5,5′-substituted indirubin-3′-oxime derivatives as potent cyclin-dependent kinase inhibitors with anticancer activity

Article abstract of DOI:10.1021/jm100080z

To enhance the ability of indirubin derivatives to inhibit CDK2/cyclin E, a target of anticancer agents, we designed and synthesized a new series of indirubin-3′-oxime derivatives with combined substitutions at the 5 and 5′ positions. A molecular docking

Full text of DOI:10.1021/jm100080z

3696 J. Med. Chem. 2010, 53, 3696–3706
DOI: 10.1021/jm100080z
5,50-Substituted Indirubin-30-oxime Derivatives as Potent Cyclin-Dependent Kinase Inhibitors with
Anticancer Activity
Soo-Jeong Choi,† Jung-Eun Lee,† Soon-Young Jeong,‡ Isak Im,† So-Deok Lee,† Eun-Jin Lee,§ Sang Kook Lee,§,#
Seong-Min Kwon, Sang-Gun Ahn, Jung-Hoon Yoon, Sun-Young Han,^ Jae-Il Kim,†,‡ and Yong-Chul Kim*,†,‡
†Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea, ‡Division of Drug Discovery,
Anygen Co., Ltd., Gwangju 500-712, Republic of Korea, §College of Pharmacy, Ewha Womans University, Seoul 120-759, Republic of Korea,
Department of Pathology, School of Dentistry, Chosun University, Gwangju 501-759, Republic of Korea, and ^Pharmacology Research Center,
Korea Research Institute of Chemical Technology, Daejon 305-600, Republic of Korea. #Present address: College of Pharmacy,
Seoul National University, Seoul, Republic of Korea
Received January 19, 2010
To enhance the ability of indirubin derivatives to inhibit CDK2/cyclin E, a target of anticancer agents, we
designed and synthesized a new series of indirubin-30-oxime derivatives with combined substitutions at the
5 and 50 positions. A molecular docking study predicted the binding of derivatives with OH or halogen
substitutions at the 50 position to the ATP binding site of CDK2, revealing the critical interactions that may
explain the improved CDK2 inhibitory activity of these derivatives. Among the synthesized derivatives, the
5-nitro-50-hydroxy analogue 3a and the 5-nitro-50-fluoro analogue 5a displayed potent inhibitory activity against
CDK2, with IC50 values of 1.9 and 1.7 nM, respectively. These derivatives also showed antiproliferative activity
against several human cancer cell lines, with IC50 values of 0.2-3.3 μM. A representative analogue, 3a, showed
greater than 500-fold selectivity for CDK relative to selected kinase panel and potent in vivo anticancer activity.
Introduction
in the regulation of transcription initiation and elongation via
phosphorylation of RNA polymerase.8,9 Therefore, CDKs
affect cell growth and survival through several mechanisms,
making the appropriateregulation of CDK activityimportant
in various cellular processes.
Deregulation of CDKs by abnormally high expression of
cyclins, such as cyclins D and E, or by mutation, has been
observed in many human tumors.10 For example, the expres-
sion and catalytic activity of CDK2/cyclin E complexes is
increased in colorectal, ovarian, breast, and prostate cancers,
and elevated expression of cyclin E in primary tumors has
been shown to correlate with poor survival rates in breast
cancer patients.11,12 Abnormal expression of CDK1/cyclin B
complexes has also been observed in some patients with
prostate adenocarcinoma and lung cancer.13,14
Although a report has shown that CDK2 may not be
required for cell cycle progression and proliferation, recent
reports suggested that melanocytes and hepatocytes may be
dependent on CDK2 for proliferation and survival.15-17 Also,
an investigation of simultaneous depletion of CDK1 and
CDK2 was reported to provide increased efficacy in antipro-
liferation of tumor cell lines, compared with targeting either
CDK1 or CDK2 alone.18 Furthermore, emerging evidence
indicates that certain tumor cells may require specific inter-
phase CDKs for proliferation.7,15 Thus, inhibition of CDKs
may provide an effective strategy for controlling tumor growth,
making CDKs attractive targets for anticancer therapy.
At present, several small-molecule CDK inhibitors are un-
dergoing clinical trials. These inhibitors are flat, small hetero-
cyclic molecules that act by competing with ATP at the kinase
ATP-binding site. Among these, flavopiridol was the first
CDK inhibitor to enter clinical evaluations.19 R-Roscovitine
Cyclin-dependent kinases (CDKs) belong to a group of
serine/threonine kinases involved in the regulation of cell cycle
progression, neuronal function, differentiation, and apoptosis.1
Their activity is tightly regulated by multiple mechanisms inclu-
ding binding to corresponding cyclins, of which level of expres-
sion oscillates throughout the different phases of the cell cycle.2
Thus, different CDK/cyclin complexes are activated during each
cell cycle step through G1, S, G2, and M phases. Sequential
phosphorylation of the retinoblastoma protein (pRb) by CDK4/
cyclin D and CDK6/cyclin D in early G1 phase and by CDK2/
cyclin E in late G1 phase causes the release of the E2F proteins.
These transcription factors, in turn, activate a set of genes
required for entry into the S-phase of the cell cycle.3,4 CDK2 is
subsequently activated by cyclin A during the late stages of DNA
replication in S-phase, and CDK2 promotes appropriately timed
deactivation of E2F to prevent apoptosis triggered by persistent
E2F activity.5,6 Finally, CDK1, in a complex with cyclin A or B,
is thought to be involved in regulating the G2/M checkpoint and
driving cells through mitosis.2,7
Inaddition toplaying rolesincell cyclecontrol, CDK2, 7, 8,
and 9 have been found to have other activities. For example,
CDK2/cyclin E is important in the p53-mediated DNA
damage response pathway, and CDK7, 8, and 9 are involved
*To whom correspondence should be addressed. Phone: 82-62-970-
2502. Fax: 82-62-970-2484. E-mail: yongchul@gist.ac.kr.
Abbreviations: ATP, adenosine 50-triphosphate; CDK, cyclin depen-
dent kinase; DMEM, Dulbecco’s Modified Eagle Medium; EI, electron
impact; ESI, electro spray ionization; FAB, fast atom bombardment;
HPLC, high-pressure liquid chromatography; HRMS, high-resolution
mass spectroscopy; MOPS, 3-(N-morpholino)propanesulfonic acid;
pRb, retinoblastoma protein; RPMI, Roswell Park Memorial Institute
medium; SAR, structure-activity relationship; SRB sulforhodamine B.
r
pubs.acs.org/jmc
Published on Web 04/05/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3697
Figure 1. Structures of indirubin derivatives.
Figure 2. Binding mode of 2 and 3a in the ATP binding site of CDK2. (a) Network of hydrogen bonds (green dashed lines) between 2 and the
backbone amino acids in the ATP binding pocket of CDK2. Binding mode of 2 with CDK2 displays three hydrogen bonds between two
nitrogen of 2 and Leu83 and Glu81 in the CDK2 hinge region, additional hydrogen bond between the oxime moiety and carbonyl oxygen of
Gln131, and a salt bridge formed between 5-nitro group and the side chains of Lys33, Asp145. (b) van der Waals surface of CDK2 with 2 in CPK
representation. 50-Bromo group is projected toward the outer aqueous space of ATP binding pocket that presents a positively charged amino
acid residue, Lys89. (c) Network of hydrogen bonds (green dashed lines) between 3a and the backbone amino acids in the ATP binding pocket
of CDK2. 3a forms an additional hydrogen bond between the 50-hydroxy group and the carboxylic acid of Asp86. This may further improve
CDK2 inhibitory activity. (d) Molecular surface representation of CDK2 with 3a bound in the ATP binding site. Some important amino acids
lying within binding surface are displayed. The 50-hydroxy group is pointing out of the pocket toward the solvent.
(a trisubstituted purine analogue)20 and BMS-387032 (an
aminothiazole)21 are selective for CDK2/cyclin E, and PD-
0332991 (a pyridopyrimidine)22 is highly selective for CDK4/
cyclin D and CDK6/cyclin D. Indirubin, a bis-indole scaffold
and its derivatives, have been investigated with considerable
interest as potent inhibitors targeting important protein kinases
such as CDK, GSK-3β, and aurora kinases.23-26 Our research
group previously described the synthesis of a series of novel
indirubin analogues and the evaluation of their antiprolife-
rative and CDK2 inhibitory activities.23 Recently, we also
reported other activities of the indirubin derivatives including
their regulation of cellular signaling through the inhibition
of Notch-1 or activation of β-catenin-mediated Wnt signa-
ling27-29 and enhancing effect of differentiation of HL-60
leukemia cells induced by conventional activators such as
1,25-dihydroxyvitamin D3 and all-trans retinoic acid.30
activity (IC50 = 0.79-2.9 μM) against several cancer cell lines,
with the additional substitution at the 50 position enhancing
activity when compared with the 5-nitro-indirubin-30-oxime
analogue, 1 (IC50 = 1.2-25.5 μM) (Figure 1). However, there
are lack of reports regarding the synthesis and biological evalua-
tions exploring the effect of various substitutions at the
50 position of indirubin skeleton. We therefore performed
a molecular docking study to design and synthesize novel
5,50-substituted indirubin-30-oxime analogues and assessed the
biological properties of these molecules, including their CDK
inhibitory, antiproliferative, and in vivo anticancer activities.
Results and Discussion
Molecular Docking for Designing Strategy. To address the
mechanism behind the enhanced antiproliferative effect of
compound 2, we performed a molecular docking study on
the ATP binding site of CDK2 (Figure 2) using CDOCKER,
a CHARMm-based molecular dynamics docking program
Among the previously reported derivatives, 5-nitro-50-bromo-
indirubin-30-oxime, 2, showed the most potent antiproliferative

3698 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Scheme 1. Synthesis of 5-Substituted Indoxyl-N,O-diacetatea
Choi et al.
a Reagents and conditions. (i) ethyl glyoxalate, NaCNBH3, 1% acetic acid, methanol, room temp, 3 h, 85-95%; (ii) 1N NaOH (aq), methanol, room
temp, 1 h, 90%; (iii) acetic anhydride, Na2CO3, reflux, 4 h, 65-72%.
Scheme 2. Synthesis of 5,50-Substituted Indirubin-30-oxime Derivativesa
a Reagents and conditions. (i) Na2CO3, methanol or methanol: water (2:1), room temp, 2-3 h, 35-65%; (ii) H2NOH HCl, pyridine, reflux, 0.5-3 h,
3
65-70%.
(Discovery Studio 2.0). The X-ray cocrystal structure of
CDK2 with 5-bromoindirubin was obtained from the PDB
data bank (PDB code: 2BHE).31 After removing the ligand
from the structure of complex, a binding sphere was con-
and thus contribute to the more potent antiproliferative
activity of 2 compared with the 50-unsubstituted-indirubin-
30-oxime analogue, 1. Results of the docking study show that
derivatization at the 50-position, along with 5-substituents
(Cl, NO2, F, and OCF3) on the indirubin scaffold, may be an
ideal strategy for optimizing binding affinity to CDK2.
To investigate the structure-activity relationships at the 50
position of indirubin skeleton for CDK inhibitory and
antiproliferative activities, following groups were selected
for the derivatization: (1) 50-F and 50-Cl, which can form
similar electrostatic interaction with Lys89 in a manner
similar to that of the 50-Br analogue, 2, (2) 50-OH, which
can provide an additional hydrogen bond with CDK2
and introduce hydrophilic character at indirubin scaffold,
(3) 50-OCH3 and 50-CH3, expecting contrast effects com-
pared with halogen-substituted analogues.
˚
structed with a radius set as 8 A. The final binding con-
formation of 2 was determined through molecular dynamics
and final energy minimization. We found that the nitrogen at
position 10, the carbonyl oxygen at position 2, and the
nitrogen at position 1 of compound 2 each formed a hydro-
gen bond with the hinge segments (Glu81, Leu83) of the ATP
binding site (Figure 2a). In addition, the 30-hydroxyl group
of the oxime moiety formed a hydrogen bond with the
backbone carbonyl of Gln131 and the 5-nitro group con-
tributed to a salt bridge with the side chains of Lys33 and
Asp145. A depiction of the binding surface with electrostatic
˚
potential within 4 A around the ligand, 2, showed that the
50-Br group may be projected toward the outer aqueous
space of the ATP binding pocket in which a positively
charged amino acid residue, Lys89, may be a virtual binding
partner (Figure 2b). It could be hypothesized that an addi-
tional electrostatic interaction between the lone pair elec-
trons of the 50-Br group and the positively charged Lys89
residue may result in a higher binding affinity with CDK2
A docking model of a representative molecule, 5-nitro-
50-hydroxy-indirubin-30-oxime derivative (3a), in the ATP
binding site of CDK2 (Figure 2c) showed that a new hydro-
gen bond may be formed between the 50-OH group of 3a
and Asp86 in the solvent-accessible region of CDK2, with
the oxime moiety of 3a interacting with Ile10 instead of
Gln131. A depiction of the binding surface with electrostatic

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3699
Scheme 3. Synthesis of 5-Substituted-50-methoxy-indirubin-30-oxime Derivativesa
a Reagents and conditions. (i) CH3I, K2CO3, acetone, room temp, 4 h, 58-65%; (ii) H2NOH HCl, pyridine, reflux, 0.5-3 h, 65-70%.
3
Table 1. CDK2/Cyclin E iInhibitory Activities of 5,50-Substituted
Indirubin-30-oxime Derivatives
compd
R1
OH
R2
IC50 (nM)a
3a
3b
3c
NO2
Cl
1.91 ( 0.16b
5.27 ( 0.51
2.25 ( 0.25
8.32 ( 0.27
23.5 ( 4.34b
11.1 ( 0.89
10.1 ( 0.86
76.2 ( 2.31
1.71 ( 0.22
8.01 ( 0.31
1.83 ( 0.23
60.3 ( 11.5
8.68 ( 1.48
2,950 ( 530
4,120 ( 450
8,620 ( 870
7.35 ( 0.21
OH
OH
OH
Cl
F
3d
4a
4b
4c
OCF3
NO2
Cl
Cl
Cl
F
4d
5a
5b
5c
Cl
F
OCF3
NO2
Cl
F
Figure 3. CDK1 and CDK2 % activity in the presence of 1 μM
of 5,50-substituted indirubin-30-oxime derivatives. New indirubin
derivatives (1 μM) were tested for their inhibitory effects in the
kinase activity of CDK1/cyclin B and CDK2/cyclin E. 5-Nitroin-
dirubin-30-oxime (1) was also tested for comparison. Results are
presented as the mean ( standard error of the mean (SEM) of the %
kinase activity of CDK1 and CDK2.
F
F
5d
6a
16a
16b
16c
1
F
OCF3
NO2
NO2
F
CH3
OCH3
OCH3
OCH3
H
OCF3
NO2
0
˚
potential within 4 A around 3a showed that the 5 -OH
protrudes into the outer aqueous surface, solvent-accessible
region (Figure 2d).
a A series of indirubin derivatives were tested at 10 concentrations in
CDK2/cyclin E kinase assay, as described in the Experimental Section.
IC50 values were calculated from the dose-response curves. b IC50 values
of 3a and 4a in CDK1/cyline B kinase assay are 13 nM and 195 nM,
respectively .
Chemistry. To synthesize the 5,50-substituted-30-indirubin
oxime derivatives, the 5-substituted indoxyl-N,O-diacetates,
9a-d were prepared from their corresponding 5-substituted
anthranilic acids, 7a-d with hydroxyl, chloro, fluoro, and
methyl groups, respectively (Scheme 1). Briefly, each com-
pound of 7a-d was subjected to conditions of reductive
alkylation with ethylglyoxalate, followed by hydrolysis to
yield the dicarboxylic acids, 8a-d. These latter compounds
were finally cyclized in acetic anhydride32 to yield acetoxy,
chloro, fluoro, and methyl substituted compounds, 9a-d.
For the 5-hydroxy analogue, the cyclized compound 9a was
obtained as a triacetate form as the phenolic hydroxyl group
was also acetylated under the cyclization conditions.
As described previously,23 the 5,50-substituted-30-indiru-
bin oxime derivatives 3-6 were synthesized from conjugate
reactions of the 5-substituted indoxyl-N,O-diacetates 9a-d
with 5-nitro, chloro, fluoro, and trifluoromethoxy substi-
tuted isatin analogues 10a-d under basic conditions to yield
the 5,50-substituted indirubin derivatives 11-14, followed
by the reaction of each of the latter with hydroxylamine
to convert the ketone groups to 30-oxime (Scheme 2). The
5-substituted-50-methoxy-30-oxime compounds 16a-c were
synthesized from their corresponding 5-nitro, fluoro, and
trifluoromethoxy substituted 50-hydroxy-indirubin deriva-
tives 15a-c by methylation of the phenolic hydroxyl groups
and subsequent reaction with hydroxylamine (Scheme 3).
CDK Inhibitory Activity and Kinase Selectivity Profile. The
5,50-substituted-indirubin-30-oxime derivatives were initially
evaluated for their inhibitory activities against CDK1/
cyclinB and CDK2/cyclinE at 1 μM (Figure 3). 5-Nitroin-
dirubin-30-oxime, 1, was tested together for the comparison
of the effects of substitutions at the 50-position. The inhibi-
tory activity and selectivity of the synthesized compounds
for CDK2 over CDK1 were dependent on the nature of the
50-substitutents at the R1 position. In general, 5,50-substi-
tuted indirubin-30-oxime derivatives showed potent inhibi-
tory activities against CDK2 with more than 90% inhibitory
potency in 1 μM, except for 4d, 16a-c. Especially, 50-OH
analogues, 3a-d showed the highest inhibitory activities
against both CDK1 and CDK2. Although a substitution of
hydroxyl group at the R1 position resulted in decreased

3700 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Choi et al.
selectivity for CDK2 over CDK1, simultaneous inhibition of
CDK1 and CDK2 may have additional benefits in terms of
anticancer activity according to the literature.15,18,33 More-
over, the 50-OH group improved the water solubility of
compound 3a about 30-fold compared with compounds 1
or 2 (the data of water solubility of compounds 1, 2, and 3a
was shown in Supporting Information, Table S3). Low-level
water solubility has been a major problem among the
physicochemical properties of indirubin analogues (data
not shown). In contrast to the 50-OH-substituted indirubin-
30-oxime derivatives, the 50-chloro and 50-fluoro-substituted
derivatives 4a-d and 5a-d showed potent CDK2 inhibitory
activities, with 6-10-fold selectivity compared to CDK1.
For example, the 5-nitro-50-chloro-indirubin-30-oxime 4a
was about 8-fold more potent against CDK2 than CDK1
(Table 1). The decreased CDK1 inhibition resulting from
50-halogenation is further supported by results showing that
the 5-nitro-50-bromo-indirubin-30-oxime, 2, had decreased
CDK1 inhibitory activity (IC50 = 190 nM) when compared
with the 5-nitro-indirubin-30-oxime, 1.34 Among the series of
compounds with electron-donating groups at the 50 posi-
tion, 6a, with a 50-CH3 group, showed potent CDK2 inhibi-
tory activity, with 7-fold selectivity over CDK1. However, at
1 μM, the 50-OCH3-substituted derivatives (16a-c) displayed
weak or no inhibitory activities against CDK1 and CDK2.
The structure-activity relationships of 5,50-indirubin-30-
oxime derivatives for their CDK2 inhibitory activities were
analyzed with IC50 values (Table 1). We found that most
derivatives, except for the 50-OCH3 compounds 16a-c,
showed potent inhibitory activities, with IC50 values of
1-70 nM. Regarding the effects of 50-substituents at R1
Figure 4. Kinase panel screening of 3a, 4a. Compounds 3a and 4a
(10μM) were tested against more diverse panel of kinases comprising of
receptor tyrosine kinases, serine/threonine kinases, and lipid kinase.
Results are presented as the mean ( standard error of the mean (SEM)
of the % kinase activity. 3a showed 9% inhibitory activity against TrkA
at 1 μM. IC50 values of 3a against two serine/threonine kinases, Aurora
A (1 μM) and IKKβ (10 μM), and a receptor tyrosine kinase, TrkA
(4 μM) were calculated from the dose-response curves.
Table 2. Antiproliferative Activities of 5,50-Substituted Indirubin-30-oxime Derivatives on Different Human Cancer Cell Linesa
IC50 (μM)
compd
R1
OH
R2
A549b
HT1080c
HCT116d
K562e
SNU638f
KBg
1.33
10.2
4.62
MCF-7h
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d
NO2
Cl
3.33
16.4
4.09
16.9
15.6
>20
18.9
>20
0.57
>20
10.3
19.9
>20
>20
>20
>20
>20
6.71
>20
16.9
14.7
0.45
2.92
0.68
2.28
1.25
12.2
3.62
17.7
0.64
5.33
8.62
3.95
3.92
>20
>20
>20
>20
4.84
>20
1.45
16.8
0.44
3.51
1.66
2.25
0.33
1.86
1.72
3.79
0.28
3.31
13.5
1.51
1.41
18.8
>20
>20
>20
3.59
>20
0.45
17.8
0.91
12.2
1.42
5.91
0.56
17.2
14.7
>20
0.46
7.14
15.9
1.53
0.81
>20
>20
>20
>20
5.29
>20
0.77
>20
1.03
5.14
2.11
2.96
1.16
2.57
2.28
2.71
0.94
6.32
2.23
1.89
1.06
12.7
11.5
>20
>20
13.6
>20
1.14
9.77
1.21
10.7
2.61
4.23
0.51
6.14
3.52
4.23
0.91
7.13
6.57
4.12
1.06
8.79
10.5
7.31
>20
>20
13.2
0.95
14.7
OH
OH
OH
Cl
F
OCF3
NO2
Cl
5.81
1.11
Cl
Cl
11.9
F
3.62
2.83
0.92
9.11
Cl
F
OCF3
NO2
Cl
F
F
F
13.2
F
OCF3
NO2
Cl
3.81
2.11
5.18
CH3
CH3
CH3
CH3
OCH3
OCH3
OCH3
Br
F
10.6
8.13
>20
OCF3
NO2
F
16a
16b
>20
20
16c
2i
Roscovitinei
OCF3
NO2
1.16
30.1
a A series of indirubin derivatives were tested at five concentrations for their effects on various human cancer cell lines using SRB assay, as described in
the Experimental Section. The IC50 ( μM) values were calculated from dose response curves and SD was described in the Supporting Information.
b Human lung cancer cell. c Human fibro sarcoma cell. d Human colon cancer cell. e Human leukemia cell. f Human stomach cancer cell. g Human
nasopharyngeal cancer cell. h Human breast cancer cell. i Experiments were also performed with 2 and Roscovitine as positive control.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3701
displayed negligible inhibitory activities against all tested
kinases. As the IC50 values of 3a and 4a against CDK1 and
CDK2 were in the low nanomolar range, the kinase selecti-
vity profiles of these compounds may be significantly favor-
able for CDK1 and CDK2.
Antiproliferative Activity. We tested the ability of the
5,50-substituted indirubin-30-oxime derivatives to inhibit
the proliferation of several cancer cell lines, including A549,
HT1080, HCT116, K562, SNU638, KB, and MCF-7, using
SRB assay, and compared the data with the positive controls
5-nitro-50-bromo-indirubin-30-oxime (2) and Roscovitine
(Table 2).
We found that the antiproliferative activities of the
50-hydroxy derivatives 3a-d were well correlated with their
ability to inhibit CDK2, with activities depending on the
substituents at the 5 position, in the order NO2 > F > Cl,
OCF3. In particular, the 5-nitro-50-hydroxy-indirubin-30-
oxime 3a showed similar or greater inhibitory activity than
did compound 2, depending on the cell line tested, with IC50
values of ∼0.4 μM against the HT1080 and HCT116 cell
lines. Among compounds in which the 50-bromo group of 2
was replaced by other halogen groups, such as Cl or F, the
5-nitro analogues 4a and 5a were especially effective against
the HCT116 human colon cancer cell line in which cyclin D1
is deregulated,35 with IC50 values of approximately 0.3 μM.
The 5-nitro-50-fluoro-indirubin-30-oxime 5a showed the
broadest spectrum of inhibitory activity against all cancer
cell lines, with IC50 values of 0.28-0.94 μM, and the values
correlated with activity against CDK2. Compound 5a was
the only derivative to show a nanomolar IC50 value against
the A549 human lung cancer cell line, in contrast to the
weak antiproliferative activities of most other derivatives.
Although the majority of the indirubin-30-oxime derivatives
substituted with electron-donating groups such as 50-CH3 or
50-OCH3 (6 and 16) were very weak inhibitors of cancer cell
lines, the 5-nitro-50-methyl-30-oxime 6a had antiproliferative
IC50 values in the range of 0.8-1 μM. Regarding the sub-
stitutions at the 5-position (R2), the 5-NO2 group in combi-
nation with 50-OH, Cl, F, or CH3 showed the most potent
antiproliferative activity among the 5,50-disubsituted deri-
vatives tested. These findings are in good agreement with our
previous results23 and are supported by the hypothesized
binding mode of 2 and 3a, in which an additional salt bridge
may form between the 5-nitro group and Asp145 and Lys33
in the ATP binding site of CDK2. Among the 50-OCH3
derivatives, only the 5-F substituted analogue 16b displayed
moderate antiproliferative activity, indicating that this ana-
logue may have a different mechanism of action. Our
combined results suggest that 50-hydroxyl or halogen groups
may enhance the inhibition of CDK and cancer cell growth,
possibly by forming additional hydrogen bond or electro-
static interaction at the ATP binding site of CDK2.
Figure 5. Inhibition of tumor growth by 3a. (a) The SD-rats were
injected with RK3E-ras-Luc cells (5 ꢀ 106/100 μL). When the tumor
reached 5 mm in size, the rats were injected intravenously with 3a
(5 mg/kg) in nonanesthesia states every other day for a total of five
times. The tumor size was measured by caliper as described in the
Experimental Section. (b) Representative excised subcutaneous
tumor from the control and 3a treated rats. (c) In vivo biolumine-
scence imaging assay from the control and 3a treated rats.
position, the CDK2 inhibitory activities increased in the
following order: -OCH3 <-Cl < -H, -CH3 < -OH, -F.
For example, the CDK2 inhibitory activities of the 50-chloro-
substituted-indirubin-30-oxime compounds 4a-d were 10-fold
lower (IC50 = 10-76 nM) than were those of the 50-fluoro-
indirubin-30-oxime derivatives 5a-c (IC50 = 1-8 nM). Sub-
stitution of an electron-donating group, 50-OCH3 (16a-c),
dramatically reduced CDK2 inhibitory activities with IC50
values of 2-8 μM, whereas substitution of another electron-
donating group, 50-CH3 (6a), enhanced inhibitory activity
270-fold compared with 16b. Particularly, the most potent
CDK2 inhibitors, 3a, 5a, and 5c had IC50 values against
CDK2 of approximately 2 nM, thus demonstrating about a
4-fold greater potency than the 5-nitro-50-unsubstituted-indir-
ubin-30-oxime, 1 (IC50 = 7 nM). Analysis of the effects of
5-substitution at the R2 position of indirubin-30-oxime deriva-
tives showed that nitro or fluoro group generally enhanced
CDK2 inhibition, compared with chloro or trifluoromethoxy
group substitution. Together, our findings show that indirubin-
30-oxime analogues with combination of substituents providing
electronic effects, such as OH or F at the 50-position and NO2
or F at the 5-position, displayed potent inhibitory activities
against CDK2.
In Vivo Anticancer Activity of 3a. To assess the in vivo
activity of the indirubin analogue, we utilized an animal
tumor model in which Sprague-Dawley (SD) rats were
subcutaneously transplanted with RK3E-ras rat kidney cells
harboring the k-ras and GFP/Luc genes, as published by our
group.36 The 5-nitro-50-hydroxy analogue, 3a, was selected
for in vivo test because the improved water solubility of 3a
while maintaining the potent CDK inhibitiory and antipro-
liferative activity compared with other derivatives, which
usually precipitated in the bloodstream of animals when
administered through tail vein. Five days after transplanta-
tion, 5 mg/kg 3a was administered iv to each animal every
We further evaluated the representative potent and selec-
tive CDK2 inhibitors 3a and 4a against a diverse panel
of receptor tyrosine kinases, serine/threonine kinases, and
lipid kinase (Figure 4). Compound3a showed weak inhibitory
activity only against Aurora A (IC50=1 μM), IKKβ (IC50
=
10 μM), and TrkA (IC50 = 4 μM), whereas compound 4a

3702 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Choi et al.
Figure 6. Histology and immunohistochemistry of tumor tissue. RK3E-ras-Luc cells were inoculated sc on the left flank of rats. 3a (5 mg/kg) was
injected intravenously into the tumor bearing rats every other day beginning from day 5. The rats were sacrificed on day 11. (a) H&E staining of tumor
sections (left), TUNEL assay on paraffin sections from solid tumor (center), and immunohistochemical staining for PCNA (right), (b) positive cells for
TUNEL assay, (c) positive cells for PCNA immunostaining. Cells were counted in triplicate. Columns, mean; bars, SD.
other day with a total of five times and the rats were
monitored daily. We found that tumor volume was signifi-
cantly decreased, by about 84.3%, compared with control
rats (Figure 5a,b). There was no significant difference in
body weight between 3a-treated and control rats (data not
shown). Bioluminescence images obtained on day 10 after
treatment with 3a indicated that luminescence intensity, an
indicator of tumor growth, was significantly decreased
(Figure 5c).
Histological analysis showed that untreated control solid
tumors were anaplastic undifferentiated carcinomas, show-
ing many mitotic figures, multifocal necrosis, and hemor-
rhage. Tumor treatment with 3a, however, led to extensive
cell death (Figure 6a, left) and a 6-fold increase in the number
of TUNEL-positive apoptotic cells (Figure 6a, center, and
Figure 6b). In contrast, the level of expression of PCNA, a
cell proliferation marker, was lower in 3a-treated animals
than in controls (Figure 6a, right, and Figure 6c). In addi-
tion, a result of cell cycle analysis using FACS showed that 3a
inhibited cell cycle progression at the G0/G1 phase in RK3E-
ras rat kidney cells (The data of cell cycle distribution is
shown in Supporting Information, Figure S1).
These results indicate that 3a suppresses tumor growth in
vivo by inhibiting cell proliferation and activating apoptosis.
Although the in vitro and in vivo anticancer activity of 3a
was correlated with the CDK inhibitory activity, a possibility
of combined mechanisms of the action could be speculated
from several reports of molecular mechanisms of indirubin
derivatives in anticancer activity such as inhibition of
Notch-1 or Stat3 signaling and promoting caspase activity
via a mitochondria-mediated pathway.27,28,37,38
Conclusions
In summary, we describe here the design and synthesis of
several novel 5,50-substituted-indirubin-30-oxime compounds
as potent inhibitors of CDK1 and CDK2 and with antiproli-
ferative activities against several cancer cell lines. The enhanced
inhibitory activities of these 5,50-substituted derivatives, com-
pared with the 50-unsubstituted indirubin-30-oxime 1, could be
interpreted based on the results of a molecular docking study at
the ATP binding site of CDK2, showing that the substi-
tuted derivates showed additional projections into the solvent-
accessible region and additional interactions with the amino
acid residues in the ATP binding pocket, as exemplified by the
50-hydroxy analogue 3a. The most potent CDK2 inhibitors
were 5-nitro-50-hydroxy-indirubin-30-oxime (3a) and 5-nitro-
50-fluoro-indirubin-30-oxime (5a), which had IC50 values of
1.91 and 1.71 nM, respectively, and IC50 values in the range
of 0.2-3.3 μM when tested as antiproliferative agents against
several human cancer cell lines. Structure-activity relationship
analysis showed that OH and halogen groups were preferred to
the electron donating groups such as 50-CH3, 50-OCH3 at 50
position. A representative analogue, 5-nitro-50-hydroxy-
indirubin-30-oxime (3a), showed greater than 500-fold
selectivity for CDK relative to selected kinase panel, and
significant in vivo anticancer activity. These findings may
contribute to the design and development of CDK inhibi-
tors and facilitate clinical application.
Experimental Section
Chemistry. 1H NMR spectra were determined with a
JEOL JNM-LA 300WB spectrometer at 300 MHz or JEOL

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3703
JNM-ECX 400P spectrometer at 400 MHz, and spectra were
taken in CDCl3 or DMSO-d6 or acetone-d6. Unless other-
wise noted, chemical shifts are expressed as ppm downfield
from internal tetramethylsilane, or relative ppm from DMSO
(2.5 ppm), acetone (2.04 ppm). Mass spectroscopy was carried
out on electrospray and high-resolution mass spectra (m/z) were
recorded on a FAB, EI (JEOL: mass range 2600 amu, 10 kV
acceleration) and ESI. High-resolution mass analysis was per-
formed at Korea Basic Science Institute (Daegu).
(6.6 mg, 0.095 mmol) was added. The reaction mixture was
heated under reflux at 120 ꢀC for 2 h. After cooling, the solution
was acidified with 1N HCl and the resulting precipitate was
filtered and washed with water to afford quantitatively the
corresponding 30-oxime selectively in a (20Z,30E) form. The
residue was purified by silica gel column chromatography
(chloroform:methanol = 20:1) to give 3-6 (7 mg, 66%).
Data for (20Z,30E)-5-Nitro-50-hydroxy-indirubin-30-oxime
(3a). 1H NMR (DMSO, 300 MHz, δ ppm, J in Hz) 13.87 (1H,
s, NOH), 11.78 (1H, s, N-H), 11.35 (1H, s, N0-H), 9.41 (1H, d,
J = 2.8 Hz, H-4), 9.32 (1H, s, O-H), 8.05 (1H, dd, J = 11.6, 2.8
Hz, H-6), 7.76 (1H, d, J = 3.2 Hz, H-40), 7.29 (1H, d, J = 11.6 Hz,
H-7), 7.04 (1H, d, J = 11.2 Hz, H-70), 6.86 (1H, dd, J = 11.2,
3.2 Hz, H-60). HRMS (EI) [M]þ• (C16H10N4O5): calcd 338.0651,
found 338.0648.
The purity of all final products was determined by HPLC
(at least 95% purity unless otherwise noted). The determination
of purity was performed on a Shimadzu SCL-10A VP HPLC
system using a Shimadzu Shim-pack C18 analytical column
˚
(250 mm ꢀ 4.6 mm, 5 mm, 100 A) in linear gradient solvent
systems. Solvent system was H2O:CH3CN = 65:35 to 5:95 over
30 min at a flow rate = 1 mL/min. Peaks were detected by UV
absorption using a diode array detector.
Data for (20Z,30E)-5-Chloro-50-hydroxy-indirubin-30-oxime
(3b). 1H NMR (DMSO, 400 MHz, δ ppm, J in Hz) 11.71 (1H,
s, N-H), 10.78 (1H, s, N0-H), 9.27 (1H, s, O-H), 8.59 (1H, d, J =
2 Hz, H-4), 7.74 (1H, d, J = 2 Hz, H-40), 7.24 (1H, d, J = 8.4 Hz,
H-7), 7.11 (1H, dd, J = 8, 2 Hz, H-60), 6.87 (1H, d, J = 8.4 Hþz•,
H-6), 6.85 (1H, d, J = 8 Hz, H-70). HRMS (EI) [M]
(C16H10ClN3O3): calcd 327.0411, found 327.0408. Purity 92%.
Data for (20Z,30E)-5-Fluoro-50-hydroxy-indirubin-30-oxime
(3c). 1H NMR (DMSO, 400 MHz, δ ppm, J in Hz) 11.69 (1H,
s, N-H), 10.66 (1H, s, N0-H), 9.25 (1H, s, O-H), 8.42 (1H, dd, J =
11.4, 2.8 Hz, H-6), 7.73 (1H, d, J = 2.4 Hz, H-40), 7.24 (1H, d,
J = 8.4 Hz, H-60), 6.81-6.99 (3H, m, H-70, H-4, H-7). HRMS
(EI) [M]þ• (C16H10FN3O3): calcd 311.0706, found 311.0707.
Purity 93%.
General Procedure for the Preparation of 5-Substituted indoxyl-
N,O-diacetate 9a-d. To a solution of 5-substituted anthranilic
acid (1.0 g, 6.45 mmol) in 50 mL of methanol was added 0.5 mLof
acetic acid, followed by ethyl glyoxalate (1 mL, 9.7 mmol) and
NaCNBH3 (609.5 mg, 9.7 mmol). The reaction mixture was
stirred for 3 h at room temperature. Then the methanol was
removed by evaporation. The residue was taken up in solution of
saturated NH4Cl in water and extracted with ethyl acetate. The
combined extracts were dried over sodium sulfate, filtered, and
evaporated. The residue was purified by silica gel column chro-
matography (CH3Cl:methanol = 30:1) to give ester product
(1.4 g, 95%) and then this compound (1.4 g, 5.8 mmol) was
hydrolyzed in 25 mL of 1 N NaOH(aq) and 10 mL of methanol.
The reaction mixture was stirred for 1 h at roomtemperature, and
the resulting solution was acidified by 1N HCl. The resulting
precipitate (1.2 g, 90%) was collected by filtration and washed
with water. To the obtained diacid compounds 8a-d (1.2 g,
4.97 mmol) was added acetic anhydride (15 mL) and Na2CO3
(1.3 g, 12.4 mmol). The reaction mixture was refluxed for 4 h, and
the product was extracted with ethyl acetate and washed with
water. The combined extracts were dried over sodium sulfate,
filtered, and evaporated. The residue was purified by silica gel
column chromatography (hexane:ethyl acetate =2:1) to give
9a-d with 5-acetoxy, chloro, fluoro, and methyl groups, respec-
tively (800 mg, 68%).
Data for (20Z,30E)-5-Trifluoromethoxy-50-hydroxy-indirubin-
1
30-oxime (3d). H NMR (DMSO, 400 MHz, δ ppm, J in Hz)
11.70 (1H, s, N-H), 10.78 (1H, s, N0-H), 9.22 (1H, s, O-H), 8.50
(1H, s, H-4), 7.70 (1H, d, J = 2 Hz, H-40), 7.21 (1H, d, J =
8.8 Hz, H-70), 7.03 (1H, d, J = 8.4 Hz, H-7), 6.88 (1H, d, J = 8.4
Hz, H-6), 6.80 (1H, dd, J = 8.8, 2.4 Hz, H-60). HRMS (ESI)
[M - H]- (C17H9F3N3O4): calcd 376.0545, found 376.0546.
Data for (20Z,30E)-5-Nitro-50-chloro-indirubin-30-oxime (4a).
1H NMR (DMSO, 400 MHz, δ ppm, J in Hz) 11.98 (1H, s,
N-H), 11.39 (1H, s, N0-H), 9.45 (1H, s, H-4), 8.25 (1H, s, H-40),
8.08 (1H, d, J = 7.6 Hz, H-6), 7.50 (2H, m, H-60, H-7), 7.05 (1H,
d, J = 8.4 Hz, H-70). HRMS (ESI) [M - H]- (C16H8ClN4O4):
calcd 355.0234, found 355.0242.
Data for (20Z,30E)-5-Chloro-50-chloro-indirubin-30-oxime (4b).
1H NMR (acetone, 300 MHz, δ ppm, J in Hz) 13.05 (1H, s,
NOH), 11.82 (1H, s, N-H), 9.81 (1H, s, N0-H), 8.67 (1H, s, H-4),
8.35 (1H, d, J = 1.8 Hz, H-40), 7.45 (2H, m, H-6, H-7), 7.16 (1H,
d, J = 8.4 Hz, H-60), 6.97 (1H, d, J = 8.4 Hz, H-70). HRMS (EI)
[M]þ• (C16H9Cl2N3O2): calcd 345.0072, found 345.0075.
Data for (20Z,30E)-5-Fluoro-50-chloro-indirubin-30-oxime (4c).
1H NMR (acetone, 300 MHz, δ ppm, J in Hz) 12.96 (1H, s,
NOH), 11.78 (1H, s, N-H), 9.71 (1H, s, N0-H), 8.41 (1H, d, J =
11.4 Hz, H-7), 8.32 (1H, s, H-4), 7.40-7.50 (2H, m, H-40, H-60),
6.88-6.91 (2H, m, H-6, H-70). HRMS (ESI) [M - H]- (C16H8-
ClFN3O2): calcd 328.0289, found 328.0291.
Data for (20Z,30E)-5-Trifluoromethoxy-50-chloro-indirubin-30-
oxime (4d). 1H NMR (acetone, 300 MHz, δ ppm, J in Hz) 13.02
(1H, s, NOH), 11.82 (1H, s, N-H), 9.88 (1H, s, N0-H), 8.63 (1H, s,
H-4), 8.34 (1H, d, J = 2.1 Hz, H-40), 7.47 (2H, m, H-6, H-7),
7.11 (1H, d, J = 8.4 Hz, H-60), 7.03 (1H, d, J = 8.4 Hz, H-70).
HRMS (ESI) [M - H]- (C17H8ClF3N3O3): calcd 394.0206,
found 394.0215.
Data for 1-Acetyl-1H-indole-3, 5-diyl Diacetate (9a). 1H NMR
(CDCl3, 300 MHz, δ ppm, J in Hz) 8.46 (1H, d, J = 9.3 Hz), 7.76
(1H, s), 7.30 (1H, d, J = 2.4 Hz), 7.11 (1H, dd, J = 9.3, 2.4 Hz),
2.67 (3H, s), 2.36 (3H, s), 2.31 (3H, s). ESI [M - H]-: 273.83.
Data for 1-Acetyl-5-chloro-1H-indol-3-yl Acetate (9b). 1H
NMR (CDCl3, 400 MHz, δ ppm, J in Hz) 8.40 (1H, d, J =
8.8 Hz), 7.75 (1H, s), 7.53 (1H, d, J = 2 Hz), 7.34 (1H, dd, J =
8.8, 2 Hz), 2.61 (3H, s), 2.39 (3H, s). ESI [M - H]-: 249.83.
Data for 1-Acetyl-5-fluoro-1H-indol-3-yl Acetate (9c). 1H
NMR (CDCl3, 400 MHz, δ ppm, J in Hz) 8.42 (1H, m), 7.75
(1H, s), 7.19 (1H, dd, J = 8.2, 2.4 Hz), 7.10 (1H, td, J = 8.8, 2.4
Hz), 2.59 (3H, s), 2.37 (3H, s). ESI [M - H]-: 233.88.
Data for 1-Acetyl-5-methyl-1H-indol-3-yl Acetate (9d). 1H
NMR (CDCl3, 400 MHz, δ ppm, J in Hz) 8.31 (1H, d, J =
7.6 Hz), 7.65 (1H, s), 7.31 (1H, s), 7.19 (1H, d, J = 7.6 Hz), 2.57
(3H, s), 2.44 (3H, s), 2.36 (3H, s). ESI [M - H]-: 229.80.
General Procedure for the Preparation of 5,50-Substituted
Indirubin-30-oxime Derivatives 3-6. To a solution of 5-nitro,
chloro, fluoro, and trifluoromethoxy substituted isatin ana-
logues 10a-d (77 mg, 0.425 mmol) in methanol (5 mL) were
added 5-substituted indoxyl-N,O-diacetate 9a-d (100 mg,
0.425 mmol) and the mixture was stirred for 5 min. Anhydrous
Na2CO3 (112.5 mg, 1.06 mmol) was added, and the stirring was
continued for 3 h at room temperature. The dark precipitate was
filtered and washed with cold water and dried under reduced
pressure to give derivatives of 5,50-substituted indirubin (63 mg,
47%), 11-14. The indirubin derivative (10 mg, 0.032 mmol) was
dissolved in pyridine (0.3 mL), and hydroxylamine hydrochloride
Data for (20Z,30E)-5-Nitro-50-fluoro-indirubin-30-oxime (5a).
1H NMR (DMSO, 400 MHz, δ ppm, J in Hz) 11.89 (1H, s,
N-H), 11.40 (1H, s, N0-H), 9.43 (1H, d, J = 2 Hz, H-4), 8.08 (1H,
dd, J = 8.6, 2 Hz, H-6), 8.00 (1H, dd, J = 8.6, 2 Hz, H-7), 7.51
(1H, m, H-40), 7.33 (1H, td, J = 9, 2.8 Hz, H-60), 7.06 (1H, d, J =
8.4 Hz, H-70). HRMS (EI) [M]þ• (C16H9FN4O4): calcd 340.0608,
found 340.0610.
Data for (20Z,30E)-5-Chloro-50-fluoro-indirubin-30-oxime (5b).
1H NMR (DMSO, 400 MHz, δ ppm, J in Hz) 11.81 (1H, s,

3704 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Choi et al.
N-H), 10.85 (1H, s, N0-H), 8.62 (1H, d, J = 2.4 Hz, H-4), 7.97 (1H,
dd, J = 8.2, 2.4 Hz, H-6), 7.47 (1H, m, H-40), 7.31 (1H, m, H-7),
7.15 (1H, dd, J = 8.4, 2 Hz, H-60), 6.88 (1H, d, J = 8.4 Hz, H-70).
HRMS (ESI) [M - H]- (C16H8ClFN3O2): calcd 328.0289, found
328.0295.
Data for (20Z,30E)-5-Fluoro-50-fluoro-indirubin-30-oxime (5c).
1H NMR (DMSO, 400 MHz, δ ppm, J in Hz) 11.76 (1H, s,
N-H), 10.68 (1H, s, N0-H), 8.41 (1H, dd, J = 11.2, 2.4 Hz, H-4),
7.93 (1H, dd, J = 8.8, 2.4 Hz, H-40), 7.39 (1H, m, H-7), 7.30
(1H, td, J = 8.8, 2 Hz, H-6), 6.93 (1H, td, J = 9, 2.4 Hz, H-60),
6.84 (1H, m, H-70). HRMS (EI) [M]þ• (C16H9F2N3O2): calcd
313.0663, found 313.0666. Purity 93%.
Data for (20Z,30E)-5-Trifluoromethoxy-50-fluoro-indirubin-30-
oxime (5d). 1H NMR (DMSO, 400 MHz, δ ppm, J in Hz) 12.02
(1H, s, N-H), 10.72 (1H, s, N0-H), 8.59 (1H, s, H-4), 8.02 (1H, dd,
J = 8.8, 2.4 Hz, H-6), 7.41 (1H, m, H-40), 7.18 (1H, td, J = 9.2, 2
Hz, H-6), 7.00 (1H, d, J = 8.4 Hz, H-7), 6.9 (1H, d, J = 8.4 Hz,
H-70). HRMS (FAB) (C17H9O3N3F4): calcd 379.0579, found
379.0580.
Data for (20Z,30E)-5-Nitro-50-methyl-indirubin-30-oxime (6a).
1H NMR (DMSO, 400 MHz, δ ppm, J in Hz) 11.87 (1H, s,
N--H), 11.38 (1H, s, N0-H), 9.46 (1H, d, J = 2.4 Hz, H-4), 8.11
(1H,s, H-40), 8.07 (1H, dd, J = 8, 2.4 Hz, H-6), 7.37 (1H, d, J =
8 Hz, H-7), 7.26 (1H, d, J = 8.8 Hz, H-60), 7.05 (1H, d, J = 8.8 Hz,
H-70), 2.32 (3H, s, CH3). HRMS (ESI) [M - H]- (C17H11N4O4):
calcd 335.0780, found 335.0787. Purity 93%.
J = 7.8, 2.4 Hz, H-60), 3.39 (3H, s, OCH3). HRMS (ESI) [M - H]-
(C17H11N4O5): calcd 351.0729, found 351.0724. Purity 94%.
Data for (20Z,30E)-5-Fluoro-50-methoxy-indirubin-30-oxime
(16b). 1H NMR (DMSO, 400 MHz, δ ppm, J in Hz) 11.68
(1H, s, N-H), 9.22 (1H, s, N0-H), 8.44 (1H, m, H-4), 7.70 (1H, d,
J = 2.4 Hz, H-40), 7.21 (1H, d, J = 8.8 Hz, H-6), 6.96 (2H, m,
H-7, H-70), 6.79 (1H, dd, J = 8.6, 2.4 Hz, H-60), 3.26 (3H, s,
OCH3). HRMS (EI) [M]þ• (C17H12FN3O3): calcd 325.0863,
found 325.0859.
Data for (20Z,30E)-5-Trifluoromethoxy-50-methoxy-indirubin-
1
30-oxime (16c). H NMR (acetone, 400 MHz, δ ppm, J in Hz)
11.70 (1H, s, N-H), 8.62 (1H, s, H-4), 8.28 (1H, s, N0-H), 7.90
(1H, d, J = 2.4 Hz, H-40), 7.24 (1H, d, J = 8.4 Hz, H-6), 7.12
(1H, m, H-70), 7.04 (1H, d, J = 8.4 Hz, H-7), 6.99 (1H, dd, J =
8.4, 2.4 Hz, H-60), 3.33 (3H, s, OCH3). HRMS (EI) [M]þ•
(C18H12F3N3O4): calcd 391.0780, found 391.0782.
Molecular Docking. Molecular docking was performed using
CDOCKER, a CHARMm-based molecular dynamics docking
algorithm on Discovery Studio 2.0 (Accelrys). The CDK2
structure cocrystallized with 5-bromo-indirubin was obtained
from the PDB data bank (PDB code: 2BHE).31 A protein clean
process and a CHARMm-force field were sequentially applied.
The area around 5-bromo-indirubin was chosen as the active
˚
site, with a radius set as 8 A. After removing the ligand from the
structure of the complex, a binding sphere in the three axis
directions was constructed around the active site. All default
parameters were used in the docking process. CHARMm-based
molecular dynamics (1000 steps) were used to generate random
ligand 3a conformations, and the position of any ligand 3a was
optimized in the binding site using rigid body rotation followed
by simulated annealing at 700 K. Final energy minimization was
set as the full potential mode. The final binding conformation of
3a was determined on the basis of energy. Docking study of
ligand 2 was carried out as described above. The most stable
binding mode among the top 10 of docking poses of each
compound was presented in Figure 2. The other binding modes
of compounds 2, 3a were not identified because of their similar
docking poses, and the differences of calculated CDOCKER
interaction energy were in the range of 4 kcal/mol.
Biological Methods. Enzyme Assay. CDK1/cyclin B, CDK2/
cyclin E activities were assayed by using radiometric filter
binding format.39 CDK1/cyclin B was purchased from Milli-
pore (Billerica, MA). In final reaction volume of 25 μL, CDK1/
cyclin B was incubated in buffer (8 mM MOPS pH 7.0, 0.2 mM
EDTA, 10 mM Mg acetate), with 0.1 mg/mL histone H1 and
45 μM [γ-33P] ATP (500 cpm/pmol). The reaction was initiated
by the addition of the MgATP mix. After incubation for 40 min
at 30 ꢀC, the reaction was stopped by the addition of 5 μL of 3%
phosphoric acid solution. Ten μL aliquots of supernatant were
spotted onto 2.5 cm ꢀ3 cm pieces of Whatman P30 phospho-
cellulose paper, and 20 s later, the filters were washed three times
for 5 min in 75 mM phosphoric acid and once in methanol prior
to drying. The wet filters were counted in the presence of 1 mL of
ACS (Amersham) scintillation fluid.
CDK2/cyclin E was purchased from Millipore (Billerica,
MA). The kinase activity was tested in buffer (8 mM MOPS
pH 7.0, 0.2 mM EDTA, 10 mM Mg acetate), with 0.1 mg/mL
histone H1, in the presence of 120 μM [γ-33P] ATP (500 cpm/
pmol) in a final reaction volume of 25 μL. The reaction was
initiated by the addition of the MgATP mix. After incubation
for 40 min at 30 ꢀC, the reaction was stopped by the addition of
5 μL of 3% phosphoric acid solution. Ten μL aliquots of
supernatant were processed as described above.
Inhibition of kinase activity against a variety of recombinant
kinases (c-Met, Met M1250T, Ron, VEGFR2, EGFR, TrkA,
PI3Kγ, IKKβ, Insulin receptor, Aurora A) was determined by
measuring the phosphorylation of substrate peptide or lipid in
homogeneous time-resolved fluorescence (HTRF) assays.40 The
inhibition of tyrosine kinases, c-Met, Met M1250T, Ron,
VEGF2, EGFR, TrkA, and insulin receptor was determined
Data for (20Z,30E)-5-Chloro-50-methyl-indirubin-30-oxime (6b).
1H NMR (DMSO, 400 MHz, δ ppm, J in Hz) 11.85 (1H, s, N-H),
10.76 (1H, s, N0-H), 8.64 (1H, s, H-4), 8.10 (1H,s, H-40), 7.29 (1H,
d, J = 8.4 Hz, H-6), 7.20 (1H, d, J = 8.4 Hz, H-7), 7.10 (1H, d,
J = 8.2 Hz, H-60), 6.86 (1H, d, J = 8.4 Hz, H-70), 2.31 (3H, s,
CH3). HRMS (EI) [M]þ• (C17H12ClN3O2): calcd 325.0618, found
325.0623.
Data for (20Z,30E)-5-Fluoro-50-methyl-indirubin-30-oxime (6c).
1H NMR (DMSO, 400 MHz, δ ppm, J in Hz) 11.78 (1H, s, N-H),
10.61 (1H, s, N0-H), 8.43 (1H, dd, J = 11.2, 2.4 Hz, H-4), 8.06
(1H,s, H-40), 7.26 (1H, d, J = 8.4 Hz, H-7), 7.17 (1H, d, J = 8 Hz,
H-70), 6.86 (1H, td, J = 8.4, 2.4 Hz, H-6), 6.8þ0•(1H, d, J = 8 Hz,
H-60), 2.28 (3H, s, CH3). HRMS (EI) [M] (C17H12FN3O2):
calcd 309.0914, found 309.0912.
Data for (20Z,30E)-5-Trifluoromethoxy-50-methyl-indirubin-
1
30-oxime (6d). H NMR (DMSO, 400 MHz, δ ppm, J in Hz)
11.80 (1H, s, N-H), 10.87 (1H, s, N0-H), 8.59 (1H, d, J = 2 Hz,
H-4), 8.09 (1H,s, H-40), 7.34 (1H, d, J = 8.4 Hz, H-6), 7.25 (1H,
d, J = 8 Hz, H-7), 7.10 (1H, d, J = 7.4 Hz, H-60), 6.94 (1H, d,
J = 8.4 Hz, H-70), 2.33 (3H, s, CH3). HRMS (EI) [M]þ• (C18H12-
F3N3O3): calcd 375.0831, found 375.0833.
General Procedure for the Preparation of 5-Substituted-50-
methoxy-indirubin-30-oxime Derivatives 16a-c. To a solution
of 50-hydroxy-indirubin analogues, 11d (10 mg, 0.028 mmol)
in acetone (1 mL) were added dropwise methyl iodide (17 μL,
0.276 mmol) and K2CO3 (19 mg, 0.138 mmol). The mixture was
stirred for 4 h at room temperature. The dark precipitate was
filtered and washed with cold water and dried under reduced
pressure to give derivatives of 5-substituted-50-methoxy-
indirubin (6 mg, 60%), 15c. The indirubin derivative (6 mg,
0.016 mmol) was dissolved in pyridine (0.3 mL), and hydro-
xylamine hydrochloride (3.4 mg, 0.048 mmol) was added. The
reaction mixture was heated under reflux at 120 ꢀC for 1 h.
After cooling, the product was acidified with 1N HCl and the
precipitate was filtered and washed with water to afford
quantitatively the corresponding 30-oxime selectively in a
(20Z,30E) form. The residue was purified by silica gel column
chromatography (hexane:ethyl acetate = 2:1) to give 16a-c
(4 mg, 65%).
Data for (20Z,30E)-5-Nitro-50-methoxy-indirubin-30-oxime
1
(16a). H NMR (DMSO, 400 MHz, δ ppm, J in Hz) 11.82(1H,
s, N-H), 9.46 (1H, d, J = 2.4Hz, H-4), 9.33 (1H, s, N0-H), 8.12 (1H,
dd, J = 8.4, 2.4 Hz, H-6), 7.77 (1H, d, J = 2.4 Hz, H-40), 7.30 (1H,
d, J = 8.4 Hz, H-7), 7.24 (1H, d, J = 9.2 Hz, H-70), 6.85 (1H, dd,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3705
by measuring the phosphorylation of poly-Glu-Tyr-biotin
(pGT-biotin, Cisbio) peptide, HTRF KinEASE kit. Recombi-
nant proteins containing kinase domain were purchased from
Millipore (Billerica, MA). Into a black 384-well Costar plate was
added 2 μL/well of serially diluted compound with kinase buffer
(250 mM HEPES (pH 7.0), 0.5 mM orthovanadate, 0.05% BSA,
0.1% NaN3). Next, 2 μL of TK peptide substrate and 1 μL of
kinase (10 ng) were added to each well. After 15 min preincuba-
tion, the kinase reaction was initiated by the addition of 5 μL of
ATP (2ꢀ the required final concentration) in reaction buffer,
after which the plate was incubated at room temperature for
30 min. The reaction was stopped by the addition of 5 μL of
TK-antibody-Eu(K) (Eu-labeled antiphosphotyrosine anti-
body, Cisbio) in HTRF detection buffer (50 mM HEPES
(pH 7.0), 0.05% BSA, 0.1% NaN3, and 100 mM EDTA) and
5 μL of streptavidin-XL-665 in HTRF detection buffer. After
additional 1 h incubation at room temperature, the plate
was read in an EnVision multilabel reader (Perkin-Elmer,
Waltham, MA).
Recombinant proteins for IKKβ and Aurora A were pur-
chased from Millipore (Billerica, MA). Assay was processed as
described above, except for using the STK substrate-biotin-2
peptide (Aurora A), STK substrate-biotin-3 peptide (IKKβ).
The followings are ATP and supplement concentration for
each enzyme: VEGFR2 (15 μM ATP, 5 mM MgCl2, 1 mM
MnCl2, and 1 mM DTT), c-MET and MET M1250T (10 μM
ATP, 5 mM MgCl2, and 1 mM DTT), Ron (5 μM ATP, 5 mM
MgCl2, 1 mM MnCl2, and 1 mM DTT), TrkA (1 μM ATP,
5 mM MgCl2, and 1 mM DTT), insulin receptor (20 μM ATP,
5 mM MgCl2, 1 mM MnCl2, and 1 mM DTT), IKKβ (2 μM
ATP, 2 mM MnCl2, and 1 mM DTT), Aurora A (100 μM ATP,
5 mM MnCl2, and 1 mM DTT), and EGFR (2 μM ATP, 5 mM
MgCl2, 1 mM MnCl2, and 1 mM DTT).
report.36 Male SD rats (6 weeks of age) were used to examine
the inhibition of tumor growth in vivo. RK3E-ras-Luc cells (5 ꢀ
106) were implanted subcutaneously into the flank of the rats on
day 0, and rats were grouped randomly (5 mice/group) on day 5
as previously described.36 A solution of 3a in a mixture of
PEG400, EtOH, and DW (30:33:37, 3 mg/mL, 5 mg/kg) were
delivered intravenously in a nonanesthesia state every other day
for a total of five times. The animals were sacrificed 48 h after
the final administration, and rat weights and tumor volumes
were measured. The tumor growth volumes were calculated
as follows: V = (ab2)/2, where, a is the longest diameter and b
is the shortest diameter of the tumor.36 All experiments were
conducted under protocols approved by the Animal Care and
Use Committee at Chosun University School of Dentistry
(Gwangju, South Korea).
Bioluminescence Imaging Assay. For bioluminescence ima-
ging, the rats received an ip injection of luciferin (Molecular
Probes, Palo Alto, CA) with Rompun/ketamine (1:1) anesthe-
sia. The rats were imaged using a LAS-1000 plus imaging System
(Fuji film. Tokyo, Japan) to record the bioluminescent signal
emitted from the tumor. The LAS-1000 system equipped with a
CCD camera was used for acquisition of the emitted light, and
Living Image software (Multi Gauge v3.0) was used for data
analysis.
Histology, Immunohistochemistry, and TUNEL Assay. The
excised solid tumors were fixed in 10% buffered formalin and
embedded in paraffin. For optical microscopic examinations,
4 μm sectioned tissues were stained with H&E. Immunohi-
stochemical staining was performed with the avidin-biotin
complex method using anti-PCNA antibodies. The immune
reactions were visualized with 3,30-diaminobenzidine and coun-
terstained with Mayer’s hematoxylin. A TUNEL assay was
performed using an ApopTag Plus peroxidase in situ apoptosis
detection kit (Intergen, Purchase, NY) according to the manu-
facturer’s instructions. Briefly, the slides were deparaffinized
and treated with 20 μg/mL proteinase K at 37 ꢀC for 15 min to
enhance staining. After immersion in 3% hydrogen peroxide
to block the endogenous peroxidase, the slides were incubated
with a reaction buffer containing terminal deoxynucleotidyl
transferase at 37 ꢀC for 1 h. The slides were then incubated
with a peroxidase-conjugated antidigoxigenin antibody for
30 min, and the reaction products were visualized with a
0.03% 3,30-diaminobenzidine solution containing 2 mM hydro-
gen peroxide. The slides were counterstained with 0.5% methyl
green. The PCNA and TUNEL-positive cells were counted and
represented as the average of the five highest areas within a
single ꢀ200 field.36
Inhibition of PI3Kγ activity was measured by commercial kit
from upstate (PI3-kinase (human) HTRF assay, catalogue no.
33-016). PI3K catalyzes the phosphorylation of phosphoinositol
4,5-biphosphate (PIP2) to phosphoinositol 3,4,5-triphosphate
(PIP3). The PIP3 product formed by PI3K activity displaces
biotin-PIP3 resulting in a loss of energy transfer and thus a
decrease in signal. Kinase reaction was performed exactly as
described in the manufacturer’s instruction.
Cell Proliferation Assay. Cells (A549, HT1080, HCT116,
K562, SNU638, KB, MCF-7) were counted, diluted to 5 ꢀ 104
cells/mL with fresh medium (MEME, DMEM, or RPMI con-
taining 10% FBS), and added 190 μL of cell suspension to
96-well plates containing various concentrations of test com-
pounds (10 μL in 10% aq DMSO).41 Test plates were incubated
for 3 days at 37 ꢀC in CO2 incubator. For zero day controls, cells
were incubated for 30 min at 37 ꢀC in CO2 incubator. All
treatments were performed in triplicate. After the incubation
periods, cells were fixed with 50 μL of cold 50% TCA at 4 ꢀC for
30 min, washed 5 times with tap water, and air-dried. The fixed
cells were stained with 0.4% SRB solution in 1% aqueous acetic
acid at room temperature for 1 h. Free SRB solution was then
removed by rinsing 5 times with 1% acetic acid, and air-dried.
The bound dye was dissolved with 200 μL of 10 mM Tris-base
(pH 10.0), and absorbance was determined at 515 nm using
an ELISA microplate reader. Finally, the absorbance values
obtained with each of the treatment procedures were averaged,
and the average values obtained with the zero day control were
subtracted. These results were expressed as a percentage, relative
to solvent treated control incubations, and IC50 values were
calculated using nonlinear regression analysis (percent survival
versus concentration).
Acknowledgment. This work was supported by a grant of
the National R&D Program for Cancer Control, Ministry of
Health & Welfare, Republic of Korea (0720430) and by a
grant from the Institute of Medical System Engineering
(iMSE) in the GIST, Korea. The work performed by S.-M.
Kwon, S.-G. Ahn, and J.-H. Yoon was supported by the
Korea Science and Engineering Foundation (KOSEF) grant
funded by the Korea government (MOST) (no. R13-2008-
010-01001-0).
Supporting Information Available: IC50 values of antiproli-
ferative activity including mean ( SD, FACS analysis data of
3a, analytical data of HPLC analysis of final compounds for
purity, solubility data of compound 1, 2, 3a. This material is
available free of charge via the Internet at http://pubs.acs.org.
Animal Tumor Model. The k-ras-transformed rat kidney
epithelial cell line (RK3E-ras) were maintained in DMEM
supplemented with 10% FCS, 100 units/mL penicillin, and
100 Ag/mL streptomycin. RK3E-ras cells were kindly provided
by Dr. Eric Fearon (University of Michigan Medical School,
Ann Arbor, MI) and have been described in the previous
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