7-(Pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-based derivatives for kinase inhibition: Co-crystallisation studies with Aurora-A reveal distinct differences in the orientation of the pyrazole N1-substituent

Article abstract of DOI:10.1016/j.bmcl.2015.08.003

Introduction of a 1-benzyl-1H-pyrazol-4-yl moiety at C7 of the imidazo[4,5-b]pyridine scaffold provided 7a which inhibited a range of kinases including Aurora-A. Modification of the benzyl group in 7a, and subsequent co-crystallisation of the resulting analogues with Aurora-A indicated distinct differences in binding mode dependent upon the pyrazole N-substituent. Compounds 7a and 14d interact with the P-loop whereas 14a and 14b engage with Thr217 in the post-hinge region. These crystallographic insights provide options for the design of compounds interacting with the DFG motif or with Thr217.

Full text of DOI:10.1016/j.bmcl.2015.08.003

Bioorganic & Medicinal Chemistry Letters 25 (2015) 4203–4209
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
7-(Pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-based derivatives for
kinase inhibition: Co-crystallisation studies with Aurora-A reveal
distinct differences in the orientation of the pyrazole N1-substituent
a
b
a
Vassilios Bavetsias a, , Yolanda Pérez-Fuertes , Patrick J. McIntyre , Butrus Atrash ,
⇑
Magda Kosmopoulou c, Lisa O’Fee a, Rosemary Burke a, Chongbo Sun a, Amir Faisal a,y, Katherine Bush a,
Sian Avery a, Alan Henley a, Florence I. Raynaud a, Spiros Linardopoulos a,d, Richard Bayliss b,c,
,
⇑
Julian Blagg a,
⇑
a Cancer Research UK Cancer Therapeutics Unit at The Institute of Cancer Research, London, United Kingdom
b University of Leicester, Department of Biochemistry, Lancaster Road, Leicester LE1 9HN, United Kingdom
c Division of Structural Biology, The Institute of Cancer Research, London, United Kingdom
d Breakthrough Breast Cancer Research Centre at The Institute of Cancer Research, London, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Introduction of a 1-benzyl-1H-pyrazol-4-yl moiety at C7 of the imidazo[4,5-b]pyridine scaffold provided
7a which inhibited a range of kinases including Aurora-A. Modification of the benzyl group in 7a, and
subsequent co-crystallisation of the resulting analogues with Aurora-A indicated distinct differences in
binding mode dependent upon the pyrazole N-substituent. Compounds 7a and 14d interact with the
P-loop whereas 14a and 14b engage with Thr217 in the post-hinge region. These crystallographic insights
provide options for the design of compounds interacting with the DFG motif or with Thr217.
Ó 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
Received 9 June 2015
Revised 29 July 2015
Accepted 3 August 2015
Available online 6 August 2015
Keywords:
Aurora-A
Imidazo[4,5-b]pyridine
Aurora kinase
Over the last decade, extensive research has been directed
towards the discovery of small molecule inhibitors of the Aurora
protein kinases as anticancer agents. This effort led to the identifi-
cation of multiple structurally diverse chemotypes for Aurora
kinase modulation.1–5 In addition to small molecules inhibiting
all three Aurora isoforms A, B and C, several compounds displaying
Aurora isoform selectivity have also been reported including
AZD1152 which selectively inhibits Aurora-B,6 and the selective
Aurora-A inhibitor MLN8237.7 Several of these small-molecule
inhibitors (e.g., PHA-739358,8,9 AZD1152 and MLN8237) have pro-
gressed through preclinical development into clinical evaluation
for the treatment of a range of human malignancies.1–5
our work related to the discovery of 3, modification of the imi-
dazo[4,5-b]pyridine scaffold at C7 included the introduction of a
1-benzyl-1H-pyrazol-4-yl moiety that provided entry into 7-(pyra-
zol-4-yl)-3H-imidazo[4,5-b]pyridine-based derivatives. Herein, we
report our medicinal chemistry effort aimed at improving the
pharmacological profile for this class of compounds, and present
kinome profiling data that indicate promiscuous kinase inhibition
for this subseries. In addition, we report ligand/Aurora-A protein
crystallographic data that show different orientations for the sub-
stituent on the pyrazole ring suggesting that the 7-(pyrazol-4-yl)-
3H-imidazo[4,5-b]pyridine scaffold could be utilised for the design
of compounds that additionally interact with the DFG motif or with
Thr217, the latter tactic has been demonstrated to enhance Aurora-
A over Aurora-B selectivity.12
Synthesis of 7-substituted imidazo[4,5-b]pyridine derivatives
7a–e (Table 1) is shown in Scheme 1. Key intermediates 5a–e were
obtained via Suzuki reaction between 4-chloro-3-nitropyridin-2-
amine (4) and the requisite boronic acid or boronic acid pinacol
ester coupling partner. 1-(3,4-Difluorobenzyl)-, 1-(4-fluoroben-
zyl)-, and 1-(4-chlorobenzyl)-1H-pyrazole-4-boronic acid pinacol
esters were prepared by heating 4-(4,4,5,5-tetramethyl-1,3,2-diox-
aborolan-2-yl)-1H-pyrazole (11) with the appropriate benzyl
We have previously reported imidazo[4,5-b]pyridine-based
inhibitors of Aurora kinases including 1 (CCT137690),10 the dual
FLT3/Aurora kinase inhibitor 2 (CCT241736),11 and compound 3
which selectively inhibits Aurora-A over Aurora-B12 (Fig. 1). In
⇑
Corresponding authors. Tel.: +44 (0)20 87224158 (V.B.), +44 (0)116 2297100
(R.B.), +44 (0)20 87224051 (J.B.).
E-mail addresses: vassilios.bavetsias@icr.ac.uk (V. Bavetsias), richard.bayliss@
leicester.ac.uk (R. Bayliss), julian.blagg@icr.ac.uk (J. Blagg).
y
Current address: SBA School of Science and Engineering, Lahore University of
Management Sciences, D.H.A., Lahore, Pakistan.
http://dx.doi.org/10.1016/j.bmcl.2015.08.003
0960-894X/Ó 2015 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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V. Bavetsias et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4203–4209
Figure 1. Imidazo[4,5-b]pyridine-based inhibitors of Aurora kinases.
Table 1
C7-Pyrazole modifications
The 2-amino-3-nitro-pyridine derivative 9 (Scheme 2), key
intermediate for the synthesis of 14a and 14b (Table 2), was
obtained from 4-chloro-3-nitropyridin-2-amine (4) by a Suzuki
cross-coupling reaction to (1-(3-(methoxycarbonyl)benzyl)-1H-
pyrazol-4-yl)boronic acid pinacol ester, prepared by reacting 11
with methyl 3-(bromomethyl)benzoate13, followed by C5-pyridine
chlorination with N-chlorosuccinimide (Scheme 2). An attempt to
prepare 10a directly from the methyl ester intermediate 9 upon
treatment with dimethylamine under microwave irradiation
resulted in formation of the corresponding carboxylic acid.
Coupling of this acid with dimethylamine via HATU carboxyl acti-
vation provided 10a (Scheme 2). Starting from the methyl ester
intermediate 9, access to 10b was achieved by alkaline ester
hydrolysis followed by HATU-mediated coupling with
1-methylpiperazine (Scheme 2). Imidazo[4,5-b]pyridine ring
formation to afford 14a and 14b (Table 2) was effected by reacting
10a and 10b with 1,3-dimethyl-1H-pyrazole-4-carbaldehyde in
the presence of Na2S2O4 as previously described.11,12,14 The ortho-
substituted derivative 14c (Table 2) was prepared by a procedure
analogous to that described for its meta-isomer 14b (Scheme 2,
Table 2).
Access to 2-amino-3-nitro-pyridine derivative 13 (Scheme 3),
key intermediate for the preparation of 14e–g, was achieved by
alkylation of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
pyrazole (11) with 3-(bromomethyl)benzaldehyde13 followed by
a Suzuki cross-coupling reaction with 4-chloro-3-nitropyridin-2-
amine (4) (Scheme 3). Reductive amination of the benzaldehyde
moiety in 13 was accomplished under standard conditions (NaBH
(OAc)3/AcOH with methylamine or pyrrolidine, and NaBH3CN with
dimethylamine). The synthesis of 14e–g (Table 2) was finalised by
the C5-pyridine chlorination of the requisite intermediate followed
by imidazo[4,5-b]pyridine ring formation as described for the
preparation of 7a–e (Scheme 1). Finally, the isoxazole derivative
14d (Table 2) was prepared from 4 by a route analogous to that
described for the synthesis of 7a–e (Scheme 1). For this synthetic
Compd
R
Aurora-A
IC50 M)
Aurora-B IC50 MLM/HLM%
M) met/30 min
(l
(l
7a
7b
0.212 0.107 0.461 0.147 40/43
0.182
0.322
0.347
0.458
52/39
31/28
7c
7d
7e
0.190
>10
0.271
>10
37/16
31/20
IC50s are mean values of two independent determinations or mean ( SD) for n > 2.17
MLM/HLM: percentage of compound metabolised after a 30 min incubation.18
bromide at 80 °C for 2–3 h in acetonitrile in the presence of Cs2CO3,
reaction conditions previously reported for the synthesis of 1-ben-
zyl-3-heterocyclic pyrazoles.13 (1-Benzyl-1H-pyrazol-4-yl)boronic
acid and (1-(4-fluorophenyl)-1H-pyrazol-4-yl)boronic acid,
required for the synthesis of 5a and 5e respectively, were commer-
cially available. N-chlorosuccinimide-mediated C5-pyridine
chlorination was followed by reaction with 1,3-dimethyl-1H-pyra-
zole-4-carbaldehyde in the presence of Na2S2O4, as previously
described,11,12,14 to afford the imidazo[4,5-b]pyridine derivatives
7a–e (Scheme 1, Table 1).
sequence,
(1-((5-methylisoxazol-3-yl)methyl)-1H-pyrazol-4-yl)
boronic acid pinacol ester, required for the Suzuki cross-coupling
reaction, was prepared by alkylation of 4-(4,4,5,5-tetramethyl-
Scheme 1. Reagents and conditions: (a) boronic acid or boronic acid pinacol ester, THF/H2O, Pd(dppf)Cl2, Na2CO3, 80 °C, 3.5–26 h or DME, Pd(dppf)Cl2, 1 M aqueous Na2CO3,
150 °C, microwave irradiation, 15 min (for the introduction of 1-(4-fluorophenyl)-1H-pyrazol-4-yl); (b) N-chlorosuccinimide, CH3CN, reflux, 3.5–7 h; (c) EtOH, 1,3-dimethyl-
1H-pyrazole-4-carbaldehyde, 1 M aqueous Na2S2O4, 80 °C, 16–20 h.

V. Bavetsias et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4203–4209
4205
Scheme 2. Reagents and conditions: (a) (1-(3-(methoxycarbonyl)benzyl)-1H-pyrazol-4-yl)boronic acid pinacol ester, DMF/H2O, Pd(dppf)Cl2, Na2CO3, 120 °C, microwave
irradiation, 0.5 h; (b) N-chlorosuccinimide, CH3CN, reflux, 3.5 h; (c) for 10a: (i) 2 M Me2NH in THF, 105 °C, microwave irradiation, 37 h then remove volatiles; (ii) DMF, HATU,
i
iPr2NEt, room temp, 1.7 h then 2 M Me2NH in THF, room temp, 2.3 h; for 10b: (i) 1 M aqueous KOH, MeOH, 50 °C, 4.5 h; (ii) DMF, HATU, Pr2NEt, room temp, 1 h then 1-
methylpiperazine, room temp, 6 h.
1,3,2-dioxaborolan-2-yl)-1H-pyrazole (11) with 3-(bromomethyl)-
5-methylisoxazole.13
Table 2
Extended C7-pyrazole modifications
As part of our medicinal chemistry programme to discover
selective inhibitors of Aurora-A,12 we introduced the 1-benzyl-
1H-pyrazol-4-yl moiety at C7 of the imidazo[4,5-b]pyridine
scaffold to provide 7a (Table 1). We previously reported
1,3-dimethyl-1H-pyrazol-4-yl as a preferred substituent at the C2
position of the imidazo[4,5-b]pyridine scaffold11 and this moiety
was therefore incorporated into the design of 7a and analogues
presented in this study. In biochemical assays, 7a inhibited both
Compd
R
Aurora-A Aurora-B MLM/HLM
Aurora-A and Aurora-B with IC50 values of 0.212 and 0.461 lM,
IC50
(lM) IC50
(
lM) % met/
respectively. A slightly improved level of target inhibition was
observed in HeLa cervical cancer cells where 7a inhibited the
autophosphorylation of Aurora-A at T288 (a cell-based biomarker
30 min
for Aurora-A inhibition)12,15,16 with an IC50 value of 0.087
lM
14a
14b
0.036
0.046
0.296
0.235
72/72
97/87
and histone H3 phosphorylation at S10 (a cell-based biomarker
for Aurora-B inhibition)12,15,16 with an IC50 value of 0.223
lM.
Regarding cell growth inhibition, 7a inhibited the growth of
SW620 and HCT116 human colon carcinoma cells (GI50 = 0.18
and 0.15 l
M, respectively).19 Compound 7a displayed moderate
mouse and human liver microsomal stability (40% and 43% meta-
bolised, respectively, after a 30 min incubation). This in vitro pro-
file prompted us to investigate this C7-pyrazole substituted
imidazo[4,5-b]pyridine subseries in more detail. Firstly, we
attempted to increase biochemical potency against Aurora-A, and
also to improve microsomal stability.
14c
0.126
0.354
98/82
Introduction of a p-F substituent in the phenyl ring of 7a pro-
vided compound 7b which displayed a similar Aurora-A/B inhibi-
tory profile, and human liver microsomal stability to that of 7a.
No improvement in Aurora-A inhibitory potency was observed
with the introduction of an additional fluorine (compound 7c,
Table 1), however the p-chlorobenzyl counterpart (compound 7d,
Table 1) displayed similar Aurora inhibitory activities to that of
7b with improved human liver microsomal stability (16% metabo-
lised after 30 min incubation with human liver microsomes). For a
better understanding of the microsomal stability data, the protein
binding for 7a, 7d and 7e in microsomal incubations was mea-
sured. The percentage of 7a unbound in MLM and HLM was deter-
mined as 6.71 and 4.89, respectively. Compounds 7d and 7e were
more highly bound: for 7d the percentage unbound was 0.50 in
both MLM and HLM and for 7e was 0.16 and 0.10, respectively.
Based on these data, it is possible that the observed improvement
in HLM metabolic stability for 7d compared to 7a is due to lower
fraction unbound for the former; notably 7a and 7d display similar
14d
14e
0.035
0.014
0.075
0.017
35/37
97/95
14f
0.017
0.018
0.031
0.032
100/92
53/38
14g
IC50s are mean values of two independent determinations or mean ( SD) for n > 2.17
MLM/HLM: percentage of compound metabolised after a 30 min incubation.18

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V. Bavetsias et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4203–4209
Scheme 3. Reagents and conditions: (a) 3-(bromomethyl)benzaldehyde, CH3CN, Cs2CO3, 80 °C, 2 h; (b) THF/H2O, Pd(dppf)Cl2, Na2CO3, 80 °C, 2 h.
MLM stabilities despite the lower fraction unbound for 7d. In HeLa
cells, 7d inhibited both Aurora-A (p-T288 IC50 = 0.040 M) and
Aurora-B (p-HH3 IC50 = 0.330 M); potencies similar to those
l
l
observed with compound 7a. The direct attachment of a p-fluo-
rophenyl group to the N1-pyrazole was detrimental to Aurora inhi-
bition, with compound 7e inhibiting Aurora-A and Aurora-B with
IC50 values greater than 10 lM (Table 1). Cell growth inhibition
studies revealed that compounds 7b–d maintained the inhibitory
potencies seen with 7a; for example, 7d inhibited the growth of
SW620 and HCT116 cells (GI50 = 0.26 and 0.24
Likewise, 7b and 7c showed potent HCT116 cell growth inhibition
(GI50 = 0.20 and 0.13 M, respectively). We postulated that potent
lM, respectively).
l
cell growth inhibitory activity relative to biochemical Aurora-A
and Aurora-B modulation may be attributable to gain of off-target
kinase inhibition in this sub-series, although we acknowledge that
translation to more potent cellular inhibition of Aurora-A versus
Aurora-A biochemical potency may also be a contributing factor.
The potent cell-based activity prompted us to investigate kinome
selectivity profiles for this class of compound by screening 7a
Figure 2. Imidazo[4,5-b]pyridine-based inhibitors of Aurora kinases co-crystallised
with Aurora-A: compound 15: PDB ID 2X6D;10 compound 16: PDB ID 4B0G.11
low oral bioavailability. In an attempt to improve the aqueous sol-
ubility, the Aurora inhibitory potency and kinase selectivity of this
sub-series, we explored the introduction of basic substituents such
as 1-methylpiperazine and pyrrolidine as well as replacement of
the phenyl ring with a more polar heterocycle. This approach
was guided by the ligand/protein interactions observed in the pro-
tein crystal structure of 7a, which showed that the N-benzyl sub-
stituent on the C7-pyrazole is oriented towards the P-loop
(Fig. 4). It was anticipated that the introduction of small sub-
stituents on the phenyl ring in 7a would be well tolerated without
altering its orientation in the kinase active site. Alternatively, we
envisaged that Thr217 in Aurora-A may be accessed via an appro-
priate pyrazole N1-benzyl derivatisation such as a bulky amido
substituent, which would form a favourable hydrogen bond inter-
action with the side chain hydroxyl of Thr217. Similar approaches
were previously applied by us in the design of selective inhibitors
of Aurora-A.12,16
and 7d in a 102-kinase panel at a concentration of 1 l
M.20
Indeed, both compounds inhibited a range of kinases including
ERK8, GSK3b, MLK1, JAK2, TrkA and VEGFR greater than 80%
(Table S1, Supplementary data) with Gini coefficients21 of 0.273
for 7a and 0.364 for 7d. These Gini coefficient values are signifi-
cantly lower to those of previously reported 7-(piperazin-1-yl)-
3H-imidazo[4,5-b]pyridine- and 7-phenoxy-3H-imidazo[4,5-b]
pyridine-based inhibitors of Aurora kinases.10,12 The Gini coeffi-
cient for compound 3 was reported as 0.719,12 and the correspond-
ing value for compound 1 is 0.560. To understand the potential for
further improvement, we obtained the crystal structure of 7a
bound to Aurora-A22 (Fig. 3, Table S3, Supplementary data). This
structure shows the ligand in the ATP binding site with the pyri-
dine nitrogen atom hydrogen bonded to the backbone NH of
Ala213 and the imidazole NH interacting with the carbonyl of
Ala213, consistent with previous reports of the hinge binding
mode for the imidazo[4,5-b]pyridine scaffold.10–12 Similar to crys-
tal structures of previous compounds 1, 15 and 16 (Figs. 1 and 2;
compounds 51 and 40c in Ref. 10, and compound 21a in Ref. 11,
respectively) the N-benzyl substituent on the C7-pyrazole is ori-
ented towards the P-loop, making Van der Waals contacts with
Val147 and Gly142 (Fig. 3).
In vivo mouse pharmacokinetic profiling of 7d revealed low oral
bioavailability (16%) with moderate clearance (0.016 L/h, 13.3 mL/
min/kg) and volume of distribution (0.02 L, 1.0 L/kg). Similar phar-
macokinetic parameters were observed for 7a with low oral
bioavailability (13%), moderate clearance (0.012 L/h, 10.0 mL/
min/kg) and volume of distribution (0.01 L, 0.5 L/kg). The mouse
plasma protein binding for 7a and 7d was determined as 99.48%
and >99.9%, respectively. We determined low kinetic solubility
for both 7a and 7d (<0.0001 mg/mL in phosphate buffer,
pH = 6.8)23 which may be a contributing factor to the observed
Replacement of the phenyl ring in 7a with 5-methylisoxazole
(compound 14d) led to a significant improvement in Aurora inhibi-
tion in our biochemical assays (Aurora-A IC50 = 0.035
IC50 = 0.075 M; Table 2) but promiscuous kinase inhibition
remained. In a panel of 105 kinases at a compound concentration
of 1 M, 22 proteins including ERK8, MLK1, JAK2, TrkA and
lM, Aurora-B
l
l
VEGFR were inhibited by P80% (Gini coefficient = 0.320,
Table S2, Supplementary data). Compound 14d inhibited the
growth of SW620 and HCT116 cells with GI50 values of 0.35 and
0.34 lM respectively, similar to the cell growth inhibition
observed with 7d. The crystal structure of 14d bound to Aurora-
A22,24,25 (Fig. 5, Table S3, Supplementary data) shows a similar
binding mode to that adopted by 7a; however, the P-loop of
Aurora-A adopts a different conformation, stabilised by interac-
tions with the 5-methylisoxazole; this P-loop conformation is very
similar to that previously observed for compound 1 (compound 51
in Ref. 10).

V. Bavetsias et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4203–4209
4207
Figure 3. Crystal structure of 7a bound to Aurora-A at 3.1 Å resolution. (A) The ATP binding site viewed from above. The P-loop has been removed to provide a clear view of
the ligand, shown with carbon atoms coloured yellow, and the surrounding protein, shown with carbon atoms coloured teal. The final 2mFo-DFc electron density map is
shown as a wire-mesh, contoured at 1
r. (B) Side view with the P-loop shown and key interacting residues labelled. (C) Crystal structure of 16 (compound 21a in Ref. 11;
carbon atoms coloured white) bound to Aurora-A with the P-loop shown and key interacting residues labelled. Structure taken from the Protein Data Bank (PDB) ID 4B0G.
Figure 4. Comparison of crystal structure of Aurora-A bound to 7a and published crystal structures from this series. (A) Overlay of structures showing superposed protein
backbones (all coloured teal) with 7a (carbon atoms coloured yellow), and published compounds 15, 1 and 16 taken from PDB IDs 2X6D, 2X6E and 4B0G, respectively. (B–E)
Magnified view of the interactions between compounds 7a, 15, 1 and 16 respectively and the P-loop with the protein surface shown.
Figure 5. Crystal structure of 14d bound to Aurora-A at 3.1 Å resolution. (A) The ATP binding site viewed from above. The P-loop has been removed to provide a clear view of
the ligand, shown with carbon atoms coloured orange and the surrounding protein, shown with carbon atoms coloured teal. The final 2mFo-DFc electron density map is
shown as a wire-mesh, contoured at 1
r. (B) Side view with the P-loop shown and key interacting residues labelled. (C) Overlay of the structures of Aurora-A bound to
compound 14d (coloured as before) and compound 1 (compound and protein coloured grey).
The m-dimethylbenzamide derivative 14a (Table 2) was a more
potent inhibitor of Aurora-A compared to 7a but its human liver
microsomal stability was lower (72% metabolised after a 30 min
stability in both mouse and human liver microsomes (Table 2).
Both 14a and 14b were more potent (by 8- and 5-fold, respec-
tively) inhibitors of Aurora-A relative to Aurora-B (Table 2). The
crystal structures of 14a and 14b bound to Aurora-A22,24,25 were
determined (Table S3, Supplementary data; Figs. 6 and 7) and
revealed a consistent difference in the binding mode of these
incubation).
A similar trend was observed with the m-1-
methylpiperazine derivative 14b which was also a more potent
inhibitor of Aurora-A compared to 7a but with poor metabolic

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V. Bavetsias et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4203–4209
Figure 6. Crystal structure of 14a bound to Aurora-A at 2.8 Å resolution. (A) The ATP binding site viewed from above. The P-loop has been removed to provide a clear view of
the ligand, shown with carbon atoms coloured pink and the surrounding protein, shown with carbon atoms coloured teal. The final 2mFo-DFc electron density map is shown
as a wire-mesh, contoured at 1
r
. (B) Side view with the P-loop shown and Ala213, Thr217 labelled. (C) Modelling a glutamic acid in position 217 (Glu*), as found in Aurora-B,
leads to steric clashes with the ligand, modelled clash shown as red dashes.
Figure 7. Crystal structure of 14b bound to Aurora-A at 2.9 Å resolution. (A) The ATP binding site viewed from above. The P-loop has been removed to provide a clear view of
the ligand, shown with carbon atoms coloured brown and the surrounding protein, shown with carbon atoms coloured teal. The final 2mFo-DFc electron density map is
shown as a wire-mesh, contoured at 1
found in Aurora-B, leads to steric clashes with the ligand, modelled clash shown as red dashes.
r
. (B) Side view with the P-loop shown and Ala213, Thr217 residues labelled. (C) Modelling a glutamic acid in position 217 (Glu*), as
ligands when compared to 7a and 14d. Bulkier substituents on the
pyrazole group no longer interact with the pocket formed by the
two b-strands of the P-loop. Instead, these bulkier groups are posi-
tioned between the b1 strand of the P-loop and the post-hinge
region. This orientation places them in close proximity to Thr
217, with a minimum interaction distance of 3.8 Å, which may
explain the selectivity of these compounds towards Aurora-A.
Notably, Aurora-B and Aurora-C have a glutamic acid at the equiv-
alent position to Thr217, and computational modelling of this Thr
to Glu replacement suggests that the bulkier R-groups of 14a and
14b may invoke a steric clash with a glutamic acid at position
217 (Fig. 6C, Fig. 7C).12
The o-regioisomer of 14b (compound 14c) displayed a similar
profile to that of 14b although its potency against Aurora-A was
marginally lower and selectivity over Aurora-B was eroded
(Table 2). The introduction of the smaller m-dimethylamino basic
substituent (compound 14e) led to potent inhibition of both
Aurora-A and Aurora-B although mouse and human liver microso-
mal stability was poor (Table 2). Likewise, the m-pyrrolidine
at a concentration of 1 lM revealed 30 kinases including ERK8,
GSK3b, MLK1, JAK2, TrkA and VEGFR that were inhibited by
P80% (Gini coefficient = 0.267, Table S2, Supplementary data).
Based on these findings, our interest in this 7-(pyrazol-4-yl)-3H-
imidazo[4,5-b]pyridine-based sub-series was discontinued and
our efforts focussed on compound 3 (Fig. 1) as a highly selective
inhibitor of Aurora-A kinase.12 However, the kinase inhibitory pro-
files of compounds 7a, 7d, 14d, and 14g suggest that the 7-(pyra-
zol-4-yl)-3H-imidazo[4,5-b]pyridine scaffold could serve as the
basis for a multitargeted kinase inhibitor design. In addition, the
ligand/Aurora-A protein crystallographic data indicate distinct dif-
ferences in the orientation of the pyrazole N-substituent. The phe-
nyl group in 7a and the 5-methylisoxazole in 14d point towards
the P-loop, whereas the bulkier benzamido substituted phenyl
rings in 14a and 14b position between the b1 strand of the P-loop
in close proximity to Thr217 and also to the DFG motif. These
ligand/protein crystallographic insights could therefore be further
exploited in the design of compounds interacting with the DFG
motif or in enhancing the selectivity for Aurora-A inhibition over
Aurora-B.
derivative 14f potently inhibited both Aurora-A (IC50 = 0.017
lM)
and Aurora-B (IC50 = 0.031 M) but was also highly metabolised
l
(Table 2). The potent Aurora-A and Aurora-B activity of these com-
pounds suggests that they do not adopt a binding mode involving
interaction with Thr217 in Aurora-A similar to 14a and 14b, but
might bind similarly to 7a and 14d. Unfortunately, we have been
unable to resolve the binding mode of compounds 14e and 14f
by co-crystal structure determination to verify this hypothesis.
The m-methylamino counterpart of 14e (compound 14g) also dis-
Acknowledgments
This work was supported by CRUK Grant numbers C309/A8274
and C309/A11566. S.L. is also supported by Breakthrough Breast
Cancer. R.B. acknowledges the support of
Research Fellowship, Breakthrough Breast Cancer Project Grant
AURA 05/06 and MRC CASE studentship MR/K016903/1. We
acknowledge NHS funding to the NIHR Biomedical Research
Centre at The Institute of Cancer Research and the Royal Marsden
NHS Foundation Trust. We also thank Dr Amin Mirza, Mr Meirion
Richards, Dr Maggie Liu for their assistance with NMR, mass spec-
trometry and HPLC, and Dr Charlotte Dodson for Aurora-A protein
a Royal Society
played potent inhibition of both Aurora-A (IC50 = 0.018
lM) and
Aurora-B (IC50 = 0.032 M) and demonstrated improved metabolic
l
stability compared to 14e and 14f (Table 2). Compound 14g inhib-
ited the growth of SW620 and HCT116 cells (GI50 = 0.34 and
0.23 lM, respectively); however, profiling in a 105-kinase panel

V. Bavetsias et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4203–4209
4209
Valenti, M.; de Haven Brandon, A.; Raynaud, F. I.; Workman, P.; Eccles, S. A.;
Bayliss, R.; Linardopoulos, S.; Blagg, J. J. Med. Chem. 2012, 55, 8721.
12. Bavetsias, V.; Faisal, A.; Crumpler, S.; Brown, N.; Kosmopoulou, M.; Joshi, A.;
Atrash, B.; Pérez-Fuertes, Y.; Schmitt, J. A.; Boxall, K. J.; Burke, R.; Sun, C.; Avery,
S.; Bush, K.; Henley, A.; Raynaud, F. I.; Workman, P.; Bayliss, R.; Linardopoulos,
S.; Blagg, J. J. Med Chem. 2013, 56, 9122.
production. We thank Diamond Light Source for beamtime and the
staff of beamlines I03 and I04 for assistance with data collection.
Supplementary data
13. Curtis, M. P.; Sammons, M. F.; Piotrowski, D. W. Tetrahedron Lett. 2009, 50,
5479.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.08.
003.
14. Yang, D.; Fokas, D.; Li, J.; Yu, L.; Baldino, C. M. Synthesis 2005, 47.
15. Manfredi, M. G.; Ecsedy, J. A.; Meetze, K. A.; Balani, S. K.; Burenkova, O.; Chen,
W.; Galvin, K. M.; Hoar, K. M.; Huck, J. J.; LeRoy, P. J.; Ray, E. T.; Sells, T. B.;
Stringer, B.; Stroud, S. G.; Vos, T. J.; Weatherhead, G. S.; Wysong, D. R.; Zhang,
M.; Bolen, J. B.; Claiborne, C. F. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 4106.
16. Bouloc, N.; Large, J. M.; Kosmopoulou, M.; Sun, C.; Faisal, A.; Matteucci, M.;
Reynisson, J.; Brown, N.; Atrash, B.; Blagg, J.; McDonald, E.; Linardopoulos, S.;
Bayliss, R.; Bavetsias, V. Bioorg. Med. Chem. Lett. 2010, 20, 5988.
17. Aurora-A and Aurora-B IC50 values were determined by using a microfluidic
assay which monitors the separation of a phosphorylated product from its
substrate, as described in Ref. 12.
18. MLM and HLM microsomal stabilities were determined as described in Ref. 12.
19. GI50 values were determined after 72 h incubation as described in the
following reference: Chan, F.; Sun, C.; Perumal, M.; Nguyen, Q.-D.; Bavetsias,
V.; McDonald, E.; Martins, V.; Wilsher, N.; Raynaud, F. I.; Valenti, M.; Eccles, S.;
te Poele, R.; Workman, P.; Aboagye, E. O.; Linardopoulos, S. Mol. Cancer Ther.
2007, 6, 3147.
References and notes
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M.; Stroud, S. G.; Chen, W.; Shinde, V.; Huck, J. J.; Wysong, D. R.; Janowick, D. A.;
Hyer, M. L.; LeRoy, P. J.; Gershman, R. E.; Silva, M. D.; Germanos, M. S.; Bolen, J.
B.; Claiborne, C. F.; Sells, T. B. Clin. Cancer Res. 2011, 17, 7614.
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10. Bavetsias, V.; Large, J. M.; Sun, C.; Bouloc, N.; Kosmopoulou, M.; Matteucci, M.;
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20. Kinase selectivity profiling for compounds 7a, 7d, 14d and 14g was carried out
at the International Centre for Protein Kinase Profiling, Division of Signal
Transduction Therapy, University of Dundee.
21. Graczyk, P. P. J. Med. Chem. 2007, 50, 5773.
22. The crystal structure of Aurora-A in complex with compound 7a was
determined as in Ref. 12. Other co-crystal structures used a mutant form of
Aurora-A kinase domain (C290A, C393A), which has enhanced expression and
generates superior crystals (Refs. 24,25). Hexagonal bipyramidal shaped crystals
of Aurora
A
C290A,C393A were grown at 18 °C by hanging drop vapour
diffusion in a 1
l
L + 1 L mixture of mother liquor (0.1 M Tris, pH 8.9; 0.2 M
l
lithium sulfate; 30% w/v PEG 400) and Aurora A (12 mg/mL, 1 mM inhibitor
compound, 5 mM MgCl2). Crystals were cryoprotected using 25% (v/v) ethylene
glycol in mother liquor before cryo-cooling in liquid nitrogen. The structure
was solved using molecular replacement with PDB:4CEG as the model and
refinement was carried out as described in Ref. 12. Coordinates and structure
factors have been deposited in the protein data bank with accession codes
5AAD, 5AAE, 5AAF, 5AAG (corresponding to the Aurora-A co-crystal structures
of 7a, 14d, 14a and 14b, respectively).
23. Solubility measurements were performed by PharmorphixÒ Solid State
Services, Member of the Sigma–Aldrich Group, Cambridge, UK.
24. Rowan, F. C.; Richards, M.; Bibby, R. A.; Thompson, A.; Bayliss, R.; Blagg, J. ACS
Chem. Biol. 2013, 8, 2184.
25. Burgess, S. G.; Bayliss, R. Acta Crystallogr. F Struct. Biol. Commun. 2015, 71, 315.