Aurora isoform selectivity: Design and synthesis of imidazo[4,5- B ]pyridine derivatives as highly selective inhibitors of aurora-A kinase in cells

Article abstract of DOI:10.1021/jm401115g

Aurora-A differs from Aurora-B/C at three positions in the ATP-binding pocket (L215, T217, and R220). Exploiting these differences, crystal structures of ligand-Aurora protein interactions formed the basis of a design principle for imidazo[4,5-b]pyridine-

Full text of DOI:10.1021/jm401115g

Article
pubs.acs.org/jmc
Aurora Isoform Selectivity: Design and Synthesis of
Imidazo[4,5‑b]pyridine Derivatives as Highly Selective Inhibitors of
Aurora‑A Kinase in Cells
Vassilios Bavetsias,*,† Amir Faisal,† Simon Crumpler,† Nathan Brown,† Magda Kosmopoulou,‡
Amar Joshi,§ Butrus Atrash,† Yolanda Perez-Fuertes,† Jessica A. Schmitt,† Katherine J. Boxall,†
́
Rosemary Burke,† Chongbo Sun,† Sian Avery,† Katherine Bush,† Alan Henley,† Florence I. Raynaud,†
Paul Workman,† Richard Bayliss,‡,§ Spiros Linardopoulos,†,∥ and Julian Blagg*,†
†Cancer Research UK Cancer Therapeutics Unit, Division of Cancer Therapeutics, The Institute of Cancer Research, London SM2
5NG, United Kingdom
‡Division of Structural Biology, The Institute of Cancer Research, Chester Beatty Laboratories, London SW3 6JB, United Kingdom
§Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, United Kingdom
∥The Breakthrough Breast Cancer Research Centre, Division of Breast Cancer Research, The Institute of Cancer Research, London
SW3 6JB, United Kingdom
S
* Supporting Information
ABSTRACT: Aurora-A differs from Aurora-B/C at three
positions in the ATP-binding pocket (L215, T217, and R220).
Exploiting these differences, crystal structures of ligand−
Aurora protein interactions formed the basis of a design
principle for imidazo[4,5-b]pyridine-derived Aurora-A-selec-
tive inhibitors. Guided by a computational modeling approach,
appropriate C7-imidazo[4,5-b]pyridine derivatization led to
the discovery of highly selective inhibitors, such as compound 28c, of Aurora-A over Aurora-B. In HCT116 human colon
carcinoma cells, 28c and 40f inhibited the Aurora-A L215R and R220K mutants with IC50 values similar to those seen for the
Aurora-A wild type. However, the Aurora-A T217E mutant was significantly less sensitive to inhibition by 28c and 40f compared
to the Aurora-A wild type, suggesting that the T217 residue plays a critical role in governing the observed isoform selectivity for
Aurora-A inhibition. These compounds are useful small-molecule chemical tools to further explore the function of Aurora-A in
cells.
Aurora-A is involved in the initiation of mitosis by promoting
centrosome maturation and bipolar mitotic spindle formation,
and its inhibition leads to increased G2/M accumulation and
spindle defects.17,22,23 Aurora-B is part of the chromosomal
passenger complex (CPC), which also includes borealin,
survivin, and the inner centromere protein (INCENP), and
plays a critical role in the regulation of chromosome alignment
and also in cytokinesis.23 Inhibition of Aurora-B leads to
abrogation of the spindle assembly checkpoint, so that cells
rapidly undergo chromosome segregation even in the presence of
spindle defects.22 The effects of Aurora-B inhibition mask the
effects of Aurora-A inhibition, and therefore, selective inhibitors
of both Aurora-A and Aurora-B are needed to carry out detailed
cell-based and in vivo studies on this kinase family.22 The role of
Aurora-C during mitosis is not well understood; however, it is
established that Aurora-C plays a specific role in spermato-
genesis.24
INTRODUCTION
■
Over the past decade there has been extensive interest in Aurora
kinases as anticancer targets.1−3 Aurora proteins, a family of three
kinases designated as Aurora-A, -B, and -C, play key and distinct
roles in different stages of mitosis4−8 and are overexpressed in a
wide range of human malignancies.9−15
Research toward the discovery of Aurora kinase modulators as
cancer therapeutics led to the identification of a significant
number of small-molecule inhibitors of Aurora kinases, the
majority of which inhibit all three isoforms A, B, and C.1
Compounds displaying isoform selectivity have also been
reported, including AZD1152,16 a selective Aurora-B inhibitor,
and 1,17 2,18 3,19 4,20 and 5,21 which selectively inhibit Aurora-A
(Figure 1). Many inhibitors of Aurora kinases have reached
clinical evaluation.1−3 In relation to isoform selectivity, the ideal
inhibitor profile (i.e., selective isoform inhibition versus pan-
Aurora inhibition) is still unclear, but the ongoing clinical
evaluation of Aurora inhibitors may provide new insights to
better answer this question. However, it should be noted that the
Aurora kinases have distinct cellular functions and their
inhibition has divergent effects on cells undergoing mitosis.22
Received: July 23, 2013
© XXXX American Chemical Society
A
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CHEMISTRY
■
The synthesis of 7-azaindoles 16a−d is shown in Schemes 2 and
3, with the key step being formation of the azaindole ring via a
Pd-catalyzed heteroannulation reaction.29−31
Starting from 8, access was readily gained to the alkyne 10
using standard Boc protection followed by a Sonogashira cross-
coupling reaction (Scheme 1). 5-Chloro-3-iodo-2-aminopyr-
a
Scheme 1
Figure 1. Selective inhibitors of Aurora-A kinase.
a
t
Other key differences between the Aurora kinases are
emerging. Aurora-A alone forms a complex with N-Myc, an
oncoprotein that induces neuroblastoma, which protects N-Myc
from proteosomal degradation,25,26 and Aurora-A-selective
inhibitors MLN8054 and MLN8237 disrupt the Aurora-A/N-
Myc complex.26 Significantly, the Aurora-A/N-Myc complex was
not disrupted by the Aurora-A-selective inhibitor MK-5108,
which belongs to a different chemotype.26 This demonstrates the
importance of having access to a range of structurally diverse
selective Aurora isoform inhibitors, in addition to the existing
pan-Aurora modulators. They can serve as useful small-molecule
chemical tools to further explore the cellular function of Aurora
proteins toward their eventual therapeutic application in cancer
treatment.
Reagents and conditions: (a) THF, BuOH, Boc2O, room temper-
ature, 1 h; (b) PdCl2(PPh3)2, CuI, THF, Et3N, 100 °C, microwave
irradiation, 20 min.
idine (11; Scheme 2) was obtained from 2-amino-5-chloropyr-
idine upon treatment with N-iodosuccinimide and p-toluene-
sulfonic acid. Azaindole ring formation (compound 12) was
effected by reacting 11 with alkyne 10 in the presence of DABCO
and Pd(PPh3)2Cl2 (Scheme 2).29−31 The silyl/iodo exchange
proceeded in good yield using N-iodosuccinimide as previously
described.31,32 Subsequent Boc group removal followed by
sulfonamide formation afforded the key intermediate 15. The
final products 16a,b were then obtained via a Suzuki cross-
coupling reaction (Scheme 2).
Starting from key intermediate 14, access to the acetamide
derivatives 16c,d (Scheme 3, Table 1) was accomplished by
acetylation of the amino functionality in 14 followed by the
introduction of 1-methylpyrazol-4-yl and 4-methoxylphenyl via a
Suzuki cross-coupling reaction (Scheme 3).
We have previously reported the discovery of novel imidazo-
[4,5-b]pyridine derivatives as potent, orally bioavailable
inhibitors of Aurora kinases (Figure 2),27,28 with a compound
The imidazo[4,5-b]pyridine derivatives 21a−f, 28a−c, and
40a−f (Tables 2, 3, and 5) and 34 (Table 4) were prepared by
reacting 1,3-dimethyl-1H-pyrazole-4-carbaldehyde with the
requisite 2-amino-3-nitropyridine precursor in the presence of
Na2S2O4 as reported previously (Schemes 4−9).28,33
The key 2-amino-3-nitropyridine intermediates 20a,c,d,f were
obtained by nucleophilic displacement of the C4-chloride of 4,5-
dichloro-3-nitropyridin-2-amine (18) with the appropriately
substituted pyrrolidine (Schemes 4 and 5) or piperazine
(Scheme 6) in the presence of diisopropylethylamine in 2-
propanol. Intermediates 20b and 20e were obtained by
procedures analogous to those described for their enantiomeric
forms 20a and 20d, respectively. Starting from 4,5-dichloro-3-
nitropyridin-2-amine (18), the benzylamino derivative 27 and N-
phenylbenzamide derivative 29 were readily prepared by a
nucleophilic substitution reaction, though for the synthesis of 29
the temperature had to be raised to 90 °C to ensure efficient
displacement by the poorly nucleophilic aniline (Scheme 7).
Access to the 2-amino-3-nitropyridine derivative 31 was
achieved upon treatment of 4-hydroxy-N-phenylbenzamide
(30), prepared as previously described,34 with KOtBu in DMF
followed by the addition of 4,5-dichloro-3-nitropyridin-2-amine
(Scheme 8). The C2-unsubstituted analogue of 28c, compound
35, was prepared from 31 by first reducing the nitro group with
Figure 2. Imidazo[4,5-b]pyridine-based inhibitors of Aurora
kinases.27,28
in this series being identified as a dual Aurora/FLT3 preclinical
development candidate.28 In parallel to this work, we also
investigated the design and synthesis of Aurora-A-selective
inhibitors based on the imidazo[4,5-b]pyridine scaffold. Herein,
we report our medicinal chemistry program aimed at the
identification of imidazo[4,5-b]pyridine- and 7-azaindole-based
inhibitors displaying a high degree of selectivity for Aurora-A
over Aurora-B.
B
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a
Scheme 2
a
Reagents and conditions: (a) DABCO, Pd(PPh3)2Cl2, DMF, 100 °C, 92 h; (b) N-iodosuccinimide, DCE, 80 °C, microwave irradiation, 30 min; (c)
TFA, CH2Cl2, room temperature, 2.5 h; (d) methanesulfonyl chloride, Et3N, DMF, 0 °C, 4.5 h; (e) 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole, toluene, EtOH, PdCl2(dppf), 2 M aq Na2CO3, 80 °C, 18 h; (f) 4-methoxyphenylboronic acid, toluene, EtOH,
PdCl2(dppf), 2 M aq Na2CO3, 80 °C, 16 h.
a
Scheme 3
a
Reagents and conditions: (a) CH2Cl2, acetyl chloride, iPr2NEt, −10 °C, 4 h; (b) 1-methyl-1H-pyrazole-4-boronic acid pinacol ester, toluene, EtOH,
PdCl2(dppf), 2 M aq Na2CO3, 80 °C, 16 h; (c) 4-methoxyphenylboronic acid, toluene, EtOH, PdCl2(dppf), 2 M aq Na2CO3, 80 °C, 16 h.
a
1H-pyrazole-4-carbaldehyde in a manner similar to that
described for 28c (Scheme 8).
Scheme 4
The 2-amino-3-nitro-4-phenoxypyridine derivatives 39a−f,
precursors to 40a−f (Scheme 9, Table 5), were accessed by
reacting 4,5-dichloro-3-nitropyridin-2-amine (18) with the
requisite 4-hydroxybenzamide using potassium tert-butoxide as
the base in DMF (Scheme 9). 4-Hydroxybenzamide derivatives
38a,b and 38d−f were all prepared from 4-acetoxybenzoic acid
(36)34 in a two-step sequence (Scheme 9); 4-hydroxy-N-
(pyridin-3-yl)benzamide (38c) was commercially available.
Amide bond formation was effected via acid chloride carboxyl
activation, and ester hydrolysis was accomplished under alkaline
conditions (Scheme 9).
a
i
Reagents and conditions: (a) 2-propanol, Pr2NEt, 45 °C, 18 h; (b)
1,3-dimethyl-1H-pyrazole-4-carbaldehyde, EtOH, 1 M aq Na2S2O4, 80
°C, 20 h.
Na2S2O4 and subsequently condensing the resulting diamine
product 33 with trimethyl orthoformate under acidic conditions
(Scheme 8). For the synthesis of the deschloro analogue of 28c
(compound 34), the 2-amino-3-nitropyridine derivative 32,
obtained from 30 and 4-chloro-3-nitropyridin-2-amine28 via an
SNAr substitution reaction, was condensed with 1,3-dimethyl-
RESULTS AND DISCUSSION
■
Aurora-A, -B, and -C kinases share a high degree of primary
sequence homology; there are only three sequence differences in
the ATP-binding site.35 In Aurora-A, these residues are Leu215
C
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a
Scheme 5
a
i
Reagents and conditions: (a) acetic anhydride, Pr2NEt, CH2Cl2, 0 °C to room temperature, 4 h; (b) (i) TFA, CH2Cl2, room temperature, 1.5 h;
(ii) 4,5-dichloro-3-nitropyridin-2-amine, 2-propanol, iPr2NEt, 45 °C, 20 h; (c) EtOH, 1,3-dimethyl-1H-pyrazole-4-carbaldehyde, 1 M aq Na2S2O4, 80
°C, 20 h.
a
Scheme 6
a
Reagents and conditions: (a) methanesulfonyl chloride, pyridine, CH2Cl2, room temperature, 5.5 h; (b) (i) TFA, CH2Cl2, room temperature, 1.5 h;
(ii) 4,5-dichloro-3-nitropyridin-2-amine, 2-propanol, iPr2NEt, 45 °C, 20 h; (c) EtOH, 1,3-dimethyl-1H-pyrazole-4-carbaldehyde, 1 M aq Na2S2O4, 80
°C, 18 h; (d) DMF, NaH, MeI, room temperature, 1 h; (e) (i) TFA, CH2Cl2, room temperature, 1.5 h; (ii) 4,5-dichloro-3-nitropyridin-2-amine, 2-
propanol, Pr2NEt, 45 °C, 20 h; (f) EtOH, 1,3-dimethyl-1H-pyrazole-4-carbaldehyde, 1 M aq Na2S2O4, 80 °C, 18 h.
i
a
Scheme 7
elicit Aurora isoform selectivity. For example, in the case of the
imidazo[1,2-a]pyrazine derivative 3 (Figure 1), it was proposed
that the observed selectivity for Aurora-A inhibition is driven by
the interaction of a sulfonamide with the side chain of Thr217 in
Aurora-A and a steric clash with the equivalent Glu residue in
Aurora-B/C.19 In the crystal structure of 3 bound to Aurora-A,
the Leu215 side chain points away from the active site, whereas
the Arg220 is highly mobile and disordered.19 Exploitation of
these two residues (i.e., Leu215 and Arg220) to drive selectivity
for Aurora-A inhibition over Aurora-B/C inhibition is therefore
considered to be challenging. We have previously reported the
discovery of imidazo[4,5-b]pyridine derivatives as inhibitors of
Aurora kinases, and compounds in this series were cocrystallized
with Aurora-A, providing us with a clear insight into the
interactions of this class of compounds with Aurora kinases.27,28
The structural knowledge on the ligand−protein interactions
across the imidazo[1,2-a]pyrazine19 and imidazo[4,5-b]-
pyridine27,28 series formed the basis of a design principle for
imidazo[4,5-b]pyridine-derived Aurora-A-selective inhibition.
Overlay of the imidazo[4,5-b]pyridine-based inhibitor 627 with
the selective inhibitor 319 bound to Aurora-A (Figure 3)
indicated that Thr217 could be accessed via an appropriate
C7-imidazo[4,5-b]pyridine derivatization (e.g., compounds 21c
in Scheme 5 and 21d,f in Scheme 6) or elaboration through the
N1-position of the imidazopyridine ring (e.g., 7-azaindole
structures 16a−d in Schemes 2 and 3). With regard to the
former approach, we recognized that Thr217 targeting for
Aurora-A-selective inhibition via C7-imidazo[4,5-b]pyridine
a
Reagents and conditions: (a) N-benzylmethylamine, 2-propanol,
iPr2NEt, 60 °C, 18 h; (b) 1,3-dimethyl-1H-pyrazole-4-carbaldehyde,
EtOH, 1 M aq Na2S2O4, 80 °C; (c) 4-amino-N-phenylbenzamide, 2-
i
propanol, Pr2NEt, 45 °C, 18 h, then at 90 °C for 7 h.
(Arg in Aurora-B/C) and Thr217 (Glu in Aurora-B/C) on the
C-lobe extension of the hinge region and Arg220 (Lys in Aurora-
B/C) on the αD helix. The majority of reported Aurora-A-
selective inhibitors exploit the Thr217 ATP-binding site
sequence difference to drive selectivity for Aurora-A inhibition
over Aurora-B inhibition.18−20,36 Crystallographic analysis of
Aurora-A in complex with small-molecule ligands provides
insight into the attractiveness of targeting the Thr217 residue to
D
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a
Scheme 8
a
Reagents and conditions: (a) (i) DMF, KOtBu, room temperature; (ii) 4,5-dichloro-3-nitropyridin-2-amine, room temperature; (b) 1,3-dimethyl-
1H-pyrazole-4-carbaldehyde, DMF, 1 M aq Na2S2O4, 84 °C, 20 h; (c) (i) DMF, KOtBu, room temperature, 20 min; (ii) 4-chloro-3-nitropyridin-2-
amine, 80 °C, 24 h; (d) DMSO/H2O, Na2S2O4, 80 °C, 2 h; (e) 1,3-dimethyl-1H-pyrazole-4-carbaldehyde, DMSO/H2O, Na2S2O4, 130 °C, 18 h; (f)
trimethyl orthoformate, concd HCl, room temperature, 24 h.
a
active site of Aurora-A indicated a mode of binding to the
hinge region similar to that observed for the imidazo[4,5-
b]pyridine-based inhibitors of Aurora27,28 and an interaction of
the sulfonamide with Thr217.
Scheme 9
It was also envisaged that the introduction of a 4-amino-N-
phenylbenzamide or 4-hydroxy-N-phenylbenzamide substituent
at the 7-position of the imidazo[4,5-b]pyridine ring could be
beneficial for Aurora-A isoform selectivity. A 4-amino-N-
phenylbenzamide fragment is present in 2-anilinopyrimidine-
based Aurora-A-selective inhibitors reported by Aliagas-Martin
et al. (see compound 2 in Figure 1).18 Docking of structure 28c
(Scheme 8) into the active site of Aurora-A (PDB code 3H0Z)18
and a comparison with 2 bound to Aurora-A indicated a good
overlay of the N-phenylbenzamide fragments (Figure 4).
Our initial efforts focused on the evaluation of the 7-azaindole
scaffold (Table 1) in relation to Aurora inhibitory potency and
Aurora-A isoform selectivity. Assuming a mode of binding to the
hinge region similar to that observed for the imidazo[4,5-
b]pyridine-based inhibitors,27,28 p-methoxyphenyl and 1-meth-
ylpyrazol-4-yl were chosen as optimal representative R2
substituents for the 7-azaindole scaffold.27,28 1-Methylpyrazol-
4-yl derivatives 16a and 16c displayed higher Aurora-A and -B
inhibitory potencies compared with their p-methoxyphenyl
counterparts 16b and 16d (Table 1). On the other hand, both
the sulfonamide and the acetamide groups appear to have similar
Aurora-A inhibitory effects (16a vs 16c and 16b vs 16d, Table 1).
Regarding selectivity for Aurora-A inhibition, although a trend to
more potent Aurora-A inhibition was seen with all compounds
listed in Table 1, the level of the observed isoform selectivity was
not considered significant.
a
Reagents and conditions: (a) (i) oxalyl chloride, CH2Cl2, DMF, room
temperature; (ii) amine/aniline, CH2Cl2, room temperature; (b)
MeOH or THF/H2O, aq NaOH, room temperature; (c) (i) DMF,
KOtBu, room temperature; (ii) 4,5-dichloro-3-nitropyridin-2-amine,
room temperature or heating; (d) 1,3-dimethyl-1H-pyrazole-4-
carbaldehyde, DMF or DMSO, aq Na2S2O4, heating.
Attempts to cocrystallize compounds of this 7-azaindole class
with Aurora-A failed to provide quality crystals; as a result we
were unable to rationalize these results on a structural basis. At
this point, we revisited the docking of 16a,b into the active site of
Aurora-A, which indicated an additional binding mode to that
previously observed. The preferred binding mode for 16a was
similar to that observed with the imidazo[4,5-b]pyridine-based
inhibitors of Aurora with the 2-aryl/heteroaryl substituent, in
both imidazo[4,5-b]pyridine and 7-azaindole scaffolds, pointing
to the solvent-accessible area27,28 (see Figure 5A and compound
Figure 3. Overlay of 6 (PDB code 2X6D)27 with 3 (PDB code 2XNG)19
bound to the ATP-binding site of Aurora-A. The hinge-binding residue
Ala213 and the Thr217 residue are highlighted.
derivatization could be a challenging task due to the conforma-
tional flexibility of the proposed C7-pyrrolidine- and -piperidine-
based substituents. For the latter approach, docking of C3-N-
benzylmethanesulfonamide-derivatized 7-azaindoles into the
E
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Figure 4. (A) Docking of 28c into the active site of Aurora-A (PDB code
3H0Z)18 and (B) overlay of 28c with the Aurora-A-selective inhibitor
2.18
Figure 5. (A) Docking of 16a into the active site of Aurora-A (PDB code
2X6D)27and (B) docking of 16b into the active site of Aurora-A (PDB
code 2X6D).27
a
Table 1. 7-Azaindole Derivatives
findings into account, we decided to terminate our interest in the
7-azaindole scaffold and focus our efforts on evaluating the
imidazo[4,5-b]pyridine-based approaches for Aurora-A-selective
inhibition.
Targeting Aurora-A Thr217 for selective Aurora-A isoform
inhibition was continued by investigation of C7-imidazo[4,5-
b]pyridine derivatization, i.e., introduction of pyrrolidine- and
piperidine-based substituents (Table 2). This approach afforded
highly potent inhibitors of both Aurora-A and -B (21a−f, Table
2), with all compounds displaying similar inhibitory effects
against these two Aurora isoforms. On this occasion, structural
insight into ligand−protein interactions was gained by
cocrystallization of 21c and 21a with Aurora-A. The complexes
formed crystals in space group P6122, with one Aurora/
compound complex per asymmetric unit.
Compound 21c occupies the ATP-binding site, with the
pyridine N and imidazole NH forming hydrogen bonds to
Ala213 in the hinge region of Aurora-A consistent with previous
reports (Figure 6A).27,28 The amide substituent of the
pyrrolidine in 21c points to Thr217 but is neither in the right
orientation nor close enough (5.9 Å distance between the −OH
of Thr217 and the amide carbonyl of compound 21c) to form a
a
The results are mean values of two independent determinations
( SD).
6 in Figure 3). However, an additional flipped binding mode was
observed in docking experiments with compound 16b compared
to 16a (compare Figure 5B with Figure 5A). It should also be
noted that the p-methoxyphenyl derivatives 16b and 16d
exhibited low Aurora-A inhibitory potencies. Taking all these
F
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28a, Table 3) and 4-amino-N-phenylbenzamide substituent
(compound 28b, Table 3). The benzylamino derivative 28a
Table 2. C7-Imidazo[4,5-b]pyridine Derivatization:
Pyrrolidine and Piperidine Substituents
a
Table 3. C7-Imidazo[4,5-b]pyridine Derivatization:
a
Benzylamino, Anilino, and Phenoxy Substituents
a
The results are mean values of at least two independent
determinations ( SD) unless specified otherwise. A superscript
Roman “a” indicates results from a single experiment (each
concentration run in duplicate).
inhibited Aurora-A and -B with similar IC50 values (Table 3).
However, the N-phenylbenzamide derivative 28b was a
significantly more potent inhibitor of Aurora-A compared to
Aurora-B, the IC50 values were determined as 0.075 and 4.12 μM,
respectively (Table 3). A similar trend was observed in Hela
cervical cancer cells; 28b inhibited the autophosphorylation of
Aurora-A at T288 (a cell-based biomarker for Aurora-A
inhibition)17,19 with an IC50 value of 0.32 μM and histone H3
phosphorylation at S10 (a cell-based biomarker for Aurora-B
inhibition)17,19 with a significantly higher IC50 value of 18.6 μM
(58-fold difference). Subsequent replacement of the C7-NH
linker in 28b with an oxygen gave compound 28c, which
displayed a higher degree of selectivity in inhibiting Aurora-A
over Aurora-B in biochemical assays (Table 3) and in cells. In the
biochemical assays, the IC50 values for inhibition of Aurora-A and
-B were determined as 0.067 and 12.71 μM, respectively (Table
3). In Hela cervical cancer cells, 28c inhibited the autophosphor-
ylation of Aurora-A at T288 (p-T288 IC50 = 0.16 μM) 480-fold
more potently compared with Aurora-B inhibition (p-HH3 IC50
= 76.84 μM). A similar trend was also observed in HCT116
human colon carcinoma cells; the IC50 values for the inhibition of
autophosphorylation of Aurora-A at T288 and histone H3
phosphorylation at S10 were determined as 0.065 and 24.65 μM,
respectively. The selectivity of 28c against the kinome was also
determined by profiling in a 110-kinase panel at a concentration
of 1 μM. In this panel, 28c inhibited only three kinases at a level
higher than 80%, namely, Aurora-A, VEGFR (VEGFR1), and
GSK3b (Table S3, Supporting Information). On this basis,
compound 28c is a highly selective kinase inhibitor with a Gini
coefficient37 calculated as 0.719. In an attempt to understand
possible reasons for the observed inhibition of VEGFR1 and
GSK3b by 28c, we examined conserved residues in the binding
site between Aurora-A and VEGFR1 and between Aurora-A and
GSK3b. We identified E211, Y212, G216, and L263 (residue
numbers for Aurora-A) as conserved binding motifs between
Aurora-A and VEGFR1 and residues L210, Y212, P214, and
a
Results are mean values of two independent determinations ( SD)
unless specified otherwise. A superscript Roman “a” indicates results
from a single experiment (each concentration run in duplicate).
Figure 6. Crystal structures of C7-imidazo[4,5-b]pyridine compounds
bound to Aurora-A: (A) compound 21c, (B) compound 21a.
strong hydrogen bond with the side chain of Thr217. Likewise,
compound 21a binds to the hinge region as previously observed
with closely related derivatives in this series. However, the
substituted pyrrolidine ring adopts an unfavorable conformation
for interaction with Thr217, with the acetamido group pointing
away from this residue (Figure 6B). These crystal structures were
consistent with the lack of selectivity for compounds 21a and
21c, and we therefore, investigated alternative substitutions at
C7.
We continued our attempts to target Thr217 for Aurora-A-
selective inhibition via C7-imidazo[4,5-b]pyridine derivatization
by the introduction of a benzylamino substituent (compound
G
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a
T217 (residue numbers for Aurora-A) as conserved binding
motifs between Aurora-A and GSK3b. However, determination
of whether these conserved regions influence the inhibition of
VEGFR1 and GSK3b would require additional crystallography
beyond the scope of this study. The antiproliferative activity in
human tumor cell lines was also studied, and 28c inhibited the
growth of HCT116 human colon carcinoma cells with a GI50
value of 2.30 μM. We have previously shown that compounds of
the imidazo[4,5-b]pyridine class exhibit potent dual Aurora-A/
FLT3 inhibition,28 and consistent with this finding, we observed
FLT3 inhibition in vitro (IC50 = 0.162 μM)38 and potent
Table 5. Aniline Replacements
antiproliferative activity in MV4-11 human AML cells (GI50
=
0.299 μM).
Compound 28c displayed high mouse and liver microsomal
stability (after a 30 min incubation with mouse and human liver
microsomes, only 22% and 9% of 28c was metabolized,
respectively). This promising stability prompted in vivo mouse
pharmacokinetic profiling that revealed 28c as a highly orally
bioavailable compound (F = 100%) with moderate clearance
(0.053 L/h, 44.16 mL/min/kg) and volume of distribution
(0.036 L, 1.8 L/kg).
The effects of the 6-Cl and C2-pyrazolyl substituents on
Aurora-A inhibition were subsequently studied by preparing
compounds 34 and 35, respectively (Scheme 8, Table 4). Both
a
Table 4. C6-Cl and C2-Pyrazolyl Aurora-A Inhibitory Effect
a
The results are mean values of at least two independent
determinations ( SD).
versus p-HH3 IC50 = 19.74 μM, 34-fold difference). Compound
40f displayed the highest potency inhibiting Aurora-A in the
biochemical assay (IC50 = 0.015 μM, Table 5), with Aurora-B
inhibition being determined as 3.05 μM (Table 5). In Hela cells,
40f inhibited Aurora-A 346 times more potently compared to
Aurora-B (p-T288 IC50 = 0.070 μM versus p-HH3 IC50 = 24.24
μM). Profiling of 40f in a 50-kinase panel at a concentration of 1
μM revealed a highly selective inhibitor; only one kinase, namely,
VEGFR (VEGFR1), was inhibited higher than 80% (Table S4,
Supporting Information). Compound 40f exhibited high mouse
and liver microsomal stability (after a 30 min incubation with
mouse and human liver microsomes, 28% and 22% of 40f was
metabolized, respectively). However, an in vivo pharmacokinetic
profiling in mouse revealed a lower oral bioavailability (14%)
compared to that for 28c (100%).
Many attempts to cocrystallize 28c and 40f with Aurora-A
were unsuccessful. However, the docking of 28c into the active
site of Aurora-A suggested that the aniline moiety resides in close
proximity to Thr217 (Figure 4). On this basis, we probed
whether Thr217 (Glu in Aurora-B) is the main residue governing
the selectivity for Aurora-A inhibition. Testing of 28c against the
Aurora-A wild type and its T217E mutant expressed in Hela cells
revealed that the Aurora-A T217E mutant was significantly less
sensitive to inhibition (40-fold) compared to the Aurora-A wild
type (p-T288 IC50 = 4.11 and 0.107 μM, respectively).
Subsequently, both 28c and 40f were tested against the
Aurora-A wild type and its T217E, L215R, and R220K mutants
in HCT116 cells (Table 6, Figure 7, and Figure S1 in the
Supporting Information). Both 28c and 40f inhibited the
Aurora-A L215R and R220K mutants with IC50 values similar
to those seen for the Aurora-A wild type (Table 6, Figure 7, and
Figure S1). On the other hand, the Aurora-A T217E mutant was
significantly less sensitive to inhibition by 28c and 40f compared
to the wild type (33-fold and 64-fold, respectively; Table 6,
a
The results are mean values of two independent determinations
( SD).
34 and 35 were significantly less potent inhibitors of Aurora-A
compared to 28c (Tables 3 and 4), indicating the requirement
for both C6-Cl and C2-aromatic or -heteroaromatic substituents
and consistent with previously reported SARs.27,28,39
Having identified 28c as a highly selective Aurora-A inhibitor,
our efforts focused on replacing the aniline moiety in 28c, a
potential toxicophore,40,41 with a range of aliphatic and
heteroaryl amines (Table 5). All replacements were well
tolerated in relation to Aurora-A inhibitory potency, and the
selectivity for Aurora-A over Aurora-B inhibition was generally
maintained (Table 5). Compounds were also tested for the
cellular inhibition of both Aurora-A and -B, and 40a inhibited
Aurora-A in HCT116 cells significantly more potently compared
to Aurora-B (p-T288 IC50 = 0.095 μM versus p-HH3 IC50 = 4.93
μM, 52-fold difference). Likewise, 40c was a more potent
inhibitor of Aurora-A than Aurora-B in Hela cells (p-T288 IC50 =
0.28 μM versus p-HH3 IC50 = 19.72 μM, 70-fold difference). A
similar trend was seen with 40b; in Hela cells it inhibited Aurora-
A more potently compared to Aurora-B (p-T288 IC50 = 0.58 μM
H
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These compounds could therefore serve as useful small-molecule
tools44,45 to further explore the function of Aurora-A in cells;
indeed, the need for structurally diverse inhibitors displaying
Aurora isoform selectivity has recently been highlighted by
Brockmann et al.26 In their study, the Aurora-A-selective
inhibitors MLN8054 and MLN8237 disrupted the Aurora-A/
N-Myc complex and induced apoptosis in neuroblastoma cell
lines and tumors, a phenotype different from that seen with the
structurally differentiated Aurora-A-selective inhibitor MK-5108
from a different chemotype.26 These data suggest differences in
the mechanism of action and also indicate a specific target patient
population for treatment with an Aurora-A-selective inhibitor.
Table 6. Inhibition of the Aurora-A Wild Type and Mutants in
HCT116 Cells
Aur-A, WT
p-T288
compd IC50 (μM)
Aur-A T217E
Aur-A L215R
Aur-A R220K
mutant, p-T288 mutant, p-T288 mutant, p-T288
IC50 (μM)
IC50 (μM)
IC50 (μM)
28c
40f
0.029
0.036
0.96
2.29
0.027
0.043
0.027
0.042
Figure 7, and Figure S1). This body of evidence suggests that the
Thr217 residue (Glu in Aurora-B/C) plays an important role in
governing the observed selectivity for Aurora-A inhibition. In the
above experiment, the inhibition of Aurora-B by 40f was also
investigated by measuring the reduction in the phosphorylation
of histone H3 at S10. As shown in Figure S2 (Supporting
Information), inhibition of histone H3 phosphorylation at S10
was only achieved at high concentrations of 40f (partial
inhibition at 25 μM and complete inhibition at 50 μM).
Interestingly, at concentrations where phosphorylation of
Aurora-A was completely inhibited (for example, at 1.5 μM),
there was an increase in histone H3 phosphorylation (Figure S2),
most likely due to an increase in the percentage of mitotic cells as
previously reported for other Aurora-A-selective inhibitors.17,42
However, at higher concentrations, histone H3 phosphorylation
was inhibited, indicating onset of Aurora B inhibition (Figure
S2).
EXPERIMENTAL SECTION
■
Aurora Kinase Assays. Aurora-A and Aurora-B kinase IC50 values
were determined as follows: Kinase activity was measured in a
microfluidic assay which monitors the separation of a phosphorylated
product from its substrate. The assay was performed on an EZ Reader II
(PerkinElmer Life Sciences, Waltham, MA) using separation buffer (no.
760367, PerkinElmer Life Sciences) containing CR-8 (500 nM, no.
760278, PerkinElmer Life Sciences). For each compound, a 10 mM
stock concentration in 100% DMSO was used. Compounds were
dispensed with an ECHO 550 acoustic dispenser (Labcyte Inc.,
Sunnyvale, CA) to generate duplicate 8 pt dilution curves directly into
384 LV polystyrene plates (no. 3676, Corning Inc., Corning, NY) to give
final assay concentrations in the range of 0.25 pM to 200 μM in either
1.0% or 2% (v/v) DMSO.
In conclusion, structural knowledge of imidazo[4,5-b]pyridine
and imidazo[1,2-a]pyrazine-based ligand−Aurora-A protein
interactions formed the basis of a design principle for
imidazo[4,5-b]pyridine-derived Aurora-A-selective inhibition.
This led to the discovery of imidazo[4,5-b]pyridine derivatives
displaying a high degree of selectivity for inhibition of Aurora-A
over Aurora-B in both biochemical and cellular assays. In
particular, 28c and 40f were highly selective in inhibiting Aurora-
A in both Hela cervical cancer and HCT116 human colon
carcinoma cells. Testing of 28c and 40f against the Aurora-A wild
type and its T217E, L215R, and R220K mutants suggested that
the Aurora-A Thr217 residue (Glu in Aurora-B/C) plays an
important role in governing the observed selectivity for Aurora-A
inhibition. In addition, 28c and 40f are a different chemotype
compared to other known Aurora-A-selective inhibitors, and 28c
is highly selective within the tested kinome, with a kinase
profiling differing from that of MK-510842 and MLN8237.43
For the Aurora-A kinase the enzyme reaction (total volume 10 μL)
was carried out with 5 nM N-terminal HIS-tagged AurA, which was
expressed and purified as previously described,46 1.5 μM fluorescently
labeled peptide (no. 760365, FL-Peptide 21, PerkinElmer, sequence
5FAM-LRRASLG-CONH2), 20 μM ATP in assay buffer (50 mM Tris
(pH 7.4), 200 mM NaCl, 5 mM MgCl2, 2 mM DTT, 0.1% Tween 20),
and the test compound or DMSO control.
For the Aurora-B kinase the enzyme reaction (total volume 10 μL)
was carried out with 1 nM full-length AurB/INCENP complex (no. 05-
102, Carna Biosciences, Inc., Kobe, Japan), 1.5 μM fluorescently labeled
peptide (no. 760365, FL-Peptide 21, PerkinElmer, sequence 5FAM-
LRRASLG-CONH2), 15 μM ATP in assay buffer (50 mM Tris (pH
7.4), 200 mM NaCl, 5 mM MgCl2, 2 mM DTT, 0.1% Tween 20), and
either the test compound or a DMSO control.
For both enzymes the reaction was carried out for 60 min at room
temperature and stopped by the addition of 10 μL of stop buffer
containing 100 mM HEPES-buffered saline (pH 7.3), 20 mM EDTA,
and 0.05% (v/v) Brij-35. The plate was read on a Caliper EZ reader II.
Figure 7. Inhibition of Aurora-A wild-type and mutant proteins expressed in HCT116 cells by compound 40f.
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The reader provides a software package (“Reviewer”) which converts
the peak heights into percent conversion by measuring both product and
substrate peaks. The percent inhibition was calculated relative to the
total wells (containing enzyme) and blank wells (containing no enzyme
plus DMSO). IC50 values were calculated from a four-parameter logistics
fit of percentage inhibition versus log concentration using GraphPad
Prism 5 (GraphPad Software, Inc., La Jolla, CA) or the Studies package
(Dotmatics, Bishops Stortford, U.K.).
Kinase Selectivity Profiling. Compound 28c was profiled against a
panel of 110 kinases at the International Centre for Protein Kinase
Profiling, Division of Signal Transduction Therapy, University of
Dundee. Compound 40f was profiled against a panel of 50 kinases
(MRC-PPU Express Screen) also at the International Centre for Protein
Kinase Profiling.
and 1, 2, 4, 6, and 24 h. Blood was removed by cardiac puncture and
centrifuged to obtain plasma samples. The plasma samples (100 μL)
were added to the analytical internal standard (IS; olomoucine),
followed by protein precipitation with 300 μL of methanol. Following
centrifugation (1200g, 30 min, 4 °C), the resulting supernatants were
analyzed for 28c levels by LC/MS using a reversed-phase X-Bridge C18
(Waters, 50 × 2.1 mm) analytical column and positive ion mode ESI
MRM on a Waters 2795 liquid chromatography system coupled to a
Quattro Ultima triple-quadrupole mass spectrometer (Waters).
In Vivo Mouse PK (Compound 40f). Mice (female Balb/C) were
dosed po or iv with 40f (5 mg kg−1) in 10% DMSO and 5% Tween 20 in
saline. After administration, the mice were sacrificed at 5, 15, and 30 min
and 1, 2, 4, 6, and 24 h. Blood was removed by cardiac puncture and
centrifuged to obtain plasma samples. The plasma samples (100 μL)
were added to the analytical IS (olomoucine), followed by protein
precipitation with 300 μL of methanol. Following centrifugation (1200g,
30 min, 4 °C), the resulting supernatants were analyzed for 40f levels by
LC/MS using a reversed-phase Polar RP (Phenomenex, 50 × 2 mm)
analytical column and positive ion mode ESI MRM on a Waters 2795
liquid chromatography system coupled to a Quattro Ultima triple-
quadrupole mass spectrometer (Waters).
Cell Viability Assay. GI50 values (50% cell growth inhibitory
concentration) were determined as previously described.27,47
Inhibition of Aurora-B and the Aurora-A Wild Type and
Mutants in Cells. Determination of cellular inhibition of Aurora-A (at
T288) and histone H3 (at S10) has been described previously.19 Briefly,
cells were transfected with the Myc-tagged Aurora-A wild type or
mutants and treated with different concentrations of the inhibitors for 2
h followed by lysis in LDS sample buffer. The samples were sonicated
and boiled, and the proteins were separated by LDS−PAGE and
transferred to nitrocellulose membranes. The upper and lower parts of
the membranes were probed with anti-P-T288 (Aurora-A autophos-
phorylation/activation) and anti-P-histone H3 (Aurora-B substrate
phosphorylation) antibodies, respectively. The blots were quantified
and IC50 values determined using Graphpad Prism. Mammalian cell
expression constructs encoding Aurora-A T217E, L215R, and R220K
mutants with N-terminal Myc tags were produced using Quikchange
mutagenesis (Stratagene).
Computational Chemistry. The protein−ligand cocrystal (PDB
2X6D) was prepared in MOE (Moleular Operating Environment) using
Protonate3D, and the waters were removed.48 GOLD (Genetic
Optimization for Ligand Docking) was used for all docking experiments
using the ChemScore (chemscore_kinase) scoring function.49 The
docked poses were analyzed on the basis of their ChemScore and also
the consistency of the docked poses. The higher scored poses were
selected as the preferred solutions if the majority of these solutions were
consistent in binding mode. The binding site was defined as those atoms
within 6 Å of the cocrystallized ligand in PDB 2X6D and the cocrystal
ligand removed. The GOLD algorithm was set to explore an extensive
binding mode space by setting the search efficiency to 200%, or very
flexible. No constraints were used in the docking experiments.
Cocrystallization of Aurora-A with Ligand. Cocrystals of
Aurora-A bound to compounds 21a and 21c were generated as
previously described.28 Data were integrated using XDS and processed
using the CCP4 package.50 Structures were solved by molecular
replacement using Aurora-A (PDB code 1MQ4) as a model. Ligand
fitting and model rebuilding were carried out using Coot,50 and
refinement was carried out using Phenix.51
Mouse Liver Microsomal Stability. The compounds (10 μM)
were incubated with male CD1 mouse liver microsome (1 mg mL−1)
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 of compound remaining was determined after
analysis by LC/MS.
Human Liver Microsomal Stability. The compounds (10 μM)
were incubated with mixed-gender pooled human liver microsome (1
mg mL−1) 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 of compound remaining was
determined after analysis by LC/MS.
Chemistry. General Procedures. Commercially available starting
materials, reagents, and dry solvents were used as supplied.
Flash column chromatography 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.
Preparative TLC was performed on Analtech or Merck plates. Ion
exchange chromatography was performed using acidic Isolute Flash
SCX-II cartridges.
1H NMR spectra were recorded on a Bruker Avance-500. Samples
were prepared as solutions in a deuterated solvent and referenced to the
appropriate internal nondeuterated solvent peak or tetramethylsilane.
Chemical shifts were recorded in parts per million (δ) downfield of
tetramethylsilane.
LC/MS analysis, method A: Analysis was performed on a Waters
LCT with a Waters Alliance 2795 separation module and Waters 2487
dual-wavelength absorbance detector coupled to a Waters/Micromass
LCT time-of-flight mass spectrometer with an ESI source. Analytical
separation was carried out at 30 °C on a Merck Chromolith SpeedROD
column (RP-18e, 50 × 4.6 mm) using a flow rate of 2 mL/min in a 3.5
min gradient elution with detection at 254 nm. The mobile phase was a
mixture of methanol (solvent A) and water containing formic acid at
0.1% (solvent B). Gradient elution was 1:9 (A/B) to 9:1 (A/B) over
2.25 min, 9:1 (A/B) for 0.75 min, then reversion back to 1:9 (A/B) over
0.3 min, and finally 1:9 (A/B) for 0.2 min. LC/MS analysis, method B:
Analysis was performed on an Agilent 1200 series HPLC instrument
with a diode array detector coupled to a 6210 time-of-flight mass
spectrometer with a dual multimode APCI/ESI source. Analytical
separation was carried out at 30 °C on a Merck Chromolith SpeedROD
column (RP-18e, 50 × 4.6 mm) using a flow rate of 2 mL/min in a 4 min
gradient elution with detection at 254 nm. The mobile phase was a
mixture of methanol (solvent A) and water containing formic acid at
0.1% (solvent B). Gradient elution was 1:9 (A/B) to 9:1 (A/B) over 2.5
min, 9:1 (A/B) for 1 min, then reversion back to 1:9 (A/B) over 0.3 min,
and finally 1:9 (A/B) for 0.2 min. For LC/MS analyses, if the method is
not stated, then method A was followed.
LC/HRMS analysis was performed on an Agilent 1200 series HPLC
instrument with a diode array detector coupled to a 6210 time-of-flight
mass spectrometer with a dual multimode APCI/ESI source. Analytical
separation was carried out at 30 °C on a Merck Chromolith SpeedROD
column (RP-18e, 50 × 4.6 mm) using a flow rate of 2 mL/min in a 4 min
gradient elution with detection at 254 nm. The mobile phase was a
mixture of methanol (solvent A) and water containing formic acid at
0.1% (solvent B). Gradient elution was 1:9 (A/B) to 9:1 (A/B) over 2.5
min, 9:1 (A/B) for 1 min, then reversion back to 1:9 (A/B) over 0.3 min,
and finally 1:9 (A/B) for 0.2 min. The following references masses were
used for HRMS analysis: caffeine [M + H]+ 195.087652, (hexakis-
(1H,1H,3H-tetrafluoropentoxy)phosphazene [M + H]+ 922.009798),
In Vivo Mouse PK (Compound 28c). Mice (female Balb/C) were
dosed po or iv with 28c (5 mg kg−1) in 10% DMSO and 5% Tween 20 in
saline. After administration, the mice were sacrificed at 5, 15, and 30 min
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and hexakis(2,2-difluoroethoxy)phosphazene [M + H]+ 622.02896 or
reserpine [M + H]+ 609.280657.
azeotroped with toluene to leave a colorless oily residue, (R)-N-
(pyrrolidin-3-ylmethyl)acetamide as a TFA salt. This material
(supposedly 1.08 mmol) was dissolved in 2-propanol (14 mL), and to
this solution was added 4,5-dichloro-3-nitropyridin-2-amine (0.214 g,
1.03 mmol) followed by N,N-diisopropylethylamine (0.503 g, 0.68 mL,
3.89 mmol). The reaction mixture was placed into an oil bath preheated
to 45 °C and stirred at this temperature for 20 h; a nearly clear solution
was obtained. Approximately half of the solvent volume was then
removed in vacuo to give a precipitate which was collected by filtration
and washed with 2-propanol and diethyl ether. The product was
obtained as a yellow solid (0.205 g, 64%): 1H NMR (500 MHz, DMSO-
d6) 1.62 (m, 1H, pyrrolidine CH), 1.79 (s, 3H, COCH3), 1.95 (m, 1H,
pyrrolidine CH), 2.32 (m, 1H, pyrrolidine CH), 3.03 (m, 1H), 3.12 (m,
1H), 3.21 (dd, J = 6.3, 10.4 Hz, 1H), and 3.38−3.52 (m, 3H)
(pyrrolidine CH and CH2NH), 6.84 (s, 2H, NH2), 7.87 (s, 1H, pyridine
6-H), 7.93 (br t, J = 5.4 Hz, 1H, CONH); LC/MS (ESI, m/z) tR = 1.26
min, 314, 316 [(M + H)+, Cl isotopic pattern].
(S)-N-((1-(6-Chloro-2-(1,3-dimethyl-1H-pyrazol-4-yl)-3H-imidazo-
[4,5-b]pyridin-7-yl)pyrrolidin-3-yl)methyl)acetamide (21c). To a
mixture of (S)-N-((1-(2-amino-5-chloro-3-nitropyridin-4-yl)-
pyrrolidin-3-yl)methyl)acetamide (0.055 g, 0.18 mmol) and ethanol
(3.5 mL) was added 1,3-dimethyl-1H-pyrazole-4-carbaldehyde (0.024 g,
0.19 mmol) followed by a freshly prepared aqueous solution of Na2S2O4
(1 M, 0.75 mL, 0.75 mmol), and the reaction mixture was stirred at 80
°C for 20 h. The reaction mixture was then concentrated in vacuo, the
residue was absorbed on silica gel (1.7 g), and the free-running powder
was placed on a 10 g Isolute silica column which was eluted with
dichloromethane/ethyl acetate (1:1, v/v), 5% methanol in dichloro-
methane/ethyl acetate (1:1, v/v), and 9% methanol in dichloro-
methane/ethyl acetate (1:1, v/v). Trituration of the resultant residue
with diethyl ether gave a precipitate which was collected by filtration,
washed with diethyl ether, water, and diethyl ether, and then dried. The
title compound was obtained as an off-white solid (0.016 g, 23%): 1H
NMR (500 MHz, DMSO-d6) 1.64 (m, 1H, pyrrolidine CH), 1.81 (s, 3H,
COCH3), 2.01 (m, 1H, pyrrolidine CH), 2.35 (m, 1H, pyrrolidine CH),
3.83 (s, 3H, pyrazole N-CH3), 3.13 (t, J = 6.3 Hz, 2H), 3.85 (m, 1H), and
4.14 (m, 3H) (pyrrolidine CH and CH2NH), 7.86 (s, 1H, pyrazole 5-H
or imidazo[4,5-b]pyridine 5-H), 7.94 (br t, J = 5.1 Hz, 1H, CONH), 8.12
(s, 1H, imidazo[4,5-b]pyridine 5-H or pyrazole 5-H), 12.76 (s, 1H,
imidazo[4,5-b]pyridine NH); LC/MS (ESI, m/z) tR = 1.78 min, 388,
390 [(M + H)+, Cl isotopic pattern]; HRMS m/z found 388.1649, calcd
for C18H23ClN7O (M + H)+ 388.1647.
4-((2-Amino-5-chloro-3-nitropyridin-4-yl)amino)-N-phenylbenza-
mide (29). To a mixture of 4,5-dichloro-3-nitropyridin-2-amine (0.104
g, 0.50 mmol) and 2-propanol (7.0 mL) was added 4-aminobenzanilide
(0.114 g, 0.54 mmol) followed by N,N-diisopropylethylamine (0.105
mL, 0.078 g, 0.60 mmol). The reaction mixture was heated at 45 °C for
18 h, then the temperature was raised to 90 °C, and stirring was
continued at this temperature for 7 h. The reaction mixture was then
allowed to cool to room temperature and diluted with 2-propanol (8.0
mL); a precipitate was obtained which was collected by filtration,
washed with 2-propanol (2 × 5 mL) and diethyl ether (2 × 5 mL), and
dried to afford the title compound as an orange solid (0.070 g, 37%): 1H
NMR (500 MHz, DMSO-d6) 7.02 (d, J = 8.7 Hz, 2H, ArH), 7.08 (t, J =
7.2 Hz, 1H, p-PhH), 7.26 (s, 2H, NH2), 7.33 (t, J = 8.3 Hz, 2H, m-PhH),
7.75 (d, J = 7.8 Hz, 2H, ArH), 7.86 (d, J = 8.7 Hz, 2H, ArH), 8.18 (s, 1H,
pyridine 6-H), 9.14 (s, 1H, NHAr), 10.07 (s, 1H, NHAr); LC/MS (ESI,
m/z) tR = 2.31 min, 384, 386 [(M + H)+, Cl isotopic pattern].
4-((6-Chloro-2-(1,3-dimethyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]-
pyridin-7-yl)amino)-N-phenylbenzamide (28b). To a mixture of 4-
((2-amino-5-chloro-3-nitropyridin-4-yl)amino)-N-phenylbenzamide
(0.065 g, 0.17 mmol) and ethanol (5.5 mL) was added 1,3-dimethyl-1H-
pyrazole-4-carbaldehyde (0.024 g, 0.19 mmol) followed by a freshly
prepared aqueous solution of Na2S2O4 (1M; 0.90 mL, 0.90 mmol). The
reaction mixture was stirred at 80 °C for 22 h, then allowed to cool to
room temperature, and concentrated in vacuo. The residue was
absorbed on silica gel (1.7 g), and the free-running powder was placed
on a 10 g Isolute silica column which was eluted with CH2Cl2,
dichloromethane/ethyl acetate (1:1, v/v), 2% methanol in dichloro-
methane/ethyl acetate (1:1, v/v), and finally 3% methanol in
Analytical HPLC analysis was performed on a Thermo-Finnigan
Surveyor HPLC system or an Agilent Technologies 1200 series HPLC
system at 30 °C using a Phenomenex Gemini C18 column (5 μm, 50 ×
4.6 mm) and 10 min gradient of 10% → 90% MeOH/0.1% formic acid,
visualizing at 254, 309, or 350 nm. The purity of the final compounds
was determined by analytical HPLC as described above and is ≥95%
unless specified otherwise.
(S)-N-(1-(2-Amino-5-chloro-3-nitropyridin-4-yl)pyrrolidin-3-yl)-
acetamide (20a). To a mixture of 4,5-dichloro-3-nitropyridin-2-amine
(0.152 g, 0.73 mmol) and 2-propanol (13.0 mL) was added (S)-3-
acetamidopyrrolidine (0.098 g, 0.77 mmol) followed by N,N-
diisopropylethylamine (0.17 mL, 0.97 mmol). The reaction mixture
was placed in an oil bath preheated at 45 °C and stirred at this
temperature for 18 h; it was then allowed to cool to room temperature
and diluted with 2-propanol (8 mL). The precipitate was collected by
filtration, washed with 2-propanol (2 × 5 mL) and diethyl ether (5 mL),
and dried to give the title compound as a yellow solid (0.133 g, 61%): 1H
NMR (500 MHz, DMSO-d6) 1.80 (s, 3H, COCH3), 1.83 (m, 1H,
pyrrolidine CH), 2.08 (m, 1H, pyrrolidine CH), 3.18 (dd, J = 5.1, 10.5
Hz, 1H, pyrrolidine CH), 3.56 (m, 2H, pyrrolidine CH), 3.64 (m, 1H,
pyrrolidine CH), 4.20 (m, 1H, pyrrolidine CH), 6.85 (s, 2H, NH2), 7.88
(s, 1H, pyridine 6-H), 8.09 (d, J = 6.1 Hz, 1H, CONH); LC/MS (ESI,
m/z) tR = 1.32 min, 300, 302 [(M + H)+, Cl isotopic pattern].
(S)-N-(1-(6-Chloro-2-(1,3-dimethyl-1H-pyrazol-4-yl)-3H-imidazo-
[4,5-b]pyridin-7-yl)pyrrolidin-3-yl)acetamide (21a). To a mixture of
(S)-N-(1-(2-amino-5-chloro-3-nitropyridin-4-yl)pyrrolidin-3-yl)-
acetamide (0.045 g, 0.15 mmol) and ethanol (3.5 mL) was added 1,3-
dimethyl-1H-pyrazole-4-carbaldehyde (0.020 g, 0.16 mmol) followed
by a freshly prepared aqueous solution of Na2S2O4 (1 M, 0.65 mL, 0.65
mmol). The reaction mixture was stirred at 80 °C for 20 h; it was then
concentrated in vacuo. The residue was absorbed on silica gel (1.7 g),
and the free-running powder was placed on a 10 g Isolute silica column.
Elution with dichloromethane/ethyl acetate (1:1, v/v), 4% methanol in
dichloromethane/ethyl acetate (1:1, v/v), and 6% methanol in
dichloromethane/ethyl acetate (1:1, v/v) afforded a solid residue
which was triturated with diethyl ether. The precipitate was collected by
filtration and washed with diethyl ether, water (4 × 2 mL), and diethyl
ether (3 × 3 mL) to afford the title compound as a pale yellow solid
(0.022 g, 39%): 1H NMR (500 MHz, DMSO-d6) 1.81 (s, 3H, COCH3),
1.85 (m, 1H, pyrrolidine CH), 2.08 (m, 1H, pyrrolidine CH), 3.83 (s,
3H, pyrazole N-CH3), 3.92 (m, 1H, pyrrolidine CH), 4.16 (m, 1H,
pyrrolidine CH), 4.25 (m, 3H, pyrrolidine CH), 7.86 (s, 1H) and 8.12
(s, 2H) (pyrazole 5-H, imidazo[4,5-b]pyridine 5-H and CONH), 12.78
(s, 1H, imidazo[4,5-b]pyridine NH); LC/MS (ESI, m/z) tR = 1.90 min,
374, 376 [(M + H)+, Cl isotopic pattern]; HRMS m/z found 374.1496,
calcd for C17H21ClN7O (M + H)+ 374.1491; HPLC tR = 6.18 min, 94%
at λ = 354 nm.
(S)-tert-Butyl 3-(Acetamidomethyl)pyrrolidine-1-carboxylate (23).
To a solution of (S)-tert-butyl 3-(aminomethyl)pyrrolidine-1-carbox-
ylate (0.30 g, 1.50 mmol) in anhydrous CH2Cl2 (4 mL) cooled in an ice
bath under argon was added DIPEA (0.213 g, 0.29 mL, 1.65 mmol)
followed by a dropwise addition of acetic anhydride (0.168 g, 1.65
mmol). The reaction mixture was stirred for 4 h under argon while being
allowed to warm to room temperature. The clear solution was diluted
with ethyl acetate (50 mL), washed with saturated NaHCO3 (20 mL),
brine (20 mL), 1 M aq HCl (20 mL), and brine (20 mL), then dried
(Na2SO4), and concentrated in vacuo. The product was obtained as a
colorless oil (0.300 g, 83%): 1H NMR (500 MHz, DMSO-d6) 1.39 (s,
9H, C(CH3)3), 1.53 (m, 1H, pyrrolidine CH), 1.80 (s, 3H, COCH3),
1.85 (m, 1H), 2.23 (m, 1H), 2.89 (dd, J = 7.2, 10.8 Hz, 1H), 3.00 (m,
1H), 3.07 (m, 1H), 3.16 (m, 1H), and 3.32 (m, obscured by water peak)
(pyrrolidine CH and CH2NH), 7.89 (s, 1H, CONH); LC/MS (ESI, m/
z) compound is non-UV-absorbent, 265 (M + Na)+.
(S)-N-((1-(2-Amino-5-chloro-3-nitropyridin-4-yl)pyrrolidin-3-yl)-
methyl)acetamide (20c). A solution of (S)-tert-butyl 3-
(acetamidomethyl)pyrrolidine-1-carboxylate (0.263 g, 1.09 mmol) in
dichloromethane (7 mL) and trifluoroacetic acid (3.0 mL) was stirred at
room temperature for 1.5 h. Solvents were removed in vacuo and
K
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Journal of Medicinal Chemistry
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dichloromethane/ethyl acetate (1:1, v/v). The title compound was
obtained as an off-white solid after trituration with diethyl ether/CH2Cl2
(1:1, v/v) (0.012 g, 15%): 1H NMR (500 MHz, DMSO-d6) 2.22 (s, 3H,
pyrazole 3-Me), 3.82 (s, 3H, pyrazole N-Me), 7.08 (t, J = 7.4 Hz, 1H, p-
PhH), 7.19 (d, J = 7.2 Hz, 2H, ArH), 7.33 (t, J = 7.8 Hz, 2H, m-PhH),
7.78 (d, J = 7.9 Hz, 2H, ArH), 7.89 (d, J = 8.7 Hz, 2H, ArH), 8.16 (s, 1H,
pyrazole 5-H or imidazo[4,5-b]pyridine 5-H), 8.17 (s, 1H, pyrazole 5-H
or imidazo[4,5-b]pyridine 5-H), 8.74 (s, 1H, NHAr), 9.97 (s, 1H,
NHAr), 13.03 (s, 1H, imidazo[4,5-b]pyridine NH); LC/MS (ESI, m/z)
tR = 2.36 min, 458, 460 [(M + H)+, Cl isotopic pattern]; HRMS m/z
found 458.1493, calcd for C24H21ClN7O (M + H)+ 458.1491.
absorbed on silica gel (1.6 g), and the free-running powder was placed
on a 20 g Isolute silica column which eluted with dichloromethane and
then 5%, 10%, and 15% EtOAc in dichloromethane. The obtained
product was dissolved in EtOAc (150 mL), and the solution was washed
with saturated aqueous NaHCO3 (3 × 20 mL), dried (Na2SO4), and
concentrated in vacuo to give the title compound as a white solid (0.150
g, 38%): 1H NMR (500 MHz, DMSO-d6) 1.66 (m, 2H, cyclobutylamine
CH), 2.06 (m, 2H, cyclobutylamine CH), 2.20 (m, 2H, cyclobutylamine
CH), 2.28 (s, 3H, COCH3), 4.39 (m, 1H, NHCH), 7.20 (d, J = 8.6 Hz,
2H, ArH), 7.87 (d, J = 8.6 Hz, 2H, ArH), 8.57 (d, J = 7.4 Hz, 1H,
CONH); LC/MS (ESI, m/z) tR = 1.91 min, 234 (M + H)+.
4-((2-Amino-5-chloro-3-nitropyridin-4-yl)oxy)-N-phenylbenza-
mide (31). To a solution of 4-hydroxy-N-phenylbenzamide34 (0.068g,
0.32 mmol) in anhydrous DMF (2.3 mL) was added potassium tert-
butoxide (0.036 g, 0.32 mmol). The solution was stirred at room
temperature for 20 min under argon, and then 4,5-dichloro-3-
nitropyridin-2-amine (0.062 g, 0.30 mmol) was added portionwise
over a 5 min period. Stirring was continued at room temperature for 1 h
and 10 min under argon; the reaction mixture was then partitioned
between ethyl acetate (110 mL) and H2O (20 mL). The organic layer
was washed with H2O (15 mL) and brine (20 mL), dried (Na2SO4), and
concentrated in vacuo to give a yellow solid. This was triturated with
dichloromethane, the precipitate was collected by filtration and washed
with dichloromethane to obtain the title compound as a yellow solid
(0.087 g, 76%): 1H NMR (500 MHz, DMSO-d6) 7.09 (t, J = 7.4 Hz, 1H,
p-PhH), 7.13 (d, J = 8.8 Hz, 2H, ArH), 7.34 (t, J = 7.7 Hz, 2H, m-PhH),
7.62 (br s, 2H, NH2), 7.75 (d, J = 7.7 Hz, 2H, ArH), 7.95 (d, J = 8.8 Hz,
2H, ArH), 8.46 (s, 1H, pyridine 6-H), 10.24 (s, 1H, CONH); LC/MS
(ESI, m/z) tR = 2.50 min, 385, 387 [(M + H)+, Cl isotopic pattern].
4-((6-Chloro-2-(1,3-dimethyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]-
pyridin-7-yl)oxy)-N-phenylbenzamide (28c). To a mixture of 4-((2-
amino-5-chloro-3-nitropyridin-4-yl)oxy)-N-phenylbenzamide (0.077 g,
0.20 mmol) and DMF (4.2 mL) was added 1,3-dimethyl-1H-pyrazole-4-
carbaldehyde (0.030 g, 0.24 mmol) followed by a freshly prepared
aqueous solution of Na2S2O4 (1 M, 1.0 mL, 1.0 mmol). The reaction
mixture was stirred at 84 °C for 20 h, then allowed to cool to room
temperature, and partitioned between EtOAc (180 mL) and water (20
mL). The organic layer washed with more H2O (15 mL), and brine (15
mL), dried (Na2SO4), and concentrated in vacuo. The resulting residue
was triturated with diethyl ether (15 mL); the precipitate was collected
by filtration and washed with diethyl ether. This solid was absorbed on
silica gel (1.4 g), and the free-running powder was placed on a 10 g
Isolute silica column which was eluted with dichloromethane,
dichloromethane/ethyl acetate (1:1, v/v), 2.5% methanol in dichloro-
methane/ethyl acetate (1:1, v/v), and finally 4% methanol in
dichloromethane/ethyl acetate (1:1, v/v). The obtained residue was
triturated with diethyl ether; the precipitate was collected by filtration
and washed with diethyl ether to give the title compound as an off-white
N-Cyclobutyl-4-hydroxybenzamide (38f). To a mixture of 4-
(cyclobutylcarbamoyl)phenyl acetate (0.145 g, 0.62 mmol) in methanol
(4.0 mL) was added an aqueous solution of NaOH (1M, 1.6 mL, 1.6
mmol) followed by water (0.4 mL); a clear solution was obtained. The
reaction mixture was stirred at room temperature for 1 h and 20 min.
Approximately half of the solvent was then removed under reduced
pressure, and the remaining solution was diluted with water (2.0 mL).
The pH was adjusted to about 7 with 1 M HCl; a white suspension was
obtained which was diluted with water (5 mL). This was extracted with
EtOAc (3 × 50 mL), and the combined extracts were dried (Na2SO4)
and concentrated in vacuo to give the title compound as a white solid
(0.105 g, 89%): 1H NMR (500 MHz, DMSO-d6) 1.67 (m, 2H,
cyclobutylamine CH), 2.05 (m, 2H, cyclobutylamine CH), 2.18 (m, 2H,
cyclobutylamine CH), 4.37 (m, 1H, NHCH), 6.77 (d, J = 8.7 Hz, 2H,
ArH), 7.70 (d, J = 8.7 Hz, 2H, ArH), 8.28 (d, J = 7.5 Hz, 1H, CONH),
9.87 (s, 1H, OH); LC/MS (ESI, m/z) tR = 1.66 min,192 (M + H)+.
4-((2-Amino-5-chloro-3-nitropyridin-4-yl)oxy)-N-cyclobutylben-
zamide (39f). To a solution of N-cyclobutyl-4-hydroxybenzamide
(0.100 g, 0.52 mmol) in anhydrous DMF (3.3 mL) was added potassium
tert-butoxide (0.058 g, 0.52 mmol). The solution was stirred at room
temperature for 20 min under argon. Then 4,5-dichloro-3-nitropyridin-
2-amine (0.102 g, 0.49 mmol) was added portionwise over a 5 min
period. Stirring was continued at room temperature for 1 h and 30 min
under argon; the reaction mixture was then partitioned between ethyl
acetate (200 mL)/dichloromethane (5 mL) and H2O (30 mL). The
organic layer was washed with H2O (20 mL), brine (30 mL), saturated
aqueous NaHCO3 (20 mL), and brine (20 mL), dried (Na2SO4), and
concentrated in vacuo to give a yellow solid. This solid was triturated
with EtOAc/dichloromethane (1:1, v/v; approximately 10 mL); the
precipitate was collected by filtration and washed with dichloromethane
to afford the title compound as a yellow solid (0.120 g, 67%): 1H NMR
(500 MHz, DMSO-d6) 1.66 (m, 2H, cyclobutylamine CH), 2.05 (m,
2H, cyclobutylamine CH), 2.20 (m, 2H, cyclobutylamine CH), 4.38 (m,
1H, NHCH), 7.04 (d, J = 8.9 Hz, 2H, ArH), 7.54 (s, 2H, NH2), 7.83 (d, J
= 8.9 Hz, 2H, ArH), 8.43 (s, 1H, pyridine 6-H), 8.53 (d, J = 7.5 Hz, 1H,
CONH); LC/MS (ESI, m/z) tR = 2.31 min, 363, 365 [(M + H)+, Cl
isotopic pattern].
1
solid (0.010 g, 11%): H NMR (500 MHz, DMSO-d6) 2.21 (s, 3H,
4-((6-Chloro-2-(1,3-dimethyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]-
pyridin-7-yl)oxy)-N-cyclobutylbenzamide (40f). To a mixture of 4-
((2-amino-5-chloro-3-nitropyridin-4-yl)oxy)-N-cyclobutylbenzamide
(0.094 g, 0.26 mmol) and DMF (5.9 mL) was added 1,3-dimethyl-1H-
pyrazole-4-carbaldehyde (0.038 g, 0.31 mmol) followed by a freshly
prepared aqueous solution of Na2S2O4 (1 M, 1.3 mL,1.30 mmol). The
reaction mixture was stirred at 85 °C for 24 h, then allowed to cool to
room temperature, and partitioned between EtOAc (220 mL) and water
(30 mL). The organic layer was washed with more H2O (20 mL) and
brine (20 mL), dried (Na2SO4), and concentrated in vacuo. The residue
was treated with diethyl ether (approximately 15 mL); a brown
precipitate was obtained which was collected by filtration and washed
with diethyl ether. This solid was absorbed on silica gel (1.2 g), and the
free-running powder was placed on a 10 g Isolute silica column which
was eluted with dichloromethane, dichloromethane/ethyl acetate (1:1,
v/v), 2.5% methanol in dichloromethane/ethyl acetate (1:1, v/v), and
finally 4% methanol in dichloromethane/ethyl acetate (1:1, v/v). The
obtained residue was triturated with diethyl ether; the precipitate was
collected by filtration and washed with diethyl ether to give the title
pyrazole 3-Me), 3.82 (s, 3H, pyrazole N-Me), 7.09 (t, J = 7.4 Hz, 1H, p-
PhH), 7.18 (br d, J = 7.5 Hz, 2H, ArH), 7.34 (t, J = 7.8 Hz, 2H, m-PhH),
7.75 (d, J = 8.0 Hz, 2H, ArH), 7.98 (d, J = 8.8 Hz, 2H, ArH), 8.21 (br s,
1H, pyrazole 5-H or imidazo[4,5-b]pyridine 5-H), 8.38 (br s, 1H,
pyrazole 5-H or imidazo[4,5-b]pyridine 5-H), 10.16 (s, 1H, CONH),
13.35 (s, 1H, imidazo[4,5-b]pyridine NH); LC/MS (ESI, m/z) tR = 2.56
min, 459, 461 [(M + H)+, Cl isotopic pattern]; HRMS m/z found
459.1348, calcd for C24H20ClN6O2 (M + H)+ 459.1331.
4-(Cyclobutylcarbamoyl)phenyl Acetate (37f). To a round-bottom
flask containing 4-acetoxybenzoic acid (0.306 g, 1.70 mmol) was slowly
added a solution of oxalyl chloride (0.430 g, 3.40 mmol) in anhydrous
dichloromethane (2.0 mL) under argon. To this mixture was slowly
added anhydrous DMF (0.030 mL), and a clear solution was obtained.
The solution was stirred at room temperature for 45 min under argon
and then concentrated in vacuo, and the residue was dissolved in
anhydrous dichloromethane (3.0 mL). Cyclobutylamine (0.363 g, 5.10
mmol) was slowly added; a precipitate had formed by completion of the
amine addition. Stirring was continued at room temperature for 20 min.
Then the reaction mixture was diluted with anhydrous dichloromethane
(2.0 mL), and the precipitate was removed by filtration and washed with
dichloromethane (4 × 3 mL). The filtrate was concentrated in vacuo and
1
compound as an off-white solid (0.016 g, 14%): H NMR (500 MHz,
DMSO-d6) 1.67 (m, 2H, cyclobutylamine CH), 2.06 (m, 2H,
cyclobutylamine CH), 2.19 (m, 5H, cyclobutylamine CH and pyrazole
L
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3-Me), 3.82 (s, 3H, pyrazole N-Me), 4.40 (m, 1H, NHCH), 7.10 (d, J =
8.1 Hz, 2H, ArH), 7.85 (d, J = 8.7 Hz, 2H, ArH), 8.19 (s, 1H) and 8.34
(s, 1H) (pyrazole 5-H and imidazo[4,5-b]pyridine 5-H), 8.51 (d, J = 7.2
Hz, 1H, CONH), 13.31 (s, 1H, imidazo[4,5-b]pyridine NH); LC/MS
(ESI, m/z) tR = 2.44 min, 437, 439 [(M + H)+, Cl isotopic pattern];
HRMS m/z found 437.1492, calcd for C22H22ClN6O2 (M + H)+
437.1487.
tography; SAR, structure−activity relationship; TFA, trifluoro-
acetic acid
REFERENCES
■
(1) Pollard, J. R.; Mortimore, M. Discovery and Development of
Aurora Kinase Inhibitors as Anticancer Agents. J. Med. Chem. 2009, 52,
2629−2651.
(2) Green, M. R.; Woolery, J. E.; Mahadevan, D. Update on Aurora
Kinase Targeted Therapeutics in Oncology. Expert Opin. Drug Discovery
2011, 6, 291−307.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures for compounds 9−15, 16a−d, 17,
20b,d−f, 21b,d−f, 25−27, 28a, 32−35, 37a,b,d,e, 38a,b,d,e,
39a−e, and 40a−e, summary of crystallographic analysis of
compounds 21c (Table S1) and 21a (Table S2), kinase
selectivity profiling of compounds 28c (Table S3) and 40f
(Table S4), inhibition of the Aurora-A wild type and mutants in
HCT116 cells by compound 28c (Figure S1), inhibition of
Aurora-B in HCT116 cells by compound 40f (Figure S2), and
omit electron density maps for compounds 21a,c (Figure S3).
This material is available free of charge via the Internet at http://
pubs.acs.org.
(3) Cheung, C. H. A.; Coumar, M. S.; Chang, J.-Y.; Hsieh, H.-P. Aurora
Kinase Inhibitor Patents and Agents in Clinical Testing: An Update
(2009−10). Expert Opin. Ther. Patents 2011, 21, 857−884.
(4) Carmena, M.; Earnshaw, W. C. The Cellular Geography of Aurora
Kinases. Nat. Rev. Mol. Cell Biol. 2003, 4, 842−854.
(5) Ducat, D.; Zheng, Y. Aurora Kinases in Spindle Assembly and
Chromosome Segregation. Exp. Cell Res. 2004, 301, 60−67.
(6) Marumoto, T.; Zhang, D.; Saya, H. Aurora-A, a Guardian of Poles.
Nat. Rev. Cancer 2005, 5, 42−50.
(7) Barr, A. R.; Gergely, F. Aurora-A: The Maker and Breaker of
Spindle Poles. J. Cell Sci. 2007, 120, 2987−2996.
(8) Bayliss, R.; Sardon, T.; Vernos, I.; Conti, E. Structural Basis of
Aurora-A Activation by TPX2 at the Mitotic Spindle. Mol. Cell 2003, 12,
851−862.
(9) Tanaka, T.; Kimura, M.; Matsunaga, K.; Fukada, D.; Mori, H.;
Okano, Y. Centrosomal Kinase AIK1 Is Overexpressed in Invasive
Ductal Carcinoma of the Breast. Cancer Res. 1999, 59, 2041−2044.
(10) Bischoff, J. R.; Anderson, L.; Zhu, Y.; Mossie, K.; Ng, L.; Souza, B.;
Schryver, B.; Flanagan, P.; Clairvoyant, F.; Ginther, C.; Chan, C. S. M.;
Novotny, M.; Slamon, D. J.; Plowman, G. D. A Homologue of
Drosophila Aurora Kinase Is Oncogenic and Amplified in Human
Colorectal Cancers. EMBO J. 1998, 17, 3052−3065.
(11) Gritsko, T. M.; Coppola, D.; Paciga, J. E.; Yang, L.; Sun, M.;
Shelley, S. A.; Fiorica, J. V.; Nicosia, S. V.; Cheng, J. Q. Activation and
Overexpression of Centrosome Kinase BTAK/Aurora-A in Human
Ovarian Cancer. Clin. Cancer Res. 2003, 9, 1420−1426.
Accession Codes
Atomic coordinates and structure factors for the crystal structure
of Aurora-A with compounds 21a and 21c can be accessed using
PDB codes 4BYJ and 4BYI, respectively.
AUTHOR INFORMATION
Corresponding Authors
*Phone: +44 (0) 20 87224158. E-mail: vassilios.bavetsias@icr.
ac.uk.
■
*Phone: +44 (0) 20 87224051. E-mail: julian.blagg@icr.ac.uk.
Notes
The authors declare the following competing financial
interest(s): Please note that all authors who are, or have been,
employed by The Institute of Cancer Research are subject to a
“Rewards to Inventors Scheme” which may reward contributors
to a program that is subsequently licensed. P.W. was a founder
and shareholder in Chroma Therapeutics. P.W. is a former
employee of AstraZeneca. J.B. is a former employee and
shareholder of Pfizer Inc.
(12) Reichardt, W.; Jung, V.; Brunner, C.; Klein, A.; Wemmert, S.;
Romeike, B. F. M.; Zang, K. D.; Urbschat, S. The Putative Serine/
Threonine Kinase Gene STK15 on Chromosome 20q13.2 Is Amplified
in Human Gliomas. Oncol. Rep. 2003, 10, 1275−1279.
(13) Chieffi, P.; Troncone, G.; Caleo, A.; Libertini, S.; Linardopoulos,
S.; Tramontano, D.; Portella, G. Aurora B Expression in Normal Testis
and Seminonas. J. Endocrinol. 2004, 181, 263−270.
(14) Araki, K.; Nozaki, K.; Ueba, T.; Tatsuka, M.; Hashimoto, N. High
Expression of Aurora-B/Aurora and Ipl1I-like Midbody-Associated
Protein (AIM-1) in Astrocytomas. J Neurooncol. 2004, 67, 53−64.
(15) Sorrentino, R.; Libertini, S.; Pallante, P. L.; Troncone, G.;
Palombini, L.; Bavetsias, V.; Cernia, D. S.; Laccetti, P.; Linardopoulos,
S.; Chieffi, P.; Fusco, A.; Portella, G. Aurora B Overexpression
Associates with the Thyroid Carcinoma Undifferentiated Phenotype
and Is Required for Thyroid Carcinoma Cell Proliferation. J. Clin.
Endocrinol. Metab. 2005, 90, 928−935.
(16) Mortlock, A. A.; Foote, K. M.; Heron, N. M.; Jung, F. H.; Pasquet,
G.; Lohmann, J-J. M.; Warin, N.; Renaud, F.; De Savi, C.; Roberts, N. J.;
Johnson, T.; Dousson, C. B.; Hill, G. B.; Perkins, D.; Hatter, G.;
Wilkinson, R. W.; Wedge, S. R.; Heaton, S. P.; Odedra, R.; Keen, N. J.;
Crafter, C.; Brown, E.; Thompson, K.; Brightwell, S.; Khatri, L.; Brady,
M. C.; Kearney, S.; McKillop, D.; Rhead, S.; Parry, T.; Green, S.
Discovery, Synthesis, and in Vivo Activity of a New Class of
Pyrazoloquinazolines as Selective Inhibitors of Aurora B Kinase. J.
Med. Chem. 2007, 50, 2213−2224.
ACKNOWLEDGMENTS
■
This work was supported by Cancer Research UK [CUK] Grants
C309/A8274 and C309/A11566. P.W. is a Cancer Research UK
Life Fellow. S.L. is supported by Breakthrough Breast Cancer.
R.B. is a Royal Society University Research Fellow and
acknowledges the support of Breakthrough Breast Cancer
Project Grant AURA 05/06 and Cancer Research UK Grant
C24461/A13231 and infrastructural support for Structural
Biology at The Institute of Cancer Research from Cancer
Research UK. We acknowledge National Health Service (NHS)
funding to the National Institute for Health Research (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, and Dr.
Charlotte Dodson for their assistance with NMR, mass
spectrometry, HPLC, and Aurora-A protein production.
(17) 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. Antitumour Activity of MLN8054, an Orally Active Small-
Molecule Inhibitor of Aurora A kinase. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 4106−4111.
ABBREVIATIONS USED
■
Boc, tert-butoxycarbonyl; DABCO, 1,4-diazabicyclo[2,2,2]-
octane; DIPEA, N,N-diisopropylethylamine; ESI, electrospray
ionization; HPLC, high-performance liquid chromatography;
HRMS, high-resolution mass spectrometry; LC, liquid chroma-
M
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(18) Aliagas-Martin, I.; Burdick, D.; Corson, L.; Dotson, J.;
Drummond, J.; Fields, C.; Huang, O. W.; Hunsaker, T.; Kleinheinz,
T.; Krueger, E.; Liang, J.; Moffat, J.; Phillips, G.; Pulk, R.; Rawson, T. E.;
Ultsch, M.; Walker, L.; Wiesmann, C.; Zhang, B.; Zhu, B.-Y.; Cochran,
A. G. A Class of 2,4-Bisanilinopyrimidine Aurora A Inhibitors with
Unusually High Selectivity against Aurora B. J. Med. Chem. 2009, 52,
3300−3307.
(19) 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. Structure-Based Design
of Imidazo[1,2-a]pyrazine Derivatives as Selective Inhibitors of Aurora-
A Kinase in Cells. Bioorg. Med. Chem. Lett. 2010, 20, 5988−5993.
(20) Coumar, M. S.; Leou, J.-S.; Shukla, P.; Wu, J.-S.; Dixit, A. K.; Lin,
W-H; Chang, C.-Y.; Lien, T.-W.; Tan, U.-K.; Chen, C.-H.; Hsu, J. T-A.;
Chao, Y.-S.; Wu, S.-Y.; Hsieh, H.-P. Structure-Based Drug Design of
Novel Aurora Kinase A Inhibitors: Structural Basis for Potency and
Specificity. J. Med. Chem. 2009, 52, 1050−1062.
(21) Prime, M. E.; Courtney, S. M.; Brookfield, F. A.; Marston, R. W.;
Walker, V.; Warne, J.; Boyd, A. E.; Kairies, N. A.; von der Saal, W.;
Limberg, A.; Georges, G.; Engh, R. A.; Goller, B.; Rueger, P.; Rueth, M.
Phthalazinone Pyrazoles as Potent, Selective, and Orally Bioavailable
Inhibitors of Aurora-A Kinase. J. Med. Chem. 2011, 54, 312−319.
(22) Girdler, F.; Gascoigne, K. E.; Eyers, P. A.; Hartmuth, S.; Crafter,
C.; Foote, K. M.; Keen, N. J.; Taylor, S. S. Validating Aurora B as an Anti-
Cancer Drug Target. J. Cell Sci. 2006, 119, 3664−3675.
(23) Kelly, K. R.; Ecsedy, J.; Mahalingam, D.; Nawrocki, S. T.;
Padmanabhan, S.; Giles, F. J.; Carew, J. S. Targeting Aurora Kinases in
Cancer Treatment. Curr. Drug Targets 2011, 12, 2067−2078.
(24) Malumbres, M. Physiological Relevance of Cell Cycle Kinases.
Physiol. Rev. 2011, 91, 973−1007.
(25) Otto, T.; Horn, S.; Brockmann, M.; Eilers, U.; Schuttrumpf, L.;
Popov, N.; Kenney, A. M.; Schulte, J. H.; Beijersbergen, R.; Christiansen,
H.; Berwanger, B.; Eilers, M. Stabilization of N-Myc Is a Critical
Function of Aurora A in Human Neuroblastoma. Cancer Cell 2009, 15,
67−78.
(26) Brockmann, M.; Poon, E.; Berry, T.; Carstensen, A.; Deubzer, H.
E.; Rycak, L.; Jamin, Y.; Thway, K.; Robinson, S. P.; Roels, F.; Witt, O.;
Fischer, M.; Chesler, L.; Eilers, M. Small Molecule Inhibitors of Aurora-
A Induce Proteasomal Degradation of N-Myc in Childhood Neuro-
blastoma. Cancer Cell 2013, 24, 1−15.
(27) Bavetsias, V.; Large, J. M.; Sun, C.; Bouloc, N.; Kosmopoulou, M.;
Matteucci, M.; Wilsher, N. E.; Martins, V.; Reynisson, J.; Atrash, B.;
Faisal, A.; Urban, F.; Valenti, M.; de Haven Brandon, A.; Box, G.;
Raynaud, F. I.; Workman, P.; Eccles, S. A.; Bayliss, R.; Blagg, J.;
Linardopoulos, S.; McDonald, E. Imidazo[4,5-b]pyridine Derivatives as
Inhibitors of Aurora Kinases: Lead Optimization Studies toward the
Identification of an Orally Bioavailable Preclinical Development
Candidate. J. Med. Chem. 2010, 53, 5213−5228.
(28) Bavetsias, V.; Crumpler, S.; Sun, C.; Avery, S.; Atrash, B.; Faisal,
A.; Moore, A. S.; Kosmopoulou, M.; Brown, N.; Sheldrake, P. W.; Bush,
K.; Henley, A.; Box, G.; Valenti, M.; de Haven Brandon, A.; Raynaud, F.
I.; Workman, P.; Eccles, S. A.; Bayliss, R.; Linardopoulos, S.; Blagg, J.
Optimization of Imidazo[4,5-b]pyridine-Based Kinase Inhibitors:
Identification of a Dual FLT3/Aurora Kinase Inhibitor as an Orally
Bioavailable Preclinical Development Candidate for the Treatment of
Acute Myeloid Leukemia. J. Med. Chem. 2012, 55, 8721−8734.
(29) Larock, R. C.; Yum, E. K. Synthesis of Indoles via Palladium-
Catalyzed Heteroannulation of Internal Alkynes. J. Am. Chem. Soc. 1991,
113, 6689−6690.
(30) Ujjainwalla, F.; Warner, D. Synthesis of 5-, 6-, and 7-Azaindoles
via Palladium-Catalysed Heteroannulation of Internal Alkynes.
Tetrahedron Lett. 1998, 39, 5355−5358.
(31) Aadal Nielsen, P.; Brimert, T.; Kristoffersson, A.; Linnanen, T.;
Sjo, P. Substituted Pyrrolopyridines. WO2004016609 A1, 2004.
(32) Finaru, A.; Berthault, A.; Besson, T.; Guillaumet, G.; Berteina-
Raboin, S. Microwave-Assisted Solid-Phase Synthesis of 5-Carbox-
amido-N-acetyltryptamine Derivatives. Org. Lett. 2002, 4, 2613−2615.
(33) Yang, D.; Fokas, D.; Li, J.; Yu, L.; Baldino, C. M. A Versatile
Method for the Synthesis of Benzimidazoles from o-Nitroanilines and
Aldehydes in One Step via a Reductive Cyclization. Synthesis 2005, 47−
56.
(34) Davidson, A. H.; Davies, S. J.; Moffat, D. F. C. Quinoline and
Quinoxaline Derivatives as Inhibitors of Kinase Enzymatic Activity.
WO2006117552 A1, 2006.
(35) Bolanos-Garcia, V. M. Aurora Kinases. Int. J. Biochem. Cell Biol.
2005, 37, 1572−1577.
(36) Dodson, C. A.; Kosmopoulou, M.; Richards, M. W.; Atrash, B.;
Bavetsias, V.; Blagg, J.; Bayliss, R. Crystal Structure of an Aurora-A
Mutant That Mimics Aurora-B Bound to MLN8054: Insights into
Selectivity and Drug Design. Biochem. J. 2010, 427, 19−28.
(37) Graczyk, P. P. Gini Coefficient: A New Way To Express
Selectivity of Kinase Inhibitors against a Family of Kinases. J. Med. Chem.
2007, 50, 5773−5779.
(38) Invitrogen’s SelectScreen Profiling. www.invitrogen.com/
drugdiscovery (accessed July 22, 2013).
(39) Bavetsias, V.; Sun, C.; Bouloc, N.; Reynisson, J.; Workman, P.;
Linardopoulos, S.; McDonald, E. Hit Generation and Exploration:
Imidazo[4,5-b]pyridine Derivatives as Inhibitors of Aurora Kinases.
Bioorg. Med. Chem. Lett. 2007, 17, 6567−6571.
(40) Blagg, J. Structure-Activity Relationships for in Vitro and in Vivo
Toxicity. Annu. Rep. Med. Chem. 2006, 41, 353−369.
(41) Kalgutkar, A. S.; Dalvie, D. K.; O’Donnell, J. P.; Taylor, T. J.;
Sahakian, D. C. On the Diversity of Oxidative Bioactivation Reactions
on Nitrogen-Containing Xenobiotics. Curr. Drug Metab. 2002, 3, 379−
424.
(42) Shimomura, T.; Hasako, S.; Nakatsuru, Y.; Mita, T.; Ichikawa, K.;
Kodera, T.; Sakai, T.; Nambu, T.; Miyamoto, M.; Takahashi, I.; Miki, S.;
Kawanishi, N.; Ohkubo, M.; Kotani, H.; Iwasawa, Y. MK-5108, a Highly
Selective Aurora-A Kinase Inhibitor, Shows Antitumour Activity Alone
and in Combination with Docetaxel. Mol. Cancer Ther. 2010, 9, 157−
166.
(43) Manfredi, M. G.; Ecsedy, J. A.; Chakravarty, A.; Silverman, L.;
Zhang, M.; Hoar, K. 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. Characterization of Alisertib (MLN8237), an Investigational Small-
Molecule Inhibitor of Aurora A Kinase Using Novel in Vivo
Pharmacodynamic Assays. Clin. Cancer Res. 2011, 17, 7614−7624.
(44) Workman, P.; Collins, I. Probing the Probes: Fitness Factors for
Small Molecule Tools. Chem. Biol. 2010, 17, 561−577.
(45) Knapp, S.; Arruda, P.; Blagg, J.; Burley, S.; Drewry, D. H.;
Edwards, A.; Fabbro, D.; Gillespie, P.; Gray, N. S.; Kuster, B.; Lackey, K.
E.; Mazzafera, P.; Tomkinson, N. C. O.; Willson, T. M.; Workman, P.;
Zuercher, W. J. A Public-Private Partnership To Unlock the Untargeted
Kinome. Nat. Chem. Biol. 2013, 9, 3−7.
(46) Dodson, C. A.; Bayliss, R. Activation of Aurora-A Kinase by
Protein Partner Binding (TPX2) and Phosphorylation Are Independent
and Synergistic. J. Biol. Chem. 2012, 287, 1150−1157.
(47) Chan, F.; Sun, C.; Perumal, M.; Nguyen, Q.-D.; Bavetsias, V.;
McDonald, E.; Martins, V.; Wilsher, N.; Valenti, M.; Eccles, S.; te Poele,
R.; Workman, P.; Aboagye, E. O.; Linardopoulos, S. Mechanism of
Action and in Vivo Quantification of Biological Activity of the Aurora
Kinase Inhibitor CCT129202. Mol. Cancer Ther. 2007, 6, 3147−3157.
(48) Molecular Operating Environment (MOE), version 2012.10;
Chemical Computing Group Inc.: Montreal, Quebec, Canada, 2011.
(49) Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R.
Development and Validation of a Genetic Algorithm for Flexible
Docking. J. Mol. Biol. 1997, 267, 727−748.
(50) CCP4. The CCP4 (Collaborative Computational Project
Number 4) Suite: Programmes for Protein Crystallography. Acta
Crystallogr., D: Biol. Crystallogr. 1994, 50, 760-763.
(51) Adams, P. D.; Grosse-Kunstleve, R. W.; Hung, L. W.; Ioerger, T.
R.; McCoy, A. J.; Moriarty, N. W.; Read, R. J.; Sacchettini, J. C.; Sauter,
N. K.; Terwilliger, T. C. PHENIX: Building New Software for
Automated Crystallographic Structure Determination. Acta Crystallogr.,
D: Biol. Crystallogr. 2002, 58, 1948−1954.
N
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