Antileishmanial Activity of 4,8-Dimethoxynaphthalenyl Chalcones on Leishmania amazonensis

Leishmaniasis is a neglected tropical disease caused by Leishmania species. Available therapeutic options have several limitations. The drive to develop new, more potent, and selective antileishmanial agents is thus a major goal. Herein we report the synthesis and the biological activity evaluation against promastigote and amastigote forms of Leishmania amazonensis of nine 4,8-dimethoxynaphthalenyl chalcones. Compound ((E)-1-(4,8-dimethoxynaphthalen-1-yl)-3-(4-nitrophenyl)prop-2-en-1-one), 4f, was the most promising with an IC50 = 3.3 ± 0.34 μM (promastigotes), a low cytotoxicity profile (CC50 = 372.9 ± 0.04 μM), and a high selectivity index (SI = 112.6). Furthermore, 4f induced several morphological and ultrastructural changes in the free promastigote forms, loss of plasma membrane integrity, and increased reactive oxygen species (ROS). An in silico analysis of drug-likeness and ADME parameters suggested high oral bioavailability and intestinal absorption. Compound 4f reduced the number of infected macrophages and the number of amastigotes per macrophage, with an IC50 value of 18.5 ± 1.19 μM. Molecular docking studies with targets, ARG and TR, showed that compound 4f had more hydrogen bond interactions with the ARG enzyme, indicating a more stable protein-ligand binding. These results suggest that 4,8-dimethoxynaphthalenyl chalcones are worthy of further study as potential antileishmanial drugs.


Introduction
The leishmaniases are a group of neglected diseases widely distributed among tropical and subtropical countries. Leishmaniases are caused by the protozoan parasite of the genus Leishmania and transmitted to humans by the bite of infected female phlebotomine sandflies. This parasitic disease presents mainly four clinical manifestations: visceral leishmaniasis (VL, also known as kala-azar), cutaneous leishmaniasis (CL), diffuse cutaneous leishmaniasis (DCL), and mucocutaneous leishmaniasis (MCL). According to the World Health Organization (WHO), 700,000 to 1 million new cases and 26,000 to 65,000 deaths occur yearly, constituting a major global public health problem [1,2].  In the search for new biological agents, an important aspect is the identification of molecular targets to provide selective inhibition. For Leishmania spp., one such target is the enzyme arginase (ARG), responsible for L-arginine hydrolysis, which provides the necessary substrate for biosynthesis of polyamines required for growing cellular, replication, and infectivity of the parasite. [11]. Another important enzyme to target is trypanothione reductase (TR). This NADPH-dependent enzyme catalyzes trypanothione disulfide reduction in trypanothione dithiol, representing an essential system to combat oxidative stress Antibiotics 2022, 11, 1402 3 of 23 caused by reactive oxygen species (ROS). A further reason for targeting TR is its absence in mammals [12].
We have synthesized and evaluated the antileishmanial potential of a series of nine new chalcone derivatives containing the 4,8-dimethoxynaphthalenyl moiety. The antipromastigote activity and cytotoxicity were assessed on murine macrophage cell lines (J774A.1). The potential of compounds was based on their IC 50 and selectivity index (SI) values. In addition, the effect against amastigote forms, as well as such biological assays, as mitochondrial depolarization, reactive oxygen species generation, morphological, and ultrastructural analysis to understand the mechanisms of action in promastigote forms. Finally, we performed molecular docking on two targets of L. amazonensis, ARG and TR (Figure 2). molecular targets to provide selective inhibition. For Leishmania spp., one such target is the enzyme arginase (ARG), responsible for L-arginine hydrolysis, which provides the necessary substrate for biosynthesis of polyamines required for growing cellular, replication, and infectivity of the parasite. [11]. Another important enzyme to target is trypanothione reductase (TR). This NADPH-dependent enzyme catalyzes trypanothione disulfide reduction in trypanothione dithiol, representing an essential system to combat oxidative stress caused by reactive oxygen species (ROS). A further reason for targeting TR is its absence in mammals [12].
We have synthesized and evaluated the antileishmanial potential of a series of nine new chalcone derivatives containing the 4,8-dimethoxynaphthalenyl moiety. The antipromastigote activity and cytotoxicity were assessed on murine macrophage cell lines (J774A.1). The potential of compounds was based on their IC50 and selectivity index (SI) values. In addition, the effect against amastigote forms, as well as such biological assays, as mitochondrial depolarization, reactive oxygen species generation, morphological, and ultrastructural analysis to understand the mechanisms of action in promastigote forms. Finally, we performed molecular docking on two targets of L. amazonensis, ARG and TR ( Figure 2).
The final products 4a-i were purified by flash chromatography and characterized by 1 H, 13 C, COSY, and HSQC NMR, IR, and HR mass spectrometry. In general, 1 H NMR spectra showed two characteristic doublets between δ 7.38-7.00 and δ 7.11-6.90 with a coupling constant (J Hα-Hβ ) around 16.0 Hz, indicating the formation of the (E)-diastereoisomer. 13 C NMR spectra exhibited signals related to the C=O carbons between δ 199. 23-197.01. In addition, the IR spectra showed two absorption bands around 3020 cm −1 and 1665 cm −1 , which were assigned to the stretching vibrations of =C−H and carbonyl band (n C=O), respectively. The novel compounds showed similar IR and NMR spectroscopic signals, and the HRMS unequivocally confirmed their molecular formulas.
The final products 4a-i were purified by flash chromatography and characterized by 1 H, 13 C, COSY, and HSQC NMR, IR, and HR mass spectrometry. In general, 1 H NMR spectra showed two characteristic doublets between δ 7.38-7.00 and δ 7.11-6.90 with a coupling constant (JHα-Hβ) around 16.0 Hz, indicating the formation of the (E)diastereoisomer. 13 C NMR spectra exhibited signals related to the C=O carbons between δ 199. 23-197.01. In addition, the IR spectra showed two absorption bands around 3020 cm −1 and 1665 cm −1 , which were assigned to the stretching vibrations of =C−H and carbonyl band (n C=O), respectively. The novel compounds showed similar IR and NMR spectroscopic signals, and the HRMS unequivocally confirmed their molecular formulas.
Crystal Structure of Compound 4f and comparisons with those of 4c and 4i The atom arrangement and numbering scheme for compound 4f are shown in Figure  3A. The molecule has a distinctive shape, between a "T" and a "Y", with an interplanar angle between the naphthalene and phenyl rings of 51.08°, see Figure 3B. The nitro group is almost coplanar with its attached phenyl ring, and an E geometry about the exo C=C group is present. The bond lengths in the linker chain between the phenyl and naphthalene rings indicate a degree of delocalization, with bond lengths being intermediate between those expected for single and double bonds, as expected for chalcones, as reported earlier for compounds 4c and 4i [13]. The atom arrangement and numbering scheme for compound 4f are shown in Figure 3A. The molecule has a distinctive shape, between a "T" and a "Y", with an interplanar angle between the naphthalene and phenyl rings of 51.08 • , see Figure 3B. The nitro group is almost coplanar with its attached phenyl ring, and an E geometry about the exo C=C group is present. The bond lengths in the linker chain between the phenyl and naphthalene rings indicate a degree of delocalization, with bond lengths being intermediate between those expected for single and double bonds, as expected for chalcones, as reported earlier for compounds 4c and 4i [13]. The final products 4a-i were purified by flash chromatography and characterized by 1 H, 13 C, COSY, and HSQC NMR, IR, and HR mass spectrometry. In general, 1 H NMR spectra showed two characteristic doublets between δ 7.38-7.00 and δ 7.11-6.90 with a coupling constant (JHα-Hβ) around 16.0 Hz, indicating the formation of the (E)diastereoisomer. 13 C NMR spectra exhibited signals related to the C=O carbons between δ 199. 23-197.01. In addition, the IR spectra showed two absorption bands around 3020 cm −1 and 1665 cm −1 , which were assigned to the stretching vibrations of =C−H and carbonyl band (n C=O), respectively. The novel compounds showed similar IR and NMR spectroscopic signals, and the HRMS unequivocally confirmed their molecular formulas.
Crystal Structure of Compound 4f and comparisons with those of 4c and 4i The atom arrangement and numbering scheme for compound 4f are shown in Figure  3A. The molecule has a distinctive shape, between a "T" and a "Y", with an interplanar angle between the naphthalene and phenyl rings of 51.08°, see Figure 3B. The nitro group is almost coplanar with its attached phenyl ring, and an E geometry about the exo C=C group is present. The bond lengths in the linker chain between the phenyl and naphthalene rings indicate a degree of delocalization, with bond lengths being intermediate between those expected for single and double bonds, as expected for chalcones, as reported earlier for compounds 4c and 4i [13].  Comparisons of the molecular structures of 4c and 4i [13] with those of 4f indicate that different rotations about the C1-C11 bonds have resulted in different separations of the O4 methoxy oxygen and the O11 carbonyl oxygen, which provides the anti-periplanar arrangements in 4i and 4f and the syn-periplanar arrangement in 4c within the bridging prop-2-en-1-one units, see Figure 4. The interplanar angles between the naphthalene and phenyl rings in 4c and 4i are 70.01(8) and 50.20(7) • , respectively, which also places the molecular structure of 4f close to that of 4i. Calculations [13] indicated that the stabilities of the antiand syn-periplanar molecular arrangements are very similar, with intermolecular interactions being significant factors in the crystal structures. arrangements in 4i and 4f and the syn-periplanar arrangement in 4c within the bridging prop-2-en-1-one units, see Figure 4. The interplanar angles between the naphthalene and phenyl rings in 4c and 4i are 70.01(8) and 50.20(7) o , respectively, which also places the molecular structure of 4f close to that of 4i. Calculations [13] indicated that the stabilities of the anti-and syn-periplanar molecular arrangements are very similar, with intermolecular interactions being significant factors in the crystal structures. Molecules of 4f are linked in the crystalline state into a three-dimension array by a variety of interactions, including C-H---O hydrogen bonds and π---π, C-H---π, and N-O---π interactions. However, the methoxy oxygens (O4 and O9) are not involved in the intermolecular interactions, unlike the carbonyl oxygen (O11) and the nitro group oxygens (O17A and O17B). The antileishmanial activities of compounds 4a-i were initially evaluated in vitro with promastigote forms of L. amazonensis, related to American cutaneous leishmaniasis. Furthermore, as macrophages are the host cells critical for Leishmania spp., the cytotoxicities were tested against murine macrophages (J774A.1) by the method developed by Mosmann [14], based on mitochondrial oxidation. The antipromastigote activities varied widely, with IC50 values ranging from 3.3 ± 0.34 to 264.1 ± 0.12 μM. However, all compounds 4a-i showed no toxicity to mammalian cells, showing CC50 values >100 μM, see Table 1. Molecules of 4f are linked in the crystalline state into a three-dimension array by a variety of interactions, including C-H-O hydrogen bonds and π-π, C-H-π, and N-O-π interactions. However, the methoxy oxygens (O4 and O9) are not involved in the intermolecular interactions, unlike the carbonyl oxygen (O11) and the nitro group oxygens (O17A and O17B). The antileishmanial activities of compounds 4a-i were initially evaluated in vitro with promastigote forms of L. amazonensis, related to American cutaneous leishmaniasis. Furthermore, as macrophages are the host cells critical for Leishmania spp., the cytotoxicities were tested against murine macrophages (J774A.1) by the method developed by Mosmann [14], based on mitochondrial oxidation. The antipromastigote activities varied widely, with IC 50 values ranging from 3.3 ± 0.34 to 264.1 ± 0.12 µM. However, all compounds 4a-i showed no toxicity to mammalian cells, showing CC 50 values > 100 µM, see Table 1.

Biological Evaluation
The results obtained from the preliminary analysis of the structure-activity relationship (SAR) indicate that the presence of electron-donating groups in the phenyl ring in 4a-b, such as methoxy groups, leads to a decrease in the antileishmanial activity compared to the unsubstituted derivative 4h, (IC 50 = 26.1 ± 0.09 µM). However, this decrease was less for the meta-methoxyl derivative, 4b, (IC 50 = 67.1 ± 0.16 µM) than for the para derivative, 4a, (IC 50 = 264.1 ± 0.12 µM). The effects of different electron-withdrawing groups, such as Br (in 4i), Cl (in 4c, 4d, and 4e), and NO 2 (in 4f and 4g), were also investigated. For compound 4i, the presence of bromine led to a small increase in activity (IC 50 = 24.3 ± 0.05 µM). Concerning the disubstituted chlorinated derivatives (4c-e), it was observed that the presence of at least one chlorine atom in an ortho position (4d and 4e) promotes an increase in IC 50 values (IC 50 = 94.1 ± 0.06 and 37.7 ± 0.06 µM, respectively), which could be associated with the ortho effect on the biological activity [15]. This is more evident for the compound, which has two chlorine atoms at ortho positions, being the least active of the chloro-substituted compounds. Compound 4c, without ortho-chlorine, showed the best activity of the chlorine-containing substituents and was even more active than the unsubstituted 4h and the brominated 4i derivatives. For the nitro-derivatives 4f-g, good activities were found, with the para nitro derivative 4f being the most active derivative of the series (IC 50 = 3.3 ± 0.34 µM), only a slight reduction was found for the meta-nitro derivative (IC 50 = 14.5 ± 0.09 µM).   The results obtained from the preliminary analysis of the structure-activity relationship (SAR) indicate that the presence of electron-donating groups in the phenyl ring in 4a-b, such as methoxy groups, leads to a decrease in the antileishmanial activity compared to the unsubstituted derivative 4h, (IC50 = 26.1 ± 0.09 μM). However, this decrease was less for the meta-methoxyl derivative, 4b, (IC50 = 67.1 ± 0.16 μM) than for the para derivative, 4a, (IC50 = 264.1 ± 0.12 μM). The effects of different electron-withdrawing groups, such as Br (in 4i), Cl (in 4c, 4d, and 4e), and NO2 (in 4f and 4g), were also investigated. For compound 4i, the presence of bromine led to a small increase in activity (IC50 = 24.3 ± 0.05 μM). Concerning the disubstituted chlorinated derivatives (4c-e), it was observed that the presence of at least one chlorine atom in an ortho position (4d and 4e) promotes an increase in IC50 values (IC50 = 94.1 ± 0.06 and 37.7 ± 0.06 μM, respectively), which could be associated with the ortho effect on the biological activity [15]. This is more evident for the compound, which has two chlorine atoms at ortho positions, being the least active of the chloro-substituted compounds. Compound 4c, without ortho-chlorine, showed the best activity of the chlorine-containing substituents and was even more active than the unsubstituted 4h and the brominated 4i derivatives. For the nitro-derivatives 4fg, good activities were found, with the para nitro derivative 4f being the most active derivative of the series (IC50 = 3.3 ± 0.34 μM), only a slight reduction was found for the meta-nitro derivative (IC50 = 14.5 ± 0.09 μM).
This analysis shows that compound 4f is the most promising in terms of activity (IC50 = 3.3 ± 0.34 μM) and selectivity index (SI = 112.6). In drug discovery for leishmaniases, the hit and lead criteria to elect a promising compound are an IC50 < 10 μM and a selectivity index ≥ 10 against parasitic protozoa [16]. Compound 4f was selected for further in vitro assays, which included the ability to generate reactive oxygen species (ROS) and the depolarization of the mitochondrial membrane, as well as in morphological and This analysis shows that compound 4f is the most promising in terms of activity (IC 50 = 3.3 ± 0.34 µM) and selectivity index (SI = 112.6). In drug discovery for leishmaniases, the hit and lead criteria to elect a promising compound are an IC 50 < 10 µM and a selectivity index ≥ 10 against parasitic protozoa [16]. Compound 4f was selected for further in vitro assays, which included the ability to generate reactive oxygen species (ROS) and the depolarization of the mitochondrial membrane, as well as in morphological and ultrastructural analysis. All these were carried out to fully understand the mechanisms of eliminating promastigote forms and the anti-amastigote evaluation. Finally, a molecular docking study was performed on potential molecular targets (ARG and TR).

Mechanism of Action in Promastigotes of L. amazonensis
The most effective response of macrophages against Leishmania spp. is mediated by producing toxic microbicide molecules, such as ROS and NO. These species trigger a well-established mechanism for eliminating the parasite, leading to infection resolution and parasite elimination without damaging host cells [17]. It is well known that high concentrations of ROS molecules can cause harmful effects in mitochondria, leading to a reduction in the membrane potential. Since this organelle is essential to ATP production, its dysfunctionality leads to parasite death [18].
A substantial rise in the intracellular ROS levels on treatment with compound 4f was indeed found at both the tested concentrations using the method of conversion of the non-fluorescent probe (H 2 CFDA) to fluorescent (DCF), Figure 5A.
As shown in Figure 5B, the tetramethylrhodamine ethyl ester (TMRE) cationic probe indicated that compound 4f reduces the potential of the mitochondrial membrane (∆Ψm) compared to the two controls, leading to depolarization. However, no difference was found in the two tested concentrations.
A substantial rise in the intracellular ROS levels on treatment with compound 4f was indeed found at both the tested concentrations using the method of conversion of the nonfluorescent probe (H2CFDA) to fluorescent (DCF), Figure 5A.
As shown in Figure 5B, the tetramethylrhodamine ethyl ester (TMRE) cationic probe indicated that compound 4f reduces the potential of the mitochondrial membrane (ΔΨm) compared to the two controls, leading to depolarization. However, no difference was found in the two tested concentrations.

Morphological and Ultrastructural Changes in Promastigotes
SEM and TEM were used to detect the morphological and ultrastructural changes induced by the treatment with 4f on the promastigote form ( Figures 6 and 7). SEM showed that untreated parasites ( Figure 6A) and those treated with DMSO (0.01%) ( Figure 6B) exhibited normal characteristics, compatible with an elongated body, flagellum proportional to body size, smooth and intact cell surface, and well-preserved morphology. However, parasites treated with 4f at both 3.3 μM ( Figure 6C-F) and 6.6 μM ( Figure 6G-I) for 24 h showed morphological changes, such as a rounded shape and reduction of cell body size, cell surface roughness, and damage in the flagellum region.

Morphological and Ultrastructural Changes in Promastigotes
SEM and TEM were used to detect the morphological and ultrastructural changes induced by the treatment with 4f on the promastigote form ( Figures 6 and 7). SEM showed that untreated parasites ( Figure 6A) and those treated with DMSO (0.01%) ( Figure 6B) exhibited normal characteristics, compatible with an elongated body, flagellum proportional to body size, smooth and intact cell surface, and well-preserved morphology. However, parasites treated with 4f at both 3.3 µM ( Figure 6C-F) and 6.6 µM ( Figure 6G-I) for 24 h showed morphological changes, such as a rounded shape and reduction of cell body size, cell surface roughness, and damage in the flagellum region.

In Silico Study to Predict Pharmacokinetic and Toxicity Parameters ADME Prediction
We also conducted in silico analysis of the physicochemical and pharmacokinetic properties to predict the behavior of compound 4f in living organisms (Table 2).  Our results are similar to those from other studies on amastigote forms of L. braziliensis [19], L. amazonensis, L. braziliensis, and L. peruviana [20], L. donovani [21], confirming that chalcones, in general, possess good antileishmanial activity.

In Silico Study to Predict Pharmacokinetic and Toxicity Parameters ADME Prediction
We also conducted in silico analysis of the physicochemical and pharmacokinetic properties to predict the behavior of compound 4f in living organisms ( Table 2).
The analysis of physicochemical properties indicates that derivative 4f does not violate Lipinski's rule and thus demonstrates that 4f could have good bioavailability after being administered orally [22] and meets the parameters proposed by Veber, indicating good permeability through membranes [23]. Furthermore, the value of Log S [24] showed that compound 4f is moderately soluble in water. macrophages were treated for 24 h with 10, 25, 50, and 130 μM, and (A) the percentage of infected macrophages and (B) the number of amastigotes per macrophage were evaluated. Control (infected macrophage), vehicle (DMSO 0.1%) and AmB (1 μM) were used as positive controls. The values represent the mean ± SEM of three independent experiments performed in triplicate. * Significant difference compared to control (p ≤ 0.05), **** (p ≤ 0.0001).

In Silico Study to Predict Pharmacokinetic and Toxicity Parameters ADME Prediction
We also conducted in silico analysis of the physicochemical and pharmacokinetic properties to predict the behavior of compound 4f in living organisms (Table 2). Regarding the pharmacokinetic properties, our study has shown that compound 4f has high gastrointestinal absorption and cannot permeate the blood-brain barrier (BBB), whose function is in the maintenance of homeostasis in the central nervous system [25]. P-glycoprotein (P-gp) is an efflux transporter that functions as a pump, sending xenobiotics out of the cell, playing a significant role in the pharmacokinetic and pharmacodynamic properties of drugs, such as reducing bioavailability [26]. This compound does not act as a substrate for P-gp. Therefore, no significant changes in absorption, distribution, and elimination of compound 4f are expected.
Cytochrome P450 (CYP) enzymes are essential in metabolizing various xenobiotics, including drugs [27]. Herein, we used a forecast model with five CYP450 isoforms of CYP1, CYP2, and CYP3 families (CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4). Compound 4f showed the ability to inhibit three isoforms, CYP2C19, CYP2C9, and CYP3A4. CYP2C19 is primarily responsible for the metabolism of drugs such as proton pump inhibitors and antidepressants [28], so it is unlikely that important drug interactions will occur in patients with leishmaniasis. However, CYP3A4 is the most abundant hepatic and intestinal phase I enzyme, responsible for the metabolism of more than 50% of drugs [29], and CYP2C9 is responsible for approximately 15% of drug metabolism, including nonsteroidal anti-inflammatory [30], used in the treatment of cutaneous leishmaniasis. The inhibition of these isoforms can decrease the efficacy and increase the side effects and toxicity of co-administered drugs, indicating the need for further studies to verify the interaction with drugs metabolized by these CYP's enzymes.
In addition, we also verified if the compound 4f could act as a possible substrate for CYPs. This prediction is useful because if a substance behaves as a substrate of CYPs, it can lead to a low oral bioavailability caused by a pre-systemic metabolism. Our predictions showed that compound 4f could be a substrate of CYP1A2, CYP2C9, CYP2D6, and CYP3A4 isoforms. In addition, through analyzing sites of metabolism (SoMs), we predicted the positions of metabolically labile atoms in the substrate, where metabolic reac-tions can be initiated. The study showed that the carbons of the methoxy groups, at 4 and 8 positions of the naphthalene ring, are susceptible to the action of CYPs since these enzymes are known to perform hydroxylation of α-carbons to heteroatoms that will result in O-demethylation [31].

Molecular Docking
ARG [32] and TR [33] are two important molecular targets of L. amazonensis since they play a pivotal role in the growth and survival of the parasite. Therefore, we decided to investigate the ligand-protein interactions between chalcone 4f and these targets through a molecular docking study.
Owing to the lack of experimental 3D structures of ARG and TR of L. amazonensis, our 3D models were built by homologic modeling. Therefore, the models were built with consideration of the structures of the ARG in L. mexicana (95.4% identity) and TR in C. fasciculata (76.8% identity).
Consensus scoring is a useful validation method as it improves the scoring's fitting performance and improves the prediction of bound conformations and poses [34]. We applied it in our study to predict and visualize the most favorable interactions between 4f and active sites, considering the consensus between at least two scoring functions (RMSD < 2.0). The analysis of the consensus generated from the best pose obtained by different scoring functions used in this study presented an RMSD of 0.7463 between the Lamarckian genetic algorithm and genetic algorithm for the ARG enzyme and an RMSD of 1.762 between the same functions for the TR enzyme.
ARG is a homotrimer ( Figure 9A), with each subunit containing a binuclear Mn (II) center critical for catalytic activity. In our docking study, only the active site of chain A was considered to indicate protein-ligand interactions. The docking analysis for the monomeric model of ARG indicated that compound 4f is hydrogen bonded to the residues Asn143, His139, and His114. These last two are pivotal since His139 is conserved in the ARG family and is involved in coordination with the binuclear manganese cluster in the active site [35], and His114 directly participates in the catalytic mechanism of this enzyme [36]. The hydrogen bonds between 4f and the protein contribute significantly to the stability of the protein-ligand complex. Furthermore, our studies also demonstrated that the nitro group is involved in coordination with the Mn (II) centers ( Figure 9B). This is clearly of importance not only to the complex stability, but also to the inhibition of the enzymatic activity involving the Mn (II) centers [37]. It is also important to note that compound 4f exhibited hydrophobic interactions with residues Asp141, Ser150, His154, Gly155, and Glu197 ( Figure 9C).
The active TR structure is homodimeric ( Figure 10A), with each monomer composed of three domains-a domain that contains an active binding site for T(S) 2 and allosteric binding domains for FAD and NADPH binding [38]. We defined the docking site only as the substrate-binding site, not including the allosteric regions for our molecular docking study. Since FAD is strongly linked to TR, this site would not be available for inhibitors in vitro [39].
TR's active site is a large crevice located at the interface of chain A and chain B. In this interface, there is a channel that connects the two active sites [40]. Given this structural characteristic and analyzing the complex between compound 4f and the TR enzyme, we can observe that the nitro group is partially inserted in this channel, forming two hydrogen bonds with residues Lys530 and Thr534. On the other hand, the methoxy group of 4,8-dimethoxynaphthalenyl lies outside the channel and interacts with the side chain of Thr390 ( Figure 10B). In addition to the hydrogen bond interactions, the compound also interacts hydrophobically with residues Leu392, Met393, Phe389, Leu531, and Ile575 ( Figure 10C). These interactions observed in the protein-ligand complex may be related to enzyme inhibition, leading to the observed increase in ROS levels since TR plays a unique role in trypanothione-based redox metabolism and oxidative defense. Antibiotics 2022, 11, x FOR PEER REVIEW 13 of 24  The binding energy calculated in the docking study for the 4f-ARG enzyme interaction (−11.94 kcal/mol) indicates greater stability than that for the 4f-TR enzyme (−7.61 kcal/mol), i.e., these results suggest that 4f interacts more efficiently with critical residues at the active site of ARG, suggesting that derivate 4f may be considered a possible pharmacophoric group to inhibit this target. Consequently, these results suggest that this compound should be indicated for further antileishmanial studies. The binding energy calculated in the docking study for the 4f-ARG enzyme interaction (−11.94 kcal/mol) indicates greater stability than that for the 4f-TR enzyme (−7.61 kcal/mol), i.e., these results suggest that 4f interacts more efficiently with critical residues at the active site of ARG, suggesting that derivate 4f may be considered a possible pharmacophoric group to inhibit this target. Consequently, these results suggest that this compound should be indicated for further antileishmanial studies.

General Information
Solvents and reagents were purified following the literature procedures [41]. Melting points were determined on a Fisatom 431 hot plate apparatus and were uncorrected. HRMS spectra of the title compounds in methanol (MS degree) solutions (1 mg/mL) with acetic acid 0.1% were obtained on a high-resolution quadrupole-TOF electrospray mass spectroscopy (Bruker, model Impact HD). Infrared spectra (400-4000 cm −1 ) were recorded as KBr discs on a Nicolet 6700. NMR spectra were obtained on a Bruker spectrometer Model Avance III operating at 500 MHz for 1 H and 125 MHz for 13 C or a Bruker spectrometer Model AVHD operating at 400 MHz for 1 H and 100 MHz for 13 C. NMR resonances were registered using CDCl 3 (Merck, Darmstadt, Germany) as solvent and TMS as the internal standard in CDCl 3 . Chemical shifts (δ in ppm) were referenced to the TMS signal in CDCl 3 at δ 0.00. The splitting of proton resonances in the reported 1 H NMR spectra is defined as singlet (s), doublet (d), triplet (t), quadruplet (qua), quintuplet (qui), and complex pattern (m). Coupling constants (J) are reported in Hz (Hertz). Iodomethane, potassium carbonate, acetic anhydride, boron trifluoride diethyl etherate and the aromatic aldehydes (3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 3, 4-dichlorobenzaldehyde, 2, 6-dichlorobenzaldehyde, 2, 4-dichlorobenzaldehyde, 3-nitrobenzaldehyde, 4-nitrobenzaldehyde, 4-bromobenzaldehyde) were purchased from Sigma Aldrich (São Paulo, Brazil). Analytical thin-layer chromatography was carried out with E. Merck silica gel 60 F254 coated 0.25 plates and visualized with a long-and short-wavelength UV lamp. Flash column chromatography was conducted over Merck silica gel 60 (0.040-0.063 mm). Ethyl acetate and n-hexane were used as eluent.

Synthesis of 1,5-Dimethoxynaphthalene (2)
A glass pressure vessel was charged with 1,5-dihydroxynaphtalene (1) (5.05 g, 31.5 mmol), K 2 CO 3 (4.76 g, 34.3 mmol), iodomethane (8.94 g, 63.0 mmol) and acetonitrile (120 mL). The reaction vessel was sealed with a PTFE bushing, the solution was heated to 80 • C with magnetic stirring for 24 h, cooled to room temperature, acetonitrile was removed under a vacuum, and then water (200 mL) was added. The aqueous phase was extracted with chloroform (3 × 50 mL). The organic phase was dried over sodium sulfate and concentrated under a vacuum. Product (2) was obtained in an 86% yield and used without further purification.

Evaluation of Cytotoxicity on Murine Macrophages
The cytotoxic effect of compounds 4a-i on murine macrophage cell lines J774A.1 (TIB-67, ATCC, Manassas, VA, USA) was assessed using the MTT assay (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, St. Louis, MO, USA), [14]. Macrophages were seeded in 96-well plates at a density of 10 4 cells/well and were cultured with 4a-i at different concentrations (10,25,50, and 130 µM) for 24 h (37 • C, 5% CO 2 ). The cells were washed, MTT (5 mg/mL) added, and the cells were incubated for 4 h. The MTT product was diluted with 100 µL of DMSO (Sigma-Aldrich, St. Louis, MO, USA) and analyzed at 550 nm in a spectrophotometer (Thermo Fisher Scientific, Multiskan GO, Waltham, MA, USA) at 550 nm. 0.5% H 2 O 2 (Merck, Darmstadt, Germany) was used as a positive control. The results were expressed as the percentage of viable cells compared to the control group. The cytotoxic concentration for 50% of the cells (CC 50 ) was calculated by nonlinear regression to the dose-response curve using GraphPad Prism statistical software (GraphPad Software, Inc., San Diego, CA, USA, 500.288).

Selectivity Index (SI)
The half-maximal inhibitory concentration for 50% of parasites (IC 50 ) was determined on L. amazonensis promastigotes treated with the nine compounds, and the cytotoxic concentration that causes the death of 50% of cells (CC 50 ) on murine macrophages (J774A.1). IC 50 and CC 50 were calculated by nonlinear regression using GraphPad Prism statistical software (GraphPad Software, Inc., USA, 500.288). The tested compounds' selectivity index was expressed as SI = CC 50 on peritoneal macrophages/IC 50 on promastigotes forms.

Statistical Analysis
Data were expressed as mean ± standard error of the mean (SEM). Three independent experiments were performed, each with duplicate datasets. Data were analyzed using the GraphPad Prism statistical software (GraphPad Software, Inc., USA, 500.288). Significant differences between the groups were determined by a t-test and one-way ANOVA, followed by Tukey's test for multiple comparisons. Differences were considered statistically significant when p ≤ 0.05.

Proteins and Ligands Preparation for Docking
The structural models of ARG and TR from L. amazonensis were built by homology modeling with the aid of the SwissModel server (https://swissmodel.expasy.org/, accessed on 1 September 2022) [52]. The protein sequence of LaARG and LaTR was obtained from the UniProt database (accession number: O96394 and Q0GU43, respectively) [53]. The 3D structures used as a template were ARG of L. mexicana [32] (PDB: 4ITY, resolution: 1.80 Å) and TR from Crithidia fasciculata [33] (PDB ID: 1TYP, Resolution: 2.80 Å), obtained from the Protein Data Bank (PDB). These models were validated according to Camargo and co-workers [54].

Consensus Molecular Docking
Molecular docking was performed using AutoDock v. 4.2 [57] and AutoDock Vina [58]. The protein and ligand preparations were performed using AutoDockTools (ADT) v.1.5.6 [59]. All water molecules were removed, polar hydrogens atoms were added, and the Kollman and Gasteiger methods, respectively, assigned atoms with charges to protein and ligand. The center of the grid box from ARG for AutoDock was centered between Manganese ions at x: 15.141, y: −15.1248, z: −5.40, size 40 × 50 × 38 Å 3 points, and spacing of 0.375 Å, for the AutoDock Vina the same coordinates were set, with a size of 20 × 23 × 19 Å 3 . For TR, the grid box for AutoDock was centered on the active binding site for T(S) 2 at x: 71.856, y: 10.434, z: 12.967, size 60 × 60 × 60 Å 3 points, and spacing of 0.375 Å, for AutoDock Vina, the size was set to 24 × 24 × 24 Å 3 .
Molecular docking calculations were carried out considering the hybrid scoring function, implemented in Vina, and the genetic (GA) and Lamarckian genetic algorithms (LGA) [57], implemented in Autodock. The ligands performed 10 iterative runs and the best-scored pose for which docking resulted was considered the root-mean-squaredeviation (RMSD) calculation.

Conclusions
Nine 4,8-dimethoxynaphthalenyl chalcone derivatives (4a-i) were obtained from 15-85%. Among them, compounds 4b, 4d, 4e, 4f, 4g, and 4i had not been previously reported. Compound 4f exhibited the best in vitro activity against promastigote forms of L. amazonensis. Regarding its mechanism of action, in vitro assays indicated that 4f produced several morphological and ultrastructural cellular changes, increased intracellular ROS levels, and induced mitochondrial depolarization with selective cytotoxicity at non-toxic concentrations to mammalian cells. In addition, 4f caused a reduction in infected macrophages and the number of amastigotes per cell, culminating in eliminating intracellular parasites. In silico studies of physicochemical and pharmacokinetic properties indicated that 4f could present good oral bioavailability and intestinal absorption. The molecular docking study indicated that 4f showed a greater affinity for ARG than TR. These findings are key to revealing promising molecules for new antileishmanial therapies based on ARG inhibition, directing us to further drug design studies aimed at optimization by structural modifications to provide a more active analog with better pharmacokinetic properties.