Design, synthesis, and evaluation of 3-aryl-4-pyrrolyl-maleimides as glycogen synthase kinase-3β inhibitors

Article abstract of DOI:10.1002/ardp.201300008

A series of 3-aryl-4-pyrrolyl-maleimides were designed, synthesized, and evaluated for their glycogen synthase kinase-3β (GSK-3β) inhibitory activity. Most compounds exhibited potent activity against GSK-3β. Among them, compounds 11a, 11c, 11h, 11i, and 11j significantly reduced Aβ-induced Tau hyperphosphorylation, showing the inhibition of GSK-3β at the cellular level. Structure-activity relationships were discussed based on the experimental data obtained. Most compounds in a new series of 3-aryl-4-pyrrolyl-maleimides exhibited potent glycogen synthase kinase-3β (GSK-3β) inhibitory activity. Compounds 11a, 11c, 11h, 11i, and 11j reduced Aβ-induced Tau hyperphosphorylation, showing inhibition of GSK-3β at the cellular level. Copyright

Full text of DOI:10.1002/ardp.201300008

Arch. Pharm. Chem. Life Sci. 2013, 346, 349–358
349
Full Paper
Design, Synthesis, and Evaluation of 3-Aryl-4-pyrrolyl-
maleimides as Glycogen Synthase Kinase-3b Inhibitors
Qing Ye1, Meng Li1, Yu-Bo Zhou2, Jia-Yu Cao2, Lei Xu2, Yu-Jin Li1, Liang Han1, Jian-Rong Gao1,
Yong-Zhou Hu3, and Jia Li2
1 State Key Laboratory Breeding Base of Green Chemistry-Synthesis Technology, Zhejiang University of
Technology, Hangzhou, China
2 ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang
University, Hangzhou, China
3 The National Center for Drug Screening, Shanghai, China
A series of 3-aryl-4-pyrrolyl-maleimides were designed, synthesized, and evaluated for their glycogen
synthase kinase-3b (GSK-3b) inhibitory activity. Most compounds exhibited potent activity against
GSK-3b. Among them, compounds 11a, 11c, 11h, 11i, and 11j significantly reduced Ab-induced Tau
hyperphosphorylation, showing the inhibition of GSK-3b at the cellular level. Structure–activity
relationships were discussed based on the experimental data obtained.
Keywords: 3-Aryl-4-pyrrolyl-maleimides / Cellular activity / Enzymatic activity / GSK-3b inhibitors
Received: January 9, 2013; Revised: February 26, 2013; Accepted: March 1, 2013
DOI 10.1002/ardp.201300008
Introduction
pharmacology [12, 13]. Various bisindolylmaleimides such as GF
109203X and Ro 31-8820 have been developed as potent GSK-3b
inhibitors based on staurosporine, a microbial alkaloid that was
identified as an early GSK-3b inhibitor (Fig. 1) [14–17]. However,
most of these bisindolylmaleimides have suffered from
issues including toxicity, bad solubility, and poor selectivity,
especially against PKC, which make them unsuitable for the
treatment of diseases such as diabetes and Alzheimer’s
disease [18, 19]. Recent efforts in replacing one indolyl ring
of bisindolylmaleimides with other heteroaryl substituents
such as imidazo[1,2-a]pyridinyl, 4-azaindolyl and benzofur-
anyl led to the emergence of some monoindolylmaleimides
(Fig. 1) [20–22]. Among them, GSK-3b inhibitor 603281-31-8
has reached preclinical trials for the treatment of diabetes.
These facts encouraged us to design a new series of mono-
indolylmaleimide analogs to find more potent and selective
GSK-3b inhibitors. In this paper, we describe the synthesis
and evaluation of 3-aryl-4-pyrrolyl-maleimides as potent and
selective GSK-3b inhibitors. Their structure–activity relation-
ship and in silico molecular modeling study in a homology
GSK-3b protein are discussed as well.
Glycogen synthase kinase-3 (GSK-3) is a multifunctional ser-
ine/threonine protein kinase that was first identified in the
late 1970s as a consequence of its phosphorylation activity
toward glycogen synthase (GS), the rate limiting enzyme of
glycogen biosynthesis [1, 2]. Today, it is known that GSK-3
plays an important role in various cellular and physiological
events, such as the Wnt signaling pathway [3], insulin signal
transduction [4], cell survival, response to DNA damage [5],
hyphosphorylation of protein Tau (one of the diagnostic
features of Alzheimer’s disease) [6]. From a drug discovery stand-
point, inhibition of GSK-3 may provide therapy for several
diseases such as cancer [7], diabetes type-2 [8], chronic inflam-
matory processes [9], stroke [10], bipolar disorders, and
Alzheimer’s disease [11]. Accordingly, searching for GSK-3
inhibitors is a very active area in both academic centers and
pharmaceutical companies. GSK-3 is encoded by two isoforms in
mammals, termed GSK-3a and GSK-3b, which are 98% identical
within the catalytic domain, but which have shown distinct
Correspondence: Jian-Rong Gao, State Key Laboratory Breeding Base
of Green Chemistry-Synthesis Technology, Zhejiang University of
Technology, Hangzhou 310032, China.
E-mail: gdgjr@zjut.edu.cn
Fax: þ86-571-88320544
Additional corresponding authors:
Yong-Zhou Hu, E-mail: huyz@zju.edu.cn
Jia Li, E-mail: jli@mail.shcnc.ac.cn
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Q. Ye et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 349–358
H
N
O
H
N
H
N
H
N
O
O
O
O
O
O
N
N
O
R1
R2
N
N
N
N
N
O
H
CH3
H
H
OC
3
NH
S
C
N
H
3C
HN
Ro 31-8220
NH2
Benzofuranyl-
indolylmaleimides
GF 109203X
Staurosporine
H
N
O
O
H
N
F
O
O
H
N
R2
R3
O
O
N
N
R1
N
N
N
N
N
N
N
N
R2
O
N
O
N
603281-31-8
R1
N
H
Imidazo[1,2-a]pyridinylindolylmaleimides
4-Azaindolyl-indolylmaleimides
Figure 1. GSK-3b inhibitors.
Results and discussion
Chemistry
Biological activity and molecular modeling
Enzymatic activity
The GSK-3b inhibitory potency of all the target compounds
was examined. In addition, the assays of inhibitory activity
toward PKCE, IKK2, Aurora A, MEK1, and ERK1 were also
conducted to determine the selectivity of the tested com-
pounds. Staurosporine, a well-known kinase inhibitor was
used as the reference compound. The results are summarized
in Tables 1 and 2.
The synthetic approach to compounds 10 and 11a–k is out-
lined in Scheme 1. Indole derivatives 1a–e were reacted with
oxalyl chloride in Et2O, followed by sodium methoxide to
give 2a–e. N-Alkylation of 2a–e with different alkyl halides in
the presence of NaH in DMF resulted in key intermediates 3a–
j. Treatment of indole with 1,4-dibromobutane afforded 4.
Substitution reaction of 4 with morpholine by using K2CO3 as
hydrogen bromide capture resulted in 5, which was then
treated with oxalyl chloride, followed by sodium methoxide
to give another key intermediate 3k. Condensation of glyoxilic
esters 3a–k with 2-(1H-pyrrol-3-yl)acetamide 9 [23, 24] in the
presence of t-BuOK in THF afforded the target compounds
11a–k. Similarly, compound 10 can be obtained by reaction of
2a with 9.
As shown in Table 1, most compounds displayed potent
inhibitory activity against GSK-3b. Different aryl substituents
in the maleimide ring could affect the inhibitory potency
greatly. Replacement of an indole ring in 10 (IC50 ¼ 1.92 mM)
with a naphthyl afforded 13b (IC50 ¼ 11.86 mM), leading to a
sixfold loss of potency for GSK-3b. Substitution of pyrrole in
11a (IC50 ¼ 0.10 mM) with other five-member-aryls including
imidazole (15a, IC50 ¼ 2.04 mM) and 1,3,4-triazole (15b,
IC50 ¼ 11.6 mM) resulted in a 20- and 116-fold loss of potency,
respectively.
The target compounds 13a,b were prepared by the conden-
sation of ethyl pyrrole-3-glyoxalate 6 with aryl acetamides
12a,b, respectively, following the same procedure described
above. Similarly, compounds 15a,b were obtained by conden-
sation of glyoxilic esters 3a with the corresponding com-
pounds aryl acetamides 14a,b (Scheme 2).
Comparing the inhibitory activity of compound 10 with
11a, 11c, and 11i, it appeared that introduction of
suitable hydrophilic side chains such as morpholinopropyl,
imidazol-1-ylpropyl, and hydroxypropyl at the 1N-position of
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Arch. Pharm. Chem. Life Sci. 2013, 346, 349–358
3-Aryl-4-pyrrolyl-maleimides as GSK-3b Inhibitors
351
O
O
O
O
R1
a
OCH3
b
OCH3
R1
N
H
1a e
N
N
_
H
(CH2)n
R2
_
R1=H; 5-CH3O; 6-Cl;
_
2a e
3a j
6-Br; 6-F
O
O
OCH3
e
c
d
N
N
1a
N
(CH2)4
N
(CH2)4
Br
(CH2)4
N
4
3k
5
O
O
O
O
O
O
O
h
g
f
OH
OC2H5
NH2
OC2H5
N
H
N
H
N
H
N
H
9
8
6
7
Scheme 1. Synthetic route to compounds
11a–i. Reagents and conditions: (a) i: (COCl)2,
Et2O; ii: CH3ONa, CH3OH. (b) NaH, DMF,
X(CH2)nR2; (c) NaH, DMF, Br(CH2)4Br;
(d) K2CO3, DMF, morpholine; (e) i: HCl,
(COCl)2, Et2O; ii: CH3ONa, CH3OH; (f) 10%
Pd/C, NaHPO2, 1,4-dioxane/H2O; (g) i:
NaOH, CH3OH/H2O, ii: concentrated HCl.
(h) DCC, NH3, 1,4-dioxane; i: i t-BuOK, THF;
ii: concentrated HCl.
H
N
H
N
O
N
O
O
O
i
i
_
2a
3a k
+
+
9
9
N
H
N
N
H
H
(CH2)n
R2
10
_
11a k
Ar:
H
N
O
O
13a:
O
O
a
Ar CH2CONH2
Ar
+
OCH3
13b:
N
H
N
H
12a,b
6
13a,b
O
O
H
N
O
N
O
Ar:
OCH3
a
Ar
N
15a:
N
Ar CH2CONH2
+
N
N
(CH2)3
N
N
(CH2)3
N
14a,b
15b:
N
15a,b
O
3a
Scheme 2. Synthetic route to compounds
13a,b, and 15a,b. Reagents and conditions:
(a) i: t-BuOK, THF; ii: concentrated HCl.
O
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352
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Arch. Pharm. Chem. Life Sci. 2013, 346, 349–358
Table 1. GSK-3b inhibitory activity of the target compounds.
H
N
H
N
O
O
O
O
H
N
H
N
H
N
O
O
H
N
O
O
O
O
O
4
O
5
N
N N
N
R1
6
N
N
N
N
N
7
H )
H
(C
N
C
(
)
2 3
2 3
H
N
N
N
N
(CH2)n
R2
N
H
H
H
H
O
O
_
a
13
10
11a k
13b
15a
15b
Compds.
R1
R2
n
IC50 (mM) ꢀ SEa)
Staurosporine
10
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
13a
13b
15a
15b
0.028 ꢀ 0.0b)
1.92 ꢀ 0.29
0.10 ꢀ 0.005
6.45 ꢀ 0.25
0.21 ꢀ 0.01
1.29 ꢀ 0.08
0.57 ꢀ 0.032
0.36 ꢀ 0.023
1.01 ꢀ 0.02
0.16 ꢀ 0.009
0.15 ꢀ 0.01
0.26 ꢀ 0.02
0.53 ꢀ 0.024
4.24 ꢀ 0.17
11.86 ꢀ 1.07
2.04 ꢀ 0.15
11.6 ꢀ 0.54
H
H
H
Morpholino
Morpholino
Imidazol-1-yl
Piperidin-1-yl
Morpholino
Morpholino
Morpholino
Morpholino
Hydroxyl
3
2
3
3
3
3
3
3
3
1
4
H
5-OCH3
6-Cl
6-Br
6-F
H
H
H
H
Morpholino
a)
SE, standard error mean.
Lit. [25]. IC50 ¼ 0.056 ꢀ 0.0069 mM.
b)
the indole ring gave an obvious enhancement of GSK-3b
inhibitory activity. Compound 11a (IC50 ¼ 0.1 mM) with a
morpholinopropyl on the indole ring was about 19-fold more
potent than 10 (IC50 ¼ 1.92 mM). Replacement of the termi-
nal morpholine group in 11a with a hydroxy group (11c,
IC50 ¼ 0.15 mM) and imidazol-1-yl (11i, IC50 ¼ 0.21 mM) led
to a slight drop in potency. However, replacement of
the morpholine group with piperidin-1-yl (11d, 1.29 mM)
resulted in 13-fold loss in potency. Changing the length of
the alkyl linker could affect the ability of the molecule to
interact with GSK-3b thereby influencing the potency of
the compounds. Compounds 11a (IC50 ¼ 0.10 mM) with a
three methylene linker showed better inhibitory activities
than 11b (IC50 ¼ 0.53 mM) with a four methylene linker
and compound 11c (IC50 ¼ 6.45 mM) with a two methylene
linker.
Table 2. The selectivity to tested kinases of target compounds 11a
and 11i.a)
Kinase assay
IC50 (mM) ꢀ SEb)
Different substituents on the indole ring could affect the
GSK-3b inhibitory activity (11a, 11e–h). Fluorine at 6-position
of the indole ring (11h) led to a slight drop in potency toward
GSK-3b as compared to 11a, while bromine at 6-position (11g)
showed a 10-fold loss of activity.
Staurosporine
11a
11i
GSK-3b
PKCE
IKK2
Aurora A
MEK1
ERK1
0.028 ꢀ 0.0
0.0015 ꢀ 0.0001
1.41 ꢀ 0.26
0.10 ꢀ 0.005
>40
>40
0.15 ꢀ 0.01
>40
>40
0.018 ꢀ 0.002
0.67 ꢀ 0.035
1.31 ꢀ 0.035
>40
>40
>40
>40
>40
>40
As reported in the literature [14], staurosporine was found
to be a potent and nonselective kinase inhibitor. Although
staurosporine potently inhibits GSK-3b (IC50 ¼ 0.028 mM), it
also potently inhibits many other kinases (e.g., PKCE, IKK2,
Aurora A, MEK1, and ERK1). The data of inhibitory activity
‘‘>40’’ means <50% inhibition at 40 mM of compound.
SE, standard error mean.
a)
b)
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Arch. Pharm. Chem. Life Sci. 2013, 346, 349–358
3-Aryl-4-pyrrolyl-maleimides as GSK-3b Inhibitors
353
against PKCE, IKK2, Aurora A, MEK1, and ERK1 showed that
compounds 11a and 11i displayed high selectivity for GSK-3b
over other tested kinases (see Table 2).
Cellular activity
Among the multiple cellular processes in which GSK-3b has
been implicated, the ability to hyperphosphorylate the Tau
protein and induce neurofibrillary tangle was intensively
studied. Therefore, the cell-based assay examining Tau phos-
phorylation at Serine 396 represents a direct functional assay
to measure the cellular activity of GSK-3b inhibitors.
Compounds 11a, 11c, 11e, 11f, 11h, 11i, 11j, and 11k were
tested for the ability to reduce Tau phosphorylation at Ser
396 in human neuroblastoma SH-SY5Y cells. LiCl is a known
inhibitor of GSK-3b and reduces Tau phosphorylation at Ser
396 in SH-SY5Y cells [26], which was used as a reference
compound in this assay. As shown in Fig. 2, compounds
11a, 11c, 11h, 11i, and 11j significantly reduced Ab-induced
Tau hyperphosphorylation, showing the inhibition of GSK-3b
at the cell level, while 11e, 11f, and 11k have no significant
cellular activity.
Molecular modeling
Figure 3. Docking of 11a to the GSK-3b crystal structure.
To explore the possible binding mode of a GSK-3b inhibitor a
molecular modeling study of the optimum compound 11a
was performed using the Tripos FlexiDock program [27, 28],
based on the published GSK-3b crystal structure (1Q3D) [14].
As it can be seen in Fig. 3, there are some important inter-
actions between ligand and GSK-3b. The maleimide portion
of 11a makes key hydrogen bonding contacts with residues
Asp-133 and Val-135 backbone carbonyl and amide hydrogen,
respectively. The oxygen atom of morpholine forms another
hydrogen bond with Lys-183.
tional assay revealed that selected compounds 11a, 11c,
11h, 11i, and 11j could significantly reduce Ab-induced
Tau hyperphosphorylation by inhibiting GSK-3b. The prelimi-
nary structure–activity relationships and molecular model-
ing study provided further insight into interactions between
the enzyme and its ligand. The results provide valuable
information for the design of GSK-3b inhibitors.
Experimental
Chemistry
Melting points were determined with a BUCHI Melting Point
B-450 apparatus (Bu¨chi Labortechnik, Flawil, Switzerland). The
1H NMR spectra were recorded in DMSO-d6 or CDCl3 on a Bruker
Avance DMX 500 at 500 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 performed on an Eager 300 instrument. All reactions were
Conclusion
¨
In summary, a series of 3-aryl-4-indolyl-maleimides were
designed, synthesized, and tested for their biological activity,
and most of them showed potent activity against GSK-3b with
11a being the most potent compound. Among them, com-
pounds 11a and 11i exhibited high selectivity against PKCE,
IKK2, Aurora A, MEK1, and ERK1. Further cell-based func-
Figure 2. Effects of GSK-3b inhibitors on Tau
phosphorylation (Ser396) in SH-SY5Y cells.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 349–358
monitored by thin-layer chromatography (TLC). All reagents were
obtained from commercial sources and used without further
purification unless stated. Et2O and THF were distilled from
sodium-benzophenone. DMF was distilled from calcium hydride.
(3 ꢂ 100 mL). The organic phase was combined, washed with
brine (3 ꢂ 300 mL), dried over Na2SO4, 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
1
3.30 g (67.5%) 3a as a light yellow solid, mp: 103–1048C. H NMR
(500 MHz, CDCl3): d 8.47–8.43 (m, 2H), 7.46–7.41 (m, 1H), 7.38–
7.32 (m, 2H), 4.32 (t, J ¼ 6.5 Hz, 2H), 3.95 (s, 3H), 3.78–3.72 (m, 4H),
2.44–2.38 (m, 4H), 2.28 (t, J ¼ 6.5 Hz, 2H), 2.08–2.02 (m, 2H). ESI-MS:
m/z [MþH]þ 331. Anal. calcd. for C18H22N2O4: C, 65.44; H, 6.71;
N, 8.48. Found: C, 65.27; H, 6.68; N, 8.58.
Methyl 2-(1H-indol-3-yl)-2-oxoacetate 2a
Oxalyl chloride (3.40 g, 26 mmol) in Et2O (5 mL) was added
dropwise to
a solution of indole (3.01 g, 26 mmol) in
Et2O (30 mL) at 0–58C. The reaction mixture kept stirring under
the same conditions for 1 h, and a 20 wt% solution of CH3ONa in
MeOH (14.05 g, 52 mmol) was added dropwise at ꢁ30 to ꢁ208C.
After addition, the mixture was stirred for 30 min at room
temperature, poured into cold water (100 mL). The product
was isolated with filtration, washed with dichloromethane,
and dried to afford 2.24 g (86.3%) 2a as a light yellow solid,
mp: 208–2108C. 1H NMR (500 MHz, DMSO-d6): d 12.48 (brs, 1H),
8.46 (d, J ¼ 3.5 Hz, 1H), 8.16 (d, J ¼ 7.0 Hz, 1H), 7.55
(d, J ¼ 7.0 Hz, 1H), 7.32–7.26 (m, 2H), 3.90 (s, 3H).
Methyl 2-(1-(2-morpholinoethyl)-1H-indol-3-yl)-2-
oxoacetate (3b)
According to the procedure used to prepare 3a, reaction of 2a
with 4-(2-chloroethyl)morpholine provided 3b in 52.4% yield
as a light yellow solid, mp: 110–1128C. 1H NMR (500 MHz,
CDCl3): d 8.51 (s, 1H), 8.49–8.44 (m, 1H), 7.43–7.39 (m, 1H),
7.38–7.35 (m, 2H), 4.29 (t, J ¼ 6.5 Hz, 2H), 3.97 (s, 3H), 3.75–
3.69 (m, 4H), 2.81 (t, J ¼ 6.5 Hz, 2H), 2.54–2.47 (m, 4H). ESI-MS:
m/z [MþH]þ 317. Anal. calcd. for C17H20N2O4: C, 64.54; H, 6.37;
N, 8.86. Found: C, 64.71; H, 6.68; N, 8.68.
Methyl 2-(5-methoxy-1H-indol-3-yl)-2-oxoacetate (2b)
According to the procedure used to prepare 2a, reaction of
5-methoxyindole with oxalyl chloride followed by CH3ONa pro-
vided 2b in 71.0% yield as a light yellow solid, mp: 221–2238C.
1H NMR (500 MHz, DMSO-d6): d 12.34 (brs, 1H), 8.37 (d, J ¼ 3.0 Hz,
1H), 7.66 (d, J ¼ 2.0 Hz, 1H), 7.45 (d, J ¼ 9.0 Hz, 1H), 6.93
(dd, J ¼ 9.0, 2.0 Hz, 1H), 3.89 (s, 3H), 3.81 (s, 3H).
Methyl 2-(1-(3-(1H-imidazol-1-yl)propyl)-1H-indol-3-yl)-2-
oxoacetate (3c)
According to the procedure used to prepare 3a, reaction of 2a
with 1-(3-chloropropyl)-1H-imidazole provided 3c in 52.8% yield
1
as a light yellow solid, mp: 72–738C. H NMR (500 MHz, CDCl3):
d 8.51–8.46 (m, 1H), 8.36 (s, 1H), 7.53 (s, 1H), 7.42–7.34 (m, 2H),
7.30–7.27 (m, 1H), 7.17 (s, 1H), 6.96 (s, 1H), 4.20 (t, J ¼ 7.0 Hz,
2H), 4.00 (t, J ¼ 7.0 Hz, 2H), 3.98 (s, 3H), 2.48–2.40 (m, 2H). ESI-MS:
m/z [MþH]þ 312. Anal. calcd. for C17H17N3O3: C, 65.58; H, 5.50;
N, 13.50. Found: C, 65.59; H, 4.88; N, 13.68.
Methyl 2-(6-chloro-1H-indol-3-yl)-2-oxoacetate (2c)
According to the procedure used to prepare 2a, reaction of 6-
chloroindole with oxalyl chloride followed by CH3ONa provided
2c in 62.8% yield as a light yellow solid, mp: 246–2488C. 1H NMR
(500 MHz, DMSO-d6): d 12.52 (brs, 1H), 8.51 (d, J ¼ 3.5 Hz, 1H),
8.15 (d, J ¼ 8.5 Hz, 1H), 7.62 (d, J ¼ 2.0 Hz, 1H), 7.31 (dd, J ¼ 8.5,
2.0 Hz, 1H), 3.90 (s, 3H).
Methyl 2-oxo-2-(1-(3-(piperidin-1-yl)propyl)-1H-indol-3-yl)-
acetate (3d)
According to the procedure used to prepare 3a, reaction of 2a with
1-(3-chloropropyl)piperidine provided 3d in 46.5% yield as a light
yellow solid, mp: 66–678C. 1H NMR (500 MHz, DMSO-d6): d 8.47
(s, 1H), 8.20 (d, J ¼ 7.5 Hz, 1H), 7.68 (d, J ¼ 7.5 Hz, 1H), 7.39–7.27
(m, 2H), 4.34 (t, J ¼ 7.0 Hz, 2H), 3.90 (s, 3H), 2.29–2.18 (m, 4H), 2.13
(t, J ¼ 7.0 Hz, 2H), 1.96–1.90 (m, 2H), 1.55–1.41 (m, 4H), 1.40–1.30
(m, 2H). ESI-MS: m/z [MþH]þ 329. Anal. calcd. for C19H24N2O3: C,
69.49; H, 7.37; N, 8.53. Found: C, 69.69; H, 7.68; N, 8.48.
Methyl 2-(6-bromo-1H-indol-3-yl)-2-oxoacetate (2d)
According to the procedure used to prepare 2a, reaction of
6-bromoindole with oxalyl chloride followed by CH3ONa pro-
vided 2d in 58.9% yield as a light yellow solid, mp: 207–2098C.
1H NMR (500 MHz, DMSO-d6): d 12.50 (brs, 1H), 8.50 (s, 1H), 8.10
(d, J ¼ 8.5 Hz, 1H), 7.75 (d, J ¼ 2.0 Hz, 1H), 7.43 (dd, J ¼ 8.5,
2.0 Hz, 1H), 3.90 (s, 3H).
Methyl 2-(6-fluoro-1H-indol-3-yl)-2-oxoacetate (2e)
According to the procedure used to prepare 2a, reaction of
6-fluoroindole with oxalyl chloride followed by CH3ONa pro-
vided 2e in 60.3% yield as a light yellow solid, mp: 182–1848C.
1H NMR (500 MHz, DMSO-d6): d 12.48 (brs, 1H), 8.48 (s, 1H), 8.15
(dd, J ¼ 8.5, 5.5 Hz, 1H), 7.36 (dd, J ¼ 9.5, 2.0 Hz, 1H), 7.15
(td, J ¼ 9.5, 2.0 Hz, 1H), 3.90 (s, 3H).
Methyl 2-(5-methoxy-1-(3-morpholinopropyl)-1H-indol-3-yl)-
2-oxoacetate (3e)
According to the procedure used to prepare 3a, reaction of 2b with
4-(3-chloropropyl)morpholine provided 3e in 64.7% yield as a light
yellow solid, mp: 67–688C. 1H NMR (500 MHz, CDCl3): d 8.38 (s, 1H),
7.95 (d, J ¼ 2.5 Hz, 1H), 7.32 (d, J ¼ 9.0 Hz, 1H), 6.97 (dd, J ¼ 9.0,
2.5 Hz, 1H), 4.28 (t, J ¼ 7.0 Hz, 2H), 3.95 (s, 3H), 3.91 (s, 3H), 3.77–
3.70 (m, 4H), 2.42–2.38 (m, 4H), 2.27 (t, J ¼ 6.5 Hz, 2H), 2.06–2.01
(m, 2H). ESI-MS: m/z [MþH]þ 361. Anal. calcd. for C19H24N2O5: C,
63.32; H, 6.71; N, 7.77. Found: C, 63.49; H, 6.68; N, 7.49.
Methyl 2-(1-(3-morpholinopropyl)-1H-indol-3-yl)-2-
oxoacetate (3a)
Seventy percent NaH (0.51 g, 14.8 mmol) was added portionwise
to a solution of 2a (3.0 g, 14.8 mmol) in DMF (30 mL) at 0–58C.
The reaction mixture was warmed to room temperature and
stirred for 30 min. After that, 4-(3-chloropropyl)morpholine
(1.14 g, 19.2 mmol) was added. Then the mixture was heated
to 55–608C and reacted for 12 h. After cooling, the mixture was
poured into cold water (300 mL) and extracted with ethyl acetate
Methyl 2-(6-chloro-1-(3-morpholinopropyl)-1H-indol-3-yl)-
2-oxoacetate (3f)
According to the procedure used to prepare 3a, reaction of 2c
with 4-(3-chloropropyl)morpholine provided 3f in 59.1% yield
1
as a light yellow solid, mp: 115–1168C. H NMR (500 MHz, CDCl3):
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Arch. Pharm. Chem. Life Sci. 2013, 346, 349–358
3-Aryl-4-pyrrolyl-maleimides as GSK-3b Inhibitors
355
d 8.42 (s, 1H), 8.34 (d, J ¼ 8.5 Hz, 1H), 7.49 (d, J ¼ 1.5 Hz, 1H), 7.30
(dd, J ¼ 8.5, 1.5 Hz, 1H), 4.28 (t, J ¼ 6.5 Hz, 2H), 3.94 (s, 3H), 3.80–
3.70 (m, 4H), 2.43–2.37 (m, 4H), 2.24 (t, J ¼ 6.5 Hz, 2H), 2.04–
2.00 (m, 2H). ESI-MS: m/z [MþH]þ 365. Anal. calcd. for
C18H21ClN2O4: C, 59.26; H, 5.80; N, 7.68. Found: C, 59.44;
H, 5.78; N, 7.45.
1-(4-Bromobutyl)-1H-indole (4)
Seventy percent NaH (1.77 g, 51.6 mmol) was added portionwise
to a solution of indole (5.0 g, 43 mmol) in DMF (50 mL) at 0–58C.
The mixture was warmed to room temperature and stirred for
30 min. After that, it was added dropwise to a mixture of 1,4-
dibromobutane (46.42 g, 215 mmol) in DMF (10 mL) at room
temperature, and stirred for 12 h. Then the mixture was poured
into cold water (300 mL) and extracted with ethyl acetate
(3 ꢂ 100 mL). The organic phase was combined, washed with
brine (3 ꢂ 300 mL), dried over Na2SO4, and concentrated
in vacuo. The residue was purified by flash column chromatog-
raphy on silica gel using petroleum ether/ethyl acetate (80:1 v/v)
Methyl 2-(6-bromo-1-(3-morpholinopropyl)-1H-indol-3-yl)-
2-oxoacetate (3g)
According to the procedure used to prepare 3a, reaction of 2d
with 4-(3-chloropropyl)morpholine provided 3g in 54.9% yield
as a light yellow solid, mp: 102–1038C. 1H NMR (500 MHz, CDCl3):
d 8.43 (s, 1H), 8.31 (d, J ¼ 8.5 Hz, 1H), 7.67 (d, J ¼ 1.5 Hz, 1H),
7.45 (dd, J ¼ 8.5, 1.5 Hz, 1H), 4.29 (t, J ¼ 6.5 Hz, 2H), 3.95 (s, 3H),
3.86–3.69 (m, 4H), 2.43–2.39 (m, 4H), 2.25 (t, J ¼ 6.5 Hz, 2H),
2.05–2.01 (m, 2H). ESI-MS: m/z [MþH]þ 409. Anal. calcd.
for C18H21BrN2O4: C, 52.82; H, 5.17; N, 6.84. Found: C, 52.68;
H, 5.26; N, 6.68.
1
as eluent to afford 7.6 g (71.2%) 4 as a colorless liquid. H NMR
(500 MHz, CDCl3): d 7.66 (d, J ¼ 8.0 Hz, 1H), 7.36 (d, J ¼ 8.0 Hz,
1H), 7.25–7.22 (m, 1H), 7.14–7.11 (m, 2H), 6.53 (d, J ¼ 3.0 Hz, 1H),
4.19 (t, J ¼ 7.0 Hz, 2H), 3.40 (t, J ¼ 6.5 Hz, 2H), 2.07–2.00 (m, 2H),
1.90–1.85 (m, 2H).
4-(4-(1H-Indol-1-yl)butyl)morpholine (5)
A mixture of 4 (2.0 g, 7.9 mmol), morpholine (6.9 g, 79.0 mmol),
and potassium carbonate (1.9 g, 13.8 mmol) in DMF (30 mL) was
stirred at 508C for 6 h [29–37]. After cooling, the mixture was
poured into cold water (200 mL) and extracted with ethyl acetate
(3 ꢂ 100 mL). The organic phase was combined, washed with
brine (3 ꢂ 300 mL), dried over Na2SO4, and concentrated in
vacuum. The residue was purified by flash column chromatog-
raphy on silica gel using petroleum ether/ethyl acetate/triethyl-
amine (20:100:1 by volume) as eluent to afford 1.63 g (80.1%) 5 as
a colorless liquid. 1H NMR (500 MHz, CDCl3): d 7.65 (d, J ¼ 8.0 Hz,
1H), 7.37 (d, J ¼ 8.0 Hz, 1H), 7.24–7.21 (m, 1H), 7.14–7.11 (m, 2H),
6.51 (d, J ¼ 3.0 Hz, 1H), 4.17 (t, J ¼ 7.0 Hz, 2H), 3.75–3.70 (m, 4H),
2.39–2.33 (m, 6H), 1.93–1.87 (m, 2H), 1.57–1.50 (m, 2H). ESI-MS:
m/z [MþH]þ 259. Anal. calcd. for C16H22N2O: C, 74.38; H, 8.58;
N, 10.84. Found: C, 74.53; H, 8.64; N, 10.93.
Methyl 2-(6-fluoro-1-(3-morpholinopropyl)-1H-indol-3-yl)-
2-oxoacetate (3h)
According to the procedure used to prepare 3a, reaction of 2e
with 4-(3-chloropropyl)morpholine provided 3h in 64.8% yield
as a light yellow solid, mp: 116–1178C. 1H NMR (500 MHz, CDCl3):
d 8.44 (s, 1H), 8.38 (dd, J ¼ 8.5, 5.5 Hz, 1H), 7.16 (dd, J ¼ 9.0,
2.0 Hz, 1H), 7.13–7.06 (m, 1H), 4.27 (t, J ¼ 6.5 Hz, 2H), 3.95 (s, 3H),
3.79–3.70 (m, 4H), 2.43–2.39 (m, 4H), 2.27 (t, J ¼ 6.5 Hz, 2H),
2.06–2.00 (m, 2H). ESI-MS: m/z [MþH]þ 349. Anal. calcd.
for C18H21FN2O4: C, 62.06; H, 6.08; N, 8.04. Found: C, 62.18;
H, 6.23; N, 8.25.
Methyl 2-(1-(3-(tert-butyldimethylsilyloxy)propyl)-1H-indol-
3-yl)-2-oxoacetate (3i)
According to the procedure used to prepare 3a, reaction of 2a
with (3-bromopropoxy) (tert-butyl)dimethylsilane provided 3i
in 62.5% yield as a light yellow solid, mp: 69–718C. 1H NMR
(500 MHz, CDCl3): d 8.40–8.36 (m, 1H), 8.31 (s, 1H), 7.39–
7.34 (m, 1H), 7.31–7.24 (m, 2H), 4.27 (t, J ¼ 7.0 Hz, 2H), 3.88
(s, 3H), 3.52 (t, J ¼ 7.0 Hz, 2H), 2.06–1.92 (m, 2H), 0.87 (s, 9H),
0.01 (s, 6H). ESI-MS: m/z [MþH]þ 349. Anal. calcd. for
C20H29NO4Si: C, 63.97; H, 7.78; N, 3.73. Found: C, 64.05;
H, 7.86; N, 3.65.
Methyl 2-(1-(4-morpholinobutyl)-1H-indol-3-yl)-2-
oxoacetate (3k)
One molar HCl in dioxane (5.5 mL, 5.5 mmol) was added to a
solution of 5 (1.29 g, 5 mmol) in 30 mL CAN and 30 mL Et2O at
room temperature and stirred for 30 min. After that, oxalyl
chloride (0.76 g, 6.0 mmol) in Et2O (5 mL) was added dropwise
at 0–58C and the reaction mixture was stirred for 2 h at the same
temperature. MeOH (10 mL) was added dropwise to the mixture
at 0–58C and the result solution was stirred for 2 h at room
temperature, then poured into a cold aqueous NaHCO3 solution
and extracted with ethyl acetate (3 ꢂ 50 mL). The organic phase
was combined, washed with brine (150 mL), dried over Na2SO4,
and concentrated in vacuum. The residue was purified by
flash column chromatography on silica gel using ethyl
acetate/methanol (50:1 v/v) as eluent to afford 0.94 g (54.6%) 5
Methyl 2-(1-methyl-1H-indol-3-yl)-2-oxoacetate (3j)
Seventy percent NaH (0.17 g, 4.9 mmol) was added portionwise
to a solution of 2a (1.0 g, 4.9 mmol) in DMF (10 mL) at 0–58C. The
reaction mixture was warmed to room temperature and stirred
for 30 min. After that, iodomethane (0.85 g, 5.9 mmol) was
added at 08C and stirred for 1 h at room temperature. The
mixture was then poured into cold water (100 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 con-
centrated in vacuo. The residue was purified by flash column
chromatography on silica gel using petroleum ether/ethyl
acetate (3:1 v/v) as eluent to afford 0.81 g (76.8%) 3j as a light
yellow solid, mp: 73–748C. 1H NMR (500 MHz, CDCl3): d 8.51–8.42
(m, 1H), 8.35 (s, 1H), 7.39–7.36 (m, 3H), 3.96 (s, 3H), 3.88 (s, 3H).
ESI-MS: m/z [MþH]þ 218. Anal. calcd. for C12H11NO3: C, 66.35;
H, 5.10; N, 6.45. Found: C, 66.55; H, 5.20; N, 6.59.
1
as a light yellow solid, mp: 84–858C. H NMR (500 MHz, CDCl3):
d 8.48–8.44 (m, 1H), 8.40 (s, 1H), 7.44–7.38 (m, 1H), 7.39–7.34
(m, 2H), 4.23 (t, J ¼ 7.5 Hz, 2H), 3.97 (s, 3H), 3.73–3.65 (m, 4H),
2.39–2.34 (m, 6H), 2.02–1.92 (m, 2H), 1.59–1.51 (m, 2H). ESI-MS:
m/z [MþH]þ 345. Anal. calcd. for C19H24N2O4: C, 66.26; H, 7.02;
N, 8.13. Found: C, 66.40; H, 7.11; N, 8.32.
3-(1H-Indol-3-yl)-4-(1H-pyrrol-3-yl)-1H-pyrrole-2,5-dione (10)
A solution of t-BuOK (134 mg, 1.2 mmol) in THF was added
dropwise to a solution of 2a (106 mg, 0.52 mmol) and 9
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356
Q. Ye et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 349–358
(50 mg, 0.4 mmol) in THF (20 mL) at ꢁ5 to 08C. After stirring for
2 h at at room temperature, 5 mL concentrated hydrochloric
acid was added and the result mixture was stirred for 30 min
at room temperature, then poured into 10% NaHCO3 aqueous
solution (100 mL) and extracted with ethyl acetate (3 ꢂ 50 mL).
The organic phase was combined and washed with brine
(3ꢂ 150 mL), dried over Na2SO4 and concentrated in vacuo.
The residue was purified by flash column chromatography on
silica gel using dichloromethane/methanol (50:1 v/v) as eluent to
afford 12.4 mg (11.2%) 10 as a red solid, mp: 169–1708C. 1H NMR
(500 MHz, DMSO-d6): d 11.85 (brs, 1H), 11.28 (brs, 1H), 10.73 (brs,
1H), 7.66 (d, J ¼ 3.0 Hz, 1H), 7.46 (d, J ¼ 8.0 Hz, 1H), 7.34 (brs, 1H),
7.11 (t, J ¼ 8.0 Hz, 1H), 6.98 (d J ¼ 8.0 Hz, 1H), 6.91 (t, J ¼ 8.0 Hz,
1H), 6.67 (brs, 1H), 6.08 (brs, 1H). ESI-MS: m/z [MþH]þ 278.
Anal. calcd. for C16H11N3O2: C, 69.31; H, 4.00; N, 15.15. Found:
C, 69.55; H, 4.11; N,15.02.
(2H, m), ESI-MS: m/z [MþH]þ 386. Anal. calcd. for C22H19N5O2: C,
68.56; H, 4.97; N, 18.17. Found: C, 68.41; H, 4.83; N, 18.33.
3-(1-(3-(Piperidin-1-yl)propyl)-1H-indol-3-yl)-4-(1H-pyrrol-
3-yl)-1H-pyrrole-2,5-dione (11d)
According to the procedure used to prepare 11a, reaction of 3d
with 9 provided 11d in 10.6% yield as a red solid, mp: 235–2378C.
1H NMR (500 MHz, CDCl3 þ DMSO-d6): d 11.20 (brs, 1H), 10.75
(brs, 1H), 7.72 (s, 1H), 7.57 (d, J ¼ 8.0 Hz, 1H), 7.36 (brs, 1H), 7.17
(t, J ¼ 8.0 Hz, 1H), 6.95–6.98 (m, 2H), 6.67 (brs, 1H), 6.07 (brs, 1H),
4.33 (t, J ¼ 7.0 Hz, 2H), 2.39–2.34 (m, 6H), 2.09–2.07 (m, 2H), 1.62–
1.59 (4H, m), 1.42–140 (m, 2H). ESI-MS: m/z [MþH]þ 403. Anal.
calcd. for C24H26N4O2: C, 71.62; H, 6.51; N, 13.92. Found: C, 71.49;
H, 6.71; N, 13.86.
3-(5-Methoxy-1-(3-morpholinopropyl)-1H-indol-3-yl)-4-
(1H-pyrrol-3-yl)-1H-pyrrole-2,5-dione (11e)
3-(1-(3-Morpholinopropyl)-1H-indol-3-yl)-4-(1H-pyrrol-3-yl)-
1H-pyrrole-2,5-dione (11a)
According to the procedure used to prepare 11a, reaction of 3e
with 9 provided 11e in 15.6% yield as a red solid, mp: 186–1888C.
1H NMR (500 MHz, CDCl3): d 8.44 (brs, 1H), 7.72 (s, 1H), 7.42
(brs, 1H), 7.37 (brs, 1H), 7.30 (d, J ¼ 8.5 Hz, 1H), 6.85
(dd, J ¼ 8.5, 2.0 Hz, 1H), 6.72 (d, J ¼ 2.0 Hz, 1H), 6.44 (brs, 1H),
6.38 (brs, 1H), 4.29 (t, J ¼ 7.0 Hz, 2H), 3.77–3.72 (m, 4H), 3.50
(s, 3H), 2.47–2.35 (m, 6H), 2.09–2.06 (2H, m). ESI-MS: m/z [MþH]þ
433. Anal. calcd. for C24H26N4O4: C, 66.34; H, 6.03; N, 12.89.
Found: C, 66.42; H, 6.09; N, 12.62.
A solution of t-BuOK (134 mg, 1.2 mmol) in THF was added
dropwise to a solution of 3a (172 mg, 0.52 mmol) and 9
(50 mg, 0.4 mmol) in THF (20 mL) at ꢁ5 to 08C. After stirring
for 2 h at room temperature, 5 mL concentrated hydrochloric
acid was added and the result mixture was stirred for 30 min at
room. The mixture was then poured into 10% NaHCO3 aqueous
solution (100 mL) and extracted with ethyl acetate (3 ꢂ 50 mL).
The organic phase was combined and washed with brine
(3 ꢂ 150 mL), dried over Na2SO4, and concentrated in vacuo.
The residue was purified by flash column chromatography
on silica gel using dichloromethane/methanol/triethylamine
(90:3:1 by volume) as eluent to afford 20.7 mg (12.8%) 11a as a
red solid, mp: 133–1358C. 1H NMR (500 MHz, CDCl3 þ DMSO-d6):
d 10.19 (brs, 1H), 9.47 (brs, 1H), 7.62 (s, 1H), 7.44–7.42 (m, 2H), 7.19
(t, J ¼ 8.0 Hz, 1H) 7.09 (d, J ¼ 8.0 Hz, 1H), 6.97 (t, J ¼ 8.0 Hz, 1H),
6.62 (brs, 1H), 6.27 (brs, 1H), 4.32 (t, J ¼ 7.0 Hz, 2H), 3.76–
3.72 (4H, m), 2.48–2.45 (4H, m), 2.35 (t, J ¼ 7.0 Hz, 2H),
2.07–2.05 (2H, m). ESI-MS: m/z [MþH]þ 405. Anal. calcd. for
C23H24N4O3: C, 68.30; H, 5.98; N, 13.85. Found: C, 68.53;
H, 6.03; N, 14.13.
3-(6-Chloro-1-(3-morpholinopropyl)-1H-indol-3-yl)-4-
(1H-pyrrol-3-yl)-1H-pyrrole-2,5-dione (11f)
According to the procedure used to prepare 11a, reaction of 3f
with 9 provided 11f in 18.5% yield as a red solid, mp: 102–1048C.
1H NMR (500 MHz, CDCl3): d 8.55 (brs, 1H), 7.81 (brs, 1H), 7.65
(s, 1H), 7.51 (brs, 1H), 7.45 (s, 1H), 6.98–6.92 (m, 2H), 6.66 (brs, 1H),
6.25 (brs, 1H), 4.27 (t, J ¼ 7.0 Hz, 2H), 3.80–3.72 (m, 4H),
2.48–2.40 (m, 4H), 2.31 (t, J ¼ 7.0 Hz, 2H), 2.07–2.04 (m, 2H).
ESI-MS: m/z [MþH]þ 439. Anal. calcd. for C23H23ClN4O3: C,
62.94; H, 5.28; N, 12.77. Found: C, 66.71; H, 5.35; N, 12.62.
3-(6-Bromo-1-(3-morpholinopropyl)-1H-indol-3-yl)-4-
(1H-pyrrol-3-yl)-1H-pyrrole-2,5-dione (11g)
3-(1-(2-Morpholinoethyl)-1H-indol-3-yl)-4-(1H-pyrrol-3-yl)-
1H-pyrrole-2,5-dione (11b)
According to the procedure used to prepare 11a, reaction of 3g
with 9 provided 11g in 13.8% yield as a red solid, mp: 83–858C.
1H NMR (500 MHz, CDCl3): d 8.47 (brs, 1H), 7.69–7.65 (m, 2H), 7.53
(s, 1H), 7.40 (brs, 1H), 7.01 (d, J ¼ 8.5 Hz, 1H), 6.93 (d, J ¼ 8.5 Hz,
1H), 6.68 (brs, 1H), 6.27 (brs, 1H), 4.29 (t, J ¼ 7.0 Hz, 2H), 3.83–3.75
(m, 4H), 2.49–2.43 (m, 4H), 2.31 (t, J ¼ 7.0 Hz, 2H), 2.08–2.03
(m, 2H). ESI-MS: m/z [MþH]þ 483. Anal. calcd. for C23H23BrN4O3:
C, 57.15; H, 4.80; N, 11.59. Found: C, 57.29; H, 4.89; N, 11.78.
According to the procedure used to prepare 11a, reaction of 3b
with 9 provided 11b in 14.5% yield as a red solid, mp: 212–2148C.
1H NMR (500 MHz, DMSO-d6): d 11.26 (brs, 1H), 10.75 (brs, 1H),
7.74 (s, 1H), 7.56 (d, J ¼ 8.5 Hz, 1H), 7.37 (brs, 1H) 7.16
(t, J ¼ 8.5 Hz, 1H), 6.99 (d, J ¼ 8.0 Hz, 1H), 6.93 (t, J ¼ 8.0 Hz,
1H), 6.27 (brs, 1H), 6.10 (brs, 1H), 4.38 (t, J ¼ 7.0 Hz, 2H),
3.56–3.52 (m, 4H), 2.71 (t, J ¼ 7.0 Hz, 2H), 2.47–2.43 (m, 4H).
ESI-MS: m/z [MþH]þ 391. Anal. calcd. for C22H22N4O3: C, 67.68;
H, 5.68; N, 14.35. Found: C, 67.51; H, 5.82; N, 14.19.
3-(6-Fluoro-1-(3-morpholinopropyl)-1H-indol-3-yl)-4-(1H-
pyrrol-3-yl)-1H-pyrrole-2,5-dione (11h)
3-(1-(3-(1H-Imidazol-1-yl)propyl)-1H-indol-3-yl)-4-
(1H-pyrrol-3-yl)-1H-pyrrole-2,5-dione (11c)
According to the procedure used to prepare 11a, reaction of 3h
with 9 provided 11h in 14.9% yield as a red solid, mp: 149–1508C.
1H NMR (500 MHz, CDCl3): d 8.49 (brs, 1H), 7.67 (s, 1H), 7.58 (brs,
1H), 7.53 (brs, 1H), 7.13 (dd, J ¼ 9.0, 2.5 Hz, 1H), 7.01–6.98 (m, 1H),
6.76 (td, J ¼ 9.0, 2.5 Hz, 1H), 6.67 (brs, 1H), 6.27 (brs, 1H), 4.27
(t, J ¼ 7.0 Hz, 2H), 3.80–3.74 (m, 4H), 2.48–2.42 (m, 4H), 2.34
(t, J ¼ 7.0 Hz, 2H), 2.08–2.04 (m, 2H). ESI-MS: m/z [MþH]þ 423.
According to the procedure used to prepare 11a, reaction of 3c
with 9 provided 11c in 11.2% yield as a red solid, 234–2368C.
1H NMR (500 MHz, DMSO-d6): d 11.22 (brs, 1H), 10.79 (brs, 1H),
7.75–7.68 (m, 2H), 7.48 (d, J ¼ 7.5 Hz, 1H), 7.37 (s, 1H), 7.28 (brs,
1H), 7.18 (t, J ¼ 7.5 Hz, 1H), 6.90–6.70 (3H, m), 6.67 (brs, 1H), 6.07
(brs, 1H), 4.26 (t, J ¼ 7.0 Hz, 2H), 4.03 (t, J ¼ 7.0 Hz, 2H), 2.35–2.30
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Arch. Pharm. Chem. Life Sci. 2013, 346, 349–358
3-Aryl-4-pyrrolyl-maleimides as GSK-3b Inhibitors
357
Anal. calcd. for C23H23FN4O3: C, 65.39; H, 5.49; N, 13.26. Found: C,
65.58; H, 5.56; N, 13.03.
222–2248C. 1H NMR (500 MHz, CDCl3): d 8.05 (s, 1H), 7.88 (s, 1H),
7.76 (brs, 1H), 7.42 (d, J ¼ 8.0 Hz, 1H), 7.25–7.21 (m, 2H), 7.15
(s, 1H), 6.98 (t, J ¼ 8.0 Hz, 1H), 6.35 (d, J ¼ 8.0 Hz, 1H), 4.35
(t, J ¼ 7.0 Hz, 2H), 3.82–3.75 (4H, m), 2.48–2.40 (4H, m), 2.34
(t, J ¼ 7.0 Hz, 2H), 2.09–2.01 (m, 2H). ESI-MS: m/z [MþH]þ 406.
Anal. calcd. for C22H23N5O3: C, 65.17; H, 5.72; N, 17.27. Found:
C, 65.28; H, 5.81; N, 17.16.
3-(1-(3-Hydroxypropyl)-1H-indol-3-yl)-4-(1H-pyrrol-3-yl)-
1H-pyrrole-2,5-dione (11i)
According to the procedure used to prepare 11a, reaction of 3i
with 9 provided 11i in 15.6% yield as a red solid, mp: 160–1628C.
1H NMR (500 MHz, CDCl3 þ DMSO-d6): d 10.23 (brs, 1H), 9.79 (brs,
1H), 7.33 (s, 1H), 7.14–7.12 (m, 2H), 6.87 (t, J ¼ 8.0 Hz, 1H), 6.79
(d, J ¼ 8.0 Hz, 1H), 6.64 (t, J ¼ 8.0 Hz, 1H), 6.30 (brs, 1H), 5.97
(brs, 1H), 4.05–4.02 (m, 3H), 3.29–3.27 (m, 2H), 1.78–1.76 (m, 2H).
ESI-MS: m/z [MþH]þ 336. Anal. calcd. for C19H17N3O3: C, 68.05;
H, 5.11; N, 12.53. Found: C, 68.26; H, 5.18; N, 12.70.
3-(1-(3-Morpholinopropyl)-1H-indol-3-yl)-4-(1H-1,2,4-
triazol-1-yl)-1H-pyrrole-2,5-dione (15b)
According to the procedure used to prepare 11a, reaction of 6
with 14a provided 15b in 9.6% yield as an orange solid, m.p. 94–
968C. 1H NMR (500 MHz, DMSO-d6): d 11.45 (brs, 1H), 8.88 (s, 1H),
8.26 (s, 1H), 8.22 (s, 1H), 7.59 (d, J ¼ 8.0 Hz, 1H), 7.18 (t, J ¼ 8.0 Hz,
1H), 6.86 (t, J ¼ 8.0 Hz, 1H), 6.12 (d, J ¼ 8.0 Hz, 1H), 4.36
(t, J ¼ 6.5 Hz, 2H), 3.62–3.56 (m, 4H), 2.37–2.28 (m, 4H), 2.21
(t, J ¼ 6.5 Hz, 2H), 1.96–1.90 (m, 2H). ESI-MS: m/z [MþH]þ 407.
Anal. calcd. for C21H22N6O3: C, 62.06; H, 5.46; N, 20.68. Found: C,
62.21; H, 5.31; N, 20.55.
3-(1-Methyl-1H-indol-3-yl)-4-(1H-pyrrol-3-yl)-1H-pyrrole-
2,5-dione (11j)
According to the procedure used to prepare 10, reaction of 3j
with 9 provided 11j in 25.2% yield as a red solid, mp: 179–1818C.
1H NMR (500 MHz, DMSO-d6): d 11.18 (brs, 1H), 10.75 (brs, 1H),
7.69 (s, 1H), 7.50 (d, J ¼ 8.0 Hz, 1H), 7.36 (brs, 1H), 7.17
(t, J ¼ 8.0 Hz, 1H), 6.98–6.95 (m, 2H), 6.68 (brs, 1H), 6.08 (brs, 1H),
3.89 (s, 3H). ESI-MS: m/z [MþH]þ 292. Anal. calcd. for C19H17N3O3:
C, 70.09; H, 4.50; N, 14.42. Found: C, 70.26; H, 4.48; N, 14.61.
Pharmacology
GSK-3b purification and activity assay
The GSK-3b cDNA was obtained from UniGene (3667B03) and
inserted into the pGEX-KG vector. The recombinant GST-GSK-3b
protein was expressed in Escherichia coli strain BL21-CodonPlus
(DE3), purified by GSTrap affinity chromatography, and cleaved
by thrombin. The GSK-3b kinase assay was carried out with the
Invitrogen Z’-LYTETM Kinase Assay kit (Ser/Thr 9 Peptide sub-
strate), with a final enzyme concentration of 50 nM. All reactions
were carried out in triplicate. IC50 values (concentration at which
50% of enzyme inhibition is shown) were derived from a non-
linear regression model (curvefit) based on a sigmoidal dose
response curve (variable slope) and computed using Graghpad
Prism version 5.02, Graphpad Software. Data were expressed as
mean ꢀ SEM.
3-(1-(4-Morpholinobutyl)-1H-indol-3-yl)-4-(1H-pyrrol-3-yl)-
1H-pyrrole-2,5-dione (11k)
According to the procedure used to prepare 11a, reaction of 3k
with 9 provided 11k in 12.6% yield as a red solid, m.p. 135–1378C.
1H NMR (500 MHz, CDCl3 þ DMSO-d6): d 9.90 (brs, 1H), 9.27
(brs, 1H), 7.49 (s, 1H) 7.38 (brs, 1H), 7.19 (d, J ¼ 8.0 Hz, 1H), 7.08
(t, J ¼ 8.0 Hz, 1H), 6.99 (d, J ¼ 8.0 Hz, 1H), 6.87 (t, J ¼ 8.0 Hz, 1H),
6.52 (brs, 1H), 6.16 (brs, 1H), 4.12 (t, J ¼ 7.0 Hz, 2H), 3.59–3.50
(m, 4H), 2.35–2.20 (m, 6H), 1.85–1.82 (m, 2H), 1.49–1.45 (m, 2H).
ESI-MS: m/z [MþH]þ 419. Anal. calcd. for C24H26N4O3: C, 68.88;
H, 6.26; N, 13.39. Found: C, 68.65; H, 6.29; N, 13.55.
Cell culture and Western blot
3-Phenyl-4-(1H-pyrrol-3-yl)-1H-pyrrole-2,5-dione (13a)
According to the procedure used to prepare 10, reaction of 6 with
12a provided 13a in 30.6% yield as an orange solid, m.p. 508C.
1H NMR (500 MHz, DMSO-d6): d 11.35 (brs, 1H), 10.87 (brs, 1H),
7.52 (brs, 1H), 7.47–7.42 (5H, m), 6.99 (brs, 1H), 6.73 (brs, 1H).
ESI-MS: m/z [MþH]þ 239. Anal. calcd. for C14H10N2O2: C, 70.58;
H, 4.23; N, 11.76. Found: C, 70.83; H, 4.28; N, 11.64.
SH-SY5Y human neuroblastoma cells were obtained from ATCC
(The American Type Culture Collection). Cells were cultured in
1:1 DMEM/Ham’s F12 containing 10% v/v fetal bovine serum
(HyClone), 1% penicillin, and 1% streptomycin at a humidified
atmosphere with 5% CO2. The medium was changed every 2 days.
For experiments, cells were grown in 12-well plates until ꢃ80%
confluence, serum-deprived for 12 h, incubated with GSK-3b
inhibitors for 1 h and Ab25–35 (amyloid beta peptide 25–35,
Sigma) for another 6 h. Cells were rinsed twice with ice-cold
PBS and lysed with 1ꢂ SDS loading buffer. Samples were electro-
phoresed on 10% SDS–polyacrylamide gels, and transferred to
PVDF membranes. The membranes were blocked for 1 h with 5%
w/v milk, incubated with rabbit anti-Tau [pS396] phosophospe-
cific antibody (Abcam) for 2 h and the anti-rabbit secondary
antibody for 1 h. Antigen–antibody complexes were detected
by the ECL Kit.
3-(Naphthalen-1-yl)-4-(1H-pyrrol-3-yl)-1H-pyrrole-2,5-
dione (13b)
According to the procedure used to prepare 10, reaction of 6 with
12b provided 13b in 24.8% yield as an orange solid, m.p. 239–
2418C. 1H NMR (500 MHz, DMSO-d6): d 10.60 (brs, 1H), 10.26
(brs, 1H), 7.94–7.92 (m, 2H), 7.70 (d, J ¼ 8.0 Hz, 1H), 7.58
(d, J ¼ 8.0 Hz, 1H), 7.51–7.48 (m, 3H), 7.41 (t, J ¼ 8.0 Hz, 1H),
6.46 (brs, 1H), 5.72 (brs, 1H). ESI-MS: m/z [MþH]þ 289. Anal. calcd.
for C18H12N2O2: C, 74.99; H, 4.20; N, 9.72. Found: C, 74.76; H, 4.31;
N, 9.91.
PKCE, IKK2, Aurora A, MEK1, and ERK1 assays
The recombinant PKCE and IKK2 were expressed in the Bac-to-Bac
baculovirus system, and recombinant Auroa A, MEK1, and
ERK1 were expressed in the E. coli system. All these kinase assays
were carried out by using the Invitrogen Z’-LYTETM Kinase Assay
kits.
3-(1H-Imidazol-1-yl)-4-(1-(3-morpholinopropyl)-1H-indol-
3-yl)-1H-pyrrole-2,5-dione (15a)
According to the procedure used to prepare 11a, reaction of 6
with 14a provided 13b in 10.6% yield as an orange solid, m.p.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

358
Q. Ye et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 349–358
We gratefully acknowledge the financial support from the Natural Science
Foundation of China (21176223) and the National Natural Science
Foundation of Zhejiang (Y407306).
Y. Li, T. D. Lindstrom, A. L. Marquart, J. McLean, D. Mendel,
E. Misener, D. Briere, J. C. O’Toole, W. J. Porter, S. Queener, J. K.
Reel, R. A. Owens, R. A. Brier, T. E. Eessalu, J. R. Wagner, R. M.
Campbell, R. Vaughn, J. Med. Chem. 2004, 47, 3934–3937.
The authors have declared no conflict of interest.
[21] Q. Ye, G. Xu, D. Lv, Z. Cheng, J. Li, Y. Hu, Bioorg. Med. Chem.
2009, 17, 4302–4312.
[22] I. N. Gaisina, F. Gallier, A. V. Ougolkov, K. H. Kim, T. Kurome,
S. Guo, D. Holzle, D. N. Luchini, S. Y. Blond, D. D. Billadeau,
A. P. Kozikowski, J. Med. Chem. 2009, 52, 1853–1863.
References
[1] N. Embi, D. B. Rylatt, P. Cohen, Eur. J. Biochem. 1980, 107, 519–
527.
[23] F. Nobuhiro, I. Yosuke, K. Hiroaki, N. Aiko, S. Kazuharu,
U. Hiroshi, Y. Shinichi, N. Kazumi, F. Shigeo, S. Yumiko,
Y. Masateru, T. Toshiya, Bioorg. Med. Chem. Lett. 2012, 22,
4358–4361.
[2] A. J. Harwood, Cell 2001, 105, 821–824.
[3] I. Dominguez, J. B. Green, Dev. Biol. 2001, 235, 303–313.
[24] H. C. Zhang, C. K. Derian, D. F. McComsey, K. B. White, H. Ye,
L. R. Hecker, J. Li, M. F. Addo, D. Croll, A. J. Eckardt, C. E.
Smith, Q. Li, W. M. Cheung, B. R. Conway, S. Emanuel, K. T.
Demarest, P. Andrade-Gordon, B. P. Damiano, B. E. Maryanoff,
J. Med. Chem. 2005, 48, 1725–1728.
[4] D. A. Cross, D. R. Alessi, P. Cohen, M. Andjelkovich, B. A.
Hemming, Nature 1995, 378, 785–789.
[5] M. Hetman, A. Kaleta, M. Chlystun, M. Higgins, J. Cavanaugh,
Z. Xia, Cell. Mol. Biol. Lett. 2001, 6, 494.
[6] S. Lovestone, C. H. Reynolds, D. Latimer, D. R. Davis, B. H.
Anderton, J.-M. Gallo, D. Hanger, S. Mulot, B. Marquardt,
Curr. Biol. 1994, 4, 1077–1086.
[25] T. A. Engler, J. R. Henry, S. Malhotra, B. Cunningham,
K. Furness, J. Brozinick, T. P. Burkholder, M. P. Clay,
J. Clayton, C. Diefenbacher, E. Hawkins, P. W. Iversen,
Y. H. Li, T. D. Lindstrom, A. L. Marquart, J. McLean,
D. Mendel, E. Misener, D. Briere, J. C. O’Toole, W. J.
Porter, S. Queener, J. K. Reel, R. A. Owens, R. A. Brier,
T. E. Eessalu, J. R. Wagner, R. M. Campbell, R. Vaughn,
J. Med. Chem. 2004, 47, 3934–3937.
[7] L. Kim, A. R. Kimmel, Curr. Opin. Genet. Dev. 2000, 10, 508–514.
[8] A. S. Wagman, J. M. Nuss, Curr. Pharm. Des. 2001, 7, 417–450.
[9] P. Cohen, Eur. J. Biochem. 2001, 268, 5001–5010.
[10] C. Sasaki, T. Hayashi, W. R. Zhang, H. Warita, Y. Manabe,
K. Sakai, K. Abe, Neurol. Res. 2001, 23, 588–592.
[26] Z. K. Sun, H. Q. Yang, J. Pan, H. Zhen, Z. Q. Wang, S. D. Chen,
J. Q. Ding, J. Neurosci. Res. 2008, 86, 3018–3027.
[11] A. Castro, A. Martinez, Exp. Opin. Ther. Pat. 2000, 10, 1519–
1527.
[27] SYBYL Molecular Modeling Systems, versions 6.9; Tripos
Associates, St. Louis, MO.
[12] B. W. Doble, J. R. Woodgett, J. Cell. Sci. 2003, 116, 1175–1186.
[13] C. J. Phiel, C. A. Wilson, V. M. Y. Lee, P. S. Klein, Nature 2003,
423, 435–439.
[28] X. H. Liu, L. Pan, Y. Ma, J. Q. Weng, C. X. Tan, Y. H. Li, Y. X. Shi,
B. J. Li, Z. M. Li, Y. G. Zhang, Chem. Biol. Drug Des. 2011, 78, 689–
694.
[14] J. A. Bertrand, S. Thieffine, A. Vulpetti C. Cristiani, B.
Valsasina, S. Knapp, H. M. Kalisz, M. Flocco, J. Mol. Biol.
2003, 333, 393–407.
[29] X. H. Liu, C. X. Tan, J. Q. Weng, Phosphorus Sulfur Silicon Relat.
Elem. 2011, 186, 558–564.
[15] I. Hers, J. M. Tavare, R. M. Denton, FEBS Lett. 1999, 460, 433–
436.
[30] C. X. Tan, Y. X. Shi, J. Q. Weng, X. H. Liu, B. J. Li, W. G. Zhao,
Lett. Drug Des. Discov. 2012, 9, 431–435.
[16] H. C. Zhang, K. B. White, H. Ye, D. F. McComsey, C. K. Derian,
M. F. Addo, P. Andrade-Gordon, A. J. Eckardt, B. R. Conway,
L. Westover, J. Z. Xu, R. Look, K. T. Demarest, S. Emanuel,
B. E. Maryanoff, Bioorg. Med. Chem. Lett. 2003, 13, 3049–3053.
[31] X. H. Liu, J. Q. Weng, B. L. Wang, Y. H. Li, C. X. Tan,
Z. M. Li, Res. Chem. Intermed. 2013. DOI 10.1007/s11164-013-
1113-4
[32] X. H. Liu, L. Pan, J. Q. Weng, C. X. Tan, Y. H. Li, B. L. Wang,
Z. M. Li, Mol. Divers. 2012, 16, 251–260.
[17] G. H. Kuo, C. Prouty, A. DeAngelis, L. Shen, D. J. O’Neill,
C. Shah, P. J. Connolly, W. V. Murray, B. R. Conway,
P. Cheung, L. Westover, J. Z. Xu, R. A. Look, K. T.
Demarest, S. Emanuel, S. A. Middleton, L. Jolliffe, M. P.
Beavers, X. Chenet, J. Med. Chem. 2003, 46, 4021–4031.
[33] C. X. Tan, Y. X. Shi, J. Q. Weng, X. H. Liu, W. G. Zhao, B. J. Li,
J. Heterocycl. Chem. 2013. DOI: 10.1002/jhet.1656
[34] X. H. Liu, J. Q. Weng, C. X. Tan, J. Chem. 2013, 2013, 306361.
[35] J. Q. Weng, L. Wang, X. H. Liu, J. Chem. Soc. Pak. 2012, 34, 1248–
1252.
[18] O. A. B. S. Gani, R. A. Engh, Nat. Prod. Rep. 2010, 27, 489–498.
[19] B. Pajak, S. Orzechowska, B. Gajkowska, A. Orzechowski,
Adv. Med. Sci. 2008, 53, 21–31.
[36] X. H. Liu, W. G. Zhao, B. L. Wang, Z. M. Li, Res. Chem. Intermed.
2012, 38, 1999–2008.
[20] T. A. Engler, J. R. Henry, S. Malhotra, B. Cunningham,
K. Furness, J. Brozinick, T. P. Burkholder, M. P. Clay,
J. Clayton, C. Diefenbacher, E. Hawkins, P. W. Iversen,
[37] J. Y. Tong, Y. X. Shi, X. H. Liu, N. B. Sun, B. J. Li, Chin. J. Org.
Chem. 2012, 32, 2373–2377.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com