Synthesis, biological evaluation and molecular modeling studies of imidazo[1,2-a]pyridines derivatives as protein kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.07.040

We report here the synthesis, the biological evaluation and the molecular modeling studies of new imidazo[1,2-a]pyridines derivatives designed as potent kinase inhibitors. This collection was obtained from 2-aminopyridines and 2-bromoacetophenone which af

Full text of DOI:10.1016/j.ejmech.2016.07.040

European Journal of Medicinal Chemistry 123 (2016) 105e114
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis, biological evaluation and molecular modeling studies of
imidazo[1,2-a]pyridines derivatives as protein kinase inhibitors
a
a
b
b
ꢀ b
Marie Lawson , Jordi Rodrigo , Blandine Baratte , Thomas Robert , Claire Delehouze ,
b
b
b
a
ꢀ
Olivier Lozach , Sandrine Ruchaud , Stephane Bach , Jean-Daniel Brion ,
,
, *
Mouad Alami a **, Abdallah Hamze a
a
ꢀ
ꢀ
ꢀ
^
BioCIS, Univ. Paris-Sud, CNRS, equipe labellisee Ligue Contre le Cancer, Universite Paris-Saclay, 92290, Chatenay-Malabry, France
Sorbonne Universites, UPMC Univ Paris 06, CNRS USR3151, “Protein Phosphorylation and Human Disease” Unit, Plateforme de criblage KISSf, Station
b
ꢀ
Biologique de Roscoff, Place Georges Teissier, 29688, Roscoff, France
a r t i c l e i n f o
a b s t r a c t
Article history:
We report here the synthesis, the biological evaluation and the molecular modeling studies of new
imidazo[1,2-a]pyridines derivatives designed as potent kinase inhibitors. This collection was obtained
from 2-aminopyridines and 2-bromoacetophenone which afforded final compound in only one step. The
bioactivity of this family of new compounds was tested using protein kinase and ATP competition assays.
The structure-activity relationship (SAR) revealed that six compounds inhibit DYRK1A and CLK1 at a
micromolar range. Docking studies provided possible explanations that correlate with the SAR data. The
Received 18 May 2016
Received in revised form
23 June 2016
Accepted 19 July 2016
Available online 21 July 2016
most active compound 4c inhibits CLK1 (IC50 of 0.7
m
M) and DYRK1A (IC50 of 2.6
© 2016 Elsevier Masson SAS. All rights reserved.
mM).
Keywords:
CLK1
DYRK1A
Kinase inhibitors
Imidazo[1,2-a]pyridines
1. Introduction
harmine is b-carboline compound that is naturally found in several
medicinal plants including Peganum harmala (Zygophyllaceae) and
Banisteriopsis caapi (Malpighiaceae) [10,11]. Leucettine L41 [12] a
synthetic analogue of Leucettamine B, a compound extracted from
the sponge Leucetta microporis in 1993 [13], inhibit various kinases
and especially CLK1 (CDC2-like Kinase 1) and DYRK1A (dual-
specificity tyrosine phosphorylation-regulated kinase1A) at a sub-
micromolar range. Synthesis of a range of analogues of harmine and
leucettine L41, including 1 [14] and 2 [15] was undertaken to un-
derstand and study biological activities of these molecules (Fig. 1).
Compounds 1 and 2 have a moderate inhibitory activity on CLK1
and DYRK1A are less potent inhibitors of these kinases than refer-
ence compounds. Furthermore, a new series of compounds of type
imidazo[1,2-a]pyridine 3 [16], has nanomolar activity against CDK-
(Cyclin-Dependent Kinase) 1 and 2, leading to compound 3 block-
ing the cell cycle and hence 3 could have utility in cancer treatment.
High-throughput screening (HTS) has become increasingly
important for hit identification in drug discovery [17]. The
increasing number of compounds available for screening and
recent advances in robotic are the driving forces for the develop-
ment of elaborated assay systems. This new technology led scien-
tists to discover new active compounds. High-throughput kinase-
based screening of small molecules is an attractive strategy to
Protein kinases are a ubiquitous group of enzymes sharing
structural similarities and are involved in various biological pro-
cesses within the cell [1,2]. They catalyze the transfer of phosphoryl
group from a phosphate donor (usually ATP) to a receptor substrate,
which can be a small molecule, lipid, or protein substrate. Kinase
deregulation is frequently observed during the development of
human diseases such as cancer [3], neurodegenerative disease
including Alzheimer's disease (AD) [4,5], inflammation or diabetes
[6]. Protein kinases are attractive targets for structure-based drug
discovery projects because of their crucial role in physiological
pathways. Over the past decades, protein kinases become attractive
targets for structure-based drug discovery projects and are a sub-
stantial and growing part of most major pharmaceutical com-
panies' drug target portfolios [2,7e9].
Nature is a source of new chemical scaffolds which could be an
excellent basis for rational structure-based design. In this context,
* Corresponding author.
** Corresponding author.
E-mail address: abdallah.hamze@u-psud.fr (A. Hamze).
http://dx.doi.org/10.1016/j.ejmech.2016.07.040
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

106
M. Lawson et al. / European Journal of Medicinal Chemistry 123 (2016) 105e114
Fig. 1. Structures of Harmine, its analogues 1, Leucettine L41, its analogue 2 and imidazo[1,2-a]pyridines 3 and our new structures 4.
Table 1
Synthesis of imidazo[1,2-a]pyridines 4a-4j by changing the nature of ring A.
Entry
1
2-Aminopyridine 5
Imidazo[1,2-a]pyridine 4
Yields (%)a
65
5a
5b
5c
5d
5e
5f
4a
4b
4c
4d
4e
4f
2
3
4
5
6
7
8
35
40
30
75
16
8
5g
5h
4g
4h
50
9
5i
5j
4i
4j
25
12
10
a
Isolated yields. General conditions: 2-bromoacetophenone 6 (1.05 mmol), 2-aminopyridine 5 (1.0 mmol) and NaHCO3 (2.0 mmol) in EtOH (20 mL), reflux, overnight.

M. Lawson et al. / European Journal of Medicinal Chemistry 123 (2016) 105e114
107
Table 2
Synthesis of imidazo[1,2-a]pyridines 4k-4r by changing the nature of ring B.
Entry
1
2-Bromoacetophenone 6
Imidazo[1,2-a]pyridine 4
Yields (%)a
6b
6c
4k
4l
20
2
40
3
4
5
6
6d
6e
6f
4m
4n
4o
4p
22
10
62
46
6g
7
8
6h
6i
4q
4r
40
38
a
Isolated yield.
identify such agents. We screened a library of 110 compounds of
our synthesized in-house collection against a panel of kinases. A
distinct kinase selectivity profile was observed for the molecule
containing the imidazo[1,2-a]pyridines scaffold. Therefore, com-
pound 4a (Table 1) was identified as a selective kinase inhibitor
with micromolar activity against CLK1 (CDC2-like Kinase 1) and
DYRK1A kinases. It is noteworthy that DYRK1A received recently
much attention due to its implication in the development of spo-
radic Alzheimer's disease [18,19] and Down syndrome [20,21],
where it promotes neurodegeneration by hyperphosphorylation of
the microtubule-associated protein tau. CLKs share a strong struc-
tural homology and some physiological functions with the DYRKs
family [22]. CLK1 is also involved in the pathophysiology of Alz-
heimer's disease by phosphorylating the serine residue in serine
and arginine-rich proteins. Hence, the inhibition of DYRK1A and
CLK1 may be used as a therapeutic strategy for Alzheimer's disease
and related disorders [22].
modification on the nucleus A or on ring B (Fig. 1).
2. Results and discussion
2.1. Chemistry
A series of 6- or 2-substituted imidazo[1,2-a]pyridines (Tables 1
and 2) were prepared from a variety of commercially available 5-
substituted
2-aminopyridines
with
5-(2-bromoacetyl)-2-
hydroxybenzamide derivatives. In a single step a large variety of
imidazo[1,2-a]pyridines bearing various substituents was readily
obtained [33,34].
In order to optimize the synthetic procedure, we used 5-(2-
bromoacetyl)-2-hydroxybenzamide
6a
and
5-chloro-2-
aminopyridin 5a as a model system (Table 1, entry 1). Reacting 1
equiv. of 5a with 1.05 equiv. of 6a in the presence of 2 equiv. of
K2CO3 in EtOH [35] gave 4a with a yield of 31%. The influence of
different mineral bases on reaction yield was investigated, with
sodium bicarbonate (NaHCO3) proving optimal, giving 4a in an
improved yield of 65% (Table 1, entry 1).
With this modified conditions, we prepared a small library of
imidazo[1,2-a]pyridines varying the substituents on ring A. Halo-
genated imidazo[1,2-a]pyridines (entries 1e4) were obtained in
moderate yield ranging from 30 to 75% with the highest yield
arising with unsubstituted aminopyridine (entry 5). A decrease in
the yield was noticed in the case of analogues bearing an electron
donating or hydrogen-bond donating group at the 5-position (en-
tries 6 and 7).
Imidazo[1,2-a]pyridines exhibit a broad range of biological
proprieties [23e25] including antiviral [26], anticancer [27,28],
antifungal [29], anti-inflammatory activities [30] and they have also
been incorporated into compounds useful for imaging A
gates in patients with Alzheimer's disease [31,32].
b aggre-
Due to the biological importance of these compounds, we aimed
to investigate new representatives on imidazo[1,2-a]pyridines
heterocyclic scaffold able to modulate a kinase activity. To optimize
the potency of this analogous and to gain further insight into its
structureꢀactivity relationships (SARs), we designed and synthe-
sized several new imidazo[1,2-a]pyridine derivatives with

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M. Lawson et al. / European Journal of Medicinal Chemistry 123 (2016) 105e114
Table 3
,b
Kinase inhibitory activity of imidazo[1,2-a]pyridines derivatives.a
Compound
CK1
GSK-3
DYRK1A
CLK1
Compound
CK1
GSK-3
DYRK1A
CLK1
4a
4b
4c
4d
4e
4f
4g
4h
4i
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
8.9
4.7
2.6
NAd
4
1.4
1.4
4k
4l
4m
4n
4o
4p
4q
4r
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
1.50
e
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
>10
0.040
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
0.029
0.040
NAd
NAd
NAd
NAd
NAd
NAd
NAd
NAd
0.072
0.090
0.7
NAd
1
3
0.89
NAd
NAd
NAd
1.7
NAd
NAd
NAd
6
Harmine
4j
Leucettine L41c
a
IC50 values are reported in
m
M. The most significant results are presented in bold.
Kinases activities were assayed in triplicate. Typically, the standard deviation of single data points was below 10%.
Leucettine L41 (IC50 in M) [12,37].
NA, not active. A compound was considered as not active (NA) when its inhibitory activity on kinases showing less than 50% inhibition (IC50 > 10
b
c
m
d
m
M).
For studies investigating modification on ring B, 5-chloro-2-
determined from the dose response curves (Sigma-Plot). The re-
sults of the in vitro kinase assay were summarized in Table 3.
Among the compounds tested, besides the first identified
compound 4a, the three most interesting molecules are 4c, 4e and
aminopyridin 5a was used as the amine partner, with the varia-
tion of the nature of the 2-bromoacetophenone 6. The imidazo[1,2-
a]pyridines (4k-4r) were obtained using the conditions described
above in modest yield (10e62%) to provide a library of 18 novel
compounds.
4f
which
M ꢁ IC50 ꢁ 4
M ꢁ IC50 ꢁ 1
M ꢁ IC50 ꢁ 6
micromolar
M) and submicromolar IC50 values against CLK1
M). 4b and 4j are a slightly less active against
M) and CLK1 (1.4 M)
M ꢁ IC50 ꢁ 1.7
activities
against
DYRK1A
(2.6
(0.7
m
m
m
m
DYRK1A (4.7
m
m
m
m
2.2. Biological results
and no activity was observed for the other compounds of the series.
This appears to define a specific role of the aromatic substituents in
the 6-position of imidazo[1,2-a]pyridines, indicating that this aro-
matic ring substituted by a hydroxyl group in position 40 and a
carboxamide at 50 position. Replacement of these substituents by a
methoxy or amino group (4k-4r) leads to a loss of the inhibitory
activity against CLK1 and DYRK1A. Nevertheless, the 6-position of
imidazo[1,2-a]pyridines tolerated various substituents such as
chlorine, fluorine, bromine, hydrogen and hydroxyl (4a-c, 4e, and
4f). The imidazo[1,2-a]pyridine scaffold could be successfully
replaced by an imidazo[1,2-a]pyrazine core (compound 4j) and
retain similar kinase inhibition activity. The introduction of bulky
phenyl substituent (4h and 4i) at the 6-position of imidazo[1,2-a]
pyridine was unfavorable for compound 4 activity. Imidazo[1,2-a]
pyridine compounds (4c, 4e, and 4f) were less potent in compari-
son with harmine and leucettine L41 against DYRK1A and CLK1
[36]. However, they constituted a promising starting point for the
In our screening efforts to discover new scaffolds for the inhi-
bition of disease-relevant protein kinases, we initially screened a
library of over 110 low molecular weight compounds against the
kinases DYRK1A and CLK1. This library comprised numerous syn-
thetic products including molecules containing imidazo [1,2-a]
pyridines ring. Among these, compound 4a was found to be a
modest but significant inhibitor of these disease-associated
kinases.
Thereafter, the synthetic imidazo [1,2-a]pyridines derivatives
4a-r were thus tested on a small panel of four kinases: (i) CK1
(casein kinase 1); (ii) GSK-3 (Glycogen Synthase Kinase-3
(iii) DYRK1A; (iv) CLK1. All compounds were first tested at a final
concentration of 10 M. Those showing less than 50% inhibition
were considered as inactive (IC50 > 10 M). Compounds displaying
more than 50% inhibition at 10 M were next tested over a wide
range of concentrations (usually 0.01e10 M) and IC50 values were
d/ε
a/
b
a/b
);
m
m
m
m
Fig. 2. Inhibitory activity of 4a, 4c and 4f against a panel of 13 protein kinases. Kinase activities in the presence of 10
measured in the absence of inhibitor). ATP concentration used in the kinase assays was 15
mol/l n ¼ 2 (mean range). Kinases are from human origin unless specified: Ssc, Sus
Scrofa; Rn, Rattus norvegicus; Mm, Mus musculus; Lm, Leishmania major; Ld, Leishmania donovani.
mM of compound are presented as % of maximal activity (i.e.
m

M. Lawson et al. / European Journal of Medicinal Chemistry 123 (2016) 105e114
109
Fig. 3. Double reciprocal plots of the inhibition caused by 4c compound on MmCLK1.
The experiment was carried out as described in material and methods section using a
fixed concentration of substrate peptide (6.75 ng/ l) and ATP concentrations varying
from 3.125 to 100
M. Data are mean standard deviation, n ¼ 3.
m
Fig. 5. Docking of compounds 4a (cyan), 4c (yellow) and 4f (orange) on DYRK1 (PDB:
4MQ1). Important amino acid residues are labeled. Putative hydrogen bonding was
depicted as yellow lines. Molecular graphics were performed using the UCSF Chimera
package. The closest hydrogen bond complementary atom to side chain ammonium
group of Lys188 and side chain carbonyl oxygen of Asn292 are indicated by dashed
lines. (Distances are respectively 3.27 and 2.64 Å). (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
m
measured the inhibitory activity of this new molecule against a
panel of 13 protein kinases (Fig. 2). The rational for testing these
kinases is their involvement in human pathologies or disorders
(notably in cancer). Some of these kinases are structurally related as
they are members of the same group of the kinome [38]. In the
tested panel, CDK1, 2, 5 and 9 are indeed members of the CMGC
cluster (for CDK, MAPK, GSK3, and CLK). As reported in Fig. 2,
members of this group are the more affected by the tested com-
pounds, the greatest selectivity was obtained toward CLK1. Among
the three active compounds, only, compound 4c was moderately
active against CDK9. Inhibitors of CDK9 can be used as an inductor
of caspase-dependent apoptosis [39].
Studies to investigate the mechanism of kinase inhibition by this
class of molecules were performed using compound 4c and
MmCLK1, the more affected target of the panel. Kinetic experiments
were performed by varying both ATP levels and 4c concentrations
(Fig. 3). Double-reciprocal plotting of the experimental data shows
that 4c is a typical ATP-competitive inhibitor for MmCLK1.
2.3. Molecular modeling
Fig. 4. Docking binding mode of compounds 4a (cyan), 4c (yellow) and 4f (orange)
over CLK1 (PDB: 1Z57) and superimposed to the co-crystallized hymenialdisine (HMD)
(green). Important amino acid residues are labeled. Putative hydrogen bonding was
depicted as yellow lines. Molecular graphics were performed with the UCSF Chimera
package. (For interpretation of the references to colour in this figure legend, the reader
is referred to the web version of this article.)
Molecular docking was accomplished for compounds 4a, 4c, and
4f in order to investigate their possible binding mode in the ATP
binding site (as demonstrated by ATP competition assay, Fig. 3).
These molecules were docked into the X-ray crystal structure of
CLK1 (resolution
¼
1.7 Å) [40] and also into the DYRK1A
(resolution ¼ 2.35 Å) [41]. Both structures were retrieved from the
Protein Data Bank (PDBs ID: 1Z57 and 4MQ1, respectively). The
structure of the kinase inhibitor hymenialdisine (HMD) co-
crystallized with CLK1 and of pyrido[2,3-d]pyrimidine co-
crystallized with DYRK1A kinase was used as references in order
to define the binding site cavity. Docking results on CLK1 showed
discovery of novel bioactive molecules, this study allows improved
understanding SARs in this series which should enable the design
of more efficient inhibitors of these disease-related kinases.
To confirm the selectivity of the more potent imidazo [1,2-a]
pyridines 4a, 4c and 4f derivatives toward DYRK1A and CLK1, we

110
M. Lawson et al. / European Journal of Medicinal Chemistry 123 (2016) 105e114
that all these three molecules present a partial superposition with
the co-crystallized HMD (Fig. 4), and may form some similar
hydrogen bonds interactions. The imidazo[1,2-a]pyridine cyclic
systems of molecules 4a, 4c and 4f are superposed to the pyrrolo
[2,3-c]azepine of HMD. In 4f, the hydroxyl from the imidazo[1,2-a]
pyridine cycle establishes two hydrogen bonds with the backbone
carbonyl of Glu242 and the NH of Leu244 as in the crystallized
HMD. The chlorine atom of 4a and the bromine atom of 4c occupy
the same region of space. These chlorine and bromine atoms are
positioned close to the location occupied by a chlorine atom in ethyl
3-(2-amino-1-cyanoethenyl)-6,7-dichloro-1-methylindole-2-
carboxylate (KHCB19) that has also been co-crystallized in the CLK1
kinase (PDB ID: 2VAG) (Supplementary material FS1) [42]. In both
cases, the chlorine and bromide are placed at 2.7 Å from backbone
carbonyl of Glu242 at the same position as the hydrogen bond
donor of common kinase inhibitors, mimicking the NH2 group of
ATP. Although when halogen bonds are significantly weaker than
hydrogen bonds their interactions can lead to establishing this
conformation and to clear gains in binding affinity [43]. The
hydroxybenzamide cycle, common to all the compounds, forms
two hydrogen bonds with residues in the binding site. The amide
NH2 forms a hydrogen bond with the side chain carbonyl of Asn293,
an amino acid also involved in the binding of HMD, and the phenol
forms a hydrogen bonding interaction with the backbone carbonyl
of Glu169 (Fig. 4). Finally, an edge-to-face t-stacking interaction
occurs between the hydroxybenzamide cycle and Phe172 for all
compounds.
Docking results on DYRK1A (Fig. 5) show again a very similar
binding mode as in CLK1 kinase. However, the Phe170 in DYRK1A
(Phe172 in CLK1) does not point inside the cavity and it is facing the
outside of the loop. This opens the possibility for some different
interactions between the compounds and the protein. The docking
suggests that the nitrogen of the imidazo[1,2-a]pyridine cycle
forms in all cases a hydrogen bond with Lys188 (Lys191 in CLK1). It
should be noted that in CLK1, Lys191 also interacts with the co-
crystallized HMD (not shown for clarity in Fig. 5) and KHCB19, via
hydrogen bonds. However, in the case of CLK1, taking into account
the presence of the Phe172, our molecules doesn't establish any
interaction with this Lys191. When comparing the co-crystallized
pyrido[2,3-d]pyrimidine in the DYRK1A kinase with our com-
pounds, we find again a nice superposition especially around one of
the chlorophenyl cycles and the imidazo[1,2-a]pyridine cycle
(Supplementary material FS2). In this fitting, chlorine from the co-
crystallized pyrido[2,3-d]pyrimidine and chlorine from 4a as well
as bromide from 4c, can establish a halogen bond interaction with
the carbonyl oxygen of Glu239 (Glu242 in CLK1 kinase). However,
the hydroxyl group belonging to 4f is located at the same position
but doesn't appear to be able to establish any hydrogen bonding
interaction with this carbonyl. As in CLK1, the hydroxybenzamide
cycle forms two hydrogen bonds, one with Asp307 (Asp325 in CLK1
kinase that also participates in the binding of HMD), and a second
one with the backbone carbonyl of Lys167 (Glu169 in CLK1 kinase)
(Fig. 5).
possible binding mode, but the docking results can't explain the
very similar activity of these molecules. For instance, despite the
fact that the hydroxyl group belonging to 4f compound doesn't
establish any hydrogen bonds interactions, a slight positive effect
was observed for the inhibition of DYRK1A.
3. Conclusions
We have described the synthesis of a new series of imidazo[1,2-
a]pyridine derivatives generated from 2-aminopyridine and 2-
bromoacetophenone in a single reaction. Kinase assays deter-
mined that six of these molecules can inhibit the enzymatic activity
of DYRK1A and CLK1 with IC50s in a micromolar range. Compound
4c was the most potent compound identified, with an IC50 of 0.7
mM
against CLK1 and 2.6 M against DYRK1A, and may provide a new
m
lead compound for molecular targeted drug discovery. Molecular
modeling studies and kinetic experiments have shown that mole-
cules 4a, 4c, and 4f have a good fit well in the ATP binding site of
these disease-related kinases, and are able to establish several
hydrogen bonds with critical residues in the ATP-binding site of
these targets. Interestingly, CDK5/p25 (Cyclin-dependent kinase 5),
one of the major tau kinase associated with neurodegenerative
disease [44], is poorly affected by 4a, 4c, and 4f. Further chemical
modifications of the structure may extend the selectivity profile.
Inhibition of kinases CLK1, DYRK1A, and CDK5 by such multi-target
compounds may produce synergistic therapeutic effects. These
results constitute a starting point enabling a better understanding
of structure-activity relationships of this new series of compounds,
and should facilitate future elaboration to more efficient and se-
lective inhibitors of DYRK1A and CLK1 which may have applications
in affecting the pathogenesis of Alzheimer's disease and related
disorders including Down's syndrome.
4. Experimental
4.1. Chemistry
4.1.1. General considerations
Melting points (mp) were recorded on a Büchi B-450 apparatus
and were uncorrected. NMR spectra were performed on a Bruker
AVANCE 300. Chemical shifts d are given in ppm, and the following
abbreviations are used: singlet (s), doublet (d), doublet of doublet
(dd), triplet (t), multiplet (m) and broad singlet (brs). IR spectra
were measured on a Bruker Vector 22 spectrophotometer (neat,
cmꢀ1). Low-resolution mass spectra (m/z) were recorded on a
Bruker Esquire electrospray ionization apparatus. High-resolution
mass spectra were recorded on a MicrotofQ Bruker Daltonics. Re-
action courses and product mixtures were routinely monitored by
TLC on silica gel (precoated F254 Merck plates), and compounds
were visualized with under a UVP Mineralight UVGL-58 lamp
(254 nm) and with phosphomolybdic acid, p-anisaldehyde, or
vanillin. Flash chromatography was performed using silica gel 60
(40e63 mm, 230e400 mesh ASTM) at medium pressure
(200 mbar). Organic extracts were dried over magnesium sulfate.
All products reported showed 1H NMR spectra in agreement with
the assigned structures.
The amide in the hydroxybenzamide cycle is positioned be-
tween the Asp307 and the Asn292 (Asn293 in CLK1 kinase) making
possible hydrogen bonds interactions. Once more, a very good t-
stacking interaction for all compounds is present but this time
between the imidazo[1,2-a]pyridine cycle and Phe238, an interac-
tion that is also present into the co-crystallized pyrido[2,3-d]py-
rimidine compound. We have shown that the binding mode of our
compounds shares several interactions with the same amino acids
as the co-crystallized ligands of CLK1 and DYRK1A. The majority of
kinase inhibitors known for CLK1 form a hydrogen bond to the
backbone NH located at the center of the active site of the hinge
strand as it is the case for our compounds. These results suggest a
4.1.2. General procedure for the synthesis of imidazo [1,2-a]
pyridines
A
mixture of 2-bromoacetophenone 6 (1.05 mmol), 2-
aminopyridine 5 (1.0 mmol) and NaHCO3 (2.0 mmol) in EtOH
(20 mL) was refluxed overnight. After cooling, the mixture was
evaporated to dryness and the residue was taken up with H2O. The
aqueous solution was acidified with 3 N HCl and the insoluble solid
was filtered off. The filtrate was basified with aqueous 5% NaOH and

M. Lawson et al. / European Journal of Medicinal Chemistry 123 (2016) 105e114
111
extracted with dichloromethane (3 ꢂ 20 mL). The collected organic
layers were dried over Na2SO4 and evaporated under reduced
pressure. The crude residue was purified by flash chromatography
on silica gel using a mixture of dichloromethane/ethyl acetate as
eluent to afford the compound 4.
found (ESI þ) m/z 254.0935, calcd for C14H12N3O2 (MþH)þ:
254.0936.
4.1.8. 2-Hydroxy-5-(6-hydroxyimidazo [1,2-a]pyridin-2-yl)
benzamide (4f)
Yield 16%; white solid; mp: 190e191 ꢃC; 1H NMR (300 MHz,
4.1.3. 5-(6-Chloroimidazo [1,2-a]pyridin-2-yl)-2-
hydroxybenzamide (4a)
MeOD)
d
8.30 (d, J ¼ 2.1 Hz, 1H), 8.00 (s, 1H), 7.94 (dd, J ¼ 8.6, 2.2 Hz,
2H), 7.43 (d, J ¼ 9.6 Hz, 1H), 7.07 (dd, J ¼ 9.6, 2.2 Hz, 1H), 7.00 (d,
Yield 65%; yellow solid; mp: 274e275 ꢃC; 1H NMR (300 MHz,
J ¼ 8.6 Hz, 1H); 13C NMR (75 MHz, Acetone-d6)
d 174.1, 162.8, 146.8,
DMSO-d6)
d
13.13 (s, 1H, OH), 8.89 (dd, J ¼ 2.1, 1 Hz, 1H, Ar), 8.59 (s,
132.6,132.2,126.4,125.5,121.0, 120.5,118.9,117.6,114.9,111.2,109.2;
1H, CONH), 8.46 (d, J ¼ 2.1 Hz, 1H, Ar), 8.26 (s, 1H, Ar), 8.03 (dd,
J ¼ 8.6, 2.1 Hz, 1H, Ar), 7.97 (s, 1H, CONH), 7.61 (d, J ¼ 9.6 Hz, 1H, Ar),
7.29 (dd, J ¼ 9.6, 2.1 Hz, 1H, Ar), 6.98 (d, J ¼ 8.6 Hz, 1H, Ar); 13C NMR
IR (n
, cmꢀ1):2384, 2218, 1609, 1333, 1255, 1176, 1125, 1075; HMRS:
found (ESI þ) m/z 270.0883, calcd for C14H12N3O3 (M þ H)þ:
270.0879.
(75 MHz, DMSO-d6) d 171.9 (CO), 160.9 (C), 145.2 (C), 143.2 (C), 131.4
(CH), 125.5 (2 CH), 124.7 (CH), 124.2 (C), 118.8 (C), 117.8 (CH), 117.0
4.1.9. 5-(6-Aminoimidazo [1,2-a]pyridin-2-yl)-2-
hydroxybenzamide (4g)
(CH), 114.6 (C), 108.8 (CH); IR (
n
, cmꢀ1):3454 (OeH), 3429, 3400
(NH amide), 3331, 3192, 3135, 2949, 2340, 2121, 2292, 2064, 2033,
Yield 8%; pink solid; mp: 186e187 ꢃC; 1H NMR (400 MHz,
2012, 1073; HMRS: found (ESI þ) m/z 288.0538, calcd for
Acetone-d6)
1H), 8.35 (d, J ¼ 2.6 Hz, 1H), 8.25 (d, J ¼ 8.0 Hz, 1H), 8.02e7.83 (m,
3H), 3.58 (s, 4H); 13C NMR (101 MHz, Acetone-d6)
163.2, 138.2,
133.8, 133.2, 130.5, 129.4, 126.5, 125.7, 123.6, 122.6, 120.7, 119.9,
116.1, 112.7; IR (
, cmꢀ1): 2989, 2960, 2361, 1676, 1624, 1594, 1594,
d
11.22 (s, 1H), 8.63 (d, J ¼ 7.8 Hz, 1H), 8.55e8.42 (m,
C
14H11ClN3O2 (MþH)þ: 288.0538.
d
4.1.4. 5-(6-Fluoroimidazo [1,2-a]pyridin-2-yl)-2-
hydroxybenzamide (4b)
n
Yield 35%; white solid; mp: 288e289 ꢃC; 1H NMR (300 MHz,
1494, 1411, 1362, 1275, 1115, 1062, 1034; HMRS: found (ESI þ) m/z
DMSO-d6)
d 13.10 (s, 1H), 8.83e8.76 (m, 1H), 8.58 (s, 1H), 8.45 (d,
269.1041, calcd for C14H13N4O2 (M þ H)þ: 269.1039.
J ¼ 2.1 Hz, 1H), 8.27 (s, 1H), 8.01 (dd, J ¼ 8.6, 2.1 Hz, 1H), 7.95 (s, 1H),
7.61 (dd, J ¼ 9.8, 5.3 Hz, 1H), 7.36e7.24 (m, 1H), 6.96 (d, J ¼ 8.6 Hz,
4.1.10. 2-Hydroxy-5-(6-(4-methoxyphenyl)imidazo [1,2-a]pyridin-
2-yl)benzamide (4h)
1H); 13C NMR (75 MHz, DMSO-d6)
d 177.1, 166.1, 157.8 (d,
J ¼ 230 Hz),150.7, 147.9, 136.5,130.6,129.6,123.0,122.2 (d, J ¼ 9 Hz),
Yield 50%; pink solid; mp: 245e246 ꢃC; 1H NMR (300 MHz,
121.6 (d, J ¼ 22.5 Hz), 119.9, 118.8 (d, J ¼ 22.5 Hz), 114.9; 19F NMR
DMSO-d6)
d
13.11 (s, 1H), 8.82 (d, J ¼ 1.6 Hz,1H), 8.61 (s, 1H), 8.48 (d,
(188 MHz, DMSO)
d
ꢀ139.94; IR (
n
, cmꢀ1): 3435, 3429, 2361, 2202,
J ¼ 2.0 Hz, 1H), 8.26 (s, 1H), 8.03 (dd, J ¼ 8.6, 2.0 Hz, 1H), 7.95 (s, 1H),
7.68e7.63 (m, 2H), 7.61 (d, J ¼ 9.3 Hz, 1H), 7.54 (dd, J ¼ 9.4, 1.8 Hz,
1H), 7.10e7.03 (m, 2H), 6.97 (d, J ¼ 8.6 Hz,1H), 3.81 (s, 3H); 13C NMR
1678, 1603, 1568, 1535, 1477, 1369, 1255, 1207, 1169; HMRS: found
(ESI þ) m/z 272.0833, calcd for C14H11FN3O2 (MþH)þ: 272.0835.
(75 MHz, DMSO-d6)
d 172.0, 160.7, 159.0, 144.5, 143.9, 131.3, 128.9,
4.1.5. 5-(6-Bromoimidazo [1,2-a]pyridin-2-yl)-2-
hydroxybenzamide (4c)
127.7, 127.7, 125.3, 124.9, 124.7, 124.7, 123.1, 117.7, 116.2, 114.6, 114.5,
114.5, 108.5, 55.2; IR (
n
, cmꢀ1):3418, 3253, 2277, 2361, 2207, 2149,
Yield 40%; yellow solid; mp: 254e255 ꢃC; 1H NMR (300 MHz,
2013, 1998, 1632, 1405, 1242, 1181, 1017; HMRS: found (ESI þ) m/z
DMSO-d6)
d 13.13 (s, 1H), 9.01e8.85 (m, 1H), 8.60 (s, 1H), 8.44 (d,
360.1366, calcd for C21H18N3O3 (M þ H)þ: 360.1363.
J ¼ 2.1 Hz, 1H), 8.23 (s, 1H), 8.00 (dd, J ¼ 8.6, 2.1 Hz, 1H), 7.97 (s, 1H),
7.53 (d, J ¼ 9.5 Hz, 1H), 7.34 (dd, J ¼ 9.5, 2.1 Hz, 1H), 6.95 (d,
4.1.11. 2-Hydroxy-5-(6-(4-nitrophenyl)imidazo [1,2-a]pyridin-2-yl)
benzamide (4i)
J ¼ 8.6 Hz, 1H);
d d 171.9, 161.0, 144.9,
13C NMR (75 MHz, DMSO-d6)
143.2,131.4, 127.7, 126.9, 125.5, 124.2, 117.8, 117.3, 114.6, 108.6,105.8;
Yield 25%; pink solid; mp: 148e149 ꢃC; 1H NMR (300 MHz,
IR (
n
, cmꢀ1):2361, 2341, 1671, 1624, 1590, 1516, 1472, 1441, 1336,
DMSO-d6)
d
13.13 (s, 1H), 9.13 (s, 1H), 8.61 (s, 1H), 8.48 (dd, J ¼ 11.6,
1283, 1140, 1059; HMRS: found (ESI þ) m/z 332.0033, calcd for
2.0 Hz, 1H), 8.37 (s, 1H), 8.33 (d, J ¼ 4.0 Hz, 2H), 8.06 (dd, J ¼ 8.5,
C
14H11BrN3O2 (MþH)þ: 332.0035.
2.1 Hz, 2H), 8.05 (d, J ¼ 8.9 Hz, 2H), 7.97 (s, 1H), 7.70 (d, J ¼ 1.0 Hz,
1H), 6.99 (d, J ¼ 8.6 Hz, 1H); 13C NMR (75 MHz, DMSO-d6)
d 171.9,
4.1.6. 5-(6-Iodoimidazo [1,2-a]pyridin-2-yl)-2-hydroxybenzamide
(4d)
161.2, 160.9, 152.5, 146.7, 145.2, 144.2, 143.4, 131.4, 127.5, 127.5,
125.6, 125.5, 124.2, 124.2, 122.9, 117.8, 116.6, 114.6, 108.9; IR (n,
Yield 30%; yellow solid; mp: 181e182 ꢃC; 1H NMR (300 MHz,
cmꢀ1):2926, 2853,2360, 1658, 1592, 1471, 1376, 1247, 1109, 1034;
HMRS: found (ESI þ) m/z 375.1102, calcd for C20H15N4O4 (M þ H)þ:
375.1093.
DMSO-d6)
d
13.12 (s, 1H), 8.95 (s,1H), 8.60 (s,1H), 8.43 (d, J ¼ 2.0 Hz,
1H), 8.19 (s, 1H), 7.99 (dd, J ¼ 8.6, 2.0 Hz, 1H), 7.98 (s, 1H), 7.40 (d,
J ¼ 0.9 Hz, 2H), 6.95 (d, J ¼ 8.6 Hz,1H); 13C NMR (75 MHz, DMSO-d6)
d
171.9, 160.9, 144.4, 143.3, 132.3, 131.4, 131.4, 125.5, 124.2, 117.8,
4.1.12. 2-Hydroxy-5-(imidazo [1,2-a]pyrazin-2-yl)benzamide (4j)
Yield 12%; yellow solid; mp: 264e265 ꢃC; 1H NMR (300 MHz,
117.6, 114.6, 108.0, 75.9; IR (n
, cmꢀ1):3425, 3347, 3217, 2483, 2207,
2166, 2028, 2011, 1482; HMRS: found (ESI þ) m/z 379.9896, calcd
DMSO-d6)
d
9.04 (s, 1H), 8.64 (d, J ¼ 4.5 Hz, 1H), 8.54 (d, J ¼ 1.9 Hz,
for C14H11IN3O2 (MþH)þ: 379.9896.
1H), 8.46 (s, 1H), 8.08 (d, J ¼ 10.6 Hz, 1H), 7.89 (d, J ¼ 4.6 Hz, 1H),
7.00 (d, J ¼ 8.6 Hz, 1H); 13C NMR (75 MHz, DMSO-d6)
d 171.8, 161.3,
4.1.7. 5-(Imidazo [1,2-a]pyridin-2-yl)-2-hydroxybenzamide (4e)
Yield 75%; yellow solid; mp: 238e239 ꢃC; 1H NMR (300 MHz,
146.0, 142.1, 140.3, 134.6, 131.6, 130.4, 129.1, 126.0, 123.7, 117.9, 114.7;
IR (n
, cmꢀ1): 3396, 2360, 1707, 1669, 1593, 1493, 1283, 1237, 1037,
DMSO-d6)
d
13.89 (s, 1H), 9.38 (s, 1H), 9.33 (d, J ¼ 6.7 Hz, 1H), 9.27
1022; HMRS: found (ESI þ) m/z 255.0883, calcd for C13H11N4O2
(d, J ¼ 2.1 Hz, 1H), 9.05 (s, 1H), 8.81 (dd, J ¼ 8.6, 2.1 Hz, 1H), 8.75 (s,
1H), 8.34 (d, J ¼ 9.1 Hz,1H), 8.01 (ddd, J ¼ 9.1, 6.7, 1.2 Hz,1H), 7.76 (d,
J ¼ 8.6 Hz, 1H), 7.66 (td, J ¼ 6.7, 1.2 Hz, 1H); 13C NMR (75 MHz,
(M þ H)þ: 255.0882.
4.1.13. 3-(6-Chloroimidazo [1,2-a]pyridin-2-yl)benzamide (4k)
Yield 20%; brownish solid; mp: 176e177 ꢃC; 1H NMR (300 MHz,
Acetone-d6): 8.76e8.71 (m, 1H), 8.67 (s, 1H), 8.60 (td, J ¼ 1.7, 0.6 Hz,
1H), 8.41e8.30 (m, 3H), 8.30e8.20 (m, 2H), 7.80e7.73 (m, 1H), 7.70
DMSO-d6)
117.7, 116.3, 114.6, 112.1, 108.1; IR (
2208, 2167, 1665, 1629, 1590, 1429, 1370, 1278, 1127, 1075; HMRS:
d 172.0,160.7,144.7,144.2,131.3,126.8,125.4,124.7,124.7,
n
, cmꢀ1):3427, 3347, 3215, 2483,

112
M. Lawson et al. / European Journal of Medicinal Chemistry 123 (2016) 105e114
(s, 1H); 13C NMR (75 MHz, DMSO-d6)
d
192.2, 166.7, 166.5, 137.2,
cmꢀ1):2838, 1586, 1463, 1427, 1414, 1339, 1242, 1229, 1125, 1072,
1003; HMRS: found (ESI þ) m/z 319.0850, calcd for C16H16ClN2O3
(M þ H)þ: 319.0849.
134.7,134.1,133.5,132.4,131.5,130.0,129.6,129.2,128.6,128.3; IR (n,
cmꢀ1): 3398, 2260, 1715, 1669, 1583, 1288, 1247, 1047, 1021; HMRS:
found (ESI þ) m/z 272.0595, calcd for C14H11ClN3O (M þ H)þ:
272.0591.
4.1.20. 4-(6-Chloroimidazo [1,2-a]pyridin-2-yl)-2-methoxyphenol
(4r)
4.1.14. 2-Bromo-4-(6-chloroimidazo [1,2-a]pyridin-2-yl)aniline (4l)
Yield 40%; orange solid; mp: 227e228 ꢃC; 1H NMR (300 MHz,
Yield 38%; yellow solid; mp: 205e206 ꢃC; 1H NMR (300 MHz,
DMSO-d6)
d
9.11 (s, 1H), 8.76 (dd, J ¼ 2.0, 0.7 Hz, 1H), 8.19 (s, 1H),
DMSO-d6)
d
8.76 (dd, J ¼ 2.0, 0.7 Hz, 1H), 8.21 (s, 1H), 7.97 (d,
7.58 (d, J ¼ 9.6 Hz, 1H), 7.40 (d, J ¼ 2.0 Hz, 1H), 7.35 (dd, J ¼ 8.3,
2.1 Hz, 1H), 7.25 (dd, J ¼ 9.6, 2.1 Hz, 1H), 6.97 (d, J ¼ 8.3 Hz, 1H), 3.80
J ¼ 1.9 Hz, 1H), 7.66 (dd, J ¼ 8.4, 2.0 Hz, 1H), 7.57 (d, J ¼ 9.5 Hz, 1H),
7.26 (dd, J ¼ 9.5, 2.1 Hz, 1H), 6.87 (d, J ¼ 8.4 Hz, 1H), 5.51 (s, 2H); 13
C
(s, 3H); 13C NMR (75 MHz, DMSO-d6)
d 147.8, 146.7, 145.7, 143.1,
NMR (75 MHz, DMSO-d6)
d
145.7, 145.0, 143.1, 129.4, 125.9, 125.2,
126.4, 125.3, 124.5, 118.6, 117.1, 116.8, 113.0, 112.4, 108.7, 55.6; IR (n,
124.4,122.9, 118.6, 116.9, 115.4, 108.1, 107.5; IR (
n
, cmꢀ1): 2924, 1619,
cmꢀ1):2364, 1557, 1469, 1423, 1338, 1264, 1246, 1223, 1173, 1132,
1554, 1480, 1406, 1336, 1220, 1156, 1072, 1033; HMRS: found (ESI þ)
1073, 1013; HMRS: found (ESI þ) m/z 275.0582, calcd for
m/z 323.9738, calcd for C13H10ClN3 (M þ H)þ: 323.9741.
C
14H12ClN2O2 (M þ H)þ: 275.0587.
4.1.15. 4-(6-Chloroimidazo [1,2-a]pyridin-2-yl)phenol (4m)
4.2. Kinase inhibition assay
Yield 22%; yellow solid; mp: 257e258 ꢃC; 1H NMR (300 MHz,
DMSO-d6)
d
9.58 (s, 1H), 8.74 (dd, J ¼ 2.1, 0.8 Hz, 1H), 8.16 (s, 1H),
Kinase activities were assayed in appropriate kinase buffer (A or
B, described below), with either protein or peptide as substrate in
7.74 (d, J ¼ 8.6 Hz, 2H), 7.54 (d, J ¼ 9.5 Hz, 1H), 7.22 (dd, J ¼ 9.5,
2.1 Hz, 1H), 6.80 (d, J ¼ 8.6 Hz, 2H); 13C NMR (75 MHz, DMSO-d6)
the presence of 15
mM [
g-33P] ATP (3000 Ci/mmol; 10 mCi/ml) in a
d
157.5, 145.9, 143.1, 127.1, 127.1, 125.2, 124.5, 124.4, 118.6, 117.0,
final volume of 30 ml following the assay described in Bach et al.,
115.5, 115.5, 108.2; IR (
n
, cmꢀ1):2959, 2928, 2364, 1729, 1683, 1601,
[45]. Controls were performed with appropriate dilutions of
dimethylsulfoxide. Full-length kinases are used unless specified.
Peptide substrates were obtained from Proteogenix (Oberhaus-
bergen, France).
1502, 1423, 1237, 1169, 1113, 1077; HMRS: found (ESI þ) m/z
245.0471, calcd for C13H10ClN2O (M þ H)þ: 245.0482.
4.1.16. 6-Chloro-2-(4-methoxyphenyl)imidazo [1,2-a]pyridine (4n)
Yield 10%; yellow solid; mp: 231e233 ꢃC; 1H NMR (300 MHz,
Buffers:
DMSO-d6)
d
8.78 (dd, J ¼ 2.1, 0.8 Hz, 1H), 8.27 (s, 1H), 7.89 (d,
J ¼ 8.9 Hz, 2H), 7.60 (d, J ¼ 9.6 Hz, 1H), 7.27 (dd, J ¼ 9.6, 2.1 Hz, 1H),
(A) 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 25 mM Tris-HCl pH
7.01 (d, J ¼ 8.9 Hz, 2H), 3.80 (s, 3H); 13C NMR (75 MHz, DMSO-d6)
7.5, 50
nitrophenyl-phosphate, 25 mM MOPS (pH 7), 5 mM EGTA, 15 mM
MgCl2, 1 mM DTT, 0.1 mM sodium orthovanadate; (C) 60 mM b-
mg/ml heparin; (B) 60 mM b-glycerophosphate, 30 mM p-
d
159.2, 145.5, 143.2, 127.0, 127.0, 126.0, 125.4, 124.6, 118.7, 117.1,
114.2, 114.2, 108.7, 55.1; IR (
n
, cmꢀ1):3003, 1612, 1553, 1503, 1486,
1434, 1369, 1277, 1106, 1074, 1032; HMRS: found (ESI þ) m/z
glycerophosphate, 15 mM p-nitrophenyl-phosphate, 25 mM Mops
(pH 7.2), 5 mM EGTA, 15 mM MgCl2, 1 mM DTT; (H) MOPS 25 mM
pH 7.5, 10 mM MgCl2; (K) Tris 50 mM pH 7.5, 20 mM MgCl2, 2 mM
259.0631, calcd for C14H12ClN2O (M þ H)þ: 259.0638.
4.1.17. 6-Chloro-2-(3-methoxyphenyl)imidazo [1,2-a]pyridine (4o)
Yield 62%; yellow solid; mp: 145e146 ꢃC; 1H NMR (300 MHz,
MnCl2; (R) 5 mM MOPS pH 7.2, 2.5 mM
MgCl2, 2.5 mM MnCl2, 1 mM EGTA, 0.4 mM EDTA, 50
b
-glycerophosphate, 4 mM
mg/ml BSA,
DMSO-d6)
d
8.80 (dd, J ¼ 2.1, 0.8 Hz, 1H), 8.39 (s, 1H), 7.64 (d,
0.05 mM DTT; (G) Tris 40 mM pH 7.5, 20 mM MgCl2, 0.1 mg/ml BSA.
HsRIPK3 (human, recombinant, expressed by baculovirus in Sf9
J ¼ 9.6 Hz, 1H), 7.57e7.49 (m, 2H), 7.35 (t, J ¼ 8.1 Hz, 1H), 7.29 (dd,
J ¼ 9.6, 2.1 Hz,1H), 6.91 (ddd, J ¼ 8.1, 2.1, 0.8 Hz,1H), 3.83 (s, 3H); 13
C
insect cells) was assayed in buffer R with 0.1
mg/ml of MBP as sub-
NMR (75 MHz, CDCl3) d 159.6,145.2,143.2,134.8,129.9,125.8,124.8,
strate. HsPIM1 (human proto-oncogene, recombinant, expressed in
119.0, 118.0, 117.4, 113.9, 110.7, 110.0, 55.1; IR (
n
, cmꢀ1): 2835, 2360,
bacteria) was assayed in buffer B with 0.8
(Sigma #H5505) as substrate.
mg/ml of histone H1
1613, 1521, 1469, 1367, 1314, 1253, 1197, 1074, 1046; HMRS: found
(ESI þ) m/z 259.0631, calcd for C14H12ClN2O (M þ H)þ: 259.0638.
HsHaspin-kd (human, kinase domain, amino acids 470 to 798,
recombinant, expressed in bacteria) was assayed in buffer H with
4.1.18. 6-Chloro-2-(3,5-dimethoxyphenyl)imidazo [1,2-a]pyridine
(4p)
0.007 mg/ml of Histone H3 (1e21) peptide (ARTKQTARKSTGG-
KAPRKQLA) as substrate; HsCDK1/CyclinB1 (human, recombinant,
expressed by baculovirus in Sf9 insect cells, SignalChem, product
Yield 46%; yellow solid; mp: 133e134 ꢃC; 1H NMR (300 MHz,
Acetone-d6)
d
9.07 (dd, J ¼ 2.0, 0.9 Hz, 1H), 8.75 (d, J ¼ 0.5 Hz, 1H),
#C22-10G) was assayed in buffer G with 0.8
substrate.
HsCDK2/CyclinA (cyclin-dependent kinase-2, human, kindly
provided by Dr. A. Echalier-Glazer, Leicester, UK) was assayed in
mg/ml of histone H1 as
8.01 (d, J ¼ 9.6 Hz, 1H), 7.69 (dd, J ¼ 9.6, 2.0 Hz, 1H), 7.66 (d,
J ¼ 2.3 Hz, 2H), 6.92 (t, J ¼ 2.3 Hz, 1H), 4.30 (s, 6H); 13C NMR
(75 MHz, Acetone-d6) d 162.3, 162.3, 147.3, 144.7, 136.9, 126.5, 125.5,
120.6, 118.6, 110.9, 104.8, 104.8, 101.1, 55.8, 55.8; IR (
2361, 1611, 1543, 1457, 1418, 1341, 1288, 1204, 1154, 1073, 1049;
n
, cmꢀ1):2838,
buffer A (þ0.15 mg/ml of BSA þ 0.23 mg/ml of DTT) with 0.8
mg/ml of
histone H1 as substrate.
HMRS: found (ESI þ) m/z 289.0743, calcd for
(M þ H)þ: 289.0744.
C
15H14ClN2O2
HsCDK9/CyclinT (human, recombinant, expressed by baculovi-
rus in Sf9 insect cells) was assayed in buffer A (þ0.15 mg/ml of
BSA þ 0.23 mg/ml of DTT) with 0.27
mg/ml of the following peptide:
YSPTSPSYSPTSPSYSPTSPSKKKK, as substrate.
4.1.19. 6-Chloro-2-(3,5-dimethoxyphenyl)imidazo [1,2-a]pyridine
(4q)
HsCDK5/p25 (human, recombinant, expressed in bacteria) was
Yield 40%; yellow solid; mp: 131e132 ꢃC; 1H NMR (300 MHz,
assayed in buffer B, with 0.8
SscGSK-3 (glycogen synthase kinase-3, porcine brain, native,
affinity purified) was assayed in buffer
(þ0.15 mg/ml of
BSA þ 0.23 mg/ml of DTT), with 0.010 g/ l of GS-1 peptide, a GSK-
3-selective substrate (YRRAAVPPSPSLSRHSSPHQSpEDEEE, “Sp”
mg/ml of histone H1 as substrate.
CDCl3)
d
8.15 (dd, J ¼ 2.0, 0.8 Hz, 1H), 7.79 (s, 1H), 7.58 (d, J ¼ 9.6 Hz,
a/b
1H), 7.16 (s, 2H), 7.13 (d, J ¼ 2.0 Hz, 1H), 3.95 (s, 6H), 3.88 (s, 3H); 13
C
A
NMR (75 MHz, CDCl3)
d
153.8,153.8,146.6,143.9,138. 7,128.7,126.5,
m m
123.5, 120.9, 117.7, 108.5, 103.5, 103.5, 61.1, 56.4, 56.4; IR (n,

M. Lawson et al. / European Journal of Medicinal Chemistry 123 (2016) 105e114
113
stands for phosphorylated serine).
SscCK1 /ε (casein kinase 1 /ε, porcine brain, native, affinity
purified) was assayed in buffer B, with 0.022 g/ l of the following
d’Avenir program by means of the OCEANOMICs project (grant #
ANR-11-BTBR-0008). Our laboratory (BioCIS UMR 8076) is a
member of the laboratory of excellence LERMIT supported by a
grant from ANR (ANR-10-LABX-33).
d
d
m
m
peptide: RRKHAAIGSpAYSITA as CK1-specific substrate.
RnDYRK1A-kd (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 (þ0.5 mg/ml of BSA þ 0.23 mg/ml of DTT)
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.07.040.
with 0.033
substrate.
mg/ml of the following peptide: KKISGRLSPIMTEQ as
MmCLK1 (from Mus musculus, recombinant, expressed in bac-
References
teria) was assayed in buffer A (þ0.15 mg/ml of BSA þ 0.23 mg/ml of
DTT) with 0.027
substrate.
mg/ml of the following peptide: GRSRSRSRSRSR as
[1] S. Cheek, H. Zhang, N.V. Grishin, Sequence and structure classification of ki-
nases, J. Mol. Biol. 320 (2002) 855e881.
[2] S. Sharma, J. Singh, R. Ojha, H. Singh, M. Kaur, P.M.S. Bedi, K. Nepali, Design
strategies, structure activity relationship and mechanistic insights for purines
as kinase inhibitors, Eur. J. Med. Chem. 112 (2016) 298e346.
[3] Z.A. Stewart, M.D. Westfall, J.A. Pietenpol, Cell-cycle dysregulation and anti-
cancer therapy, Trends Pharmacol. Sci. 24 (2003) 139e145.
[4] M.H. Flight, Neurodegenerative diseases: new kinase targets for Alzheimer's
disease, Nat. Rev. Drug Discov. 12 (2013), 739e739.
LdTLK (tousled-like kinase, from Leishmania donovani, recom-
binant, expressed in bacteria), was assayed in buffer K with 0.6 g/
l of casein dephosphorylated from bovine milk (Sigma #C4032) as
substrate.
LmCK1 (from Leishmania major, recombinant, expressed in
bacteria) was assayed in buffer B (adjusted at pH 8) with 0.028 g/
m
m
m
ml
[5] C.-H. Lin, Y.-S. Hsieh, Y.-R. Wu, C.-J. Hsu, H.-C. Chen, W.-H. Huang, K.-H. Chang,
H.M. Hsieh-Li, M.-T. Su, Y.-C. Sun, G.-C. Lee, G.-J. Lee-Chen, Identifying GSK-3
kinase inhibitors of Alzheimer's disease: virtual screening, enzyme, and cell
assays, Eur. J. Pharm. Sci. 89 (2016) 11e19.
b
of the following peptide: RRKHAAIGSpAYSITA as CK1-specific
substrate.
[6] H. Noh, G.L. King, The role of protein kinase C activation in diabetic ne-
phropathy, Kidney Int., 72(0000) S49eS53.
[7] S.K. Grant, Therapeutic protein kinase inhibitors, Cell. Mol. Life Sci. 66 (2009)
1163e1177.
4.2.1. ATP competition assay using ADP-GloTM bioluminescent
assay
The activity of recombinant mouse CLK1 was measured using
the ADP-GloTM bioluminescent kinase assay kit (Promega, Madi-
son, WI) according to the recommendations of the manufacturer
and described in Zegzouti et al., 2009 [46]. Reactions were carried
[8] J. Zhang, P.L. Yang, N.S. Gray, Targeting cancer with small molecule kinase
inhibitors, Nat. Rev. Cancer 9 (2009) 28e39.
[9] S. Sharma, S. Mehndiratta, S. Kumar, J. Singh, P.M.S. Bedi, K. Nepali, Purine
analogues as kinase inhibitors: a review, Recent Pat. Anticancer Drug Discov.
10 (2015) 308e341.
out in a final volume of 5
(40 mM Tris pH 7.5, 20 mM MgCl2 and 0.1 mg/ml of BSA). After that,
l of ADP-GloTM Kinase Reagent was added to stop the kinase
reaction. After an incubation time of 50 min at room temperature,
10 l of Kinase Detection Reagent was added for 1 h at room
m
l for 30 min at 30 ꢃC in ADP-Glo buffer
ꢀ
ꢀ
ꢀ
[10] T. Herraiz, D. Gonzalez, C. Ancín-Azpilicueta, V.J. Aran, H. Guillen, b-Carboline
alkaloids in Peganum harmala and inhibition of human monoamine oxidase
(MAO), Food Chem Toxicol. 48 (2010) 839e845.
5
m
[11] V. Samoylenko, M.M. Rahman, B.L. Tekwani, L.M. Tripathi, Y.-H. Wang,
S.I. Khan, I.A. Khan, L.S. Miller, V.C. Joshi, I. Muhammad, Banisteriopsis caapi, a
unique combination of MAO inhibitory and antioxidative constituents for the
activities relevant to neurodegenerative disorders and Parkinson's disease,
J. Ethnopharmacol. 127 (2010) 357e367.
[12] M. Debdab, F. Carreaux, S. Renault, M. Soundararajan, O. Fedorov,
P. Filippakopoulos, O. Lozach, L. Babault, T. Tahtouh, B. Baratte, Y. Ogawa,
M. Hagiwara, A. Eisenreich, U. Rauch, S. Knapp, L. Meijer, J.-P. Bazureau,
Leucettines, a class of potent inhibitors of cdc2-like kinases and dual speci-
ficity, tyrosine phosphorylation regulated kinases derived from the marine
sponge leucettamine b: modulation of alternative Pre-RNA splicing, J. Med.
Chem. 54 (2011) 4172e4186.
m
temperature. The transmitted signal was measured using the
Envision (PerkinElmer, Waltham, MA) microplate luminometer and
expressed in Relative Light Unit (RLU). Assays were performed in
the absence or presence of 4c (4, 2 or 0.5
concentrations of ATP (3.125e100 M). The peptide:
GRSRSRSRSRSR (6.75 ng/ l of reaction) was used as substrate. To
mM) and increasing
m
m
plot and analyze the data Prism from GraphPad Software (La Jolla,
CA) was used.
[13] G.W. Chan, S. Mong, M.E. Hemling, A.J. Freyer, P.H. Offen, C.W. DeBrosse,
H.M. Sarau, J.W. Westley, New leukotriene B4 receptor antagonist: leucett-
amine a and related imidazole alkaloids from the marine sponge leucetta
microraphis, J. Nat. Prod. 56 (1993) 116e121.
4.3. Molecular modeling
ꢀ
[14] A. Bruel, C. Loge, M.-L.D. Tauzia, M. Ravache, R. Le Guevel, C. Guillouzo, J.-
ꢀ
ꢀ
F. Lohier, J.S.-D. Oliveira Santos, O. Lozach, L. Meijer, S. Ruchaud, H. Benedetti,
J.-M. Robert, Synthesis and biological evaluation of new 5-benzylated 4-oxo-
Coordinates for compounds 4a, 4c and 4f were generated using
CORINA v3.44 software [47]. Molecules were then docked in the
ATP binding site from the CLK1 and DYRK1 kinases retrieved from
the Protein Data Bank (PDBs ID: 1Z57 and 4MQ1, respectively),
using GOLD v5.2.2 docking package software [48]. CHEMPLP with
default parameters was used as an objective score function [49].
Structures of the complexes were exported for further examination
and depiction with Chimera v1.9 software [50], including hydrogen
bonds detection and close contact analysis.
3,4-dihydro-5H-pyridazino[4,5-b]indoles as PI3K
a inhibitors, Eur. J. Med.
Chem. 57 (2012) 225e233.
[15] G. Burgy, E. Limanton, F. Carreaux, E. Durieu, L. Meijer, J.-P. Bazureau,
Exploring the Synthesis of New 1-(4-Substitutedphenylamino)imidazo[1,5-a]
indol-3-one derivatives as cyclized analogs of leucettines, J. App. Pharm. Sci. 4
(2014) 025e032.
[16] K.F. Byth, J.D. Culshaw, S. Green, S.E. Oakes, A.P. Thomas, Imidazo[1,2-a]pyr-
idines. Part 2: SAR and optimisation of a potent and selective class of cyclin-
dependent kinase inhibitors, Bioorg. Med. Chem. Lett. 14 (2004) 2245e2248.
[17] R.P. Hertzberg, A.J. Pope, High-throughput screening: new technology for the
21st century, Curr. Opin. Chem. Biol. 4 (2000) 445e451.
ꢀ
[18] I. Ferrer, M. Barrachina, B. Puig, M. Martínez de Lagran, E. Martí, J. Avila,
M. Dierssen, Constitutive Dyrk1A is abnormally expressed in alzheimer dis-
ease, down syndrome, pick disease, and related transgenic models, Neurobiol.
Dis. 20 (2005) 392e400.
Acknowledgments
The authors gratefully acknowledge the support of this project
by CNRS, University Paris-Sud and “La Ligue Contre le Cancer”
throughout an “Equipe Labellisee 2014” grant. We also thank the
[19] M. Mariano, R.W. Hartmann, M. Engel, Systematic diversification of benzyli-
dene heterocycles yields novel inhibitor scaffolds selective for Dyrk1A, Clk1
and CK2, Eur. J. Med. Chem. 112 (2016) 209e216.
ꢀ
ꢁ
[20] W.-J. Song, L.R. Sternberg, C. Kasten-Sportes, M.L.V. Keuren, S.-H. Chung,
ꢀ
^
Canceropole Grand Ouest (axis: natural sea products in cancer
A.C. Slack, D.E. Miller, T.W. Glover, P.-W. Chiang, L. Lou, D.M. Kurnit, Isolation
of human and murine homologues of thedrosophilaminibrain gene: human
homologue maps to 21q22.2 in the down syndrome “critical region”, Geno-
mics 38 (1996) 331e339.
treatment), IBiSA (French Infrastructures en sciences du vivant: bio-
ꢀ
logie, sante et agronomie) and Biogenouest (Western France life
science and environment core facility network) for supporting
KISSf screening facility. S.B. is supported by ANR/Investissements
[21] Y. Ogawa, Y. Nonaka, T. Goto, E. Ohnishi, T. Hiramatsu, I. Kii, M. Yoshida,
T. Ikura, H. Onogi, H. Shibuya, T. Hosoya, N. Ito, M. Hagiwara, Development of

114
M. Lawson et al. / European Journal of Medicinal Chemistry 123 (2016) 105e114
a novel selective inhibitor of the Down syndrome-related kinase Dyrk1A, Nat.
Commun. 1 (2010) 86.
[22] J. Princi, K. Chandrabose, N.S.H.N. Moorthy, W. Digambar Kumar, J. Arvind
Kumar, T. Piyush, Human CDC2-Like Kinase 1 (CLK1): a novel target for Alz-
heimer's disease, Curr. Drug Targets 15 (2014) 539e550.
[23] Y. Sato, Y. Onozaki, T. Sugimoto, H. Kurihara, K. Kamijo, C. Kadowaki,
T. Tsujino, A. Watanabe, S. Otsuki, M. Mitsuya, M. Iida, K. Haze, T. Machida,
Y. Nakatsuru, H. Komatani, H. Kotani, Y. Iwasawa, Imidazopyridine derivatives
as potent and selective Polo-like kinase (PLK) inhibitors, Bioorg. Med. Chem.
Lett. 19 (2009) 4673e4678.
[24] K.S. Gudmundsson, S.D. Boggs, J.G. Catalano, A. Svolto, A. Spaltenstein,
M. Thomson, P. Wheelan, S. Jenkinson, Imidazopyridine-5,6,7,8-tetrahydro-8-
quinolinamine derivatives with potent activity against HIV-1, Bioorg. Med.
Chem. Lett. 19 (2009) 6399e6403.
[25] W. An, W. Wang, T. Yu, Y. Zhang, Z. Miao, T. Meng, J. Shen, Discovery of novel
2-phenyl-imidazo[1,2-a]pyridine analogues targeting tubulin polymerization
as antiproliferative agents, Eur. J. Med. Chem. 112 (2016) 367e372.
[26] A. Gueiffier, M. Lhassani, A. Elhakmaoui, R. Snoeck, G. Andrei, O. Chavignon, J.-
C. Teulade, A. Kerbal, E.M. Essassi, J.-C. Debouzy, M. Witvrouw, Y. Blache,
J. Balzarini, E. De Clercq, J.-P. Chapat, Synthesis of acyclo-c-nucleosides in the
imidazo[1,2-a]pyridine and pyrimidine series as antiviral agents, J. Med.
Chem. 39 (1996) 2856e2859.
[27] N. Dahan-Farkas, C. Langley, A.L. Rousseau, D.B. Yadav, H. Davids, C.B. de
Koning, 6-Substituted imidazo[1,2-a]pyridines: synthesis and biological ac-
tivity against colon cancer cell lines HT-29 and Caco-2, Eur. J. Med. Chem. 46
(2011) 4573e4583.
A. Backlund, B. Masereel, R. Kiss, J. Wouters, Novel trisubstituted harmine
derivatives with original in vitro anticancer activity, J. Med. Chem. 55 (2012)
6489e6501.
[37] T. Tahtouh, J.M. Elkins, P. Filippakopoulos, M. Soundararajan, G. Burgy,
E. Durieu, C. Cochet, R.S. Schmid, D.C. Lo, F. Delhommel, A.E. Oberholzer,
L.H. Pearl, F. Carreaux, J.-P. Bazureau, S. Knapp, L. Meijer, Selectivity, cocrystal
structures, and neuroprotective properties of leucettines, a family of protein
kinase inhibitors derived from the marine sponge alkaloid leucettamine B,
J. Med. Chem. 55 (2012) 9312e9330.
[38] G. Manning, D.B. Whyte, R. Martinez, T. Hunter, S. Sudarsanam, The protein
kinase complement of the human genome, Science 298 (2002) 1912e1934.
[39] T. Yin, M.J. Lallena, E.L. Kreklau, K.R. Fales, S. Carballares, R. Torrres,
G.N. Wishart, R.T. Ajamie, D.M. Cronier, P.W. Iversen, T.I. Meier, R.T. Foreman,
D. Zeckner, S.E. Sissons, B.W. Halstead, A.B. Lin, G.P. Donoho, Y. Qian, S. Li,
S. Wu, A. Aggarwal, X.S. Ye, J.J. Starling, R.B. Gaynor, A. de Dios, J. Du, A novel
CDK9 inhibitor shows potent antitumor efficacy in preclinical hematologic
tumor models, Mol. Cancer Ther. 13 (2014) 1442e1456.
ꢀ
[40] A.N. Bullock, S. Das, J.E. Debreczeni, P. Rellos, O. Fedorov, F.H. Niesen, K. Guo,
E. Papagrigoriou, A.L. Amos, S. Cho, B.E. Turk, G. Ghosh, S. Knapp, Kinase
domain insertions define distinct roles of CLK kinases in SR protein phos-
phorylation, Structure 17 (2009) 352e362.
[41] K. Anderson, Y. Chen, Z. Chen, R. Dominique, K. Glenn, Y. He, C. Janson, K.-
C. Luk, C. Lukacs, A. Polonskaia, Q. Qiao, A. Railkar, P. Rossman, H. Sun,
Q. Xiang, M. Vilenchik, P. Wovkulich, X. Zhang, Pyrido[2,3-d]pyrimidines:
discovery and preliminary SAR of a novel series of DYRK1B and DYRK1A in-
hibitors, Bioorg. Med. Chem. Lett. 23 (2013) 6610e6615.
[28] W. Han, D.L. Menezes, Y. Xu, M.S. Knapp, R. Elling, M.T. Burger, Z.-J. Ni,
A. Smith, J. Lan, T.E. Williams, J. Verhagen, K. Huh, H. Merritt, J. Chan,
S. Kaufman, C.F. Voliva, S. Pecchi, Discovery of imidazo[1,2-a]-pyridine in-
hibitors of pan-PI3 kinases that are efficacious in a mouse xenograft model,
Bioorg. Med. Chem. Lett. 26 (2016) 742e746.
[42] O. Fedorov, K. Huber, A. Eisenreich, P. Filippakopoulos, O. King, A.N. Bullock, D.
Szklarczyk, L.J. Jensen, D. Fabbro, J. Trappe, U. Rauch, F. Bracher, S. Knapp,
Specific CLK inhibitors from a novel chemotype for regulation of alternative
splicing, Chem. Biol., 18 67e76.
[43] C. Bissantz, B. Kuhn, M. Stahl, A medicinal chemist's guide to molecular in-
teractions, J. Med. Chem. 53 (2010) 5061e5084.
[29] Y. Rival, G. Grassy, A. Taudou, R. Ecalle, Antifungal activity in vitro of some
imidazo[1,2-a]pyrimidine derivatives, Eur. J. Med. Chem. 26 (1991) 13e18.
[30] K.C. Rupert, J.R. Henry, J.H. Dodd, S.A. Wadsworth, D.E. Cavender, G.C. Olini,
B. Fahmy, J.J. Siekierka, Imidazopyrimidines, potent inhibitors of p38 MAP
kinase, Bioorg. Med. Chem. Lett. 13 (2003) 347e350.
[44] S.-L. Liu, C. Wang, T. Jiang, L. Tan, A. Xing, J.-T. Yu, The role of Cdk5 in Alz-
heimer's disease, Mol. Neurobiol. (2015) 1e15.
[45] S. Bach, M. Knockaert, J. Reinhardt, O. Lozach, S. Schmitt, B. Baratte, M. Koken,
S.P. Coburn, L. Tang, T. Jiang, D.-c. Liang, H. Galons, J.-F. Dierick, L.A. Pinna,
€
€
[31] Z.-P. Zhuang, M.-P. Kung, A. Wilson, C.-W. Lee, K. Plossl, C. Hou,
F. Meggio, F. Totzke, C. Schachtele, A.S. Lerman, A. Carnero, Y. Wan, N. Gray,
D.M. Holtzman, H.F. Kung, Structureꢀactivity relationship of imidazo[1,2-a]
L. Meijer, Roscovitine targets, protein kinases and pyridoxal kinase, J. Biol.
Chem. 280 (2005) 31208e31219.
pyridines as ligands for detecting
b-Amyloid plaques in the brain, J. Med.
Chem. 46 (2003) 237e243.
[46] H. Zegzouti, M. Zdanovskaia, K. Hsiao, S.A. Goueli, ADP-Glo: a bioluminescent
and homogeneous ADP monitoring assay for kinases, Assay. Drug Dev.
Technol. 7 (2009) 560e572.
[47] J. Sadowski, J. Gasteiger, G. Klebe, Comparison of automatic three-dimensional
model builders using 639 X-ray structures, J. Chem. Inf. Comput. Sci. 34 (1994)
1000e1008.
[32] Y. Uto, Imidazo 1,2-a pyridines as cholesterol 24-hydroxylase (CYP46A1) in-
hibitors: a patent evaluation (WO2014061676), Expert Opin. Ther. Pat. 25
(2015) 373e377.
€
[33] N.P. Buu-HoI, P. Jacquignon, N.D. Xuong, D. Lavit, 2-Arylpyrrocolines and 2-
arylpyrimidazoles, J. Org. Chem. 19 (1954) 1370e1375.
[34] S.-Y. Takizawa, J.-I. Nishida, T. Tsuzuki, S. Tokito, Y. Yamashita, Synthesis and
characterization of novel iridium complexes with ligands of 2-phenylimidazo
[1,2-a]pyridine derivatives and application to organic light-emitting diode,
Chem. Lett. 34 (2005) 1222e1223.
[48] G. Jones, P. Willett, R.C. Glen, A.R. Leach, R. Taylor, Development and valida-
tion of a genetic algorithm for flexible docking1, J. Mol. Biol. 267 (1997)
727e748.
[49] O. Korb, T. Stützle, T.E. Exner, Empirical scoring functions for advanced pro-
teinꢀligand docking with PLANTS, J. Chem. Inf. Model. 49 (2009) 84e96.
[50] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt,
E.C. Meng, T.E. Ferrin, UCSF ChimeradA visualization system for exploratory
research and analysis, J. Comput. Chem. 25 (2004) 1605e1612.
[35] J. Koubachi, S. El Kazzouli, S. Berteina-Raboin, A. Mouaddib, G. Guillaumet,
Synthesis of polysubstituted imidazo[1,2-a]pyridines via microwave-assisted
one-pot cyclization/suzuki coupling/palladium-catalyzed heteroarylation,
J. Org. Chem. 72 (2007) 7650e7655.
ꢀ
ꢀ
ꢁ
[36] R. Frederick, C. Bruyere, C. Vancraeynest, J. Reniers, C. Meinguet, L. Pochet,