Aromatic amine N-oxide organometallic compounds: Searching for prospective agents against infectious diseases

Article abstract of DOI:10.1039/c5dt00557d

In search of prospective agents against infectious diseases, 1,1′-bis(diphenylphosphino)ferrocene pyridine-2-thiolato-1-oxide M(II) hexafluorophosphate compounds [M(mpo)(dppf)](PF₆), where M = palladium or platinum, were synthesized and fully characterized in the solid state and in solution using experimental and DFT computational techniques. The compounds are isomorphous and the M(II) transition metal ions are in a nearly planar trapezoidal cis-coordination bound to the pyridine-2-thiolato-1-oxide (mpo) and to the 1,1′-bis(diphenylphosphino)ferrocene molecules, both acting as bidentate ligands. Both compounds showed high cytotoxic activity on Trypanosoma cruzi and Mycobacterium tuberculosis (MTB) and acceptable selectivities towards MTB, but good to excellent selectivity index values as anti-T. cruzi compounds. The inclusion of the ferrocene moiety (dppf ligand) improved the selectivity towards the parasite when compared to the previously reported [M(mpo)₂] complexes. Related to the probable mechanism of action of the complexes, molecular docking studies on modelled T. cruzi NADH-fumarate reductase (TcFR) predicted that both be very good inhibitors of the enzyme. The effect of the compounds on the enzyme activity was experimentally confirmed using T. cruzi protein extracts. According to all obtained results, both [M(mpo)(dppf)](PF₆) compounds could be considered prospective anti-trypanosomal agents that deserve further research.

Full text of DOI:10.1039/c5dt00557d

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M. F. Mosquillo, L. Perez, G. Etcheverria, O. E. Piro, A. Merlino, E. L. Coitiño, C. Maríngolo Ribeiro, C. Q.F.
Leite, F. R. Pavan, L. Otero and D. Gambino, Dalton Trans., 2015, DOI: 10.1039/C5DT00557D.
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1,1'ꢀbis(dipheny1phosphino)ferrocene pyridineꢀ2ꢀthiolatoꢀ1ꢀoxide Pd(II) and Pt(II)
hexafluorophosphate compounds, [M(mpo)(dppf)](PF ), were synthesized and
6
characterized. Both showed high cytotoxicity on Trypanosoma cruzi and
Mycobacterium tuberculosis and good to excellent selectivity indexes towards T. cruzi.
Experimental and molecular docking studies on modeled T. cruzi NADHꢀfumarate
reductase showed that both are very good inhibitors of the parasite enzyme.
[
Pd(mpo)(dppf)](PF
6
)
T. cruzi: IC50 = 0.64 µM
M. tuberculosis: MIC = 2.8 µM
Vero cells: IC50 24 µM
N
O
SH
[Pt(mpo)(dppf)](PF
6
)
T. cruzi: IC50 = 0.28 µM
M. tuberculosis: MIC = 1.6 µM
Vero cells: IC50 5 µM
Hmpo

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Aromatic amine N-oxide organometallic compounds: searching for prospective agents
against infectious diseases
a
b
b
Esteban Rodríguez Arce, M. Florencia Mosquillo, Leticia PérezꢀDíaz, Gustavo A.
c
c
d
d
Echeverría, Oscar E. Piro, Alicia Merlino, E. Laura Coitiño, Camila Maríngolo
e
e
e
a*
a*
Ribeiro, Clarice Q. F. Leite, Fernando R. Pavan, Lucía Otero, Dinorah Gambino
a
Cátedra de Química Inorgánica, Departamento Estrella Campos, Facultad de Química,
Universidad de la República, Gral. Flores 2124, 11800 Montevideo, Uruguay
b
c
Laboratorio de Interacciones Moleculares, Facultad de Ciencias, Universidad de la República,
Iguá 4225, 11400 Montevideo, Uruguay
Departamento de Física, Facultad de Ciencias Exactas, Universidad Nacional de La Plata
and Institute IFLP (CONICET, CCTꢀLa Plata), C. C. 67, 1900 La Plata, Argentina
d
Laboratorio de Química Teórica y Computacional, Instituto de Química Biológica,
Facultad de Ciencias, Universidad de la República, Iguá 4225, 11400 Montevideo,
Uruguay.
e
Facultade de Ciencias Farmacêuticas, Unesp, 14801ꢀ902 Araraquara (SP), Brazil
*
To whom correspondence should be addressed. Eꢀmail: dgambino@fq.edu.uy;
luotero@fq.edu.uy; Tel. +5982ꢀ9249739; fax: +5982ꢀ9241906
Keywords: Mycobacterium tuberculosis; Trypanosoma cruzi; pyridineꢀ2ꢀthiol 1ꢀoxide;
ferrocene compounds; palladium and platinum
1

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Abstract
Searching for prospective agents against infectious diseases, 1,1'ꢀbis (dipheny1phosphino)
ferrocene pyridineꢀ2ꢀthiolatoꢀ1ꢀoxide M(II) hexafluorophosphate compounds
[
6
M(mpo)(dppf)](PF ), where M(II) = palladium or platinum, were synthesized and fully
characterized in the solid state and in solution using experimental and DFT computational
techniques. The compounds are isomorphous and the M(II) transition metal ions are in a nearly
planar trapezoidal cisꢀcoordination bound to the pyridineꢀ2ꢀthiolatoꢀ1ꢀoxide (mpo) and to the
1,1'ꢀbis (dipheny1phosphino)ferrocene molecules, both acting as bidentate ligands. Both
compounds showed high cytotoxic activity on Trypanosoma cruzi and Mycobacterium
tuberculosis (MTB) and acceptable selectivities towards MTB, but good to excellent selectivity
index values as antiꢀT.cruzi compounds. The inclusion of the ferrocene moiety (dppf ligand)
improved the selectivity towards the parasite when compared to the previously reported
[
2
M(mpo) ] complexes. Related to the probable mechanism of action of the complexes,
molecular docking studies on modeled T. cruzi NADHꢀfumarate reductase (TcFR) predicted
that both should be very good inhibitors of the enzyme. The effect of the compounds on the
enzyme activity was experimentally confirmed using T. cruzi protein extracts. According to all
obtained results, both [M(mpo)(dppf)](PF ) compounds could be considered prospective antiꢀ
6
trypanosomal agents that deserve further research.
Introduction
Infectious diseases caused by pathogenic microorganisms such as bacteria, viruses, parasites or
fungi, are the most prevalent illnesses worldwide and constitute an onꢀgoing threat to global
1
health security. Among them, Chagas disease (American trypanosomiasis) is a potentially lifeꢀ
threatening illness caused by the protozoan parasite Trypanosoma cruzi (T. cruzi) and
2

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transmitted to the human host by infected triatomine bugs. It is an ancient, endemic disease in
Latin America. It originally affected people living in rural areas where environmental
conditions favor the installation of the bug. However, rural exodus, deforestation and
2
urbanization have rendered it to a urban or periꢀurban illness. Currently, Chagas disease has
spread to nonꢀendemic countries in other continents mainly because of growing migratory
flows. Globally, more than 10 million people are infected worldwide, mostly in Latin America.
Only two old drugs are available for the treatment of the disease, namely Nifurtimox and
Benznidazole. Both are poorly tolerated and show controversial efficacy. Hence the
2
ꢀ6
development of more efficacious and less toxic drugs is urgently needed.
Tuberculosis (TB) is a highly contagious infectious disease caused by Mycobacterium
tuberculosis (MTB). Although curable and preventable, it remains one of the leading causes of
death in the world. In 2013, 9 million people felt ill and 1.5 million died from the disease. Over
95% of these deaths occurred in lowꢀ and middleꢀincome countries. The risk of infection is
higher in persons with impaired immune system; TB is a leading killer of HIVꢀpositive people.
About oneꢀthird of the world's population is infected but is not yet ill, bearing latent TB,
7,8
working as reservoir of the disease. Only few drugs are active against M. tuberculosis bacilli.
Moreover, there is an increasing emergence of multiꢀdrug resistant (MDR) and extensively
9
ꢀ12
drug resistant (XDR) MTB strains.
Our group has been particularly devoted to the search for prospective metalꢀbased drugs against
1
3ꢀ19
T. cruzi and, more recently, M. tuberculosis.
In the case of antiꢀtrypanosomal agents the
main design strategy has consisted in the metal complexation of antiꢀparasitic organic
1
3,14
compounds in an attempt to modulate their activity and global biological properties.
In this sense, some of us have been studying the effect of metal coordination on the bioactivity
16,20ꢀ22
of the aromatic amine Nꢀoxide, pyridineꢀ2ꢀthiol 1ꢀoxide (Figure 1, Hmpo).
3

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Figure 1. Pyridineꢀ2ꢀthiol 1ꢀoxide (Hmpo, left) and its Pt(II) and Pd(II) complexes ([M(mpo)2],
right).
II
Square planar complexes [M (mpo)
2
], where M(II) = Pd(II) or Pt(II) (Fig. 1) and the Au(I)
I
2
homodinuclear mixedꢀligand complex, [Au
(mpo)
2
(PPh
3
)
2
], showed high activity on T. cruzi.
Although they may have other intraparasite targets, the free ligand and the metal complexes
inhibit the activity of the parasite specific enzyme NADHꢀfumarate reductase (TcFR), which is
responsible for the conversion of fumarate to succinate as energy source. Whereas TcFR is
essential for the survival of T. cruzi, being present in all the life cycle stages of the parasite, its
20ꢀ
absence in the human host makes it a promising target for selective antiꢀtrypanosomal drugs.
2
1,23
Generation of succinate through a similar enzymatically catalyzed process has also been
described in latent TB lung granulomas where the oxygen amount is severely limited. The use
24
of the produced succinate enables MTB to maintain its basic physiological functions.
Accordingly, previously established mpo complexes with Bi(III), Fe(III) and Ga(III) showed to
16
be potent antiꢀM. tuberculosis prospective agents.
Among the previously developed mpo antiꢀtrypanosomal metal compounds, the [M(mpo)
complexes, with M = Pd or Pt, demonstrated to be the most promising ones (Fig. 1).
2
]
Nevertheless, both showed only moderate selectivity towards T. cruzi in respect to mammalian
4

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2
0
cell model (J774 macrophages). In order to further address the therapeutic potential of these
metalꢀmpo complexes, we rationally designed two new compounds including a ferrocene
derivative (1,1'ꢀbis(dipheny1phosphino) ferrocene; dppf) in the metal coordination sphere. The
“
sandwich type” ferrocene moiety has shown high potentiality in the development of novel
organometallic drugs. For instance, compounds including it, like the antitumoral ferrocifen and
the antimalarial ferroquine, have entered the phase of clinical trials. Although being
organometallics, ferrocene derivatives are usually stable in air and in solution and, in addition,
they usually show low cytotoxicity and confer the molecules were they are included adequate
2
5ꢀ27
lipophilicity for trespassing cell membranes.
In this work, 1,1'ꢀbis(dipheny1phosphino) ferrocene pyridineꢀ2ꢀthiolatoꢀ1ꢀoxide M(II)
hexafluorophosphate compounds [M(mpo)(dppf)](PF ), where M(II) = palladium or platinum,
6
were synthesized and fully characterized in the solid state and in solution. Both species were
evaluated on T. cruzi and M. tuberculosis as well as on VERO epithelial cells taken as
mammalian cell model. The inhibitory effect of the newly developed compounds against TcFR,
was predicted by molecular docking studies conducted on a model structure of the enzyme
previously constructed by some of us. In addition, the inhibition of TcFR activity by the
compounds was experimentally confirmed on T. cruzi protein extracts.
Experimental
General considerations. All common laboratory chemicals were purchased from commercial
sources and used without further purification. Pyridineꢀ2ꢀthiol 1ꢀoxide sodium salt (Na mpo)
and 1,1´‐bis(diphenylphosphine) ferrocene (dppf) were commercially available. [MCl
2
(dppf)]
precursors were synthesized according to a previously reported procedure by heating for 30
2
8
min an equimolar mixture of [MCl
2
(dmso)
2
3
] and dppf in CHCl .
5

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Chemical and physical measurements. C, H, N and S analyses were carried out with a
Thermo Scientific Flash 2000 elemental analyzer. Conductimetric measurements were done
o
−3
over time (7 days) at 25 C in 10 M DMSO solutions using a Conductivity Meter 4310
Jenway to determine the type of electrolyte and to assess the stability of the complexes in such
29
medium.
–1
The FTIR absorption spectra (4000ꢀ300 cm ) were measured as KBr pellets with a Shimadzu
1
13
o
6
IR Prestigeꢀ21 instrument. H NMR and C NMR spectra were recorded in DMSOꢀd at 30 C
on a Bruker DPXꢀ400 instrument (at 400 MHz and 100 MHz, respectively). Homoꢀnuclear
correlation experiments (COSY) and heteroꢀnuclear correlation experiments (2DꢀHETCOR),
HSQC (heteroꢀnuclear single quantum correlation) and HMBC (heteroꢀnuclear multiple bond
correlation), were carried out with the same instrument. Tetramethylsilane was used as the
31
internal standard. P NMR spectra were recorded on a Bruker Advance 400 MHz spectrometer
(at 160.984 MHz) equipped with a 5 mm multinuclear broadꢀband dual probehead,
31
incorporating a zꢀgradient coil. P{1H} spectra were calibrated respect to the external pattern
H PO 10% (0.0 ppm). Chemical shifts are reported in ppm. Electrochemical behaviour was
3
4
studied by cyclic voltammetry. Cyclic voltammograms were obtained with an Epsilon
Electrochemical Analyzer. A standard electrochemical three electrode cell of 10 mL volume
completed the system. A glassy carbon electrode was employed as a working electrode. A
platinum wire was used as counter electrode while a Ag/AgCl electrode was used as a reference
electrode. Measurements were performed at room temperature in 1mM acetonitrile solutions of
the complexes using tetrabutyl amonium hexafluorophosphate (c.a. 0.1 M) as supporting
electrolyte. Solutions were deoxygenated via purging with nitrogen for 15 min prior to the
measurements. A continuous gas stream was passed over the solution during the measurements.
6

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Syntheses of [M(mpo)(dppf)](PF ), where M = Pd(II) or Pt(II). The complexes were
6
synthesized by the following procedure: 50 mg of the precursor [MCl (dppf)] (0.068 mmol
2
[
2 2
PdCl (dppf)] or 0.061 mmol [PtCl (dppf)]) were dissolved in 10 mL methanol. To this
solution, an equimolar amount of Na mpo (10.2 mg for the Pd compound and 9.1 mg for the Pt
compound) dissolved in 5 mL methanol was added. The mixture was shaken at room
temperature for 1 h. The solution was evaporated up to 5 mL and then kept in a refrigerator for
2
4 h. After adding NaPF dissolved in a minimal volume of methanol (11.39 mg, 0.068 mmol
6
for Pd and 10.22 mg, 0.061 mmol for Pt) the solution was kept again in refrigerator during 24
hs. The Pd compound was isolated as a reddish brown solid and the Pt compound as a greenish
brown one. Single crystals suitable for Xꢀray diffraction studies were obtained in both cases by
slow diffusion of hexane into a dichloromethane solution of the compounds.
[
Pd(mpo)(dppf)](PF ). Yield: 40mg, 63%. Found: C, 50.13; H, 3.45; N, 1.50; S, 3.43.Calc. for
6
2
–1 1
C
39
H
32
F
6
FeNOP
3
PdS: C, 50.26; H, 3.46; N, 1.50; S, 3.44.Λ
M
(DMSO): 34.7 Scm mol . Hꢀ
NMR: δ = 7.92 (1H, d, H5 mpo), 7.86 (4H, m, PPh
2
), 7.68 (12H, m, PPh
2
), 7.63 (1H, H2 mpo),
7
.52 (1H, H3 mpo), 7.50 (4H, m, PPh ), 7.17(1H, t, H4 mpo), 4.95 (2H, s, H Cp), 4.80 (2H, s,
2
a
13
H
b
Cp), 4.64 (2H, s, H
c
Cp), 3.90 (2H, s, H Cp) ppm. CꢀNMR: δ = 154.9 (C1, mpo), 138.1
d
(C5, mpo), 132.1 (C3, mpo), 127.5 (C2, mpo),120.1 (C4, mpo), 78.13 (CꢀP Cp), 78.01 (CꢀH
a
31
Cp), 75.85 (CꢀH
b
Cp), 74.48 (CꢀH
c
, CꢀH
d
2
Cp) ppm. PꢀNMR: δ = 37.2 (d, PPh ), 29.3(d,
PPh ), ꢀ143.0 (sept, PF ) ppm.
2
6
6
[Pt(mpo)(dppf)](PF ). Yield: 31 mg, 50%. Found: C, 45.75; H, 3.17; N, 1.37; S, 3.15.Calc. for
2
–1 1
C H F FeNOP PtS: C, 45.90; H, 3.16; N, 1.37; S, 3.14. Λ (DMSO): 33.1 Scm mol . Hꢀ
39
32
6
3
M
2 2
NMR: δ = 7.92 (1H, d, H5 mpo), 7.82 (1H, H2 mpo), 7.81 (4H, m, PPh ), 7.67 (12H, m, PPh ),
7
.57 (1H, H3 mpo), 7.50 (4H, m, PPh ), 7.14(1H, t, H4 mpo), 4.78 (2H, s, H Cp), 4.76 (2H, s,
2
a
13
H
b
Cp), 4.62 (2H, s, H
c
Cp), 4.00 (2H, s, H
d
Cp) ppm. CꢀNMR: δ = 151.8 (C1, mpo), 139.1
7

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(
C5, mpo), 132.1 (C3, mpo), 128.2 (C2, mpo), 120.7 (C4, mpo), 76.89 (CꢀH Cp), 75.85 (CꢀH
a b
31
Cp), 75.39 (CꢀH
c
Cp), 75.02 (CꢀH
d
2 2
Cp) ppm. PꢀNMR: δ = 21.6 (d, PPh ), 9.8 (d, PPh ), ꢀ
1
43.0 (sept, PF ) ppm.
6
6
X-ray diffraction study of [M(mpo)(dppf)](PF ), where M = Pd(II) or Pt(II). The
measurements were performed on an Oxford Xcalibur Gemini, Eos CCD diffractometer with
graphiteꢀmonochromated MoKα (λ=0.71073 Å) radiation. Xꢀray diffraction intensities were
3
0
collected (ω scans with ϑ and κꢀoffsets), integrated and scaled with CrysAlisPro suite of
programs. The unit cell parameters were obtained by leastꢀsquares refinement (based on the
angular settings for all collected reflections with intensities larger than seven times the standard
deviation of measurement errors) using CrysAlisPro. Data were corrected empirically for
absorption employing the multiꢀscan method implemented in CrysAlisPro. The complexes are
isomorphous to each other and their common structure was solved by direct methods with the
3
1
SHELXSꢀ97 program of the SHELX package and the molecular model developed by
alternated cycles of Fourier methods and fullꢀmatrix leastꢀsquares refinement with SHELXLꢀ97
of the same suit of programs.
The hydrogen atom were positioned on stereoꢀchemical basis and refined with the riding
model. Crystal data, data collection procedure, structure determination methods and refinement
results are summarized in Table 1. Crystallographic structural data have been deposited at the
Cambridge Crystallographic Data Centre (CCDC). Any request to the CCDC for this material
should quote the full literature citation and the reference number CCDC 1035234 (Pd) and
CCDC 1035235 (Pt).
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Table 1. Crystal data and structure refinement results for the [M(mpo)(dppf)]PF (M = Pd, Pt)
6
isomorphous complexes.
[Pd(mpo)(dppf)]PF6
[Pt(mpo)(dppf)]PF6
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
C H F FeNOP PdS
C H F FeNOP PtS
39
32
6
3
39 32
6
3
931.88
295(2)
0.71073
1020.57
295(2)
0.71073
Monoclinic
P2 /c
Monoclinic
P2 /c
1
1
10.345(2)
22.319(3)
17.307(3)
106.23(2)
10.3631(5)
22.363(1)
17.3974(9)
106.240(6)
b (Å)
c (Å)
β (°)
3
Volume (Å )
3836.6(10)
3871.0(3)
4, 1.751
3
4
, 1.613
.089
Z, density (calculated, Mg/m )
ꢀ
1
1
4.224
Absorption coefficient (mm )
F(000)
1872
2000
Crystal shape / color
Needle / purple
Needle / yellow
3
0.399 x 0.042 x 0.027
0.259 x 0.048 x 0.036
Crystal size (mm )
ϑꢀrange (º) for data collection
Index ranges
3.00 to 26.00
2.99 to 26.00
ꢀ12≤h≤12, ꢀ26≤k≤27, ꢀ21≤l≤21
18298
ꢀ12≤h≤12, ꢀ27≤k≤17, ꢀ21≤l≤21
17086
Reflections collected
Independent reflections
7532 [R(int) = 0.1320]
7599 [R(int) = 0.0528]
9

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Observed reflections [I>2σ(I)]
3308
99.8 (to ϑ = 26.00°)
Semiꢀempirical from equivalents Semiꢀempirical from equivalents
5221
Completeness (%)
99.8 (to ϑ = 26.00°)
Absorption correction
Max. and min. transmission
Refinement method
0.9712 and 0.6707
1.000 and 0.851
2
2
Fullꢀmatrix leastꢀsquares on F
7532 / 0 / 478
Fullꢀmatrix leastꢀsquares on F
Data / restraints / parameters
7599 / 0 / 478
2
0.995
1.018
Goodnessꢀofꢀfit on F
a
Final R indices [I>2σ(I)]
R1 = 0.0761, wR2 = 0.1348
R1 = 0.1826, wR2 = 0.1847
1.157 and ꢀ0.716
R1 = 0.0452, wR2 = 0.0730
R1 = 0.0829, wR2 = 0.0864
1.244 and ꢀ0.822
R indices (all data)
ꢀ3
Largest diff. peak and hole (e.Å )
a
2
2 2
2 2 1/2
R
1
=Σ||F
o
|ꢀ|F
c
||/Σ|F
o
2
|, wR =[Σw(|F
o
| ꢀ|F
c
| ) /Σw(|F
o
| ) ]
Biological studies
Determination of anti-MTB Minimal Inhibitory Concentration (MIC). The antiꢀMTB activity
3
2
of the compounds was determined by the REMA (Resazurin Microtiter Assay) method. Stock
solutions of the tested compounds were prepared in DMSO and diluted in Middlebrook 7H9
broth (Difco) supplemented with oleic acid, albumin, dextrose and catalase (OADC enrichment
ꢀ
BBL/BectonꢀDickinson), to obtain final drug concentration ranges of 0.09ꢀ25 µg/mL.
Isoniazid was dissolved in distilled water and rifampicin in DMSO, and both were used as
standard drugs. A suspension of MTB H37Rv ATCC 27294 was cultured in Middlebrook 7H9
broth supplemented with OADC and 0.05% Tween 80. The cultures were frozen at ꢀ80ºC in
aliquots. After two days the CFU/mL (colony formation unit/mL) of analiquot was determined.
5
The concentrations were adjusted by 5x10 CFU/mL and 100 µL of the inoculum were added to
each well of a 96ꢀwell microꢀplate together with 100 µL of the compounds. Samples were set
up in triplicate. The plates were incubated for 7 days at 37°C. Resazurin (solubilized in water)
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was added (30 µL of 0.01%). The fluorescence of the wells was read after 24 h with a TECAN
®
Spectrafluor . The MIC was defined as the lowest concentration resulting in 90% inhibition of
growth of MTB.
In vitro anti-Trypanosoma cruzi activity. T. cruzi epimastigotes of the Dm28c strain were
maintained in exponential growth at 28 °C in liver infusion tryptose (LIT) medium
complemented with 10 % (v/v) fetal calf serum (FCS). The effect on cell growth was analyzed
6
–1
incubating an initial concentration of 1×10 cells mL with various concentrations (from 0 to 6
ꢀM) of the compounds for 5 days in 96 well plates. Compounds were added as stock DMSO
solutions immediately after the preparation of these solutions. The final DMSO concentration
in the culture media never exceeded 0.4 % (v/v) and had no effect by itself on the proliferation
33,34
of the parasites.
For the succinate protection assays, epimastigotes were also cultured for 5
days in presence or absence of 5 mM succinate. The percentage of cell growth was followed by
measuring the absorbance, A, of the culture at 595 nm and calculated as follows: % = (Apꢀ
A
0
p)/(AcꢀA
the culture containing the drug at day 0; Ac = A595 of the culture in the absence of any drug
control) at day 5; A c = A595 in the absence of the drug at day 0. Doseꢀresponse curves were
0
0
c)×100, where Ap = A595 of the culture containing the drug at day 5; A p = A595 of
(
0
recorded and the IC50 values (50 % inhibitory concentration) were determined. The results are
presented as averages ± SD (standard deviation) and correspond to assays performed in
triplicates. Nifurtimox (Nfx) was used as the reference trypanosomicidal drug.
Cytotoxicity assay. In vitro cytotoxicity assays were performed on VERO epithelial cells
(ATCC CCL81). This lineage is widely used for phenotypic screening of drugs being regarded
35
as a normal cell derived from normal epithelial tissue. The cells were routinely maintained in
DMEM complete medium supplemented with 10% heat inactivated fetal bovine serum (FBS)
plus gentamicin (50 mg/L) and amphotericin B (2 mg/L) at 37 °C, in a humidified 5% CO2
atmosphere. After reaching confluence, the cells were detached and counted. For the
1
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5
cytotoxicity assay, 1 × 10 cells/mL was seeded in 200ꢁL of complete medium in 96ꢀwell
plates (NUNC™). The plates were incubated under the same conditions for 24 h to allow cell
adhesion prior to drug testing. The compounds were dissolved in DMSO (5%) and subjected to
twoꢀfold serial dilution from 500 to 3.9ꢁg/mL. The cells were exposed to the compounds at
various concentrations for 24 h. Resazurin solution was then added to the cell cultures and
incubated for 6 h. Cell respiration (as an indicator of cell viability) was detected by reduction of
resazurin to resorufin whose pink color and fluorescence indicate cell viability. A persistent
blue color of resazurin is a sign of cell death. The fluorescence measurements (530 nm
excitation filter and 590 nm emission filter) were performed in a SPECTRA fluor Plus (Tecan)
microfluorimeter. The IC50 value was defined as the highest drug concentration at which 50%
of the cells are viable relative to the control. Each test was set up in triplicate.
T. cruzi NADH-fumarate reductase activity measurements. For TcFR activity
quantification T. cruzi epimastigotes (Dm28c clone) were grown at 28 ºC in liver
infusion tryptose (LIT) medium supplemented with 10% heat inactivated fetal bovine
7
serum (FBS). T. cruzi protein extracts were prepared from 5x10 exponentially grown
20
7
parasites as previously described. Briefly, parasites (1x10 /mL) were harvested at
500xg and resuspended in MST buffer (0.23 M mannitol, 0.07 M sucrose, 5 mM Tris
HCl, pH 7.4) supplemented with protease inhibitors tablets (SIGMAFAST Protease
Inhibitor tablets, SIGMA). Epimastigotes were homogenized in the presence of 0.1%
Triton Xꢀ100 and 200 mM KCl on ice using a PotterꢀElvehjem Teflonꢀglass
homogenizer to disrupt the cell membranes and extract the enzyme. The preparation
was immediately used to spectrophotometrically test NADHꢀfumarate reductase activity
as the fumarateꢀdependent rate of NADH oxidation at 340 nm (molar absorption
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ꢀ1
ꢀ1
coefficient = 6.22 mM cm ) using 250 ꢁM NADH in Buffer HEPES 30 mM pH 7.0
36
containing 125 mM KCl and 0.5 mM fumarate as previously described.
Computational modeling
DFT characterization. Electronic structure calculations on the crystallographic geometry of
+
each [M(mpo)(dppf)] complex were conducted by applying the BP86 GGA density
37,38
39,40
functional
with the 6ꢀ31+G(d) basis set for all the nonꢀmetallic atoms
and the
LANL2DZ relativistic effective core potential and the associated basis set for all the metal
4
1
atoms (Pt/Pd/Fe). Natural atomic NPA charges and Wiberg bond indices were derived for
42,43
each structure from Weinhold’s Natural Bond Orbital (NBO) analysis.
All the calculations
44
were performed with the suite of programs Gaussian09.
Molecular docking studies on T. cruzi NADH-fumarate reductase. The crystallographic
structures of both Mꢀdppfꢀmpo complexes (after hydrogen atoms removal) and the 3D
structure of the central domain of T. cruzi fumarate reductase previously predicted by some of
22
us combining homology modeling/molecular dynamics simulations were used here for
4
5
conducting ligandꢀprotein docking with AutoDock 4.2. The binding site of both complexes
into TcFR was predicted by flexibleꢀligand docking using a grid box of 126×126×126 points
(0.650 Å spacing) centered on the macromolecule to cover the entire protein surface (blind
docking). GasteigerꢀMarsilli charges were used and parameters for Pt(II) and Pd(II) were
included in AutoDock’s parameters file (Rii and εii values for Pt(II) were based on AutoDock
4
6
parameters for transition metals, a set of parameters developed by Divsalar et al. were used
47
for Pd(II), with R = 2.50 Å and ε = 0.458 kcal/mol ). A Lamarckian genetic algorithm with
ii
ii
6
population size of 150 individuals and 2.5x10 energy evaluations was applied for 50 search
runs. Default values were employed for the rest of the parameters. Proteinꢀligand structures
differing by less than 2.0 Å in rootꢀsquare deviation were grouped in the same cluster, choosing
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the conformation with lowest binding energy from the most populated cluster for further
analysis.
Results and discussion
Syntheses and characterization of the complexes
Two new organometallic heterobimetallic compounds of the bioactive ligand pyridineꢀ2ꢀ
thiolatoꢀ1ꢀoxide (mpo) with the formula [M(mpo)(dppf)](PF ), where M = Pd(II) or Pt(II),
6
were obtained with good purity and good yield and they were exhaustively characterized in the
solid state and in solution.
Characterization of the complexes in the solid state
Most relevant IR vibration bands of both complexes were assigned taking into account
1
6,20,21
previous assignments performed on related mpo compounds (Table 2).
The ν(NꢀO), δ(Nꢀ
O) and ν(CꢀS) bands shift upon coordination to the metal in agreement with the bidentate
coordination of the ligand through the oxygen of the NꢀO group and the sulfur, as shown by the
corresponding crystal structures.
Table 2. Tentative assignment of the main characteristic
6
bands of [M(mpo)(dppf)](PF ). Bands for mpo sodium salt,
2
0
ꢀ1
were included for comparison. (wave numbers in cm )
ν (N-O)
δ (N-O)
ν (C-S)
706 (s)
694 (m)
694 (m)
Nampo
1263 (s)
833 (s)
*
*
[
[
Pd(mpo)(dppf)](PF
6
) 1246 (m) 834 (vs)
Pt(mpo)(dppf)](PF
6
) 1252 (m) 840 (vs)
ν: stretching; δ: bending; s: strong; m: medium; vs: very
ꢀ
strong.*superimposed with the strong ν(PꢀF) of the PF
6
counterion.
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Crystal structure of isomorphous Pd-dppf-mpo and Pt-dppf-mpo complexes. Single crystals
suitable for Xꢀray diffraction studies were obtained for both 1,1'ꢀ
bis(diphenylphosphino)ferrocene pyridineꢀ2ꢀthiolatoꢀ1ꢀoxide M(II) hexafluorophosphate
compounds, where M(II) is palladium or platinum (Pdꢀdppfꢀmpo and Ptꢀdppfꢀmpo). Figure 2
4
8
shows an ORTEP drawing of the platinum complex and its intraꢀmolecular bond distances
and angles around the metal along with the corresponding ones of the isomorphous palladium
complex are given in Table 3. For this pair of isomorphous compounds a change in the nature
of M precludes comparing the strength of the interactions at each metal coordination sphere
by direct comparison of the corresponding MꢀL bond lengths. Therefore, for reaching that
goal, a selection of Wiberg bond indices (WBI) calculated on the corresponding crystal
structure at the BP87/6ꢀ31+G(d) level of theory are also included in Table 3 (See the
complete set of calculated data in the supplementary information, Tables S1 and S2).
The M(II) transition metal ions are in a quasi-planar trapezoidal environment [rms deviation
of atoms from the best least squares plane is less than 0.054 Å], coordinated to a nearly
coplanar pyridineꢀ2ꢀthiolatoꢀ1ꢀoxide anion acting as a bidentate ligand through its sulfur [MꢀS
bond distances of 2.293(2) Å for Pd and 2.303(2) Å for Pt] and oxygen atoms [MꢀO lengths
of 2.059(6) Å for Pd and 2.071(4) Å for Pt], and to the 1,l'ꢀbis (dipheny1phosphino)ferrocene
neutral moiety acting as a bidentate ligand through its phosphorus atoms along their free
tetrahedral binding direction [MꢀP distances of 2.240(3) Å and 2.335(2)Å for Pd and 2.239(2)
Å and 2.316(2) Å for Pt]. Higher WBI values were found for all the MꢀS/MꢀO/MꢀP
coordinative interactions in the Pt(II) species in comparison to their counterparts in the Pd(II)
one; a feature that confers less reactivity to the Ptꢀdppfꢀmpo compound.
A similar planar coordination around Pd(II) was previously reported for the related
4
9
[
2
Pd(dppf)Cl ] complex [PdꢀP distances of 2.283(1) and 2.301(1) Å], but with a less marked
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asymmetry between the two PdꢀP bonds with respect to Pdꢀdppfꢀmpo. Platinum-mpo binding
2
in Ptꢀdppfꢀmpo complex agrees with the one observed in the transꢀcoordinated [Pt(mpo) ]
planar compound [mean PtꢀS and PtꢀO bond distances of 2.270(3) and 2.007(3) Å,
50
respectively]. Similarly, palladiumꢀmpo binding in Pdꢀdppfꢀmpo complex agrees with that
observed in [Pd(mpo) ] [mean PdꢀS and PdꢀO bond distances of 2.242(2) and 2.043(5) Å,
2
51
respectively]. In both Mꢀdppfꢀmpo complexes the dppf ligand shows the expected
Archimedean pentagonal antiꢀprism conformation for the coordination around iron in the
ferrocene group, with the 5ꢀmembered Cp rings staggered to each other [FeꢀC distances in the
range from 2.014(6) to 2.079(6) Å for Ptꢀdppfꢀmpo and from 2.016(9) to 2.068(9) Å for Pdꢀ
dppfꢀmpo]. PhosphorusꢀC(Ph) bond distances are in the 1.808(6)ꢀ1.821(6) Å interval, while
PꢀC(Cp) lengths are slightly shorter [1.794(6) and 1.798(6) Å].
Figure 2. Drawing of platinum(II) complex in the [Pt(mpo)(dppf)]PF
atomic displacement ellipsoids at the 20% probability level. For clarity, only a few
representative atoms have been labeled and the PF counterꢀion has been omitted.
6
solid showing the
6
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Table 3. Crystallographic bond lengths [Å] and angles [°] and DFT Wiberg bond
a
indices [a.u.] around the metal in the [M(mpo)(dppf)]PF (M
=
Pd, Pt) compounds.
6
[
Pd(mpo)(dppf)]PF
6
[Pt(mpo)(dppf)]PF
6
PdꢀO(4)
PdꢀP(2)
PdꢀP(3)
PdꢀS(4)
2.059(6) [0.39]
PtꢀO(4)
PtꢀP(2)
PtꢀP(3)
PtꢀS(4)
2.071(4) [0.43]
2.239(2) [0.77]
2.316(2) [0.66]
2.303(2) [0.70]
2.240(3) [0.66]
2.335(2) [0.58]
2.293(2) [0.66]
O(4)ꢀPdꢀP(2)
O(4)ꢀPdꢀS(4)
P(2)ꢀPdꢀS(4)
O(4)ꢀPdꢀP(3)
P(2)ꢀPdꢀP(3)
S(4)ꢀPdꢀP(3)
174.9(2)
84.4(2)
O(4)ꢀPtꢀP(2)
O(4)ꢀPtꢀS(4)
P(2)ꢀPtꢀS(4)
O(4)ꢀPtꢀP(3)
P(2)ꢀPtꢀP(3)
S(4)ꢀPtꢀP(3)
175.5(1)
84.4(1)
91.73(9)
87.0(2)
92.42(6)
85.7(1)
97.03(9)
170.9(1)
97.68(6)
169.51(6)
a
values in italics between brackets.
Characterization of the complexes in solution
1
No conductivity changes were observed during at least 7 days at 25°C. In addition, H NMR
spectra were measured during seven days. Neither changes in intensity of the signals nor new
peaks due to release of free ligand were detected. Hence results suggest that the complexes are
stable at air in DMSO solution.
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NMR results
1
Table 4. H NMR chemical shift values (
δ, in ppm) of the ligands and the complexes in
6
DMSOꢀd .
Hd
Ha
3
4
5
2
1
δ
H
(multiplicity)(integration)
Hc
P
N
O
SH
Hb
a
a
Ligand
H
2
Free Ligand [Pd(mpo)(dppf)](PF
)
6
ꢁδ
[Pt(mpo)(dppf)](PF ) ꢁδ
6
(b)
(b)
7.32 (d) (1) 7.63 (1)
0.31
0.78
0.62
7.82 (1)
0.50
(b)
(b)
3
6.74 (t) (1) 7.52 (1)
6.55 (t) (1) 7.17 (t)(1)
7.97 (d) (1) 7.92 (d) (1)
4.95 (s) (2)
7.57 (1)
0.83
0.59
ꢀ0.05
0.84
0.06
0.49
0.35
Mpo
4
7.14 (t) (1)
7.92 (d) (1)
4.78 (s) (2)
4.00(s) (2)
4.76 (s) (2)
4.62 (s) (2)
7.81 (m) (4)
7.67 (m) (12)
7.50 (m) (4)
5
ꢀ0.05
1.01
ꢀ0.04
0.53
0.37
Ha
3
.94 (s) (4)
Dppf
H
d
3.90 (s) (2)
4.80 (s) (2)
4.64 (s) (2)
(
Cp ring)
Hb
4
.27 (s) (4)
H
c
7.86 (m) (4)
7.68 (m) (12)
7.50 (m) (4)
Dppf
7.23 (s) (8)
ꢀ
ꢀ
(
PPh2)
7.34 (s) (12)
a
ꢁ
δ= (δcomplex ꢀ δligand)
b
2
overlapped with PPh protons
Multiplicity: s: singlet, d: doublet, t: triplet, m: multiplet
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1
13
31
H, C and P NMR spectra were recorded to completely characterize the obtained
complexes in DMSOꢀd solution. Twoꢀdimensional NMR experiments such as COSY
6
1
and HSQC aided in the assignment of the spectra. Results of the H NMR experiments
for the ligands and the complexes are shown in Table 4. As expected, both complexes
showed a similar pattern of signals corresponding to the protons of mpo and dppf
ligands. Obtained integration and multiplicity are in accordance with the obtained
stoichiometry of the complexes. A deshielding effect was observed on most of the
protons as a consequence of metal coordination of mpo (which induces significant
charge reorganization in the ligand, as can be appreciated from the changes in NPA
atomic charge and WBI values collected in Table S2a). As previously observed for
2
0,21
other mpo complexes,
protons 2 and 5, respectively vicinal to the S and O
coordinating atoms, were the less deshielded (actually, 2 was slightly shielded in both
Mꢀdppfꢀmpo complexes) due to backꢀdonation of electron density from the SꢀMꢀO
moiety. Two proton signals (singlets, four proton integration) at 3.94 and 4.27 ppm
were observed for the Cp rings of the dppf free ligand. These signals have been assigned
to equivalent HaꢀHd and HbꢀHc protons of both Cp rings that are coplanar to each other
5
2
and with an average eclipsed conformation at room temperature (Table 4). In the new
compounds, these two signals split into four. The equivalence of the Cp protons is
broken under formation of the Mꢀdppfꢀmpo complexes due to the loss of fluxionality
caused by the geometric requirements of the MꢀP bonding in the presence of the
53,54
bidentate mpo ligand.
At higher ppm values, multiplets corresponding to protons of
the PPh moieties were also observed.
2
13
Through 2DꢀHSQC experiments, the signals of the C NMR spectra could be partially
assigned in spite of their observed complexity (Table S7). The most noticeable feature
of the spectra is the change in the chemical shift of C1 carbon because of complexation
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(
ꢁδ
= δcomplex ꢀ δligand = ꢀ13.01 and ꢀ16.11 ppm for palladium and platinum compounds,
13
respectively). This is in accordance with the C NMR spectroscopic behavior
16,20,21
previously reported for other mpo complexes.
3
1
PꢀNMR spectra present two doublets corresponding to the PPh
2
moieties at 37.2 and
29.3 ppm for the palladium compound and at 21.6 and 9.98 ppm for the platinum one.
This clearly shows that the two phosphorus atoms are nonꢀequivalent in the new
complexes, in consistency with the crystallographic study and the asymmetries found in
their NPA atomic charges (Table S2b) and the WBI indices for the chemical
environment of each one in the Mꢀdppfꢀmpo species (Table S1). For the platinum one,
two satellites at the sides of the phosphorus signal due to the coupling with platinum
55
atom were observed (JPtP = 1605 and 1840 Hz). In addition, the typical septet at ꢀ143.0
ꢀ
ppm corresponding to the PF
6
counter ion was observed for both complexes.
Cyclic voltammetry studies
The electrochemical properties of the complexes were investigated by cyclic voltammetry
using a glassy carbon disc as working electrode, a platinum wire as counter electrode, and
Ag/AgCl as reference electrode. Obtained voltammograms of the complexes (recorded at a
potential scan rate of 100 mV/s in acetonitrile solution) are shown in Fig. 3.
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4
3
2
1
0
1
2
3
4
5
I
II
)
-
-
-
-
-
Pd-dppf-mpo
Pt-dppf-mpo
III
1,0
0,5
0,0
-0,5
-1,0
-1,5
-2,0
E (V)
Figure 3. Cyclic voltammograms of the Mꢀdppfꢀmpo (M=Pd, Pt) complexes at 100mV/s.
56
Couple III corresponds to an adsorption process.
Both complexes showed a similar electrochemical behaviour. On the forward cathodic scan, a
reduction peak (I) was observed at ꢀ1.04 and ꢀ1.45 V for the palladium and platinum
complexes, respectively. According to the behaviour previously reported for the
[M(mpo)2]
complexes with M = Pd or Pt, this peak could be assigned to the irreversible reduction of the
20
metal center. This is also consistent with the features of the corresponding KohnꢀSham
2
2
LUMO orbitals (σ* character, mainly involving M dx -y contributions combined with p-orbitals
from the P, O, S coordinative atoms at the two bidentate ligands, as shown in Fig. S1) obtained
at the DFT level for the Mꢀddpfꢀmpo, with energies of ꢀ5.45 and ꢀ4.95 eV for M = Pd and Pt,
respectively.
III
II
On the reverse scan, a reversible redox couple (II) characteristic of the Fe /Fe couple of the
dppf ligand was observed at 1.07 and 1.06 V for the palladium and platinum complexes,
respectively (again in agreement with the finding of HOMO KS orbitals –shown in Fig. S1ꢀ
located on the ferrocenyl moiety for both compounds and very close in energy between them,
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1

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with values of ꢀ7.40 eV for the Pd and of ꢀ7.39 eV for the Pt species). The effect of the scan
rate on the electrochemical response of this couple was investigated between 50 and 1000
mV/s. For both complexes the anodic and cathodic peak potentials (Epa and Epc) were not
1
/2
dependent on the scan rate and the ꢂE
p
values were around 60 mV. In addition, the ipa/v
value was invariant with changing scan rates and the ratio of cathodic to anodic peak currents
(ipc/ipa) remained near to unit (Fig. 4). This behaviour is expected for a reversible charge
57
transfer process.
Figure 4. Cyclic voltammograms of Mꢀdppfꢀmpo (M=Pd, Pt) complexes in the potential range
of couple II at different scan rates: (a) Pdꢀdppfꢀmpo and (b) Pdꢀdppfꢀmpo.
Biological results
Anti-Mycobacterium tuberculosis activity. The metal compounds were evaluated in their
activity on Mycobacterium tuberculosis, strain H37Rv ATCC 27294 (panꢀsusceptible). MIC
values are collected in Table 5. Both species showed good to excellent activity against the
bacillus when compared to the four firstꢀline antiꢀTB drugs (MIC values: isoniazid 0.18 µM,
rifampicin 0.49 µM, ethambutol 2.45 µM, pyrazinamide 48.74µM), secondꢀline antiꢀTB drugs,
1
6,58
and some new molecules in clinical trials.
They displayed higher activity than some drugs
already used in the treatment. The Pt compound resulted to be more active than the Pd
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isostructural compound, reaffirming the important role played by the metal center in drug
16
design, and also more active than Na mpo (MIC = 2.42 ± 0.07 µM).
Table 5. In vitro activity of Mꢀdppfꢀmpo (M=Pd, Pt) complexes against T. cruzi (Dm28c
epimastigotes), M. tuberculosis H37Rv ATCC27294, cytotoxicity on Vero epithelial cells (
CCL81) and selectivity index (SI) values.
a
b
Compound
Vero cells
M. tuberculosis SI (fold)
MIC / ꢀM
T. cruzi
SI (fold)
IC / ꢀM
IC / ꢀM
5
0
50
Pd-dppf-mpo 24 ± 12
2.8± 0.7
1.6 ±0.3
9
3
0.64 ± 0.03
0.28 ± 0.01
39
18
Pt-dppf-mpo 5 ± 3
a
b
SI: IC Vero cells / MICM. tuberculosis. SI: IC Vero cells / IC T. cruzi.
50
50
50
Results are the means of three different experiments.
In vitro anti-Trypanosoma cruzi activity. The effect of both Mꢀdppfꢀmpo compounds on the
epimastigote form of T. cruzi, Dm28c strain, was evaluated and compared with that of Na mpo
and the reference tripanosomicidal drug Nifurtimox. The results for the new compounds are
shown in Table 5. Both showed high cytotoxic activity on the parasite with IC50 values in the
submicromolar range. A twoꢀ to fiveꢀfold increase of activity was observed with respect to free
Na mpo (IC50= 1.33 ± 0.08
ꢀM) for Pdꢀdppfꢀmpo and Ptꢀdppfꢀmpo, respectively. In addition,
the complexes showed about 10ꢀ20 times higher activity than the reference drug Nifurtimox
59
(IC50 = 6.0 µM).
Unselective cytotoxicity. In order to get further insight into the potentiality of both metal
compounds as drugs, their corresponding cytotoxicity on VERO epithelial cells (ATCC
CCL81) was assayed in vitro. The IC values on these cells are shown in Table 5, together
5
0
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3

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with the selectivity index (SI) values calculated by dividing them by the MIC for M.
tuberculosis H37Rv or by the IC50 for T. cruzi, all of them expressed in M units. Usually a
compound is considered promising for further testing against M. tuberculosis if a SI value close
ꢀ
6
0
or higher than 10 is found. Thus from this study Pdꢀdppfꢀmpo emerges as a promising
compound for further drug development.
Related to the T. cruzi selectivity, the new compounds showed very good SI values (Table 5).
Even though compounds obtained in this work showed similar antiꢀT. cruzi activity than that
of the previouly developed [Pd(mpo)
M on Tulahuen 2 strain) , Mꢀdppfꢀmpo ones were much
10; tested on J774 macrophages). In conclusion,
2 2
] and [Pt(mpo) ] compounds (IC50[Pd(mpo)2] = 0.067 ±
20
0.015, IC50[Pt(mpo)2] = 0.200 ± 0.018
ꢀ
less toxic (SI[Pd(mpo)2]= 4.9; SI[Pt(mpo)2]
>
although homoleptic and heteroleptic metal compounds showed similar activities, the inclusion
of the ferrocene moiety seemed to decrease the cytotoxicity on mammalian cells increasing the
selectivity against the parasite.
As pointed out by the aforementioned features of the “HOMO/LUMO” frontier KS orbitals of
the new Mꢀdppfꢀmpo species and their corresponding energy gaps (1.95 vs. 2.44 eV for M = Pd
and Pt, respectively), whereas the palladium derivative appears as intrinsically more reactive
and more prone to be reduced at the metal center M (in consistency with the comparative
6
1,62
behaviour found for other Pd/Pt pairs of prospective square planar cytotoxic agents
) both
species display the same tendency to undergo oxidation at the ferrocene moiety forming
oxidized openꢀshell derivatives at the dppf ligand that could contribute to their biological
action. Connected to the fact that Ptꢀdppfꢀmpo consistently displayed higher activity against
both parasites and mammalian cells together with a lower selectivity, all these chemical
features would be indicative of the involvement of multiple mechanisms and targets in the
biological action of the Mꢀdppfꢀmpo species. Whether production of cytotoxic radical species
would be the main course of biological action, both compounds should in principle display
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similar activity and unselective cytotoxicity patterns, which is not the case here, as shown by
the data collected in Table 5. On the other hand, whether a chemical reaction involving ligandꢀ
exchange and/or reduction of M by biomolecular targets would be the main mechanism of
action of these two new compounds, an unobserved higher activity should be expected for Pdꢀ
dppfꢀmpo, unless sideꢀreactions with other components present in the biological assays
(condition excluding fumarate reductases, which are not present in Vero cells, as the sole target
of action) selectively capture the more reactive Pd species early before reaches its molecular
target, thus explaining the observed modulation of activity/selectivity resulting by the change in
the nature of the central metal M. The greater selectivity found against T. cruzi, makes it of
special interest for further exploration of more specific mechanisms of antiꢀparasitic action, as
the inhibition of TcFR addressed in the next section by a combination of biological and docking
approaches.
Interaction with TcFR: biological studies and molecular docking. Since we have previously
shown that other mpo compounds inhibit fumarate reductase activity as part of their probable
mechanism of antitrypapanosomal action, we analyzed the effect of the newly developed Pd
and Pt mpo complexes on this enzyme activity. Figure 5 shows the results of the inhibition test
at a single dose (28 ꢁM). In the conditions assayed, the free ligand and both metalꢀmpo
complexes inhibited T. cruzi´s NADHꢀfumarate reductase activity. Both complexes showed a
marked increase in the inhibitory effect respect to the free ligand, showing the Pt compound the
highest inhibition. Interestingly a similar trend was observed when analyzing the IC of these
50
metal compounds against T. cruzi epimastigotes.
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Dalton Transactions
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DOI: 10.1039/C5DT00557D
2
.5
.0
.5
.0
.5
.0
2
1
1
0
0
Figure 5. Inhibitory effect of metalꢀmpo complexes and Na mpo on T. cruzi NADHꢀ
fumarate reductase activity. Protein extracts of T. cruzi epimastigotes were incubated
for 5 minutes at 30 ºC in the absence (control) or presence of 28 ꢁM of Na mpo, Ptꢀ
dppfꢀmpo and Pdꢀdppfꢀmpo prior to the spectrophotometrical measurement of NADHꢀ
fumarate reductase activity as mentioned in Experimental. The total enzyme inhibition
produced by Na mpo (■) was used to calculate the relative fold of inhibition produced
by Ptꢀdppfꢀmpo (■) and Pdꢀdppfꢀmpo (■). S.D. for each compound is shown for two
independent studies.
In order to further characterize the enzyme inhibition by both Mꢀdppfꢀmpo complexes, the
dependence of their inhibitory effect on concentration was studied. Pdꢀdppfꢀmpo and Ptꢀdppfꢀ
mpo inhibit the enzyme activity in a doseꢀdependent manner (Fig. 6).
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DOI: 10.1039/C5DT00557D
Figure 6. Inhibitory effect of Pdꢀdppfꢀmpo (A) and Ptꢀdppfꢀmpo (B) on NADH fumarate
reductase activity from T. cruzi epimastigotes. Protein extracts were incubated with various
concentrations of the complexes (3, 6, 9 and 12 µM) for 1 h at 28 °C prior to the
spectrophotometrical measurement of NADHꢀfumarate reductase activity as described in
Experimental. Average of two replicas is shown with their SD.
Since TcFR is the enzyme responsible for the conversion of fumarate to succinate, which is one
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3
of the main respiratory substrates required by the parasite for energy production, we sought to
analyze the protective effect of succinate in presence of the aforementioned compounds to
analyze the involvement of TcFR as a target in their mechanism of action. The effect of
succinate on parasite proliferation was analyzed as described in Experimental, by measuring
absorbance at 595 nm at day 5 of incubation using Ptꢀdppfꢀmpo and Pdꢀdppfꢀmpo
concentrations in the range of their IC50 values (0.3 and 0.6 ꢀM, respectively). The ability of
succinate to protect the parasites from the effect of the compounds was evaluated in triplicates
and in comparison with a control with no succinate. As shown in Fig. 7, a clear relief of the
growth inhibition produced by the compounds was observed in the presence of added succinate
in culture medium.
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Figure 7. Protective effect of succinate against the inhibition of parasite growth caused by Pdꢀ
dppfꢀmpo and Ptꢀdppfꢀmpo. Epimastigotes were incubated with 0.6 and 0.3 ꢀM of Pdꢀdppfꢀ
mpo and Ptꢀdppfꢀmpo, respectively, in presence or absence of 5 mM succinate. The percentage
of cell growth was plotted comparing both conditions: succinate (dotted pattern) or no added
succinate (filled pattern). Averages of three replicas are shown with their SD values. **: P
values <0.01 t-test.
Though we cannot discard that both metalꢀmpo complexes could be affecting other
processes contributing to the blockage of T. cruzi´s growth, they clearly have an
inhibitory effect on NADHꢀfumarate reductase activity.
Molecular docking shows that like the other Pt(II)ꢀ and Pd(II)ꢀmpo complexes previously
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+
studied by our group, both [M(mpo)(dppf)] species bind in a lobby cavity of the enzyme,
close to the cofactor binding site (Fig. 8), and positioned next to residues Ser763 and Lys714.
Since Ser763 is adjacent to the catalytic Arg762, such binding could affect the proper
positioning of this residue for catalysis.
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