Antikinetoplastid SAR study in 3-nitroimidazopyridine series: Identification of a novel non-genotoxic and potent anti-T. b. brucei hit-compound with improved pharmacokinetic properties

Article abstract of DOI:10.1016/j.ejmech.2020.112668

To study the antikinetoplastid 3-nitroimidazo[1,2-a]pyridine pharmacophore, a structure-activity relationship study was conducted through the synthesis of 26 original derivatives and their in vitro evaluation on both Leishmania spp and Trypanosoma brucei brucei. This SAR study showed that the antitrypanosomal pharmacophore was less restrictive than the antileishmanial one and highlighted positions 2, 6 and 8 of the imidazopyridine ring as key modulation points. None of the synthesized compounds allowed improvement in antileishmanial activity, compared to previous hit molecules in the series. Nevertheless, compound 8, the best antitrypanosomal molecule in this series (EC₅₀ = 17 nM, SI = 2650 & E° = ?0.6 V), was not only more active than all reference drugs and previous hit molecules in the series but also displayed improved aqueous solubility and better in vitro pharmacokinetic characteristics: good microsomal stability (T_1/2 > 40 min), moderate albumin binding (77%) and moderate permeability across the blood brain barrier according to a PAMPA assay. Moreover, both micronucleus and comet assays showed that nitroaromatic molecule 8 was not genotoxic in vitro. It was evidenced that bioactivation of molecule 8 was operated by T. b. brucei type 1 nitroreductase, in the same manner as fexinidazole. Finally, a mouse pharmacokinetic study showed that 8 displayed good systemic exposure after both single and repeated oral administrations at 100 mg/kg (NOAEL) and satisfying plasmatic half-life (T_1/2 = 7.7 h). Thus, molecule 8 appears as a good candidate for initiating a hit to lead drug discovery program.

Full text of DOI:10.1016/j.ejmech.2020.112668

Journal Pre-proof
Antikinetoplastid SAR study in 3-nitroimidazopyridine series: identification of a
novel non-genotoxic and potent anti-T. b. brucei hit-compound with improved
pharmacokinetic properties
Cyril Fersing, Clotilde Boudot, Romain Paoli-Lombardo, Nicolas Primas, Emilie
Pinault, Sébastien Hutter, Caroline Castera-Ducros, Youssef Kabri, Julien Pedron,
Sandra Bourgeade-Delmas, Alix Sournia-Saquet, Jean-Luc Stigliani, Alexis Valentin,
Amaya Azqueta, Damián Muruzabal, Alexandre Destere, Susan Wyllie, Alan H.
Fairlamb, Sophie Corvaisier, Marc Since, Aurélie Malzert-Fréon, Carole Di Giorgio,
Pascal Rathelot, Nadine Azas, Bertrand Courtioux, Patrice Vanelle, Pierre Verhaeghe
PII:
S0223-5234(20)30640-1
DOI:
https://doi.org/10.1016/j.ejmech.2020.112668
EJMECH 112668
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 May 2020
Revised Date: 13 July 2020
Accepted Date: 14 July 2020
Please cite this article as: C. Fersing, C. Boudot, R. Paoli-Lombardo, N. Primas, E. Pinault, S. Hutter,
C. Castera-Ducros, Y. Kabri, J. Pedron, S. Bourgeade-Delmas, A. Sournia-Saquet, J.-L. Stigliani, A.
Valentin, A. Azqueta, D. Muruzabal, A. Destere, S. Wyllie, A.H. Fairlamb, S. Corvaisier, M. Since,
A. Malzert-Fréon, C. Di Giorgio, P. Rathelot, N. Azas, B. Courtioux, P. Vanelle, P. Verhaeghe,
Antikinetoplastid SAR study in 3-nitroimidazopyridine series: identification of a novel non-genotoxic and
potent anti-T. b. brucei hit-compound with improved pharmacokinetic properties, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112668.
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Antikinetoplastid SAR study in 3-nitroimidazopyridine series: identification
of a novel non-genotoxic and potent anti-T. b. brucei hit-compound with
improved pharmacokinetic properties.
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Cyril Fersing1#, Clotilde Boudot2#, Romain Paoli-Lombardo1, Nicolas Primas1, Emilie Pinault3,
Sébastien Hutter4, Caroline Castera-Ducros1, Youssef Kabri1, Julien Pedron5, Sandra Bourgeade-
Delmas6, Alix Sournia-Saquet5, Jean-Luc Stigliani5, Alexis Valentin6, Amaya Azqueta7, Damián
Muruzabal7, Alexandre Destere8, Susan Wyllie9, Alan H. Fairlamb9, Sophie Corvaisier10, Marc
Since10, Aurélie Malzert-Fréon10, Carole Di Giorgio11, Pascal Rathelot1, Nadine Azas4, Bertrand
Courtioux2, Patrice Vanelle1 and Pierre Verhaeghe5*.
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11
Aix Marseille Univ, CNRS, ICR UMR 7273, Equipe Pharmaco-Chimie Radicalaire, Faculté de
Pharmacie, 27 Boulevard Jean Moulin, CS30064, 13385, Marseille Cedex 05, France.
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2 Université de Limoges, UMR Inserm 1094, Neuroépidémiologie Tropicale, Faculté de Pharmacie, 2 rue
du Dr Marcland, 87025 Limoges, France.
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3 Université de Limoges, BISCEm, US 042 INSERM – UMS 2015 CNRS, Mass Spectrometry Platform,
CBRS, 2 rue du Pr. Descottes, F-87025 Limoges, France.
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Aix Marseille Univ, IHU Méditerranée Infection, UMR VITROME - Tropical Eukaryotic Pathogens,
19-21 Boulevard Jean Moulin, 13005 Marseille, France.
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5 LCC-CNRS Université de Toulouse, CNRS, UPS, Toulouse, France.
6 UMR 152 PHARMA-DEV, Université de Toulouse, IRD, UPS, Toulouse, France.
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Department of Pharmacology and Toxicology, Faculty of Pharmacy and Nutrition, University of
Navarra, C/ Irunlarrea 1, CP 31008, Pamplona, Navarra, Spain.
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Department of Pharmacology, Toxicology and Pharmacovigilance, CHU Limoges, Limoges, France,
INSERM, UMR 1248, University of Limoges, France.
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University of Dundee, School of Life Sciences, Division of Biological Chemistry and Drug Discovery,
Dow Street, Dundee DD1 5EH, Scotland, United Kingdom.
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10 Normandie Univ, UNICAEN, CERMN, 14000 Caen, France.
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Institut Méditerranéen de Biodiversité et d’Ecologie marine et continentale (IMBE), Aix-Marseille
Université, UMR CNRS IRD Avignon Université, Campus Timone – Faculté de Pharmacie, 27 boulevard
Jean-Moulin, F13385 Marseille cedex 05.
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#Co first-authors
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*Corresponding author:
E-mail address: pierre.verhaeghe@lcc-toulouse.fr (P. Verhaeghe)
Postal address: Université Paul Sabatier - CNRS UPR 8241, Laboratoire de Chimie de Coordination, 205
Route de Narbonne, 31077 Toulouse cedex 04, France.

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Abstract:
To study the antikinetoplastid 3-nitroimidazo[1,2-a]pyridine pharmacophore, a structure-activity
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relationship study was conducted through the synthesis of 26 original derivatives and their in
vitro evaluation on both Leishmania spp and Trypanosoma brucei brucei. This SAR study
showed that the antitrypanosomal pharmacophore was less restrictive than the antileishmanial
one and highlighted positions 2, 6 and 8 of the imidazopyridine ring as key modulation points.
None of the synthesized compounds allowed improvement in antileishmanial activity, compared
to previous hit molecules in the series. Nevertheless, compound 8, the best antitrypanosomal
molecule in this series (EC50 = 17 nM, SI = 2650 & E° = -0.6 V), was not only more active than
all reference drugs and previous hit molecules in the series but also displayed improved aqueous
solubility and better in vitro pharmacokinetic characteristics: good microsomal stability (T1/2
>
40 min), moderate albumin binding (77 %) and moderate permeability across the blood brain
barrier according to a PAMPA assay. Moreover, both micronucleus and comet assays showed
that nitroaromatic molecule 8 was not genotoxic in vitro. It was evidenced that bioactivation of
molecule 8 was operated by T. b. brucei type 1 nitroreductase, in the same manner as
fexinidazole. Finally, a mouse pharmacokinetic study showed that 8 displayed good systemic
exposure after both single and repeated oral administrations at 100 mg/kg (NOAEL) and
satisfying plasmatic half-life (T1/2 = 7.7 h). Thus, molecule 8 appears as a good candidate for
initiating a hit to lead drug discovery program.
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Keywords: Imidazo[1,2-a]pyridine; Nitroaromatic; Nitroreductases; Kinetoplastids; Redox
potentials; SARs.

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1. Introduction
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Kinetoplastid diseases are vectorial parasitoses caused by flagellated blood protozoa and
transmitted to their hosts by specific insect vectors. These diseases are found in developing
countries in intertropical regions, especially where health facilities are inadequate [1]. They
threaten nearly one billion people, causing an estimated 30 000 deaths annually [2] The World
.
Health Organization (WHO) thereby classifies them as Neglected Tropical Diseases (NTDs) [3].
These infectious diseases are caused by two parasitic genera: Leishmania spp. and Trypanosoma
spp. The first is responsible for leishmaniasis (caused by several Leishmania species) which can
evolve in different forms (visceral, cutaneous or cutaneomucous) [4].
The second genus contains most of Trypanosoma species especially T. cruzi causing Chagas
disease in South and Central America, and T. brucei spp. responsible for Human African
Trypanosomiasis (HAT, or sleeping sickness) [5,6]. HAT is found in 36 countries throughout
sub-Saharan Africa. It is estimated that 70 million people would be at risk of contracting it [7].
Two subspecies, T. brucei gambiense (T.b.g.) and T. brucei rhodesiense (T.b.r.), are at the origin
of different symptoms and two clinical forms: chronic disease for T.b.g. and acute disease for
T.b.r. Following the bite of the blood sucking fly (tsetse fly or glossina), the parasite penetrates
into the derma and disseminates into the whole organism. In a first step, blood and lymph are
affected by the bloodstream form, and patients show headaches, anemia, joint pain and various
organ damages occur. This stage is called hemolymphatic stage. Then, without treatment, the
parasites are able to cross the blood brain barrier (BBB) and invade the central nervous system
(stage 2, or nervous stage), causing various neurological changes such as sleeping disorders
(responsible for the name of the disease), abnormal tone and mobility, ataxia, psychiatric
disorders, seizures, coma and finally, death, as all kinetoplastid diseases are lethal if untreated
[7,8]. Nervertheless, very few efficient, safe and affordable drugs are proposed to treat the
infected populations. Regarding HAT, treatments are complex because they are stage and species
dependent. For early stage HAT, suramin and pentamidine, molecules presenting severe side

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effects, must be given by injection (IV or IM) for a prolonged period against T.b.r. and T.b.g.
infections, respectively. For the late-stage, melarsoprol is given in T.b.r. infections despite the
high toxicity of this arsenic derivative (it can cause encephalopathies in 10% of cases). Against
T.b.g. infections, the NECT (Nifurtimox Eflornithine Combination Therapy) is prescribed
despite the genotoxic character of nifurtimox [9]. Since the end of 2018, a new safer
nitroaromatic drug, fexinidazole, is available to treat HAT [10,11]. Concerning the new chemical
entities that are clinically studied by the Drug for Neglected Disease initiative (DNDi), only
acoziborole (benzoxaborole derivative) is in phase IIb/III against HAT [10]. This quite limited
drug portfolio calls for the research of new molecules that could strengthen the therapeutic
arsenal and reduce the risk of emergence of resistant parasites.
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Concerning fexinidazole, a 2-substituted-5-nitroimidazole derivative, it is a prodrug which is
activated through the reduction of its nitro group by a NADH-specific type 1 nitroreductase
(NTR), to generate electrophilic cytotoxic metabolites (nitroso and hydroxylamine derivatives)
able to form covalent adducts with DNA and that are cytotoxic for the parasite [9,12]. Only one
mitochondrial type 1 nitroreductase was identified in Trypanosoma brucei [13]. The particularity
of nitroreductases is that they are absent in mammalian cells and then, constitute important
targets for the development of new selective antiparasitic treatments. In this context,
nitroaromatic compounds are key derivatives that have been and remain studied to fight against
kinetoplastids, as shown with the structures of nifurtimox, fexinidazole and DNDI-0690, a novel
4-nitroimidazole derivative that is clinically studied against visceral leishmaniasis [14] (Figure
1).
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Figure 1. Structures of nitroheterocyclic drug-compounds that are marketed or clinically studied
against Trypanosoma brucei and Leishmania infections.
Within the framework of our research program, focused on the development of original
nitroheterocyclic antikinetoplastid molecules, we formerly reported a first antileishmanial hit
compound in 8-halogeno-3-nitroimidazo[1,2-a]pyridine series (hit A, Figure 2) [15].
Pharmacomodulation studies at position 8 of the scaffold were conducted and showed that
introducing a 4-chlorophenylthio moiety improved in vitro antikinetoplastid activity (hit B,
Figure 2) [16]. However, hit-molecule A displayed limited water solubility while hit B showed a
poor mouse liver microsomal stability (T1/2 = 3 min), even if some probable sulfoxide and
sulfone metabolites remained active. Thus, to improve hydrosolubility, microsomal stability and
antikinetoplastid activity of this nitroheterocyclic series, we explored deeper the structure
activity relationships by studying the influence of substituents at position 2 and 6 of the
imidazo[1,2-a]pyridine ring and comparing to the activities of hits A and B.
Figure 2. Structures and biological profiles of previously identified antikinetoplastid hit-
compounds in 3-nitroimidazo[1,2-a]pyridine series [15–17].
Hit A
1.8
5.5
4.4
2.9
Hit B
1.0
1.3
1.7
1.3
EC50 L. donovani promastigotes (µM)
EC50 L. donovani amastigotes (µM)
EC50 L. infantum axenic amastigotes (µM)
EC50 T. b. brucei trypomastigotes (µM)
CC50 HepG2 (µM)
>31
>100
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2. Results and discussion
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To evaluate if redox potentials had an influence on the bioactivation of this chemical series by
parasitic NTRs, analogs of hit A bearing a methyl group (electron donating) or a trifluoromethyl

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group (electron withdrawing) at position 6 of the imidazo[1,2a]pyridine ring were synthesized. A
bromine atom was not included in the pharmacomodulation study considering that we previously
noted that molecules belonging to this series and bearing a bromine atom at position 6 showed
the same level of antiparasitic activity as their chlorinated analogs, these latter presenting the
advantage to show a better aqueous solubility. Thus, in parallel with the previously described 6-
chloro analogue 1c of hit A, new derivatives 2c and 3c were obtained through a 3-steps synthesis
procedure (Scheme 1) that was previously reported [18,19].
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Scheme 1. Synthesis of compounds 1a-c to 3a-c.
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Reagents and conditions: (i) 1,3-Dichloroacetone 1.1 equiv, EtOH or DME, 80°C, 31% to 60%;
(ii) HNO3 6 equiv, H2SO4, 0°C→RT, 51% to 85%; (iii) Sodium benzenesulfinate 3 equiv,
DMSO, RT, 3 h, 57% to 89%.
To study the influence of the substituent at position 2 of the imidazo[1,2-a]pyridine ring, the
phenylsulfonylmethyl group encountered in the structures of hit A and B was modulated
(Scheme 2). First, a 2-unsubstituted derivative (4b) was obtained through a cyclocondensation
reaction of 2-amino-3-bromo-5-chloropyridine with chloroacetaldehyde, followed by a nitration
reaction. Then, the 2-methyl-substituted derivative 5b was synthesized by dehalogenation of 1a
followed by a nitration reaction of 5a at position 3. Finally, the 2-hydroxymethyl-derivative 6
was obtained by hydrolysis of 1b in the presence of copper sulfate in aqueous DMSO [20].

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Scheme 2. Synthesis of compounds 4a-b, 5a-b and 6.
Reagents and conditions: (i) Chloroacetaldehyde 1.5 equiv, NaHCO3 2 equiv, EtOH, 80 °C,
31%; (ii) HNO3 6 equiv, H2SO4, 0°C→RT, 78% to 93%; (iii) NaBH4 1 equiv, DMSO, RT, 63%;
(iv) CuSO4 1 equiv, DMSO/H2O (7.5:2.5), 100 °C, 32%.
In order to explore the role of the sulfone moiety, an original 2-phenylthiomethyl derivative 7
was prepared from 1b by a substitution reaction with sodium thiophenolate, formed in situ with
NaH in DMSO at RT (Scheme 3), according to a protocol that we previously reported [21].
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Scheme 3. Synthesis of compound 7.
Reagents and conditions: (i) Thiophenol 1 equiv, NaH 1.1 equiv, DMSO, N2, RT, 28%.

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Then, the phenyl ring of the phenylsulfonylmethyle moiety was replaced by a methyl, cyclohexyl
or pyridin-3-yl group, using a previously described reaction [15] and leading to original
derivatives 8-10 respectively, according to Scheme 4.
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Scheme 4. Synthesis of compounds 8-10.
Reagents and conditions: (i) Appropriate sulfonyl chloride 2 equiv, NaHCO3 2 equiv, Na2SO3 2
equiv, EtOH/H2O (1:1), N2, 120 °C, MW, 22% to 47%.
To complete SAR data and allow comparison with hit B, nine derivatives (11–19) bearing a 4-
chlorophenylthio moiety at position 8 were synthesized, applying a protocol that we previously
reported in various nitroaromatic series with antiparasitic potential [22,23] (Scheme 5).
R6
R2
R8 = -Br R8 = -S-Ph-Cl
(2c)
(3c)
(4b)
(5b)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
CH3
CF3
Cl
Cl
Cl
Cl
Cl
Cl
Cl
CH2-SO2-Ph
CH2-SO2-Ph
H
CH3
CH2OH
CH2-S-Ph
CH2-SO2-CH3
CH2-SO2-Cyclohexyl
CH2-SO2-Pyridin-3-yl
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Scheme 5. Synthesis of compounds 11-19.
Reagents and conditions: (i) 4-Chlorothiophenol 1 equiv, NaH 1 equiv, DMSO, N2, RT, 20% to
90%.
Still by analogy with hit B, the influence of the heteroatom at position 8 was investigated deeper.
First, the synthesis of 8-anilino derivatives via a Buchwald-Hartwig cross-coupling reaction was
investigated (Scheme 6). The reaction conditions were studied by varying several parameters

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such as nature of base, nature and amount of catalyst and ligand, nature of solvent, and reaction
temperature. Three derivatives bearing an aniline moiety at position 8 were synthesized (Scheme
6) in moderate yields (42% to 52%). Then, a selenium analogue (23) of hit B was also prepared
according to a previously described protocol [24], as presented in Scheme 6.
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Scheme 6. Synthesis of 8-anilino derivatives 20-22 and 8-phenylselanyl derivative 23.
Reagents and conditions: (i) Appropriate aniline 1.2 equiv, Pd(OAc)2 0.08 equiv, rac-BINAP
0.15 equiv, K2CO3 1.8 equiv, toluene, N2, 140 °C, MW, 42% to 52 %; (ii) Bis(4-
chlorophenyl)diselenide 0.5 equiv, NaBH4 2 equiv, PEG-400, N2, 60 °C, 9 %.
Globally, ten molecules (analogues of hit A) bearing a bromine atom at position 8 of the
imidazo[1,2-a]pyridine ring and 13 molecules (analogues of hit B) bearing a phenylthio,
phenylamino or phenylselanyl group at the same position were evaluated in vitro. Their
influence on cell viability (cytotoxic concentration 50% = CC50) was assessed on the HepG2 cell
line, using doxorubicin as a positive control. In vitro antileishmanial activity (measured through
effective concentration 50% = EC50) was also measured on both the promastigote and axenic
amastigote form of L. donovani and L. infantum, respectively. In parallel, the in vitro
antitrypanosomal activity (EC50) of these derivatives was determined on the trypomastigote
blood stream form (BSF) of T. b. brucei. For all molecules, selectivity indices (SI) were
calculated. Their antikinetoplastid activity was compared to previous hit molecules and to
commercially available reference drugs amphotericin B, miltefosine, fexinidazole, suramine,
eflornithine and nifurtimox. The results are summarized in Table 1.

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Table 1. In vitro evaluation of molecules 1c-23 on L. donovani promastigotes, L. infantum
axenic amastigotes, T. b. brucei trypomastigotes (BSF) and on the human hepatocyte HepG2 cell
line.
Cell viability
Activity
EC50 L. infantum
EC50 L.
donovani
pro. (µM)
SI
L. donovani
pro.
SI
CC50 HepG2
(µM)
EC50 T. b. brucei SI T. b.
Compd
R2
R6
R8
L. infantum
axenic ama.
axenic ama. (µM)
BSF (µM)
brucei
1c
2c
3c
4b
5b
6
-CH2-SO2-Ph
-Cl
-Br
-Br
-Br
-Br
-Br
-Br
-Br
-Br
>15.6a
>31.2a
>15.6a
16.2
5.3 ± 0.3
5.6 ± 1.4
6.2 ± 0.7
9.3 ± 1.7
>12.5c
>2.9
>5.6
>2.5
1.7
-
16.4 ± 0.2
>3.1
>1
-
1.4 ± 0.4
1.2 ± 0.5
4.0 ± 2.7
0.4 ± 0.01
>50c
>11.1
>26
>3.9
40.5
-
-CH2-SO2-Ph -CH3
-CH2-SO2-Ph -CF3
>3.1
-
-H
-Cl
-Cl
-Cl
-Cl
15.9 ± 0.7
32.1 ± 0.7
46.3 ± 1.4
17.8 ± 0.8
>25c
1
-CH3
>125a
>125a
>31.2a
45
>3.9
>5.4
>1.8
<1.8
-CH2-OH
-CH2-S-Ph
>12.5c
-
>50c
-
7
>12.5c
-
>50c
-
8
-CH2-SO2-CH3 -Cl
5.7 ± 1.4
7.9
0.017 ± 0.002
2647
-CH2-SO2-
-Cl
9
-Br
-Br
>15.6a
>62.5a
0.9 ± 0.3
0.8 ± 0.3
>17.3
>78
> 100c
-
0.5 ± 0.07
>31.2
2500
Cyclohexyl
-CH2-SO2-
-Cl
77.8 ± 0.8
>0.8
0.025 ± 0.005
10
Pyridin-3-yl
11
12
13
14
15
16
17
18
-CH2-SO2-Ph -CH3
4-Cl-Ph-S-
4-Cl-Ph-S-
4-Cl-Ph-S-
4-Cl-Ph-S-
4-Cl-Ph-S-
4-Cl-Ph-S-
4-Cl-Ph-S-
>62.5a
>7.8a
2.6 ± 0.4
1.9 ± 0.2
2.5 ± 0.2
>12.5c
>24
>4.1
>3.1
-
5.0 ± 0.3
12.5
1.8 ± 0.5
0.17 ± 0.02
0.085 ± 0.02
-
>34.7
>45.9
>92.8
-
-CH2-SO2-Ph -CF3
-
-
-H
-Cl
-Cl
-Cl
-Cl
>7.8a
-
-
-
-
-CH3
>7.8a
-CH2-OH
-CH2-S-Ph
>15.6a
>15.6a
>31.2a
>31.2a
4.0 ± 0.3
7.4 ± 0.4
1.2 ± 0.1
1.9 ± 0.1
>3.9
>2.1
>26
>16.4
7.9 ± 0.3
-
>2
-
>50
-
>50
-
-CH2-SO2-CH3 -Cl
10.3 ± 0.4
1.6 ± 0.2
>3
>19.5
0.067 ± 0.007
1.5 ± 0.1
>465.7
>20.8
-CH2-SO2-
-Cl
4-Cl-Ph-S-
Cyclohexyl
-CH2-SO2-
19
20
21
22
23
-Cl
4-Cl-Ph-S-
Ph-NH-
>31.2a
>3.9a
>3.9a
>3.9a
>7.8a
0.5 ± 0.1
>62.4
0.8 ± 0.2
>39
1.2 ± 0.2
>26
Pyridin-3-yl
-CH2-SO2-Ph
-CH2-SO2-Ph
-CH2-SO2-Ph
-CH2-SO2-Ph
-Cl
-Cl
-
-
-
-
-
-
-
4-Cl-Ph-NH-
-
-
-
-
-
-
-
-Cl 4-CH3O-Ph-NH-
-Cl 4-Cl-Ph-Se-
-
-
-
-
0.7 ± 0.1
>11.1
0.9 ± 0.1
>8.7
0.3 ± 0.03
>26
Ref. 1
Ref. 2
Ref. 3
Ref. 4
Ref. 5
Ref. 6
Ref. 7
Ref. 8
Ref. 9
Hit A molecule
Hit B molecule
Doxorubicinb
>31a
>100
1.8 ± 0.8
>17.2
4.4 ± 0.8
>7.1
2.9 ± 0.5
1.3 ± 0.1
-
>10.7
>76.9
-
1.0 ± 0.3
>100
1.7 ± 0.3
>58.8
0.2 ± 0.02
8.8 ± 0.3
85 ± 8.8
>200c
-
-
-
-
Amphotericin Bd
Miltefosined
Fexinidazoled,e
Suramine
0.07 ± 0.01
125.7
0.06 ± 0.001
146.7
-
-
3.1 ± 0.2
27.4
0.8 ± 0.2
106.3
-
-
1.2 ± 0.2
>166.7
3.4 ± 0.8
>58.8
0.6 ± 0.2
0.03 ± 0.009
13.3 ± 2.1
2.6 ± 0.8
>333
>3333
>7.5
17.4
>100c
-
-
-
-
-
-
-
-
-
-
-
-
Eflornithinee
>100c
Nifurtimoxe
45.2 ± 1.3
212
213
214
215
216
217
a 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 EC50 or CC50 value was not reached at the highest tested concentration
d Amphotericin B, Miltefosine and Fexinidazole were used as antileishmanial reference drugs
e Fexinidazole, Suramin, Eflornithine and Nifurtimox were used as anti-Trypanosoma brucei reference drugs
f SI = CC50 HepG2 / EC50 L. infantum

218
219
g SI = CC50 HepG2 / EC50 T. brucei brucei
Bold: New antitrypanosomal hit
220
221
222
223
224
225
Out of the 23 tested molecules, 7 showed a poor aqueous solubility (< 10 µM) limiting their in
vitro evaluation. Among them, 8-anilino-derivatives 20-22 were particularly insoluble in
biological culture media, indicating that the aniline moiety should be avoided at position 8 of the
antitrypanosomal scaffold. Regarding the effect on the cell viability of the HepG2 human cells,
all molecules appeared as weakly cytotoxic (15.6 ≤ CC50 < 125 µM) in comparison with
doxorubicin (CC50 = 0.2 µM).
226
227
228
229
230
231
Then, 20 molecules were screened in vitro for their activity against L. donovani promastigotes
and compared both to previous hit molecules and to amphotericin B, miltefosine and
fexinidazole. Among them, only compounds 9, 10, 17, 18 and 19, bearing an alkyl- or pyridin-3-
yl-sulfonylmethyl group at position 2, showed both good EC50 values (0.5 ≤ EC50 ≤ 1.9 µM) and
good selectivity indices (16 < SI < 78). This suggests that a sulfonyl group in position 2 is
needed to reach a good antileishmanial activity level on L. donovani promastigotes.
232
233
234
235
236
237
238
239
When considering the screening against L. infantum axenic amastigotes, out of the 5 molecules
that were active on L. donovani promastigotes, only compounds 18 and 19 remained active (0.8
≤ EC50 ≤ 1.6 µM) and selective (19.5 < SI < 39). These results confirmed that, in addition to a
sulfonylmethyl substituent at position 2 of the imidazopyridine ring, antileishmanial activity was
also favored when substituting position 8 by a thiophenol moiety. Unfortunately, compounds 18
and 19 were then evaluated on the intramacrophage amastigote stage of L. donovani and were
not active (EC50 > 10 µM). Consequently, hit B still remains the most active antileishmanial
molecule in this chemical series.
240
241
242
243
244
Regarding antitrypanosomal activity, molecules were tested on the blood stream form of T. b.
brucei and compared to reference drugs fexinidazole, suramin, eflornithine and nifurtimox. Out
of the 20 tested molecules, 8 displayed submicromolar activities (0.017 ≤ EC50 ≤ 0.5 µM), quite
significantly better than the ones of hit A and B (EC50 = 2.9 and 1.3 µM respectively). Contrary
to what was noted for antileishmanial activity, these 8 antitrypanosomal molecules are equally

245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
distributed between hit A (8-Br) and hit B (8-thiophenol) derivatives, showing that the
antitrypanosomal pharmacophore is less restrictive than the antileishmanial pharmacophore.
Among these submicromolar active molecules, 8, 9, 10, 12, 17 and 23 presented a
sulfonylmethyl moiety at position 2 whereas molecules 4b and 13 were not substituted.
Nevertheless, 4b was the most cytotoxic derivative in the series and 13 showed a poor aqueous
solubility. Then, as with antileishmanial activity, the sulfonylmethyl moiety seems to play a
favorable role toward antitrypanosomal activity. When looking at hit A derivatives, methyl-,
cyclohexyl- and pyridin-3-yl-sulfonylmethyl groups provided good to excellent activities
(molecules 8-10), showing that the substituent of the sulfonylmethyl moiety is a key modulation
zone. Thus, molecule 8 appeared as the most active (EC50 = 17 nM) and selective (SI = 2647)
antitrypanosomal molecule in the series. It was a lot more active than hits A and B (EC50 = 2.9
and 1.3 µM respectively) but also more active than fexinidazole (EC50 = 0.6 µM), suramin (EC50
= 30 nM), eflornithine (EC50 =13.3 µM) and nifurtimox (EC50 =2.6 µM). The selenium
containing analog 23 of hit B appeared a lot less soluble in aqueous biological media than hit B
despite being 4 times more active. Because of the same lack of water solubility, the 6-
trifluoromethylated analogue 12 of hit B appeared less promising than other derivatives.
Nevertheless, the comparison of the close activities of 11 and 12 toward hit B but also 2c and 3c
toward 1c and hit A, permitted to conclude that the antitrypanosomal pharmacophore allows
small electron-donating or electron-withdrawing substituents such as CH3, Cl, Br or CF3 at
position 6.
From antitrypanosomal hit-molecule 8, to deeper evaluate the influence of the sulfonylmethyl
moiety, a 2-methylthiomethyl analog (24) was synthesized from substrate 1b, using sodium
methanethiolate as reagent (Table 2). Partial oxidation of the sulfur atom of 24 using m-CPBA in
dichloromethane led to the sulfoxide derivative 25 (Table 2). The activities of 24 and 25 were
evaluated in vitro for their activity against T. b. brucei and for their effect on cell viability on the
HepG 2 cell line (Table 2). Thioether derivative 24 showed limited water solubility and modest
antitrypanosomal activity (EC50 = 2.2 µM) whereas sulfoxide derivative 25 was very active

272
273
274
275
276
against T. b. brucei (EC50 = 20 nM) and displayed excellent selectivity index (SI = 2570). These
results confirmed that position 2 was key to the antiparasitic activity and indicated that the
antitrypanosomal pharmacophore included either a sulfone or a sulfoxide functional group at
position 2 of the imidazopyridine ring.
277
Table 2. Synthesis and biological activities of compounds 24 and 25.
T. brucei brucei BSF
Compound
HepG2 CC50 (µM)
>15.6a
SI T. b. brucei
>7.1
EC50 (µM)
2.20 ± 0.24
24
25
0.020 ± 0.006
51.4 ± 8.3
2570
a The product could not be tested at higher concentrations due to a poor solubility in aqueous
medium.
Reagents and conditions: (i) CH3SNa 1 equiv, DMSO, RT, 52% (ii) mCPBA 1 equiv, CH2Cl2,
0 °C, 36%.
278
279
280
281
282
283
284
285
286
287
288
To assess the influence of the aforementioned modulations at position 2, 6 and 8 on the reduction
potential of this nitroaromatic scaffold, an electrochemical study was conducted on several 3-
nitroimidazo[1,2-a]pyridine derivatives using cyclic voltammetry. Experimental reduction
potentials (Figure 3) were compared to the ones of fexinidazole, nifurtimox, and hit A (for 8-Br-
derivatives) or hit B (for 8-phenylthio-derivatives). For all tested compounds, a reversible single
electron reduction was observed and attested the formation of a nitro radical anion. The redox
potentials were measured and the values were corrected versus Normal Hydrogen Electrode
(NHE). These values ranged from -0.58 V to -0.79 V, in comparison with the ones of nifurtimox
(E0 = -0.61 V) and fexinidazole (E0 = -0.83 V). There was almost no difference in redox
potential values when comparing hit A with hit B analogues, bearing the same substituents at

289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
positions 2 and 6: the electronic effect of the bromine atom or of the p-chlorothiophenol group at
position 8 were almost the same. Introducing an aniline moiety at position 8 of the
imidazopyridine ring (molecule 20, E0 = -0.69 V) had the same effect toward the redox potential
value as a phenyl moiety [17] but allowed to reach a lower E° value than with a p-
chlorothiophenol moiety (hit B, E0 = -0.63 V). Modifying the substituent at position 6 of the
imidazopyridine ring had a strong influence toward the reducibility of the nitro group: 6-
trifluoromethyl-derivatives (3c and 12) displayed high E0 values (-0.60 to -0.58 V) whereas 6-
methyl-derivatives (2c and 11) presented the lowest E0 values (-0.76 V) among the derivatives
presenting a phenylsulfonylmethyl side chain at position 2. There was also an important
influence of the substituent at position 2 toward E0 values. Switching from phenylsulfonylmethyl
derivatives 1c or hit B (E0 values = -0.65 V and -0.63 V) to their phenylthiomethyl analogues 7
or 16 provided molecules with lower E0 values (-0.70 and -0.72 V respectively). This effect that
was rather comparable to the one noted with the hydrogen atom when comparing 16 to 13 (E0 = -
0.74 V). This decrease in E0 value was even more acute when introducing a methyl group at
position 2 (molecule 14, E0 = -0.79 V). Finally, by comparing the E0 values of compounds 1c
and 8, it appeared that replacing the phenylsulfonylmethyl group by a methylsulfonylmethyl one
was responsible for a significant increase in the redox potential values from -0.65 to -0.60 V.
Nevertheless, no correlation between redox potential values and antiparasitic EC50 values could
be noted in the studied series.
308

309
310
311
312
313
Figure 3. Redox potentials (E0) of Hit A (left) and Hit B (right) derivatives determined by cyclic
voltammetry and given versus NHE. Conditions: selected compounds (10-3 mol.L-1) in non-
aqueous medium (DMSO + 0.1 mol.L-1 (n-Bu4N)[PF6]) on GC microdisk (r = 0.5 mm) at room
temperature. Scan rate: 0.2 V.s-1.
314
315
These molecules being probable substrates of parasitic nitroreductases, the mechanism of
bioactivation of molecule 8 in the Trypanosoma cell was investigated by comparing the EC50
values measured on a wild-type strain with the one measured on a NTR-overexpressing strain.
This assay confirmed that, like for nifurtimox and for all previous hit molecules studied in 3-
nitroimidazopyridine series, molecule 8 was bioactivated by T. b. brucei type 1 NTR as the EC50
value of 8 on the NTR-overexpressing strain was 4 times lower than on the wild-type strain.
(Figure 4). Noticeably, compound 8 was almost thirty times more active on T. b. brucei wild-
type strain than nifurtimox.
316
317
318
319
320
321
322

323
324
325
Figure 4. Sensitivity of wild-type and NTR-overexpressing T. brucei BSF trypomastigotes
strains toward hit molecule 8 and nifurtimox control.
326
327
328
329
330
331
332
333
334
335
336
337
338
339
The 3-amino-analogue 26 of hit compound 8 was also synthesized and evaluated in vitro to
confirm the essentiality of the nitro group for antikinetoplastid activity. Molecule 26 was
prepared by reduction of 8 in the presence of iron powder in refluxing acetic acid (Table 3). As
expected, 26 did not show any antileishmanial activity and displayed a poor antitrypanosomal
activity (EC50 = 7.2 µM). These results are consistent with the hypothesis that these 3-
nitroimidazo[1,2-a]pyridines require bioactivation by parasitic NTRs, making the nitro group an
essential element of the antikinetoplastid pharmacophore.

340
Table 3. Synthesis and biological activities of compounds 26.
L. donovani
promastigotes
EC50 (µM)
L. infantum axenic T. brucei brucei
HepG2 CC50
(µM)
Compound
amastigotes
EC50 (µM)
BSF
EC50 (µM)
>12.5a
>100a
7.2 ± 0.8
>125b
26
a The EC50 or CC50 value was not reached at the highest tested concentration.
b The product could not be tested at higher concentrations due to a poor solubility in aqueous medium.
Reagents and conditions: (i) Fe0 powder 10 equiv, AcOH, reflux, 30 min, 40 %.
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
The poor microsomal stability of hit molecules A and B was a limiting PK parameter for further
development of the series (hit A T1/2 = 9 min and hit B T1/2 = 3 min; T1/2 of propranolol control =
26 min), although some metabolites of hit B remained active in vitro against Leishmania spp
[16]. For that reason, more polar, ionizable or less lipophilic pyridin-3-yl- or methyl-
sulfonylmethyl-derivatives 8, 10 and 17 of hit B and 1c were evaluated in vitro for their
microsomal stability (Table 4). As expected, microsomal stability was greatly improved when
changing the phenyl ring (1c, T1/2 = 12 min) with a pyridine one (10, T1/2 = 27 min) and even
better with a methyl group (8, T1/2 > 40 min). Effectively, molecules 10 and 8 were less
lipophilic and more water-soluble than compound 1c, but some microsomal N-oxidation can
possibly be anticipated on the pyridine moiety of 10. Even if real, this improvement in
microsomal stability was less concluding comparing hit B to 17, most probably because the
sulfur atom of the thiophenol moiety remains actively oxidized by microsomes in 17. Thus,
positions 2 and 6 of the imidazopyridine ring appeared as essential modulation sites for
increasing microsomal stability. Besides, the binding of molecule 8 to human albumin was
logically much lower than all other derivatives as the two aromatic rings that substitute the
imidazopyridine ring in hit B (thiophenol and phenylsulfonylmethyl moieties) were avoided,
leading to a decarbonated scaffold with lower molecular weight. Reducing the binding to human

359
360
361
albumin could be an important parameter to control so as to optimize future in vivo activity that
can be restricted by insufficient distribution.
362
Table 4. In vitro pharmacokinetic data of selected key compounds.
1c
Hit B
10
8
17
Binding to
human albumin
(%)
96.5
99.9
87.0
76.9
98.9
Microsomal
stability: T1/2
(min)
12
3
27
>40
12
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
Some important physicochemical parameters of hit molecule 8 were also calculated or measured
(Table 5): molecule 8 showed average lipophilicity (cLogP = 1.14) and was 20 times more
soluble in water than hit B (thermodynamic solubility of 8 = 26 µM versus 1.4 µM for hit B)
[16].
The mutagenicity of many nitroheterocyclic molecules towards bacteria or parasites has been
known for many years [25], the reduction of the nitro group being at the origin of this property.
For fexinidazole, nitroreductase-dependent mutagenic activity was observed in the Ames test.
However, its genotoxicity evaluation in the micronucleus assay was negative for the native
molecule and its two metabolites [26]. This result suggests that the Ames test, the international
reference test for measuring the mutagenicity of a molecule in vitro [27], is not suitable for the
evaluation of nitroheterocyclic molecules. Indeed, since they undergo metabolic activation by
Salmonella type 1 NTRs during the test, it appears difficult to extrapolate their genotoxicity
profile to humans. Thus, to overcome the presence of bacterial nitroreductases expressed by the
Salmonella strains used in the Ames test, a study of the genotoxicity of compound 8 was carried

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380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
out using two complementary assays. First, a micronucleus test was conducted on the CHO-K1
cell line, both with and without addition of the metabolizing S9 mixture (see supplementary
material). Molecule 8 showed no significant increase in micronucleated cell rates for all the
tested concentrations (0.01 mM to 0.5 mM), either in the absence or presence of S9 mix,
indicating that molecule 8 does not exert cytogenetic effects in CHO cells in culture and does not
produce metabolites with cytogenetic effects. Then a comet assay was carried out on the HepG2
cell line at 5, 10 and 20 µM, exposing cells with compound 8 for 2, 24 or 72 h and using methyl
methanesulfonate as a positive control (supplementary material): the values of % DNA in tail
were similar to the ones obtained in control cells. The negative results of these 2 assays are
consistent with those obtained with previously studied hit molecules in the series and also with
those of fexinidazole that is also devoid of mutagenic properties.
Finally, an in vivo toxicity study of molecule 8 was carried out to determine its maximal
tolerated dose. A group of 4 mice received a once daily repeated oral (intragastrical gavage) dose
of molecule 8 at 100 mg/kg for 5 days, and a follow-up was performed. As no side effect was
detected, the No Observed Adverse Event Level (NOAEL) in mice was set at 100 mg/kg/day. A
post-mortem examination revealed no lesion on different organs (brain, lung, heart, liver and
kidney). Thereafter, a pharmacokinetic analysis was performed to identify the behavior of
molecule 8 after oral administration. The main pharmacokinetic parameters are presented in
Table 5. Compound 8 was orally absorbed and showed good systemic exposure. It displayed a
long half-time (7.7 h) and, with a Tmax of 5 h, a single daily administration could be considered
for further experiments. A PAMPA BBB assay finally showed the possibility for molecule 8 to
cross the blood-brain barrier by a moderate passive diffusional way, in accordance with its CNS
MPO score (5.8) [28].

406
Table 5. Physicochemical, pharmacokinetic and genotoxic data regarding hit compound 8.
cLogPa
1.14
Thermodynamic solubility (µM)
PAMPA blood-brain barrier permeability assay : Pe (nm/s)
CNS MPO score
25.8 ± 0.6
84.2 ± 2.9
5.8
Cmax in mouse (ng/mL)
Tmax in mouse (h)
Plasma half-life in mouse (h)
AUC0-inf (ng.h/mL)
Clearance (mL/h)
741 ± 580
5.0 ± 5.2
7.7 ± 1.3
7060 ± 693
0.51 ± 0.05
Negative
Negative
Micronucleus assay (@ 10, 50, 100 and 500 µM +/- S9mix)
Comet assay (@ 5, 10 and 20 µM for 2, 24 and 72 h)
a Weighted clogP was computed by Marvin® (ChemAxon)
407
408
409
410
3. Conclusion
This antikinetoplastid SAR study was conducted through the synthesis and evaluation of 26
original imidazo[1,2-a]pyridine derivatives. Although most of these molecules displayed modest
to good in vitro antileishmanial activity on the promastigote form of L. donovani, they were
ineffective on the axenic amastigote form of L. infantum and/or the intramacrophage amastigote
form of L. donovani. However, nine of these compounds showed submicromolar EC50 values on
T. b. brucei bloodstream form and low cytotoxicities on the HepG2 cell line, including 3
molecules with a selectivity index ≥2500. In this series, positions 2 and 6 appeared as key
positions for modulating the reduction potential of these nitroheterocycles while maintaining
their antitrypanosomal activity. Position 2 also allowed to improve mouse liver microsomal
stability, as illustrated by the best molecule in this series (8) bearing a methylsulfonylmethyl
group instead of the phenylsulfonylmethyl group found in previous hit molecules A and B.
Indeed, in addition to its very good in vitro activity on T. b. brucei BSF (better than the ones of
all reference compounds), its in vitro microsomal stability was >40 min and therefore greatly
411
412
413
414
415
416
417
418
419
420
421
422

423
424
425
426
427
428
429
430
431
increased in comparison with previous imidazopyridine hit molecules that we reported.
Compound 8 also displayed increased water solubility, appeared BBB-permeable in a PAMPA
assay and was shown to be bioactivated by type 1 parasitic NTR. Moreover, this derivative was
not genotoxic, neither the micronucleus assay nor in the comet assay, which could represent a
significant improvement over nitroaromatic antitypanosomal drugs like nifurtimox. In the
mouse, molecule 8 was orally absorbed and well tolerated after repeated administrations of 100
mg/kg for 5 days. Its long plasma half-life (7.7 h) is encouraging and opens the way for a hit to
lead program searching for novel antikinetoplastid drug candidates.
432
433
4. Experimental section
4.1. Organic synthesis and characterizations
434
435
436
437
438
439
440
441
442
443
444
445
446
447
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
IconNMR 400 MHz spectrometer at the Laboratoire de Chimie de Coordination, Toulouse (1H-
NMR: 200, 250, 300 or 400 MHz, 13C-NMR: 50, 63, 75 or 100 MHz). NMR references were the
following: 1H: CHCl3 δ = 7.26, DMSO-d6 δ = 2.50 and 13C: CHCl3 δ = 76.9, DMSO-d6 δ = 39.5.
Solvents were dried by conventional methods. 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 aluminum plates coated with silica gel 60F-254 (Merck) in
an appropriate eluent. Visualization was made with ultraviolet light (254 nm). HRMS spectra

448
449
450
451
452
453
454
455
456
457
458
459
460
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 (tR) of
the molecules analyzed are indicated in min.
461
462
Compounds 1a, 1b and 1c were prepared as previously described [17].
4.1.1. 8-Bromo-2-chloromethyl-6-methylimidazo[1,2-a]pyridine (2a)
463
464
465
466
467
468
469
470
471
472
To a solution of 3-bromo-5-methylpyridin-2-amine (2 g, 10.1 mmol, 1 equiv.) in ethanol (100
mL), 1,3-dichloroacetone (1.46 g, 11.8 mmol, 1.1 equiv.) was added. The reaction mixture was
stirred and heated under reflux for 72 h. The solvent was then evaporated in vacuo. Compound
2a was obtained, after purification by chromatography on silica gel (eluent: cyclohexane–ethyl
acetate 6.5:3.5) and recrystallization from propan-2-ol as a beige solid in 31% yield (0.82 g). mp:
151 °C. 1H NMR (300 MHz, CDCl3) δ: 2.31 (3H, s), 4.79 (2H, s), 7.33 (1H, d, J = 1.4 Hz), 7.63
(1H, s), 7.84 (1H, t, J = 1.2 Hz). 13C NMR (100 MHz, CDCl3) δ: 18.0 (CH3), 39.7 (CH2), 111.0
(C), 112.53 (CH), 122.9 (C), 123.0 (CH), 130.7 (CH), 142.4 (C), 143.9 (C). LC/MS ESI+ tR 1.77
min, (m/z) [M+H]+ 259.08/261.09/263.12. HRMS (+ESI): 260.9609 (M + H+). Calcd for
C9H8BrClN2: 260.9610.

473
4.1.2. 8-Bromo-2-chloromethyl-6-methyl-3-nitroimidazo[1,2-a]pyridine (2b)
474
475
476
477
478
479
480
481
482
483
484
To a solution of 8-bromo-2-chloromethyl-6-methylimidazo[1,2-a]pyridine 2a (0.8 g, 3.09 mmol,
1 equiv.) in concentrated sulfuric acid (8 mL) cooled by an ice-water bath, nitric acid 65% (850
µL, 18.5 mmol, 6 equiv.) was added. The reaction mixture was stirred for 1 h at room
temperature. Then, the mixture was slowly poured into an ice-water mixture and the desired
product precipitated. The beige solid was collected by filtration, dried under reduced pressure
and recrystallized from propan-2-ol to give the expected product 2b in 85% yield (0.8 g). mp 179
1
°C. H NMR (400 MHz, CDCl3) δ: 2.51 (3H, d, J = 0.8 Hz), 5.10 (2H, s), 7.79 (1H, d, J = 1.1
13
Hz), 9.24–9.25 (1H, m). C NMR (100 MHz, DMSO-d6) δ: 17.6 (CH3), 31.1 (CH2), 110.8 (C),
125.4 (CH), 126.1 (C), 128.0 (C), 136.5 (CH), 140.8 (C), 146.5 (C). LC/MS ESI+ tR 2.04 min,
(m/z) [M+H]+ 304.44/305.80/307.06. HRMS (+ESI): 305.9461 (M + H+). Calcd for
C9H7BrClN3O2: 305.9461.
485
486
4.1.3. 8-Bromo-6-methyl-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-a]pyridine
(2c)
487
488
489
490
491
492
493
494
495
496
497
498
To a solution of 8-bromo-2-chloromethyl-6-methyl-3-nitroimidazo[1,2-a]pyridine 2b (0.5 g,
1.64 mmol, 1 equiv.) in dimethylsulfoxide (30 mL), sodium benzenesulfinate (0.81 g, 4.93
mmol, 3 equiv.) was added. The reaction mixture was stirred for 3 h at room temperature. The
reaction mixture was then slowly poured into an ice-water mixture, making the desired product
precipitate. The pale yellow solid was collected by filtration, dried under reduced pressure and
recrystallized from acetonitrile to give the expected product 2c in 89% yield (0.6 g). mp 202 °C.
1H NMR (200 MHz, CDCl3) δ: 2.50 (3H, d, J = 0.8 Hz), 5.17 (2H, s), 7.52–7.57 (2H; m), 7.67
(1H, tt, J = 1.3 and 7.4 Hz), 7.76 (1H, d, J = 1.5 Hz), 7.86–7.89 (2H, m), 9.19–9.20 (1H, m). 13C
NMR (50 MHz, CDCl3) δ: 18.6 (CH3), 57.0 (CH2), 112.4 (C), 124.9 (CH), 128.0 (C), 128.7
(2CH), 129.3 (2CH), 131.7 (C), 134.3 (CH), 136.1 (CH), 139.3 (C), 139.4 (C), 141.7 (C).
LC/MS ESI+ tR 1.81 min, (m/z) [M+H]+ 409.90/411.89/412.98. HRMS (+ESI): 411.9750 (M +
H+). Calcd for C15H12BrN3O4S: 411.9785.

499
4.1.4. 8-Bromo-2-chloromethyl-6-(trifluoromethyl)imidazo[1,2-a]pyridine (3a)
500
501
502
503
504
505
To a solution of 3-bromo-5-trifluoromethylpyridin-2-amine (1 g, 4.15 mmol, 1 equiv.) in 1,2-
dimethoxyethane (60 mL), 1,3-dichloroacetone (0.58 g, 4.56 mmol, 1.1 equiv.) was added. The
reaction mixture was stirred and heated under reflux for 72 h. The solvent was then evaporated in
vacuo and the product 3a was directly involved in the next synthesis step, without being isolated.
1H NMR (200 MHz, CDCl3) δ: 5.55 (2H, s), 7.62–7.71 (1H, m), 7.84–7.95 (1H, m), 8.22–8.32
(1H, m). LC/MS ESI+ tR 2.49 min, (m/z) [M+H]+ 312.9/314.99/316.93.
506
507
4.1.5. 8-Bromo-2-chloromethyl-3-nitro-6-(trifluoromethyl)imidazo[1,2-a]pyridine
(3b)
508
509
510
511
512
513
514
515
516
517
518
519
To a solution of 8-bromo-2-chloromethyl-6-(trifluoromethyl)imidazo[1,2-a]pyridine 3a (1 g,
3.19 mmol, 1 equiv.) in concentrated sulfuric acid (12 mL) cooled by an ice-water bath, nitric
acid 65% (1 mL, 19.14 mmol, 6 equiv.) was added. The reaction mixture was stirred for 1 h at
room temperature. Then, the mixture was slowly poured into an ice-water mixture with sodium
carbonate and extracted three times with dichloromethane. The organic layer was washed three
times with brine, dried over MgSO4, filtered and evaporated. The crude residue was purified by
column chromatography on silica gel (eluent: dichloromethane) and compound 3b was isolated
1
as a white solid in 51% yield (0.58 g). mp 143 °C. H NMR (200 MHz, CDCl3) δ: 5.11 (2H, s),
13
8.06 (1H, d, J = 1.5 Hz), 9.75–9.77 (1H, m). C NMR (50 MHz, CDCl3) δ: 38.1 (CH2), 114.3
(C), 121.6 (C, q, J = 35.5 Hz), 122.1 (C, q, J = 272.9 Hz), 125.6 (CH, q, J = 5.5 Hz), 129.1 (CH,
d, J = 2.6 Hz), 130.5 (C), 142.5 (C), 148.8 (C). LC/MS ESI+ tR 2.83 min, (m/z) [M+H]+
357.97/358.95. HRMS (+ESI): 357.9202 (M + H+). Calcd for C9H4BrClF3N3O2: 357.9200.
520
521
4.1.6. 8-Bromo-3-nitro-2-(phenylsulfonylmethyl)-6-(trifluoromethyl)imidazo[1,2-
a]pyridine (3c)
522
523
To a solution of 8-bromo-2-chloromethyl-3-nitro-6-(trifluoromethyl)imidazo[1,2-a]pyridine 3b
(0.5 g, 1.4 mmol, 1 equiv.) in dimethylsulfoxide (40 mL), sodium benzenesulfinate (0.69 g, 4.18

524
525
526
527
528
529
530
531
532
533
534
mmol, 3 equiv.) was added. The reaction mixture was stirred for 2 h at room temperature. Then,
the mixture was slowly poured into an ice-water mixture and extracted three times with
dichloromethane. The organic layer was washed five times with brine, dried over MgSO4,
filtered and evaporated. The crude residue was recrystallized from acetonitrile to obtain the
1
expected product 3c as a white solid in 57% yield (0.37 g). mp 210 °C. H NMR (200 MHz,
CDCl3) δ: 5.20 (2H, s), 7.55–7.62 (2H, m), 7.68–7.72 (1H, m), 7.88–7.93 (2H, m), 8.05–8.06
(1H, m), 9.74–9.75 (1H, m). 13C NMR (50 MHz, CDCl3) δ: 56.6 (CH2), 114.1 (C), 121.5 (C, q, J
= 35.5 Hz), 122.1 (C, q, J = 273.4 Hz), 125.3 (CH, q, J = 5.4 Hz), 128.5 (2CH), 129.1 (CH, d, J
= 2.5 Hz), 129.3 (2CH), 132.5 (C), 134.4 (CH), 139.0 (C), 140.6 (C), 142.4 (C). LC/MS ESI+ tR
2.76 min, (m/z) [M+H]+ 463.74/465.78/468.23. HRMS (+ESI): 465.9499 (M + H+). Calcd for
C15H9BrF3N3O4S: 465.9502.
535
4.1.7. 8-Bromo-6-chloroimidazo[1,2-a]pyridine (4a)
536
537
538
539
540
541
542
543
544
545
546
547
To a solution of 3-bromo-5-chloropyridin-2-amine (10 g, 48.2 mmol, 1 equiv.) in ethanol (150
mL), chloroacetaldehyde (6.69 g, 72.3 mmol, 1.5 equiv.) and sodium bicarbonate (6.86 g, 81.6
mmol, 2 equiv.) were added. The reaction mixture was stirred and heated under reflux for 96 h.
Then, the mixture was slowly poured into an ice-water mixture and extracted three times with
dichloromethane. The organic layer was washed three times with brine, dried over MgSO4,
filtered and evaporated. Compound 4a was obtained, after purification by chromatography on
silica gel (eluent: dichloromethane) and recrystallization from propan-2-ol as a pale beige solid
in 31% yield (3.48 g). mp: 131 °C. 1H NMR (300 MHz, DMSO-d6) δ: 7.68 (1H, d, J = 1.2 Hz),
7.74 (1H, d, J = 1.8 Hz), 8.05 (1H, d, J = 1.2 Hz), 8.90 (1H, d, J = 1.8 Hz). 13C NMR (75 MHz,
DMSO-d6) δ: 110.9 (C), 111.8 (C), 116.0 (CH), 118.3 (C), 124.8 (CH), 127.3 (CH), 134.4 (CH).
LC/MS ESI+ tR 1.21 min, (m/z) [M+H]+ 231.09/233.09/235.10. HRMS (+ESI): 232.9300 (M +
H+). Calcd for C7H4BrClN2: 232.9297.
548

549
4.1.8. 8-Bromo-6-chloro-3-nitroimidazo[1,2-a]pyridine (4b)
550
551
552
553
554
555
556
557
558
559
560
561
To a solution of 8-bromo-6-chloroimidazo[1,2-a]pyridine 4a (0.5 g, 2.16 mmol, 1 equiv.) in
concentrated sulfuric acid (5 mL) cooled by an ice-water bath, nitric acid 65% (500 µL, 13
mmol, 6 equiv.) was added. The reaction mixture was stirred for 1 h at room temperature. Then,
the mixture was slowly poured into an ice-water mixture with potassium carbonate and extracted
three times with dichloromethane. The organic layer was washed three times with brine, dried
over MgSO4, filtered and evaporated. Compound 4b was obtained, after purification by
chromatography on silica gel (eluent: dichloromethane) and recrystallization from propan-2-ol as
a beige solid in 93% yield (0.56 g). mp 145 °C. 1H NMR (400 MHz, CDCl3) δ: 7.90 (1H, d, J =
1.8 Hz), 8.65 (1H, s), 9.46 (1H, d, J = 1.8 Hz). 13C NMR (100 MHz, CDCl3) δ: 113.4 (C), 124.3
(CH), 125.0 (C), 130.2 (C), 133.4 (CH), 138.1 (CH), 148.0 (C). LC/MS ESI+ tR 1.42 min, (m/z)
[M+H]+ 275.98/276.96/277.58. HRMS (+ESI): 277.9149 (M + H+). Calcd for C7H3BrClN3O2:
277.9148.
562
4.1.9. 8-Bromo-6-chloro-2-methylimidazo[1,2-a]pyridine (5a)
563
564
565
566
567
568
569
570
571
572
573
574
To a solution of sodium borohydride (0.07 g, 1.79 mmol, 1 equiv.) in dimethylsulfoxide (60
mL), 8-bromo-6-chloro-2-chloromethylimidazo[1,2-a]pyridine 1a (0.5 g, 1.79 mmol, 1 equiv.)
was added. The reaction mixture was stirred for 72 h at room temperature. Then, the mixture was
slowly poured into an ice-water mixture and extracted three times with dichloromethane. The
organic layer was washed five times with brine, dried over MgSO4, filtered and evaporated.
Compound 5a was obtained, after purification by chromatography on silica gel (eluent:
dichloromethane/cyclohexane/diethyl ether 7:2.5:0.5) and recrystallization from acetonitrile as a
white solid in 63% yield (0.28 g). mp 140 °C. 1H NMR (300 MHz, CDCl3) δ: 2.48 (3H, s), 7.39–
13
7.41 (2H, m), 8.70 (1H, d, J = 1.8 Hz). C NMR (75 MHz, CDCl3) δ: 14.7 (CH3), 111.1 (C),
112.0 (CH), 119.5 (C), 122.5 (CH), 127.5 (CH), 141.8 (C), 145.5 (C). LC/MS ESI+ tR 1.71 min,
(m/z) [M+H]+ 245.05/247.04/249.07. HRMS (+ESI): 246.9453 (M + H+). Calcd for
C8H6BrClN2: 246.9453.

575
4.1.10. 8-Bromo-6-chloro-2-methyl-3-nitroimidazo[1,2-a]pyridine (5b)
576
577
578
579
580
581
582
583
584
585
To a solution of 8-bromo-6-chloro-2-methylimidazo[1,2-a]pyridine 5a (0.3 g, 1.22 mmol, 1
equiv.) in concentrated sulfuric acid (5 mL) cooled by an ice-water bath, nitric acid 65% (500
µL, 7.3 mmol, 6 equiv.) was added. The reaction mixture was stirred for 30 min at room
temperature. Then, the mixture was slowly poured into an ice-water mixture and the desired
product precipitated. The yellow solid was collected by filtration, dried under reduced pressure
and recrystallized from acetonitrile to give the expected product 5b in 78% yield (0.28 g). mp
192 °C. 1H NMR (300 MHz, DMSO-d6) δ: 2.74 (3H, s), 8.36 (1H, d, J = 1.8 Hz), 9.34 (1H, d, J
= 1.8 Hz). 13C NMR (75 MHz, DMSO-d6) δ: 14.0 (CH3), 111.3 (C), 122.6 (C), 125.2 (CH),
131.2 (C), 133.5 (CH), 140.7 (C), 150.1 (C). LC/MS ESI+ tR 2.13 min, (m/z) [M+H]+
289.90/291.90/293.90. HRMS (+ESI): 291.9302 (M + H+). Calcd for C8H5BrClN3O2: 291.9304.
586
4.1.11. (8-Bromo-6-chloro-3-nitroimidazo[1,2-a]pyridin-2-yl)methanol (6)
587
588
589
590
591
592
593
594
595
596
597
598
599
To a solution of copper sulfate pentahydrate (0.77 g, 3.08 mmol, 1 equiv.) in a
dimethylsulfoxide/water mixture (7.5/2.5, 80 mL), 8-bromo-6-chloro-2-chloromethyl-3-
nitroimidazo[1,2-a]pyridine 1b (1 g, 3.08 mmol, 1 equiv.) was added. The reaction mixture was
stirred and heated at 100 °C for 24 h. Then, the mixture was slowly poured into an ice-water
mixture with sodium carbonate and extracted three times with dichloromethane. The organic
layer was washed five times with brine, dried over MgSO4, filtered and evaporated. The crude
residue was purified by column chromatography on silica gel (eluent: dichloromethane/ethyl
acetate 9.5:0.5) and compound 6 was isolated as a white solid in 32% yield (0.3 g). mp 202 °C.
1H NMR (300 MHz, DMSO-d6) δ: 4.92 (2H, d, J = 6.3 Hz), 5.57 (1H, t, J = 6.3 Hz), 8.38 (1H, d,
13
J = 1.8 Hz), 9.36 (1H, d, J = 1.8 Hz). C NMR (75 MHz, DMSO-d6) δ: 58.3 (CH2), 111.9 (C),
122.9 (C), 125.2 (CH), 129.7 (C), 133.6 (CH), 141.0 (C), 153.2 (C). LC/MS ESI+ tR 1.01 min,
(m/z) [M+H]+ 305.84/307.83/309.91. HRMS (+ESI): 307.9255 (M + H+). Calcd for
C8H5BrClN3O3: 307.9253.