A pentacyclic aurora kinase inhibitor (AKI-001) with high in vivo potency and oral bioavailability

Article abstract of DOI:10.1021/jm800052b

Aurora kinase inhibitors have attracted a great deal of interest as a new class of antimitotic agents. We report a novel class of Aurora inhibitors based on a pentacyclic scaffold. A prototype pentacyclic inhibitor 32 (AKI-001) derived from two early lead

Full text of DOI:10.1021/jm800052b

J. Med. Chem. 2008, 51, 4465–4475
4465
A Pentacyclic Aurora Kinase Inhibitor (AKI-001) with High in Vivo Potency and Oral
Bioavailability∞
Thomas E. Rawson,† Matthias Ru¨th,‡ Elizabeth Blackwood,§ Dan Burdick,† Laura Corson,# Jenna Dotson,† Jason Drummond,†
Carter Fields,§ Guy J. Georges,‡ Bernhard Goller, Jason Halladay,† Thomas Hunsaker,# Tracy Kleinheinz,† Hans-Willi Krell,|
Jun Li,† Jun Liang,† Anja Limberg,‡ Angela McNutt,§ John Moffat,† Gail Phillips,§ Yingqing Ran,† Brian Safina,†
Mark Ultsch, Leslie Walker,† Christian Wiesmann, Birong Zhang,† Aihe Zhou,† Bing-Yan Zhu,† Petra Ru¨ger,| and
Andrea G. Cochran*,
Departments of Small Molecule Drug DiscoVery, Cell Cycle and Global Regulators, Translational Oncology, and Protein Engineering,
Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, and Departments of Medicinal Chemistry, Cell Biology, and HTS and
Biochemistry, Pharma Research, Roche Diagnostics GmbH, D-82372 Penzberg, Germany
ReceiVed January 18, 2008
Aurora kinase inhibitors have attracted a great deal of interest as a new class of antimitotic agents. We
report a novel class of Aurora inhibitors based on a pentacyclic scaffold. A prototype pentacyclic inhibitor
32 (AKI-001) derived from two early lead structures improves upon the best properties of each parent and
compares favorably to a previously reported Aurora inhibitor, 39 (VX-680). The inhibitor exhibits low
nanomolar potency against both Aurora A and Aurora B enzymes, excellent cellular potency (IC50 < 100
nM), and good oral bioavailability. Phenotypic cellular assays show that both Aurora A and Aurora B are
inhibited at inhibitor concentrations sufficient to block proliferation. Importantly, the cellular activity translates
to potent inhibition of tumor growth in vivo. An oral dose of 5 mg/kg QD is well tolerated and results in
near stasis (92% TGI) in an HCT116 mouse xenograft model.
Introduction
Aurora C, has a kinase domain most similar in sequence to
that of Aurora B,18 and Aurora C appears to have functions
that overlap with those of Aurora B.19,20 However, the
expression pattern of Aurora C is very restricted,18 and a
comprehensive view of its function has yet to emerge.
Aurora kinases are a family of mitotic serine/threonine
kinases conserved from yeast to humans, and they have
received significant recent attention as new targets for
anticancer therapy.1–4 The two major Aurora kinases, Aurora
A and Aurora B, have distinct functions in mitosis.5–7 Aurora
A localizes predominantly to the centrosome, and it is
important for proper centrosome maturation and separation.
The kinesin HsEg5 (KSP) is also required for centrosome
separation,8 and Eg5, at least in frog extracts, is a direct
substrate of Aurora A.9 HsEg5 has itself been the target of
anticancer drug discovery efforts.3 Loss or inhibition of either
Aurora A10 or Eg511 arrests replicating cells in early mitosis
with a characteristic monopolar spindle phenotype. Aurora
B is required for phosphorylation of histone H3 at serine 10
in early mitosis, prior to chromosome condensation.12 Aurora
B is also a critical component of the spindle assembly
checkpoint13 and maintains a ”wait anaphase” signal until
all replicated chromosomes achieve correct biorientation and
attachment.14 Accordingly, loss of Aurora B function can
lead to premature anaphase and associated errors in chromo-
some segregation.15,16 Finally, Aurora B is required to
complete cytokinesis.17 Pharmacological inhibition or siRNA
knockdown of Aurora B leads to very large multinucleated
cells, resulting from repeated rounds of DNA synthesis with
no intervening cell division.15,16 The third Aurora kinase,
The initial motivation to direct drug discovery efforts to
Aurora kinases arose from early findings that Aurora A in
particular is highly expressed in many tumor types and that
this overexpression is in some cases driven by amplification
of the chromosomal region (20q13) on which the Aurora A
gene resides.21,22 In some cell lines, overexpression of Aurora
A is reported to be transforming.22,23 It does not, however,
appear that Aurora A can be considered a classic oncogene.
There are cell lines that are not transformed by Aurora A,22,24
and transgenic mice inducibly overexpressing Aurora A do
not develop tumors with high frequency.25 Instead, it appears
that elevation of Aurora A may play a supporting role in
tumor progression, perhaps in concert with p53 loss-of-
function or other mutations, or that a more extended period
of overexpression may be required for tumor development.26
Alternatively, a high Aurora A level may be simply a
consequence of a high mitotic index in a particular tumor
type.3 These questions have yet to be resolved and are under
active investigation and debate.
Despite uncertainty surrounding the role of Aurora kinases
in tumorigenesis, it is clear that both Aurora A and Aurora B
are critical for completion of mitosis and for accurate cell cycle
progression. As such, drugs targeting these kinases might be
expected to have potent antimitotic and antitumor activity. The
concept of targeting Aurora kinases with small molecule
inhibitors has been validated recently in mouse xenograft studies
from several groups.27–31 Advanced programs exist at many
companies,32 and several of these Aurora inhibitors have recently
entered the clinic.3 We report here a new Aurora kinase inhibitor
∞
Coordinates and structure factors have been deposited in the RCSB
Protein Data Bank (access code 3COH).
* To whom correspondence should be addressed. Phone: 650-225-5943.
Fax: 650-225-3734. E-mail: andrea@gene.com.
†
Department of Small Molecule Drug Discovery, Genentech, Inc.
Department of Medicinal Chemistry, Roche Diagnostics GmbH.
Department of Translational Oncology, Genentech, Inc.
Department of Cell Cycle and Global Regulators, Genentech, Inc.
‡
§
#
Department of Cell Biology, Roche Diagnostics GmbH.
|
Department of HTS and Biochemistry, Roche Diagnostics GmbH.
Department of Protein Engineering, Genentech, Inc.
10.1021/jm800052b CCC: $40.75
2008 American Chemical Society
Published on Web 07/17/2008

4466 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Rawson et al.
Scheme 1. Progression and Combination of Benzimidazole
Scheme 3. General Synthesis of Tetracyclic Corea
(Left) and Tetracycle (Right) Lead Series
a (a) NaNO2, HCl, H2O, cold; (b) 10, 11, or 12; NaOAc; H2O/MeOH;
(c) HCl, HOAc, heat; (d) DMFDMA (R ) H) or [(CH3)3Si]2NLi,
electrophile, THF; (e) N2H4, EtOH; (f) LiAlH4, THF.
overall yield from 7. Condensation of 9 with the appropriate
pyrazole carboxylic acid gave the final product 2 in 20% yield
after purification.
Construction of the tetracyclic core (Scheme 3) began with
nucleophilic addition of the appropriate ꢀ-ketoaldehyde
(10-12)35–37 to anilino diazonium chloride generated in situ,
producing, in a Japp-Klingemann reaction, 13-15 in 43-92%
yield. Subsequent Fischer cyclization under warm acidic condi-
tions gave the corresponding ketoindoles 16-1838–40 in 64-77%
yields. The unsubstituted pyrazole analogue 19 was formed by
thermal condensation of 17 with N,N-dimethylformamide dim-
ethylacetal and hydrazine to give the desired product in 11%
yield. 5-Methylpyrazole analogues 20-23 were prepared from
enolates obtained by treatment of either commercially available
2-acetylindole (for the acyclic analogue 20) or cyclic compounds
16-18 with lithium bis(trimethylsilyl)amide. Electrophilic ad-
dition of acetic anhydride followed by immediate reaction of
the ꢀ-diketone intermediates with hydrazine then gave com-
pounds 20-23 in 7-24% yield. The electrophiles cyclopro-
panoyl chloride and diethyloxylate were used in analogous
fashion to generate compounds 24 and 25 in 47% and 54%
yields, respectively. Reduction of 25 with LAH gave compound
26 in 75% yield.
Synthesis of the pentacyclic series required the 5-amino
regioisomer of lactam 5. Regioselective nitration of 1,3-dihydro-
3,3-dimethyl-2H-indol-2-one41 (27) was accomplished in 63%
yield (Scheme 4). Nitrolactam 28 was either directly reduced
(H2, Pd/C) to afford the aniline 29 or, alternatively, ethylated
before reduction to afford 31. Synthesis of pentacycles 32 and
33 then proceeded as described above for the tetracycles via
keto intermediates 34 and 35, respectively (Scheme 5). Synthesis
of analogue 36 proceeded likewise via 37, synthesized from
aniline 38 (Scheme 2).
Scheme 2. General Synthesis of Benzimidazolesa
a (a) H2, Pd/C; (b) NaH, EtBr; (c)H2, Pd/C; (d) Ac2O; (e)Ac2O, HNO3,
(f) HCl; (g) H2, PtO2; (h) 5-methyl-1H-pyrazole-3-carboxylic acid, poly-
phosphoric acid, P2O5, heat.
with excellent oral availability and very potent activity in mouse
xenograft models.
Chemical Synthesis
The synthesis of benzimidazole 1 has been described.33,34 The
conceptually parallel synthesis of the ethyl analogue 2 is outlined
in Scheme 2. Treatment of 6-nitrolactam 333 with NaH and
bromoethane yielded N-ethyl lactam 4. Hydrogenation of the
nitro group, followed by acylation of resulting aniline 5, yielded
the protected intermediate 6 in 78% overall yield for the three
steps. Nitration of 6 in a mixture of acetic anhydride and nitric
acid gave the 5-nitro derivative 7 in good yield (78%).
Hydrolysis of the acetamide followed by hydrogenation of
nitroaniline 8 yielded the 5,6-diamino intermediate 9 in 83%
Results and Discussion
We undertook a screening campaign against Aurora A kinase
to identify promising lead compounds. Among the initial
screening hits were the related structures 1 and 19 (Scheme 1).
Upon being retested, these compounds exhibited modest IC50
values of 0.18 and 0.30 µM, respectively, in an Aurora A

Orally AVailable Pan-Aurora Kinase Inhibitor
Scheme 4. Construction of Lactama
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4467
Table 1. Lactam Analogues of Compound 1
R
AurA IC50, µM AurB IC50, µM Cmax, µM AUClast, h µM
1
2
H
Et
0.18
0.069
0.15
0.13
0.88
51
1.5
180
Table 2. Pyrazole Analogues of Compound 19
a (a) Fuming HNO3, H2SO4; (b) NaH, EtBr; (c) H2, Pd/C.
R
AurA IC50, µM
Scheme 5. Completion of Pentacyclesa
19
22
24
25
26
H
Me
cyclopropyl
COOEt
CH2OH
0.30
0.051
0.11
5.7
0.34
Table 3. Bridging Ring Analogues of Compound 22
AurA IC50,
AurB IC50,
HT29 IC50,
MCF7 IC50,
n
µM
µM
µM
µM
20
21
22
23
0a
1
2
1.1
0.51
0.88
0.22
0.022
ndb
>20
>20
2.6
ndb
>20
4.5
0.87
0.051
0.042
a (a) NaNO2, HCl, H2O, cold; (b) 12, NaOAc, H2O/MeOH; (c) HCl,
HOAc, heat; (d) [(CH3)3Si]2NLi, Ac2O, THF; (e) N2H4, EtOH.
3
0.67
a No bridge. b Not determined.
pounds 1 and 19, with the particular goal of improving cellular
potency and oral exposure.
Exploration of the lactam portion of compound 1 (Table 1)
revealed that N-alkylation had little effect on enzymatic potency
and modestly improved cellular potency (∼10-fold, not shown).
However, this modification, especially N-ethylation, had a
profound effect on oral PK properties (Figure 1). By comparison
of the N-ethyl analogue 2 to the parent 1, Cmax increased by a
factor of 57 while AUClast increased 117-fold. Other N-alkyl
lactam derivatives explored showed improvement relative to 1,
but none had properties as favorable as 2 (not shown).
Our investigation of the tetracycle 19 began with an assess-
ment of the effects of substitution on the pyrazole. On the basis
of structures of reported Aurora inhibitors (and, in particular,
the structure of compound 1), we introduced a group of
relatively small substituents at position 5 of the pyrazole ring
(Table 2). Analogues containing hydrophilic or sterically
demanding groups showed a decrease in activity (examples 25
and 26), while the relatively small and lipophilic methyl
substitution (22) appeared to optimize potency. Accordingly,
the methylpyrazole was fixed in subsequent analogues.
We then explored the role of the hydrocarbon bridge joining
the pyrazole to the indole and varied its length (Table 3). As
expected, the lack of a constraining ring significantly reduced
potency. Constriction to a five-membered ring also reduced
potency compared to the six-membered ring present in com-
pounds 19 and 22. However, we found that expansion to a
seven-membered ring appeared slightly beneficial, particularly
Figure 1. Comparison of pharmacokinetics of compounds 1 (open
symbols) and 2 (closed symbols) after oral dosing (30 mg/kg) in mouse.
Compounds were formulated as suspensions in gelatin.
enzymatic assay. However, cellular potency was relatively poor
for compound 1 (5-20 µM against several cell lines) and
undetectable for compound 19 (>20 µM). Pharmacokinetic
studies in mouse gave promising results for compound 19, which
showed significant oral bioavailability and a moderate rate of
clearance (not shown), whereas compound 1 was cleared rapidly
and exhibited a low Cmax after oral dosing (Figure 1). We
therefore undertook a preliminary SAR investigation of com-

4468 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Table 4. Exploration of Lactam Orientation
Rawson et al.
AurA IC50, µM AurB IC50, µM HT29 IC50, µM MCF7 IC50, µM
33
36
0.0020
0.072
<0.002
0.086
0.030
2.6
0.10
1.5
Table 5. Activities of 32 and Its Parents in Various Assays
values in µM
IC50, Aurora A
IC50, Aurora B
IC50, HCT116
IC50, HT29
2
23
32
39b
0.069
0.13
3.3
4.3
2.1
0.042
0.022
nda
2.6
0.67
2.2
0.004
0.005
0.070
0.070
0.10
0.006
0.030
0.053
0.15
0.048
nda
IC50, MCF7
G2/M block
2.2
0.11
p-Aurora A, HCT116
centrosome separation, HCT116
p-histone H3, HT29
p-histone H3, HCT116
3.9
1.3
10
5.0
6.6
10
0.16
0.08
0.10
0.31
0.31
0.31
0.74
0.31
6.6
nda
a Not determined. b Previously described pan-Aurora inhibitor27 included
as a positive control.
Figure 2. Comparison of cocrystal structures of 32 bound to Aurora
A and the Aurora kinase inhibitor 39 bound to mutant Abl kinase (PDB
code 2F4J42): (A) hydrogen bonds between 32 (shown in green) and
the “hinge” region in the ATP-binding pocket of Aurora A; (B)
comparison of orientation of the conserved Asp residue at the beginning
of the activation loop for Abl (blue) bound to 39 (white) and Aurora
A bound to 32 (same color scheme as in part A).
when assayed against Aurora B (compound 23). Gratifyingly,
in addition to this improvement in enzymatic potency, 23 was
significantly improved in cellular potency.
After limited optimization, our two lead series had improved
in certain target properties, namely, oral availability for benz-
imidazole 2 and measurable cellular potency for tetracycle 23.
Nevertheless, potency (enzymatic and cellular) was still rather
less than we desired. We were intrigued by the structural
similaritybetweenthetwoseries.Eachhasa“donor-acceptor-donor”
arrangement of hydrogen-bonding groups frequently seen in
kinase inhibitors that bind to the adenine-binding “hinge” region
of the ATP-binding pocket, and it seemed likely that these would
be critical affinity determinants. It also appeared that both the
hydrocarbon bridge of 23 and the lactam portion of 2 contributed
favorably to binding (compare 20 to 23 and 20 to 2; Tables 3
and 1). We therefore decided to merge 2 and 23 into a single
pentacyclic analogue 32 (AKI-001, Scheme 1). Two orientations
of the lactam ring are possible. Our decision to orient the ring
as in 32 was based on comparison of the related des-ethyl
compounds 33 and 36 (Table 4).
Inclusion of both the lactam ring and the hydrocarbon
constraint in 32 led to an increase in enzymatic potency of ∼10-
fold compared to 2 and 23 (Table 5). The binding mode was
determined from a 2.7 Å X-ray cocrystal structure (Figure 2).
As expected, the indole and pyrazole nitrogens arrayed along
the edge of 32 participate in hydrogen-bonding interactions with
the amide backbone of the Aurora A hinge region (Figure 2A).
The methylpyrazole occupies the back of the ATP pocket, where
it is rather tightly accommodated and binds in the same
orientation seen for the methylpyrazole of 39 (Chart 1) in
complex with a Gleevec-resistant mutant (His396Pro) of Abl
kinase (PDB code 2F4J, Figure 2B).42 The pyrazole pocket is
defined in part by the Aurora A “gatekeeper” residue Leu210,
which appears to block access from the pyrazole methyl group
to a deeper hydrophobic pocket, consistent with the observed
preference for small pyrazole substituents (Table 2). The lactam
portion of 32 extends toward solvent and overlays the solubi-
lizing methylpiperazine of 39.
Chart 1. Structure of the Pan-Aurora Kinase Inhibitor 39
(VX-680)27
at the beginning of the activation loop. In the 39/Abl structure,
the side chain Asp381 points in toward the active site and forms
a hydrogen bond with the NH of the cyclopropylamide of 39.
This “DFG-in” orientation of Asp381 is consistent with an
activated conformation of the kinase and differs from that
observed when Gleevec is bound to wild-type Abl.42 Binding
of Gleevec instead causes the side chain of Asp381 to rotate
away from the active site and an associated reorientation of
Phe382, leading to the so-called “DFG-out” inactive conforma-
tion of Abl. In the complex structure of 32 bound to Aurora A,
a segment of the activation loop is disordered (residues
275-288). The lack of order in this loop may be influenced by
the mutation of the phosphorylation sites T287 and T288 to
alanine. Nevertheless, Asp274 would appear to adopt the “DFG-
out” orientation (Figure 2B), suggesting that 32 can bind to an
inactive conformation of Aurora A.
32 was greatly improved in antiproliferative activity against
several tumor cell lines (Table 5). This antiproliferative effect
coincided with the compound concentration required to produce
a G2/M block (24 h assay), as measured by flow cytometry.
We used several more specific cellular assays to judge whether
the observed antiproliferative effect was driven by inhibition
of Aurora A, Aurora B, or both (Figure 3). For inhibition of
Aurora A, we used two cellular readouts in the HCT116 colon
cancer cell line: loss of autophosphorylation of Aurora A at
One difference between the two cocrystal structures is the
conformation of the conserved Asp residue in the “DFG” motif

Orally AVailable Pan-Aurora Kinase Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4469
Table 6. Kinase Selectivity of 32
kinase
IC50, µMa
% activity at 1 µMb
CDK2/cyclinA
CDK3/cyclinE
CDK5/p25
CDK6/cyclinD3
CDK7/cyclinH/MAT1
CDK9/cyclinT1
Chk1
15
106
8
103
82
87
0.085
NEK2
Plk3
Abl
98
103
10
Flt3
GSK3ꢀ
KDR
0.002
7.7
0.012
0.25
Src
TrkB
2
a IC50 determination in-house at [ATP] ) Km. b Single point activity
determinations from a commercial selectivity panel (Upstate; 100% cor-
responds to no inhibition of the target enzyme).
failure of cytokinesis from inhibition of Aurora B. It is difficult
to assess the relative contributions of Aurora A and Aurora B
inhibitory activities to the block in cellular proliferation, but it
is clear that with 32, an early mitotic block cannot be maintained
indefinitely; eventually cells bypass the spindle checkpoint,
likely through inhibition of Aurora B, and undergo endoredu-
plication (repeated replication of DNA without cell division).
Given the improvements in potency we observed for 32, we
decided to evaluate it further in vivo (Figure 4). Pentacycle 32
showed significant and sustained exposure after a single oral
dose of 5 mg/kg in mouse, exceeding the threshold required
for cellular inhibition (∼0.1 µM) for at least 10 h (Figure 4A).
Oral exposure was evaluated also in rat and dog (Table 7) and
found to be significant. We therefore tested the efficacy of 32
in an HCT-116 mouse xenograft model. Substantial inhibition
of tumor growth was observed for QD doses of 2.5 (82% TGI;
p < 0.0001) and 5 mg/kg (92% TGI; p < 0.0001), while a lesser
effect was observed at the 1 mg/kg dose (52% TGI; p ) 0.0002)
(Figure 4B). The compound was well tolerated at 1, 2.5, and 5
mg/kg doses, as judged by minimal loss of body weight (Figure
4C), while dosing at 10 mg/kg QD led to unacceptable weight
loss, marked bone marrow depletion, and gastrointestinal
hypocellularity. These effects are similar to those observed with
other Aurora inhibitors and antimitotic agents generally.
Figure 3. Cellular effects of 32. HCT116 cells were incubated with
(A) DMSO or (B) 100 nM 32 for 16 h, then fixed and stained for
phosphohistone H3 (Ser10) (green), Aurora A (red), and DNA (gray).
Note that inhibitor-treated mitotic cells (arrows) have poorly separated
centrosomes, indicating inhibition of Aurora A, and fail to stain for
phosphohistone H3, indicating inhibition of Aurora B. After ∼2 h, these
aberrant mitotic cells undergo chromatin decondensation, forming
multilobed nuclei (indicated by an asterisk).
Thr 288 and failure of centrosomes to separate. These occurred
with thresholds for complete inhibition at 0.16 and 0.08 µM
32, respectively. To assess inhibition of Aurora B, we deter-
mined the threshold for complete loss of phosphohistone H3
staining in mitotic cells. This effect was seen at 0.31 µM in the
HCT116 cell line and at 0.10 µM in the colon cancer line HT29.
Overall, 32 is approximately equipotent in cellular inhibition
of Aurora A and Aurora B (consistent with the equivalent
enzymatic IC50 values), and the compound concentrations
required to produce these effects are fairly close to the
antiproliferative IC50 values. This suggests that the antiprolif-
erative effect is likely to be a consequence of Aurora inhibition.
To explore this further, we assayed 32 in a panel of kinase assays
(Table 6 and Supporting Information Table S2). Importantly,
there is little inhibition of non-Aurora kinases important for cell
cycle progression (for example, CDK or PLK family members).
However, 32 does show off-target activities, most notably
against receptor tyrosine kinases and Src family members as
has been reported for other Aurora kinase inhibitors.27,30,42
There has been much discussion in the literature of the
dominance of Aurora B inhibition phenotypes over those of
Aurora A. We can clearly see both effects in the short inhibitor
exposure times (∼60 min) necessary for the immunofluorescence
assays. (Similar observations of dual inhibition have recently
been reported for cells treated with 39.43) Over the longer
exposure times needed for antiproliferative assays (4-5 days),
we see extensive polyploidization similar to that previously
reported for Aurora A/B inhibitors. This is consistent with a
Conclusion
Pentacycle 32 represents a new class of pan-Aurora kinase
inhibitors with excellent oral availability and high potency
against tumor xenografts in mouse. These properties compare
favorably with those of other reported Aurora kinase inhibitors.
Cellular studies support Aurora kinase inhibition as the mech-
anism of antiproliferative activity and, presumably, of tumor
growth inhibition.
Experimental Section
Expression and Purification of Aurora Kinases. Both Auroras
A and B were cloned into pET21a (EMD Biosciences) and
expressed in BL21-DE3 pLysS-Rosetta (EMD Biosciences). Trans-
formants were grown with shaking at 37 °C in LB broth
supplemented with 50 µg/mL carbenicillin and 12.5 µg/mL
chloramphenicol until cultures reached an A600 reading of 0.45-0.6.
Expression was induced with 0.4 mM isopropyl-ꢀ-D-1-thiogalac-
topyranoside (IPTG), and the cultures were transferred to a shaker
at room temperature (∼18-22 °C) for an additional 12-16 h. The
construct for Aurora A kinase domain surrogate included residues
124-391 with the following amino acid substitutions: K124A,
K153R, V206I, R251K, L289M, and K339R. Aurora B surrogate

4470 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Rawson et al.
Table 7. Pharmacokinetic Properties of 32
species
CL, mL min-1 kg-1 a
F,b
%
mouse
rat
dog
25
22
34
178
97
56
a Formulation of iv dosing solutions differs in each case and is given
(along with additional details) in the Experimental Section. b Oral dosing
was performed in all cases with solutions of 32 in 80% polyethylene glycol.
After the sample was passed through a microfluidizer, lysates were
centrifuged for 30 min at 10 000 rpm. Supernatant was then loaded
on Ni-NTA Superflow resin (Qiagen) previously equilibrated in
lysis buffer. The column was washed with 4 × 8 column volumes
of lysis buffer containing 5 mM imidazole. Kinase was eluted with
0.5 M imidazole, pH 7.0, 0.5 M NaCl, 20% v/v glycerol, 5 mM
ꢀ-ME, either in a step fashion (4 × 1 column volume) or by linear
gradient from wash buffer for larger scale work.
Aurora A was further purified by dilution with 20 mM HEPES,
pH 7.5, 5 mM EDTA, 20% v/v glycerol, 5 mM dithiothreitol (DTT)
to a final NaCl concentration of 50 mM. This solution was loaded
onto an SP Sepharose fast flow column (GE Healthcare) previously
equilibrated in dilution buffer. After the sample was washed with
the same buffer, kinase was eluted in a linear gradient from 0.05
to 0.5 M NaCl in dilution buffer over 5 column volumes. Final
purification of the Aurora A for crystallography was performed on
a Superdex 75 column (26/60, GE Healthcare) equilibrated in 0.5
M NaCl, 20 mM HEPES, pH 7.2, 20% v/v glycerol, 5 mM DTT,
1 mM EDTA. Purity was assessed by SDS-PAGE. Mass spec-
trometry confirmed that Aurora A isolated from E. coli was
autophosphorylated and that the major species contained two
phosphates. Subsequent peptide mapping identified Thr277 and
Thr288 as the major autophosphorylation sites (not shown),
consistent with published observations.44
Aurora B fractions eluted from the Ni column were combined
and loaded onto anti-FLAG M2 agarose (Sigma) previously
equilibrated in lysis buffer lacking ꢀ-ME. The column was washed
with 6 × 2.5 column volumes of the same buffer. Kinase was then
eluted with FLAG peptide (Sigma; 100 µg/mL in the same buffer,
5 × 0.75 column volumes). The most concentrated fractions of
highly purified Aurora B were identified by SDS-PAGE and
combined. Like Aurora A, Aurora B was purified in active form
from E. coli, albeit at ∼10-fold lower yield because of its limited
solubility.
Crystallography. Aurora A was desalted prior to crystallization
using a NAP 5 column (Amersham) equilibrated with 40 mM
HEPES, pH 7.2, 50 mM NaCl, 5 mM DTT. The final concentration
of protein was 20 mg/mL. Inhibitor stock (20 mM) was prepared
in DMSO. The compound was added to Aurora A at a final
concentration of 0.1 mM and incubated on ice for 30 min.
Crystallization was carried out using sitting drops containing an
equal volume of complex plus reservoir. The reservoir contained
30% w/v PEG 3350, 0.2 M ammonium acetate, and 0.1 M HEPES,
pH 7.5. Crystals appeared within 3-4 days at 18 °C and grew to
0.1 mm × 0.1 mm × 0.1 mm within a week.
Figure 4. In vivo evaluation of 32: (A) pharmacokinetics after dosing
orally (po, 5 mg/kg, closed symbols) or intravenously (iv, 1 mg/kg,
open symbols); (B) tumor growth in an HCT116 xenograft model at 0,
1, 2.5, 5, and 10 mg/kg daily (QD) oral doses of 32. (Five mice were
terminated during study; see Experimental Section.) (C) Body weight
of mice in the xenograft study shown in part B.
Data were collected at the Stanford Synchrotron Radiation
Laboratory and processed using Denzo and Scalepack from the
HKL Suite.45 Crystals contained two molecules in the asymmetric
unit; cell parameters and data statistics are summarized in Sup-
porting Information Table 1. The structure was solved by molecular
replacement using AmoRe46 using the coordinates of 1MUO (RCSB
Protein DataBank) as a search model. Model building and refine-
ment were performed using the programs O47 and Refmac,48
respectively.
Inhibition Assays. Both Auroras A and B were assayed in
ELISA format using a GST fusion (pGEX-4T, GE Healthcare) of
the N-terminus of Histone H3 (aa 1-18) as substrate. Plates were
coated with 2 µg/mL substrate in PBS then blocked with 1 mg/mL
I-block (Tropix) in PBS. Kinase reactions were run for 40 min with
5 ng/mL (0.16 nM) Aurora A or 45 ng/mL (1.1 nM) Aurora B at
30 µM ATP (∼Km) in kinase buffer (Cell Signaling). Final DMSO
was N-terminally FLAG-tagged (Sigma) full length protein with
the following amino acid substitutions: S61K, K97R, I150V,
M233L, K195R, R283K. All substitutions were at solvent-exposed
sites well away from the ATP-binding pocket. Specific activities
of the mutant proteins (in the assays described below) were
indistinguishable from those of wild-type (see Figure S1). The
construct of Aurora A used for crystallography was the same as
that described above but with the substitutions K124A, Q154N,
A203S, R251K, T287A, T288A, and E336D.
Purification of both Auroras was facilitated after we observed
that they were retained on Ni-NTA columns despite lacking His6
tags. All purification steps were performed at 4 °C. Frozen cell
pellets were thawed and resuspended in 20 mM HEPES, pH 7.5,
0.5 M NaCl, 20% v/v glycerol, 5 mM ꢀ-mercaptoethanol (ꢀ-ME).

Orally AVailable Pan-Aurora Kinase Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4471
concentration was 4%. Product was detected by incubation with
antiphosphohistone H3 (Ser10) 6G3 mouse monoclonal antibody
(Cell Signaling, 1:4000) and sheep-antimouse HRP conjugate
(Amersham, 1:4000), followed by washing and addition of TMB
substrate (Kirkegaard & Perry Laboratories). After quenching with
1 M phosphoric acid, plates were read at 450 nM.
Cell-Based Antiproliferative Assays. Inhibitors were tested for
activity against the human colon cancer cell lines HCT116 and
HT29 and against the human breast cancer cell line MCF7. Cells
were seeded in 384-well plates on day 0 in 50 µL of complete
medium (1:1 mixture of Ham’s F12 and DMEM-high glucose
supplemented with 10% fetal bovine serum and 2 mM L-glutamine)
and incubated overnight in a 5% CO2 atmosphere at 37 °C. On
day 1, 10 µL of diluted inhibitor (DMSO solution of desired
concentration diluted 20-fold with the above medium lacking serum
and glutamine) was added to cells. Cells were incubated as
described above. On day 4, plates were allowed to reach room
temperature, and 30 µL Cell Titer-Glo reagent (Promega) was added
to each well (to measure total ATP levels). Plates were read after
shaking 15 min at room temperature.
Cell-Cycle Analysis. Human MOLT-4 leukemia cells were
treated with 3-fold serial dilutions of inhibitors for 24 h at 37 °C.
Cells were collected by centrifugation and resuspended in 0.1 M
Tris, pH 7.4, 154 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2, 0.2%
bovine serum albumin, 0.1% Nonidet P 40. Resuspended cells were
treated on ice for 30 min with 60 U/mL RNase A (Calbiochem)
and 20 µg/mL propidium iodide (Sigma). DNA content was then
determined by FACS.
Aurora A and Aurora B Phenotypic Assays. HCT116 or HT29
cells were treated with 2-fold serial dilutions of inhibitors for 4 h,
fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton
X-100, and stained with the antibodies indicated below. Slides were
then imaged on the high-content screening ImageXpress system
and quantitated in MetaXpress software (Molecular Devices).
Approximate threshold concentrations for complete failure of
centrosome separation and loss of phospho-Aurora A staining served
to estimate Aurora A inhibition in cells. Approximate concentrations
for the complete loss of phosphohistone staining of mitotic cells
(MPM2 positive cells) served to estimate Aurora B inhibition in
cells.
Primary antibodies used include total Aurora A (Santa Cruz, SC-
14321; 1:500 dilution), phospho-Aurora A (Cell Signaling, 3091;
1:350 dilution), MPM2 (Upstate, 05-368; 1:4000 dilution), and
phosphohistone H3 (serine 10) (Cell Signaling, 9701; 1:350
dilution).
Pharmacokinetics Evaluation of 32. For the mouse study shown
in Figure 4A, three female CD-1 mice were dosed for each time
point. For iv dosing (1 mg/kg), drug was formulated as a solution
in 5% DMSO, 5% Cremophor. For po dosing (5 mg/kg), the drug
was given as a solution in 80% polyethylene glycol (PEG). For
the rat study, three male SD rats were dosed per route. The po
dosing was as described above for mouse, and the iv dose (1 mg/
kg) was also administered as an 80% solution in PEG. For the dog
study, three male beagles were dosed per route. Again, oral dosing
was as described above for mouse, while the iv dose (1 mg/kg)
was delivered as a solution in 60% PEG. Averages of serum levels
(N ) 3) were used to calculate PK parameters.
In Vivo Efficacy Study. For the HCT-116 xenograft study,
female athymic nude mice were injected subcutaneously in the right
flank with 5 × 106 cells. When tumors reached a mean volume of
∼150 mm3, mice with near average-sized tumors were randomly
assigned to groups (N ) 10) for dosing. 32 was formulated as a
suspension in 20% HP-ꢀ-cylodextrin. Mice were dosed orally QD
with 100 µL of vehicle containing the appropriate dose of inhibitor
(0, 1, 2.5, 5, or 10 mg/kg). Three mice from each group were
sacrificed for PD analysis after 2 days of dosing; the remainder
were dosed for 21 days. Mice were euthanized if tumors became
excessively large (g2000 mm3) or if 20% of body weight was
lost. Significance (p-value) of observed differences in average
end-of-study tumor volumes was calculated using Dunnett’s
method. Percent tumor growth inhibition (% TGI) was calculated
from the ratio of the change in average tumor volumes for 32
vs vehicle treatment: TGI ) 100(1 - [{Vday21 - Vday0
}dose/{Vday21
- Vday0 vehicle]).
}
General Synthetic Procedures. All reactions were carried out
under a nitrogen atmosphere with dry solvents under anhydrous
conditions, unless otherwise noted. Reagents were purchased at the
highest commercial quality available and were used without further
purification unless otherwise stated. Reactions were monitored by
thin-layer chromatography (TLC) carried out on 0.25 mm E. Merck
silica gel plates (60F-254) using UV light as visualizing agent.
Alternatively, an ethanolic solution of phosphomolybdic acid or
cerium sulfate, followed by heating, was used as a developing agent.
E. Merck silica gel 60 (particle size 0.040-0.063 mm) was used
for flash column chromatography. NMR spectra were recorded on
Varian 400 instruments and were calibrated using chemical shifts
of residual undeuterated solvent. The following abbreviations were
used to explain the multiplicities: s )singlet, d ) doublet, t ) triplet,
q ) quartet, m ) multiplet, br ) broad. Electrospray ionization
(ESI) mass spectrometry (MS) experiments were performed on an
API100 Perkin-Elmer SCIEX single quadrupole mass spectrometer
at 4000 V emitter voltage. Yields refer to chromatographically and
spectroscopically (1H NMR) homogeneous materials, unless oth-
erwise stated.
7,7-Dimethyl-2-(5-methyl-1H-pyrazol-3-yl)-6-oxo-1H-imidazo[4,5-
f]indole (1). Compound 1 was synthesized as described.33,34 HPLC
purity 99.8%.
1-Ethyl-3,3-dimethyl-6-nitroindol-2-one (4). A solution of 333
(6 g, 29.10 mmol) in anhydrous N,N-dimethylformamide (DMF,
35 mL) was treated with sodium hydride (0.9 g, 37.8 mmol). The
resulting suspension was stirred for 1 h at 60 °C. A solution of
bromoethane (2.17 mL, 3.17 g, 29.10 mmol) in DMF (10 mL) was
added. The mixture was allowed to cool to room temperature and
stirred for 1 h. After removal of solvent, the mixture was quenched
with water (100 mL) and extracted with ethyl acetate (3 × 100
mL). The extract was dried over Na2SO4 and evaporated, and the
crude product was purified by column chromatography on silica
gel. Elution with ethyl acetate/n-heptane (1:3) yielded 5.94 g (87%)
1
as a yellow solid. MS (ESI+) m/z ) 235.3 (M + 1); H NMR
(400 MHz, DMSO-d6) δ 7.97 (d, J ) 8.3 Hz, 1H), 7.86 (s, 1H),
7.66 (d, J ) 8.3 Hz, 1H), 3.81 (q, J ) 7.0 Hz, 2H), 1.32 (s, 6 H),
1.16 (t, J ) 7.0 Hz, 3H).
6-Amino-1-ethyl-3,3-dimethylindol-2-one (5). To a solution of
4 (5.9 g, 25.19 mmol) in methanol/tetrahydrofuran (THF) (1:1, 80
mL) palladium on charcoal (10%, 1.2 g) was added, and the mixture
was hydrogenated at ambient pressure and room temperature for
4 h. After filtration and evaporation of the solvents, 5.05 g (98%)
of 5 was isolated as a white solid. MS (ESI+) m/z ) 205.0 (M +
1
1); H NMR (400 MHz, DMSO-d6) δ 6.92 (d, J ) 7.8 Hz, 1H),
6.25 (s, 1H), 6.21 (d, J ) 7.8 Hz, 1H), 5.12 (br, 2H), 3.58 (q, J )
7.1 Hz, 2H), 1.17 (s, 6H), 1.11 (t, J ) 7.1 Hz, 3H).
6-Acetamido-1-ethyl-3,3-dimethylindol-2-one (6). A solution of
5 (5.05 g, 24.72 mmol) in acetic anhydride (80 mL) was stirred at
room temperature for 4 h. The mixture was poured into ice-water
(150 mL), allowed to warm to room temperature, and was stirred
again for 2 h. After extraction with ethyl acetate (3 × 100 mL),
the combined organic layers were washed with saturated NaHCO3
solution (3 × 100 mL) and brine (100 mL), then dried over sodium
sulfate. After removal of solvent, the crude product was purified
by column chromatography on silica gel (ethyl acetate/n-heptane
1:1) yielding 5.6 g (91%) of 6 as a light-yellow solid. MS (ESI+)
m/z ) 247.1 (M + 1); 1H NMR (400 MHz, DMSO-d6) δ 9.97 (br,
1H), 7.37 (s, 1H), 7.23 (d, J ) 8.0 Hz, 1H), 7.12 (d, J ) 8.0 Hz,
1 H), 3.63 (q, J ) 7.1 Hz, 2H), 2.04 (s, 3H), 1.23 (s, 6H), 1.13 (t,
J ) 7.1 Hz, 3H).
6-Acetamido-1-ethyl-3,3-dimethyl-5-nitroindol-2-one (7). To a
solution of 6 (5.6 g, 22.73 mmol) in acetic anhydride (70 mL) at
0 °C, nitric acid (100%, 1.96 g, 1.29 mL, 31.2 mmol) was added.
The mixture was stirred for 30 min, then poured onto ice-water
(150 mL). After being stirred for 4 h, the mixture was extracted
with ethyl acetate (3 × 100 mL). The combined organic layers
were washed with sodium hydroxide solution (1 M, 100 mL) and

4472 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Rawson et al.
water (100 mL), dried over sodium sulfate, and concentrated. The
crude product was purified by column chromatography on silica
gel (ethyl acetate/n-heptane 1:1) to yield 5.2 g (78%) 7 as a yellow
solid. MS (ESI+) m/z ) 292.0 (M + 1); 1H NMR (400 MHz,
DMSO-d6) δ 10.39 (br, 1H), 8.12 (s, 1H), 7.54 (s, 1 H), 3.71 (m,
2H), 2.13 (s, 3H), 1.31 (s, 6H), 1.16 (t, J ) 7.1 Hz, 3H).
6-Amino-1-ethyl-3,3-dimethyl-5-nitroindol-2-one (8). Compound
7 (5.2 g, 17.85 mmol) was dissolved in ethanol (40 mL). After
addition of hydrochloric acid (25%, 8 mL, 81.44 mmol), the mixture
was stirred under reflux for 3 h. The reaction mixture was allowed
to cool to room temperature and then was quenched with water
(80 mL). A yellow precipitate was isolated by filtration and washed
with ethanol/water (1:1). The solid was dissolved in ethyl acetate,
dried over sodium sulfate, and concentrated to yield 4.15 g (93%)
of 8 as an orange solid. MS (ESI+) m/z ) 250.0 (M + 1); 1H
NMR (400 MHz, DMSO-d6) δ 7.95 (s, 1H), 7.67 (br, 2H), 6.54 (s,
1 H), 3.64 (m, 2H), 1.27 (s, 6H), 1.15 (t, J ) 7.1 Hz, 3H).
5,6-Diamino-1-ethyl-3,3-dimethylindol-2-one (9). To a solution
of 8 (4.15 g, 16.65 mmol) in ethanol (80 mL), PtO2 (0.4 g) was
added and the mixture hydrogenated at ambient pressure and room
temperature for 3.5 h. After filtration and evaporation of solvent,
3.25 g (89%) of 9 was isolated as an orange solid. MS (ESI+) m/z
) 220.0 (M + 1); 1H NMR (400 MHz, DMSO-d6) δ 6.50 (s, 1H),
6.27 (s, 1H), 4.48 (br, 2H), 4.08 (br, 2H), 1.13 (s, 6H), 3.53 (m,
2H), 1.10 (t, J ) 7.1 Hz, 3H).
room temperature. Ethyl acetate was added, and the organic solution
was washed with water and brine, dried over Na2SO4, and
evaporated. The isolated crude intermediate was taken up in 5 mL
of ethanol, which was added to 5 mL of hydrazine hydrate in 20
mL of ethanol. The solution was stirred for 12 h at room
temperature. After removal of solvent and an extractive workup in
ethyl acetate, the concentrated product was purified by flash column
chromatography (silica gel, DCM/MeOH, 97:3) to give the desired
pyrazole.
2-(5-Methyl-1H-pyrazol-3-yl)-1H-indole (20). 20 was synthesized
from commercially obtained 2-acetylindole in 73% yield according
to general procedure. MS (ESI+) m/z ) 198.2 (M + 1); 1H NMR
(500 MHz, DMSO-d6) δ 11.30 (s, 1H), 7.48 (d, J ) 7.9 Hz, 1H),
7.36 (dd, J ) 8.0 Hz, 1H), 7.10-6.90 (m, 2H), 6.67 (s, 1H), 6.44
(s, 1H), 2.28 (s, 3H); HRMS m/z ) 198.1033 (M + H), calcd
(C12H11N3 + H) ) 198.1031. HPLC purity 100%.
2,9-Dihydro-3-methylpyrazolo[4′,3′:4,5]cyclopenta[1,2-b]in-
dole (21). Synthesized from 16 in 53% yield according to general
procedure. MS (ESI+) m/z ) 210.2 (M + 1); 1H NMR (500 MHz,
DMSO-d6) δ 11.52 (s, 1H), 7.53-7.45 (m, 1H), 7.39-7.32 (m,
1H), 7.06-6.99 (m, 2H), 3.37 (s, 2H), 2.29 (s, 3H); HRMS m/z )
210.1032 (M + H), calcd (C13H11N3 + H) ) 210.1031. HPLC
purity 98.4%.
2,4,5,10-Tetrahydro-3-methylpyrazolo[3,4-a]carbazole (22). 22
was synthesized from 17 in 16% yield according to general
procedure. MS (ESI+) m/z ) 224.2 (M + 1); 1H NMR (500 MHz,
DMSO-d6) δ 11.30 (s, 1H), 7.44 (d, J ) 7.8 Hz, 1H), 7.32 (d, J )
8.0 Hz, 1H), 7.06-7.00 (m, 1H), 7.00-6.94 (m, 1H), 2.90 (t, J )
7.8 Hz, 2H), 2.72 (t, J ) 7.7 Hz, 2H), 2.21 (s, 3H); HRMS m/z )
224.1187 (M + H), calcd (C14H13N3 + H) ) 224.1188. HPLC
purity 96.1%.
2,5,6,11-Tetrahydro-3-methylpyrazolo[4′,3′:6,7]cyclohepta[1,2-
b]indole (23). 23 was synthesized from 18 in 7% yield according
to general procedure. MS (ESI+) m/z ) 238.3 (M + 1); 1H NMR
(500 MHz, DMSO-d6) δ 10.85 (s, 1H), 7.40 (d, J ) 7.8 Hz, 1H),
7.31 (d, J ) 8.0 Hz, 1H), 7.05-6.98 (m, 1H), 6.97-6.89 (m, 1H),
3.07-2.96 (m, 2H), 2.17 (s, 3H), 2.04-1.92 (m, 2H), 2.77-2.66
(m, 2H). HPLC purity 100%.
8-Ethyl-2,5,6,12-tetrahydro-3,10,10-trimethyl-9-oxopyrazolo[4′3′:
6,7]cyclohepta[1,2-b]pyrrolo[2,3-f]indole (32). 32 was synthesized
from 34 in 62% yield according to general procedure. MS (ESI+)
m/z ) 349.3 (M + 1); 1H NMR (400 MHz, DMSO-d6) δ 12.37 (s,
1H), 10.87 (s, 1H), 7.22 (s, 1H), 6.97 (s, 1H), 3.74 (q, J ) 7.0 Hz,
2H), 3.09-2.95 (m, 2H), 2.79-2.67 (m, 2H), 2.17 (s, 3H),
2.02-1.92 (m, 2H), 1.19 (t, J ) 7.1 Hz, 4H), 1.34-1.24 (m, 6H);
HRMS m/z ) 349.2039 (M + H), calcd (C21H24N4O + H) )
349.2047. HPLC purity 98.2%.
2,5,6,8,12-Pentahydro-3,10,10-trimethyl-9-oxopyrazolo[4′3′:6,7]cy-
clohepta[1,2-b]pyrrolo[2,3-f]indole (33). 33 was synthesized from
35 in 24% yield according to general procedure. MS (ESI+) m/z
) 321.1 (M + 1); 1H NMR (400 MHz, DMSO-d6) δ 12.35 (s,
1H), 10.80 (s, 1H), 10.10 (s, 1 H), 7.15 (s, 1H), 6.70 (s, 1H),
3.00-2.87 (m, 2H), 2.75-2.62 (m, 2H), 2.15 (s, 3H), 2.00-1.87
(m, 2H), 1.35 (s, 6H). HPLC purity 100%.
2,5,6,10,12-Pentahydro-3,8,8-trimethyl-9-oxopyrazolo[4′3′:6,7]cy-
clohepta[1,2-b]pyrrolo[3,2-f]indole (36). 36 was synthesized from
37 in 24% yield according to general procedure. MS (ESI+) m/z
) 321.3 (M + 1); 1H NMR (400 MHz, DMSO-d6) δ 12.23 (s,
1H), 10.65 (s, 1H), 10.10 (s, 1H), 7.21 (s, 1H), 6.72 (s, 1H),
3.00-2.87 (m, 2H), 2.70-2.60 (m, 2H), 2.15 (s, 3H), 2.00-1.87
(m, 2H), 1.25 (s, 6H). HPLC purity 98.9%.
5-Ethyl-7,7-dimethyl-2-(5-methyl-1H-pyrazol-3-yl)-6-oxo-1H-
imidazo[4,5-f]indole (2). 5-Methyl-1H-pyrazole-3-carboxylic acid
(0.23 g, 1.82 mmol) and 9 (0.4 g, 1.82 mmol) were heated to 160
°C in a mixture of polyphosphoric acid (6 g) and P2O5 (1.68 g,
11.8 mmol) for 6 h. The mixture was poured into water (50 mL),
and stirring continued until a precipitate formed. After filtration
the crude product was suspended in water and neutralized by the
addition of aqueous ammonia (25%). The precipitate was collected,
washed with water, and dried in vacuo. Purification was achieved
by chromatography on silica gel (dichloromethane/methanol 95:
5), yielding 115 mg (20%) of 2 as slightly yellow solid. MS (ESI+)
1
m/z ) 310.1 (M + 1); H NMR (400 MHz, DMSO-d6) δ 12.90
(br, 1H), 12.60 (br, 1H), 7.39 and 7.55 (s, 1H, two tautomeric
forms), 6.95 and 7.22 (s, 1H, two tautomeric forms), 6.58 (s, 1H),
3.75 (q, J ) 7.1 Hz, 2H), 2.31 (s, 3H), 1.31 (s, 6H), 1.17 (t, J )
7.1 Hz, 3H). Anal. Calcd for C17H19N5O: C, 66.0; H, 6.19; N, 22.64.
Found C, 64.58; H, 6.09; N, 21.95.
Synthesis of Cycloalkylindolones (16-18). ꢀ-Ketoaldehydes
10-12 were synthesized as described.35–37 Transformation through
the monophenylhydrazones 13-15 to the indole derivatives 16-18
was achieved as previously reported.38–40
2,4,5,10-Tetrahydropyrazolo[3,4-a]carbazole (19). 17 (0.650 g,
3.51 mmol) was diluted with 20 mL of toluene and 5 mL of N,N-
dimethylformamide dimethylacetal. The solution was heated to
reflux for 20 h and then was allowed to cool to room temperature
and concentrated. The residue was diluted with 20 mL of ethyl
ether, and the crystalline product was collected by filtration to give
264 mg of the intermediate enamine (31%). Crude enamine (400
mg) was taken up in 50 mL of ethanol and 5 mL of hydrazine
hydrate. The solution was allowed to stir for 1 week at room
temperature. After removal of solvent, the residue was taken up in
ethyl acetate and then washed with water and brine. The organic
solution was dried and concentrated, and the residue was purified
by flash column chromatography (silica gel, DCM/MeOH, 97:3)
1
to give 19 (340 mg, 98%). MS (ESI+) m/z ) 210.1 (M + 1); H
NMR (400 MHz, DMSO-d6) δ 12.45 (s, 1H), 11.41 (s, 1H), 7.54
(s, 1H), 7.42 (d, J ) 8.0 Hz, 1H), 7.30 (d, J ) 8.0 Hz, 1H), 7.42
(d, J ) 8.1 Hz, 1H), 7.06-6.94 (m, 2H), 2.94-2.80 (m, 4H);
HRMS m/z ) 210.1034 (M + H), calcd (C13H11N3 + H) )
210.1031. HPLC purity 100%.
3-Cyclopropyl-2,4,5,10-tetrahydropyrazolo[3,4-a]carbazole (24).
17 (0.200 mg, 1.08 mmol) was dissolved in 10 mL of dry THF
at 0 °C. Lithium bis-trimethylsilylamide (1.30 mL, 1.30 mmol,
1 M THF solution) was added dropwise to the solution, and the
mixture was stirred for 15 min. Cyclopropanoyl chloride (0.119
mL, 1.30 mmol) was slowly added and the viscous mixture
stirred for 2 h at room temperature. The crude mixture was
diluted with ethyl acetate, and the solution was washed with
water and brine. After the mixture was dried over Na2SO4,
solvent was removed. The residue was taken up directly in 2
General Procedure for Methylpyrazole Formation. The ap-
propriate ketone (0.500 mmol) was dissolved in 2.5-10 mL of dry
THF at 0 °C. Lithium bis-trimethylsilylamide (1.25 mmol, 1 M
THF solution) was added dropwise, and the mixture was stirred
for 15 min at 0 °C. Acetic anhydride (1.50-7.50 mmol) was slowly
added. The resulting viscous mixture was stirred for 1-12 h at

Orally AVailable Pan-Aurora Kinase Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4473
mL of ethanol, which was added to 3 mL of hydrazine hydrate
in 10 mL of ethanol. The solution was stirred for 12 h at room
temperature. The reaction mixture was concentrated, and the
residue was then taken up in ethyl acetate. The solution was
washed with water and brine, and then it was dried and
concentrated. The crude product was purified by flash column
chromatography (silica gel, DCM/MeOH, 97:3) to give 24 (0.127
g, 47%). MS (ESI+) m/z ) 250.2 (M + 1); 1H NMR (500 MHz,
DMSO-d6) δ 11.30 (s, 1H), 7.45-7.31 (m, 2H), 7.10-6.91 (m,
2H), 2.85 (dq, J ) 8.4 Hz, 4H), 1.90 (tt, J ) 8.5, 5.2 Hz, 1H),
1.09-0.76 (m, 4H); HRMS m/z ) 250.1349 (M + H), calcd
(C16H15N3 + H) ) 250.1344. HPLC purity 98.1%.
Ethyl 3-Carboxylate-2,4,5,10-tetrahydropyrazolo[3,4-a]carba-
zole (25). 17 (0.700 mg, 3.78 mmol) was dissolved in 40 mL of
dry THF at 0 °C. Lithium bis-trimethylsilylamide (9.45 mL, 9.45
mmol, 1 M THF solution) was added dropwise to the solution,
and the mixture was stirred for 15 min at 0 °C. Diethyl oxylate
(0.615 mL, 4.54 mmol) was slowly added. Stirring of the viscous
mixture was continued for 2 h at room temperature. After
addition of ethyl acetate, the organic extract was washed with
water and brine and then dried. Solvent was removed, and the
residue was taken up in 2 mL of ethanol. This was added to 3
mL of hydrazine hydrate in 10 mL of ethanol. The solution was
stirred 3 h at room temperature. The solution was concentrated,
and the resulting residue was taken up in ethyl acetate and
subjected to an extractive workup. The isolated crude material
was purified by flash column chromatography (silica gel, DCM/
EtOH, 97:3) to give 25 (0.570 g, 54%). MS (ESI+) m/z ) 280.2
(M + 1); 1H NMR (400 MHz, DMSO-d6) δ 13.78 (s, 1H), 11.32
(s, 1H), 8.18 (d, J ) 7.5 Hz, 1H), 8.05 (d, J ) 8.6 Hz, 1H),
7.85 (d, J ) 8.4 Hz, 1H), 7.81-7.71 (m, 1H), 7.42 (t, J ) 6.9
Hz, 1H), 7.25 (t, J ) 7.2 Hz, 1H), 4.44 (dd, J ) 13.4 Hz, 3H),
3.31 (s, 3H), 1.42 (t, J ) 6.9 Hz, 4H). HPLC purity 100%.
3-Hydroxymethyl-2,4,5,10-tetrahydropyrazolo[3,4-a]carbazole
(26). 25 (0.025 mg, 0.089 mmol) was dissolved in 5 mL of dry
THF at 0 °C. Lithium aluminum hydride (0.178 mL, 0.178 mmol,
1 M THF solution) was added dropwise to the solution, which
was then stirred for 15 min at 0 °C. The reaction was quenched
with 1 M HCl and then concentrated. The residue was purified
by HPLC (Vydac C-18 column (5 cm × 25 cm), flow rate 20
mL/min, gradient 0-100% (0.1% TFA in water/acetonitrile), UV
detector 254 nm, flow rate 20 mL/min, tR ) 52 min) to give 26
(0.016 g, 75%). MS (ESI+) m/z ) 240.2 (M + 1); 1H NMR
(500 MHz, DMSO-d6) δ 11.33 (s, 1H), 7.45 (d, J ) 7.8 Hz,
1H), 7.32 (d, J ) 8.0 Hz, 1H), 7.08-6.99 (m, 1H), 7.00-6.92
(m, 1H), 4.50 (s, 2H), 2.91 (dd, J ) 11.6 Hz, 2H), 2.80 (dd, J
) 11.7 Hz, 2H); HRMS m/z ) 240.1143 (M + H), calcd
(C14H13N3O + H) ) 240.1137. HPLC purity 100%.
2H), 2.51 (td, J ) 3.7, 1.83 Hz, 2H), 1.4 (s, 6H), 1.17 (t, J )
7.2 Hz, 3H).
8-Ethyl-3,4,5,6-tetrahydro-10,10-dimethylcyclohepta[1,2-b]pyr-
rolo[2,3-f]-1H-indole, 2,9-Dione (34). 30 (8.52 g, 36.4 mmol) was
suspended in 100 mL of methanol, and 1 g of 10% Pd/C catalyst
was added. The resulting suspension was hydrogenated at 1 atm
for 18 h. The catalyst was removed by filtration. Evaporation of
the solvent gave the crude aniline 31, which was immediately
suspended in 75 mL of water. Concentrated HCl (2.3 mL) was
slowly added. The resulting solution was cooled to 0 °C, and a
solution of sodium nitrite (1.55 g, 22.5 mmol) in 20 mL of water
was added dropwise over 10 min with vigorous stirring. This
solution was added slowly to a cold (0 °C) dispersion of 12
(2.89 g, 20.6 mmol) and sodium acetate (6.9 g, 84.1 mmol) in
200 mL of water and 50 mL of methanol. After the mixture
was stirred for 2 h at 0 °C, the hydrazone (4.68 g, 84%) was
collected by filtration as a yellow solid. To this crude material
(4.68 g, 14.3 mmol) was added 300 mL of concentrated acetic
acid followed by 20 mL of concentrated HCl, and the reaction
mixture was heated to 70 °C for 20 min. The solution was cooled
and ∼80% of the solvent removed by rotary evaporation under
vacuum. The mixture was partitioned between 9:1 ethyl acetate/
hexanes and washed sequentially with water, saturated sodium
bicarbonate, and brine before drying over Na2SO4. The solvent
was removed, and the crude product was recrystallized from ethyl
acetate to afford 34 (2.25 g, 42%). MS (ESI+) m/z ) 311.3 (M
1
+ 1); H NMR (400 MHz, DMSO-d6) δ 9.0 (br, 1H), 7.21 (s,
1H), 6.87 (s, 1H), 3.77 (q, J ) 8.0 Hz, 2H), 3.12 (t, J ) 8.0 Hz,
2H), 2.85 (t, J ) 8.0 Hz, 2H), 1.93 (m, 4H), 1.37 (s, 6H), 1.25
(t, J ) 8.1 Hz, 3H).
3,4,5,6-Tetrahydro-10,10-dimethylcyclohepta[1,2-b]-1H-pyrrolo[2,3-
f]-1H-indole, 2,9-Dione (35). Compound 35 was synthesized in
an overall yield of 82% according to the procedure given above
for 34, beginning with nitrolactam 28 and proceeding through
aniline 29. MS (ESI+) m/z ) 283.3 (M + 1); 1H NMR (400
MHz, DMSO-d6) δ 11.32 (s, 1H), 10.3 (s, 1H), 7.25 (s, 1H),
6.94 (s, 1H), 3.03 (t, J ) 5.6 Hz, 2H), 2.74 (m, 2H), 2.52 (m,
2H), 1.99-1.84 (m, 4H), 1.28 (s, 6H).
3,4,5,6-Tetrahydro-8,8-dimethylcyclohepta[1,2-b]-1H-pyrrolo[3,2-
f]-1H-indole, 2,9-Dione (37). Compound 37 was synthesized in
an overall yield of 61% as described above above for 34,
beginning with nitrolactam 3 and proceeding through aniline 38.
MS (ESI+) m/z ) 283.1 (M + 1); 1H NMR (400 MHz, DMSO-
d6) δ 11.15 (s, 1H), 10.18 (s, 1H), 7.51 (s, 1H), 6.72 (s, 1H),
3.00 (m, 2H), 2.68 (m, 2H), 1.62 (m, 4H), 1.20 (s, 6H).
Acknowledgment. Portions of this research were carried out
at the Stanford Synchrotron Radiation Laboratory, a national
user facility operated by Stanford University on behalf of the
U.S. Department of Energy, Office of Basic Energy Sciences.
The SSRL Structural Molecular Biology Program is supported
by the Department of Energy, Office of Biological and
Environmental Research, and by the National Institutes of
Health, National Center for Research Resources, Biomedical
Technology Program, and the National Institute of General
Medical Sciences. We thank members of the DMPK and
Purification groups within Genentech Small Molecule Drug
Discovery for analytical support and thank also the Genentech
Oligonucleotide and DNA Sequencing groups.
3,3-Dimethyl-5-nitro-1H-indol-2-one (28). Compound 2741 (38.4
g, 0.238 mol) was dissolved in 300 mL of concentrated sulfuric
acid using mechanical stirring and cooled in a -40 °C bath until
the reaction mixture thickened and stirring became difficult. A
solution of 10.1 mL of fuming nitric acid and 50 mL of
concentrated sulfuric acid was added dropwise and the mixture
allowed to warm to ambient temperature with stirring over 12 h.
The reaction mixture was poured into ice-water, and the
precipitate was collected by filtration and dried to afford 28 (31
g, 63%). MS (ESI+) m/z ) 207.2 (M + 1); 1H NMR (500 MHz,
DMSO-d6) δ11.02 (s, 1H), 8.27 (d, J ) 2.4 Hz, 1H), 8.16 (dd,
J ) 8.6 Hz, 1H), 7.04 (d, J ) 8.6 Hz, 1H), 1.33 (s, 6H).
1-Ethyl-3,3-dimethyl-5-nitro-1H-indol-2-one (30). Compound 28
(9.38 g, 40.0 mmol) was dissolved in 100 mL of DMF and added
dropwise to a stirred suspension of sodium hydride (2 g, 50.0
mmol) in 25 mL of DMF. When hydrogen evolution ceased,
ethyl iodide (4.00 mL, 50.0 mmol) was added. After 12 h the
reaction mixture was partitioned between ethyl acetate and water.
The organic extract was concentrated, and the residual solid was
Supporting Information Available: X-ray data collection and
refinement statistics, ELISA activity comparison for wild-type and
surrogate Aurora kinases, additional kinase selectivity data for
inhibitor 32, HPLC analytical data for compounds 19-26, 32, 33,
and 36, and 1H NMR spectra with assignments for compounds 23,
25, 33, and 36. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
recrystallized from ether/hexanes to give 30 (8.52 g, 91%). H
NMR (500 MHz, DMSO-d6) δ 8.33 (d, J ) 2.4 Hz, 1H), 8.24
(dd, J ) 8.7 Hz, 1H), 7.33-7.29 (m, 1H), 3.79 (q, J ) 7.2 Hz,

4474 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Rawson et al.
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