Structure-activity relationship of the 7-hydroxy benzimidazole analogs as glycogen synthase kinase 3β inhibitor

Article abstract of DOI:10.1016/j.bmcl.2012.01.065

Design, synthesis and the GSK3β inhibitory activities of the 7-hydroxy benzimidazole analogs are described. The solid-phase synthetic route was also developed for preparation of the analogs consisting of the novel ATP competitive scaffold. In addition, th

Full text of DOI:10.1016/j.bmcl.2012.01.065

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1891–1894
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship of the 7-hydroxy benzimidazole analogs
as glycogen synthase kinase 3b inhibitor
Seung-Chul Lee a,b, Dongkyu Shin a, Joong Myung Cho a, Sunggu Ro a, , Young-Ger Suh b,
⇑
⇑
a CrystalGenomics, Inc., 5F, Tower A, Korea Bio Park 694-1, Sampyeong-dong, Bundang-gu Seongnam-si Gyeonggi-do 463-400, Republic of Korea
b College of Pharmacy, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Design, synthesis and the GSK3b inhibitory activities of the 7-hydroxy benzimidazole analogs are
described. The solid-phase synthetic route was also developed for preparation of the analogs consisting
of the novel ATP competitive scaffold. In addition, the structure–activity relationship of the 7-hydroxy
benzimidazole analogs and their biological activities are reported.
Received 21 December 2011
Revised 16 January 2012
Accepted 19 January 2012
Available online 28 January 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
GSK3 inhibitor
7-Hydroxy benzimidazole
Glycogen synthase kinase 3 (GSK3) is a multi-functional serine/
threonine kinase involved in many biological processes. GSK3
activity relationship (SAR). The analogs were synthesized by the
solid phase parallel synthetic method. The synthetic route to the
modified 7-hydroxy benzimidazole is shown in Scheme 1. 3-
Amino-4-methoxybenzoic acid 1 was converted into 2 via sequen-
tial esterification, imidation13 and HCl-salt formation. Reaction of 2
with NaOCl followed by cyclization in the presence of Na2CO3
afforded the benzimidazole intermediate, which was further trea-
ted with AlCl3 in toluene for the concomitant hydrolysis of ester
and methoxy groups. Finally, the benzimidazole 3 was obtained
by an esterification of the corresponding acid intermediate. The
benzimidazole 3 was coupled with Wang resin14 in the presence
a
(51 kDa) and GSK3b (47 kDa) are known two isomers, which are
highly homologous each other in their kinase domain. GSK3 con-
trols the relevant substrates, such as glycogen synthase (GS), p53,
p27, and b-catenin, which are associated with many diseases such
as type II diabetes mellitus (T2DM), cancer, bipolar disorder,
Alzheimer’s disease, and schizophrenia.1–3 GSK3 is a ubiquitous
kinase that participates in a multitude of cellular processes,
including cell membrane-to-nucleus signaling, gene transcription,
translation, cytoskeletal structuring and cell cycle progression
and survival. Thus, modulation of GSK3 has been considered as
one of the promising therapeutic ways for the treatment of the
corresponding human diseases.4–6
H
N
O
Up to date, a number of orthosteric GSK3b inhibitors have been
proposed with a wide range of potencies at the discovery stage. In
addition, the non-ATP competitive (allosteric) inhibitors in clinical
trial have been reported in literature (Fig. 1).7–11 We have recently
reported 7-hydroxy benzimidazole scaffold as the novel ATP com-
petitive GSK3b inhibitor through X-ray crystallography and dock-
ing studies.12 The scaffold possesses an important hydroxyl
group, which is involved in hydrogen-bonding with NH of Val135
and carbonyl of Asp133.
This implied that the 7-hydroxyl group functioned as a hydro-
gen bonding donor and as an acceptor.
Herein we described structure optimization of the 7-hydroxy
benzimidazole scaffold, their biological evaluation and structure–
O
N
N
NH
N
H
O
H
O
O
HN
Staurosporine
Indirubin
O
N
O
N
S
N
O2N
S
N
H
N
H
O
O
⇑
Corresponding authors. Tel.: +88 31 628 2801; fax: +88 31 628 2701 (S.R.), tel.:
+88 2 880 7875; fax: +88 2 888 0649 (Y.-G.S.).
AR-AO014418
Tideglusib (NP-12)
Figure 1. Examples of the known GSK3 inhibitors.
E-mail addresses: sgro@cgxinc.com (S. Ro), ygsuh@snu.ac.kr (Y.-G. Suh).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.

1892
S.-C. Lee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1891–1894
Table 1
O
O
O
O
O
OH
Inhibitory activities of the 7-hydroxy benzimidazole analogs against GSK3b
N
a, b, c
HCl
NH
d,e,f
H
R1
O
N
R2
N
N
H
N
H
R1
NH2
OMe
OH
OMe
3
2
1
N
H
H
N
OH
O
R2
N
O
O
Analogs R2
Ref. 16
Remaining activity at
M (%)
IC50
M)
N
R1
h, i, j
R1
g
5
l
(
l
N
H
N
H
ꢀ1
75
63
0.070
ND
ND
OH
O
6
7
H
Butyl
5
4
OH
8
9
53
32
ND
2.0
Scheme 1. Reagents and conditions: (a)MeOH, H2SO4, reflux, 90%; (b) R1-CN, p-TSA,
heat to 180–210 °C; (c) HCl (g), bubbling, rt; (d) NaOCl, NaHCO3, 50% aq MeOH, rt
30–70% of three steps; (e) AlCl3, toluene, reflux, 50–80%; (f) MeOH, H2SO4, reflux,
80–90%; (g) Wang bromo resin (black dot), Cs2CO3, KI, DMF, 60 °C; (h) LiOHÁH2O,
MeOH, aq THF, rt, 90–95%; (i) R2-NH2, EDC, HOBt, DMAP, DMF, rt; (j) 10–50% TFA in
DCM solution, rt, 20–80% of two steps.
NO2
O
10
11
36
18
2.8
1.6
S
N
O
H
of Cs2CO3 and KI in DMF at 60 °C. Hydrolysis of the ester 4 with
LiOH and amidation of the resin supporting acid, in the presence
of EDC, HOBt and DMAP, in DMF provided a variety of amide
analogs. Finally, the solid resin was washed with MeOH, DMF
and MC several times and then TFA treatment in DCM yielded
the free 7-hydroxy benzimidazole analog 5.
O
S
N
O
H
ND, IC50 not determined.
addition of the freely rotatable sp3 carbon substituent to the aro-
matic moiety by amidation seemed to enhance the inhibitory
activities of the analogs 8 and 9. The analog 11 exhibited the best
Initially, the synthetic route including solid phase chemistry
was successfully developed for the facile derivatization of R1 and
R2 of 7-hydroxy benzimidazole 5. The linker for R2 was fixed as
amide, which has the optimal direction to the sugar and triphos-
phate binding site of ATP.
GSK3b inhibitory activity with IC50 of 1.6 lM. We turned our atten-
tion to the R1-substituents effect on the inhibitory activity on the
structural basis of 11. The results were summarized in Table 2.
Interestingly, the analogs (13, 14, 15, 17, 18 and 19) directly
linked to the cyclic R1-substituents exhibited more potent GSK3b
inhibitory activities compared to the analog 11. In particular, the
analogs 15 and 17 exhibited excellent inhibitory activities.
The cell based assay was also performed to confirm the intrinsic
cell permeability and potency in cellular environment. The result
of cellular assay revealed the similar tendency as shown in the en-
zyme assay as anticipated, although the analog 19 with an EC50 of
Our docking study (Fig. 2) revealed the oxygen of phenethyl sul-
fonamide moiety, which is linked to amide at the 4-position of the
benzimidazole, would enhance the inhibitory activity by a hydro-
philic interaction with Arg141 at the triphosphate and substrate
binding region of the catalytic domain. Accordingly, the synthesis
was commenced with preparation of the R1 and R2-diversified
analogs by use of solid phase parallel reaction. The GSK3b kinase
inhibitory activity was determined using the known method.15
We first conducted the R2-variation with R1 fixed as a benzyl
group. The GSK3b inhibitory activities were summarized in Table 1.
Initial SAR of the synthesized analogs revealed that introduction
of the relatively small substituents for R2 (6 and 7) provided the
low inhibitory activities. However, introduction of the bulky
aromatic moieties improved the inhibitory potency. In particular,
25 lM of cellular inhibitory activity showed an impermeable
character. Other analogs directly linked to the substituted aromatic
system (15, 16, 17, and 18) showed good cell permeable character
and the analog 15 exhibited the best potency in both enzymatic
and cellular assays. Based on preliminary examination of the R1
Table 2
Enzyme and cellular (Rd cells) inhibitory activities of the 7-hydroxy benzimidazole
analogs against GSK3b
H
O
N
O
S
N
N
H
O
R1
N
H
OH
Analogs
R1
IC50
(l
M)
EC50
(lM)
11
12
13
14
15
16
17
18
19
Benzyl
Methyl
Cyclopropyl
Cyclopentyl
4-Cl-phenyl
2,4-Di-F-phenyl
4-F-phenyl
2-Cl, 4-F-phenyl
4-Pyridyl
1.600
ND
0.140
0.044
0.007
ND
0.001
0.020
0.110
3.90
21.00
4.80
2.70
0.12
0.58
0.40
0.23
25.00
Figure 2. Binding mode of the analog 11 in the GSK3b binding pocket. Interaction of
OH and NH of the hydroxy benzimidazole moiety with Asp133, Val135, and Pro136
with distances of 2.52, 2.82, and 2.86 Å are shown. The oxygen of the sulfonamide
group also interacts with NH of Arg141 with distance of 3.01 Å.
ND, IC50 not determined.

S.-C. Lee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1891–1894
1893
Table 3
Enzymatic and cellular (Rd cells) inhibition of the 7-hydroxy benzimidazole analogs against GSK3b
H
O
N
(CH2
N
)
Ar R3
n
R1
N
H
OH
R3
4-NHSO2CH3
Analogs
R1
n
Ar
IC50
(
lM)
EC50
0.12
(lM)
CCIC50
(
lM)
TIa
20
21
22
23
24
25
26
27
28
29
4-Cl-phenyl
4-F-phenyl
4-Cl-phenyl
2,4-Di-Cl-phenyl
2,4-Di-Cl-phenyl
3-Cl, 4-F-phenyl
2-Thiophenyl
2-Thiophenyl
2-Thiophenyl
2-Furanyl
2
2
3
3
3
3
3
2
3
3
Phenyl
Phenyl
Phenyl
1-Imidazole
1-Imidazole
1-Imidazole
1-Imidazole
Phenyl
0.007
0.026
0.031
0.015
0.006
0.003
0.050
0.046
0.008
0.010
28
17.5
17
45
60
>100
>100
51
>100
70
233
184
68
187
545
>204
>322
127
>2941
1400
4-NHSO2-4-tosyl
3-OCH2CO2H
—
4-Cl, 5-Cl
—
4-Methyl
4-CON(C2H4)2NC2H5
4-Cl, 5-Cl
4-Cl, 5-Cl
0.095
0.25
0.24
0.11
0.49
0.31
0.40
0.034
0.05
1-Imidazole
1-Imidazole
a
In vitro therapeutic index (IC50 of cytotoxicity/IC50 of enzyme inhibition).
synthase and reduces the blood glucose level in mammalian.17
Accordingly, we tested the glycogen synthase (GS) activation of
the representative analogs 16, 22, 24, 25 and 29 to confirm the
pathophysiological relation of GSK3b with T2DM.18 This assay
was performed in Rd cell with the 24 h starving serum before the
test. The results were summarized in Table 4. Considering that
the tested analogs effectively activated the glycogen synthase
(GS), the benzimidazole analogs seemed to inhibit the negative
modulator (GSK3b) in the GS activation assay system. These results
also imply that the benzimidazole analogs lower the blood glucose
level of the systemic circulation and accumulate the glycogen in
organs.
Table 4
Glycogen synthase activation assay
Analogs
EC50 (lM) (GS activation, Rd cells)
16
22
24
25
29
0.013
0.080
0.170
0.100
0.040
Table 5
Selectivity assay of the representative analogs
GSK3b is well known to have multifunction in the many physi-
ological pathways and is ubiquitously expressed in the many types
of mammalian tissue, such as liver and muscle tissue.16 Thus, the
kinase selectivity using a broad panel of seven protein kinases
was examined for the representative analogs, which depicted
excellent activities against GSK3b. The results were summarized
in Table 5. In general, the tested benzimidazole analogs exhibited
high kinase selectivity and low inhibitory activities against the se-
Analogs
Selectivity (remaining activity at 5
GSK3b IKKb ERK1 ERK2 KDR CDK4 CDK1 AKT
91.4 114
104.5 54.5 58.6
102.2 108.9 74.9
lM) (%)
16
22
24
25
27
29
À0.58
0.3
0.0
93.1
87.3
80.7
80.0
72.7
74.7
73.4
80.0
97.9
66.5
80.9
53.9
49.2
ND
ND
100.5
ND
90.9
ND
1.0
76.0
80.7
70.6
85.3 52.4
60.8 68.2
83.6 38.6
71.3
70.4
65.2
83.9
96.0
92.3
0.9
À1.7
ND, not determined.
ven protein kinases at 5 lM level. However, the analogs 16 and 29
partially inhibited CDK4. This is likely due to the structural similar-
and R2-substituents, the aromatic systems for the R1 and R2-
substituents including the length of linker as well as the substitu-
ent effects of both aromatic systems on the inhibitory activities
were further investigated. The results were summarized in Table
3. Generally, the propyl linker provided the better enzymatic and
cellular inhibitory activities.
ity of the ATP binding pockets of both kinases.19
In summary, we established the structure–activity relationship
of the 7-hydroxy benzimidazole analogs, which were prepared by
the solid phase parallel synthesis. The synthesized benzimidazole
analogs exhibited excellent inhibitory activities against GSK3b in
both enzyme and cell based assays. In addition, they exhibited
the high selectivity in the assay of the seven protein kinases. Cur-
rently, further studies for the therapeutic implication of the se-
lected analogs are in progress.
The analogs possessing the phenyl substituent for R1 showed
the similar inhibitory potencies regardless of substituent and sub-
stitution pattern (20, 21, and 22 for mono-substitution; 23, 24 and
25 for di-substitution). In addition, the enzyme inhibitory activities
were well correlated with the cell based inhibitory activities.
Among the analogs possessing the hetero-aromatic moieties for
R1, the analogs possessing the 2-thiophenyl (28) and the 2-furanyl
(29) moieties provided the excellent cellular inhibitory activities.
We also examined cellular toxicity of the synthesized analogs.
Acknowledgments
This work was partially supported by Yuyu, Inc., the National
Research Laboratory Program of MOST (Ministry of Science and
Technology) and MOCIE (Ministry of Commerce, Industry and En-
ergy) of Korea and Medium-term Strategic Technology Develop-
ment Program of MOICE.
The MTS assay revealed CCIC50s of higher than 100
lM for the
analogs 25, 26 and 28 and the cytotoxic effects at 17
lM for
the analogs 21 and 22. The MTS assay result implied that the 7-
hydroxy benzimidazole scaffold is quite safe in terms of the
in vitro cytotoxicity.
GSK3b is one of the main mediators of the blood glucose and
negatively controls the glycogen synthase. The competitive inhibi-
tion of GSK3b against ATP causes an activation of the glycogen
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