Synthesis and Evaluation of 3-(furo[2,3-b]pyridin-3-yl)-4-(1H-indol-3-yl)-maleimides as Novel GSK-3β Inhibitors and Anti-Ischemic Agents

Article abstract of DOI:10.1111/cbdd.12546

A series of novel 3-(furo[2,3-b]pyridin-3-yl)-4-(1H-indol-3-yl)-maleimides were designed, synthesized, and biologically evaluated for their GSK-3β inhibitory activities. Most compounds showed favorable inhibitory activities against GSK-3β protein. Among them, compounds 5n, 5o, and 5p significantly reduced GSK-3β substrate tau phosphorylation at Ser396 in primary neurons, indicating inhibition of cellular GSK-3β activity. In the in vitro neuronal injury models, compounds 5n, 5o, and 5p prevented neuronal death against glutamate, oxygen-glucose deprivation, and nutrient serum deprivation which are closely associated with cerebral ischemic stroke. In the in vivo cerebral ischemia animal model, compound 5o reduced infarct size by 10% and improved the neurological deficit. The results may provide new insights into the development of novel GSK-3β inhibitors with potential neuroprotective activity against brain ischemic stroke.

Full text of DOI:10.1111/cbdd.12546

Chem Biol Drug Des 2015; 86: 746–752
Research Article
Synthesis and Evaluation of 3-(furo[2,3-b]pyridin-3-yl)-
4-(1H-indol-3-yl)-maleimides as Novel GSK-3b Inhibitors
and Anti-Ischemic Agents
1
,2
1
2
2
Qing Ye , Qiu Li , Yubo Zhou , Lei Xu ,
of effective treatment for ischemia stroke. Glycogen
synthase kinase-3b (GSK-3b) is a serine/threonine protein
kinase which phosphorylates and regulates many impor-
tant metabolic and signaling proteins, structural proteins,
and transcription factors (3,4). The amounting evidence
indicate that increased GSK-3b activity contributes to neu-
ronal death and pathogenesis of ischemic stroke. There is
an increased GSK-3b phosphorylation at tyrosine 216 resi-
due following middle cerebral artery occlusion (MCAO),
indicating the involvement of active GSK-3b (5). Recent
studies have shown that inhibition of GSK-3b attenuates
apoptotic signals and prevents neuronal death (6,7).
Recently, it has been shown that GSK-3b inhibition by lith-
ium, Chir025, or TDZD-8 was protective against cerebral
ischemia and prevents neurons from glutamate-induced
apoptosis, suggesting that GSK-3b inhibition improves
brain neurons survival (8–12). Considering important roles
of GSK-3b in neuronal apoptosis, GSK-3b inhibition may
be a therapeutic strategy for ischemic neuroprotection. As
previously reported, several kinds of monoindolylmaleimide
derivatives such as imidazo[1,2-a]pyridinyl-indolyl-malei-
mides, 4-azaindolyl-indolyl-maleimides, benzisoxazolyl-ind-
olyl-maleimides, and 7-azaindazolyl-indolyl-maleimides
have been identified as potent and selective GSK-3b inhib-
itors (13–16). These studies encouraged us to design and
synthesize a novel series of 3-(furo[2,3-b]pyridin-3-yl)-4-
1
3
3
Weili Mao , Yuanxue Gao , Chenhui Li ,
3
3
3
Yuan Xu , Yazhou Xu , Hong Liao ,
3
1,
2,
Luyong Zhang , Jianrong Gao *, Jia Li * and
Tao Pang *
3
,
1
State Key Laboratory Breeding Base of Green Chemistry-
Synthesis Technology, Zhejiang University of Technology,
Hangzhou 310032, China
State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China
Jiangsu Key Laboratory of Drug Screening, China
Pharmaceutical University, Nanjing 210009, China
Corresponding authors: Jianrong Gao, gdgjr@zjut.edu.cn;
Jia Li, jli@simm.ac.cn; Tao Pang, tpang@cpu.edu.cn
2
3
*
A series of novel 3-(furo[2,3-b]pyridin-3-yl)-4-(1H-indol-
-yl)-maleimides were designed, synthesized, and bio-
logically evaluated for their GSK-3b inhibitory activities.
Most compounds showed favorable inhibitory activities
against GSK-3b protein. Among them, compounds 5n,
3
5
o, and 5p significantly reduced GSK-3b substrate tau
phosphorylation at Ser396 in primary neurons, indicat-
ing inhibition of cellular GSK-3b activity. In the in vitro
neuronal injury models, compounds 5n, 5o, and 5p
prevented neuronal death against glutamate, oxygen–
glucose deprivation, and nutrient serum deprivation
which are closely associated with cerebral ischemic
stroke. In the in vivo cerebral ischemia animal model,
compound 5o reduced infarct size by 10% and
improved the neurological deficit. The results may pro-
vide new insights into the development of novel GSK-
(1H-indol-3-yl)-maleimides using bioisostere and ring
substitution principle to find more potent and selective
inhibitors of GSK-3b which could be used for the treat-
ment of cerebral ischemia disease. In addition, the struc-
ture–activity relationships (SAR) and molecular modeling
study in GSK-3b protein were also discussed in this study.
3
b inhibitors with potential neuroprotective activity
against brain ischemic stroke.
Key words: anti-ischemic agents, GSK-3b inhibitors, kinase
Experimental Section
activity, maleimides
Chemistry
Received 29 November 2014, revised 18 January 2015 and
accepted for publication 9 February 2015
Melting points were determined with a B U€ CHI Melting
Point B-450 apparatus (Buchi Labortechnik, Flawil, Swit-
€
1
zerland). The H NMR spectra were recorded on a Bruker
Avance DMX 500 at 500 MHz (chemical shifts are
expressed as d values relative to TMS as internal stan-
dard). ESI (positive) was recorded on an Esquire-LC-
Stroke becomes the worldwide burden as its high mortality
and morbidity, causing about 6 000 000 deaths each
year. In the total number of strokes, ischemic stroke
accounts for more than 80% (1,2). At present, there is lack
0
0075 spectrometer. Element analyses were performed on
an Eager 300 instrument. All reactions were monitored by
7
46
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12546

Maleimides GSK-3b Inhibitors Anti-Ischemic Agents
thin-layer chromatography (TLC). All reagents were
obtained from commercial sources and used without fur-
was poured into cold water (200 mL) and extracted with
ethyl acetate (3 9 100 mL). The organic phase was com-
ther purification unless stated. Et
2
O and THF were distilled
2 4
bined, washed with brine (3 9 300 mL), dried over Na SO ,
from sodium-benzophenone. DMF was distilled from cal-
cium hydride. Preparation methods and physicochemical
properties for compounds 2a, 2d, 2f–i, 3a, 3b, 3l, 3n, 3q,
and 3r were reported in Ref (15).
and concentrated in vacuo. The residue was purified by
flash column chromatography on silica gel using ethyl
acetate/methanol (50:1, v/v) as eluent to afford 3o and 3p.
General procedure for the preparation of 5a–r
General procedure for the preparation of 2b, 2c,
and 2e
A solution of t-BuOK in t-BuOH (0.84 mL, 0.84 mmol) was
added dropwise to a solution of 3a–r (0.37 mmol) and 2-
(furo[2,3-b]pyridin-3-yl)acetamide 4 (0.28 mmol) in dry THF
(10 mL) at À5 to 0 °C. After stirring for 2 h at room tem-
perature, concentrated hydrochloric acid (5 mL) was
added and the resultant mixture was stirred for 30 min at
room temperature, poured into 10% NaHCO3 aqueous
solution (100 mL), and extracted with ethyl acetate
(3 9 50 mL). The organic phase was combined and
Oxalyl chloride (3.4 g, 26 mmol) in dry Et
added dropwise to a solution of indole derivatives 1b, 1c,
and 1e (26 mmol) in dry Et O (30 mL) at 0–5 °C. The
reaction mixture was stirred under the same conditions for
2
O (5 mL) was
2
1
5
h, and 17.5 wt% solution of CH ONa in MeOH (16.3 g,
2 mmol) was added dropwise at À30 to À20 °C. After
3
addition, the mixture was stirred for 30 min at room tem-
perature and poured into cold water (100 mL). The prod-
uct was isolated with filtration, washed with
dichloromethane, and dried to afford 2b, 2c, and 2e.
2 4
washed with brine (3 9 150 mL), dried over Na SO , and
concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel using dichlorome-
thane/methanol as eluent to give 5a–r.
General procedure for the preparation of 3c–k
Yield, physical–chemical data, and bioassay method were
70% NaH (0.17 g, 4.9 mmol) was added portionwise to a
recorded in Appendix S1.
solution of 2a–i (4.9 mmol) in dry DMF (10 mL) at 0–5 °C.
The reaction mixture was then warmed to room tempera-
ture and stirred for 30 min. After that, alkyl halide
Results and Discussion
(
5.9 mmol) was added at 0 °C and stirred for 1 h at room
temperature. The mixture was then poured into cold water
100 mL) and extracted with ethyl acetate (3 9 50 mL).
The organic phase was combined, washed with brine
3 9 150 mL), dried over Na SO , and concentrated in
Chemistry
(
The synthetic route of target compounds 5a–r is illustrated
in Scheme 1. Indole derivatives 1a–i were reacted with ox-
(
2
4
alyl chloride, followed CH
3
ONa to afford 2a–i. The reaction
vacuo. The residue was purified by flash column chroma-
tography on silica gel using petroleum ether/ethyl acetate
of 2a with (Boc) O in the presence of catalytic amount of
2
DMAP in THF afforded 3a. N-Alkylation of 2a–i with differ-
ent alkyl halides using NaH as a base resulted in other key
intermediates 3b–r. Condensation of 3a–r with 2-(furo[2,3-
b]pyridin-3-yl)acetamide 4 in the presence of t-BuOK in t-
BuOH afforded the target compounds 5a–r.
(3:1, v/v) as eluent to afford 3c–k.
Procedure for the preparation of 3m
A mixture of 2a (3 mmol), cesium carbonate (6 mmol), 2-
bromopropane (6 mmol), and dry DMF (20 mL) was stirred
at 50 °C for 8 h. After cooling, the mixture was poured
into water (200 mL) and extracted with ethyl acetate
Biological activity and molecular modeling study
(3 9 50 mL). The organic phase was combined, washed
Enzymatic activity
with brine (3 9 100 mL), dried over Na SO , and concen-
2
4
The GSK-3b inhibitory potency of all target compounds
was examined. In addition, the assays of inhibitory activity
toward PKCe, JAK2, BRAF, IKK2, Drak2, PKA, Akt1, and
CDK2 were also conducted to determine the selectivity of
tested compounds. A well-known kinase inhibitor stauro-
sporine was used as the positive control. The results are
summarized in Tables 1 and 2.
trated in vacuum. The residue was purified by flash col-
umn chromatography on silica gel using petroleum ether/
ethyl acetate (3:1, v/v) as eluent to afford 3m.
General procedure for the preparation of 3o and
3p
7
0% NaH (7.4 mmol) was added portionwise to a solution
As indicated in Table 1, most of the tested compounds
displayed moderate-to-high GSK-3b inhibitory potency.
Introduction of an alkyl substituent, such as methyl (5b,
IC50 = 0.28 lM), allyl (5k, IC50 = 0.53 lM), butyl (5l,
of 2g and 2e (7.4 mmol) in DMF (20 mL) at 0–5 °C. The
reaction mixture was warmed to room temperature and stir-
red for 30 min. After that, 1-(3-chloropropyl)-1H-imidazole
1
(9.6 mmol) was added. Then the mixture was headed to
IC50 = 0.57 lM), and isopropyl (5m, IC50 = 0.19 lM) at N -
55–60 °C and reacted for 12 h. After cooling, the mixture
position of the indole ring, led to about three- to ninefold
Chem Biol Drug Des 2015; 86: 746–752
747

Ye et al.
2 3 3 2
Scheme 1: Synthetic route to compounds 5a–r. Reagents and conditions: (a) i (COCl) , diethyl ether; ii CH ONa, CH OH; (b) (Boc) O,
DMAP, THF; (c) NaH, R X, DMF; (d) i t-BuOK, t-BuOH; ii concentrated HCl.
2
Table 1: GSK-3b inhibitory activity of the target compounds
Table 2: The selectivity to tested kinases of target compounds
n, 5o, and 5p
5
Compounds
Staurosporine
R
1
R
2
IC50 (lM) Æ SE
IC50 (nM) Æ SE or inhibition% at ~2 lM
Kinases Staurosporine 5n 5o
0.038 Æ 0.0002
1.78 Æ 0.12
0.28 Æ 0.01
0.29 Æ 0.03
0.39 Æ 0.04
0.56 Æ 0.16
0.17 Æ 0.02
1.84 Æ 0.10
0.07 Æ 0.02
1.94 Æ 0.10
1.31 Æ 0.11
0.53 Æ 0.07
0.57 Æ 0.11
0.19 Æ 0.02
0.21 Æ 0.02
0.19 Æ 0.01
0.24 Æ 0.02
0.31 Æ 0.03
0.69 Æ 0.01
5p
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
GSK-3b 38.0 Æ 2.4
210.0 Æ 20.0 190.0 Æ 10.0 240.0 Æ
7-Me
5-F
20.0
PKCe
JAK2
BRAF
IKK2
0.6 Æ 0.02
2.3 Æ 0.1
5.0 Æ 0.8
0.9 Æ 0.2
25.8 Æ 2.1
10.0 Æ 0.1
17.6 Æ 1.5
3.2 Æ 0.1
13.9%
15.1%
8.9%
25.9%
16.9%
13.2%
32.7%
5.9%
20.2%
11.7%
10.1%
14.6%
1.9%
6-F
5-Cl
6-Cl
5-Br
6-Br
g
h
i
12.9%
3.5%
Drak2
PKA
Akt1
20.4%
3.8%
7.4%
5.7%
4.9%
1.6%
j
5-Meo Me
k
l
H
Allyl
CDK2
1.3%
4.1%
2.2%
H
Butyl
m
n
o
p
q
r
H
Isopropyl
SE, standard error mean.
H
3-(1H-imidazol-1-yl)propyl
3-(1H-imidazol-1-yl)propyl
3-(1H-imidazol-1-yl)propyl
Hydroxypropyl
5-Br
5-Cl
H
hydrogen bond interactions with amino acids such as
Asp-200, Lys-183, and Gln-185 of GSK-3b) at N -position
of the indole ring could give an obvious increase of the
1
H
3-morpholinopropyl
activity (i.e. 5a, 5n–p). Among them, compound 5n with
an imidazole group at the end of N -propyl chain exhibited
SE, standard error mean.
1
potent GSK-3b inhibitory activity with IC50 of 0.21 lM,
which was about eightfold increase compared to that of
compound 5a.
increase in potency compared to the case of 5a
IC50 = 1.78 lM). It suggested that these substituents may
(
form hydrophobic interactions with the hydrophobic
pocket of GSK-3b. In addition, introduction of a suitable
hydrophilic side chain (which may be adapted to form
The inhibitory activities of the compounds with different
substituents on the indole ring (i.e. 5b–j, 5n–p) revealed
7
48
Chem Biol Drug Des 2015; 86: 746–752

Maleimides GSK-3b Inhibitors Anti-Ischemic Agents
Table 3: The neuroprotective effects of selected compounds on
glutamate-induced neurotoxicity in primary rat cerebellar granule
neuronal cells (CGCs)
mate, nutrient serum deprivation (-B27), or oxygen–glu-
cose deprivation (OGD). The results are shown in Table 3
and Figures 1–3.
a
Compounds
Cell viability (%) at 10 lM
As indicated in Table 3, compounds 5n, 5o, and 5p with
1
5
5
5
5
5
5
5
c
f
52.4 Æ 2.9
40.6 Æ 3.9
48.0 Æ 5.8
33.7 Æ 0.9
83.6 Æ 2.8
81.2 Æ 6.6
84.8 Æ 5.6
a 3-(1H-imidazol-1-yl)propyl at N -position of the indole
ring showed potent neuroprotection against glutamate-
induced cells death, which correlates well with their activi-
ties on GSK-3b inhibition. Moreover, compounds 5n, 5o,
and 5p dose dependently prevented neurons from gluta-
mate-induced neuronal death (Figure 1). As shown in
Figures 2 and 3, compounds 5n, 5o, and 5p can also
reduce nutrient serum deprivation (-B27)- or OGD-induced
neuronal death at 10 lM. However, potent GSK-3b inhibi-
h
m
n
o
p
a
The neuroprotective effects of these compounds on glutamate-
induced neurotoxicity in cerebellar granule neurons. The cell viabil-
ity in control was taken as 100%, and the average value of cell
viability under glutamate exposure was 41.3%.
1
tors 5c, 5f, 5h, and 5m with an alkyl substituent at N -
position of the indole ring did not exhibit neuroprotection
against glutamate-induced cells death.
that introduction of a hydrophobic substituent on the 5-
position (i.e.,5-F, 5-Cl, 5-Br) of the indole ring was tolerate
for the activity, while fluorine, chlorine, and bromine at 6-
position or methoxyl, a hydrophilic group, at 5-position led
to a loss of potency.
To demonstrate whether the neuroprotection of com-
pounds 5n, 5o, and 5p is mediated by inhibition of cellu-
lar GSK-3b, we measured the effects of compounds on
cellular GSK-3b substrate tau phosphorylation at residue
Ser396 which is GSK-3b dependent, determined by Wes-
tern blotting using a phosphospecific antibody. The low lev-
els of cellular tau phosphorylation at residue Ser396 indicate
the inhibitory activity of cellular GSK-3b. As shown in
Figure 4, the phosphorylation of tau (Ser396) in primary neu-
rons was inhibited by compounds 5n, 5o, and 5p, indicating
the inhibition of cellular GSK-3b activity, which may be
responsible for the neuroprotective effects of these com-
pounds. These data suggested that these compounds
could exhibit potential neuroprotective activities through
GSK-3b inhibition.
The data in Table 2 showed that selected compounds 5n,
5o, and 5p displayed high selectivity for GSK-3b over
other tested kinases including PKCe, JAK2, BRAF, IKK2,
Drak2, PKA, Akt1, and CDK2. Staurosporine, a positive
control, exhibited as a potent and non-selective kinase
inhibitor, which not only inhibits GSK-3b (IC50 = 38.0 nM),
but also potently inhibits other kinases (i.e. PKCe, JAK2,
BRAF, IKK2, Drak2, PKA, Akt1, CDK2).
Cellular activity
The cerebral ischemia is characterized by excess gluta-
mate release, insufficient nutrient supply, low oxygen, and
glucose supply in the brain which result in neuronal death
at last. Therefore, selected compounds with high GSK-3b
inhibitory activity were further investigated for their in vitro
neuroprotective effects on neurotoxicity induced by gluta-
In vivo anti-ischemic activity
Compound 5o with good cellular neuroprotective activity
was chosen for in vivo evaluation in animal brain ische-
mic model. The widely accepted transient middle cere-
bral artery occlusion (tMCAO) stroke model was used in
Figure 1: Neuroprotective effects of
compounds 5n, 5o, and 5p on glutamate-
induced neuronal injury. Compounds at
1
0 lM show potent protection against
glutamate-induced cells death. Data are
expressed as mean Æ SD (n = 4),
#
#
*
**p < 0.001 versus Control, p < 0.01,
#
##
p < 0.001 versus Glutamate group.
Chem Biol Drug Des 2015; 86: 746–752
749

Ye et al.
Figure 2: Neuroprotective effects of compounds 5n, 5o, and 5p
on nutrient B27 deprivation (-B27)-induced neuronal injury.
Compounds (10 lM) significantly attenuated the reduced cell
viability. Data are expressed as mean Æ SD (n = 4), ***p < 0.001
#
###
versus Control with B27, p < 0.05,
p < 0.001 versus -B27
group.
Figure 5: Effect of compound 5o on acute cerebral ischemia in
rats at 24 h after MCAO. 5o ameliorated ischemia-induced infarct
volume and the neurological deficits. *p < 0.05 versus Vehicle
(MCAO group).
treated rats. There was no statistical difference in infarct
size and the neurological deficit between vehicle-treated
and saline-treated rats (data not shown) ruling out a pro-
tective effect of the vehicle. Until now, there is no GSK-
3b inhibitor with in vivo anti-ischemic effects having been
approved for clinical trials or clinical use. Therefore,
in vivo studies with compound 5o indicated that these
series of GSK-3b inhibitors as novel neuroprotective
agents may have beneficial effects on the treatment of
brain ischemic stroke.
Figure 3: Neuroprotective effects of compounds 5n, 5o, and 5p
on oxygen–glucose deprivation (OGD)-induced neuronal injury.
Compounds (10 lM) show potent protection against OGD insult.
Data are expressed as mean Æ SD (n = 4), ***p < 0.001 versus
Molecular modeling study
The binding modes between the active maleimides and
GSK-3b [1Q3D (17)] were proposed as exemplified in the
case of representative compounds with different side
##
###
Control, p < 0.01,
p < 0.001 versus OGD group.
1
chains at N -position of the indole ring (i.e. 5a, 5m, and
5
n) using the Discovery studio 2.5/LigandFit. Figure 6
shows that 5a, 5m, and 5n can tightly occupy ATP bind-
ing site of GSK-3b, and the docking results are consistent
with their GSK-3b potencies (IC50 of 5a, 5m, and 5n are
1
.78, 0.19, and 0.21 lM, respectively). NH and carbonyl
group in maleimide ring of 5a, 5m, and 5n can form two
hydrogen bonds with Asp133 and Val135 in hinge domain
of GSK-3b. Besides, indole ring of 5a, 5m, and 5n can
insert into the hydrophobic pocket of GSK-3b which is
embraced by Val70, Leu132, and Cys199. Comparing to
Figure 4: Effects of GSK-3b inhibitors on tau phosphorylation in
primary cerebellar granule neurons. The phosphorylated tau
(Ser396) and total tau protein were determined by Western
compound 5a, additional interaction can be observed in
blotting. Compounds (10 lM) show inhibition of tau (Ser396)
phosphorylation in neurons.
1
5
m (isopropyl group at N -position of the indole ring
inserts into the small pocket embraced by Val70, Gly63,
and Gly65) and 5n (3-position nitrogen atom of the imid-
azole ring of 5n forms hydrogen bond with Lys-183),
which resulted in the significant enhancement of the GSK-
3b inhibitory activities of 5m (~36 folds) and 5n (~13
folds).
this study (18–20). As shown in Figure 5, compound
5
o-treated rats displayed significantly smaller average
infarct size and the neurological deficit than vehicle-
7
50
Chem Biol Drug Des 2015; 86: 746–752

Maleimides GSK-3b Inhibitors Anti-Ischemic Agents
A
B
C
D
Figure 6: Docking of 5a, 5m, and 5n to GSK-3b crystal structure. (A) Compound 5a bound to GSK-3b; (B) compound 5m bound to
GSK-3b; (C) compound 5n bound to GSK-3b; (D) the alignment of proposed docking conformation of 5a, 5m, and 5n. The hydrogen
bonds were highlighted by green dash lines.
Acknowledgments
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