Complexes of different nitrogen donor heterocyclic ligands with SbCl 3 and PhSbCl2 as potential antileishmanial agents against SbIII-sensitive and -resistant parasites

Article abstract of DOI:10.1016/j.jinorgbio.2013.12.001

Novel trivalent antimony complexes with the nitrogen donor heterocyclic ligand 2,2′-bipyridine (bipy), 1,10-phenanthroline (phen) or dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq) have been synthesized by the reaction with SbCl₃ or PhSbCl₂. The crystal structures of [Sb(phen)Cl₃] and [PhSb(phen)Cl₂]CH₃COOH were determined and shown to adopt a distorted square pyramid geometry with a five-coordinated Sb center. Surprisingly, all the complexes, the ligands and PhSbCl₂ showed very high antileishmanial activities, with IC ₅₀ in the nanomolar range against Sb^III-sensitive and -resistant Leishmania infantum (syn. Leishmania chagasi) and Leishmania amazonensis strains. These compounds were much more active against these Leishmania strains than the old trivalent drug potassium antimonyl tartrate. [PhSb(phen)Cl₂]CH₃COOH complex was found to be the most active compound and the lack of cross-resistance of PhSbCl₂ suggests that the transport pathways of this compound across the cell membrane differ from those responsible for the resistance of Leishmania to Sb(OH)₃. In the case of the complexes with PhSbCl₂, our data supports the model that both ligand and metal contributed to the overall activity of the complex. Furthermore, among the complexes with SbCl₃, only bipy showed an improved activity upon complexation. Cytotoxicity evaluations of these compounds against murine peritoneal macrophages showed high selective indexes in the range of 7-70 for [Sb(phen)Cl₃], [Sb(bipy)Cl₃] and [Sb(dpq)Cl₃] complexes, being much more selective than potassium antimonyl tartrate. In conclusion, this study presents a set of new antileishmanial agents including one of the most active Sb-based compounds ever reported, which can contribute to the development of new chemotherapeutic strategies against leishmaniasis including Sb-resistant cases.

Full text of DOI:10.1016/j.jinorgbio.2013.12.001

Journal of Inorganic Biochemistry 132 (2014) 30–36
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Complexes of different nitrogen donor heterocyclic ligands with SbCl3
and PhSbCl2 as potential antileishmanial agents against SbIII-sensitive
and -resistant parasites
Edgar H. Lizarazo-Jaimes a, Priscila G. Reis b, Filipe M. Bezerra b, Bernardo L. Rodrigues a, Rubens L. Monte-Neto c,
Maria N. Melo d, Frédéric Frézard b, Cynthia Demicheli a,
⁎
a
Department of Chemistry, Institute of Exact Sciences, Federal University of Minas Gerais (UFMG), Av. Antônio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil
Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), Av. Antônio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil
Infectious Disease Research Centre, Laval University, 2705 Boulevard Laurier, RC-709, G1V 4G2 Québec, QC, Canada
Department of Parasitology, Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), Av. Antônio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 June 2013
Received in revised form 6 December 2013
Accepted 10 December 2013
Available online 19 December 2013
Novel trivalent antimony complexes with the nitrogen donor heterocyclic ligand 2,2′-bipyridine (bipy), 1,10-
phenanthroline (phen) or dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq) have been synthesized by the reaction with
SbCl3 or PhSbCl2. The crystal structures of [Sb(phen)Cl3] and [PhSb(phen)Cl2]CH3COOH were determined and
shown to adopt a distorted square pyramid geometry with a five-coordinated Sb center. Surprisingly, all the com-
plexes, the ligands and PhSbCl2 showed very high antileishmanial activities, with IC50 in the nanomolar range
against SbIII-sensitive and -resistant Leishmania infantum (syn. Leishmania chagasi) and Leishmania amazonensis
strains. These compounds were much more active against these Leishmania strains than the old trivalent drug po-
tassium antimonyl tartrate. [PhSb(phen)Cl2]CH3COOH complex was found to be the most active compound and
the lack of cross-resistance of PhSbCl2 suggests that the transport pathways of this compound across the cell
membrane differ from those responsible for the resistance of Leishmania to Sb(OH)3. In the case of the complexes
with PhSbCl2, our data supports the model that both ligand and metal contributed to the overall activity of the
complex. Furthermore, among the complexes with SbCl3, only bipy showed an improved activity upon complex-
ation. Cytotoxicity evaluations of these compounds against murine peritoneal macrophages showed high selec-
tive indexes in the range of 7–70 for [Sb(phen)Cl3], [Sb(bipy)Cl3] and [Sb(dpq)Cl3] complexes, being much more
selective than potassium antimonyl tartrate. In conclusion, this study presents a set of new antileishmanial agents
including one of the most active Sb-based compounds ever reported, which can contribute to the development of
new chemotherapeutic strategies against leishmaniasis including Sb-resistant cases.
Keywords:
Antileishmanial
Leishmania
1,10-Phenanthroline
Antimony
2,2′-Bipyridine
Dipyridoquinoxaline
© 2013 Elsevier Inc. All rights reserved.
1. Introduction
Other mechanisms such as a diminished biological reduction of SbV to
SbIII [8], the loss of an aquaglyceroporin (AQP1) allele or its down regu-
Pentavalent antimonials such as sodium stibogluconate and
meglumine antimoniate, have been used in the treatment of all forms
of leishmaniasis for more than half a century [1,2]. Although the mech-
anism of action of pentavalent antimonial is not fully understood, it is
generally accepted that the active form of the metal is the reduced
form SbIII [3,4].
A major problem in antimonial chemotherapy is the emergence of
clinical resistance against pentavalent antimony drugs that has reached
epidemic proportions in parts of India [5]. The ATP binding cassette
(ABC) protein MRPA plays a major role in metal resistance in Leishmania
parasites [6] and its localization in intracellular vesicle membranes sug-
gests that it sequesters SbIII-thiol complexes into these vesicles [7].
lation [9,10] and hypoxic conditions [11] have been reported to cause an
increase in resistance to pentavalent antimonials. In this context, there
is a great need for new safe and effective drugs that do not exhibit
cross-resistance with conventional antimonial drugs.
It was suggested that the mode of action of trivalent antimonial
compounds involves some pathways similar to apoptosis, as DNA frag-
mentation [12,13], which is preceded by an increase in reactive oxygen
species caused by alterations of the redox potential [14]. As an
antileishmanial agent, SbIII inhibits and forms a complex with the en-
zyme trypanothionereductase which acts by recycling trypanothione
disulfides in trypanothione, the major antioxidant thiol in Leishmania
[15,16]. Furthermore, zinc finger domains in proteins, which were asso-
ciated with protein–nucleic acid interactions [17,18], were recently pro-
posed as potential targets for SbIII, due to the ability of SbIII to compete
with ZnII for its binding to the CCHC and CCCH zinc finger domains
[19,20].
⁎
Corresponding author. Tel.: +55 31 3409 5755; fax: +55 31 3409 5700.
E-mail address: demichel@netuno.lcc.ufmg.br (C. Demicheli).
0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2013.12.001

E.H. Lizarazo-Jaimes et al. / Journal of Inorganic Biochemistry 132 (2014) 30–36
31
In the search for safer and more effective SbIII complexes, our group
recently investigated dipyrido[3,2-a:2′,3′-c]phenazine (dppz) as a
polypyridyl ligand [21]. The resulting antimony complex showed a
high antileishmanial activity in the micromolar range, but the metal
did not seem to be active by itself. It was proposed that complexation
decreased the lipophilicity of dppz, resulting in enhanced dppz activity.
As a continuation, the present study investigates for the first time
the antileishmanial activity of SbIII complexes with three other nitrogen
donor heterocyclic ligands: a less lipophilic polypyridyl ligand dipyrido
[3,2-d:2′,3′-f]quinoxaline (dpq), 2,2′-bipyridine (bipy) and 1,10-
phenanthroline (phen). PhSbCl2 was also evaluated as a new active
form of SbIII, as an attempt to enhance the biological activity of the
metal complex.
It is noteworthy that phen was shown to exhibit antileishmanial ac-
tivity against Leishmania braziliensis and Leishmania amazonensis
[22,23], which was attributed to its ability to inhibit Leishmania protein-
ases through chelation of Zn ion [23–27]. More recently, ternary
copper(II) as well as oxidovanadium(IV) complexes bearing phen ligand
were also presented as promising antileishmanial compounds [28,29].
The present work reports the synthesis, the structural and physico-
chemical characterization of the complexes between the nitrogen donor
heterocyclic ligands and SbCl3 or PhSbCl2. The ligands (dpq, bipy and
phen), SbCl3, PhSbCl2 and their resulting complexes were tested against
both SbIII-sensitive and -resistant Leishmania infantum (syn. Leishmania
chagasi) and L. amazonensis promastigotes. The cytotoxicity of these com-
pounds towards murine peritoneal macrophages is also reported.
Table 1
Crystal data and structure refinement parameters for [Sb(phen)Cl3] and [PhSb(phen)Cl2]
CH3COOH.
Complex
[Sb(phen)Cl3]
[PhSb(phen)Cl2]CH3COOH
Chemical formula
Formula weight
Temperature (K)
Crystal system
Space group
a (Å)
b (Å)
c (Å)
α (o)
β (o)
C12H8Cl3N2Sb
408.30
293 (2)
Triclínic
P-1
8.4284 (2)
8.5966 (2)
9.9016 (3)
82.401 (2)
72.928 (3)
76.798 (2)
666.05 (3)
2
392
2.036
0.38 × 0.38 × 0.15
2.16–32.82
25,514
4680
0.0344
1.000/0.706
C22H19Cl2N2O2Sb
510.03
150 (2)
Monoclínic
C2/c
19.4337 (17)
13.8280 (4)
18.9136 (13)
90
129.481 (12)
90
3923.0 (5)
4
2016
1.727
γ (o)
V (Å3)
Z
F (000)
Dcalc (mg/m3)
Crystal dimensions mm3
θ Range (°)
Reflections collected
Independent reflection
Rint
Maximum/minimum
transmission
Data/restraints/
parameters
Goodness-of-fit on F2
Final R indices
[I N 2σ(I)]
0.30 × 0.29 × 0.09
2.00–29.5
10,400
4686
0.025
1.000/0.742
4680/0/163
4680/0/246
1.131
1.190
R1 = 0.0254,
wR2 = 0.064
R1 = 0.0354,
wR2 = 0.0644
0.5 and −0.79
R1 = 0.0335,
wR2 = 0.0935
R1 = 0.0458,
wR2 = 0.1085
1.53 and −0.81
R indices (all data)
2. Experimental
Largest difference in
peak/hole (e·Å−3
)
2.1. Materials
phen, bipy, SbIII chloride (SbCl3), SbPh3 and potassium antimonyl(III)
tartrate were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq) was synthesized according
to Liang et al. [30].
Phenyl antimony(III) dichloride (PhSbCl2) was prepared as described
previously [31] by reorganization of 1/2 molar mixtures of SbPh3 and
SbCl3 at 25 °C. The solid PhSbCl2 was isolated from the resulting viscous
liquid through crystallization from glacial acetic acid. All the preparations
were done under anhydrous conditions.
Atlas Gemini ultra. Crystallographic data and experimental details of
the structure determinations are listed in Table 1.
The stability of the complexes in aqueous solution was checked by
UV absorption spectroscopy. For this purpose, the complexes and their
respective ligand were diluted at 40 μM in phosphate-buffered saline
(PBS: 0.15 M KCl, 0.05 M phosphate, pH 7.0) from 4 mM stock
solutions in DMSO. UV spectra were registered just after dilution and
after 4-h of incubation at 25 °C. The spectra of the complexes were dif-
ferent from those of their respective ligands and did not suffer signifi-
cant change after 4-h of incubation (see Supplementary materials).
This data indicates that the complexes do not readily dissociate upon di-
lution in aqueous solution.
2.2. Synthesis of the complexes
A solution of SbCl3 or PhSbCl2 (0.2 mmol) dissolved in chloroform
(10 mL) was added dropwise to a chloroform solution (15 mL) contain-
ing 0.2 mmol of polypyridyl ligand (2,2′-bipyridine, 1,10-phenanthroline
or dipyrido[3,2-d:2′,3′-f]quinoxaline). The mixture was stirred in reflux
system for 1 h at room temperature. Then the mixture was filtered, the
precipitate was collected, washed with 20 mL of fresh chloroform and
then dried in vacuo. The phen complexes were recrystallized in DMSO.
2.3.1. [Sb(bipy)Cl3]
The bipy complex was characterized by infrared spectra, 1H NMR, 13
C
NMR and elemental analysis. Yield: 87% (67.0 mg). IR (KBr, cm−1): 1600,
1584, 1526 ν(C_C, C_N); 760 δ(Csp2-H). 1H NMR (DMSO-d6 400 MHz)
δ: 8.72 (d, H6,9, J = 4 Hz); 8.41 (d, H3,12, J = 8 Hz); 7.99 (t, H4,11
,
J = 8Hz); and 7.49 (t, H5,10, J = 6 Hz). 13C NMR (DMSO-d6) δ: 120.83
(C3,12), 124.58 (C5,10), 137.98 (C4,11), 148.95 (C6,9), and 154.30 (C2,7).
Elemental analysis for C10H8Cl3N2Sb, calc. (found): C% 31.25 (31.24);
H% 2.10 (2.04); and N% 7.29 (7.60).
2.3. Physicochemical characterizations
Elemental analyses for C, H and N were carried out using a
Perkin Elmer 2400 CHNS/O elemental analyzer. IR spectra in the
4000–400 cm−1 region were obtained from KBr pellets using the Perkin
Elmer RX-83303 FTIR spectrophotometer.
2.3.2. [Sb(phen)Cl3]
Conductivity of the complexes was measured in DMSO at 10−3
M
The phen complex was characterized by infrared spectra, 1H NMR, 13
C
using DM31 Digimed apparatus equipped with a conductivity cell
(1.185 cm−1). All measurements were carried out at room temperature
with freshly prepared solutions.
NMR spectra were recorded at 200 MHz using Bruker DPX-200
spectrometer. 1H and 13C NMR chemical shifts were measured relative
to tetramethylsilane (TMS) and dimethyl sulfoxide-d6 (DMSO-d6) as
the solvent. Intensity data for the X-ray were collected with Xcalibur
NMR, elemental analysis and X-ray diffraction. Yield: 88% (71.5 mg). IR
(KBr, cm−1): 1574, 1516, 1424 ν(C_C, C_N); 716 δ(Csp2-H). 1H NMR
(DMSO-d6 400 MHz) δ: 9.60 (dd, H1,10, J = 4 Hz); 8.96 (dd, H3,8
,
J = 8 Hz); 8.29 (s, H5,6); and 8.18 (dd, H2,9, J = 8 Hz). 13C NMR
(DMSO-d6) δ: 125.72 (C2,9), 127.62 (C5,6), 129.72 (C4,7), 138.60 (C3,8),
141.32 (C11,12), and 147.75 (C1,10). Elemental analysis for C12H8Cl3N2Sb,
calc. (found): C% 35.30 (35.67); H% 1.97 (1.98); and N% 6.86 (6.96).

32
E.H. Lizarazo-Jaimes et al. / Journal of Inorganic Biochemistry 132 (2014) 30–36
Table 3
2.3.3. [PhSb(bipy)Cl2]
The bipy complex was characterized by infrared spectra, 1H NMR, 13
C
Selected bond length (Å) and angles (°) for complex [PhSb(phen)Cl2]CH3COOH.
NMR, and elemental analysis. Yield: 85% (72.0 mg). IR (KBr, cm−1):
1597, 1592, 1528 ν(C_C, C_N); 764 δ(Csp2-H). 1H NMR (DMSO-d6
400 MHz) δ: 8.72 (d, H6,9, J = 4 Hz); 8.42 (d, H3,12, J = 8 Hz); 7.94–
8.08 (m, H14,18,4,11); and 7.51–7.28 (m, H5,10,15,17,16). 13C NMR
(DMSO-d6) δ: 120.59 (C3,12), 124.35 (C5,10), 127.57 (C15,17), 128.44
(C16), 134.57 (C14,18), 137.56 (C4,11), 149.17 (C6,9), 154.89 (C13), and
160.19 (C2,7). Elemental analysis for C16H13Cl2N2Sb, calc. (found): C%
45.12 (44.34); H% 3.08 (3.10); and N% 6.58 (6.64).
Ligation
Distance(Å)
Angle
(°)
87.37 (9)
Sb(1)–Cl(1)
Sb(1)–Cl(2)
Sb(1)–N(1)
Sb(1)–N(2)
Sb(1)–C(21)
N(1)–C(1)
N(1)–C(5)
N(2)–C(9)
N(2)–C(12)
2.6168 (9)
2.5723 (10)
2.418 (3)
2.377 (3)
2.178 (4)
1.323 (4)
1.366 (4)
1.370 (4)
1.328 (4)
C(21)–Sb(1)–Cl(1)
Cl(2)–Sb(1)–Cl(1)
N(1)–Sb(1)–Cl(1)
N(2)–Sb(1)–Cl(1)
C(21)–Sb(1)–Cl(2)
N(1)–Sb(1)–Cl(2)
N(2)–Sb(1)–Cl(2)
C(21)–Sb(1)–N(1)
C21–Sb1–N2
104.78 (3)
162.33 (7)
94.41 (7)
88.52 (10)
90.47 (7)
158.64 (8)
84.12 (11)
83.00 (11)
69.23 (9)
N(2)–Sb(1)–N(1)
2.3.4. [PhSb(phen)Cl2]CH3COOH
The phen complex was characterized by infrared spectra, 1H
NMR,13C NMR, elemental analysis and X-ray diffraction. Yield: 86%
(87.4 mg). IR (KBr, cm−1): 1576, 1518, 1428 ν(C_C, C_N); 724
δ(Csp2-H). 1H NMR (DMSO-d6 400 MHz) δ: 11.92 (s, OH); 9.38 (dd,
H1,12, J = 4 Hz); 8.81 (d, H3,10, J = 12 Hz); 8.18 (s, H6,7); 8.09–8.02
(m, H22,26 and H2,11); 7.33–7.16 (m, H24 and H23,25); and 1.91 (s, H31).
13C NMR (DMSO-d6) δ: 125.01 (C2,11), 127.22 (C23,25), 127.35 (C6,7),
127.84 (C24), 129.34 (C4,8), 135.10 (C22,26), 139.93 (C3,10), 140.54
(C5,9), 148.54 (C1,12), and 161.13 (C21). Elemental analysis for
2.5. In vitro antileishmanial assay
2.5.1. Parasite strains
L. (Leishmania) amazonensis (strains MHOM/BR/1989/BA199,
sensitive and resistant to SbIII at concentration up to 2700 μM) and
L. (Leishmania) infantum (syn. L. chagasi) (strains MCAN/BR/2002/
BH400, sensitive and resistant to SbIII at concentration up to 2700 μM)
promastigotes were maintained in minimum essential culture medium
(alpha-MEM) (Gibco, Invitrogen NY, USA) supplemented with 10% (v/
v) heat inactivated fetal calf serum (FBS, Cultilab, Campinas, SP,
Brazil), 100 mg/mL kanamycin, 50 mg/mL ampicillin, 2 mM L-gluta-
mine, 5 mg/mL hemin, 5 mM biopterin (Sigma-Aldrich, St Louis, USA),
at pH 7.0 and incubated at 25 °C. L. amazonensis and L. infantum (syn.
L. chagasi) were selected for SbIII resistance as described previously
[33]. The SbIII-resistant mutants L. amazonensis BA199 SbIII 2700.2 and
L. infantum (syn. L. chagasi) BH400 SbIII 2700.2 were selected in
25 cm2 flasks containing 5 mL of alpha-MEM in the presence of increas-
ing SbIII concentrations up to 2700 μM. The concentration that inhibits
the parasite growth by 50% (IC50) was calculated by non-linear regres-
sion using the GraphPad Prism 5 software.
C20H17Cl2N2O2Sb, calc. (found): C% 47.10 (47.14); H% 3.36 (3.20); and
N% 5.49 (5.42).
2.3.5. [Sb(dpq)Cl3]
The dpq complex was characterized by infrared spectra, 1H NMR, 13
C
NMR and elemental analysis and. Yield: 77% (71.0 mg). IR (KBr, cm−1):
1620, 1582, 1536 ν(C_C, C_N); 1124 δ(Csp2-H). 1H NMR (DMSO-d6
400 MHz) δ: 9.72 (d, H7,16, 8 Hz); 9.37 (d, H5,18, 4 Hz); 9.30 (s,
H
11,12); and 8.29 (t, H6,17, 6 Hz). 13C NMR (DMSO-d6) δ: 126.60 (C6,17),
128.02 (C8,15), 136.99 (C7,16), 138.79 (C9,14), 140.69 (C2,3), 146.67
(C11,12), and 149.45 (C5,18). Elemental analysis for C14H8Cl3N4Sb, calc.
(found): C% 36.52 (36.10); H% 1.75 (2.00); and N% 12.17 (12.10).
2.4. X-ray crystallography
2.5.2. Antileishmanial activity
Single crystal X-ray diffraction data were obtained at room tem-
perature on a Xcalibur Atlas Gemini Ultra diffractometer, using
graphite mono chromate MoKα radiation (λ = 0.71069 Å). Final
unit cell parameters and the integration of the collected reflections
were performed using the CRYSALISPRO software (version
1.171.33.55 release 05-01-2010 CrysAlis171.NET). The structure
solutions and full-matrix least-squares refinements based on F2
were performed with the SHELXS-97 and SHELXL-97 program pack-
age [32]. All atoms except hydrogen were refined anisotropically.
Although many hydrogen atoms could be identified in a Fourier dif-
ference map all of them were geometrically added to the structure
and then refined by the riding model in the final stages. Details of
data collection and structure refinement are given in Table 2. Select-
ed distances and angles are given in Table 3. The crystallographic
data were deposited at the Cambridge Crystallographic Data Centre
(CCDC 946085 and 946086—www.ccdc.cam.ac.uk).
Compounds were evaluated in vitro for their activity against both
SbIII-sensitive and -resistant Leishmania parasites, as described
previously [21]. Briefly, log-phase L. amazonensis and L. infantum (syn.
L. chagasi) promastigotes (1 × 106 parasites/mL) were seeded in
24-well cell culture plates with 1.5 mL of alpha-MEM. The cells were in-
cubated under shaking at 25 °C for 72 h in the presence of seven differ-
ent concentrations of the compound to be tested. Non-treated parasites
were established for growth comparison. Stock solutions of the drugs
were prepared in DMSO and diluted in alpha-MEM cell culture medium
to obtain the range of tested concentrations. The final DMSO concentra-
tion did not exceed 0.2%, which is known to be nontoxic to Leishmania
parasites [34]. For drug susceptibility assays, Leishmania growth curves
were constructed by measuring absorbance at 600 nm. Antileishmanial
activity is expressed as IC50/72 h, which is the compound concentration
that reduces cell growth by 50% compared to untreated control (relative
growth). All experiments were performed in triplicate.
2.6. Cytotoxicity assay against murine peritoneal macrophages
Table 2
Selected bond length (Å) and angles (°) for complex [Sb(phen)Cl3].
The cytotoxicity of the compounds towards murine peritoneal mac-
rophages was evaluated using the classical 3-(4,5-dimethylthiazol-2-
yl)-2,5 diphenyltetrazolium bromide (MTT) method [35]. Briefly, mac-
rophages were obtained by lavage of the peritoneal cavities of Swiss
mice with 10 mL cold RPMI-1640 (Gibco, Invitrogen NY, USA) without
FBS. After washing, the cell suspension (4.0 × 106/mL) was seeded
(0.1 mL) in 96-well flat bottom plates. Macrophages were allowed to
adhere for 1 h at 37 °C and non-adherent cells were removed by wash-
ing with RPMI. The compounds to be studied were then added to the
wells at varying concentrations and the cells were further cultured in
RPMI supplemented with 10% FBS for 24 h at 38 °C in a humidified 5%
Ligation
Distance(Å)
Angle
(°)
Sb(1)–Cl(1)
Sb(1)–Cl(2)
Sb(1)–Cl(3)
Sb(1)–N(1)
N(1)–C(1)
N(1)–C(12)
Sb(1)–N(2)
N(2)–C(10)
C(11)–N(2)
2.5805 (6)
2.5013 (6)
2.5493 (7)
2.3963 (17)
1.332 (3)
1.355 (2)
2.2408 (16)
1.334 (2)
Cl(3)–Sb(1)–Cl(1)
Cl(2)–Sb(1)–Cl(1)
Cl(2)–Sb(1)–Cl(3)
N(1)–Sb(1)–Cl(3)
N(2)–Sb(1)–Cl(3)
N(1)–Sb(1)–Cl(2)
N(2)–Sb(1)–Cl(2)
N(1)–Sb(1)–Cl(1)
N(2)–Sb(1)–Cl(1)
N(2)–Sb(1)–N(1)
161.38 (2)
86.87 (2)
95.61 (3)
89.78 (5)
80.01 (5)
155.13 (4)
85.97 (4)
80.69 (4)
81.76 (5)
71.05 (6)
1.364 (2)

E.H. Lizarazo-Jaimes et al. / Journal of Inorganic Biochemistry 132 (2014) 30–36
33
CO2 atmosphere. Thereafter, the medium was replaced with fresh RPMI
containing 0.5 mg/mL of MTT and the plates were incubated for an ad-
ditional 4 h. Supernatants were aspirated, and the formazan crystals
formed were dissolved in 100 μL of DMSO. After 15 min of incubation
at room temperature, the absorbance of solubilized MTT formazan
product was measured spectrophotometrically at 570 nm. Cell viability
was calculated from the ratio of the absorbance of the well treated with
the drug to that of the non-treated well. The concentration of drug that
decreased cell viability by 50% (CC50) was determined.
3. Results and discussion
3.1. Synthesis and characterization of antimony complexes
The SbIII complexes were prepared by direct reaction of ligands (bipy,
phen or dpq) with 1:1 mole ratio of SbCl3 or PhSbCl2 in chloroform under
inert conditions. The conductivity of the complexes in DMSO (10−3 M)
at room temperature (13.70–27.00 μS/cm) showed that they are non-
electrolytes. The elemental analysis of [Sb(bipy)Cl3], [PhSb(bipy)Cl2],
[Sb(phen)Cl3], [PhSb(phen)Cl2]CH3COOH and [Sb(dpq)Cl3] was in agree-
ment with the proposed molecular formulas and structures (Figs. 1, 2 and
3). The IR spectra of the complexes in the 1600–400 cm−1 region were
consistent with coordinated bipy, phen and dpq [36,37]. The polypyridyl
ligand bands corresponding to C\C and C\N stretching (1562, 1506,
1422 cm−1 for phen; 1578, 1556, 1530 cm−1 for bipy; 1582, 1572,
1520 cm−1 for dpq) showed considerable changes in the IR spectra of
the complexes. Moreover, the strong band of bipy at 758 cm−1 with a
shoulder at 740 cm−1 splitted upon coordination to 772 and 760 cm−1
in [Sb(bipy)Cl3] complex, and to 764 and 742 cm−1 in [PhSb(bipy)Cl2]
complex. The 725 cm−1 band of phen, assigned to the out of plane mo-
tion of the three adjacent hydrogen atoms on the heterocyclic rings,
also exhibited marked changes upon the formation of the complexes.
The unequivocal proton NMR assignment was achieved by concerted
analysis of 1D 1H-, 1D 13C-, 2D 1H 1H COSY and 2D 1H 13C-HMQC. The
formation of the complexes was also supported by downfield shift in
the proton resonance for the protons adjacent to the coordinating nitro-
gen atoms of all ligands: Δδ 0.06–0.11 ppm in [Sb(bipy)Cl3], Δδ 0.03–
0.06 ppm in [PhSb(bipy)Cl2], Δδ 0.51–0.59 ppm in [Sb(phen)Cl3], Δδ
0.31–0.44 ppm in [PhSb(phen)Cl2]CH3COOH and Δδ 0.13–0.32 ppm in
[Sb(dpq)Cl3].
Fig. 2. Molecular structure of [Sb(phen)Cl3].
to the antimony center for complexes [Sb(phen)Cl3] and [PhSb(phen)
Cl2]CH3COOH are listed in Tables 2 and 3, respectively.
The geometry around the metal center, in both complexes, is
distorted square pyramidal (SP), with N(2) as the apex and N(1),
Cl(1), Cl(2) and Cl(3) occupying the equatorial plane for [Sb(phen)Cl3]
(Fig. 2) and with C(21) as the apex and Cl(1), Cl(2), N(1) and N(2) oc-
cupying the equatorial plane for [PhSb(phen)Cl2]CH3COOH (Fig. 3).
The root mean square deviation of the plane through atoms Sb, N(1),
Cl(1), Cl(2) and Cl(3) of [Sb(phen)Cl3] is equal to 0.182 Å, with the Sb
atom distant 0.3528(4) Å from this plane. This suggests some distortion
from an ideal square pyramidal geometry.
In contrast, The root mean square deviation of the plane through
atoms Sb, N(1), N(2), Cl(1) and Cl(2) of complex [PhSb(phen)Cl2]CH3-
COOH is equal to 0.069 Å, with the Sb atom being the most distant
from the mean plane. The distance from this atom to the plane is
equal to 0.1373(8) Å.
From these values, it is possible to conclude that the equatorial coor-
dination of antimony in [PhSb(phen)Cl2]CH3COOH is almost planar, in
contrast to the coordination in [Sb(phen)Cl3]. The sums of equatorial
angles for the two complexes corroborate this conclusion: for
[Sb(phen)Cl3] this sum is equal to 352.95° (Cl(3)–Sb–Cl(2) 95.61 (3)°,
Cl(3)–Sb–N(1) 89.78 (5)°, N(1)–Sb–Cl(1) 80.69 (4)°, and Cl(1)–Sb–
Cl(2) 86.87 (2)°), while for [PhSb(phen)Cl2]CH3COOH the sum is
equal to 358.89° (N(1)–Sb(1)–Cl(2) 90.47 (7)°, Cl(2)–Sb(1)–Cl(1)
104.78 (3)°, N(2)–Sb(1)–Cl(1) 94.41 (7)°, and N(2)–Sb(1)–N(1) 69.23
(9)°), suggesting a slight distortion from an ideal square pyramidal
[38]. As shown in Fig. 3, the asymmetrical unit in the crystal contains
one molecule of acetic acid besides the [PhSb(phen)Cl2] molecule.
3.2. Crystal structures
The structure of [Sb(phen)Cl3] and [PhSb(phen)Cl2]CH3COOH was
characterized by X-ray crystallography. The precursor, phenyl
antimony(III) dichloride (PhSbCl2) was prepared in acetic acid and
used without further purification. The presence of acetic acid in the re-
action medium led to cocrystallization of this solvent with [PhSb(phen)
Cl2] complex. Crystal parameters and structure refinement details are
summarized in Table 1. The selected bond lengths and angles relevant
Fig. 1. Molecular structures of [Sb(bipy)Cl3], [PhSb(bipy)Cl2] and [Sb(dpq)Cl3] complexes; ligand atoms are numbered for NMR spectral assignments.

34
E.H. Lizarazo-Jaimes et al. / Journal of Inorganic Biochemistry 132 (2014) 30–36
IC50 greater than 80 μM for WT lines, and was almost inactive against
Sb-resistant mutants (Table 4). It is noteworthy that these complexes
are much more active than [Sb(dppz)Cl3]∙H2O∙CH3OH (Sb-dppz com-
plex) [21] and this difference may be due to the less lipophilic character
of the actual compounds.
Surprisingly, the ligands and PhSbCl2 also showed very high
antileishmanial activities (Table 4), with IC50 values in the range of
0.6–22 nM and 0.7–1.8 nM, respectively. The lipophilicity of PhSbCl2
can explain its higher activity, when compared to SbCl3 or TA (Table 4).
Another interesting feature is the lack of cross-resistance of PhSbCl2
with SbCl3. These results suggest that PhSbCl2 is not recognized by
the membrane transporters involved in the resistance of Leishmania to
SbIII [6].
To evaluate the impact of antimony complexation of the nitrogen
donor ligands on their antileishmanial activity, IC50 values were com-
pared between the ligand and its respective complexes (Table 4).
In the case of phen ligand, complexation resulted in enhanced
antileishmanial activity, especially with PhSbCl2 compound. The fact
that both PhSbCl2 and phen showed IC50 in the nanomolar range sug-
gests that both phen and the metal contributed to the overall activity
of the complex. On the other hand, the activity of [Sb(phen)Cl3] may
be attributed mainly to the ligand.
Fig. 3. Molecular structure of [PhSb(phen)Cl2]CH3COOH.
3.3. Antileishmanial activity of nitrogen donor heterocyclic ligands and
their antimony complexes
In the case of dpq ligand, only small changes in antileishmanial activ-
ity were observed upon complexation with antimony. A slight increase
of activity was observed only in L. amazonensis strains following com-
plexation with PhSbCl2. Thus, it is suggested that essentially the ligand
may contribute to the activity of dpq complexes with SbCl3.
In the case of the less active bipy ligand, a marked increase of activity
was observed in the L. infantum strains following complexation with
both metal forms and, in the L. amazonensis strains, following complex-
ation with PhSbCl2 only. This data supports the model that both bipy
and the metal contributed to the activity of the complex. It also suggests
that L. amazonensis and L. infantum species exhibit differences in their
drug metabolism.
Thus, our data strongly suggest that PhSbCl2-derived complexes
exert their cytotoxicity through both the ligand and the metal. On the
other hand, the mode of action of SbCl3-derived complexes is less
clear, since the ligands are cytotoxic in the same concentration range
as the complexes and are much more active than SbCl3 or TA. Overall
the ligands bipy, phen and dpq are small, planar and non-polar mole-
cules that can readily cross the Leishmania cell membrane and form
complexes with intracellular transition metal ions triggering their in-
trinsic antileishmanial effect reported here. In the case of [Sb(bipy)Cl3]
that is more active than bipy, the ligand could act as a Sb carrier facilitat-
ing its delivery, leading to a synergistic effect of both ligand and metal
against the parasite. However, the lack of cross-resistance of [Sb(bipy)
Cl3] with SbCl3 or TA, i.e. [Sb(bipy)Cl3] is equally effective against Sb-
The ligands, the antimony(III) chlorides and their complexes
were evaluated for their activity against SbIII-sensitive and -resistant
Leishmania strains from two different New World Leishmania species:
L. infantum (syn. L. chagasi), the etiological agent for visceral leishmani-
asis [39] and L. amazonensis, related to the cutaneous form of the disease
in the New World [40]. These two species lead to different clinical man-
ifestations, suggesting different metabolism and consequently distinct
drug sensitivity. This observation highlights the importance of using dif-
ferent Leishmania species in drug screening [41], as well as strains with
different susceptibilities to antimonials.
Before starting the biological assays, we first checked the stability of
the complexes in water by UV absorption spectroscopy. The UV spectra
of the complexes at 40 μM in water were found to differ from those of
their respective ligand and did not suffer significant change upon incu-
bation for 30 min at 25 °C (data not shown).
All the antimony complexes were found to be highly active against
all Leishmania strains with IC50 in the 0.25–19 nM range (Table 4).
[PhSb(phen)Cl2]CH3COOH compound was the most effective complex.
Strikingly, the IC50 values for Sb complexes were similar when compar-
ing the Sb-resistant mutants with their respective parental sensitive WT
strains, except for [PhSb(phen)Cl2]CH3COOH and [Sb(dpq)Cl3] that
were 5 and 2.5 times less active in the resistant L. infantum strain, re-
spectively. These data are in contrast with that obtained with the con-
ventional SbIII complex potassium antimonyl tartrate which showed
Table 4
Growth inhibitory concentrations of the nitrogen donor heterocyclic ligands and their SbIII complexes towards antimony-sensitive and -resistant New World Leishmania species.
aIC50
SEM (nM)
Compound
L. infantum
L. infantum
L. amazonensis
L. amazonensis
SbS
SbR
SbS
SbR
[Sb(phen)Cl3]
[PhSb(phen)Cl2]CH3COOH
[Sb(bipy)Cl3]
[PhSb(bipy)Cl2]
[Sb(dpq)Cl3]
Phen
Bipy
Dpq
SbCl3
1.66
0.26
1.34
2.21
0.43
7.8
15.8
0.69
(341
1.79
(86
0.36
1.93
1.26
1.87
2.54
1.15
2.29
21.7
0.85
(462
1.48
0.28
0.78
0.47
18.9
1.69
2.28
0.84
16.2
2.23
(362
0.97
(110
0.18
0.08
2.0
0.14
0.19
0.13
1.6
0.47
0.28
9.1
1.65
1.62
0.49
7.4
0.22
0.04
2.4
0.30
0.28
0.08
1.8
0.40
2) × 103
0.07
0.12
0.23
0.28
0.10
2.4
1.5
0.32
0.14
0.13
0.32
0.49
2.7
0.13
0.23
3) × 103
0.08
0.20
1.52
(1502
0.74
2) × 103
0.56
2) × 103
0.07
PhSbCl2
bTA
2) × 103
N3000 × 103
1) × 103
N3000 × 103
a
IC50, concentration of the compound that inhibits 50% of parasite growth; SEM, standard error of the mean. SbS: Sb-sensitive; SbR: Sb-resistant.
TA: potassium antimonyl tartrate as SbIII source.
b

E.H. Lizarazo-Jaimes et al. / Journal of Inorganic Biochemistry 132 (2014) 30–36
35
Table 5
and [PhSb(phen)Cl2]CH3COOH organometallic complex showed a
distorted square pyramid geometry with a five-coordinated Sb center.
All the complexes, the ligands and PhSbCl2 showed very high
antileishmanial activities, with IC50 in the nanomolar range against
SbIII-sensitive and -resistant L. infantum (syn. L. chagasi) and
L. amazonensis strains. These compounds were much more active against
these Leishmania strains than potassium antimonyl tartrate. In the case
of the complexes with PhSbCl2, both the ligand and the metal contribut-
ed to the overall activity of the complex. Among the complexes formed
from SbCl3, only bipy showed an improved activity upon complexation.
PhSbCl2 emerges as a new highly active form of SbIII capable of bypassing
the resistance mechanisms of Leishmania to antimony. However, prog-
ress towards new antimonial drugs still depends on the achievement
of reduction of the cytotoxicity of the metal against the host mammalian
cells.
Cytotoxicity of the nitrogen donor heterocyclic ligands and their SbIII complexes against
mouse peritoneal macrophages and resulting selective index.
Compound
aCC50 (nM)
bSI
bSI
macrophages
(L. infantum)
(L. amazonensis)
[Sb(phen)Cl3]
[PhSb(phen)Cl2]
CH3COOH
[Sb(bipy)Cl3]
[PhSb(bipy)Cl2]
[Sb(dpq)Cl3]
Phen
Bipy
Dpq
PhSbCl2
TA
12.4
0.91
1.2
0.10
7.5
3.5
16
1.9
38.3
2.11
30.1
37.4
1.1
0.14
3.2
28.6
0.95
70
2.0
1.2
13.2
3.9
4.8
44
N20 × 103
N8 × 103
N1265
N11,594
2.4
N1234
N3587
4.4
4.29
(63
0.36
1)x103
0.73
0.57
a
CC50: concentration of the compound that kills 50% of macrophages according to MTT
assay.
b
SI: selective index, calculated as the ratio between CC50 in murine macrophages and
Abbreviations
IC50 in the WT Leishmania strains.
RPMI
MTT
Roswell Park Memorial Institute (cell culture medium)
3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide
sensitive and -resistant strains, argues against a cytotoxic action of the
metal as Sb(OH)3 which is recognized by the cellular mechanisms of re-
sistance. As alternative explanations for the high activity of [Sb(bipy)
Cl3], the complex may act as a new chemical entity or the metal may im-
prove the bioavailability of the ligand through for instance the increase
of its stability in the biological medium.
alpha-MEM minimum essential culture medium
phen
dpq
bipy
dppz
PBS
1,10-phenanthroline
dipyrido[3,2-d:2′,3′-f]quinoxaline
2,2′-bipyridine
dipyrido[3,2-a:2′,3′-c]phenazine
phosphate-buffered saline
3.4. Cytotoxicity of nitrogen donor heterocyclic ligands and their antimony
complexes against murine peritoneal macrophages
Acknowledgments
The antimony complexes and their ligand were also evaluated for
their cytotoxicity against macrophages, which is one of the host mam-
malian cell in which Leishmania parasite resides, in order to determine
their parasite-to-host selectivity index (SI).
The authors are grateful to the Brazilian agencies CNPq, FAPEMIG
and CAPES for the financial support.
Appendix A. Supplementary data
When compared to their respective ligand, all complexes showed an
enhanced cytotoxicity, the effect being much more pronounced in the
case of bipy and dpq ligands which were not cytotoxic in the concentra-
tion range tested (Table 5). The SbIII complexes displayed nanomolar
cytotoxic concentrations, with CC50 values in the range of 0.9–2.1 nM
and 12–39 nM for the complexes obtained from PhSbCl2 and SbCl3, re-
spectively. Considering the high cytotoxicity of PhSbCl2 (CC50 ~ 4 nM),
this metal species was most probably responsible for the toxicity of de-
rived complexes. The increase of cytotoxicity in the case of the com-
plexes formed from SbCl3, to a much higher extent than that of
potassium antimonyl tartrate, suggests that the lipophilic complex
may enhance the delivery of toxic SbIII to macrophages (compared to
hydrophilic tartaric acid) or that these new chemical entities may be
more toxic by themselves. The lipophilicity conferred by the phenyl
group (Ph) in the case of the complexes derived from PhSbCl2, may
also contribute to the increase in cytotoxicity when compared to those
obtained from SbCl3or TA (Table 5).
As a consequence of their lower cytotoxicity, the complexes obtain-
ed from SbCl3 were found to be more selective than those made from
PhSbCl2. In that sense, [Sb(dpq)Cl3] and [Sb(phen)Cl3] with SI in the
range of 7–70 appear as the most promising compounds, being much
more selective than potassium antimonyl tartrate with a SI less than 1
(Table 5).
Another unexpected result emerging from this study is the extremely
high selectivity indexes of dpq and bipy ligands, revealing that these
compounds are also promising drug candidates by themselves for treat-
ment of Sb-resistant leishmaniasis.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jinorgbio.2013.12.001.
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