Evaluation of 7-arylaminopyrazolo[1,5-a]pyrimidines as anti-Plasmodium falciparum, antimalarial, and Pf-dihydroorotate dehydrogenase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.09.073

Malaria remains one of the most serious global infectious diseases. An important target for antimalarial chemotherapy is the enzyme dihydroorotate dehydrogenase from Plasmodium falciparum (PfDHODH), which is responsible for the conversion of dihydroorotat

Full text of DOI:10.1016/j.ejmech.2016.09.073

European Journal of Medicinal Chemistry 126 (2017) 72e83
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Evaluation of 7-arylaminopyrazolo[1,5-a]pyrimidines as anti-
Plasmodium falciparum, antimalarial, and Pf-dihydroorotate
dehydrogenase inhibitors
,
,
Luís Felipe S.P. Azeredo a b, Julia P. Coutinho c d, Valquiria A.P. Jabor e,
Patricia R. Feliciano e, Maria Cristina Nonato e, Carlos R. Kaiser b, Carla Maria S. Menezes a,
Amanda S.O. Hammes f, Ernesto Raul Caffarena f, Lucas V.B. Hoelz a, Nicolli B. de Souza c,
Glaecia A.N. Pereira , Isabela P. Ceravolo , Antoniana U. Krettli d, Nubia Boechat a
c
c
c
,
, b, *
ꢀ
ꢀ
a
ꢀ
ꢀ
Departamento de Síntese de Farmacos, Instituto de Tecnologia em Farmacos, Farmanguinhos-FIOCRUZ, Rua Sizenando Nabuco 100, Manguinhos, Rio de
Janeiro, RJ 21041-250, Brazil
b
ꢀ
~
Programa de Pos Graduaçao em Química, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
c
ꢀ
Centro de Pesquisas Rene Rachou, CPqRR-FIOCRUZ, Belo Horizonte, MG 30190-002, Brazil
d
e
ꢀ
~
Programa de Pos Graduaçao em Medicina Molecular, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
ꢀ
^
^
~
~
ꢀ
Laboratorio de Cristalografia de Proteínas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Avenida do Cafe s/n Monte
~
Alegre, 14040-903 Ribeirao Preto, SP, Brazil
f
~
Grupo de Biofísica Computacional e Modelagem Molecular, Programa de Computaçao Científica (PROCC), FIOCRUZ, Av. Brasil 4365, Rio de Janeiro, CEP
21040-360, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Malaria remains one of the most serious global infectious diseases. An important target for antimalarial
chemotherapy is the enzyme dihydroorotate dehydrogenase from Plasmodium falciparum (PfDHODH),
which is responsible for the conversion of dihydroorotate to orotate in the de novo pyrimidine biosyn-
thetic pathway. In this study, we have designed and synthesized fifteen 7-arylpyrazolo[1,5-a]pyrimidine
derivatives using ring bioisosteric replacement and molecular hybridization of functional groups based
on the highly active 5-methyl-N-(naphthalen-2-yl)-2-(trifluoromethyl)- [1,2,4]triazolo[1,5-a]pyrimidin-
7-amine. The compounds were tested against Plasmodium falciparum, as antimalarials in mice with
P. berghei, and as inhibitors of PfDHODH. Thirteen compounds were found to be active against
Received 3 August 2016
Received in revised form
21 September 2016
Accepted 22 September 2016
Available online 30 September 2016
Keywords:
Malaria
P. falciparum
Pyrazolo[1,5-a]pyrimidine
Dihydroorotate dehydrogenase (DHODH)
Molecular docking
P. falciparum, with IC50 values ranging from 1.2 0.3 to 92 26
assays. Four compounds showed the highest selective index (SI), which is a ratio between cytotoxicity
and activity in vitro. The inhibition of PfDHODH showed that compound 30 (R2 ¼ CH3; R5 ¼ CF3; Ar ¼ 7-
naphthyl) displayed higher and selective inhibitory activity, with IC50 ¼ 0.16 0.01 M, followed by 25
(R2 ¼ CH3; R5 ¼ CH3; Ar ¼ 7- -Naphthyl) and 19 (R2 ¼ CF3; R5 ¼ CF3; Ar ¼ 7- -naphthyl), with
IC50 ¼ 4 M and 6 M, respectively. The trifluoromethyl group at the 2- or 5-positions of the
mM in the anti-HRP2 and hypoxanthine
b-
m
b
b
1
m
1 m
pyrazolo[1,5-a]pyrimidine ring led to increased drug activity. The docking results agreed with the values
obtained from enzymatic assays.
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
AIDS [1]. According to epidemiological data, it is present in more
than 90 countries [2,3]. This means that approximately half of the
Among the current devastating infectious diseases, malaria is
the third leading cause of death in the world after tuberculosis and
world's population is exposed to this disease, which has resulted in
approximately 198 million cases of malaria in 2013, leading to
584,000 deaths [4].
The emergence of Plasmodium falciparum multidrug resistance
to chloroquine (CQ) and mefloquine (MQ) [5,6] and the confirma-
tion of cases of artemisinin derivatives resistance [7] justifies the
need for research to expand the range of therapeutic targets [8].
ꢀ
* Corresponding author. Departamento de Síntese de Farmacos, Instituto de
ꢀ
Tecnologia em Farmacos, Farmanguinhos-FIOCRUZ, Rua Sizenando Nabuco 100,
Manguinhos, Rio de Janeiro, RJ 21041-250, Brazil.
E-mail address: boechat@far.fiocruz.br (N. Boechat).
http://dx.doi.org/10.1016/j.ejmech.2016.09.073
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

L.F.S.P. Azeredo et al. / European Journal of Medicinal Chemistry 126 (2017) 72e83
73
The enzyme dihydroorotate dehydrogenase (DHODH) has been
investigated as a pharmacological target for new therapeutic agents
to treat cancer, rheumatoid arthritis and parasitic diseases such as
malaria, Chagas disease and leishmaniasis [9e12].
antimalarial compounds mefloquine and amodiaquine (11) [20].
We observed that a trifluoromethyl group at the 2-position of the
triazolopyrimidine ring plays an important role in antiplasmodial
activity (Fig. 2).
The DHODH enzyme participates in the fourth step of the de
novo pyrimidine biosynthesis pathway. This flavoenzyme catalyses
the stereospecific oxidation of (S)-dihydroorotate (DHO) to orotate
based on the ping-pong mechanism of catalysis [13]. In the first
half-reaction comprising the oxidation of dihydroorotate to orotate,
electrons are transferred to the flavin mononucleotide moiety
(FMN) that is reduced to dihydroflavin mononucleotide (FMNH2).
After dissociation of orotate from the enzyme, FMNH2 is regener-
ated by an appropriate electron receptor [13].
There are two classes of DHODHs that are classified based on the
difference in their amino acid sequences and the preference for
electron acceptors. Both human (HsDHODH) and P. falciparum
(PfDHODH) enzymes belong to class 2, that use ubiquinone as a
terminal oxidant agent and are located in the inner mitochondrial
membrane [10e12].
Following the good results obtained with 2-(trifluoromethyl)
[1,2,4]triazolo[1,5-a]pyrimidines [20], in this study, we planned to
use the ring bioisosterism replacement (green) of triazolopyr-
imidine 10 (Fig. 3) to obtain 7-arylaminopyrazolo[1,5-a]pyrimi-
dines. . Molecular hybridization (blue) of 7-b-naphtylamino of 10
was also used. Different arylamines (blue) were incorporated into
the structure to investigate the importance of the substituent at the
7-position of the pyrazolopyrimidine nucleus. Investigation of the
impact of the substituents CF3 and CH3 (red) in the 2- or 5-positions
of the pyrazolo[1,5-a]pyrimidine scaffold was prioritized. These
compounds were evaluated in vitro against P. falciparum, in vivo as
antimalarial, and in vitro as PfDHODH inhibitors.
In addition, a molecular docking study was performed to eval-
uate the possible binding mode of the 7-arylaminopyrazolo[1,5-a]
pyrimidine compounds to PfDHODH [21].
HsDHODH inhibitors are not clinically significant and have
demonstrated high potency as anti-inflammatory and anti-
proliferative activity [14]. Unlike human cells, which can acquire
pyrimidine bases through pathway reuse, the P. falciparum de novo
pyrimidine biosynthetic pathway is essential for the maintenance
of life. The inhibition of PfDHODH has been proved to be an
attractive strategy to search for new substances with anti-
plasmodial activity [9,10,12]. Synthetic compounds have been
identified as inhibitors of class 2 DHODH enzymes (Fig. 1) such as
brequinar and its analogues 1 [15]. Some triazolopyrimidine com-
pounds such as 5-methyl-N-(naphthalen-2-yl)- [1,2,4]triazolo[1,5-
a]pyrimidin-7-amine (JZ8; 2) and 3, and N-phenylbenzamide 4
were discovered by Phillips and co-workers through enzyme-based
high-throughput screening [11,16,17]. Other compounds, such as 5
and 6, were obtained through pharmacophoric group modification
[18,19].
In 2012, we evaluated a series of 2-(trifluoromethyl) [1,2,4]tri-
azolo[1,5-a]pyrimidines against P. falciparum that were designed
based on bioisosteric replacement of functional groups in the
Fig. 2. 2-(trifluoromethyl) [1,2,4]triazolo[1,5-a]pyrimidines 7e10 reported as having
antiplasmodial activity.
Fig. 1. Brequinar and compounds 1e6 as PfDHODH inhibitors.

74
L.F.S.P. Azeredo et al. / European Journal of Medicinal Chemistry 126 (2017) 72e83
Table 1
Yields of the synthesized 7-arylaminopyrazolo[1,5-a]pyrimidines 16e30.
Compounds Reaction conditions Ar R1 R2 Yield (%)
CF3 CH3 51
16
17
EtOH/rt
EtOH/rt
CF3 CH3 90
18
19
20
Toluene/ref.
EtOH/rt
CF3 CH3 92
CF3 CH3 85
CF3 CH3 83
Toluene/ref.
21
22
Toluene/ref.
EtOH/rt
CF3 CH3 44
CH3 CH3 78
Fig. 3. Rational approach to the design of 7-arylaminopyrazolo[1,5-a]pyrimidines,
using ring bioisosterism replacement (green), molecular hybridization and different
arylamino groups (blue) as well as the groups CF3 and CH3 (red) in the 2- or 5-positions
of the pyrazolo[1,5-a]pyrimidine scaffold. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
23
EtOH/rt
CH3 CH3 77
24
25
26
EtOH/rt
EtOH/rt
EtOH/rt
CH3 CH3 68
CH3 CH3 89
CH3 CH3 90
2. Results and discussion
2.1. Chemistry
Fifteen compounds (16e30) were synthesized with different
groups in the 2-, 5- and 7-positions of the pyrazolo[1,5-a]pyrimi-
dine ring, as shown in Table 1. All are new except 24 [22].
The first step of the synthetic route to obtain the pyrazolo[1,5-a]
pyrimidine derivatives (Scheme 1) was a condensation of 3-amino-
27
28
EtOH/rt
CH3 CH3 60
CH3 CF3 58
pyrazoles 12a-b with the corresponding
b-ketoesters 13a-b. Re-
actions were carried out in acetic acid under reflux to produce
pyrazolo[1,5-a]pyrimidinones 14a-c with 71e93% yields after
5e16 h [23].
Toluene/NaH/ref.
The pyrimidinones 14a-c were submitted to a chlorodehy-
droxylation reaction with phosphorus oxychloride at 85 ꢀC for
1e4 h to produce the respective 7-chloro-pyrazolo[1,5-a]pyrimi-
dines 15a-c with 50e82% yields [20,23]. 15a-c underwent aromatic
nucleophilic substitution with the corresponding anilines to pro-
duce the desired 7-arylaminopyrazolo[1,5-a]pyrimidines (16e30)
at 44e92% yields (Table 1). Spectroscopic data from the in-
termediates and final products agreed with the proposed
structures.
29
30
Toluene/NaH/ref.
Toluene/NaH/ref.
CH3 CF3 74
CH3 CF3 81
2.2. Molecular docking
series 2-CF3/5-CH3 (16e21), 2-CH3/5-CH3 (22e27) and 2-CH3/5-CF3
(28e30) docked at the same binding pocket as the inhibitor JZ8 did.
For the compound 21 (2-CF3/5-CH3-7-(2-CF3Ph)), the lowest energy
pose occupied an adjacent pocket, whereas the second lowest en-
ergy superposed the JZ8 inhibitor.
The poses were obtained by superposing the crystallographic
structure, which interacted with the same residues in the pocket of
the PfDHODH enzyme that is composed of the H-bond pocket
(His185 and Arg265), the co-crystallized HOH15 and the
Molecular docking studies were performed as an initial evalu-
ation of the possible binding mode of the pyrazolo[1,5-a]pyrimi-
dine compounds to PfDHODH (PDB code: 3i65) [11].
The redocking of the co-crystallized inhibitor (JZ8, compound 2,
Fig. 1) showed an RMSD (root-mean-square deviation) equal to
0.5 Å [24], which validated the selected parameters to evaluate the
binding mode of the pyrazolo[1,5-a]pyrimidine compounds.
According to results, pyrazolo[1,5-a]pyrimidine compounds of

L.F.S.P. Azeredo et al. / European Journal of Medicinal Chemistry 126 (2017) 72e83
75
Scheme 1. Reagents and conditions: (i) AcOH, 5e16 h, reflux, 71e93 ꢀC (ii) POCl3, 1e4 h, 50e82 ꢀC; (iii) appropriate amine, EtOH or toluene, 14e26 h *See information in Table 1.
hydrophobic pocket (Phe188 and Phe227) [21]. Fig. 4 illustrates the
binding modes of the corresponding 7- -naphthyl derivatives (19,
25, and 30). The lowest binding energy values varied from ꢁ11.6
to ꢁ7.6 kcal/mol, which was higher than the calculated value for JZ8
(Table 2).
observed for the lowest energy pose of compound 21. Compound 10
superposed to compound JZ8. In a radius of 5 Å, both pockets
shared some important residues, such as Phe227 and His185.
Considering the importance of Arg265 on the inhibitory effect [21]
and the worst energy binding value, it is possible to infer that 11, in
opposition to compound 10 that showed interesting antiplasmodial
activity [20], could not inhibit PfDHODH”.
b
When the double change between the methyl (CH3) or tri-
fluoromethyl (CF3) groups at the 2- and 5-positions were consid-
ered, maintaining the substituent at the 7-amino position, no
significant differences were observed between the binding energy
values. However, according to an analysis of the ligand-protein
complexes, the nearest distances to the complexes of JZ8 were
found for the series 2- CH3/5-CF3 (28e30), followed by 2- CH3/5-
CH3 (22e27) and 2-CF3/5-CH3 (16e21) (Table 3). Moreover,
similar to JZ8, only the series 2-CH3/5-CF3 compounds were able to
establish equivalent electrostatic interactions with Arg265, His185
(H-bond pocket) and HOH15 (Fig. 5C). The hydrogen bond between
the N1 and HOH15 may be the reason this water molecule also
interacts with the hydroxyl group of Tyr528 (Fig. 4). Furthermore,
HOH15 was hypothesized to participate in conjunction with in-
teractions that lead to a narrow and unique cavity in which FMN,
ORO, and the inhibitor could enter into the enzyme [21]. The two
other series showed a specific interaction with Arg265 (Fig. 5A and
B). Additionally, the CF3 group at the position 5 favoured hydro-
phobic contacts with adjacent residues (Fig. 5C).
2.3. Biological evaluation
Compounds (16e30) were assayed against P. falciparum (W2
clone, chloroquine-resistant) for cytotoxicity in BGM cells and
percent inhibition of the PfDHODH enzyme (Table 4). Three of the
better in vitro results against P. falciparum were used for in vivo
assays against P. berghei. The results were compared to references
5-methyl-N-(naphthalen-2-yl)-2-(trifluoromethyl)- [1,2,4]triazolo
[1,5-a]pyrimidin-7-amine (10) [20] and the antimalarial amodia-
quine (11). Compound 10 was re-synthesized in this work in order
to be tested in vitro in the same conditions of the 7-
arylaminopyrazolo[1,5a]pyrimidines.In the antiplasmodial evalua-
tion, three of the four most potent compounds against the W2
clone were those that contained 7-b-naphthyl (19, 25, 30) and a
3,5-dimetoxy (28) group in the 7-position. The compounds 19, 28,
30 and 25 also showed high selectivity index (SI) values of 467.8,
330.3, 234.1 and 79.6, respectively. Compounds containing an
unsubstituted phenyl group in 7-position (18, 24 and 29) were less
potent for this series, which indicates that bulkier groups in this
region were important for increased potency. In relation to sub-
Therefore, the major differences in the binding energy values
seem to be associated with the intrinsic characteristics of the
substituent at the 7-amino position of the pyrazolo[1,5-a]pyrimi-
dine nucleus. The presence of 7-b-naphthyl was associated with the
lowest values of binding energy, followed by 4-CF3Ph and 4-ClPh
and then by the Ph and 3,5-Di-CH3OPh groups, respectively
(Table 2). The highest values of binding energy corresponded to
derivatives containing the 2-CF3Ph group. The observed values
could be related to the intrinsic characteristics of each substituent
stituents at the 2- and 5-positions when Ar ¼ 7-
b-naphthyl, the
following order of activity was observed: 2-CF3/5-CH3 (19) > 2-CH3/
5-CH3 (25) > 2-CH3/5-CF3 (30). Similar to the triazolopyrimidine
series [25] the most potent 19 possessed a CF3 group in the pyrazole
ring and 7-b-naphthyl as substituent. However, the activities
(
b
-naphthyl, phenyl or phenyl substituted) and/or the position of
observed were lower than the control drugs 11 and triazolopyr-
imidine 10. The results are summarized in Table 4.
substitution at the phenyl group, especially regarding van der
Waals contacts within the hydrophobic pocket.
The lowest binding energy values calculated for 11 and com-
pound 10 were ꢁ7.3 and ꢁ12.4 kcal/mol, respectively (Table 2).
These values could be related to their poses in the enzyme. 11
docked at the adjacent pocket of the binding site, which was
Evaluating the inhibition of PfDHODH, the compounds 10, 16, 17,
19, 20, 23, 25, 26 and 30 showed the highest percentage inhibition
(Table 4). For that reason, these compounds were subjected to an
IC50 assay. The amodiaquine (11) was highly potent against the W2
strain in antiplasmodial evaluation. However, it showed a low

76
L.F.S.P. Azeredo et al. / European Journal of Medicinal Chemistry 126 (2017) 72e83
Fig. 4. Superposition of the 7-
b
-naphthyl derivatives (19, 25 and 30) to the co-crystallized inhibitor (JZ8). Hydrogen bonds are shown for the 30 molecule in the binding site of
PfDHODH. Carbon atoms of 19, 25 and 30 are shown in yellow, cyan and magenta, respectively. Carbon atoms of JZ8 are shown in green. Carbon atoms of FMN and ORO are shown in
orange. The hydrogen atoms were omitted for clarity. The red sphere represents HOH15. Hydrogen bond distances are measured in angstroms. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
(Table 4). Compound 20 was insoluble at concentrations above
100 mM in the medium used in the enzymatic reaction and was not
tested. Additionally, it was not possible to determine the IC50 of
compounds 17 and 20 because these compounds did not show a
Table 2
Binding energy values between the 7-arylaminopyrazolo[1,5-a]py-
rimidines at the binding pocket of PfDHODH relative to co-crystallized
inhibitor (JZ8), compound 10 and amodiaquine (11).
sufficient inhibitory response in this test up to 400
Due to the high SI of compounds 19, 25 and 30 and the presence
of the same substituent at the 7-position ( -naphthylamine), these
compounds were selected for testing the antimalarial activity in
P. berghei infected mice. Compounds 18 and 25 inhibited P. berghei-
parasitemia by 50% on day 5 after inoculation, whereas compound
30 was partially active with up to 25% parasitemia inhibition. All
three tested compounds showed an increase of parasitemia inhi-
bition on day 11. Control antimalarial drugs that were tested in
parallel, inhibited parasitemia by 100% (Table 5).
mM (Table 4).
Compound
Binding energy (kcal/mol)
JZ8
10
11
ꢁ11.9
ꢁ12.4
ꢁ7.3
b
Series
7-Amine substitution
Ph 4-
b
-
4-
3,5-Di CH3O 2-CF3
Naphthyl
CF3Ph ClPh Ph
Ph
2-CH3/5-
CH3
ꢁ11.6
ꢁ9.0 ꢁ10.2 ꢁ9.3 ꢁ8.9
ꢁ7.9
2- CH3/5- ꢁ11.2
ꢁ8.8 e
e
ꢁ8.8
e
CF3
3. Conclusions
2-CF3/5-
CH3
ꢁ11.6
ꢁ9.0 ꢁ10.3 ꢁ9.4 ꢁ8.8
ꢁ7.3a
Among 15 arylaminopyrazolo[1,5-a]pyrimidines (16e30) syn-
thesized with different combinations of substituents at positions 2-
, 5- and 7- of the pyrazolo[1,5-a]pyrimidine ring, 13 exhibited anti-
P. falciparum activity in vitro against chloroquine-resistant para-
a
Second lowest binding energy pose.
percentage of inhibition of PfDHODH, which indicates that this
antimalarial drug did not act inhibiting this enzyme. Compound 30
sites, with IC50 values ranging from 1.2 to 92.4
with -naphthyl (19, 25 and 30) and 3,5-dimethoxyphenyl (28)
groups at the 7-position were the most active.
The compounds 19, 28, 30 and 25 exhibited low toxicity and low
IC50 values and consequently the highest SI values of 467.8, 330.3,
234.1 and 79.6, respectively. When evaluated against P. berghei
malaria, compounds 19 and 25 were active, and both reduced
mM. Compounds
b
displayed better activity in this assay with IC50 ¼ 0.16 0.01
m
m
M,
M,
followed by 25 and 19 with IC50 ¼ 4
mM
1 and IC50 ¼ 6
1
respectively, and both compounds were also active against
P. falciparum. Moreover, 30 was highly selective against the parasite
enzyme when tested against the human homologue enzyme

L.F.S.P. Azeredo et al. / European Journal of Medicinal Chemistry 126 (2017) 72e83
77
Table 3
Interatomic distances between the 7-
b
-naphthyl derivatitives of the pyrazolo[1,5-a]pyrimidines series and residues of the binding pocket of PfDHODH relative to JZ8.
Compound
Interatomic distances (Å)a
N4-Nh
N8-Nd
N1eO (HOH15)
C40-Cg
C40-Cz
Phe227
Arg265
His185
Phe188
JZ8
19
25
30
2.98
3.17
3.05
2.93
2.93
3.34
3.29
3.10
3.06
3.86
3.64
3.33
4.44
4.62
4.58
4.59
3.57
3.57
3.54
3.52
a
Atoms showed in Fig. 5 (C).
Fig. 5. Two-dimensional diagrams of the molecular interactions between the 7-b-naphthyl derivatives of the series 2-CF3/5-CH3 (A), 2-CH3/5-CH3 (B) and 2- CH3/5-CF3 (C) within
the binding site of PfDHODH. Black dashed lines indicate hydrogen bonds. Green solid lines show hydrophobic interactions, while green dashed lines show
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
p-p interactions. (For
parasitemia by 50% on day 5 after inoculation, whereas 30 was
partially active.
was performed using Merck TLC Silica gel 60 F254 aluminium sheets
20 ꢂ 20 cm (eluent ethyl acetate/hexane). The melting points (m.p.)
were determined using a Büchi model B-545 apparatus. Column
chromatography was performed using silica gel 60 (0.040e0.063 mm).
The 1H, 13C and 19F nuclear magnetic resonance (NMR) spectra were
generated at 400, 100 and 376 MHz, respectively, using a BRUKER
Avance instrument equipped with a 5 mm probe. Tetramethylsilane
Compounds that had better activity against the PfDHODH
enzyme were also the most active against
a P. falciparum
chloroquine-resistant W2 clone, which indicated that inhibition of
this enzyme was one of the mechanisms of action of 7-arylamine-
pyrazolo[1,5-a]pyrimidines. However, the antimalarial 11 showed a
low percentage of inhibition against PfDHODH, which supported
that this drug had a different mode of action [25].
The proposed bioisosteric replacements of 1,2,4-triazolo[1,5-a]
pyrimidine on compound 10 with the pyrazolo[1,5-a]pyrimidine
was shown to be a positive proposition according to the molecular
docking study. As found for triazolopyrimidines the 2-CF3 played an
important role in the antiplasmodial activity in pyrazolopyr-
imidines. Meanwhile, 11 did not seem to inhibit PfDHODH. The
results demonstrated the potential of 7-arylpyrazolo[1,5-a]pyrim-
idine derivatives as inhibitors of PfDHODH and may represent new
leads for developing drugs against malaria.
was used as an internal standard. The chemical shifts (d) are reported
in ppm, and the coupling constants (J) are reported in Hertz. The
Fourier transform infrared (FTIR) absorption spectra were recorded on
a Shimadzu mode IR Prestige-21 spectrophotometer using an atten-
uated total reflection (ATR) technique. The analysis with high perfor-
mance liquid chromatography (HPLC) was performed with Shimadzu
liquid chromatography LC-10AD using a Supelcosil LC-8 column
(150 mm ꢂ 4.6 mm x 2.6
mm) and a 2.5 mM acetonitrile mobile phase
acetate buffer (pH 5.8) at 30ΊC and with a flow of 1.0 mL/min.
4.2. Synthesis
4. Materials and methods
The aminopyrazoles substrates (12a-b) were purchased or
synthesized following published procedures. The 3-amino-5-(tri-
fluoromethyl)-1H-pyrazol (12a) was prepared as described in a
previous report [26,27] and 3-amino-5-methyl-1H-pyrazol (12b)
4.1. Chemistry
All reagents and solvents used in this study were analytical grade.
All reactions were tested with TLC (thin layer chromatography), which
was commercially available. The substrate
were also commercially available.
b-ketoesters (13a-b)

78
L.F.S.P. Azeredo et al. / European Journal of Medicinal Chemistry 126 (2017) 72e83
Table 4
Evaluation of anti-Plasmodium falciparum, W2 clone, chloroquine-resistant (IC50), cytotoxicity (MLD50) against a kidney epithelial cell line (BGM) selectivity index (SI ¼ MLD50
/
IC50) in vitro and inhibitory percentage of HsDHODH, PfDHODH and IC50 against PfDHODH.
Compound Substituents Cytotoxicity
R1/R2 MLD50 M)
IC50
Anti-HRPII
(
m
M)
IC50
Hypoxanthine
(
m
M) [3H]-
SI (MLD50/IC50
Anti-HRPII
)
SI (MLD50/IC50) [3H]- (%) Activity
hypoxanthine
(%) Activity
IC50 against
PfDHODH (mM)
(m
HsDHODHa
PfDHODHa
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
10
11
2-CF3/5-Me 67.5 11
255.7 16.5
856.2 37.7
561.4 49.7
108.3 36.1
227.8 36.1
2-CH3/5-CH3 566.2 180
187.9 50.3
315.0 67.2
406 216
67.5 28.5
>142
>171.2
50.3 30.7
>142
>171.2
1.0
e
1.3
e
121
95
113
6
52
57
88
5
7
7
23
>400
Np
5
8
e
e
6
1.2 0.3b
30.6 6.9
44.4 16.4
23.5 2.2
21.5 1.7
92.4 26
5.1 1.5b
8.5 0.1
49.0 10.5
8.6 1.9b
17.1 5.0b
10 3.7b
1.2 0.3c
47.2 14.4
50.0 16.9
25.7 9.2
26.8 12.4
67.2 40
5.1 1.5c
15.7 4.6
42.5 12.4
8.6 1.9c
17.1 5.0c
10 3.7c
467.8d
3.5
467.8d
2.3
56.837 0.003 67.474 0.002 6
95
93
95
80
104
71.92 0.02
88
96
104
122
101
np
1
4
5
5
5
33
48
74 10
42
66
41
9
4
>100
Np
Np
>400
Np
4
5.1
24.1
8.7
4.6
22.0
7.0
4
8
3
3.4
4.7
6
79.6d
10
79.6d
5.4
1
85.0 5.6
5
5
34.343 0.004 24
3
104.6 32.7
2-CH3/5-CF3 >2840.9
261 6,1
2.1
2.5
94
77
77
60
37
79
8
1
5
3
3
4
Np
Np
Np
0.16 0.01
0.70 0.08
>400
330.3d
15.2d
234.1d
18.478
e
>330.3d
15.2d
234.1d
18.478
e
8
6
8
2341 105
425
0.023 0.002 0.023 0.002
e
0.006 0.002c 0.006 0.002c
np
IC50 ¼ 50% inhibitory concentration of the parasite growth.
MLD50 ¼ minimal lethal dose for 50% of cells.
np, not performed.
a
Using 50 mM of each compound.
IC50 determined by SYBR test.
Selectivity index based on SYBR test.
Insoluble at concentrations above 100
b
c
d
mM.
H-6) 2.24 (s, 6H, 2CH3). 13C NMR (100 MHz, DMSO-d6,
156.15, 151.78, 149.68, 142.17, 94.91, 88.03, 18.55, 14.07.
d
in ppm):
Table 5
Percent inhibition of P. berghei-parasitemia (NK65 strain) in mice orally treated with
antimalarial and the most selective compounds of in vitro testsa.
Compound (dose)
Days after inoculation
4.2.1.3. 2-Methyl-5-(trifluoromethyl)pyrazolo
[1,5-a]pyrimidin-
7(4H)-one (14c). Yield: 71%. m.p. degradation at 281.2 ꢀC. IR (KBr,
cmꢁ1): 3174, 2904, 1705, 1631, 1585, 1537, 1460, 1409, 1278, 1220,
1186, 1151, 835, 744. ESI-MS (Neg): m/z 216.3 (M ꢁ 1).1H NMR
5 (%)
7 (%)
9 (%)
11 (%)
19 (5 mg/kg)
25 (5 mg/kg)
30 (5 mg/kg)
11 (5 mg/kg)
50
50
25
61
40
1
47
53
4
59
65
29
100
(400 MHz, DMSO-d6, d in ppm): 13.24 (s,1H, NH),12.18 (s,1H, Ar-H),
6.47 (s, 1H, Ar-H), 2.41 (s, 3H, CH3)$13C NMR (100 MHz, DMSO-d6,
100
100
100
d
in ppm (J in Hz)): 161.94, 150.35, 134.16, 133.05 (q, 2J ¼ 33.9, C-
a
Percent inhibition calculated in relation to the control untreated group. Mice
were treated for 3 consecutive days 24 h after receiving the i.p. inoculum containing
106 parasitized red blood cells.
CF3), 122.22 (q, 1J ¼ 273.4, CF3), 115.17, 97.06, 10.78. 19F NMR
(376 MHz, DMSO-d6,
d in ppm): -63.66.
4.2.2. General procedure for the preparation of 7-chloropyrazolo
[1,5-a]pyrimidines 15a-c
4.2.1. General procedure for the preparation of pyrazolo [1,5-a]
pyrimidinones 14a-c
A mixture of a 3-aminopyrazole derivative (12a-b) (8 mmoL)
and the appropriate b-ketoester (13a-b) (8 mmoL) was refluxed in
acetic acid (20 mL) for 5e16 h. The resulting mixture was poured
into ice (10 mL) and pH was adjusted to 8 with NaOH (2.5 M aq.)
then ethyl acetate (2 ꢂ 30 mL) was extracted. The combined organic
layers were washed with water (2 ꢂ 30 mL), dried with anhydrous
sodium sulphate, filtered and then the solvent was evaporated
under vacuum to afford 14a-c.
A
suspension of pyrazolo[1,5-a]pyrimidinone intermediate
(14a-c) (2 mmoL) in phosphorus oxychloride (3 mL, 32.1 mmoL)
was heated to 85 ꢀC and stirred for up to 4 h. The mixture was
carefully dripped into crushed ice (20 mL) under stirring and then
neutralized to pH 7 with NaOH (2.5 M aq.). The resulting emulsion
was extracted with ethyl acetate (2 ꢂ 20 mL). The combined
organic layers were dried with anhydrous sodium sulphate, filtered
and then the solvent was removed under vacuum to afford 15-a-c.
4.2.2.1. 7-Chloro-5-methyl-2-(trifluoromethyl)pyrazolo [1,5-a] py-
rimidine (15a). Yield: 82%. m.p.: 110.8e114.3 ꢀC. IR (KBr, cmꢁ1):
3062, 1614, 1523, 1487, 1062, 1261, 1215, 1190, 1157, 752. MS-EI
(70 eV) m/z (%): 235 (100); 237 (33); 166 (14); 216 (12); 200 (8);
185 (9).
4.2.1.1. 5-Methyl-2-(trifluoromethyl)pyrazolo
[1,5-a]pyrimidin-
7(4H)-one (14a). Yield: 89%. m.p.: degradetion at 262.5 ꢀC. IR (KBr,
cmꢁ1): 3672; 3200; 3142; 2847; 2789; 1681; 1631; 1555; 1518;
1502; 1242; 1194; 837; 808; 779; 742. 1H NMR (400 MHz, DMSO-d6,
d
in ppm): 6.16 (s, 1H, Ar-H), 5.38 (s, 1H, Ar-H), 2.15 (s, 3H, CH3). 13
C
NMR (100 MHz, DMSO-d6,
d in ppm (J in Hz)): 160.46, 158.20,
4.2.2.2. 7-Chloro-2,5-dimethylpyrazolo [1,5-a]pyrimidine (15b).
Purified by chromatography column with ethyl acetate/hexane
(3:7) as eluent. Yield: 53%. m.p.: 161.0e163.7 ꢀC. IR (KBr, cmꢁ1):
3062, 2929,1614,1521,1477,1365, 1250, 1193, 860. MS-EI (70 eV) m/
z (%): 181(100), 183 (32), 146 (31), 180 (30), 182(19).
152.30, 142.18 (q, 2J ¼ 36.3, C-CF3), 122.88 (q, 1J ¼ 269.0, CF3), 93.32,
89.66, 24.47. 19F NMR (376 MHz, DMSO-d6,
d in ppm): -64.97.
4.2.1.2. 2,5-Dimethylpyrazolo [1,5-a]pyrimidin-7(4H)-one (14b).
Yield: 93%. m.p. degradation at 241.4 ꢀC. IR (KBr, cmꢁ1): 3415, 1712,
1641, 1566, 1554, 1280, 817. ESI-MS (Neg): m/z 162.4 (M ꢁ 1). 1H
4.2.2.3. 7-Chloro-2-methyl-5-(trifluoromethyl)pyrazolo [1,5-a] py-
rimidine (15c). Yield: 50%. m.p.: 133.5e136.6 ꢀC. IR (KBr, cmꢁ1):
NMR (400 MHz, DMSO-d6,
d in ppm): 5.89 (s, 1H; H-3) 5.47 (s, 1H,

L.F.S.P. Azeredo et al. / European Journal of Medicinal Chemistry 126 (2017) 72e83
79
3130, 1639, 1557, 1502, 1428, 1316, 1273, 1181, 1019, 823, 724, 625.
4.2.3.5. 7-(3,5-Dimethoxyphenylamine)-2,5-dimethylpyrazolo [1,5-a]
pyrimidine (23). Yield: 77%. m.p.: 138.8e141.3 ꢀC. IR (KBr, cmꢁ1):
3290, 3078, 3006, 2969, 2939, 2915, 2840, 1623, 1602, 1557, 1489,
1417, 1387, 1206, 1156, 1071, 1056, 817, 722. MS-EI (70 eV) m/z (%):
298 (100), 283 (43), 297 (21), 299 (18), 122 (14). 1H NMR (400 MHz,
MS-EI (70 eV) m/z (%): 235 (100), 234 (97), 236 (39), 237 (32), 181
(8). 1H NMR (400 MHz, Acetone-d6,
d
in ppm): 7.55 (s, 1H, Ar-H),
in ppm (J in
2.59 (s, 3H, CH3). 13C NMR (100 MHz, Acetone-d6,
d
Hz)): 170.93, 154.03, 150.64, 141.19, 133.57 (q, 2J ¼ 34.9, C-CF3),
123.26 (q, 1J ¼ 271.1, CF3), 114.34 (q, 3J ¼ 5.6, HC-C-CF3), 108.32,
Acetone-d6,
1H, J ¼ 4.4, Ph-H), 6.37 (s, 1H, Ar-H), 6.13 (s, 1H, Ar-H), 3.83 (s, 6H,
2OCH3), 2.40 (2s, 6H, 2CH3). 13C NMR (100 MHz, Acetone-d6,
in
d
in ppm (J in Hz)): 6.70 (d, 2H, J ¼ 4.4, Ph-H), 6.40 (t,
79.21, 60.55, 20.83,14.36. 19F NMR (376 MHz, Acetone-d6,
d in ppm):
-61.89.
d
ppm): 161.78, 159.08, 153.22, 150.07, 143.82, 139.42, 101.59, 97.32,
93.86, 86.54, 55.02, 24.30, 13.81. HPLC: 98%.
4.2.3. General procedure for the preparation of 7-arylamine
pyrazolo [1,5-a]pyrimidines 16, 17, 19 and 22e23, 25e27
4.2.3.6. 2,5-Dimethyl-7-(naphthalen-2-ylamine)pyrazolo [1,5-a]py-
rimidine (25). Yield: 89%. m.p.: 135.6e139.0 ꢀC. IR (KBr, cmꢁ1):
3322, 3033, 2920, 1626, 1607, 1591, 1562, 1511, 1417, 1363, 793, 754,
723, 674. MS-EI (70 eV) m/z (%): 288 (100), 287 (44), 273 (26), 289
A mixture of the corresponding 7-chloropyrazolo[1,5-a]pyrim-
idines derivative 15a-b (1 mmol) and of the appropriate amine
(1 mmol) in ethanol (10 mL) was stirred at room temperature for
18e20 h. The reaction mixture was concentrated and poured into
30 mL of ice-cold water with 2 drops of NaOH (2.5 M aq.) added.
The precipitate was collected by filtration and washed with cold
water (10 mL).
(20), 127 (19). 1H NMR (400 MHz, Acetone-d6,
d
in ppm): 7.96 (m,
4H, Naph-H), 7.67 (m, 2H, Naph-H), 7.25 (m, 2H, Naph-H), 6.39 (s,
1H, Ar-H), 6.17 (s, 1H, Ar-H), 2.43 (s, 3H, CH3), 2.38 (s, 3H, CH3). 13
NMR 100 MHz, Acetone-d6, in ppm: 129.46, 127.79, 127.58, 126.78,
125.72, 123.18, 120.59, 93.89, 86.20, 24.28, 13.87. HPLC: 99%.
C
d
4.2.3.1. 7-(4-Chlorophenylamine)-5-methyl-2-(trifluoromethyl)pyr-
azolo [1,5-a]pyrimidine (16). Yield: 51%. m.p.: 137.6e138.8 ꢀC. IR
(KBr, cmꢁ1): 3373, 3349, 3124, 1626, 1589, 1560, 1492, 1409, 1255,
1237, 1120, 1089, 957, 808, 742, 586. MS-EI (70 eV) m/z (%): 326
(100), 176 (36), 328 (33), 327 (21), 120 (20). 1H NMR (400 MHz,
4.2.3.7. 2,5-Dimethyl-7-(4-(trifluoromethyl)phenylamine)-pyrazolo
[1,5-a]pyrimidine (26). Yield: 90%. m.p.: 77.3e79.0 ꢀC. IR (KBr,
cmꢁ1): 3188, 2928, 1603, 1560, 1504, 1374, 1326, 1165, 1100, 1067,
1014, 842, 794, 770, 646, 594. MS-EI (70 eV) m/z (%): 306 (100), 305
(36), 307 (17), 266 (15), 145 (13). 1H NMR (400 MHz, Acetone-d6,
Acetone-d6, d in ppm): 7.57 (m, 4H, Ph-H), 6.73 (s, 1H, Ar-H), 6.50 (s,
1H, Ar-H), 2.45 (s, 3H, CH3). 13C NMR (100 MHz, Acetone-d6,
d in
d
in ppm): 7.78 (s, 4H, Ph-H), 6.48 (s, H, Ar-H), 6.17 (s, 1H, Ar-H) 2.41
ppm (J in Hz)): 162.49, 156.28, 152.12, 150.68, 145.51 (q, 2J ¼ 37.8; C-
CF3), 131.81, 130.51, 127.02, 122.58 (q, 1J ¼ 265.3, CF3), 93.68 (q,
3J ¼ 3.3, HC-C-CF3), 89.56, 25.14. 19F NMR (376 MHz, Acetone-d6,
(s, 6H, 2CH3). 13C NMR (100 MHz, Acetone-d6,
d in ppm (J in Hz)):
160.11, 154.34, 150.96, 143.94, 142.72, 127.58 (q, 3J ¼ 7.6, HC-C-CF3),
126.80 (q, 2J ¼ 64.7; C-CF3),123.65,122.73, 95.06, 87.80, 25.16,14.71.
d
in ppm): -62.89. HPLC: 98%.
19F NMR (376 MHz, Acetone-d6,
d in ppm): - 67.23. HPLC: 88%.
4.2.3.2. 7-(3,5-Dimethoxyphenylamine)-5-methyl-2-(tri-
fluoromethyl)pyrazolo [1,5-a]pyrimidine (17). Yield: 90%. m.p.:
143.0e144.1 ꢀC. IR (KBr, cmꢁ1): 3265, 3148, 3097, 3003, 2942, 2845,
1628, 1601, 1567, 1503, 1406, 1208, 1154, 1122, 1057, 958, 793, 674.
MS-EI (70 eV) m/z (%): 352 (100), 337 (37), 176 (30), 353 (18), 321
4.2.3.8. 2,5-Dimethyl-7-(2-(trifluoromethyl)phenylamine)-pyrazolo
[1,5-a]pyrimidine (27). Yield: 60%. m.p.: 145.9e147.3 ꢀC. IR (KBr,
cmꢁ1): 3365, 3140, 3081, 2928, 1625, 1569, 1457, 1417, 1369, 1317,
1305, 1109, 1090, 1058, 1034, 768, 652, 534. MS-EI (70 eV) m/z (%):
306 (100), 237 (93), 307 (18), 238 (16), 145 (15). 1H NMR (400 MHz,
(10). 1H NMR (400 MHz, Acetone-d6,
d in ppm (J in Hz)): 6.75 (d, 2H,
Acetone-d6,
d in ppm): 7.87 (m, 3H, Ph-H), 7.58 (m, 1H, Ph-H), 6.18
J ¼ 4.4, Ph-H), 6.71 (s, 1H, Ph-H), 6.60 (s, 1H, Ar-H), 6.45 (t, 1H,
(s, 1H, Ar-H), 6.02 (s, 1H, Ar-H), 2.43 (s, 3H, CH3), 2.36 (s, 3H, CH3).
J ¼ 4.4, Ar-H), 3.83 (s, 6H, 2OCH3), 2.48 (s, 3H, CH3). 13C NMR
13C NMR (100 MHz, Acetone-d6,
d in ppm (J in Hz)): 160.01, 154.56,
(100 MHz, Acetone-d6,
d in ppm): 162.69, 162.48, 150.58, 145.87,
150.73, 145.20, 136.36, 134.81, 128.93, 128.19 (q, 3J ¼ 10.3, HC-C-
145.48, 139.63, 125.43, 103.20, 98.87, 93.66, 90.02, 55.91, 25.23. 19
NMR (376 MHz, Acetone-d6, in ppm): -67.19. HPLC: 94%.
F
CF3), 127.99, 127.63, 126.60, 126.01, 122.22, 95.01, 87.17, 25.12, 14.72.
d
19F NMR (376 MHz, Acetone-d6,
d in ppm): -67.20. HPLC: 98%.
4.2.3.3. 5-Methyl-7-(naphthalen-2-ylamine)-2-(trifluoromethyl)pyr-
azolo [1,5-a]pyrimidine (19). Yield: 85%. m.p.: 188.1e190.5 ꢀC. IR
(KBr, cmꢁ1): 3341, 3111, 1634, 1602, 1563, 1505, 1403, 1256, 1176,
1113, 956, 803, 758, 556. MS-EI (70 eV) m/z (%): 342 (100), 341 (39),
4.2.4. General procedure for the preparation of 7-arylamine
pyrazolo [1,5-a]pyrimidines 18, 20 and 21
A mixture of the corresponding 7-chloropyrazolo[1,5-a]pyrim-
idines derivative 15a-b (1 mmoL) and the appropriate amine (1.2
equivalent) in toluene (20 mL) was stirred under reflux. After
6e17 h, the solvent was evaporated under vacuum, and the residue
was dissolved in 2 mL EtOAc and then cold hexane was added until
precipitation. The suspension was filtered to produce 18, 20 and
21 at 44e92% yields.
127 (28), 327 (24), 176 (23). 1H NMR (400 MHz, Acetone-d6,
d
in
ppm): 8.01 (m, 4H, Naph-H), 7.73 (m, 1H, Naph-H), 7.56 (m, 2H,
Naph-H), 6.75 (s, 1H, Ar-H), 6.63 (s, 1H, Ar-H), 2.46 (s, 3H, CH3). 13
NMR (100 MHz, Acetone-d6, in ppm): 162.56, 150.68, 146.24,
C
d
135.57, 134.97, 132.74, 130.50, 128.74, 128.60, 127.76, 126.96, 124.39,
122.68, 93.77, 89.70, 25.25. 19F NMR (376 MHz, Acetone-d6,
d in
ppm): -67,47. HPLC: 90%.
4.2.4.1. 5-Methyl-7-phenylamine-2-(trifluoromethyl)pyrazolo [1,5-a]
pyrimidine (18). Yield: 92%. m.p.: 156.2e159.8 ꢀC. IR (KBr, cmꢁ1):
3363, 3132, 3065, 2926, 1695, 1630, 1597, 1566, 1500, 1408, 1311,
1285, 1180, 1161, 1128, 837, 802, 727. MS-EI (70 eV) m/z (%): 292
(100), 291 (22), 176 (19), 293 (16), 77 (16). 1H NMR (400 MHz,
4.2.3.4. 7-(4-Chlorophenylamine)-2,5-dimethylpyrazolo [1,5-a]py-
rimidine (22). Yield: 78%. m.p.: 155.0e158.8 ꢀC. IR (KBr, cmꢁ1):
3178, 3049, 2993, 2931, 1622, 1560, 1483, 1411, 1380, 1320, 1215,
1018, 992, 846, 758, 722, 650, 584. MS-EI (70 eV) m/z (%): 272 (100),
274 (33), 271 (28), 273 (24), 232 (16). 1H NMR (400 MHz, Acetone-
Acetone-d6,
d
in ppm (J in Hz)): 7.59 (d, 3J ¼ 7.6, 2H, Ph-H), 7.52 (t,
3J ¼ 7.8, 2H, Ph-H), 7.34 (t, 3J ¼ 7.3, 1H, Ph-H), 6.72 (s, 1H, Ar-H), 6.47
d6,
2.40 (s, 3H, CH3) 2.37 (s, 3H, CH3). 13C NMR (100 MHz, Acetone-d6,
in ppm): 159.99, 154.24, 150.96, 144.87, 137.61, 131.30, 130.42,
126.30, 94.78, 86.96, 25.10, 14.69. HPLC: 99%.
d
in ppm): 7.52 (m, 4H, Ph-H), 6.26 (s,1H, Ar-H), 6.14 (s,1H, Ar-H)
(s, 1H, Ar-H), 2.45 (s, 3H, CH3). 13C NMR (100 MHz, Acetone-d6,
d in
ppm (J in Hz)): 162.45, 150.68, 146.28, 145.94 (q, 2J ¼ 37.7, C-CF3),
d
138.04, 130.59, 127.18, 125.43, 122.84 (q, 1J ¼ 268.7, CF3), 93.69 (q,
3J ¼ 3.3, HC-C-CF3), 89.37, 25.25. 19F NMR (376 MHz, Acetone-d6,
d in

80
L.F.S.P. Azeredo et al. / European Journal of Medicinal Chemistry 126 (2017) 72e83
ppm): -62.88. HPLC: 99%.
Acetone-d6, d in ppm (J in Hz)): 155.71, 141.66, 140.60, 132.19 (q,
2J ¼ 33.8, C-CF3), 129.56, 123.99 (q, 1J ¼ 270.1, CF3), 122.97, 119.96,
4.2.4.2. 5-Methyl-2-(trifluoromethyl)-7-(4-(trifluoromethyl) phenyl-
119.85, 105.20 (q, 3J ¼ 5.5, HC-C-CF3), 102.46, 14.38. 19F NMR
amine)pyrazolo
[1,5-a]pyrimidine
(20). Yield:
83%.
m.p.:
(376 MHz, Acetone-d6, d in ppm): -62.82. HPLC: 96%.
146.2e147.6 ꢀC. IR (KBr, cmꢁ1): 3375, 3130, 2928, 1680, 1629, 1595,
1566, 1501, 1412, 1321, 1253, 1161, 1132, 842, 808, 740. MS-EI
(70 eV) m/z (%): 360 (100), 176 (21), 359 (18), 120 (15), 145 (14),
4.2.5.3. 2-Methyl-7-(naphthalen-2-ylamine)-5-(trifluoromethyl)
pyrazolo [1,5-a]pyrimidine (30). Yield: 81%. m.p.: degrades at
253.5 ꢀC. IR (KBr, cmꢁ1): 3385, 3052, 2931, 2856, 1635, 1616, 1504,
1435, 1396, 1246, 1164, 1119, 997, 850, 811, 745, 724, 665. MS-EI
(70 eV) m/z (%): 342 (100), 341 (85), 343 (18), 127 (9), 115 (8). 1H
341 (13). 1H NMR (400 MHz, Acetone-d6,
d
in ppm): 7.85 (m, 4H, Ph-
H), 6.77 (s, 1H, Ar-H), 6.75 (s, 1H, Ar-H), 2.46 (s, 3H, CH3). 13C NMR
(100 MHz, Acetone-d6,
d in ppm (J in Hz)): 162.62, 150.53, 145.09,
145.95 (q, 2J ¼ 37.8, C-CF3), 141.96, 129.77 (q, 1J ¼ 246.4, CF3), 127.62
NMR (400 MHz, Acetone-d6, d in ppm): 8.73 (m, 1H, Ar-H), 7.83 (m,
(q, 3J ¼ 3.8, HC-C-CF3), 124.48, 120.74 (q, 1J ¼ 272.1, CF3), 93.93 (q,
3H, Naph-H), 7.75 (m, 1H, Naph-H), 7.48 (m, 1H, Naph-H), 7.38 (m,
3J ¼ 3.3, HC-C-CF3), 90.34, 25.17. 19F NMR (376 MHz, Acetone-d6,
d in
1H, Naph-H), 7.12 (s, 1H, Ar-H), 2.50 (m, 3H, CH3). 13C NMR
ppm): -63.90, -67.22. HPLC: 98%.
(100 MHz, Acetone-d6, d in ppm (J in Hz)): 155.67, 154.64, 140.66,
139.21, 135.28, 132.22 (q, 2J ¼ 33.8, C-CF3), 130.69, 129.23, 128.43,
128.05, 127.20, 124.92, 124.01 (q, 1J ¼ 270.4, CF3), 121.29, 115.44,
105.37 (q, 3J ¼ 5.3, HC-C-CF3), 102.64, 14.41. 19F NMR (376 MHz,
4.2.4.3. 5-Methyl-2-(trifluoromethyl)-7-(2-(trifluoromethyl) phenyl-
amine)pyrazolo
[1,5-a]pyrimidine
(21). Yield:
44%.
m.p.:
83.9e89.1 ꢀC. IR (KBr, cmꢁ1): 3622, 3196, 1623, 1574, 1497, 1409,
1317, 1256, 1172, 1128, 1088, 1039, 959, 844, 784, 634, 595. MS-EI
(70 eV) m/z (%): 360 (100), 291 (39), 321 (26), 176 (24), 290 (24).
Acetone-d6, d in ppm): -62.76. HPLC: 99%.
4.3. Molecular docking
1H NMR (400 MHz, Acetone-d6,
d
in ppm): 7.80 (m, 4H, Ph-H), 6.77,
(s, 1H, Ar-H), 6.11 (s, 1H, Ar-H), 2.42 (s, 3H, CH3). 13C NMR (100 MHz,
The tridimensional geometries of the pyrazolo[1,5-a]pyrimidine
compounds (16e30), amodiaquine (11) and compound 10 were
drawn and optimized according to the default setup conditions
defined in the Marvin platform [28]. Then, they were reoptimized
to adjust them to the MMFF94 force field [29] incorporated in the
Spartan software (wavefunction,Inc.). The structure of P. falciparum
dihydroorotate dehydrogenase (PfDHODH) in complex with the
inhibitor JZ8 was retrieved from the PDB (PDB ID: 3I65) and used as
a receptor (We emphasize that JZ8 corresponded to DSM1 in this
reference and was previously shown as compound 2) [21]. Note
that the His185 residue was unprotonated at the Nd nitrogen atom
and Arg265 was protonated to contribute to the electron delocal-
ization in the ligand structure, which was suggested by Deng et al.
[21]. Additionally, the co-crystalized water molecules (HOH) 11e13,
15, 16 and 20 were taken into account because of their interaction
with the substrate orotate (ORO) and/or the cofactor flavin mono-
nucleotide (FMN) or interaction with JZ8, such as the case for
HOH15. Automated docking was performed with the AutoDock
Vina program [24] while keeping the protein fixed and allowing full
flexibility for the ligands. The grid box was centred at the centre of
mass for JZ8, which encompassed a volume of 2187 Å3 (X ¼ 36,
Y ¼ 32 and Z ¼ 36 points). Discretization was set at 0.375 Å along
with an energy range of 5 kcal/mol, and the number of models ¼ 10
and exhaustiveness ¼ 150. All the remaining parameters were used
as defaults. The visual analyses of ligand-protein complexes were
made with the PyMOL program [30]. Two-dimensional diagrams
were obtained through the Pose View program [31].
Acetone-d6,
d in ppm (J in Hz)): 162.54, 150.40, 146.99, 146.23 (q,
2J ¼ 75.1, C-CF3), 135.74, 135.06, 130.70, 129.24, 128.38 (q, 3J ¼ 10.2,
HC-C-CF3), 127.98 (q, 2J ¼ 58.5, C-CF3), 127.46, 125.42, 122.04,
120.08, 116.61, 93.92, 89.97, 25.15. 19F NMR (376 MHz, Acetone-d6,
d
in ppm): -65.83, -68.22. HPLC: 89%.
4.2.5. General procedure for the preparation of 7-arylamine
pyrazolo [1,5-a]pyrimidines 28e30
A mixture of the appropriate amine (1.2 mmol), 10 mmol of
sodium hybrid 60% suspension (in oil) and toluene (15 mL) were
stirred at reflux until a colour change occurred (1e4 h). Afterwards,
a solution containing 1 mmol of intermediate 7-chloro-2-methyl-5-
(trifluoromethyl)pyrazolo[1,5-a]pyrimidine (15c) in toluene
(15 mL) was slowly added to the reaction mixture in reflux. Then,
the mixture was stirred at reflux for 1e5 h. The cooled reaction
mixture was concentrated and poured into cold ethanol (50 mL).
Solvents were evaporated under vacuum, and the residual solid
was washed with cold water. The final product was extracted from
the aqueous mixture using ethyl acetate. The organic layer was
dried over anhydrous sodium sulphate and then filtered. The
filtered solution was concentrated and purified with a flash chro-
matography column (silica-gel with ethyl acetate:hexane 1:4).
4.2.5.1. 7-(3,5-Dimethoxyphenylamine)-2-methyl-5-(tri-
fluoromethyl)pyrazolo [1,5-a]pyrimidine (28). Yield: 58%. m.p.:
239.4e240.1 ꢀC. IR (KBr, cmꢁ1): 3365, 3306, 3143, 2966, 2843, 1603,
1541, 1484, 1428, 1396, 1316, 1204, 1145, 1122, 1026, 841, 796, 680.
MS-EI (70 eV) m/z (%): 351 (100), 352 (93), 353 (16), 336 (12), 337
4.4. Biological evaluation
(11). 1H NMR (400 MHz, Acetone-d6,
d in ppm (J in Hz)): 8.92 (s, 1H,
Ar-H), 7.18 (t, 2H, 4J ¼ 2.2, Ph-H), 7.03 (s, 1H, Ar-H), 6.20 (t, 1H,
4.4.1. Continuous cultures and in vitro assays with P. falciparum-
Infected erythrocytes
4J ¼ 2.2, Ph-H), 3.80 (s, 6H, 2OCH3), 2.48 (m, 3H, CH3). 13C NMR
(100 MHz, Acetone-d6,
d
in ppm (J in Hz)): 162.10, 155.61, 143.25,
P. falciparum (W2 clone, chloroquine resistant) parasites were
cultured in human red blood cells under conditions established by
Trager and Jensen [32] with minor modifications [33]. Briefly, the
parasites were kept as described [32] at 37 ꢀC in human erythro-
cytes (Aþ) in complete medium (RPMI 1640 supplemented with
10% human sera blood group Aþ, 2% glutamine, and 7.5% NaHCO3)
either in Petri dishes in a candle jar or in 75-cm2 culture flasks in an
environment containing a gas mixture atmosphere (3% O2, 5% CO2
and 91% N2). Prior to testing, the ring-stage parasites were syn-
chronized using sorbitol [34].
140.64, 132.14 (q, 2J ¼ 33.9, C-CF3), 123.98 (q, 1J ¼ 270.3, CF3), 105.35
(q, 3J ¼ 5.6, HC-C-CF3), 102.42, 99.46, 98.30, 94.93, 14.39. 19F NMR
(376 MHz, Acetone-d6,
d in ppm): -62.82. HPLC: 99%.
4.2.5.2. 2-Methyl-7-phenylamine-5-(trifluoromethyl)pyrazolo [1,5-a]
pyrimidine (29). Yield: 74%. m.p.: 202.5e208.1 ꢀC. IR (KBr, cmꢁ1):
3210, 3143, 2921, 2850, 1615, 1598, 1496, 1424, 1393, 1274, 1170,
1130, 995, 817, 730, 695, 662, 534. MS-EI (70 eV) m/z (%): 291 (100),
292 (93), 293 (13), 77 (9), 263 (7). 1H NMR (400 MHz, Acetone-d6,
d
in ppm): 8.94 (s, 1H, Ar-H), 7.87 (m, 2H, Ph-H), 7.34 (m, 2H, Ph-H),
The infected red blood cells were distributed in a 96-well mi-
crotiter plate (Corning, Santa Clara, CA, USA),180 mL/well with 20 mL
7.02 (m, 2H, Ph-H and Ar-H), 2.48 (m, 3H, CH3). 13C NMR (100 MHz,

L.F.S.P. Azeredo et al. / European Journal of Medicinal Chemistry 126 (2017) 72e83
81
of different concentrations of test drugs and controls that had
previously been added. The maximum concentration, 50 g/mL,
was tested two or three times, and compounds were considered
inactive at equal or higher doses.
of up to 30 mice were inoculated i.p. with 1 ꢂ 106 infected eryth-
rocytes, kept together for approximately 24 h, and then randomly
distributed into groups of five per cage. The mice were treated daily
for three consecutive days with compounds freshly diluted in 3%
DMSO (Sigma-Aldrich) in RPMI medium and administered orally at
5 mg/kg. The control groups received either the drug vehicle or the
antimalarial amodiaquine administered at 5 mg/kg. On days 5, 7, 9
and 11 after the parasite inoculation, blood was taken from the tail
of each mouse and used to prepare thin smears that were
methanol-fixed, Giemsa-stained, and examined microscopically to
determine parasitemia. The inhibition of parasite growth was
determined in relation to parasitemia in the untreated mice, which
was considered to be 100% parasite growth. Compounds reducing
the parasitemia by ꢃ 40% were considered active, between 20% and
39% partially active, and by less than 20% were considered inactive.
m
The effects of compounds against the P. falciparum blood cul-
tures were evaluated through incorporation of [3H]-hypoxanthine
(Perkin Elmer, Waltham, MA, USA) by the parasites [35]. These
compounds were also tested using the anti-HRPII assay. The latter
was an assay with monoclonal antibodies against a parasite protein
rich in histidine and alanine, as previously described [36].
The [3H]-hypoxanthine assay was performed with 1% para-
sitemia and 1% haematocrit, and the level of isotope incorporation
was read after 42 h in a beta-counter (Perkin Elmer). Alternatively,
some compound effects were also examined using the SYBR test as
previously described by Smilkstein et al. [37] with the parasite
suspensions (0.5% parasitemia and 2% haematocrit). Briefly, the test
compounds, in serial dilutions, were incubated in 96-wells plates
(‘U’ bottom). After 48 h at 37 ꢀC, the culture supernatant was
4.4.4. Recombinant production of human and Plasmodium
falciparum DHODH
removed and replaced by 100
(20 mM; pH 7.5), EDTA (5 mM), saponin (0.008%; wt/vol), and
Triton X-100 (0.08%; vol/vol)] followed by addition of 0.2 l/mL
Sybr Safe (SigmaeAldrich, Carlsbad, CA, USA). The plate content
was transferred to a flat bottom plate and incubated in the dark for
30 min. Then, the plate was read in a fluorometer (Synergy H4
Hibrid Reader, BioteK) with excitation at 485 nm and an emission of
535 mm.
m
L of lysis buffer solution [Tris
N-terminally truncated PfDHODH (aa 159e569) gene was syn-
thesized by GenScript with codons optimized for expression in
E. coli. The PfDHODH gene was amplified by PCR with primers 50-
m
GACGACGGATCCGAATCCTATAACCCGGAATTT-30
and
50-GAC-
GACCTCGAGTTACGATTTGGAGTGTTTGCG-3, and cloned into a pET-
28a-SUMO vector between the BamHI and XhoI restriction sites. A
single colony of E. coli BL21(DE3) cells harbouring a recombinant
pET-28a-SUMO-PfDHODH plasmid was used to inoculate 10 mL of
In all tests, the compounds activities were expressed by the 50%
inhibitory concentration of the parasite growth (IC50) compared to
the drug-free controls and estimated using the curve-fitting soft-
ware Origin 8.0 (OriginLab Corporation, Northampton, MA, USA)
[38].
rich medium supplemented with 30
cells were grown for 16 h at 37 ꢀC at 180 rpm. The cells were then
1:100 diluted with rich medium containing 30 g/mL kanamycin
and grown at 37 ꢀC at 180 rpm until OD600nm reached 0.5e0.6. The
cells were then induced with 0.25 mM isopropyl -thio-
mg/mL kanamycin, and the
m
b-D
galactopyranoside (IPTG), and growth continued for 4 h at 25 ꢀC
and 180 rpm. The induced cells were harvested by centrifugation at
5000ꢂg for 10 min and frozen at ꢁ20 ꢀC. The cells were suspended
in buffer A (50 mM NaH2PO4, pH 8, 300 mM NaCl, 10% glycerol,
0.05% Thesit®) with 1 mM phenylmethylsulphonyl (PMSF) and
lysed by sonication with fifteen bursts of 30 s (with 30 s intervals
between each burst). The crude extract was clarified by centrifu-
gation at 16,000ꢂg for 30 min at 4 ꢀC. The soluble fraction was
loaded on a 2 mL column of NieNTA resin (Qiagen) equilibrated
with buffer A. The column was then washed with a step gradient of
imidazole from 0 to 50 mM in buffer A. The recombinant PfDHODH
was eluted with buffer A containing 500 mM imidazole, and then
loaded on a HiTrap Desalting column (GE Healthcare Life Science)
for buffer exchange (buffer A containing 10 mM imidazole). After-
wards, the fractions containing PfDHODH were incubated overnight
at 8 ꢀC with histidine tagged protease ULP1 and then loaded on a
Ni-NTA column. The tag-free PfDHODH was eluted with buffer A
containing 10 mM imidazole, and then purified by size exclusion
chromatography on a Superdex 200 column (GE Healthcare Life
Science) equilibrated with buffer B (50 mM Hepes, pH 7.7, 400 mM
NaCl, 10% glycerol, 0.05% Thesit®). The purity of the enzyme was
checked by SDSePAGE for all purification steps.
4.4.2. Cell cultures and cytotoxicity tests
For the cytotoxicity test, a monkey kidney cell line (BGM),
originally acquired from ATCC (American Type Culture Collection,
Manassas, VA, USA) by the University of Minas Gerais, Brazil [20],
was cultured in 75 cm2 sterile flasks containing RPMI 1640 medium
supplemented with 10% heat-inactivated foetal serum and 40 mg/L
of gentamicin in a 5% CO2 atmosphere at 37 ꢀC. When the cell
monolayer was confluent, it was trypsinized, washed with culture
medium distributed in a flat-bottomed 96-well plate (1 ꢂ 105 cells/
mL) and incubated for 18 h at 37 ꢀC to ensure cell adherence. The
cells were properly diluted and incubated with 20 mL of the com-
pounds at different concentrations (1000-1 mg/mL) for 24 h in a 5%
CO2 air atmosphere at 37 ꢀC. The neutral red assay (NR) was used to
evaluate cell viability [39]. Briefly, the cells were incubated for 2 h
with 100
discarded, 200
tion were added, and followed by another 5 min incubation. Then
the supernatant was removed and 100 L of an alcohol-acetic acid
mL of a NR solution (100 mg/mL), the supernatants were
mL of formaldehyde (0.5% v/v) and CaCl2 (1%) solu-
m
(50-1%) solution was added to the sediment. The absorbance was
read at 540 nm on an ELISA reader (SpectraMax340PC384, Molec-
ular Devices).
Cell viability was expressed as the percentage of control absor-
bance obtained in untreated cells after subtracting the absorbance
from due to background signals. Lastly, the minimum lethal dose
for 50% of the cells (MLD50) was determined as previously
described in the literature [40]. The ratio between MLD-50 and
drug activity (IC50), in vitro was used to determine the selectivity
index (SI).
HsDHODH was obtained as previously described [42].
4.4.4.1. DHODH activity assay. 24. Dihydroorotate dehydrogenase
was assayed by using a colorimetric continuous assay that moni-
tored 2,6-dichloroindophenol (DCIP) reduction. Changes in absor-
bance at 600 nm and 610 nm for PfDHODH and HsDHODH,
respectively, were monitored over a period of 60 s at 25 ꢀC using a
microplate reader (Molecular Devices, SpectraMax 384 Plus, Cali-
4.4.3. P. Berghei and antimalarial tests in mice
fornia, USA). The enzymatic reaction in a total volume of 195
contained 50 mM Tris buffer, pH 8.15; 150 mM KCl; 0.1% Triton X-
100; 1 mM -dihydroorotate, 100 M CoQ0 (2,3-Dimethoxy-5-
methyl-p-benzoquinone); and 60 M DCIP. The assay was started
mL
The suppressive test was performed as described [41] with some
modifications [33]. The P. berghei NK65 parasites used were main-
tained through weekly blood passages. For the experiments, groups
L
m
m

82
L.F.S.P. Azeredo et al. / European Journal of Medicinal Chemistry 126 (2017) 72e83
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De Gruyter, Berlin, 2013, pp. 297e312.
respectively, from stock enzyme solutions prepared in 50 mM
HEPES, pH 7.7, 400 mM NaCl, 10% glycerol and 0.05% Thesit® to give
a final enzyme concentration of 50 nM and 20 nM for PfDHODH and
HsDHODH, respectively. A reference measurement was obtained by
preparing the same solution without the enzyme solution.
The inhibitory effects of 10, 16, 17, 19, 23, 25, 26 and 30 were
examined. All compounds were tested with four replicates for each
concentration. Different inhibitor concentrations were used. Con-
trol enzyme activity in the absence of an inhibitor was considered
to be 100%. For each inhibitor, Activity (%) versus log [Inhibitor]
graphs were drawn. The half maximal inhibitory concentration
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(IC50
) values were calculated from nonlinear fitting of the
concentration-response data to equation (1):
ꢀ
ꢁ
€
[18] T. Heikkila, C. Ramsey, M. Davies, C. Galtier, A.M.W. Stead, A.P. Johnson,
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C.W.G. Fishwick, A.N. Boa, G.A. McConkey, Design and synthesis of potent
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%ðactivityÞ ¼ bottom þ
(1)
10log½Iꢄꢁlog½IC ꢄ þ 1
50
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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-
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8285e8302.
Equation (1): Calculation of the half maximal inhibitory con-
centration values (IC50) from nonlinear fitting of the
concentration-response data; where bottom is the % (activity) at the
bottom plateau, and top is the % (activity) at the top plateau.
Acknowledgements
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The authors thank the Coordination of Improvement of Higher
Education (CAPES) and the National Council of R&D of Brazil (CNPq,
310450/2013-6; 407741/2012-7; 404455/2012-3) for the fellow-
ships granted to the authors. We also thank the Foundation for
Research of the State of Rio de Janeiro (FAPERJ, E-26/201.375/2014-
BBP; E-26/111.401/2013), the Foundation for Research of the State
of Minas Gerais (FAPEMIG; CBBAPQ01355-14), the Technological
Development Program on Products for Health (PDTIS) and CNPq-
MCT/MS (PRONEX Rede Malaria) and FAPESP (Foundation of Sup-
~
port to Research of the State of Sao Paulo (FAPESP, grants #2012/
25075-0) for financial support.
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