Targeting the human parasite Leishmania donovani: Discovery of a new promising anti-infectious pharmacophore in 3-nitroimidazo[1,2-a]pyridine series

Article abstract of DOI:10.1016/j.bmc.2013.09.002

We report herein the discovery of antileishmanial molecules based on the imidazo[1,2-a]pyridine ring. In vitro screenings of imidazopyridines belonging to our chemical library, toward the promastigotes stage of Leishmania donovani, J774A.1 murine and HepG2 human cells, permitted to identify three selective hit-compounds (12, 20 and 28). New derivatives were then synthesized to allow structure-activity and -toxicity relationships analyses, enabling to characterize a lead-compound (44) displaying both a high potency (IC ₅₀ = 1.8 μM) and a good selectivity index, in comparison with three antileishmanial reference drug-compounds (amphotericin B, miltefosine and pentamidine). Moreover, lead-compound 44 also exhibits good in vitro activity against the intracellular amastigote stage of L. donovani. Thus, the 6-halo-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-a]pyridine scaffold appears as a new promising selective antileishmanial pharmacophore, especially when substituted at position 8 by a bromine atom.

Full text of DOI:10.1016/j.bmc.2013.09.002

Accepted Manuscript
Targeting the human parasite Leishmania donovani: Discovery of a new prom‐
ising anti-infectious pharmacophore in 3-nitroimidazo[1,2-a]pyridine series
Caroline Castera-Ducros, Lucie Paloque, Pierre Verhaeghe, Magali Casanova,
Christophe Cantelli, Sébastien Hutter, Floriane Tanguy, Michèle Laget, Vincent
Remusat, Anita Cohen, Maxime D. Crozet, Pascal Rathelot, Nadine Azas,
Patrice Vanelle
PII:
S0968-0896(13)00773-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.09.002
BMC 11086
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
13 April 2013
29 August 2013
1 September 2013
Please cite this article as: Castera-Ducros, C., Paloque, L., Verhaeghe, P., Casanova, M., Cantelli, C., Hutter, S.,
Tanguy, F., Laget, M., Remusat, V., Cohen, A., Crozet, M.D., Rathelot, P., Azas, N., Vanelle, P., Targeting the
human parasite Leishmania donovani: Discovery of a new promising anti-infectious pharmacophore in 3-
nitroimidazo[1,2-a]pyridine series, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.
2013.09.002
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Graphical Abstract
.
Targeting the human parasite Leishmania
donovani: Discovery of a new promising anti-
infectious pharmacophore in 3-nitroimidazo[1,2-
a]pyridine series
Leave this area blank for abstract info.
C. Castera-Ducros; L. Paloque; P. Verhaeghe; M. Casanova; C. Cantelli; S. Hutter; F. Tanguy; M. Laget; V.
Remusat; A. Cohen; M. D. Crozet; P. Rathelot; N. Azas; P. Vanelle
Aix-Marseille Université, Faculté de Pharmacie, 27 Bd Jean Moulin, 13385 Marseille cedex 05, France.
O2S
N
N
X
NO2
X = Cl, Br or I

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Targeting the human parasite Leishmania donovani: Discovery of a new promising
anti-infectious pharmacophore in 3-nitroimidazo[1,2-a]pyridine series
Caroline Castera-Ducrosa, Lucie Paloqueb, Pierre Verhaeghea, Magali Casanovab, Christophe Cantellia,
Sébastien Hutterb, Floriane Tanguya, Michèle Lagetb, Vincent Remusata, Anita Cohenb, Maxime D. Crozeta,
Pascal Rathelota, Nadine Azasb* and Patrice Vanellea*
aEquipe Pharmacochimie Radicalaire, Faculté de Pharmacie, Aix-Marseille Université – UMR CNRS 7273, Institut de Chimie Radicalaire, 27 Boulevard Jean
Moulin-CS30064, 13385 Marseille Cedex 05, France
b Infections Parasitaires, Transmission, Physiopathologie et Thérapeutique, UMR MD3, Faculté de Pharmacie, Aix-Marseille Université, 27 Boulevard Jean
Moulin-CS30064, 13385 Marseille Cedex 05, France
*Corresponding author for chemistry: patrice.vanelle@univ-amu.fr; tel: +33 491 835 580, fax: +33 491 794 677
*Corresponding author for biology: nadine.azas@univ-amu.fr; tel: +33 491 835 564
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
We report herein the discovery of antileishmanial molecules based on the imidazo[1,2-
a]pyridine ring. In vitro screenings of imidazopyridines belonging to our chemical library,
toward the promastigotes stage of Leishmania donovani, J774A.1 murine and HepG2 human
cells, permitted to identify 3 selective hit-compounds (12, 20, 28). New derivatives were then
synthesized to allow structure-activity and -toxicity relationships analyses, enabling to
characterize a lead-compound (44) displaying both a high potency (IC50 = 1,8 µM) and a good
selectivity index, in comparison with 3 antileishmanial reference drug-compounds (amphotericin
B, miltefosine and pentamidine). Moreover, lead-compound 44 also exhibits good in vitro
activity against the intracellular amastigote stage of L. donovani. Thus, the 6-halo-3-nitro-2-
(phenylsulfonylmethyl)imidazo[1,2-a]pyridine scaffold appears as a new promising selective
antileishmanial pharmacophore, especially when substituted at position 8 by a bromine atom.
Available online
Keywords:
Leishmania donovani
Imidazo[1,2-a]pyridine
Structure-activity relationships
HepG2 in vitro cytotoxicity
2013 Elsevier Ltd. All rights reserved.
1. Introduction
There are very few drugs available for treating
leishmaniasis: pentavalent antimonials (sodium stibogluconate
Leishmaniasis is a parasitic infection second to malaria
and meglumine antimoniate), liposomal amphotericin B,
pentamidine, paromomycin and miltefosine4,5. Some other drugs
are in clinical trials: sitamaquine, imiquimod and antifungal
azoles6,7. Currently, the drugs used in the treatment of
leishmaniasis present major limitations such as non-oral routes of
administration (apart for miltefosine), high toxicity of antimonials
and pentamidine, and teratogenicity for miltefosine8. Moreover,
the expensive cost of liposomal amphotericin B, and the socio-
economic problems encountered in endemic areas, lead to patients
withdrawing from treatment and global emergence of resistant
strains9. During the last decade, antimonial resistance reached
epidemic dimension in Bihar, India, where about 60% of newly
diagnosed visceral leishmaniasis do not respond to these
molecules10. Evaluation of the in vitro susceptibility of Indian L.
donovani patient isolates to antimonials, amphotericin B and
miltefosine indicates that cross-resistance may be emerging
among these three drugs. Despite the absence of reported cases of
clinical resistance to miltefosine, the preliminary data of clinical
trials suggest a doubling of the relapse rates, leading to predict a
fast development of the resistance11. Thus, efficient, safe and
in terms of parasite-related mortality. Leishmaniasis pathologies
include cutaneous leishmaniasis (CL), muco-cutaneous
leishmaniasis (MCL) and visceral leishmaniasis (VL), depending
on Leishmania species. Visceral leishmaniasis is the most severe
clinical form of the disease, lethal in untreated patients. There are
an estimated 14 million people infected by leishmaniasis in 98
countries worldwide, mainly in tropical and sub-tropical regions,
but also in southern Europe, especially around the Mediterranean
area. The annual incidence of leishmaniasis is 2 million with
about 50.000 estimated deaths due to its visceral form. The
number of cases is certainly under-evaluated as leishmaniasis are
reportable diseases in only 40 countries1. These parasitic
infections are caused by a protozoan of the Leishmania genus
transmitted to its mammal hosts (humans, dogs, monkeys,
rodents…) by the bite of an infected sandfly (Phlebotominae).
Leishmania parasites exist in two major morphological stages:
extracellular flagellated promastigotes in the digestive tract of
their sandfly vector and non-motile intracellular amastigotes in
cells of their host mononuclear phagocytic system2,3.

cheap oral antileishmanial agents are needed to safeguard against
the expanding resistance problem and to overcome miltefosine’s
shortcomings12.
antileishmanial spectrum of activity was more completely
highlighted.
Twenty-one compounds with a 3-nitroimidazo[1,2-a]-
A bibliographical review about molecules exhibiting pyridine backbone from our library were randomly screened
antileishmanial activity shows that nitroaromatic compounds have against L. donovani. Based on the activity of these compounds,
already been reported13,14 as interesting agents (Figure 1), we wanted to further investigate the influence of the nitro group.
displaying inhibitory concentrations 50% (IC50) in the micromolar Therefore, an additional 23 compounds were prepared and
range toward the promastigote and/or the amastigote stages of the screened.
parasite, in particular in 5-nitroimidazole (A and C)15,16
,
dinitrodiphenylsulfane (B)17, nitroquinolin-2(1H)-one (D)18 and
2. Results and Discussion
dinitrodiphenylamine (E)19 series.
2.1. Chemistry
Figure 1. Structures of some nitroaromatic compounds displaying
antileishmanial properties.
Among these 44 synthesized compounds, 21 were
previously described (labeled* in the result and discussion part)
and came from our internal chemical library, part of the French
National Chemical Library (Chimiothèque Nationale, CNRS),
while 23 were newly prepared for studying structure-activity
relationships.
N
N
S
N
H3CO
O2N
S
CN
N
O2N
N
CH3
N
Cl
A
B
S
NO2
O
As initially reported29,30, imidazo[1,2-a]pyridine and 2-
methylimidazo[1,2-a]pyridine can be nitrated at room
temperature to afford, respectively, compounds 1* and 2* in 90%
yield (Scheme1).
IC50 L. major pro. = 9.3 µM
IC50 L. donovani axenic ama. = 0.7 µM
N
O
H3C
S
NO2
N
C
CH3
IC50 L. donovani pro. = 5.6 µM
IC50 L. donovani axenic ama. = 2.8 µM
OH
Scheme 1. Synthesis of compounds 1* and 2*
H
N
HNO3/H2SO4
N
N
O2N
NO2
N
O
1 (R = H): 90%
2 (R = CH3): 90%
H
R
R
NO2
N
N
R.T.
NO2
CF3
D
E
IC50 L. donovani pro. = 6.6 µM
IC50 L. donovani ama. = 6.5 µM
IC50 L. donovani pro. = 0.6 µM
IC50 L. infantum ama. = 1.8 µM
Following an operating procedure which we previously
reported31,32, 2-chloromethylimidazo[1,2-a]pyridines 3*, 7, 15*
and 23* were obtained by cyclo-condensation of 1,3-
dichloroacetone with corresponding 2-aminopyridines in
refluxing ethanol (Scheme 2).
Thanks to the sequencing of the parasite genome20, new
targets were identified in order to come up with new powerful
antileishmanial drugs21. Among them, Leishmania kinases appear
to be of great interest. Thus, some imidazo[1,2-a]pyridines
substituted at positions 2 and 3 have demonstrated antileishmanial
activity, notably due to the inhibition of the parasite casein-kinase
Scheme 2. Formation of the imidazopyridine ring by a cyclo-
condensation reaction
1 (Figure 2)22,23
.
R
3 (R = H): 40%
7 (R = I): 70%
15 (R = Cl): 62%
23 (R = Br): 58%
Cl
EtOH
78 °C
N
Cl
Cl
+
NH2
N
N
O
R
Figure 2. Structure of previously described antileishmanial
imidazo[1,2-a]pyridines.
1.1 equiv.
H3C
N
N
2-Chloromethyl-3-nitroimidazo[1,2-a]pyridines 5*, 11,
N
F
CH3
N
N
19* and 27* were then obtained by nitration at room
temperature33,34 (Scheme 3).
N
NH2
N
Scheme 3. Nitration reactions in 2-chloromethylimidazo[1,2-
a]series.
NO2
IC50 L. major pro. = 4.9 µM
IC50 L. major pro. = 0.2 µM
HNO3/H2SO4
R.T.
5 (R = H): 80%
11 (R = I): 80%
19 (R = Cl): 70%
27 (R = Br): 100%
Cl
N
Cl
N
From these bibliographical data, and in continuation
with our efforts toward the conception and preparation of original
molecules bearing anti-infectious potential24,25, focusing on the
development of new antiplasmodial26,27 and antileishmanial18,28
agents, we recently investigated the antileishmanial potential of
N
N
R
R
NO2
2-Chloromethylimidazo[1,2-a]pyridines
bearing
a
hydrogen or a halogen atom at position 6 were involved into a
microwave-assisted reaction with various sulfonylchlorides,
according to a procedure which we previously reported,35,36
affording corresponding sulfones in 25 to 100% yield (Scheme
4). The obtained molecules are presented in Table 1.
new imidazo[1,2-a]pyridine derivatives.
A large series of
molecules was prepared and screened in vitro on the promastigote
stage of Leishmania donovani, in parallel with 3 antileishmanial
drug-compounds. To assess the selectivity of action of these
molecules, cytotoxicity evaluations were also conducted on the
murine J774A.1 macrophage cell line and on the human HepG2
hepatocyte cell line. This medicinal chemistry study allowed us to
identify a new potent antileishmanial lead-compound and also to
Scheme 4. Preparation of sulfone derivatives under microwave
irradiation
characterize
the
corresponding pharmacophore
whose

1) Sodium sulfite (2 equiv.)
NaHCO3 (2 equiv.)
O2S
R
N
Finally, molecule 44* which synthesis was first
described in our lab by Szabo et al.40 was prepared in 4 steps from
2-amino-5-bromopyridine with a 54% global yield (Scheme 7).
O
S
M.W. 120 °C
N
+
H2O
R
Cl
R'
2)
Cl
N
O
R''
2 equiv.
N
R'
R''
1 equiv.
Scheme 7. Four-step synthesis of lead-compound 44*
Table 1. Sulfone synthesized according to Scheme 4
Br
NBS 1 equiv.
CH3CN, 80 °C
Br
Br
95%
Br
R-
C6H5-
C6H5-
C6H5-
R’-
-H
-H
-I
-I
-I
-I
-I
-I
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Br
-Br
-Br
-Br
-Br
-Br
-Br
-Br
-Br
-Br
-Br
-Br
-Br
R’’-
-H
-NO2
-H
-H
-H
-NO2
-NO2
-NO2
-H
-H
-H
-NO2
-NO2
-NO2
-H
-H
-H
-NO2
-NO2
-NO2
-NO2
-NO2
-NO2
-NO2
-NO2
-NO2
-NO2
Yield
60%
60%
74%
80%
53%
90%
100%
90%
69%
88%
88%
80%
86%
76%
100%
94%
89%
90%
90%
60%
80%
30%
44%
60%
46%
25%
40%
N
NH2
N
NH2
4
6
8
Br
Br
NH2
Cl
N
EtOH
78 °C
Cl
Cl
O
+
N
4-CH3-C6H4-
4-Cl-C6H4-
C6H5-
4-CH3-C6H4-
4-Cl-C6H4-
C6H5-
4-CH3-C6H4-
4-Cl-C6H4-
C6H5-
4-CH3-C6H4-
4-Cl-C6H4-
C6H5-
9
Br
N
63%
1.1 equiv.
10
12
13
14
16*
17
18
20*
21*
22
24*
25
26
28*
29*
30*
31
32
33
34
35
36
37
Br
Br
Cl
N
Cl
N
HNO3 / H2SO4
RT
N
N
Br
96%
Br
NO2
1) Na2SO3 (2 equiv.)
NaHCO3 (2 equiv.)
Br
O2S
N
M.W. 120 °C
+
H2O
N
2)
Br
44
94%
O
S
O
Br
NO2
Cl
N
Cl
2 equiv.
N
Br
NO2
4-CH3-C6H4-
4-Cl-C6H4-
C6H5-
1 equiv.
2.2. Biological activity
4-CH3-C6H4-
4-Cl-C6H4-
4-Br-C6H4-
4-I-C6H4-
4-F-C6H4-
4-CF3-C6H4-
4-OCH3-C6H4-
CH3-
All molecules were first screened in vitro on the
promastigote stage of L. donovani by determining their inhibitory
concentrations 50% (IC50) and comparing them to pentamidine,
miltefosine and amphotericin B, chosen as antileishmanial
reference drugs. In order to assess their selectivity of action, all
molecules were also evaluated in vitro in regards to their
cytotoxicity toward two complementary cell lines, measured by
the cytotoxic concentrations 50% (CC50) on the murine J774A.1
macrophage and metabolizing human hepatocyte HepG2 cell
lines (positive control = doxorubicin), giving access to the
corresponding selectivity indexes (SI = CC50 / IC50). The results
are presented in Table 2.
4-C6H5-C6H4-
Sulfane derivative 38* was prepared by reacting 27*
with thiophenol (Scheme 5) in the presence of sodium hydride in
dry DMSO.33
A global analysis of the results first shows that neither
the imidazo[1,2-a]pyridine ring nor its nitrated counterpart 1
display any antileishmanial properties. Among all 45 tested
molecules, only thirteen (5*, 12-14, 17, 20*, 22, 27*-31 and 44*)
presented measurable activity against the parasite, all of them
including a nitro group at position 3 of the imidazo[1,2-a]pyridine
ring, except molecule 17. However, molecules 5*, 27* and 29*
appeared to be toxic toward, at least, one of the cell lines used for
measuring cytotoxicity. Then, out of the nine remaining nitrated
molecules, only four (12, 20*, 28* and 44*) displayed significant
antileishmanial activities (IC50 < 20 µM) objectivized by
significantly higher selectivity indexes (> 5 by considering the
HepG2 human cell line). From a structural point of view, these
four hit-molecules appear to be very closely related, sharing the
Scheme 5. Preparation of sulfane derivative 38*
SH
Cl
S
N
N
1 equiv.
N
N
Br
Br
NaH (1.1 equiv.)
DMSO, N2
27
1 equiv.
38
NO2
NO2
80%
6-Bromo-3-nitro-2-phenylsulfonylmethylimidazo[1,2-
a]pyridine 28* was involved into microwave-assisted Suzuki-
Miyaura cross-coupling reactions37 with boronic acids (Scheme
6), to afford compounds 39*, 40*, 41*, 42, 43* in good yields38,39
.
same
a]pyridine scaffold.
When comparing the activities of the hit-molecules to
the one of compound 6, it can be concluded that a halogen atom at
position of the scaffold is necessary for providing
6-halo-3-nitro-2-phenylsulfonyl-methylimidazo[1,2-
Scheme 6. Suzuki-Miyaura coupling reactions leading to
derivatives 39*-43*
6
Ar
antileishmanial activity to this series. Comparison of the hit-
molecules to non-nitrated analogs 8, 16* and 24* shows that the
nitro group is mandatory for conferring activity in the studied
series. Moreover, sulfane analog 38* of sulfone hit-molecule 28*
appears very toxic, indicating that the sulfone group is also
required for designing selective antileishmanial molecules.
Finally, when studying the influence of the nature of the sulfone
moiety (compounds 28* to 37), it can be concluded that the
O2S
B
N
HO
OH
O2S
N
N
1.3 equiv.
Ar
N
NO2
Na2CO3 (5 equiv.)
Pd(PPh3)4 (0.1 equiv.)
TBABr (1 equiv.)
H2O
Br
28
39 (Ar = 2-CH3-C6H4): 67%
40 (Ar = 3-CF3-C6H4): 69%
41 (Ar = 4-OCH3-C6H4): 69%
42 (Ar = 3,4,5-tri-OCH3-C6H2): 60%
43 (Ar = naphthalen-2-yl ): 78%
NO2
1 equiv.
100°C / M.W.

unsubstituted phenylsulfonyl moiety is the only one which
confers satisfying antileishmanial profile.
100
90
Thus, from all these structure-activity relationships data,
the 6-halo-3-nitro-2-phenylsulfonylmethylimidazo[1,2-a]pyridine
scaffold corresponds to the minimal structural skeleton required
for displaying in vitro antileishmanial activity against the
promastigotes stage of Leishmania donovani and then, appears as
a new antileishmanial pharmacophore. Molecule 28*, chosen as a
good representative of this pharmacophore, has an IC50 value of
14 µM, 2.3 times higher than the one of pentamidine and 4.5
times higher than the one of miltefosine. Its CC50 values make it
less toxic than pentamidine and amphotericin B and globally as
slightly toxic as miltefosine, reaching a HepG2 selectivity index
>7, much better than the one of pentamidine, and about 2 times
inferior to the one of miltefosine.
80
70
60
50
40
30
20
0
2
4
6
8
10
Concentration (µM)
Indeed,
6,8-dibrominated
imidazopyridine
44*,
(B)
corresponding to the previously described pharmacophore bearing
an additional bromine atom at position 8, appeared to be the lead-
compound in the tested series. Such molecule is about eight times
more active than hit-molecule 28*, presents a very good activity
(IC50 = 1.8 µM), better than the ones of both pentamidine and
miltefosine and a selectivity index (SI >17.2, considering the
HepG2 human cell line) standing higher than the ones of
pentamidine and miltefosine.
100
90
80
70
60
50
40
30
20
In order to investigate and define more precisely the
antileishmanial potential of hit-molecule 28* and lead-molecule
44*, these two molecules were tested on the promastigote stage of
Leishmania infantum (second causing agent of visceral
leishmaniasis worldwide and species responsible for visceral
leishmaniasis in the endemic Mediterranean area) and on the
amastigote stage of Leishmania donovani. Remembering that only
amastigotes develop in the mammalian hosts of the parasite, this
last evaluation is fundamental so as to identify molecules which
could be used for treating human visceral leishmaniasis. The
intracellular amastigotes assay was done according to the method
of da Luz et al.41 which uses a brief exposure of promastigotes to
an acidic pH to insure optimal metacyclogenesis42,43 just prior to
macrophages infection, leading to a higher final cell density and
10
0
0
0.1
0.2
0.3
0.4
0.5
Concentration (µM)
Hit-compound 28* does not show any interesting activity toward
either L. infantum promastigotes or L. donovani amastigotes
whereas lead-compound 44* exhibits quite good in vitro activity
against both of them. Like all antileishmanial reference drugs,
44* is slightly less active toward the amastigote stage of the
parasite (IC50 = 5.5 µM) than the promastigote one (IC50 = 1.8
µM). Nevertheless, concerning the amastigote stage of L.
more reproducible intracellular infection41,44
. Results were
donovani, amphotericin B remains the most active drug (IC50
=
compared to the IC50 values obtained with three antileishmanial
reference-drugs: pentamidine, miltefosine and amphotericin B
(Table 3, Figure 3). Finally, to complete the evaluation of these
two molecules, their selectivity of action among protozoa was
assessed by determining their in vitro antiplasmodial IC50 on the
K1 multi-resistant strain of Plasmodium falciparum, in
comparison with atovaquone, chosen as an antimalarial reference
drug.
0.07 µM), but the activity of 44* is interesting (Figure 3). Indeed,
44* is much more active than pentamidine (IC50 > 20 µM) and
approximately as active as miltefosine (IC50 = 6.8 µM) (Table 3).
Moreover, concerning antiprotozoal profile, 44* appears rather
selective, in comparison with reference drug-compounds, being 3
to 9 times less active toward the Plasmodium genus (IC50 = 16
µM) than the Leishmania one.
3. Conclusions
Figure 3. Dose-response curves of compound 44* (A) and
amphotericine B (B) toward the amastigote stage of L. donovani.
In vitro screening of a series of imidazo[1,2-a]pyridines
permitted us to identify selective molecules targeting the
promastigote stage of Leishmania donovani and displaying low
cytotoxicity. From numerous derivatives, structure-activity
relationships could be studied and allowed the characterization of
(A)
the
corresponding
pharmacophore:
6-halo-3-nitro-2-
phenylsulfonyl-methylimidazo[1,2-a]pyridine. Substitution of this
pharmacophore at position 8 by a bromine atom generates lead-
compound 44* which appears as
a potent and selective
antileishmanial molecule targeting both the pro- and intracellular
amastigote stages of the human parasite. Molecule 44* will next
be the starting point for additional pharmaceutical chemistry work
exploring both position 6 and key-position 8, trying to improve,
among others, solubility in biological media. Enzymatic assays on
several Leishmania kinases will also be carried out.

Table 2. In vitro antileishmanial activity and cytotoxicity of imidazo[1,2-a]pyridine derivatives.
R4
N
R3
N
R1
R2
L. donovani
J774
HepG2
Cytotoxicity
CC50 (µM)a
>100
Selectivity
Indexb
Selectivity
Indexc
N°
-R1
-R2
-R3
-R4 promastigotes Cytotoxicity
IC50 (µM)a
>50
CC50 (µM)a
>100
-
-H
-H
-NO2
-NO2
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-Br
NDg
NDg
NDg
NDg
NDg
0.3
NDg
NDg
NDg
NDg
NDg
0.6
1*
-H
-H
>50
>100
>100
2*
-H
-CH3
>50
>100
>100
3*
-H
Cl-CH2-
>50
>100
>100
4
-H
-H
C6H5-SO2-CH2-
Cl-CH2-
>50
>100
>100
5*
-H
-NO2
-NO2
-H
15.1 (± 1.2)
>50
4.8 (± 0.7)
>100
8.7 (± 1.2)
>62.5d
6
-H
C6H5-SO2-CH2-
C6H5-SO2-CH2-
4-CH3-C6H4-SO2-CH2-
4-Cl-C6H4-SO2-CH2-
C6H5-SO2-CH2-
4-CH3-C6H4-SO2-CH2-
4-Cl-C6H4-SO2-CH2-
C6H5-SO2-CH2-
4-CH3-C6H4-SO2-CH2-
4-Cl-C6H4-SO2-CH2-
C6H5-SO2-CH2-
4-CH3-C6H4-SO2-CH2-
4-Cl-C6H4-SO2-CH2-
Cl-CH2-
NDg
<0.1
<0.5
<0.8
2.1
NDg
<0.1
NDg
<0.8
>7.9
>1.3
>1.7
<0.3
2.0
8
-I
>50
5.2 (± 0.6)
25 (± 0.8)
44.2 (± 5.4)
27 (± 0.6)
34.8 (± 1.3)
43.6 (± 1.7)
13.3 (± 0.7)
33.3 (± 1.2)
68.7
5.0 (± 0.9)
>12.5d
9
-I
-H
>50
10
-I
-H
>50
39.1 (± 4.0)
>100
12
-I
-NO2
-NO2
-NO2
-H
12.6 (± 0.8)
39.2 (± 0.4)
29.1 (± 1.0)
>50
13
-I
0.9
>50
14
-I
1.5
>50
16*
17
-Cl
<0.3
0.9
16.5 (± 1.1)
68.3 (± 1.3)
71.5
-Cl
-H
33.5 (± 0.9)
>50
18
-Cl
-H
<1.3
>5.5
NDg
1.8
<1.4
>5.5
NDg
1.1
20*
21*
22
-Cl
-NO2
-NO2
-NO2
-H
18.2 (± 0.6)
>50
>100
>100
-Cl
>94.0d
>100
-Cl
23.0 (± 0.8)
>50
42.4 (± 1.6)
68.4 (± 2.3)
6.4 (± 0.3)
20.9 (± 1.8)
43.9 (± 2.1)
4.9 (± 0.9)
34.3 (± 2.3)
44.3 (± 4.6)
51.1 (± 6.1)
28.2 (± 1.4)
16.2 (± 0.7)
>92.8d
>25d
23*
24*
25
-Br
<1.4
<0.1
<0.4
<0.9
0.5
>100
NDg
0.15
<0.6
<0.5
1.6
-Br
-H
C6H5-SO2-CH2-
4-CH3-C6H4-SO2-CH2-
4-Cl-C6H4-SO2-CH2-
Cl-CH2-
>50
7.3 (± 0.9)
28.9 (± 1.5)
23.0 (± 0.4)
15.6 (± 1.1)
>100
-Br
-H
>50
26
-Br
-H
>50
27*
28*
29*
30*
31
-Br
-NO2
-NO2
-NO2
-NO2
-NO2
-NO2
-NO2
-NO2
9.6 (± 0.8)
14.0 (± 0.8)
44.1 (± 2.8)
45.4 (± 3.6)
24.3 (± 1.1)
>50
-Br
C6H5-SO2-CH2-
4-CH3-C6H4-SO2-CH2-
4-Cl-C6H4-SO2-CH2-
4-Br-C6H4-SO2-CH2-
4-I-C6H4-SO2-CH2-
4-F-C6H4-SO2-CH2-
4-CF3-C6H4-SO2-CH2-
2.5
>7.1
0.13
0.12
1.6
-Br
1.0
5.9 (± 0.8)
5.5 (± 0.3)
39 (± 2.1)
20.3 (± 0.2)
>100
-Br
1.1
-Br
1.1
32
-Br
<0.3
NDg
NDg
NDg
NDg
<1.1
NDg
<0.5
NDg
NDg
<0.1
NDg
>13.4
<0.4
NDg
<0.6
NDg
NDg
NDg
<0.05
NDg
NDg
NDg
<0.7
NDg
>17.2
33
-Br
-Br
>50
34
>50
>25d
31.7 (± 1.1)
>100
35
-Br
-NO2 4-OCH3-C6H4-SO2-CH2-
>50
>92.5d
36
-Br
-NO2
-NO2
-NO2
-NO2
-NO2
-NO2
CH3-SO2-CH2-
4-C6H5-C6H4-SO2-CH2-
C6H5-S-CH2-
>50
>100
>100
37
-Br
>50
54.4 (± 3.4)
>6d
>100
38*
39*
40*
41*
42
-Br
>10d
0.53 (± 0.06)
>9.4d
2-CH3-C6H4-
3-CF3-C6H4-
4-OCH3-C6H4-
C6H5-SO2-CH2-
C6H5-SO2-CH2-
C6H5-SO2-CH2-
C6H5-SO2-CH2-
C6H5-SO2-CH2-
C6H5-SO2-CH2-
>10d
5.0 (± 0.6)
NSh
NSh
NSh
NSh
NSh
NSh
3,4,5-tri-OCH3-C6H2- -NO2
>50
5.0 (± 0.8)
NSh
34.7 (± 1.7)
NSh
43*
44*
2-Naphthyl-
-NO2
NSh
-Br
-NO2
1.8 (± 0.8)
>25d
>31d

Ref.
Ref.
Ref.
Ref.
Amphotericin Be
Miltefosinee
0.06 (± 0.02)
3.1 (± 0.06)
6.0 (± 0.8)
-
3.1 (± 0.3)
94.6 (± 2.1)
1.0 (± 0.7)
51.6
30.5
0.1
-
8.8 (± 0.6)
50.3 (± 1.5)
2.3 (± 0.5)
0.2 (± 0.05)
147
16.2
0.4
-
Pentamidinee
Doxorubicinf
0.02 (± 0.03)
aThe values are means ± SD of three independent experiments.
bSelectivity Index was calculated according to the formula : SI = (J774 CC50) / (L. donovani IC50)
CSelectivity Index was calculated according to the formula : SI = (HepG2 CC50) / (L. donovani IC50)
dDetermination of the IC50 or CC50 value was limited by lack of solubility in the culture medium
eAmphotericin B, pentamidine and miltefosine were used as antileishmanial drug compounds of reference.
fDoxorubicin was used as a cytotoxic drug compound of reference.
gND = not determinable
hNS = non-soluble
Table 3. In vitro antileishmanial profile and antiplasmodial activity of hit compounds 28* and 44*.
28: R = H
44: R = Br
R
O2S
N
N
Br
NO2
L. infantum promastigotes
IC50 (µM)a
>25d
L. donovani promastigotes
IC50 (µM)a
14.0 (± 0.8)
1.8 (± 0.8)
L. donovani amastigotes
P. falciparum (K1)
Compound
IC50 (µM)a
>25d
IC50 (µM)a
28*
41.2 (± 1.3)
44*
3.3 (± 0.7)
8.2 (± 0.1)
11.6 (± 0.4)
0.04 (± 0.01)
-
5.5 (± 0.2)
>20
16.0 (± 0.7)
Pentamidineb
Miltefosineb
Amphotericin Bb
Atovaquonec
6.0 (± 0.8)
-
3.1 (± 0.06)
0.06 (± 0.02)
-
6.8 (± 0.3)
0.07 (± 0.02)
-
-
-
0.0015 (± 0.0007)
a The values are means ± SD of three independent experiments.
bAmphotericin B, pentamidine and miltefosine were used as antileishmanial drug compounds of reference.
cAtovaquone was used as a reference antiplasmodial drug compound.
dMolecule 28 could not be tested at higher concentrations due to lack of solubility in the culture mediu

processed on dose–response curves, using TableCurve 2D V5
software. IC50 values represent the mean value calculated from three
independent experiments.
4. Experimental
4.1. Biology
4.1.2. Antiplasmodial evaluation
4.1.1. Antileishmanial evaluation
In this study, a K1 culture-adapted P. falciparum strain resistant to
chloroquine, pyrimethamine and proguanil was used in an in vitro
culture. Maintenance in continuous culture was done as described
previously by Trager and Jensen46. Cultures were maintained in fresh
A+ human erythrocytes at 2.5% hematocrit in complete medium
(RPMI 1640 with 25 mM HEPES, 25 mM NaHCO3, 10% of A+
human serum) at 37 C under reduced O2 atmosphere (gas mixture
5% O2, 5% CO2, and 90% N2). Parasitaemia was maintained daily
between 1% and 6%. The P. falciparum drug susceptibility test was
carried out by comparing quantities of DNA in treated and control
cultures of parasite in human erythrocytes according to a SYBR
Green I fluorescence-based method47 using a 96-well fluorescence
plate reader. Parasite culture was synchronized at ring stage with 5%
sorbitol. Compounds were incubated in a total assay volume of 200
μL (RPMI, 2% hematocrit and 1% parasitaemia) for 72 h in a
humidified atmosphere (5% O2 and 5% CO2) at 37 °C, in 96-well flat
bottom plates. Triplicate assays were performed for each sample.
After incubation, 170 μL supernatant was discarded and cells were
washed with 150 μL 1X PBS. 15 μL re-suspended cells were
transferred to 96-well flat bottom non-sterile black plates (Greiner
Bio-one) already containing 15 μL of the SYBR Green I lysis buffer
(2XSYBR Green, 20 mM Tris base pH 7.5, 20 mM EDTA, 0.008%
w/v saponin, 0.08% w/v Triton X-100). Negative control, treated by
solvents (DMSO or H2O) and positive control (atovaquone) were
added to each set of experiments. Plates were incubated for 15 min at
37 °C and then read on a TECAN Infinite F-200 spectrophotometer
with excitation and emission wavelengths at 497 and 520 nm,
respectively. The concentrations of compounds required to induce a
50% decrease of parasite growth (IC50 K1) were calculated from
three independent experiments.
Leishmania species used in this study were L. donovani
MHOM/IN/00/DEVI and L. infantum MCAN/ES/98/LLM-877, all
provided by the CNR Leishmania (Montpellier, France).
Antileishmanial activity on promastigotes
The effects of the tested compounds on the growth of Leishmania
donovani and Leishmania infantum promastigotes were assessed by
MTT assay45. Briefly, promastigotes in log-phase in Schneider’s
medium supplemented with 20% fetal calf serum (FCS), 2 mM L-
glutamine and antibiotics (100 U/mL penicillin and 100 µg/mL
streptomycin), were incubated at an average density of
106 parasites/mL in sterile 96-well plates with various concentrations
of compounds dissolved in DMSO (final concentration less than
0.5% v/v), in duplicate. Appropriate controls treated by DMSO,
pentamidine, miltefosine or amphotericin B (reference drugs
purchased from Sigma Aldrich) were added to each set of
experiments. After a 72 h incubation period at 27 °C, parasite
metabolic activity was determined. Each plate-well was then
microscope-examined for detecting possible precipitate formation.
Plates were centrifuged at 900 g for 10 min and the supernatant
removed. After the addition of MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide), 0.5 mg/mL in Schneider’s
medium, 100 µL/well, plates were incubated for 6 h at 27 °C. The
plates were subsequently centrifuged at 900 g for 10 min and the
supernatant removed. The pellet was dissolved in 100 µl of DMSO
and the absorbance measured in a plate reader at 570 nm. Inhibitory
concentration 50% (IC50) was defined as the concentration of drug
required to inhibit by 50% the metabolic activity of Leishmania
donovani and Leishmania infantum promastigotes compared to the
control. IC50 were calculated by non-linear regression analysis
processed on dose–response curves, using TableCurve 2D V5
software. IC50 values represent the mean value calculated from three
independent experiments.
4.1.3. Cytotoxicity evaluation
The evaluation of the tested molecules cytotoxicity on the HepG2
(hepatocarcinoma cell line purchased from ATCC, ref HB-8065)
J774A.1 (mouse macrophage cell line ECACC, Salisbury UK) and
HFF (human foreskin fibroblast) cell lines was done according to the
method of Mosmann45 with slight modifications. Briefly, cells in 100
µL of complete medium, [RPMI supplemented with 10% FCS, 1%
L-glutamine (200 mM) and penicillin (100 U/mL) / streptomycin
(100 µg/mL)] were inoculated into each well of 96-well plates and
incubated at 37 °C in a humidified 6% CO2 with 95% air atmosphere.
After a 24 h incubation, 100 µL of medium with various product
concentrations were added and the plates were incubated for 72 h at
37 °C. Each plate-well was then microscope-examined for detecting
possible precipitate formation before the medium was aspirated from
the wells. 100 µL of MTT solution (0.5 mg / mL in PBS) were then
added to each well with 100 µl of medium without FCS. Cells were
incubated for 2 h at 37 °C. After this time, the MTT solution was
removed and DMSO (100 µL) was added to dissolve the resulting
blue formazan crystals. Plates were shaken vigorously (300 rpm) for
5 min. The absorbance was measured at 570 nm with 630 nm as
reference wavelength with a microplate spectrophotometer. DMSO
was used as blank and doxorubicin (purchased from Sigma Aldrich)
as positive control. Cell viability was calculated as percentage of
control (cells incubated without compound). The 50% cytotoxic
concentration was determined from the dose–response curve by using
the TableCurve 2D V5 software.
Antileishmanial activity on intracellular amastigotes
The effects of the tested compounds on the growth of Leishmania
amastigotes were assessed according to the method of da Luz et al41.
in the following way. 500 µL of J774A.1 cells were seeded in sterile
chamber-slides at an average density of 5x104 cells/mL and incubated
for 24 h at 37 °C and 6% CO2. Leishmania donovani promastigotes
were centrifuged at 900 g for 10 min and the supernatant replaced by
the same volume of Schneider 20% FCS pH 5.4 and incubated for 24
h at 27 °C. J774A.1 cells were then infected by acidified
promastigotes at an average density of 5x105 cells/mL (10:1 ratio)
and chamber-slides incubated for 24 h at 37 °C. Then, in duplicate,
the medium containing various concentrations of tested-compounds
was added (final DMSO concentration being inferior to 0.5% v/v).
Appropriate controls treated with or without solvent (DMSO), and
various concentrations of pentamidine, miltefosine and amphotericin
B were added to each set of experiments. After 120 h incubation at 37
°C and 6% CO2, well supernatant was removed. Cells were fixed
with analytical grade methanol and stained with 10% Giemsa. The
percentage of infected macrophages in each assay was determined
microscopically by counting at least 200 cells in each sample (31% ±
0.5%). IC50 was defined as the concentration of drug necessary to
produce a 50% decrease of infected macrophages compared to the
control. IC50 were calculated by non-linear regression analysis
4.2. Chemistry

4.2.1. General
4.2.1.3. General procedure for the preparation of sulfone
derivatives 4, 6, 8, 9, 10, 12-14, 16, 17, 18, 22, 25, 26, 31-37
from 2-chloromethylated imidazo[1,2-a]pyridines
Melting points were determined in open capillary tubes with a
Büchi apparatus and are uncorrected. H and 13C NMR spectra
1
were determined on a Bruker Avance 200 MHz or 400 MHz A aqueous solution (15 mL) containing 4 mmol of corresponding
instrument, at the Faculté de Pharmacie de Marseille and Faculté sulfonyl chloride, 4 mmol of sodium sulfite, 4 mmol of sodium
des Sciences de St Jérôme. Chemical shifts are given in δ values
referenced to the solvent. High resolution mass spectra were
recorded on a QStar Elite at the Spectropôle department of the
Faculté des Sciences de St Jérôme. Elemental analyses were
hydrogenocarbonate
and
2
mmol
corresponding
2-
chloromethylated imidazo[1,2-a]pyridines was heated under
microwave irradiation (120 °C, 5 bars) in a sealed vial. After
disappearance of the starting materials (about 1 hour), monitored
carried out with a Thermo Finnigan EA 1112 apparatus at the by TLC, the solid was collected by filtration and dried under
Spectropole department of the Faculté des Sciences de St Jérôme.
Silica Gel 60 (Merck 70-230) was used for column
chromatography. The progress of the reactions was monitored by
thin layer chromatography using Kieselgel 60 F254 (Merck)
plates.
reduced pressure to give, after purification by column
chromatography on silica gel and/or recrystallization, the
expected product in 25 to 100% yield.
4.2.1.4. 2-Phenylsulfonylmethylimidazo[1,2-a]pyridine 4
Imidazo[1,2-a]pyridine was purchased from Sigma Aldrich.
Compound 4 was obtained, after recrystallization from ethanol, as
a white solid in 60% yield (0.33 g), mp 170-171 °C (Ethanol). 1H
Compounds 129, 230, 332, 533, 1533, 1633, 1933, 2033, 2148, 2333, 2433, NMR (200 MHz, CDCl3) δ (ppm): 4.59 (2H, s), 6.75-6.82 (1H,
2733, 2833, 2948, 3049, 3833, 3934, 4034, 4134, 4334, 4440 were m), 7.11-7.19 (1H, m), 7.42-7.50 (3H, m), 7.56-7.64 (1H, m),
prepared as previously describe and are presented with a * in the 7.70-7.80 (3H, m), 8.06-8.09 (1H, m). 13C NMR (50 MHz,
result and discussion part.
CDCl3) δ (ppm): 57.0 (CH2), 112.8 (CH), 112.9 (C), 117.6 (CH),
125.0 (CH), 125.7 (CH), 128.3 (CH*2), 129.0 (CH*2), 133.7
(CH), 133.8 (C), 138.7 (CH), 144.9 (C). Anal. Calcd. For
C14H12N2O2S: C, 61.75; H, 4.44; N, 10.29%. Found: C, 61.82; H,
4.2.1.1. 2-Chloromethyl-6-iodoimidazo[1,2-a]pyridine 7
To a solution of 2.95 mmol of 1,3-dichloroacetone in absolute 4.49; N, 10.12%.
ethanol (120 mL) were added 2.27 mmol of 2-amino-5-
iodopyridine. The mixture was stirred and heated under reflux for 4.2.1.5. 3-Nitro-2-phenylsulfonylmethylimidazo[1,2-a]pyridine
4 h. The solvent was evaporated in vacuo. The residue was
poured into water and basified with a saturated aqueous solution
of Na2CO3. The solution was extracted three times with
6
Compound
6
was obtained, after recrystallization from
chloroform. The organic layer was dried over anhydrous Na2SO4 isopropanol, as a beige solid in 60% yield (0.38 g), mp 208-209
and concentrated in vacuo. The crude residue was purified by °C (Isopropanol). 1H NMR (200 MHz, CDCl3) δ (ppm): 5.16 (2H,
chromatography on a silica gel, eluting with dichloromethane- s), 7.27-7.35 (1H, m), 7.49-7.56 (2H, m), 7.63-7.71 (2H, m), 7.83-
ethyl acetate (8-2) and recrystallized from isopropanol to give, the
7.90 (3H, m), 9.36-9.40 (1H, m). 13C NMR (50 MHz, CDCl3) δ
expected product as a white solid in 70% yield (0.46 g).
White solid, mp 150-151 °C (Isopropanol). H NMR (200 MHz, (CH*2), 129.2 (CH*2), 131.1 (CH), 134.2 (CH), 138.7 (C), 139.1
(ppm): 56.8 (CH2), 117.2 (CH), 118.7 (CH), 127.7 (CH), 128.3
1
CDCl3) δ (ppm): 4.74 (2H, s), 7.35-7.36 (2H, m), 7.56 (1H, s),
(C), 139.5 (C), 145.0 (C). Anal. Calcd. For C14H11N3O4S: C,
8.35-8.36 (1H, m). 13C NMR (50 MHz, CDCl3) δ (ppm): 39.3 52.99; H, 3.49; N, 13.24%. Found: C, 52.92; H, 3.47; N, 13.14%.
(CH2), 75.4 (C), 110.5 (CH), 118.8 (CH), 130.6 (CH), 133.1
(CH), 143.7 (C), 143.9 (C). Anal. Calcd. For C8H6ClIN2: C, 4.2.1.6. 6-Iodo-2-phenylsulfonylmethylimidazo[1,2-a]pyridine
32.85; H, 2.07; N, 9.58%. Found: C, 32.88; H, 2.07; N, 9.63%.
8
4.2.1.2. 2-Chloromethyl-6-iodo-3-nitroimidazo[1,2-a]pyridine Compound
8
was obtained, after recrystallization from
11
isopropanol, as a beige solid in 74% yield (0.59 g), mp 242-243
°C (Isopropanol). H NMR (200 MHz, DMSO-d6) δ (ppm): 4.81
1
To a solution of 1.71 mmol of 2-chloromethyl-6-iodoimidazo[1,2- (2H, s), 7.30 (1H, d, J = 9.4 Hz), 7.39 (1H, dd, J = 1.5 and 9.4
a]pyridine in concentrated sulfuric acid (30 mL) cooled by an ice- Hz), 7.54-7.78 (6H, m), 8.94-8.95 (1H, m). 13C NMR (50 MHz,
water bath, nitric acid 65% (5.1 mmol.) was added. The reaction DMSO-d6) δ (ppm): 55.7 (CH2), 76.4 (C), 113.5 (CH), 118.0
mixture was stirred for 3 h at room temperature. The reaction
(CH), 128.1 (CH*2), 129.3 (CH*2), 131.6 (CH), 132.5 (CH),
mixture was then slowly poured into a water-ice-mixture making 133.9 (CH), 134.5 (C), 139.0 (C), 142.7 (C). Anal. Calcd. For
the desired product precipitate. The yellow solid was collected by C14H11IN2O2S: C, 42.23; H, 2.78; N, 7.03%. Found: C, 42.23; H,
filtration, dried under reduced pressure and recrystallized from
isopropanol to give, the expected product in 80% yield (0.46 g).
Yellow solid, mp 211-212 °C (Isopropanol). H NMR (200 MHz,
2.79; N, 6.90%.
1
4.2.1.7. 6-Iodo-2-tosylmethylimidazo[1,2-a]pyridine 9
CDCl3) δ (ppm): 5.10 (2H, s), 7.62 (1H, dd, J = 0.8 and 9.3 Hz),
7.87 (1H, dd, J = 1.5 and 9.3 Hz), 9.69 (1H, dd, J = 0.8 and 1.5
Hz). 13C NMR (50 MHz, CDCl3) δ (ppm): 38.6 (CH2), 80.8 (C),
119.4 (CH), 132.3 (CH), 138.7 (C), 139.2 (CH), 143.4 (C), 147.4
(C). Anal. Calcd. For C8H5ClIN3O2: C, 28.47; H, 1.49; N,
12.45%. Found: C, 28.53; H, 1.47; N, 12.60%.
Compound
9
was obtained, after recrystallization from
isopropanol, as a beige solid in 80% yield (0.66 g), mp 241-242
°C (Isopropanol). H NMR (200 MHz, DMSO-d6) δ (ppm): 2.38
1
(3H, s), 4.75 (2H, s), 7.29-7.42 (4H, m), 7.64 (2H, d, J = 8.3 Hz),
7.78 (1H, s), 8.94-8.95 (1H, m). 13C NMR (50 MHz, DMSO-d6) δ
(ppm): 21.2 (CH3), 55.8 (CH2), 76.4 (C), 113.4 (CH), 118.0 (CH),
128.1 (CH*2), 129.7 (CH*2), 131.6 (CH), 132.5 (CH), 134.7 (C),
136.2 (C), 142.7 (C), 144.4 (C). Anal. Calcd. For C15H13IN2O2S:
C, 43.70; H, 3.18; N, 6.80. Found: C, 43.84; H, 3.20; N, 6.80%.

4.2.1. 8. 2-[(4-Chlorophenylsulfonyl)methyl]-6-iodoimidazo-
[1,2-a]pyridine 10
(C), 142.6 (C), 144.4 (C). Anal. Calcd. For C15H13ClN2O2S: C,
56.16; H, 4.08; N, 8.73%. Found: C, 55.91; H, 4.07; N, 8.79%.
Compound 10 was obtained, after recrystallization from 4.2.1.13. 6-Chloro-2-[(4-chlorophenylsulfonyl)methyl]-
isopropanol, as a grey solid in 53% yield (0.46 g), mp 179-180 °C imidazo[1,2-a]pyridine 18
(Isopropanol). 1H NMR (200 MHz, DMSO-d6) δ (ppm): 4.86 (2H,
s), 7.30 (1H, d, J = 9.5 Hz), 7.40 (1H, dd, J = 1.6 and 9.5 Hz), Compound 18 was obtained, after recrystallization from
7.63-7.79 (5H, m), 8.94-8.95 (1H, m). 13C NMR (50 MHz, isopropanol, as a pale grey solid in 88% yield (0.60 g), mp 185-
1
DMSO-d6) δ (ppm): 55.6 (CH2), 76.4 (C), 113.6 (CH), 118.0 186 °C (Isopropanol). H NMR (200 MHz, DMSO-d6) δ (ppm):
(CH), 129.4 (CH*2), 130.2 (CH*2), 131.7 (CH), 132.6 (CH),
134.4 (C), 137.8 (C), 139.0 (C), 142.8 (C). Anal. Calcd. For
C14H10ClIN2O2S: C, 38.86; H, 2.33; N, 6.47%. Found: C, 38.90;
H, 2.43; N, 6.79%.
4.87 (2H, s), 7.27 (1H, dd, J = 1.9 and 9.6 Hz), 7.52 (1H, d, J =
9.6 Hz), 7.64-7.78 (4H, m), 7.85 (1H, s), 8.92 (1H, dd, J = 0.5 and
1.9 Hz). 13C NMR (50 MHz, DMSO-d6) δ (ppm): 55.6 (CH2),
114.4 (CH), 117.6 (CH), 119.3 (CH), 125.0 (CH), 126.0 (CH),
129.4 (CH*2), 130.2 (CH*2), 135.1 (C), 137.8 (C), 139.0 (C),
4.2.1.9. 6-Iodo-3-nitro-2-phenylsulfonylmethylimidazo[1,2-a]- 142.7 (C). Anal. Calcd. For C14H10Cl2N2O2S: C, 49.28; H, 2.95;
pyridine 12 N, 8.21%. Found: C, 49.33; H, 2.92; N, 8.21%.
Compound 12 was obtained, after recrystallization from 4.2.1.14. 6-Chloro-2-[(4-chlorophenylsulfonyl)methyl]-3-nitro-
isopropanol, as a beige solid in 90% yield (0.79 g), mp 237-238 imidazo[1,2-a]pyridine 22
1
°C (Isopropanol). H NMR (200 MHz, DMSO-d6) δ (ppm): 4.85
(2H, s), 7.31 (1H, d, J = 9.4 Hz), 7.40 (1H, dd, J = 1.6 and 9.4 Compound 22 was obtained, after recrystallization from benzene,
Hz), 7.63-7.79 (5H, m), 8.95 (1H, dd, J = 0.9 and 1.6 Hz). 13C as a white solid in 76% yield (0.58 g), mp 199-200 °C (benzene).
NMR (50 MHz, DMSO-d6) δ (ppm): 55.6 (CH2), 76.5 (C), 113.6 1H NMR (200 MHz, CDCl3) δ (ppm): 5.12 (2H, s), 7.49-7.82 (6H,
(CH), 118.1 (CH), 129.4 (CH*2), 130.2 (CH*2), 131.7 (CH), m), 9.44-9.45 (1H, m). 13C NMR (50 MHz, CDCl3) δ (ppm): 56.7
132.6 (CH), 134.4 (C), 137.8 (C), 139.0 (C), 142.8 (C). Anal. (CH2), 118.9 (CH), 125.7 (CH), 126.0 (C), 129.6 (CH*2), 129.8
Calcd. For C14H10IN3O4S: C, 37.94; H, 2.27; N, 9.48%. Found: C,
37.99; H, 2.21; N, 9.46%.
(CH*2), 132.5 (CH), 137.6 (C), 139.0 (C), 139.4 (C), 141.1 (C),
143.1 (C). Anal. Calcd. For C14H9Cl2N3O4S: C, 43.54; H, 2.35; N,
10.88%. Found: C, 43.62; H, 2.34; N, 10.90%.
4.2.1.10. 6-Iodo-3-nitro-2-tosylmethylimidazo[1,2-a]pyridine
13
4.2.1.15. 6-Bromo-2-(tosylmethyl)imidazo[1,2-a]pyridine 25
Compound 13 was obtained, after recrystallization from Compound 25 was obtained, after recrystallization from
isopropanol, as a yellow solid in 100% yield (0.91 g), mp 210-211 isopropanol, as a grey solid in 94% yield (0.68 g), mp 197-198 °C
1
°C (Isopropanol). H NMR (200 MHz, DMSO-d6) δ (ppm): 2.40 (Isopropanol). 1H NMR (200 MHz, DMSO-d6) δ (ppm): 2.38 (3H,
(3H, s), 5.17 (2H, s), 7.39 (2H, d, J = 8.0 Hz), 7.62 (2H, d, J = 8.0 s), 4.77 (2H, s), 7.32 (1H, dd, J = 1.8 and 9.6 Hz), 7.35-7.67 (4H,
Hz), 7.75 (1H, dd, J = 0.8 and 9.2 Hz), 8.05 (1H, dd, J = 1.6 and m), 7.45 (1H, d, J = 9.6 Hz), 7.83 (1H, s), 8.92 (1H, dd, J = 0.7
9.2 Hz), 9.44 (1H, dd, J = 0.8 and 1.6 Hz). 13C NMR (50 MHz, and 1.8 Hz). 13C NMR (50 MHz, DMSO-d6) δ (ppm): 21.2 (CH3),
DMSO-d6) δ (ppm): 21.3 (CH3), 56.2 (CH2), 83.0 (C), 119.1 55.8 (CH2), 106.3 (C), 114.0 (CH), 117.8 (CH), 127.0 (CH),
(CH), 128.2 (CH*2), 129.9 (CH*2), 132.3 (CH), 136.2 (C), 139.0 127.9 (CH), 128.1 (CH*2), 129.7 (CH*2), 135.2 (C), 136.2 (C),
(C), 139.3 (C), 139.6 (CH), 143.2 (C), 144.9 (C). Anal. Calcd. For
C15H12IN3O4S: C, 39.40; H, 2.65; N, 9.19%. Found: C, 39.55; H,
2.66; N, 9.21%.
142.6 (C), 144.4 (C). Anal. Calcd. For C15H13BrN2O2S: C, 49.33;
H, 3.59; N, 7.67%. Found: C, 49.33; H, 3.65; N, 7.96%.
4.2.1.16. 6-Bromo-2-(4-chlorophenylsulfonylmethyl)imidazo-
4.2.1.11. 2-[(4-Chlorophenylsulfonyl)methyl]-6-iodo-3-nitro-
[1,2-a]pyridine 26
imidazo[1,2-a]pyridine 14
Compound 26 was obtained, after recrystallization from
Compound 14 was obtained, after recrystallization from isopropanol, as a grey solid in 89% yield (0,68 g), mp 187-188 °C
isopropanol, as a yellow solid in 90% yield (0.86 g), mp 208-209 (Isopropanol). 1H NMR (200 MHz, DMSO-d6) δ (ppm): 4.87 (2H,
1
°C (Isopropanol). H NMR (200 MHz, DMSO-d6) δ (ppm): 5.25 s), 7.33 (1H, dd, J = 1.9 and 9.6 Hz), 7.45 (1H, d, J = 9.6 Hz),
(2H, s), 7.64-7.77 (5H, m), 8.05 (1H, dd, J = 1.6 and 9.3 Hz),
9.44-9.45 (1H, m). 13C NMR (50 MHz, DMSO-d6) δ (ppm): 56.1 13C NMR (50 MHz, DMSO-d6) δ (ppm): 55.6 (CH2), 106.3 (C),
(CH2), 83.1 (C), 119.1 (CH), 129.6 (CH*2), 130.3 (CH*2), 132.4 114.2 (CH), 117.9 (CH), 127.1 (CH), 128.1 (CH), 129.4 (CH*2),
7.64-7.78 (4H, m), 7.83 (1H, s), 8.92 (1H, dd, J = 0.7 and 1.9 Hz).
(CH), 137.7 (C), 138.7 (C), 139.0 (C), 139.5 (C), 139.7 (CH), 130.2 (CH*2), 134.9 (C), 137.8 (C), 139.0 (C), 142.7 (C). Anal.
143.2 (C). Anal. Calcd. For C14H9ClIN3O4S: C, 35.20; H, 1.90; N, Calcd. For C14H10BrClN2O2S: C, 43.60; H, 2.61; N, 7.26%.
8.80%. Found: C, 35.27; H, 1.97; N, 9.20%.
Found: C, 43.62; H, 2.62; N, 7.55%.
4.2.1.12. 6-Chloro-2-(tosylmethyl)imidazo[1,2-a]pyridine 17
Compound 17 was obtained, after recrystallization from
4.2.1.17. 6-Bromo-2-[(4-bromophenylsulfonyl)methyl]-3-nitro-
imidazo[1,2-a]pyridine 31
isopropanol, as a pale blue solid in 88% yield (0.56 g), mp 179- Compound 31 was obtained, after purification by column
1
180 °C (Isopropanol). H NMR (200 MHz, DMSO-d6) δ (ppm): chromatography on silica gel (eluent: CH2Cl2-ACOEt 8/2) and
2.38 (3H, s), 4.78 (2H, s), 7.26 (1H, dd, J = 1.8 and 9.6 Hz), 7.36- recrystallization from isopropanol, as a beige solid in 80% yield
7.67 (4H, m), 7.51 (1H, d, J = 9.6 Hz), 7.84 (1H, s), 8.84 (1H, dd,
(0.76 g), mp 228-229 °C (Isopropanol). 1H NMR (200 MHz,
J = 0.7 and 1.8 Hz). 13C NMR (50 MHz, DMSO-d6) δ (ppm): 21.2 CDCl3) δ (ppm): 5.12 (2H, s), 7.65-7.79 (6H, m), 9.54-9.55 (1H,
(CH3), 55.8 (CH2), 114.2 (CH), 117.6 (CH), 119.3 (C), 124.9 m). 13C NMR (50 MHz, CDCl3) δ (ppm): 56.6 (CH2), 112.6 (C),
(CH), 125.9 (CH), 128.1 (CH*2), 129.7 (CH*2), 135.4 (C), 136.2
119.1 (CH), 127.7 (CH), 129.8 (C), 129.9 (CH*2), 132.6 (CH*2),

134.7 (CH), 138.1 (C), 138.7 (C), 139.2 (C), 143.3 (C). Anal. Compound 36 was obtained, after purification by column
Calcd. For C14H9Br2N3O4S: C, 35.39; H, 1.91; N, 8.84%. Found:
C, 35.28; H, 1.87; N, 8.95%.
chromatography on silica gel (eluent: CH2Cl2-ACOEt 9/1) and
recrystallization from isopropanol, as a beige solid in 25% yield
(0.17 g), mp 233-234 °C (Isopropanol). 1H NMR (200 MHz,
CDCl3) δ (ppm): 3.12 (3H, s), 5.03-5.04 (2H, m), 7.74-7.76 (2H,
m), 9.59-9.60 (1H, m). 13C NMR (50 MHz, CDCl3) δ (ppm): 41.3
(CH3), 54.5 (CH2), 111.6 (C), 119.1 (CH), 128.1 (CH), 130.9 (C),
135.0 (CH), 140.4 (C), 143.2 (C). Anal. Calcd. For
4.2.1.18. 6-Bromo-2-[(4-iodophenylsulfonyl)methyl]-3-nitro-
imidazo[1,2-a]pyridine 32
Compound 32 was obtained, after purification by column C9H8BrN3O4S: C, 32.25; H, 2.41; N, 12.58%. Found: C, 32.64; H,
chromatography on silica gel (eluent: CH2Cl2) and 2.43; N, 12.54%.
recrystallization from toluene, as a white solid in 30% yield (0.31
1
g), mp 260-261 °C (Toluene). H NMR (200 MHz, CDCl3) δ 4.2.1.23. 2-(Biphenyl-4-ylsulfonyl)methyl-6-bromo-3-nitro-
(ppm): 5.12 (2H, s), 7.55-7.60 (2H, m), 7.73-7.75 (2H, m), 7.88- imidazo[1,2-a]pyridine 37
7.93 (2H, m), 9.55-9.56 (1H, m). 13C NMR (50 MHz, CDCl3) δ
(ppm): 56.1 (CH2), 103.4 (C), 111.7 (C), 119.1 (CH), 128.0 (CH),
129.9 (CH*2), 130.7 (C), 135.0 (CH), 138.4 (CH*2), 138.5 (C),
Compound 37 was obtained, after purification by column
chromatography on silica gel (eluent: CH2Cl2-ACOEt 9/1) and
139.4 (C), 143.1 (C). Anal. Calcd. For C14H9BrIN3O4S: C, 33.21; recrystallization from toluene, as a yellow solid in 40% yield
1
H, 1.74; N, 8.05%. Found: C, 32.56; H, 1.72; N, 8.13%.
(0.38 g), mp 199-200 °C (Toluene). H NMR (200 MHz, CDCl3)
δ (ppm): 5.17 (2H, s), 7.43-7.53 (3H, m), 7.59-7.63 (2H, m), 7.74-
7.76 (4H, m), 7.91-7.95 (2H, m) 9.54-9.55 (1H, m). 13C NMR (50
MHz, CDCl3) δ (ppm): 56.8 (CH2), 112.5 (C), 119.1 (C), 127.4
(CH*2), 127.7 (CH), 127.9 (CH*2), 128.8 (CH), 128.9 (CH*2),
4.2.1.19. 6-Bromo-2-[(4-fluorophenylsulfonyl)methyl]-3-nitro-
imidazo[1,2-a]pyridine 33
Compound 33 was obtained, after purification by column 129.1 (CH*2), 134.6 (CH), 137.5 (CH), 138.7 (C), 139.0 (C),
chromatography on silica gel (eluent: CH2Cl2-ACOEt 9/1) and 139.5 (C), 143.3 (C), 147.2 (C). Anal. Calcd. For C20H14BrN3O4S:
C, 50.86; H, 2.99; N, 8.90%. Found: C, 51.14; H, 3.03; N, 8.77%.
recrystallization from toluene, as a beige solid in 44% yield (0.36
g), mp 243-244 °C (Toluene). H NMR (200 MHz, DMSO-d6) δ
1
(ppm): 5.22 (2H, s), 7.37-7.45 (2H, m), 7.76-8.01 (4H, m), 9.36- 4.2.1.24. 3-Nitro-2-(phenylsulfonylmethyl)-6-(3,4,5-tri-
9.37 (1H, m). 13C NMR (50 MHz, DMSO-d6) δ (ppm): 56.2 methoxyphenyl)imidazo[1,2-a]pyridine 42
(CH2), 111.7 (C), 116.8 (CH*2, d, J = 23 Hz), 119.1 (CH), 128.1
(CH), 131.7 (CH*2, d, J = 10 Hz), 133.0 (C, d, J = 225 Hz), 135.0
A
solution
of
1.26
mmol
of
6-bromo-3-nitro-2-
(CH), 135.1 (C, d, J = 9 Hz), 139.0 (C), 139.5 (C), 143.1 (C). phenylsulfonylmethylimidazo[1,2-a]pyridine, 1.63 mmol of
Anal. Calcd. For C14H9BrFN3O4S: C, 40.60; H, 2.19; N, 10.14%.
Found: C, 40.47; H, 2.25; N, 10.18%.
3,4,5-trimethoxyphenylboronic acid, 6.3 mmol of Na2CO3, 0.12
mmol of Pd(PPh3)4, 1.26 mmol of TBAB and water (15 mL) were
heated under microwave irradiation (105 °C, 300 W) for 1 h.
After disappearance of the starting materials (monitored by TLC),
the solid was collected by filtration and dried under reduced
pressure to give, after purification by column chromatography on
silica gel (eluent: CH2Cl2-ethyl acetate 8/2) and recrystallization
4.2.1.20. 6-Bromo-3-nitro-2-(4-trifluoromethyl)phenyl-
sulfonylmethylimidazo[1,2-a]pyridine 34
Compound 34 was obtained, after purification by column
from isopropanol, the expected product in 60% yield (0.37 g).
recrystallization from toluene, as a pale yellow solid in 60% yield Beige solid, mp 216-217 °C (Isopropanol). H NMR (200 MHz,
chromatography on silica gel (eluent: CH2Cl2-ACOEt 9/1) and
1
1
(0.55 g), mp 239-240 °C (Toluene). H NMR (200 MHz, CDCl3) CDCl3) δ (ppm): 3.92 (3H, s), 3.95 (6H, s), 5.17 (2H, s), 6.77 (2H,
δ (ppm): 5.16 (2H, s), 7.68-7.85 (4H, m), 8.01-8.06 (2H, m), 9.56- s), 7.45-7.71 (5H, m), 7.88-7.92 (2H, m) 9.51-9.52 (1H, m). 13C
9.57 (1H, m). 13C NMR (50 MHz, CDCl3) δ (ppm): 56.6 (CH2), NMR (50 MHz, CDCl3) δ (ppm): 56.4 (CH3*2), 56.9 (CH2), 61.0
112.7 (C), 119.1 (CH), 123.0 (C, q, J = 272.0 Hz), 126.4 (CH*2,
q, J = 3.7 Hz), 127.7 (CH), 129.1 (CH*2), 134.8 (CH), 135.9 (C,
q, J = 33.3 Hz), 138.7 (C), 138.9 (C), 142.7 (C), 143.3 (C). Anal.
(CH3), 104.9 (CH), 118.3 (CH), 124.8 (CH), 128.3 (CH*2), 129.2
(CH*2), 131.4 (C), 131.6 (CH), 131.9 (CH), 132.1 (C), 134.1
(CH), 138.7 (C), 139.2 (C), 139.3 (C), 139.7 (C), 144.1 (C), 154.0
Calcd. For C15H9BrF3N3O4S: C, 38.81; H, 1.95; N, 9.05%. Found: (C*2). Anal. Calcd. For C23H21N3O7S: C, 57.14; H, 4.38; N,
C, 38.78; H, 1.83; N, 9.37%.
8.69%. Found: C, 57.08; H, 4.39; N, 8.43%.
4.2.1.21. 6-Bromo-2-[(4-methoxyphenylsulfonyl)methyl]-3-
nitroimidazo[1,2-a]pyridine 35
Acknowledgments
Compound 35 was obtained, after purification by column This work was supported by the Centre National de la Recherche
chromatography on silica gel (eluent: CH2Cl2-ACOEt 9/1) and Scientifique (CNRS) and Aix-Marseille Université. L. Paloque
recrystallization from toluene, as a pale yellow solid in 46% yield thesis was financed by a Region PACA/Yelen BDE grant. The
1
(0.39 g), mp 228-229 °C (Toluene). H NMR (200 MHz, DMSO- authors also thank the French Leishmaniasis Reference Center of
d6) δ (ppm): 3.83 (3H, s), 5.13 (2H, s), 7.04-7.09 (2H, m), 7.61- Montpellier for providing the Leishmania strains and M. Piednoël
7.66 (2H, m), 7.85-7.99 (2H, m), 9.36-9.37 (1H, m). 13C NMR
for her technical assistance.
(50 MHz, DMSO-d6) δ (ppm): 56.0 (CH3), 56.5 (CH2), 111.6 (C),
114.7 (CH*2), 119.1 (CH), 128.0 (CH), 130.5 (CH*2), 130.7 (C),
134.9 (CH), 139.0 (C), 139.8 (C), 143.0 (C), 163.7 (C). Anal.
Calcd. For C15H12BrN3O5S: C, 42.27; H, 2.84; N, 9.86%. Found:
C, 42.53; H, 2.84; N, 9.81%.
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