Synthesis and leishmanicidal activities of 1-(4-X-phenyl)-N′-[(4-Y- phenyl)methylene]-1H-pyrazole-4-carbohydrazides

Article abstract of DOI:10.1016/j.ejmech.2005.10.007

1H-pyrazole-4-carbohydrazides were synthesized and their leishmanicidal in vitro activities and cytotoxic effects were investigated. The drugs prototypes of these new compounds (ketoconazole, benznidazole, allopurinol and pentamidine) were also tested. It

Full text of DOI:10.1016/j.ejmech.2005.10.007

European Journal of Medicinal Chemistry 41 (2006) 80–87
http://france.elsevier.com/direct/ejmech
Short communication
Synthesis and leishmanicidal activities of 1-(4-X-phenyl)-
N′-[(4-Y-phenyl)methylene]-1H-pyrazole-4-carbohydrazides
Alice M.R. Bernardino a, Adriana O. Gomes a, Karen S. Charret b, Antônio C.C. Freitas c,
Gérzia M.C. Machado d, Marilene M. Canto-Cavalheiro d, Leonor L. Leon d,
Veronica F. Amaral b,*
a
Departamento de Química Orgânica, Instituto de Química, Programa de Pós-graduação em Química Orgânica,
Universidade Federal Fluminense, Outeiro de São João Baptista, CEP 24020-150, Niterói, RJ, Brazil
Departamento de Imunobiologia, Instituto de Biologia, Universidade Federal Fluminense, Outeiro de São João Baptista, CEP 24020-150, Niterói, RJ, Brazil
b
c
Faculdade de Farmácia, Universidade Federal Fluminense, Outeiro de São João Baptista, CEP 24020-150, Niterói, RJ, Brazil
d
Departamento de Imunologia, IOC, Fundação Oswaldo Cruz, RJ, Brazil
Received 7 March 2005; received in revised form 21 September 2005; accepted 6 October 2005
Available online 21 November 2005
Abstract
1H-pyrazole-4-carbohydrazides were synthesized and their leishmanicidal in vitro activities and cytotoxic effects were investigated. The drugs
prototypes of these new compounds (ketoconazole, benznidazole, allopurinol and pentamidine) were also tested. It was found that among all the
1H-pyrazole-4-carbohydrazides derivatives examined, the most active compounds were those with X = Br, Y = NO2 (27) and X = NO2, Y = Cl
(15) derivatives which showed to be most effective on promastigotes forms of L. amazonensis than on L. chagasi and L. braziliensis species.
When tested against murine peritoneal macrophages as mammalian host cell controls of toxicity, 1-(4-Br-phenyl)-N′-[(4-NO2-phenyl)methylene]-
1H-pyrazole-4-carbohydrazides (27) (EC50 = 50 µM l–1) and 1-(4-NO2-phenyl)-N′-[(4-Cl-phenyl)methylene]-1H-pyrazole-4-carbohydrazides (15)
EC50 = 80 µM l–1)] was reasonably toxic. However, both compounds were less toxic than pentamidine and ketoconazole. These results provide
new perspectives on the development of drugs with activities against Leishmania parasite.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Synthesis; 1-(4-X-phenyl)-N′-[(4-Y-phenyl)methylene]-1H-pyrazole-4-carbohydrazides; Antileishmania activity; Cytotoxicity
1. Introduction
the cutaneous and visceral clinical presentation. It has also
been considered as an emerging disease with specific difficul-
ties in terms of treatment [4,5].
Leishmania is a pathogenic protozoa that causes a broad
spectrum of infectious diseases in mammalian hosts ranging
from self-healing cutaneous ulceration to progressive and lethal
visceral infection that are transmitted by phlebotomine sand-
flies. Leishmania has two evolutive forms: an extracellular fla-
gellated promastigotes in the vector and intracellular amasti-
gotes within mononuclear phagocytes in mammalian host.
Leishmaniasis is endemic in 88 countries in the world and af-
fects about 2 million people per year, with approximately 350
million individuals at risk of infection [1]. Lately, in Brazil [2]
and in South-western Europe [3], the number of cases of Leish-
mania/HIV co-infection has been increasing, including both
The difficulty to control the parasitic disease remains a ser-
ious problem principally to the diversity of the mammalian re-
servoirs (wild and domestic animals), the variety of species of
vectors and complexity of Leishmania species [6]. The severity
of clinical manifestations depends, mainly, on the Leishmania
species involved. In Brazil, Leishmania (Leishmania) amazo-
nensis was found in different regions and it has been described
an association with various clinical forms such as: cutaneous,
mucosal, diffuse cutaneous and visceral leishmaniasis consid-
ered a species with an epidemiological significance [7]. Addi-
tionally, Leishmania (Leishmania) chagasi is responsible for
visceral clinical form and Leishmania (Viannia) braziliensis is
evolved with cutaneous or mucosal lesions [6].
* Corresponding author. Tel.: +55 21 2629 2316; fax: +55 21 2629 2376.
E-mail addresses: alice@rmn.uff.br (A.M.R. Bernardino), vfa@vm.uff.br
(V.F. Amaral).
The absence of an alternative chemotherapeutic approach to
the treatment of Leishmania infection requires urgent attention.
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.10.007

A.M.R. Bernardino et al. / European Journal of Medicinal Chemistry 41 (2006) 80–87
81
There are presently no vaccines [8] and chemotherapy is
unsatisfactory and high-priced [9]. Therefore, in this work we
report the synthesis and the in vitro leishmanial activity of new
pyrazolic system series, 1-(4-X-phenyl)-N′-[(4-Y-phenyl)
methylene]-1-H-pyrazole-4-carbohydrazides (1–30) have been
investigated on their inhibitory effects against the extracellular
promastigote stage of L. amazonensis, L. chagasi and
L. braziliensis parasites, as its direct toxic effect on macro-
phage host cells. The drugs prototypes (ketoconazole, benzni-
dazole, allopurinol and pentamidine) were also examined.
azole-3-carboxylic acid hydrazide analogs and some derived 4-
substituted-1,2,4-triazolin-3-thiones, 2-substituted-1,3,4-thia-
diazole and 2-substituted-1,3,4-oxadiazoles were synthesized
and showed antitumoral and antiviral activities [17].
The principal prototype compounds were ketoconazole, al-
lopurinol, pentamidine and benznidazole. Therefore, ketocona-
zole drug inhibits sterol biosynthesis on ergosterol in Leishma-
nia species [18] and is reported that is not active on
L. braziliensis [19,20]. In trypanosomatids, which are deficient
in xanthine oxidase, allopurinol acts as a purine analogue and
is incorporated, through hypoxanthine-guanine phosphoribo-
syltransferase (HGPRT), obstructing the proteins synthesis
[21,22]. Benznidazole is a nitroimidazole derivative, which
has an important activity against Trypanosoma, and it is the
drug employed in Brazil for the treatment of T. cruzi infection.
The anaerobic reduction of benznidazole to reactive intermedi-
ates involve covalent bind to proteins and lipids [23] and also
induce oxidative stress within the parasite [24]. Pentamidine is
the standard drug used in leishmaniasis and early stage of
sleeping sickness. Different mechanisms of action have been
proposed for pentamidine in kinetoplastid parasites including
inhibition of topoisomerase II [25] and serine proteases [26]
and disturbance of polyamine metabolism [27]. In this report,
new pyrazolic system series, 1-(4-X-phenyl)-N′-[(4-Y-phenyl)
methylene]-1H-pyrazole-4-carbohydrazides (1–30) and proto-
type drugs have been investigated on their inhibitory effects
against the extracellular promastigote stage of L. amazonensis,
L. chagasi and L. braziliensis parasites, besides its direct toxic
effects on macrophage host cells.
2. Chemistry
Drugs containing acylhydrazone derivatives have showed
some important biological effects as analgesic, antiplatelets
and anti-inflammatory activities. The evaluation of this drug
profile leads us to identify a new potent prototype of biological
activity [10,11].
In this work it is taking into consideration this molecular
characteristic, the new arylpyrazolic-attached acyllhydrazone
derivatives were synthesized exploring the molecular hybridi-
zation approach. Additionally, the change of para-substituent
group of acylhydrazone and arylpyrazolic framework permitted
to identify electronic and hydrophobic derivatives effect. The
nature of these substituents in phenyl and arylic groups (OH,
NCH3, CH3, OCH3, SCH3, H, F, Cl, Br, CN, NO2) could be
correlated to an important electronic variation (Hammett values
σ ranging from –0.18 to 0.78) which could be useful to inves-
tigate the contribution of this structural sub-unit on its bioac-
tivity profile. Therefore, we recognized that the aza-vinylogous
groups in the molecule pyrazole-carbohydrazides possibly
would be functioned as hydrophobic anchors for active sites
of protozoa [11].
4. Results and discussion
The new series of 1-(4-X-phenyl)-N′-[(4-Y-phenyl)methy-
lene]-1H-pyrazole-4-carbohydrazides (1–30) were synthesized
following the previous procedure, as showed in Fig. 1. The
literature describe synthesis and pharmacological evaluation
of some 1-(4-X-phenyl)-N′-[(4-Y-phenyl)methylene]-1H-pyra-
zole-4-carbohydrazides [28–30]. The structure of these new
compounds has been characterized by a lateral chain of 1H-
pyrazole-4-carbohydrazide, which probably contributes to their
biological activities [31].
3. Pharmacology
Pentavalent antimonials, available as sodium stibogluconate
(pentostam) or N-methylglucamine antimoniate (glucantime),
have been the standard drugs for treatment of leishmaniasis
recognized as being expensive and need to be given parenter-
ally, often for prolonged periods [12]. Moreover, the classical
and alternative drugs (pentamidine, amphotericin B) often
cause toxic side effects and induction of parasitic resistance
[13,14]. Miltefosine, is the first effective orally administered
treatment for visceral leishmaniasis [15], but possible emer-
gence of miltefosine resistance and side effects should be con-
sidered.
Studies with acylhydrazone series have been showed some
important biological effects as analgesic, antiplatelets and anti-
inflammatory activities [10]. Some enzymes identified in para-
sites have been accepted as targets for antiparasite chemother-
apy developing protease inhibitors for HIV, Plasmodium falci-
parum and Trypanosoma cruzi [16]. It was also recognized that
iminic insaturation of acylhydrazone could be had the hydro-
phobic anchors function for protozoa enzymatic active site. In
addition, a novel series of 1-(chlorophenyl)-4-hydroxy-1H-pyr-
The 1-arilpyrazoles-4carboxaldehydes (X = OCH3, NO2 and
Br) (31–33) were prepared by Vilsmeier-Haack reaction [32].
The acids (34–36) were prepared by Jones oxidation. The 34–
36 compounds were treated with SOCl2. The freshly prepared
4-(4′-phenyl) pyrazolylchlorides were converted to the corre-
sponding hydrazides intermediates (40–42) by treatment with
hydrazine hydrate. Finally, compounds (1–30) were obtained
by condensing of (40–42) with functionalized aromatic alde-
hydes (Y = H, p-OH, p-CN, p-OCH3, p-Br, p-Cl, p-F, p-
SCH3, p-NO2, p-CH3, p-N(CH3)2, p-OC2H5) at reflux in etha-
nol to produce the desired of 1-(4-X-phenyl)-N′-[(4-Y-phenyl)
methylene]-1H-pyrazole-4-carbohydrazides (1–30) in good
yields. The structure of these stable and crystalline compounds
was fully characterized by usual methods (IR, 1H-, 13C-NMR).

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A.M.R. Bernardino et al. / European Journal of Medicinal Chemistry 41 (2006) 80–87
Fig. 1. General procedure for the synthesis of 1-(4-X-phenyl)-N′-[(4-Y-phenyl)methylene]-1H-pyrazole-4-carbohydrazides.
4.1. Anti-leishmanial assays
results indicate that, when compared with reference drug like
ketoconazole the compounds 15 and 27 demonstrated similar
leishmanicidal activity (Table 2). In the literature there have
been studies among azole drugs demonstrating the superior in
vitro inhibitory activity on L. donovani, L. braziliensis and on
L. amazonensis than on L. aethiopica, L. major, L. tropica and
L. mexicana promastigotes [18]. In other studies, ketoconazole
was considered efficient on treatment of cutaneous leishmania-
sis patients with L. panamensis [33], L. major [34,35] and
L. mexicana although presented unsatisfactory activity on
L. braziliensis infection [36].
The biological effects of 1H-pyrazole-4-carbohydrazides
(1–30), on the viability of Leishmania sp. were tested and com-
pared with references drugs (ketoconazole pentamidine, benz-
nidazole and allopurinol). Some of these 30 compounds have
activities against infective promastigotes forms of
L. amazonensis (Table 1). Therefore, two substituents, the 27
and 15 have a potent activity, which inhibited the growth of
L. amazonensis (90% and 66%) at very low concentrations,
with EC50/24h of 50 and 80 µM l–1, respectively. Others com-
pounds displayed in vitro activity against promastigotes forms
only at very high concentrations, like demonstrated in Table 1.
In our experiments, the effect of these principal components
(27 and 15) were also investigated on others Leishmania spe-
cies like L. chagasi and L. braziliensis and demonstrated to be
less sensitive than on L. amazonensis. Taken together, these
These differences accentuate the importance of speciation in
the treatment of leishmaniasis. American cutaneous leishmaniasis
is associated to at least 13 species of Leishmania pathogenic to
human with biochemical and molecular variation [4,37].
In order to evaluate possible structure–activity relationships
of these 1H-pyrazole-4-carbohydrazides derivatives it was ob-

A.M.R. Bernardino et al. / European Journal of Medicinal Chemistry 41 (2006) 80–87
83
Table 1
The toxic effects of these compounds were also evaluated
on mouse peritoneal macrophages (Table 1). Compounds 15
and 27 which demonstrated better leishmanicidal activity dis-
played toxicity index on mammalian cells correspondent to
EC50 of > 270.4 and 213.9 µM l–1, respectively. When com-
pared with a pattern drug as pentamidine (227 µM l–1) or ke-
toconazole (130 µM l–1), the results showed these compounds
are more toxic to cells than these new compounds increasing
the importance to continue this studies.
In conclusion, this study confirms the marked leishmanicidal
activity of new 1H-pyrazole-4-carbohydrazides on L. amazon-
ensis and less important effect on L. chagasi and
L. braziliensis. Fortunately, it will be necessary in vitro and in
vivo studies on amastigotes intracellular forms of Leishmania.
EC50 values of 1-(4-X-phenyl)-N′-[(4-Y-phenyl)methylene]-1H-pyrazole-4-
carbohydrazides on promastigotes of L. amazonensis and murine macrophage
Leishmanicidal
activity
Cytotoxicity
Compound
1
2
3
4
5
6
7
8
9
10
11
X
Y
H
OH
CN
OCH3,
Br
Cl
(µM l–1
ND
)
(µM l–1
ND
)
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
> 600
> 600
> 600
ND
480 360
No activity
No activity
> 600
ND
ND
> 285.4
184.1 2.2
> 281.8
> 295.5
> 273.0
> 273.7
ND
F
SCH3
NO2
CH3
N
ND
> 600
> 275.1
(CH3)2
OC2H5
OCH3
Br
Cl
F
5. Experimental protocols
12
13
14
15
16
17
OCH3
NO2,
NO2
NO2
NO2
NO2
440
> 320
560 80
80
> 600
> 600
5
> 274.4
> 273.7
222.6 13.7
> 270.44
188.8 2.0
ND
Chemicals employed were obtained from commercial sup-
plies and used without purification, unless otherwise noted.
7
N
(CH3)2
OC2H5
H
OH
CN
OCH3
Br
Cl
5.1. Chemistry
18
19
20
21
22
23
24
25
26
27
28
29
NO2
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
> 600
> 600
> 600
No activity
> 600
> 263.6
> 270.8
> 259.6
229.0 28.6
> 250.5
5.1.1. 1-(4-X-phenyl)-1H-pyrazole-4-carboxilic acids (34–36)
The preparation of these compounds was carried out in accor-
dance with published procedure [32]. The purity of the compounds
was verified by means of thin layer chromatography (TLC) using
silica gel plate with fluorescent indicator and CHCl3/methanol
> 600
> 223.1
No activity
No activity
No activity
192.7 9.1
203.2 1.7
> 240.8
213.9 26.5
ND
F
1
(8:2) as eluent, melting point, IR spectra and H-NMR.
SCH3
NO2
CH3
N
(CH3)2
OC2H5
Compound (34): IR (KBr, cm–1) 3200–2500 (ν, OH), 1660
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 3.9 (s, 1H,
OCH3) 9.1 (s, 1H, H3); 8.2 (s, 1H, H5), 7.2; 7.3 (d, 2H, H2′,
H6′); 8.0; 8.1 (d, 2H, H3′, H5′), 12,8 (s, 1H, OH). Melting
point: 235–237 °C. Yield: 91%.
50
2
ND
340 230
215.6 2.4
30
Br
> 600
167.7 16.6
Compound (35): IR (KBr, cm–1) 3200–2500 (ν, OH), 1680
(ν, C=O), 1520, 1340 (NO2). H-NMR (300 MHz; DMSO-d6,
1
served that the nature and position of substituent on the mole-
cule to improve biological functions. It seems that positions of
substituent on molecule 27 which X = Br, Y = NO2 was more
effective in relation to leishmanicidal behavior than what was
observed on molecule 14 (X = NO2, Y = Br). This fact could
be associated to the mesomeric electronic effect of Br substitu-
ent (where substituent constant σ = 0.23) and NO2 substituent
(where substituent constant σ = 0.78) in Y position to confer
increased electrophilic character to iminic group. This last
group could be useful to bind on nucleophilic site of protozoa
cys-protease [16]. Therefore, it is likely that electronic and hy-
drophobic factors on molecule structure would be important as
hydrophobic anchors to the enzymatic site of parasite.
δ). Melting point: 163–165 °C. Yield: 82%.
Compound (36): IR (KBr, cm–1) 3200–2400 (ν, OH), 1660
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 9.2 (s, 1H, H3);
8.2 (s, 1H, H5), 7.8; 7.9 (d, 2H, H2´, H6´); 8,0; 8,1 (d, 2H,
H3´, H5´), 12.8 (s. 1H, OH). Melting point: 202–203 °C.
Yield: 91%.
5.1.2. Methyl 1-(4-X-phenyl)-1H-pyrazole-4-carboxylates
(37–39)
The corresponding freshly prepared 1-(4-X-phenyl)-1H-pyr-
azole-4-carbonylchloride (10 mmol) was added to a stirred
methanol (20 ml). The reaction mixture was maintained under
Table 2
Effect of 1H-pyrazole-4-carbohydrazides derivatives and prototypes drugs on promastigotes of Leishmania sp and murine peritoneal macrophages
Compound
15
27
Allopurinol
Ketoconazole
Benznidazole
Pentamidine
X
NO2
Br
–
–
–
Y
Cl
NO2
–
–
–
L. braziliensis
540 100
210 20
370 100
190 20
> 600
L. chagasi
270 70
220 20
> 600
60 10
160 10
140 10
L. amazonensis
Macrophage
> 270.4
210 30
> 600
130 10
330 20
227 80
80
50
7
2
> 600
70
2
No activity
–
–
50
6
10
3
Mean of EC50 (µM l–1
)
S.D. for three determination.

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A.M.R. Bernardino et al. / European Journal of Medicinal Chemistry 41 (2006) 80–87
reflux for 2 h. In the next step, the reaction mixture was poured
in cold water neutralized with 10% aqueous sodium bicarbo-
nate solution and the precipitate formed was filtered out and
dried. The purity of the compounds was checked by means of
TLC using silica gel plate with fluorescent indicator and
CHCl3/methanol (8:2) as eluent, melting point, IR spectra and
1H-NMR.
benzaldehyde derivative in the presence of catalytic amount of
35% hydrochloric acid. The mixture was maintained under re-
flux for 2 h, until TLC indicated the end of reaction. Then, the
reaction mixture was poured in cold water, and the precipitate
formed was filtered out washed with ethanol and recrystallized
from ethanol to afford crystals. This process was completed
and the following compounds (1–30) were prepared.
Compound (1) IR (KBr, cm–1) 3500–2800 (ν, N–H), 1620
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 3.9 (s, 3H,
OCH3) 8.8 (s, 1H, H3); 8.2 (s, 1H, H5); 7.2; 7.3 (d, 2H, H2´,
H6´); 7.9, 8.0 (d, 2H, H3´, H5´); 7.2–8.0 (m, 1H, N=C–H); 7.6;
8.0 (m, 5H, H2´´, H6´´); 7.6; 8.0 (m, 5H, H3´´, H5´´); 9.0 (s,
1H, N–H). Melting point: 135–137 °C. Yield: 80%.
Compound (37): IR (KBr, cm–1) 1710 (ν, C=O); 1240, 1130
1
(ν, C–O). H-NMR (300 MHz; DMSO-d6, δ): 3.86 (s, 1H,
OCH3); 3.85 (s, 1H, OCH3) 8.3 (s, 1H, H3); 8.0 (s, 1H, H5),
6.9; 7.0 (d, 2H, H2´, H6´); 7.5; 7.6 (d, 2H, H3´, H5´). Melting
point: 134–136 °C. Yield: 86%.
Compound (38): IR (KBr, cm–1) 1730 (ν, C=O); 1270, 1110
1
Compound (2): IR (KBr, cm–1) 3500–2500 (ν, N–H, OH),
1640 (ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 3.9 (s, 3H,
OCH3) 9.0 (s, 1H, H3), 8.4 (s, 1H, H5), 6.9; 7.0 (d, 2H, H2´,
H6´); 7.7; 7.73 (d, 2H, H3´, H5´); 8.1 (s, 1H, N=C–H); 7.2; 7.3
(d, 2H, H2´´; H5´´); 7.9; 8.0 (d, 2H, H3´´, H5´´); 10.0 (s, 1H,
OH); 11.5 (s, 1H, N–H). Melting point: 245–247 °C, 86%.
Compound (3): IR (KBr, cm–1) 3500–2600 (ν, NH), 2220
(ν, C–O); 1520; 1430 (ν, NO2). H-NMR (300 MHz; DMSO-
d6, δ): 4.1 (s, 1H, OCH3); 9.5 (s, 1H, H3); 8.9 (s, 1H, H5), 8.3;
8.4 (d, 2H, H2´, H6´); 8.6; 8.4 (d, 2H, H3´, H5´). Melting
point: 168–170 °C. Yield: 75%.
Compound (39): IR (KBr, cm–1) 1720 (ν, C=O); 1250, 1140
(ν, C–O). 1H-NMR (300 MHz; DMSO-d6, δ): 3.8 (s, 1H,
OCH3); 8.3 (s, 1H, H3); 8.0 (s, 1H, H5), 7.6; 7.7 (d, 2H,
H2´, H6´); 7.8; 7.9 (d, 2H, H3´, H5´). Melting point: 160–
162 °C. Yield: 78%.
1
(ν, CN) 1640 (ν, C=O). H-NMR (300 MHz; DMSO-d6, δ):
3.8 (s, 3H, OCH3); 9.0 (s, 1H, H3); 8.4 (s, 1H, H5); 7.1; 7.2 (d,
2H, H3´, H5´); 7.9; 8.0 (d, 2H, H3´, H5´); 7.8; 7.9 (d, 2H,
H2´´, H6´´); 7.9; 8.0 (d, 2H, H3´´, H5´´); 8.2 (s, 1H, N=C–
H); 11.5 (s, 1H, N–H). Melting point: 220–222 °C. Yield:
62%.
Compound (4): IR (KBr, cm–1) 3500–2800 (ν, NH), 1640
(ν, C=O), 1255 and 1169 (ν, C–O). (DMSO-d6, δ): 3.5 (s, 3H,
OCH3); 3.9 (s, 3H, OCH3); 9.1 (s, 1H, H3); 8.7 (s, 1H, H5);
7.1; 7.2 (d, 2H, H2´, H6´), 7.2; 7.21 (d, 2H, H3´, H5´); 8.2 (s,
1H, N=C–H); 7.7; 7.8 (d, 2H, H2´´; H6´´); 7.9; 7.92 (d, 2H,
H3´´, H5´´); 11.9 (s, 1H, N–H). Melting point: 186–188 °C.
Yield: 64%.
Compound (5): IR (KBr, cm–1) 3500–2800 (ν, NH), 1650
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 4.0 (s, 3H,
OCH3); 9.2 (s, 1H, H3); 8.6 (s, 1H, H5); 7.3; 7.4 (d, 2H,
H2´, H6´); 7.9; 8.0 (d, 2H, H3´, H5´); 7.9; 8.1 (d, 2H, H2´´,
H6´´); 8.0 8.1 (d, 2H, H3´´, H5´´); 8.0 (s, 1H, N=C–H); 11.9
(s, 1H, N–H). Melting point: 205–207 °C. Yield: 75%.
Compound (6): IR (KBr, cm–1) 3500–2800 (ν, NH), 1650
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 4.0 (s, 3H,
OCH3); 9.1 (s, 1H, H3); 8.6 (s, 1H, H5); 7.3; 7.34 (d, 2H,
H2´, H6´); 8.1; 8.12 (d, 2H, H3´, H5`); 7.7; 7.8 (d, 2H, H2´´,
H6´´); 7.9; 8.0 (d, 2H, H3´´, H5´´); 8.0 (s, 1H, N=C–H); 11.9
(s, 1H, N–H). Melting point: 205–208 °C. Yield: 67%.
Compound (7): IR (KBr, cm–1) 3500–2800 (ν, NH), 1660
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 3.9 (s, 3H,
OCH3); 9.1 (s, 1H, H3); 8.5 (s, 1H, H5); 7.2; 7.3 (d, 2H,
H2´, H6´); 7.4; 7.5 (d, 2H, H3´, H5´); 7.9; 8.0 (d, 2H, H2´´,
H6´´); 7.9; 8.0 (d, 2H, H3´´, H5´´), 8.4 (s, 1H, N=C–H); 11.8
(s, 1H, N–H). Melting point: 180–182 °C. Yield: 67%.
Compound (8): IR (KBr, cm–1) 3500–2800 (ν, NH), 1638
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 3.5 (s, 3H,
OCH3); 9.0 (s, 1H, H3); 8.4 (s, 1H, H5); 7.2; 7.25 (D, 2h,
h2´, h6´); 7.4; 7.43 (d, 2H, H3´, H5´); 7.7; 7.8 (d, 2H, H2´´,
5.1.3. General procedure for the preparation
of 1-(4-X-phenyl)-1H-pyrazole-4-carbohydrazides (40–42)
To a stirred solution of 1 mmol of the methyl 1-(4-X-phe-
nyl)-1H-pyrazole-4-carboxylate (37–39) in ethanol (10 ml),
2 ml of 80% hydrazine monohydrate was added. The reaction
mixture was maintained under reflux for 5–8 hours, until TLC
indicated the end of reaction. After this time, the reaction mix-
ture was poured on ice and the solid formed was collected by
filtration, washed with cold water and recrystallized from etha-
nol. As a result of this process the following compounds were
prepared (40–42).
Compound (40): IR (KBr, cm–1) 3400–2800 (ν, NH), 1650
1
(ν, C=O); 1260, 1090 (ν, C–O). H-NMR (300 MHz; DMSO-
d6, δ): 3.9 (s, 1H, OCH3); 8.8 (s, 1H, H3); 8.1 (s, 1H, H5), 7.2;
7.3 (d, 2H, H2´, H6´); 7.8; 7.9 (d, 2H, H3´, H5´), 9.5 (s, 1H,
NH), 4.5 (s, 1H, NH2). Melting point: 200–202 °C. Yield:
95%.
Compound (41): IR (KBr, cm–1) 3500–3000 (ν, NH), 1660
1
(ν, C=O); 1550, 1440 (ν, NO2). H-NMR (300 MHz; DMSO-
d6, δ): 8.7 (s, 1H, H3); 8.1 (s, 1H, H5), 6.8; 6.9 (d, 2H, H2´,
H6´); 7.5; 7.6 (d, 2H, H3´, H5´), 9.5 (s, 1H, NH), 4.6 (s, 1H,
NH2). Melting point: 230–231 °C. Yield: 72%.
Compound (42): IR (KBr, cm–1) 3320–3100 (ν, NH), 1640
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 8.9 (s, 1H, H3);
8.1 (s, 1H, H5), 7.7; 7.73 (d, 2H, H2´, H6´); 7.8; 7.84 (d, 2H,
H3´, H5´), 9.5 (s, 1H, NH), 4.4 (s, 1H, NH2). Melting point:
205–209 °C. Yield: 94%.
5.1.4. General procedure for the preparation
of 1-(4-X-phenyl)-N′-[(4-Y-phenyl)methylene]-1H-
pyrazole-4-carbohydrazides (1–30)
To a solution of derivatives (40–42) (1 mmol) in 10 ml of
ethanol, it was added an equimolar amount of the appropriate

A.M.R. Bernardino et al. / European Journal of Medicinal Chemistry 41 (2006) 80–87
85
H6´´); 7.9; 8.0 (d, 2H, H3´´, H5´´); 8.2 (s, 1H, N=C–H); 11.7
(s, 1H, N–H). Melting point: 143–145 °C. Yield: 71%.
Compound (9): IR (KBr, cm–1) 3500–2800 (ν, NH), 1670
(ν, C=O), 1530 and 1350 (ν, NO2). 1H-NMR (300 MHz;
DMSO-d6, δ): 3.8 (s, 3H, OCH3); 8.8 (s, 1H, H3); 8.3 (s,
1H, H5); 7.1; 7.2 (d, 2H, H2´; H6´); 7.8; 7.9 (d, 2H, H3´,
H5´); 7.7; 7.8 (d, 2H, H2´´, H6´´); 8.1; 8.2 (d, 2H, H3´´,
H5´´); 8.1 (s, 1H, N=C–H); 9.0 (s, 1H, N–H). Melting point:
225–226 °C. Yield: 83%.
N(CH3)28.4 (s, 1H, H3); 8.2 (s, 1H, H5); 6.6; 6.7 (d, 2H,
H2´, H6´); 7.8; 7.9 (d, 2H, H3´, H5´); 6.7, 6.8 (d, 2H, H2´´,
H6´´); 7.6; 7.7 (d, 2H, H3´´, H5´´); 8.1(s, 1H, N=C–H); 11.9
(s, 1H, N–H). Melting point: 235–237 °C. Yield: 70%.
Compound (18): IR (KBr, cm–1) 3500–2800 (ν, NH), 1650
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 4.2; 1.4 (m, 5H,
OCH2CH3); 8.9 (s, 1H, H3); 8.7 (s, 1H, H5); 6.9; 7.0 (d, 2H,
H2´, H6´); 7.0; 7.1 (d, 2H, H3´; H5´); 7.7; 7.73 (d, 2H, H2´´,
H6´´); 7.9; 8.0 (d, 2H, H3´´, H5´´); 8.3 (s, 1H, N=C–H); 11.5
(s, 1H, N–H). Melting point: 231–232 °C. Yield: 40%.
Compound (19): IR (KBr, cm–1) 3500–2800 (ν, NH), 1653
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 8.7 (s, 1H, H3);
8.1 (s, 1H, H5); 7.5; 7.6 (d, 2H, H2´, H6´); 7.9; 8.0 (d, 2H,
H3´, H5´); 7.4–7.7 (m, 5H, H´Ar); 7.5 (s, 1H, N=C–H); 9.1
(s, 1H, N–H). Melting point: 228–229 °C. Yield: 75%.
Compound (20): IR (KBr, cm–1) 3500–2400 (ν, NH), 1628
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 9.2 (s, 1H, H3);
8.5 (s, 1H, H5); 7.0; 7.1 (d, 2H, H3´, H6´); 7.8; 7.9 (d, 2H,
H3´, H5´); 7.96; 7.93 (d, 2H, H2´´; H6´´); 8.0; 8.1 (d, 2H,
H3´´, H5´´); 8.1 (s, 1H, N=C–H); 10.1 (s, 1H, OH); 11.6 (s,
1H, N–H). Melting point: 258–261 °C. Yield: 76%.
Compound (10): IR (KBr, cm–1) 3500–2800 (ν, NH), 1643
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 3.3 (s, 3H,
CH3); 3.7 (s, 3H, OCH3); 8.9 (s, 1H, H3); 8.3 (s, 1H, H5);
7.0; 7.1 (d, 2H, H2´, H6´); 7.7; 7.8 (d, 2H, H3´, H5´); 7.2;
7.3 (d, 2H, H2´´, H6´´); 7.6, 7.63 (d, 2H, H3´´, H5´´); 8.0 (s,
1H, N=C–H); 11.5 (s, 1H, N–H). Melting point: 194–196 °C.
Yield: 75%.
Compound (11): IR (KBr, cm–1) 3500–2800 (ν, NH), 1652
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 3.3 (s, 3H,
CH3); 3.8 (s, 3H, OCH3); 8.9 (s, 1H, H3); 8.2 (s, 1H, H5);
6.8; 6.83 (d, 2H, H2´, H6´); 7.1; 7.2 (d, 2H, H3´, H5´); 7.5;
7.6 (d, 2H, H2´´, H6´´); 7.8; 7.8 (d, 2H, H3´´, H5´´); 8.0 (s, 1H,
N=C–H); 11.3 (s, 1H, N–H). Melting point: 170–172 °C.
Yield: 76%.
Compound (21): IR (KBr, cm–1) 3500–2800 (ν, NH), 2222
1
(ν, CN) 1640 (ν, C=O). H-NMR (300 MHz; DMSO-d6, δ):
Compound (12): IR (KBr, cm–1) 3500–2800 (ν, NH), 1637
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 1.3; 4.0 (m, 5H,
OCH2CH3); 3.2 (s, 3H, OCH3); 8.8 (s, 1H, H3); 8.2 (s, 1H,
H5); 6.8; 6.9 (d, 2H, H2´, H6´); 7.63; 7.6 (d, 2H, H3´, H5´);
7.0; 7.1 (d, 2H, H2´´, H6´´); 7.7; 7.8 (d, 2H, H3´´, H5´´); 8.0
(s, 1H, N=C–H); 11.4 (s, 1H, N–H). Melting point: 163–
165 °C. Yield: 71%.
9.3 (s, 1H, H3); 8.6 (s, 1H, H5); 7.9; 8.0 (d, 2H, H2´; H6´);
8.0; 8.1 (d, 2H, H3´, H5´); 8.0; 8.1 (d, 2H, H2´´, H6´´); 8.0; 8.1
(d, 2H, H3´´, H5´´); 8.2 (s, 1H, N=C–H); 12.1 (s, 1H, N–H).
Melting point: 250–252 °C. Yield: 65%.
Compound (22): IR (KBr, cm–1) 3500–2700 (ν, NH), 1652
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 3.8 (s, 3H,
OCH3); 9.1 (s, 1H, H3); 8.4 (s, 1H, H5); 7.07; 7.1 (d, 2H,
H2´, H6´); 7.8; 7.7 (d, 2H, H3´, H5´); 7.72; 7.75 (d, 2H,
H2´´, H6´´); 7.9; 7.94 (d, 2H, H3´´; H5´´); 8.1 (s, 1H, N=C–
H); 11.5 (s, 1H, N–H). Melting point: 227–232 °C. Yield:
85%.
Compound (13): IR (KBr, cm–1) 3500–2900 (ν, NH), 1650
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 3.9 (s, 3H,
OCH3); 9.1 (s, 1H, H3); 8.7 (s, 1H, H5); 7.1; 7.2 (d, 2H,
H2´, H6´); 7.5; 7.6 (d, 2H, H3´; H5´); 7.6; 7.7 (d, 2H, H2´´,
H6´´); 7.8; 7.83 (d, 2H, H2´´, H6´´); 8.3 (s, 1H, N=C–H); 12.0
(s, 1H, N–H). Melting point: 221–223 °C. Yield: 60%.
Compound (14): IR (KBr, cm–1) 3500–2500 (ν, NH), 1650
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 9.2 (s, 1H, H3);
8.9 (s, 1H, H5); 7.1; 7.2 (d, 2H, H2´, H6´); 7.95; 7.97 (d, 2H,
H3´, H5´); 7.8; 7.9 (d, 2H, H2´´, H6´´); 8.0; 8.1 (d, 2H, H3´´;
H5´´); 8.6 (s, 1H, N=C–H); 12.1 (s, 1H, N–H). Melting point:
230–232 °C. Yield: 73%.
Compound (23): IR (KBr, cm–1) 3500–2500 (ν, NH), 1633
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 9.0 (s, 1H, H3);
8.3 (s, 1H, H5); 7.6; 7.7 (d, 2H, H2´, H6´); 7.8, 7.83 (d, 2H,
H3´; H5´); 7.6; 7.7 (d, 2H, H2´´, H6´´); 7.7; 7.8 (d, 2H, H3´´,
H5´´); 8.0 (s, 1H, N=C–H); 11.6 (s, 1H, N–H). Melting point:
248–250 °C. Yield: 78%.
Compound (24): IR (KBr, cm–1) 3500–2900 (ν, NH), 1634
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 9.2 (s, 1H, H3);
8.4 (s, 1H, H5); 7.6; 7.64 (d, 2H, H2´; H6´); 7.9; 8.0 (d, 2H,
H3´, H5´); 7.8; 7.83 (d, 2H, H2´´, H6´´), 7.8; 7.7 (d, 2H, H3´´,
H5´´); 8.0 (s, 1H, N=C–H); 11.8 (s, 1H, N–H). Melting point:
215–216 °C. Yield: 70%.
Compound (15): IR (KBr, cm–1) 3500–2800 (ν, NH), 1640
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 9.1 (s, 1H, H3);
8.7 (s, 1H, H5); 7.1; 7.2 (d, 2H, H2´, H6´); 7.5; 7.6 (d, 2H,
H3´; H5´); 7.6; 7.7 (d, 2H, H2´´, H6´´); 7.8; 7.83 (d, 2H,
H2´´, H6´´); 8.3 (s, 1H, N=C–H); 12.0 (s, 1H, N–H). Melting
point: 240–241 °C. Yield: 68%.
Compound (25): IR (KBr, cm–1) 3500–3000 (ν, NH), 1651
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 9.1 (s, 1H, H3);
8.7 (s, 1H, H5); 7.3; 7.37 (d, 2H, H2´, H6´); 7.75; 7.78 (d, 2H,
H3´; H5´); 7.8; 7.9 (d, 2H, H2´´, H6´´), 8.0; 7.9 (d, 2H, H3´´,
H5´´); 8.1 (s, 1H, N=C–H); 11.7 (s, 1H, N–H). Melting point:
255–258 °C. Yield: 60%.
Compound (16): IR (KBr, cm–1) 3500–2800 (ν, NH), 1650
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 9.2 (s, 1H, H3);
8.9 (s, 1H, H5); 6.9; 6.91 (d, 2H, H2´, H6´); 8.0; 8.1 (d, 2H,
H3´; H5´); 7.4; 7.5 (d, 2H, H2´´, H6´´); 7.6; 7.7 (d, 2H, H3´´,
H5´´); 8.5 (s, 1H, N=C–H); 11.7 (s, 1H, N–H). Melting point:
200–202 °C. Yield: 61%.
Compound (26): IR (KBr, cm–1) 3500–2600 (ν, NH), 1650
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 3.4 (s, 3H,
CH3); 9.2 (s, 1H, H3); 8.4 (s, 1H, H5); 7.4; 7.5 (d, 2H, H2´,
H6´); 7.8; 7.9 (d, 2H, H3´; H5´); 7.7; 7.8 (d, 2H, H2´´; H6´´);
Compound (17): IR (KBr, cm–1) 3500–280 (ν, NH), 1640
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 3.0 (s, 6H,

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A.M.R. Bernardino et al. / European Journal of Medicinal Chemistry 41 (2006) 80–87
7.9; 8.0 (d, 2H, H3´´, H5´´); 8.2 (s, 1H, N=C–H); 11.7 (s, 1H,
N–H). Melting point: 258–260 °C. Yield: 62%.
tutional Committee of the Center for Biological Evaluation and
Care of Research Animals (CEUA-Fiocruz).
Compound (27): IR (KBr, cm–1) 3300–2700 (ν, NH), 1660
(ν, C=O), 1550 and 1320 (ν, NO2). 1H-NMR (300 MHz;
DMSO-d6, δ): 9.3 (s, 1H, H3); 8.7 (s, 1H, H5); 7.9; 8.0 (d,
2H, H2´, H6´); 8.5; 8.6 (d, 2H, H3´, H5´); 8.0; 8.1 (d, 2H,
H2´´, H6´´); 8.2; 8.24 (d, 2H, H3´´; H5´´); 8.6 (s, 1H, N=C–
H); 12.1 (s, 1H, N–H). Melting point: 273–275 °C. Yield:
67%.
5.2.3. Drug assay
The drugs tested within
a concentration range of
40–320 µg ml–1 were solubilized in dimethylsulfoxide
(DMSO, Sigma Chemical Co.), with the final concentration
of the solvent in the experiments never exceeding 1.6%, then
added to a 96 wells micro plate and incubated at 26 °C for 24 h
with the parasites in their metacyclic phase in a concentration
of 4 × 106 cells per ml [39].
Compound (28): IR (KBr, cm–1) 3500–2600 (ν, NH), 1637
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 3.4 (s, 3H,
CH3); 9.3 (s, 1H, H3); 8.5 (s, 1H, H5); 7.4; 7.5 (d, 2H, H2´;
H6´); 7.8; 7.9 (d, 2H, H3´; H5´); 7.9; 8.0 (d, 2H, H2´´; H6´´);
8.1; 8.11 (d, 2H, H3´´; H5´´); 8.3 (s, 1H, N=C–H); 11.8 (s, 1H,
N–H). Melting point: 228–231 °C. Yield: 68%.
The procedure used to observe the drug effects was a count-
ing of the remaining parasites in Neubauer’s chamber. The per-
centage of inhibition was estimated compared with the control
and the results were expressed as EC50/24h, i.e. the concentra-
tion of a compound that caused a 50% reduction in survival/
viability in comparison to identical cultures without this com-
pound. All tests were carried out in triplicate and ketoconazole,
allopurinol or pentamidine was used as the reference drugs.
Compound (29): IR (KBr, cm–1) 3500–2600 (ν, NH), 1600
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 3.5 (s, 6H,
N(CH3)2); 9.2 (s, 1H, H3); 8.5 (s, 1H, H5); 6.9; 7.0 (d, 2H,
H2´, H6´); 7.8; 7.9 (d, 2H, H3´; H5´); 7.6; 7.7 (d, 2H, H2´´,
H6´´); 8.0; 8.1 (d, 2H, H3´´; H5´´); 8.4 (s, 1H, N=C–H); 11.5
(s, 1H, N–H). Melting point: 245–246 °C. Yield: 82%.
Compound (30): IR (KBr, cm–1) 3500–2800 (ν, NH), 1639
(ν, C=O). 1H-NMR (300 MHz; DMSO-d6, δ): 1.4; 4.2 (m, 5H,
OCH2CH3); 9.1 (s, 1H, H3); 8.4 (s, 1H, H5); 7.1; 7.13 (d, 2H,
H2´; H6´); 7.7; 7.8 (d, 2H, H3´; H5´); 7.8; 7.9 (d, 2H, H2´´,
H6´´); 7.9; 8.0 (d, 2H, H3´´; H5´´); 8.3 (s, 1H, N=C–H); 11.6
(s, 1H, N–H). Melting point: 228–233 °C. Yield: 90%.
Melting points were obtained with a Fischer apparatus and
5.2.4. Cytotoxicity assays
The cytotoxicity effect of the main compounds: 27 and 15
expressed as cell viability was assayed on the mice’s peritoneal
macrophages. The cells were isolated from peritoneal cavity of
Balb/c mice with cold RPMI 1640 medium, supplemented with
1 mmol l–1 L-glutamine, 1 mol l–1 HEPES, penicillin G
(105 IU l–1), streptomycin sulfate (0.10 g l–1). The 2 × 105 cells
per well were cultivated on microplate and incubated at 37 °C
in a humidified 5% CO2 atmosphere. After 2 h of incubation
no adherent cells were then removed, and the adhered macro-
phages were washed twice with RPMI. Compounds were
added to the cell culture at the respective EC50/24h for
L. amazonensis and cells incubated at 24 h. Then, the 3-[4,5-
dymethilthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide, MTT
(Sigma) was added and the results could be read on an ELISA
reader [40].
1
were not corrected. H- and 13C-NMR spectra were recorded
on a Varian Unity plus 300 MHz, employing tetramethysilane
as the internal reference at room temperature. The chemical
shifts (δ) are reported in ppm and the coupling constant (J) in
hertz. Infrared (IR) spectra were recorded on a Perkin–Elmer
model 1420 FT-IR Spectrophotometer in KBr pellets. Microa-
nalyses were performed on a Perkin–Elmer Model 2400 instru-
ment and all values were within 0.4% of the calculated com-
positions.
Acknowledgements
5.2. Pharmacology
This work is supported by grants from CNPq, CAPES, FIO-
CRUZ, and fellowships from CAPES and PIBIC/UFF, Brazil.
5.2.1. Leishmanicidal assays
Parasites: L. amazonensis (MHOM/BR/77LTB0016 strain),
L. chagasi (MCAN/BR/97/P142 strain) and L. braziliensis
(MCAN/BR/98/R619 strain) were maintained by animal pas-
sage and cryopreserved in liquid nitrogen. Promastigotes were
cultured in Schneider’s Drosophila medium pH 7.2 (Sigma,
MO, USA), supplemented with 10–20% (v/v) of heat-inacti-
vated foetal calf serum (FCS). The characterization of the
strains was performed by molecular techniques such as isoen-
zyme electrophoresis [38].
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