Synthesis and SAR of 1-acetanilide-4-aminopyrazole-substituted quinazolines: Selective inhibitors of Aurora B kinase with potent anti-tumor activity

Article abstract of DOI:10.1016/j.bmcl.2008.02.002

A new class of 1-acetanilide-4-aminopyrazole-substituted quinazoline Aurora kinase inhibitors has been discovered possessing highly potent cellular activity. Continuous infusion into athymic mice bearing SW620 tumors of the soluble phosphate derivative 2

Full text of DOI:10.1016/j.bmcl.2008.02.002

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1904–1909
Synthesis and SAR of 1-acetanilide-4-aminopyrazole-
substituted quinazolines: Selective inhibitors of Aurora B
kinase with potent anti-tumor activity
a
a
b
Kevin M. Foote,a, Andrew A. Mortlock, Nicola M. Heron, Frederic H. Jung,
George B. Hill,a Georges Pasquet,b Madeleine C. Brady,a Stephen Green,a
Simon P. Heaton,a Sarah Kearney,a Nicholas J. Keen,a, Rajesh Odedra,a
Stephen R. Wedgea and Robert W. Wilkinsona
*
´ ´
aAstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom
bAstraZeneca, Centre de recherches, Parc Industriel, Pompelle, BP1050, Chemin de Vrilly, 51689 Reims Cedex2, France
Received 21 December 2007; revised 31 January 2008; accepted 2 February 2008
Available online 7 February 2008
Abstract—A new class of 1-acetanilide-4-aminopyrazole-substituted quinazoline Aurora kinase inhibitors has been discovered pos-
sessing highly potent cellular activity. Continuous infusion into athymic mice bearing SW620 tumors of the soluble phosphate deriv-
ative 2 led to dose-proportional exposure of the des-phosphate compound 8 with a high-unbound fraction. The combination of
potent cell activity and high free-drug exposure led to pharmacodynamic changes in the tumor at low doses, indicative of Aurora
B-kinase inhibition and a reduction in tumor volume.
Ó 2008 Elsevier Ltd. All rights reserved.
The Aurora proteins are a small family of serine/threo-
nine kinases that are expressed during mitosis and have
roles in chromosome segregation and cytokinesis.
Humans express three Aurora kinase paralogues—
Aurora A, B and C, the biology of which has been
reviewed extensively.1–4 With their key role in mitosis
and being aberrantly over-expressed in tumor cells, the
Aurora kinases have been suggested to be attractive
drug targets.1 Inhibition of either Aurora A or Aurora
B leads to very different cellular phenotypes. When
Aurora B is inhibited in tumor cells, the cells are forced
through a catastrophic mitotic exit leading to polyploid
cells that rapidly lose viability. In contrast, selective
inhibition of Aurora A-kinase activity results in an
accumulation in mitosis and abnormalities in centro-
some separation leading to the formation of monopolar
spindles.4
A number of small molecule inhibitors of the Aurora
kinases have demonstrated anti-tumor activity and have
subsequently entered clinical evaluation. MK-0457
(VX-680),5 and PHA-7395358,6,7 inhibit Aurora A and
Aurora B (whilst being more potent inhibitors of
Aurora A-kinase activity) and have some activity
against other, non-Aurora, kinases.5–7 Most recently,
MLN8054 a selective Aurora A-kinase inhibitor with
potent anti-tumor activity has been reported,8,9 and we
have described the discovery and pre-clinical activity
of AZD1152, the first Aurora B selective inhibitor to
enter clinical evaluation.10,11
AZD1152 1 (Fig. 1) resulted from optimization of a
series of 5-acetanilide-3-aminopyrazole (3-pyrazole)-
substituted quinazolines which are selective for Aurora
B over Aurora A and achieve high levels of solubility
by virtue of their ability to be delivered as readily
activated phosphate derivatives making them suitable
for parenteral administration.10 AZD1152 possesses
robust anti-tumor activity in a range of pre-clinical mod-
els and is currently in phase I clinical trials.11 In this paper
we report the synthesis and properties of a series of 1-
acetanilide-4-aminopyrazole-substituted quinazolines that
Keywords: Aurora; Kinase; Quinazoline; Pyrazole.
*
Corresponding author. Tel.: +44 (0)1625 518629; fax: +44 (0)1625
510097; e-mail: kevin.foote@astrazeneca.com
Present address: AstraZeneca Pharmaceuticals LP, 35 Gatehouse
Drive, Waltham MA 02451, USA.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.002

K. M. Foote et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1904–1909
1905
H
N
N
N
N
HO
HO
N
HO
O
O
H
N
H
N
P
HN
N
HN
N
HO
P
O
F
O
O
O
N
F
F
N
O
N
O
AZD1152, 1
2
Figure 1. Structures of AZD1152, 1 and 4-pyrazole compound, 2.
led to the discovery of compound 2 (Fig. 1) and which
complements the other series we have described previ-
ously.10–15
nazoline ring is replaced with a proton. In matched pair
analogues, compounds lacking the C-6 methoxy substi-
tuent achieve significantly increased inhibition of his-
tone-H3 phosphorylation in human SW620 colorectal
adenocarcinoma tumor cells (compare compounds 5 vs
7 and 3 vs 8 in Table 1). Histone-H3 is a cellular sub-
strate of Aurora B and a marker of Aurora B-kinase
inhibition in vivo. The cell activity for the 4-pyrazole
compounds is particularly noteworthy and even greater
than the corresponding 3-pyrazole analogues.10 In the 4-
pyrazole series multiple compounds were discovered
with a cellular EC50 < 10 nM and some, such as 8, with
a cellular EC50 < 1 nM.
Compounds 2–8 were prepared as shown in Scheme 1.16
The amino pyrazoles 15 and 16,16,17 were reacted with
the 4-chloroquinazoline intermediates 9 and 10,10 to give
the chloropropoxy pyrazole acetanilide intermediates
11–14. The drug compounds 3–8 were then prepared
by displacing the chloro-leaving group in 11–14 with
the appropriate amine under standard conditions. The
phosphate derivative 2 was prepared using an analogous
procedure described previously.10
The SAR in the 1-acetanilide-4-aminopyrazole (4-pyra-
zole) series followed the same broad trends we have re-
ported for the related 5-acetanilide-2-aminothiazole
(thiazole),15 and 3-pyrazole series.10 A representative
set of compounds is shown in Table 1.16 In summary,
the 4-pyrazole compounds are highly potent Aurora B-
kinase inhibitors but show less activity against Aurora
A. Fluoro groups are particularly favorable especially
in the ortho- and meta-positions and a number of basic
side chains attached to the C-7 position of the quinazo-
line ring delivered potent cellular activities. Selectivity
for Aurora B over Aurora A is enhanced in compounds
where the methoxy group at the C-6 position on the qui-
In contrast to the 3-pyrazole series, compounds from the
4-pyrazole series potently inhibit a small number of
other kinases while retaining generally high specificity
for Aurora B. The kinase activity profile for compound
8 in a panel of 62 kinases is shown in Figure 2. Four
kinases (PDGFRa, b, CSF-1R and c-KIT) are inhibited
with IC50 values within 100-fold of the Aurora B-IN-
CENP value (IC50 values = 0.069, 0.006, 0.036 and
0.009 lM, respectively). Despite the existence of other
non-Aurora kinase activities, SW620 cells treated with
compound 8 show reduced histone-H3 phosphorylation
and undergo endoreduplication, a phenotype consistent
with the inhibition of Aurora B kinase.4,12
N
N
Cl
H
HN
R1
N
R3
a
N
O
R1
O
N
Cl
O
N
F
Cl
N
9
R1 = MeO
11 R1 = MeO; R3 = H
12 R1 = MeO; R3 = F
13 R1 = H; R3 = H
14 R1 = H; R3 = F
10 R1 = H
N
N
H
N
H2N
R3
O
b
F
c
15 R3 = H
16 R3 = F
3 - 8
2
Scheme 1. Reagents and conditions: (a) 15 or 16, 4.0 N HCl (dioxane), DMA, 50–90 °C, 69–95% yield; (b) Amine, KI, DMA, 90 °C, 58–91% yield;
(c) di-tert-butyldiethylphosphoramidite, tetrazole, DMF, rt; H2O2, ꢀ10 °C ! rt; HCl, dioxane, rt; 68% yield over 3 steps.

1906
K. M. Foote et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1904–1909
Table 1. Summary of SAR in the 4-pyrazole series
N
N
H
N
HN
N
R3
O
R1
O
N
F
R2
a,b
Cell EC50 (lM)
Compound
R1
R2
R3
F
Aurora A Kia,c (lM)
Aurora B-INCENP Kia (lM)
HO
3
MeO
0.34
<0.001
0.023
0.010
0.089
0.032
0.007
<0.001
]
N
N
]
]
HO
HO
N
4
5
6
7
8
MeO
MeO
MeO
H
F
0.54
1.3
1.1
3.0
<0.001
0.001
H
H
H
F
]
HO
HO
N
0.001
<0.001
<0.001
]
]
N
HO
H
N
a Average of at least two independent dose–response curves. Variation was generally <25%.
b Inhibition of histone-H3 phosphorylation following 24-h incubation in SW620 tumor cells. Assays were carried out as described in Ref. 10.
c Ki value for compound 8 was not measured.
Compounds from the 4-pyrazole, like the 3-pyrazole ser-
ies,10 possess attractive physicochemical, pharmacoki-
netic and safety-pharmacology properties; selected
data for compounds 8 and 2 are summarized in Table
2. In our previous reports we have shown a role for
the charged side chain, in compounds such as 8, in
reducing LogD and concomitantly enhancing po-
tency.10,13,15 In addition to the charged side chain, the
heterocycle linking the hinge (quinazoline) to the lipo-
philic pocket (benzamide)-binding group, has a further
effect on physicochemical-related properties. Both pyra-
zole-based series have significantly reduced protein
binding compared to the corresponding thiazole ana-
logues (Fig. 3). Lipophilicity is likely to play a key role
in this observed difference with the potential for an addi-
tional structural component.
100
90
80
70
60
50
40
30
20
10
0
Kinase IC50 μM
0.001-0.01
0.01-0.1
0.1-1
1-10
>10
Figure 2. Activity profile for compound 8 in a panel of 62 serine/
threonine and tyrosine kinases.
Table 2. Physicochemical, pharmacokinetic and safety-pharmacology properties of compounds 8 and 2
Compound LogD7.4 pKa
Solubilitya (mg/mL) % Boundb Rat Clc (mL/min/kg) 3A4 inhibition IC50 (lM) hErg IC50 (lM)
d
e
8
2
2.1
8.9, 5.4
91
40
>10
6.1
>100
>100
10
a Tris buffer, pH 9.
b Rat serum albumin.
c Compounds were dosed to male Han Wistar rats at 5 mg/kg formulated in a mixture of 10% DMSO in water.
d The activity of human CYP3A4 determined by inhibiting the biotransformation of 7-benzyloxy-4-(trifluoromethyl)-coumarin to the fluorescent
metabolite 7-hydroxy-4-trifluoromethyl-coumarin.
e Activity against the human ether-a-go-go-related gene (hERG)-encoded potassium channel was determined using automated whole-cell
electrophysiology.

K. M. Foote et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1904–1909
1907
100
Compound 2
Compound 8
H
N
HN
N
R3
O
10
1
R1
O
N
F
R2
6
5.5
5
0.1
0.01
0.001
0
2
4
6
8
10
12
Time (hours)
4.5
Figure 5. Group mean plasma concentration ( SEM) of compound 2
and compound 8 in male Wistar Hannover rats following single
intravenous bolus dosing of compound 2 at 5 mg/kg.
4
3.5
H
N
N
the phosphate 2 have potent anti-proliferative effects in
SW620 cells (IC50 < 0.001 lM).18 However, compounds
bearing phosphate groups are considered to be imper-
meable due to the highly charged nature of the phos-
phate moiety at physiological pH.19 The cellular
activity observed for the phosphate 2 can be rationalized
by conversion to the parent compound 8 during the 48-h
incubation period of the assay.20
N
N
]
N
]
]
[
S
[
[
Figure 3. LogK values measured in rat plasma for matched-pair
analogues in the thiazole (j),15 3-pyrazole (d),10 and 4-pyrazole (m)
series. Each marker represents an individual compound matching the
substructure shown. Lines connect exact (R1, R2, R3) matches.
Average LogK values for thiazole, 3-pyrazole and 4-pyrazole com-
pound sets in the study = 5.2, 4.3 and 4.1, respectively.
The combination of high cellular potency, a high-un-
bound fraction and good exposure for compound 8 fol-
lowing dosing of the phosphate prodrug 2 led to in-vivo
activity at low doses. Inhibition of histone-H3 phos-
phorylation at doses of 2 from 0.5 mg/kg/d was observed
reaching a maximum of 75–85% inhibition from doses
of 2.5 mg/kg/d onwards (Fig. 6). At all of the doses
tested, inhibition of histone-H3 phosphorylation corre-
lated with an accumulation of cells with 4 N DNA con-
tent compared with vehicle-treated controls. These
Following subcutaneous infusion of the phosphate 2,
the plasma concentration of the phosphate 2 and the
parent drug 8 increased in parallel with increasing dose
(Fig. 4). To determine the rate and extent of conversion,
an iv-bolus of phosphate 2 was administered in rats. The
peak mean plasma concentration (Cmax) of both 2 and
the parent 8 occurred at 2 min postdose (tmax), indicat-
ing that 2 was rapidly converted to the parent drug 8
(Fig. 5). When compound 2 was dosed as an iv-bolus,
it provided 98% of the AUC of the parent 8 compared
with direct dosing of 8. These data demonstrate that
conversion of the phosphate prodrug to the parent drug
is both rapid and complete following parenteral admin-
istration. Interestingly, both the parent compound 8 and
100
10
phospho-histone H3
4N
9
8
7
6
5
4
3
2
1
0
90
80
70
60
50
40
30
20
10
10
Compound 2 Compound 8
1
0.1
0.01
Control
0.5
2.5
10
50
0.001
Dose mg/kg/d
0.5
2.5
10
50
Figure 6. Pharmacodynamic activity of compound 2 in SW620 tumor
xenografts established in male nude mice. Compound 2 at the doses
indicated or vehicle were administered as a constant infusion for 48 h
using a subcutaneous osmotic mini-pump. Figure shows flow-cyto-
metric analysis for phospho-histone-H3 (PhH3) (Columns, percentage
of PhH3 positive cells gated in G2/M phase of cell cycle; bars, SD) and
4 N DNA content (Points, percentage 4 N DNA cells; bars, SD).11
Dose mg/kg/d
Figure 4. Plasma concentration of compounds 2 and 8 following
dosing of compound 2 in male Swiss nude (nu/nu genotype) mice.
Compound 2 was administered as a constant infusion for 48 h from a
subcutaneous osmotic mini-pump. Columns, plasma concentration
measured immediately at end of infusion period lM; bars, SEM.

1908
K. M. Foote et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1904–1909
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0.8
0.6
0.4
0.2
0
Control
Compound 2
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H.; Pasquet, G.; Lohmann, Jean-Jacques 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.;
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Keen, N. J.; Crafter, C.; Foster, J. R.; Brady, M. C.;
Bigley, A.; Brown, E.; Byth, K.; Barrass, N. C.; Mundt,
K.; Foote, K. M.; Heron, N. M.; Jung, F.; Mortlock, A.
A.; Boyle, F. T.; Green, S. Clin. Can. Res. 2007, 13, 3682.
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5
7
9
11
13
15
17
19
Day post tumour inoculation
Figure 7. In vivo tumor-growth inhibition activity of compound 2 in
SW620 tumor xenografts. Male nude mice (n = 9–10 per group) were
dosed with either vehicle or a single cycle of compound 2 (50 mg/kg/day)
as a constant infusion for 24 h using a subcutaneous osmotic mini-pump
from day 6 to day 7. Points, mean tumor volume cm3; bars, SEM.11
results are consistent with inhibition of Aurora B-kinase
activity in the tumors and recapitulate the in-vitro phe-
notypic observations.10,11
The robust pharmacodynamic activity of compound 2
led to significant inhibition of SW620 tumor growth in
nude mice. At the higher doses, mechanism-related
and reversible myelosuppression was found to be the
dose-limiting toxicity. However, anti-tumor activity
was seen for compound 2 using a tolerated schedule of
50 mg/kg/d infusing for 24 h which gave a reduction in
tumor size of 89% at the end of the study compared to
tumors in vehicle-treated control animals (Fig. 7). A
reduction in tumor size was also seen at doses as low
as 2.5 mg/kg/d dosed for 48 h (data not shown).
In summary, we have discovered a new series of amino-
pyrazole-substituted quinazoline Aurora kinase inhibi-
tors. Optimization led to the discovery of compound
2, which in pre-clinical in-vivo models shows activity
at lower doses compared with other series we have pre-
viously reported.
13. Mortlock, A. A.; Keen, N. J.; Jung, F. H.; Heron, N. M.;
Foote, K. M.; Wilkinson, R.; Green, S. Curr. Top. Med.
Chem. 2005, 5, 807.
14. Heron, N. M.; Anderson, M.; Blowers, D. P.; Breed, J.;
Eden, J. M.; Green, S.; Hill, G. B.; Johnson, T.; Jung, F.
H.; McMiken, H. H. J.; Mortlock, A. A.; Pannifer, A. D.;
Pauptit, R. A.; Pink, J.; Roberts, N. J.; Rowsell, S. Bioorg.
Med. Chem. Lett. 2006, 16, 1320.
15. Jung, F. H.; Pasquet, G.; Lambert-van der Brempt, C.;
Lohmann, J-J. M.; Warin, N.; Renaud, F.; Germain, H.;
De Savi, C.; Roberts, N.; Johnston, T.; Dousson, C.; Hill,
G. B.; Mortlock, A. A.; Heron, N.; Wilkinson, R. W.;
Wedge, S. R.; Heaton, S. P.; Odedra, R.; Keen, N. J.;
Green, S.; Brown, E.; Thompson, K.; Brightwell, S.
J. Med. Chem. 2006, 49, 955.
Acknowledgments
We acknowledge the excellent technical expertise of the
following scientists: Chris De Savi, Trevor Johnson,
Nicola J. Roberts, Glenn Hatter, Kristy Readman,
Fabrice Renaud, Patrice Koza, Jean-Jacques Lohmann,
Liz Khatri, Elaine Brown, Katherine Thompson,
Stephen Brightwell, Sharon Barnett and the CDMG
team.
16. Heron, N. M.; Pasquet, G. R.; Mortlock, A. A.; Jung, F.
H. WO2004094410.
References and notes
17. Hill, G. B.; Mortlock, A. A. Synthesis 2007, 11, 1697.
18. Compounds were incubated with SW620 tumor cells in the
presence of 5% foetal-calf serum for 48-h. The antiprolif-
1. Keen, N.; Taylor, S. Nat. Rev. Can. 2004, 4, 927.
2. Andrews, P. D. Oncogene 2005, 24, 5005.

K. M. Foote et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1904–1909
1909
erative effect of compounds was measured by inhibition of
DNA synthesis using a 2-h BrdU uptake assay.
Med. Chem. 2003, 11, 885; (b) Jones, R. J.; Bischofberger,
N. Antiviral Res. 1995, 27, 1.
19. Pharmaceutical interest in phosphate esters (especially in
the antiviral area) has necessitated the development of
prodrugs of the phosphate moiety to deliver such com-
pounds into target cells e.g. see: (a) Schultz, C. Bioorg.
20. The phosphonate analogue of 1 retains activity vs Aurora
B kinase but does not have significant activity in cellular
assays (IC50 > 1 lM) confirming that highly charged
compounds have low cell permeability.