Design, synthesis and biological evaluation of N-alkyl or aryl substituted isoindigo derivatives as potential dual cyclin-dependent kinase 2 (CDK2)/glycogen synthase kinase 3β (GSK-3β) phosphorylation inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.08.049

A series of N-alkyl or aryl substituted isoindigo derivatives have been synthesized and their anti-proliferative activity was evaluated by Sulforhodamine B (SRB) assay. Some of the target compounds exhibited significant antitumor activity, including compounds 6h and 6k (against K562 cells), 6i (against HeLa cells) and 6j (against A549 cells). N-(p-methoxy-phenyl)-isoindigo (6k) exhibited a high and selective anti-proliferative activity against K562 cells (IC₅₀ 7.8 μM) and induced the apoptosis of K562 cells in a dose-dependent manner. Compound 6k arrested the cell cycle at S phase in K562 cells by decreasing the expression of cyclin A and CDK2, which played critical roles in DNA replication and passage through G2 phase. Moreover, compound 6k down-regulated the expression of p-GSK-3β (Ser9), β-catenin and c-myc proteins, up-regulated the expression of GSK-3β, consequently, suppressed Wnt/β-catenin signaling pathway and induced the apoptosis of K562 cells. The binding mode of compound 6k with GSK-3β was simulated using molecular docking tools. All of these studies gave a better understanding to the molecular mechanisms of this class of agents and clues to develop dual CDK2/GSK-3β (Ser9) phosphorylation inhibitors applied in cancer chemotherapy.

Full text of DOI:10.1016/j.ejmech.2014.08.049

European Journal of Medicinal Chemistry 86 (2014) 165e174
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of N-alkyl or aryl
substituted isoindigo derivatives as potential dual cyclin-dependent
kinase 2 (CDK2)/glycogen synthase kinase 3
phosphorylation inhibitors
b (GSK-3b)
Ping Zhao a, Yanzhong Li a, Guangwei Gao a, Shuai Wang a, Yun Yan a, Xiaoping Zhan a,
,
Zenglu Liu a, Zhenmin Mao a *, Shaoxiong Chen b, Liqun Wang b
a School of Pharmacy, Shanghai Jiaotong University, No. 800, Dongchuan Road, Shanghai 200240, China
b Strategic Alliance of Biomedical Technology, No. 333, Cailun Road, Shanghai 201210, 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 N-alkyl or aryl substituted isoindigo derivatives have been synthesized and their anti-
proliferative activity was evaluated by Sulforhodamine B (SRB) assay. Some of the target compounds
exhibited significant antitumor activity, including compounds 6h and 6k (against K562 cells), 6i (against
HeLa cells) and 6j (against A549 cells). N-(p-methoxy-phenyl)-isoindigo (6k) exhibited a high and se-
lective anti-proliferative activity against K562 cells (IC50 7.8 mM) and induced the apoptosis of K562 cells
in a dose-dependent manner. Compound 6k arrested the cell cycle at S phase in K562 cells by decreasing
Received 3 May 2014
Received in revised form
9 August 2014
Accepted 14 August 2014
Available online 15 August 2014
the expression of cyclin A and CDK2, which played critical roles in DNA replication and passage through
Keywords:
G2 phase. Moreover, compound 6k down-regulated the expression of p-GSK-3
myc proteins, up-regulated the expression of GSK-3 , consequently, suppressed Wnt/
pathway and induced the apoptosis of K562 cells. The binding mode of compound 6k with GSK-3
simulated using molecular docking tools. All of these studies gave a better understanding to the mo-
b
(Ser9),
-catenin signaling
was
b-catenin and c-
Isoindigo derivative
Antitumor
Mechanism
Apoptosis
b
b
b
CDKs
GSK-3
lecular mechanisms of this class of agents and clues to develop dual CDK2/GSK-3
lation inhibitors applied in cancer chemotherapy.
b (Ser9) phosphory-
b
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
exhibited a higher solubility, antitumor activity and fewer adverse
side effects than indirubin. Meisoindigo has replaced indirubin and
become a crucial chemotherapy drug in treatment of CML.
It is well known that the occurrence and development of cancer
is a multi-factor and step involved process. The clinical researches
also verify that single-target drugs such as Imatinib, are more likely
to produce drug-resistance when applied for a long term, and the
curative effects are also less evident [6]. Therefore, to develop drugs
bearing multi-target inhibitory activity and drugs which is unlikely
to produce drug-resistance has become an important direction in
antitumor agent development.
Indigo, indirubin and isoindigo (Fig. 1), containing a bis-indole
framework, have been found in a number of natural products
such as indigo-producing plants, several species of Gastropod
molluscs, urine of healthy and diseased human, and various bac-
teria [1]. Indirubin have been recognized as the main active
ingredient of a traditional Chinese medicinal recipe, Danggui Luhui
Wan, and was used to treat chronic myelocytic leukemia (CML) in
clinic [2]. However, indirubin could cause severe gastrointestinal
side effects due to the poor water solubility. Moreover, its long term
use may lead to bone marrow suppression and drug-resistance [3].
To address the solubility problem of this promising compound, a
series of derivatives have been designed and synthesized by sci-
entists [4,5]. Among them, meisoindigo (1-methyl-isoindigo, Fig. 1)
Cell cycle dysregulation is a hallmark of tumor cells and con-
tributes to the abnormal proliferation of cells [7]. Cyclin-dependent
kinases (CDKs) combining with various cyclins plays vital roles in
cell cycle progression [8]. It is also well known that glycogen syn-
thase kinase 3b (GSK-3b) has been associated with a host of high-
profile diseases such as cancer, diabetes, neurodegeneration and
inflammation due to its involvement in a great number of signaling
pathways [9,10]. Plenty of studies suggest that indirubin, the isomer
* Corresponding author.
E-mail address: zmmao@sjtu.edu.cn (Z. Mao).
http://dx.doi.org/10.1016/j.ejmech.2014.08.049
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

166
P. Zhao et al. / European Journal of Medicinal Chemistry 86 (2014) 165e174
Fig. 1. Structure of indigo (A), isoindigo (B), meisoindigo (C) and indirubin (D).
of isoindigo, displays potent inhibitory activities against CDKs [11]
and GSK-3 [12,13] by competing with ATP for binding at the cat-
spectrum data of the target compounds synthesized herein have
been given in part 4, Experimental.
b
alytic site. However, the specific antitumor mechanism of isoindigo
derivatives is not illustrated very clearly so far. Ji et al. reported that
meisoindigo inhibited DNA synthesis and led to the accumulation
of L1210 cells at S phase, and could also act on microtubule protein,
affect spindle formation and finally lead to mitotic arrest [14].
Xiao's findings suggested that meisoindigo could inhibit angiogenic
process by lowering the VEGF secretion in leukemic cells and also
inhibiting the proliferation, adhesion and differentiation of endo-
thelial cells [15].
In this paper, we synthesized a series of N-alkyl or aryl
substituted isoindigo derivatives, some of which have never been
reported before. The anti-proliferative activity of all target com-
pounds against leukemia cell lines (K562, L1210), several human
solid tumor cell lines (HCT-116, MGC80-3, A549, HeLa) and non-
cancerous cell line (VEC) were examined by SRB assay. The anti-
tumor mechanisms have been explored in detail by evaluation of
morphological changes, cell cycle distribution, apoptosis ratio and
2.2. Anti-proliferative activity
The anti-proliferative activity of all target compounds against
six cancer cell lines (K562, HCT-116, MGC80-3, L1210, A549 and
HeLa) and one non-cancerous cell line vascular endothelial cell
(VEC) was tested by SRB assay. Meisoindigo 6a was used as a pos-
itive control. The results have been shown in Table 2. It suggested
that most of these isoindigo derivatives showed significant anti-
proliferative activity to these six cancer cell lines especially to
K562 and A549 cells. With one exception, compound 6f 1,10-
diacetyl-isoindigo showed no anti-proliferative activity in any cell
line. This indicated that substitution of two lactam NH in isoindigo
might lead to the antitumor activity to disappear. The activity
depended on the presence of at least one lactam NH without sub-
stitution in isoindigo skeleton. Compound 6i and 6k exhibited a
selective inhibitory activity against HeLa and K562 cells, the IC50
the expression of relevant proteins (CDK2, cyclin A, GSK-3
, c-myc, -catenin). Furthermore, the binding mode of compound
6k with GSK-3 was evaluated by docking assay. All of these studies
b, p-GSK-
3
b
b
values were 11.2 mM and 7.8 mM, respectively. For K562 cells, iso-
b
indigo derivatives with different R1 exhibited different anti-
proliferative activity. The activity sequence can be summarized as
p-CH3OePhe > PheC2H5e > i-C4H9e > n-C4H9e > C2H5e, i-C3H7e,
Phe, p-FePhe. This may indicate that larger alkyl and aryl with
electron-donating group were helpful to the inhibitory activity.
Major R1 group may produce favorable steric effect to interact with
the amino residues around. It also seemed that clogP had an
inconsistent relationship with the activity (compound 6i had a
similar clogP with compound 6k, but showed no activity against
K562 cells). The target compounds also affected the proliferation of
the non-cancerous VEC cells in varying degree. Noticeably, the most
promising isoindigo derivatives 6k and 6h slightly affected the VEC
cells but selectively inhibited K562 cells.
gave a deeper insight into the anti-proliferative mechanisms of this
class of agents and also provided an admirable direction to the
development of novel dual CDK2/GSK-3
inhibitors.
b
phosphorylation
2. Result and discussion
2.1. Synthesis of the target compounds
All of the target compounds were synthesized according to the
procedure presented in Scheme 1. Compound 3 was prepared via
Sandmeyer's method previously described in the literature [16].
Compound 4 was obtained by reduction of compound 3 with hy-
drazine hydrate [17]. In this reduction reaction, instead of using a
strong base as the catalyst, a weak base sodium acetate was used as
the catalyst and the reaction gave an unusually good yield 95%. N-
alkyl substituted indole-2,3-dione 5aee and 5geh were obtained
by alkylation of 3 with corresponding alkyl halide using sodium
hydride as dehydrogenating agent. N-aryl substituted indole-2,3-
dione 5iek were synthesized by arylation of 3 with correspond-
ing aryl halide and two equivalent cupric oxide in N,N0-dime-
thylformamide (DMF) under reflux. The physical characters and
spectrum data of 5aee and 5gek have been shown in Table 1.
Target compounds 6aee and 6gek were obtained by conden-
sation of 4 and 5 in glacial acetic acid catalyzed by concentrated
hydrochloric acid. Compound 6f was synthesized via acetylation of
isoindigo (B) in acetic anhydride under reflux overnight. The
2.3. Mechanism studies
2.3.1. Cell morphological assessment
Cell morphological assessment was carried out by Hoechst
33342 and propidium iodide (PI) dual staining. As shown in Fig. 2,
the untreated cells showed round and homogeneous blue nuclei
stained with Hoechst 33342, the necrotic cells showed round pink
nuclei stained with both Hoechst 33342 and PI, and apoptotic cells
showed brilliant blue or shrunken and fragmented nuclei. The
brilliant blue indicated the chromatin condensation and nuclei
compaction in early apoptosis cells. Destructive fragmented nuclei
and apoptosis body could be observed in late apoptosis cells. These
results suggested that compound 6k induced the apoptosis and
necrosis of K562 cells.

P. Zhao et al. / European Journal of Medicinal Chemistry 86 (2014) 165e174
167
Scheme 1. Synthetic route for target compounds 6aek. Reagents and conditions: (i) chloral hydrate, anhydrous Na2SO4, NH2OH$HCl, Hþ, 80e90 ꢁC, 2 h; (ii) 86% H2SO4, 60 ꢁC,
50 min; 80 ꢁC, 15 min; (iii) H2N4$H2O, NaOAc, DMF, 100 ꢁC, 8 h; (iv) 1) NaH, DMF, N2, rt, 0.5 h; 2) R1X, rt; (v) ArX, CuO, DMF, reflux, 8 h; (vi) concentrated HCl, AcOH, reflux; (vii)
concentrated HCl, AcOH, reflux; (viii) Ac2O, reflux.
2.3.2. Cell cycle assay
contrast to 24 h treatment, which due to the proliferative effect
counteracted the inhibitory effect after treated for 48 h. But in
higher concentration groups in 48 h treatment, the inhibitory effect
played a dominate role in cells growth. In a word, both concen-
tration of compound and treatment-time could affect the growth of
cells, which also provided some instruction for clinical treatment.
Flow cytometric analyzer was applied to analyze the effect of
compound 6k on the cell cycle of K562 cells after treatment for
24 h. As shown in Fig. 3, compound 6k induced a dose-dependent
cell cycle arrest at S phase and a corresponding decrease of cell
numbers in G0/G1 and G2/M phase in K562 cells. These results
indicated that compound 6k could inhibit the growth of cancer
cells by inhibiting the cell cycle progression.
2.3.4. Analysis of proteins related to cell cycle and Wnt/
signaling pathway
b-catenin
2.3.3. Cells apoptosis assay
Inducing apoptosis is considered as one of the major strategies
for antitumor agent development [18]. In this study, the quantita-
tive analysis of early apoptotic cells and late apoptotic/necrotic cells
were carried out using Flow cytometer assay. As presented in Fig. 4,
K562 cells underwent both early apoptosis (Annexin Vþ/PIꢀ, lower
right quadrant) and late apoptosis/necrosis (Annexin Vþ/PIþ, upper
right quadrant). The proportion of both apoptotic cells and necrotic
cells increased in a dose-dependent manner (Fig. 4). Treatment-
time (24 h or 48 h) displayed different effect on the growth of
K562 cells. When treated for 24 h, the proportion of early apoptotic
cells was consistently slightly higher than late apoptotic/necrotic
cells in different concentration groups. When the exposure time
was extended to 48 h, there was a reduction in both apoptotic and
It is well known that CDK2/cyclin A complex plays critical role in
cell cycle progression by driving cells through S-phase and it is also
active at G2 phase [19]. In this study, western blot assay was carried
out after K562 cells exposed to compound 6k for 24 h. As shown in
Fig. 5, compound 6k down-regulated the expression of cyclin A and
CDK2 in a dose-dependent manner. The decrease of cyclin A and
CDK2 could prevent cells from passing S phase into G2 phase,
subsequently, arrest the cell cycle at S phase, which was consistent
with the results in Fig. 3.
Modern molecular biology studies also display that aberrant
activation of Wnt/
ation of cells [20]. Wnt/
Wnt molecule, Dishevelled (DVL), APC, Axin, GSK-3
TCF-4. GSK-3 participates in the formation of -catenin destruc-
tion complex, APC/GSK-3 /Axin, which can phosphorylate -cat-
enin. After phosphorylated, -catenin is degraded by ubiquitin-
proteasome pathway. Thus, the amount of -catenin passing into
b
-catenin pathway can cause excessive prolifer-
-catenin pathway is mainly comprised by
-catenin and
b
b, b
necrotic cells in lower concentration groups (0, 5.0, 10.0, 15.0
contrast to 24 h treatment. But there was a significant increase of
necrotic cells in higher concentration group (30.0 M group, from
20.02% to 42.09%). Moreover, a trend of cells shifting from early
apoptosis to late apoptosis/necrosis could be seen in 20.0 M and
30.0 M groups, especially in 30.0 M group, after treatment for
mM) in
b
b
b
b
m
b
b
m
the nucleus is decreased, which causes the lower transcription of
genes, including oncogenes, such as c-myc, cyclin D1 and c-jun, and
m
m
48 h. We could conclude that when treated for 24 h, compound 6k
affected the growth of K562 cells mainly by inducing the apoptosis
of cells. But when treated for 48 h, two different situations could be
seen as compared with 24 h groups. Firstly, in lower concentration
groups, there was a reduction in apoptotic and necrotic cells in
induces the apoptosis of cells [20,21]. In short, GSK-3
suppressive role in tumor cells growth by Wnt/ -catenin pathway.
However, the suppressive activity of GSK-3 to cells' proliferation
can be inhibited if GSK-3 (Ser9) is phosphorylated by other ki-
nases, such as upstream kinase Akt [22].
b plays a
b
b
b

168
P. Zhao et al. / European Journal of Medicinal Chemistry 86 (2014) 165e174
Table 1
6k possibly suppressed the phosphorylation of GSK-3
GSK-3 (Ser9) and caused the level of GSK-3 increased, subse-
quently, inhibited Wnt/ -catenin signaling pathway and induced
the apoptosis of K562 cells. It is worth mentioning that the role of
GSK-3 in cancer is somewhat enigmatic. The overall role of GSK-3
as tumor suppressor or promoter still remains inconclusive.
Depending on the cancer cell types, GSK-3 inhibitor is beneficial
(e.g. pancreatic, renal cancer) while activation of GSK-3 is bene-
ficial for example in oral cancer [23]. This could be possible because
GSK-3 participates in a complex array of critical cellular processes
b (Ser9) to p-
Physical characters and spectrum data of 5aee, 5gek.
b
b
Yield mp. (ꢁC) Solvent 1H NMR (400 MHz,
(%)
d ppm)
b
No. Reagent
b
b
5a CH3I
86
132e134 CDCl3 7.64e7.59 (m, 2H), 7.14 (t, 1H,
J ¼ 10.0 Hz), 6.90 (d, 1H, J ¼ 10.8 Hz),
3.27 (s, 3H)
b
5b C2H5Br
80
86e87 CDCl3 7.62e7.57 (m, 2H), 7.12 (t, 1H,
J ¼ 10.0 Hz), 6.91 (d, 1H, J ¼ 9.6 Hz),
3.83e3.76 (m, 2H), 1.32 (t, 3H,
b
b
J ¼ 10.0 Hz)
5c i-C3H7Br
5d n-C4H9Br
23
56
76e77 CDCl3 7.62e7.53 (m, 2H), 7.09 (t, 1H,
J ¼ 9.6 Hz), 7.02 (d, 1H, J ¼ 10.8 Hz),
4.59e4.50 (m, 1H), 1.52 (d, 6H,
and is regulated by multiple factors.
2.4. Docking study
J ¼ 9.2 Hz)
41e42 CDCl3 7.62e7.57 (m, 2H), 7.12 (t, 1H,
J ¼ 10.0 Hz), 6.91 (d, 1H, J ¼ 10.8 Hz),
3.73 (t, 2H, J ¼ 9.6 Hz), 1.72e1.65 (m,
2H), 1.46e1.30 (m, 2H), 0.98 (t, 3H,
J ¼ 9.6 Hz)
Molecular docking studies were performed using Autodock
Tools (ADT) v1.5.6 and Autodock v4.2 program to seek a possible
binding mode of compound 6k with GSK-3
b. Crystal structure of
GSK-3 in complex with the non-hydrolyzable ATP analog AMP-
b
5e i-C4H9Br
60
133e135 CDCl3 7.61e7.55 (m, 2H), 7.11 (t, 1H,
J ¼ 10.0 Hz), 6.90 (d, 1H, J ¼ 10.4 Hz),
3.53 (d, 2H, J ¼ 9.6 Hz), 2.20e2.07 (m,
1H), 0.99 (d, 6H, J ¼ 8.4 Hz)
PNP (PDB code: 1J1B) was selected for the docking studies [24].
Firstly, from Fig. 7a and b, we can see that the complementary
shape of compound 6k allows it to bury the bis-indole section in
the tight-fitting ATP binding pocket. The pocket sandwiches com-
pound 6k between Ile62, Val70, Ala83 on the top and Val110,
Leu188 on the bottom [25]. All of these five amino residues are
contributed to produce potent hydrophobic interaction between
5g C6H5CH2Br 89
5h C6H5C2H4Br 70
131e132 CDCl3 7.62 (d, 1H, J ¼ 9.6 Hz), 7.49 (t, 1H,
J ¼ 10.0 Hz), 7.34 (m, 5H), 7.10 (t, 1H,
J ¼ 10.0 Hz), 6.78 (d, 1H, J ¼ 10.4 Hz),
4.94 (s, 2H)
133e134 DMSO- 7.63 (t, 1H, J ¼ 10.4 Hz), 7.53 (d, 1H,
d6
J ¼ 10.0 Hz), 7.28e7.24 (m, 4H), 7.22
compound 6k and GSK-3b. Secondly, the complementary shape of
e7.09 (m, 3H), 3.89 (t, 2H,
the GSK-3 hinge segment Leu132-Asp133-Tye134-Val135-Pro136
b
J ¼ 10.0 Hz), 2.91 (t, 2H, J ¼ 10.0 Hz)
5i C6H5Br
53
137e139 CDCl3 7.70 (d, 1H, J ¼ 9.2 Hz), 7.59e7.52 (m,
3H), 7.48e7.41 (m, 3H), 7.18 (t, 1H,
forms another portion of the pocket [26]. In this hinge segment,
important hydrogen bonds can be seen between compound 6k and
J ¼ 9.6 Hz), 6.90 (d, 1H, J ¼ 10.8 Hz)
GSK-3b
. The hydrogen atom of 10-NH of 6k, acting as hydrogen
5j p-FeC6H5I 84
230e232 DMSO- 7.67e7.51 (m, 4H), 7.44 (t, 2H,
donor, formed a hydrogen bond with carbonyl oxygen atom from
Asp133 (distance: 1.8 Å). The carbonyl oxygen atom of 20-position
of compound 6k, was involved to form the second hydrogen bond
with amide hydrogen from Val135 (distance: 2.8 Å). Finally, the
oxygen atom in p-methoxyphenyl of compound 6k forms the third
d6
J ¼ 8.0 Hz), 7.18 (t, 1H, J ¼ 10.0 Hz),
6.79 (d, 1H, J ¼ 10.8 Hz)
5k p-CH3O
eC6H5I
48
168e171 DMSO- 7.65e7.57 (m, 2H), 7.42e7.37 (m,
d6
2H), 7.19e7.12 (m, 3H), 6.74 (d, 1H,
J ¼ 10.4 Hz), 3.83 (s, 3H)
Table 2
Anti-proliferative activity of compound 6aek against cell lines K562, HCT-116, MGC80-3, L1210, A549, HeLa and VEC.
Compd.
IC50
(m
M)a
clogPc
K562
HCT-116
MGC80-3
L1210
A549
HeLa
VEC
6ab
6b
6c
6d
6e
6f
6g
6h
6i
23.3 1.8
>200
>200
114.3 6.8
74.4 6.8
>200
>200
17.1 1.9
>200
>200
7.8 0.5
27.0 10.3
51.0 4.3
148.9 13.5
34.2 4.2
62.2 5.6
>200
37.1 3.7
85.2 7.5
49.9 2.6
40.1 2.0
>200
128.0 14.5
67.2 3.7
175.5 20.3
87.9 6.7
>200
>200
97.9 4.5
>200
167.0 6.3
47.5 6.7
>200
75.4 6.1
73.8 4.9
>200
113.8 8.1
158.3 11.2
156.6 11.0
>200
57.3 4.8
61.0 4.6
182.6 13.2
30.2 1.5
52.8 3.4
>200
62.4 2.8
85.4 6.1
41.4 2.4
22.6 2.3
31.9 3.2
15.7 2.1
194.3 10.8
>200
80.7 3.6
>200
>200
147.6 12.9
>200
122.0 0.8
73.0 5.4
>200
58.1 5.2
97.4 6.5
>200
86.8 6.5
183.5 9.1
52.6 4.4
93.5 4.8
>200
2.71
3.24
3.55
4.30
4.17
0.94
4.01
4.81
2.93
3.07
2.85
109.3 7.2
108.1 8.7
121.6 9.2
11.2 7.9
>200
165.6 14.1
6j
6k
102.1 7.9
a
IC50 value (mean SEM) was determined by three independent experiments by SRB assay.
Positive control.
Calculated using ChemBioDraw Ultra 12.0.
b
c
From the above, we deduce that if we increase the level of GSK-
or block the phosphorylation of GSK-3 (Ser9) to p-GSK-3
hydrogen bond with Gln185 (distance: 3.3 Å) which can fix the
compound at the outer end of GSK-3 pocket and prevent the
3
b
b
b
b
(Ser9) by competing with ATP for binding site, the suppressive
activity of GSK-3 to cells growth can be played preferably. Here,
we investigated Wnt/ -catenin pathway proteins including GSK-3
p-GSK-3 (Ser9), -catenin and c-myc. As presented in Fig. 6, the
expression of p-GSK-3 (Ser9), -catenin and c-myc were down-
regulated and the expression of GSK-3 was up-regulated in a
dose-dependent manner. These results indicated that compound
dissociation of compound 6k with GSK-3b.
b
b
b,
3. Conclusion
b
b
b
b
In conclusion, a series of N-alkyl or aryl substituted isoindigos
have been designed and synthesized in this study. Many of the
target compounds exhibited acceptable inhibitory activity against
b

P. Zhao et al. / European Journal of Medicinal Chemistry 86 (2014) 165e174
169
Fig. 2. Morphological changes of K562 cells observed under fluorescent inverted microscope. NC: negative control (DMSO, 0.5%, v/v), MIO: meisoindigo, as positive control. Cells
were exposed to compound 6k with different concentration for 24 h.
various cancer cells, especially to K562 and A549 cells. Further
mechanism research of the most potential compound 6k on
K562 cells was examined in detail. Compound 6k could arrest cell
cycle at S phase in K562 cells by down-regulating the expression of
CDK2 and cyclin A. Moreover, compound 6k down-regulated the
the reaction was completed, the solvent was distilled off from re-
action mixture, and the remaining mixture was added with 10 mL
of water. The resulting mixture was extracted by 30 mL of toluene
(10 mL ꢂ 3). The organic layer was dried over Na2SO4 and
concentrated under vacuum to give about 1.4 g of yellowish crude
4. The crude solid was then decolorized by activated carbon in
dichloroethane and afterward crystallized from the solution. The
solid was separated from the solution and dried to give 1.35 g of
compound 4 as gray white solid, yield 92%, mp. 126e128 ꢁC. 1H
expression of Wnt/
(Ser9), -catenin, and c-myc, and up-regulated the expression of
GSK-3 , which played critical role in cells apoptosis. Molecular
b-catenin signaling pathway proteins p-GSK-3b
b
b
docking studies indicated that three hydrogen bonds and potent
hydrophobic interaction played crucial rules in the binding of
NMR (400 MHz, CDCl3,
d ppm): 7.68 (br s, 1H), 7.26e7.21 (m, 2H),
compound 6k with GSK-3
compound 6k could serve as a potential dual CDK2/GSK-3
b
. Taken together, the new synthetic
7.04 (t, 1H, J ¼ 10.0 Hz), 6.87 (d, 1H, J ¼ 10.4 Hz), 3.56 (s, 2H).
b
(Ser9)
phosphorylation inhibitor candidate in antitumor agent
development.
4.1.2. General procedure for the synthesis of N-alkyl substituted
indole-2,3-dione (5aee, 5geh)
Sodium hydride (12 mmol) was added to compound
3
4. Experimental
(10 mmol) in 20 mL of DMF under nitrogen atmosphere protection.
After stirring for 30 min at room temperature, the solution of cor-
responding alkyl halide (11 mmol) in 2 mL of DMF was added to the
reaction mixture and the mixture was stirred at room temperature
to give a dark red solution. The reaction process was monitored by
TLC. After the completion of reaction, the reaction was quenched by
careful addition of water (30 mL) and was extracted with
dichloromethane. The organic layer was washed with distilled
water, dried over Na2SO4, concentrated under vacuum and purified
by column chromatography to afford the title compounds 5aee and
5geh. The physical characters and spectrum data have been shown
in Table 1.
4.1. Chemistry
Melting points were determined with an SGW X-4 digital micro-
melting point apparatus without further correction. Column chro-
matography was performed with a column packed with silica gel 60
(200e300 mesh). 1H NMR (400 MHz) and 13C NMR (100 MHz)
spectra were recorded on a Bruker 400 UltraShield™ spectrometer
with DMSO-d6 or CDCl3 as solvent and TMS as internal standard.
High resolution mass spectra (HRMS) data were obtained by an AB
SCIEX TripleTOF® 4600 mass spectrometer using electrospray
ionization (ESI) technique.
4.1.3. General procedure for the synthesis of N-aryl substituted
indole-2,3-dione (5iek)
4.1.1. Procedure for the synthesis of 2-oxindole (4)
Compound 3 was obtained by Sandmeyer's method as described
in detail in the literature [15]. 1.66 g (11 mmol) of compound 3 was
dissolved in 10 mL of DMF in a flask. 0.7 mL of 80% hydrazine hy-
drate (11 mmol) mixed with 2 mL of DMF was added in drops into
the flask. Afterward, 0.083 g of sodium acetate (1 mmol) was added
into the flask, and the temperature of the reaction mixture was
raised to 100 ꢁC. The reaction process was monitored by TLC. After
N-aryl substituted indole-2,3-dione (5iek) were obtained by
Coppola's method [27]. A mixture of 10 mmol of indole-2,3-dione
(3), 14 mmol of the corresponding aryl halide and 20 mmol of
cupric oxide in 25 mL of DMF was stirred under reflux for 5e12 h.
While hot, any insoluble material was filtered from the mixture.
The filtrate was poured into cold water and the resulting precipitate
was filtered. The crude product was purified by column

170
P. Zhao et al. / European Journal of Medicinal Chemistry 86 (2014) 165e174
J ¼ 8.0 Hz), 7.06e6.99 (m, 3H), 6.85 (d, 1H, J ¼ 8.0 Hz), 3.23 (s, 3H).
13C NMR (100 MHz, DMSO-d6,
ppm): 168.9, 167.1, 144.9, 144.2,
d
133.8, 132.9, 132.5, 132.2, 129.4, 129.0, 121.7, 121.6, 121.1, 120.8,
109.5, 108.3, 26.0. HRMS-EI m/z [MþH]þ calcd for C17H12N2O2:
277.0972, found: 277.0964.
4.1.4.2. 1-Ethyl-isoindigo (6b). Purple red solid. Yield: 64%, mp.
180e182 ꢁC. 1H NMR (400 MHz, CDCl3,
d ppm): 9.30 (d, 1H,
J ¼ 10.4 Hz), 9.24 (d, 1H, J ¼ 11.6 Hz), 7.75 (br s, 1H), 7.51e7.41 (m,
2H), 7.17 (t, 2H, J ¼ 10.0 Hz), 6.92 (d, 2H, J ¼ 10.0 Hz), 4.00e3.93 (m,
2H), 1.43 (t, 3H, J ¼ 9.6 Hz). 13C NMR (100 MHz, DMSO-d6,
d ppm):
169.0, 166.8, 144.2, 143.9, 133.8, 132.8, 132.6, 132.3, 129.5, 129.1,
121.6, 121.1, 120.9, 120.8, 109.6, 108.4, 34.1, 12.4. HRMS-EI m/z
[MþH]þ calcd for C18H14N2O2: 291.1128, found: 291.1129.
4.1.4.3. 1-(i-Propyl)-isoindigo (6c). Purple red solid. Yield: 57%, mp.
>300 ꢁC. 1H NMR (400 MHz, DMSO-d6,
d ppm): 11.00 (br s, 1H, NH),
9.20 (d, 1H, J ¼ 1.2 Hz), 9.18 (d, 1H, J ¼ 1.2 Hz), 7.53e7.41 (m, 2H),
7.30 (d, 1H, J ¼ 10.4 Hz), 7.14e7.04 (m, 2H), 6.93 (d, 1H, J ¼ 10.0 Hz),
4.78e4.66 (m, 1H), 1.57e1.55 (d, 6H, J ¼ 9.2 Hz). 13C NMR (100 MHz,
DMSO-d6,
d ppm): 168.8, 164.4, 144.2, 143.7, 133.6, 132.9, 132.8,
132.6, 132.5, 129.4, 129.1, 129.0, 121.3, 121.1, 109.6, 109.4, 43.5, 29.0,
19.0. HRMS-EI m/z [MþH]þ calcd for C19H16N2O2: 305.1285, found:
305.1278.
4.1.4.4. 1-(n-Butyl)-isoindigo (6d). Purple red solid. Yield: 52%, mp.
140e142 ꢁC. 1H NMR (400 MHz, DMSO-d6,
d ppm): 11.01 (br s, 1H,
NH), 9.21e9.16 (m, 2H), 7.55e7.43 (m, 2H), 7.18e7.05 (m, 3H), 6.94
(d, 1H, J ¼ 10.0 Hz), 3.87 (t, 2H, J ¼ 10.0 Hz), 1.76e1.66 (m, 2H),
1.47e1.40 (m, 2H), 1.01 (t, 3H, J ¼ 10.0 Hz). 13C NMR (100 MHz,
DMSO-d6,
d ppm): 168.9, 167.1, 144.3, 144.2, 133.8, 132.8, 132.5,
132.1, 129.5, 129.1, 121.62, 121.60, 121.1, 120.8, 109.6, 108.5, 49.2,
29.0, 19.6, 13.6. HRMS-EI m/z [MþH]þ calcd for C20H18N2O2:
319.1441, found: 319.1438.
4.1.4.5. 1-(i-Butyl)-isoindigo (6e). Purple red solid. Yield: 60%, mp.
172e175 ꢁC. 1H NMR (400 MHz, CDCl3,
d ppm): 9.20e9.11 (m, 2H),
7.63 (br s, 1H), 7.42e7.31 (m, 2H), 7.09e7.04 (m, 2H), 6.84e6.80 (m,
2H), 3.62 (d, 2H, J ¼ 10.0 Hz), 2.25e2.15 (m, 1H), 1.03e1.01 (d, 6H).
13C NMR (100 MHz, DMSO-d6,
d ppm): 168.9, 167.4, 144.7, 144.2,
134.0, 132.9, 132.6, 132.1, 129.5, 129.0,121.6, 121.1, 120.7, 115.5, 109.6,
108.8, 46.6, 26.6, 20.0 (ꢂ2). HRMS-EI m/z [MþH]þ calcd for
Fig. 3. Cell cycle analysis of K562 cells after treatment with compound 6k for 24 h.
*p < 0.05, compared with negative control group (DMSO, 0.5%, v/v).
C
20H18N2O2: 319.1441, found: 319.1435.
4.1.4.6. 1-Benzyl-isoindigo (6g). Purple red solid. Yield: 46%, mp.
238e240 ꢁC. 1H NMR (400 MHz, CDCl3,
ppm): 9.33 (d, 1H,
chromatography to afford the title compounds (5iek). The physical
characters and spectrum data have been shown in Table 1.
d
J ¼ 10.8 Hz), 9.24 (d, 1H, J ¼ 11.2 Hz), 7.72 (br s, 1H, NH), 7.45e7.39
(m, 6H), 7.21e7.13 (m, 3H), 6.93 (d, 1H, J ¼ 10.8 Hz), 6.83 (d, 1H,
J ¼ 11.2 Hz), 5.13 (s, 2H). 13C NMR (100 MHz, DMSO-d6,
d ppm):
4.1.4. General procedure for the synthesis of target compounds
(6aee, 6gek)
168.8, 167.4, 144.4, 143.9, 136.4, 134.3, 133.0, 132.4, 131.8, 130.1,
129.5,129.1, 128.7,127.4, 127.2, 121.9, 121.6, 121.2,120.9,120.2,109.6,
109.0, 42.7. HRMS-EI m/z [MþH]þ calcd for C23H16N2O2: 353.1285,
found: 353.1274.
2-Oxindole (4) and N-substituted indole-2,3-dione (5) were
dissolved in glacial acetic acid and three drops of concentrated
hydrochloric acid was added. The solution was stirred under reflux
overnight. The resultant mixture was concentrated under vacuum.
Toluene was added to aid the removal of glacial acetic acid. Ethyl
acetate was added to the residue, the suspension was filtered under
reduced pressure, washed with chilled ethyl acetate and dried in
the oven. The target compounds were obtained usually as a purple
red powder.
4.1.4.7. 1-Phenethyl-isoindigo (6h). Purple red solid. Yield: 58%, mp.
215e217 ꢁC. 1H NMR (400 MHz, DMSO-d6,
d ppm): 10.93 (br s, 1H,
NH), 9.09 (d, 1H, J ¼ 11.2 Hz), 8.98 (d, 1H, J ¼ 10.8 Hz), 7.42e7.20 (m,
7H), 7.09e6.93 (m, 3H), 6.83 (d, 1H, J ¼ 9.2 Hz), 3.99 (t, 2H,
J ¼ 10.0 Hz), 2.93 (t, 2H, J ¼ 10.0 Hz). 13C NMR (100 MHz, MHz,
DMSO-d6,
d ppm): 169.7, 168.8, 144.2, 140.1, 138.3, 129.4, 128.8,
4.1.4.1. Meisoindigo (6a). Purple red solid. Yield: 67%, mp.
128.8 (ꢂ2), 128.4 (ꢂ2), 126.5, 126.4, 125.4, 124.4, 123.1, 121.6 (ꢂ2),
228e230 ꢁC. 1H NMR (400 MHz, DMSO-d6,
d
ppm): 10.85 (br s, 1H,
121.1, 120.8, 115.8, 110.8, 41.0, 32.9. HRMS-EI m/z [MþH]þ calcd for
NH), 9.11e9.08 (m, 2H), 7.43 (t, 1H, J ¼ 8.0 Hz), 7.35 (t, 1H,
C24H18N2O2: 367.1441, found: 367.1435.

P. Zhao et al. / European Journal of Medicinal Chemistry 86 (2014) 165e174
171
Fig. 4. Flow cytometer analysis for Annexin VeFITC and PI staining, after exposure to compound 6k for 24 h and 48 h, respectively. (A) Negative control, DMSO (0.5%, v/v); (B)e(F)
were the different concentration groups of compound 6k, (B) 5.0 M, (C) 10.0 M, (D) 15.0 M, (E) 20.0 M, (F) 30.0 M; UR (upper right): late apoptotic cells and necrotic cells
labeled with PI and Annexin VeFITC, LL (lower left): fully viable cells, LR (lower right): early apoptotic cells labeled with Annexin VeFITC but not with PI.
m
m
m
m
m
4.1.4.8. 1-Phenyl-isoindigo (6i). Dark red solid. Yield: 73%, mp.
272e274 ꢁC. 1H NMR (400 MHz, DMSO-d6,
ppm): 10.95 (br s, 1H,
4.1.5. General procedure for the synthesis of 6f
d
1,10-Diacetyl-isoindigo (6f) was prepared by reacting isoindigo
with acetic anhydride under reflux overnight. The product was
precipitated out in reaction solution. The precipitate was filtered,
washed with acetone and dried at 40 ꢁC overnight to give the title
compound 6f. Purple red solid. Yield: 77%, mp. 259e262 ꢁC. 1H
NH), 9.16 (d, 1H, J ¼ 10.4 Hz), 8.95 (d, 1H, J ¼ 9.6 Hz), 7.62e7.57 (m,
2H), 7.51e7.48 (m, 3H), 7.38e7.31 (m, 2H), 7.08 (t, 1H, J ¼ 10.0 Hz),
6.93 (t, 1H, J ¼ 10.0 Hz), 6.83 (d, 1H, J ¼ 10.8 Hz), 6.66 (d, 1H,
J ¼ 10.0 Hz). 13C NMR (100 MHz, DMSO-d6,
d ppm): 168.8, 167.3,
146.8, 145.2, 134.7, 133.9, 133.4, 133.0, 131.9, 130.0, 129.8, 129.7 (ꢂ2),
128.4, 127.5 (ꢂ2), 123.3, 121.6, 121.2, 121.0, 109.6, 108.9. HRMS-EI m/
z [MþH]þ calcd for C22H14N2O2: 339.1089, found: 339.1078.
NMR (400 MHz, DMSO-d6,
d
ppm): 8.91 (d, 2H, J ¼ 11.6 Hz), 8.31 (d,
2H, J ¼ 11.2 Hz), 7.64 (t, 2H, J ¼ 9.6 Hz), 7.39 (t, 2H, J ¼ 9.2 Hz), 2.77
(s, 6H). 13C NMR (100 MHz, DMSO-d6,
d ppm): 170.3, 167.8, 141.9,
133.6, 132.7, 128.6, 124.8, 122.2, 116.2, 27.0. HRMS-EI m/z [MþH]þ
calcd for C20H14N2O4: 347.1026, found: 347.1029.
4.1.4.9. 1-(p-Fluoro-phenyl)-isoindigo (6j). Dark red solid. Yield:
57%, mp. 276e279 ꢁC. 1H NMR (400 MHz, CDCl3,
d ppm): 9.20 (d,1H,
J ¼ 10.8 Hz), 9.14 (d, 1H, J ¼ 11.2 Hz), 7.62 (br s, 1H, NH), 7.47e7.30
(m, 6H), 7.13 (t, 1H, J ¼ 9.6 Hz), 7.03 (t, 1H, J ¼ 9.2 Hz), 6.84 (d, 1H,
J ¼ 10.4 Hz), 6.71 (d, 1H, J ¼ 11.2 Hz). 13C NMR (100 MHz, DMSO-d6,
4.2. Biology
d
ppm): 168.8, 166.9, 156.6, 144.9, 144.4, 134.5, 133.2, 132.6, 131.8,
4.2.1. Cell culture
129.9, 129.2, 126.7, 122.3, 121.5, 121.2, 121.0 (ꢂ2), 116.7 (ꢂ2), 116.4,
109.7, 108.7. HRMS-EI m/z [MþH]þ calcd for C22H13FN2O2: 357.1034,
found: 357.1022.
K562, HCT-116, MGC80-3, L1210, A549, HeLa and VEC cells were
cultured at 37 ꢁC in an atmosphere containing 5% CO2, with RPMI
1640 medium (Hyclone, SH30027 LS, USA) supplemented with 10%
heat-inactivated fetal bovine serum (Hyclone), 100 IU penicillin and
100 mg/mL streptomycin.
4.1.4.10. 1-(p-Methoxy-phenyl)-isoindigo (6k). Purple red solid.
Yield: 76%, mp. 223e226 ꢁC. 1H NMR (400 MHz, DMSO-d6,
d ppm):
10.94 (br s, 1H, NH), 9.16 (d, 1H, J ¼ 7.6 Hz), 8.98 (d, 1H, J ¼ 8.0 Hz),
7.41 (d, 2H, J ¼ 8.4 Hz), 7.35 (t, 2H, J ¼ 8.0 Hz), 7.14 (d, 2H, J ¼ 8.4 Hz),
7.08 (t, 1H, J ¼ 8.0 Hz), 6.94 (t, 1H, J ¼ 7.6 Hz), 6.86 (d, 1H, J ¼ 8.0 Hz),
6.62 (d, 1H, J ¼ 7.6 Hz), 3.84 (s, 3H). 13C NMR (100 MHz, DMSO-d6,
4.2.2. Drug treatment
Compounds were dissolved in DMSO and stored at ꢀ20 ꢁC. For
each experiment, the stock solutions were further diluted in me-
dium to obtain the desired concentration. The final DMSO content
in cell culture was ꢃ0.5% (v/v), which was found to be nontoxic to
cells. Parallel culture of cells in medium with DMSO was used as
negative control.
d
ppm): 168.9, 166.8, 159.0, 145.3, 144.5, 134.3, 133.0, 132.5, 132.1,
128.8 (ꢂ2), 127.9, 126.5, 124.6, 123.1, 122.1, 121.6, 121.0, 114.8 (ꢂ2),
109.6, 108.7, 55.4. HRMS-EI m/z [MþH]þ calcd for C23H16N2O3:
369.1234, found: 369.1232.

172
P. Zhao et al. / European Journal of Medicinal Chemistry 86 (2014) 165e174
a FACScan flow cytometer (FACSCalibun, Becton Dickinson). The
data was analyzed using Cell Quest (BD Biosciences) and Modfit
(Verity Software, Topsham, ME, USA) software.
4.2.6. Apoptosis assay
The apoptosis of K562 cells was determined by Annexin VeFITC/
PI assay [29]. After exposed to different concentration of compound
6k for 24 h, K562 cells were collected and incubated with fluo-
rescein isothiocyanate (FITC)-conjugated Annexin V according to
the manufacture's instruction (Lianke Bio, China). The nuclei were
then counterstained with PI. After the dual staining, the cells were
screened by a FACScan flow cytometer (FACSCalibun, Becton Dick-
inson). Annexin Vþ/PIe cells were considered as early apoptotic
while Annexin Vþ/PIþ cells as late apoptotic/necrotic.
4.2.7. Western blot assay
After treatment with DMSO or drugs, cells were collected and
lysed with ice-cold lysis buffer (P0013, Beyotime). The lysates were
clarified by centrifugation at 12,000 rpm at 4 ꢁC for 5 min, then
mixed with 5 ꢂ loading buffer (4% SDS, 10% 2-mercaptoethanol,
20% glycerol, and 0.2 mg/mL bromphenol blue in 0.1 M TriseHCl,
pH 6.8, 20% v/v in final volume), and boiled for 5 min immediately.
The boiled lysates containing equal amount of proteins were elec-
trophoresed on 10% SDS-polyacrylamide gel and transferred to
nitrocellulose membrane (Millipore). After blocking with 5%
skimmed milk in PBS with 0.1% Tween-20 for 1 h, the membranes
Fig. 5. Effects of compound 6k on the expression of cell cycle related proteins (cyclin A
and CDK2) in K562 cells after treatment for 24 h.
control. MIO: meisoindigo, 10.0 M, as positive control, *p < 0.05, compared with
negative control (DMSO, 0.5%, v/v).
b-actin was used as equal loading
m
4.2.3. Anti-proliferation assay
The effects of target compounds on cells proliferation were
examined by SRB assay. Cells suspension (5 ꢂ 103 cells/well) was
seeded into 96-well plates and incubated for 12 h. The cells were
then treated with different concentration of drugs in their respec-
tive culture medium and maintained with the treatment for addi-
tional 48 h. Thereafter, the cells were fixed with ice-cold
trichloroacetic acid (TCA) for 1 h at 4 ꢁC. The TCA solution was
removed lightly and the plates were washed five times in distilled
water and dried in the air. 50 mL of 0.4% SRB solution was added to
each well and stained at room temperature for 30 min. The SRB
solution was removed by washing the plates quickly five times with
1% (v/v) acetic acid. The washed plates were dried in the air. 100 mL
of 10 mM unbuffered Tris Base solution (pH 10.5) was added to each
well to dissolve the bound SRB. The absorbance was measured at
570 nm using a plate reader (MultiScan MK3, Thermo).
4.2.4. Hoechst 33342/PI staining assay
The cells were treated with the 0.5% DMSO alone and with
various concentrations of compound 6k for 24 h. Then the cells
were collected and centrifuged at 1000 rpm for 5 min, washed
twice with PBS. Staining was performed with 1 mL of Hoechst
33342 (5.0
at 1000 rpm for 5 min at 4 ꢁC. The supernatant was discarded and
1 mL of PI solution (10.0 g/mL) was added. Resuspended cells and
m
g/mL) for 10 min at 37 ꢁC. The mixture was centrifuged
m
incubated at room temperature in dark for 10 min. The apoptotic
and necrotic cells were observed under fluorescence microscopy
(IX71-32PH, Olympus) and microphotographs were taken by a
coupled digital camera.
4.2.5. Cell cycle assay
Cell cycle assay was carried out using method previously
described [28]. Briefly, cells suspension (4 ꢂ 105 cells/well) were
seeded into six-well plates and treated with drugs. After drug
treatment, the cells were fixed with 75% ethanol at ꢀ20 ꢁC over-
night, Removed the ethanol by centrifugation and resuspended
Fig. 6. Effects of compound 6k on the expression of Wnt/
related proteins (c-myc, p-GSK-3 (Ser9), GSK-3 -catenin) in K562 cells after
treatment for 24 h. -actin was used as equal loading control. MIO: meisoindigo,
M, as positive control, *p < 0.05, compared with negative control (DMSO, 0.5%, v/
b-catenin signaling pathway
b
b, b
b
cells in 1 mL of PBS containing 0.1 mg/mL RNase and PI (40
mg/mL)
10.0
v).
m
to stain DNA for 30 min at 37 ꢁC, DNA contents were analyzed using

P. Zhao et al. / European Journal of Medicinal Chemistry 86 (2014) 165e174
173
Fig. 7. Compound 6k in the ATP binding pocket of GSK-3
b
. a. Overall structure of GSK-3
b
with compound 6k bound to the ATP binding site; b. Hydrogen bonds interaction between
and yellow for compound 6k; Hydrogen
compound 6k and GSK-3 ; Color coding: nitrogen atoms blue, oxygen atoms red, hydrogen atoms gray, carbon atoms green for GSK-3
b
b
bonds were showed by black dash-line, the distance was denoted by Å. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of
this article.)
were incubated with antibodies against
b
-actin, p-GSK-3
b
(Ser9)
Acknowledgments
(Beyotime), c-myc, -catenin, GSK-3 , CDK2 and cyclin A (BOSTER)
b
b
at room temperature for 2 h. Primary antibodies were bound with
secondary antibody IRDye 680LT Goat anti-Rabbit IgG (H þ L) or
IRDye 800CW Goat anti-Mouse IgG (H þ L) (KPL, USA) by incuba-
tion at room temperature for 1 h. Then images were obtained by
Two Color Infrared Imaging System (Odyssey, CLx-L I-COR). The
gray value of protein bands was analyzed by Quantity One 4.6
software (BIO-RAD).
This work was supported by the National Significant and Special
Project of New Created Drugs (No. 2010ZX09401-404). We are
particularly grateful to Ph. D Hema Negi for giving us careful essay
revising and polishing.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.08.049.
4.3. Docking studies
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carbons and the internal degrees of freedom and torsions were set.
For the preparation of ligand, three dimensional structures of
compound 6k was constructed using Chem3D Ultra 12.0 software,
then it was energetically minimized by using MOPAC. Polar
hydrogen atoms were added to the ligand by Chimera software.
Before docking, a search grid was used to extend over the binding
site in GSK-3b. A standard set of genetic algorithm with the pop-
ulation size 100, the number of operations 100,000 were applied.
As the result, 10 binding poses with different binding energy were
obtained. The results were visualized by PyMOL 1.6 (DeLano Sci-
entific LLC).
4.4. Statistical analysis
All of the quantitative data in this study were reported as
means standard from at least three independent experiments.
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16.0 software. A p-value ꢃ0.05 was thought to have a significant
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