Synthesis and evaluation of the antiparasitic activity of bis-(arylmethylidene) cycloalkanones

Article abstract of DOI:10.1016/j.ejmech.2013.11.011

A series of bis-(arylmethylidene)-cycloalkanones was synthesized by cross-aldol condensation. The activity of the compounds was evaluated against amastigotes forms of Trypanosoma cruzi and promastigotes forms of Leishmania amazonensis. The cytotoxicity of the active compounds on uninfected fibroblasts or macrophages was established in vitro to evaluate the selectivity of their antiparasitic effects. Six compounds displayed trypanocidal activity against amastigotes intracellular forms of T. cruzi with IC₅₀ values ranging from 7.0 to 249 μM. Besides these six compounds, eight other molecules exhibited significant leishmanicidal activity (IC₅₀ values ranging from 0.6 to 110.4 μM). Two compounds can be considered as promising antiparasitic lead molecules because they showed IC₅₀ values in the low-micromolar range (≤1.2 μM) with an adequate SI (≥19.9). To understand the mechanism of action of these compounds, two possible molecular targets were investigated: trypanothione reductase (TR) and cruzain.

Full text of DOI:10.1016/j.ejmech.2013.11.011

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European Journal of Medicinal Chemistry 71 (2014) 282e289
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and evaluation of the antiparasitic activity
of bis-(arylmethylidene) cycloalkanones
Saulo F.P. Braga a, Érika V.P. Alves a, Rafaela S. Ferreira b, Jordana R.B. Fradico b,
Paula S. Lage e, Mariana C. Duarte e, Tatiana G. Ribeiro a, Policarpo A.S. Júnior c,
Alvaro J. Romanha c, Maiko L. Tonini d, Mário Steindel d, Eduardo F. Coelho e,
,
Renata B. de Oliveira a
*
a Departamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Avenida Antônio Carlos 6627,
Belo Horizonte 31.270-901, MG, Brazil
b Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos 6627,
Belo Horizonte 31.270-901, MG, Brazil
c Centro de Pesquisas René Rachou, FIOCRUZ, Avenida Augusto de Lima, 1.715, Barro Preto, Belo Horizonte 30.190-002, MG, Brazil
d Departamento de Microbiologia, Imunologia e Parasitologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Setor F, Bloco A,
Florianópolis 88.040-970, SC, Brazil
e Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais,
Belo Horizonte, Minas Gerais, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2013
Received in revised form
2 November 2013
Accepted 7 November 2013
Available online 16 November 2013
A series of bis-(arylmethylidene)-cycloalkanones was synthesized by cross-aldol condensation. The
activity of the compounds was evaluated against amastigotes forms of Trypanosoma cruzi and promasti-
gotes forms of Leishmania amazonensis. The cytotoxicity of the active compounds on uninfected fibroblasts
or macrophages was established in vitro to evaluate the selectivity of their antiparasitic effects. Six com-
pounds displayed trypanocidal activity against amastigotes intracellular forms of T. cruzi with IC50 values
ranging from 7.0 to 249
leishmanicidal activity (IC50 values ranging from 0.6 to 110.4
promising antiparasitic lead molecules because they showed IC50 values in the low-micromolar range
(ꢀ1.2 M) with an adequate SI (ꢁ19.9). To understand the mechanism of action of these compounds, two
possible molecular targets were investigated: trypanothione reductase (TR) and cruzain.
Ó 2013 Elsevier Masson SAS. All rights reserved.
m
M. Besides these six compounds, eight other molecules exhibited significant
m
M). Two compounds can be considered as
Keywords:
Bis-(arylmethylidene)-cycloalkanones
Antileishmanial activity
Leishmania amazonensis
Trypanocidal
m
Trypanosoma cruzi
1. Introduction
addition, drug resistant parasite strains have become more preva-
lent [2e6]. The search for more effective and less toxic drugs to
treat both diseases have became mandatory.
Bis-(arylmethylidene)-cycloalkanones have been found to
display a range of biological effects including antineoplastic [7],
antioxidant [8e10], antitubercular [11] and antifertility activities
[12]. It has been proposed that the activity of this class of com-
pounds is related to their ability to act as Michael acceptors with
nucleophilic biological macromolecules, especially nucleic acids
and enzymes [13].
To the best of our knowledge, there is no report on the appli-
cation of this class of compounds for the treatment of parasitic
infections. Therefore, we decided to evaluate the in vitro anti-
leishmanial and antitrypanosomal activities of a series of bis-
(arylmethylidene)cycloalkanones. The cytotoxicity of compounds
to non-infected cells was also evaluated.
Chagas’ disease and Leishmaniasis are endemic protozoan dis-
eases recognized as epidemiological priorities by the World Health
Organization (WHO). Chagas’ disease is found mainly in Latin
America and it is estimated to affect 12 to 14 million people.
Leishmaniasis affects 12 million people in 88 countries, 72 of which
are developing countries [1]. Chagas’ disease and Leishmaniasis are
caused by the trypanosomatid parasites Trypanosoma cruzi and
Leishmania sp, respectively. There are limited drugs available for the
treatment of both diseases, and the currently used chemothera-
peutic agents are still inadequate due to their extreme toxicity. In
* Corresponding author. Tel.: þ55 31 34096395; fax: þ55 31 34096935.
E-mail address: renatabo@farmacia.ufmg.br (R.B. de Oliveira).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.11.011

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To understand the mechanism of action of these compounds,
two possible molecular targets were investigated: trypanothione
reductase (TR) and cruzain.
Trypanothione Reductase (TR) [14,15] is a vital enzyme for the
antioxidant defenses in trypanosomatids. TR exerts a key role in
combating oxidative stress, regenerating the main antioxidant
present in these protozoa, trypanothione [16]. TR is involved in the
reduction of trypanothione disulfite T[S]2 to the dithiol T[SH]. This
enzyme also is considered an attractive due to its high conservation
between Leishmania and trypanosomes [17e19], since it is expected
that a TR inhibitor will be potentially active against both Trypano-
soma and Leishmania parasites.
Besides participating in an essential pathway for parasite sur-
vival, the remarkable structural differences with respect to the
human enzyme, Glutathione Reductase (GR), makes TR as potential
target for the development of selective inhibitors. The main dif-
ferences between TR and GR are related to the size, charge and
distribution of hydrophilic/hydrophobic regions in the active site of
the enzymes. The active site of TR is larger than that of GR and is
negatively charged with surrounding hydrophobic residues to
accommodate the positively charged spermidine portion of T[S]2
[20]. In contrast, the GR active site has three positively charged
arginine residues (Arg37, Arg38 and Arg347), required for interac-
tion with its substrate, GSSG, which has a formal charge of ꢂ2 at
physiological pH [21].
We hypothesized that bis-(arylmethylidene)-cycloalkanones
could interact strongly with the active site of TR due to the presence
of bulky and hydrophobic substituents. As human GR has a
hydrophilic and smaller binding site, low affinity would be
expected between these compounds and GR. To confirm this
hypothesis, enzyme inhibition assays were carried out against TR
and GR.
Cruzain, the major cysteine protease from T. cruzi, which is
among the most studied therapeutic targets for Chagas’ disease
[22e24], was also evaluated as a possible mechanism of trypano-
cidal activity. Cysteine proteases play a key role at various stages in
the life cycle of the parasites and are involved in host protein
degradation by the parasite, facilitating the evasion of host defense
mechanism, invasion cellular, replication and differentiation of the
parasite [23,25]. These enzymes contain a catalytic cysteine residue
in its active site responsible for the hydrolysis of peptide bonds
[26,27].
Scheme 1. General synthetic route to bis-(arylmethylidene)cycloalkanones.
performed under microwave irradiation in solvent-free condition
[33] and the product was obtained in low yield (17%), even after
optimization attempts (Scheme 2).
The first attempt to obtain compounds 17 and 18 (Scheme 3)
consisted of a five-step procedure. Initially, reaction of p-tolunitrile
with N-bromosuccinimide (NBS) in the presence of radical initiator
(PhCOO)2 provided the bromo derivative 19 in 58% yield [34].
Reduction of the nitrile group of 19 with diisobutylaluminum
hydride (DIBALH) followed by displacement of the derived benzylic
bromide 20 with sodium azide afforded 4-(azidomethyl)benzal-
dehyde 21 in 97% yield [34,35]. Cross-aldol condensation of 21 with
cyclopentanone led to diazide derivative 22 in quantitative yield.
However, the attempted copper-catalyzed 1,3-dipolar cycloaddi-
tions between azide derivative 22 and 3-butyn-1-ol or phenyl-
acetylene were unsuccessful, probably because of the low solubility
of the 21 in the reaction medium. To overcome this problem, we
decided to repeat the reaction replacing azide 22 by azide 21,
considerably more soluble in organic solvents. Thus, in the pres-
ence of the sodium ascorbate, CuSO4$5H2O and terminal alkyne,
the azide 21 was completely consumed, and the 1,4-disubstituted
triazole products 23 and 24 were formed in 61 and 86% yield,
respectively. Finally, the last step consisted in the aldol condensa-
tion of the aldehydes 23 and 24 with cyclopentanone, resulting in
the formation of the desired products 17 and 18 (Scheme 4).
There are several classes of cruzain inhibitors [24,28,29], but
typically cysteine protease inhibitors possess an electrophilic a,b-
unsaturated moiety, such as, vinyl ketone, vinyl sulfones, vinyl
amide, vinyl ester and vinyl nitrile, acting as a Michael acceptor for
the nucleophilic sulfhydryl groups of catalytic cysteine residue
[30,31]. Based on the potential of the bis-(arylmethylidene)-
cycloalkanones to act as Michael acceptor, the evaluation of the
ability of these compounds to inhibit cruzain is of interest.
3. Biological assays
The compounds were evaluated in vitro for their ability to inhibit
recombinant T. cruzi TR using the colorimetric assay described by
Hamilton et al. (2003) [36]. Inhibition of human GR activity was
carried out in parallel according to Carlberg and Mannervik (1985)
[37]. Cruzain inhibition was measured based on a fluorimetric
2. Chemistry
The bis-(arylmethylidene)cycloalkanones 2e16 were synthe-
sized by cross-aldol condensation of cycloalkanones (cyclo-
pentanone or cyclohexanone) with a variety of commercially
available aldehydes as outlined in Scheme 1. The main advantages
of this synthesis are that the compounds can be obtained by a
simple and rapid one-step procedure in good yield from readily
available starting materials. The only exception was the synthesis of
the dinitro derivative 8, which cannot be obtained using the usual
method under basic conditions [32]. The synthesis of 8 was
repeated several times varying the reaction temperature and time
and the ratio of the reagents, but in all cases, only the unreacted
starting materials were recovered. Then, the reaction was
Scheme 2. Synthetic route for the preparation of 8.

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S.F.P. Braga et al. / European Journal of Medicinal Chemistry 71 (2014) 282e289
Scheme 3. Synthetic route for the preparation of compounds 17 and 18. Reagents and conditions: (a) benzoyl peroxide, NBS, benzene, reflux, 4 h. (b) (i) DIBALH, toluene, 0 ꢃC, N2
atm, 2 h. (ii) HCl 10%, CHCl3, rt, 1 h. (c) NaN3, THF/water, reflux, 24 h. (d) NaOH 0.2 M, EtOH 95%, rt, 2 h. (e) Terminal alkyne, CuSO4$5H2O, sodium ascorbate, CH2Cl2/water, rt, 24 h.
assay, as previously described [38]. The compounds were also
assayed in vitro against promastigotes forms of Leishmania (L.)
amazonensis [39] and amastigotes forms of T. cruzi [40,41]. Results
are expressed as percentage of inhibition of parasite growth after
treatment with each compound. Cell viability test was performed to
evaluate the cytotoxicity of the compounds against normal cell, the
murine fibroblast L-929 and murine peritoneal macrophages
[39,42]. The concentrations that caused 50% inhibition of the
enzymes activity and parasite growth (IC50) were evaluated for the
more potent compounds (those presenting more than 70% inhibi-
tion in the initial screening at 100 mM).
4. Results and discussion
Initially, the in vitro antiprotozoal activities of the synthesized
compounds were assayed against T. cruzi and L. amazonensis. IC50
values are summarized in Table 1.
Scheme 4. Alternative route for the preparation of 17 and 18. Reagents and conditions:
(a) terminal alkyne, CuSO4$5H2O, sodium ascorbate, CH2Cl2/water, r.t., 24 h. (b) NaOH
0.2 M, EtOH 95%, r.t., 4e20 h.
Six compounds (2, 5, 11, 13, 14 and 16) displayed in vitro trypa-
nocidal activity with IC50 values ranging from 7.0 to 249
mM
(Table 1). There was a good correlation between leishmanicidal and
trypanocidal activity. All compounds active against intracellular
Table 1
Biological data for bis-(arylmethylidene)cycloalkanones.
Compound
IC50 T. cruzi (
m
M)
CC50 fibroblast (
m
M)
SIa
IC50 L. amazonensis (
mM)
CC50 macrophage (
mM)
SIb
1.6
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
73 ꢄ 18
>250
>250
375 ꢄ 177
5.1
1.1
46.6 ꢄ 4.3
8.4 ꢄ 0.3
52.6 ꢄ 0.7
110.4 ꢄ 1.6
44.1 ꢄ 5.5
18.6 ꢄ 1.1
NDc
76.2 ꢄ 6.9
50.6 ꢄ 8.0
39.4 ꢄ 4.2
19.7 ꢄ 3.3
17.6 ꢄ 1.6
20.8 ꢄ 3.8
e
e
e
e
6
e
0.7
0.2
0.4
1.1
230.5 ꢄ 27.6
250 ꢄ 0
>250
e
e
e
e
e
e
>250
>250
>250
>250
e
e
e
NDc
e
e
1.2 ꢄ 0.0
4.9 ꢄ 0.9
0.6 ꢄ 0.1
0.9 ꢄ 0.2
8.4 ꢄ 2.7
14.7 ꢄ 0.9
5.3 ꢄ 1.8
50.9 ꢄ 13.3
NDc
23.9 ꢄ 9.6
3.7 ꢄ 0.9
0.6 ꢄ 0.5
21.4 ꢄ 5.3
78.1 ꢄ 9.9
55.8 ꢄ 5.0
83.2 ꢄ 3.3
60.4 ꢄ 3.8
e
19.9
0.8
1
23.8
9.3
3.8
15.7
1.2
e
7.0 ꢄ 2.1
25.3 ꢄ 12.2
3.6
>250
e
e
e
31.3 ꢄ 22.2
68.7 ꢄ 8.8
>250
500 ꢄ 0
16
2.7
187.5 ꢄ 88.4
e
249 ꢄ 104
375 ꢄ 177
1.5
>250
e
e
e
e
e
18
22
>250
>250
e
e
NDc
e
e
Amphotericin Bd
Benznidazoled
e
e
0.08 ꢄ 0.03
0.7 ꢄ 0.21
e
8.8
e
3.8 ꢄ 0.8
>1000
>262
e
a
Selectivity index (SI) is the ratio of murine fibroblast viability (CC50) to the IC50 on T. cruzi.
SI is the ratio of murine macrophage viability (CC50) to the IC50 on L. amazonensis.
ND ¼ not determined due to low solubility.
b
c
d
Positive control.

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Table 2
The cytotoxicity of the active compounds on uninfected fibro-
blasts or macrophages was established in vitro to evaluate the
selectivity of their antiparasitic effects. The selectivity index (SI)
was calculated as the ratio of the CC50 value determined on the
uninfected cells (cytotoxicity) to IC50 determined on parasite cells.
Among the active compounds, compound 13 was identified as the
promising compound with high SI (ꢁ16) and remarkable activity
against both parasites (Table 1).
Inhibitory activities of bis-(arylmethylidene)cycloalkanones against T. cruzi TR and
human GR.
Compound
n
R
TR % inhibition IC50 TR (
m
M) IC50 GR
M)
In an attempt to investigate a possible mechanism of action, all
the compounds were first screened at 100 mM for TR, GR inhibi-
at 100
m
M
(m
tory activity. The compounds 3 and 10 were able to inhibit more
than 75% TR activity and then their IC50 values were determined
2
1
2
1
2
1
2
1
1
2
1
2
1
2
1
2
0
75.3
0
>100
>100
as 45.8 and 14.7
presence of cyclohexanone ring is essential for activity, since the
corresponding cyclopentyl analogs and were inactive
mM, respectively. These results suggest that the
3
45.8 ꢄ 11.2 >100
2
9
(Table 2). A possible explanation for the difference in the activity
between 3e2 and 10e9 analogs may be related to solubility dif-
ferences. The cyclohexanone derivatives are always more soluble
than corresponding cyclopentanone. All compounds exhibited
weak inhibitory activity against GR, which is a good evidence of
the selectivity of the compounds active against TR. The results are
summarized in Table 2. These results are consistent since the
active site of GR is narrow and cannot accommodate bulky hy-
drophobic substrates.
Compounds 3 and 10 exhibit good association between TR
inhibitory activity and antileishmanial potency (extracellular forms
of the parasite promastigotes), while showing almost no activity
against amastigote forms of T. cruzi. The low trypanocidal activity of
these two compounds may be due to their inability to cross the
fibroblast and parasite’s cell membranes and to reach their intra-
cellular target. In an intracellular assay, any active compound must
enter the host cell and the parasite in sufficient concentration to
inhibit TR.
4
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
5
0
6
0
7
0.7
0
8
9
2.2
97.0
18.6
18.5
9
10
11
12
13
14
15
16
14.7 ꢄ 4.2 >100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Compound 10, as well as compound 13, can be considered as a
promising antileishmanial lead candidate because it showed IC50
values in the low-micromolar range (1.2
(19.9).
mM) with an adequate SI
Given that several trypanocidal compounds were not TR
inhibitors, their activity against cruzain was evaluated as an alter-
native mechanism of action. Due to the low solubility of most
compounds in the assay buffer, only eight were evaluated against
17.4
0
this enzyme and none showed significant activity at 100
mM
(Table 3). We speculate that, although these compounds are
potential Michael acceptors, they have low affinity for the active
site of the enzyme. In other words, despite their potential as elec-
trophiles, these compounds probably show low chemical comple-
mentarity to the enzyme’s active site. There results show that the
antiparasitic activity of the molecules described in this study is not
due to cruzain inhibition, and further studies are necessary to
determine their mechanism of action.
0
17
18
1
1
0
0
>100
>100
>100
>100
22
1
2.5
>100
228 ꢄ 11.2
5. Conclusions
Clomipraminea e e
e e
e
e
12.3 ꢄ 2.3
e
A new class of antiparasitic compounds has been discovered in
this study, including molecules active at low micromolar concen-
trations against the parasites T. cruzi and Leishmania (L.) ama-
zonensis. Based on its antiparasitic activity and low toxicity to
mammalian cells, compounds 10 and 13 were identified as the best
candidates for further studies for the development of novel anti-
parasitic drugs. A clear correlation between enzyme inhibition (TR
or cruzain) and the antiparasitic activity was not observed to the
active compounds of this series, except for compound 10, which
probably acts by inhibiting the enzyme TR. Further studies are
needed to establish the mechanism of action of other active
compounds.
Carmustinea
e
8.1 ꢄ 1.9
a
Positive control.
forms of T. cruzi were also active against extracellular promastigote
forms of L. amazonensis. However, all compounds were more potent
against L. amazonensis (IC50 values ranging from 0.6 to 110.4 mM)
than against T. cruzi. Besides these six compounds, eight other
molecules (3, 4, 6, 7, 10, 12, 15 and 17) exhibited significant leish-
manicidal activity, demonstrating that the differences in suscepti-
bility can be associated with the species parasites life stage.