Antimalarial and anti-inflammatory activities of new chloroquine and primaquine hybrids: Targeting the blockade of malaria parasite transmission

Article abstract of DOI:10.1016/j.bmc.2020.115832

Malaria is a disease that requires new drugs not only to fight Plasmodium but also to reduce symptoms of infection such as fever and inflammation. A series of 21 hybrid compounds were designed from chloroquine (CQ) and primaquine (PQ) linked to the pharmacophoric group present in phenylacetic anti-inflammatory drugs. These compounds were designed to have dual activity: namely, to be capable of killing Plasmodium and still act on the inflammatory process caused by malaria infection. The compounds were assayed with nine different biological methods. The carbonylated CQ derivative 6 (n = 3; R₁ = Cl) was more potent than CQ in vitro, and 8 (n = 4; R₁ = H) reduced P. berghei parasitemia up to 37% on day 7. The carbonylated PQ derivative 17 (R = Br) was slightly less potent than PQ. The gem-difluoro PQ derivative 20 (R = Cl) exhibited high transmission blockade of the malaria sporogonic cycle in mosquitoes. Compounds 6 and 20 dose-dependently reduced nitric oxide (NO) production and inhibited TNFα production by LPS-stimulated J774A.1 macrophages. Our results indicate a viable and interesting approach in planning new chemical entities that act as transmission-blocking drugs for treating malaria caused by P. falciparum and P. vivax and the anti-inflammatory process related to this disease.

Full text of DOI:10.1016/j.bmc.2020.115832

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Bioorg. Med. Chem. 28 (2020) 115832
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antimalarial and anti-inflammatory activities of new chloroquine and
primaquine hybrids: Targeting the blockade of malaria
parasite transmission
,
Nubia Boechata *, Rita C.C. Carvalhoa, Maria de Lourdes G. Ferreiraa, Julia Penna Coutinhob,
Paula M. Saa, Leonardo N. Seitoc, Elaine C. Rosasc, Antoniana U. Krettlib, Monica M. Bastosa,
Luiz C.S. Pinheiroa
a Laboratorio de Sintese de Farmacos, Instituto de Tecnologia em Farmacos, Farmanguinhos - FIOCRUZ, Fundacao Oswaldo Cruz. Rua Sizenando Nabuco 100,
Manguinhos, Rio de Janeiro, RJ 21041-250, Brazil
b Centro de Pesquisas Rene Rachou, CPqRR - FIOCRUZ, Fundacao Oswaldo Cruz, Belo Horizonte, MG 30190-002, Brazil
c Departamento de Farmacologia, Instituto de Tecnologia em Farmacos, Farmanguinhos - FIOCRUZ, Fundacao Oswaldo Cruz, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Malaria is a disease that requires new drugs not only to fight Plasmodium but also to reduce symptoms of infection
such as fever and inflammation. A series of 21 hybrid compounds were designed from chloroquine (CQ) and
primaquine (PQ) linked to the pharmacophoric group present in phenylacetic anti-inflammatory drugs. These
compounds were designed to have dual activity: namely, to be capable of killing Plasmodium and still act on the
inflammatory process caused by malaria infection. The compounds were assayed with nine different biological
Malaria
Primaquine
Chloroquine
Anti-inflammatory
Antiplasmodial
methods. The carbonylated CQ derivative 6 (n = 3; R1 = Cl) was more potent than CQ in vitro, and 8 (n = 4; R1
=
H) reduced P. berghei parasitemia up to 37% on day 7. The carbonylated PQ derivative 17 (R = Br) was slightly
less potent than PQ. The gem-difluoro PQ derivative 20 (R = Cl) exhibited high transmission blockade of the
malaria sporogonic cycle in mosquitoes. Compounds 6 and 20 dose-dependently reduced nitric oxide (NO)
production and inhibited TNFα production by LPS-stimulated J774A.1 macrophages. Our results indicate a viable
and interesting approach in planning new chemical entities that act as transmission-blocking drugs for treating
malaria caused by P. falciparum and P. vivax and the anti-inflammatory process related to this disease.
1. Introduction
Plasmodium and has been the first treatment choice.
However, its abusive use has led to the emergence of chloroquine-
Malaria is still the most important parasitic disease worldwide and a
major public health problem. Data from the World Health Organization
(WHO) have revealed a significant reduction in the number of cases of
malaria across the globe. In 2018, a total of 228 million cases of malaria
were reported by the WHO, compared with 251 million cases in 2010
and 231 million cases in 2017, with an estimated 405,000 deaths yearly1
However, 405,000 malaria deaths were still recorded worldwide in
2018.1
resistant strains (CQR), making it ineffective in many parts of the
world.3–6 There is a widespread resistance of the parasite, which
threatens the effectiveness of drug treatment for malaria.7 This resis-
tance is defined as a delay in the parasite elimination rate after the
administration of artemisinin derivatives,8–11 the which justifies the
development of new antimalarials. The 8-aminoquinoline PQ and
recently approved tafenoquine are both the only drug options for
treating relapsing P. vivax malaria and act as transmission blocking
drugs, despite their side effects.12,13
Quinolines are among the most widely used drugs for malaria
treatment. Quinine was first used to treat malaria followed by other
quinoline synthetic derivatives, such as chloroquine (CQ) and prima-
quine (PQ) (Fig. 1).2 For treating malaria, CQ is one of the safest,
financially affordable and effective drugs against the different species of
Killing the parasite that causes malaria is the main goal of any
antimalarial drug candidate. However, the most serious manifestation of
this disease is cerebral malaria (CM), which is associated with increased
levels of inflammatory mediator cytokines and chemokines.14 Severe
* Corresponding author.
E-mail address: nubia.boechat@far.fiocruz.br (N. Boechat).
https://doi.org/10.1016/j.bmc.2020.115832
Received 2 September 2020; Received in revised form 22 October 2020; Accepted 26 October 2020
Available online 2 November 2020
0968-0896/© 2020 Elsevier Ltd. All rights reserved.

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
N. Boechat et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115832
cases of malaria are life threatening and demand effective treatment.15
Thus, a new chemical entity with dual activities that can combat the
parasitic Plasmodium spp and attenuate the hazardous inflammation
caused by the parasitic infection could be considered as an interesting
approach in innovative pharmaceutical research. One drug with this
scope in the market for malaria chemotherapy could allow a revolution
in the treatment of patients, ensuring an improvement in the quality of
their lives. We previously used this approach in developing some hy-
brids from aminoquinolines and atorvastatin16 since this statin drug
reduces systemic and brain inflammation and is used as an adjuvant in
CM treatment.17
To plan our hybrids (1–21, Fig. 1), compounds (I and II) were used as
bioisosteres of anti-inflammatory drugs such as ibuprofen and diclofe-
nac. They contain the 2-(2-acetamidophenyl)-2,2-gem-difluor-
oacetamide (I) or 2-(2-acetamidophenyl)-2-oxoacetamide (II) moieties,
which have previously shown anti-inflammatory activity.26 As they are
easy to prepare and have relatively low cost, the molecular hybridiza-
tion of these scaffolds was performed by connecting the quinolinic
pharmacophoric group of CQ to linker groups containing 2–4 (CH2)
units to yield compounds 1–13. The acetamidic skeletons were also
directly bound to PQ to yield compounds 14–21. This study aims to
obtain compounds capable of killing Plasmodium that can still act on the
inflammatory process caused by malaria infection. Novel compounds
derived from PQ may represent a broad spectrum of action against all
forms of Plasmodium involved in the biological cycle of malaria in the
human host and be new alternatives for the treatment of P. vivax ma-
laria. These PQ derivatives can also block malaria transmission.
Additionally, PQ is a drug that also prevents the transmission of the
parasite, acting against gametocytes, which is the sexual form of the
parasite responsible for its development in mosquito vectors.18 Dual-
activity antimalarial drugs able to kill the parasite in the human blood
and block transmission, i.e., those that can target both asexual parasites
and gametocytes, should be value and required.19
Molecular hybridization consists of linking two pharmacophoric
groups of distinct compounds into a single molecule.20 This new mole-
cule, now called a hybrid, may exhibit equal or better activity than the
precursor compound. It can act by the same mechanism of action as the
precursor substance or in different ways, thus making it possible to avoid
the mechanisms of resistance.21 These characteristics are important in
regard to obtaining drugs for the treatment of endemic diseases in
neglected populations, as is the case among patients with malaria. We
have exhaustively used the hybridization approach to obtain many
different hybrids against P. falciparum.22 CQ and sulfadoxine hybrids
were designed, which gave rise to a prototype that was more active than
both precursor drugs.23 Among the nonquinoline derivatives, we ob-
tained new compounds, namely, inhibitors of the P. falciparum dihy-
2. Results and discussion
2.1. Chemistry
The synthetic route to prepare the 2-(2-acetamidophenyl)-N-(3-((7-
chloroquinolin-4-yl)amino)alkyl)-2-oxoacetamides (1–10) and 2-(2-
acetamidophenyl)-N-(3-((7-chloroquinolin-4-yl)amino)alkyl)-2,2-
difluoroacetamides (11–13) is shown in Scheme 1.
Initially, the intermediates N1-(7-chloroquinolin-4-yl)alkyldiamines
(22–24) were synthesized with 85–90% yield by the nucleophilic sub-
stitution reaction between 4,7-dichloroquinoline (25) with the corre-
sponding diamines: ethane-1,2-diamine, propane-1,3-diamine and
butane-1,4-diamine without the use of a solvent under reflux for 4
hours.23 The acetylation of isatins (26a-d) with acetic anhydride under
reflux for 4 h provided the 1-acetylindoline-2,3-diones (27a-d) with
89–82% yield. The acetylated isatins (27a-d) were characterized by
droorotate
dehydrogenase
(PfDHODH)
enzyme.
These
triazolopyrimidine and pyrazolopyrimidine derivatives are prototypes
used to synthesize new compounds.24,25
Figure 1. Rational approach to the design of compounds 1–21.
2

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
N. Boechat et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115832
Scheme 1. Synthetic route used to prepare compounds 1–21.
melting point and mass spectrometry, and the data were consistent with
the literature.27 The deoxofluorination of (27a-d) was performed using
sulfur diethylaminosulfur trifluoride (DAST) in anhydrous dichloro-
methane under an argon atmosphere for 16 h at 25 ◦C and furnished 1-
acetyl-3,3-difluoroindolin-2-ones (28a-d) with 96–75% yield.27 The
reaction of aminoquinolines (22–24) with 1-acetylindoline-2,3-diones
(27a-d) or 1-acetyl-3,3-difluoroindolin-2-ones (28a-d) in acetonitrile
at 25 ◦C for 2–3 h furnished the compounds the 2-(2-acetamidophenyl)-
N-(3-((7-chloroquinolin-4-yl)amino)alkyl)-2-oxoacetamides (1–10) or
2-(2-acetamidophenyl)-N-(3-((7-chloroquinolin-4-yl)amino)alkyl)-2,2-
difluoroacetamides (11–13), respectively, with 60–18% yield. PQ was
obtained by the simple neutralization of PQ diphosphate with 10%
aqueous NaOH solution at room temperature.16
J774A.1 macrophages and enzyme-linked immunosorbent assay
(ELISA). The assay details are described below in experimental section.
2.2.1. Continuous cultures of P. falciparum, in vitro assays with P.
falciparum-infected erythrocyte cell cultures and cytotoxicity tests
Twenty-one new CQ and PQ synthesized derivatives (1–21) with
different substituents in the 5- position of the acetamide moiety were
tested in vitro against a P. falciparum CQR clone (W2), and the results are
shown in Table 1. The carbonylated CQ derivatives 1–10 presented
activity with IC50 values ranging from 0.12 to 3.18 µM with high
selectivity index (SI) in the range of 270 - >700, excluding 10 (n = 4; R
= Cl; SI = 29), 5 (n = 3; R = CH3) and 9 (n = 4; R = CH3), which were not
determined. The most potent of the series was compound 6 (n = 3; R =
Cl) with an IC50 = 0.12 µM and SI = 258. However, compound 8 (n = 4;
R = H) was also potent (0.18 µM) and showed the best selectivity
(>700).
The reaction of PQ with 1-acetylindoline-2,3-diones (27a-d) or 1-
acetyl-3,3-difluoroindolin-2-ones (28a-d) using the same conditions
described previously furnished the compounds the 2-(2-acet-
amidophenyl)-N-(4-((6-methoxyquinolin-8-yl)amino)pentyl)-2-oxoace-
tamides (14–17) or 2-(2-acetamidophenyl)-2,2-difluoro-N-(4-((6-
methoxyquinolin-8-yl)amino)pentyl)acetamides (18–21), respectively,
with 45–3% yield.
The gem-difluorinated CQ derivatives (11–13) were also active, with
IC50 values ranging from 0.40 to 2.70 µM. Compound 12 (n = 3; R = Cl)
showed the largest SI (>1308) (Table 1). When comparing them by
carbon numbers as linkers, it was not possible to say that one series was
much better than the other. The gem-difluorinated CQ derivatives 11–13
did not showed significant differences when compared with the car-
bonylated derivatives.
2.2. Biology
The carbonylated PQ derivatives 14–17 and gem-difluorinated de-
rivatives 18–19 were the least potent CQ derivatives, with IC50 values
between 2.50 and 26.8 µM and 3.50 to 25.5 µM, respectively. Compound
17 (R = Br) and its gem-difluorinated analogue 21 (R = Br) were the best
of this series, with IC50 values of 2.50 and 3.50 µM, respectively. The SIs
of this series were lower than those of the CQ series, suggesting the
Compounds were assayed with nine different biological methods
such as continuous cultures of P. falciparum, in vitro assays with
P. falciparum-infected erythrocytes, cell cultures and cytotoxicity tests,
P. berghei and antimalarial tests in mice, P. gallinaceum sporogonic as-
says, resazurin cell viability assay for J774A.1 macrophages, nitric oxide
production by J774A.1 macrophages, evaluation of TNFα production by
3

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
N. Boechat et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115832
Table 1
In vitro activity (IC50) of compounds 1–21, CQ and PQ, tested against a P. falciparum CQR (clone W2), cytotoxicity against BGM cell line (MDL50), and the selectivity
index (SI).
Compounds
Anti-P. falciparum activity
IC50* (µM)
Cytotoxicity to BGM cells
SI**
MDL50 (µM)
[MDL50 / IC50
]
CQ derivatives
1
0.71 ± 0.05
0.49 ± 0.12
261 ± 61.0
>84
>300
>170
2
3
4
0.52 ± 0.03
0.20 ± 0.06
152.0 ± 9.0
91.7 ± 20.8
292
>400
5
6
7
0.22
ND
ND
0.12 ± 0.02
0.38 ± 0.01
30.9 ± 1.4
102.6 ± 2.0
258
270
8
9
0.18 ± 0.08
0.84 ± 0.17
123.8 ± 35.3
>700
ND
ND
10
3.18 ± 1.0
91.1
29
gem-difluorinated CQ derivatives
11
0.40 ± 0.2
0.50 ± 0.3
99.0 ± 84.0
>250
12
654.0 ± 461.0
>1300
13
2.70 ± 3.3
140.0 ± 119.0
457 ± 22
>50
CQ
0.20 ± 0.04
2285
PQ derivatives
14
26.8
ND
ND
15
25.3
> 2163.6
> 80
4
(continued on next page)