6-Aryl-pyrazolo[3,4-b]pyridines: Potent inhibitors of glycogen synthase kinase-3 (GSK-3)

Article abstract of DOI:10.1016/S0960-894X(03)00645-0

A novel series of 6-aryl-pyrazolo[3,4-b]pyridines has been identified that are potent inhibitors of glycogen synthase kinase-3 (GSK-3).

Full text of DOI:10.1016/S0960-894X(03)00645-0

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3055–3057
6-Aryl-pyrazolo[3,4-b]pyridines: Potent Inhibitors of
Glycogen Synthase Kinase-3 (GSK-3)
Jason Witherington,* Vincent Bordas, Alessandra Gaiba, Neil S. Garton,
Antoinette Naylor, Anthony D. Rawlings, Brian P. Slingsby, David G. Smith,
Andrew K. Takle and Robert W. Ward
Department of Medicinal Chemistry, Neurology & GI Centre of Excellence for Drug Discovery, GlaxoSmithKline Research Limited,
New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK
Received 31 March 2003; accepted 22 April 2003
Abstract—A novel series of 6-aryl-pyrazolo[3,4-b]pyridines has been identified that are potent inhibitors of glycogen synthase
kinase-3 (GSK-3).
# 2003 Elsevier Ltd. All rights reserved.
Glycogen synthase kinase-3 (GSK-3) is a serine/threo-
nine kinase implicated in the control of several reg-
ulatory proteins.1 It was first discovered by virtue of its
ability to phosphorylate and inactivate glycogen syn-
thase, the regulatory enzyme of mammalian glycogen
synthesis. Since then a number of other substrates have
been identified, implicating GSK-3 in the regulation of
several physiological processes. Recently we identified
pyridazine 1 through pharmacophore searching of the
SmithKline Beecham in house database.2a Simplifica-
tion of pyridazine 1 afforded a series of pyrazolo[3,4-
b]pyridines2a and pyrazolo[3,4-b]pyridazines2b that were
potent inhibitors of GSK-3 (Fig. 1).
potency compared to the parent phenyl analogue 4.
Frommodelling studies, it was predicted that the phe-
nol moiety was involved in a H-bonding interaction
with Glu97 and Asp200 (Fig. 2b). The para methoxy
analogue 6 was inactive, possibly due to a steric clash
with Glu97.
Interestingly, the meta phenol analogue 8 displayed
comparable potency to the para phenol analogue 5.
Modelling studies predicted this also to be as a result of
a hydrogen bonding network with Glu97 and Asp200 as
proposed for the para phenol analogue 5. Support for
this rationale comes from the finding that analogue 7,
which has a hydroxyl moiety at both the 3- and 4-posi-
tions, displays comparable but not additive, potency to
both 8 and 5, suggesting these analogues do indeed
interact with the same residues. Although the ortho
phenol analogue 10 is more potent than the parent
phenyl analogue 4 it is less potent than either the para
or meta analogues. Having investigated the SAR
around the C-6 position of the pyrazolopyridine
nucleus, we sought to explore the C-5 position to see if
additional potency could be achieved (Table 2). Inter-
estingly, insertion of bromine at the C-5 position affor-
ded an improvement in potency (cf 5 and 13, 4 and 17).
Several hypotheses could explain the increase in potency
observed. Firstly, the group at the C-5 position further
occupies the ATP binding site and thus the inhibitor has
greater surface contact with the enzyme. Secondly, there
is a specific hydrophobic interaction with the bromine
The proposed rationale for this increased potency of the
pyrazolo[3,4-b]pyridazines was confirmed by X-ray
crystallography to be due to a hydrogen bond between
the N-6 position of the inhibitor and a water lattice
(Fig. 2a). Analysis of the binding orientation suggested
to us that it maybe possible to displace the structural
waters fromthe binding site via insertion of a suitably
functionalised group at the 6-position of the pyr-
azolo[3,4-b]pyridine nucleus. In order to test this
hypothesis, a small set of functionalised 6-aryl pyridines
were designed and prepared (Table 1). The para phenol
derivative 5, demonstrated a dramatic improvement in
*Corresponding author. Tel.: +44-1279-627832; e-mail: jason_
witherington@gsk.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00645-0

3056
J. Witherington et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3055–3057
Figure 1. Optimisation of pyridazine 1.
Table 1. Inhibition of hGSK-3a by selected 6-aryl-pyrazolo[3,4-
b]pyridine analogues3
Compd
R
GSK-3a (IC50 nM)
4
5
6
7
8
9
10
11
Ph
4-OH
4-OMe
3,4-di-OH
3-OH
3-OMe
2-OH
2-OMe
425
8Æ1
>5000
8Æ1
12Æ3
125Æ26
36Æ11
1593
that a phenyl or methyl group at the 5-position does not
lead to an increase in potency (cf 5 with 12, 4 with 16),
while introduction of alternative electron withdrawing
groups such as chlorine or a nitrile group also affords an
increase in potency (cf 5 with 14, 4 with 19).
Figure 2. (a) X-ray co-crystallisation of pyrazolo[3,4-b]pyridazines
with GSK-3; (b) model of 6-Aryl pyrazolo[3,4-b]pyridines.
Previously, in the structurally related 5-aryl-pyr-
azolo[3,4-b]pyridazine series, we had identified the
amide moiety as a position of the molecule that could
tolerate the incorporation of tertiary amines and thus
potentially increase aqueous solubility.2b Attachment of
basic amines onto the side chain of the amide moiety
was also in tolerated in this series without significant
loss of potency (Table 3).
atomat the C-5 position and the ATP binding site.
Thirdly, the substituent at the C-5 position modulates
the acidity of the pyrazolo ring, and thus further opti-
mises the hydrogen bonding interaction with Asp133 of
the hinge region of the ATP binding site. The SAR
obtained to date may support the latter argument, given
Scheme 1. ꢀPreparation 6-aryl-pyrazolopyrazolo[3,4-b]pyridine 13. Reagents: (a) (i) DMF–DMA reflux, 12 h; (ii) 2-cyanoacetamide, NaOMe,
ꢀ
.
MeCN, 80 C (90%, two steps); (b) NBS, DMF, reflux (88%); (c) POCl3, reflux (100%); (d) N2H2 H2O, EtOH, reflux, 12 h (77%); (e) BBr3, 25 C,
16 h (75%); (f) cyclopropyl carbonyl chloride, pyridine, reflux (90%).

J. Witherington et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3055–3057
3057
Table 2. Inhibition of hGSK-3a by selected C-5 substituted 6-aryl-
pyrazolo[3,4-b]pyridine analogues
Introduction of a nitrogen atominto the 6-position of a
series of pyrazolo[3,4-b]pyridines had previously been
demonstrated to lead to a dramatic improvement in
GSK-3 potency. Subsequent X-ray crystallography
studies with these inhibitors indicated a hydrogen bond
between the nitrogen at the 6-position and the water
lattice. Utilising this structural knowledge, modification
of the template to incorporate functionality at the 6-
position capable of displacing these structural water
molecules afforded a novel series of potent GSK-3 inhi-
bitors.
Compd
R1
R2
GSK-3a, (IC50 nM)
4
5
H
4-OH
4-OH
4-OH
4-OH
4-OH
H
H
H
H
H
H
Ph
Br
Cl
Me
Ph
Br
Cl
425
8Æ1
12
13
14
15
16
17
18
19
24Æ3
0.8Æ0.4
1Æ1
Acknowledgements
6Æ1
The authors acknowledge the assistance of Glaxo-
SmithKline colleagues from the Department of Bio-
technology and Genetics for the recombinant human
enzymes and Molecular Screening Technologies for
assay of the compounds.
415Æ123
75Æ10
234Æ32
87Æ19
CN
Table 3. Inhibition of hGSK-3a by 6-aryl-pyrazolopyridines con-
taining basic side chains
References and Notes
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minatti, H. Arch. Biochem. Biophys. 1959, 81, 508. (b) Man-
darino, L.; Wright, K.; Verity, L.; Nichols, J.; Bell, J.;
Kolterman, O.; Beck-Nielsen, H. J. Clin. Invest. 1987, 80, 655.
(c) Roach, P. FASEB J. 1990, 4, 2961. (d) Skurat, A. V.;
Roach, P. J. Biochem. J. 1996, 313, 45. (e) Zhang, W.;
DePaoli-Roach, A. A.; Roach, P. J. Arch. Biochem. Biophys.
1993, 304, 219. (f) Eldar-Finkelmann, H.; Argast, G. M.;
Foord, O.; Fischer, E. H.; Krebs, E. G. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 10228. (g) DeFronzo, R. A.; Bonadonna,
R. C.; Ferrannini, E. Diabetes Care 1992, 15, 318. (h) Thor-
burn, A. W.; Gumbiner, B.; Bulacan, F.; Wallace, P.; Henry,
R. R. J. Clin. Invest. 1990, 85, 522. (j) Beck-Nielsen, H.; Vaag,
A.; Damsbo, P.; Handberg, A.; Neilsen, O. H.; Henriksen,
J. E.; Thye-Ronn, P. Diabetes Care 1992, 15, 418. (k) Bogar-
dus, C.; Lillioja, S.; Stone, K.; Mott, D. J. Clin. Invest. 1984,
73, 1185. (l) Lovestone, S.; Reynolds, C. H.; Latimer, D.;
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8455.
Compd
R1
H
R2
Br
R3
GSK-3a, (IC50 nM)
20
21
22
23
24
4-Piperidine-N-Me
383Æ59
4Æ1
4-OH Br (CH2)3piperazinyl-N-Et
4-OH
4-OH Br
3-OH
H
4-Piperidine-N-Me
4-Piperidine-N-Me
(CH2)3piperazinyl-N-Et
12Æ2
1Æ1
H
21Æ3
Chemistry4,5
The pyrazolo[3,4-b]pyridine analogues were prepared
following the general procedure outlined for analogue
13 in Scheme 1. Treatment of the commercially avail-
able ketone 25 with DMF–DMA at reflux afforded the
intermediate dimethylaminopropenone, which was
cyclised without purification to afford the pyridone 26.
Selective bromination at the 3-position of the pyridone
employing NBS at reflux afforded 27 in excellent yield.
Subsequent treatment with phosphorus oxychloride at
reflux, followed by cyclisation of the intermediate
chloronitrile with hydrazine afforded the amine 28.
Demethylation employing BBr3 followed by subsequent
selective N-3 acylation afforded the desired pyr-
azolo[3,4-b]pyridine 13 in excellent overall yield.
2. (a) Witherington, J.; Bordas, V.; Garland, S. L.; Hickey, D.
M.; Ife, R. J.; Liddle, J.; Saunders, M.; Smith, D. G. Bioorg.
Med. Chem. Lett. In press. (b) Witherington, J.; Bordas, V.;
Haigh D.; Hickey, D. M.; Ife, R. J.; Rawlings, A. R.; Slingsby,
B.; Smith, D. G.; Ward, R. Bioorg. Med. Chem. Lett. In press.
3. Results are a mean of at least two determinations run in
duplicate (n=4) and are given as mean. MeanÆSEM are also
quoted for compounds of specific interest.
4. Assay conditions described in ref 2a.
5. All novel compounds gave satisfactory analytical data in
full agreement with their proposed structures.