4-Nitroacetophenone-derived thiosemicarbazones and their copper(II) complexes with significant in vitro anti-trypanosomal activity

Article abstract of DOI:10.1016/j.ejmech.2007.06.020

N⁴-Methyl-4-nitroacetophenone thiosemicarbazone (H4NO₂Ac4M, 1), N⁴,N⁴-dimethyl-4-nitroacetophenone thiosemicarbazone (H4NO₂Ac4DM, 2) and N⁴-piperidyl-4-nitroacetophenone thiosemicarbazone (H4NO₂Ac4Pip, 3) and their copper(II) complexes [Cu(4NO₂Ac4M)₂] (4), [Cu(4NO₂Ac4DM)₂] (5) and [Cu(4NO₂Ac4Pip)₂] (6) were tested for their in vitro ability to inhibit the growth of Trypanosoma cruzi epimastigote forms. H4NO₂Ac4DM (2), [Cu(4NO₂Ac4M)₂] (4) and [Cu(4NO₂Ac4DM)₂] (5) proved to be as active as the clinical reference drugs nifurtimox and benznidazol. Taking into consideration the serious side effects and the poor efficacy of the reference drugs, as well as the appearance of resistance during treatment, the studied compounds could constitute a new class of anti-trypanosomal drug candidates.

Full text of DOI:10.1016/j.ejmech.2007.06.020

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 939e948
http://www.elsevier.com/locate/ejmech
Original article
4-Nitroacetophenone-derived thiosemicarbazones and their copper(II)
complexes with significant in vitro anti-trypanosomal activity
a
b
c
d
Anayive Perez-Rebolledo , Letıcia R. Teixeira , Alzir A. Batista , Antonio S. Mangrich ,
´
Gabriela Aguirre , Hugo Cerecetto , Mercedes Gonzalez , Paola Hernandez ,
´
e
e
e
e
´
´
Ana M. Ferreira f, Nivaldo L. Speziali g, Heloisa Beraldo a,
*
a Departamento de Qu´ımica, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil
b Departamento de Qu´ımica, Pontif´ıcia Universidade Cato´lica do Rio de Janeiro, 22453-900 Rio de Janeiro, Brazil
c Departamento de Qu´ımica, Universidade Federal de S~ao Carlos, 1365-905 S~ao Carlos, Brazil
d Departamento de Qu´ımica, Universidade Federal do Parana´, 81.531-990 Curitiba, Brazil
e Departamento de Qu´ımica Orga´nica, Universidad de la Repu´blica, 11400 Montevideo, Uruguay
f Ca´tedra de Inmunolog´ıa, Universidad de la Repu´blica, 11600 Montevideo, Uruguay
g Departamento de F´ısica, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil
Received 26 April 2007; received in revised form 20 June 2007; accepted 22 June 2007
Available online 10 July 2007
Abstract
N4-Methyl-4-nitroacetophenone thiosemicarbazone (H4NO2Ac4M, 1), N4,N4-dimethyl-4-nitroacetophenone thiosemicarbazone (H4NO2
Ac4DM, 2) and N4-piperidyl-4-nitroacetophenone thiosemicarbazone (H4NO2Ac4Pip, 3) and their copper(II) complexes [Cu(4NO2Ac4M)2]
(4), [Cu(4NO2Ac4DM)2] (5) and [Cu(4NO2Ac4Pip)2] (6) were tested for their in vitro ability to inhibit the growth of Trypanosoma cruzi epi-
mastigote forms. H4NO2Ac4DM (2), [Cu(4NO2Ac4M)2] (4) and [Cu(4NO2Ac4DM)2] (5) proved to be as active as the clinical reference drugs
nifurtimox and benznidazol. Taking into consideration the serious side effects and the poor efficacy of the reference drugs, as well as the
appearance of resistance during treatment, the studied compounds could constitute a new class of anti-trypanosomal drug candidates.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: 4-Nitroacetophenone thiosemicarbazones; Copper(II) complexes; Anti-trypanosomal activity
1. Introduction
is responsible for an infected population of nearly 20 millions
and more than 200 millions are at risk [1,2].
Parasitic diseases affect hundreds of millions of people
around the world, mainly in underdeveloped countries. Since
parasitic protozoa are eukaryotic they share many common
features with their mammalian hosts making the development
of effective and selective drugs a hard task. Despite the great
effort that has been made in the discovery of unique targets
that afford selectivity, many of the drugs used today have se-
rious side effects. Diseases caused by Trypanosomatidae,
which share a similar state regarding drug treatment, include
Chagas’ disease, caused by Trypanosoma cruzi. T. cruzi alone
Current treatment of the disease has been based on nifurti-
mox (nfx, 4-[(5-nitrofurfurylidene)-amino]-3-methylthio mor-
pholine-1,1-dioxide), a nitrofuran derivative produced only in
El Salvador by Bayer, and benznidazole (bnz, N-benzyl-2-nitro-
1-imidazoleacetamide), a nitroimidazole derivative produced
by Roche (see Fig. 1a). They were introduced empirically
over three decades ago. Both drugs are active in the acute phase
of the disease but their efficacies are very low in the established
chronic phase. These compounds cause serious undesirable side
effects and show poor clinical efficacy [2].
Both drugs act via reduction of the nitro group. In the case
of nfx, reduction generates an unstable nitro anion radical,
which produces highly toxic reduced oxygen species
* Corresponding author. Tel.: þ55 31 3499 5740; fax: þ55 31 3499 5700.
E-mail address: hberaldo@ufmg.br (H. Beraldo).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.06.020

940
A. Pe´rez-Rebolledo et al. / European Journal of Medicinal Chemistry 43 (2008) 939e948
Fig. 2. Chemical structures of 4-nitroacetophenone thiosemicarbazones, show-
ing the numbering system used: (a) H4NO2Ac4M (1), (b) H4NO2Ac4DM (2)
and (c) H4NO2Ac4Pip (3).
2. Results and discussion
2.1. NMR studies of H4NO2Ac4M (1), H4NO2Ac4DM (2)
and H4NO2Ac4Pip(3)
Fig. 1. (a) Chemical structures of nifurtimox (nfx, 4-[(5-nitrofurfurylidene)-
amino]-3-methylthio morpholine-1,1-dioxide) and benznidazole (bnz, N-benzyl-
2-nitro-1-imidazoleacetamide); (b) proposed mechanism of action of nfx.
1
The H resonances were assigned on the basis of chemical
shifts, multiplicities and coupling constants. The carbon type
(C, CH) was determined by using DEPT 135 experiments.
The NMR spectra of 1 were studied previously [12]. In the
1H NMR (DMSO-d6) spectrum of the thiosemicarbazones the
signal of N(3)eH is found in the d 10.46e9.84 range. This
high frequency signal is due to hydrogen bonding with the sol-
vent [12,13]. In the spectra of 2 and 3 one doublet attributed to
H(3,5) was observed at d 8.29 and d 8.25, respectively, and
a second doublet attributed to H(2,6) was found at d 8.05
and d 8.01, respectively, resulting from the vicinal coupling
of H(2) with H(3) and of H(5) with H(6). In the spectrum of
1 only one sharp singlet had been observed for the aromatic
hydrogens at d 8.20, indicating equivalency of the four hydro-
gens. In the 13C NMR spectrum only small differences were
observed in the chemical shifts of C]N and C]S for the
three thiosemicarbazones (see Section 4.3).
(Fig. 1b), whereas the mechanism of action of bnz involves
covalent modification of macromolecules by nitro reduction
intermediates [3].
Thiosemicarbazones, semicarbazones and their metal com-
plexes represent an interesting class of compounds with a wide
range of pharmacological applications [4]. However, few
works deal with nitro derivatives. Recently, the literature has
described that some semicarbazones and thiosemicarbazones
exhibit trypanocidal activity against parasites in cell culture.
Hence 5-nitro-2-furaldehyde and 5-nitrothiophene-2-carboxal-
dehyde semicarbazone derivatives and 5-nitrofuryl-containing
thiosemicarbazones were found to be active against T. cruzi
[2,5,6].
It has been shown that some metal complexes of anti-
trypanosomal drugs resulted to be more active than the corre-
sponding free ligands [7,8], suggesting that the preparation of
metal complexes with ligands bearing anti-trypanosomal activ-
ity could be a successful strategy for the development of new
anti-Chagas’ disease agents. As a first stage, palladium and
platinum have been selected as central metals because of
the postulated metabolic similarities between tumor cells and
T. cruzi cells [9]. On the other hand some copper compounds
have shown the ability to bind DNA as the main mechanism of
anti-tumoral action [10]. In addition, copper complexes have
shown activity as anti-parasites against Entamoeba histolytica,
and as anti-microbials against Mycobacterium tuberculosis,
acting through different mechanisms [11].
2.2. Microanalyses, molar conductivity and magnetic
susceptibility studies of the copper(II) complexes
Microanalyses and molar conductivity data suggest the for-
mation of [Cu(4NO2Ac4M)2] (4), [Cu(4NO2Ac4DM)2] (5)
and [Cu(4NO2Ac4Pip)2] (6). The effective magnetic moments
in the 1.50e1.80 BM range are compatible with the theoretical
spin only value of 1.73 BM for copper(II) complexes.
2.3. Infrared and electronic spectral studies
To our knowledge the anti-trypanosomal activity of nitroa-
cetophenone thiosemicarbazones has not been investigated. In
most cases N(4)-alkyl or N(4)-aryl thiosemicarbazones exhibit
higher pharmacological activity in comparison to their unsub-
stituted analogues, probably due to increased lipophilicity [4].
Therefore, in the present work N4-methyl- (H4NO2Ac4M, 1),
N4,N4-dimethyl- (H4NO2Ac4DM, 2) and N4-piperidyl-4-nitro-
acetophenone thiosemicarbazone (H4NO2Ac4Pip, 3) (Fig. 2)
and their copper(II) complexes were prepared and tested for
their in vitro ability to inhibit the growth of T. cruzi epimasti-
gote forms.
In the infrared spectra, the n(C]N) stretching vibration of
the thiosemicarbazones at 1590 cmꢀ1 shifts to 1585e1568 cmꢀ1
in the complexes, indicating coordination of the imine nitrogen
[14e16]. The absorption at 818e806 cmꢀ1 in the spectra of
the free bases, attributed to the n(C]S) vibration, shifts to
751e719 cmꢀ1 in the spectra of the complexes, indicating
complexation through a thiolate sulfur [17]. Absorptions at
452e432 cmꢀ1 and 368e322 cmꢀ1 in the spectra of the com-
plexes were attributed to the n(CueN) and n(CueS) vibrations
[18]. Hence the infrared spectra indicate coordination through
the NeS chelating system.

A. Pe´rez-Rebolledo et al. / European Journal of Medicinal Chemistry 43 (2008) 939e948
941
The nep* transitions at 27,951e27,225 cmꢀ1 attributed to
the azomethine and thioamide functions in the electronic spectra
of the thiosemicarbazones [19], overlap in the 29,939e
29,561 cmꢀ1 range in the spectra of the complexes. The Ne
CuII and SeCuII ligand-to-metal charge transfer transitions
were found at 25,060e24,312 cmꢀ1 and the ligand field transi-
tions at 17,126e16,664 cmꢀ1 [20].
Table 1
EPR parameters g, A (ꢂ10ꢀ4 cmꢀ1) and g /A ratio for [Cu(4NO2Ac4M)2] (4),
k
[Cu(4NO2Ac4DM)2] (5) and [Cu(4NO2Ac4Pip)2] (6)
k
Compound
g
k
A
g
k
A
k
g /A
k
k
k
4
5
6
2.0300
2.0360
2.0380
30
25
25
2.1530
2.1500
2.1365
153
158
160
141
136
133
2.5. Electrochemistry
2.4. EPR spectra of the copper(II) complexes
Fig. 4 shows the voltammogram and the differential pulse
voltammogram of H4NO2Ac4M, and Fig. 5 shows the voltam-
mogram of complex 4.
The X-band EPR spectra of frozen solutions of complexes
4, 5 and 6 in DMF are shown in Fig. 3. The Hamiltonian
parameters are resumed in Table 1.
The voltammograms of the thiosemicarbazones show
a quasi-reversible process around ꢀ1.00 V, which has been at-
tributed to the formation of the AreNOꢁ2ꢀ radical. The second
process between ꢀ1.28 and ꢀ1.48 V has been assigned to the
formation of the AreNHOH species and the irreversible
oxidation at 0.08e0.17 V to the formation of AreNO (see
Table 2). The observed signals correspond to the well known
mechanism for the nitro aromatic reduction, as proposed be-
fore in many different works [23e26]. Eqs. (1)e(3) describe
the suggested electrochemical mechanism.
The EPR spectra of the three complexes indicate axial sym-
metry. For all compounds g > gt > 2, in accordance with ap-
k
proximately square-planar geometry. Moreover, the g and A
k
k
values of 4e6 are found in the region characteristic of
CuN2S2 chromophores in the g vs. A diagram. Sakaguchi
k
k
and Addison [21] showed that the g /A ratio can be used as
k
k
a convenient empirical index of tetrahedral distortion in
CuN4 units. This value ranges from ca. 105 to 135 cm for
the square-planar structure, and this quotient increases on
the introduction of tetrahedral distortion to the chromophore.
Further, tetrahedral distortion of a square-planar chromophore
AreNO2 þ eꢀ # AreNOꢁ2ꢀ ðpeaks I and I0Þ
AreNOꢁ2ꢀ þ 3eꢀ þ 4Hþ / AreNHOH
ð1Þ
is observed when any (N, O, S) donors reduce A and increase
k
g . Using that relationship and the results in Table 1, com-
k
þ H2O ðpeaks II and II0Þ
ð2Þ
ð3Þ
pounds 4e6 present tetrahedral distortions from the square-
planar geometry. The crystal structures of 4 and 5 (see Section
2.6) reveal that the geometries of these complexes are distorted
square-planar or distorted tetrahedral. From the EPR data we
may infer that the geometry of 6 is probably closer to the
AreNHOH # AreNO þ 2eꢀ þ 2Hþ ðpeak IIIÞ
The voltammograms of complexes 4e6show a quasi-reversible
process attributed to the CuII/CuI reduction (between ꢀ0.62 and
ꢀ0.49 V) followed by the CuI/CuII oxidation (between ꢀ0.43
and ꢀ0.26 V) (see Table 3). The process occurring in the
square-planar arrangement. The increased g values and de-
k
creased A values for the studied compounds, in the order 6,
k
5, and 4, respectively, show that the ligand field strength
decreases in these compounds in the same order [22].
Fig. 4. Cyclic voltammogram and differential pulse voltammogram of N(4)-
methyl-4-nitroacetophenone thiosemicarbazone (H4NO2Ac4M, 1) (CH2Cl2,
0.1 mol Lꢀ1 TBAP).
Fig. 3. X-band EPR spectra of frozen solutions of [Cu(4NO2Ac4M)2] (4),
[Cu(4NO2Ac4DM)2] (5) and [Cu(4NO2Ac4Pip)2] (6), in DMF.

942
A. Pe´rez-Rebolledo et al. / European Journal of Medicinal Chemistry 43 (2008) 939e948
Table 3
Cyclic voltammetry data for [Cu(4NO2Ac4M)2] (4), [Cu(4NO2Ac4DM)2] (5)
and [Cu(H4NO2Ac4Pip)2] (6) (0.100 V sꢀ1, CH2Cl2, 0.1 mol Lꢀ1 TBAP)
Compound
CuII/CuI (V)
CuI/CuII (V)
AreNHOH (V)
4
5
6
ꢀ0.49
ꢀ0.57
ꢀ0.62
ꢀ0.26
ꢀ0.35
ꢀ0.43
ꢀ1.15
ꢀ1.20
ꢀ1.20
The bond distances are very similar in 2 and 1. However,
O1eN1 is 1.217(5) in 2 and 1.233(4) in 1, probably because
these atoms are involved in intermolecular hydrogen bonding
in the latter but not in the former compound. The N3eC8eN4,
N3eC8eS and C7eN2eN3 bond angles are different in the
two compounds. Steric effects could be responsible for the dif-
ferences but electronic effects of two methyl groups instead of
one could also have some contribution to these variations.
The crystal structure of complex 4 shows one independent
molecule per asymmetric unit (see Fig. 7). One of the coordi-
nated thiosemicarbazones presents a disorder at the terminal
nitrogen N4. The difference Fourier map showed a residual
electron density that could be interpreted in terms of a disor-
dered acetone solvent molecule.
Fig. 5. Cyclic voltammogram of [Cu(4NO2Ac4M)2] (4) (0.100 V sꢀ1, CH2Cl2,
0.1 mol Lꢀ1 TBAP).
ꢀ1.15 and ꢀ1.20 V range has been attributed to the formation
of AreNHOH. The remaining processes have been assigned to
the thiosemicarbazone moiety, as previously proposed by us
[27]. The formation of the AreNOꢁ2ꢀ radical has not been ob-
served separately in the voltammograms of the complexes, in-
dicating that in this case, once formed the radical reacts
immediately.
The foregoing results show that within the nitro-aryl moiety
the electrochemical behaviors of the three thiosemicarbazones
are very similar, as are the behaviors of the complexes.
Variations in bond distances and angles were observed upon
complexation of 1 (see Table 5). The C8eS bonddistancechanges
˚
from 1.682(3) A in the free thiosemicarbazone to 1.749(6) A and
˚
˚
1.776(7) A in complex 4, as a consequence of deprotonation at N3
and formation of an extensively delocalized system involving the
thiosemicarbazone moiety and the aromatic ring attached to C7.
Therefore the CeS bond changes from mainly double in 1 to
predominantly single in 4. The C8eN3 bond changes from
˚
˚
1.364(5) A in the thiosemicarbazone to 1.313(7) A and
˚
1.282(8) A in complex 4, indicating the formation of a new pre-
dominant double bond in each ligand. The N2eN3 bond goes
2.6. Crystal structures of H4NO24DM (2),
˚
˚
˚
[Cu(4NO2Ac4M)2] (4) and [Cu(4NO2Ac4DM)2] (5)
from 1.377(4) A in 1 to 1.396(5) A and 1.372(6) A in 4.
Copmpound 1 crystallizes in the EE configuration in relation
to the C7eN2 and N3eC8 bonds, respectively. Compound 4
crystallizes in the EZ configuration, in which a twisting of ap-
proximately 180ꢃ in the N3eC8 bond occurs, to match the steric
requirements of coordination through the N2eS system.
The angles in the thiosemicarbazone chain undergo signif-
icant changes on coordination (see Table 5). For example, the
N3eC8eS angle goes from 119.7(3)ꢃ in 1 to 126.5(4)ꢃ and
126.8(5)ꢃ in 4; the N3eN2eC7 angle changes from
119.6(3)ꢃ in 1 to 115.7(4)ꢃ and 116.0(5)ꢃ in 4 and the N2e
N3eC8 angle changes from 118.3(3)ꢃ in 1 to 112.2(4)ꢃ and
111.8(5)ꢃ in 4. Upon coordination a variation occurs in the an-
gle between the aromatic ring and the thiosemicarbazone
chain, which changes from 24.7ꢃ [12] in the free thiosemicar-
bazone to 40.3(1)ꢃ and 41.3(1)ꢃ in complex 4.
Variations in some bond distances and angles also occur
upon coordination of 2 with formation of complex 5 (see Table
5 and Fig. 8).
Comparison between the crystal structures of complexes 4
and 5 reveals that in both the sulfurs are approximately in cis
position to each other. Interestingly, the crystal structure of
[Ni(4NO2Ac4M)2] previously reported by us [12] shows that
in this case the sulfurs are cis to each other as well.
Crystal data and refinement results of 2, 4 and 5 are shown
in Table 4. ORTEP3 views [28] of the structures can be seen in
˚
Figs. 6e8. Table 5 shows selected bond distances [A] and an-
gles [ꢃ] for the studied compounds.
Single crystals of 2 and complexes 4 and 5 adequate for
structural X-ray diffraction work were obtained by slow evap-
oration of the solvent (ethanol in the case of 2 and acetone in
the case of 4 and 5).
The crystal structure of 1 has been reported previously by
us [12]. Compound 2 crystallizes in the orthorhombic system,
Pcab space group with Z ¼ 8 (see Table 4 and Fig. 6). Unlike
1, which crystallizes in the EE configuration in relation to
C7eN2 and N3eC8, 2 adopts the EZ configuration.
Table 2
Cyclic voltammetry data for H4NO2Ac4M (1), H4NO2Ac4DM (2) and
H4NO2Ac4Pip (3) (0.100 V sꢀ1, CH2Cl2, 0.1 mol Lꢀ1 TBAP)
Compound
AreNOꢁ2ꢀ(V)
AreNHOH (V)
AreNO (V)
Epc
Epa
Epc
Epa
Epa
3
1
2
ꢀ0.95
ꢀ1.02
ꢀ1.14
ꢀ0.96
ꢀ0.98
ꢀ0.99
ꢀ1.28
ꢀ1.34
ꢀ1.48
ꢀ1.14
ꢀ1.09
ꢀ1.09
0.08
0.17
0.17

A. Pe´rez-Rebolledo et al. / European Journal of Medicinal Chemistry 43 (2008) 939e948
943
Table 4
Crystal data, structure solution methods and refinement results for H4NO2Ac4DM (2), [Cu(4NO2Ac4M)2] (4) and [Cu(4NO2Ac4DM)2] (5)
Compound
H4NO2Ac4DM
[Cu(4NO2Ac4M)2]$CH3COCH3
[Cu(4NO2Ac4DM)2]
Empirical formula
Formula weight
Temperature, K
Crystal system
Space group
C11H14N4O2S
266.32
C23H28CuN8O5S2
624.19
C22H26CuN8O4S2
594.20
293 (2)
Orthorhombic
Pbca
Orthorhombic
Pbca
Monoclinic
P21/n
Unit cell dimensions
˚
a, A
˚
13.910(1)
7.766(1)
23.834(2)
90
13.554(1)
15.271(1)
8.892(3)
24.386(3)
12.316(3)
90
b, A
˚
c, A
ꢃ
27.132(3)
90
a, ꢃ
b,
90
90
90
90
92.43(2)
90
ꢃ
g,
3
˚
Volume, A
2574.7(4)
8, 1.374
0.252
5615.9(8)
8, 1.476
2668.2(12)
4, 1.484
1.020
Z, Density calc., Mg/m3
Absorption coefficient, mmꢀ1
F(000)
0.975
2552
1120
0.20 ꢂ 0.15 ꢂ 0.10
Orange, prismatic
1236
0.15 ꢂ 0.20 ꢂ 0.40
Dark red, prismatic
Crystal size, mm3
0.15 ꢂ 0.20 ꢂ 0.20
Dark green, prismatic
Crystal color, shape
Rad., graph. Monoch.
q range for data coll.,
˚
Mo Ka, l ¼ 0.71073 A
2.25e26.00
ꢃ
1.50e25.50
ꢀ4 ꢄ h ꢄ 16
1.67e25.00
ꢀ1 ꢄ h ꢄ 10
Index range, q
0 ꢄ h ꢄ 17
ꢀ5 ꢄ k ꢄ 9
ꢀ1 ꢄ k ꢄ 18
ꢀ1 ꢄ k ꢄ 29
ꢀ29 ꢄ l ꢄ 0
ꢀ1 ꢄ l ꢄ 32
ꢀ14 ꢄ l ꢄ 14
100% (to w ¼ 25.00ꢃ)
Completeness
Refinement method
Goodness-of-fit on F2
Reflections collected/unique (Rint
99.9% (to w ¼ 26.00ꢃ)
Full-matrix least-squares on F2
0.940
100% (to w ¼ 25.50ꢃ)
0.998
6862/5239 (0.0415)
2912/0/371
1.048
5451/4703 (0.0517)
2872/0/334
)
3187/2524 (0.0686)
1346/0/163
0.0600/0.1324
Data [I > 2s(I )]/restraints/parameters
obs, Rall
wR2obs, wR2all
Larg. peak and hole, e A
R
0.0611/0.1328
0.1361/0.1700
0.537 and ꢀ0.438
0.0675/0.1236
0.1624/0.1933
0.450 and ꢀ0.332
0.1489/0.1916
0.220 and ꢀ0.234
ꢀ3
˚
2.7. In vitro anti-trypanosomal activity
3. Conclusion
Table 6 shows the effect of the thiosemicarbazones and their
corresponding copper complexes on the growth of the epimas-
tigote form of T. cruzi at 25 mM at day 5 of exposure (percentage
of growth inhibition, PGI). At this dose 2 and complexes 4 and 5
proved to be as active as the reference drugs, nifurtimox (nfx)
and benznidazole (bnz). Complex 6 did not inhibit T. cruzi
growth under the assayed conditions. For the most active com-
pounds (2 and complexes 4 and 5) doseeresponse curves were
recorded and the dose that inhibits 50% of T. cruzi growth
(ID50,T. cruzi) was calculated (Fig. 9). The effect of copper(II)
as the acetate and sulfate salts was evaluated and the low bioac-
tivity of these salts as well as that of complex 6 indicates that
copper(II) does not possess anti-T. cruzi activity per se.
Compound 2 proved to be the most active of the three as-
sayed thiosemicarbazones. From the determined values of
ID50,T. cruzi, 2 as well as complexes 4 and 5 proved to be as ac-
tive as the reference drugs nfx and bnz. Complex 5 exhibited
high activity at very low dose (0.056 mM). However, although
it presents a good therapeutic index (TI), the therapeutic index
of the free thiosemicarbazone (2) is higher. The therapeutic in-
dexes (TI) of 2 and complex 5 are much higher than those of
the clinically used drugs nfx and bnz, indicating that the thio-
semicarbazone and the copper(II) complexes could be anti-
trypanosomal drug candidates.
The significant enhancement of activity upon complexation
could be due either to changes in lipophilicity, to the rigid con-
formation of the ligand in the complex, which could facilitate its
interaction with the biological target, to a greater stability of the
thiosemicarbazone in the complex or to redox effects involving
the thiosemicarbazone and copper. It is clear that copper(II)
per se is not responsible for the studied anti-T. cruzi activity.
As already mentioned, the mechanisms of action of nfx and
bnz involve intracellular reduction of the nitro group. Nfx acts
via reduction of the nitro group to an unstable nitro anion radi-
cal, which produces highly toxic reduced oxygen species (ROS)
such as superoxide [8]. The mode of action of bnz involves
2.8. In vitro mammal cytotoxicity
After the first screening, new in vitro studies were per-
formed to analyze the nonspecific toxicity of the best anti-T.
cruzi compounds using THP-1 human macrophages as model.
The ID50,macrophage values are presented in Table 6 and the
therapeutic index (TI) defined as the ratio ID50,macrophage
/
ID50,T. cruzi is reported. Among the best anti-T. cruzi com-
pounds 2 presented the highest therapeutic index.

944
A. Pe´rez-Rebolledo et al. / European Journal of Medicinal Chemistry 43 (2008) 939e948
Fig. 6. Perspective view of the structure of H4NO2Ac4DM (2).
covalent modification of biomacromolecules by nitro reduction
intermediates which cause cellular damage. Similar mecha-
nisms could be proposed for nitro thiosemicarbazones [2,29].
Our results suggest that probably the differences in activity
between the three thiosemicarbazones cannot be attributable to
variations in the electrochemical potentials involving the nitro
group. Similarly, the different activities of the complexes are
probably not due to distinct electrochemical behaviors involv-
ing the nitro-aryl moiety.
Interestingly, other authors observed a similar effect of en-
hancement of the anti-trypanosomal activity upon coordina-
tion to copper(II), of imidazole derivatives not containing
the nitro group, which therefore cannot undergo electrochem-
ical processes involving this group [30].
Fig. 8. Perspective view of the structure of [Cu(4NO2Ac4DM)2] (5).
8453 spectrometer in dimethyl formamide (DMF) solutions
using 1 cm cells. NMR spectra were obtained at room temper-
ature with a Bruker DRX-400 Avance (400 MHz) spectrome-
ter using deuterated dimethyl sulfoxide (DMSO-d6) as the
solvent and tetramethylsilane (TMS) as internal reference.
Splitting patterns are designated as follows; s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet.
The electrochemical experiments were carried out at room
temperature in dichloromethane containing 0.1 mol Lꢀ1 tetra-
butylammonium perchlorate (TBAP) purchased from Fluka.
These measurements were performed by using an electro-
chemical analyzer from Bioanalytical Systems Inc. (BAS),
model 100BW. The working and auxiliary electrodes were sta-
tionary Pt foils, and the reference electrode was Ag/AgCl
a medium in which ferrocene is oxidized at 0.48 V (Fcþ/Fc).
EPR spectra of the copper(II) complexes were obtained at
77 K (liquid N2 temperature) on a VARIAN E109-110 equip-
ment operating at about 9.5 GHz (X-band) using DMF as
solvent.
Taking into consideration the serious side effects and the
poor efficacy of the clinical reference drugs, as well as the ap-
pearance of resistance during treatment, acetophenone thiose-
micarbazones and their copper(II) complexes could constitute
a new class of anti-trypanosomal drugs.
4. Experimental
4.1. Physical measurements
Infrared spectra were recorded on a PerkineElmer spec-
trum GX CDRH FTIR system using nujol mulls between
CsI plates; partial elemental analyses were obtained with a Per-
kineElmer PE 2400 analyzer. An YSI model 31 conductivity
bridge was employed for molar conductivity measurements.
Electronic spectra were acquired with a Hewlett Packard
4.2. X-ray crystallography
The crystal structures of H4NO24DM (2), [Cu(4-
NO2Ac4M)2] (4), and [Cu(4NO2Ac4DM)2] (5) were investi-
gated using a SIEMENS P4 diffractometer at 273 K with
a graphite monochromator and Mo Ka radiation l ¼ 0.71073
˚
A. Data were obtained using the XSCANS software [31].
The lattice parameters were obtained by least-squares fit
from 21/43/42 accurately centered reflections in the 2q range
8.5e12.2/5.3e12.5/8.5e10.4 for H4NO24DM (2), [Cu(4-
NO2Ac4M)2] (4), and [Cu(4NO2Ac4DM)2] (5), respectively.
Data were corrected for Lorentz and polarization effects, but
not for absorption. The structures were solved by direct
methods and refined by full-matrix-least-squares on F2 using
SHELX97 [32]. Although some of the hydrogen atoms could
be identified in a Fourier difference map, in the final model
they were geometrically positioned and refined using a riding
model. All non-H atoms were refined anisotropically.
Fig. 7. Perspective view of the structure of [Cu(4NO2Ac4M)2] (4).

A. Pe´rez-Rebolledo et al. / European Journal of Medicinal Chemistry 43 (2008) 939e948
945
Table 5
Selected bond lengths [A] and angles [ ] for H4NO2Ac4DM (2), [Cu(4NO2Ac4M)2] (4) and [Cu(4NO2Ac4DM)2] (5)
ꢃ
˚
2 [ꢃ]
4 [ꢃ]
4 [ꢃ]
5 [ꢃ]
5 [ꢃ]
˚
2 [A]
˚
4 [A]
˚
4 [A]
˚
5 [A]
˚
5 [A]
Bond
Angle
Ligand 1
Ligand 2
Ligand 1
Ligand 2
Ligand 1
Ligand 2
Ligand 1
Ligand 2
SeC8
1.680(4)
1.372(5)
1.340(5)
1.374(4)
1.284(5)
1.217(5)
1.214(5)
e
1.749(6)
1.313(7)
1.351(7)
1.396(5)
1.288(6)
1.223(6)
1.211(6)
2.018(4)
2.238(2)
1.776(7)
1.282(8)
e
1.372(6)
1.294(6)
1.212(7)
1.207(6)
2.007(4)
2.232(2)
1.750(6)
1.318(7)
1.347(7)
1.379(6)
1.287(7)
1.215(9)
1.219(9)
2.027(5)
2.226(2)
1.735(6)
1.307(7)
1.355(7)
1.403(6)
1.288(7)
1.236(9)
1.192(9)
2.018(5)
2.224(2)
C8eN4eC10
N4eC8eS
N4eC8eN3
N3eC8eS
N2eN3eC8
C7eN2eN3
N2eCueN20
N20eCueS0
N2eCueS
N2eCueS0
N20eCueS
SeCueS0
122.7(4)
123.5(3)
114.0(3)
122.5(3)
120.1(3)
117.2(3)
e
123.5(5)
116.9(5)
116.6(5)
126.5(4)
112.2(4)
115.7(4)
100.5(2)
84.6(1)
e
123.8(6)
117.8(5)
117.6(5)
124.6(4)
113.3(4)
114.7(4)
105.1(2)
84.9(1)
123.1(6)
117.6(5)
117.0(5)
125.3(4)
113.3(5)
114.5(5)
C8eN3
C8eN4
N3eN2
C7eN2
N1eO1
N1eO2
CueN2
CueS
e
e
126.8(5)
111.8(5)
116.0(5)
e
e
e
85.2(1)
84.5(1)
e
148.0(1)
150.9(1)
105.7(1)
156.4(2)
155.7(2)
95.0(1)
e
e
0: Atom of the second ligand.
4.3. Synthesis of H4NO2Ac4M (1), H4NO2Ac4DM (2)
and H4NO2Ac4Pip(3)
4.3.2. N(4)-Dimethyl 4-nitroacetophenone thiosemicarba-
zone (H4NO2Ac4DM, 2)
Orange solid; yield: 77%; mp: 182.0e183.0 ꢃC. Anal.
Calcd (C11H14N4O2S): C, 49.61; H, 5.30; N, 21.04. Found:
C, 49.16; H, 5.35; N, 21.02%. UV: lmax (cmꢀ1): 36,900,
27,951. IR (nujol mulls, cmꢀ1): nassNO2 1535, nsNO2 1329,
n(NH) 3330, n(C]N) 1590, n(C]S) 809. 1H NMR
(DMSO-d6): (d, ppm) 9.76 (1H, s, N(3)H), 8.29 (2H, d, aryl
H(3,5), J ¼ 8.93), 8.05 (2H, d, aryl H(2,6), J ¼ 8.93), 3.32
(6H, s, H(10,11)), 2.35 (3H, s, H(9)), C8]S 182.08, C4
147.43, C1 147.31, C7]N 144.31, C2 and C6 127.08, C3
and C5 123.56, C10 and C14 42.20, C9 13.78.
N(4)-Methyl thiosemicarbazide was purchased from Al-
drich and used without further purification. N(4)-Dimethyl
and N(4)-piperidyl thiosemicarbazides were prepared as
previously reported [33]. The thiosemicarbazones were ob-
tained by mixing equimolar amounts of 4-nitroacetophenone
(6.6 ꢂ 10ꢀ3 mol) with the desired N(4)-substituted thiosemi-
carbazide in absolute ethanol (40 mL) with addition of 2e4
drops of concentrated sulfuric acid as catalyst. The reaction
mixture was kept under reflux for 6e7 h. The resulting solids
were filtered off and re-crystallized from ethanol.
4.3.3. N(4)-Piperidyl 4-nitroacetophenone thiosemicarba-
zone (H4NO2Ac4Pip, 3)
4.3.1. N(4)-Methyl 4-nitroacetophenone thiosemicarbazone
(H4NO2Ac4M, 1)
N(4)-Methyl
Yellow solid; yield: 72%; mp: 144.9e146.0 ꢃC. Anal.
Calcd (C14H18N4O2S): C, 54.88; H, 5.92; N, 18.29. Found:
C, 54.05; H, 5.83; N, 18.72%. UV: lmax (cmꢀ1): 36,900,
4-nitroacetophenone
(H4NO2Ac4M, 1) has been reported previously [12].
thiosemicarbazone
Table 6
In vitro anti-T. cruzi activities and human macrophages cytotoxicities for H4NO2Ac4M (1), H4NO2Ac4DM (2), H4NO2Ac4Pip (3) and their copper(II) complexes
[Cu(4NO2Ac4M)2] (4) and [Cu(4NO2Ac4DM)2] (5) and [Cu(4NO2Ac4Pip)2] (6)
Thiosemicarbazones
Copper complexes
Compound
Compound
PGIa
(%)
ID50,T. cruzi
(mM)
ID50,macrophage
(mM)
TIe
PGIa
(%)
ID50,T. cruzi
(mM)
ID50,macrophage
(mM)
TIe
b,c
c,d
b,c
c,d
1
2
3
31
>25.0
0.28
>>25.0
>400.0
>400.0
>16
>1428
ND
4
5
6
100
100
0
2.0
0.056
>>25.0
4.0
2
100
12
50.0
ND
893
ND
f
ND
Nfx
Bnz
a
100
100
7.7
7.4
>300.0j
>300.0j
>38
>40
Copper(II) acetateg
>>2.0h
>>2.0i
g
Copper(II) sulfate
PGI: percentage of growth inhibition at 25 mM, epimastigote form of T. cruzi Tulahuen 2.
ID50,T. cruzi: dose that inhibits 50% of T. cruzi growth.
b
c
d
e
f
The results are the mean values of three different experiments with a SD less than 10% in all cases.
ID50,macrophage: dose that inhibits 50% of THP-1 human macrophages growth.
TI: therapeutic index, see text for definition.
ND: not determined.
g
h
i
The following doses were studied: 2.0, 1.0, 0.5 and 0.1 mM.
The salt resulted inactive in the 0.1e1.0 mM range; PGI at 2.0 mM ¼ 15%.
The salt resulted inactive in the 0.1e1.0 mM range; PGI at 2.0 mM ¼ 5%.
Higher doses were not assayed due to solubility problems.
j

946
A. Pe´rez-Rebolledo et al. / European Journal of Medicinal Chemistry 43 (2008) 939e948
4.4.1. [Cu(4NO2Ac4M)2] (4)
Green solid; yield: 83%. Anal. Calcd (C20H22CuN8O4S2):
C, 42.43; H, 3.92; N, 19.79. Found: C, 42.06; H, 3.84;
N, 19.66%. UV: lmax (cmꢀ1): 29,939, 25,060, 16,664. IR
(nujol mulls, cmꢀ1): nassNO2 1521, nsNO2 1340, n(NH)
3438, n(C]N) 1568, n(C]S) 751, n(MeN) 432,
n(MeS) 350. Molar conductivity (1 ꢂ 10ꢀ3 mol Lꢀ1 DMF):
1.44 Uꢀ1 cm2 molꢀ1. Magnetic susceptibility: 1.50 m (BM).
4.4.2. [Cu(4NO2Ac4DM)2] (5)
Brown solid; yield: 82%. Anal. Calcd (C22H26CuN8O4S2):
C, 44.47; H, 4.41; N, 18.86. Found: C, 43.81; H, 4.40; N,
18.72%. UV: lmax (cmꢀ1): 29,939, 24,413, 16,820. IR (nujol
mulls, cmꢀ1): nsNO2 1331, n(C]N) 1572, n(C]S) 725,
n(MeN) 448, n(MeS) 322. Molar conductivity
(1 ꢂ 10ꢀ3 mol Lꢀ1 DMF): 2.00 Uꢀ1 cm2 molꢀ1. Magnetic sus-
ceptibility: 1.80 m (BM).
4.4.3. [Cu(4NO2Ac4Pip)2] (6)
Yellowish green solid; yield: 78%. Anal. Calcd (C28H34Cu-
N8O4S2): C, 49.87; H, 5.08; N, 16.62. Found: C, 48.09; H,
5.04; N, 16.50%. UVevis (cmꢀ1): 29,561, 24,312, 17,126.
IR (nujol mulls, cmꢀ1): nsNO2 1330, n(C]N) 1585,
n(C]S) 719, n(MeN) 452, n(MeS) 368. Molar conductivity
(1 ꢂ 10ꢀ3 mol Lꢀ1 DMF): 7.21 Uꢀ1 cm2 molꢀ1. Magnetic sus-
ceptibility: 1.80 m (BM).
4.5. In vitro anti-trypanosomal activity
T. cruzi epimastigotes (Tulahuen 2 strain) were grown at
28 ꢃC in an axenic medium (BHI-Tryptose) as previously de-
scribed [2,6e8], complemented with 5% fetal calf serum.
Cells were harvested in the late log phase, re-suspended in
fresh medium, counted in Neubauer’s chamber and placed in
24-well plates (2 ꢂ 106/mL). Cell growth was measured as
the absorbance of the culture at 590 nm, which was proved
to be proportional to the number of cells present [34]. Before
inoculation, the media were supplemented with the indicated
amount of the studied compound from a stock solution in
DMSO. The final concentration of DMSO in the culture media
never exceeded 1% and the control was run in the presence of
1% DMSO and in the absence of any compound. No effect on
epimastigotes growth was observed by the presence of up to
1% DMSO in the culture media. Nfx and bnz were used as
the reference trypanocidal drugs. Copper(II) acetate and cop-
per(II) sulfate were included in these assays to provide infor-
mation on the trypanocidal effect of copper, as copper(II)
entity. The percentage of growth inhibition (PGI), was calcu-
lated as follows: PGI ¼ {1 ꢀ [(Ap ꢀ A0p)/(Ac ꢀ A0c)]} ꢂ 100,
where Ap ¼ A600 of the culture containing the studied com-
pound at day 5; A0p ¼ A600 of the culture containing the stud-
ied compound right after addition of the inocula (day 0);
Ac ¼ A600 of the culture in the absence of any compound (con-
trol) at day 5; A0c ¼ A600 in the absence of the compound at
day 0. To determine ID50,T. cruzi values, parasite growth was
followed in the absence (control) and presence of increasing
concentrations of the corresponding compound. The ID50,T.
Fig. 9. Curves doseeanti-T. cruzi activity for (a) H4NO2Ac4M and complex
(4); (b) H4NO2Ac4DM and complex (5).
27,225. IR (nujol mulls, cmꢀ1): nassNO2 1538, nsNO2 1332,
n(C]N) 1590, n(C]S) 806. NMR (DMSO-d6): (d, ppm)
9.84 (1H, s, N(3)H), 8.25 (2H, d, aryl H(3,5), J ¼ 8.97), 8.01
(2H, d, aryl H(2,6), J ¼ 8.97), 3.87 (4H, s, H(10,14)), 2.35
(3H, s, H(9)), 1.65 (6H, s, H(11,12,13)), C8]S 182.00, C4
147.27, C1 146.81, C7]N 144.22, C2 and C6 127.03, C3
and C5 123.56, C10 and C14 51.46, C11 and C13 25.78,
C12 23.87, C9 13.94.
4.4. Synthesis of [Cu(4NO2Ac4M)2] (4),
[Cu(4NO2Ac4DM)2] (5) and [Cu(4NO2Ac4Pip)2] (6)
The copper(II) complexes were obtained by reacting the
desired thiosemicarbazone (0.002 mol) in methanol (30 mL)
with copper(II) acetate (0.001 mol) in 30 mL of methanol.
The reaction mixture was kept at 3 ꢃC under stirring while
the thiosemicarbazones were added. The resulting solids
were filtered, washed with anhydrous diethyl ether and
re-crystallized from ethanol.

A. Pe´rez-Rebolledo et al. / European Journal of Medicinal Chemistry 43 (2008) 939e948
947
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been deposited at Cambridge Crystallographic Data Centre
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available material can be obtained on application to CCDC,
12 Union Road, Cambridge CB2 1Ez, UK (fax: þ44 1223
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Acknowledgements
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