Benzodipyrazoles: A new class of potent CDK2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.01.023

The synthesis and the preliminary expansion of this new class of CDK2 inhibitors are presented. The synthesis was accomplished using a solution-phase protocol amenable to rapid parallel expansion and suitable to be scaled-up in view of possible lead devel

Full text of DOI:10.1016/j.bmcl.2005.01.023

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1315–1319
Benzodipyrazoles: a new class of potent CDK2 inhibitors
Roberto DÕAlessio,a,* Alberto Bargiotti,a Suzanne Metz,c M. Gabriella Brasca,a
Alexander Cameron,a Antonella Ermoli,a Aurelio Marsiglio,b Paolo Polucci,a
Fulvia Roletto,b Marcellino Tibolla,a Michael L. Vazquez,c
Anna Vulpettia and Paolo Pevarelloa
aChemistry Department, Nerviano Medical Sciences, Oncology Business Unit, Viale Pasteur 10, 20014 Nerviano (MI), Italy
bBiology Department, Nerviano Medical Sciences, Oncology Business Unit, Viale Pasteur 10, 20014 Nerviano (MI), Italy
cChemistry Department, Pfizer Inc., St. Louis, MI, USA
Received 21 October 2004; revised 10 January 2005; accepted 12 January 2005
Abstract—The synthesis and the preliminary expansion of this new class of CDK2 inhibitors are presented. The synthesis was
accomplished using a solution-phase protocol amenable to rapid parallel expansion and suitable to be scaled-up in view of possible
lead development. Following a medicinal chemistry program aimed at improving cell permeability and selectivity, a series of com-
pounds with nanomolar activity in the biochemical assay and able to efficiently inhibit tumor cell proliferation has been obtained.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Recent discoveries in diverse fields of biomedical re-
search have elucidated the molecular basis of cell cycle
control and have shown how alterations to this homeo-
static mechanism play an important role in cancer devel-
opment.1,2 Cyclin-dependent kinase
2 (CDK2) in
complex with cyclins E and/or A is a key cell cycle reg-
ulator and continues to be an attractive target for the
discovery of new anti-tumor agents.3 In particular,
inhibitors of CDK2/cyclin A/E have already progressed
into clinical trials with encouraging early results.4,5 Ben-
zodipyrazoles (BDPs) as CDK2 inhibitors originated
from high throughput screening of our internal chemical
collection. The hit compound of this class (1, Fig. 1), a
tetrahydro-benzodipyrazole (TH-BDP) derivative bear-
ing a sulfamidophenyl moiety was found to possess a
remarkable activity on the CDK2/cyclinA complex
(IC50 = 7 nM) and to be an ATP competitive inhibitor.
Figure 1. The structure of 1 and the general formula of BDPs.
the key features for binding of the ligand to the adenine,
phosphate and ribose regions of the ATP pocket,
respectively.
In spite of the notable potency in the biochemical assay,
the anti-proliferative activity of this compound, mea-
sured as inhibition of the A2780 ovarian tumor cell line,
was disappointing (IC50 > 20lM). This is likely due to
the presence of both the sulfamoyl and carbamoyl moi-
eties which, although important for binding, may pre-
vent this compound from penetrating cells efficiently.
A medicinal chemistry program was therefore started
with the aim to improve cell permeability, while main-
taining activity on CDK2/cyclin A/E in the nanomolar
range.
The crystal structure of CDK2/cyclin A in complex with
the compound 1 was determined (Fig. 2). The pyrazole
ring A, the carboxamido and the sulfamido groups are
Keywords: CDK2; Cyclins; Kinase selectivity; Tumor cell proliferation
inhibition.
*
Corresponding author. Tel.: +39 0331 58 1523; fax: +39 0331 58
1757; e-mail: roberto.dalessio@nervianoms.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.023

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R. D’Alessio et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1315–1319
for ring A was particularly profitable, allowing us to in-
crease the overall yield and to reduce reaction times.
Interestingly, only one regioisomer was formed under
tritylation and the protecting group was directly re-
moved during the closure of the second pyrazole ring
C. After condensation with diethyl oxalate, the diketo-
ester 5 was reacted with a suitably substituted hydrazine
to form the BDP 6. The trityl-protecting group is nor-
mally lost during the cyclization reaction if a salified
form of the hydrazine is used (i.e., hydrochloride).
Optionally, diluted hydrochloric acid can be added to
complete the deprotection, once the cyclization has oc-
curred. In the final step, the ester was then converted
to the TH-BDP amide 7 by treatment with ammonium
hydroxide in methanol. All the intermediates were crys-
talline and could be obtained in very good yields with-
out chromatographic purification. The ethoxycarbonyl
and aminocarbonyl groups of compounds 6 and 7 could
be further manipulated to obtain compounds 8–16 as
shown in Scheme 2.
Figure 2. The crystal structure of CDK2/cyclin A in complex with
compound 1.
2. Chemistry
The synthetic methodology used to produce this class of
compounds (Scheme 1) started from the commercially
available cyclohexanedione that was condensed with
N,N-dimethylformamide dimethyl acetal to obtain the
adduct 2.6
The aromatization of the central ring B of TH-BDPs to
obtain the corresponding dihydrobenzo-dipyrazole
(DH-BDP) counterparts could be conveniently accom-
plished by the use of 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (Scheme 1).
In the second step, the adduct 2 was reacted with hydra-
zine dihydrochloride to obtain the cyclic intermediate 3,
that was then protected with trityl chloride to give the
intermediate 4. The use of trityl as the protecting group
Finally, the preparation of the 4,4-gem-dimethyl deriva-
tive 17 (Table 3) was accomplished, according to the
general synthetic scheme, using dimedone as a starting
material.
N
OMe
OMe
O
O
O
O
NH2-NH2 . 2HCl
TrCl/Et3N
N
N
Tr
N
NaOH/MeOH
reflux, 3 h
98%
N
H
reflux, 1 h
90%
DCM
94%
N
O
O
2
3
4
(COOEt)2
93%
OEt
(Me3Si)2NLi
O
O
R1
R1
NH4OH/MeOH
25-60°C/1-2 days
N
N
R1-NH-NH2
AcOH
N
N
OEt
NH2
Tr
N
O
O
N
O
N
N
65°C/3h
81-92%
75-78%
N
H
N
H
7
6
5
R1
R1
DDQ/dioxane
N
N
N
N
N
N
NH2
100°C-2.5h
(prepared from
dimedone)
R2
R2
N
N
O
N
73-87%
N
H
N
H
N
H
TH-BDPs
DH-BDPs
17
Scheme 1. General scheme for the synthesis of TH-BDPs and DH-BDPs.

R. D’Alessio et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1315–1319
1317
3. Results and discussion
R2
OH
Conditions
Low lipophilicity and the presence of several NH bonds
may contribute to the poor cell permeability of the
parent compound 1. In order to improve these features,
some modifications were designed, trying to retain those
functionalities directly involved in the binding to the
protein as much as possible.
NaOH/MeOH,
8
9
reflux-1.5h; 85-92%
O
6 (R2 = COOEt)
H
N
MeNH2/MeOH,
65°C-1 day; 90%
O
H
N
NH2NH2.H2O/MeOH,
reflux-3h; 89-90%
NH2
10
O
As a first approach, we focused our attention on R1 that,
according to X-ray data, points towards the ribose re-
gion of the ATP pocket and offered wider room for
modifications. As shown in Table 1, the progressive sub-
stitution at sulfonamido (compd 7a–e) or its replace-
ment with other groups, while reducing the activity on
CDK2, led to an increase of the anti-proliferative activ-
ity, both in TH-BDP and DH-BDP series.
H
N
NH2OH.HCl/CDI/DMF,
R.T.-1 day; 45%
R1
OH
O
11
12
N
N
O
O
R2
H
N
RONH2/HOBT/EDC,
R.T-1 day; 55%
N
N
H
CH3MgCl/TEA,
0°C - 1h; 22%
13
14
O
7 (R2 = CONH2)
TFAA/Py,
Particularly, phenyl 7h was better than benzyl 7w, while
compound 7u, bearing a 2-pyridyl moiety was better
than the phenyl derivative. Some aliphatic substituents
were also explored: among them trifluoroethyl group
7y appeared to be the most promising.
R.T.-30'; 85%
N
NH2
OH
NH2OH.HCl/K2CO3/
EtOH, reflux-4h; 76%
15
N
NaOH/NaClO,
100°C-15 min; 76%
16
NH2
Thus, looking at the potency against CDK2/cyclin A
combined with the anti-proliferative activity, the sulfo-
namido could be conveniently replaced by other groups.
Generally, more lipophilic substituents led to com-
pounds that were less potent on CDK2/cyclin A (due
to the loss of H-bond interactions involving the
Scheme 2. Synthetic manipulations at R2.
Table 1. Variations at R1 (refer to the general formula in Fig. 1)
Compd
R2 = –CONH2
R1
CDK2/cyclin A IC50 (lM)
A2780 cells IC50 (lM)
R3
–CH2–CH2–
–CH@CH–
–CH2–CH2–
–CH@CH–
7a
7b
7c
7d
7e
7f
–Ph–4-SO2NH2
–Ph–4-SO2NHMe
–Ph–4-SO2NMe2
–Ph–4-SO2NHBu
–Ph–4-SO2N(CH2–CH2)2N–Me
–Ph–4-SO2Me
–H
0.004
0.04
0.54
0.21
>10
0.03
0.46
0.15
0.14
0.14
0.16
0.27
0.08
0.51
0.19
0.56
0.25
0.16
0.36
0.42
0.26
1.60
2.20
0.29
0.02
1.15
0.0003
0.002
0.010
0.002
0.150
0.001
0.078
0.014
0.015
0.009
0.008
0.036
0.013
0.037
0.005
0.018
0.075
0.009
0.029
0.028
0.008
0.062
0.140
0.018
0.002
0.028
>20
>20
8.74
>20
1.89
1.25
>20
8.81
3.54
3.86
0.88
1.46
2.73
1.65
1.77
2.53
1.94
4.07
>20
0.40
2.10
1.50
0.65
4.48
2.98
0.58
0.26
0.92
13.92
9.76
>20
18.19
5.27
8.05
6.05
>20
8.50
>20
>20
>20
8.75
>20
1.88
4.50
4.10
4.06
7g
7h
7i
–Ph
–Ph–4-Me
7j
–Ph–4-OMe
–Ph–4-Cl
–Ph–4-F
7k
7l
7m
7n
7o
7p
7q
7r
7s
7t
–Ph–4-CF3
–Ph–4-OCF3
–Ph–4-CN
–Ph–4-N(CH2–CH2)2O
–Ph–4-(2-imidazolo)
–Ph–3-Me
–Ph–3-Cl
–Ph–3-F
7u
7v
7w
7x
7y
7z
–2-pyridyl
–3-pyridyl
–Bn
–Me
13.45
2.67
–CH2–CF3
–CH2–CH2–OH
Values are means of at least three experiments.
Only compd with IC50 (CDK2/cyclin A) <1lM were tested on A2780 cell line.

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R. D’Alessio et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1315–1319
sulfonamido group), but more effective on cells, proba-
bly as a result of their improved permeability.
the aromatization of the central ring B, the flatter scaf-
fold is less able to discriminate amongst the various
shapes of kinase ATP pockets.
Some variations were also performed at R2, in an at-
tempt to conveniently replace the carboxamido group
(Table 2). Primary carboxamides (7j, d, i) proved to be
the most active derivatives with a reduction of the activ-
ity upon N-alkylation (9d). Esters (6j, d, i) were consid-
erably less active on CDK2 than the corresponding
amides, while acids (8j, d, i) and the ketone 16i retained
some activity, according to the binding mode of the par-
ent compound. Among other functionalities explored,
the hydroxamic acid 11j was the most promising one,
showing activity comparable to the parent amide and,
at the same time, conferring higher solubility to the
scaffold.
In this view, the 4,4-gem-dimethyl derivative subclass, a
representative of which is shown in Table 3, has been re-
cently designed to improve selectivity for CDK2 versus
GSK3b kinase.
This has been achieved exploiting a key difference be-
tween the two kinases. Figure 3 shows the 4,4-gem-di-
methyl derivative 17 docked in CDK2 and GSK3b
crystal structure solved in house.7 The larger size of
GSK3-Cys 199 compared with CDK2-Ala 144 and the
nonplanar nature of the GSK3-Leu 132 as compared
to CDK2-Phe 80 would be responsible for the selectivity
due to gem-dimethyl clashing. As shown in Table 3,
compound 17 was able to inhibit the CDK2/cyclin A
complex at concentrations comparable to those of the
corresponding DH-BDP 7i without affecting GSK3b
kinase.
A considerable improvement on activity and selectivity
was achieved through modifications of the central ring
B (R3). DH-BDPs (R3 = –CH@CH–) were always more
active than their TH-BDP counterparts (R3 = –CH2–
CH2–) both in terms of potency on CDK2/cyclin A
complex and cytostatic activity on A2780 cell line (Ta-
bles 1 and 2). On the other hand, the higher activity of
DH-BDPs corresponded to a marked reduction of their
selectivity evaluated on a panel of different kinases (data
not shown). It seems, therefore, that although a higher
potency against CDK2/cyclin A could be obtained by
4. Conclusions
Starting from compound 1, a sulfonamido derivative en-
dowed with good potency as a CDK2/cyclin A inhibitor,
but devoid of anti-proliferative activity on cells, a series
Table 2. Variations at R2 (refer to the general formula in Fig. 1)
Compd
R1
R2
CDK2/cyclin A IC50 (lM)
A2780 cells IC50 (lM)
R3
–CH2–CH2–
–CH@CH–
–CH2–CH2–
6.05
–CH@CH–
6j
–Ph–4-OMe
–COOEt
–CONH2
–COOH
8.10
0.14
2.10
2.10
0.69
4.50
7.50
7j
0.009
0.21
1.30
1.46
>20
8j
10j
11j
12j
14j
15j
16j
–CONHNH2
–CONHOH
–CONHOCH2CH@CH2
–CN
0.029
7.50
2.82
4.60
1.75
0.31
–C@N(OH)NH2
–NH2
>10
6d
7d
8d
9d
12d
–Ph–4-SO2NHBu
–COOEt
–CONH2
10
0.21
1.20
0.60
2.85
0.002
9.76
1.25
–COOH
–CONHMe
–CONHOCH2CH@CH2
0.16
0.20
10.20
2.90
8.70
6i
–Ph–4-Me
–COOEt
–CONH2
–COMe
8.50
0.14
0.90
7i
16i
0.015
8.05
>20
0.88
Values are means of at least three experiments.
Only compd with IC50 (CDK2/cyclin A) <1 lM were tested on A2780 cell line.
Table 3. 4,4-gem-dimethyl series (refer to the general formula in Fig. 1)
Compd
R1
R2
R3
CDK2/cyclin A IC50 (lM)
GSK3b IC50 (lM)
A2780 cells IC50 (lM)
7i
7i
17
–Ph–4-Me
–CONH2
–CH2–CH2–
–CH@CH–
–CH2–C(CH3)2À
0.140
0.015
0.087
0.460
0.023
>10
8.05
0.88
1.65
Values are means of at least three experiments.

R. D’Alessio et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1315–1319
1319
more reasonable lipophilicity and an improved cell
permeability.
The new derivatives, still maintaining potency on
CDK2/cyclin A in the nanomolar range, are able to
block tumor cell growth at low micromolar concentra-
tions. In addition, a subset of highly selective benzodipy-
razoles was designed exploiting the structural diversity
existing among kinase ATP pockets. Ongoing tests to
assess the selectivity of this class on a wider panel of ki-
nases and preliminary in vivo efficacy studies on tumor
models will tell more about the real potential of this
new class of CDK2 inhibitors as anti-cancer agents.
References and notes
1. Stein, G. S.; Baserga, R.; Giordano, A.; Denhard, D. T. In
The Molecular Basis of Cell Cycle and Growth Control;
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Kinase Inhibitors. In Progress in Cell Cycle Research;
´ ´
Meijer, L., Jezequel, A., Roberge, M., Eds.; 2003; Vol. 5,
pp 235–248.
3. For recent reviews, see: Huwe, A.; Mazitschek, R.; Giannis,
A. Angew. Chem. Int. Ed. 2003, 42(19), 2122–2138; Kong,
N.; Fotouhi, N.; Wovkulich, P. M.; Roberts, J. Drugs
Future 2003, 28(9), 881–896.
4. Sausville, E. A. Curr. Med. Chem.: Anti-Cancer Agents
2003, 3(1), 47–56.
5. Fischer, P. M.; Gianella-Borradori, A. Exp. Opin. Invest.
Drugs 2003, 12(6), 955–970.
Figure 3. Compound 17 docked in CDK2 and GSK3 crystal structure.
6. Peet, N. P.; LeTourneau, M. E. Heterocycles 1991, 32(1),
41.
7. Bertrand, J. A.; Thieffine, S.; Vulpetti, A.; Cristiani, C.;
Valsasina, B.; Knapp, S.; Kalisz, H. M.; Flocco, M. J. Mol.
Biol. 2003, 333(2), 393–407.
of new derivatives was synthesized through chemical
modifications designed to give the compounds both a