Synthesis, antileishmanial activity and cytotoxicity of 2,3-diaryl- and 2,3,8-trisubstituted imidazo[1,2-a]pyrazines

Article abstract of DOI:10.1016/j.ejmech.2015.09.002

A series of original 2-phenyl-3-(pyridin-4-yl)imidazo[1,2-a]pyrazines and the 3-iodo precursors, bearing a polar moiety at the C-8 position, was synthesized and evaluated for their antileishmanial activities. Two derivatives exhibited very good activity a

Full text of DOI:10.1016/j.ejmech.2015.09.002

European Journal of Medicinal Chemistry 103 (2015) 381e395
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis, antileishmanial activity and cytotoxicity of 2,3-diaryl- and
2,3,8-trisubstituted imidazo[1,2-a]pyrazines
,
*
a
b
a
Pascal Marchand a , Marc-Antoine Bazin , Fabrice Pagniez , Guillaume Riviere ,
ꢀ
a, b
a
a
a
ꢁ
Lizeth Bodero , Sophie Marhadour , Marie-Renee Nourrisson , Carine Picot
,
c
c
c
d, e
ꢁ
Sandrine Ruchaud , Stephane Bach , Blandine Baratte , Michel Sauvain
,
Denis Castillo Pareja f, Abraham J. Vaisberg f, Patrice Le Pape b
a
ꢁ
ꢁ
ꢁ
ꢁ
Universite de Nantes, Nantes Atlantique Universites, Laboratoire de Chimie Therapeutique, Cibles et Medicaments des Infections et du Cancer, IICiMed
UPRES EA 1155, UFR de Sciences Pharmaceutiques et Biologiques, 1 rue Gaston Veil, 44035 Nantes, France
b
ꢁ
ꢁ
ꢁ
ꢁ
Universite de Nantes, Nantes Atlantique Universites, Laboratoire de Parasitologie et Mycologie Medicale, Cibles et Medicaments des Infections et du
Cancer, IICiMed UPRES EA 1155, UFR de Sciences Pharmaceutiques et Biologiques, 1 rue Gaston Veil, 44035 Nantes, France
c
ꢁ
Sorbonne Universites, UPMC Univ Paris 06, CNRS USR3151, “Protein Phosphorylation & Human Diseases” Group, Station Biologique, Place Georges Teissier,
29688 Roscoff, France
d
ꢁ
Institut de Recherche pour le Developpement (IRD), UMR152, Mission IRD Casilla, 18-1209 Lima, Peru
e
ꢁ
ꢁ
Universite de Toulouse, UPS, UMR152 (Laboratoire de Pharmacochimie et Pharmacologie pour le Developpement, Pharma-DEV), F-31062 Toulouse Cedex
9, France
f
ꢁ
Departamento de Microbiología y Laboratorios de Investigacion y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia,
Lima, Peru
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of original 2-phenyl-3-(pyridin-4-yl)imidazo[1,2-a]pyrazines and the 3-iodo precursors, bearing
a polar moiety at the C-8 position, was synthesized and evaluated for their antileishmanial activities. Two
derivatives exhibited very good activity against the promastigote and the amastigote forms of Leishmania
major in the micromolar to submicromolar ranges, coupled with a low cytotoxicity against macrophages
and 3T3 mouse fibroblast cells. Through LmCK1 inhibition assay, investigations of the putative molecular
target of these promising antileishmanial compounds will be discussed.
Received 22 July 2015
Received in revised form
1 September 2015
Accepted 3 September 2015
Available online 9 September 2015
© 2015 Elsevier Masson SAS. All rights reserved.
Keywords:
Antileishmanial activity
Imidazo[1,2-a]pyrazines
Direct arylation
Casein kinase 1
1. Introduction
Leishmania infantum is the most severe form of leishmaniasis and is
usually fatal in the absence of treatment whereas CL caused by
According to a recent report from the World Health Organiza-
tion (WHO) [1], leishmaniases - visceral leishmaniasis (VL), cuta-
neous leishmaniasis (CL), and mucocutaneous leishmaniasis (MCL)
- collectively affect 12 million people in 98 countries, and 350
million more are at risk of infection. Moreover, there are 1.3 million
new cases and 40,000 deaths attributed to leishmaniases each year
and leishmaniasis is included in the neglected tropical diseases
(NTDs) [2]. Clinical forms differ in immunopathologies and degree
of morbidity and mortality. VL caused by Leishmania donovani and
Leishmania major, Leishmania amazonensis, Leishmania mexicana,
Leishmania braziliensis, and Leishmania panamensis is significantly
associated with morbidity. Cutaneous leishmaniasis is more widely
distributed, with about one-third of cases occurring in each of three
epidemiological regions, the Americas, the Mediterranean basin,
and western Asia from the Middle East to Central Asia. The ten
countries with the highest estimated case counts are: Afghanistan,
Algeria, Brazil, Colombia, Costa Rica, Ethiopia, Iran, Peru, Sudan and
Syria, and together account for 70e75% of global estimated CL
incidence. More than 90% of global VL cases occur in six countries:
Bangladesh, Brazil, Ethiopia, India, South Sudan and Sudan [1e9].
These parasitic infections are caused by a protozoan of the
Leishmania genus transmitted to its mammal hosts (humans, dogs,
* Corresponding author.
E-mail address: pascal.marchand@univ-nantes.fr (P. Marchand).
http://dx.doi.org/10.1016/j.ejmech.2015.09.002
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

382
P. Marchand et al. / European Journal of Medicinal Chemistry 103 (2015) 381e395
monkeys, rodents,…) by the bite of an infected sandfly (Phleboto-
minae). Leishmania parasites present two morphological stages:
extracellular flagellated promastigotes in the digestive tract of their
sandfly vector and non-motile amastigotes inside the cells of their
hosts' mononuclear phagocytic system [1]. Currently, there are no
effective vaccines and a number of drugs are used in the treatment
of leishmaniasis. These drugs include pentavalent antimonials,
amphotericin B, miltefosine, pentamidine and paromomycin. Un-
fortunately, many of these drugs cause side effects and high tox-
icities, and display a high rate of treatment failure in HIV co-
infected patients. In addition, an inevitable resistance has devel-
oped in recent times in Leishmania parasites towards some of these
drugs. Otherwise, they are costly and require long-term treatment
[2e9]. Consequently, there is an urgent need to speed up the
development of a new generation of more effective and safe
antileishmanials.
Various classes of natural products have shown promising
antileishmanial activity [10] and compounds of synthetic origin
comprising a diverse group of chemical structures have been re-
ported as antileishmanial agents [3,5,11]. These include mostly the
nitrogen heterocycles for instance as quinolines [12], quinolinones
[13], quinazolines [14], quinoxalines [15], acridines [16], pyrimi-
dines [17], thienopyrimidines [18] or azoles [9].
pyrazines have been gaining attention in drug discovery but no
biological application related to Leishmania diseases was found in
the literature [38]. Consequently, we planned to explore such
scaffold for the design of new antileishmanial agents associated
with LmCK1 inhibition (Fig. 1, Series A). In addition, to try to
enhance LmCK1 inhibition, our second strategy was to introduce a
variety of polar groups, namely amines [33], at the imidazo[1,2-a]
pyrazine C8 position, by holding constant the 4-pyridyl at the C3
position along with a phenyl or a 4-fluorophenyl substituent at the
C2 position (Fig. 1, Series B).
2. Results and discussion
2.1. Chemistry
For the design of imidazo[1,2-a]pyrazines substituted at 2,3-ring
positions, we were interested in preparing 2-phenylimidazo[1,2-a]
pyrazine 2 as intermediate (Scheme 1).
To this end, a generally established method was the condensa-
tion of a-bromoacetophenone with 2-aminopyrazine 1 [39]. The
low yield observed for the cyclization step (30%) prompted us to
prepare imidazo[1,2-a]pyrazin-2-yl triflate 3, as recently described
by our team in imidazo[1,2-a]pyridine series [21,36], followed by
Suzuki coupling to give the desired product 2 (Scheme 1). Thus, 2-
aminopyrazine 1 reacted with ethyl bromoacetate and a subse-
quent treatment with N-phenylbis(trifluoromethanesulfonimide)
led to 3 in only 33% yield. This could be due to the competitive
reactivity of the N-4 nitrogen of starting 2-aminopyrazine, involved
in the nucleophilic substitution mechanism, thereby preventing
following cyclisation. Suzuki coupling was then performed in the
presence of phenylboronic acid to produce compound 2 in satis-
factory yield (67%). Considering the overall yield of the two steps of
this sequence (22% vs 30%), this second approach was less effective.
Nevertheless, the original introduction of a leaving group (OTf) at
the position 2 of the imidazo[1,2-a]pyrazine core will allow metal-
catalyzed chemistry for a broad pharmacomodulation since the
corresponding halogenated derivatives are almost not described in
the literature. In the aim of the obtention of 2-arylimidazo[1,2-a]
pyrazines, a wide variety of boronic acids are commercially avail-
During the last few years, our strategy to fight against leish-
maniasis was the design of small heterocyclic compounds in azo-
lylindole series [19,20] and, more recently, 2,3-diarylimidazo[1,2-a]
pyridine series [21].
Interestingly,
2,3-diarylimidazo[1,2-a]pyridine
derivatives
exhibited good activity against the promastigote form and/or the
amastigote form of L. major, coupled with a low cytotoxic activity
against the HeLa human cell line. We studied LmCK1 as a potential
molecular target responsible of antiparasitic properties but, in a
general approach, antileishmanial activities of the imidazo[1,2-a]
pyridines were not clearly correlated with LmCK1 inhibition.
Nevertheless, the 4-pyridyl derivative I (Fig. 1) displayed LmCK1
kinase inhibition in the submicromolar range (IC50 value of
0.30 mM) and IC50 value of 6.5 mM on L. major promastigote stage,
constituting a good starting point for the development of new
antileishmanial agents [21].
Indeed, among the different targets such as sterol biosynthetic
pathway, trypanothione or methylglyoxal metabolism, parasite
glycolysis, purine salvage pathway, folate biosynthesis or top-
oisomerases [4e6,10,22], the involvement of several parasitic pro-
tein kinases was found essential for parasite proliferation and
viability [23,24]. These enzymes were validated as potential mo-
lecular targets for drug development, namely cyclin-dependent
kinases (CDKs) [25e27], mitogen-activated protein kinases
(MAPKs) [28e30], Aurora kinase [31], Protein kinase C (PKC) [32] or,
in our field of interest, casein kinase 1 (CK1) [33e35].
able for Suzuki coupling in the contrary of
for heterocyclization reaction.
a-bromoacetophenones
The direct arylation reaction at the 3-position was then exam-
ined using 2-phenylimidazo[1,2-a]pyrazine 2 and aryl halides,
applying Fagnou's procedure [40,41]. In the presence of Pd(OAc)2
(2 mol %), PCy3$HBF4 (4 mol %), PivOH (0.3 equiv), K2CO3 (1.5 equiv),
the reaction proceeded smoothly (18e40 h) in DMA at 100 ꢀC to
afford 3-aryl-2-phenylimidazo[1,2-a]pyrazines 4aeg in low yields
due to degradation of the reaction mixture (Scheme 1, Table 1).
In the second series of compounds, the purpose of the synthetic
scheme was to introduce a chlorine atom at the position 8 of the
imidazo[1,2-a]pyrazine scaffold for further pharmacomodulation.
Thus, 2,3-dichloropyrazine 5 was the starting material subjected to
amination and cyclization (Scheme 2, Table 2) [42,43].
In the course of our ongoing synthetic and screening programs
for new biologically active imidazo[1,2-a]azines [21,36,37], we
decided to develop bioisostere analogues of the previously
described 2,3-diarylimidazo[1,2-a]pyridines [21]. Imidazo[1,2-a]
N
N
N
(Het)Ar
= 6.5 µM; LmCK1 IC = 0.30 µM
SERIES A
SERIES B
Pentamidine: L. major promastigotes IC = 4.8 µM (reference)
Fig. 1. Structures of target compounds.

P. Marchand et al. / European Journal of Medicinal Chemistry 103 (2015) 381e395
383
N
NH2
N
N
(a)
N
(d)
N
N
N
N
(Het)Ar
2
1
4a-g
N
(b)
N
(c)
OTf
N
3
Scheme 1. Reagents and conditions: (a) BrCH2Ph, NaHCO3, EtOH, 17 h, reflux, 30%; (b) BrCH2CO2Et, iPrOH, 90 ꢀC, 6 h then PhNTf2, Et3N, toluene, reflux, 6 h, 33%; (c) PhB(OH)2,
Pd(PPh3)4, Na2CO3, EtOH, sealed tube, MW 100 ꢀC, 10 min, 67%; (d) (Het)ArX (X ¼ Br, I), Pd(OAc)2, PCy3$HBF4, PivOH, K2CO3, DMA, sealed tube, 100 ꢀC.
Table 1
Selective arylation of the 3,8-dihalogenated precursor 8a was
achieved by the displacement of the iodine atom to furnish 2,3-
Structures of 3-aryl-2-phenylimidazo[1,2-a]pyrazines 4aeg.
Compound
(Het)Ar
Time
Yield (%)a
diaryl derivative 11 in 51% yield. Only traces (<5%) of the 2,3,8-
triaryl counterpart were detected by UPLC-MS analysis. To avoid
the iodination step, direct arylation procedure was applied to
starting compound 7a but this alternative strategy provided com-
pound 11 in low yield (27%) in comparison to the first route (43% for
2 steps). Indeed, the mobility of chlorine atom at the 8-position in
metal-catalyzed reaction conditions gave dechlorinated by-
products. Finally, compound 11 reacted with 4-(morpholin-4-yl)
ethanamine to synthesize product 10c in 49% yield. From com-
pound 7a, the overall yield for the obtention of 10c was divided into
two using the second route (21% vs 43%) but, in a general approach,
this pathway offers the possibility of a wide diversity for further
structureeactivity relationship study at the 8-position considering
the more convergent synthetic scheme.
4a
4b
4c
4d
4e
4f
C6H5
40 h
18 h
22 h
21 h
28 h
28 h
30 h
15
20
20
18
13
20
22
4-MeOC6H4
4-(NO2)C6H4
4-(CO2Et)C6H4
3-ClC6H4
3,5-(Cl)2C6H3
4-Pyridyl
4g
a
Isolated yields.
By comparison to unsubstituted aminopyrazine 1, the hetero-
cyclisation proceeded in better yield (55e58% vs 30%) in the pres-
ence of 3-chloropyrazin-2-amine
6
and suitable 2-
bromoacetophenone to furnish compounds 7aeb. Interestingly,
placing a chlorine atom adjacent to the amino group in the ring can
reduce the nucleophilicity of the N-4 nitrogen and therefore can
prevent the formation of by-product. Subsequent iodination at the
C-3 position of imidazo[1,2-a]pyrazines 7aeb provided the key
intermediates 8aeb, which facilitated installation of different
functional groups at C-3 by Suzuki coupling and at C-8 by nucleo-
philic substitution [38]. Thus different amines were introduced
leading to heterocyclic amines 9aef in acceptable yields. In the last
step, the C-3 palladium-catalyzed arylation in the presence of 4-
pyridylboronic acid pinacol ester allowed preparing the target
compounds 10aef in moderate to good yields.
2.2. Biological evaluation
2.2.1. In vitro antileishmanial activity against promastigotes
All compounds 4aeg, 9aef and 10aef were evaluated for their
in vitro antileishmanial activity upon the L. major strain. Pentami-
dine was used as the reference drug. The screening data are pre-
sented in Table 3.
When compared to the previously reported 2,3-diarylimidazo
[1,2-a]pyridine analogues displaying IC50 values ranging from 4.9
to 22.5
[21], it could be noticed that the new 2,3-diarylimidazo[1,2-a]pyr-
azines 4aeg, with IC50 values from 10.3 to 27.6 M, were globally
mM on promastigote stage of the same Leishmania strain
Keeping 4-pyridyl moiety as important pharmacophore to
obtain CK1 inhibition, we studied the opportunity to introduce the
diversity at the C-8 position in the last step (Scheme 3).
m
found less active (Table 3). In this sub-series the SAR study was not
Cl
Cl
N
N
N
Cl
N
N
Cl
Cl
N
N
R1
(b)
(a)
(c)
N
R1
N
N
NH2
I
(R1 = H )
(R1 = F)
(R1 = H )
(R1 = F)
7a
7b
8a
8b
5
6
NR2R3
NR2R3
N
N
N
R1
(d)
N
(e)
R1
N
N
I
9a-f
10a-f
N
Scheme 2. Reagents and conditions: (a) NH4OH, sealed tube, 100 ꢀC, 17 h, 70%; (b) BrCH2(4-R1C6H4), CH3CN, 80 ꢀC, 11 h, 58% (7a) and 18 h, 55% (7b); (c) NIS, CH3CN, 80 ꢀC, 40 h, 85%
(8a) and 73% (8b); (d) R2R3NH, K2CO3, DMF, 100 ꢀC, 5e12 h, 55e77%; (e) (4-Pyridyl)B(pin), PdCl2(dppf), K3PO4, dioxane/H2O (9/1), sealed tube, 100 ꢀC, 8 h, 44e84%.

384
P. Marchand et al. / European Journal of Medicinal Chemistry 103 (2015) 381e395
Table 2
Structures of 2-aryl-3-iodoimidazo[1,2-a]pyrazines 9aef and 2-aryl-3-(pyridin-4-yl)imidazo[1,2-a]pyrazines 10aef.
R1
H
NR2R3
Compound
Time
8 h
Yield (%)a
55
Compound
Yield (%)a
64
9a
10a
H
H
N(CH3)2
9b
9c
5 h
12 h
73
77
10b
10c
63
66
F
9d
8 h
52
10d
44
F
F
N(CH3)2
9e
9f
5 h
12 h
70
60
10e
10f
84
52
a
Isolated yields.
O
N
HN
Cl
Cl
Cl
N
N
N
N
N
N
N
(a)
(d)
N
(b)
N
N
N
N
I
7a
8a
11
10c
N
N
(c)
Scheme 3. Reagents and conditions: (a) NIS, CH3CN, 80 ꢀC, 40 h, 85%; (b) (4-Pyridyl)B(pin), PdCl2(dppf), K3PO4, dioxane/H2O (9/1), sealed tube, 100 ꢀC, 8 h, 51%; (c) 4-Iodopyridine,
Pd(OAc)2, PPh3, Cs2CO3, DMF, sealed tube, 90 ꢀC, 16 h, 27%; (d) 4-(Morpholin-4-yl)ethanamine, K2CO3, DMF, 100 ꢀC, 10 h, 49%.
easy to establish since, from the 3-phenyl derivative 4a displaying
IC50 value of 26.2 M, the introduction at the para position of an
electron donating group (OMe, IC50 ¼ 17.4 M, compound 4b) or
electron withdrawing groups (NO2, IC50 ¼ 27.6 M, compound 4c
M compound 4d) provided analogues with
derivative 9f was the most active compound of the series displaying
better IC50 value than the reference. In a general trend for each sub-
series, N,N-dimethylamino and (morpholin-4-yl)ethylamino ap-
pendages afforded more potent compound than their N,N-dime-
thylethane-1,2-diamine analogues with the exception of compound
9e that remained inactive in the L. major promastigote assay
without clear explanation. In the iodine sub-series, comparing IC50s
m
m
m
and CO2Et, IC50 ¼ 17.4
m
the same level of activity but also slightly more active. Neverthe-
less, the presence of a chlorine atom on the phenyl ring for com-
pound 4e (3-Cl, IC50 ¼ 14.5
IC50 ¼ 10.3 M) enhanced the antileishmanial potency. Finally, the
replacement of the phenyl ring at the C-3 position of 2,3-
diphenylimidazo[1,2-a]pyrazine 4a by 4-pyridyl moiety
M, compound 4g) allowed retaining the low anti-
m
M) and compound 4f (3,5-diCl,
of compounds 9c (10.6 mM) and 9f (2.8 mM), the introduction of a
m
fluorine atom enhanced the activity. As previously discussed, this
feature was found in the 4-pyridyl sub-series. Finally, the intro-
duction of a polar moiety at the C-8 position of the scaffold pro-
vided the best active compounds 9b, 9c, 9f and 10e, in comparison
to unsubstituted molecules 4aeg. Such substitution allowed
recovering the level of activity previously obtained in 2,3-
diarylimidazo[1,2-a]pyridine series [21], validating the interest of
this new series of compounds as antileishmanial agents.
a
(IC50 ¼ 20.1
m
parasitic activity.
The introduction of a polar substituent in C-8 position of 2-
phenyl-3-(pyridin-4-yl)imidazo[1,2-a]pyrazine
4g
did
not
enhance the antileishmanial activity of compounds 10a, 10b and
the (morpholin-4-yl)ethylamino counterpart 10c. Interestingly, a
slight modification of these three compounds by the design of the
corresponding 4-fluorophenyl derivatives 10def led to moderate
2.2.2. Cytotoxicity and selectivity index
Toxicity study was first performed on murine peritoneal mac-
rophages in order to identify drugs which were less toxic and as a
prelude to select drugs for in vitro assay on the more relevant
clinical Leishmania amastigote stage [44]. The results concerning
the cytotoxicity and selectivity index (SI) data are presented in
increase in activity for 10d and 10 f (IC50 values of 35.3, 16.5
68.8, 23.5 M, respectively) but significant increase for the N,N-
dimethylamino derivative 10e (IC50 values of 6.4 vs 31.3 M) that
mM vs
m
m
showed the same level of activity as pentamidine, chosen as the
antileishmanial reference drug.
Table 3. IC50 values ranged from 29.2 to 153.7
mM and most of drugs
We also investigated the antileishmanial potential of precursors
9aef, bearing a iodine atom at the C-3 position of the imidazo[1,2-
a]pyrazine heterocycle instead of a 4-pyridyl moiety. Compounds
9b, 9c and 9f exhibited very promising activities with IC50 values
had IC50 more than 50 M, indicating low level of cytotoxicity
m
against macrophages. The SI was calculated as the ratio of cyto-
toxicity (IC50 value on macrophages) to activity (IC50 value on
promastigotes). This in vitro therapeutic index showed that com-
pounds 4g and 10e possessed a moderate safety profile (SI of 7.7
ranging from 6.8, 10.6 and 2.8
mM, respectively. Especially,

P. Marchand et al. / European Journal of Medicinal Chemistry 103 (2015) 381e395
385
Table 3
In vitro antileishmanial activities against L. major (MHOM/IL/81/BNI) promastigotes and cytotoxicity evaluation of 3-aryl-2-phenylimidazo[1,2-a]pyrazines 4aeg, 2-aryl-3-
iodoimidazo[1,2-a]pyrazines 9aef and 2-aryl-3-(pyridin-4-yl)imidazo[1,2-a]pyrazines 10aef.
Compd
R1 R2
R3
L. major promastigotes
Cytotoxicity on macrophages
Selectivity
indexc
Cytotoxicity on 3T3 cells Selectivity
IC50 SEM (
m
M)a
IC50 SEM (
m
M)a
IC50
(
m
M)a
indexd
Pentamidine
4a
4b
4.6 1.1
152.7 1.7
41.4 6.1
60.2 8.8
33.2
1.6.
3.5
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
H
H
C6H5
4-
H
H
26.2 11.1 (22.5 0.6)b
17.4 3.1 (7.2 0.3)b
MeOC6H4
4-(NO2)
C6H4
4-(CO2Et)
C6H4
4c
H
H
H
H
27.6 8.3 (4.9 0.2)b
17.5 11.2 (5.2 1.0)b
113.6 4.0
49.8 7.2
4.2
2.9
n.d.
n.d.
n.d.
n.d.
4d
4e
4f
H
H
3-ClC6H4
3,5-
H
H
14.5 8.2 (n.d.)b
51.5 7.1
35.6 4.8
3.5
3.4
n.d.
n.d.
n.d.
n.d.
10.3 4.2 (7.7 0.2)b
(Cl)2C6H3
4-Pyridyl
I
4g
9a
H
H
H
20.1 12.2 (6.5 1.6)b
24.2 5.9
153.7 12.3
68.6 6.4
7.7
2.8
n.d.
31.9
n.d.
1.3
9b
9c
H
H
I
I
N(CH3)2
6.8 0.6
10.6 3.3
29.2 2.4
31.4 4.5
4.3
3.0
35.7
235.9
5.3
22.3
9d
F
I
24.7 13.2
33.8 2.2
1.4
18.8
0.8
9e
9f
F
F
I
I
N(CH3)2
>100
2.8 0.4
n.d.
93.2 9.5
n.d.
33.3
99.4
>267.5
n.d.
>95.6
10a
H
4-Pyridyl
68.8 6.2
64.9 11.4
0.9
86.5
1.3
10b
10c
H
H
4-Pyridyl N(CH3)2
4-Pyridyl
31.3 14.3
23.5 2.9
143.9 5.6
51.7 4.1
4.6
2.2
155.4
n.d.
5.0
n.d.
10d
F
4-Pyridyl
35.3 13.2
57.8 4.3
1.6
100.9
2.9
10e
10f
F
F
4-Pyridyl N(CH3)2
4-Pyridyl
6.4 0.2
16.5 1.3
42.0 4.5
40.3 3.7
6.6
2.4
>375.0
47.8
>58.6
2.9
n.d.: not determined.
SEM: Standard Error of the Mean.
a
Mean from at least two or three determinations.
IC50 values of the 2,3-diarylimidazo[1,2-a]pyridine analogues [21].
b
c
SI ¼ Cytotoxicity on murine peritoneal macrophages (IC50)/Promastigotes (IC50).
d
SI ¼ Cytotoxicity on BALB/3T3 clone A31 embryonic mouse fibroblast cells (IC50)/Promastigotes (IC50).
and 6.6, respectively) and 9f a high safety profile (SI of 33.3), in all
sub-series. It is worth mentioning that the SI of 9f was equivalent to
pentamidine (SI of 33.2). Considering the SI of 9f (>95.6) and 10e
(>58.6) on non-tumorogenic mouse fibroblast cell line 3T3, the
selective activity of the compounds towards L. major strain was
confirmed. In addition, molecules 9f and 10e were found among the
most active antileishmanial compounds on promastigote stage of
stage. Furthermore, the selectivity indexes varied between 2.4 and
4.6 for the majority of compounds justifying the interest of the
series for the design antiparasitic agents.
2.2.3. In vitro antileishmanial activity against amastigotes
In view to study antileishmanial activity against amastigotes,
compounds were selected according to their high level of activity
against L. major promastigotes and/or their selectivity index.
Considering these criteria, compounds 4g, 9f and 10e were selected
L. major with IC50 values of 2.8 and 6.4
mM, respectively. Conse-
quently, they were suitable for further investigation on amastigote

386
P. Marchand et al. / European Journal of Medicinal Chemistry 103 (2015) 381e395
for testing against intracellular L. major amastigotes. The data are
indicated in Table 4. Molecule 10b was added to the set of com-
pounds in order to check the influence of the presence of a fluorine
atom on the scaffold towards the antileishmanial activity on
amastigotes, as previously discussed in the context of promastigote
assay.
of activity by lipophilic consideration.
For comparison with another strain, all the compounds were
also evaluated against axenic amastigotes of a cloned line of
L. amazonensis strain (MHOM/BR/76/LTB-012) but unfortunately no
promising activity was denoted since compounds displayed IC50
values from 31.4 to 153.7 mM and low SI ranging from 1 to 5 [18].
Compounds 4g, 9f and 10e provided very high activities against
the intracellular amastigote stage of L. major. Compound 4g dis-
The lack of activity on axenic amastigotes of L. amazonensis does
not bode inefficiency of the molecule on intracellular amastigotes
of this species given the metabolic and environmental differences
of clinical stage [45,46].
played IC50 value at the level of the reference (IC50 of 2.7
vs IC50 of 3.0 M for pentamidine) and compounds 10e (IC50 of
0.8 M) and 9f (IC50 of 0.2 M) were found 4e15-fold more active
mM for 4g
m
m
m
than the pentamidine, respectively. In addition, very low toxicity
was measured for the molecules affording significant SI ranging
from 52.5 to 466 and better than pentamidine (SI of 50.9). More-
over, the values were enhanced in comparison with the promasti-
gote stage (Table 4). It is noteworthy to highlight that the variation
of IC50 values is conserved from promastigotes to amastigotes.
Considering structure-activity relationships, it was difficult to
conclude about the importance of the presence of the polar moiety
at the C-8 position of imidazo[1,2-a]pyrazine ring towards amas-
2.2.4. Kinase inhibitory activities
To investigate mechanism of action, compounds were tested for
L. major CK1 inhibition [33e35]. The results presented in Table 5
showed that 4-pyridyl moiety at the C-3 position of the imidazo
[1,2-a]pyrazine ring is a structural requirement to obtain LmCK1
inhibition.
Indeed, 4-pyridyl derivatives 4g and 10aef displayed IC50 values
ranging from 0.48 to 1.3 mM whereas the corresponding arylated or
iodinated analogues remained inactive. Comparing the level of
activities between unsubstituted compound 4g (IC50 value of
tigote testing. Indeed, compound 10b (IC50 value of 9.4
a N,N-dimethylamino group, was less active than its unsubstituted
analogue 4g (IC50 value of 2.7 M), and the most active compound
9f (IC50 value of 0.2 M) was substituted by a iodine atom at the C-3
mM), bearing
1.3
mM) and the C-8 substituted counterparts 10aef (IC50 values
m
from 0.48 to 2.4
mM), an increase of activity was observed for
m
molecules 10a, 10b, 10d and 10f exhibiting 2-fold more potent
LmCK1 inhibition. The two other analogues 10c (IC50 value of
2.4 mM) and 10e (IC50 value of 0.99 mM) were less active than 4g or
as active as this reference compound, respectively. These results
prompted us to conclude that LmCK1 inhibition was not related to
the nature of the amine side chain in C-8 position of the imidazo
[1,2-a]pyrazine based scaffold.
position and a (morpholin-4-yl)ethylamino appendage at the C-8
position. An interesting structural feature for enhancing anti-
leishmanial activity of this series is clearly the presence of a fluorine
atom on the molecule since, once again, compound 10b (IC50 value
of 9.4
m
M) was 12-fold less active than its fluoro-substituted
M). During the process of in-
counterpart 10e (IC50 value of 0.8
m
vasion, the parasite initiates the formation of a membrane, the
socalled parasitophorous vacuole membrane, which surrounds the
intracellular parasite. Penetration of the two membranes (i.e.
plasmatic and vacuole) is therefore an important criterion for ac-
tivity and can differ from a compound to the other, depending on its
hydrophobic character. To discuss this point of view, Log P values
close to 2.9 (10b) and 3.0 (10e) were calculated using Log P Plugin of
MarvinSketch software from ChemAxon. Taking into account these
data, the difference of Log P was not enough to explain the variation
Taking into account the first results, antileishmanial activities
against promastigotes and amastigotes were not correlated with
the inhibition of LmCK1, except for compounds 10e and 4g. In
addition, no selectivity was observed against the corresponding
protozoa model since the selected compounds were also able to
inhibit porcine brain Sus scrofa CK1 (SsCK1) in the micromolar
range. Finally, through biological data of the most active compound
9f against both promastigotes and amastigotes (Table 5), it clearly
appeared that a different target is to be investigated to explain
Table 4
In vitro antileishmanial activities of 2-arylimidazo[1,2-a]pyrazines 4g, 9f,10b and 10e against promastigotes and intracellular amastigotes of a strain of L. major, and cytotoxicity
evaluation.
Compd
R1 R2
R3
H
L. major 1 promastigotes
L. major 1 amastigotes
IC50 SEM (
M)a
Cytotoxicity on macrophages
IC50 SEM (
Selectivity index
amastigotesb
IC50 SEM (
m
M)a
m
m
M)a
Pentamidine
4g
4.6 1.1
20.1 12.2
3
1
152.7 1.7
50.9 (33.2)d
57.0 (7.7)d
H
F
4-
Pyridyl
I
2.7c
153.7 12.3
93.2 9.5
9f
2.8 0.4
0.2 0.1
466 (33.3)d
10b
10e
H
F
4-
Pyridyl
4-
N(CH3)2
N(CH3)2
31.3 14.3
6.4 0.2
9.4 7.9
0.8c
143.9 5.6
42.0 4.5
15.3 (4.6)d
52.5 (6.6)d
Pyridyl
SEM: Standard Error of the Mean.
a
Mean from two determinations.
b
SI ¼ Cytoxicity (IC50)/Amastigotes (IC50).
c
In one of the two experiments, IC50 was below the lower tested concentration, so that SEM calculation was not possible.
d
SI ¼ Cytoxicity (IC50)/Promastigotes (IC50).

P. Marchand et al. / European Journal of Medicinal Chemistry 103 (2015) 381e395
387
Table 5
In vitro antileishmanial activities against promastigotes and intracellular amastigotes of a L. major strain and inhibition of Leishmania major CK1 and porcine brain Sus scrofa
CK1 activities of 3-aryl-2-phenylimidazo[1,2-a]pyrazines 4aeg, 2-aryl-3-iodoimidazo[1,2-a]pyrazines 9aef and 2-aryl-3-(pyridin-4-yl)imidazo[1,2-a]pyrazines 10aef.
Compd
R1 R2
R3
L. major promastigotes IC50 SEM L. major amastigotes IC50 SEM L. major CK1 IC50 Native porcine brain SsCK1
d
/ε
(
m
M)a
(
m
M)a
(
m
M)a
IC50 (m
M)a
Pentamidine
4.6 1.1
3
1
4a
4b
4c
H
H
H
C6H5
H
H
H
26.2 11.1
17.4 3.1
27.6 8.3
>10
>10
>10
n.d.
n.d.
n.d.
4-MeOC6H4
4-(NO2)
C6H4
4d
H
4-(CO2Et)
C6H4
H
17.5 11.2
>10
n.d.
4e
4f
H
H
3-ClC6H4
3,5-
H
H
14.5 8.2
10.3 4.2
n.d.
>10
n.d.
n.d.
(Cl)2C6H3
4-Pyridyl
I
4g
9a
H
H
H
20.1 12.2
24.2 5.9
2.7b
1.3
>10
3.3
>10
9b
9c
H
H
I
I
N(CH3)2
6.8 0.6
10.6 3.3
>10
>10
>10
>10
9d
F
I
24.7 13.2
>10
>10
9e
9f
F
F
I
I
N(CH3)2
>100
2.8 0.4
>10
>10
>10
>10
0.2 0.1
10a
H
4-Pyridyl
68.8 6.2
0.51
1.6
10b
10c
H
H
4-Pyridyl
4-Pyridyl
N(CH3)2
31.3 14.3
23.5 2.9
9.4 7.9
0.48
2.4
2.4
3.3
10d
F
4-Pyridyl
35.3 13.2
0.52
1.6
10e
10f
F
F
4-Pyridyl
4-Pyridyl
N(CH3)2
6.4 0.2
16.5 1.3
0.8b
0.99
0.62
2.1
2.7
n.d.: not determined.
SEM: Standard Error of the Mean.
a
Mean from at least two or three determinations.
In one of the two experiments, IC50 was below the lower tested concentration, so that SEM calculation was not possible.
b
promising activities of these new antileishmanial agents.
[1,2-a]pyrazines 9a, 9b, 9d, 9e, 10a and 10d, bearing N,N-dime-
thylethane-1,2-diamino or N,N-dimethylamino groups at the C-8
position of the scaffold and independently of the substitution at the
position C-3 position (iodine atom or 4-pyridyl). Nevertheless, with
2.2.5. Antiproliferative activity
In order to explore more in depth the biological profile of the
new imidazo[1,2-a]pyrazines 9aef and 10aef, the antiproliferative
activity of these compounds was also studied against a panel of
seven human cancer cell lines, as depicted in Table 6.
Considering the GI50 value of 5-fluorouracil (5FU), used as
reference in the assay, all the compounds displayed very low
cytotoxicity towards the studied cell lines, with the exception of
derivatives 9a and 9d, and MCF-7 (human breast adenocarcinoma)
tumour cell line. Indeed, MCF-7 cell line was sensitive to imidazo
GI50 values ranging from 3 to 9
mg/mL, they remained 10-folde30-
fold less active than the reference (GI50 values of 0.3
mg/mL). In
addition, it was highlighted that N'-(3-iodo-2-phenylimidazo[1,2-
a]pyrazin-8-yl)-N,N-dimethylethane-1,2-diamine 9a and especially
its fluorine analogue 9d showed the best activity against all the
panel of cell lines, exhibiting GI50 values from 4 to 13 mg/mL but
except DU145 for 9a and K562 for 9d.
Finally, as very interesting result, no antiproliferative activity

388
P. Marchand et al. / European Journal of Medicinal Chemistry 103 (2015) 381e395
Table 6
Antiproliferative activity of 2-aryl-3-iodoimidazo[1,2-a]pyrazines 9aef and 2-aryl-3-(pyridin-4-yl)imidazo[1,2-a]pyrazines 10aef against various cancer cell lines.
Compd
R1
H
R2
R3
Human cancer cell linesa GI50 g/mL)b
(m
H460
HuTu80
DU145
MCF-7
M-14
HT-29
K562
5FUc
9a
0.3
13
0.4
12
1.0
20
0.3
9
0.7
4
0.4
4
0.9
5
I
9b
9c
H
H
I
I
N(CH3)2
47
121
44
33
39
47
3
29
>125
179
38
22
16
18
9d
F
I
9
6
13
5
4
4
43
9e
9f
F
F
I
I
N(CH3)2
25
>125
22
73
40
>125
3
>125
>125
>125
>125
>125
>125
>125
10a
H
4-Pyridyl
40
32
47
6
27
6
15
10b
10c
H
H
4-Pyridyl
4-Pyridyl
N(CH3)2
70
n.d.
50
n.d.
90
n.d.
32
n.d.
84
n.d.
56
n.d.
48
n.d.
10d
F
4-Pyridyl
34
24
52
8
19
8
14
10e
10f
F
F
4-Pyridyl
4-Pyridyl
N(CH3)2
>125
20
77
28
>125
39
52
19
>125
36
>125
27
>125
21
n.d.: not determined.
a
Mean from three determinations.
b
H460, human large cell lung cancer; HuTu 80, human duodenal carcinoma; DU145, human prostate carcinoma; MCF-7, human breast adenocarcinoma; Mꢁ14, human
melanoma; HT-29, human colon adenocarcinoma; K562, human chronic myelogenous leukaemia cells.
c
5FU: 5-Fluorouracil.
was detected towards all tumour cell lines and non-tumour cells
3T3 when treating by the compounds 10e and especially 9f, high-
lighting the interest of such molecules as candidates for the
development of safe and effective antileishmanial drugs.
antileishmanial compounds were highlighted with 3-iodoimidazo
[1,2-a]pyrazine 9f and 3-(pyridin-4-yl)imidazo[1,2-a]pyrazine 10e
as hit compounds exhibiting very good activity and high thera-
peutic index, especially on amastigote stage of the parasite. More-
over, the presence of an additional fluorine atom in the molecules,
in terms of physicochemical behaviour, was denoted as a relevant
criterion to enhance antiparasitic activity. In the aim to identify
putative mechanisms of action involved in antileishmanial prop-
erties, 4-pyridyl moiety at the C-3 position of the heterocyclic core
was found as the pharmacophore to target LmCK1 but unfortu-
nately no selective inhibition towards mammalian SsCK1 was ob-
tained. Further pharmacomodulation will be dedicated to
selectivity approach and will be developed to reach nanomolar
inhibition that could be necessary to link clearly LmCK1 inhibition
to antileishmanial properties since CK1 was established as one of
the major protein kinase families expressed and active in Leish-
mania at both stages.
3. Conclusion
To the best of our knowledge, we have developed for the first
time very promising imidazo[1,2-a]pyrazine derivatives, acting as
potent antileishmanial agents on both promastigote and amasti-
gote forms of L. major and displaying high SI. From previous results,
the design of 2-phenyl-3-(pyridin-4-yl)imidazo[1,2-a]pyrazine 4g
provided interesting starting point to obtain antiamastigotes ac-
tivity. The modulation at the C-3 position of the scaffold was
envisaged by Suzuki coupling from iodinated precursors or by
direct arylation. Following these pathways and by introducing po-
lar moiety at the C-8 position, two sub-series of interesting

P. Marchand et al. / European Journal of Medicinal Chemistry 103 (2015) 381e395
389
Finally, the possibility is obvious that inhibition of another
target contributes to the antileishmanial effect of the most active
compound 9f, bearing a iodine atom at the C-3 position of the
imidazo[1,2-a]pyrazine ring instead of a 4-pyridyl group. Taking
into account the level of activity on Leishmania coupled with a
putative original mechanism of action and high protozoa selectivity
of the compound 9f, we plan to evaluate its activity in an experi-
mental murine model of leishmaniasis, and to extend the assess-
ment to other species of protozoa, i.e., trypanosomatidae and not
trypanosomatidae.
8.64 (s, 1H, H3), 9.12 (d, 1H, J ¼ 1.3 Hz, H8); 13C NMR (100 MHz,
DMSO-d6) d 110.5 (CH), 120.0 (CH), 125.9 (2 CH), 128.5 (CH), 128.8 (2
CH), 129.1 (CH), 132.9 (C), 140.3 (C), 142.6 (CH), 146.2 (C); MS (ESI)
m/z (%): 196.1 (100) [MþH]þ. Anal. calcd for C12H9N3: C 73.83, H
4.65, N 21.52. Found: C 74.02, H 4.66, N 21.59.
4.1.2. Imidazo[1,2-a]pyrazin-2-yl trifluoromethanesulfonate (3)
To a stirred solution of 2-aminopyrazine 1 (300 mg, 3.0 mmol) in
isopropanol (6 mL) was added ethyl bromoacetate (0.40 mL,
3.6 mmol). The mixture was heated at 90 ꢀC for 6 h, then it was
cooled to room temperature and the solvent was evaporated in
vacuo. The residue was diluted with toluene (40 mL), N-phenyl-
bis(trifluoromethanesulfonimide) (2.14 g, 6.0 mmol) and triethyl-
amine (0.75 mL) were added. The solution was refluxed for 6 h,
cooled to room temperature and poured into water. The aqueous
layer was extracted with ethyl acetate (2 x 50 mL), the combined
organic extracts were dried over sodium sulfate, filtered and
concentrated in vacuo. The crude product was purified by silica gel
column chromatography using dichloromethane/methanol (95/5)
as eluent to afford the compound 3 (262 mg, 33%) as a yellow oil. Rf
4. Experimental protocols
4.1. Chemistry
All reactions were carried out under argon. All reactions were
monitored by TLC analysis using Merck silicagel 60F-254 thin-layer
plates. Column chromatography was carried out on silicagel Merck
60 (70e230 mesh ASTM). Melting points were determined on an
Electrothermal IA 9000 melting point apparatus and are uncor-
rected. 1H and 13C NMR spectra were performed in DMSO-d6 using a
Bruker AVANCE 400 MHz spectrometer. Chemical shifts are re-
0.48 (CH2Cl2/MeOH 95:5); 1H NMR (400 MHz, DMSO-d6):
d 8.10 (d,
1H, J ¼ 4.6 Hz, H5), 8.42 (s, 1H, H3), 8.67 (dd, 1H, J ¼ 4.6 and 1.5 Hz,
ported as
d values in parts per million (ppm) relative to tetrame-
H6), 9.16 (d, 1H, J ¼ 1.5 Hz, H8); 13C NMR (100 MHz, DMSO-d6):
thylsilane as internal standard and coupling constants (J) are given
in hertz (Hz). The following abbreviations are used to describe peak
patterns when appropriate: s (singlet), d (doublet), dd (doublet of
doublets), t (triplet), q (quartet), m (multiplet). Mass spectra were
recorded using an Electrospray Ionization Method with Waters ZQ
2000 spectrometer. Microwave reactions were carried out in a CEM
Discover microwave reactor in sealed vessels (monowave,
maximum power 300 W, temperature control by IR sensor, fixed
temperature). Elemental analyses were performed on a Thermo
Scientific Elemental Analyser Flash EA 1112 and were found within
0.4% of the theoretical values.
d
103.7 (C), 118.5 (q, JC-F ¼ 319 Hz, CF3), 120.7 (CH), 130.6 (CH), 135.2
(C), 142.9 (CH), 147.2 (C); MS (ESI) m/z (%): 268.0 (100) [MþH]þ.
Anal. calcd for C7H4F3N3O3S: C 31.47, H 1.51, N 15.73. Found: C 31.59,
H 1.52, N 15.77.
4.1.3. Representative direct arylation procedure for the synthesis of
3-aryl-2-phenylimidazo[1,2-a]pyrazines 4aeg
2-Phenylimidazo[1,2-a]pyrazine 2 (1.0 mmol) was introduced
into a 10 mL vial with (hetero)arylhalide (2.0 mmol), palladium
acetate (0.04 mmol), tricyclohexylphosphine tetrafluoroborate
(0.08 mmol), pivalic acid (0.6 mmol) and potassium carbonate
(3.0 mmol) in N,N-dimethylacetamide (4 mL). The vial was purged
with argon for 10 min and then sealed. The mixture was heated at
100 ꢀC for 18e40 h and then it was cooled to room temperature.
Water was added and the aqueous layer was extracted with ethyl
acetate (2 x 10 mL). The combined organic extracts were washed
with brine and water, dried over sodium sulfate, filtered, concen-
trated in vacuo and purified by silica gel column chromatography.
Trituration with diisopropylic ether afforded the compounds 4aeg.
4.1.1. 2-phenylimidazo[1,2-a]pyrazine (2)
4.1.1.1. Method A. To a solution of 2-aminopyrazine 1 (2.38 g,
25.0 mmol) in ethanol (500 mL) were added 2-
bromoacetophenone (4.98 g, 25.0 mmol) and sodium hydro-
genocarbonate (6.30 g, 75.0 mmol). The mixture was refluxed
overnight, cooled to room temperature and the solvent was evap-
orated in vacuo. The residue was diluted with ethyl acetate, then
water was added and the aqueous layer was extracted with ethyl
acetate (2 x 100 mL). The combined organic extracts were dried
over sodium sulfate, filtered and concentrated in vacuo. The crude
product was purified by neutral alumina column chromatography
using dichloromethane/methanol (98/2) as eluent to give the
compound 2 (1.46 g, 30%) as a yellow solid.
4.1.3.1. 2,3-diphenylimidazo[1,2-a]pyrazine (4a). Compound 4a was
obtained following the representative procedure, using 2-
phenylimidazo[1,2-a]pyrazine
2 (300 mg, 1.5 mmol), bromo-
benzene (0.15 mL, 3.0 mmol) and heating for 40 h. The crude
product was purified by silica gel column chromatography using
petroleum ether/ethyl acetate (1/1) as eluent to give 4a (61 mg,
15%) as an orange solid. Rf 0.28 (PET/EtOAc 1:1); Mp: 138 ꢀC; 1H
4.1.1.2. Method B. Imidazo[1,2-a]pyrazin-2-yl trifluoromethane
sulfonate 3 (100 mg, 0.4 mmol) was introduced into a 10 mL sealed
tube with phenylboronic acid (98 mg, 0.8 mmol), sodium carbonate
(169 mg, 1.6 mmol) and tetrakis(triphenylphosphine)palladium(0)
(23 mg, 5 mol%) in ethanol (3 mL). The mixture purged with argon
and heated at 100 ꢀC for 10 min under microwave irradiation
(70 W). After cooling, the resulting solution was poured into water
and the organic layer was extracted with ethyl acetate (2 x 10 mL).
The combined organic extracts were washed with brine and water,
dried over sodium sulfate, filtered, and concentrated in vacuo. The
crude product was purified by neutral alumina column chroma-
tography using dichloromethane/methanol (99/1) as eluent to give
the compound 2 (49 mg, 67%) as a yellow powder. Rf 0.40 (CH2Cl2/
MeOH 98:2); Mp: 102 ꢀC (lit. [39]: 100e102 ꢀC); 1H NMR (400 MHz,
NMR (400 MHz, DMSO-d6):
d 7.31e7.37 (m, 2H, Har), 7.52e7.63 (m,
8H, Har), 7.87 (d, 1H, J ¼ 4.6 Hz, H5), 8.09 (dd, 1H, J ¼ 4.6 and 1.2 Hz,
H6), 9.16 (d, 1H, J ¼ 1.2 Hz, H8); 13C NMR (100 MHz, DMSO-d6):
d
117.0 (CH), 122.2 (C), 127.7 (2CH), 128.0 (C), 128.2 (CH), 128.5
(2CH), 129.6 (CH), 129.7 (3CH), 130.4 (2CH), 133.2 (C), 139.4 (C),
142.9 (CH), 143.3 (C); MS (ESI) m/z (%): 272.1 (100) [MþH]þ. Anal.
calcd for C18H13N3: C 79.68, H 4.83, N 15.49. Found: C 79.42, H 4.84,
N 15.45.
4.1.3.2. 3-(4-methoxyphenyl)-2-phenylimidazo[1,2-a]pyrazine (4b).
Compound 4b was obtained following the representative proce-
dure, using 2-phenylimidazo[1,2-a]pyrazine 2 (100 mg, 0.5 mmol),
4-bromoanisole (0.12 mL, 1.0 mmol) and heating for 18 h. The crude
product was purified by silica gel column chromatography using
petroleum ether/ethyl acetate (1/1) as eluent to give 4b (31 mg,
DMSO-d6):
d
7.40e7.55 (m, 3H, Har), 7.93 (d, 1H, J ¼ 4.5 Hz, H5), 8.07
(dd, 2H, J ¼ 7.2 and 1.2 Hz, Har), 8.62 (dd, 2H, J ¼ 4.5 and 1.3 Hz, H6),

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P. Marchand et al. / European Journal of Medicinal Chemistry 103 (2015) 381e395
20%) as a brown powder. Rf 0.30 (PET/EtOAc 1:1); Mp: 109 ꢀC; 1H
NMR (400 MHz, DMSO-d6): 3.86 (s, 3H, CH3), 7.16 (d, 2H,
procedure, using 2-phenylimidazo[1,2-a]pyrazine 2 (200 mg,
d
1.0 mmol), 1,3-dichloro-5-iodobenzene (680 mg, 2.0 mmol) and
heating for 28 h. The crude product was purified by silica gel col-
umn chromatography using petroleum ether/ethyl acetate (1/1) as
eluent to afford 4f (68 mg, 20%) as a brown powder. Rf 0.34 (PET/
J ¼ 8.6 Hz, Har), 7.30e7.37 (m, 3H, Har), 7.46 (d, 2H, J ¼ 8.6 Hz, Har),
7.65 (dd, 2H, J ¼ 8.3 and 1.6 Hz, Har), 7.85 (d, 1H, J ¼ 4.6 Hz, H5), 8.05
(dd, 1H, J ¼ 4.6 and 1.2 Hz, H6), 9.13 (d, 1H, J ¼ 1.2 Hz, H8); 13C NMR
(100 MHz, DMSO-d6):
d
55.3 (CH3), 115.2 (2CH), 117.0 (CH), 119.8 (C),
EtOAc 1:1); Mp: 199 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d 7.35e7.42
122.1 (C), 127.6 (2CH), 128.0 (CH), 128.5 (2CH), 129.6 (CH), 131.8
(2CH), 133.4 (C), 139.3 (C), 142.8 (CH), 143.0 (C), 160.0 (C); MS (ESI)
m/z (%): 302.1 (100) [MþH]þ. Anal. calcd for C19H15N3O: C 75.73, H
5.02, N 13.94. Found: C 75.98, H 5.04, N 13.97.
(m, 3H, Har), 7.59e7.61 (m, 2H, Har), 7.66 (d, 2H, J ¼ 1.9 Hz, Har), 7.84
(t,1H, J ¼ 1.9 Hz, Har), 7.92 (d, 1H, J ¼ 4.6 Hz, H5), 8.24 (dd, 1H, J ¼ 4.6
and 1.3 Hz, H6), 9.18 (d, 1H, J ¼ 1.3 Hz, H8); 13C NMR (100 MHz,
DMSO-d6):
d 117.6 (CH), 119.4 (C), 127.9 (2CH), 128.4 (CH), 128.6
(2CH), 129.2 (2CH), 129.3 (CH), 129.8 (CH), 131.6 (C), 132.8 (C), 135.1
(2C), 139.6 (C), 142.9 (CH), 143.9 (C); MS (ESI) m/z (%): 340.1 (100)
[MþH]þ, 342.1 (65) [MþHþ2]þ, 344.1 (11) [MþHþ4]þ. Anal. calcd
for C18H11Cl2N3: C 63.55, H 3.26, N 12.35. Found: C 63.41, H 3.24, N
13.31.
4.1.3.3. 3-(4-nitrophenyl)-2-phenylimidazo[1,2-a]pyrazine
(4c).
Compound 4c was obtained following the representative proce-
dure, using 2-phenylimidazo[1,2-a]pyrazine 2 (100 mg, 0.5 mmol),
1-bromo-4-nitrobenzene (202 mg, 1.0 mmol) and heating for 22 h.
The crude product was purified by silica gel column chromatog-
raphy using dichloromethane/ethanol (95/5) as eluent to afford 4c
(31 mg, 20%) as a brown powder. Rf 0.37 (CH2Cl2/EtOH 95:5); Mp:
4.1.3.7. 2-phenyl-3-(pyridin-4-yl)imidazo[1,2-a]pyrazine
(4g).
Compound 4g was obtained following the representative proce-
dure, using 2-phenylimidazo[1,2-a]pyrazine 2 (200 mg, 1.0 mmol),
4-bromopyridine hydrochloride (389 mg, 2.0 mmol) and heating
for 30 h. The crude product was purified by silica gel column
chromatography using dichloromethane/ethanol (99/1) as eluent
to afford 4g (60 mg, 22%) as a beige powder. Rf 0.10 (CH2Cl2/EtOH
140 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d 7.37e7.39 (m, 3H, Har),
7.57e7.60 (m, 2H, Har), 7.85 (d, 2H, J ¼ 8.7 Hz, Har), 7.94 (d, 1H,
J ¼ 4.6 Hz, H5), 8.28 (dd, 1H, J ¼ 4.6 and 1.1 Hz, H6), 8.42 (d, 2H,
J ¼ 8.7 Hz, Har), 9.21 (d, 1H, J ¼ 1.4 Hz, H8); 13C NMR (100 MHz,
DMSO-d6):
d 112.7 (C), 115.3 (C), 117.29 (CH), 124.6 (2CH), 128.1
99:1); Mp: 126 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d 7.37e7.40 (m,
(2CH), 128.5 (CH), 128.7 (2CH), 130.0 (CH), 131.7 (2CH), 132.6 (C),
135.0 (C), 140.3 (C), 143.1 (CH), 144.7 (C); MS (ESI) m/z (%): 317.2
(100) [MþH]þ. Anal. calcd for C18H12N4O2: C 68.35, H 3.82, N 17.71.
Found: C 68.52, H 3.79, N 17.61.
3H, Har), 7.56e7.60 (m, 4H, Har and Hpyr), 7.93 (d, 1H, J ¼ 4.6 Hz, H5),
8.31 (dd, 1H, J ¼ 4.6 and 1.2 Hz, H6), 8.78 (d, 2H, J ¼ 5.2 Hz, Hpyr),
9.21 (d, 1H, J ¼ 1.2 Hz, H8); 13C NMR (100 MHz, DMSO-d6):
d 117.3
(CH), 119.6 (C), 124.7 (2CH), 128.1 (2CH), 128.5 (CH), 128.6 (2CH),
130.0 (CH), 132.8 (C), 136.0 (C), 139.9 (C), 143.1 (CH), 144.4 (C), 150.8
(2CH); MS (ESI) m/z (%): 273.2 (100) [MþH]þ. Anal. calcd for
4.1.3.4. Ethyl 4-(2-phenylimidazo[1,2-a]pyrazin-3-yl)benzoate (4d).
Compound 4d was obtained following the representative proce-
dure, using 2-phenylimidazo[1,2-a]pyrazine 2 (100 mg, 0.5 mmol),
ethyl 4-bromobenzoate (0.16 mL, 1.0 mmol) and heating for 21 h.
The crude product was purified by silica gel column chromatog-
raphy using petroleum ether/ethyl acetate (1/1) as eluent to give 4d
(30 mg, 18%) as a brown powder. Rf 0.35 (PET/EtOAc 1:1); Mp:
C17H12N4: C 74.98, H 4.44, N 20.58. Found: C 75.21, H 4.46, N 20.65.
4.1.4. 3-chloropyrazin-2-amine (6)
2,3-Dichloropyrazine 5 (5.0 g, 33.6 mmol) in 28% aqueous
ammonia solution (20 mL) in a reactor Parr was stirred at 100 ꢀC for
17 h. After cooling, the resulting mixture was filtered, the solid was
washed with water and dried in vacuo to afford compound 6 (3.0 g,
70%) as a white powder. Rf 0.37 (cyHex/EtOAc 1:1); Mp: 170 ꢀC (lit.
123 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d
1.35 (t, 3H, J ¼ 8.0 Hz, CH3),
4.32 (q, 2H, J ¼ 8.0 Hz, CH2), 7.34e7.37 (m, 3H, Har), 7.59 (dd, 2H,
J ¼ 6.9 and 1.8 Hz, Har), 7.70 (d, 2H, J ¼ 8.1 Hz, Har), 7.90 (d, 1H,
J ¼ 4.6 Hz, H5), 8.14 (d, 2H, J ¼ 8.1 Hz, Har), 8.20 (d,1H, J ¼ 4.6 Hz, H6),
[43]: 169.4e170.1 ꢀC); 1H NMR (400 MHz, DMSO-d6):
d 6.83 (bs, 2H,
9.18 (s, 1H, H8); 13C NMR (100 MHz, DMSO-d6):
d 14.1 (CH3), 61.0
NH2), 7.60 (d,1H, J ¼ 2.4 Hz, H5), 7.98 (d,1H, J ¼ 2.4 Hz, H6); 13C NMR
(CH2), 117.1 (CH), 121.1 (C), 127.9 (2CH), 128.3 (CH), 128.6 (2CH),
129.9 (CH), 130.2 (2CH), 130.4 (C), 130.6 (2CH), 132.7 (C), 132.9 (C),
139.7 (C), 143.0 (CH), 143.9 (C), 165.2 (C]O); MS (ESI) m/z (%): 344.2
(100) [MþH]þ. Anal. calcd for C21H17N3O2: C 73.45, H 4.99, N 12.24.
Found: C 73.64, H 5.01, N 13.28.
(100 MHz, DMSO-d6):
d 130.6 (CH), 132.6 (C), 141.4 (CH), 152.7 (C);
MS (ESI) m/z (%): 129.8 (100) [MþH]þ, 131.8 (40). [MþHþ2]þ.
4.1.5. Cyclization step for the synthesis of 8-chloro-2-phenylimidazo
[1,2-a]pyrazine derivatives 7a and 7b
4.1.3.5. 3-(3-chlorophenyl)-2-phenylimidazo[1,2-a]pyrazine
(4e).
4.1.5.1. 8-chloro-2-phenylimidazo[1,2-a]pyrazine
(7a). To
3-
Compound 4e was obtained following the representative proce-
dure, using 2-phenylimidazo[1,2-a]pyrazine 2 (100 mg, 0.5 mmol),
3-chlorobromobenzene (0.12 mL, 1.0 mmol) and heating for 28 h.
The crude product was purified by silica gel column chromatog-
raphy using petroleum ether/ethyl acetate (1/1) as eluent to give 4e
(20 mg, 13%) as a brown solid. Rf 0.36 (PET/EtOAc 1:1); Mp: 221 ꢀC;
chloropyrazin-2-amine 6 (500 mg, 3.9 mmol) in acetonitrile
(7 mL) was added 2-bromoacetophenone (920 mg, 4.6 mmol). The
reaction mixture was stirred at 80 ꢀC for 11 h. After cooling, the
resulting mixture was diluted with dichloromethane and neutral-
ized by a saturated sodium hydrogenocarbonate aqueous solution.
The organic layer was dried over sodium sulfate, filtered and
concentrated under vacuum. The crude product was purified by
silica gel column chromatography using cyclohexane, then cyclo-
hexane/ethyl acetate (1/1) as eluent to give 7a (521 mg, 58%) as a
white powder. Rf 0.42 (cyHex/EtOAc 1:1); Mp: 206 ꢀC (lit. [42]:
1H NMR (400 MHz, DMSO-d6):
d 7.33e7.40 (m, 3H, Har), 7.49e7.52
(m, 1H, Har), 7.59e7.68 (m, 5H, Har), 7.89 (d, 1H, J ¼ 4.6 Hz, H5), 8.16
(dd, 1H, J ¼ 4.6 and 1.4 Hz, H6), 9.17 (d, 1H, J ¼ 1.4 Hz, H8); 13C NMR
(100 MHz, DMSO-d6):
d 117.3 (CH), 120.7 (C), 127.8 (2CH), 128.3
(CH), 128.6 (2CH), 129.3 (CH), 129.6 (CH), 129.8 (CH), 130.1 (CH),
130.2 (C), 131.5 (CH), 133.0 (C), 134.1 (C), 139.5 (C), 142.9 (CH), 143.7
(C); MS (ESI) m/z (%): 306.1 (100) [MþH]þ, 308.2 (35) [MþHþ2]þ.
Anal. calcd for C18H12N3Cl: C 70.71, H 3.96, N 13.74. Found: C 70.95,
H 3.98, N 13.66.
206e208 ꢀC); 1H NMR (400 MHz, DMSO-d6):
d
7.45 (t,1H, J ¼ 7.6 Hz,
Har), 7.54 (t, 2H, J ¼ 7.6 Hz, Har), 7.77 (d, 1H, J ¼ 4.4 Hz, H6), 8.08 (d,
2H, J ¼ 7.2 Hz, Har), 8.66 (d, 1H, J ¼ 4.4 Hz, H5), 8.77 (s, 1H, H3); 13
C
NMR (100 MHz, DMSO-d6):
d 113.2 (CH), 120.6 (CH), 126.1 (2CH),
127.7 (CH), 128.9 (CH), 129.03 (2CH), 132.5 (C), 137.3 (C), 141.3 (C),
146.5 (C); MS (ESI) m/z (%): 230.0 (100) [MþH]þ, 232.0 (35)
[MþHþ2]þ. Anal. calcd for C12H8ClN3: C 62.76, H 3.51, N 18.30.
Found: C 62.99, H 3.52, N 18.35.
4.1.3.6. 3-(3,5-dichlorophenyl)-2-phenylimidazo[1,2-a]pyrazine (4f).
Compound 4f was obtained following the representative

P. Marchand et al. / European Journal of Medicinal Chemistry 103 (2015) 381e395
391
4.1.5.2. 8-chloro-2-(4-fluorophenyl)imidazo[1,2-a]pyrazine
(7b).
dimethylethylenediamine (184
mL, 1.7 mmol). The crude product
Compound 7b was obtained using the same procedure as 7a by
reaction of 3-chloropyrazin-2-amine 6 (500 mg, 3.9 mmol) with 2-
bromo-4’-fluoroacetophenone (1.00 g, 4.6 mmol) and heating for
18 h to afford 7b (529 mg, 55%) as white powder. Rf 0.46 (cyHex/
EtOAc 1:1); Mp: 191 ꢀC (lit. [42]: 188e190 ꢀC); 1H NMR (400 MHz,
was purified by silica gel column chromatography using dichloro-
methane then dichloromethane/ethanol (95/5) as eluent to afford
9a (190 mg, 55%) as a beige powder. Rf 0.12 (CH2Cl2/EtOH 9:1); Mp:
126 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d 2.23 (s, 6H, 2CH3), 2.56 (t,
2H, J ¼ 6.4 Hz, CH2), 3.62 (q, 2H, J ¼ 6.3 Hz, CH2), 7.33 (t, 1H,
J ¼ 5.5 Hz, NH), 7.39e7.44 (m, 2H, Har and H5), 7.51 (t, 2H, J ¼ 7.5 Hz,
Har), 7.60 (d, 1H, J ¼ 4.7 Hz, H6), 8.02 (d, 2H, J ¼ 7.5 Hz, Har); 13C NMR
DMSO-d6):
d
7.38 (t, 2H, J ¼ 8.8 Hz, Har), 7.78 (d, 1H, J ¼ 4.6 Hz, H6),
8.13 (dd, 2H, J ¼ 8.8 and 5.2 Hz, Har), 8.67 (d, 1H, J ¼ 4.6 Hz, H5), 8.77
(s, 1H, H3); 13C NMR (100 MHz, DMSO-d6):
d
113.2 (CH), 116.1 (d, 2JC-
(100 MHz, DMSO-d6): d 37.8 (CH2), 45.0 (2CH3), 57.5 (CH2), 67.0 (C),
¼ 22 Hz, 2CH), 120.7 (CH), 127.8 (CH), 128.4 (d, 3JC-F ¼ 9 Hz, 2CH),
109.7 (CH), 127.7 (2CH), 128.0 (CH), 128.2 (2CH), 129.2 (CH), 133.3
(C), 135.4 (C), 145.0 (C), 148.6 (C); MS (ESI) m/z (%): 408.0 (100)
[MþH]þ. Anal. calcd for C16H18IN5: C 47.19, H 4.45, N 17.20. Found: C
47.35, H 4.47, N 17.28.
F
129.2 (d, 4JC-F ¼ 3 Hz, C), 137.4 (C), 141.3 (C), 145.6 (C), 162.6 (d, 1JC-
¼ 245 Hz, C); MS (ESI) m/z (%): 248.0 (100) [MþH]þ, 250.0 (35)
F
[MþHþ2]þ. Anal. calcd for C12H7ClFN3: C 58.20, H 2.85, N 16.97.
Found: C 58.40, H 2.87, N 16.92.
4.1.7.2. N0-(3-iodo-2-phenylimidazo[1,2-a]pyrazin-8-yl)-N,N-dime-
4.1.6. Iodination step for the synthesis of 8-chloro-3-iodo-2-
thylamine (9b). Compound 9b was obtained following the repre-
phenylimidazo[1,2-a]pyrazine derivatives 8a and 8b
4.1.6.1. 8-chloro-3-iodo-2-phenylimidazo[1,2-a]pyrazine
sentative procedure for
phenylimidazo[1,2-a]pyrazine 8a (200 mg, 0.56 mmol) and N,N-
dimethylamine (57 L, 1.1 mmol). The crude product was purified
by silica gel column chromatography using cyclohexane/ethyl ac-
etate (95/5) as eluent to afford 9b (150 mg, 73%) as a white powder.
Rf 0.48 (cyHex/EtOAc 6:4); Mp: 142 ꢀC; 1H NMR (400 MHz, DMSO-
5
h, using 8-chloro-3-iodo-2-
(8a).
To 8-chloro-2-phenylimidazo[1,2-a]pyrazine 7a (500 mg,
2.2 mmol) in acetonitrile (10 mL) was added N-iodosuccinimide
(489 mg, 2.2 mmol). The reaction mixture was stirred at 80 ꢀC for
40 h. After cooling, the resulting mixture was diluted with ethyl
acetate and washed with water. The organic layer was dried over
sodium sulfate, filtered and concentrated under vacuum. The crude
product was purified by silica gel column chromatography using
cyclohexane, then cyclohexane/ethyl acetate (1/1) as eluent to
afford 8a (655 mg, 85%) as a yellow powder. Rf 0.69 (cyHex/EtOAc
m
d6):
d
3.52 (s, 6H, 2CH3), 7.42 (t, 1H, J ¼ 7.5 Hz, Har), 7.46 (d, 1H,
J ¼ 4.5 Hz, H5), 7.52 (t, 2H, J ¼ 7.5 Hz, Har), 7.69 (d, 1H, J ¼ 4.5 Hz, H6),
8.04 (d, 2H, J ¼ 7.5 Hz, Har); 13C NMR (100 MHz, DMSO-d6):
d 39.4
(2CH3), 67.1 (C), 110.4 (CH), 127.7 (2CH), 128.2 (CH), 128.5 (2CH),
129.1 (CH), 133.3 (C), 136.3 (C), 144.2 (C), 149.5 (C); MS (ESI) m/z (%):
365.1 (100) [MþH]þ. Anal. calcd for C14H13IN4: C 46.17, H 3.60, N
15.38. Found: C 46.02, H 3.58, N 15.33.
1:1); Mp: 155 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d 7.52 (t, 1H,
J ¼ 7.2 Hz, Har), 7.59 (t, 2H, J ¼ 7.2 Hz, Har), 7.89 (d,1H, J ¼ 4.6 Hz, H6),
8.11 (d, 2H, J ¼ 7.2 Hz, Har), 8.55 (d, 1H, J ¼ 4.6 Hz, H5); 13C NMR
(100 MHz, DMSO-d6):
d
70.2 (C), 121.0 (CH), 128.4 (2CH), 128.7 (CH),
4.1.7.3. 3-iodo-N-[2-(morpholin-4-yl)ethyl]-2-phenylimidazo[1,2-a]
pyrazin-8-amine (9c). Compound 9c was obtained following the
representative procedure for 12 h, using 8-chloro-3-iodo-2-
phenylimidazo[1,2-a]pyrazine 8a (100 mg, 0.28 mmol) and 4-
128.8 (2CH), 129.1 (CH), 132.70 (C), 140.1 (C), 141.5 (C), 148.6 (C); MS
(ESI) m/z (%): 355.9 (100) [MþH]þ, 357.9 (35) [MþHþ2]þ. Anal.
calcd for C12H7ClIN3: C 40.54, H 1.98, N 11.82. Found: C 40.67, H 1.99,
N 11.85.
(morpholin-4-yl)ethanamine (36 mL, 0.56 mmol). The crude prod-
uct was purified by silica gel column chromatography using
cyclohexane/ethyl acetate (95/5) then cyclohexane/ethyl acetate (1/
1) as eluent to afford 9c (100 mg, 77%) as a beige powder. Rf 0.10
(cyHex/EtOAc 1:1); Mp: 146 ꢀC; 1H NMR (400 MHz, DMSO-d6):
4.1.6.2. 8-chloro-3-iodo-2-(4-fluorophenyl)imidazo[1,2-a]pyrazine
(8b). Compound 8b was obtained using the same procedure as 8a
by reaction of 8-chloro-2-(4-fluorophenyl)imidazo[1,2-a]pyrazine
7b (1.00 g, 4.0 mmol) with N-iodosuccinimide (908 mg, 4.0 mmol)
to give 8b (1.10, 73%) as beige powder. Rf 0.76 (cyHex/EtOAc 1:1);
d
2.42e2.46 (m, 4H, 4Hmorph), 2.57 (t, 2H, J ¼ 6.4 Hz, CH2),
3.55e3.62 (m, 6H, CH2 and 4Hmorph), 7.39e7.44 (m, 3H, Har, H5 and
NH), 7.51 (t, 2H, J ¼ 7.5 Hz, Har), 7.59 (d, 1H, J ¼ 4.7 Hz, H6), 8.01 (d,
Mp: 194 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d
7.41 (t, 2H, J ¼ 8.8 Hz,
Har), 7.86 (d, 1H, J ¼ 4.6 Hz, H6), 8.12 (dd, 2H, J ¼ 8.8 and 5.5 Hz, Har),
2H, J ¼ 7.5 Hz, Har); 13C NMR (100 MHz, DMSO-d6):
d 37.3 (CH2),
8.52 (d, 1H, J ¼ 4.6 Hz, H5); 13C NMR (100 MHz, DMSO-d6):
d
70.1
53.4 (2CH2), 57.1 (CH2), 66.3 (2CH2), 66.9 (C), 109.8 (CH), 128.0
(2CH), 128.3 (CH), 128.6 (2CH), 129.6 (CH), 133.3 (C), 135.6 (C), 145.1
(C), 148.7 (C); MS (ESI) m/z (%): 450.0 (100) [MþH]þ. Anal. calcd for
(C), 115.5 (d, 2JC-F ¼ 26 Hz, 2CH), 120.8 (CH), 128.4 (CH), 129.0 (d, 4JC-
¼ 3 Hz, C), 130.4 (d, 3JC-F ¼ 9 Hz, 2CH), 139.9 (C), 141.2 (C), 147.5 (C),
F
162.4 (d, 1JC-F ¼ 245 Hz, C); MS (ESI) m/z (%): 373.9 (100) [MþH]þ,
375.9 (35) [MþHþ2]þ. Anal. calcd for C12H6ClFIN3: C 38.58, H 1.62,
N 11.25. Found: C 38.70, H 1.63, N 11.22.
C18H20IN5O: C 48.12, H 4.49, N 15.59. Found: C 48.30, H 4.51, N
15.63.
4.1.7.4. N0-[2-(4-fluorophenyl)-3-iodoimidazo[1,2-a]pyrazin-8-yl]-
N,N-dimethylethane-1,2-diamine (9d). Compound 9d was obtained
following the representative procedure for 8 h, using 8-chloro-3-
iodo-2-(4-fluorophenyl)imidazo[1,2-a]pyrazine 8b (300 mg,
4.1.7. Representative amination procedure for the synthesis of 2-
aryl-3-iodoimidazo[1,2-a]pyrazines 9aef
To 8-chloro-3-iodo-2-phenylimidazo[1,2-a]pyrazine derivatives
8a or 8b (1.0 mmol) in N,N-dimethylformamide (12 mL) was added
amine (2.0 mmol) and potassium carbonate (1.0 mmol). The reac-
tion mixture was purged with argon through the septum and then
stirred at 100 ꢀC for 5e12h. The resulting mixture was diluted with
ethyl acetate and washed with water. The organic layer was dried
over sodium sulfate, filtered and concentrated under vacuum. The
crude product was purified by silica gel column chromatography.
0.80 mmol) and N,N-dimethylethylenediamine (175 mL, 1.6 mmol).
The crude product was purified by silica gel column chromatog-
raphy using dichloromethane then dichloromethane/ethanol (95/
5) as eluent to afford 9d (177 mg, 52%) as a beige powder. Rf 0.11
(CH2Cl2/EtOH 9:1); Mp: 127 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d
2.26 (s, 6H, 2CH3), 2.60 (t, 2H, J ¼ 6.3 Hz, CH2), 3.59 (q, 2H,
J ¼ 6.3 Hz, CH2), 7.33e7.40 (m, 3H, Har and NH), 7.43 (d, 1H,
J ¼ 4.7 Hz, H5), 7.59 (d, 1H, J ¼ 4.7 Hz, H6), 8.04 (dd, 2H, J ¼ 8.7 and
4.1.7.1. N0-(3-iodo-2-phenylimidazo[1,2-a]pyrazin-8-yl)-N,N-dime-
thylethane-1,2-diamine (9a). Compound 9a was obtained following
the representative procedure for 8 h, using 8-chloro-3-iodo-2-
phenylimidazo[1,2-a]pyrazine 8a (300 mg, 0.85 mmol) and N,N-
5.6 Hz, Har); 13C NMR (100 MHz, DMSO-d6):
d 37.5 (CH2), 44.7
(2CH3), 57.2 (CH2), 67.3 (C), 109.6 (CH), 115.2 (d, 2JC-F ¼ 22 Hz, 2CH),
4
3
129.3 (CH), 129.6 (d, JC-F ¼ 3 Hz, C), 129.7 (d, JC-F ¼ 8 Hz, 2CH),
135.6 (C), 144.4 (C),148.8 (C), 162.1 (d, 1JC-F ¼ 243 Hz, C); MS (ESI) m/

392
P. Marchand et al. / European Journal of Medicinal Chemistry 103 (2015) 381e395
z (%): 426.1 (100) [MþH]þ. Anal. calcd for C16H17FIN5: C 45.19, H
H5 and NH), 7.44 (d, 1H, J ¼ 4.7 Hz, H6), 7.49 (d, 2H, J ¼ 5.7 Hz, Hpyr),
7.54 (d, 2H, J ¼ 7.5 Hz, Har), 8.74 (d, 2H, J ¼ 5.7 Hz, Hpyr); 13C NMR
4.03, N 16.47. Found: C 45.31, H 4.05, N 16.52.
(100 MHz, DMSO-d6):
d 37.4 (CH2), 44.7 (2CH3), 57.3 (CH2), 106.8
4.1.7.5. 2-(4-fluorophenyl)-3-iodo-N,N-dimethylimidazo[1,2-a]pyr-
azin-8-amine (9e). Compound 9e was obtained following the
representative procedure for 5 h, using 8-chloro-3-iodo-2-(4-
fluorophenyl)imidazo[1,2-a]pyrazine 8b (300 mg, 0.80 mmol) and
(CH), 120.6 (C), 124.4 (2CH), 127.7 (2CH), 127.8 (CH), 128.3 (2CH),
129.0 (CH), 132.4 (C), 133.2 (C), 136.7 (C), 141.4 (C), 148.9 (C), 150.5
(2CH); MS (ESI) m/z (%): 359.2 (100) [MþH]þ. Anal. calcd for
C
21H22N6: C 70.37, H 6.19, N 23.45. Found: C 70.60, H 6.21, N 23.52.
N,N-dimethylamine (81 mL, 1.6 mmol). The crude product was pu-
rified by silica gel column chromatography using cyclohexane/ethyl
acetate (95/5) as eluent to afford 9e (215 mg, 70%) as a white
powder. Rf 0.48 (cyHex/EtOAc 6:4); Mp: 129 ꢀC; 1H NMR (400 MHz,
4.1.8.2. N,N-dimethyl-2-phenyl-3-(pyridin-4-yl)imidazo[1,2-a]pyr-
azin-8-amine 10b. Compound 10b was obtained following the
representative procedure, using N'-(3-iodo-2-phenylimidazo[1,2-a]
pyrazin-8-yl)-N,N-dimethylamine 9b (200 mg, 0.55 mmol). The
crude product was purified by silica gel column chromatography
using cyclohexane/ethyl acetate (95/5) then cyclohexane/ethyl ac-
etate (1/1) as eluent to afford 10b (110 mg, 63%) as a beige powder.
DMSO-d6):
d
3.48 (s, 6H, 2xCH3), 7.33 (t, 2H, J ¼ 8.9 Hz, Har), 7.43 (d,
1H, J ¼ 4.6 Hz, H5), 7.64 (d, 1H, J ¼ 4.6 Hz, H6), 8.05 (dd, 2H, J ¼ 8.7
and 5.7 Hz, Har); 13C NMR (100 MHz, DMSO-d6):
d 39.5 (2CH3), 67.0
(C), 110.4 (CH), 115.4 (d, 2JC-F ¼ 21 Hz, 2CH), 129.1 (CH), 129.7 (d, 3JC-
¼ 8 Hz, 2CH),129.8 (d, 4JC-F ¼ 3 Hz, C),136.2 (C),143.3 (C),149.3 (C),
Rf 0.22 (AcOEt); Mp: 185 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d 3.35
F
162.0 (d, 1JC-F ¼ 244 Hz, C); MS (ESI) m/z (%): 383.0 (100) [MþH]þ.
Anal. calcd for C14H12FIN4: C 44.00, H 3.16, N 14.66. Found: C 43.85,
H 3.17, N 14.70.
(s, 6H, 2CH3), 7.27e7.40 (m, 4H, 3Har and H5), 7.43 (d, 1H, J ¼ 4.6 Hz,
H6), 7.48e7.56 (m, 4H, 2Hpyr and 2Har), 8.76 (d, 2H, J ¼ 5.7 Hz, Hpyr);
13C NMR (100 MHz, DMSO-d6):
d 39.5 (2CH3), 107.5 (CH), 119.9 (C),
124.9 (2CH), 127.6 (2CH), 127.9 (CH), 128.5 (2CH), 128.9 (CH), 133.2
(C), 133.3 (C), 136.9 (C), 140.4 (C), 149.7 (C), 150.8 (2CH); MS (ESI) m/
z (%): 316.2 (100) [MþH]þ. Anal. calcd for C19H17N5: C 72.36, H 5.43,
N 22.21. Found: C 72.51, H 5.45, N 22.28.
4.1.7.6. 2-(4-fluorophenyl)-3-iodo-N-[2-(morpholin-4-yl)ethyl]imi-
dazo[1,2-a]pyrazin-8-amine (9f). Compound 9f was obtained
following the representative procedure for 12 h, using 8-chloro-3-
iodo-2-(4-fluorophenyl)imidazo[1,2-a]pyrazine 8b (300 mg,
0.80 mmol) and 4-(morpholin-4-yl)ethanamine (211
m
L, 1.6 mmol).
4.1.8.3. N-[2-(morpholin-4-yl)ethyl]-2-phenyl-3-(pyridin-4-yl)imi-
dazo[1,2-a]pyrazin-8-amine 10c
4.1.8.3.1. Method A. Compound 10c was obtained following the
representative procedure, using 3-iodo-N-[2-(morpholin-4-yl)
The crude product was purified by silica gel column chromatog-
raphy using cyclohexane/ethyl acetate (95/5) then cyclohexane/
ethyl acetate (1/1) as eluent to afford 9f (225 mg, 60%) as a white
powder. Rf 0.10 (cyHex/EtOAc 1:1); Mp: 167 ꢀC; 1H NMR (400 MHz,
ethyl]-2-phenylimidazo[1,2-a]pyrazin-8-amine
9c
(70
mg,
DMSO-d6):
d
2.43e2.59 (m, 6H, CH2 and 4Hmorph), 3.55e3.62 (m,
0.15 mmol). The crude product was purified by silica gel column
chromatography using dichloromethane then dichloromethane/
ethanol (95/5) as eluent to afford 10 c (40 mg, 66%) as a beige
powder.
6H, CH2 and 4Hmorph), 7.36 (t, 2H, J ¼ 8.8 Hz, Har), 7.43 (d, 1H,
J ¼ 4.7 Hz, H5), 7.44e7.48 (m, 1H, NH), 7.59 (d, 1H, J ¼ 4.7 Hz, H6),
8.04 (dd, 2H, J ¼ 8.8 and 5.6 Hz, Har); 13C NMR (100 MHz, DMSO-d6):
d
30.4 (CH2), 53.3 (2CH2), 56.9 (CH2), 66.2 (2CH2), 66.8 (C), 109.8
4.1.8.3.2. Method B. Compound 10c was obtained following the
representative procedure used for the synthesis of compounds
9aef with 8-chloro-2-phenyl-3-(pyridin-4-yl)imidazo[1,2-a]pyr-
azine 11 (80 mg, 0.26 mmol) as starting material for 10 h. The crude
product was purified by silica gel column chromatography using
dichloromethane then dichloromethane/ethanol (95/5) as eluent to
afford 10 c (51 mg, 49%) as a beige powder. Rf 0.68 (CH2Cl2/EtOH
(CH), 115.3 (d, 2JC-F ¼ 25 Hz, 2CH), 129.5 (CH), 129.7 (d, 4JC-F ¼ 3 Hz,
C),129.9 (d, 3JC-F ¼ 8 Hz, 2CH),135.4 (C),144.3 (C),148.6 (C),162.0 (d,
1JC-F ¼ 243 Hz, C); MS (ESI) m/z (%): 468.0 (100) [MþH]þ. Anal. calcd
for C18H19FIN5O: C 46.27, H 4.10, N 14.99. Found: C 46.43, H 4.13, N
15.03.
4.1.8. Representative Suzuki coupling procedure for the synthesis of
2-aryl-3-(pyridin-4-yl)imidazo[1,2-a]pyrazines 10aef
95:5); Mp: 159 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d 2.42e2.46 (m,
4H, 4Hmorph), 2.60 (t, 2H, J ¼ 6.4 Hz, CH2), 3.56e3.64 (m, 6H, CH2
and 4Hmorph), 7.32e7.39 (m, 4H, 3Har and NH), 7.43 (d, 1H,
J ¼ 4.6 Hz, H5), 7.49e7.55 (m, 5H, 2Hpyr, 2Har and H6), 8.74 (d, 2H,
To N-substituted 2-aryl-3-iodoimidazo[1,2-a]pyrazin-8-amines
9aef (1.0 mmol) in dioxane/water (9:1) (10 mL) was added 4-
pyridylboronic acid pinacol ester (1.1 mmol), potassium phos-
phate tribasic (2.5 mmol) and [1,1’-bis(diphenylphosphino)ferro-
cene]dichloropalladium (0.05 mmol). The vial was purged with
argon for 10 min and then sealed. The mixture was heated at 100 ꢀC
for 8 h and then it was cooled to room temperature. Water was
added and the aqueous layer was extracted with ethyl acetate (2 x
10 mL). The combined organic extracts were washed with brine and
water, dried over sodium sulfate, filtered, concentrated in vacuo.
The crude product was purified by silica gel column chromatog-
raphy to afford the compounds 10 aef.
J ¼ 5.6 Hz, Hpyr); 13C NMR (100 MHz, DMSO-d6):
d 37.2 (CH2), 53.5
(2CH2), 57.2 (CH2), 66.4 (2CH2), 107.2 (CH), 120.8 (C), 124.8 (2CH),
128.1 (2CH), 128.2 (CH), 128.7 (2CH), 129.5 (CH), 132.7 (C), 133.4 (C),
136.9 (C), 141.6 (C), 149.1 (C), 150.9 (2CH); MS (ESI) m/z (%): 401.2
(100) [MþH]þ. Anal. calcd for C23H24N6O: C 68.98, H 6.04, N 20.99.
Found: C 68.75, H 6.02, N 20.92.
4.1.8.4. N0-[2-(4-Fluorophenyl)-3-(pyridin-4-yl)imidazo[1,2-a]pyr-
azin-8-yl]-N,N-dimethylethane-1,2-diamine 10d. Compound 10d
was obtained following the representative procedure, using N'-[2-
(4-fluorophenyl)-3-iodoimidazo[1,2-a]pyrazin-8-yl]-N,N-dime-
thylethane-1,2-diamine 9d (100 mg, 0.24 mmol). The crude product
was purified by silica gel column chromatography using dichloro-
methane then dichloromethane/ethanol (95/5) as eluent to afford
10d (40 mg, 44%) as a beige powder. Rf 0.11 (CH2Cl2/EtOH 9:1); Mp:
4.1.8.1. N,N-Dimethyl-N0-[2-phenyl-3-(pyridin-4-yl)imidazo[1,2-a]
pyrazin-8-yl]ethane-1,2-diamine 10a. Compound 10a was obtained
following the representative procedure, using N'-(3-iodo-2-
phenylimidazo[1,2-a]pyrazin-8-yl)-N,N-dimethylethane-1,2-
diamine 9a (90 mg, 0.22 mmol). The crude product was purified by
silica gel column chromatography using dichloromethane then
dichloromethane/ethanol (95/5) as eluent to afford 10a (51 mg,
64%) as a beige powder. Rf 0.10 (CH2Cl2/EtOH 9:1); Mp: 126 ꢀC; 1H
137 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d 2.26 (s, 6H, 2CH3), 2.59 (t,
2H, J ¼ 6.2 Hz, CH2), 3.61 (q, 2H, J ¼ 6.2 Hz, CH2), 7.22 (t, 2H,
J ¼ 8.8 Hz, Har), 7.34 (d, 1H, J ¼ 4.7 Hz, H5), 7.40 (t, 1H, J ¼ 5.5 Hz,
NH), 7.44 (d, 1H, J ¼ 4.7 Hz, H6), 7.49 (d, 2H, J ¼ 5.9 Hz, Hpyr), 7.55
(dd, 2H, J ¼ 8.6 and 5.6 Hz, Har); 8.74 (d, 2H, J ¼ 5.9 Hz, Hpyr); 13C
NMR (400 MHz, DMSO-d6):
J ¼ 6.3 Hz, CH2), 3.61 (q, 2H, J ¼ 6.3 Hz, CH2), 7.31e7.39 (m, 5H, 3Har,
d 2.25 (s, 6H, 2CH3), 2.58 (t, 2H,
NMR (100 MHz, DMSO-d6):
d 37.4 (CH2), 44.7 (2CH3), 57.3 (CH2),

P. Marchand et al. / European Journal of Medicinal Chemistry 103 (2015) 381e395
393
106.8 (CH), 115.2 (d, 2JC-F ¼ 21 Hz, 2CH), 120.5 (C), 124.5 (2CH), 129.1
(CH), 129.5 (d, 4JC-F ¼ 3 Hz, C), 129.7 (d, 3JC-F ¼ 8 Hz, 2CH), 132.4 (C),
136.5 (C), 140.5 (C), 148.9 (C), 150.7 (2CH), 161.9 (d, 1JC-F ¼ 243 Hz,
C); MS (ESI) m/z (%): 377.2 (100) [MþH]þ. Anal. calcd for C21H21FN6:
C 67.00, H 5.62, N 22.33. Found: C 67.20, H 5.63, N 22.40.
column chromatography using dichloromethane then dichloro-
methane/ethanol (95/5) as eluent to afford 11 (36 mg, 27%) as a
beige powder.
Rf 0.42 (CH2Cl2/EtOH 9:1); Mp: 212 ꢀC; 1H NMR (400 MHz,
DMSO-d6): d 7.49e7.53 (m, 3H, Har), 7.61e7.63 (m, 4H, Har and Hpyr),
7.79 (d, 1H, J ¼ 4.4 Hz, H6), 8.35 (d, 1H, J ¼ 4.4 Hz, H5), 8.84 (d, 2H,
4.1.8.5. 2-(4-fluorophenyl)-N,N-dimethyl-3-(pyridin-4-yl)imidazo
[1,2-a]pyrazin-8-amine 10e. Compound 10e was obtained following
the representative procedure, using 2-(4-fluorophenyl)-3-iodo-
J ¼ 5.6 Hz, Hpyr); 13C NMR (100 MHz, DMSO-d6):
d 118.3 (CH), 122.2
(C), 124.9 (2CH), 128.3 (2CH), 128.5 (CH), 128.9 (2CH), 129.0 (CH),
132.5 (C), 136.0 (C), 137.1 (C), 141.9 (C), 144.6 (C), 151.1 (2CH); MS
(ESI) m/z (%): 307.1 (100) [MþH]þ, 309.1 (35) [MþHþ2]þ. Anal.
calcd for C17H11ClN4: C 66.56, H 3.61, N 18.26. Found: C 66.76, H
3.59, N 18.31.
N,N-dimethylimidazo[1,2-a]pyrazin-8-amine
9e
(150
mg,
0.39 mmol). The crude product was purified by silica gel column
chromatography using cyclohexane/ethyl acetate (9/1) then cyclo-
hexane/ethyl acetate (1/1) as eluent to afford 10e (110 mg, 84%) as a
white powder. Rf 0.22 (AcOEt); Mp: 141 ꢀC; 1H NMR (400 MHz,
4.2. Biological evaluation
DMSO-d6):
d
3.53 (s, 6H, 2CH3), 7.20 (t, 2H, J ¼ 8.8 Hz, Har), 7.35 (d,
1H, J ¼ 4.5 Hz, H5), 7.41 (d, 1H, J ¼ 4.7 Hz, H6), 7.50 (d, 2H, J ¼ 5.7 Hz,
4.2.1. In vitro antileishmanial activity
H
H
pyr), 7.55 (dd, 2H, J ¼ 8.6 and 5.6 Hz, Har); 8.76 (d, 2H, J ¼ 5.7 Hz,
Compounds were diluted in dimethylsulfoxide (DMSO) in view
to obtain stock solutions (10 mM). Dilutions of each compound
were realized in medium in accordance with cell line culture and at
a maximum final concentration of 1% DMSO.
pyr); 13C NMR (100 MHz, DMSO-d6):
d 39.1 (2CH3), 107.2 (CH),
115.2 (d, 2JC-F ¼ 21 Hz, 2CH), 119.7 (C), 124.6 (2CH), 128.7 (CH), 129.3
(d, 3JC-F ¼ 8 Hz, 2CH), 129.4 (d, JC-F ¼ 3 Hz, C), 133.2 (C), 136.7 (C),
4
139.4 (C), 149.6 (C), 150.6 (2CH), 1618 (d, 1JC-F ¼ 244 Hz, C); MS (ESI)
m/z (%): 334.2 (100) [MþH]þ. Anal. calcd for C19H16FN5: C 68.46, H
4.84, N 21.01. Found: C 68.69, H 4.86, N 21.07.
4.2.1.1. Strains. L. major (MHOM/IL/81/BNI) promastigotes were
maintained by weekly subpassages in Schneider's insect medium
(Sigma chemical Co. St Louis, Mo) supplemented with 13% heat-
inactivated foetal bovine serum (FBS, SigmaeAldrich) at 26 ꢀC
[19]. Axenically grown amastigote forms of L. amazonensis (MHOM/
BR/76/LTB-012) were maintained by weekly subpassages in MAA/
20 medium at 32 1 ꢀC [47].
4.1.8.6. 2-(4-fluorophenyl)-N-[2-(morpholin-4-yl)ethyl]-3-(pyridin-
4-yl)imidazo[1,2-a]pyrazin-8-amine 10f. Compound 10f was ob-
tained following the representative procedure, using 2-(4-
fluorophenyl)-3-iodo-N-[2-(morpholin-4-yl)ethyl]imidazo[1,2-a]
pyrazin-8-amine 9f (150 mg, 0.32 mmol). The crude product was
purified by silica gel column chromatography using dichloro-
methane then dichloromethane/ethanol (95/5) as eluent to afford
10f (70 mg, 52%) as a beige powder. Rf 0.17 (CH2Cl2/EtOH 9:1); Mp:
4.2.1.2. Antileishmanial activity against promastigote stage of Leish-
mania major. Promastigote susceptibility testing was performed
with a spectrofluorimetric micromethod previously described [48].
156 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d
2.42e2.46 (m, 4H,
Briefly, 100
into wells of a 96-well microplate (Nunc®). The cultures were
exposed for 96 h at 26 ꢀC to the drugs (100,10 and 1
M). Four hours
before measurement, 10 L of resazurin solution (700 M) were
m
L of a 106 promastigotes/mL suspension were placed
4Hmorph), 2.59 (t, 2H, J ¼ 6.1 Hz, CH2), 3.53e3.67 (m, 6H, CH2 and
4Hmorph), 7.22 (t, 2H, J ¼ 8.8 Hz, Har), 7.33 (d, 1H, J ¼ 4.7 Hz, H5), 7.43
(d, 1H, J ¼ 4.7 Hz, H6), 7.47e7.50 (m, 1H, NH), 7.49 (d, 2H, J ¼ 5.8 Hz,
m
m
m
H
pyr), 7.55 (dd, 2H, J ¼ 8.6 and 5.6 Hz, Har); 8.74 (d, 2H, J ¼ 5.8 Hz,
added. Then fluorescence was measured at 590 nm with an exci-
tation at 550 nm. Appropriate controls treated by DMSO, pentam-
idine (reference drugs purchased from SigmaeAldrich) were added
to each two sets of experiments.
H
pyr); 13C NMR (100 MHz, DMSO-d6):
d 37.0 (CH2), 53.3 (2CH2), 56.9
(CH2), 66.2 (2CH2),107.0 (CH),115.5 (d, 2JC-F ¼ 22 Hz, 2CH),120.5 (C),
4
3
124.6 (2CH), 129.4 (CH), 129.7 (d, JC-F ¼ 3 Hz, C), 129.9 (d, JC-
¼ 8 Hz, 2CH), 132.4 (C), 136.5 (C), 140.5 (C), 148.9 (C), 150.8 (2CH),
F
161.9 (d, 1JC-F ¼ 244 Hz, C); MS (ESI) m/z (%): 419.2 (100) [MþH]þ.
Anal. calcd for C23H23FN6O: C 66.01, H 5.54, N 20.08. Found: C 66.24,
H 5.56, N 20.14.
4.2.1.3. Antileishmanial activity against amastigote stage of L. major.
Activity against the intracellular amastigote stage of the parasite
was determined after infection of Balb/c mice peritoneal macro-
phages (CE Janvier, Le Genest, France). 100
mL of a peritoneal
4.1.9. 8-chloro-2-phenyl-3-(pyridin-4-yl)imidazo[1,2-a]pyrazine 11
4.1.9.1. Method A. Compound 11 was obtained following the
representative procedure used for the synthesis of compounds
10aef with 8-chloro-3-iodo-2-phenylimidazo[1,2-a]pyrazine 8a
(200 mg, 0.56 mmol) as starting material. The crude product was
purified by silica gel column chromatography using dichloro-
methane then dichloromethane/ethanol (95/5) as eluent to afford
11 (88 mg, 51%) as a beige powder.
macrophage suspension were placed into a 24-well plate (Nunc®)
on glass slides (10 mm diameter) at 1.5 105 cell/mL in RPMI 1640
with 15% FBS at 37 ꢀC and 5% CO2. Following a 24 h-incubation to
allow attachment, macrophages were infected with 100
mL of a
stationary phase promastigotes (1.5 ꢂ 106 promastigotes/mL in
RPMI 1640 medium plus 15% FBS) and then incubated for a 24 h-
period at 37 ꢀC and 5% CO2 for infection. Macrophage culture was
washed and exposed to drugs at concentrations of 100, 10, 1 and
0.1 mM. After 4 days, cultures were fixed with methanol, stained
4.1.9.2. Method B. To
a
stirred solution of 8-chloro-2-
with May-Grunwald-Giemsa and microscopically examined. The
average number of amastigotes per macrophage was determined
by counting the number of amastigotes in 100 randomly chosen
macrophages in each duplicate well. IC50 values were calculated by
using the values of the number of amastigotes per macrophage [19].
Pentamidine was used as a reference at the concentrations of 100,
phenylimidazo[1,2-a]pyrazine 7a (100 mg, 0.43 mmol) in DMF
(2 mL) was added 4-iodopyridine (179 mg, 0.86 mmol), cesium
carbonate (213 mg, 0.65 mmol), triphenylphosphine (46 mg,
0.17 mmol) and palladium acetate (20 mg, 0.09 mmol). The vial was
purged with argon for 10 min and then sealed. The mixture was
heated at 90 ꢀC for 16 h and then it was cooled to room tempera-
ture. Water was added and the aqueous layer was extracted with
ethyl acetate (2 x 20 mL). The combined organic extracts were
washed with brine and water, dried over sodium sulfate, filtered,
concentrated in vacuo. The crude product was purified by silica gel
10 and 1 mM. Experiments were done twice.
4.2.1.4. Antileishmanial activity against axenic amastigote stage of
Leishmania amazonensis. The in vitro leishmanicidal activity of
compounds was determined in axenic cultures of amastigotes of

394
P. Marchand et al. / European Journal of Medicinal Chemistry 103 (2015) 381e395
L. amazonensis. To estimate the 50% inhibitory concentration (IC50
)
4.2.3.4. Cytotoxicity assays. To determine the cytotoxicity of the
compounds, cells were plated into 96-well tissue culture plates and
in their corresponding growth medium, and incubated at 37 ꢀC in a
5% CO2 and 95% air humidified atmosphere for 24 h to allow cells to
attach. A plate containing each of these cells was fixed in situ with
trichloroacetic acid (TCA) in order to obtain the cell values at cero
time before adding the compounds. The rest of the plates con-
taining the different cell lines received serial 4-fold dilutions of the
compound to be tested. The plates were then incubated at 37 ꢀC in a
5% CO2 and 95% air humidified atmosphere for 48 h. The assay was
terminated by the addition of cold TCA. TCA treated plates were
incubated at 4 ꢀC for 1 h and then washed 5 times with tap water to
remove TCA and air dried. Background optical densities were
measured in wells incubated with growth medium without cells.
TCA-fixed cells were stained for 30 min with 0.2% (w/v) SRB dis-
solved in 1% acetic acid. At the end of the staining period unbound
dye was removed by washing 4 times with 1% acetic acid. After air
drying the plates, bound dye was solubilized with 10 mM Tris base
(pH 10.5) and the absorbance read on an automated plate reader at
a wavelength of 510 nm. The GI50 value was defined as the con-
centration of test sample resulting in a 50% reduction of absorbance
as compared with untreated controls that received a serial dilution
of the solvent in which the test samples were dissolved, and was
determined by linear regression analysis. For K562 cells, which
grow in suspension, instead of fixing and staining with SRB, cells
were counted using a Coulter counter. 5-Fluorouracil (5-FU) was
used as a reference compound for the testing.
of the compounds, the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) micromethod was used as
previously described [49].
4.2.2. SsCK1 and LmCK1 (lmjF35.1010) kinases inhibitory activities
4.2.2.1. Buffer. Buffer A: 60 mM
nitrophenylphosphate, 25 mM Mops (pH 7.2), 5 mM EGTA, 15 mM
MgCl2, mM DTT, 0.1 mM sodium vanadate, mM
phenylphosphate.
b-glycerophosphate, 30 mM p-
1
1
4.2.2.2. Kinase preparations and assays. Kinase activities were
assayed in Buffer A, at 30 ꢀC, at a final ATP concentration of 15
M.
m
Blank values were subtracted and activities expressed in % of the
maximal activity, i.e. in the absence of inhibitors. Controls were
performed with appropriate dilutions of DMSO. SsCK1 and LmCK1
peptide substrates were obtained from Proteogenix (Oberhaus-
bergen, France).
SsCK1
fold diluted buffer A, as previously described [50,51] but using
25 M CKS peptide (RRKHAAIGpSAYSITA), a SsCK1-specific sub-
strate [52]. Its kinase activity was assayed in buffer A, with 1 mg
histone H1/mL, in the presence of 15 M [
33P] ATP (3000 Ci/
mmol; 10 mCi/mL) in a final volume of 30 L. After incubation for
30 min at 30 ꢀC, the reaction was stopped by deposing 25 mL onto
P81 phosphocellulose papers (Whatman) using FilterMate
d/ε (S. scrofa porcine brain, native) was assayed in three-
m
m
g-
m
a
harvester (PerkineElmer) and were washed in 1% phosphoric acid.
Scintillation fluid was added and the radioactivity measured in a
PerkineElmer counter. LmCK1.2 (LmjF35.1010, L. major, recombi-
nant, His-fusion protein in Escherichia coli) [34] was purified by
affinity chromatography on cobalt beads and its kinase activity was
assayed as described for SsCK1.
Acknowledgements
We thank ChemAxon Ltd for providing the MarvinSketch soft-
ware. For LmCK1 and SsCK1 evaluation, this work was supported by
ꢁ
^
Canceropole Grand Ouest e Line “Valorisation des produits de la
mer”.
4.2.3. Cell cytotoxicity bioassays
4.2.3.1. Cells. Cytotoxicity of the compounds was evaluated in
various tumour cell lines and in a normal cell lines using the sul-
forhodamine B (SRB) assay method [18]. Cell lines tested include
BALB/3T3 (non-tumorogenic, BALB/c mouse embryo cells), HuTu 80
(human duodenal carcinoma), H460 (human lung large cell carci-
noma), Mꢁ14 (human amelanotic melanoma), DU145 (human
prostate carcinoma), MCF-7 (human breast adenocarcinoma), HT-
29 (human colon adenocarcinoma), and K562 (human chronic
myelogenous leukaemia).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.09.002.
References
[1] World Health Organization, Leishmaniasis, Fact sheet Nꢀ375 updated February
2015. Information site: http://www.who.int/mediacentre/factsheets/fs375/en/
.
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