The discovery of the potent aurora inhibitor MK-0457 (VX-680)

Article abstract of DOI:10.1016/j.bmcl.2009.04.136

The identification of a novel series of Aurora kinase inhibitors and exploitation of their SAR is described. Replacement of the initial quinazoline core with a pyrimidine scaffold and modification of substituents led to a series of very potent inhibitors of cellular proliferation. MK-0457 (VX-680) has been assessed in Phase II clinical trials in patients with treatment-refractory chronic myelogenous leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL) containing the T315I mutation.

Full text of DOI:10.1016/j.bmcl.2009.04.136

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3586–3592
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery of the potent aurora inhibitor MK-0457 (VX-680)
David Bebbington, Hayley Binch, Jean-Damien Charrier, Simon Everitt, Damien Fraysse, Julian Golec,
*
David Kay, Ronald Knegtel, Chau Mak, Francesca Mazzei, Andrew Miller, Michael Mortimore ,
Michael O’Donnell, Sanjay Patel, Francoise Pierard, Joanne Pinder, John Pollard, Sharn Ramaya,
Daniel Robinson, Alistair Rutherford, John Studley, James Westcott
Vertex Pharmaceuticals (Europe) Ltd, 88 Milton Park, Abingdon OX14 4RY, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
The identification of a novel series of Aurora kinase inhibitors and exploitation of their SAR is described.
Replacement of the initial quinazoline core with a pyrimidine scaffold and modification of substituents
led to a series of very potent inhibitors of cellular proliferation. MK-0457 (VX-680) has been assessed
in Phase II clinical trials in patients with treatment-refractory chronic myelogenous leukemia (CML) or
Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL) containing the T315I
mutation.
Received 24 March 2009
Revised 24 April 2009
Accepted 27 April 2009
Available online 3 May 2009
Keywords:
Aurora
Ó 2009 Elsevier Ltd. All rights reserved.
Kinase inhibitor
SAR
Cellular proliferation is a tightly regulated process. A number of
protein kinases have been assigned as critical mediators of cell-cy-
cle progression. The Aurora family of highly homologous serine/
thronine protein kinases (consisting of Aurora-A, -B and -C) regu-
late many of the processes that are pivotal to the final stages of cell
division or mitosis. Inappropriate completion of mitosis leads to
genetic instability and often to cells that contain non-diploid
DNA content, a common hallmark of cancer.1 The Aurora kinases
were first recognised in 19952 and since then there has been an
increasing body of evidence linking Aurora-A and -B expression
with cancer.3 Though the Aurora family members are structurally
similar their biological functions are distinct. These differences
have been extensively reviewed.4–8
Aurora-A is involved in regulating entry into mitosis and in
early mitotic events. Its roles involve the bringing together and
assembly of important components for the process of cell division.
This includes recruiting microtubule spindle components to the
centrosome to enable centrosome maturation. After maturation
the centrosomes migrate apart. This process and formation of the
mitotic spindle is mediated by kinesin motor protein Eg5 an Aur-
ora-A substrate. Aurora-A depletion often results in delayed entry
into mitosis, defects in centrosome maturation and microtubule
organisation resulting in disruption of spindle formation leading
to mono- or multi-polar spindles. Aurora-B also plays multiple
roles in cell-division. It is involved in chromosome condensation,
spindle formation and subsequent attachment of microtubules to
the middle of the chromosome (kinetochore). The fidelity of this
process is controlled by the spindle checkpoint which in turn is
regulated by Aurora-B. Only when the kinetochores are correctly
attached can the final stage of cell division (cytokinesis) take place.
Cells depleted of Aurora-B fail cytokinesis and prematurely exit
mitosis without division, leaving polyploid cells. Aurora-C is less
well studied. It has a localisation pattern during cell division sim-
ilar to that of Aurora-B. Aurora-C is required for spermatogenesis,
but conclusive proof that it controls cell cycle progression in tu-
mour cells is lacking.
A number of studies have demonstrated that depletion or inhi-
bition of Aurora-A or - B by siRNA, dominant negative kinase mu-
tant or neutralising antibodies results in critical disruption of
mitosis and a block in proliferation leading to cell death in human
cancer cell-lines (recently reviewed in Ref. 8). These observations
have highlighted the Aurora kinases as promising targets for
anti-cancer therapy. In this Letter, we describe the discovery and
optimisation of a new series of Aurora inhibitors that led to MK-
0457 (VX-680), the first Aurora inhibitor to enter clinical trials.
MK-0457 (VX-680) is an Aurora inhibitor that disrupts mitosis,
inhibits proliferation and promotes apoptosis in cycling cells while
leaving non-cycling cells unaffected.9 Since the discovery of MK-
0457 (VX-680), many other Aurora inhibitors have also progressed
to clinical evaluation.5,10–15
The Aurora-A gene is located on chromosome 20q13.2 which is
frequently amplified in multiple human cancers and Aurora-A over
expression has been observed in many tumours.16 With these con-
siderations in mind we initiated an oncology project with Aurora-A
as our target. A screening campaign against full-length Aurora-A
* Corresponding author. Tel.: +44 0 1235 438805.
E-mail address: mike_mortimore@vrtx.com (M. Mortimore).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.136

D. Bebbington et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3586–3592
3587
Table 1
identified compound 1, an amino pyrazole linked to a 2-substi-
tuted quinazoline, as a lead. Compound 1 is a potent inhibitor of
Aurora-A (Ki = 81 nM) but also inhibits other kinases including
Src (Ki = 181 nM) and GSK3b (Ki = 73 nM). These two kinases were
used as counterscreens to gauge selectivity in the optimisation
process; GSK3b was regarded as the kinase being most similar in
sequence to the Auroras and Src kinase was used as a guide to gen-
eral kinase inhibition. A co-complex crystal structure of compound
2, a close analogue of compound 1, with Aurora-A (Fig. 1) showed
that the aminopyrazole provides three hydrogen bonds to the ki-
nase hinge region and the 2-phenyl group on the quinazoline en-
ters a lipophilic pocket, formed by the activation loop that is
capped by Phe275 from the DFG motif and Trp277.17 Since this
lipophilic pocket did not appear to be a common feature of kinases,
we hypothesised that maximising non-bonding interactions in this
region would provide an opportunity for increasing potency and
selectivity.
Effect of linker between quinazoline and aromatic ring on kinase activity and
selectivity
NH
N
HN
N
N
X
Compound
X
Aur-A Ki (nM)18
GSK3b Ki (nM)
Src Ki (nM)
3
4
5
6
7
Bond
NH
NMe
O
58
24
17
36
20
22
24
312
51
81
393
1059
1288
800
S
171
NH
NH
N
N
profile and this, coupled with the ease with which such compounds
could be synthesised, prompted us to base our immediate future
studies on the thioether system.
HN
N
HN
N
The introduction of simple lipophilic substituents onto the thio-
phenylethers was found to increase potency against Aurora-A by
up to ten fold. In some cases, for instance the 3,4-dimethoxythio-
phenylether 17 and the 2-naphthylthioether 18 (Table 2), gains
in selectivity against both Src and GSK3b were also observed. Per-
haps more remarkably, some compounds showed good selectivity
against Aurora-B as well. Despite many of these compounds giving
Ki values of less than 10 nM against Aurora-A, none were able to
inhibit the proliferation of Colo205 cells, as measured by 3H thymi-
N
N
N
Compound 1
Compound 2
Examination of the crystal structure of the compound 2/Aurora-A
co-complex suggested that placing a linking atom between the qui-
nazoline and the phenyl group of compound 2 would lead to more
favourable interactions with the lipophilic pocket created by
Phe275 and Trp277. In addition, this tactic would simplify the syn-
thetic chemistry and allow greater scope for exploration. Of the four
linkers examined, NH, NMe, O and S (Table 1), none gave a signifi-
cant improvement in potency compared to compound 3 where a
phenyl ring is directly attached to the quinazoline. However, all
showed improvements in selectivity profile as judged by their
cross-reactivity with Src and GSK3b. The nitrogen-linked com-
pounds were found to inhibit a number of CyP450s and were not
considered further. The thioether 7 gave an encouraging selectivity
dine uptake, at concentrations below 12 lM. This disappointing re-
sult was attributed to poor physical properties (e.g., the 2-
naphthylthioether 18 has cLog P = 6.5) and low cellular penetra-
tion. Therefore, attempts were made to decrease the lipophilicity
of the molecules while maintaining the overall shape of the 2-
naphthylthioether 18. Amides of 4-aminophenylthioethers were
found to be suitable isosteres (compounds 21, 23–26, Table 3). In
general, the amides retained or improved upon the potency against
Aurora-A with respect to the naphthyl compound 18, retained
selectivity versus Src and GSK3b and inhibited proliferation of
Colo205 cells at sub-lM concentrations.
Compound 21, from the series of amides (Table 3), was found to
have ADME properties (T1/2 ꢀ 1.5 h, F = 40% in rats) considered suf-
ficient to enable it to be used as a proof of principle compound in
xenograft models of cancer and was therefore characterised more
fully. Compound 21 was found to block proliferation of three dif-
ferent cancer cells lines with similar IC50 values of ꢀ500 nM, inhi-
bit Auroras-A, -B, and -C with Ki values of 4, 27 and 11 nM,
respectively, and show >100-fold selectivity over 53 kinases in
addition to Src and GSK3b. In addition, it was used to show that
blocking of mitosis by an Aurora inhibitor results in cell death
(IC50 for Colo205 cellular viability at 48 h using MTS stain-
ing = 8.5 lM). The cell death was due to apoptosis as shown by an-
nexin V binding, DNA fragmentation ELISA and TUNEL staining. In a
two-week xenograft experiment, the compound inhibited the
growth of the MCF-7 tumour by 50% when administered at its
maximum tolerated dose (MTD) (300 mg/kg bid po). Animal body
weights were largely unaffected, though, as expected, total white
blood cell counts were reduced. Taken together, these results in-
creased our belief in Aurora kinase inhibition as a target for cancer
therapy.
Although quinazoline compounds such as 21 had properties
adequate for demonstrating pharmacological activity, better po-
tency and improved physical properties, especially solubility, were
required for clinical development. Examination of co-complex
Figure 1. Co-complex crystal structure of Compound 2 in Aurora-A solved at 2.7 Å
resolution with crystallographic R-factor = 0.26. The amino pyrazole makes three
hydrogen bonds to the hinge region and the phenyl group enters lipophilic pocket
towards the back of the ATP binding site made up of Phe 275 and Trp 277.

3588
D. Bebbington et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3586–3592
Table 2
focussed on 5-methylpyrazoles and amides of small aliphatic acids
as the thiophenyl substituent.
Effect of SAr substituents on kinase activity and selectivity
NH
N
Lipophilic substitution at the 6-position of the new pyrimidine
scaffold, as illustrated by the sequence of inhibitors 27–35 in Table
4, led to compounds that were equal in potency against Aurora-A
to quinazoline 21 but without gains in cellular potency. Introduc-
tion of heterocycles as the 6-substituent on the pyrimidine, as
illustrated by the series of compounds 36–43, had little effect in
terms of potency against Aurora-A and only a marginal improve-
ment against Aurora-B. However, a dramatic improvement in cel-
lular potency was observed within the series. For instance,
compound 37 with Aur-A Ki = 1.6 nM and Aur-B Ki = 8 nM inhibited
Colo205 proliferation with IC50 = 400 nM, while compound 40,
with a similar Aurora inhibition profile inhibited cellular prolifera-
tion with IC50 = 24 nM. A 200-fold increase in cellular activity is ob-
served for compound 43 over 37, again with little difference
between their respective ability to inhibit Aurora. While these re-
sults were broadly in line with the concept that that inhibition of
Aurora-B rather than Aurora-A is more important for the inhibition
of cell proliferation,20,21 the remarkable increases in cellular po-
tency were difficult to explain in terms of changes to physical
properties or improvements in potency against Aurora-B.
A co-complex crystal structure of compound 44 (VX-680/MK-
0457) with Aurora-A showed the compound bound to a closed
and, what might be considered to be, an inactive conformation of
the enzyme.22,23 The cyclopropyl group of the amide makes excel-
lent interactions with a lipophilic pocket derived from Phe275 of
the DFG loop (Fig. 2) that is not present in an open ‘active’ confor-
mation. These observations led us to study the enzyme inhibition
kinetics more deeply. It was noted that compounds that inhibited
cellular proliferation most potently, the N-alkylpiperazine com-
pounds, inhibited Aurora-B through a time dependent mechanism,
with a long residence time for the compound on the enzyme (for
compound 43: Aurora-B Kiꢁ ¼ 0:8 nM; T1/2 = 8 h). Since the enzyme
assay used for the structure activity studies assumed rapid equilib-
rium kinetics, the potency of such compounds on Aurora-B was
underestimated. Extended analysis of the enzyme kinetics showed
a two-step binding process. It is likely that the first step is the for-
mation of a complex between the inhibitor and an open conforma-
tion of the active enzyme followed by a conformational change to
deliver a tight binding complex. These inhibitors exhibit normal,
rapid equilibrium, reversible kinetics with Aurora-A. The anoma-
lies between enzyme Ki and Colo205 IC50 can now be explained,
not in terms of Aurora-A affinity or lipophilicity, but cellular po-
HN
N
Ar
N
S
Compound Ar
Aur-A Ki
(nM)
Aur-B Ki
(nM)
GSK3b Ki
(nM)
Src Ki
(nM)
7
8
Ph
2-ClPh
20
5
4
6
3
2
5
2
24
9
590
445
185
>1100
675
>1100
>1100
>40
995
575
171
126
33
44
48
800
201
9
3-ClPh
10
11
12
13
14
15
16
17
4-ClPh
2,3-DiClPh
2,4-DiClPh
2,6-DiClPh
3,4-DiClPh
2-OMe
8
113
>15
216
124
1168
>38
>1000
4-OMe
3,4-
17
120
2180
>500
DiOMePh
2-Naphthyl
18
1
>40
>150
crystal structures led to the hypothesis that the lipophilic binding
provided by the fused benzene portion of the quinazoline could be
replaced by suitable substitution at the 6-position of a pyrimidine
ring. The strategy of using a pyrimidine rather than a quinazoline
as a core scaffold was particularly attractive in that it was expected
to provide the scope for preparing more soluble molecules. Further
lipophilic binding might be obtained by making better interactions
in the pocket utilised by the 5-methyl group on the pyrazole of
compound 21 or by increasing the size or nature of the amide
group.
A series of compounds designed to optimise the substitution at
the 5-position of the pyrazole (R1 in Table 3) and the size of the
amide on the thiophenyl ether group showed that while gains in
potency might be achieved, a comparison between compounds
21, 23–26 (Table 3) illustrates this point, it was usually at the ex-
pense of general kinase selectivity (data not shown). As a conse-
quence, optimisation of pyrimidine-based Aurora inhibitors was
Table 3
Optimisation of S-naphthyl mimics for kinase activity and cellular potency
NH
R1
N
HN
N
Ar
S
N
Compound
R1
Ar
Aur-A Ki (nM)
Aur-B Ki (nM)
GSK3b Ki (nM)
Src Ki (nM)
Colo205 IC50 (l
M)19
18
19
20
21
22
23
24
25
26
Me
Me
Me
Me
Me
Me
Me
H
2-Naphthyl
1
2
9
4
<1
1
<1
24
<1
>40
230
250
27
105
57
51
>1100
15
>150
67
>4000
1034
17
1500
1314
605
>500
681
>3000
1258
796
720
461
277
1735
>12
4-(NHSO2Me)Ph
4-(NHC(O)OtBu)Ph
4-(NHC(O)Me)Ph
4-(NMeC(O)Me)Ph
4-(NHC(O)Et)Ph
4-(NHC(O)cPr)Ph
4-(NHC(O)Me)Ph
4-(NHC(O)Me)Ph
0.78
2.87
0.48
5.77
0.83
0.91
2.20
0.21
cPr
338

D. Bebbington et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3586–3592
3589
Table 4
Effect of 6-pyrimidine substituents on kinase activity and cellular potency
NH
N
HN
H
N
R3
N
O
R2
cLog P
N
S
Compound
R2
R3
Aq. solubility at pH 7.4 (
l
g/ml)
Aur-A Ki (nM)
Aur-B Ki (nM)
Colo205 IC50 (lM)
27
28
29
30
31
32
33
34
35
H
Me
Ph
Me
CyPr
tBu
Ph
3-Py
4-Py
Me
Me
Me
Et
Et
Et
Et
Et
Et
3.0
3.5
5.1
4.0
4.4
5.3
5.6
4.2
4.2
13.8
<1
86
18
2.4
5.1
3.9
10
<1
207
220
83
153
59
115
145
52
4.05
2.00
4.2
11.2
<1
0.72
1.42
2.00
0.87
0.47
1.00
3.9
<3
49
36
37
38
39
40
41
Et
Et
Et
Et
Et
Et
4.0
5.1
3.7
3.7
4.1
4.8
2.4
1.6
1.7
3.7
1.6
<1
23
8
0.73
N
N
N
2.5
0.400
0.135
0.079
0.024
0.024
5.4
14
18
20
25
O
N
296
63.4
114.5
HN
N
N
N
N
N
N
42
Et
4.6
12.8
<1
9.5
0.075
N
43
44
Et
5.4
4.3
220.8
1.3
0.6
11
18
0.002
0.019
N
N
CyPr
6.8
N

3590
D. Bebbington et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3586–3592
tency correlated with the compounds’ Kꢁi and time-dependent
kinetics with Aurora-B. A full description of the structural and ki-
netic studies together with the cellular activity and pharmacody-
namic implications of such Aurora inhibitors will be reported
elsewhere.
Compound 44 (MK-0457 (VX-680)) was considered to have the
best combination of potency and pharmaceutical properties in this
series of compounds and it was nominated for further develop-
ment. It is a potent inhibitor of all three Aurora kinases with Ki
of 0.6 nM against Aurora-A, 18 nM against Aurora-B and 5 nM
against Aurora-C and Kꢁi of 1.8 nM against Aurora-B. It shows selec-
tivity over 190 other protein kinases although it does cross-react
with a small number of unrelated kinases that are themselves
interesting cancer targets, including Flt-3, Abl and the T315I Abl
mutant, one of the most prevalent and resistant mutations of
Abl.22,24 The inhibition of these seemingly distinct kinases can be
explained by compound 44 being able to bind to their closed, inac-
tive conformations and so exploit lipophilic pockets that are not
thought to be available in an open or active kinase conformation.
MK-0457 (VX-680) is a potent anti-proliferative agent that is active
Figure 2. Co-complex crystal structure of compound 44, MK-0457 (VX-680), in
Aurora-A, solved at 2.9 Å resolution with crystallographic R-factor = 0.24, showing
the cyclopropyl group of the amide packing against Phe 275.
O
O
OH
NH2
NH2
N
NaOH / EtOH
PhC(O)Cl / Et3N
NH2
NH
N
X
O
X
NH
N
R1
H
N
Cl
N
HN
N
R1
N
P(O)Cl3 / Pr3N
H2N
DMF
N
N
X
X
Scheme 1. X = N, CH. Preparation of quinazoline-based inhibitor compounds 1–3.
NH
NH
N
N
R1
R1
H
N
Cl
N
HN
N
HN
N
R1
N
R
HX
N
N
H2N
R
EtOH
N
Cl
Cl
X
Scheme 2. Preparation of quinazoline-based inhibitor compounds 4–26.
NH
NH
N
N
H
N
Cl
N
HN
N
HN
N
N
NHC(O)R3
HS
NHC(O)R3
H2N
N
N
DMF
R2
N
Cl
R2
Cl
R2
S
Scheme 3. Preparation of 6-substituted pyrimidine-based inhibitor compounds 27–35.

D. Bebbington et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3586–3592
3591
Cl
Cl
Cl
N
NHC(O)R3
HS
NHC(O)R3
N
N
N
mCPBA
CH2Cl2
Cl
N
SO2Me
Cl
N
S
Cl
SMe
NH
NH
N
N
N
H
N
HN
HN
N
N
NHC(O)R3
NHC(O)R3
R4R5NH
4R5RN
H2N
DMF
N
S
Cl
N
S
Scheme 4. Preparation of 6-substituted pyrimidine-based inhibitor compounds 36–44.
10. Gautschi, O.; Mack, P. C.; Davies, A. M.; Lara, P. N.; Gandra, D. R. Clin. Lung
Cancer 2006, 8, 93.
11. Mortlock, A.; Keen, N. J.; Jung, F. H.; Heron, N. M.; Foote, K. M.; Wilkinson, R.;
Green, S. Curr. Top. Med. Chem. 2005, 5, 199.
12. Carpinelli, P.; Moll, J. Expert Opin. Ther. Targets 2008, 12, 69.
13. Naruganahalli, K. S.; Lakshmanan, M.; Dastidar, S. G.; Ray, A. Current Opin.
Invest. Drugs 2006, 7, 1044.
14. Oslob, J. D.; Romanowski, M. J.; Allen, D. A.; Baskaran, S.; Bui, M.; Elling, R. A.;
Flanagan, W. M.; Fung, A. D.; Hanan, E. J.; Harris, S.; Heumann, S. A.; Hoch, U.;
Jacobs, J. W.; Lam, J.; Lawrence, C. E.; McDowell, R. S.; Nannini, S. A.; Shen, W.;
Silverman, J. A.; Sopko, M. M.; Tangonan, B. T.; Teague, J.; Yoburn, J. C.; Yu, C. H.;
Zhong, M.; Zimmerman, K. M.; O’Brien, T.; Lew, W. Bioorg. Med. Chem. Lett.
2008, 18, 4880.
15. Howard, S.; Berdini, V.; Boulstridge, J. A.; Carr, M. G.; Cross, D. M.; Curry, J.;
Devine, L. A.; Early, T. R.; Fazal, L.; Gill, A. L.; Heathcote, M.; Maman, S.;
Matthews, J. E.; McMenamin, R. L.; Navarro, E. F.; O’Brien, M. A.; O’Reilly, M.;
Rees, D. C.; Reule, M.; Tisi, D.; Williams, G.; Vinkovi, M.; Wyatt, P. G. J. Med.
Chem. 2009, 52, 379.
16. For reviews of early Aurora research see: Biscoff, J. R.; Plowman, G. D. Trends
Cell Biol. 1999, 9, 454–459; Giet, R.; Prigent, C. J. Cell Sci. 1999, 112, 3591.
17. Cheetham, G. M. T.; Knegtel, R. M. A.; Coll, J. T.; Renwick, S. B.; Swenson, L.;
Weber, P.; Lippke, J. A.; Austen, D. A. J. Biol. Chem. 2002, 45, 42419.
18. Compounds were screened for their ability to inhibit Aurora-A using a standard
coupled enzyme assay (Fox et al., Protein Sci. 1998, 7, 2249). Assays were
carried out in a mixture of 100 mM Hepes (pH7.5), 10 mM MgCl2, 1 mM DTT,
against all cycling cells (IC50 value ranges from 15 nM to 113 nM).
Treatment of proliferating cells with MK-0457 (VX-680) leads to
accumulation of cells with 4 N DNA and in many cases extensive
endoreduplication in the absence of cell division. A detailed
description of its biological characteristics has been pub-
lished.9,25–29 MK-0457 (VX-680) has been assessed in a number
of clinical studies. In particular it has been studied in a dose esca-
lating Phase I/II study in refractory leukemias. It is interesting to
note that of the 14 patients with chronic myelogenous leukaemia
(CML) that were evaluated, 9 expressed the refractory T315I Abl
mutation and significantly, of these, 8 showed a haematological
or cytogenic response.30–32 Although clinical studies on MK-0457
(VX-680) have now been stopped, alternative Aurora kinase inhib-
itors continue to be studied.
From an initial strategy of investigating the effect of inhibiting
Aurora-A, the importance of inhibiting Aurora-B for preventing cel-
lular proliferation and causing cell death became apparent.
Through detailed inhibitor kinetic characterization and crystallo-
graphic investigation, the cellular potency and, to some extent,
the selectivity profile of MK-0457 (VX-680) has been explained.
It is fully expected that this understanding, together with what is
learnt in the clinic, will be utilized in the design of further classes
of Aurora inhibitors that will add to the armory of cancer therapies.
The synthesis of the Aurora inhibitors is shown in General
Schemes 1–4 below. The quinazoline-based inhibitors described
can be prepared according to Schemes 1 and 2.
25 mM NaCl, 2.5 mM phosphoenolpyruvate, 300
kinase and 10 g/ml lactate dehydrogenase. Final substrate concentrations in
the assay were 400 M ATP (Sigma Chemicals) and 570 M peptide (Kemptide,
lM NADH, 30 lg/ml pyruvate
l
l
l
American Peptide, Sunnyvale, CA). Assays were carried out at 30 °C and in the
presence of 40 nM Aurora-A. Ki data were calculated from non-linear
regression analysis using the Prism software package (GRAPHPAD PRISM version
3.0cx for Macintosh, GraphPad Software, San Diego California, USA).
19. Compounds were screened for their ability to inhibit cellular proliferation
using Colo205 cells obtained from ECACC. Colo205 cells were seeded in 96 well
plates and serially diluted compound was added to the wells in duplicate.
Control groups included untreated cells, the compound diluent (0.1% DMSO
alone) and culture medium without cells. The cells were then incubated for 72
or 96 h at 37 °C in an atmosphere of 5% CO2/95% humidity. Three hours prior to
The pyrimidine-based inhibitors described can be prepared
according to Schemes 3 and 4.
the end of the experiment 0.5 lCi of 3H thymidine was added to each well.
Acknowledgement
Cells were then harvested and the incorporated radioactivity counted on a
Wallac microplate beta-counter. Dose response curves were calculated using
either PRISM 3.0 (GRAPHPAD) or SoftMax Pro 4.3.1 LS (molecular devices) software.
20. Cochran, A. G. Chem. Biol. 2008, 15, 525.
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2008, 15, 552.
The authors thank Dr. Graham Cheetham for determining the
crystal structures.
22. Cheetham, G. M. T.; Charlton, P. A.; Golec, J. M. C.; Pollard, J. R. Cancer Lett. 2007,
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