Discovery of a potent, selective, and orally bioavailable pyridinyl-pyrimidine phthalazine aurora kinase inhibitor

Article abstract of DOI:10.1021/jm100394y

The discovery of aurora kinases as essential regulators of cell division has led to intense interest in identifying small molecule aurora kinase inhibitors for the potential treatment of cancer. A high-throughput screening effort identified pyridinyl-pyrimidine 6a as a moderately potent dual inhibitor of aurora kinases -A and -B. Optimization of this hit resulted in an anthranilamide lead (6j) that possessed improved enzyme and cellular activity and exhibited a high level of kinase selectivity. However, this anthranilamide and subsequent analogues suffered from a lack of oral bioavailability. Converting the internally hydrogen-bonded six-membered pseudo-ring of the anthranilamide to a phthalazine (8a-b) led to a dramatic improvement in oral bioavailability (38-61%F) while maintaining the potency and selectivity characteristics of the anthranilamide series. In a COLO 205 tumor pharmacodynamic assay measuring phosphorylation of the aurora-B substrate histone H3 at serine 10 (p-histone H3), oral administration of 8b at 50 mg/kg demonstrated significant reduction in tumor p-histone H3 for at least 6 h.

Full text of DOI:10.1021/jm100394y

6368 J. Med. Chem. 2010, 53, 6368–6377
DOI: 10.1021/jm100394y
Discovery of a Potent, Selective, and Orally Bioavailable Pyridinyl-Pyrimidine Phthalazine
Aurora Kinase Inhibitor
Victor J. Cee,*,† Laurie B. Schenkel,† Brian L. Hodous,† Holly L. Deak,† Hanh N. Nguyen,† Philip R. Olivieri,†
Karina Romero,† Annette Bak,‡ Xuhai Be,§ Steve Bellon, Tammy L. Bush,^ Alan C. Cheng, Grace Chung,^
Steve Coats,^ Patrick M. Eden,^ Kelly Hanestad,^ Paul L. Gallant,# Yan Gu, Xin Huang, Richard L. Kendall,^
Min-Hwa Jasmine Lin,§ Michael J. Morrison,# Vinod F. Patel,† Robert Radinsky,^ Paul E. Rose, Sandra Ross,# Ji-Rong Sun,^
Jin Tang, Huilin Zhao, Marc Payton,^ and Stephanie D. Geuns-Meyer†
Departments of †Medicinal Chemistry, ‡Pharmaceutics, §Pharmacokinetics and Drug Metabolism, Molecular Structure, ^Oncology Research,
and #Molecular Pharmacology, Amgen Inc., 360 Binney Street, Cambridge, Massachusetts 02142 and Amgen Inc., One Amgen Center Drive,
Thousand Oaks, California 91320
Received March 29, 2010
The discovery of aurora kinases as essential regulators of cell division has led to intense interest in identifying
small molecule aurora kinase inhibitors for the potential treatment of cancer. A high-throughput screening
effort identified pyridinyl-pyrimidine 6a as a moderately potent dual inhibitor of aurora kinases -A and -B.
Optimization of this hit resulted in an anthranilamide lead (6j) that possessed improved enzyme and cellular
activity and exhibited a high level of kinase selectivity. However, this anthranilamide and subsequent
analogues suffered from a lack of oral bioavailability. Converting the internally hydrogen-bonded six-
membered pseudo-ring of the anthranilamide to a phthalazine (8a-b) led to a dramatic improvement in oral
bioavailability (38-61%F) while maintaining the potency and selectivity characteristics of the anthranil-
amide series. In a COLO 205 tumor pharmacodynamic assay measuring phosphorylation of the aurora-B
substrate histone H3 at serine 10 (p-histone H3), oral administration of 8b at 50 mg/kg demonstrated
significant reduction in tumor p-histone H3 for at least 6 h.
Introduction
A considerable research effort from both the pharmaceu-
tical industry and academic laboratories has led to many
reports of small molecule aurora kinase inhibitors.5 Both
pan-aurora kinase inhibitors and subtype-selective inhibitors
are currently under evaluation in human clinical trials. The
earliest example of a pan-aurora kinase inhibitor in clinical
development was MK-0457 (VX-680) (1), which inhibits
aurora-A and -B as well as a number of other kinases
implicated in cancer (Figure 1).5 The earliest examples of
isoform-selective aurora kinase inhibitors in clinical develop-
ment are MLN8054, an aurora-A selective inhibitor (2),6 and
AZD-1152 (3a), a phosphate prodrug of the selective aurora-
B inhibitor 3b.7 Herein we report the discovery of a pyridinyl-
pyrimidine series of pan-aurora kinase inhibitors and describe
the optimization of this series, which culminated in phthala-
zine 8b, a potent and selective inhibitor of aurora-A and -B
with properties suitable for oral dosing.
The aurora family of serine/threonine kinases is represented
by three paralogous genes (aurora-A, -B, and -C).1 Aurora-A
and -B are essential mitotic regulators required for chromosome
alignment, segregation, and cytokinesis, while aurora-C func-
tion appears to be restricted mainly to spermatogenesis. Despite
the high degree of structural similarity, aurora-A and -B have
distinct subcellular localizations and functions during mitosis.
As cells enter mitosis, aurora-A is localized to the centrosomes
and the proximal mitotic spindle, whereas aurora-B is localized
to the kinetochore/centromeric region. At the onset of anaphase,
as sister-chromatids separate, aurora-B localizes to the spindle
midzone where it coordinates exit from mitosis.1,2 Aurora-A can
function as an oncogene capable of transforming rodent fibro-
blast cells in culture.1,3 Expression of aurora-A and -B is elevated
in a variety of human cancers, with increased expression corre-
lating with poor prognosis in many cases.1,4
In tumor cells, inhibition of aurora-B or dual inhibition of
aurora-A and -B leads to premature exit from mitosis, result-
ing in undivided 4N DNA-containing cells in the G1-phase of
the cell cycle. These cells can progress through further rounds
of genome replication without cellular division, a process
referred to as endoreduplication, which ultimately results in
cell death.1 The described mechanism of action for an aurora-
A/B kinase inhibitor is distinct from that of traditional
antimitotic agents in that death of tumor cells is driven by
continued cell-cycle progression rather than by arrest in
mitosis (e.g., paclitaxel and docetaxel).
Chemistry
Pyridinyl-pyrimidine chloride derivative 48 was reacted
with 4-aminophenol or a substituted 4-aminophenol in the
presence of cesium carbonate at elevated temperature to
produce penultimate aniline intermediates 5a-c (Scheme 1).
These aniline intermediates (5a-c) were converted to amides
by HATU-mediateda reactions with a variety of carboxylic
a Abbreviations: ATP, adenosine triphosphate; Cl, clearance; HATU,
O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluoro-
phosphate; HTS, high throughput screening; KDR, kinase insert do-
main receptor; RLM, rat liver microsome; Tie-2, tyrosine kinase with
extracellular epidermal growth factor homology domains 2; TPX2,
targeting protein for Xenopus plus end-directed kinesin-like protein 2.
*To whom correspondence should be addressed. Phone: 805-313-
5500. Fax: 805-480-1337. E-mail: vcee@amgen.com.
r
pubs.acs.org/jmc
Published on Web 08/04/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6369
Scheme 1. Preparation of Amide Targetsa
a Reagents and conditions: (a) 4-aminophenol, Cs2CO3, DMSO, 130 ꢀC (90-95%); (b) ArCO2H, HATU, DIPEA, CHCl3, rt (33-84%).
Scheme 2. Preparation of Phthalazine Targetsa
a Reagents and conditions: (a) 1,4-dichlorophthalazine, s-BuOH, 100 ꢀC (88%); (b) boronic acid, Pd(dppf)Cl2, aq Na2CO3, dioxane, 100 ꢀC
(44-60%).
Table 1. Potency and Selectivity Summary for HTS Hit 6a
enzyme IC50 (nM)a
aurora-A/TPX2
aurora-B
Lck
586
66
c-kit
c-fms
KDR
Src
1350
1580
3270
5750
8000
385
503
630
Abl
Tie-2
p38R
IC50 >8000 nM: EGFR, EphB4, IGFR-1, Zap70, Jak2, Jak3, BTK,
JNK2, p70s6, PLK1, Erk1, PKBR, CDK5.
a All kinase assays were run at the Km for ATP and represent an
average of at least two determinations. For statistical analysis of
averaged data, see the Supporting Information.
Figure 1. Representative aurora kinase inhibitors and pyridinyl-
pyrimidine phthalazine 8b.
measuring the ability of a test compound to induce an accu-
mulation of cells with g4N DNA content, compound 6a was
not able to increase the number of HeLa cells with g4N DNA
content relative to control in a 24 h time period at concentra-
tions up to 1200 nM. Therefore, our initial goal was to improve
the aurora cellular potency of the screening hit 6a.
acids to give target amides 6a-6l. Phthalazines 8a and 8b were
also derived from intermediate 5a, which was treated with 1,4-
dichlorophthalazine to provide chloride 7. Suzuki reaction of
7 with phenyl- or 4-fluorophenylboronic acid provided 8a and
8b, respectively (Scheme 2).
Table 2 illustrates selected structure-activity relationships
for variations of the terminal aryl ring. A positional scan of a
chlorine substituent revealed that substitution at the 3- and
4-position (6c and 6d) was superior to 2-substitution (6b),
although cellular activity was still not achieved. It was hypo-
thesized that the larger phenoxy substituent might improve
inhibitory activity by increased van der Waals interactions
between ligand and protein, so phenoxy-substituted analo-
gues were also investigated. In contrast to chlorine substitu-
tion, the 2-substituted 6e showed the most potent activity
Discussion
High throughput screening (HTS) of a kinase-focused pro-
prietary compound collection identified pyridinyl pyrimidine
amide 6a as a moderately potent inhibitor of both aurora-A/
TPX2 (aurora-A in complex with a fragment [1-43] of partner
protein TPX29) and aurora-B enzymes, with a reasonable level
of selectivity against a small panel of kinases (Table 1). In an
aurora-B cellular assay (hereafter termed 24 h DNA ploidy)

6370 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Table 2. Potency Data for Pyridinyl-Pyrimidine Amides
Cee et al.
Table 3. Potency Data for Pyridinyl-Pyrimidine Anthranilamides
enzyme, IC50 (nM)a
aurora-A/TPX2 aurora-B
586 66
cell, EC50 (nM)b
R
DNA ploidy
>1200c
ND
>1200c
>1200c
>1200c
>1200c
ND
6a
6b
6c
6d
6e
6f
3-CF3
2-Cl
3-Cl
>25000
1546
2342
1544
964
>25000
430
335
56
4-Cl
2-OPh
3-OPh
4-OPh
2-NHPh
251
542
17
6g
6h
ND
62
89
a All kinase assays were run at the Km for ATP and represent an
average of at least two determinations. b Average of at least two
determinations, unless otherwise specified. c One determination. For
statistical analysis of averaged data, see the Supporting Information.
toward aurora-B enzyme in this series, although cellular
activity was still not observed. Replacement of the 2-OPh
substituent with 2-NHPh produced anthranilamide (6h),
which was an important breakthrough, as this compound
exhibited double-digit nanomolar potency against both aurora-
A/TPX2 and aurora-B enzymes as well as an 89 nM EC50
value in the 24 h DNA ploidy cellular assay. Although 6e and
6h differ by only a single atom, the conformational differences
imposed by intramolecular hydrogen bonding between the
ether oxygen and amide NH of salicylamide 6e and aniline
NH and carbonyl O of anthranilamide 6h are significant, and
this may contribute to the difference in potency between 6e
and 6h.
a All kinase assays were run at the Km for ATP and represent an
average of at least two determinations. b Average of at least two
determinations, unless otherwise specified. c One determination. For
statistical analysis of averaged data, see the Supporting Information.
Table 4. Potency, Selectivity, and Pharmacokinetic Properties of 6j
The structure-activity relationship of the central para-
aminophenoxy portion of the molecule is illustrated in Table 3.
Moving the methyl group to the position adjacent to the
aniline nitrogen (6i) caused a significant loss of aurora-A/
TPX2 and aurora-B enzyme potency as well as poor cellular
activity. Removing the methyl group (6j) led to an improve-
ment in both intrinsic potency and cellular activity. These
data suggest that an unsubstituted central aminophenoxy
ring provides optimal potency, and further efforts focused
on this series.
The selectivity profile of 6j against a panel of phylogene-
tically diverse kinases and the pharmacokinetic properties
of 6j in Sprague-Dawley rats are presented in Table 4.
Aside from aurora-A and -B, only Tie-2 and KDR enzymes
were inhibited at submicromolar concentrations (IC50=
440 nM and 794 nM, respectively), resulting in a mini-
mum selectivity factor of 29-fold over the EC50 of the 24 h
DNA ploidy cellular assay (440 nM against Tie-2 enzyme
vs 15 nM DNA ploidy). Unfortunately, 6j exhibited sub-
optimal rat pharmacokinetics, with clearance (Cl = 3.6 L/h/kg)
approaching rat liver blood flow. The high clearance was
consistent with rapid turnover of 6j in rat liver microsomes
(RLM Cl = 663 μL/min/mg). When dosed orally, 6j was
not detectable in rat plasma, indicating a lack of oral
bioavailability.
Enzyme IC50 (nM)a
aurora-A/TPX2
aurora-B
Tie2
16
9
440
794
KDR
IC50 > 1000 nM: p38R, JNK2, cMET, Jak3, c-kit, Src, Lck, Abl(T315I),
BTK, IGFR-1, CDK1, CDK2, CDK5, PKBR, p70s6k, PLK1, Erk1
Rat Pharmacokineticsb
Cl (L/h/kg)
Vss (L/kg)
t1/2 (h)
3.6
4.8
2.8
%F
<1
a All kinase assays were run at the Km for ATP and represent an
average of at least two determinations. b Administered intravenously at
0.5 mg/kg and orally at 2 mg/kg to male Sprague-Dawley rats (N = 3).
blocking two likely sites of oxidative metabolism.10 Both
analogues maintained comparable potency to 6j and showed
lower rates of rat liver microsome metabolism (6k, RLM Cl =
467 μL/min/mg, and 6l, RLM Cl = 338 μL/min/mg). A
Two fluorinated anthranilamide analogues, 6k and 6l
(Table 5), were synthesized in order to assess the impact of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6371
Table 5. Potency, Pharmacokinetic Properties, and Solubility of Anthranilamide Analogues
a Average of at least two determinations; for statistical analysis of averaged data, see the Supporting Information. b Administered intravenously at 0.5
mg/kg and orally at 2 mg/kg to male Sprague-Dawley rats (N = 3). ND = not determined. c Equilibrium solubility determined in 0.01 N HCl.
corresponding reduction in clearance was also observed follow-
ingivdosinginrat,with6l characterized by a clearance of 0.87 L/
h/kg. Unfortunately, moderating the in vitro metabolism and
lowering the in vivo clearance in this series did not result in any
improvement in oral bioavailability (6l, F<1%), suggestingthat
poor absorption was likely responsible for the lack of oral
bioavailability of 6l. This was further substantiated by equilib-
rium solubility measurements, which established that 6l exhibi-
ted poor solubility in aqueous media (0.016 mg/mL, 0.01 N HCl).
Figure 2 illustrates X-ray cocrystal structures of 911 and 10,12
two analogues representative of the cell-inactive initial screening
hit 6a and the cell-active anthranilamide derivative 6j, respec-
tively, bound to the ATP site of the aurora-A kinase domain.13
In the aurora-A/9 cocrystal structure (PDB ID: 3O50), the ligand
binds with the activation loop in a DFG-in conformation14 and
the terminal trifluoromethyl group is flanked by lipophilic side-
chain residues of the C-helix (Val174 and Ile178) and P-loop
(Phe144). Key hydrogen-bonding interactions exist between the
Figure 2. X-ray cocrystal structure of representative inhibitors bound
pyrimidine nitrogen and kinase hinge NH (Ala213), the amide
carbonyl and the amino terminus of the catalytic lysine (Lys162),
to the kinase domain of aurora-A. (a) Benzamide 9; (b) anthrani-
lamide 10.
and the amide NH of the ligand and the side chain of Asn261
(water-mediated). The cocrystal structure of aurora-A/10 (PDB
ID: 3O51) shows a similar orientation in the ATP-binding site,
but the conserved DFG motif of the activation loop occupies a
strikingly different DFG-out conformation, with the aromatic
ring of the anthranilamide partially occupying the space vacated
by Phe275 and the terminal phenyl ring flanked by lipophilic
side-chain residues of the C-helix (Val174 and Ile178) and P-loop
(Phe144). The anthranilamide adopts an internally hydrogen-
bonded conformation, with the plane of the amide nearly
parallel to Phe275. While the amide carbonyl oxygens of 9 and
10 occupy very similar space, Lys162 is disordered in the aurora-
A/10 structure, precluding any assessment of a hydrogen-bond-
ing interaction. The conformation of the kinase activation loop
in the aurora-A/10 cocrystal structure appears to be induced by
the anthranilamide, as the N-phenyl portion of the molecule is
incompatible with the protein conformation observed in the
aurora-A/9 cocrystal structure.
The aurora-A/10 cocrystal structure verified that the anthra-
nilamide portion of the molecule adopts a pseudo-six-membered
ring conformation with an intramolecular hydrogen bond. This
information supported the replacement of the pseudo-ring with
a six-membered aromatic ring. Gratifyingly, phthalazine 8a, a
corollary to anthranilamide 6j, andthepara-fluoro derivative 8b,
a corollary to anthranilamide 6k, demonstrated good cellular

6372 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Cee et al.
Table 6. Emergence of the Phthalazine Inhibitors from Anthranilamide 6j
potency (Table 6). Both 8a and 8b show significantly improved
solubility in aqueous media (0.01 N HCl) versus 6j, and both
were selected for rat pharmacokinetic experiments. Both 8a
and 8b (Table 7) showed significantly lower clearance as well as
greatly improved oral bioavailability relative to the anthranil-
amides 6j and 6l (Table 5).
Compound 8b was further profiled against a panel of
diverse kinases (Table 8). Aside from aurora-A and -B, only
Tie-2 and p38R enzymes were inhibited with IC50 values less
than 25 μM (IC50 = 872 nM and 1700 nM, respectively),
resulting in a minimum selectivity factor of 36-fold over the
EC50 of the 24 h DNA ploidy cellular assay (872 nM against
Tie-2 enzyme vs 24 nM 24 h DNA ploidy).
The impact of 8b on aurora kinase function was further
established by experiments in the human cervical cancer cell line
HeLa. Exposure of synchronized cells to 8b (0.0049 to 5 μM) and
measurement of phosphorylated aurora-A (Thr 288) and aurora-
B (Thr 232) by Western blotting revealed near-complete suppres-
sion of p-aurora-A and p-aurora-B at 0.313 μM 8b (Figure 3A).
In this experiment, 0.5 μM MLN8054 (2, aurora-A kinase
enzyme, IC50 (nM)a
cell, EC50 (nM)b solubility (mg/mL)
aurora-A/TPX2 aurora-B
DNA ploidy
0.01 N HCl
6j
16
149
41
9
22
15
15
15
24
0.005
0.159
>0.200
8a
8b
a All kinase assays were run at the Km for ATP and represent an
average of at least two determinations. b Average of at least three determi-
nations. For statistical analysis of averaged data, see the Supporting
Information.
Table 8. Kinase Selectivity of 8ba
enzyme
aurora-A/TPX2
IC50 (nM)
41
15
Table 7. Pharmacokinetic Properties of 8a and 8ba
aurora-B
Tie-2
872
1700
Cl (L/h/kg)
Vss (L/kg)
t1/2(h)
%F
p38R
8a
8b
0.80
0.44
0.69
0.42
1.3
1.1
61
38
IC50 > 25000 nM: KDR, Lck, Src, BTK, IGFR-1, Jak3, JNK2, CDK5,
Erk1, c-Met, p706s6k, PLK1, PKBR, PKBβ, MSK1, Abl(T315I), c-Kit
a All kinase assays were run at the Km for ATP and represent an
average of at least two determinations.
a Administered intravenously at 0.5 mg/kg and orally at 2 mg/kg to
male Sprague-Dawley rats (N = 3).
Figure 3. Compound 8b inhibits aurora-A and aurora-B activity and induces polyploidy in human tumor cells. (A) Western blotting of cell
lysates from HeLa cells synchronized in mitosis and treated with DMSO, compound 8b at the indicated concentrations, MLN8054 (aurora-A
kinase inhibitor) at 0.5 μM, or AZD1152 (aurora-B kinase inhibitor) at 1 μM for 3 h. (B) HeLa cells were treated with DMSO or 8b at 0.313 μM
for 24 h. The levels of p-histone H3 Ser 10 (red) and DNA content (2N, 4N, and 8N) were determined by flow cytometry. (C) Representative
merged images of HeLa cells treated with DMSO or compound 8b at 0.313 μM for 48 h. Cells were immunostained with anti-R-tubulin (red)
and anti-pericentrin (green) antibodies and DNA counterstained with DAPI (blue).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6373
not assessed, but our experience with related pyridyl-pyrimidine
phthalazines indicates that g6 h/day suppression of tumor
p-histone H3 results in robust inhibition of tumor growth.15
Conclusion
The evolution of a pyridinyl-pyrimidine phthalazine series of
aurora kinase inhibitors has been described in detail. Structure-
activity relationship studies beginning with benzamide 6a, a
dual inhibitor of aurora-A and -B with poor cellular activity, led
to the discovery of anthranilamide 6j. This lead was also a dual
aurora inhibitor that displayed potent cellular activity (EC50
=
15 nM) and good kinase selectivity. An X-ray cocrystal struc-
ture of close analogues of 6a and 6j bound to aurora-A revealed
that the cell-inactive benzamides bind aurora-A with the kinase
activation loop in a DFG-in conformation, whereas the cell-
active anthranilamides bind aurora-A with the kinase activation
loop in a DFG-out conformation. Lead anthranilamide 6j
exhibited high clearance and a lack of oral bioavailability in
rat, and whereas improvements in in vivo clearance were
possible, our ability to advance the anthranilamide series was
ultimately hindered by poor oral bioavailability. Converting
the internally hydrogen bonded, six-membered pseudo-ring of
the anthranilamide (6j) to a phthalazine (8a-b) led to an
unexpected improvement in equilibrium solubility in aqueous
media (0.01 N HCl), reduced in vivo clearance, and a significant
improvement in oral bioavailability (from <1% to g38% F). In
addition, this phthalazine series maintained the desirable cell
potency and high kinase selectivity of the anthranilamide series.
In a COLO 205 tumor pharmacodynamic assay measuring
phosphorylation of the aurora-B substrate histone H3 at serine
10 (p-histone H3), oral administration of 8b at 50 mg/kg
demonstrated significant reduction in tumor p-histone H3 for
at least 6 h. The phthalazine series marks a significant break-
through for the program and further optimization of this series
will be reported in due course.
Figure 4. Mice bearing established COLO 205 xenograft tumors
were administered a single oral dose of vehicle alone or 8b at 50 mg/
kg. Tumors were collected at 3, 6, and 24 h after treatment (N = 4
per time-point) and the level of p-histone H3 was determined by
immunofluorescence-based flow cyotmetry. Pharmacodynamic re-
sponse is represented as the percentage of p-histone H3 positive cells
in G2/M (bars, mean þ SD) plotted against concentration of 8b in
mouse plasma (b) (mean ( SD). The controls (black columns)
represent vehicle-treated tumors. Statistical significance was deter-
mined using ANOVA followed by Scheffe’s post hoc analysis
(*P < 0.0001 vs vehicle-treated control).
inhibitor)6 and 1 μM AZD1152 (3a, aurora-B kinase inhibitor)7
selectively blocked the autophosphorylation of either aurora-A
or aurora-B, respectively, as expected. Flow cytometry experi-
ments (Figure 3B) established that HeLa cells incubated with
0.313 μM 8b had reduced phosphorylation of histone H3 on
Ser 10 (a proximal substrate of aurora-B) and an increased per-
centage of cells with >4N DNA content, a direct measure of
polyploidy. To investigate the effects of 8b on cellular morpho-
logy, HeLa cells were treated with compound for 48 h and
stained with DAPI (40,6-diamidino-2-phenylindole) and anti-
bodies to R-tubulin and to percentrin. As anticipated with inhibi-
tion of aurora-B, 8b treatment increased the nuclear size of
mitotic and interphase cells and induced irregular spindle geo-
metry (Figure 3C). Although cell viability studies were not con-
ducted with 8b, our general experience with pyridinyl-pyrimidine
phthalazines like 8b is that inhibition of cell proliferation
correlates well with inhibition of histone H3 phosphorylation.15
On the basis of excellent potency, selectivity, and acceptable
rat pharmacokinetics, 8b was selected for further evaluation in a
tumor pharmacodynamic assay (Figure 4). To assess whether 8b
can inhibit phosphorylation of the aurora-B substrate histone
H3 at serine 10 in vivo, mice with established COLO 205
xenograft tumors of ∼300 mm3 were orally administered a
single dose of vehicle or 8b at 50 mg/kg (N = 12 per group).
At 3, 6, and 24 h post-treatment (N = 4 per time-point), plasma
samples were collected and analyzed for 8b, and tumor tissues
were harvested and analyzed for p-histone H3 by immunofluor-
escence-based flow cytometry. After 3 and 6 h, 8b treatment
resulted in statistically significant inhibition of p-histone H3 at
50 mg/kg (g96% inhibition, P e 0.0001) with plasma concen-
trations of 8b g20000 ng/mL (g38 μM). By 24 h, p-histone H3
returned to baseline levels with a plasma concentration of 8b
equal to 2460 ng/mL (4.8 μM). A qualitatively similar result was
also observed in mouse bone marrow (data not shown). As 8b is
highly bound to mouse plasma proteins (>99.9% bound), the
unbound concentration at the 6 h time point is <20 ng/mL
(<39 nM), similar in magnitude to the DNA ploidy EC50
(24 nM), while the unbound concentration at the 24 h time
point is <2.5 ng/mL (<5 nM), at least 5-fold below the DNA
ploidy EC50. The impact of 8b on tumor growth inhibition was
Experimental Section
Unless otherwise noted, all materials were obtained from
commercial suppliers and used without further purification.
Anhydrous solvents were obtained from Aldrich and used di-
rectly. All reactions involving air- or moisture-sensitive reagents
were performed under a nitrogen or argon atmosphere. Silica gel
chromatography was performed using either glass columns
packed with silica gel (200-400 mesh, Aldrich Chemical) or
medium pressure liquid chromatography (MPLC) on a Combi-
Flash Companion (Teledyne Isco) with RediSep normal-phase
silica gel (35-60 μm) columns and UV detection at 254 nm.
Preparative reversed-phase HPLC was performed on a Gilson
(215 liquid handler), YMC-Pack Pro C18, 150 mm ꢀ 30 mm I.
D. column, eluting with a binary solvent system A and B using a
gradient elusion (A: H2O with 0.1% TFA; B: CH3CN with 0.1%
TFA) with UV detection at 254 nm. All final compounds were
purified to g95% purity as determined by Agilent 1100 series high
performance liquid chromatography (HPLC) with UV detection at
254 nm (method A: Zorbax SB-C8, 4.6 mm ꢀ 150 mm, 15 min; 1.5
mL/min flow rate; 0-100% 0.1% TFA in CH3CN/0.1% TFA in
H2O; method B: Phenomenex Synergi, 2 mm ꢀ 50 mm, 3 min, 1.0
mL/min flow rate, 5-95% 0.1% formic acid in CH3CN/0.1%
formic acid in H2O). NMR spectra were determined with either a
Bruker AV-400 (400 MHz) spectrometer or a Varian 400 MHz or a
300 MHz spectrometer at ambient temperature. Low-resolution
mass spectral (MS) data were determined on an Agilent 1100 series
LCMS with UV detection at 254 nm and a low resonance electro-
spray mode (ESI). Chemical shifts are reported in ppm from the
solvent resonance (DMSO-d6 2.49 ppm). Data are reported as

6374 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Cee et al.
follows: chemical shift, multiplicity (s=singlet, d=doublet, t=
triplet, q=quartet, br=broad, m=multiplet), coupling constants,
and number of protons. High-resolution mass spectra (HRMS)
were obtained on a high resonance electrospray time-of-flight mass
spectrometer.
1H), 7.17 (m, 1H), 7.09 (d, J = 8.8 Hz, 1H), 2.89 (d, J = 4.9 Hz,
3H), 2.08 (s, 3H). HRMS (C24H20ClN5O2)þ calcd 446.13783,
found 446.13909.
4-Chloro-N-(3-methyl-4-(3-(2-(methylamino)pyrimidin-4-yl)-
pyridin-2-yloxy)phenyl)benzamide (6d). Following the proce-
dure described for 6j, 4-chlorobenzoic acid and 5b provided
the title compound (86 mg, 65%) as an off-white solid. 1H NMR
(400 MHz, DMSO-d6) δ 10.29 (s, 1H), 8.42 (br s, 1H), 8.39 (m,
1H), 8.16 (m, 1H), 8.00 (d, J = 6.3 Hz, 2H), 7.69 (m, 1H), 7.62
(m, 4H), 7.32 (m, 1H), 7.26 (m, 1H), 7.17 (m, 1H), 7.09 (d, J =
8.5 Hz, 1H), 2.88 (s, 3H), 2.08 (s, 3H). HRMS (C24H20ClN5O2)þ
calcd 446.13783, found 446.13927.
N-(3-Methyl-4-(3-(2-(methylamino)pyrimidin-4-yl)pyridin-2-
yloxy)phenyl)-2-phenoxybenzamide (6e). Following the proce-
dure described for 6j, 2-phenoxybenzoic acid and 5b provided
the title compound (85 mg, 80%) as a white solid. 1H NMR (400
MHz, DMSO-d6) δ ppm 10.26 (s, 1 H), 8.45 (br s, 1 H), 8.38 (d,
J = 5.2 Hz, 1 H), 8.15 (dd, J = 4.8, 2.0 Hz, 1H), 7.68 (dd, J =
7.7, 1.6 Hz, 1H), 7.62 (d, J = 2.3 Hz, 1H), 7.46-7.54 (m, 2H),
7.36-7.44 (m, 2H), 7.21-7.33 (m, 3H), 7.12-7.19 (m, 2H), 7.08
(dd, J = 8.6, 1.0 Hz, 2H), 7.03 (d, J = 8.6 Hz, 1H) 6.96-7.00 (m,
1H), 2.89 (d, J = 4.8 Hz, 3H), 2.05 (s, 3 H). HRMS (C30H25-
N5O3)þ calcd 504.20302, found 504.20379.
N-(3-Methyl-4-(3-(2-(methylamino)pyrimidin-4-yl)pyridin-2-
yloxy)phenyl)-3-phenoxybenzamide (6f). Following the procedure
described for 6j, 3-phenoxybenzoic acid and 5b provided the title
compound (105 mg, 80%) as a white solid. 1H NMR (400 MHz,
DMSO-d6) δ ppm 10.25 (s, 1 H), 8.46 (br s, 1 H), 8.39 (d, J = 4.9
Hz, 1 H), 8.17 (dd, J = 4.8, 2.0 Hz, 1 H), 7.74-7.83 (m, 1 H), 7.70
(d, J = 2.4 Hz, 1 H) 7.59-7.63 (m, 2 H), 7.57 (t, J = 7.9 Hz, 1 H),
7.40-7.48 (m, 2 H), 7.32 (d, J = 5.1 Hz, 1 H), 7.25-7.29 (m, 1 H),
7.22-7.25 (m, 1 H), 7.18-7.21 (m, 1 H), 7.14-7.18 (m, 1 H),
7.07-7.12 (m, 3 H), 2.89 (d, J = 4.8 Hz, 3 H), 2.09 (s, 3 H). HRMS
(C30H25N5O3)þ calcd 504.20302, found 504.20456.
N-(3-Methyl-4-(3-(2-(methylamino)pyrimidin-4-yl)pyridin-2-
yloxy)phenyl)-4-phenoxybenzamide (6g). Following the proce-
dure described for 6j, 4-phenoxybenzoic acid and 5b provided
the title compound (85 mg, 80%) as a white solid. 1H NMR (400
MHz, DMSO-d6) δ ppm 10.15 (s, 1 H), 8.45 (br s, 1 H), 8.38 (d,
J = 5.2 Hz, 1 H), 8.16 (dd, J = 4.8, 2.0 Hz, 1 H), 7.98-8.05 (m,
2 H), 7.70 (d, J = 2.2 Hz, 1 H), 7.58-7.63 (m, 1 H), 7.41-7.50
(m, 2 H), 7.31 (d, J = 5.1 Hz, 1 H), 7.19-7.28 (m, 2 H), 7.08-
7.17 (m, 5 H), 7.06 (d, J = 8.6 Hz, 1 H), 2.88 (d, J = 4.8 Hz, 3 H),
2.08 (s, 3 H). HRMS (C30H25N5O3)þ calcd 504.20302, found
504.20825.
N-(3-Methyl-4-(3-(2-(methylamino)pyrimidin-4-yl)pyridin-2-
yloxy)phenyl)-2-(phenylamino)benzamide (6h). Following the
procedure described for 6j, N-phenylanthranilic acid and 5b
provided the title compound (75 mg, 54%) as a light-yellow
solid. 1H NMR (400 MHz, DMSO-d6) δ 10.33 (s, 1H), 9.18 (s,
1H), 8.48 (br s, 1H), 8.39 (m, 1H), 8.19 (m, 1H), 7.80 (d, J = 7
Hz, 1H), 7.67 (s, 1H), 7.58 (m, 1H), 7.31 (m, 6H), 7.18 (m, 3H),
7.06 (m, 1H), 6.95 (m, 2H), 2.89 (s, 3H), 2.08 (s, 3H). HRMS
(C30H26N6O2)þ calcd 503.21900, found 503.21903.
N-(2-Methyl-4-(3-(2-(methylamino)pyrimidin-4-yl)pyridin-2-
yloxy)phenyl)-2-(phenylamino)benzamide (6i). Following the
procedure described for 6j, N-phenylanthranilic acid and 5c
provided the title compound (123 mg, 84%) as a light-yellow
solid. 1H NMR (400 MHz, DMSO-d6) δ 10.04 (s, 1H), 9.42 (s,
1H), 8.44 (br s, 1H), 8.37 (m, 1H), 8.23 (m, 1H), 7.89 (m, 1H),
7.30 (m, 6H), 7.16 (m, 5H), 6.95 (m, 3H), 2.87 (s, 3H), 2.23 (s,
3H). HRMS (C30H26N6O2)þ calcd 503.21900, found 503.21869.
2-(4-Fluorophenylamino)-N-(4-(3-(2-(methylamino)pyrimidin-
4-yl)pyridin-2-yloxy)phenyl)benzamide (6k). Following the pro-
cedure described for 6j, 2-(4-fluorophenylamino)benzoic acid
and 5a provided the title compound (35 mg, 33%) as a light-
yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 10.36 (s, 1H),
9.11 (s, 1H), 8.44 (br s, 1H), 8.37 (m, 1H), 8.21 (m, 1H), 7.77
(m, 3H), 7.39 (m, 1H), 7.17 (m, 10H), 6.92 (m, 1H), 2.88 (s, 3H).
HRMS (C29H23FN6O2)þ calcd 507.19393, found 507.19384.
4-(2-(4-Aminophenoxy)pyridin-3-yl)-N-methylpyrimidin-2-
amine (5a). 4-Aminophenol (1.50 g, 13.6 mmol) was combined
with cesium carbonate (8.90 g, 27.2 mmol) in DMSO (18 mL) at
100 ꢀC for 5 min. 4-(2-Chloropyridin-3-yl)-N-methylpyrimidin-
2-amine (4, 3.00 g, 13.6 mmol) was added, and the mixture was
heated at 130 ꢀC for 16 h. The cooled mixture was diluted with
water, and the resulting precipitate was filtered, washed with
water, and dried to yield 3.75 g (94%) of the title compound as a
light-brown solid. 1H NMR (300 MHz, DMSO-d6) δ 8.40 (br s,
1H), 8.35 (d, J = 5.4 Hz, 1H), 8.15 (m, 1H), 7.30 (m, 1H), 7.21
(m, 2H), 6.82 (m, 2H), 6.58 (m, 2H), 4.98 (s, 1H), 2.86 (d, J = 4.8
Hz, 3H). MS: m/z 280.1 ([M þ H]þ).
4-(2-(4-Amino-2-methylphenoxy)pyridine-3-yl)-N-methylpyri-
midin-2-amine (5b). Following the procedure for 5a, 4 and
4-amino-2-methylphenol provided the title compound (1.56 g,
90%). 1H NMR (400 MHz, DMSO-d6) δ 8.42 (br s, 1H), 8.37 (d,
J = 4.0 Hz, 1H), 8.14-8.12 (m, 1H), 7.31 (d, J = 4.0 Hz, 1H),
7.21-7.18 (m, 1H), 7.13-7.11 (m, 1H), 6.72 (d, J = 8.0 Hz, 1H),
6.47-6.41 (m, 2H), 4.87 (br s, 2H), 2.88 (d, J = 4.0 Hz, 3H), 1.92
(s, 3H). MS: m/z 294.1 ([M þ H]þ).
4-(2-(4-Amino-3-methylphenoxy)pyridine-3-yl)-N-methylpyri-
midin-2-amine (5c). Following the procedure for 5a, 4 and
4-amino-3-methylphenol provided the title compound (2.63 g,
95%), 1H NMR (400 MHz, DMSO-d6) δ 8.32 (br s, 1H), 8.35 (d,
J = 4.0 Hz, 1H), 8.16-8.14 (m, 1H), 7.27 (d, J = 4.0 Hz, 1H),
7.22-7.19 (m, 1H), 7.10-7.14 (m, 1H), 6.74-6.75 (m, 1H), 6.69-
6.71 (m, 1H), 6.60-6.62 (m, 1H), 4.70 (br s, 2H), 2.87 (d, J = 8.0
Hz, 3H), 2.05 (s, 3H). MS: m/z 294.1 ([M þ H]þ).
N-(4-(3-(2-(Methylamino)pyrimidin-4-yl)pyridin-2-yloxy)phe-
nyl)-2-(phenylamino)benzamide (6j). 4-(2-(4-Aminophenoxy)-
pyridin-3-yl)-N-methylpyrimidin-2-amine (5a, 75 mg, 0.26 mmol),
N-phenylanthranilic acid (65 mg, 0.31 mmol), and HATU (136
mg, 0.36 mmol) were combined in chloroform (2.0 mL) followed
by the addition of DIPEA (0.089 mL, 0.51 mmol). The mixture was
stirred at room temperature for 5 h. After concentration, the crude
mixture was purified by flash chromatography (15-45% EtOAc/
hex) to give 100 mg (79%) of the title compound as a light-yellow
solid. 1HNMR(400 MHz, DMSO-d6) δ10.41 (s, 1H), 9.17 (s, 1H),
8.42 (br s, 1H), 8.37 (s, 1H), 8.20 (m, 1H), 7.75 (m, 3H), 7.30 (m,
6H), 7.17 (m, 5H), 6.94 (m, 2H), 2.87 (s, 3H). HRMS (C29H24-
N6O2)þ calcd 489.20335, found 489.20319.
N-(3-Methyl-4-(3-(2-(methylamino)pyrimidin-4-yl)pyridin-2-
yloxy)phenyl)-3-(trifluoromethyl)benzamide (6a). Following the
procedure described for 6j, 3-trifluoromethylaniline and 5b
provided the title compound (60 mg, 77%) as a white solid. 1H
NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1H), 8.50 (br s, 1H), 8.39
(m, 1H), 8.30 (m, 2H), 8.17 (m, 1H), 7.98 (m, 1H), 7.80 (m, 1H),
7.71 (s, 1H), 7.65 (m, 1H), 7.22 (m, 3H), 7.10 (d, J = 8.8 Hz, 1H),
2.83 (d, J = 4.4 Hz, 3H), 2.09 (s, 3H). HRMS (C25H20F3N5O2)þ
calcd 480.16419, found 480.16424.
2-Chloro-N-(3-methyl-4-(3-(2-(methylamino)pyrimidin-4-yl)-
pyridin-2-yloxy)phenyl)benzamide (6b). Following the proce-
dure described for 6j, 2-chlorobenzoic acid and 5b provided
the title compound (74 mg, 56%) as an off-white solid. 1H NMR
(400 MHz, DMSO-d6) δ 10.33 (s, 1H), 8.44 (br s, 1H), 8.38 (m,
1H), 8.16 (m, 1H), 8.02 (s, 1H), 7.91 (m, 1H), 7.60 (m, 4H), 7.32
(m, 1H), 7.26 (m, 1H), 7.17 (m, 1H), 7.09 (d, J = 8.8 Hz, 1H),
2.89 (d, J = 4.9 Hz, 3H), 2.09 (s, 3H). HRMS (C24H20ClN5O2)þ
calcd 446.13783, found 446.13923.
3-Chloro-N-(3-methyl-4-(3-(2-(methylamino)pyrimidin-4-yl)-
pyridin-2-yloxy)phenyl)benzamide (6c). Following the procedure
described for 6j, 3-chlorobenzoic acid and 5b provided the title
1
compound (68 mg, 52%) as an off-white solid. H NMR (400
MHz, DMSO-d6) δ 10.48 (s, 1H), 8.42 (br s, 1H), 8.40 (m, 1H),
8.15 (m, 1H), 7.69 (m, 1H), 7.50 (m, 5H), 7.32 (m, 1H), 7.26 (m,

Article
5-Fluoro-N-(4-(3-(2-(methylamino)pyrimidin-4-yl)pyridin-2-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6375
(23 μM) in 50 mM HEPES pH 7.5, 5 mM NaCl, 0.5 mM MgCl2,
0.5 mM MnCl2, 2 mM DTT, 0.05% BSA. Enzyme assays were
generally performed in 40 μL for 90-120 min at ambient
temperature before stopping the reaction with 160 μL of a
solution containing detection reagents (SA-APC at a final
concentration of 0.002 mg/mL, and either europilated antipho-
spho PLK (Ser137) antibody for aurora-A assay (0.080 nM) or
antiphospho histone H3(Ser10)(6G3) antibody for aurora-B
assay (0.057 nM)) and 50 mM Tris, pH 7.5, 100 mM NaCl,
3 mM EDTA, 0.05% BSA, and 0.1% Tween 20. Antibodies
were custom labeled by Perkin-Elmer Life Sciences. The assay
plate was read using a BMG RUBYStar fluorescence plate
reader with excitation at 320 nm and emission at 615 and
655 nm. Other enzyme kinase assays. Assays for other kinases
were performed in a similar way as described above, varying the
concentrations of enzyme, peptide substrate, and ATP added to
the reaction, depending on the specific activity of the kinase and
measured Km’s for the substrates.
Cellomics Aurora-B Cellular Assay (DNA Ploidy). HeLa cell
line was obtained from the American Type Culture Collection.
HeLa cells were plated at a density of 1.0 ꢀ 104 cells per well in a
96-well plate in 100 μL of complete media (DMEM with 10%
FBS). The following day, cells were treated with DMSO or com-
pound over a 10-point concentration range (0.0024-1.25 μM)
with a final DMSO concentration of 0.1% in complete media.
After 24 h, cells were fixed for 10 min at room temperature by
adding 110 μL of 2ꢀ fixative solution (6.8% formaldehyde and
0.24% glutaraldehyde in 1ꢀ PBS). Cells were then permeabli-
lized with wash buffer (0.5% Triton X-100 and 1% BSA in 1ꢀ
PBS) at room temperature for 30 min. Cells were stained with
0.5 μg/mL Hoechest DNA dye (Invitrogen) and incubated at
room temperature for 30 min. Cells were washed twice with
100 μL of wash buffer. The 96-well cell assay plates were ana-
lyzed on an ArrayScan VTi HCS reader using a Cell-cycle V2
algorithm (Cellomics, Thermo Fisher Scientific). The percen-
tage of cells with g4N DNA content were determined and used
to generate a concentration-response curve and EC50 value
using a four-parameter equation.
yloxy)phenyl)-2-(phenylamino)benzamide (6l). Following the
procedure described for 6j, 4-fluoro-2-(phenylamino)benzoic
acid16 and 5a provided the title compound (49 mg, 40%) as a
1
light-brown solid. H NMR (400 MHz, DMSO-d6) δ 10.42 (s,
1H), 8.82 (s, 1H), 8.44 (br s, 1H), 8.35 (m, 1H), 8.19 (m, 1H), 7.74
(m, 2H), 7.63 (m, 1H), 7.27 (m, 6H), 7.16 (m, 5H), 6.89 (m, 1H),
2.86 (s, 3H). HRMS (C29H23FN6O2)þ calcd 507.19393, found
507.19407.
4-Chloro-N-(4-(3-(2-(methylamino)pyrimidin-4-yl)pyridin-2-
yloxy)phenyl)phthalazin-1-amine (7). 4-(2-(4-Aminophenoxy)-
pyridin-3-yl)-N-methylpyrimidin-2-amine (5a, 8.0 g, 27.3 mmol)
and 1,4-dichlorophthalazine (11.9 g, 60.0 mmol) and 136 mL
s-BuOH were placed in a 350 mL pressure bottle and heated to
100 ꢀC for 3 h. Upon cooling to ambient temperature, the solids
were filtered and washed with MeOH. The crude material was
triturated in CH2Cl2, filtered, washed with CH2Cl2, and air-
dried to provide the title compound as a tan solid (11.0 g, 88%
yield). 1H NMR (400 MHz, DMSO-d6) δ 10.30 (br s, 1H),
8.90-8.88 (m, 1 H), 8.60-8.54 (br s, 1H), 8.45 (d, J = 4.0 Hz,
1H), 8.31-8.30 (m, 1H), 8.27-8.25 (m, 1H), 8.19-8.17 (m, 2H),
7.82 (d, J = 8.0 Hz, 2H), 7.57-7.53 (m, 1H), 7.37-7.34 (m, 1H),
7.38 (d, J = 8.0 Hz, 2H), 2.96 (s, 3H). HRMS (C24H18N7OCl)þ
calcd 455.12814, found 455.13341.
N-(4-(3-(2-(Methylamino)pyrimidin-4-yl)pyridin-2-yloxy)phe-
nyl)-4-phenylphthalazin-1-amine (8a). Following the procedure
described for 8b, phenylboronic acid and 7 provided the title
compound (130 mg, 60%) as an off-white solid. 1H NMR (400
MHz, DMSO-d6) δ 9.33 (s, 1H), 8.69 (m, 1H), 8.51 (br s, 1H),
8.44 (m, 1H), 8.21 (s, 1H), 7.98 (m, 5H), 7.62 (m, 5H), 7.27 (m,
5H), 2.89 (s, 3H). HRMS (C30H23N7O)þ calcd 498.20368, found
498.20376.
4-(4-Fluorophenyl)-N-(4-(3-(2-(methylamino)pyrimidin-4-yl)-
pyridin-2-yloxy)phenyl)phthalazin-1-amine (8b). 4-Chloro-N-
(4-(3-(2-(methylamino)pyrimidin-4-yl)pyridin-2-yloxy)phenyl)-
phthalazin-1-amine (7, 360 mg, 0.790 mmol), 4-fluorophenyl-
boronic acid (276 mg, 1.97 mmol), and 1,10-bis(diphenylphos-
phino)ferrocene-palladium dichloride (116 mg, 0.158 mmol)
were placed in a pressure bottle under nitrogen and taken up
in 1,4-dioxane (3.9 mL). Aqueous sodium carbonate (2.0 M)
(0.869 mL, 1.73 mmol) was added, and the reaction vessel was
sealed and heated to 90 ꢀC overnight. The reaction was cooled to
ambient temperature, and the mixture was diluted with dichloro-
methane and filtered through Celite. The filtrate was concentrated
under reduced pressure and the residue purified by silica gel chro-
matography (2-5% methanol/dichloromethane) to afford 4-(4-
fluorophenyl)-N-(4-(3-(2-(methylamino)pyrimidin-4-yl)pyridin-2-
In Vitro Metabolism. The in vitro intrinsic clearances, CLint
,
of the test compounds were determined in incubations of rat liver
microsomes. The 400 μL incubations contained 0.25 mg of micro-
somal protein/mL, 1 mM NADPH, 2 mM MgCl2 in 50 mM
potassium phosphate buffer, pH 7.4. To begin the reactions, test
compounds were added to the prewarmed (37 ꢀC) incubation
mixtures at the final concentration of 1 μM. At 0, 10, 20, 30, and
40 min following addition of test compound, aliquots of incubation
mixture (35 μL) were collected into an equal volume of acetonitrile þ
internal standard (1 μM tolbutamide). The samples were
centrifuged at 3000g for 15 min and analyzed on a liquid chroma-
tography tandem mass spectrometry system consisting of two
Shimadzu LC-10AD HPLC pumps and a DGU-14A degasser
(Shimadzu, Columbia, MD), a CTC PAL autoinjector (Leap
Technologies, Carrboro, NC) and a API3000 LC-MSMS system.
Chromatography was conducted on a Sprite Armor C18 (20 mmꢀ
2.1 mm, 10 μm) analytical column (Analytical Sales and Products,
Pompton Plains, NJ) with a 0.5 μm PEEK guard filter, using the
following mobile phase gradient program: MPA = H2O with 0.1%
formic acid; MPB = acetonitrile with 0.1% formic acid; 0 min =
98% MPA, 2% MPB; 0.3 min = 98% MPA, 2% MPB; 0.7 min =
5% MPA, 95% MPB; 1.3 min = 5% MPA, 95% MPB; 1.4 min =
98% MPA, 2% MPB; 1.7 min = end of run; approximately 2 min
between sample injections. For each compound, peak areas at each
time point were converted to the natural log of the % remaining
relative to the 0 min samples. The resulting slope of these values
1
yloxy)phenyl)phthalazin-1-amine (180 mg, 44%). H NMR (400
MHz, DMSO-d6) δ 9.35 (s, 1H), 8.69 (d, J = 12.0 Hz, 1H), 8.45 (br
s, 1H), 8.40 (d, J = 4.0 Hz, 1H), 8.23-8.25 (m, 1H), 8.04-8.07 (m,
1H), 7.94-7.99 (m, 3H), 7.87-7.89 (m, 1H), 7.71-7.75 (m, 2H),
7.39-7.44 (m, 2H), 7.30-7.33 (m, 2H), 7.19-7.21 (m, 3H), 2.89
(d, J = 4, 3H). HRMS (C30H22N7OF)þ calcd 516.18699, found
516.19314.
Solubility Determination. Aqueous equilibrium solubility was
determined according to an automated procedure.17
Homogenous Time-Resolved Fluorescence Enzyme Assays.
IC50’s for the inhibition of the aurora A/TPX2 complex, aurora
B, and all other kinase enzymes for individual compounds were
measured using an HTRF assay.8 Aurora enzyme kinase assays.
The Aurora HTRF kinase assays involved ATP dependent
phosphorylation of a biotinylated substrate peptide in the
presence or absence of an inhibitor. Aurora-A (a glutathione-
S-transferase aurora-A catalytic domain fusion protein com-
plexed with TPX2 [1-43]9) was assayed at 10 nM using a
biotinyl-PLK peptide substrate (1 μM) and ATP at its apparent
Km (1 μM) in 60 mM HEPES pH 7.5, 25 mM NaCl, 10 mM
MgCl, 2 mM DTT, 0.05% BSA. Aurora-B (full-length aurora-B
with N-terminal (His)6) was assayed at 1 nM with biotinyl-
histone H3 peptide (0.1 μM) and ATP at its apparent Km
relative to time (k) was converted to in vitro t1/2 where t1/2 =
-0.693/k. CLint was calculated using the following relationship:
CLint = (0.693/T1/2) ꢀ (1/0.25 mg/mL).
Plasma Protein Binding. Mouse plasma protein binding was
determined in filtered mouse plasma by ultrafiltration18 at 37 ꢀC
with a 10 μM concentration of 8b.

6376 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Cee et al.
Pharmacokinetic Studies. Male Sprague-Dawley rats with
surgically implanted femoral vein and jugular vein cannulae
were obtained from Charles River Laboratories (Boston, MA).
Animals were fasted overnight and the following day com-
pounds were administered either by oral gavage or by intra-
venous bolus injection (N = 3 animals per study). Oral (OraPlus,
pH = 2.2) formulations were made 24-48 h prior to dosing,
while intravenous formulations (DMSO) were made on the day
of dosing. Blood samples were collected over 24 h via jugular
cannula into a heparinized tube. Following centrifugation,
plasma samples were stored in a freezer to maintain -70 ꢀC
until analysis. Lithium heparinized plasma samples (50 μL) were
precipitated with 100% acetonitrile containing the internal
standard (IS). The supernatant was transferred into a 96-well
plate, and an aliquot of 20 μL was injected onto an LC-MS/MS
system. The analytes were separated by HPLC using a C-18
analytical column. The analyte ions were generated by an electro-
spray ionization (ESI) source and detected by a Sciex API3000
triple quadrupole mass spectrometer operated in the multiple
reaction monitoring (MRM) mode. Study sample concentra-
tions were determined from a weighted (1/x2) linear regression
of peak area ratios (analyte peak area/IS peak area) versus
the theoretical concentrations of the calibration standards.
Pharmacokinetic parameters were calculated using an Inna-
phase computer program Small Molecules Discovery Assay
(SMDA) Watson.
Characterization of 8b on Aurora End Points in HeLa Cells.
Nocodazole Block. HeLa cells were treated with 0.1 μg/mL
nocodazole for 12 h. The cells were gently washed with PBS to
remove the mitotic cell fraction. Western blot analysis. Cell
lysates were processed for Western blot analysis as previously
described.19 Immunoblots were probed with the following
antibodies: anti-aurora-A, anti-aurora-B (BD Biosciences),
anti-p-aurora-A (Thr 288), anti-p-aurora-B (Thr 232) (Cell Sig-
naling), and anti-β-actin (Sigma). Next, the immunoblots were
probed with either anti-rabbit or anti-mouse IgG (Vectastain
kit, Vector Laboratories), and the protein bands were detected
using the Lightning-enhanced chemiluminescence kit from
PerkinElmer. Immunofluorescence cell staining. HeLa cells
were plated at 40000 cells per well in a 4-well LabTek chamber
slide (Nalge Nunc) in 1 mL of complete media. The following
day, cells were treated with DMSO or compound 8b at 0.313
μM. After 48 h, cells were fixed and permeabilized as pre-
viously described.19 Cells were blocked in 1 mL of wash buffer
(PBS, 1% BSA, 0.2% Triton X-100 supplemented with 4 drops
of horse serum (Vector Laboratories)) for 1 h at room tem-
perature. The cells were then immunostained in wash buffer
supplemented with rabbit anti-pericentrin (Abcam) and mouse
anti-R-tubulin (Sigma) antibodies for 2 h at room temperature.
Detection was performed with anti-mouse IgG-alexa-568 and
anti-rabbit IgG-alexa-488 antibodies (Invitrogen) for 1 h at
room temperature. Cells were washed and stained with 40,6-
diamidino-2-phenylindole (DAPI, Invitrogen) for 5 min at
room temperature. One drop of ProLong Gold antifade
(Invitrogen) was added to the chamber slides before affixing
the glass coverslips. Representative images of cells were ac-
quired with a Nikon upright microscope using a 40ꢀ objective.
Images were collected and analyzed using MetaMorph soft-
ware (MDS). DAPI nuclear staining (blue) was further en-
hanced using Photoshop CS4 (Adobe). Cell-cycle assay. Flow
cytometry was performed on a LSRII using BD FACS Diva
software (BD Biosciences). HeLa cells treated with DMSO or
compound 8b at 0.313 μM for 24 h. Cells were processed for
cell-cycle analysis as previously described.19 Cells were stained
with anti-p-histone H3 (Ser 10) (Millipore) antibody and pro-
pidium iodide (BD Biosciences). To effectively detect the DNA
content of polyploidy cells, the flow cytometer voltage settings
were decreased to accommodate the 2N, 4N, and 8N cell
populations.
COLO 205 Tumor Xenograft Pharmacodynamic Assay. Fe-
male CD 1 nude mice (4-6 weeks old) were obtained from
Charles River, Inc. All mice were housed in sterilized cages,
provided Sterilizable Rodent Diet 8656 (Harlan Teklad) and
reverse osmosis water ad libitum and maintained under aseptic
conditions in a ventilated rack system. All studies were con-
ducted under the Amgen Animal Care and Use Committee
protocol and satisfied all Association for Assessment and
Accreditation of Laboratory Animal Care specifications. Hu-
man colon cancer carcinoma cells, COLO 205, were obtained
from the American Type Culture Collection. Cells [2 ꢀ 106 cells/
100 μL in a solution of 50% matrigel (Becton Dickenson
Biosciences) in serum-free media] were injected subcutaneously
on the right flank area of the mice. After tumors were allow
establishment and growth (approximately 14-21 days post-
injection) to an average size of ∼300 mm3, mice were treated
orally with a single dose of vehicle (OraPlus pH 2.2) or 8b (50
mg/kg). Tumor and plasma samples were collected at 3, 6, and
24 h after treatment (N = 4 per time-point). Excised tumors
were immediately minced into fine pieces with a razor blade and
transferred into 10 mL of ice-cold cell dissociation buffer
(Hank’s buffer supplemented with Dispase II, collagenase V
and XI, DNase I, and 1% BSA Fraction V) in a 50 mL conical
tube containing a small magnetic stir bar. To facilitate tumor
dissociation into a single-cell suspension, the tubes containing
tumor pieces were stirred for 15 min at 37 ꢀC. The cells were
filtered through 70 μm filter and transferred into a new 50 mL
conical tube. The filtered cells were centrifuged at 2000 rpm
using a tabletop centrifuge for 4 min at 18 ꢀC, and the super-
natant was aspirated off the cell pellets. The cells were washed in
1 mL of Versene and transferred to 1.5 mL Eppendorf tube. To
fix the cells, 800 μL of ice-cold 90% methanol was added
dropwise and stored at -20 ꢀC for at least 24 h. Before
intracellular staining, the fixed cells were permeabilized with
200 μL of wash buffer (0.2% Triton X-100 and 1% BSA
Fraction V in 1ꢀ PBS). Cells were stained in 200 μL of wash
buffer with 4 μg/mL of anti-p-histone H3 antibody (Millipore)
and 30 μL anti-cytokeratin-FITC antibody (Vector Labora-
tories) for 2 h at room temperature in the dark. Cells were then
washed and stained with 1.5 μg/mL of anti-rabbit-Alexa
647 antibody (Invitrogen) for 45 min at room temperature in
the dark. Cells were washed and counterstained with 600 μL of
propidium iodide (PI) overnight at 4 ꢀC in the dark. The samples
were filtered through 35 μm cell strainer and transferred into
5 mL polystyrene flow cytometry tubes. Data was acquired
using a LSRII flow cytometer running FACSDiva software.
Stained cells were analyzed and gated according to DNA
content and cytokeratin (human epithelial cell marker) to
acquire a cell cycle profile. Tumor cells in the G2/M phase were
evaluated for levels of p-histone H3. Tumors from mice treated
with the vehicle alone served as the p-histone H3 staining
control. The effect of 8b on p-histone H3 was assessed by
analysis of variance (ANOVA), followed by Scheffe post hoc
testing using StatView 5.0 software. In all analysis, P < 0.05 was
considered statistically significant.
Acknowledgment. We thank Helming Tan and Maggie
Reed (equilibrium solubility assay), Loren Berry, Daniel
Waldon, and Jenne Fretland (PKDM support), Melanie
Cooke (pharmaceutics support), Beth Ziegler (in vivo phar-
macology support), and Peter Jaeckel (PLK1 assay).
Supporting Information Available: Statistical analysis of
Aurora A/TPX2, Aurora B, and DNA Ploidy data. Synthesis
of compound 10. This material is available free of charge via the
Internet at http://pubs.acs.org.
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