Synthesis and biological evaluation of glycogen synthase kinase 3 (GSK-3) inhibitors: An fast and atom efficient access to 1-aryl-3-benzylureas

Article abstract of DOI:10.1016/j.bmcl.2011.06.131

The glycogen synthase kinase 3 (GSK-3) is implicated in multiple cellular processes and has been linked to the pathogenesis of Alzheimer's disease (AD). In the course of our research topic we synthesized a library of potent GSK-3 inhibitors. We utilized the urea scaffold present in the potent and highly selective GSK-3 inhibitor AR-A014418 (AstraZeneca). This moiety suits both (a) a convergent approach utilizing readily accessible building blocks and (b) a divergent approach based on a microwave heating assisted Suzuki coupling. We established a chromatography-free purification method to generate products with sufficient purity for the biological assays. The structure-activity relationship of the library provided the rationale for the synthesis of the benzothiazolylurea 66 (IC₅₀ = 140 nM) and the pyridylurea 62 (IC ₅₀ = 98 nM), which displayed two to threefold enhanced activity versus the reference compound 18 (AR-A014418: IC₅₀ = 330 nM) in our assays.

Full text of DOI:10.1016/j.bmcl.2011.06.131

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5610–5615
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of glycogen synthase kinase 3 (GSK-3)
inhibitors: An fast and atom efficient access to 1-aryl-3-benzylureas
Fabio Lo Monte a, , Thomas Kramer a, , Alexander Boländer a, Batya Plotkin b, Hagit Eldar-Finkelman b,
Ana Fuertes c, Juan Dominguez c, Boris Schmidt a,
⇑
a Clemens Schöpf—Institute of Organic Chemistry and Biochemistry, Technische Universität Darmstadt, 64287 Darmstadt, Germany
b Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
c Noscira, Tres Cantos, 28760 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The glycogen synthase kinase 3 (GSK-3) is implicated in multiple cellular processes and has been linked
to the pathogenesis of Alzheimer’s disease (AD). In the course of our research topic we synthesized a
library of potent GSK-3 inhibitors. We utilized the urea scaffold present in the potent and highly selective
GSK-3 inhibitor AR-A014418 (AstraZeneca). This moiety suits both (a) a convergent approach utilizing
readily accessible building blocks and (b) a divergent approach based on a microwave heating assisted
Suzuki coupling. We established a chromatography-free purification method to generate products with
sufficient purity for the biological assays. The structure–activity relationship of the library provided
the rationale for the synthesis of the benzothiazolylurea 66 (IC50 = 140 nM) and the pyridylurea 62
(IC50 = 98 nM), which displayed two to threefold enhanced activity versus the reference compound 18
(AR-A014418: IC50 = 330 nM) in our assays.
Received 15 April 2011
Revised 11 June 2011
Accepted 13 June 2011
Available online 18 July 2011
Keywords:
Glycogen synthase kinase 3 (GSK-3)
Alzheimer disease
Structure–activity relationship (SAR)
Suzuki coupling
Ó 2011 Published by Elsevier Ltd.
Microwave irradiation
Alzheimer’s disease (AD) is a neurodegenerative disorder defined
by progressive memory loss and cognitive impairment.1 The definite
diagnosis of AD is possible postmortem only. It is based on the pres-
ence of extracellular plaques of b-amyloid (Ab), and intracellular
neurofibrillary tangles (NFTs) consisting of hyperphosphorylated
tau protein.2 Glycogen synthase kinase 3 (GSK-3) interacts with sev-
eral neuronal proteins that are directly linked to AD.3 There are two
GSK-3 genes, GSK-3a and GSK-3b. Both are ubiquitously expressed
and constitutively active proline-directed serine/threonine
kinases.1 Several GSK-3 inhibitors have been studied in kinase as-
says and cellular test panels. Among these inhibitors are paullones,
indirubin, aminomaleimides and other small molecules. The potent
and specific GSK-3 inhibitor AR-A014418 (18) was reported by
AstraZeneca and its interactions with the essential amino acids of
GSK-3b are denoted in Figure 1.4
AR-A014418 (18) is selective against Cdk2, Cdk5 and 26 other
kinases tested. It inhibits GSK-3 activitywith a strongly assay depen-
dent IC50, the highest activity was reported as IC50 = 104 nM.4
AR-A014418 constitutes a lead compound in our kinase inhibitor
programe targeting neurodegenerative diseases.5
Figure 1. Potent and highly selective GSK-3 inhibitor AR-A014418 (AstraZeneca) in
the ATP binding pocket of GSK-3b; important protein–inhibitor interactions are
shown. The distance is denoted in Å. PDB code 1Q5K (Accelrys Discovery Studio
Visualizer 2.5).
There is a need of a robust and inexpensive approach to AR-
A014418 (18), suitable for combinatorial chemistry, which
provides straightforward access to related ureas displaying en-
hanced GSK-3 inhibition. The generation of chemical diversity
and the limitation by commercial building blocks stimulated a
search for mild reaction conditions, rapid purification strategy
and access to the reactive intermediate isocyanates without destil-
lation.6 The two step protocol utilized the in situ generation of
⇑
Corresponding author. Tel.: +49 6151 164531; fax: +49 6151 163278.
E-mail address: schmidt_boris@t-online.de (B. Schmidt).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2011.06.131

F. L. Monte et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5610–5615
5611
R2
R2
triphosgene,
R1
R1
sat. NaHCO3,
O
C
DCM,
0 °C,
NH2
N
II
I
2.5 h
RNH2,
THF, rt,
12 h
Comp.
17-22
Comp.
23-29
Comp.
1-16
R2
R2
R2
R1
R1
R1
H
N
H
N
H
N
H
N
H
N
H
N
N
R3
R4
S
S
R3
O
III
O
N
O
N
R3
IV
V
R4
R5
Scheme 1. Mild synthesis and rapidly purified ureas.
Table 1
Mild synthesis provides crystalline ureas free of chromatography
R
R
H
N
H
N
Compd
R1
R2
R3
R4
R5
Yielda (%)
c Log Pc
1
2
3
4
5
6
7
8
F
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
Br
Br
H
H
H
H
H
H
H
H
H
H
H
H
CN
NO2
NO2
COOEt
COOEt
H
H
Br
F
CF3
CF3
NO2
NO2
CN
I
H
H
Br
Br
H
H
H
H
NO2
NO2
CN
CN
CN
CN
H
H
H
Me
Me
4-Me-Ph
4-Me-Ph
—
—
—
—
—
—
—
H
H
H
H
11
52
41
54
48
75
31
34
3.74
3.48
3.25
3.48
3.25
3.25
3.33
3.10
2.00
2.51
2.28
2.22
2.00
2.94
3.10
O
MeO
F
MeO
MeO
F
MeO
MeO
F
MeO
F
MeO
Br
OCF3
–OCH2O–
F
MeO
F
MeO
F
VI
H
Scheme 2. Commonly observed dimerization product.
Br
Cl
Cl
CN
H
H
H
H
H
Table 2
Dimerization derived products
9
63b
21b
13b
63b
58b
34b
13b
2.04
21b
26b
35b
24b
29b
46b
24b
52b
31b
50b
26b
36b
70b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Compd
R
Yielda (%)
c Log Pb
306,7
316
4-F
4-MeO
37
43
3.17
2.72
H
a
Yields of analytically pure products; products recrystallized from methanol.
Determined by CS ChemOffice 2008.
19b
—
—
—
—
—
—
—
—
—
—
—
—
—
b
2.34
2.11
3.02
2.80
5.00
4.77
4.87
3.92
4.96
4.74
3.64
3.93
3.34
Using n-BuLi for the deprotonation of the heterocyclic amines
solved this problem. Furthermore, high-pressure liquid chroma-
tography (HPLC) controls showed that the n-BuLi preactivated
amines reacted with the isocyanates immediately, but neverthe-
less the reactions were stirred 12 h at room temperature.
Afterward the reaction solution was suspended in vigorously stir-
red water and precipitation occured promptly. The purification of
the crude urea was achieved by filtration and recrystallization.
These reaction and purification conditions were standardized for
a variety of amines, including benzylamines, aminopyridines and
aminothiazoles.
AR-A014418 (18) was synthesized without heating and chro-
matographic purification, which were employed by AstraZeneca
2003.4 The initial yields of the standard methodology were moder-
ate at best, but they did not require chromatography and the yield
of compound 6 obtained by the standard protocol was optimized in
scaled up experiments to 75%. Thus the atom efficiency of the total
process, which includes the purification, is actually sufficiently
high as it produces very little waste material.
MeO
F
MeO
F
MeO
MeO
F
MeO
H
a
Yields of analytically pure products; products recrystallized from methanol or
ethanol.
b
Amines preactivated with n-BuLi.
Determined by CS ChemOffice 2008.
c
isocyanates from benzylamine and triphosgene. These crude inter-
mediates were dissolved in dry N,N-dimethylformamide or dry tet-
rahydrofurane and reacted with nonactivated or n-buthyllithium
(n-BuLi) activated amines (Scheme 1 and Table 1).
Initially, the reaction of aminopyridines was dominated by the
dimerization of benzylamines. This resulted in the isolation of
known benzylamine adducts 30 and 31 (Scheme 2 and Table 2).7,8
The required diversity was introduced by the Suzuki-coupling
of arylbromides and boronic acids (Scheme 3 and Table 3).

5612
F. L. Monte et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5610–5615
R1
R1
Pd-cat.,
H
N
H
N
H
N
H
N
Rx-boronic acid,
NaOEt,
EtOH,
110 °C,
R2
R3
N
N
O
O
Br
VII
VIII
R4
0.5 - 3 h
Comp. 32-62
Scheme 3. Suzuki reactions.
Table 3
Urea enlargement supported by Suzuki reaction
Compd
R1
R2
R3
R4
Yielda (%)
c Log Pd
32
33
34
35
36
37
38
39
40
41
42
43
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
F
Ph
Ph
3-F-Ph
3-MeO-Ph
3-MeO-Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
40b
71b
29b
31b
34b
51b
67b
50b
70c
47c
34c
32c
32c
68c
33c
37c
34c
43c
61c
26c
19c
27c
83c
26c
85c
87c
77b
57b
62b
47c
33c
67
4.58
4.36
4.75
6.37
6.13
6.37
3.26
3.04
4.37
4.15
4.87
4.65
4.65
4.54
4.32
4.39
4.17
5.23
5.01
6.16
5.94
5.07
4.62
4.40
5.09
4.87
4.15
4.32
4.17
3.61
4.06
3.03
MeO
F
F
MeO
F
F
MeO
F
MeO
F
MeO
MeO
F
MeO
F
MeO
F
MeO
F
MeO
MeO
F
MeO
F
MeO
MeO
MeO
MeO
MeO
MeO
MeO
3-BnO-Ph
4-Py
4-Py
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph
Ph
4-Me-Ph
4-Me-Ph
4-Me-Ph
3-F-Ph
3-F-Ph
3-MeO-Ph
3-MeO-Ph
3-Isopro-poxy-Ph
3-Isopro-poxy-Ph
3-BnO-Ph
3-BnO-Ph
3-CF3-Ph
2,3-di-F-Ph
2,3-di-F-Ph
4-Vinyl-Ph
4-Vinyl-Ph
H
H
H
Ph
3-F-Ph
3-MeO-Ph
2-MeO-Ph
2-Thio-phen
2-F-Py
H
H
H
H
H
H
a
b
c
Yields of analytically pure products; products were recrystallized from methanol.
Pd(OAc)2.
Pd(PPh3)4.
Determined by CS ChemOffice 2008.
d
The Suzuki coupling of phenylboronic acids and brominated
Two derivatives (66 and 62) are more potent than our reference
compound AR-A014418 (18). Five derivatives (29, 63, 11, 60, and
13) display comparable inhibitory activity and four of them are
not associated with toxicity alerts (Fig. 2). It should be noted that
the tetrazoles, for example, compound 63 with a topological polar
surface area of 111.83 Å (ChemOffice2008) may not permeate the
blood brain barrier.
A docking study of compound 66 and PDB structure 1Q5K of
GSK-3b suggested a binding mode along the hinge region of the
ATP-binding pocket approximately like AR-A014418 (18)
(Fig. 3).4,14 There are two hydrogen bond interactions between
66 and the amino acid Glu137. Furthermore we assume two other
interactions, one between the Asp133 carbonyl and a hydrogen of
the phenyl group and the other between the tetrazol moiety and
the polar pocket consisting of Lys85, Glu97 and Asp200. We
hypothesize that the latter interaction is the reason for the en-
hanced potency of derivative (66).
arylureas was catalyzed by Pd(OAc)2 under standard conditions
or subtle modifications thereof.9,10 Some reactions required cata-
lyst exchange to Pd(PPh3)4. The boronic acids were activated by so-
dium ethanolate. The starting materials were suspended in dry
ethanol and the reactions were carried out in a sealed, argon
flushed vessel. Again, the crude products of the Suzuki reaction
were purified by atom efficient recrystallization.
Finally, we obtained four tetrazole derivatives (63–66) using
microwave radiation in a Biotage Initiator 300. These products were
purified by recrystallization and isolated in very good yields requir-
ing no further optimization at this stage (Scheme 4 and Table 4),
although the reaction conditions can be improved by using ionic
liquids.11 Be careful while working with azides, they can be explo-
sive and should be handled with great care. During our study we
encountered no adverse events.12
The arylureas were evaluated for their inhibition of GSK-3 activ-
ity. Several compounds were identified to reduce in vitro GSK-3b
Via the expeditious structure–activity relationship (SAR) we
ascertained that the aminothiazole, used by AstraZeneca, can be
activity beneath 50% at a concentration of 10 l
M (Table 5).13

F. L. Monte et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5610–5615
5613
R
Comp.
H
H
N
63
R
N
N
N
H
N
H
N
N
N
O
XI
O
NaN3,
NH4Cl,
DMF,
CN
N
NH
IX
R
R
N
N
Comp.
64, 65
H
N
H
N
or
100 °C,
2 h, mw
R
H
N
O
H
H
N
N
N
XII
N
N
Comp.
66
O
S
H
N
H
N
N
X
CN
O
S
Nitriles: 9, 12, 13, 29
XIII
NH
N
N
N
Scheme 4. Microwave reactions.
Table 4
Urea aryl tetrazole formation by microwave reaction
O
H
H
N
Compd
R
Yielda (%)
c Log Pb
N
S
63
64
65
66
MeO
F
MeO
MeO
87
81
88
91
1.75
1.98
1.75
3.26
NO2
O
N
Figure 2. Potential toxic alerts: 1. Nitro group; 2. P450 associated-hepatotoxicity
a
Yields of analytically pure products; products recrystallized from methanol.
Determined by CS ChemOffice 2008.
alert.
b
position R4 of structure III as long as no acceptor is allocated in po-
sition R5. Our future research is focused on structure V and position
R5 of structure III from which we expect to obtain improved
activity.
replaced by aminopyridines and aminobenzothiazoles. We found
that an elongation in position R3 and R4 of structure III entailed re-
duced activity. However, an acceptor like NO2 or CN is needed in
Table 5
GSK-3b inhibitory activity of selected compounds
Compd
Structure
GSK-3b activity ina (%)
1
lM of compd
10
12
lM of compd
O
H
N
H
N
N
O
62
—
F
N
O
O
H
N
H
N
S
H
N
66
18
69
55
18
20
N
N
O
O
N
N
N
H
N
H
N
S
NO2
(continued on next page)

5614
F. L. Monte et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5610–5615
Table 5 (continued)
Compd
Structure
GSK-3b activity ina (%)
1
l
M of compd
10
24
lM of compd
O
H
N
H
N
S
29
57
O
O
N
CN
O
H
N
H
N
N
63
11
60
13
82
75
91
79
24
31
32
N
NH
N
N
O
O
H
N
H
N
N
N
O
O
NO2
H
N
H
N
O
O
O
H
N
H
N
N
40
42
O
O
CN
H
N
H
N
N
9
101
CN
a
Conditions for GSK-3b in vitro assay see Supplementary data.10
Figure 3. Docking of compound 66 into PDB structure 1Q5K of GSK-3b; Surface illustration of the ATP-binding pocket with 66 (left); Hydrogen bond interactions of 66 with
the amino acids of the ATP-binding pocket (right).4,14

F. L. Monte et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5610–5615
5615
Figure 4. Exposure of zebrafish embryos to 1% DMSO (left), 100 mM 18 (AR-A14418, middle), and 100 mM 66 (right). The embryos were collected and maintained in E2
medium at 30 °C, compounds were added at 8–15 somites, and the phenotypes were compared after 25 h.
selectivity. A preview of the biological activity and selectivity of
the synthesized compounds is depicted in Tables 5 and 6. They
are of comparable potency and selectivity to the reference com-
pound 18 (AR-A014418). The aminothiazole used by AstraZeneca
was replaced by other heterocycles. We have also shown that a
selection of our compounds compared to 18 (AR-A014418) were
less toxic in a zebrafish embryo phenotype assay, albeit some of
these are not expected to be brain penetrant. This is subject of
ongoing and future research in animal models of Alzheimer’s
disease.
Table 6
GSK3 selectivity panel
Compound
IC50 values (mM)
Cdk5/p35 CKI
GSK-3b GSK-3
a
e
AurKA PKCa
18 (AR-A014418) 0.33
9
27
63
66
0.07
1.12
0.22
0.37
0.13
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1.43
3.64
0.51
0.14
>100
>100
>100
26.4
15.3
4.8
24.2
To evaluate the specificity of the family against GSK-3b, four of
the synthesized compounds (9, 27, 63 and 66) were selected and
Acknowledgment
tested against five human protein kinases (GSK-3
Cdk5/p35 and CKI ) (Table 6).
Compound 66 with an IC50 of 140 nM for GSK3b was the only
one able to inhibit all of the kinases tested (PKC , AurKA, Cdk5/
p35 and CKI ) but the IC50 values were one or two orders of mag-
a, PKCa, AurKA,
This work was supported by a collaborative project financed by
the 7th Framework Program of the European Union.
e
a
e
Supplementary data
nitude above those found for the GSK-3 isoforms, indicating a fair
degree of selectivity for this compound towards these latter en-
zymes. Such selectivity is even larger for the other three com-
pounds tested, which were inactive (or poorly active) against the
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.06.131.
former kinases when tested up to 100 lM.
References and notes
In order to demonstrate the utility and nontoxicity of the pyri-
dine and benzothiazole moiety in whole organisms, we performed
a zebrafish embryo phenotype assay.15,16 We exposed the zebrafish
embryos to the compounds 66, 18 (AR-A014418) and 63 at early
stages of development (8 till 15 somites, Fig. 4).
1. Hooper, C.; Killick, R.; Lovestone, S. J. Neurochem. 2008, 104, 1433.
2. Bhat, R. V.; Haeberlein, S. L. B.; Avila, J. J. Neurochem. 2004, 89, 1313.
3. Eldar-Finkelman, H. Trends in Mol. Med. 2002, 8, 126.
4. Bhat, R.; Xue, Y.; Berg, S.; Hellberg, S.; Ormö, M.; Nilsson, Y.; Radesäter, A.;
Jerning, E.; Markgren, P.; Borgegard, T.; Nylöf, M.; Giménez-Cassina, A.;
Hernández, F.; Lucas, J.; Díaz-Nido, J.; Avila, J. J. Biol. Chem. 2003, 278, 45937.
5. Hoettecke, N.; Liebeck, M.; Baumann, K.; Schubenel, R.; Winkler, E.; Steiner, H.;
Schmidt, B. Bioorg. Med. Chem. Lett. 2010, 20, 2958.
Controls after 18 h and 25 h showed that 18 (AR-A014418) is in-
deed toxic. This toxicity could be diminished by replacement of the
6. Tsai, J. H.; Takaoka, L. R.; Powell, N. A.; Nowick, J. S. Org. Synth. 2002, 78, 220.
7. Wiley, R.; Beasley, P.; Knabeschuh, L. J. Am. Chem. Soc. 1954, 76, 311.
8. Shelton, P.; Zhang, Y.; Nguyen, T.; McElwee-White, L. Chem. Commun. 2009,
947.
thiazole with
(CHIR98014 is not toxic and compound 19 precipitates—data not
shown). We observed some deformation in the fishtail at 100
(66, 63 and at 25 M of CHIR98014—data not shown) and after
eclosion we observed these fishes were swimming in circles only.
This correlates with the observation that Wnt signaling, and thus
GSK-3b, plays a crucial role in the development of metazoan and
that known GSK-3 inhibitors like LiCl and the ruthenium complex
(R)-7 perturbe the zebrafish development.15
We have established a fast and atom efficient approach to gen-
erate kinase targeting urea derivatives. Hereby, it is feasible to
diversify the scaffold in a time efficient manner. Thereby we expect
to identify novel GSK-3 inhibitors displaying enhanced activity and
a benzothiazole (66) or pyridine (63) moiety
lM
9. Miyauro, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
l
10. Kotha, S.; Lahiri, K.; Kashinath, D. Tetrahedron 2002, 48, 9633.
11. Schmidt, B.; Meid, D.; Kieser, D. Tetrahedron 2007, 63, 492.
12. (a) Prudent Practice for Handling Hazardous Chemicals in Laboratories,
National Academic: Washington, DC, 1983, 87.; (b) Bräse, S.; Gil, C.; Knepper,
K.; Zimmermann, V. Angew. Chem. 2005, 117, 5320.
13. Liberman, Z.; Eldar-Finkelman, H. J. Biol.Chem. 2005, 280, 4422.
14. Molegro Virtual Docker, Version 4.2.0 (October 4th, 2010).
15. Atilla-Gokcumen, G. E.; Williams, D. S.; Bregman, H.; Pagano, N.; Meggers, E.
ChemBioChem 2006, 7, 1443.
16. Paquet, D.; Bath, R.; Sydow, A.; Mandelkow, E.; Berg, S.; Hellberg, S.; Fälting, J.;
Distel, M.; Köster, R.; Schmid, B.; Haass, C. J. Clin. Invest. 2009, 119, 1382.