Discovery of a new series of Aurora inhibitors through truncation of GSK1070916

Article abstract of DOI:10.1016/j.bmcl.2010.02.091

Novel Aurora inhibitors were identified truncating clinical candidate GSK1070916. Many of these truncated compounds retained potent activity against Aurora B with good antiproliferative activity. Mechanistic studies suggested that these compounds, dependi

Full text of DOI:10.1016/j.bmcl.2010.02.091

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2552–2555
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a new series of Aurora inhibitors through truncation of
GSK1070916
*
Jesus R. Medina , Seth W. Grant, Jeffrey M. Axten, William H. Miller, Carla A. Donatelli,
Mary Ann Hardwicke, Catherine A. Oleykowski, Qiaoyin Liao, Ramona Plant, Hong Xiang
Oncology Research, GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, PA 19426, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel Aurora inhibitors were identified truncating clinical candidate GSK1070916. Many of these trun-
cated compounds retained potent activity against Aurora B with good antiproliferative activity. Mecha-
nistic studies suggested that these compounds, depending on the substitution pattern, may or may not
exert their antiproliferative effects via inhibition of Aurora B. The SAR results from this investigation will
be presented with an emphasis on the impact structural changes have on the cellular phenotype.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 7 January 2010
Revised 22 February 2010
Accepted 24 February 2010
Available online 1 March 2010
Keywords:
Aurora inhibitors
BEI
Lead generation
FBDD
FACS
Cellular phenotype
The generation of viable chemical leads is a crucial step in the
drug discovery process. The quality of a lead compound can have
a profound effect on the lead-to-candidate phase of the drug
discovery process, as well as on the chances that the respective
candidate might be successful in the clinic.1 Recently, fragment-
based screening technologies have provided a new source of lead
compounds, making fragment-based drug discovery (FBDD) an
important tool in the drug discovery process.2 One of the most
important concepts in FBDD is the use of ligand efficiency indices
such as BEI3 (binding efficiency index = pIC50 ꢀ 1000/MW), which
provides a metric for assessing the quality of initial screening hits.
BEI can also be used to help assess the additional contributions
that new functionality makes to the overall activity of a compound
as it is optimized. This concept can be applied to any drug discov-
ery methodology, even retrospectively.2d The deconstruction of
high molecular weight lead compounds can be used to identify
the minimal core fragments with high BEI, which can serve as
new starting points for lead generation.
development.4a,5 Studies have shown that mitotic defects follow-
ing exposure of cells to Aurora kinase inhibitors are largely due
to the inhibition of Aurora B.6 Since Aurora B function is critical
for cytokinesis, its inhibition forces the cells through a mitotic exit
leading to polyploidy cells that ultimately lose viability.7
Recently the Aurora B inhibitor GSK1070916 was advanced as
an iv agent for the treatment of cancer.8,9 As part of our effort to
find a suitable back-up series, we wanted to identify a new lead
with a lower MW and higher BEI. To discover new leads, we em-
barked on an investigation to determine the minimum pharmaco-
phore of GSK1070916 and use it as a fragment for optimization.
GSK1070916 evolved from compound 1, a lead generated from
cross-screening and subsequent SAR refinement (Fig. 1).10 Since
we recognized the pyrazoloazaindole ‘fragment’ to be present in
both compounds, we started our investigation by preparing the
un-substituted 4-pyrazoloazaindole 2 and the corresponding N-
methylpyrazole derivative 3. The synthetic route to the pyrazoloaz-
aindoles 2 and 3 is illustrated in Scheme 1. Suzuki coupling of 4-
bromoazaindole 4 with (1H-pyrazol-4-yl)boronic acid pinacol ester
gave the corresponding phenylsulfonamide protected pyrazoloaz-
aindole 5. Removal of the phenylsulfonamide protecting group un-
der basic conditions afforded 4-pyrazoloazaindole 2. Alternatively,
methylation of intermediate 5 followed by deprotection of the phe-
nylsulfonamide group gave the N-methylpyrazole derivative 3.
In addition, we prepared the truncated versions of GSK1070916
where either the arylamine group (compound 6) or the phenylurea
group (compound 9) was removed. Compound 6 was prepared as
The Aurora protein kinases (A, B, and C) are a small family of
serine/threonine kinases that are expressed during mitosis and
have roles in chromosome segregation and cytokinesis.4 Because
overexpression of Aurora A and Aurora B is frequently associated
with tumorigenesis, these proteins have been targeted for therapy,
and a number of small molecule inhibitors have been progressed to
* Corresponding author. Tel.: +1 610 917 5889.
E-mail address: jesus.r.medina@gsk.com (J.R. Medina).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.091

J. R. Medina et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2552–2555
2553
nitro group gave the corresponding intermediate 8. Reaction of
aniline 8 with dimethylcarbamoyl chloride afforded compound 6.
The syntheses of compound 9 and its respective isomer 10 were
initiated by sequential Suzuki couplings of azaindole 11 with the
appropriate boronic acid or ester followed by N-methylpyrazolyl
boronic ester. Reductive amination of intermediate 12a followed
by phenylsulfonamide deprotection afforded 9. Compound 10
was obtained from the direct deprotection of 12b (Scheme 3).
Compounds 2 and 3 showed relatively weak activity in the Aur-
ora B enzyme assay (pIC50 = 6.1) but excellent binding efficiency
(BEI >30), which defined the baseline level of activity of the azain-
dole–pyrazole template (Table 1). Compound 6, which represents
the truncated version of GSK1070916 lacking the arylamine group,
exhibited a 32-fold increase in enzyme potency relative to the pyr-
azoloazaindoles 2 and 3, but with a significant decrease in binding
efficiency (BEI = 20). In addition, the enzyme potency did not cor-
respond with a similar level of activity in the cell proliferation as-
say. Interestingly, compounds 9 and 10, which lack the phenylurea
but contained an arylamine group, both exhibited good activity in
the Aurora B enzyme assay (pIC50 = 8.2), good binding efficiency
(BEI = 25), and excellent activity in the A549 proliferation assay
(Table 1). Compound 10 does show oral exposure (DNAUC = 139
ng h/mL/mg/kg) and 72% bioavailability in rats, although it has
high clearance (CLb = 86 mL/min/kg).
N N
N
O
N
N
H
H
N
1
H
Aurora B pIC50 8.7
MW
BEI
422.5
21
Phenylurea
N N
N
O
N
N
N
H
N
H
Arylamine
GSK1070916
Aurora B pIC50 8.5
MW
BEI
507.6
17
Figure 1. Discovery of GSK1070916.
H
To investigate the mechanism of the antiproliferative activity,
we evaluated A549 lung tumor cell lines treated with 9 and 10
using fluorescence-activated cell sorting (FACS). Given that com-
pounds had different potencies against tumor cell growth (Table
1), in order to compare them in the FACS analysis they were all
tested at 5ꢀ their EC50 values in the cell proliferation assay.
A549 lung tumor cell lines treated with 10 showed an increase
in sub-2N DNA and little or no increase in 4N/>4N DNA compared
to the DMSO control (Table 2). Since inhibition of Aurora B should
cause an increase in 4N/>4N DNA,7,9 this result strongly suggested
that 10 exerts its antiproliferative effects by a non-Aurora B
mechanism of action. We attribute these observations to the de-
crease in kinase selectivity associated with the removal of the
phenylurea group.12 In contrast, FACS analysis on 9 (Table 2),
exhibited an accumulation of sub-2N and 4N DNA (but not >4N
DNA). Although this phenotype was not entirely consistent with
that of an Aurora B inhibitor, we reasoned that 9 may be acting
partially via an Aurora B mechanism of action based on the ob-
served increase in 4N DNA.
N N
Br
N
a
N
N
N
SO2Ph
SO2Ph
4
5
c
b, c
H
N N
N N
N
N
N
N
H
H
2
3
Scheme 1. Reagents and conditions: (a) (1H-pyrazol-4-yl)boronic acid pinacol
ester, PdCl2(PPh3)2, 1,4-dioxane, aq NaHCO3, 100 °C, 73%; (b) MeI, Cs2CO3, DMF,
72%; (c) 6 N NaOH, CH3OH, reflux, 15% for 2, 85% for 3.
illustrated in Scheme 2. Suzuki coupling of N-ethylpyrazolyl boro-
nic ester 711 with 4-bromoazaindole followed by reduction of the
N N
Br
a
b
I
R
N
N
N N
B
N N
N
SO2Ph
N
a
SO2Ph
O2N
H2N
11
12a: R = meta-CHO
O
O
12b: R = para-CH2NMe2
N
N
N N
N
H
7
8
3 N
4
N N
O
b
N
N
H
N
H
9 (meta), 10 (para
)
N
N
H
Scheme 3. Reagents and conditions: (a) (1) Pd(PPh3)4, 1,4-dioxane, aq NaHCO3,
100 °C [for 12a: (3-formylphenyl)boronic acid; for 12b: {4-[(dimethyl-
amino)methyl]phenyl}boronic acid pinacol ester]; (2) (1-methyl-1H-pyrazol-4-
yl)boronic acid pinacol ester, Pd(PPh3)4, 1,4-dioxane, aq NaHCO3, 100 °C, 40% for
12a, 45% for 12b; (b) for 12a to 9: (1) Me2NH, NaBH(OAc)3, THF; (2) 6 N NaOH,
CH3OH, reflux, 26%; for 12b to 10: 6 N NaOH, CH3OH, reflux, 54%.
6
Scheme 2. Reagents and conditions: (a) (1) 4-bromoazaindole, Pd(PPh3)4, 1,4-
dioxane, 2 N aq K2CO3, 100 °C, 80%; (2) Zn, AcOH, 85%; (b) dimethylcarbamoyl
chloride, pyridine, 38%.

2554
J. R. Medina et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2552–2555
Table 1
SAR summary
R1
N N
R2
R3
R4
N
N
H
Compounds
GSK1070916
R1
R2
R3
H
R4
MW
Aurora Aa
IC50
Aurora Ba
IC50 (pIC50
BEIb
17
pHH3c,d
IC50
A549d
EC50
)
O
N
N
N
CH3CH2
507.6
1100
3.2 (8.5)
32
7
H
2
3
H
CH3
H
H
H
H
H
H
184.2
198.2
584
982
782 (6.1)
802 (6.1)
33
31
13,398
6161
2573
11,660
O
6
CH3CH2
CH3
H
H
374.5
>10,000
26 (7.6)
20
NA
1530
134
N
N
H
N
9
H
H
331.4
128
5.7 (8.2)
25
104
N
N
10
13
CH3
CH3
H
H
H
331.4
365.9
76
5.9 (8.2)
25
25
169
9
24
13
Cl
7.9
0.61 (9.2)
N
14
CH3
H
Cl
365.9
6.2
0.51 (9.3)
25
67
21
a
Values are averages of at least two experiments (nM). Experimental details describing the in vitro AurB/INCENP and AurA/TPX2 inhibition assays for IC50 determination
are described in Ref. 8.
b
BEI = (Aurora B pIC50 ꢀ 1000)/MW.
c
Cellular Mechanistic Assay. ELISA developed to measure intracellular activity of Aurora B kinase activity (nM) by determining the levels of histone-H3 (pHH3) at serine 10,
a specific Aurora B substrate.
d
Values for the cellular proliferation assay are averages of at least two experiments (nM). The percentage of human serum in the A549 proliferation assay is 10%. Further
details on the cellular proliferation (A549 cells) and mechanistic pHH3 assays are described in Ref. 9. NA = not available.
Table 2
FACS analysis in A549 cellsa
Compounds
Concd (nM)
Sub-2N DNA
2N DNA
2N to 4N DNA
4N DNA
>4N DNA
DMSO
GSK1070916
10
9
13
14
NA
40
135
670
65
2
21
30
36
27
34
63
6
32
13
14
6
13
3
7
4
5
20
23
29
43
40
26
2
47
2
3
15
30
100
4
a
The cell cycle profile was evaluated after 48 h. For a description of the cell cycle analysis see Ref. 9.
Since compounds 9 and 10 do not have the phenylurea moiety,
cyclization generated the desired chloroazaindole compounds 13
and 14.13
we speculated that a substituent at the 5-position of the azaindole
moiety could fill some space in the binding pocket and/or break the
co-planarity of the pyrazole and azaindole rings to mimic more
closely the conformation of GSK1070916. 5-Chloroazaindoles 13
and 14 were chosen as our first targets to test the viability of our
strategy. Their synthesis is described in Scheme 4. Chlorination
of position-5 of 4-bromo-2-aminopyridine, followed by Suzuki
cross-coupling with N-methylpyrazole boronic ester, afforded the
aminopyridine intermediate 16. Halogenation at the 3 position
with NBS in DMF at room temperature, or ICl in DMF at 40 °C, gave
intermediates 17a and 17b, respectively. Sonogashira cross-cou-
pling with the corresponding alkyne, followed by base induced
Gratifyingly, both compounds 13 and 14 exhibited high potency
in the Aurora B enzyme, phosphorylation of histone-H3, and A549
proliferation assays (Table 1). FACS analysis demonstrated that 13
causes an increase in 4N/>4N DNA (40/15%) compared to the
DMSO control, suggestive of an Aurora B mode of action (Table
2). In addition, multipolar spindles were observed at 12 nM and
37 nM by immunofluorescent microscopy, also consistent with
an Aurora B mode of action (Fig. 2). Compound 14, which combines
the 5-chloroazaindole hinge binder and the meta-aminomethyl
group in the tail region, has a phenotype more consistent with
Aurora B inhibition. FACS analysis of 14 exhibited the highest

J. R. Medina et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2552–2555
2555
accumulation of >4N DNA (30%) of the four compounds, and was
similar to GSK1070916 (Table 2). In addition, analysis by immuno-
fluorescent microscopy showed multipolar spindles at 37 nM, and
a mixture of malformed bipolar spindles (phenotype associated
with Aurora B inhibition) and monopolar spindles (phenotype
associated with Aurora A inhibition) at 110 nM (Fig. 2).
N N
a
Br
b
Cl
N
NH2
N
NH2
15
16
Compared to GSK1070916, compound 14 has similar Aurora B
enzyme and cellular activity, and exhibits a cellular phenotype
consistent with Aurora B inhibition. Since compound 14 has a
relatively low MW (365) and high binding efficiency (BEI = 25),
it offers a valuable starting point for the development of another
class of Aurora B inhibitors. This work illustrates the value of
deconstructing and reconstructing an advanced compound to
generate new, more ligand efficient leads for further
optimization.
N N
N N
N
4
3
c
Cl
X
Cl
N
H
N
NH2
N
17a: X = Br
17b: X = I
13 (para), 14 (meta)
Scheme 4. Reagents and conditions: (a) (1) NCS, DMF, 53%; (2) (1-methyl-1H-
pyrazol-4-yl)boronic acid pinacol ester, Pd(PPh3)2Cl2, 1,4-dioxane, aq NaHCO3,
100 °C, 45%; (b) for 17a: NBS, DMF, 52%; for 17b: ICl, DMF, 40 °C, 78%; (c) (1) Zn, NaI,
DMSO, Et3N, DBU, Pd(PPh3)4, 80 °C [for 13: {4-[(dimethylamino)methyl]phenyl}eth-
yne; for 14: {3-[(dimethylamino)methyl]phenyl}ethyne]; (2) KOt-Bu, DMF, 6% for
13 from 17a, 25% for 14 from 17b.
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10. A description of the lead optimization efforts culminating in the identification
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12. Compound 10 was assayed in a panel of 51 kinases. For 23 of 51 kinases tested,
the IC50 was within 50-fold the Aurora B activity.
13. Crisp, G. T.; Turner, P. D.; Stephens, K. A. J. Organomet. Chem. 1998, 570, 219.
Figure 2. Mitotic phenotype of 13- and 14-treated A549 tumor cells by immuno-
fluorescent staining. A549 cells were treated with 12, 37, or 110 nM of 13 and 14 for
24 h and stained with
a-tubulin antibody (red staining) to visualize mitotic
spindles, pericentrin antibodies for centrosomes (yellow staining), and 40,6-
diamino-2-phenylindole for DNA (blue staining).