Discovery of a potent and orally bioavailable benzolactam-derived inhibitor of polo-like kinase 1 (MLN0905)

Article abstract of DOI:10.1021/jm2011172

This article describes the discovery of a series of potent inhibitors of Polo-like kinase 1 (PLK1). Optimization of this benzolactam-derived chemical series produced an orally bioavailable inhibitor of PLK1 (12c, MLN0905). In vivo pharmacokinetic-pharmacodynamic experiments demonstrated prolonged mitotic arrest after oral administration of 12c to tumor bearing nude mice. A subsequent efficacy study in nude mice achieved tumor growth inhibition or regression in a human colon tumor (HT29) xenograft model.

Full text of DOI:10.1021/jm2011172

Article
pubs.acs.org/jmc
Discovery of a Potent and Orally Bioavailable Benzolactam-Derived
Inhibitor of Polo-Like Kinase 1 (MLN0905)
Matthew O. Duffey,* Tricia J. Vos, Ruth Adams, Jennifer Alley, Justin Anthony, Cynthia Barrett,
Indu Bharathan, Douglas Bowman, Nancy J. Bump, Ryan Chau, Courtney Cullis, Denise L. Driscoll,
Amy Elder, Nancy Forsyth, Jonathan Frazer, Jianping Guo, Luyi Guo, Marc L. Hyer, David Janowick,
Bheemashankar Kulkarni, Su-Jen Lai, Kerri Lasky, Gang Li, Jing Li, Debra Liao, Jeremy Little, Bo Peng,
Mark G. Qian, Dominic J. Reynolds, Mansoureh Rezaei, Margaret Porter Scott, Todd B. Sells,
Vaishali Shinde, Qiuju Judy Shi, Michael D. Sintchak, Francois Soucy, Kevin T. Sprott, Stephen G. Stroud,
Michelle Nestor, Irache Visiers, Gabriel Weatherhead, Yingchun Ye, and Natalie D’Amore
Millennium Pharmaceuticals, Inc., 40 Landsdowne Street, Cambridge, Massachusetts 02139, United States
*
S Supporting Information
ABSTRACT: This article describes the discovery of a series of potent inhibitors of Polo-like kinase 1
PLK1). Optimization of this benzolactam-derived chemical series produced an orally bioavailable
(
inhibitor of PLK1 (12c, MLN0905). In vivo pharmacokinetic−pharmacodynamic experiments
demonstrated prolonged mitotic arrest after oral administration of 12c to tumor bearing nude mice.
A subsequent efficacy study in nude mice achieved tumor growth inhibition or regression in a human
colon tumor (HT29) xenograft model.
INTRODUCTION
(Figure 1) after progression of our corporate compound library
through a PLK1 flash plate-based high-throughput screen.
■
Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase
that plays a key role in cell cycle control. PLK1 controls entry
into and progression through mitosis at multiple stages by
regulation of centrosome maturation, activation of initiating
factors, degradation of inhibitory components, chromosome
1
condensation, and exit from mitosis. PLK1 has been reported
to be overexpressed in numerous cancers such as melanoma,
prostate, ovarian, colorectal, pancreatic, non-small-cell lung,
esophageal, endometrial, glioma, squamous cell carcinoma of
2
the head and neck, and non-Hodgkins lymphoma. Increased
levels of PLK1 expression are additionally correlated with poor
2
b,3
prognosis and survival.
Overexpression of this kinase
transforms cells, rendering them oncogenic such that they
4
acquire the ability to form tumors in mice. PLK1 protein levels
Figure 1. PLK1 screening hit.
are also elevated in tumor relative to normal cell lines in
culture. Down-regulation of PLK1 protein expression by
RNA interference in tumor cell lines results in a reduction of
cell proliferation, mitotic arrest at prometaphase, and rapid
Compound 1 demonstrated good inhibition of the PLK1
enzyme but suffered from suboptimal characteristics including
modest cell-based activity and low solubility. Our goals were
first and foremost to improve enzyme potency and understand
the SAR of PLK1 inhibition. Secondarily, we hoped to create
5
progression into apoptosis. This effect was not observed in
6
normal cell lines. Moreover, down-regulation of PLK1 by short
hairpin expression in mice with human xenografts reduced7
tumor growth to 18% of that observed for a control shRNA.
Its key role in mitotic progression, its overexpression in a wide
range of malignancies, and the antiproliferative effect observed
upon its depletion, underscore the merits of evaluating PLK1 as
an anticancer target. Indeed, a number of PLK1 inhibitors have
inhibitors of PLK1 that would possess good cellular activity as9
well as improved physicochemical properties (i.e., solubility).
Ultimately, we hoped to identify selective inhibitors that would
have adequate in vivo properties to achieve a desirable
8
advanced into clinical development.
As such, efforts were initiated to identify a small molecule
PLK1 inhibitor with drug-like properties. We identified 1
Received: August 19, 2011
Published: November 9, 2011
©
2011 American Chemical Society
197
dx.doi.org/10.1021/jm2011172 | J. Med. Chem. 2012, 55, 197−208

Journal of Medicinal Chemistry
Article
pharmacodynamic response resulting in tumor growth
inhibition.
became clear that modification of the benzolactam core was
tolerated at the C-9 position, and substitution at this carbon
was essential for enzyme potency. However, the series still
suffered from a lack of cell potency and poor solubility (data
not shown).
RESULTS AND DISCUSSION
■
Initial investigation into SAR began with substitution of the
benzolactam core. A series of analogues that focused on
modification at the C-8, C-10, and C-5 sites of the benzolactam
resulted in a decrease in enzyme potency (data not shown).
Modification at the C-9 position was tolerated, however (Table 1).
Scheme 1 details the synthetic route used for construction of
the benzolactam core. In a straightforward manner, aminoesters
(
2) substituted at C-3 of the phenyl ring were converted to
their corresponding 7-membered benzolactams (3). The 3-step
sequence began with addition of 3-(carbomethoxy)propionyl
chloride to form the amide intermediate. Then, a Dieckmann
condensation followed by decarboxylation achieved by reflux-
a
Table 1. SAR of 9-Position on the Benzolactam Core
ing in DMSO/H O provided 3. The benzolactam was next
2
transformed to the corresponding enamine 4 by heating in the
presence of DMF-DMA. A subsequent base promoted
condensation with N-(3,4-dimethoxyphenyl)guanidine pro-
vided aminopyrimidine analogues of general structure 5. For
the syntheses of analogues that required further chemical
manipulation, the requisite iodinated analogues of 3 (R = I) or
5 (R = I) were transformed to the desired final compounds (see
Supporting Information for further details).
R
PLK1 IC50 (nM)
1
5
5
5
5
5
5
5
9-Cl
9-I
31 ± 10
10 ± 2
18
In addition to understanding SAR and improving enzyme
potency, we also sought to improve the cell activity and
solubility of this chemical series. As we were unable to achieve
these goals by modification of the benzolactam core, we took
aim at other regions of the scaffold. Our strategy focused on
creating analogues that varied substitution on the upper aryl
ring while keeping chloro constant at the 9-position of the
a
b
c
d
e
f
9-CF3
9-H
450 ± 80
200
9-F
9-Me
9-CN
9-Et
65 ± 13
83
g
31 ± 6
10
a
benzolactam core. Appropriately substituted anilines (6) were
converted to the corresponding guanidines (7) by one of two
methods (Scheme 2). Either the addition of N,N′-Di-BOC-S-
methylisothiourea followed by removal of the BOC protecting
groups or the addition of cyanamide in the presence of nitric
acid was employed. Condensation of the guanidine with
Standard deviations are reported for N ≥ 3. Otherwise IC50 values are
mean values of two determinations.
Chloro was replaced with several other small functional groups,
and potency against PLK1 was retained. The dechlorinated
analogue (5c), however, was significantly less potent. Thus, it
Scheme 1. General Synthetic Scheme for the Synthesis of Benzolactam Analogues
Scheme 2. General Synthetic Scheme for the Synthesis of Analogues in Table 2
1
98
dx.doi.org/10.1021/jm2011172 | J. Med. Chem. 2012, 55, 197−208

Journal of Medicinal Chemistry
Article
intermediate 4 (R = Cl) allowed generation of analogues of
general structure 10. If the appropriate aniline was not available
or easily constructed, then condensation of 4 (R = Cl) with
guanidine was employed to provide amino pyrimidine 8. A
Buchwald-type coupling with an appropriate aryl halide (9)
was then utilized to complete the synthesis and also furnish
analogues of general structure 10.
or i-propyl (11g) reside at C-6′, a high degree of selectivity for
PLK1 was achieved. At the same time, we also observed a slight
decrease in cell activity as the steric bulk was increased.
Substitution at other sites on the phenyl ring was also
investigated (Table 3). Installation of chloro at C-5′ produced a
compound (11h) that was equipotent with 11a but not
selective for PLK1 over PLK3. It was also poorly soluble.
Introduction of various functionalities at C-4′ of the phenyl ring
also provided a series of analogues that had good enzyme and
cell potency but showed no improvement in selectivity (11i−
11k). Additionally, placement of a methoxy substituent at C-2′
produced a significant decrease in enzyme and cell potency
(11l). When the phenyl ring was replaced with a pyridine ring
(12a), an increase in enzyme and cell potency was realized, but
selectivity still remained low. By installing a methyl group at the
C-6′ position of this pyridine ring (12b) an increase in
selectivity was achieved similar to what was observed for the
phenyl analogues (11a−g). Although the solubility of 12b
was moderate, its excellent cell activity made it an attractive
compound.
11
The compounds in Table 2 were prepared by these methods.
Cell activities of analogues were measured in a cell viability
12
assay (HT29) and in a mitotic index assay (MIA). In addi-
tion, we developed a Cdc25C-T96 cell-based assay as a direct
readout of PLK1 inhibition. Threonine-96 on Cdc25C is a
13
specific phosphorylation site for PLK1. Readout for the assay
reported the decrease in the levels of phosphorylated Cdc25C-
T96 in treated versus untreated cells.
Analysis of the SAR of the prepared structures showed that
systematic removal and repositioning of the methoxy groups on
the phenyl ring resulted in analogues with either decreased
enzyme potency or decreased cell activity, or both (10a−10d).
Substitution of the phenyl ring with carboxylic acid groups also
decreased cell activity (10e, 10f). A variety of analogues was
also constructed to evaluate substitution at the para position
1
4
Following the procedure established by Kothe, we
determined the crystal structure of the PLK1 kinase domain
(residues 13−345, T210 V mutant) in complex with 12b at 2.6 Å
resolution. Compound 12b binds in the hinge region of the
kinase domain, forming key hydrogen bonds between the
aminopyrimidine and the main chain atoms of Cys-133, as well
as an unusual heterocyclic CH−O hydrogen bond with the
main chain carbonyl of Glu-131 (Figure 2). Similar hinge-
binding interactions have been reported in previous PLK1
(
C-4′) on the phenyl ring. Introduction of iodide at this site
produced a loss of cell activity (10g) while installation of
amines generally improved solubility but decreased enzyme
potency (10h, 10i). Amides at C-4′ gave mixed results (10j,
10k). Although cyclopentyl amide (10k) provided increases in
enzyme and cell potency, it was poorly soluble. A sulfonamide
produced a significant decrease in enzyme potency (10l) as did
the methylmorpholinoamide substituted at the meta position
1
5
crystal structures with inhibitors bound. Also, the 3-
(
C-3′) (10m). However, when the 3-dimethylaminopropyl
dimethylaminopropyl group forms a hydrogen bond with the
16
group was installed at C-3′, a significant increase in cell activity
and solubility was realized while maintaining enzyme potency
side chain carboxylate of Glu-140. This key interaction helps
to characterize the SAR observed around the upper phenyl ring
and the increased activity observed for 10n over related phenyl
analogues (Table 2). Another important interaction involves a
hydrogen bond between the thiolactam NH group and the side
chain of Asp194 from the DFG motif of this kinase. In addition,
the 6′-Me substituent on the pyridine ring fits into a small
selectivity pocket near residue Leu-132. This hinge residue was
identified previously as a key determinant of PLK1 inhibitor
selectivity over related kinases that possess a bulkier residue
(10n). Further investigation at the C-3′ position revealed that a
variety of alkylamino groups decreased enzyme potency (10o−
q), although acyclic alkylamines gave good solubility (10p,
1
enzyme and cell activity (10r). From these investigations, 10n
was identified as an important analogue. It exhibited significant
improvements in both cellular activity and solubility compared
to 1.
Further optimization of the scaffold led to conversion of the
amide of 10n to its corresponding thioamide by use of phos-
phorus pentasulfide (Lawesson’s reagent could also be used)
0q). Additionally, morpholine at C-3′ produced a decrease in
15
(Phe or Tyr) at this position. This structural feature of PLK1
helps inform the SAR and selectivity observed by increased
steric bulk at C-6′ of the phenyl and pyridine ring described
previously (see Table 3).
(
Scheme 3). The newly constructed analogue (11a) achieved
an increase in potency against PLK1 and a significant boost in
cell potency over any compound that had been synthesized
to date in this chemical series. However, while the thio-
amide offered an improvement in potency, it also resulted in
decreased solubility.
Having achieved a significant increase in cell activity versus
the original HTS hit (1), we next focused on attaining
selectivity over related kinases. To accomplish this, we focused
on further chemical manipulation of the upper phenyl ring.
Compounds were constructed by making use of the general
method outlined in Scheme 2, followed by the thioamide
formation detailed in Scheme 3. We found that substitution at
C-6′ of the phenyl ring generally resulted in increased selectivity
for PLK1 over a small internally available panel of kinases
Having discovered a compound (12b) with excellent in vitro
activities and a promising selectivity profile, we hoped to build
upon this discovery and further optimize this series. Previously,
we determined that a variety of functional groups were
tolerated at the C-9 position of the benzolactam core (see
Table 1). After discovery of 12b, the methylpyridine func-
tionality became the favored appendage off of the amino-
pyrimidine for analogue construction. Consequently, we
constructed analogues that combined the methylpyridine
group with a number of functionalities at C-9. Out of this
17
effort we identified 12c (MLN0905), a trifluoromethyl
replacement of the chloro substituent (Table 4). Like its
precursor, 12c displayed excellent enzyme and cell potency but
achieved a significant increase in solubility. Additionally, both
of these compounds were screened in an extensive kinase panel
(Ambit) to determine a wider selectivity profile. Evaluation of
12b and 12c against a panel of 359 kinases revealed them to be
(
Table 3). As the steric bulk of the substituent at C-6′
was increased, the selectivity for PLK1 over the kinase panel
also increased (11a−11g). When R = H (11a), little to no
selectivity was observed. However, when trifluoromethyl (11f)
18
reasonably selective inhibitors of PLK1.
1
99
dx.doi.org/10.1021/jm2011172 | J. Med. Chem. 2012, 55, 197−208

Journal of Medicinal Chemistry
Article
b
Table 2. SAR of Upper Phenyl Ring
a
b
pH = 6.8. Standard deviations are reported for N ≥ 3. Otherwise IC , EC , and LD values are mean values of two determinations.
50
30
50
2
00
dx.doi.org/10.1021/jm2011172 | J. Med. Chem. 2012, 55, 197−208

Journal of Medicinal Chemistry
Article
Scheme 3. Conversion of Lactam to Thiolactam
Figure 2. Crystal structure of PLK1 bound to 12b. The crystal
structure is available from the RCSB PDB with access code 3THB and
is further described in the Supporting Information.
We then advanced these compounds into an evaluation of
their in vivo properties. While chemical modification of the C-9
position resulted in little effect on the in vitro potency profiles
of these compounds, evaluation of the in vivo pharmacokinetic
Compounds 12b and 12c were administered as single oral
doses (50 mg/kg) to nude mice bearing HT29 xenograft
tumors and plasma and tumor compound concentrations were
measured at 24, 48, and 72 h post dose. PD effects were
analyzed in tumor tissue by immunofluorescent staining to
determine the percentage of tumor area staining positive for
pH3. Administration of both compounds resulted in sustained
(PK) properties and pharmacodynamic (PD) responses to
these inhibitors did allow for differentiation.
PLK1 inhibitors elicit mitotic arrest as a pathway effect.
19
This effect has been confirmed in both cells and xenografts
based on the mitotic marker histone H3 (Ser10), as the
phosphorylation of histone H3 (pH3) is a readout of PLK1
(
24 h) tumor tissue concentrations of >1 μM (Figure 3). A
corresponding robust PD effect (>30%) was also noted at 24
and 48 h for both compounds. These data suggest a trend
whereby efficient and sustained distribution of inhibitor to the
tumor is important for a significant and prolonged PD
response. Overall, 12c showed a higher sustained PD response
as it impressively generated a robust PD response up to 72 h
20
pathway modulation. By inhibiting PLK1, we expected that
cells would be arrested/delayed in mitosis and this could be
measured by an increase in pH3. Accordingly, accumulation of
pH3 as a pathway biomarker and PD readout of PLK1 inhi-
bition was exploited in these studies.
a
Table 3. SAR of Upper Aryl Ring and Kinase Selectivity
cell activity (nM)
kinase selectivity IC50 (nM)
solubility
PLK1 IC50
(nM)
HT29
LD50
Cdc25c
EC50
b
R
(μg/mL)
MIA EC30
PLK3
KDR PDGFRb FLT3 FGFR1
KIT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1a
1b
1c
1d
1e
1f
H
24
100
52
5 ± 10
2 ± 0.4
1 ± 0.8
10
18 ± 5
32
72 ± 37
44 ± 15
96 ± 20
180 ± 48
150
44
64
12
18
10
10
19
49
28
720
12
1600
2800
1000
2000
4100
63
160
6′-Me
6′-Cl
53 ± 14
22
440
130
73
56
93
970
720
6′-OMe
6′-OCF3
6′-CF3
6′-i-Pr
5′-Cl
12
140
260
1500
1100
5
54
190
1100
550
1600
590
1100
2600
2300
28
7
48
190
310
1400
1600
5600
28
4
160
150
37
250
1g
1h
1i
8
1
380
3
13
6
120
150
38
4′-F
2
17
55
7
21
1
23
27
72
1j
4′-OMe
4′-CF3
2′-OMe
H
1 ± 0.4
1
18
26 ± 1
83
32
5
1k
1l
4
59
9
51
51
22
50
470
5400
2a
2b
1 ± 0.5
2 ± 0.8
11 ± 5
9 ± 3
34 ± 9
17 ± 7
53
31
64
11
27
49
95
11
20
6′-Me
33 ± 21
130
730
200
610
a
b
Standard deviations are reported for N ≥ 3. Otherwise IC , EC , and LD values are mean values of two determinations. pH = 6.8.
50
30
50
2
01
dx.doi.org/10.1021/jm2011172 | J. Med. Chem. 2012, 55, 197−208

Journal of Medicinal Chemistry
Article
a
Table 4. Methylpyridine Analogues
solubility
PLK1 IC50
(nM)
Cdc25C EC50 cell viability HT29 LD50 kinase selectivity (359 kinases screened at 1 μM
b
R
(μg/mL)
MIA EC30 (nM)
(nM)
(nM)
inhibitor)
1
1
2b Cl
50
2 ± 0.8
9 ± 3
33 ± 21
17 ± 7
37 kinases ≥65% inh
1
4 kinases ≥90% inh
2c
CF3
105
2 ± 0.8
9 ± 3
29 ± 20
22 ± 10
28 kinases ≥65% inh
0 kinases ≥90% inh
1
a
b
Standard deviations are reported for N ≥ 3. Otherwise IC , EC , and LD values are mean values of two determinations. pH = 6.8.
50
30
50
Figure 3. Comparison of PD response of 12b and 12c following a single 50 mg/kg po dose (vehicle: 10% HPβCD/2.5% dextrose in water).
after a single 50 mg/kg dose. In contrast, pH3 levels for 12c
had returned back to near baseline levels at this time point.
monopolar spindle formation, is consistent with that of PLK1
RNAi treatment and small molecule PLK1 inhibition (data not
20b,21
12c also proved itself to be a potent inhibitor of PLK1 in
shown).
Combined with its excellent in vitro potency,
cells (Figure 4) in addition to being a reasonably selective
good solubility, and ability to generate a prolonged PD
response in vivo, 12c differentiated itself sufficiently to become
our lead compound and warrant further investigation into its in
vivo properties.
The PK properties of 12c were further characterized in
female nude NCR mice bearing HT29 tumors and naive male
Sprague−Dawley (SD) rats after intravenous (IV) and oral
dosing (Table 5). In general, the PK of 12c was characterized
by moderate to high systemic clearance (CL ), high volume of
P
distribution (V ), moderate apparent elimination half-life
SS
(
t1/2), and moderate bioavailability (%F) in both species. The
plasma protein binding of 12c was moderate to high (87.4−
9
5
4.3%) and concentration-independent in the range of 0.5−
μM in mice and rats. In nude NCR mice bearing HT29
xenografts, 12c is rapidly and extensively distributed to tumor
with a tumor-to-plasma AUC0−24h ratio of 10.4 after a single PO
dose of 12.5 mg/kg.
Encouraged by the initial PD response observed with 12c
(50 mg/kg dose in Figure 3) and its efficient distribution to
tumor tissue, additional PD studies were conducted in nude
mice bearing HT29 xenograft tumors. Following single oral
Figure 4. In vitro activity profile of 12c.
inhibitor of PLK1 (Table 4). From the kinase selectivity screen,
none of the inhibited kinases would be expected to contribute
to the cellular and PD effects observed. Moreover, the cellular
18
phenotype observed with 12c treatment, G2-M arrest and
2
02
dx.doi.org/10.1021/jm2011172 | J. Med. Chem. 2012, 55, 197−208

Journal of Medicinal Chemistry
Article
Table 5. Summary of 12c Pharmacokinetic Properties
CLP
VSS
t1/2
Cmax
b
a
a
a
b
c
species
rat, SD
mouse, NCR nude
(L/h/kg) (L/kg) (h) (nM) Tmax (h) F (%)
2.3
2.5
10.2
6.2
4.7
3.4
335
720
4.0
4.0
72
45
a
CL , V , and t are from IV studies (dosed at 2 mg/kg in mice,
P
SS
1/2
b
1
1
mg/kg in rats). Cmax and Tmax are from PO studies (dosed at
c
2.5 mg/kg in mice, 5 mg/kg in rats). F (%) was calculated relative to
the listed respective IV doses versus PO dose (12.5 mg/kg in mice,
5
mg/kg in rats). Vehicle: 10% HPβCD/2.5% dextrose in water.
doses of 25, 12.5, and 6.25 mg/kg, a PD dose response was
observed (Figure 5). A robust PD effect was sustained for the
Figure 6. Tumor growth Inhibition following oral administration of
2
5 and 12.5 mg/kg dosing groups for 48 h after administration
1
2c in mice bearing HT29 xenograft tumors (vehicle: 10% HPβCD/
of 12c, and a lesser PD response was observed in the 6.25 mg/kg
group. Comparison with the 50 mg/kg dosing group discussed
previously (data from Figure 3) demonstrated a proportional
dose response across all the dosing groups. Accordingly, the
concentration of 12c observed in tumor samples was also
proportional across the dosing groups (Figure 5). These
data indicate a concentration dependent PD response
whereby greater tumor exposure of 12c yields a greater PD
response.
Ensuing tolerability studies were initiated in nude mice to
determine the maximum tolerated dose (MTD) for 12c dosed
on a variety of different schedules. Results from these experi-
ments determined the following MTDs: daily (QD), 12.5 mg/kg;
QD×1/week, 50 mg/kg; and QD×3/week, 25 mg/kg (data not
shown).
2
.5% dextrose in water).
course of the study with mean body weight loss less than 10%.
While 50 mg/kg dosed once/week achieved tumor growth
inhibition (T/C = 0.53, p < 0.005), tumor stasis and regression
were achieved with more continuous exposure to drug, even at
low doses. By altering the administration schedule of 12c to
3 days on/4 days off (QD×3/week, 25 mg/kg), a significant
decrease in T/C was observed (0.14, p < 0.0001). Following a
schedule of daily administration of 6.25 mg/kg, a significant
inhibition of tumor growth was also accomplished (T/C = 0.15,
p < 0.0001). Ultimately, tumor regressions were observed
through daily treatment with 12.5 mg/kg of 12c (T/C = 0.03,
p < 0.0001). In the 12.5 mg/kg group, the mean body weight
loss for the group was <5%, however one animal was removed
from the study between days 10−14 due to body weight loss of
Having established MTDs for 12c, the antitumor effect of
12c was next evaluated in nude mice bearing HT29 xenograft
tumors. Mice (10 animals/group) were treated orally with 12c
for 21 days at doses based on tolerability results: QD (6.25 and
>
16%. In the 12.5 mg/kg QD group, three of the nine mice
1
(
0
2.5 mg/kg), QD×1/week (50 mg/kg), and QD×3/week
25 mg/kg) (Figure 6). Significant antitumor activity (p <
.005) was observed using all doses and schedules in this study,
and all doses and schedules were tolerated throughout the
which completed the 21 day treatment phase had no
measurable tumor (i.e., complete response) out to 70 days
post treatment initiation.
Figure 5. Dose dependent pH3 PD modulation in HT29 xenograft tumors following a single oral dose of 12c in nude mice (vehicle: 10% HPβCD/
.5% dextrose in water).
2
2
03
dx.doi.org/10.1021/jm2011172 | J. Med. Chem. 2012, 55, 197−208

Journal of Medicinal Chemistry
Article
(
(
dd, J = 8.7, 2.3 Hz, 1H), 6.88 (d, J = 8.8 Hz, 1H), 3.74 (s, 3H), 3.70
s, 3H), 3.36 (s, 2H). LCMS (FA) m/z = 489 (M + H).
CONCLUSION
■
In conclusion, a series of small molecule inhibitors of PLK1 was
identified and described in this report. Optimization of an HTS
hit (1) led to the discovery of 12c, a potent inhibitor of PLK1
in cells confirmed by inhibition of Cdc25C-T96 phosphor-
ylation, a direct cellular readout of PLK1 inhibition. 12c also
demonstrated the ability to impede cell proliferation in
numerous human tumor cell lines. Growth inhibition in these
cell lines was consistent with mitotic arrest or delay, which are
known to be a result of PLK1 inhibition. This effect was
confirmed by in vivo PD experiments that showed increased
levels of phosphorylation of the mitotic pathway marker
histone H3 (Ser10) in xenografts after exposure to 12c. In vivo
antitumor activity of 12c was observed in the HT29 colon
xenograft model, with tumor stasis or regression observed
at oral doses that were well-tolerated. Additionally, there
was flexibility in the administration of 12c because various
treatment schedules (intermittent and continuous) demon-
strated antitumor activity in this model. Taken together these
data demonstrate that 12c is a pharmacologically active
compound whose effects are consistent with PLK1 inhibition.
9
-Trifluoromethyl-2-(3,4-dimethoxyphenylamino)-5H-
benzo[b]pyrimido[4,5-d]azepin-6(7H)-one (5b). Prepared by
general procedure A using 3 (R = CF ) in step 1.
3
1
H NMR (300 MHz, DMSO) δ 10.44 (s, 1H), 9.63 (s, 1H), 8.51 (s,
1
7
3
H), 8.27 (d, J = 8.1 Hz, 1H), 7.66 (d, J = 8.6 Hz, 2H), 7.54 (s, 1H),
.22 (dd, J = 8.8, 2.4 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H), 3.73 (s, 3H),
.69 (s, 3H), 3.41 (s, 2H). LCMS (FA) m/z = 431 (M + H).
2
-(3,4-Dimethoxyphenylamino)-5H-benzo[b]pyrimido[4,5-
d]azepin-6(7H)-one (5c). Prepared by general procedure A using 3
(R = H) in step 1.
1H NMR (400 MHz, DMSO) δ 10.22 (s, 1H), 9.53 (s, 1H), 8.47 (s,
1H), 8.10 (dd, J = 7.9, 1.4 Hz, 1H), 7.72 (s, 1H), 7.63−7.50 (m, 1H),
7.38−7.28 (m, 1H), 7.28−7.18 (m, 2H), 6.89 (d, J = 8.8 Hz, 1H),
3
(
.76 (s, 3H), 3.72 (s, 3H), 3.35 (s, 2H). LCMS (FA) m/z = 363.2
M + H).
9
-Fluoro-2-(3,4-dimethoxyphenylamino)-5H-benzo[b]-
pyrimido[4,5-d]azepin-6(7H)-one (5d). Prepared by general pro-
cedure A using 3 (R = F) in step 1.
1
H NMR (300 MHz, DMSO) δ 10.34 (s, 1H), 9.56 (s, 1H), 8.46 (s,
1H), 8.14 (dd, J = 8.8, 6.7 Hz, 1H), 7.68 (s, 1H), 7.21 (ddd, J = 10.8,
5.3, 2.5 Hz, 2H), 7.02 (dd, J = 10.4, 2.5 Hz, 1H), 6.88 (d, J = 8.8 Hz,
1H), 3.74 (s, 3H), 3.71 (s, 3H), 3.38 (s, 2H). LCMS (FA) m/z =
3
81.1 (M + H).
9
-Methyl-2-(3,4-dimethoxyphenylamino)-5H-benzo[b]-
EXPERIMENTAL SECTION
NMR spectra were recorded in the solvent reported on a Bruker
00 MHz Avance 1 or 400 MHz Avance 2 (5 mm QnProbe) using
■
3
pyrimido[4,5-d]azepin-6(7H)-one (5e). Prepared by general pro-
cedure A using 3 (R = Me) in step 1.
1
H NMR (300 MHz, DMSO) δ 10.16 (s, 1H), 9.52 (s, 1H), 8.44 (s,
residual solvent peaks as the reference. Compound purity was
determined by analysis of the diode array UV trace of an LC-MS
spectrum using the following procedure: compounds were dissolved in
DMSO, methanol, or acetonitrile, and the solutions were analyzed
using an Agilent 1100 LC interfaced to a micromass Waters
Micromass Zspray mass detector (ZMD). One of two gradients was
used to elute the compounds; either a formic acid (FA) gradient
1H), 7.99 (d, J = 8.0 Hz, 1H), 7.72 (s, 1H), 7.18 (dd, J = 23.7, 8.4 Hz,
2H), 7.01 (s, 1H), 6.88 (d, J = 8.8 Hz, 1H), 3.75 (s, 3H), 3.71 (s, 3H),
3.33 (s, 2H), 2.36 (s, 3H). LCMS (FA): m/z = 377.4 (M + H).
9-Cyano-2-(3,4-dimethoxyphenylamino)-5H-benzo[b]-
pyrimido[4,5-d]azepin-6(7H)-one (5f). A mixture of 5a (100 mg,
0.21 mmol), Zn(CN) (14 mg, 0.12 mmol), Pd (dba) (9.5 mg,
0.01 mmol), and dppf (14 mg, 0.02 mmol) was stirred in DMF (2 mL). A
drop of water was added and the mixture heated at 120 °C overnight
with stirring. The reaction was then allowed to cool to room tem-
perature and diluted with EtOAc and a solution of saturated NaHCO3
(aq). The organic layer was separated and washed with brine and
2
2
3
(
acetonitrile containing 0−100% 0.1% formic acid in water) or an
ammonium acetate (AA) gradient (acetonitrile containing zero to
00% 10 mM ammonium acetate in water). All compounds were
determined to be >95% pure unless otherwise noted.
-Chloro-2-(3,4-dimethoxyphenylamino)-5H-benzo[b]-
pyrimido[4,5-d]azepin-6(7H)-one (1). General Procedure A. Step
1
9
water, then dried over MgSO , filtered, and concentrated. The crude
4
1
product was purified on silica gel to give 5f (75 mg, 92%). H NMR
1
8
0
: To a 250 mL round-bottom flask with magnetic stirrer was added
-chloro-3,4-dihydro-1H-1-benzazepine-2,5-dione (3; R = Cl) (52.0 g,
.248 mol) and THF (364 mL). 1,1-Dimethoxy-N,N-dimethylmethan-
(300 MHz, DMSO) δ 10.46 (s, 1H), 9.65 (s, 1H), 8.53 (s, 1H), 8.24
(d, J = 8.2 Hz, 1H), 7.76 (dd, J = 8.2, 1.6 Hz, 1H), 7.66 (s, 1H), 7.61
(d, J = 1.4 Hz, 1H), 7.20 (dd, J = 8.7, 2.4 Hz, 1H), 6.88 (d, J = 8.8 Hz,
1H), 3.74 (s, 3H), 3.70 (s, 3H), 3.41 (s, 2H). LCMS (FA) m/z =
388.2 (M + H).
amine (175 mL, 1.24 mol) was added, and then the flask was fitted
with a reflux condenser. The resulting reaction mixture was heated at
6
0 °C under an atmosphere of argon for 2 h. The light-orange solution
2-Amino-9-chloro-5H-benzo[b]pyrimido[4,5-d]azepin-6(7H)-
one (8). To a solution of 4 (R = Cl) (11.8 g, 44.6 mmol) in EtOH
(300 mL) was added guanidine hydrochloride (4.68 g, 49.0 mmol)
and potassium carbonate (20.0 g, 145 mmol). The reaction mixture
was allowed to stir, overnight, at 75 °C. Most of the solvent was
removed in vacuo, and the remaining mixture was diluted in water
(500 mL). The resulting solution was stirred, at room temperature, for
30 min. The cloudy mixture was filtered and washed with water and
with a suspended solid was allowed to cool to room temperature.
Ether (250 mL) was added, and the precipitated material was collected
via suction filtration, washed with ether, and dried in vacuum oven
1
(
40 °C) for 2 days to give 62 g (94%) of 4 (R = Cl) as a gray solid. H
NMR (400 MHz, DMSO) δ 10.05 (s, 1H), 7.72 (d, J = 8.5 Hz, 1H),
7
.62 (s, 1H), 7.23−7.16 (m, 1H), 7.08 (d, J = 2.1 Hz, 1H), 3.33
(
s, 2H), 3.23 (s, 6H). LCMS (FA) m/z = 265.2 (M + H).
1
Step 2: A mixture of 4 (R = Cl) (101 mg, 0.382 mmol), N-(3,4-
MeOH to give 8 (10.5 g, 90%) as a white solid. H NMR (400 MHz,
dimethoxyphenyl)guanidine·HNO3 (118 mg, 0.458 mmol), and
potassium carbonate (132 mg, 0.954 mol) in ethanol (2.7 mL) was
heated to reflux overnight. The mixture was cooled to room tem-
perature and added to water (20 mL). The resulting suspension was
stirred for 2 h, and the solid was collected by filtration and dried to
DMSO) δ 10.24 (s, 1H), 8.25 (s, 1H), 7.99−7.89 (m, 1H), 7.33 (dd,
J = 8.5, 2.1 Hz, 1H), 7.22 (d, J = 2.1 Hz, 1H), 6.74 (s, 2H), 3.27 (s,
2H). LCMS (FA): m/z = 261.1 (M + H)
9-Chloro-2-(phenylamino)-5H-benzo[b]pyrimido[4,5-d]-
azepin-6(7H)-one (10d). To a solution of 8 (148 mg, 0.568 mmol),
1
give 136 mg (90%) of 1. H NMR (400 MHz, DMSO) δ 10.35
1-bromobenzene (0.072 mL, 0.681 mmol), Cs
mmol), and BINAP (53 mg, 0.085 mmol) in 4 mL of toluene was
added Pd(OAc) (12.8 mg, 0.057 mmol), and the reaction was stirred
at 100 °C for 12 h. The reaction mixture was then treated with H
and extracted with CH Cl (2 × 30 mL). The organic fractions were
combined, filtered, dried over MgSO , and concentrated in vacuo to
2 3
CO (333 mg, 1.02
(
s, 1H), 9.60 (s, 1H), 8.49 (s, 1H), 8.11 (d, J = 8.5 Hz, 1H), 7.69 (s,
H), 7.41 (dd, J = 8.5, 2.1 Hz, 1H), 7.28 (d, J = 2.1 Hz, 1H), 7.22 (dd,
J = 8.7, 2.4 Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H), 3.75 (s, 3H), 3.72 (s,
H), 3.39 (s, 2H). LCMS (FA) m/z = 397.1 (M + H).
1
2
O
2
3
2
2
9
-Iodo-2-(3,4-dimethoxyphenylamino)-5H-benzo[b]-
4
pyrimido[4,5-d]azepin-6(7H)-one (5a). Prepared by general pro-
cedure A using 3 (R = I) in step 1.
give an orange oil, which was purified by HPLC to afford 10d (1.8 mg,
1
0.9%). H NMR (300 MHz, DMSO) δ 10.35 (s, 1H), 9.77 (s, 1H),
1
H NMR (300 MHz, DMSO) δ 10.24 (s, 1H), 9.57 (s, 1H), 8.46 (s,
8.53 (s, 1H), 8.08 (d, J = 8.6 Hz, 1H), 7.80 (d, J = 7.9 Hz, 2H), 7.43
(dd, J = 8.5, 2.0 Hz, 1H), 7.29 (m, 3H), 6.95 (t, J = 7.2 Hz, 1H), 3.41
1H), 7.83 (d, J = 8.3 Hz, 1H), 7.74−7.62 (m, 2H), 7.59 (s, 1H), 7.22
2
04
dx.doi.org/10.1021/jm2011172 | J. Med. Chem. 2012, 55, 197−208

Journal of Medicinal Chemistry
Article
(
(
s, 2H). HRMS calcd for C H ClN O, 337.0856; found, 337.0853
washed with water and brine. The solution was then dried over
Na SO , filtered, and concentrated. The residue was triturated with
18
13
4
M + H).
-(3-(3-Aminopropyl)phenylamino)-9-chloro-5H-benzo[b]-
2
4
2
ether to give a yellow solid, which was redissolved in THF. 2N HCl in
ether was added to give a yellow solid which was collected by filtration.
pyrimido[4,5-d]azepin-6(7H)-one (10n). To a microwave tube was
added 3-(3-bromophenyl)-N,N-dimethylpropan-1-amine (1.53 g, 6.31
mmol), 8 (1.64 g, 6.31 mmol), cesium carbonate (2.88 g, 8.83 mmol),
1
Obtained 11d as the HCl salt (160 mg, 85%). H NMR (400 MHz,
MeOD) δ 8.04 (s, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.48 (s, 1H), 7.15
(dd, J = 8.5, 2.0 Hz, 1H), 7.04 (d, J = 2.0 Hz, 1H), 6.80 (d, J = 8.3 Hz,
1H), 6.70 (d, J = 8.4 Hz, 1H), 3.68 (s, 2H), 3.60 (s, 3H), 2.79 (m,
2H), 2.53 (s, 6H), 2.43 (m, 2H), 1.80 (m, 2H). LCMS (FA): m/z
4
,5-bis(diphenylphosphino)-9,9-dimethylxanthene (0.22 g, 0.38 mmol),
and tris(dibenzylideneacetone)dipalladium (0) (0.23 g, 0.25 mmol)
and dioxane (30 mL). The resulting mixture was heated at
1
50 °C for 70 min in a microwave reactor. The reaction mixture was
4
68.9 (M + H).
- C h l o r o - 2 - { [ 5 - [ 3 - ( d i m e t h y l a m i n o ) p r o p y l ] - 2 -
cooled to room temp, then 80 mL of THF was added to the mixture. It
was then filtered, and the filtrate was concentrated. The crude residue
was purified on silica gel to give 10n (1.92 g; 72%). 1H NMR
9
(trifluoromethoxy)phenyl]amino}-5,7-dihydro-6H-pyrimido-
[5,4-d][1]benzazepine-6-thione (11e). General procedure C using
Lawesson’s reagent was used to give 11e as the formate salt after
(
(
2
400 MHz, DMSO) δ 10.35 (s, 1H), 9.69 (s, 1H), 8.51 (s, 1H), 8.08
d, J = 8.5 Hz, 1H), 7.73 (s, 1H), 7.60−7.50 (m, 1H), 7.40 (dd, J = 8.5,
1
HPLC purification (60 mg, 32%). H NMR (300 MHz, DMSO)
.1 Hz, 1H), 7.27 (d, J = 2.1 Hz, 1H), 7.17 (t, J = 7.8 Hz, 1H), 6.78 (d,
δ 9.27 (s, 1H), 8.46 (s, 1H), 8.16 (s, 1H), 7.99 (d, J = 8.5 Hz, 1H),
7.78 (s,1H), 7.49 (d, J = 8.5, 1H), 7.40 (S, 1H), 7.28 (d, J = 8.4, 1H),
7.05 (d, J = 8.41H), 3.85 (s, 2H), 3.76−3.67 (m, 1H), 2.65−2.54 (m,
2H), 2.27 (s, 6H), 1.75 (m, 2H). LCMS (FA): m/z 522.1 (M + H).
9 - C h l o r o - 2 - ( 5 - ( 3 - ( d i m e t h y l a m i n o ) p r o p y l ) - 2 -
(trifluoromethyl)phenylamino)-5H-benzo[b]pyrimido[4,5-d]-
azepine-6(7H)-thione (11f). General procedure C using Lawesson’s
reagent was used to give 11f as the formate salt after HPLC
J = 7.6 Hz, 1H), 3.39 (s, 2H), 2.60−2.51 (m, 2H), 2.20 (t, J = 7.2 Hz,
2
4
H), 2.10 (s, 6H), 1.69 (dt, J = 14.7, 7.5 Hz, 2H). LCMS (FA): m/z
22.0 (M + H).
9
-Chloro-2-({3-[3-(dimethylamino)propyl]phenyl}amino)-
5
,7-dihydro-6H-pyrimido[5,4-d][1]benzazepine-6-thione
(
11a). General Procedure B. Thioamide formation using phospho-
rus pentasulfide.
1
A mixture of 10s (940 mg, 2.2 mmol) and phosporous pentasulfide
purification (8 mg, 20%). H NMR (300 MHz, DMSO) δ 8.97 (s,
(
1.14 g, 5.12 mmol) in pyridine (17 mL) was allowed to stir overnight
at 60 °C. At this point, additional phosporous pentasulfide (66 mg,
.30 mmol) was added and the reaction mixture was allowed to stir for
1H), 8.38 (s, 1H), 8.17 (s, 1H), 7.90 (d, J = 8.5 Hz, 1H), 7.63 (d, J =
8.0 Hz, 1H), 7.56 (s, 1H), 7.45 (d, J = 8.5 Hz, 1H), 7.38 (s, 1H), 7.24
(d, J = 7.6 Hz, 1H), 3.82 (s, 2H), 2.65 (m, 2H), 2.31 (m, 2H), 2.18 (s,
6H), 1.74 (m, 2H). LCMS (FA): m/z 506.1 (M + H).
0
another 1 h. The reaction mixture was then added dropwise into a
stirred soln of 1:1 water/1 M aq NaHCO solution (160 mL) and
9 - C h l o r o - 2 - ( { 5 - [ 3 - ( d i m e t h y l a m i n o ) p r o p y l ] - 2 -
isopropylphenyl}amino)-5,7-dihydro-6H-pyrimido[5,4-d][1]-
benzazepine-6-thione (11g). General procedure C using Law-
esson’s reagent was used to give 11g as the formate salt after HPLC
3
allowed to stir for an hour. The resulting solid was obtained after
filtration and a wash with water and ether. The yellowish−tan solid
obtained was vacuum-dried to give 11a (980 mg; 100%). LCMS (FA):
m/z 438.2 (M + H).
1
purification (22 mg, 44%). H NMR (400 MHz, DMSO) δ 9.00 (s,
1
1a (920 mg, 2.1 mmol) was then dissolved in tetrahydrofuran
1H), 8.34 (d, J = 9.3 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.47 (dd, J =
8.5, 2.0 Hz, 1H), 7.39 (d, J = 2.0 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H),
7.19 (s, 1H), 7.03 (d, J = 7.8 Hz, 1H), 3.78 (s, 2H), 3.76−3.69 (m,
1H), 3.28 (m, 1H), 3.19 (m, 1H), 2.54 (m, 2H), 2.33 (s, 6H),
1.82−1.69 (m, 2H), 1.11 (d, J = 6.8 Hz, 6H). LCMS (FA): m/z 480.4
(M + H).
(
37 mL), and to this 2.00 M of HCl in ether (2.10 mL, 4.20 mmol)
was added and allowed to stir for 10 min. Ether (170 mL) was added
and then allowed to stir for 30 min. The resulting solid was filtered and
washed with ether and dried under vacuum to give 11a as the bis-HCl
1
salt (944 mg; 88%). H NMR (400 MHz, DMSO) δ 12.27 (s, 1H),
1
0.22 (s, 1H), 9.82 (s, 1H), 8.49 (s, 1H), 8.09 (d, J = 8.5 Hz, 1H), 7.67
9-Chloro-2-({3-chloro-5-[3-(dimethylamino)propyl]phenyl}-
amino)-5,7-dihydro-6H-pyrimido[5,4-d][1]benzazepine-6-thi-
one (11h). General procedure C using Lawesson’s reagent was used
to give 11h as the formate salt after HPLC purification (0.16 g, 41%).
(
s, 1H), 7.62 (d, J = 8.1 Hz, 1H), 7.56 (dd, J = 8.5, 1.9 Hz, 1H), 7.42
(
3
1
d, J = 1.8 Hz, 1H), 7.22 (t, J = 7.8 Hz, 1H), 6.84 (d, J = 7.4 Hz, 1H),
.85 (s, 2H), 3.03 (m, 2H), 2.72 (s,3H), 2.71(s, 3H), 2.60 (m, 2H),
.95 (m, 2H). LCMS (FA): m/z 438.2 (M + H).
1
H NMR (400 MHz, DMSO) δ 10.01 (s, 1H), 8.55 (s, 1H), 8.25 (s,
1
H), 8.09 (t, J = 6.7 Hz, 1H), 7.79 (s, 1H), 7.63 (s, 1H), 7.54 (dd, J =
9
-Chloro-2-(5-(3-(dimethylamino)propyl)-2-methylphenyla-
mino)-5H-benzo[b]pyrimido[4,5-d]azepine-6(7H)-thione
11b). General procedure B using P S was used to give 11b as the
8.5, 2.0 Hz, 1H), 7.43 (d, J = 2.0 Hz, 1H), 6.87 (s, 1H), 3.88 (s, 2H),
2.60−2.54 (m, 2H), 2.45 (s, 2H), 2.29 (s, 6H), 1.76 (s, 2H). LCMS
(FA): m/z 472.1 (M + H).
(
2
5
1
formate salt after HPLC purification (115 mg, 29%). H NMR (300
MHz, DMSO) δ 8.94 (s, 1H), 8.37 (s, 1H), 7.94 (m, 1H), 7.46 (dd,
J = 8.5, 1.9 Hz, 1H), 7.41−7.33 (m, 2H), 7.11 (d, J = 7.7 Hz, 1H), 6.89
(
(
(
9-Chloro-2-({3-[3-(dimethylamino)propyl]-4-fluorophenyl}-
amino)-5,7-dihydro-6H-pyrimido[5,4-d][1]benzazepine-6-thi-
d, J = 7.5 Hz, 1H), 3.80 (s, 2H), 3.74 (d, J = 5.3 Hz, 1H), 2.59−2.51
m, 2H), 2.45−2.34 (m, 2H), 2.23 (s, 6H), 2.18 (s, 3H), 1.81−1.64
m, 2H). LC-MS (FA): m/z 452.2 (M + H).
one (11i). General procedure B using P S was used to give 11i as the
2 5
1
formate salt after HPLC purification (80 mg, 40%). H NMR (300
MHz, DMSO) δ 9.79 (s, 1H), 8.48 (s, 1H), 8.18 (s, 1H), 8.08 (d, J =
8.5 Hz, 1H), 7.76 (d, J = 4.6 Hz, 1H), 7.55 (m, 1H), 7.53−7.48 (m,
1H), 7.41 (d, J = 1.9 Hz, 1H), 7.06 (t, J = 9.3 Hz, 1H), 3.85 (s, 2H),
2.63−2.54 (m, 2H), 2.34 (t, J = 7.1 Hz, 2H), 2.18 (s, 6H), 1.80−1.61
(m, 2H). LCMS (FA): m/z 456.3 (M + H).
9
-Chloro-2-({2-chloro-5-[3-(dimethylamino)propyl]phenyl}-
amino)-5,7-dihydro-6H-pyrimido[5,4-d][1]benzazepine-6-thi-
one (11c). General procedure B using P S was used to give 11c as
the formate salt after HPLC purification (1.9 g, 46%). H NMR (400
MHz, DMSO) δ 9.03 (s, 1H), 8.46 (s, 1H), 8.23 (s, 1H), 7.99 (d, J =
2
5
1
9 - C h l o r o - 2 - ( { 3 - [ 3 - ( d i m e t h y l a m i n o ) p r o p y l ] - 4 -
8.5 Hz, 1H), 7.68 (d, J = 1.6 Hz, 1H), 7.49 (dd, J = 8.5, 2.0 Hz, 1H),
7.41 (s, 1H), 7.40 (d, J = 5.1 Hz, 1H), 7.02 (dd, J = 8.2, 1.8 Hz, 1H),
3.86 (s, 2H), 2.59 (m, 2H), 2.36 (m, 2H), 2.22 (s, 6H), 1.81−1.68 (m,
2H). LCMS (FA): m/z 472.1 (M + H).
methoxyphenyl}amino)-5,7-dihydro-6H-pyrimido[5,4-d][1]-
benzazepine-6-thione (11j). General procedure B using P S was
2 5
used to give 11j as the formate salt after HPLC purification (54 mg,
1
29%). H NMR (400 MHz, DMSO) δ 9.57 (s, 1H), 8.43 (s, 1H), 8.08
9
- C h l o r o - 2 - ( { 5 - [ 3 - ( d i m e t h y l a m i n o ) p r o p y l ] - 2 -
(d, J = 8.5 Hz, 1H), 7.56 (s, 1H), 7.55−7.47 (m, 2H), 7.41 (d, J = 2.1
methoxyphenyl}amino)-5,7-dihydro-6H-pyrimido[5,4-d][1]-
benzazepine-6-thione (11d). General Procedure C. Thioamide
formation using Lawesson’s reagent.
Hz, 1H), 6.88 (d, J = 8.9 Hz, 1H), 3.82 (s, 2H), 3.74 (s, 3H), 2.56−
2
.51 (m, 4H), 2.39−2.28 (m, 2H), 2.20 (s, 6H), 1.67 (dt, J = 14.9, 7.6
Hz, 2H). LCMS (FA): m/z 468.4 (M + H).
Into a round-bottomed flask was added SI-25 (0.18 g, 0.40 mmol),
9
- C h l o r o - 2 - { [ 3 - [ 3 - ( d i m e t h y l a m i n o ) p r o p y l ] - 4 -
2
(
6
(
,4-bis(4-methoxyphenyl)-2,4-dithioxo-1,3,2,4-dithiadiphosphetane
0.33 g, 0.81 mmol), and THF (6 mL). The mixture was stirred at
0 °C for 1 h. The mixture was then partitioned between DCM
50 mL) and satd NaHCO (aq) (20 mL). The organic phase was
(trifluoromethyl)phenyl]amino}-5,7-dihydro-6H-pyrimido[5,4-
d][1]benzazepine-6-thione (11k). General procedure B using P S
2
5
was used to give 11k as the formate salt after HPLC purification
1
(67 mg, 49%). H NMR (400 MHz, DMSO) δ 10.22 (s, 1H), 8.59 (s, 1H),
3
2
05
dx.doi.org/10.1021/jm2011172 | J. Med. Chem. 2012, 55, 197−208

Journal of Medicinal Chemistry
Article
8
.18 (s, 1H), 8.14 (d, J = 8.5 Hz, 1H), 8.02 (s, 1H), 7.78 (d, J = 8.8
Hz, 1H), 7.63−7.50 (m, 2H), 7.44 (d, J = 2.1 Hz, 1H), 3.91 (s, 2H),
.80−2.63 (m, 2H), 2.48−2.41 (m, 2H), 2.26 (s, 6H), 1.77 (m, 2H).
LCMS (FA): m/z 506.1 (M + H).
- C h l o r o - 2 - ( { 3 - [ 3 - ( d i m e t h y l a m i n o ) p r o p y l ] - 2 -
methoxyphenyl}amino)-5,7-dihydro-6H-pyrimido[5,4-d][1]-
benzazepine-6-thione (11l). General procedure B using P S was
Perkin-Elmer). The wells were washed, and then the bound europium
was liberated using 50 μL of Enhancement Solution (Perkin-Elmer).
Quantification of europium was done using a Pherastar (BMG
Labtech)
2
9
Cellular Assays. Cdc25C-T96 Assay. HeLa cells were seeded in
3
a 96-well cell culture plate (4.5 × 10 cells/well) and incubated over-
2
5
night at 37 °C. pSGcdc25C (0.05 ug) and pCDNA3.1 PLK T210D
used to give 11l as the formate salt after HPLC purification (10 mg,
(0.02 ug) DNA was transfected using 0.15 mL of Fugene 6 (Roche)
1
49%). H NMR (400 MHz, DMSO) δ 8.58 (s, 1H), 8.47 (d, J =
5.5 Hz, 1H), 8.22 (s, 1H), 8.04 (d, J = 8.5 Hz, 1H), 7.84 (dd, J = 8.0,
1.5 Hz, 1H), 7.50 (dd, J = 8.5, 2.1 Hz, 1H), 7.40 (d, J = 2.1 Hz, 1H),
7.04 (t, J = 7.8 Hz, 1H), 6.95 (dd, J = 7.6, 1.5 Hz, 1H), 3.85 (s, 2H),
3.67 (s, 3H), 2.66−2.55 (m, 2H), 2.40−2.31 (m, 2H), 2.21 (s, 6H),
1.72 (m, 2H). LCMS (FA): m/z = 468.0 (M + H).
transfection reagent in each well. Cells were incubated with PLK1
inhibitors for 2 h at 37 °C, fixed with 4% paraformaldehyde for 15 min,
and then permeabilized 15 min with 0.5% Triton X-100 in PBS. Then
1
00 mL of Roche blocking buffer was added to the wells prior to
incubation with rabbit anti-pcdc25c T96 (1:2500, Millennium Phar-
maceuticals) and mouse antimyc (clone 9E10) (1:250, Millennium
Pharmaceuticals Inc.) antibodies overnight at 4 °C. After washing with
PBS, the cells were stained with antirabbit IgG Alexa 488 (1:500,
Molecular Probes) and antimouse IgG Alexa 660 (1:500) for 45 min at
room temperature. DNA was then stained with Hoechst solution
9
-Chloro-2-({5-[3-(dimethylamino)propyl]pyridin-3-yl}-
amino)-5,7-dihydro-6H-pyrimido[5,4-d][1]benzazepine-6-thi-
one (12a). General procedure B using P S was used to give 12a as
the formate salt after HPLC purification (46 mg, 20%). H NMR (400
MHz, DMSO) δ 9.99 (s, 1H), 8.73 (d, J = 2.3 Hz, 1H), 8.54 (s, 1H),
8
2
5
1
(
2 μg/mL). The percentage of pcdc25c and antimyc positive cells were
quantified using the Opera instrument and Acapella Image analysis
Perkin-Elmer.)
.18 (s, 1H), 8.14 (s, 1H), 8.09 (d, J = 8.5 Hz, 1H), 8.03 (d, J = 1.6
Hz, 1H), 7.53 (dd, J = 8.5, 2.0 Hz, 1H), 7.42 (d, J = 2.0 Hz, 1H), 3.88
(
(
(
s, 2H), 2.57 (dd, J = 18.2, 10.7 Hz, 2H), 2.28 (t, J = 7.2 Hz, 2H), 2.15
s, 6H), 1.78−1.64 (m, 2H). LCMS (FA): m/z 423.2 (M + H).
3
Mitotic Index Assay. HT29 cells (2.5 × 10 cells/well) in 75 μL of
McCoy’s 5A media (Invitrogen) supplemented with 10% fetal bovine
9
-Chloro-2-({5-[3-(dimethylamino)propyl]-2-methylpyridin-
serum (Invitrogen) was seeded in wells of a 96-well Optilucx plate
3
-yl}amino)-5,7-dihydro-6H-pyrimido[5,4-d][1]benzazepine-6-
(BD bioscience) and incubated overnight at 37 °C. Then 25 μL of
thione (12b). General procedure B using P S was used to give 12b
2
5
1
serially diluted test compounds in McCoy’s 5A media supplemented
with 10% Fetal Calf Serum (Gibco) were added to the wells. Cells
were incubated for 24 h at 37C and then fixed with the addition of
as the formate salt after HPLC purification (240 mg, 53%). H NMR
400 MHz, DMSO) δ 9.16 (s, 1H), 8.42 (s, 1H), 8.21 (s, 1H), 8.07 (d,
J = 1.8 Hz, 1H), 7.95 (d, J = 8.5 Hz, 1H), 7.81 (d, J = 1.4 Hz, 1H),
(
5
0 μL of 4% paraformaldehyde for 10 min and permeabilized with the
7
2
.47 (dd, J = 8.5, 2.1 Hz, 1H), 7.39 (d, J = 2.1 Hz, 1H), 3.83 (s, 2H),
.55 (dd, J = 13.1, 5.5 Hz, 2H), 2.40 (s, 3H), 2.35 (dd, J = 15.2, 7.7
addition of 50 μL of 0.5% TritonX-100 in PBS. After washing with
PBST, 50 μL of 0.5% Roche blocking buffer was added to the wells
and incubated for 1 h at room temperature. Cells were then incubated
with mouse anti-pHistone H3 (1:500, Millennium Pharmaceuticals)
for 1 h at room temperature. After washing with PBST, the cells were
stained with antimouse IgG Alexa 594 (1:200) for 1 h at room tem-
perature. DNA was then stained with Hoechst solution (2 μg/mL).
The percentage of pHistone H3 cells were quantified using Discovery-1
and MetaMorph (Molecular Devices.)
Hz, 2H), 2.21 (s, 6H), 1.78−1.66 (m, 2H). LCMS (FA): m/z 453.2
(
M + H).
9
-Trifluoromethyl-2-({5-[3-(dimethylamino)propyl]-2-meth-
ylpyridin-3-yl}amino)-5,7-dihydro-6H-pyrimido[5,4-d][1]-
benzazepine-6-thione (12c). General procedure B using P S was
used to give 12c as the formate salt after HPLC purification (4.94 g,
2
5
1
7
2%). H NMR (400 MHz, DMSO) δ 9.23 (s, 1H), 8.47 (s, 1H), 8.19
(
s, 1H), 8.14 (d, J = 8.2 Hz, 1H), 8.08 (d, J = 1.8 Hz, 1H), 7.82 (s,
1H), 7.75 (d, J = 8.3 Hz, 1H), 7.68 (s, 1H), 3.87 (s, 2H), 2.55 (dd, J =
14.2, 6.4 Hz, 2H), 2.41 (s, 3H), 2.38 (d, J = 7.7 Hz, 2H), 2.22 (s, 6H),
1.80−1.66 (m, 2H). LCMS (FA): m/z 487.2 (M + H).
Antiproliferation Assays. Eight μL of serially diluted test com-
4
pounds were added to75 μL of HT29 (2.66 × 10 cells/well) cells in
McCoy’s 5A media supplemented with 10% Fetal Calf Serum (Gibco)
in Biocoat Poly-D lysine 384 well Black/Clear plates (BD
Biosciences). Cells were incubated for 72 h at 37 °C. Supernatant
was aspirated from the wells, leaving 25 μL in each well. ATP-lite
1 step reagent (25 μL, Roche) was added to each well, and lumi-
nescence for each plate was read on the LeadSeeker (Amersham
Biosciences). Percent inhibition was calculated using the values from a
DMSO control set to 100%.
Protein Kinase Enzyme Assays. PLK1 Flash Plate Assay. The
human PLK1 enzymatic reaction totaling 30 μL contained 50 mM
Tris-HCl (pH 8.0), 10 mM MgCl , 0.02% BSA, 10% glycerol, 1 mM
DTT, 100 mM NaCl, 3.3% DMSO, 8 μM ATP, 0.2 μCi [γ- P]-ATP,
4
2
33
μM peptide substrate (Biotin-AHX-LDETGHLDSSGLQEVHLA-
CONH ), and 10 nM recombinant human PLK1[2−369]T210D. The
2
enzymatic reaction mixture, with or without PLK inhibitors, was
incubated for 2.5 h at 30 °C before termination with 20 μL of 150 mM
EDTA. Then 25 μL of the stopped enzyme reaction mixture was
transferred to a 384-well streptavidin coated Image FlashPlate (Perkin-
Elmer) and incubated at room temperature for 3 h. The Image Flash
Plate wells were washed three times with 0.02% Tween-20 and then
read on the Perkin-Elmer Viewlux.
In Vivo Studies. Cell Culture and Tumor Model. HT29 cells
were obtained from ATCC and were cultured in McCoy’s 5A medium
6
supplemented with 10% FBS. HT29 cells (2 × 10 ) were resuspended
in Hanks buffer and injected subcutaneously into the flanks of female
Nude mice (Taconic). All animals were housed and handled in
accordance with the Guide for the Care and Use of Laboratory
Animals.
PLK1 DELFIA Kinase Assay. The human PLK1 enzymatic reaction
totaled 30 μL contained 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2,
Pharmacokinetics: Sample Harvest and Analysis. All compounds
were formulated in a mixture of 2-hydroxypropyl-β-cyclodextrin
(HPβCD) (w/w 10%) and dextrose (w/w 2.5%). HT29 tumor
bearing animal were dosed orally with compound. At indicated time
0
5
.02% BSA, 10% glycerol, 1 mM DTT, 100 mM NaCl, 3.3% DMSO,
0 μM ATP, 2 μM peptide substrate (Biotin-AHX-LDETGHL-
DSSGLQEVHLA-CONH ), and 0.3 nM recombinant human
2
PLK1[2−369]T210D. The enzymatic reaction mixture, with or
without PLK inhibitors, was incubated 90 min at room temperature
before termination with 50 μL of STOP buffer containing 1% BSA,
points, animals were euthanized with CO asphyxiation and cardiac
puncture was used to obtain whole blood. Collected blood was quickly
2
transferred into K EDTA labeled tubes, centrifuged, and plasma was
2
0
.05% Tween 20, 100 mM EDTA. Then 50 μL of the stopped enzyme
analyzed for compound concentration. Tumor tissue was surgically
removed, weighed, and homogenized in mouse plasma (1:4, equivalent
weight in milligrams vs plasma volume in microliters) using a
FastPrep-24 homogenizer (MP Biomedicals, Solon, OH) for con-
centration analysis. The quantification of compound was conducted by
a method based on a liquid chromatography tandem mass
spectrometry (LC/MS/MS) methodology. Compound was extracted
from the protein matrix by precipitating the proteins in samples with
reaction mixture was transferred to a Neutravidin-coated 384-well
plate (Pierce) and incubated at room temperature for 60 min. The
wells were washed with wash buffer (25 mM Tris, 150 mM sodium
chloride, and 0.1% Tween 20) and incubated for 1 h with 50 μL of
antibody reaction mixture containing 1% BSA, 0.05% Tween 20,
antiphospho-cdc25c rabbit monoclonal antibody (325 pM, Millen-
nium Pharmaceuticals), and europium labeled antirabbit IgG (2 nM,
2
06
dx.doi.org/10.1021/jm2011172 | J. Med. Chem. 2012, 55, 197−208

Journal of Medicinal Chemistry
Article
acetonitrile at a ratio of 1:4 (v/v, sample/acetonitrile). The internal
standard used for analysis was a stable isotope labeled MLN0905. The
dynamic range of the assay was 1−1000 nM. The pharmacokinetic
analysis was performed using Winonlin software (Pharsight, St. Louis,
Missouri).
value was then calculated as:
mean(AUC ) − mean(AUC )
ctl
trt
dAUC =
× 100
mean(AUCctl)
Immunohistochemistry. HT29 tumor bearing mice were dosed
orally with compound and at indicated time points tumor tissue
harvested and placed in 10% neutral buffered formalin. Immuno-
fluoresence was performed on 5 μm of paraffin embedded tumor
sections using the Discovery XT automated staining system (Ventana
Medical Systems, Tucson, AZ). Sections were deparaffinized, followed
by epitope unmasking with cell conditioning 1 solution (Ventana
Medical Systems) for 20 min. Tumor sections were stained for
pHisH3 (Cell Signaling Technologies, Danvers, MA). The deoxy-
ribonucleic acid (DNA) stain 4′,6-diamidino-2-phenylindole (DAPI,
Vector Laboratories, Inc., Burlingame, CA) was used to estimate the
total number of cells/field. One tissue section was used for each of the
three animals in a treatment group. Images were acquired using a Leica
DMLB microscope (Leica Microsystems, Wetzlar, Germany) with a
Photometrics Cool Snap HQ Nikon Eclipse E800 camera. Five images
from each slide were captured using a 20× Leica Plan objective (Leica
Microsystems, Wetzlar Germany) and analyzed on Metamorph
This assumes AUCctl was positive. In instances where AUCctl
was negative, the above formula was multiplied by −1. All P
values <0.05 in these analyses were considered statistically
significant.
ASSOCIATED CONTENT
Supporting Information
■
*
S
Synthetic details and characterization data for all compounds
reported in this manuscript are described in full. In addition,
crystallographic methods for the crystal structure of 12b are
also available. This material is available free of charge via the
Internet at http://pubs.acs.org.
Accession Codes
PDB:ID: Figure 2 crystal structure, 12b in PLK1, 3THB.
6
.3r7 image processing software (Molecular Devices, Downingtown,
AUTHOR INFORMATION
Corresponding Author
Phone: 617-551-7832. E-mail: matthew.duffey@mpi.com.
■
PA) using a custom image processing application module (Molecular
Devices Inc., City, State). The number of pHisH3-positive cells were
counted and averaged in the five fields of view, and DAPI Vectashield
HardSet Medium (Vector Laboratories, Burlingame, CA) was used as
a chromatin counter stain.
Efficacy Studies. Tumor growth was monitored using vernier
calipers, and the mean tumor volume was calculated using the formula
V = W × L/2. When the mean tumor volume reached approximately
*
ACKNOWLEDGMENTS
■
We thank Ashok Patil, Nanda Gulavita, Nelson Troupe, and
David Matheka for assistance in purification, David Lok and
Nina Molchanova for determining the HPLC purity of our
compounds, and Matt Jones, Ling Xu, and David Lok for
bioanalysis of PK samples.
2
3
200 mm , the animals were randomized into treatment groups (n = 10
animals/group). Tumor growth and body weights were measured
twice per week.
In Vivo Statistical Analysis. Treatment over Control (T/C). The
mean volume on day x for the treatment group was divided by the
mean volume for the control group on day x.
Area under the Tumor Growth Curve (AUC) Analysis (during
Treatment). The differences in the tumor growth trends (tumor
volume or photon flux) over time between pairs of groups were
assessed using linear mixed effects regression models. To compare
pairs of groups, the following mixed-effects linear regression model
was fit to the data using the maximum likelihood method:
ABBREVIATIONS USED
■
PLK1, Polo-like kinase 1; shRNA, short hairpin RNA; SAR,
structure−activity relationship; HT29, human colon adenocar-
cinoma grade II cell line; MIA, mitotic index assay; Cdc25C,
Cell division cycle 25 homologue C; pH3, phosphohistone H3;
HPβCD, (2-hydroxypropyl)-β-cyclodextrin; RNAi, RNA inter-
ference; V , steady state volume of distribution; CL , plasma
SS
P
clearance; Cmax, maximum concentration in plasma; Tmax, time
of measurement of Cmax; %F, percent bioavailability; t1/2,
elimination half-life; MTD, maximum tolerated dose; QD, daily
2
Y
− Yi0k = Yi0k + treat + day + day + day
ijk
i
i
j
j
2
+
(treat × day) + (treat × day ) + ε
ij
ij
ijk
REFERENCES
where Yijk is the log10 tumor value at the jth time point of the
kth animal in the ith treatment, Yi0k is the day 0 (baseline) log10
■
(
1) Barr, F. A.; Sillje, H. H. W.; Nigg, E. A. Polo-like kinases and the
orchestration of cell division. Nature Rev. Mol. Cell. Biol. 2004, 5, 429−
tumor value in the kth animal in the ith treatment, day was the
j
2
441.
(
median-centered time point and (along with day ) was treated
j
2) (a) Kneisel, L.; Strebhardt, K.; Bernd, A.; Wolter, M.; Binder, A.;
as a continuous variable, and ε is the residual error. A spatial
ijk
Kaufmann, R. Expression of polo-like kinase (PLK1) in thin
melanomas: a novel marker of metastatic disease. J. Cutaneous
Pathol. 2002, 29, 354−358. (b) Takai, N.; Miyazaki, T.; Fujisawa,
K.; Nasu, K.; Hamanaka, R.; Miyakawa, I. Polo-like kinase (PLK)
expression in endometrial carcinoma. Cancer Lett. 2001, 164, 41−49.
(c) Takahashi, T.; Sano, B.; Nagata, T.; Kato, H.; Sugiyama, Y.;
Kunieda, K.; Kimura, M.; Okano, Y.; Saji, S. Polo-like kinase 1 (PLK1)
is overexpressed in primary colorectal cancers. Cancer Sci. 2003, 94,
48−152. (d) Macmillan, J. C.; Hudson, J. W.; Bull, S.; Dennis, J. W.;
Swallow, C. J. Comparative expression of the mitotic regulators SAK
and PLK in colorectal cancer. Ann. Surg. Oncol. 2001, 8, 729−740.
e) Gray, P. J.; Bearss, D. J.; Han, H.; Nagle, R.; Tsao, M.-S.; Dean, N.;
Von Hoff, D. D. Identification of human polo-like kinase 1 as a
potential therapeutic target in pancreatic cancer. Mol. Cancer. Ther.
2004, 3, 641−646. (f) Dietzmann, K.; Kirches, E.; von Bossanyi, P.;
Jachau, K.; Mawrin, C. Increased human polo-like kinase-1 expression
in gliomas. J. Neurooncol. 2001, 53, 1−11. (g) Ito, Y.; Miyoshi, E.;
power law covariance matrix was used to account for the
repeated measurements on the same animal over time. Inter-
2
j
action terms as well as day terms were removed if they were
not statistically significant. A likelihood ratio test was used to
assess whether a given pair of treatment groups exhibited
differences which were statistically significant. The −2 log
likelihood of the full model was compared to one without any
treatment terms (reduced model) and the difference in the
values were tested using a χ-squared test. The degrees of
freedom of the test were calculated as the difference between
the degrees of freedom of the full model and that of the
reduced model. The predicted differences in the log tumor
1
(
values (Yijk − Y , which can be interpreted as log (fold
i0k
10
change from day 0)) were taken from the above models to
calculate mean AUC values for each treatment group. A dAUC
2
07
dx.doi.org/10.1021/jm2011172 | J. Med. Chem. 2012, 55, 197−208

Journal of Medicinal Chemistry
Article
Sasaki, N.; Kakudo, K.; Yoshida, H.; Tomoda, C.; Uruno, T.;
Takamura, Y.; Miya, A.; Kobayashi, K.; Matsuzuka, F.; Matsuura, N.;
Kuma, K.; Miyauchi, A. Polo-like kinase 1 overexpression is an early
event in the progression of papillary carcinoma. Br. J. Cancer 2004, 90,
(9) Solubility in this study was determined by a turbidimetric
solubility assay which provides the kinetic solubility of compounds in
50 mM potassium phosphate buffer at pH 6.8. Our target for an
acceptable solubility profile was 100 mg/mL in this assay.
(10) While the 9-iodo (5a) and 9-trifluoromethyl (5b) analogues of 1
did show slight improvements in enzyme activity, these differences in
activity were minimal. We chose to create analogues with the 9-chloro
substituent because of better accessibility to starting material, and we
were confident that after optimization of the upper aryl ring we would
be able to survey chloride replacements for a fully optimized scaffold.
414−418. (h) Weichert, W.; Schmidt, M.; Jacob, J.; Gekeler, V.;
Langrehr, J.; Neuhaus, P.; Bahra, M.; Denkert, C.; Dietel, M.;
Kristiansen, G. Overexpression of Polo-Like Kinase 1 Is a Common
and Early Event in Pancreatic Cancer. Pancreatology 2005, 5, 259−265.
(
i) Mito, K.; Kashima, K.; Kikuchi, H.; Daa, T.; Nakayama, I.;
Yokoyama, S. Expression of Polo-Like Kinase (PLK1) in Non-
Hodgkin’s Lymphomas. Leuk. Lymphoma 2005, 46, 225−231. (j) Liu,
L.; Zhang, M.; Zou, P. Polo-like kinase 1 as a new target for non-
Hodgkin’s lymphoma treatment. Oncology 2008, 74, 96−103.
(11) Huang, X.; Anderson, K. W.; Zim, D.; Jiang, L.; Klapars, A.;
Buchwald, S. L. Expanding Pd-Catalyzed C−N Bond-Forming
Processes: The First Amidation of Aryl Sulfonates, Aqueous Amina-
tion, and Complementarity with Cu-Catalyzed Reactions. J. Am. Chem.
Soc. 2003, 125, 6653−6655.
(
k) Yamamoto, Y.; Matsuyama, H.; Kawauchi, S.; Matsumoto, H.;
Nagao, K.; Ohmi, C.; Sakano, S.; Furuya, T.; Oga, A.; Naito, K.; Sasaki,
K. Overexpression of Polo-Like Kinase 1 (PLK1) and Chromosomal
Instability in Bladder Cancer. Oncology 2006, 70, 231−237.
(12) The MIA screens small molecule inhibitors of human PLK. The
inhibition of PLK results in cell cycle arrest in pro-metaphase, which is
detected by a phospho-histone H3 (ser10) monoclonal antibody
(
l) Weichert, W.; Ullrich, A.; Schmidt, M.; Gekeler, V.; Noske, A.;
(
pH3). This IF assay measures an EC30 or accumulation of 30%
positive pH3 in cells.
13) Toyoshima-Morimoto, F.; Toyoshima-Morimoto, E.; Nishida,
Niesporek, S.; Buckendahl, A.-C.; Dietel, M.; Denkert, C. Expression
patterns of polo-like kinase 1 in human gastric cancer. Cancer Sci.
(
2
(
006, 97, 271−276.
3) Wolf, G.; Elez, R.; Doermer, A.; Holtrich, U.; Ackermann, H.;
Stutte, H. J.; Altmannsberger, H.-M.; Rubsamen-Waigmann, H.;
E. Plk1 promotes nuclear translocation of human Cdc25C during
prophase. EMBO Rep. 2002, 3, 341−348.
̈
(14) Kothe, M.; Kohls, D.; Low, S.; Coli, R.; Cheng, A. C.; Jacques,
Strebhardt, K. Prognostic significance of polo-like kinase (PLK)
S. L.; Johnson, T. L.; Lewis, C.; Loh, C.; Nonomiya, J.; Sheils, A. L.;
Verdries, K. A.; Wynn, T. A.; Kuhn, C.; Ding, Y.-H. Structure of the
Catalytic Domain of Human Polo-like Kinase 1. Biochemistry 2007, 46,
expression in non-small cell lung cancer. Oncogene 1997, 14, 543−549.
(
b) Knecht, R.; Elez, R.; Oechler, M.; Solbach, C.; von Ilberg, C.;
Strebhardt, K. Prognostic Significance of Polo-like Kinase (PLK)
Expression in Squamous Cell Carcinomas of the Head and Neck.
Cancer Res. 1999, 59, 2794−2797. (c) Strebhardt, K.; Kneisel, L.;
Linhart, C.; Bernd, A.; Kaufmann, R. Prognostic value of pololike
kinase expression in melanomas. JAMA, J. Am. Med. Assoc. 2000, 283,
5
(
960−5971.
15) (a) Kothe, M.; Kohls, D.; Low, S.; Coli, R.; Rennie, G. R.; Feru,
F.; Kuhn, C.; Ding, Y. Selectivity-determining Residues in Plk1. Chem.
Biol. Drug Des. 2007, 70, 540−546. (b) Beria, I.; Ballinari, D.; Bertrand,
J. A.; Borghi, D.; Bossi, R. T.; Brasca, M. G.; Cappella, P.; Caruso, M.;
Ceccarelli, W.; Ciacolella, A.; Cristiani, C.; Croci, V.; De Ponti, A.;
Fachin, G.; Ferguson, R. D.; Lansen, J.; Moll, J. K.; Pesenti, E.; Posteri,
H.; Perego, R.; Rocchetti, M.; Storici, P.; Volpi, D.; Valsasina, B.
Identification of 4,5-Dihydro-1H-pyrazolo[4,3-h]quinazoline
Derivatives as a New Class of Orally and Selective Polo-Like Kinase
1 Inhibitors. J. Med. Chem. 2010, 53, 3532−3551.
(16) Beria, I.; Valsasina, B.; Brasca, M. G.; Ceccarelli, W.; Colombo,
M.; Cribioli, S.; Fachin, G.; Ferguson, R. D.; Fiorentini, F.; Gianellini,
L. M.; Giorgini, M. L.; Moll, J. K.; Posteri, H.; Pezzetta, D.; Roletto,
F.; Sola, F.; Tesei, D.; Caruso, M. 4,5-Dihydro-1H-pyrazolo[4,3-h]-
quinazolines as potent and selective Polo-like kinase 1 (PLK1)
inhibitors. Bioorg. Med. Chem. Lett. 2011, 21, 2969−2974.
479−480. (d) Weichert, W.; Kristiansen, G.; Schmidt, M.; Gekeler, V.;
Noske, A.; Niesporek, S.; Dietel, M.; Denkert, C. Polo-like kinase 1
expression is a prognostic factor in human colon cancer. World J.
Gastroenterol. 2005, 11, 5644−5650. (e) Tokumitsu, Y.; Mori, M.;
Tanaka, S.; Akazawa, K.; Nakano, S.; Niho, Y. Prognostic significance
of polo-like kinase expression in esophageal carcinoma. Int. J. Oncol.
1
999, 15, 687−692. (f) Weichert, W.; Schmidt, M.; Gekeler, V.;
Denkert, C.; Stephan, C.; Jung, K.; Loening, S.; Dietel, M.; Kristiansen,
G. Polo-like kinase 1 is overexpressed in prostate cancer and linked to
higher tumor grades. Prostate 2004, 60, 240−245. (g) Kanaji, S.; Saito,
H.; Tsujitani, S.; Matsumoto, S.; Tatebe, S.; Kondo, A.; Ozaki, M.; Ito,
H.; Ikeguchi, M. Expression of polo-like kinase 1 (PLK1) protein
predicts the survival of patients with gastric carcinoma. Oncology 2006,
(17) Bharathan, I.; Duffey, M. O.; Elder, A. M.; Guo, J.; Li, G.;
Reynolds, D.; Soucy, F.; Vos, T. J. 5,7-Dihydro-6H-pyrimido[5,4-d][1]-
benzazepin-6-thiones as PLK inhibitors. Patent WO 2010/065134
A1, 2010.
7
(
0, 126−133.
4) Smith, M. R.; Wilson, M. L.; Hamanaka, R.; Chase, D.; Kung, H.;
Longo, D. L.; Ferris, D. K. Malignant transformation of mammalian
cells initiated by constitutive expression of the polo-like kinase.
Biochem. Biophys. Res. Commun. 1997, 234, 397−405.
(
18) Additional kinase binding data for 12b and 12c can be found in
Supporting Information Tables 1 and 2.
19) Degenhardt, Y.; Lampkin, T. Targeting Polo-like kinase in
cancer therapy. Clin. Cancer Res. 2010, 16, 384−389.
20) (a) Scutt, P. J.; Chu, M. L. H.; Sloane, D. A.; Cherry, M.;
(
(
̈
5) (a) Spankuch-Schmitt, B.; Bereiter-Hahn, J.; Kaufmann, M.;
Strebhardt, K. Effect of RNA silencing of polo-like kinase-1 (PLK1) on
(
apoptosis and spindle formation in human cancer cells. J. Natl. Cancer
Bignell, C. R.; Williams, D. H.; Eyers, P. A. Discovery and Exploitation
of Inhibitor-Resistant Aurora and Polo Kinase Mutants for the Analysis
of Mitotic Networks. J. Biol. Chem. 2009, 284, 15880−15893.
Inst. 2002, 94, 1863−1877. (b) Span
̈
kuch-Schmitt, B.; Wolf, G.;
ller, M.; von Minckwitz, G.;
Solbach, C.; Loibl, S.; Knecht, R.; Stegmu
̈
Kaufmann, M.; Strebhardt, K. Downregulation of human polo-like
kinase activity by antisense oligonucleotides induces growth inhibition
in cancer cells. Oncogene 2002, 21, 3162−3171.
(
b) Steegmaier, M.; Hoffmann, M.; Baum, A.; Len
M.; Krssak, M.; Gu
Grauert, M.; Adolf, G. R.; Kraut, N.; Peters, J. M.; Rettig, W. J. BI
536, A Potent and Selective Inhibitor of Polo-like Kinase 1, Inhibits
Tumor Growth in Vivo. Curr. Biol. 2007, 17, 316−322.
21) (a) Gilmartin, A. G.; Bleam, M. R.; Richter, M. C.; Erskine,
́ ́
t, P.; Petronczki,
ar
́
̈
rtler, U.; Garin-Chesa, P.; Lieb, S.; Quant, J.;
(
6) Liu, X.; Lei, M.; Erikson, R. L. Normal cells, but not cancer cells,
survive severe Plk1 depletion. Mol. Cell. Biol. 2006, 26, 2093−2108.
7) Spankuch, B; Matthess, Y; Knecht, R; Zimmer, B; Kaufmann, M;
2
(
̈
(
Strebhardt, K. Cancer inhibition in nude mice after systemic
application of U6 promoter-driven short hairpin RNAs against
PLK1. J. Natl. Cancer Inst 2004, 96, 862−872.
S. G.; Kruger, R. G.; Madden, L.; Hassler, D. F.; Smith, G. K.;
Gontarek, R. R.; Courtney, M. P.; Sutton, D.; Diamond, M. A.;
Jackson, J. R.; Laquerre, S. G. Distinct Concentration-Dependent
Effects of the Polo-like Kinase 1-Specific Inhibitor GSK461364A,
Including Differential Effect on Apoptosis. Cancer Res. 2009, 69,
(
8) (a) Schoffski, D. Polo-like kinase (PLK) inhibitors in preclinical
and early clinical development in oncology. Oncologist 2009, 14, 559−
70. (b) Christoph, D. C.; Schuler, M. Polo-like kinase 1 inhibitors in
5
6
969−6877.
mono- and combination therapies: a new strategy for treating
malignancies. Expert Rev. Anticancer Ther. 2011, 11, 1115−1130.
2
08
dx.doi.org/10.1021/jm2011172 | J. Med. Chem. 2012, 55, 197−208