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Rhenium(I) tricarbonyl compounds of bioactive thiosemicarbazones: Synthesis, characterization and activity against Trypanosoma cruzi
Accepted Manuscript Rhenium(I) tricarbonyl compounds of bioactive thiosemicarbazones: Synthesis, characterization and activity against Trypanosoma cruzi
Esteban Rodríguez Arce, Ignacio Machado, Belén Rodríguez, Michel Lapier, María Carolina Zúñiga, Juan Diego Maya, Claudio Olea Azar, Lucía Otero, Dinorah Gambino PII: DOI: Reference:
S0162-0134(16)30328-2 doi: 10.1016/j.jinorgbio.2017.01.011 JIB 10153
To appear in:
Journal of Inorganic Biochemistry
Received date: Revised date: Accepted date:
11 October 2016 29 December 2016 20 January 2017
Please cite this article as: Esteban Rodríguez Arce, Ignacio Machado, Belén Rodríguez, Michel Lapier, María Carolina Zúñiga, Juan Diego Maya, Claudio Olea Azar, Lucía Otero, Dinorah Gambino , Rhenium(I) tricarbonyl compounds of bioactive thiosemicarbazones: Synthesis, characterization and activity against Trypanosoma cruzi. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jib(2017), doi: 10.1016/j.jinorgbio.2017.01.011
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ACCEPTED MANUSCRIPT Rhenium(I) tricarbonyl compounds of bioactive thiosemicarbazones: synthesis, characterization and activity against Trypanosoma cruzi
Esteban Rodríguez Arce,a# Ignacio Machado,a# Belén Rodríguez,a Michel Lapier,b María Carolina Zúñiga,c Juan Diego Maya,b Claudio Olea Azar,c Lucía Otero,a
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Química Inorgánica, Facultad de Química, Universidad de la República, Gral. Flores
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a
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Dinorah Gambino *a
2124, 11800 Montevideo, Uruguay
c
ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile
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b
Departamento de Química Inorgánica y Analítica, Facultad de Ciencias Químicas y
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Farmacéuticas, Universidad de Chile, Santiago, Chile
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# Both authors have equally contributed.
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*Corresponding author: Tel +598-29249739; fax +598-29241906
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e-mail address: [email protected]
Keywords: fac-rhenium(I) tricarbonyl compounds; 5-nitrofuryl derived
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thiosemicarbazones; Trypanosoma cruzi; Chagas disease; bioactive ligands
Abstract American Trypanosomiasis is a chronic infection discovered and described in 1909 by the brazilian scientist Carlos Chagas. It is caused by the protozoan parasite Trypanosoma cruzi. Although it affects about 10 million people in Latin America, the current chemotherapy is still inadequate. The discovery of new drugs is urgently
ACCEPTED MANUSCRIPT needed. Our group is focused on the development of prospective metal-based drugs mainly based on bioactive ligands and pharmacologically interesting metal ions. In this work three new rhenium(I) tricarbonyl compounds fac-[ReI(CO)3Br(HL)] where HL = 5-nitrofuryl containing thiosemicarbazones were synthesized and fully characterized in solution and in the solid state. The in vitro evaluation of the compounds on T. cruzi
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trypomastigotes (Dm28c strain) showed that the Re(I) compounds are 8 to 15 times
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more active than the reference drug Nifurtimox and show a 4 to 17 fold increase in activity in respect to the free (HL) ligands. Obtained compounds also show good
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selectivity indexes (IC50 endothelial cells Ea.hy926 / IC50 T. cruzi (Dm28c tripomastigotes)). 1H NMR and
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MS studies, performed with time, showed that the fac-[Re(CO)3Br(HL)] species convert into the dimers [Re2(CO)6(L)2] in solution. Crystal structure of [ReI2(CO)6(L2)2], the
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product of complexes´ dimerization, was solved. Related to the mechanism of action, the studied compounds do not generate ROS in the parasite (as 5-nitrofuryl derived
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thiosemicarbazones do) probably due to the unfavorable nitro reduction potential of the
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generated dimeric species. On the contrary, the compounds produce a decrease of the oxygen consumption rate of the parasites may be inhibiting their mitochondrial
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Introduction
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respiration.
American Trypanosomiasis (Chagas disease) is an ancient and endemic disease in Latin America that was discovered and described in 1909 by the Brazilian scientist Carlos Chagas. The disease is caused by the protozoan parasite Trypanosoma cruzi and it is mainly transmitted through the bite of a blood-sucking bug. Up to 30 % of chronically infected people develop cardiac alterations that finally lead to the death. Its socioeconomic impact makes Chagas disease the most important parasitic disease in
ACCEPTED MANUSCRIPT America. Although it affects about 6-7 million people and causes more than 10,000 deaths each year, the current chemotherapy is old and inadequate. The illness is included in the group of seventeen diseases considered by the World Health Organization as neglected tropical diseases (NTDs). In addition, Chagas disease has spread to non-endemic regions (North America, Europe, Asia, Australia) due to the
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mobility of unknowingly infected people from Latin America and the lack of controls in
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these countries of other ways of transmission, like blood transfusion, organ transplant
and less toxic drugs is urgently needed [1-5].
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and passage from infected mother to child. Hence, the development of more efficacious
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The development of metal-based drugs has shown to be a promising approach to find a pharmacological answer to parasitic diseases [6-11].
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In particular, our group is focused on the development of prospective metal-based drugs, mainly based on bioactive ligands and pharmacologically interesting metal ions. This
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strategy could lead to improved chemical and pharmacological profiles. The aim of our
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research has been the generation of metal-based agents that could modulate multiple targets in the parasite: those related to the bioactive ligands and those related to the
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metal [6,7,11,12]. Among the bioactive ligands we have extensively studied a family of thiosemicarbazones (Figure 1) whose main mode of trypanosomicidal action is the
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intraparasite bioreduction of their 5-nitrofuryl pharmacophore, leading to toxic free radical oxygen species (ROS) [13].
Figure 1. Bioactive 5-nitrofuryl derived thiosemicarbazone ligands .
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Around sixty complexes of these ligands have been rationally designed by changing the nature and oxidation state of the metal centre or by including in the coordination sphere of the metal ion different co-ligands that could modulate physicochemical properties of the designed complexes. Most complexes resulted more or at least equally active than
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the corresponding ligands HL. Their activities could be correlated to properties, like
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solubility, stability, lipophilicity and protein interaction, and their mechanisms of action
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against T. cruzi have been extensively studied. Classical Pd(II), Pt(II) and Ru(II/III) coordination compounds as well as organometallic Ru(II) p-cymene and Ru(II)
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cyclopentadienil compounds showed to generate in the parasite the nitro anion radical and ROS by bioreduction of the 5-nitrofuryl moiety, as main part of their mechanism of
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antiparasitic action [11,14-26].
Related to the organometallic compounds, their biological profile showed to be highly
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dependent on the nature of the organometallic moiety [14-16]. Therefore, we expanded
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our research on the effect of metal coordination of these bioactive ligands by including the fac-Re(I) tricarbonyl organometallic moiety. The coordination chemistry of the fac-
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{Re(CO)3}+ core has been widely explored for the development of radiotherapeutic bioorganometallic agents for nuclear medicine purposes [27-29] and, more recently, as a
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new class of promising antiproliferative compounds [30,31]. Moreover, rhenium compounds have been scarcely explored as antitrypanosomal agents. In particular, our group evaluated fac-Re(I) tricarbonyl complexes with salicylaldehyde semicarbazone ligands [32]. Fricker et al. tested a series of classical ‘3+1’ mixed-ligand oxidorhenium(V) complexes [10]. In addition, organometallic cyrhetrenyl complexes derived from 5-nitrofurane were in vitro evaluated on T. cruzi [33].
ACCEPTED MANUSCRIPT In this work three new fac-rhenium(I) tricarbonyl compounds, [fac-Re(CO)3Br(HL)], were synthesized and characterized in solution and in the solid state using different techniques. The selected ligands, HL1-HL3 (Figure 2), include two similar compounds with different chain length (HL1 and HL3) and one of the more bulky compounds of the series bearing the phenyl substituent (HL2). The rhenium(I) compounds were
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evaluated in vitro on Trypanosoma cruzi trypomastigotes (Dm28c strain) and on EaHy
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endothelial mammalian model cells to get IC50 values on the parasite and the selectivity
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towards it. In order to get an insight into the mechanism of action, spin trapping ESR experiments and respiration studies were performed on T. cruzi epimastigotes. In 1
H NMR and cyclic voltammetry experiments, performed with time,
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addition,
supplemented with other experiments, allowed us to get an explanation for the
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compounds´ behaviour in solution. Crystal structure of [ReI2(CO)6(L2)2], the product of
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complexes´ dimerization, was solved.
ACCEPTED MANUSCRIPT Figure 2. Selected bioactive ligands and synthetic procedure for the rhenium compounds.
2. Materials and methods 2.1. Materials All common laboratory chemicals were purchased from commercial sources and were
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used without further purification. The 5-nitrofuryl containing thiosemicarbazones were
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synthesized using a previously reported methodology [13]. [ReBr(CO)5] was prepared
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according to a published procedure by reacting [Re2(CO)10] and Br2 in hexane under N2. Solid [ReBr(CO)5] was isolated by evaporating hexane at low pressure and was purified
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by sublimation [34,35].
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2.2. Syntheses of the rhenium(I) complexes, [ReI(CO)3Br(HL)] The new [ReI(CO)3Br(HL)] complexes, where HL = HL1-HL3, were prepared by
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mixing ReBr(CO)5 (44 mg, 0.108 mmol) with HL (0.108 mmol, 23 mg HL1, 28 mg
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HL2, 22 mg HL3) in toluene (15 mL) and refluxing for 24 h. In each case a brown solid was isolated by centrifugation and washed with two portions of toluene.
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[ReI(CO)3Br(HL1)], 1. Yield: 36 mg, 61 %. Anal. calc. for C9H6BrN4O6SRe (%): C, 19.15; H, 1.07; N, 9.93; S, 5.68. Found: C, 19.25; H, 1.03; N, 9.94; S, 5.67. FTIR
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(KBr): ν/cm−1 1558 (C=N), 1358 s(NO2), 1129 (N-N), 816 δ(NO2)+furan, 2026, 1925, 1906 (CO). 1H NMR (DMSO-d6, /ppm): 7.99 (1H, d, furan-H), 7.86 (1H, d, furan- H), 7.73 (1H, s, CH =N) and ((CD3)2CO, /ppm): 8.93 (2H, br, NH) ppm. 13C NMR (DMSO-d6, /ppm): 150.86 (furan-C), 120.89 (furan-C), 115.72 (furan-C), 149.74 (furan- C), 136.11 (-C=N), 185.21 (CO) ppm. [ReI(CO)3Br(HL2)], 2. Yield: 36 mg, 58 %. Anal. calc. for C15H10BrN4O6SRe (%): C, 28.13; H, 1.57; N, 8.75; S, 5.01. Found: C, 28.12; H, 1.59; N, 8.70; S, 5.02. FTIR
ACCEPTED MANUSCRIPT (KBr): ν/cm−1 1594 (C=N), 1347 s(NO2), 1116 (N-N), 808 δ(NO2)+furan, 2026, 1958, 1914 (CO). 1H NMR (DMSO-d6, /ppm): 7.82 (1H, d, furan-H), 7.78 (1H, d, furan-H), 7.91 (1H, s, CH=N), 10.04 (1H, s, NH), 7.57 (2H, d, phenyl-H), 7.35 (2H, t, phenyl-H), 7.09 (1H, t, phenyl-H) and ((CD3)2CO, /ppm): 11.07 (1H, s, NH) ppm. 13C NMR (DMSO-d6, ppm): 151.236 (furan-C), 115.120 (furan-C), 118.39 (furan-C),
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149.10 (furan- C), 138.36 (-C=N), 122.35 (phenyl-C), 129.01 (phenyl-C), 123.95
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(phenyl-C), 183.83 (CO) ppm.
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[ReI(CO)3Br(HL3)], 3. Yield: 38 mg, 53 %. Anal. calc. for C11H8BrN4O6SRe (%): C, 22.38; H, 1.37; N, 9.49; S, 5.43. Found: C, 22.37; H, 1.36; N, 9.52; S, 5.48. FTIR
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(KBr): ν/cm−1 1561 (C=N), 1347 s(NO2), 1144 (N-N), 813 δ(NO2)+furan, 2029, 1921br (CO). 1H NMR (DMSO-d6, /ppm): 7.53 (1H, d, furan-H), 7.58 (1H, d, furan-
/ppm): 12.48 (2H, s, NH) ppm.
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H), 8.12 (1H, s, CH=N), 7.24 (1H, m, -CH=), 7.80 (1H, m, =CH-) and ((CD3)2CO, C NMR (DMSO-d6, /ppm): 152.76 (furan-C),
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115.40 (furan-C), 129.38 (furan-C), 149.88 (furan- C), 128.01 (-CH=), 124.91 (=CH-), 153.46 (-C=N), 178.98 (CO) ppm.
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2.3. Physicochemical characterization
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C, H, N and S analyses were carried out with a Thermo Scientific Flash 2000 elemental analyzer. Bromide was semi-quantitatively determined by refluxing for several hours a sample of the [Re(CO)3Br(HL)] complexes in toluene in presence of an equimolar amount of silver triflate, according to a previously established procedure [36]. The FTIR absorption spectra (4000-300 cm–1) of the complexes and free ligands were measured as KBr pellets with a Shimadzu IR Prestige-21 instrument. 1H NMR and 13C NMR spectra were recorded in DMSO-d6 or acetone-d6 at 30 oC on a Bruker DPX-400 instrument (at 400 MHz and 100 MHz, respectively). Homo-nuclear correlation
ACCEPTED MANUSCRIPT experiments (COSY) and hetero-nuclear correlation experiments (2D-HETCOR), HSQC (hetero-nuclear single quantum correlation) and HMBC (hetero-nuclear multiple bond correlation), were carried out with the same instrument. Tetramethylsilane was used as the internal standard. Chemical shifts are reported in ppm. Mass spectra were obtained in a Perkin Elmer AxION 2 TOF MS Instrument. Direct sample analyses
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(DSA) with atmospheric pressure chemical ionization (APCI) and time of flight
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detection were used (DSA-APCI-TOF/MS). Compounds dissolved in acetonitrile (10μl)
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were added directly onto a stainless mesh. Samples were run in positive ionization trap mode with a flight tube voltage of 10KV. The capillary exit voltage was set to 110 V for
and an acquisition rate of 2 spectra/sec.
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normal MS analysis. Mass spectra were acquired with a mass range of 100–1300 m/z
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Cyclic voltammograms were obtained with an Epsilon Electrochemical Analyzer. A standard electrochemical three electrode cell of 10 mL volume completed the system. A
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glassy carbon electrode was employed as a working electrode. A platinum wire was
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used as counter electrode while a Ag/AgCl electrode was used as a reference electrode. Measurements were performed at room temperature in 1mM DMSO solutions of the
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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
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the measurements. A continuous gas stream was passed over the solution during the measurements.
2.4. X-ray diffraction study of [ReI2(CO)6(L2)2] The measurements were performed on a Bruker D8 Venture diffractometer with multilayer mirror monochromated CuKα (=1.54178Å) radiation. X-ray diffraction intensities were collected and integrated with APEX2 v2014.5-0 (Bruker AXS). These intensities were
ACCEPTED MANUSCRIPT scaled and the data were corrected empirically for absorption (employing the multi-scan method) with SADABS V2014/2 (Bruker AXS Inc.) program. The structure was solved by direct methods with the SHELXT-2014 [37] and the molecular model developed by alternated cycles of Fourier methods and full-matrix least-squares refinement with Olex2 [38].
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The hydrogen atoms were positioned on stereo-chemical basis and refined with the
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riding model. Crystal data, data collection procedure, structure determination methods
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and refinement results are summarized in Table 1. Crystallographic structural data have been deposited at the Cambridge Crystallographic Data Centre (CCDC). Any request to
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the CCDC for this material should quote the full literature citation and the reference
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number CCDC 1507435.
Formula weight
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Temperature (K)
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Empirical formula
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Table 1. Crystal data and structure refinement results for [Re2(CO)6(L2)2]. C36H30N8O14Re2S2 1235.22 298(2) 1.54178
Crystal system
monoclinic
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Wavelength (Å)
Space group
C 2/c
Unit cell dimensions a (Å)
27.145(5)
b (Å)
12.132(2)
c (Å)
14.098(3)
β (°)
113.37(1)
ACCEPTED MANUSCRIPT 4261.79(14)
Z, density (calculated, Mg/m3)
4, 1.925
Absorption coefficient (mm-1)
12.505
F(000)
2384
Crystal shape/color
rectangular prism/ brownish
Crystal size (mm3)
0.029 x 0.060 x 0.176
-range (º) for data collection
3.547 to 72.249
Index ranges
-33≤h≤33, -14≤k≤14, -17≤l≤17
Reflections collected
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Independent reflections
4198 [R(int) = 0.0399]
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Volume (Å3)
3752
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Observed reflections [I>2(I)]
99.9 (to = 72.249°)
Completeness (%)
multi-scan
Refinement method
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Max. and min. transmission
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Absorption correction
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Data / restraints / parameters
0.7536 and 0.5021 Full-matrix least-squares on F2 4198/0/282 1.144
Final R indicesa [I>2(I)]
R1= 0.0184, wR2 = 0.0437
R indices (all data)
R1= 0.0234, wR2 = 0.0455
Largest diff. peak and hole (e.Å-3)
0.297 and -0.674
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Goodness-of-fit on F2
a
R1=ΣFo-Fc/ΣFo, wR2=[Σw(Fo2-Fc2)2/Σw(Fo 2)2]1/2
2.5. Lipophilicity studies Reversed-phase TLC experiments were done on precoated TLC plates SIL RP-
ACCEPTED MANUSCRIPT 18W/UV254 and eluted with MeOH : DMF : 10 mM aqueous buffer Tris pH 7.4 (20:20:60 v/v/v). Stock solutions were prepared in pure methanol (Aldrich) prior to use. The plates were developed in a closed chromatographic tank, dried and the spots were located under UV light. The Rf values were averaged from two to three determinations,
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and converted to RM via the relationship: RM = log10 [(1/Rf) –1] [39-42].
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2.6. Biological studies
2.6.1. Viability on T. cruzi (Dm28c clone) trypomastigotes. Vero cells were infected
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with T. cruzi metacyclic trypomastigotes from 15 days old Dm28c clone epimastigote cultures. Subsequently, the trypomastigotes harvested from this culture were used to
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infect further Vero cell cultures at a multiplicity of infection (MOI) of 10. These trypomastigote-infected Vero cell cultures were incubated at 37 °C in humidified air
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and 5% CO2 for 5 to 7 days. After this time, culture media were collected and
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centrifuged at 3,000 x g for 5 min. The trypomastigote-containing pellets were resuspended in RPMI media supplemented with 5 % fetal bovine serum and penicillin-
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streptomycin at a final density of 1 x 107 parasites/mL. 2.10 x 108 trypomastigotes are equivalent to 1 mg of protein or 12 mg of wet weight. Viability assays were performed
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by using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction method as previously described [43,44]. Briefly, 1 x 107 trypomastigotes were incubated in fetal bovine serum-RPMI culture medium at 37 °C for 24 h with and without the complexes under study at different concentrations. An aliquot of the parasite suspension was extracted and it was incubated in a flat-bottom 96-well plate and MTT was added at a final concentration of 0.5 mg/mL, incubated at 28 °C for 4 h, and then solubilized with 10% sodium dodecyl sulfate 0.1 mM HCl and incubated overnight.
ACCEPTED MANUSCRIPT Formazan formation was measured at 570 nm with the reference wavelength at 690 nm in a multiwell reader (Biochrom® Asys Expert Plus, Biochrom, USA). Untreated parasites were used as negative controls (100% of viability). Finally, a non-linear regression analysis, using Log concentration vs normalized response fit, by Graph Pad
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prism® software was performed.
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2.6.2 Cytotoxicity on endothelial mammalian cells
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The endothelial cell lines EA.hy926 (permanent human cell line derived by fusing human umbilical vein endothelial cells–HUVEC with human lung cells-A549) were
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maintained in the nutrient medium, Iscove's modified Dulbecco's medium (IMDM) (Sigma–Aldrich), was supplemented with 10% fetal bovine serum (25 mM), penicillin
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(100 units/ml), and streptomycin (100 mg/ml). Cells were maintained as a monolayer culture in tissue culture flasks (Thermo Scientific Nunc™) in an incubator at 37 °C in a
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humidified atmosphere composed of 5 % CO2. Viability assays were performed by
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assay (see above).
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using the MTT reduction method as previously described for T. cruzi trypomastigote
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2.6.3. Generation of free radical species in T. cruzi The free radical production capacity of the new complexes was assessed in the parasite with ESR (electron spin resonance) using DMPO (5,5-dimethyl-1-pirroline-N-oxide) for spin trapping. Each tested compound was dissolved in DMSO (spectroscopy grade, approx. 1 mM) and the solution was added to a mixture containing the epimastigote form of T. cruzi (Dm28c strain; final protein concentration, 4– 8 mg/mL) and DMPO (final concentration, 250 mM). The mixture was transferred to a 50-μL capillary. ESR spectra were recorded in the X band (9.85 GHz) using a Bruker ECS 106 spectrometer
ACCEPTED MANUSCRIPT with a rectangular cavity and 50-kHz field modulation. All the spectra were registered in the same scale, after 15 scans [26,45].
2.6.4 Effect on oxygen consumption by the parasite Dm28c strain T. cruzi epimastigotes were harvested by 500g centrifugation, followed
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by washing and re-suspension in 0.05 M sodium phosphate buffer, pH 7.4, and
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containing 0.107 M sodium chloride. Respiration measurements were carried out
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polarographically with a Clark electrode No. 5331 (Yellow Springs Instruments, 53 YSI model) connected to a 100 mV single channel Goerz RE 511 recorder [17,18,24]. The
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chamber volume was 0.6 mL and the temperature was 28ºC. Compounds, dissolved in DMSO, were added in 40 µM final concentration. The amount of parasite used was
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3. Results and discussion
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effect produced by DMSO alone.
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equivalent to 1.2 mg of protein/mL. Results were corrected according to the observed
3. 1. Synthesis and characterization
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Three complexes including the fac-{Re(CO)3}+ moiety and bidentate bioactive 5nitrofuryl containing thiosemicarbazones as ligands were synthesized with reasonable
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yields according to the proposed synthetic scheme shown in Figure 2. The new compounds were characterized in the solid state and in solution using different techniques. Analytical (including semi quantitative determination of bromide), FTIR and NMR spectroscopic results are in agreement with the formula [ReI(CO)3Br(HL)], where HL HL1- HL3 (Figure 2). All complexes showed a similar FTIR band pattern. Based on our previous experience on vibrational behavior of HL ligands and HL metal complexes [14,16,46], relevant
ACCEPTED MANUSCRIPT vibration bands were tentatively assigned (Table 2). After coordination clear changes in the wavenumber of the (C=N) and (N–N) bands of the free thiosemicarbazone ligands were observed. These modifications have been related to the coordination of the ligand through the iminic nitrogen [46]. In addition, (C=S) bands at around 820–850 cm–1, should shift to lower wave numbers when thiosemicarbazones act as N,S bidentate
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ligands, but as it had been previously stated, they could not be unambiguously assigned
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for the complexes. Nevertheless, significant changes are observed in this region after
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coordination [26,46].
In addition, IR spectra display three strong carbonyl stretching bands, (CO), in the
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2029–1906 cm–1 region, which are characteristic of monomeric pseudooctahedral fac-
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{Re(CO)3}+ complexes [32,47-50].
Table 2. Tentative assignment of selected IR bands of the [ReI(CO)3Br(HL)] complexes.
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positions are given in cm–1.
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Bands of the free thiosemicarbazone ligands HL1-HL3 are included for comparison. Band
(C=N)
s(NO2)
(N-N)
δ(NO2)+furan
(CO)
1602
1355
1108
811
-
1558
1358
1129
816
2026, 1925,1906
1595
1344
1104
811
-
[ReI(CO)3Br(HL2)]
1601
1347
1116
808
2026, 1958, 1914
HL3 a
1586
1353
1081
811
-
[ReI(CO)3Br(HL3)]
1561
1347
1144
813
2029, 1921br
HL1 a
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Compound
HL2 a
a
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[ReI(CO)3Br(HL1)]
from reference [46]
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H NMR and 13C NMR experiments supported the proposed structures of the
synthesized complexes. Two-dimensional NMR experiments such as COSY and HSQC aided in the assignment of the spectra. The 1H NMR spectra of all the complexes showed a similar pattern of signals for the common portions of the nitrofuryl thiosemicarbazone ligands (Table S1). Three signals, corresponding to the two H of
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furan ring (H2 and H3) and H7 of the CH=N moiety of the thiosemicarbazone ligand,
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are observed (see numbering scheme in figure 2). The signals show the expected
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chemical shifts, integration and multiplicity [14,24]. In addition, [Re(CO)3Br(HL2)] spectrum shows the three characteristic signals of the phenyl substituent. For all the
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complexes, H8, being an easily exchangeable proton, could not be detected in DMSO-d6 solution due to the presence of water in the solvent. Nevertheless, a singlet
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corresponding to H8 was easily assigned in acetone-d6 solution spectra of both the complexes and the free ligands. This confirms that the thiosemicarbazone ligand is
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coordinated to Re(I) in a neutral protonated manner. As usual the signals of the ligands
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where shifted due to coordination to the metal centre [14,24]. 13
C-NMR and HSQC experiments allowed to assign the signals of the different
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thiosemicarbazone carbon atoms (Table S2). In addition, the carbonyl carbons signals were identified in the 13C-NMR experiments with the guide of previously reported Re(I)
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tricarbonyl compounds [51,52].
3.2. Solution behavior 1
H NMR experiments on DMSO solutions of the compounds followed with time were
performed in order to study complexes´ behavior in solution. . The behavior of [Re(CO)3Br(HL1)] is shown as an example in Figure 3. Figure 3 (a) shows the spectrum obtained shortly after dissolution in DMSO-d6. The characteristic
ACCEPTED MANUSCRIPT three signals (two doublets and one singlet) of coordinated HL1 in [Re(CO)3Br(HL1)] described above are observed. Besides, two additional doublets at 7.90 and 8.09 ppm, with very low intensity could be detected. After 24 h, an intensity increase of these two additional signals and a modification of the integration and multiplicity of the signal at 7.73 ppm were observed (Figure 3 (b)). These changes reveal that the original
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compound is converting with time into another one with the same spectral pattern, two
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doublets and one singlet (the latter is overlapped by the H3 signal of
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[Re(CO)3Br(HL1)]). Based on previously reported dimerization of chemically related systems we proposed the generation in solution of [Re2(CO)6(L1)2] compound [53-57].
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The chemical shifts of the new signals corresponding to those of the protons of the
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dimer [Re2(CO)6(L1)2] are shown in Table S1.
Figure 3. 1H-NMR spectrum of [Re(CO)3Br(HL1)] (a) zero time (b) after 24 hours.
A similar behavior was observed for the other two [Re(CO)3Br(HL)] HL = HL2 and HL3 compounds (Table S1). However, in both cases an intermediary compound was also detected in the 1H NMR spectra. Since it is well known that bromide could be substituted by a coordinating solvent, we propose that this intermediary emerges from
ACCEPTED MANUSCRIPT the substitution of bromide by DMSO that finally leads to the corresponding dimer [Re2(CO)6(L)2] [27]. Knowing that the original compounds are modified in solution, different experiments were performed to confirm the presence of the dimeric species in solution. Mass spectrometry experiments on solutions of the complexes [Re(CO)3Br(HL)] showed the
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presence in solution of the proposed dimers. Figure S1 depicts part of the spectrum
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obtained for a [Re(CO)3Br(HL2)] solution.
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In addition, we managed to isolate single crystals of [Re2(CO)6(L2)2] from an acetone dissolution of the monomer [Re(CO)3Br(HL2)] by very slow evaporation. The crystals
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were suitable to perform X-ray diffraction studies.
The compound crystalizes in the monoclinic C 2/c space group (Figure 3). The dimeric
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structure is composed of two distorted fac-[Re(CO)3(L2)] subunits with two μ-S bridging both metal centers. The structure is similar to that reported for other Re(I)
D
complexes [53-57].
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The dimer is centrosymmetric. Each rhenium (I) atom lies in a distorted octahedral environment, with a nitrogen and two bridging sulfur atoms coordinated in a facial
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arrangement to the fac-[Re(CO)3]+ moiety. The dimer core is an asymmetric rectangular parallelogram with (μ-S)2Re2 unit at the centre (Figure 3). Each edge of it consists of
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Re-S bonds with different lengths: Re-S1 (2.4490(6) Å) and Re-S1a (2.5513(7) Å). These distances fall in the range normally observed for sulfur atoms coordinated to a fac-[Re(CO)3]+ core [53,54]. The Re-C (carbonyl) distances also fall in the commonly observed range [53-57]. The asymmetry of the distorted octahedron and of the Re-S bond lengths is related with the presence of a more constricted S1-Re-S1a (81.58 (2)°) angle when compared with those of other similar sulphur bridged complexes [53]. The bond lengths and angles around metal centre are summarized in Table 3.
ACCEPTED MANUSCRIPT The bidentate coordination of the thiosemicarbazone ligand through S1 and N2 atoms is typical for complexes with this type of ligands [24] However, only rarely dimeric compounds have been reported [14,15]. The structure is stabilized by hydrogen bonds between the acetone molecule and the
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proton of the nitrogen atom (N4) of thiosemicarbazone ligand (Figure 3).
Re – C15
Re – C16
1.9274 (31)
Re – N2
Re – S1
2.4490 (6)
2.1896 (21)
Re – S1a
2.5513 (7)
97.21 (10)
S1a – Re – C14
172.41 (10)
1.9395 (35)
NU
1.9178 (35)
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Re - C14
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Bond Lengths
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Table 3. Bond lengths [Å] and angles [°] around metal center in [Re2(CO)6(L2)2].
Bond Angles
90.09 (13)
N2 – Re – C15
C14 – Re - C15
89.35 (15)
S1 – Re – C14
92.46 (10)
S1a – Re – C16
86.06 (10)
C15 - Re – C16
87.12 (13)
S1 – Re – C16
97.46 (9)
S1a – Re – C15
96.98 (11)
N2 –Re –C14
93.50 (11)
S1 – Re – C15
175.06 (9)
S1a – Re – N2
89.88 (6)
S1 – Re – N2
78.10 (6)
S1a – Re – S1
81.58 (2)
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N2 – Re – C16
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C14 – Re – C16
174.39 (11)
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ACCEPTED MANUSCRIPT
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Figure 3. ORTEP diagram of fac-[Re2(CO)6(L2)2] showing the atomic displacement
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3.3. Biological results
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ellipsoids at the 50% probability level. Inset: nearly square Re2S2 ring.
3.3.1. Anti-T. cruzi activity
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The new rhenium(I) tricarbonyl compounds were active against T. cruzi (trypomastigotes Dm28c) with IC50 values in the low micromolar range (Table 4). The
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compounds showed a 4- to 17- fold increase of activity in respect to the corresponding thiosemicarbazone ligand. In addition, they are 8 to 15 times more active than the reference trypanocidal drug Nifurtimox (IC50 = 20.1 ± 0.8 µM) [25]. The precursor [Re(CO)5Br] was previously tested showing very low activity on the parasite (IC50 100M) [32].
ACCEPTED MANUSCRIPT Table 4. In vitro activity (measured as the IC50 value, the half inhibitory concentration) against T. cruzi tripomastigotes (Dm28c), cytotoxicity on EA.hy926
endothelial
cells
and
selectivity
index
(SI)
values
of
[Re(CO)3Br(HL)] and HL (included for comparison) and RM values of the complexes and the corresponding ligands (MeOH : DMF : Tris buffer pH 7.4
36.9 ± 0.7
15
0.91
100
10
- 0.29
IC50 / M
HL1
9.8 ± 1.5
[ReI(CO)3Br(HL2)]
1.3 ± 0.3
14.3 ± 0.6
11
1.38
HL2
22.7 ± 1.6
100
4
0.60
[ReI(CO)3Br(HL3)]
2.01 ± 0.03
43.6 ± 1.1
22
0.87
HL3
12.7 ± 1.7
44.9 ± 5.3
3
-0.37
20.1 ± 0.8
-
-
-
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D
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2.4 ± 0.2
SI: IC50 endothelial cells Ea.hy926 / IC50 T. cruzi (Dm28c tripomastigotes) The Rf values were averaged from two to three determinations, and
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b
RM b
[ReI(CO)3Br(HL1)]
Nifurtimoxc a
IC50 / M
SI(fold) a
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Compound
Ea.hy926
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T. cruzi
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(20:20:60 v/v/v)).
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converted to RM via the relationship: RM = log10 [(1/Rf) –1] c
From reference [26]
3.3.2. Cytotoxicity on mammalian cells The specificity of the antitrypanosomal activity of the new compounds was evaluated by analyzing their cytotoxicity against a human-derived endothelial cell line (EA.hy926). All the new compounds showed moderate to good selectivity towards the parasites when compared to the selected mammalian cell model (Table 4). In all cases
ACCEPTED MANUSCRIPT the coordination to the rhenium(I) center improved the selectivity in respect to the free ligands.
3.3.3. Lipophilicity Lipophilicity is a very important physicochemical property of prospective drugs that
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controls biological behaviour, particularly transmembrane transport and interaction with
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biological receptors. Therefore, its correlation with the observed biological activity is
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usually relevant [59]. The effect of complex formation on lipophilicity was experimentally determined using reversed-phase TLC, where the stationary phase
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(precoated TLC-C18) may be considered to simulate lipids of biological membranes or receptors, and the mobile phase (MeOH : DMF : Tris buffer pH 7.4 (20:20:60 v/v/v))
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resembles the aqueous biological milieu. The composition of the mobile phase was tuned-up in order to allow differentiating complexes according to their lipophilicity and
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comparing with the free thiosemicarbazones.
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The complexes were more lipophilic than the corresponding thiosemicarbazone ligands (Table 4). As expected, the lipophilicity of the complexes increases as the N-substituent
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in the thiosemicarbazone ligand changes from hydrogen to phenyl. [ReI(CO)3Br(HL2)], 2, is the more lipophilic and the more active compound but being the series of
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compounds too short this is only a preliminar qualitative observation. To establish a correlation between lipophilicity and antiparasitic activity a larger series of structurally related compounds should be considered.
3.4. Insight into the mechanism of action 3.4.1. Generation of free radical species in T. cruzi
ACCEPTED MANUSCRIPT As previously stated, the main mechanism of anti T. cruzi action of the selected 5nitrofuryl derived thiosemicarbazones involves the intraparasite bioreduction leading to toxic free radicals. The first step of this mechanism involves the one electron reduction of the nitro moiety by parasite enzymes. Through redox cycling, the formed nitro anion radical generates other radical species that are toxic for the parasite [13]. We previously
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demonstrated that this mechanism of action is retained by all the other previously
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reported metal complexes of these bioactive ligands [14,16-18,24,25]. As usual, the free
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radical production capacity of the new rhenium(I) compounds was studied by ESR. The compounds were incubated with T. cruzi (Dm28c strain) epimastigotes. DMPO was
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added as spin trapping agent to detect free radical species having short half-lives. For the first time signals were not observed in the ESR spectra of these complexes showing
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that the expected free radicals were not generated in the parasite by effect of them.
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3.4.2. Effect on oxygen consumption by the parasite
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parasites
ReHL4 40
435nm O2 12 min
83.2% inhibition
Figure 4. Effect of [Re(CO)3Br(HL4)] on the rate of respiration of the parasite.
ACCEPTED MANUSCRIPT The involvement of the obtained rhenium complexes in redox cycling processes should increase the parasite oxygen consumption as reported for the thiosemicarbazone ligands and their previously reported complexes [17,18,24]. Thus, oxygen uptake, was measured for the obtained complexes. However, results showed that the compounds decrease the rate of oxygen consumption of the parasite
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([Re(CO)3Br(HL1)], 3.7 %; [Re(CO)3Br(HL2)], 83.2 %; [Re(CO)3Br(HL3)], 12.3 %)
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(Figure 4). This is in agreement with the lack of generation of ROS observed in the ESR
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experiments indicating a different mechanism of action for the obtained rhenium
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compounds that would involve the inhibition of mitochondrial respiration.
3.4.3. Cyclic voltammetry studies
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Hence, these compounds act through a different mechanism than the one known for the free ligands and for all their previously developed metal compounds. In this regard, the
D
reduction potential of the nitro group, first step in the bioreduction, is a very important
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parameter to be determined in order to predict how easy this reduction process in vivo could be. The electrochemical characterization of the complexes was performed at room
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temperature by cyclic voltammetry in DMSO solutions using a carbon disk electrode. The obtained voltammograms (recorded at a scan rate of 100 mV/s) are depicted in
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Figure 5. Selected electrochemical data are shown in Table 5. All obtained complexes showed a similar voltammetric response in both cathodic and anodic directions.
ACCEPTED MANUSCRIPT
12
II
I
ReHL1 ReHL3 ReHL2
III
10
I ( A )
8 6 4
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2 0
0,0
-0,2
-0,4
-0,6
-0,8
-1,0
-1,2
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E (V)
-1,4
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-2
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Figure 5 Cyclic voltammograms of 1mM DMSO solutions of the [Re(CO)3Br(HL)] complexes, codes ReHL1, ReHL2 and ReHL3, measured in the cathodic direction, scan
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rate100 mV/s. Working electrode: glassy carbon. Potentials are measured vs. Ag/AgCl.
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The voltammograms show three processes centered on the thiosemicarbazone ligand.
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As previously described, the complexes show a quasi reversible reduction process around -0.7 V (vs Ag/AgCl), corresponding to the generation of a nitro anion radical
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(NO2·-) by reduction of the nitro group of the thiosemicarbazone (couple II) (Figure 5). Near -1.3 V, an irreversible signal is observed (couple III) that corresponds to further
AC
reduction of the nitro anion radical to hydroxylamine [16,17,60]. When moving to less negative potentials, an irreversible reduction process is detected (couple I). In order to assign this signal the electrochemical behavior of the complexes in DMSO solution was studied with time during 24 h after preparation of the DMSO solution. Changes in the relative intensities of couple I and couple II were observed with time for the three complexes. Figure 6 shows the behavior of ReHL1 as an example. A
ACCEPTED MANUSCRIPT decrease of the intensity of couple II with time was observed, with a simultaneous increase in the intensity of the cathodic peak of redox process I.
I
II
12 10
6
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(A)
8
4
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2
-2 0,0
-0,2
-0,4
-0,6
-0,8
-1,0
-1,4
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E (V)
-1,2
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0
Figure 6. Cyclic voltammograms of a 1mM DMSO solution of ReHL1 measured in the
D
cathodic direction, scan rate100 mV/s, at different times: (-) t = 0, (-) 1 h and (-) 24 h
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after preparation of the solution. Working electrode: glassy carbon, scan rate 100 mV/s.
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Potentials are measured vs. Ag/AgCl.
These results are in agreement with the conversion in solution of each monomeric
AC
compound [Re(CO)3Br(HL)] into the dimeric one [Re2(CO)6(L)2] (Table 5). In these species, a less negative reduction potential of the nitro moiety was observed.
Table 5. Electrochemical data of the compounds [Re(CO)3Br(HL)] and HL with a glassy carbon working electrode at a scan rate of 100 mV/s. Potentials are measured
ACCEPTED MANUSCRIPT vs. Ag/AgCl. ReI/ReII Compound
Couple I
Couple II (HL)
(dimer)
(monomer)
Epc (V)
Epa (V)
Epc (V)
[Re(CO)3Br(HL1)
0.93
-0.50
-0.64 (-0.64)
-0.74 (-0.72)
[Re(CO)3Br(HL2)
0.97
-0.40
-0.61 (-0.62)
-0.70 (-0.70)
[Re(CO)3Br(HL3)
0.83
-0.43
-0.61 (-0.64)
-0.71 (-0.71)
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Epa (V)
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Previous cyclic voltammetry studies demonstrated that coordination of the 5-nitrofuryl thiosemicarbazone ligands to different metal ions lead to a slight displacement of the
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potential of the nitro group – nitro anion radical couple to less negative values. This change would favor the generation of radical species in the parasite [14,16-18,24,25].
D
However, in this case, the new species generated in solution show a high shift of the
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reduction potential of the nitro group that could fall outside the potential window of the enzymes involved in the reduction in the parasite. This fact could explain the lack of
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formation of ROS in the parasite.
A similar behavior with time was observed in the anodic direction. The anodic scan
AC
shows an irreversible process around 1.0 V assigned to the redox couple ReI/ReII, characteristic of the fac-Re(I) tricarbonyl compounds [61,62]. The observed differences in Epa values for the three compounds (Table 5), show that the nature of the substituent R and/or the chain length of the hydrocarbon linker between the pharmacophore and the thiosemicarbazone moiety are relevant for the oxidation process. This is in agreement with reported values for other fac-Re(I)Br complexes with bidentate ligands where the redox potential varies with the electrodonating capacity of the ligand [62].
ACCEPTED MANUSCRIPT The conversion of the [Re(CO)3Br(HL) complex into the dimeric species in solution also affects the oxidation of the metal center. The voltammograms obtained in the study with time show a displacement of Epa to more positive values (Figure S2). The new species shows a less favorable oxidation of the metal center than the original
PT
[Re(CO)3Br(HL) compound.
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4. Conclusions
Coordination of the fac-Re(CO)3 moiety to bioactive HL thiosemicarbazone ligands led
SC
to compounds with improved biological properties. The fac-[Re(CO)3Br(HL)]
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compounds showed high activities on bloodstream T. cruzi, with IC50 values in the low M range, and good selectivities towards the parasite. The Re(I) compounds are 8 to 15
MA
times more active than Nifurtimox and show a 4 to 17 fold increase in activity in respect to the free ligands.
D
The nature of the species generated in solution has been studied by NMR, MS, CV and
[Re2(CO)6(L)2].
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X-ray diffraction studies. The fac-[Re(CO)3Br(HL)] species convert into the dimers
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The compounds do not generate ROS in the parasite probably due to the unfavorable nitro reduction potential of the generated dimeric species. The compounds inhibit
AC
mitochondrial respiration of the parasite, effect shown by the decrease of the oxygen consumption rate.
Acknowledgements ERA thanks ANII (Uruguay) for the doctoral grant POS_NAC_2015_1_110215. IM thanks ANII (Uruguay) for the research grant INI_X_2011_1_3902. Authors thank PEDECIBA and ANII-SNI, Uruguay and FONDECYT 1150175, Chile.
ACCEPTED MANUSCRIPT
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[58] R. Schiifer, W. Kahn, J. Fiedled, Inorg. Chem. 32 (1993) 3199-3200. [59] E.H. Kerns, L. Di, Drug-like properties: Concepts, structure design and methods from ADME to toxicity optimization. Academic Press, Amsterdam, 2008. [60] C. Rigol, C. Olea-Azar, F. Mendizábal, L. Otero, D. Gambino, M. González, Hugo Cerecetto, Spectrochim Acta A 61 (2005) 2933- 2938.
ACCEPTED MANUSCRIPT [61] R. Silva, B. Liddle, S. Lindeman, M. Smith, J. Gardinier, Inorg. Chem. 45 (2006) 6794-6802. [62] D. Reger, K. Brown, J. Gardinier, M. Smith J. Organomet. Chem. 690 (2005)
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Graphical abstract
ACCEPTED MANUSCRIPT Coordination of the fac-Re(CO)3 moiety to bioactive thiosemicarbazones HL led to fac[Re(CO)3Br(HL)] compounds with high activities on bloodstream Trypanosoma cruzi and good selectivities towards the parasite. Complexes convert into [Re2(CO)6(L)2] in solution and inhibit parasite mitochondrial respiration, effect shown by decrease of
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oxygen consumption rate, without generating radical oxygen species.
ACCEPTED MANUSCRIPT Highlights New fac-[Re(CO)3Br(HL)] compounds with bioactive HL thiosemicarbazones were synthesized.
The compounds showed high activities on bloodstream Trypanosoma cruzi.
They showed good selectivities towards the parasite in respect to mammalian cell
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model.
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They inhibit parasite mitochondrial respiration without generating radical oxygen
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species.
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The fac-[Re(CO)3Br(HL)] species convert into the dimers [Re2(CO)6(L)2] in solution.
Esteban Rodríguez Arce, Ignacio Machado, Belén Rodríguez, Michel Lapier, María Carolina Zúñiga, Juan Diego Maya, Claudio Olea Azar, Lucía Otero, Dinorah Gambino PII: DOI: Reference:
S0162-0134(16)30328-2 doi: 10.1016/j.jinorgbio.2017.01.011 JIB 10153
To appear in:
Journal of Inorganic Biochemistry
Received date: Revised date: Accepted date:
11 October 2016 29 December 2016 20 January 2017
Please cite this article as: Esteban Rodríguez Arce, Ignacio Machado, Belén Rodríguez, Michel Lapier, María Carolina Zúñiga, Juan Diego Maya, Claudio Olea Azar, Lucía Otero, Dinorah Gambino , Rhenium(I) tricarbonyl compounds of bioactive thiosemicarbazones: Synthesis, characterization and activity against Trypanosoma cruzi. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jib(2017), doi: 10.1016/j.jinorgbio.2017.01.011
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ACCEPTED MANUSCRIPT Rhenium(I) tricarbonyl compounds of bioactive thiosemicarbazones: synthesis, characterization and activity against Trypanosoma cruzi
Esteban Rodríguez Arce,a# Ignacio Machado,a# Belén Rodríguez,a Michel Lapier,b María Carolina Zúñiga,c Juan Diego Maya,b Claudio Olea Azar,c Lucía Otero,a
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Química Inorgánica, Facultad de Química, Universidad de la República, Gral. Flores
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a
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Dinorah Gambino *a
2124, 11800 Montevideo, Uruguay
c
ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile
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b
Departamento de Química Inorgánica y Analítica, Facultad de Ciencias Químicas y
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Farmacéuticas, Universidad de Chile, Santiago, Chile
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# Both authors have equally contributed.
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*Corresponding author: Tel +598-29249739; fax +598-29241906
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e-mail address: [email protected]
Keywords: fac-rhenium(I) tricarbonyl compounds; 5-nitrofuryl derived
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thiosemicarbazones; Trypanosoma cruzi; Chagas disease; bioactive ligands
Abstract American Trypanosomiasis is a chronic infection discovered and described in 1909 by the brazilian scientist Carlos Chagas. It is caused by the protozoan parasite Trypanosoma cruzi. Although it affects about 10 million people in Latin America, the current chemotherapy is still inadequate. The discovery of new drugs is urgently
ACCEPTED MANUSCRIPT needed. Our group is focused on the development of prospective metal-based drugs mainly based on bioactive ligands and pharmacologically interesting metal ions. In this work three new rhenium(I) tricarbonyl compounds fac-[ReI(CO)3Br(HL)] where HL = 5-nitrofuryl containing thiosemicarbazones were synthesized and fully characterized in solution and in the solid state. The in vitro evaluation of the compounds on T. cruzi
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trypomastigotes (Dm28c strain) showed that the Re(I) compounds are 8 to 15 times
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more active than the reference drug Nifurtimox and show a 4 to 17 fold increase in activity in respect to the free (HL) ligands. Obtained compounds also show good
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selectivity indexes (IC50 endothelial cells Ea.hy926 / IC50 T. cruzi (Dm28c tripomastigotes)). 1H NMR and
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MS studies, performed with time, showed that the fac-[Re(CO)3Br(HL)] species convert into the dimers [Re2(CO)6(L)2] in solution. Crystal structure of [ReI2(CO)6(L2)2], the
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product of complexes´ dimerization, was solved. Related to the mechanism of action, the studied compounds do not generate ROS in the parasite (as 5-nitrofuryl derived
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thiosemicarbazones do) probably due to the unfavorable nitro reduction potential of the
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generated dimeric species. On the contrary, the compounds produce a decrease of the oxygen consumption rate of the parasites may be inhibiting their mitochondrial
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Introduction
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respiration.
American Trypanosomiasis (Chagas disease) is an ancient and endemic disease in Latin America that was discovered and described in 1909 by the Brazilian scientist Carlos Chagas. The disease is caused by the protozoan parasite Trypanosoma cruzi and it is mainly transmitted through the bite of a blood-sucking bug. Up to 30 % of chronically infected people develop cardiac alterations that finally lead to the death. Its socioeconomic impact makes Chagas disease the most important parasitic disease in
ACCEPTED MANUSCRIPT America. Although it affects about 6-7 million people and causes more than 10,000 deaths each year, the current chemotherapy is old and inadequate. The illness is included in the group of seventeen diseases considered by the World Health Organization as neglected tropical diseases (NTDs). In addition, Chagas disease has spread to non-endemic regions (North America, Europe, Asia, Australia) due to the
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mobility of unknowingly infected people from Latin America and the lack of controls in
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these countries of other ways of transmission, like blood transfusion, organ transplant
and less toxic drugs is urgently needed [1-5].
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and passage from infected mother to child. Hence, the development of more efficacious
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The development of metal-based drugs has shown to be a promising approach to find a pharmacological answer to parasitic diseases [6-11].
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In particular, our group is focused on the development of prospective metal-based drugs, mainly based on bioactive ligands and pharmacologically interesting metal ions. This
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strategy could lead to improved chemical and pharmacological profiles. The aim of our
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research has been the generation of metal-based agents that could modulate multiple targets in the parasite: those related to the bioactive ligands and those related to the
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metal [6,7,11,12]. Among the bioactive ligands we have extensively studied a family of thiosemicarbazones (Figure 1) whose main mode of trypanosomicidal action is the
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intraparasite bioreduction of their 5-nitrofuryl pharmacophore, leading to toxic free radical oxygen species (ROS) [13].
Figure 1. Bioactive 5-nitrofuryl derived thiosemicarbazone ligands .
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Around sixty complexes of these ligands have been rationally designed by changing the nature and oxidation state of the metal centre or by including in the coordination sphere of the metal ion different co-ligands that could modulate physicochemical properties of the designed complexes. Most complexes resulted more or at least equally active than
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the corresponding ligands HL. Their activities could be correlated to properties, like
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solubility, stability, lipophilicity and protein interaction, and their mechanisms of action
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against T. cruzi have been extensively studied. Classical Pd(II), Pt(II) and Ru(II/III) coordination compounds as well as organometallic Ru(II) p-cymene and Ru(II)
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cyclopentadienil compounds showed to generate in the parasite the nitro anion radical and ROS by bioreduction of the 5-nitrofuryl moiety, as main part of their mechanism of
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antiparasitic action [11,14-26].
Related to the organometallic compounds, their biological profile showed to be highly
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dependent on the nature of the organometallic moiety [14-16]. Therefore, we expanded
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our research on the effect of metal coordination of these bioactive ligands by including the fac-Re(I) tricarbonyl organometallic moiety. The coordination chemistry of the fac-
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{Re(CO)3}+ core has been widely explored for the development of radiotherapeutic bioorganometallic agents for nuclear medicine purposes [27-29] and, more recently, as a
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new class of promising antiproliferative compounds [30,31]. Moreover, rhenium compounds have been scarcely explored as antitrypanosomal agents. In particular, our group evaluated fac-Re(I) tricarbonyl complexes with salicylaldehyde semicarbazone ligands [32]. Fricker et al. tested a series of classical ‘3+1’ mixed-ligand oxidorhenium(V) complexes [10]. In addition, organometallic cyrhetrenyl complexes derived from 5-nitrofurane were in vitro evaluated on T. cruzi [33].
ACCEPTED MANUSCRIPT In this work three new fac-rhenium(I) tricarbonyl compounds, [fac-Re(CO)3Br(HL)], were synthesized and characterized in solution and in the solid state using different techniques. The selected ligands, HL1-HL3 (Figure 2), include two similar compounds with different chain length (HL1 and HL3) and one of the more bulky compounds of the series bearing the phenyl substituent (HL2). The rhenium(I) compounds were
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evaluated in vitro on Trypanosoma cruzi trypomastigotes (Dm28c strain) and on EaHy
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endothelial mammalian model cells to get IC50 values on the parasite and the selectivity
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towards it. In order to get an insight into the mechanism of action, spin trapping ESR experiments and respiration studies were performed on T. cruzi epimastigotes. In 1
H NMR and cyclic voltammetry experiments, performed with time,
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addition,
supplemented with other experiments, allowed us to get an explanation for the
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compounds´ behaviour in solution. Crystal structure of [ReI2(CO)6(L2)2], the product of
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complexes´ dimerization, was solved.
ACCEPTED MANUSCRIPT Figure 2. Selected bioactive ligands and synthetic procedure for the rhenium compounds.
2. Materials and methods 2.1. Materials All common laboratory chemicals were purchased from commercial sources and were
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used without further purification. The 5-nitrofuryl containing thiosemicarbazones were
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synthesized using a previously reported methodology [13]. [ReBr(CO)5] was prepared
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according to a published procedure by reacting [Re2(CO)10] and Br2 in hexane under N2. Solid [ReBr(CO)5] was isolated by evaporating hexane at low pressure and was purified
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by sublimation [34,35].
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2.2. Syntheses of the rhenium(I) complexes, [ReI(CO)3Br(HL)] The new [ReI(CO)3Br(HL)] complexes, where HL = HL1-HL3, were prepared by
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mixing ReBr(CO)5 (44 mg, 0.108 mmol) with HL (0.108 mmol, 23 mg HL1, 28 mg
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HL2, 22 mg HL3) in toluene (15 mL) and refluxing for 24 h. In each case a brown solid was isolated by centrifugation and washed with two portions of toluene.
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[ReI(CO)3Br(HL1)], 1. Yield: 36 mg, 61 %. Anal. calc. for C9H6BrN4O6SRe (%): C, 19.15; H, 1.07; N, 9.93; S, 5.68. Found: C, 19.25; H, 1.03; N, 9.94; S, 5.67. FTIR
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(KBr): ν/cm−1 1558 (C=N), 1358 s(NO2), 1129 (N-N), 816 δ(NO2)+furan, 2026, 1925, 1906 (CO). 1H NMR (DMSO-d6, /ppm): 7.99 (1H, d, furan-H), 7.86 (1H, d, furan- H), 7.73 (1H, s, CH =N) and ((CD3)2CO, /ppm): 8.93 (2H, br, NH) ppm. 13C NMR (DMSO-d6, /ppm): 150.86 (furan-C), 120.89 (furan-C), 115.72 (furan-C), 149.74 (furan- C), 136.11 (-C=N), 185.21 (CO) ppm. [ReI(CO)3Br(HL2)], 2. Yield: 36 mg, 58 %. Anal. calc. for C15H10BrN4O6SRe (%): C, 28.13; H, 1.57; N, 8.75; S, 5.01. Found: C, 28.12; H, 1.59; N, 8.70; S, 5.02. FTIR
ACCEPTED MANUSCRIPT (KBr): ν/cm−1 1594 (C=N), 1347 s(NO2), 1116 (N-N), 808 δ(NO2)+furan, 2026, 1958, 1914 (CO). 1H NMR (DMSO-d6, /ppm): 7.82 (1H, d, furan-H), 7.78 (1H, d, furan-H), 7.91 (1H, s, CH=N), 10.04 (1H, s, NH), 7.57 (2H, d, phenyl-H), 7.35 (2H, t, phenyl-H), 7.09 (1H, t, phenyl-H) and ((CD3)2CO, /ppm): 11.07 (1H, s, NH) ppm. 13C NMR (DMSO-d6, ppm): 151.236 (furan-C), 115.120 (furan-C), 118.39 (furan-C),
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149.10 (furan- C), 138.36 (-C=N), 122.35 (phenyl-C), 129.01 (phenyl-C), 123.95
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(phenyl-C), 183.83 (CO) ppm.
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[ReI(CO)3Br(HL3)], 3. Yield: 38 mg, 53 %. Anal. calc. for C11H8BrN4O6SRe (%): C, 22.38; H, 1.37; N, 9.49; S, 5.43. Found: C, 22.37; H, 1.36; N, 9.52; S, 5.48. FTIR
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(KBr): ν/cm−1 1561 (C=N), 1347 s(NO2), 1144 (N-N), 813 δ(NO2)+furan, 2029, 1921br (CO). 1H NMR (DMSO-d6, /ppm): 7.53 (1H, d, furan-H), 7.58 (1H, d, furan-
/ppm): 12.48 (2H, s, NH) ppm.
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H), 8.12 (1H, s, CH=N), 7.24 (1H, m, -CH=), 7.80 (1H, m, =CH-) and ((CD3)2CO, C NMR (DMSO-d6, /ppm): 152.76 (furan-C),
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115.40 (furan-C), 129.38 (furan-C), 149.88 (furan- C), 128.01 (-CH=), 124.91 (=CH-), 153.46 (-C=N), 178.98 (CO) ppm.
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2.3. Physicochemical characterization
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C, H, N and S analyses were carried out with a Thermo Scientific Flash 2000 elemental analyzer. Bromide was semi-quantitatively determined by refluxing for several hours a sample of the [Re(CO)3Br(HL)] complexes in toluene in presence of an equimolar amount of silver triflate, according to a previously established procedure [36]. The FTIR absorption spectra (4000-300 cm–1) of the complexes and free ligands were measured as KBr pellets with a Shimadzu IR Prestige-21 instrument. 1H NMR and 13C NMR spectra were recorded in DMSO-d6 or acetone-d6 at 30 oC on a Bruker DPX-400 instrument (at 400 MHz and 100 MHz, respectively). Homo-nuclear correlation
ACCEPTED MANUSCRIPT experiments (COSY) and hetero-nuclear correlation experiments (2D-HETCOR), HSQC (hetero-nuclear single quantum correlation) and HMBC (hetero-nuclear multiple bond correlation), were carried out with the same instrument. Tetramethylsilane was used as the internal standard. Chemical shifts are reported in ppm. Mass spectra were obtained in a Perkin Elmer AxION 2 TOF MS Instrument. Direct sample analyses
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(DSA) with atmospheric pressure chemical ionization (APCI) and time of flight
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detection were used (DSA-APCI-TOF/MS). Compounds dissolved in acetonitrile (10μl)
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were added directly onto a stainless mesh. Samples were run in positive ionization trap mode with a flight tube voltage of 10KV. The capillary exit voltage was set to 110 V for
and an acquisition rate of 2 spectra/sec.
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normal MS analysis. Mass spectra were acquired with a mass range of 100–1300 m/z
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Cyclic voltammograms were obtained with an Epsilon Electrochemical Analyzer. A standard electrochemical three electrode cell of 10 mL volume completed the system. A
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glassy carbon electrode was employed as a working electrode. A platinum wire was
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used as counter electrode while a Ag/AgCl electrode was used as a reference electrode. Measurements were performed at room temperature in 1mM DMSO solutions of the
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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
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the measurements. A continuous gas stream was passed over the solution during the measurements.
2.4. X-ray diffraction study of [ReI2(CO)6(L2)2] The measurements were performed on a Bruker D8 Venture diffractometer with multilayer mirror monochromated CuKα (=1.54178Å) radiation. X-ray diffraction intensities were collected and integrated with APEX2 v2014.5-0 (Bruker AXS). These intensities were
ACCEPTED MANUSCRIPT scaled and the data were corrected empirically for absorption (employing the multi-scan method) with SADABS V2014/2 (Bruker AXS Inc.) program. The structure was solved by direct methods with the SHELXT-2014 [37] and the molecular model developed by alternated cycles of Fourier methods and full-matrix least-squares refinement with Olex2 [38].
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The hydrogen atoms were positioned on stereo-chemical basis and refined with the
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riding model. Crystal data, data collection procedure, structure determination methods
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and refinement results are summarized in Table 1. Crystallographic structural data have been deposited at the Cambridge Crystallographic Data Centre (CCDC). Any request to
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the CCDC for this material should quote the full literature citation and the reference
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number CCDC 1507435.
Formula weight
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Temperature (K)
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Empirical formula
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Table 1. Crystal data and structure refinement results for [Re2(CO)6(L2)2]. C36H30N8O14Re2S2 1235.22 298(2) 1.54178
Crystal system
monoclinic
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Wavelength (Å)
Space group
C 2/c
Unit cell dimensions a (Å)
27.145(5)
b (Å)
12.132(2)
c (Å)
14.098(3)
β (°)
113.37(1)
ACCEPTED MANUSCRIPT 4261.79(14)
Z, density (calculated, Mg/m3)
4, 1.925
Absorption coefficient (mm-1)
12.505
F(000)
2384
Crystal shape/color
rectangular prism/ brownish
Crystal size (mm3)
0.029 x 0.060 x 0.176
-range (º) for data collection
3.547 to 72.249
Index ranges
-33≤h≤33, -14≤k≤14, -17≤l≤17
Reflections collected
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Independent reflections
4198 [R(int) = 0.0399]
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Volume (Å3)
3752
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Observed reflections [I>2(I)]
99.9 (to = 72.249°)
Completeness (%)
multi-scan
Refinement method
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Max. and min. transmission
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Absorption correction
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Data / restraints / parameters
0.7536 and 0.5021 Full-matrix least-squares on F2 4198/0/282 1.144
Final R indicesa [I>2(I)]
R1= 0.0184, wR2 = 0.0437
R indices (all data)
R1= 0.0234, wR2 = 0.0455
Largest diff. peak and hole (e.Å-3)
0.297 and -0.674
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Goodness-of-fit on F2
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R1=ΣFo-Fc/ΣFo, wR2=[Σw(Fo2-Fc2)2/Σw(Fo 2)2]1/2
2.5. Lipophilicity studies Reversed-phase TLC experiments were done on precoated TLC plates SIL RP-
ACCEPTED MANUSCRIPT 18W/UV254 and eluted with MeOH : DMF : 10 mM aqueous buffer Tris pH 7.4 (20:20:60 v/v/v). Stock solutions were prepared in pure methanol (Aldrich) prior to use. The plates were developed in a closed chromatographic tank, dried and the spots were located under UV light. The Rf values were averaged from two to three determinations,
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and converted to RM via the relationship: RM = log10 [(1/Rf) –1] [39-42].
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2.6. Biological studies
2.6.1. Viability on T. cruzi (Dm28c clone) trypomastigotes. Vero cells were infected
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with T. cruzi metacyclic trypomastigotes from 15 days old Dm28c clone epimastigote cultures. Subsequently, the trypomastigotes harvested from this culture were used to
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infect further Vero cell cultures at a multiplicity of infection (MOI) of 10. These trypomastigote-infected Vero cell cultures were incubated at 37 °C in humidified air
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and 5% CO2 for 5 to 7 days. After this time, culture media were collected and
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centrifuged at 3,000 x g for 5 min. The trypomastigote-containing pellets were resuspended in RPMI media supplemented with 5 % fetal bovine serum and penicillin-
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streptomycin at a final density of 1 x 107 parasites/mL. 2.10 x 108 trypomastigotes are equivalent to 1 mg of protein or 12 mg of wet weight. Viability assays were performed
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by using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction method as previously described [43,44]. Briefly, 1 x 107 trypomastigotes were incubated in fetal bovine serum-RPMI culture medium at 37 °C for 24 h with and without the complexes under study at different concentrations. An aliquot of the parasite suspension was extracted and it was incubated in a flat-bottom 96-well plate and MTT was added at a final concentration of 0.5 mg/mL, incubated at 28 °C for 4 h, and then solubilized with 10% sodium dodecyl sulfate 0.1 mM HCl and incubated overnight.
ACCEPTED MANUSCRIPT Formazan formation was measured at 570 nm with the reference wavelength at 690 nm in a multiwell reader (Biochrom® Asys Expert Plus, Biochrom, USA). Untreated parasites were used as negative controls (100% of viability). Finally, a non-linear regression analysis, using Log concentration vs normalized response fit, by Graph Pad
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prism® software was performed.
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2.6.2 Cytotoxicity on endothelial mammalian cells
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The endothelial cell lines EA.hy926 (permanent human cell line derived by fusing human umbilical vein endothelial cells–HUVEC with human lung cells-A549) were
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maintained in the nutrient medium, Iscove's modified Dulbecco's medium (IMDM) (Sigma–Aldrich), was supplemented with 10% fetal bovine serum (25 mM), penicillin
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(100 units/ml), and streptomycin (100 mg/ml). Cells were maintained as a monolayer culture in tissue culture flasks (Thermo Scientific Nunc™) in an incubator at 37 °C in a
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humidified atmosphere composed of 5 % CO2. Viability assays were performed by
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assay (see above).
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using the MTT reduction method as previously described for T. cruzi trypomastigote
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2.6.3. Generation of free radical species in T. cruzi The free radical production capacity of the new complexes was assessed in the parasite with ESR (electron spin resonance) using DMPO (5,5-dimethyl-1-pirroline-N-oxide) for spin trapping. Each tested compound was dissolved in DMSO (spectroscopy grade, approx. 1 mM) and the solution was added to a mixture containing the epimastigote form of T. cruzi (Dm28c strain; final protein concentration, 4– 8 mg/mL) and DMPO (final concentration, 250 mM). The mixture was transferred to a 50-μL capillary. ESR spectra were recorded in the X band (9.85 GHz) using a Bruker ECS 106 spectrometer
ACCEPTED MANUSCRIPT with a rectangular cavity and 50-kHz field modulation. All the spectra were registered in the same scale, after 15 scans [26,45].
2.6.4 Effect on oxygen consumption by the parasite Dm28c strain T. cruzi epimastigotes were harvested by 500g centrifugation, followed
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by washing and re-suspension in 0.05 M sodium phosphate buffer, pH 7.4, and
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containing 0.107 M sodium chloride. Respiration measurements were carried out
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polarographically with a Clark electrode No. 5331 (Yellow Springs Instruments, 53 YSI model) connected to a 100 mV single channel Goerz RE 511 recorder [17,18,24]. The
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chamber volume was 0.6 mL and the temperature was 28ºC. Compounds, dissolved in DMSO, were added in 40 µM final concentration. The amount of parasite used was
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3. Results and discussion
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effect produced by DMSO alone.
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equivalent to 1.2 mg of protein/mL. Results were corrected according to the observed
3. 1. Synthesis and characterization
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Three complexes including the fac-{Re(CO)3}+ moiety and bidentate bioactive 5nitrofuryl containing thiosemicarbazones as ligands were synthesized with reasonable
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yields according to the proposed synthetic scheme shown in Figure 2. The new compounds were characterized in the solid state and in solution using different techniques. Analytical (including semi quantitative determination of bromide), FTIR and NMR spectroscopic results are in agreement with the formula [ReI(CO)3Br(HL)], where HL HL1- HL3 (Figure 2). All complexes showed a similar FTIR band pattern. Based on our previous experience on vibrational behavior of HL ligands and HL metal complexes [14,16,46], relevant
ACCEPTED MANUSCRIPT vibration bands were tentatively assigned (Table 2). After coordination clear changes in the wavenumber of the (C=N) and (N–N) bands of the free thiosemicarbazone ligands were observed. These modifications have been related to the coordination of the ligand through the iminic nitrogen [46]. In addition, (C=S) bands at around 820–850 cm–1, should shift to lower wave numbers when thiosemicarbazones act as N,S bidentate
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ligands, but as it had been previously stated, they could not be unambiguously assigned
RI
for the complexes. Nevertheless, significant changes are observed in this region after
SC
coordination [26,46].
In addition, IR spectra display three strong carbonyl stretching bands, (CO), in the
NU
2029–1906 cm–1 region, which are characteristic of monomeric pseudooctahedral fac-
MA
{Re(CO)3}+ complexes [32,47-50].
Table 2. Tentative assignment of selected IR bands of the [ReI(CO)3Br(HL)] complexes.
PT E
positions are given in cm–1.
D
Bands of the free thiosemicarbazone ligands HL1-HL3 are included for comparison. Band
(C=N)
s(NO2)
(N-N)
δ(NO2)+furan
(CO)
1602
1355
1108
811
-
1558
1358
1129
816
2026, 1925,1906
1595
1344
1104
811
-
[ReI(CO)3Br(HL2)]
1601
1347
1116
808
2026, 1958, 1914
HL3 a
1586
1353
1081
811
-
[ReI(CO)3Br(HL3)]
1561
1347
1144
813
2029, 1921br
HL1 a
CE
Compound
HL2 a
a
AC
[ReI(CO)3Br(HL1)]
from reference [46]
ACCEPTED MANUSCRIPT 1
H NMR and 13C NMR experiments supported the proposed structures of the
synthesized complexes. Two-dimensional NMR experiments such as COSY and HSQC aided in the assignment of the spectra. The 1H NMR spectra of all the complexes showed a similar pattern of signals for the common portions of the nitrofuryl thiosemicarbazone ligands (Table S1). Three signals, corresponding to the two H of
PT
furan ring (H2 and H3) and H7 of the CH=N moiety of the thiosemicarbazone ligand,
RI
are observed (see numbering scheme in figure 2). The signals show the expected
SC
chemical shifts, integration and multiplicity [14,24]. In addition, [Re(CO)3Br(HL2)] spectrum shows the three characteristic signals of the phenyl substituent. For all the
NU
complexes, H8, being an easily exchangeable proton, could not be detected in DMSO-d6 solution due to the presence of water in the solvent. Nevertheless, a singlet
MA
corresponding to H8 was easily assigned in acetone-d6 solution spectra of both the complexes and the free ligands. This confirms that the thiosemicarbazone ligand is
D
coordinated to Re(I) in a neutral protonated manner. As usual the signals of the ligands
PT E
where shifted due to coordination to the metal centre [14,24]. 13
C-NMR and HSQC experiments allowed to assign the signals of the different
CE
thiosemicarbazone carbon atoms (Table S2). In addition, the carbonyl carbons signals were identified in the 13C-NMR experiments with the guide of previously reported Re(I)
AC
tricarbonyl compounds [51,52].
3.2. Solution behavior 1
H NMR experiments on DMSO solutions of the compounds followed with time were
performed in order to study complexes´ behavior in solution. . The behavior of [Re(CO)3Br(HL1)] is shown as an example in Figure 3. Figure 3 (a) shows the spectrum obtained shortly after dissolution in DMSO-d6. The characteristic
ACCEPTED MANUSCRIPT three signals (two doublets and one singlet) of coordinated HL1 in [Re(CO)3Br(HL1)] described above are observed. Besides, two additional doublets at 7.90 and 8.09 ppm, with very low intensity could be detected. After 24 h, an intensity increase of these two additional signals and a modification of the integration and multiplicity of the signal at 7.73 ppm were observed (Figure 3 (b)). These changes reveal that the original
PT
compound is converting with time into another one with the same spectral pattern, two
RI
doublets and one singlet (the latter is overlapped by the H3 signal of
SC
[Re(CO)3Br(HL1)]). Based on previously reported dimerization of chemically related systems we proposed the generation in solution of [Re2(CO)6(L1)2] compound [53-57].
NU
The chemical shifts of the new signals corresponding to those of the protons of the
AC
CE
PT E
D
MA
dimer [Re2(CO)6(L1)2] are shown in Table S1.
Figure 3. 1H-NMR spectrum of [Re(CO)3Br(HL1)] (a) zero time (b) after 24 hours.
A similar behavior was observed for the other two [Re(CO)3Br(HL)] HL = HL2 and HL3 compounds (Table S1). However, in both cases an intermediary compound was also detected in the 1H NMR spectra. Since it is well known that bromide could be substituted by a coordinating solvent, we propose that this intermediary emerges from
ACCEPTED MANUSCRIPT the substitution of bromide by DMSO that finally leads to the corresponding dimer [Re2(CO)6(L)2] [27]. Knowing that the original compounds are modified in solution, different experiments were performed to confirm the presence of the dimeric species in solution. Mass spectrometry experiments on solutions of the complexes [Re(CO)3Br(HL)] showed the
PT
presence in solution of the proposed dimers. Figure S1 depicts part of the spectrum
RI
obtained for a [Re(CO)3Br(HL2)] solution.
SC
In addition, we managed to isolate single crystals of [Re2(CO)6(L2)2] from an acetone dissolution of the monomer [Re(CO)3Br(HL2)] by very slow evaporation. The crystals
NU
were suitable to perform X-ray diffraction studies.
The compound crystalizes in the monoclinic C 2/c space group (Figure 3). The dimeric
MA
structure is composed of two distorted fac-[Re(CO)3(L2)] subunits with two μ-S bridging both metal centers. The structure is similar to that reported for other Re(I)
D
complexes [53-57].
PT E
The dimer is centrosymmetric. Each rhenium (I) atom lies in a distorted octahedral environment, with a nitrogen and two bridging sulfur atoms coordinated in a facial
CE
arrangement to the fac-[Re(CO)3]+ moiety. The dimer core is an asymmetric rectangular parallelogram with (μ-S)2Re2 unit at the centre (Figure 3). Each edge of it consists of
AC
Re-S bonds with different lengths: Re-S1 (2.4490(6) Å) and Re-S1a (2.5513(7) Å). These distances fall in the range normally observed for sulfur atoms coordinated to a fac-[Re(CO)3]+ core [53,54]. The Re-C (carbonyl) distances also fall in the commonly observed range [53-57]. The asymmetry of the distorted octahedron and of the Re-S bond lengths is related with the presence of a more constricted S1-Re-S1a (81.58 (2)°) angle when compared with those of other similar sulphur bridged complexes [53]. The bond lengths and angles around metal centre are summarized in Table 3.
ACCEPTED MANUSCRIPT The bidentate coordination of the thiosemicarbazone ligand through S1 and N2 atoms is typical for complexes with this type of ligands [24] However, only rarely dimeric compounds have been reported [14,15]. The structure is stabilized by hydrogen bonds between the acetone molecule and the
PT
proton of the nitrogen atom (N4) of thiosemicarbazone ligand (Figure 3).
Re – C15
Re – C16
1.9274 (31)
Re – N2
Re – S1
2.4490 (6)
2.1896 (21)
Re – S1a
2.5513 (7)
97.21 (10)
S1a – Re – C14
172.41 (10)
1.9395 (35)
NU
1.9178 (35)
MA
Re - C14
SC
Bond Lengths
RI
Table 3. Bond lengths [Å] and angles [°] around metal center in [Re2(CO)6(L2)2].
Bond Angles
90.09 (13)
N2 – Re – C15
C14 – Re - C15
89.35 (15)
S1 – Re – C14
92.46 (10)
S1a – Re – C16
86.06 (10)
C15 - Re – C16
87.12 (13)
S1 – Re – C16
97.46 (9)
S1a – Re – C15
96.98 (11)
N2 –Re –C14
93.50 (11)
S1 – Re – C15
175.06 (9)
S1a – Re – N2
89.88 (6)
S1 – Re – N2
78.10 (6)
S1a – Re – S1
81.58 (2)
PT E
CE
AC
N2 – Re – C16
D
C14 – Re – C16
174.39 (11)
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
MA
Figure 3. ORTEP diagram of fac-[Re2(CO)6(L2)2] showing the atomic displacement
PT E
3.3. Biological results
D
ellipsoids at the 50% probability level. Inset: nearly square Re2S2 ring.
3.3.1. Anti-T. cruzi activity
CE
The new rhenium(I) tricarbonyl compounds were active against T. cruzi (trypomastigotes Dm28c) with IC50 values in the low micromolar range (Table 4). The
AC
compounds showed a 4- to 17- fold increase of activity in respect to the corresponding thiosemicarbazone ligand. In addition, they are 8 to 15 times more active than the reference trypanocidal drug Nifurtimox (IC50 = 20.1 ± 0.8 µM) [25]. The precursor [Re(CO)5Br] was previously tested showing very low activity on the parasite (IC50 100M) [32].
ACCEPTED MANUSCRIPT Table 4. In vitro activity (measured as the IC50 value, the half inhibitory concentration) against T. cruzi tripomastigotes (Dm28c), cytotoxicity on EA.hy926
endothelial
cells
and
selectivity
index
(SI)
values
of
[Re(CO)3Br(HL)] and HL (included for comparison) and RM values of the complexes and the corresponding ligands (MeOH : DMF : Tris buffer pH 7.4
36.9 ± 0.7
15
0.91
100
10
- 0.29
IC50 / M
HL1
9.8 ± 1.5
[ReI(CO)3Br(HL2)]
1.3 ± 0.3
14.3 ± 0.6
11
1.38
HL2
22.7 ± 1.6
100
4
0.60
[ReI(CO)3Br(HL3)]
2.01 ± 0.03
43.6 ± 1.1
22
0.87
HL3
12.7 ± 1.7
44.9 ± 5.3
3
-0.37
20.1 ± 0.8
-
-
-
PT E
D
MA
NU
2.4 ± 0.2
SI: IC50 endothelial cells Ea.hy926 / IC50 T. cruzi (Dm28c tripomastigotes) The Rf values were averaged from two to three determinations, and
CE
b
RM b
[ReI(CO)3Br(HL1)]
Nifurtimoxc a
IC50 / M
SI(fold) a
RI
Compound
Ea.hy926
SC
T. cruzi
PT
(20:20:60 v/v/v)).
AC
converted to RM via the relationship: RM = log10 [(1/Rf) –1] c
From reference [26]
3.3.2. Cytotoxicity on mammalian cells The specificity of the antitrypanosomal activity of the new compounds was evaluated by analyzing their cytotoxicity against a human-derived endothelial cell line (EA.hy926). All the new compounds showed moderate to good selectivity towards the parasites when compared to the selected mammalian cell model (Table 4). In all cases
ACCEPTED MANUSCRIPT the coordination to the rhenium(I) center improved the selectivity in respect to the free ligands.
3.3.3. Lipophilicity Lipophilicity is a very important physicochemical property of prospective drugs that
PT
controls biological behaviour, particularly transmembrane transport and interaction with
RI
biological receptors. Therefore, its correlation with the observed biological activity is
SC
usually relevant [59]. The effect of complex formation on lipophilicity was experimentally determined using reversed-phase TLC, where the stationary phase
NU
(precoated TLC-C18) may be considered to simulate lipids of biological membranes or receptors, and the mobile phase (MeOH : DMF : Tris buffer pH 7.4 (20:20:60 v/v/v))
MA
resembles the aqueous biological milieu. The composition of the mobile phase was tuned-up in order to allow differentiating complexes according to their lipophilicity and
D
comparing with the free thiosemicarbazones.
PT E
The complexes were more lipophilic than the corresponding thiosemicarbazone ligands (Table 4). As expected, the lipophilicity of the complexes increases as the N-substituent
CE
in the thiosemicarbazone ligand changes from hydrogen to phenyl. [ReI(CO)3Br(HL2)], 2, is the more lipophilic and the more active compound but being the series of
AC
compounds too short this is only a preliminar qualitative observation. To establish a correlation between lipophilicity and antiparasitic activity a larger series of structurally related compounds should be considered.
3.4. Insight into the mechanism of action 3.4.1. Generation of free radical species in T. cruzi
ACCEPTED MANUSCRIPT As previously stated, the main mechanism of anti T. cruzi action of the selected 5nitrofuryl derived thiosemicarbazones involves the intraparasite bioreduction leading to toxic free radicals. The first step of this mechanism involves the one electron reduction of the nitro moiety by parasite enzymes. Through redox cycling, the formed nitro anion radical generates other radical species that are toxic for the parasite [13]. We previously
PT
demonstrated that this mechanism of action is retained by all the other previously
RI
reported metal complexes of these bioactive ligands [14,16-18,24,25]. As usual, the free
SC
radical production capacity of the new rhenium(I) compounds was studied by ESR. The compounds were incubated with T. cruzi (Dm28c strain) epimastigotes. DMPO was
NU
added as spin trapping agent to detect free radical species having short half-lives. For the first time signals were not observed in the ESR spectra of these complexes showing
MA
that the expected free radicals were not generated in the parasite by effect of them.
PT E
D
3.4.2. Effect on oxygen consumption by the parasite
AC
CE
parasites
ReHL4 40
435nm O2 12 min
83.2% inhibition
Figure 4. Effect of [Re(CO)3Br(HL4)] on the rate of respiration of the parasite.
ACCEPTED MANUSCRIPT The involvement of the obtained rhenium complexes in redox cycling processes should increase the parasite oxygen consumption as reported for the thiosemicarbazone ligands and their previously reported complexes [17,18,24]. Thus, oxygen uptake, was measured for the obtained complexes. However, results showed that the compounds decrease the rate of oxygen consumption of the parasite
PT
([Re(CO)3Br(HL1)], 3.7 %; [Re(CO)3Br(HL2)], 83.2 %; [Re(CO)3Br(HL3)], 12.3 %)
RI
(Figure 4). This is in agreement with the lack of generation of ROS observed in the ESR
SC
experiments indicating a different mechanism of action for the obtained rhenium
NU
compounds that would involve the inhibition of mitochondrial respiration.
3.4.3. Cyclic voltammetry studies
MA
Hence, these compounds act through a different mechanism than the one known for the free ligands and for all their previously developed metal compounds. In this regard, the
D
reduction potential of the nitro group, first step in the bioreduction, is a very important
PT E
parameter to be determined in order to predict how easy this reduction process in vivo could be. The electrochemical characterization of the complexes was performed at room
CE
temperature by cyclic voltammetry in DMSO solutions using a carbon disk electrode. The obtained voltammograms (recorded at a scan rate of 100 mV/s) are depicted in
AC
Figure 5. Selected electrochemical data are shown in Table 5. All obtained complexes showed a similar voltammetric response in both cathodic and anodic directions.
ACCEPTED MANUSCRIPT
12
II
I
ReHL1 ReHL3 ReHL2
III
10
I ( A )
8 6 4
PT
2 0
0,0
-0,2
-0,4
-0,6
-0,8
-1,0
-1,2
SC
E (V)
-1,4
RI
-2
NU
Figure 5 Cyclic voltammograms of 1mM DMSO solutions of the [Re(CO)3Br(HL)] complexes, codes ReHL1, ReHL2 and ReHL3, measured in the cathodic direction, scan
MA
rate100 mV/s. Working electrode: glassy carbon. Potentials are measured vs. Ag/AgCl.
D
The voltammograms show three processes centered on the thiosemicarbazone ligand.
PT E
As previously described, the complexes show a quasi reversible reduction process around -0.7 V (vs Ag/AgCl), corresponding to the generation of a nitro anion radical
CE
(NO2·-) by reduction of the nitro group of the thiosemicarbazone (couple II) (Figure 5). Near -1.3 V, an irreversible signal is observed (couple III) that corresponds to further
AC
reduction of the nitro anion radical to hydroxylamine [16,17,60]. When moving to less negative potentials, an irreversible reduction process is detected (couple I). In order to assign this signal the electrochemical behavior of the complexes in DMSO solution was studied with time during 24 h after preparation of the DMSO solution. Changes in the relative intensities of couple I and couple II were observed with time for the three complexes. Figure 6 shows the behavior of ReHL1 as an example. A
ACCEPTED MANUSCRIPT decrease of the intensity of couple II with time was observed, with a simultaneous increase in the intensity of the cathodic peak of redox process I.
I
II
12 10
6
PT
(A)
8
4
RI
2
-2 0,0
-0,2
-0,4
-0,6
-0,8
-1,0
-1,4
MA
NU
E (V)
-1,2
SC
0
Figure 6. Cyclic voltammograms of a 1mM DMSO solution of ReHL1 measured in the
D
cathodic direction, scan rate100 mV/s, at different times: (-) t = 0, (-) 1 h and (-) 24 h
PT E
after preparation of the solution. Working electrode: glassy carbon, scan rate 100 mV/s.
CE
Potentials are measured vs. Ag/AgCl.
These results are in agreement with the conversion in solution of each monomeric
AC
compound [Re(CO)3Br(HL)] into the dimeric one [Re2(CO)6(L)2] (Table 5). In these species, a less negative reduction potential of the nitro moiety was observed.
Table 5. Electrochemical data of the compounds [Re(CO)3Br(HL)] and HL with a glassy carbon working electrode at a scan rate of 100 mV/s. Potentials are measured
ACCEPTED MANUSCRIPT vs. Ag/AgCl. ReI/ReII Compound
Couple I
Couple II (HL)
(dimer)
(monomer)
Epc (V)
Epa (V)
Epc (V)
[Re(CO)3Br(HL1)
0.93
-0.50
-0.64 (-0.64)
-0.74 (-0.72)
[Re(CO)3Br(HL2)
0.97
-0.40
-0.61 (-0.62)
-0.70 (-0.70)
[Re(CO)3Br(HL3)
0.83
-0.43
-0.61 (-0.64)
-0.71 (-0.71)
SC
RI
PT
Epa (V)
NU
Previous cyclic voltammetry studies demonstrated that coordination of the 5-nitrofuryl thiosemicarbazone ligands to different metal ions lead to a slight displacement of the
MA
potential of the nitro group – nitro anion radical couple to less negative values. This change would favor the generation of radical species in the parasite [14,16-18,24,25].
D
However, in this case, the new species generated in solution show a high shift of the
PT E
reduction potential of the nitro group that could fall outside the potential window of the enzymes involved in the reduction in the parasite. This fact could explain the lack of
CE
formation of ROS in the parasite.
A similar behavior with time was observed in the anodic direction. The anodic scan
AC
shows an irreversible process around 1.0 V assigned to the redox couple ReI/ReII, characteristic of the fac-Re(I) tricarbonyl compounds [61,62]. The observed differences in Epa values for the three compounds (Table 5), show that the nature of the substituent R and/or the chain length of the hydrocarbon linker between the pharmacophore and the thiosemicarbazone moiety are relevant for the oxidation process. This is in agreement with reported values for other fac-Re(I)Br complexes with bidentate ligands where the redox potential varies with the electrodonating capacity of the ligand [62].
ACCEPTED MANUSCRIPT The conversion of the [Re(CO)3Br(HL) complex into the dimeric species in solution also affects the oxidation of the metal center. The voltammograms obtained in the study with time show a displacement of Epa to more positive values (Figure S2). The new species shows a less favorable oxidation of the metal center than the original
PT
[Re(CO)3Br(HL) compound.
RI
4. Conclusions
Coordination of the fac-Re(CO)3 moiety to bioactive HL thiosemicarbazone ligands led
SC
to compounds with improved biological properties. The fac-[Re(CO)3Br(HL)]
NU
compounds showed high activities on bloodstream T. cruzi, with IC50 values in the low M range, and good selectivities towards the parasite. The Re(I) compounds are 8 to 15
MA
times more active than Nifurtimox and show a 4 to 17 fold increase in activity in respect to the free ligands.
D
The nature of the species generated in solution has been studied by NMR, MS, CV and
[Re2(CO)6(L)2].
PT E
X-ray diffraction studies. The fac-[Re(CO)3Br(HL)] species convert into the dimers
CE
The compounds do not generate ROS in the parasite probably due to the unfavorable nitro reduction potential of the generated dimeric species. The compounds inhibit
AC
mitochondrial respiration of the parasite, effect shown by the decrease of the oxygen consumption rate.
Acknowledgements ERA thanks ANII (Uruguay) for the doctoral grant POS_NAC_2015_1_110215. IM thanks ANII (Uruguay) for the research grant INI_X_2011_1_3902. Authors thank PEDECIBA and ANII-SNI, Uruguay and FONDECYT 1150175, Chile.
ACCEPTED MANUSCRIPT
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D
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AC
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CE
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Graphical abstract
ACCEPTED MANUSCRIPT Coordination of the fac-Re(CO)3 moiety to bioactive thiosemicarbazones HL led to fac[Re(CO)3Br(HL)] compounds with high activities on bloodstream Trypanosoma cruzi and good selectivities towards the parasite. Complexes convert into [Re2(CO)6(L)2] in solution and inhibit parasite mitochondrial respiration, effect shown by decrease of
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oxygen consumption rate, without generating radical oxygen species.
ACCEPTED MANUSCRIPT Highlights New fac-[Re(CO)3Br(HL)] compounds with bioactive HL thiosemicarbazones were synthesized.
The compounds showed high activities on bloodstream Trypanosoma cruzi.
They showed good selectivities towards the parasite in respect to mammalian cell
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model.
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They inhibit parasite mitochondrial respiration without generating radical oxygen
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species.
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The fac-[Re(CO)3Br(HL)] species convert into the dimers [Re2(CO)6(L)2] in solution.