Exploration of the imidazo[1,2-b]pyridazine scaffold as a protein kinase inhibitor

Article abstract of DOI:10.1016/j.ejmech.2016.09.064

3,6-Disubstituted imidazo[1,2-b]pyridazine derivatives were synthesized to identify new inhibitors of various eukaryotic kinases, including mammalian and protozoan kinases. Among the imidazo[1,2-b]pyridazines tested as kinase inhibitors, several derivatives were selective for DYRKs and CLKs, with IC₅₀?50?=?82?nM), CLK4 (IC₅₀?=?44?nM), DYRK1A (IC₅₀?=?50?nM), and PfCLK1 (IC₅₀?=?32?nM). The compounds were also tested against Leishmania amazonensis. Several compounds showed anti-leishmanial activity at rather high (10?μM) concentration, but were not toxic at 1?μM or 10?μM, as judged by viability assays carried out using a neuroblastoma cell line.

Full text of DOI:10.1016/j.ejmech.2016.09.064

Accepted Manuscript
Exploration of the imidazo[1,2-b]pyridazine scaffold as a protein kinase inhibitor
Lyamin Z. Bendjeddou, Nadège Loaëc, Benoît Villiers, Eric Prina, Gerald Späth,
Hervé Galons, Laurent Meijer, Nassima Oumata
PII:
S0223-5234(16)30797-8
10.1016/j.ejmech.2016.09.064
EJMECH 8930
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 August 2015
Revised Date: 19 September 2016
Accepted Date: 20 September 2016
Please cite this article as: L.Z. Bendjeddou, N. Loaëc, B. Villiers, E. Prina, G. Späth, H. Galons, L.
Meijer, N. Oumata, Exploration of the imidazo[1,2-b]pyridazine scaffold as a protein kinase inhibitor,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.064.
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ACCEPTED MANUSCRIPT
C3-optimization
20a
N
N
CF
3
IC50: CLK1 = 0.082 µM
DYRK1A = 0.05 µM
N
H
N H
N
N
O
S
O
O H
O
11v
O H
O
H
O
OEt
IC50: CLK1 = 0.023 µM
DYRK1A = 0.025 µM
C6-optimization

Bendjeddou et al.
imidazo[1,2-b]pyridazine kinase inhibitors
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Exploration of the imidazo[1,2-b]pyridazine
scaffold as a protein kinase inhibitor
Lyamin Z. Bendjeddoua, b, Nadège Loaëca, Benoît Villiersa, Eric Prinac, Gerald Späthc, Hervé
Galons,b Laurent Meijera* and Nassima Oumataa**
aManRos Therapeutics, Perharidy Research Center, 29680 Roscoff, Bretagne, France,
bUnité de Technologies Chimiques et Biologiques pour la Santé, Université Paris Descartes
UMR-S 1022 Inserm, 4 avenue de l’Observatoire, 75270 Paris cedex 06, France
c.Institut Pasteur, Unité INSERM U1201, Département des Parasites et Insectes Vecteurs,
Unité de Parasitologie Moléculaire et Signalisation, 28 rue du Dr. Roux, 75015 Paris,
France
** Correspondence should be addressed to:
Biology: L. Meijer (<meijer@manros-therapeutics.com>)
Chemistry: N. Oumata (<oumata@manros-therapeutics.com>)
Running Title: imidazo[1,2-b]pyridazine kinase inhibitors
Keywords: kinase inhibitor, imidazo[1,2-b]pyridazine, unicellular parasites, Leishmania,
DYRK1A, CLKs
Abbreviations:
AD, Alzheimer’s disease; CDKs, cyclin-dependent kinases; CK1, casein kinase 1; CLKs,
cdc2-like kinases; CMGC (cyclin-dependent kinase [CDK], mitogen-activated protein kinase
[MAPK], glycogen synthase kinase [GSK3], CDC-like kinase [CLK]); CML, chronic
myelocytic leukemia; DAD, diode array detector; DEPC, diethyl pyrocarbonate; DMEM,
Dulbecco’s modified Eagle’s medium; DCM, dichloromethane; DMSO, dimethylsulfoxide;
DS, Down syndrome; DTT, dithiothreitol; DYRKs, dual-specificity tyrosine phosphorylation
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regulated kinases; FBS, fetal bovine serum; GSH, glutathione; GSK-3, glycogen synthase
kinase-3; GST, glutathione-S-transferase; IPTG, isopropyl-β-D-thiogalactopyranoside; LC,
Liquid chromatography; MAP kinases, mitogen-activated protein kinases; NFTs,
neurofibrillary tangles; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium; PBS, phosphate-buffered saline; SAR, structure activity
relationship; SRp, serine/arginine-rich proteins; TLC, Thin-layer chromatography; TMS,
tetramethylsilane.
ABSTRACT
3,6-Disubstituted imidazo[1,2-b]pyridazine derivatives were synthesized to identify new
inhibitors of various eukaryotic kinases, including mammalian and protozoan kinases.
Among the imidazo[1,2-b]pyridazines tested as kinase inhibitors, several derivatives were
selective for DYRKs and CLKs, with IC50 <100 nM. The characterization of the kinome of
several parasites, such as Plasmodium and Leishmania, has pointed out profound divergences
between protein kinases of the parasites and those of the host. This led us to investigate the
activities of the prepared compounds against 11 parasitic kinases. 3,6-Disubstituted
imidazo[1,2-b]pyridazines showed potent inhibition of Plasmodium falciparum CLK1
(PfCLK1). Compound 20a was found to be the most selective product against CLK1 (IC50 =
82 nM), CLK4 (IC50 = 44 nM), DYRK1A (IC50 = 50 nM), and PfCLK1 (IC50 = 32 nM). The
compounds were also tested against Leishmania amazonensis. Several compounds showed
anti-leishmanial activity at rather high (10 µM) concentration, but were not toxic at 1 µM or
10 µM, as judged by viability assays carried out using a neuroblastoma cell line.
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1. Introduction
Reversible protein phosphorylation is the most common post-translational modification
occurring in many fundamental cellular processes, such as differentiation, division,
proliferation, apoptosis, and signal transduction mechanisms. Due to their strong involvement
in essentially all physiological processes, dysfunctional phosphorylation can generate many
human diseases, such as cancer [1], inflammation, auto-immune and neurodegenerative
diseases, and parasite infections. Among the human kinome described by Manning et al. [2] in
2002, the CMGC group, which comprises nine kinase families, has been extremely well-
studied as a group of therapeutic targets. Cdc2-like kinases (CLKs) and dual-specificity
tyrosine-phosphorylation-regulated kinases (DYRKs) are families of conserved groups of
dual-specificity kinases belonging to the CMGC group, including CDKs (cyclin-dependent
kinases), MAP kinases (mitogen-activated protein kinases), GSK-3 (glycogen synthase
kinase), and CLKs (Cdc2-like kinases). These two families of kinases auto-phosphorylate at
tyrosine residues in their activation loop, but exclusively phosphorylate serine/threonine
residues. The CLK family consists of four isoforms: CLK1, CLK2, CLK3, and CLK4. CLKs
phosphorylate serine/arginine-rich splicing proteins (SRp), which are implicated in the
regulation of alternative splicing [3]. CLK1 is involved in Alzheimer’s disease (AD) [4]
(splicing of microtubule-associated protein Tau pre-mRNA). The DYRK family comprises
five isoforms, DYRK1A, DYRK1B, DYRK2, DYRK3, and DYRK4. The human DYRK1A
gene is located on chromosome 21 in the “Down syndrome (DS) critical region,” and
overexpression of DYRK1A in DS has been associated with neurodegenerative disease [5].
DYRK1A and DYRK1B are also implicated in diverse types of cancers [6,7]. Orthologs of
mammalian CLKs and DYRKs are also found in unicellular parasites, such as Leishmania,
Trypanosoma, Cryptosporidium, Giardia, Toxoplasma, and Plasmodium. Deregulation and
dysfunction of CLKs and DYRKs have been linked to several diseases, which make these
kinases attractive potential therapeutic targets.
In the present paper, we describe potent and selective CLK and DYRK inhibitors. We
optimized the C-3 position of disubstituted imidazo[1,2-b]pyridazines as CLK and DYRK
kinase inhibitors. Our interest in this scaffold is based on the fact that imidazo[1,2-
b]pyridazines are frequently described as kinase inhibitors. Imidazo[1,2-b]pyridazines have
been reported to be inhibitors of CDK2 [8], Pim [9], IKKβ [10-12], VEGFR2 [13], and Syk
[14] (Figure 1). We have identified more than 20 patents issued since 2007 that describe
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imidazo[1,2-b]pyridazines as kinase inhibitors, the most advanced product being Ponatinib
[15], which in 2012 was launched on the market as a BCR-ABL inhibitor in the treatment of
chronic myelocytic leukemia (CML). The MRC (Medical Research Council) technology team
has described PfPK7 [16] and PfCDPK1 [17,18] inhibitors as being potential antimalarial
agents. More recently, diarylimidazo[1,2-b]pyridazines have been identified and evaluated for
antiplasmodial activity [19] (Figure 1).
(Figure 1)
Alzheimer’s disease (AD) is a neurodegenerative disease responsible for the most common
form of dementia. It is characterized by extracellular accumulation of amyloid plaque (Aβ)
and intracellular deposition of neurofibrillary tangles (NFTs). NFTs mainly result from the
aggregation of hyperphosphorylated microtubule-associated Tau protein. Aberrant
hyperphosphorylation of Tau induces decreased microtubule binding, causing loss of function
and aggregation. CLK1 [4] and DYRK1A [20] have been shown to be involved in
neurodegenerative disease, particularly AD. Many CLK and DYRK inhibitors have been
described over the last few years, including harmine, TG003 [21] and Indy [22], leucettines
[23,24], quinazolines [25-27], KH-CB19 [3], meriolins [28], meridianins [29],
imidazopyridazines [30], and most recently, furo- or thieno[3,2-d]pyrimidin-4-amines [31]
derived from harmine. We describe the synthesis strategy and biological evaluation of a new
series of disubstituted imidazo[1,2-b]pyridazine inhibitors of CLKs and DYRKs. The aim of
this work is first to identify new mixed inhibitors of DYRK1A and CLKs, which could be
further evaluated in cell and animal AD models. A second objective is to identify inhibitors of
orthologous parasitic kinases which could be useful in the treatment of Leishmania and
Plasmodium infections.
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2. Results and discussion
2.1. Chemistry
A large group of disubstituted imidazo[1,2-b]pyridazines was synthesized, following routes
A, B, or C, as depicted in Scheme 1. The synthesis strategy developed provides access to a
broad family of disubstituted imidazo[1,2-b]pyridazines. 6-Chloroimidazo[1,2-b]pyridazine 1
was obtained by the cyclisation of 3-amino-6-chloropyridazine and chloroacetaldehyde at
reflux in butanol [8], followed by bromination by N-bromosuccinimide in CHCl3, which
yielded 4. A Suzuki cross-coupling reaction at the C-3 position and nucleophilic substitution
[32] at C-6 yielded the desired compounds. The aromatic ring can also be introduced by direct
arylation on 6-chloroimidazo[1,2-b]pyridazine [33] (route B). An alternative is to introduce
the amine at C-6 and then carry out the Suzuki reaction or direct arylation. This route C
synthesis allows the introduction of more sensitive groups. (Scheme 1). Indoles were
introduced via route A at position C-3 by a Suzuki reaction, after -NH protection by Boc.
Nucleophilic substitution at position C-6 was then carried out from (S)-2-amino-1-butanol in
EtOH. Methoxy and sulfonyl groups were obtained by nucleophilic substitution, starting from
5-6. Pyridines were then introduced at the C-3 position. 6-Chloro-3-(3-pyridyl)imidazo[1,2-
b]pyridazine 6e was prepared using route A in a 66% yield. (Scheme 1). The carbamate 14
was synthesized using route C, the ethyl carbamate being introduced on 1 by a Buchwald
reaction, which afforded 7a. Bromination of 7a yielded 8a, which eventually led to 14 after a
Suzuki coupling reaction (Scheme 2). The acetanilide derivative 15 was prepared by reacting
6h with 3-aminoacetanilide under Buchwald conditions (Scheme 3).
(Scheme 2 and 3)
Several pyrimidines were obtained through CH-arylation of 6-chloroimidazo[1,2-b]pyridazine
1, using the the S-methyl derivative 23. (Scheme 4). Oxidation of the S-methyl group
provided a mixture of the corresponding sulfone and sulfoxide. Indirect amination of the
sulfones 10d and 21g was performed by 2,4-dimethoxybenzylamine followed by acidic
removal of the dimethoxybenzyl protective group. Similarly, the oxidation step was also
achieved with 11o bearing the aminobutanol group and with 21e substituted by OCH3. Finally,
indirect amination of the pyrimidine yielded 11r and 21i.
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(Scheme 4)
3-Formylphenyl was linked with an excellent yield (90%) on position 3 of 3. Imine 20a was
the main product obtained from aldehyde 6m and (R)-2-amino-1-butanol (Scheme 7). An
alternative route to imines presented in scheme 5 gave access to acid and imine derivatives in
three steps, starting from 6-chloro-3-bromoimidazo[1,2-b]pyridazine. We used the same
procedure for a C-6 position for compounds 11v, but we used route A for chlorine (6n) and
methoxy (21j) groups. Amide synthesis was the last one we used to complete substituent
optimization. The four possible stereoisomers SS(11x), SR(11y), RR(20c) and RS(20d) were
prepared (Scheme 5). Several oximes such as 11v were synthesized from an aldehyde in the
presence of hydroxylamine and sodium acetate in methanol.
(Schemes 5)
2.2.Evaluation against kinases.
Our studies originated from testing the biological activity of product A (Figure 1). Among
several others, this compound was prepared in the aim of identifying new DYRK and CLK
inhibitors. We therefore decided to analyze the structure-activity relationship (SAR) of this
class of molecules, particularly by introducing an aromatic ring at the C-3 position. Leucettine
L41 was used as a reference compound in the evaluation of DYRK/CLK inhibitors. In the
context of this article, IC50 values obtained with mammalian kinases were in accordance with
those reported in a previous paper [24]. IC50 values of Leucettine L41 on unicellular parasite
DYRK/CLK homologs are shown in table 13, which is added in the supplementary material
.
The indole series revealed a slight inhibition preference for CLKs and DYRKs (Table 1). Two
derivatives displaying an IC50 of less than 0.1 µM, were obtained, with substitutions on the
indole C-5 position (0.092 µM for 22b against CLK4 and 0.098 µM for 11a against
LdDYRK1B). With the substituted indole in position C-2 (11b), activity is increased for
CLKs/DYRKs, but a bromine atom in compound 11c decreased the activity (Table 1). The
introduction of a 3-pyridyl group at the 3-position led to rather potent inhibitors of CLKs as
soon as an amino-alcohol was in position 6 (0.059 µM for 11d against CLK4 and 0.090 µM
for 12 against CLK4). (Table 2).
The optimization was pursued by introducing an arylhalide and a trifluromethylphenyl group.
Comparing the various C-6 substituents, (S)-2-amino-1-butanol 11e (IC50 CLK1 = 0.12 µM)
appeared to be the most active (Table 3). 4-Trifluoromethylbenzene derivatives 10a, 21c, and
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11f synthesized using route B displayed decreased activity and elongation of the ring, further
decreasing the activity, but increasing selectivity towards CLK1 and CLK4. (Table 3).
(Table 3)
The trifluoromethyl group was replaced in the same position by chlorine, the most active
compound against CLKs and DYRKs, is substituted by amino alcohol (compound 11h, table
4). In order to increase activity and selectivity towards mammalian kinases, the C-6 position
was modulated with heterocyclic aromatic and heterocyclic aliphatic amine substitutions.
(Table 4)
N-(3-Aminophenyl)acetamide derivative (15) and 2-piperazin-1-ylethanol derivative (16)
displayed good inhibitory activity against CLK1 (IC50 values of 86 nM and 84 nM,
respectively). Compound 15 also inhibited PfCLK1 (IC50 =78 nM) (Table 4).
Converting the amine (11i) to an acetamide (11k) led to increased activity toward CLKs and
DYRKs as well as toward CDK1. Compound 11k inhibited CLK1, CLK2, CLK4, DYRK1A,
DYRK1B, DYRK2, and CDK1, with IC50 values of less than 100 nM (Table 5).
Compound 11m was more active than compound 11l on CLK1, CLK2, CLK4, DYRK1A, and
DYRK2 (Table 6).
In general, compared to phenyl, the pyrimidines were less active. Pyrimidine appeared not to
be the most efficient aryl substitution. Compound 11m (Table 6) was 10-fold more active than
11q the product (Table 7) on CLK1 and DYRK1A (Table 7).
(Tables 5, 6, and 7)
The formyl-containing derivative 11u showed good inhibitory activity against CLK1 (41 nM),
CLK4 (41 nM), DYRK1A (79 nM), DYRK1B (53 nM), and DYRK2 (63 nM). Next, chlorine
was replaced by fluorine to compare inhibitory activities and to observe the increased
reactivity of fluorine over chlorine in nucleophilic substitution. Comparison of compounds 5b
and 6m (Table 8) revealed decreased inhibitory activity against CDK5, DYRK1A, and
DYRK2 for the fluorine derivative, but its inhibitory activity was maintained for CLKs and
CK1.
Derivative 20a selectively inhibited CLK1, CLK4, and DYRK1A with a low IC50. Among all
the compounds tested, 20a seems to be the most potent; we therefore synthesized its isomer
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(S)-2-amino-1-butanol via route C. Compound 11s revealed activity, particularly against the
CLK and DYRK families, but it also inhibited CDK1 and CDK2
The S-isomer of amino alcohol 11w was more active than isomer R(20a) against CLK1 and
CLK4, but less active against DYRK1A. Compound 20a also inhibited PfCLK1 (32 nM),
whereas compound 11w was 10-fold less active.
Among all the products described here, compound 11v was the most active against CLK1 (23
nM) and CLK4 (25 nM). DYRK1A was inhibited at 33 nM by compound 21j. It is interesting
to observe the differences in activity of compounds 11w and 20a (Table 9) All formamides
were found to be less active than the imine analogues (Table 10).
(Tables 9 and 10)
2.3. Cytotoxicity
Because these derivatives are intended to be used in the treatment of neurodegenerative
diseases, it is important that they not be cytotoxic. All compounds were tested on the SH-
SY5Y human neuroblastoma cell-line in a cell-survival assay. The results showed that no
compound was cytotoxic at 1µM and 10µM.
Since many kinases are involved in proliferation, it may be estimated that the present
compounds are not potent inhibitors of other kinases, and may therefore be considered rather
selective inhibitors of the kinases under study. (See Supplementary Material, Table 12).
2.4. Antiparasitic activities
Finally, in respect to the tests on parasitic kinases, the compounds were assayed against L.
amazonensis. Several compounds were able to reduce parasite proliferation, but at the rather
high concentration of 10 µM (Table 11).
(Table 11)
3. Conclusion
In conclusion, based on the imidazo[1,2-b]pyridazine scaffold, we have identified and
optimized novel CLK1 and DYRK1A inhibitors, and have established a detailed SAR.
Investigation of aryl groups at the C-3 position led us to the finding that the imine group is the
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most active substituent. A large number of substituents were explored, compound 20a
representing the most selective compound against CLK1 and DYRK1A, but also against
PfCLK1. Since parasite kinases have recently been shown to be potential targets for anti-
microbial chemotherapy, we also carried out enzymatic assays with our compounds, using
recombinant or purified kinases from various protozoa, including T. brucei, T. cruzi, T.
gondii, C. parvum, G. lamblia, L. donovani, and L. major. Furthermore, the compounds were
also assessed for anti-leishmanial activity, using a viability assay with Leishmania
amazonensis parasites and a phenotypic HCA with intra-macrophagic parasites. Our
derivatives showed much less inhibitory activity against Leishmania and Trypanosoma
parasite kinases. Alternatively, the oxime derivatives 21j and 11v showed significant
inhibitory activity against mammalian CLK1 and DYRK1A. Moreover, cell-survival
evaluation showed no cytotoxicity, at least in a neuroblastoma cell line. Finally, several
compounds were found to be highly potent against L. donovani kinases. However, no
correlation was found between this enzymatic inhibitory activity and the effect on Leishmania
survival. In fine, the imidazo[1,2-b]pyridazine scaffold presents a wide potential for future use
in vivo evaluation. The co-crystal structure determination of 11v with several kinases, as
previously described in the case of leucettine L41 [24], should be undertaken. These results
will guide future design in this family of imidazo[1,2-b]pyridazines.
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4. Experimental
4.1. Buffers and chemicals
Buffer A: 10 mM MgCl2, 1 mM EGTA (MW 380.4), 1 mM dithiothreitol (DTT) (MW 154.2),
25 mMTris/HCl (MW 121.1) and 50 µg/mL heparin.
Buffer C: 60 mM ß-glycerophosphate, 30 mM p-nitrophenylphosphate, 25 mM MOPS, 5 mM
EGTA, 15 mM MgCl2, 1 mM DTT and 0.1 mM sodium vanadate.
All chemicals were purchased from Sigma, unless otherwise stated; the protease inhibitor
cocktail was obtained from Roche.
4.2. Protein kinase assays
Kinase activities were assayed in buffer A or C at 30 °C at a final ATP concentration of 15
µmol/L. Blank values were subtracted and activities expressed in percent of maximal activity,
i.e., in the absence of inhibitors. Controls were carried out with appropriate DMSO dilutions.
The GS-1, CKS, CDK7/9 tide, and RS peptide substrates were obtained from Proteogenix
(Oberhausbergen, France).
CDK1/cyclin B (M phase starfish oocytes, native), CDK2/cyclin A and CDK5/p25 (human,
recombinant) were prepared as previously described [33]. Their kinase activity was assayed in
buffer A with 1 mg histone H1/mL, in the presence of 15 µmol/L [γ-33P] ATP (3,000
Ci/mmol; 10 mCi/mL) in a final volume of 30 µL. After 30 min incubation at 30 °C, the
reaction was stopped by harvesting onto P81 phosphocellulose supernatant (Whatman) using
a FilterMate harvester (Packard) and were washed in 1% phosphoric acid. Scintillation fluid
was added and the radioactivity measured in a Packard counter.
CDK9/cyclin T (human, recombinant, expressed in insect cells) was assayed as described for
CDK1/cyclin B, but using CDK7/9-tide (YSPTSPSYSPTSPSYSPTSPSKKKK) (8.1
µg/assay) as a substrate.
GSK-3 (porcine brain, native) and PfGSK3 (Plasmodium falciparum, recombinant, expressed
in E. coli as glutathione-S-transferase (GST) fusion proteins) was assayed as described for
CDK1 with 0.5 mg BSA /mL + 1 mM DTT and using a GSK-3-specific substrate (GS-1:
YRRAAVPPSPSLSRHSSPHQSpEDEEE) (Sp stands for phosphorylated serine) [33].
CK1δ/ε (porcine brain, native) and LmCK1 (Leishmania major, recombinant, expressed in E.
coli as HIS fusion proteins) was assayed as described for CDK1, but in buffer C, and using 25
µM CKS peptide (RRKHAAIGpSAYSITA), a CK1-specific substrate [33].
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DYRK1A, 1B, 2, and 3 (human, recombinant, expressed in E. coli as GST fusion proteins)
were purified by affinity chromatography on glutathione-agarose and assayed as described for
CDK1/cyclin B with 0.5 mg BSA /mL + 1 mM DTT and using Woodtide
(KKISGRLSPIMTEQ) (1.5µg/assay) as a substrate, a residue of transcription factor FKHR.
CLK1, 2, 3, and 4 (mouse, recombinant, expressed in E. coli as GST fusion proteins) were
assayed as described for CDK1/cyclin B with 0.5 mg BSA /mL + 1 mM DTT and RS peptide
(GRSRSRSRSRSR) (1µg/assay) as a substrate.
Protozoan DYRKs and CLKs (recombinant, expressed in E. coli) were assayed as described
for CDK1/cyclin B with 0.5 mg BSA /mL + 1 mM DTT and Woodtide or RS peptide for
DYRKs or CLKs isoforms.
4.3. Production and purification of the parasitic kinases
The parasitic kinase genes (Table 13) were optimized for expression in E. coli, synthesized
and cloned in pGEX-6P-1 (GE Healthcare) with the type II restriction enzymes BamHI and
XhoI for glutathione S-transferase (GST) fusion at the N-terminus (GenScript).
The plasmids were transformed into chemically competent E. coli BL21(DE3), which were
grown overnight at 37°C in 2xYT medium containing 100 ꢀg/mL ampicillin. These cultures
were used to inoculate 1 L volumes of 2xYT medium (containing 100 ꢀg/mL ampicillin) in
5L flasks. The cultures were allowed to grow at 37 °C before the temperature was decreased
to 18 °C. At an optical density (OD600) of about 1.0, protein expression was induced
overnight at 18°C with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The bacteria
were harvested by centrifugation and frozen at -20 °C.
Cells were resuspended in lysis buffer (PBS pH 7.4, 1% NP40, 1 mM DTT, 1 mM EDTA, 1
mM PMSF) in the presence of protease inhibitor cocktail (Roche), and lysozyme was added to
1 mg/mL and incubated for 1h at 4 °C with gentle agitation. Cells were then sonicated on ice
before MgCl2 (6 mM) and benzonase (Novagen, 25 U/mL) were added to degrade nucleic
acids and decrease sample viscosity. After centrifugation at 4 °C, the soluble fraction was
collected and proteins were bound to glutathione (GSH) sepharose 4B beads (GE Healthcare).
Beads were washed three times with lysis buffer and once with buffer C (25 mM MOPS pH
7.0, 60 mMβ-glycerophosphate, 15 mM p-nitrophenylphosphate, 15 mM EGTA, 15 mM
MgCl2, 2 mM DTT, 1 mM sodium vanadate). Proteins were eluted with elution buffer (buffer
C containing 30 mM reduced GSH, 15% glycerol, and pH adjusted to 8.5).
4.4. Cell culture conditions; cell-survival quantification
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SH-SY5Y cells were grown in DMEM medium from Invitrogen (Cergy-Pontoise, France).
All media were supplemented with antibiotics (penicillin-streptomycin) from Lonza and 10%
volume of fetal calf serum from In vitrogen. Cells were cultured at 37 °C with 5% CO2. Drug
treatments were carried out on exponentially growing cultures at the indicated concentrations.
Control experiments were also carried out, using appropriate dilutions of DMSO (maximum
1% DMSO). Cell Titer 96® containing the 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent was purchased
from Promega (Madison, WI, USA). Cell viability was determined by measuring the
reduction of MTS as described [34].
4.5. Antileishmanial activity
4.5.1. Parasites
L. amazonensis strain LV79 (MPRO/BR/1972/M1841) expressing the mCherry fluorescent
reporter was propagated in Swiss nu/nu mice. Amastigotes were isolated from mouse lesions
and used for HCA. Promastigotes were differentiated from lesion-derived amastigotes and
grown in M199 medium at 26 °C for use in dye reduction assay to assess compound toxicity
on host cell-free parasites [35].
4.5.2. Viability assay on host cell-free Leishmania
An enzymatic assay which incorporates a REDOX indicator (resazurin), which fluoresces in
response to cellular metabolic reduction (resofurin), was used to quantify the toxic effect of
selected compounds on L. amazonensis promastigotes in axenic conditions. Compounds were
tested at three final concentrations (20, 4, and 0.8 µM) in quadruplicate.
Supporting information,
1. Supplementary material 1. Experimental procedures for the synthesis of all compounds.
Table 12: cell survival after treatment by prepared compounds. Table 13: Inhibition of
parasitic kinases by Leucettine L41. Table 14: Parasitic kinase genes used for the
expression of tested kinases.
2. Supplementary material 2 and 3. 1H NMR, 13C NMR, and MS spectra.
12

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imidazo[1,2-b]pyridazine kinase inhibitors
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Acknowledgements
This research was supported by grants from the ‘Fonds Unique Interministériel” (FUI)
PHARMASEA, and TRIAD projects (HG, LM), the “Association France-Alzheimer
(Finistère)” (LM), the “Fondation Jérôme Lejeune” (LM), ANR grant TRANSLEISH and
EEC FP7 European Union 7th Framework Program Knowledge-based Bioeconomy (FP7-
KBBE) grant BLUEGENICS (LM).
13

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List of tables figure and schemes:
a
Table 1. Kinase inhibition values: IC50 in µM for indole derivatives. Leucettine L41 was
used as reference compoundb
Table 2. Kinase inhibition values: IC50 a in µM for pyridine derivatives.
Table 3. Kinase inhibition values: IC50 a in µM for halogens and CF3 derivatives
Table 4. Kinase inhibition values: IC50a in µM for chlorine derivatives.
Table 5. Kinase inhibition values IC50a in µM) for amino and acetamido derivatives.
Table 6. Kinase inhibition values IC50a in µM for compound 11m, 11n and 11o.
Table 7. Kinase inhibition values IC50a in µM for pyrimidine derivatives.
Table 8. Kinase inhibition values: IC50a in µM for aldehyde, acid and ester derivatives.
Table 9. Kinase inhibition values (IC50 in µM) for imine and oxime derivatives.
Table 10. Kinase inhibition values: IC50a in µM for amide derivatives.
Table 11. Anti-leishmanial activity on promastigotes. A miniaturized dye reduction assay
(resazurin-based assay) was used to estimate the leishmanicidal activity of each compound.
Three compound concentrations (20, 4, and 0.8 µM) were tested in quadruplicate for three
days on promastigotes. Following subtraction of the mean background fluorescence value
from the medium controls, data were expressed as percent of inhibition (PI) as compared to
untreated parasite culture wells. The mean +/- standard deviation is shown for every tested
compound. PI above 30% and 75% are highlighted in bold and underlined, respectively.
Figure 1. The imidazo[1,2-b]pyridazine scaffold: selected kinase inhibitors and initial hit A.
Scheme 1. General synthetic strategy, C3, C6-disubstituted imidazo[1,2-b]pyridazines. Reagents and
conditions: (a) i nBuOH, 18h, reflux, ii H2O, NaOH; (b) NBS, CHCl3, 8 h, 20 °C; (c) Boronic acid or
ester, Pd[P(C6H5)3]4, Na2CO3 (2 M), 1,4-dioxane, 100 °C; (d) 2-amino-1-butanol, 160-180 °C; (e)
Pd(OAc)2, xantphos, t-BuOK, H2O, 1,4-dioxane, 100 °C, 48 h; (f) NaOMe, MeOH, 48 h, reflux; (g)
NaSMe, DMSO, r.t or 40 °C, 2 h ; (h) Aryl/heteroaryl halide, Pd(OAc)2, P(C6H5)3, K2CO3, toluene,
110 °C; (i) KF, DMSO/toluene, 170 °C, 6 h.
Scheme 2. Reagents and conditions: (a) Ethyloxycarbamate or 3-aminoacetanilide, Pd(OAc)2,
xantphos, t-BuOK, H2O, 1,4-dioxane, 100 °C, 24 h, 27%; (b) NBS, CHCl3, 20 °C, 4 h, 100%; (c) 3-
Chlorophenylboronic pinacol ester, Pd[P(Ph)3]4, Na2CO3 (2 M), 1,4-dioxane, 100 °C.
19

Bendjeddou et al.
imidazo[1,2-b]pyridazine kinase inhibitors
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Scheme 3. Reagents and conditions: (a) Acetic anhydride, DMAP, Et3N, DCM, 20 °C, 2 h; (b) (S)-2-
Amino-1-butanol, 160 °C, 7 h; (b’) (S)-2-Amino-1-butanol,180 °C, 7 h; (a’) Acetic anhydride, DMAP,
Et3N, DCM, r.t, 2 h.
Scheme 4. Reagents and conditions: (a) TMSBr, CH3CN, 40 °C, 27 h; (b) 6-Chloro-imidazo[1,2-
b]pyridazine, Pd(OAc)2, PPh3, K2CO3, toluene, 2 days, 110 °C, 38%; (c) mCPBA, DCM, r.t; (d)
MeONa, MeOH, 6 h, 80 °C; (e) (S)-2-Amino-1-butanol, 170 °C, 4 h. (f) 2,4-Dimethoxybenzylamine 3
Eq. 1,4-dioxane, 8 h, reflux; (g) TFA, DCM, 20 °C.
Scheme 5. Reagents and conditions : (a) (R) or (S)-2-Amino-1-butanol, 8 h, 180 °C; (c) Ethyl
3-bromobenzoate, Pd(OAc)2, P(C6H5)3, K2CO3, toluene, 8 h, 110 °C, 10 %; (c) i: NaOH (1N),
THF/EtOH, ii: HCl (1N), H2O, 28%; (d) 3-Formylphenylboronic acid, Pd[P(C6H5)3]4,
Na2CO3 (2 M), 1,4-dioxane, 12 h, 100 °C. (e) (S)-2-Amino-1-butanol 2.5 Eq., 3 days 20 to 60
°C. (f ) (S or R)-2-Amino-1-butanol, DCC/HOBt, Et3N, THF/DCM, 20 °C, 24 h. (g) (S or R)-
2-Amino-1-butanol, DCC/HOBt, Et3N, THF/DCM, 20 °C, 24 h; (b) (S or R)-2-Amino-1-
butanol, 180 °C, 12 h; (h) KF, DMSO/Toluene, 8 h, 180 °C; (i) NH2OH.HCl, NaOAc 1.1 Eq,
MeOH-H2O 10%, 20 °C, 16 h.
20

ACCEPTED MANUSCRIPT
a
Table 1. Kinase inhibition values : IC50 in µM for indole derivatives. Leucettine L41 was
used as reference compound.b
N
N
Cl
N
N
H
Compd
6a
4.1
5.9
6.7
5
21a
4.3
22b
2.7
11a
2.7
11b
1.8
4.1
1.2
1.1
2.1
0.11
0.38
2.4
0.12
0.34
0.22
0.21
0.12
2.3
0.15
10
11c
1.7
9.1
8.3
0.22
10
CDK1
>10
5.1
9.1
41
CDK2
CDK5
4.2
3.1
3.1
2
2.8
CDK9
2.8
1.2
1.2
5
CK1
0.13
1
0.22
0.71
3.1
0.13
0.22
5.8
0.27
2.2
1.2
1.9
10
CLK1
CLK2
5.2
0.12
2.4
1.2
0.61
4.7
5.6
1.8
>10
>10
>10
>10
1.2
5.9
2.8
4
21
CLK3
0.18
0.47
1.2
0.092
0.58
1.4
0.21
0.35
0.96
0.38
1.8
1
CLK4
0.71
2.1
1.2
0.61
10
DYRK1A
DYRK1B
DYRK2
DYRK3
GSK-3
0.32
0.91
4.3
0.44
1.2
>10
3.1
2.7
1.2
0.098
˃10
˃10
˃10
˃10
3.1
0.13
10
LdDYRK1B
LdDYRK3
LdDYRK4
TbCLK1
TcCLK1
CpCLK1
GlCLK
9.2
>10
>10
>10
>10
4.2
>10
>10
>10
2.7
10
10
10
10
10
10
10
10
5.2
8.1
1.1
2.1
10
2.9
10
4.1
4.1
4.1
TgCLK
LmDYRK2
LmCLK
PfCLK1
5
>10
>10
0.9
3.9
4.1
10
10
>10
1.6
>10
1.3
10
10
2.2
2.2
10
a IC50 values were estimated from dose-response curves and are shown in µM. >10, less than
50% inhibition at 10 µM.
b Leucettine L41 [24] DYRK1A: IC50 = 0.040 µM; CLK1: IC50 = 0.090 µM (See also table
the table in supplementary material.

ACCEPTED MANUSCRIPT
Table 2. Kinase inhibition values: IC50 a in µM for pyridine derivatives.
Compd
6e
11d
12
13
>10
71
3
3.4
2.3
3.2
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
CDK1
CDK2
4.7
>10
4.1
0.89
4.1
42
1.7
1.4
CDK5
2.1
1.2
CDK9
5.1
1.2
CK1
0.19
0.71
3.9
0.18
0.59
3.3
CLK1
CLK2
CLK3
0.4
13
0.059
0.87
0.51
0.6
0.09
0.39
0.13
0.19
0.32
5.1
CLK4
DYRK1A
DYRK1B
DYRK2
DYRK3
GSK-3
20
11
16
0.95
3
51
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.69
˃10
˃10
˃10
˃10
2.2
0.39
>10
>10
>10
>10
3.8
LdDYRK1B
LdDYRK3
LdDYRK4
TbCLK1
TcCLK1
CpCLK1
GlCLK
0.71
4.9
4.2
5.2
TgCLK
LmDYRK2
LmCLK
PfCLK1
5.7
6.5
˃10
3
>10
1.3
a IC50 values were estimated from dose-response curves and are shown in µM >10, less than
50% inhibition at 10 µM.

ACCEPTED MANUSCRIPT
Table 9. Kinase inhibition values: aIC50 in µM for imine and oxime derivatives.
Cl
OMe
R
11w
20a
11v
Compd
CDK1
6n
>10
>10
>10
>10
0.12
0.12
0.13
0.62
>10
0.078
0.68
0.73
0.51
>10
4.1
21j
1.1
0.59
1.2
5.3
5.9
0.71
1.4
2.8
CDK2
0.59
0.9
>10
1.3
1.8
1
CDK5
2.2
2
CDK9
2.7
0.19
0.082
0.16
2.3
0.25
0.25
0.059
0.31
2.2
2.1
CK1
0.066
0.16
1.9
0.023
0.052
1.4
CLK1
CLK2
CLK3
0.042
0.12
0.11
0.5
0.044
0.05
0.11
0.38
3.1
0.025
0.11
0.08
0.39
0.92
>10
0.41
3.3
CLK4
0.033
0.17
0.17
0.14
11
DYRK1A
DYRK1B
DYRK2
DYRK3
GSK-3
1.4
>10
0.48
3.1
3.2
1
0.98
6
LdDYRK1B
LdDYRK3
LdDYRK4
TbCLK1
TcCLK1
CpCLK1
GlCLK
TgCLK
LmDYRK2
LmCLK
PfCLK1
3.8
>10
>10
>10
>10
2.2
>10
>10
>10
1.8
>10
>10
>10
0.46
3
>10
>10
>10
1.5
>10
>10
>10
1.2
2.3
2.4
2.9
2
1.4
1.7
>10
7.8
6.3
0.73
0.98
>10
0.31
1.7
0.81
>10
0.032
4.2
>10
0.37
>10
0.83
>10
1.1
a
IC50 values were estimated from dose-response curves and are shown in µM. >10, less than
50% inhibition at 10 µM.

ACCEPTED MANUSCRIPT
Table 3. Kinase inhibition values: IC50 a in µM for halogens and CF3 derivatives
N
N
R
N
CF
3
Cl
F
OMe
SMe
Cl
OMe
Cl
OMe
R
10a
Compd
CDK1
6f
7.5
5a
21b
3.3
22c
1.4
7.8
0.75
4
11e
1.3
21c
5.1
11f
1.7
6g
>10
>10
>10
>10
5.1
21d
>10
>10
>10
>10
>10
0.51
0.89
>10
0.22
>10
>10
>10
>10
>10
>10
6.4
11g
>10
>10
>10
3.7
>10
>10
>10
>10
>10
2.8
>10
>10
>10
>10
3.6
45
12
82
5.1
5.3
CDK2
4.7
18
50
>10
>10
2.4
7.1
CDK5
>10
8.1
8.1
1.7
7.5
CDK9
10
5.9
0.33
0.54
12
˃10
0.12
0.38
39
3.8
>10
0.79
9.1
CK1
0.58
2.8
0.79
1.5
2.7
1.1
0.18
1.4
0.59
1.3
CLK1
6.3
5.9
1.8
CLK2
>100
0.57
7.8
>10
1.5
12
8.8
>10
0.98
2.8
>10
0.18
1.1
>10
0.32
8.9
>10
0.41
>10
>10
>10
>10
>10
>10
2.2
CLK3
0.61
1.3
0.23
1
0.17
0.22
0.19
0.18
˃100
91
2.1
CLK4
4.2
7.3
DYRK1A
DYRK1B
DYRK2
DYRK3
GSK-3
0.82
0.48
>10
>100
2.1
>10
6.3
3.4
1.7
1.8
3
>10
8.6
3.6
1.1
7.1
1.2
2.5
0.78
2.1
7.1
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
5.3
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
4.5
4.9
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
7
22
12
>10
3.1
>10
0.42
>10
>10
>10
>10
>10
3.9
2.2
4.1
>10
>10
>10
>10
3
1.2
LdDYRK1B
LdDYRK3
LdDYRK4
TbCLK1
TcCLK1
CpCLK1
GlCLK
TgCLK
LmDYRK2
LmCLK
PfCLK1
>10
>10
>10
>10
>10
>10
>10
>10
>10
8
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
˃10
˃10
˃10
˃10
2.3
>10
>10
>10
>10
>10
>10
>10
7.8
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
2
˃10
˃10
˃10
˃10
8.6
>10
>10
>10
3.9
7.1
>10
2.9
>10
4.5
a IC50 values were estimated from dose-response curves and are shown in µM. >10, less than
50% inhibition at 10 µM.

ACCEPTED MANUSCRIPT
Table 4. Kinase inhibition values: IC50a in µM for chlorine derivatives.
Cl
R
Compd
CDK1
6h
6.7
10
11h
1.1
14
15
0.89
4
16
8.2
17
>10
>10
>10
0.62
0.4
>10
>10
>10
>10
>10
0.63
1.2
>10
>10
>10
1
CDK2
6.8
4.8
6.7
0.23
2.1
>10
0.15
1.4
1.7
1.3
3
0.61
0.22
1.1
0.39
2.1
CDK5
CDK9
>10
0.086
0.13
1.8
0.9
CK1
0.048
0.22
>10
0.084
1.2
1.8
CLK1
1.9
CLK2
>10
0.52
0.42
1.1
>10
0.51
0.51
0.52
0.44
0.43
>10
1.8
>10
0.8
CLK3
0.043
0.075
0.051
0.05
0.081
>10
0.1
CLK4
0.11
0.18
0.11
5.3
2.8
DYRK1A
DYRK1B
DYRK2
DYRK3
GSK-3
3.6
1.6
3.2
7.1
1.1
>10
6.2
>10
>10
>10
>10
5.1
>10
>10
>10
>10
>10
>10
>10
>10
0.59
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.19
>10
LdDYRK1B
LdDYRK3
LdDYRK4
TbCLK1
TcCLK1
CpCLK1
>10
>10
>10
>10
>10
>10
>10
>10
6.1

ACCEPTED MANUSCRIPT
>10
7.1
8.3
>10
3.4
>10
>10
>10
>10
1.2
>10
>10
>10
>10
>10
>10
>10
0.5
>10
>10
8.9
>10
>10
>10
>10
4.1
GlCLK
TgCLK
LmDYRK2
LmCLK
PfCLK1
>10
0.078
>10
1.2
aIC50 values were estimated from dose-response curves and are shown in µM. >10, less than
50% inhibition at 10 µM.

ACCEPTED MANUSCRIPT
Table 5. Kinase inhibition values IC50a in µM) for amino and acetamido derivatives.
Cl
Cl
R
Compd
CDK1
CDK2
CDK5
CDK9
CK1
6i
>10
41
11i
6.1
7.1
3.8
3.8
>10
6j
5.1
61
11j
3.2
22
11k
0.071
0.41
0.19
0.8
18
0.52
1.3
1.1
3.5
19
0.17
0.58
0.22
2.1
29
71
17
7.3
>10
>10
>10
3.4
>10
1.2
3
1.8
0.028
0.028
0.28
0.027
0.039
0.038
0.043
0.11
5.2
>10
0.038
0.18
8.2
>10
0.061
0.03
1.8
0.38
1.9
0.57
1.4
1
CLK1
CLK2
3.9
43
19
6.1
2.8
1.3
11
CLK3
0.12
9.4
0.44
2.5
0.54
14
0.04
0.19
0.13
0.12
0.93
>10
2.2
0.065
0.063
0.032
0.04
0.52
5.2
CLK4
DYRK1A
DYRK1B
DYRK2
DYRK3
GSK-3
9.1
1.8
28
13
7
1.9
19
21
15
5.1
41
17
61
41
69
65
8.9
2.8
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.11
1.1
0.69
7
LdDYRK1B
LdDYRK3
LdDYRK4
TbCLK1
TcCLK1
CpCLK1
GlCLK
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
3
>10
>10
>10
3
>10
>10
3.9
8.2
1.1
4.4
6.1
>10
3.3
TgCLK
3.2
LmDYRK2
LmCLK
PfCLK1
>10
3.2
>10
3.1
>10
2.1
a
IC50 values were estimated from dose-response curves and are shown in µM. >10, less than
50% inhibition at 10 µM.

ACCEPTED MANUSCRIPT
Table 6. Kinase inhibition values IC50a in µM for compound 11m, 11n and 11o.
Compd
CDK1
11l
0.51
1.1
11m
0.27
0.39
0.89
1.1
11n
0.8
2.2
CDK2
0.82
0.32
0.61
0.081
0.18
2.5
0.59
0.6
CDK5
CDK9
>10
0.088
0.15
0.52
4.7
CK1
0.041
0.047
0.37
0.048
0.093
0.14
0.082
2.1
CLK1
CLK2
CLK3
0.1
0.042
0.47
0.41
0.6
CLK4
0.12
0.09
0.12
1.3
DYRK1A
DYRK1B
DYRK2
DYRK3
GSK-3
1.7
>10
0.42
0.42
>10
>10
>10
>10
>10
>10
0.88
>10
>10
>10
>10
3.3
1.6
LdDYRK1B
LdDYRK3
LdDYRK4
TbCLK1
TcCLK1
CpCLK1
3.3
>10
>10
>10
4.1