5-Aryl-4-carboxamide-1,3-oxazoles: Potent and selective GSK-3 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.01.034

5-Aryl-4-carboxamide-1,3-oxazoles are a novel, potent and selective series of GSK-3 inhibitors. The optimization of the series to yield compounds with cell activity and brain permeability is described.

Full text of DOI:10.1016/j.bmcl.2012.01.034

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1989–1994
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5-Aryl-4-carboxamide-1,3-oxazoles: Potent and selective GSK-3 inhibitors
Gabriella Gentile a, , Giancarlo Merlo a, , Alfonso Pozzan b, , Giovanni Bernasconi a, Benjamin Bax c,
Paul Bamborough c, Angela Bridges c, Paul Carter c, Margarete Neu c, Gang Yao c, Caroline Brough c,
Geoffrey Cutler c, Aaron Coffin c, Svetlana Belyanskaya c
⇑
⇑
⇑
a Department of Medicinal Chemistry, Neuroscience Centre of Excellence of Drug Discovery, GlaxoSmithKline, Via A Fleming 4, 37135 Verona, Italy
b Molecular Discovery Research, GlaxoSmithKline, Via A Fleming 4, 37135 Verona, Italy
c Molecular Discovery Research, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
5-Aryl-4-carboxamide-1,3-oxazoles are a novel, potent and selective series of GSK-3 inhibitors. The opti-
mization of the series to yield compounds with cell activity and brain permeability is described.
Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
Received 9 December 2011
Revised 10 January 2012
Accepted 11 January 2012
Available online 21 January 2012
Keywords:
GSK-3 inhibitors
Glycogen synthase kinase-3 (GSK-3)
Bipolar disorder
Phenyloxazole carboxamide
H
Glycogen synthase kinase-3 (GSK-3),
serine/threonine kinase existing as two isoforms (GSK-3
a
multifunctional
and
N
O
N
a
1
GSK-3b) is a key regulator of numerous signalling pathways.1 One
function is to phosphorylate glycogen synthase (GS), implicating
GSK-3 in type-2 diabetes.2 In addition, GSK-3 inhibition is a
therapeutic target for the treatment of neurodegenerative diseases
such as Alzheimers,3 and neurological diseases such as bipolar
disorder.4,5 Lithium is indicated as a preferential treatment for
bipolar disorders, and the ability of this cation to inhibit GSK-3
has been proposed as a potential mechanism of action.6 Due to this
therapeutic potential, identification of GSK-3 inhibitors is a focus of
research for both pharmaceutical companies and academic
centres.7 The availability of GSK-3b crystal structures8,9 enables
structure based lead discovery and optimization.
O
H
N
Me
O
O
Figure 1. Hit 1, pIC50 = 6.5 in the GSK-3b FP binding assay.16
were synthesised according to a general route (Scheme 1) which
was used for all the compounds reported in this Letter.
In our effort to discover novel GSK-3 inhibitors through affinity
based screening of ultra-large DNA encoded libraries,10 phenylox-
azole carboxamide 1 was identified as a potent inhibitor of
GSK-3 (Fig. 1). This represents a novel kinase inhibitor scaffold.
However, one concern about compound 1 was its unfavourable
physico-chemical properties, and potential low CNS permeabil-
ity.11–13 The polar surface area (PSA)14,15 of CNS-penetrant
molecules is usually 660–70 Å2, whilst that of compound 1 was
93 Å2. To improve the CNS drug-like characteristics, analogues
Substituted carboxylic acids 2 were converted, directly or via
acyl chloride formation, to phenyloxazole diethyl ester intermedi-
ates 3 by reaction with ethyl-isocyanoacetate. Hydrolysis to
carboxylic acids 4 followed by amide coupling with appropriate
primary amines led to the final compounds. Results are shown in
Table 1.The N,N-dimethyl analogue 5 and the methylbenzamide 6
were of comparable potency to the hit 1. Surprisingly, N-methyla-
tion led to the completely inactive N,N0-dimethylbenzamide 7. This
suggested that a hydrogen bond donor on the amide nitrogen next
to the oxazole ring was essential for activity. The lower activity of
the benzylamide 8 compared to 6 suggested that the hydrogen
bond acceptor provided by the methylbenzamide group of 6 may
be beneficial. Therefore, replacement of the benzamide with
⇑
Corresponding authors. Tel.: +39 0458219355; fax: +39 0458218196 (G.G.).
E-mail addresses: gabriella.gentile@aptuit.com (G. Gentile), giancarlo.merlo@
alice.it (G. Merlo), alfonso.pozzan@aptuit.com (A. Pozzan).
0960-894X/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.034

1990
G. Gentile et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1989–1994
R1
hydrogen-bond containing heterocycles was attempted in order to
improve upon 6 by reducing its polar surface area (PSA).
The pyridyl analogues 9–11 were prepared as analogues of 6
with reduced PSA. Gratifyingly, although the 2-pyridyl analogue
9 was 10-fold less active, compounds 10 and 11 showed similar
activity to 6 (Table 1). Attempts to replace the methoxyphenyl ring
of 11 with simple substituted phenyls in which the methoxy group
was replaced or moved generally led to compounds with lower po-
tency, for example, 12–16 (Table 1).
2
3
O
OH
a or b
O
R1
O
N
O
c
Compound 11 proved to be particularly interesting due to its
favourable selectivity profile against a panel of protein kinases.
From 17 kinases tested, all but one (AKT1, AKT2, ALK5, ASK1,
AurA, EGFR, IKKb, InsR, ITK, JNK1, LCK, PAK1, PDK1, ROCK1, SYK
R1
R1
R2
NH2
4
and VEGFR2) had IC50 greater than 10
was CDK-2 (pIC50 = 6.1), which is not surprising since apart from
GSK-3 this is one of the most closely related kinases to GSK-3b,
lM. The one exception
O
d
O
H
N
R2
a
N
OH
sharing 35% sequence identity. Because of this encouraging selec-
tivity and the lower PSA with predicted improvement in CNS-per-
meability over 1, 11 was chosen as the starting point for further
lead optimization.
The binding mode of compound 11 (Fig. 2) was determined
from a 1.98 Å X-ray crystal structure.17 The essential hydrogen-
bonding interaction typically donated to inhibitors by the hinge
N
O
O
Scheme 1. General route to phenyl oxazole derivatives. Reagents and conditions:
(a) (i) SOCl2, reflux; (ii) ethyl isocyanoacetate, TEA, THF, rt; (b) ethyl isocyanoac-
etate, (EtO)2P(O)CN, DIPEA, DMF, 0 °C to rt; (c) NaOH, EtOH, H2O, rt; (d) HATU,
DIPEA, DMF, rt.
Table 1
16
GSK-3b FP binding activity of 5–16. pIC50 = ꢀlog10 (IC50
)
R1
N
O
O
R2
Compound
R1
R2
GSK-3b pIC50
PSA (Å2)
O
O
O
N
5
4-OMe
6.5
85
H
N
N
H
6
7
4-OMe
4-OMe
6.6
93
85
H
N
N
<4.6
N
H
N
8
9
4-OMe
4-OMe
4-OMe
6.0
5.7
6.6
64
77
77
H
N
N
H
N
10
N

G. Gentile et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1989–1994
1991
Table 1 (continued)
Compound
R1
R2
GSK-3b pIC50
PSA (Å2)
N
H
N
11
12
13
14
15
16
4-OMe
6.4
77
88
N
H
N
3-OH
6.2
5.2
5.5
5.6
5.1
N
H
N
4-CF3
68
N
H
N
4-CN
92
N
H
N
3-OMe
77
N
H
N
3-CONH2
111
(residue Val135 in GSK-3b) is accepted by the oxazole nitrogen
atom. This interaction of the nitrogen of a monocyclic five-mem-
bered azole ring has been observed on other molecular scaffolds,
for example in the 2-anilino 5-aryloxazole class of VEGFR2 kinase
inhibitors.18 The closest public GSK-3b crystal structure complex is
with 5-nitro 2-urea thiazole AR-A014418, PDB code 1q5k.19 The
exact orientation of such hinge-binding rings is strongly influenced
by the sterics of its substituents. In the above two examples the li-
gand atom adjacent to the acceptor nitrogen is substituted directly
with an NH-R group, which donates a hydrogen bond to the hinge
residue carbonyl (Val135). In contrast to this common substitution
pattern, 11 has an unusual C(@O)NH-R group at this position, and
it is this NH group that interacts with the carbonyl of Val135. This
results in a rotation of the ring with respect to the typical binding
mode exemplified by AR-A104418. As a consequence, the C-2 atom
0
of the oxazole is in close proximity (2.9 ÅA) to the carbonyl of
Asp133, acting as an unusual C–H hydrogen bond donor. The C-4
position amide substituent projects towards the solvent side of
the site, in the region of Ile62, against which the pyridyl ring packs.
The C-5-position phenyl substituent lies within the ATP-site in the
vicinity of Val70, with the C-4 methoxy group in the region of the
catalytic residues Lys85 and Asp200.0Although reasonably close to
the sidechain nitrogen of Lys85 (3.8 ÅA), the methoxy oxygen is not
close enough to form a hydrogen bond, but it is possible that an
electrostatic interaction contributes to the potency of 11.
Two water molecules participate in a small buried network at
the back of the site, interacting with the backbone NH atoms of
Asp200 and Phe201 and the sidechain of Glu97. These are present
in other complexes and can interact directly with ligands,20 but in
this case their closest interaction with 12 is edge-on to the meth-
oxy-phenyl ring.
Based on the X-ray structural information it seemed that the
interaction of the 4-methoxyphenyl with the pocket might be
improved by optimising interactions with Phe67 and Val70. As
the equivalent residue to Phe67 in CDK-2 is Tyr15, in principle this
could also improve the selectivity. Because of this, an exploration
of different substituents in the meta and para positions of the
phenyl ring at the oxazole five-position was carried out following
Figure 2. Top: X-ray structure of 11 in GSK-3b. Below: With AR-A014418
superimposed by protein coordinates (yellow).

1992
G. Gentile et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1989–1994
Table 2
GSK-3b catalytic activity, cellular activity (CRMP-2), and CDK-2 activity of compounds 17–27.16
R1
GSK-3b pIC50
CRMP-2 pIC50
CDK-2 pIC50
PSA (Å2)
4-OMe
3-Cl, 4-OMe
11
17
7.1
7.9
5.7
6.1
6.3
6.3
77
77
N
3-F, 4-OMe
2-F, 4-OMe
2-Me, 4-OMe
3-(4-Pyridyl), 4-OMe
3(4-Morpholinyl-4-OMe
3-Cl, 4-OPh
18
19
20
21
22
23
7.5
6.4
5.5
5.9
5.7
6.1
6.0
5.2
64.3
4.4
4.5
4.5
6.3
5.3
<4.8
<4.8
<4.8
<4.8
77
77
77
90
90
77
NH
N
O
R1
O
R2
24
25
26
27
7.1
7.2
6.4
5.0
5.7
5.7
5.5
4.2
5.3
5.3
<4.8
<4.8
90
90
90
90
N
OMe
OMe
N
N
24
25
Cl
OMe
NH
OMe
26
N
N
27
O
Cl
N
Cl
O
R2
Table 3
GSK-3b catalytic activity, cellular activity (CRMP-2), and CDK-2 activity of compounds 28–3516
H
N
O
N
R
O
Cl
MeO
R
GSK-3b pIC50
CRMP-2 pIC50
6.1
CDK-2 pIC50
PSA (Å2)
77
N
17
28
7.9
6.3
6.2
Cl
N
N
7.7
7.5
5.3
5.6
77
77
29
30
5.9
6.2
O
N
7.5
5.2
86

G. Gentile et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1989–1994
1993
Table 3 (continued)
R
GSK-3b pIC50
CRMP-2 pIC50
CDK-2 pIC50
PSA (Å2)
CF3
N
31
32
7.0
5.3
5.7
77
90
N
N
7.7
6.2
6.3
N
33
34
35
7.4
7.7
8.3
5.7
5.7
6.2
5.5
5.5
6.3
90
77
82
N
N
N
N
the synthetic route of Scheme 1. To overcome the tight-binding
limit of the FP assay, these compounds were evaluated using a
GSK-3b AlphaLISA assay and a cellular phospho-CRMP-2 assay
(Table 2).16
Table 4
Rat pharmacokinetic parameters for compound 29 (iv dose 0.5 mg/kg, po dose 1 mg/
kg)
Clb (ml/min/Kg)
(h)
Vd (l/Kg)
F (%)
Brain/blood ratio
51
0.7
2.4
18
2.9
H
N
The most potent compounds were those in which the methoxy
at the phenyl C-4 position was accompanied by halogen groups at
the C-3 position. For instance, the 3-chloro analogue 17 was best,
closely followed by the 3-fluoro derivative 18. Compared to 11
both showed significant improvements in GSK-3b potency, cellular
activity, and CDK-2 selectivity. The beneficial halogen effect was
strongly position-dependent: for example, the 2-fluoro analogue
19 was 10-fold less potent than the 3-fluoro compound 18. The
2-methyl derivative 20 proved to be almost inactive, possibly be-
cause of disruption of the planarity in the aryl-oxazole system
due to steric hindrance.
t
½
N
O
Me
O
N
Cl
MeO
the methyl-(4-pyridyl) moiety and the amide was tolerated and
gave a small increase in CDK-2 selectivity (34). These findings sug-
gested that a bulkier moiety at that position might increase po-
tency, and indeed the imidazopyridine derivative 35 showed
increased GSK-3b activity with respect to 17.
Compound 29 showed an acceptable profile in terms of activity,
selectivity and PSA. This compound was selected as an exemplar of
the 5-aryl-4-carboxamide-1,3-oxazole series for rat in vivo phar-
macokinetic evaluation (Table 4).
The pharmacokinetic profile of 29 proved to be acceptable, with
a low half life and volume of distribution, and moderate clearance
and bioavailability. Brain penetration was good, validating the
optimization strategy to reduce polar surface area.
In summary, optimization of the initial hit 1 led to the discovery
of the novel 5-aryl-4-carboxamide-1,3-oxazoles series. Its binding
mode has been elucidated by X-ray crystallography. Most of the
exemplars showed good GSK-3b activity, cellular activity and a
promising selectivity profile. Compound 29 proved to be brain pe-
netrant, with a suitable pharmacokinetic profile for use in in vivo
animal models.
Compounds with larger groups at the C-3 position were also
prepared (e.g., 21, 22) but these also had lower activity. The addi-
tion of a bulkier phenoxy group instead of the methoxy at the C-4
position was also unfavourable (23).
We next investigated substitution of the phenyl ring at the oxa-
zole C-5 position with heterocyclic rings, preserving the position of
the methoxy group at the C-4 position relative to the oxazole.
Introduction of a pyridyl nitrogen at the 3-position relative to the
attachment point was tolerated but modestly detrimental to
GSK-3b activity (24–25), regardless of the presence of the chlorine
substituent. Introduction of the pyridyl nitrogen adjacent to the
oxazole substitution bond was unfavourable (26), even more so
when it was placed on the opposite side of the ring to the chlorine
(27).
Up to this point, 17 seemed to be the optimal compound for
GSK-3b activity and PSA. We next returned to the oxazole C-4 po-
sition with further exploration of the heteroaromatic carboxamide
while retaining the 3-chloro-4-methoxyphenyl moiety at the C-5
position. This was carried out by the route of Scheme 1. Decoration
of the pyridine (28–31) and alternative heterocycles (32–35) were
both attempted (Table 3). The 2-chloro, 2-methyl and 2-methoxy
pyridyl compounds 28–30 were as potent as 17, although the cor-
responding trifluoromethyl compound 31 was somewhat less ac-
tive. The pyridazine 32 and methylpyrimidine 33 analogues also
retained GSK-3b activity. Introducing a methylene spacer between
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8 and co-crystals grown with
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