Coordination of nitro-thiosemicarbazones to ruthenium(II) as a strategy for anti-trypanosomal activity improvement

Article abstract of DOI:10.1016/j.ejmech.2010.03.005

Complexes [RuCl(H4NO₂Fo4M)(bipy)(dppb)]PF₆ (1), [RuCl(H4NO₂Fo4M)(Mebipy)(dppb)]PF₆ (2), [RuCl(H4NO ₂Fo4M)(phen)(dppb)]PF₆ (3), [RuCl(H4NO₂Ac4M) (bipy)(dppb)]PF₆ (4), [RuCl(H4NO₂Ac4M)(Mebipy)(dppb)] PF₆ (5) and [RuCl(H4NO₂Ac4M)(phen)(dppb)]PF₆ (6) with N⁴-methyl-4-nitrobenzaldehyde thiosemicarbazone (H4NO ₂Fo4M) and N⁴-methyl-4-nitroacetophenone thiosemicarbazone (H4NO₂Ac4M) were obtained from [RuCl₂(bipy)(dppb)], [RuCl₂(Mebipy)(dppb)], and [RuCl₂(phen)(dppb)], (dppb = 1,4-bis(diphenylphospine)butane; bipy = 2,2′-bipyridine; Mebipy = 4,4′-dimethyl-2,2′-bipyridine; phen = 1,10-phenanthroline). In all cases the thiosemicarbazone is attached to the metal center through the sulfur atom. Complexes (1-6), together with the corresponding ligands and the Ru precursors were evaluated for their ability to in vitro suppress the growth of Trypanosoma cruzi. All complexes were more active than their corresponding ligands and precursors. Complexes (1-3) and (5) revealed to be the most active among all studied compounds with ID₅₀ = 0.6-0.8 μM. In all cases the association of the thiosemicarbazone with ruthenium, dppb and bipyridine or phenanthroline in one same complex proved to be an excellent strategy for activity improvement.

Full text of DOI:10.1016/j.ejmech.2010.03.005

European Journal of Medicinal Chemistry 45 (2010) 2847e2853
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Coordination of nitro-thiosemicarbazones to ruthenium(II) as a strategy
for anti-trypanosomal activity improvement
Claudia Rodrigues a, Alzir A. Batista a, Javier Ellena b, Eduardo E. Castellano b, Diego Benítez c,
,
d
Hugo Cerecetto c, Mercedes González c, Letícia R. Teixeira d , Heloisa Beraldo
*
a Departamento de Química, Universidade Federal de São Carlos, 13565-905 São Carlos (SP), Brazil
b Instituto de Física de São Carlos, Universidade de São Paulo, 13560-970 São Carlos (SP), Brazil
c Laboratorio de Química Orgânica, Facultad de CienciaseFacultad de Química, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay
d Departamento de Química, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte (MG), Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Complexes [RuCl(H4NO2Fo4M)(bipy)(dppb)]PF6 (1), [RuCl(H4NO2Fo4M)(Mebipy)(dppb)]PF6 (2), [RuCl
(H4NO2Fo4M)(phen)(dppb)]PF6 (3), [RuCl(H4NO2Ac4M)(bipy)(dppb)]PF6 (4), [RuCl(H4NO2Ac4M)
(Mebipy)(dppb)]PF6 (5) and [RuCl(H4NO2Ac4M)(phen)(dppb)]PF6 (6) with N4-methyl-4-nitrobenzalde
hyde thiosemicarbazone (H4NO2Fo4M) and N4-methyl-4-nitroacetophenone thiosemicarbazone (H4NO2
Ac4M) were obtained from [RuCl2(bipy)(dppb)], [RuCl2(Mebipy)(dppb)], and [RuCl2(phen)(dppb)], (dppb ¼
1,4-bis(diphenylphospine)butane; bipy ¼ 2,20-bipyridine; Mebipy ¼ 4,40-dimethyl-2,20-bipyridine; phen ¼
1,10-phenanthroline). In all cases the thiosemicarbazone is attached to the metal center through the sulfur
atom.
Received 7 December 2009
Received in revised form
2 March 2010
Accepted 4 March 2010
Available online 11 March 2010
Keywords:
Thiosemicarbazones
Ruthenium(II) complexes
Anti-trypanosomal activity
Complexes (1e6), together with the corresponding ligands and the Ru precursors were evaluated for
their ability to in vitro suppress the growth of Trypanosoma cruzi. All complexes were more active than
their corresponding ligands and precursors. Complexes (1e3) and (5) revealed to be the most active
among all studied compounds with ID50 ¼ 0.6e0.8
mM.
In all cases the association of the thiosemicarbazone with ruthenium, dppb and bipyridine or phe-
nanthroline in one same complex proved to be an excellent strategy for activity improvement.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
least 12.5 times more active than the thiosemicarbazone counter-
part [7].
Trypanosoma cruzi (T. cruzi) is the etiologic agent of Chagas
disease or American trypanosomiasis [1,2]. Cruzain was shown to
be one of the most relevant proteases in T. cruzi [2]. Thio-
semicarbazones represent an interesting class of compounds with
a wide range of pharmacological applications [3] and have been
identified as lead scaffolds of cruzain inhibitors [1,2]. Considering
that many anti-trypanosomal drugs contain a nitro group, which
produces the nitro anion radical, highly toxic to the parasite [4e6],
we recently started an investigation of the pharmacological profile
of 4-nitroacetophenone-derived thiosemicarbazones. We demon-
strated that these thiosemicarbazones and their copper(II)
complexes present significant in vitro anti-trypanosomal activity,
the complexes resulting to be at least 5 times more active than the
free ligands. [Cu(4NO2Ac4M)2], with N4-methyl-4-nitro-
acetophenone thiosemicarbazones (H4NO2Ac4M) proved to be at
It has been shown that ruthenium complexes with inhibitors of
sterol biosyntheses such as clotrimazole and ketoconazole are more
active against T. cruzi than the corresponding free ligands [8]. [Ru
(Cl2(CTZ)2] (CTZ ¼ clotrimazole) was able to inhibit 90% of the
proliferation of epimastigote form of T. cruzi at a concentration
where the parent compound presented a modest effect. The
complex was able to eradicate experimental infection by the highly
infective intracellular amastigotes of T. cruzi grown on mammalian
cells at concentrations as low as10ꢀ8 mol Lꢀ1, which represented
a 10-fold enhancement of CTZ’s activity [8].
A series of ruthenium(II) complexes derived from 5-nitro-
furylsemicarbazones (HL), [RuIICl2(DMSO)2HL], were developed by
some of us, which were able to produce free radicals and redox
cycling into the parasite [9]. However, their high protein binding
capacity and hydrophilicity did not allow identifying in vitro active
compounds against T. cruzi.
The strategy of linking a nitro-thiosemicarbazone to ruthenium
could in principle lead to new anti-trypanosomal drug candidates.
* Corresponding author. Tel.: þ55 31 3409 5764; fax: þ55 31 3409 5700.
E-mail address: lregina@qui.ufmg.br (L.R. Teixeira).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.03.005

2848
C. Rodrigues et al. / European Journal of Medicinal Chemistry 45 (2010) 2847e2853
In the present work ruthenium(II) complexes containing N4-
methyl-4-nitrobenzaldehyde thiosemicarbazone (H4NO2Fo4M), or
N4-methyl-4-nitroacetophenone thiosemicarbazone (H4NO2Ac4M)
and 1,4-bis(diphenylphospine)butane (dppb), 2,20-bipyridine
(bipy), 4,40-dimethyl-2,20-bipyridine (Mebipy) or 1,10-phenan-
throline (phen) as co-ligands were prepared (Fig. 1).
The nitro-thiosemicarbazones, the new complexes and the
ruthenium(II) precursors [RuCl2(bipy)(dppb)], [RuCl2(Mebipy)
(dppb)], and [RuCl2(phen)(dppb)], were evaluated for their ability
to in vitro suppress the growth of T. cruzi Tulahuen 2 strain [10].
2. Results and discussion
2.1. Microanalyses and molar conductivity studies
Fig. 2. 31P{1H} NMR spectrum of [RuCl(H4NO2Fo4M)(bipy)(dppb)]PF6 (1).
Microanalyses suggest the formation of [RuCl(H4NO2Fo4M)
(bipy)(dppb)]PF6 (1), [RuCl(H4NO2Fo4M)(Mebipy)(dppb)]PF6 (2),
[RuCl(H4NO2Fo4M)(phen)(dppb)]PF6 (3), [RuCl(H4NO2Ac4M)
(bipy)(dppb)]PF6 (4), [RuCl(H4NO2Ac4M)(Mebipy)(dppb)]PF6 (5)
and [RuCl(H4NO2Ac4M)(phen)(dppb)]PF6 (6). The molar conduc-
tivity data indicate that all complexes are 1:1 electrolytes in
accordance with the proposed formulations.
43.6e43.3) and the second phosphorous atom is trans to the
nitrogen of bipyridine, methylbipyridine or phenanthroline (
41.6e41.0). For complexes (4e6) the doublets were observed at ca.
36 and 31, indicating again that one of the phosphorous atoms is
trans to a chlorine (
d
d
d
d
37.5e35.8) and the second phosphorous atom
is trans to a nitrogen (32.4e30.2) [13]. The difference in the 31P{1H}
NMR chemical shifts (ca. 7 ppm) between (1e3) and (4e6) is
probably due to the different electronic effects of H4NO2Fo4M and
H4NO2Ac4M.
2.2. Infrared spectral studies
In the infrared spectra, the n(C]N) stretching vibrations of
H4NO2Fo4M and H4NO2Ac4M at 1599 cmꢀ1 and 1590 cmꢀ1
respectively [7,11] remain practically unaltered in the spectra of the
complexes, indicating that the imine nitrogen is not involved in
coordination. In contrast, the absorption at 830 and 823 cmꢀ1 in the
spectra of H4NO2Fo4M and H4NO2AC4M respectively, attributed to
2.4. Electrochemistry
The voltammograms of the thiosemicarbazones show a quasi-
reversible process between ꢀ0.93 and ꢀ1.02 V, which has been
attributed to the formation of the Ar-NOꢁ2ꢀ radical. The second
process between ꢀ1.11 and ꢀ1.34 V has been assigned to the
formation of the Ar-NHOH species, and the irreversible oxidation at
0.17e0.21 V to the formation of Ar-NO. The observed processes
correspond to the well known mechanism for nitro-aromatic
reduction, as previously proposed by us [7,11] and by other authors
[14e17].
The voltammograms of complexes (1e6) show a quasi-revers-
ible process (ipa/ipc w 1.1) attributed to the RuII/RuIII oxidation
(between 0.94 V and 1.09 V) followed by the RuIII/RuII reduction
(between 0.82 and 0.96 V) (see Table 2 and Fig. 3). The high value of
the n
(C]S) vibration, shifts to 811e815 cmꢀ1 in the spectra of
complexes (1e6), indicating coordination through the sulfur [12].
Hence, the infrared data suggest coordination of the thio-
semicarbazones as monodentate S-donor ligands, as confirmed by
the crystal structure determination of complex (3) (see Section 2.5).
2.3. 31P{1H} NMR studies
In the 31P{1H} NMR spectra of the complexes two doublets were
observed in all cases (see Fig. 2 and Table 1). For complexes (1e3)
the doublets occur at ca.
d 43 and d 41 suggesting that one of the
phosphorous atoms of dppb is trans to the chlorine atom (
d
Fig. 1. Synthetic route for complexes (1)e(6).

C. Rodrigues et al. / European Journal of Medicinal Chemistry 45 (2010) 2847e2853
2849
Table 1
31P{1H} NMR spectral data for complexes (1e6).
Complex
d
2JPeP (Hz)
[RuCl(H4NO2Fo4M)(bipy)(dppb)]PF6 (1)
43.3
41.0
43.6
41.2
43.4
41.6
35.9
30.2
35.8
31.8
37.5
32.4
33.2
[RuCl(H4NO2Fo4M)(Mebipy)(dppb)]PF6 (2)
[RuCl(H4NO2Fo4M)(phen)(dppb)]PF6 (3)
[RuCl(H4NO2Ac4M)(bipy)(dppb)]PF6 (4)
[RuCl(H4NO2Ac4M)(Mebipy)(dppb)]PF6 (5)
[RuCl(H4NO2Ac4M)(phen)(dppb)]PF6 (6)
32.4
34.0
30.9
32.4
34.0
the RuII/RuIII potential for all complexes indicates electrochemical
stability. The formation of the Ar-NOꢁ2ꢀ radical has been observed
separately in the voltammograms of complexes (3) and (6) but not
in those of complexes (1), (2), (4) and (5), indicating that in the
latter, once formed the radical reacts immediately. For complexes
(1e6) the process occurring between ꢀ1.04 and ꢀ1.17 V has been
attributed to the formation of Ar-NHOH. For all complexes the
remaining processes have been assigned to the thiosemicarbazone
moiety, as previously proposed by us [18].
Experiments carried out on the main drugs used in Chagas
disease treatment, nifurtimox (Nfx, 3-methyl-4-[(5-nitro-
furfurylidene)amino]thiomorpholine-1,1-dioxide),
derivative, and benznidazole (2-nitro-N-(phenylmethyl)-1H-imid-
azole-1-acetamide), nitroimidazole derivative, suggest that
a
nitrofuran
a
intracellular nitro-moiety reduction followed by redox cycling
yielding reactive oxygen species may be their major mode of action
against T. cruzi [9].
In the present work the quasi-reversible process between ꢀ0.93
and ꢀ1.02 V has been attributed to the formation of the Ar-NO2ꢁꢀ
radical. The reported value for the same process in Nfx [9] falls into
this same range. Interestingly, complexes (1e6) have adequate
NO2-reduction potential to act against T. cruzi via a redox cycling
process. Coordination to ruthenium(II), like in the case of copper(II)
[7], affects positively the value of the ligand NO2-reduction
potential (compare Epc of H4NO2Ac4M, ꢀ1.02 V [7], to Epc of
complex (6), ꢀ0.91 V, Table 2), generating compounds which are
more easily reduced in biological media.
Fig. 3. Cyclic voltammogram of [RuCl(H4NO2Fo4M)(phen)(dppb)]PF6 (3) (CH2Cl2,
0.1 mol Lꢀ1 TBAP). Inset: peaks of the ruthenium redox process.
However some bond angles undergo significant variations upon
coordination. The C7eN2eN3 bond angle is 116.70(3)ꢂ in
H4NO2Fo4M and 113.7(3)ꢂ in complex (3). In addition the
N3eC8eS and N4eC8eS angles are 118.05(14)ꢂ and 124.82(14)ꢂ in
H4NO2Fo4M and 121.1(3)ꢂ and 121.2(3)ꢂ respectively, in
complex (3).
2.5. Crystal structure of complex (3)
Crystal data and refinement results for complex (3) are shown in
Table 3. The ORTEP3 view [19] of the structure can be seꢂen in Fig. 4.
ꢀ
Table 4 shows selected bond distances [A] and angles [ ].
3 crystallizes in the monoclinic system, P21/n space group with
Z ¼ 4. In 3 the thiosemicarbazone in the EE configuration is attached
to the metal center through the sulfur atom. As expected, all bond
distances are very similar in H4NO2Fo4M and in complex (3).
2.6. In vitro anti-T. cruzi activity
Table 5 shows the effect of all evaluated compounds on the
growth of the epimastigote form of T. cruzi (Tulahuen 2 strain),
expressed as ID50. The ligands proved to be less active than both the
reference drug, Nfx, and the ruthenium precursors. H4NO2Fo4M
was more active than H4NO2Ac4M. The ruthenium(II) complexes
(1e6) were more active than their corresponding ligands, nifurti-
mox (Nfx), and the synthetic ruthenium(II) precursors.
Table 2
Cyclic voltammetry data (V) for complexes (1e6) (0.100 V sꢀ1, CH2Cl2, 0.1 mol Lꢀ1
,
TBAP).
Complex
RuII/RuIII RuII/RuIII Ar-NOꢁ2ꢀ Ar-
NHOH
All complexes (1e6) were at least 10 times more active than
their corresponding ligands. Complexes (1e3) and (5) revealed to
be the most active among all studied compounds. Complexes (1e3)
and (5) were 20 times more active than the free thio-
semicarbazones. The greatest amount of dose reduction upon
coordination to ruthenium was observed for complex (5).
[RuCl(H4NO2Fo4M)(bipy)(dppb)]PF6 (1)
[RuCl(H4NO2Fo4M)(Mebipy)(dppb)]PF6 (2) 0.94
[RuCl(H4NO2Fo4M)(phen)(dppb)]PF6 (3)
[RuCl(H4NO2Ac4M)(bipy)(dppb)]PF6 (4)
[RuCl(H4NO2Ac4M)(Mebipy)(dppb)]PF6 (5) 1.02
[RuCl(H4NO2Ac4M)(phen)(dppb)]PF6 (6) 1.06
0.98
0.83
0.82
0.96
0.93
0.87
0.90
e
e
ꢀ1.05
ꢀ1.16
1.09
1.02
ꢀ0.90 ꢀ1.04
e
e
ꢀ1.17
ꢀ1.13
ꢀ0.91 ꢀ1.11

2850
C. Rodrigues et al. / European Journal of Medicinal Chemistry 45 (2010) 2847e2853
Table 3
Table 4
ꢂ
ꢀ
Crystal data for H4NO2Fo4M and [RuCl(H4NO2Fo4M)(phen)(dppb)]PF6 (3).
Selected bond lengths (A) and angles ( ) for H4NO2Fo4M and [RuCl(H4NO2Fo4M)
(phen)(dppb)]PF6 (3) (standard deviations in parentheses).
Compound
H4NO2Fo4M
[RuCl(H4NO2Fo4M)
(phen)(dppb)]PF6 (3)
Bond
lengths (A) Fo4M
H4NO2
(3)
Angle (ꢂ)
H4NO2
Fo4M
(3)
ꢀ
Empirical formula
Formula weight
Temperature, K
Crystal system
Space group
C9H12N4O3S
256.29
293(2)
Triclinic
P ꢀ 1
C51H50 N6O2F6P3SCl5Ru
1296.26
150(2)
Monoclinic
P21/n
C(8)eS(1)
C(8)eN(4)
C(8)eN(3)
N(2)eN(3)
C(1)eC(7)
N(1)eO(1)
N(1)eO(2)
1.7000(18) 1.703(4) N(3)eC(8)eS(1) 118.05(14) 121.1(3)
1.323(2)
1.354(2)
1.374(2)
1.466(2)
1.228(2)
1.223(2)
1.328(4) N(4)eC(8)eS(1) 124.82(14) 121.2(3)
1.343(4) C(7)eN(2)eN(3) 116.70(16) 113.7(3)
1.381(4) C(8)eN(3)eN(2) 119.55(16) 120.5(3)
1.453(5) N(2)eC(7)eC(1) 120.77(16) 120.8(3)
1.227(4) O(2)eN(1)eO(1) 122.79(18) 123.9(3)
1.223(4)
Unit cell dimensions
ꢀ
a, A
7.8686(16)
11.236(2)
13.795(3)
83.37(3)
81.90(3)
85.10(3)
12.0023(3)
26.0686(6)
17.6136(3)
ꢀ
b, A
ꢀ
c, A
ꢂ
ꢂ
ꢂ
a
,
b
,
100.093(2)
Table 5
g
,
In vitro anti-T. cruzi activities for ligands, ruthenium complexes and synthetic
precursors.
3
ꢀ
Volume, A
1196.4(4)
5425.7(2)
4, 1.587
0.73
Z, Density calc., mg/m3 4, 1.423
ID50 (m
M)a,b,c
Absorption
0.274
coefficient, mmꢀ1
F(000)
[RuCl2(bipy)(dppb)]
[RuCl2(Mebipy)(dppb)]
[RuCl2(phen)(dppb)]
H4NO2Fo4M
[RuCl(H4NO2Fo4M)(bipy)(dppb)]PF6 (1)
[RuCl(H4NO2Fo4M)(Mebipy)(dppb)]PF6 (2)
[RuCl(H4NO2Fo4M)(phen)(dppb)]PF6 (3)
H4NO2Ac4M
[RuCl(H4NO2Ac4M)(bipy)(dppb)]PF6 (4)
[RuCl(H4NO2Ac4M)(Mebipy)(dppb)]PF6 (5)
[RuCl(H4NO2Ac4M)(phen)(dppb)]PF6 (6)
Nfx
13.7
6.9
8.1
14.2
0.7
0.6
536
2632
Crystal size, mm3
0.199 ꢃ 0.543 ꢃ 0.231
0.14 ꢃ 0.1 ꢃ 0.04
q
range for data coll., ꢂ 3.09e25.00
3.08e26.06
Index ranges
ꢀ8 ꢄ h ꢄ 9,ꢀ13 ꢄ k ꢄ 13,ꢀ ꢀ14 ꢄ h ꢄ 14,ꢀ32 ꢄ
16 ꢄ l ꢄ 16
k ꢄ 32,ꢀ17 ꢄ l ꢄ 19
34 021
Reflections collected
28 253
0.7
Independent reflections 4188 [R(int) ¼ 0.0367]
10 092 [R(int) ¼ 0.0652]
>25.0 (31%, [7])
Completeness
99.5% (to
q
¼ 25.00ꢂ)
94.8% (to
None
q
¼ 25.00ꢂ)
2.3
0.8
1.2
7.7
Absorption correction None
Refinement method
Full-matrix least-squares
on F2
4188/0/341
Full-matrix
least-squares on F2
Data/restraints/
parameters
10 092/0/686
a
ID50: dose that inhibits 50% of T. cruzi growth.
The results are the mean values of three different experiments with an SD less
b
Goodness-of-fit on F2
Final R indices
1.031
1.038
than 10% in all cases.
R1 ¼ 0.0365, R2 ¼ 0.0875
R1 ¼ 0.0442, R2 ¼ 0.1063
c
Values in parenthesis are PGI (percentage of growth inhibition) at 25 mM.
[I > 2sigma(I)]
R indices (all data)
Largest diff. peak
and hole, e A
R1 ¼ 0.0516, R2 ¼ 0.0945
0.199 and ꢀ0.167
R1 ¼ 0.0728, R2 ¼ 0.1203
0.734 and ꢀ0.882
ꢀꢀ3
Comparison of the activities of the previously developed copper
(II) complex, [Cu(4NO2Ac4M)2] (ID50 ¼ 2.0 M) [7], with those of
the presently studied ruthenium(II) complexes (4e6) with the
M) reveals that coordination
m
All ruthenium precursors revealed to be active, [RuCl2(Mebipy)
(dppb)] being the most effective. Interestingly the greatest amount
of activity improvement upon association of the precursor with
a nitro-thiosemicarbazone was verified for complex (1), which is 20
times more active than its ruthenium precursor. The anti-trypa-
nosomal activity of the studied ruthenium precursors was
demonstrated for the first time in the present work.
In all cases association of the thiosemicarbazone with ruthe-
nium, dppb and bipyridine or phenanthroline in one same complex
resulted to be an excellent strategy for activity improvement.
same ligand (ID50 ¼ 2.3, 0.8, and 1.2
m
to ruthenium is a better strategy of dose reduction.
2.7. Haemolytic assay
Red blood cell lysis is a simple method widely used to study the
damage caused by a compound to a human cell. It provides
a quantitative measure of haemoglobin release. In the present work
haemolysis was controlled spectrophotometrically 24 h after
Table 6
In vitro haemolytic activities for ligands, ruthenium complexes and synthetic
precursors.
Doses (
mM)
Haemolysis
percentage (%)
[RuCl2(Mebipy)(dppb)]
H4NO2Fo4M
50.0a
200.0b
50.0
50.0a
200.0
100.0
50.0
50.0a
e
21.6
42.3
9.0
11.1
2.6
0.0
0.0
100.0
100.0
34.4
0.0
[RuCl(H4NO2Fo4M)(Mebipy)(dppb)]PF6 (2)
H4NO2Ac4M
[RuCl(H4NO2Ac4M)(Mebipy)(dppb)]PF6 (5)
Positive control (water)
Amphotericin B
Nfx
1.5
50.0
a
Solubility problems, in the assayed milieu, were observed at higher concen-
M.
Some solubility problems, in the assayed milieu, were observed at this
concentration.
trations than 50.0
m
b
Fig. 4. Perspective view of the structure of [RuCl(H4NO2Fo4M)(phen)(dppb)]PF6 (3).

C. Rodrigues et al. / European Journal of Medicinal Chemistry 45 (2010) 2847e2853
2851
treatment with the studied compounds. We performed the assay
for selected compounds (see Table 6). The assayed compounds,
4.2. X-ray crystallography
except ruthenium complex 5, displayed lower lysis at 50.0
mM
Room temperature X-ray diffraction data collection (
4
scans and
doses than the red blood damaging agent amphotericin B at 1.5
m
M.
u
scans with
k offsets) of [RuCl(H4NO2Fo4M)(phen)(dppb)]PF6 was
Solubility problems did not allow us to determine the exact ID50
values; however the approximate ID50 values for ligands, ruthe-
nium complexes and synthetic precursors were similar to or higher
to that of Nfx.
performed on an Enraf-Nonius Kappa-CCD diffractometer (95 mm
CCD camera on
radiation (0.71073 A). Data were collected up to 50 in 2
k
-goniostat) using graphite-monochromated MoK
a
ꢂ
ꢀ
q, with
a redundancy of 4. The final unit cell parameters were based on all
reflections. Data collections were carried out using the COLLECT
program [23]; integration and scaling of the reflections were per-
formed with the HKL DenzoeScalepack system of programs [24].
Analytical absorption correction was applied [25].
3. Conclusions
The developed ruthenium complexes (1e6) present a mono-
dentate thiosemicarbazone, which is rarely found in the literature.
The foregoing results indicate that the studied compounds might
be investigated in vivo as anti-trypanosomal agents, since they are
able to in vitro inhibit the growth of the parasite. Especially complex
2, with the lowest ID50 against T. cruzi, resulted non-toxic in the red
blood cells assay, presenting a selectivity index (SI), defined as
SI ¼ ID50, red blood cells/ID50, T. cruzi > 83. Its SI was higher than the
estimated SI values for the corresponding ligand (SI w 14) and the
ruthenium synthetic precursor (SI > 7) (Table 6). Complex 5
resulted to be more toxic in this assay.
The structure was solved by direct methods with SHELXS-97
[26a]. The model was refined by full-matrix least-squares on F2 with
SHELXL-97 [26b]. All hydrogen atoms were stereochemically posi-
tioned and refined with the riding model [26b]. Hydrogen atoms of
the CH groups were set isotropic with a thermal parameter 20%
greater than the equivalent isotropic displacement parameter of the
atom to which each one was bonded. This percentage was set to 50%
for the hydrogen atoms of the CH3 group. The programs SHELXL-97
[26b], and ORTEP3 [19] were used within WinGX[27].
4.3. Syntheses of [RuCl2(bipy)(dppb)], [RuCl2(Mebipy)(dppb)] and
[RuCl2(phen)(dppb)]
Complexes 1e6 are capable to form the Ar-NOꢁ2ꢀ radical in
a potential range similar to that of nitro-containing anti-trypano-
somal drugs. Therefore intracellular nitro-moiety reduction
followed by redox cycling yielding reactive oxygen species may be
one of their modes of action against T. cruzi. However, since the
ruthenium precursors also present anti-trypanosomal activity
other modes of action could take place as well.
It has been suggested for ruthenium complexes with azoles that
RueDNA binding provides a means of damaging the parasite, and
that rapid metaleDNA binding might be a key feature of their
therapeutic action [20]. In addition, it has been suggested that in
order for DNA binding to take place, a chloro ligand must be
replaced by water [21,22].
Hence we may hypothesize that a similar mechanism of
RueDNA binding could take place in the present case. Moreover,
a synergistic effect involving both the nitro-containing ligand and
ruthenium was observed, which favored the activity improvement
upon coordination. Considering that the thiosemicarbazone binds
to the metal center as a monodentate ligand, its release is facili-
tated, which probably allows both metal and ligand to be easily
directed to their biological targets.
[RuCl2(bipy)(dppb)], [RuCl2(Mebipy)(dppb)] and [RuCl2(phen)
(dppb)] were prepared according to previously described proce-
dures [28].
4.4. Synthesis of N4-methyl-4-nitrobenzaldehyde
thiosemicarbazone (H4NO2Fo4M) and N4-methyl-4-
nitroacetophenone thiosemicarbazone (H4NO2Ac4M)
The thiosemicarbazones were prepared as described by some of
us [7,11]. Briefly, equimolar amounts of 4-nitrobenzaldehyde and
4-nitrobenzophenone (6.6 ꢃ 10ꢀ3 mol) were mixed with N4-methyl
thiosemicarbazide 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 solids which
precipitated were filtered and washed with diethyl ether and dried.
4.5. Syntheses of the complexes
The complexes were obtained by dissolving the desired thio-
semicarbazone (0.079 mmol) in CH2Cl2 (20 mL) with gentle heating
and stirring under argon atmosphere. After cooling the solution to
room temperature [RuCl2(bipy)(dppb)], [RuCl2(Mebipy)(dppb)] or
[RuCl2(phen)(dppb)] (0.079 mmol) and NH4PF6 (0.079 mmol) were
added. The reaction was stirred at room temperature for 24 h. The
solids which precipitated were filtered and washed with diethyl
ether and dried in vacuo.
4. Experimental
4.1. Physical measurements
Elemental analyses were performed on a Fison equipment,
model EA 1108. A Radiometer Copenhagen Meter Lab., model CDM
230 was employed for molar conductivity measurements. Infrared
spectra (KBr pellets) were obtained using a BOMEM MICHELSON
instrument, model 102. NMR spectra were obtained at room
temperature with a Bruker DRX-400 Avance (400 MHz) spec-
trometer. For 31P{1H} NMR (161 MHz) measurements CH2Cl2 was
used as solvent and H3PO4 85% as external reference.
The electrochemical experiments were carried out at room
temperature in CH2Cl2 containing 0.1 mol Lꢀ1 tetrabutylammo-
niumperchlorate (TBAP, Fluka Purum) using an electrochemical
analyzer from Bioanalytical Systems Inc. (BAS), model 100BW. The
working and auxiliary electrodes were stationary Pt foils, and the
reference electrode was Ag/AgCl, a medium in which ferrocene is
oxidized at 0.48 V (Fcþ/Fc). The voltammogram was performed at
a scan rate of 0.100 V sꢀ1, at 298 K.
4.5.1. [RuCl(H4NO2Fo4M)(bipy)(dppb)]PF6 (1)
Red solid. Anal. Calc. (C47H46ClF6RuN6O2P3S): C, 51.21; H, 4.21;
N, 7.62; S, 2.91%. Found: C, 51.47; H, 4.21; N, 7.66; S, 2.95%. IR (KBr,
cmꢀ1): nassNO2 1540, nsNO2 1332,
n(C]N) 1589, n(C]S) 815. Molar
conductivity (1 ꢃ 10ꢀ3 mol Lꢀ1 CH3COCH3): 84.4
m
S cmꢀ1 31P{1H}
NMR (CH2Cl2 d/ppm): 43.3 (d, 2JPeP/Hz 33.2); 41.0 (d, 2JPeP/Hz 32.2).
4.5.2. [RuCl(H4NO2Fo4M)(Mebipy)(dppb)]PF6 (2)
Red solid. Anal. Calc. (C49H50ClF6RuN6O2P3S): C, 52.06; H, 4.46;
N, 7.43; S, 2.84%. Found: C, 52.52; H, 4.50; N, 7.47; S, 2.85%. IR (KBr,
cmꢀ1): nassNO2 1538, nsNO2 1331,
n
(C]N) 1597,
n
(C]S) 814. Molar
conductivity (1 ꢃ 10ꢀ3 mol Lꢀ1 CH2Cl2): 40.6
m
S cmꢀ1 31P{1H} NMR
(CH2Cl2 d/ppm): 43.6 (d, 2JPeP/Hz 32.4); 41.2 (d, 2JPeP/Hz 32.4).

2852
C. Rodrigues et al. / European Journal of Medicinal Chemistry 45 (2010) 2847e2853
4.5.3. [RuCl(H4NO2Fo4M)(phen)(dppb)]PF6 (3)
any released haemoglobin. Once the supernatant was removed after
the last wash, the cells were suspended in PBS to get a 2% w/v red
Red solid. Anal. Calc. (C49H48ClF6RuN6O2P3S): C, 52.15; H, 4.29;
N, 7.45; S, 2.84%. Found: C, 52.46; H, 4.31; N, 7.48; S, 2.86%. IR (KBr,
blood cell solution. 400
and 200 M), negative control (solution of PBS), or amphotericin B
(1.5 M) were added to 400 L of the 2% w/v red blood cell solution in
mL of the studied compounds in PBS (50,100
cmꢀ1): nassNO2 1543, nsNO2 1344,
n
(C]N) 1597,
n
(C]S) 814. Molar
m
conductivity (1 ꢃ 10ꢀ3 mol Lꢀ1 CH2Cl2): 40.1
m
S cmꢀ1 31P{1H} NMR
m
m
(CH2Cl2
d
/ppm): 43.4 (d, 2JPeP/Hz 34.0); 41.6 (d, 2JPeP/Hz 34.0).
ten microcentrifuge tubes for each concentration and incubated for
24 h at 37 ꢂC. Complete haemolysis was attained using neat water
yielding the 100% control value (positive control). After incubation,
the tubes werecentrifuged and the supernatants weretransferredto
new tubes. The release of haemoglobin was determined by spec-
trophotometric analysis of the supernatant at 405 nm using EL 301
MICROWELL STRIP READER. Results were expressed as percentage
of the total amount of haemoglobin released by action of the
compounds. This percentage is calculated using the equation Hae-
molysis percentage (%) ¼ [(A1 ꢀ A0)/A1 water] ꢃ 100, where A1 is the
absorbance at 405 nm of the test sample at t ¼ 24 h, A0 is the
absorbance at 405 nm of the test sample at t ¼ 0 h, and A1 water is the
absorbance at 405 nm of the positive control (water) at t ¼ 24 h. The
experiments were performed in quintuplicate.
4.5.4. [RuCl(H4NO2Ac4M)(bipy)(dppb)]PF6 (4)
Orange solid. Anal. Calc. (C48H48ClF6RuN6O2P3S): C, 51.64; H, 4.33;
N, 7.53; S, 2.87%. Found: C, 51.90; H, 4.35; N, 7.58; S, 2.90%. IR (KBr,
cmꢀ1): nassNO2 1523, nsNO2 1338,
n(C]N) 1603, n(C]S) 811. Molar
conductivity (1 ꢃ 10ꢀ3 mol Lꢀ1 CH3COCH3): 100.6
m
S cmꢀ1 31P{1H}
NMR (CH2Cl2 d/ppm): 35.9 (d, 2JPeP/Hz 30.8); 30.2 (d, 2JPeP/Hz 30.9).
4.5.5. [RuCl(H4NO2Ac4M)(Mebipy)(dppb)]PF6 (5)
Orange solid. Anal. Calc. (C50H52ClF6RuN6O2P3S): C, 52.47; H,
4.58; N, 7.34; S, 2.80%. Found: C, 52.68; H, 4.59; N, 7.37; S, 2.82%. IR
(KBr, cmꢀ1): nassNO2 1520, nsNO2 1344,
n
(C]N) 1608,
n
(C]S) 813.
Molar conductivity (1 ꢃ10ꢀ3 mol Lꢀ1 CH2Cl2): 48.9
m
S cmꢀ1 31P{1H}
NMR (CH2Cl2 d/ppm): 35.8 (d, 2JPeP/Hz 32.4); 31.8 (d, 2JPeP/Hz 32.4).
Supplementary material available
4.5.6. [RuCl(H4NO2Ac4M)(phen)(dppb)]PF6 (6)
Orange solid. Anal. Calc. (C50H50ClF6RuN6O2P3S): C, 52.56; H,
4.41; N, 7.36; S, 2.81%. Found: C, 52.76; H, 4.43; N, 7.41; S, 2.84%. IR
CCDC 702060 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
(KBr, cmꢀ1): nassNO2 1519, nsNO2 1342,
n
(C]N) 1610,
n
(C]S) 812.
Molar conductivity (1 ꢃ10ꢀ3 mol Lꢀ1 CH2Cl2): 48.8
m
S cmꢀ1 31P{1H}
NMR (CH2Cl2 d/ppm): 37.5 (d, 2JPeP/Hz 34.0); 32.4 (d, 2JPeP/Hz 34.0).
Acknowledgments
4.6. In vitro anti-trypanosomal activity
Financial support from FAPEMIG, CNPq Instituto do Milênio-
Inovação e Desenvolvimento de Novos Fármacos e Medicamentos
(IM-INOFAR, Proc. CNPq 420015/05-1), CNPq-PROSUL and Fapesp
from Brazil, RIDIMEDCHAG-CYTED is acknowledged.
T. cruzi epimastigotes (Tulahuen 2 strain) were grown at 28 ꢂC in
an axenic medium (BHI-Tryptose) as previously described [7,9,10],
complemented with 5% fetal calf serum. Cells were harvested in the
late log phase, re-suspended in fresh medium, counted in Neu-
bauer’s chamber and placed in 24-well plates (2 ꢃ 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.
Before inoculation, the media were supplemented with the indi-
cated 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. Nifurtimox (Nfx) was used as the reference trypa-
nocidal drugs. The ruthenium precursors were included in these
assays to provide information on the trypanocidal effect of these
complexes. The percentage of growth inhibition was calculated as
follows {1 ꢀ [(Ap ꢀ A0p)/(Ac ꢀ A0c)]} ꢃ 100, where Ap ¼ A590 of
the culture containing the studied compound at day 5; A0p ¼ A590
of the culture containing the studied compound right after addition
of the inocula (day 0); Ac ¼ A590 of the culture in the absence of
any compound (control) at day 5; A0c ¼ A590 in the absence of the
compound at day 0. To determine ID50 values, parasite growth was
followed in the absence (control) and presence of increasing
concentrations of the corresponding compound. The ID50 values
were determined as the drug concentrations required to reduce by
half the absorbance of that of the control (without compound).
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