Soluble 3′,6-substituted indirubins with enhanced selectivity toward glycogen synthase kinase -3 alter circadian period

Article abstract of DOI:10.1021/jm800648y

Glycogen synthase kinase -3 (GSK-3) is a key enzyme involved in numerous physiological events and in major diseases, such as Alzheimer's disease, diabetes, and cardiac hypertrophy. Indirubins are bis-indoles that can be generated from various natural sources or chemically synthesized. While rather potent and selective as GSK-3 inhibitors, most indirubins exhibit low water solubility. To address the issue of solubility, we have designed novel analogues of 6-bromo-indirubin-3′-oxime with increased hydrophilicity based on the GSK-3/indirubins cocrystal structures. The new derivatives with an extended amino side chain attached at position 3′ showed potent GSK-3 inhibitory activity, enhanced selectivity, and dramatically increased water solubility. Furthermore, some of them displayed little or no cytotoxicity. The new indirubins inhibit GSK-3 in a cellular reporter model. They alter the circadian period measured in rhythmically expressing cell cultures, suggesting that they might constitute tools to investigate circadian rhythm regulation.

Full text of DOI:10.1021/jm800648y

J. Med. Chem. 2008, 51, 6421–6431
6421
Soluble 3′,6-Substituted Indirubins with Enhanced Selectivity toward Glycogen Synthase Kinase
-3 Alter Circadian Period
Konstantina Vougogiannopoulou,†,‡ Yoan Ferandin,†,§ Karima Bettayeb,§ Vassilios Myrianthopoulos,| Olivier Lozach,§
Yunzhen Fan, Carl Hirschie Johnson, Prokopios Magiatis,‡ Alexios-Leandros Skaltsounis,*,‡ Emmanuel Mikros,| and
Laurent Meijer*,§
Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, UniVersity of Athens, Panepistimiopolis Zografou,
GR-15771 Athens, Greece, C.N.R.S., Cell Cycle Group & UPS2682, Station Biologique, B.P. 74, 29682 Roscoff Cedex, Bretagne, France,
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, UniVersity of Athens, Panepistimiopolis Zografou, GR-15771 Athens, Greece,
Vanderbilt UniVersity, Department of Biological Sciences, U-8214 BSB/MRB III, 465 21st AVenue South, NashVille, Tennessee 37232, USA
ReceiVed May 29, 2008
Glycogen synthase kinase -3 (GSK-3) is a key enzyme involved in numerous physiological events and in
major diseases, such as Alzheimer’s disease, diabetes, and cardiac hypertrophy. Indirubins are bis-indoles
that can be generated from various natural sources or chemically synthesized. While rather potent and selective
as GSK-3 inhibitors, most indirubins exhibit low water solubility. To address the issue of solubility, we
have designed novel analogues of 6-bromo-indirubin-3′-oxime with increased hydrophilicity based on the
GSK-3/indirubins cocrystal structures. The new derivatives with an extended amino side chain attached at
position 3′ showed potent GSK-3 inhibitory activity, enhanced selectivity, and dramatically increased water
solubility. Furthermore, some of them displayed little or no cytotoxicity. The new indirubins inhibit GSK-3
in a cellular reporter model. They alter the circadian period measured in rhythmically expressing cell cultures,
suggesting that they might constitute tools to investigate circadian rhythm regulation.
Introduction
for the treatment of specific diseases, and for the maintenance
of pluripotent stem cells in the absence of feeder cells.20
Numerous GSK-3 inhibitory scaffolds have been described.21-24
Interestingly, many of these inhibitors also interact with cyclin-
dependent kinases (CDKs), another family of well-studied key
regulatory enzymes.25
Among the 518 protein kinases that constitute the human
kinome, glycogen synthase kinase -3 (GSK-3a) stands out as a
particularly interesting and well-studied family of serine/
threonine kinases.1-5 There are only two GSK-3 forms (GSK-
3R and GSK-3ꢀ), which share extensive similarity (84% overall
identity, 98% within the catalytic domain), the main difference
coming from an extra Gly-rich stretch in the N terminal domain
of GSK-3R. GSK-3s are highly conserved protein kinases
present from unicellular parasites6,7 to yeast8 up to mammals.
These kinases are involved in numerous critical physiological
events such as Wnt and Hedgehog signaling, embryonic
development (pattern specification and axial orientation), tran-
scription, insulin action, cell division cycle, cell death, cell
survival, differentiation, multiple neuronal functions, circadian
rhythm regulation, stem cell differentiation, etc. In addition,
GSK-3s are implicated in a large diversity of human diseases,
including nervous system disorders such as Alzheimer’s
disease,9-11 schizophrenia,12 bipolar disorder,13 diabetes,14 heart
hypertrophy,15,16 renal diseases,17 shock and inflammation,18
cancers,19 etc. There is thus a strong rationale supporting the
search for potent and selective GSK-3 inhibitors for their use
as pharmacological tools in basic research, as potential drugs
Among GSK-3 inhibitors, derivatives of the bis-indole
indirubin (collectively referred to as indirubins) appear as a class
of original and promising tools and agents.26 Their moderate
selectivity might be an inconvenient when used as a research
reagent, but their combined effects on several disease-relevant
targets (in particular CDKs and GSK-3) may constitute an
advantage for potential therapeutic applications.24 Among many
indirubins, 6-bromo-indirubin-3′-oxime (6BIO)27-29 has been
widely used to investigate the physiological role of GSK-3 in
various cellular settings and to alter the fate of embryonic stem
cells.20 While highly potent and relatively selective kinase
inhibitory indirubins have been developed, they usually exhibit
low water solubility. To address the solubility problem of these
promising compounds, we have designed novel analogues of
6BIO with increased hydrophilicity. Improvement of the hy-
drophilic character of a molecule may be approached by several
ways. The decrease of the aromatic character of indirubin
scaffold by changing the hybridization state of an aromatic
carbon atom to sp3 has been proposed as a way to enhance
solubility.30 An alternative method is the introduction of
hydrophilic groups on the molecule.31 Obviously, it is essential
that the optimization of hydrophilicity does not negatively
impact on either the potency or on the selectivity of the molecule
toward the target kinase. The choice of the substitution position
is thus highly significant because there are two important areas
of the molecule that cannot be altered without dramatic decrease
of efficacy on kinases. The first one is the pharmacophore
consisting of the lactam nitrogen and carbonyl and the hetero-
cyclic nitrogen of the bis-indole core that form the key hydrogen
bonding interaction pattern with the active site of the kinase
* To whom correspondence should be addressed. For L.M.: phone,
+33.(0)2.98.29.23.39; fax, +33.(0)2.98.29.25.26; E-mail, meijer@
sb-roscoff.fr. For A.L.S.: phone and fax, +30.210.727.45.94; E-mail,
skaltsounis@pharm.uoa.gr.
†
These authors contributed equally.
Department of Pharmacognosy and Natural Products Chemistry, Faculty
‡
of Pharmacy, University of Athens.
§
C.N.R.S., Cell Cycle Group & UPS2682, Station Biologique.
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
|
University of Athens.
Vanderbilt University, Department of Biological Sciences.
a Abbreviations: 6BIO, 6-bromoindirubin-3′-oxime; CDK, cyclin-de-
pendent kinase; FCS, fetal calf serum; GSK-3, glycogen synthase kinase-
3; IO, indirubin-3′-oxime; MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-car-
boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium).
10.1021/jm800648y CCC: $40.75
2008 American Chemical Society
Published on Web 09/25/2008

6422 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Vougogiannopoulou et al.
targets.27,28,32-36 The second is the bromine substitution at
position 6, which is the selectivity determinant of 6BIO toward
GSK-3ꢀ.27,28,37 A detailed analysis of the crystal structure of
GSK-3ꢀ in complex with 6BIO27,28 provided critical information
suggesting that the 3′ position might be ideal for carrying out
chemical modifications on the indirubin scaffold. We thus
designed and synthesized a series of 6-bromo-indirubins with
various substitutions on position 3′. Some of these molecules
displayed high potency toward GSK-3, enhanced selectivity,
and much increased water solubility. These molecules were
evaluated for their GSK-3 inhibitory actions in several cellular
systems.
Results and Discussion
Rationale: Binding Cavity Mapping. The biochemical and
potential therapeutic importance of 6BIO being a rather potent
and selective GSK-3ꢀ ATP-competitive inhibitor is undermined
by its poor water solubility (<5 mg/l). Chemical synthesis was
chosen to address the question of improving this physicochem-
ical property. Modifications of the 6BIO molecule had to be
done with care, as the aim was to introduce substitutions that
might improve solubility and simultaneously maintain affinity
and selectivity.
Figure 1. Mapping of the hydrophobic (green), positive (blue), and
negative (red) regions of the binding pocket of GSK-3. Three distinct
regions are visible: the hydrophobic active site where 6BIO molecule
(magenta) is bound, the ribose subsite where the sugar of ATP forms
stabilizing interactions with the kinase, and the phosphate subsite where
the three phosphate groups interact with catalytic residues of the kinase.
The ribose and phosphate subsites provide a polar environment that
could accommodate favorable electrostatic or hydrogen bonding
interactions with the inhibitor molecule.
Both affinity and selectivity are attributed to particular sites
of the 6BIO molecule that, as a consequence, should not be
altered. The pharmacophore is an essential part of the molecule
that needs to be kept intact. It accommodates the main stabilizing
interactions of the ligand, which is anchored at the kinase active
site through three hydrogen bonds. Furthermore, substitution
at position 6 is crucial for selectivity. A bulky group at that
position takes advantage of the difference in the active site
“gatekeeper” residue between GSK-3ꢀ (leu132) and the CDKs
(phe80CDK2). Selectivity results from the steric clash between
the substituent at position 6 and the phenylalanine residue of
the CDKs. Positions 4 and 7 are not suitable for substitution.
An atom larger than hydrogen at position 4 distorts the planarity
of the molecule. Substitution on position 7 causes unfavorable
contacts with the protein backbone.37,38 An introduction of
hydrophilic groups at position 5 has already been performed.39,40
Nevertheless, an unfavorable interaction with the catalytic
residues lys85 and asp200 of the active site is possible.
Substituents at position 5′ and 6′ would be oriented toward the
side chain of arg141, forming π-π face-to-edge stacking
interactions, but analogues carrying bulky substitutions at these
positions have demonstrated a diminished potency.25,27,28,40,41
The study of crystal structures of indirubin 3′-oxime bound
to CDK5/p25 (1UNH)34 and 6BIO bound to GSK3ꢀ (1UV5)27,28
indicated the oxime moiety as an ideal site for introducing
groups that will increase the hydrophilic character of 6BIO. The
oxime of the bound ligand lies at the periphery of the binding
cleft being partly exposed to the solvent (Figure 1). As a
consequence, the introduction of bulky groups or chains at this
position seems to be sterically acceptable. Most importantly,
the rich polar environment provided by the ribose subsite where
the oxime is positioned could facilitate novel favorable interac-
tions. The binding mode of ATP to CDK2 as it appears in crystal
structures (1B38, 1HCK) demonstrates the significance of
interactions formed at the ribose subsite on stabilizing the
inhibitor-receptor complex (Figure 2). The ribose moiety of
ATP is anchored by the side chain carboxyl of asp86 and by
the backbone carbonyl of gln131 through hydrogen bonds
(Figure 1). In the structure of ATP bound to CDK2/cyclin A
(1FIN), a conformational change is induced by cyclin binding
that results in a reorientation of ATP in the cavity. However,
Figure 2. Superposition of crystal structures of ATP bound to
monomeric CDK2 (orange) and CDK2/cyclin A (blue). In both cases,
two hydrogen bonds formed between the adenine and the receptor
(yellow dashed lines) stabilize the complex. An additional set of
favorable interactions exists at the ribose subsite of the binding pocket,
where the sugar moiety of ATP interacts through hydrogen bonds with
residues that constitute the periphery of the binding pocket, namely
asp86 and gln131 in the monomeric kinase and ile10 in the CDK2/
cyclin A dimer. Visible is also the CR trace of the glycine loop at the
top of the binding pocket.
ribose retains a strong interaction with the cavity environment,
in this case with the backbone carbonyl of ile10 of the glycine
loop. The ribose subsite of the GSK-3ꢀ binding cavity is similar
to that of CDK2. Ile10CDK2 corresponds to ile62GSK3ꢀ
,
gln131CDK2 to gln185GSK3ꢀ, and asp86CDK2 to thr138GSK3ꢀ. The

3′,6-Substituted Indirubins Alter Circadian Period
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6423
Scheme 1a
a Reagents: (i) dibromoethane, triethylamine, DMF anhyd, 25 °C; (ii) 2-bromoethanol, triethylamine, DMF anhyd, 25 °C; (iii) 3-bromo-1,2-propanediol,
triethylamine, DMF anhyd, 25 °C; (iv) N,N-diethylcarbamyl chloride, triethylamine, DMF anhyd, 25 °C. (v) DMF anhyd, 25 °C, amine a-k.
ribose subsite thus provides a rich environment of polar groups
that could be targeted as interaction partners of improved 6BIO
analogues.
chain containing appropriate chemical groups that could interact
with residues of the ribose and the phosphate sites of the receptor
cavity was considered as suitable (Figure 1). Such a substitution
is expected to resolve the solubility problem of indirubins
without affecting the selectivity toward GSK-3ꢀ as the 6Br
substitution remains unaltered. Introducing hydrophilic chemical
groups on 6BIO could have undesirable consequences on
binding affinity, as an increase of the polar character of a
molecule results in a larger desolvation energy penalty upon
binding. In addition, conformational entropy cost proportional
to molecular flexibility could further decrease binding affinity.
Energetics of binding imply that the finest way to overcome
the impact of introducing flexible hydrophilic groups on the
affinity of 6BIO for GSK-3ꢀ is the formation of novel stabilizing
interactions between these groups and the kinase. Maximizing
the attractive interactions in an otherwise highly flexible moiety
could equilibrate the expected entropic loss in binding affinity
due to the degrees of freedom inherent to such a system as well
as the desolvation energy loss and optimize the affinity of the
inhibitors.
By examining crystal structures of ATP or ATP-competitive
inhibitors bound to various kinases, an additional part of the
cavity was located where favorable interactions could be
facilitated. It is the place where the phosphate groups of ATP
interact with the receptor, called the phosphate subsite. Key
interactions between the ATP phosphates and the kinase are
formed there and more specifically between the R-phosphate
and the side chain of lys33, between the ꢀ-phosphate and
the side chains of lys129 and gln131, and between the
γ-phosphate and the side chains of lys129 and thr14. These
residues along with asn132 and the catalytic asp145 (the D of
the DFG conserved motif) form the phosphate subsite in CDK2.
In GSK-3ꢀ, the homologous residues are ser66 (thr14CDK2),
lys85 (lys33CDK2), lys183 (lys129CDK2), gln185 (gln131CDK2),
asn186 (asn132CDK2), and asp200 (asp145CDK2). A grid-based
mapping of the hydrophilic and hydrophobic sites of the binding
cavity of GSK-3ꢀ was performed by the Sitemap module of
Maestro. Finally, two water molecules (PDB 1UV5, waters 49
and 74) that are proposed to bridge the interaction between
thr138 and the oxime were considered as part of the cavity and
possible mediators of favorable interactions.28
Synthesis of 3′-Substituted, 6-Bromo-indirubins. In an
effort to prepare derivatives consistent with the above rationale,
we synthesized 3′-substituted oximes of 6BIO bearing side
chains with amine groups 5-15 and the corresponding salts
16-26 (Scheme 1). The synthesis was based on the reaction of
the 3′-[O-(2-bromoethyl)oxime] intermediate 1 with the ap-
Rationale: Energetics. On the basis of the GSK-3ꢀ active
site inspection, the substitution of the 3′ oxime by a hydrophilic

6424 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Vougogiannopoulou et al.
Table 1. Effects of Indirubins 1-26 on Three Protein Kinases and on the Survival of Human Neuroblastoma SH-SY5Y Cellsa
a Indirubins were tested at various concentrations on GSK-3R/ꢀ, CDK1/cyclin B, CDK5/p25, as described in Experimental Section. IC50 values, calculated
from the dose-response curves, are reported in µM. The compounds were tested at various concentrations for their effects on SH-SY5Y cells survival after
48 h incubation as estimated using the MTS reduction assay. IC50 values, calculated from the dose-response curves, are reported in µM.
propriate amine: pyrrolidine, morpholine, piperazine, N-meth-
ylpiperazine, hydroxyethylpiperazine, methoxyethylpiperazine,
dimethylamine and diethylamine, N,N-bis-2-hydroxyethylamine,
N-2,3-dihydroxypropyl-N-methyl amine, and N-2-hydroxyethox-
yethyl piperazine. The intermediate 1 was prepared by the
reaction of 6BIO with 1,2-dibromoethane in DMF and Et3N at
room temperature. In addition, the carbamate 4 was prepared
by the reaction of 6BIO with N,N-diethylcarbamyl chloride. The
alcohols 2 and 3 were prepared by the reaction of 6BIO with
the appropriate bromo alcohol in DMF and Et3N at room
temperature. Indirubin and 6BIO were synthesized as previously
reported.28
Building Analogues: Kinase Inhibitory Properties. Several
chemical groups were introduced on the oxime oxygen of 6BIO
and designed analogues along with their IC50 values for three
different kinases (GSK-3R/ꢀ, CDK1/cyclin B, CDK5/p25) are
summarized in Table 1. As the initial objective was to increase
solubility, these groups were chosen according to their polarity
and their potential to ionize. The biological results of the new
6BIO analogues have provided interesting information about
structure-activity relations concerning GSK-3. In addition,
docking calculations have provided insight in the binding mode
of the novel analogues. The physicochemical properties of
representative compounds such as calculated pKa, clogD, and
water solubility are summarized in Table 2.
Analogue 3 carries a side chain with two aliphatic hydroxyls
at a distance of two carbon atoms from the oxime oxygen, which
were expected to mimic the 2′ and 3′ hydroxyl groups of ATP
ribose. It demonstrated a 6-fold decrease of activity compared
to 6BIO. This decrease was accompanied by a mediocre
improvement of solubility. This result was indicative of the
unfavorable influence of chain flexibility on affinity. As flex-
ibility was implicit for groups that would be used to improve
solubility, the amplification of their stabilizing interactions was

3′,6-Substituted Indirubins Alter Circadian Period
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6425
Table 2. Water Solubility of Indirubin Salts and Calculated
Physicochemical Properties pKa and logD at pH 7.4 of Corresponding
Bases
no.
solubility (g/L)
pKa
LogD
6BIO
16
17
18
21
22
23
24
25
<0.005
0.141
0.192
0.195
1.45
1.61
1.50
1.14
0.57
2.59
1.69
1.19
1.16
1.79
-0.87
1.74
1.41
1.90
1.46
8.59
9.38
9.18
7.48
8.85
7.65
7.65
7.47
7.58
26
4.253
considered as a way to equalize the entropic loss. A series of
analogues carrying a tertiary amine two carbons from the oxime
was prepared in order to enhance the strength of hydrogen
bonding interactions that are expected to form at the ribose
subsite. Analogue 5 carries a dimethylamino group two carbon
atoms from the oxime oxygen. Analogue 6, carrying a bulkier
diethylamino group, was designed to scan the extent by which
the ATP channel could accommodate larger hydrophobic
interactions. The ethyl carbons of 6 were fused to a pyrrolidine
ring in analogue 7 to reduce conformational entropy. Their
activity was only slightly improved. Docking calculations
demonstrated that the main interaction formed by those ana-
logues was a charge-assisted hydrogen bond between the
protonated secondary amine nitrogen and the side chain of the
catalytic asp200. However, the geometry of this bond was not
optimal. As a result, alternative binding modes were observed
where residues asn64 (glycine loop), asn186 (ribose-site), and
water 47 participated with polar interactions. Keeping the tertiary
amine unaltered was encouraged by this observed binding mode.
As a result, further scanning of the cavity toward the phosphate
subsite was performed by the extended chains of 9, 10 and 8.
The first two carry a short aliphatic extension bearing two
hydroxyl groups. The free rotation of the extended chain was
restricted in 8, where the two hydroxyethyl groups were fused
to a morpholine ring. Compounds 8 and 9 demonstrated a
noteworthy decrease of affinity (8-fold and 12-fold). According
to these results and modeling, it was deduced that affinity could
not be optimized by introduction of hydroxyl groups. Interac-
tions formed by hydroxyls at the ribose subsite included
hydrogen bonds with gln185 and a water-mediated bridge with
thr138. Docking calculations suggested that these bonds could
not compensate for the hydroxy-carrying group inherent flex-
ibility. The conversion of the heterocyclic oxygen of the rigid
morpholine to nitrogen would thus add a tertiary positively
charged amine group closer to asp200. Analogue 11/22, carrying
a piperazine ring, demonstrated the highest affinity (3.3 nM/
1.3 nM). The proposed binding mode of 11 is depicted in Figure
3a and shows an optimal pattern of interactions between
the ligand and the active site of GSK-3ꢀ, accounting for the
excellent affinity of this compound. The 4-nitrogen of the
piperazine ring is protonated and donates a high-quality charge-
assisted hydrogen bond to the carboxylate oxygen of asp200.
The piperazine ring adopts the lowest energy chair conformation,
which permits an optimal approach and orientation between
donor and acceptor atoms (1.8 Å). In addition, the oxime oxygen
forms a hydrogen bond with water49 in a similar way to the
other compounds mode. However, the high ionizing potential
of 11 has a negative impact on its physicochemical profile,
which is reflected on the calculated logD value (-0.87). A
conversion of the secondary 4-nitrogen to tertiary was utilized
as a way to limit its basicity. This conversion was combined
Figure 3. Binding mode of analogues 11 (A) and 13 (B) to the ATP
binding pocket of GSK-3ꢀ. The piperazine substitution of both
analogues interacts with asp200 and residues located at the phosphate
subsite of the binding pocket in addition to the hydrogen bonds (yellow
dashed lines) formed between the indirubin scaffold and the kinase
backbone.
with a gradual extension of the molecule that assisted on
mapping the full extent by which the cavity can accommodate
bulky ligands. The piperazine ring was substituted by a methyl,
a hydroxyethyl, a methoxyethyl, and a hydroxyethoxyethyl
group in analogues 12, 13, 14, and 15, respectively (and
corresponding salts: 23, 24, 25, 26). Within this last group of
analogues, 12 and 13 share the best consensus pharmacological
and physicochemical profile. They demonstrate affinity similar
to that of 6BIO, improved selectivity of 1 order of magnitude
for GSK-3, increased water solubility, and acceptable lipophi-
licity. The proposed binding mode of 13 is depicted in Figure
3b.
Cytotoxicity of the 6BIO Analogues. All compounds were
next tested for their effects on survival of SH-SY5Y neuro-
blastoma cells using an MTS reduction assay. These assays
revealed that increased potency on GSK-3 was not associated
with enhanced cell death (Table 1). Analogues 13, 14, and 15
(and their corresponding salts, 24, 25, 26) had little cell death
inducing activities. The IC50 values were respectively 28 µM,
>100 µM, 94 µM (salts: 17 µM, >100 µM, 98 µM) to compare
with the IC50 of 6BIO, 9 µM. Therefore, the substituted
piperazine ring extension not only favors selectivity and efficacy
toward GSK-3, allows better solubility, but also reduces their
cytotoxicity. These features are particularly favorable for the
use of these compounds in the study of GSK-3 in cellular

6426 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Vougogiannopoulou et al.
Figure 4. ꢀ-Catenin phosphorylation at GSK-3 phosphorylation sites is inhibited by the indirubin derivatives. (A) SH-SY5Y neuroblastoma
cells were exposed for 6 h to 10 µM of each indirubin in the presence of a constant 2 µM level of the proteasome inhibitor MG132. The level
of GSK-3-phosphorylated ꢀ-catenin was estimated by Western blotting following SDS-PAGE, using an antibody that specifically cross-
reacts with ꢀ-catenin phosphorylated by GSK-3. Lack or reduction of the signal indicates that the indirubin has been able to inhibit GSK-3
within the neuroblastoma cells. C, Control (DMSO); 6B, 6BIO; M6B, methyl-6BIO (a control inactive analogue of 6BIO27); K, kenpaullone,
a structurally unrelated GSK-3 inhibitor.49 (B) Dose-response curves for a selection of indirubins were run in an ELISA assay using the
same antibodies directed against GSK-3 phosphorylated ꢀ-catenin. SH-SY5Y cells were exposed for 6 h to a range of concentrations of each
indirubin, in the presence of MG132, and extracts were assessed in the ELISA assay. Activity was expressed as percentage of phosphorylated
ꢀ-catenin in untreated control cells.
systems and also as potential therapeutic leads in the context
of neurodegenerative diseases and diabetes.
show a robust circadian rhythm of luminescence as a gauge of
clock-controlled Per2 promoter (PPer2). Cells were cultured and
treated first with 10 µM indirubin 15 as described in the
Experimental Section, and their circadian rhythm of Per
expression dependent luminescence was monitored during 4
days. A gradual shortening of the period length was clearly
observed (Figure 5a). Similar experiments were next performed
with a small selection of indirubins, and the period length was
calculated as in Figure 5a. The most efficient compounds in
shortening the period length (Figure 5b) were also the most
efficient at inhibiting ꢀ-catenin phosphorylation in the cellular
assay (Figure 4), supporting the hypothesis that the action of
the indirubins in shortening the circadian period is dependent
upon GSK-3. Previous studies have suggested a key action of
GSK-3 in regulating the circadian rhythm of mammalian cells
by using lithium as a pharmacological tool.45 Lithium lengthens
the period of the circadian rhythm, whereas indirubins shorten
the period (Figure 5). However, the concentrations of lithium
used in the previous investigations of circadian rhythms and
GSK-3 were 10-20 mM,46 whereas our results with indirubins
were obtained with concentrations 1000× lower (10 µM). These
comparisons suggest that the circadian period-lengthening effects
of lithium may be due to side effects of lithium. Therefore,
indirubins might constitute a useful pharmacological tool to
investigate the role of GSK-3 in the regulation of the circadian
rhythm.
Confirmation of Intracellular Inhibition of GSK-3 by
Indirubins. To investigate whether the new indirubins were
effective at inhibiting GSK-3 in a cellular context, we measured
their effects on the phosphorylation of ꢀ-catenin at GSK-3
specific sites in SH-SY5Y neuroblastoma cells. Cells were
exposed to various concentrations of 10 µM of each indirubin
in the presence of a constant level of MG132 (an inhibitor of
the proteasome, which prevented the rapid degradation of
ꢀ-catenin once phosphorylated by GSK-3). The level of GSK-
3-phosphorylated ꢀ-catenin was estimated either by Western
blotting (with an antibody that specifically cross-reacts with
ꢀ-catenin when phosphorylated at a GSK-3 site) following SDS-
PAGE (Figure 4a) or by an ELISA assay (Figure 4b). Results
revealed a dose-dependent inhibition of GSK-3 selective phos-
phorylation sites on ꢀ-catenin, demonstrating that these com-
pounds are actually able to inhibit GSK-3 in cells. The most
efficient compounds were 6BIO, 3, 5, 9, 11, 12, and 13 (and
their salts when available, i.e., 16, 20, 22, 23, 24). Dose-response
curves were obtained with ELISA assay (Figure 4b). The kinase
inactive derivative 1-methyl-6BIO was ineffective in the cellular
assay.
Effects on Circadian Rhythm in Mammalian Cell
Cultures. GSK-3 is a key regulator of the circadian rhythm
(aka the daily biological clock).44,45 The circadian rhythm can
be partially reproduced in a cellular system, which is an excellent
model system for circadian clocks in non-neural, peripheral
tissues.46 We therefore used this system to explore the possibility
that GSK-3 inhibition could affect the circadian rhythm. Rat-1
fibroblasts stably transfected with a PPer2::Fluc reporter construct
Conclusions
Through a rationale analysis of the key interactions of
indirubins at the ATP-binding site of the disease-relevant
glycogen synthase kinase -3 and the synthesis and biological

3′,6-Substituted Indirubins Alter Circadian Period
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6427
Figure 5. Indirubins alter the circadian period in mammalian fibroblasts. Rat-1 fibroblasts stably transfected with a PPer2::Fluc reporter construct
show a robust circadian rhythm of luminescence as a gauge of clock-controlled Per2 promoter (PPer2) activity. Cells were cultured up to 100%
confluence and treated for 2 h with 0.1 µM dexamethasone to synchronize the oscillators. The medium was then replaced with assay medium
supplemented with 0.1 mM luciferin, and the luminescence rhythm was monitored for 4 days or more. Compounds were added to the culture dishes
at 10 µM and left continuously. DMSO was used as a solvent control. Regression analyses were used to determine period and phase of the luminescence
rhythms. (A) Time-course of a typical luminescence rhythm recorded in control cells (O) or cells treated with indirubin 15 (b). White arrows
indicate the peaks of luminescence in control cells, black arrows indicate the peaks of luminescence in indirubin 15 treated cells. (B) Period lengths
calculated in cells exposed to various compounds. C, control; K, kenpaullone.
(1H, d, J ) 8.3 Hz, H-4), 8.21 (1H, d, J ) 7.8 Hz, H-4′), 7.45
(2H, brs, H-6′, H-7′), 7.16 (1H, dd, J ) 8.3/2.0 Hz, H-5), 7.07
(2H, m, H-5′, H-7), 4.93 (2H, t, J ) 5.6 Hz, H-1′′), 3.97 (2H, t, J
) 5.6 Hz, H-2′′). APCI-MS m/z 462, 464, 466 (M + H)+. Anal.
(C18H13N3O2Br2) C, H, N.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-[O-(2-hydroxyethyl)-
oxime] (2). Yield: 96%; mp >300 °C. 1H NMR (400 MHz, DMSO-
d6, δ ppm, J in Hz) 11.70 (1H, brs, H-1′), 10.90 (1H, brs, H-1),
8.54 (1H, d, J ) 8.4 Hz, H-4), 8.18 (1H, d, J ) 7.6 Hz, H-4′), 7.44
(2H, m, H-7′, H-6′), 7.16 (1H, dd, J ) 8.4/1.9 Hz, H-5), 7.05 (2H,
m, H-5′, H-7), 5.02 (1H, t, J ) 5.4 Hz, -OH), 4.62 (2H, t, J ) 4.8
Hz, H-1′′), 3.89 (2H, m, H-2′′). CI-MS m/z 400, 402 (M + H)+.
Anal. (C18H14N3O3Br) C, H, N.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-[O-(2,3-dihydroxypro-
pyl)-oxime] (3). Yield: 95%; mp >300 °C. 1H NMR(400 MHz,
DMSO-d6, δ ppm, J in Hz) 11.70 (1H, s, H-1′), 10.90 (1H, s, H-1),
8.56 (1H, d, J ) 8.5 Hz, H-4), 8.17 (1H, d, J ) 7.7 Hz, H-4′), 7.44
(2H, m, H-6′, H-7′), 7.15 (1H, dd, J ) 8.5, 1.9 Hz, H-5), 7.06 (1H,
m, H-5′), 7.03 (1H, d, J ) 1.9 Hz, H-7), 5.14 (1H, d, J ) 5.0 Hz,
-CHOH), 4.84 (1H, t, J ) 5.6 Hz, -CH2OH), 4.65 (1H, dd, J )
10.9, 3.7 Hz, H-1′′a), 4.50 (1H, dd, J ) 10.9, 6.6 Hz, H-1′′b), 3.99
(1H, m, H-2′′), 3.50 (2H, m, H-3′′). APCI-MS (+) m/z 430, 432
(M + H)+. Anal. (C19H16N3O4Br2) C, H, N.
evaluation of analogues exploring various modifications at the
3′ site, we were able to uncover new interaction sites, offering
further stabilization to the inhibitor/GSK-3 complexes. Conse-
quently, extensions at this site provide enhanced activity and
selectivity toward GSK-3 and also provided the opportunity to
introduce substitutions favoring enhanced water solubility. The
novel indirubin analogues were less cytotoxic than the parent
6BIO compound and demonstrated potent GSK-3 inhibition in
two cellular models. These results open new directions toward
the design of pharmacologically favorable indirubins with
potential development against Alzheimer’s disease, diabetes,
heart hypertrophy, or in the field of embryonic stem cell
pluripotency maintenance.
Experimental Section
Chemistry. General Chemistry Experimental Procedures. All
chemicals were purchased from Aldrich Chemical Co. NMR spectra
were recorded on Bruker DRX 400 and Bruker AC 200 spectrom-
eters (1H (400 and 200 MHz) and 13C (50 MHz)); chemical shifts
1
are expressed in ppm downfield from TMS. The H-1H and the
1H-13C NMR experiments were performed using standard Bruker
microprograms. Melting points were determined with a Sanyo
Gallencamp apparatus. MS spectra were determined on a MSQ
Thermofinnigan spectrometer. All UV/vis spectra were recorded
on a Shimadzu UV-160A spectrophotometer.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-[O-(N,N-diethylcarbamyl)-
oxime] (4). To a solution of 6-bromoindirubin-3′-oxime (36 mg,
0.11 mmol) in DMF (10 mL) were added triethylamine (0.5 mL)
and 0.3 mL (3.66 mmol) of N,N-diethylcarbamylchloride under Ar,
and the mixture was stirred for 12 h at room temperature. Then
water (40 mL) was added and the precipitate formed was collected
by filtration and washed with water to give 4 quantitatively. Yield:
Solubility Measurements. Equilibrium solubilities were deter-
mined by adding an excess amount of solid to the medium (water,
double distilled), followed by 5 min of sonification and overnight
equilibration by stirring at ambient temperature (25 ( 0.1 °C). The
samples were centrifuged, and aliquots were removed. Standard
solutions were prepared for each compound in order to quantify
the aforementioned saturated solutions, and reference curves were
plotted for each compound. The absorbance of each saturated and
standard solution was measured with a UV/vis spectrophotometer
at wavelength that varied between 515 and 518 nm.
General Procedure for the Preparation of the Ethers 1-3.
To a solution of 6-bromoindirubin-3′-oxime (1.0 g, 2.83 mmol) in
DMF (90 mL) were added triethylamine (0.5 mL) and the
appropriate bromide (2 equiv) under Ar, and the mixture was stirred
for 17 h at room temperature. Then water (300 mL) was added
and the precipitate formed was collected by filtration and washed
with water.
1
90%; mp 237 °C. H NMR (400 MHz, pyridine-d5, δ ppm, J in
Hz) 12.36 (1H, s, H-1′), 12.15 (1H, s, H-1), 9.93 (1H, d, J ) 8.6
Hz, H-4), 8.13 (1H, d, J ) 7.8 Hz, H-4′), 7.61 (1H, dd, J ) 8.6/
1.9 Hz, H-5), 7.43 (1H, m, H-6′), 7.35 (1H, d, J ) 1.9 Hz, H-7′),
7.14-7.06 (2H, m, H-5′, H-7), 3.44 (4H, brs, -N(CH2CH3)2), 1.18
(6H, t, J ) 7.0 Hz, -N(CH2CH3)2). APCI-MS (+) m/z 455, 457
(M + H)+. Anal. (C21H19N4O3Br) C, H, N.
General Procedure for the Preparation of the Amines 5-15.
First, 100 mg of 6-bromoindirubin-3′-[O-(2′′-bromoethyl)-oxime] (1)
was dissolved in 5 mL of anhydrous DMF. An excess of the
appropriate amine was added under magnetic stirring and the mixture
was then heated at 50 °C. After the completion of the reaction, the
mixture was poured into water (30 mL) and the precipitate was filtered
and washed with water and cyclohexane. Dimethylamine, diethylamine,
pyrrolidine, morpholine, diethanolamine, 3-methylamine-1,2-pro-
panediole, piperazine, 1-methylpiperazine, 1-(2-methoxyethyl) pip-
Data for (2′Z-3′E)-6-Bromoindirubin-3′-[O-(2-bromoethyl)-
oxime] (1). Yield: 95%; mp 252 °C. 1H NMR (400 MHz, DMSO-
d6, δ ppm, J in Hz) 11.67 (1H, s, H-1′), 10.93 (1 h, s, H-1), 8.49

6428 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Vougogiannopoulou et al.
erazine, 1-(2-hydroxyethyl) piperazine, and 1-[2-(2-hydroxyethoxy)-
ethyl] piperazine afforded products (5-15), correspondingly, in
qualitative yields.
m, H-5′), 7.02 (1H, d, J ) 1.9 Hz, H-7), 4.68 (2H, t, J ) 5.9 Hz,
H-1′′), 2.85 (2H, t, J ) 5.9 Hz, H-2′′), 2.50 (4H, brs, H-3′′, H-6′′,
overlapped with DMSO), 2.31 (4H, brs, H-4′′, H-5′′), 2.13 (3H, s,
-NCH3). APCI-MS (+) m/z 482, 484 (M + H)+. Anal.
(C23H24N5O2Br) C, H, N.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-[O-(2-dimethylamino-
1
ethyl)-oxime] (5). mp 230 °C. H NMR (400 MHz, DMSO-d6, δ
ppm, J in Hz) 11.71 (1H, brs, H-1′), 10.92 (1H, brs, H-1), 8.55
(1H, d, J ) 8.5 Hz, H-4), 8.14 (1H, d, J ) 7.5 Hz, H-4′), 7.44
(2H, d, J ) 4.1 Hz, H-6′, H-7′), 7.15 (1H, dd, J ) 8.5/2.0 Hz,
H-5), 7.05 (2H, m, H-5′, H-7), 4.69 (2H, t, J ) 5.8 Hz, H-1′′),
2.80 (2H, t, J ) 5.8 Hz, H-2′′), 2.27 (6H, s, -N(CH3)2). APCI-MS
m/z 427, 429 (M + H)+. Anal. (C20H19N4O2Br) C, H, N.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-(O-{2-[4-(2-hydroxy-
ethyl)piperazin-1-yl]ethyl}oxime) (13). mp 187 °C. 1H NMR (400
MHz, DMSO-d6, δ ppm, J in Hz) 8.52 (1H, d, J ) 8.5 Hz, H-4),
8.16 (1H, d, J ) 7.6 Hz, H-4′), 7.43 (2H, m, H-6′, H-7′), 7.13 (1H,
dd, J ) 8.5/1.8 Hz, H-5), 7.05 (1H, m, H-5′), 7.02 (1H, d, J ) 1.8
Hz, H-7), 4.69 (2H, t, J ) 5.7 Hz, H-1′′), 3.45 (2H, t, J ) 6.3 Hz,
H-8′′), 2.85 (2H, t, J ) 5.7 Hz, H-2′′), 2.50 (4H, H-3′′, H-6′′,
overlapped with DMSO), 2.42 (4H, H-4′′, H-5′′), 2.34 (2H, t, J )
6.3 Hz, H-7′′). APCI-MS (+) m/z 512, 514 (M + H)+. Anal.
(C24H26N5O3Br) C, H, N.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-(O-{2-[4-(2-methoxy-
ethyl)piperazin-1-yl]ethyl}oxime) (14). mp 184 °C.1H (400 MHz,
DMSO-d6, δ ppm, J in Hz) 11.70 (1H, s, H-1′), 10.90 (1H, s, H-1),
8.50 (1H, d, J ) 8.5 Hz, H-4), 8.16 (1H, d, J ) 7.6 Hz, H-4′), 7.44
(2H, m, H-6′, H-7′), 7.15 (1H, dd, J ) 8.5, 1.7 Hz, H-5), 7.07 (2H,
m, H-5′, H-7), 4.70 (2H, t, J ) 5.6 Hz, H-1′′), 3.40 (2H, H-8′′,
overlapped with water), 3.21 (3H, s, -OCH3), 2.87 (2H, brt, H-2′′),
2.66-2.40 (H-4′′, H-5′′, H-3′′, H-6′′, H-7′′, overlapped with
DMSO). APCI-MS (+) m/z 526, 528 (M + H)+. Anal.
(C25H28N5O3Br) C, H, N.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-[O-(2-{4-[2-(2-hydroxy-
ethoxy)-ethyl]piperazin-1-yl}ethyl)oxime] (15). mp 183 °C. 1H
NMR (400 MHz, pyridine-d5, δ ppm, J in Hz) 12.31 (1H, s, H-1′),
12.25 (1H, s, H-1), 8.89 (1H, d, J ) 8.3 Hz, H-4), 8.39 (1H, d, J
) 7.9 Hz, H-4′), 7.45-7.33 (3H, m, H-5, H-7′, H-6′), 7.09 (2H,
m, H-5′, H-7), 4.78 (2H, t, J ) 5.8 Hz, H-1′′), 3.96 (2H, t, J ) 5.0
Hz, H-10′′), 3.70 (2H, t, J ) 5.0 Hz, H-9′′), 3.66 (2H, t, J ) 5.8
Hz, H-8′′), 2.94 (2H, t, J ) 5.8 Hz, H-2′′), 2.68 (2H, brs, H-3′′,
H-6′′), 2.57 (8H, t, J ) 5.8 Hz, H-4′′, H-5′′, H-7′′). APCI-MS (+)
m/z 556, 558 (M + H)+. Anal. (C26H30N5O4Br) C, H, N.
General Procedure for the Preparation of the Amine Salts
16-26. The appropriate indirubin derivative 5-15 (0.10 mmol) was
dissolved in anhydrous THF (50 mL) and 0.2 mL of a saturated
solution of hydrochloric acid in ether was added dropwise. The
reaction mixture was left to cool in an ice bath and the precipitate
formed was collected by filtration.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-[O-(2-diethylaminoet-
hyl)-oxime] (6). mp 232 °C. 1H NMR (400 MHz, DMSO-d6, δ ppm,
J in Hz) 11.71 (1H, s, H-1′), 10.92 (1H, s, H-1), 8.55 (1H, d, J )
8.2 Hz, H-4), 8.16 (1H, d, J ) 7.8 Hz, H-4′), 7.44 (2H, d, J ) 3.4
Hz, H-6′, H-7′), 7.13 (1H, dd, J ) 8.2/2.1 Hz, H-5), 7.09-7.02
(2H, m, H-7, H-5′), 4.65 (2H, t, J ) 6.0 Hz, H-1′′), 2.94 (2H, t, J
) 6.0 Hz, H-2′′), 2.58 (2H, q, J ) 7.2 Hz, -N(CH2CH3)2), 0.98
(6H, t, J ) 7.2 Hz, -N(CH2CH3)2). APCI-MS (+) m/z 455, 457
(M + H)+. Anal. (C22H23N4O2Br) C, H, N.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-[O-(2-pyrrolidin-1-yl-
1
ethyl)oxime] (7). mp 208 °C. H NMR (400 MHz, DMSO-d6, δ
ppm, J in Hz) 11.70 (1H, s, H-1′), 10.93 (1H, s, H-1), 8.54 (1H, d,
J ) 8.5 Hz, H-4), 8.14 (1H, d, J ) 7.7 Hz, H-4′), 7.45 (2H, m,
H-6′, H-7′), 7.14 (1H, d, J ) 8.5/1.9 Hz, H-5), 7.05 (2H, m, H-5′,
H-7), 4.70 (2H, t, J ) 5.8 Hz, H-1′′), 2.98 (2H, brt, J ) 5.8 Hz,
H-2′′), 2.57 (4H, brs, H-3′′, H-6′′), 1.69 (4H, m, H-4′′, H-5′′). APCI-
MS (+) m/z 453, 455 (M + H)+. Anal. (C22H21N4O2Br) C, H, N.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-[O-(2-morpholin-1-yl-
1
ethyl)oxime] (8). mp 235 °C. H NMR (400 MHz, DMSO-d6, δ
ppm, J in Hz) 11.70 (1H, s, H-1′), 10.90 (1H, s, H-1), 8.52 (1H, d,
J ) 8.5 Hz, H-4), 8.15 (1H, d, J ) 7.6 Hz, H-4′), 7.43 (2H, m,
H-6′, H-7′), 7.14 (1H, dd, J ) 8.5/1.9 Hz, H-5), 7.05 (1H, m, H-5′),
7.02 (1H, d, J ) 1.9 Hz, H-7), 4.70 (2H, t, J ) 5.8 Hz, H-1′′),
3.57 (4H, t, J ) 4.5 Hz, H-4′′, H-5′′), 2.86 (2H, t, J ) 5.8 Hz,
H-2′′), 2.50 (4H, m, H-3′′, H-6′′, overlapped with DMSO). APCI-
MS (+) m/z 469, 471 (M + H)+. Anal. (C22H21N4O3Br) C, H, N.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-[O-(2-(N,N-(2-hydroxy-
ethyl)aminoethyl)oxime] (9). mp 201 °C. 1H NMR (400 MHz,
pyridine-d5, δ ppm, J in Hz) 12.31 (1H, brs, H-1′), 12.25 (1H, brs,
H-1), 8.93 (1H, d, J ) 8.2 Hz, H-4), 8.42 (1H, d, J ) 7.8 Hz,
H-4′), 7.47 (1H, dd, J ) 8.2, 1.8 Hz, H-5), 7.39 (1H, d, J ) 1.8
Hz, H-7), 7.34 (1H, t, J ) 7.2 Hz, H-6′), 7.04 (2H, m, H-5′, H-7′),
5.89 (1H, brs, -OH), 4.86 (2H, t, J ) 6.3 Hz, H-1′′), 4.00 (4H, m,
-N(CH2CH2OH)2), 3.38 (2H, t, J ) 6.3 Hz, H-2′′), 3.08 (4H, t, J
) 5.9 Hz, -N(CH2CH2OH)2). APCI-MS (+) m/z 487, 489 (M +
H)+. Anal. (C22H23N4O4Br) C, H, N.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-[O-(2-dimethylamino-
1
ethyl)oxime] Hydrochloride (16). Sw (g/L) 0.141. H NMR (400
MHz, DMSO-d6, δ ppm J in Hz) 11.70 (1H, s, H-1′), 10.97 (1H,
s, H-1), 8.49 (1H, d, J ) 8.3 Hz, H-4), 8.22 (1H, J ) 7.4 Hz,
H-4′), 7.46 (2H, m, H-7, H-6′), 7.20 (1H, dd, J ) 8.3/1.7 Hz, H-5),
7.05 (2H, m, H-5′, H-7′), 4.95 (2H, brs, H-1′′), 3.58 (2H, m, H-2′′),
2.81 (6H, brs, -N(CH3)2). Anal. (C20H20N4O2BrCl) C, H, N.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-(O-{2-[N-methyl,N-(2,3-
1
dihydroxypropyl)amino]ethyl}oxime] (10). mp 195 °C. H NMR
Data for (2′Z-3′E)-6-Bromoindirubin-3′-[O-(2-diethylaminoet-
hyl)oxime] Hydrochloride (17). Sw (g/L) 0.192. 1H NMR (400 MHz,
DMSO-d6, δ ppm, J in Hz) 11.70 (1H, s, H-1′), 10.98 (1H, s, H-1),
8.49 (1H, d, J ) 8.6 Hz, H-4), 8.21 (1H, d, J ) 7.4 Hz, H-4′), 7.47
(2H, m, H-7, H-6′), 7.21 (1H, dd, J ) 8.6/1.9 Hz, H-5), 7.09-7.04
(2H, m, H-5′, H-7′), 5.00 (2H, brs, H-1′′), 3.58 (2H, brs, H-2′′),
3.24 (4H, brs, -N(CH2CH3)2), 1.21 (6H, t, J ) 7.0 Hz,
-N(CH2CH3)2). Anal. (C22H24N4O2BrCl) C, H, N.
(400 MHz, pyridine-d5, δ ppm, J in Hz) 12.27 (2H, m, H-1, H-1′),
8.90 (1H, d, J ) 8.8 Hz, H-4), 8.41 (1H, d, J ) 7.5 Hz, H-4′), 7.46
(1H, dd, J ) 8.8,1.8 Hz, H-5), 7.38 (1H, d, J ) 1.8 Hz, H-7′), 7.36
(1H, t, J ) 7.5 Hz, H-6′), 7.05 (2H, m, H-5′, H-7), 4.80 (2H, t, J
) 6.1 Hz, H-1′′), 4.29 (1H, m, H-4′′), 4.11 (1H, dd, J ) 11.0, 4.6
Hz, H-5′′a), 4.04 (1H, dd, J ) 11.0, 5.5 Hz, H-5′′b), 3.14 (2H, t,
J ) 6.1 Hz, H-2′′), 2.93 (2H, m, H-3′′), 2.49 (3H, s, -NCH3).
CI-MS m/z 487, 489 (M + H)+. Anal. (C22H23N4O4Br) C, H, N.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-[O-(2-piperazine-1-yl-
ethyl)oxime] (11). mp 255 °C (dec). 1H NMR (400 MHz, DMSO-
d6, δ ppm, J in Hz) 11.69 (1H, s, H-1′), 10.92 (1H, s, H-1), 8.53
(1H, d, J ) 8.5 Hz, H-4), 8.15 (1H, d, J ) 7.4 Hz, H-4′), 7.43
(2H, m, H-6′, H-7′), 7.14 (1H, d, J ) 8.5 Hz, H-5), 7.03 (2H, m,
H-5′, H-7), 4.69 (2H, br t, H-1′′), 2.83 (2H, br t, H-2′′), 2.71 (4H,
brs, H-4′′, H-5′′), 2.46 (4H, brs, H-3′′, H-6′′, partially overlapped
with DMSO). APCI-MS (+) m/z 468, 470 (M + H)+. Anal.
(C22H22N5O2Br) C, H, N.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-[O-(2-pyrrolidin-1-yl-
1
ethyl)oxime] Hydrochloride (18). Sw (g/L) 0.195. H NMR (400
MHz, DMSO-d6, δ ppm, J in Hz) 11.71 (1H, s, H-1′), 10.97 (1H,
s, H-1), 8.48 (1H, d, J ) 8.6 Hz, H-4), 8.22 (1H, d, J ) 7.4 Hz,
H-4′), 7.44 - 7.52 (2H, m, H-7, H-6′), 7.20 (1H, dd, J ) 8.6/1.9
Hz, H-5), 7.07 (2H, m, H-5′, H-7′), 4.94 (2H, brs, H-1′′), 3.64 (2H,
brs, H-2′′), 3.13 (4H, m, H-3′′, H-6′′), 2.02 (4H, m, H-4′′, H-5′′).
Anal. (C22H22N4O2BrCl) C, H, N.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-[O-(2-morpholin-1-yl-
ethyl)oxime] Hydrochloride (19). 1H NMR(400 MHz, DMSO-d6,
δ ppm, J in Hz) 11.69 (1H, s, H-1′), 10.98 (1H, s, H-1), 8.47 (1H,
d, J ) 8.5 Hz, H-4), 8.22 (1H, d, J ) 7.8 Hz, H-4′), 7.45 (2H, m,
H-7, H-6′), 7.21 (1H, dd, J ) 8.3, 1.8 Hz, H-5), 7.06 (2H, m, H-5′,
H-7′), 5.05 (2H, brs, H-1′′), 3.95 (2H, m, H-2′′), 3.75 (4H, m, H-4′′,
Data for (2′Z-3′E)-6-Bromoindirubin-3′-{O-[2-(4-methyl-piper-
azin-1-yl)ethyl]oxime} (12). mp 222 °C. 1H NMR (400 MHz,
DMSO-d6, δ ppm, J in Hz) 11.68 (1H, s, H-1′), 10.90 (1H, s, H-1),
8.40 (1H, d, J ) 8.5 Hz, H-4), 8.14 (1H, d, J ) 7.7 Hz, H-4′), 7.42
(2H, m, H-6′, H-7′), 7.13 (1H, dd, J ) 8.5/1.9 Hz, H-5), 7.04 (1H,

3′,6-Substituted Indirubins Alter Circadian Period
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6429
H-5′′), 3.27 (4H, H-3′′, H-6′′, overlapped with water). Anal.
(C22H22N4O3BrCl) C, H, N.
was minimized in order to relieve the crystallographic strain in the
absence of the ligand and then all molecules were manually docked
in the active site by superimposition on crystallographic 6BIO
coordinates. Docking was performed with the Monte Carlo Multiple
Minimum search algorithm as implemented in Macromodel 9
software.43 The three key hydrogen bonds of each complex were
restrained at their crystallographic values by application of harmonic
distance and angle constraints. Force constraints were applied on
waters 49 and 79. The remaining atoms within 6 Å around the
ligand were free to move. All calculations were performed with a
truncated Newton conjugate gradient minimizer, a convergence
criterion of 0.05 kJ/Å-mol, the AMBER* forcefield, including
protein partial charges, and the GB/SA implicit solvent model.
Values of LogD and pKa were calculated by Pallas 3.1 software.
Biology. Kinase Preparation and Assays. Kinase activities were
assayed in buffer A (10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 25
mM Tris-HCl pH 7.5, 50 µg heparin/mL) or C (homogenization
buffer but 5 mM EGTA, no NaF and no protease inhibitors), at 30
°C, at a final ATP concentration of 15 µM. Blank values were
subtracted and activities were expressed in % of the maximal
activity, i.e., in the absence of inhibitors. Controls were performed
with appropriate dilutions of dimethylsulfoxide. Phosphorylation
of the substrate was assessed by the P81 phosphocellulose binding
assay.
CDK1/cyclin B was extracted in homogenization buffer (60 mM
ꢀ-glycerophosphate, 15 mM p-nitrophenylphosphate, 25 mM Mops
(pH 7.2), 15 mM EGTA, 15 mM MgCl2, 1 mM DTT, 1 mM sodium
vanadate, 1 mM NaF, 1 mM phenylphosphate, 10 µg leupeptin/
mL, 10 µg aprotinin/mL, 10 µg soybean trypsin inhibitor/mL, and
100 µM benzamidine) 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.25 The kinase activity was assayed
in buffer C, with 1 mg 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/liter of water. The wet filters were counted in the
presence of 1 mL of ACS (Amersham) scintillation fluid.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-[O-(2-(N,N-(2-hydroxy-
1
ethyl)aminoethyl)oxime] (20). H NMR (400 MHz, DMSO-d6, δ
ppm, J in Hz) 11.71 (1H, s, H-1′), 10.98 (1H, s, H-1), 8.48 (1H, d,
J ) 8.3 Hz, H-4), 8.22 (1H, d, J ) 7.9 Hz, H-4′), 7.46 (2H, m,
H-7, H-6′), 7.21 (1H, dd, J ) 8.3/1.8 Hz, H-5), 7.06 (2H, m, H-5′,
H-7′), 5.35 (2H, brs, OH), 5.03 (2H, brs, H-1′′), 3.84 (2H, brs,
H-2′′), 3.78 (4H, brs, -N(CH2CH2OH)2), 3.38 (4H, m,
-N(CH2CH2OH)2, overlapped with water). Anal. (C22H24N4O4BrCl)
C, H, N.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-(O-{2-[N-methyl,N-(2,3-
dihydroxypropyl)amino]ethyl}oxime] Hydrochloride (21). Sw (g/
1
L) 1.45. H NMR (400 MHz, DMSO-d6, δ ppm, J in Hz) 11.69
(1H, s, H-1′), 10.96 (1H, s, H-1), 8.48 (1H, d, J ) 8.5 Hz, H-4),
8.20 (1H, d, J ) 7.9 Hz, H-4′), 7.4 (2H, m, H-7, H-6′), 7.19 (1H,
dd, J ) 8.5/1.8 Hz, H-5), 7.05 (2H, m, H-5′, H-7′), 4.95 (2H, brs,
H-1′′), 3.89 (1H, brs, H-4′′), 3.38 (4H, H-3′′, H-5′′, overlapped with
water), 2.83 (2H, brs, H-3′′), 2.50 (3H, -N(CH3), overlapped with
DMSO). Anal. (C22H24N4O4BrCl) C, H, N.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-[O-(2-piperazine-1-yl-
1
ethyl)oxime] Dihydrochloride (22). Sw (g/L) 1.61. H NMR (400
MHz, D2O, δ ppm, J in Hz) 7.65 (1H, d, J ) 8.5 Hz, H-4), 7.55
(1H, d, J ) 7.5, H-4′), 7.26 (1H, t, J ) 7.5 Hz, H-6′), 6.85 (1H, t,
J ) 7.2 Hz, H-5′), 6.76 (1H, d, J ) 8.5 Hz, H-5), 6.72 (1H, d, J
) 7.5 Hz, H-7′), 6.54 (1H, s, H-7), 4.42 (2H, brt, H-1′′), 3.38 (4H,
brt, H-4′′, H-5′′), 3.11 (6H, brs, H-3′′, H-6′′, H-2′′). Anal.
(C22H24N5O2BrCl2) C, H, N.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-{O-[2-(4-methylpiper-
azin-1-yl)ethyl]oxime} Dihydrochloride (23). Sw (g/L) 1.50. 1H
NMR (400 MHz, D2O, δ ppm, J in Hz) 7.63 (1H, d, J ) 8.2 Hz,
H-4), 7.53 (1H, d, J ) 7.5, H-4′), 7.25 (1H, t, J ) 7.6 Hz, H-6′),
6.84 (1H, t, J ) 7.6 Hz, H-5′), 6.75 (1H, d, J ) 8.2 Hz, H-5), 6.70
(1H, d, J ) 7.5 Hz, H-7′), 6.53 (1H, s, H-7), 4.39 (2H, brs, H-1′′),
3.39 (4H, brs, H-3′′, H-6′′), 3.11 (6H, brs, H-2′′, H-4′′, H-5′′), 2.90
(3H, s, -NCH3). Anal. (C23H26N5O2BrCl2) C, H, N.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-(O-{2-[4-(2-hydroxy-
ethyl)piperazin-1-yl]ethyl}oxime) Dihydrochloride (24). Sw (g/L)
1.14. 1H NMR (400 MHz, D2O, δ ppm, J in Hz) 7.68 (1H, d, J )
8.5 Hz, H-4), 7.56 (1H, d, J ) 7.4, H-4′), 7.27 (1H, brt, J ) 7.2
Hz, H-6′), 6.85 (1H, brt, J ) 7.2 Hz, H-5′), 6.78 (1H, d, J ) 8.5
Hz, H-5), 6.73 (1H, d, J ) 7.4 Hz, H-7′), 6.57 (1H, s, H-7), 4.42
(2H, brs, H-1′′), 3.89 (2H, brs, H-8′′), 3.39 (4H, brs, H-3′′, H-6′′),
3.26 (2H, brs, H-7′′), 3.12 (6H, brs, H-2′′, H-4′′, H-5′′). Anal.
(C24H28N5O3BrCl2) C, H, N.
CDK5/p25 was reconstituted by mixing equal amounts of
recombinant human CDK5 and p25 expressed in E. coli as GST
(Glutathione-S-transferase) fusion proteins and purified by affinity
chromatography 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.
GSK-3R/ꢀ was purified from porcine brain by affinity chroma-
tography on immobilized axin.47 It was assayed, following a 1/100
dilution in 1 mg BSA/mL 10 mM DTT, with 4 µM GS-1
(YRRAAVPPSPSLSRHSSPHQSpEDEEE), a GSK-3 specific sub-
strate obtained from Millegen (Labege, France), 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.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-(O-{2-[4-(2-methoxy-
ethyl)piperazin-1-yl]ethyl}oxime) Dihydrochloride (25). Sw (g/L)
1
0.57. H NMR (400 MHz, D2O, δ ppm, J in Hz) 7.70 (1H, brs,
H-4), 7.61 (1H, brs, H-4′), 7.28 (1H, brt, J ) 7.2 Hz, H-6′), 6.88
(1H, brt, J ) 7.5 Hz, H-5′), 6.77 (2H, brs, H-5, H-7′), 6.58 (1H, s,
H-7), 4.52 (2H, brs, H-1′′), 3.74 (2H, brs, H-8′′), 3.55-3.22 (12H,
H-2′′, H-3′′, H-4′′, H-5′′, H-6′′, H-7′′), 3.35 (3H, s, -OCH3). Anal.
(C25H30N5O3BrCl2) C, H, N.
Data for (2′Z-3′E)-6-Bromoindirubin-3′-[O-(2-{4-[2-(2-hydroxy-
ethoxy)-ethyl]piperazin-1-yl}ethyl)oxime] Dihydrochloride (26). Sw
(g/L) 4.253. 1H NMR (400 MHz, D2O, δ ppm, J in Hz) 7.54 (1H,
d, J ) 8.1 Hz, H-4), 7.44 (1H, d, J ) 7.2 Hz, H-4′), 7.21 (1H, brt,
J ) 7.6 Hz, H-6′), 6.78 (1H, brt, J ) 7.2 Hz, H-5′), 6.69 (1H, d,
J ) 7.2 Hz, H-7′), 6.61 (1H, d, J ) 8.1 Hz, H-5), 6.34 (1H, s,
H-7), 4.28 (2H, brs, H-1′′), 3.82, 3.72, 3.63, (6H, H-8′′, H-9′′,
H-10′′), 3.46-2.72 (12H, H-2′′, H-3′′, H-4′′, H-5′′, H-6′′, H-7′′).
Anal. (C26H32N5O4BrCl2) C, H, N.
Molecular Modeling. All compounds were designed and mini-
mized. Compounds carrying nitrogen in their side chain were
designed in their free and protonated states as well. Ligand partial
charges were calculated in a semiempirical level with the AM1
Hamiltonian using MOPAC6.42 The receptor was prepared from
the PDB entry of 6BIO bound to GSK-3ꢀ (1UV5). Protons were
added, and all crystallographic water molecules were deleted except
waters 47 and 79, which mediate the interaction of the oxime with
thr138 and water 1, which is buried in the cavity.28 The kinase
Cellular Assays. Cell Culture Conditions and Cell Survival
Assessment. SH-SY5Y human neuroblastoma cell line (kindly
provided by Dr. Jacint Boix, Lleida) was grown at 37 °C with 5%
CO2 in DMEM supplemented with 2 mM L-glutamine (Invitrogen,
Cergy Pontoise, France), plus antibiotics (penicillin-streptomycin)
from Lonza, and a 10% volume of fetal calf serum (FCS)
(Invitrogen). Drug treatments were performed on exponentially
growing cultures at the indicated time and concentrations. Control
experiments were carried also using appropriate dilutions of DMSO.
Cell viability was determined by means of the MTS (3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe-
nyl)-2H -tetrazolium) method after 48 h of treatment as previously
described.48

6430 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Vougogiannopoulou et al.
ꢀ-Catenin Phosphorylation in SH-SY5Y Human Neuroblas-
toma Cells. Nearly confluent SH-SY5Y human neuroblastoma cells
were grown in 96 plates in DMEM (supplemented with 10% FCS
and antibiotics). Cells were cotreated with tested compounds and
2 µM MG132 (to allow accumulation of phosphorylated ꢀ-catenin)
for 6 h. Final DMSO concentration did not exceed 1%. Cells were
then subjected to an ELISA assay using antibodies directed against
Ser33/Ser37/Thr41-phosphorylated (1:1000) ꢀ-catenin obtained
from Cell Signaling Technology. Results are expressed in percent-
age of maximal ꢀ-Catenin phosphorylation, i.e., in untreated cells
exposed to MG132 only as positive control (100% phosphorylation).
References
(1) Doble, B. W.; Woodgett, J. R. GSK-3: tricks of the trade for a
multitasking kinase. J. Cell Sci. 2003, 116, 1175–1186.
(2) Jope, R. S.; Johnson, G. V. W. The glamour and gloom of glycogen
synthase kinase-3. Trends Biochem. Sci. 2004, 29, 95–102.
(3) Dajani, R.; et al. Crystal structure of glycogen synthase kinase 3ꢀ:
structural basis for phosphate-primed substrate specificity and auto-
inhibition. Cell 2001, 105, 721–732.
(4) ter Haar, E.; et al. Structure of GSK3ꢀ reveals a primed phosphory-
lation mechanism. Nat. Struct. Biol. 2001, 8, 593–596.
(5) Kockeritz, L.; Doble, B.; Patel, S.; Woodgett, J. R. Glycogen synthase
kinase-3: an overview of an overachieving protein kinase. Curr. Drug
Targets 2006, 7, 1377–88.
Cell Culture and Luminescence Assay of Circadian Rhythmic-
ity. These experiments used Rat-1 fibroblasts that have been stably
transfected with a PPer2::Fluc reporter construct that shows a robust
circadian rhythm of luminescence as a gauge of clock-controlled
Per2 promoter (PPer2) activity.46 The cells were cultured in DMEM
(11965-092, GIBCO/Invitrogen) supplemented with 5% FBS, 50
units/mL penicillin, and 50 µg/mL streptomycin in a 5% CO2
incubator at 37 °C. Approximately 5 × 105 cells were seeded in a
35 mm dish at least 5 days before the experiment. Three days after
the cells reached 100% confluence, the cells were treated with 0.1
µM dexamethasone (Sigma) for 1 h to synchronize the oscillators
among the cells in the population. At the end of the treatment, the
medium was replaced with assay medium (DMEM without phenol
red, supplemented with bicarbonate (350 mg/L), 5% FBS, 10 mM
HEPES (pH 7.2), antibiotics (25 units/mL penicillin, 25 µg/mL
streptomycin), and 0.1 mM luciferin (Promega)). Culture dishes
were sealed with a 40 mm microscope glass coverslip and high-
vacuum grease to prevent the evaporation of culture medium. The
luminescence rhythm was monitored in a LumiCycle (Actimetrics
Inc., Evanston, IL). Before being sealed, drugs were added to the
culture dishes to different final concentrations and left continuously
with the cells thereafter while the luminescence patterns were
recorded for 5 days or more. DMSO was used as a solvent control.
Regression analyses to determine period and phase of the lumi-
nescence rhythms were performed with the Chrono II program
(courtesy of Dr. Till Roenneberg).
Electrophoresis and Western blotting. Cells were resuspended,
lysed for 30 min at 4 °C in homogenization buffer, and sonicated.
After centrifugation (14000 rpm for 15 min at 4 °C), the protein
concentration was determined in the supernatants by the Bradford
protein assay (Bio-Rad). Following heat denaturation for 5 min,
proteins were separated by 10% NuPAGE precast Bis-Tris acetate
polyacrylamide mini gel (Invitrogen) with MOPS SDS running
buffer. Proteins were transferred to 0.45 µm nitrocellulose filters
(Schleicher and Schuell). These were blocked with 5% low fat milk
in Tris-buffered saline-Tween-20, incubated overnight at 4 °C with
antibodies directed against Ser33/Ser37/Thr41-phosphorylated ꢀ-cate-
nin (1:1000) (Cell Signaling Technology), and analyzed by
enhanced chemiluminescence (ECL, Amersham).
(6) Droucheau, E.; Primot, A.; Thomas, V.; Mattei, D.; Knockaert, M.;
Richardson, C.; Sallicandro, P.; Alano, P.; Jafarshad, A.; Baratte, B.;
Kunick, C.; Parzy, D.; Pearl, L.; Doerig, C.; Meijer, L. Plasmodium
falciparum glycogen synthase kinase-3, molecular model, expression,
intracellular localisation and selective inhibitors. Biochim. Biophys.
Acta: Proteins and Proteomics 2004, 1697, 181–196.
(7) Xingi, E.; Smirlis, D.; Bisti, S.; Myrianthopoulos, V.; Magiatis, P.;
Meijer, L.; Mikros, E.; Alexios-Skaltsounis, A. L.; Soteriadou, K. Role
of Leishmania donoVani glycogen synthase kinase-3ꢀ in parasite
cellular processes: a potential drug target. Mol. Pharmacol. 2008, in
press.
(8) Kassir, Y.; Rubin-Bejerano, I.; Mandel-Gutfreund, Y. The Saccharo-
myces cereVisiae GSK-3 beta homologs. Curr. Drug Targets 2006,
7, 1455–1465.
(9) Muyllaert, D.; Kremer, A.; Jaworski, T.; Borghgraef, P.; Devijver,
H.; Croes, S.; Dewachter, I.; Van Leuven, F. Glycogen synthase kinase-
3beta, or a link between amyloid and tau pathology. Genes Brain
BehaV. 2008, 7 (Suppl 1), 57–66.
(10) Hooper, C.; Killick, R.; Lovestone, S. The GSK3 hypothesis of
Alzheimer’s disease. J. Neurochem. 2008, 104, 1433–1439.
(11) Avila, J.; Herna´ndez, F. GSK-3 inhibitors for Alzheimer’s disease.
Expert ReV. Neurother. 2007, 7, 1527–1533.
(12) Lovestone, S.; Killick, R.; Di Forti, M.; Murray, R. Schizophrenia as
a GSK-3 dysregulation disorder. Trends Neurosci. 2007, 30, 142–
149.
(13) Rowe, M. K.; Wiest, C.; Chuang, D. M. GSK-3 is a viable potential
target for therapeutic intervention in bipolar disorder. Neurosci.
BiobehaV. ReV. 2007, 31, 920–931.
(14) Tanabe, K.; Liu, Z.; Patel, S.; Doble, B. W.; Li, L.; Cras-Me´neur, C.;
Martinez, S. C.; Welling, C. M.; White, M. F.; Bernal-Mizrachi, E.;
Woodgett, J. R.; Permutt, M. A. Genetic deficiency of glycogen
synthase kinase-3beta corrects diabetes in mouse models of insulin
resistance. PLoS Biol. 2008, 6, e37.
(15) Sugden, P. H.; Fuller, S. J.; Weiss, S. C.; Clerk, A. Glycogen synthase
kinase 3 (GSK3) in the heart: a point of integration in hypertrophic
signalling and a therapeutic target? A critical analysis. Br. J.
Pharmacol. 2008, 153 (Suppl 1), S137–S153.
(16) Kerkela¨, R.; Woulfe, K.; Force, T. Glycogen synthase kinase-3beta:
actively inhibiting hypertrophy. Trends CardioVasc. Med. 2007, 17,
91–96.
(17) Obligado, S. H.; Ibraghimov-Beskrovnaya, O.; Zuk, A.; Meijer, L.;
Nelson, P. J. CDK/GSK-3 inhibitors as therapeutic agents for
parenchymal renal diseases. Kidney Int. 2008, 73, 684–690.
(18) Dugo, L.; Collin, M.; Thiemermann, C. Glycogen synthase kinase 3beta
as a target for the therapy of shock and inflammation. Shock 2007,
27, 113–123, Erratum in: Shock 2007, 27, 709.
(19) Ougolkov, A. V.; Billadeau, D. D. Targeting GSK-3: a promising
approach for cancer therapy. Future Oncol. 2006, 2, 91–100.
(20) Sato, N.; Meijer, L.; Skaltsounis, L.; Greengard, P.; Brivanlou, A.
Maintenance of pluripotency in human and mouse embryonic stem
cells through activation of Wnt signaling by a pharmacological GSK-3
specific inhibitor. Nat. Med. 2004, 10, 55–63.
(21) Martinez, A. Preclinical efficacy on GSK-3 inhibitors: towards a future
generation of powerful drugs. Med. Res. ReV. 2008, 28, 773–796.
(22) Patel, D. S.; Dessalew, N.; Iqbal, P.; Bharatam, P. V. Structure-based
approaches in the design of GSK-3 selective inhibitors. Curr. Protein
Pept. Sci. 2007, 8, 352–364.
(23) Cohen, P.; Goedert, M. GSK3 inhibitors: development and therapeutic
potential. Nat. ReV. Drug DiscoVery 2004, 3, 479–487.
(24) Meijer, L.; Flajolet, M.; Greengard, P. Pharmacological inhibitors of
glycogen synthase kinase 3. Trends Pharmacol. Sci. 2004, 25, 471–
480.
(25) Leclerc, S.; Garnier, M.; Hoessel, R.; Marko, D.; Bibb, J. A.; Snyder,
G. L.; Greengard, P.; Biernat, J.; Mandelkow, E.-M.; Eisenbrand, G.;
Meijer, L. Indirubins inhibit glycogen synthase kinase-3ꢀ and CDK5/
p25, two kinases involved in abnormal tau phosphorylation in
Alzheimer’s disease: a property common to most CDK inhibitors.
J. Biol. Chem. 2001, 276, 251–260.
Acknowledgment. We are grateful to Dr. J. Boix for SH-
SY5Y cell line. This research was supported by grants from
the EEC (FP6-2002-Life Sciences & Health, PRO-KINASE
Research Project) (L.M.), and TEMPO (FP6 program, LSHG-
CT-2006-037543) (L.M.), PENED program (V.M.), the Can-
ceropole Grand-Ouest (L.M.), the “Association France-Alzhe-
imer” (Finiste`re), and the ACCAMBA project (CT500485)).
K.B. was supported by a fellowship from the “Ministe`re de la
Recherche”. Work in C. H. Johnson’s laboratory was funded
by a grant from the National Institutes of Health (grant number
R21 NS054991) and a Vanderbilt Institute for Chemical Biology
Pilot Project Grant.
Supporting Information Available: Elemental analysis. This
material is available free of charge via the Internet at http://
pubs.acs.org.

3′,6-Substituted Indirubins Alter Circadian Period
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6431
(26) Meijer, L., Guyard, N., Skaltsounis, L. A., Eisenbrand, G., Eds.
Indirubin: The Red Shade of Indigo; Life in Progress: Station
Biologique, Roscoff, France, 2006; 297 pp, 27 chapters.
(27) Meijer, L.; Skaltsounis, A. L.; Magiatis, P.; Polychonopoulos, P.;
Knockaert, M.; Leost, M.; Ryan, X. P.; Vonica, C. D.; Brivanlou, A.;
Dajani, R.; Tarricone, A.; Musacchio, A.; Roe, S. M.; Pearl, L.;
Greengard, P. GSK-3 selective inhibitors derived from Tyrian purple
indirubins. Chem. Biol. 2003, 10, 1255–1266.
(38) Ferandin, Y.; Bettayeb, K.; Kritsanida, M.; Lozach, O.; Polychro-
nopoulos, P.; Magiatis, P.; Skaltsounis, A. L.; Meijer, L. 3′-Substituted,
7-halogenoindirubins, a new class of cell death inducing agents. J. Med.
Chem. 2006, 49, 4638–4649.
(39) Beauchard, A.; Ferandin, Y.; Fre`re, S.; Lozach, O.; Blairvacq, M.;
Meijer, L.; Thie´ry, V.; Besson, T. Synthesis of novel 5- substituted
indirubins as potential inhibitor of protein kinases. Bioorg. Med. Chem.
2006, 14, 6434–6443.
(28) Polychronopoulos, P.; Magiatis, P.; Skaltsounis, L.; Myrianthopoulos,
V.; Mikros, E.; Tarricone, A.; Musacchio, A.; Roe, S. M.; Pearl, L.;
Leost, M.; Greengard, P.; Meijer, L. Structural basis for the synthesis
of indirubins as potent and selective inhibitors of glycogen synthase
kinase-3 and cyclin-dependent kinases. J. Med. Chem. 2004, 47, 935–
946.
(29) Ribas, J.; Bettayeb, K.; Ferandin, Y.; Garrofe´-Ochoa, X.; Knockaert,
M.; Totzke, F.; Scha¨chtele, C.; Mester, J.; Polychronopoulos, P.;
Magiatis, P.; Skaltsounis, A. L.; Boix, J.; Meijer, L. 7-Bromoindirubin-
3′-oxime induces caspase-independent cell death. Oncogene 2006, 25,
6304–6318.
(30) Jautelat, R.; Brumby, T.; Schafer, M.; Briem, H.; Eisenbrand, G.;
Schwahn, S.; Kruger, M.; Lucking, U.; Prien, O.; Siemeister, G. From
the insoluble dye indirubin towards highly active, soluble CDK2-
inhibitors. Chem. Biochem. 2005, 6, 531–540.
(31) Merz, K. H.; Schwahn, S.; Hippe, F.; Mu¨hlbeyer, S.; Jakobs, S.;
Eisenbrand, G. Novel indirubin derivatives, promising antitumor agents
inhibiting cyclin-dependent kinases. Int. J. Clin. Pharmacol. Ther.
2004, 42, 656–658.
(32) Hoessel, R.; Leclerc, S.; Endicott, J.; Noble, M.; Lawrie, A.; Tunnah,
P.; Leost, M.; Damiens, E.; Marie, D.; Marko, D.; Niederberger, E.;
Tang, W.; Eisenbrand, G.; Meijer, L. Indirubin, the active constituent
of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases.
Nat. Cell Biol. 1999, 1, 60–67.
(33) Davies, T. G.; Tunnah, P.; Meijer, L.; Marko, D.; Eisenbrand, G.;
Endicott, J. A.; Noble, M. E. M. Inhibitor binding to active and inactive
CDK2. The crystal structure of a CDK2-cyclin A/indirubin-5-
sulphonate. Structure 2001, 9, 389–397.
(34) Mapelli, M.; Massimiliano, L.; Crovace, C.; Seeliger, M.; Tsai, L. H.;
Meijer, L.; Musacchio, A. Mechanism of CDK5/p25 binding by CDK
inhibitors. J. Med. Chem. 2005, 48, 671–679.
(35) Holton, S.; Merckx, A.; Burgess, D.; Doerig, C.; Noble, M.; Endicott,
J. Structures of P. falciparum PfPK5 test the CDK regulation paradigm
and suggest mechanisms of small molecule inhibition. Structure 2003,
11, 1329–1337.
(40) Moon, M. J.; Lee, S. K.; Lee, J. W.; Song, W. K.; Kim, S. W.; Kim,
J. I.; Cho, C.; Choi, S. J.; Kim, Y. C. Synthesis and structure-activity
relationships of novel indirubin derivatives as potent antiproliferative
agents with CDK2 inhibitory activities. Bioorg. Med. Chem. 2006,
14, 237–246.
(41) Knockaert, M.; Blondel, M.; Bach, S.; Leost, M.; Elbi, C.; Hager, G.;
Naggy, S. R.; Han, D.; Denison, M.; French, M.; Ryan, X. P.; Magiatis,
P.; Polychronopoulos, P.; Greengard, P.; Skaltsounis, L.; Meijer, L.
Independent actions on cyclin-dependent kinases and aryl hydrocarbon
receptor mediate the antiproliferative effects of indirubins. Oncogene
2004, 23, 4400–4412.
(42) Stewart, J. J. P. MOPAC-A semiempirical molecular orbital program.
J. Comput.-Aided Mol. Des. 1990, 4, 1–105.
(43) Mohamadi, F.; Richards, N. G.; Guida, W. C.; Liskamp, R.; Lipton,
M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. Macromodel:
an intergrated software system for modeling organic and bioorganic
molecules using molecular mechanics. J. Comput. Chem. 1990, 11,
440–467.
(44) Martinek, S.; Inonog, S.; Manoukian, A. S.; Young, M. W. A role for
the segment polarity gene shaggy/GSK-3 in the Drosophila circadian
clock. Cell 2001, 105, 769–779.
(45) Iitaka, C.; Miyazaki, K.; Akaike, T.; Ishida, N. A role for glycogen
synthase kinase-3beta in the mammalian circadian clock. J. Biol. Chem.
2005, 280, 29397–29402.
(46) Izumo, M.; Sato, T. R.; Straume, M.; Johnson, C. H. Quantitative
analyses of circadian gene expression in mammalian cell cultures. PLoS
Comput. Biol. 2006, 2, e136.
(47) Primot, A.; Baratte, B.; Gompel, M.; Borgne, A.; Liabeuf, S.; Romette,
J. L.; Costantini, F.; Meijer, L. Purification of GSK-3 by affinity
chromatography on immobilised axin. Protein Expr. Purif. 2000, 20,
394–404.
(48) Ribas, J.; Boix, J. Cell differentiation, caspase inhibition, and
macromolecular synthesis blockage, but not BCL-2 or BCL-XL
proteins, protect SH-SY5Y cells from apoptosis triggered by two CDK
inhibitory drugs. Exp. Cell Res. 2004, 295, 9–24.
(49) Leost, M.; Schultz, C.; Link, A.; Wu, Y.-Z.; Biernat, J.; Mandelkow,
E.-M.; Bibb, J. A.; Snyder, G. L.; Greengard, P.; Zaharevitz, D. W.;
Gussio, R.; Senderovitz, A.; Sausville, E. A.; Kunick, C.; Meijer, L.
Paullones are potent inhibitors of glycogen synthase kinase-3ꢀ and
cyclin-dependent kinase 5/p25. Eur. J. Biochem. 2000, 267, 5983–5994.
(36) Bertrand, J. A.; Thieffine, S.; Vulpetti, A.; Cristiani, C.; Valsasina,
B.; Knapp, S.; Kalisz, H. M.; Flocco, M. Structural characterization
of the GSK-3ꢀ active site using selective and non-selective ATP-
mimetic inhibitors. J. Mol. Biol. 2003, 33, 393–407.
(37) Myrianthopoulos, V.; Magiatis, P.; Ferandin, Y.; Skaltsounis, A. L.;
Meijer, L.; Mikros, E. An integrated computational approach to the
phenomenon of potent and selective inhibition of Aurora kinases B
and C by a series of 7-substituted indirubins. J. Med. Chem. 2007,
50, 4027–4037.
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