Design, synthesis and evaluation of 7-azaindazolyl-indolyl-maleimides as glycogen synthase kinase-3β (GSK-3β) inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.07.046

A series of 7-azaindazolyl-indolyl-maleimides were designed, synthesized and evaluated for their GSK-3β inhibitory activity. Most compounds exhibited potent activity against GSK-3β. Among them, compounds 17a, 17b, 17g, 17i, 29a and 30 significantly reduced Aβ-induced Tau hyperphosphorylation, showin;g the inhibition of GSK-3β at the cell level. Preliminary structure-activity relationships were discussed based on the experimental data obtained.

Full text of DOI:10.1016/j.ejmech.2013.07.046

European Journal of Medicinal Chemistry 68 (2013) 361e371
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and evaluation of 7-azaindazolyl-indolyl-
maleimides as glycogen synthase kinase-3
b
(GSK-3
b) inhibitors
,
,
Qing Ye a b, Yanhong Shen d, Yubo Zhou c, Dan Lv a, Jianrong Gao b, Jia Li c
**
,
Yongzhou Hu a
,
*
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
b State Key Laboratory Breeding Base of Green Chemistry-Synthesis Technology, Zhejiang University of Technology, Hangzhou 310032, China
c The National Center for Drug Screening, Shanghai 201203, China
d Department of Chemical Engineering, Anyang Institute of Technology, Anyang 455000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 7-azaindazolyl-indolyl-maleimides were designed, synthesized and evaluated for their GSK-
Received 25 March 2013
Received in revised form
5 July 2013
Accepted 12 July 2013
Available online 9 August 2013
3
b
inhibitory activity. Most compounds exhibited potent activity against GSK-3
pounds 17a, 17b, 17g, 17i, 29a and 30 significantly reduced A -induced Tau hyperphosphorylation,
showin;g the inhibition of GSK-3 at the cell level. Preliminary structureeactivity relationships were
discussed based on the experimental data obtained.
b. Among them, com-
b
b
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
7-Azaindazolyl-indolyl-maleimides
GSK-3b inhibitors
Enzymatic activity
Cellular activity
1. Introduction
on staurosporine, a microbial alkaloid that was identified as an
early GSK-3 inhibitor (Fig. 1) [14e18]. Most of these bisindo-
b
Glycogen synthase kinase-3 (GSK-3) is a multifunctional
serine/threonine protein kinase which was identified in the late
lylmaleimides have suffered from issues including toxicity, bad
solubility and poor selectivity, which make them unsuitable for the
treatment of diseases such as diabetes and Alzheimer’s disease
[19,20]. Recent efforts in replacing one indole with other heteroaryl
substituents such as imidazo[1,2-a]pyridinyl and benzofuranyl led
to the emergence of some monoindolylmaleimides (Fig. 1) exhib-
1970s. Mammalian GSK-3 exists in two isomers, GSK-3
a and
GSK-3 , which share high homology at the catalytic domain but
b
significantly differ in their N-terminal domain (84% overall and
98% in catalytic domain). Both isomers ubiquitously exist in
cells and tissues and have similar biochemical properties [1e4].
GSK-3 plays a critical role in glycogen metabolism, embryo-
genesis, mitotic regulation, inflammation and neuroplasticity
[5e8]. Inhibition of GSK-3 may provide therapy for several dis-
eases such as cancer, diabetes type-2, chronic inflammatory
processes, stroke, bipolar disorders and Alzheimer’s disease and
so on [9e13]. Accordingly, searching for GSK-3 inhibitors is a
very active area in both academic centers and pharmaceutical
companies.
iting potent and selective GSK-3
b inhibitory activity [21,22].
Among which, compound 603288-22-8, an imidazo[1,2-a]pyr-
idinyl-indolyl-maleimide derivative, has reached preclinical trials
for the treatment of diabetes. In our previous studies, we have
developed a series of 4-azaindolyl-indolyl-maleimides, and some of
them showed potent inhibitory potency against GSK-3b [23]. The
results revealed that the introduction of a nitrogen atom at 4-
position of the indole ring could remarkably increase the selec-
tivity for GSK-3b. Herein a series of 7-azaindazolyl-indolyl-mal-
Various bisindolylmaleimides such as GF 109203X and Ro 31-
eimdes was designed and synthesized by introducing modified
indole and 7-azaindazole pharmacophore into the maleimide
scaffold. All synthesized compounds were evaluated for their GSK-
8820 have been developed as potent GSK-3
b inhibitors based
3b inhibitory activities. Selected compounds 17g, 17i and 29a were
also tested for their inhibitory potency against other kinases (PKCE,
IKK2 and Aurora A et al.) to assess kinase selectivity. Furthermore,
the structureeactivity relationships were discussed.
* Corresponding author. Fax: þ86 0571 88208460.
** Corresponding author. Fax: þ86 021 50801313.
E-mail addresses: jli@mail.shcnc.ac.cn (J. Li), huyz@zju.edu.cn (Y. Hu).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.07.046

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Q. Ye et al. / European Journal of Medicinal Chemistry 68 (2013) 361e371
2. Results and discussion
2.2. Biological activity and molecular modeling
2.1. Chemistry
2.2.1. Enzymatic activity
The GSK-3 inhibitory potency of all the target compounds was
b
The synthetic route of target compounds 8, 9 and 12aef is
outlined in Scheme 1. 3-(Tributylstanny)-1H-pyrazolo[3,4-b]pyr-
idine 6 was prepared following the reported method by using 2-
chloronicotinonitrile 1 as a raw material [24e26]. Stille coupling
of 6 with 3-chloro-4-(1H-indol-3-yl)-1-phenyl-1H-pyrrole-2,5-
dione 7 [27,28] in the presence of Pd(PPh3)2Cl2 obtained 7-
azaindazolyl-indolyl-maleimide 8 [29]. Compound 9 were pre-
pared by treatment of 8 with neat NH4OAc [28]. Reaction of 8
with methylamine in methanol afforded 10, which was reacted
with 5 mol/L KOH in ethanol to give 11 [30,31]. Compounds 12aef
were obtained by reaction of 11 with corresponding primary
amines in DMF [32].
The preparation of target compounds 15, 17aei and 20 is
illustrated in Scheme 2. Stille coupling of compound 6 with 13 in
the presence of Pd(PPh3)2Cl2 yielded compound 14, which was
reacted with alkyl halides to afford 16aei. Compound 18 was
prepared by treating 14 with excessive 1,4-dibromobutane in the
presence of Cs2CO3 [29]. Treatment of 18 with morpholine
resulted in 19. Compounds 15, 17aei and 20 were prepared by
treatment of 14, 16aei and 19 with neat NH4OAc at 140 ꢀC
respectively [30].
Compounds 27, 29aee and 30 were prepared as shown in
Scheme 3. Stille coupling of 6 with 21 afforded the intermediate 22,
which was subsequently reacted with 4-(3-chloropropyl)morpho-
line to yield 23 [29]. The deprotection of N-Boc group of 23 with
concentrated hydrochloric acid afforded 24. Alkylation of 24 with
alkyl halides yielded 25 and 28aee. Reaction of 25 with potassium
t-butoxide afforded compound 26. Compounds 27, 29aee and 30
were obtained by treatment of 26, 28aee and 24 with NH3$H2O in
DMF respectively [33].
examined. In addition, the assays of inhibitory activity toward
PKCE, IKK2, Aurora A, MEK1 and ERK1 were also conducted to
determine the selectivity of tested compounds. Staurosporine, a
well known kinase inhibitor was used as the reference compound
[21]. The results are summarized in Tables 1 and 2.
As shown in Table 1, most compounds displayed moderate to
potent inhibitory activity against GSK-3b. The potency of GSK-3b
inhibition of tested compounds was mainly influenced by the
substitutions on the maleimide nitrogen as well as the side chains
on the nitrogen of the 7-azaindazole or indole ring. Replacement of
the hydrogen on the nitrogen in maleimide ring with various
substituents led to significant decrease in the potency, by
comparing of 8, 12aef with 9, which indicated that the NH of the
imide moiety is an essential element of the pharmacophore.
As expected, the hydrophilic side chain at N1-position of the 7-
azaindazole ring was a key feature for enhancing inhibitory po-
tency (i.e. 17aei, 20). Among them, compound 17a, with a mor-
pholine group at the end of the N1-propyl chain, exhibited potent
GSK-3
16-fold more potent than N1-position no substituted compound 15
M). Replacement of the N1-terminal morpholine
b inhibitory activity with IC50 of 0.75 mM, which was about
(IC50 ¼ 12.27
m
group in 17a with a hydroxy group (17b) led to a slight drop in
potency. Furthermore, replacement of the morpholine group with
other substituents such as piperidine (17c), 4-methylpiperazine
(17d), imidazole (17e) and dimethylamine (17f) resulted in 4e8-
fold loss in potency.
A view on inhibitory data of compounds 17e, 17g and 17h
showed that changing the length of the N1-alkyl linker could
affect GSK-3
b
inhibitory potency. For example, compounds 17g
(IC50 ¼ 0.81
m
M) with a (CH2)2 linker and 17h (IC50 ¼ 0.96 M) with
m
Fig. 1. GSK-3
b
inhibitors.

Q. Ye et al. / European Journal of Medicinal Chemistry 68 (2013) 361e371
363
Sn(Bu)3
N
I
NH2
N
N2BF4
CN
Cl
a
b
e
c
d
N
N
N
N
N
N
H
N
N
N
N
N
H
N
N
N
H
H
H
1
4
3
2
6
5
H
N
O
O
g
Ph
N
Ph
N
N
N
N
H
N
O
O
O
O
H
9
f
6
Cl
N
N
N
O
O
N
H
N
N
H
H
h
7
8
N
N
N
N
H
H
10
R
N
12a
12b
R: NH2
O
O
O
O
O
R: CH2CH2(CH3)2
R: NHCHO
j
i
12c
10
N
N
N
N
N
H
N
12d R: NHCONH2
N
N
H
H
H
12e
R: NHCSNH2
11
12a-f
12f
R: CH2COOCH3
Scheme 1. The synthetic route for compounds 8, 9 and 12aef. Reagents and conditions: (a) NH2NH2$H2O, EtOH, reflux, 78%; (b) CHMe2CH2CH2NO2, HBF4, CH3OH, 0e5 ꢀC, 70%; (c)
HPO2, H2O, rt, 79%; (d) I2, KOH, DMF, rt, 88%; (e) EtMgBr, (Bu)3SnCl, THF, ꢁ5e0 ꢀC, 61%; (f) Pd(PPh3)2Cl2, LiCl, toluene, 100 ꢀC, 55%; (g) NH4OAc, 140 ꢀC, 86%; (h) CH3NH2, CH3OH, rt,
83%; (i) 5 mol/L KOH, EtOH, 30e35 ꢀC, 70%; (j) ReNH2, DMF or ReNH2.HCl, DMF, base, 35e68%.
a (CH2)4 linker showed better inhibitory activities than compounds
M) with a (CH2)3 linker. The same conclusion
could also be draw from comparison the inhibitory potency of 17i
and 20 with 17a.
and induce neurofibrillary tangle was intensively studied.
Therefore, the cell-based assay examining Tau phosphorylation
at Serine 396 represents a direct functional assay to measure the
17e (IC50 ¼ 4.89
m
cellular activity of GSK-3b inhibitors. Compounds 17a, 17b, 17g,
Comparison of GSK-3
various N-substituents on indole ring also revealed that com-
pounds with N-methyl (17a, IC50 ¼ 0.75 M) or N-isopropyl (29a,
IC50 ¼ 0.38 M) on indole ring were tolerated for GSK-3 inhibitory
activity. However, the introduction of a large benzyl group (29b,
IC50 ¼ 1.90 M) or a vinyl group (27, IC50 ¼ 8.84 M) on the indole
nitrogen led to 2-fold and 10-fold loss in potency as compared to
M) respectively.
b
inhibitory data of compounds with
17i, 29a, 29d, 29e and 30 were tested for the ability to reduce
Tau phosphorylation at Ser 396 in human neuroblastoma SH-
m
SY5Y cells. LiCl, a known inhibitor of GSK-3
control in this assay. As shown in Fig. 2, compounds 17a, 17b,
17g, 17i, 29a and 30 significantly reduced A -induced Tau
hyperphosphorylation, showing the inhibition of GSK-3 at
b [34], was used as
m
b
b
m
m
b
the cell level, while 29d and 29e have no significant cellular
activity.
the case of compound 30 (IC50 ¼ 0.83
m
As reported in the literature [14], staurosporine was found to be
a potent and nonselective kinase inhibitor. Staurosporine not only
2.2.3. Molecular modeling
inhibits GSK-3
b
(IC50 ¼ 0.2
m
M), but also potently inhibits many
To explore the possible binding mode of the target compounds,
a molecular modeling study of the optimum compound 17i was
performed on SYBYL [35] using the Tripos FlexiDock program [36],
other kinases (e.g. PKCE, IKK2, Aurora A, MEK1 and ERK1). In order
to determine whether target compounds had selection to various
kinases, compounds 17g, 17i and 29a were evaluated for their
inhibitory activities against PKCE, IKK2, Aurora A, MEK1 and ERK1
by using the Invitrogen Z0-LYTETM Kinase Assay kits. The results
based on the reported GSK-3
seen in Fig. 3, there are some important interactions between
ligand and GSK-3 . The maleimide portion of 17i makes key
b structure (PDB ID: 1Q3D). As it can be
b
showed that 17g, 17i and 29a displayed high selectivity for GSK-3
over other tested kinases (Table 2).
b
hydrogen bonding contacts with residues Asp-133 and Val-135
ꢀ
backbone carbonyl (distance: 2.20 A) and amide hydrogen (dis-
ꢀ
tance: 2.02 A), respectively. The oxygen atom of morpholine forms
ꢀ
2.2.2. Cellular activity
another hydrogen bond with Lys-183 (distance: 3.12 A). The ni-
Among the multiple cellular processes in which GSK-3
been implicated, the ability to hyperphosphorylate Tau protein
b
has
trogen of morpholine moiety of 17i formed additional ionic inter-
action with the carboxyl group of Asp-200 (2.13 A).
ꢀ

364
Q. Ye et al. / European Journal of Medicinal Chemistry 68 (2013) 361e371
H
N
O
O
b
c
N
N
N
N
H
15
Ph
N
H
N
Ph
N
Ph
N
O
O
O
O
O
O
O
O
a
b
6
Cl
N
N
N
N
N
N
R
N
N
R
N
N
N
N
N
H
16a-i
17a-i
Ph
13
14
Ph
N
N
O
O
O
O
d
e
N
N
N
N
N
N
N
N
(CH2)4
N
(CH2)4
Br
18
O
19
H
N
O
O
(CH ) NH(CH )
16a, 17a R: (CH )
2 3N
16f, 17f R:
16g, 17g R:
2 3
3 2
O
b
N
N
19
N
N
(CH )
2 2 N
16b, 17b R: (CH2)3OH
N
(CH2)4
N
16c, 17c R: (CH )
2 3N
16h, 17h R: (CH )
2 4 N
N
20
O
16d, 17d R:
(CH )
2 3N
N
N
16i, 17i R: (CH )
2 3N
O
16e, 17e R: (CH )
2 3 N
Scheme 2. The synthetic route for compounds 15, 17a-i and 20. Reagents and conditions: (a) Pd(PPh3)2Cl2, LiCl, toluene, 100 ꢀC, 55%; (b) NH4OAc, 140 ꢀC, 49e94%; (c) alkyl halides,
K2CO3, DMF, 65e75 ꢀC, 68e76%; (d) 1,4-dibromobutane, K2CO3, DMF, 60 ꢀC, 77%; (e) morpholine, K2CO3, DMF, 60 ꢀC, 66%.
3. Conclusion
purification unless stated. Et2O, THF and benzene were distilled
from sodiumebenzophenone. DMF was distilled from calcium
hydride. Preparation methods and physicochemical properties
for compounds 6, 14, 15, 16a-h and 17a-h were reported in
Ref. [26].
In summary, a number of 7-azaindazolyl-indolyl-maleimides
were designed, synthesized and tested for their GSK-3
activity, mostof them showedpotentactivityagainstGSK-3
based functional assay, selected compounds 17a,17b,17g,17i, 29a and
30 significantly reduced A -induced Tau hyperphosphorylation by
inhibiting GSK-3 . The preliminary structureeactivity relationships
b
inhibitory
b. Inacell-
b
4.1.1. 3-(1H-Indol-3-yl)-1-phenyl-4-(1H-pyrazolo[3,4-b]pyridine-
3-yl)-1H-pyrrole-2,5-dione (8)
b
and molecular modeling study provided further insight into in-
teractions between the enzyme and its ligand. The results provide
A mixture of 3-(tributylstanny)-1H-pyrazolo[3,4-b]pyridine 6
(2.0 g, 4.9 mmol), LiCl (0.48 g, 11.4 mmol), Pd(PPh3)2Cl2 (0.48 g,
0.75 mmol), 3-chloro-4-(1H-indol-3-yl)-1-phenyl-1H-pyrrole-2,5-
dione 7 (1.66 g, 5.14 mmol) and toluene (70 mL) was stirred at
100 ꢀC for 10 h under N2 atmosphere. After cooling, the reaction
mixture was poured into water (500 mL) and extracted with ethyl
acetate (3 ꢂ 100 mL). The organic phase was combined, washed
with brine (3 ꢂ 100 mL), dried over Na2SO4 and concentrated in
vacuum. The residue was purified by flash column chromatography
on silica gel using dichloromethane/methanol (30:1, v/v) as eluent
to afford compound 8 (1.09 g, 55%) as a red solid, mp: >250 ꢀC. 1H
NMR (DMSO-d6, d): 6.39 (1H, d, J ¼ 8.0 Hz), 6.69 (1H, t, J ¼ 8.0 Hz),
7.04 (1H, t, J ¼ 8.0 Hz), 7.15e7.18 (1H, m), 7.42e7.46 (2H, m), 7.51e
7.55 (4H, m), 8.15 (1H, dd, J ¼ 1.6, 8.4 Hz,), 8.25 (1H, d, J ¼ 3.2 Hz),
8.54 (1H, dd, J ¼ 1.6, 4.0 Hz), 12.04 (1H, s), 14.05 (1H, s). ESI-MS: m/
z ¼ 406 [M þ H] þ. Anal. Calcd for C24H15N5O2: C, 71.10; H, 3.73; N,
17.27. Found: C, 71.38; H, 3.59; N, 17.13.
valuable information for the design of GSK-3b inhibitors.
4. Experimental
4.1. Chemistry
Melting points were determined with a BÜCHI Melting Point
B-450 apparatus (Büchi Labortechnik, Flawil, Switzerland). The
1H NMR spectra were recorded on a Bruker Avance DMX 400 at
400 MHz (chemical shifts are expressed as
d values relative to
TMS as internal standard). ESI (positive) was recorded on an
Esquire-LC-00075 spectrometer. Element analyses were per-
formed on an Eager 300 instrument. All reactions were moni-
tored by thin-layer chromatography (TLC). All reagents were
obtained from commercial sources and used without further

Q. Ye et al. / European Journal of Medicinal Chemistry 68 (2013) 361e371
365
Scheme 3. The synthetic route for compounds 27, 29aee and 30. Reagents and conditions: (a) Pd(PPh3)2Cl2, LiCl, toluene, 100 ꢀC, 40%; (b) 4-(3-chloropropyl)morpholine, K2CO3,
DMF, 60 ꢀC, 52%; (c) concentrated HCl, EtOH, 30e35 ꢀC, 68%; (d) 1,2-dibromoethane, Cs2CO3, DMF, 60 ꢀC, 55%; (e) potassium tert-butoxide, THF, 0e5 ꢀC, 54%; (f) 28% NH3.H2O, DMF,
90 ꢀC, 79e89%; (g) alkyl halides, NaH, DMF, 56e71%.
4.1.2. 3-(1H-Indol-3-yl)-4-(1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-
pyrrole-2,5-dione (9)
After that, the solvent was removed in vacuum and the residue was
purified by flash column chromatography on silica gel using
dichloromethane/methanol (30:1, v/v) as eluent to afford 10 (0.4 g,
A mixture of 8 (0.05 g, 0.12 mmol) and ammonium acetate
(0.92 g, 12.0 mmol) was reacted at 140 ꢀC for 6 h. After cooling, the
mixture was poured into water (50 mL), adjusted to weak alkalinity
with Na2CO3 and extracted with ethyl acetate (3 ꢂ 50 mL). The
organic layer was combined, washed with brine (3 ꢂ 150 mL), dried
over Na2SO4 and concentrated in vacuum. The residue was purified
by flash column chromatography on silica gel using dichloro-
methane/methanol (30:1, v/v) as eluent to afford compound 9
83%) as a red solid, mp: >250 ꢀC. 1H NMR (DMSO-d6,
d): 3.08 (3H, s),
6.34 (1H, d, J ¼ 8.0 Hz), 6.66 (1H, t, J ¼ 7.6 Hz), 7.02 (1H, t, J ¼ 8.0 Hz),
7.13e7.15 (1H, m), 7.40 (1H, d, J ¼ 8.4 Hz), 8.03 (1H, d, J ¼ 8.0 Hz),
8.19 (1H, d, J ¼ 3.2 Hz), 8.51(1H, d, J ¼ 4.4 Hz), 11.97 (1H, s), 14.00
(1H, s). ESI-MS: m/z ¼ 344 [M þ H]þ Anal. Calcd for C19H13N5O2: C,
66.47; H, 3.82; N, 20.40. Found: C, 66.31; H, 3.89; N, 20.62.
(34.8 mg, 86%) as a red solid, mp: >250 ꢀC. 1H NMR (DMSO-d6,
d):
4.1.4. 3-(1H-Indol-3-yl)-4-(1H-pyrazolo[3,4-b]pyridine-3-yl)furan-
2,5-dione (11)
6.35 (1H, d, J ¼ 8.0 Hz), 6.66 (1H, t, J ¼ 8.0 Hz), 7.02 (1H, t, J ¼ 7.6 Hz),
7.12e7.15 (1H, m), 7.39 (1H, d, J ¼ 8.0 Hz), 8.05 (1H, d, J ¼ 7.6 Hz), 8.16
(1H, s), 8.52 (1H, d, J ¼ 4.0 Hz), 11.18 (1H, brs), 11.94 (1H, brs), 13.97
(1H, brs). ESI-MS: m/z ¼ 330 [M þ H]þ. Anal. Calcd for C18H11N5O2: C,
65.65; H, 3.37; N, 21.27. Found: C, 65.38; H, 3.47; N, 21.33.
A mixture of 10 (0.4 g, 1.2 mmol), 5 M aqueous potassium hy-
droxide (17 mL) and ethanol (50 mL) was stirred at 25e30 ꢀC for
5 h. After that, water (250 mL) was added to the mixture. The
mixture was acidified with 2 M HCl and extracted with ethyl ace-
tate (3 ꢂ 60 mL). The organic phase was combined, washed with
brine (3 ꢂ 120 mL), dried over Na2SO4 and concentrated in vacuum.
The residue was purified by flash column chromatography on silica
gel using petroleum ether/ethyl acetate (8:1, v/v) as eluent to afford
4.1.3. 3-(1H-Indol-3-yl)-1-methyl-4-(1H-pyrazolo[3,4-b]pyridine-
3-yl)-1H-pyrrole-2,5-dione (10)
A mixture of 8 (0.57 g, 1.4 mmol) and a solution of methylamine
in methanol (32.0%, 30 mL) was stirred at room temperature for 2 h.
11 (0.27 g, 70%) as a red solid, mp: >250 ꢀC. 1H NMR (DMSO-d6,
d):

366
Q. Ye et al. / European Journal of Medicinal Chemistry 68 (2013) 361e371
Table 1
GSK-3ba inhibitory activity of 7-azaindazolyl-indolyl-maleimides.
R1
N
O
O
N
N
N
N
R2
R3
Copmd.
R1
R2
H
R3
GSK-3
IC50
b
(m
M) ꢃ SEb
%Inhibition@20 mg/mL
ST
8
0.20 ꢃ 0.01
H
4.14 ꢃ 7.49
9
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
7.31 ꢃ 3.16
12a
12b
12c
12d
12e
12f
15
CH2CH2(CH3)2
NH2
28.56 ꢃ 2.84
45.73 ꢃ 10.04
ꢁ7.42 ꢃ 24.79
ꢁ6.81 ꢃ 9.95
33.70 ꢃ 8.96
51.54 ꢃ 13.76
NHCHO
NHCONH2
NHCSNH2
CH2COOCH3
H
12.27 ꢃ 2.24
0.75 ꢃ 0.20
17a
H
CH3
(CH2)3
N
O
17b
17c
H
H
CH3
CH3
1.09 ꢃ 0.07
2.66 ꢃ 0.23
(CH2)3OH
(CH2)3
N
17d
17e
H
H
CH3
CH3
3.34 ꢃ 0.07
4.89 ꢃ 0.24
(CH2)3
(CH2)3
N
N
N
N
17f
H
H
CH3
CH3
5.69 ꢃ 0.41
0.81 ꢃ 0.01
(CH2)3N(CH3)2
-
(CH2)2
N
17g
N
N
(CH2)4
17h
H
CH3
0.96 ꢃ 0.04
N
17i
20
H
H
H
H
H
H
H
H
CH3
0.36 ꢃ 0.01
0.46 ꢃ 0.01
8.84 ꢃ 0.11
0.38 ꢃ 0.01
1.90 ꢃ 0.06
0.96 ꢃ 0.01
1.29 ꢃ 0.11
0.68 ꢃ 0.01
(CH2)2
(CH2)4
N
N
N
N
N
N
N
N
O
O
O
O
O
O
O
O
CH3
27
CH ¼ CH2
CH(CH3)2
CH2Ph
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
29a
29b
29c
29d
29e
CH2eCH ¼ CH2
(CH2)3
(CH2)3
N
O
N
N
30
H
H
0.83 ꢃ 0.08
(CH2)3
N
O
a
Assay details were described in the Experimental section.
SE: standard error mean.
b

Q. Ye et al. / European Journal of Medicinal Chemistry 68 (2013) 361e371
367
Table 2
The selectivity to tested kinases of target compounds 17g, 17i and 29a.a
Kinase assay
IC50 ꢃ SEb
(mM)
Staurosporine
17g
17i
29a
GSK-3
PKCE
IKK2
Aurora A
MEK1
b
0.20 ꢃ 0.0
0.0015 ꢃ 0.0001
1.41 ꢃ 0.26
0.81 ꢃ 0.01
>40
>40
>40
>40
0.36 ꢃ 0.01
>40
>40
>40
>40
0.38 ꢃ 0.01
>40
>40
>40
>40
0.018 ꢃ 0.002
0.67 ꢃ 0.035
1.31 ꢃ 0.035
ERK1
>40
>40
>40
a
‘>40’ means<50% inhibition at 40 lM of compound.
SE: standard error mean.
b
6.45 (1H, d, J ¼ 8.0 Hz), 6.75 (1H, t, J ¼ 7.6 Hz), 7.07e7.13 (2H, m),
7.42 (1H, d, J ¼ 8.0 Hz), 8.08 (1H, d, J ¼ 8.0 Hz), 8.34 (1H, d,
J ¼ 3.2 Hz), 8.54 (1H, dd, J ¼ 1.6, 4.8 Hz), 11.91 (1H, s), 13.95 (1H, s).
ESI-MS: m/z ¼ 331 [M þ H]þ. Anal. Calcd for C18H10N4O3: C, 65.45;
H, 3.05; N, 16.96. Found: C, 65.39; H, 3.09; N, 16.77.
Fig. 3. Docking of 17i to GSK-3b crystal structure.
4.1.5. 1-Amino-3-(1H-indol-3-yl)-4-(1H-pyrazolo[3,4-b]pyridine-
3-yl)-1H-pyrrole-2,5-dione (12a)
0.94 (3H, s), 2.05e2.08 (1H, m), 3.41 (2H, d, J ¼ 7.6 Hz), 6.35 (1H, d,
J ¼ 8.0 Hz), 6.67 (1H, t, J ¼ 7.6 Hz), 7.02 (1H, t, J ¼ 8.0 Hz), 7.13e7.14
(1H, m), 7.40 (1H, d, J ¼ 8.0 Hz), 8.02 (1H, d, J ¼ 8.4 Hz), 8.20 (1H, d,
J ¼ 3.2 Hz), 8.52 (1H, d, J ¼ 4.4 Hz), 11.98 (1H, s), 14.01(1H, s). ESI-
MS: m/z ¼ 386 [M þ H]þ. Anal. Calcd for C22H19N5O2: C, 68.56; H,
4.97; N, 18.17. Found: C, 68.69; H, 4.91; N, 18.02.
A mixture of 11 (30 mg, 0.09 mmol), hydrazine dihydrochloride
(66 mg, 0.63 mmol), potassium carbonate (174 mg, 1.26 mmol) and
DMF (2 mL) was stirred at room temperature for 4 h. After that, the
mixture was poured into water (50 mL) and extracted with ethyl
acetate (3 ꢂ 30 mL). The organic phase was combined, washed with
brine (3 ꢂ 60 mL), dried over Na2SO4 and concentrated in vacuum.
The residue was purified by flash column chromatography on silica
gel using dichloromethane/methanol (30:1, v/v) as eluent to afford
12a (21.1 mg, 68%) as a red solid, mp: >250 ꢀC. 1H NMR (DMSO-d6,
4.1.6.2. 1-Formamido-3-(1H-indol-3-yl)-4-(1H-pyrazolo[3,4-b]pyri-
dine-3-yl)-1H-pyrrole-2,5-dione (12c). According to the general
method, reaction of 11 with formohydrazide afforded 12c in 36%
d
): 4.91(2H, s), 6.33 (1H, d, J ¼ 8.0 Hz), 6.66 (1H, t, J ¼ 7.6 Hz), 7.02
yield, mp: >250 ꢀC. 1H NMR (DMSO-d6,
d
): 6.27 (1H, d, J ¼ 8.0 Hz),
(1H, t, J ¼ 7.6 Hz), 7.13e7.16 (1H, m), 7.40 (1H, d, J ¼ 8.4 Hz), 7.99 (1H,
d, J ¼ 8.4 Hz), 8.19 (1H, d, J ¼ 3.2 Hz), 8.51 (1H, dd, J ¼ 1.6, 4.8 Hz),
11.97 (1H, s), 14.00 (1H, s). ESI-MS: m/z ¼ 345 [M þ H]þ. Anal. Calcd
for C18H12N6O2: C, 62.79; H, 3.51; N, 24.41. Found: C, 62.93; H, 3.58;
N, 24.52.
6.66 (1H, t, J ¼ 7.6 Hz), 7.04 (1H, t, J ¼ 7.6 Hz), 7.13e7.16 (1H, m), 7.42
(1H, d, J ¼ 8.0 Hz), 7.93 (1H, dd, J ¼ 1.6, 8.0 Hz), 8.23 (1H, d,
J ¼ 3.2 Hz), 8.38 (1H, s), 8.51 (1H, dd, J ¼ 1.6, 4.8 Hz). ESI-MS: m/
z ¼ 373 [M þ H]þ. Anal. Calcd for C19H12N6O3: C, 61.29; H, 3.25; N,
22.57. Found: C, 61.17; H, 3.29; N, 22.68.
4.1.6. General procedure for the synthesis of 12b-e
4.1.6.3. 3-(1H-Indol-3-yl)-4-(1H-pyrazolo[3,4-b]pyridine-3-yl)-1-
ureido-1H-pyrrole-2,5-dione (12d). According to the general
method, reaction of 11 with hydrazinecarboxamide afforded 12d in
A mixture of 11 (0.09 mmol), ReNH2 (0.63 mmol) and DMF
(2 mL) was stirred at 70 ꢀC for 10 h. After cooling, the mixture was
poured into water (50 mL) and extracted with ethyl acetate
(3 ꢂ 30 mL). The organic phase was combined, washed with brine
(3 ꢂ 60 mL), dried over Na2SO4 and concentrated in vacuum. The
residue was purified by flash column chromatography on silica gel
using dichloromethane/methanol (30:1, v/v) as eluent to afford
12b-e.
60% yield, mp: >250 ꢀC. 1H NMR (DMSO-d6,
d): 6.33 (1H, d,
J ¼ 8.0 Hz), 6.43 (2H, brs), 6.67 (1H, t, J ¼ 8.0 Hz), 7.03 (1H, t,
J ¼ 8.4 Hz), 7.12e7.16 (1H, m), 7.42 (1H, d, J ¼ 8.0 Hz), 7.96 (1H, d,
J ¼ 8.4 Hz), 8.20 (1H, d, J ¼ 3.2 Hz), 8.50e8.53 (2H, m), 12.02 (1H, s),
14.06 (1H, s). ESI-MS: m/z ¼ 388 [M þ H]þ. Anal. Calcd for
C19H13N7O3: C, 58.91; H, 3.38; N, 25.31. Found: C, 59.05; H, 3.41; N,
25.17.
4.1.6.1. 3-(1H-Indol-3-yl)-1-isobutyl-4-(1H-pyrazolo[3,4-b]pyridine-
3-yl)-1H-pyrrole-2,5-dione (12b). According to the general method,
reaction of 11 with 2-methylpropan-1-amine afforded 12b in 65%
4.1.6.4. 3-(1H-Indol-3-yl)-4-(1H-pyrazolo[3,4-b]pyridine-3-yl)-1-
thioureido-1H-pyrrole-2,5-dione (12e). According to the general
method, reaction of 11 with hydrazinecarbothioamide afforded 12e
yield as a red solid, mp: >250 ꢀC. 1H NMR (DMSO-d6,
d): 0.92 (3H, s),
in 52% yield, mp: >250 ꢀC. 1H NMR (DMSO-d6,
d): 6.34 (1H, d,
J ¼ 8.0 Hz), 6.67 (1H, t, J ¼ 7.6 Hz), 7.04 (1H, t, J ¼ 7.6 Hz), 7.13e7.16
(1H, m), 7.42 (1H, d, J ¼ 8.0 Hz), 7.96 (1H, d, J ¼ 7.6 Hz), 8.20 (1H, d,
J ¼ 3.2 Hz), 8.52 (1H, dd, J ¼ 1.6, 4.4 Hz). ESI-MS: m/z ¼ 404
[M þ H]þ. Anal. Calcd for C19H13N7O2S: C, 56.57; H, 3.25; N, 24.30;
Found: C, 56.39; H, 3.29; N, 24.17.
4.1.7. Methyl 2-(3-(1H-indol-3-yl)-2,5-dioxo-4-(1H-pyrazolo[3,4-
b]pyridin-3-yl)-2,5-dihydro-1H-pyrrol-1-yl)acetate (12f)
A mixture of 11 (30 mg, 0.09 mmol), methyl glycinate hydro-
chloride (113 mg, 0.9 mmol), triethylamine (91 mg, 0.9 mmol) and
DMF (4 mL) was stirred at 70 ꢀC for 10 h. After cooling, the mixture
was poured into water (80 mL) and extracted with ethyl acetate
Fig. 2. Effects of GSK-3b inhibitors on tau phosphorylation (ser396) in SH-SY5Y cells.

368
Q. Ye et al. / European Journal of Medicinal Chemistry 68 (2013) 361e371
(3 ꢂ 30 mL). The organic phase was combined, washed with brine
(3 ꢂ 60 mL), dried over Na2SO4 and concentrated in vacuum. The
residue was purified by flash column chromatography on silica gel
using dichloromethane/methanol (30:1, v/v) as eluent to afford 12f
(15 mg, 41%) as an orange solid, mp: >250 ꢀC. 1H NMR (DMSO-d6,
J ¼ 6.8 Hz), 3.94 (3H, s), 4.45 (2H, t, J ¼ 6.8 Hz), 6.34 (1H, d,
J ¼ 8.4 Hz), 6.74 (1H, t, J ¼ 8.4 Hz), 7.12 (1H, t, J ¼ 7.6 Hz), 7.21e7.25
(1H, m), 7.43e7.46 (1H, m), 7.49e7.55 (5H, m), 8.21 (1H, d,
J ¼ 8.0 Hz), 8.29 (1H, s), 8.54 (1H, d, J ¼ 4.4 Hz). ESI-MS: m/z ¼ 554
and 556 [M þ H]þ. Anal. Calcd for C29H24BrN5O2: C, 62.82; H, 4.36;
N, 12.63; Found: C, 62.69; H, 4.24; N, 12.41.
d
): 3.73 (3H, s), 4.48 (2H, s), 6.34 (1H, d, J ¼ 7.6 Hz), 6.68 (1H, t,
J ¼ 8.0 Hz), 7.02 (1H, t, J ¼ 8.0 Hz), 7.14e7.16 (1H, m), 7.42 (1H, d,
J ¼ 8.4 Hz), 7.97 (1H, d, J ¼ 8.0 Hz), 8.23 (1H, d, J ¼ 3.2 Hz), 8.52 (1H,
d, J ¼ 4.4 Hz), 12.04 (1H, s), 14.07 (1H, s). ESI-MS: m/z ¼ 402
[M þ H]þ Anal. Calcd for C21H15N5O4: C, 62.84; H, 3.77; N, 17.45.
Found: C, 63.05; H, 3.59; N, 17.33.
4.1.11. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(4-morpholinobutyl)-1H-
pyrazolo[3,4-b]pyridine-3-yl)-1-phenyl-1H-pyrrole-2,5-dione (19)
A mixture of 18 (150 mg, 0.27 mmol), potassium carbonate
(112 mg, 0.81 mmol), morpholine (238 mg, 2.7 mmol) and DMF
(30 mL) was stirred at 60 ꢀC for 1 h. After cooling, the mixture was
poured into water (200 mL) and extracted with ethyl acetate
(3 ꢂ 50 mL). The organic phase was combined, washed with brine
(3 ꢂ 150 mL), dried over Na2SO4 and concentrated in vacuum. The
residue was purified by flash column chromatography on silica gel
using dichloromethane/methanol/triethylamine (120:4:1, v/v/v) as
eluent to afford 19 (100.0 mg, 66%) as a red solid, mp: 166e168 ꢀC.1H
4.1.8. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(2-morpholinoethyl)-1H-
pyrazolo[3,4-b]pyridine-3-yl)-1-phenyl-1H-pyrrole-2,5-dione (16i)
A
mixture of 14 (0.24 mmol) [26], potassium carbonate
(0.48 mmol), 4-(2-chloroethyl)morpholine (0.36 mmol) and DMF
(10 mL) was stirred at 70 ꢀC for 8 h. After cooling, the mixture was
poured into water (200 mL) and extracted with ethyl acetate
(3 ꢂ 50 mL). The organic phase was combined, washed with brine
(3 ꢂ 100 mL), dried over Na2SO4 and concentrated in vacuum. The
residue was purified by flash column chromatography on silica gel
using dichloromethane/methanol/triethylamine (120:4:1, v/v/v) as
eluent to afford 16i in 67% yield as a red solid, mp: 140e142 ꢀC. 1H
NMR (CDCl3, d): 1.41e1.48 (2H, m), 1.67e1.74 (2H, m), 2.28 (2H, t,
J ¼ 6.8 Hz), 2.35e2.43 (4H, m), 3.63e3.74 (4H, m), 3.90 (3H, s), 4.44
(2H, t, J ¼ 6.8 Hz), 6.32 (1H, d, J ¼ 8.0 Hz), 6.76 (1H, t, J ¼ 7.6 Hz), 7.11e
7.16 (2H, m), 7.32 (1H, d, J ¼ 8.0 Hz), 7.36e7.42 (1H, m), 7.50e7.54 (4H,
m), 8.18 (1H, s), 8.25 (1H, dd, J ¼ 1.6, 8.0 Hz), 8.55 (1H, dd, J ¼ 1.6,
4.0 Hz). ESI-MS: m/z ¼ 561 [M þ H]þ. Anal. Calcd for C33H32N6O3: C,
70.70; H, 5.75; N, 14.99; Found: C, 70.56; H, 5.63; N, 14.81.
NMR (CDCl3,
d
): 2.52e2.58 (4H, m), 2.75 (2H, t, J ¼ 6.8 Hz), 3.68e
3.76 (4H, m), 3.92 (3H, s), 4.68 (2H, t, J ¼ 6.8 Hz), 6.34 (1H, d,
J ¼ 8.0 Hz), 6.78 (1H, t, J ¼ 7.6 Hz), 7.14e7.19 (2H, m), 7.34 (1H, d,
J ¼ 8.0 Hz), 7.38e7.42 (1H, m), 7.49e7.54 (4H, m), 8.16 (1H, s), 8.29
(1H, dd, J ¼ 1.6, 8.0 Hz), 8.56 (1H, dd, J ¼ 1.6, 4.0 Hz). ESI-MS: m/
z ¼ 533 [M þ H]þ. Anal. Calcd for C31H28N6O3: C, 69.91; H, 5.30; N,
15.78; Found: C, 69.76; H, 5.11; N, 15.93.
4.1.12. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(4-morpholinobutyl)-1H-
pyrazolo[3,4-b]pyridine-3-yl)-1H-pyrrole-2,5-dione (20)
Accordingtothe procedureused toprepare17a, the reaction of 19
with ammonium acetate afforded 20 in 87% yield as a red solid, mp:
126e128 ꢀC. 1H NMR (CDCl3,
d): 1.44e1.51 (2H, m), 1.69e1.75 (2H,
4.1.9. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(2-morpholinoethyl)-1H-
pyrazolo[3,4-b]pyridine-3-yl)-1H-pyrrole-2,5-dione (17i)
m), 2.32 (2H, t, J ¼ 6.8 Hz), 2.38e2.49 (4H, m), 3.67e3.75 (4H, m),
3.90 (1H, s), 4.45 (2H, t, J ¼ 6.8 Hz), 6.26 (1H, d, J ¼ 8.0 Hz), 6.74 (1H, t,
J ¼ 7.6 Hz), 7.10e7.15 (2H, m), 7.30 (1H, d, J ¼ 8.0 Hz), 8.06 (1H, brs),
8.11 (1H, s), 8.14 (1H, dd, J ¼ 1.6, 8.0 Hz), 8.54 (1H, dd, J ¼ 1.6, 4.0 Hz).
Under N2 atmosphere, a mixture of 16i (0.057 mmol) and
ammonium acetate (57.0 mmol) was reacted at 140 ꢀC for 6 h. After
cooling, the mixture was poured into water (20 mL), adjusted to
weak alkalinity with Na2CO3 and extracted with ethyl acetate
(3 ꢂ 20 mL). The organic layer was combined, washed with brine
(3 ꢂ 50 mL), dried over Na2SO4 and concentrated in vacuum. The
residue was purified by flash column chromatography on silica gel
using dichloromethane/methanol/triethylamine (120:4:1, v/v/v) as
eluent to afford 17i in 85% yield as a red solid, mp: 112e114 ꢀC. 1H
13C NMR (CDCl3,
d):171.94, 171.60, 150.49, 148.90, 137.35, 135.56,
134.39, 134.07, 131.87, 125.25, 122.50, 122.08, 121.77, 120.73, 117.44,
115.44, 109.76, 104.89, 66.61 (2), 58.15, 53.36 (2), 47.05, 33.40, 27.47,
23.22. ESI-MS: m/z ¼ 485 [M þ H]þ. Anal. Calcd for C27H28N6O3: C,
66.93; H, 5.82; N, 17.34; Found: C, 66.79; H, 5.88; N, 17.56.
4.1.13. 3-(1-(tert-Butyloxycarbonyl)-1H-indol-3-yl)-4-chloro-1-
phenyl-1H-pyrrole-2,5-dione (21)
NMR (CDCl3,
d
): 2.40e2.52 (4H, m), 2.63 (2H, t, J ¼ 6.8 Hz), 3.60e
3.72 (4H, m), 3.92 (3H, s), 4.59 (2H, t, J ¼ 6.8 Hz), 6.28 (1H, d,
J ¼ 7.6 Hz), 6.75 (1H, t, J ¼ 8.0 Hz), 7.13e7.17 (2H, m), 7.33 (1H, d,
J ¼ 8.4 Hz), 7.96 (1H, brs), 8.11 (1H, s), 8.19 (1H, dd, J ¼ 1.6, 8.0 Hz),
To a solution of 7 (6.4 g, 20.0 mmol) in THF (250 mL), di-tert-
butyl dicarbonate (5.5 g, 25.0 mmol) and catalytic amount of DMAP
(0.5%) were added and the mixture was stirred for 3 h at room
temperature. After removal of the solvent in vacuum, the residue
was purified by flash column chromatography on silica gel using
dichloromethane/methanol (40:1, v/v) as eluent to afford 21 (7.8 g,
8.56 (1H, dd, J ¼ 1.6, 4.0 Hz). 13C NMR (CDCl3,
d):171.90, 171.58,
150.61, 148.92, 137.33, 135.54, 134.33, 134.26, 131.95, 125.21, 122.46,
122.11, 121.68, 120.74, 117.48, 115.38, 109.76, 104.89, 66.61 (2), 57.13,
53.31 (2), 44.27, 33.35. ESI-MS: m/z ¼ 457 [M þ H]þ. Anal. Calcd for
C25H24N6O3: C, 65.78; H, 5.30; N, 18.41; Found: C, 65.59; H, 5.25; N,
18.53.
92%) as a yellow solid, mp: 150e152 ꢀC. 1H NMR (DMSO-d6,
d): 1.70
(9H, s), 7.35e7.36 (1H, m), 7.40e7.43 (4H, m), 7.48e7.50 (2H, m),
7.90 (1H, d, J ¼ 6.4 Hz), 8.25e8.27 (2H, m). ESI-MS: m/z ¼ 423
[M þ H]þ. Anal. Calcd for C23H19ClN2O4: C, 65.33; H, 4.53; N, 6.62;
Found: C, 65.44; H, 4.68; N, 6.59.
4.1.10. 3-(1-Methyl-1H-indol-3-yl)-4-(1-(4-bromobutyl)-1H-
pyrazolo[3,4-b]pyridine-3-yl)-1-phenyl-1H-pyrrole-2,5-dione (18)
A mixture of 14 (200 mg, 0.48 mmol), potassium carbonate
(199 mg, 1.44 mmol),1,4-dibromobutane (1.03 g, 4.8 mmol) and
DMF (20 mL) was stirred at 60 ꢀC for 1 h [37e45]. After cooling, the
mixture was poured into water (200 mL) and extracted with ethyl
acetate (3 ꢂ 50 mL). The organic phase was combined, washed with
brine (3 ꢂ 150 mL), dried over Na2SO4 and concentrated in vacuum.
The residue was purified by flash column chromatography on silica
gel using dichloromethane/methanol (50:1, v/v) as eluent to afford
compound 18 (205.0 mg, 77%) as a red solid, mp: 158e160 ꢀC. 1H
4.1.14. 3-(1-(tert-Butyloxycarbonyl)-1H-indol-3-yl)-1-phenyl-4-
(1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-pyrrole-2,5-dione (22)
According to the procedure used to prepare 8, the reaction of 6
with 21 afforded 22 in 40% yield as a yellow solid, mp: 215e218 ꢀC.
1H NMR (CDCl3,
d): 1.69 (9H, s), 6.50 (1H, d, J ¼ 7.6 Hz), 6.83 (1H, t,
J ¼ 8.0 Hz), 7.17e7.21 (2H, m), 7.41e7.44 (1H, m), 7.50e7.55 (4H, m),
8.17 (1H, d, J ¼ 8.4 Hz), 8.30 (1H, d, J ¼ 8.0 Hz), 8.49 (1H, s), 8.58 (1H,
dd, J ¼ 1.6, 4.0 Hz), 12.48 (1H, brs). ESI-MS: m/z ¼ 506 [M þ H]þ.
Anal. Calcd for C29H23N5O4: C, 68.90; H, 4.59; N, 13.85; Found: C,
68.83; H, 4.61; N, 13.99.
NMR (CDCl3, d): 1.63e1.71 (2H, m), 1.74e1.80 (2H, m), 3.43 (2H, t,

Q. Ye et al. / European Journal of Medicinal Chemistry 68 (2013) 361e371
369
4.1.15. 3-(1-(tert-Butyloxycarbonyl)-1H-indol-3-yl)-1-phenyl-4-(1-
(3-morpholinopropyl)-1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-
pyrrole-2,5-dione (23)
dropwise at 0e5 ꢀC. After addition, the mixture was stirred at 0e
5 ꢀC for 2 h. After that, it was concentrated in vacuum. The residue
was purified by flash column chromatography on silica gel using
dichloromethane/methanol/triethylamine (120:4:1, v/v/v) as
eluent to afford 26 (42.6 mg, 54%) as a red solid, mp: 123e125 ꢀC. 1H
A mixture of 22 (443 mg, 1.0 mmol), potassium carbonate
(276 mg, 2.0 mmol), 4-(3-chloropropyl)morpholine (245 mg,
1.5 mmol) and DMF (30 mL) was stirred at 60 ꢀC for 10 h. After
cooling, the mixture was poured into water (200 mL) and extracted
with ethyl acetate (3 ꢂ 50 mL). The organic phase was combined,
washed with brine (3 ꢂ 100 mL), dried over Na2SO4 and concen-
trated in vacuum. The residue was purified by flash column chro-
matography on silica gel using dichloromethane/methanol/
triethylamine (120:4:1, v/v/v) as eluent to afford 23 (295.0 mg, 52%)
NMR (CDCl3, d): 2.04e2.06 (2H, m), 2.39e2.48 (6H, m), 3.68e3.77
(4H, m), 4.51 (2H, t, J ¼ 6.8 Hz), 5.04 (1H, t, J ¼ 8.0 Hz), 5.48 (1H, t,
J ¼ 16.0 Hz), 6.43 (1H, d, J ¼ 8.0 Hz), 6.83 (1H, t, J ¼ 8.0 Hz), 7.18e
7.26 (3H, m), 7.39e7.43 (1H, m), 7.47 (1H, d, J ¼ 8.0 Hz), 7.50e7.54
(4H, m), 8.32 (1H, d, J ¼ 8.0 Hz), 8.46 (1H, s), 8.57 (1H, d, J ¼ 4.0 Hz).
ESI-MS: m/z ¼ 559 [M þ H]þ. Anal. Calcd for C33H30N6O3: C, 70.95;
H, 5.41; N, 15.04; Found: C, 71.11; H, 5.55; N, 15.18.
as a yellow solid, mp: 122e124 ꢀC. 1H NMR (CDCl3,
d): 1.70 (9H, s),
1.84e1.89 (2H, m), 2.30e2.37 (6H, m), 3.64e3.72 (4H, m), 4.47 (2H,
t, J ¼ 6.8 Hz), 6.47 (1H, d, J ¼ 8.0 Hz), 6.87 (1H, t, J ¼ 8.0 Hz), 7.19e
7.24 (2H, m), 7.40e7.43 (1H, m), 7.50e7.54 (4H, m), 8.20 (1H, d,
J ¼ 8.0 Hz), 8.38 (1H, dd, J ¼ 1.6, 8.0 Hz), 8.47 (1H, s), 8.57 (1H, dd,
J ¼ 1.6, 4.0 Hz). ESI-MS: m/z ¼ 633 [M þ H]þ. Anal. Calcd for
C36H36N6O5: C, 68.34; H, 5.74; N, 13.28; Found: C, 68.62; H, 5.71; N,
13.39.
4.1.19. General procedure for the synthesis of 28a and 28b
To a solution of 24 (53 mg, 0.1 mmol) in DMF (25 mL), 60% NaH
(4.2 mg, 0.105 mmol) was added portionwise at room temperature.
After stirring for 30 min, alkyl halide (0.15 mmol) was added and
the mixture was then reacted at 60 ꢀC for 10 h. After cooling, the
mixture was poured into water (100 mL) and extracted with ethyl
acetate (3 ꢂ 50 mL). The organic phase was combined, washed with
brine (3 ꢂ 100 mL), dried over Na2SO4 and concentrated in vacuum.
The residue was purified by flash column chromatography on silica
gel using dichloromethane/methanol/triethylamine (120:4:1, v/v/
v) as eluent to afford 28a and 28b.
4.1.16. 3-(1H-Indol-3-yl)-4-(1-(3-morpholinopropyl)-1H-pyrazolo
[3,4-b]pyridine)-3-yl)-1-phenyl-1H-pyrrole-2,5-dione (24)
To a solution of 23 (275 mg, 0.5 mmol) in ethanol (30 mL),
concentrated hydrochloric acid (15 mL) was added. The mixture was
stirred at 30e35 ꢀC for 12 h. After that, it was poured into water
(300 mL), adjusted to weak alkalinity with Na2CO3 and extracted
with ethyl acetate (3 ꢂ 50 mL). The organic layer was combined,
washed with brine (3 ꢂ 100 mL), dried over Na2SO4 and concen-
trated in vacuum. The residue was purified flash column chroma-
tography on silica gel using dichloromethane/methanol/
triethylamine (120:4:1, v/v/v) as eluent to afford 24 (182 mg, 68%) as
4.1.19.1. 3-(1-Isopropyl-1H-indol-3-yl)-4-(1-(3-morpholinopropyl)-
1H-pyrazolo[3,4-b]pyridine-3-yl)-1-phenyl-1H-pyrrole-2,5-dione
(28a). According to the general method, the reaction of 24 with 2-
bromopropane afforded 28a in 61% yield as a red solid, mp: 116e
118 ꢀC. 1H NMR (CDCl3,
d
): 1.63 (6H, d, J ¼ 6.5 Hz),1.85e1.92 (2H, m),
2.21e2.29 (6H, m), 3.59e3.65 (4H, m), 4.51(2H, t, J ¼ 6.8 Hz), 4.70e
4.76 (1H, m), 6.37 (1H, d, J ¼ 8.0 Hz), 6.77 (1H, t, J ¼ 8.0 Hz), 7.11e
7.15 (2H, m), 7.37e7.42 (2H, m), 7.49e7.54 (4H, m), 8.25 (1H, d,
J ¼ 8.0 Hz), 8.38 (1H, s), 8.55 (1H, d, J ¼ 4.0 Hz). ESI-MS: m/z ¼ 575
[M þ H]þ. Anal. Calcd for C34H34N6O3: C, 71.06; H, 5.96; N, 14.62;
Found: C, 71.33; H, 6.05; N, 14.87.
a red solid, mp: 123e125 ꢀC. 1H NMR (CDCl3,
d): 1.85e1.89 (2H, m),
2.22e2.31 (6H, m), 3.57e3.66 (4H, m), 4.50 (2H, t, J ¼ 6.8 Hz), 6.42
(1H, d, J ¼ 8.0 Hz), 6.77 (1H, t, J ¼ 7.6 Hz), 7.08e7.12 (2H, m), 7.34 (1H,
d, J ¼ 7.6 Hz), 7.38e7.42 (1H, m), 7.49e7.55 (4H, m), 8.20 (1H, d,
J ¼ 3.2 Hz), 8.25 (1H, d, J ¼ 8.0 Hz), 8.55 (1H, d, J ¼ 4.4 Hz), 9.13 (1H,
brs). ESI-MS: m/z ¼ 533 [M þ H]þ. Anal. Calcd for C31H28N6O3: C,
69.91; H, 5.30; N, 15.78; Found: C, 67.13; H, 5.41; N, 15.99.
4.1.19.2. 3-(1-Benzyl-1H-indol-3-yl)-4-(1-(3-morpholinopropyl)-1H-
pyrazolo[3,4-b]pyridine-3-yl)-1-phenyl-1H-pyrrole-2,5-dione (28b).
According to the general method, the reaction of 24 with (chlor-
omethyl) benzene afforded 28b in 71% yield as a red solid, mp:
4.1.17. 3-(1-(2-Bromoethyl-1H-indol-3-yl)-4-(1-(3-
morpholinopropyl)-1H-pyrazolo[3,4-b] pyridine-3-yl)-1-phenyl-
1H-pyrrole-2,5-dione (25)
142e144 ꢀC. 1H NMR (CDCl3,
d): 2.16e2.23 (2H, m), 2.42e2.58 (6H,
m), 3.72e3.81 (4H, m), 4.53 (2H, t, J ¼ 6.8 Hz), 5.41 (2H, s), 6.41 (1H,
d, J ¼ 8.0 Hz), 6.79 (1H, t, J ¼ 8.4 Hz), 7.09e7.16 (2H, m), 7.23e7.27
(2H, m), 7.30e7.42 (5H, m), 7.48e7.53 (4H, m), 8.22 (1H, d,
J ¼ 8.0 Hz), 8.28 (1H, s), 8.57 (1H, d, J ¼ 4.4 Hz). ESI-MS: m/z ¼ 623
[M þ H]þ. Anal. Calcd for C38H34N6O3: C, 73.29; H, 5.50; N, 13.50;
Found: C, 73.45; H, 5.34; N, 13.64.
A mixture of 24 (159 mg, 0.3 mmol), cesium carbonate (196 mg,
0.6 mmol), 1,2-dibromoethane (564 mg, 3.0 mmol) and DMF
(20 mL) was stirred at 60 ꢀC for 2 h. After cooling, the mixture was
poured into water (200 mL) and extracted with ethyl acetate
(3 ꢂ 50 mL). The organic phase was combined, washed with brine
(3 ꢂ 100 mL), dried over Na2SO4 and concentrated in vacuum. The
residue was purified by flash column chromatography on silica gel
using dichloromethane/methanol/triethylamine (120:4:1, v/v/v) as
eluent to afford 25 (105 mg, 55%) as a red solid, mp: 84e86 ꢀC. 1H
4.1.20. 3-(1-Allyl-1H-indol-3-yl)-4-(1-(3-morpholinopropyl)-1H-
pyrazolo[3,4-b]pyridine-3-yl)-1-phenyl-1H-pyrrole-2,5-dione (28c)
To a solution of 24 (53 mg, 0.1 mmol) in DMF (25 mL), 60% NaH
(4.2 mg, 0.105 mmol) was added portionwise at room temperature.
After stirring for 30 min, 3-bromoprop-1-ene (18 mg, 0.15 mmol)
was added and the mixture was then stirred at room temperature
for 2 h. After that, it was poured into water (100 mL) and extracted
with ethyl acetate (3 ꢂ 50 mL). The organic phase was combined,
washed with brine (3 ꢂ 100 mL), dried over Na2SO4 and concen-
trated in vacuum. The residue was purified by flash column chro-
matography on silica gel using dichloromethane/methanol/
triethylamine (120:4:1, v/v/v) as eluent to afford 28c (33 mg, 58%)
NMR (CDCl3,
d): 1.88e1.90 (2H, m), 2.24e2.33 (6H, m), 3.61e
3.67(4H, m), 3.74 (2H, t, J ¼ 6.8 Hz), 4.51 (2H, t, J ¼ 6.8 Hz), 4.63 (2H,
t, J ¼ 6.8 Hz), 6.43 (1H, d, J ¼ 8.0 Hz), 6.78 (1H, t, J ¼ 8.0 Hz), 7.12e
7.14 (2H, m), 7.33 (1H, d, J ¼ 8.0 Hz), 7.41e7.42 (1H, m), 7.50e7.54
(4H, m), 8.17 (1H, dd, J ¼ 1.6, 8.0 Hz), 8.24 (1H, s), 8.54 (1H, dd,
J ¼ 1.6, 4.4 Hz); ESI-MS: m/z ¼ 639 and 641 [M þ H]þ. Anal. Calcd for
C33H31BrN6O3: C, 61.97; H, 4.89; N,13.14; Found: C, 67.13; H, 5.41; N,
15.99.
4.1.18. 3-(1-(3-Morpholinopropyl)-1H-pyrazolo[3,4-b]pyridine-3-
yl)-1-phenyl-3-(1-vinyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (26)
To a solution of 25 (90 mg, 0.14 mmol) in THF (2 mL), a solution
of potassium tert-butoxide in THF (0.15 mL, 0.15 mmol) was added
as a red solid, mp: 71e73 ꢀC. 1H NMR (CDCl3,
d): 1.83e1.92 (2H, m),
2.18e2.32 (6H, m), 4.01e4.13 (4H, m), 4.49 (2H, t, J ¼ 6.8 Hz), 4.82
(2H, d, J ¼ 4.8 Hz), 5.25e5.29 (2H, m), 6.05e6.12 (1H, m), 6.37 (1H,
d, J ¼ 8.0 Hz), 6.77 (1H, t, J ¼ 7.6 Hz), 7.10e7.15 (2H, m), 7.32 (1H, d,

370
Q. Ye et al. / European Journal of Medicinal Chemistry 68 (2013) 361e371
J ¼ 8.0 Hz), 7.36e7.41 (1H, m), 7.49e7.55 (4H, m), 8.22 (1H, s), 8.25
(1H, dd, J ¼ 1.6, 8.0 Hz), 8.55 (1H, dd, J ¼ 1.6, 4.4 Hz). ESI-MS: m/
z ¼ 573 [M þ H]þ. Anal. Calcd for C34H32N6O3: C, 71.31; H, 5.63; N,
14.68; Found: C, 71.59; H, 5.54; N, 14.61.
4.1.22.2. 3-(1-Isopropyl-1H-indol-3-yl)-4-(1-(3-morpholinopropyl)-
1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-pyrrole
ꢁ2,5-dione (29a).
According to the general method, the reaction of 28a with aqueous
ammonia afforded 29a in 84% yield as a red solid, mp: 99e101 ꢀC.
1H NMR (CDCl3,
d
): 1.65 (6H, d, J ¼ 6.5 Hz), 1.89e1.97 (2H, m), 2.24e
4.1.21. General procedure for the synthesis of 28d and 28e
To a solution of 24 (53 mg, 0.1 mmol) in DMF (25 mL), 60% NaH
(4.2 mg, 0.105 mmol) was added portionwise at room temperature.
After stirring for 30 min, alkyl halide (0.15 mmol) was added and
the mixture was then reacted at 80 ꢀC for 10 h. After cooling, the
mixture was poured into water (100 mL) and extracted with ethyl
acetate (3 ꢂ 50 mL). The organic phase was combined, washed with
brine (3 ꢂ 100 mL), dried over Na2SO4 and concentrated in vacuo.
The residue was purified by flash column chromatography on silica
gel using dichloromethane/methanol/triethylamine (120:4:1, v/v/
v) as eluent to afford 28d and 28e.
2.34 (6H, m), 3.63e3.68 (4H, m), 4.52 (2H, t, J ¼ 6.8 Hz), 4.69e4.76
(1H, m), 6.36 (1H, d, J ¼ 8.0 Hz), 6.76 (1H, t, J ¼ 8.0 Hz), 7.10e7.15
(2H, m), 7.36 (1H, d, J ¼ 8.0 Hz), 7.83 (1H, brs), 8.15 (1H, dd, J ¼ 1.6,
8.0 Hz), 8.28 (1H, s), 8.56 (1H, dd, J ¼ 1.6, 4.0 Hz). 13C NMR (CDCl3,
d): 171.41, 171.15, 150.66, 149.06, 136.44, 134.79, 134.18, 131.87,
131.41, 125.65, 122.43, 122.32, 121.55, 120.89, 117.58, 115.52, 110.12,
105.20, 66.65 (2), 55.71, 53.27 (2), 48.02, 45.52, 26.15, 22.66 (2). ESI-
MS: m/z ¼ 499 [M þ H]þ. Anal. Calcd for C28H30N6O3: C, 67.45; H,
6.06; N, 16.86; Found: C, 67.38; H, 6.12; N, 16.98.
4.1.22.3. 3-(1-Benzyl-1H-indol-3-yl)-4-(1-(3-morpholinopropyl)-
1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-pyrrole-2,5-dione (29b).
According to the general method, the reaction of 28b with aqueous
ammonia afforded 29b in 86% yield as a red solid, mp: 174e176 ꢀC.1H
4.1.21.1. 3-(1-Morpholinopropyl-1H-indol-3-yl)-4-(1-(3-
morpholinopropyl)-1H-pyrazolo[3,4-b]pyridine-3-yl)-1-phenyl-1H-
pyrrole-2,5-dione (28d). According to the general method, the re-
action of 24 with 4-(3-chloropropyl)morpholine afforded 28d
NMR (CDCl3, d): 1.88e1.96 (2H, m), 2.25e2.33 (6H, m), 3.58e3.66
(4H, m), 4.51 (2H, t, J ¼ 6.8 Hz), 5.38 (2H, s), 6.43 (1H, d, J ¼ 8.0 Hz),
6.77 (1H, t, J ¼ 8.0 Hz), 7.07e7.13 (2H, m), 7.20e7.26 (3H, m), 7.32e
7.35 (3H, m), 8.13 (1H, d, J ¼ 8.0 Hz), 8.17e8.24 (2H, m), 8.55 (1H, d,
J ¼ 4.0 Hz). ESI-MS: m/z ¼ 547 [M þ H]þ. Anal. Calcd for C32H30N6O3:
C, 67.45; H, 6.06; N, 16.86; Found: C, 67.38; H, 6.12; N, 16.98.
(40 mg, 58%) as a red solid, mp: 156e158 ꢀC. 1H NMR (CDCl3,
d):
2.07e2.12 (2H, m), 2.18e2.22 (2H, m), 2.52e2.64 (12H, m), 3.80e
3.89 (8H, m), 4.38 (2H, t, J ¼ 6.8 Hz), 4.54 (2H, t, J ¼ 6.8 Hz), 6.41 (1H,
d, J ¼ 8.0 Hz), 6.79 (1H, t, J ¼ 7.6 Hz), 7.12e7.17 (2H, m), 7.38e7.41
(2H, m), 7.48e7.53 (4H, m), 8.12 (1H, dd, J ¼ 1.6, 8.0 Hz), 8.28 (1H, s),
8.56 (1H, dd, J ¼ 1.6, 4.4 Hz). ESI-MS: m/z ¼ 660 [M þ H]þ. Anal.
Calcd for C38H41N7O4: C, 69.18; H, 6.26; N, 14.86; Found: C, 69.37;
H,6.44; N, 14.69.
4.1.22.4. 3-(1-Allyl-1H-indol-3-yl)-4-(1-(3-morpholinopropyl)-
1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-pyrrole-2,5-dione (29c).
According to the general method, the reaction of 28c with aqueous
ammonia afforded 29c in 79% as a red solid, mp: 148e150 ꢀC.1H NMR
4.1.21.2. 3-(1-(3-(1H-Imidazol-1-yl)propyl)-1H-indol-3-yl)-4-(1-(3-
morpholinopropyl)-1H-pyrazolo[3,4-b]pyridine-3-yl)-1-phenyl-1H-
pyrrole-2,5-dione (28e). According to the general method, the re-
action of 24 with 1-(3-chloropropyl)-1H-imidazole afforded 28e in
(CDCl3, d): 1.88e1.99 (2H, m), 2.25e2.41 (6H, m), 3.60e3.73 (4H, m),
4.52 (2H, t, J ¼ 7.2 Hz), 4.82 (2H, d, J ¼ 5.6 Hz), 5.24 (1H, d, J ¼ 17.2 Hz),
5.33(1H, d, J¼ 10.0 Hz), 6.01e6.11(1H, m), 6.34(1H, d, J¼ 8.0Hz), 6.75
(1H, t, J ¼ 8.0 Hz), 7.10e7.15 (2H, m), 7.31 (1H, d, J ¼ 8.0 Hz), 8.06 (1H,
brs), 8.14e8.18 (2H, m), 8.55 (1H, dd, J ¼ 1.6, 4.0 Hz). 13C NMR (CDCl3,
56% yield as a scarlet solid, mp: 68e70 ꢀC. 1H NMR (CDCl3,
d): 1.89e
1.93 (2H, m), 2.27e2.35 (6H, m), 2.41e2.45 (2H, m), 3.61e3.67 (4H,
m), 4.01 (2H, t, J ¼ 6.8 Hz), 4.20 (2H, t, J ¼ 6.8 Hz), 4.49 (2H, t,
J ¼ 6.8 Hz), 6.37 (1H, d, J ¼ 8.0 Hz), 6.79 (1H, t, J ¼ 8.0 Hz), 6.98 (1H,
brs), 7.12e7.17 (3H, m), 7.22 (1H, d, J ¼ 8.0 Hz), 7.39e7.42 (1H, m),
7.50e7.55 (4H, m), 7.56 (1H, brs), 8.17 (1H, s), 8.25 (1H, dd, J ¼ 1.6,
8.0 Hz), 8.55 (1H, dd, J ¼ 1.6, 4.4 Hz). ESI-MS: m/z ¼ 641 [M þ H]þ.
Anal. Calcd for C37H36N8O3: C, 69.36; H, 5.66; N, 17.49; Found: C,
69.55; H,5.58; N, 17.61.
d):171.73, 171.46, 150.55, 148.96, 136.74, 134.65, 134.43, 134.04,
132.23, 131.85, 125.53, 122.57, 122.23, 122.13, 120.84, 118.49, 117.51,
115.47, 110.22, 105.26, 66.56 (2), 55.62, 53.16 (2), 49.44, 45.48, 26.02.
ESI-MS: m/z ¼ 497 [M þ H]þ. Anal. Calcd for C28H28N6O3: C, 67.73; H,
5.68; N, 16.92; Found: C, 67.58; H, 5.72; N, 17.03.
4.1.22.5. 3-(1-Morpholinopropyl-1H-indol-3-yl)-4-(1-(3-
morpholinopropyl)-1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-pyrrole-
2,5-dione (29d). According to the general method, the reaction of
compound 28d with aqueous ammonia afforded 29d in 89% as a
4.1.22. General procedure for the synthesis of 27, 29aee and 30
A mixture of 26, 28aee and 24 (0.05mol), 28% aqueous
ammonia (1 mL) and DMF (2 mL) was stirred at 90 ꢀC for 4 h. After
cooling, it was poured into water (100 mL) and extracted with ethyl
acetate (3 ꢂ 50 mL). The organic phase was combined, washed with
brine (3 ꢂ 100 mL), dried over Na2SO4 and concentrated in vacuum.
The residue was purified by flash column chromatography on silica
gel using dichloromethane/methanol/triethylamine (120:4:1, v/v/
v) as eluent to afford 27, 29aee and 30 respectively.
jacinth solid, mp: 115e117 ꢀC. 1H NMR (CDCl3,
d): 1.97e2.03 (2H, m),
2.09e2.14 (2H, m), 2.36e2.51 (12H, m), 3.70e3.81 (8H, m), 4.34 (2H,
t, J ¼ 6.8 Hz), 4.52 (2H, t, J ¼ 6.8 Hz), 6.26 (1H, d, J ¼ 8.0 Hz), 6.73 (1H,
t, J ¼ 8.0 Hz), 7.10e7.15 (2H, m), 7.37 (1H, d, J ¼ 8.0 Hz), 8.10 (1H, d,
J ¼ 8.0 Hz), 8.17 (1H, brs), 8.21 (1H, s), 8.55 (1H, d, J ¼ 4.0 Hz). ESI-MS:
m/z ¼ 584 [M þ H]þ. Anal. Calcd for C32H37N7O4: C, 65.85; H, 6.39; N,
16.80; Found: C, 65.88; H, 6.31; N, 17.08.
4.1.22.6. 3-(1-(3-(1H-Imidazol-1-yl)propyl)-1H-indol-3-yl)-4-(1-(3-
morpholinopropyl)-1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-pyrrole-
2,5-dione (29e). According to the general method, the reaction of
28e with aqueous ammonia afforded compound 29e in 81% as a red
4.1.22.1. 3-(1-(3-Morpholinopropyl)-1H-pyrazolo[3,4-b]pyridine-3-
yl)-4-(1-vinyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (27). According to
the general method, the reaction of 26 with aqueous ammonia
afforded 27 in 87% yield as a red solid, mp: 165e167 ꢀC. 1H NMR
solid, mp: 84e86 ꢀC. 1H NMR (CDCl3,
d): 1.85e1.91 (2H, m), 2.23e
(CDCl3,
d
): 1.81e1.87 (2H, m), 2.20e2.28 (6H, m), 3.59e3.65 (4H, m),
2.31 (6H, m), 2.41e2.46 (2H, m), 3.58e3.65 (4H, m), 3.99 (2H, t,
J ¼ 6.4 Hz), 4.18 (2H, t, J ¼ 6.4 Hz), 4.48 (2H, t, J ¼ 6.8 Hz), 6.32 (1H, d,
J ¼ 8.0 Hz), 6.76 (1H, t, J ¼ 8.4 Hz), 7.00 (1H, brs), 7.16e7.21 (4H, m),
7.62 (1H, brs), 8.09 (1H, s), 8.14e8.19 (2H, m), 8.55 (1H, d,
J ¼ 4.0 Hz). ESI-MS: m/z ¼ 565 [M þ H]þ. Anal. Calcd for
C31H32N8O3: C, 65.94; H, 5.71; N, 19.85; Found: C, 65.88; H, 5.41; N,
19.98.
4.49 (2H, t, J ¼ 6.8 Hz), 5.02 (1H, t, J ¼ 8.8 Hz), 5.45 (1H, t, J ¼ 16.0 Hz),
6.41 (1H, d, J ¼ 8.0 Hz), 6.79 (1H, t, J ¼ 8.0 Hz), 7.19e7.24 (3H, m), 7.44
(1H, d, J ¼ 8.0 Hz), 7.85 (1H, brs), 8.23 (1H, d, J ¼ 8.0 Hz), 8.39 (1H, s),
8.55 (1H, d, J ¼ 4.0 Hz). ESI-MS: m/z ¼ 483 [M þ H]þ. Anal. Calcd
for C27H26N6O3: C, 67.21; H, 5.43; N, 17.42; Found: C, 67.38; H, 5.48;
N, 17.65.

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4.1.22.7. 3-(1H-Indol-3-yl)-4-(1-(3-morpholinopropyl)-1H-pyrazolo
References
[3,4-b]pyridine-3-yl)-1H-pyrrole-2,5-dione (30). According to the
general method, compound 24 reacted with aqueous ammonia to
afford compound 30 in 81% as a jacinth solid, mp: 218e220 ꢀC. 1H
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NMR (CDCl3, d): 1.91e1.97 (2H, m), 2.28e2.37 (6H, m), 3.63e3.70
(4H, m), 4.53 (2H, t, J ¼ 6.8 Hz), 6.45 (1H, d, J ¼ 8.0 Hz), 6.78 (1H, t,
J ¼ 8.0 Hz), 7.11 (1H, t, J ¼ 8.0 Hz), 7.14e7.17 (1H, m), 7.33 (1H, d,
J ¼ 8.0 Hz), 8.15e8.19 (3H, m), 8.57 (1H, d, J ¼ 4.4 Hz), 9.19 (1H, brs).
Anal. Calcd for C25H24N6O3: C, 65.78; H, 5.30; N, 18.41; Found: C,
18.59; H, 5.35; N, 18.57.
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4.2. Pharmacology
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The GSK-3
inserted into pGEX-KG vector. The recombinant GST-GSK-3
b
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cDNA was obtained from UniGene (3667B03) and
protein
b
b
was expressed in Escherichia coli strain BL21-CodonPlus (DE3), pu-
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.07.046.
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