Optimization of 6,6-dimethyl pyrrolo[3,4-c]pyrazoles: Identification of PHA-793887, a potent CDK inhibitor suitable for intravenous dosing

Article abstract of DOI:10.1016/j.bmc.2010.01.042

We have recently reported CDK inhibitors based on the 6-substituted pyrrolo[3,4-c]pyrazole core structure. Improvement of inhibitory potency against multiple CDKs, antiproliferative activity against cancer cell lines and optimization of the physico-chemical properties led to the identification of highly potent compounds. Compound 31 (PHA-793887) showed good efficacy in the human ovarian A2780, colon HCT-116 and pancreatic BX-PC3 carcinoma xenograft models and was well tolerated upon daily treatments by iv administration. It was identified as a drug candidate for clinical evaluation in patients with solid tumors.

Full text of DOI:10.1016/j.bmc.2010.01.042

Bioorganic & Medicinal Chemistry 18 (2010) 1844–1853
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Optimization of 6,6-dimethyl pyrrolo[3,4-c]pyrazoles: Identification
of PHA-793887, a potent CDK inhibitor suitable for intravenous dosing
a
a
b
a
a
Maria Gabriella Brasca a, , Clara Albanese , Rachele Alzani , Raffaella Amici , Nilla Avanzi , Dario Ballinari ,
James Bischoff c, Daniela Borghi a, Elena Casale a, Valter Croci a, Francesco Fiorentini d, Antonella Isacchi a,
Ciro Mercurio b, Marcella Nesi a, Paolo Orsini a, Wilma Pastori a, Enrico Pesenti a, Paolo Pevarello e,
Patrick Roussel f, Mario Varasi b, Daniele Volpi a, Anna Vulpetti g, Marina Ciomei a
*
a Nerviano Medical Sciences Srl, Business Unit Oncology, Viale Pasteur 10, 20014 Nerviano (MI), Italy
b Congenia S.r.l., Milano, Italy
c Centro Nacional de Investigaciones Oncologicas (CNIO), Spain
d Accelera, viale Pasteur 10, 20014 Nerviano (MI), Italy
e Newron, Milan, Italy
f Basilea Pharmaceutica Ltd, Switzerland
g Novartis Pharma AG, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
We have recently reported CDK inhibitors based on the 6-substituted pyrrolo[3,4-c]pyrazole core struc-
ture. Improvement of inhibitory potency against multiple CDKs, antiproliferative activity against cancer
cell lines and optimization of the physico-chemical properties led to the identification of highly potent
compounds. Compound 31 (PHA-793887) showed good efficacy in the human ovarian A2780, colon
HCT-116 and pancreatic BX-PC3 carcinoma xenograft models and was well tolerated upon daily treat-
ments by iv administration. It was identified as a drug candidate for clinical evaluation in patients with
solid tumors.
Received 7 September 2009
Revised 15 January 2010
Accepted 18 January 2010
Available online 25 January 2010
Keywords:
Kinase
Cyclin dependent kinases
CDK
Ó 2010 Elsevier Ltd. All rights reserved.
Kinase inhibitor
Tumor cell proliferation inhibition
Cell cycle
Anti-cancer
1. Introduction
results not only in proliferative advantages, but also in increased
susceptibility to genetic alterations. Despite recent genetic studies
Cyclin dependent kinases (CDKs) are a family of serine/threo-
nine kinases that, in concert with cyclins (positive regulators)
and natural inhibitors (CDKI), control the cell cycle progression.1
Deregulation of the activity of CDKs, mainly due to alterations of
expression and/or genetic mutations of cyclins, CDKs, CDKIs and
other components of the retinoblastoma protein (pRB) pathway,
has been reported in more than 90% of human neoplasms. For
example, cyclins E and A have been found over expressed in 50%
of breast and lung cancer whereas decreased levels of the inhibitor
p27 predict for a poor prognosis in breast, prostate, colon, gastric,
lung and esophageal cancer.2 The high frequency of alterations
found in the core members of this pathway in human tumors led
to the suggestion that its deregulation, leading to unscheduled pro-
liferation, is an obligatory event for cancer development, because it
in mice indicating that normal cells are not dependent on inter-
phase CDKs (CDK4 and 2) for their growth, certain tumor cells,
depending on their origin and their pathogenic spectrum of muta-
tion, may be sensitive to the inhibition of CDKs.1 Observations of
functional redundancy within the CDK family have led to the belief
that highly selective inhibitors of individual CDKs may not be ther-
apeutically effective. The best combination of CDK activities that
will lead to the greatest efficacy with minimal toxicity is still under
debate. Several small molecules which inhibit the function of mul-
tiple CDKs such as Roscovitine/CYC-202,3 BMS-387032/SNS-0324
(both mainly CDK2 but also CDK7 and 9 inhibitors), PD03329915
(a selective CDK4 and 6 inhibitor) and recently R5476 (CDK1, 2, 4
inhibitor), SCH-7279657 (CDK1, 2, 5, 9 inhibitor), AT75198 (CDK1,
2, 4, 5 inhibitor) and AZD55979 (CDK1, 2 and 9 inhibitor) are in
clinical development, confirming that agents which inhibit the
function of multiple CDKs may be clinically more successful than
very selective inhibitors of CDKs.10
* Corresponding author. Tel.: +39 0331 581533; fax: +39 0331 581347.
E-mail address: gabriella.brasca@nervianoms.com (M.G. Brasca).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.042

M. G. Brasca et al. / Bioorg. Med. Chem. 18 (2010) 1844–1853
1845
o[3,4-c]pyrazole B, prepared as previously described,11 was treated
either with an acyl chloride in the presence of a tertiary amine or
with an isocyanate to give, respectively, amides or ureas C. Re-
moval of the N-Boc protection and treatment of the secondary
amines D with either an acyl chloride or a carboxylic acid in the
presence of a coupling agent or triphosgene followed by addition
of an amine generated compounds of types E. Final deprotection
of the ethyl carbamate group with triethylamine in methanol gave
final compounds 1–36.
H
N
H
N
6
N
N
O
N
O
3
N
5
HN
HN
R2
O
O
R1
F
A
1
F
Figure 1. From 6,6-dimethyl pyrrolo[3,4-c]pyrazole core structure A to compound 1.
3. Results and discussion
As previously reported,11 optimization of 6,6-dimethyl pyrrol-
o[3,4-c]pyrazole core structure led to the identification of the
tert-butyl moiety as potentially best R2 group, although the most
promising compounds (1 and 2) were characterized by poor solu-
bility in neutral buffer.
We have recently reported CDK inhibitors based on the 6-
substituted pyrrolo[3,4-c]pyrazole core structure A11 (Fig. 1). This
versatile adenino mimetic scaffold has been exploited for the con-
ventional and polymer-assisted solution phase synthesis of a series
of CDK2/cyclin A inhibitors. In this study, we identified compound
1 displaying nanomolar activity against CDK2/cyclin A and other
CDKs, and selectivity against a panel of serine-threonine and tyro-
sine kinases (see Section 5, kinase assays). Within this class, selec-
tivity was attained by the introduction of dimethyl group on
position 6 of the bicyclic pyrrolo-pyrazole skeleton, exploiting
the different size and shape of the buried region of CDK2 (defined
by Ala31, Val64, Phe80, and Ala144) with respect to other ki-
nases.11–13
Furthermore, the high binding affinity of compound 1 was also
accompanied by inhibition of CDK2-mediated cell proliferation
in vitro and by in vivo antitumor activity. These results prompted
us to start a medicinal chemistry program with the aim to identify
an analogue with a better profile with respect to potency and phys-
ico-chemical properties. Particular attention was focused on the
solubility of the new compounds. In fact, notwithstanding the
favorable pharmacodynamic profile of compound 1, its low solubil-
ity in 5% dextrose solution (3.4 mg/mL) prevented its use by intra-
venous administration. In the present communication, we
illustrate the synthesis and the biological evaluation of a set of
compounds related to compound 1.
As previously reported,11 optimization of 6,6-dimethyl pyrrol-
o[3,4-c]pyrazole core structure led to the identification of the
tert-butyl moiety as potentially the best R2 group (Fig. 1), although
the most promising compounds (1 and 2) were characterized by
poor solubility in neutral buffer.
Therefore, in order to improve this property, we explored a
series of amides at position 3 bearing an additional basic center
(Table 1). 3-Pyridine and 4-pyridine carboxamide (3 and 4)
maintained CDK2 inhibitory activity and potency in the cellular
antiproliferative assay, in particular compound 4, without a clear
improvement in the solubility in neutral buffer that instead was
achieved moving from aryl or heteroaryl moieties to heterocyclic
or linear alkyl ones (5–7). Unfortunately, a small decrease in inhib-
itory activity against CDK2/cyclin A was also observed, except for
compound 7, which showed an interesting enzymatic activity. On
the other hand, no significant improvement in A2780 cell prolifer-
ation assays was observed.
These results prompted us to introduce a basic moiety at posi-
tion 5 (pointing towards the relatively spacious phosphate binding
region) while keeping unvaried 4-fluoro benzamido group at posi-
tion 3 (Table 2).
2. Chemistry
An initial set of piperidino amides 8–11 was synthesized and
compared to compound 1. No substitution (8) or insertion of small
substituents such as methyl, ethyl, and cyclopropyl (9–11) pro-
vided compounds with good activity against CDK2/cyclin A. Inter-
esting potency in the A2780 cell proliferation assay as well as good
The general synthetic approach that allowed the generation in a
parallel fashion of a wide array of derivatives of general formula A
is represented in Scheme 1. Doubly protected 3-amino-pyrrol-
N
N
N
a
b
EtO2C
N
N
Boc
EtO2C
N
N
Boc
EtO2C
N
NH HCl
H2N
HN
R1
HN
O
O
R1
B
C
D
c
H
O
N
O
N
d
N
HN
R1
N
EtO2C
N
N
R2
R2
HN
R1
O
1-36
O
E
Scheme 1. Reagents and conditions: (a) R1COCl, DIPEA, THF or R0NCO, THF; (b) HCl 4 N in dioxane; (c) R2COCl, DIPEA, THF or R2COOH, TBTU, DIPEA, DCM or triphosgene then
NHR0R00; (d) TEA, MeOH.

1846
M. G. Brasca et al. / Bioorg. Med. Chem. 18 (2010) 1844–1853
Table 1
Table 3
R1 modification
R1, R3 modifications
H
N
H
N
N
N
O
O
N
N
N
HN
R1
HN
R1
O
O
Compd R1
CDK2/A
IC50
A2780
IC50
Solubility
lM, pH 7
R3
,
l
M
,
l
M
Compd R1
R3
CDK2/A A2780
Solubility
M, pH 7
1
2
3
4
5
6
7
3,5-Difluorophenyl
4-Fluorophenyl
3-Pyridinyl
4-Pyridinyl
4-Piperidinyl
0.060
0.030
0.050
0.049
0.169
0.214
0.31
0.27
0.69
0.11
0.65
0.35
0.58
186
235
207
205
>225
>225
>225
IC50 IC50
,
l
M
,
l
l
M
17
18
19
20
21
22
Phenyl
3-Fluorophenyl
3,4-Difluorophenyl
3,5-Difluorophenyl
4-Chlorophenyl
4-
Me
Me
Me
Me
Me
Me
0.023
0.014
0.014
0.016
0.015
0.026
0.16
0.20
0.10
0.13
0.12
0.06
>225
>225
>225
>225
>225
191
1-Methyl-piperidinyl
Dimethylaminomethyl 0.077
Trifluoromethylphenyl
2-Naphthyl
1-Naphthyl
4-Pyridinyl
2-Thienyl
3-Thienyl
2-Furyl
Cyclopropyl
Cyclobutyl
i-Bu
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
0.027
0.016
0.025
0.010
0.005
0.033
0.015
0.009
0.008
0.014
0.27
0.06
0.50
0.03
0.14
>5
1.42
0.14
0.09
0.41
0.32
2.68
0.65
0.35
219
200
>225
155
Table 2
R3 modification
H
N
N
>225
>225
>225
>225
>225
>225
>225
>225
>225
>225
O
N
HN
X
O
i-Bu
i-Bu
N
R3
Cyclopropyl 0.024
Me
Me
Benzylamino
Propylamino
3-Fluorophenylamino Me
0.021
0.011
0.010
F
Compd R3
X
CDK2/A IC50
M
,
A2780 IC50
lM
,
Solubility
pH 7
lM,
l
8
9
H
CH 0.030
CH 0.023
CH 0.030
CH 0.068
CH 4.80
0.65
0.12
0.29
0.38
>10
0.18
>5
204
against CDK2/cyclin A, in particular the naphthyl-1-carboxamide
24, but without an improvement of solubility in neutral buffer.
Among the heteroaryl carboxamide derivatives (25–28), the 2-
thienyl (26) was potent against CDK2/cyclin A and in antiprolifer-
ative assay, but less soluble in neutral buffer. The cyclopropyl and
the cyclobutyl groups also gave potent CDK2/cyclin A inhibitors
(29–30). Interestingly, the branched iso-butyl group gave the po-
tent enzyme inhibitor 31 with an improvement of the antiprolifer-
ative activity in A2780 human ovarian carcinoma cells as well as
solubility in neutral buffer, with respect to the parent compound
1. Introduction of ethyl or cyclopropyl instead of methyl on the
nitrogen of piperidine group (32–33) was compatible with the
maintenance of the CDK2 inhibitory activity but potency in the cel-
lular antiproliferative assay was diminished, with respect to ana-
logue 1. A small set of ureas (34–36) was tested against CDK2/
cyclin A, showing interesting enzymatic activity, but a drop in
the antiproliferative activity, in particular for benzylurea 34.
The crystal structure of compound 31, in complex with CDK2/
cyclin A has been completely elucidated (Fig. 2).14 The pyrrolo-pyr-
azole system occupies the adenine region of the ATP pocket, while
the iso-butyl group points towards the solvent accessible region.
This compound binds into the ATP pocket of CDK2/cyclin A by
three hydrogen bonds with the protein backbone of the hinge re-
gion: the NH of the pyrazole binds to the carbonyl of Glu81, the
nitrogen atom of the pyrazole core and the adjacent NH interact
with the NH and the carbonyl oxygen of Leu83. The dimethyl group
on position 6 occupies the buried region formed in CDK2 by Ala31,
Val64, Phe80 and Ala144. In addition, the carbonyl group is within
hydrogen bond distance of the side chain of Lys33 and the nitrogen
atom of the piperidine binds to the side chain of Asp145. This addi-
tional hydrogen bond, not present in compound 1, contributes to
the improvement of activity against CDK2/cyclin A observed for
compound 31 and, more in general, for compounds bearing an N-
alkyl piperidin-4-yl moiety as R2 group (Fig. 1).
Methyl
Ethyl
Cyclopropyl
Acetyl
Methyl
Hydroxyethyl
Acetyl
Phenyl
>225
>225
>225
204
>225
>225
>225
55
10
11
12
13
14
15
16
N
N
N
N
0.046
0.060
0.54
3.5
>10
nt
solubility in neutral buffer was observed, except for compound 8
which showed lower potency on cells with respect to compound 1.
Acylation of the piperidino nitrogen (12) led to a drop in po-
tency against CDK2/cyclin A and in cells. Moreover, the role of
substituted piperazino ureas was explored. Compound 13 as well
as hydroxyethyl derivative 14 displayed interesting enzymatic
activity, whereas a bulkier residue such as phenyl (16) caused a
marked decrease in activity against CDK2/cyclin A. Acylation of
the piperazino nitrogen (15) led to a drop in potency in biological
assay, even if less dramatic with respect to the corresponding acet-
yl piperidino 12. No improvement in terms of cellular antiprolifer-
ative activity was achieved moving from methyl piperazino 13 to
ureas 14–16.
The interesting results obtained with N-substituted piperidino
moiety (9–11) prompted us to expand this subclass. Thus, we pre-
pared a set of 5-piperidino carboxamides to further explore the sol-
vent accessible region of the ATP binding site of CDK2/cyclin A
(Table 3).
A phenyl ring eventually bearing halogen substituents (17–22)
gave compounds with strong activity against CDK2/cyclin A and
in A2780 cell proliferation assays. In particular, the p-trifluoro-
methyl phenyl derivative 22 was more potent in the antiprolifera-
tive assay than the previous ones, but less soluble in neutral buffer.
Also naphthyl derivatives (23–24) showed interesting potency

M. G. Brasca et al. / Bioorg. Med. Chem. 18 (2010) 1844–1853
1847
Table 5
Effects on cell cycle progression and DNA synthesis of compounds 1 and 31 vs control
cells
Compd
G0/G1%
S%
G2/M%
BrdU incorporation%
1a
+22
ꢀ11
+25
ꢀ52
ꢀ48
ꢀ76
+26
+263
+62
ꢀ96
ꢀ100
ꢀ85
31a
31b
a
At 3
At 1
l
l
M.
M.
b
says compound 31 showed to be more potent than the parent com-
pound 1.
In addition, the effect on the phosphorylation status of a known
CDK substrate such as retinoblastoma protein (pRb) was analyzed
in cells treated with compound 31 at concentrations of 1 and 3 lM.
A clear reduction of the hyperphosphorylated form of pRb and an
accumulation of the hypophosphorylated form of pRb was ob-
served in the extracts of treated cells in comparison with the un-
treated cells, indicative of an effect on the activity of CDK (Fig. 3).
As far as preliminary in vitro and in vivo pharmacokinetic
parameters are concerned, compound 31 displayed better solubility
in vehicle (10 mg/mL in 5% dextrose solution as hydrochloride salt)
with respect to compound 1 (3.4 mg/mL in 5% dextrose solution),
stability to human cytochrome CYP4503A4 (91% remaining), and
low permeability in the Caco-2 cell permeability assay (Table 6),
which could explain the lack of oral bioavailability found in the
pharmacokinetic experiment performed on healthy nude mice
(F <1%). The plasma protein binding was relatively low. In vivo
pharmacokinetic results in healthy nude mice (10 mg/kg by iv
administration in 5% dextrose solution as hydrochloride salt) indi-
cated a volume of distribution higher than the total body water sug-
gesting tissue distribution, with a clearance in the range of 9% of the
hepatic blood flow. The half-life is approximately 4.1 h. Further-
more, compound 31 demonstrated potent antiproliferative activity
(Table 7) in several tumor cell lines, including sarcoma, melanoma,
ovarian, colon, breast and prostate carcinoma cells. These results
indicate a broad spectrum of activity of compound 31.
Figure 2. Crystal structure of compound 31 in complex with CDK2/cyclin A (PDB
code 2WPA).
Based on the biochemical assay, cellular potency and prelimin-
ary physico-chemical properties compound 31 was selected for
further assessment. Table 4 reports the selectivity profile of com-
pounds 1 and 31 on a panel of 44 serine–threonine and tyrosine
kinases.
Among the members of the CDK family that were assessed,
compound 31 turned out to be a potent inhibitor of CDK2/cyclin
A, CDK2/cyclin E, CDK5/p25 and CDK7/cyclin H at comparable lev-
els, but also, albeit less potent, an inhibitor of CDK1/cyclin B and
CDK4/cyclin D1 (ratio vs CDK2/cyclin A: 7.5 x). Among all the other
enzymes in the panel, only GSK3 was inhibited (ratio vs CDK2/cy-
clin A: 10 x) by compound 31, which generally demonstrated to be
more potent than the parent compound 1.
The effects of compound 31 on the cell cycle progression and
DNA synthesis, measured as BrdU incorporation (Table 5), were
analyzed using Flow cytometry and immunocytochemistry,
respectively, on A2780 ovarian carcinoma cells in exponential
growth in the presence or absence of compound, for 24 h, at the
C
treated
concentration of 3
ted cells showed a mixed effect at 1
l
M and 1
l
M. The cell cycle profile of the trea-
1
3
μM
lM: a decrease in the S phase
population and a subsequent increase of the G1 population, as ex-
pected for a CDK2/cyclin A inhibitor, and a slight accumulation of
pRb
G2/M phase population compared to the control cells. At 3 lM only
β -tubulin
a strong G2/M increase was observed, which can be ascribed to
inhibition of CDK1/cyclin B. The presence of cells with sub G1
DNA content (from 2.6% in control cells to 26.2% in cells treated
Figure 3. Compound 31 decreases the amount of pRB phosphorylation in treated
cells in comparison with untreated cells (b-tubulin as control cellular protein).
with compound 31 at 3 lM) clearly implied cell death and apopto-
sis. A clear reduction in DNA synthesis was observed, compared to
the control, as measured by BrdU incorporation. Also in these as-
Table 6
In vitro physico-chemical ADME properties and in vivo pharmacokinetic parameters
of compound 31
Table 4
In vitro physico-chemical ADME properties
Solubility 5% dextrose (mg/mL)
CYP4503A4 (% remaining)
PPB (%)
31
10
91
54
Low
31a
10 mg/kg
36.5
19.2
Selectivity profile of compounds 1 and 31 (IC50, lM)
Protein Kinase
1
31
CDK2/cyclin A
CDK2/cyclin E
CDK5/p25
0.060
0.090
0.14
Nd
0.45
Nd
Nd
3.63
>10
0.008
0.008
0.005
0.010
0.060
0.062
0.138
0.079
>10
CaCO2 cell permeability
In vivo pharmacokinetic parameters
IV administration
CDK7/cyclin H
CDK1/cyclin B
CDK4/cyclin D1
CDK9/cyclin T1
GSK3b
Cmax
(l
M)
AUC (
CL (mL/min/kg)
l
Mꢁh)
23.9
4.1
1360
t1/2 (h)
Vss (mL/Kg)
Other kinasesa
a
a
See Section 5.
Dosed in 5% dextrose as hydrochloride salt.

1848
M. G. Brasca et al. / Bioorg. Med. Chem. 18 (2010) 1844–1853
Table 7
5.0
a
Inhibition of cell proliferation by compound 31
Cell line
IC50, lM
4.0
3.0
2.0
1.0
0.0
A2780 human ovarian carcinoma
HCT-116 human colon carcinoma
COLO-205 human colon carcinoma
C-433 Ewing’s sarcoma
DU-145 human prostate carcinoma
A375 human melanoma
PC3 human prostate carcinoma
MCF7 human breast carcinoma
BX-PC3 human pancreatic carcinoma
0.088 0.038
0.163 0.057
0.188 0.096
0.285 0.073
0.303 0.142
0.396 0.100
0.601 0.324
1.287 0.546
3.444 0.827
8
9
10
11
12
13
14
15
16
17
18
days
On the basis of these data compound 31 was evaluated for its
in vivo antitumor activity in the human ovarian A2780 xenograft
mouse model. The doses of 10, 20 and 30 mg/kg were selected
and administered by iv route, once a day, for 10 consecutive days,
on the basis of the plasma levels reached in the preliminary in vivo
PK study. Compound 31 was dissolved in 5% dextrose solution and
caused a dose-dependent inhibition of the A2780 tumor growth up
to 76% at the dose of 30 mg/kg at the end of treatment (Fig. 4a).
With the same schedule, compound 31 was tested at the doses of
10 and 20 mg/kg also on a model of human colon carcinoma
(HCT-116) causing a dose-dependent inhibition of the tumor
growth up to 81% at the dose of 20 mg/kg at the end of treatment
(Fig. 4b) and on a model of human pancreatic carcinoma (BX-PC3)
with a 84% of tumor growth inhibition at the end of treatment with
20 mg/kg (Fig. 4c). Compound 31 was well tolerated upon daily
treatments by iv administration. In fact, in all experiments, body
weight reduction was always marginal (<10% vs control mice)
and no toxic effects were reported after gross autopsy.
2.0
1.5
1.0
0.5
0.0
b
8
10
12
14
16
18
20
22
24
26
28
days
1.5
1.0
0.5
0.0
c
In order to verify the mechanism of action of compound 31 also
in vivo, A2780 xenograft tumors from mice treated at the dose of
30 mg/kg iv for 5 days were analyzed for BrdU incorporation and
phosphorylation status of pRb by immunohistochemistry. Prolifer-
ation and CDK related markers were significantly reduced in trea-
ted tumors (p = 0.0022 and p = 0.005, respectively, for BrdU and
phosho-pRb) compared to vehicles (Fig. 5).
8
13
18
days
23
28
4. Conclusion
Starting from compound 1, which showed good potency as
CDK2/cyclin A inhibitor, a series of new analogues was synthesized
with the aim of improving CDK inhibitor activity and antiprolifer-
ative activity against A2780 human ovarian carcinoma cells.
Optimization of the physico-chemical properties led to the identi-
fication of compound 31 (PHA-793887), highly potent CDK inhibi-
tor suitable for intravenous dosing that exhibited inhibition of
tumor growth ranging from 76% to 84% in preclinical xenograft
tumor models, well tolerated upon repeated daily treatments.
The mechanism of action of compound 31 was demonstrated both
in vitro and in vivo. PHA-793887 was selected for clinical evalua-
tion as anticancer agent in patients with solid tumors. Further
studies will be reported in the future.
Figure 4. Compound 31 inhibits tumor growth of human ovarian A2780 (a), colon
HCT-116 (b) and pancreatic BX-PC3 (c) carcinoma xenograft models. It was iv
administered respectively at doses of 10 (square), 20 (triangle) and 30 (cross) mg/kg
for 10 consecutive days starting from day 8. Vehicle-treated curve (control) is
reported with open circle.
Column chromatography was conducted either under medium
pressure on silica (Merck Silica Gel 40–63 lm) or on prepacked sil-
ica gel cartridges (Biotage). Components were visualized by UV
light (k = 254 nm) and by iodine vapor. 1H NMR spectra were
recorded in DMSO-d6 at 28 °C on a Varian Inova 400 spectrometer
operating at 400.50 MHz and equipped with a 5 mm 1H{15N–31P}
z-axis-PFG indirect detection probe. DMSO-d6 residual solvent
signal was used as internal reference (d = 2.50 ppm for 1H). Chem-
ical shifts (d) are reported in parts per million (ppm) and coupling
constant (J) in Hz. The following abbreviations are used for multi-
plicities: s = singlet; br s = broad singlet; d = doublet; t = triplet;
m = multiplet; dd = doublet of doublets. Electrospray (ESI) mass
spectra were obtained on a Finnigan LCQ ion trap. HPLC–UV–MS
analyses were carried out combining the ion trap MS instrument
with HPLC system SSP4000 (Thermo Separation Products)
equipped with an autosampler LC Pal (CTC Analytics) and
UV6000LP diode array detector (UV detection 215–400 nm).
Instrument control, data acquisition and processing were
performed by using Xcalibur 1.2 software (Finnigan). HPLC
5. Experimental
5.1. Chemistry
All solvents and reagents, unless otherwise stated, were com-
mercially available, of the best grade and were used without fur-
ther purification. All experiments dealing with moisture-sensitive
compounds were carried out under dry nitrogen or argon atmo-
sphere. Organic solutions were evaporated using a Heidolph WB
2001 rotary evaporator at 15–20 mmHg. Thin-layer chromatogra-
phy was performed on Merck Silica Gel 60 F254 pre-coated plates.

M. G. Brasca et al. / Bioorg. Med. Chem. 18 (2010) 1844–1853
1849
BrdU
Phospho pRb
700
600
500
400
300
200
40
35
30
25
20
15
10
5
0
vehicle
compound 31
vehicle
compound 31
Groups
Groups
Figure 5. Compound 31 decreases the number of BrdU and phospho pRb positive cells in A2780 xenograft tumors as determined by immunohistochemistry.
chromatography was run at room temperature, and 1 mL/min flow
rate, using a Waters X Terra RP 18 column (4.6 ꢂ 50 mm; 3.5 m).
J = 7.1 Hz, 2H), 1.67 (s, 6H), 1.37 (t, J = 7.1 Hz, 3H); LC–MS (ESI) m/z
347 [M+H]+. This intermediate (2.08 g, 5.33 mmol), in DCM
(70 mL), was treated with N,N-diisopropylethylamine (1.6 mL,
9.2 mmol) and at 0 °C with pivaloyl chloride (0.78 mL, 6.3 mmol).
Gradually the reaction was brought to room temperature and stirred
overnight. The solution was washed with saturated sodium hydro-
gen carbonate aqueous solution and brine. The organic phase was
dried over sodium sulfate, evaporated and purified by flash
chromatography (DCM/EtOAc 90:10) to afford ethyl 5-(2,2-dimeth-
ylpropanoyl)-3-{[(4-fluorophenyl)carbonyl]amino}-6,6-dimethyl-
5,6-dihydropyrrolo[3,4-c]pyrazole-2(4H)-carboxylate (2.03 g, 88%).
1H NMR (400 MHz, DMSO-d6) d 10.83 (s, 1H), 8.09–8.15 (m, 2H),
7.30–7.36 (m, 2H), 4.96 (s, 2H), 4.46 (q, J = 7.2 Hz, 2H), 1.68 (s, 6H),
1.36 (t, J = 7.2 Hz, 3H), 1.24 (s, 9H); LC–MS (ESI) m/z 431 [M+H]+. This
intermediate (2.0 g, 4.64 mmol) was dissolved in methanol (60 mL),
treated with triethylamine (6.45 mL, 46.4 mmol) and stirred over-
night at room temperature. After evaporation, the solid was treated
with diethyl ether/hexane and filtered to afford the title compound 2
(1.43 g, 86%). 1H NMR (400 MHz, DMSO-d6) d 12.44 (br s, 1H), 10.93
(s, 1H), 8.08 (dd, J = 5.5, 8.8 Hz, 2H), 7.33 (t, J = 8.8 Hz, 2H), 4.86 (br s,
2H), 1.66 (s, 6H), 1.23 (s, 9H); LC–MS (ESI) m/z 359 [M+H]+; HRMS
(ESI): m/z calcd for C19H23FN4O2+H+ 359.1878, found 359.1889;
Anal. Calcd for C19H23FN4O2: C, 63.67; H, 6.47; N, 15.63. Found: C,
63.63; H, 6.44; N, 15.55.
l
Mobile phase A was ammonium acetate 5 mM buffer (pH 5.5 with
acetic acid): acetonitrile 90:10, and mobile phase B was ammo-
nium acetate 5 mM buffer (pH 5.5 with acetic acid): acetonitrile
10:90; the gradient was from 0% to 100% B in 7 min then hold
100% B for 2 min before requilibration. Mass are given as m/z ratio.
ESI(+) high resolution mass spectra (HRMS) were obtained on a
Waters Q-Tof Ultima directly connected with micro HPLC 1100
Agilent as previously described.15 Elemental analyses were per-
formed on a Carlo Erba 1110 instrument, and C, H, and N results
were within 0.4% of theoretical values unless specified.
5.1.1. General procedure A
General procedure for the preparation of compounds 1–7 is
illustrated below for the preparation of 2.
5.1.1.1. N-[5-(2,2-Dimethylpropanoyl)-6,6-dimethyl-1,4,5,6-tet-
rahydropyrrolo[3,4-c]pyrazol-3-yl]-4-fluorobenzamide (2).
5-tert-Butyl 2-ethyl 3-amino-6,6-dimethyl-4,6-dihydropyrrol-
o[3,4-c]pyrazole-2,5(4H,6H)-dicarboxylate (2.0 g, 6.16 mmol) was
dissolved in THF (40 mL), treated with N,N-diisopropylethylamine
(5.4 mL, 30.80 mmol) and then, at 0 °C dropwise, with 4-fluor-
obenzoyl chloride (0.80 mL, 6.77 mmol) dissolved in THF (8 mL).
The reaction mixture was stirred at room temperature for 5 h, con-
centrated and dissolved in DCM, washed with saturated sodium
hydrogen carbonate aqueous solution and with brine. The organic
phase was dried over sodium sulfate, evaporated and purified by
flash chromatography (hexane/EtOAc 80:20) to afford 5-tert-butyl
2-ethyl 3-{[(4-fluorophenyl)carbonyl]amino}-6,6-dimethylpyrrol-
o[3,4-c]pyrazole-2,5(4H,6H)-dicarboxylate (2.50 g, 90%). 1H NMR
(400 MHz, DMSO-d6) d 10.80 (s, 1H), 7.97–8.03 (m, 2H), 7.41–7.48
(m, 2H), 4.52 and 4.54 (2 ꢂ s, 2H, conformers), 4.45 (q, J = 7.1 Hz,
2H), 1.61 and 1.63 (2 ꢂ s, 6H, conformers), 1.46 and 1.48 (2 ꢂ s,
9H, conformers), 1.36 (t, J = 7.1 Hz, 3H); LC–MS (ESI) m/z 447
[M+H]+. This intermediate (2.49 g, 5.58 mmol) was dissolved in
dioxane (50 mL) and treated with HCl 4 M in dioxane (28 mL,
20 equiv). After 2 h at 40 °C the reaction mixture was concentrated
and the residue was treated with diethyl ether, filtered to afford
ethyl 3-{[(4-fluorophenyl)carbonyl]amino}-6,6-dimethyl-5,6-dihy
dropyrrolo[3,4-c]pyrazole-2(4H)-carboxylate hydrochloride (2.09
g, 98%). 1H NMR (400 MHz, DMSO-d6) d 10.90 (s, 1H), 9.88 (br s,
2H), 7.98–8.03 (m, 2H), 7.45–7.50 (m, 2H), 4.60 (br s, 2H), 4.48 (d,
The following compounds 1, 3–7 were prepared according to
the general procedure A described above.
5.1.1.2. N-[5-(2,2-Dimethylpropanoyl)-6,6-dimethyl-1,4,5,6-tet-
rahydropyrrolo[3,4-c]pyrazol-3-yl]-3,5-difluorobenzamide (1).
1H NMR (400 MHz, DMSO-d6) d 12.52 (br s, 1H), 11.10 (s, 1H),
7.68–7.76 (m, 2H), 7.45–7.55 (m, 1H), 4.87 (br s, 2H), 1.67 (s,
6H), 1.23 (s, 9H); LC–MS (ESI): m/z 377 [M+H]+; HRMS (ESI): m/z
calcd for C19H22F2N4O2+H+ 377.1784, found 377.1786; Anal. Calcd
for C19H22F2N4O2: C, 60.63; H, 5.89; N, 14.88. Found: C, 60.56; H,
5.91; N, 14.76.
5.1.1.3. N-[5-(2,2-Dimethylpropanoyl)-6,6-dimethyl-1,4,5,6-tet-
rahydropyrrolo[3,4-c]pyrazol-3-yl]pyridine-3-carboxamide (3).
1H NMR (400 MHz, DMSO-d6) d 12.49 (br s, 1H), 11.15 (s, 1H),
9.12 (d, J = 2 Hz, 1H), 8.74 (d, J = 4.0 Hz, 1H), 8.32 (d, J = 7.9 Hz,
1H), 7.50–7.55 (m, 1H), 4.89 (br s, 2H), 1.67 (s, 6H), 1.23 (s, 9H);
LC–MS (ESI): m/z 342 [M+H]+; Anal. Calcd for C18H23N5O2: C,
63.32; H, 6.79; N, 20.51. Found: C, 62.95; H, 6.83; N, 20.32.

1850
M. G. Brasca et al. / Bioorg. Med. Chem. 18 (2010) 1844–1853
5.1.1.4. N-[5-(2,2-Dimethylpropanoyl)-6,6-dimethyl-1,4,5,6-tet-
rahydropyrrolo[3,4-c]pyrazol-3-yl]pyridine-4-carboxamide (4).
1H NMR (400 MHz, DMSO-d6) d 12.53 (br s, 1H), 11.23 (s, 1H),
8.74 (d, J = 5 Hz, 2H), 7.90 (d, J = 5 Hz, 2H), 4.89 (s, 2H), 1.67 (s,
6H), 1.23 (s, 9H); LC–MS (ESI): m/z 342 [M+H]+; Anal. Calcd for
C18H23N5O2: C, 63.32; H, 6.79; N, 20.51. Found: C, 63.57; H, 6.89;
N, 20.24.
5.1.2.2. N-[6,6-Dimethyl-5-(piperidin-4-ylcarbonyl)-1,4,5,6-tet-
rahydropyrrolo[3,4-c]pyrazol-3-yl]-4-fluorobenzamide (8).
Compound 8 was obtained in 77% yield after deprotection
of ethyl 5-({1-[(9H-fluoren-9-ylmethoxy)carbonyl]piperidin-4-yl}-
carbonyl)-3-{[(4-fluorophenyl)carbonyl]amino}-6,6-dimethyl-
5,6-dihydropyrrolo[3,4-c]pyrazole-2(4H)-carboxylate
(prepared
according the general procedure B) with 20% piperidine in metha-
nol. 1H NMR (400 MHz, DMSO-d6) d 12.43 (br s, 1H), 10.95 (br s,
1H), 8.07 (dd, J = 5.5, 8.9 Hz, 2H), 7.34 (t, J = 8.9 Hz, 2H), 4.72 (br
s, 2H), 2.96–3.04 (m, 2H), 2.54–2.64 (m, 2H), 2.46–2.56 (m, 1H),
1.66 (m, 6H), 1.58–1.66 (m, 2H), 1.45–1.58 (m, 2H); LC–MS (ESI):
m/z 386 [M+H]+.
5.1.1.5. N-[5-(2,2-Dimethylpropanoyl)-6,6-dimethyl-1,4,5,6-tet-
rahydropyrrolo[3,4-c]pyrazol-3-yl]piperidine-4-carboxamide (5).
Compound 5 was obtained in 82% yield after deprotection of
ethyl
5-(2,2-dimethylpropanoyl)-3-[({1-[(9H-fluoren-9-ylmeth-
oxy)carbonyl]piperidin-4-yl}carbonyl)amino]-6,6-dimethyl-5,6-di
hydropyrrolo[3,4-c]pyrazole-2(4H)-carboxylate (prepared accor
ding the general procedure A) with 20% piperidine in methanol.
1H NMR (400 MHz, DMSO-d6) d 12.26 (br s, 1H), 10.38 (br s, 1H),
4.80 (br s, 2H), 3.02–3.09 (m, 2H), 2.54–2.62 (m, 2H), 2.44–2.54
(m, 1H), 1.67–1.77 (m, 2H), 1.64 (s, 6H), 1.51–1.62 (m, 2H), 1.23
(s, 9H); LC–MS (ESI): m/z 348 [M+H]+.
5.1.2.3. N-{5-[(1-Ethylpiperidin-4-yl)carbonyl]-6,6-dimethyl-1,4,
5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}-4-fluorobenzamide (10).
1H NMR (400 MHz, DMSO-d6) d 12.47 (br s, 1H), 10.95 (s, 1H),
8.01–8.13 (m, 2H), 7.28–7.40 (m, 2H), 4.74 (br s, 2H), 2.88–3.04
(m, 2H), 2.28–2.47 (m, 3H), 1.85–2.10 (m, 2H), 1.66 (s, 6H), 1.55–
1.76 (m, 4H), 1.01 (t, J = 6.8 Hz, 3H); LC–MS (ESI): m/z 414 [M+H]+;
Anal. Calcd for C22H28FN5O2: C, 63.90; H, 6.83; N, 16.94. Found: C,
63.54; H, 6.98; N, 16.65.
5.1.1.6. N-[5-(2,2-Dimethylpropanoyl)-6,6-dimethyl-1,4,5,6-tet-
rahydropyrrolo[3,4-c]pyrazol-3-yl]-1-methylpiperidine-4-car-
boxamide (6). 1H NMR (400 MHz, DMSO-d6) d 12.26 (br s, 1H),
10.37 (s, 1H), 4.78 (s, 2H), 2.79–2.91 (m, 2H), 2.26–2.36 (m, 1H),
2.20 (br s, 3H), 1.85–2.03 (m, 2H), 1.55–1.79 (m, 4H), 1.62 (s,
6H), 1.21 (s, 9H); LC–MS (ESI): m/z 362 [M+H]+; Anal. Calcd for
C19H31N5O2: C, 63.13; H, 8.64; N, 19.37. Found: C, 62.84; H, 8.61;
N, 19.12.
5.1.2.4. N-{5-[(1-Cyclopropylpiperidin-4-yl)carbonyl]-6,6-dimet-
hyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}-4-fluorobenz-
amide (11). 1H NMR (400 MHz, DMSO-d6) d 12.47 (br s, 1H),
10.95 (s, 1H), 8.03–8.11 (m, 2H), 7.28–7.39 (m, 2H), 4.74 (br s,
2H), 2.92–3.07 (m, 3H), 2.36–2.46 (m, 1H), 2.11–2.33 (m, 2H),
1.46–1.74 (m, 4H), 1.65 (s, 6H), 0.26–0.50 (m, 4H); LC–MS (ESI):
m/z 426 [M+H]+; Anal. Calcd for C23H28FN5O2: C, 64.92; H, 6.63;
N, 16.46. Found: C, 64.95; H, 6.98; N, 16.65.
5.1.1.7. N-[5-(2,2-Dimethylpropanoyl)-6,6-dimethyl-1,4,5,6-tet-
rahydropyrrolo[3,4-c]pyrazol-3-yl]-N2,N2-dimethylglycinamide
(7). 1H NMR (400 MHz, DMSO-d6) d 12.35 (br s, 1H), 10.06 (s, 1H),
4.80 (s, 2H), 3.13 (br s, 2H), 2.31 (s, 6H), 1.63 (s, 6H), 1.21 (s, 9H);
LC–MS (ESI): m/z 322 [M+H]+; Anal. Calcd for C16H27N5O2: C, 59.79;
H, 8.47; N, 21.79. Found: C, 59.94; H, 8.51; N, 21.43.
5.1.2.5. N-{5-[(1-Acetylpiperidin-4-yl)carbonyl]-6,6-dimethyl-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}-4-fluorobenzam-
ide (12). 1H NMR (400 MHz, DMSO-d6) d 12.48 (br s, 1H), 10.96 (s,
1H), 8.04–8.11 (m, 2H), 7.28–7.38 (m, 2H), 4.77 (br s, 2H), 4.34–
4.42 and 3.79–3.86 (2 ꢂ m, 2H), 3.06–3.16 and 2.56–2.66 (2 ꢂ m,
2H), 2.66–2.74 (m, 1H), 1.99 (s, 3H), 1.66 (s, 6H), 1.63–1.77 (m,
2H), 1.32–1.57 (2 ꢂ m, 2H); LC–MS (ESI): m/z 428 [M+H]+; Anal.
Calcd for C22H26FN5O3: C, 60.07; H, 6.10; N, 15.71. Found: C,
60.45; H, 6.13; N, 16.02.
5.1.2. General procedure B
General procedure for the preparation of compounds 8–12 and
17–33 is illustrated below for the preparation of 9.
5.1.2.1. N-{6,6-Dimethyl-5-[(1-methylpiperidin-4-yl)carbonyl]-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}-4-fluorobenzam-
ide (9). Ethyl 3-{[(4-fluorophenyl)carbonyl]amino}-6,6-dimethyl-
5,6-dihydropyrrolo[3,4-c]pyrazole-2(4H)-carboxylate hydrochlo-
ride (prepared according to the general method A) (0.5 g,
1.3 mmol) in DCM (25 mL), was treated with N,N-diisopropylethyl-
amine (1.13 mL, 6.5 mmol) and TBTU (0.542 g, 1.69 mmol), at room
temperature for 1 h, and then 1-methyl-piperidine-4-carboxylic
acid hydrochloride (0.29 g, 1.61 mmol) was added. The reaction
was stirred overnight. The solution was washed with saturated so-
dium hydrogen carbonate aqueous solution and brine, the organic
phase was dried over sodium sulfate and concentrated. The residue
was dissolved in methanol (16 mL), treated with triethylamine
(2 mL, 14.3 mmol) and stirred overnight at room temperature.
After evaporation, the solid was purified by flash chromatography
(DCM/MeOH/NH3 aq 90:10:2). The solid was treated with diiso-
propylether and filtered to afford the title compound 9 (0.36 g,
69%). 1H NMR (400 MHz, DMSO-d6) d 12.46 (br s, 1H), 10.95 (s,
1H), 8.02–8.12 (m, 2H), 7.28–7.40 (m, 2H), 4.73 (br s, 2H), 2.80–
2.94 (m, 2H), 2.30–2.43 (m, 1H), 2.22 (br s, 3H), 1.92–2.15 (m,
2H), 1.55–1.75 (m, 4H), 1.66 (s, 6H); LC–MS (ESI): m/z 400
[M+H]+; HRMS (ESI): m/z calcd for C21H26FN5O2 + H+ 400.2144,
found 400.2149; Anal. Calcd for C21H26FN5O2: C, 63.14; H, 6.56;
N, 17.53. Found: C, 62.78; H, 6.83; N, 17.32.
5.1.2.6. N-{6,6-Dimethyl-5-[(1-methylpiperidin-4-yl)carbonyl]-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}benzamide (17).
1H NMR (400 MHz, DMSO-d6) d 12.46 (br s, 1H), 10.93 (br s, 1H),
7.96–8.01 (m, 2H), 7.56–7.62 (m, 1H), 7.48–7.54 (m, 2H), 4.73 (br s,
2H), 2.82–2.90 (m, 2H), 2.34–2.43 (m, 1H), 2.21 (s, 3H), 1.96–2.07
(m, 2H), 1.66 (s, 6H), 1.58–1.63 (m, 4H); LC–MS (ESI): m/z 382
[M+H]+.
5.1.2.7. N-{6,6-Dimethyl-5-[(1-methylpiperidin-4-yl)carbonyl]-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}-3-fluorobenzam-
ide (18). 1H NMR (400 MHz, DMSO-d6) d 12.46 (br s, 1H), 11.07 (s,
1H), 7.85 (dt, J = 1.7, 8.0 Hz, 1H), 7.80 (ddd, J = 1.7, 2.4, 9.9 Hz, 1H),
7.57 (td, J = 5.8, 8.0 Hz, 1H), 7.42–7.50 (m, 1H), 4.79 (s, 2H), 2.75–
2.83 (m, 2H), 2.23–2.34 (m, 1H), 2.14 (s, 3H), 1.83–1.94 (m, 2H),
1.67 (s, 6H), 1.59–1.68 (m, 4H); LC–MS (ESI): m/z 400 [M+H]+;
HRMS (ESI): m/z calcd for C21H26FN5O2+H+ 400.2143, found
400.2142.
5.1.2.8. N-{6,6-Dimethyl-5-[(1-methylpiperidin-4-yl)carbonyl]-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}-3,4-difluoroben-
zamide (19). 1H NMR (400 MHz, DMSO-d6) d 12.57 (br s, 1H),
11.09 (br s, 1H), 8.01–8.10 (m, 1H), 7.85–7.93 (m, 1H), 7.54–7.65
(m, 1H), 4.78 (br s, 2H), 2.75–2.84 (m, 2H), 2.25–2.35 (m, 1H),
2.15 (s, 3H), 1.82–1.93 (m, 2H), 1.62 (s, 6H), 1.54–1.65 (m, 4H);
LC–MS (ESI): m/z 418 [M+H]+.
The following compounds 8, 10–12 and 17–33 were prepared
according to the general procedure B described above.

M. G. Brasca et al. / Bioorg. Med. Chem. 18 (2010) 1844–1853
1851
5.1.2.9. N-{6,6-Dimethyl-5-[(1-methylpiperidin-4-yl)carbonyl]-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}-3,5-difluoroben-
zamide (20). 1H NMR (400 MHz, DMSO-d6) d 12.51 (br s, 1H),
11.17 (s, 1H), 7.67–7.75 (m, 2H), 7.48–7.55 (m, 1H), 4.78 (s, 2H),
2.74–2.84 (m, 2H), 2.26–2.34 (m, 1H), 2.16 (s, 3H), 1.82–1.93 (m,
2H), 1.62 (s, 6H), 1.56–1.67 (m, 4H); LC–MS (ESI): m/z 418
[M+H]+; HRMS (ESI): m/z calcd for C21H25F2N5O2+H+ 418.2049,
found 418.2055.
J = 0.8, 1.7 Hz, 1H), 7.42 (dd, J = 0.8, 3.5 Hz, 1H), 6.68 (dd, J = 1.7,
3.5 Hz, 1H), 4.75 (s, 2H), 2.80–2.87 (m, 2H), 2.34–2.42 (m, 1H),
2.17 (s, 3H), 1.91–2.01 (m, 2H), 1.66 (s, 6H), 1.58–1.64 (m, 4H);
LC–MS (ESI): m/z 372 [M+H]+; HRMS (ESI): m/z calcd for
C19H25N5O3+H+ 372.2030, found 372.2013.
5.1.2.18. N-{6,6-Dimethyl-5-[(1-methylpiperidin-4-yl)carbonyl]-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}cyclopropanecarb-
oxamide (29). 1H NMR (400 MHz, DMSO-d6) d 12.28 (br s, 1H),
10.70 (br s, 1H), 4.61 (s, 2H), 2.81–2.90 (m, 2H), 2.27–2.36 (m,
1H), 2.23 (s, 3H), 1.97–2.07 (m, 2H), 1.83 (m, 1H), 1.61 (m, 6H),
1.56–1.68 (m, 4H), 0.72–0.80 (m, 4H); LC–MS (ESI): m/z 346
[M+H]+.
5.1.2.10. 4-Chloro-N-{6,6-dimethyl-5-[(1-methylpiperidin-4-yl)-
carbonyl]-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}benzam-
ide (21). 1H NMR (400 MHz, DMSO-d6) d 12.53 (br s, 1H), 11.05 (br
s, 1H), 8.01 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.4 Hz, 2H), 4.79 (br s, 2H),
2.76–2.82 (m, 2H), 2.24–2.34 (m, 1H), 2.14 (s, 3H), 1.83–1.93 (m,
2H), 1.67 (s, 6H), 1.58–1.73 (m, 4H); LC–MS (ESI): m/z 416 [M+H]+.
5.1.2.19. N-{6,6-Dimethyl-5-[(1-methylpiperidin-4-yl)carbonyl]-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}cyclobutancarbox-
amide (30). 1H NMR (400 MHz, DMSO-d6) d 12.25 (br s, 1H), 10.25
(br s, 1H), 4.67 (br s, 2H), 3.22 (m, 1H), 2.77–2.85 (m, 2H), 2.28–2.37
(m, 1H), 2.17 (s, 3H), 2.14–2.23 (m, 2H), 2.02–2.11 (m, 2H), 1.87–
1.98 (m, 3H), 1.73–1.82 (m, 1H), 1.62 (s, 6H), 1.56–1.67 (m, 4H);
LC–MS (ESI): m/z 360 [M+H]+; HRMS (ESI): m/z calcd for
5.1.2.11. N-{6,6-Dimethyl-5-[(1-methylpiperidin-4-yl)carbonyl]-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}-4-(trifluoromethyl)-
benzamide (22). 1H NMR (400 MHz, DMSO-d6) d 12.58 (br s, 1H),
11.23 (br s, 1H), 8.18 (d, J = 8.2 Hz, 2H), 7.89 (d, J = 8.2 Hz, 2H), 4.81
(br s, 2H), 2.73–2.84 (m, 2H), 2.25–2.34 (m, 1H), 2.14 (s, 3H), 1.67 (s,
6H), 1.54–1.69 (m, 4H); LC–MS (ESI): m/z 450 [M+H]+.
C19H29N5O2
+
H+ 360.2394, found 360.2396; Anal. Calcd for
C19H29N5O2: C, 63.48; H, 8.13; N, 19.48. Found: C, 63.25; H, 8.30;
N, 19.12.
5.1.2.12. N-{6,6-Dimethyl-5-[(1-methylpiperidin-4-yl)carbonyl]-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}naphthalene-2-car-
boxamide (23). 1H NMR (400 MHz, DMSO-d6) d 12.49 (br s, 1H),
11.10 (br s, 1H), 8.65 (s, 1H), 7.97–8.09 (m, 4H), 7.58–7.69 (m,
2H), 4.78 (br s, 2H), 2.82–2.90 (m, 2H), 2.35–2.44 (m, 1H), 2.21 (s,
3H), 1.95–2.05 (m, 2H), 1.67 (s, 6H), 1.59–1.75 (m, 4H); LC–MS
(ESI): m/z 432 [M+H]+.
5.1.2.20. N-{6,6-Dimethyl-5-[(1-methylpiperidin-4-yl)carbonyl]-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}-3-methylbutana-
mide (31). 1H NMR (400 MHz, DMSO-d6) d 12.27 (br s, 1H), 10.34
(br s, 1H), 4.64 (br s, 2H), 2.82–2.89 (m, 2H), 2.30–2.37 (m, 1H), 2.21
(s, 3H), 2.12–2.18 (m, 2H), 2.15 (d, J = 7.1 Hz, 2H), 1.96–2.09 (m,
3H), 1.62 (s, 6H), 1.54–1.67 (m, 4H), 0.90 (d, J = 6.6 Hz, 6H); LC–
MS (ESI): m/z 362 [M+H]+; HRMS (ESI): m/z calcd for
C19H31N5O2+H+ 362.2551, found 362.2555; Anal. Calcd for
C19H31N5O2: C, 63.13; H, 8.64; N, 19.37. Found: C, 63.01; H, 8.78;
N, 19.25.
5.1.2.13. N-{6,6-Dimethyl-5-[(1-methylpiperidin-4-yl)carbonyl]-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}naphthalene-1-car-
boxamide (24). 1H NMR (400 MHz, DMSO-d6) d 12.42 (br s, 1H),
11.11 (br s, 1H), 8.20–8.27 (m, 1H), 8.04–8.10 (m, 1H), 7.97–8.03
(m, 1H), 7.71–7.77 (m, 1H), 7.55–7.63 (m, 3H), 4.80 (br s, 2H),
2.80–2.87 (m, 2H), 2.34–2.42 (m, 1H), 2.17 (s, 3H), 1.90–2.00 (m,
2H), 1.68 (s, 6H), 1.59–1.74 (m, 4H); LC–MS (ESI): m/z 432 [M+H]+.
5.1.2.21. N-{5-[(1-Ethylpiperidin-4-yl)carbonyl]-6,6-dimethyl-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}-3-methylbutana-
mide (32). 1H NMR (400 MHz, DMSO-d6) d 12.32 (br s, 1H), 10.39
(s, 1H), 4.70 (s, 2H), 2.88–3.04 (m, 2H), 2.28–2.47 (m, 3H), 2.15 (d,
J = 7.1 Hz, 2H), 1.98–2.08 (m, 1H), 1.85–2.10 (m, 2H), 1.63 (s, 6H),
1.55–1.76 (m, 4H), 1.01 (t, J = 6.8 Hz, 3H), 0.90 (d, J = 6.6 Hz, 6H);
LC–MS (ESI): m/z 376 [M+H]+.
5.1.2.14. N-{6,6-Dimethyl-5-[(1-methylpiperidin-4-yl)carbonyl]-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}pyridine-4-carbox-
amide (25). 1H NMR (400 MHz, DMSO-d6)
d 11.37 (s, 1H),
8.73–8.76 (m, 2H), 7.88–7.93 (m, 2H), 4.81 (br s, 2H), 2.78–2.85
(m, 2H), 2.28–2.37 (m, 1H), 2.17 (s, 3H), 1.90–2.01 (m, 2H), 1.68
(s, 6H), 1.58–1.74 (m, 4H); LC–MS (ESI): m/z 383 [M+H]+.
5.1.2.22. N-{5-[(1-Cyclopropylpiperidin-4-yl)carbonyl]-6,6-di-
methyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}-3-methyl-
butanamide (33). 1H NMR (400 MHz, DMSO–d6) d 12.33 (br s,
1H), 10.39 (s, 1H), 4.71 (s, 2H), 2.92–3.07 (m, 3H), 2.36–2.46 (m,
1H), 2.16 (d, J = 7.1 Hz), 2.11–2.33 (m, 2H), 1.97–2.08 (m, 1H),
1.46–1.74 (m, 4H), 1.63 (s, 6H), 0.90 (d, J = 6.6 Hz, 6H), 0.26–0.50
(m, 4H); LC–MS (ESI): m/z 388 [M+H]+.
5.1.2.15. N-{6,6-Dimethyl-5-[(1-methylpiperidin-4-yl)carbonyl]-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}thiophene-2-car-
boxamide (26). 1H NMR (400 MHz, DMSO-d6) d 12.50 (br s, 1H),
11.06 (s, 1H), 8.10 (d, J = 3.8 Hz, 1H), 7.84 (dd, J = 1.0, 5.0 Hz, 1H),
7.20 (dd, J = 3.8, 5.0 Hz, 1H), 4.77 (s, 2H), 2.75–2.83 (m, 2H),
2.23–2.35 (m, 1H), 2.16 (s, 3H), 1.82–1.90 (m, 2H), 1.66 (s, 6H),
1.54–1.64 (m, 4H); LC–MS (ESI): m/z 388 [M+H]+.
5.1.3. General procedure C
General procedure for the preparation of compounds 13–16 is
illustrated below for the preparation of 13.
5.1.2.16. N-{6,6-Dimethyl-5-[(1-methylpiperidin-4-yl)carbonyl]-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}thiophene-3-car-
boxamide (27). 1H NMR (400 MHz, DMSO-d6) d 12.45 (br s, 1H),
10.77 (s, 1H), 8.42 (br s, 1H), 7.61–7.70 (m, 2H), 4.73 (br s, 2H),
2.86–3.00 (m, 2H), 2.36–2.46 (m, 1H), 2.29 (br s, 3H), 2.07–2.22
(m, 2H), 1.59–1.77 (m, 4H), 1.66 (s, 6H); LC–MS (ESI): m/z 388
[M+H]+; HRMS (ESI): m/z calcd for C19H25N5O2S+H+ 388.1802,
found 388.1806.
5.1.3.1. N-{6,6-Dimethyl-5-[(4-methylpiperazin-1-yl)carbonyl]-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}-4-fluorobenzamide
(13). Ethyl 3-{[(4-fluorophenyl)carbonyl]amino}-6,6-dimethyl-
5,6-dihydropyrrolo[3,4-c]pyrazole-2(4H)-carboxylate hydrochlo-
ride (prepared according to the general method A) (442 mg,
1.15 mmol) in DCM (30 mL) was added to a solution of triphosgene
(195 mg, 0.65 mmol) in DCM (15 mL), followed by N,N-diisopro-
5.1.2.17. N-{6,6-Dimethyl-5-[(1-methylpiperidin-4-yl)carbonyl]-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}furan-2-carboxam-
ide (28). 1H NMR (400 MHz, DMSO-d6) d 10.85 (s, 1H), 7.91 (dd,
pylethylamine (0.76 mL, 4.31 mmol). After 3 h, a solution of
N-methylpiperazine (0.195 mL, 1.72 mmol) and diisopropylethyl-
amine (0.30 mL, 1.72 mmol) in DCM (8 mL) was added. The

1852
M. G. Brasca et al. / Bioorg. Med. Chem. 18 (2010) 1844–1853
reaction was stirred overnight at room temperature. The solution
was washed with brine, the organic phase was dried over sodium
sulfate and concentrated. The residue was dissolved in methanol
(16 mL), treated with triethylamine (1.6 mL, 11.5 mmol) and stir-
red overnight at room temperature. After evaporation, the solid
was purified by flash chromatography (DCM/MeOH 90:10). The so-
lid was treated with diisopropylether and filtered to afford the title
compound 13 (0.294 g, 64%). 1H NMR (400 MHz, DMSO-d6) d 12.39
(br s, 1H), 10.89 (s, 1H), 8.04 (dd, J = 5.5, 8.6 Hz, 2H), 7.33 (t,
J = 8.6 Hz, 2H), 4.55 (br s, 2H), 2.99–3.12 (m, 4H), 2.32–2.48 (m,
4H), 2.24 (br s, 3H), 1.62 (s, 6H); LC–MS (ESI): m/z 401 [M+H]+;
HRMS (ESI): m/z calcd for C20H25FN6O2 + H+ 401.2096, found
401.2091; Anal. Calcd for C20H25FN6O2: C, 59.99; H, 6.29; N,
20.99. Found: C, 59.70; H, 6.40; N, 20.66.
7.24 mmol) was added. The reaction was stirred overnight. The solu-
tion was washed with saturated sodium hydrogen carbonate aque-
ous solution and brine, the organic phase was dried over sodium
sulfate and concentrated. The residue was dissolved in methanol
(72 mL), treated with triethylamine (8 mL, 14.3 mmol) and stirred
overnight at room temperature. After evaporation, the solid was
purified by flash chromatography (eluent: DCM/MeOH/NH3 aq
90:10:2). The solid was treated with diisopropylether and filtered
to afford the title compound 36 (1.80 g, 75%). 1H NMR (400 MHz,
DMSO-d6) d 12.24 (br s, 1H), 9.03 (br s, 1H), 7.46 (dt, J = 2.2,
11.6 Hz, 1H), 7.28–7.34 (m, 1H), 7.10 (d, J = 9.5 Hz, 1H), 6.79 (ddd,
J = 2.8, 8.0, 9.5 Hz, 1H), 4.66 (br s, 2H), 2.85–2.93 (m, 2H), 2.34–
2.43 (m, 1H), 2.25 (br s, 3H), 2.03–2.15 (m, 2H), 1.63 (s, 6H), 1.57–
1.73 (m, 4H); LC–MS (ESI): m/z 415 [M+H]+.
The following compounds 14–16 were prepared according to
the general procedure C described above.
The following compounds 34–35 were prepared according to
the general procedure D described above.
5.1.3.2. 4-Fluoro-N-(5-{[4-(2-hydroxyethyl)piperazin-1-yl]car-
bonyl}-6,6-dimethyl-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-
yl)benzamide (14). 1H NMR (400 MHz, DMSO-d6) d 12.41 (br s,
1H), 10.91 (s, 1H), 8.06 (dd, J = 5.5, 8.8 Hz, 2H), 7.33 (t, J = 8.8 Hz,
2H), 4.55 (s, 2H), 3.48–3.57 (m, 2H), 3.03–3.11 (m, 4H), 2.44–
2.64 (m, 6H), 1.62 (s, 6H); LC–MS (ESI): m/z 431 [M+H]+; HRMS
(ESI): m/z calcd for C21H27FN6O3 + H+ 431.2202, found 431.2200.
5.1.4.2. 1-Benzyl-3-{6,6-dimethyl-5-[(1-methylpiperidin-4-yl)car-
bonyl]-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}urea (34). 1H
NMR (400 MHz, DMSO-d6) d 12.06 (br s, 1H), 8.83 (s, 1H), 7.22–
7.36 (m, 5H), 6.89 (br s, 1H), 4.59 (br s, 2H), 4.31 (d, J = 5.9 Hz,
2H), 2.82–2.89 (m, 2H), 2.29–2.37 (m, 1H), 2.21 (s, 3H), 1.96–
2.06 (m, 2H), 1.61 (s, 6H), 1.55–1.68 (m, 4H); LC–MS (ESI): m/z
411 [M+H]+.
5.1.3.3. N-{5-[(4-Acetylpiperazin-1-yl)carbonyl]-6,6-dimethyl-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}-4-fluorobenzam-
ide (15). 1H NMR (400 MHz, DMSO-d6) d 12.45 (br s, 1H), 10.92 (s,
1H), 8.06 (dd, J = 5.5, 8.9 Hz, 2H), 7.33 (t, J = 8.9 Hz, 2H), 4.58 (br s,
2H), 3.44–3.49 (m, 4H), 3.05–3.10 (m, 2H), 2.99–3.04 (m, 2H), 2.00
(s, 3H), 1.63 (s, 6H); LC–MS (ESI): m/z 429 [M+H]+.
5.1.4.3. 1-{6,6-Dimethyl-5-[(1-methylpiperidin-4-yl)carbonyl]-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}-3-propylurea
(35). 1H NMR (400 MHz, DMSO-d6) d 11.98 (br s, 1H), 8.66 (s, 1H),
6.44 (br s, 1H), 4.58 (s, 2H), 3.00–3.07 (m, 2H), 2.79–2.86 (m, 2H),
2.26–2.35 (m, 1H), 2.18 (br s, 3H), 1.90–1.98 (m, 2H), 1.60 (s, 6H),
1.56–1.66 (m, 4H), 1.37–1.46 (m, 2H), 0.86 (t, J = 7.6 Hz, 3H); LC–
MS (ESI): m/z 363 [M+H]+.
5.1.3.4. N-{6,6-Dimethyl-5-[(4-phenylpiperazin-1-yl)carbonyl]-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}-4-fluorobenzam-
ide (16). 1H NMR (400 MHz, DMSO-d6) d 12.48 (br s, 1H), 10.92 (s,
1H), 8.06 (dd, J = 5.5, 8.8 Hz, 2 H), 7.32 (t, J = 8.8 Hz, 2 H), 7.19–7.26
(m, 2H), 6.93–6.98 (m, 2H), 6.77–6.82 (m, 1H), 4.62 (s, 2 H), 3.14–
3.24 (m, 8H), 1.65 (s, 6 H); LC–MS (ESI): m/z 463 [M+H]+.
5.2. Crystallographic methods
Expression, purification, crystallization and soaking procedures
of the CDK2/cyclin A complex was carried out as previously de-
scribed.16 The final R-factor for the complex with compound 31
was 22.3% (Rfree 26.1%). The coordinates have been deposited in
the Protein Data Bank with code 2WPA together with structure fac-
tors and detailed experimental conditions.
5.1.4. General procedure D
General procedure for the preparation of compounds 34–36 is
illustrated below for the preparation of 36.
5.3. Kinase assays
5.1.4.1. 1-{6,6-Dimethyl-5-[(1-methylpiperidin-4-yl)carbonyl]-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}-3-(3-fluoro-
Kinase assays were performed as previously described.16 The
panel includes: c-ABL, AKT1, ALK, Aur-A, Aur-B, CDC7, CDK2/A,
CDK1/B, CDK2/E, CDK4/D1, CDK5/p25, CDK7/H, CDK9/T1, CHK1,
CK2, EGFR, ERK2, FGFR1, FLT3, GSK3b, IGF1R, IKK2, IR, JAK2, C-
phenyl)urea (36). 5-tert-Butyl 2-ethyl 3-amino-6,6-dimethyl-4,6-
dihydropyrrolo[3,4-c]pyrazole-2,5(4H,6H)-dicarboxylate (3.0 g, 9.24
mmol) was dissolved in anhydrous THF (50 mL), treated at room
temperature with 3-fluorophenyl-isocyanate (1.4 g, 10.21 mmol)
and stirred overnight. Then the reaction mixture was evaporated, ta-
ken up with DCM and washed with brine. The organic phase was
dried over sodium sulfate and evaporated to dryness. Purification
by flash chromatography (DCM/MeOH 90:10) afforded 5-tert-butyl
2-ethyl 3-{[(3-fluorophenyl)carbamoyl]amino}-6,6-dimethyl-4,6-
dihydropyrrolo[3,4-c]pyrazole-2,5-dicarboxylate (3.05 g, 71%). LC–
MS (ESI): m/z 462 [M+H]+. This intermediate (2.80 g, 6.06 mmol)
was dissolved in dioxane (50 mL) and treated with HCl 4 M in
dioxane (30 mL, 20 equiv). After 2 h at 40 °C the reaction mixture
was concentrated and the residue was treated with diethyl ether,
filtered to afford ethyl 3-{[(3-fluorophenyl)carbamoyl]amino}-6,6-
dimethyl-5,6-dihydropyrrolo[3,4-c]pyrazole-2(4H)-carboxylate hy-
drochloride (2.30 g, 95%). LC–MS (ESI): m/z 362 [M+H]+. This
intermediate (2.30 g, 5.78 mmol), in DCM (100 mL), was treated
with N,N-diisopropylethylamine (5.0 mL, 28.9 mmol) and TBTU
(2.50 g, 7.78 mmol), at room temperature for 1 h, and then 1-
methyl-piperidine-4-carboxylic acid hydrochloride (1.30 g,
KIT, LCK, MAPKAPK2, MET, NEK-6, NIM, PAK4, PDGFR, PDK1, PKA
PKC , PLK1, PLK2, P38 , RET, STLK2, SULU1, TRKA, VEGFR2,
VEGFR3.
a,
a
a
5.4. In vitro pharmacology. A2780 cells proliferation assay
Cells were seeded into 96- or 384-wells plates at final concentra-
tion ranging from 10000 to 30000 cells per cm2 in appropriate med-
ium plus 10% FCS. After 24 h cells were treated using serial dilution
of compounds in two replicates. At 72 h after the treatment the
amount of cells were evaluated using the Cell Titer_Glo assay (Pro-
mega). IC50 values were calculated using a sygmoidal fitting (Assay
Explorer MDL). Experiments were replicated at least two times.
5.5. Flow cytometry analysis and BrdU incorporation
A2780 cells (human adenocarcinoma ovary, from ECACC), were
seeded in T-75 tissue culture flasks, 25000 cells/cm2 in RPMI 1640,

M. G. Brasca et al. / Bioorg. Med. Chem. 18 (2010) 1844–1853
1853
pH 7.4, 10% FBS, 2 mM
and maintained in 5% CO2 at 37 °C with 96% relative humidity.
After 24 h, cells were treated with compounds at 1 M and 3
for 24 h. Cells in the supernatant and adherent cells were collected
using 0.25% Trypsin, 0.02% EDTA. Cells were washed with PBS and
were divided into three samples for flow cytometry analysis and
for immunoblot and BrdU incorporation, as previously described.16
L
-glutamine, 1 ꢂ penicillin–streptomycin
with hematoxylin. Automated quantitative analysis was performed
by two observers using a Image Pro Plus software. Five fields at
100ꢂ magnification were analyzed and results are reported as
the mean of positive cells/field. Statistical analysis was performed
by Mann–Withney test.
l
lM
Acknowledgments
5.6. In vivo pharmacology
We thank Luca Corti and Fabrizio Orzi for their skilled synthetic
chemistry support. We thank Maristella Colombo for skillful assis-
tance in MS spectra recording and interpretation, and Roberta Cer-
uti for her contribution in immunohistochemistry analysis.
Evaluation of antitumor efficacy was performed as previously
described.16
5.7. High-throughput solubility
References and notes
Solubility at pH 7 was performed as previously described.16
1. Malumbres, M.; Barbacid, M. Nat. Rev. Cancer 2009, 9, 153.
2. Malumbres, M.; Barbacid, M. Nat. Rev. Cancer 2001, 1, 222.
3. Meijer, L.; Bettayeb, K.; Galons, H. In Inhibitors of Cyclin-Dependent Kinases as
Anti-Tumor Agents; CRC: Boca Raton, FL, 2007; Vol. 187.
4. Misra, R. N.; Xiao, H.; Kim, K. S.; Lu, S.; Han, W.; Barbosa, S. A.; Hunt, J. T.;
Rawlins, D. B.; Shan, W.; Ahmed, S. Z.; Qian, L.; Chen, B.; Zhao, R.; Bednarz, M.
S.; Kellar, K. A.; Mulheron, J. G.; Batorsky, R.; Roongta, U.; Kamath, A.; Marathe,
P.; Ranadive, S. A.; Sack, J. S.; Tokarski, J. S.; Pavletich, N. P.; Lee, F. Y. F.;
Webster, K. R.; Kimball, S. D. J. Med. Chem. 2004, 47, 1719.
5. Toogood, P. L.; Harvey, P. J.; Repine, J. T.; Sheehan, D. J.; VanderWel, S. N.; Zhou,
H.; Keller, P. R.; McNamara, D. J.; Sherry, D.; Zhu, T.; Brodfuehrer, J.; Choi, C.;
Barvian, M. R.; Fry, D. W. J. Med. Chem. 2005, 48, 2388.
6. Chu, X. J.; DePinto, W.; Bartkovitz, D.; So, S. S.; Vu, B. T.; Packman, K.; Lukacs, C.;
Ding, Q.; Jiang, N.; Wang, K.; Goelzer, P.; Yin, X.; Smith, M. A.; Higgins, B. X.;
Chen, Y.; Xiang, Q.; Moliterni, J.; Kaplan, G.; Graves, B.; Lovey, A.; Fotouhi, N. J.
Med. Chem. 2006, 49, 6549.
5.8. Metabolic stability
Compounds were dissolved in DMSO at 10 lM concentration.
Human cDNA expressed cytochrome P450 isoforms (supersomes)
were purchased from Gentest (Woburn, MA). All chemicals used
were of analytical grade and commercially available. The potential
inhibitory effect was investigated against cDNA expressed human
CYP4503A4 supersome using typical substrates incubated at their
respective Km concentration. The known inhibitor ketoconazole
was included to check the inhibition response. Analysis of both
substrate and metabolite was done by LC/MS/MS.
7. Parry, D.; Guzi, T.; Seghezzi, W.; Paruch, K.; Doll, R.; Nomeir, A.; Wang, Y.; Oft,
M.; Kirschmeier, P.; Chen, T.; Lees, E. Poster no. 4371, American Association
Cancer Research, Los Angeles, CA, April 14–18, 2007.
8. Wyatt, P. G.; Woodhead, A. J.; Berdini, V.; Boulstridge, J. A.; Carr, M. G.; Cross, D.
M.; Davis, D. J.; Devine, L. A.; Early, T. R.; Feltell, R. E.; Lewis, E. J.; McMenamin,
R. L.; Navarro, E. F.; O’Brien, M. A.; O’Reilly, M.; Reule, M.; Saxty, G.; Seavers, L.
C. A.; Smith, D.-M.; Squires, M. S.; Trewartha, G.; Walker, M. T.; Woolford, A. J.-
A. J. Med. Chem. 2008, 51, 4986.
5.9. Plasma protein binding
Plasma protein binding was performed as previously
described.16
5.10. In vivo pharmacokinetics
9. Jones, C. D.; Andrews, D. M.; Barker, A. J.; Blades, K.; Daunt, P.; East, S.; Geh, C.;
Graham, M. A.; Johnson, K. M.; Loddick, S. A.; McFarland, H. M.; McGregor, A.;
Moss, L.; Rudge, D. A.; Simpson, P. B.; Swain, M. L.; Tam, K. Y.; Tucker, J. A.;
Walker, M. Bioorg. Med. Chem. Lett. 2008, 18, 6369.
10. Lapenna, S.; Giordano, A. Nat. Rev. Drug Discovery 2009, 8, 547.
11. Brasca, M. G.; Albanese, C.; Amici, R.; Ballinari, D.; Corti, L.; Croci, V.; Fancelli,
D.; Fiorentini, F.; Nesi, M.; Orsini, P.; Orzi, F.; Pastori, W.; Perrone, E.; Pesenti,
E.; Pevarello, P.; Riccardi-Sirtori, F.; Roletto, F.; Roussel, P.; Varasi, M.; Vulpetti,
A.; Mercurio, C. ChemMedChem. 2007, 6, 841.
12. D’Alessio, R.; Bargiotti, A.; Metz, S.; Brasca, M. G.; Cameron, A.; Ermoli, A.;
Marsiglio, A.; Polucci, P.; Roletto, F.; Tibolla, M.; Vazquez, M. L.; Vulpetti, A.;
Pevarello, P. Bioorg. Med. Chem. Lett. 2005, 15, 1315.
13. Vulpetti, A.; Pevarello, P. Curr. Med. Chem.: Anti-Cancer Agents 2005, 5,
561.
The pharmacokinetic profile of the compounds was investigated
in overnight fasted male Nu/Nu mice following a single dose given
intravenously (iv) or orally (po). The vehicle used was 5% dextrose
solution. A total of six mice were treated (three for each leg). Blood
samples of each mouse were collected from the saphenous vein at
predose, 0.083, 0.5, 1, 6, and 24 h postdosing following iv dosing,
and at predose, 0.25, 0.5, 1, 6 and 24 h following oral dosing. Sam-
ples were centrifuged at 10,000g for 3 min at 4 °C and the plasma
was stored at ꢀ80 °C until analysis. Samples were analyzed by LC/
MS/MS technique.
14. Coordinates of the CDK2 complex with compound 31 have been deposited in
the Protein Data Base under accession code 2WPA, together with the
corresponding structure factor files.
5.11. Immunohistochemistry
15. Colombo, M.; Riccardi-Sirtori, F.; Rizzo, V. Rapid Commun. Mass Spectrom. 2004,
18, 511.
Immunohistochemistry was performed on serial sections of for-
malin fixed-paraffin embedded A2780 tumors. After dewaxing and
heat-induced epitope retrival, sections were processed as previ-
ously described17 for phosphor-pRb (pRb phosphorylated in Threo-
nin 821 rabbit polyclonal antibody, Biosource cat 44–582). For
BrdU staining, tumors, denaturated and pretreated with HCl 4 N
and proteinase K, were incubated with Dako ARK kit following
manufacturer’s procedure. All samples were then counterstained
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