Synthesis and activity of novel tetrazole compounds and their pyrazole-4-carbonitrile precursors against Leishmania spp

Article abstract of DOI:10.1016/j.bmcl.2013.09.062

A new series of 5-(1-aryl-3-methyl-1H-pyrazol-4-yl)-1H-tetrazole derivatives (4a-m) and their precursor 1-aryl-3-methyl-1H-pyrazole-4- carbonitriles (3a-m) were synthesized and evaluated as antileishmanials against Leishmania braziliensis and Leishmania amazonensis promastigotes in vitro. In parallel, the cytotoxicity of these compounds was evaluated on the RAW 264.7 cell line. The results showed that among the assayed compounds the substituted 3-chlorophenyl (4a) (IC₅₀/24 h = 15 ± 0.14 μM) and 3,4-dichlorophenyl tetrazoles (4d) (IC₅₀/24 h = 26 ± 0.09 μM) were the most potent against L. braziliensis promastigotes, as compared the reference drug pentamidine, which presented IC₅₀ = 13 ± 0.04 μM. In addition, 4a and 4d derivatives were less cytotoxic than pentamidine. However, these tetrazole derivatives (4) and pyrazole-4- carbonitriles precursors (3) differ against each of the tested species and were more effective against L.braziliensis than on L. amazonensis.

Full text of DOI:10.1016/j.bmcl.2013.09.062

Accepted Manuscript
Synthesis and activity of novel tetrazole compounds and their pyrazole-4-car-
bonitrile precursors against Leishmania spp
Jéssica V. Faria, Maurício S. dos Santos, Alice M.R. Bernardino, Klaus M.
Becker, Gérzia M.C. Machado, Raquel F. Rodrigues, Marilene M. Canto-
Cavalheiro, Leonor L. Leon
PII:
S0960-894X(13)01136-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.09.062
BMCL 20914
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 August 2013
20 September 2013
23 September 2013
Please cite this article as: Faria, J.V., dos Santos, M.S., Bernardino, A.M.R., Becker, K.M., Machado, G.M.C.,
Rodrigues, R.F., Canto-Cavalheiro, M.M., Leon, L.L., Synthesis and activity of novel tetrazole compounds and
their pyrazole-4-carbonitrile precursors against Leishmania spp, Bioorganic & Medicinal Chemistry Letters (2013),
doi: http://dx.doi.org/10.1016/j.bmcl.2013.09.062
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Synthesis and activity of novel tetrazole compounds and their pyrazole-4-
carbonitrile precursors against Leishmania spp.
Jéssica V. Faria1, Maurício S. dos Santos2, Alice M.R.Bernardino1, Klaus M. Becker3, Gérzia M. C.
Machado3, Raquel F. Rodrigues3, Marilene M. Canto-Cavalheiro3, Leonor L. Leon3.
1Departamento de Química Orgânica, Programa de Pós-Graduação em Química, Instituto de Química, Universidade
Federal Fluminense, 24020-150, Niterói, RJ, Brazil
2Instituto de Física e Química, Centro de Estudos e Inovação em Materiais Biofuncionais Avançados, Universidade
Federal de Itajubá, 37500-903, Itajubá, MG, Brazil
3Laboratório de Bioquímica de Tripanosomatídeos, IOC, Fundação Oswaldo Cruz, 21040-900, Rio de Janeiro, RJ,
Brazil
*Corresponding author. Tel.: +55 35 3629-1628; Fax: +55 35 3629-1140
E-mail address: mauriciounifei@yahoo.com.br
ABSTRACT
A new series of 5-(1-aryl-3-methyl-1H-pyrazol-4-yl)-1H-tetrazole derivatives (4a-m) and their
precursor 1-aryl-3-methyl-1H-pyrazole-4-carbonitriles (3a-m) were synthesized and evaluated
as antileishmanials against Leishmania braziliensis and Leishmania amazonensis
promastigotes in vitro. In parallel, the cytotoxicity of these compounds was evaluated on the
RAW 264.7 cell line. The results showed that among the assayed compounds the substituted 3-
chlorophenyl (4a) (IC50/24h =15 0.14 µM) and 3,4-dichlorophenyl tetrazoles (4d)
(IC50/24h=26 0.09µM) were the most potent against L. braziliensis promastigotes, as compared
the reference drug pentamidine, which presented IC50=13 0.04µM. In addition, 4a and 4d
derivatives were less cytotoxic than pentamidine. However, these tetrazole derivatives (4) and
pyrazole-4-carbonitriles precursors (3) differ against each of the tested species and were more
effective against L.braziliensis than on L. amazonensis.
Keywords: Antileishmanial activity, Leishmania braziliensis, Leishmania amazonensis, Tetrazole
The parasitic protozoans of the genus Leishmania are the causative agents of several
forms of cutaneous, mucocutaneous, and visceral leishmaniasis. It is transmitted to human
beings and other a mammals by the bite of an infected female Phlebotomine sandfly.1
Leishmania has two evolutive forms: an extracellular flagellated promastigotes within the
Phlebotomine vector and the intracellular amastigotes within mononuclear phagocytes in
mammalian host. 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 public health problem. It is estimated that 1.6 million new cases each year, of which
an estimated 400 000 are visceral and 1.2 million are cutaneous.2,3 Cutaneous leishmaniasis is
mainly caused by Leishmania major in the Old World, and by L. braziliensis or L. amazonensis
in the Americas, in particular in Brazil.4 L. braziliensis cause a disease which is characterized by
its chronicity and the possibility to metastasize leading to the mucocutaneous clinical form. On
the other hand, L. amazonensis is found in different regions and it has been described
associated with various clinical forms such as: cutaneous, mucosa, diffuse cutaneous and
visceral leishmaniasis, therefore it is considered a specie with an epidemiological significance.5,6
Despite the high worldwide prevalence, few advances were made in the treatment of this
disease. There are no vaccines for leishmaniasis and chemotherapy is the main control
strategy. For more than fifty years, pentavalent antimonials have been the first-line drugs in the
treatment for all types of leishmaniasis, however these drugs require long-term and painful
treatment which cause serious side effects and the rate of treatment failure of cutaneous
leishmaniasis (CL) is increasing due to emerging of drug resistance. In the case of relapse,
patients need treatment with second-line medicines, such as pentamidine and amphotericin B
which have demonstrated nephrotoxicity. The recent and first oral drug miltefosine is really a
progress, but drug resistance is at risk.7,8 In addition, several reports regarding natural and
synthetic new antileishmanial compounds have been described,9 including some by our
research group.10 Among the several other compounds at various stages of development there
are the azoles derivatives, which have been revealed as potential new drugs against L.
amazonensis infection.11,12 We have also demonstrated the leishmanicidal activity of the 5-
amino-1-(3,5-dichlorophenyl)-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles in vivo against
L.amazonensis and exhibited significant inhibition in comparison to untreated control.13 Based
on these promising results obtained by our research group with azoles compounds, we planned
to synthesize pyrazole-tetrazole hybrids, keeping the pyrazole ring in the structure of the target
molecules. Thus, a new class of the 5-(1-aryl-3-methyl-1H-pyrazol-4-yl)-1H-tetrazole derivatives
(4a-m) and their respective precursors (3a-m) were synthesized and evaluated against L.
braziliensis and L. amazonensis.
The synthesis of the 5-(1-aryl-3-methyl-1H-pyrazol-4-yl)1H-tetrazoles (4a-m) is
summarized in Scheme 1. Arylhydrazine hydrochlorides (1a-m) were reacted with 1-
ethoxyethylidenemalononitrile and sodium acetate in ethanol, under reflux to form 5-amino-1-
aryl-3-methyl-1H-pyrazole-4-carbonitriles (2a-m). Then, the compounds (2a-m) were converted
to 1-aryl-3-methyl-1H-pyrazole-4-carbonitriles (3a-m) by the aprotic deamination using t-butyl
nitrite and tetrahydrofuran (THF), under reflux.11,14 Ultimately, the target 5-(1-aryl-3-methyl-1H-
pyrazol-4-yl)-1H-tetrazoles (4a-m) were obtained by the reaction of 1-aryl-3-methyl-1H-
pyrazole-4-carbonitriles (3a-m) with sodium azide, ammonium chloride and dimethylformamide
(DMF) at 130°C.14

The in vitro biological activities of tetrazole derivatives and their pyrazole-4-carbonitriles
precursors were assayed against L. amazonensis (MHOM/BR/77/LTB0016 strain) and L.
braziliensis (MCAN/BR/98/R619 strain), using pentamidine isethionate as reference drug. Table
1 displays IC50 (inhibitory concentration-the concentration of a compound to inhibit the growth of
50% of parasites), CC50 (cytotoxic concentration-the concentration of a compound which cause
a 50% reduction in cell viability) and SI (selective index between CC50/IC50 in L. braziliensis)
values of synthesized 5-(1-aryl-3-methyl-1H-pyrazol-4-yl)-1H-tetrazoles (4a-m) and pyrazole-4-
carbonitriles (3a-m) against Leishmania promastigotes and macrophage cell line (RAW 264.7).
Interestingly, the activity of the tetrazoles and 4-carbonitriles differs for each of the tested
species and were more effective against L. braziliensis than on L. amazonensis. The substituted
3-chlorophenyl tetrazole 4a (IC50=15 0.14 µM) and 3,4-dichlorophenyl (IC50=26 0.09µM) 4d
were found to be the most potent in inhibiting promastigotes forms of L. braziliensis.
Furthermore, the CC50 (cell line RAW 264.7) and SI for L. braziliensis indicate that these two
compounds exhibited low levels of the cytotoxicity, with a CC50 of 151.20 and 244.0 µM,
respectively. On the other hand, the reference drug pentamidine presented IC50=13 0,04 µM
with SI of 1.96 for L. braziliensis. The overall activity profile of the tested compounds
demonstrated that the biological activities were highly influenced by the tetrazole ring. This can
be clearly seen through the corresponding tetrazole 4a (IC50 of 15.0 and 123.0 µM) and the
precursor 4b (IC50 of 147.0 and >1000 µM) for L. braziliensis and L. amazonensis, respectively.
Additionally, in our previous structure activity studies we found that there is no significant
difference in the antileishmanial effect related to tetrazole and imidazole rings.13 However, the
antipromastigote activity was also noticeably influenced by the type of substituent attached on
the phenyl nucleus. Compounds 4a, 4c and 4d consisting a 3-chloro substituted group showed
the lowest IC50 and low toxicity (table 1). Moreover, the replacement of 3-chloro group with 3-
fluoro (4h) or 3-bromo (4l) resulted in decreased of antileishmanial activity, while that non-
halogen derivative with a 4-OMe (4m) displayed a negligible in vitro activity against both
Leishmania species.
In conclusion, this study indicates that the pyrazole-tetrazole are a new structural class
of azoles with antileishmanial activity. Among all the tested compounds it was found that 3-
chlorophenyl tetrazole derivative (4a) should be further investigated for the development of a
highly selective antileishmanial compound emphasizing that this compound was more effective
against L. braziliensis. Here it has also been reported the positive effect of introducing tetrazole
group, leading to increased activity of Leishmania spp.
Acknowledgements
We thank the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ),
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Programa

Estratégico de Apoio à Pesquisa em Saúde (PAPES), Coordenação de Aperfeiçoamento de
Pessoal Docente (CAPES), Universidade Federal Fluminense (PROPP/UFF), the Instituto
Oswaldo Cruz/Fundação Oswaldo Cruz(IOC/Fiocruz), Rede Mineira de Química (RMQ) and
Centro de Estudos e Inovação em Materiais Biofuncionais Avançados (UNIFEI) for fellowships
and financial support.
Supplementary data
Supplementary data associated with this article can be found in the online version.
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1#
Table 1. IC50 (µM) values of 5-(1-aryl-3-methyl-1H-pyrazol-4-yl)-1H-tetrazoles (4a-m) and
pyrazole-4-carbonitriles precursors (3a-m) on promastigotes of Leishmania spp., cytotoxicity
(CC50) in macrophage cell line (RAW 264.7) and selectivity index (SI).
Compounds
L. braziliensis
L. amazonensis
Cytotoxicity
SI
(IC50 µM)
(IC50 µM)
(CC50 µM)
(CC50/ IC50L.
braziliensis)
3a(3-Cl)
147.0 0.10
119.0 0.26
180.0 0.12
ND
>900
404.25 0.77
264.04 0.93
440.0 0.09
ND
2.75
3b(2,4-diCl)
3c(3,5-diCl)
3d (3,4-diCl)
3e(4-Cl)
>900
2.21
2.4
-
277.78 0.38
ND
658.99 0.30
>900
221.20 0.29
>900
ND
-
3f(2-F)
ND
-
3g(4-F)
378.11 0.41
144.28 0.61
706.11 0.21
137.40 2.99
694.66 0.17
361.50 3.20
15.38 0.14
152.54 0.13
85.41 0.12
26.69 0.09
111.54 0.49
225.41 0.27
459.02 0.18
77.87 0.21
111.48 0.12
68.75 0.38
109.84 0.37
660.16 0.22
13.0 0.04
>900
ND
-
3h(3-F)
417.91 0.33
137.40 0.38
206.11 0.24
274.81 0.14
>900
363.42 0.98
ND
2.52
-
3i(2-Br)
3j(4-Br)
219.53 0.48
ND
1.5
-
3l(3-Br)
3m(4-OMe)
4a(3-Cl)
ND
-
123.08 0.44
105.08 0.18
160.14 0.22
266.90 0.35
246.15 0.23
401.64 0.10
512.30 2.21
270.49 0.15
144.26 0.13
295.08 0.09
134.43 0.12
660.16 0.32
3.38 0.02
151.2 0.06
ND
9.83
-
4b(2,4-diCl)
4c(3,5-diCl)
4d(3,4-diCl)
4e(4-Cl)
272 0,05
244 0,08
>1000
3.18
9.14
9.0
-
4f(2-F)
ND
4g(4-F)
ND
-
4h(3-F)
284.0 0.06
90.34 2.81
295.0 0.06
>1000
3.6
0.81
4.29
9.1
-
4i(2-Br)
4j(4-Br)
4l(3-Br)
ND
4m(4-OMe)
Pentamidine
25.5 0.24
1.96
1#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.
ND: not done.

a: 3-Cl; b: R=2,4-diCl; c: R=3,5-diCl; d: R=3,4-diCl; e: R=4-Cl; f: R=2-F; g: R=4-F; h: R=3-F; i: R=2-Br; j: R=4-
Br; l: R=3-Br; m: R=4-OCH3.
Scheme 1. Reagents and conditions: i) sodium acetate, ethanol, 0,5h reflux; ii) 1-ethoxyethylidenemalononitrile, ethanol, 1h, reflux;
iii) t-butyl nitrite, THF, 2h, reflux; iv) sodium azide, ammonium chloride, DMF at 130°C, 14h.

A series of 5-(1-aryl-3-methyl-1H-pyrazol-4-yl)-1H-tetrazole derivatives (4a-m) and their 1-aryl-3-
methyl-1H-pyrazole-4-carbonitriles (3a-m) precursors were synthesized and evaluated for their
antileishmanial activity.
Compound
4a (3-Cl)
IC50 (µM)
15 0.14
26 0.09
4d (3,4-diCl)
3a-m
4a-m