Hit generation and exploration: Imidazo[4,5-b]pyridine derivatives as inhibitors of Aurora kinases

Article abstract of DOI:10.1016/j.bmcl.2007.09.076

A hit generation and exploration approach led to the discovery of 31 (2-(4-(6-chloro-2-(4-(dimethylamino)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)-N-(thiazol-2-yl)acetamide), a potent, novel inhibitor of Aurora-A, Aurora-B and Aurora-C kinases

Full text of DOI:10.1016/j.bmcl.2007.09.076

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6567–6571
Hit generation and exploration: Imidazo[4,5-b]pyridine
derivatives as inhibitors of Aurora kinases
*
´
Vassilios Bavetsias, Chongbo Sun, Nathalie Bouloc, Johannes Reynisson, Paul Workman,
Spiros Linardopoulos and Edward McDonald*
Department of Chemistry, Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research,
Cancer Research UK Laboratory, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK
Received 8 August 2007; revised 21 September 2007; accepted 23 September 2007
Available online 22 October 2007
Abstract—A hit generation and exploration approach led to the discovery of 31 (2-(4-(6-chloro-2-(4-(dimethylamino)phenyl)-3H-
imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)-N-(thiazol-2-yl)acetamide), a potent, novel inhibitor of Aurora-A, Aurora-B and Aur-
ora-C kinases with IC50 values of 0.042, 0.198 and 0.227 lM, respectively. Compound 31 inhibits cell proliferation and has good
microsomal stability.
Ó 2007 Elsevier Ltd. All rights reserved.
The Aurora proteins A, B and C are serine/threonine ki-
nases that play a key role in the regulation of mitosis,
and are implicated in cancer initiation and progres-
sion.1,2 Aurora-A and Aurora-B are overexpressed in a
broad range of tumours1,2 including breast,3 colorectal,4
testis,5 ovarian6 and glioma.7 In addition, Aurora-A can
transform cells when ectopically expressed in vitro.4 The
Aurora proteins have therefore emerged as attractive
anticancer targets for the development of small molecule
inhibitors as cancer therapeutic agents.
allel, we initiated a hit generation programme by pro-
ducing a small, kinase-focused piperazinylquinazoline
library via SNAr substitution reactions on 4-chloro-
6,7-dimethoxyquinazoline (Scheme 1 and Table 1).
Members of this library (Table 1) displayed Aurora-A
inhibitory activity, with compound 2f being the most
potent inhibitor (IC50 = 5.9 lM, Table 1). Despite the
modest levels of inhibition observed, we were encour-
aged by the identification of the (piperazin-1-yl)-N-(thia-
zol-2-yl)acetamide substituent as a novel Aurora kinase
inhibitor motif, which might be utilised in hit
exploration.
Several structurally diverse inhibitors of Aurora kinases
with anti-tumour activity have been reported including
quinazoline ZM447439,8 VX-6809 and the tetrahydro-
pyrrolo[3,4-c]pyrazole PHA-680632.10,11 More recently
inhibitors selective for Aurora-A kinase (MLN805412),
and for Aurora-B (pyrazoloquinazoline AZD115213),
have been reported but it is not yet clear what is the ideal
profile for therapeutic use. In this paper, we describe a
novel class of imidazo[4,5-b]pyridines with activity
against Aurora kinases.
From the HTS campaign the imidazo[4,5-b]pyridine 3
was identified (Fig. 1) as a hit. Compound 3 inhibited
Aurora-A with an IC50 value of 0.57 lM, whilst its 6-
H counterpart 4 (Fig. 1) was considerably less potent
with an IC50 value of 4.3 lM, highlighting the 6-halo
substituent as an important contributor to potency
and an area of focus for further SAR work.
Our main approach was based on high-throughput
screening (HTS) of our in-house compound library
against recombinant human Aurora-A kinase.14 In par-
Keywords: Imidazo[4,5-b]pyridines; Aurora; Kinases.
*
Corresponding authors. Tel.: +44 20 87224294; fax: +44 20
87224047; e-mail addresses: Vassilios.Bavetsias@icr.ac.uk;
Ted.McDonald@icr.ac.uk
Scheme 1. Reagent and condition: (a) isopropyl alcohol, 105 °C, 7 h.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.076

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V. Bavetsias et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6567–6571
Table 2. Effect of N4 and N3–H on Aurora-A inhibition
Table 1. Piperazinylquinazoline library: inhibition of Aurora-A
Compound
Structure
Aurora-A IC50 (lM)
4
4.3 ( 0.29)
7
8
9
12.5, 35% at 10 lM
8% at 100 lM
Compound
R
Aurora-A IC50 (lM)
2a
76
2b
2c
2d
2e
2f
na
26% at 100 lM
na
Results shown are mean values for samples run in triplicate. The IC50
value for 4 is the mean of three independent IC50 determinations.
98
Table 3. Effect of substitution in aryl substituent
16.5
5.9
Compound
X
Aurora-A, IC50 (lM)
4
p-NMe2
4.3 ( 0.29)a
10.0a
10
11
12
13
14
15
16
17
m-NMe2
o-NMe2
p-OMe
20% at 10 lMb
6.6 ( 3.18)a
16.0a
2g
2h
2i
na
40
35
na
25
9.6
m-OMe
o-OMe
29b
p-Pyrrolidin-1-yl
p-Pyrid-2-yl
p-CN
4.6a
4.4a
38% at 10 lMb
a Mean of two independent IC50 determinations or mean ( SD) for
n > 2.
b Results are mean values for samples run in triplicate.
2k
2l
(Scheme 2).15 Alternatively, compounds of this type
(examples 10, 11, and 18) could be produced by the cyc-
lisation of 2,3-diaminopyridines with benzoic acids in
POCl3 (Scheme 2).16 The N3-ethyl derivative 9 (Table
2) was obtained by reacting 3-amino-2-ethylaminopyri-
dine with 4-(dimethylamino)benzaldehyde in nitroben-
zene. Compound 31 (Table 5) was prepared from the
corresponding 2-amino-3-nitropyridine precursor and
4-(dimethylamino)benzaldehyde via a reductive cyclisa-
tion,17 following a procedure reported by Yang et al.18
This reductive cyclisation methodology was also applied
to prepare compound 19 (Table 4) from 2-amino-3-
nitro-5-trifluoromethylpyridine.19
2m
Results shown are mean values for samples run in triplicate. The value
for 2f is the mean of two independent IC50 determinations. na, no
activity at 100 lM.
Having confirmed the activity of 3 and 4, we embarked
on a hit-to-lead exploration programme focusing firstly
on the importance of N4 and N3–H for inhibiting the en-
zyme, and then in turn the 2-aryl moiety, 6-substituent,
7-substituent and 6,7-combinations. Our aim was to ex-
plore SAR trends and identify lead series for further
optimisation.
Figure 1. Inhibition of Aurora-A by compounds 3 and 4.
Access to 3, 4 and compounds presented in Tables 2–5
was gained mainly by the oxidative condensation of
2,3-diaminopyridines with aldehydes in nitrobenzene
The importance of the pyridine N and the imidazole NH
was investigated by preparing the imidazo[4,5-c]pyridine
7, the benzimidazole analogue 8 and the N-ethyl deriva-

V. Bavetsias et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6567–6571
Table 4. Effect of substituent at 6-position
6569
Scheme 2. Reagents and conditions: (a) aryl aldehyde, nitrobenzene,
150 °C, overnight; (b) POCl3, aryl carboxylic acid, 120 °C, 6 h.
Compound
X (6-substituent)
Aurora-A IC50 (lM)
3
Cl
0.57 ( 0.03)a
4.3 ( 0.29)a
0.49 ( 0.24)a
0.74a
4
H
Br
tant role in binding to Aurora-A. A requirement for a
hydrogen bonding interaction with the hinge region of
the kinase is a plausible explanation, as this is commonly
observed with ATP competitive inhibitors of kinases.20
Docking studies based on the crystal structure of Aur-
ora-A in complex with adenosine21,22 confirmed that 3
can be accommodated at the ATP-binding site with
bidentate H-bonds to Ala 213 in the hinge region of
Aurora-A.
18
19
20
CF3
Me
6.9a
21
22
23
20b
33% at 100 lMb
18,b 31% at 10 lMb
Investigation of the positional effect of the NMe2 substi-
tuent revealed that the para-isomer (compound 4) was a
slightly more potent inhibitor of the enzyme than the
meta-isomer 10, and both are significantly more potent
than the ortho-isomer 11. A similar trend was observed
with the OMe substituent (Table 3, compounds 12–
14). In addition to OMe, pyrrolidin-1-yl and pyrid-2-yl
substituents were well tolerated (Table 3, compounds
12, 15, 16).
24
9.0a
25
26
28% at 10 lMb
34% at 10 lMb
a Mean of two independent IC50 determinations or mean ( SD) for
n > 2.
b Results are mean values for samples run in triplicate.
The Aurora-A inhibitory effect of the 6-substituent was
explored in more detail. The 6-Cl in 3 was replaced first
by Br and CF3 with retention of Aurora-A inhibitory
activity (Table 4, compounds 18, 19). In contrast, the
replacement of the 6-Cl in 3 by a Me group led to a loss
of potency, and compound 20 inhibited Aurora-A with
an IC50 value similar to that of the 6-unsubstituted ana-
logue 4 (Table 4, compounds 4, 20). A 6-aryl substituent
also had a detrimental effect on enzyme inhibition, with
compounds 21–26 all being less potent than 3, suggest-
ing that any interaction at this position is size limited.
Table 5. Effect of substituent at 7-position, and 6,7-combinations
Compound
X
Y
Aurora-A IC50 (lM)
3
Cl
H
Cl
H
H
H
0.57 ( 0.03)
4.3 ( 0.29)
0.25
4
H
Access to the 6-aryl derivatives 21–26 was gained via a
Suzuki cross-coupling reaction that worked well either
under thermal conditions (ArB(OH)2, PdCl2(dppf),
DME, 1 M aq Na2CO3, 80 °C, 2.5–7 h) or microwave
irradiation (ArB(OH)2, PdCl2(dppf), DME, 1 M aq
Na2CO3, 150 °C, 6 min) provided the N3–H in 18 is pro-
tected. The MEM protecting group was used since it
could be readily removed under acidic conditions
(HCl, H2O/THF).
27
28
29
Cl
Me
Cl
7.0
2.4
30
31
H
0.87
The final part of this SAR exploration was focused on
substitutions at the 7-position, and 6,7-combinations.
The 7-Me derivative 28 had similar potency to the par-
ent compound 4 (Table 5, compounds 4, 28). A marginal
improvement in potency was seen with the 7-Cl substi-
tuted analogue 29 (Table 5, compounds 4, 29). Introduc-
tion of a 6-Cl substituent in 29 gave the 6,7-dichloro
analogue 27 (Table 5)23 that was approximately 10-fold
more potent as an inhibitor of Aurora-A than 29, a sim-
ilar trend to that observed with compounds 3 and 4.
Cl
0.042 ( 0.022)
Values are mean of two independent IC50 determinations or mean
( SD) for n > 2.24
tive 9 (Table 2). The imidazo[4,5-c]pyridine derivative 7
was a slightly less potent inhibitor of Aurora-A than 4,
but compounds 8 and 9 were significantly less potent
(Table 2) indicating that N4 and N3–H play an impor-
Comparison of structure 4 and structure 2f (a member
of the piperazinylquinazoline library, Table 1) prompted

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V. Bavetsias et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6567–6571
tics is funded primarily by Cancer Research UK
[CUK] Grant C309/A2187. We thank F. Urban, Dr.
N. E. Wilsher and Dr. F. Raynaud for metabolism
and cytochrome P450 isoform experiments. Paul Work-
man is a Cancer Research UK Life Fellow. We also
thank Dr. A. Mirza, A. Hayes and M. Richards for
assistance with compound characterisation.
References and notes
Scheme 3. Reagents and conditions: (a) 2-(piperazin-1-yl)-N-(thiazol-
2-y)acetamide, isopropanol, 90 °C, 5 h; (b) EtOH/EtOAc, 10% Pd/C,
1 h; (c) 4-(dimethylamino)benzaldehyde, nitrobenzene, 140 °C,
overnight.
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the synthesis of the novel piperazinyl imidazo[4,5-b]pyr-
idine derivative 30 (Table 5 and Scheme 3),25 aiming at
gaining potency from the additional interactions of the
(piperazin-1-yl)-N-(thiazol-2-yl)acetamide moiety. In-
deed, the incorporation of this fragment into 4 led to a
ꢀ5-fold potency improvement, compound 30 having
an IC50 value of 0.87 lM (Table 5). Subsequent intro-
duction of a 6-Cl substituent in 30 led to an additional
15-fold improvement in potency, and compound 31
had an IC50 = 0.042 lM. This gain in potency is similar
to that observed earlier, when a 6-Cl substituent was
introduced into structures 4 and 29 (see compounds 3,
4 and 27, 29; Table 5).
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In vitro kinase assays using purified recombinant
proteins, showed that 31 inhibited Aurora-B and
Aurora-C with IC50 values of 0.198 0.050 and
0.227 0.064 lM, respectively. Compound 31 was
shown to inhibit HCT116 cell growth with a GI50 of
0.350 lM, and HeLa cell growth with a GI50 of
0.200 lM. It also displayed good metabolic stability
with 75% of 31 remaining after a 30-min incubation with
mouse liver microsomes. Regarding inhibition of cyto-
chrome P450 isoforms, 31 showed IC50 > 10 lM for
CYP1A2, CYP2A6, CYP2C19, CYP2D6 and ꢀ10 lM
for CYP3A4 and CYP2C9.26,27
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In summary, two distinct chemical series of Aurora-A
inhibitors were discovered; namely the imidazo[4,5-
b]pyridines via HTS and quinazolines carrying N-substi-
tuted piperazines. SAR studies were undertaken and led
to the hybrid structure 31 which is a potent, novel inhib-
itor of Aurora-A, Aurora-B and Aurora-C kinases with
IC50 values of 0.042, 0.198 and 0.227 lM, respectively.
In addition, 31 blocks proliferation of HCT116 colon
cancer cells, (GI50 = 0.350 lM), has good microsomal
stability and comparatively weak inhibitory activity ver-
sus cytochrome P450 isoforms in vitro, a balance of
properties that led to the adoption of 31 as a lead com-
pound for further optimisation studies.
Acknowledgments
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The authors are grateful to Chroma Therapeutics, and
in particular Dr. A. Davidson, Dr. A. Drummond and
Dr. D. Moffat for valuable discussions. The work of
the Cancer Research UK Centre for Cancer Therapeu-

V. Bavetsias et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6567–6571
6571
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2004016611 A1. Likewise, 4-chloro-2,3-diaminopyridine,
required for the synthesis of 29, was prepared from 2-
amino-4-chloro-3-nitropyridine by applying the same
methodology.
17. Bavetsias, V.; McDonald, E.; Linardopoulos, S. Patent
WO 2007/072017 A2, 2007.
18. Yang, D.; Fokas, D.; Li, J.; Yu, L.; Baldino, C. M.
Synthesis 2005, 47.
24. IC50 values were determined using either the Flashplate
assay as described in Ref. 14, or the Filterplate assay as
described in Ref. 17. ATP concentration in the assay is
20 lM.
19. For clarity, imidazo[4,5-b]pyridines are represented in a
single tautomeric form.
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thesis of 27, was prepared by reducing 2-amino-4,5-
dichloro-3-nitropyridine as described in: Johanson, H.;
25. 2-Amino-4-chloro-3-nitropyridine was prepared from 2-
amino-4-chloropyridine by nitration (concd H2SO4, 70%
HNO3).
26. Inhibition of human liver CYP isozymes was assessed in
human liver microsomes (pool of 50 individuals) as
described in Ref. 27 with the following modifications:
microsomal protein concentration 0.5 mg/ml, incubation
time 10 min, mephenytoin as the CYP2C19 substrate and
metabolite detection by LC–MS–MS ESI+ on a Shimadzu
LC system connected to
Biosystems).
a QTRAP 4000 (Applied
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