Synthesis and evaluation of pyrazolo[1,5-b]pyridazines as selective cyclin dependent kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.09.069

A novel series of pyrazolo[1,5-b]pyridazines have been synthesized and identified as cyclin dependant kinase inhibitors potentially useful for the treatment of solid tumors. Modification of the hinge-binding amine or the C(2)- and C(6)-substitutions on the pyrazolopyridazine core provided potent inhibitors of CDK4 and demonstrated enzyme selectivity against VEGFR-2 and GSK3β.

Full text of DOI:10.1016/j.bmcl.2008.09.069

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5758–5762
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of pyrazolo[1,5-b]pyridazines as selective cyclin
dependent kinase inhibitors
a
a
b
c
Kirk L. Stevens a, , Michael J. Reno , Jennifer B. Alberti , Daniel J. Price , Laurie S. Kane-Carson ,
*
Victoria B. Knick d, Lisa M. Shewchuk b, Anne M. Hassell b, James M. Veal a, , Stephen T. Davis d,à
,
Robert J. Griffin e, Michael R. Peel a,§
a Department of Oncology, Medicinal Chemistry, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
b Department of Computational and Structural Chemistry, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
c Department of Molecular Biochemistry, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
d Department of Cancer Biology, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
e Department of Drug Metabolism and Pharmacokinetics, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of pyrazolo[1,5-b]pyridazines have been synthesized and identified as cyclin dependant
kinase inhibitors potentially useful for the treatment of solid tumors. Modification of the hinge-binding
amine or the C(2)- and C(6)-substitutions on the pyrazolopyridazine core provided potent inhibitors of
CDK4 and demonstrated enzyme selectivity against VEGFR-2 and GSK3b.
Received 8 September 2008
Revised 16 September 2008
Accepted 19 September 2008
Available online 24 September 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Cyclin-dependant kinase inhibitor
CDK2
CDK4
VEGFR-2
GSK3b
Pyrazolo[1,5-b] pyridazine
Cancer
The current treatments for cancer have a highly fragmented
market since these agents are only active in specific patient popu-
lations or tumor types. The accumulation of genetic and biological
information from solid tumors is providing additional targets that
have the potential of yielding drugs with broad activity and less
toxicity than current therapies. Human genetic evidence supports
the cyclin dependant kinase (CDK) family as key therapeutic tar-
gets for disease modification. In particular, the cyclin D/CDK4/
pRb tumor suppressor pathway of cell growth control is frequently
deregulated in human cancers.1 Several lines of evidence suggest
that perturbation of this pathway is on the critical path toward
tumorigenesis.2 The CDK4 pathway of cell growth control is fre-
quently altered by overexpression of cyclin D1, the activation sub-
unit of CDK4, in breast, lung, and pancreatic cancers.3 Also,
antisense RNA to cyclin D1 sensitizes tumor cells to chemother-
apy4 and cyclin D1 overexpression correlates with poor disease
outcome.5 An estimated 60–70% of human cancers including
breast, non-small cell lung, and colon cancer are pRb positive
and could respond to selective inhibition of CDK4.6
N
N
6
5
N
2
3
4
N
N
N
H
1
Following a screening campaign on CDK4 and subsequent cross-
screening of hits against a panel of kinases, pyrazolopyridazine 1
was identified as having compelling CDK family activity, being
roughly equipotent on the 3 isoforms screened at approximately
100 nM.7 Screening hit 1 was attractive in that, despite its relatively
modest molecular weight (252 amu), it was >100-fold selective over
* Corresponding author. Tel.: +1 919 483 7707; fax: +1 919 483 6053.
E-mail address: Kirk.L.Stevens@gsk.com (K.L. Stevens).
Present address: Serenex Inc., Durham, NC, USA.
à
Present address: TransTech Pharma Inc., High Point, NC, USA.
Present address: Scynexis Inc., Durham, NC, USA.
§
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.069

K. L. Stevens et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5758–5762
5759
I-
acceptor pair to the hinge region of the ATP binding site and the
pyrazolopyridazine presents its extended electronegative edge to
the catalytic lysine. Subsequent crystal structures of compounds
within the series complexed with CDK2 suggested the binding
mode was invariant to the substitutions discussed here. To affect
CDK4 potency, we targeted three areas of the of the ATP binding
pocket that historically have improved potency in other kinases:
(1) the inner hydrophobic area near the phenylalanine gatekeeper,
(2) the hydrophobic pocket beneath the G-rich loop, and (3) the
hinge/solvent exposed area.12,13
Our crystal structure suggested that the space near the gate-
keeper was accessible from the 2-position of the pyrazolopyrid-
azine. The CDK family of kinases possesses a sterically imposing
phenylalanine gatekeeper that was expected to be unaccommodat-
ing to larger substituents. Indeed, compounds were prepared
which verified that even a methyl group lowers CDK4 activity 5-
to 10-fold (Table 1, 7 and 11).14 This trend is in stark contrast to
VEGFR-2, which contains a more permissive valine gatekeeper
and sees increasing activity with increasingly large substitutions
at this position (Table 1), 7–9, 11–13).
Crystallography also suggested that adding bulk either directly
at the 6-position or with a 1-atom linker would access the area un-
der the G-rich loop. In order to affect substitution at the 6-position
we needed a synthetic handle. We envisioned a cross coupling
reaction or direct displacement of a halogen. Since we did not ex-
pect a halogen to survive the cycloaddition chemistry to 4, due to
displacement by water, we used a methoxy group on the pyrida-
zine (Schemes 1 and 2, R1 = OMe) which we expected could be con-
verted to a leaving group for subsequent chemistry. Standard
demethylation conditions using mineral acids with heating on 4
or 1 gave only moderate conversion to the respective hydroxyl
compounds in about 50% isolated yield. Therefore, alternative
chemistry was developed to convert this methoxy group to a cross
coupling partner or appropriate leaving group. Nucleophilic condi-
tions using morpholine for deprotection proved to be the most effi-
cient and highest yielding (14, Scheme 2) providing the
pyrazolopyridazinone in 85–90% yield.15 We converted this com-
pound to the triflate utilizing N-phenyl triflimide in 75–85% yield.
We found the triflate leaving group to be more effective than con-
version to a halogen in the subsequent displacement reactions to
prepare amine substitutions (16) or as a cross coupling partner
to prepare aryl or vinyl substitutions (17).
Unfortunately, improvement of CDK4 potency was not achieved
with these substitutions (Table 2); however, there are notable ki-
nase selectivity trends. Specifically, GSK3b is less tolerant of bulk
at this position. Even a two carbon vinyl substitution (17) reduces
GSK3b enzyme potency as compared to 1. Anything larger, such as
a cycloalkyl (16), an aryl or heteroaryl (18, 19), or heteroalkyl (20)
removes measurable GSK3b activity (Table 2). Electronic factors
are also involved as all oxygen linked substituents had reduced po-
tency (14, 22, 23).
R1
NH2
N
R1
N
+
N
N
2
c
N
a
b
N
N
R1
O
3
4
R1
R1
N
N
N
N
N
N
6
2
d
5
3
NH
4
N
O
H2N
N
H
N
N
N
6
H
5
1, R1 = H
Scheme 1. Reagents and conditions: (a) HOSA, KHCO3, KI, H2O (use product
without purification); (b) butyne-2-one, KOH, CH2Cl2/H2O, (80–90%); (c) DMF/DMA
(75–90%); (d) K2CO3, 6, DMF (55–77%).
12 of the 18 non-CDK family kinases screened and ꢀ10-fold selec-
tive over the rest. However, developability profiling determined 1
suffered from high intrinsic clearance as measured by stability in
mouse liver S9 fractions. To be considered a good starting point
for the program, it was necessary to demonstrate that the scaffold
had the capacity for improved potency, CDK2 selectivity, and met-
abolic stability. A preliminary synthetic chemistry effort was mobi-
lized to determine if improvements in these properties were
achievable among close analogs.
Pyrazolopyridazines were prepared in an efficient manner by
initial reaction of pyridazines (2) with hydroxyl amine sulfonic
acid (HOSA) to afford the aminated pyridazines (3, Scheme 1). This
was followed by a formal [3+2] cycloaddition between the desired
1-amino-pyridazines and butyn-2-one under basic conditions to
give pyrazolo[1,5-b]pyridazines (4). Condensation with DMF/
DMA afforded enamines (5), which gave the corresponding pyrim-
idine 1 upon exposure to guanidine (6) under basic conditions at
elevated temperatures.8
With an efficient synthesis in hand, we focused our attention on
optimizing kinase potency and selectivity. A crystal structure of 1
bound to the kinase domain of human CDK2 was obtained
(Fig. 1).9,10
CDK4 is very difficult to express and purify and, to our knowl-
edge, there are no CDK4 structures in the literature. Therefore,
CDK2 was utilized as a surrogate for CDK4 due to the close homol-
ogy within the active site and based on information gathered from
ongoing efforts with CDK2.11 The crystal structure revealed that
the amino-pyrimidine acts as a two-point hydrogen-bond donor/
We considered a structural rationale for the selectivity of com-
pounds 16–23. One publicly available crystal structure of GSK3b
(pdb code 1O9U) aligns well with our internal crystal structures
bound to a variety of ligands.16 These structures show Phe67 ex-
tended down, and tucked under, the G-rich loop, thereby occupy-
ing the same space accessible from the 6-position of the
pyrazolopyridazine. Admittedly, there are multiple publicly avail-
able crystal structures of ligand-bound GSK3b showing Phe67 in
a variety of positions, however it is unclear from static structures
the relative stability of these conformations. It is postulated here
that CDK-family kinases, which have a tyrosine at this position
(Tyr17 in CDK4 and Tyr15 in CDK2), more stably adopt this Phe/
Tyr ‘out’ conformation that would be necessary to accommodate
a 6-substituent by directly coordinating the catalytic lysine and
the proximal conserved glutamate (Lys33 and Glu51 in CDK2).
Figure 1. CDK2 crystal structure of 1 showing interaction of the pyrazolopyridazine
with catalytic lysine and phenylalanine gatekeeper.

5760
K. L. Stevens et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5758–5762
Table 1
Comparison of CDK4 and VEGFR-2 enzyme potency
N
4
N
4
N
N
6
6
N
N
N
N
R
R
2
2
5
5
N
3
3
N
N
N
N
H
N
H
a
a
a
a
R
Compound #
CDK4 IC50
(l
M)
VEGFR-2 IC50
(lM)
Compound #
CDK4 IC50
(
lM)
VEGFR-2 IC50 (lM)
H
1
7
8
9
0.080
0.800
2.00
>20
>20
3.2
10
11
12
13
0.012
0.050
0.025
0.800
0.400
0.063
0.063
0.025
Methyl
Ethyl
n-Butyl
>40
2.5
a
Values are the mean of two or more experiments.
Figure 2 shows the overlay of a ‘Phe-in’ GSK3b structure with the
‘Tyr-out’ conformation observed with compound 41 bound to
CDK2.10
Table 2
CDK4 and GSK3b enzyme activity—6-position substitutions
N
R1
N
It is known that many kinases prefer an aromatic group along
the hinge,12 as opposed to the cyclopropylamino substitution at
the 2-position of the pyrimidine as described. Indeed, potency is
improved by replacing the cyclopropylamine with a substituted
aniline, however, it also significantly erodes the selectivity against
other kinases, such as GSK3b (Table 3, 27–34). Alkyl amino substit-
uents other than cyclopropyl made poor CDK4 inhibitors (Table 3,
24–26). Aniline substituents had a wide variety of effects on the
CDK activity and selectivity. The para-N-methylpiperazine aniline
substitution (Table 3, 10) gave moderate CDK2 selectivity (ꢀ8Â)
however addition of an additional meta-substituent resulted in loss
of CDK2 selectivity (Table 3), 28). While several extremely potent
meta- and para-substituted anilines were identified (Table 3), 30–
34), highlighting the scaffold’s capacity for high CDK4 potency,
they also had less desirable selectivity against both CDK-family
members and other kinases.
N
N
6
5
2
3
4
N
N
H
Compound #
R1
H
CDK4
GSK3b
a
a
IC50
(
l
M)
IC50
(
l
M)
1
0.080
2.00
1.600
25
25
3.2
>32
>32
25
25
25
25
14
16
17
18
19
20
21
22
23
–OCH3
–NH-Cyclopentyl
–CH@CH2
2-Thiophene
p-Fluorophenyl
N-morpholine
Pyrrolidine
0.160
0.200
0.250
0.250
0.400
0.500
1.30
Selectivity over CDK2 at the expense of broader kinase inhibi-
tion profile is not an acceptable profile for a CDK4 inhibitor. How-
ever, the selectivity trends we observed suggested that proper
combinations of substitutions on the template could be expected
to reassert the selectivity over GSK3b while maintaining CDK2
–OH
–O-Isopropyl
1.60
a
Values are the mean of two or more experiments.
H
N
N
N
N
6
5
2
3
4
N
OMe
OTf
N
N
N
N
N
N
c
N
N
H
16
a, b
d
N
N
N
N
N
N
N
N
N
6
5
H
H
2
14
3
15
4
N
N
N
H
17
Scheme 2. Reagents and conditions: (a) morpholine, 90 °C, 24 h (85–90%); (b) NaH, Tf2NPh, DMF (75–85%) (c) DIEA, cyclopentylamine, DMF (66%); (d) vinyltributylstannane,
Pd2(dba)3, DMF (55%).

K. L. Stevens et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5758–5762
5761
Table 4
CDK4 and GSK3b enzyme activity—double substituted analogs
N
R1
N
N
N
R2
N
N
H
Compound R1
#
R2
CDK4
IC50
CDK2
IC50
GSK3b
IC50
a
a
a
(
lM)
(lM)
(
lM)
1
H
H
NA, cyclopropylamine
0.080
0.120
1.600
N
10
0.012
0.100
0.200
N
Figure 2. CDK2 crystal structure with (41) (green) and overlay with ‘Phe-in’ (pink)
of GSK3b (pdb code 1O9U). GSK3b ligand and all residues except Phe67/Tyr15
removed for clarity.
N
39
40
41
p-F-Phenyl-
–O-Isopropyl
–Morpholine
0.026
0.045
0.038
0.740
0.230
0.450
>25
N
Table 3
CDK4, CDK2, and GSK3b enzyme activity—substitution at R2
N
0.850
N
N
N
N
OH
0.690
O
N
N
a
R2
Values are the mean of two or more experiments.
N
N
H
respectively). Table 4 illustrates select analogs with substituted
anilino hinge-binders crossed with 6-position substitutions that
achieve both selectivity goals. Compound 39 (CDK4 IC50 = 26 nM)
highlights this effort, achieving >10-fold CDK2 selectivity and
>100-fold GSK3b selectivity.
Compound R2
#
CDK4 IC50
CDK2 IC50
M)
GSK3b IC50
(lM)
a
a
a
(l
M)
(l
1
–Cyclopropyl
–CH2CF3
–CH2-(p-chlorophenyl)
–CH2CH2CH2-(N-
morpholine)
0.080
1.00
1.00
3.20
0.120
1.00
1.00
NA
1.600
>32
10
24
25
26
The majority of the compounds described were profiled for
metabolic stability in mouse liver S9 fractions. While modest
improvements were seen for some anilines and some 6-substitu-
tions relative to the original hit (1), most compounds profiled
showed more than 85% turnover after 60-min incubation in mouse
liver S9 fractions. As expected from the high turnover, representa-
tive examples did not show acceptable oral exposure in mice. Com-
pounds 20 and 23 had dose normalized AUC > 1000 ng h/mL/mg/
kg. While the morpholine and –O-iso-propyl derivatives were the
best of the cyclopropylamine substituted series, this trend did
not hold for the p-(N-methyl)-N-piperazine aniline series. Com-
pound 41,17 however, represented an improvement in turnover
in mouse liver S9 fractions (46% after 60-min incubation). This
compound also demonstrated acceptable selectivity over CDK2
and GSK3b, although it failed to achieve any oral exposure. Com-
pound 41 displayed much greater exposure when delivered i.p.
(DNAUC > 7000 ng h/mL/mg/kg) suggesting that poor adsorption
rather than metabolism is most likely the cause of poor oral
exposure.
In summary, a flexible, efficient synthesis was used to prepare
analogs which varied at the 6-position of the pyrazolopyridazine
and the amine on the pyrimidine hinge binder. Based on X-ray
studies, analogs were prepared which improved or maintained
CDK4 enzyme activity while improving kinase selectivity over
VEGFR-2 and GSK3b. With limited, iterative chemistry, analogs
were quickly identified that demonstrated the pyrazolopyridazine
template’s capacity for acceptable CDK2 selectivity and improved
metabolic stability. Further lead optimization details will be re-
ported in a subsequent communication.
32
N
N
N
R2
N
N
N
H
27
10
H
0.012
0.012
0.008
0.100
0.032
0.200
p-(N-methyl)-N-
piperazine
28
m-CF3, p-(N-methyl
piperazine)
m-CH2N(CH2CH3)2
p-NO2
m-NO2
p-CN
0.006
0.004
0.016
29
30
31
32
33
34
35
36
37
38
0.012
0.008
0.0003
0.025
0.004
0.016
0.160
0.790
2.000
0.008
0.050
0.003
0.0003
0.002
0.002
0.003
0.016
0.040
0.040
0.040
0.079
0.005
0.002
0.005
0.010
0.008
0.020
0.050
0.016
0.020
m-CN
m-(4-oxazolyl)
p-CONH2
p-CO2H
p-Isopropyl
m-
CONHCH2CH2N(CH2CH3)2
a
Values are the mean of two or more experiments.
selectivity, for example, by using p-fluorophenyl at R1 and 1-
methyl-4-phenyl piperazine at R2 (from compounds 19 and 10,

5762
K. L. Stevens et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5758–5762
(c) Vadivelan, S.; Sinha, B.; Irudayam, S.; Jagarlapudi, S. A. R. P. J. Chem. Inf.
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14. Radioactive Glutathione plate-binding assay of CDK4. Fifty microliters of reaction
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lM GST-RB protein, 1 lCi/mL
[
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-33P]ATP,10 mM MgCl2, 2.5 mM EDTA, 0.2 mg/mL BSA, 1 mM DTT and
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1.13
0.1 mg/mL BSA, 1 mM DTT, 2.5
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l
s. SPA assay of GSK3b. Thirty microliters reaction mixtures containing,
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l
l
l
l
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10. Image generated with PyMol (Delano Scientific).
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