Synthesis and biological evaluation of 3-benzisoxazolyl-4-indolylmaleimides as potent, selective inhibitors of glycogen synthase kinase-3β

Article abstract of DOI:10.3390/molecules18055498

A series of novel 3-benzisoxazolyl-4-indolyl-maleimides were synthesized and evaluated for their GSK-3β inhibitory activity. Most compounds exhibited high inhibitory potency towards GSK-3β. Among them, compound 7j with an IC₅₀ value of 0.73 nM was the most promising GSK-3β inhibitor. Preliminary structure-activity relationships were examined and showed that different substituents on the indole ring and N¹-position of the indole ring had varying degrees of influence on the GSK-3β inhibitory potency. Compounds 7c, 7f, 7j-l and 7o-q could obviously reduce Aβ-induced Tau hyperphosphorylation by inhibiting GSK-3β in a cell-based functional assay.

Full text of DOI:10.3390/molecules18055498

Molecules 2013, 18, 5498-5516; doi:10.3390/molecules18055498
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Evaluation of
3-Benzisoxazolyl-4-indolylmaleimides as Potent,
Selective Inhibitors of Glycogen Synthase Kinase-3β
Qing Ye 1, Meng Li 1, Yubo Zhou 2, Tao Pang 2, Lei Xu 2, Jiayi Cao 2, Liang Han 1, Yujin Li 1,
Weisi Wang 3, Jianrong Gao 1,* and Jia Li 2,*
1
State Key Laboratory Breeding Base of Green Chemistry-Synthesis Technology,
Zhejiang University of Technology, Hangzhou 310032, Zhejiang, China;
E-Mails: yeqing1975@yeah.net (Q.Y.); limeng71085110@126.com (M.L.);
hanliang@zjut.edu.cn (L.H.); lyjzjut@zjut.edu.cn (Y.L.)
2
The National Center for Drug Screening, Shanghai 201203, China;
E-Mails: ybzhou@mail.shcnc.ac.cn (Y.Z.); tyne_pang@hotmail.com (T.P.);
lxu@mail.shcnc.ac.cn (L.X.); jycao@mail.shcnc.ac.cn (J.C.)
3
ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences,
Zhejiang University, Hangzhou 310058, Zhejiang, China, E-Mail: pipilu1985@zju.edu.cn
* Authors to whom correspondence should be addressed; E-Mails: gdgjr@zjut.edu.cn (J.G.);
jli@mail.shcnc.ac.cn (J.L.); Tel./Fax: +86-571-8832-0891 (J.G.).
Received: 21 March 2013; in revised form: 2 May 2013 / Accepted: 7 May 2013/
Published: 13 May 2013
Abstract: A series of novel 3-benzisoxazolyl-4-indolyl-maleimides were synthesized and
evaluated for their GSK-3β inhibitory activity. Most compounds exhibited high inhibitory
potency towards GSK-3β. Among them, compound 7j with an IC50 value of 0.73 nM was
the most promising GSK-3β inhibitor. Preliminary structure-activity relationships were
examined and showed that different substituents on the indole ring and N1-position of
the indole ring had varying degrees of influence on the GSK-3β inhibitory potency.
Compounds 7c, 7f, 7j–l and 7o–q could obviously reduce Aβ-induced Tau
hyperphosphorylation by inhibiting GSK-3β in a cell-based functional assay.
Keywords: 3-benzisoxazolyl-4-indolylmaleimides; synthesis; GSK-3β; biological activity;
docking; SAR

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1. Introduction
Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase which was identified in
the late 1970s. Mammalian GSK-3 exists as two isoforms, GSK-3α and GSK-3β which share high
homology at the catalytic domain but 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 [1–4]. GSK-3β plays a critical role in glycogen metabolism, embryogenesis,
mitotic regulation, inflammation and neuroplasticity [5–8]. Inhibition of GSK-3β may provide therapy
approach for several diseases such as cancer, type 2 diabetes, chronic inflammatory processes, stroke,
bipolar disorders and Alzheimer's disease [9–13]. Accordingly, searching for novel and selective
GSK-3β inhibitors is a very active area in both academic centers and pharmaceutical companies.
Staurosporine, a microbial alkaloid, was identified as a potent but nonselective GSK-3β inhibitor.
Various bisindolylmaleimides such as GF 109203X and Ro 31-8220 have also been developed as
potent GSK-3β inhibitors based on staurosporine (Figure 1) [14–16]. However, most of these
bisindolylmaleimides are not suitable for the treatment of diseases such as diabetes and Alzheimer’s
disease due to their toxicity, poor solubility and low selectivity, especially against PKC family [17,18].
The replacement of one indole with other heteroaryl groups resulted in a series of
monoindolylmaleimides such as 4-azaindolyl-indolyl-maleimides, benzofuranyl-indolyl-maleimides
and imidazo[1,2-a]pyridinyl-indolyl-maleimides (Figure 1), which showed potent and selective
GSK-3β inhibitory activities [19–21].
Figure 1. GSK-3β inhibitors.
H
N
H
H
H
O
N
N
N
O
O
O
O
O
O
N
N
R2
R1
O
N
N
N
N
N
O
CH3
H
H
OCH3
NH
S
C
N
H3C
HN
Ro 31-8220
NH2
Benzofuranyl-
indolylmaleimides
GF 109203X
Staurosporine
H
N
O
O
H
N
O
O
H
R2
R3
N
O
O
N
N
R1
N
N
N
N
N
N
N
N
R2
O
N
O
N
603281-31-8
R1
O
Imidazo[1,2-a]pyridinyl-indolylmaleimides
4-Azaindolyl-indolylmaleimides
Among them, GSK-3β inhibitor 603281-31-8 developed by Eli Lilly & Co had reached preclinical
studies for the treatment of diabetes and was proved efficacy in ZDF rats [19]. In view of these facts

Molecules 2013, 18
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and also as a part of our work on the development of potent and selective GSK-3β inhibitors, herein we
report the synthesis and biological evaluation of a new series of 3-benzisoxazolyl-4-indolyl-maleimides as
GSK-3β inhibitors. Their structure-activity relationship and in silico molecular modeling study are also
discussed in this study.
2. Results and Discussion
2.1. Chemistry
The general synthetic approach to target compounds 7a–q is outlined in Scheme 1.
Scheme 1. Synthetic route to compounds 7a–q.
Reagents and conditions: (a) i (COCl)2, Et2O; ii CH3ONa, CH3OH; (b) (Boc)2O, DMAP, THF;
(c) NaH, DMF, R2X; (d) NaH, Br(CH2)4Br, DMF; (e) K2CO3, morpholine, DMF; (f) i HCl,
(COCl)2, Et2O; ii CH3ONa, CH3OH; (g) i t-BuOK, THF; ii concentrated HCl.

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Indole derivatives 1a–f were reacted with oxalyl chloride in Et2O, followed by sodium methoxide to
give compounds 2a–f. Reaction of 2a with (Boc)2O in the presence of a catalytic amount of DMAP in
THF afforded 3a. N-alkylation of 2a–f with different alkyl halides resulted in key intermediates 3b–h
and 3j–q. In addition, treatment of indole with 1,4-dibromobutane afforded 4. N-substitution reaction
of 4 with morpholine using K2CO3 as acid-trapping agent resulted in 5, which was then treated with
oxalyl chloride, followed by sodium methoxide to give another key intermediate 3i. Condensation of
glyoxylic esters 3a–q with 2-(benzo[d]isoxazol-3-yl)acetamide 6 [22] in the presence of t-BuOK in
THF afforded the target compounds 7a–q.
2.2. Biological Activity and Molecular Modeling
2.2.1. Enzymatic Activity
The GSK-3 inhibitory potency of all target compounds was examined. In addition, selected
compounds 7c, 7j and 7o were also tested for their inhibitory potency against other kinases (PKC-
epsilon, JAK2, BRAF, IKK2, Drak2) to assess kinase selectivity. Staurosporine, a well known kinase
inhibitor was used as the reference compound [19]. The results are listed in Tables 1 and 2. As
indicated in Table 1, most of the tested compounds showed similar or more potent GSK-3β inhibitory
activity as compared to that of staurosporine. The potency of GSK-3β inhibition of tested compounds
was mainly influenced by the substitutions on the indole ring and N1-position of the indole ring.
As expected, the introduction of suitable hydrophilic side chains at N1-position of the indole ring
gave an obvious enhancement of GSK-3β inhibitory activity (i.e., 7a, 7h, 7j–l, 7q). Among them,
compound 7j with an imidazole group at the end of N1-propyl chain exhibited the most potent GSK-3
inhibitory activity, with an IC50 of 0.73 nM, which was about 460-fold more potent than that of
compound 7a (IC50 = 332.2 nM). Replacement of the terminal imidazole group in 7j with other
substituents such as 1,2,4-triazole (7l, IC50 = 20.9 nM), hydroxyl (7q, IC50 = 38.9 nM), piperidine
(7m, IC50 = 511.8 nM) and pyrrolidine (7o, IC50 = 658.8 nM) resulted in a 29- to 915-fold less
inhibitory potency.
The results of inhibitory activities of compounds 7g–i, 7j and 7k showed that the length of the
N1-alkyl linker affected GSK-3 inhibitory potency. For example, compound 7j (IC50 = 0.73 nM) with
a (CH2)3 linker showed better inhibitory activity than compound 7k (IC50 = 89.8 nM) with a (CH2)4
linker. The same conclusion could also be drawn from comparison of the inhibitory potency of 7h and 7i.
Interestingly, the introduction of a hydrophobic methyl group on the N1-position of the indole ring
in 7a resulted in a 15-fold increase in inhibitory potency for GSK-3, while the replacement of the
methyl with a large butyl group showed a 3-fold decrease in potency for GSK-3β inhibition.
When comparing the inhibitory activity of 7b–7f with 7h, it suggested that different substituents on
the indole ring affected the inhibitory potency for GSK-3β. Compound 7c (IC50 = 10.2 nM) with
bromine at 5-position of the indole ring showed a 14-fold increase in inhibitory activity toward
GSK-3 as compared to that of 7h (IC50 = 137.7 nM). Fluorine at 6-position of the indole ring did not
influence activity of 7h, while bromine or chlorine at 6-position or methoxy at the 5-position showed
less inhibitory potency.

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Table 1. GSK-3β inhibitory activity of the target compounds.
Compounds
R 1
R 2
IC50 (nM) ± SE a
72.2 ± 3.6
Staurosporine
7a
7b
7c
7d
7e
7f
H
5-OMe
5-Br
6-Br
6-Cl
6-F
H
H
332.2 ± 25.7
357.2 ± 17.1
10.2 ± 5.9
3-morpholinopropyl
3-morpholinopropyl
3-morpholinopropyl
3-morpholinopropyl
3-morpholinopropyl
2-morpholinoethyl
3-morpholinopropyl
4-morpholinobutyl
3-(1H-imidazol-1-yl)propyl
4-(1H-imidazol-1-yl)butyl
3-(1H-1,2,4-triazol-1-yl)propyl
3-(piperidin-1-yl)propyl
3-(pyrrolidin-1-yl)propyl
methyl
209.6 ± 8.9
510.0 ± 20.1
126.7 ± 3.2
615.6 ± 6.1
137.7 ± 6.0
410.7 ± 8.2
0.73 ± 0.02
89.8 ± 4.5
7g
7h
7i
H
H
7j
H
7k
7l
H
H
20.9 ± 5.1
7m
7n
7o
7p
7q
H
511.8 ± 15.6
658.8 ± 15.7
22.1 ± 1.5
H
H
H
butyl
58.7 ± 2.8
H
hydroxypropyl
38.9 ± 2.6
a SE: standard error mean.
Table 2. The selectivity to tested kinases of target compounds 7c, 7j and 7o.
Kinases
IC50 (nM) ± SE a or inhibition% at 0.8 μg/mL (~2 μM)
Staurosporine
72.2 ±3.6
7c
10.2 ± 5.9
12.1%
7j
0.7 ± 0.02
10.9%
4.9%
7o
22.1 ± 1.5
21.2%
9.4%
GSK-3β
PKC-epsilon
JAK2
0.88 ± 0.03
2.26 ± 0.14
14.37 ± 0.86
1.21 ± 0.13
25.82 ± 2.11
0.1%
BRAF
3.4%
0.8%
1.1%
IKK2
13.1%
3.5%
2.9%
Drak2
1.67%
4.47%
13.76%
a SE: standard error mean.
The data in Table 2 showed that staurosporine was a potent and nonselective kinase inhibitor as
reported in the literature [14], which inhibits not only GSK-3β (IC50 = 72.2 nM) but also many other
kinases (e.g., PKC-epsilon, JAK2, BRAF, IKK2 and Drak2). Selected compounds 7c, 7j and 7o with
high potency for GSK-3β inhibition were also evaluated for kinase selectivity against PKC-epsilon,
JAK2, BRAF, IKK2 and Drak2. The results indicated that they displayed high selectivity for GSK-3β
over other tested kinases.

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2.2.2. Cellular Activity
It has been implicated that GSK-3β is involved in multiple cellular processes, and its ability to
hyperphosphorylate Tau protein 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 cellular activity of GSK-3β inhibitors [23]. Compounds 7c, 7f, 7j–l and 7o–q were
tested for the ability to reduce Tau phosphorylation at Ser 396 in human neuroblastoma SH-SY5Y
cells. LiCl, a well-known inhibitor of GSK-3β [24], was used as a positive control in this assay. As shown
in Figure 2, all selected compound significantly reduced Aβ25-35-induced Tau hyper-phosphorylation,
showing that these compounds can inhibit GSK-3β activity at the cellular level. The Aβ-induced Tau
hyperphosphorylation results in neurofibrillary tangle formation, which plays an important role in
Alzheimer’s disease. Our data suggests that these novel GSK-3β inhibitors may have potential actions
on inhibition of neurofibrillary tangle formation and would be tested for the treatment of Alzheimer’s
disease. In addition, the predictions about these compounds’ brain permeability were performed using
ADME module with Discovery Studio 2.1 software package. According to the prediction results (data
not shown), most of them exhibited moderate blood-brain barrier permeability and would be further
investigated for the treatment of Alzheimer’s disease.
Figure 2. Effects of GSK-3β inhibitors on Tau phosphorylation (ser396) in
SH-SY5Y cells.
2.2.3. Molecular Modeling
To examine possible binding modes of compounds bearing different side chains at N1-position of
the indole ring (e.g., 7j and 7n) with GSK-3β, a docking analysis utilizing the C-DOCKER program
within the Discovery Studio 2.1 software package was performed. The published X-ray crystal
structure of GSK-3β (PDB ID: 1Q3D) [14] was used for the docking calculation. Figure 3 shows that
both 7j and 7n could occupy the ATP binding site of GSK-3β with similar binding modes as a few
other ATP-competitive inhibitors of GSK-3β [14,25], and they could thus serve as ATP-competitive
inhibitors of GSK-3β. The NH and carbonyl group in maleimide ring of 7j and 7n could form two key
hydrogen bonds with Asp133 and Val135 of GSK-3β. Besides, the 3-position nitrogen atom of the
imidazole ring of 7j could form another hydrogen bond with Lys-183, which was not observed in 7n.
Furthermore, the CDOCKERENERAGE of 7j (−20.821 kcal/mol) was much lower than that of 7n

Molecules 2013, 18
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(2.971 kcal/mol). Thus, the molecular docking study results could explain the fact that 7j showed
significantly improved potency compared to that of 7n.
Figure 3. Docking of 7j to GSK-3β crystal structure. (A) Ribbon show of compound 7j
bound to GSK-3β; (B) Surface show of compound 7j dock into GSK-3β. Main text
paragraph; (C) Ribbon show of compound 7n bound to GSK-3β; (D) Surface show of
compound 7n dock into GSK-3β.
3. Experimental
3.1. General
Melting points were determined with a BÜCHI Melting Point B-450 apparatus (Büchi
1
Labortechnik, Flawil, Switzerland). The H-NMR spectra were recorded in DMSO-d6 or CDCl3 on
Bruker Avance DMX 500 at 500 MHz (chemical shifts are expressed as δ values relative to TMS as
internal standard). ESI spectra (positive ion mode) were recorded on an Esquire-LC-00075
spectrometer. Elemental analyses were performed on an Eager 300 instrument. All reactions were
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.

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3.2. General Procedure for the Preparation of 2a–f
Oxalyl chloride (3.40 g, 26 mmol) in Et2O (5 mL) was added dropwise to a solution of indole
adducts 1a–f (26 mmol) in Et2O (30 mL) at 0~5 °C [26,27]. The reaction mixture was stirred 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 °C~−20 °C. After addition, the mixture was stirred for 30 min at room temperature,
and poured into cold water (100 mL). The product was isolated with filtration, washed with
dichloromethane and dried to afford 2a–f.
Methyl 2-(1H-indol-3-yl)-2-oxoacetate (2a). Light yellow solid, 86.3% yield, mp: 208–210 °C.
1H-NMR (DMSO-d6): δ 12.48 (brs, 1H), 8.46 (d, J = 3.5 Hz, 1H), 8.16 (d, J = 7.0 Hz, 1H), 7.55 (d,
J = 7.0 Hz, 1H), 7.32–7.26 (m, 2H), 3.90 (s, 3H).
Methyl 2-(5-methoxy-1H-indol-3-yl)-2-oxoacetate (2b). Light yellow solid, 71.0% yield, mp: 221–223 °C.
1H-NMR (DMSO-d6): δ 12.34 (brs, 1H), 8.37 (d, J = 3.0 Hz, 1H), 7.66 (d, J = 2.0 Hz, 1H), 7.45 (d,
J = 9.0 Hz, 1H), 6.93 (dd, J = 9.0, 2.0 Hz, 1H), 3.89 (s, 3H), 3.81 (s, 3H).
Methyl 2-(5-bromo-1H-indol-3-yl)-2-oxoacetate (2c). Light yellow solid, 55.3% yield, mp: 215–217 °C.
1H-NMR (DMSO-d6) δ 12.59 (brs, 1H), 8.52 (d, J = 3.0 Hz, 1H), 8.30 (d, J = 2.0 Hz, 1H), 7.54 (d,
J = 8.5 Hz, 1H), 7.45 (dd, J =8.5, 2.0 Hz, 1H), 3.90 (s, 3H).
Methyl 2-(6-bromo-1H-indol-3-yl)-2-oxoacetate (2d). Light yellow solid, 58.9% yield, mp: 207–209 °C.
1H-NMR (DMSO-d6): δ 12.50 (brs, 1H), 8.50 (s, 1H), 8.10 (d, J = 8.5 Hz, 1H), 7.75 (d, J = 2.0 Hz,
1H), 7.43 (dd, J =8.5, 2.0 Hz, 1H), 3.90 (s, 3H).
Methyl 2-(6-chloro-1H-indol-3-yl)-2-oxoacetate (2e). Light yellow solid, 62.8% yield, mp: 246–248 °C.
1H-NMR (DMSO-d6): δ 12.52 (brs, 1H), 8.51 (d, J = 3.5 Hz, 1H), 8.15 (d, J = 8.5 Hz, 1H), 7.62 (d,
J = 2.0 Hz, 1H), 7.31 (dd, J =8.5, 2.0 Hz, 1H), 3.90 (s, 3H).
Methyl 2-(6-fluoro-1H-indol-3-yl)-2-oxoacetate (2f). Light yellow solid, 60.3% yield, mp: 182–184 °C.
1H-NMR (DMSO-d6): δ 12.48 (brs, 1H), 8.48 (s, 1H), 8.15 (dd, J = 8.5, 5.5 Hz, 1H), 7.36 (dd, J = 9.5,
2.0 Hz, 1H), 7.15 (td, J = 9.5, 2.0 Hz, 1H), 3.90 (s, 3H).
3.3. tert-Butyl 3-(2-methoxy-2-oxoacetyl)-1H-indole-1-carboxylate (3a)
A solution of 2a (2.0 g, 9.8 mmol), Boc anhydride (2.7 g, 12.3 mmol) and DMAP (0.01 g) in dry
THF (100 mL) was reacted for 3 h at room temperature. The solvent was removed under vacuum, and
the residue was recrystallized from petroleum ether/ethyl acetate to give 2.0 g (67.1%) 3a as a white
solid, mp: 132–133 °C. 1H-NMR (CDCl3) δ 8.81 (s, 1H), 8.40 (dd, J = 6.5, 2.0 Hz, 1H), 8.17 (dd, 1H,
J = 6.5, 1.5 Hz, 1H), 7.41–7.38 (m, 2H), 3.98 (s, 3H), 1.71 (s, 9H).
3.4. General Procedure for the Preparation of 3b–h, 3j–n and 3q
70% NaH (0.51 g, 14.8 mmol) was added portionwise to a solution of 2a–f (14.8 mmol) in DMF
(30 mL) at 0~5 °C. The reaction mixture was warmed to room temperature and stirred for 30 min.

Molecules 2013, 18
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After that, the appropriate R2X [4-(3-chloropropyl)morpholine, 4-(2-chloroethyl)morpholine, 1-(3-
chloropropyl)-1H-imidazole, 1-(4-chlorobutyl)-1H-imidazole, 1-(3-chloropropyl)-1H-1,2,4-triazole,
1-(3-chloropropyl)piperidine, 1-(3-chloropropyl)pyrrolidine or (3-bromopropoxy)(tert-butyl)dimethyl-
silane)] (19.2 mmol) was added. Then the mixture was headed to 55~60 °C and reacted for 12 h. After
cooling, the mixture was poured into cold water (300 mL) and extracted with ethyl acetate (3 × 100 mL).
The organic phase was combined, washed with brine (3 × 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 3b–h, 3j–n and 3q.
Methyl 2-(5-methoxy-1-(3-morpholinopropyl)-1H-indol-3-yl)-2-oxoacetate (3b). Light yellow solid,
64.7% yield, mp: 67–68 °C. 1H-NMR (CDCl3): δ 8.38 (s, 1H), 7.95 (d, J = 2.5 Hz, 1H), 7.32 (d, J = 9.0 Hz,
1H), 6.97 (dd, J = 9.0, 2.5 Hz, 1H), 4.28 (t, J =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 = 6.5 Hz, 2H), 2.06–2.01 (m, 2H). ESI-MS: m/z [M+H]+ 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-(5-bromo-1-(3-morpholinopropyl)-1H-indol-3-yl)-2-oxoacetate (3c). Light yellow solid,
1
56.6% yield, mp: 112–113 °C. H-NMR (CDCl3) δ 8.43 (s, 1H), 8.31 (d, J = 8.5 Hz, 1H), 7.67 (d,
J = 2.0 Hz, 1H), 7.45 (dd, J = 8.5, 2.0 Hz, 1H), 4.29 (t, J = 7.0 Hz, 2H), 3.95 (s, 3H), 3.81–3.75
(m, 4H), 2.43–2.39 (m, 4H), 2.25 (t, J = 6.5 Hz, 2H), 2.05-2.01 (m, 2H). ESI-MS: m/z [M+H]+ 409.
Anal. Calcd for C18H21BrN2O4: C, 52.82; H, 5.17; N, 6.84. Found: C, 52.96; H, 5.23; N, 6.76.
Methyl 2-(6-bromo-1-(3-morpholinopropyl)-1H-indol-3-yl)-2-oxoacetate (3d). Light yellow solid,
1
54.9% yield, mp: 102–103 °C. H-NMR (CDCl3): δ 8.43 (s, 1H), 8.31 (d, J = 8.5 Hz, 1H), 7.67 (d,
J = 1.5 Hz, 1H), 7.45 (dd, J = 8.5, 1.5 Hz, 1H), 4.29 (t, J = 6.5 Hz, 2H), 3.95 (s, 3H), 3.86–3.69
(m, 4H), 2.43–2.39 (m, 4H), 2.25 (t, J = 6.5 Hz, 2H), 2.05–2.01 (m, 2H). ESI-MS: m/z [M+H]+ 409.
Anal. Calcd for C18H21BrN2O4: C, 52.82; H, 5.17; N, 6.84. Found: C, 52.68; H, 5.26; N, 6.68.
Methyl 2-(6-chloro-1-(3-morpholinopropyl)-1H-indol-3-yl)-2-oxoacetate (3e). Light yellow solid,
1
59.1% yield, mp: 115–116 °C. H-NMR (CDCl3): δ 8.42 (s, 1H), 8.34 (d, J = 8.5 Hz, 1H), 7.49 (d,
J = 1.5 Hz, 1H), 7.30 (dd, J = 8.5, 1.5 Hz, 1H), 4.28 (t, J = 6.5 Hz, 2H), 3.94 (s, 3H), 3.80–3.70
(m, 4H), 2.43–2.37 (m, 4H), 2.24 (t, J = 6.5 Hz, 2H), 2.04–2.00 (m, 2H). ESI-MS: m/z [M+H]+ 365.
Anal. Calcd for C18H21ClN2O4: C, 59.26; H, 5.80; N, 7.68. Found: C, 59.44; H, 5.78; N, 7.45.
Methyl 2-(6-fluoro-1-(3-morpholinopropyl)-1H-indol-3-yl)-2-oxoacetate (3f). Light yellow solid,
1
64.8% yield, mp:116–117 °C. H-NMR (CDCl3): δ 8.44 (s, 1H), 8.38 (dd, J = 8.5, 5.5 Hz, 1H), 7.16
(dd, J = 9.0, 2.0 Hz, 1H), 7.13–7.06 (m, 1H), 4.27 (t, J = 6.5 Hz, 2H), 3.95 (s, 3H), 3.79–3.70 (m, 4H),
2.43–2.39 (m, 4H), 2.27 (t, J = 6.5 Hz, 2H), 2.06–2.00 (m, 2H). ESI-MS: m/z [M+H]+ 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-(2-morpholinoethyl)-1H-indol-3-yl)-2-oxoacetate (3g). Light yellow solid, 52.4% yield,
mp: 110–112 °C. 1H-NMR (CDCl3): δ 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 = 6.5 Hz, 2H), 3.97 (s, 3H), 3.75–3.69 (m, 4H), 2.81 (t, J = 6.5 Hz, 2H), 2.54–2.47

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(m, 4H). ESI-MS: m/z [M+H]+ 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-(1-(3-morpholinopropyl)-1H-indol-3-yl)-2-oxoacetate (3h). Light yellow solid, 67.5% yield,
mp: 103–104 °C. 1H-NMR (CDCl3): δ 8.47–8.43 (m, 2H), 7.46–7.41 (m, 1H), 7.38–7.32 (m, 2H), 4.32
(t, J = 6.5 Hz, 2H), 3.95 (s, 3H), 3.78–3.72 (m, 4H), 2.44–2.38 (m, 4H), 2.28 (t, J = 6.5 Hz, 2H),
2.08–2.02 (m, 2H). ESI-MS: m/z [M+H]+ 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-(1-(3-(1H-imidazol-1-yl)propyl)-1H-indol-3-yl)-2-oxoacetate (3j). Light yellow solid, 52.8%
yield, mp: 72–73 °C. 1H-NMR (CDCl3) δ 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 = 7.0 Hz, 2H), 4.00 (t, J = 7.0 Hz, 2H),
3.98 (s, 3H), 2.48–2.40 (m, 2H). ESI-MS: m/z [M+H]+ 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-(1-(4-(1H-imidazol-1-yl)butyl)-1H-indol-3-yl)-2-oxoacetate (3k). Light yellow solid, 41.6%
1
yield, H-NMR (CDCl3) δ 8.50–8.42 (m, 1H), 8.35 (s, 1H), 7.44 (s, 1H), 7.38–7.32 (m, 3H), 7.06 (s,
1H), 6.85 (s, 1H), 4.19 (t, J = 7.0 Hz, 2H), 3.96 (s, 3H), 3.93 (t, J = 7.0 Hz, 2H), 1.95–1.86
(m, 2H), 1.87–1.77 (m, 2H). ESI-MS: m/z [M+H]+ 326. Anal. Calcd for C18H19N3O3: C, 66.45; H,
5.89; N, 12.91. Found: C, 66.62; H, 5.77; N, 12.85.
Methyl 2-(1-(3-(1H-1,2,4-triazol-1-yl)propyl)-1H-indol-3-yl)-2-oxoacetate (3l). Light yellow solid,
64.5% yield, mp: 98–99 °C. 1H-NMR (CDCl3) δ 8.49–8.45 (m, 1H), 8.44 (s, 1H), 8.08 (s, 1H), 8.05 (s,
1H), 7.40–7.36 (m, 2H), 7.35–7.32 (m, 1H), 4.31 (t, J = 7.0 Hz, 2H), 4.18 (t, J = 7.0 Hz, 2H), 3.97 (s,
3H), 2.59–2.49 (m, 2H). ESI-MS: m/z [M+H]+ 313. Anal. Calcd for C16H16N4O3: C, 61.53; H, 5.16; N,
17.94. Found: C, 61.69; H, 5.31; N, 17.99.
Methyl 2-oxo-2-(1-(3-(piperidin-1-yl)propyl)-1H-indol-3-yl)acetate (3m). Light yellow solid, 52.4%
yield, mp: 66–67 °C. 1H-NMR (DMSO-d6) δ 8.47 (s, 1H), 8.20 (d, J = 7.5 Hz, 1H), 7.68 (d, J = 7.5 Hz,
1H), 7.39–7.27 (m, 2H), 4.34 (t, J = 7.0 Hz, 2H), 3.90 (s, 3H), 2.29–2.18 (m, 4H), 2.13 (t, J = 6.5 Hz,
2H), 1.96–1.90 (m, 2H), 1.55–1.41 (m, 4H), 1.40–1.30 (m, 2H). ESI-MS: m/z [M+H]+ 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-oxo-2-(1-(3-(pyrrolidin-1-yl)propyl)-1H-indol-3-yl)acetate (3n). Light yellow solid, 48.5%
yield, mp: 56–58 °C. 1H-NMR (DMSO-d6) δ 8.50 (s, 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.0 Hz,
1H), 7.39–7.27 (m, 2H), 4.37 (t, J = 7.0 Hz, 2H), 3.90 (s, 3H), 2.42–2.39 (m, 4H), 2.35 (t, J = 6.5 Hz,
2H), 2.00–1.92 (m, 2H), 1.72–1.67 (m, 4H). ESI-MS: m/z [M+H]+ 311. Anal. Calcd for C18H18N2O3:
C, 69.66; H, 5.85; N, 9.03. Found: C, 69.78; H,5.96; N, 9.22.
Methyl 2-(1-(3-((tert-butyldimethylsilyl)oxy)propyl)-1H-indol-3-yl)-2-oxoacetate (3q). Light yellow
solid, 62.5% yield, mp: 69–71 °C. 1H-NMR (CDCl3): δ 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 = 7.0 Hz, 2H), 3.88 (s, 3H), 3.52 (t, J = 7.0 Hz, 2H), 2.06–1.92 (m,
2H), 0.87 (s, 9H), 0.01 (s, 6H). ESI-MS: m/z [M+H]+ 349. Anal. Calcd for C20H29NO4Si: C, 63.97; H,
7.78; N, 3.73. Found: C, 64.05; H, 7.86; N, 3.65.

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3.5. 1-(4-Bromobutyl)-1H-indole (4)
70% NaH (1.77 g, 51.6 mmol) was added portionwise to a solution of indole (5.0 g, 43.0 mmol) in
DMF (50 mL) at 0~5 °C. 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 (46.4 g, 215 mmol) in DMF (10 mL) at
room temperature, and stirred for 12 h. The mixture was then poured into cold water (300 mL) and
extracted with ethyl acetate (3 × 100 mL). The organic phase was combined, washed with brine
(3 × 300 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel using petroleum ether/ethyl acetate (80:1, v/v) as eluent to afford 7.6 g
1
(71.2%) of 4 as a colorless liquid. H-NMR (CDCl3): δ 7.66 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 8.0 Hz,
1H), 7.25–7.22 (m, 1H), 7.14–7.11 (m, 2H), 6.53 (d, J = 3.0 Hz, 1H), 4.19 (t, J = 7.0 Hz, 2H), 3.40
(t, J = 6.5 Hz, 2H), 2.07–2.00 (m, 2H), 1.90–1.85 (m, 2H).
3.6. 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 50 °C for 6 h[28–40]. After cooling, the mixture was
poured into cold water (200 mL) and extracted with ethyl acetate (3 × 100 mL). The organic phase was
combined, washed with brine (3 × 300 mL), dried over Na2SO4 and concentrated in vacuum. The
residue was purified by flash column chromatography on silica gel using petroleum ether/ethyl
acetate/triethylamine (20:100:1, v/v/v) as eluent to afford 1.63 g (80.1%) of 5 as a colorless liquid,
1H-NMR (CDCl3): δ 7.65 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.24–7.21 (m, 1H), 7.14–7.11
(m, 2H), 6.51 (d, J = 3.0 Hz, 1H), 4.17 (t, J = 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 [M+H]+ 259. Anal. Calcd for C16H22N2O: C,
74.38; H, 8.58; N, 10.84. Found: C, 74.53; H, 8.64; N, 10.93.
3.7. Methyl 2-(1-(4-morpholinobutyl)-1H-indol-3-yl)-2-oxoacetate (3i)
1M HCl in dioxane (5.5 mL, 5.5 mmol) was added to a solution of 5 (1.29 g, 5.0 mmol) in CH3CN
(30 mL) and Et2O (30 mL) 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 0~5 °C and the reaction mixture was stirred for 2 h
at the same temperature. MeOH (10 mL) was added dropwise to the mixture at 0~5 °C 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 × 50 mL). The organic phase was combined, washed with brine
(150 mL), dried over Na2SO4 and concentrated under 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
1
(54.6%) of 3i as a light yellow solid, mp: 84–85 °C. H-NMR (CDCl3): δ 8.48–8.44 (m, 1H), 8.40 (s,
1H), 7.44–7.38 (m, 1H), 7.36–7.34 (m, 2H), 4.23 (t, J = 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 [M+H]+ 345. Anal. Calcd for
C19H24N2O4: C, 66.26; H, 7.02; N, 8.13. Found: C, 66.40; H, 7.11; N, 8.32.

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3.8. General Procedure for the Preparation of 3o and 3p
70% 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 0~5 °C. The reaction mixture was warmed to room temperature and stirred for 30 min.
After that, iodomethane or 1-bromobutane (5.9 mmol) was added at 0 °C and stirred for 1 h at room
temperature. The mixture was then poured into cold water (100 mL) and extracted with ethyl acetate
(3 × 50 mL). The organic phase was combined, washed with brine (3 × 150 mL), dried over Na2SO4
and concentrated 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 3o and 3p.
Methyl 2-(1-methyl-1H-indol-3-yl)-2-oxoacetate (3o). Light yellow solid, 76.8% yield, mp: 73–74 °C.
1H-NMR (CDCl3) δ 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 [M+H]+ 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
Methyl 2-(1-butyl-1H-indol-3-yl)-2-oxoacetate (3p). White solid, 80.5% yield, mp: 81–82 °C. H-NMR
(CDCl3) δ 8.48–8.44 (m, 1H), 8.39 (s, 1H), 7.45–7.38 (m, 1H), 7.37–7.32 (m, 2H), 4.20 (t, J = 7.5 Hz,
2H), 3.96 (s, 3H), 1.86–1.82 (m, 2H), 1.42–1.38 (m, 2H), 0.98 (t, J = 7.5 Hz, 3H). ESI-MS: m/z
[M+H]+ 260. Anal. Calcd for C15H17NO3: C, 69.48; H, 6.61; N, 5.40. Found: C, 69.62; H,6.52; N, 5.65.
3.9. General Procedure for the Preparation of 7a–q
A solution of t-BuOK (94.0 mg, 0.84 mmol) in THF (5mL) was added dropwise to a solution of 3a–q
(0.36 mmol) and 2-(benzo[d]isoxazol-3-yl)acetamide 6 (0.28 mmol) in dry THF (10 mL) at −10~0 °C.
After stirring for 2 h at room temperature, concentrated hydrochloric acid (5 mL) 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 × 50 mL). The organic phase was combined and
washed with brine (3 × 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 7a–q.
3-(Benzo[d]isoxazol-3-yl)-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione (7a). Red solid, 44.4% yield, mp:
227–228 °C. 1H-NMR (DMSO-d6) δ 12.14 (brs,1H), 11.44 (brs, 1H), 8.23 (s, 1H), 7.83 (d, J = 8.5 Hz,
1H), 7.69–7.58 (m, 2H), 7.44 (d, J = 8.0 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.08–7.04 (m, 1H), 6.72 (t,
J = 8.0 Hz, 1H), 6.51 (d, J = 8.5 Hz, 1H). ESI-MS: m/z [M+H]+ 330. Anal. Calcd for C19H11N3O3: C,
69.30; H, 3.37; N, 12.76. Found: C, 69.06; H, 3.45; N, 12.64
3-(Benzo[d]isoxazol-3-yl)-4-(5-methoxy-1-(3-morpholinopropyl)-1H-indol-3-yl)-1H-pyrrole-2,5-dione
(7b). Saffron yellow solid, 11.6% yield, mp: 191–193 °C. 1H-NMR (CDCl3) δ 8.27 (s, 1H), 7.97 (brs,
1H), 7.66–7.62 (m, 2H), 7.57 (td, J = 8.0, 1.5 Hz, 1H), 7.25–7.23 (m, 2H), 6.76 (dd, J = 9.0, 2.5 Hz,
1H), 5.94 (d, J = 2.5 Hz, 1H), 4.29 (t, J = 6.5 Hz, 2H), 3.77 (t, J = 4.5 Hz, 4H), 3.11 (s, 3H), 2.48–2.42
(m, 4H), 2.32 (t, J = 6.5 Hz, 2H), 2.06–2.01(m, 2H). ESI-MS: m/z [M+H]+ 487. Anal. Calcd for
C27H26N4O5: C, 66.65; H, 5.39; N, 11.52. Found: C, 66.76; H, 5.47; N, 11.62.

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3-(Benzo[d]isoxazol-3-yl)-4-(5-bromo-1-(3-morpholinopropyl)-1H-indol-3-yl)-1H-pyrrole-2,5-dione
(7c). Saffron yellow solid, 21.3% yield, mp: 180–182 °C. 1H-NMR (CDCl3) δ 8.32 (s, 1H), 7.97 (brs,
1H), 7.68 (t, J = 8.5 Hz, 2H), 7.61 (td, J = 8.5, 1.5 Hz, 1H), 7.32 (t, J = 8.0 Hz, 1H), 7.26–7.25 (m,
2H), 6.81 (s, 1H), 4.29 (t, J = 7.0 Hz, 2H), 3.75 (t, J = 4.5 Hz , 4H), 2.42–2.39 (m, 4H), 2.30 (t, J = 6.6 Hz,
2H), 2.03–2.01 (m, 2H). ESI-MS: m/z [M+H]+ 535. Anal. Calcd for C26H23N4O4Br: C, 58.33; H, 4.33;
N, 10.46. Found: C, 58.21; H, 4.49; N, 10.53.
3-(Benzo[d]isoxazol-3-yl)-4-(6-bromo-1-(3-morpholinopropyl)-1H-indol-3-yl)-1H-pyrrole-2,5-dione
(7d). Saffron yellow solid, 12.7% yield, mp: 226–228 °C. 1H-NMR (CDCl3) δ 8.27 (s, 1H), 7.97 (brs,
1H), 7.68–7.64 (m, 2H), 7.62 (d, J = 2.0 Hz, 1H), 7.59 (td, J = 7.0, 1.0 Hz, 2H), 7.29 (t, J = 8.0 Hz,
1H), 6.99 (dd, J = 8.0, 2.0Hz, 1H), 6.59 (d, J = 8.0 Hz, 1H), 4.29 (t, J = 6.5 Hz, 2H), 3.79 (t, J = 4.5 Hz,
4H), 2.47–2.40 (m, 4H), 2.29 (t, J = 6.5Hz, 2H), 2.05–2.02 (m, 2H). ESI-MS: m/z [M+H]+ 535. Anal.
Calcd for C26H23N4O4Br: C, 58.33; H, 4.33; N, 10.46. Found: C, 58.39; H, 4.51; N, 10.42.
3-(Benzo[d]isoxazol-3-yl)-4-(6-chloro-1-(3-morpholinopropyl)-1H-indol-3-yl)-1H-pyrrole-2,5-dione
(7e). Red solid, 11.5% yield, mp: 216–218 °C. 1H-NMR (CDCl3) δ 8.29 (s, 1H), 7.97 (brs, 1H), 7.65 (t,
J = 7.5 Hz, 2H), 7.59 (t, J = 8.0 Hz, 1H), 7.45 (d, J = 1.5 Hz, 1H), 7.28 (t, J = 7.5 Hz, 1H), 6.85 (dd,
J = 8.5, 1.5 Hz, 1H), 6.64 (d, J = 8.5 Hz, 1H), 4.29 (t, J = 6.5 Hz, 2H), 3.78 (t, J = 4.5 Hz, 4H),
2.45–2.41 (m, 4H), 2.29 (t, J = 6.5 Hz, 2H), 2.06–2.02 (m, 2H). ESI-MS: m/z [M+H]+ 491. Anal.
Calcd for C26H23N4O4Cl: C, 63.61; H, 4.72; N, 11.41. Found: C, 63.52; H, 4.64; N, 11.51.
3-(Benzo[d]isoxazol-3-yl)-4-(6-fluoro-1-(3-morpholinopropyl)-1H-indol-3-yl)-1H-pyrrole-2,5-dione
1
(7f). Yellow solid, 29.9% yield, mp: 219–221 °C. H-NMR (CDCl3) δ 8.29 (s, 1H), 8.06 (brs, 1H),
7.66–7.66 (m, 2H), 7.58 (t, J = 7.5 Hz, 1H), 7.29 (t, J = 7.5 Hz, 1H), 7.11 (d, J = 9.5 Hz, 1H),
6.68–6.62 (m, 2H), 4.27 (t, J = 6.5 Hz, 2H), 3.77 (t, J = 4.5 Hz, 4H), 2.46–2.41 (m, 4H), 2.31 (t,
J = 6.5 Hz, 2H), 2.06–2.03 (m, 2H). ESI-MS: m/z [M+H]+ 475. Anal. Calcd for C26H23N4O4F: C,
65.81; H, 4.89; N, 11.81. Found: C, 65.99; H, 4.75; N, 11.87.
3-(Benzo[d]isoxazol-3-yl)-4-(1-(2-morpholinoethyl)-1H-indol-3-yl)-1H-pyrrole-2,5-dione (7g). Yellow
solid, 24.0% yield, mp: 205–207 °C. 1H-NMR (CDCl3) δ 8.39 (s, 1H), 7.79 (brs, 1H), 7.57 (t, J = 7.5 Hz,
2H), 7.35 (d, J = 8.0 Hz, 1H), 7.25 (t, J = 7.5 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 6.90 (t, J = 7.5 Hz,
1H), 6.73 (d, J = 8.0 Hz, 1H), 4.31 (t, J = 6.5Hz, 2H), 3.73 (t, J = 7.5 Hz , 4H), 2.82 (t, J = 6.5 Hz,
2H), 2.56–2.42 (m, 4H). ESI-MS: m/z [M+H]+ 443. Anal. C25H22N4O4: C, 67.86; H, 5.01; N, 12.66.
Found: C, 67.96; H, 5.11; N, 12.85.
3-(Benzo[d]isoxazol-3-yl)-4-(1-(3-morpholinopropyl)-1H-indol-3-yl)-1H-pyrrole-2,5-dione (7h). Red
1
solid, 38.1% yield, mp: 197–198 °C. H-NMR (CDCl3) δ 8.89 (brs, 1H), 8.33 (s, 1H), 7.66–7.60 (m,
2H), 7.55 (td, J = 7.0, 1.0 Hz 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.24 (t, J = 7.5 Hz, 1H), 7.16 (t, J = 7.5 Hz,
1H), 6.86 (t, J = 7.5 Hz, 1H), 6.67 (d, J = 8.0 Hz, 1H), 4.32 (t, J = 6.5 Hz, 2H), 3.79 (t, J = 4.5 Hz,
4H), 2.52–2.42 (m, 4H), 2.37 (t, J = 7.0 Hz, 2H), 2.13–2.05 (m, 2H). ESI-MS: m/z [M+H]+ 457. Anal.
C26H24N4O4: C, 68.41; H, 5.30; N, 12.27. Found: C, 68.69; H, 5.25; N, 12.41.

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3-(Benzo[d]isoxazol-3-yl)-4-(1-(4-morpholinobutyl)-1H-indol-3-yl)-1H-pyrrole-2,5-dione (7i). Red
solid, 22.6% yield, mp: 89–91 °C. 1H-NMR (CDCl3) δ 8.28 (s, 1H), 8.25(brs, 1H), 7.66–7.62 (m, 2H),
7.56 (t, J = 7.5 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.24 (t, J = 8.0 Hz, 1H), 7.18 (t, J = 7.0 Hz, 1H), 6.88
(t, J = 7.5 Hz, 1H), 6.72 (d, J = 8.0 Hz, 1H), 4.23 (t, J = 6.5 Hz, 2H), 3.71 (t, J = 4.5 Hz, 4H), 2.45–2.35
(m, 6H), 2.01–1.90 (m, 2H), 1.62–1.55 (m, 2H). ESI-MS: m/z [M+H]+ 471. Anal. C27H26N4O4: C,
68.92; H, 5.57; N, 11.91. Found: C, 68.75; H, 5.62; N, 11.82.
3-(1-(3-(1H-Imidazol-1-yl)propyl)-1H-indol-3-yl)-4-(benzo[d]isoxazol-3-yl)-1H-pyrrole-2,5-dione
(7j).
1
Red solid, 16.1% yield, mp: 208–210 °C. H-NMR (CDCl3) δ 8.32 (brs, 1H), 8.24 (s, 1H), 7.67 (d,
J = 8.5 Hz,1H), 7.85 (d, J = 8.5 Hz,1H), 7.61–7.53 (m, 2H), 7.29 (t, J = 7.0 Hz, 1H), 7.24–7.18 (m,
2H), 7.17 (s, 1H), 6.98 (s, 1H), 6.94 (td, J = 7.0, 1.0 Hz, 1H), 6.81 (d, J = 8.0 Hz, 1H), 4.20 (t, J = 6.5 Hz,
2H), 3.99 (t, J = 6.5 Hz, 2H), 2.47–2.40 (m, 2H). ESI-MS: m/z [M+H]+ 438. Anal. C25H19N5O3: C,
68.64; H, 4.38; N, 16.01. Found: C, 68.81; H, 4.35; N, 16.26.
3-(1-(4-(1H-Imidazol-1-yl)butyl)-1H-indol-3-yl)-4-(benzo[d]isoxazol-3-yl)-1H- pyrrole-2,5-dione (7k).
1
Red solid, 15.6% yield, mp: 203–205 °C. H-NMR (CDCl3) δ 8.35 (brs, 1H), 8.24 (s, 1H), 7.69–7.63
(m, 2H), 7.57 (td, J = 7.0, 1.5Hz, 1H), 7.47 (s, 1H), 7.29 (d, J = 6.0 Hz, 1H), 7.25 (d, J = 7.5 Hz, 1H),
7.19 (t, J = 7.5 Hz, 1H), 7.08 (s, 1H), 6.92 (t, J = 7.5 Hz, 1H), 6.87 (s, 1H), 6.78(d, J = 8.0 Hz, 1H),
4.21 (t, J = 6.5 Hz, 2H), 3.92 (t, J = 6.5 Hz, 2H), 1.95–1.87 (m, 2H), 1.86–1.80 (m, 2H). ESI-MS: m/z
[M+H]+ 452. Anal. C26H21N5O3: C, 69.17; H, 4.69; N, 15.51. Found: C, 68.96; H, 4.65; N, 15.73.
3-(1-(3-(1H-1,2,4-Triazol-1-yl)propyl)-1H-indol-3-yl)-4-(benzo[d]isoxazol-3-yl)-1H-pyrrole-2,5-dione
1
(7l). Red solid, 21.3% yield, mp: 214–216 °C. H-NMR (DMSO-d6) δ 11.46 (brs, 1H), 8.54 (s, 1H),
8.28 (s, 1H), 8.01 (s, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.66-7.61(m, 2H), 7.52 (d, J = 8.0 Hz, 1H), 7.29 (t,
J = 7.5 Hz, 1H), 7.13 (t, J = 7.5 Hz, 1H), 6.79 (t, J = 8.0 Hz, 1H), 6.57 (d, J = 8.0 Hz, 1H), 4.36 (t,
J = 6.5 Hz, 2H), 4.20 (t, J = 6.5 Hz, 2H), 2.36–2.28 (m, 2H). ESI-MS: m/z [M+H]+ 439. Anal.
C24H18N6O3: C, 65.75; H, 4.14; N, 19.17. Found: C, 65.58; H, 4.12; N, 19.34.
3-(Benzo[d]isoxazol-3-yl)-4-(1-(3-(piperidin-1-yl)propyl)-1H-indol-3-yl)-1H-pyrrole-2,5-dione (7m).
Red solid, 15.6% yield, mp: 73–75 °C. 1H-NMR (CDCl3) δ 8.32 (s, 1H), 7.64 (d, J = 8.5 Hz, 1H), 7.59
(d, J = 8.0 Hz, 1H), 7.55 (td, J = 8.0, 1.0 Hz, 1H), 7.38 (d, J = 8.5 Hz, 1H), 7.23 (t, J = 8.0 Hz, 1H),
7.16 (t, J = 8.0 Hz, 1H), 6.85 (t, J = 8.0 Hz, 1H), 6.67 (d, J = 8.0 Hz, 1H), 4.31 (t, J = 7.0 Hz, 2H),
2.55–2.35 (m, 6H), 2.17–2.10 (m, 2H), 1.73–1.61 (m, 4H), 1.52–1.47 (m, 2H). ESI-MS: m/z [M+H]+
455. Anal. C27H26N4O3: C, 71.35; H, 5.77; N, 12.33. Found: C, 71.13; H, 5.63; N, 12.46.
3-(Benzo[d]isoxazol-3-yl)-4-(1-(3-(pyrrolidin-1-yl)propyl)-1H-indol-3-yl)-1H-pyrrole-2,5-dione (7n).
Saffron yellow solid, 20.8% yield, mp: 171–173 °C. 1H-NMR (CDCl3) δ 8.41 (s, 1H), 7.63 (d, J = 8.5,
1H), 7.59 (d, J = 8.5, 1H), 7.54 (td, J = 8.5, 1H, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.22 (t, J = 7.5 Hz, 1H),
7.15 (t, J = 7.5 Hz, 1H), 6.83 (t, J = 7.5 Hz, 1H), 6.62 (d, J = 8.5 Hz, 1H), 4.32 (t, J = 7.0 Hz, 2H),
2.65–2.55 (m, 6H), 2.25–2.15 (m, 2H), 1.89–1.83 (m, 4H). ESI-MS: m/z [M+H]+ 441. Anal.
C26H24N4O3: C, 70.89; H, 5.49; N, 12.72. Found: C, 70.68; H, 5.34; N, 12.97.

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3-(Benzo[d]isoxazol-3-yl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (7o). Red solid, 27.8%
yield, mp: 221–222 °C. 1H-NMR (CDCl3) δ 8.24 (s, 1H), 7.81 (brs, 1H), 7.65 (d, J = 9.5 Hz, 2H), 7.57
(t, J = 8.0 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H), 6.97 (t, J = 8.0 Hz, 1H), 6.88 (t,
J = 7.5 Hz, 1H), 6.65 (d, J = 8.5 Hz, 1H), 3.90 (s, 3H). ESI-MS: m/z [M+H]+ 344. Anal. C20H13N3O3:
C, 69.96; H, 3.82; N, 12.24. Found: C, 70.11; H, 3.77; N, 12.05.
3-(Benzo[d]isoxazol-3-yl)-4-(1-butyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione(7p). Saffron yellow solid,
1
35.2% yield, mp: 180–182 °C. H-NMR (CDCl3) δ 8.25 (s, 1H), 7.82 (brs,1H), 7.65 (d, J = 8.5, 1H),
7.61 (d, J =8.5, 1H), 7.56 (t, J = 8.0 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.24 (t, J = 7.5 Hz, 1H), 7.18
(t, J = 7.5 Hz, 1H), 6.89 (t, J = 7.5 Hz, 1H), 6.75 (d, J =7.5 Hz, 1H), 4.22 (t, J = 7.5, 2H), 1.95–1.83
(m, 2H), 1.42–1.37 (m, 2H), 0.98 (t, J = 7.5 Hz, 3H). ESI-MS: m/z [M+H]+ 386. Anal. C23H19N3O3:
C, 71.67; H, 4.97; N, 10.90. Found: C, 71.55; H, 4.84; N, 11.03.
3-(Benzo[d]isoxazol-3-yl)-4-(1-(3-hydroxypropyl)-1H-indol-3-yl)-1H-pyrrole-2,5-dione (7q). Yellow
1
solid, 10.0% yield, mp: 219–220 °C. H-NMR (CDCl3) δ 8.29 (s, 1H), 7.64 (t, J = 8.0 Hz, 2H), 7.57
(t, J = 7.5 Hz, 1H), 7.40 (d, J = 8.5 Hz, 1H), 7.24 (t, J = 8.0 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H), 6.91
(t, J = 7.5 Hz, 1H), 6.79 (d, J = 7.5 Hz, 1H), 4.39 (t, J =7.0 Hz, 2H), 3.67 (t, J = 7.0 Hz, 2H), 2.17–2.10
(m, 2H). ESI-MS: m/z [M+H]+ 388. Anal. C22H17N3O4: C, 68.21; H, 4.42; N, 10.85. Found: C, 68.07;
H, 4.51; N, 11.09.
3.10. Biological Activity Assay
3.10.1. Kinase Assay
Human kinome, consisting of 518 kinases, is classified into CMGC, AGC, TK, TKL, CAMK, STE
and other 7 subfamilies, according to the DNA sequence and evolution. GSK3β belongs to CMGC
family. Here, the other family members were used to evaluate the selectivity of GSK3β inhibitors,
including PKC-epsilon (AGC family), JAK2 (TK family), Braf (TKL family), DRAK2 (CAMK
family) and IKKβ (other family).The recombinant GST-GSK-3βprotein was expressed in Escherichia
coli strain BL21-Codon Plus (DE3), purified by GSTrap affinity chromatography, and cleaved by
thrombin. The GSK-3β kinase assay was carried out with the Z´-LYTETM Kinase Assay kit Ser/Thr 9
Peptide substrate (Invitrogen, Grand, NY, USA) in 10 μL reaction volume containing 50 nM enzyme,
30 μM ATP and 2 μM substrate peptide. Drak2 Proteins were presented by professor Jiang-ping Wu
(Laboratory of Immunology, Research Centre, CHUM, Notre Dame Hospital, Pavilion DeSève). The
Drak2 kinase reaction was performed in a final assay volume of 3.4 μL using the ADP-GLO Kinase
Assay Kit (Promega, Madison, WI, USA), according the ADP-GLO protocol and read on an EnVision
plate reader. The recombinant PKC-epsilon, IKK, and JAK2 with N-terminal His-tag were expressed
using baculovirus expression system and purified with Ni-Beads. BRAF protein was from Carna
Biosciences, Inc. (Kobe Port Island, Japan). And the related kinase reactions were performed in a final
assay volume of 10 μL using the related HTRF Assay Kit (Cisbio, Codolet, France). Reactions were
according the HTRF protocol and read on an EnVision plate reader. All reactions were carried out in
triplicate. IC50 values (concentration at which a 50% of enzyme inhibition is shown) were derived from
a nonlinear regression model (curvefit) based on sigmoidal dose response curve (variable slope) and

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computed using Graphpad Prism version 5.02, (Graphpad Software, La Jolla, CA, USA). Data were
expressed as mean ± SE.
3.10.2. Cell Culture and Western Blotting
SH-SY5Y human neuroblastoma cells were obtained from the American Type Culture Collection
(ATCC, Manassas, VA, USA). Cells were cultured in 1:1 DMEM:Ham's F12 containing 10% (v/v)
fetal bovine serum (HyClone, Logan, UT, USA), 1% penicillin, and 1% streptomycin at a humidified
atmosphere with 5% CO2. The medium was changed every 2 days. For experiments, cells were and
grown in 12-well plates until ~80% confluence, serum-deprived for 12 hours, incubated with GSK-3β
inhibitors for 1 hour and Aβ25-35 (amyloid beta peptide 25–35, Sigma, St. Louis, MO, USA) for another
6 hours. Cells were rinsed twice with ice-cold PBS and lysed with 1 × SDS loading buffer. Samples
were electrophoresed on 10% SDS-polyacrylamide gels, and transferred onto PVDF membranes. The
membranes were blocked for 1 h with 5% (w/v) milk, incubated with rabbit anti-Tau [pS396]
phosopho-specific antibody (Abcam, Cambridge, UK) for 2 h and the anti-rabbit secondary antibody
for 1 h. Antigen-antibody complexes were detected by the ECL Kit.
4. Conclusions
In conclusion, a series of novel 3-benzisoxazolyl-4-indolyl-maleimides were synthesized and
identified as potent and selective GSK-3β inhibitors. Among them, compound 7j was the most
promising GSK-3β inhibitor, with an IC50 of 0.73 nM, which was about 140-fold more potent than
staurosporine. Futher cell-based functional assays showed that all selected compounds such as 7c, 7f,
7j–l and 7o–q could obviously reduce Tau phosphorylation at 100 nM by inhibiting GSK-3β.
Preliminary SAR and molecular modeling studies provided further insights into interactions between
GSK-3β and its inhibitors. The results are useful for the design of novel and selective GSK-3β
inhibitors. Future progress on related series will be reported in due course.
Acknowledgments
We gratefully acknowledge the financial support from the Natural Science Foundation of China
(21176223) and the National Natural Science Foundation of Zhejiang (Y407306).
Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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