Bioactivity of Novel Pyrazole-Thiazolines Scaffolds against Trypanosoma cruzi: Computational Approaches and 3D Spheroid Model on Drug Discovery for Chagas Disease

Chagas disease, a century-old disease that mainly affects the impoverished population in Latin America, causes high morbidity and mortality in endemic countries. The available drugs, benznidazole (Bz) and nifurtimox, have limited effectiveness and intense side effects. Drug repurposing, and the development of new chemical entities with potent activity against Trypanosoma cruzi, are a potential source of therapeutic options. The present study describes the biological activity of two new series of pyrazole-thiazoline derivatives, based on optimization of a hit system 5-aminopyrazole-imidazoline previously identified, using structure–activity relationship exploration, and computational and phenotype-based strategies. Promising candidates, 2c, 2e, and 2i derivatives, showed good oral bioavailability and ADMET properties, and low cytotoxicity (CC50 > 100 µM) besides potent activity against trypomastigotes (0.4–2.1 µM) compared to Bz (19.6 ± 2.3 µM). Among them, 2c also stands out, with greater potency against intracellular amastigotes (pIC50 = 5.85). The selected pyrazole-thiazoline derivatives showed good permeability and effectiveness in the 3D spheroids system, but did not sustain parasite clearance in a washout assay. The compounds’ mechanism of action is still unknown, since the treatment neither increased reactive oxygen species, nor reduced cysteine protease activity. This new scaffold will be targeted to optimize in order to enhance its biological activity to identify new drug candidates for Chagas disease therapy.


Introduction
Chagas disease (CD) is a potentially fatal infection, caused by the flagellated protozoan Trypanosoma cruzi, that affects about 6-7 million people worldwide, leading to more than 10,000 annual deaths [1]. This pathology is considered a serious public health problem, representing one of the biggest causes of heart failure and sudden death in many endemic countries in Latin America [2].
With the increase in migratory mobility, the disease has been increasingly reported in non-endemic countries, such as Canada, Australia, Spain, Japan, and the United States of America, among others [3]. As a result of areas with active transmission, and the high number of people without diagnosis and treatment, it is estimated that 75 million people are at risk of infection [1].
Although vector-borne transmission, which occurs during the blood meal of triatomine bugs, has been mostly controlled in some endemic countries [4], T. cruzi oral infection, A mixture of 2-aminoethanethiol hydrochloride (2 mmol), potassium carbonate (1 mmol), and methanol (5 mL) was stirred and refluxed for 30 min. Subsequently, the key intermediate, 4(a-l) or 5(a-l) (1 mmol), was added, and the reaction was accompanied by TLC. After 3-6 h, the mixture was poured into cold water. The product was filtered out and washed with cold water. In order to facilitate the assignment of signals in 1 H NMR spectra, the hydrogen atoms of compounds 1(a-l) and 2(a-l) were highlighted (Figures 1 and 2).
Pharmaceutics 2022, 14, x FOR PEER REVIEW 3 of 24 In this study, we demonstrate the trypanocidal activity and computational data of two new series of pyrazole-thiazoline derivatives, 1(a-l) and 2(a-l). A three-dimensional (3D) in vitro model and reversibility assay were also performed to evaluate the permeability and efficacy in microtissue and parasite recrudescence.

Cell Culture
Vero cells, obtained from the Rio de Janeiro Cell Bank (BCRJ code 0245), were dissociated with trypsin-EDTA solution (0.025%), and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 • C in a humidified atmosphere of 5% CO 2 .
Tridimensional Vero cell cultures were obtained as previously described [31]. Briefly, the isolated cells (2 × 10 4 /well) were cultured in 96-well U-bottom plates previously coated with 1% agarose. The spheroids were kept for 5 days at 37 • C in RPMI 1640 medium supplemented with 10% FBS for complete three-dimensional organization. Both 2D and 3D culture models were used for trypanocidal activity assays.

Parasite
The screening assays were performed with T. cruzi clone Dm28c (TcI) genetically modified to express luciferase gene (Dm28c-Luc), kindly provided by Dr. Cristina Henriques [34,35]. Trypomastigotes were isolated from T. cruzi-infected Vero cell monolayers (10:1 parasites/host cell ratio). On the 4th day post-infection (dpi), free trypomastigotes in the culture supernatant were harvested, and the number of parasites/mL was quantified in a Neubauer chamber [36].

Cytotoxicity In Vitro Assay
The compound toxicity was evaluated in Vero cell cultures. Briefly, Vero cells were seeded in 96-well white culture plates at a density of 1.5 × 10 4 cells/well for 24 h at 37 • C and 5% CO 2 . Thereafter, the cell monolayers were washed with phosphate-buffered saline (PBS) and 200 µL of fresh medium with a serial dilution of the pyrazole-thiazoline derivatives and Bz (1.9-500 µM) added. The cell viability was determined by luminescent assay using the CellTiter-Glo Kit (Promega Corporation, Madison, WI, USA) based on the ATP level [31,36]. After 72 h of treatment, 20 µL of CellTiter-Glo solution was added per well, and cultures were kept for 2 min under agitation. The luminescent signal was read on the GloMax reader (Promega Corporation, Madison, WI, USA). The concentration of compounds that reduced 50% of cell viability (CC 50 ) was calculated by linear regression. At least three independent assays were performed in duplicate. All compound stock solutions were prepared at 100 mM in dimethyl sulfoxide (DMSO). The final DMSO concentration for the controls and drug treatment did not exceed 1%.

Antiparasitic Assay
The trypanocidal activity of the pyrazole-thiazoline derivatives was evaluated against trypomastigote and intracellular amastigote forms of T. cruzi (Dm28c-Luc). Tissue-derived trypomastigotes (1 × 10 6 parasites/well) were incubated with pyrazole-thiazoline derivatives and Bz in different concentrations (0.41-100 µM) for 24 h at 37 • C in a humidified atmosphere and 5% CO 2 . The parasite viability was determined by the addition of luciferin (300 µg/mL), followed by luminescence detection on the GloMax microplate reader (Promega Corporation, Madison, WI, USA) [31,36]. The susceptibility of intracellular amastigotes to pyrazole-thiazoline derivatives (series 1 and 2) was evaluated using 24 h T. cruzi-infected Vero cell monolayers (10:1 parasite/host cell ratio). Then, the cultures were washed with PBS to remove non-internalized parasites, and 200 µL of fresh medium with a serial dilution of pyrazole-thiazoline derivatives and Bz (0.41-100 µM) was added for 72 h at 37 • C.
After 72 h of treatment, luciferin (300 µg/mL) was added to the culture, and the viability of the parasites was assessed by reading the luminescent signal on the GloMax reader (Promega Corporation, Madison, WI, USA). Controls were also performed in nontoxic concentrations of DMSO (≤ 1%). The concentration that reduces the number of viable parasites by 50% (IC 50 ) or 90% (IC 90 ) was calculated by linear regression. The selectivity index (SI) was calculated as CC 50 /IC 50 for each parasite form.

Reversibility Assays
Vero cell cultures infected with T. cruzi Dm28c-Luc, as previously described, were incubated for 72 h at 37 • C, with the promising derivatives at concentrations higher than IC 90 [31]. Then, the cultures were washed with PBS, and maintained for another 72 h at 37 • C in a drug-free medium. Trypomastigotes released in the culture supernatant were detected by transferring the culture medium to 96-well plates, followed by luciferin (300 µg/mL) addition. Viable intracellular amastigotes were also accessed by adding luciferin solution to the cell monolayers. The luminescence signal, revealing viable parasites, was read on the GloMax microplate reader (Promega Corporation, Madison, WI, USA).

3D Culture Model
Vero cell spheroids were infected for 24 h with 2 × 10 5 trypomastigotes (Dm28c-Luc). After washing, to remove the non-internalized parasites, the 3D cultures were treated for 72 h at 37 • C, with the promising derivatives in concentrations higher than IC 90 [31]. Parasite load was assessed by the luminescence reaction after adding the luciferase substrate (luciferin 300 µg/mL). In addition, fluorescence microscopy images of untreated and treated T. cruzi-infected spheroids were acquired after fixing the cultures with 4% paraformaldehyde in PBS and 4 ,6-diamidino-2-phenylindole (DAPI) staining, a DNA dye. The samples were observed under the Zeiss Axio Imager M2 fluorescence microscope (Carl Zeiss, Baden-Württemberg, Germany).

In Vitro Combination Therapy
In vitro combination treatment between promising candidates and Bz was performed against intracellular amastigotes (Dm28c-Luc) using a modified fixed-ratio isobologram method [37]. The highest concentrations of the compounds were determined by ensuring that the IC 50 concentrations of the compounds in monotherapy were close to half of a series of eight serial dilutions (1:3). The first concentrations were prepared in the following ratios: 5:0, 5:1, 2:1, 1:2, 1:5, and 0:5 of Bz and promising candidates, respectively. Then, serial dilutions (1:3) of the proportions were made at eight points of dilution. Vero cell cultures infected with T. cruzi Dm28c-Luc were incubated with the compound combinations for 72 h. Two independent experiments in triplicate were performed. In each ratio, the IC 50 of each compound was determined. The FICI (fractional inhibitory concentration index) of each IC 50 was calculated as follows: IC 50 when in combination/IC 50 of compound alone. The FICI sum (ΣFICI) was determined as: FICI drug A + FICI drug B. xΣFICI is the mean of the FICI sums (FICI). The isobologram graph was built plotting the FICI for each proportion of the compounds. The xΣFICI was used to classify the interaction as synergistic xΣFICI ≤ 0.5, no interaction xΣFICI > 0.5-4, and antagonistic for xΣFICI > 4 [38,39].

Measurement of Reactive Oxygen Species (ROS) Levels
The production of intracellular ROS was evaluated in trypomastigotes (7 × 10 6 parasites/well) treated for 1 h, 3 h, and 24 h at 37 • C, with promising derivatives in IC 50 concentration. Then, the parasites were washed in PBS, and incubated for 30 min in the dark with 20 µM of 2 ,7 -dichlorodihydrofluorescein diacetate (H 2 DCFDA). The fluorescence signal, generated by the oxidation of ROS sensitive probes, was measured using 485/535 nm excitation/emission wavelengths with a SpectraMax M2 microplate reader (Molecular Devices, San Jose, CA, USA). The fluorescence intensity of the blank was subtracted from all samples evaluated. Parasites incubated with 0.5% DMSO and 20 µM H 2 O 2 were used as negative and positive controls, respectively [40].

In Silico Analysis
Physicochemical properties of promising candidates were calculated using OSIRIS DataWarrior software version 5.5.0 [41] and FAFDrugs4 [42]. The maps of molecular lipophilicity potential (MLP) were generated using Molinspiration Galaxy 3D Structure Generator v2021.01 beta [43]. DataWarrior was also used for the target search. First, molecules were retrieved within the ChEMBL database, annotated to target the T. cruzi parasite and proteins. Duplicity was removed, and then, scaffolds of pyrazole-thiazoline were queried against the small library of compounds. ADMET parameters (adsorption, distribution, metabolism, elimination, and toxicity) were acquired by inserting compounds' molecular structures in the ADMETsar platform [44].

Proteinase Activity in Solution
Trypomastigote protein extracts (Dm28c-Luc; 10 8 parasites/mL) were obtained as previously described [30]. The cysteine protease activity of the total protein extracts (5 µg) was measured using the reaction buffer (CH 3 COONa 10 mM, pH 5.0 containing 1 mM DTT), in the presence of the fluorogenic peptide substrate (60 µM of 7-Amino-4-Methylcoumarin hydrochloride), CBZ-L-Phenylalanyl-l-Arginine amide (Z -FR-AMC), and serial dilutions of pyrazole-thiazolines derivatives (100-6.25 µM). The samples were incubated for 45 min at room temperature. The variation in arbitrary fluorescence units (AFU), corresponding to the enzymatic cleavage of the fluorogenic substrate, was monitored in a SpectraMax M2e reader (Molecular Devices, Sunnyvale, CA, USA) for 1 h, with excitation values at 370 nm and emission values at 460 nm. Extracts incubated with 0.5% DMSO or with cysteine protease inhibitor E-64 were used as positive and negative controls, respectively. Substrate self-degradation was monitored, and did not produce significant effects. The inhibitory activity was expressed as percentage inhibition [31].

Physicochemical and ADMET Properties
The identification of three derivatives of series 2 (2c, 2e, and 2i) with potential trypanocidal activity, based on the IC 50 < 10 µM and SI > 20 criteria, led us to assess whether the selected candidates had a good oral bioavailability prediction. The in silico analysis showed no violation of Lipinski's rule of five (RO5), with molecular weight (MW) ranging from 308.20 to 298.19; a maximum lipophilicity (cLogP) value of 2.83; no more than 5 and 10 hydrogen bond donors (HBD) and acceptors, respectively; a reduced number of rotatable bonds (RB=2); and a topological polar surface area (TPSA) of 55.48 Å2 (Figure 3). Distinct physicochemical parameters were noticed between promising pyrazole-thiazoline derivatives and Bz (Figure 3). Bz revealed low lipophilicity, and a high number of rotatable bonds (RB = 5), and did not satisfy the drug-likeness criteria. The radar chat showed that all derivatives fit the drug-likeness criteria, except for the saturation values (sp3 carbon fraction <0.25), suggestive of good oral bioavailability (Figure 3). 10 hydrogen bond donors (HBD) and acceptors, respectively; a reduced number of rotatable bonds (RB=2); and a topological polar surface area (TPSA) of 55.48 Å2 (Figure 3). Distinct physicochemical parameters were noticed between promising pyrazole-thiazoline derivatives and Bz (Figure 3). Bz revealed low lipophilicity, and a high number of rotatable bonds (RB=5), and did not satisfy the drug-likeness criteria. The radar chat showed that all derivatives fit the drug-likeness criteria, except for the saturation values (sp3 carbon fraction <0.25), suggestive of good oral bioavailability (Figure 3).  2c, 2e, 2i) and Bz. (A) DataWarrior software was used to calculate and plot in 2D graphs the following physicochemical parameters: rotatable bonds, aqueous solubility (cLogS), hydrogen bond donors (HBD) and acceptors (HBA), lipophilicity (cLogP), molecular weight (MW), topological polar surface area (tPSA), and drug-likeness. (B) Radar plot representation of six physicochemical parameters (lipophilicity, size, polarity, solubility, flexibility, and saturation), calculated using SwissADME. In red, the threshold values to achieve the drug-likeness criteria.
Differences in the molecular lipophilicity potential (MLP) were observed between the pyrazole-thiazoline derivatives. Lipophilic regions coded with blue and violet color were mostly visualized in 2c and 2e, showing a prevalence of domains with a prediction of Differences in the molecular lipophilicity potential (MLP) were observed between the pyrazole-thiazoline derivatives. Lipophilic regions coded with blue and violet color were mostly visualized in 2c and 2e, showing a prevalence of domains with a prediction of hydrophobic interactions which can favor the ligand-target binding ( Figure 4). Interestingly, the polarity and lipophilicity are distinct between selected highly active compounds (2c, 2e, and 2i) and their lower active analogues (1c, 1e, and 1i), suggesting that, similar to cLogP, the distribution of hydrophilic and lipophilic domains in the molecular surface also interferes with the biological activity.
The in silico pharmacokinetic analysis of drug absorption revealed a prediction of diffusion across the blood-brain barrier (BBB), and good Caco-2 cell permeability and human intestinal (HIA) absorption (Supplementary Table S1). Pyrazole-thiazoline derivatives show a high probability to inhibit cytochrome P450 (CYP450) enzymes, such as CYP1A2, CYP2C19, and CYP2D6, with a prediction of low clearance. Most compounds showed a likelihood of hepatoxicity, but only 2e exhibited mutagenicity prediction. None of the compounds are predictive of human ether-à-go-go-related gene (hERG) cardiac potassium channel inhibition. Thus, the favorable prediction of physicochemical and ADMET parameters motivated us to validate the trypanocidal effect using more complex biological systems. hydrophobic interactions which can favor the ligand-target binding (Figure 4). Interestingly, the polarity and lipophilicity are distinct between selected highly active compounds (2c, 2e, and 2i) and their lower active analogues (1c, 1e, and 1i), suggesting that, similar to cLogP, the distribution of hydrophilic and lipophilic domains in the molecular surface also interferes with the biological activity. The in silico pharmacokinetic analysis of drug absorption revealed a prediction of diffusion across the blood-brain barrier (BBB), and good Caco-2 cell permeability and human intestinal (HIA) absorption (Supplementary Table S1). Pyrazole-thiazoline derivatives show a high probability to inhibit cytochrome P450 (CYP450) enzymes, such as CYP1A2, CYP2C19, and CYP2D6, with a prediction of low clearance. Most compounds showed a likelihood of hepatoxicity, but only 2e exhibited mutagenicity prediction. None of the compounds are predictive of human ether-à-go-go-related gene (hERG) cardiac potassium channel inhibition. Thus, the favorable prediction of physicochemical and AD-MET parameters motivated us to validate the trypanocidal effect using more complex biological systems.

Drug Efficacy on 3D Culture Model
The low success rate in the translation from in vitro biological activity into in vivo model efficacy led us to introduce the 3D culture model in our drug screening platform. Then, a 3D spheroids culture, which more accurately reflected human tissue complexity and the microenvironment, was employed to evaluate the efficacy of promising compound candidates. T. cruzi-infected 3D Vero cells were treated for 72 h with 2c, 2e, and 2i, and parasite load was evaluated after luciferin addition. Parasite viability, assessed by luciferase activity, was determined by luminescence signal, represented as an arbitrary luminescence unit (ALU). A high parasite burden was observed in untreated spheroids (ALU = 166,678 ± 22,824), which was significantly reduced after treatment with all promising candidates ( Figure 5). Potent activity was demonstrated by 2c at concentrations of 25 (ALU = 3329 ± 250) and 50 μM (ALU = 1648 ± 343), resulting in approximately 98% inhibition compared to untreated T. cruzi-infected microtissue. Also, 2c showed better efficacy even at lower concentrations (1.5-3 times IC90). Compounds 2e (ALU = 15,110 ±

Drug Efficacy on 3D Culture Model
The low success rate in the translation from in vitro biological activity into in vivo model efficacy led us to introduce the 3D culture model in our drug screening platform. Then, a 3D spheroids culture, which more accurately reflected human tissue complexity and the microenvironment, was employed to evaluate the efficacy of promising compound candidates. T. cruzi-infected 3D Vero cells were treated for 72 h with 2c, 2e, and 2i, and parasite load was evaluated after luciferin addition. Parasite viability, assessed by luciferase activity, was determined by luminescence signal, represented as an arbitrary luminescence unit (ALU). A high parasite burden was observed in untreated spheroids (ALU = 166,678 ± 22,824), which was significantly reduced after treatment with all promising candidates ( Figure 5). Potent activity was demonstrated by 2c at concentrations of 25 (ALU = 3329 ± 250) and 50 µM (ALU = 1648 ± 343), resulting in approximately 98% inhibition compared to untreated T. cruzi-infected microtissue. Also, 2c showed better efficacy even at lower concentrations (1.5-3 times IC 90 ). Compounds 2e (ALU = 15,110 ± 1990) and 2i (ALU = 25,788 ± 2776) also demonstrated effectiveness in reducing parasite viability in T. cruzi-infected microtissue, similarly to Bz-treated spheroids (ALU = 20,024 ± 2796) ( Figure 5).
The ability of promising compounds to eliminate intracellular parasites was also analyzed by fluorescence microscopy. DAPI staining images revealed a high parasite load in infected and untreated microtissues ( Figure 6). A large number of intracellular amastigotes was clearly visualized throughout the untreated spheroid, even in the innermost layers. In contrast, Bz-treated microtissues demonstrated a drastic reduction in the infection profile, with few intracellular parasites visualized in the spheroids ( Figure 6). Treatment with 2c, 2e, and 2i also impacted the parasite clearance. Few parasites were visualized in the spheroid treated with 2c, indicative of good permeability and activity of the compound in the microtissue. All pyrazole-thiazoline derivatives markedly reduced the parasite load in the microtissue (Figure 6), as revealed by arbitrary luminescent data ( Figure 5).  The ability of promising compounds to eliminate intracellular parasites was also analyzed by fluorescence microscopy. DAPI staining images revealed a high parasite load in infected and untreated microtissues ( Figure 6). A large number of intracellular amastigotes was clearly visualized throughout the untreated spheroid, even in the innermost layers. In contrast, Bz-treated microtissues demonstrated a drastic reduction in the infection profile, with few intracellular parasites visualized in the spheroids ( Figure 6). Treatment with 2c, 2e, and 2i also impacted the parasite clearance. Few parasites were visualized in the spheroid treated with 2c, indicative of good permeability and activity of the compound in the microtissue. All pyrazole-thiazoline derivatives markedly reduced the parasite load in the microtissue (Figure 6), as revealed by arbitrary luminescent data ( Figure 5). Figure 5. Effect of promising candidates, 2c, 2e, and 2i, in T. cruzi-infected microtissue. Trypanocidal activity, represented as arbitrary luminescence unit (ALU), correlates to the luminescent signal proportional to the number of viable parasites (Dm28c-Luc). All compounds significantly decreased the parasite load in microtissue compared to control, showing a similar efficacy to Bz treatment. Statistical significance, in relation to the untreated group, was determined using the One-Way ANOVA test, with p < 0.001 (***).

Potential of Pyrazole-Thiazoline Derivatives to Prevent Parasite Resurgence and the Effect in Combination Therapy
An important issue was to assess the compound activity reversibility. Then, the selected candidates (2c, 2e, and 2i) were evaluated for a prolonged period of time in order to assess their efficacy under drug-free conditions. Drug treatment was performed for 72 h at concentrations ranging from 1.5 to 7.2-fold the IC 90 value, respecting their toxicity levels in mammalian cells (CC 50 ). Most compounds were tested at 100 µM, except for 2c, whose maximum concentration reached 50 µM. The trypanocidal effect was analyzed after drug washing and 3 days exposure in the absence of compounds. None of the pyrazole-thiazoline derivatives led to the suppression of parasitism, but all selected compounds significantly reduced the parasite load ( Figure 7).  Bz treatment (100 μM) sustained the parasite clearance in both supernatant and cell monolayers. Compounds 2c (50 μM) and 2i (100 μM) were the most effective in controlling the intracellular amastigotes proliferation, inhibiting 78% and 89% of cell monolayer infection, respectively, compared to 2e (69% inhibition) (Figure 7). Among the pyrazolethiazoline derivatives, 2c stands out for reducing by 85% the release of trypomastigotes in the culture supernatant (Figure 7). It is possible that prolonged treatment with the drug- Figure 7. Analysis of 2c, 2e, and 2i treatment on parasite recrudescence in vitro. After 72 h treatment, the compounds were removed, and after culture washing, drug-free medium was added for an additional 72 h culture. The luminescent signal, corresponding to parasite viability, was performed on both supernatant (A) and T. cruzi-infected Vero cell monolayers (B). Statistical significance, in relation to the untreated group, was determined using the One-Way ANOVA test, with p < 0.001 (***).
Bz treatment (100 µM) sustained the parasite clearance in both supernatant and cell monolayers. Compounds 2c (50 µM) and 2i (100 µM) were the most effective in controlling the intracellular amastigotes proliferation, inhibiting 78% and 89% of cell monolayer infection, respectively, compared to 2e (69% inhibition) (Figure 7). Among the pyrazolethiazoline derivatives, 2c stands out for reducing by 85% the release of trypomastigotes in the culture supernatant (Figure 7). It is possible that prolonged treatment with the drug-containing medium replacement may improve the trypanocidal activity of promising candidates. It was shown that Bz treatment for 16 days of T. cruzi-infected Vero cell monolayers (strain Silvio X10/7) resulted in the prevention of spontaneous parasite relapse for up to 60 days, whereas posaconazole was unable to achieve parasite clearance [45].
The 2c showing better activity against trypomastigotes (IC 50 = 0.4 µM) and intracellular amastigotes (IC 50 = 1.4 µM), and even better potency (pIC 50 = 5.85) than Bz (pIC 50 = 5.48), motivated us to investigate whether the combination of 2c and Bz could potentiate the trypanocidal activity. The in vitro effect of the Bz and 2c combined treatment against intracellular amastigotes was analyzed by a luminescent assay, using T. cruzi Dm28c-Luc. The drug pairs were combined at four different ratios in accordance with the previously determined IC 50

Drug Target and Mechanism of Action
To evaluate if the insertion of the thiazoline group could change the pyrazole derivatives target that was previously identified as cruzipain, the compound scaffolds were queried in the ChEMBL database for similarity with different compounds with curated T. cruzi targets. One hit compound (IC 50 < 10 µM) was similar (>0.8) to compound 2a, and was annotated to target cruzipain ( Figure 9). Despite similarity with 2a, two other compounds had low activity against T. cruzi. Figure 9. In silico analysis of pyrazole-thiazoline scaffolds using fragment-based search of molecular targets. The image shows matched scaffolds of pyrazole-thiazoline to trypanocidal compounds with different IC50 (nM) against annotated targets in the ChEMBL database. The analysis was performed using DataWarrior software, and three inhibitors of cruzipain were retrieved with similarity to pyrazole-thiazoline derivatives.
The prediction of cruzipain as a potential target of promising pyrazole-thiazoline derivatives motivated us to investigate whether the ligand-target interaction would promote inhibition of cysteine protease activity. Thus, the enzymatic activity was measured in the total protein extract of trypomastigotes, using Z-FR-AMC as fluorogenic substrate. Incubation with promising compounds did not induce significant changes in enzymatic activity. Derivatives 2c, 2e, and 2i showed a maximum inhibition profile of 10% of cysteine protease activity, even at high concentrations of the compounds (100 μM) ( Figure 10). In contrast, E-64, a selective inhibitor of cysteine protease, inhibited the enzyme activity by more than 80% from the lowest concentration analyzed (6.25 μM). Thus, the replacement of imidazoline by thiazoline in 5-amino-1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazole (hit compound) did not favor binding with the active site of cruzipain, despite the prediction of an extensive hydrophobic domain associated with the phenyl ring containing 2,4-and 3,4-diCl, 2c and 2e, respectively (Figure 4), as previously evidenced in the hit compound [30]. Figure 9. In silico analysis of pyrazole-thiazoline scaffolds using fragment-based search of molecular targets. The image shows matched scaffolds of pyrazole-thiazoline to trypanocidal compounds with different IC 50 (nM) against annotated targets in the ChEMBL database. The analysis was performed using DataWarrior software, and three inhibitors of cruzipain were retrieved with similarity to pyrazole-thiazoline derivatives.
The prediction of cruzipain as a potential target of promising pyrazole-thiazoline derivatives motivated us to investigate whether the ligand-target interaction would promote inhibition of cysteine protease activity. Thus, the enzymatic activity was measured in the total protein extract of trypomastigotes, using Z-FR-AMC as fluorogenic substrate. Incubation with promising compounds did not induce significant changes in enzymatic activity. Derivatives 2c, 2e, and 2i showed a maximum inhibition profile of 10% of cysteine protease activity, even at high concentrations of the compounds (100 µM) ( Figure 10). In contrast, E-64, a selective inhibitor of cysteine protease, inhibited the enzyme activity by more than 80% from the lowest concentration analyzed (6.25 µM). Thus, the replacement of imidazoline by thiazoline in 5-amino-1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazole (hit compound) did not favor binding with the active site of cruzipain, despite the prediction of an extensive hydrophobic domain associated with the phenyl ring containing 2,4-and 3,4-diCl, 2c and 2e, respectively (Figure 4), as previously evidenced in the hit compound [30]. The failure to inhibit cysteine protease activity led us to assess whether the mechanism of action could be related to the induction of oxidative stress, since pyrazole derivatives have already been reported as iron-superoxide dismutase inhibitors [28,46]. Thus, the levels of reactive oxygen species (ROS) were evaluated using the 2, 7-dichlorofluorescein diacetate (H2DCFDA) probe. The intracellular ROS level in trypomastigotes was measured after 1, 3, and 24 h of compound treatment. None of the derivatives were able to induce redox imbalance in the IC50 concentration (Figure 11), indicating that another mechanism of action, not yet identified, is responsible for parasite death. Similar to Bz, Figure 10. Analysis of the inhibitory activity of promising compounds on T. cruzi cysteine proteases. Protein extracts (5 µg) of trypomastigote forms of T. cruzi were incubated with a series of dilutions (100-6.25 µM) of pyrazole-thiazoline derivatives (2c, 2e, and 2i) and E-64, a specific inhibitor of cysteine proteases. The fluorescent signal resulting from the degradation of the fluorogenic substrate Z-FR-AMC (60 µM) was quantified to determine the enzymatic activity. Results are presented as mean and standard deviation (±) of percent (%) enzyme activity from at least three independent experiments. The failure to inhibit cysteine protease activity led us to assess whether the mechanism of action could be related to the induction of oxidative stress, since pyrazole derivatives have already been reported as iron-superoxide dismutase inhibitors [28,46]. Thus, the levels of reactive oxygen species (ROS) were evaluated using the 2, 7-dichlorofluorescein diacetate (H 2 DCFDA) probe. The intracellular ROS level in trypomastigotes was measured after 1, 3, and 24 h of compound treatment. None of the derivatives were able to induce redox imbalance in the IC 50 concentration (Figure 11), indicating that another mechanism of action, not yet identified, is responsible for parasite death. Similar to Bz, which undergoes reductive metabolism to generate toxic intermediates that can act on redox metabolism, causes damage to DNA and RNA, and inhibits protein synthesis [47][48][49], the pyrazolethiazoline derivatives or their metabolites may also act on multiple targets by interacting with proteins, or even forming adducts with nucleotides. The H2DCFDA fluorescent probe was measured spectrofluorimetrically at 535 nm after excitation at 485 nm. Untreated and H2O2(20 μM)-treated parasites were used as negative and positive controls, respectively. Data are expressed as normalized values for the negative (untreated) control. Statistical significance, in relation to the untreated group, was determined using the One-Way ANOVA test, with p < 0.001 (***).
The induction of apoptosis through ROS production and the activation of caspase 3 by pyrazole derivatives has been reported in breast cancer cells, surpassing the activity of the first-line drug, paclitaxel [24]. However, a potent antioxidant activity has also been highlighted for pyrazole hybrids through increased antioxidant enzymes, and reduced lipid peroxidation [50].

Conclusions
A new series of twenty-four pyrazole-thiazoline derivatives were synthetized and analyzed against Trypanosoma cruzi. Three analogues, 2c, 2e, and 2i, showed potent activity against trypomastigotes (IC50 ≤ 2.1 μM), and greater potency against intracellular amastigotes (pIC50 > 5.16). The promising candidates showed good pharmacokinetic prediction and drug-likeness properties. The SAR analysis highlights the improvement of trypanocidal activity after removal of the amino group, and the inclusion of halogen substituents, such as chlorine and bromine, in the para position of the phenyl ring. Despite the inability to sterilize infected cultures in the washout assay, the selected derivatives, 2c, 2e, and 2i, showed improved potency and marked trypanocidal activity in the microtissue, encouraging the design of novel pyrazole-thiazoline scaffolds to advance in the drug discovery pipeline for Chagas disease.
Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Figures S1: NMR spectra of the compounds; Table S1: ADMET properties.  The H 2 DCFDA fluorescent probe was measured spectrofluorimetrically at 535 nm after excitation at 485 nm. Untreated and H 2 O 2 (20 µM)-treated parasites were used as negative and positive controls, respectively. Data are expressed as normalized values for the negative (untreated) control. Statistical significance, in relation to the untreated group, was determined using the One-Way ANOVA test, with p < 0.001 (***).
The induction of apoptosis through ROS production and the activation of caspase 3 by pyrazole derivatives has been reported in breast cancer cells, surpassing the activity of the first-line drug, paclitaxel [24]. However, a potent antioxidant activity has also been highlighted for pyrazole hybrids through increased antioxidant enzymes, and reduced lipid peroxidation [50].

Conclusions
A new series of twenty-four pyrazole-thiazoline derivatives were synthetized and analyzed against Trypanosoma cruzi. Three analogues, 2c, 2e, and 2i, showed potent activity against trypomastigotes (IC 50 ≤ 2.1 µM), and greater potency against intracellular amastigotes (pIC 50 > 5.16). The promising candidates showed good pharmacokinetic prediction and drug-likeness properties. The SAR analysis highlights the improvement of trypanocidal activity after removal of the amino group, and the inclusion of halogen substituents, such as chlorine and bromine, in the para position of the phenyl ring. Despite the inability to sterilize infected cultures in the washout assay, the selected derivatives, 2c, 2e, and 2i, showed improved potency and marked trypanocidal activity in the microtissue, encouraging the design of novel pyrazole-thiazoline scaffolds to advance in the drug discovery pipeline for Chagas disease.