Synthesis and antileishmanial evaluation of 1-aryl-4-(4,5-dihydro-1H- imidazol-2-yl)-1H-pyrazole derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.09.134

A series of 1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles (4a-g) and 5-amino-1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles (5a-g) were synthesized and evaluated in vitro against three Leishmania species: L. amazonensis, L. braziliensis and L. infantum (L. chagasi syn.). The cytotoxicity was assessed. Among the derivatives examined, six compounds emerged as the most active on promastigotes forms of L. amazonensis with IC₅₀ values ranging from 15 to 60 μM. The reference drug pentamidine presented IC ₅₀ = 10 μM. However, these new compounds were less cytotoxic than pentamidine. Based on these results, the more promising derivative 5d was tested further in vivo. This compound showed inhibition of the progression of cutaneous lesions in CBA mice infected with L. amazonensis relative to an untreated control.

Full text of DOI:10.1016/j.bmcl.2011.09.134

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7451–7454
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antileishmanial evaluation of 1-aryl-4-(4,5-dihydro-1H-imidazol-
2-yl)-1H-pyrazole derivatives
Maurício S. dos Santos a, , Mariana L.V. Oliveira b, Alice M.R. Bernardino b, Rosa M. de Léo c,
⇑
Veronica F. Amaral d, Flavia T. de Carvalho d, Leonor L. Leon c, Marilene M. Canto-Cavalheiro c
a Departamento de Física e Química, Instituto de Ciências Exatas, Universidade Federal de Itajubá, 37500-903 Itajubá, MG, Brazil
b Departamento de Química Orgânica, Programa de Pós-Graduação em Química, Instituto de Química, Universidade Federal Fluminense, 24020-150 Niterói, RJ, Brazil
c Laboratório de Bioquímica de Tripanosomatídeos, IOC, Fundação Oswaldo Cruz, 21040-900 Rio de Janeiro, RJ, Brazil
d Departamento de Imunobiologia, Instituto de Biologia, Universidade Federal Fluminense, 24020-150 Niterói, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles (4a–g) and 5-amino-1-aryl-4-(4,5-
dihydro-1H-imidazol-2-yl)-1H-pyrazoles (5a–g) were synthesized and evaluated in vitro against three
Leishmania species: L. amazonensis, L. braziliensis and L. infantum (L. chagasi syn.). The cytotoxicity was
assessed. Among the derivatives examined, six compounds emerged as the most active on promastigotes
Received 25 August 2011
Revised 28 September 2011
Accepted 30 September 2011
Available online 18 October 2011
forms of L. amazonensis with IC50 values ranging from 15 to 60
lM. The reference drug pentamidine pre-
sented IC50 = 10 M. However, these new compounds were less cytotoxic than pentamidine. Based on
l
Keywords:
these results, the more promising derivative 5d was tested further in vivo. This compound showed inhi-
bition of the progression of cutaneous lesions in CBA mice infected with L. amazonensis relative to an
untreated control.
Antileishmanial activity
Leishmania
Azoles
Ó 2011 Elsevier Ltd. All rights reserved.
Pyrazoles
Imidazolines
Amidines
Leishmaniasis is a group of tropical diseases caused by protozoan
parasites of the genus Leishmania. It is transmitted to human beings
and others mammals by the bite of an infected female Phlebotomine
sandfly.1 The clinical manifestations are particularly diverse repre-
senting a complex of diseases: visceral leishmaniasis (VL) or kala
azar is usually fatal when untreated, mucocutaneous (MCL) is a
mutilating disease, diffuse cutaneous leishmaniasis (DCL) is a
long-lasting diseasedue to a deficient cellular-mediatedimmunere-
sponse and cutaneous leishmaniasis (CL) is disabling when lesions
are multiple.2 Visceral leishmaniasis (VL) is the most severe form,
in which vital organs of the body are affected. The cutaneous form
often results in self cure, although consequences like permanent
scars and deformations of the nose, mouth and throat. Patients with
these forms of the disease may also suffer from discrimination and
prejudice.3
and 1.1 million cutaneous or mucocutaneous. Of the 1.6 million
estimated cases, only about 600000 are reported.3
Unfortunately, there is no effective treatment against various
forms of leishmaniasis. There are no vaccines and the chemother-
apeutic agents for treating these diseases are deficient and most of
the drugs cause many adverse and side effects.3,5 For more than
50 years, pentavalent antimonials have been the first-line drugs
in the treatment for all types of leishmaniasis in most countries.
Two organic salts, meglumine antimoniate (Glucantime) and
sodium stibogluconate (Pentostam) are mainly used.5 The treat-
ment is lengthy, potentially toxic and painful.3 In the case of re-
lapse, patients need treatment with second-line medicines such
as pentamidine and amphotericin B. These drugs are toxic and
may show a serious of side effects such as nephrotoxicity.5,6 Newly
developed, liposomal amphotericin B is highly effective, has almost
no side effects and requires only a short course of treatment but it
is too expensive to be a viable treatment option in most developing
nations.3 Other effective medicine is miltefosine, an alkylphosph-
ocholine derivative originally developed as an anticancer drug,
which has been registered in India, Germany and Colombia for oral
treatment of visceral leishmaniasis (VL). However, it is contraindi-
cated in pregnant women and shows severe gastrointestinal side
effects.5,7 In the light of these facts, there is an urgent need for
the development of more efficient, inexpensive, nontoxic and inno-
vative drugs for the treatment of leishmaniases.
According to WHO estimations, leishmaniasis affect almost 12
million people in 88 countries on five continents (Africa, Asia,
Europe, North and South America), representing a worldwide pub-
lic health problem.4 The disease is estimated to cause 1.6 million
new cases each year, of which an estimated 500000 are visceral
⇑
Corresponding author. Tel.: +55 35 3629 1554; fax: +55 35 3629 1140.
E-mail address: mauriciounifei@yahoo.com.br (M.S. dos Santos).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.134

7452
M. S. dos Santos et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7451–7454
There are several other compounds at various stages of develop-
77/LTB0016), L. infantum (MCAN/BR/98R619) and L. braziliensis
(MCAN/BR/97/P142) parasites, at various concentrations, taking
pentamidine as a control. The cytotoxicity responses were assayed
on the mice’s peritoneal macrophages. The IC50/24 h values were
determined by linear regression from the percentages of inhibition.
All tests were done in triplicate. The results are shown in Table 1.
In general, these compounds presented lower cytotoxicity in mur-
ine macrophages than the reference drug pentamidine. Six deriva-
tives 4a, 4b, 4e, 4g, 5d and 5f were more effective against L.
amazonensis promastigotes, comparing to the others studied spe-
cies, as L. infantum and L. braziliensis. It can be observed that the
activity of the derivatives expressed as IC50/24 h was in a range
ment, such as azoles derivatives, which have been revealed as
potential new drugs for therapy or an additional therapy to that
already existing.5,8 Among azoles, many compounds containing
pyrazole ring have been synthesized and evaluated. In recent
works, we have reported the synthesis of a series of 1-aryl-1H-pyr-
azole-4-carboximidamides derivatives as potential antileishmanial
agents.9 Concomitantly, imidazoline ring, also known as cyclic
amidine, has been found in several compounds with antileishma-
nial activity.10
Based on the above report and in the continuation of our studies
on chemotherapy of leishmaniasis, herein we described synthesis
of some 1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles
(4a–g) and 5-amino-1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-
pyrazoles (5a–g) and their evaluation against Leishmania amazon-
ensis, Leishmania braziliensis and Leishmania infantum (Leishmania
chagasi syn.) promastigotes containing a high percentage of meta-
cyclic forms.
of 15–60
IC50 >250
l
M, while for the other two species showed
lM. In the literature the in vitro inhibitory effect of the
pentamidine on L. amazonensis is also superior compared to L.
infantum and L. braziliensis.15,16 Interestingly, the relationships be-
tween structure and anti-L. amazonensis activity revealed that
compound 5d with chlorine (–Cl) substituents attachment at two
meta-positions of the aryl nucleus of the aminopyrazole derivatives
was most active while similar structure without amino group 4d
presented unsatisfactory activity on the Leishmania species as-
sayed. These results also indicated the positive effect of introduc-
ing p-bromine 4g and m-bromine 5f on L. amazonensis activity.
On the other hand, the presence of only one m-Cl position attached
at aminopyrazole derivative 5b or without amino group 4b confers
moderate activity. From these results, the most attractive com-
pound 5d was further evaluated for in vivo evaluated.
The synthesis of the compounds 1-aryl-4-(4,5-dihydro-1H-imi-
dazol-2-yl)-1H-pyrazoles (4a–g) and 5-amino-1-aryl-4-(4,5-dihy-
dro-1H-imidazol-2-yl)-1H-pyrazoles (5a–g) is summarized in
Scheme 1. Arylhydrazine hydrochlorides (1a–g) reacted with eth-
oxymethylenemalononitrile and sodium acetate in ethanol, under
reflux, to form 5-amino-1-aryl-1H-pyrazole-4-carbonitriles
(2a–g).11 Soon after, the compounds 2a–g were converted to the
1-aryl-1H-pyrazole-4-carbonitriles (3a–g) by the aprotic deamina-
tion using t-butyl nitrite and tetrahydrofuran (THF), under reflux.12
Finally, the targets 1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-
pyrazoles (4a–g) were obtained by the reaction of 1-aryl-1H-pyra-
zole-4-carbonitriles (3a–g) with carbon disulfide and 1,2-diamin
To examine the therapeutic efficacy, CBA mice infected with L.
amazonensis was treated orally with this compound 5d (29 mg/
kg/day, 100 l
mol/kg/day),17 and the reference drug ketoconazole
oethane
(ethylenediamine).13
Similarly,
the
compounds
(50 mg/Kg/day).9c The use of ketoconazole in the in vivo experi-
ments is due to it similarity to the tested compound, concerning
the imidazole ring.
5-amino-1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles
(
5a–g) were synthesized from 5-amino-1-aryl-1H-pyrazole-4-car-
bonitriles (2a–g). All these final compounds 4a–g and 5a–g are
news, except 1-phenyl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyr-
azole (4a), which was obtained by De La Hoz and co-workers.14
The compounds 4a–g and 5a–g were evaluated in vitro against
the extracellular promastigote stage of L. amazonensis (MHOM/BR/
In our study, the treatment was started in the fourth week after
infection and continued for 30 days without interruption. It was
observed in the groups treated with derivative 5d or ketoconazole,
a smaller development of cutaneous lesion compared to the
untreated control group and infected (Fig. 1). Additionally, the tox-
N
N
CN
CN
H
NHNH2.HCl
N
N
N
NH2
N
N
N
i, ii
iii
iv
R
R
R
R
1a-g
2a-g
3a-g
4a-g
v
a: R= H
b: R= 3-Cl
c: R= 3,4-diCl
d: R= 3,5-diCl
e: R= 3-F
N
N
H
f: R= 3-Br
g: R= 4-Br
N
NH2
N
R
5a-g
Scheme 1. Reagents and conditions: (i) sodium acetate, ethanol, 0.5 h, reflux; (ii) ethoxymethylene-malononitrile, ethanol, 1 h, reflux; (iii) t-butyl nitrite, THF, 2 h, reflux; (iv)
ethylenediamine, CS2, 12–14 h, 110 °C; (v) ethylenediamine, CS2, 14–15 h, 115 °C.

M. S. dos Santos et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7451–7454
7453
Table 1
a
IC50
(lM) values of 1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles (4a–g) and 5-amino-1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles (5a–g) on promastigotes
of Leishmania spp. and percentage of cytoxicity in murine macrophages
Compd No.
L. amazonensis IC50
(
lM)
L. braziliensis IC50
(l
M)
L. infantum IC50
(l
M)
Cytotoxicityb%
4a
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
5e
5f
5g
43 4.2
60 3.2
297 6.0
>500
54
>500
30 5.5
365 3.5
>500
381 29
15.5 6.8
>500
31.3 5.4
>500
10 4.44
>500
>500
—
459 1.69
—
—
—
433 2.2
>500
—
437 1.6
>500
—
>500
>500
>500
>500
>500
>500
>500
>500
>500
—
>500
>500
—
>500
100 2.5
11.26
6.46
5.32
3.97
—
11.98
3.52
—
15.2
15.5
—
—
—
272 2.9
50
—
31
Pentamidine
a
IC50 values were obtained from the drug concentration-response curve, and the results were expressed as the mean standard deviation determined from three
independent experiments.
b
Percentage of cytoxicity values from IC50 of L. amazonensis.
In vitro results showed that these derivatives were less cytotoxic
than pentamidine. The compound 5-amino-1-(3,5-dichloro-
phenyl)-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazole (5d) was
tested further in vivo and exhibited significant inhibition relative
to an untreated control. In addition, no apparent hepatic or renal
toxicity due to these compounds was found. This finding indicates
that derivative should be investigated for the development of
selective antileishmanial compounds.
Non treated
5d
400
350
300
250
200
150
100
50
ketoconazole
Acknowledgments
We thank the Fundação de Amparo à Pesquisa do Estado do Rio
de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Cientí-
fico e Tecnológico (CNPq), Programa Estratégico de Apoio à Pesqu-
isa em Saúde (PAPES), Coordenação de Aperfeiçoamento de Pessoal
Docente (CAPES) and Universidade Federal Fluminense (PROPP-
UFF) for fellowships and financial support.
0
2
11
16
21
Weeks post-infection
Figure 1. Mean lesion diameter of CBA mice infected with Leishmania amazonensis
(MHOM/BR/77LTB0016) and treated with 5-amino-1-(3,5-dichlorophenyl)-4-(4,
5-dihydro-1H-imidazol-2-yl)-1H-pyrazole (5d), ketoconazole or left untreated.
Treatment started at the fourth week post-infection and continued up to 30 days
post-infection (n = 8 per group).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.134.
icity of this compound in mice body weight was determined and
samples of blood were taken at different times during compound
administration from the tails of both uninfected and infected mice
left untreated or treated. The total number of leukocytes was esti-
mated by counting in a Neubauer chamber. The sera collected were
assayed colorimetrically for alanine aminotransferase (ALT), aspar-
tate aminotransferase (AST), and creatinine with commercial kits
(Labtest Diagnostica, Brazil). No apparent signs of drug toxicity,
weight loss, or lymphocyte, monocyte, or neutrophil alterations
were observed in any experiment, and AST, ALT, and creatinine
concentrations showed no apparent hepatic or renal toxicity after
the treatment with 5-amino-1-aryl-4-(4,5-dihydro-1H-imidazol-
2-yl)-1H-pyrazoles (5d), compared with uninfected mice left un-
treated. All in vivo data were analyzed by the Student’s t-test. P
values <0.05 were considered significant. The InStat program
(Graph Pad Software, San Diego, CA) was used for these tests. All
experiments were repeated at least three times.
References and notes
1. Sharma, U.; Singh, S. J. Vector Borne Dis. 2008, 45, 255.
2. Desjeux, P. Comp. Immun. Microbiol. Infect. Dis. 2004, 27, 305.
3. Crompton, D. W. T.; Peters, P. Working to Overcome the Global Impact of
Neglected Tropical Diseases: First WHO Report on Neglected Tropical Diseases;
WHO Press, 2010. pp 91–96.
4. Santos, D. O.; Coutinho, C. E. R.; Madeira, M. F.; Bottino, C. G.; Vieira, R. T.;
Nascimento, S. B.; Bernardino, A. M. R.; Bourguignon, S. C.; Corte-Real, S.; Pinho,
R. T.; Castro, H. C.; Rodrigues, C. R. Parasitol. Res. 2008, 103, 1.
5. (a) Cavalli, A.; Bolognesi, M. L. J. Med. Chem. 2009, 52, 7339; (b) Graebin, C. S.;
Uchoa, F. D.; Bernardes, L. S. C.; Campo, V. L.; Carvalho, I.; Eifler-Lima, V. L. Anti-
Infect. Agents Med. Chem. 2009, 8, 345; (c) Mitropoulos, P.; Konidas, P.; Durkin-
Konidas, M. J. Am. Acad. Dermatol. 2010, 63, 309; (d) Croft, S. L.; Seifert, K.;
Yardley, V. Indian J. Med. Res. 2006, 123, 399.
6. Croft, S. L.; Yardley, V. Curr. Pharm. Des. 2002, 8, 319.
7. Polonio, T.; Efferth, T. Int. J. Mol. Med. 2008, 22, 277.
8. (a) Silva, E. F.; Canto-Cavalheiro, M. M.; Braz, V. R.; Cysne-Finkelstein, L.; Leon,
L. L.; Echevarria, A. Eur. J. Med. Chem. 2002, 37, 979; (b) Alrajhi, A. A.; Ibrahim, E.
A.; De Vol, E. B.; Khairat, M.; Faris, R. M.; Maguire, J. H. N. Engl. J. Med. 2002, 346,
891; (c) Bhandari, K.; Srinivas, N.; Marrapu, V. K.; Verma, A.; Srivastava, S.;
Gupta, S. Bioorg. Med. Chem. Lett. 2010, 20, 291.
9. (a) Santos, M. S.; Gomes, A. O.; Bernardino, A. M. R.; Souza, M. C.; Khan, M. A.;
Brito, M. A.; Castro, H. C.; Abreu, P. A.; Rodrigues, C. R.; Léo, R. M. M.; Leon, L. L.;
Canto-Cavalheiro, M. M. J. Braz. Chem. Soc. 2011, 22, 352; (b) Charret, K. S.;
Rodrigues, R. F.; Bernardino, A. M. R.; Gomes, A. O.; Carvalho, A. V.; Canto-
Cavalheiro, M. M.; Leon, L. L.; Amaral, V. F. Am. J. Trop. Med. Hyg. 2009, 80, 568;
In conclusion, this study showed that the compounds 5-amino-
1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles (4a–g) and
1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles (5a–g) pre-
sented strong activities against promastigotes of L. amazonensis
and lower activities against those of L. braziliensis and L. infantum.

7454
M. S. dos Santos et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7451–7454
(c) Bernardino, A. M. R.; Gomes, A. O.; Charret, K. S.; Freitas, A. C.; Machado, G.
11. Cheng, C. C.; Robins, R. K. J. Org. Chem. 1956, 21, 1240.
12. Cadogan, J. I. G.; Molina, G. A. J. Chem. Soc. Perkin Trans. 1 1973, 541.
13. El-Emary, T. I.; El-Dean, A. M. K.; El-Kashef, H. S. Farmaco 1998, 53, 383.
14. De La Hoz, A.; Díaz-Ortiz, A.; Carmen, M. M.; Moral, M.; Moreno, A.; Elguero, J.;
Foces-Foces, C.; Rodríguez, M. L.; Sánches-Migállon, A. Tetrahedron 2006, 62,
5868.
15. Alves, L. V.; Canto-Cavalheiro, M. M.; Cysne-Finkelstein, L.; Leon, L. L. Biol.
Pharm. Bull. 2003, 26, 453.
16. Rodrigues, R. F.; Silva, E. F.; Echevarria, A.; Fajardo-Bonin, R.; Amaral, V. F.;
Leon, L. L.; Canto-Cavalheiro, M. M. J. Med. Chem. 2007, 42, 1039.
17. Lima, L. M.; Frattani, F. S.; Santos, J. L.; Castro, H. C.; Fraga, C. A. M.; Zingali, R.
B.; Barreiro, E. J. Eur. J. Med. Chem. 2008, 43, 348.
M.; Canto-Cavalheiro, M. M.; Leon, L. L.; Amaral, V. F. Eur. J. Med. Chem. 2006,
41, 80; (d) Mello, H.; Echevarria, A.; Bernardino, A. M. R.; Canto-Cavalheiro, M.
M.; Leon, L. L. J. Med. Chem. 2004, 47, 5427.
10. (a) Patrick, D. A.; Bakunov, S. A.; Bakunova, S. M.; Kumar, E. V. K. S.; Chen, H.;
Jones, S. K.; Wenzler, T.; Barzcz, T.; Werbovetz, K. A.; Brun, R.; Tidwell, R. R. Eur.
J. Med. Chem. 2009, 44, 3543; (b) Torres-Gómes, H.; Hernández-Núñez, E.;
Léon-Rivera, I.; Guerrero-Alvarez, J.; Cedillo-Rivera, R.; Moo-Puc, R.; Argotte-
Ramos, R.; Rodríguez-Gutiérrez, M. C.; Chan-Bacab, M. J.; Navarrete-Vásquez,
G. Bioorg. Med. Chem. Lett. 2008, 18, 3147; (c) Mayence, A.; Pietka, A.; Collins, M.
S.; Cushion, M. T.; Tekwani, B. L.; Huang, T. L.; Eynde, J. J. V. Bioorg. Med. Chem.
Lett. 2008, 18, 2658; (d) Brendle, J. J.; Outlaw, A.; Kumar, A.; Boykin, D. W.;
Patrick, D. A.; Tidwell, R. R.; Werbovetz, K. A. Antimicrob. Agents Chemother.
2002, 46, 797.