Synthesis and structure-activity relationships of novel indirubin derivatives as potent anti-proliferative agents with CDK2 inhibitory activities

Article abstract of DOI:10.1016/j.bmc.2005.08.008

Indirubin, an active ingredient of a traditional Chinese recipe Danggui Longhui Wan, has been known as a CDK inhibitor competing with ATP for binding to the catalytic site of cyclin-dependent kinases (CDKs). Since CDKs, a group of serine/threonine kinases forming active heterodimeric complexes with cyclins, are key regulators of the cell cycle regulation, therapeutic interventions targeting CDKs have been stimulated for the treatment of proliferative diseases, such as cancer, psoriasis, and for the prevention of chemotherapy-associated side effects, such as alopecia. A series of novel indirubin analogs was synthesized and evaluated for anti-proliferative and CDK2 inhibitory activities. Among the indirubin derivatives tested in the growth inhibitions against several human cancer cell lines, 5-nitro, halide, and bulky group containing acylamino substituted analogs showed high anti-proliferative effects. Selected analogs showing potent anti-proliferative activities were evaluated further in the CDK2 enzyme assay, which resulted in the discovery of potent CDK2 inhibitors.

Full text of DOI:10.1016/j.bmc.2005.08.008

Bioorganic & Medicinal Chemistry 14 (2006) 237–246
Synthesis and structure–activity relationships of novel
indirubin derivatives as potent anti-proliferative agents
with CDK2 inhibitory activities
Myoung Ju Moon,a Sang Kook Lee,b Jong-Won Lee,b Woo Keun Song,a Si Wouk Kim,c
Jae Il Kim,a Chunghee Cho,a Soo Jeong Choia and Yong-Chul Kima,*
aDepartment of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea
bCollege of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
cDepartment of Environmental Engineering, Chosun University, Gwangju 501-759, Republic of Korea
Received 4 July 2005; revised 1 August 2005; accepted 2 August 2005
Available online 22 September 2005
Abstract—Indirubin, an active ingredient of a traditional Chinese recipe Danggui Longhui Wan, has been known as a CDK inhib-
itor competing with ATP for binding to the catalytic site of cyclin-dependent kinases (CDKs). Since CDKs, a group of serine/
threonine kinases forming active heterodimeric complexes with cyclins, are key regulators of the cell cycle regulation, therapeutic
interventions targeting CDKs have been stimulated for the treatment of proliferative diseases, such as cancer, psoriasis, and for
the prevention of chemotherapy-associated side effects, such as alopecia. A series of novel indirubin analogs was synthesized and
evaluated for anti-proliferative and CDK2 inhibitory activities. Among the indirubin derivatives tested in the growth inhibitions
against several human cancer cell lines, 5-nitro, halide, and bulky group containing acylamino substituted analogs showed high
anti-proliferative effects. Selected analogs showing potent anti-proliferative activities were evaluated further in the CDK2 enzyme
assay, which resulted in the discovery of potent CDK2 inhibitors.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
inhibitors as potential pharmacological agents to treat
proliferative diseases, such as cancer, psoriasis, and reste-
nosis,10–12 and for the prevention of chemotherapy-asso-
ciated side effects, such as alopecia.13 A number of
selective low-molecular weight inhibitors of CDK1,
CDK2, and CDK4, such as Flavopiridol, Roscovitine,
UCN-01, and Indirubin14,15 (Fig. 1), have been developed
toward the clinical trials against various cancer diseases.
Abnormal control of cell cycle has been recognized as one
of the mechanisms of cancerization. Indeed, oncogenic
alterations of cyclins, cyclin-dependent kinases (CDKs),
and other upstream regulators of retinoblastoma (Rb)
protein, which function as important components of cell
cycle take place in a variety of human tumors.1 CDKs
are key regulators among which many of them are related
to the cell cycle, the complex process by which cells
divide.2–5 Cell cycle progression is stimulated and
controlled by the sequential activation of a series of
CDK complexes6,7 which are binding with specific cyclin
through the four phases of the cell cycle.2,8 A recent report
has suggested that CDK2 or cyclin E1/E2-deficient cells
could proliferate normally without causing any centro-
some duplication defect.9 These results gave a strong
impetus to the research on the development of CDK
Among these inhibitors, indirubin, the active ingredient
of Danggui Longhui Wan, a traditional Chinese medic-
inal recipe, has been used to treat chronic myelocytic
leukemia.16 Recently, indirubin and a few indirubin
derivatives have been reported to inhibit CDKs by com-
peting in the ATP-binding sites with high selectivity
among several kinase families.17 Although the detailed
mechanism of indirubin in the cells is not known com-
pletely, the anti-proliferative effect on the human cancer
cells was reported to have resulted from inhibiting the
genes or proteins of regulating cell cycle progression,
arresting G2/M phase of the cell cycle.18 Additional ser-
ies of indirubin derivatives were developed aiming at the
great potential for the treatment of Alzheimerꢀs disease
Keywords: Indirubin; Anti-proliferative activity; Cyclin-dependent
kinases.
*
Corresponding author. Tel.: +82 62 970 2502; fax: +82 62 970
2484; e-mail: yongchul@gist.ac.kr
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.08.008

238
M. J. Moon et al. / Bioorg. Med. Chem. 14 (2006) 237–246
ing anti-proliferative activity against several cancer cell
lines (A549, SNU-638, Col2, HT1080, HL-60, and
MCF-7) and CDK2 inhibitory activity for selected
indirubin derivatives.
2. Results and discussion
2.1. Chemistry
The general synthetic approach for the substituted indiru-
bin derivatives is shown in Scheme 1. The substituted
indirubins (3a–j) were prepared by condensation of
appropriately substituted (mainly 5- and 7-substituted)
isatins (1a–j) with indoxyl acetates (2) in the presence of
Na2CO3 in methanol. Since indirubin-30-monoxime was
reported to exhibit an enhanced inhibitory activity on
CDKs compared to indirubin,17 indirubin-30-monoxime
analogs (4a–l) were prepared from reactions of the substi-
tuted indirubins (3a–l) with hydroxylamine in pyridine.
Figure 1.
Indirubin-30-hydrazone derivative, 4m, was also synthe-
sized from the reaction with hydrazine monohydrate for
a comparison with the corresponding monoxime, 4a.
Acetylated compounds 5a,b were prepared to confirm
the importance of the proton of secondary amine. To
synthesize more diverse analogs with 5-substitutions,
for example, 5-acylamino groups, the 5-nitroindirubin,
3c was first reduced to 5-aminoindirubin, 6 by tin(II)
chloride, followed by acylation with acid chlorides in
pyridine and subsequent reaction with hydroxylamine
to afford various 5-acylamino-indirubin-30-oxime deriv-
atives (Scheme 2).
(AD) by inhibiting GSK-3b and CDK5, which are relat-
ed to the control of abnormal hyperphosphorylation of
the microtubule-binding protein tau, one of the diagnos-
tic features of AD.19,20 In addition, indirubin-30-monox-
ime was reported to up-regulate p27Kip1 transcription by
acting as an agonist of the aryl hydrocarbon recep-
tor21–23 and inhibiting c-Jun NH2-terminal kinase,
which is an important regulator of neuronal apoptosis.24
Moreover, recent reports provide an evidence that
indirubin-30-monoxime can selectively abrogate induced
aberrant centriole and centrosome duplication in tumor
cells without affecting normal centrosome duplication.25
2.2. Anti-proliferative activity
In the present report, we describe the synthesis of novel
indirubin derivatives and biological evaluations includ-
The synthesized indirubin derivatives were tested for
anti-proliferative activities against several cancer cell
Scheme 1. Reagents and conditions: (a) Na2CO3, MeOH, rt; (b) H2NOH Æ HCl, pyridine, reflux; (c) H2NNH2 Æ H2O, 70–80 ꢁC; (d) acetic anhydride,
reflux.

M. J. Moon et al. / Bioorg. Med. Chem. 14 (2006) 237–246
239
Scheme 2. Reagents and conditions: (e) SnCl2 Æ 2H2O, DMF, 70 ꢁC; (f) ClCOR6, pyridine, 0 ꢁC; (g) H2NOH Æ HCl, pyridine, reflux.
lines including A594, SNU-638, Col2, HT 1080, HL-60,
and MCF-7 cells. Tables 1 and 2 show the IC50 values of
cancer cell growth inhibition by the new indirubin ana-
logs derivatized at various positions of the bis-indole
skeleton. Ellipticine, a well-known anticancer agent,
was tested together as a positive control in the assays.
of the cancer cells, with IC50 values in the range of 1–
10 lM. Particularly, it represented an IC50 value of
1.2 lM against human stomach cancer cell line (SNU-
638) and 1.4 lM against human breast cancer cell line
(MCF-7), which are comparable with those of the positive
control Ellipticine. However, compound 3j, the potent
CDK inhibitor reported in the literature17 and its monox-
ime derivative, 4j, showed no inhibitory activities, proba-
bly because of the low membrane permeability due to the
ionic character of 5-sulfonate. Thus, it can be assumed
that the partial negative charge by the electron-rich nitro
group, which can compete with the phosphate of ATP in
the active sites of CDKs, and the membrane permeable
character of 4c enable the potent inhibitory activity. In-
deed, further study has demonstrated that 4c arrested
Although literatures17,19 have reported that indirubin-30-
monoxime, 4a, inhibited CDKs much more efficiently
than indirubin, 3a, with IC50 values of 100–200 nM, the
anti-proliferative activities of 3a, 4a, and the hydrazone
analog, 4m, were similar, except for HT1080 cell lines.
Among the series of 5-substituted indirubin-30-monoxime
derivatives given in Table 1, 5-nitro-indiruin-30-monox-
ime, 4c, showed potent inhibitory activity against most
Table 1. Anti-proliferative effects of indirubin derivatives
Compound
R1
R2
R3
R4
R5
IC50 (lM)
A549a
SNU-638b
Col2c
HT1080d
HL60e
MCF-7f
3a
3b
3c
3d
3j
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
Br
H
H
H
H
H
O
H
31
68
>100
>100
>100
40
>100
>100
>100
>100
>100
>100
>100
25.5
42
>100
>100
>100
>100
>100
>100
>100
9.2
>100
>100
40
CF3O
NO2
F
Na+SO3
H
O
H
>100
>100
>100
>100
4.8
H
O
H
>100
97
H
O
H
>100
>100
10
ꢀ
H
O
H
>100
62
9.6
>100
>100
23
4a
4b
4c
4d
4e
4f
H
H
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NHNH2
O
H
CF3O
NO2
F
H
H
32
5.9
>100
1.4
9.0
H
H
5.4
13
1.2
2.1
H
H
>100
>100
>100
>100
17
3.4
37
89
65
CH3
CH3
Cl
H
H
20
18
91
33
3.5
CH3
CH3
H
H
>100
>100
12
>100
>100
11
>100
>100
4.8
4g
4h
4i
H
>100
6.2
34
2.5
Cl
I
Na+SO3
H
H
H
>100
>100
39
>100
>100
33
>100
>100
11
>100
>100
74
>100
47
25
>100
>100
12
ꢀ
4j
H
H
4k
4l
H
H
H
NO2
H
H
H
2.9
98
0.79
>100
>100
>100
1.6
1.8
1.6
41
2.6
1.3
4m
5a
5b
H
H
>100
>100
>100
1.5
>100
>100
>100
3.1
>100
>100
>100
0.99
H
CF3O
H
COCH3
COCH3
>100
>100
0.28
>100
>100
3.6
H
Ellipticine (positive control)
O
a Human lung cancer cell line.
b Human stomach cell line.
c Human colon cancer cell line.
d Human fibro sarcoma cell line.
e Human leukemia cell line.
f Human breast cancer cell line.

240
M. J. Moon et al. / Bioorg. Med. Chem. 14 (2006) 237–246
Table 2. Anti-proliferative effects of 5-N-acyl indirubin derivatives
Compound
R6
IC50 (lM)
HT1080
A549
SNU-638
Col2
HL60
MCF-7
7
8
–CH3
–CH2CH3
10
13
38
61
19
48
8.4
20
57
5.4
14
>100
>100
>100
21
9
–(CH2)2CH3
–(CH2)3CH3
–C(CH3)3
>100
>100
6.4
>100
68
6.5
>100
>100
14
52
8.2
14
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
>100
4.2
11
6.7
6.3
>100
38
–CH2C(CH3)3
–CH@C(CH3)2
–Cyclopropyl
11
25
14
>100
>100
>100
>100
>100
32
>100
>100
>100
>100
90
>100
>100
>100
71
>100
>100
>100
>100
73
70
71
–Cyclohexyl
–2-Furane
>100
65
73
>100
4.5
12
–2-Thiophene
–Phenyl
54
>100
>100
>100
97
>100
>100
>100
73
>100
>100
>100
>100
>100
63
>100
>100
>100
55
>100
>100
>100
>100
>100
94
19
58
–2-Chlorophenyl
–4-Chlorophenyl
2-Fluorophenyl
–4-Fluorophenyl
–2-Trifluoromethylphenyl
–3-Nitrophenyl
–4-Nitrophenyl
–3,5-Dinitrophenyl
–4-Methoxyphenyl
–4-Methylphenyl
–3-Chlorophenoxymethyl
–4-Chlorophenoxymethyl
–CH@CH-Ph (trans)
–CH2CH2Ph
20
>100
13
50
>100
17
58
>100
28
15
>100
17
99
>100
>100
>100
>100
>100
77
>100
>100
>100
>100
>100
97
13
11
58
98
>100
12
>100
16
2.6
21
94
>100
79
52
>100
>100
45
>100
12
18
13
5.1
6.8
15
82
53
76
35
72
96
21
37
>100
>100
6.3
>100
>100
11
44
5.6
29
6.6
4.5
3.5
8.1
0.99
–Diphenylmethyl
–1-Naphthyl
Ellipticine (positive control)
2.7
16
>100
2.9
11
2.0
44
1.4
>100
15
1.3
the cell cycle progression at the G2/M phase and induced
apoptosis via p53- and mitochondria-dependent path-
way.26 Other lone pair electron-rich group substituted
analogs, such as 4b (5-OCF3), 4d (5-F), and 4h (5-Cl), also
displayed relatively higher inhibitory activities in general.
However, 5-iodo analog, 4i, did not inhibit the cell
growth. Bromo substitution (4k) at 50 position (R1) of
indirubin-30-monoxime, 4a, resulted in an appreciable in-
crease in activity. Further substitution with nitro group at
5 position (R2) of 4k afforded synergistic effect of the anti-
proliferative activity, showing 1–2 lM IC50 values at all of
the tested cancer cell lines (compound 41). On the other
hand, acetyl group substituents at R5 position resulted
in a significant decrease in inhibitory activities against
A549 and HT1080 cell lines (3a versus 5a, 3b versus 5b).
These results suggested that the hydrogen bond between
secondary amine proton of indirubin and the backbone
oxygen of Leu83 of CDK2 as described in the literature17
was also important for the anti-proliferative activity.
acylamino derivatives of indirubin-monoximes, bulky
tertiary butyl group containing compounds, 11 and 12
were particularly active than other alkyanoyl and cyclo-
alkanoyl analogs. Substituted benzoyl analogs were
generally inactive, except for trifluoromethyl and
dinitro substituted compounds, 23 and 26. Especially,
26 showed potent anti-proliferative activity against hu-
man breast cancer cell line in this series of compounds.
Among the series of compounds listed in Table 2, diph-
enylacetyl analog, 33, was the most potent compound
showing 2–10 lM IC50 values at all the tested cancer cell
lines. Considering the different electronic environment
around the 5 position of the representative potent anti-
proliferative indirubin derivatives in Tables 1 and 2,
such as 4c and 33, we could suppose that similar activity
could have resulted from different factors, such as differ-
ent solubility and cellular uptake, as well as possibly
inhibiting different target protein molecules.
In general, the various acylamino derivatives showed bet-
ter solubility than the compounds listed in Table 1. To
investigate the relationship between the anti-proliferative
potency and CDK2 inhibitory activity, we further studied
Diversity at 5 position was introduced by acylation of
5-amino-indirubin, 6, which was obtained by the reduc-
tion of 3c with tin(II) chloride. Among the aliphatic

M. J. Moon et al. / Bioorg. Med. Chem. 14 (2006) 237–246
241
the inhibitory activity of the representative derivatives at
CDK2 catalyzed Rb protein phosphorylation.
cules affecting cell proliferation, such as aryl hydrocarbon
receptor (AhR),23 JNK,24 Src kinase27, and Stat3.27 N1-
methylated indirubins were reported as potent AhR
agonists by leading to a G1 arrest in a strictly AhR-depen-
dent manner.23 Also, indirubin-30-alkyloxime derivatives
were recently reported to directly block the Src-Stat3
signaling pathway, which has an important role in
oncogenesis and is a promising anticancer target system,
inducing apoptosis of human cancer cells.27
2.3. CDK2 inhibitory activity
To determine the CDK inhibitory activity in a set of
breast cancer cell lines (MCF-7), cells were treated with
representative indirubin derivatives (3a, 4a, 4b, 4c, 11,
and 33). CDK2, 4, and 6 from whole cell lysates were
immunoprecipitated with a monoclonal antibody and
the kinase activity was measured using a C-terminal frag-
ment of pRb as a substrate. Indirubin (3a) showed no
CDK2, 4, and 6 inhibitory activity, even at 500 lM con-
centration, whereas indirubin-30-monoxime (4a) inhibit-
ed CDK2 more efficiently with 50% inhibition at 30–
40 lM concentration (Fig. 2A). In the case of 4b and 4c,
the kinase activity of CDK2 was significantly blocked
up to 0.1–1 lM concentration of the compounds. Howev-
er, despite similar high potency of anti-proliferative activ-
ity with 4b and 4c, compounds 11 and 33 did not affect the
CDK2 activity in the cancer cell line, even at 10 lM con-
centration, and only showed decreased CDK2 activity at
100 lM or higher concentrations of the compounds
(Fig. 2B). This study demonstrated that compounds 11
and 33 might modulate other kinases or signaling mole-
Further study to figure out the molecular mechanism of
anti-proliferative activity of compound 11 or 33 is in
progress.
3. Conclusion
Novel indirubin derivatives were developed with struc-
ture–activity relationship studies, determining anti-
proliferative activity with a positive control, Ellipticine.
5-Substituted indirubin-30-oxime analogs, 4b and 4c, as
the potent cancer cell growth inhibitors displayed potent
inhibition of CDK2, which should be one of the mech-
anisms of anti-proliferative activity. However, 5-acyla-
mino analogs, 11 and 33, which are equally potent
Figure 2. Inhibition of CDK catalyzed Rb protein phosphorylation by indirubin derivatives. Recombinant human Rb protein was phosphorylated
in vitro with CDK2, 4, and 6 (A), CDK2 (B) in the presence of indirubin derivatives (3a, 4a, 4b, 4c, 11, and 33) and resolved by SDS–PAGE, followed
by autoradiography.

242
M. J. Moon et al. / Bioorg. Med. Chem. 14 (2006) 237–246
anti-proliferative derivatives did not inhibit CDK2
activity. The indirubin derivatives discovered in this
study may provide valuable therapeutic intervention
for the treatment of anti-proliferative diseases.
(1H, dd, J = 2.6, 11.1 Hz), 8.23 (1H, d, J = 7.5 Hz),
7.43 (2H, m), 7.00 (3H, m); MS (MALDI-TOF) m/z:
295.
4.1.2.5. 5-Methyl-indirubin-30-oxime (4e). Yield: 78%
1H NMR (300 MHz, DMSO-d6) d (ppm) 13.40 (1H, s,
NOH), 11.72 (1H, s, N0–H), 10.59 (1H, s, N–H), 8.48
(1H, s), 8.24 (1H, d, J = 7.5 Hz), 7.39 (2H, m), 7.00
(2H, m), 6.77 (1H, d, J = 7.8 Hz), 2.34 (3H, s, CH3);
MS (MALDI-TOF) m/z: 291.
4. Experimental
4.1. Chemistry
All chemicals were purchased from Aldrich Chemical
Co. Proton nuclear magnetic resonance spectroscopy
was performed on Bruker AVANCE 600 and JEOL
JNM-LA 300WB spectrometers, and spectra were taken
in DMSO-d6. Unless noted, chemical shifts are ex-
pressed in ppm downfield from TMS as the internal
standard, and J values are given in Hz. Mass spectrosco-
py was carried out on a MALDI-TOF. Elemental and
high-resolution mass analyses were performed in Seoul
Branch Analytical Laboratory of Korea Basic Science
Institute.
4.1.2.6. 5,7-Dimethyl-indirubin-30-oxime (4f). Yield:
97% 1H NMR (300 MHz, DMSO-d6) d (ppm) 13.38
(1H, s, NOH), 11.77 (1H, s, N0–H), 10.61 (1H, s, N–
H), 8.34 (1H, s), 8.24 (1H, d, J = 7.5 Hz), 7.39 (2H,
m), 7.02 (1H, s), 6.77 (1H, s), 2.30 (3H, s, CH3), 2.22
(3H, s, CH3); MS (MALDI-TOF) m/z: 305.
4.1.2.7. 5-Chloro-7-methyl-indirubin-30-oxime (4g).
1
Yield: 97.2% H NMR (300 MHz, DMSO-d6) d (ppm)
13.65 (1H, s, NOH), 11.88 (1H, s, N0–H), 10.88 (1H, s,
N–H), 8.52 (1H, s), 8.24 (1H, d, J = 7.2 Hz), 7.43 (2H,
m), 7.04 (2H, m), 2.22 (3H, s, CH3); MS (MALDI-
TOF) m/z: 325.
4.1.1. General procedure for the synthesis of indirubin
derivatives (3a–l). Under nitrogen, 176 mg (1 mmol) of
indoxyl acetate and 1 mmol of isatin analogs were dis-
solved in 5 ml methanol with 256 mg (2.5 mmol)
Na2CO3 and stirring was continued for 2–3 h at room
temperature. The dark violet precipitation was filtered,
washed twice with methanol and several times with cold
water, and dried under reduced pressure (yield 50–60%).
4.1.2.8. 5-Chloro-indirubin-30-oxime (4h). Yield: 37%
1H NMR (300 MHz, DMSO-d6) d (ppm) 13.68 (1H, s,
NOH), 11.83 (1H, s, N0–H), 10.85 (1H, s, N–H), 8.65
(1H, s), 8.24 (1H, d, J = 7.5 Hz), 7.42 (2H, m), 7.10
(2H, m), 6.88 (1H, d, J = 8.1 Hz); MS (MALDI-TOF)
m/z: 311.
4.1.2. General procedure for the preparation of oxime
derivatives (4a–l). One millimole of the appropriate
indirubin analogs 3a–l and 6 was added to 10 ml pyri-
dine with 6 mmol of hydroxylamine hydrochloride.
The mixture was refluxed for 2–3 h at 120 ꢁC. After
cooling, the product was neutralized with 1 N HCl.
The precipitation was filtered and washed with water.
1
4.1.2.9. 5-Iodo-indirubin-30-oxime (4i). Yield: 90% H
NMR (300 MHz, DMSO-d6) d (ppm) 13.67 (1H, s,
NOH), 11.83 (1H, s, N0–H), 10.84 (1H, s, N–H), 8.90
(1H, s), 8.25 (1H, d, J = 7.8 Hz), 7.40 (3H, m), 7.06
(1H, s), 6.74 (1H, d, J = 7.8 Hz); MS (MALDI-TOF)
m/z: 403.
4.1.2.1. Indirubin-30-oxime (4a). Yield: 44% 1H NMR
(300 MHz, DMSO-d6) d (ppm) 13.49 (1H, s, NOH),
11.74 (1H, s, N0–H), 10.74 (1H, s, N–H), 8.65 (1H, d,
J = 7.8 Hz), 8.23 (1H, d, J = 7.8 Hz), 7.20 (2H, m),
7.13 (1H, t, J = 7.5 Hz), 6.92 (4H, m); MS (MALDI-
TOF) m/z: 276.
4.1.2.10. Indirubin-30-oxime-5-sulfonic acid sodium
salt (4j). Yield: 30% H NMR (300 MHz, DMSO-d6) d
(ppm) 13.70 (1H, s, NOH) 11.81 (1H, s, N–H), 10.79
(1H, s, N0–H), 8.87 (1H, s), 8.25 (1H, d, J = 7.5 Hz),
7.43 (3H, m), 7.03 (1H, m), 6.81 (1H, d, J = 7.8 Hz);
MS (MALDI-TOF) m/z: 380.
1
4.1.2.2. 5-Trifluoromethoxy-indirubin-30-oxime (4b).
4.1.2.11. 50-Bromo-indirubin-30-oxime (4k). Yield: 43%
1H NMR (300 MHz, DMSO-d6) d (ppm) 13.74 (1H, s,
NOH), 11.75 (1H, s, N0–H), 10.75 (1H, s, N–H), 8.61
(1H, d, J = 10.2 Hz), 8.32 (1H, s), 7.57 (1H, d,
J = 7.8 Hz), 7.41 (1H, d, J = 7.5 Hz), 7.14 (1H, t,
J = 2.4 Hz), 6.92 (2H, m); MS (MALDI-TOF) m/z: 356.
1
Yield: 71% H NMR (300 MHz, DMSO-d6) d (ppm)
13.65 (1H, s, NOH), 11.85 (1H, s, N0–H), 10.91 (1H, s,
N–H), 8.60 (1H, s), 8.24 (1H, d, J = 7.5 Hz), 7.40 (2H,
m), 7.01 (2H, m), 6.94 (1H, d, J = 8.4 Hz); MS (MAL-
DI-TOF) m/z: 403.
4.1.2.3. 5-Nitro-indirubin-30-oxime (4c). Yield: 94.1%
1H NMR (300 MHz, DMSO-d6) d (ppm) 13.92 (1H, s,
NOH), 11.90 (1H, s, N0–H), 11.44 (1H, s, N–H), 9.47
(1H, s), 8.27 (1H, d, J = 7.5 Hz), 8.10 (1H, dd, J = 2.1,
8.7 Hz), 7.48 (2H, m), 7.09 (2H, m); MS (MALDI-
TOF) m/z: 321.
4.1.2.12. 50-Bromo-5-nitro-indirubin-30-oxime (4l). Yield:
33% 1H NMR (300 MHz, DMSO-d6) d (ppm) 14.17 (1H,
s, NOH), 11.90 (1H, s, N–H), 11.42 (1H, s, N0–H), 9.43
(1H, s), 8.36 (1H, s), 8.10 (1H, d, J = 8.4 Hz), 7.60 (1H,
d, J = 7.8 Hz), 7.46 (1H, d, J = 8.4 Hz), 7.06 (1H, d,
J = 8.4 Hz); MS (MALDI-TOF) m/z: 401.
4.1.2.4. 5-Fluoro-indirubin-30-oxime (4d). Yield: 54.4%
1H NMR (300 MHz, DMSO-d6) d (ppm) 13.66 (1H, s,
NOH), 11.80 (1H, s, N0–H), 10.75 (1H, s, N–H), 8.48
4.1.2.13. Indirubin-30-hydrazone (4m). Eighty milli-
grams (0.3 mmol) of indirubin (3a) was dissolved in
3 ml H2NNH2 Æ H2O and heated under 70–80 ꢁC for

M. J. Moon et al. / Bioorg. Med. Chem. 14 (2006) 237–246
243
24 h. The blue solid was filtered and washed with diethyl
ether. The appropriate product was dissolved in N-
methylpyrrolidone and purified by column chromatog-
raphy on silica gel (n-hexanes/EtOAc, 1:1) to afford
the product as a solid. The first time, NMP must be re-
moved with constant hexane solvent. 1H NMR
(300 MHz, DMSO-d6) d (ppm) 11.86 (1H, s, NH),
11.86 (1H, s, N0–H), 11.37 (1H, s, N–H), 8.24 (1H, d,
J = 7.8 Hz), 8.13 (1H, d, J = 7.8 Hz), 7.52 (2H, s,
NH2), 7.45 (2H, m), 7.38 (3H, m), 7.22 (1H, m); MS
(MALDI-TOF) m/z: 276.
4.1.3.2. 5-Propionamido-indirubin-30-oxime (8). 1H
NMR (300 MHz, DMSO-d6) d (ppm) 13.33 (1H, s,
NOH), 11.73 (1H, s, N0–H), 10.64 (1H, s, N–H), 9.48
(1H, s, amide-NH), 8.53 (1H, d, J = 1.8 Hz), 8.23 (1H,
d, J = 7.8 Hz), 7.39 (2H, m), 7.31 (1H, dd, J = 8.1,
1.8 Hz), 7.03 (1H, m), 6.81 (1H, d, J = 8.1 Hz), 2.30
(2H, q, J = 7.5 Hz, CH2), 1.11 (3H, t, J = 7.5 Hz,
CH3); MS (MALDI-TOF) m/z: 348.
4.1.3.3. 5-Butyramido-indirubin-30-oxime (9). 1H NMR
(300 MHz, DMSO-d6) d (ppm) 13.34 (1H, s, NOH),
11.73 (1H, s, N0–H), 10.65 (1H, s, N–H), 9.49 (1H, s,
amide-NH), 8.53 (1H, s), 8.23 (1H, d, J = 7.8 Hz), 7.33
(3H, m), 7.02 (1H, m), 6.81 (1H, d, J = 8.4 Hz), 2.27
(2H, t, J = 7.2 Hz, CH2), 1.64 (2H, m, CH2), 0.92 (2H,
t, J = 7.2 Hz, CH3); MS (MALDI-TOF) m/z: 377.
4.1.2.14. N10-acetyl-indirubin (5a,b). 314.4 mg
(1.2 mmol) of indirubin (3a,b) was added to 15 ml of ace-
tic anhydride and refluxed for 10 h. The precipitate was
filtered and washed with water several times. Then the res-
idue was dried.
4.1.3.4. 5-Pantanamido-indirubin-30-oxime (10). 1H
NMR (300 MHz, DMSO-d6) d (ppm) 13.27 (1H, s,
NOH), 11.73 (1H, s, N0–H), 10.65 (1H, s, N–H), 9.49
(1H, s, amide-NH), 8.51 (1H, s), 8.23 (1H, s), 7.44
(3H, m), 7.02 (2H, m), 2.28 (2H, m, CH2), 1.58 (2H,
m, CH2), 1.32 (2H, m, CH2), 0.89 (3H, m, CH3); MS
(MALDI-TOF) m/z: 376.
Compound 5a: (yield 98%) 1H NMR (300 MHz, DMSO-
d6) d (ppm) 10.52 (1H, s, N–H), 9.00 (1H, dd, J = 7.8,
1.2 Hz), 8.29 (1H, d, J = 7.5 Hz), 7.73 (1H, d,
J = 7.5 Hz), 7.52 (1H, t, J = 7.5 Hz), 7.31 (2H, m), 7.03
(2H, m), 2.76(3H, s, CH3);MS(MALDI-TOF)m/z: 304.2.
Compound 5b: (yield 95%) 1H NMR (300 MHz, DMSO-
d6) d (ppm) 11.49 (1H, s, N–H), 8.98 (1H, s), 8.31 (1H, d,
J = 9.3 Hz), 7.69 (1H, d, J = 7.2 Hz), 7.62 (1H, t,
J = 7.2 Hz), 7.45 (1H, d, J = 8.1 Hz), 7.32 (1H, d,
J = 9.3Hz), 7.09 (1H, t, J = 7.2 Hz); MS (MALDI-TOF)
m/z: 388.
4.1.3.5. 5-Trimethylacetamido-indirubin-30-oxime (11).
1H NMR (300 MHz, DMSO-d6) d (ppm) 13.40 (1H, s,
NOH), 11.73 (1H, s, N0–H), 10.67 (1H, s, N–H), 8.89
(1H, s, amide-NH), 8.46 (1H, s), 8.25 (1H, d,
J = 7.5 Hz), 7.40 (2H, m), 7.24 (1H, d, J = 8.4 Hz),
7.03 (1H, m), 6.81 (1H, d, J = 8.4 Hz), 1.26 (9H, s,
(CH3)3); MS (MALDI-TOF) m/z: 376.
4.1.2.15. 5-Amino-indirubin (6). Five hundred milli-
grams (1.629 mmol) of indirubin-5-nitro (3c) and 1.8 g
(8.145 mmol) of SnCl2 Æ 2H2O were dissolved in 5 ml
DMF and heated under 70 ꢁC for 2–4 h. The mixture
was acidified with 1 N HCl and extracted with ethyl ace-
tate. Then, the solvent was evaporated and the residue
was dried under reduced pressure. Yield: 43% 1H
NMR (300 MHz, DMSO-d6) d (ppm) 10.95 (1H, s,
N0–H), 10.45 (1H, s, N–H), 8.15 (1H, s), 7.63 (1H, d,
J = 7.5 Hz), 7.55 (1H, t, J = 7.5 Hz), 7.39 (1H, d,
J = 7.5 Hz), 6.99 (1H, t, J = 7.5 Hz), 6.59 (1H, d,
J = 8.4 Hz), 6.53 (1H, d, J = 8.4 Hz), 4.75 (2H, s,
NH2); MS (MALDI-TOF) m/z: 277.
4.1.3.6. 5-(3,3-Dimethylbutanamido)-indirubin-30-oxime
(12). 1H NMR (300 MHz, DMSO-d6) d (ppm) 13.37 (1H,
s, NOH), 11.73 (1H, s, N0–H), 10.63 (1H, s, N–H), 9.43
(1H, s, amide-NH), 8.55 (1H, s), 8.23 (1H, d,
J = 7.5 Hz), 7.39 (2H, m), 7.31 (1H, d, J = 7.8 Hz), 7.03
(1H, m), 6.81 (1H, d, J = 7.8 Hz), 2.18 (2H, s, CH2)
1.05 (9H, s, (CH3)3); MS (MALDI-TOF) m/z: 390.
4.1.3.7. 5-(3-Methylbut-2-enamido)-indirubin-30-oxime
(13). 1H NMR (300 MHz, DMSO-d6) d (ppm) 13.33
(1H, s, NOH), 11.72 (1H, s, N0–H), 10.64 (1H, s, N–
H), 9.44 (1H, s, amide-NH), 8.53 (1H, d, J = 1.5 Hz),
8.23 (1H, d, J = 7.5 Hz), 7.39 (2H, m), 7.33 (1H, dd,
J = 8.4, 1.5 Hz), 7.03 (1H, m), 6.81 (1H, d, J = 8.4 Hz),
5.88 (1H, s, CH) 2.15 (3H, s, CH3), 1.86 (3H, s, CH3);
MS (MALDI-TOF) m/z: 374.
4.1.3. General procedure for the preparation of oxime
derivatives (7–34). One millimole of 5-amino-indirubin (6)
was dissolved in pyridine, 1.2 equiv of acyl chloride was
dropwise added at 0 ꢁC, and the mixture was stirred for
30 min. After workup, 5-NH acyl indirubins (7–39) were
added to pyridine with 6 equiv of hydroxylamine hydro-
chloride. The mixture was refluxed for 2–3 h at
80–90 ꢁC. After cooling, the product was neutralized with
1 N HCl. The precipitate was filtered and washed with
water.
4.1.3.8.
5-(Cyclopropanecarboxamido)-indirubin-30-
1
oxime (14). H NMR (300 MHz, DMSO-d6) d (ppm)
13.37 (1H, s, NOH), 11.71 (1H, s, N0–H), 10.62 (1H, s,
N–H), 9.85 (1H, s, amide-NH), 8.56 (1H, s), 8.23 (1H,
d, J = 7.5 Hz), 7.43 (2H, m), 7.33 (1H, d, J = 8.4 Hz),
7.03 (1H, m), 6.80 (1H, d, J = 8.4 Hz), 1.25 (1H, m,
CH) 0.81 (4H, m, CH2), MS (MALDI-TOF) m/z: 360.
4.1.3.1. 5-Acetamido-indirubin-30-oxime (7). 1H NMR
(300 MHz, DMSO-d6) d (ppm) 13.33 (1H, s, NOH),
11.73 (1H, s, N0–H), 10.63 (1H, s, N–H), 9.47 (1H, s,
amide-NH), 8.52 (1H, d,J = 1.8 Hz), 8.24 (1H, d,
J = 7.5 Hz), 7.42 (2H, m), 7.22 (1H, dd, J = 1.8,
1.8 Hz), 7.05 (1H, m), 6.82 (1H, d, J = 8.1 Hz), 1.23
(3H, s); MS (MALDI-TOF) m/z: 334.
4.1.3.9. 5-(Cyclohexanecarboxamido)-indirubin-30-ox-
ime (15). 1H NMR (300 MHz, DMSO-d6) d (ppm) 13.29
(1H, s, NOH), 11.71 (1H, s, N0–H), 10.62 (1H, s, N–H),
9.35 (1H, s, amide-NH), 8.50 (1H, d, J = 1.5 Hz), 8.23
(1H, d, J = 7.2 Hz), 7.39 (3H, m), 7.03 (1H, m), 6.79

244
M. J. Moon et al. / Bioorg. Med. Chem. 14 (2006) 237–246
(1H, d, J = 8.1Hz), 2.28 (H, m, CH) 1.80 (4H, m, CH2),
1.26 (6H, m, CH2); MS (MALDI-TOF) m/z: 402.
(1H, s), 7.78 (4H, m), 7.40 (3H, m), 7.03 (2H, m); MS
(MALDI-TOF) m/z: 464.
4.1.3.10. 5-(Furan-2-carboxamido)-indirubin-30-oxime
(16). 1H NMR (300 MHz, DMSO-d6) d (ppm) 13.37
(1H, s, NOH), 11.75 (1H, s, N0–H), 10.71 (1H, s, N–
H), 9.84 (1H, s, amide-NH), 8.63 (1H, d, J = 1.8 Hz),
8.23 (1H, d, J = 7.5 Hz), 7.92 (1H, s), 7.40 (2H, m),
7.32 (2H, m), 7.03 (1H, m), 6.87 (1H, d, J = 8.1 Hz),
6.70 (1H, m); MS (MALDI-TOF) m/z: 386.
4.1.4.3. 5-(3-Nitrobenzamido)-30-oxime (24). 1H NMR
(300 MHz, DMSO-d6) d (ppm) 13.42 (1H, s, NOH),
11.79 (1H, s, N0–H), 10.74 (1H, s, N–H), 10.44 (1H, s,
amide-NH), 8.87 (1H, s), 8.72 (1H, s), 8.46 (2H, m),
8.24 (1H, d, J = 7.2 Hz), 7.85 (1H, t, J = 8.1 Hz), 7.41
(2H, m), 7.33 (1H, d, J = 8.1 Hz), 7.03 (1H, m), 6.91
(1H, d, J = 8.1 Hz); MS (MALDI-TOF) m/z: 442.
4.1.3.11. 5-(Thiophene-2-carboxamido)-indirubin-30-
oxime (17). H NMR (300 MHz, DMSO-d6) d (ppm)
4.1.4.4. 5-(4-Nitrobenzamido)-indirubin-30-oxime (25).
1H NMR (300 MHz, DMSO-d6) d (ppm) 13.35 (1H, s,
NOH), 11.75 (1H, s, N0–H), 10.73 (1H, s, N–H), 10.36
(1H, s, amide-NH), 8.73 (1H, d, J = 1.8 Hz), 8.38 (2H,
m), 8.24 (2H, m), 7.69 (1H, m), 7.40 (2H, m), 7.35
(1H, dd, J = 8.1, 1.8 Hz), 7.03 (1H, m), 6.90 (1H, d,
J = 8.1 Hz); MS (MALDI-TOF) m/z: 441.
1
13.40 (1H, s, NOH), 11.77 (1H, s, N0–H), 10.74 (1H, s,
N–H), 10.06 (1H, s, amide-NH), 8.61 (1H, d,
J = 1.8 Hz), 8.23 (1H, d, J = 7.8 Hz), 8.02 (1H, d,
J = 3.6 Hz), 7.83 (1H, d, J = 3.8 Hz), 7.40 (2H, m),
7.28 (1H, dd, J = 8.1, 1.8 Hz), 7.23 (1H, t, J = 3.8 Hz),
7.03 (1H, m), 6.89 (1H, d, J = 8.1 Hz); MS (MALDI-
TOF) m/z: 402.
4.1.4.5. 5-(3,5-Dinitrobenzamido)-indirubin-30-oxime
(26). 1H NMR (300 MHz, DMSO-d6) d (ppm) 13.42
(1H, s, NOH), 11.77 (1H, s, N0–H), 10.77 (2H, s, N–
H, amide-NH), 9.29 (2H, s), 9.01 (1H, s), 8.75 (1H, s),
8.23 (1H, d, J = 7.5 Hz), 7.40 (3H, m), 7.03 (1H, m),
6.92 (1H, d, J = 8.1 Hz); MS (MALDI-TOF) m/z: 486.
4.1.3.12. 5-Benzamido-indirubin-30-oxime (18). 1H NMR
(300 MHz, DMSO-d6) d (ppm) 13.42 (1H, s, NOH), 11.76
(1H, s, N0–H), 10.71 (1H, s, N–H), 10.06 (1H, s, amide-
NH), 8.78 (1H, s), 8.23 (1H, d, J = 7.5 Hz), 7.99 (2H,
m), 7.56 (3H, m), 7.37 (3H, m), 7.03 (1H, t, J = 7.5 Hz),
6.91 (1H, d, J = 8.1 Hz); MS (MALDI-TOF) m/z: 396.
4.1.4.6. 5-(4-Methoxybenzamido)-indirubin-30-oxime
(27). 1H NMR (300 MHz, DMSO-d6) d (ppm) 13.42
(1H, s, NOH), 11.74 (1H, s, N0–H), 10.70 (1H, s, N–
H), 9.86 (1H, s, amide-NH), 8.68 (1H, s), 8.23 (1H, d,
J = 7.8 Hz), 8.04 (2H, m), 7.41 (3H, m), 7.05 (3H, m),
6.87 (1H, d, J = 8.4 Hz), 3.85 (3H, s, CH3); MS (MAL-
DI-TOF) m/z: 426.
4.1.3.13. 5-(2-Chlorobenzamido)-indirubin-30-oxime (19).
1H NMR (300 MHz, DMSO-d6) d (ppm) 13.25 (1H, s,
NOH), 11.74 (1H, s, N0–H), 10.70 (1H, s, N–H), 10.04
(1H, s, amide-NH), 8.74 (1H, s), 8.24 (1H, d, J =
7.5 Hz), 7.53 (7H, m), 7.03 (1H, m), 6.87 (1H, d, J =
8.1 Hz); MS (MALDI-TOF) m/z: 430.
4.1.4.7.
5-(4-Methylbenzamido)-indirubin-30-oxime
4.1.3.14.
5-(4-Chlorobenzamido)-indirubin-30-oxime
(28). 1H NMR (300 MHz, DMSO-d6) d (ppm) 13.35
(1H, s, NOH), 11.76 (1H, s, N0–H), 10.69 (1H, s, N–
H), 9.89 (1H, s, amide-NH), 8.66 (1H, d, J = 1.8 Hz),
8.24 (1H, d, J = 7.5 Hz), 7.94 (2H, m), 7.36 (5H, m),
7.03 (1H, m), 6.89 (1H, d, J = 8.1 Hz); MS (MALDI-
TOF) m/z: 410.
(20). 1H NMR (300 MHz, DMSO-d6) d (ppm) 13.35
(1H, s, NOH), 11.75 (1H, s, N0–H), 10.71 (1H, s, N0–
H), 10.09 (1H, s, amide-NH), 8.68 (1H, d, J = 1.5 Hz),
8.23 (1H, d, J = 7.8 Hz), 8.06 (2H, d, J = 8.4 Hz), 7.61
(2H, d, J = 8.4 Hz), 7.40 (2H, m), 7.3 (1H, dd, J = 8.4,
1.5 Hz), 7.03 (1H, m), 6.89 (1H, d, J = 8.4 Hz); MS
(MALDI-TOF) m/z: 430.
4.1.4.8. 5-(2-(3-Chlorophenoxy)acetamido)-indirubin-
30-oxime (29). 1H NMR (300 MHz, DMSO-d6) d
(ppm) 13.38 (1H, s, NOH), 11.76 (1H, s, N0–H), 10.70
(1H, s, N–H), 9.85 (1H, s, amide-NH), 8.65 (1H, s),
8.25 (1H, d, J = 7.5 Hz), 7.65 (1H, dd, J = 7.2, 7.5 Hz),
7.37 (5H, m), 7.02 (2H, m), 6.88 (1H, J = 7.2 Hz), 4.69
(2H, s, CH2); MS (MALDI-TOF) m/z: 460.
1
4.1.4. 5-(2-Fluorobenzamido)-indirubin-30-oxime (21). H
NMR (300 MHz, DMSO-d6) d (ppm) 13.30 (1H, s,
NOH), 11.75 (1H, s, N0–H), 10.72 (1H, s, N–H), 9.95
(1H, s, amide-NH), 8.72 (1H, s), 8.23 (1H, d,
J = 7.8 Hz), 7.75 (1H, t, J = 7.8 Hz), 7.58 (1H, m),
7.37 (5H, m), 7.03 (1H, m), 6.88 (1H, d, J = 8.4 Hz);
MS (MALDI-TOF) m/z: 414.
4.1.4.9. 5-(2-(4-Chlorophenoxy)acetamido)-indirubin-
30-oxime (30). 1H NMR (300 MHz, DMSO-d6) d
(ppm) 13.40 (1H, s, NOH), 11.75 (1H, s, N0–H), 10.71
(1H, s, N–H), 9.72 (1H, s, amide-NH), 8.60 (1H, s),
8.23 (1H, d, J = 7.5 Hz), 7.36 (5H, m), 7.02 (4H, m),
4.68 (2H, s, CH2); MS (MALDI-TOF) m/z: 460.
4.1.4.1. 5-(4-Fluorobenzamido)-indirubin-30-oxime (22).
1H NMR (300 MHz, DMSO-d6) d (ppm) 13.36 (1H, s,
NOH), 11.76 (1H, s, N0–H), 10.73 (1H, s, N–H), 10.04
(1H, s, amide-NH), 8.66 (1H, s), 8.23 (1H, d,
J = 8.4 Hz), 8.10 (2H, m), 7.34 (5H, m), 7.03 (1H, m),
6.89 (1H, d, J = 8.1 Hz); MS (MALDI-TOF) m/z: 414.
4.1.4.10. 5-(Cinnamamido)-indirubin-30-oxime (31). 1H
NMR (300 MHz, DMSO-d6) d (ppm) 13.36 (1H, s,
NOH), 11.76 (1H, s, N0–H), 10.68 (1H, s, N–H), 9.89
(1H, s, amide-NH), 8.63 (1H, s), 8.24 (1H, d,
J = 7.5 Hz), 7.59 (3H, m), 7.44 (6H, m), 7.03 (1H, m),
6.87 (2H, m); MS (MALDI-TOF) m/z: 422.
4.1.4.2. 5-(2-Trifluoromethylbenzamido)-indirubin-30-
1
oxime (23). H NMR (300 MHz, DMSO-d6) d (ppm)
13.33 (1H, s, NOH), 11.74 (1H, s, N0–H), 10.71 (1H, s,
N–H), 10.13 (1H, s, amide-NH), 8.70 (1H, s), 8.23

M. J. Moon et al. / Bioorg. Med. Chem. 14 (2006) 237–246
245
4.1.4.11. 5-(3-Phenylpropanamido)-indirubin-30-oxime
(32). 1H NMR (300 MHz, DMSO-d6) d (ppm) 13.35
(1H, s, NOH), 11.73 (1H, s, N0–H), 10.65 (1H, s, N–
H), 9.60 (1H, s, amide-NH), 8.56 (1H, s), 8.23 (1H, d,
J = 7.5 Hz), 7.39 (2H, m), 7.32 (5H, m), 7.21 (1H, m),
7.04 (1H, m), 6.81 (1H, d, J = 8.1 Hz), 2.94 (2H, t,
J = 7.2 Hz, CH2), 2.61 (2H, t, J = 7.2 Hz, CH2); MS
(MALDI-TOF) m/z: 424.
Pharmacia Biotech UK Ltd., Buckinghamshire, UK)
and then incubated with anti-CDK2 antibody (Santa
Cruz, CA) at 4 ꢁC for 2 h. After incubation, immune
complexes were collected with protein G–Sepharose.
The kinase reaction using CDK2 immune complexes
was carried out for 30 min in 30 ll containing 20 mM
HEPES, pH 7.5, 1 mM DTT, 20 mM MgCl2, 2 mM
EGTA, 0.1 mM vanadate, 0.2 lCi [c-33P]ATP (3000
Ci mmolꢀ1, 25 lM ATP, and 1 lg substrate (GST-Rb fu-
sion protein: amino acids 761–928)) at 30 ꢁC. The reac-
tions were stopped by adding Laemmli buffer and
heated to 100 ꢁC for 5 min. The proteins were separated
by SDS–PAGE on 10% gels, dried, and exposed to an
X-ray-sensitive film.
4.1.4.12. 5-Diphenylacetamido-indirubin-30-oxime (33).
1H NMR (300 MHz, DMSO-d6) d (ppm) 13.33 (1H, s,
NOH), 11.71 (1H, s, N0–H), 10.65 (1H, s, N–H), 10.13
(1H, s, amide-NH), 8.55 (1H, s), 8.23 (1H, d,
J = 6.6 Hz), 7.34 (13H, m), 7.02 (1H, m), 6.81 (1H, d,
J = 8.4 Hz), 5.21 (1H, s, CH); MS (MALDI-TOF) m/z:
486.
Acknowledgments
4.1.4.13. 5-(1-Naphthamido)-indirubin-30-oxime (34).
1H NMR (300 MHz, DMSO-d6) d (ppm) 13.36 (1H, s,
NOH), 11.76 (1H, s, N0–H), 10.72 (1H, s, N–H), 10.11
(1H, s, amide-NH), 8.79 (1H, s), 8.32 (1H, m), 8.23
(1H, d, J = 7.2 Hz), 8.04 (2H, m), (1H, d, J = 6.9 Hz),
7.62 (3H, m), 7.52 (1H, d, J = 8.4 Hz), 7.03 (1H, m),
6.91 (1H, d, J = 8.4 Hz); MS (MALDI-TOF) m/z: 446.
This work was supported by the 21C Frontier Microbial
Genomics and Applications Center Program, Ministry
of Science and Technology (Grant MG05-0304-3-0).
Dr. Woo Keun Song was supported in part by grants
from the National Research Laboratory (The Ministry
of Science and Technology). This research was support-
ed by research grants from the Korea Health 21 R&D
Project, Ministry of Health & Welfare, Republic of Kor-
ea. (grant No. 0405-NS01-0704-0001-13).
4.1.5. Anti-proliferative activity using sulforhodamine B
assay. Cells (A549, Col2, HT1080, HL-60, MCF-7, and
Snu-638) were counted, diluted to 5 · 104 cells/ml with
fresh medium (MEME, DMEM, or RPMI containing
10% FBS), and 190 ll of cell suspension was added to
96-well plates containing various concentrations of test
compounds (10 ll in 10% aqueous DMSO).28 Test
plates were incubated for 3 days at 37 ꢁC in a CO2 incu-
bator. For zero day controls, cells were incubated for
30 min at 37 ꢁC in a CO2 incubator. All treatments were
performed in triplicate. After the incubation periods,
cells were fixed with 50 ll cold 50% TCA at 4 ꢁC for
30 min, washed five times with tap water, and air-dried.
The fixed cells were stained with 0.4% sulforhodamine B
(SRB) solution in 1% aqueous acetic acid at room tem-
perature for 1 h. Free SRB solution was then removed
by rinsing five times with 1% acetic acid and air-dried.
The bound dye was dissolved with 200 ll 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 treat-
ment 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 non-linear regression analysis
(percent survival versus concentration).
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