Design, synthesis, biological evaluation and cellular imaging of imidazo[4,5-b]pyridine derivatives as potent and selective TAM inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.09.031

The TAM kinase family arises as a new effective and attractive therapeutic target for cancer therapy, autoimmune and viral diseases. A series of 2,6-disubstituted imidazo[4,5-b]pyridines were designed, synthesized and identified as highly potent TAM inhib

Full text of DOI:10.1016/j.bmc.2018.09.031

Accepted Manuscript
Design, Synthesis, Biological Evaluation and Cellular Imaging of Imidazo[4,5-
b]pyridine Derivatives as Potent and Selective TAM inhibitors
Tom Baladi, Jessy Aziz, Florent Dufour, Valentina Abet, Véronique Stoven,
François Radvanyi, Florent Poyer, Ting-Di Wu, Jean-Luc Guerquin-Kern,
Isabelle Bernard-Pierrot, Sergio Marco Garrido, Sandrine Piguel
PII:
S0968-0896(18)31380-4
https://doi.org/10.1016/j.bmc.2018.09.031
BMC 14556
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
2 August 2018
17 September 2018
23 September 2018
Please cite this article as: Baladi, T., Aziz, J., Dufour, F., Abet, V., Stoven, V., Radvanyi, F., Poyer, F., Wu, T-D.,
Guerquin-Kern, J-L., Bernard-Pierrot, I., Garrido, S.M., Piguel, S., Design, Synthesis, Biological Evaluation and
Cellular Imaging of Imidazo[4,5-b]pyridine Derivatives as Potent and Selective TAM inhibitors, Bioorganic &
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.09.031
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Design, Synthesis, Biological Evaluation and
Cellular Imaging of Imidazo[4,5-b]pyridine
Derivatives as Potent and Selective TAM
inhibitors.
Tom Baladi a, b, Jessy Aziz , Florent Dufour , Valentina Abet , Véronique Stoven d, e,
a
c
a
c
a
a
a
François Radvanyi , Florent Poyer , Ting-Di Wu , Jean-Luc Guerquin-Kern , Isabelle
c
a
a, b,*
Bernard-Pierrot , Sergio Marco Garrido , Sandrine Piguel
Corresponding author
*
E-mail address: sandrine.piguel@curie.fr
a
Institut Curie, PSL Research University, CNRS, INSERM, UMR9187-U1196, F-91405,
Orsay, France
b
Université Paris Sud, Université Paris-Saclay, F-91405, Orsay, France
c
Institut Curie, PSL Research University, CNRS, UMR 144, F-75248 Paris 05, France
d
Institut Curie, PSL Research University, INSERM, U900, F-75248 Paris 05, France
e
Mines ParisTech, CBIO - Centre for Computational Biology, 35 rue Saint-Honoré, F-77300
Fontainebleau, France
Abstract: The TAM kinase family arises as a new effective and attractive therapeutic target
for cancer therapy, autoimmune and viral diseases. A series of 2,6-disubstituted imidazo[4,5-
b]pyridines were designed, synthesized and identified as highly potent TAM inhibitors.
Despite remarkable structural similarities within the TAM family, compounds 28 and 25
demonstrated high activity and selectivity in vitro against AXL and MER, with IC value of
5
0
0
.77 nM and 9 nM respectively and a 120- to 900-fold selectivity. We also observed an
unexpected nuclear localization for compound 10Bb, thanks to nanoSIMS technology, which
could be correlated to the absence of cytotoxicity on three different cancer cell lines being
sensitive to TAM inhibition.
1

Keywords: kinase inhibitors, AXL, TYRO3, MER, imidazopyridines, NanoSIMS imaging.
1
. Introduction
Receptor tyrosine kinases (RTKs) are transmembrane proteins which transduce molecular
signals from the extracellular medium to the cytoplasm. They regulate diverse cellular
functions such as growth, differentiation or motility. The 58 known RTKs are classified in the
kinome into 20 families, based on sequence and structure similarities.[1] Being overexpressed
in a large number of human cancers, RTKs have therefore emerged as attractive therapeutic
targets in the past 20 years. Small molecule kinase inhibitors can target very specific
molecular pathways, as opposed to traditional non-selective chemotherapies, heretofore more
than 40 compounds have been approved by the Food & Drug Administration, illustrating the
,
,
,
benefits of such drugs.[2] [3] [4] [5] However, such inhibitors only cover 10 – 15 % of all
known kinases.[6] Among the uncovered part of the kinome, the TAM family regroups three
proteins, TYRO3, AXL and MER, which are characterized by the presence of a common
KW-I/L-A-I/L-ES sequence in their intracellular kinase domains.[7] The TAM RTKs play
major parts in cancer development, autoimmune reactions
and viral
,
,
,
infections.[8] [9] [10] [11] To date, only a few small molecules have been intentionally
,
,
,
designed to target TAM kinases, with a major interest in AXL and MER.[12] [13] [14] [15]
Most of the known inhibitors of the family were in fact developed for other protein kinase
targets and identified through selectivity profiles. Given the high sequence similarity between
TYRO3, AXL and MER (52 – 57 % in the extracellular region and 72 – 75 % in the
intracellular domain), the design of specific inhibitors of either one of them remains a
5
challenge. More precisely, kinase domains consist of a -stranded N-terminal lobe and a
helical C-terminal lobe connected by a short strand named the hinge region. The ATP binding
site is sandwiched between these two lobes. In the active form of the kinase, a characteristic
Asp-Phe-Gly DFG motif adopts the so-called DFG-in conformation. In the inactive form, a
large conformational change of this loop creates a large hydrophobic pocket adjacent to the
active site, the allosteric site, which is not accessible in the active form. Two main classes of
inhibitors are therefore known for kinases. Type I inhibitors mainly occupy the ATP-binding
pocket (the active site), and bind to the DFG-in active form of the kinase domain.[16] Type II
inhibitors bind both to the ATP active site and to the allosteric site in the DFG-out form.[17]
Type II inhibitors are generally expected to display better selectivity profiles within the whole
kinome than type I inhibitors, because residues of the allosteric site are less conserved than
those of the active site.
2

As part of an ongoing project aiming to develop novel TAM inhibitors, the structure of our
two previous reported compounds I and II have been modified to gain insight into the
,
selectivity profile (Figure 1).[18] [19] Based on the imidazo[4,5-b]pyridine scaffold, this new
series of molecules led to the identification of highly potent and selective TAM inhibitors by
exploring the volume and electronic properties of various substituents at positions 2 and 6.
2
. Results and discussion
2
.1. Design strategy
The two series of purine-I and pyrimidine-II based type II inhibitors of the TAM family
enzymes shared similar features: the main heteroaromatic platform carried two nitrogen atoms
to form hydrogen bonds with the active site of the proteins, and a side chain to interact with a
proximal allosteric pocket. While a number of variations in the structure of the side chain
were explored, the most active compounds in both series featured a pyridone motif linked to
two fluorine-containing phenyl rings. The most efficient linkage between the side chain and
the heterocycle was an oxygen atom.
The presence of the
amidopyridone system is
important for activity
H
F
N
N
An ether linkage
should be maintained
O
N
O
O
F
Heterocycle
H
N
H
H
N
F
N
F
N
N
F
O
O
O
O
O
O
O
F
O
F
O
F
R2
6
N
1
N
Introduction of diversity
to tune the selectivity
within the TAM family?
N
N
R1
2
N
H
N
H
NH2
N
N
N
I
II
III
Previous work
This work
Figure 1. In-house TAM inhibitors
In 2013, the group of Powell reported that 2,4-diaminopyrimidine-5-carboxamide IV
derivative was 14- and 12-fold more selectivite for murine TYRO3 as compared to AXL and
MER (Figure 2).[20] This inhibitor was a type I inhibitor that bounds to the active DFG-in
form of the kinase domain. The crystal structure of this inhibitor in murine TYRO3 showed
that its benzyl moiety was oriented towards an available space in front of the Ala 571 residue
(PDB ID: 4FF8, corresponding to Ala 581 in human Tyro3).
3

Ala571
H
N
H
N
N
N
O
O
H CO
3
N
HN
IV
Figure 2. Structure of compound IV, indicating the chemical moiety that point towards Ala
71 in murine TYRO3 crystal structure.
5
Interestingly, the Ala 571 residue in TYRO3 is replaced by bulkier residues in other TAM
proteins, namely a methionine in AXL and isoleucine in MER. Therefore, it was postulated
that steric hindrance of the phenyl ring with bulkier amino acids at the same position could
explain the lower activities on MER and AXL. This structural specificity of the protein
TYRO3 was thereafter called the “alanine pocket”.
Building on these interesting results, we hypothesized that modulation of type II inhibitors
within the TAM family kinases could be achieved with compounds that would occupy the
alanine pocket also available in TYRO3 in the DFG-out conformation. The absence of X-ray
crystallographic data for the TYRO3 kinase domain in DFG-out form led us to build a
structure model for it, by homology with the X-ray DFG-out structures of proteins Met and c-
Kit (PDB ID: 3F82 and 1T46). Its topological quality, including bond lengths and angles, was
carefully checked, as detailed in the experimental section. Previous molecular docking studies
in the DFG-out model of human TYRO3 showed that the nitrogen at position 1 of the purine
derivative I faced the alanine pocket (Figures 1 and 3). Thus, we hypothesized that the
compound selectivity for the members of the TAM family could be tuned by modifying the
volume of an R-group inserted at N-1 position of compound I. From this perspective, bulky
substituents could lead to selectivity for TYRO3 while a progressive decrease in their volume
could enable better interactions and affinities for MER and AXL. Indeed, a closer look to the
superimposition of the X-ray crystal structure of Powell’s compound IV in complex with
murine TYRO3 and the purine I in the model of human TYRO3 seemed to confirm this
tendency. The benzyl substituent of compound IV occupies and points out towards the
“
alanine pocket” in the same way as the N-1 of purine I (Figure 3).
4

Figure 3. Crystal structure of DFG-in crystal structure of murine TYRO3 with its bound
molecule IV (PDB code 4FF8) superposed to the DFG-out model of human TYRO3 docked
with molecule I. For sake of clarity, only human TYRO3 is shown in blue ribbon (this image
was made with VMD software developed with NIH support by the Theoretical and
Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-
Champaign).
To further study this potential way towards selectivity among the TAM family kinases, we
decided to develop a new series of molecules, based on the imidazo[4,5-b]pyridine moiety III
(Figure 1). While being a purine isostere, this platform allows the introduction of structural
diversity in the place of the N1 nitrogen in the purine ring. In addition, the imidazo[4,5-
b]pyridine heterocycle is increasingly present in biologically active molecules for the
treatment
of
cancer,
neurodegenerative
diseases
or
diabetes
for
,
,
,
,
,
,
,
,
example.[21] [22] [23] [24] [25] [26] [27] [28] [29] In the present work, we explored the
influence of the volume and electronic properties of various substituents at positions 2 and 6
of the imidazo[4,5-b]pyridine scaffold on the selectivity profile of the corresponding
compounds on the TAM family. The synthetic strategy to access these potential inhibitors will
be described. In vitro evaluation of their inhibitory activity and biological effect on viability
of TAM dependent cancer cell lines will be also discussed, with the support of imaging
experiments in cancer cells by NanoSIMS.
2
.2. Chemistry
5

During the structure-activity relationship (SAR) study, we mainly focused our attention on the
substitution at the 6-position of the imidazo[4,5-b]pyridine moiety III (Figure 1), while
leaving the 2-position untouched or merely substituted with short and flexible alkyl
substituents (hydrogen, ethyl and isopropyl). As aforementioned, substitution at position 6 by
a variety of sterically hindered groups such as halogen or heteroaryl, could be crucial in
controlling the selectivity within the TAM family. Based on this, synthetic strategies were
developed in order to provide a wide selection of imidazo[4,5-b]pyridine analogues for the
SAR study. Cyclization of the commercially available 5-bromopyridine-2,3-diamine 1 with
the adequate carboxylic acids gave compounds 2A and 2B (Scheme 1). Formation of the
corresponding N4-oxide intermediate in the presence of m-CPBA turned out to be essential
for the activation of compounds 2 to the chlorination reaction using POCl .[30] It should be
3
noticed that an inseparable mixture of two regioisomers (chlorination at position 7 or 5) was
obtained with an NMR ratio of 3:1 respectively. Benzyl (Bn) protection of this mixture
allowed the separation of the N1-protected regioisomers and pure compounds 4A and 4B were
obtained with moderate yields. Nucleophilic aromatic substitution (S Ar) allowed the
N
introduction of a phenol group at the 7-position of the imidazo[4,5-b]pyridine 5A and 5B with
concomitant cleavage of the tert-butoxycarbonyl group. A Suzuki-Miyaura coupling at the 6-
position followed by condensation of compounds 6Aa and 6Ba with a pyridone carboxylic
1
7
acid derivative gave products 7Aa and 7Ba with satisfying yields. Compounds 8Aa and 8Ba
were obtained by the deprotection of the N-benzyl group in the presence of Pd/C and under
hydrogen atmosphere. Product 10Bb was formed from the amidation of compound 5B
followed by the simultaneous debenzylation and debromination under palladium-catalyzed
hydrogenolysis.
6

F
NH2
R1
Cl
N
Cl
N
O
Br
At 2-position A = Et
NH2
NH2
Br
Br
N
iii
Br
N
N
Br
i
N
ii
iv
N
N
R1
R1
R1
N
H
N
H
N
N
N
B = i-Pr
Bn
Bn
1
3A
2
2
A
B
4
4
A
B
3B
5A
5B
At 6-position a = Ph
b = H
H
N
H
N
F
N
F
N
F
NH2
O
O
O
O
O
F
O
F
O
vii
vi
v
N
N
N
N
N
2
R1
R1
R1
6
N
H
N
N
N
Bn
Bn
8
8
Aa
Ba
7
Aa
6Aa
6Ba
7Ba
H
N
H
F
N
F
N
N
O
O
O
O
O
N
F
O
N
F
H
6
vii
Br
N
vi
N
2
N
N
Bn
H
10Bb
9B
Scheme 1. Synthesis of compounds 8Aa, 8Ba and 10Bb. Reagents and conditions: i)
Carboxylic acid, NH Cl, POCl , 100 °C, 2d; ii) m-CPBA, AcOH, RT, 3d then POCl , 80°C
4
3
3
µW, 10 min; iii) Benzyl bromide, NaH, DMF, 0 °C to RT; iv) tert-butyl (3-fluoro-4-
hydroxyphenyl)carbamate, K CO , DMSO,100 °C, 16h; v) PhB(OH) , PdCl dppf, Cs CO
3
2
3
2
2
2
2
M, 1,4-dioxane,100 °C, 16h; vi) 1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic
acid, DIPEA, TBTU, DMF, 0 °C, 6h; vii) Pd/C, H , MeOH, RT, 4d.
2
At this point, we aimed for a higher diversity of substituents at 2- and 6-positions following
the same synthetic pathway as for compounds 8. However, many attempts for the deprotection
of the N-Benzyl group by palladium-catalyzed hydrogenation were not successful.[31] For
this reason, we switched to p-methoxybenzyl (PMB) as a protecting group, which was
introduced in the same manner as the Bn group. We subsequently applied the same synthetic
pathway as described before (Scheme 2). Satisfyingly, we were able to separate compounds
1
1 from their corresponding 5-chlorinated regioisomers after the protection step. S Ar
N
followed by Suzuki-Miyaura coupling, amide condensation and subsequent PMB cleavage
under acidic conditions gave the multi-substituted 3H-imidazo[4,5-b]pyridine analogues 15. It
should be mentioned that for a 4-hydroxyphenyl substitution at the 6-position in compounds
1
8, a slightly different synthetic approach was used. Compounds 12A and 12B were
submitted to amide condensation before the Suzuki-Miyaura coupling with the (4-
hydroxyphenyl)boronic acid, in order to avoid potential competitive reactivity between the
7

amine and the hydroxyl groups towards the carboxylic acid used in the amidation step.
Palladium-catalyzed cross-coupling reaction on compounds 16A and 16B furnished
intermediates 17Ah and 17Bh. At last, deprotection of these compounds allowed the
formation of the final products 18Ah and 18Bh.
F
NHR
Cl
Cl
O
A = Et
B = i-Pr
C = H
Br
i
Br
N
Br
N
ii
N
N
iii
Br
R1
R1
R1
R1
N
N
R1
N
H
N
N
H
N
N
N
PMB
PMB
12A R = H
12B R = H
2
2
2
A
B
C
3A
3B
3
1
1
11C
1A
1B
c = 4-NMe2(C6H4)
d = 3-furyl
C
12C R = Boc
R2
e = 4-pyridyl
f = 3-pyridyl
g = Me
H
H
N
F
N
N
F
NH2
F
N
h = 4-OH(C6H4)
O
O
O
O
O
N
F
O
O
F
R2
R2
R2
vi
N
N
v
N
N
N
iv
R1
R1
R1
N
H
N
N
PMB
PMB
1
1
1
5Ac-f
5Bc-f
5Cg
14Ac-f
14Bc-f
14Cg
13Ac-f
13Bc-f
13Cg
H
H
N
H
N
F
N
F
N
N
F
N
HO
O
O
O
O
HO
O
O
O
N
F
O
F
O
F
Br
N
N
N
iv
v
N
vi
R1
R1
R1
N
PMB
N
N
H
N
PMB
1
1
8Ah
8Bh
17Ah
17Bh
16A
16B
Scheme 2. Synthesis of compounds 15 and 18. Reagents and conditions: i) m-CPBA, AcOH,
RT, 3d then POCl , 80°C µW, 10 min; ii) p-methoxybenzyl bromide, NaH, DMF, 0 °C to RT;
3
iii) tert-butyl (3-fluoro-4-hydroxyphenyl)carbamate, K CO , DMSO, 100 °C, 16h; iv)
2
3
2
R B(OH) , [Pd], [Base], 1,4-dioxane,100 °C, 16h; v) 1-(4-fluorophenyl)-2-oxo-1,2-
2
dihydropyridine-3-carboxylic acid, DIPEA, TBTU, DMF, 0 °C, 6h; vi) H SO , TFA, anisole,
2
4
RT, 18h.
Parallel to this synthesis, the formation of analogues with no substitution at the 2-position and
displaying a phenyl ring, bromine or hydrogen at the 6-position was performed using 2-
tetrahydropyranyl (THP) as a protecting group (Scheme 3). It should be noticed that there was
no rationale in the use of PMB (Scheme 2) or THP (Scheme 3) as protecting groups, with the
main scope of performing an efficient deprotection. The common intermediate 20 for the
three final products 23, 25 and 28 was obtained by the N-THP protection of the compound 3C
followed by S Ar on position 7 with a phenol derivative. A sequence of Suzuki-Miyaura
N
coupling with phenylboronic acid, deprotection and amidation gave compound 23.
8

F
NHBoc
F
NH2
H
H
N
F
N
O
O
O
O
O
F
iii
N
N
iv
N
v
N
H
H
N
N
H
N
N
H
N
THP
21
22
23
F
NHBoc
F
NH2
H
H
N
Cl
N
Cl
N
F
N
O
O
Br
N
Br
N
N
i
ii
Br
N
N
iv
Br
N
v
O
O
H
H
H
O
F
N
H
N
N
N
Br
N
THP
H
THP
H
3C
19
20
N
N
H
24
25
H
N
F
NHBoc
F
NH2
F
N
O
N
O
O
O
O
F
vi
H
N
iv
H
N
v
H
N
H
H
H
N
H
N
H
N
N
N
H
26
27
28
Scheme 3. Synthesis of compounds 23, 25 and 28. Reagents and conditions: i) 3,4-dihydro-
H-pyran, PTSA, EtOAc, Reflux, 18h; ii) tert-butyl (3-fluoro-4-hydroxyphenyl)carbamate,
K CO , DMSO, 100 °C, 16h; iii) PhB(OH) , PdCl dppf, Cs CO 2M, 1,4-dioxane,100 °C,
2
2
3
2
2
2
3
1
6h; iv) TFA, DCM, 0 °C - RT, 2h; v) 1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-
carboxylic acid, DIPEA, TBTU, DMF, 0 °C, 6h; vi) Pd/C, H , MeOH, RT, 18h.
2
Compound 25 was formed by the deprotection of both the aniline and the imidazo[4,5-
b]pyridine of intermediate 20 under acidic conditions and subsequent amidation. Finally,
simultaneous reduction of the C-Br bond and THP cleavage by hydrogenation gave product
2
6. Deprotection of the aniline and subsequent amide formation provided compound 28.
2
.3. Biochemical tyrosine kinase inhibitory assays
The library of the above synthesized compounds was submitted to a number of biological
assays in order to evaluate their inhibition potency against the TAM family kinases.
A first screening of the 17 synthesized molecules was performed by an ELISA assay that
allows evaluating the phosphorylation of poly Glu-Ala-Tyr peptide by the three TAM
kinases.16 As commercially available positive controls, we used a non-selective kinase
inhibitor Vandetanib, along with the BMS-777607, a MET inhibitor displaying also good IC50
values against the TAM receptors family.[32] Molecule 28 featuring a non-substituted
imidazol[4,5-b]pyridine ring at the 2- and 6-positions appeared to be the most effective,
showing no selectivity over the three proteins (Figure 4). Compound 10Bb, only substituted
by an isopropyl group at position 2 exhibited an interesting activity on MER and seemed to
have selectivity for this protein, as no significant effects were observed on TYRO3 and AXL
9

(Figure 4). Eventually, molecules bearing groups bulkier than a mere hydrogen at the 6-
position of the imidazo[4,5-b] pyridine, such as 15Ac-f, 15Bc-f, 18Ah, 18Bh and 23, did not
display any inhibitory activity on the TAM family kinases as illustrated for compounds 25
and 15Cg (Figure 4).
Figure 4. ELISA assay of compounds 10Bb, 15Cg, 25, 28 and Vandetanib and BMS-777607
as positive controls against the three TAM kinases: TYRO3 (A), AXL (B), MER (C).
Based on these preliminary studies, the four compounds substituted at position 6 by the
smallest R-groups, namely 10Bb, 15Cg, 25 and 28, were selected for determining their IC50
values against the three TAM kinases by measuring ADP formation in presence of a poly Gly-
Ala-Tyr substrate (Chart 1). As anticipated with the ELISA assay, compound 10Bb showed
an interesting IC50 of 40 nM on MER, along with a 120-fold selectivity as compared to
TYRO3. Molecule 28 exhibited a particularly low IC on AXL (0.77 nM) and showed a 120-
5
0
to 900-fold selectivity as compared to the two other TAM proteins. It is worth of notice that a
good IC was measured for compound 25 on MER (9 nM), although this molecule was not
5
0
active in the ELISA assay. Satisfyingly, a high selectivity was again observed within the
TAM family kinases (IC = 270 nM and 2000 nM for Tyro3 and AXL). Unfortunately, due
5
0
1
0

to solubility issues, the IC values for compound 15Cg had to be extrapolated but are in
5
0
agreement with the results of the ELISA assay.
H
H
H
H
F
N
N
F
N
N
F
N
N
F
N
N
O
O
O
O
O
O
O
O
O
F
O
F
O
F
O
F
Br
N
N
N
N
10Bb
25
28
15Cg
N
H
N
N
H
N
N
H
N
N
H
N
IC50 (Tyro3) = 270 nM
IC50 (Axl) = 2000 nM
IC50 (Mer) = 9 nM
IC50 (Tyro3) = 690 nM
IC50 (Axl) = 0.77 nM
IC50 (Mer) = 90 nM
IC50 (Tyro3) = 4700 nM
IC50 (Axl) = 70 nM
IC50 (Mer) = 40 nM
IC50 (Tyro3) > 100 µM
IC50 (Axl) > 100 µM
IC50 (Mer) > 100 µM
Chart 1. IC values of compounds 10Bb, 15Cg, 25 and 28.
5
0
The activity of the four compounds was finally evaluated against a panel of 11 kinases using a
competition binding assay to immobilized ligand on beads, developed and conducted by the
KinomeScan Company. This test delivers true thermodynamic affinity data without ATP
required as opposed to IC . The fraction of unbound kinase to the substrate in presence of
5
0
compound reflects the affinity of the inhibitor for that kinase. The results obtained for kinases
Abl1, AurKA, BRaf, CDK2, EGFR, FGFR1, GSK3, Met, TYRO3, AXL and MER are given
in Table 1. Satisfyingly, no affinity was observed for kinases outside the TAM family, except
for MET, which is the closest to the TAM family and in a targeted way to CDK2 for
compound 10Bb. It appeared that affinities for proteins MET, AXL and MER were generally
higher than for protein TYRO3. A very good correlation exists between the affinities and the
IC values measured on TYRO3 and MER for compounds 10Bb (4700 nM and 40 nM,
5
0
respectively) and 28 (690 nM and 90 nM, respectively) (Chart 1 and Table 1). However, it
should be noticed that the affinity of compound 28 for AXL is lower than for MER, despite a
lower IC50 for this protein (0.77 nM) (Chart 1 and Table 1). In agreement with the two
previous tests, compound 15Cg displayed much lower scores for the TAM family kinases.
Surprisingly, compound 25 showed no affinity for TYRO3 and MER in this setting while it
prevented substrate phosphorylation in the ADP formation assay (Chart 1).
1
1

Conc.
µM)
Compound
Abl1 AurKA Braf CDK2 EGFR FGFR1 GSK3B Met Tyro3 Axl Mer
(
0
1
1
0
1
1
0
1
1
0
1
1
,1
1
1
0
41
95,5
99,7
0
2
0
8
0
0
0
1
0
0
0
0
1
7
0
0
0
0
5
6
2
5
0
0
1
0
83
1
61
90
89
78
92,3
99,6
99,1
87
1
2
1
2
0Bb
3
2
8
1
96,7 39
0
17
2
12
5
43
9
1
96,6 92,4
,1
1
83
96
97
54
17
81
99,8
0
8
13
34
1
2
12
58
0
0
2
90,7 99,3
93,3 99,6
0
13
1
48
0
6
,1
8
22
67
17
78
5Cg
1
6
2
0
2
94,5 0
96,2 5
0
3
41
7
18
0
3
15
7
90,1 99,1
,1
0
0
12
82
15
11
4
4
5
0
9
0
0
1
52
60
0
0
45
10
32
11
96,4 10
90,4 98,2
Table 1: Selectivity profile on 11 kinases. Fraction of unbound kinase to substrate reflect (0-
00) reflects the affinity of the molecule for the kinase. The affinity is illustrated by a range of
colors from red (low) to green (high).
1
2
.4 Docking experiments
The above results showed that compounds 10Bb and 25 are active against the TAM family
kinases. Interestingly, they are both mono-substituted imidazo[4,5-b]pyridine compounds (see
compound III in Figure 1), respectively at positions 2 and 6, and therefore, we wondered
whether these two compounds could adopt similar binding modes in the protein pocket. We
docked these two compounds in our DGF-out model of TYRO3. As shown in Figure 5, the
imidazo[4,5-b]pyridine rings do not adopt the same orientation in the binding site and are
flipped of 180° around the O-C bond that links them to the rest of the molecule.
Consequently, the key H-bonds that form in the ATP binding pocket are different for the two
1
2

molecules. In the case of 10Bb, the isopropyl group at position 2 is oriented towards the exit
of the pocket, in a region that is accessible to the solvent. Two H-bonds are formed between
Met 606 and 10Bb, as classically observed in kinase inhibitors. In the case of 25, an
orientation in which the Br atom at position 6 is also oriented towards the exit of the pocket is
also preferred, which leads to the 180° flip of the imidazo[4,5-b]pyridine rings. Two H-bonds
are still observed, involving Pro 604 and Met 606, ensuring molecule 25 anchorage.
A)
B)
Figure 5. Panels A and B: schematic views of molecule 10Bb and 25 respectively docked in
the TYRO3 model (in yellow ribbon).
These results seem to indicate that, in compounds with mono-substituted imidazo[4,5-
b]pyridine rings and whatever the substituents positions (2 or 6) on the imidazo[4,5-
b]pyridine ring, they point towards the exit of the pocket avoiding potential clashes within the
1
3

pocket, rather than towards the alanine pocket. This may explain the lack of specificity of
molecules 25 and 10Bb towards TYRO3, within the TAM family kinases.
2
.5. Cellular viability assays
Given the good results observed in the three in vitro biochemical assays, we decided to
evaluate the ability of four compounds 10Bb, 15Cg, 25 and 28 to inhibit TAM receptors in
cancer cells and therefore to reduce the cell viability of TAM receptors-dependent cell lines.
We selected two different bladder cancer cell lines, MGH-U3 and RT112, that we
demonstrated, using siRNAs, to be dependent on TYRO3 expression for their growth.[33] In
addition, RT112 was shown to be also dependent, to a lesser extent, to AXL expression. We
also considered a lung carcinoma derived cell line, A549 that expresses the three TAM
receptors and that has been proven to be sensitive to both AXL inhibition/ depletion and MER
,
inhibition.[34] [35] As commercially available positive controls, we used Vandetanib, along
with the BMS-777607, and the Cabozantinib, a MET/AXL/VEGFR2 inhibitor. Compound
1
8Ah was used as a negative control, as it showed no inhibition of TAM proteins in the
ELISA assay. The viability of the three cell lines was evaluated after 72 h of incubation in the
presence of the selected molecules at concentrations ranging from 0.15 to 10 µM (Figure 6).
As expected, the three cell lines were sensitive to Vandetanib with IC ranging from 2.5 µM
5
0
to 5 µM, whereas the two AXL-dependent cell lines (RT112 and A549) were more sensitive
to Cabozantinib treatment. Both cell lines were slightly sensitive to BMS777607. However,
none of the AXL-dependent and/or MER-dependent cell lines were sensitive to our four
compounds 10Bb, 28, 15Cg and 18Aa, despite the fact that compounds 10Bb, 28 and 15Cg
had shown a good affinity for AXL and MER in the biochemical in vitro assays using the
kinase domains (Figure 4, chart 1 and table 1). Unfortunately, compound 25 was poorly
soluble in 0.2% DMSO and could not be tested even with increasing amounts of DMSO
which were detrimental to the intrinsic viability of the cell lines.
1
4

Figure 6. Cellular viability of TAM receptors-dependent cell lines after 72h of treatment with
different drugs. Cell viability was measured using the CellTiter-Glo® Luminescent Cell
Viability Assay. For each cell line, TAM receptors expression was evaluated by western-blot
and FACS and is indicated.
2
.6. Cellular imaging by NanoSIMS
To try to understand such a discrepancy between the IC50 values measured using the
recombinant kinase domains of the TAM receptors and the absence of effect observed on
cellular viability for cells expressing the native full length receptors, we focused on
compound 10Bb, presenting a high affinity for both AXL and MER (Chart 1 and Table 1). Its
cellular localization was studied in A549 cells expressing the three TAM receptors and in
MGH-U3 cells expressing only TYRO3 receptors using a specific cellular imaging.
The use of ion microscopy based on SIMS-technique (Secondary Ion Mass Spectrometry) to
life sciences was attempted by Pierre Galle more than 40 years ago, to investigate the cellular
localisation of a molecule.[36] In the present study, by using NanoSIMS-50, a new generation
1
9
of high spatial resolution ion microscopy, we imaged the cellular distribution of F which
reflects directly the cellular localisation of 10Bb, by taking advantage of the presence of two
1
5

,
fluorine atoms.[37] [38] Experiments were performed on two cell lines (A549 and MGH-U3)
with an incubation time of 24 hours at concentration of 10 M for compound 10Bb. As
positive controls, we used the A549 cell line alone but also the A549 cell lines incubated with
BMS-777607 for 24h.
For A549, as illustrated in figure 7.1, the 19F image suggests a high concentration of
compound 10Bb inside the nucleus, in correlation with the chromatin localization (indicated
3
1
by P, which represents mainly the localization of nucleic acids) while a few hot spots were
1
9
also observed in the cytoplasm. Comparatively, the F images of the positive controls showed
no specific accumulation in the nucleus (see supporting data). Such accumulation in the
nucleus might impede an interaction with the intracellular kinase domains of AXL and MER,
which could account for the absence of cytotoxicity of the molecule on this cell line despite
its high in vitro activity measured on the recombinant kinase domains of the receptors. This
first result demonstrated that, without addition of tag (fluorophore, etc…) which could modify
the molecular structure and thus its cellular localization, it is possible to directly localize our
1
9
compound within the cell, although an evenly distributed, non-specific background in F is
also detected inside and outside the cells.
1
9
In comparison, slightly different F distribution was observed in MGH-U3 cells that express
neither AXL nor MER but TYRO3 for which the compound had only a very low affinity
(Figure 7.2). Compound 10Bb is again more concentrated in the nucleus, however, high
1
9
extracellular F content around the cells is also observed in this cell line together with a
relatively weak signal detected in the cytoplasm. This phenomenon may be related either to
the retention of compound 10Bb by the cell pseudopods, or to a possible membrane fixation.
From these results, the predominant localisation of compound 10Bb inside the nucleus seems
to be independent of the presence of the targeted tyrosine kinase receptors at the cell surface.
1
)
2
)
1
6

Figure 7. NanoSIMS imaging showing the localization of compound 10Bb (10 µM) in 2
different cell lines after 24 hours of incubation. 1) A549 cells incubated with 10Bb (A, B and
C). 2) MGH-U3 cells incubated with 10Bb (D, E, F). Left panels (A and D) correspond to the
1
9
31
images of F; central panels (B and E) images of P; right panels (C and F) superimposition
1
9
31
of F (Red) and P (Blue) images, respectively. Scale bars are 5µm.
3
. Conclusion
A family of 2,6-disubstituted imidazo[4,5-b]pyridines have been synthesized with the aim of
tuning the selectivity within the TAM family kinases by exploiting a structural difference in
the ATP pocket to induce TYRO3 inhibition efficacy. These molecules were then evaluated in
vitro and in cellulo. Although the results were not in accordance with the initial hypothesis,
we have nevertheless highlighted two compounds with high activity and selectivity within the
TAM transmembrane receptors. This result is quite relevant due to the lack of high selective
inhibitors within the TAM family. For instance, compound 28 exhibits an IC value of 0.77
5
0
nM against AXL and a 120- to 900-fold superior selectivity over MER and TYRO3 whereas
compound 25 has an IC50 value of 9 nM against MER and a 38- to 200-fold superior
selectivity over AXL and TYRO3. However, these in vitro results are counterbalanced by the
results of the cellular viability performed on three different cancer cell lines that are sensitive
to TAM kinase inhibition. Indeed, no cytotoxicity was observed for the most in vitro potent
and selective compounds. Thanks to nanoSIMS imaging, an unexpected nuclear localization
of compound 10Bb was observed in two different cell lines, which may be correlated to the
absence of cytotoxicity. Interestingly, this nuclear distribution also highlights the ability of
our compounds to be cell penetrant, which could be not trivial at first sight. In the future, the
design of bioconjugates with targeting agents such as sugars or peptides could be the next step
towards overcoming the nuclear distribution issue and relocating the molecules to the cellular
membrane.
1
7

4
. Materials and Methods
4
4
.1. Chemistry
.1.1. General remarks
Commercially available reagents and solvents were used without further purification. Yields
refer to isolated and purified products by column chromatography, performed on a Teledyne
ISCO Combiflash Companion using pre-packed silica 60 columns. Reactions were monitored
by TLC carried out on 60F-254 silica gel plates and visualized under UV light at 254 and 365
1
13
nm. Chemical shifts (δ) of H and C NMR are reported in ppm and residual non deuterated
solvents were used as references. Multiplicities are designated by the following abbreviations:
s = singlet, d = doublet, t = triplet, q = quadruplet, bs = broad singlet, m = multiplet. High-
resolution mass spectra (HRMS) were measured by a TOF spectrometer, using electrospray
ionization (ESI). LC-MS spectra (ESI in the positive ion mode) were recorded on a Waters
Micromass ZQ instrument (source voltage 20-40 kV) coupled to a Binary Gradient Module
2
525 and a Waters Column Fluidic Organizer, equipped with a Waters XTerra MS C18 - 5µm
column and a Waters PDA 2996. All indicated masses for bromine- and chlorine-containing
derivatives correspond to 79Br and 35Cl isotopes. Melting points were measured with a Stuart
SMP30. To determine the purity of the final compounds, XBridge C18, 3.5μm, 3.0 x 100 mm
(Waters) column was used. HPLC injection: i) Method I: flow 0.75 mL/min; gradient elution:
eluent A % H O 0.1 % HCOOH, eluent B % MeCN: 0 min (98/2), 3 min (50/50), 6 min
2
(50/50), 10 min (0/100), 11 min (98/2), 16 min (98/2); ii) Method II: flow 0.75 mL/min;
gradient elution: eluent A % H O 0.1 % HCOOH, eluent B % MeCN 0.1 % HCOOH: 0 min
2
(98/2), 1 min (98/2), 7 min (0/100), 9 min (100/0), 13 min (98/2).
4
4
2
.1.2. General procedures
.1.2.1. General procedures A and B for the synthesis of 3H-imidazo[4,5-b]pyridines from
,3-diaminopyridines:
A: A mixture of 2,3-diaminopyridine (1 equiv.), NH Cl (6 equiv.) and the corresponding
4
carboxylic acid (1.5 equiv.) in POCl (0.6 M) was heated at 100 °C for 24 h. The mixture was
3
subsequently poured on ice and neutralized with 3 M NaOH aqueous solution. The aqueous
layer was extracted with EtOAc and the combined organic layers were dried on MgSO and
4
concentrated under reduced pressure. The residue was purified by chromatography eluting
with 0 to 10% EtOH in DCM gradient to give the corresponding product.
1
8

B: To a mixture of 2,3-diaminopyridine (1 equiv.) in methyl-orthoformate was added
dropwise a 10 M HCl aqueous solution (2 equiv.). The reaction was stirred at RT overnight.
Subsequently, the reaction mixture was dissolved in H O, neutralized by addition of a 3 M
2
NaOH aqueous solution and extracted with EtOAc. The combined organic layers were dried
with anhydrous MgSO and filtered. Evaporation of the solvent under reduced pressure gave
4
the corresponding product that we used with no further purification.
4
.1.2.2. General procedure C for the chlorination at the 7-position of the 3H-imidazo[4,5-
b]pyridine core: To a mixture of 6-bromo-3H-imidazo[4,5-b]pyridine (1 equiv.) in AcOH was
added m-chloroperbenzoic acid (2 equiv.). The reaction mixture was stirred at RT for 3 d. The
solid precipitate was collected by filtration affording a first fraction of 6-bromo-3H-
imidazo[4,5-b]pyridine 4-oxide. The filtrate was concentrated under reduced pressure,
suspended in EtOAc and filtered again to give a second fraction of the N-oxide. The
combined fractions of 6-bromo-3H-imidazo[4,5-b]pyridine 4-oxide were suspended in EtOAc
and refluxeded for 1 h. Subsequent filtration through sintered glass disk afforded 6-bromo-
3
H-imidazo[4,5-b]pyridine-4-oxide with no trace of benzoic acid. The solid was distributed in
microwave tubes in 250 mg portions and POCl (1.5 mL) was added in each tube. The
3
reaction mixture was heated under microwave irradiation at 80 °C for 10 min. Subsequently,
the reaction mixture was poured on ice, neutralized with a 3 M NaOH aqueous solution and
the aqueous layer was extracted with EtOAc. The combined organic layers were dried on
anhydrous MgSO , filtered and concentrated under reduced pressure to give the desired
4
product as an inseparable mixture (ratio 85:15) with the 5-chloro-3H-imidazo[4,5-b]pyridine
isomer.
4
.1.2.3. General procedure D for the benzyl protection of the imidazo[4,5-b]pyridine core: To
a solution of the imidazo[4,5-b]pyridine (1 equiv.) in dry DMF at 0 °C under argon
atmosphere was added NaH (60 % dispersion in mineral oil, 1.1 equiv.) portion wise. The
mixture was left stirring at 0 °C for 30 min then benzyl bromide (1.1 equiv.) was added
dropwise. The reaction mixture was then allowed to warm up to room temperature and stirred
overnight. Subsequently, the solvent was removed under reduced pressure and the resulting
oil was taken in EtOAc and washed with water. The combined organic layers were dried on
MgSO and concentrated under reduced pressure. The residue was then purified by column
4
chromatography eluting with a 0 to 50% EtOAc in cyclohexane gradient to give the
corresponding product.
1
9

4
.1.2.4. General procedure E for the PMB protection of the imidazo[4,5-b]pyridine core: To
a mixture of the 3H-imidazo[4,5-b]pyridine (1 equiv.) in dry DMF at 0 °C under argon
atmosphere was added NaH (60 % dispersion in mineral oil, 1.1 equiv) portionwise. The
mixture was stirred at 0 °C for 30 min, then p-methoxybenzyl bromide (1.1 equiv.) was added
dropwise. The reaction mixture was then allowed to warm up to RT and stirred overnight.
Subsequently, the DMF was evaporated under reduced pressure and the residue was taken in
H O and extracted with EtOAc. The combined organic layers were dried on MgSO and
2
4
concentrated under reduced pressure. The crude product was purified by column
chromatography eluting with a 0 to 100% EtOAc in cyclohexane gradient to give the
corresponding product.
4
.1.2.5. General procedure F for the aromatic nucleophilic substitution at the 7-position on
the imidazo[4,5-b]pyridine core: In a sealed tube, dry K CO (2 equiv.) was added to a
2
3
solution of t-butyl (3-fluoro-4-hydroxyphenyl)carbamate (1.5 equiv.) in DMSO. The resulting
suspension was stirred for 10 min at RT before the corresponding N3-protected 5-chloro-3H-
imidazo[4,5-b]pyridine (1 equiv.) was added. The tube was then warmed up to 70 °C for 16 h.
After cooling down to RT, brine was added and the mixture was extracted with EtOAc. The
combined organic layers were dried on MgSO and concentrated under reduced pressure. The
4
residue was then purified by chromatography eluting with a 0 to 100% EtOAc in cyclohexane
gradient to give the corresponding product.
4
.1.2.6. General Procedure G for the Suzuki coupling: To a mixture of the N3-protected 6-
bromo-3H-imidazo[4,5-b]pyridine (1 equiv.), PdCl (dppf) (5 mol %) and the corresponding
2
boronic acid (1.5 equiv.) in 1,4-dioxane was added a 2 M Cs CO aqueous solution (2 equiv.).
2
3
The reaction mixture was warmed up to 100 °C under argon atmosphere for 18 h.
Subsequently, water was added and the mixture was extracted with EtOAc. The combined
organic layers were dried on anhydrous MgSO , filtered and concentrated under reduced
4
pressure. The crude product was purified by column chromatography eluting with a 0 to 60%
EtOAc in cyclohexane gradient to give the corresponding product.
4
.1.2.7. General procedure H for the condensation of anilines: In a round bottom flask under
argon atmosphere, 1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid (1 equiv.)
and TBTU (1.3 equiv.) were dissolved in DMF and the resulting solution was cooled down to
0
°C. After 15 min, DIPEA (3 equiv.) was added and the mixture was stirred for further 15
min. The corresponding aniline (1 equiv.) was subsequently added and the resulting solution
was stirred at 0 °C for 4 h. Solvent was removed under reduced pressure and the residue was
2
0

taken in EtOAc and washed with a saturated NaHCO aqueous solution. The organic layer was
3
dried on MgSO , filtered and purified by chromatography eluting with a 0 to 10% EtOH in
4
DCM gradient to give the corresponding product.
4
.1.2.8. Typical procedure I for the deprotection of N3-PMB-3H-imidazo[4,5-b]pyridines: In
a round-bottom flask under argon atmosphere, the N3-protected imidazo[4,5-b]pyridine (1
equiv.) was solubilized in anisole (2.3 equiv.) before TFA (33 equiv.) was added dropwise.
Subsequently, sulfuric acid (58 %, 2.3 equiv.) was added dropwise and the mixture is stirred
at RT for 16 h. The mixture was then cooled down to 0 °C and neutralized with a saturated
NaHCO aqueous solution. The aqueous layer was extracted with EtOAc then DCM. The
3
combined organic layers were dried on MgSO , filtered and concentrated under reduced
4
pressure. The resulting material was purified by chromatography eluting with a 0 to 10%
EtOH in DCM gradient to give the corresponding product.
4
.1.3. 6-Bromo-2-ethyl-3H-imidazo[4,5-b]pyridine 2A. The reaction was performed following
procedure A on 2,3-diamino-5-bromopyridine 1 (4 g, 20.4 mmol) to give the corresponding
1
product 2A as a brown solid (3.2 g, 70 %). Mp: 237 – 239 °C. H NMR (300 MHz, DMSO-
d6) δ = 12.91 (bs, 1H), 8.31 (d, J = 2.0 Hz, 1H), 8.13 (d, J = 2.0 Hz, 1H), 2.85 (q, J = 7.6 Hz,
1
3
2
1
2
H), 1.31 (t, J = 7.6 Hz, 3H). C NMR (75 MHz, DMSO-d ) δ = 160.1, 142.8, 112.2, 22.3,
6
+
+
1.7. MS (ES ) m/z (%): 226.0 (100) [M+H] . HRMS (ESI) calculated for C H BrN
3
8
9
25.9980; found 225.9983.
4
.1.4. 6-Bromo-2-isopropyl-3H-imidazo[4,5-b]pyridine 2B. The reaction was performed
following procedure A on 2,3-diamino-5-bromopyridine 1 (4.17 g, 21.30 mmol) to give the
1
corresponding product 2B as a brown solid (3.6 g, 70 %). Mp: 190 – 192 °C. H NMR (300
MHz, DMSO-d ) δ = 12.83 (d, J = 121.0 Hz, 1H), 8.33 (d, J = 19.6 Hz, 1H), 8.12 (d, J = 37.8
6
1
3
Hz, 1H), 3.29 – 2.98 (m, 1H), 1.35 (s, 3H), 1.33 (s, 3H). C NMR (75 MHz, DMSO-d ) δ =
6
+
+
1
63.3, 147.7, 142.7, 136.2, 127.8, 112.2, 28.7, 20.9. MS (ES ) m/z (%): 240.0 (100) [M+H] .
HRMS (ESI) calculated for C H N Br 240.0136; found 240.0141.
9
11
3
4
.1.5. 6-Bromo-3H-imidazo[4,5-b]pyridine 2C. Reaction was performed following procedure
B on commercial 2,3-diamino-5-bromopyridine 1 (12 g, 64.00 mmol) to give the
corresponding product 2C as a grey solid (3.6 g, 99 %) which was used without any further
1
purification. H NMR (300 MHz, DMSO-d ) δ = 8.47 (s, 1H), 8.41 (d, J = 1.6 Hz, 1H), 8.27
6
+
+
(
d, J = 1.2 Hz, 1H). MS (ES ) m/z (%): 198.0 (100) [M+H] . Spectroscopic data were in
agreement with those reported in the literature.[39]
2
1

4
.1.6. 6-Bromo-7-chloro-2-ethyl-3H-imidazo[4,5-b]pyridine 3A. The reaction was performed
following procedure C on 6-bromo-2-ethyl-3H-imidazo[4,5-b]pyridine 2A (1.10 g, 4.90
1
mmol) to give a mixture of two regioisomers ( H NMR ratio 75:25; chlorine at position 7 and
5
respectively) with 6-bromo-7-chloro-2- ethyl-3H-imidazo[4,5-b]pyridine 3A as the major
1
regioisomer, a beige solid (890 mg, 70 %). Mp: 79 – 81 °C. H NMR (300 MHz, DMSO-d ) δ
6
1
3
=
8.44 (s, 1H), 8.33 (s, 0.34H), 3.04 – 2.70 (m, 2H), 1.33 (s, 3H). C NMR (75 MHz, DMSO-
d6) δ = 161.4, 160.8, 150.9, 149.9, 144.1, 141.3, 132.5, 131.4, 129.5, 127.9, 112.5, 110.3,
+
+
2
2
2.4, 11.8. MS (ES ) m/z (%): 260.0 (100) [M+H] . HRMS (ESI) calculated for C H ClBrN
8 8 3
59.9590; found 259.9591.
4
.1.7. 6-Bromo-7-chloro-2-isopropyl-3H-imidazo[4,5-b]pyridine 3B. The reaction was
performed following procedure C on 6-bromo-2-isopropyl-3H-imidazo[4,5-b]pyridine 2B
1
(
3.4 g, 14.3 mmol) to give a mixture of two regioisomers ( H NMR ratio 75:25; chlorine at
position 7 and 5 respectively) with 6-bromo-7-chloro-2- isopropyl-3H-imidazo[4,5-b]pyridine
1
3
B as the major regioisomer, a beige solid (2.75 g, 70 %). Mp: 123 – 125 °C. H NMR (300
MHz, DMSO-d ) δ = 8.41 (s, 1H), 8.31 (s, 0.25H), 3.33 – 3.00 (m, 1H), 1.35 (s, 3H), 1.33 (s,
6
1
3
3
1
H). C NMR (75 MHz, DMSO-d ) δ = 165.2, 164.6, 151.0, 150.1, 144.0, 141.2, 132.7,
6
+
31.5, 129.7, 128.0, 112.3, 110.1, 28.9, 28.8, 21.0, 20.9. MS (ES ) m/z (%): 276.0 (100)
+
[
M+H] . HRMS (ESI) calculated for C H N BrCl 273.9747; found 273.9760.
9
10
3
4
.1.8. 6-Bromo-7-chloro-3H-imidazo[4,5-b]pyridine 3C. The reaction was performed
following procedure C using 6-bromo-3H-imidazo[4,5-b]pyridine 2C (3.6 g, 18.5 mmol) to
give a mixture of two regioisomers (1H NMR ratio 85:15; chlorine at position 7 and 5
respectively) with the 6-bromo-7-chloro-3H-imidazo[4,5-b]pyridine isomer 3C as the major
1
regioisomer, a beige solid (3.1 g, 73 %). Mp: 199-201 °C. H NMR (300 MHz, CD OD) δ =
3
1
3
8
1
.51 (s, 2H), 8.46 (s, 0.33H, 5-Cl isomer). C NMR (75 MHz, DMSO-d ) δ = 146.6, 145.8,
6
+
45.5, 142.3, 133.7, 133.2, 132.3, 130.4, 128.8, 127.8, 113.1, 111.1. MS (ES ) m/z (%): 231.9
+
+
(
100) [M+H] , 273.0 (20) [M+CH CN+H] . HRMS (ESI) calculated for C H N ClBr
3 6 4 3
2
31.9277; found 231.9287. Spectroscopic data were in agreement with those reported in the
literature.[40]
4
.1.9. 3-Benzyl-6-bromo-7-chloro-2-isopropyl-3H-imidazo[4,5-b]pyridine 4A. The reaction
was performed following procedure D on the mixture of the two regioisomers (see 3A) (350
mg, 1.30 mmol) to give the corresponding product 4A as a light yellow solid (200 mg, 40 %).
1
H NMR (300 MHz, CDCl ) δ = 8.45 (s, 1H), 7.38 – 7.21 (m, 3H), 7.17 – 7.06 (m, 2H), 5.46
3
2
2

1
3
(
s, 2H), 2.85 (q, J = 7.4 Hz, 2H), 1.34 (t, J = 7.5 Hz, 3H). C NMR (75 MHz, CDCl ) δ =
3
1
–
3
63.2, 147.4, 145.1, 135.9, 133.9, 133.4, 129.0, 128.1, 126.6, 114.5, 45.7, 27.4, 21.2. Mp: 118
+
+
120 °C. MS (ES ) m/z (%): 352.0 (100) [M+H] . HRMS (ESI) calculated for C H N ClBr
1
5
14
3
50.0060; found 350.0066.
4
.1.10. 3-Benzyl-6-bromo-7-chloro-2-isopropyl-3H-imidazo[4,5-b]pyridine 4B. The reaction
was performed following procedure D on the mixture of the two regioisomers (see 3B) (500
mg, 1.8 mmol) to give the corresponding product 4B as a light yellow solid (270 mg, 40 %).
1
Mp: 123 – 125 °C. H NMR (300 MHz, CDCl ) δ = 8.44 (s, 1H), 7.28 (m, 3H), 7.09 (m, 2H),
3
1
3
5
.49 (s, 2H), 3.13 (hept, J = 6.6 Hz, 1H), 1.34 (d, J = 6.7 Hz, 6H). C NMR (75 MHz,
CDCl ) δ = 163.5, 146.4, 143.4, 135.7, 134.7, 131.9, 128.9, 128.0, 126.7, 112.4, 45.5, 27.2,
3
+
+
2
3
1.1.MS (ES ) m/z (%): 366.0 (100) [M+H] . HRMS (ESI) calculated for C H N ClBr
16 16 3
64.0216; found 364.0209.
4
.1.11. 4-((3-Benzyl-6-bromo-2-ethyl-3H-imidazo[4,5-b]pyridin-7-yl)oxy)-3-fluoroaniline 5A.
The reaction was performed following procedure F on 3-benzyl-6-bromo-7-chloro-2-ethyl-
3
H-imidazo[4,5-b]pyridine 4A (640 mg, 1.83 mmol) to give the corresponding product 5A as
1
a brown solid (440 mg, 55 %). Mp: 157 – 159 °C. H NMR (300 MHz, CDCl ) δ = 8.40 (s,
3
1
H), 7.39 – 7.20 (m, 3H), 7.20 – 7.05 (m, 2H), 6.95 (t, J = 8.8 Hz, 1H), 6.48 (dd, J = 12.1, 2.5
Hz, 1H), 6.37 (d, J = 7.2 Hz, 1H), 5.39 (s, 2H), 3.67 (s, 2H), 2.67 (q, J = 7.4 Hz, 2H), 1.17 (t,
1
3
J = 7.4 Hz, 3H). C NMR (75 MHz, CDCl ) δ = 157.0, 154.5 (d, J = 246.3 Hz), 151.0, 150.4,
3
1
2
46.1, 144.3 (d, J = 9.5 Hz), 136.7 (d, J = 12.4 Hz), 136.0, 128.9, 128.0, 126.9, 121.8 (d, J =
.1 Hz), 116.7, 110.2 (d, J = 2.9 Hz), 104.3, 103.4 (d, J = 21.3 Hz), 45.6, 21.4, 10.8. MS
+
+
(
ES ) m/z (%): 441.1 (100) [M+H] . HRMS (ESI) calculated for C H N OBrF 441.0726;
21 19 4
found 441.0733.
4
.1.12.
4-((3-Benzyl-6-bromo-2-isopropyl-3H-imidazo[4,5-b]pyridin-7-yl)oxy)-3-
fluoroaniline 5B. The reaction was performed following procedure F on 3-benzyl-6-bromo-7-
chloro-2-isopropyl-3H-imidazo[4,5-b]pyridine 4B (450 mg, 1.23 mmol) to give the
1
corresponding product 5B as a brown solid (310 mg, 55 %). Mp: 156 – 158 °C. H NMR (300
MHz, CDCl ) δ = 8.38 (s, 1H), 7.39 – 7.18 (m, 5H), 7.18 – 7.04 (m, 2H), 6.96 (t, J = 8.7 Hz,
3
1
H), 6.48 (d, J = 12.0 Hz, 1H), 6.43 – 6.32 (m, 1H), 5.43 (s, 2H), 3.39 (b, 2H), 3.03 – 2.92
1
3
(
m, 1H), 1.14 (d, J = 6.8 Hz, 6H). C NMR (75 MHz, CDCl ) δ = 160.5, 154.6 (d, J = 246.0
3
Hz), 151.0, 150.2,146.0, 144.3 (d, J = 9.4 Hz), 136.6 (d, J = 12.5 Hz), 136.3, 128.8, 127.8,
26.7, 126.5, 121.9 (d, J = 2.0 Hz), 109.9 (d, J = 2.9 Hz), 104.0, 103.2 (d, J = 21.4 Hz), 45.4,
1
2
3

+
+
2
7.1, 21.1.MS (ES ) m/z (%): 455.1 (100) [M+H] . HRMS (ESI) calculated for
C H N OBrF 455.0883; found 455.0872.
2
2
21
4
4
6
3
.1.13.
4-((3-Benzyl-2-ethyl-6-phenyl-3H-imidazo[4,5-b]pyridin-7-yl)oxy)-3-fluoroaniline
Aa. The reaction was performed following procedure G on 4-((3-Benzyl-6-bromo-2-ethyl-
H-imidazo[4,5-b]pyridin-7-yl)oxy)-3-fluoroaniline 5A (110 mg, 0.25 mmol) with
phenylboronic acid (46 mg, 0.37 mmol) to give the corresponding product 6Aa as a beige
1
solid (70 mg, 65 %). Mp: 168 – 170 °C. H NMR (300 MHz, MeOD) δ = 8.37 (s, 1H), 7.89
(s, 1H), 7.53 – 7.42 (m, 2H), 7.41 – 7.18 (m, 7H), 6.92 (m, 1H), 6.83 – 6.68 (m, 2H), 5.72 (m,
1
3
1
H), 5.61 (s, 2H), 2.87 (q, J = 7.1 Hz, 2H), 1.19 (t, J = 7.0 Hz, 3H). C NMR (75 MHz,
CDCl ) δ = 156.6, 154.1 (d, J = 245.6 Hz), 150.9, 150.8, 145.9, 143.6 (d, J = 9.4 Hz), 137.1
3
(d, J = 12.1 Hz), 136.3, 135.5, 129.8, 128.8, 128.2, 127.8, 127.1, 126.8, 125.9, 123.1, 121.2
+
(
d, J = 2.3 Hz), 110.0(d, J = 2.9 Hz), 103.3 (d, J = 21.4 Hz), 45.4, 21.4, 11.0. MS (ES ) m/z
+
(
%): 439.2 (100) [M+H] . HRMS (ESI) calculated for C H BrN OF 439.1934; found
2
7
24
4
4
39.1938.
4
.1.14.
4-((3-Benzyl-2-isopropyl-6-phenyl-3H-imidazo[4,5-b]pyridin-7-yl)oxy)-3-
fluoroaniline 6Ba. The reaction was performed following procedure G on 4-((3-Benzyl-6-
bromo-2-isopropyl-3H-imidazo[4,5-b]pyridin-7-yl)oxy)-3-fluoroaniline 5B (100 mg, 0.22
mmol) with phenylboronic acid (40 mg, 0.33 mmol) to give the corresponding product 6Ba as
1
a beige solid (80 mg, 80 %). Mp: 157 – 159 °C. H NMR (300 MHz, CDCl ) δ = 8.27 (s, 1H),
3
7
6
.62 – 7.47 (m, 2H), 7.47 – 7.20 (m, 6H), 7.15 (d, J = 6.1 Hz, 2H), 6.79 (t, J = 8.7 Hz, 1H),
.34 (dd, J = 12.4, 2.8 Hz, 1H), 6.24 (d, J = 8.1 Hz, 1H), 5.49 (s, 2H), 3.55 (bs, 2H), 3.02
1
3
(
hept, J = 6.7 Hz, 1H), 1.18 (d, J = 6.7 Hz, 6H). C NMR (75 MHz, CDCl ) δ= 160.3, 154.4
3
(d, J = 245.9 Hz), 151.3, 150.7, 146.0, 143.6 (d, J = 9.3 Hz), 137.4 (d, J = 12.1 Hz), 136.7,
1
35.7, 129.9,129.0, 128.3, 127.9, 127.2, 126.8, 126.0, 123.0, 121.7 (d, J = 2.3 Hz), 110.1 (d, J
+
+
=
3.0 Hz), 103.3 (d, J = 21.4 Hz), 45.5, 27.3, 21.4. MS (ES ) m/z (%): 453.2 (100) [M+H] .
HRMS (ESI) calculated for C H N OF 453.2091; found 453.2088.
2
8
26
4
4
.1.15.
N-(4-((3-Benzyl-2-ethyl-6-phenyl-3H-imidazo[4,5-b]pyridin-7-yl)oxy)-3-
fluorophenyl)-1-(4-fluoro- phenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide 7Aa. The
reaction was performed following procedure H on 4-((3-benzyl-2-ethyl-6-phenyl-3H-
imidazo[4,5-b]pyridin-7-yl)oxy)-3-fluoroaniline 6Aa (100 mg, 0.23 mmol) to give the
1
corresponding product 7Aa as a white solid (105 mg, 71 %). Mp: 136 – 138 °C. H NMR
(
300 MHz, CDCl ) δ= 11.77 (s, 1H), 8.71 (dd, J = 7.3, 2.1 Hz, 1H), 8.32 (s, 1H), 7.72 (dd, J =
3
2
4

1
1
6
2.5, 1.7 Hz, 1H), 7.58 (dd, J = 6.9, 1.9 Hz, 1H), 7.54 (d, J = 7.2 Hz, 2H), 7.41 – 7.25 (m,
0H), 7.17 (d, J = 7.1 Hz, 2H), 7.09 (d, J = 8.8 Hz, 1H), 6.85 (t, J = 8.9 Hz, 1H), 6.58 (t, J =
1
3
.9 Hz, 1H), 5.47 (s, 2H), 2.71 (q, J = 7.5 Hz, 2H), 1.18 (t, J = 7.5 Hz, 3H). C NMR (75
MHz, CDCl ) δ = 162.8 (d, J = 226.38 Hz), 162.4, 161.1 (d, J = 5.2 Hz), 157.0, 153.0 (d, J =
3
2
46.4 Hz), 150.7, 150.1,146.0, 144.9, 141.4, 141.2 (d, J = 11.9 Hz), 136.2, 135.8 (d, J = 3.3
Hz), 135.2, 134.5 (d, J = 9.5 Hz), 129.7, 128.9, 128.4 (d, J = 8.9 Hz), 128.3, 127.9, 127.3,
1
1
26.8, 126.3, 123.5, 122.2, 119.9 (d, J = 1.7 Hz), 116.8 (d, J = 23.2 Hz), 115.3 (d, J = 3.3 Hz),
+
+
08.9 (d, J = 23.3 Hz), 107.2, 45.5, 21.5, 11.1. MS (ES ) m/z (%): 654.2 (100) [M+H] .
HRMS (ESI) calculated for C H N O F 2654.2317; found 654.2303.
3
9
30
5
3
4
.1.16.
N-(4-((3-Benzyl-2-isopropyl-6-phenyl-3H-imidazo[4,5-b]pyridin-7-yl)oxy)-3-
7Ba. The
fluorophenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide
reaction was performed following procedure H on 4-((3-benzyl-2-isopropyl-6-phenyl-3H-
imidazo[4,5-b]pyridin-7-yl)oxy)-3-fluoroaniline 6Ba (100 mg, 0.22 mmol) to give the
1
corresponding product 7Ba as a light yellow solid (88 mg, 60 %). Mp: 134 – 136 °C. H
NMR (300 MHz, CDCl ) δ = 11.76 (s, 1H), 8.71 (d, J = 5.4 Hz, 1H), 8.30 (s, 1H), 7.70 (dd, J
3
=
12.1, 1.6 Hz, 1H), 7.58 (d, J = 4.2 Hz, 1H), 7.56 – 7.50 (m, 2H), 7.43 – 7.20 (m, 10H), 7.15
(d, J = 5.9 Hz, 2H), 7.09 (d, J = 8.6 Hz, 1H), 6.86 (t, J = 8.6 Hz, 1H), 6.58 (t, J = 6.6 Hz, 1H),
1
3
5
1
1
1
.50 (s, 2H), 3.11 – 2.88 (m, 1H), 1.17 (d, J = 6.6 Hz, 6H). C NMR (75 MHz, CDCl ) δ=
3
62.7 (d, J = 250.3 Hz), 162.4, 161.1, 160.7, 153.1 (d, J = 246.3 Hz), 150.6, 150.2, 145.9,
44.9, 141.5, 141.1 (d, J = 11.9 Hz), 136.5, 135.8 (d, J = 3.3 Hz), 135.2, 134.6 (d, J = 9.6 Hz),
29.7, 128.8, 128.4 (d, J = 8.9 Hz), 128.2, 127.8, 127.3, 126.6, 126.2, 123.3, 122.1, 120.3 (d,
J = 1.6 Hz), 116.8 (d, J = 23.2 Hz), 115.1 (d, J = 3.2 Hz), 108.7 (d, J = 23.3 Hz), 107.2, 45.4,
+
+
2
6
7.2, 21.2.MS (ES ) m/z (%): 668.2 (100) [M+H] . HRMS (ESI) calculated for C H N O F
40 32 5 3 2
68.2473; found 668.2480.
4
.1.17. N-(4-((2-Ethyl-6-phenyl-3H-imidazo[4,5-b]pyridin-7-yl)oxy)-3-fluorophenyl)-1-(4-
fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide 8Aa. To a solution of N-(4-((3-
benzyl-2-ethyl-6-phenyl-3H-imidazo[4,5-b]pyridin-7-yl)oxy)-3-fluorophenyl)-1-(4-
fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide 7Aa (60 mg, 0.09 mmol) in EtOH
(10 mL) wasadded Pd 10 % on carbon (98 mg, 0.09 mmol). The resulting suspension was
flushed with argon and purged twice before flushing with hydrogen. The reaction was then
stirred at RT overnight. The suspension was subsequently filtered on a celite plug and the
filtrate was concentrated under reduce pressure. The residue was purified by chromatography
eluting with a 0 to 10% EtOH in DCM gradient to give the corresponding product 8Aa as a
2
5

1
white solid (27 mg, 52 %). Mp: 173 – 175 °C. H NMR (300 MHz, DMSO-d ) δ = 13.00 (s,
6
1
7
1
H), 11.93 (s, 1H), 8.55 (dd, J = 7.2, 1.8 Hz, 1H), 8.26 (s, 1H), 8.10 (dd, J = 6.6, 1.5 Hz, 1H),
.85 (dd, J = 14.1, 1.6 Hz, 1H), 7.62 – 7.49 (m, 4H), 7.46 – 7.32 (m, 5H), 7.18 (d, J = 8.2 Hz,
H), 6.89 (t, J = 9.2 Hz, 1H), 6.71 (t, J = 6.9 Hz, 1H), 2.73 (q, J = 7.6 Hz, 2H), 1.20 (t, J = 7.7
1
3
Hz, 3H). C NMR (125 MHz, DMSO-d ) δ = 161.8 (d, J = 246.1 Hz), 161.7, 161.2, 157.3,
6
1
1
51.8 (d, J = 245.5 Hz), 151.3, 148.1, 145.0, 144.7, 143.9, 140.5, 136.2 (d, J = 2.2 Hz), 134.9,
34.2, 129.3, 129.2 (d, J = 9.1 Hz), 128.3, 127.2,126.0, 122.4, 120.2, 119.5, 116.0 (d, J = 23.0
+
+
Hz), 115.4,108.0 (d, J = 24.1 Hz), 107.0, 28.6, 20.8.MS (ES ) m/z (%): 564.2 (100) [M+H] .
HRMS (ESI) calculated for C H N O F 564.1847; found 564.1854. HPLC purity (method
3
2
24
5
3 2
I): 96 %.
4
.1.18. N-(3-Fluoro-4-((2-isopropyl-6-phenyl-3H-imidazo[4,5-b]pyridin-7-yl)oxy)phenyl)-1-
(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide 8Ba. To a solution of N-(4-((3-
benzyl-2-isopropyl-6-phenyl-3H-imidazo[4,5-b]pyridin-7-yl)oxy)-3-fluorophenyl)-1-(4-
fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide7Ba (60 mg, 0.09 mmol) in EtOH
(10 mL) was added Pd 10 % on carbon (96 mg, 0.09 mmol). The resulting suspension was
flushed with argon and purged twice before flushing with hydrogen. The reaction was then
stirred at RT overnight. The suspension was subsequently filtered on a celite plug and the
filtrate was concentrated under reduce pressure. The residue was purified by chromatography
eluting with a 0 to 10% EtOH in DCM gradient to give the corresponding product 8Ba as a
1
white solid (33 mg, 64 %). Mp: 177 – 179 °C. H NMR (500 MHz, DMSO-d ) δ = 12.96 (s,
6
1
7
7
=
H), 11.93 (s, 1H), 8.55 (dd, J = 7.3, 2.1 Hz, 1H), 8.26 (s, 1H), 8.10 (dd, J = 6.6, 2.1 Hz, 1H),
.84 (dd, J = 13.2, 2.4 Hz, 1H), 7.59 (dd, J = 8.9, 4.9 Hz, 2H), 7.53 (d, J = 7.2 Hz, 2H), 7.46 –
.37 (m, 3H), 7.34 (d, J = 7.4 Hz, 1H), 7.20 – 7.13 (m, 1H), 6.90 (t, J = 9.1 Hz, 1H), 6.71 (t, J
1
3
14.0 Hz, 1H), 3.04 (hept, J = 7.0 Hz, 1H), 1.24 (d, J = 7.0 Hz, 6H). C NMR (125 MHz,
DMSO-d ) δ= 161.8 (d, J = 246.1 Hz), 161.7, 161.2, 157.7, 151.7 (d, J = 243.8 Hz), 151.2,
6
1
9
2
47.9, 144.9, 144.7, 143.9, 140.6 (d, J = 11.3 Hz), 136.2 (d, J = 3.1 Hz), 134.9, 134.1 (d, J =
.4 Hz), 129.3, 129.2 (d, J = 9.0 Hz), 128.4, 127.3, 126.2, 122.6, 120.2, 119.3, 116.0 (d, J =
+
3.1 Hz), 115.6, 108.1 (d, J = 23.0 Hz), 107.0, 22.2, 11.8. MS (ES ) m/z (%): 578.2 (100)
+
[
M+H] ; 1177.4 (10) [2M+Na]+. HRMS (ESI) calculated for C H N O F 578.2004; found
3
3
26
5
3 2
5
78.2020. HPLC purity (method I): 98 %.
4
.1.19. N-(4-((3-Benzyl-6-bromo-2-isopropyl-3H-imidazo[4,5-b]pyridin-7-yl)oxy)-3-
fluorophenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide 9B.
2
6

The reaction was performed following procedure H on 4-((3-benzyl-6-bromo-2-isopropyl-3H-
imidazo[4,5-b]pyridin-7-yl)oxy)-3-fluoroaniline 5B (80 mg, 0.18mmol) to give the
1
corresponding product 9B as a beige solid (72 mg, 61 %). Mp: 219 – 221 °C. H NMR (300
MHz, CDCl ) δ = 11.88 (s, 1H), 8.73 (dd, J = 7.3, 2.2 Hz, 1H), 8.39 (s, 1H), 7.85 (dd, J =
3
1
7
2
2.7, 2.4 Hz, 1H), 7.60 (dd, J = 6.6, 2.2 Hz, 1H), 7.44 – 7.35 (m, 2H), 7.31 – 7.20 (m, 6H),
.09 (dd, J = 7.8, 2.1 Hz, 2H), 7.03 (t, J = 8.8 Hz, 1H), 6.60 (t, J = 7.0 Hz, 1H), 5.43 (s, 2H),
1
3
.97 (hept, J = 6.7 Hz, 1H), 1.11 (d, J = 6.8 Hz, 6H). C NMR (75 MHz, CDCl ) δ = 162.7
3
(
d, J = 250.4 Hz), 162.4, 161.2, 160.9, 153.5 (d, J = 246.8 Hz), 150.2, 150.2,146.0, 145.0,
41.6, 140.5 (d, J = 12.2 Hz), 136.2, 135.8 (d, J = 3.3 Hz), 135.4 (d, J = 9.6 Hz), 128.9, 128.4
1
(d, J = 8.8 Hz), 127.9, 126.8, 126.6, 122.1, 120.8 (d, J = 1.1 Hz), 116.8 (d, J = 23.1 Hz), 115.3
+
(
d, J = 3.3 Hz), 108.9 (d, J = 23.2 Hz), 107.3, 104.5, 45.5, 27.2, 21.1. MS (ES ) m/z (%):
+
6
70.1 (100) [M+H] . HRMS (ESI) calculated for C H N O F Br 670.1265; found 670.1280.
3
4
27
5
3 2
4
.1.20.
N-(3-Fluoro-4-((2-isopropyl-3H-imidazo[4,5-b]pyridin-7-yl)oxy)phenyl)-1-(4-
fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide 10Bb. To a solution of N-(4-((3-
benzyl-6-bromo-2-isopropyl-3H-imidazo[4,5-b]pyridin-7-yl)oxy)-3-fluorophenyl)-1-(4-
fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide 9B (50 mg, 0.07 mmol) in EtOH (10
mL) was added Pd 10 % on carbon (80 mg, 0.07 mmol). The resulting suspension was flushed
with argon and purged twice before flushing with hydrogen. The reaction was then stirred at
RT overnight. The suspension was subsequently filtered on a celite plug and the filtrate was
concentrated under reduce pressure. The residue was purified by chromatography eluting with
a 0 to 10% EtOH in DCM gradient to give the corresponding product 10Bb as a white solid
1
(
18 mg, 48 %). Mp: 244 – 246 °C. H NMR (500 MHz, DMSO-d ) δ = 12.89 (s, 1H), 12.10
6
(s, 1H), 8.60 (dd, J = 7.3, 2.0 Hz, 1H), 8.14 (dd, J = 6.6, 2.0 Hz, 1H), 8.09 – 8.00 (m, 2H),
7
1
.65 – 7.60 (m, 2H), 7.49 (d, J = 8.2 Hz, 1H), 7.43 (t, J = 8.8 Hz, 2H), 7.37 (t, J = 8.7 Hz,
H), 6.74 (t, J = 6.9 Hz, 1H), 6.42 (d, J = 5.5 Hz, 1H), 3.21 – 3.10 (m, 1H), 1.36 (d, J = 6.7
1
3
Hz, 6H). C NMR (125 MHz, DMSO-d ) δ = 161.9 (d, J = 246.1 Hz), 161.8, 161.6, 160.2,
6
1
53.8, 153.5 (d, J = 245.6 Hz), 151.4,145.0, 144.8, 144.2 (d, J = 16.5 Hz), 136.7 (d, J = 9.3
Hz), 136.2 (d, J = 2.5 Hz), 129.3, 129.2, 124.2, 123.6, 120.1, 116.5, 116.1 (d, J = 23.1 Hz),
+
+
1
1
5
08.6 (d, J = 22.9 Hz), 107.0, 102.7, 28.6, 21.0. MS (ES ) m/z (%): 502.2 (100) [M+H] ;
025.3 (20) [2M+Na]+. HRMS (ESI) calculated for C H N O F 502.1691; found
2
7
22
5
3 2
02.1710. HPLC purity (method II): 97 %.
4
.1.21. 6-Bromo-7-chloro-2-ethyl-3-(4-methoxybenzyl)-3H-imidazo[4,5-b]pyridine 11A. The
reaction was performed following procedure E on the mixture of the two regioisomers (see
2
7

3
A) (1.3 g, 4.95mmol) in DMF (15 mL) to give the corresponding product 11A as a white
1
solid (780 mg, 40 %). Mp: 104 – 106 °C. H NMR (300 MHz, CDCl ) δ = 8.43 (s, 1H), 7.07
3
(d, J = 8.4 Hz, 2H), 6.80 (d, J = 8.4 Hz, 2H), 5.37 (s, 2H), 3.75 (s, 3H), 2.85 (q, J = 7.5 Hz,
1
3
2
1
H), 1.33 (t, J = 7.5 Hz, 3H). C NMR (75 MHz, CDCl ) δ = 159.6, 159.4, 147.6, 145.1,
3
+
33.8, 133.3, 128.4, 127.7, 114.4, 114.4, 55.4, 45.5, 21.9, 11.6. MS (ES ) m/z (%): 380.0
+
(
100) [M+H] .HRMS (ESI) calculated for C H BrClN O 380.0165; found 380.0174.
1
6
16
3
4
.1.22. 6-Bromo-7-chloro-2-isopropyl-3-(4-methoxybenzyl)-3H-imidazo[4,5-b]pyridine 11B.
The reaction was performed following procedure E on the mixture of the two regioisomers
(see 3B) (3.1 g, 11.29mmol) in DMF (30 mL) to give the corresponding product 11B as a
1
white solid (1.76 g, 40 %). Mp: 106 – 108 °C. H NMR (300 MHz, CDCl ) δ = 8.44 (s, 1H),
3
7
6
1
3
.07 (d, J = 8.7 Hz, 2H), 6.82 (d, J = 8.7 Hz, 2H), 5.42 (s, 2H), 3.76 (s, 3H), 3.17 (hept, J =
1
3
.8 Hz, 1H), 1.34 (d, J = 6.8 Hz, 6H). C NMR (75 MHz, CDCl ) δ = 163.3, 159.5, 147.5,
3
+
45.1, 134.0, 133.4, 128.3, 128.1, 114.5, 114.4, 55.4, 45.4, 27.6, 21.4.MS (ES ) m/z (%):
+
94.0 (100) [M+H] . HRMS (ESI) calculated for C H N OClBr 394.0322; found 394.0319.
1
7
18
3
4
.1.23. 6-Bromo-7-chloro-3-(4-methoxybenzyl)-3H-imidazo[4,5-b]pyridine 11C. The reaction
was performed following procedure E on the mixture of the two regioisomers (see 3C) (2.5 g,
1
0.75 mmol) in DMF (25 mL) to give the corresponding product 11C as a light yellow solid
1
(
1.71 g, 45 %). Mp: 99 – 101 °C. H NMR (300 MHz, CDCl ) δ = 8.51 (s, 1H), 8.03 (s, 1H),
3
1
3
7
.25 (d, J = 8.5 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 5.36 (s, 2H), 3.77 (s, 3H). C NMR (75
MHz, CDCl ) δ = 159.7, 146.4, 146.1, 144.9, 134.8, 134.4, 129.4, 127.0, 114.8, 114.4, 55.3,
3
+
+
4
3
7.2. MS (ES ) m/z (%): 354.0 (100) [M+H] . HRMS (ESI) calculated for C H N O BrCl
14 12 3 4
51.9852; found 351.9863.
4
.1.24. 4-((6-Bromo-2-ethyl-3-(4-methoxybenzyl)-3H-imidazo[4,5-b]pyridin-7-yl)oxy)-3-
fluoroaniline 12A. The reaction was performed following procedure F on 6-bromo-7-chloro-
-ethyl-3-(4-methoxybenzyl)-3H-imidazo[4,5-b]pyridine 11A (110 mg, 0.29 mmol) in DMSO
2
(3 mL) to give the corresponding product 12A as a beige solid (750 mg, 55 %). Mp: 122 –
1
1
24 °C. H NMR (300 MHz, CDCl ) δ = 8.39 (s, 1H), 7.08 (d, J = 8.4 Hz, 2H), 6.91 (t, J = 8.6
3
Hz, 1H), 6.80 (d, J = 8.4 Hz, 2H), 6.52 – 6.37 (dd, J = 3, 8.6 Hz, 1H), 6.33 (d, J = 8.6 Hz,
1
3
1
H), 5.31 (s, 2H), 3.75 (s, 3H), 2.67 (q, J = 7.4 Hz, 2H), 1.16 (t, J = 7.4 Hz, 3H). C NMR
(
75 MHz, CDCl ) δ = 159.3, 157.0, 154.5 (d, J= 243.8 Hz), 150.9, 150.5, 146.0, 144.4 (d, J=
3
9
.5 Hz), 136.6 (d, J= 12.4 Hz), 128.5, 128.1, 126.6, 121.7 (d, J= 1.9 Hz), 114.3, 110.1 (d, J=
2
8

+
2
.9 Hz), 104.3, 103.4 (d, J= 21.3 Hz), 55.4, 45.13, 21.5, 10.8. MS (ES ) m/z (%): 471.1 (100)
+
[
M+H] . HRMS (ESI) calculated for C H BrFN O 471.0832; found 471.0829.
2
2
21
4
2
4
.1.25. 4-((6-Bromo-2-isopropyl-3-(4-methoxybenzyl)-3H-imidazo[4,5-b]pyridin-7-yl)oxy)-3-
fluoroaniline 12B. The reaction was performed following procedure F on 6-bromo-7-chloro-
-isopropyl-3-(4-methoxybenzyl)-3H-imidazo[4,5-b]pyridine 11B (814 mg, 2.06mmol) in
2
DMSO (4 mL) to give the corresponding product 12B as a beige solid (550mg, 55 %). Mp:
1
1
17 – 119 °C. H NMR (300 MHz, CDCl ) δ = 8.37 (s, 1H), 7.06 (d, J = 8.6 Hz, 2H), 6.88 (t,
3
J = 8.8 Hz, 1H), 6.81 (d, J = 8.6 Hz, 2H), 6.40 (d, J = 3 Hz, 1H), 6.32 (d, J = 8.8 Hz, 1H),
1
3
5
.35 (s, 2H), 3.73 (s, 3H), 2.99 (m, 1H), 1.14 (d, J = 6.8 Hz, 6H). C NMR (75 MHz, CDCl )
3
δ = 160.6, 159.2,154.7 (d, J = 246.1 Hz), 151.0, 150.3, 146.0, 144.4 (d, J = 9.4 Hz), 136.7 (d,
J = 12.4 Hz), 128.5, 128.2, 126.7, 122.0 (d, J = 1.9 Hz), 114.3, 110.1 (d, J = 2.9 Hz), 104.1,
+
+
1
03.3 (d, J = 21.4 Hz), 55.3, 45.1, 27.2, 21.3. MS (ES ) m/z (%): 485.1 (100) [M+H] . HRMS
(
ESI) calculated for C H BrFN O 485.0988; found 485.0974.
2
3
23
4
2
4
.1.26. tert-Butyl(4-((6-bromo-3-(4-methoxybenzyl)-3H-imidazo[4,5-b]pyridin-7-yl)oxy)-3-
fluorophenyl)- carbamate 12C. The reaction was performed following procedure F on 6-
bromo-7-chloro-3-(4-methoxybenzyl)-3H-imidazo[4,5-b]pyridine 11C (1.4 g, 3.97mmol) in
DMSO (3 mL) to give the corresponding product 12C as a light red solid (1.1 g, 51 %). Mp:
1
1
7
2
1
62 – 164 °C. H NMR (300 MHz, CDCl ) δ = 8.50 (s, 1H), 7.73 (s, 1H), 7.50 – 7.39 (m, 1H),
3
.22 (d, J = 8.6 Hz, 2H), 7.08 (t, J = 8.8 Hz, 1H), 6.92 (d, J = 8.5 Hz, 1H), 6.85 (d, J = 8.6 Hz,
1
3
H), 6.57 (bs, 1H), 5.31 (s, 2H), 3.78 (s, 3H), 1.49 (s, 9H). C NMR (75 MHz, CDCl ) δ =
3
59.7, 153.4 (d, J = 296.2 Hz), 152.4, 148.8, 147.4, 143.1, 139.2 (d, J = 12.4 Hz), 136.2 (d, J
=
=
9.6 Hz), 129.4, 127.3, 126.7, 121.2 (d, J = 1.6 Hz), 114.4, 113.5 (d, J = 2.6 Hz), 107.2 (d, J
+
+
24.0 Hz), 104.4, 80.8, 55.3, 47.0, 28.3. MS (ES ) m/z (%): 545.1 (100) [M+H] . HRMS
(
ESI) calculated for C H N O BrF 543.1043; found 543.1042.
25 25 4 4
4
.1.27.
4-(7-(4-Amino-2-fluorophenoxy)-3-(4-methoxybenzyl)-2-ethyl-3H-imidazo[4,5-
b]pyridin-6-yl)-N,N-dimethylaniline 13Ac. The reaction was performed following procedure
G with double equivalents of catalyst and base on 4-((6-bromo-2-ethyl-3-(4-methoxybenzyl)-
3
H-imidazo[4,5-b]pyridin-7-yl)oxy)-3-fluoroaniline 12A (77 mg, 0.16 mmol) in 1,4-dioxane
(3 mL) to give the corresponding product 13Ac as a brown solid (68 mg, 78 %). Mp: 180 –
1
1
8
82 °C. H NMR (300 MHz, CDCl ) δ = 8.32 (s, 1H), 7.48 (d, J = 8.5 Hz, 2H), 7.13 (d, J =
3
.5 Hz, 2H), 6.42 – 6.30 (m, 5H), 6.37 (dd, J = 3, 8.4 Hz, 1H), 6.23 (d, J = 8.4 Hz, 1H), 5.37
1
3
(
s, 2H), 3.76 (s, 3H), 2.96 (s, 6H), 2.70 (q, J = 7.5 Hz, 2H), 1.19 (t, J = 7.5 Hz, 3H). C NMR
2
9