Design and synthesis of 7-hydroxy-1H-benzoimidazole derivatives as novel inhibitors of glycogen synthase kinase-3β

Article abstract of DOI:10.1016/j.bmcl.2007.07.056

A hydroxy functional group was introduced as the hydrogen bond donor and acceptor at the hinge region of protein kinase in order to develop novel ATP-competitive inhibitors. Several derivatives of 7-hydroxyl-1H-benzoimidazole were designed as inhibitors o

Full text of DOI:10.1016/j.bmcl.2007.07.056

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5686–5689
Design and synthesis of 7-hydroxy-1H-benzoimidazole
derivatives as novel inhibitors of glycogen synthase kinase-3b
Dongkyu Shin,a,b Seung-Chul Lee,a Yong-Seok Heo,c Woon-Young Lee,a
Yong-Soon Cho,a Yong Eun Kim,a Young-Lan Hyun,a Joong Myung Cho,a
Yoon Sup Leeb and Seonggu Roa,*
aCrystalGenomics, Inc., Asan Institute for Life Sciences, 388-1 Pungnap-2dong, Songpa-gu, Seoul 138-736, Republic of Korea
bDepartment of Chemistry and School of Molecular Science (BK 21), KAIST, Daejeon 305-701, Republic of Korea
cDepartment of Chemistry, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea
Received 9 January 2007; revised 5 April 2007; accepted 17 July 2007
Available online 19 August 2007
Abstract—A hydroxy functional group was introduced as the hydrogen bond donor and acceptor at the hinge region of protein
kinase in order to develop novel ATP-competitive inhibitors. Several derivatives of 7-hydroxyl-1H-benzoimidazole were designed
as inhibitors of glycogen synthase kinase-3b with the help of ab initio calculations and a docking study. Enzymatic assay and an
X-ray complex study showed that these designed compounds were highly potent ATP-competitive inhibitors.
Ó 2007 Elsevier Ltd. All rights reserved.
Glycogen synthase kinase-3b (GSK-3b) is a serine/thre-
onine protein kinase that inhibits the activity of glyco-
gen synthase (GS), tau, and numerous other substrates
by means of phosphorylation.1 On the basis of such bio-
chemical actions, there is much evidence that it may be a
good target for diabetes and several central nervous sys-
tem diseases, such as dementia and bipolar disease. For
example, in the fatty tissue of mice suffering from fatty
diabetes, the GSK-3b activity has been observed to be
twice that of normal mice.2 Transgenic mice pro-
grammed to express GSK-3b in the brain have abnor-
mal neurons caused by the hyperphosphorylating tau
of the neurofibrillary tangle, which plays an important
role in dementia.3 Moreover, patients with type II diabe-
tes4 and dementia5 show a higher level of GSK-3b
expression than normal controls. Bipolar disorder has
been treated with lithium and valproic acid, well-known
GSK-3b inhibitors.6 Thus, we have designed novel
GSK-3b inhibitors using proprietary structure-based
technologies.
As was done for other known kinase inhibitors,7,8 we
targeted the adenosine triphosphate (ATP) binding site,
Figure 1. Scheme for designing new kinase inhibitors. (a) Pharmaco-
phore representation of inhibitor at the hinge region in protein kinase,
HBD represents hydrogen bond donor and HBA represents hydrogen
bond acceptor. (b) A secondary amine group is adopted as the HBD
and an hydroxy group is adopted as the HBD and HBA. (c) 7-
Hydroxy indole is designed as a skeleton. (d) For synthetic feasibility,
7-hydroxy benzoimidazole is selected as a scaffold.
Keywords: Kinase inhibitor; Drug design; Glycogen synthase kinase;
GSK; 7-Hydroxy-1H-benzoimidazole.
*
Corresponding author. Tel.: +82 2 3010 8618; fax: +82 2 3010
8601; e-mail: sgro@cgxinc.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.056

D. Shin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5686–5689
5687
and 6-31G* basis sets in the Gaussian 03 package10 on
the Linux system. The results are summarized in Table
1. However, conformers c and d cannot make a multiple
hydrogen bond network properly at the hinge region of
the kinase. Thus, conformer a is the lowest-energy con-
former for a new scaffold, considering the hydrogen
bond network.
Figure 2. Four possible conformers a, b, c, and d of designed scaffold.
specifically the hydrogen bond network at the hinge re-
gion of the kinase. To bind to this region, most known
kinase inhibitors include a hydrogen bond acceptor
flanked by two hydrogen bond donors on the basis of
pharmacophoric analysis (Fig. 1).9 In our design, a hy-
droxy functional group (AOH) is expected to act as both
a hydrogen bond donor and an acceptor at the same
time. A secondary amine (ANHA) is considered as an-
other hydrogen bond donor. Initially, we adopted 7-hy-
droxyl indole as a skeleton, but changed to 7-hydroxy
benzimidazole because of synthetic difficulties. More-
over, to facilitate the derivatization, we decided to intro-
duce an amide into the para-position of the phenyl ring
of the skeleton.
A docking study was performed with the published
structure (PDB code: 1I09)11 using the Affinity module
of InsightII12 employing the cvff force field. All solvent
molecules were removed and explicit hydrogen atoms
were generated at pH 7.4. The binding site atoms were
˚
defined as the residue atoms within 5 A from the initial
position of the ligand. During the docking process, the
binding site atoms and the ligand atoms were flexible.
The Monte Carlo and the Simulated Annealing methods
were applied to generate and minimize the possible
poses. A representative result is depicted in Figure 3
using the structure of compound 6a (Scheme 1 and Ta-
ble 2). The compound fits well in the GSK-3b protein
structure with a reasonable conformation like con-
former a. It binds to the hinge region to make hydrogen
bonds. As expected, the hydrogen bond distance was
Considering the internal hydrogen bond of the designed
scaffold, only four conformers are possible as summa-
rized in Figure 2. Structural optimization was performed
for each conformer using the DFT method with B3LYP
˚
measured to be 2.88 A between the Val135 C@O and
imidazole NH of the compound, 2.83 A between the
˚
Val135 NH and the oxygen of the hydroxy group and
˚
2.54 A between the Asp133 C@O and the oxygen of
OH. Additionally, the corresponding hydrogen bond
angles are 172°, 148° and 137°, respectively. These re-
sults indicate that the designed scaffold can bind to the
ATP binding site like most other kinase inhibitors.
Table 1. Ab initio optimization results of conformers a, b, c and d
Conformer
Energy difference
(Kcal/mol)
Torsion angle
(degree)
d
a
c
0.0
4.1
176
0
The designed inhibitors were synthesized using the meth-
ods in Scheme 1. 3-Amino-4-methoxybenzoic acid (1) was
reacted with MeOH under acidic conditions to give
6.0
13.9
25
140
b
Scheme 1. Reagents and conditions: (i) SOC12/MeOH, reflux, 2 h
(yield: 98%); (ii) p-TSA, 180 °C; (iii) NaOCl, 50% aq MeOH, 0.5 h
(yield: 30–70%); (iv) Na2CO3 solution, 60 °C, 1 h (yield: 70–95%); (v)
LiOHÆH2O, THF/MeOH/H2O (3:1:1), reflux, 4 h (yield: 90–98%); (vi)
EDC, HOBt, DMAP, DMF, rt, 8 h (yield: 30–60%); (vii) AlCl3,
toluene, reflux, 5 h (yield: 20–60%).
Figure 3. Docking result of compound 6a. The key interaction
between GSK-3b and compound 6a is the hydrogen bond network
in the hinge region depicted by a white dotted line. The carbon atom in
the ligand is colored white, the carbon atom in the protein is green,
oxygen is red, and nitrogen is blue.

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D. Shin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5686–5689
Table 2. GSK-3b inhibition results for synthesized compounds
OH
H
N
R1
N
R1
HN
R2
O
Compound
R1
R2
% inhibition at 5 lM (%)
IC50 (nM)
N
O
O
O
Ref.
6a
6b
6c
99
86
70
N
N
H
H
93
870
580
25
H
91
O
S
O
6d
6e
H
100
64
NH
Cl
Cl
Cl
Cl
6f
64
6g
6h
87
1000
15
O
O
99
S
NH
methyl 3-amino-4-methoxybenzoate (2). Amidine inter-
mediates (3) were prepared by treating p-TSA and NaOCl
at 180 °C and rt, respectively. These amidines were cyc-
lized with sodium carbonate solution to give methyl 7-
methoxy-2-o-, p-substituted phenyl-1H-benzimidazole-
4-carboxylate (4) and hydroxylated by LiOHÆH2O to give
a benzimidazole-4-carboxylic acid (5) in good yield. Var-
ious final compounds (6) were prepared by treating R2-
amine with EDC coupling reagents. We incorporated
phenyl, benzyl, phenethyl and p-methanesulfoneamide-
phenethyl amine as summarized in Table 2.
ular, compound 6h was evaluated as having an IC50 va-
lue of 15 nM, probably due to the hydrophilic
interaction between two oxygens of the methanesulfo-
neamide and three NHs in the side chains of Arg141,
Gln185 and Arg220.
To confirm the calculated binding mode of the designed
scaffold, the X-ray structure of GSK-3b bound with
compound 6h was determined at 3.2 A resolution.15 This
˚
showed that the designed hydrogen bond network in the
hinge region is realized (Fig. 4a). The hydrogen bond
˚
distances are measured to be 3.31 A between the
The inhibitory activities of the synthesized compounds
were determined using the published method.13 We used
3-(1-methylindol-3-yl)-4-[3-(2-aminoethoxy)phenyl]-1H-
pyrrole-2,5-dione as a reference compound, of which
IC50 value was reported as 20 nM in the patent14 and
was determined as 70 nM in this system. Compound
6a, the first synthesized basic structure, was evaluated
as giving 86% inhibition at 5 lM concentration. To in-
crease the steric attraction, two chlorides were intro-
duced at R1 but had no effect on the inhibitory
activity. As the size of R2 increased, the potency of the
inhibitor was improved in compounds 6b–6h. In partic-
Val135 C@O and imidazole NH of compound 6h,
3.36 A between the Val135 NH and oxygen of the hy-
droxy group and 2.55 A between the Asp133 C@O
˚
˚
and oxygen of OH. Additionally, the corresponding
hydrogen bond angles between GSK-3b and compound
6h are 130°, 129° and 132°, respectively. Moreover, the
torsion angle between the benzimidazole ring and amide
is in concordance with our expectation based on ab initio
calculations. When the two structures from X-ray stud-
ies and calculations are superimposed along the back-
bone of the kinase hinge region, the benzimidazole
rings almost overlap (Fig. 4b).

D. Shin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5686–5689
5689
CIE (Ministry of Commerce, Industry and Energy) of
Korea and Medium-term Strategic Technology Devel-
opment Program of MOICE.
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Figure 4. (a) X-ray complex structure of GSK-3b and compound 6 h.
The hydrogen bond network in the hinge region is depicted by a white
dotted line. The carbon atom in the ligand is colored white, the carbon
atom in the protein is green, oxygen is red, and nitrogen is blue. (b)
Comparison between X-ray and docking structures. The docking
structure with 6a is colored red, the corresponding protein is purple,
the X-ray complex structure 6 h is white and the corresponding protein
is cyan.
In summary, we have designed new kinase inhibitors by
considering the hydrogen bond network between the ki-
nase protein and ligands. The binding conformation and
positioning of the designed inhibitors were predicted
using ab initio calculation and a molecular docking
study and confirmed through enzymatic assay and X-
ray crystallography.
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Y.-L.; Lee, S. C.; Kim, J. H.; Jeon, Y. H. PCT Int. App.
WO2004065370, 2004.
Acknowledgments
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15. Coordinates for the structure have been deposited at
Protein Data Bank (Access Code: 2O5K).
This work was partially supported by Yuyu, Inc. as well
as by the National Research Laboratory Program of
MOST (Ministry of Science and Technology) and MO-