that it targets lipid metabolism, while this metabolism is absent in
proliferating parasites. By contrast, it has been shown that parasite lipid
metabolism takes place during induced quiescence of the parasites by
dihydroartemisinin treatment.13 In these conditions, TCS would inhibit
the FabI enzyme of the Plasmodium FAS-II pathway and delay the
recrudescence of quiescent parasites.13 Moreover, good selectivity of
these molecules towards mammalian cells (Table 2) led us to pursue
their evaluation in an artemisinin resistance context.
1
9
thanks to two specific tests, the recrudescence assay,
and the
Quiescent-stage Survival Assay (QSA) for the best combination.17 In
addition, the very high antiplasmodial activities reported with these
combinations lead us to a hybridization strategy. Hybrid molecules,
combining at least two pharmacophoric subunits with distinct modes of
action, are considered as original compounds which offer the potential
of improved biological activity, reduced risk of drug resistance emer-
gence and better patient compliance. Compared to simple drug combi-
nations, they also offer easier formulation, as well as more predictable
The Plasmodium artemisinin resistance mechanism consists in a
quiescence phenomenon based on a cell cycle arrest of a sub-population
of the parasites during artemisinin or its derivatives treatment. When the
pharmacokinetic and pharmacodynamic relationships.2
0,21
Several re-
views dealing with recent development of antimalarial hybrids are
available in the literature.2
2,23
Combinations of selected compounds
drug is eliminated, the parasites are able to develop again normally.
10,32
that showed activity both on proliferating parasites and on quiescent
parasites were thus translated to corresponding hybrid molecules: ATQ-
TCS, ATQ-MQ and GW844520-TCS. The synthesis of these original
compounds is reported herein, together with their evaluation for in vitro
antiparasitic activity against ART-susceptible and ART-resistant strains
of P. falciparum (chemosensitivity, recrudescence assay, and QSA for the
best hybrid).
This quiescence is characterized by a DNA and RNA synthesis arrest
under treatment.13 A standard chemosensitivity assay, based on the
measurement of the inhibition of parasite proliferation, is thus not
relevant for the evaluation of compounds against artemisinin-resistant
parasites. Indeed, there is no difference in IC50 values between
1
0,19
artemisinin-resistant and artemisinin-susceptible strains.
In this
context, the recrudescence assay, based on the comparison of recru-
descence capacities between the artemisinin-resistant strain F32-ART5
and its twin artemisinin-sensitive strain F32-TEM after 48 h of expo-
sure with the molecule of interest, was used to determine if a cross-
resistance exists with artemisinin. Potential cross-resistance is evi-
denced by a faster resumption of the ART-resistant strain (F32-ART5)
compared to the sensitive F32-TEM parasites. Interestingly, no signifi-
cant differences of recrudescence were observed between F32-ART5 and
F32-TEM neither for the compounds tested alone nor for the three
Drug combinations targeting different pathways were first evaluated
for antiplasmodial effect on proliferating P. falciparum. With an IC50
value of 2 nM, atovaquone (ATQ) displayed a very high activity on
proliferating parasites. GW844520 and mefloquine (MQ) also confirmed
their antiplasmodial activity with IC50 values in the 40–90 nM range
(
Table 2). Significantly less active, triclosan had a low antiplasmodial
activity with an IC50 of 5 µM. Interestingly the 1:1 combination ATQ +
MQ supports the strategy to target different parasite pathways, with an
Table 1
1
3,16
Structures and targets of compounds reported as active on ART- or DHA-induced quiescent parasites (ATQ, TCS, haloxyfop)
or which have showed activity on
a
17
ARTs-resistant parasites maintained in a quiescent state (ATQ, MQ, GW844520).
Drug
Structure
Main targets (P. falciparum)
Ref
Atovaquone (ATQ)
Mitochondrial electron transport chain (ETC)
15,24
1 o
Cytochrome bc (Q site)
Triclosan (TCS)
haloxyfop
Apicoplast FAS-II pathway
FabI
25,26
27
Apicoplast FAS-II pathway
Acetyl-CoA carboxylase
GW844520
Mitochondrial electron transport chain (ETC)
28
1 i
Cytochrome bc (Q site)
Mefloquine (MQ)
Metacaspase; Phospholipids/Ferriprotoporphyrin IX; 80S-ribosome
29,30,31
a
Activity measured by Quiescent-stage Survival Assay (QSA).
2

M. Ouji et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127884
Table 2
Antimalarial and cytotoxic activities of selected molecules and their (1:1) combinations.
Compound
Hybrid pharmacophoric
units
Antiplasmodial activity on P. falciparum IC50
± SEM (nM)
Cytotoxicity on Vero Cells CC50
±
Selectivity index CC50 (Vero Cells) / IC50
SEM (µM)
(P. falciparum)
ATQ
–
–
–
–
–
–
2.1 ± 0.6
42 ± 10.5
87 ± 18
0.5 ± 0.1
8 ± 1
21 ± 7
25 ± 9
–
257
190
241
5
GW844520
MQ
3
3
TCS
5 10 ± 1 10
ATQ + TCS
GW844520 +
TCS
1.8 ± 0.8
38 ± 6
–
ATQ + MQ
Hybrid 4
Hybrid 8
Hybrid 12
Hybrid 13
Artemisinin
–
0.9 ± 0.3
0.6 ± 0.5
169 ± ±37
0.6 ± 0.3
1.1 ± 1.0
18 ± 2.5
–
ATQ/TCS
GW844520/TCS
ATQ/MQ
ATQ/MQ
–
6 ± 2
>120
4 ± 1
5 ± 2
130
10 000
>710
6660
4545
7222
IC50 values on Plasmodium falciparum F32-TEM strain were obtained using SYBR Green assay. Cytotoxicity activities were evaluated against Vero cell line. Artemisinin
was used as antiplasmodial control drug.
ATQ: atovaquone; MQ: mefloquine; TCS: triclosan.
Number of different independent experiments performed: n = 4 for the cytotoxicity assay; at least n = 4 for the antiplasmodial activity. For each one, a triplicate
(
technical repeats) was carried out.
combinations (Table 3), thus demonstrating the absence of cross-
resistance with artemisinin.
presented in Scheme 3. The first step consisted in the protection of the
piperidinyl amine of MQ by a Boc group, followed by treatment of the
resulting Boc-mefloquine 9 with succinic or glutaric anhydride to give
compounds 1039 and 11, with overall yields of 94% and 92%. The free
carboxylic acids of these two compounds were then esterified by ATQ
after an activation step with thionyl chloride. During the course of the
reaction, the piperidinyl amine was (partially) deprotected, affording
hybrids 12 (and 12-Boc) and 13, respectively. 12-Boc, isolated with a
yield of 21%, was finally converted to hybrid 12 by treatment with HCl.
Hybrids were first evaluated for their activity on proliferating par-
asites. Hybrid 4 (ATQ/TCS) showed a sub-nanomolar IC50 value (0.6
nM) against P. falciparum, better than the corresponding pharmaco-
phores alone and their combination (Table 2). Similarly, hybrids 12 and
13 (ATQ/MQ) displayed IC50 values of 0.6 and 1.1 nM, respectively,
comparable to the one of the ATQ + MQ combination and better than
the activities of the individual drugs. By contrast hybrid 8, resulting
from the association of GW844520 and TCS, was significantly less active
than the corresponding combination and GW844520 alone (Table 2).
One explanation for this reduced activity could be a default of aqueous
solubility of the hybrid, lower than the one of GW844520, which was
already described as poorly soluble.40 Otherwise, the linker attachment
in hybrid 8 compared to GW844520 may affect target interaction.
Indeed, the modification introduced in hybrid 8 locked the 4-pyridone
moiety in the less favored pyridinol tautomeric form,37 preventing H-
bond formation between the pyridine carbonyl group and Ser35 residue
The good IC50 values for the three combinations, associated with
good selectivity indexes and absence of cross-resistance with artemisi-
nin, motivated the design of hybrid compounds inspired from such
combinations. The first hybrid was thus based on the association of ATQ
and TCS bound via an 8-carbon chain, in order to limit steric hindrance.
Since ester derivatives of ATQ and TCS have shown antimalarial activ-
ities comparable to the one of their parent drugs, 3
3,34
the free –OH
groups of both drugs were selected for linker attachment through ester
bonds from octanedioic acid. Ethers or carbamate bonds have also been
considered for linker attachment to the –OH groups. However, El Hage
et al. reported that ester analogues of atovaquone were more active than
the corresponding ethers33 and carbamate derivatives of atovaquone
were not selected because such compounds are reported as chemically
unstable.3
5,36
Hybrid 4 was thus prepared in a four steps synthesis
(
Scheme 1). Octanedioic acid was monoprotected via a Steglich esteri-
fication reaction performed with benzylic alcohol, in the presence of
dicyclohexylcarbodiimide (DCC) and a catalytic amount of 3,4-dimethy-
laminopyridine (DMAP), with a yield of 71%. The remaining carboxylic
acid function of 1 was then activated in the presence of thionyl chloride
and reacted with TCS, to afford the intermediate 2 in 61% yield. Finally,
quantitative deprotection of the benzylic ester by catalytic hydrogena-
tion on Pd/C, activation of the resulting carboxylic acid with thionyl
chloride and subsequent esterification with ATQ afforded hybrid 4,
which was isolated in 89% yield.
2
8
1
of cytochrome bc .
A second hybrid, for which the ATQ moiety of 4 was replaced by the
GW844520 unit, was prepared according to a similar sequence (Scheme
Interestingly, the cytotoxicity of the four hybrids was very low
(Table 2), resulting in excellent selectivity indexes.
2
). GW844520 (7)37 was first prepared via a Suzuki-Miyaura cross-
The recrudescence assay (Table 3) showed a loss of activity of all
hybrids compared to their corresponding combinations with more days
necessary for parasites to reach initial parasitemia when they are treated
by combinations than for parasites treated by the corresponding hybrids.
Surprisingly, this activity decrease of hybrids is only noted when high
concentrations are tested (recrudescence assay, Table 3) but not for
lower ones (IC50 values, Table 2). This result could indicate that the
hybrids are less soluble than the corresponding combinations. Interest-
ingly, for all the hybrid compounds, no cross-resistance with artemisinin
was reported since no significant difference in recrudescence capacity of
the parasites was observed between the two strains, like for the com-
pounds alone and their combinations. The difference of 12.5 days be-
tween the two strains, F32-ART5 and F32-TEM, can be noted for the
control drug artemisinin as the sign of the resistance of the strain F32-
ART5 (Table 3).
coupling reaction between 3-chloro-5-iodo-2,6-dimethylpyridin-4(1H)-
3
8
37
one 5 and boronic acid 6, under microwave conditions. Compound
was then treated with sodium hydride and the resulting pyridinolate
7
intermediate was reacted with the acyl chloride of compound 3, to give
hybrid 8 with a yield of 30%.
The two last hybrids 12 and 13 were designed by associating the two
antimalarial drugs ATQ and MQ, bound via a diester linker, as for the
ATQ-containing hybrid 4. The selection of an ester bond for MQ linking
allowed the direct transposition of the previous synthesis scheme using a
symmetrical linker, and was supported by the antiplasmodial and anti-
malarial activity of a trifluoromethylartemisinin–mefloquine hybrid
displaying similar linker attachment.3 Our first attempts to use octa-
nedioic acid as linker precursor, as for hybrids 4 and 8, having been
unsuccessful, we envisaged shorter linkers accessible from sucininc and
glutaric anhydrides. The synthesis pathway for hybrids 12 and 13 is
9
As the mitochondrial electron transfer chain and the apicoplast FAS-
3

M. Ouji et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127884
Table 3
Recrudescence capacity of Plasmodium falciparum F32-ART5 and F32-TEM parasites after 48 h of drug exposure.
a
b
Compounds
ATQ
Doses
Median recrudescence days
Delay in recrudescence time
P-value
Mean ± SEM
F32-ART5
F32-TEM
1 µM
11
21
2
14
23
8
3
–
7
µM
1 ± 0.6
3 ± 1.2
–
0.8 ± 0.9
2.3 ± 1.1
3.3 ± 1.4
–
0.486
0.325
–
0.417
0.175
0.418
–
0.265
0.093
–
GW844520
7 µM
1 µM
7 µM
7 µM
7 µM
1 µM
7 µM
7 µM
MQ
>30
5
>30
6
TCS
ATQ + TCS (1:1)
GW844520 + TCS (1:1)
ATQ + MQ (1:1)
Hybrid 4 (ATQ/TCS)
Hybrid 8 (GW844520/TCS)
17
8
19.5
12
>30
12
4
>30
9
4 ± 2.4
0.6 ± 0.4
1
3
2
0 µM
2
3
Hybrid 12 (ATQ/MQ)
Hybrid 13 (ATQ/MQ)
1 µM
1 µM
18 µM
14
14
9
14
14
22
0
–
–
0
c
Artemisinin
12.5 ± 1.8
< 0.0001
Each experiment was performed for F32-ART5 and F32-TEM cultivated in parallel in the same conditions (adjusted to the same initial parasitemia and cultivated with
the same batch of erythrocytes and same batch of human serum) to generate paired results.
Synchronized ring-stage parasites have undergone 48 h of drug treatment. After culture washing, the parasitemia was monitored during 30 days or until reaching the
initial parasitemia, defined as the recrudescence day. If no parasites were observed at the end of the experiment, the culture was considered as no recrudescent, and the
recrudescence day was noted as > 30. The doses tested for ATQ correspond to concentrations pharmacologically relevant in patients. The same doses were then used
for all the compounds tested. The results correspond to data obtained in at least 3 independent experiments except for the molecules 12, 13, ATQ (1 µM), MQ and ATQ
+
MQ (1:1), 1 time. The same number of experiments was performed for each parasite lineage and statistically analyzed.
ATQ: atovaquone; MQ: mefloquine; TCS: triclosan.
a
The delay of recrudescence days corresponds to the mean of the differences, obtained for each experiment, between the day of recrudescence of F32-ART5
compared to F32-TEM, after 48 h of molecule exposure.
b
A log-rank (Mantel-Cox) test was used for statistical analysis of recrudescence days; a significant difference between F32-ART5 and F32-TEM is validated for P <
0
.05.
c
19
The concentration of artemisinin at 18 µM was shown as the most relevant to discriminate both strains F32-ART5 and F32-TEM regarding artemisinin sensitivity.
II pathway are described as maintained active during
of 13 days in the recrudescence time observed between DHA 6 h/(DHA
+ (ATQ + TCS) 48 h) compared to DHA (DHA 6 h/DHA 48 h) means that
the 1:1 combination ATQ + TCS is active on quiescent parasites. This can
be correlated with the activity on quiescent parasites of ATQ alone with
a difference of 12 days between (DHA 6 h/(DHA + ATQ) 48 h) compared
1
3
dihydroartemisinin-induced quiescence, and because of their very
high antiplasmodial activity reported, the ATQ + TCS combination
(
IC50 = 1.8 nM) and its corresponding hybrid molecule 4 (IC50 = 0.6 nM)
were selected for evaluation on artemisinin-resistant quiescent para-
sites, using the Quiescent-stage Survival Assay (QSA) (Table 4). The
parent molecules (ATQ and TCS) were also evaluated for comparison. In
the QSA, quiescence is first induced with 6 h-DHA treatment then par-
asites are exposed to the drug to be tested for 48 h in the presence of
DHA to maintain the quiescence state. QSA interpretation is based on the
difference in recrudescence days after exposure of DHA-induced quies-
cent parasites to the compound being tested (DHA 6 h/(DHA + mole-
to DHA (DHA 6 h/DHA 48 h) and the maintained mitochondrial activity
under the quiescence state.1
3,16,17
TCS alone didn’t show any activity
neither on proliferating parasites, nor on quiescent parasites with no
delay observed between both first conditions. At the same dose tested (7
µM), hybrid 4, compared to the 1:1 ATQ + TCS combination, showed a
reduced activity in the quiescent parasites evidenced by a delay of only
3 days between conditions (DHA 6 h / (DHA + 4) 48 h) and DHA (DHA 6
h/DHA 48 h). This loss of activity of the hybrid 4 is also illustrated, on
proliferating parasites, by 10 days necessary for parasites to reach initial
parasitemia comparatively to 20 days when they are treated by the
corresponding combination ATQ + TCS (Table 4, third column).
In conclusion, 1:1 combinations of three compounds having good
antiplasmodial activities, atovaquone, mefloquine, GW844520, and of
triclosan, an inhibitor of the lipid metabolism, were evaluated against
1
7
cule) 48 h) compared to DHA alone treatment (DHA 6 h/DHA 48 h).
A
cut-off of 6 days-delay is reported as significant.17 The control drug
chloroquine shows that a molecule can be active on proliferative para-
sites (Table 4, third column) but lose its activity on quiescent parasites,
with no significant difference between the two first columns. The delay
Scheme 1. Synthesis of hybrid 4. Reagents and reaction conditions: (a). Benzyl
◦
alcohol, DMAP, DCC, DCM, rt, 1 h, 71%; (b). i) SOCl
2
, 50 C, 3 h, ii) TCS,
◦
pyridine, DCM, 0 C to rt, 8 h, 61%; (c). H
2
, Pd/C, EtOAc, rt, overnight, 100%;
Scheme 2. Synthesis of hybrid 8. Reagents and reaction conditions: (a). Pd
◦
◦
(
d). i) SOCl
2
, 50 C, 3 h, ii), ATQ, pyridine, DCM, rt, overnight, 89%.
(OAc)
2
, K
2
CO
3
, n-Bu
4
NBr, EtOH, 100 C, 1 h, MW, 29%; (b). i) NaH, THF, rt, 10
2
min, ii) (3 + SOCl ), THF, rt, overnight, 30%.
4

M. Ouji et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127884
tested, further studies will be carried out on these hybrids focusing on
linker pharmacomodulation. Thus, varying the nature of the linker with
an oxygenated chain, modifying its length and the nature of the chem-
ical bond for drug linkage (e.g. O-alkylation versus O-esterification)
would certainly impact the solubility, the stability and more generally
the activity of the resulting hybrids. Furthermore, the use of a combi-
nation active on quiescent parasites such as atovaquone combined with
triclosan represents an encouraging prospect for the development of
effective treatment of malaria cases caused by artemisinin-resistant
parasites.
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.
Scheme 3. Synthesis of hybrids 12 and 13. Reagents and reaction conditions:
Acknowledgments
(
a). Boc
Et N, CHCl
1% for 12 (+21% 12-Boc), 27% for 13; (d). HCl, MeOH, 96%.
2
O, Et
3
N, DCM, 95%; (b) succinic anhydride or glutaric anhydride,
3
3
, 99% and 97%, respectively; (c). i) SOCl
2
, ii) ATQ, pyridine, DCM,
This study was supported in part by the French “Agence Nationale de
la Recherche” (ANR grant INMAR ANR16 CE35 0003), the “Centre
National de la Recherche Scientifique” and the Sanofi-Institut Pasteur
award.
1
P. falciparum parasites. Corresponding hybrid molecules combining
atovaquone and triclosan, GW844520 and triclosan and, atovaquone
and mefloquine were synthesized, focusing on rapid and efficient drugs
linking, and studied in similar conditions to allow a first comparison
between drug combination and drug hybridization. All combinations
and hybrids showed high activity on proliferative P. falciparum para-
sites, with IC50 values in the low- or sub-nanomolar range for ATQ +
TCS, GW844520 + TCS, ATQ + MQ and hybrids 4, 12 and 13, together
with very good selectivity towards mammalian cells. Interestingly, in a
context of artemisinin resistance, no cross-resistance with artemisinin
was observed for these combinations and hybrids. Moreover, the com-
bination ATQ + TCS showed a strong activity against parasites at the
DHA-induced quiescence state. However, at high concentrations, the
corresponding hybrid 4 showed a weaker activity than the combination
on proliferative and on quiescent parasites, suggesting a lower solubil-
ity. This result does not call into question the value of using hybrid
molecules to combat resistant parasites. Indeed, the combination of two
molecules, each having different targets and presenting no cross-
resistance with artemisinin, in a hybrid compound would be a good
approach to prevent cross-resistance and kill quiescent parasites. That is
why, based on the very promising results obtained with all combinations
Christian Bijani from the Laboratoire de Chimie de Coordination and
Catherine Claparols, Nathalie Martins-Froment, Val ´e rie Bourdon and
Eric Leroy from the Mass spectrometry service of the ICT-FR 2599
(
Toulouse) are acknowledged for their support, respectively for NMR
and HR-MS experiments.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.127884.
References
1
2
World Health Organisation. World Malaria Report.; 2020. ISBN 978-92-4-001579-1.
World Health Organisation. Status Report on Artemisinin and ACT Resistance. World
Health Organisation; 2015. https://apps.who.int/iris/handle/10665/338493.
Wongsrichanalai C, Meshnick SR. Declining artesunate-mefloquine efficacy against
falciparum malaria on the Cambodia-Thailand border. Emerg Infect Dis. 2008;14:
716–719. https://doi.org/10.3201/eid1405.071601.
3
4
5
6
7
8
Noedl H, Se Y, Schaecher K, Smith BL, Socheat D, Fukuda MM. Evidence of
Artemisinin-resistant malaria in Western Cambodia. N Engl J Med. 2008;359:
2
619–2620. https://doi.org/10.1056/NEJMc0805011.
Ashley EA, Dhorda M, Fairhurst RM, et al. Spread of Artemisinin resistance in
Plasmodium falciparum malaria. N Engl J Med. 2014;371:411–423. https://doi.org/
Table 4
1
0.1056/NEJMoa1314981.
QSA: Recrudescence time of the P. falciparum ART-resistant line F32-ART5.
Dondorp AM, Nosten F, Yi P, et al. Artemisinin resistance in Plasmodium falciparum
malaria. N Engl J Med. 2009;361:455–467. https://doi.org/10.1056/
NEJMoa0808859.
Compounds
Median recrudescence days
DHA 6 h
then
DHA 6 h
then
Nothing 6 h
then
M ´e nard D, Khim N, Beghain J, et al. A worldwide map of Plasmodium falciparum K13-
propeller polymorphisms. N Engl J Med. 2016;374:2453–2464. https://doi.org/
1
0.1056/NEJMoa1513137.
DHA
(DHA + Molecule)
48 h
Molecule
48 h
Amaratunga C, Lim P, Suon S, et al. Dihydroartemisinin–piperaquine resistance in
Plasmodium falciparum malaria in Cambodia: a multisite prospective cohort study.
Lancet Infect Dis. 2016;16:357–365. https://doi.org/10.1016/S1473-3099(15)00487-
9.
4
8 h
ATQ
6
7
7
7
6
18
7
17
6
TCS
ATQ + TCS (1:1)
Hybrid 4
20
10
8
20
9
Duru V, Khim N, Leang R, et al. Plasmodium falciparum dihydroartemisinin-
piperaquine failures in Cambodia are associated with mutant K13 parasites
presenting high survival rates in novel piperaquine in vitro assays: retrospective and
prospective investigations. BMC Med. 2015;13:305. https://doi.org/10.1186/
s12916-015-0539-5.
10
a
Chloroquine (200 nM)
>30
Synchronized ring-stage parasites have different drug treatment during 54 h.
After culture washing, the parasitemia was monitored until reaching the initial
parasitemia, defined as the recrudescence day. Delay in recrudescence times
corresponds to the difference between the recrudescence day after DHA 6 h /
1
0
1
2
Witkowski B, Lelievre J, Lopez Barragan MJ, et al. Increased tolerance to artemisinin
in Plasmodium falciparum is mediated by a quiescence mechanism. Antimicrob Agents
Chemother. 2010;54:1872–1877. https://doi.org/10.1128/AAC.01636-09.
Ariey F, Witkowski B, Amaratunga C, et al. A molecular marker of artemisinin-
resistant Plasmodium falciparum malaria. Nature. 2014;505:50–55. https://doi.org/
10.1038/nature12876.
1
1
(
(
DHA + molecule) 48 h exposure and the recrudescence day after DHA alone
54 h) treatment. All the molecules were tested at 7 µM (except chloroquine at
Teuscher F, Gatton ML, Chen N, Peters J, Kyle DE, Cheng Q. Artemisinin-induced
dormancy in Plasmodium falciparum: duration, recovery rates, and implications in
treatment failure. J Infect Dis. 2010;202:1362–1368. https://doi.org/10.1086/
2
00 nM), in 3 independent experiments. The control condition (nothing 6 h /
molecule 48 h) allowed the confirmation of the antiplasmodial activity of the
molecules tested alone at this concentration.
6
56476.
ATQ: atovaquone; TCS: triclosan.
1
3
Chen N, LaCrue AN, Teuscher F, et al. Fatty acid synthesis and pyruvate metabolism
pathways remain active in dihydroartemisinin-induced dormant ring stages of
a
17
data from reference
.
5

M. Ouji et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127884
Plasmodium falciparum. Antimicrob Agents Chemother. 2014;58:4773–4781. https://
diclofop. Proc Natl Acad Sci USA. 2004;101:5910–5915. https://doi.org/10.1073/
pnas.0400891101.
doi.org/10.1128/AAC.02647-14.
1
1
4
5
Paloque L, Ramadani AP, Mercereau-Puijalon O, Augereau J-M, Benoit-Vical F.
Plasmodium falciparum: multifaceted resistance to artemisinins. Malar J. 2016;15:
28 Capper MJ, O’Neill PM, Fisher N, et al. Antimalarial 4(1H)-pyridones bind to the Qi
site of cytochrome bc1. Proc Natl Acad Sci. 2015;112:755–760. https://doi.org/
10.1073/pnas.1416611112.
29 Gunjan S, Singh SK, Sharma T, et al. Mefloquine induces ROS mediated programmed
cell death in malaria parasite: Plasmodium. Apoptosis. 2016;21:955–964. https://doi.
org/10.1007/s10495-016-1265-y.
1
49. https://doi.org/10.1186/s12936-016-1206-9.
Nixon GL, Pidathala C, Shone AE, et al. Targeting the mitochondrial electron
transport chain of Plasmodium falciparum: New strategies towards the development of
improved antimalarials for the elimination era. Future Med Chem. 2013;5:
1
573–1591. https://doi.org/10.4155/fmc.13.121.
30 Chevli R, Fitch CD. The antimalarial drug mefloquine binds to membrane
phospholipids. Antimicrob Agents Chemother. 1982;21:581–586. https://doi.org/
10.1128/aac.21.4.581.
31 Wong W, Bai X-C, Sleebs BE, et al. Mefloquine targets the Plasmodium falciparum 80S
ribosome to inhibit protein synthesis. Nat Microbiol. 2017;2, 17031. https://doi.org/
10.1038/nmicrobiol.2017.31.
32 Witkowski B, Amaratunga C, Khim N, et al. Novel phenotypic assays for the detection
of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: In-vitro and ex-
vivo drug-response studies. Lancet Infect Dis. 2013;13:1043–1049. https://doi.org/
10.1016/S1473-3099(13)70252-4.
33 El Hage S, Ane M, Stigliani J-L, et al. Synthesis and antimalarial activity of new
atovaquone derivatives. Eur J Med Chem. 2009;44:4778–4782. https://doi.org/
10.1016/j.ejmech.2009.07.021.
1
1
6
7
Peatey CL, Chavchich M, Chen N, et al. Mitochondrial membrane potential in a small
subset of Artemisinin-induced dormant Plasmodium falciparum parasites in vitro.
J Infect Dis. 2015;212:426–434. https://doi.org/10.1093/infdis/jiv048.
Reyser T, Paloque L, Ouji M, et al. Identification of compounds active against
quiescent artemisinin-resistant Plasmodium falciparum parasites via the quiescent
stage survival assay (QSA). J Antimicrob Chemother. 2020;75:2826–2834. https://doi.
org/10.1093/jac/dkaa250.
Capper MJ, O’Neill PM, Fisher N, et al. Antimalarial 4(1H)-pyridones bind to the Qi
site of cytochrome bc1. Proc Natl Acad Sci. 2015;112. https://doi.org/10.1073/
pnas.1416611112, 755 LP - 760.
1
1
8
9
M ´e nard S, Ben Haddou T, Ramadani AP, et al. Induction of multidrug tolerance in
Plasmodium falciparum by extended artemisinin pressure. Emerg Infect Dis. 2015;21:
1
733–1741. https://doi.org/10.3201/eid2110.150682.
34 Mishra S, Karmodiya K, Parasuraman P, Surolia A, Surolia N. Design, synthesis, and
application of novel triclosan prodrugs as potential antimalarial and antibacterial
agents. Bioorg Med Chem. 2008;16:5536–5546. https://doi.org/10.1016/j.
bmc.2008.04.006.
35 Comley JC, Yeates CL, Frend TJ. Antipneumocystis activity of 17C91, a prodrug of
atovaquone. Antimicrob Agents Chemother. 1995;39:2217–2219. https://doi.org/
10.1128/aac.39.10.2217.
36 Romeo S, Parapini S, Dell’Agli M, et al. Atovaquone-Statine “Double-Drugs” with
high antiplasmodial activity. ChemMedChem. 2008;3:418–420. https://doi.org/
10.1002/cmdc.200700166.
37 Yeates CL, Batchelor JF, Capon EC, et al. Synthesis and structure–activity
relationships of 4-pyridones as potential antimalarials. J Med Chem. 2008;51:
2845–2852. https://doi.org/10.1021/jm0705760.
2
2
2
0
1
2
Morphy R, Rankovic Z. Designed multiple ligands. An emerging drug discovery
paradigm. J Med Chem. 2005;48:6523–6543. https://doi.org/10.1021/jm058225d.
Morphy R, Kay C, Rankovic Z. From magic bullets to designed multiple ligands. Drug
Discov Today. 2004;9:641–651. https://doi.org/10.1016/S1359-6446(04)03163-0.
Agarwal D, Gupta RD, Awasthi SK. Are antimalarial hybrid molecules a close reality
or a distant dream? Antimicrob Agents Chemother. 2017;61:e00249–e317. https://doi.
org/10.1128/AAC.00249-17.
Feng L-S, Xu Z, Chang Le, et al. Hybrid molecules with potential in vitro
antiplasmodial and in vivo antimalarial activity against drug-resistant Plasmodium
falciparum. Med Res Rev. 2020;40:931–971. https://doi.org/10.1002/med.
v40.310.1002/med.21643.
2
2
3
4
Fry M, Pudney M. Site of action of the antimalarial hydroxynaphthoquinone, 2-
[
trans-4-(4’-chlorophenyl) cyclohexyl]-3- hydroxy-1,4-naphthoquinone (566C80).
Biochem Pharmacol. 1992;43:1545–1553. https://doi.org/10.1016/0006-2952(92)
0213-3.
38 Bueno JM, Calderon F, Chicharro J, et al. Synthesis and structure–activity
relationships of the novel antimalarials 5-pyridinyl-4(1H)-pyridones. J Med Chem.
2018;61:3422–3435. https://doi.org/10.1021/acs.jmedchem.7b01256.
39 Grellepois F, Grellier P, Bonnet-Delpon D, B ´e gu ´e J-P. Design, synthesis and
antimalarial activity of trifluoromethylartemisinin–mefloquine dual molecules.
ChemBioChem. 2005;6:648–652. https://doi.org/10.1002/cbic.200400347.
40 Bueno JM, Manzano P, García MC, et al. Potent antimalarial 4-pyridones with
improved physico-chemical properties. Bioorg Med Chem Lett. 2011;21:5214–5218.
https://doi.org/10.1016/j.bmcl.2011.07.044.
9
2
2
2
5
6
7
Perozzo R, Kuo M, Sidhu ABS, et al. Structural elucidation of the specificity of the
antibacterial agent triclosan for malarial enoyl acyl carrier protein reductase. J Biol
Chem. 2002;277:13106–13114. https://doi.org/10.1074/jbc.M112000200.
Surolia N, Surolia A. Triclosan offers protection against blood stages of malaria by
inhibiting enoyl-ACP reductase of Plasmodium falciparum. Nat Med. 2001;7:167–173.
https://doi.org/10.1038/84612.
Zhang H, Tweel B, Tong L. Molecular basis for the inhibition of the
carboxyltransferase domain of acetyl-coenzyme-A carboxylase by haloxyfop and
6