8-Alkynyl-3-nitroimidazopyridines display potent antitrypanosomal activity against both T. b. brucei and cruzi

Article abstract of DOI:10.1016/j.ejmech.2020.112558

An antikinetoplastid pharmacomodulation study was done at position 8 of a previously identified pharmacophore in 3-nitroimidazo[1,2-a]pyridine series. Twenty original derivatives bearing an alkynyl moiety were synthesized via a Sonogashira cross-coupling reaction and tested in vitro, highlighting 3 potent (40 nM ≤ EC₅₀ blood stream form≤ 70 nM) and selective (500 ≤ SI ≤ 1800) anti-T. brucei brucei molecules (19, 21 and 22), in comparison with four reference drugs. Among these hit molecules, compound 19 also showed the same level of activity against T. cruzi (EC₅₀ amastigotes = 1.2 μM) as benznidazole and fexinidazole. An in vitro comet assay showed that nitroaromatic derivative 19 was not genotoxic. It displayed a low redox potential value (?0.68 V/NHE) and was shown to be bioactivated by type 1 nitroreductases both in Leishmania and Trypanosoma. The SAR study indicated that an alcohol function improved aqueous solubility while maintaining good activity and low cytotoxicity when the hydroxyl group was at position beta of the alkyne triple bond. Hit-compound 19 was also evaluated regarding in vitro pharmacokinetic data: 19 is BBB permeable (PAMPA assay), has a 16 min microsomal half-life and a high albumin binding (98.5percent). Moreover, compound 19 was orally absorbed and was well tolerated in mouse after both single and repeated administrations at 100 mg/kg. Its mouse plasma half-life (10 h) is also quite encouraging, paving the way toward further efficacy evaluations in parasitized mouse models, looking for a novel antitrypanosomal lead compound.

Full text of DOI:10.1016/j.ejmech.2020.112558

Journal Pre-proof
8-Alkynyl-3-nitroimidazopyridines display potent antitrypanosomal activity against
both T. b. brucei and cruzi
Cyril Fersing, Clotilde Boudot, Caroline Castera-Ducros, Emilie Pinault, Sébastien
Hutter, Romain Paoli-Lombardo, Nicolas Primas, Julien Pedron, Line Seguy, Sandra
Bourgeade-Delmas, Alix Sournia-Saquet, Jean-Luc Stigliani, Jean-Yves Brossas,
Luc Paris, Alexis Valentin, Susan Wyllie, Alan Fairlamb, Élisa Boutet-Robinet, Sophie
Corvaisier, Marc Since, Aurélie Malzert-Fréon, Alexandre Destere, Dominique Mazier,
Pascal Rathelot, Bertrand Courtioux, Nadine Azas, Pierre Verhaeghe, Patrice Vanelle
PII:
S0223-5234(20)30530-4
DOI:
https://doi.org/10.1016/j.ejmech.2020.112558
EJMECH 112558
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 March 2020
Revised Date: 9 June 2020
Accepted Date: 9 June 2020
Please cite this article as: C. Fersing, C. Boudot, C. Castera-Ducros, E. Pinault, S. Hutter, R. Paoli-
Lombardo, N. Primas, J. Pedron, L. Seguy, S. Bourgeade-Delmas, A. Sournia-Saquet, J.-L. Stigliani,
J.-Y. Brossas, L. Paris, A. Valentin, S. Wyllie, A. Fairlamb, É. Boutet-Robinet, S. Corvaisier, M. Since,
A. Malzert-Fréon, A. Destere, D. Mazier, P. Rathelot, B. Courtioux, N. Azas, P. Verhaeghe, P. Vanelle,
8-Alkynyl-3-nitroimidazopyridines display potent antitrypanosomal activity against both T. b. brucei and
cruzi, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112558.
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8
-Alkynyl-3-nitroimidazopyridines display potent antitrypanosomal activity
against both T. b. brucei and cruzi
1
#
2#
1
3
4
Cyril Fersing , Clotilde Boudot , Caroline Castera-Ducros , Emilie Pinault , Sébastien Hutter ,
1
1
5
5
Romain Paoli-Lombardo , Nicolas Primas , Julien Pedron , Line Seguy , Sandra Bourgeade-
Delmas , Alix Sournia-Saquet , Jean-Luc Stigliani , Jean-Yves Brossas , Luc Paris , Alexis
Valentin , Susan Wyllie , Alan Fairlamb , Élisa Boutet-Robinet , Sophie Corvaisier , Marc
Since , Aurélie Malzert-Fréon , Alexandre Destere , Dominique Mazier , Pascal Rathelot ,
6
5
5
7
7
6
8
8
9
10
1
0
10
11
12
1
2
4
5*
1
Bertrand Courtioux , Nadine Azas , Pierre Verhaeghe and Patrice Vanelle .
1
Aix Marseille Univ, CNRS, ICR UMR 7273, Equipe Pharmaco-Chimie Radicalaire, Faculté de
Pharmacie, 27 Boulevard Jean Moulin, CS30064, 13385, Marseille Cedex 05, France.
2
Université de Limoges, UMR Inserm 1094, Neuroépidémiologie Tropicale, Faculté de Pharmacie, 2 rue
du Dr Marcland, 87025 Limoges, France.
3
Université de Limoges, BISCEm Mass Spectrometry Platform, CBRS, 2 rue du Pr. Descottes, F-87025
Limoges, France.
4
Aix Marseille Univ, IHU Méditerranée Infection, UMR VITROME - Tropical Eukaryotic Pathogens,
1
9-21 Boulevard Jean Moulin, 13005 Marseille, France.
5
LCC-CNRS Université de Toulouse, CNRS, UPS, Toulouse, France.
6
7
8
UMR 152 PHARMA-DEV, Université de Toulouse, IRD, UPS, Toulouse, France.
AP-HP, Groupe Hospitalier Pitié-Salpêtrière, Service de Parasitologie Mycologie, Paris, France.
University of Dundee, School of Life Sciences, Division of Biological Chemistry and Drug Discovery,
Dow Street, Dundee DD1 5EH, Scotland, United Kingdom.
9
Toxalim (Research Centre in Food Toxicology), Université de Toulouse, INRA, ENVT, INP-Purpan,
UPS, Toulouse, France.
10
Normandie Univ, UNICAEN, CERMN, 14000 Caen, France
11
Department of Pharmacology, Toxicology and Pharmacovigilance, CHU Limoges, Limoges, France,
INSERM, UMR 1248, University of Limoges, France.
1
#
*
2
CIMI-Paris, Sorbonne Université 91 boulevard de l'Hôpital 75013 Paris, France.
Co first-authors
Corresponding author:
E-mail address: pierre.verhaeghe@lcc-toulouse.fr (P. Verhaeghe)
Postal address: Université Paul Sabatier, Faculté des Sciences Pharmaceutiques - CNRS UPR 8241,
Laboratoire de Chimie de Coordination, 205 Route de Narbonne, 31077 Toulouse cedex 04, France.

Abstract:
An antikinetoplastid pharmacomodulation study was done at position 8 of a previously
identified pharmacophore in 3-nitroimidazo[1,2-a]pyridine series. Twenty original derivatives
bearing an alkynyl moiety were synthesized via a Sonogashira cross-coupling reaction and tested
in vitro, highlighting 3 potent (40 nM ≤ EC blood stream form≤ 70 nM) and selective (500 ≤ SI
5
0
≤
1800) anti-T. brucei brucei molecules (19, 21 and 22), in comparison with four reference
drugs. Among these hit molecules, compound 19 also showed the same level of activity against
T. cruzi (EC amastigotes = 1.2 µM) as benznidazole and fexinidazole. An in vitro comet assay
50
showed that nitroaromatic derivative 19 was not genotoxic. It displayed a low redox potential
value (-0.68 V/NHE) and was shown to be bioactivated by type 1 nitroreductases both in
Leishmania and Trypanosoma. The SAR study indicated that an alcohol function improved
aqueous solubility while maintaining good activity and low cytotoxicity when the hydroxyl
group was at position beta of the alkyne triple bond. Hit-compound 19 was also evaluated
regarding in vitro pharmacokinetic data: 19 is BBB permeable (PAMPA assay), has a 16 min
microsomal half-life and a high albumin binding (98.5%). Moreover, compound 19 was orally
absorbed and was well tolerated in mouse after both single and repeated administrations at 100
mg/kg. Its mouse plasma half-life (10 h) is also quite encouraging, paving the way toward further
efficacy evaluations in parasitized mouse models, looking for a novel antitrypanosomal lead
compound.
Keywords: Imidazo[1,2-a]pyridine; Nitroaromatic; Nitroreductases; Kinetoplastids; Comet
assay; SARs.

1. Introduction
Neglected tropical diseases (NTDs) constitute a heterogeneous group of communicable diseases
that occur mainly in developing countries and are closely related to poverty [1]. Among the 20
NTDs listed to date by the WHO, three are caused by trypanosomatid protozoa: leishmaniasis
(caused by several Leishmania species) [2], human African trypanosomiasis (HAT, consequence
of an infection by Trypanosoma brucei) [3] and Chagas disease (CD, related to an infection by
Trypanosoma cruzi) [4]. Half a billion people, mainly in tropical and sub-tropical areas, are at
risk of contracting one of these 3 NTDs. The number of individuals infected with these
pathogens is estimated at nearly 20 million, causing up to 50,000 deaths per year [5]. Moreover,
it is very likely that these values are underestimated due to difficulties in data collection in the
most rural and isolated areas. In the absence of human vaccines or chemoprophylaxis to prevent
the transmission of these parasitic diseases in human, the control of trypanosomatid infections
relies on eradication of vectors, management of animal reservoirs and chemotherapy. Only a
small number of molecules are currently available as antitrypanosomatid treatments, most of
them associated with significant drawbacks such as a lack of efficacy, toxicities, constraining
dosing regimens or non-oral route of administration. Likewise, very few new chemical entities
have currently reached the clinical stage of development, even if significant results have been
achieved in the treatment of HAT since the marketing authorization of the nitroaromatic drug
fexinidazole in 2018 (Figure 1) [6]. Thus, there are currently only three new antileishmanial
drugs in clinical trials according to DNDi, all being studied in the early phase 1 [7]. Regarding
sleeping sickness, only acoziborole remains in phase IIb/III clinical trial and, more worryingly,
no original anti-Trypanosoma cruzi molecule is under evaluation in humans at this time, despite
major needs. Only fexinidazole is currently used in a phase II proof-of-concept study as a
possible treatment of Chagas disease. Thus, efficient, safe and cheap orally available
antikinetoplastid agents are awaited [8], particularly against VL and CD [9], considering both
mortality but also the genotoxic character of the two molecules used against CD: nifurtimox and

benznidazole [10]. Nevertheless, nitroaromatic derivatives are a major group of antikinetoplastid
molecules (Figure 1) and the recent introduction of fexinidazole (a non-genotoxic 5-
nitroimidazole derivative) as an oral drug for the treatment of HAT is certainly a pivotal
milestone [6] which illustrates the interest of developing novel nitroheterocyclic drugs to fight
against trypanosomatid infections [11]. In general, anti-infective nitroheterocycles act as
prodrugs requiring a bioactivation step [12]. The accepted mechanism of action for these
compounds involves, first of all, their entry into the parasite by passive diffusion, their reduction
into various reactive metabolites (nitroso, hydroxylamine), and finally the reaction of these
electrophilic metabolites with cellular components such as DNA or proteins, to form covalent
adducts that are cytotoxic [13].
Figure 1. Structures of nitroheterocyclic drug-compounds used in the treatments of
trypanosomatid infections.
The enzymes catalyzing nitrodrugs activation are nitroreductases (NTRs). Two NTRs have been
identified in Leishmania (mitochondrial type 1 NTR1 [14], responsible for the activation of 5-
nitroimidazoles such as fexinidazole [15,16]; and cytosolic type 2 NTR2 [17], responsible for the
activation of bicyclic nitroheterocycles such as delamanid or pretomanid [18]), whereas only one

NTR was discovered in Trypanosoma (type 1 NTR, initiating the activation of nifurtimox [19]
and benznidazole [20]). As NTRs are absent from mammalian cells, substrates of these enzymes
can be envisaged as selective antikinetoplastid candidates. Unfortunately, no X-ray structure of
parasitic NTRs was reported and their low degree of homology with bacterial isoforms restricts
the use of most classical rational medicinal chemistry approaches, such as molecular docking, for
the design of new nitrodrug substrates of these enzymes.
Looking for original nitroheterocyclic antikinetoplastid molecules, our group formerly identified
a first antileishmanial hit molecule in 8-halogeno-3-nitroimidazo[1,2-a]pyridine series (hit A,
Figure 2) [21]. Subsequent work demonstrated the key role of the substituent in position 8 of the
scaffold. Introduction of an heteroaryl moiety in this position improved in vitro activity against
Trypanosoma b. brucei but was not satisfying regarding water solubility (hit B, Figure 2) [22].
Introduction of a phenylthio moiety at position 8 slightly improved in vitro antileishmanial
activity and aqueous solubility (hit C, Figure 2) [23]. Nevertheless, hit-compound C showed a
poor mouse liver microsomal stability (T1/2 = 3 min). Then, in a view to improve both
antitrypanosomal activity and microsomal stability of this series, we present herein the benefit of
introducing a hydroxyalkynyl group at position 8 of the 3-nitroimidazo[1,2-a]pyridine scaffold,
using the Sonogashira cross-coupling reaction.
Figure 2. Structures and biological profiles of previously identified antikinetoplastid hit-
compounds in 3-nitroimidazo[1,2-a]pyridine series [21-23].

2. Results and discussion
The substrate 8-bromo-6-chloro-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-a]pyridine 4 was
prepared in four steps as previously described [22]. The use of the Sonogashira cross-coupling
reaction for the antitrypanosomatid pharmacomodulation of a nitroaromatic scaffold is an
interesting option that was already reported by our team [24]. Hence, the Sonogashira reaction
conditions were optimized from previously described protocols [25-26] to afford 20 original 8-
alkynylimidazo[1,2-a]pyridine derivatives (Scheme 1). Primary alkyne reagents were chosen to
display a large panel of lipophilicity and flexibility, varying from rigid and highly lipophilic
aromatic derivatives to more flexible and hydrophilic hydroxyaliphatic derivatives. Coupling
products were isolated with moderate to good yields (30−90%). Finally, the reduction of the
nitro group of 19 afforded the amino derivative 25, considered as a negative control.
Scheme 1. Synthesis of compounds 1-25.
Reagents and conditions: (i) NBS 1 equiv, ACN, 80°C, 1 h, 71%; (ii) 1,3-dichloroacetone 1.1
equiv, EtOH, 80°C, 96 h, 60%; (iii) HNO 6 equiv, H SO , 0°C→RT, 1 h, 60%; (iv) Sodium
3
2
4
benzenesulfinate 3 equiv, DMSO, RT, 3 h, 80%. (v) appropriate alkyne 1.5 equiv, Pd(PPh ) 0.1
3
4

equiv, CuI 0.1 equiv, diisopropylamine 12 equiv, THF, RT, 1 to 24 h, 30% to 90%. (vi) Fe 10
equiv, AcOH, reflux, 30 min, 75%.
All 21 new compounds were evaluated in vitro, first on the HepG2 cell line to determine their
influence on cell viability (cytotoxic concentration 50% = CC ), using doxorubicin as control. In
5
0
vitro activity of these compounds was then measured on the promastigote form of L. donovani
and on the trypomastigote blood stream form (BSF) of T. b. brucei. Their efficacy was compared
to commercial reference drugs amphotericin B, miltefosine, fexinidazole, suramin, eflornithine
and fexinidazole. The results obtained are presented in Table 1.
Table 1. In vitro bioevaluation of molecules 5-25 on L. donovani promastigotes, T. b. brucei
BSF trypomastigotes and on the human HepG2 cell line.
Activity
Cell viability
EC50 L.
donovani
EC50 T. b.
brucei
Compd
R
CC50 HepG2 (µM)
SI Leishmania
SI Trypanosoma
pro. (µM)
BSF (µM)
a
5
6
>3.9
-
-
-
-
-
-
-
-
a
>3.9
a
7
>3.9
-
-
-
-
H3CO
a
8
9
>3.9
-
-
-
-
-
-
-
-
-
-
-
-
a
>3.9
a
1
1
0
1
>3.9
a
>3.9
-
-
-
-
a
1
1
1
2
3
4
>3.9
-
-
-
-
-
-
-
-
-
-
-
-
a
>3.9
a
>3.9
1
5
8.4 ± 2.3
1.3 ± 0.1
6.5
0.16 ± 0.03
52.5

a
1
1
6
7
>15.6
1.6 ± 0.1
1.0 ± 0.2
>9.8
18.8
0.16 ± 0.03
0.13 ± 0.04
>97.5
18.8 ± 2.2
10.1 ± 1.4
>144.6
1
1
8
9
3.9 ± 0.1
7.4 ± 0.5
2.6
0.12 ± 0.03
0.07 ± 0.01
84.2
a
>125
>16.9
>1785.7
2
2
2
0
1
2
44.2 ± 11.2
4.7 ± 0.2
3.0 ± 0.4
1.6 ± 0.7
9.4
0.04 ± 0.01
0.12 ± 0.03
0.04 ± 0.008
1105
a
>62.5
>20.8
>39.1
>520.8
>1562.5
a
>62.5
a
2
2
2
3
4
5
>31.3
1.7 ± 0.2
1.0 ± 0.4
>18.4
>25
-
0.10 ± 0.03
0.10 ± 0.04
6.8 ± 1.05
>313
>250
>9.2
a
>25
a
a
>62.5
>62.5
a
Ref. 1
Ref. 2
Ref. 3
Ref. 4
Ref. 5
Ref. 6
Ref. 7
Ref. 8
Ref. 9
Ref. 10
Hit A molecule
Hit B molecule
>31
1.8 ± 0.8
>17.2
2.9 ± 0.5
0.25 ± 0.01
1.3 ± 0.1
-
>10.7
>200
>76.9
-
>50
>100
1.2 ± 0.4
>41.7
Hit C molecule
1.0 ± 0.3
>100
b
Doxorubicin
0.2 ± 0.02
8.8 ± 0.3
-
-
d
Amphotericin B
0.07 ± 0.01
125.7
-
-
d
Miltefosine
85 ± 8.8
3.1 ± 0.2
27.4
-
-
d,e
c
>200
c
Fexinidazole
1.2 ± 0.2
>166.7
0.6 ± 0.2
0.03 ± 0.009
13.3 ± 2.1
2.6 ± 0.8
>333
>3333
>7.5
17.4
e
Suramin
>100
c
-
-
-
-
-
-
e
Eflornithine
e
>100
Nifurtimox
45.2 ± 1.3
a
b
c
d
e
f
The product could not be tested at higher concentrations due to a poor solubility in aqueous medium
Doxorubicin was used as a cytotoxic reference drug
The EC50 or CC50 value was not reached at the highest tested concentration
Amphotericin B, Miltefosine and Fexinidazole were used as antileishmanial reference drugs
Fexinidazole, Suramin, Eflornithine and Fexinidazole were used as anti-Trypanosoma brucei reference drugs
SI = CC50 HepG2 / EC50 L. infantum
SI = CC50 HepG2 / EC50 T. brucei brucei
g
Compounds 5-14, which did not bear an alcohol function, could not be evaluated in vitro because
of a lack of aqueous solubility. Most compounds in the series did not show a cytotoxic character
in comparison with doxorubicin: four compounds (15 to 18) showed some decrease in cell
viability with CC values below 20 µM whereas 6 molecules (19 to 24) displayed low values on
50
the HepG2 cell line (>25 to >125 µM). Eleven molecules (15-25) were tested on L. donovani
promastigotes: their EC ranged from 1 µM to 7.4 µM, values comparable to the previously
5
0
identified hit molecules in the series. These activities are also very similar to those of
fexinidazole and miltefosine, respectively 1.2 µM and 3.1 µM. Good CC values in this series,
50
due to better water solubility of hydroxylated molecules, allowed to reach selectivity indices (SI)
that are comparable or superior to that of miltefosine, as for example with the two best
antileishmanial molecules in this series, compounds 22 and 24 (>39.1 and >25, respectively). All

soluble compounds in the experimental conditions (15-24) were evaluated on T. b. brucei BSF
trypomastigotes. Quite interestingly, they all displayed significantly improved EC values (40 ≤
5
0
EC ≤ 160 nM) compared to previously identified hit molecules A, B and C (25 ≤ EC ≤ 2900
50
50
nM); these activity values were all better than the ones of fexinidazole (EC = 0.6 µM) and
5
0
nifurtimox (EC50 = 2.6 µM). High selectivity indices were achieved with most of these
derivatives, hit molecule 19 reaching the highest value (> 1786). This SI value appeared as quite
significant when comparing to those of suramin (> 3333), fexinidazole (> 333), nifurtimox (17.4)
and eflornithine (> 7.5) and hit B (best antitrypanosomal hit previously identified in the series
with SI > 200). Derivatives with a tertiary alcohol function (17, 21, 23, 24) tend to show reduced
activities on Trypanosoma brucei BSF trypomastigotes than those bearing a secondary alcohol
function (20, 22). Likewise, molecules bearing a hydroxy group in alpha position of the alkyne
function (17, 18, 20) tend to decrease cell viability on the HepG2 cell line more than those
bearing a hydroxyl group in beta position of the triple bond (19, 22). As expected, compared
with nitrated molecule 19, the amino-derivative 25 showed a 2 log decrease in antitrypanosomal
activity (EC = 6,8 µM), pointing out the key role of the nitro group in the pharmacophore.
5
0
Thus, because molecule 19 presented the best compromise between in vitro activity, water
solubility, cytotoxicity and selectivity index, it was selected as the hit-compound of this novel
series.
To deeper evaluate the antitrypanosomatid potential of 19, its activity was measured against L.
infantum axenic amastigotes and T. cruzi amastigotes, in addition to its cytotoxicity on the
human macrophage THP1 cell line (Table 2). Thus, molecule 19 did not show a very promising
antileishmanial profile (EC = 7.4 & 10.7 µM) whereas it displayed a good anti-T. cruzi activity
50
(EC50 = 1.2 µM), similar to those of benznidazole and fexinidazole (0.5 and 3.0 µM,
respectively).

Table 2. Summary of the in vitro antitrypanosomatid profile of hit compound 19.
EC (µM)
CC (µM)
50
5
0
Compound
L. donovani
L. inf. axenic
T. b. brucei
T. cruzi
HepG2
THP1
promast.
amast.
BSF
amast.
d
d
1
9
7.4 ± 0.5
10.7 ± 0.7
0.07 ± 0.01
1.2 ± 0.8
>125
>100
a
Doxorubicin
-
-
-
-
0.2 ± 0.02
85 ± 8.8
0.7 ± 0.07
35 ± 2.4
b
Miltefosine
3.1 ± 0.2
1.2 ± 0.2
-
0.8 ± 0.2
3.4 ± 0.8
-
-
-
b,c
e
e
Fexinidazole
0.6 ± 0.2
1.8 ± 0.3
3.0 ± 0.1
0.5 ± 0.1
>200
>200
c
e
e
Benznidazole
>200
>200
a
b
c
d
e
Doxorubicin was used as a cytotoxic reference drug
Miltefosine and Fexinidazole were used as antileishmanial reference drugs
Fexinidazole and Benznidazole were used as antitrypanosomal reference drugs
The product could not be tested at higher concentrations in aqueous medium
The CC50 or EC50 value was not reached at the highest tested concentration
In order to ensure that this novel series contained compounds that were substrates of parasitic
NTRs (Table 3), hit molecules 19 and 22 were assayed against L. donovani promastigotes
corresponding to the wild type, NTR1- and NTR2-overexpressing strains. Molecules 19 and 22
were twenty to forty times more effective against the strain overexpressing (OE) NTR1 than on
the NTR2-overexpressing strain, indicating that these compounds are selectively bioactivated by
L. donovani type 1 NTR. In the same way in T. b. brucei, it was also demonstrated that
trypanosomal type 1 NTR was responsible for the bioactivation of 19 and 22, these latter being 3
to 5 times more potent on the NTR-overexpressing strain than on the wild type. These results
were consistent with those obtained with previous hit molecules, suggesting that introducing an
alkynyl group at position 8 of the imidazo[1,2-a]pyridine pharmacophore preserves its
bioactivation by type 1 NTRs.

Table 3. Sensitivity of wild-type and NTR-overexpressing L. donovani promastigotes and T.
brucei BSF trypomastigotes strains to hit molecules 19 and 22.
L. donovani promastigote EC (µM)
50
Compound
OE
OE
Wild-type
21.4 ± 6
NTR1
NTR2
1
2
9
2
0.5 ± 0.05
0.4 ± 0.01
24.5 ± 5.9
8.5 ± 0.3
3.0 ± 0.08
2.0 ± 0.2
0.3 ± 0.01
7.3 ± 0.6
[22]
[22]
[23]
Hit A
Hit B
Hit C
1.9 ± 0.08
1.5 ± 0.05
0.26 ± 0.01
0.07 ± 0.002
0.04 ± 0.009
0.033 ± 0.007
T. brucei BSF trypomastigote EC (nM)
50
Compound
OE
Wild-type
124.3 ± 7.2
200.0 ± 10.5
1870 ± 0.05
NTR1
1
2
9
2
26.5 ± 1.4
66.3 ± 4.8
600 ± 0.05
Nifurtimox
With regard to a mechanism of action involving an initial reductive bioactivation by NTRs, an
electrochemistry study was carried out by measuring, in DMSO, the redox potentials of six 8-
alkynylimidazo[1,2-a]pyridine derivatives, using cyclic voltammetry (Figure 3). The redox
potentials values measured were corrected with respect to the normal hydrogen electrode (NHE).
They correspond to a reversible one electron reduction/oxidation (redox couple = nitro
group/anion radical counterpart).

Figure 3: Redox potentials (E°) determined by cyclic voltammetry and given versus NHE.
-
3
-1
-1
Conditions: selected compounds (10 mol.L ) in non-aqueous medium (DMSO + 0.1 mol.L
-
1
(
n-Bu N)[PF ]) on GC microdisk (r = 0.5 mm) at room temperature. Scan rate: 0.2 V.s .
4
6
Corriger N° des molécules
Redox potentials within this series appeared to be very homogeneous, with values of -0.67
V/NHE (21, 24) and -0.68 V/NHE (5, 19, 22, 23). The nature of the alkyne group at position 8
does not have an influence on the redox potential of the pharmacophore. However, in
comparison with hit A (-0.59 V/NHE), hit B (-0.64 V/NHE) and hit C (-0.63 V/NHE), molecules
with an alkynyl group at position 8 displayed lower redox potential values, ranging in between
the ones of nifurtimox (- 0.61 V/NHE) and fexinidazole (- 0.83 V/NHE).
The mutagenicity of many nitroaromatic molecules has always been a major concern, limiting
the development of many of these derivatives. For fexinidazole, NTR-dependent mutagenic

activity was observed in Ames test, related to the expression of these enzymes by the Salmonella
typhimurium strains used in this assay. However, evaluation of its genotoxicity by micronucleus
test (in vitro on human cells and in vivo in rats) was negative [27]. Thus, a positive Ames test
when evaluating nitroaromatic compounds has limited predictive values for humans, considering
that there are no NTRs in mammalian cells and that most of nitroaromatics only display
genotoxic properties after being bioactivated into reduced metabolites. Although the Ames test
remains the most common method for assessing the mutagenicity of a substance, it is nowadays
accepted that the comet assay or the micronucleus assay, using mammalian cells, are better in
vitro alternatives for evaluating the potential genotoxicity of nitroheterocyclic molecules.
Considering molecule 19, an Ames test in metabolizing conditions and a comet assay were
performed simultaneously and showed that, despite presenting mutagenic properties (Ames test)
at 0.25 or 2.5 mM, compound 19 was not genotoxic in the comet assay after a 2 or 72 h
exposure, at 20 or 30 µM. These results are consistent with those obtained with fexinidazole and
with previously identified hit molecules in 3-nitroimidazo[1,2-a]pyridine series, representing an
great improvement over nifurtimox and benznidazole which are known to be genotoxic over
mammalian cells [9].
Then, some in vitro physicochemical and pharmacokinetic parameters of hit compound 19 were
determined (Table 4): 19 is a lipophilic molecule (cLogP = 2.5) that shows good aqueous
solubility (thermodynamic solubility = 71 µM), due to its hydroxyl group, that strongly binds to
human albumin (98.5%) and whose microsomal stability was significantly improved (T1/2 = 16
min), in comparison with previously identified hit-compounds in the series (T1/2 = 3 to 9 min).
Hit compound 19 was also shown to cross the blood brain barrier according to a PAMPA BBB
assay, in accordance with its high CNS MPO score (4.35) [28].

Table 4. Physicochemical, pharmacokinetic and toxicological data regarding hit compound 19
Compound 19
a
cLogP
2.49
70.7 ± 2.8
98.5
Thermodynamic solubility (µM)
Binding to human albumin (%)
Microsomal stability: T1/2 (min)
16
PAMPA Blood-brain barrier permeability assay : P (nm/s)
CNS MPO score
346.1 ± 89.2
4.35
e
Cmax in mouse (ng/mL)
Tmax in mouse (h)
Plasma half-life in mouse (h)
AUC0-inf (ng.h/mL)
Clearance (mL/h)
393.96 ± 156.36
0.83 ± 0.29
10.01 ± 3.02
1433.05 ± 69.92
2.44 ± 0.03
Positive
Ames test (0.25 and 2.5 mM + S9mix on 4 strains)
Comet assay (after 2 and 72 h & 20 and 30 µM)
Negative
a
®
Weighted clogP was computed by Marvin (ChemAxon)
Finally, compound 19 was administered orally to mice to determine its maximal tolerated dose
and its main in vivo pharmacokinetic parameters. A once daily repeated oral administration
(intragastrical gavage) of 19 at 100 mg/kg for 5 days was well tolerated in mice. Thus, the No
Observed Adverse Event Level (NOAEL) in mice was set at 100 mg/kg/day. After euthanasia,
no lesions were found on the different organs (kidney, liver, brain, heart and lung).
Pharmacokinetics parameters were determined to understand the behavior of 19 after oral
administration. The main pharmacokinetics parameters are shown in Table 4. Hit compound 19
is orally absorbed and shows a long plasma half-life (10 h), parameters that are encouraging for
further development.

3. Conclusion
Pharmacomodulation at position 8 of the imidazo[1,2-a]pyridine scaffold, using the Sonogashira
cross-coupling reaction, led to 20 original alkynyl derivatives. These displayed modest in vitro
antileishmanial activity, whereas several hydroxylated derivatives showed very good in vitro
activities on T. b. brucei bloodstream forms and low cytotoxicities against a human hepatocyte
cell line (7 molecules with SI > 100 and 2 molecules with SI > 1000). The lead compound of this
series (19) was also tested in vitro on T. cruzi and showed good EC values, two times lower
5
0
than that of fexinidazole. Its mechanism of action involves activation by the parasite NTR1.
Moreover, nitroaromatic hit-molecule 19 was not genotoxic in the comet assay which represents
a significant improvement over nifurtimox and benznidazole. Study of in vitro physicochemical
and pharmacokinetic parameters of molecule 19 showed improved properties in the imidazo[1,2-
a]pyridine series, regarding in particular water solubility and microsomal stability. In the mouse,
19 was orally absorbed and well tolerated after repeated administrations of 100 mg/kg for 5 days.
Its plasma half-life (10 h) is encouraging and allows further determination of its activity in an
infected mouse model, to try to identify a novel antitrypanosomal lead compound.
4
4
4
. Experimental section
.1. Chemistry
.1.1. Synthesis
Commercial reagents were used as received without additional purification. Melting points were
determined in open capillary tubes with a Büchi apparatus and are uncorrected. Elemental
analysis and HRMS were carried out at the Spectropole, Faculté des Sciences et Techniques de
Saint-Jérôme, Marseille, France. NMR spectra were recorded on a Bruker ARX 200
spectrometer or a Bruker AV 250 spectrometer at the Faculté de Pharmacie de Marseille, or a
BRUKER Avance III nanobay 400 spectrometer at the the Spectropole, Faculté des Sciences et
Techniques de Saint-Jérôme, Marseille, or on a Bruker UltraShield 300 MHz or a Bruker
1
IconNMR 400 MHz spectrometer at the Laboratoire de Chimie de Coordination, Toulouse ( H-
1
3
NMR: 200, 250, 300 or 400 MHz, C-NMR: 50, 63, 75 or 100 MHz). NMR references were the

1
13
following: H: CHCl δ = 7.26, DMSO-d δ = 2.50, Acetone-d δ = 2.05 and C: CHCl δ =
3
6
6
3
7
6.9, DMSO-d δ = 39.5, Acetone-d δ = 29.9. Solvents were dried by conventional methods.
6 6
The following adsorbent was used for column chromatography: silica gel 60 (Merck, particle
size 0.063–0.200 mm, 70–230 mesh ASTM). TLC was performed on 5 cm × 10 cm aluminium
plates coated with silica gel 60F-254 (Merck) in an appropriate eluent. Visualization was made
with ultraviolet light (254 nm). HRMS spectra were recorded on QStar Elite (Applied
Biosystems SCIEX) spectrometer. PEG was the matrix for HRMS. The experimental exact mass
was given for the ion which has the maximum isotopic abundance. Purity of synthetized
compounds was checked with LC-MS analyses which were realized at the Faculté de Pharmacie
®
de Marseille with a Thermo Scientific Accela High Speed LC System coupled with a single
®
quadrupole mass spectrometer Thermo MSQ Plus . The RP-HPLC column used is a Thermo
®
Hypersil Gold 50 × 2.1 mm (C18 bounded), with particles of 1.9 µm diameter. The volume of
sample injected on the column was 1 µL. The chromatographic analysis, total duration of 8 min,
is made with the gradient of following solvents: t = 0 min, water/methanol 50/50; 0 < t < 4 min,
linear increase in the proportion of methanol to a ratio water/methanol 5/95; 4 < t < 6 min,
water/methanol 5/95; 6 < t < 7 min, linear decrease in the proportion of methanol to return to a
ratio 50/50 water/methanol; 6 < t < 7 min, water/methanol 50/50. The water used was buffered
with 5 mM ammonium acetate. The retention times (t ) of the molecules analyzed are indicated
R
in min.
Molecules 1-4 were previously described [22].
4.1.1.1. General procedure for the preparation of 8-alkynylimidazo[1,2-a]pyridine
derivatives (5 to 24)
A mixture of 8-bromo-6-chloro-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-a]pyridine 4 (400
mg, 1 equiv.), tetrakis(triphenylphosphine)palladium(0) (107.3 mg, 0.1 equiv.), copper iodide
(17.7 mg, 0.1 equiv.), diisopropylamine (1.57 mL, 12 equiv.), appropriate alkyne (1.5 equiv.), in
THF (15 mL) was stirred under N at room temperature until complete disappearance of the
2
starting material (as monitored by LC/MS or TLC). Water was then added and the mixture was
extracted three times with dichloromethane. The organic layer was washed three times with
water, dried over MgSO , filtered and evaporated. The crude residue was purified by column
4
chromatography on silica gel (with appropriate eluent) and recrystallized from the appropriate
solvent, affording compounds 1 to 20.
4.1.1.2.
6-Chloro-3-nitro-8-(phenylethynyl)-2-(phenylsulfonylmethyl)imidazo[1,2-
a]pyridine (5)

Compound
5
was obtained after purification by chromatography (eluent:
dichloromethane/cyclohexane/diethyl ether 6.9/3/0.1) and recrystallization from acetonitrile as a
1
yellow solid in 90% yield (0.38 g). mp 228 °C. H NMR (400 MHz, CDCl ) δ: 5.19 (2H, s),
3
7.40–7.47 (3H, m), 7.50–7.54 (2H, m), 7.59–7.63 (3H, m), 7.79 (1H, d, J = 1.9 Hz), 7.88–7.91
1
3
(
2H, m), 9.41 (1H, d, J = 1.9 Hz). C NMR (100 MHz, CDCl ) δ: 56.9 (CH ), 81.5 (C), 99.8
3 2
(
C), 115.7 (C), 121.6 (C), 124.9 (CH), 125.5 (C), 128.7 (2 CH), 128.8 (2 CH), 129.3 (2 CH),
30.0 (CH), 131.3 (C), 132.4 (2 CH), 134.2 (CH), 134.5 (CH), 139.3 (C), 139.8 (C), 143.0 (C).
1
+
+
+
LC/MS ESI t 3.99 min, (m/z) [M+H] 451.83/453.84. HRMS (+ESI): 452.0462 [M+H] . Calcd
R
for C H ClN O S: 452.0466.
2
2
14
3
4
4.1.1.3.
6-Chloro-8-(4-methoxyphenylethynyl)-3-nitro-2-
(phenylsulfonylmethyl)imidazo[1,2-a]pyridine (6)
Compound
6
was obtained after purification by chromatography (eluent:
dichloromethane/cyclohexane 9/1) and recrystallization from acetonitrile as a yellow solid in
1
5
6
1% yield (0.29 g). mp 210 °C. H NMR (400 MHz, CDCl ) δ: 3.88 (3H, s), 5.19 (2H, s), 6.92–
3
.94 (2H, m), 7.50–7.55 (4H, m), 7.60–7.64 (1H, m), 7.74 (1H, d, J = 1.9 Hz), 7.88–7.91 (2H,
1
3
m), 9.37 (1H, d, J = 1.9 Hz). C NMR (100 MHz, CDCl ) δ: 55.6 (CH ), 56.9 (CH ), 80.7 (C),
3
3
2
100.4 (C), 113.6 (C), 114.3 (2 CH), 116.1 (C), 124.5 (CH), 125.5 (C), 128.8 (2 CH), 129.3 (2
CH), 131.3 (C), 134.0 (CH), 134.1 (2 CH), 134.2 (CH), 139.3 (C), 139.8 (C), 143.0 (C), 161.0
+
+
+
(
C). LC/MS ESI t 4.09 min, (m/z) [M+H] 480.61/483.87. HRMS (+ESI): 482.0569 (M + H ).
R
Calcd for C H ClN O S: 482.0572.
2
3
16
3
5
4.1.1.4.
6-Chloro-8-(3-methoxyphenylethynyl)-3-nitro-2-
(phenylsulfonylmethyl)imidazo[1,2-a]pyridine (7)
Compound 7 was obtained after purification by chromatography (eluent: chloroform/diethyl
1
ether 9.9/0.1) as a yellow solid in 47% yield (0.21 g). mp 171 °C. H NMR (400 MHz, Acetone-
d6) δ: 3.89 (3H, s), 5.22 (2H, s), 7.08–7.12 (2H, m), 7.17–7.19 (1H, m), 7.40–7.44 (1H, m), 7.58–
7.62 (2H, m), 7.69–7.73 (1H, m), 7.84–7.86 (2H, m), 8.04 (1H, d, J = 1.9 Hz), 9.42 (1H, d, J =
1
3
1
.9 Hz). C NMR (100 MHz, Acetone-d ) δ: 54.9 (CH ), 56.1 (CH ), 81.5 (C), 97.9 (C), 114.5
6
3
2
(
(
(
C), 116.1 (CH), 116.6 (CH), 122.6 (C), 124.1 (C), 124.2 (CH), 125.6 (CH), 128.5 (2 CH), 129.0
2 CH), 129.84 (CH), 131.5 (C), 133.9 (CH), 134.1 (CH), 139.5 (C), 139.7 (C), 142.7 (C), 159.7
+
+
+
C). LC/MS ESI t 4.09 min, (m/z) [M+H] 480.70/483.65. HRMS (+ESI): 482.0569 (M + H ).
R
Calcd for C H ClN O S: 482.0572.
2
3
16
3
5
4.1.1.5.
6-Chloro-8-(2-methoxyphenylethynyl)-3-nitro-2-
(phenylsulfonylmethyl)imidazo[1,2-a]pyridine (8)

Compound
8
was obtained after purification by chromatography (eluent:
dichloromethane/cyclohexane/diethyl ether 6.9/3/0.1) and recrystallization from acetonitrile as a
1
yellow solid in 67% yield (0.30 g). mp 204 °C. H NMR (400 MHz, CDCl ) δ: 3.96 (3H, s), 5.19
3
(2H, s), 6.94–7.01 (2H, m), 7.39–7.44 (1H, m), 7.49–7.54 (3H, m), 7.58–7.62 (1H, m), 7.80 (1H,
1
3
d, J = 1.9 Hz), 7.89–7.91 (2H, m), 9.38 (1H, d, J = 1.9 Hz). C NMR (100 MHz, DMSO-d ) δ:
6
5
6.3 (CH ), 56.4 (CH ), 86.0 (C), 95.4 (C), 110.4 (C), 112.1 (CH), 114.4 (C), 121.1 (CH), 124.2
3 2
(C), 126.3 (CH), 128.7 (2 CH), 129.7 (2 CH), 131.7 (C), 132.2 (CH), 134.0 (CH), 134.6 (CH),
+
+
1
34.9 (CH), 139.2 (C), 139.8 (C), 142.9 (C), 160.5 (C). LC/MS ESI t 3.86 min, (m/z) [M+H]
R
+
4
80.62/483.88. HRMS (+ESI): 482.0570 (M + H ). Calcd for C H ClN O S: 482.0572.
2
3
16
3
5
4.1.1.6. 6-Chloro-8-(4-fluorophenylethynyl)-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-
a]pyridine (9)
Compound
9
was obtained after purification by chromatography (eluent:
dichloromethane/cyclohexane 7/3) and recrystallization from acetonitrile as a yellow solid in
1
4
9% yield (0.21 g). mp 245 °C. H NMR (250 MHz, DMSO-d ) δ: 5.31 (2H, s), 7.36–7.43 (2H,
6
1
3
m), 7.57–7.67 (4H, m), 7.70–7.73 (1H, m), 7.76–7.81 (2H, m), 8.26 (1H, s), 9.35 (1H, s). C
NMR (100 MHz, DMF-d ) δ: 56.7 (CH ), 82.3 (C), 97.1 (C), 114.4 (C), 116.7 (2 CH, d, J = 22.6
7
2
Hz), 118.3 (C, d, J = 3.3 Hz), 124.5 (C), 126.5 (CH), 128.9 (2 CH), 129.7 (2 CH), 132.1 (C),
34.6 (CH), 134.8 (2 CH, d, J = 8.9 Hz), 135.0 (CH), 139.8 (C), 140.1 (C), 143.2 (C), 164.9 (C).
1
+
+
+
LC/MS ESI t 4.01 min, (m/z) [M+H] 469.70/471.23. HRMS (+ESI): 470.0371 (M + H ).
R
Calcd for C H ClFN O S: 470.0372.
2
2
13
3
4
4.1.1.7. 6-Chloro-8-(3-fluorophenylethynyl)-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-
a]pyridine (10)
Compound 10 was obtained after purification by chromatography (eluent:
dichloromethane/cyclohexane/diethyl ether 6.8/3/0.2) and recrystallization from acetonitrile as a
1
yellow solid in 38% yield (0.17 g). mp 240 °C. H NMR (400 MHz, CDCl ) δ: 5.19 (2H, s),
3
1
3
7
.16–7.18 (1H, m), 7.28–7.65 (6H, m), 7.79 (1H, s), 7.89 (2H, d, J = 7.6 Hz), 9.42 (1H, s). C
NMR (100 MHz, CDCl ) δ: 56.8 (CH ), 82.2 (C), 98.1 (C), 115.2 (C), 117.4 (CH, d, J = 21.2
3
2
Hz), 119.1 (CH, d, J = 23 Hz), 123.3 (C, d, J = 9.43 Hz), 125.3 (CH), 125.4 (C), 128.2 (CH, d, J
=
3.23 Hz), 128.2 (2 CH), 129.3 (2 CH), 130.3 (CH, d, J = 8.54 Hz), 131.3 (C), 134.3 (CH),
+
1
34.6 (CH), 139.3 (C), 139.9 (C), 142.9 (C), 162.5 (C, d, J = 247.6 Hz). LC/MS ESI t 4.03
R
+
+
min, (m/z) [M+H] 469.85/471.98. HRMS (+ESI): 470.0372 (M + H ). Calcd for
C H ClFN O S: 470.0372.
22
13
3
4
4.1.1.8. 6-Chloro-8-(2-fluorophenylethynyl)-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-
a]pyridine (11)

Compound 11 was obtained after purification by chromatography (eluent:
dichloromethane/cyclohexane 8/2) and recrystallization from acetonitrile as a yellow solid in
1
6
5% yield (0.28 g). mp 229 °C. H NMR (400 MHz, DMSO-d ) δ: 5.30 (2H, s), 7.35–7.45 (2H,
6
m), 7.56–7.72 (5H, m), 7.80 (2H, d, J = 7.1 Hz), 8.28 (1H, d, J = 1.9 Hz), 9.36 (1H, d, J = 1.9
1
3
Hz). C NMR (100 MHz, DMSO-d ) δ: 55.8 (CH ), 86.7 (C), 90.7 (C), 109.4 (C, d, J = 15.4
6
2
Hz), 112.9 (C), 116.0 (CH, d, J = 20.2 Hz), 123.6 (C), 125.1 (CH, d, J = 3.58 Hz), 126.4 (CH),
128.2 (2 CH), 129.2 (2 CH), 131.3 (C), 132.5 (CH, d, J = 8.26 Hz), 133.8 (CH), 134.1 (CH),
+
1
35.1 (CH), 138.7 (C), 139.4 (C), 142.3 (C), 161.9 (C, d, J = 251.5 Hz). LC/MS ESI t 3.95
R
+
+
min, (m/z) [M+H] 469.83/471.49. HRMS (+ESI): 470.0371 (M + H ). Calcd for
C H ClFN O S: 470.0372.
22
13
3
4
4.1.1.9.
4-[6-Chloro-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-a]pyridin-8-
yl]ethynylaniline (12)
After washing the crude residue with acetone and filtration, compound 12 was obtained as an
1
orange solid in 72% yield (0.31 g). mp 255 °C. H NMR (400 MHz, DMSO-d ) δ: 5.29 (2H, s),
6
1
3
5
.90 (2H, s), 6.63 (2H, s), 7.22 (2H, s), 7.60–7.81 (5H, m), 8.06 (1H, s), 9.24 (1H, s). C NMR
(
100 MHz, DMSO-d ) δ: 55.8 (CH ), 80.2 (C), 101.1 (C), 106.2 (C), 113.6 (2 CH), 114.8 (C),
6
2
123.8 (C), 124.6 (CH), 128.2 (2 CH), 129.2 (2 CH), 131.2 (C), 133.1 (CH), 133.2 (2 CH), 134.1
+
+
(
CH), 138.7 (C), 139.2 (C), 142.4 (C), 150.8 (C). LC/MS ESI t 3.28 min, (m/z) [M+H]
R
+
4
66.80/468.68. HRMS (+ESI): 467.0572 (M + H ). Calcd for C H ClN O S: 467.0575.
2
2
15
4
4
4.1.1.10. 6-Chloro-3-nitro-2-(phenylsulfonylmethyl)-8-(thiophen-2-ylethynyl)imidazo[1,2-
a]pyridine (13)
Compound 13 was obtained after purification by chromatography (eluent:
dichloromethane/cyclohexane/diethyl ether 5/4.5/0.5) and recrystallization from acetonitrile as a
1
yellow solid in 65% yield (0.28 g). mp 220 °C. H NMR (400 MHz, CDCl ) δ: 5.19 (2H, s),
3
1
3
7
.08–7.11 (1H, m), 7.44–7.66 (5H, m), 7.77 (1H, s), 7.89 (2H, d, J = 7.7 Hz), 9.40 (1H, s). C
NMR (100 MHz, CDCl ) δ: 56.8 (CH ), 85.3 (C), 93.2 (C), 115.4 (C), 121.4 (C), 124.9 (CH),
3
2
125.5 (C), 127.6 (CH), 128.8 (2 CH), 129.3 (2 CH), 129.8 (CH), 131.3 (C), 134.2 (CH), 134.3
+
+
(
CH), 134.5 (CH), 139.2 (C), 139.8 (C), 142.6 (C). LC/MS ESI t 3.79 min, (m/z) [M+H]
R
+
4
57.72/459.88. HRMS (+ESI): 458.0031 (M + H ). Calcd for C H ClN O S : 458.0031.
2
0
12
3
4 2
4.1.1.11.
6-Chloro-3-nitro-2-(phenylsulfonylmethyl)-8-(pyridin-3-ylethynyl)imidazo[1,2-
a]pyridine (14)
Compound 14 was obtained after purification by chromatography (eluent: chloroform/diethyl
1
ether 8/2) as a yellow solid in 36% yield (0.15 g). mp 240 °C. H NMR (400 MHz, DMSO-d ) δ:
6

5.31 (2H, s), 7.56–7.61 (3H, s), 7.68–7.72 (1H, s), 7.79–7.81 (2H, m), 7.99–8.02 (1H, m), 8.30
(1H, d, J = 1.9 Hz), 8.70 (1H, dd, J = 1.6 Hz and 4.9 Hz), 8.77 (1H, d, J = 1.6 Hz), 9.37 (1H, d, J
13
=
1.9 Hz). C NMR (100 MHz, DMSO-d ) δ: 55.8 (CH ), 85.0 (C), 94.2 (C), 112.8 (C), 118.2
6 2
(C), 123.6 (C), 123.9 (CH), 126.5 (CH), 128.3 (2 CH), 129.2 (2 CH), 131.3 (C), 134.1 (CH),
+
135.1 (CH), 138.7 (C), 138.9 (CH), 139.4 (C), 142.2 (C), 150.1 (CH), 151.8 (CH). LC/MS ESI
+
+
t 2.91 min, (m/z) [M+H] 452.77/454.84. HRMS (+ESI): 453.0417 (M + H ). Calcd for
R
C H ClN O S: 453.0419.
21
13
4
4
4.1.1.12.
6-Chloro-3-nitro-8-(3-phenoxyprop-1-yn-1-yl)-2-
(phenylsulfonylmethyl)imidazo[1,2-a]pyridine (15)
Compound 15 was obtained after purification by chromatography (eluent: dichloromethane/ethyl
1
acetate 9.8/0.2) as a dark solid in 74% yield (0.33 g). mp 72 °C. H NMR (250 MHz, DMSO-d )
6
δ: 5.17 (2H, s), 5.27 (2H, s), 6.98–7.09 (2H, m), 7.32–7.39 (3H, m), 7.56–7.62 (2H, m), 7.70–
13
7
.80 (3H, m), 8.16 (1H, d, J = 1.7 Hz), 9.31 (1H, d, J = 1.9 Hz). C NMR (62.5 MHz, DMSO-
d ) δ: 55.8 (CH ), 56.0 (CH ), 79.0 (C), 93.8 (C), 112.6 (C), 114.9 (2 CH), 121.4 (CH), 123.5
6
2
2
(C), 126.3 (CH), 128.1 (2 CH), 129.2 (2 CH), 129.5 (2 CH), 131.2 (C), 134.1 (CH), 135.2 (CH),
+
+
1
38.7 (C), 139.2 (C), 142.5 (C), 157.3 (C). LC/MS ESI t 3.73 min, (m/z) [M+H]
R
+
4
80.60/483.76. HRMS (+ESI): 482.0572 (M + H ). Calcd for C H ClN O S: 482.0572.
2
3
16
3
5
4.1.1.13.
6-Chloro-8-[(4-hydroxymethyl)phenylethynyl]-3-nitro-2-
(phenylsulfonylmethyl)imidazo[1,2-a]pyridine (16)
Compound 16 was obtained after purification by chromatography (eluent: dichloromethane/ethyl
acetate 9/1) and recrystallization from propan-2-ol as a pale yellow solid in 68% yield (0.30 g).
1
mp 187 °C. H NMR (250 MHz, DMSO-d ) δ: 4.58 (2H, d, J = 5.4 Hz), 5.31 (2H, s), 5.38 (1H, t,
6
J = 5.6 Hz), 7.44–7.48 (2H, m), 7.52–7.63 (4H, m), 7.70–7.75 (1H, m), 7.79–7.82 (2H, m), 8.24
1
3
(
1H, d, J = 1.7 Hz), 9.33 (1H, d, J = 1.8 Hz). C NMR (62.5 MHz, DMSO-d ) δ: 55.8 (CH ),
6 2
6
2.4 (CH ), 81.7 (C), 98.0 (C), 113.6 (C), 119.0 (C), 123.6 (C), 125.8 (CH), 126.7 (2 CH), 128.2
2
(2 CH), 129.2 (2 CH), 131.2 (C), 131.5 (2 CH), 134.1 (CH), 134.5 (CH), 138.7 (C), 139.3 (C),
+
+
1
4
42.3 (C), 144.8 (C). LC/MS ESI t 3.29 min, (m/z) [M+H] 480.61/483.75. HRMS (+ESI):
R
+
82.0571 (M + H ). Calcd for C H ClN O S: 482.0572.
2
3
16
3
5
4.1.1.14.
4-[6-Chloro-3-nitro-2-(phenylsulfonylmethyl]imidazo[1,2-a]pyridin-8-yl)-2-
phenylbut-3-yn-2-ol (17)
Compound 17 was obtained after purification by chromatography (eluent:
dichloromethane/methanol 9.8/0.2) and recrystallization from propan-2-ol as a yellow solid in
1
7
2% yield (0.33 g). mp 160 °C. H NMR (250 MHz, DMSO-d ) δ: 1.77 (3H, s), 5.30 (2H, s),
6

6.48 (1H, s), 7.31–7.46 (3H, m), 7.56–7.62 (2H, m), 7.70–7.75 (3H, m), 7.81–7.84 (2H, m), 8.18
1
3
(
(
(
(
(
1H, s), 9.35 (1H, s). C NMR (62.5 MHz, DMSO-d ) δ: 33.5 (CH ), 56.0 (CH ), 68.7 (C), 76.0
6 3 2
C), 103.4 (C), 113.5 (C), 123.6 (C), 125.0 (2 CH), 125.8 (CH), 127.2 (CH), 128.0 (2 CH), 128.1
2 CH), 129.2 (2 CH), 131.2 (C), 134.1 (CH), 134.7 (CH), 138.8 (C), 139.3 (C), 142.5 (C), 145.8
+
+
C). LC/MS ESI t 3.66 min, (m/z) [M+NH ] 511.53/512.76/514.32. HRMS (+ESI): 518.0547
R
4
+
+
M + Na ). Calcd for C H ClN O S, Na : 518.0548.
2
4
18
3
5
4.1.1.15. 3-[6-Chloro-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-a]pyridin-8-yl]prop-2-
yn-1-ol (18)
Compound 18 was obtained after purification by chromatography (eluent: dichloromethane/ethyl
1
acetate 9/1) as a white solid in 60% yield (0.23 g). mp 225 °C. H NMR (300 MHz, DMSO-d )
6
δ: 4.42 (2H, d, J = 5.54 Hz), 5.56 (2H, s), 5.58 (1H, t, J = 6.2 Hz), 7.58–7.63 (2H, m), 7.74–7.80
1
3
(
3H, m), 8.11 (1H, s), 9.30 (1H, s). C NMR (75 MHz, DMSO-d ) δ: 49.6 (CH ), 55.9 (CH ),
6 2 2
7
1
6.4 (C), 99.0 (C), 113.5 (C), 123.6 (C), 125.9 (CH), 128.1 (2 CH), 129.3 (2 CH), 131.2 (C),
+
34.2 (CH), 134.8 (CH), 138.7 (C), 139.1 (C), 142.6 (C). LC/MS ESI t 1.79 min, (m/z)
R
+
+
[
M+NH ] 422.88/425.02. HRMS (+ESI): 406.0259 (M + H ). Calcd for C H ClN O S:
4
17 12
3
5
406.0259.
4.1.1.16. 4-[6-Chloro-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-a]pyridin-8-yl]but-3-yn-
1-ol (19)
Compound 19 was obtained after purification by chromatography (eluent:
dichloromethane/methanol 9.5/0.5) and recrystallization from acetonitrile as a beige solid in 67%
1
yield (0.26 g). mp 185 °C. H NMR (400 MHz, CDCl ) δ: 2.78–2.86 (3H, m), 3.89 (2H, s), 5.15
3
13
(
2H, s), 7.55–7.67 (4H, m), 7.87–7.90 (2H, m), 9.37 (1H, s). C NMR (100 MHz, CDCl ) δ:
3
2
4.6 (CH ), 56.8 (CH ), 60.7 (CH ), 75.1 (C), 100.1 (C), 115.8 (C), 124.7 (CH), 125.6 (C), 128.6
2 2 2
(2 CH), 129.4 (2 CH), 131.3 (C), 134.1 (CH), 134.3 (CH), 139.3 (C), 139.4 (C), 143.6 (C).
+
+
+
LC/MS ESI t 2.13 min, (m/z) [M+H] 419.81/421.93. HRMS (+ESI): 420.0416 (M + H ).
R
Calcd for C H ClN O S: 420.0415.
1
8
14
3
5
4.1.1.17. 4-[6-Chloro-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-a]pyridin-8-yl]but-3-yn-
2-ol (20)
Compound 20 was obtained after purification by chromatography (eluent:
dichloromethane/methanol 9.8/0.2) and recrystallization from propan-2-ol as a beige solid in
1
6
7% yield (0.26 g). mp 196 °C. H NMR (300 MHz, CDCl ) δ: 1.60 (3H, d, J = 6.1 Hz), 3.62
3
(1H, s), 4.88 (1H, bs), 5.18 (2H, s), 7.51–7.56 (2H, m), 7.64–7.70 (2H, m), 7.85–7.87 (2H, m),
1
3
9
.36 (1H, s). C NMR (75 MHz, CDCl ) δ: 23.6 (CH ), 56.6 (CH ), 58.5 (CH), 75.9 (C), 102.3
3 3 2
(C), 114.8 (C), 125.0 (CH), 125.3 (C), 128.4 (2 CH), 129.3 (2 CH), 131.1 (C), 134.2 (CH), 134.7

+
+
(
CH), 139.0 (C), 139.4 (C), 142.9 (C). LC/MS ESI t 2.28 min, (m/z) [M+NH ] 436.88/438.9.
R 4
+
HRMS (+ESI): 420.0418 (M + H ). Calcd for C H ClN O S: 420.0415.
1
8
14
3
5
4.1.1.18.
4-[6-chloro-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-a]pyridin-8-yl]-2-
methylbut-3-yn-2-ol (21)
Compound 21 was obtained after purification by chromatography (eluent: dichloromethane/ethyl
acetate 9/1) and recrystallization from propan-2-ol as a pale yellow solid in 78% yield (0.31 g).
1
mp 193 °C. H NMR (400 MHz, DMSO-d ) δ: 1.50 (6H, s), 5.27 (2H, s), 5.71 (1H, s), 7.58–7.64
6
1
3
(
2H, m), 7.72–7.81 (3H, m), 8.04 (1H, s), 9.30 (1H, s). C NMR (100 MHz, DMSO-d ) δ: 31.2
6
(
2 CH ), 55.9 (CH ), 63.8 (C), 73.5 (C), 105.0 (C), 113.8 (C), 123.6 (C), 125.6 (CH), 128.1 (2
3
2
CH), 129.2 (2 CH), 131.2 (C), 134.2 (CH), 134.8 (CH), 138.8 (C), 139.2 (C), 142.4 (C). LC/MS
+
+
+
ESI t 2.73 min, (m/z) [M+NH ] 450.37/452.80. HRMS (+ESI): 434.0571 (M + H ). Calcd for
R
4
C H ClN O S: 434.0572.
19
16
3
5
4
3
.1.1.19. 6-[6-Chloro-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-a]pyridin-8-yl]hex-5-yn-
-ol (22)
Compound 22 was obtained after purification by chromatography (eluent: cyclohexane/ethyl
acetate 4/5) and recrystallization from propan-2-ol as a white solid in 60% yield (0.25 g). mp 154
1
°
C. H NMR (400 MHz, DMSO-d ) δ: 0.93 (3H, t, J = 7.4 Hz), 1.39–1.70 (2H, m), 2.63 (2H, d, J
6
=
5.9 Hz), 3.59–3.69 (1H, m), 4.91 (1H, d, J = 5.2 Hz), 5.25 (2H, s), 7.58–7.63 (2H, m), 7.73–
1
3
7
.79 (3H, m), 8.04 (1H, d, J = 1.9 Hz), 9.28 (1H, d, J = 1.9 Hz). C NMR (100 MHz, DMSO-d )
6
δ: 10.0 (CH ), 27.9 (CH ), 28.9 (CH ), 55.9 (CH ), 69.9 (CH), 74.5 (C), 98.7 (C), 114.4 (C),
3
2
2
2
123.6 (C), 125.3 (CH), 128.1 (2 CH), 129.3 (2 CH), 131.1 (C), 134.2 (CH), 134.6 (CH), 138.8
+
+
(
C), 139.1 (C), 142.8 (C). LC/MS ESI t 2.88 min, (m/z) [M+H] 447.51/448.78/450.54. HRMS
R
+
(
+ESI): 448.0728 (M + H ). Calcd for C H ClN O S: 448.0728.
2
0
18
3
5
4.1.1.20.
1-{[6-Chloro-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-a]pyridin-8-
yl]ethynyl}cyclopentanol (23)
Compound 23 was obtained after purification by chromatography (eluent:
dichloromethane/methanol 9.8/0.2) and recrystallization from propan-2-ol as a white solid in
1
6
1% yield (0.26 g). mp 214 °C. H NMR (400 MHz, DMSO-d ) δ: 1.68–1.81 (4H, m), 1.87–1.99
6
(4H, m), 5.27 (2H, s), 5.55 (1H, s), 7.59–7.63 (2H, m), 7.73–7.80 (3H, m), 8.05 (1H, d, J = 1.9
1
3
Hz), 9.30 (1H, d, J = 1.9 Hz). C NMR (100 MHz, DMSO-d ) δ: 23.1 (2 CH ), 41.9 (2 CH ),
6
2
2
5
5.9 (CH ), 72.9 (C), 74.4 (C), 104.3 (C), 113.9 (C), 123.6 (C), 125.6 (CH), 128.1 (2 CH), 129.3
2
+
(
2 CH), 131.2 (C), 134.2 (CH), 134.7 (CH), 138.8 (C), 139.2 (C), 142.5 (C). LC/MS ESI t 3.35
R

+
+
min, (m/z) [M+NH4] 476.91/478.71. HRMS (+ESI): 460.0727 (M + H ). Calcd for
C H ClN O S: 460.0728.
21
18
3
5
4.1.1.21.
1-{[6-Chloro-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-a]pyridin-8-
yl]ethynyl}cyclohexanol (24)
Compound 24 was obtained after purification by chromatography (eluent:
dichloromethane/methanol 9.6/0.4) and recrystallization from acetonitrile as a white solid in 77%
1
yield (0.34 g). mp 232 °C. H NMR (400 MHz, DMSO-d ) δ: 1.26 (1H, bs), 1.49–1.65 (8H, m),
6
1.84–1.90 (2H, m), 5.25 (2H, s), 7.56–7.63 (2H, m), 7.71–7.80 (3H, m), 8.07 (1H, d, J = 1.83
13
Hz), 9.31 (1H, d, J = 1.81 Hz). C NMR (100 MHz, DMSO-d ) δ: 22.6 (2 CH ), 24.8 (CH ),
6
2
2
3
9.4 (2 CH ), 56.0 (CH ), 67.3 (C), 75.7 (C), 104.2 (C), 113.9 (C), 123.6 (C), 125.6 (CH), 128.0
2 2
(2 CH), 129.2 (2 CH), 131.2 (C), 134.1 (CH), 134.6 (CH), 138.8 (C), 139.3 (C), 142.5 (C).
+
+
+
LC/MS ESI t 3.58 min, (m/z) [M+NH ] 490.94/492.91. HRMS (+ESI): 474.0887 (M + H ).
R
4
Calcd for C H ClN O S: 474.0885.
2
2
20
3
5
4.1.1.22. Preparation of 4-[3-amino-6-chloro-2-(phenylsulfonylmethyl)imidazo[1,2-
a]pyridin-8-yl]but-3-yn-1-ol (25)
A mixture of 8-bromo-6-chloro-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-a]pyridine 4 (400
mg, 1 equiv.) in acetic acid (60 mL), iron powder (519 mg, 10 equiv.) was stirred and heated
under reflux for 30 min. The mixture was then filtered through celite and the solvent was
evaporated in vacuo. The resulting residue was diluted with H O and basified with saturated
2
aqueous NaHCO . The mixture was extracted three times with dichloromethane, dried over
3
MgSO , filtered and evaporated. Then, a mixture of the previous residue (200 mg, 1 equiv.),
4
tetrakis(triphenylphosphine)palladium(0) (57.8 mg, 0.1 equiv.), copper iodide (9.5 mg, 0.1
equiv.), diisopropylamine (843 µL, 12 equiv.) and but-3-yn-1-ol (57 µL, 1.5 equiv.) in THF (7
mL) was stirred under N at room temperature for 24 h. The reaction mixture was then slowly
2
poured into an ice-water mixture. The resulting suspension was filtered. Compound 25 was
obtained after purification by flash chromatography (eluent: dichloromethane/ethyl acetate
1
2
2
7
8
7
1
0/80) as a yellow solid in 57 % yield (111 mg). mp 190 °C. H NMR (400 MHz, DMSO-d ) δ:
6
.63 (2H, t, J = 6.8 Hz), 3.60 (2H, dd, J = 10.6, 6.4 Hz), 4.79 (2H, s), 4.94 (1H, s), 5.28 (2H, s),
.12 (1H, d, J = 1.8 Hz), 7.59 (2H, t, J = 7.7 Hz), 7.71 (1H, t, J = 7.4 Hz), 7.82 – 7.76 (2H, m),
1
3
.20 (1H, d, J = 1.8 Hz). C NMR (100 MHz, DMSO-d ) δ: 23.6 (CH ), 54.5 (CH ), 59.6 (CH ),
6
2
2
2
5.9 (CH), 95.2 (CH), 113.0 (C), 115.9 (CH), 117.5 (C), 120.0 (C), 124.8 (CH), 128.1 (2 CH),
+
29.0 (2 CH), 132.3 (C), 133.6 (C), 136.1 (CH), 139.5 (C). LC/MS ESI t 1.86 min, (m/z)
R
+
+
[
M+H] 390.02/392.02. HRMS (+ESI): 390.0671 [M+H] ; Calcd for C H ClN O S :
18 16 3 3
390.0674.

4.1.2. Electrochemistry
Voltammetric measurements were carried out with a potentiostat Autolab PGSTAT100 (ECO
Chemie, The Netherlands) controlled by GPES 4.09 software. 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 cm² apparent
surface. The working electrode was GC microdisk (1.0 mm of diameter – Bio-logic SAS). The
supporting electrolyte (nBu N)[PF ] (Fluka, 99% puriss electrochemical grade) and the solvent
4
6
DMSO (Sigma-Aldrich puriss p.a. dried <0.02% water) were used as received and simply
-3
degassed under argon. The solutions used during the electrochemical studies were typically 10
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). Under these experimental conditions employed in this work, the half-wave
potential (E ) of the ferrocene Fc+/Fc couple in DMSO was E = 0.45 V vs SCE. Experimental
1
/2
1/2
peak potentials have been measured versus SCE and converted to NHE by adding 0.241 V.
4
4
.2. Biology
.2.1. Antileishmanial activity against L. donovani promastigotes
Leishmania species used in this study were L. donovani (MHOM/IN/00/DEVI) purchased from
CNR Leishmania (Montpellier, France). Leishmania promastigotes 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 antileishmanial
activity on promastigote forms of the tested compound was carried out by an MTT assay
according to the protocol of Mosmann with some modifications. [29] Briefly, promastigotes in
6
log-phase were incubated at an average density of 10 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 by DMSO, miltefosine, amphotericin B,
fexinidazole and doxorubicin (reference drugs purchased from Sigma-Aldrich, Saint-Louis,
Missouri, USA) were added to each set of experiments. After a 72h incubation period at 27 °C,
parasitic metabolic activity was determined. Each plate-well was then microscope-analyzed for
detecting possible 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 followed and incubated 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 finally measured at 570 nm
in a BIO-TEK Elx808 (Biotek, Colmar, France) absorbance microplate reader. Inhibitory
concentration 50% (EC ) was defined as the concentration of drug required to inhibit by 50%
5
0
the metabolic activity of Leishmanial promastigotes forms compared to the control. EC were
5
0
calculated by non-linear regression analysis processed dose-response curves, using TableCurve
D V5.0 software. EC50 values represent the mean value calculated from at least three
independent experiments.
2
4.2.2. Antileishmanial activity on L. infantum axenic amastigotes [30]
L. infantum promastigotes (MHOM/MA/67/ITMAP-263, CNR Leishmania, Montpellier, France,
expressing luciferase activity) were cultivated in RPMI 1640 medium supplemented with 10%
foetal calf serum (FCS), 2 mM L-glutamine and antibiotics (100 U/mL penicillin and 100 µg/mL
streptomycin) and 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 promastigotes 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 for confirming the
overexpression of ATG8 and amastin genes in amastigotes, compared to promastigotes. The
effects of the tested compounds on the growth of L. infantum axenic amastigotes were assessed
6
as follows. L. infantum amastigotes were incubated at a density of 2 × 10 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 DMSO, amphotericin B,
miltefosine and fexinidazole (reference drugs purchased from Sigma Aldrich) 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. To estimate the luciferase activity
of axenic amastigotes, 80 µL of each well were transferred to white 96-well plates, Steady
Glow® reagent (Promega) was added according to manufacturer's instructions, and plates were
incubated for 2 min. The luminescence was measured in Microbeta Luminescence Counter
(
PerkinElmer). Efficient concentration 50% (EC ) was defined as the concentration of drug
50
required to inhibit by 50% the metabolic activity of L. infantum amastigotes compared to control.
EC50 values were calculated by non-linear regression analysis processed on dose response
curves, using TableCurve 2D V5 software. EC values represent the mean of three independent
5
0
experiments.

4.2.3. 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. [31] 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.
®
[
32] 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 . They were incubated at an
2
®
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 treated by
DMSO on sterile water, suramin, eflornithine and fexinidazole (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) was
then added to each well, and the plates were incubated for 5 h. The plates were read in a
ENSPIRE microplate reader (PerkinElmer) using an excitation wavelength of 530 nm and an
emission wavelength of 590 nm. EC was defined as the concentration of drug necessary to
5
0
inhibit by 50% the activity of T. b. brucei compared to the control. EC were calculated by
5
0
nonlinear regression analysis processed on dose-response curves, using GraphPad Prism
software (USA). EC values were calculated from three independent experiments.
5
0
4.2.4. Antitrypanosomal evaluation on the development of T. cruzi amastigotes
Vero cells (normal kidney epithelial cells of Cercopithecus aethiops) were obtained from the
Virology Laboratory of the Pitié Salpêtrière Hospital (Paris, France). At late exponential growth
phase, trypsin-treated Vero cells were subcultured every seven days in RPMI-1640 medium (Life
technologies) supplemented with streptomycin/penicillin (Life technologies) and 5% heat-
inactivated fœtal bovine serum (FBS) (Life technologies). Subcultures were maintained at 37 °C
in a humidified atmosphere of 5% CO . CL Brener strain (collection number: MNHN-CEU-
2
2016-0159) was a gift from Pr. P. Grellier of the Muséum National d’Histoire Naturelle (Paris,
France). T. cruzi stocks were maintained by weekly passage in Vero cells. Infectious
trypomastigotes were collected from culture supernatants.
Then, sterile, 6-well plates were seeded with exponentially growing Vero cells (40,000 cells per
2
cm in 2, 2 mL RPMI with serum per well) harvested from the preceding subcultures, were
added to each well. After incubation at 37 °C for 2 days in 5% CO in air, the cells were infected
2
with T. cruzi trypomastigotes in ratio 3:1 (parasites : host cells). After 24 h, the non-infecting
2
+
2+
trypomastigotes removed by washing twice with HBSS buffer (without Ca and Mg ) and the
chemical compounds in completed RPMI media were added immediately, to be tested to their
inhibitory effects on parasite growth and development. Culture plates were incubated for an

additional 120 h at 37 °C with 5% CO .Three replicate wells for each condition were done. On
2
days 5 and 6 post-infection, trypomastigotes were released from the cells. On day 6, the culture
medium was removed and transferred to a centrifuge tube. Attached infected cells were washed
with 5 mL of HBSS buffer. The culture medium and wash containing trypomastigotes were
mixed and centrifuged at 1000 g for 15 min at room temperature. Subsequently, trypomastigotes
re-suspended in 2 mL and counted in a haemocytometer (Kova cells) using a light microscope.
For 50% effective concentration (EC ) determinations, compounds were serially diluted 2 to 4-
5
0
fold in RPMI media, with final assay concentrations ranging from 0.1 to 25 µM. The 50%
inhibiting concentrations (EC ), defined as the drug concentration that resulted in a 50%
5
0
reduction of trypomastigotes compared to the non-treated controls was estimated by non-linear
regression analysis. EC50 values represent the mean value calculated from two independent
experiments that were performed in triplicate.
4.2.5. Antileishmanial activity on L. donovani promastigotes NTR1 and NTR2 over-
expressing strain.
The clonal Leishmania donovani cell line LdBOB (derived from MHOM/SD/62/1S-CL2D) was
grown as promastigotes at 26 °C in modified M199 media, as previously described [33]. LdBOB
promastigotes overexpressing NTR1 (LinJ.05.0660) [16] and NTR2 (LinJ.12.0730) [17] were
grown in the presence of nourseothricin (100 µg/mL). To examine the effects of test compounds
4
on growth, triplicate promastigote cultures were seeded with 5 × 10 parasites/mL. Parasites
were grown in 10 mL cultures in the presence of drug for 72 h, after which 200 μL aliquots of
each culture were added to 96-well plates, 50 μM resazurin was added to each well and
fluorescence (excitation of 528 nm and emission of 590 nm) measured after a further 4 h
incubation [15]. Data were processed using GRAFIT (version 5.0.4; Erithacus software) and
fitted to a 2-parameter equation, where the data are corrected for background fluorescence, to
obtain the effective concentration inhibiting growth by 50% (EC ):
5
0
1
00
y =
m

[I] 
1
+ 




EC50

In this equation, [I] represents inhibitor concentration and m is the slope factor. Experiments
were repeated at least two times and the data is presented as the mean plus standard deviation.
4.2.6 Antitrypanosomal activity on T. brucei NTR1 over-expressing strain.
Trypanosoma brucei bloodstream-form 'single marker' S427 (T7RPOL TETR NEO) and drug-
resistant cell lines were cultured at 37 °C in HMI9-T medium [34] supplemented with 2.5 μg/mL
G418 to maintain expression of T7 RNA polymerase and the tetracycline repressor protein.