Nongenotoxic 3‐Nitroimidazo[1,2‐a]pyridines Are NTR1 Substrates That Display Potent in Vitro Antileishmanial Activity

Twenty nine original 3-nitroimidazo[1,2-a]pyridine derivatives, bearing a phenylthio (or benzylthio) moiety at position 8 of the scaffold, were synthesized. In vitro evaluation highlighted compound 5 as an antiparasitic hit molecule displaying low cytotoxicity for the human HepG2 cell line (CC50 > 100 μM) alongside good antileishmanial activities (IC50 = 1−2.1 μM) against L. donovani, L. infantum, and L. major; and good antitrypanosomal activities (IC50 = 1.3−2.2 μM) against T. brucei brucei and T. cruzi, in comparison to several reference drugs such as miltefosine, fexinidazole, eflornithine, and benznidazole (IC50 = 0.6 to 13.3 μM). Molecule 5, presenting a low reduction potential (E° = −0.63 V), was shown to be selectively bioactivated by the L. donovani type 1 nitroreductase (NTR1). Importantly, molecule 5 was neither mutagenic (negative Ames test), nor genotoxic (negative comet assay), in contrast to many other nitroaromatics. Molecule 5 showed poor microsomal stability; however, its main metabolite (sulfoxide) remained both active and nonmutagenic, making 5 a good candidate for further in vivo studies.

T rypanosomatids are flagellated protozoan parasites that belong to the Kinetoplastid order. These unicellular parasites are responsible for several vector borne diseases in mammals. Among the trypanosomatids, Leishmania spp and Trypanosoma spp are the two main parasites encountered in human pathology and responsible for lethal infections: visceral leishmaniasis (VL), 1 human African trypanosomiasis (HAT), 2 or Chagas disease (CD). 3 These neglected tropical diseases threaten millions of people worldwide with current total deaths estimated at 30,000 per annum, mainly in tropical and subtropical areas. 4 New efficient, safe, and cheap oral anti-Kinetoplastid agents are urgently needed due to the major limitations of the few currently available drugs (i.e., toxicity, mode of administration, cost) and the emergence of resistant parasites (mainly toward antimony derivatives and miltefosine). 5 In this context, small azaheterocyclic molecules, including imidazo[1,2-a]pyridine derivatives, 6 are studied to enrich the antitrypanosomatid drug discovery pipeline. 7,8 Recently, nitroaromatic molecules, such as fexinidazole 9 and delamanid, 10 showed a renewed interest against infectious diseases such as HAT, VL, CD, and even tuberculosis. 11 Indeed, these compounds act as prodrugs, requiring the bioactivation of their nitro group into reduced reactive intermediates, in order to display their antiparasitic properties. This bioactivation step is catalyzed by nitroreductases (NTR), enzymes found in some bacteria and parasites, but not in mammalian cells. Thus, the concept of exploiting NTR to activate prodrugs is an attractive strategy to target selectively pathogen parasites and bacteria.
Two NTRs have been identified and characterized in Leishmania spp: (i) an essential mitochondrial type 1 NTR (NTR1) 12 catalyzing a two-electron reduction reaction and that bioactivates 5-nitroimidazoles like fexinidazole 13,14 and (ii) a cytosolic type 2 NTR (NTR2) 15 catalyzing a monoelectronic reduction reaction. NTR2 more particularly activates bicyclic nitroheterocycles containing a 4-nitroimidazole moiety, such as delamanid and pretomanid. 16 In Trypanosoma brucei spp, only the type 1 NTR was discovered, mediating the bioactivation of the nitroaromatic drugs benznidazole 17 and nifurtimox. 18 To date, the structures of parasitic NTRs remain unknown, as no X-ray diffraction data is available. Thus, drug design techniques such as docking cannot be applied for designing new NTR substrates.
Previously, we reported the antileishmanial activity of 3nitroimidazo[1,2-a]pyridine derivatives. After identifying a hit molecule bearing bromine atoms at positions 6 and 8 of the imidazopyridine ring (hit A), 19 a pharmacomodulation study led to a series of 8-aryl-6-chloro-3-nitroimidazo[1,2-a]pyridines that displayed an improved activity against Leishmania spp and Trypanosoma b. brucei, 20 suggesting the key role of the substituent at position 8 of the scaffold ( Figure  1).
In a continuation of this project, we wanted to explore the influence of the insertion of a heteroatomic bridge between the imidazopyridine ring and an aryl group at position 8, using a synthetic route to functionalize antiparasitic azaheterocycles by a phenylthio moiety. 21,22 We applied this synthetic procedure to a previously described substrate: 8-bromo-6-chloro-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-a]pyridine. 20 Twenty-six original nitroaromatic derivatives bearing a sulfur group at position 8 were then synthesized, using various thiophenol or benzylthiol reagents. The majority of these compounds were prepared with moderate to good yields (58−95%). The use of disubstituted thiophenols (compounds 20−22) led to lower yields (31−38%).
All synthesized molecules were first screened in vitro against the promastigote form of L. donovani and potencies were compared to those achieved with commercial reference drugs (i.e., amphotericin B, miltefosine, and pentamidine) along with drug-candidate fexinidazole (Table 1). To assess antiparasitic selectivity, the cytotoxicity of all compounds was measured against the HepG2 human cell line, using doxorubicin as control.
About half of the tested molecules showed poor solubility in the culture medium and could not be tested against the HepG2 cell line above 4−6 μM. This is particularly noted with the four benzylthioether derivatives 23 to 26, which could not be tested. Three compounds (10, 18, and 20) showed moderate solubility in the culture medium, ranging from 15.6 to 25 μM. Finally, four molecules, all presenting a para-substituted thiophenyl moiety, showed satisfactory CC 50 values, above 50 μM, indicating both good solubility in the culture medium and a low cytotoxicity on the tested cell line. Among the 22 molecules evaluated on the promastigote stage of L. donovani, only compound 19 was found to be devoid of antileishmanial activity, perhaps due to its charged carboxylate group. The active molecules display IC 50 values ranging between 1.0 and 2.3 μM, slightly lower than miltefosine and comparable to fexinidazole. Two compounds (2 and 5) functionalized with a p-halophenylsulfane group are particularly promising and can be considered as hit compounds, with respective selectivity index of >62.5 and >100 (2 to 4 times better than miltefosine). The 3D structure of hit compound 5 was confirmed by X-ray diffraction (see Supporting Information).
A secondary screening was conducted on hit molecules 2 and 5, against the intramacrophage amastigote stage of L. donovani and L. major, on the promastigote stage of L. major and on L. infantum axenic amastigotes. The CC 50 values for these molecules (Table 2) were also determined against THP1 monocyte cell line. None of these compounds showed cytotoxic effects on THP1 cells up to 62.5 μM. Molecule 5 showed the best activities against intracellular L. donovani and L. major amastigotes (IC 50 values of 1.3 and 2.1 μM, respectively), three times better than miltefosine against L. donovani. Compound 5 also returned an IC 50 value of 1.7 μM against L. infantum axenic amastigotes, about two times lower than fexinidazole. This compound also demonstrated promising activity against the promastigote stage of L. major (IC 50 = 1.3 μM). Moreover, molecules 2 and 5 also demonstrated in vitro activity against Trichomonas vaginalis (IC 50 = 4.6−5.0 μM), comparable to that of the reference drug metronidazole (IC 50 = 4.2 μM). This efficacy against T. vaginalis, also found with fexinidazole, 23 is probably due to similar modes of bioactivation by parasitic nitroreductases. 24 Nevertheless, compared with atovaquone (IC 50 = 1 nM), the activity of

ACS Medicinal Chemistry Letters
Letter compound 5 against Plasmodium falciparum (K1 strain) is negligible (IC 50 = 19 μM), demonstrating its selectivity for the parasites expressing nitroreductases. Finally, the in vitro antitrypanosomal activity of compounds 2 and 5 were evaluated against the bloodstream form of T. b. brucei and the epimastigote form of T. cruzi and compared to reference drugs (i.e., eflornithine, suramin, benznidazole, and nifurtimox) and to drug candidate fexinidazole ( Table 2). With IC 50 values ranging from 1.3 to 6.2 μM, these molecules showed a better activity than eflornithine against T. b. brucei and a better activity than fexinidazole against T. cruzi, while molecule 5 was two times less active than drug candidate fexinidazole on T. b. brucei and two times less active than benznidazole on T. cruzi. Finally, the amino derivative 27 of hit-compound 5, prepared by reduction of the nitro group, was tested in vitro ( Table 3). As expected, 27 did not show antileishmanial activity (IC 50 = 32 to 124 μM) and presented a limited antitrypanosomal activity (IC 50 = 11 μM), in accordance with the hypothesis that these nitroaromatic molecules require bioactivation by parasitic NTRs, making the nitro group a key element of the pharmacophore.
To investigate the pharmaceutical potential of compound 5 (best molecule in the series) further, its microsomal stability was measured after incubation with hepatic microsomes isolated from female mice. Molecule 5 exhibited a poor stability with a half-life (T 1/2 of propranolol control = 26 min) of 3 min. An LC/MS/MS study (see Supporting Information) enabled us to identify that the primary metabolite of 5 was a sulfoxide derivative. This sulfoxide metabolite (28) was then synthesized from 5, along with a probable secondary sulfone metabolite (29), respectively, by partial or complete oxidation of the sulfur atom, using m-CPBA (Table 3). The activities of metabolite 28 and molecule 29 were evaluated in vitro against Leishmania spp and T. b. brucei. Both molecules demonstrated promising activity against the promastigote stage of L. donovani and of L. infantum axenic amastigotes. Likewise, both molecules also demonstrated antitrypanosomal activity, thus compound 5 appears to resemble fexinidazole in that it is similarly metabolized into bioactive sulfoxide and sulfone derivatives. 13 These results suggest that the poor microsomal stability of 5 should not be a barrier to further in vivo evaluation.
The mutagenicity of many nitroaromatic molecules has been known for many years. 25 Nevertheless, considering the therapeutic potential of these molecules, many attempts have been made to dissociate their anti-infective activity from their mutagenic character. 26 However, the difficulty remains that the anti-infective mechanism(s) of these compounds involves the formation of mutagenic derivatives within microorganisms. 27 The Salmonella typhimurium strains used in the Ames test, the most common method for assessing the mutagenicity of a substance, also express nitroreductases, 28 particularly type 1. This may explain why nitrated molecules are generally identified as mutagens in this test. The mutagenic properties of hit A, molecule 5, and sulfoxide metabolite 28 were then evaluated by the Ames test, using four classical strains of S. typhimurium expressing NTRs, both with and without addition of the metabolizing S9 mixture. Hit A shows a mutagenic character in the absence of S9 mix on each strain of S. typhimurium, as well as on the TA97a strain in metabolizing conditions. Interestingly, the Ames test was negative on all four strains of Salmonella exposed to molecules 5 or 28, with or without S9 mix (Table 4). Molecules 5 and its principal metabolite 28 therefore appeared as nonmutagenic nitroaromatic derivatives in the Ames test. Thus, the introduction of a p-chlorophenylthioether group at position 8 of the imidazo[1,2-a]pyridine ring, along with the oxidation of the sulfur atom, seem to favor selectivity for parasitic NTRs.
To complete the toxicological profile of hit compound 5, a comet assay using a HepG2 human hepatic cell line, which does not express any NTR, was carried out on hit A and molecule 5. HepG2 cells were incubated with the drugs for 2 Reagents and conditions: thiophenol or benzylthiol reagent (1 equiv), NaH (1 equiv), DMSO, N 2 , RT, 0.5−2 h. b Doxorubicin was used as a cytotoxic reference drug. c Amphotericin B, Miltefosine, Pentamidine, and Fexinidazole were used as antileishmanial reference drugs. d The product could not be tested at higher concentrations in the culture medium. e The CC 50 value was not reached at the highest tested concentration. f SI = CC 50 HepG2/IC 50 L. donovani promastigote. g NS = nonsoluble in the culture medium. In bold: hit molecules.

ACS Medicinal Chemistry Letters
Letter or 72 h (Table 4). No increase in the percentage of DNA laddering was observed for these two compounds at the three tested concentrations: 1, 10, and 20 μM (see Supporting Information). These molecules are therefore not genotoxic according to this test. This assay reveals, for hit A, a profile identical to fexinidazole or metronidazole: a mutagenic character shown by the Ames test and no genotoxic properties objectified on mammalian cells (micronucleus test or comet assay). 29 However, molecule 5 was neither mutagenic nor genotoxic.
Among the accepted criteria for the selection of antiparasitic candidate for preclinical evaluation, 30 the percentage of albumin binding may affect both the distribution and the elimination of the molecule under consideration. The binding percentage to human albumin of compound 5 was measured by LC/MS, after incubation in the presence of human plasma, and compared with four reference drugs. With a bound fraction of 99.95% (log(f u ) = −3.28), this molecule is therefore strongly bound to albumin (Table 4). The chromatographic hydrophobicity index (CHI) of molecule 5 was also determined and provides a logD 7.4 value of 4.26, making this molecule very lipophilic, in accordance with a high albumin binding (Table 4). However, the binding of a drug to plasma proteins was shown to have little effect on the in vivo efficacy of this drug when its mechanism of action results in a covalent binding to its target, 31 which is the likely mode of action proposed for anti-infective nitroaromatics. Moreover, the high lipophilicity of 5 may facilitate its activity against the amastigote stage, by increasing its penetration into both macrophages and parasites.
In order to check that compounds 5 and 29 were substrates of parasitic NTRs, their IC 50 values were determined on three strains of L. donovani promastigotes: a wild type strain, a strain overexpressing NTR1, and a strain overexpressing NTR2. The IC 50 of 5 and 29 are about eight times lower in the strain overexpressing NTR1 than the two other strains (Table 4). These data demonstrate that these compounds are selectively bioactivated by NTR1 in L. donovani, the same type 1 Amphotericin B, miltefosine, and fexinidazole were used as antileishmanial reference drugs. b Eflornithine, suramin, and fexinidazole were used as anti-T. b. brucei reference drugs. c Fexinidazole, benznidazole, and nifurtimox were used as anti-Trypanosoma cruzi reference drugs. d Molecule was not tested at higher concentrations. e Molecules could not be tested at higher concentrations because of a lack of solubility in the culture media.

ACS Medicinal Chemistry Letters
Letter nitroreductase that is responsible for the bioactivation of fexinidazole and its associated metabolites.
On the basis that molecules 5, 28, and 29 could be substrates of parasitic nitroreductases, we determined their reduction potential to examine whether the absence of mutagenicity of molecules 5 and 28 (and probable selectivity for L. donovani NTR1 versus S. typhimurium NTRs) was associated with a lower reduction potential. For the three tested molecules (as for the two hit molecules A and B), the cyclic voltammogram showed a reversible single electron reduction for a 1 V·s −1 scan rate, corresponding to the formation of a nitro radical anion. The measured reduction potentials of compounds 5, 28, and 29 ranged from −0.63 to −0.59 V/NHE. As expected, the more oxidized the sulfur atom in position 8, the higher the reduction potential ( Figure 2). Nonmutagenic molecules 5 and 28 presented a lower reduction potential than mutagenic Hit A, but the difference in E°values was not significant. Thus, steric parameters should be explored in order to explain the atypical behavior of the current series that is selectively bioactivated by L. donovani NTR1 and not by S. typhimurium type 1 NTR. These data suggest that the introduction of a thiophenyl moiety at position 8 of the scaffold maintains affinity for the parasitic type 1 NTR while decreasing the affinity for the bacterial counterpart.
Finally, a preliminary in vivo toxicity study was conducted on swiss mice (three mice per group) that were treated by intraperitoneal administration (50 μL) of molecule 5 at either 1 (group 1) or 10 (group 2) mg/kg for 5 days. There was no sign of acute toxicity noted on living mice. In each group, a mouse was sacrificed at D7, D15, or D30 and autopsied. Histology analysis (brain, liver, kidney, spleen, heart, lung, adipose tissues, and muscles) did not reveal any sign of chronic toxicity in group 1. In group 2, only a discrete inflammatory infiltrate was detected in the liver, classified as nonspecific, and a hypodermic lymph node was observed in the skin in addition to some fibrosis and inflammation at the injection site (see Supporting Information).
In summary, introducing a para-chlorophenylthioether moiety at position 8 of the antileishmanial 3-nitroimidazo-[1,2-a]pyridine pharmacophore led to hit molecule 5 that displayed improved in vitro antikinetoplastid activity against both Leishmania (donovani and infantum) and Trypanosoma (brucei brucei and cruzi) parasites, in comparison with the previously identified hit molecules in the series. Moreover, 5 was the first compound in the series that was neither mutagenic nor genotoxic. It presented a short microsomal half-life due to transformation into the sulfoxide metabolite (28) that remains active and nonmutagenic. Hit compound 5 presents a low reduction potential and is selectively bioactivated by L. donovani NTR1. These results make molecule 5 a good candidate for a hit to lead program focused on developing novel and safe nitroaromatic drug candidates that selectively target flagellated protozoans.

Author Contributions
All authors have given approval to the final version of the manuscript.

Notes
The authors declare no competing financial interest.