In vitro identification of imidazo[1,2-a]pyrazine-based antileishmanial agents and evaluation of L. major casein kinase 1 inhibition

Article abstract of DOI:10.1016/j.ejmech.2020.112956

Leishmaniasis constitutes a severe public health problem, with an estimated prevalence of 12 million cases. This potentially fatal disease has a worldwide distribution and in 2012, the fatal Visceral Leishmaniasis (VL) was declared as new emerging disease in Europe, mainly due to global warming, with expected important public health impact. The available treatments are toxic, costly or lead to parasite resistance, thus there is an urgent need for new drugs with new mechanism of action. Previously, we reported the discovery of CTN1122, a potent imidazo[1,2-a]pyrazine-based antileishmanial hit compound targeting L-CK1.2 at low micromolar ranges. Here, we described structurally related, safe and selective compounds endowed with antiparasitic properties, better than miltefosine, the reference therapy by oral route. L-CK1.2 homology model gave the first structural explanations of the role of 4-pyridyl (CTN1122) and 2-aminopyrimidin-4-yl (compound 21) moieties, at the position 3 of the central core, in the low micromolar to nanomolar L-CK1.2 inhibition, whereas N-methylpyrazole derivative 11 remained inactive against the parasite kinase.

Full text of DOI:10.1016/j.ejmech.2020.112956

Journal Pre-proof
In vitro identification of imidazo[1,2-a]pyridine-based antileishmanial agents and
evaluation of L. major casein kinase 1 inhibition
Marc-Antoine Bazin, Sandrine Cojean, Fabrice Pagniez, Guillaume Bernadat,
Christian Cavé, Isabelle Ourliac-Garnier, Marie-Renée Nourrisson, Cathy Morgado,
Carine Picot, Olivier Leclercq, Blandine Baratte, Thomas Robert, Gerald F. Späth,
Najma Rachidi, Stéphane Bach, Philippe M. Loiseau, Patrice Le Pape, Pascal
Marchand
PII:
S0223-5234(20)30928-4
DOI:
https://doi.org/10.1016/j.ejmech.2020.112956
EJMECH 112956
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 September 2020
Revised Date: 19 October 2020
Accepted Date: 19 October 2020
Please cite this article as: M.-A. Bazin, S. Cojean, F. Pagniez, G. Bernadat, C. Cavé, I. Ourliac-Garnier,
M.-R. Nourrisson, C. Morgado, C. Picot, O. Leclercq, B. Baratte, T. Robert, G.F. Späth, N. Rachidi, S.
Bach, P.M. Loiseau, P. Le Pape, P. Marchand, In vitro identification of imidazo[1,2-a]pyridine-based
antileishmanial agents and evaluation of L. major casein kinase 1 inhibition, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112956.
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Graphical abstract

In vitro identification of imidazo[1,2-a]pyridine-based antileishmanial agents and
evaluation of L. major casein kinase 1 inhibition
a
b
b
Marc-Antoine Bazin , Sandrine Cojean , Fabrice Pagniez a, Guillaume Bernadat , Christian Cavé b, Isabelle
a
a
a
a
c
Ourliac-Garnier , Marie-Renée Nourrisson , Cathy Morgado , Carine Picot , Olivier Leclercq , Blandine
c
c
b
Baratte d,e, Thomas Robert d,e, Gerald F. Späth , Najma Rachidi , Stéphane Bach d,e, Philippe M. Loiseau ,
Patrice Le Pape a, Pascal Marchand a,
*
a Université de Nantes, Cibles et médicaments des infections et du cancer, IICiMed, EA 1155, F-44000, Nantes, France
b
BioCIS Biomolécules: Conception, Isolement, Synthèse, Chimiothérapie Antiparasitaire, UMR CNRS 8076, Univ. Paris-
Sud, Université Paris-Saclay, Faculté de Pharmacie, F-92296 Châtenay-Malabry, France
c
Institut Pasteur and Institut National de Santé et Recherche Médicale INSERM U1201, Unité de Parasitologie Moléculaire
et Signalisation, F-75015 Paris, France
d Sorbonne Université, CNRS, UMR8227, Integrative Biology of Marine Models Laboratory (LBI2M), Station Biologique de
Roscoff, F-29680 Roscoff, France
e Sorbonne Université, CNRS, FR2424, Kinase Inhibitor Specialized Screening facility - KISSf, Station Biologique, F-29680
Roscoff, France
Keywords: Antileishmanial agents, Casein kinase 1, L-CK1.2 inhibitors, Imidazo[1,2-a]pyrazines, Homology
model.
* Corresponding author:
Université de Nantes, Cibles et Médicaments des Infections et du Cancer, Département de Chimie Thérapeutique, IICiMed,
EA 1155, Institut de Recherche de la Santé 2, 22 Boulevard Bénoni Goullin 44200 Nantes, France. E-mail address:
pascal.marchand@univ-nantes.fr (P. Marchand)
Abstract:
Leishmaniasis constitutes a severe public health problem, with an estimated prevalence of 12 million cases. This
potentially fatal disease has a worldwide distribution and in 2012, the fatal Visceral Leishmaniasis (VL) was
declared as new emerging disease in Europe, mainly due to global warming, with expected important public
health impact. The available treatments are toxic, costly or lead to parasite resistance, thus there is an urgent
need for new drugs with new mechanism of action. Previously, we reported the discovery of CTN1122, a potent
imidazo[1,2-a]pyrazine-based antileishmanial hit compound targeting L-CK1.2 at low micromolar ranges. Here,
we described structurally related, safe and selective compounds endowed with antiparasitic properties, better
than miltefosine, the reference therapy by oral route. L-CK1.2 homology model gave the first structural
explanations of the role of 4-pyridyl (CTN1122) and 2-aminopyrimidin-4-yl (compound 21) moieties, at the
position 3 of the central core, in the low micromolar to nanomolar L-CK1.2 inhibition, whereas N-
methylpyrazole derivative 11 remained inactive against the parasite kinase.

1. Introduction
Leishmaniases constitute one of the 20 diseases included in the Neglected Tropical Diseases (NTDs) program of
the World Health Organisation (WHO). According to a recent report from WHO [1], the three clinical forms,
cutaneous (CL), mucocutaneous (MCL) and visceral leishmaniasis (VL), collectively affect 12 million people in
98 countries, and 1 billion are at risk of infection. Moreover, there are almost 1 million new cases and 30 000
deaths attributed to leishmaniases each year [2]. Clinical forms differ in immunopathologies, degree of morbidity
and mortality. VL is the most severe form of Leishmaniasis, fatal in the absence of treatment, whereas CL is
significantly associated with morbidity. Indeed, leishmaniasis-related disabilities impose a great social burden
and impair economic productivity; the social stigma associated with the deformities and disfiguring scars often
keeps patients hidden. Leishmaniasis has so become a disease, which impedes socio-economic development. CL
is more widely distributed than VL, with 70 to 75% of the global estimated CL incidence occurring in ten
countries: Afghanistan, Algeria, Brazil, Colombia, Costa Rica, Ethiopia, Iran, Peru, Sudan and Syria. In contrast,
more than 90% of VL cases occur in six countries: Bangladesh, Brazil, Ethiopia, India, South Sudan and Sudan
[3]. In 2012, VL was declared as new emerging disease in Europe, due mainly to global warming, with an
expected important public health impact [4]. According to WHO, despite the measures taken to eradicate
leishmaniasis, the number of cases is increasing as well as the mortality. The expansion of leishmaniasis and the
alarming rise in number of cases is related to environmental changes such as deforestation, building of dams,
new irrigation schemes and migration of non-immune people to endemic areas. The causative agent of
Leishmaniasis is a protozoan of the Leishmania genus transmitted to its mammalian hosts (humans, dogs,
monkeys, rodents...) by the bite of an infected sandfly (Phlebotominae). The form of the disease depends upon
the species; VL is caused by Leishmania donovani and L. infantum, CL by species such as L. major, L.
amazonensis, L. tropica or L. mexicana and MCL by L. braziliensis [5]. 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 host mononuclear phagocytic system [2,5].
Despite efforts of the community, there is as yet no effective vaccine in widespread clinical use for prevention of
human leishmaniasis [6] and vector control approaches remain extremely difficult [7]. Moreover, only a limited
number of drugs are used in the treatment of Leishmaniasis, including pentavalent antimonials, amphotericin B,
pentamidine, paromomycin and miltefosine. Miltefosine is the only orally active drug and is highly effective (up
to 98% cure rates) [1-3]. Unfortunately, most of these drugs cause side effects, high toxicities and display a high
rate of treatment failure in HIV co-infected patients. They are costly, require long-term treatment and lead to
parasite resistance [1-3].
In the last decade, efforts have been made by governmental, non-governmental and non-profit organizations to
develop leads that moved forward to preclinical and clinical stages of drug discovery, especially against VL, but
the drug portfolio is still quite limited [8,9].
In this context, Drug for Neglected Disease initiative (DNDi), through drug repurposing strategy, studied
nitroaromatic compounds including nitroimidazole, nitroimidazo-oxazole and nitroimidazo-oxazine derivatives.
7-Substituted nitroimidazooxazine compound, identified DNDI-0690 (Figure 1), displaying very good activity
against intramacrophage forms of both L. donovani and L. infantum (EC50 = 60 nM and 93 nM, respectively),

non-toxicity (SI > 1000) for mammalian cells and in vivo efficacy with high oral bioavailability, entered
preclinical trials in September 2015, with the first-in-man studies being carried out by the end 2018 [8,9].
[insert Figure 1]
In 2016, the 6-carboxamide benzoxaborole DNDI-6148 (Figure 1) concluded preclinical VL studies with in vivo
efficacy compared to miltefosine in mice at a minimum dose of 25 mg/kg/day b.i.d. after a 10-day treatment
[8,9]. Nevertheless, its antileishmanial mode of action remains unclear.
DNDi, with support from pharma industry, produced the aminopyrazole urea lead candidate DNDI-5561 to enter
in Phase I clinical trials against VL. Its structure remains unknown but analogues such as DNDI-1044 and
compound I (Figure 1) are described in the literature [8,9].
The University of Dundee in collaboration with Glaxo Smith Kline (GSK) developed a novel series of
pyrazolopyrimidines. Among them, GSK-3186899/DDD853651 (Figure 1) showed excellent in vivo activity in a
mouse model of visceral leishmaniasis with good oral availability and safety profile. In addition, Cdc2-related
kinase 12 (CRK12) was identified as druggable target for further optimizations. Its biological data support
progression to definitive preclinical studies and further phase I clinical trials [8,9].
Novel concepts towards selection of target and novel drugs are thus urgently needed to overcome the current
situation and more efficiently combat and eventually eradicate this deadly disease.
In this context, we identified Leishmania casein kinase I paralog 2 (L-CK1.2) as the potential target of 2,3-
diarylimidazo[1,2-a]azine series [10,11] with antileishmanial activity against L. major. Especially, we showed
that imidazo[1,2-a]pyrazine derivative CTN1122 (Figure 2) killed intracellular amastigotes at the
submicromolar dose and was highlighted as an efficient L-CK1.2 inhibitor (IC50 value of 0.72 µM) with a good
selectivity index (SI = 52.5) towards macrophages [11].
[insert Figure 2]
Interestingly, L-CK1.2 was validated as a drug target for antileishmanial therapy [12,13]. L-CK1.2 is essential
for intracellular parasite survival and is released in macrophages via extracellular vesicles (EVs) [12,14].
Moreover, several evidences suggest that L-CK1.2 has been evolutionary selected to interact with and to
phosphorylate host proteins subverting the biological and immune functions of the macrophage [12,15,16]. Thus,
targeting L-CK1.2 not only kills the intracellular parasite but because of its role outside of the parasite, could
also limit the emergence of parasite resistance.
In previous work, the 4-pyridyl in the C3 position of the imidazo[1,2-a]pyrazine central core was proved to be
the best substitution to retain CK1 inhibition compared to substituted phenyl rings [11]. Starting from the
structure of CTN1122, the introduction of other aza(hetero)aryl appendages was considered to study their
influence on antileishmanial properties and kinase inhibitory activity (Figure 2). The biological activity of
advanced precursors was also evaluated for obtaining further structure activity relationship (SAR) data.
Additional information of ligand-protein interactions was provided through homology model of LmCK1. We
also compared inhibitory activity of L-CK1.2 versus mammalian CK1 in order to foresee potential toxicity due
to lack of selectivity (Lm vs Hs). In parallel, in vitro cytotoxicity was measured towards two cell lines (human

fibroblast MRC-5 and murine macrophage RAW264.7). In complement to the study, the determination of in vivo
toxicity using Galleria mellonella larvaes guided the choice of the most promising antiparasitic compounds [17].
2. Results and discussion
2.1. Chemistry
The design of the first series of imidazo[1,2-a]pyrazines 7-11 (Scheme 1) is considered as the extension of the
synthetic work previously described by our group [11].
[insert Scheme 1]
Thus, derivatives of the reference compound CTN1122 were prepared by modifying the nature of the ring at the
position 3 of the imidazopyrazine scaffold. Our initial SAR efforts were directed toward the introduction of
diverse azaheterocyclic cores to compare with 4-pyridyl ring present on CTN1122. Briefly, the obtention of the
dihalo key intermediate 5 allowed the sequential functionalization at C-8 by nucleophilic substitution and at C-3
by Suzuki coupling. In the last step, different azaheterocyclic moieties such as 3-pyridine, 5-pyrimidine, 5-(2-
aminopyrimidine), 5-indazole or 4-(1-methylpyrazole) were introduced to provide five final compounds 7-11 in
low to good yields (Table 1). Poor yields were associated with problems of purification mainly due to the
polarity of compounds 7 and 9.
[insert Table 1]
Next, we hypothesized that the introduction of a 2-aminopyrimidin-4-yl moiety, in position 3 of the imidazo[1,2-
a]pyrazine central core, could lead to significant L-CK1.2 inhibition and parasite cell death [18]. The
introduction of such substituent was not possible directly from iodo precursor 6 (Scheme 1) since the
corresponding boronic acid was not easily available. As depicted in schemes 2 and 3, the functionalization at the
level of the 8-chloro analogs 3 and 4 by direct arylation procedure was interesting since it allowed obtaining
various advanced derivatives for broader SAR study both at the C8 and C3 positions of the imidazopyrazine.
[insert Scheme 2]
[insert Scheme 3]
The synthesis began with the preparation of the 4-iodo-2-(methylsulfanyl)pyrimidine 13 following the procedure
described by Majeed and coworkers [19]. Once this iodo derivative in hand we could perform a regioselective
direct arylation using Fagnou’s conditions [20,21] with compounds 3 and 4 as starting materials. Subsequent
amination reaction was carried out with dimethylamine and allowed chlorine displacement at the position 8 and
isolation of compounds 16 and 17. Oxidation of the sulfide group with Oxone® afforded sulfones 18 and 19
which were treated with ammonium hydroxide to give 2-aminopyrimidine derivatives 20 and 21 in moderate
yields (scheme 2) [22].
We further incorporated an amino group (NH2) in the position 8 to verify the influence of the substitution at the
level of the nitrogen on the binding mode of the molecule. Indeed, we assume that donor-acceptor sequence

between NH2/N-7 would behave differently than the N-dimethyl substitution in the binding site of the target
protein. Compound 15 underwent an amination with ammonia under microwave irradiation to furnish 22 in a
quantitative yield. The same oxidation / amination sequence as previously used led to the desired compound 24
(Scheme 3).
2.2. Biological evaluation
2.2.1. In vitro antileishmanial activity against intracellular amastigotes
The biological evaluation of compounds 7−11, 14−17, 20−22 and 24 was conducted against five different
species of Leishmania parasite, namely L. major and L. amazonensis for CL, L. braziliensis for MCL (Table 2),
L. infantum and L. donovani for fatal VL (Table 3) in the intramacrophage form. Miltefosine was used as
reference drug. The intracellular amastigote model is one of the most important assay for the in vitro
antileishmanial evaluation, because the amastigote stage is found in mammalian host and its infection causes the
clinical manifestations of leishmaniasis [2].
To identify new bioactive compounds against Leishmania, CTN1122 was used as a hit with IC50 values of 0.8
µM and 2.74 µM against L. major and L. donovani strains, respectively [11]. Low toxicity against macrophages
(CC50=42.03 µM) and human MRC-5 cells (CC50>100 µM) was also highlighted. Nevertheless, CTN1122
showed a modest or poor anti-amastigote response against the three other strains. Notably, differences in the in
vitro drug sensitivities among Leishmania species are a well-known fact that has been reported for current
antileishmanial drugs [23].
To investigate the SAR for this potential new chemical class of antiparasitic drugs, the 4-pyridyl part at the C3
position of the imidazo[1,2-a]pyrazine ring was selected for modification (compounds 7−11, 17 and 21). In
addition, the synthetic scheme for the obtention of compound 21 allowed us to produce the intermediate 15
which gave access to compounds 22 and 24, all the molecules varying from CTN1122 by the C3 and C8
positions (Scheme 2). We were also interested in verifying the influence of the fluorine present on the 2-phenyl
ring (R=F, Scheme 2) towards antiparasitic activity by synthesizing the unsubstituted counterparts (R=H for
compounds 14, 16 and 20, Scheme 2).
[insert Table 2]
[insert Table 3]
In a general trend for compounds 7−11, the replacement of 4-pyridyl ring in CTN1122 by other azaheterocycle
(3-pyridyl, 5-pyrimidinyl, 2-aminopyrimidin-5-yl, 5-indazolyl, 1-methylpyrazol-4-yl) furnished poor active
compounds on L. major, except N-methylpyrazole derivative 11 displaying an IC50 value of 8.99 µM. In the
pyrimidin-4-yl sub-series, all the compounds 14−17, 20, 22 and 24 remained inactive. Interestingly, only
compound 21 that showed improved potency (IC50=8.24 µM) was considered as a close analogue of CTN1122
by exchanging the 4-pyridyl ring with the 2-aminopyrimidin-4-yl appendage. Its analogue 20 (IC50=28.32 µM),
without fluorine atom (R=H), was 3.4 fold less active.

Considering L. amazonensis, compound 21 (IC50=6.12 µM) was 4.6 fold more potent than the original hit
CTN1122 (IC50=28.36 µM) and was at the level of the control drug miltefosine (IC50=4.22 µM) (Table 2). All
the other tested compounds were proved to be inactive, except the 2-(methylthio)pyrimidin-4-yl derivative 14
(IC50=3.20 µM, R8=Cl), more potent than the reference, and 16 (IC50=5.99 µM, R8=N(CH3)2). It should be noted
that the results on L. amazonensis encompass a reverse structure−activity relationship with respect to the
presence of fluorine atom in the pyrimidin-4-yl sub-series. Indeed, when compared to compound 20 (IC50=28.35
µM), its fluoro analogue 21 showed a 3.8 fold increased antileishmanial activity, as previously denoted for L.
major results. Neverthless, the introduction of a fluorine atome on compounds 14 and 16 was deleterious for the
activity when observing the potency of their respective analogues 15 (IC50>100 µM), unactive and 17
(IC50=12.49 µM), twice less active.
No activity was observed against L. braziliensis amastigotes for all the compounds. This strain was also not very
sensitive to the reference compound Miltefosine (IC50=8.33 µM) (Table 2).
Table 3 depicted the results obtained against visceral leishmaniasis by using classicaly L. infantum and L.
donovani strains for the assays. Briefly, before discussing more in depth the antileishmanial activities, it is
encouraging to see that four IC50 values ranged from 1.27 to 2.74 µM, including CTN1122 and similar to the
reference drug Miltefosine on both VL strains. In addition, two compounds displayed IC50s around 6 µM against
L. infantum. Indeed, VL is the deadly form of Leishmaniasis, if untreated, and it needs consequently to require
particular attention when designing new antileishmanial agents.
Among the seven C3 derivatives of CTN1122 (compounds 7−11, 17 and 21) tested against L. infantum, only
three compounds showed decreased activity compared to the hit (IC50=12.80 µM), indicating that heteroaromatic
modifications on this position could be a promising strategy for enhancing antileishmanial properties. Indeed,
introducing 3-pyridyl (7), 5-pyrimidinyl (8), 2-aminopyrimidin-5-yl (9) moieties resulted in unactive compounds
(IC50>50 µM) but other modulations were in favour of antiparasitic properties. Indazole derivative 10 displayed
the same potency as CTN1122 whereas its N-methylpyrazole counterpart 11 exhibited high potency with an IC50
value of 2.60 µM, slightly lower than the reference drug miltefosine. In addition, 2-(methylthio) and 2-
aminopyrimidin-4-yl derivatives 17 and 21, respectively, showed IC50s around 6 µM, twice more active than
CTN1122. Imidazopyrazine 14, bearing a chlorine atom at the position C8 and a 2-(methylthio)pyrimidine at the
position C3, was highlighted as the most potent compound against L. infantum (IC50=1.93 µM), 1.5 fold more
active than the reference drug. Again, the incidence of the fluorine atom in para position of the C3 phenyl ring
remained unclear since it was detrimental when introduced on compound 14 (IC50>100 µM for compound 15)
and favourable for compounds 16 (IC50=12.54 µM versus 17) and 20 (IC50>100 µM versus 21).
Regarding L. donovani strain, the profile of antiparasitic activity of the tested compounds was almost the same as
encountered for L. infantum strain but with decreased level of potency for compounds 14, 16, 17 and 21 with
IC50 values ranging from 9.82 to 17.57 µM. It must be denoted that the most active compound 14 against L.
infantum was 5 fold less active against L. donovani. Interestingly, N-methylpyrazole derivative 11 remained very
active with an IC50 value of 1.27 µM, 1.4 times more potent than Miltefosine. Whereas displaying poor activity
against L. infantum strain, CTN1122 exhibited a promising IC50 value of 2.74 µM.
2.2.2. Cytotoxicity and selectivity index

In order to validate the biological interest of the described series as a source of antileishmanial agents, low
cytotoxicity profile is mandatory towards the host cells. In this context, all the compounds were tested on RAW
264.7 macrophages and MRC-5 human fibroblast cells. Tables 2 and 3 show the selectivity index (SI), calculated
as the ratio of cytotoxicity (CC50 RAW or CC50 MRC-5) to activity (IC50 value on intracellular amastigotes), and
SI>10 is usually required for the candidate to progress to the in vivo evaluation [24].
Unfortunately, 8-amino-imidazo[1,2-a]pyrazines 22 and 24 showed high cytotoxicity for macrophages with IC50
values of 11.51 and 4.69 µM, respectively. Consequently, such toxicity did not allow measuring
intramacrophage antileishmanial activity for compound 24 and must induce some concerns with the
interpretation of the biological results for compound 22. The indazolyl moiety on compound 10 also brought
important cytotoxicity on macrophages. Except the three compounds discussed before, no general cytotoxicity
was observed on MRC5 cells for all the series and the toxicity on macrophages ranged from 15.95 to 50.64 µM.
We previously denoted the interest of N-methylpyrazole derivative 11 as antileishmanial agent active against VL
at low micromolar range. Even if its cytotoxicity on macrophages has a significant impact (IC50=15.95 µM), the
SI reached 6.1 and 12.6 towards L. infantum and L. donovani, respectively. In addition, no toxicity was measured
on MRC-5 cells. The second promising compound to consider is the 2-aminopyrimidine derivative 21,
structurally close to CTN1122. This compound showed phenotypic activity on almost all the strains and was
accompanied with a SI from 4.2 to 6.9. 2-(Methylthio)pyrimidine derivative 14 showed a good antiparasitic
activity together with an appealing SI versus macrophages (SI=15.8 for L. amazonensis and SI=26.2 for L.
infantum).
The greater wax moth, G. mellonella, is an insect in the order Lepidoptera which larvae have been widely used
for acute toxicity testing as a great alternative to testing in rodents [25]. The results obtained with this alternative
model are considered to be comparable to those obtained using mammalian models due to G. mellonella’s
immune system that is functionally and structurally similar to the mammalian innate immune system [26].
All compounds were tested for their toxicity in G. mellonella larvae (Figure 3). Groups of 10 larvae were
injected with 10 µL of compound at a dose varying from 10 mg/kg to 50 mg/kg. No compounds were tested at a
higher dose due to poor solubility of the compounds and limitation of DMSO percentage in the solvent (<40%).
The mortality was recorded daily for 7 days. No reduction of viability (or non significative reduction) and no
sign of cuticular darkening were observed for any compounds at a dose of 50 mg/kg. These results indicated no
toxic effect.
[insert Figure 3]
2.2.3. Kinase inhibitory activity
We identified L-CK1.2 as potential target of the reference compound CTN1122, displaying inhibitory potency
of 0.72 µM against this parasitic kinase [11]. Thus, as part of our continuous efforts to investigate the
mechanism of action responsible of antileishmanial properties and to discover new ligands targeting L-CK1.2,
all the compounds were tested for L. major CK1 inhibition [10−13]. Besides IC50 values against LmCK1, the
results presented in Table 4 showed the selectivity profile of the compounds versus a human CK1 and, an array
of 10 representative human and mammalian protein kinases. Interestingly, the activity on these "off-targets"
could allow foreseeing toxicity of the described series in the frame of the therapeutic approach relying on host-
pathogen interaction.

[insert Table 4]
2.2.3.1. Comparative results between phenotypic screening and CK1 inhibition assays of the antileishmanial
imidazo[1,2-a]pyrazines
For compounds 7−9, the results clearly indicated that the replacement of 4-pyridyl ring in CTN1122 by 3-
pyridyl, 5-pyrimidinyl and 2-aminopyrimidin-5-yl moieties dramatically reduced the effectiveness of CTN1122
for both antileishmanial and anti-CK1 activities. The promising 1-methylpyrazol-4-yl derivative 11, as
antiparasitic agent against VL (intracellular assays on L. infantum: IC50=2.60 µM and L. donovani: IC50=1.27
µM), displayed poor activity against LmCK1 (IC50=12.42 µM), suggesting that its potency is not mediated
through CK1 inhibition. In that sub-series, the introduction of a 5-indazolyl azaheterocycle in compound 10
allowed retaining micromolar LmCK1 inhibition, but 2.6 fold less potent than the reference with deleterious
impact on antileishmanial potency.
In the pyrimidin-4-yl sub-series, compound 14 behaved nearly as compound 11 since, despite micromolar
antileishmanial activity against L. infantum and L. amazonensis, it showed a moderate IC50 value of 5.00 µM on
the targeted protein CK1. Moreover, when comparing 2-(methylthio)pyrimidine derivatives 16, 17 and 22 with
their respective 2-aminopyrimidine counterparts 20, 21 and 24, it appears clearly that the amino substitution
enhances the CK1 inhibitory potency (100, 24 and 16 times, respectively).
As expected, the introduction of a fluorine atom on the C2 phenyl ring of imidazopyrazine scaffold did not
interfere with kinase inhibition since compounds 16 versus 17 and 20 versus 21 showed close potency.
Considering the slight difference in the structure, the only result that remains unexplained is the broad
antileishmanial activity of compound 21 (IC50s < 10 µM against four strains over five) in comparison to the
unactivity of compound 20. As pharmacokinetic hypothesis, the improved lipophilicity (cLog P (20)=2.39 vs
cLog P (21)=2.53 [27]), due to the presence of the additional fluorine atom, could favour the passive diffusion
through the cell membranes to reach the parasite. Finally, 8-amino-imidazo[1,2-a]pyrazines 22 and 24 displayed
sub-micromolar and nanomolar activities against LmCK1 but, as previously denoted, were too toxic for further
consideration. Their levels of inhibitory activity were in the same range as the corresponding 8-(N,N-
dimethylamino)imidazo[1,2-a]pyrazines 17 and 21, respectively.
2.2.3.2. Selectivity profile over a panel of 11 human and mammalian protein kinases including HsCK1
In order to check in early stage kinase selectivity issue, since CTN1122 displayed IC50 of 0.92 µM against
HsCK1ε of the host (SI= 1.3 over LmCK1, Table 4), and to foreseen potential toxicity, all the compounds were
tested against a panel of 11 human and mammalian representative protein kinases including HsCK1.
Among the active or very potent compounds 10, 16, 20, 17, 21, 22 and 24, against LmCK1, all the analogues
showed emerging or high selectivity over HsCK1, at the exception of compounds 10 and 22 furnishing poor
selectivity. The SI ranged from 3 to 30, for the best compound 20 considering this criterion. It must be denoted
that, in the contrary to antiparasitic activity, the presence of an additional fluorine atom in compound 21
decreased the selectivity from 30 to 3.8.

The screening assays over the panel of 10 supplementary kinases highlighted a good selectivity of all the
compounds except compounds 22 and 24 displaying micromolar or submicromolar activities against CDK2,
CDK5, CDK9, GSK3 and CLK1. Interestingly, compounds 16, 20 and 21 were also very active on CLK1.
Thus, we assumed that the poor selectivity of compounds 22 and 24 could explain their enhanced cytotoxicity.
2.2.3.3. In silico binding study
In order to interpret SAR in the framework of the enzyme inhibition assay, we devised a homology model of
LmCK1 based on a crystal structure of Oryza sativa CK1 (sharing 75% of sequence identity with LmCK1
ignoring the C-terminal flexible loop, see Figure S1 in Supplementary data). This model building phase was
followed by a short molecular dynamics simulation in order to refine the overall structure of the model, as well
as to explore ATP binding site conformational space (under the hypothesis that active compounds are
competitive inhibitors), so as to take protein flexibility into account during the subsequent docking phase.
Ligands were then docked against a representative subset of binding site conformations obtained by active site
residues RMSD-based clustering. Combinations exhibiting the best score were retained for further analysis (see
Figure S2 in Supplementary data).
Examination of the CK1 binding site showed that a significant part is quite hydrophobic due to the presence of
several Leu and Ile residues (Figure 4).
[insert Figure 4]
Analysis of the structure calculated for one of the best-ranked complex with CTN1122 suggests that the 4-
pyridyl moiety could penetrate the binding site first and occupy one of the most buried parts of the cavity, where
the lone pair beared by the nitrogen atom would interact with a strongly polarized N−H group belonging to the
side chain of the Lys40 residue (which is also involved in a salt bridge with Glu54). This hypothesis could
explain why altering nitrogen atom location and thus the corresponding lone pair orientation would weaken this
interaction for compounds 7, 8, 9, 11, as a consequence of dramatic decrease of CK1 inhibition. Moreover, the
fluorinated aromatic phenyl ring would be accomodated by the above-mentioned lipophilic region of the binding
site.
Ligand-bound state molecular dynamics calculations are currently in progress to further assess the stability and
likelyhood of this binding mode. Similar modelling studies on the structure of the human ortholog of CK1 are
also being carried out in order to rationalize in vitro finding regarding selectivity.
A similar procedure with compounds 17, 20, 21, 22 and 24 confirmed that substituted pyrimidin-4-yl as R3
groups (Table 4) could still be accomodated by the buried pocket in which they could similarly interact with Lys
40, albeit with a not exactly identical imidazopyrine scaffold position. This is likely due to the slight extra bulk
brought by the amino or methylthio substituents. However, the possibility to induce further opening of the
pocket in question (which would allow the binding mode of compounds from this subset to accurately match that
of CTN1122) is expected to be confirmed by the aforementioned in progress ligand bound-state molecular
dynamics simulations.
3. Conclusion

Optimization of the hit compound CTN1122 led to generate 13 new imidazo[1,2-a]pyrazine analogues to
investigate SAR in the fields of antileishmanial potency and LmCK1 inhibition, mostly by modifying the
substitution at the level of the positions 3 and/or 8 of the scaffold. Among the tested compounds on the three
clinical forms of leishmaniasis, four of them (compounds 11, 14, 17 and 21) demonstrated promising efficacy on
VL with micromolar activity against intracellular L. infantum and L. donovani strains, better than the reference.
Considering CL, the modulations decreased drastically the potency against L. major strain but furnished
significantly more active compounds (14, 16 and 21) against L. amazonensis strain. Interestingly, all the
compounds were characterized by very good safety profile displaying low cytotoxicity in vitro, except 8-
aminoimidazo[1,2-a]pyrazine derivatives 22 and 24, and in vivo on Galleria mellonella model. As regards
parasitic target, the results of LmCK1 inhibition assays coupled with binding mode study, through molecular
homology model, gave the structural requirement to retain kinase inhibition: whether for the pyridine or
pyrimidine ring at the position 3 of the central core, the presence of a nitrogen atom at the position 4’ is essential
to establish polar interaction with Lys 40 of the protein ATP-binding site. Interestingly, two of the most active
compounds 20 and 21 on LmCK1, at nanomolar range, showed the highest selectivity towards their human
ortholog, in favour of further development. Nevertheless, additional study is mandatory to explain the lack of
activity of compound 20 on whole parasite. In this context, efforts are also in progress to lead to a detailed
understanding of the mechanism of antileishmanial activity of N-methylpyrazole derivative 11 since it was
highlighted as the most active compound of the series, on both VL strains, without displaying any kinases
inhibition. Thus, LmCK1 proved to be a valuable parasitic target to work on because compound 21, for instance,
inhibiting LmCK1 with an IC50 value of 42 nM, with emerging selectivity profile versus HsCK1 and other nine
kinases, displayed a broad antileishmanial potency against all the tested strains of CL and VL.
In addition, the results of the biological evaluation demonstrated that imidazo[1,2-a]pyrazine heterocycle can be
considered as suitable central core in the search for new drugs for the therapy of leishmaniasis.
All the structural findings will contribute to establishing structure activity relationships to guide further design of
new compounds, which may include new motifs at the positions 3 and/or 8 of the scaffold to promote
antiparasitic activity induced through LmCK1 inhibition and to enhance selectivity indexes.
Finally, assessment of the bioavailability in vitro (ADME) is in progress, for the most promising compounds of
this study, to go ahead in their potential development.
4. Experimental protocols
4.1. Chemistry
All commercial reagents were used without further purification. All solvents were reagent or HPLC grade.
Analytical TLC was performed on silica gel 60 F254 plates. Open column chromatography was performed on
silica gel 60 (70–230 mesh ASTM). The flash column chromatography was performed using a Reveleris® X2
Buchi system, with Buchi cartridge (FlashPure 12g, 40 µm irregular silica). Yields refer to chromatographically
and spectroscopically pure compounds. Melting points were determined on an Electrothermal melting point
apparatus. 1H NMR and 13C NMR spectra were recorded in CDCl3 or in DMSO-d6 on a 400 MHz spectrometer.

Chemical shifts are reported as δ values in parts per million (ppm) relative to tetramethylsilane as internal
standard and coupling constants (J) are given in hertz. Multiplicities are reported as follows: s = singlet, d =
doublet, dd = doublet of doublets, t = triplet, mt = multiplet, bs = broad singlet, tapp = apparent triplet. Low
resolution mass spectra were recorded using an Electrospray Ionization (ESI) Method with Waters ZQ 2000
spectrometer. UPLC column used was an Acquity UPLC® BEH Phenyl (2.1 mm i.d., 50 mm length, 1.7 µm
particle size) from Waters. A linear mobile phase gradient was used with a mobile phase A as 100% of
acetonitrile in water (at 2%) and mobile phase B as 100% acetonitrile. The gradient table was: 0-0.5 min, 0% B;
0.5-4.0 min 0→100% B; 4.0-5.5% 100% B; 5.5-5.7 min 100→0% B; 5.7-7.5 min 0% B. at flow rate 0.5
mL.min-1 and column temperature 35 °C. Formic acid (0.1%) was added in diluent to improve ionization. Pure
compounds 7-11 were analyzed at 1 mg/mL in UPLC grade MeOH (Biosolve), following a 5 µL injection on a
UFLC-ESIHRMS (IT-TOFMS) Shimadzu instrument (Prominence Ultra Fast Liquid Chromatography coupled
to High Resolution Electrospray Ionization Mass Spectrometry combining Ion trap and Time of Flight
analyzers). Analyses were performed by Flow Injection (FIA) in the isocratic flow consisting of
H2O/Acetonitrile 85:15 (V/V) at 300 µL/min. The detection using ESI was performed both in positive and
negative ionization modes in the mass range m/z 100–1000 with a mass accuracy of 7 ppm and a resolution of
10,000 at m/z 500. For compounds 14-17 and 20-24, high resolution mass spectrometry (HRMS) was recorded
on a Waters Xevo G2-XS Qtof spectrometer (coupled with an HPLC Acquity H-Class) for ESI. Microwave
reactions were carried out in a CEM Discover microwave reactor in sealed vessels (monowave, maximum power
300 W, temperature control via IR-sensor, fixed temperature).
Compounds 2-6 were synthesized following procedures previously described [9].
4.1.1. General procedure for the synthesis of compounds 7–11
In a vial were added 2-(4-fluorophenyl)-3-iodo-N,N-dimethylimidazo[1,2-a]pyrazin-8-amine 6 (1 equiv.),
boronic acid (1.1 equiv.), K3PO4 (2.5 equiv.) in a 1,4-dioxane/water mixture (9:1, 15 mL). The vial was purged
twice with N2 for 10 minutes, befor and after addition of PdCl2(dppf) (0.05 equiv.). The vial was sealed and the
suspension was then heated at 100 °C for 19–23 h. After cooling, the resulting mixture was diluted with EtOAc
and water and the organic layer was extracted twice with EtOAc. The combined organic layers were washed
with brine, dried over Na2SO4, filtered and concentrated under vacuum. The crude product was then purified by
chromatography to provide 7–11.
4.1.1.1. 2-(4-Fluorophenyl)-N,N-dimethyl-3-(pyridin-3-yl)imidazo[1,2-a]pyrazin-8-amine (7)
The reaction was carried out using compound 6 (0.147 g, 0.39 mmol), 3-pyridylboronic acid (0.052 g, 0.42
mmol), K3PO4 (0.200 g, 0.96 mmol) and PdCl2(dppf) (0.020 g, 0.04 mmol). in a 1,4-dioxane/water mixture (9:1,
15 mL) and the reaction was stirred for 22 h. The product was purified by silica gel column chromatography
using DCM/EtOH (98:2) as eluent and the compound 7 (34% yield) was obtained as a grey powder. Mp: 139.8
°C; 1H NMR (400 MHz, CDCl3) δ 8.75 (d, 3J = 4.0 Hz, 1H), 8.69 (s, 1H), 7.76 (ddd, 3J = 8.0 Hz, 4J = 2.0 Hz, 4J
= 2.0 Hz, 1H), 7.57 (mt, 2H), 7.48 (dd, 3J = 8.0 Hz, 4J = 4.0 Hz, 1H), 7.35 (d, 3J = 4.4 Hz, 1H), 7.17 (d, 3J = 4.4
Hz, 1H), 6.99 (mt, 2H), 3.65 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ 161.72 (1JC-F = 243 Hz), 150.88, 150.26,

149.60, 139.40, 138.38, 133.05, 129.88 (4JC-F = 3 Hz), 129.29 (3JC-F = 8 Hz, 2C), 128.78, 125.11, 124.47, 119.15,
115.48 (2JC-F = 22 Hz, 2C), 107.45, 39.35 (2C); MS (ESI) m/z (%): 334.2 (100) [M+H]+; UPLC purity = 100%,
Rt 1.84 min; HRMS (TOF MS ES+): calcd. for C19H16FN5 [M+H]+ 334.1463, found: 334.1461.
4.1.1.2. 2-(4-Fluorophenyl)-N,N-dimethyl-3-(pyrimidin-5-yl)imidazo [1,2-a]pyrazin-8-amine (8)
The reaction was carried out using compound 6 (0.350 g, 0.91 mmol), 5-pyrimidylboronic acid (0.125 g, 1.01
mmol), K3PO4 (0.486 g, 2.29 mmol) and PdCl2(dppf) (0.034 g, 0.05 mmol). in a 1,4-dioxane/water mixture (9:1,
15 mL) and the reaction was stirred for 22 h. The product was purified by silica gel column chromatography
using DCM/EtOH (99:1) as eluent and the compound 8 (67% yield) was obtained as a beige powder. Mp: 124.4
°C; 1H NMR (400 MHz, DMSO-d6) 9.36, (s, 1H), 8.97 (s, 2H), 7.55 (mt, 2H), 7.48 (d, 3J = 4.4 Hz, 1H), 7.36 (d,
3J = 4.4 Hz, 1H), 7.21 (mt, 2H), 3.55 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ 161.80 (1JC-F = 244 Hz), 158.55,
158.46 (2C), 149.54, 140.32, 133.38, 129.32 (4JC-F = 3 Hz), 129.51 (3JC-F = 8 Hz, 2C), 128.88, 123.91, 116.09,
115.58 (2JC-F = 21 Hz, 2C), 107.75, 39.80 (2C); MS (ESI) m/z (%): 335.1 (100) [M+H]+; UPLC purity = 100%,
Rt 1.92 min; HRMS (TOF MS ES+): calcd. for C18H15FN6 [M+H]+ 335.1415, found: 335.1419.
4.1.1.3. 3-(2-Aminopyrimidin-5-yl)-2-(4-fluorophenyl)-N,N-dimethylimidazo[1,2-a]pyrazin-8-amine (9)
The reaction was carried out using compound 6 (0.300 g, 0.79 mmol), 2-amino-5-pyrimidylboronic acid (0.120
g, 0.86 mmol), K3PO4 (0.410 g, 1.96 mmol) and PdCl2(dppf) (0.030 g, 0.04 mmol). in a 1,4-dioxane/water
mixture (9:1, 15 mL) and the reaction was stirred for 22 h. The product was purified by flash column
chromatography using cyclohexane/AcOEt (1:1) as eluent and the compound 9 (24% yield) was obtained as a
1
3
beige powder. Mp: 200.4 °C; H NMR (400 MHz, DMSO-d6) 8.30 (s, 2H), 7.66 (mt, 2H), 7.36 (d, J = 4.4 Hz,
3
1H), 7.31 (d, J = 4.4 Hz, 1H), 7.23 (mt, 2H), 7.14 (s, 2H), 3.53 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ
163.50, 160.04 (1JC-F = 242 Hz), 159.67 (2C), 149.51, 139.32, 132.95, 130.17 (4JC-F = 3 Hz), 129.06 (3JC-F = 8
Hz, 2C), 128.42, 117.65, 115.44 (2JC-F = 21 Hz, 2C), 110.98, 107.78, 39.02 (2C); MS (ESI) m/z (%): 350.1 (100)
[M+H]+; UPLC purity = 100%, Rt 1.74 min; HRMS (TOF MS ES+): calcd. for C18H16FN7 [M+H]+ 350.1524,
found: 350.1520.
4.1.1.4. 2-(4-Fluorophenyl)-3-(1H-indazol-5-yl)-N,N-dimethylimidazo[1,2-a]pyrazin-8-amine (10)
The reaction was carried out using compound 6 (0.200 g, 0.52 mmol), 1H-indazole-5-boronic acid (0.093 g, 0.58
mmol), K3PO4 (0.278 g, 1.36 mmol) and PdCl2(dppf) (0.019 g, 0.026 mmol). in a 1,4-dioxane/water mixture
(9:1, 15 mL) and the reaction was stirred for 23 h. The product was purified by silica gel column
chromatography using DCM/EtOH (98:2) as eluent and the compound 10 (77% yield) was obtained as a beige
powder. Mp: 131.3 °C; 1H NMR (400 MHz, DMSO-d6) 13.35 (s, 1H), 8.18 (s, 1H), 7.93 (s, 1H), 7.75 (d, 3J = 8.4
3
3
3
4
3
Hz, 1H), 7.59 (dd, J = 8.8 Hz, J = 5.6 Hz, 2H), 7.35 (dd, J = 8.4 Hz, J = 1.2 Hz, 1H), 7.28 (d, J = 4.8 Hz,
3
1H), 7.23 (d, J = 4.8 Hz, 1H), 7.13 (mt, 2H), 3.56 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ 161.47 (1JC-F
=
243 Hz), 149.55, 139.82, 138.26, 134.12, 132.44, 130.32 (4JC-F = 3 Hz), 128.89 (3JC-F = 8 Hz, 2C), 128.34,
128.12, 123.50, 123.23, 122.70, 120.31, 115.24 (2JC-F = 22 Hz, 2C), 111.59, 107.49, 39.06 (2C); MS (ESI) m/z

(%): 373.1 (100) [M+H]+; UPLC purity = 100%, Rt 2.14 min; HRMS (TOF MS ES+): calcd. for C21H17FN6
[M+H]+ 371.1426, found: 371.1425.
4.1.1.5. 2-(4-Fluorophenyl)-N,N-dimethyl-3-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyrazin-8-amine (11)
The reaction was carried out using compound 6 (0.250 g, 0.65 mmol), 1-methyl-1H-pyrazole-4-boronic acid
(0.150 g, 0.72 mmol), K3PO4 (0.347 g, 1.63 mmol) and PdCl2(dppf) (0.024 g, 0.032 mmol). in a 1,4-
dioxane/water mixture (9:1, 15 mL) and the reaction was stirred for 19 h. The product was purified by silica gel
column chromatography using petroleum ether/EtOAc (3:1) as eluent and the compound 11 (85% yield) was
1
obtained as a light beige powder. Mp: 125.0 °C; H NMR (400 MHz, DMSO-d6) 8.07 (s, 1H), 7.73 (mt, 2H),
7.61 (s, 1H), 7.31 (s, 2H), 7.20 (mt, 2H), 3.95 (s, 3H), 3.52 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ 161.60
(1JC-F = 243 Hz), 149.48, 139.32, 138.94, 132.78, 131.63, 130.43 (4JC-F = 3 Hz), 128.98 (3JC-F = 8 Hz, 2C),
128.26, 115.26 (2JC-F = 21 Hz, 2C), 114.65, 107.77, 39.25 (2C), 38.82; MS (ESI) m/z (%): 337.1 (100) [M+H]+;
UPLC purity = 100%, Rt 1.93 min; HRMS (TOF MS ES+): calcd. for C18H17FN6 [M+H]+ 373.1571, found:
373.1570.
4.1.2. 4-Iodo-2-(methylsulfanyl)pyrimidine (13)
4-Chloro-2-(methylsulfanyl)pyrimidine 12 (10.00 g, 62.3 mmol) was added to HI (57% in water, 50 mL). The
reaction mixture was stirred at room temperature for 72 h. After filtration, the precipitate was dissolved in water
(50 mL), and the pH mixture was adjusted to 8 with NaHCO3. The aqueous layer was extracted with CH2Cl2 and
the organic layer was washed with saturated aqueous solution of Na2S2O3 and water, dried over Na2SO4, filtered,
and concentrated under reduced pressure. Trituration with petroleum ether afforded 13 (14.85 g, 95% yield) as a
1
white powder. Mp: 51–52 °C (lit. 52–53 °C) [17]; H NMR (400 MHz, CDCl3) δ 7.99 (d, 3J = 5.1 Hz, 1H, H6),
7.39 (d, 3J = 5.1 Hz, 1H, H5), 2.53 (s, 3H, SCH3).
4.1.3. General procedure for the synthesis of compounds 14–15
In a vial were added 2-aryl-8-chloroimidazo[1,2-a]pyrazine (1 equiv.), compound 13 (2 equiv.), Pd(OAc)2 (0.2
equiv.), PPh3 (0.4 equiv.), Cs2CO3 (1.5 equiv.) in DMF. The vial was sealed and purged with N2 through the
septum inlet for 10 min. The suspension was then heated at 90 °C for 15–20 h. After cooling, the resulting
mixture was diluted with EtOAc and water and the organic layer was extracted twice with EtOAc. The combined
organic layers were washed with water, dried over Na2SO4, filtered and concentrated under vacuum. The crude
product was then purified by flash column chromatography to provide 14–15.
4.1.3.1. 8-Chloro-3-[2-(methylsulfanyl)pyrimidin-4-yl]-2-phenylimidazo[1,2-a]pyrazine (14)
The reaction was carried out using 8-chloro-2-phenylimidazo[1,2-a]pyrazine 3 (2.00 g, 8.7 mmol), compound 13
(4.39 g, 17.4 mmol), Pd(OAc)2 (0.39 g, 1.7 mmol), PPh3 (0.91 g, 3.5 mmol), Cs2CO3 (4.26 g, 13.1 mmol) in
DMF (20 mL) and the reaction mixture was heated for 20 h. Purification by flash column chromatography using
cyclohexane/AcOEt (90:10) as eluent afforded 14 (0.97 g, 31% yield) as a yellow powder. Mp: 178–179 °C; 1H

NMR (400 MHz, CDCl3) δ 9.39 (d, 3J = 4.8 Hz, 1H), 8.39 (d, 3J = 5.2 Hz, 1H), 7.86 (d, 3J = 4.8 Hz, 1H), 7.67–
7.65 (m, 2H), 7.48–7.45 (m, 3H), 6.89 (d, 3J = 5.6 Hz, 1H), 2.66 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 173.10,
157.24, 156.31, 150.68, 143.87, 138.64, 132.98, 129.62, 129.54 (2C), 129.20, 128.97 (2C), 120.44, 119.49,
115.21, 14.23; MS (TOF MS ES+) m/z (%): 354.06 (100) [M+H]+, 376.04 [M+H+Na]+; UPLC purity = 98%, Rt
2.89 min; HRMS (TOF MS ES+): calcd. for C17H12ClN5S [M+H]+ 354.0580, found: 354.0569.
4.1.3.2. 8-Chloro-2-(4-fluorophenyl)-3-[2-(methylsulfanyl)pyrimidin-4-yl]imidazo[1,2-a]pyrazine (15)
The reaction was carried out using 8-chloro-2-(4-fluorophenyl)imidazo[1,2-a]pyrazine 4 (0.50 g, 2.0 mmol),
compound 13 (1.02 g, 4.0 mmol), Pd(OAc)2 (0.09 g, 0.4 mmol), PPh3 (0.21 g, 0.8 mmol), Cs2CO3 (0.99 g, 3.0
mmol) in DMF (5 mL) and the reaction mixture was heated for 15 h. Purification by flash column
chromatography using cyclohexane/AcOEt (1:1) as eluent afforded 15 (0.35 g, 47% yield) as an orange powder.
Mp: 188–189 °C; 1H NMR (400 MHz, CDCl3) δ 9.36 (d, 3J = 4.8 Hz, 1H), 8.43 (d, 3J = 5.2 Hz, 1H), 7.88 (d, 3J
3
= 4.8 Hz, 1H), 7.68–7.64 (m, 2H), 7.20–7.16 (m, 2H), 6.90 (d, J = 5.2 Hz, 1H), 2.68 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 173.15, 163.68 (1JC-F = 248 Hz), 156.94, 156.37, 149.70, 143.90, 131.50 (3JC-F = 8 Hz, 2C),
129.41, 128.94, 120.35, 119.40, 116.21 (2JC-F = 21 Hz, 2C), 115.04, 14.28; MS (TOF MS ES+) m/z (%): 372.05
(100) [M+H]+; UPLC purity = 99%, Rt 3.08 min; HRMS (TOF MS ES+): calcd. for C17H11ClFN5S [M+H]+
372.0486, found: 372.0475.
4.1.4. General procedure for the synthesis of compounds 16–17
In a vial were added compound 14 or 15 (1 equiv.), dimethylamine (40% in water, 20 equiv.), K2CO3 (1 equiv.)
in DMF. The vial was sealed and the suspension was heated at 90 °C for 4–17 h. After cooling, the resulting
mixture was diluted with EtOAc and water and the organic layer was extracted twice with EtOAc. The combined
organic layers were washed with water, dried over Na2SO4, filtered and concentrated under vacuum. The crude
product was then purified by flash column chromatography to provide 16–17.
4.1.4.1. N,N-Dimethyl-3-[2-(methylsulfanyl)pyrimidin-4-yl]-2-phenylimidazo[1,2-a]pyrazin-8-amine (16)
The reaction was carried out using compound 14 (500 mg, 1.4 mmol), dimethylamine (40% in water, 1.4 mL,
28.2 mmol), K2CO3 (195 mg, 1.4 mmol) in DMF (15 mL) and the reaction mixture was heated for 17 h.
Purification by flash column chromatography using cyclohexane/AcOEt (90:10) as eluent afforded 16 (282 mg,
55% yield) as a yellow powder. Mp: 136–137 °C; 1H NMR (400 MHz, CDCl3) δ 8.57 (d, 3J = 4.6 Hz, 1H), 8.33
(d, 3J = 5.3 Hz, 1H), 7.64–7.61 (m, 2H), 7.48 (d, 3J = 4.7 Hz, 1H), 7.42–7.41 (m, 3H), 6.86 (d, 3J = 5.3 Hz, 1H),
3.59 (s, 6H, -N(CH3)2), 2.64 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 173.05, 157.33, 157.04, 150.26, 146.63,
134.95, 134.06, 129.37 (2C), 128.97, 128.90 (2C), 119.33, 116.00, 110.11, 77.36, 40.46 (2C), 14.37; MS (TOF
MS ES+) m/z (%): 363.14 (100) [M+H]+; UPLC purity = 98%, Rt 2.50 min; HRMS (TOF MS ES+): calcd. for
C19H18N6S [M+H]+ 363.1392, found: 363.1381.

4.1.4.2. 2-(4-Fluorophenyl)-N,N-dimethyl-3-[2-(methylsulfanyl)pyrimidin-4-yl]imidazo[1,2-a]pyrazin-8-amine
(17)
The reaction was carried out using compound 15 (220 mg, 0.6 mmol), dimethylamine (40% in water, 0.6 mL,
11.8 mmol), K2CO3 (82 mg, 0.6 mmol) in DMF (10 mL) and the reaction mixture was heated for 4 h.
Purification by flash column chromatography using cyclohexane/AcOEt (1:1) as eluent afforded 17 (131 mg,
58% yield) as a yellow powder. Mp: 141–142 °C; 1H NMR (400 MHz, CDCl3) δ 8.52 (d, 3J = 5.2 Hz, 1H), 8.39
(d, 3J = 5.2 Hz, 1H), 7.62–7.59 (m, 2H), 7.51 (d, 3J = 5.2 Hz, 1H), 7.14–7.11 (m, 2H), 6.86 (d, 3J = 5.2 Hz, 1H),
3.68 (s, 6H), 2.63 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 173.17, 163.18 (1JC-F = 247 Hz), 157.06, 156.94,
149.76, 145.43, 134.62, 131.00 (3JC-F = 8 Hz, 2C), 129.88, 128.09, 119.31, 115.84 (2JC-F = 22 Hz, 2C), 115.73,
109.89, 40.49 (2C), 14.28; MS (TOF MS ES+) m/z (%): 381.13 (100) [M+H]+; UPLC purity = 100%, Rt 2.38
min; HRMS (TOF MS ES+): calcd. for C19H17FN6S [M+H]+ 381.1298, found: 381.1284.
4.1.5. General procedure for the synthesis of compounds 18–19
In a three-neck round-bottom flask was added compound 16 or 17 (1 equiv.) in methanol. Oxone® dissolved in
water was then added dropwise and the suspension was stirred vigorously at room temperature for 17–24h. The
resulting mixture was evaporated under reduced pressure. The residue was diluted with EtOAc and the organic
layer was washed with a saturated aqueous NaHCO3 solution, water, dried over Na2SO4, filtered and
concentrated under vacuum. The crude product was used for the next step without further purification.
4.1.5.1. N,N-Dimethyl-3-[2-(methylsulfonyl)pyrimidin-4-yl]-2-phenylimidazo[1,2-a]pyrazin-8-amine (18)
The reaction was carried out using compound 16 (0.40 g, 1.1 mmol) in MeOH (19 mL), Oxone® (1.02 g, 3.3
mmol) in water (18 mL) and the reaction was stirred for 17 h. The crude product (426 mg, 98% yield) was
obtained as a yellow powder. 1H NMR (400 MHz, CDCl3) δ 8.87 (d, 3J = 4.0 Hz, 1H), 8.62 (d, 3J = 4.0 Hz, 1H),
7.64–7.60 (m, 3H), 7.50–7.49 (m, 3H), 7.38 (d, 3J = 5.6 Hz, 1H), 7.26 (d, 3J = 5.6 Hz, 1H), 3.66 (s, 6H), 3.42 (s,
3H); MS (ESI) m/z (%): 395.1 (100) [M+H]+; UPLC purity = 99%, Rt 2.09 min.
4.1.5.2. 2-(4-Fluorophenyl)-N,N-dimethyl-3-[2-(methylsulfonyl)pyrimidin-4-yl]imidazo[1,2-a]pyrazin-8-amine
(19)
The reaction was carried out using compound 17 (130 mg, 0.34 mmol) in MeOH (5.9 mL), Oxone® (315 mg,
1.03 mmol) in water (5.5 mL) and the reaction was stirred for 24 h. The crude product (42 mg, 30% yield) was
obtained as a yellow powder. 1H NMR (400 MHz, CDCl3) δ 8.84 (d, 3J = 4.8 Hz, 1H), 8.63 (d, 3J = 4.9 Hz, 1H),
7.63–7.59 (m, 3H), 7.36 (d, 3J = 4.9 Hz, 1H), 7.18 (tapp, 3Japp = 8.8 Hz, 2H), 3.60 (s, 6H), 3.43 (s, 3H); MS (ESI)
m/z (%): 413.1 (100) [M+H]+; UPLC purity = 97%, Rt 2.07 min.
4.1.6. General procedure for the synthesis of compounds 20–21

In a vial were added compound 18 or 19, ammonium hydroxide (25% in water) in 1,4-dioxane. The vial was
sealed and the suspension was heated at 110 °C for 1 h 30–9 h. After cooling, the resulting mixture was diluted
with EtOAc and water and the organic layer was washed with water, dried over Na2SO4, filtered and
concentrated under vacuum. The crude product was then purified by chromatography to provide 20–21.
4.1.6.1. 3-(2-Aminopyrimidin-4-yl)-N,N-dimethyl-2-phenylimidazo[1,2-a]pyrazin-8-amine (20)
The reaction was carried out using compound 18 (200 mg, 0.51 mmol), ammonium hydroxide (25% in water, 5
mL) in 1,4-dioxane (5 mL) and the reaction mixture was heated for 9 h. Purification by flash column
chromatography using cyclohexane/AcOEt (1:1) as eluent afforded 20 (85 mg, 51% yield) as a white powder.
Mp: 224–225 °C; 1H NMR (400 MHz, CDCl3) δ 8.47 (d, 3J = 4.8 Hz, 1H), 8.17 (d, 3J = 4.9 Hz, 1H), 7.68–7.65
(m, 2H), 7.45 (d, 3J = 4.8 Hz, 1H), 7.42–7.39 (m, 3H), 6.57 (d, 3J = 4.9 Hz, 1H), 5.25 (bs, 2H), 3.60 (s, 6H); 13
C
NMR (100 MHz, CDCl3) δ 162.87, 158.40, 158.26, 150.53, 145.37, 134.71, 134.19, 129.20 (2C), 128.90, 128.53
(2C), 128.46, 119.32, 111.92, 109.82, 39.98 (2C); MS (TOF MS ES+) m/z (%): 332.16 (100) [M+H]+; UPLC
purity = 98%, Rt 1.77 min; HRMS (TOF MS ES+): calcd. for C18H17N7 [M+H]+ 332.1624, found: 332.1614.
4.1.6.2. 3-(2-Aminopyrimidin-4-yl)-2-(4-fluorophenyl)-N,N-dimethylimidazo[1,2-a]pyrazin-8-amine (21)
The reaction was carried out using compound 19 (84 mg, 0.20 mmol), ammonium hydroxide (25% in water, 3
mL) in 1,4-dioxane (3 mL) and the reaction mixture was heated for 1 h 30. Purification by flash chromatography
(dichloromethane/ethanol, 98:2) afforded 21 (52 mg, 73% yield) as a white powder. Mp: 172–173 °C; 1H NMR
3
3
3
(400 MHz, CDCl3) δ 8.44 (d, J = 4.8 Hz, 1H), 8.20 (d, J = 5.6 Hz, 1H), 7.66–7.63 (m, 2H), 7.46 (d, J = 4.4
Hz, 1H), 7.12–7.08 (m, 2H), 6.56 (d, 3J = 5.2 Hz, 1H), 5.24 (bs, 2H), 3.60 (s, 6H); 13C NMR (100 MHz, CDCl3)
δ 163.00 (1JC-F = 247 Hz), 162.90, 158.40, 158.24, 150.41, 144.31, 134.62, 130.94 (3JC-F = 8 Hz, 2C), 130.22,
128.89, 119.28, 115.59 (2JC-F = 22 Hz, 2C), 111.77, 109.74, 40.02 (2C); MS (TOF MS ES+) m/z (%): 350.15
(100) [M+H]+; UPLC purity = 100%, Rt 1.83 min; HRMS (TOF MS ES+): calcd. for C19H18N6S [M+H]+
350.1529, found: 350.1517.
4.1.7. 2-(4-Fluorophenyl)-3-[2-(methylsulfanyl)pyrimidin-4-yl]imidazo[1,2-a]pyrazin-8-amine (22)
In a vial were added compound 15 (0.125 g, 0.34 mmol), ammonium hydroxide (3.3 mL, 25% in water) and
isopropanol (1.6 mL). The vial was sealed and heated under microwave irradiation at 120 °C for 1 h (P = 80 W).
After cooling, water was added and the resulting precipitate was filtered off. The filtrate was extracted with
dichloromethane and the organic layer was dried over Na2SO4, filtered and concentrated under vacuum. The
combined solids were washed with diisopropylic ether to provide 22 (0.118 g, 100% yield) as a beige powder.
Mp: 185–186 °C; 1H NMR (400 MHz, CDCl3) δ 8.70 (d, 3J = 5.0 Hz, 1H), 8.37 (d, 3J = 5.0 Hz, 1H), 7.61 (dd, 3J
= 8.4 Hz, 3J = 5.2 Hz, 2H), 7.47 (d, 3J = 4.8 Hz, 1H), 7.16 (mt, 2H), 6.83 (d, 3J = 5.2 Hz, 1H), 5.72, (s, 2H), 2.65
(s, 3H); 13C NMR (100 MHz, CDCl3) δ 173.10, 163.32 (1JC-F = 248 Hz), 157.00, 156.80, 149.78, 146.93, 133.37,
131.11 (3JC-F = 8 Hz, 2C), 129.84 (4JC-F = 4 Hz), 129.43, 120.08, 116.07 (2JC-F = 22 Hz, 2C), 114.98, 111.92,
14.21; MS (TOF MS ES+) m/z (%): 353.10 (100) [M+H]+; UPLC purity = 97%, Rt 2.09 min; HRMS (TOF MS
ES+): calcd. for C17H13FN6S [M+H]+ 353.0985, found: 353.0979.

4.1.8. 2-(4-Fluorophenyl)-3-[2-(methylsulfonyl)pyrimidin-4-yl]imidazo[1,2-a]pyrazin-8-amine (23)
In a round-bottom flask was dissolved compound 22 (100 mg, 0.34 mmol) in MeOH (4.6 mL). Oxone® (263
mg, 0.85 mmol) in water (4.6 mL) was added and the reaction was stirred at room temperature for 24 h. The
resulting mixture was evaporated under reduced pressure. Water was added and the mixture was extracted with
EtOAc and the organic layer was dried over Na2SO4, filtered and concentrated under vacuum. The crude product
1
(81 mg, 74% yield) was used for the next step without further purification. H NMR (400 MHz, CDCl3) δ 8.97
(d, 3J = 4.6 Hz, 1H), 8.65 (d, 3J = 5.6 Hz, 1H), 7.61 (dd, 3J = 8.6 Hz, 3J = 5.4 Hz, 2H), 7.56 (d, 3J = 4.6 Hz, 1H),
3
7.35 (d, J = 5.6 Hz, 1H), 7.24 (mt, 2H), 6.02 (s, 2H), 3.42 (s, 3H); MS (ESI) m/z (%): 385.1 (100) [M+H]+;
UPLC purity = 79%, Rt 1.92 min.
4.1.9. 3-(2-Aminopyrimidin-4-yl)-2-(4-fluorophenyl)imidazo[1,2-a]pyrazin-8-amine (24)
In a vial were added compound 23 (70 mg, 0.18 mmol), ammonium hydroxide (3 mL, 25% in water) in 1,4-
dioxane (3 mL). The vial was sealed and the suspension was heated at 110 °C for 1 h 15. After cooling, water
was added the resulting mixture was extracted with EtOAc. The organic layer was dried over Na2SO4, filtered
and concentrated under vacuum. Purification by flash chromatography (EtOAc) afforded 24 (25 mg, 43% yield)
3
as a white powder. Mp: 128.5 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.57 (d, J = 4.6 Hz, 1H), 8.16 (d, 3J = 5.4
Hz, 1H), 7.66 (mt, 2H), 7.35 (d, 3J = 4.6 Hz, 1H), 7.29 (mt, 2H), 7.07 (s, 2H), 6.92 (s, 2H), 6.34 (d, 3J = 5.4 Hz,
1H); 13C NMR (100 MHz, DMSO-d6) δ 163.47, 162.15 (1JC-F = 245 Hz), 158.56, 156.75, 150.32, 144.10, 132.44,
131.06 (3JC-F = 8 Hz, 2C), 130.58 (4JC-F = 3 Hz), 129.29, 120.09, 115.45 (2JC-F = 22 Hz, 2C), 110.35, 108.89; MS
(TOF MS ES+) m/z (%): 322.12 (100) [M+H]+; UPLC purity = 100%, Rt 1.52 min; HRMS (TOF MS ES+):
calcd. for C16H12FN7 [M+H]+ 322.1216, found: 322.1208.
4.2. Molecular modelling
4.2.1 Homology modelling
The 2.0 Å resolution X-ray crystal structure of Oryza sativa CK1 with accession code 3SV0 was retrieved from
the Protein Data Bank, and used as template for the construction of a homology model for the amino-acids
sequence associated with entry Q9NHE1 from the UniProt database with the MODELLER v9.22 software
package [28]. A range of structures of the ab initio C-terminal loop (not covered by the template) was generated
using the same software, and the solution exhibiting the best score (and folding) was merged with the part
obtained by homology to yield a complete model of the protein.
4.2.2 Molecular dynamics
The resulting homology model was placed within a simulation periodic box with a 1 nm margin. The protein was
solvated with water molecules taken from a library of pre-equilibrated pure solvent model, and sodium chloride
was added in sufficient quantity to reach electric neutrality as well as an ionic strength corresponding to a c =

150 mM salt concentration. Simulation was performed with GROMACS v2019.1 software package [29], using
AMBER99SB-ILDN force field [30] in combination with theTIP3P water model [31].
The so-built system was energy-minimized using a steepest descent algorithm then subjected to two consecutive
100 ps MD simulations for equilibration purposes (the first one with Berendsen [32] and the second one with
Nosé-Hoover [33] thermostat). Production MD simulation was then conducted with Nosé–Hoover thermostat
and Parrinello–Rahman pressure coupling [34] for a total simulated time of 10ꢀns. Atom coordinates were saved
every simulated 20 ps for trajectory analysis.
10 representative conformations of the binding site were selected after binding site residues RMSD-based
clustering of the trajectory performed using the GROMOS algorithm [35] with a cut-off value of 0.66 Å.
4.2.3 Molecular docking
Initial coordinates for ligands were generated using the MarvinSketch v19.26 software package [36], and
compounds were freely docked in a parallelepipedic box circumscribed to the ATP binding site using the
AutoDock Vina v1.1.2 software package [37] in each of the 10 representative protein structures derived from the
molecular dynamics calculation. Best-ranked solutions in terms of scoring function were retained for analysis
and depiction using the UCSF Chimera v1.14 software package [38].
4.3. Biological evaluation
4.3.1 Antileishmanial activity
4.3.1.1 Cell lines and cultures
The mouse monocyte/macrophage cell line RAW264.7 was maintained in culture in DMEM supplemented with
10% heat-inactivated fetal bovine serum.
The Leishmania strains were obtained from CNRLeish (Centre National de Référence des Leishmania). Strains
used for in vitro experiments were Leishmania donovani (MHOM/ET/67/HU3), L. infantum
(MHOM/FR/2008/LEM5700), L. major (MHOM/PT/92/CRE26), L. amazonensis (MHOM/BR/73/M2269) and
L. braziliensis (MHOM/BR/75/M2903b).
These strains were used for in vitro experiments. Promastigotes forms were grown in M-199 medium (Sigma-
Aldrich) supplemented with 40 mM HEPES, 100 µM adenosine, 0.5 mg/mL haemin, 10% heat-inactivated fœtal
bovine serum (FBS) (Invitrogen Life Technologies) at 25°C in a dark environment under an atmosphere of 5%
CO2. For differentiation of promastigotes into axenic amastigotes, plateau-phase cultures of promastigotes were
used and a suspension of parasites was prepared at 107 parasites / mL in axenic amastigote medium (1 x M-199,
supplemented with 40 mM HEPES, 100 µM adenosine, 0.5 mg/mL haemin, 10% FBS; 2 mM MgCl2, 2 mM
CalCl2). The pH was adjusted to pH 5.5. Axenic amastigotes were grown at 37 °C in 5% CO2. The axenic
amastigote experiments were performed only after parasite differentiation.
Leishmania 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, Sigma–Aldrich) at 26 °C [39].
4.3.1.2 In vitro antileishmanial evaluation on intramacrophage amastigotes
The mouse monocyte/macrophage cell line RAW 264.7 was maintained in DMEM supplemented with 10% heat-
inactivated fetal bovine serum. RAW 264.7 cells were seeded into a 96-well microtiter plate at a density of
100,000 cells/well in 100 µL of DMEM. After incubation in a 5% CO2 incubator at 37 °C for 24 h, the culture
medium was replaced with 100 µL of fresh DMEM containing a suspension of plateau-phase culture of
promastigotes to reach a ratio parasite/macrophage of 16:1. In each plate, 8 wells contained axenic amastigotes
(control of the parasite growth), 8 wells contained only macrophages (control of the macrophage growth) and
finally 8 wells contained infected macrophages (control of the growth of intramacrophage parasites). After
incubation in a 5 % CO2 incubator at 37 °C for 24 h (the time needed by the parasite to infect the macrophage),
cells were visualized using an inverted microscope to check the presence of inside parasites. Then, the culture
medium was replaced with fresh DMEM to realize the serial dilution. Final concentrations of test compound
were obtained by serial dilution ranging between 391 nM and 100 µM for pure compounds. After incubation,
cells were visualized using an inverted microscope to check the cell lysis, their morphology and the presence of
outside parasites. The viability of the amastigotes into macrophages was then assessed using the SYBR1 Green I
(Invitrogen, France) incorporation method. Thus, the medium was removed and the cells were lysed in 100 µL
lysis buffer. The plates were then subjected to 3 freeze-thaw cycles to achieve complete lysis. The parasite lysis
suspension was diluted 1:1 in lysis buffer with SYBR Green I as previously described. The IC50 value was
calculated by nonlinear regression using icestimator website 1.2 version: http://www.antimalarial-
icestimator.net/MethodIntro.htm. Fluorescence obtained was compared to those from the range obtained with
parasite, infected cell and non-infected cell densities. The activity of the compounds was expressed as IC50
(concentration of drug inhibiting the parasite growth by 50%, comparatively to the controls treated with the
excipient only). Miltefosine was used as the reference drug.
For Leishmania major (MHOM/IL/81/BNI), 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. Activity against the intracellular amastigote
stage of the parasite was determined after infection of Balb/c mice peritoneal macrophages (CE Janvier, Le
Genest, France). 100 µL of a peritoneal 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 µL of a stationary phase
promastigotes (1.5 x 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 µM. After 4 days, cultures were fixed with methanol, stained 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 [39]. Miltefosine
was used as a reference at the concentrations of 100, 10 and 1 µM. Experiments were done twice.

4.3.2 Cytotoxicity determination
4.3.2.1 Evaluation of compounds cytotoxicity using SYBR Green method
Cytotoxicity was evaluated on mouse monocyte/macrophage-like cell line RAW 264.7 (Cell collection CNRS
UMR 8076 BioCis, University Paris-Saclay). The RAWW 264.7 cells were cultured in Dulbecco’s modified
Eagle's minimal essential medium (DMEM) (Invitrogen Life Technologies) supplemented with 10% heat-
inactivated fetal bovine serum, at 37 °C in a humidified atmosphere containing 5% CO2. Cells were seeded into a
96-well microtitration plate at a density of 100,000 cells/well in 100 µL. After incubation in a 5 % CO2 incubator
at 37 °C for 24 h, the culture medium was replaced with 100 µL of fresh DMEM containing two-fold serial
dilutions of the tested compounds. Final concentrations of tested compounds or extracts were obtained by serial
dilution ranging between 49 nM and 100 µM for pure compounds and between 49 ng/mL and 100 µg/mL for
extracts. Miltefosine was used as the reference drug and cells without drug treatment as growth control. After
incubation, cells were visualized using an inverted microscope to check their morphology and to control the drug
solubility. After a 48 h incubation time at 37 °C with 5% CO2, growth was determined by using SYBR Green 1
as previously described [40]. The cytotoxicity of the compounds was expressed as CC50 (Cytotoxic
Concentration 50%: concentration inhibiting the macrophages growth by 50%). The CC50 was calculated by
nonlinear
regression
using
icestimator
website
1.2
version:
http://www.antimalarial-
icestimator.net/MethodIntro.htm.
4.3.2.2. Cytotoxicity assay on MRC-5 fibroblat cells
Cytotoxicity of compounds was studied with human fibroblast (MRC-5). Cells were grown in RPMI 1640
medium (Sigma-aldrich) supplemented with 10% foetal bovin serum (Sigma-aldrich). Drugs were tested at three
concentrations (100, 10 and 1 µM) in triplicate for a 96 h-incubation time. Cytotoxicity was measured on the
Fluorolite 1000 (Dynatech, France) after a 4-h incubation time with 10 µL of resazurin solution (700 µM).
Inhibitory concentration 50 (IC50) is a mean of triplicate values [41,42].
4.3.2.3. Evaluation of compounds cytotoxicity using in vivo Galleria mellonella model
G. mellonella larvae were bred in IICiMed laboratory at 30-32 °C in a mixture of flour, honey, oat meal, glycerol
and pollen until 260 to 320 mg (6th developmental stage). Groups of ten larvae were randomly selected for the
experiments. Each larva was injected in the 4th left pro-leg with 10 µL of antifungal compounds or an equivalent
volume of PBS/DMSO for control group at a dose varying between 10 mg/kg and 50 mg/kg using a 0.3 mL -
30G-Insulin syringe. Larvae were incubated in the dark at 37 °C and survival was evaluated daily for 7 days.
Death was assessed by the lack of movement in response to stimulus, with or without discolouration. Percentage
survival was plotted using GraphPad Prism and survival analyses were determined using the log-rank test and
the Kaplan-Meier survival curves. Two independent exepriments were performed.

4.3.3 Kinase inhibition assays
Two different methods were used to study the effect of the compounds on kinase activities: (i) a radiometric
assay using [γ-33P] ATP and (ii) a luminescent ADP detection assay (ADP-Glo assay, Promega, Madison, WI).
4.3.3.1. Experimental details on the radiometric assay
Kinase activities were assayed in appropriate kinase buffer, with either protein or peptide as substrate in the
presence of 15 µM [γ-33P] ATP (3,000 Ci/mmol; 10 mCi/mL) in a final volume of 30 µL following the assay
described in [43]. Controls were performed with appropriate dilutions of dimethylsulfoxide (DMSO). Full-length
kinases are used unless specified. Peptide substrates were obtained from ProteoGenix (Schiltigheim, France) or
Sigma for Histone H1 (#H5505), Casein (#C4032) and MBP (#M1891).
4.3.3.2 Experimental details on ADP-GloTM assay
Kinase enzymatic activities were assayed in 384-well plates using the ADP-GloTM assay kit (Promega, Madison,
WI) according to manufacturer’s recommendations ([44] for details on this method). Briefly, reactions were
carried out in a final volume of 6 µL for 30 min at 30 °C in appropriate kinase buffer, with either protein or
peptide as substrate in the presence of 10 µM ATP. The experimental conditions used to perform the various
kinase assays are reported in [45]. In order to determine the half maximal inhibitory concentration (IC50), the
assays were performed in duplicate in the absence or presence of increasing doses of the tested compounds.
Kinase activities are expressed in % of maximal activity, i.e. measured in the absence of inhibitor. IC50 values
were determined from the dose response curves using Prism-GraphPad (GraphPad Software, San Diego, CA,
USA).
Buffers: (A) 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 25 mM Tris-HCl pH 7.5, 50 µg/mL heparin; (B) 5 mM
MOPS pH 7.2, 2.5 mM β-glycerophosphate, 4 mM MgCl2, 2.5 mM MnCl2, 1 mM EGTA, 0.5 mM EDTA, 50
µg/mL BSA, 0.05 mM DTT; (C) 25 mM MOPS, pH7.2, 12.5 mM β-glycerophosphate, 25 mM MgCl2, 5 mM
EGTA, 2 mM EDTA, 0.25 mM DTT.
Native or recombinant protein kinases used during this study were:
LmCK1 (LmjF35.1010, from Leishmania major, recombinant, expressed in bacteria [12], was assayed in buffer
A with 170 µM of the following peptide: RRKHAAIGSpAYSITA (“Sp” stands for phosphorylated serine) as
CK1-specific substrate.
HsCK1ε (human, recombinant, expressed by baculovirus in Sf9 insect cells) was assayed in buffer A with 170
µM of the following peptide: RRKHAAIGSpAYSITA.
HsCDK2/CyclinA (human, cyclin-dependent kinase-2, kindly provided by Dr. A. Echalier-Glazer, Leicester,
UK) was assayed in buffer A with 37.2 µM of histone H1 as substrate.
HsCDK5/p25 (human, recombinant, expressed in bacteria) was assayed in buffer A, with 37.2 µM of histone H1
as substrate.

HsCDK9/CyclinT (human, recombinant, expressed by baculovirus in Sf9 insect cells) was assayed in buffer A
with 83 µM of the following peptide: YSPTSPSYSPTSPSYSPTSPSKKKK, as substrate.
SscGSK-3α/β (Sus scrofa domesticus, native porcine enzyme, affinity purified from porcine brain) was assayed
in
buffer
A
with
20
µM
of
GS-1
peptide,
a
GSK-3-selective
substrate
(YRRAAVPPSPSLSRHSSPHQSpEDEEE).
HsPIM1 (human proto-oncogene, recombinant, expressed in bacteria) was assayed in buffer A with 18.6 µM of
histone H1 (Sigma #H5505) as substrate.
HsRIPK3 (human, recombinant, expressed by baculovirus in Sf9 insect cells) was assayed in buffer B with 8.7
µM of MBP as substrate.
HsHASPIN (human, kinase domain, amino acids 470 to 798, recombinant, expressed in bacteria) was assayed in
buffer A with 8 µM of Histone H3 (1-21) peptide (ARTKQTARKSTGGKAPRKQLA) as substrate.
HsAURKB (human Aurora kinase B, recombinant, expressed by baculovirus in Sf9 insect cells, SignalChem,
product #A31-10G) was assayed in buffer C with 8.7 µM of MBP as substrate.
MmCLK1 (from Mus musculus, recombinant, expressed in bacteria) was assayed in buffer A with 57.3 µM of
the following peptide: GRSRSRSRSRSR as substrate.
RnDYRK1A (Rattus norvegicus, amino acids 1 to 499 including the kinase domain, recombinant, expressed in
bacteria, DNA vector kindly provided by Dr. W. Becker, Aachen, Germany) was assayed in buffer A with 10.7
µM of the following peptide : KKISGRLSPIMTEQ as substrate.
Controls inhibitors:
To validate each kinase assay, the following model inhibitors were used under the same conditions than the
tested compounds: Barasertib (AZD1152-HQPA, #S1147, Selleckchem) for AuroraB; Staurosporine from
Streptomyces sp. (#S5921, purity 95%, Sigma-Aldrich) for CK1ε and LmCK1; Indirubin-3’-oxime (#I0404,
Sigma-Aldrich) for CDK2/CyclinA, CDK5/p25, CDK9/CyclinT, SscGSK-3α/β, RnDYRK1A and MmCLK1;
CHR-6494 (#SML0648, Sigma-Aldrich) for HASPIN; GSK’872 (GSK2399872A, #S8465, Selleckchem) for
RIPK3; SGI-1776 (#S2198, Selleckchem) for PIM1.
Acknowledgements
The authors gratefully acknowledge the Consortium against Parasites and Fungi (CaPF) for the scientific
exchanges.
Authors acknowledge the Corsaire-ThalassOMICS Metabolomics Facility (Biogenouest, University of Nantes,
France) and UMR 6230 CEISAM Laboratory for HRMS analyses.
The authors also thank GIS IBiSA (Infrastructures en Biologie Santé et Agronomie, France) and Biogenouest
(Western France life science and environment core facility network) for supporting kinase screening facility
(KISSf, Roscoff, France).
Supplementary data
Supplementary data related to this article can be found at
…

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Captions
Figure 1. Chemical structures of some antileishmanial compounds entering Phase I clinical trials
Figure 2. Modulation from the hit compound CTN1122
Scheme 1. Synthesis of compounds 2-11.
Reagents and conditions: (a) NH4OH, sealed tube, 100 °C, 17 h, 70%; (b) BrCH2CO(4-RC6H4), CH3CN, 80 °C,
11 h, 58% (3) and 18 h, 55% (4); (c) NIS, DMF, 60 °C, N2, 7 h, 84%; (d) Dimethylamine 40% aq., K2CO3,
DMF, sealed tube, 100 °C, 5 h, 70%; (e) (Het)ArB(OH)2, PdCl2(dppf), K3PO4, dioxane/H2O (9:1), sealed tube,
100 °C, 20 h, 24–85%.
Table 1 Yields of Suzuki coupling reaction to obtain imidazo[1,2-a]pyrazines 7-11 from compound 6.
Scheme 2. Synthesis of compounds 13-21.
Reagents and conditions: (a) 1) HI (57% aq), rt, 72 h; 2) H2O, NaHCO3, 95%; (b) 13, Pd(OAc)2, PPh3, Cs2CO3,
DMF, 90 °C, 20 h, 31% (14) and 15 h, 47% (15); (c) Dimethylamine 40% aq., K2CO3, DMF, sealed tube, 90 °C,
4–17 h, 55–58%; (d) Oxone®, MeOH/H2O, rt, 17–24 h, 25–98%; (e) NH4OHaq, 1,4-dioxane, sealed tube, 110
°C, 9 h, 51% (20) and 1 h 30, 73% (21).
Scheme 3. Synthesis of compounds 22-24.
Reagents and conditions: (a) NH4OHaq, isopropanol, µ-wave, 120 °C, 1 h, 100%; (b) Oxone®aq, MeOH, rt, 24
h, 74 %; (c) NH4OHaq, 1,4-dioxane, sealed tube, 110 °C, 1 h 15, 43%.
Table 2 In vitro antileishmanial activities against intracellular L. major, L. amazonensis and L. braziliensis
amastigotes and cytotoxicity evaluation of imidazo[1,2-a]pyrazines CTN1122, 7-11, 14-17, 20-22 and 24.
Table 3 In vitro antileishmanial activities against intracellular L. infantum and L. donovani amastigotes and
cytotoxicity evaluation of imidazo[1,2-a]pyrazines CTN1122, 7-11, 14-17, 20-22 and 24.
Figure 3. Evaluation of in vivo cytotoxicity of imidazo[1,2-a]pyrazines CTN1122, 7-11, 14-17, 20-22 and 24 on
G. mellonella model. Percentage of survival 7 days after injection of a dose of 50 mg/kg for each compound.
Mean of two independent experiments
Figure 4. Putative binding mode of CTN1122 within the ATP binding site of a homology model of LmCK1.
Table 4 Inhibition of Leishmania major CK1 and human CK1 activities of imidazo[1,2-a]pyrazines CTN1122,
7-11, 14-17, 20-22 and 24, and their selectivity profile against an array of 10 native or recombinant protein
kinasesa.

N
F
N
O H
H
N
N
B
N
N
O
O
O
N
N
N
NH
N
DNDI-6148
NH
N
O
NH
N
N
O
N
N
H N
NH
O
S
O
N
CF3
O
NH
N
H
N
N
O
N
NO2
GSK-3186899/DDD853651
F
O
F
DNDI-0690
Compound I
DNDI-1044
Figure 1. Chemical structures of some antileishmanial compounds entering Phase I clinical trials
Cl, N(CH3)2, NH2
F
N
8
N
N
CTN1122
H, F
expanding spectrum of activity
(CL, MCL, VL)
improving activity against LmCK1
N
3
IC50 (amastigotes L. major) = 0.80 µM
SI (RAW cells/amastigotes L. major) = 52.5
IC50 (LmCK1) = 0.72 µM
(het)aryl
N
Figure 2. Modulation from the hit compound CTN1122
Cl
1
Cl
2
Cl
Cl
NH2
N
N
N
N
a
b
R
N
N
N
3, R = H
4, R = F
N
Cl
N
N
N
N
N
N
N
N
e
c
d
F
F
F
4
N
N
(het)Ar
7-11
I
I
5
6
7, (het)Ar = 3-pyridyl
8, (het)Ar = 5-pyrimidyl
9, (het)Ar = 5-(2-aminopyrimidyl)
10, (het)Ar = 5-indazolyl
11, (het)Ar = 4-(1H-methylpyrazolyl)
Scheme 1. Synthesis of compounds 2-11.
Reagents and conditions: (a) NH4OH, sealed tube, 100 °C, 17 h, 70%; (b) BrCH2CO(4-RC6H4), CH3CN, 80 °C, 11 h, 58% (3) and 18 h, 55%
(4); (c) NIS, DMF, 60 °C, N2, 7 h, 84%; (d) Dimethylamine 40% aq., K2CO3, DMF, sealed tube, 100 °C, 5 h, 70%; (e) (Het)ArB(OH)2,
PdCl2(dppf), K3PO4, dioxane/H2O (9:1), sealed tube, 100 °C, 20 h, 24–85%.
Table 1 Yields of Suzuki coupling reaction to obtain imidazo[1,2-a]pyrazines 7-11 from compound 6.
Entry
(het)Ar
Compound Yield (%)a
7
34
N
1