Antimalarial activity of 4-(5-trifluoromethyl-1H-pyrazol-1-yl)-chloroquine analogues

Article abstract of DOI:10.1016/j.bmcl.2005.10.033

The antimalarial activity of chloroquine-pyrazole analogues, synthesized from the reaction of 1,1,1-trifluoro-4-methoxy-3-alken-2-ones with 4-hydrazino-7-chloroquinoline, has been evaluated in vitro against a chloroquine resistant Plasmodium falciparum clone. Parasite growth in the presence of the test drugs was measured by incorporation of [³H]hypoxanthine in comparison to controls with no drugs. All but one of the eight (4,5-dihydropyrazol-1-yl) chloroquine 2 derivatives tested showed a significant activity in vitro, thus, are a promising new class of antimalarials. The three most active ones were also tested in vivo against Plasmodium berghei in mice. However, the (pyrazol-1-yl) chloroquine 3 derivatives were mostly inactive, suggesting that the aromatic functionality of the pyrazole ring was critical.

Full text of DOI:10.1016/j.bmcl.2005.10.033

Bioorganic & Medicinal Chemistry Letters 16 (2006) 649–653
Antimalarial activity of 4-(5-trifluoromethyl-1H-pyrazol-1-yl)-
chloroquine analogues
Wilson Cunico,b Cleber A. Cechinel,c Helio G. Bonacorso,c
Marcos A. P. Martins,c Nilo Zanatta,c Marcus V. N. de Souza,b
Isabela O. Freitas,a Rodrigo P. P. Soaresa and Antoniana U. Krettlia,*
aCentro de Pesquisas Rene´ Rachou, Fiocruz e Departamento de Parasitologia-Universidade Federal de Minas Gerais (UFMG),
Belo Horizonte, MG, Brazil
bFioCruz, Fundac¸a˜o Oswaldo Cruz, Instituto de Tecnologia em Fa´rmacos, FarManguinhos,
Rua Sizenando Nabuco 100, Manguinhos, 21041-250 Rio de Janeiro-RJ, Brazil
cNu´cleo de Quı´mica de Heterociclos, NUQUIMHE, Departamento de Quı´mica, Universidade Federal de Santa Maria,
97105-900 Santa Maria, RS, Brazil
Received 26 August 2005; revised 10 October 2005; accepted 12 October 2005
Available online 27 October 2005
Abstract—The antimalarial activity of chloroquine-pyrazole analogues, synthesized from the reaction of 1,1,1-trifluoro-4-methoxy-
3-alken-2-ones with 4-hydrazino-7-chloroquinoline, has been evaluated in vitro against a chloroquine resistant Plasmodium
falciparum clone. Parasite growth in the presence of the test drugs was measured by incorporation of [3H]hypoxanthine in compar-
ison to controls with no drugs. All but one of the eight (4,5-dihydropyrazol-1-yl) chloroquine 2 derivatives tested showed a
significant activity in vitro, thus, are a promising new class of antimalarials. The three most active ones were also tested in vivo
against Plasmodium berghei in mice. However, the (pyrazol-1-yl) chloroquine 3 derivatives were mostly inactive, suggesting that
the aromatic functionality of the pyrazole ring was critical.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Malaria remains one of the most important diseases of
human with over half of the world population at risk
of infection. It affects mainly those living in tropical
and subtropical areas with an incidence of 500 million
cases per year globally.1 Despite the importance of this
disease, treatment investment and malaria control are
modest, especially in Africa where it kills over 2.5 mil-
lion children per year, according to the World Health
Organization data.2 In Brazil, it is estimated that
500,000 cases occur annually, mainly in the Amazon
Rain Forest.3
Figure 1. Structures of chloroquine and amodiaquine, the drugs most
frequently used to treat malaria.
Chloroquine (CQ) and other quinoline antimalarials
have been mainstays of malaria chemotherapy for much
of the past 40 years (Fig. 1). The success of these drugs
was based on limited host toxicity especially of CQ, ease
of use, with very few side effects, low cost and effective
synthesis. The inhibition of parasite growth by CQ
and related antimalarials is caused by formation of a
complex with hematin (Fe(III)FPIX), a toxic lytic moi-
ety that must be sequestered as an inert pigment, the
hemozoin.4
The use of such drugs has been seriously eroded in
recent years, mainly as a result of the development of
parasite resistance to CQ.5 The molecular basis for CQ
resistance is not fully understood. However, it is clear
Keywords: Antimalarial; 4,5-Dihydropyrazole; Chloroquine analogues.
*
Corresponding author. Tel.: +55 31 3295 3566; fax: +55 31 3295
3115; e-mail: akrettli@cpqrr.fiocruz.br
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.033

650
W. Cunico et al. / Bioorg. Med. Chem. Lett. 16 (2006) 649–653
that the resistance is a consequence of decreased accu-
mulation of the drug in the parasite, due to enhanced ef-
flux, reduced uptake or a combination of both.6 Several
studies indicate that point mutations in the multidrug
resistance 1 (pfmdr1),7 candidate (cg2)8 and CQ resis-
tance transporter (pfcrt) gene9 are involved in the mech-
anism of resistance. Recently, it was also demonstrated
that resistance to mefloquine in Southeast Asia was
due to an increase in the gene copy numbers of pfmdr1.10
each experiment. Inhibition of parasite growth was eval-
uated through the levels of [3H]–hypoxanthine incorpo-
ration plotted to generate dose–response curves. The
half-maximal inhibitory response (IC50) compared with
parasite growth in the drug-free controls was estimated
by curve fitting using a software program [Microcal,
Origin Software (Northampton, MA, USA)].
The synthesis of 1,1,1-trifluoro-4-methoxy-3-alken-2-
ones 1a–h was performed as previously reported.19
The reaction of ketones with trimethyl orthoformate in
the presence of p-toluenosulfonic acid delivered the
respective acetals. The intermediate acetals react with
It has been shown that close analogues of CQ and CQ-
derivatives are active against CQ-resistant parasites,11
suggesting that the mechanism of resistance does not
involve any changes to their targets but involves a
compound specific resistance.
Pyrazoles and 4,5-dihydropyrazoles are important bio-
logical agents with a wide range of pharmaceutical
(anti-inflammatory, antifungal, antibacterial, antitumor
and antiviral) and agrochemical activities.12,13
Table 1. Antimalarial activity of 4-(5-trifluoromethyl-5-hydroxy-4,5-
dihydropyrazol-1-yl)-7- chloroquinolines 2a–h and 4-(pyrazole-1-yl)-7-
chloroquinolines 3a–j against Plasmodium falciparum W2 clone in vitro
a
Compound
IC50 (lg/mL)
2a
2b
1.39
3.04
2.13
1.69
1.55
>50.0
5.71
2.12
0.19
9.53
>50
The aim of this study was to assess in vitro activity of
pyrazole chloroquinoline analogues against Plasmodium
falciparum and in vivo activity against Plasmodium
berghei. The parasite clone W2, chloroquine resistant,
was used14 for the in vitro tests, as described in our
previous work,15 and the activity compared to that of
CQ, used in parallel in each test.
2c
2d
2e
2f
2g
2h
Chloroquin
3a
3b
Parasites were cultured with human erythrocytes (blood
group O+) at 5% haematocrit in RPMI 1640 supple-
mented with 10% human plasma as previously
described.16 Molecules were solubilized in ethanol prior
to in vitro tests. The antiparasitic effects of the molecules
were measured by the [3H]hypoxanthine incorporation
3c
3d
27.62
>50
>50
3e
3f
3g
>50
>50
assay.17 Briefly, trophozoite stages in sorbitol-synchro-
3h
3i
18.53
4.29
>50
18
nized blood
were cultured at 2% parasitaemia and
3j
Chloroquine
2.5% haematocrit, in the presence of the test molecules
(at various concentrations), diluted with culture medium
(RPMI 1640) without hypoxanthine; a chloroquine
control (as a reference antimalarial drug) was used in
0.22
a The IC50 represents concentration inhibitory dose of the parasite
growth in relation to control cultures with no drugs.
g
a
b
c
d
e
f
h
R
H
Me
F
Cl
Br
OMe
NO2
phenyl
Scheme 1. Reagents and conditions: (a) TsOH, HC(OCH3)3, MeOH, rt, 24 h; (b) (CF3CO)2O, Py, CHCl3, 0–45 ꢁC, 16 h; (c) 4-hydrazine-7-
chloroquinoline, MeOH, 68 ꢁC, 15–30 min; (d) AcOH, reflux, 4–10 h.

W. Cunico et al. / Bioorg. Med. Chem. Lett. 16 (2006) 649–653
651
2e
2a
1.0
0.8
0.6
0.4
1.0
0.8
0.6
0.4
0.2
0.0
IC50=1.39 µg/ml
IC50=1.55 µg/ml
0.2
0.0
0.1
1
10
0.1
1
10
Concentration (µg/ml)
Concentration (µg/ml)
Figure 2. Dose–response curves of 4-(3-substituted-5-trifluoromethyl-5-hydroxy-4,5-dihydro-1H-pyrazol-1-yl)-7-chloroquinolines analogues 2a and
e against Plasmodium falciparum in vitro.
(1H and 13C NMR) of all compounds are in full agreement
with the proposed structure.23,24
Table 1 shows the antimalarial activities of all com-
pounds expressed as 50% inhibitory concentration
(IC50) of P. falciparum growth.
All (4,5-dihydropyrazol-1-yl) chloroquine derivatives
2a–h but one (2f, inactive) inhibited growth of P. falcipa-
i
j
rum in vitro at concentrations of micromolar range
(Table 1). The compound 2a that has no substituent
group (R = H) was the most active one (IC50 = 1.39 lg/
mL) (Fig. 2). The halogen substituted compounds 2c–e
have also shown good activity based on the low IC50 val-
ues. The compound 2g, that has a strong electron-with-
drawing group (R = NO2), showed a little decrease of
antimalarial activity (IC50 = 5.71 lg/mL). Even the bulky
biphenyl compound 2h was active (IC50 = 2.12 lg/mL).
Despite the fact that the more active compounds in vitro
are at least 10- to 15 times less active than chloroquine,
their IC50 values are in the micromolar concentration
range comparable to recently reported results.25
R1
Me
naphtyl
Scheme 2. Reagents and conditions: (a) 4-hydrazine-7-chloroquino-
line, MeOH, 68 ꢁC, 15–30 min.
trifluoroacetic anhydride in pyridine using chloroform
as solvent to obtain compounds 1a–h. The cycloconden-
sation reaction of 1,3-dielectrophilic compounds 1a–h
with 4-hydrazino-7-chloroquinoline was carried out in
methanol for 15–30 min under reflux to give the 4-(5-
hydroxy-4,5-dihydropyrazol-1-yl)-7-chloroquinolines 2a–h
in good yields (Scheme 1).20,21 Compounds 2a–h were
converted to the respective aromatic system 3a–h by treat-
ment of 2a–h with acetic acid at reflux for 4 h in 73–96%
yields (Scheme 1).20,22 Both analytical and spectral data
As shown for compound 2f, the presence of a strong elec-
tron-releasing (R = OMe) group results in complete loss
Table 2. Antimalarial activity of 4-(4,5-dihydropyrazol-1-yl)-7-chloroquinolines 2a, d and e against Plasmodium berghei in Balb/c mice infected with
blood parasites, treated by oral or subcutaneous routes (20 mg/kg)
Compound
Route of treatment
% parasitaemia mean SD
Reduction of parasite growtha (%)
2a
Oral
Subcutaneous
Oral
8.7 5.8
11.2 2.5
11.4 5.4
3.9 3.5
9.7 3.5
1.3 0.9
3.4 4.6
0
0
2d
0
Subcutaneous
Oral
47
0
2e
Subcutaneous
Oral
82
53
100
0
Chloroquineb
Subcutaneous
—
0
7.3 4.0
0
Non-treated
a Calculated in comparison to non-treated mice (6 per group), at day 7 after parasite inoculation.
b Dose of 15 mg/kg.

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of activity at the maximum concentration (50 lg/mL)
used.
The aromatic functionality of pyrazole ring is critical
since water elimination of 5-hydroxy-4,5-dihydropyraz-
oles 2a–h results in a significant drop in potency for all
pyrazoles 3, including 3a (IC50 = 9.53 lg/mL), 3c
(IC50 = 27.62 lg/mL) and 3h (IC50 = 18.53 lg/mL). For
compounds 3b,d,e,f and g, the IC50 values were higher
than 50 lg/mL, thus considered inactive.
11. (a) Ridley, R. G.; Hofheinz, Z.; Matile, H.; Jaquet, C.;
Dorn, A.; Masciadri, R.; Jolidon, S.; Richter, W. F.;
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Casali, V. W. D.; Njaine, B.; Zalis, M. G.; Oliveira, L. A.;
Krettli, A. U. Phytother. Res. 2004, 18, 634.
The 3-methyl and 3-naphthyl pyrazole derivatives 3i
and j were pursued in an attempt to gain insight into
the importance of 3-substituent group at pyrazole ring
(Scheme 2).20 The introduction of a methyl group in-
stead of an aryl group had an increasing effect on anti-
malarial activity (IC50 = 4.29 lg/mL), however lower
than that of 3-aryl-5-hydroxy-4,5-dihydopyrazole ser-
ies. The compound with the bulky group R1 = naph-
thyl (3j) was inactive at high concentration (50 lg/
mL). No traces of the corresponding intermediate 5-hy-
droxy-4,5-dihydropyrazoles are detected in these
reactions.
The most effective in vitro analogues (2a, d and e) were
selected for in vivo tests against NK-65 strain of P. berg-
hei in infected mice.26 Orally, compounds 2a, d and e did
not inhibit the parasite growth at the dose of 20 mg/kg
(Table 2). However, after subcutaneous injection, 2a
was still inactive and compounds 2d and e inhibited par-
asite growth by 47% and 82%, respectively. Chloroquine
controls inhibited parasite growth by 53% (oral route)
and 100% (subcutaneous route).
16. Trager, W.; Jensen, J. B. Science 1976, 193, 673.
17. Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay,
J. D. Antimicrob. Agents Chemother. 1979, 16, 710.
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21. General procedure for the preparation of 4-(3-substi-
tuted-5-trifluoromethyl-5-hydroxy-4,5-dihydro-1H-pyrazol-
1-yl)-7-chloroquinolines (2a–h): to a magnetic stirred
solution of 1a–h (1 mmol) in MeOH (10 mL), 7-chloro-
4-hydrazinoquinoline (1 mmol) was added at room
temperature. The mixture was stirred under reflux
(64 ꢁC) for 15–30 min. The solvent was evaporated
under reduced pressure and the solid products 2a–h
were recrystallized from a mixture of acetone and water
5:1.
Assayed as antimalarial for the first time, these 4-(5-tri-
fluoromethyl-5-hydroxy-4,5-dihydropyrazol-1-yl)-7- chlo-
roquinolines 2d and e have exhibited promising
antimalarial activity in vitro and in vivo.
Acknowledgments
The authors are thankful to CNPq, CAPES, FAPERGS,
and FAPEMIG for financial support.
22. General procedure for the preparation of 4-(3-substituted-
5-trifluoromethyl-1H-pyrazol-1-yl)-7-chloroquinolines (3a–
h): to a magnetic stirred pure acetic acid (10 mL),
compounds 2a–h (1 mmol) were added at room temper-
ature. The mixture was stirred under reflux (116 ꢁC) for
4–10 h. The solvent was evaporated under reduced
pressure and the solid products 3 were recrystallized from
a mixture of ethanol and water 5:1.
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´
23. Selected data for compound 4-[3-(4-bromophenyl)-5-tri-
fluoromethyl-5-hydroxy-4,5-dihydro-1H-pyrazol-1-yl]-7-
´
1
chloroquinoline 2e: mp 220–222 ꢁC. H NMR (200 MHz,
DMSO) (pyrazol) d = 8.75 (s, OH); 4.05 (d, J = 18.5, Ha);
3.68 (d, J = 18.5, Hb); (quinolyl) d = 8.85 (d, H2, J = 4.9);
8.34 (d, H8, J = 9.1); 8.06 (d, H6, J = 2.2); 7.79 (d, H5,
J = 5.0); 7.60 (d, H3, J = 2.3); (phenyl) d = 7.63–7.62 (m,
4 H). 13C NMR (50 MHz, DMSO) (pyrazol) d = 148.1
2
(C3); 124.9 (q, JCF = 285.3, CF3); 94.1 (q, JCF = 31.7,
C5); 43.5 (C4); (quinolyl) d = 151.8 (C2); 149,7 (C8a);
146.2 (C4); 133.8 (C7); 130.0 (C8); 127.7 (C6); 126.1 (C5);
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J.; Forsyth, K.; Kemp, D. J.; Cowman, A. F. Nature 1990,

W. Cunico et al. / Bioorg. Med. Chem. Lett. 16 (2006) 649–653
653
122.1 (C4a); 113.9 (C3); (phenyl) d = 131.7; 127.6; 127.3;
122.9 (6C).
intraperitoneal route with P. berghei-infected red blood
cells (1 · 105), were daily treated by oral or subcutaneous
route, during 4 days. The samples were dissolved in an
aqueous solution of DMSO 0.02% (v:v) and this solution
was, later, diluted to final volume with saline and given in
a 200 lL volume per animal. Two control groups (n5)
were used each time, one treated with non-curative doses
of chloroquine (625 mg/kg), one untreated or treated with
saline, as specified in the results. On day 5 after parasite
inoculation blood smears were prepared, methanol fixed,
stained with Giemsa and microscopically examined by
counting parasitaemia in 1000 up to 6000 erythrocytes.
Overall mortality was daily monitored until all controls
died. The inhibition of parasite growth in the drug-treated
groups was calculated in relation to non-treated
controls.28
24. Selected data for compound 4-[3-(4-bromophenyl)-5-tri-
fluoromethyl-1H-pyrazol-1-yl]-7-chloroquinoline 3e: mp
1
162–164 ꢁC. H NMR (200 MHz, DMSO) (phenyl pyra-
zol) d = 7.96–7.91; 7.72–7.68 (m, 5 H, H4 + 4 HAr); (quin-
olyl) d = 9.24 (d, H2, J = 4.4); 8.33 (d, H8, J = 2.0); 7.99
(d, H6); 7.75 (d, H5, J = 2.0); 7.50 (d, H3, J = 8.9). 13C
NMR (50 MHz, DMSO) (pyrazol) d = 151.2 (C3); 134.5
2
(q, JCF = 39.5, C5) 121.7 (JCF = 269.1, CF3); 107.2 (C4);
(quinolyl) d = 152.3 (C2); 149,0 (C8a); 142.1 (C4); 135.4
(C7); 129.9 (C8); 128.0 (C6); 124.5 (C5); 122.3 (C4a); 120.0
(C3); (phenyl) d = 131.8; 129.3; 127.7; 124.9 (6C).
25. (a) Andrade-Neto, V. F.; Goulart, M. O. F.; Silva Filho, J.
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adult Swiss outbred mice, 20 2 g weight, inoculated by
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