Improving Aqueous Solubility and In Vitro Pharmacokinetic Properties of the 3-Nitroimidazo[1,2-a]pyridine Antileishmanial Pharmacophore

An antileishmanial structure–activity relationship (SAR) study focused on positions 2 and 8 of the imidazo[1,2-a]pyridine ring was conducted through the synthesis of 22 new derivatives. After being screened on the promatigote and axenic amastigote stages of Leishmania donovani and L. infantum, the best compounds were tested against the intracellular amastigote stage of L. infantum and evaluated regarding their in vitro physicochemical and pharmacokinetic properties, leading to the discovery of a new antileishmanial6-chloro-3-nitro-8-(pyridin-4-yl)-2-[(3,3,3-trifluoropropylsulfonyl)methyl]imidazo[1,2-a]pyridine hit. It displayed low cytotoxicities on both HepG2 and THP1 cell lines (CC50 > 100 µM) associated with a good activity against the intracellular amastigote stage of L. infantum (EC50 = 3.7 µM versus 0.4 and 15.9 µM for miltefosine and fexinidazole, used as antileishmanial drug references). Moreover, in comparison with previously reported derivatives in the studied series, this compound displayed greatly improved aqueous solubility, good mouse microsomal stability (T1/2 > 40 min) and high gastrointestinal permeability in a PAMPA model, making it an ideal candidate for further in vivo studies on an infectious mouse model.


Introduction
Neglected tropical diseases (NTDs) are defined by the World Health Organization (WHO) as a heterogeneous group of infections mainly found in developing countries, especially in tropical and subtropical environments, and are of little interest to the pharmaceutical industry [1,2]. Among these NTDs, leishmaniases are vector-borne parasitic diseases caused by several species of flagellated protozoa of the genus Leishmania. More than 1 billion people in 98 countries are at risk of infection, and nearly 1 million new cases occur annually [3]. There are three main forms of the disease, cutaneous leishmaniasis (CL), In order to develop new original nitroheterocyclic antikinetoplastid molecules, our team identified a first antileishmanial hit compound in 8-halogeno-3-nitroimidazo[1,2a]pyridine series (Hit A) [19]. Introduction of a 4-chlorophenylthioether moiety at position 8 led to a new derivative (Hit B) with improved in vitro antileishmanial activity but showing a poor aqueous solubility (thermodynamic solubility = 1.4 µ M) and a poor mouse liver microsomal stability (T1/2 = 3 min) (Table 1) [20]. Its sulfoxide and sulfone metabolites were identified and appeared active, but were also rapidly metabolized in vitro (T1/2 = 3 min). In order to develop new original nitroheterocyclic antikinetoplastid molecules, our team identified a first antileishmanial hit compound in 8-halogeno-3-nitroimidazo[1,2a]pyridine series (Hit A) [19]. Introduction of a 4-chlorophenylthioether moiety at position 8 led to a new derivative (Hit B) with improved in vitro antileishmanial activity but showing a poor aqueous solubility (thermodynamic solubility = 1.4 µM) and a poor mouse liver microsomal stability (T 1/2 = 3 min) (Table 1) [20]. Its sulfoxide and sulfone metabolites were identified and appeared active, but were also rapidly metabolized in vitro (T 1/2 = 3 min). The para position of the phenyl ring at position 2 was also identified as potentially oxidized.  In order to develop new original nitroheterocyclic antikinetoplastid molecules, our team identified a first antileishmanial hit compound in 8-halogeno-3-nitroimidazo[1,2a]pyridine series (Hit A) [19]. Introduction of a 4-chlorophenylthioether moiety at position 8 led to a new derivative (Hit B) with improved in vitro antileishmanial activity but showing a poor aqueous solubility (thermodynamic solubility = 1.4 µ M) and a poor mouse liver microsomal stability (T1/2 = 3 min) (Table 1) [20]. Its sulfoxide and sulfone metabolites were identified and appeared active, but were also rapidly metabolized in vitro (T1/2 = 3 min). The para position of the phenyl ring at position 2 was also identified as potentially oxidized.  11 3 Thermodynamic solubility at pH 7.4 (µ M) 6.9 1.4 With a view to identify an optimal antileishmanial derivative which could reach in vivo studies, we explored the structure-activity relationships, investigating the influence of the substituents at position 2 of the 3-nitroimidazo[1,2-a]pyridine scaffold, including structures of probable oxidized metabolites and by synthesizing analogues bearing analogues with metabolic blockers at para position. Attempts were also made to replace the sulfone group by a sulfoximine group.

Synthesis
Using a previously reported synthetic route developed in our lab [16,17], intermediates 1a and 2a, were obtained by the cyclocondensation of 1,3-dichloroacetone with commercially available 2-amino-5-chloropyridine in refluxing ethanol, followed by a nitration In order to develop new original nitroheterocyclic antikinetoplastid molecules, our team identified a first antileishmanial hit compound in 8-halogeno-3-nitroimidazo[1,2a]pyridine series (Hit A) [19]. Introduction of a 4-chlorophenylthioether moiety at position 8 led to a new derivative (Hit B) with improved in vitro antileishmanial activity but showing a poor aqueous solubility (thermodynamic solubility = 1.4 µ M) and a poor mouse liver microsomal stability (T1/2 = 3 min) (Table 1) [20]. Its sulfoxide and sulfone metabolites were identified and appeared active, but were also rapidly metabolized in vitro (T1/2 = 3 min). The para position of the phenyl ring at position 2 was also identified as potentially oxidized.  11 3 Thermodynamic solubility at pH 7.4 (µ M) 6.9 1.4 With a view to identify an optimal antileishmanial derivative which could reach in vivo studies, we explored the structure-activity relationships, investigating the influence of the substituents at position 2 of the 3-nitroimidazo[1,2-a]pyridine scaffold, including structures of probable oxidized metabolites and by synthesizing analogues bearing analogues with metabolic blockers at para position. Attempts were also made to replace the sulfone group by a sulfoximine group.

Synthesis
Using a previously reported synthetic route developed in our lab [16,17], intermediates 1a and 2a, were obtained by the cyclocondensation of 1,3-dichloroacetone with commercially available 2-amino-5-chloropyridine in refluxing ethanol, followed by a nitration With a view to identify an optimal antileishmanial derivative which could reach in vivo studies, we explored the structure-activity relationships, investigating the influence of the substituents at position 2 of the 3-nitroimidazo[1,2-a]pyridine scaffold, including structures of probable oxidized metabolites and by synthesizing analogues bearing analogues with metabolic blockers at para position. Attempts were also made to replace the sulfone group by a sulfoximine group.

Synthesis
Using a previously reported synthetic route developed in our lab [16,17], intermediates 1a and 2a, were obtained by the cyclocondensation of 1,3-dichloroacetone with commercially available 2-amino-5-chloropyridine in refluxing ethanol, followed by a nitration reaction at position 3, giving the 2-chloromethyl-3-nitro-8-halogeno-imidazo[1,2-a]pyridines 1b and 2b (Scheme 1). harmaceuticals 2022, 15,998 reaction at position 3, giving the 2-chloromethyl-3-nitro-8-halogeno-imida dines 1b and 2b (Scheme 1). The increasingly common incorporation of sulfoximine in antineoplastic and antiinfective agents in clinical trials often enhances potency and improves both physicochemical and pharmacokinetic properties [21]. The attempts to synthesize new sulfoximine analogues either by starting from sulfide substrate 4, using phenyliodine diacetate and ammonium carbamate [22], or by starting from sulfoxide substrate 5, using sodium azide and Eaton's reagent [23], were not successful. Only the sulfone derivative (corresponding to Hit A) was obtained with the second reaction instead of the sulfoximine, because of sulfur's strong propensity to be oxidized (Scheme 3). The increasingly common incorporation of sulfoximine in antineoplastic and antiinfective agents in clinical trials often enhances potency and improves both physicochemical and pharmacokinetic properties [21]. The attempts to synthesize new sulfoximine analogues either by starting from sulfide substrate 4, using phenyliodine diacetate and ammonium carbamate [22], or by starting from sulfoxide substrate 5, using sodium azide and Eaton's reagent [23], were not successful. Only the sulfone derivative (corresponding to Hit A) was obtained with the second reaction instead of the sulfoximine, because of sulfur's strong propensity to be oxidized (Scheme 3). Previous works showed that the sulfur atom of the 4-chlorophenylthioether moiety was easily oxidized by CYP450 [20]. As compound 3i showed improved, but not yet sufficient, microsomal stability, sulfoxide 6a and sulfone 6b metabolites were then synthesized by partial and complete oxidation of the sulfur atom, in the same reaction, using m-CPBA at room temperature (Scheme 4). Finally, in order to improve microsomal stability, aqueous solubility and gastrointestinal permeability, we replaced the 4-chlorophenylthioether moiety by a pyridin-4-yl substituent at position 8 of the gem-trifluoropropyl derivative. This group was already introduced at this position before, on a 2-phenylsulfonylmethyl derivative [24], showing poor, but 2 times better aqueous solubility than Hit B (thermodynamic solubility = 3.3 µ M), good mouse microsomal stability (T1/2 > 40 min) and high gastrointestinal permeability in a PAMPA model (PAMPA GIT = 452 nm/s). Under conditions adapted from a previously reported Suzuki-Miyaura coupling reaction in our lab, compound 7 was prepared in good yield starting from 2j in presence of 4-pyridinylboronic acid, Pd(dppf)Cl2 as catalyst and potassium carbonate as base, under microwave (MW) heating, in a sealed tube (Scheme 5). Finally, in order to improve microsomal stability, aqueous solubility and gastrointestinal permeability, we replaced the 4-chlorophenylthioether moiety by a pyridin-4-yl substituent at position 8 of the gem-trifluoropropyl derivative. This group was already introduced at this position before, on a 2-phenylsulfonylmethyl derivative [24], showing poor, but 2 times better aqueous solubility than Hit B (thermodynamic solubility = 3.3 µM), good mouse microsomal stability (T 1/2 > 40 min) and high gastrointestinal permeability in a PAMPA model (PAMPA GIT = 452 nm/s). Under conditions adapted from a previously reported Suzuki-Miyaura coupling reaction in our lab, compound 7 was prepared in good yield starting from 2j in presence of 4-pyridinylboronic acid, Pd(dppf)Cl 2 as catalyst and potassium carbonate as base, under microwave (MW) heating, in a sealed tube (Scheme 5).
was easily oxidized by CYP450 [20]. As compound 3i showed improved, but not ficient, microsomal stability, sulfoxide 6a and sulfone 6b metabolites were then sized by partial and complete oxidation of the sulfur atom, in the same reaction, u CPBA at room temperature (Scheme 4). Finally, in order to improve microsomal stability, aqueous solubility and gast tinal permeability, we replaced the 4-chlorophenylthioether moiety by a pyridin-4 stituent at position 8 of the gem-trifluoropropyl derivative. This group was alread duced at this position before, on a 2-phenylsulfonylmethyl derivative [24], showin but 2 times better aqueous solubility than Hit B (thermodynamic solubility = 3 good mouse microsomal stability (T1/2 > 40 min) and high gastrointestinal permea a PAMPA model (PAMPA GIT = 452 nm/s). Under conditions adapted from a pre reported Suzuki-Miyaura coupling reaction in our lab, compound 7 was prepared yield starting from 2j in presence of 4-pyridinylboronic acid, Pd(dppf)Cl2 as cata potassium carbonate as base, under microwave (MW) heating, in a sealed tube ( 5).

Antileishmanial Structure-Activity Relationships Study
The antileishmanial activities of the twenty-two new derivatives synthesized were evaluated in vitro against the promastigote form of L. donovani and against both the promastigote and axenic amastigote forms of L. infantum (measured through effective concentration 50% = EC 50 ). Their influence on cell viability (cytotoxic concentration 50% = CC 50 ) was assessed on the HepG2 and THP1 cell lines, using doxorubicin as a positive control. The best compounds were also assessed in vitro against the intramacrophage (intra-THP1) amastigote form of L. infantum. Selectivity indices (SI) were calculated as SI = CC 50 /EC 50 , for both HepG2 and THP1 cell lines. Their antileishmanial activities were compared to the ones of previous hit molecules (Hit A and Hit B) and to reference drugs (amphotericin B, miltefosine, fexinidazole and fexinidazole sulfone). For all molecules, redox potentials (E 0 ) of the nitro group were measured using cyclic voltammetry, and the values were corrected versus Normal Hydrogen Electrode (NHE). Weighted clogP were also computed by the SwissADME online tool [25] (Table 2). Table 2. In vitro evaluation of molecules 1c-7 on L. donovani promastigotes, L. infantum promastigotes, L. infantum axenic amastigotes, L. infantum intracellular amastigotes, the human hepatocyte HepG2 and human leukemia monocytic THP1 cell lines. Redox potentials (E 0 ) of the nitro group were measured using cyclic voltammetry and corrected versus Normal Hydrogen Electrode (NHE). Weighted clogP were computed by the SwissADME online tool. data confirm that the substituent at position 8 played an essential role in the antileishmanial activity. Table 2. In vitro evaluation of molecules 1c-7 on L. donovani promastigotes, L. infantum promastigotes, L. infantum axenic amastigotes, L. infantum intracellular amastigotes, the human hepatocyte HepG2 and human leukemia monocytic THP1 cell lines. Redox potentials (E0) of the nitro group were measured using cyclic voltammetry and corrected versus Normal Hydrogen Electrode (NHE). Weighted clogP were computed by the SwissADME online tool.  The product could not be tested at higher concentrations due to a lack solubility in the culture medium. b Doxorubicin was used as a cytotoxic reference drug. c The EC 50 or CC 50 value was not reached at the highest tested concentration. d Amphotericin B, Miltefosine, Fexinidazole and Fexinidazole sulfone were used as antileishmanial reference drugs.
Of the ten 8-halogeno derivatives tested, hydroxyl derivative 2c and some of the analogues with metabolic blockers 1c, 1d and 2e showed poor solubility in the culture media and a loss of activity against the axenic amastigote form of L. infantum. Compound 2d, with a chlorine atom, showed good EC 50 values on the promastigote form of L. donovani and against both the promastigote and axenic amastigote forms of L. infantum, but at the cost of a loss of solubility in the tested culture media. Derivative 2g with a methoxy group displayed a good anti-leishmanial activity but was cytotoxic on the HepG2 cell line. Compound 2f with a trifluoromethoxy group presented a good biological profile compared to Hit A, with comparable cell viability and activity against both the promastigote form of L. donovani and L. infantum, even with a better activity on the axenic amastigote form of L. infantum and a better solubility in the aqueous culture media. Compounds 2h and 2i, bearing an isopropyl-and a cyclopropyl-sulfonylmethyl group at position 2, respectively, proved to be inactive on both the promastigote form of L. donovani and L. infantum, and toward L. infantum axenic amastigotes. Finally, the gem-trifluoropropyl derivative 2j displayed a better activity than Hit A against L. infantum promastigotes, but lower activities against L. donovani promastigotes and L. infantum axenic amastigotes. These results were promising, indicating for the first time that a compound with an alkyl substituent at this position could be active against Leishmania spp., since the isopropyl derivative 2h, the cyclopropyl derivative 2i and also the methyl derivative previously described [26] did not show any activity.
All new 2-subsituted derivatives bearing a 4-chlorophenylthioether moiety at position 8 showed good EC 50 values, similar to or even better than Hit B (Table 2). Compound 3a, which was confirmed to be a metabolite of Hit B by LC-MS/MS study after in vitro mouse microsomal incubation [20], maintained its promising activity against the promastigote form of L. donovani and the axenic amastigote form of L. infantum. Furthermore, except for compounds 3b and 3g, all the derivatives showed satisfactory CC 50 values on the THP1 cell line (>50 µM), indicating both a low cytotoxicity and a good solubility in the culture medium. Unfortunately, on the HepG2 cell line, all these derivatives showed lower CC 50 values than Hit B, some displaying significantly increased cytotoxicity (compound 3b) and some with a slightly increased cytotoxicity although remaining weakly cytotoxic in comparison with doxorubicine (compounds 3a, 3g and 3h). Others (compounds 3c, 3d, 3e, 3f, 3i) had poor solubility in the aqueous culture medium (compounds 3c, 3d, 3e, 3f, 3i). In contrast to 8-halogeno derivatives, compounds 3g and 3h, with the isopropyl-and the cyclopropyl-sulfonylmethyl group, respectively, appeared to be active on the promastigote form of L. donovani and against both the promastigote and axenic amastigote forms of L. infantum. Finally, the sulfinyl 6a and sulfonyl 6b metabolites of 3i, as well as the 4-pyridyl derivative 7, showed a loss of activity against the promastigote and axenic amastigote forms of L. infantum but remained active on the promastigote form of L. donovani. Moreover, compound 7 showed the best CC 50 values of this series on both HepG2 and THP1 cell lines, better than Hits A and B values. All these data confirm that the substituent at position 8 played an essential role in the antileishmanial activity.
In order to find the most active compounds of this series, only derivatives with low cytotoxicity on the THP1 cell line, good solubility in THP1 culture medium, and displaying better activity than Hit A against L. infantum axenic amastigotes (EC 50 < 16 µM) were tested against the intramacrophage (obtained from THP1 monocytes) amastigote form of L. infantum (Table 2). Compared to Hit A and Hit B, hydroxy-metabolite 3a and trifluoromethyl-derivative 3d showed low activities, together with low selectivity indices. While chlorine derivative 3b was the most active compound of this series on the intracellular amastigote form of L. infantum, its selectivity indices were low, due to its cytotoxicity on the HepG2 cell line and to a low solubility in THP1 aqueous culture medium. The selectivity indices of derivatives 2f, 2g, 3b, 3c, 3e, 3f, 3h and 3i were roughly comparable to those of hit compounds and to fexinidazole, except on the HepG2 cell line, where their selectivity indices were below 10. The sulfinyl 6a and sulfonyl 6b metabolites of 3i presented a slight loss of activity against the L. infantum intracellular amastigotes form, especially for 6b, but remained active on this form. In the end, only the 4-pyridyl derivative 7 showed comparable or better selectivity indices than the hit compounds and fexinidazole, with selectivity indices on the HepG2 cell line of above 27, together with renewed activity against the intracellular amastigote form of L. infantum, a selectivity index on THP1 better than the ones of the two hit compounds, fexinidazole and miltefosine, and the best selectivity indices on the HepG2 cell line of all the newly synthesized molecules.
Another objective was to study how the substituent at position 2 might affect the reduction potential (E 0 ) of the nitro group. The lowest the reduction potential, the lowest is the risk of the nitro group to be reduced by host enzymes and be responsible for genotoxicity. It should be noted that it was not possible to determine the E 0 of compounds 2c and 3a. Unfortunately, these modifications did not significantly decrease the reduction potential of the nitro group, the values remaining quite close to those of Hit A (E 0 = −0.65 V) and B (E 0 = −0.63 V). The variations produced by these modifications depended on the substituent at position 8: the E 0 of the 8-halogeno derivatives was between −0.60 for compounds 1c, 1d and 2j and −0.65 V for compound 2h, that of the 4-chlorothiophenyl derivatives was between −0.56 for the trifluoromethyl-derivative 3d and −0.68 V for the chlorine derivative 3b, while the introduction of a 4-pyridyl group (compound 7) led to a E 0 of −0.61 V.
Finally, the clogP of the synthesized compounds, Hit A, Hit B and the reference drugs were computed by the SwissADME ( Table 2). The antileishmanial activity of the new derivatives and clogP were compared, but no correlation was observed.  [20]. However, with marketing authorization for Fexinidazole in late 2018 as the first all-oral treatment for T. b. gambiense, the search for new antitrypanosomal molecules is of much less interest. Thus, EC 50 values of only four derivatives were determined (Table 3).
The product could not be tested at higher concentrations due to a lack solubility in the culture medium. b Suramin and Fexinidazole were used as anti-Trypanosomoa brucei reference drugs. c SI = CC 50 HepG2/EC 50 T. brucei brucei.
Submicromolar activities (0.40 ≥ EC 50 ≥ 0.89 µM) of compounds 2c, 3a, 3c and 3d were better than the one of the reference drug fexinidazole, and better than those of Hit A and Hit B, evidence that a para-substituent on the phenyl ring improved antitrypanosomal activity for both 8-bromo and 8-phenylthio derivatives. However, as seen before, 2c, 3c and 3d showed low solubility in HepG2 aqueous culture medium, restricting their selectivity indices.

In Vitro Physicochemical and Pharmacokinetic Study of Selected Key Compounds
In vitro physicochemical and pharmacokinetic properties of selected key compounds were evaluated (Table 4). First, thermodynamic aqueous solubility at pH = 7.4 was measured. Despite good solubility values in aqueous culture media, Hit A (thermodynamic solubility = 6.9 µM) and especially Hit B (thermodynamic solubility = 1.4 µM) showed low thermodynamic solubility at pH = 7.4. Of all the synthesized derivatives, only three compounds showed a good thermodynamic aqueous solubility (>10 µM): compounds 3i and 7 both with a gem-trifluoropropyl chain and bearing a 4-chlorophenylthioether moiety and a 4-pyridyl group at position 8, respectively (thermodynamic solubility = 12.4 µM and 31.1 µM, respectively), and compound 3e with a trifluoromethoxy group (thermodynamic solubility = 64.7 µM). As expected, the replacement of the 4-chlorophenylthioether moiety at position 8 by a 4-pyridyl group resulted in an improved thermodynamic solubility, which is correlated to the solubility values obtained in HepG2 and THP1 culture media. Surprisingly, the substitution of the para position on the phenyl ring by a trifluoromethoxy group also greatly improved solubility compared to Hit B without any substituent and compared to compound 3d bearing a trifluoromethyl group.
12.4 ± 3.0 9. In vitro mouse microsomal stability (Table 4) did not appear to be significantly improved by introducing metabolic blockers (compounds 3b and 3c, T 1/2 = 4.5 min and 5.7 min, respectively) when compared to Hit B (T 1/2 = 3.0 min), which suggested that other phase 1 metabolism reactions may be involved, such as oxidation at other positions of the phenyl ring. Fortunately, the replacement of the phenyl ring at position 2 by the gem-trifluoropropyl chain (compound 3i) resulted in a slight improvement in microsomal stability (T 1/2 = 9.1 min); gem-Trifluoropropyl derivative 6b, with complete oxidation of the sulfur atom of the 4-chlorophenylthioether moiety, led to poor microsomal stability (T 1/2 = 7.9 min), implying that other positions were metabolized, probably on the 4-chlorophenyl ring. In the end, replacement of metabolically labile phenylthioether moiety by the electron-deficient pyridine (compound 7) addressed the metabolic liability (T 1/2 > 40 min).
In addition to water solubility and microsomal stability issues with hit compounds A and B, permeability across the gastrointestinal tract, a critical parameter in oral formulations, presented another challenge. Despite Hit A's good permeability (PAMPA GIT = 173.2 nm/s), the introduction of the 4-chlorophenylthioether moiety (Hit B) led to a significant permeability decrease (PAMPA GIT = 4.0 nm/s). Modifying position 2 did not improve these values (compounds 3i, PAMPA GIT = 6.3 nm/s). Fortunately, the 4-pyridine derivative 7 showed very good permeability (twice as good as Hit A and 100 times better than Hit B), confirming that 4-chlorophenylthioether is a restraining moiety for both microsomal stability and gastrointestinal permeability.
The last pharmacokinetic parameter investigated was the percentage of human albumin binding. This parameter was not the most critical parameter (many drugs highly bound to albumin (>99%) were marketed [27]) but it was worth trying to decrease albumin binding, to increase the proportion of the unbound fraction exerting pharmacological activity. Once again, the introduction of a 4-pyridyl group led to a significant decrease in the percentage of albumin binding from 99.8% for compound 3i to 88.6% for compound 7.
To be considered as an antileishmanial hit, a molecule must meet several criteria which were defined by a panel of drug experts convened by the Japanese Global Health Innovative Technology (GHIT) Fund [28]. Thus, compound 7, by validating all key hit selection criteria, appeared as a new antileishmanial hit compound (Table 5). ( 1 H NMR: reference CHCl 3 δ = 7.26 ppm, reference DMSO-d 6 δ = 2.50 ppm and 13 C NMR: reference CHCl 3 δ = 76.9 ppm, reference DMSO-d 6 δ = 39.52 ppm). The following adsorbent was used for column chromatography: silica gel 60 (Merck KGaA, Darmstadt, Germany, particle size 0.063-0.200 mm, 70-230 mesh ASTM). TLC was performed on 5 cm x 10 cm aluminum plates coated with silica gel 60F-254 (Merck) in an appropriate eluent. Visualization was performed with ultraviolet light (234 nm). Purity determination of synthesized compounds was checked by LC/MS analyses, which were realized at the Faculté de Pharmacie of Marseille with a Thermo Scientific Accela High Speed LC System ® (Waltham, MA, USA) coupled using a single quadrupole mass spectrometer Thermo MSQ Plus ® (Waltham, MA, USA). The purity of all the synthesized compounds was > 95%, except for compounds 3c, 6a and 6b for which the purity was > 90%. The RP-HPLC column is a Thermo Hypersil Gold ® (Waltham, MA, USA) 50 × 2.1 mm (C 18 bounded), with particles of a diameter of 1.9 mm. The volume of sample injected on the column was 1 µL. Chromatographic analysis, total duration of 8 min, was on the gradient of the following solvents: t = 0 min, methanol/water 50:50; 0 < t < 4 min, linear increase in the proportion of methanol to a methanol/water ratio of 95:5; 4 < t < 6 min, methanol/water 95:5; 6 < t < 7 min, linear decrease in the proportion of methanol to return to a methanol/water ratio of 50:50; 6 < t < 7 min, methanol/water 50:50. The water used was buffered with ammonium acetate 5 mM. The flow rate of the mobile phase was 0.3 mL/min. The retention times (tR) of the molecules analyzed were indicated in min. Microwave reactions were performed using monomode reactors: Biotage Initiator ® classic (Uppsala, Sweden) in sealed vials with a power output of 0 to 400 W. Reagents were purchased from Sigma-Aldrich or Fluorochem and used without further purification. Compound 4 was synthesized according to the previous reported procedure and its physical characteristics agreed with the published data [26]. Copies of 1 H and 13 C NMR spectra are available in the Supplementary Materials.

General Procedure for the Preparation of Sodium Sulfinate
Appropriate sulfonyl chloride (1 equivalent), sodium sulfite (2 equivalent) and sodium bicarbonate (2 equivalent) were dissolved in distilled water (8 mL). The reaction mixture was stirred at 80 • C for 6 h and was then cooled to room temperature. Water was removed under vacuum and the desired sodium sulfinate was obtained and used in the next step without further purification.

Electrochemistry
Voltammetric measurements were carried out with a potentiostat Autolab PGSTAT100 (ECO Chemie, Utrecht, The Netherlands) controlled by GPES 4.09 software (ECO Chemie, Utrecht, The Netherlands). Experiments were performed at room temperature in a homemade airtight three-electrode cell connected to a vacuum/argon line. The reference electrode consisted of a saturated calomel electrode (SCE) separated from the solution by a bridge compartment. The counter electrode was a platinum wire of 1 cm 2 apparent surface. The working electrode was a GC microdisk (1.0 mm in diameter-Bio-logic SAS). The supporting electrolyte (nBu 4 N)[PF 6 ] (Fluka, 99% puriss electrochemical grade) and the solvent DMSO (Sigma-Aldrich puriss p.a. dried <0.02% water) were used as received and simply degassed under argon. The solutions used during electrochemical analyses were typically 10 −3 M in compound and 0.1 M in supporting electrolyte. Before each measurement, the solutions were degassed by bubbling Ar, and the working electrode was polished with a polishing machine (Presi P230, Eybens, France). Under the experimental conditions adopted in this work, the half-wave potential (E 1/2 ) of the ferrocene Fc+/Fc couple in DMSO was E 1/2 = 0.45 V vs. SCE. Experimental peak potentials were measured versus SCE and converted to NHE by adding 0.241 V.

Antileishmanial Activity against L. donovani Promastigotes
The Leishmania species used in this study were L. donovani (MHOM/IN/00/DEVI) purchased from CNR Leishmania (Montpellier, France). Leishmania promastigote forms were grown in Schneider's Drosophila medium (Life Technologies, Saint-Aubin, France) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine and 20% FCS (Life Technologies, Saint-Aubin, France) at 27 • C. The in vitro evaluation of the tested compound's antileishmanial activity on promastigote forms was carried out by MTT assay according to the Mosmann protocol [29] with some modifications. Briefly, promastigotes in log-phase were incubated at an average density of 10 6 parasites/mL in sterile 96-well plates with various concentrations of compound dissolved in DMSO (final concentration less than 0.5% v/v), in duplicate. Appropriate controls treated with DMSO, Miltefosine, Amphotericin B, Fexinidazole, and Fexinidazole sulfone (reference drugs purchased from Sigma-Aldrich, Saint-Louis, Missouri, USA) were added to each set of experiments. After a 72 h incubation period at 27 • C, parasitic metabolic activity was determined. Each plate-well was then microscope-analyzed to detect any precipitate formation. 20 µL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, Saint-Louis, Missouri, USA) solution (5 mg/mL in PBS) were added to each well and incubated for 4 h at 27 • C. The enzyme reaction was then stopped by addition of 100 µL of 50% isopropanol-10% sodium dodecyl sulfate. Plates were shaken vigorously at 300 rpm for 10 min. The absorbance was measured at 570 nm in a BIO-TEK Elx808 (Biotek, Colmar, France) absorbance microplate reader. Inhibitory concentration 50% (EC 50 ) was defined as the concentration of drug required to inhibit the metabolic activity of Leishmanial promastigote forms by 50% relative to the control. EC 50 values were calculated by non-linear regression analysis processed dose-response curves, using TableCurve 2D V5.0 software (Systat Software Inc. (Palo Alto, CA, USA)). EC 50 values represent the mean value calculated from at least three separate experiments.
3.3.2. Antileishmanial Activity against L. infantum Promastigotes L. infantum promastigotes (MHOM/MA/67/ITMAP-263, CNR Leishmania, Montpellier, France, expressing luciferase activity) were used in the following tests. The effects of the tested compounds on the growth of L. infantum promastigotes were assessed by Luciferase Assay. Briefly, promastigotes in log-phase in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine and antibiotics (100U/mL penicillin, 100 µg/mL streptomycin and 50µg/mL geneticin), were incubated at an average density of 10 6 parasites/mL (100 µL/well) in sterile 96-well plates with various concentrations of compounds dissolved in DMSO (final concentration less than 0.5% v/v), in duplicate. Appropriate controls (100 µL/well) treated by DMSO and reference drugs (purchased for the majority from Sigma Aldrich) were added to each set of experiments. After a 72 h incubation period at 24 • C, each plate-well was then microscope-examined for detecting possible precipitate formation. To estimate the luciferase activities of promastigotes, 80 µL of each well are transferred in white 96-well plates, Steady Glow reagent (Promega) was added according to manufacter's instructions, and plates were incubated for 2 min. The luminescence was measured in a FLUOstar Omega (BMG Labtech, Champigny-sur-Marne, France). Efficient concentration 50% (EC 50 ) was defined as the concentration of drug required to inhibit by 50% the metabolic activity of L. infantum promastigotes compared to the control. EC 50 were calculated by non-linear regression analysis processed on dose-response curves, using TableCurve 2D V5 software.

Antileishmanial Activity on L. infantum Axenic Amastigotes
L. infantum promastigotes were harvested in logarithmic phase of growth by centrifugation at 900× g for 10 min. The supernatant was removed carefully and was replaced by the same volume of RPMI 1640 complete medium at pH 5.4 and incubated for 24 h at 24 • C. The acidified promastigotes were then incubated for 24 h at 37 • C in a ventilated flask to transform them into axenic amastigotes. The amastigote stage was checked both by electron microscopy (short flagellum with small bulbous tip extending beyond a spherical cell body) and RT-PCR to confirm the overexpression of ATG8 and amastin genes in amastigotes, relative to promastigotes. The effects of the tested compounds on the growth of L. infantum axenic amastigotes were assessed as follows. L. infantum amastigotes were incubated at a density of 2 × 10 6 parasites/mL in sterile 96-well plates with various concentrations of compounds dissolved in DMSO (final concentration less than 0.5% v/v), in duplicate. Appropriate controls treated with DMSO, amphotericin B, miltefosine, fexinidazole and fexinidazole sulfone were added to each set of experiments. After a 48 h incubation period at 37 • C, each plate-well was then microscopically examined to detect any precipitate formation.
Luciferase activity and determination of EC 50 were performed as above.

Antileishmanial Activity on L. infantum Intracellular Amastigotes
The undifferentiated THP1 human monocyte cells (acute monocytic leukemia cell line purchased from ATCC, ref TIB-202) were 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 1.10 6 cells/mL. The in vitro evaluation of the antileishmanial activity on intracellular amastigote forms of the tested compound was assessed as below.
Briefly, 200 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 0.77 × 10 5 cells/mL and incubated for 96 h at 37 • C, 5% CO 2 . Promastigotes were centrifuged at 3000 rpm for 10 min and the supernatant replaced by the same volume of complete RPMI pH 5.4 and incubated for 24 h at 27 • C. Differentiated THP-1 cells were then infected by acidified promastigotes with an infection ratio of twenty parasites for one macrophage and incubated for 24 h at 37 • C, 5% CO 2 . Plates were rinsed three times and then, in duplicate, medium containing various concentrations of reference drugs and DMSO was added (final DMSO concentrations being inferior to 0.5% v/v) and plates were incubated for 120 h.
Luciferase activity and determination of EC 50 were performed as above.

Antitrypanosomal Evaluation on T. b. brucei BSF Trypomastigotes
The effects of the tested compounds on the growth of T. b. brucei were assessed by Alamar Blue ® assay described by Räz et al. [30] T. b. brucei AnTat 1.9 (IMTA, Antwerpen, Belgium) was cultured in MEM with Earle's salts, supplemented according to the protocol of Baltz et al. [31] with the following modifications: 0.5 mM mercaptoethanol (Sigma Aldrich ® , France), 1.5 mM L-cysteine (Sigma Aldrich ® ), 0.05 mM bathocuproïne sulfate (Sigma Aldrich ® ) and 20% heat-inactivated horse serum (Gibco ® , France), at 37 • C and 5% CO 2 . They were incubated at an average density of 2000 parasites/100 µL in sterile 96-wells plates (Fisher ® , France) with various concentrations of compounds dissolved in DMSO, in duplicate. Appropriate controls suramin, treated with sterile water, and fexinidazole, treated with DMSO on sterile water (reference drugs purchased from Sigma Aldrich, France and Fluorochem, UK) were added to each set of experiments. After a 69 h incubation period at 37 • C, 10 µL of the viability marker Alamar Blue ® (Fisher, France) were then added to each well, and the plates were incubated for 5 h. The plates were read in a ENSPIRE microplate reader (PerkinElmer, Waltham, MA, USA) using an excitation wavelength of 530 nm and an emission wavelength of 590 nm. EC 50 was defined as the concentration of drug necessary to inhibit by 50% the activity of T. b. brucei relative to the control. EC 50 were calculated by nonlinear regression analysis processed on dose-response curves, using GraphPad Prism software (USA). EC 50 values were calculated from three separate experiments.

Cytotoxicity Evaluation on 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% foetal 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 [25] 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. 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 BIO-TEK ELx808 Absorbance Microplate Reader. DMSO was used as blank and doxorubicin (purchased from Sigma Aldrich) as positive control. Cell viability was calculated as percentage of control (cells incubated without compound). The 50% cytotoxic concentration Cells in 100 µL of complete RPMI medium, were incubated at an average density of 5 × 10 4 cells/mL in sterile 96-well plates with various concentrations of compounds dissolved in DMSO (final concentration less than 0.5% v/v), and references in duplicate. The plates were incubated for 72 h at 37 • C. Each well plate was then microscope-examined for detecting possible precipitate formation before the medium was aspirated from the wells. 100 µL of MTT solution (0.5 mg/mL in medium) were then added to each well. Cells were incubated for 2 h at 37 • C. After this time, the MTT solution was removed and DMSO (100 µL) was added to dissolve the resulting blue formazan crystals. Plates were shaken vigorously (300 rpm) for 10 min. The absorbance was measured at 570 nm using a BIO-TEK ELx808 Absorbance Microplate Reader. DMSO was used as blank and doxorubicin (purchased from Sigma Aldrich) as positive control. Cell viability was calculated as percentage of control (cells incubated with DMSO). The 50% cytotoxic concentration (CC 50 ) was determined from the dose-response curve by using the TableCurve 2D V5 software Systat Software Inc. (Palo Alto, CA, USA). CC 50 values represent the mean value calculated from three separate experiments.

Plasma Protein Binding
Plasma doped with the tested compound was incubated at 37 • C in triplicate in one of the compartments of the insert, the other compartment containing a phosphate buffer solution at pH 7.2. After stirring for 4 h at 300 rpm, a 25 µL aliquot of each compartment was taken and diluted; the dilution solution was adapted so as to obtain an identical matrix for all the compartments after dilution. Parallel, reprocessing of a plasma doped but not incubated was used to assess recovery. The LC-MS used for this study was a Waters ® Acquity I-Class/Xevo TQD, equipped with a Waters ® Acquity BEH C18 column, 50 × 2.1 mm, 1.7 µM. The mobile phases were (A) ammonium acetate 10 mM and (B) acetonitrile with 0.1% formic acid. The injection volume was 1 µL and the flow rate 600 µL/min. Chromatographic analysis, total duration of 4 min, was performed along the following gradient: 0 < t < 0.2 min, 2% (B); 0.2 < t < 2 min, linear increase to 98% (B); 2 < t < 2.5 min, 98% (B); 2.5 < t < 2.6 min, linear decrease to 2% (B); 2.6 < t < 4 min, 2% (B). Carbamazepine, oxazepam, warfarin and diclofenac were used as reference drugs and propranolol as internal standard. The unbound fraction (f u ) was calculated according to the following formula: f u = A Plasma, 4h −A PBS,4h A Plasma, 4h × 100. The percentage of recovery was calculated according to the following formula: where A is the ratio of the area under peak of the studied molecule and the area under peak of the internal standard (propranolol 200 nM). V is the volume of solution present in the compartments (V PBS = 350 µL and V plasma = 200 µL).

Microsomal Stability
The tested product and propranolol, used as reference, were incubated in duplicate (reaction volume of 0.5 mL) with female mouse microsomes (CD-1, 20 mg/mL, BD Gen-test™) at 37 • C in a 50 mM phosphate buffer, pH 7.4, in the presence of MgCl 2 (5 mM), NADP (1 mM), glucose-6-phosphate dehydrogenase (0.4 U/mL) and glucose-6-phosphate (5 mM). To access intrinsic clearance, 50 µL aliquot at 0, 5, 10, 20, 30 and 40 min were collected and the reaction was stopped with 4 volumes of acetonitrile (ACN) containing the internal standard. After centrifugation at 10,000× g, 10 min, 4 • C, the supernatants were maintained at 4 • C for immediate analysis or placed at -80 • C for later analysis. Controls (t 0 and t final ) in triplicate were prepared by incubation of the internal standard with microsomes denatured by acetonitrile. The LC-MS used for this study was a Waters ® Acquity I-Class/Xevo TQD, equipped with a Waters ® Acquity BEH C18 column, 50 × 2.1 mm, 1.7 µm. The mobile phases were (A) ammonium acetate 10 mM and (B) acetonitrile with 0.1% formic acid. The injection volume was 1 µL and the flow rate 600 µL/min. Chromatographic analysis, total duration of 4 min, was perform along the following gradient: 0 < t < 0.2 min, 2% (B); 0.2 < t < 2 min, linear increase to 98% (B); 2 < t < 2.5 min, 98% (B); 2.5 < t < 2.6 min, linear decrease to 2% (B); 2.6 < t < 4 min, 2% (B). 8-Bromo-6-chloro-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-a]pyridine Hit A is used as internal standard. Each compound was quantified by converting the average of the ratios of the analyte/internal standard surfaces to the percentage of product consumed. The ratio of the control at t 0 corresponded to 0% of product consumed. The half-life (T 1/2 ) of each compound in the presence of microsomes was calculated according to the equation: t 1 2 = ln (2) k , where k is the first-order degradation constant (the slope of the logarithm of compound concentration versus incubation time). The intrinsic clearance in vitro (Cl int expressed in µL/min/mg) is calculated according to the equation: