Antileishmanial Activity, Toxicity and Mechanism of Action of Complexes of Sodium Usnate with Lanthanide Ions: Eu(III), Sm(III), Gd(III), Nd(III), La(III) and Tb(III)

Leishmaniases are neglected diseases with limited therapeutic options. Diffuse cutaneous leishmaniasis can occur in Brazil due to Leishmania amazonensis. This study details the antileishmanial activity and cytotoxicity of complexes of sodium usnate (SAU) with lanthanide ions ([LnL3 (H2O)x] (Ln = La(III), Nd(III), Gd(III), Tb(III), Eu(III) and Sm(III); L = SAU). All lanthanide complexes were highly active and more potent than SAU against L. amazonensis promastigotes and intracellular amastigotes (Pro: IC50 < 1.50 μM; Ama: IC50 < 7.52 μM). EuL3·3H2O and NdL3·3H2O were the most selective and effective on intracellular amastigotes, with a selectivity index of approximately 7.0. In silico predictions showed no evidence of mutagenicity, tumorigenicity or irritation for all complexes. Treatment with EuL3·3H2O triggered NO release even at the lowest concentration, indicating NO production as a mechanism of action against the parasite. Incubating promastigotes with the lanthanide complexes, particularly with SmL3·4H2O and GdL3·3H2O, led to a change in the mitochondrial membrane potential, indicating the ability of these complexes to target this essential organelle. The same complexes caused cell death through cell membrane disruption, but their relationship with early or late apoptotic processes remains unclear. Thus, the inclusion of lanthanide ions in SAU improves selectivity with a promising mechanism of action targeting the mitochondria.


Introduction
The World Health Organization (WHO) defines neglected tropical diseases (NTDs) as a group of diseases that are prevalent in the poorest regions of the world, where access to healthcare, sanitation and safe drinking water is inadequate [1].During the COVID-19 pandemic, there has been a rise in mortality rates from NTDs due to insufficient support for affected individuals, who are often among the most vulnerable populations [2].This reinforces what the Drugs for Neglected Diseases initiative (DNDi) has confirmed: there are no neglected diseases, only neglected patients [3].
Leishmaniases are among the top ten NTDs.These are a group of vector-borne infectious diseases that affect more than 12 million people worldwide, with 0.9-1.6 million new cases each year [4].The potentially fatal visceral manifestation or even the potentially disfiguring and stigmatizing cutaneous and/or mucocutaneous forms are caused by Leishmania protozoan parasites.This broad spectrum of clinical manifestations is related to the variety of parasite species: more than 20 species can cause the disease, and these are transmitted by a diversity of phlebotomine species in various epidemiological cycles [5].
Cutaneous leishmaniasis (CL) produces skin lesions with a tendency to ulceration and can evolve into destructive lesions on the mucous membranes (mucosal or mucocutaneous leishmaniasis, ML).Of the cases, 95% occur in the Americas, the Mediterranean basin, the Middle East and Central Asia [4].Leishmania (Leishmania) amazonensis Lainson and Shaw, 1972 is one of the pathogens responsible for cutaneous leishmaniasis (CL) in Brazil.It causes either localized lesions or the diffuse form of the disease, in which the parasite spreads due to an impaired cell-mediated immune response [6].Consequently, lesions tend to be more resistant to treatment and do not heal spontaneously [7].
The antileishmanial drugs currently used include pentavalent antimonials (N-methyl glucamine antimonate and sodium stibogluconate), amphotericin B (deoxycholate and the less toxic liposomal formulation), the antibiotic pentamidine and miltefosine, an oral anticancer drug currently used to treat cutaneous and visceral leishmaniasis [8].In addition, controlled released drug systems carried out by nanoparticles became an important tool to improve antileishmanial chemotherapy [9][10][11].Therapeutic options for leishmaniases are limited, toxic and costly, in addition to being poorly accessible to patients due to the repetitive parenteral route of administration [8].Thus, the development of safer and shorter treatments is highly desirable.Mitochondria are considered potential targets for the development of antileishmanial drug candidates, as they are important organelles with unique properties and proteins that differ from those of the mammalian host [12].As an example, paromomycin (paromomycin sulfate, C 23 H 47 O 18 S) is an intramuscularly administered aminoglycoside antibiotic for the treatment of leishmaniasis, whose mechanism of action involves mitochondria [13].
The biological activities of usnic acid have been widely described, among which we can highlight the antibacterial, antiprotozoal, antiviral, anti-inflammatory, antipyretic and antitumor activities [16,18].To enhance these activities, several changes have been made in the structure of the molecule, as its different functional groups make it a good target for structural modification [19].Derivatization was also performed to overcome the low solubility of usnic acid and optimize the production of the compounds, as the acquisition of natural lichen biomass to supply secondary metabolites on a large scale is neither practical nor environmentally friendly [20].
Derici et al. [18] have shown that usnic acid has an apoptotic effect on several species of Leishmania.In order to increase the solubility of this compound in water, sodium usnate (SAU) 1 with the general formula NaL 2 •5H 2 O (Figure 1) was synthesized and com- plexed with metals to improve the activity [21].The coordination compounds containing lanthanide ions that we present in this study have been obtained from the molecular modification of SAU 1.Because of their high chemical reactivity, lanthanides never occur as pure elements in nature but only as sparsely distributed compounds that form rare minerals.Lanthanide compounds often have magnetic, catalytic and optical properties, and therefore they are currently widely used in industry and medicine as chemical markers and optical sensors [22].
In this work, the lanthanides samarium-Sm(III), gadolinium-Gd(III), europium-Eu(III), neodymium-Nd(III), lanthanum-La(III) and terbium-Tb(III) were complexed with SAU 1 [21], resulting in complexes that were tested for antileishmanial activity and selectivity.In addition to elucidating their mechanisms of leishmanicidal action, these promising results may contribute to more viable and effective therapeutic options against cutaneous leishmaniasis.Derici et al. [18] have shown that usnic acid has an apoptotic effect on several species of Leishmania.In order to increase the solubility of this compound in water, sodium usnate (SAU) 1 with the general formula NaL2.5H2O (Figure 1) was synthesized and complexed with metals to improve the activity [21].The coordination compounds containing lanthanide ions that we present in this study have been obtained from the molecular modification of SAU 1.Because of their high chemical reactivity, lanthanides never occur as pure elements in nature but only as sparsely distributed compounds that form rare minerals.Lanthanide compounds often have magnetic, catalytic and optical properties, and therefore they are currently widely used in industry and medicine as chemical markers and optical sensors [22].
In this work, the lanthanides samarium-Sm(III), gadolinium-Gd(III), europium-Eu(III), neodymium-Nd(III), lanthanum-La(III) and terbium-Tb(III) were complexed with SAU 1 [21], resulting in complexes that were tested for antileishmanial activity and selectivity.In addition to elucidating their mechanisms of leishmanicidal action, these promising results may contribute to more viable and effective therapeutic options against cutaneous leishmaniasis.

Antileishmanial Activity
Sodium usnate (SAU 1) was synthesized to increase the solubility of usnic acid in water (Figure 1), looking for better suitability for biological in vitro and in vivo assays.
Biological applications of lanthanides have recently increased because they are abundant in the environment and exhibit chemistry similar or superior to other biologically useful metals because of their higher Lewis acidity.They have sufficient coordination chemistry to allow selective uptake, transport and incorporation into

Antileishmanial Activity
Sodium usnate (SAU 1) was synthesized to increase the solubility of usnic acid in water (Figure 1), looking for better suitability for biological in vitro and in vivo assays.
Biological applications of lanthanides have recently increased because they are abundant in the environment and exhibit chemistry similar or superior to other biologically useful metals because of their higher Lewis acidity.They have sufficient coordination chemistry to allow selective uptake, transport and incorporation into enzymes.Their biological coordination chemistry is analogous to that of other metals, most notably Ca II and Fe III , but includes cooperative metal bonding to amplify the effects of small differences in ionic radius and allow selectivity [23][24][25][26][27][28].
In this work, SAU 1 and the lanthanide complexes 2-7 were tested against L. (L.) amazonensis promastigote forms, and cell viability was assessed by MTT metabolization, using the principle of tetrazolium-formazan conversion by living cell mitochondrial enzymes.All complexes were found to be highly active against promastigote forms (Table 1), more potent than the original compound SAU 1 (IC 50 = 2.09 µM) and even more effective than the reference drug, pentamidine (IC 50 = 1.50 µM).EuL 3 •3H 2 O 4 was more potent than the reference drug amphotericin B (IC 50 = 0.20 versus 0.145 µM).The complex with Tb(III) 6 showed impressive activity (IC 50 = 0.0023 µM), 63 times higher than amphotericin B and almost a thousand times higher than SAU 1 (Table 1).The optimization of the cytotoxic effect of the same lanthanide complexes over SAU was observed by Nunes et al. on MCF-7 tumor cells [21].[16] found an IC 50 /72 h = 18.30µg•mL −1 for L. (L.) infantum promastigotes treated with usnic acid.In our work, sodium usnate SAU 1 was found to be highly active against L. (L.) amazonensis promastigotes, with an IC 50 /72 h = 2.09 µM (=0.77 µg•mL −1 ), showing that improved solubility in aqueous medium may have resulted in optimized antileishmanial activity.
All compounds were found to be more active against promastigotes than against amastigote forms.To understand this pharmacological behavior, it is important to emphasize that amastigotes are the intracellular stage of Leishmania parasites.Therefore, a drug candidate must be able to cross the macrophage cell membrane to reach these forms.In this context, biopharmaceutical aspects are crucial for predicting the biological tendencies of a molecule.The topological polar surface area (TPSA) is an important biopharmaceutical parameter considering molecular complexes and is directly related to the number of polar groups exposed to solvents.Complexes and peptoids with low TPSA values are already known to have a higher permeability ratio [29].All complexes tested showed moderate TPSA values (Table 2).Considering that the in vitro activity on intracellular amastigotes was demonstrated in RPMI 1640 medium, the intermolecular interaction between the polar groups of the complexes and the aqueous medium may have reduced their permeability across the membrane, which explains the inferior results.In addition, biopharmaceutical parameters are useful to indicate whether a compound may have oral viability.This route of administration is desirable for the treatment of most diseases, such as leishmaniasis.Advantages include ease of administration, cost-effective manufacturing, easy storage conditions, high patient compliance and more accurate selfadministered dose [30].In particular, the logP value has been widely discussed in the postulation of the rule of five and is still considered an important parameter of a prototype.This parameter can be used to predict whether a compound will penetrate the cell membrane or be easily soluble in an aqueous medium such as blood [31].According to Lipinski et al. [32], a logP ≤ 5 is considered necessary for an ideal prototype.Meanwhile, logS values are directly related to the water solubility of a compound, and values close to −4 have been defined as the standard for a drug candidate [33].All tested compounds exhibited logS values close to −4 and logP ≤ 5, following the postulated characteristics and indicating that the complexes can be administered orally (Table 2).
Intracellular amastigote activity was evaluated according to the following arbitrary scale: compounds were considered active when IC 50 < 20 µM, moderately active when 20 < IC 50 < 50 µM, and potentially inactive when IC 50 > 50 µM [34].Therefore, all lanthanide complexes were active against L. (L.) amazonensis amastigotes (Table 1), with a greater effect than SAU 1. Two-way analysis of variance (ANOVA) followed by the Tukey test has confirmed that NdL 3 •3H 2 O 5 was statistically different from SAU 1 (p ≤ 0.0001) at all concentrations tested.EuL 3 •3H 2 O 4 was more potent than SAU at the lowest concentrations (p ≤ 0.0001) and TbL 3 •2H 2 O 6 only at the higher concentration (50 µg/mL; p ≤ 0.0001); all the other complexes were more potent than SAU 1 at all the concentrations tested excepting 6.25 µg/mL (p ≤ 0.0001).
These results lead to the conclusion that the insertion of the lanthanide ion led to the optimization of the antileishmanial activity.Because of their high chemical reactivity, it is not possible to analyze the activity of isolated lanthanides.This is the case with other drugs in medicine, such as the use of cyclodextrin to promote the stability of gold nanoparticles and the programmed release of antitumor drugs [35,36].For example, the stability of trivalent antimonials (SbIII) is possible when linked to meglumine and gluconate, as in the antileishmanial drugs Glucantime ® and Pentostam ® [37].
Among the complexes tested, those with Eu(III) 4 and Nd(III) 5 were the most active in intracellular amastigotes (IC 50 = 2.98 and 2.83 µM, respectively; Figure 2), while the compound with Tb(III) was the least active (IC 50 = 7.52 µM).Studies with other lanthanide complexes on other parasite species have shown similar results.Caballero et al. [38] observed that the complexes with Eu(III) and Nd(III) had the same effect as the reference drug Glucantime ® ; on the other hand, the complex with La(III) was the most active on L. (L.) infantum and on L. (L.) braziliensis amastigotes.In the present study, LaL 3 •3H 2 O 7 was active on L. (L.) amazonensis (IC 50 = 4.58 µM), opening a promising perspective for application against several species of Leishmania.
Among the complexes tested, those with Eu(III) 4 and Nd(III) 5 were the most active in intracellular amastigotes (IC50 = 2.98 and 2.83 µM, respectively; Figure 2), while the compound with Tb(III) was the least active (IC50 = 7.52 µM).Studies with other lanthanide complexes on other parasite species have shown similar results.Caballero et al. [38] observed that the complexes with Eu(III) and Nd(III) had the same effect as the reference drug Glucantime ® ; on the other hand, the complex with La(III) was the most active on L. (L.) infantum and on L. (L.) braziliensis amastigotes.In the present study, LaL3•3H2O 7 was active on L. (L.) amazonensis (IC50 = 4.58 µM), opening a promising perspective for application against several species of Leishmania.

Cytotoxicity and Selectivity Index in Mammalian Cells
In silico predictions were used to estimate the risk of mutagenicity, tumorigenicity and irritation, and the complexes showed no evidence of these side effects (Table 2).To demonstrate host cell safety, SAU 1 and lanthanide complexes 2-7 were tested on NIH/3T3 fibroblasts to assess their cytotoxicity and estimate their selectivity, as recommended by the Organisation for Economic Co-operation and Development (OECD) [39].

Nitric Oxide (NO) Production
The induction of a strong oxidative response by lanthanides on human cells has already been described, with the release of reactive oxygen species (ROS) that interfere with the signaling that regulates the immune response to an infection [24].Macrophages are the host cells of Leishmania sp. and also play a role in the action against the parasite.The beneficial function of the ROS oxidative burst and NO is associated with the eradication of the parasite, in addition to the clinical improvement of the patient [42].
Intracellular parasites rely on the ability to undergo replication cycles within host cells.Once the cellular environment of macrophages becomes inviable, the parasite cannot replicate.NO production is one of the main mechanisms for the intracellular killing of the parasite.In addition to more immediate effects, such as reduced parasite metabolism and

Cytotoxicity and Selectivity Index in Mammalian Cells
In silico predictions were used to estimate the risk of mutagenicity, tumorigenicity and irritation, and the complexes showed no evidence of these side effects (Table 2).To demonstrate host cell safety, SAU 1 and lanthanide complexes 2-7 were tested on NIH/3T3 fibroblasts to assess their cytotoxicity and estimate their selectivity, as recommended by the Organisation for Economic Co-operation and Development (OECD) [39].

Nitric Oxide (NO) Production
The induction of a strong oxidative response by lanthanides on human cells has already been described, with the release of reactive oxygen species (ROS) that interfere with the signaling that regulates the immune response to an infection [24].Macrophages are the host cells of Leishmania sp. and also play a role in the action against the parasite.The beneficial function of the ROS oxidative burst and NO is associated with the eradication of the parasite, in addition to the clinical improvement of the patient [42].
Intracellular parasites rely on the ability to undergo replication cycles within host cells.Once the cellular environment of macrophages becomes inviable, the parasite cannot replicate.NO production is one of the main mechanisms for the intracellular killing of the parasite.In addition to more immediate effects, such as reduced parasite metabolism and proliferation, it is associated with immune regulation of infection [43,44].Paradoxically, NO alone may not be sufficient to control the infection and may contribute to the tissue damage observed in human CL [42].
Treatment with lanthanide complexes at some concentrations induced an increase in NO release from infected macrophages (Figure 3).EuL 3 •3H 2 O 4 had the strongest effect at the very lowest concentration (6.25 µg•mL −1 ).This concentration is close to the half-maximal inhibitory concentration on the intracellular amastigotes (IC 50 = 2.98 µM or 3.687 µg•mL −1 ), suggesting NO production as a mechanism of action of this complex against L. (L.) amazonensis.
Treatment with lanthanide complexes at some concentrations induced an increase in NO release from infected macrophages (Figure 3).EuL3•3H2O 4 had the strongest effect at the very lowest concentration (6.25 µg•mL −1 ).This concentration is close to the half-maximal inhibitory concentration on the intracellular amastigotes (IC50 = 2.98 µM or 3.687 µg•mL −1 ), suggesting NO production as a mechanism of action of this complex against L. (L.) amazonensis.

Flow Cytometric Analysis for Detection of Changes in Mitochondrial Membrane Potential (ΔΨm)
Promastigotes treated with SAU 1 and lanthanide complexes 2-7 were stained with TMRE to evaluate changes in mitochondrial membrane potential (ΔΨm).Figure 4 and Table 3 show the histograms (%) of TMRE-labeled promastigotes after incubation with 1-7 at the calculated IC50 (Table 1).The complex with Sm(III) showed a marked effect on the mitochondrial membrane potential, as we observed a reduction in labeling of 40.7% and 41.7% after 48 and 72 h of treatment, respectively.Similarly, the complex with Gd(III) was able to reduce the ΔΨm by 36.2% and 24.5% after 48 and 72 h treatment, respectively.Complexes with La(III) and Nd(III) affected the ΔΨm in 22.3% and 11.35%, respectively,

Flow Cytometric Analysis for Detection of Changes in Mitochondrial Membrane Potential (∆Ψm)
Promastigotes treated with SAU 1 and lanthanide complexes 2-7 were stained with TMRE to evaluate changes in mitochondrial membrane potential (∆Ψm).Figure 4 and Table 3 show the histograms (%) of TMRE-labeled promastigotes after incubation with 1-7 at the calculated IC 50 (Table 1).The complex with Sm(III) showed a marked effect on the mitochondrial membrane potential, as we observed a reduction in labeling of 40.7% and 41.7% after 48 and 72 h of treatment, respectively.Similarly, the complex with Gd(III) was able to reduce the ∆Ψm by 36.2% and 24.5% after 48 and 72 h treatment, respectively.Complexes with La(III) and Nd(III) affected the ∆Ψm in 22.3% and 11.35%, respectively, after 72 h of treatment.Thus, we can assume that the complexes with these lanthanides cause mitochondrial dysfunction, an important and recognized target of antileishmanial drugs.
SAU 1 had a mild effect on ∆Ψm 72 h after treatment, with a 12% reduction in labeling at the concentration tested (IC 50 = 2.09 µM).There are few reports on the effect of usnic acid on trypanosomatid mitochondria.Carvalho et al. [45] showed damage to the mitochondria of Trypanosoma cruzi epimastigotes treated with 30-50 µg•mL −1 usnic acid, with marked swelling and branching, as well as loss of organization of the mitochondrial cristae observed in ultrastructural analysis.Similarly, Luz et al. [16] observed changes in the mitochondrial morphology of L. (L.) infantum promastigotes treated with 25 µg•mL −1 usnic acid, with marked swelling.

Flow Cytometric Analysis for Detection of Apoptosis and Membrane Permeability
L. (L.) amazonensis promastigote forms were treated with SAU 1 and the lanth complexes 2-7 at the calculated IC50 (Table 1) for 24, 48 and 72 h and then stained w Annexin V and 7-AAD for FACS analysis.This measurement can track cells ove from PE Annexin V and 7-AAD negative (viable or without measurable apoptosis) Annexin V and 7-AAD positive (early apoptosis, membrane integrity present) and fi to PE Annexin V and 7-AAD positive (terminal apoptosis and death).The movem  Unst.= unstained parasites; An. = PE Annexin V-stained parasites; 7-AAD = 7-AAD-stained parasites; An./7-AAD = double stained parasites.

Flow Cytometric Analysis for Detection of Apoptosis and Membrane Permeability
L. (L.) amazonensis promastigote forms were treated with SAU 1 and the lanthanide complexes 2-7 at the calculated IC 50 (Table 1) for 24, 48 and 72 h and then stained with PE Annexin V and 7-AAD for FACS analysis.This measurement can track cells over time from PE Annexin V and 7-AAD negative (viable or without measurable apoptosis) to PE Annexin V and 7-AAD positive (early apoptosis, membrane integrity present) and finally to PE Annexin V and 7-AAD positive (terminal apoptosis and death).The movement of parasites through these three stages is indicative of apoptosis.On the other hand, cells that are both PE Annexin V and 7-AAD positive may have undergone apoptotic death or died by a necrotic pathway.
Figure 5 and Table 3 show the percentage of cell labeling after incubation with PE annexin and 7-AAD.Again, the effect of the complexes with the lanthanides Sm(III) and Gd(III) on the parasites can be highlighted.SmL 3 •4H2O 2 caused an earlier cell death effect since 24 h after treatment, there was a 17.2% reduction in viability, with 10.2% 7-AAD staining and 5.5% double labeling.On the other hand, GdL 3 •3H2O 3 had its greatest effect 48 h after treatment, also reducing cell viability by 17.4%, with 12.7% 7-AAD staining and 6% double labeling.Despite the disruption of cell membrane integrity in both cases, it was not possible to establish a relationship with either early or late apoptotic processes, as PE Annexin V labeling was low at all times studied.staining and 5.5% double labeling.On the other hand, GdL3•3H2O 3 had its greatest e 48 h after treatment, also reducing cell viability by 17.4%, with 12.7% 7-AAD staining 6% double labeling.Despite the disruption of cell membrane integrity in both cases, it not possible to establish a relationship with either early or late apoptotic processes, a Annexin V labeling was low at all times studied.

Study Limitations
The complexity of understanding the mechanisms of leishmanicidal action, nam the induction of apoptosis, may be related to the use of IC50 as the inhibitory concentra

Study Limitations
The complexity of understanding the mechanisms of leishmanicidal action, namely the induction of apoptosis, may be related to the use of IC 50 as the inhibitory concentration for promastigote forms.The use of IC 90 or IC 99 , or higher concentrations, would improve these analyses by promoting complete parasitic inhibition.Likewise, investigating the mechanisms of action of the complexes on the intracellular amastigote forms could greatly enhance our understanding despite the current methodology's challenging nature.

Lanthanide Complexes
Compounds 1-7 (Figure 1) were synthesized in the Institute of Chemistry of the Federal University of Mato Grosso do Sul [19].Sodium usnate (SAU) 1 was prepared as described in the literature [46] from the reaction of the ethanolic suspension of usnic acid (3 mmol) with a sodium carbonate solution (1.5 mmol) with stirring at 40 • C for 4 h, followed by filtration and evaporation of the solvent to obtain a yellow solid.Lanthanide complexes were then synthesized and characterized [21].Briefly, sodium usnate (0.3 mmol) was dissolved in water and ethanol (3:1); 0.1 mmol of lanthanide ions: Sm(III), Gd(III), Eu(III), Nd(III), Tb(III) and La(III) were added to this solution and kept under stirring for 8 h at 50 • C. The resulting yellow precipitate was washed with ice water and dried in a vacuum desiccator (Figure 1).The general coordination formula was used: [LnL 3 (H 2 O) x ], where L= C 18 H 15 O 7 , Ln = Tb(III), x = 2; Ln = Gd(III), Eu(III), Nd(III), La(III), x = 3 and Ln = Sm(III), x = 4.

Animals
Four-week-old female BALB/c mice were obtained from the Central Animal Facility of the Federal University of Mato Grosso do Sul (UFMS).The animals were housed in mini-isolators connected to a ventilated rack, fed a balanced diet (Nuvital ® , Nuvilab ® , Seoul, Republic of Korea) and given ad libitum access to filtered water.This study received approval from the local Animal Experimentation Ethical Committee (CEUA/UFMS) under protocol 1,172/2021.

In Vitro Antileishmanial Activity on L. (L.) amazonensis Intracellular Amastigotes
Peritoneal macrophages were obtained from BALB/c mice in RPMI 1640 culture medium (Sigma-Aldrich ® São Paulo, Brazil).After exclusion of inviable cells with trypan blue staining (Sigma-Aldrich ® São Paulo, Brazil), cells were transferred to 24-well plates with 13 mm round glass coverslips (10 6 cells/well) and kept at 37 • C/5% CO 2 .After adhesion for one hour, cells were infected with promastigote forms of L. (L.) amazonensis in the stationary phase (4 × 10 6 cells/well).After 4 h of incubation, samples (complexes 1-7) were added in six replicates (6.25-50.0µg•mL −1 ).Nontreated infected cells were used as the negative control, and AMB (0.125-1.0 µg•mL −1 ) and Pentamidine (6.25-50.0µg•mL −1 ) were used as the positive controls.Coverslips were removed after 24 h, subjected to a dehydration battery with increasing concentrations of acetone: xylol and stained with Giemsa (Sigma-Aldrich ® , São Paulo, Brazil) diluted 1:10 in distilled water [34].After mounting on slides, 200 cells per coverslip were analyzed by light microscopy and the average number of amastigotes/cells was calculated.IC 50 was determined using a nonlinear dose-response regression curve as described above.The concentrations of complexes were compared by Two-way ANOVA followed by the Tukey test.

In Silico ADMET Predictions
All compounds were subjected to the OSIRIS Property Explorer program to estimate the risk of side effects such as mutagenicity, tumorigenicity and irritation.Furthermore, drug-related properties were measured, including clogP, logS, MW, drug similarity and overall drug score [33].

Cytotoxicity in Mammalian Cells
The cytotoxic effect of compounds 1-7 was evaluated on NIH/3T3 fibroblasts [48] obtained from the Rio de Janeiro Cell Bank (Duque de Caxias, Brazil).Fibroblasts were seeded at 5 × 10 5 cells/well in 96-well plates.After 24 h of fixation, cells were incubated for 48 h with 1-7 at 2.5-250 µg•mL −1 , in three replicates.Cell growth was evaluated using the sulforhodamine B colorimetric method [48], and the optical with absorbances read on the PT-READER microplate instrument (Spectramax 190, Molecular Devices, Sunnyvale, CA, USA) at 540 nm.DMSO (dimethyl sulfoxide, Sigma-Aldrich ® São Paulo, Brazil) was used as a negative control at a concentration necessary to solubilize the highest concentration of the test sample and did not interfere with cell viability.The growth inhibition percentage-GI (%) of each test sample was calculated in Microsoft Office Excel 2007 software [49].IC 50 was determined graphically in the Microcal Origin Version 6.00 program [50].The selectivity index (SI) was calculated as the ratio between the cytotoxicity on cells (IC 50 ) and the activity on the parasite forms (IC 50 ).

NO Production by Infected Cells
Nitric oxide (NO) production by infected and treated peritoneal macrophages was estimated in the supernatants from the intracellular amastigotes assay by the Griess reaction [51].Aliquots of the supernatants (50 mL) were incubated with Griess reagent (1:1) for 10 min at room temperature.Absorbance was read on a microplate reader (HumanReader HS, Wiesbaden, Germany) at 550 nm.Conversion of nitrite (NO −2 ) to micromolar (µM) was performed by comparing the samples to a standard curve obtained with known concentrations (1-10 µM) of sodium nitrite diluted in RPMI 1640 medium (Sigma-Aldrich ® São Paulo, Brazil).The average and standard error of the mean (SEM) were calculated from three replicates.The data were analyzed using ANOVA and subsequently subjected to the Bonferroni test.

Flow Cytometric Analysis to Detect Changes in Mitochondrial Membrane Potential (∆Ψm)
The mitochondrial membrane potential (∆Ψm) can change in cells undergoing apoptosis, oxidative stress, necrosis and other cellular processes.Cationic and lipophilic ∆Ψmsensitive dyes accumulate within the mitochondria of healthy cells but not in mitochondria that have lost ∆Ψm.In this work, L. (L.) amazonensis promastigote forms (1 × 10 6 mL −1 ) were treated for 48 and 72 h with SAU 1 and the lanthanide complexes 2-7 at the calculated IC 50 (Table 1), in Schneider's insect medium (Sigma-Aldrich ® ) supplemented with 20% FCS (Cultilab ® , São Paulo, Brazil), 10,000 U•mL −1 penicillin and 10 mg•mL −1 streptomycin (Sigma-Aldrich ® ).Heat-killed parasites (60 • C bath/10 min) were used as positive control, and nontreated parasites were used as negative control [52].Promastigotes were stained with 50 nM tetramethylrhodamine, ethylester (TMRE) (MitoStatus TMRE, BD Pharmingen TM , San Diego, CA, USA) for 15 min at room temperature and subjected to flow cytometric analysis (BD FACSCanto TM II, Kingston, ON, Canada).Then, 10,000 events were acquired in the region previously determined to correspond to the parasites.Fluorescence was quantified and histograms were generated.

Flow Cytometry Analysis for Detection of Apoptosis
PE Annexin V staining precedes the loss of membrane integrity observed in the final stages of cell death, resulting from either apoptotic or necrotic processes.PE Annexin V is used in conjunction with the vital dye 7-Amino-Actinomycin (7-AAD) to identify early apoptotic cells (7-AAD negative, PE Annexin V positive).Viable cells with intact membranes exclude 7-AAD, but the membranes of dead and damaged cells are permeable to 7-AAD.Cells that are in late apoptosis or already dead are both PE Annexin V and 7-AAD positive.In this work, L. (L.) amazonensis promastigote forms (1 × 10 6 mL −1 ) were treated for 24, 48 and 72 h with 1-7 at the calculated IC 50 (Table 1) in Schneider's insect medium (Sigma-Aldrich ® ) supplemented with 20% FCS (Cultilab ® ), 10,000 U•mL −1 penicillin and 10 mg•mL −1 streptomycin (Sigma-Aldrich ® ).Heat-killed parasites (60 • C bath/10 min) were used as positive control, and untreated parasites were used as negative control [52].Promastigotes were stained with 5 µL of PE Annexin V and 5 µL 7-AAD (PE Annexin V Apoptosis Detection Kit I, BD Biosciences TM , Franklin Lakes, NJ, USA) and immediately subjected to flow cytometric analysis (BD FACSCanto TM II).Then, 10,000 events were captured in the region previously determined to correspond to the parasites.Fluorescence was quantified and histograms were generated.
3 •2H 2 O 6 and EuL 3 •3H 2 O 4. EuL 3 •3H 2 O 4 and NdL 3 •3H 2 O 5 were the most selective and effective on intracellular amastigotes, with an SI of approximately 7.0.Treatment with EuL 3 •3H 2 O 4 triggered NO release even at the lowest concentration (6.25 µg•mL −1 ), indicating NO production as a mechanism of action against L. (L.) amazonensis.Incubation of promastigotes with the lanthanide complexes at the calculated IC 50 , especially with SmL 3 •4H 2 O 2 and GdL 3 •3H 2 O 3, was found to modulate the mitochondrial membrane potential, suggesting that these complexes are able to target this crucial organelle.The aforementioned complexes caused cell death through cell membrane disruption, but their relationship with early or late apoptotic processes remains unclear.Further research is being conducted to understand the mechanisms of leishmanicidal action at different concentrations.

Table 1 .
In vitro antileishmanial activity, cytotoxic effect on mammalian cells and selectivity index of SAU 1 and lanthanide complexes 2

Table 2 .
In silico absorption, distribution, metabolism, excretion and toxicity (ADMET) predictions of lanthanide complexes.No mutagenic, tumorigenic or irritating effects were observed for any of the complexes.
LogP = partition coefficient; LogS = water solubility; TPSA = topological polar surface area; Druglikeness = similarity of the properties between compounds and existing drugs; Drug-score = compilation of ADMET parameters to judge the compound's overall potential to qualify for a drug.

Table 3 .
Flow cytometry of L. (L.) amazonensis to evaluate the mitochondrial membrane potential (∆Ψ m ) and apoptosis after treatment with SAU 1 and lanthanide complexes 2-7.Promastigotes captured in the gated region and representative histograms (% labeled cells) of promastigotes incubated with TMRE, PE Annexin V and 7-AAD.