Synthesis of 4-(4-chlorophenyl)thiazole compounds: in silico and in vitro evaluations as leishmanicidal and trypanocidal agents

Abstract Neglected tropical diseases are a diverse group of communicable pathologies that mainly prevail in tropical and subtropical regions. Thus, the objective of this work was to evaluate the biological potential of eight 4-(4-chlorophenyl)thiazole compounds. Tests were carried out in silico to evaluate the pharmacokinetic properties, the antioxidant, cytotoxic activities in animal cells and antiparasitic activities were evaluated against the different forms of Leishmania amazonensis and Trypanosoma cruzi in vitro. The in silico study showed that the evaluated compounds showed good oral availability. In a preliminary in vitro study, the compounds showed moderate to low antioxidant activity. Cytotoxicity assays show that the compounds showed moderate to low toxicity. In relation to leishmanicidal activity, the compounds presented IC50 values that ranged from 19.86 to 200 µM for the promastigote form, while for the amastigote forms, IC50 ranged from 101 to more than 200 µM. The compounds showed better results against the forms of T. cruzi with IC50 ranging from 1.67 to 100 µM for the trypomastigote form and 1.96 to values greater than 200 µM for the amastigote form. This study showed that thiazole compounds can be used as future antiparasitic agents.


INTRODUCTION
Neglected tropical diseases (NTDs) are a group of infectious diseases of protozoan, helminthic, bacterial, viral, and fungal origin, among others, common in tropical and subtropical regions where situations of poverty are common, although there are also records of their presence in non-endemic areas, that is, in developed countries (Elphick-Pooley & Engels 2022, Zhou 2022).These diseases affect more than 1.5 billion people worldwide and are responsible for more than 530000 deaths each year (Ahmed et al. 2022, Naqvi et al. 2022).In this scenario, research on NTDs has increased due to the need to establish monitoring systems and improve existing treatment programs, aiming to control the impacts of these diseases (Otte & Pica- Ciamarra 2021, Souza et al. 2021, Ahmed et al. 2022, Elphick-Pooley & Engels 2022, Naqvi et al. 2022, Zhou 2022).
Among the different NTDs, there is Leishmaniasis, a disease caused by a parasite of the Leishmania genus, which affects about 12 million people a year (Santos et al. 2020).There are still no vaccines available for humans and the drugs used for treatment (pentavalent antimonials, amphotericin B, and miltefosine) can promote parasite resistance in addition to being highly toxic to patients (Santos et al. 2020, Chanda 2021).Like leishmaniasis, Chagas disease is part of the NTD group and affects about 10 million people, it is caused by a parasite called Trypanosoma cruzi, which is endemic mainly in the Americas and spreads in different European countries, Australia, and Japan (Chanda 2021).The drugs used for the treatment of this disease are benznidazole and nifurtimox, which have limited efficiency in the chronic phase of the disease and have significant side effects, in addition to being contraindicated during pregnancy (Santos et al. 2020, Chanda 2021).
In this context, the search for new treatment therapies for Chagas disease and leishmaniasis has increased, to obtain drugs with low toxicity, which promote fewer side effects and have a shorter treatment time and low cost (Chanda 2021).Therefore, different compounds have been isolated or synthesized to act as new ways of treating NTDs caused by parasites.Among the variety of compounds, there are the thiazoles, pentagonal heteroaromatics, with three carbon atoms, one sulfur, and one nitrogen atom (Petrou et al. 2021).The arrangement of these atoms in the heterocyclic ring can vary in two ways leading to the existence of 1,2-thiazole or 1,3-thiazole.Thiazole is a functional group that occupies a prominent place in medicinal chemistry due to its reactivity and biological activity, is widely found in drugs applied in the fight against diseases in general ( Chhabria et al. 2016, Petrou et al. 2021).
D i f fe re n t s t u d i e s e val u a t i n g t h e leishmanicidal and trypanocidal activity have been carried out, among which we can mention Haroon et al. (2021) evaluating the leishmanicidal and trypanocidal activity of 1, 3-thiazole, and 4-thiazolidinone ester compounds.Oliveira et al. (2020)  González et al. (2021) performed QSAR studies to assess the potential of different thiazoles also against Trypanosoma cruzi.Oliveira-Filho et al. (2017) and Silva et al. (2017) evaluated the action of thiazole compounds against different forms of Trypanosoma cruzi.In addition to the potential effect of thiazoles in monotherapy, they can be combined with other drugs to increase the efficiency of the treatment (Scarim et al. 2019, Perdomo et al. 2021).
Therefore, this work aimed to synthesize new thiazole compounds, to evaluate in a preliminary way the biological potential through a pharmacokinetic study (ADME) and to evaluate the activities, cytotoxicity in animal cells and antiparasitic activity against the promastigote and amastigote forms of Leishmania amazonensis and against the trypomastigote and amastigote forms of Trypanosoma cruzi in vitro (Figure 1).
All reagents used were obtained commercially (Fluka and Merck).The development of the reactions was monitored using thin layer chromatography (Merck, silica gel F254 on aluminum foil).The melting points were determined from capillary tubes with the Fisatom device (model 431D 60W, Brazil).The IR spectrum was generated from the Spectrum 400 equipment (Perkin Elmer).NMR spectra were obtained using Bruker AMX-300 MHz devices (300 MHz for 1 H and 75.5 MHz for 13 C).Chemical shifts were recorded in δ units and coupling constants (J) were recorded in Hertz (Hz).The multiplicities were displayed as s (singlet), d (doublet), t (triplet), m (multiplet), dd (double doublet).

Preparation of solutions of compounds
All compounds evaluated in this study were solubilized in dimethylsulfoxide (DMSO), according to the methodology proposed by Jacob et al. (2021) with few modifications to obtain a concentration of 800 µM.Then, they were diluted in different concentrations.Dilutions were performed in phosphate-buffered saline to obtain a final concentration of 1% DMSO.

Prediction of Pharmacokinetic Properties
The in silico study of the compounds was carried out in order to predict pharmacokinetic and physicochemical properties (Norinder &Bergström 2006).Knowledge of properties such as absorption, distribution, metabolism and excretion (ADME) are indispensable in the process of developing new drug candidate molecules and help predict the oral bioavailability of new drug candidates (Pires et al. 2015).For this, we use the software available online SwissADME (http://www.swissadme.ch) and pkCSM (http:// biosig.unimelb.edu.au/pkcsm/prediction).
The parameters evaluated were: molecular weight, number of hydrogen bond acceptors, number of hydrogen bond donors, log P, total polar surface area, LogS, permeability in Caco-2, intestinal absorption, volume of distribution, unbound and total fraction clearance.

ABTS radical capture method
Antioxidant activity by the ABTS [2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] method was performed according to the methodology described by Salar et al. (2017) with modifications.The ABTS radical was formed by reacting 5 mL of the ABTS solution 7mM with 88 µL of 140mM potassium persulfate solution, incubated at 25ºC and in the absence of light, for 16 hours.Once formed, the radical was diluted with ethanol P.A. until obtaining the absorbance value of 0.70 ± 0.020 at 734 nm.
Compounds in a volume of 0.5 mL at different concentrations (6 -5000 µM) were added to 3.5 mL of the reagent, then the system was kept in the dark for 30 minutes.Then the assays were analyzed in a spectrophotometer at 734 nm.The equipment blank was ethanol.The standards used were ascorbic acids and butylated hydroxytoluene (BHT) under the same conditions as the compounds.The percentage of radical capture was determined through Equation 1. (1) Where: ABS control is the absorbance of the control and ABS Sample is the absorbance of the sample containing the compounds after testing.
The IC 50 (concentration capable of capturing 50% of the radicals) was determined by nonlinear regression analysis of data obtained by SPSS 8.0 software for Windows.The results were expressed in concentrations of µM.

DPPH radical capture method
The assay to determine the antioxidant activity by the 1,1-diphenyl-2-picrylhydrazine (DPPH) method was performed according to the methodology described by Andreani et al. ( 2013) with modifications.The technique consists of the reaction of the free radical DPPH 0.03mM with the compounds in ethanolic solution, for 30 minutes.Compounds in a volume of 0.5 mL at different concentrations (6 -5000 µM) were added to 3.5 mL of the reagent, then the system was kept in the dark for 30 minutes.Then the assays were analyzed in a spectrophotometer at 517 nm.The equipment blank was ethanol.The standards used were ascorbic acids and butylated hydroxytoluene (BHT) under the same IRANILDO JOSÉ DA CRUZ FILHO et al.Bras Cienc (2023) 95(1) e20220538 7 | 22 conditions as the compounds.The percentage of radical capture was determined through Equation 1.

An Acad
The IC 50 (concentration capable of capturing 50% of the radicals) was determined by nonlinear regression analysis of data obtained by SPSS 8.0 software for Windows.The results were expressed in concentrations of µM.

In vitro hemolytic activity
The assay was performed according to Queiroz et al. ( 2020) and Ansari et al. (2020) with some modifications.Hemolytic activity was performed in 96-well microplates.Erythrocytes were isolated by centrifugation (1500 rpm, 10 min at 4°C).Subsequently, they were washed three times with phosphate-buffered saline (PBS; pH 7.4).Then, each tube received 1.1 mL of erythrocyte suspension (1%) and 0.4 mL of various concentrations of compounds (0 to 200 µM).Distilled water (negative) and Triton X100 (0.0025%, positive) were used as controls.After 60 minutes of incubation, the cells were centrifuged and the absorbance of the supernatant was recorded at 540 nm.The hemolytic activity results were expressed by the following Equation 4. (1) Where: ABS sample: Sample absorbance, ABS blanck: negative control absorbance, ABS Triton X: positive control absorbance.
The effective concentration that promotes 50% of hemolysis (IC 50 ) was determined.Three independent experiments were performed in triplicate.The study was approved by the Ethics Committee on the Use of Animals of the Aggeu Magalhães Institute/Oswaldo Cruz Foundation, protocol number 164/2020.  , corresponding to 250 mL), and maintained in RPMI 1640 medium with phenol red.For supplementation of the culture medium, 10% fetal bovine serum was used, the antibiotics penicillin, 100 U/mL, and streptomycin, 100 µg/mL, and the buffer solution HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).
Assays were performed in 96-well culture plates and monitored under a microscope.100 µL of RPMI-1640 medium, supplemented with fetal bovine serum, containing 10 4 cells in each well was added to the plates.The plates were incubated in a 5% CO 2 oven at 37 °C for 24 hours.After this incubation period, compounds were added to the cells at concentrations from 0 to 200 µM in triplicate on each plate.The plates were incubated again in a CO 2 oven for 72 hours.
A f t e r t h i s p e r i o d , 2 0 µ L o f 3 -( 4 , 5 -d i m e t h y l t r i a z o l -2 -y l ) -2 , 5diphenyltetrazolium bromide (MTT) solution at 0.5 mg/mL was added to each well.The plates were incubated for another 3 hours for the MTT to react with the cells and form the insoluble crystals of Formazan.After 3 hours, 100 µL of supernatant were removed from each well and discarded, and later, 130 µL of sodium lauryl sulfate solution was added to dissolve the formed crystals.After 24 hours it was possible to read the absorbance in a plate spectrophotometer at 570 nm.With the absorbance values obtained by reading, it was possible to obtain the percentage of cell viability (amount of live cells) resulting from the treatment with each tested compound in its proper concentrations.In addition to the compounds studied here, tests were also carried out for comparison purposes using the standard drug amphotericin B, miltefosine, and benznidazole (dissolved under the same conditions as the compounds).The experiments were performed in triplicate and biological replication and cell viability and inhibition were calculated using Equations 2 and 3.
(2) Inhibition of cel Growth(%)= 100 -cell viability (3) Where: VC is the number of cells at different concentrations, TC is the concentration of cells in the control which represents 100% viability.
The effective concentration that promotes 50% of cell viability (CC 50 ) was determined by non-linear regression analysis of data obtained by SPSS 8.0 software for Windows.

Cultivation and maintenance of parasites
The experiments were carried out according to Gouveia et al. (2022) with few modifications.The promastigote forms of Leishmania amazonensis were maintained in supplemented Schneider medium (20% fetal bovine serum and 1% streptomycin penicillin solution), pH 7.2 at 26 ºC.Parasites in the exponential phase of growth were used in all experiments with re-raising every three days.Then, they were subjected to three cycles of washing with cold sterile saline, with centrifugation at 3000 rpm, for 15 minutes at 4 °C and adjusted with Schneider medium to the desired concentrations in each experiment.The viability of the parasites was analyzed by light microscopy.The amastigote forms were obtained after incubation and internalization of the promastigote forms in macrophage culture.

Growth kinetics of promastigote forms
The parasites in the promastigote form were maintained at 26 °C in supplemented Schneider medium (20% fetal bovine serum and 1% penicillin-streptomycin solution), pH 7.2.The growth curve was performed according to Gouveia et al. (2022) and Silva et al. ( 2020) with modifications.To carry out the experiments, an initial concentration of 1x10 6 cells/well of parasites was used, grown in 96-well plates in an atmosphere of 5% CO 2 at 37 °C at times ranging from 0 to 120 hours.Culture growth was monitored by counting in a Neubauer chamber.The experiments were carried out in triplicate.

Cytotoxicity of promastigote forms in vitro
The cytotoxicity experiments in promastigote forms were carried out according to a methodology adapted from Gouveia et al. (2022) and Silva et al. ( 2020) with few modifications.The experiments were performed on 96well plates.Promastigote forms were grown in Schneider's medium supplemented for 72 hours and counted in a Neubauer chamber at a final concentration of 1x10 6 cells/well.Then these forms were incubated in the presence of different concentrations (0 to 200 µM) of the compounds for 72 hours.The positive control used was the drugs Amphotericin B and Miltefosine (under the same experimental conditions as the compounds) and the negative control was the culture medium and only cells, respectively.Viability was evaluated by counting in a Neubauer chamber.Through graphs of inhibition against concentrations, it was possible to determine the IC 50 (concentration of inhibition of growth by 50%) through non-linear regression analysis by SPSS 8.0 software for Windows.Assays were performed in biological and technical triplicate.

Cytotoxicity of amastigote forms in vitro
The tests were performed according to Gouveia et al. (2022) and Silva et al. ( 2020) with modifications.Initially, RAW.264.7 macrophage cells at a concentration of 1.0x10 5 cells/ well were grown in 96-well microplates and incubated at 37°C and 5% CO 2 for 24 hours.After growth, macrophages were infected with the promastigote forms grown in 72 hours, in a ratio of 10:1 parasite macrophage for 24 hours.After this period, the parasites that did not infect the macrophages were removed by 10 washes with RPMI 1640 medium.Then, the infected cells were exposed to different concentrations of the compounds (0 to 200 µM).as a positive control, the drugs Amphotericin B and Miltefosine were used under the same conditions as the compounds.Assays were incubated for 72 hours at 37 °C and 5% CO 2 .After 72 hours of treatment, the plates were washed with PBS, fixed with methanol and stained with Giemsa.
The percentage of infected macrophages was determined by counting 100 cells in triplicate.IC 50 was determined by non-linear regression analysis of data obtained by SPSS 8.0 software for Windows.Assays were performed in biological and technical triplicate.

Culture of Trypomastigotes
After reaching 100% confluence in culture, RAW.264.7 macrophage cells were infected with 1 x 10 7 trypomastigotes (Y strain) and cultured in RPMI medium + 5% fetal bovine serum.After 7 days, cells began to release new trypomastigotes and new RAW.264.7 macrophage cell culture bottles were infected.

Evaluation of the cytotoxicity of the compounds against trypomastigote forms
The tests were carried out according to Oliveira-Cardoso et al. (2014) with modifications, the trypomastigote forms (Y strain) were obtained from the in vitro infection (1x10 7 parasites) of the macrophage cell line RAW.264.7, after they reached confluence in culture.To determine the antiproliferative effect for trypomastigote forms of strain Y (1x10 6 parasites/well), maintained in RPMI medium + 1 % antibiotic + 5 % fetal bovine serum, were seeded in 96-well plates at 37 ºC, together with different concentrations. of compounds (0 to 200 µM) for 24 hours in a 5% CO 2 atmosphere.Each compound was tested in triplicate.Untreated wells were obtained as a negative control of the reaction and the reference drug used as a positive control was Benznidazole.The parasite viability was determined by direct counting in a Neubauer chamber and, from these values, the IC 50 was obtained through a simple linear regression using the Prisma 5.0 Graphpad software.

Evaluation of the cytotoxicity of the compounds against amastigote forms
The tests were carried out according to Oliveira-Cardoso et al. (2014) with modifications.The trypomastigote forms were obtained from the in vitro infection of the macrophage strain RAW.264.7.To obtain amastigotes, RAW.264.7 macrophages were seeded in 96-well plates, and incubated for 24 hours at 37°C and an atmosphere with 5% CO 2 .Parasites were added at the rate of 10 trypomastigotes/cell.After 24 hours, the non-internalized parasites were removed, and the plates were incubated for 48 hours.The compounds were added at different concentrations (0 to 200 µM), and the plates were incubated for 96 hours.Wells containing only culture medium and cells were the negative control and wells containing Benznidazole (diluted under the same experimental conditions as the compounds) the positive control.At the end of the incubation, Chlorophenol red-β-Dgalactopyranoside (CPRG), 500 µM, 0.5% Nonidet P-40, in PBS, was added and incubated for 18 hours at 37°C.The absorbance was read at 570 nm on the Thermo Scientific Multiskan FC spectrophotometer.IC 50 values were calculated by regression analysis using GraphPad Prism software.Each assay was performed in triplicate.

Determination of the selectivity index (SI) and specificity index (SPI) of the compounds for the different forms of Leishmania amazonensis and Trypanosoma cruzi
The SI demonstrates the relationship of compound toxicity between mammalian cells and parasitic forms.It was obtained by the ratio between the value of the cytotoxic concentration (CC 50 ) of the compound in mammalian cells, and the inhibitory concentration (IC 50 ) in promastigote/trypomastigote and amastigotes (Silva et al. 2020, Gouveia et al. 2022).The SPI establishes the specificity of the compound between the two forms of the parasite, promastigote and amastigote.It was calculated by the ratio between the compound IC 50 for promastigote/trypomastigote forms and the amastigote IC 50 (Gouveia et al. 2022).

Statistical analysis
The results obtained were expressed as mean ± standard deviation and submitted to analysis of variance (ANOVA) and the means were submitted to Tukey's test (p ≤ 0.05) using the GraphPad Prism 5.0 software (test version).

In silico pharmacokinetic properties
Theoretical in silico pharmacokinetics is an approach currently widely used in the initial study of ADME properties (absorption, distribution, metabolism and excretion) that aims to reduce unnecessary expenditure in biological assays of compounds (Fowler et al. 2022).A good pharmacokinetic profile increases the likelihood that a promising drug candidate will offer a successful therapy (Liu & Shah 2022).Two common ways to assess the potential oral bioavailability of a compound are through the rules proposed by Lipinski (hydrogen donors ≤ 5, hydrogen bond acceptors ≤ 10, molecular weight < 500 g/mol, logP < 5 (or MLogP < 4.15) and Veber (rotational bonds ≤ 10 and polar surface area (TPSA) ≤ 140 Å2) are described in Table I (Domínguez-Villa et al. 2021, Bilen et al. 2022, Tabti et al. 2022).
The results show that compounds 1a, 1b and 1f obeyed Lipinski and Veber's rules, but compounds 1c, 1d, 1e, 1g and 1h showed high values of lipophilicity, violating Lipinski's rule in this parameter.Therefore, the compound may violate only one of these parameters to be a drug candidate.Therefore, these rules allow a good prediction of the oral bioavailability profile for new molecules.Other important properties were also evaluated, such as aqueous solubility, expressed numerically by LogS (Tabti et al. 2022).This parameter significantly affects the absorption and distribution characteristics.In general, a compound with low solubility is not well absorbed by the body (Domínguez-Villa et al. 2021, Bilen et al. 2022).Aqueous solubility is the inverse of lipophilicity, the ligands used in this study are classified in the following order: insoluble <-10 < slightly soluble < -6 < moderately soluble < -4 < soluble < -2 < very soluble < 0 < highly soluble (Pires et al. 2015).All compounds have low solubility and are classified as slightly soluble.
Through the pkCMS platform, some important pharmacokinetic properties were predicted for the ligands under study, such as the permeability value of Caco-2 cells, which provides an estimate for the absorption of the compound in the human intestinal mucosa.Thus, the results allow classifying the compounds as poorly absorbed (< 1x10 -6 cm/s), moderately absorbed (1 between 10x10 -6 cm/s) and well absorbed (> 10x10 -6 cm/s).Compound 1h is classified as poorly absorbed and the other compounds are classified as moderately absorbed.The compounds showed a percentage of absorption through the intestine of greater than 90% (Pires et al. 2015).
The volume of distribution (VDss) is the parameter that describes the extent of drug distribution in tissues and plasma.VDss values (log VDss) > 0.45 indicate that the drug will be distributed into the tissue.Log VDss values <−0.15 indicate that the drug will be distributed in plasma (Pires et al. 2015, Tabti et al. 2022).The results show that the compounds have a higher affinity to be distributed in tissues, since all VDss values were >-0.15 with the exception of compound 1g, which presented a VDss value of -0.26 with greater distribution in plasma.
The compounds have a low fraction bound to serum protein.In addition, they present a low clearance rate between 0.07 and 0.28.Therefore, the evaluated compounds showed good ADME results indicating potential oral bioavailability.

In vitro antioxidant activity
The antioxidant activity was evaluated through the ABTS and DPPH radical scavenging assay In the literature, different studies report that thiazole compounds are molecules capable of promoting antioxidant activity both in vitro and in vivo, and this potential activity is directly related to the chemical structure of these compounds (Salar et al. 2017, Khamees et al. 2019, Dincel et al. 2020).However, the 2-chlorophenylthiazoles compounds did not show antioxidant activity.
Cytotoxicity assays in animal cells: erythrocytes, macrophages RAW.264.7,fibroblasts (V79) and hepatoma (HepG2) Cytotoxicity in erythrocytes is one of the experimental models of in vitro toxicity that stands out as a screening method (Amin & Dannenfelser 2006).This is because it is an easy, fast and efficient method to evaluate the effects of compounds on the cell membrane of erythrocytes (Amin & Dannenfelser 2006, Marques-Garcia 2020).Figure 2 presents the results of hemolytic activity promoted by the compounds at different concentrations.The negative control consisted only of erythrocytes (0% hemolysis) and the positive one contained Triton-X (100% hemolysis).(2023) 95(1) e20220538 13 | 22 The results show that the compounds and standards (Miltefosine, Amphotericin B and Benznidazole) showed a percentage of hemolysis lower than 10% at the concentrations evaluated.Therefore, the compounds are not able to promote hemolysis in vitro (Amin & Dannenfelser 2006).The literature presents results of hemolytic activity for different thiazole derivatives.2020) evaluating thiazol-2 (3H)-thiones containing the fraction 4- (3,4,5-trimethoxyphenyl) observed that they were not able to promote hemolysis in erythrocytes.
These findings reinforce that the compounds evaluated in our study are not considered hemolytic in in vitro assays.In addition to the hemolysis assays performed on erythrocytes, other mammalian cells were also evaluated in order to assess the cytotoxic potential of the compounds.Table II presents the results of percentage of growth inhibition at a concentration of 200 µM and CC 50 values for each of the compounds against the evaluated cells (RAW.264.7 macrophages, V79 fibroblasts and hepatoma (HepG2) cells).
The results show different IC 50 results for the compounds.IC 50 results for macrophage cells ranged from 45.12 to values greater than 200 µM.For fibroblast cells (V79) they ranged from 68.9 to greater than 200 µM, while for HepG2 cells they ranged from 73.4 to greater than 200 µM.Lower IC 50 values indicate that the compounds are more toxic.Thus, we can highlight compounds 1b and 1d, the other compounds (1a, 1c, 1e, 1f, 1g and 1h) were not able to promote cytotoxicity in the different cells evaluated.
The literature presents different works evaluating different thiazole derivatives against mammalian cells.Rodrigues et al. ( 2018) evaluating the cytotoxic effect of 4-Phenyl-1, Table II.Cytotoxicity results were expressed in percentage of growth inhibition at the highest concentration (200 µM) for the assays and in CC 50 (capacity to inhibit growth by 50%) promoted by the compounds against RAW.264.7 macrophage cells, V79 fibroblasts and liver cells HepG2 respectively.These results reinforce that cytotoxicity is directly related to the chemical structure and cell type evaluated (Oliveira Cardoso et al. 2014, Rodrigues et al. 2018, Teixeira et al. 2020, Vra et al. 2019).Thus, the compounds evaluated in this study were able to promote moderate to low cytotoxicity against animal cells.

In vitro antipromastigote activity
Parasites of the Leishmania genus are digenetic (heteroxenes) and present in their life cycle only two evolutionary forms: the promastigote form, which is flagellated and extracellular, and the amastigote form, which is intracellular and without movements.Promastigotes have an elongated body, measuring between 14 and 20 mm and a free flagellum.Amastigotes have an ovoid body, measuring between 2.1 and 3.2 mm and an internal flagellum (Pessoa & Martins 1982).
To evaluate the leishmanicidal potential of the different compounds tested in the promastigote forms of L. amazonensis, after 72 hours of treatment, the total number of promastigotes was counted under an optical microscope.Table III presents the results of the percentage of inhibition of growth of the promastigote forms at a concentration of 200 µM and IC 50 values (concentration that inhibits the growth of the parasite by 50%) and the selectivity index between CC 50 (mammal cells) and the IC 50 (of the parasites) promoted by the compounds under study.
The compounds presented IC 50 values that ranged from 19.86 to 200 µM and were classified according to scales adapted from the studies proposed by Upegui et al. (2014) and Gouveia et al. (2022) where compounds that presented IC 50 < 50 µM were considered active, those that presented IC 50 between 50 and 200 µM moderately active and inactive with IC 50 > 200 µM.Thus, compounds 1c, 1d, 1f, 1g and 1h were considered active and compound 1a moderately active and finally compounds 1b and 1e were considered inactive.The compounds when compared to Amphotericin B and Miltefosine showed low selectivity.
The results presented showed that the compounds evaluated in this work are promising against the promastigote form of Leishmania amazonensis found in the sand fly (insect vector).However, even the compounds showing potential, assays need to be carried out with the amastigote forms.

In vitro anti-amastigote activity
In addition to the promastigote forms, the cytotoxic effect of the compounds against the amastigote forms of L. amazonensis, after 72 hours of treatment, the viability was evaluated by optical microscope.Table IV presents the results of growth inhibition of intracellular amastigotes, IC 50 values in addition to the selectivity and specificity indices in relation to macrophage cells.
The compounds presented IC 50 values that ranged from 101 to greater than 200 µM and were also classified according to scales adapted from the studies proposed by Upegui et al. (2014) and Gouveia et al. (2022) previously used for the amastigote forms where compounds that presented IC 50 < 50 µM were considered active, those that presented IC 50 between 50 and 200 µM moderately active and inactive with IC 50 > 200 µM.Thus, compounds 1a, 1c, 1d, 1e, 1f, 1g and 1h were considered moderately active, with very close IC 50 values, compound 1b was considered inactive.
The compounds showed low selectivity and compounds 1a, 1c, 1e, 1f, 1g and 1h were more toxic to the amastigote forms when compared to macrophage cells, that is, they showed lower IC 50 values.Regarding specificity, compound 1e showed greater specificity, that is, it was more specific for the amastigote form.The others were more specific for the promastigote forms, with the exception of compound 1b, which did not show activity for any of the forms.
It is important to point out that the action on the amastigote form is hampered by the need for the compound to permeate through the macrophage membrane before reaching the parasite inside the cell, thus, there may be a loss of effectiveness of the compound for this form (Gouveia et al. 2022).
The literature presents different values of IC 50 promoted for the thiazole compounds to different species of leishmania.Santos-Aliança et al. (2017) evaluating the leishmanicidal activity of phthalimido-thiazole derivatives obtained IC 50 The results found in the literature confirm that the compounds evaluated in this study promote moderate leishmanicidal activity against the amastigote forms of Leishmania amazonensis.Therefore, in order to choose the best compounds, cytotoxicity against mammalian cells and cytotoxicity against amastigote forms were evaluated.A compound with potential leishmanicidal activity must have low cytotoxicity against mammalian cells (high IC 50 values) and high cytotoxicity against the parasitic form (low IC 50 values).Thus, compounds 1a, 1c, 1e, 1f, 1g and 1h showed potential in vitro results against the amastigote forms.

Evaluation of cytotoxic activity against the forms of Trypanosoma cruzi in vitro Cytotoxic activity against the trypomastigote form in vitro
The compounds were also evaluated against the trypomastigote form of Trypanosoma cruzi.Trypomastigote forms are elongated, with a kinetoplast with a rounded shape located in the region posterior to the nucleus, flagellum emerging from the flagellar pocket that is located laterally, in the posterior region of the parasite (Contreras et al. 2002, Takagi et al. 2022).The flagellum emerges and adheres along the body of the parasite, becoming free in the anterior region.This form is highly infectious, and can be found: in the insect vector; blood and intercellular space of vertebrate hosts I have macrophages as the main host (Contreras et al. 2002, Silva-Júnior et al. 2022, Takagi et al. 2022).
Table V presents the results of the percentage of inhibition of the growth of the trypomastigote forms at a concentration of 200 µM and IC 50 values (concentration that inhibits the growth of the parasite by 50%) and the selectivity index between CC 50 (mammal cells) and the IC 50 (of the parasites) promoted by the compounds under study.The results presented in the Table V show that the compounds presented IC 50 ranging from 1.67 to 100 µM.Compounds 1c, 1e and 1h were considered active and presented IC 50 values lower than the standard benznidazole.Furthermore, the compounds in ascending order 1c, 1e and 1h showed a high selectivity index, that is, they were more selective for the trypomastigote forms when compared to mammalian cells.The other compounds (1a, 1b, 1d, 1f and 1g) were considered moderately active.
The thiazoles show promising activity against the trypomastigote forms.Among the different studies, we can mention those carried out by Gomes et al. (2016) evaluating Phthalimidothiazoles compounds against trypomastigote forms of Trypanosoma cruzi obtained IC 50 values ranging from 0.5 to 107.5µM.Oliveira-Cardoso et al. (2014) obtained IC 50 values ranging from 1.1 to 36.7 µM for the 2-Pyridyl thiazoles compounds.These results show that compounds 1c 1e and 1f show promising activity against the trypomastigote forms of Trypanosoma cruzi.

Cytotoxic activity against the amastigote form in vitro
The compounds were also evaluated against the amastigote form of Trypanosoma cruzi.The results of anti-amastigote activity are shown in Table VI.
The results presented in Table VI show that the compounds showed anti-amastigote activity ranging from 1.96 to values greater than 200 µM.Compounds 1c, 1e and 1g were considered active, in addition, they showed the highest levels of selectivity.Compounds 1d and 1f moderately active and the others inactive.Regarding specificity, only compound 1g showed greater specificity against the amastigote form when compared to the trypomastigote form.The literature reports that thiazoles are compounds with potential anti-amastigote activity.Vra et al. (2019) obtained IC 50 values ranging from 5.28 to 52.99 µM for 4-thiazolidinone and 1,3-thiazole compounds. Álvarez et al. (2015a) evaluating the activity of a thiazole containing an amide group against the amastigote form, they obtained an IC 50 of 0.72 µM.Álvarez et al. (2015b) evaluating the activity of a bis-thiazole compound, they obtained IC 50 results of 1.2 µM.Haroon et al. (2021) obtained IC 50 values for the amastigote forms ranging from 16.85 to 75.39 µM.
The results found in the literature show that the compounds evaluated in this work showed promising results against the amastigote form of Trypanosoma cruzi.Therefore, the choice of the best compounds against the forms of Trypanosoma cruzi was carried out.Trypomastigote and amastigote forms are found in the vertebrate host.With this, the selection proceeded on compounds that presented low cytotoxicity against animal cells (high IC 50 ) and high cytotoxicity (low IC 50 ) against the trypomastigote and amastigote forms.The selected compounds were compounds 1c and 1e effective for both forms of the parasite.Again, it was observed that for the amastigote form, an increase in concentration when compared to the trypomastigote form, this is because, for the compound to promote activity, it is necessary that its cross different membranes until it reaches the parasite that is intracellular (Gouveia et al. 2022).

CONCLUSIONS
This study presented a screening to verify the biological potential of eight thiazole compounds.The study showed that they had good pharmacokinetic profiles, moderate to low antioxidant activity.In addition, they also have moderate to low cytotoxicity.Antiparasitic assays for Leishmania amazonensis and Trypanosoma cruzi in vitro show promising results, with emphasis on compounds 1c for leishmanicidal activity and 1e for trypanocidal activity, which, in addition to presenting low toxicity against mammalian cells.Therefore, this study generally showed that the compounds evaluated may be good candidates for antiparasitic drugs.

Figure 2 .
Figure 2. hemolytic activity results in different concentrations promoted by the compounds.
IRANILDO JOSÉ DA CRUZ FILHO et al.THE BIOLOGICAL ACTIVITIES OF THIAZOLESAn Acad Bras

Table I .
In silico pharmacokinetic properties: adsorption, distribution, metabolism and excretion for the compounds under study.Activity results were presented at IC 50 (concentration capable of capturing radicals by 50%).For the ABTS assays, the values ranged from 322.4 to 2091.1 µM, showing low antioxidant activity when compared to the standard's ascorbic acid (76.9 µM) and butylated hydroxytoluene (28.6 µM).Only compounds 1a (322.4 ± 0.1 µM), 1d (1836.2±1.9 µM), 1e (2091.1 ± 1.5 µM) and 1f (1823.4±1.0 µM) showed IC 50 .ABTS results for each of the compounds are shown in the supplementary material (TableSI).In relation to the DPPH assays none of the compounds presented IC 50 values, in the highest concentration the percentage of capture varied from 23.7 to 35%.This difference in results may be related to the versatility of the ABTS assay.
a SwissADME Molecular Weight; b SwissADME Number H-bonds acceptors; c SwissADME Number H-bonds donors; d SwissADME Moriguchi log of octanol-water partition coefficient; e SwissADME Ali log of aqueous solubility; f SwissADME calculation of Topological Polar Surface Area (TPSA); g pkCSM prediction of Caco-2 cell permeability as estimation of absorption at human intestinal mucosa; h pkCSM prediction of the proportion of compound absorption though the human small intestine; i pkCSM prediction of the log of steady state volume of distribution (VDss); j pkCSM prediction of compound fraction unbound in plasma (not bound to serum proteins); k pkCSM prediction of the log of total drug clearance.IRANILDO JOSÉ DA CRUZ FILHO et al.THE BIOLOGICAL ACTIVITIES OF THIAZOLES An Acad Bras Cienc (2023) 95(1) e20220538 12 | 22 of compounds 1a -1h.This is capable of evaluating compounds of a hydrophobic nature (the compounds were more hydrophobic as shown in the in silico study) and hydrophilic, unlike the DPPH test, which presents good results for compounds that have a mostly hydrophilic character (Shalaby & Shanab 2013, Moharram & Youssef 2014).

Table III .
Antipromastigote activity results obtained by the compounds expressed as percentage of inhibition at a concentration of 200 µM in addition to IC 50 values and selectivity index.

Table IV .
Results of anti-amastigote activity promoted by the compounds, percentage of inhibition of parasite growth at a concentration of 200µM, IC 50 values, selectivity and specificity index.Mean ± Standard Deviation; SI: CC 50 Macrophages RAW.264.7 /IC 50 amastigote; SPI: IC 50 promastigote /IC 50 amastigote.Selectivity and specificity results in absolute values.

Table V .
Antitrypomastigote activity results obtained by the compounds expressed as percentage of inhibition at a concentration of 200 µM in addition to IC 50 values and selectivity index.

Table VI .
Antiamastigote activity results obtained by the compounds expressed as percentage of inhibition at a concentration of 200 µM in addition to IC 50 values and selectivity index.Mean ± Standard Deviation; SI: CC 50 Macrophages RAW.264.7 /IC 50 amastigote; SPI: IC 50 promastigote /IC 50 amastigote.Selectivity and specificity results in absolute values.