Synthesis of Antiprotozoal 2-(4-Alkyloxyphenyl)-Imidazolines and Imidazoles and Their Evaluation on Leishmania mexicana and Trypanosoma cruzi

Twenty 2-(4-alkyloxyphenyl)-imidazolines and 2-(4-alkyloxyphenyl)-imidazoles were synthesized, with the former being synthesized in two steps by using MW and ultrasonication energy, resulting in good to excellent yields. Imidazoles were obtained in moderate yields by oxidizing imidazolines with MnO2 and MW energy. In response to the urgent need to treat neglected tropical diseases, a set of 2-(4-alkyloxyphenyl)- imidazolines and imidazoles was tested in vitro on Leishmania mexicana and Trypanosoma cruzi. The leishmanicidal activity of ten compounds was evaluated, showing an IC50 < 10 µg/mL. Among these compounds, 27–31 were the most active, with IC50 values < 1 µg/mL (similar to the reference drugs). In the evaluation on epimastigotes of T. cruzi, only 30 and 36 reached an IC50 < 1 µg/mL, showing better inhibition than both reference drugs. However, compounds 29, 33, and 35 also demonstrated attractive trypanocidal activities, with IC50 values < 10 µg/mL, similar to the values for benznidazole and nifurtimox.


Introduction
According to the World Health Organization (WHO), more than one billion people around the world suffer from neglected tropical diseases, which include Leishmaniasis and Chagas disease.Leishmaniasis affects ~12 million people worldwide, with ~1-2 million new cases per year.Chagas disease is mostly found in Latin America, where it threatens the lives of 8-10 million people [1].
The etiologic agent of leishmaniasis is Leishmania spp.[2,3], a vector-born flagellate protozoan parasite.In the American continents, it is transmitted to humans from other vertebrate hosts by Lutzomyia spp.sand flies [3].The disease has three principal forms: cutaneous leishmaniasis (characterized by a localized ulcerative lesion or diffuse nodular lesions), mucocutaneous leishmaniasis (manifesting in the destruction of oral mucosal tissue), and visceral leishmaniasis (a life-threatening condition involving severe organ damage).This disease depends mainly on the infecting species.For example, an infection with Leishmania mexicana tends to trigger either localized or diffuse cutaneous leishmaniasis [4].
Chagas disease, caused by the hemoflagellate protozoan Trypanosoma cruzi, is a parasitic disease endemic to tropical and subtropical countries of Latin America.Infection is Chagas disease, caused by the hemoflagellate protozoan Trypanosoma cruzi, is a parasitic disease endemic to tropical and subtropical countries of Latin America.Infection is mainly initiated by hematophagous insects that excrete parasite-laden feces near the bite site during feeding [5,6].The first (acute) phase of the disease manifests in a high level of parasitemia, with the parasites accumulating in peripheral blood and tissue.This phase is frequently asymptomatic or can produce nonspecific symptoms (e.g., nausea, diarrhea, and rashes), making it difficult to diagnose.In the second (chronic) phase, parasites predominantly reside in certain tissues, such as those of the heart, esophagus, colon, and peripheral nervous system.Even though the infection may remain latent for decades, 30% of infected individuals eventually develop cardiac and/or intestinal complications [5,6].
Regarding Chagas disease, the only drugs known to provide successful treatment are nifurtimox (10) [9] and benznidazole (11) [10] (Figure 3).Unfortunately, these two compounds are expensive and highly toxic [11].Their multiple adverse effects are often intensified by the long-term administration required for an efficacious treatment.Furthermore, both nifurtimox and benznidazole are only effective at the onset of the disease, not during the chronic phase [12].Hence, it is important to develop new therapeutic drugs with a low toxicity that are capable of treating the chronic phase as well as the onset of Chagas disease.
Contradictory reports have been published on the toxicity and side effects of pentamidine isethionate (9), employed in the treatment of trypanosomiasis and leishmaniasis (Figure 2).Piccica et al. [13] stated that there are very few cases of adverse effects, although fatalities are among them.According to Kuhlmann et al. [14], 50% of patients undergo side effects, which include arrhythmia, syncope, vomiting, hyperglycemia, and reversible kidney damage.
Regarding Chagas disease, the only drugs known to provide successful treatment are nifurtimox (10) [9] and benznidazole (11) [10] (Figure 3).Unfortunately, these two compounds are expensive and highly toxic [11].Their multiple adverse effects are often intensified by the long-term administration required for an efficacious treatment.Furthermore, both nifurtimox and benznidazole are only effective at the onset of the disease, not during the chronic phase [12].Hence, it is important to develop new therapeutic drugs with a low toxicity that are capable of treating the chronic phase as well as the onset of Chagas disease.Figure 2. Other drugs tested against Leishmania: amphotericin (3), miltefosine (4), allopurinol (5), ketoconazole (6), metronidazole (7), pentamidine (8), and pentamidine isethionate (9).Contradictory reports have been published on the toxicity and side effects of pentamidine isethionate (9), employed in the treatment of trypanosomiasis and leishmaniasis (Figure 2).Piccica et al. [13] stated that there are very few cases of adverse effects, although fatalities are among them.According to Kuhlmann et al. [14], 50% of patients undergo side effects, which include arrhythmia, syncope, vomiting, hyperglycemia, and reversible kidney damage.
The aim of the current contribution was to synthesize new 2-(4-alkyloxyphenyl)-imidazoles and imidazolines and evaluate them in vitro on L. mexicana and T. cruzi.(10) and benznidazole (11).

Chemical Basis of Bioisosteres
Since pentamidines have an amidine group in their chemical structure, they are easily modified to promote the formation of nonclassical bioisosteres (imidazolines and imidazoles).The central chain can occupy the lateral position, still as an alkyl group with an aromatic ring.The amidine group closes in imidazolines or imidazoles, thus generating nonclassical bioisosteres of pentamidine.These new compounds could possibly possess better properties than pentamidine.Indeed, the treatment of T. cruzi and L. mexicana with imidazoles as antiprotozoal compounds has already been reported [15].Moreover, our group has synthesized some imidazolines and imidazoles and tested them on two bacteria, Chromobacterium violaceum and Serratia marcescens [16][17][18].To our knowledge, the compounds herein synthesized have not been examined as either leishmanicidal or trypanocidal agents.

Synthesis of Imidazolines
The synthesis of imidazolines has been the focus of many studies because numerous compounds with this ring have important applications, ranging from pharmacological therapy to sensors [19,20].The basis of the present synthesis of imidazolines is the procedure previously described by our group [17,18], improved by adopting MW and ultrasound as energy sources.Firstly (step a), an n-alkyl chain was introduced through alkylation of the phenol group of para-hydroxy benzaldehyde (Table 1).idazoles and imidazolines and evaluate them in vitro on L. mexicana and T. cruzi.

Chemical Basis of Bioisosteres
Since pentamidines have an amidine group in their chemical structure, they are easily modified to promote the formation of nonclassical bioisosteres (imidazolines and imidazoles).The central chain can occupy the lateral position, still as an alkyl group with an aromatic ring.The amidine group closes in imidazolines or imidazoles, thus generating nonclassical bioisosteres of pentamidine.These new compounds could possibly possess better properties than pentamidine.Indeed, the treatment of T. cruzi and L. mexicana with imidazoles as antiprotozoal compounds has already been reported [15].Moreover, our group has synthesized some imidazolines and imidazoles and tested them on two bacteria, Chromobacterium violaceum and Serratia marcescens [16][17][18].To our knowledge, the compounds herein synthesized have not been examined as either leishmanicidal or trypanocidal agents.

Synthesis of Imidazolines
The synthesis of imidazolines has been the focus of many studies because numerous compounds with this ring have important applications, ranging from pharmacological therapy to sensors [19,20].The basis of the present synthesis of imidazolines is the procedure previously described by our group [17,18], improved by adopting MW and ultrasound as energy sources.Firstly (step a), an n-alkyl chain was introduced through alkylation of the phenol group of para-hydroxy benzaldehyde (Table 1).With the intermediate on hand, the synthesis of imidazolines from aldehydes is a relatively simple process involving ethylenediamine and an oxidizing agent.When K 2 CO 3 , I 2 , and t-BuOH were combined at 70 • C, the reaction took 4-5 h.On the other hand, the reaction of NBS and CH 2 Cl 2 at room temperature (rt) lasted up to 16 h [21].These methods afford good to excellent yields, but the reaction times are long.From the chemical point of view, almost no reaction or product is surprising, considering the numerous existing catalysts and novel methods developed to synthesize sophisticated compounds.Given the broad range of options, many researchers in organic synthesis select synthetic routes that employ the principles of green chemistry, with the aim of having the lowest impact on the environment.Accordingly, MW and ultrasound were chosen as energy sources and compared to conventional heating.
As mentioned above, Sant' Anna et al. [22] described the synthesis of an imidazoline by utilizing ultrasound energy, NBS, and H 2 O.They managed to decrease the reaction time from hours to just minutes.Taking this work as the starting point, some experiments were carried out to synthesize 8-octyloxyphenylimidazoline (Table 2).impact on the environment.Accordingly, MW and ultrasound were chosen as energy sources and compared to conventional heating.
As mentioned above, Sant' Anna et al. [22] described the synthesis of an imidazoline by utilizing ultrasound energy, NBS, and H2O.They managed to decrease the reaction time from hours to just minutes.Taking this work as the starting point, some experiments were carried out to synthesize 8-octyloxyphenylimidazoline (Table 2).The lack of reaction found in experiments B and C could be due to the insolubility of the raw material in water and even in ethanol water.The other outcomes were also unattractive, even though experiment F gave almost the same yield with a drastic reduction in reaction time.
Subsequently, MW and ultrasound energy were used.Imidazolines containing short chains (22)(23)(24)(25)(26) were not included because these compounds provide good yields with both conventional heating and MW.The results of reacting compounds with carbon chains C5-C9 are shown in Table 3 (27)(28)(29)(30)(31), showing almost the same yields with MW and ultrasound energy, but in half the time with the latter.routes that employ the principles of green chemistry, with the aim of having the lowest impact on the environment.Accordingly, MW and ultrasound were chosen as energy sources and compared to conventional heating.As mentioned above, Sant' Anna et al. [22] described the synthesis of an imidazoline by utilizing ultrasound energy, NBS, and H2O.They managed to decrease the reaction time from hours to just minutes.Taking this work as the starting point, some experiments were carried out to synthesize 8-octyloxyphenylimidazoline (Table 2).The lack of reaction found in experiments B and C could be due to the insolubility of the raw material in water and even in ethanol water.The other outcomes were also unattractive, even though experiment F gave almost the same yield with a drastic reduction in reaction time.
Subsequently, MW and ultrasound energy were used.Imidazolines containing short chains (22)(23)(24)(25)(26) were not included because these compounds provide good yields with both conventional heating and MW.The results of reacting compounds with carbon chains C5-C9 are shown in Table 3 (27)(28)(29)(30)(31), showing almost the same yields with MW and ultrasound energy, but in half the time with the latter.The lack of reaction found in experiments B and C could be due to the insolubility of the raw material in water and even in ethanol water.The other outcomes were also unattractive, even though experiment F gave almost the same yield with a drastic reduction in reaction time.
Subsequently, MW and ultrasound energy were used.Imidazolines containing short chains (22)(23)(24)(25)(26) were not included because these compounds provide good yields with both conventional heating and MW.The results of reacting compounds with carbon chains C 5 -C 9 are shown in Table 3 (27)(28)(29)(30)(31), showing almost the same yields with MW and ultrasound energy, but in half the time with the latter.

Synthesis of Imidazoles
Imidazoles were synthesized by starting from imidazolines and oxidizing the imidazoline ring.Although NBS can work well to oxidize oxazolines to oxazoles, it did not work in this case.The other oxidant utilized was MnO2, which has been reported for the synthesis of imidazoles [23,24].Since the number of equivalents used in the reaction was not specified, the current experiments started with five equivalents, and the concentration was gradually increased to try to improve the yield (Table 4).

Synthesis of Imidazoles
Imidazoles were synthesized by starting from imidazolines and oxidizing the imidazoline ring.Although NBS can work well to oxidize oxazolines to oxazoles, it did not work in this case.The other oxidant utilized was MnO2, which has been reported for the synthesis of imidazoles [23,24].Since the number of equivalents used in the reaction was not specified, the current experiments started with five equivalents, and the concentration was gradually increased to try to improve the yield (Table 4).With the intermediate on hand, the synthesis of imidazolines from aldehydes is a relatively simple process involving ethylenediamine and an oxidizing agent.When K2CO3, I2, and t-BuOH were combined at 70 °C, the reaction took 4-5 h.On the other hand, the reaction of NBS and CH2Cl2 at room temperature (rt) lasted up to 16 h [21].These methods afford good to excellent yields, but the reaction times are long.From the chemical point of view, almost no reaction or product is surprising, considering the numerous existing catalysts and novel methods developed to synthesize sophisticated compounds.Given the broad range of options, many researchers in organic synthesis select synthetic routes that employ the principles of green chemistry, with the aim of having the lowest impact on the environment.Accordingly, MW and ultrasound were chosen as energy sources and compared to conventional heating.
As mentioned above, Sant' Anna et al. [22] described the synthesis of an imidazoline by utilizing ultrasound energy, NBS, and H2O.They managed to decrease the reaction time from hours to just minutes.Taking this work as the starting point, some experiments were carried out to synthesize 8-octyloxyphenylimidazoline (Table 2).The lack of reaction found in experiments B and C could be due to the insolubility of the raw material in water and even in ethanol water.The other outcomes were also unattractive, even though experiment F gave almost the same yield with a drastic reduction in reaction time.
Subsequently, MW and ultrasound energy were used.Imidazolines containing short chains (22)(23)(24)(25)(26) were not included because these compounds provide good yields with both conventional heating and MW.The results of reacting compounds with carbon chains C5-C9 are shown in Table 3 (27-31

Synthesis of Imidazoles
Imidazoles were synthesized by starting from imidazolines and oxidizing the imidazoline ring.Although NBS can work well to oxidize oxazolines to oxazoles, it did not work in this case.The other oxidant utilized was MnO 2 , which has been reported for the synthesis of imidazoles [23,24].Since the number of equivalents used in the reaction was not specified, the current experiments started with five equivalents, and the concentration was gradually increased to try to improve the yield (Table 4).

Synthesis of Imidazoles
Imidazoles were synthesized by starting from imidazolines and oxidizing the imidazoline ring.Although NBS can work well to oxidize oxazolines to oxazoles, it did not work in this case.The other oxidant utilized was MnO2, which has been reported for the synthesis of imidazoles [23,24].Since the number of equivalents used in the reaction was not specified, the current experiments started with five equivalents, and the concentration was gradually increased to try to improve the yield (Table 4).Based on the data in Table 4, imidazoles 32-36 were synthesized (Table 5).Based on the data in Table 4, imidazoles 32-36 were synthesized (Table 5).

Synthesis of the Diol
The diol (37) was prepared according to our previously reported methodology [25] (Figure 4).

Synthesis of the Diol
The diol (37) was prepared according to our previously reported methodology [25] (Figure 4).Based on the data in Table 4, imidazoles 32-36 were synthesized (Table 5).

Synthesis of the Diol
The diol (37) was prepared according to our previously reported methodology [25] (Figure 4).
On the other hand, activity against T. cruzi has been found for several heterocycle compounds.These include 2-, 4-, and 5-substituted imidazoles with good activity [15] and Some compounds containing five-membered heterocycles have been investigated for their leishmanicidal activity.For instance, Fluconazole and Itraconazole are active against leishmaniasis, but the former has serious side effects and the latter showed low effectiveness [4].Pyrazolo-pyridazinones are active against leishmaniasis when the disease is caused by Leishmania amazonensis [26].In addition, benzimidazole derivatives are selective inhibitors of arginase from Leishmania.The latter derivatives show biological activity against promastigotes and amastigotes [27].
On the other hand, activity against T. cruzi has been found for several heterocycle compounds.These include 2-, 4-, and 5-substituted imidazoles with good activity [15] and imidazole-containing nitrophthalazine derivatives [28].The activity of quinones possessing triazine derivatives is attributed to the reducing power of quinone rather than the heterocycle [29].

Biological Evaluation
In Vitro Evaluation of Some Compounds on Leishmania mexicana and Trypanosoma cruzi Some of the synthesized compounds (22-23, 26-31, 33, 35-38) were chosen to assess their in vitro effect on L. mexicana promastigotes and T. cruzi epimastigotes, expressed as the half inhibitory concentration (IC 50 ).Compounds 37 and 38 were tested only on T. cruzi.The cytotoxicity of the compounds was examined on murine macrophages.Based on the resulting data, the selectivity index (SI) was calculated for all thirteen compounds (Table 6).As known, a compound with acceptable activity should have an SI above 10.
The leishmanicidal activity of ten of the thirteen compounds was expressed as an IC 50 < 10 µg/mL.Among these compounds, 27, 28, 29, 30, and 31 were the most active, with IC 50 values < 1 µg/mL (similar to the reference drugs).By relating the leishmanicidal activity to the cytotoxic effect, the SI can be determined.Various researchers suggest that compounds of interest should present an SI ≥ 10 [30], which was the case for all ten compounds.The compound with the greatest leishmanicidal activity was 28, followed by 30, 29, and 31 in descending order.
In the evaluation on epimastigotes of T. cruzi, only 30 and 36 reached an IC 50 < 1 µg/mL, showing better inhibition than both reference drugs.However, compounds 29, 33, and 35 also demonstrated attractive trypanocidal activities, with IC 50 values < 10 µg/mL, similar to the values for Bnz and Nfx.By analyzing the relation of trypanocidal activity to toxicity, a low SI was found for 29, indicating that it should be discarded.The SI values of 30, 33, 35, and 36 were above 10, with the latter compound being the most selective, surpassing both reference drugs.Compounds 30, 33, 35, and 36 were active against T. cruzi and L. mexicana.In contrast, 27 and 28 only exhibited leishmanicidal activity.
It is important to measure the cytotoxic effect along with the antiparasitic activity in order to understand the intensity of the effect of compounds on parasite versus mammalian cells (herein represented by a murine macrophage cell line).Mammals constitute the hosts of both parasites.Compounds 22 and 23 exhibited the least cytotoxic effect but are not of interest due to their high IC 50 values.The values in parentheses represent confidence interval determined by the Probit method with 96% confidence; ND: not determined; AmB = amphotericin; Bnz = benznidazole; Nfx = nifurtimox.All tests were performed in triplicate.

Structure-Activity Relationship
When considering the distinct alkyloxy substitution of each imidazoline derivative (22-28 and 30), the resulting activity on L. mexicana promastigotes displayed a clear trend in relation to the molecular structure.Molecules with carbon chains longer than four carbons exhibited IC 50 values under 1 µg/mL.Compounds 27 (C5), 28 (C6), 29 (C7), and 30 (C8) showed the lowest values, at 0.808, 0.175, 0.2022, and 0.2020 µg/mL, respectively.The trend was similar for T. cruzi epimastigotes, although 27 and 28 had a lesser effect.The methyloxy (22) and ethyloxy (23) derivatives of imidazoline are regarded as inactive because of their elevated IC 50 values.Compounds with a carbon chain length between four and six carbons (26-28) afforded double-digit IC 50 values.The best IC 50 (0.628 µg/mL) was obtained with the octyloxy derivative (30), but 36 also presented a very good IC 50 (0.6337 µg/mL).
The evaluation of the safety of the compounds on macrophages resulted in significantly higher 50% cellular cytotoxicity concentration (CC 50 ) values than the IC 50 values with respect to L. mexicana.Therefore, the SI values were good (>10) in all cases.Given that the IC 50 values were greater on T. cruzi, the corresponding SI values were lower.Of all the imidazolines tested on T. cruzi, only 30 (C8) had an SI value > 10, which was better than the 8.31 value found for benznidazole (one of the reference drugs).In contrast, the three imidazoles (33, 35, and 36) tested on T. cruzi gave an SI > 10.
A trend could be observed in the group of hexyl (33), octyl (35), and nonyloxy (36) imidazole derivatives.Longer alkyl chains consistently produced good IC 50 values against L. mexicana and T. cruzi.Thus, 36 exhibited the best IC 50 values for L. mexicana (1.095 µg/mL) and T. cruzi (0.6337 µg/mL).Another pattern identified was the increased cytotoxicity associated with a greater number of carbons in alkyl substituents.Three imidazoles (33-36) exhibited a very positive outcome, each with an SI value over 10.The most attractive compound was 36, with an SI superior to that of each of the two reference drugs.
For the purpose of structural screening, the present study included a diol compound (37) containing the previously described hexyloxyphenyl fragment as a dimer, connected through a vicinal alcohol.Taking the inactivity of 37 into account, this fragment was not responsible for the antiprotozoal activity of the other compounds, pointing to the probable importance of the imidazoline and imidazole moieties.Finally, a 3-substituted quinoxalin-2(1H)-one derivative (38) was examined to determine the selectivity of the method of activity evaluation on T. cruzi and L. mexicana; as expected, no effect was detected.

General
Reagents and solvents were purchased from Sigma Aldrich (Toluca, Mexico) and used without further purification.The reactions were monitored via thin-layer chromatography (TLC) on Merck F253 silica gel aluminum sheets.Spots were visualized with UV light (254 nm) and iodine.Energy was provided by a Prendo chemical microwave oven MIC-1 (Puebla, Mexico) with a maximum power of 600 W and an ND Scientific ultrasonicator (model A150) with a maximum power of 150 W and frequency of 20-25 KHz.Melting points were determined on an Electrothermal MELT-THEMP apparatus (Electrothermal, Burlington, NJ, USA) and were uncorrected. 1H and 13 C NMR spectra of the compounds were recorded on a Varian NMR System (500 MHz and 125 MHz) and a Varian Mercury (300 MHz and 75 MHz), respectively, assigning the peaks with input from 2D experiments (gHSQC and gHMBC).The chemical shifts (δ) are expressed in ppm.MS spectra were acquired on Bruker Amazon Speed apparatus (ESI) (Bruker, Bremen, Germany) via the direct insertion probe-electrospray ionization-mass spectrometry (DIP-ESI-MS) technique.IR spectra were captured on a Perkin Elmer FT-IR Spectrum 2000 spectrometer (Shelton, CT, USA) at the ENCB-IPN spectroscopy instrumentation center.Selected spectra are attached in the Supplementary Materials.

Synthesis of 4-Alkyloxybenzaldehydes
To 30 mL of freshly distilled acetone were added 1 g (8.2 mmol, 1 eq.) of 4-hydroxy benzaldehyde and 2.59 g (16.4 mmol, 2 eq.) of potassium carbonate, and the solution was placed in an ultrasound apparatus (P = 60 W, f = 20-25 KHz) at 40 • C for 30 min.Subsequently, 0.92 mL (9.84 mmol, 1.1 eq.) of haloalkyl was injected and the reaction was continued for 1.5 h under the same conditions.Upon completion of the reaction, the solution was cooled to room temperature.Potassium carbonate was filtered and washed with 150 mL of dichloromethane.The combined organic layers were evaporated under reduced pressure.The crude material was purified with column chromatography on silica gel, using a polarity gradient with different hexane/ethyl acetate ratios (95:5, 9:1, 8:2, and 7:3).The fractions with a pure product were evaporated under reduced pressure and finally dried under vacuum, yielding a yellow liquid that was characterized via 1 H and 13 C NMR as well as IR and mass spectrometry.Data from the characterization of the compounds are described hereafter.

In Vitro Evaluation of the Metabolic Inhibition of Leishmania mexicana by Fluorometric Analysis with Resazurin
A promastigote culture of L. mexicana was harvested in the stationary phase after 7 days of growth in culture.To each well of a sterile 96-well microplate, 5 × 10 5 promastigotes and the compound to be assessed (or the reference drug) were added to a final volume of 100 µL in RPMI culture medium supplemented with 10% SFB and 1% ampicillin/streptomycin.Each compound was examined at different concentrations, obtained by serial two-fold dilutions starting from 50 µg/mL.The microplate was incubated in the dark at 27 • C for 24 h.Promastigotes with 0.5% DMSO (at the vehicle concentration of the greatest dilution of the compound) served as the positive viability control and amphotericin B at a final concentration of 5 µg/mL served as the negative viability control.Each condition was assayed in triplicate.
After 24 h of incubation, 10 µL of 0.01% resazurin was added to each of the wells and incubation continued for another 24 h under the same conditions.The plate was read via a fluorometer (Spectramax M3, Molecular Devices, LLC, San Jose, CA, USA) at 544 nm excitation and 590 nm emission.Each treated well was compared to the positive viability control, and metabolic inhibition was expressed as a percentage.The IC 50 value was determined with the Probit statistical tool version 4.1 [31][32][33].
Resazurin is a blue, permeable, and non-fluorescent compound.Upon entering cells, it is reduced to resorufin due to the reducing environment of the cytosol in living cells.Resorufin is red and highly fluorescent.The number of living cells is proportional to the fluorescence intensity of resorufin [31].Epimastigotes of T. cruzi INC-5 were harvested after 7 days of growth in culture.In each well of a sterile 96-well microplate, 1 × 10 6 epimastigotes and the compound to be tested (or the reference drug) were added to a final volume of 100 µL in brain heart infusion (BHI) culture medium supplemented with 10% FBS and 1% ampicillin/streptomycin.Each compound was examined at various concentrations prepared through serial two-fold dilutions starting from 50 µg/mL.The microplate was incubated in the dark at 27 • C for 24 h.Epimastigotes with 0.5% DMSO served as the positive viability control and the reference drugs nifurtimox (Lampit, Bayer, Germany) and benznidazole (Rochagan, Roche, Brazil) served as the negative viability control.Each condition was assayed in triplicate.
Upon completion of the incubation time, 10 µL of MTT solution at 5 mg/mL was added to each well, and incubation continued in the dark at 27 • C for 20 h.Then, 100 µL of a solution containing 10% SDS and 0.01 M HCl was added to stop the reaction.To dissolve the formazan crystals formed by the metabolism of viable epimastigotes, the mixture was left to stand for 4 h.The plates were read on a spectrophotometer (Spectramax Plus, Molecular Devices) at an absorbance of 570 nm.The result found in each well with a given concentration of the respective compound was compared to the positive viability control and the metabolic inhibition was expressed as a percentage.The IC 50 was determined using the Probit statistical tool [33].

:
Ultrasound (P = 60 W, f = 20-25 KHz); NR, no reaction and the recovery of the raw materials.
conventional heating at 70 °C; b, : Ultrasound (P = 60 W, f = 20-25 KHz); NR, no reaction and the recovery of the raw materials.

Table 2 .
(30)arison of the results of the synthesis of 8-octyloxyphenylimidazoline(30)with conventional heating and microwave energy.

Table 2 .
Comparison of the results of the synthesis of 8-octyloxyphenylimidazoline (30) with conventional heating and microwave energy.

Table 2 .
Comparison of the results of the synthesis of 8-octyloxyphenylimidazoline (30) with conventional heating and microwave energy.

Table 2 .
Comparison of the results of the synthesis of 8-octyloxyphenylimidazoline (30) with conventional heating and microwave energy.

Table 6 .
Effect of the synthesized compounds on L. mexicana promastigotes, T. cruzi epimastigotes, and murine macrophages.