New Amino Naphthoquinone Derivatives as Anti-Trypanosoma cruzi Agents Targeting Trypanothione Reductase

To develop novel chemotherapeutic alternatives for the treatment of Chagas disease, in this study, a set of new amino naphthoquinone derivatives were synthesised and evaluated in vitro on the epimastigote and trypomastigote forms of Trypanosoma cruzi strains (NINOA and INC-5) and on J774 murine macrophages. The design of the new naphthoquinone derivatives considered the incorporation of nitrogenous fragments with different substitution patterns present in compounds with activity on T. cruzi, and, thus, 19 compounds were synthesised in a simple manner. Compounds 2e and 7j showed the lowest IC50 values (0.43 µM against both strains for 2e and 0.19 µM and 0.92 µM for 7j). Likewise, 7j was more potent than the reference drug, benznidazole, and was more selective on epimastigotes. To postulate a possible mechanism of action, molecular docking studies were performed on T. cruzi trypanothione reductase (TcTR), specifically at a site in the dimer interface, which is a binding site for this type of naphthoquinone. Interestingly, 7j was one of the compounds that showed the best interaction profile on the enzyme; therefore, 7j was evaluated on TR, which behaved as a non-competitive inhibitor. Finally, 7j was predicted to have a good pharmacokinetic profile for oral administration. Thus, the naphthoquinone nucleus should be considered in the search for new trypanocidal agents based on our hit 7j.


Introduction
American trypanosomiasis (also known as Chagas disease) is one of the most important neglected diseases affecting Latin America and is caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), which is transmitted to humans by the bite of a triatomine bug [1,2]. Owing to migratory processes, the disease can be found in non-endemic places such as the United States and Europe, affecting approximately 11 million people worldwide [3]. However, there are only two approved drugs used to treat the infection, benznidazole (Bzn) and nifurtimox, both of which are frequently associated with unpleasant side effects and low effectiveness [4,5]. Thus, there is a need to find new compounds with trypanocidal activity which are safer and more effective than the canonical drugs [6,7].
Compounds derived from natural or synthetic naphthoquinones have been considered as privileged scaffolds for obtaining novel agents with antiparasitic activity [8][9][10]. Our research group previously reported the synthesis and trypanocidal evaluation of a series of 2-anilino-1,4-naphthoquinones, highlighting compound I (Figure 1), which exhibited potent activity against epimastigotes of the Tulahuén strain (IC 50 = 1.72 µM) and high selectivity against Vero cells [11]. Subsequently, we reported the synthesis and evaluation of a series of aryloxy-quinones against the Y strain of T. cruzi epimastigote, where 2-(3-nitrophenoxy)-1,4-naphthoquinone II (Figure 1) displayed remarkable nanomolar inhibitory activity (IC 50 = 20 nM) and a high selectivity index (SI = 625) compared with J774 murine macrophages cells [12]. To evaluate our quinone derivatives on other T. cruzi strains as well as trypomastigote forms, we recently designed and synthetised a small series of aryloxy-naphthoquinones. Our best results were for compound III, which showed high potency on the NINOA strain of T. cruzi trypomastigotes (IC 50 = 9.38 µM), low toxicity on J774 cells (IC 50 = 117 µM), and was more potent and selective than Bzn (40.67 and 352 µM, respectively) [13].
Pharmaceutics 2022, 14, x FOR PEER REVIEW 3 of 21 a pivotal role in the redox metabolism of the parasite. This possible target has been previously studied by our group [21,22]. Finally, for the most promising trypanocidal quinone, we conducted an inhibition kinetics evaluation in terms of TcTR.

General
The reagents and chemicals used in this work were obtained from Sigma Aldrich (San Louis, MO, USA). Those compounds that had already been published previously were synthesised according to the respective procedures, and this was indicated with the relevant references. The purity of all synthesised compounds was checked by NMR and TLC. In the case of TLC, silica gel 60 F254 25 aluminium foils 20 × 20 C (purchased from Merck, Burlington, VT, USA) were used as a stationary phase and a mobile phase composed of petroleum ether and ethyl acetate in a ratio of 75: 25. In the NMR spectra, the chemical shifts of each signal were reported in parts per million (ppm) and the coupling constants (J) in hertz (Hz). Also, for the multiplicity of the NMR signals, they are expressed as s (singlet), d (doublet), t (triplet), dd (doublet doublet), and bs (broad singlet), as appropriate.

Instrumentation
The following instruments were used for the identification and characterisation of It is well-known that some antiparasitic quinones also have potent antitumoral activity and share the same biological targets, likely due to their mechanisms of action [11,14]. One example of this behaviour was observed for compound IV, whose design was inspired by the antimalarial agent atovaquone and showed cytotoxic effects on several cancer cell lines and induced apoptosis in these cells [15].
In the search for new chemical entities to obtain anti-T. cruzi agents, the use of nitrogenous scaffolds has been well documented [16]. For example, Braga et al. [17], synthesised derivatives of quinolines, indoles, and pyrimidines substituted with aminoalkylmorpholines, which were shown to be active against this parasite. Compound V (Figure 1) exhibited an IC 50 = 67.7 µM in trypomastigote cultures (Y strain). Another example of the use of nitrogenous heterocycles was presented by Araujo et al. [18], where the Pharmaceutics 2022, 14, 1121 3 of 20 use of aryl-piperazines linked to the imidazole ring resulted in derivatives with activity on T. cruzi and T. brucei; derivative VI ( Figure 1), in particular, showed high potency on T. cruzi trypomastigotes (IC 50 = 2.2 µM), with a low toxicity on MCR-5 human lung fibroblasts. The use of arylpiperazine isosteres linked to different scaffolds has also provided compounds with activity on T. cruzi. Interesting examples include the 1-benzyl-1,4-diazepane fragment (VII, Figure 1), which exhibited powerful activity on epimastigotes, tripomastigotes and amastigotes [19], and the 1-benzylpiperidin-4-amine core (VIII, Figure 1), which showed activity against the intracellular amastigotes of T. cruzi (Tulahuén strain) [20].
Considering that the aforementioned compounds have provided chemical fragments to design novel anti-Chagasic agents, in this work, we synthesised and evaluated two series of hybrid molecules related to amino naphthoquinones against epimastigotes and trypomastigotes of two Mexican strains of T. cruzi (Figure 1). The design of Series I was based on some modifications of the naphthoquinone scaffold: (i) the incorporation of a nitrogen fragment at the C-2 or C-3 position, such as those present in compounds V, VI, and VIII, and (ii) the substitution of the hydrogen atom at the C-2 position by a halogen atom (chlorine or bromine), thanks to our previous observation that this modification enhanced selectivity against T. cruzi compared with mammalian cells [11,12]. For Series II, we decided to replace the nitrogenated fragment considered in Series I by a N-(piperidin-4yl) benzamide moiety, with different substituents in the benzene core and the maintaining of the chlorine at the C-2 position. Subsequently, the biological effects on INC-5 and NINOA strains of all of the synthetised quinones were evaluated. In addition, to determine a possible mechanism of action for these quinone derivatives, molecular docking studies were performed on T. cruzi trypanothione reductase (TcTR), as this protein plays a pivotal role in the redox metabolism of the parasite. This possible target has been previously studied by our group [21,22]. Finally, for the most promising trypanocidal quinone, we conducted an inhibition kinetics evaluation in terms of TcTR.

General
The reagents and chemicals used in this work were obtained from Sigma Aldrich (San Louis, MO, USA). Those compounds that had already been published previously were synthesised according to the respective procedures, and this was indicated with the relevant references. The purity of all synthesised compounds was checked by NMR and TLC. In the case of TLC, silica gel 60 F 254 25 aluminium foils 20 × 20 C (purchased from Merck, Burlington, VT, USA) were used as a stationary phase and a mobile phase composed of petroleum ether and ethyl acetate in a ratio of 75:25. In the NMR spectra, the chemical shifts of each signal were reported in parts per million (ppm) and the coupling constants (J) in hertz (Hz). Also, for the multiplicity of the NMR signals, they are expressed as s (singlet), d (doublet), t (triplet), dd (doublet doublet), and bs (broad singlet), as appropriate.

Instrumentation
The following instruments were used for the identification and characterisation of each of the synthesised compounds: (1) Kofler Thermogerate apparatus (Reichert, Werke A.G., Wien, Austria) for the determination of the melting points (m.p.), which are expressed in degrees Celsius ( • C) without corrections; (2) BRUKER AVANCE III HD-400 spectrometers (400 MHz ( 1 H) and 100 MHz ( 13 C)) or 200 MHz )200 MHz ( 1 H) and 50 MHz ( 13 C)) to obtain the 1 H and 13 C NMR spectra (tetramethylsilane, TMS, was used as an internal reference); (3) BRUKER COMPACT QTOF MS + Elute UHPLC with a constant nebuliser temperature of 250 • C, for the acquisition of HRMS-ESI data. For this, samples dissolved in acetonitrile were injected directly into the ESI source through an injection valve and by means of a syringe pump with a flow rate of 5 µL min −1 . Measurements were carried out in the positive ion mode, with a scanning range of m/z 300.00-1510.40 and a resolution of 140,000. In a reaction flask, the suitable amine, halo-1,4-naphthoquinone (1.0 mmol), and DIEA (2.0 mmol) were suspended in acetonitrile (10 mL). The mixture was stirred for 4 h at room temperature. Then, the solvent was removed under a vacuum, and the crude product was purified by column chromatography on silica gel, using methylene chloride/acetone (8:1) as a mobile phase. In a reaction flask, the respective acyl chlorides 4a-j (1.5 mmol), tert-butyl-4-aminopiperidine-1-carboxylate (1.0 mmol), and triethylamine (3.0 mmol) were added using anhydrous THF as a solvent (10 mL). The reaction mixture was stirred at room temperature for 3 h. The solvent was then removed under reduced pressure and the reaction crude was purified in a silica gel chromatographic column using methylene chloride as a mobile phase [25].

General Procedure for the Synthesis for Compounds 6a-j
Derivatives 5a-j (1.0 mmol) dissolved in methylene chloride were added to a reaction flask. Then, trifluoroacetic acid (3.0 mmol) was added and the reaction mixture was allowed to stir for two hours at room temperature. Complete conversion to the respective free amine was confirmed by TLC. Subsequently, the solvent was removed under reduced pressure and the crude obtained without purification was used for the subsequent step.

General Procedure for the Synthesis for Compounds 7a-j
2,3-Dichloro-1,4-naphthoquinone (1.0 mmol) and DIEA (3.0 mmol) dissolved in acetonitrile were added to the crude obtained in the previous synthetic step (6a-j). The mixture was stirred for 4 h at room temperature. Once the reaction time was complete, the solvent was evaporated, and the crude obtained was purified by a silica gel column using a 6:1 chloroform/acetone mixture as a mobile phase.     18 (d, J = 11.8 Hz, 2H), 1.78 (ddd, J = 23.9, 11.9, 3.9 Hz, 2H). 13 13 13  Epimastigotes from T. cruzi strains NINOA and INC-5 were cultured in BHI (brain heart infusion broth, 89%) axenic medium, as reported in [26], at an initial concentration of 1 × 10 6 parasites/mL, at 28 • C, supplemented with 10% foetal bovine serum and 1% sodium penicillin-streptomycin. Epimastigote growth was supervised by measuring the absorbance at 630 nm of the culture in an ELISA Epoch reader (Epoch 2 Microplate Spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA) daily. The assayed drugs were initially dissolved in DMSO at a concentration of 25 mM. This stock solution was then diluted with culture medium, ensuring that the final DMSO concentration did not exceed 0.4%. The control contained only 0.4% DMSO in the culture medium. Bzn was used as the reference compound, and epimastigote growth as the control. The concentrations of Bzn and each series of corresponding drug were monitored daily for five days. On the last day, the absorbance of the cultures was measured and related to the control. All measurements were carried out in triplicate. No toxic effect was attributable to DMSO. Since parasite growth is exponential, the data were analysed according to a first order equation, and the respective growth constant (k), corresponding to the slope, was calculated for controls and parasites treated with different drug concentrations. From these measurements, percentage growth inhibition (%GI) and half-maximal inhibitory concentration (IC 50 ) values were calculated.

Ex Vivo Evaluation of INC-5 and NINOA Strain Trypomastigotes
Those compounds with the lowest IC 50 values in the epimastigote studies of each strain were evaluated in trypomastigotes. For this purpose, bloodstream trypomastigotes of INC-5 and NINOA strains were used for peritoneal injection into 6−8-week-old CD1 mice. The peak of parasitemia was achieved after 4-6 weeks, at which time parasitised blood samples were obtained by cardiac puncture using sodium heparin as an anticoagulant and diluted to a concentration of 1 × 10 6 trypomastigotes/mL. 10 µL of each of the dilutions of the study compounds and Bzn, with these being placed in a 96-well plate, and 90 µL of infected blood was added, providing a final volume of 100 µL per well. The microplates were incubated at 4 • C for 24 h, and concentrations of 20 µM, 10 µM, and 5 µM of each compound were considered in these experiments. Each evaluation was performed in triplicate. Wells with untreated blood trypomastigotes were used as a negative lysis control and Bzn as a positive control. Then, the degree of lysis of trypomastigotes was determined by their reduction in number using an optical microscope according to the Brener-Pizzi method. For this purpose, 5 µL of blood was placed between a slide and a 13 × 13 coverslip. Mobile trypomastigotes were counted in 15 microscope fields at 40× magnification. Quantification of the degree of lysis for each case was expressed as a percentage considering the number of viable trypomastigotes with respect to the negative control [27]. The half-maximal lytic concentration (LC 50 ) was determined by linear regression. The experiments were carried out in accordance with the recommendations and approval of the local Ethics and Research Committee (Approval number: ENCB/CEI/078/2020).

Cytotoxicity Assays
Cytotoxicity assays for the compounds selected for their trypanocidal activity were performed on J774 macrophage cells using the MTT method [28]. For this purpose, 50,000 cells/well resuspended in RPMI medium and 2% foetal bovine serum were seeded in a 96-well plate and incubated for 24 h at 37 • C. From the stock solutions of each of the compounds and Bzn, dilutions were made sequentially with PBS so that the final concentration in the well of each of these did not exceed 1% DMSO. After 24 h of incubation, the existing culture medium was replaced in the plate with fresh culture medium along with 5 mL each of the compound dilutions (100 to 1.25 µM) and Bzn to a final volume of 100 µL per well. This plate was then incubated in 5% CO 2 for 24 h at 37 • C. The controls used were cells with medium as a positive control and cells without culture medium as a negative control. After the 24 h incubation, the morphology of the macrophages was observed under a microscope and then the culture medium was removed again and the MTT solution (5 mg/mL) was added. After 1 h incubation at 37 • C, the absorbance at 570 nm was recorded on an Elisa Epoch reader. The following formula was used to calculate the % cytotoxicity: %cytotoxicity = (100 − (mean number of cells with treatment/cells without treatment) × 100).

Ligand Preparation
Synthesised compounds 2a-i and 7a-j were initially drawn in the open access software Marvin Sketch 18.10 and were then saved in 2D.mol2 files. These 2D files were further-on energy minimised and converted to 3D.mol2 format using the open access software Open Babel 2.4.1. Next, the .mol2 file for each compound was processed using the software ACPYPE/AmberTools 17 to generate .pdb files and to prepare other files required to run molecular dynamics simulations in a later stage of the in silico analysis. Lastly, pdb files were converted to.pdbqt format utilizing MGLTools 1.5.6 executable prepare_ligand4.py. Along with these compounds, mepacrine, a known TR inhibitor, and three lead structures from Vera et al. [21], a previous study with naphthoquinones, were prepared for molecular docking.

Predicted Bindings Site
The Site Finder tool of the MOE software was used to predict the binding sites for the tested compounds. Residues present at the site were considered to locate the docking Grid Box. PyMol software was used to locate the Grid Box centre using the centerofmass function for residues present at the predicted site.

Protein Preparation
Protein crystal was obtained from the Protein Data Bank (PDB) in .pdb format with access code 1BZL. The protein was prepared for molecular docking using open-source software. Initially, the proteins were cleaned using the USCFChimera 1.12 software, and any atom that was co-crystalised in structure, bound ligand, and other ions were removed from structure. The Dock Prep tool was then used for each of the cleaned proteins to add hydrogens and any charges to amino acids to prepare for molecular docking. The prepared crystal was exported in .pdb format for further use.

Molecular Docking
Using the open source software Auto Dock Tools, the receptor structure was converted to .pdbqt format and the ligands were set up to be docked at the Z-site with the centre at X, Y, and Z-coordinates 41.825, 4.351, and −28.343, respectively, with 24 × 24 × 24 Å dimensions in each axis. The AutoDock Vina docking protocol was used with a spacing of 1 Å, generating nine models for each docked compound. All of the compounds were analysed to determine the interactions present in the receptor-ligand complex obtained by molecular docking analysis. The interactions were determined using an online server, the Protein-Ligand Interaction Profiler (PLIP). The interaction profiles for the tested compounds were compared to the interactions observed for the naphthoquinones previously reported by our group: NQ-d, NQ-g, and NQ-h.

Trypanothione Reductase Inhibition Assay
The inhibition of TcTR activity by 7j was evaluated by monitoring the trypanothione disulphide (TS 2 )-dependent NADPH oxidation by using the assay described before [29]. The reaction mixture contained assay buffer (40 mM HEPES pH 7.4, 1 mM EDTA), recombinant TcTR (88-168 ng/mL), NADPH (the concentration depended on experiment), and different 7j concentrations when required, in a volume of 500 µL. The mixture was incubated at 37 • C for 3 min, and TS 2 was added to start the reaction. The absorbance at 340 nm was recorded in real time for 10 min, and the activity was calculated from the slope of the curves as µmol of NADPH oxidised per min (e 340 = 6.22 mM −1 cm −1 ).
Å dimensions in each axis. The AutoDock Vina docking protocol was used with a spacing of 1 Å, generating nine models for each docked compound. All of the compounds were analysed to determine the interactions present in the receptor-ligand complex obtained by molecular docking analysis. The interactions were determined using an online server the Protein-Ligand Interaction Profiler (PLIP). The interaction profiles for the tested com pounds were compared to the interactions observed for the naphthoquinones previously reported by our group: NQ-d, NQ-g, and NQ-h.

Trypanothione Reductase Inhibition Assay
The inhibition of TcTR activity by 7j was evaluated by monitoring the trypanothione disulphide (TS2)-dependent NADPH oxidation by using the assay described before [29] The reaction mixture contained assay buffer (40 mM HEPES pH 7.4, 1 mM EDTA), recom binant TcTR (88-168 ng/mL), NADPH (the concentration depended on experiment), and different 7j concentrations when required, in a volume of 500 μL. The mixture was incu bated at 37 °C for 3 min, and TS2 was added to start the reaction. The absorbance at 340 nm was recorded in real time for 10 min, and the activity was calculated from the slope o the curves as μmol of NADPH oxidised per min (e 340 = 6.22 mM −1 cm −1 ).

Chemistry
The synthesis of derivatives from Series I (2a-i) was conducted according to a previ ously reported procedure [12,27,30], and as shown in Scheme 1, using mono-or dihalo naphthoquinones 1a-c as starting material. The nucleophilic substitution of 1a-c with 2 morpholinoethanamine, 1-cyclopentylpiperazine, or 4-(piperidin-4-yl)morpholine in basic medium, with acetonitrile as a solvent at room temperature, yielded the quinone derivatives 2a-i (18-86%). The derivatives from Series II (7a-j) were obtained through the synthetic pathway shown in Scheme 2. First, N-(piperidin-4-yl)benzamides 6a-j were obtained by a nucleophilic substitution reaction between tert-butyl-4-aminopiperidin-1-carboxilate 3 and the respective acyl chlorides 4a-j in triethylamine (Et 3 N) to yield the piperidine amide derivatives 5a-j [25]. Then, 5a-j were treated with trifluoracetic acid (TFA) and dichloromethane as a solvent at room temperature to yield the amides 6a-j, which, without further purification, reacted with 1b, using acetonitrile as a solvent and N,N-diisopropylethylamine (DIEA) as a base, yielding the target compounds 7a-j. All of the synthetised compounds were purified by column chromatography, and their structures were established based on their spectroscopic properties (IR, MS, 1 H NMR, and 13 C NMR, see the Experimental Section and Supplementary Information).
atives 5a-j [25]. Then, 5a-j were treated with trifluoracetic acid (TFA) and dichloromethane as a solvent at room temperature to yield the amides 6a-j, which, without further purification, reacted with 1b, using acetonitrile as a solvent and N,N-diisopropylethylamine (DIEA) as a base, yielding the target compounds 7a-j. All of the synthetised compounds were purified by column chromatography, and their structures were established based on their spectroscopic properties (IR, MS, 1 H NMR, and 13 C NMR, see the Experimental Section and Supplementary Information).

Trypanocidal Effects on Epimastigote and Trypomastigote Strains
In the trypanocidal evaluation for the synthesised amino naphthoquinone derivatives 2a-i and 7a-j, the growth inhibition (GI) of the epimastigote strains NINOA and INC-5 was evaluated (%GI). In these assays, Bzn was used as the reference drug because it was approved in 2017 by the United States Food and Drugs Administration (FDA) for the treatment of Chagas disease [21,22]. The results showed that all of the compounds had a %GI value greater than 70% in both strains using a concentration of the compounds at 10 μM (Table S1, Supporting Information). Therefore, based on the results, dose-dependent assays were carried out using several concentrations (0.625-5.0 μM) to determine the half-maximal inhibitory concentration (IC50) for these quinones in each strain and for the reference drug.
The IC50 values are shown in Table 1. The results showed that most compounds induced greater inhibitory activity than Bzn in both Mexican epimastigote strains. Compounds 2e and 7j exhibited the lowest IC50 values for the NINOA and INC-5 strains (0.43 µM for both strains for 2e and 0.19 µM and 0.92 µM for 7j). From a chemical viewpoint, a simple structure-activity relationship (SAR) for the compounds of Series I, showed that the most potent compounds in both strains had the cyclopentyl-piperazine fragment, which indicated that this nitrogen ring is an interesting scaffold to consider in new trypanocidal agents. Likewise, regarding the effect of the halogen atom at C-2 on the naphthoquinone core, a chlorine atom and bromine atom increases the parasiticidal activity on the

Trypanocidal Effects on Epimastigote and Trypomastigote Strains
In the trypanocidal evaluation for the synthesised amino naphthoquinone derivatives 2a-i and 7a-j, the growth inhibition (GI) of the epimastigote strains NINOA and INC-5 was evaluated (%GI). In these assays, Bzn was used as the reference drug because it was approved in 2017 by the United States Food and Drugs Administration (FDA) for the treatment of Chagas disease [21,22]. The results showed that all of the compounds had a %GI value greater than 70% in both strains using a concentration of the compounds at 10 µM (Table S1, Supporting Information). Therefore, based on the results, dose-dependent assays were carried out using several concentrations (0.625-5.0 µM) to determine the half-maximal inhibitory concentration (IC 50 ) for these quinones in each strain and for the reference drug.
The IC 50 values are shown in Table 1. The results showed that most compounds induced greater inhibitory activity than Bzn in both Mexican epimastigote strains. Compounds 2e and 7j exhibited the lowest IC 50 values for the NINOA and INC-5 strains (0.43 µM for both strains for 2e and 0.19 µM and 0.92 µM for 7j). From a chemical viewpoint, a simple structure-activity relationship (SAR) for the compounds of Series I, showed that the most potent compounds in both strains had the cyclopentyl-piperazine fragment, which indicated that this nitrogen ring is an interesting scaffold to consider in new trypanocidal agents. Likewise, regarding the effect of the halogen atom at C-2 on the naphthoquinone core, a chlorine atom and bromine atom increases the parasiticidal activity on the NINOA strain and INC-5 strains, respectively. This evidence confirms our previous results regarding the importance of a halogen atom in this position [27]. Most of the Series II compounds were more potent than the Series I compounds. In the NINOA strain, compounds 7b, 7c, 7h, and 7j had IC 50 values lower than 1.0 µM (0.77, 0.52, 0.84, and 0.43, respectively). However, for the INC-5 strain, the best results were obtained for compounds 7c, 7h, and 7j, with the latter being the only one that had an IC 50 value lower than 1.0 µM. SAR analysis for N-(piperidin-4-yl)benzamide quinones was not possible because the trypanocidal effect was heterogeneous. However, considering that the most active compound was 7j, it appeared that a bulky group in the amide fragment (a naphthalene ring) was suitable to increase the activity in both strains.
Because these compounds had good activity on epimastigote strains, we decided to conduct our evaluation on the trypomastigote forms. Subsequently, for compounds with an IC 50 < 3.0 µM in the epimastigote strains, their capacity for cell lysis of trypomastigote was evaluated (Table 1). In these assays, compounds 7b, 7c, 7f, 7h, and 7i showed greater potency than Bzn on NINOA, whereas on INC-5, the compound with the highest activity was 7j, with a LC 50 = 59.73 µM. The significance of these results relates to the fact that trypomastigotes are the infective flagellated form of the parasite in the blood and are responsible for the acute and chronic stages of Chagas disease in mammalian hosts [31]. Epimastigotes are used for primary screening because their replication is extracellular and they are easy to handle and maintain in the laboratory [32].

Cytotoxicity in Murine Macrophages
The cytotoxic effect against J774 murine macrophages was evaluated for compounds with the highest activity on trypomastigote. Derivatives 7c, 7f, 7h, and 7j showed moderate cytotoxicity values (IC 50 =~25µM; Table 2). With these data, it was possible to determine the selectivity index (SI) against the epimastigote and trypomastigote strains ( Table 2). The SI values were higher for the epimastigote than trypomastigote form for these quinone derivatives. However, 7j was more selective than Bzn on epimastigotes and was most selective on INC-5 trypomastigote compared with the other compounds in the study. Once the in vitro characterization was conducted, an in silico study was developed on TcTR to explore this putative target in relation to the trypanocidal effect of the compounds.

Molecular Docking Studies
TR is one of the most important enzymes for the survival of parasites because it participates in redox metabolism, catalysing the reduction of trypanothione disulphide (TS 2 ) to trypanothione (TSH) [22,33,34]. Furthermore, it is considered an attractive target for the search of new anti-Chagasic agents since this enzyme is absent in mammals [33,34]. Additionally, previous studies have shown that naphthoquinones are potential TR inhibitors [21]. Thus, an in silico docking study was conducted to assess the possible inhibitory interactions of 1,4-naphthoquinone derivatives (Series I and II) on TcTR. NQ-d, NQ-g, and NQ-h, shown in Figure 2, were considered control compounds for our molecular docking studies because these TcTR inhibitors were previously reported by our research group [21]. Once the in vitro characterization was conducted, an in silico study was developed on TcTR to explore this putative target in relation to the trypanocidal effect of the compounds.

Molecular Docking Studies
TR is one of the most important enzymes for the survival of parasites because it participates in redox metabolism, catalysing the reduction of trypanothione disulphide (TS2) to trypanothione (TSH) [22,33,34]. Furthermore, it is considered an attractive target for the search of new anti-Chagasic agents since this enzyme is absent in mammals [33,34]. Additionally, previous studies have shown that naphthoquinones are potential TR inhibitors [21]. Thus, an in silico docking study was conducted to assess the possible inhibitory interactions of 1,4-naphthoquinone derivatives (Series I and II) on TcTR. NQ-d, NQ-g, and NQ-h, shown in Figure 2, were considered control compounds for our molecular docking studies because these TcTR inhibitors were previously reported by our research group [21]. The synthesised naphthoquinones were docked on a hydrophobic region of TcTR named the "Z-site", which was previously defined by el-Waer et al. [35]. In that work, the Z-site was suggested as a relevant binding pocket for TR inhibitors, and it is located at the innermost part of the dimer interphase and the nicotinamide adenine dinucleotide phosphate-binding site (Figure 3 and Table 3). The highest free energy of binding (FEB) values for the naphthoquinones are shown in Table 4; these scores were used to compare all of the compounds with the scores for NQ-d, NQ-g, and NQ-h. As shown in Table 4, nine The synthesised naphthoquinones were docked on a hydrophobic region of TcTR named the "Z-site", which was previously defined by el-Waer et al. [35]. In that work, the Z-site was suggested as a relevant binding pocket for TR inhibitors, and it is located at the innermost part of the dimer interphase and the nicotinamide adenine dinucleotide phosphate-binding site (Figure 3 and Table 3). The highest free energy of binding (FEB) values for the naphthoquinones are shown in Table 4; these scores were used to compare all of the compounds with the scores for NQ-d, NQ-g, and NQ-h. As shown in Table 4, nine quinone derivatives belonging to Series II (7a-7j, except 7c) had a FEB value with a higher affinity for TcTR than NQ-g. The remaining naphthoquinones from Series I (2a-2i) were predicted to have an FEB value with lower affinity than the control compounds. The difference in binding energies within the compounds from both Series I and II was minimal, which suggests that most of the predicted binding energies could be attributed to the naphthoquinone scaffold. The quinones from Series I with less affinity for TcTR were 2a-c, with a slightly flexible amino alkyl chain at C-3 of the naphthoquinone ring, suggesting that this flexibility was insufficient to attain a favourable position at the binding site. The other quinones of Series I (2d-f and 2g-i) had similar FEB values, both with a bicycle substituent: a piperazinyl-cyclopentyl or piperidyl-morpholinyl ring, with the latter having a slightly better affinity. For the quinones from Series II, a slight tendency was observed when the phenyl ring was substituted at position 4; 7b, 7d, 7h, and 7i had the highest FEB values. A clear difference may be attributed in 7j, as the substituent is a naphthyl group and not a phenyl group. This naphthyl fragment was present in two of the three lead quinones considered to be controls, NQ-g and NQ-h, which further supports the relevance of large aromatic groups favouring interactions with the binding site. quinone derivatives belonging to Series II (7a-7j, except 7c) had a FEB value with a higher affinity for TcTR than NQ-g. The remaining naphthoquinones from Series I (2a-2i) were predicted to have an FEB value with lower affinity than the control compounds. The difference in binding energies within the compounds from both Series I and II was minimal, which suggests that most of the predicted binding energies could be attributed to the naphthoquinone scaffold. The quinones from Series I with less affinity for TcTR were 2ac, with a slightly flexible amino alkyl chain at C-3 of the naphthoquinone ring, suggesting that this flexibility was insufficient to attain a favourable position at the binding site. The other quinones of Series I (2d-f and 2g-i) had similar FEB values, both with a bicycle substituent: a piperazinyl-cyclopentyl or piperidyl-morpholinyl ring, with the latter having a slightly better affinity. For the quinones from Series II, a slight tendency was observed when the phenyl ring was substituted at position 4; 7b, 7d, 7h, and 7i had the highest FEB values. A clear difference may be attributed in 7j, as the substituent is a naphthyl group and not a phenyl group. This naphthyl fragment was present in two of the three lead quinones considered to be controls, NQ-g and NQ-h, which further supports the relevance of large aromatic groups favouring interactions with the binding site.
To include compounds that may have a higher probability of behaving as TcTR inhibitors, all compounds with an equal or higher FEB score than the controls were considered for further analyses to determine their interaction profile.
Considering the highest values of FEB compared with NQ-g, an exhaustive study on the interaction profiles using nine models generated by VINA analysis for Series II quinones was conducted. Full details of the interactions observed between these quinones at the binding site are shown in Table 5. Hydrophobic interactions (HI), hydrogen bonds (HB), π-cation interactions (π-c), π-stacking (π-s), salt bridges (SB), and halogen interactions (HalB) were observed. Full details about the interaction profiles for each quinone are shown in Table S2. Interaction profiles observed for the predicted binding site for NQ-g ( Figure 4) were used to compare with the tested compounds 2a-7j to assess their potential as TcTR inhibitors. As shown in Table S2, there were a few common residue interactions shared between NQ-d, NQ-g, and NQ-h and the tested compounds; of these, the most represented were Gln69 , Gln69, and Asn433.

Evaluation of 7j on TcTR
Considering the results obtained regarding the parasiticidal effect on epimastigotes and trypomastigotes of these naphthoquinone derivatives, in addition to their cytotoxicity on J774 and the docking studies on TR, we conducted kinetic assays on TcTR for com pound 7j. To determine the type of enzyme inhibition, a study was carried out at differen substrate concentrations, and the kinetic parameters of the enzyme were calculated using Lineweaver-Burk plots ( Figure 5).
7j behaved as a simple mixed-type inhibitor with respect to both substrates, TS2 and NADPH, according to the patterns of intersecting lines in the double reciprocal plots (Fig  ure 5c,d). Its most important effect was a decrease in Vmax (Figure 5a,b); the Km values remained highly similar, since the alpha value in the mixed-type inhibitor equation (Equa tion (1)) was close to 1 ( Table 6). The kinetic data suggested that 7j and substrates bind to different sites on the enzyme, which was consistent with the docking results for this naph thoquinone as well as with our previous results for NQ-g [21]. The naphthoquinones that showed the most shared interactions with NQ-g on the predicted TcTR-binding site were 7b, 7d, 7i, 7h, and 7j (all had FEB values > 9.5 kcal/mol), which shared at least five out the eight amino acids involved in these interactions (Phe367, Pro371 , Pro435 , Gln69, and Glu436) (Figure 4).

Evaluation of 7j on TcTR
Considering the results obtained regarding the parasiticidal effect on epimastigotes and trypomastigotes of these naphthoquinone derivatives, in addition to their cytotoxicity on J774 and the docking studies on TR, we conducted kinetic assays on TcTR for compound 7j. To determine the type of enzyme inhibition, a study was carried out at different substrate concentrations, and the kinetic parameters of the enzyme were calculated using Lineweaver-Burk plots ( Figure 5).
7j behaved as a simple mixed-type inhibitor with respect to both substrates, TS 2 and NADPH, according to the patterns of intersecting lines in the double reciprocal plots (Figure 5c,d). Its most important effect was a decrease in Vmax (Figure 5a,b); the Km values remained highly similar, since the alpha value in the mixed-type inhibitor equation (Equation (1)) was close to 1 ( Table 6). The kinetic data suggested that 7j and substrates bind to different sites on the enzyme, which was consistent with the docking results for this naphthoquinone as well as with our previous results for NQ-g [21].   Figure 5 using Equation (1); b Values were estimated by replots of slope and 1/v axis intercept vs. 7j.

Calculated Physicochemical Properties
Using the SwissADME web server (http://www.swissadme.ch/index.php, accessed on 27 January 2022), compound 7j was analysed to predict its drug-likeness properties, such as its pharmacokinetic (absorption, distribution, metabolism, excretion, and ADME) and pharmacodynamic (e.g., toxicological) profiles. This is because these properties are used to guide rational drug design and to optimise a lead compound into a successful preclinical one [36]. ADME and toxicity properties are determined by some important chemical descriptors, such as the polar surface area (PSA) and molecular weight (MW) of the molecules, which are useful for predicting the oral absorption of drugs. Thus, to assess these properties and predict good oral bioavailability, there are two sets of rules, the   Figure 5 using Equation (1); b Values were estimated by replots of slope and 1/v axis intercept vs. 7j.

Calculated Physicochemical Properties
Using the SwissADME web server (http://www.swissadme.ch/index.php, accessed on 27 January 2022), compound 7j was analysed to predict its drug-likeness properties, such as its pharmacokinetic (absorption, distribution, metabolism, excretion, and ADME) and pharmacodynamic (e.g., toxicological) profiles. This is because these properties are used to guide rational drug design and to optimise a lead compound into a successful preclinical one [36]. ADME and toxicity properties are determined by some important chemical descriptors, such as the polar surface area (PSA) and molecular weight (MW) of the molecules, which are useful for predicting the oral absorption of drugs. Thus, to assess these properties and predict good oral bioavailability, there are two sets of rules, the Lipinski rule and the Veber rule, which are used for these purposes [37,38]. The Lipinski rule states that an orally bioavailable molecule must not fail to meet the following four criteria: ≤5 hydrogen bond donors (HBDs); ≤10 hydrogen bond acceptors (HBAs); MW < 500; and log p-value < 5. Additionally, Veber et al. established the role of PSA and the number of rotational bonds as criteria for estimating oral bioavailability. Veber's rule states that for the compound to be orally bioavailable, it must have a PSA ≤ 140 Å, ≤10 rotational bonds (NRB) or ≤ 12 total HBDs, and HBAs and ≤ 10 rotational bonds. As shown in Table 7, compound 7j met the criteria described by Lipinski and Veber; therefore, it is expected to have good oral bioavailability. This is also depicted in the bioavailability radar plot, which showed that 7j falls within the desired range (pink region) of five parameters from the six parameters used for oral absorption prediction: flexibility, lipophilicity, solubility, size, and polarity, while it lies in the undesirable area of saturation (Figure 6), confirming its good oral bioavailability. Lipinski rule and the Veber rule, which are used for these purposes [37,38]. The Lipinski rule states that an orally bioavailable molecule must not fail to meet the following four criteria: ≤5 hydrogen bond donors (HBDs); ≤10 hydrogen bond acceptors (HBAs); MW < 500; and log p-value < 5. Additionally, Veber et al. established the role of PSA and the number of rotational bonds as criteria for estimating oral bioavailability. Veber's rule states that for the compound to be orally bioavailable, it must have a PSA ≤ 140 Å, ≤ 10 rotational bonds (NRB) or ≤ 12 total HBDs, and HBAs and ≤ 10 rotational bonds. As shown in Table 7, compound 7j met the criteria described by Lipinski and Veber; therefore, it is expected to have good oral bioavailability. This is also depicted in the bioavailability radar plot, which showed that 7j falls within the desired range (pink region) of five parameters from the six parameters used for oral absorption prediction: flexibility, lipophilicity, solubility, size, and polarity, while it lies in the undesirable area of saturation ( Figure 6), confirming its good oral bioavailability.  The bioavailability radar plot of 7j. The pink zone represents the range of the values for optimal oral bioavailability property, and the predicted properties were displayed as red lines. The radar was generated by SwissADME (http://www.swissadme.ch/index.php, accessed on 27 January 2022).

Conclusions
In this study, two series of amino naphthoquinones were synthesised and evaluated in vitro and ex vivo against the epimastigote and trypomastigote forms of two Mexican T. cruzi strains. The trypanocidal activity of these compounds against both forms was, in some cases, better than Bzn. The most promising naphthoquinone derivative is compound 7j due to its higher trypanocidal activity on epimastigotes (INC-5 and NINOA strains) and trypomastigotes (NINOA strain) and its higher SI values against epimastigote forms compared to Bzn. Molecular docking studies and experimental kinetics assays showed that 7j binds with and behaved as a mixed-type inhibitor of TcTR, which was consistent with the interaction of 7j in a site that was not a substrate-binding site. Thus, TcTR can be a possible target of 7j in the parasite and may contribute to the compound's trypanocidal effect. On the other hand, predictions regarding ADME properties indicated that 7j should have good oral bioavailability. Therefore, in this work, we have expanded the chemical Figure 6. The bioavailability radar plot of 7j. The pink zone represents the range of the values for optimal oral bioavailability property, and the predicted properties were displayed as red lines. The radar was generated by SwissADME (http://www.swissadme.ch/index.php, accessed on 27 January 2022).

Conclusions
In this study, two series of amino naphthoquinones were synthesised and evaluated in vitro and ex vivo against the epimastigote and trypomastigote forms of two Mexican T. cruzi strains. The trypanocidal activity of these compounds against both forms was, in some cases, better than Bzn. The most promising naphthoquinone derivative is compound 7j due to its higher trypanocidal activity on epimastigotes (INC-5 and NINOA strains) and trypomastigotes (NINOA strain) and its higher SI values against epimastigote forms compared to Bzn. Molecular docking studies and experimental kinetics assays showed that 7j binds with and behaved as a mixed-type inhibitor of TcTR, which was consistent with the interaction of 7j in a site that was not a substrate-binding site. Thus, TcTR can be a possible target of 7j in the parasite and may contribute to the compound's trypanocidal effect. On the other hand, predictions regarding ADME properties indicated that 7j should have good oral bioavailability. Therefore, in this work, we have expanded the chemical library of trypanocidal compounds and validated the usefulness of naphthoquinone scaffolds as well as the incorporation of a nitrogenated fragment as a strategy for the development of anti-Chagasic agents.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pharmaceutics14061121/s1, 1 H, 13 C NMR, and HRMS of selected compounds; Table S1: Effect of amino naphthoquinone derivatives upon culture growth of T. cruzi epimastigote forms expressed as % growth inhibition at 10 µM; Table S2: Protein-ligand interaction profiles for naphthoquinones and controls.