5-(2-Amino-pyrimidin-4-yl)-1H-pyrrole and 2-(2-amino-pyrimidin-4-yl)-1,5,6, 7-tetrahydro-pyrrolo[3,2-c]pyridin-4-one derivatives as new classes of selective and orally available Polo-like kinase 1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.11.065

The discovery and characterization of two new chemical classes of potent and selective Polo-like kinase 1 (PLK1) inhibitors is reported. For the most interesting compounds, we discuss the biological activities, crystal structures and preliminary pharmacokinetic parameters. The more advanced compounds inhibit PLK1 in the enzymatic assay at the nM level and exhibit good activity in cell proliferation on A2780 cells. Furthermore, these compounds showed high levels of selectivity on a panel of unrelated kinases, as well as against PLK2 and PLK3 isoforms. Additionally, the compounds show acceptable oral bioavailability in mice making these inhibitors suitable candidates for further in vivo activity studies.

Full text of DOI:10.1016/j.bmcl.2011.11.065

Bioorganic & Medicinal Chemistry Letters 22 (2012) 96–101
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5-(2-Amino-pyrimidin-4-yl)-1H-pyrrole and 2-(2-amino-pyrimidin-4-yl)-1,5,6,7-
tetrahydro-pyrrolo[3,2-c]pyridin-4-one derivatives as new classes of selective
and orally available Polo-like kinase 1 inhibitors
Michele Caruso a, Barbara Valsasina a, Dario Ballinari a, Jay Bertrand a, Maria Gabriella Brasca a,
Marina Caldarelli a, Paolo Cappella a, Francesco Fiorentini b, Laura M. Gianellini a, Alessandra Scolaro a,
Italo Beria a,
⇑
a Nerviano Medical Sciences srl, Business Unit Oncology, Viale Pasteur 10, 20014 Nerviano, (MI), Italy
b Accelera srl, Viale Pasteur 10, 20014 Nerviano, (MI), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery and characterization of two new chemical classes of potent and selective Polo-like kinase 1
(PLK1) inhibitors is reported. For the most interesting compounds, we discuss the biological activities,
crystal structures and preliminary pharmacokinetic parameters. The more advanced compounds inhibit
PLK1 in the enzymatic assay at the nM level and exhibit good activity in cell proliferation on A2780 cells.
Furthermore, these compounds showed high levels of selectivity on a panel of unrelated kinases, as well
as against PLK2 and PLK3 isoforms. Additionally, the compounds show acceptable oral bioavailability in
mice making these inhibitors suitable candidates for further in vivo activity studies.
Received 13 September 2011
Revised 17 November 2011
Accepted 18 November 2011
Available online 23 November 2011
Keywords:
PLK1
Polo-like kinase
PLK2 and PLK3
Cell cycle
Ó 2011 Elsevier Ltd. All rights reserved.
Cellular activity
Crystal structure
Mitotic cell cycle progression is tightly regulated through
reversible covalent protein phosphorylation events coordinated
by regulatory kinases, including members of the Polo subfamily.1,2
Polo-like kinases (PLKs) are a group of highly conserved serine/
threonine kinases with an important role in the mitosis process.3,4
Currently, five members of the PLK family have been identified in
humans (PLK1–5); among them, PLK1 is the best characterized
and is recognized to be a key component of the cell cycle control
machinery with important roles in the mitotic entry, centrosome
duplication, bipolar mitotic spindle formation, transition from
metaphase to anaphase, cytokinesis and maintenance of genomic
stability.5,6 PLK1 has been found to be overexpressed in a variety
of tumors and has been shown to correlate with poor clinical out-
comes.7 Less clear is the functional role of the other PLK family
members, especially those of PLK2 and PLK3 whose inhibition
could cause toxicities associated with the nervous system or pro-
mote angiogenesis, respectively.8,9 Thus, development of small
molecules that selectively inhibit PLK1 could have reduced off-tar-
get effects.10,11
PLK1 inhibitors targeting the ATP binding pocket in the kinase
domain or the PBD region of the enzyme are under development
as potential anticancer agents.10
Recently, we disclosed the discovery and the optimization
work of a series of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline
derivatives,12–14 As part of our continuing effort in the search of
novel PLK1 inhibitors, we report here the identification and opti-
mization of both 5-(2-amino-pyrimidin-4-yl)-1H-pyrrole scaffold
N
O
NH2
H2N
N
N
H
N
O
Scaffold A
H2N
N
NH2
N
N
N
H
O
H2N
N
4,5-Dihydro-1H-pyrazolo[4,3-h]
quinazoline scaffold
NH
N
H
Scaffold B
⇑
Corresponding author. Tel.: +39 331 581516; fax: +39 331 581347.
E-mail address: italo.beria@nervianoms.com (I. Beria).
Figure 1. From pyrazolo-quinazoline to pyrimidinyl-pyrrole and pyrimidinyl-
pyrrolo[3,2-c]pyridinone scaffolds.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.065

M. Caruso et al. / Bioorg. Med. Chem. Lett. 22 (2012) 96–101
97
A and 2-(2-amino-pyrimidin-4-yl)-1,5,6,7-tetrahydro-pyrrolo[3,2-
c]pyridin-4-one scaffold B, that led to the identification of potent
and selective ATP-competitive PLK1 inhibitors ( Fig. 1). The
chemical expansion, of the two scaffolds took advantage of the
information acquired in the development of the 4,5-dihydro-1H-
pyrazolo[4,3-h]quinazoline series, that related to the pyrrole and
to the aminopyrimidine rings. In particular, three key moieties
at the 1-position of the pyrrole ring and 2,5-disubstituted phenyl
residues at the 2-amino-pyrimidin-4-yl template were identified
as suitable appendixes to obtain selective and potent PLK1
inhibitors.
Synthesis of scaffold A was simply performed by hydrolysis of
intermediate 1 and the subsequent reaction of the corresponding
acid with 1-hydroxybenzotriazole ammonium salt (HOBtꢀNH3)
and EDCI (Scheme 1).15 Alkylation of the intermediate 1 at the
1-position of the pyrrole ring by reaction with the suitable hal-
ogen derivatives afforded the versatile intermediates 2–4, that
were used for subsequent introduction of the 2,5-disubstituted
aniline residue at the 2-position of the pyrimidine ring, through
two different pathways (Scheme 1). Compounds 7 and 9 were
prepared by reacting esters 2 and 3 with 1-[3-iodo-4-(trifloro-
methoxy)phenyl]-4-methylpiperazine under Buchwald–Hartwig
conditions catalyzed by Pd2(dba)3 and 4,5-bis(diphenylphos-
phino)-9,9-dimethylxanthene (Xantphos). Then, the ester func-
tion of intermediates
5
and
6
was converted to the
corresponding amide to yield the compound 7 and the interme-
diate 8, that was in turn converted to compound 9 by removal of
the tetrahydropyranyl protecting group under acid conditions.
Alternatively, compounds 13, 14 and 15 were obtained trans-
forming the 2-amino-4-pyrimidinyl derivative 4 to the corre-
sponding 2-iodo derivative 10 via diazonium salt. Then,
coupling of 10 with 5-bromo-2-(trifluoromethoxy)aniline, again
exploiting Buchwald–Hartwig cross-coupling methodology, cata-
lyzed by Pd(OAc)2 and ( )-BINAP, afforded the bromo ester 11,
which was converted to the corresponding amide 12 in two
steps, as previously reported. Finally, further palladium mediated
C–N bond formation with the suitable piperazine derivatives,
Pd2(dba)3 and 2-dicyclohexylphosphosphino-20-(N,N-dimethyl-
amino)-biphenyl gave, directly or after removal of the protecting
group, the final compounds 13, 14 and 15, respectively.
COOEt
CONH2
N
N
a, b
N
N
N
N
H2N
H2N
H
H
1
Scaffold A
c
Scaffold B and its derivatives were prepared starting from com-
pound 16.16 Removal of Boc protecting group from compound 16
yielded directly scaffold B, while alkylation of 16 at the 1-position
f
COOEt
COOEt
N
N
N
N
N
N
H2N
I
R1
2 R1= -CH2CF3
10
O
O
g
3 R1 = -CH2-CH2-O-THP
4 R1 = -CH3
Boc
a
N
NH
N
N
COOEt
N
N
N
d
N
H
N
N
H
H2N
H2N
N
HN
16
scaffold B
COOEt
CF3O
N
N
N
b
N
O
HN
Br
R1
O
CF3O
Boc
N
11
N
Boc
e
N
N
N
a, b
N
HN
R1
N
R1
N
N
H2N
CF3O
CONH2
N
N
17 R1 = -CH2-CF3
18 R1 = -CH3
5 R1= -CH2CF3
6 R1 = -CH2-CH2-O-THP
N
N
N
HN
23 R1 = -CH3
CF3O
19 R1 = -CH2-CH2-O-THP
24 R1 = -CH2-CH2-O-THP
a, b
c
Br
O
12
Boc
N
CONH2
N
h
N
I
a
N
N
N
N
R1
HN
N
R1
CF3O
CONH2
N
20 R1 = -CH2-CF3
N
N
N
HN
d
CF3O
N
O
O
NH
N
7 R1= -CH2CF3
8 R1 = -CH2-CH2-O-THP
N
HN
Boc
N
N
N
a
N
HN
N
N
R2
e
R1
R1
9 R1 = -CH2-CH2-OH
13 R2 = -CH3
14 R2 = -Boc
CF3O
CF3O
N
N
N
N
i
15 R2 = H
21 R1 = -CH2-CF3
22 R1 = -CH2-CF3
25 R1 = -CH3
Scheme 1. Reagents and conditions: (a) 2 M KOH in EtOH, 80 °C, 3 h, quant.; (b)
DMF/DCM 1:1 mixture, EDCI, HOBt.NH3, TEA, rt, 24 h, 60–90%; (c) THF/DMSO, NaH,
R1-I or R1-Br, rt, 12 h, 70–82%; (d) 1-[3-iodo-4-(trifloromethoxy)phenyl]-4-meth-
ylpiperazine, Pd2(dba)3, Xantphos, Cs2CO3, 110 °C, 8 h, 50–75%; (e) PTSA, MeOH, rt,
6 h, 70%; (f) CsI, I2, CuI, isopentyl nitrite, DME, 70 °C, 6 h, 25%; (g) Pd(OAc)2, ( )-
BINAP, K2CO3, 5-bromo-2-(trifluoromethoxy)aniline, DMF, 80 °C, 6 h, 52%; (h) 1-
methylpiperazine or N-Boc-piperazine, Pd2(dba)3, 2-dicyclohexylphosphosphino-
20-(N,N-dimethylamino)-biphenyl, LiN(TMS)2/THF, 80 °C, 8 h, 50–60%; (i) 4 M HCl in
dioxane, rt, 6 h, 66%.
26 R1 = -CH2-CH2-OH
Scheme 2. Reagents and conditions: (a) 4 M HCl in dioxane, rt, 6 h, 30–98%; (b)
DMF, Cs2CO3, R1-I or R1-Br, rt, 4 h, 70-90%; (c) CsI, I2, CuI, isopentyl nitrite, DME,
80 °C, 4 h, 35%; (d) Pd(OAc)2, ( )-BINAP, K2CO3, 5-(4-methylpiperazin-1-yl)-2-
(trifuoromethoxy)aniline, DMF, 80 °C, 2 h, 25%; (e) 1-[3-iodo-4-(triflorometh-
oxy)phenyl]-4-methylpiperazine, Pd2(dba)3, Xantphos, Cs2CO3, dioxane, reflux,
2 h, 75–85%.

98
M. Caruso et al. / Bioorg. Med. Chem. Lett. 22 (2012) 96–101
of the pyrrole ring by reaction with the suitable halogen deriva-
tives afforded versatile intermediates 17–19 (Scheme 2).15 Two
synthetic approaches were exploited to convert intermediates
17–19 to final compounds 22, 25 and 26. The first approach fore-
saw the substitution of the amino group of intermediate 17 with
iodine through diazononium salt formation followed by Buch-
wald–Hartwig cross-coupling of iodo derivative 20 with 5-(4-
methyl-piperazin-1-yl)-2-(trifluoromethoxy)aniline catalyzed by
Pd(OAc)2 and ( )-BINAP to yield the compound 21. Finally, removal
of the Boc protecting group afforded compound 22. In the second
synthetic approach, compounds 25 and 26 were obtained in a
shorter way by reacting the protected intermediates 18 and 19
with the 2-trifluoromethoxy-iodobenzene derivatives using
Pd2(dba)3 and Xantphos and then removing the protecting groups
from intermediates 23 and 24 with trifluoroacetic acid.15
Compounds were tested in the biochemical assay against PLK1–
3, CDK2/A and Aur-A kinases and their activity in cell proliferation
assay was evaluated on A2780 cell lines. In both series, the intro-
duction of selected R1 and 5-(4-methyl-piperazin-1-yl)-2-(triflu-
oromethoxy)aniline residues transformed the inactive scaffolds A
Table 1
SAR of 5-(2-Amino-pyrimidin-4-yl)-1H-pyrrole derivatives
CONH2
N
N
N
N
R1
HN
CF3O
N
R2
Compound R1
Scaffold A
R2
PLK1 IC50
(l
M) PLK2 IC50
(
l
M) PLK3 IC50
(l
M) CDK2/A IC50
(l
M) Au-AIC50 (l
M) A2780 IC50 (lM)
a
a
a
a
a
a
>10
nd
nd
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
nd
13
7
15
9
–CH3
–CH2–CF3
–CH3
–CH3
–CH3
H
0.015
0.018
0.018
0.151
0.272
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.360
0.109
0.610
2.531
6.690
4.513
–(CH2)2–OH
–CH3
8
14
–(CH2)2–O–THP –CH3
–CH3
–C(O)OC(CH3)3 >10
a
Values are means of three experiments. Standard deviation is <20%. nd = not determined.
Table 2
SAR of 2-(2-amino-pyrimidin-4-yl)-1,5,6,7-tetrahydro-pyrrolo[3,2-c]pyridin-4-one derivatives
O
N
R2
N
N
N
R1
HN
CF3O
N
N
a
a
a
a
a
a
Compound
R1
R2
PLK1 IC50
(l
M)
PLK2 IC50
(
lM)
PLK3 IC50
(l
M)
CDK2/A IC50
(l
M)
Aur-A IC50
(l
M)
A2780 IC50 (lM)
Scaffold B
1.131
0.009
0.028
0.102
0.817
nd
nd
0.219
>10
>10
>10
>10
>10
>10
>10
>10
>10
1.53
25
22
26
23
–CH3
H
H
H
>10
>10
>10
>10
>10
>10
>10
>10
0.037
0.062
0.722
0.176
–CH2–CF3
–(CH2)2–OH
–CH3
–C(O)OC(CH3)3
a
Values are means of three experiments. Standard deviation is <20%.
Figure 2. Crystal structure of inhibitors 13 (panel A) and 25 (panel B) bound to PLK1. Hydrogen bonds are shown as red dashed lines (PBD 4A4L and 4A4O, respectively).

M. Caruso et al. / Bioorg. Med. Chem. Lett. 22 (2012) 96–101
99
Table 3
Panel A
Kinase profile of compounds 13 and 25 on a larger kinase panel
Noc
Noc
Comp. 13 (µM)
Comp. 25 (µM)
a
a
Enzyme
Compound 13 IC50
(l
M)
Compound 25 IC50
(
lM)
0
0.05 0.5
5
0
0.05 0.5
5
PLK1
CK2
0.015
0.951
nd
0.009
0.420
0.659
>10
PLK1
CyclinB1
CyclinA
FLT3
Other kinasesb
>10
a
pSer10 Histone H3
Values are means of three experiments. Standard deviation is <20.
For the list of kinases, see Ref. 18.
b
pThr199 NPM
PARP
Caspase3
Table 4
Activity of compounds 13 and 25 on different cell lines
Panel B
Comp. 13 (µM)
a
a
Cell lines
Tumor
types
Compound 13 IC50
M)
Compound 25 IC50
M)
Noc
Noc
Comp. 25 (µM)
(
l
(
l
0
0.05 0.5
5
0
0.05 0.5
5
pSer46 TCTP
A375
HCT-116
LNCaP
MDA-MB-
468
K-562
MOLM-13
MOLT-4
SUP-M2
Melanoma
Colon
Prostate
Breast
0.386
0.520
0.280
0.624
0.027
0.032
0.026
0.041
pSer10 Histone H3
Figure 4. Western Blot analysis of compounds 13 and 25 on A2780 cell lines. Cells
were treated with the compounds at three different doses (0.05 M, 0.5 M and
M) for 24 h (Panel A) or for 1 h (Panel B). Nocodazole (Noc) treated cells are used
as positive control. PLK1 specific marker (pSer46 TCTP), as well as mitotic markers
(pSer10 Histone H3 and pThr199 NPM) and apoptosis markers were evaluated.
l
l
Leukemia
Leukemia
Leukemia
Lymphoma
0.590
0.201
0.253
0.647
0.060
0.021
0.017
0.053
5
l
a
Values are means of two experiments. Standard deviation is <20%.
In order to characterize the binding modes for the two different
chemical classes, crystal structures of compounds 13 and 25 were
solved in complex with PLK1 to 2.35 and 2.70 Å resolution, respec-
tively.17 The activation loop of PLK1 in the crystals showed a DFG-
in conformation indicative of an active form of the kinase. As
expected, the two compounds bind in the ATP-pocket making sim-
ilar interactions with the protein; furthermore, these interactions
are similar to those recently reported for the related compound
NMS-P937.14 In fact, both inhibitors make donor–acceptor–donor
interactions with the PLK1 hinge residues Glu131 and Cys133, both
make hydrogen bonds with Lys182 and Asp194 and both make a
polar interaction with the sidechain of Glu140. In addition, both
inhibitors bind their 2-trifluoromethoxy groups in the pocket
formed by Arg57 and the hinge segment Leu132–Cys133–Arg134
and multipolar interactions are present between the fluorine
atoms from each inhibitor and the backbone carbonyl of Arg134
(Fig. 2). One significant difference between the two structures is
the presence of the pyridin-4-one ring in 25 that is situated under-
neath the glycine-rich loop residues Lys61 and Gly62. In contrast,
since 13 lacks a similar moiety the corresponding region under
the glycine-rich loop is left vacant.
and B into potent and selective PLK1 inhibitors, as outlined in
Tables 1 and 2. These results are in agreement with what we found
in the past for the 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline ser-
ies.12–14 Also the loss of activity of compound 14, wherein the ba-
sicity of the 4-N of the piperazine appendix is destroyed by
formation of a carbamate bond, is in agreement with the previ-
ously published results.12,13 Among the most interesting new
inhibitors, compound 13 was selected as representative of the class
for a deeper characterization.
The trend of activity observed in the 5-pyrimidin-4-yl-1H-pyr-
role series was also found in the 2-pyrimidin-4-yl-1,5,6,7-tetrahy-
dro-pyrrolo[3,2-c]pyridin-4-one series. Thus, while scaffold
B
resulted poorly active against PLK1 with high cross-reactivity ver-
sus CDK2/A (Table 2), the introduction of the key residues at the
pyrrole and at the pyrimidine nucleous on the scaffold, as in com-
pounds 22, 23, 25, and 26, led to the loss of CDK2/A cross reactivity
and a remarkable improvement of the potency against PLK1, asso-
ciated to a significant cellular activity. The best inhibitor of the ser-
ies resulted the compound 25 that showed more than 140-fold
increase in the PLK1 activity and more than 40-fold improvement
of the cellular cytotoxicity.
Figure 3. Flow cytometry analysis on A2780 cell lines treated for 24 h with 0.05, 0.5 and 5 lM of compounds 13 and 25, respectively. Nocodazole was used as a positive
control.

100
M. Caruso et al. / Bioorg. Med. Chem. Lett. 22 (2012) 96–101
Table 5
In vitro ADME parameters of the selected compounds 13 and 25
Compound
Permeability
Caco-2 Papp (10ꢁ6 cm/ PAMPAa Papp (10ꢁ6 cm/
Metabolic stability
Clint (mL/min/kg) rat hepatocytes
Clint (mL/min/kg) HLMc
(1 M) (1 M)
Solubility pH 7 (lM)
b
s)
s)
l
l
13
25
High
Moderate
50.00
5.79
408
—
14.90
16.50
174
163
a
b
c
Parallel artificial membrane permeability.
Intinsic clearance.
Human liver microsomes.
Table 6
In vivo pharmacokinetic parameters standard deviation of selected compounds 13 and 25 in Bulb Nu/Nu micea
Compound
PK data (iv), dose: 10 mg/kg
PK data (po), dose: 10 mg/kg
d
e
f
AUC
(l
Mꢀh)
CL (mL/min/kg)
Vss (L/kg)
t1/2 (h)
Cmax
(l
M)
AUC
(lMꢀh)
t1/2 (h)
F (%)g
1
1
13b
25c
5.41 2.18
5.78 3.10
74.70 35.20
66.40 27.60
2.50 0.52
2.49 0.80
0.65 0.28
0.52 0.15
0.76 0.22
0.76 0.09
1.92 0.43
2.38 0.06
1.70 0.70
1.40 0.23
36
52
a
b
c
d
e
f
n = 3 animals per study.
Dosed as trifluoroacetate salt in dextrose.
Dosed as free base in 50% PEG 400/glucosate (iv); as free base in 0.5% methocel (po).
Maximum plasma concentration.
Area under the curve.
Terminal half-life.
g
Bioavailability.
The selected compounds 13 and 25, belonging to the two differ-
caco-2 assay. Both compounds also show good solubility and good
metabolic stability, especially in human liver microsomes.
ent chemical classes were then further screened against a wide ki-
nase panel (39 kinases),18 where they confirmed the high
selectivity profile (Table 3). From this screening compound 13
Pharmacokinetic properties were evaluated in male Balb Nu/Nu,
Harlan mice at 10 mg/kg following intravenous (iv) and oral (po)
administration (Table 6). After iv dosing both compounds 13 and
25 showed similar behavior, with medium/high clearance and a
volume of distribution about 4-fold the total body water, suggest-
ing a good distribution of the compounds into tissues. After oral
administration both compounds exhibited the same Cmax value
was found to cross react only with CK2 kinase (IC50 = 0.951
while compound 25 cross reacted with both CK2 and FLT3
(IC50 = 0.420 M and IC50 = 0.659 M, respectively). Cellular activ-
lM),
l
l
ity was also evaluated in a small panel of cell lines from tumors
of different origin. Both compounds showed good activity in all
tested cell lines with a higher potency of compound 25 with re-
spect to 13 (Table 4).
(0.76 lM), that for compound 13 is reached after 30 min. post-dos-
ing, while for compound 25 after 90 min. post-dosing. Most impor-
tantly, the two compounds showed good oral bioavailability that,
for compound 25 was found to reach 52%.
The mechanism of action of compounds 13 and 25 was eval-
uated by fluorescence activated cell sorting analysis (FACS) and
by Western Blot analysis in A2780 cell lines. In agreement with
a PLK1 mechanism, a clear cell cycle block, with cell accumula-
tion in the G2–M phase, was indeed shown by FACS analysis
after 24 h treatment with both selected inhibitors (Fig. 3). The
PLK1 mechanism was also demonstrated by Western Blot. After
24 h cell treatment the modulation of different cell cycle mark-
ers including cyclin B1, cyclin A, phospho Ser10 histone H3,
phospho Thr 199 Nucleophosmin and apoptotic markers such
as PARP cleavage and caspase3 activation were evaluated. An in-
crease in phosho Ser10 histone H3 and phosho Thr 199 Nucleo-
phosmin signals, in agreement with a mitotic block mechanism,
was observed for both inhibitors in a dose dependent manner
with apoptosis induction, detected by PARP cleavage and Cas-
pase 3 activation, at higher tested doses (Fig. 4 Panel A). Addi-
tionally, 1 h treatment of cells with both compounds 13 and
25 showed down-regulation of phosphorylation of the direct
PLK1 substrate Translationally Controlled Tumor Protein (TCTP)
as well as increased pSer 10 histone H3 levels (Fig. 4 Panel B).
This behavior clearly differentiate PLK1 inhibitors form nocodaz-
ole treated cells where increase of pSer10 histone H3 is corre-
lated with increase in pSer46 TCTP signal and indicative of a
different mechanism of mitotic block.19
In summary we have reported here the development of two
new chemical classes that led to the identification of two lead com-
pounds 13 and 25; both compounds demonstrated high activity on
PLK1 and good selectivity versus a wide panel of kinases, as well as
versus the PLK2 and PLK3 isoforms. The two new inhibitors possess
good pharmacokinetic profile and good oral bioavailability in
mouse. In particular, compound 25 showed 52% oral bioavailability
that, taken together with the good activity in the cellular prolifer-
ation assay (IC50 = 37 nM on A2780) makes it a good candidate for
further in vivo activity studies.
Acknowledgments
A particular thank to Roberto Bossi for his work on the crystal
structures. We also thank the group of Assay Development and
Biochemical Screening for the biochemical assay on kinase panel,
Walter Ceccarelli and Gabriele Fachin for the compounds’ synthe-
sis, the Biochemistry Laboratory for protein production, Daniele
Donati and Eduard R. Felder for their useful comments on the
manuscript.
References and notes
The selected compounds were further profiled for Absorption,
Distribution, Metabolism and Excretion (ADME) properties and for
pharmacokinetic characteristics. As reported in Table 5, both inhib-
itors show similar profiles with compound 13 showing a slightly
better permeability profile, both in the PAMPA assay and in the
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