Antiprotozoal Activity of the Cyclopalladated Complexes Against Leishmania amazonensis and Trypanosoma cruzi

The present study describes the antiprotozoal activities of four cyclopalladated compounds, [Pd(dmba)(μ-Cl)]2, [Pd(dmba)(NCO)(isn)], [Pd(dmba)(N3)(isn)] and [Pd(dmba)(μ-NCO)]2, (dmba: N,N’-dimethylbenzylamine and isn: isonicotinamide), against the diseases leishmaniasis (Leishmania amazonensis and Leishmania infantum), Chagas disease (Trypanosoma cruzi) and human African trypanosomiasis (Trypanosoma brucei). [Pd(dmba)(μ-NCO)]2 exhibited good leishmanicidal and trypanocidal activities against L. amazonensis and T. cruzi intracellular amastigote forms, with a 50% inhibitory concentration (IC50) value of less than 9 μM and selectivity indexes of 14.47 and 28.42, respectively. Stability essays were conducted in phosphate buffer saline (PBS) pH 7.0 and showed that [Pd(dmba)(μ-NCO)]2 is the most stable molecule. These findings indicate that this compound presented higher selectivity for these parasites than the other tested compounds. The data presented here suggest that this compound should be considered in the development of new and more potent drugs for the treatment of leishmaniasis and Chagas disease.


Introduction
Leishmaniasis, Chagas disease (American trypanosomiasis, AT) and sleeping sickness (human African trypanosomiasis, HAT) are parasitic diseases caused by flagellated protozoa related to the family Trypanosomatidae.According to the World Health Organization (WHO), 1.3 million new cases of leishmaniasis and 20,000 to 30,000 deaths occur annually in all continents, 1 7 to 8 million people are infected worldwide with Chagas disease 2 and the estimated number of actual cases of HAT is currently 30,000. 3ore than 20 species of Leishmania 4 are responsible for different clinical manifestations, including cutaneous (CL), mucocutaneous (MCL) and visceral forms (VL). 5n the Old World, Leishmania major, Leishmania tropica, Leishmania aethiopica and some zymodemes from Leishmania infantum are the causative agents of CL.
In the New World, CL is mainly caused by Leishmania amazonensis, Leishmania guyanensis, Leishmania panamensis and Leishmania braziliensis. 6MCL can be caused by L. braziliensis, L. panamensis, L. guyanensis and L. amazonensis in the New World and by L. major and L. infantum in the Old World. 7Pentavalent antimonial drugs are the most frequently prescribed treatments for leishmaniasis. 8The main adverse effects that occur in systemic administration of these antimonials are arthralgias, myalgias, anorexia, and nausea, and other serious side effects include pancreatitis, liver-enzyme abnormalities, cardiac malfunctions and severe renal toxic effects. 9ther drugs, such as amphotericin B, pentamidine and miltefosine, are second choice drugs but they also produce side effects that can endanger the patient's life.
1][12] Nifurtimox and benznidazole are the only drugs available to treat patients in the acute phase of the disease. 13,14However, these drugs are not effective against the chronic phase of the disease, and they present multiple side effects, from dermatitis to bone marrow depression. 15HAT is caused by Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. 16reatment of the hemolymphatic stage of HAT relies on suramin and pentamidine.In the meningoencephalitis stage, melarsoprol, a highly toxic arsenic-based drug that is effective against both T. b. gambiense and T. b. rhodesiense is used.Eflornithine is useful only against T. b. gambiense.An eflornithine/nifurtimox combined therapy has been used, but this also causes several side effects. 17In summary, the conventional drugs that have been employed to treat these diseases are antiquated, have high toxicity and are ineffective due to drug resistance.Therefore, is critical to develop new drugs for the treatment of these trypanosomiases.
9][20] The emergence of drug resistance in tropical diseases has led to alternatives to chloroquine and its analogues, such as metallocene derivatives for the treatment of malaria. 19,20Several scaffolds of metal complexes containing palladium, platinum, gold, iridium, rhodium, osmium and iron have shown leishmanicidal [18][19][20][21] and trypanocidal 22,23 activity.For palladium compounds, it was recently verified that a cyclopalladated complex showed a high selectivity index with trypanocidal activity in the treatment of Chagas disease. 24Literature has reported a cyclopalladated compound with leishmanicidal activity 25 against L. amazonensis promastigote and amastigote forms; and the activity of some palladium(II) cyclometalated complexes 26 against T. cruzi and Leishmania, which indicate on preliminary data that the compounds inhibited the growth of intracellular amastigote forms.
Herein we tested the above mentioned cyclopalladated complexes against L. amazonensis, L. infantum, T. cruzi and T. brucei.

Experimental
Infrared spectra (IR) were recorded on a Spectrum 2000, PerkinElmer, in the spectral range 4000-370 cm -1 .Samples were prepared in KBr pellets. 1 H and 13 C{ 1 H} nuclear magnetic resonance (NMR) spectra were referred to the high field SiMe 4 signal, on a INOVA 500 spectrometer; the magnetic field applied was 11.7 T and the resonance of 1 H and 13 C{ 1 H} nucleus were at 500 and 125 MHz, respectively.Full spectroscopic data are presented in the Supplementary Information section.

Synthesis of cyclopalladated complexes
All synthesis was carried out at room temperature.All reagents were obtained from commercial suppliers and used without further purification.

Stability assay
The samples 1-4 were dissolved in acetonitrile (minimum amount to solubilize) and the solutions then prepared in phosphate buffer saline (PBS) pH 7.0 to give concentrations of 317, 312, 220 and 143 μmol L -1 .The solutions were prepared and the samples were left with occasional stirring at room temperature being analyzed aliquots of these solutions in the days immediately after the preparation (time 0) and after 12, 24, 48 and 72 hours.The experiments were performed in HPLC Shimazu LC-20AT CNM-20A UV detector SPD-20A using ODS (C-18) column, particle size 5 μm, 4.6 × 250 mm, mobile phase: methanol:water (70:30, v/v/v), flow 0.8 mL min -1 and λ = 254 nm.For more details see Supplementary Information section.

Compounds
Cyclopalladated complexes 1, 2, 3, 4 and controls (pentamidine, amphotericin B and benznidazole (Sigma-Aldrich)) were dissolved in DMSO, dimethyl sulfoxide, (the highest concentration was 1.4%, which was not hazardous to the parasites, as previously determined), added to the parasite suspension to final concentrations between 0.5 and 100.0 μmol L -1 .

Cytotoxicity using murine macrophages
To determine the cytotoxicity in murine macrophages we used a method previously described. 43Adult male Swiss albino mice (20 to 35 g) were used in the experiments.They were housed in single-sex cages under a 12 h light/12 h dark cycle in a controlled-temperature room (22 ± 2 °C).The mice had free access to food and water.Groups of three animals were used in each test group.The experiments were performed in concordance to protocol approved by the Institutional Ethics Committee-CEUA (Comissão de Ética no Uso de Animais), protocol CEUA/FCF/CAr No. 20/2013.The mice were stimulated with thioglycolate to collect peritoneal macrophages.Murine peritoneal macrophages were seeded in 96-well flat-bottom plates (TPP) at a density of 1 × 10 5 cells per well in RPMI 1640 medium 44 supplemented with 10% heatinactivated FBS, 25 mM HEPES, and 2 mM L glutamine and incubated for 4 h at 37 ± 2 °C in a 5% CO 2 -air mixture.The medium was removed, and then new medium was added to the cells, which were treated with different Vol.27, No. 6, 2016 concentrations of compounds and controls.Cells without drugs were used as a negative control.After that, plates were incubated for 24 h at 37 ± 2 °C in a 5% CO 2 -air mixture.Subsequently, the MTT (3-4(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) colorimetric assay was carried out as described further on.Absorbance was read in a 96-well plate reader (Robonik) at 595 nm.The drug concentration corresponding to 50% of cell growth inhibition was expressed as the inhibitory concentration (CC 50 , μM). 45 MTT colorimetric assay L. amazonensis and L. infantum promastigote, T. brucei procyclic and T. cruzi epimastigote forms were treated with the cyclopalladated complexes and their respective controls to calculate the half maximal inhibitory concentration (IC 50 ) by MTT colorimetric assay. 46,47The MTT assay is based on the determination of the ability of living cells to reduce 3-4(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT).To avoid interaction of compounds with the MTT, the cells were gently washed with PBS before the addition of MTT solution on the cells.The compounds were tested in different concentrations and incubated at 28 ± 2 °C for 72 h for L. amazonensis, L. infantum and T. cruzi and 24 h for T. brucei. 23,43The assays were carried out in triplicates.Absorbance was read in a 96-well plate reader (Robonik) at 490 nm for Leishmania species and 595 nm for T. cruzi and T. brucei.The drug concentration corresponding to 50% parasite growth inhibition was expressed as the IC 50 in μM.
The safety index (SI) was calculated and SI around 10 means that a compound can be better evaluate for further studies.Data obtained were processed with the software Origin 7.0. 48

Leishmanicidal and trypanocidal activities against intracellular amastigote forms
To determine the leishmanicidal and trypanocidal activities against intracellular amastigote forms we used a method previously described. 43Murine peritoneal macrophages were plated at 3 × 10 5 cells per well on coverslips (13 mm diameter) previously arranged in a 24 well plate in RPMI 1640 medium 44 supplemented with 10% inactivated FBS and allowed to adhere for 4 h at 37 ± 2 °C in 5% CO 2 .Adherent macrophages were infected with Leishmania promastigotes in the stationary growth phase or T. cruzi trypomastigote using a ratio of 5/10:1 parasites per cell (L.amazonensis or L. infantum/T.cruzi: macrophages) at 37 °C in 5% CO 2 for 4 h for L. amazonensis, 18 h for L. infantum and 24 h for T. cruzi.After that time, the non-internalized parasites were removed by washing and infected cultures were incubated in RPMI 1640 medium for 24 h at 37 ± 2 °C in 5% CO 2 to allow parasite multiplication.Then, infected cells were treated with different concentrations of compounds, amphotericin B (Sigma-Aldrich), pentamidine (Sigma-Aldrich) or benznidazole (Sigma-Aldrich) for 24 h.The cells were then fixed in a methanol solution and stained with Giemsa.The number of amastigotes/100 macrophage cells and the percent of infected cells were determined.The concentration that caused a 50% decrease of growth inhibition compared to the control was determined by regression analysis and expressed as IC 50 in μM. 45he experiments were performed in accordance with the protocol approved by the Institutional Ethics Committee-CEUA (Comissão de Ética no Uso de Animais), protocol CEUA/FCF/CAr No. 20/2013.

Differentiation of T. cruzi epimastigote to trypomastigote forms
In vitro differentiation of T. cruzi was made as previously described in literature. 49,50The epimastigote forms were collected from the LIT culture on the 7 th day when the plateau phase of the growth curve was reached and then, re-suspended in artificial triatomine urine (TAU) (190 mmol L -1 NaCI, 8 mmol L -1 phosphate buffer, pH 6.0, 17 mmol L -1 KCl, 2 mmol L -1 CaCI 2 , 2 mmol L -1 MgCI 2 ), and incubated for 2 h at room temperature.The parasites were diluted to a final concentration of 5 × 10 6 parasites mL -1 in TAU supplemented with 2.5% (v/v) sodium bicarbonate 1.4%, 500 units penicillin mL -1 , 10 mmol L -1 proline (TAUP medium) and incubated at 27 °C for 10 days.

Results and Discussion
Compounds 1-4 (Figure 1) were obtained as stable solids (yields ranging from 79% to 94%), and their structures are fully supported by infrared spectroscopy (IR) and elemental analysis.
The cyclopalladated complexes were tested against L. amazonensis, L. infantum (promastigote and amastigote), T. cruzi (epimastigote and amastigote) and T. brucei (procyclic forms) using the MTT colorimetric assay [44][45][46] and an in vitro intracellular amastigote assay 43,49,50 to evaluate the antiprotozoal activity.Amphotericin B and pentamidine were included in the assays as positive controls for Leishmania species, benznidazole for T. cruzi and pentamidine for T. brucei.The MTT assays were carried out after 72 h (L.amazonensis, L. infantum and T. cruzi) and 24 h (T.brucei) of incubation.In general, all cyclopalladated complexes showed variable leishmanicidal and trypanocidal activity.
The antiprotozoal effect of the cyclopalladated complexes on intracellular amastigotes was evaluated in murine peritoneal macrophages obtained from adult male Swiss albino mice infected with Leishmania species promastigote forms and T. cruzi trypomastigote forms.We observed a decrease in the amount of intracellular amastigote forms after treatment with the cyclopalladated complexes.A cytotoxicity assay was also carried out to evaluate the toxicity of the compounds in the peritoneal macrophages, showing lower toxic activity on mammalian cells.All data are presented in Table 1.
The ligands dmba and isn by themselves did not exhibit antiprotozoal activities (Table 1), suggesting that the observed biological activity was due to the cyclopalladated complexes.In the present study, we observed that compound 2 showed in vitro activity against T. cruzi amastigotes (IC 50 = 4.45 ± 0.45 μM), while compound 4 showed activity against both L. amazonensis (IC 50 = 9.29 ± 2.08 μM) and T. cruzi amastigotes (IC 50 = 4.73 ± 1.19 μM).It is interesting to note that for L. amazonensis amastigote, compound 4 showed almost 3 times higher leishmanicidal activity compared to compounds 2 (IC 50 = 22.45 ± 1.17 μM) and 3 (IC 50 = 28.82± 2.15 μM) (Table 1), suggesting that the isn group complexed to Pd decreases the anti-Leishmania activity but does not influence the anti-T.cruzi activity.
The replacement of chlorido by the cyanato ligand (1 → 4) did not result in any increase in leishmanicidal activity against the promastigote and amastigote forms; however compound 1 was 2 times more active than compound 4 against the T. cruzi epimastigote.On the other hand, different efficacy was observed in the T. cruzi amastigote form, where compound 4 was approximately 3 times more active than compound 1.The presence of Table 1.Antiparasitic activities (IC 50 , half-maximal inhibitory concentration, μM), mammalian cell toxicity (CC 50 , half-maximal cytotoxicity concentration, μM) and safety index (SI = CC 50 /IC 50 ) of the cyclopalladated complexes.The SI values ≥ 10 in bold indicate promising compounds, i.e., those with low mammalian toxicity and high toxicity to parasites.Each value is the mean of three experiments performed in triplicate ± standard error   NCO instead of Cl in compound 4 increased its chemical stability after 72 hours and decrease in its content which suggests a profile of Pd release/time without structure change (Figure 3).The advantage of compound 4 is its effectiveness on amastigotes forms that's present in human host while epimastigotes is present in the bugs.
Among the tested complexes, compound 4 showed good activity against the selected pathogens as well as low toxicity, with SI values of approximately 10 and 30, except for the L. infantum amastigote.In particular, for L. amazonensis promastigote and amastigote forms, compound 4 presented SI values of 9.83 and 14.47, respectively, which are safer than those of amphotericin B (7.17 For T. brucei procyclic forms, we did not observe any compound with higher trypanocidal activity than pentamidine.However, the compounds 1 and 4 were more selective (SI = 8.29 and 8.21, respectively) than pentamidine (SI = 5.54).
Cyclopalladated 2, 3 and 4 evaluated against intracellular amastigote forms of T. cruzi showed the best SI values compared to those obtained for the intracellular amastigote forms of the Leishmania species (Table 1).This data showed that compounds 2, 3 and 4 were more selective for T. cruzi when compared to other parasites.Further studies on 3 and 4 are required in order to gain a better understanding about their mechanism of action.
NMR analysis of complexes 2 and 3 were recorded in DMSO which indicated that isn is free of its dimeric precursors, [Pd(dmba)(μ-N 3 )] 2 and [Pd(dmba)(μ-NCO)] 2 ; besides we do not observe species containing DMSO coordinated to Pd II .According to the literature, 51 cyclopalladated complexes containing pyridine generate its corresponding binuclear precursors in solution at room temperature.Herein, the mononuclear derivatives of cyclopalladated with isn and pseudohalides do not maintain their structure completely intact in solution acting possibly as a prodrug since we observed anti-T.cruzi activity, therefore, the activity of compounds 2 and 3 might be the result of fractionation of these species present in solution and their precursors.Probably the observed dissociation in solution would explain the biological activity of the compound and the formation of pro-active species.This could contribute to the bioactivity displayed by compound 4.This hypothesis can be supported by cisplatin mode of action since it undergoes successive hydrolysis reactions resulting in active species that react more rapidly with the target. 52Further pharmacokinects studies are needed to prove the pro-action model of the cyclopalladed compound.Some authors 24 observed that cyclopalladated complex against T. cruzi reduced the parasitemia and mortality in vivo with very low and nontoxic doses, as well as inhibited amastigote intracellular proliferation, similar to observed for us in vitro in T. cruzi and L. amazonensis intracellular amastigote.
The mechanism of actions proposed by Matsuo et al. 24 suggests that cyclopalladated complex interacts with the mitochondria of T. cruzi, causing a collapse in the cell extrusion of protons, followed by deoxyribonucleic acid (DNA) fragmentation and exposure of phosphatidylserine on the surface, a process resembling to apoptosis in mammalian cells.Already the same authors mentioned that the cyclopalladated complexes are "much more stable and less toxic than other derivatives of palladium(II) 53 and were able to inhibit cathepsin B (CpB)". 54Other authors [55][56][57] reported that cathepsin B and L are involved in the growth of Leishmania and their virulence in vitro and in vivo.Other authors 25 observed that a palladacycle compound showed inhibition of the cysteine protease activity expressed in L. amazonensis amastigotes, being significant the inhibition of CpB activity.It was also reported that cyclometalled palladium(II) complexes 26 inhibited cathepsin B in other Leishmania species.However, the compounds did not affect the CpB activity of macrophages. 25Probably, for this latter reason, our compounds were relatively innocuous on peritoneal macrophages which is very interesting for new drugs evaluation.
Other mechanism of action by palladium complexes 58 described that "the production of oxidative stress as a result of their bioreduction and extensive redox cycling", as well as the interaction with DNA. 21,56tudies on the mechanism of action of cyclopalladated complexes 1-4 against both L. amazonensis and T. cruzi are underway, and so are the tests with other T. cruzi strains, due to genetic variability, to determine if our compounds may be following the similar mechanisms of action reported in the literature.

Conclusions
The results presented in this work suggest that the cyclopalladated compounds 1-4 (in special 4 showed to be the most active compound against L. amazonensis and T. cruzi intracellular amastigote forms whit low cytotoxicity) should be further considered as potential new hit in the search for new drugs for the chemotherapy of Chagas disease and leishmaniasis.In addition, the cyclopalladated 2 and 3 showed a promising anti-T.cruzi activity.Our biological results demonstrated that all cyclopalladated complexes presented low cytotoxicity towards mammalian cells.