3′-substituted 7-halogenoindirubins, a new class of cell death inducing agents

Article abstract of DOI:10.1021/jm060314i

Indirubins are kinase inhibitory bis-indoles that can be generated from various plant, mollusk, mammalian, and bacterial sources or chemically synthesized. We here report on the synthesis and biological evaluation of 3′-substituted 7-halogenoindirubins. M

Full text of DOI:10.1021/jm060314i

4638
J. Med. Chem. 2006, 49, 4638-4649
3′-Substituted 7-Halogenoindirubins, a New Class of Cell Death Inducing Agents
Yoan Ferandin,†,‡ Karima Bettayeb,†,‡ Marina Kritsanida,§,‡ Olivier Lozach,† Panagiotis Polychronopoulos,§ Prokopios Magiatis,§
Alexios-Leandros Skaltsounis,*,§ and Laurent Meijer*,†
Cell Cycle Group & UPS2682, C.N.R.S., Station Biologique, B.P. 74, 29682 Roscoff Cedex, Bretagne, France, and Department of Pharmacy,
DiVision of Pharmacognosy and Natural Products Chemistry, UniVersity of Athens, Panepistimiopolis Zografou, GR-15771 Athens, Greece
ReceiVed March 19, 2006
Indirubins are kinase inhibitory bis-indoles that can be generated from various plant, mollusk, mammalian,
and bacterial sources or chemically synthesized. We here report on the synthesis and biological evaluation
of 3′-substituted 7-halogenoindirubins. Molecular modeling and kinase assays suggest that steric hindrance
prevents 3′-substituted 7-halogenoindirubins from interacting with classical kinase targets of other indirubins
such as cyclin-dependent kinases and glycogen synthase kinase-3. Surprisingly 3′-substituted 7-halogeno-
indirubins induce cell death in a diversity of human tumor cell lines. Although some 3′-substituted
7-halogenoindirubins appear to induce effector caspase-independent, nonapoptotic cell death, others trigger
the landmarks of classical apoptosis. A structure-activity relationship study was performed to optimize
3′-substituted 7-halogenoindirubins with respect to solubility and cell death induction. Despite their
unidentified targets, 3′-substituted 7-halogenoindirubins constitute a new promising family of antitumor
agents.
Introduction
regulation of survival factors such as survivin and Mcl-1,
followed by cell death induction.25 While synthesizing and
testing the biological activity of new indirubins, we fortuitously
discovered that 7-bromoindirubin-3′-oxime (7BIO) displayed
potent cell death inducing properties, despite a lack of activity
toward CDKs and GSK-3.26 In contrast to indirubin-3′-oxime
(IO), 5-bromoindirubin-3′-oxime (5BIO), and 6-bromoindirubin-
3′-oxime (6BIO) (Figure 1), which at least in part induce
classical apoptosis, 7BIO induced caspase-independent, non-
apoptotic cell death.26
In this article we report on the synthesis and biological
properties of 3′-substituted 7-halogenoindirubins. Fifty-eight 3′-
substituted 7-halogenoindirubins were synthesized and tested
on various kinases as well as for their effects on neuroblastoma
SH-SY5Y cell survival. We also characterized the effects of
the most active compounds on a panel of 11 human tumor cell
lines and investigated the intracellular mechanisms of cell death
induction. Altogether, the results suggest that 3′-substituted
7-halogenoindirubins constitute a new family of potential
antitumor agents, acting through both caspase-dependent and
caspase-independent mechanisms. They also suggest that some
indirubins may interact with new, yet unidentified, target
proteins, distinct from their well characterized CDKs, GSK-3,
and AhR targets.
Indirubin (1a) is a dark-red isomer of the blue indigo. Both
are bis-indoles derived from the spontaneous, nonenzymatic
dimerization of isatin and indoxyl, two colorless precursors.
Indirubin can be extracted from four different natural sources:
indigo-producing plants, Tyrean purple-producing mollusks,
various recombinant bacterial strains, and urine from various
mammals including man.1 Indirubin has been reported as the
active ingredient of a traditional Chinese medicinal recipe,
danggui longhui wan, used to treat chronic myelocytic leuke-
mia.2,3 Interest in indirubin and derived analogues (collectively
referred to as indirubins) strongly increased when they were
discovered to inhibit cyclin-dependent kinases (CDKsa),4 gly-
cogen synthase kinase-3 (GSK-3),5 and glycogen phosphorylase
b6 and to bind and activate the aryl hydrocarbon receptor (AhR),
known also as the dioxin receptor.7-13 Indirubins have been
cocrystallized with CDK2,4 CDK2/cyclin A,14 CDK5/p25,15
PfPK5, the Plasmodium falciparum CDK1 homologue,16 GSK-
3â,17-19 and glycogen phosphorylase b.4
Indirubins display clear antiproliferative and cell death
inducing effects.20-24 Although there is evidence suggesting that
these effects originate from inhibition of CDKs, interaction with
AhR and subsequent induction of p27kip1 may also contribute
to the cellular effects of indirubins.12 Furthermore, some
indirubins have recently been shown to prevent the activity of
the transcription factor STAT3, probably by inhibition of its
src-dependent tyrosine phosphorylation.25 This leads to down-
Results
Despite considerable work on the bis-indole indirubins,
substitutions on position 7 have never been investigated. In a
previous report we have shown that, in contrast to other bromo-
substituted indirubins, 7BIO had little effect on CDK1/cyclin
B, CDK5/p25, and GSK-3R/â.26 One reason might be that 3′-
substituted 7-halogenoindirubins would be unable to bind to
CDKs and GSK-3 because of steric hindrance. To investigate
this hypothesis, we modeled 7BIO into GSK-3â, based on the
cocrystal structures of 6BIO with this kinase, as described by
Polychronopoulos et al.19 Results show a potential steric clash
between 7BIO’s bromine and the side chain of Leu 132, the
gatekeeper amino acid (Figure 2). This would most likely
prevent interaction of 3′-substituted 7-halogenoindirubins with
* To whom correspondence should be addressed. For A.-L.S.: phone,
+30 210 727 45 94; fax, 33 2 98 29 23 42; e-mail, skaltsounis@pharm.uoa.gr.
For L.M.: phone, +33 (0)2 98 29 23 39; fax, 33 2 98 29 23 42; e-mail,
meijer@sb-roscoff.fr.
† Station Biologique.
‡ These authors contributed equally.
§ University of Athens.
a Abbreviations: AhR, aryl hydrocarbon receptor; 5BIO, 5-bromo-
indirubin-3′-oxime; 6BIO, 6-bromoindirubin-3′-oxime; 7BIO, 7-bromo-
indirubin-3′-oxime; CDK, cyclin-dependent kinase; FCS, fetal calf serum;
GSK-3, glycogen synthase kinase-3; IO, indirubin-3′-oxime; LDH, lactate
dehydrogenase; Me7BIO, 1-methyl-7-bromoindirubin-3′-oxime; MTS, 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium; PBS, phosphate buffered saline.
10.1021/jm060314i CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/24/2006

3′-Substituted 7-Halogenoindirubins
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4639
The oximes 7b-e and 8b-e were prepared selectively in a
(2′Z,3′E) form following a typical procedure from the appropri-
ate indirubin derivatives 1b-e and 6b-e with hydroxylamine
hydrochloride in pyridine under reflux. A similar typical
procedure was followed for the preparation of the methoximes
9b-e and 10b-e using methoxylamine hydrochloride. The
acetoximes 11b-e and 12b-e were prepared from the oximes
with acetic anhydride in pyridine. The temperature of the
reaction was carefully kept at 0 °C to avoid bisacetylation.
In an effort to prepare derivatives with increased water
solubility, we synthesized 3′-substituted oximes of 7BIO (7d)
and Me7BIO (8d) bearing side chains with amine groups 13g-l
and 14g-l (Scheme 2). The synthesis was based on the reaction
of the 3′-[O-(2-bromoethyl)oxime] intermediate 13f or 14f with
the appropriate amine: pyrrolidine, morpholine, piperazine,
imidazole, dimethylamine, and diethylamine. The intermediates
13f and 14f were prepared by the reaction of 7BIO or Me7BIO
with 1,2-dibromoethane in DMF and Et3N at room temperature.
In addition, two carbamates 15 and 16 were prepared by the
reaction of 7BIO or Me7BIO with N,N-diethylcarbamyl chloride.
Figure 1. Structure of indirubin-3′-oxime (IO), 5-bromoindirubin-3′-
oxime (5BIO), 6-bromoindirubin-3′-oxime (6BIO), and 7-bromo-
indirubin-3′-oxime (7BIO).
Indirubin (1a), nonhalogenated indirubin derivatives (6a-
9a and 11a), 5BIO, and 6BIO were synthesized as previously
reported.19
3′-Substituted 7-Halogenoindirubins Display Little Kinase
Inhibitory Activity toward CDKs and GSK-3R/â. We first
synthesized a variety of 7-halogenoindirubins with or without
a methyl substitution at position N1 and with an oxime,
methoxime, or acetoxime substitution at position 3′ (compounds
1b-e, 6b-e, 7b-e, 8b-e, 9b-e, 10b-e, 11b-e, 12b-e). In
addition a few indirubins that are not substituted on position 7
were synthesized for comparison purposes (compounds 1a, 6a-
9a, 11a). Molecules were then tested on three kinases, CDK1/
cyclin B, CDK5/p25, and GSK-3R/â (Table 1). A complete lack
of activity was confirmed for all N1-methylated indirubins. A
weak and gradually decreasing inhibitory activity was observed
with 7-halogenoindirubin-3′-oxime when the size of the atom
at position 7 increased (H > F > Cl > Br > I) (compare
compounds 7a-e), suggesting increased hindrance at this
position 7.
Figure 2. Superimposition of 7BIO (turquoise) and 6BIO (red) docked
into the active site of GSK-3â. Only the backbone atoms of the residues
from Asp133 to Val135 interacting with the ligands are visible, while
the rest of the protein is presented as a ribbon. In the case of 7BIO a
steric clash between the bromine atom at position 7 and the Leu132
residue occurs (visualized by overlap of the VdW spheres) resulting in
a lower affinity of 7BIO for GSK-3â.
the ATP-binding site of these kinases. However, despite this
lack of interaction with classical indirubin targets, 7BIO displays
striking antiproliferative effects.26
In a second series of indirubins, the 3′ substituent was varied
on a 7-bromoindirubin scaffold (( a methyl at N1) (compounds
13f-l, 14f-l, 15, 16) (Table 2). With the exception of
compound 13h on GSK-3, none of these compounds displayed
any significant activity on any of the three kinases tested (Table
2).
Synthesis of 3′-Substituted 7-Halogenoindirubins. We thus
started to investigate this subfamily of 7-substituted indirubins
by designing a method to synthesize 7-halogenoindirubins with
various substitutions on position 3′ (Schemes 1 and 2; Tables 1
and 2). The synthesis of 7-halogenoindirubins was mainly based
on the dimerization reaction of an appropriately substituted isatin
derivative with 3-acetoxyindole, as depicted in Scheme 1. The
desired isatins were synthesized through a two-step procedure,
using the corresponding commercial 2-halogenoanilines 2b-e
as starting material. In the first step, the appropriate aniline
derivatives were reacted with chloral hydrate and hydroxylamine
hydrochloride to give the corresponding isonitrosoacetanilides
3b-e. In the second step, the intermediate isonitrosoacenilides
were heated in concentrated sulfuric acid to give the 7-halo-
genoisatins (4b-e). 7-Halogeno-N-methylisatins (5b-e) were
prepared from 4b-e, respectively, by treatment with dimethyl
sulfate and Na2CO3.
Several 3′-Substituted 7-Halogenoindirubins Induce Cell
Death in Culture. We next tested the effects of each indirubin,
at a 25 µM final concentration, on the survival of the
neuroblastoma SH-SY5Y cell line after 48 h of exposure (Tables
1 and 2). Cell survival was estimated by the MTS reduction
assay. Several compounds showed clear effects on the SH-SY5Y
cell survival rate. A complete dose response curve was
performed for these active compounds after 24 and 48 h of
exposure, and the IC50 values were calculated (Table 3). Because
MTS reduction is occasionally observed under conditions
different from the conditions of cell death, we used an
independent cell death evaluation procedure, the lactate dehy-
drogenase (LDH) release assay. This assay confirmed the
induction of cell death by our selection of indirubins, despite
their overall lack of effects on CDKs and GSK-3.
The substituted isatins, 7-halogenoisatins (4b-e) or 7-halo-
geno-N-methylisatins (5b-e), were reacted with 3-acetoxyindole
in alkaline medium to give, generally in good yields, the
corresponding bis-indoles 1b-e and 6b-e selectively in the Z
form.
We next tested the various 3′-substituted 7-bromoindirubins
on 11 cell lines, namely, HT-29 and HCT116 (colon cancer),
MDA-MB-231 (breast cancer), A549 (lung cancer), PC3
(prostate cancer), 5L and BP8 (hepatoma), F1 and Huh7

4640 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Ferandin et al.
Scheme 1. General Synthesis of 7-Substituted Indirubins and Their Corresponding 3′-Oxime, Methoxime, and Acetoxime Derivativesa
a Reagents (i) chloral hydrate, Na2SO4, H2NOH‚HCl, H2O, HCl; (ii) H2SO4, 80 °C; (iii) (CH3)2SO4, Na2CO3, DMF; (iv) 3-acetoxyindole, Na2CO3/MeOH,
25 °C; (v) H2NOCH3‚HCl, Py, 120 °C; (vi) H2NOH‚HCl, Py, 120 °C; (vii) Ac2O, Py, 0 °C.
(hepatoma), SH-SY5Y (neuroblastoma), and HEK293 (embry-
onic kidney). Like SH-SY5Y, these cell lines showed dose-
dependent induction of cell death (Table 4), suggesting the
generality of effect of these compounds on cell survival rather
than a cell type or differentiation stage-specific effect. The
similar sensitivity of 5L (AhR +/+) and BP8 (AhR -/-)
suggests that AhR does not play a major role in 3′-substituted
7-bromoindirubins induced cell death.
and caspase-independent mechanism of action (compound 14gs;
Figure 3, upper central panel). In the third category, the presence
of Q-VD-OPh essentially protects cells from cell death, sug-
gesting that these indirubins act mostly through a classical,
caspase-dependent mechanism (compound 13k; Figure 3, upper
right panel). Interestingly this is observed with the most active
indirubins. In this last category, a small fraction of cells (20%)
die despite the presence of Q-VD-OPh.
3′-Substituted 7-Halogenoindirubins Induce Apoptotic and
Nonapoptotic Cell Death. In a previous work we showed that
cell death induced by 7BIO is primarily different from apoptosis
because it does not induce or require caspase activation.26 We
thus investigated whether this was also the case with the new
indirubins presented here. To this aim, we tested the effects of
the effector caspase (3, 8, 9, 10, 12) inhibitor Q-VD-OPh27 (20
µM final concentration) on cell death induced by the selection
of indirubins. Results show that 3′-substituted 7-bromoindirubins
fall into three categories (Table 5, Figure 3). In the first category,
some indirubins are completely insensitive to the presence of
the caspase inhibitor, suggesting a caspase-independent mech-
anism. 7BIO (7d) falls into this category (Figure 3, upper left
panel). In the second category, Q-VD-OPh shifts the dose
response curves to the right and thus shifts the IC50 values
toward higher values, suggesting a mixed, caspase-dependent
The activity of caspases was next assayed over a time-course
in SH-SY5Y cells exposed to the three types of 3′-substituted
7-bromoindirubins (Figure 3, lower panel). As previously
reported26 7BIO did not trigger any caspase activation. In
contrast, compound 13k induced strong activation of caspases,
while compound 14gs induced an intermediate activation. The
level of caspase activation induced by the indirubins thus
correlated very well with the sensitivity of their cellular effects
to Q-VD-OPh.
Discussion
We here report on the synthesis and biological evaluation of
a new subfamily of indirubins substituted on positions 3′ and
7. Despite a lack of inhibitory activity toward classical targets
of indirubins such as CDKs and GSK-3, 3′-substituted 7-halo-
genoindirubins display potent cell death inducing activity. This

3′-Substituted 7-Halogenoindirubins
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4641
Table 1. Effects of Indirubin Derivatives 1, 6, and 7-12 on Three
Scheme 2. General Synthesis Scheme of 3’-Substituted
Protein Kinases and on the Survival of Neuroblastoma SH-SY5Y Cellsa
7-Bromoindirubinsa
IC50 (µM)
SH-SY5Y
GSK-3 % survival
indirubin
X (3′)
Y (7) Z (1) CDK1 CDK5
1a
7a
9a
11a
6a
8a
1b
7b
9b
11b
6b
8b
10b
12b
1c
7c
9c
11c
6c
8c
10c
12c
1d
7d
9d
11d
6d
8d
10d
12d
1e
7e
9e
11e
6e
8e
O
NOH
NOCH3
NOCOCH3
O
NOH
O
NOH
H
H
H
H
H
H
F
F
F
F
F
F
F
F
Cl
Cl
Cl
H
H
H
H
CH3
CH3
H
H
H
10
0.18
1.4
1.2
100
73
10
1.5
>10
15
10
0.10
0.4
0.7
1.0
0.022
0.3
104 ( 4
27 ( 2
86 ( 4
36 ( 2
98 ( 3
100 ( 5
70 ( 5
46 ( 2
105 ( 4
39 ( 4
101 ( 7
99 ( 1
107 ( 3
105 ( 4
92 ( 1
7 ( 0
97 ( 2
89 ( 3
94 ( 5
94 ( 2
99 ( 2
96 ( 1
92 ( 1
4 ( 0
97 ( 3
61 ( 8
98 ( 3
84 ( 2
100 ( 2
95 ( 1
96 ( 2
64 ( 3
84 ( 3
67 ( 2
93 ( 2
74 ( 0
99 ( 2
82 ( 2
0.2
>100
>100
>100
0.40
>100
g10
0.51
0.27
0.44
0.33
NOCH3
NOCOCH3
O
>100
>100
>100
>100
>100
>100
>100
6
>100
>100
>100
>100
>100
>100
>100
33
>100
>100
>100
>100
>100
>100
>100
77
>100
>100
>100
>100
>100
>100
H
CH3 >10
CH3 >100
CH3 >10
CH3 >10
H
H
H
H
>100
NOH
>100
>100
>100
>100
21
NOCH3
NOCOCH3
O
NOH
NOCH3
NOCOCH3 Cl
O
NOH
NOCH3
NOCOCH3 Cl CH3 >10
O
NOH
NOCH3
NOCOCH3 Br
>10
3.7
>10
>10
>100
>100
>100
>100
>100
>100
>100
32
>100
>100
>100
>100
>100
>100
>100
16
a Reagents: (i) dibromoethane, triethylamine, anhydrous DMF, 25 °C;
(ii) anhydrous DMF, 25 °C, amines g-l; (iii) N,N-diethylcarbamyl chloride,
triethylamine, anhydrous DMF, 25 °C.
Cl CH3 >10
Cl CH3 >100
Cl CH3 >10
Br
Br
Br
H
H
H
H
>100
22
>100
>100
100
observation poses a series of questions on the nature of the
molecular targets of these indirubins, their cellular mechanism
of action, and the possibility of further optimization and their
potential development as antitumor agents.
O
Br CH3
Br CH3 >100
Br CH3 >100
NOH
NOCH3
NOCOCH3 Br CH3
O
NOH
NOCH3
NOCOCH3
O
NOH
NOCH3
NOCOCH3
Three pieces of evidence suggest that 3′-substituted 7-halo-
genoindirubins target very specific proteins. First, substitutions
on position 7 inactivate indirubins as CDK and GSK-3 inhibitors
and, in the case of 7BIO, as an inhibitor of quite a few kinases.26
A large substituent like bromine at position 7 indeed creates a
molecular hindrance preventing the interaction with CDKs and
GSK3 (Figure 2). Second, a substitution on position 1 with a
methyl inactivates indirubins as CDK and GSK-3 inhibitors.18
This is likely the consequence of the prevention of a hydrogen
bond between the cyclic nitrogen of the lactam ring of indirubins
and the carbonyl oxygen of Asp133 (in GSK-3), Glu81 (in
CDK5),15,18,19 or Glu81 (in CDK2).4 This hydrogen bond
constitutes a key feature in the interaction between indirubins
and their kinase targets. Nevertheless some N1-methyl-, 3′-
substituted 7-halogenoindirubins (14g, 14gs, 14ks, 14ls) display
very clear cell death inducing activity, suggesting that their
interaction with an unidentified target does not involve this key
hydrogen bond (Table 2). Third, varying the substituent on
position 3′ leads to a different range of biological activity.
However at this point we are unable to correlate the cell death
inducing activity with a specific substitution on 3′. The hydroxyl
of indirubin-3′-oxime may provide an indirect link (through a
water molecule) to CDKs and GSK-3.15,18,19 In CDK/GSK-3
indirubins cocrystal structures, the 3′ position appears to face
the outside of the ATP-binding pocket and therefore is less prone
to steric hindrance.4,15,18,19 Altogether, this information suggests
that the binding mode of 3′-substituted 7-halogenoindirubins
to its (their) unknown target(s) is rather different from that of
previously described indirubins to CDK2, CDK5, or GSK-3.
Another possibility is that 3′-substituted 7-halogenoindirubins
specifically bind to non-protein kinase targets and that the
biological effects of this indirubin subclass are due to this
interaction. An example is AhR. 7-13 However, SH-SY5Y cells
appear to be devoid of AhR (data not shown) and quite sensitive
70
>10
66
>10
>10
I
I
I
I
I
I
I
I
H
H
H
H
>100
>100
>100
30
>100
>100
CH3 >10
CH3 >100
CH3 >10
CH3 >10
10e
12e
a A series of indirubin analogues were tested at various concentrations
in three kinase assays, as described in the Experimental section. IC50 values
were calculated from the dose response curves. The compounds were also
tested at 25 µM for their effects on SH-SY5Y cells. Cell survival was
estimated by the MTS reduction assay and is expressed as percent of survival
in untreated cells (average ( SE of three independent measurements;
representative of two independent experiments): boldface numbers, e15%
survival; italic numbers, e50% survival.
to 3′-substituted 7-halogenoindirubins. Furthermore, we were
unable to see any significant difference in the biological
responses of the hepatoma 5L (AhR +/+) and BP8 (AhR -/-)
cell lines to these indirubins (Table 4).26 Therefore, interaction
with AhR is unlikely to be the major cause of cell death induced
by 3′-substituted 7-halogenoindirubins, although we cannot
exclude a secondary role. Identification of the molecular target-
(s) of 3′-substituted 7-halogenoindirubins is one of our main
priorities. First, we will explore the possible substitutions that
can be added to these structures on additional positions
(especially on 5′ and 5). Second, from this information, we will
attempt to design an affinity reagent that should allow us to
purify and identify some of the interacting proteins as success-
fully performed with other kinase inhibitors.28,29
3′-Substituted 7-halogenoindirubins induce cell death via at
least three different mechanisms: some induce cell death in a
caspase-independent way, some in a clearly caspase-dependent

4642 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Ferandin et al.
Table 2. Effects of of Indirubin Derivatives 13-16 on Three Protein
Table 3. Effects of Indirubin Derivatives 7, 13, and 14 on the Survival
Kinases and on the Survival of Neuroblastoma SH-SY5Y Cellsa
and Death of Neuroblastoma SH-SY5Y Cellsa
cell survival IC50 (µM),
% cell death,
LDH release,
MTS reduction
indirubin
7a
24 h
48 h
48 h
>25
14
12
12
33
92
94
14
80
78
94
94
100
86
84
33
80
58
59
80
7c
7d (7BIO)
13g
13gs
14g
14gs
13j
13js
13k
13ks
14k
14ks
14ls
5BIO
6BIO
8.0
7.1
2.3
2.0
5.0
4.0
6.2
6.0
2.0
1.0
5.5
9.0
18
3.6
3.1
7.8
7.3
10.5
6.0
4.1
2.1
>25
21
23
18
18
12
9.5
a A series of indirubin analogues were tested at various concentrations
for their effects on SH-SY5Y cells. Cell survival was estimated 24 or 48 h
after the addition of each indirubin using the MTS reduction assay.
Experiments were also performed with 5BIO and 6BIO for comparison.
IC50 values were calculated from the dose response curves (average ( SE
of two independent measurements performed in triplicate): italic numbers,
IC50 < 10 µM. In addition, cell death was estimated 48 h after the addition
of each indirubin (25 µM) using the LDH release assay. Results are
expressed as percent cell death: boldface numbers, >85% cell death; italic
numbers, >50% cell death.
IC50 (µM)
SH- SY5Y
% survival
indirubin X (3′) Z (1)
CDK1
22
>100
CDK5
GSK3
7d
NOH
H
33
25
32
100
>10
7
4 ( 0
87 ( 5
99 ( 4
13 ( 0
12 ( 0
15 ( 1
1 ( 0
13f
f
H
14f
f
g
gs
g
gs
h
hs
h
hs
i
CH3
H
H
CH3
CH3
H
>10
>100
>100
>10
>10
>100
>100
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>100
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>100
>10
13g
13gs
14g
14gs
13h
13hs
14h
14hs
13i
3
expression and protein synthesis. If this is the case, analysis of
the pattern of gene expression should provide clues on the
mechanisms involved in cell death induction and how they
impinge on classical apoptosis or on nonapoptotic cell death
pathways. This work is currently underway.
We shall continue to explore the structure-activity relation-
ship of these 3′-substituted 7-halogenoindirubins in order to
improve their efficacy. This work may lead to the development
of potential antitumor agents acting through a caspase-dependent
or a caspase-independent pathway. Besides these cell efficacy
improvements, we have identified compounds with much better
water solubility (such as the 13gs-ls, 14gs-ls in Table 2). This
is an important progress because insolubility in biologically
compatible media has been a major issue in the development
of indirubins.22
>10
>10
0.57
>100
>10
>10
9
98 ( 1
76 ( 1
98 ( 2
90 ( 2
59 ( 1
62 ( 1
85 ( 1
70 ( 1
3 ( 1
H
CH3
CH3
H
13is
14i
14is
13j
is
i
is
j
H
11
CH3
CH3
H
>10
>10
5
13js
13k
13ks
14k
14ks
13l
js
k
ks
k
ks
l
H
H
H
CH3
CH3
H
>10
3 ( 1
8
1 ( 0
>10
>10
>10
>10
>10
>10
>10
>100
>10
0.016
0.005
5 ( 1
49 ( 1
4 ( 0
49 ( 3
72 ( 3
83 ( 2
15 ( 1
106 ( 5
105 ( 3
13 ( 0.4
5 ( 0.6
13ls
14l
14ls
15
16
5BIO
6BIO
ls
l
H
CH3
CH3
H
CH3
H
Experimental Section
ls
m
m
NOH
NOH
Chemistry. General Chemistry Experimental Procedures. All
chemicals were purchased from Aldrich Chemical Co. Melting
points were determined with a Sanyo Gallenkamp apparatus.
Infrared spectra (IR) were recorded on a Perkin-Elmer Paragon 500
FT-IR spectrometer using multiple internal reflectance (MIR) on a
KRS-5 crystal at 45°. NMR spectra were recorded on a Bruker
DRX 400. Chemical shifts are expressed in ppm downfield from
TMS. CI-MS spectra were determined on a Finnigan GCQ Plus
ion-trap mass spectrometer using CH4 as the chemical ionization
reagent. Column chomatographies were conducted using flash silica
gel 60 (40-63 µm) from Merck, with an overpressure of 300 mbars.
All the compounds gave satisfactory combustion analysis results
(C, H, N within (0.4% of calculated values).
0.045
0.320
0.028
0.083
H
a A series of indirubin analogues were tested at various concentrations
in three kinase assays, as described in the Experimental Section. IC50 values
were calculated from the dose response curves. Experiments were also
performed with 5BIO and 6BIO for comparison. Kinase data for 6BIO is
from Meijer.18 The compounds were also tested at 25 µM for their effects
on SH-SY5Y cells. Cell survival was estimated by the MTS reduction assay
and is expressed as percent of survival in untreated cells (average ( SE of
three independent measurements; representative of two independent experi-
ments): boldface numbers, e15% survival; italic numbers, e50% survival.
General Procedure for the Preparation of Isatins 4b-e and
5b-e. Chloral hydrate (5.0 g) and Na2SO4 (35.0 g) were dissolved
in water (70 mL) in a 300 mL beaker and warmed to 35 °C. A
warm solution of the appropriate commercial aniline derivative
2b-e (27.6 mmol) in water (20 mL) and an aqueous solution of
concentrated HCl (3 mL) was added (a white precipitate of the
amine sulfate was formed), followed by a warm solution of
hydroxylamine hydrochloride (6.1 g) in water (27.5 mL). The
mixture was stirred by hand and heated on a hot plate (a thick paste
manner, and some in a partially caspase-dependent manner.
There are many possible reasons for this diversity of response:
different targets, different intracellular distribution, different
kinetics of two independent mechanisms, metabolism into
different types of cell death inducers. At this point it will be
most interesting to investigate whether cell death induced by
the different 3′-substituted 7-halogenoindirubins requires gene

3′-Substituted 7-Halogenoindirubins
Table 4. Effects of Indirubin Derivatives 7, 13, and 14 on the Survival of Various Cell Linesa
cell survival IC50 (µM), MTS reduction assay
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4643
cell line
7d
13gs
14g
14gs
13j
13js
13k
13ks
14ks
HT-29 (colon)
HCT116 (colon)
MDA-MB-231 (breast)
A549 (lung)
PC3 (prostate)
5L (hepatoma)
BP8 (hepatoma)
F1 (hepatoma)
21.2
6.2
10.4
3.8
>30.0
16.0
19.8
13.6
26.8
21.0
21.6
18.8
18.8
5.6
5.4
5.1
6.3
5.4
5.3
5.9
6.1
5.2
5.6
1.7
5.3
5.4
5.0
1.9
5.1
5.1
6.0
6.0
5.4
5.4
1.4
2.0
5.1
1.9
21.8
6.5
2.4
2.0
18.8
5.8
15.0
>30.0
>30.0
1.7
11.2
0.8
20.0
15.7
17.8
20.8
25.0
24.0
19.0
5.4
10.5
21.8
19.8
20.0
19.4
8.0
9.0
7.4
12.3
12.0
14.6
19.0
13.0
7.5
2.9
5.6
5.0
8.0
26.4
21.8
27.2
25.0
19.5
5.5
19.6
5.4
14.4
30.0
>30.0
>30.0
3.1
13.6
2.1
Huh7 (hepatoma)
SH-SY5Y (neuroblastoma)
HEK293 (embryo kidney)
18.8
10.6
21.4
18.0
8.0
18.8
>30.0
3.0
a A series of indirubin analogues were tested at various concentrations for their effects on 11 different cell lines. Cell survival was estimated 48 h after
the addition of each indirubin using the MTS reduction assay. IC50 values were calculated from the dose response curves (average ( SE of measurements
performed in triplicate): italic numbers, IC50 < 10 µM; boldface numbers, IC50 < 1 µM.
Table 5. Cell Death Induced by Indirubin Derivatives 7, 13, and 14 Is
7-Chloro-N-methylisatin (5c). This compound was prepared
from 7-chloroisatin (4c) by a procedure analogous to that of 5b:
yield 76%.
Either Caspase-Independent or Caspase-Dependenta
IC50 (µM)
7-Bromo-N-methylisatin (5d). This compound was prepared
from 7-bromoisatin (4d) by a procedure analogous to that of 5b:
yield 90%.
7-Iodo-N-methylisatin (5e). This compound was prepared from
7-iodoisatin (4e) by a procedure analogous to that of 5b: yield
85%.
indirubin
7a
-Q-VD-OPh (MTS)
+Q-VD-OPh (MTS)
13.0
10.0
7.0
2.0
2.8
6.1
5.0
10.0
6.2
1.5
1.1
6.4
11.0
18.0
13.0
10.0
>25.0
11.0
7c
7d (7BIO)
13g
13gs
14g
14gs
13j
13js
13k
13ks
14k
14ks
14ls
5BIO
6BIO
7.0
- (>25.0)
- (>25.0)
>25.0
13.0
(2′Z)-7-Fluoroindirubin (1b). Methanol (25 mL) was vigorously
stirred under nitrogen for 20 min, and then 7-fluoroisatin (4b) (150
mg, 0.91 mmol) and 3-acetoxyindole (106 mg, 0.61 mmol) were
added and stirring was continued for 5 min. Anhydrous Na2CO3
(155 mg) was added, and the stirring was continued for 3 h. The
dark precipitate was filtered and washed with aqueous methanol
(1:1, 20 mL) to give 7 (130 mg, 0.46 mmol, 77%) selectively in
10.3
6.8
- (>25.0)
- (>25.0)
- (>25.0)
>25.0
>25.0
23.0
1
the Z form. Mp >300 °C; H NMR (DMSO, 400 MHz, δ ppm)
11.37 (1H, s, N′-H), 11.12 (1H, s, N-H), 8.58 (1H, d, J ) 7.7
Hz, H-4), 7.64 (1H, d, J ) 7.5 Hz, H-4′), 7.57 (1H, t, J ) 7.5 Hz
H-6′), 7.42 (1H, d, J ) 7.5 Hz, H-7′), 7.15 (1H, t, J ) 8.0 Hz,
H-6), 7.02 (2H, m, H-5′, 5). CI-MS m/z 281 (M + H)+. Anal.
(C16H9N2O2F) C, H, N.
13.0
a SH-SY5Y cells were treated with various concentrations of indirubin
analogues in the presence or absence of 20 µM Q-VD-OPh, a broad
spectrum inhibitor of caspases. Cell survival was estimated 48 h after the
addition of each indirubin using the MTS reduction assay. IC50 values were
calculated from the dose response curves (average ( SE of two independent
measurements): -, no cell death at highest concentration tested. Results
are not emphasized when Q-VD-Oph has no effect on the dose response
curve. Italic numbers: when Q-VD-Oph partially protects from cell death.
Boldface numbers: when Q-VD-Oph provides complete protection.
(2′Z)-7-Chloroindirubin (1c). This compound was prepared
from 7-chloroisatin (4c) by a procedure analogous to that of 7: yield
80%. Mp >300 °C.1H NMR (DMSO, 400 MHz, δ ppm) 11.29
(1H, s, N′-H), 11.16 (1H, s, N-H), 8.72 (1H, d, J ) 7.8 Hz, H-4),
7.66 (1H, d, J ) 7.5 Hz, H-4′), 7.59 (1H, t, J ) 7.8 Hz H-6′), 7.43
(1H, d, J ) 7.8 Hz, H-7′), 7.30 (1H, d, J ) 7.8 Hz, H-6), 7.05
(2H, m, H-5,5′). CI-MS m/z 297, 299 (M + H)+. Anal. (C16H9N2O2-
Cl) C, H, N.
formed at 75-70 °C) at 80-90 °C for 2 h, then allowed to cool
for 1 h, by which time the temperature had fallen to 50 °C, and
filtered. The pale-cream product was washed by stirring with water
(100 mL) and filtered. Drying overnight at 40 °C gave the
corresponding isonitrosoacetanilide 3b-e.
Sulfuric acid (100 mL) was heated in a 3 L beaker on a hot
plate to 60 °C and then removed. The dry isonitrosoacetanilide
3b-e was added in portions with stirring over 30 min so that the
temperature did not exceed 65 °C. The mixture was then heated to
80 °C for 15 min, allowed to cool to 70 °C, and cooled on ice. The
solution was poured onto crushed ice (500 mL) and left to stand
for 1 h before the orange-red precipitate was filtered. The product
was washed by stirring with water (100 mL) and filtered to give
the corresponding isatins. Yields: 4b, 57%; 4c, 50%; 4d, 65%;
4e, 50%.
7-Fluoro-N-methylisatin (5b). To a solution of 4b (380 mg,
2.30 mmol) in dry acetone (60 mL) was added Na2CO3 (anhydrous)
(3.5 g) and dimethyl sulfate (0.4 mL) under Ar, and the reaction
mixture was heated at 60 °C for 20 h. Then the mixture was filtered,
and the filtrate was carefully evaporated using a high vacuum pump
(under 40 °C). The solid residue was submitted to flash chroma-
tography with CH2Cl2 to afford 5b (288 mg, 1.61 mmol, 70%).
(2′Z)-7-Bromoindirubin (1d). This compound was prepared
from 7-bromoisatin (4d) by a procedure analogous to that of 7:
yield 85%. Mp >300 °C. IR 3350 (br), 1674, 1612, 1593, 1464,
1306, 1216 cm-1. 1H NMR (DMSO, 400 MHz, δ ppm) 11.18 (2H,
br s, N-H), 8.77 (1H, d, J ) 7.9 Hz, H-4), 7.67 (1H, d, J ) 7.5
Hz, H-4′), 7.59 (1H, t, J ) 7.5 Hz, H-6′), 7.44 (1H, d, J ) 7.9 Hz,
H-6), 7.43 (1H, d, J ) 7.5 Hz, H-7′), 7.04 (1H, t, J ) 7.5 Hz,
H-5′), 6.98 (1H, t, J ) 7.9 Hz, H-5). CI-MS m/z 341, 343 (M +
H)+. Anal. (C16H9N2O2Br) C, H, N.
(2′Z)-7-Iodoindirubin (1e). This compound was prepared from
7-iodoisatin (4e) by a procedure analogous to that of 7: yield 90%.
Mp >300 °C. 1H NMR (DMSO, 400 MHz, δ ppm) 8.77 (1H, d, J
) 7.5 Hz, H-4), 7.64 (1H, d, J ) 7.5 Hz, H-4′), 7.59 (2H, m,
H-6,6′), 7.41 (1H, d, J ) 7.5 Hz, H-7′), 7.04 (1H, t, J ) 7.5 Hz,
H-5′), 6.84 (1H, t, J ) 7.5 Hz, H-5). CI-MS m/z 389 (M + H)+.
Anal. (C16H9N2O2I) C, H, N.
(2′Z)-7-Fluoro-1-methylindirubin (6b). This compound was
prepared from 7-fluoro-N-methylisatin (5b) and 3-acetoxyindole
by a procedure analogous to that of 7: yield 78%. Mp 280 °C. 1H
NMR (DMSO, 400 MHz, δ ppm) 11.22 (1H, s, N′-H), 8.66 (1H,
d, J ) 8.0 Hz, H-4), 7.67 (1H, d, J ) 7.7 Hz, H-4′), 7.60 (1H, t,
J ) 7.7 Hz, H-6′), 7.44 (1H, d, J ) 7.7 Hz, H-7′), 7.22 (1H, t, J

4644 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Ferandin et al.
Figure 3. 7-Bromoindirubins induce caspase-independent or caspase-dependent cell death. (Upper panel) SH-SY5Ycells were exposed for 48 h to
increasing concentrations of three 7-bromoindirubins (7d, 14gs, 13k) in the presence (filled symbols) or absence (open symbols) of 20 µM Q-VD-
OPh. Cell survival was assessed by the MTS assay and is expressed as a percentage of untreated cells. Every point is the mean ( SE of two
independent experiments with two independent measurements per experiment. (Lower panel) The time-course of effector caspase activity was
determined in SH-SY5Y cells treated with 25 µM of three 7-bromoindirubins (7d, 14gs, 13k) for 24 h. DEVDase activity was measured as arbitrary
fluorescence units. Every point is the mean ( SE of three independent determinations.
) 8.0 Hz, H-6), 7.07 (2H, m, H-5′, 5), 3.46 (3H, s, N-CH3). CI-
MS m/z 295 (M + H)+. Anal. (C17H11FN2O2) C, H, N.
Data for (2′Z,3′E)-7-Fluoroindirubin-3′-oxime (7b). Mp 253
°C. H NMR (DMSO, 400 MHz, δ ppm) 13.61 (1H, brs, NOH),
11.85 (1H, s, N′-H), 11.19 (1H, s, N-H), 8.44 (1H, d, J ) 7.8
Hz, H-4), 8.19 (1H, d, J ) 7.5, H-4′), 7.39 (2H, m, H-6′, 7′), 7.00
(2H, m, H-5′, 6), 6.90 (1H, td, J ) 7.8 Hz, 5.0, H-5). CI-MS m/z
296 (M + H)+. Anal. (C16H10N3O2F) C, H, N.
1
(2′Z)-7-Chloro-1-methylindirubin (6c). This compound was
prepared from 7-chloro-N-methylisatin (5c) and 3-acetoxyindole
by a procedure analogous to that of 7: yield 95%. Mp 269 °C. 1H
NMR (DMSO, 400 MHz, δ ppm) 10.50 (1H, s, N′-H), 8.85 (1H,
d, J ) 7.5 Hz, H-4), 7.67 (1H, d, J ) 7.4 Hz, H-4′), 7.60 (1H, t,
J ) 7.4 Hz, H-6′), 7.44 (1H, d, J ) 7.4 Hz, H-7′), 7.32 (1H, d, J
) 7.4 Hz, H-6), 7.08 (2H, m, H-5, 5′), 3.62 (3H, s, N-CH3). CI-
MS m/z 311, 313 (M + H)+. Anal. (C17H11ClN2O2) C, H, N.
(2′Z)-7-Bromo-1-methylindirubin (6d). This compound was
prepared from 7-bromo-N-methylisatin (5d) and 3-acetoxyindole
by a procedure analogous to that of 7: yield 83%. Mp 277 °C. IR
Data for (2′Z,3′E)-7-Chloroindirubin-3′-oxime (7c). Mp 263
1
°C. H NMR (DMSO, 400 MHz, δ ppm) 13.70 (1H, brs, NOH),
11.86 (1H, s, N′-H), 11.09 (1H, s, N-H), 8.62 (1H, d, J ) 7.9
Hz, H-4), 8.23 (1H, d, J ) 7.6 Hz, H-4′), 7.44 (2H, m, H-6′, 7′),
7.17 (1H, d, J ) 7.9 Hz, H-6), 7.06 (1H, t, J ) 7.6 Hz, H-5′), 6.96
(1H, t, J ) 7.8 Hz, H-5). CI-MS m/z 312, 314 (M + H)+. Anal.
(C16H10N3O2Cl) C, H, N.
1
3312 (br), 1656, 1619, 1596, 1477, 1449, 1330, 1110 cm-1. H
Data for (2′Z,3′E)-7-Bromoindirubin-3′-oxime (7d). Mp 276
°C. IR 3096 (br), 1674, 1613, 1563, 1463, 1440, 1330, 1314, 1230,
NMR (DMSO, 400 MHz, δ ppm) 8.88 (1H, d, J ) 7.9 Hz, H-4),
7.66 (1H, d, J ) 7.5 Hz, H-4′), 7.60 (1H, t, J ) 7.5 Hz, H-6′),
7.47 (1H, d, J ) 7.5 Hz, H-7′), 7.38 (1H, d, J ) 7.9 Hz, H-6), 7.04
(2H, m, H-5, 5′), 3.61 (3H, s, N-CH3). CI-MS m/z 355, 357 (M +
H)+. Anal. (C17H11BrN2O2) C, H, N.
(2′Z)-7-Iodo-1-methylindirubin (6e). This compound was pre-
pared from 7-iodo-N-methylisatin (5e) and 3-acetoxyindole by a
procedure analogous to that of 7: yield 87%. Mp 299 °C. 1H NMR
(DMSO, 400 MHz, δ ppm) 11.26 (1H, s, N′-H), 8.93 (1H, d, J )
7.7 Hz, H-4), 7.74 (1H, d, J ) 7.4 Hz, H-4′), 7.66 (1H, d, J ) 7.5
Hz, H-6), 7.60 (1H, t, J ) 7.4 Hz, H-6′), 7.44 (1H, d, J ) 7.4 Hz,
H-7′), 7.05 (1H, t, J ) 7.4 Hz, H-5′), 6.86 (1H, t, J ) 7.7 Hz,
H-5), 3.65 (3H, s, N-CH3). CI-MS m/z 403 (M + H)+. Anal.
(C17H11N2O2I) C, H, N.
1
1186 cm-1. H NMR (DMSO, 400 MHz, δ ppm) 13.68 (1H, brs,
NOH) 11.90 (1H, s, N′-H), 10.91 (1H, s, N-H), 8.67 (1H, d, J )
7.8 Hz, H-4), 8.23 (1H, d, J ) 7.8 Hz, H-4′), 7.42 (2H, m, H-6′,
7′), 7.29 (1H, d, J ) 7.8 Hz, H-6), 7.06 (1H, t, J ) 7.8 Hz, H-5′),
6.90 (1H, t, J ) 7.8 Hz, H-5). CI-MS m/z 356, 358 (M + H)+.
Anal. (C16H10N3O2Br) C, H, N.
Data for (2′Z,3′E)-7-Iodoindirubin-3′-oxime (7e). Mp 284 °C.
1H NMR (DMSO, 400 MHz, δ ppm) 13.65 (1H, brs, NOH), 11.87
(1H, s, N′-H), 10.63 (1H, s, N-H), 8.68 (1H, d, J ) 7.8 Hz, H-4),
8.23 (1H, d, J ) 7.2 Hz, H-4′), 7.47 (1H, d, J ) 7.8 Hz, H-6), 7.43
(2H, m, H-6′, 7′), 7.06 (1H, t, J ) 7.2 Hz, H-5′), 6.76 (1H, t, J )
7.8 Hz, H- 5). CI-MS m/z 404 (M + H)+. Anal. (C16H10N3O2I)
C, H, N.
Data for (2′Z,3′E)-7-Fluoro-1-methylindirubin-3′-oxime (8b).
Mp 264 °C. H NMR (DMSO, 400 MHz, δ ppm) 13.72 (1H, brs,
General Procedure for the Preparation of the Oximes 7b-e
and 8b-e. The appropriate indirubin derivative 1b-e and 6b-e
(1 mmol) was dissolved in pyridine (10 mL). With magnetic stirring,
hydroxylamine hydrochloride (10 equiv) was added and the mixture
was heated under reflux (120 °C) for 1.5 h. Then the solvent was
evaporated under reduced pressure and the residue was washed with
water to afford quantitatively the corresponding 3′-oxime selectively
in the (2′Z,3′E) form.
1
NOH), 11.90 (1H, s, N′-H), 8.56 (1H, d, J ) 7.7 Hz, H-4), 8.23
(1H, d, J ) 7.6 Hz, H-4′), 7.44 (2H, m, H-6′, 7′), 7.07 (1H, m,
H-5′, 6), 6.97 (1H, td, J ) 7.8, 5.0 Hz, H-5), 3.60 (3H, s, N-CH3).
CI-MS m/z 310 (M + H)+. Anal. (C17H12N3O2F) C, H, N.
Data for (2′Z,3′E)-7-Chloro-1-methylindirubin-3′-oxime (8c).
1
Mp 276 °C. H NMR (DMSO, 400 MHz, δ ppm) 13.79 (1H, brs,

3′-Substituted 7-Halogenoindirubins
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4645
NOH), 11.97 (1H, s, N′-H), 8.76 (1H, d, J ) 7.8 Hz, H-4), 8.23
(1H, d, J ) 7.3 Hz, H-4′), 7.45 (2H, m, H-6′, 7′), 7.18 (1H, d, J )
7.8 Hz, H-6), 7.07 (1H, t, J ) 7.3 Hz, H-5′), 6.99 (1H, t, J ) 7.8
Hz, H-5), 3.67 (3H, s, N-CH3). CI-MS m/z 326, 328 (M + H)+.
Anal. (C17H12N3O2Cl) C, H, N.
H-4′), 7.47 (2H, m, H-6′, 7′), 7.38 (1H, d, J ) 7.9 Hz, H-6), 7.07
(1H, m, H-5′), 7.00 (1H, t, J ) 7.9 Hz, H-5), 4.40 (3H, s, OCH3),
3.68 (3H, s, N-CH3). CI-MS m/z 384, 386 (M + H)+. Anal.
(C18H14N3O2Br) C, H, N.
Data for (2′Z,3′E)-7-Iodo-1-methylindirubin-3′-methoxime
(10e). Mp 220 °C. 1H NMR (DMSO, 400 MHz, δ ppm) 11.92 (1H,
s, N′-H), 8.81 (1H, d, J ) 7.7 Hz, H-4), 8.12 (1H, d, J ) 7.7 Hz,
H-4′), 7.64 (1H, d, J ) 7.7 Hz, H-6), 7.50 (2H, m, H-6′, 7′), 7.06
(1H, m, H-5′), 6.83 (1H, t, J ) 7.7 Hz, H-5), 4.39 (3H, s, OCH3),
3.68 (3H, s, N-CH3). CI-MS m/z 432 (M + H)+. Anal.
(C18H14N3O2I) C, H, N.
General Procedure for the Preparation of the Acetoximes
11b-e and 12b-e. The appropriate indirubin-3′-oxime derivatives
7b-e and 8b-e (0.2 mmol) were dissolved in pyridine (10 mL).
Ac2O was added (0.5 mL), and the mixture was stirred for 30 min
at 0°C. Then water (1 mL) was added and the solvents were
evaporated under reduced pressure. The residue was washed with
water to afford quantitatively the corresponding 3′-acetoxime
selectively in the (2′Z,3′E) form.
Data for (2′Z,3′E)-7-Bromo-1-methylindirubin-3′-oxime (8d).
Mp 280 °C. IR 3184 (br), 1652, 1613, 1565, 1455, 1344, 1240,
1
1138 cm-1. H NMR (DMSO, 400 MHz, δ ppm) 12.00 (1H, s,
N′-H), 8.81 (1H, d, J ) 7.9 Hz, H-4), 8.23 (1H, d, J ) 7.9 Hz,
H-4′), 7.43 (2H, m, H-6′, 7′), 7.34 (1H, d, J ) 7.9 Hz, H-6), 7.07
(1H, t, J ) 7.9 Hz, H-5′), 6.93 (1H, t, J ) 7.9 Hz, H-5), 3.68 (3H,
s, N-CH3). CI-MS m/z 370, 372 (M + H)+. Anal. (C17H12N3O2-
Br) C, H, N.
Data for (2′Z,3′E)-7-Iodo-1-methylindirubin-3′-oxime (8e). Mp
280 °C. 1H NMR (DMSO, 400 MHz, δ ppm) 13.70 (1H, brs, NOH),
12.00 (1H, s, N′-H), 8.85 (1H, d, J ) 7.7 Hz, H-4), 8.24 (1H, d,
J ) 7.8 Hz, H-4′), 7.60 (1H, d, J ) 7.7 Hz, H-6), 7.43 (2H, m,
H-6′, 7′), 7.06 (1H, t, J ) 7.8 Hz, H-5′), 6.77 (1H, t, J ) 7.7 Hz,
H-5), 3.70 (3H, s, N-CH3). CI-MS m/z 418 (M + H)+. Anal.
(C17H12N3O2I) C, H, N.
General Procedure for the Preparation of the Methoximes
9b-e and 10b-e. The appropriate indirubin derivatives 1b-e and
6b-e (1 mmol) were dissolved in pyridine (10 mL). With magnetic
stirring, methoxylamine hydrochloride (10 equiv) was added and
the mixture was heated under reflux (120 °C) for 1.5 h. Then the
solvent was evaporated under reduced pressure and the residue was
washed with water to afford quantitatively the corresponding 3′-
methoxime selectively in the (2′Z,3′E) form.
Data for (2′Z,3′E)-7-Fluoroindirubin-3′-acetoxime (11b). Mp
1
253 °C. H NMR (DMSO, 400 MHz, δ ppm) 11.68 (1H, s, N′-
H), 11.33 (1H, s, N-H), 8.92 (1H, d, J ) 7.9 Hz, H-4), 8.25 (1H,
d, J ) 7.7 Hz, H-4′), 7.51 (2H, m, H-6′, 7′), 7.01 (2H, m, H-5′, 6),
6.96 (1H, td, J ) 7.8, 4.5 Hz, H-5), 2.47 (3H, s, OCOCH3). CI-
MS m/z 338 (M + H)+. Anal. (C18H12N3O3F) C, H, N.
Data for (2′Z,3′E)-7-Chloroindirubin-3′-acetoxime (11c). Mp
1
251 °C. H NMR (DMSO, 400 MHz, δ ppm) 11.70 (1H, s, N′-
H), 11.23 (1H, s, N-H), 9.07 (1H, d, J ) 8.0 Hz, H-4), 8.25 (1H,
d, J ) 7.6 Hz, H-4′), 7.52 (2H, m, H-6′, 7′), 7.24 (1H, d, J ) 8.0
Hz, H-6), 7.11 (1H, t, J ) 7.6 Hz, H-5′), 6.97 (1H, t, J ) 8.0 Hz,
H-5), 2.47 (3H, s, OCOCH3). CI-MS m/z 354, 356 (M + H)+. Anal.
(C18H12N3O3Cl) C, H, N.
Data for (2′Z,3′E)-7-Fluoroindirubin-3′-methoxime (9b). Mp
1
277 °C. H NMR (DMSO, 400 MHz, δ ppm) 11.79 (1H, s, N′-
H), 11.24 (1H, s, N-H), 8.46 (1H, d, J ) 7.5 Hz, H-4), 8.12 (1H,
d, J ) 7.6 Hz, H-4′), 7.44 (2H, m, H-6′, 7′), 7.05 (3H, m, H-5, 5′,
6), 4.39 (3H, s, OCH3). CI-MS m/z 310 (M + H)+. Anal.
(C17H12N3O2F) C, H, N.
Data for (2′Z, 3′E)-7-bromoindirubin-3′-acetoxime (11d). Mp
250 °C. IR 3239 (br), 2926, 2853, 1778, 1672, 1613, 1564, 1462,
1321, 1174, 1122 cm-1. 1H NMR (DMSO, 400 MHz, δ ppm) 11.73
(1H, s, N′-H), 11.11 (1H, s, N-H), 9.12 (1H, d, J ) 7.5 Hz, H-4),
8.27 (1H, d, J ) 7.9 Hz, H-4′), 7.53 (2H, m, H-6′, 7′), 7.37 (1H,
d, J ) 7.5 Hz, H-6), 7.11 (1H, t, J ) 7.9 Hz, H-5′), 6.92 (1H, t, J
) 7.5 Hz, H-5), 2.48 (3H, s, OCOCH3). CI-MS m/z 398, 400 (M
+ H)+. Anal. (C18H12N3O3Br) C, H, N.
Data for (2′Z,3′E)-7-Chloroindirubin-3′-methoxime (9c). Mp
1
272 °C. H NMR (DMSO, 400 MHz, δ ppm) 11.82 (1H, s, N′-
H), 11.24 (1H, s, N-H), 8.60 (1H, d, J ) 7.9 Hz, H-4), 8.12 (1H,
d, J ) 7.9 Hz, H-4′), 7.46 (2H, m, H-6′, 7′), 7.20 (1H, d, J ) 7.9
Hz, H-6), 7.05 (2H, m, H-5, 5′), 4.40 (3H, s, OCH3). CI-MS m/z
326, 328 (M + H)+. Anal. (C17H12N3O2Cl) C, H, N.
Data for (2′Z,3′E)-7-Bromoindirubin-3′-methoxime (9d). Mp
280 °C. IR 3394 (br), 2982, 2872, 1672, 1640, 1610, 1560, 1459,
1325, 1307, 1229 cm-1. 1H NMR (DMSO, 400 MHz, δ ppm) 11.84
(1H, s, N′-H), 11.02 (1H, s, N-H), 8.65 (1H, d, J ) 7.9 Hz, H-4),
8.13 (1H, d, J ) 7.9 Hz, H-4′), 7.46 (2H, m, H-6′, 7′), 7.34 (1H,
d, J ) 7.9 Hz, H-6), 7.06 (1H, m, H-5′), 6.97 (1H, t, J ) 7.9 Hz,
H-5), 4.41 (3H, s, OCH3). CI-MS m/z 370, 372 (M + H)+. Anal.
(C17H12N3O2Br) C, H, N.
Data for (2′Z,3′E)-7-Iodoindirubin-3′-acetoxime (11e). Mp 263
1
°C. H NMR (DMSO, 400 MHz, δ ppm) 11.71 (1H, s, N′-H),
10.78 (1H, s, N-H), 9.12 (1H, d, J ) 7.9 Hz, H-4), 8.25 (1H, d,
J ) 7.5 Hz, H-4′), 7.52 (3H, m, H-6, 6′, 7′), 7.10 (1H, t, J ) 7.5
Hz, H-5′), 6.77 (1H, t, J ) 7.9 Hz, H-5), 2.47 (3H, s, OCOCH3).
CI-MS m/z 446 (M + H)+. Anal. (C18H12N3O3I) C, H, N.
Data for (2′Z,3′E)-7-Fluoro-1-methylindirubin-3′-acetoxime
(12b). Mp 249 °C. 1H NMR (DMSO, 400 MHz, δ ppm) 11.76 (1H,
s, N′-H), 9.00 (1H, d, J ) 8.0 Hz, H-4), 8.26 (1H, d, J ) 7.4 Hz,
H-4′), 7.53 (2H, m, H-6′, 7′), 7.12 (2H, m, H-5′, 6), 7.00 (1H, td,
J ) 7.8, 4.5 Hz, H-5), 3.50 (3H, s, N-CH3), 2.47 (3H, s, OCOCH3).
CI-MS m/z 352 (M + H)+. Anal. (C19H14N3O3F) C, H, N.
Data for (2′Z,3′E)-7-Chloro-1-methylindirubin-3′-acetoxime
(12c). Mp 240 °C. 1H NMR (DMSO, 400 MHz, δ ppm) 11.83 (1H,
s, N′-H), 9.20 (1H, d, J ) 8.0 Hz, H-4), 8.27 (1H, d, J ) 7.5 Hz,
H-4′), 7.52 (2H, m, H-6′, 7′), 7.26 (1H, d, J ) 8.0 Hz, H-6), 7.12
(1H, t, J ) 7.5 Hz, H-5′), 7.01 (1H, t, J ) 8.0 Hz, H-5), 3.66 (3H,
s, N-CH3), 2.47 (3H, s, OCOCH3). CI-MS m/z 368, 370 (M +
H)+. Anal. (C19H14N3O3Cl) C, H, N.
Data for (2′Z,3′E)-7-Iodoindirubin-3′-methoxime (9e). Mp 297
1
°C. H NMR (DMSO, 400 MHz, δ ppm) 11.82 (1H, s, N′-H),
10.69 (1H, s, N-H), 8.66 (1H, d, J ) 7.8 Hz, H-4), 8.12 (1H, d,
J ) 7.7 Hz, H-4′), 7.50 (1H, d, J ) 7.8 Hz, H-6), 7.45 (2H, m,
H-6′, 7′), 7.06 (1H, m, H-5′), 6.82 (1H, t, J ) 7.8 Hz, H-5), 4.39
(3H, s, OCH3). CI-MS m/z 418 (M + H)+. Anal. (C17H12N3O2I) C,
H, N.
Data for (2′Z,3′E)-7-Fluoro-1-methylindirubin-3′-methoxime
(10b). Mp 227 °C. 1H NMR (DMSO, 400 MHz, δ ppm) 11.83 (1H,
s, N′-H), 8.52 (1H, d, J ) 7.4 Hz, H-4), 8.10 (1H, d, J ) 7.6 Hz,
H-4′), 7.44 (2H, m, H-6′, 7′), 7.06 (3H, m, H-5, 5′, 6), 4.39 (3H,
s, OCH3), 3.48 (3H, s, N-CH3). CI-MS m/z 324 (M + H)+. Anal.
(C18H14N3O2F) C, H, N.
Data for (2′Z,3′E)-7-Chloro-1-methylindirubin-3′-methoxime
(10c). Mp 203 °C. 1H NMR (DMSO, 400 MHz, δ ppm) 11.91 (1H,
s, N′-H), 8.72 (1H, d, J ) 7.8 Hz, H-4), 8.11 (1H, d, J ) 7.8 Hz,
H-4′), 7.46 (2H, m, H-6′, 7′), 7.21 (1H, d, J ) 7.8 Hz, H-6), 7.05
(2H, m, H-5, 5′), 4.40 (3H, s, OCH3), 3.66 (3H, s, N-CH3). CI-
MS m/z 340, 342 (M + H)+. Anal. (C18H14N3O2Cl) C, H, N.
Data for (2′Z,3′E)-7-Bromo-1-methylindirubin-3′-methoxime
(10d). Mp 212 °C. IR 2927 (br), 1646, 1613, 1555, 1463, 1332,
1234, 1161 cm-1. 1H NMR (DMSO, 400 MHz, δ ppm) 11.94 (1H,
s, N′-H), 8.80 (1H, d, J ) 7.9 Hz, H-4), 8.13 (1H, d, J ) 7.1 Hz,
Data for (2′Z,3′E)-7-Bromo-1-methylindirubin-3′-acetoxime
(12d). Mp 237 °C. IR 3229, 2936, 2853, 1778, 1651, 1610, 1557,
1461, 1330, 1233, 1202 cm-1. 1H NMR (DMSO, 400 MHz, δ ppm)
11.83 (1H, s, N′-H), 9.24 (1H, d, J ) 7.9 Hz, H-4), 8.26 (1H, d,
J ) 7.5 Hz, H-4′), 7.54 (1H, d, J ) 7.5 Hz, H-7′), 7.51 (1H, t, J
) 7.5 Hz, H-6′), 7.41 (1H, d, J ) 7.9 Hz, H-6), 7.12 (1H, t, J )
7.5 Hz, H-5′), 6.94 (1H, t, J ) 7.9 Hz, H-5), 3.67 (3H, s, N-CH3),
2.47 (3H, s, OCOCH3). CI-MS m/z 412, 414 (M + H)+. Anal.
(C19H14N3O3Br) C, H, N.
Data for (2′Z,3′E)-7-Iodo-1-methylindirubin-3′-acetoxime (12e).
1
Mp 240 °C. H NMR (DMSO, 400 MHz, δ ppm) 11.81 (1H, s,
N′-H), 9.26 (1H, d, J ) 7.8 Hz, H-4), 8.25 (1H, d, J ) 7.5 Hz,

4646 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Ferandin et al.
H-4′), 7.68 (1H, d, J ) 7.8 Hz, H-6), 7.52 (2H, m, H-6′, 7′), 7.11
(1H, t, J ) 7.5 Hz, H-5′), 6.78 (1H, t, J ) 7.8 Hz, H-5), 3.68 (3H,
s, N-CH3), 2.47 (3H, s, OCOCH3). CI-MS m/z 460 (M + H)+.
Anal. (C19H14N3O3I) C, H, N.
(4H, m, H-2′′′, 5′′′), 1.68 (4H, m, H-3′′′, 4′′′). CI-MS m/z 467, 469
(M + H)+. Anal. (C23H23N4O2Br) C, H, N.
Data for (2′Z,3′ E)-1-Methyl-7-bromoindirubin-3′-[O-(2-pyr-
1
rolidin-1-ylethyl)oxime] Hydrochloride (14gs). Mp 214 °C. H
NMR (DMSO, 400 MHz, δ ppm) 11.94 (1H, s, N′-H), 10.05 (1H,
brs, N′′′-H), 8.73 (1H, d, J ) 7.8 Hz, H-4), 8.24 (1H, d, J ) 7.8
Hz, H-4′), 7.49 (2H, m, H-6′, 7′), 7.40 (1H, d, J ) 7.8 Hz, H-6),
7.09 (1H, ddd, J ) 7.8, 4.1, 1.7 Hz, H-5′), 7.01 (1H, t, J ) 7.8 Hz,
H-5), 4.96 (2H, m, H-1′′), 3.68 (3H, s, N-CH3), 3.64 (2H, m,
H-2′′′a, 5′′′a), 3.14 (2H, m, 2′′′b, 5′′′b), 2.00 (2H, m, H-3′′′a, 4′′′a),
1.85 (2H, m, H-3′′′b, 4′′′b). Anal. (C23H24N4O2BrCl) C, H, N.
Data for (2′Z,3′E)-7-Bromoindirubin-3′-[O-(2-morpholin-1-
ylethyl)oxime] (13h). Yield: 85%. Mp 223 °C. 1H NMR (C5D5N,
400 MHz, δ ppm) 12.68 (1H, s, N′-H), 12.40 (1H, s, N-H), 9.02
(1H, d, J ) 7.7 Hz, H-4), 8.42 (1H, d, J ) 7.7 Hz, H-6), 7.54 (1H,
d, J ) 7.7 Hz, H-4′), 7.42 (1H, t, J ) 7.7 Hz, H-6′), 7.18 (2H,
overlapped, H-5′, H-7′), 7.10 (1H, t, J ) 7.7 Hz, H-5), 4.80 (2H,
t, J ) 5.9 Hz, H-1′′), 3.76 (4H, t, J ) 4.2 Hz, H-3′′′, 5′′′), 2.94
(2H, t, J ) 5.9 Hz, H-2′′), 2.60 (4H, t, J ) 4.2 Hz, H-2′′′, 6′′′).
CI-MS m/z 469, 471 (M + H)+. Anal. (C22H21N4O3Br) C, H, N.
Data for (2′Z,3′E)-7-Bromoindirubin-3′-[O-(2-morpholin-1-
ylethyl)oxime] Hydrochloride (13hs). Mp 235 °C. 1H NMR
(DMSO, 400 MHz, δ ppm) 11.82 (1H, s, N′-H), 11.04 (1H, s,
N-H), 10.71 (1H, brs, N′′′-H), 8.58 (1H, d, J ) 7.9 Hz, H-4),
8.23 (1H, d, J ) 7.7 Hz, H-4′), 7.47 (2H, m, H-6′, 7′), 7.35 (1H,
d, J ) 7.9 Hz, H-6), 7.08 (1H, ddd, J ) 7.7, 5.8, 2.3 Hz, H-5′),
6.99 (1H, t, J ) 7.9 Hz, H-5), 5.02 (2H, m, H-1′′), 3.95 (2H, m,
H-3′′′a, 5′′′a), 3.74 (4H, overlapped, H-2”, 3′′′b, 5′′′b), 3.57 (2H,
m, H-2′′′a, 6′′′a), 3.25 (2H, overlapped, 2′′′b, 6′′′b). Anal.
(C22H22N4O3BrCl) C, H, N.
(2′Z,3′E)-7-Bromoindirubin-3′-[O-(2-bromoethyl)oxime] (13f).
To a solution of 7BIO (7d) (100 mg, 0.30 mmol) in 5 mL of
anhydrous DMF, 120 µL of triethylamine (2.9 equiv) and 72 µL
of 1,2-dibromoethane (2.8 equiv) were added, and the reaction
mixture was stirred under Ar at room temperature for 48 h. Then
the solvent was evaporated under reduced pressure and the residue
was washed with water and dried at 50 °C to afford in 95% yield
the corresponding 3′-substituted oxime 13f. Mp 229 °C. 1H NMR
(DMSO, 400 MHz, δ ppm) 11.81 (1H, s, N′-H), 11.02 (1H, s,
N-H), 8.59 (1H, d, J ) 8.0 Hz, H-4), 8.22 (1H, d, J ) 8.0 Hz,
H-4′), 7.47 (2H, m, H-6′, 7′), 7.33 (1H, d, J ) 8.0 Hz, H-6), 7.08
(1H, m, H-5′), 6.95 (1H, t, J ) 8.0 Hz, H-5), 4.93 (2H, t, J ) 5.4
Hz, H-1′′), 3.98 (2H, t, J ) 5.4 Hz, H-2′′). CI-MS m/z 463, 465,
467 (M + H)+. Anal. (C18H13N3O2Br2) C, H, N.
(2′Z,3′E)-1-Methyl-7-bromoindirubin-3′-[O-(2-bromoethyl)-
oxime] (14f). This compound was prepared from Me7BIO (8d) by
a procedure analogous to that of 13f. Mp 218 °C. 1H NMR (DMSO,
400 MHz, δ ppm) 11.92 (1H, s, N′-H), 8.74 (1H, d, J ) 8.1 Hz,
H-4), 8.22 (1H, d, J ) 8.0 Hz, H-4′), 7.47 (2H, m, H-6′, 7′), 7.38
(1H, d, J ) 8.1 Hz, H-6), 7.09 (1H, m, H-5′), 6.98 (1H, t, J ) 8.1
Hz, H-5), 4.94 (2H, t, J ) 5.3 Hz, H-1′′), 3.98 (2H, t, J ) 5.3 Hz,
H-2′′), 3.68 (3H, s, N-CH3). CI-MS m/z 477, 479, 481 (M + H)+.
Anal. (C19H15N3O2Br2) C, H, N.
General Procedure for the Preparation of 3′-Substituted
Oximes of 7BIO (13g-l) or Me7BIO (14 g-l). 7-Bromoindirubin-
3′-[O-(2-bromoethyl)oxime] (13f) or 1-methyl-7-bromoindirubin-
3′-[O-(2-bromoethyl)oxime] (14f) (25 mg, 0.05 mmol) were
dissolved in 3 mL of anhydrous DMF. The corresponding amine
(pyrrolidine, morpholine, imidazole, piperazine, dimethylamine, and
diethylamine) (30 equiv) was added, and the reaction mixture was
stirred under Ar at room temperature for 48 h. Then the solvent
was evaporated under reduced pressure and the residue was washed
with water and dried at 50 °C to afford the corresponding
3′-substituted oximes 13g-l or 14g-l with 75-90% yield. For the
preparation of the hydrochloric salts of the above compounds, 10
mg of each compound was dissolved in 5 mL of anhydrous
tetrahydrofuran. Then a solution of hydrochloric acid in diethyl
ether was added slowly and the formed precipitate was filtered,
washed with dichloromethane, and dried at 50 °C to afford the
corresponding hydrochloric salts 13gs-ls and 14gs-ls.
Data for (2′Z, 3′E)-7-Bromoindirubin-3′-[O-(2-pyrrolidin-1-
ylethyl)oxime] (13g). Yield: 90%. Mp 198 °C. 1H NMR (DMSO,
400 MHz, δ ppm) 11.82 (1H, s, N′-H), 11.00 (1H, s, N-H), 8.64
(1H, d, J ) 8.0 Hz, H-4), 8.15 (1H, d, J ) 7.7 Hz, H-4′), 7.45
(2H, m, H-6′, 7′), 7.33 (1H, d, J ) 8.0 Hz, H-6), 7.07 (1H, ddd, J
) 7.7, 5.5, 3.1 Hz, H-5′), 6.94 (1H, t, J ) 8.0 Hz, H-5), 4.70 (2H,
t, J ) 5.9 Hz, H-1′′), 2.98 (2H, t, J ) 5.9 Hz, H-2′′), 2.56 (4H, m,
H-2′′′, 5′′′), 1.68 (4H, m, H-3′′′, 4′′′). CI-MS m/z 453, 455 (M +
H)+. Anal. (C22H21N4O2Br) C, H, N.
Data for (2′Z,3′E)-1-Methyl-7-bromoindirubin-3′-[O-(2-mor-
pholin-1-ylethyl)oxime] (14h). Yield: 85%. Mp 169 °C. 1H NMR
(C5D5N, 400 MHz, δ ppm) 12.40 (1H, s, N′-H), 9.11 (1H, d, J )
7.8 Hz, H-4), 8.42 (1H, d, J ) 7.7 Hz, H-6), 7.49 (1H, d, J ) 7.7
Hz, H-4′), 7.40 (1H, m, H-6′, 7′), 7.10 (1H, m, H-5, 5′), 4.81 (2H,
t, J ) 5.9 Hz, H-1′′), 3.76 (4H, t, J ) 4.5 Hz, H-3′′′, 5′′′), 3.70
(3H, s, N-CH3), 2.94 (2H, t, J ) 5.9 Hz, H-2′′), 2.60 (4H, t, J )
4.5 Hz, H-2′′′, 6′′′). CI-MS m/z 483, 485 (M + H)+. Anal.
(C23H23N4O3Br) C, H, N.
Data for (2′Z,3′E)-1-Methyl-7-bromoindirubin-3′-[O-(2-mor-
1
pholin-1-ylethyl)oxime] Hydrochloride (14hs). Mp 214 °C. H
NMR (DMSO, 400 MHz, δ ppm) 11.94 (1H, s, N′-H), 10.52 (1H,
brs, N′′′-H), 8.73 (1H, d, J ) 8.0 Hz, H-4), 8.23 (1H, d, J ) 7.7
Hz, H-4′), 7.49 (2H, m, H-6′, 7′), 7.40 (1H, d, J ) 8.0 Hz, H-6),
7.09 (1H, ddd, J ) 7.7, 4.1, 1.0 Hz, H-5′), 7.01 (1H, t, J ) 8.0 Hz,
H-5), 5.02 (2H, m, H-1′′), 3.98 (2H, m, H-3′′′a, 5′′′a), 3.72 (4H,
overlapped, H-2′′, 3′′′b, 5′′′b), 3.68 (3H, s, N-CH3), 3.55 (2H, m,
H-2′′′a, 6′′′a), 3.26 (2H, overlapped, 2′′′b, 6′′′b). Anal. (C23H24N4O3-
BrCl) C, H, N.
Data for (2′Z,3′E)-7-Bromoindirubin-3′-[O-(2-imidazol-1-yl-
1
ethyl)oxime] (13i). Yield: 75%. Mp 211 °C. H NMR (DMSO,
400 MHz, δ ppm) 11.79 (1H, s, N′-H), 10.99 (1H, s, N-H), 8.51
(1H, d, J ) 8.0 Hz, H-4), 7.99 (1H, d, J ) 7.4 Hz, H-4′), 7.67
(1H, s, H-2′′′), 7.44 (2H, m, H-6′, 7′), 7.33 (1H, d, J ) 8.0 Hz,
H-6), 7.27 (1H, s, H-4′′′), 7.02 (1H, ddd, J ) 8.0, 5.5, 3.1 Hz,
H-5′), 6.96 (1H, t, J ) 8.0 Hz, H-5), 6.87 (1H, s, H-5′′′), 4.90 (2H,
t, J ) 4.2 Hz, H-1′′), 4.54 (2H, t, J ) 4.2 Hz, H-2′′). CI-MS m/z
450, 452 (M + H)+. Anal. (C21H16N5O2Br) C, H, N.
Data for (2′Z,3′E)-7-Bromoindirubin-3′-[O-(2-imidazol-1-yl-
ethyl)oxime] Hydrochloride (13is). Mp 225 °C. 1H NMR (DMSO,
400 MHz, δ ppm) 11.79 (1H, s, N′-H), 11.02 (1H, s, N-H), 9.19
(1H, s, H-2′′′), 8.40 (1H, d, J ) 7.9 Hz, H-4), 7.95 (1H, d, J ) 7.5
Hz, H-4′), 7.86 (1H, s, H-5′′′), 7.62 (1H, s, H-4′′′), 7.44 (2H, m,
H-6′, 7′), 7.35 (1H, d, J ) 7.9 Hz, H-6), 6.94-7.04 (2H, overlapped,
H-5, 5′), 5.04 (2H, t, J ) 4.6 Hz, H-1′′), 4.77 (2H, t, J ) 4.6 Hz,
H-2′′). Anal. (C21H17N5O2BrCl) C, H, N.
Data for (2′Z,3′E)-7-Bromoindirubin-3′-[O-(2-pyrrolidin-1-
ylethyl)oxime] Hydrochloride (13gs). Mp 210 °C. 1H NMR
(DMSO, 400 MHz, δ ppm) 11.84 (1H, s, N′-H), 11.06 (1H, s,
N-H), 10.31 (1H, brs, N′′′-H), 8.58 (1H, d, J ) 7.9 Hz, H-4),
8.24 (1H, d, J ) 8.3 Hz, H-4′), 7.49 (2H, m, H-6′, 7′), 7.37 (1H,
d, J ) 7.9 Hz, H-6), 7.09 (1H, ddd, J ) 8.3, 4.4, 1.3 Hz, H-5′),
6.99 (1H, t, J ) 7.9 Hz, H-5), 4.97 (2H, brs, H-1′′), 3.77 (2H, brs,
H-2′′), 3.64 (2H, m, H-2′′′a, 5′′′a), 3.12 (2H, m, 2′′′b, 5′′′b), 2.00
(2H, m, H-3′′′a, 4′′′a), 1.86 (2H, m, H-3′′′b, 4′′′b). Anal. (C22H22N4O2-
BrCl) C, H, N.
Data for (2′Z,3′E)-1-Methyl-7-bromoindirubin-3′-[O-(2-pyr-
rolidin-1-ylethyl)oxime] (14g). Yield: 90%. Mp 169 °C. 1H NMR
(DMSO, 400 MHz, δ ppm) 11.93 (1H, s, N′-H), 8.80 (1H, d, J )
7.9 Hz, H-4), 8.16 (1H, d, J ) 8.0 Hz, H-4′), 7.46 (2H, m, H-6′,
7′), 7.37 (1H, d, J ) 7.9 Hz, H-6), 7.07 (1H, ddd, J ) 8.0, 5.5, 3.1
Hz, H-5′), 6.97 (1H, t, J ) 7.9 Hz, H-5), 4.71 (2H, t, J ) 5.9 Hz,
H-1′′), 3.68 (3H, s, N-CH3), 2.98 (2H, t, J ) 5.9 Hz, H-2′′), 2.56
Data for (2′Z,3′E)-1-Methyl-7-bromoindirubin-3′-[O-(2-imi-
1
dazol-1-ylethyl)oxime] (14i). Yield: 76%. Mp 246 °C. H NMR
(DMSO, 400 MHz, δ ppm) 11.89 (1H, s, N′-H), 8.65 (1H, d, J )
8.0 Hz, H-4), 7.99 (1H, d, J ) 8.1 Hz, H-4′), 7.69 (1H, s, H-2′′′),
7.45 (2H, m, H-6′, 7′), 7.37 (1H, d, J ) 8.0 Hz, H-6), 7.26 (1H, s,

3′-Substituted 7-Halogenoindirubins
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4647
H-4′′′), 6.97-7.05 (2H, overlapped, H-5′, 5), 6.86 (1H, s, H-5′′′),
4.90 (2H, t, J ) 4.8 Hz, H-1′′), 4.54 (2H, t, J ) 4.8 Hz, H-2′′),
3.67 (3H, s, N-CH3). CI-MS m/z 464, 466 (M + H)+. Anal.
(C22H18N5O2Br) C, H, N.
(1H, d, J ) 7.9 Hz, H-4), 8.17 (1H, d, J ) 7.8 Hz, H-4′), 7.45
(2H, m, H-6′, 7′), 7.33 (1H, d, J ) 7.9 Hz, H-6), 7.06 (1H, ddd, J
) 7.8, 5.5, 3.4 Hz, H-5′), 6.93 (1H, t, J ) 7.9 Hz, H-5), 4.66 (2H,
t, J ) 6.1 Hz, H-1′′), 2.95 (2H, t, J ) 6.1 Hz, H-2′′), 2.59 (4H, q,
J ) 7.1 Hz, N′′′(CH2CH3)2), 0.98 (6H, t, J ) 7.1 Hz, N′′′-
(CH2CH3)2). CI-MS m/z 461, 463 (M + H)+. Anal. (C22H29N4O2-
Br) C, H, N.
Data for (2′Z,3′E)-1-Methyl-7-bromoindirubin-3′-[O-(2-imi-
dazol-1-ylethyl)oxime] Hydrochloride (14is). Mp 218 °C. 1H
NMR (DMSO, 400 MHz, δ ppm) 11.90 (1H, s, N′-H), 9.11 (1H,
s, H-2′′′), 8.55 (1H, d, J ) 7.9 Hz, H-4), 7.96 (1H, d, J ) 7.6 Hz,
H-4′), 7.83 (1H, s, H-5′′′), 7.58 (1H, s, H-4′′′), 7.46 (2H, m, H-6′,
7′), 7.40 (1H, d, J ) 7.9 Hz, H-6), 6.97-7.05 (2H, overlapped,
H-5, 5′), 5.04 (2H, t, J ) 4.6 Hz, H-1′′), 4.75 (2H, t, J ) 4.6 Hz,
H-2′′), 3.67 (3H, s, N-CH3). Anal. (C22H19N5O2BrCl) C, H, N.
Data for (2′Z,3′E)-7-Bromoindirubin-3′-[O-(2-piperazin-1-
ylethyl)oxime] (13j). Yield: 84%. Mp 150 °C. IR 3229 (br), 2946,
Data for (2′Z,3′E)-7-Bromoindirubin-3′-[O-(2-diethylamino-
ethyl)oxime] Hydrochloride (13ls). Mp >300 °C. 1H NMR
(DMSO, 400 MHz, δ ppm) 11.82 (1H, s, N′-H), 11.03 (1H, s,
N-H), 10.52 (1H, brs, N′′′-H), 8.58 (1H, d, J ) 8.0 Hz, H-4),
8.23 (1H, d, J ) 7.9 Hz, H-4′), 7.48 (2H, m, H-6′, 7′), 7.35 (1H,
d, J ) 8.0 Hz, H-6), 7.07 (1H, ddd, J ) 7.9, 5.4, 2.9 Hz, H-5′),
7.00 (1H, t, J ) 8.0 Hz, H-5), 5.03 (2H, t, J ) 6.1 Hz, H-1′′), 3.68
(2H, t, J ) 6.1 Hz, H-2′′), 3.25 (4H, m, N′′′(CH2CH3)2), 1.22 (6H,
t, J ) 7.1 Hz, N′′′(CH2CH3)2). Anal. (C21H28N4O2BrCl) C, H, N.
Data for (2′Z,3′E)-1-Methyl-7-bromoindirubin-3′-[O-(2-di-
ethylaminoethyl)oxime] (14l). Yield: 88%. Mp 158 °C. 1H NMR
(DMSO, 400 MHz, δ ppm) 11.94 (1H, s, N′-H), 8.80 (1H, d, J )
7.9 Hz, H-4), 8.17 (1H, d, J ) 7.8 Hz, H-4′), 7.46 (2H, m, H-6′,
7′), 7.37 (1H, d, J ) 7.9 Hz, H-6), 7.07 (1H, ddd, J ) 7.8, 5.1, 3.1
Hz, H-5′), 6.95 (1H, t, J ) 7.9 Hz, H-5), 4.65 (2H, t, J ) 6.1 Hz,
H-1′′), 3.67 (3H, s, N-CH3), 2.94 (2H, t, J ) 6.1 Hz, H-2′′), 2.58
(4H, q, J ) 7.1 Hz, N′′′(CH2CH3)2), 0.98 (6H, t, J ) 7.1 Hz, N′′′-
(CH2CH3)2). CI-MS m/z 475, 477 (M + H)+. Anal. (C23H31N4O2-
Br) C, H, N.
1
2853, 1665, 1611, 1587, 1562, 1463, 1308, 1221 cm-1. H NMR
(DMSO, 400 MHz, δ ppm) 11.82 (1H, s, N′-H), 11.00 (1H, s,
N-H), 8.63 (1H, d, J ) 8.0 Hz, H-4), 8.17 (1H, d, J ) 7.8 Hz,
H-4′), 7.45 (2H, m, H-6′, 7′), 7.33 (1H, d, J ) 8.0 Hz, H-6), 7.06
(1H, ddd, J ) 7.8, 5.1, 3.1 Hz, H-5′), 6.94 (1H, t, J ) 8.0 Hz,
H-5), 4.71 (2H, t, J ) 5.6 Hz, H-1′′), 2.87 (2H, t, J ) 5.6 Hz,
H-2′′), 2.68 (4H, t, J ) 4.6 Hz, H-2′′′, 6′′′), 2.44 (4H, brs, H-3′′′,
5′′′). CI-MS m/z 468, 470 (M + H)+. Anal. (C22H22N5O2Br) C, H,
N.
Data for (2′Z,3′E)-7-Bromoindirubin-3′-[O-(2-piperazin-1-
1
ylethyl)oxime] Dihydrochloride (13js). Mp >300 °C. H NMR
(DMSO, 400 MHz, δ ppm) 11.82 (1H, s, N′-H), 11.05 (1H, s,
N-H), 9.32 (2H, br, piperazine N+-H), 8.59 (1H, d, J ) 8.0 Hz,
H-4), 8.25 (1H, d, J ) 7.5 Hz, H-4′), 7.48 (2H, m, H-6′, 7′), 7.35
(1H, d, J ) 8.0 Hz, H-6), 7.06 (1H, ddd, J ) 7.5, 4.1, 1.4 Hz,
H-5′), 6.99 (1H, t, J ) 8.0 Hz, H-5), 4.98 (2H, m, H-1′′), 3.70
(2H, m, H-2′′), 3.50 (8H, overlapped, H-2′′′, 3′′′, 5′′′, 6′′′). Anal.
(C22H24N5O2BrCl2) C, H, N.
Data for (2′Z,3′E)-1-Methyl-7-bromoindirubin-3′-[O-(2-di-
1
ethylaminoethyl)oxime] Hydrochloride (14ls). Mp 199 °C. H
NMR (DMSO, 400 MHz, δ ppm) 11.93 (1H, s, N′-H), 9.95 (1H,
brs, N′′′-H), 8.72 (1H, d, J ) 8.0 Hz, H-4), 8.22 (1H, d, J ) 7.8
Hz, H-4′), 7.49 (2H, m, H-6′, 7′), 7.41 (1H, d, J ) 8.0 Hz, H-6),
7.08 (1H, ddd, J ) 7.8, 4.0, 1.5 Hz, H-5′), 7.02 (1H, t, J ) 8.0 Hz,
H-5), 5.00 (2H, t, J ) 6.1 Hz, H-1′′), 3.68 (5H, m, N-CH3, H-2′′),
3.26 (4H, m, N′′′(CH2CH3)2), 1.21 (6H, t, J ) 7.3 Hz, N′′′-
(CH2CH3)2). Anal. (C22H30N4O2BrCl) C, H, N.
(2′Z,3′E)-7-Bromoindirubin-3′-[O-(N,N-diethylcarbamyl)-
oxime] (15). To a solution of 7BIO (25 mg, 0.07 mmol) in
anhydrous DMF (3 mL), 14 µL of triethylamine (1.5 equiv) and
13 µL of N,N-diethylcarbamyl chloride (1.5 equiv) were added,
and the reaction mixture was stirred under Ar at room temperature
for 48 h. Then the solvent was evaporated under reduced pressure
and the residue was washed with water and dried at 50°C to afford
quantitatively the corresponding 3′-substituted oximes. Mp 227 °C.
IR 3396 (br), 2954, 2917, 2853, 1734, 1661, 1610, 1555, 1459,
1413, 1261, 1216 cm-1. 1H NMR (C5D5N, 400 MHz, δ ppm) 12.70
(1H, s, N′-H), 12.29 (1H, s, N-H), 10.04 (1H, d, J ) 7.6 Hz,
H-4), 8.18 (1H, d, J ) 7.6 Hz, H-6), 7.49 (2H, m, H-4′, 6′), 7.34
(1H, t, J ) 7.9 Hz, H-5′), 7.22 (1H, overlapped, H-7′), 7.14 (1H,
t, J ) 7.6 Hz, H-5), 3.46 (4H, brs, N(CH2CH3)2), 1.19 (6H, t, J )
6.5 Hz, N(CH2CH3)2). CI-MS m/z 455, 457 (M + H)+. Anal.
(C21H19N4O3Br) C, H, N.
Data for (2′Z,3′E)-7-Bromoindirubin-3′-[O-(2-dimethylami-
noethyl)oxime] (13k). Yield: 90%. Mp 226 °C. 1H NMR (DMSO,
400 MHz, δ ppm) 11.82 (1H, s, N′-H), 11.00 (1H, s, N-H), 8.65
(1H, d, J ) 8.0 Hz, H-4), 8.15 (1H, d, J ) 7.8 Hz, H-4′), 7.46
(2H, m, H-6′, 7′), 7.33 (1H, d, J ) 8.0 Hz, H-6), 7.07 (1H, ddd, J
) 7.8, 5.1, 3.4 Hz, H-5′), 6.94 (1H, t, J ) 8.0 Hz, H-5), 4.70 (2H,
t, J ) 5.9 Hz, H-1′′), 2.81 (2H, t, J ) 5.9 Hz, H-2′′), 2.26 (6H, s,
N′′′(CH3)2). CI-MS m/z 433, 435 (M + H)+. Anal. (C20H25N4O2-
Br) C, H, N.
Data for (2′Z,3′E)-7-Bromoindirubin-3′-[O-(2-dimethylami-
noethyl)oxime] Hydrochloride (13ks). Mp 234 °C. 1H NMR
(DMSO, 400 MHz, δ ppm) 11.83 (1H, s, N′-H), 11.04 (1H, s,
N-H), 9.74 (1H, brs, N′′′-H), 8.58 (1H, d, J ) 8.0 Hz, H-4),
8.23 (1H, d, J ) 7.7 Hz, H-4′), 7.48 (2H, m, H-6′, 7′), 7.36 (1H,
d, J ) 8.0 Hz, H-6), 7.07 (1H, ddd, J ) 7.7, 5.0, 3.3 Hz, H-5′),
6.97 (1H, t, J ) 8.0 Hz, H-5), 4.94 (2H, t, J ) 5.9 Hz, H-1′′), 3.64
(2H, t, J ) 5.9 Hz, H-2′′), 2.85 (6H, s, N′′′(CH3)2). Anal.
(C20H26N4O2BrCl) C, H, N.
Data for (2′Z,3′E)-1-Methyl-7-bromoindirubin-3′-[O-(2-di-
1
methylaminoethyl)oxime] (14k). Yield: 90%. Mp 164 °C. H
Data for (2′Z,3′E)-1-Methyl-7-bromoindirubin-3′-[O-(N,N-
diethylcarbamyl)oxime] (16). This compound was prepared from
Me7BIO (27) by a procedure analogous to that of 63. Mp 229 °C.
IR 3351 (br), 2972, 1738, 1642, 1610, 1555, 1463, 1417, 1330,
NMR (DMSO, 400 MHz, δ ppm) 11.94 (1H, s, N′-H), 8.80 (1H,
d, J ) 7.9 Hz, H-4), 8.16 (1H, d, J ) 7.8 Hz, H-4′), 7.47 (2H, m,
H-6′, 7′), 7.38 (1H, d, J ) 7.9 Hz, H-6), 7.08 (1H, ddd, J ) 7.8,
5.5, 2.6 Hz, H-5′), 6.97 (1H, t, J ) 7.9 Hz, H-5), 4.70 (2H, t, J )
5.8 Hz, H-1′′), 3.68 (3H, s, N-CH3), 2.81 (2H, t, J ) 5.8 Hz,
H-2′′), 2.26 (6H, s, N′′′(CH3)2). CI-MS m/z 447, 449 (M + H)+.
Anal. (C21H27N4O2Br) C, H, N.
Data for (2′Z,3′E)-1-Methyl-7-bromoindirubin-3′-[O-(2-di-
methylaminoethyl)oxime] Hydrochloride (14ks). Mp >300 °C.
1H NMR (DMSO, 400 MHz, δ ppm) 11.94 (1H, s, N′-H), 10.07
(1H, brs, N′′′-H), 8.73 (1H, d, J ) 8.1 Hz, H-4), 8.25 (1H, d, J )
7.7 Hz, H-4′), 7.49 (2H, m, H-6′, 7′), 7.40 (1H, d, J ) 8.1 Hz,
H-6), 7.09 (1H, ddd, J ) 7.7, 5.5, 3.5 Hz, H-5′), 7.02 (1H, t, J )
8.1 Hz, H-5), 5.00 (2H, t, J ) 5.8 Hz, H-1′′), 3.68 (3H, s, N-CH3),
3.64 (2H, t, J ) 5.8 Hz, H-2′′), 2.85 (6H, s, N′′′(CH3)2). Anal.
(C21H28N4O2BrCl) C, H, N.
1
1229 cm-1. H NMR (C5D5N, 400 MHz, δ ppm) 12.32 (1H, s,
N′-H), 10.10 (1H, d, J ) 7.6 Hz, H-4), 8.18 (1H, d, J ) 7.6 Hz,
H-6), 7.46 (2H, m, H-4′, 6′), 7.30 (1H, t, J ) 7.8 Hz, H-5′), 7.16
(2H, overlapped, H-5, 7′), 3.66 (3H, s, N-CH3), 3.46 (4H, brs,
N(CH2CH3)2), 1.19 (6H, t, J ) 6.8 Hz, N(CH2CH3)2). CI-MS m/z
469, 471 (M + H)+. Anal. (C22H21N4O3Br) C, H, N.
Protein Kinase Assays. Biochemical Reagents. Sodium ortho-
vanadate, EGTA, EDTA, Mops, â-glycerophosphate, phenyl phos-
phate, sodium fluoride, dithiothreitol (DTT), glutathione agarose,
glutathione, bovine serum albumin (BSA), nitrophenyl phosphate,
leupeptin, aprotinin, pepstatin, soybean trypsin inhibitor, benzami-
dine, and histone H1 (type III-S) were obtained from Sigma
Chemicals. [γ-33P]ATP was obtained from Amersham. The GS-1
peptide (YRRAAVPPSPSLSRHSSPHQSpEDEEE) was synthesized
by the Peptide Synthesis Unit, Institute of Biomolecular Sciences,
University of Southampton, Southampton SO16 7PX, U.K.
Data for (2′Z,3′E)-7-Bromoindirubin-3′-[O-(2-diethylamino-
1
ethyl)oxime] (13l). Yield: 89%. Mp 208 °C. H NMR (DMSO,
400 MHz, δ ppm) 11.83 (1H, s, N′-H), 11.00 (1H, s, N-H), 8.66

4648 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Ferandin et al.
Buffers. Homogenization buffer: 60 mM â-glycerophosphate,
15 mM p-nitrophenyl phosphate, 25 mM Mops (pH 7.2), 15 mM
EGTA, 15 mM MgCl2, 1 mM DTT, 1 mM sodium vanadate, 1
mM NaF, 1 mM phenyl phosphate, 10 µg of leupeptin/mL, 10 µg
of aprotinin/mL, 10 µg of soybean trypsin inhibitor/mL, and 100
µM benzamidine.
Buffer A: 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 25 mM
Tris-HCl, pH 7.5, 50 µg of heparin/mL.
Buffer C: homogenization buffer but 5 mM EGTA, no NaF,
and no protease inhibitors.
of 37 °C. Culture dishes and other plastic disposable tools were
supplied by Corning (Corning, NY). Indirubin treatments were
performed on exponentially growing cultures at the indicated times
and concentrations. Control experiments were also carried out using
appropriate dilutions of DMSO.
MDA-MB-231 cells (derived from hormone-independent breast
cancer) were obtained from Dr. J. Mester and cultured in DMEM
supplemented with 10% FCS. For experiments, these cells were
seeded in 96-well plates (7500 cells/well) and exposed to indirubins
as indicated.
Kinase Preparations and Assays. Kinase activities were assayed
in buffer A or C at 30 °C at a final ATP concentration of 15 µM.
Blank values were subtracted and activities calculated as pmoles
of phosphate incorporated for a 10 min incubation. The activities
are usually expressed as a percentage of the maximal activity, i.e.,
in the absence of inhibitors. Controls were performed with
appropriate dilutions of dimethyl sulfoxide.
CDK1/cyclin B was extracted in homogenization buffer from
M phase starfish (Marthasterias glacialis) oocytes and purified by
affinity chromatography on p9CKShs1-sepharose beads, from which
it was eluted by free p9CKShs1 as previously described.30 The kinase
activity was assayed in buffer C, with 1 mg of histone H1/mL, in
the presence of 15 µM [γ-33P]ATP (3000 Ci/mmol, 10 mCi/mL)
in a final volume of 30 µL. After 30 min of incubation at 30 °C,
25 µL aliquots of supernatant were spotted onto 2.5 cm × 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 of phosphoric acid/L of water. The wet filters
were counted in the presence of 1 mL of ACS (Amersham)
scintillation fluid.
Cell Death and Cell Viability Assessments. Cell viability was
determined by measuring the reduction of 3-(4,5-dimethylthiazol-
2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoli-
um (MTS) as previously described in detail.31
Caspase Assay. The measurement of caspase activity is based
on determining the fluorescence released from the AcDEVDafc
synthetic substrate after its direct addition to the culture medium,
detergent lysis, and incubation at 37°C. This method is devised to
be applied to 96 multiwell plates. It allows kinetic determinations
of caspase activation and the characterization of multiple drugs
simultaneously.31
Acknowledgment. We are grateful to our colleagues M.
Gottlicher, J. Mester, L. H. Tsai, and B. Vogelstein for providing
several reagents used in this study. This research was supported
by a grant from the EEC (Grant FP6-2002, Life Sciences &
Health, PRO-KINASE Research Project) (L. M.), a grant from
the “Association pour la Recherche sur le Cancer“ (Grant
ARC3362) (L. M.) and a “Cance´ropole Grand-Ouest” grant (L.
M.). K.B. was supported by a fellowship from the “Ministe`re
de la Recherche”. A.L.S. and P.M. were supported by a
“KAPODISTRIAS” grant of the University of Athens.
CDK5/p25 was reconstituted by mixing equal amounts of
recombinant mammalian CDK5 and p25 expressed in E. coli as
GST (glutathione-S-transferase) fusion proteins and purified by
affinity chomatography on glutathione agarose (vectors kindly
provided by Dr. L. H. Tsai) (p25 is a truncated version of p35, the
35 kDa CDK5 activator). Its activity was assayed with histone H1
in buffer C as described for CDK1/cyclin B.
Supporting Information Available: Results from elemental
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org.
GSK-3R/â was purified from porcine brain by affinity chroma-
1
tography on immobilized axin.18 It was assayed, following a /100
References
dilution in 1 mg of BSA/mL 10 mM DTT, with 5 µL of 4 µM
GS-1 peptide substrate in buffer A in the presence of 15 µM [γ-33P]-
ATP (3000 Ci/mmol, 10 mCi/mL) in a final volume of 30 µL. After
30 min of incubation at 30 °C, 25 µL aliquots of supernatant were
processed as described above.
Cell Biology. Antibodies and Chemicals. AcDEVDafc and
Q-VD-OPh were purchased from MPbiomedicals (Vannes, France).
Cell Titer 96 kit containing the MTS reagent was purchased from
Promega (Madison, WI). The protease inhibitor cocktail was from
Roche. Unless otherwise stated, the nonlisted reagents were also
from Sigma.
Cell Lines and Culture Conditions. The mouse 5L hepatoma
cell line (AhR +/+) and BP8 (an AhR -/- subclone) were kindly
provided by Dr. M. Gottlicher (Forschungszentrum Karlsruhe,
Institute of Genetics, 76021 Karlsruhe, Germany). They were
cultured in Dulbecco’s modified Eagle medium (DMEM) (Bio-
whittaker) supplemented with 2 mM L-glutamine (Eurobio), 10%
fetal calf serum (FCS), and penicillin and streptomycin (Gibco BRL)
at 37 °C in an atmosphere of 5% CO2. Indirubin treatments were
performed on cultures at the indicated times and concentrations.
Control experiments were carried out using appropriate dilutions
of DMSO.
SH-SY5Y human neuroblastoma cell line was grown in DMEM
medium (Biowhittaker) supplemented with 2 mM L-glutamine from
Eurobio (Courtaboeuf, France), antibiotics, and 10% volume of FCS
from Invitrogen (Cergy Pontoise, France). HCT116 human adeno-
carcinoma cell line was kindly provided by Dr. B. Vogelstein
(Howard Hughes Medical Institute, Sidney Kimmel Comprehensive
Cancer Center, The Johns Hopkins School of Medicine, Baltimore,
MD 21231). HCT116 cells were cultured in McCoy’s 5A (Bio-
whittaker) supplemented with antibiotics and 10% FCS. General
culture conditions were an atmosphere of 5% CO2 and a temperature
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