Imidazo[4,5-b]pyridine derivatives as inhibitors of aurora kinases: Lead optimization studies toward the identification of an orally bioavailable preclinical development candidate

Article abstract of DOI:10.1021/jm100262j

Lead optimization studies using 7 as the starting point led to a new class of imidazo[4,5-b]pyridine-based inhibitors of Aurora kinases that possessed the 1-benzylpiperazinyl motif at the 7-position, and displayed favorable in vitro properties. Cocrystallization of Aurora-A with 40c (CCT137444) provided a clear understanding into the interactions of this novel class of inhibitors with the Aurora kinases. Subsequent physicochemical property refinement by the incorporation of solubilizing groups led to the identification of 3-((4-(6-bromo-2-(4-(4-methylpiperazin-1-yl)phenyl)-3H-imidazo[4,5-b] pyridin-7-yl)piperazin-1-yl)methyl)-5-methylisoxazole (51, CCT137690) which is a potent inhibitor of Aurora kinases (Aurora-A IC₅₀ = 0.015 ± 0.003 μM, Aurora-B IC₅₀ = 0.025 μM, Aurora-C IC₅₀ = 0.019 μM). Compound 51 is highly orally bioavailable, and in in vivo efficacy studies it inhibited the growth of SW620 colon carcinoma xenografts following oral administration with no observed toxicities as defined by body weight loss.

Full text of DOI:10.1021/jm100262j

J. Med. Chem. 2010, 53, 5213–5228 5213
DOI: 10.1021/jm100262j
Imidazo[4,5-b]pyridine Derivatives As Inhibitors of Aurora Kinases: Lead Optimization Studies toward
the Identification of an Orally Bioavailable Preclinical Development Candidate‡
Vassilios Bavetsias,*,† Jonathan M. Large,† Chongbo Sun,† Nathalie Bouloc,† Magda Kosmopoulou,# Mizio Matteucci,†
†
Nicola E. Wilsher, Vanessa Martins, Johannes Reynisson, Butrus Atrash, Amir Faisal, Frederique Urban,
†
†
†
†
†
ꢀ
Melanie Valenti,† Alexis de Haven Brandon,† Gary Box,† Florence I. Raynaud,† Paul Workman,† Suzanne A. Eccles,†
Richard Bayliss,# Julian Blagg,† Spiros Linardopoulos,†,§ and Edward McDonald*,†
†Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey,
SM2 5NG, United Kingdom, #Section of Structural Biology, The Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road,
London, SW3 6JB, United Kingdom, and §The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, Fulham Road,
London SW3 6JB, United Kingdom
Received February 26, 2010
Lead optimization studies using 7 as the starting point led to a new class of imidazo[4,5-b]pyridine-based
inhibitors of Aurora kinases that possessed the 1-benzylpiperazinyl motif at the 7-position, and
displayed favorable in vitro properties. Cocrystallization of Aurora-A with 40c (CCT137444) provided
a clear understanding into the interactions of this novel class of inhibitors with the Aurora kinases.
Subsequent physicochemical property refinement by the incorporation of solubilizing groups led to the
identification of 3-((4-(6-bromo-2-(4-(4-methylpiperazin-1-yl)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)-
piperazin-1-yl)methyl)-5-methylisoxazole (51, CCT137690) which is a potent inhibitor of Aurora
kinases (Aurora-A IC50 = 0.015 ( 0.003 μM, Aurora-B IC50 = 0.025 μM, Aurora-C IC50 = 0.019 μM).
Compound 51 is highly orally bioavailable, and in in vivo efficacy studies it inhibited the growth of SW620
colon carcinoma xenografts following oral administration with no observed toxicities as defined by body
weight loss.
Introduction
and glioma.9-12 In addition, Aurora-A can transform cells
when ectopically expressed in vitro.10,13 Aurora-B is also
overexpressed in human malignancies such as glioma, thyroid
carcinoma, seminoma, and colorectal cancer.14-16 Moreover,
exogenous overexpression of Aurora-B (AIM-1) in Chinese
hamster embryo cells led to increased levels of phosphoryla-
tion of histone H3 at Ser-10 which is associated with chromo-
some instability and increased tumor invasiveness, indicating a
role for Aurora-B in tumor progression.17 Overexpression of
Aurora-C has been observed in some colon cancers;18 how-
ever, its function during mitosis and its involvement in cancer
development are less well-defined.
Aurora proteins A, B, and C, a small family of serine/threonine
kinases, play distinct roles in the regulation of mitosis.1-3
Aurora-A is localized to the spindle poles and spindle micro-
tubules proximal to the centrosomes and is required for
centrosome maturation, whereas Aurora-B is localized to
kinetochores and is essential for chromosome segregation
and cytokinesis.3 Aurora-B interacts with a number of chro-
mosomal passenger proteins including the inner centromere
protein (INCENPa)4 and is known to phosphorylate histone
H3 during mitosis.5 These findings generated a considerable
interest in the involvement of Aurora kinases in cancer causa-
tion and progression.6-8 Aurora-A is overexpressed in a wide
range of human tumors including breast, colorectal, ovarian,
In recent years, the Aurora proteins have been actively
pursued as anticancer targets for the discovery of new cancer
chemotherapeutics.19 As a result, several small-molecule in-
hibitors of Aurora kinases have been identified, some of which
have reached clinical evaluation, including 1 (VX-680 (MK-
‡Atomic coordinates and structure factors for the crystal structures of
Aurora-A with compounds 40c and 51 can be accessed using PDB codes
2X6D and 2X6E, respectively.
0457)),20 2 (PHA-739358),21,22 3 (AT9283),23 4 (SNS-314),24
5
(MLN8054),25 and 6 (AZD1152)26 (Figure 1). In clinical
trials, these compounds are dosed via the iv administration
route, for example, 1, 2, 3, 4, 6, or orally, for example, 3, 5.19
The quinazoline derivative 6 has been reported as being a
selective Aurora-B inhibitor, whereas 5 is a selective inhibitor
of Aurora-A kinase activity. However, in relation to Aurora
isoform selectivity, the ideal inhibitor profile for therapeutic
use is still unclear.
*Corresponding authors. (V.B.) Telephone: þ44 (0) 20 86438901 ext
4601. E-mail: vassilios.bavetsias@icr.ac.uk. (E.M.) Telephone: þ44 (0)
20 87224231. E-mail: Ted.McDonald@icr.ac.uk.
aAbbreviations: ADP, adenosine diphosphate; AUC, area under the
curve; BOC, tert-butoxycarbonyl; dppf, 1,10-bis(diphenylphosphino)-
ferrocene; DIPEA, N,N-diisopropylethylamine; DME, 1,2-dimethox-
yethane; ESI, electrospray ionization; hERG, the human ether-a-go-go
related gene; HPLC, high pressure liquid chromatography, HRMS, high
resolution mass spectrometry; INCENP, inner centromere protein; LC,
liquid chromatography; LE, ligand efficiency; MLM, mouse liver micro-
somes; PAMPA, parallel artificial membrane permeability assay; PCy3,
tricyclohexylphosphine; PyBOP, benzotriazol-1-yloxytris(pyrrolidino)-
phosphonium hexafluorophosphate; PPB, plasma protein binding; SAR,
structure-activity relationship; TFA, trifluoroacetic acid.
We have previously reported the identification of the novel
imidazo[4,5-b]pyridine derivative 7 as a potent inhibitor of
Aurora-A (Figure 2).27,28 Herein, we report our medicinal
r
2010 American Chemical Society
Published on Web 06/21/2010
pubs.acs.org/jmc

5214 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Bavetsias et al.
Figure 1. Inhibitors of Aurora kinases.
Scheme 1a
Figure 2. Inhibition of Aurora kinases.27,28
chemistry program aimed at converting the potent lead
compound 7 to an orally bioavailable inhibitor of Aurora
kinases suitable for preclinical evaluation.
a Reagents and conditions: (a) NBS, CH3CN, 16 h; (b) conc. H2SO4,
70% HNO3, heating, (c) 2-(piperazin-1-yl)-N-(thiazol-2-yl)acetamide ꢀ
i
2HCl salt, DIPEA, PrOH, overnight; (d) EtOH, 1 M aq. Na2S2O4,
4-(dimethylamino)benzaldehyde, 80 °C; (e) DMF, Pd2dba3, dppf, Zn-
(CN)2, μW, 180 °C, 30 min.
Chemistry
The 6-Br derivative 15 was prepared as shown in Scheme 1.
The synthesis starts with 8 which was converted to 9 by
reacting with N-bromosuccinimide in acetonitrile. Nitration
of 9 was effected withthe useof conc. H2SO4/70% HNO3, and
access to the key intermediate 13 was readily gained via an
SNAr substitution reaction on 11. Formation of 15 from 13
and 4-(dimethylamino)benzaldehyde was accomplished in
one step using sodium dithionite (Na2S2O4) in ethanol.29
Starting from 10 which was prepared as previously reported,30
access to 7 was achieved in a manner similar to its 6-Br
counterpart (Scheme 1). The 6-CN derivative 16 (Table 1)
was obtained from 15 via a Pd-catalyzed cyanation reaction
using Zn(CN)2 as the cyanide source (Scheme 1).
under Pd-catalyzed conditions31 and was then converted to 20
by first removing the BOC group and then reacting 19 with
2-chloro-N-(thiazol-2-yl)acetamide. Finally, 21 was obtained
from 20 by treatment with p-methoxybenzaldehyde in the
presence of Na2S2O4 in ethanol (Scheme 2).
Access to the R2 unsubstituted analogue 23 (Figure 2, Table 2)
was gained by reducing the nitro group of 2-amino-3-nitropyr-
idine derivative 13 with Na2S2O4 in ethanol, followed by ring
formation which was achieved using trimethyl orthoformate
under acidic conditions (Scheme 3). Analogue 25 (Table 2) was
prepared by reacting 13 with tert-butyl 4-formylbenzylcarba-
mate in the presence of Na2S2O4 to effect ring formation,
followed by TFA removal of the BOC group and dimethylation
of the resulting primary amine by treatment with 38% aq.
formaldehyde and NaBH3CN (Scheme 3). Compounds 26, 27,
and 28 (Table 2) were prepared from 13 or 14 by reacting with
The 6-cyclopropyl analogue 21 (Table 1) was prepared as
shown in Scheme 2. Commencing with 11, access to the BOC-
piperazine derivative 17 was achieved via an SNAr substitution
reaction. The 5-cyclopropyl subsituted pyridine derivative 18
was obtained by reaction of 17 with cyclopropylboronic acid

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5215
Table 1. R1 Modificationsa
by reaction of the acetic acid derivative 32 with 3-chloroani-
line under standard amide bond formation conditions
(benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluoro-
phosphate, DIPEA, CH2Cl2) followed by protecting group
cleavage. The latter was obtained from 1-(pyridin-4-yl)ethanol32
by treatment with MsCl in the presence of triethylamine
followed by mesylate displacement with 1-BOC-piperazine,
and finally removal of the BOC protecting group with TFA.
Synthesis of the 4-methoxyphenyl and morpholinomethyl-
phenyl analogues 39a-h and 40a-h (Table 4) is shown in
Scheme 6 and was accomplished in a manner similar to the
dimethylaminophenyl series (Schemes 4 and 6). The key inter-
mediates 38a-h were obtained by a nucleophilic displacement
of the C-4 chloride of 11 with the requisite piperazine
(Scheme 6). Piperazines 37a, 37c, and 37d were commercially
available; 37b and 37g were prepared by a reductive amination
of the corresponding aldehyde with 1-BOC-piperazine fol-
lowed by removal of the BOC protecting group (Scheme 7).
The piperazine derivatives 37e, 37f, and 37h were obtained
via a substitution reaction on the appropriate heteroaryl-
methyl halide followed by the removal of the BOC protecting
group (Scheme 8, Table 4).
Aurora-A,
IC50 (μM)
0.042 ( 0.022c
0.055 ( 0.009
0.050 ( 0.020
0.053b
HCT116
GI50 (μM)
0.35c
0.48
1.03b
2.50
compound
R1
X
7
Cl
Br
NMe2
NMe2
NMe2
OMe
15
16
21
CN
cyclopropyl
a Results are mean values of two independent determinations or mean
((SD) for n > 2 unless specified otherwise. b Results are mean values for
samples run in triplicate. c From refs 27 and 28.
Scheme 2a
Compounds listed in Table 6 were prepared by treating 38e
or its 5-chloro counterpart with the required benzaldehyde in
the presence of Na2S2O4. For the synthesis of 44, 46, 47, and
50, the amino functionality present on the requisite benzalde-
hyde was protected with the BOC group which was removed
with TFA after ring formation. Compound 45 was obtained
from 46 by a reductive alkylation reaction (38% aq. formal-
dehyde, THF/MeOH, NaBH3CN).
Results and Discussion
As previously reported, compound 7 inhibits Aurora-A, -B,
and -C with IC50 values of 0.042, 0.198, and 0.227 μM,
respectively,27 and is also potent in inhibiting cell proliferation
in a range of cancer cell lines.28 Additionally, compound 7
displayed selectivity for inhibition of Aurora-A and Aurora-B
proteins in a small kinase panel.28 Regarding the binding mode
of 7 to Aurora-A, we had no crystallographic data on 7 or a
closely related analogue bound to Aurora-A when we initiated
the lead optimization process. However, the SAR data ob-
tained during the hit-to-lead exploration program27 and mod-
eling studies suggested that the pyridine N and imidazole NH
form hydrogen bonds to Ala213 in the hinge region of Aurora-
A with the 2-dimethylaminophenyl substituent pointing to the
solvent accessible area. The imidazole NH in 2-arylimidazo-
[4.5-b]pyridines is a strong hydrogen donor, and the pKa value
of the imidazole NH in 2-phenylimidazo[4.5-b]pyridine was
reported as 11.07.33 The hit-to-lead exploration chemistry also
suggested that substitution at the 6-position was sterically
limited; however, compound 7 was a considerably more potent
inhibitor of Aurora-A compared with the 6-unsubstituted
counterpart.27 We had no clear understanding of the interac-
tions of the (piperazin-1-yl)-N-(thiazol-2-yl)acetamide moiety
in 7 with the protein. With this in mind, we decided to explore
in more detail the role of the R3 substituent (Figure 2) in
relation to Aurora-A kinase activity. By modifying the R3
substituent, we were also aiming to reduce the molecular
weight and remove the 2-aminothiazole moiety, a potential
toxicophore.34,35 Our second objective was to replace the R2
dimethylaminophenyl moiety because of the known toxicity
liabilities associated with aniline derivatives.35,36 Finally, we
were planning to optimize the C-6 position within the steric
i
a Reagents and conditions: (a) 1-BOC-piperazine, DIPEA, PrOH,
45 °C, 20 h; (b) cyclopropylboronic acid, DME, Pd(OAc)2, PCy3,
K3PO4, μW, 150 °C, 45 min; (c) TFA,CH2Cl2; (d) 2-chloro-N-(thiazol-
2-yl)acetamide, CH2Cl2, DIPEA; (e) EtOH/DMF, 4-methoxybenzalde-
hyde, 1 M aq. Na2S2O4, 80 °C.
the appropriate aldehyde in the presence of Na2S2O4 as described
for the synthesis of 15 (Scheme 1).
Compound 31a (Table 3) was obtained from intermediate
17 by treatment with 4-(dimethylamino)benzaldehyde and
Na2S2O4 in ethanol followed by TFA removal of the BOC
protecting group (Scheme 4). Other compounds in Table 3
were readily prepared by reaction of the appropriate 2-amino-
3-nitropyridine intermediate 30b-l with 4-(dimethylamino)-
benzaldehyde under standard ring formation conditions (1 M
aq. Na2S2O4, EtOH or DMF, heating; see Scheme 4). 2-Amino-
3-nitropyridine derivatives 30b-d, 30 g-l were obtained via
an SNAr substitution reaction on 11 or 12 (Scheme 4).
Starting from 17, access to 2-amino-3-nitropyridine deriva-
tives 30e and 30f was achieved by first removing the BOC
group with TFA followed by treatment with phenyl isocya-
nate and benzenesulfonyl chloride, respectively (Scheme 4,
Table 3).
Piperazines 29b-d, 29g-l (Scheme 4) were commercially
available with the exception of 29d and 29i which were
synthesized as shown in Scheme 5. The former was accessed

5216 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Table 2. R2 Modifications#
Bavetsias et al.
# Results are mean values of two independent determinations or mean ((SD) for n > 2 unless specified otherwise. a Results are mean values for
samples run in triplicate. b From refs 27 and 28.
Scheme 3a
displaying Aurora IC50 and HCT116 GI50 (50% cell growth
inhibitory concentration) values similar to those of 7 (Table 1).
The 6-CN and 6-cyclopropyl derivatives 16 and 21, respec-
tively, (Table 1) inhibited in vitro Aurora-A kinase activity
equivalent to 7 and 27 but were less potent in inhibiting
HCT116 cell growth relative to their 6-Cl counterparts 7
(Table 1) and 27 (Table 2).
Subsequently, our chemical effort was focused on the R2
substituent (Figure 2, Table 2). Removal of the dimethylami-
no group in 15 did not significantly change the Aurora-A
inhibitory activity (compound 15 versus 26; Table 2), but the
replacement of the 2-aryl substituent in 15 with a proton led to
a ∼10-fold drop inpotency against Aurora-A(compounds 23,
15; Table 2), indicating that the phenyl ring plays an impor-
tant role in binding to the Aurora-A kinase and consequent
translation to cell based antiproliferative activity. Next, the
NMe2 moiety in 7or 15 was replaced with a methoxy, dimethyl-
aminomethyl, and a morpholinomethyl group (compounds 27,
25, and 28; Table 2), with all three analogues demonstrating
cellular potencies comparable to those of 7 or 15. Interestingly,
inhibitory activity against the Aurora-A enzyme was retained
or significantly improved, with 28 being ∼10-fold more potent
inhibitor compared with 7 (Table 2). These results were very
encouraging and clearly demonstrated that the dimethylami-
nophenyl moiety, a potential toxicophore, could be replaced
with retention or an enhancement of the Aurora-A inhibitory
activity without compromising the cellular potency.
a Reagents and conditions: (a) EtOH, 1 M aq. Na2S2O4, 80 °C, 16 h;
(b) (MeO)3CH, conc. HCl, room temp., 24 h; (c) tert-butyl 4-formyl-
benzylcarbamate, EtOH, 1 M aq. Na2S2O4, 80 °C; (d) i. TFA/CH2Cl2, ii.
38% aq. formaldehyde, THF/MeOH, NaBH3CN.
constraints already determined by our previous work. To this
end, a limited exploration of the R1 substituent was under-
taken, and it was found that Br is well tolerated, compound 15

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5217
Table 3. R3 Modifications#
# Results are mean values of two independent determinations or mean ((SD) for n > 2 unless specified otherwise. a Results are mean values for
samples run in triplicate. b From refs 27 and 28. c n.d. = not determined.
To evaluate the contribution of the undesirable N-(thiazol-
2-yl)acetamide moiety to the inhibitory potency of 7 or 15, we
prepared the unsubstituted compound 31a (Table 3). This
analogue was ∼8-fold less potent in inhibiting the Aurora-A
kinase compared with the parent 15. The replacement of the
thiazole ring in 15 or 7 with a phenyl, pyridyl, or a substituted
phenyl (compounds 31b, 31c, and 31d, respectively) was
broadly tolerated, but all three compounds were less potent
inhibitors of the HCT116 cell growth relative to 15 or 7
(Table 3). The attachment of a phenyl group to the piperazine

5218 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Bavetsias et al.
Scheme 4a
Table 4. R3 Benzyl Replacement#
a Reagents and conditions: (a) TFA/CH2Cl2; (b) CHCl3, DIPEA,
PhNCO, room temp., 12 h; (c) CHCl3, pyridine, PhSO2Cl, room temp.,
12 h; (d) 2-amino-4,5-dichloro-3-nitropyridine or 2-amino-5-bromo-4-
i
chloro-3-nitropyridine, DIPEA, PrOH, heating; (e) 4-(dimethylamino)-
benzaldehyde, EtOH or DMF, 1 M aq. Na2S2O4, 80 °C.
Scheme 5a
a Reagents and conditions: (a) 3-chloroaniline, benzotriazol-1-yloxytris-
(pyrrolidino)phosphonium hexafluorophosphate, DIPEA, CH2Cl2; (b)
TFA/CH2Cl2; (c) i. MsCl, Et3N, CH2Cl2, ii. 1-BOC-piperazine, DMSO,
60 °C, 18 h.
# Results are mean values of two independent determinations or mean
((SD) for n > 2 unless specified otherwise. a Results are mean values for
samples run in triplicate. c n.d. = not determined.
ring, directly or via an amide or a sulphonyl linker, had a
detrimental effect on enzyme inhibition (compounds 31g, 31e,
and 31f; Table 3). Likewise, the isobutyl derivative 31h was a
weaker inhibitor of Aurora-A (IC50 value of 0.210 μM
compared with that of 0.055 μM for 15). However, the
attachment of a phenyl or a 4-pyridyl group to the piperazine
ring via a CH(CH3) linker (compounds 31k and 31i, respec-
tively; Table 3) was well tolerated regarding both Aurora-A
enzyme inhibition and HCT116 cellular potency. Compounds
31i and 31k inhibited Aurora-A with IC50 values of 0.085 and
0.079 μM, respectively, prompting the introduction of the
pyridin-4-ylmethyl group as the R3-substituent (31l, Table 3).
Compound 31l was a potent inhibitor of both the Aurora-A
kinase activity and the HCT116 cell growth, with IC50/GI50
values of 0.055 and 0.70 μM, respectively. Subsequent combi-
nation of R1 = Cl, R2 = p-methoxyphenyl, and R3 = pyridin-
4-ylmethyl led to compound 36a (Figure 3), a potent Aurora-A
inhibitor (IC50 = 0.021 μM) devoid of the toxicophore
liabilities associated with 7, and of lower molecular weight
and higher ligand efficiency compared with 7 (Figure 3). On
this basis, 36a (Figure 3) was selected as a scaffold to identify
the substituents with optimum in vitro profile and evaluate the
most promising compounds for kinase selectivity and in vivo
PK properties. The 6-Br counterpart of 36a (compound 36b)
was also prepared and displayed a similar Aurora-A inhibitory
potency (IC50 = 0.015 μM). Optimization of the R3 benzyl
group was carried out with a 6-Br substituent due to avail-
ability of bulk intermediate, while the neutral p-methoxyphe-
nyl group and the weakly basic morpholinobenzyl moiety were
utilized as the R2 substituents (Figure 3, Table 4).
In the R2 = p-methoxyphenyl series (compounds 39a-h),
the introduction of 3-pyridyl or a pyrimidinyl ring was
beneficial versus inhibition of Aurora-A kinase (compound
39a, IC50 = 0.009 μM;compound 39b, IC50 = 0.008μM). The
pyrimidinyl derivative 39b exhibited higher cellular potency
relative to 39a, but lower stability in mouse liver microsomes
(35% of parent compound remaining after 30 min incubation).
The introduction of the p-chlorophenyl group provided no
Aurora-A inhibitory benefit (39c, IC50 = 0.040 μM) but 39c

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5219
Scheme 6a
Figure 3. A new class of imidazo[4,5-b]pyridine-based inhibitors of
Aurora kinases.
Table 5. Kinase Selectivity Profile of 40c
kinase
Aurora-B
EPHB4
% inhibition at 1 μM
60
4
a Reagents and conditions: (a) 2-amino-5-bromo-4-chloro-3-nitro-
pyridine, DIPEA, iPrOH, heating; (b) 4-methoxybenzaldehyde, EtOH,
1 M aq. Na2S2O4, 80 °C; (c) 4-(morpholinomethyl)benzaldehyde, EtOH,
1 M aq. Na2S2O4, 80 °C.
FLT1 (VEGFR1)
KDR (VEGFR2)
LCK
35
35
8
MET (cMET)
NTRK1 (TRKA)
PDGFR beta
Aurora-A
3
33
15
98
5
Scheme 7a
TEK (Tie2)
remaining after a 30 min incubation). In the R2 morpholino-
benzyl series (compounds 40a-h), the effect of the R3 sub-
stituent followed a similar trend with that observed in the R2 =
p-methoxyphenyl series (compounds 39a-h, Table 4) with two
notable exceptions. In line with observations in the R3 =
N-(thiazol-2-yl)acetamide series (Table 2), the morpholino-
methyl group appeared to have a higher affinity for the enzyme
as demonstrated with compounds 40c (CCT137444) and 40e
(Table 4). However, the morpholinomethyl derivatives exhibit
lower stability in mouse liver microsomes, probably a reflec-
tion of metabolic liabilities associated with the morpholine
ring.37
a Reagents and conditions: (a) pyrimidine 5-carboxaldehyde,
NaBH3CN, EtOH, AcOH, or 1-methyl-1H-imidazole-2-carbaldehyde,
1,2-dichloroethane, NaBH(OAc)3; (b) TFA/CH2Cl2.
Scheme 8a
Toobtain an indicationof the kinase selectivityforthisclass
of imidazo[4,5-b]pyridine-based inhibitors of Aurora kinases,
40c was evaluated in a panel of 10 kinases. At a compound
concentration of 1 μM, it was found that 40c inhibited only
Aurora-A and Aurora-B to a significant level (Table 5). For
selected examples, affinity for the hERG ion-channel was
determined. Compound 39e had no effect on hERG tail-
currents, but 40c was a weak inhibitor of hERG (53%
inhibition at 10 μM).
a Reagents and conditions: (a) 3-bromomethyl-5-methylisoxazole,
CH2Cl2 or 4-(chloromethyl)-2-isopropyloxazole, DIPEA, CH2Cl2 or
4-(chloromethyl)thiazole, DIPEA, CH2Cl2; (b) TFA/CH2Cl2.
was potent in cells (GI50 = 0.36 μM). The cyclopropylmethyl
analogue 39d followed the same trend seen with the isobutyl
derivative 31h (Table 3), namely, inferior inhibition of Aurora-
A (IC50 = 0.65 μM) but relatively potent in HCT116 cells
(GI50 = 0.83 μM). Subsequently, a range of 5-membered ring
aromatic heterocycles was explored. The introduction of the
5-methylisoxazole (39e, Table 4) was well tolerated. Com-
pound 39e was a potent inhibitor of the Aurora-A kinase and
the HCT116 cell growth (IC50 = 0.017, GI50 = 0.54 μM) with
good stability in mouse liver microsomes (>50% remaining
after 30 min incubation). The isopropyloxazole analogue 39f
was relatively inferior on inhibiting the kinase (IC50 =0.081μM),
and metabolically labile (only 11% remaining after 30 min
incubation with mouse liver microsomes). The N-methylimi-
dazole derivative 39g displayed inferior cellular potency
(GI50 = 1.5 μM), and the thiazole analogue 39h suffered
from a moderate stability in mouse liver microsomes (45%
To determine the binding mode of this class of inhibitors,
we cocrystallized the catalytic domain of Aurora-A (residues
122-403) with 40c. The crystal structure of compound 40c
˚
bound to Aurora-A (determined to a resolution of 2.8 A,
Figure 4A, Table S1 in Supporting Information) confirmed
our hypothesis that the pyridine N and imidazole NH are
interacting with Ala213 in the hinge region of the kinase.
Compound 40c occupies the ATP-binding site with the acti-
vation loop in a DFG-in conformation. The pyridine N is
˚
hydrogen bonded to backbone NH of Ala213 (2.9 A) and the
˚
imidazole NH to the carbonyl of Ala213 (2.9 A) as shown in
Figure 4. The 6-Br substituent occupies a small lipophilic
pocket defined by the side chains of Val147, Ala160, Leu194,
˚
and Leu210 (4-4.2 A distant). The morpholinomethylphenyl
substituent points to the solvent accessible area with the

5220 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Bavetsias et al.
Table 6. Incorporation of Solubilizing Groups#
˚
Figure 4. 2.8 A resolution crystal structure of compound 40c
bound to Aurora-A. (A) Stick representation of 40c (carbon atoms
colored cyan) bound to Aurora-A (carbon atoms colored green)
with the final Fo - Fc electron density map contoured at 1.0σ shown
as a wiremesh. (B) Cartoon representation of the Gly-rich loop and
stick representation of ligand in ADP-bound Aurora-A (gray
cartoon and carbon atoms) and 40c-bound Aurora-A (green car-
toon and carbon atoms).
phenyl ring residing at close proximity to Gly216. The R3
p-chlorobenzyl substituent interacts with the Aurora-Akinase
Gly-rich loop at the backbone of residues Gly140, Lys141,
˚
and Asn146. This interaction induces an approximately 3 A
outward conformational shift of the Gly-rich loop with
respect to the conformation observed in the ADP-bound form
of Aurora-A (Figure 4B).
Subsequently, on the basis of desirable in vitro properties, a
set of compounds was selected for in vivo PK profiling. Oral
bioavailability was 13% and 23% for compounds 40a and
39e, respectively. Though permeability, determined as 6 ꢀ
10-6 cm/s for 39e in the PAMPA assay (pH6.5), was not
excluded as the cause of the low bioavailability, this was
mainly attributed to low aqueous solubility (measured as
0.03 mg/mL for 40a, and <0.01 mg/mL for 39e). These
findings suggest that although potent inhibitors of Aurora
kinases with desirable cellular potency and mouse liver micro-
somal stability have been identified, their physicochemical
properties and in particular solubility may limit their utility.
Consequently, solubility-enhancing features such as alcohols,
heterocycles, and basic amines were designed in the subse-
quent compound set. We sought to introduce such groups at
the p-position of the R2 phenyl substituent (Table 6) with the
aim of simultaneously maintaining or improving the kinase
inhibitory activity. Our ultimate objective was to identify
compounds for in vivo PK characterization and in vivo
efficacy studies. To achieve this, we mainly focused our
chemical efforts on three R3 substituents: p-chlorobenzyl,
5-methylisoxazol-3-ylmethyl, and pyridin-3-ylmethyl (based
# Results are mean values of two independent determinations or mean
((SD) for n > 2 unless specified otherwise. a Results are mean values for
samples run in triplicate.
on the in vitro compound profiling). Particular attention was
given to R3 5-methylisoxazol-3-ylmethyl since the 5-methyl-
isoxazole moiety has the lowest contribution to overall com-
pound cLogP (0.39) relative to pyridine (0.65) and chloroben-
zene (2.86).38 The results for the 5-methylisoxazole derivatives
are shown in Table 6.
For selected examples, aqueous solubility was obtained;
it was found that X substituents bearing a basic nitrogen
fulfilled our expectations. For example, the aqueous solubility
for compounds 44 and 51 (CCT137690) was determined as
0.87mg/mL and0.23 mg/mL, respectively. Onthe other hand,
no benefit was gained from the introduction of the hydroxy-
ethoxy moiety as a solubilizing group; the measured solu-
bility for compound 48 was low (0.01 mg/mL). The vast
majority of the synthesized analogues were potent inhibitors
of Aurora-A and of HCT116 cell growth (Table 6). In addi-
tion, compounds 44-52 displayed good stabilityin mouse liver
microsomes (>50% of parent compound remaining after

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5221
30 min incubation). This desirable set of in vitro properties
(i.e., enzymatic/cellular potency, and mouse liver microsomal
stability) justified the in vivo PK characterization for many of
compounds shown in Table 6. A selection of compounds
including the piperazinyl derivative 44 (Table 6) were adminis-
tered iv in a cassette of five compounds for PK profiling.
Pharmacokinetic analysis indicated a rapid plasma clearance
for 44 with undetectable levels 1 h postadministration of 1 mg/kg.
To further accelerate the compound PK profiling, suitable
analogues (compounds 45-52, Table 6) were first subjected to
a “limited PK” evaluation in which plasma and muscle (as a
surrogate for tumor) compound concentrations were deter-
mined at 6 and 24 h following oral administration to mice (dose
of 5 mg/kg). The hydroxyethoxy derivative 49 and the benzyl-
amine derivative 47 were only detected at the 6 h time point in
plasma at a total concentration <10 nM and not in muscle.
Likewise, the N,N-dimethylbenzylamine analogue 45 was only
detected at 6 h post dose in plasma (total concentration =
90 nM). In contrast, compound 51 was detectable in both plasma
and muscle at the 6 h time point (total concentration in plasma =
250 nM). Compound 52 was present in both plasma and muscle
at 6 h post dose at levels similar to those determined for 51.
However, 52 was not detectable in muscle at 24 h, whereas 51
was present in muscle at 24 h (total concentration=10 nM).
Assuming linear pharmacokinetics, the compound concentra-
tion at the two time points (6, 24 h) was projected based upon an
oral dose of 50 or 100 mg/kg. Calculated values significantly
above the compound cellular GI50 value led to further investiga-
tion of the full in vivo PK. By following these experimental triage
procedures, compound 51 was identified having the optimal
combination of in vitro and in vivo properties. It is a completely
orally bioavailable inhibitor (F = 100%) of Aurora kinases with
desirable pharmacokinetic parameters in mouse (Cl: 0.037 L/h
(∼25 mL/min/kg), Vd: 0.087 L (∼3.5 L/kg); Table 7).
Table 7. Compound 51: Mouse Plasma Protein Binding, and PK
Parameters in Mouse (iv dosing: 1 mg/kg)
PPB
(mouse)
t1/2, AUC,
(iv) (h) Cl (L/h) (iv) h nmol/L Vd (L) F% (po)
3
994
98.0 ( 0.5
1.6
0.037
0.087
100
Compound 51 was profiled in a 94-kinase panel, and at a
concentration of 1 μM, inhibited only three kinases at a level
higher than 80%, that is, Aurora-A, FGF-R1, and VEG-FR
(see Table S2 in Supporting Information).
Compound 51 inhibited Aurora-B and Aurora-C with IC50
values of 0.025 and 0.019 μM, respectively, and displayed
antiproliferative activity in a range of human tumor cell lines,
including SW620 colon carcinoma (GI50 = 0.30 μM) and
A2780 ovarian cancer cell line (GI50 = 0.14 μM). In addition,
51 inhibited in vitro the phosphorylation of histone H3, a
biomarker for inhibition of Aurora-B kinase (data not shown -
to be reported elsewhere). Regarding key safety pharmacol-
ogy parameters, 51 was tested for inhibition of the hERG
ion-channel and cytochrome P450 isoforms. It inhibited the
major cytochrome P450 isoforms (CYP1A2, CYP2A6,
CYP2C9, CYP2C19, CYP2D6, CYP3A4) with an IC50 value
greater than 10 μM. However, 51 was a moderate inhibitor of
˚
Figure 5. 3.35 A resolution crystal structure of 51 bound to Aurora-A.
(A) Stick representation of 51 (carbon atoms colored yellow) bound to
Aurora-A (carbon atoms colored green) with the final Fo - Fc electron
density map contoured at 1.0σ shown as a wiremesh. (B) Cartoon
representation of the Gly-rich loop and stick representation of ligand
in 51-bound Aurora-A (cyan cartoon and carbon atoms) and 40c-
bound Aurora-A (green cartoon and carbon atoms).
Figure 6. In vivo efficacy of 51 against SW620 human colon carcinoma xenografts in athymic mice. (A) Mean tumor volumes ( SEM, (B)
mouse body weights.

5222 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Bavetsias et al.
51 which is a highly orally bioavailable and potent inhibitor of
Aurora kinases. In an in vivo efficacy evaluation, 51 inhibited
the growth of SW620 colon cancer cell xenografts following
oral administration with no observed toxicities as defined by
body weight loss. However, 51 has a narrow safety margin
against hERG which may limit its preclinical development and
the discovery of compounds with a wider therapeutic index
versus hERG will be the subject of future publications.
Experimental Section
Aurora Kinase Assays. Aurora kinase IC50 values were deter-
mined as previously described.28
Kinase Selectivity Profiling. Compound 51 was profiled
against a panel of 94 kinases at the National Centre for Protein
Kinase Profiling, Division of Signal Transduction Therapy,
University of Dundee.
Cell Viability Assay. GI50 values (50% cell growth inhibitory
concentration) were determined as previously described.28
Cocrystallization of Aurora-A with Ligand. Wild-type Aurora-A
catalytic domain (residues 122-403) was expressed and purified as
previously described.39 Cocrystals with 40c were produced using
Bicine pH 9.0, 2.0 M (NH4)2SO4 as crystallization buffer. Cocrys-
tals with 51 were produced using 0.1 M NaCl, 0.1 M HEPES pH
7.5, 1.6 M (NH4)2SO4 as crystallization buffer. Crystals were
briefly soaked in crystallization buffer supplemented with 25%
ethylene glycol 40c or 30% glycerol 51 before flash freezing in
liquid N2. Structures were solved by molecular replacement using
Aurora-A (PDB code 1MQ4) as a model. Ligand fitting and model
rebuilding was carried out using Coot,40 and refinement was
carried out using Phenix.41
Figure 7. Compound 51 inhibits histone H3 phosphorylation in
SW620 xenografts. Tumour samples were obtained 6 h after the
final dose and analyzed for histone H3 phosphorylation. Total
histone H3 and GAPDH were used as loading controls.
the hERG ion-channel (IC50 = 3.0 μM; n= 2). The 6-Cl coun-
terpart of 51, compound 52 (Table 6), displayed a similar
hERG inhibitory activity (IC50 = 2.3 μM).
The crystal structure of 51 bound to Aurora-A was deter-
˚
mined to a resolution of 3.35 A, and shows that 51 occupies
the ATP-binding site in a binding mode similar to that
described for 40c (Figure 5A). Compound 51 makes contacts
withtheGly-rich loop that aresimilartothatobserved for40c,
the isoxazole O and N have the potential to act as H-bond
˚
acceptors, and in our model the O sits 3.1 A away from the
backbone NH of Gly142. However, the resolution of this
structure is insufficient to determine whether H-bonding
occurs between the isoxazole and Aurora-A. Compound 51
induces a similar conformational shift in the Gly-rich loop as
40c (Figure 5B).
Mouse Liver Microsomal Stability. Compounds (10 μM) were
incubated with male CD1 mouse liver microsomes (1 mg mL-1
)
On the basis of the encouraging PK parameters of 51, the
absence of concerns related to inhibition of P450 isoforms,
desirable Aurora-A enzyme/cellular potency, and pharmaco-
dynamic effect related to inhibition of Aurora kinases, 51 was
evaluated in in vivo efficacy studies (Figure 6). Athymic mice
bearing established SW620 human colorectal tumors were
treated with either vehicle (DMSO-Tween-saline) or 51 ad-
ministered orally at a dose of 75 mg/kg twice a day for 21 days.
Compound 51 slowed the growth of the SW620 xenografts
with no observed toxicity as defined by body weight loss
(Figure 6B). The treated/control (T/C) ratio was calculated as
42.4% based on final tumor weights (not shown). A repeat
efficacy study with 51 (75 mg/kg p.o. twice daily for 17 days)
showed comparable efficacy (T/C = 37%), and inhibition of
histone H3 phosphorylation, a biomarker for Aurora-B ki-
nase inhibition (Figure 7). These findings are consistent with
target modulation in vivo by compound 51.
protein in the presence of NADPH (1 mM), UDPGA (2.5 mM),
and MgCl2 (3 mM) in phosphate buffered saline (10 mM) at
37 °C. Incubations were conducted for 0 and 30 min. Control
incubations were generated by the omission of NADPH and
UDPGA from the incubation reaction. The percentage com-
pound remaining was determined after analysis by LCMS.
Inhibition of Cytochrome P450 Isoforms. Inhibition of human
liver CYP isozymes was assessed in human liver microsomes
(pool of 50 individuals) as previously described42 with the
following modifications: microsomal protein concentration
0.5 mg/mL, incubation time 10 min, mephenytoin as the
CYP2C19 substrate and metabolite detection by LCMSMS
ESIþ on a Shimadzu LC system connected to a QTRAP 4000
(Applied Biosystems).
Aqueous Solubility. Compound kinetic aqueous solubility was
determined by Evotec, Abingdon, Oxfordshire, UK.
Inhibition of hERG. hERG IC50 values were determined by
Millipore (Millipore UK Ltd., Cambridge, UK).
In Vivo “Limited PK”. All animal studies were performed in
accordance with the UK guidelines for animal use and welfare
(UKCC). Compounds were formulated at 0.5 mg mL-1 in 10%
DMSO, 5% Tween 20 in sterile saline. Female Balb/C mice
received an oral dose of compound (5 mg/kg). After adminis-
tration, mice were bled at 6 and 24 h. Blood was removed by
cardiac puncture in heparinised syringes and centrifuged to
obtain plasma samples for PK analysis. Muscle and liver were
harvested for analysis. Plasma samples (50 μL) were extracted
by protein precipitation by addition of 150 μL of methanol
containing internal standard (IS). Tissue samples were homo-
genized with 3v/w of PBS and compound extracted from the
resulting homogenate as described for plasma. Plasma and
homogenate extracts were analyzed by LCMS using reverse-
phase Eclipse Plus C18 (Agilent, 50 ꢀ 2.1 mm) analytical column
and positive ion mode ESI MRM.
Conclusion
In the course of this work, our goal was to convert 7 to an
orally bioavailable inhibitor of Aurora kinases suitable for
preclinical evaluation. This led to a new class of imidazo[4,5-b]-
pyridine-based inhibitors of Aurora kinases possessing the
1-benzylpiperazinyl motif at the 7-position. Compounds be-
longing to this class are devoid of the potential toxicophore
liabilities associated with 7, and a significant number of ana-
logues displayed favorable in vitro properties (enzymatic/
cellular potency, and mouse liver microsomal stability).
Cocrystallization of Aurora-A with 40c bound provided a clear
understanding of the interactions of this novel class of inhibi-
tors with the Aurora-A kinase. Subsequent physicochemical
property refinement by reduction of lipophilicity and the
introduction of solubilizing groups led to the identification of
Cassette Dose Pharmacokinetic Profiling. Five compounds
were formulated at 0.1 mg/mL in 10% DMSO, 5% Tween 20 in

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5223
saline. Balb C female mice received the compound mixture via iv
administration at 1 mg/kg per compound. Following compound
administration, mice were terminated at 0.083, 0.25, 0.5, 1, 2, 4,
6, and 24 h. Blood was removed by cardiac puncture and
centrifuged to obtain plasma samples and tissues harvested for
PK analysis. Plasma samples (150 μL) were extracted by protein
precipitation by addition of 150 μL of acetonitrile containing
internal standard (IS). Tissues were homogenized with 3w/v of
PBS prior to protein precipitation with acetonitrile containing
IS. Plasma and tissue extracts were analyzed by LCMS using
reverse-phase analytical column and positive ionization mode
with multiple reaction monitoring of optimized transitions.
In Vivo Full PK (Compound 51). Full pharmacokinetic anal-
ysis was performed at 1 mg/kg (iv) and 5 mg/kg (po) as described
in ref 28.
5-Bromo-4-chloro-pyridin-2-ylamine (9). To a solution of
2-amino-4-chloropyridine (0.50 g, 3.9 mmol) in acetonitrile
(20 mL) was added dropwise a solution of N-bromosuccinimide
(0.730 g, 4.1 mmol) in acetonitrile (10 mL). The reaction mixture
was stirred at room temperature for 16 h then concentrated in
vacuo. The crude product was purified by chromatography on
silica gel (hexane/ethyl acetate 6:4) to give the title compound as
a white solid (0.65 g, 80%); 1H NMR (250 MHz, CDCl3) 4.50 (br
s, 2H, NH2), 6.63 (s, 1H) and 8.16 (s, 1H) (3-H, 6-H); LC
(Method A) - MS (ESI, m/z) Rt = 4.8 min - 207, 209, 211 [(M þ
Hþ), BrCl isotopic pattern].
5-Bromo-4-chloro-3-nitro-pyridin-2-ylamine (11). 5-Bromo-4-
chloro-pyridin-2-ylamine (0.640 g, 3.0 mmol) was added in
portions to conc. H2SO4 (6 mL) while the temperature was kept
at below 10 °C. The reaction mixture was stirred at 5 to 10 °C for
15 min. The resulting solution was then heated at 55 °C, and
HNO3 (70%; 208 μL, 3.2 mmol) was dropwise added while the
temperature was kept at 55-60 °C. The reaction mixture was
stirred at 55 °C for 30 min then cooled to room temperature and
poured slowly into ice (20 g). The pH was adjusted to 7 with 10%
aqueous NaOH. The precipitated product was filtered, washed
with water (20 mL), and dried in vacuo over P2O5 for 16 h to give
the title compound as a yellow solid (0.580 g). Further purifica-
tion of this product (0.48 g) by chromatography (hexane/ethyl
acetate; v/v 7:3) gave the title compound as a yellow solid (0.287
g, 38%); 1H NMR (250 MHz, CDCl3) 5.78 (br s, 2H, NH2), 8.29
(s, 1H, 6-H).
2-[4-(2-Amino-5-bromo-3-nitro-pyridin-4-yl)-piperazin-1-yl]-
N-thiazol-2-yl-acetamide (13). To a mixture of 5-bromo-4-
chloro-3-nitro-pyridin-2-ylamine (0.100 g, 0.39 mmol) and iso-
propanol (7 mL) was added 2-(piperazin-1-yl)-N-(thiazol-2-
yl)acetamide ꢀ 2HCl salt (0.124 g, 0.41 mmol) and dry DIPEA
(275 μL, 1.58 mmol). The mixture was heated at 50 °C for 16 h,
then allowed to cool to room temperature, and concentrated in
vacuo. The crude product was purified by chromatography on
silica gel (hexane/ethyl acetate v/v 1:1, and a gradient of methanol
(0 to 5%) in ethyl acetate) to give the title compound as a yellow
solid (0.144 g, 82%); 1H NMR (250 MHz, DMSO-d6) 2.68 (broad
t, J = 4.0 Hz, 4H, piperazine N(CH2)2), 3.10 (broad t, J = 4.0 Hz,
4H, piperazine N(CH2)2), 3.36 (s, 2H, NCH2CO), 6.95 (broad s,
2H, NH2), 7.22 (d, J = 3.6 Hz, 1H) and 7.47 (d, J = 3.6 Hz, 1H)
(thiazole 4-H, 5-H), 8.16 (s, 1H, pyridine 6-H), 11.8 (broad s, 1H,
CONH); LC (Method A) - MS (ESI, m/z) Rt = 4.69 min - 442,
444 [(M þ H)þ, Br isotopic pattern].
Human Tumor Xenograft Efficacy Study. Procedures invol-
ving animals were carried out within guidelines set out by The
Institute of Cancer Research’s Animal Ethics Committee and in
compliance with national guidelines (Workman P., Twentyman
P., Balkwill F., et al. United Kingdom Co-ordinating Commit-
tee on Cancer Research (UKCCCR) Guidelines for the Welfare
of Animals in Experimental Neoplasia (Second Edition). Br J
Cancer, 77: 1-10, 1998).
Human SW620 colon carcinoma cells (4 ꢀ 106) were injected
subcutanously (s.c.) in the right flank of female CrTac:NCr-
Fox1(nu) athymic mice. Animals (8 per group) were treated
twice daily with vehicle or 51 once tumors reached a mean
diameter of 5.3-5.4 mm. Tumour volumes and body weights
were measured three times a week, and at the end of the study
tumors were excised, weighed and snap frozen for PD analysis 6
and 24 h after the final dose.
Chemistry. Commercially available starting materials, reagents,
and dry solvents were used as supplied. Flash column chromato-
graphy was performed using Merck silica gel 60 (0.025-0.04 mm).
Column chromatography was also performed on a FlashMaster
personal unit using isolute Flash silica columns or a Biotage SP1
purification system using Biotage Flash silica cartridges. Ion
exchange chromatography was performed using acidic Isolute
1
Flash SCX-II cartridges. H NMR spectra were recorded on a
Bruker Avance dpx250 or a Bruker Avance-500. Samples were
preparedassolutions ina deuteratedsolventandreferencedtothe
appropriate internal nondeuterated solvent peak or tetramethyl-
silane. Chemical shifts were recorded in ppm (δ) downfield of
tetramethylsilane. LC-MS spectra were recorded on a Waters
LCT with a Waters Alliance 2795 separations module, using a
Phenomenex Gemini C18 column and either of the following
conditions: Method A (10 min) - nominal mass, LC injection with
a 10 min gradient (MeOH and 0.1% formic acid), positive
ionization and an injection volume of 3 μL. Column: Phenom-
enex Gemini C18 column(5 μm, 50 ꢀ 4.6mm). MethodB (6 min) -
nominal mass, LC injection with a 6 min gradient (MeOH and
0.1% formic acid), positive ionization and an injection volume of
2 μL. Column: Phenomenex Gemini C18 column (3 μm, 30 ꢀ 4.
Method C (3.5 min) - nominal mass, LC injection with a 3.5 min
gradient (MeOH and 0.1% formic acid), positive ionization
and an injection volume of 2 μL. Column: Phenomenex Gemini
C18 column (3 μm, 30 ꢀ 4.6 mm). High resolution mass spectra
were obtained using the above instrumental setup and the
following conditions: Accurate mass, LC injection with a 10
min gradient (MeOH and 0.1% formic acid), þve ionization
and an injection volume of 4 μL. Column: Phenomenex Gemini
C18 column (5 μm, 50 ꢀ 4.6 mm). Analytical HPLC analysis was
performed on a Thermo-Finnigan Surveyor HPLC system at
30 °C, using a Phenomenex Gemini C18 column (5 μm, 50 ꢀ
4.6 mm) and 10 min gradient of 10 f 90% MeOH/0.1% formic
acid, visualizing at 254, 309, or 350 nm. The purity of final
compounds was determined by analytical HPLC as described
above and is g95% unless specified otherwise. Elemental ana-
lyses were determined by Warwick Analytical Service Ltd.,
Coventry, UK.
2-{4-[6-Bromo-2-(4-dimethylaminophenyl)-3H-imidazo[4,5-b]-
pyridin-7-yl]-piperazin-1-yl}-N-thiazol-2-yl-acetamide (15). To a
mixture 2-[4-(2-amino-5-bromo-3-nitro-pyridin-4-yl)-pipera-
zin-1-yl]-N-thiazol-2-yl-acetamide (0.100 g, 0.22 mmol) and
ethanol (3 mL) was added 4-(dimethylamino)benzaldehyde
(0.044 g, 0.29 mmol) and 1 M aq. Na2S2O4 (900 μL, 0.9 mmol).
The reaction mixture was stirred at reflux for 16 h then con-
centrated in vacuo. The crude product was purified by chroma-
tography on silica gel (dichloromethane/ethyl acetate v/v 7:3,
and then 0.5% to 2% methanol in ethyl acetate) to give the title
compound as an off-white solid (0.034 g, 27%); 1H NMR
(250 MHz, DMSO-d6) 2.77 (broad s, 4H, piperazine N(CH2)2),
2.99 (s, 6H, N(CH3)2), 3.40 (s, 2H, NCH2CO), 3.67 (broad s, 4H,
piperazine N(CH2)2), 6.82 (d, J = 8.8 Hz, 2H) and 8.02 (d, J =
8.8 Hz, 2H) 2,6-ArH and 3,5-ArH, 7.23 (d, J = 3.5 Hz, 1H) and
7.49 (d, 1H, J = 3.5 Hz) (thiazole 4-H, 5-H), 8.15 (s, 1H,
imidazo[4,5-b]pyridine 5-H), 11.80 (broad s, 1H, CONH),
13.12 (broad s, 1H, imidazo[4,5-b]pyridine NH); LC (Method A)-
MS (ESI, m/z) Rt = 6.0 min - 541, 543 [(M þ Hþ), Br isotopic
pattern]; ESI-HRMS Found: 541.1132, calculated for C23H26-
BrN8OS (M þ H)þ: 541.1128.
2-Amino-4,5-dichloro-3-nitropyridine (12). To a 50 mL round-
bottomed flask containing 2-amino-4,5-dichloropyridine30
(0.275 g, 1.65 mmol) and cooled into an ice-bath was slowly
added conc. H2SO4 (2.79 g). The reaction mixture was stirred for
3 min and then HNO3 (70%; 0.186 g) was dropwise added. The

5224 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Bavetsias et al.
1
reaction mixture was stirred at 0 °C (ice-bath) for 7 min, then
heated to 55 °C and stirred at this temperature for 1 h, allowed to
cool to room temperature, and diluted with ice-water (∼15 mL),
and the pH was adjusted to ∼7.5 with 10% aqueous NaOH. The
yellow precipitate was collected by filtration, washed with water,
and dried in vacuo over P2O5, then absorbed on silica gel, and the
free running powder was placed on a 10 g isolute silica column.
Elution with 2% ethyl acetate in dichloromethane afforded
gave the pure product as a colorless solid; H NMR (500 MHz,
DMSO-d6) 2.80 (t, br, J = 4.7 Hz, 4H, piperazine N(CH2)2), 3.00
(s,6H,N(CH3)2), 3.42 (s, 2H, NCH2CO),4.11(s,br,4H, piperazine
N(CH2)2), 6.82 (d, J = 9.0 Hz, 2H, N,N-dimethylaminophenyl H-3
& H-5), 7.24 (d, J = 3.5 Hz, 1H, thiazole H-4 or H-5), 7.50 (d, J =
3.5 Hz, 1H, thiazole H-4 or H-5), 7.99 (d, J = 9.0 Hz, 2H, N,N-
dimethylaminophenyl H-2 & H-6), 8.23 (s, 1H, imidazo[4,5-b]-
pyridine H-5), 11.91 (br s, 1H, CONH), 13.46 (br s, 1H, imidazo-
[4,5-b]pyridine NH); LC (Method A) - MS (ESI, m/z): 6.87 min -
488 [(M þ H)þ, 100%]; ESI-HRMS: Found: 488.1993, calculated
for C24H26N9OS (MþH)þ: 488.1981.
1
the title compound as a yellow solid (0.090 g, 26%); H NMR
(250 Mz, DMSO-d6) 7.39 (s, 2H, NH2), 8.39 (s, 1H, 6-H).
2-(4-(2-Amino-5-chloro-3-nitropyridin-4-yl)piperazin-1-yl)-
N-(thiazol-2-yl)acetamide (14). 2-(Piperazin-1-yl)-N-(thiazol-2-yl)-
acetamide ꢀ 2HCl salt (0.209 g, 0.70 mmol) was suspended in
iPrOH (12 mL) and DIPEA (0.295 g, 2.30 mmol). To this solution,
4,5-dichloro-3-nitropyridin-2-amine (0.130 g, 0.63 mmol) was
added, and the reaction mixture was heated and stirred at 45 °C
for 17 h. The mixture was then allowed to cool to room tempera-
ture, diluted with isopropanol (10 mL), filtered, washed with
iPrOH (3 ꢀ 3 mL), Et2O (2 ꢀ 5 mL), and dried to give the title
compound as a yellow solid (0.200 g, 80%); 1H NMR (500 MHz,
DMSO-d6): 2.68 (m, 4H, piperazine N(CH2)2), 3.10 (m, 4H,
piperazine N(CH2)2), 3.38 (s, 2H, NCH2CO), 6.95 (brs, 2H,
NH2), 7.22 (d, 1H, J = 2.5 Hz), 7.47 (d, 1H, J = 2.5 Hz)
(thiazole 4-H and thiazole 5-H), 8.06 (s, 1H, pyridine 6-H), 11.98
(s, 1H, CONH); LC (Method B) - MS (ESI, m/z): Rt = 3.01 min
-398, 400 [(M þ Hþ), Cl isotopic pattern]. The reaction was also
performed using excess of 2-(piperazin-1-yl)-N-(thiazol-2-yl)-
acetamide as a free base which was obtained from its hydrochloride
salt as follows: 2-(Piperazin-1-yl)-N-(thiazol-2-yl)acetamide ꢀ
2HCl salt (0.360 g) was partitioned between saturated aqueous
NaHCO3 (40 mL) and ethyl acetate (30 mL). The aqueous layer
was extracted with ethyl acetate (2 ꢀ 30 mL) and dichloromethane
(5 ꢀ 25 mL). The combined organics were dried (Na2SO4) and
then concentrated in vacuo to give 0.165 g of the free base.
2-(4-(6-Chloro-2-(4-methoxyphenyl)-3H-imidazo[4,5-b]pyridin-
7-yl)piperazin-1-yl)-N-(thiazol-2-yl)acetamide (27). To a mixture
of 2-[4-(2-amino-5-chloro-3-nitro-pyridin-4-yl)-piperazin-1-yl]-
N-thiazol-2-yl-acetamide (0.040 g, 0.10 mmol), ethanol (3 mL),
and p-methoxybenzaldehyde (0.019 g, 0.14 mmol) was added a
freshly prepared aqueous solution of Na2S2O4 (1 M; 0.4 mL,
0.4 mmol). The reaction mixture was heated at 70 °C for 5 h, then
allowed to cool to room temperature and the solvents were
removed in vacuo. The residue was absorbed on silica gel and
the free running powder was placed on a 10 g isolute silica column
which was eluted with 50% dichloromethane in ethyl acetate and
then 2.5% methanol in ethyl acetate/dichloromethane (v/v; 1:1).
The title compound was obtained as a pale yellow solid after
trituration withdiethyl ether (0.012g, 25%); 1H NMR (500 MHz,
DMSO-d6) 2.76 (br s, 4H, piperazine N(CH2)2), 3.40 (s, 2H,
NCH2CO), 3.72 (br s, 4H, piperazine N(CH2)2), 3.83 (s, 3H,
OCH3), 7.10 (d, J = 8.8 Hz, 2H, 3,5-ArH or 2,6-ArH), 7.24 (d,
J = 3.5 Hz, 1H) and 7.50 (d, J = 3.5 Hz, 1H) (thiazole 4-H, 5-H),
8.09 (s, 1H, imidazo[4,5-b]pyridine 5-H), 8.13 (d, J = 8.8 Hz, 2H,
3,5-ArH or 2,6-ArH), 11.95 (s, 1H, CONH), 13.38 (s, 1H, imidazo-
[4,5-b]pyridineN-H) ; LC (Method A) - MS (ESI, m/z):Rt=6.05
min -484, 486 [(M þ H)þ, Cl isotopic pattern]. ESI-HRMS:
Found: 484.1324, calculated for C22H23ClN7O2S (M þ H)þ:
484.1322.
2-(4-(2-Amino-5-chloro-3-nitropyridin-4-yl)piperazin-1-yl)-N-
phenylacetamide (30b). To a mixture of 2-amino-4,5-dichloro-3-
nitropyridine (0.031 g, 0.15 mmol) and isopropanol (3.5 mL) was
added N-phenyl-2-piperazin-1-yl-acetamide ꢀ 2HCl salt (0.048 g,
0.16 mmol) followed by diisopropylethylamine (0.10 mL, 0.57
mmol). The reaction mixture was heated at 45 °C for 18 h, then
allowed to cool to room temperature and the solvents were
removed in vacuo. The residue was absorbed on silica gel and
the free running powder was placed on a 10 g isolute silica column
which was eluted with 10% to 30% ethyl acetate in dichloro-
methane. The title compound was obtained as a yellow solid
(0.041 g, 71%); 1H NMR (500 MHz, DMSO-d6) 2.64 (br s, 4H,
piperazine N(CH2)2), 3.13 (br s, 4H, piperazine N(CH2)2), 3.19 (s,
2H, NCH2CO), 7.00 (br s, 2H, 2-NH2), 7.06 (t, J = 7.3 Hz, 1H,
p-ArH), 7.31 (t, J = 8.3 Hz, 2H, m-ArH), 7.63 (d, J = 7.3 Hz, 2H,
o-ArH), 8.07 (s, 1H, 6-H), 9.76 (s, 1H, CONH); LC (Method A) -
MS (ESI, m/z): Rt = 4.50 min -391, 393 [(M þ H)þ, Cl isotopic
pattern].
2-(4-(6-Chloro-2-(4-(dimethylamino)phenyl)-3H-imidazo[4,5-b]-
pyridin-7-yl)piperazin-1-yl)-N-(thiazol-2-yl)acetamide (7). To a
mixture of 2-[4-(2-amino-5-chloro-3-nitro-pyridin-4-yl)-piperazin-
1-yl]-N-thiazol-2-yl-acetamide (0.040 g, 0.10 mmol), ethanol
(3 mL), and 4-(dimethylamino)benzaldehyde (0.019 g, 0.13 mmol)
was added a freshly prepared aqueous solution of Na2S2O4 (1M;
0.4 mL, 0.4 mmol). The reaction mixture was heated at 70 °C for
3.5 h, then allowed to cool to room temperature and the solvents
were removed in vacuo. The residue was absorbed on silica gel,
and the free running powder was placed on a 10 g isolute silica
column which was eluted with ethyl acetate/dichloromethane (v/v;
1:1), 1.5% methanol in ethyl acetate/dichloromethane (v/v; 1:1),
and finally 2% methanol in ethyl acetate/dichloromethane (v/v;
1:1). The title compound was obtained after trituration with
1
diethyl ether as a pale yellow solid (0.005 g, 10%); H NMR
(250 MHz, DMSO-d6) 2.78 (m, 4H, piperazine N(CH2)2), 3.01 (s,
6H, N(CH3)2), 3.40 (s, 2H, NCH2CO), 3.72 (m, 4H, piperazine
N(CH2)2), 6.83 (d, J = 8.8 Hz, 2H, 3,5-ArH or 2,6-ArH), 7.25 (d,
J = 3.4 Hz, 1H) and 7.51 (d, J = 3.5 Hz, 1H) (thiazole 4-H, 5-H),
8.03 (d, 3H, J = 9.9 Hz, 3,5-ArH or 2,6-ArH, and imidazo[4,5-b]-
pyridine 5-H); 11.95 (s, 1H, CONH), 13.12 (s, 1H, imidazo[4,5-b]-
pyridine N-H); LC (Method A) - MS (ESI, m/z) 6.17 min -497,
499 [(M þ H)þ, Cl isotopic pattern]; ESI-HRMS Found:
497.1652, calculated for C23H26N8ClOS (M þ H)þ: 497.1633.
2-(4-(6-Cyano-2-(4-(dimethylamino)phenyl)-3H-imidazo[4,5-b]-
pyridin-7-yl)piperazin-1-yl)-N-(thiazol-2-yl)acetamide (16). A solu-
tion of 2-(4-(6-bromo-2-(4-(dimethylamino)phenyl)-3H-imidazo-
[4,5-b]pyridin-7-yl)piperazin-1-yl)-N-(thiazol-2-yl)acetamide (75 mg,
0.14 mmol) in degassed DMF (1 mL) containing Pd2dba3 (0.05 eq,
0.0069 mmol, 6 mg), dppf (0.1 eq, 0.014 mmol, 8 mg), and Zn(CN)2
(1.5 eq, 0.21 mmol, 24 mg) was stirred with microwave heating at
180 °C for 30 min. After this time, further Pd2(dba)3 (6 mg), dppf (8
mg), and Zn(CN)2 (24 mg) were added and the mixture was stirred
under the same conditions for a further 30 min. HPLC then showed
partial conversion to the desired compound. Concentration in
vacuo and purification of a small sample by semipreparative HPLC
2-(4-(6-Chloro-2-(4-(dimethylamino)phenyl)-3H-imidazo[4,5-b]-
pyridin-7-yl)piperazin-1-yl)-N-phenylacetamide (31b). To a mixture
of 2-(4-(2-amino-5-chloro-3-nitropyridin-4-yl)piperazin-1-yl)-
N-phenylacetamide (0.040 g, 0.10 mmol), ethanol (3 mL), and
4-dimethylaminobenzaldehyde (0.019 g, 0.13 mmol) was added a
freshly prepared aqueous solution of Na2S2O4 (1 M; 0.40 mL,
0.40 mmol). The reaction mixture was heated at 70 °C for 3 h,
then allowed to cool to room temperature and the solvents were
removed in vacuo. The residue was absorbed on silica gel and the
free running powder was placed on a 10 g isolute silica column
which was eluted with 20% ethyl acetate in dichloromethane and
then 1% methanol in ethyl acetate/dichloromethane (v/v; 1:1).
The title compound was obtained as a yellow solid after tritura-
1
tion with diethyl ether (0.006 g, 12%); H NMR (500 MHz,
DMSO-d6) 2.76 (br s, 4H, piperazine N(CH2)2), 3.00 (s, 6H,
N(CH3)2), 3.23 (s, 2H, NCH2CO), 3.75 (br s, 4H, piperazine
N(CH2)2), 6.81 (d, J = 7.9 Hz, 2H), and 8.01 (d, J = 8.5 Hz, 2H)

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5225
(3,5-C6H4NMe2 and 2,6-C6H4-NMe2), 7.07 (t, J = 7.3 Hz, 1H,
p-ArH), 7.32 (t, J = 8.5 Hz, 2H, m-ArH), 7.67 (d, J = 8.5 Hz, 2H,
o-ArH), 8.06 (s, 1H, imidazo[4,5-b]pyridine 5-H), 9.77 (s, 1H,
CONH), 13.11 (s, 1H, imidazo[4,5-b]pyridine N-H); LC
(Method B) - MS (ESI, m/z): Rt = 4.00 min -490, 492 [(M þ H)þ,
Cl isotopic pattern]. ESI-HRMS: Found: 490.2128, calculated for
C26H29ClN7O (M þ H)þ: 490.2122.
(0.035 g). The filtrate was concentrated in vacuo, and purifica-
tion of the residue on a isolute silica column using 0 to 5%
methanol in ethyl acetate/dichloromethane (v/v; 1:1) as eluant
gave an additional 0.036 g of the product (total yield: 81%); 1H
NMR (500 MHz, DMSO-d6) 3.09 (br t, J = 4.4 Hz, 4H,
piperazine N(CH2)2), 3.57 (s, 2H, NCH2), 6.96 (s, 2H, NH2),
7.34 (d, J = 5.8 Hz, 2H) and 8.51 (d, J = 5.9 Hz, 2H) (pyridine
H-2 & H-6, H-3 & H-5), 8.06 (s, 1H, 6-H); LC (Method B) - MS
(ESI, m/z): Rt = 1.95 min -349, 351 [(M þ H)þ, Cl isotopic
pattern].
tert-Butyl 4-(2-amino-5-bromo-3-nitropyridin-4-yl)piperazine-
1-carboxylate (17). To a mixture of 5-bromo-4-chloro-3-nitro-
pyridin-2-ylamine (0.126 g, 0.50 mmol) and isopropanol (9 mL)
was added 1-BOC-piperazine (0.102 g, 0.55 mmol) followed by
diisopropylethylamine (0.10 mL, 0.57 mmol). The reaction
mixture was heated at 45 °C for 20 h, then allowed to cool to
room temperature, and diluted with isopropanol (3 mL). The
precipitate was collected by filtration and washed with isopro-
panol and diethyl ether. The title compound was thus obtained
as a yellow solid (0.112 g, 56%). 1H NMR (500 MHz, DMSO-
d6) 1.42 (s, 9H, C(CH3)3), 2.99 (br s, 4H, piperazine N(CH2)2),
3.45 (br s, 4H, piperazine N(CH2)2), 7.02 (s, 2H, NH2), 8.20 (s,
1H, 6-H); LC (Method B) - MS (ESI, m/z): Rt = 5.00 min -402,
404 [(M þ H)þ, Br isotopic pattern].
6-Chloro-2-(4-methoxyphenyl)-7-(4-(pyridin-4-ylmethyl)piper-
azin-1-yl)-3H-imidazo[4,5-b]pyridine (36a). To a mixture of 5-chloro-
3-nitro-4-(4-(pyridin-4-ylmethyl)piperazin-1-yl)pyridin-2-amine
(0.031 g, 0.09 mmol) and ethanol (3.0 mL) was added 4-methoxy-
benzaldehyde (0.020 g, 0.14 mmol) with the aid of ethanol (1 mL)
followed by a freshly prepared aqueous solution of Na2S2O4 (1 M;
0.36 mL, 0.36 mmol). The reaction mixture was heated at 70 °C for
5 h, then allowed to cool to room temperature and the solvents
were removed in vacuo. The residue was triturated with water, and
the precipitate was collected by filtration, washed with water,
ethanol, and diethyl ether. This material was further purified on
a 10 g isolute silica column using a gradient of methanol (0 to 5%)
in ethyl acetate/dichloromethane (v/v; 1:1) as eluant. The title
compound was obtained as a pale yellow solid (0.007 g, 18%); 1H
NMR (500 MHz, DMSO-d6) 2.61 (br s, 4H, piperazine N(CH2)2),
3.71 (br s, 4H, piperazine N(CH2)2), 3.61 (s, 2H, NCH2), 3.83 (s,
3H, OCH3), 7.09 (d, J = 8.8 Hz, 2H) and 8.12 (d, J = 8.8 Hz, 2H)
(3,5-C6H4OMe and 2,6-C6H4-OMe), 7.40 (d, J = 5.8 Hz, 2H)
and 8.54 (d, J= 4.5 Hz, 2H) (pyridine H-2 & H-6, H-3 & H-5), 8.08
(s, 1H, imidazo[4,5-b]pyridine 5-H), 13.37 (s, 1H, imidazo[4,5-b]-
pyridine N-H); LC (Method B) - MS (ESI, m/z): Rt = 3.20 min
-435, 437 [(M þ H)þ, Cl isotopic pattern]. ESI-HRMS: Found:
435.1695, calculated for C23H24ClN6O (M þ H)þ: 435.1700.
5-Bromo-3-nitro-4-(4-(pyridin-3-ylmethyl)piperazin-1-yl)pyri-
din-2-amine (38a). To a mixture of 5-bromo-4-chloro-3-nitro-
pyridin-2-ylamine (0.126 g, 0.50 mmol) and isopropanol (9 mL)
was added 1-[(3-pyridyl)-methyl]-piperazine (0.097 g, 0.55 mmol)
followed by diisopropylethylamine (0.10 mL, 0.57 mmol). The
reaction mixture was heated at 45 °C for 18 h, it was then allowed
to cool to room temperature. The precipitate was collected by
filtrationandwashedwithisopropanolanddiethylether. The title
compound was thus obtained as a yellow solid (0.160 g, 82%); 1H
NMR (500 MHz, DMSO-d6) 3.05 (br s, 4H, piperazine
N(CH2)2), 3.56 (s, 2H, NCH2), 7.02 (s, 2H, NH2), 7.36 (dd, J =
7.8, 4.8 Hz, 1H, pyridyl 5-H), 7.74 (dt, J = 7.8, 1.70 Hz, 1H,
pyridyl 4-H), 8.16 (s, 1H, 6-H), 8.47 (dd, J = 4.8, 1.6 Hz, 1H,
pyridyl 6-H), 8.50 (d, J = 1.6 Hz, 1H, pyridyl 2-H); LC (Method
B) - MS (ESI, m/z): Rt = 1.79 min -393, 395 [(M þ H)þ, Br
isotopic pattern].
4-(2-Amino-5-bromo-3-nitropyridin-4-yl)-N-phenylpiperazine-
1-carboxamide (30e). A solution of tert-butyl 4-(2-amino-5-bromo-
3-nitropyridin-4-yl)piperazine-1-carboxylate (250 mg, 0.62 mmol)
in CH2Cl2 (2.5 mL) at 0 °C was treated with TFA (2.5 mL) and
stirred at 0 °C for 1.5 h. After this time, the solvents were
evaporated in vacuo and the excess TFA was removed by azeo-
troping with toluene (3 ꢀ 10 mL). The residue was suspended in
CHCl3 (2.5 mL) and treated with DIPEA (5 eq, 3.11 mmol, 0.54
mL) and phenyl isocyanate (1.05 eq, 0.65 mmol, 0.07 mL). The
reaction was warmed to room temperature and stirred for 12 h.
The formed precipitate was filtered off and dried to give the
product as a yellow solid (221 mg, 84% for two steps); 1H NMR
(500 MHz, DMSO-d6) 3.07 (br s, 4H, piperazine N(CH2)2), 3.58
(br s, 4H, piperazine N(CH2)2), 6.94 (tt, J = 7.4, 1.1 Hz, 1H, phenyl
H-4), 7.03 (br s, 2H, NH2), 7.24 (dd, J = 8.5, 7.5 Hz, 2H, phenyl
H-3 & H-5), 7.45 (dd, J = 8.5, 1.1 Hz, 2H, phenyl H-2 & H-6), 8.21
(s, 1H, pyridine H-6), 8.59 (br s, 1H, NH); LC (Method C) - MS
(ESI, m/z): Rt = 2.34 min -421, 423 [(M þ H)þ, Br isotopic
pattern].
4-(6-Bromo-2-(4-(dimethylamino)phenyl)-3H-imidazo[4,5-b]-
pyridin-7-yl)-N-phenylpiperazine-1-carboxamide (31e). A solution
of 4-(2-amino-5-bromo-3-nitropyridin-4-yl)-N-phenylpiperazine-
1-carboxamide (100 mg, 0.25 mmol) and 4-(dimethylamino)-
benzaldehyde (1.05 eq, 0.26 mmol, 39 mg) in DMF (1.25 mL)
was treated with a freshly prepared 1 M aqueous solution of
Na2S2O4 (3 eq, 0.75 mmol, 0.75 mL) and stirred at 80 °C for 4 h.
After this time, the precipitate which had formed was filtered off
and washed with hexane to give the product (64 mg, 53%) as an off-
white solid; 1H NMR (500 MHz, DMSO-d6) 3.00(s,6H,N(CH3)2),
3.64-3.68 (m, 8H, 2 ꢀ piperazine N(CH2)2), 6.82 (d, J = 8.4 Hz,
2H, N,N-dimethylaminophenyl H-3 & H-5), 6.94 (t, J = 7.4 Hz,
1H, phenyl H-4), 7.25 (t, J = 7.5 Hz, 2H, phenyl H-3 & H-5), 7.51
(d, J = 8.2 Hz, 2H, phenyl H-2 & H-6), 8.02 (d, J = 8.2 Hz, 2H,
N,N-dimethylaminophenyl H-2 & H-6), 8.20 (s, 1H, imidazo[4,5-b]-
pyridine H-5), 8.61 (s, br, 1H, PhNH), 13.19 (s, br, 1H, imidazo[4,5-
b]pyridine NH); LC (Method A) - MS (ESI, m/z): Rt = 8.34 min
-520, 522 [(M þ H)þ, bromine isotopic pattern). ESI-HRMS:
Found: 520.1450, calculated for C25H27BrN7O (M þ H)þ:
520.1460. Analytical hplc: (λ 254 nm) Rt 8.98 min, area 94.0%.
5-Chloro-3-nitro-4-(4-(pyridin-4-ylmethyl)piperazin-1-yl)pyri-
din-2-amine (intermediate for the synthesis of 36a). To a mixture
of 2-amino-4,5-dichloro-3-nitropyridine (0.052 g, 0.25 mmol)
and isopropanol (4.5 mL) was added 1-[(4-pyridyl)-methyl]-
piperazine (0.049 g, 0.28 mmol) followed by diisopropylethyl-
amine (0.05 mL, 0.28 mmol). The reaction mixture was heated at
45 °C for 24 h, then allowed to cool to room temperature, and
diluted with isopropanol (3 mL). The formed precipitate was
collected by filtration and washed with isopropanol and diethyl
ether. The title compound was thus obtained as yellow solid
6-Bromo-2-(4-morpholin-4-ylmethyl-phenyl)-7-(4-pyridin-3-yl-
methyl-piperazin-1-yl)-3H-imidazo[4,5-b]pyridine (40a). To a mix-
ture of 5-bromo-3-nitro-4-(4-pyridin-3-ylmethyl-piperazin-1-yl)-
pyridin-2-ylamine (0.047 g, 0.12 mmol) and EtOH (3.5 mL) was
added 4-(morpholin-4-ylmethyl)benzaldehyde (0.032 g, 0.16 mmol)
followed by a freshly prepared aqueous solution of Na2S2O4 (1 M;
0.48 mL, 0.48 mmol). The reaction mixture was stirred at 80 °C for
20 h, then allowed to cool to room temperature and concentrated in
vacuo. The residue was absorbed on silica gel, the free-running
powder was placed on a 10 g isolute silica column, and elution of the
column with a gradient of methanol (2-12%) in ethyl acetate/
dichloromethane (v:v; 4:1) afforded a yellow solid which was
triturated with diethyl ether. The precipitate was collected by
filtration and was successively washed with diethyl ether, water,
1
and diethyl, then dried in vacuo (0.009 g, 14%). H NMR (500
MHz, DMSO-d6) 2.38 (br s, 4H), 2.62 (br s, 4H), 3.59 (t, J =
4.6 Hz, 4H) and 3.67 (br s, 4H) (morpholine N(CH2)2, morpholine
O(CH2)2), and piperazine N(CH2)2), 3.53 (s, 2H) and 3.62 (s, 2H)
(NCH2-pyridyl and C6H4CH2), 7.39 (dd, J = 5.3, 7.1 Hz, 1H,
pyridine 5-H), 7.47 (d, J = 7.7 Hz, 2H) and 8.14 (d, J= 8.0 Hz, 2H)
(3,5-C6H4 and 2,6-C6H4), 7.78 (d, J = 7.5 Hz, 1H, pyridine 4-H),

5226 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Bavetsias et al.
8.23 (s, 1H, imidazo[4,5-b]pyridine 5-H), 8.50 (dd, J = 1.6, 4.7 Hz,
1H, pyridine 6- H), 8.56 (d, J= 1.6 Hz, 1H, pyridine 2-H), 13.48 (br
s, 1H, imidazo[4,5-b]pyridine N-H); LC (Method B) - MS (ESI,
m/z): Rt = 1.94 min -548, 550 [(M þ H)þ, Br isotopic pattern].
ESI-HRMS: Found: 548.1776, calculated for C27H31BrN7O (M þ
H)þ: 548.1773.
5-Bromo-4-(4-(4-chlorobenzyl)piperazin-1-yl)-3-nitropyridin-
2-amine (38c). To a mixture of 5-bromo-4-chloro-3-nitro-pyri-
din-2-ylamine (0.126 g, 0.50 mmol) and isopropanol (15 mL) was
added 1-(4-chlorobenzyl)piperazine (0.115 g, 0.55 mmol) fol-
lowed by diisopropylethylamine (0.10 mL, 0.55 mmol). The
reaction mixture was heated at 45 °C for 18 h, then allowed to
cool to room temperature. The precipitate was collected by
filtration and washed with isopropanol and diethyl ether. The
title compound was thus obtained as a yellow solid (0.148 g,
70%); 1H NMR (500 MHz, DMSO-d6) 3.05 (br s, 4H, piperazine
N(CH2)2), 3.52 (s, 2H, NCH2), 7.02 (s, 2H, NH2), 7.34 (d, J =
8.5 Hz, 2H) and 7.38 (d, J = 8.5 Hz, 2H) (3,5-ArH and 2,6-ArH),
8.16 (s, 1H, 6-H); LC (Method B) - MS (ESI, m/z): Rt = 2.92 min
-426, 428, 430 [(M þ H)þ, BrCl isotopic pattern].
5-Bromo-4-[4-(5-methyl-isoxazol-3-ylmethyl)-piperazin-1-yl]-
3-nitro-pyridin-2-ylamine (38e). A solution of 4-(5-methyl-iso-
xazol-3-ylmethyl)-piperazine-1-carboxylic acid tert-butyl ester
(0.200 g, 0.71 mmol) in dichloromethane (6 mL) and TFA
(8 mL) was stirred at room temperature for 2 h then concen-
trated in vacuo, and the resulting residue was dried in vacuo to
give 37e. This material (supposedly 0.70 mmol) was dissolved in
isopropanol (13 mL) and to this solution 2-amino-5-bromo-4-
chloro-3-nitropyridine (0.157 g, 0.63 mmol) was added followed
by diisopropylethylamine (0.65 mL, 3.70 mmol). The reaction
mixture was stirred at 45 °C for 20 h, then allowed to cool to
room temperature and diluted with isopropanol (5 mL). The
resulting orange solid was collected by filtration, washed with
isopropanol (2 ꢀ 5 mL), diethyl ether (3 ꢀ 5 mL), and dried
1
(0.170 g, 68%); H NMR (500 MHz, DMSO-d6) 2.38 (s, 3H,
isoxazole 5-CH3), 2.53 (br s, 4H, piperazine N(CH2)2), 3.05 (br s,
4H, piperazine N(CH2)2), 3.55 (s, 2H, NCH2-isoxazole), 6.21 (s,
1H, isoxazole 4-H), 6.97 (s, 2H, NH2), 8.16 (s, 1H, pyridine 6-H);
LC (Method B) - MS (ESI, m/z): Rt = 2.67 min -397, 399 [(M þ
H)þ, Br isotopic pattern].
6-Bromo-2-(4-methoxyphenyl)-7-[4-(5-methyl-isoxazol-3-
ylmethyl)-piperazin-1-yl]-3H-imidazo[4,5-b]pyridine (39e). To a
mixture of 5-bromo-4-[4-(5-methyl-isoxazol-3-ylmethyl)-
piperazin-1-yl]-3-nitro-pyridin-2-ylamine (0.044 g, 0.11 mmol)
and EtOH (3 mL) was added 4-methoxybenzaldehyde (0.023 g,
0.17 mmol) with the aid of EtOH (1 mL), followed by a freshly
prepared aqueous solution of Na2S2O4 (1 M; 0.44 mL,
0.44 mmol). The reaction mixture was stirred at 70 °C for 18 h,
then allowed to cool to room temperature and concentrated in
vacuo. The resulting residue was absorbed on silica, and the free-
running powder was placed on a 10 g isolute silica column. Elution
with ethyl acetate/dichloromethane (v:v; 3:7) and then 2.5%
methanol in ethyl acetate/dichloromethane (v:v; 1:1) afforded a
yellow solid. The title compound was obtained as a pale yellow
solid after trituration with diethyl ether (0.023 g, 43%); 1H NMR
(500 MHz, DMSO-d6) 2.40 (s, 3H, isoxazole 5-CH3), 2.64 (br s,
4H, piperazine N(CH2)2), 3.60 (s, 2H, NCH2), 3.65 (br s, 4H,
piperazine N(CH2)2), 3.84 (s, 3H, OCH3), 6.25 (s, 1H, isoxazole
4-H), 7.10 (d, J = 8.9 Hz, 2H) and 8.13 (d, J = 8.8 Hz, 2H) (3,5-
C6H4OMe and 2,6-C6H4OMe), 8.20 (s, 1H, imidazo[4,5-b]-
pyridine 5-H), 13.35 (br s, 1H, imidazo[4,5-b]pyridine N-H);
LC (Method B) - MS (ESI, m/z): Rt = 3.80 min -483, 485 [(M þ
H)þ, Br isotopic pattern]. ESI-HRMS: Found: 483.1150, calcu-
lated for C22H24BrN6O2 (M þ H)þ: 483.1144.
6-Bromo-7-[4-(4-chlorobenzyl)-piperazin-1-yl]-2-(4-morpholin-
4-ylmethyl-phenyl)-3H-imidazo[4,5-b]pyridine (40c). To a mix-
ture of 5-bromo-4-[4-(4-chlorobenzyl)-piperazin-1-yl]-3-nitro-
pyridin-2-ylamine (0.060 g, 0.14 mmol) and EtOH (6.5 mL)
was added 4-(morpholin-4-ylmethyl)benzaldehyde (0.040 g,
0.19 mmol) followed by a freshly prepared aqueous solution of
Na2S2O4 (1 M; 0.63 mL, 0.63 mmol). The reaction mixture was
stirred at 80 °C for 20 h, then allowed to cool to room tempera-
ture and concentrated in vacuo. The residue was absorbed on
silica gel, the free-running powder was placed on a 10 g isolute
silica column and elution with a gradient of methanol (0-8%) in
ethyl acetate/dichloromethane (v:v; 1:1) afforded a yellow solid.
The title compound was obtained as a pale yellow solid after
trituration with diethyl ether (0.038 g, 46%). 1H NMR (500
MHz, DMSO-d6) 2.38 (br s, 4H), 2.61 (br s, 4H) 3.59 (t, J = 4.5
Hz, 4H), and 3.66 (br s, 4H) (piperazine N(CH2)2, morpholine
N(CH2)2 and morpholine O(CH2)2), 3.54 (s, 2H) and 3.57 (s, 2H)
(NCH2-C6H4Cl and C6H4CH2), 7.41 (m, 4H, C6H4Cl), 7.47 (d,
J = 8.2 Hz, 2H) and 8.14 (d, J = 8.2 Hz, 2H) (3,5-C6H4 and 2,6-
C6H4), 8.23 (s, 1H, imidazo[4,5-b]pyridine 5-H), 13.48 (br s, 1H,
imidazo[4,5-b]pyridine N-H); LC (Method B) - MS (ESI, m/z):
Rt = 2.55 min -581, 583, 585 [(M þ H)þ, BrCl isotopic pattern].
ESI-HRMS: Found: 581.1424, calculated for C28H31BrClN6O
(M þ H)þ: 581.1426. This compound was also converted to its
hydrochloride salt as follows: To a mixture of 40c (0.021 g, 0.036
mmol) and dichloromethane (1.0 mL) was slowly added a
solution of HCl in methanol (1.25 M; 1.1 mL). The resulting
clear solution was stirred at room temperature for 2 min, then
diethyl ether (15 mL) was added. The white precipitate was
collected by filtration, washed with diethyl ether and dried in
vacuo over P2O5 to afford the title compound (0.020 g) as a 3 ꢀ
HCl salt. LC (Method B) - MS (ESI, m/z): Rt = 2.55 min -581,
583, 585 [(M þ H)þ, BrCl isotopic pattern]; Anal. (C28H30-
BrClN6O ꢀ 3HCl ꢀ 2H2O) Cl; calcd 19.50, found 19.38.
4-(5-Methyl-isoxazol-3-ylmethyl)-piperazine-1-carboxylic acid
tert-butyl ester (43e). To a solution of 3-bromomethyl-5-methyl-
isoxazole (0.102 g, 0.58 mmol) in dichloromethane (6 mL) was
added 1-BOC-piperazine (0.240 g, 1.30 mmol). The reaction
mixture was stirred at room temperature for 18 h under argon,
then concentrated in vacuo. The resulting residue was absorbed
on silica and the free-running powder was placed on a 10 g isolute
silica column. Elution with a gradient of ethyl acetate (30 to 70%)
in petroleum ether (60-80 °C) afforded the title compound as a
white solid (0.124 g, 76%). 1H NMR (500 MHz, DMSO-d6) 1.39
(s, 9H, C(CH3)3), 2.32 (t, J = 5.1 Hz, 4H, piperazine N(CH2)2),
2.38 (s, 3H, isoxazole 5-CH3), 3.35 (br t, 4H, piperazine
N(CH2)2), 3.50 (s, 2H, NCH2 isoxazole), 6.17 (s, 1H, isoxazole
4-H); LC (Method B) - MS (ESI, m/z): Rt = 2.60 min -282 [(M þ
H)þ, 5%], 226 [(M - tBu)þ, 100%].
3-((4-(6-Bromo-2-(4-(4-methylpiperazin-1-yl)phenyl)-3H-imidazo-
[4,5-b]pyridin-7-yl)piperazin-1-yl)methyl)-5-methylisoxazole (51). To
a mixture of 5-bromo-4-(4-((5-methylisoxazol-3-yl)methyl)piper-
azin-1-yl)-3-nitropyridin-2-amine (0.052 g, 0.13 mmol) and EtOH
(5 mL) was added 4-(4-methylpiperazino)benzaldehyde (0.032 g,
0.16 mmol) followed by a freshly prepared aqueous solution of
Na2S2O4 (1 M; 0.52 mL, 0.52 mmol). The reaction mixture was
stirred at 80 °C for 18 h, then allowed to cool to room temperature
and concentrated in vacuo. The residue was absorbed on silica gel,
and the free-running powder was placed on a 10 g isolute silica
column. Elution with ethyl acetate/chloroform (v:v; 1:1), and then
a gradient of methanol (5-12%) in chloroform afforded a yellow
solid. This solid was triturated with diethyl ether, and the formed
yellow precipitate was collected by filtration and was successively
washed with diethyl ether, water, and diethyl ether. The title
compound was obtained as a pale yellow solid (0.022 g, 31%).
1H NMR (500 MHz, DMSO-d6) 2.23 (s, 3H, piperazine N-Me),
2.40 (s, 3H, isoxazole 5-CH3), 2.46 (br t, J =4.8 Hz, 4H), 2.63 (br s,
4H), 3.27 (br s obscured by water peak), and 3.63 (br t, J = 4.7 Hz,
4H) (piperazine NCH2), 3.60 (s, 2H, N-CH2-isoxazole), 6.25 (s,
1H, isoxazole 4-H), 7.06 (d, J = 9.0 Hz, 2H), and 8.02 (d, J = 8.9
Hz, 2H) (2,6-C6H4 and 3,5-C6H4), 8.17 (s, 1H, imidazo[4,5-b]-
pyridine 5-H), 13.20 (br s, 1H, imidazo[4,5-b]pyridine N-H); LC
(Method B) - MS (ESI, m/z): Rt = 2.33 min -551, 553 [(M þ H)þ,
Br isotopic pattern]. ESI-HRMS: Found: 551.1870, calculated for
C26H32BrN8O (M þ H)þ: 551.1877.

Article
Acknowledgment. The authors are grateful to Chroma
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5227
Chieffi, P.; Fusco, A.; Portella, G. Aurora B Overexpression Associ-
ates with the Thyroid Carcinoma Undifferentiated Phenotype and Is
Required for Thyroid Carcinoma Cell Proliferation. J. Clin. Endo-
crinol. Metab. 2005, 90, 928–935.
Therapeutics, and in particular to Dr. D. Moffat, for valuable
discussions, the kinase selectivity profiling of 40c, and the
provision of bulk intermediates 11 and 17. We acknowledge
NHS funding to the NIHR Biomedical Research Centre. This
work was supported by Cancer Research UK [CUK] grant
number C309/A8274. R.B. is a Royal Society University
Research Fellow and acknowledges the support of Break-
through Breast Cancer Project Grant AURA 05/06 and
infrastructural support for Structural Biology at the ICR
from Cancer Research UK. We also thank Dr. Amin Mirza
and Mr. Meirion Richards for their assistance with NMR,
mass spectrometry, and HPLC.
(17) Ota, T.; Suto, S.; Katayama, H.; Han, Z-B; Suzuki, F.; Maeda, M.;
Tanino, M.; Terada, Y.; Tatsuka, M. Increased Mitotic Phosphor-
ylation of Histone H3 Attributable to AIM-1/Aurora-B Over-
expression Contributes to Chromosome Number Instability.
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Ajose-Adeogun, A. O.; Nakayama, T.; Graham, J. A.; Demur, C.;
Hercend, T.; Diu-Hercend, A.; Su, M.; Golec, J. M. C.; Miller,
K. M. VX-680, a Potent and Selective Small-Molecule Inhibitor of
the Aurora Kinases, Suppresses Tumor Growth in Vivo. Nat. Med.
2004, 10, 262–267.
(21) Fancelli, D.; Moll, J.; Varasi, M.; Bravo, R.; Artico, R.; Berta, D.;
Bindi, S.; Cameron, A.; Candiani, I.; Cappella, P.; Carpinelli, P.;
Croci, W.; Forte, B.; Giorgini, M. L.; Klapwijk, J.; Marsiglio, A.;
Pesenti, E.; Roccheti, M.; Roletto, F.; Severino, D.; Soncini, C.;
Storici, P.; Tonani, R.; Zugnoni, P.; Vianello, P. 1,4,5,6-
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(22) Caprinelli., P.; Ceruti, R.; Giorgini, M. L.; Cappella, P.; Gianellini,
L.; Croci, V.; Degrassi, A.; Texido, G.; Rocchetti, M.; Vianello, P.;
Rusconi, L.; Storici, P.; Zugnoni, P.; Arrigoni, C.; Soncini, C.; Alli,
C.; Patton, V.; Marsiglio, A.; Ballinari, D.; Pesenti, E.; Fancelli, D.;
Moll, J. PHA-739358, a Potent Inhibitor of Aurora Kinases With a
Selective Target Inhibition Profile Relevant to Cancer. Mol. Can-
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(23) Howard, S.; Berdini, V.; Boulstridge, J. A.; Carr, M. G.; Cross,
D. M.; Curry, J.; Devine, L. A.; Early, T. R.; Fazal, L.; Gill, A. L.;
Heathcote, M.; Maman, S.; Matthews, J. E.; McMenamin, R. L.;
Navarro, E. F.; O’Brien, M. A.; O’Reilly., M.; Rees, D. C.; Reule,
Supporting Information Available: Experimental procedures
for compounds 18-26, 28, 29d, 29i, 30c, 30d, 30f-l, 31a, 31c,
31d, 31f-l, 33, 35, 36b, 37b,g, 42b,g, 37f,h, 43f,h, 38b,d, 38f-h,
39a-d, 39f-h, 40b, 40e, 40g, 40h, 44-50, 52-54, summary of
crystallographic analysis of compounds 40c and 51 (Table S1)
and kinase selectivity profiling of compound 51 (Table S2). This
material is available free of charge via the Internet at http://
pubs.acs.org.
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