Synthesis and tripanocidal activity of ferrocenyl and benzyl diamines against Trypanosoma brucei and Trypanosoma cruzi

Article abstract of DOI:10.1016/j.bmcl.2014.02.046

Trypanosoma brucei and Trypanosoma cruzi are the etiologic agents of sleeping sickness and Chagas disease, respectively, two of the 17 preventable tropical infectious diseases (NTD) which have been neglected by governments and organizations working in the health sector, as well as pharmaceutical industries. High toxicity and resistance are problems of the conventional drugs employed against trypanosomiasis, hence the need for the development of new drugs with trypanocidal activity. In this work we have evaluated the trypanocidal activity of a series of N1,N2-dibenzylethane-1,2-diamine hydrochlorides (benzyl diamines) and N1-benzyl,N2-methyferrocenylethane-1,2- diamine hydrochlorides (ferrocenyl diamines) against T. brucei and T. cruzi parasite strains. We show that incorporation of the ferrocenyl group into the benzyl diamines increases the trypanocidal activity. The molecules exhibit potential trypanocidal activity in vitro against all parasite strains. Cytotoxicity assay was also carried out to evaluate the toxicity in HepG2 cells.

Full text of DOI:10.1016/j.bmcl.2014.02.046

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1707–1710
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and tripanocidal activity of ferrocenyl and benzyl diamines
against Trypanosoma brucei and Trypanosoma cruzi
c
a
Angela Maria Arenas Velásquez a,b, , Acácio Ivo Francisco , Andréa Akiko Nakaima Kohatsu ,
Flavia Alves de Jesus Silva a, Danilo Fernando Rodrigues a,b, Rafaela Gomes da Silva Teixeira c,
Bruna Galdorfini Chiari d, Maria Gabriela José de Almeida d, Vera Lucia Borges Isaac d,
⇑
Maria D. Vargas c, Regina Maria Barretto Cicarelli a,
⇑
a Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista ‘Júlio de Mesquita Filho’, Rodovia Araraquara-Jaú Km 01-Campus,
Araraquara 14.801-902, Brazil
b Instituto de Química, Universidade Estadual Paulista ‘Júlio de Mesquita Filho’, Rua Prof. Francisco Degni, 55, Araraquara 14.800-900, Brazil
c Instituto de Química, Universidade Federal Fluminense, Campus do Valonguinho—Centro, Niterói 24.020-150, Brazil
d Departamento de Fármacos e Medicamentos, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista ‘Júlio de Mesquita Filho’, Rodovia Araraquara-Jaú Km 01-Campus,
Araraquara 14.801-902, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Trypanosoma brucei and Trypanosoma cruzi are the etiologic agents of sleeping sickness and Chagas dis-
ease, respectively, two of the 17 preventable tropical infectious diseases (NTD) which have been
neglected by governments and organizations working in the health sector, as well as pharmaceutical
industries. High toxicity and resistance are problems of the conventional drugs employed against try-
panosomiasis, hence the need for the development of new drugs with trypanocidal activity. In this work
we have evaluated the trypanocidal activity of a series of N1,N2-dibenzylethane-1,2-diamine hydrochlo-
rides (benzyl diamines) and N1-benzyl,N2-methyferrocenylethane-1,2-diamine hydrochlorides (ferroce-
nyl diamines) against T. brucei and T. cruzi parasite strains. We show that incorporation of the ferrocenyl
group into the benzyl diamines increases the trypanocidal activity. The molecules exhibit potential try-
panocidal activity in vitro against all parasite strains. Cytotoxicity assay was also carried out to evaluate
the toxicity in HepG2 cells.
Received 12 December 2013
Revised 13 February 2014
Accepted 18 February 2014
Available online 28 February 2014
Keywords:
Sleeping sickness
Chagas disease
Benzyl diamines
Ferrocenyl diamines
Ó 2014 Elsevier Ltd. All rights reserved.
The Trypanosomatidae family, flagellated parasitic protozoa, is
responsible for important infectious diseases in humans: sleeping
sickness, Chagas diseases and leishmaniasis. Cost, toxicity and
resistance problems of conventional drugs point to an urgent need
to identify and develop new therapeutic alternatives. Chagas dis-
ease is a zoonosis that spans the Great Lakes in North America to
the southeast of Patagonia1,2 and occurs widely in Latin America.1
The infection can be readily transmitted by blood transfusion and
migration from endemic areas. The causative agent is Trypanosoma
cruzi.3 According to the World Health Organization, WHO, an esti-
mated 10 million people are infected worldwide, mostly in Latin
America and more than 25 million people are at risk of the disease.
It is estimated that in 2008 Chagas disease killed over 10,000
people.4 The only prescription drugs in medical practice to treat
Chagas disease are nifurtimox and benznidazole.5,6 These medi-
cines are curative only in acute or early chronic infections, and in
congenital cases.7 Benznidazole is more effective than nifurtimox
in suppressing parasitemia during the chronic phase8, but neither
is effective in curing the disease at this stage.9 Both drugs have
significant side effects, probably as a consequence of reducing
oxidative damage in tissues or hosts.10
Sleeping sickness is caused by T.b. gambiense and
T.b. rhodesiense, subspecies related to T.b. brucei, which is found
in domestic animals and related to the disease called nagana.11
WHO reports that T.b. gambiense accounts for 95% of reported cases
of sleeping sickness.12 The estimated number of actual cases is cur-
rently 30.000. The symptoms of sleeping sickness can be varied,
but two clinical phases can be recognized during infection: an early
hemolymphatic stage and a late meningoencephalitic stage. Treat-
ment of the first phase of sleeping sickness relies on suramin and
pentamidine and in the late phase, melarsoprol is active against
T.b. gambiense and T.b. rhodesiense, whereas eflornithine is useful
⇑
Corresponding authors. Tel.: +55 16 33016946; fax: +55 16 3301 6940.
E-mail addresses: avellangel@gmail.com (A. M. A. Velásquez), reginacicarelli@
only against T.b. gambiense. Melarsoprol is
a highly toxic
arsenic-based drug, which can lead to patient death. A new
gmail.com (R. M. B. Cicarelli).
http://dx.doi.org/10.1016/j.bmcl.2014.02.046
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

1708
A. M. A. Velásquez et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1707–1710
combination therapy with the oral drugs nifurtimox and eflorni-
thine is more effective when compared to nifurtimox and eflorni-
thine monotherapies, but causes several side effects.13 In
addition, eflornithine reduces the number of leukocytes, erythro-
cytes and platelets.14 These drugs are mostly antiquated, scarce,
highly toxic and subject of parasite resistance.13–17 Clearly there
is an urgent need to develop new drugs to treat these diseases.
Polyamines are present in all parasitic protozoa, and in recent
years, these compounds and their associated enzymes have been
investigated as drug targets in search of novel parasitic therapy
options.18 A large number of N-alkylated polyamine analogs have
been described which selectively interfere with T. cruzi polyamine
metabolism.19 Furthermore, in a series of recent reports, Yamanaka
et al.20 (2013) Provided evidence that simpler lipophilic alkyl
diamines can be leading molecules for the development of new
antiparasitic drugs. They described the in vitro activity of several
1,2-ethane, 1,4-butane and 1,6-hexanediamine hydrochlorides
against T. cruzi, L. braziliensis and L. chagasi and showed that in the
case of T. cruzi the mitochondria is a target for the active
compounds.21 Differently, diaryl diamines [2-(3-(substituted-ben-
zylamino)propylamino)-quinolin-4(1H)-ones] were shown to
inhibit T. brucei and T. cruzi growth by selectively inhibiting their
methionyl-tRNA synthetase.21
Incorporation of transition metal fragments to the structure of a
drug often enhances its activity.22–24 Of special interest in this
work is the ferrocenyl (Fc) group25, which is derived from ferro-
cene [bis(g
5-cyclopentadienyl)iron(II)], a non-toxic and stable
organometallic compound.26 Incorporation of the Fc group into
standard drugs offers new possibilities in therapeutic applications
and reversal of drug resistances.27 For example introduction of Fc
into the lateral chain of chloroquine yielded ferroquine28, with
excellent in vitro and in vivo activity against malaria parasites—
Plasmodium spp. and particularly good activity against chloro-
quine-resistant ones.29,30 Furthermore substitution of a phenyl
group in tamoxifen with Fc resulted in improved cytotoxic activity
in breast cancer27,31,32, and modification of ethambutol with Fc
groups led to improved antimicobacterial activity (Mycobacterium
tuberculosis).28
Table 1
Antiparasitic activities and safety index of benzyl diamines and ferrocenyl diamines
a
a
a
b
a
b
Compound
R
HepG2 cells
T. brucei 427
S.I.b
T. brucei 29–13
S.I.
T. cruzi Y
S.I.
T. brucei 427
T. brucei 29–13
T. cruzi Y
Ferrocenyl diamines
2
3
4
5
6
7
8
9
C6H5
30.99
18.60
32.26
34.49
9.20
0.51
0.36
1.78
1.27
0.35
7.46
0.45
0.41
4.04
0.82
7.69
5.33
4.81
10
7.67
6.03
4.23
2.21
9.58
5.59
3.74
3.98
3.37
5.14
4.40
60.76
51.67
18.12
27.16
26.29
11.45
48.76
4-OMeC6H4
2-OMeC6H4
2-OHC6H4
Fc
22.68
4.20
14.60
3.60
6.47
1.91
8.55
4.79
1.65
Py(3Cl-)
85.45
21.94
26.20
22.68
22.85
5.51
2,4-ClC6H3
2-BrC6H4
4-NO2C6H4
4.58
1.13
7.77
63.90
9.95
1.95
23.19
6.37
3.13
10
11
2.28
10.42
3.56
6.48
6.07
12.84
3.74
1.58
4(R)-7-chloroquinoline 20.31
Benzyl diamines
2a
3a
5a
C6H5
223.74
990.62
148.21
11.08
8.25
4.61
9.99
8.63
5.16
159.61
10.9
1.40
20.19
22.40
4-OMeC6H4
2-OMeC6H4
120.08
114.79
90.88
1.36
108.9
32.15
6.26
28.72
6.16
7a
8a
py
86.91
280.57
13.88
12.35
14.11
16.78
258.97
51.44
0.34
5.45
2,4-ClC6H3
22.72
16.13
16.72
30.71
10a
4-NO2C6H4
590.18
36.58
19.22
122.01
4.84
Pentamidine
Benznidazole
—
—
159.71
2126.16
6.43
207.57
24.84
10.24
6.43
149.58
24.84
14.21
4.58
34.00
34.87
62.53
a
IC50 (lM): half maximal inhibitory concentration.
S.I.: safety index. OBS: the underlined S.I. values indicate promising compounds, that is those with low mammalian toxicity and high toxicity to parasites.
b
2Cl-
H2
H2N
1. MeOH, Ar
+
N
H
N
R
+
R-CHO
+
N
H2
Fe
2. NaBH4, H2O
3. HCl 6 mol/L
1
Fe
2-11
Py(3Cl-)
7
8
9
R = C6H5
2
3
4
5
6
2,4-ClC6H3
2-BrC6H4
4-OMeC6H4
2-OMeC6H4
2-OHC6H4
Fc
4-NO2C6H4
10
4(R)-7-chloroquinoline 11
2Cl-
R = C6H5
2a
3a
H2
1. MeOH, Ar
4-OMeC6H4
+
H2N
N
R
+
R-CHO
2-OHC6H4
py
NH2
R
5a
+
N
H
2. NaBH4, H2O
3. HCl 6 mol/L
7a
8a
10a
2,4-ClC6H3
4-NO2C6H4
Figure 1. Synthesis of benzyl and ferrocenyl diamines.

A. M. A. Velásquez et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1707–1710
1709
Herein we describe the synthesis of several N1,N2-dibenzyle-
thane-1,2-diamine hydrochlorides (benzyl diamines) and
N1-benzyl,N2-methyferrocenylethane-1,2-diamine hydrochlorides
(ferrocenyl diamines) and the evaluation of their trypanocidal activ-
ity (Table 1). Compounds 2–10 (Fig. 1) have been obtained as stable
solids (yields ranging from 66% to 80%); their structures are fully
supported by 1H and 13C NMR spectroscopy and elemental analysis.
Compounds 2a–10a (Fig. 1), described previously, have been
synthesized according to the literature.33–35 Synthesis and full
spectroscopic data are presented in the Supplementary information.
The compounds were tested against T. brucei and T. cruzi strains
using MTT colorimetric assay to evaluate the trypanocidal activ-
ity.36,37 Pentamidine and benznidazole were included in the assays
as positive controls for T. brucei and T. cruzi, respectively. MTT as-
says were carried out after 24 h (T. brucei) and 72 h (T. cruzi) incu-
bation. The methodology is detailed in the Supplementary
information. A cytotoxicity assay was also carried out to evaluate
the toxicity of different concentrations of the compounds in HepG2
cells. This hepatoma cell line is used as a model to simulate the he-
patic function of the human organism in vitro, as it exhibits similar
metabolic functions to the human liver.38,39
survival and growth, trypanosomatids possess a defense mecha-
nism against oxidative stress. Superoxide dismutase (SOD), old yel-
low enzyme (OYE) and peroxiredoxine (Prx) are involved in various
mitochondrial activities in certain life cycle stages of trypanoso-
matids.16 The ferrocenyl group could therefore interfere with the
defense mechanisms of the parasites.
According to the IC50 values in HepG2 cells (Table 1) the ferr-
ocenyl amines showed lower toxic activity on mammalian cells,
as compared with their toxicity against parasites, which is very
interesting for new drugs evaluation.
These ferrocenyl diamines could therefore be potential trypan-
ocidal drug candidates. Studies on the mechanism of action of the
ferrocenyl diamines against both T. cruzi and T. brucei are under-
way, and so are the tests with other T. cruzi strains, due to genetic
variability, and with the T. cruzi amastigote form and the blood-
stream forms of T. brucei.
Acknowledgments
The authors thank FAPESP (Fundação de Amparo à Pesquisa do
Estado de São Paulo), CAPES (Coordenação de Aperfeiçoamento de
The benzyl diamines showed higher trypanocidal effect in T.
brucei strains than in T. cruzi Y (see Table 1), in accord with a pre-
vious report on T. cruzi strains being less responsive than T. brucei
to this type of compounds.20 Especially for T. cruzi Y the nature of
the substituent strongly influenced the IC50 values. Compound 3a
bearing a 4-OMe group on the phenyl rings is by far the most active
Pessoal de Nível Superior), FUNDUNESP (Fundação para
o
Desenvolvimento da Unesp) and CNPq (Conselho Nacional de
Desenvolvimento Científico e Tecnológico) for research funding
and financial support.
Supplementary data
compound against T. cruzi Y (IC50 = 10.9 lM) and also the least
toxic one of the series, with an S.I. value of about 90, higher than
both pentamidine and benznidazole. It was also the most active
compound of the series against both strains of T. brucei, with IC50
values comparable to that of pentamidine (8.25 and 8.63 vs
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.02.
046.
6.43 lM) and lower mammalian toxicity.
References and notes
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7, 8 and 10, respectively (Fig. 1), IC50 values decreased considerably
in T. cruzi Y (Table 1), demonstrating that the presence of the Fc
group leads to increased trypanocidal activity to T. cruzi. In fact
ferrocenyl diamines exhibited higher tripanocidal activity than
the corresponding benzyl diamines on both parasites (T. brucei 427,
T. brucei 29–13 and T. cruzi Y) which might be associated to in-
creased lipophilicity and/or differences in electronic (redox proper-
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l
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