4,5-Dihydro-1H-pyrazolo[4,3-h]quinazolines as potent and selective Polo-like kinase 1 (PLK1) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.09.060

A series of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives was optimized as Polo-like kinase 1 inhibitors. Extensive SAR afforded a highly potent and selective PLK1 compound. The compound showed good antiproliferative activity when tested in a panel of tumor cell lines with PLK1 related mechanism of action and with good in vivo antitumor efficacy in two xenograft models after iv administration.

Full text of DOI:10.1016/j.bmcl.2010.09.060

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6489–6494
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4,5-Dihydro-1H-pyrazolo[4,3-h]quinazolines as potent and selective
Polo-like kinase 1 (PLK1) inhibitors
Italo Beria a, , Barbara Valsasina a, Maria Gabriella Brasca a, Walter Ceccarelli a, Maristella Colombo a,
⇑
Sabrina Cribioli a, Gabriele Fachin a, Ronald D. Ferguson a,b, , Francesco Fiorentini b, Laura M. Gianellini a,
Maria L. Giorgini a, Jurgen K. Moll a, Helena Posteri a, Daniele Pezzetta b, Fulvia Roletto a, Francesco Sola a,
Dania Tesei a, Michele Caruso a
a Nerviano Medical Sciences srl, Business Unit Oncology, Viale Pasteur 10, 20014 Nerviano, Milan, Italy
b Accelera, Viale Pasteur 10, 20014 Nerviano, Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 July 2010
Revised 10 September 2010
Accepted 10 September 2010
Available online 17 September 2010
A series of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives was optimized as Polo-like kinase 1
inhibitors. Extensive SAR afforded a highly potent and selective PLK1 compound. The compound showed
good antiproliferative activity when tested in a panel of tumor cell lines with PLK1 related mechanism of
action and with good in vivo antitumor efficacy in two xenograft models after iv administration.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
PLK1
Polo-like kinase
Kinase inhibitor
In vivo activity
Polo-like kinases (PLKs) are a group of highly conserved serine/
threonine kinases that regulate critical processes in the cell cycle.1
Four mammalian PLK family members PLK1, PLK2 (SNK), PLK3
(FNK or PRK) and PLK4 (SAK) were identified.2,3 Among them,
PLK1 is the best characterized and is recognized to be a key compo-
nent of the cell cycle control machinery with important roles in the
mitotic entry, centrosome duplication, bipolar mitotic spindle for-
mation, transition from metaphase to anaphase, cytokinesis and
maintenance of genomic stability.4,5 PLK1 is often over-expressed
in many different tumor types including lung, colon, prostate, ovary,
breast, head and neck squamous cell carcinoma, and its over-expres-
sion often correlates with poor prognosis.6,7 Furthermore recent
studies have identified a possible role of PLK1 in DNA damage
recovery by phosphorylation and destabilization of Claspin, a known
regulator of Chk1 activation.8,9 PLK1 is not expressed in differenti-
ated postmitotic cells like neurons where instead expression of
PLK2 and PLK3 was reported indicating a potentially better safety
profile for a PLK1 specific inhibitor.10 Thus, PLK1 is thought to be a
promising target for anti-cancer therapy and some PLK1 inhibitors
are currently under evaluation in clinical trials.11
In order to identify PLK1 inhibitors a high-throughput screening
(HTS) of the Nerviano Medical Sciences collection was conducted.
Prior work in the 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline ser-
ies was recently disclosed identifying the key scaffold positions
to achieve selective and potent PLK1 inhibitors.12 In order to devel-
op a PLK1 specific inhibitor suitable for clinical trials, work on this
class was continued and here we report the results obtained, start-
ing from the hit compound 1, by a focused expansion at the 50- and
1-positions of the scaffold that led to the identification of the
potent and selective compound 4 (Fig. 1).
Compounds modified at the 50-position were prepared starting
from the iodo amide 2,13 which was coupled under Buchwald–
Hartwig conditions with the suitable anilines, Pd(OAc)2 and
( )-BINAP as ligand to obtain compounds 1, 3–5 (Scheme 1).14
The bromo derivative 3 was in turn used as starting material to
N
O
NH2
N
NH
CF3
O
O
NH
N
N
CF3
O
2'
N
N
N
N
NH2
N
N
1
1
4
5'
⇑
Corresponding author. Tel.: +39 331 581516; fax: +39 331 581347.
E-mail address: italo.beria@nervianoms.com (I. Beria).
Present address: Merck Co. & Inc. 126 E. Lincoln Ave., Rahway, NJ 07065, USA.
Figure 1. Evolution from the hit compounds 1–4.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.060

6490
I. Beria et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6489–6494
N
N
N
O
NH2
O
O
a
b
CF
O
3HN
N
CF3HN
O
N
I
N
N
NH2
NH2
N
N
N
N
N
3
2
R1
Br
a
7-12
N
O
13-15
16-18
5 R1 = NO2
CF
O
3HN
N
c
NH2
N
N
HN
14 R1 =
N
R1
boc
N
boc
N
15 R1 =
1
4
5
N
N
d
e
19, 20
6
O
Scheme 1. Reagents and conditions: (a) Pd(OAc)2, ( )-BINAP, K2CO3, anilines, DMF, 80 °C, 43–55%; (b) primary or secondary amines, Pd2(dba)3, 2-dicyclohexylphosphino-
20-(N,N-dimethylamino)-biphenyl, LiN(TMS)2, THF, reflux, 5–70%; (c) 4 M HCl in dioxane, dioxane, rt, quant; (d) 0.1 M 3,3-dimethyl-dioxirane in acetone, DCM/acetone (1:1),
rt, 40%; (e) Fe, NH4Cl, MeOH/H2O, reflux, quant.
access compounds 7–15, by reaction with suitable amines,
Pd2(dba)3 and 2-dicyclohexylphosphino-20-(N,N-dimethylamino)-
biphenyl. Removal of the tert-butoxycarbonyl protecting group
from 13 to 15 under acid conditions afforded compounds 16–18
while, oxidation at the piperazine residue of 4 to yield compound
19 and 20 was performed with freshly prepared 3,3-dimethyl-
dioxirane solution. Finally, compound 6 was prepared by reduction
the nitro derivative 5 with iron and ammonium chloride.
Modifications at the 1-position of the pyrazole ring was per-
formed reacting the advanced intermediate 23 with alcohols under
Mitsunobu conditions using polimer-bound Ph3P or, alternatively
with alkyl halogens under basic conditions (Scheme 2).15 The alkyl-
ation reaction proceeds in both cases regioselectively at the posi-
tion 1 of the pyrazolo ring giving compounds 24–28. Compound
23 was obtained from deprotection with hydrochloric acid of the
trityl protected derivative 22 which was in turn prepared subject-
ing the iodo-1-trityl amide 2113 to Buchwald–Hartwig coupling
conditions with 5-(4-methyl-piperazin-1-yl)-2-trifluoromethoxy-
phenylamine, Pd(OAc)2 and ( )-BINAP. Conversion of the
chloro-ethyl derivative 28 to vinyl derivative 29 was performed
in good yield with diaza(1,3)bicyclo[5.4.0]undecene (DBU).
Table 1 summarizes the structure–activity relationship (SAR) of
derivatives modified at the 50-position of the hit compound 1. Intro-
duction at this position of simple heterocyclyl residues (4, 7, 12 and
17) resulted crucial to access good PLK1 activity, achieve selectivity
against PLK2 and PLK3 and, improve selectivity vs CDK2/A with
respect to the hit compound 1. Among them, compounds 4 and 17
resulted to show the best compromise in terms of PLK1 activity,
selectivity against PLK2, PLK3 and CDK2/A, cellular activity and
acceptable solubility. Introduction of heterocyclyl-amino moieties
(9–11 and 16) from one side contributed to increase compounds’
solubility, from the other side determined a loss of PLK1 activity
ranging from 38-fold (16) to more than 400-fold (10), a restored
cross reactivity against PLK2, PLK3 and CDK2/A and a decreased
cellular proliferation potency that in the best case (10) is 36-fold
reduced with respect to the best inhibitor 17. The worse biochemical
profile shown by these compounds in comparison with analogs 4, 7,
12 and 17 bearing a piperazine or homopiperazine residue at the
position 50 of the aniline moiety is in agreement with the key role
played by one of the piperazine nitrogens to pick-up a polar
interaction with Glu 140 in the solvent accessible region of PLK1.12
In case of compounds 9–11 and 16 the increased side chain freedom
determined by the introduction of heterocyclyl-amino moieties im-
pairs this interaction thus determining a decreased activity and
selectivity with respect to piperazine analogs 4, 12 and 17. This
hypothesis is further supported by the decreased PLK1 activity of 8
and 18 wherethe basiccenter of the50 residue was moved away with
respect to the one of the piperazine moiety of compounds 4, 12 and
17. The same behavior was found with compound 6 lacking of the
piperazino residue or, with compounds 13, 19, and 20 where the
piperazine residue is unable to establish polar interaction with the
acid residue Glu 140 and wherein the PLK1 activityis decreased from
20-fold (6) to around 1000-fold (20) with respect to compound 4.
To verify if the methyl substituent at the pyrazolo ring of the
compound 4 was the optimal group, a reduced number of deriva-
tives modified at this position were tested (Table 2). Replacement
of the methyl with trityl group (22) or 4-methoxy-benzyl moiety
(24) gave a complete loss of activity. Compounds wherein the
methyl group was removed (23) or replaced with small residues
(25–29) exhibited a good antiproliferative activity (A2780 IC50
<100 nM), a maintained PLK1 activity (IC50 <20 nM) and for some
of them (25, 26 and 28) even an improved selectivity against
PLK2 and PLK3 with respect to the compound 4. These data
confirmed the previous finding that small groups at the position
1 of the 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline template are
N
N
O
O
a
CF
3 HN
O
N
N
I
N
NH2
N
N
NH2
N
N
Tr
Tr
21
22
N
b
N
N
O
O
c or d
CF
3 HN
O
CF
3 HN
O
N
N
N
N
NH2
NH2
N
N
H
N
N
R2
23
N
N
24-27
28
e
29
Scheme 2. Reagents and conditions: (a) Pd(OAc)2, ( )-BINAP, K2CO3, amine, DMF,
80 °C, 30%; (b) TFA, DCM, rt, 71%; (c) DTBAD, Ph3P polymer-bound, alcohol R2-OH,
THF; (d); Cs2CO3, R2-Cl, DMF, rt; (e) DBU, 80 °C.

I. Beria et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6489–6494
6491
Table 1
SAR of 50-position of the aniline residue
N
O
NH2
CF3 HN
O
N
N
N
R1
R1
H
PLK1 IC50
0.117
(
lM)
PLK2 IC50
(l
M)
PLK3 IC50
0.032
(lM)
CDK2/A IC50
1.762
(l
M)
A2780 IC50
6.412
(lM)
Solubility pH 7 (
lM)
a
a
a
a
a
Compound
1
0.206
<1
4
6
N
N
0.003
0.060
3.519
1.282
1.439
0.154
>10
0.021
1.075
72
31
NH2
1.180
7
8
0.025
0.033
>10
>10
>10
>10
>10
>10
0.941
0.583
95
N
N
N
133
N
H
N
9
0.200
0.964
3.415
>10
3.451
>10
1.020
3.019
1.092
1.754
>225
147
N
N
H
N
10
H
N
11
12
0.123
0.008
0.429
3.387
0.485
1.215
0.969
>10
2.556
0.034
157
33
N
N
N
N
N
O
O
13
0.859
0.768
0.262
1.133
1.293
nd
NH
16
17
0.077
0.002
0.762
>10
0.239
1.433
0.414
>10
2.217
0.03
171
114
N
H
NH
N
N
N
NH
18
N
0.035
0.190
0.083
0.622
0.538
85
O
N+
N
19
20
0.127
2.682
>10
>10
2.410
>10
>10
>10
0.782
>10
>225
48
O
N+
N+
O
O
a
Values are means of three experiments.
tolerated by PLK1.12 A selection of the most promising compounds
(4, 17, 25 and 29) was then profiled against a larger kinase panel
(Table 3). All compounds showed good selectivity in a panel of
more than 40 kinases, with a slightly more favorable profile for 4
in comparison with 17, 25 and 29.16 Selected compounds 4, 17,
25 and 29 were further evaluated on the basis of solubility prefor-
mulation test and in vitro ADME properties (Table 4).17,18 Among
them compound 17 showed the worse profile with the lower
solubility and the lower permeability in the PAMPA assay. Com-
pounds 4 and 29 are both characterized by good solubility and
by moderate metabolic stability in human liver microsomes with
respect to 25. In addition compound 4 compared to 29 exhibited
a higher permeability value indicating a possible more favorable
oral bioavailability.
lowing intravenous (iv) and oral (po) dosing (Table 5).19 Compound
4 was considered to have the best pharmacokinetic balance with
relatively high clearance (4.21 L/h/kg), a volume of distribution
about five times the mouse total body water (3.39 L/kg), indicating
a good tissue distribution, an half-life of 0.92 h and an AUC of
4.97 lM h. After po dosing, compound 4 showed a terminal half-
life compatible to that found after iv administration and an accept-
able oral bioavailability (F = 21%).
On the basis of these favorable pharmacokinetic data, the
acceptable solubility, the high selectivity and good antiprolifera-
tive activity, compound 4 was profiled against additional cancer
cell lines in a 72 h proliferation assay (Table 6). The compound re-
sulted active in cells derived from solid tumors as well as hematol-
ological malignancies showing a cytotoxicity ranging from 2 nM
(KU812 chronic myeloid leukemia) to 195 nM (NCI-H1993 NSCLC).
Interestingly compound 4, when tested in A2780 ovary carcinoma
In view of the ADME profile results, in vivo pharmacokinetic
properties of 4, 25 and 29 were evaluated in CD1 nu/nu mice fol-

6492
I. Beria et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6489–6494
Table 2
Modifications at the 1 position of the pyrazole ring
N
O
NH2
CF3 HN
O
N
N
N
N
R2
N
R2
PLK1 IC50
(lM)
PLK2 IC50
(
lM)
PLK3 IC50
(lM)
CDK2/A IC50
(l
M)
A2780 IC50
(lM)
Solubility pH 7 (lM)
a
a
a
a
a
Compound
4
22
23
24
25
26
27
28
29
Methyl
Trityl
H
4-Methoxy-benzyl
2-Fluoro-ethyl
Ethyl
2-Methoxy-ethyl
2-Chloro-ethyl
Vinyl
0.003
>10
0.004
2.65
0.004
0.006
0.015
0.007
0.006
3.519
nd
>10
nd
>10
>10
>10
>10
0.335
1.439
nd
>10
nd
>10
>10
>10
>10
0.182
>10
>10
>10
>10
>10
>10
>10
2.01
>10
0.021
2.105
0.093
3.624
0.014
0.011
0.075
0.031
0.008
72
<1
12
<1
18
21
136
9
8
a
Values are means of three experiments.
Table 3
Table 6
Antiproliferative activity of compound 4
Expanded kinase selectivity panel
a
Kinase
IC50
(l
M)
25
Cell line
Tumor type
IC50 (l
M)a
4
17
29
HT-29
LoVo
Colon
Colon
Colon
0.008
0.007
0.041
0.052
0.126
0.038
0.051
0.021
0.005
0.195
0.016
0.002
0.014
PLK1
CK2
FLT3
NEK6
PDGFR
ALK
Others 37 kinases
0.003
0.002
0.059
0.194
0.595
>10
0.004
0.134
0.216
>10
>10
>10
0.006
0.069
0.226
0.336
1.461
2.375
>10
HCT-116
MIA-PaCa-2
PANC-1
A2780 PTX22
A2780 Adria
A2780 CIS
A2780-E6
NCI-H1993
HL-60
0.140
1.517
1.664
>10
>10
>10
Pancreas
Pancreas
Ovarian
Ovarian
Ovarian
Ovarian
NSCLC
Acute myeloid leukemia
Chronic myeloid leukemia
Multiple myeloma
>10
>10
>10
a
Values are means of three experiments.
KU812
KMS-11
Table 4
Solubility preformulation and in vitro ADME properties of selected compounds
a
IC50 values are reported as the mean of 2–3 experiments with a coefficient of
variation below 35%.
Compd
In viro ADME properties
Solubility 10% Tween
80, (mg/mL)
PAMPAa
Clint (mL/min/kg)
(Papp 10ꢀ6 cm/s)
HLMb (1
lM)
(A2780) or, defective for p53 (A2780 E6) thus indicating that the
mechanism of action of the compound is not dependent from
p53 status.
4
17
25
29
>3.2
1.1
2.1
50.0
7.22
50.0
19.6
25.7
6..9
56.1
28.8
Mechanism of action of compound 4 was first evaluated by
array scan analysis (Cellomics) measuring different cell cycle
markers in U2OS cells. Cells were treated for 24 h with different
doses of compound and extent of mitotic block was established
measuring the accumulation of cells positive for pSer10 Histone
H3 and nuclear Cyclin B1 (Fig. 2, panels A and B). The specific
PLK1 mechanism was confirmed evaluating the decrease of pSer46
TCTP signal in U2OS cells synchronized in mitosis by nocodazole
and then, treated for 1 h with different doses of 4 (Fig. 2, panel
C). Values of 260 and 320 nM, respectively, for pSer10 Histone
H3 (20% positive cells) and Cyclin B1 (20% positive cells) (Fig. 2,
>3.2
a
Parallel artificial membrane permeability.
Human liver microsomes.
b
cell line, showed similar potency on parent cell line and in cells
resistant to classical cytotoxic agents like doxorubicin (A2780
Adria), paclitaxel (A2780 PTX22) and cisplatin (A2780 CIS). In addi-
tion compound 4 was equally active on p53 proficient cells
Table 5
In vivo pharmacokinetic parameters standard deviation of selected compounds in CD1 nu/nu micea
Compound
PK data (iv), doseb: 10 mg/kg
PK data (po), doseb: 10 mg/kg
AUC1
(lM h)
CL (L/h/kg)
Vss (L/kg)
t1/2 (h)
Cmax
(
lM)
AUC1
(
lM h)
t1/2 (h)
Fc (%)
4
25
29
4.97 0.71
10.20 5.97
5.37 0.16
4.21 0.45
2.42 1.07
3.44 0.15
3.39 0.25
1.53 1.04
2.58 0.06
0.92 0.12
0.74 0.05
0.89 0.02
0.29 0.04
0.26 0.08
0.61 0.06
1.04 0.10
0.192 0.06
2.15 0.18
1.44 0.29
nd
2.58 0.06
21
2
29
nd = not determined.
a
n = 3 animals per study.
Dosed as HCl in situ salt/glucosate.
Bioavailability.
b
c

I. Beria et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6489–6494
6493
Asynchronous
Synchronous
Compound 4
40
30
20
10
0
50
40
30
20
10
0
Panel A
Panel B
Panel C
120
100
80
60
40
20
0
10-5 10-4 10-3 10-2 10-1 100
101
10-4
10-3
10-2
Dose log (µM)
10-1
100
10-5 10-4 10-3 10-2 10-1 100
101
Dose log (µM)
Dose log (µM)
Figure 2. Modulation of phosphoSer10 Histone H3 (panel A), cyclin B1 (panel B) and phosphoSer46 TCTP (panel C) in U2OS cells treated with 4.
panels A and B) and, IC50 of 95 nM for pSer46 TCTP signal inhibition
were determined (Fig. 2, panel C).
Control
Compound 4
2.0
1.0
0.0
A larger number of cell cycle and apoptosis markers were
evaluated in A2780 and in HCT116 cell lines treated for 24 h with
different doses of 4. Clear modulation of markers was already
appreciable at the dose of 50 nM with increase in PLK1 and cyclin
B1 total levels, decrease in the phosphorylation of Tyrosine 15 on
CDK1, increase in the phosphorylation of Histone H3 on Serine
10 and nucleophosmin on Threonine 199 indicating cells accumu-
lating in the G2/M phase of the cell cycle. Apoptosis induction was
observed in both cell lines as indicated by PARP cleavage signal
(Fig. 3).
0
5
10
In vivo efficacy of compound 4 was examined in CD1 nu/nu mice
xenografted with human ovarian A2780 or with HCT116 colon ade-
nocarcinoma cells. The compound 4, when administered intrave-
nously at 30 mg/kg, bid on day 1, 2, 3 on A2780 xenograft mice
model, showed a good tumor growth inhibition (TGImax = 74%, day
12) with severe but reversible body weight loss (BWLmax 19%, day
12) indicating that this dose is potentially close to the maximal tol-
erated dose (Fig. 4). Even better efficacy was shown in the HCT116
xenograft model where, after iv treatment at 30 mg/kg, bid, given
at day 1, 2 in a weekly scheduling ꢁ 3 cycles, the tumor growth inhi-
bition reached 81% at day 32 with good tolerability (BWLmax = 14%)
(Fig. 5). Antitumor efficacy of compound 4 was also evaluated orally
in HCT116 xenograft model at 60 mg/kg, daily, given at day 1, 2,
3 ꢁ 2 cycles. In this case the compound exhibited moderate activity
(TGImax = 70%) with BWLmax = 13%.
Days
Figure 4. In vivo antitumor activity of 4 in A2780 xenograft model.
Control
Compound 4
2.0
1.0
0.0
0
5
10
15
20
25
30
Days
Figure 5. In vivo antitumor activity of 4 in HCT116 xenograft model.
C
C
N
N
In summary, we have reported the identification of a new PLK1
specific inhibitor with high potency on the target and demon-
strated PLK1 mechanism of action in cells. Compound 4 showed
good ADME properties suitable for moving it to in vivo efficacy
experiments where it showed good activity in two different xeno-
graft animal models with TGImax ranging from 74% to 81% after iv
administration. Furthermore, compound 4 exhibited a moderate
activity also upon oral administration.
PLK1
CyclinB1
H3-S10P
CDK1-Tot
CDK1-Y15P
Acknowledgments
NPM-P Thr199
PARP
We thank Franco Ciprandi and Viviana Desperati for C18 pre-
parative HPLC purifications, the group of Assay Development and
Biochemical Screening for the biochemical assay on kinase panel,
Dario Ballinari and the group of Cell Screening for cell proliferation
assay, Enrico Pesenti and the Experimental Therapy group for the
in vivo experiments, Daniela Borghi for NMR data, and Daniele
Donati for discussion and support.
A0872
H611TC
Figure 3. Western blot analysis of HCT116 and A2780 cells after 24 h treatment
with three different doses of 4.

6494
I. Beria et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6489–6494
10. Kauselmann, G.; Weiler, M.; Wulff, P.; Jessberger, S.; Konietzko, U.; Scafidi, J.;
Supplementary data
Staubli, U.; Bereiter-Hahn, J.; Strebhardt, K.; Kuhl, D. EMBO J. 1999, 18, 5528.
11. Schoffski, P. Oncologist 2009, 14, 559.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.09.060.
12. Beria, I.; Ballinari, D.; Bertrand, J. A.; Borghi, D.; Bossi, R. T.; Brasca, M. G.;
Cappella, P.; Caruso, M.; Ceccarelli, W.; Ciavolella, 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. J. Med. Chem.
2010, 53, 3532.
References and notes
13. Caruso, M.; Beria, I.; Brasca, M. G.; Ferguson, R.; Posteri, H.; Valsasina, B. Patent
WO 2008074788, 2008.
1. Day, W. Oncogene 2005, 24, 215.
14. Wolfe, J. P.; Tomori, H.; Sadighi, J. P.; Yin, J.; Buchwald, S. L. J. Org. Chem. 2000,
65, 1158.
2. Garland, L. L.; Taylor, C.; Pilkington, D. L.; Cohen, J. L.; Von Hoff, D. D. Clin.
Cancer Res. 2006, 12, 5182.
15. (a) Hughes, D. L. Org. Prep. 2006, 28, 127; (b) Di Fabio, R.; Giovannini, R.;
Bertani, B.; Borriello, M.; Bozzoli, A.; Donati, D.; Falchi, A.; Ghirlanda, D.; Leslie,
C. P.; Pecunioso, A.; Rumboldt, G.; Spada, S. Bioorg. Med. Chem. Lett. 2006, 16,
1749.
16. The panel includes c-ABL, AKT1, Aur-A, Aur-B, CDC7, CDK1/B, CDK2/E, CDK4/
D1, CDK5/P25, CHK1, EGFR1, ERK2, FGFR1, GSK3b IGF1R, IKK2, IR, JAK2, KIT,
3. Santamaria, A.; Neef, R.; Eberspaecher, U.; Eis, K.; Husemann, M.; Mumberg, D.;
Prechtl, S.; Schulze, V.; Siemeister, G.; Wortmann, L.; Barr, F. A.; Nigg, E. A. Mol.
Biol. Cell. 2007, 18, 4024.
4. Barr, F. A.; Sillje, H. H.; Nigg, E. A. Nat. Rev. Mol. Cell Biol. 2004, 5, 429.
5. van Vugt, M. A.; van de Weerdt, B. C.; Vader, G.; Janssen, H.; Calafat, J.;
Klompmaker, R.; Wolthuis, R. M.; Medema, R. H. J. Biol. Chem. 2004, 279, 36841.
6. Weichert, W.; Schmidt, M.; Gekeler, V.; Denkert, C.; Stephan, C.; Jung, K.;
Loening, S.; Dietel, M.; Kristiansen, G. Prostate 2004, 60, 240.
7. Weichert, W.; Kristiansen, G.; Winzer, K. J.; Schmidt, M.; Gekeler, V.; Noske, A.;
Muller, B. M.; Niesporek, S.; Dietel, M.; Denkert, C. Virchows Arch. 2005, 446,
442.
8. Mamely, I.; van Vugt, M. A.; Smits, V. A.; Semple, J. I.; Lemmens, B.; Perrakis, A.;
Medema, R. H.; Freire, R. Curr. Biol. 2006, 16, 1950.
9. Bassermann, F.; Frescas, D.; Guardavaccaro, D.; Busino, L.; Peschiamoli, A.;
Pagano, M. Cell 2008, 134, 256.
LCK, MAP-KAPK2, MET, MPS1, STK2, NIM, P38
a, P38b, PAK4, PDK1, PKAa, PKCb,
RET, TAO3, TRKA, VEGFR2, VEGFR3 and, ZAP70.
17. Human liver microsomes was carried out as previously reported Ref. 12.
18. High-throughput solubility was carried out as previously reported Ref. 12.
19. The pharmacokinetic profiles of the compounds were investigated in overnight
fasted male CD1 nu/nu mice following a single dose given intravenously (iv) or
orally (po). Blood samples of each mouse were taken from saphenous vein at
different time and after centrifugation plasma samples were analyzed by
LC/MS/MS technique. For a more complete description see Ref. 12.