Synthesis of novel 5-substituted indirubins as protein kinases inhibitors

Article abstract of DOI:10.1016/j.bmc.2006.05.036

In an effort to identify new pharmacological inhibitors of disease-relevant protein kinases with increased potency and selectivity, we synthesized and evaluated new 5-substituted indirubins. The effects of 34 indirubin derivatives on CDK1/cyclin B, CDK5/p25, and GSK-3, as well as on SH-SY5Y human neuroblastoma cell survival, were investigated.

Full text of DOI:10.1016/j.bmc.2006.05.036

Bioorganic & Medicinal Chemistry 14 (2006) 6434–6443
Synthesis of novel 5-substituted indirubins as
protein kinases inhibitors
a
b
a
b
´
´
`
Anne Beauchard, Yoan Ferandin, Stephane Frere, Olivier Lozach,
b
b
a,
a
*
´
´
Melina Blairvacq, Laurent Meijer, Valerie Thiery and Thierry Besson
aLaboratoire de Biotechnologies et de Chimie Bio-organique, FRE CNRS 2766, UFR Sciences Fondamentales
ˆ
et Sciences pour l’Inge´nieur, Universite´ de La Rochelle, Batiment Marie Curie, 17042 La Rochelle, France
bCell Cycle Group, UMR 7150 & UPS2682, Station Biologique, B.P. 74, 29682 Roscoff cedex, Bretagne, France
Received 5 April 2006; revised 12 May 2006; accepted 18 May 2006
Available online 8 June 2006
Abstract—In an effort to identify new pharmacological inhibitors of disease-relevant protein kinases with increased potency and
selectivity, we synthesized and evaluated new 5-substituted indirubins. The effects of 34 indirubin derivatives on CDK1/cyclin B,
CDK5/p25, and GSK-3, as well as on SH-SY5Y human neuroblastoma cell survival, were investigated.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
a high selectivity for CDK1 and CDK2.4 Among indiru-
bin isomers isolated from marine organisms, the natural
product 6-bromoindirubin and its synthetic, cell-perme-
able derivative, 6-bromoindirubin-30-oxime (Fig. 1, IV),
display increased selectivity for inhibition of GSK-3 ver-
sus CDKs.7,8
Because of their involvement in all major human diseas-
es protein kinases have become a major screening target
in the pharmaceutical industry1. Cyclin-dependent ki-
nases (CDKs) constitute a family of highly conserved
protein kinases targeted for the therapy of cancer, viral
infections and neurodegenerative diseases. Glycogen
synthase kinases (GSK-3) play a key role in neurodegen-
erative disorders (Alzheimer’s disease), diabetes, and
cardiovascular diseases. Both families of kinases have
thus been extensively used as targets to identify small
molecular weight pharmacological inhibitors of poten-
tial therapeutic interest.2,3 Over 100 CDK inhibitors
and 40 GSK-3 inhibitors have been identified, most of
which act by competition with ATP for binding at the
kinase catalytic site.4,5 Among these inhibitors, the bis-
indole indirubin (Fig. 1, I) and its analogs has raised
considerable interest.5 Indirubin is a red-colored isomer
of the famous blue dye indigo (Fig. 1, II). Interestingly
indirubin is the active constituent of a traditional Chi-
nese medicine, Danggui Longhui Wan, reported as a
treatment for chronic myelocytic leukemia.5,6 Indiru-
bin-30-oxime (Fig. 1, III) and indirubin 5-sulfonate show
In the course of our studies of small molecular weight
kinase inhibitors of potential therapeutic interest, we
synthesized new series of 5-substituted indirubins, with
the aim of improving the pharmacological properties
of this promising scaffold. The effects of these new
indirubins on CDKs (CDK1, CDK5) and GSK-3 were
investigated. As this work was in progress, similar oxin-
dole-based CDK inhibitors were described.9,10
2. Results and discussion
2.1. Chemistry
In an effort to optimize new kinase inhibitors with in-
creased potency and selectivity, we synthesized various
new 5-nitro-indirubins, 5-amino-indirubins, and 5-halo-
geno-indirubins.
The preparation of substituted indirubins 12–27 was
accomplished by a route analogous to that described
in the literature, that is, by condensation of isatins 1–9
with indoxyl acetates 10 or 11 in the presence of Na2CO3
Keywords: Indirubin; Cyclin-dependent kinase; Glycogen synthase
kinase-3.
*
Corresponding author. Tel.: +33 (0)5 46 45 82 76; fax: +33 (0)5 46 45
82 47; e-mail: vthiery@univ-lr.fr
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.036

A. Beauchard et al. / Bioorg. Med. Chem. 14 (2006) 6434–6443
6435
Br
HO
5
HO
6
N
N
O
4
H
O
3'
4'
7'
N
7
3
5'
NH
NH
N
N
N
2'
NH
1
2
H
N
H
H
6'
O
1' H
O
O
O
I
II
III
IV
indirubine-3'-oxime
(IO)
6-bromoindirubin-3'-oxime
(6BIO)
indirubin
indigo
Figure 1. Structure of indirubin, its isomer indigo, and frequently indirubin derivatives.
in methanol (Scheme 1).4,8,10 The bis-indoles were ob-
O
O
R1
a
R1
tained in a Z form. The structure of the Z isomers was
unambiguously established by an X-ray diffraction
study of compound 17. Because of poor water solubility
and low bioavailability of indirubins 12–27, their oxime
analogs 28–43 were also synthesized with hydroxyl-
amine in pyridine under microwave irradiation11
(Scheme 1). According to literature data, the oximes
were selectively prepared in a (20Z, 30E) form. In order
to establish the structure of the different indirubin
regioisomers unambiguously, we prepared some previ-
ously described compounds 12 and 20 which were used
as biological references.
O
O
N
CH3
N
H
1 (R1 = NO2)
2 (R1 = Br)
4 (R1 = H)
7 (R1 = NO2)
8 (R1 = Br)
9 (R1 = H)
Scheme 3. Reagents and condition: (a) NaH, CH3I, THF, rt.
2.2. Biology
5-Aminoisatin 5 was obtained from commercially avail-
able 5-nitroisatin 1 by reduction in the presence of iron
in acidic media in 79% yield. Treatment of aminoisatin 5
with acetic anhydride yielded 91% of the corresponding
amide 6 (Scheme 2).
Indirubin analogs were next tested for their potential
inhibitory action on three protein kinases, CDK1/cyclin
B, CDK5/p25, and GSK-3a/b. Kinases were purified
and assayed in the presence of 15 lM ATP and appropri-
ate protein substrates (histone H1 for CDKs, GS-1 pep-
tide for GSK-3) as previously described.12–14 For those
compounds exhibiting some inhibitory activity at a
10 lM concentration, IC50 values were determined from
dose–response curves and are provided in Tables 1–3.
Alkaline treatment of indole-2,3-diones 1, 2, 4 with
iodomethane afforded the N-methylisatin derivatives
7–9 in quantitative yield (Scheme 3).
Scheme 1. Reagents and conditions: (a) Na2CO3, MeOH, rt, 30–95%; (b) NH2OHÆHCl, pyridine, lW, 25–90%.
O
O
O
AcHN
H2N
O2N
b
O
a
O
O
N
N
H
N
H
H
1
5
6
Scheme 2. Reagents and conditions: (a) Fe, HCl 36%, 70 ꢁC; (b) Ac2O, pyridine, 0 ꢁC.

6436
A. Beauchard et al. / Bioorg. Med. Chem. 14 (2006) 6434–6443
Table 1. Kinase inhibition by 5-nitroindirubin analogs
R2
X
R3
N
N
R1
H
O
Compound
R1
R2
R3
X
CDK1
CDK5
GSK-3
SH-SY5Y survival
24 h
48 h
12a
13
H
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
H
O
40
>10
>10
>10
25
>10
>10
>10
19
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
CH3
H
H
O
14b
15
Br
Br
H
O
CH3
H
O
28a,b
29
30b
NOH
NOH
NOH
NOH
0.019
0.006
1.4
0.0021
1.8
>10
1.8
>10
1.2
>10
1.2
1.2
CH3
H
H
2.2
0.16
8
0.53
0.055
9.3
Br
Br
0.05
3.3
31
CH3
Effects of 5-nitroindirubins on the activity of three protein kinases and on SH-SY5Y cell survival. Kinase assays: IC50 values were determined
for compounds displaying some activity at an initial 10 lM concentration. SH-SY5Y cells were exposed for 24 h to increasing concentrations of
5-nitroindirubins. Cell survival was estimated by the MTS reduction assay and is expressed in % of survival in untreated cells. Average SE of at
least four independent experiments with three independent measurements per experiment.
a CDK/GSK-3 inhibition also reported in Ref. 7.
b Anti-proliferative activity and CDK2 inhibition also reported in Ref. 10.
Table 2. Kinase inhibition by 5-aminoindirubin analogs
R2HN
X
R3
N
N
R1
H
O
Compound
R1
R2
R3
X
CDK1
CDK5
GSK-3
SH-SY5Y survival
24 h
48 h
16
17
18
19
32
33a
34
35
H
H
H
H
H
H
H
H
H
H
O
1.5
0.59
0.018
0.55
0.11
0.15
0.16
1
0.08
0.0075
0.44
0.073
0.36
0.35
6.6
>10
>10
>10
>10
6
>10
>10
>10
>10
5
Ac
H
H
O
0.05
0.6
Br
Br
H
O
Ac
H
O
0.055
0.1
NOH
NOH
NOH
NOH
Ac
H
H
0.088
0.41
1.4
10
>10
2
Br
Br
4.8
5.3
Ac
3.3
40
4
Effects of 5-aminoindirubins on the activity of three protein kinases and on SH-SY5Y cell survival. Kinase and cellular assays as described in Table 1.
a Anti-proliferative activity and CDK2 inhibition also reported in Ref. 10.
In previously published results, substitution at position
6 contributed to an increased selectivity.7 Among the
known 6-substituted indirubins, the bromo analog
(Fig. 1, III) exhibited the highest activity against GSK-
3. Although it had also been demonstrated that some
5-nitroindirubin derivatives showed an enhanced inhibi-
tory activity on CDK1 or CDK2, no indications on
GSK-3 had been reported.9,10
cyclin B, CDK5/p25, and GSK-3 (0.019, 0.006, and
0.0021 lM vs 40, 25, and 19 lM, respectively). Addition
of a bromine in position 50 led to a reduction in kinase
inhibition (compounds 30 and 31).
Unexpectedly, the 1-N-methyl-5-nitro-indirubin-30-ox-
ime 29 and the 1-N-methyl-5-nitro-30-monoxime-50-bro-
mo-indirubin 31 displayed modest but significant
inhibitory activity on the three kinases (Table 1).
1-Methyl derivatives have so far provided kinase inactive
control compounds7. The reason for the residual activity
of compounds 29 and 31 remains to be identified.
First, we considered the nitro-indirubin derivatives
(Table 1). 5-Nitroindirubins 12–15 displayed little, if
any, kinase inhibitory activity. Substitution by an oxime
on position 30 greatly enhanced the kinase inhibitory
activity (compounds 28–31). For example, compared
to 5-nitro-indirubin 12, 5-nitro-indirubin-30-oxime 28
presented a much higher inhibitory activity on CDK1/
We next considered the amino-indirubin derivatives (Ta-
ble 2). Replacement of the nitro group on position 5 by
the electron-donating amino group leads to kinase active

A. Beauchard et al. / Bioorg. Med. Chem. 14 (2006) 6434–6443
Table 3. Kinase inhibition by 5-halogenoindirubin analogs
6437
Compound
R1
R2
R3
X
CDK1
CDK5
GSK-3
SH-SY5Y
survival
24 h
48 h
20a
21
H
Br
Br
Br
Br
F
H
O
0.4
>10
>10
>10
>10
>10
>10
>10
0.53
7.1
0.25
>10
>10
>10
>10
>10
>10
>10
0.46
23
0.21
>10
>10
>10
>10
>10
>10
>10
1.3
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
5
CH3
H
H
O
22
Br
Br
H
O
23
CH3
H
O
24b
25
O
H
F
Br
Br
H
O
26
CH3
CH3
H
H
H
F
O
27
O
36b
37
H
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
H
F
Br
H
15
2
38b
39
H
Br
Br
Br
Br
H
H
0.033
>10
0.23
>10
>10
>10
0.016
>10
0.1
0.053
>10
24c
7
CH3
H
H
>10
>10
>10
>10
>10
40
41
Br
Br
H
CH3
CH3
CH3
>10
>10
>10
>10
>10
>10
42
43
Br
Effects of 5-halogenoindirubins on the activity of three protein kinases and on SH-SY5Y cell survival. Kinase and cellular assays as described in
Table 1.
a CDK/GSK-3 inhibition also reported in Ref. 7.
b Anti-proliferative activity and CDK2 inhibition also reported in Ref. 10.
c Precipitates.
indirubins (Table 2). In addition a somewhat enhanced
selectivity for GSK-3 over CDKs was generally ob-
served. Among all 5-NH-substituted indirubins reported
in Table 2, the acetamidoindirubin 17 appeared to be the
most potent GSK-3 inhibitor (IC50: 7.5 nM). In this
amino series, addition of a bromo substitution on posi-
tion 50 led to a major reduction in GSK-3 inhibition.
This contrasted with the enhancement of GSK-3 inhib-
itory potency observed with 50-bromo-5-nitro deriva-
tives (Table 1). As expected, the selectivity for CDK
versus GSK-3 inhibition was slightly enhanced for 5-
amino indirubin substituted with an oxime on 30 (com-
pounds 32–35). In all cases, addition of an amino or
acetamido group on position 5 led to an overall increase
in kinase inhibitory effects (Table 2).
SH-SY5Y cells estimated with an MTS assay as previ-
ously described.15 For those compounds exhibiting
some inhibitory activity at a 10 lM concentration,
IC50 values were determined from dose–response
curves and are provided in Tables 1–3. Results show
that: (1) all indirubins that are not substituted on 30
were inactive; (2) all N1-methylated indirubins were
inactive with the exception of compound 31 which
showed some activity after 48 h of treatment; (3)
5-nitroindirubin-30-oxime (28) and 5-nitro-50-bromo-
indirubin-30-oxime (30), 5-amino-indirubin-30-oxime
(32), 5-amino-50-bromoindirubin-30-oxime (34), and 5-
acetamino-50-bromoindirubin-30-oxime (35) comprised
cell survival, even after 24 h of treatment; (4) 5-halo-
geno-indirubin-30oximes (36–38) had modest effects;
(5) no correlation could be established between the
effects of the presented indirubins on kinases and their
effects on cell survival.
We next tested the consequence of substitution at posi-
tion 5 by a weaker electron-withdrawing group such as
a bromine or a fluorine atom. This mainly resulted in
a loss of kinase inhibitory activity, except for 5-fluoro-
indirubin-30-oxime (36) which exhibited modest kinase
inhibitory activity, and for the known, potent 5-bro-
mo-indirubin-30-oxime 38 (Table 3). Addition of the
bromine atom on position 50 or alkylation on N1 did
not increase activity. All N1-methylated compounds
were inactive.
3. Conclusion
During the past decade, several groups have been
interested in indirubin bis-indoles as a protein kinase
inhibitory scaffold. We here reported the synthesis of
5-substituted indirubin analogs. These derivatives
were evaluated against the catalytic activity of CDK1,
CDK5, and GSK-3. Compounds 17, 19, 28, 30, and
38 are potent, low nanomolar inhibitors of GSK-3.
Low but maybe significant kinase inhibitory activity
was observed with 1-N-methyl indirubin compounds
2.3. Cellular viability assays
The synthesized indirubins were next evaluated for
their effects on the survival of human neuroblastoma

6438
A. Beauchard et al. / Bioorg. Med. Chem. 14 (2006) 6434–6443
29 and 31, in contrast with previously described indiru-
bins substituted in N1.
(100 MHz, DMSO-d6) d ppm 185.31, 159.28, 44.76,
140.73, 123.46, 117.99, 112.60, 108.98. IR = 3196,
1730, 1616, 1499, 1335, 1200, 834 cmꢀ1. HRMS (EI)
[M]+Å (C8H6N2O2): calcd 162.0429, found 162.0421.
4. Experimental
4.1. Chemistry
4.1.1.2. 5-Acetamidoisatin (6). Under an inert atmo-
sphere of argon, to a solution of amine 5 (0.17 g,
1.05 mmol) in pyridine (3 mL) was added dropwise ace-
tic anhydride (0.10 mL, 1.05 mmol). After 3 h under stir-
ring at 0 ꢁC, the residue was treated with a saturated
solution of sodium carbonate NaHCO3 and extracted
with ethyl acetate. The organic layer was dried (MgSO4)
and the filtrate was concentrated under reduced pres-
sure. The crude residue was purified by chromatography
on silica gel, with dichloromethane/ethyl acetate (5:5) as
eluent to give the acetamide 3. Yield: 91%. Mp > 260 ꢁC.
1H NMR (400 MHz, DMSO-d6) d ppm 10.88 (s, NH),
9.940 (s, NH), 7.78 (d, 1H, J = 2.4 Hz, H-4), 7.64 (dd,
1H, J = 2.4, 8.4 Hz, H-6), 6.86 (d, 1H, J = 8.4 Hz, H-
7), 2.02 (s, 3H). 13C NMR (100 MHz, DMSO-d6) d
ppm 184.99, 169.32, 159.99, 146.44, 134.87, 129.46,
117.99, 115.86, 112.90, 24.07. IR = 3095, 1736, 1498,
1289, 1197 cmꢀ1. HRMS (EI) [M]+Å (C10H8N2O3): calcd
204.05349, found 204.0530.
Commercial reagents were used as received without
additional purification. Melting points were determined
using a Ko¨fler melting point apparatus and are uncor-
rected. IR spectra were recorded on a Perkin-Elmer Par-
agon 1000PC instrument. 1H and 13C NMR were
recorded on a JEOL NMR LA400 (400 MHz) spectrom-
´
eter in the ‘Centre Commun d’Analyses, Universite de la
Rochelle’. Chemical shifts (d) are reported in parts per
million (ppm) downfield from tetramethylsilane (TMS)
which was used as internal standard. Coupling constants
J are given in Hz. The mass spectra (HRMS) were
recorded on a Varian MAT311 spectrometer in the
´
‘Centre Regional de Mesures Physiques de l’Ouest’
(CRMPO), Universite de Rennes. Column chromatog-
´
raphy was performed by using Merck silica gel (70–
230 mesh) at medium pressure. Light petroleum refers
to the fraction boiling point 40–60 ꢁC. Other solvents
were used without purification. Analytical thin-layer
chromatography (TLC) was performed on Merck Kie-
selgel 60 F254 aluminium-backed plates. Focused
microwave irradiations were carried out with a CEM
DiscoverTM focused microwave reactor (300 W,
2450 MHz, monomode system), having in situ magnetic
variable speed rotation, irradiation monitored by PC
computer, infrared measurement and continuous feed-
back temperature control. Experiments may be per-
formed at atmospheric pressure or in a sealed tube in
pressure-rated reaction tubes with continuous pressure
measurement.
4.1.2. General procedure for the synthesis of 1-N-methyl
isatin derivatives. Under an inert atmosphere of argon,
to a suspension of isatin (5 mmol) and sodium hydride
at 60% dispersion in oil (0.17 g, 5 mmol) in DMF
(10 mL) was added dropwise methyl iodide (0.7 mL,
5 mmol). After 12 h under stirring at room tempera-
ture, the residue was treated with water and extracted
with ethyl acetate. The organic layer was dried
(MgSO4) and the filtrate was concentrated under re-
duced pressure. The crude residue was recrystallized
from ethanol.
4.1.2.1. 1-N-Methyl-5-nitroisatin (7). Yield: 93%. Mp
202 ꢁC. 1H NMR (400 MHz, DMSO-d6) d ppm 8.53
(dd, 1H, J = 2.4, 8.8 Hz, H-6), 8.22 (d, 1H, J = 2.4 Hz,
H-4), 7.35 (d, 1H, J = 8.8 Hz, H-7), 3.31 (s, 3H, CH3).
IR = 3110, 1717, 1656, 1507, 1335, 1276, 1132,
Starting materials 1, 2, 3, 4, and 9 are commercially
available. Spectral data for compounds 12, 20, 24, and
38 are consistent with assigned structures as previously
described.7 Spectral data for compounds 14, 28, 33, 36
are consistent with assigned structures as previously
described.10
754 cmꢀ1
.
HRMS (EI) [M]+Å (C9H6N2O4): calcd
206.03276, found 206.0322.
4.1.1. Synthesis of 5-N-substituted-isatins
4.1.2.2. 1-N-Methyl-5-bromoisatin (8). Yield: 99%.
Mp 170 ꢁC. 1H NMR (400 MHz, DMSO-d6) d ppm
7.83 (dd, 1H, J = 2.0, 8.8 Hz, H-6), 7.68 (d, 1H,
J = 8.8 Hz, H-6), 7.11 (d, 1H, J = 8.8 Hz, H-7), 3.12 (s,
3H, CH3). 13C NMR (100 MHz, DMSO-d6) d ppm
184.99, 169.32, 159.99, 146.44, 134.87, 129.46, 117.99,
115.86, 112.90, 24.07. IR = 3095, 1736, 1498, 1289,
1197 cmꢀ1. HRMS (EI) [M]+Å (C9H6NO279Br): calcd
238.95819, found 238.9565.
4.1.1.1. 5-Aminoisatin (5). Under an inert atmosphere
of argon, 5-nitroisatin 1 (0.50 g, 2.6 mmol) was dis-
solved in warm ethanol (10 mL), and reduced iron pow-
der (0.43 g, 7.8 mmol) was added. Concentrated
hydrochloric acid (2 mL) was added gradually and the
reaction mixture refluxed for 30 min. When the reaction
mixture turned colorless, the residue was treated with a
saturated solution of sodium carbonate NaHCO3 to pH
8 and extracted with ethyl acetate. The organic layer was
dried (MgSO4) and the filtrate was concentrated under
reduced pressure. The crude residue was purified by
chromatography on silica gel, with dichloromethane/
MeOH (95:5) as eluent to give the amino derivative 5.
Yield: 79%. Mp > 260 ꢁC. 1H NMR (400 MHz,
DMSO-d6) d ppm 10.60 (s, NH), 6.82 (dd, 1H, J = 2.4,
8.4 Hz, H-6), 6.71 (d, 1H, J = 2.4 Hz, H-4), 6.63 (d,
1H, J = 8.4 Hz, H-7), 5.12 (s, 2H, NH2). 13C NMR
4.1.3. General procedure for the synthesis of indirubin
derivatives. Under an inert atmosphere of argon, a solu-
tion of isatin derivatives 1–9 (3 mmol), indoxyl acetate
10 or 11 (5 mmol) in methanol (15 mL) was vigorously
stirred with Na2CO3. After 3 h under stirring at room
temperature, the dark violet residue was filtered and suc-
cessively and intensively washed with methanol and cold

A. Beauchard et al. / Bioorg. Med. Chem. 14 (2006) 6434–6443
6439
water. The solid was dried over P2O5 under reduced
pressure (30–95%).
10.91 (s, N0H), 9.85 (s, NH), 8.88 (s, 1H, H-4), 7.68
(d, 1H, J = 7.2 Hz, H-40), 7.60 (t, 2H, J = 8.0 Hz, H-50,
H-70), 7.50 (d, NH, J = 8.0 Hz, NH-acetamide), 7.41
(d, 1H, J = 8.0 Hz, H-6), 7.07 (t, 1H, J = 7.2 Hz, H-
50), 6.88 (d, 1H, J = 8.0 Hz, H-7), 2.06 (s, 3H, CH3).
4.1.3.1. (20Z)-5-Nitroindirubin (12). Yield: 89%.
1
Mp > 260 ꢁC. H NMR (400 MHz, DMSO-d6) d ppm
11.48 (s, 1H, N0H), 11.18 (s, 1H, NH), 9.68 (s, 1H,
H-4), 8.18 (d, 1H, J = 8.4 Hz, H-6), 7.70 (d, 1H,
J = 8.0 Hz, H-40), 7.61 (t, 1H, J = 8.0 Hz, H-60), 7.44
(d, 1H, J = 8.4 Hz, H-7), 7.05–7.10 (m, 2H, H-70, H-
IR = 3277, 1732, 1665, 1475, 1313, 1196, 1004 cmꢀ1
.
HRMS (EI) [M]+Å (C18H13N3O3): calcd 319.09569,
found 319.0961.
50). IR = 3314, 3094, 2874, 1687, 1593, 1522 cmꢀ1
.
4.1.3.7. (20Z)-5-Amino-50-bromoindirubin (18). Yield:
HRMS (EI) [M]+Å (C16H9N3O4): calcd 307.05931, found
307.0565.
1
97%. Mp > 260 ꢁC. H NMR (400 MHz, DMSO-d6) d
ppm 11.01 (s, 1H, N0H), 10.47 (s, 1H, NH), 8.13
(d, 1H, J = 2.0 Hz, H-4), 7.75 (d, 1H, J = 2.0 Hz,
H-40), 7.71 (dd, 1H, J = 2.0, 8.4 Hz, H-60), 7.40 (d, 1H.
J = 8.4 Hz, H-70), 6.59 (d, 1H, J = 8.4 Hz, H-7),
6.55 (dd, 1H, J = 2.0, 8.4 Hz, H-6). IR = 3278, 1664,
1464, 1278, 1195, 1120, 1015, 819 cmꢀ1. HRMS (EI)
[M]+Å (C16H10N3O279Br): calcd 354.99564, found
354.9973.
4.1.3.2. (20Z)-1-Methyl-5-nitroindirubin (13). Yield:
90%. Mp > 260 ꢁC. 1H NMR (400 MHz, CDCl3) d
ppm 10.54 (s, 1H, N0H), 9.82 (d, 1H, J = 2.0 Hz, H-4),
8.29 (dd, 1H, J = 7.6 Hz, H-6), 7.80 (d, 1H, J = 7.6 Hz,
H-40), 7.57 (t, 1H, J = 7.6 Hz, H-60), 7.10 (t, 1H,
J = 7.6 Hz, H-60), 7.04 (d, 1H, J = 7.5 Hz, H-50), 6.97
(d, 1H, J = 8.8 Hz, H-7), 3.43 (s, 3H, CH3). 13C NMR
(100 MHz, DMSO-d6) d ppm 181.98, 157.87, 152.75,
151.68, 124.80, 121.82, 118.18, 105.07, 26.30.
IR = 3294, 3098, 1675, 1612, 1522, 1473, 1089,
749 cmꢀ1. HRMS (EI) [M]+Å (C17H12N3O4): calcd
321.07496, found 321.0772.
4.1.3.8. (20Z)-5-Acetamido-50-bromoindirubin (19).
Yield: 85%. Mp > 260 ꢁC. 1H NMR (400 MHz,
DMSO-d6): 11.12 (s, 1H, N0H), 10.83 (s, 1H, NH),
9.91 (s, 1H, NH), 8.81 (s, 1H, H-40), 7.76 (s, 1H, H-4),
7.71 (dd, 1H. J = 2.0, 8.8 Hz, H-60), 7.56 (dd, 1H,
J = 2.0, 8.8 Hz, H-6), 7.37 (d, 1H, J = 8.8 Hz, H-70),
6.82 (d, 1H, J = 8.8 Hz, H-7), 2.03 (s, 3H, CH3). 13C
NMR (100 MHz, DMSO-d6) d ppm 187.26, 170.87,
167.89, 151.42, 139.09, 137.94, 137.10, 133.31, 126.41,
121.77, 121.29, 120.81, 117.23, 115.72, 112.82, 109.27,
107.73, 23.78. IR = 3267, 1733, 1667, 1463, 1190, 1121,
1016, 820 cmꢀ1. HRMS (EI) [M]+Å (C18H12N3O379Br):
calcd 397.00620, found 397.051.
4.1.3.3. (20Z)-50-Bromo-5-nitroindirubin (14). Yield:
1
93%. Mp > 260 ꢁC. H NMR (400 MHz, DMSO-d6) d
ppm 11.32 (s, 1H, N0H), 9.65 (d, 1H, J = 2.4 Hz, H-4),
8.19 (dd, 1H, J = 2.4, 8.4 Hz, H-6), 7.84 (d, 1H,
J = 2.4 Hz, H-40), 7.76 (dd, 1H. J = 2.4, 8.4 Hz, H-60),
7.42 (d, 1H, J = 8.4 Hz, H-7), 7.07 (d, 1H, J = 8.4 Hz,
H-70). IR = 3312, 1687,1605, 1464, 1287, 1193,
818 cmꢀ1. HRMS (EI) [M]+Å (C16H9N3O479Br): calcd
384.96982, found 384.9725.
4.1.3.9. (20Z)-5-Bromoindirubin (20). Yield: 95%.
1
Mp > 260 ꢁC. H NMR (400 MHz, DMSO-d6) d ppm
4.1.3.4.
(20Z)-1-Methyl-50-bromo-5-nitroindirubin
11.02 (s, 1H, N0H), 8.93 (d, 1H, J = 1.9 Hz, H-4), 7.66
(d, 1H, J = 7.3 Hz, H-40), 7.56 (t, 1H. J = 8.3 Hz, H-
6), 7.38–7.44 (m, 2H, H-50, H-70), 7.04 (t, 1H,
J = 7.3 Hz, H-60), 6.86 (d, 1H, J = 8.3 Hz, H-7).
IR = 3152, 1673, 1590, 1466, 1214, 1004, 801,
580 cmꢀ1. HRMS (EI) [M]+Å (C16H9N2O2Br): calcd
321.07496, found 321.0772.
(15). Yield: 70%. Mp > 260 ꢁC. 1H NMR (400 MHz,
DMSO-d6) d ppm 11.33 (s, 1H, N0H), 9.68 (d, 1H,
J = 2.0 Hz, H-4), 8.28 (dd, 1H, J = 2.0, 8.8 Hz, H-6),
7.85 (d, 1H, J = 2.0 Hz, H-40), 7.77 (dd, 1H, J = 2.0,
8.8 Hz, H-60), 7.43 (d, 1H, J = 8.8 Hz, H-70), 7.30 (d,
1H,.J = 8.8 Hz, H-7). IR = 3300, 1680, 1610, 1466,
1117, 824 cmꢀ1. HRMS (EI) [M]+Å (C17H11N2O279Br):
calcd 398.98547, found 398.9886.
4.1.3.10. (20Z)-1-Methyl-5-bromoindirubin (21). Yield:
1
4.1.3.5. (20Z)-5-Aminoindirubin (16). Yield: 90%.
30%. Mp > 260 ꢁC. H NMR (400 MHz, DMSO-d6) d
ppm 11.14 (s, 1H, N0H), 8.96 (s, 1H, H-4), 7.68 (d,
1H, J = 8.0 Hz, H-40), 7.60 (t, 1H, J = 8.0 Hz, H-60),
7.50 (d, 1H, J = 8.4 Hz, H-6), 7.43 (d, 1H, J = 8.4 Hz,
H-7), 7.04–7.06 (m, 2H, H-50, H-70), 3.28 (d, 3H,
CH3). IR = 3305, 1653, 1598, 1462, 1320, 1103, 1081,
753 cmꢀ1. HRMS (EI) [M]+Å (C17H11N2O279Br): calcd
354.00039, found 354.0011.
Mp > 260 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
ppm 10.96 (s, 1H, N0H), 10.48 (s, 1H, NH), 8.17 (d,
1H, J = 2.0 Hz, H-4), 7.68 (d, 1H, J = 7.2 Hz, H-40),
7.60 (t, 1H, 7.2 Hz, H-60), 7.34 ( d, 1H, J = 8.4 Hz,
H-70), 7.06 (t, 1H, J = 7.2 Hz, H-50), 6.70 (d, 1H,
J = 8.0 Hz, H-7), 6.61 (dd, 1H, J = 2.0, 8.0 Hz, H-6),
4.74 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d6)
d ppm 189.15, 171.48, 152.71, 143.46, 138.47, 138.00,
132.76, 125.06, 122.48, 122.02, 119.58, 116.70,
113.69, 112.26, 110.68,1 08.59. IR = 3226, 1767,
1730, 1713, 1612, 1480, 1295, 1195, 1002 cmꢀ1. HRMS
(EI) [M]+Å (C16H11N3O2): calcd 277.08513, found
277.0838.
4.1.3.11. (20Z)-5,50-Dibromoindirubin (22). Yield:
1
79%. Mp > 260 ꢁC. H NMR (400 MHz, DMSO-d6) d
ppm 11.11 (s, 1H, N0H), 8.90 (s, 1H, H-4), 7.77 (s, 1H,
H-40), 7.73 (d, 1H, J = 8.4 Hz, H-6), 7.41 (s, 2H, H-60,
H-70), 6.85 (d, 1H, J = 8.4 Hz, H-7). IR = 3488, 1669,
1611, 1462, 1270, 1206, 1123, 1017, 822 cmꢀ1. HRMS
(EI) [M]+Å (C16H8N2O279Br): calcd 417.89525, found
417.89892.
4.1.3.6. (20Z)-5-Acetamidoindirubin (17). Yield: 90%.
1
Mp > 260 ꢁC. H NMR (400 MHz, DMSO-d6) d ppm

6440
A. Beauchard et al. / Bioorg. Med. Chem. 14 (2006) 6434–6443
4.1.3.12. (20Z)-5-Fluoroindirubin (24). Yield: 95%.
7.2 Hz, H-60), 7.06 (d, 1H, J = 8.4 Hz, H-70).
IR = 3281, 1732, 1671, 1615, 1459, 1328, 1220, 1084,
1019, 747 cmꢀ1. HRMS (EI) [M]+Å (C16H10N4O4): calcd
322.07020, found 322.0707.
1
Mp > 260 ꢁC. H NMR (400 MHz, DMSO-d6) d ppm
11.09 (s, 1H, N0H), 10.89 (s, 1H, NH), 8.57 (dd, 1H,
J = 2.8, 7.2 Hz, H-60), 7.67 (d, 1H, J = 7.2 Hz, H-40),
7.59 (t, 1H. J = 7.2 Hz, H-50), 7.44 (d, 1H, J = 7.6 Hz,
H-4), 7.09 (td, 1H, J = 2.8, 7.6 Hz, H-6), 7.04 (d, 1H,
J = 7.2 Hz, H-70), 6.88 (m, 1H, H-7). IR = 3331, 3196,
1667, 1600, 1468, 1309, 1187, 1148, 1003, 818,
753 cmꢀ1. HRMS (EI) [M]+Å (C16H9N2O2F): calcd
280.06481, found 280.0644.
4.1.4.2. (20Z,30E)-1-Methyl-5-nitroindirubin-30-oxime
(29). Yield: 44%. Mp > 260 ꢁC. 1H NMR (400 MHz,
DMSO-d6) d ppm 13.95 (s, 1H, NOH), 11.90 (s, 1H,
N0H), 9.50 (d, 1H, J = 2.4 Hz, H-4), 8.26 (d, 1H,
J = 8.0 Hz, H-40), 8.15 (dd, 1H, J = 2.4, 8.8 Hz, H-6),
7.50 (d, 1H, J = 8.0 Hz, H-70), 7.44 (t, 1H, J = 7.6 Hz,
H-50), 7.26 (d, 1H, J = 8.8 Hz, H-7), 7.10 (t, 1H,
J = 7.6 Hz, H-60), 3.39 (s, 3H, CH3). IR = 3242, 1602,
4.1.3.13. (20Z)-50-Bromo-5-fluoroindirubin (25). Yield:
1
80%. Mp > 260 ꢁC. H NMR (400 MHz, DMSO-d6) d
ppm 11.01 (s, 1H, N0H), 8.53 (dd, 1H, J = 2.4, 8.4 Hz,
H-60), 7.77 (s, 1H, H-40), 7.73 (d, 1H, J = 8.4 Hz, H-
70), 7.39 (d, 1H, J = 8.4 Hz, H-4), 7.08 (td, 1H, J = 2.4,
4.4 Hz, H-6), 6.86 (m, 1H, H-7), 4.09 (s, 1H, NH).
IR = 3159, 1737, 1667, 1595, 1466, 1283, 1015,
819 cmꢀ1. HRMS (EI) [M]+Å (C16H8N2O279BrF): calcd
357.97532, found 357.9727.
1557, 1510, 1465, 1338, 1125, 1078, 1021, 748 cmꢀ1
.
HRMS (EI) [M]+Å (C17H12N4O4): calcd 336.08586,
found 336.0861.
4.1.4.3. (20Z,30E)-50-Bromo-5-nitroindirubin-30-oxime
(30). Yield: 78%. Mp > 260 ꢁC. 1H NMR (400 MHz,
DMSO-d6) d 14.14 (s, 1H, NOH), 11.90 (s, 1H, N0H),
11.39 (s, 1H, NH), 9.43 (d, 1H, J = 2.4 Hz, H-4), 8.36
(d, 1H, J = 2.0 Hz, H-40), 8.09 (dd, 1H, J = 2.4,
8.8 Hz, H-6), 7.61 (dd, 1H, J = 2.0, 8.8 Hz, H-60), 7.46
(d, 1H, J = 8.8 Hz, H-70), 7.05 (d, 1H, J = 8.8 Hz, H-
7). IR = 3279, 1752, 1715, 1452, 1335, 1285,
1024 cmꢀ1. HRMS (EI) [M]+Å (C16H9N4O479Br): calcd
399.98072, found 399.9789.
4.1.3.14.
(20Z)-50-Bromo-1-methylindirubin
(26).
Yield: 63%. Mp > 260 ꢁC. 1H NMR (400 MHz,
DMSO-d6) d ppm 11.11 (s, 1H, N0H), 8.77 (d, 1H,
J = 7.8 Hz, H-4), 7.77 (d, 1H, J = 1.9 Hz, H-40), 7.73
(dd, 1H, J = 1.9, 8.4 Hz, H-60), 7.40 (d, 1H,
J = 8.4 Hz, H-70), 7.35 (td, 1H, J = 1.9, 7.8 Hz, H-6),
7.07–7.11 (m, 2H, H-5, H-7). IR = 3307, 1672, 1607,
1469,
1127,
752 cmꢀ1
.
HRMS
(EI)
[M]+Å
4.1.4.4. (20Z,30E)-1-Methyl-50-bromo-5-nitroindirubin-
30-oxime (31). Yield: 49%. Mp > 260 ꢁC. 1H NMR
(400 MHz, DMSO-d6) d ppm 14.21 (s, 1H, NOH),
11.93 (s, 1H, N0H), 9.46 (s, 1H, H-4), 8.35 (s, 1H, H-
40), 8.16 (d, 1H, J = 8.4 Hz, H-60), 7.62 (d, 1H,
J = 8.4 Hz, H-70), 7.47 (d, 1H, J = 8.4 Hz, H-6), 7.25
(d, 1H, J = 8.4 Hz, H-7), 3.38 (s, 3H, CH3). IR = 3254,
1877, 1554, 1453, 1284, 1079, 1024, 817 cmꢀ1. HRMS
(EI) [M]+Å (C17H11N4O479Br): calcd 413.99637, found
413.9989.
(C17H11N2O279Br): calcd 354.00039, found 354.0022.
4.1.3.15. (20Z)-1-Methylindirubin (27). Yield: 58%.
Mp > 260 ꢁC. 1H NMR (400 MHz, DMSO-d63) d
ppm 11.04 (s, 1H, N0H), 8.81 (d, 1H, J = 8.3 Hz, H-
4), 7.66 (d, 1H, J = 7.3 Hz, H-40), 7.59 (t, 1H,
J = 7.3 Hz, H-60), 7.43 (d, 1H, J = 7.3 Hz, H-70),
7.35 (t, 1H, J = 8.3 Hz, H-6), 7.07–7.12 (m, 2H, H-5,
H-7), 7.03 (t, 1H, J = 7.3 Hz, H-50), 3.24 (s, 3H,
CH3). IR = 3305, 1656, 1608, 1482, 1102, 748 cmꢀ1
.
HRMS (EI) [M]+Å (C17H12N2O2): calcd 276.08988,
found 276.0928.
4.1.4.5. (20Z,30E)-5-Aminoindirubin-30-oxime (32).
1
Yield: 84%. Mp > 260 ꢁC. H NMR (400 MHz, CDCl3)
d ppm 13.18 (s, 1H, NOH), 11.67 (s, 1H, N0H), 10.21 (s,
1H, NH), 8.22 (d, 1H, J = 8.4 Hz, H-40), 8.03 (d, 1H,
J = 2.0 Hz, H-4), 7.37 (t, 1H, J = 7.2 Hz, H-50), 7.33
(d, 1H, J = 7.2 Hz, H-70), 6.99 (t, 1H, J = 7.2 Hz, H-
60), 6.60 (d, 1H, J = 8.4 Hz, H-7), 6.55 (dd, 1H,
J = 2.0, 8.4 Hz, H-6), 4.21 (s, 2H, NH2). 13C NMR
(100 MHz, DMSO-d6) d ppm 170.85, 151.06, 144.82,
144.51, 141.87, 131.79, 130.04, 127.79, 123.17, 120.97,
116.37, 112.54, 111.08, 110.67, 108.68, 30.67.
IR = 3296, 1737, 1714, 1573, 1473, 1325, 1238, 999,
747 cmꢀ1. HRMS (EI) [M]+Å (C16H13N4O2): calcd
293.10385, found 293.10342.
4.1.4. General procedure for the synthesis of 30-oxime
indirubin derivatives. Under an inert atmosphere of ar-
gon, to a stirred solution of indirubin derivatives 16–
35 (3 mmol) in pyridine (5 mL) was added 2.1 g
(30 mmol) of hydroxylamine hydrochloride. The mix-
ture was irradiated during 10 min. The irradiation in
CEM oven was programmed to maintain a constant
temperature (110 ꢁC) with a maximal power output of
150 W. After cooling, the pyridine was removed under
reduced pressure. The crude material was successively
and intensively washed with cold water and acetone.
The solid was dried over P2O5 under reduced pressure
(25–90%).
4.1.4.6. (20Z,30E)-5-Acetamidoindirubin-30-oxime (33).
Yield: 25%. Mp > 260 ꢁC. 1H NMR (400 MHz, DMSO-
d6) d ppm 13.26 (s, 1H, NOH), 11.69 (s, 1H, N0H), 10.35
(s, 1H, NH), 8.19 (d, 1H, J = 8.0 Hz, H-40), 8.07 (s, 1H,
H-4), 7.38 (t, 1H, J = 8.0 Hz, H-50), 7.22 (dd, 1H,
J = 4.0 Hz, H-6), 7.03 (t, 1H, J = 7.6 Hz, H-60), 6.72
(d, 1H, J = 8.0 Hz, H-7), 6.57 (d, 1H, J = 7.6 Hz, H-
70), 2. 05 (s, 3H, CH3). IR = 3420, 1740, 1660, 1466,
4.1.4.1. (20Z,30E)-5-Nitroindirubin-30-oxime (28).
Yield: 92%. Mp > 260 ꢁC. 1H NMR (400 MHz,
DMSO-d6) d ppm 13.88 (s, 1H, NOH), 11.89 (s, 1H,
N0H), 11.39 (s, 1H, NH), 9.47 (d, 1H, J = 2.4 Hz, H-
4), 8.27 (d, 1H, J = 8.4 Hz, H-7), 8.09 (dd, 1H, J = 2.4,
8.4 Hz, H-6), 7.49 (d, 1H, J = 7.2 Hz, H-40), 7.44 (td,
1H, J = 1.6, 7.2 Hz, H-50), 7.10 (td, 1H, J = 1.6,
1319, 1200, 1003 cmꢀ1
.

A. Beauchard et al. / Bioorg. Med. Chem. 14 (2006) 6434–6443
6441
4.1.4.7. (20Z,30E)-50-Bromo-5-aminoindirubin-30-ox-
ime (34). Yield: 78%. Mp > 260 ꢁC. 1H NMR
(400 MHz, DMSO-d6) d ppm 11.71 (s, 1H, NOH),
10.41 (s, 1H, N0H), 8.32 (d, 1H, J = 2.0 Hz, H-40), 8.24
(s, 1H, H-4), 7.56 (dd, 1H, J = 2.0, 8.4 Hz, H-60), 7.36
(d, 1H, J = 8.4 Hz, H-70), 6.72 (d, 1H, J = 8.4 Hz, H-
7), 6.63 (dd, 1H, J = 2.0, 8.4 Hz, H-6), 3.25 (s, 2H,
NH2). 13C NMR (100 MHz, DMSO-d6) d ppm 196.31,
170.64, 167.60, 166.82, 163.55, 149.99, 144.46, 144.00,
134.21, 129.64, 123.10, 117.04, 113.49, 112.38, 108.90,
99.92. IR = 3218, 1746, 1665, 1457, 1303, 1007,
805 cmꢀ1. HRMS (EI) [M]+Å (C18H15N4O279BrNa):
calcd 421.02761, found 421.0283.
4.1.4.12. (20Z,30E)-1-Methyl-5-bromoindirubin-30-ox-
ime (39). Yield: 84%. Mp > 260 ꢁC. 1H NMR
(400 MHz, DMSO-d6) d 13.73 (s, 1H, NOH), 11.83 (s,
1H, N0H), 8.79 (d, 1H, J = 2.0 Hz, H-4), 8.23 (d, 1H,
J = 7.6 Hz, H-40), 7.39–7.44 (m, 2H, H-60, H-70), 7.36
(dd, 1H, J = 2.0, 8.0 Hz, H-6), 7.05 (t, 1H, J = 7.6 Hz,
H-50), 7.01 (d, 1H, J = 8.0 Hz, H-7), 3.31 (s, 3H, CH3).
IR = 3473, 3240, 1769, 1649, 1465, 1333, 1117, 1015,
973, 799 cmꢀ1. HRMS (EI) [M]+Å (C17H12N3O279Br):
calcd 369.0113, found 369.0091.
4.1.4.13. (20Z,30E)-5,50-Bromoindirubin-30-oxime (40).
Yield: 58%. Mp > 260 ꢁC. 1H NMR (400 MHz, DMSO-
d6) d ppm 13.91 (s, 1H, NOH), 11.83 (s, 1H, N0H), 10.84
(s, 1H, NH), 8.71 (s, 1H, H-40), 8.34 (s, 1H, H-4), 7.59
(dd, 1H, J = 2.0, 8.4 Hz, H-60), 7.42 (d, 1H,
J = 8.4 Hz, H-70), 7.28 (dd, 1H, J = 2.0, 8.4 Hz, H-6),
6.83 (d, 1H, J = 8.4 Hz, H-7). 13C NMR (100 MHz,
DMSO-d6) d ppm 170.88, 150.88, 145.99, 144.33,
137.89, 134.87, 130.32, 128.75, 125.36, 125.01, 118.64,
114.27, 113.43, 113.12, 110.96, 98.91. IR = 3209, 1726,
1661, 1609, 1568, 1304, 1011, 799 cmꢀ1. HRMS (EI)
[M]+Å (C16H9N3O279Br2): calcd 432.90615, found
432.9035.
4.1.4.8. (20Z,30E)-5-Acetamido-50-bromoindirubin-30-
oxime (35). Yield: 41%. Mp > 260 ꢁC. 1H NMR
(400 MHz, DMSO-d6) d ppm 11.68 (s, 1H, NOH),
10.25 (s, 1H, N0H), 8.33 (s, 1H, H arom, H-4), 8.01 (s,
1H, H arom, H-40), 7.53 (dd, 1H, H arom, J = 2.0,
8.4 Hz, H-60), 7.25 (d, 1H, H arom, J = 8.4 Hz, H-70),
6.63 (d, 1H, H arom, J = 8.0 Hz, H-7), 6.47 (dd, 1H,
H arom, J = 2.0, 8.0 Hz, H-6), 2.05 (s, 3H, CH3).
IR = 3243, 2939. 2314. 1735, 1663, 1604, 1456, 1295,
1005, 805 cmꢀ1
.
4.1.4.9. (20Z,30E)-5-Fluoroindirubin-30-oxime (36).
Yield: 90%. Mp > 260 ꢁC. 1H NMR (400 MHz,
DMSO-d6) d 13.56 (s, 1H, NOH), 11.78 (s, 1H,
N0H), 10.65 (s, 1H, NH), 8.46 (dd, 1H, H arom,
J = 2.4, 10.8 Hz), 8.23 (d, 1H, H arom, J = 8.0 Hz),
7.40 (t, 1H, H arom, J = 7.2 Hz), 7.37 (t, 1H, H arom,
J = 7.2 Hz), 7.06 (t, 1H, H arom, J = 7.2 Hz), 6.93 (td,
1H, H arom, J = 2.4, 8.0 Hz), 6.85 (dd, 1H, H arom,
J = 5.2, 8.0 Hz). IR = 3179, 2360, 1741, 1666, 1573,
1465, 1321, 1234, 1174, 968,753 cmꢀ1. HRMS (EI)
[M]+Å (C16H10N3O2F): calcd 295.07570, found
295.0767.
4.1.4.14. (20Z,30E)-1-Methyl-5,50-bromoindirubin-30-
oxime (41). Yield: 67%. Mp > 260 ꢁC. 1H NMR
(400 MHz, DMSO-d6) d ppm 14.00 (s, 1H, NOH),
11.86 (s, 1H, N0H), 8.77 (s, 1H, H-4), 8.34 (s, 1H, H-
40), 7.60 (dd, 1H, J = 2.0, 8.0 Hz, H-60), 7.43 (d, 1H,
J = 8.0 Hz, H-70), 7.38 (dd, 1H, J = 2.0, 8.4 Hz, H-6),
7.02 (d, 1H, J = 8.4 Hz, H-7), 3.27 (s, 3H, CH3).
IR = 3141, 1653, 1568, 1458, 1162, 1023, 797 cmꢀ1
HRMS (EI) calcd
[M]+Å (C17H11N3O279Br2):
446.92180, found 446.9193.
.
4.1.4.15. (20Z,30E)-1-Methyl-indirubin-30-oxime (42).
Yield: 85%. Mp > 260 ꢁC. 1H NMR (400 MHz, DMSO-
d6) d ppm 13.52 (s, 1H, NOH), 11.735 (s, 1H, N0H), 8.68
(d, 1H, J = 7.8 Hz, H-4), 8.23 (d, 1H, J = 7.2 Hz, H-40),
7.40 (m, 2H, H-60, H-70), 7.21 (t, 1H, J = 7.8 Hz, H-6),
7.025 (m, 3H, H-50, H-5, H-7), 3.32 (s, 3H, CH3).
IR = 2979, 1646, 1596, 1454, 1340, 1136, 1011,
748 cmꢀ1. HRMS (EI) [M]+Å (C17H13N3O2): calcd
291.10078, found 291.1019.
4.1.4.10. (20Z,30E)-50-Bromo-5-fluoroindirubin-30-ox-
ime (37). Yield: 68%. Mp > 260 ꢁC. 1H NMR
(400 MHz, DMSO-d6) d ppm 13.89 (s, 1H, NOH),
11.81 (s, 1H, N0H), 10.74 (s, 1H, NH), 8.44 (dd, 1H,
H arom, J = 1.6, 10.8 Hz), 8.32 (s, 1H, H arom), 7.59
(dd, 1H, H arom, J = 1.6, 8.0 Hz), 7.42 (d, 1H, H arom,
J = 8.0 Hz), 6.95 (td, 1H, H arom, J = 2.4, 8.8 Hz), 6.84
(dd, 1H, H arom, J = 5.2, 8.8 Hz). 13C NMR (100 MHz,
DMSO-d6) d ppm 171.31, 159.09, 156.78, 150.84, 150.02,
145.80, 144.40, 135.29, 134.85, 130.25, 117.60, 114.24,
113.34, 112.66, 112.42, 110.36, 110.09, 109.57, 109.49.
4.1.4.16. (20Z,30E)-1-Methyl-50-bromoindirubin-30-ox-
ime (43). Yield: 86%. Mp > 260 ꢁC. 1H NMR
(400 MHz, DMSO-d6) d ppm 13.70 (s, NOH), 11.73
(s, NH), 8.65 (d, 1H, H arom, J = 8.4 Hz), 8.33 (s,
IR = 3165, 2792, 1732, 1466, 1290, 1016, 796 cmꢀ1
.
HRMS (EI) [M]+Å (C16H9N3O2): calcd372.98622, found
372.9867.
1H,
H
arom, H40), 7.67 (d, 1H,
H
arom,
J = 8.4 Hz), 7.38 (d, 1H, H arom, J = 8.4 Hz), 7.23
(t, 1H, H arom, J = 7.2 Hz, H4 or H5), 7.04 (d, 1H,
H arom, J = 7.2 Hz, H2 or H5), 7.00 (d, 1H, H arom,
J = 7.2 Hz, H2 or H5), 3.30 (s, 3H, CH3). IR = 2990,
2846. 1741, 1652, 1600, 1560, 1452, 1303, 1159,
1016 cmꢀ1. HRMS (EI) [M]+Å (C17H12N3O279Br): calcd
369.01129, found 369.0091.
4.1.4.11. (20Z,30E)-5-Bromoindirubin-30-oxime (38).
Yield: 72%. Mp > 260 ꢁC. 1H NMR (400 MHz,
DMSO-d6) d ppm 11.82 (s, 1H, NOH), 10.76 (s,
1H, N0H), 8.71 (s, 1H, H-4), 8.19 (d, 1H,
J = 8.0 Hz, H-6), 7.39 (t, 1H, J = 7.2 Hz, H-60), 7.25
(m, 2H, H-40, H-70), 7.05 (t, 1H, J = 7.2 Hz, H-50),
6.88 (d, 1H, J = 8.0 Hz, H-7). IR = 3118, 1726,
1666, 1570, 1467, 1321, 1227, 1152, 748 cmꢀ1. HRMS
(EI) [M]+Å (C16H10N3O279Br): calcd 354.99564, found
354.9940.
4.2. Preparation and assay of protein kinases
CDKs and GSK-3 were first assayed in the presence of
10 lM of each indirubin. For molecules showing inhib-

6442
A. Beauchard et al. / Bioorg. Med. Chem. 14 (2006) 6434–6443
itory activity at 10 lM, dose–response curves were per-
formed to calculate the IC50 value.
Control experiments were also carried out using appro-
priate dilutions of DMSO.
Kinase activities were assayed in buffer A or C (unless
otherwise stated), at 30 ꢁC, at a final ATP concentration
of 15 lM. Blank values were subtracted and activities
calculated as picomoles of phosphate incorporated for
a 10 min. incubation. The activities are usually ex-
pressed in % of the maximal activity, that is, in the ab-
sence of inhibitors. Controls were performed with
appropriate dilutions of dimethylsulfoxide.
Cell viability was determined by measuring the reduc-
tion of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeth-
oxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) as
previously described in detail.15
Acknowledgments
`
We thank the Ministere de la Recherche et de l’Enseign-
ement Superieur for a PhD grant (AB), the ‘Comite de
Charente-Maritime de la Ligue Nationale Contre le
´
´
GSK-3a/b was purified from porcine brain by affinity
chromatography on immobilized axin.12 It was assayed,
following a 1/100 dilution in 1 mg BSA/mL of 10 mm
DTT, with 5 ll of 40 lM GS-1 peptide as a substrate,
in buffer A, in the presence of 15 lM [c-33P]ATP
(3000 Ci/mmol; 1 mCi/mL) in a final volume of 30 ll.
After 30-min incubation at 30 ꢁC, 25 ll aliquots of
supernatant were spotted onto 2.5 · 3 cm pieces of
Whatman P81 phosphocellulose paper, and, 20 s later,
the filters were washed five times (for at least 5 min each
time) in a solution of 10 mL phosphoric acid/liter of
water. The wet filters were counted in the presence of
1 mL ACS (Amersham) scintillation fluid.
´
ˆ
Cancer’ and the ‘Canceropole Grand Ouest’ for finan-
cial support. The work presented here was also support-
ed by a grant from the EEC (FP6-2002-Life Sciences &
Health, PRO-KINASE Research Project) (L.M.) and
´ ˆ
the Canceropole Grand-Ouest (L.M.).
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4.3. Cell biology
Cell Titer 96ꢂ kit containing the MTS reagent was pur-
chased from Promega (Madison, WI, USA). The prote-
ase inhibitor cocktail was from Roche.
SH-SY5Y human neuroblastoma cell line was grown in
DMEM (Biowhittaker) supplemented with 2 mM L-glu-
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