Synthesis of Nitrostyrylthiazolidine-2,4-dione Derivatives Displaying Antileishmanial Potential

A series of 61 thiazolidine-2,4-diones bearing a styryl group at position 5 was synthesized in 2–5 steps and their structure was proved by elemental and spectral analyses. The compounds obtained were evaluated in vitro against the promastigote stage of the kinetoplastid parasite Leishmania infantum and the human HepG2 cell line, to determine selectivity indices and to compare their activities with those of antileishmanial reference drugs. The study of structure–activity relationships indicated the potential of some derivatives bearing a nitro group on the phenyl ring, especially when located at the meta position. Thus, among the tested series, compound 14c appeared as a hit compound with good antileishmanial activity (EC50 = 7 µM) and low cytotoxicity against both the hepatic HepG2 and macrophage THP-1 human cell lines (CC50 = 101 and 121 µM, respectively), leading to good selectivity indices (respectively, 14 and 17), in comparison with the reference antileishmanial drug compound miltefosine (EC50 = 3.3 µM, CC50 = 85 and 30 µM, SI = 26 and 9). Regarding its mechanism of action, among several possibilities, it was demonstrated that compound 14c is a prodrug bioactivated, predominantly by L. donovani nitroreductase 1, likely leading to the formation of cytotoxic metabolites that form covalent adducts in the parasite. Finally, compound 14c is lipophilic (measured CHI LogD7.7 = 2.85) but remains soluble in water (measured PBS solubility at pH7.4 = 16 µM), highlighting the antileishmanial potential of the nitrostyrylthiazolidine-2,4-dione scaffold.


Introduction
Leishmaniasis is a neglected tropical disease, caused by a parasite belonging to the genus Leishmania.The infection is transmitted through a bite of a female Phlebotomine sandfly infected with the protozoan.According to the World Health Organization (WHO), leishmaniasis is the second major tropical parasitic killer disease after malaria [1].Currently, it affects 12 million people in 98 countries mainly in tropical and sub-tropical regions, and in southern Europe, especially around the Mediterranean area.There are three clinical forms of leishmaniasis: cutaneous (CL), mucocutaneous (MCL), and visceral (VL).The first type is the most common while the third one is the most severe, mainly caused by Leishmania donovani and Leishmania infantum, targeting visceral organs such as the spleen and liver [1].Currently, there is no vaccine available for any form of human leishmaniasis [1].Moreover, there is a limited number of available drugs representing major therapeutic limitations including severe side effects and increasing parasite drug-resistance issues.
The available drugs for the treatment of VL include sodium stibogluconate (pentavalent antimonials), miltefosine, pentamidine, paromomycin, and amphotericin B. However, these drugs are outdated, showing serious side effects such as renal or cardiac toxicity, teratogenicity, and parasite resistance.Moreover, today, miltefosine remains the only orally available antileishmanial drug against VL.This matter calls for the discovery of new antileishmanial chemical entities, with both original mechanisms of action and oral route of administration, to improve and facilitate the therapeutic care of the infected populations, especially in endemic developing countries [2][3][4].
Thiazolidinediones (TZDs), also known as Glitazones, are an important class of heterocycles abundantly reported in the literature.Initially, certain derivatives were used as insulin sensitizers for the treatment of Type 2 diabetes mellitus (T2DM) due to their PPAR-γ activation properties [27].However, the TZDs, clinically used as anti-diabetes mellitus drugs, were later reported to suffer from several serious side effects and thus many of them were withdrawn from the market [28].However, pioglitazone is still marketed in the USA and Lobeglitazone was recently approved in India for the treatment of Type 2 diabetes.
A literature survey revealed that TZDs possess a very wide range of biological activities, which are included in the reference [29], such as antidiabetic, anti-infectious, antiinflammatory, analgesic, antioxidant, immunomodulatory, overactive bladder inhibitory and insecticide activities.TZDs also possess inhibitory activity against tyrosinase, hyperlipidemia, and acute liver injury.Furthermore, it was reported that TZDs affected cancer development, progression and metastasis, among which the Raf/MEK/extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K)/AKT, Wnt signal transduction pathways, PIM-1 and PIM-2 protein kinases overexpression, receptor-stimulated genes and peroxisome proliferator-activated receptors (PPARs) signaling cascades which are the most commonly up-regulated in human cancers [29].It is important to note that the TZD derivative S49076 has reached Phase I clinical trials against solid tumors [30].On the other hand, the potential of TZD against leishmaniasis has not yet been explored, with the exception of a single study [31].
In the present work, we synthesized a library of sixty-one 5-stryryl-3-substituted thiazolidine-2,4-dione derivatives, in an endeavor to evaluate their in vitro antileishmanial activity.
Table 1.1,3-Thiazolidine-2,4-diones (12a-e to 16a-e).Compound 1 is a versatile heterocycle that allows the synthesis of various thiazolidinediones by virtue of its two reactive centers: (A) the acidic NH hydrogen at position 3 and (B) the active-methylene group at position 5. Thus, when this 5-methylene group was allowed to react, under Knoevenagel reaction conditions, with the aromatic aldehydes; benzaldehyde, o-, m-, p-nitrobenzaldehyde, and 3,4,5-trimethoxy benzaldehyde, the corresponding 5-stryryl-1,3-thiazolidine-2,4-diones 2-6 were obtained (Scheme 1) in 75-90% yields.These latter compounds 2-6, could be easily converted into the corresponding potassium salts 7-11 in 69-90% yields upon treatment with KOH in boiling ethanol or ethanol-dioxane mixture (Scheme 1).The reaction of the nucleophilic N-anion of the latter potassium salts 7-11 with a series of halo-compounds namely ethyl chloroacetate, chloroacetone, chloroacetonitrile, 2-chloroacetamide, and phenacyl bromide in refluxing DMF, resulted in the formation of a series of the corresponding target (Z)-N-substituted 5-styrylthiazolidinediones 12a-e to 16a-e (Scheme 2, Table 1) in 62-95% yields.2.  2. All the obtained thiazolidinediones were found to possess exclusively a Z-configuration as evidenced by their 1 H-NMR spectra.The methylidene =CH-proton of these compounds appeared as a deshielded singlet at 8.25-7.79ppm.This deshielding is caused by the anisotropic effect of the neighboring C=O group at the 4-position.On the other hand, no signal indicating the presence of the E-isomer was observed.The =CH-proton of the E-isomer should appear around 6.2-6.3 ppm, due to the less deshielding effect of the S atom of the TZD ring [29].Moreover, the Z-configuration is more thermodynamically stable due to the hydrogen bonding between methylidene hydrogen and the 4-C=O group.The X-ray single crystal analysis of compound 6 is a further unambiguous confirmation of the Z-configuration of 2-6 [32].All the obtained thiazolidinediones were found to possess exclusively a Z-configuration as evidenced by their 1 H-NMR spectra.The methylidene =CH-proton of these compounds appeared as a deshielded singlet at 8.25-7.79ppm.This deshielding is caused by the anisotropic effect of the neighboring C=O group at the 4-position.On the other hand no signal indicating the presence of the E-isomer was observed.The =CH-proton of the E-isomer should appear around 6.2-6.3 ppm, due to the less deshielding effect of the S atom of the TZD ring [29].Moreover, the Z-configuration is more thermodynamically stable due to the hydrogen bonding between methylidene hydrogen and the 4-C=O group The x-ray single crystal analysis of compound 6 is a further unambiguous confirmation of the Z-configuration of 2-6 [32].
Finally, for discussing structure-activity relationships, the ethyl ester 12c was hydrolyzed into the corresponding carboxylic acid 29 (Scheme 5), following a procedure reported by Maccari et al. [33].Finally, for discussing structure-activity relationships, the ethyl ester 12c was hydrolyzed into the corresponding carboxylic acid 29 (Scheme 5), following a procedure reported by Maccari et al. [33].It is of interest to note that the 1 H-NMR spectrum of acid 29 did not show the signal corresponding to the carboxylic acid OH group.This could be explained by a rapid exchange of the acidic proton with the deuterium of the deuterated solvent DMSOd6.However, its 13 C-NMR spectrum showed the expected 12 carbons which includes the carbonyl carbon of the -COOH group (cf.Supplementary Material).

In Vitro Assays
All the compounds belonging to the 5-stryryl-1,3-thiazolidine-2,4-dione series (apart from corrosive potassium salts 7-11) were first evaluated in vitro on the promastigote stage of L. infantum, to determine their half-maximal effective concentrations (EC50), and to compare the obtained results with those of some antileishmanial reference drug compounds: amphotericin B (the most potent of all antileishmanial drugs), miltefosine (the unique orally available antileishmanial drug) and fexinidazole sulfone (the active metabolite of fexinidazole).Out of these 61 tested compounds, 22 were found to be insufficiently soluble in the aqueous culture medium to be properly evaluated.Among the 38 remaining molecules, 21 showed moderate to good antileishmanial activity with EC50 values ≤ 30 µM, including 19 nitroaromatic derivatives, as presented in Table 3.
It is of interest to note that the 1 H-NMR spectrum of acid 29 did not show the signal corresponding to the carboxylic acid OH group.This could be explained by a rapid exchange of the acidic proton with the deuterium of the deuterated solvent DMSO d6 .However, its 13 C-NMR spectrum showed the expected 12 carbons which includes the carbonyl carbon of the -COOH group (cf.Supplementary Material).

In Vitro Assays
All the compounds belonging to the 5-stryryl-1,3-thiazolidine-2,4-dione series (apart from corrosive potassium salts 7-11) were first evaluated in vitro on the promastigote stage of L. infantum, to determine their half-maximal effective concentrations (EC 50 ), and to compare the obtained results with those of some antileishmanial reference drug compounds: amphotericin B (the most potent of all antileishmanial drugs), miltefosine (the unique orally available antileishmanial drug) and fexinidazole sulfone (the active metabolite of fexinidazole).Out of these 61 tested compounds, 22 were found to be insufficiently soluble in the aqueous culture medium to be properly evaluated.Among the 38 remaining molecules, 21 showed moderate to good antileishmanial activity with EC 50 values ≤ 30 µM, including 19 nitroaromatic derivatives, as presented in Table 3. showed low cytotoxicity (CC50 ≥ 50 µM) and 4 hit compounds bearing a nitro group on the benzene ring of the styryl moiety (5, 12c, 12d, 14c) were thus highlighted, presenting good activities (5.5 ≤ EC50 ≤ 14 µM), low cytotoxicities (101 ≤ CC50 ≤ 125 µM) and good selectivity index values (10 ≤ SI ≤ 23), as shown in Table 3.This in vitro biological activity profile appeared rather similar to that of the miltefosine reference drug (EC50 = 3.3 µM, CC50 = 85 µM, SI = 26) but remained far from reaching the high potency of amphotericin B (EC50 = 0.06 µM).Table 3. Results of the in vitro evaluations (activity and cytotoxicity on HepG2) of the 21 compounds presenting both good solubility in the culture medium (Schneider's Drosophila Medium) and antileishmanial EC50 activity values < 30 µM.In terms of structure-activity relationships (SARs), it was initially observed that out of the 21 stryrylthiazolidinone derivatives that showed antileishmanial activity (EC50 ≤ 30 µM), had a nitro group on the benzene ring of the styryl moiety (Table 3).The pharmacophore appeared to be a 5-styrylthiazolidin-2,4-dione scaffold, substituted at positions meta or para of the phenyl ring by a nitro group (compounds 4 and 5).Compounds 12a and 27e were the only ones without a nitro group.Compound 27e showed a moderate selectivity index (SI = 3.9), whereas compound 12a was more promising (SI = 9.4), due to In terms of structure-activity relationships (SARs), it was initially observed that out of the 21 stryrylthiazolidinone derivatives that showed antileishmanial activity (EC50 ≤ 30 µM), had a nitro group on the benzene ring of the styryl moiety (Table 3).The pharmacophore appeared to be a 5-styrylthiazolidin-2,4-dione scaffold, substituted at positions meta or para of the phenyl ring by a nitro group (compounds 4 and 5).Compounds 12a and 27e were the only ones without a nitro group.Compound 27e showed a moderate selectivity index (SI = 3.9), whereas compound 12a was more promising (SI = 9.4), due to In terms of structure-activity relationships (SARs), it was initially observed that out of the 21 stryrylthiazolidinone derivatives that showed antileishmanial activity (EC50 ≤ 30 µM), had a nitro group on the benzene ring of the styryl moiety (Table 3).The pharmacophore appeared to be a 5-styrylthiazolidin-2,4-dione scaffold, substituted at positions meta or para of the phenyl ring by a nitro group (compounds 4 and 5).Compounds 12a and 27e were the only ones without a nitro group.Compound 27e showed a moderate selectivity index (SI = 3.9), whereas compound 12a was more promising (SI = 9.4), due to Then, the cytotoxicity of these 21 bioactive molecules was evaluated against the human hepatic HepG2 cell line and compared to the one of doxorubicin (chosen as a reference cytotoxic drug compound), by determining their 50% cytotoxic concentrations (CC 50 ), to calculate the corresponding selectivity indices (SI = CC 50 /EC 50 ).Eight molecules showed low cytotoxicity (CC 50 ≥ 50 µM) and 4 hit compounds bearing a nitro group on the benzene ring of the styryl moiety (5, 12c, 12d, 14c) were thus highlighted, presenting good activities (5.5 ≤ EC 50 ≤ 14 µM), low cytotoxicities (101 ≤ CC 50 ≤ 125 µM) and good selectivity index values (10 ≤ SI ≤ 23), as shown in Table 3.This in vitro biological activity profile appeared rather similar to that of the miltefosine reference drug (EC 50 = 3.3 µM, CC 50 = 85 µM, SI = 26) but remained far from reaching the high potency of amphotericin B (EC 50 = 0.06 µM).

Cpd
In terms of structure-activity relationships (SARs), it was initially observed that 19 out of the 21 stryrylthiazolidinone derivatives that showed antileishmanial activity (EC 50 ≤ 30 µM), had a nitro group on the benzene ring of the styryl moiety (Table 3).The pharmacophore appeared to be a 5-styrylthiazolidin-2,4-dione scaffold, substituted at positions meta or para of the phenyl ring by a nitro group (compounds 4 and 5).Compounds 12a and 27e were the only ones without a nitro group.Compound 27e showed a moderate selectivity index (SI = 3.9), whereas compound 12a was more promising (SI = 9.4), due to its low cytotoxicity, indicating a positive contribution of the ethyloxycarbonylmethyl group at position 3 of the thiazolidine-2,4-dione ring.
All the derivatives bearing a nitro group at the ortho position of the phenyl ring were moderately active and/or selective.Logically, combining an ethyloxycarbonylmethyl group at position 3 of the thiazolidine-2,4-dione ring and a nitro group at position meta of the phenyl ring led to a first hit compound (12c) with good efficacy (EC 50 = 5.51 µM) and selectivity (SI > 22.7).Maintaining the meta-nitrostyryl moiety and introducing a cyanomethyl group at position 3 of the thiazolidine-2,4-dione ring led to a second hit compound (14c), showing both activity (EC 50 = 7 µM) and selectivity (SI = 14.4).The paranitro isomers of 12c and 14c; 12d and 14d, were less active and less selective.Among all other substituents introduced at position 3 of the thiazolidine-2,4-dione ring, none allowed any improvement, neither regarding antileishmanial activity, nor cytotoxicity.Introducing a second nitroaromatic moiety on the pharmacophore (compounds 26b-d) was not favorable.
Examining the structure of the two hit compounds 12c and 14c revealed that 12c bears an ester function which is usually quite labile in vivo.Consequently, it was crucial to determine whether its carboxy metabolite was active or not.Thus, the ester compound 12c was subjected to acid hydrolysis to give the corresponding acid 29 (Scheme 5), and this latter was evaluated in vitro (Table 3).Unfortunately, despite being non-cytotoxic (CC 50 > 250 µM), carboxylic acid 29 was not active on L. infantum (EC 50 > 50 µM), making the ester 12c a poor candidate for further development.Thus, compound 14c was regarded as the best molecule in the series.Accordingly, it was assessed for its cytotoxicity on a second human cell line (THP-1) along with its lipophilicity and aqueous solubility, to evaluate its potential for further hit to lead chemistry (Table 4).Compound 14c was not cytotoxic on the THP-1 cell line (CC 50 = 121 µM), and presented a measured logD 7.4 value of 2.85, making it a lipophilic molecule.However, 14c showed a PBS (pH 7.4) solubility of 16 µM (logS = 4.8).These complementary parameters were in favor of pursuing the development of this chemical series to search for a new derivative with improved potency.
For decades, antiparasitic (more precisely antiprotozoal) properties were reported for many nitroaromatic compounds, including commercial drug compounds such as metronidazole, tinidazole, secnidazole, ornidazole (anti-T.vaginalis, anti-G.intestinalis, anti-E.histolytica), nifurtimox and benznidazole (anti-T.cruzi) or nitazoxanide (anti-C.parvum).These nitroaromatic compounds were shown to behave as prodrugs which are bioactivated in the parasite cell by nitroreductase enzymes (type 1 and/or 2), leading to lethal cytotoxic metabolites [4,34].Indeed, nitroaromatics remain attractive chemotypes for the conception of selective and potent novel antiparasitic candidates, such as recently approved fexinidazole (anti-T.brucei gambiense), but also to develop novel antibacterial compounds, as recently illustrated with the approval of antitubercular drug compounds delamanid and pretomanid.human cell line (THP-1) along with its lipophilicity and aqueous solubility, to potential for further hit to lead chemistry (Table 4).Compound 14c was not c the THP-1 cell line (CC50 = 121 µM), and presented a measured logD7.4value o ing it a lipophilic molecule.However, 14c showed a PBS (pH 7.4) solubility of = 4.8).These complementary parameters were in favor of pursuing the deve this chemical series to search for a new derivative with improved potency.For decades, antiparasitic (more precisely antiprotozoal) properties were many nitroaromatic compounds, including commercial drug compounds suc nidazole, tinidazole, secnidazole, ornidazole (anti-T.vaginalis, anti-G.intestin histolytica), nifurtimox and benznidazole (anti-T.cruzi) or nitazoxanide (anti These nitroaromatic compounds were shown to behave as prodrugs which vated in the parasite cell by nitroreductase enzymes (type 1 and/or 2), leadi cytotoxic metabolites [4,34].Indeed, nitroaromatics remain a ractive chemot conception of selective and potent novel antiparasitic candidates, such as r proved fexinidazole (anti-T.brucei gambiense), but also to develop novel antiba pounds, as recently illustrated with the approval of antitubercular drug delamanid and pretomanid.
To date, two leishmanial nitroreductases have been characterized: an es 1 mitochondrial NTR1 (PMID: 23208716) and a cytosolic type 2 NTR2 (PMID that catalyze the 1-electron reduction in nitroaromatics.To determine if NT bio-activation played a role in the mechanism of action of compounds in thi potency of 14c was assessed against both wild-type L. donovani (LdBOB)and t donovani overexpressing either NTR1 or NTR2.Indeed, parasites overexpressi troreductases were hyper-sensitive to 14c compared to wild-type, mo To date, two leishmanial nitroreductases have been characterized: an essential type 1 mitochondrial NTR1 (PMID: 23208716) and a cytosolic type 2 NTR2 (PMID: 27812217), that catalyze the 1-electron reduction in nitroaromatics.To determine if NTR-mediated bio-activation played a role in the mechanism of action of compounds in this series, the potency of 14c was assessed against both wild-type L. donovani (LdBOB)and transgenic L. donovani overexpressing either NTR1 or NTR2.Indeed, parasites overexpressing these nitroreductases were hyper-sensitive to 14c compared to wild-type, most notably promastigotes expressing elevated levels of NTR1 (Table 5).This strongly suggests that 14c is a substrate of Leishmania NTRs, and that bioactivation by these enzymes is a key factor in its antileishmanial activity.However, beyond the bioactivation of these nitroaromatic derivatives by parasitic NTRs, it is possible that stryrylthiazolidin-2,4-diones interact with other antileishmanial targets, such as reported in the literature dealing with inhibition of pteridine reductase 1 [31], perhaps explaining the antileishmanial activity noted with non-nitrated compounds 12a and 27e.

General Methods
The starting materials were purchased from Sigma-Aldrich (Saint Louis, MO, USA).All melting points were determined on a Stuart melting point apparatus SMP3 and are uncorrected.IR spectra were recorded on a Nicolet iS10 FT-IR spectrometer using KBr wafer technique.The 1 H-NMR spectra were recorded on a Bruker Avance 400 spectrometer operating at 400 MHz ( 1 H) or 100 MHz ( 13 C). 1 H-and 13 C-NMR chemical shifts (δ) were reported in parts per million (ppm) and were referenced to the residual proton peaks in the deuterated solvent DMSO-d 6 (2.50 ppm for 1 H and 39.70 ppm for 13 C).Multiplicities are represented by s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet).Coupling constants (J) are reported in Hertz (Hz).Elemental analyses (C, H, N) were carried out using a Perkin Elmer 240 C Micro analyzer at the Microanalytical Laboratory at Assiut University, and the results obtained were found in good agreement with the theoretical values within ±0.4%.All the compounds involved in biological evaluation showed a level of purity above 95%.Thiazolidine-2,4-dione (1) was prepared according to a literature procedure [31].
Lipophilicity was evaluated by the Chromatographic Hydrophobicity Index (CHI) [35] on a Restek Ultra C 18 column (5 µm particle size, 100 A porosity, 50 × 2.1 mm internal dimensions) using a gradient of A: 10 mM ammonium acetate, adjusted to pH 7.4; B: acetonitrile, at a flow rate of 0.300 mL/min and 0.5-µL injections.
The validity of the CHI versus retention time was established on a reference mixture containing theophylline, phenyltetrazole, benzimidazole, acetophenone, propiophenone, butyrophenone and valerophenone.The correlation between CHI and logD was taken from literature data: logD 7.4 CHI = 0.054 × CHI-1.467[35].

Organic Synthesis and Product Characterization
General Procedure for the Synthesis of the Potassium 5-Arylidenethiazolidin-2,4-dion-3-ides 7-11 A solution of potassium hydroxide (0.123 g, 2.2 mmol) in ethanol (7 mL) was added to a hot solution of compounds 2-6 (2 mmol) in an ethanol-dioxane mixture (30 mL).The reaction mixture was then heated under stirring for 20 min.The solid products obtained were filtered, washed with ethanol, dried, and crystallized.

In Vitro Cytotoxicity on the Human Hepatic HepG2 Cell Line
HepG2 cell line (hepatocarcinoma cell line purchased from ATCC, ref HB-8065) was maintained at 37 • C, 5% CO 2 with 90% humidity in MEM supplemented with 10% fetal bovine serum, 1% L-glutamine (200 mM) and penicillin (100 U/mL)/streptomycin (100 mg/mL) (complete MEM medium).The tested molecules' cytotoxicity was assessed according to the method of Mosmann [37] with slight modifications.Briefly, 5 × 10 3 cells in 100 µL of complete medium were inoculated into each well of 96-well plates and incubated at 37 • C in a humidified 5% CO 2 .After 24 h incubation, 100 µL of medium with various product concentrations dissolved in DMSO (final concentration less than 0.5% v/v) were added and the plates were incubated for 72 h at 37 • C. Triplicate assays were performed for each sample.Each plate well was then microscope-examined to detect possible precipitate formation before the medium was aspirated from the wells.Then, 100 µL of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) solution (0.5 mg/mL in medium without FCS) were then added to each well.Cells were incubated for 2 h at 37 • C, following which the MTT solution was removed and DMSO (100 µL) was added to dissolve the resulting blue formazan crystals.Plates were shaken vigorously (700 rpm) for 10 min.The absorbance was measured at 570 nm with 630 nm as reference wavelength using a TECAN Infinite F-200 spectrophotometer (Tecan Trading AG, Männedorf, Switzerland).DMSO was used as blank and doxorubicin (purchased from Sigma Aldrich, Saint Louis, MI, USA) as positive control.Cell viability was calculated as a percentage of control (cells incubated without compound).The 50% cytotoxic concentration (CC 50 ) was determined from the dose-response curve by using the Table Curve 2D v5.0 software (Systat Software Inc.; Palo Alto, CA, USA).CC 50 values represent the mean value calculated from three separate experiments.

In Vitro Cytotoxicity on the Human Monocyte THP-1 Cell Line
The undifferentiated THP1 human monocyte cells (acute monocytic leukemia cell line purchased from ATCC, ref TIB-202) were routinely grown in RPMI 1640 medium (Life Technologies, Saint-Aubin, France) supplemented with 10% FCS (Life Technologies, Saint-Aubin, France), 100 U/mL penicillin, 100 µg/mL streptomycin and 2 mM L-glutamine at 37 • C, 5% CO 2 .The culture was maintained between 3.10 5 and 8.10 5 cells/mL.The in vitro evaluation of the cytotoxicity of the tested compound was assessed according to the method of Mosmann [37] with slight modifications.Briefly, 100 µL of THP-1 cells with Phorbol 12-Myristate 13-Acetate (final concentration 50 ng/mL) were seeded in 96-well plates at an average density of 1 × 10 6 cells/mL and incubated for 24 h at 37 • C, 5% CO 2 .Wells were washed with RPMI and 100 µL of fresh complete medium was added.Then,

Table 3 .
Results of the in vitro evaluations (activity and cytotoxicity on HepG2) of the 21 compounds presenting both good solubility in the culture medium (Schneider's Drosophila Medium) and antileishmanial EC 50 activity values < 30 µM.

Table 5 .
Sensitivity of wild-type and NTR-overexpressing L. donovani promastigotes to compound

14c. Cell Line EC 50 Values, nM 14c Nifurtimox Delamanid
Data represent the weighted mean ± standard deviation for five independent experiments.