From a natural product lead to the identification of potent and selective benzofuran-3-yl-(indol-3-yl)maleimides as glycogen synthase kinase 3βinhibitors that suppress proliferation and survival of pancreatic cancer cells

Article abstract of DOI:10.1021/jm801317h

Recent studies have demonstrated that glycogen synthase kinase 3β (GSK-3β) is overexpressed in human colon and pancreatic carcinomas, contributing to cancer cell proliferation and survival. Here, we report the design, synthesis, and biological evaluation of benzofuran-3-yl-(indol-3-yl) maleimides, potent GSK-3β inhibitors. Some of these compounds show picomolar inhibitory activity toward GSK-3β and an enhanced selectivity against cyclin-dependent kinase 2 (CDK-2). Selected GSK-3β inhibitors were tested in the pancreatic cancer cell lines MiaPaCa-2, BXPC-3, and HupT3. We determined that some of these compounds, namely compounds 5, 6, 11, 20, and 26, demonstrate antiproliferative activity against some or all of the pancreatic cancer cells at low micromolar to nanomolar concentrations. We found that the treatment of pancreatic cancer cells with GSK-3β inhibitors 5 and 26 resulted in suppression of GSK-3β activity and a distinct decrease of the X-linked inhibitor of apoptosis (XIAP) expression, leading to significant apoptosis. The present data suggest a possible role for GSK-3β inhibitors in cancer therapy, in addition to their more prominent applications in CNS disorders.

Full text of DOI:10.1021/jm801317h

J. Med. Chem. 2009, 52, 1853–1863
1853
From a Natural Product Lead to the Identification of Potent and Selective
Benzofuran-3-yl-(indol-3-yl)maleimides as Glycogen Synthase Kinase 3ꢀ Inhibitors That
Suppress Proliferation and Survival of Pancreatic Cancer Cells
Irina N. Gaisina,† Franck Gallier,† Andrei V. Ougolkov,‡ Ki H. Kim,† Toru Kurome,† Songpo Guo,† Denise Holzle,§
Doris N. Luchini,‡ Sylvie Y. Blond,§ Daniel D. Billadeau,*,‡ and Alan P. Kozikowski*,†
Drug DiscoVery Program, College of Pharmacy, Department of Medicinal Chemistry and Pharmacognosy, UniVersity of Illinois at Chicago,
833 South Wood Street, Chicago, Illinois 60612, Department of Medicinal Chemistry and Pharmacognosy and the Center for Pharmaceutical
Biotechnology, UniVersity of Illinois at Chicago, 900 South Ashland, Chicago, Illinois 60607, and College of Medicine, Department of
Immunology and DiVision of Oncology Research, Mayo Clinic, 13-42 Guggenheim, 200 First Street SW, Rochester, Minnesota 55905
ReceiVed October 17, 2008
Recent studies have demonstrated that glycogen synthase kinase 3ꢀ (GSK-3ꢀ) is overexpressed in human
colon and pancreatic carcinomas, contributing to cancer cell proliferation and survival. Here, we report the
design, synthesis, and biological evaluation of benzofuran-3-yl-(indol-3-yl)maleimides, potent GSK-3ꢀ
inhibitors. Some of these compounds show picomolar inhibitory activity toward GSK-3ꢀ and an enhanced
selectivity against cyclin-dependent kinase 2 (CDK-2). Selected GSK-3ꢀ inhibitors were tested in the
pancreatic cancer cell lines MiaPaCa-2, BXPC-3, and HupT3. We determined that some of these compounds,
namely compounds 5, 6, 11, 20, and 26, demonstrate antiproliferative activity against some or all of the
pancreatic cancer cells at low micromolar to nanomolar concentrations. We found that the treatment of
pancreatic cancer cells with GSK-3ꢀ inhibitors 5 and 26 resulted in suppression of GSK-3ꢀ activity and a
distinct decrease of the X-linked inhibitor of apoptosis (XIAP) expression, leading to significant apoptosis.
The present data suggest a possible role for GSK-3ꢀ inhibitors in cancer therapy, in addition to their more
prominent applications in CNS disorders.
Introduction
phosphorylated constitutively at Tyr-216; this phosphorylation
step appears to take place through autophosphorylation and plays
a role in stabilizing the enzyme. In addition, there are different
extracellular stimuli and proteins that can regulate this enzyme.
For example, GSK-3ꢀ is inhibited by the presence of secreted
glycoproteins, the so-called Wnts, that function in a pathway
that is crucial for the determination of the cell’s fate during
embryonic development.4 The Wnt signaling targets a particular
subcellular pool of GSK-3ꢀ, in which it is complexed to axin,
ꢀ-catenin, and the adenomatous polyposis coli (APC) proteins.
This protein complex is disrupted during Wnt signaling as a
result of the displacement of axin by other proteins, such as
disheveled and FRAT (frequently rearranged during advanced
T-cell lymphomas, also called GBP (GSK-3-binding protein))
proteins. The resulting dephosphorylation of ꢀ-catenin leads to
its accumulation and translocation to the nucleus, where it forms
a transcriptional complex with Tcf and Lef family proteins
subsequently stimulating activation of several oncogenes. In
earlier publications, it was indicated that GSK-3ꢀ plays the role
of tumor suppressor by down-regulating various proto-onco-
proteins.5-7 Consequently, GSK-3ꢀ inhibitors have been con-
sidered to possibly mimic the Wnt signaling pathway and to be
potentially oncogenic.3 However, in spite of these concerns, it
is well-known that long-term use of lithium, a nonspecific GSK-
3ꢀ inhibitor, for the treatment of bipolar disorder is not
associated with an increased risk of cancer.8 Moreover, lithium
actually increases the survival rates of patients with adenocar-
cinomas.9 The administration of the GSK-3ꢀ inhibitor 6-{2-[4-
(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-
2-ylamino]ethylamino}nicotinonitrile (CHIR 99021)10 in Zucker
diabetic fatty (ZDF) rats for up to 20 h was found not to cause
an observable increase in ꢀ-catenin or cyclin D1 mRNA in brain,
liver, lung, adipose tissue, or colon. Thus, it is possible that the
Glycogen synthase kinase 3 (GSK-3a) was identified in the
late 1970s and originally found to regulate glycogen metabo-
lism.1 Later, this enzyme has attracted immense interest due to
its diverse roles in cellular events. It is well established that
GSK-3 affects a variety of biological processes such as cell cycle
progression, proliferation, apoptosis, signaling, and transcription
by phosphorylation of many different substrates. In mammals,
GSK-3 consists of two distinct isoforms, R and ꢀ,2 which are
highly homologous within their ATP binding domain, whereas
they are significantly different in their terminal regions. This
serine/threonine kinase is constitutively active in resting cells
and physiologically inhibited by distinct signaling pathways at
multiple levels in stimulated cells. GSK-3ꢀ is negatively
regulated, for example, by post-translational phosphorylation
of Ser-9 located in the N-terminal domain.3 GSK-3ꢀ is also
* To whom correspondence should be addressed. A.P.K.: phone, +1 312
9967577; fax, +1 312 9967107; e-mail: kozikowa@uic.edu. D.D.B.: phone,
+1 507 2664334; fax, +1 507 2665146; e-mail, Billadeau.Daniel@
mayo.edu.
†
Drug Discovery Program, College of Pharmacy, Department of
Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago.
‡
Mayo Clinic.
§
Department of Medicinal Chemistry and Pharmacognosy and the Center
for Pharmaceutical Biotechnology, University of Illinois at Chicago, 900
South Ashland, Chicago, IL 60612.
a Abbreviations: APC, adenomatous polyposis coli; CDK-2, cyclin-
dependent kinase 2; CDKs, cyclin-dependent kinases; DMF-DMA, N,N-
dimethylformamide dimethyl acetal; DMF-DIPA, N,N-dimethylformamide
diisopropyl acetal; FRAT, frequently rearranged in advanced T-cell lym-
phoma; GBP, GSK-3-binding protein; GS, glycogen synthase; GSK-3,
glycogen synthase kinase 3; GSK-3ꢀ, glycogen synthase kinase 3 isoform
type ꢀ; NFκB, nuclear factor kappa B; pGS, phosphorylated glycogen
synthase; P-gp, permeability-glycoprotein; PKC, protein kinase C; TPSA,
total polar surface area; XIAP, X-linked inhibitor of apoptosis; ZDF rat,
Zucker diabetic fatty rat.
10.1021/jm801317h CCC: $40.75
2009 American Chemical Society
Published on Web 03/16/2009

1854 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Gaisina et al.
inhibition of GSK-3ꢀ by itself may be unable to elevate
ꢀ-catenin levels in primary cells; such elevations may require
that the cell lines have already undergone some prior transform-
ing events such as APC protein truncation.11 Recently, a line
of evidence led to an indication that GSK-3ꢀ is pathologically
active in different types of gastrointestinal cancer.12,13 It was
shown that this enzyme is overexpressed in colon and pancreatic
cancer cells where it has been implicated in NFκB (nuclear
factor kappa B) activation.14,15 In fact, GSK-3ꢀ is found to
accumulate in the nucleus of cancer cells and has recently been
shown to regulate chromatin structure and the binding of NFκB
to its target gene promoters in chronic lymphocytic leukemia
cells.16 Importantly, inhibition of GSK-3ꢀ activity in several
cancer cell types results in diminished NFκB transcriptional
activity of antiapoptotic genes, resulting in apoptosis.15,16 Thus,
GSK-3ꢀ has emerged as a promising target in development of
new drugs for the treatment of chronic and progressive
diseases.3,17 Over the past decades, a number of small molecule
GSK-3 inhibitors have been designed from natural products or
known kinase inhibitors17 for diabetes and neurodegenerative
disorders, but only a few of these were tested in cancer cell
lines. In the present work we have directed our attention to the
possibility to use inhibitors of GSK-3ꢀ in the treatment of
pancreatic cancer. Our work in this area was influenced by the
maleimide-bearing natural product staurosporine, first identified
as a protein kinase C (PKC) inhibitor, although it is now known
to be able to target other protein kinases, including GSK-3ꢀ.
From our original screening studies of staurosporine and its
analogs, we were led to the design of benzofuran-3-yl-(indol-
3-yl)maleimides as GSK-3ꢀ inhibitors.18 In continuation of this
work and to gain better insights into the SAR of this benzofuran
containing scaffold, a small library of substituted maleimides
has been generated. Using structure-based design methods, we
have optimized our lead compounds so as to arrive at subna-
nomolar potency GSK-3ꢀ inhibitors that are relatively selective
for GSK-3ꢀ versus homologous kinases.
and subsequent amidation of the intermediate ester provided
the acetamide 47. The alcohol functionality was protected as
its tert-butyldiphenylsilyl ether (compound 48) or alkylated with
different alkyl and aryl halides (compounds 49-54). Finally,
condensation with the appropriate indolylglyoxylic acid esters
followed by deprotection of the silyl ether if needed gave a
variety of 6-hydroxy substituted maleimides (4, 9, 10, 16-18)
as shown in Table 1.
For the introduction of hydroxymethyl and methoxymethyl
functionality at the 6-position of the benzofuran moiety, the
3-methoxybenzyl alcohol 55 was converted into the acetate 56
(Scheme 3). Chloroacetylation of 56 and subsequent cyclization
of the corresponding aryl ketone 57 yielded the 6-substituted
3-benzofuranone 58. The general procedure was followed to
obtain the 6-hydroxymethylbenzofuranyl-3-acetamide 59. The
acetamide 59 was converted in turn to the methyl ether 60 or
silyl ether 61 and then to the maleimides 2, 3, 7, 15, 23, 32,
and 34 (Table 1).
Next, our effort was directed to the introduction of a hydroxym-
ethyl, methoxymethyl, or carboxylic acid group at the 7-position
of the indole moiety (Scheme 4). Accordingly, the (1H-indol-7-
yl)methanol 62 was used to synthesize glyoxalates 63 and 64. The
general procedure to form the maleimide was followed to obtain
the desired compounds 31 and 33. The oxidation of the alcohol
33 led to the corresponding aldehyde 65, which was then subjected
to a Horner-Emmons reaction to give the ester 66 (Scheme 5).
Treatment with nickel boride, which was generated in situ, resulted
in the selective reduction of the double bond and formation of the
ester 35. The latter was converted into the acid 36 by the treatment
with sodium hydroxide.
The SAR work was further extended by exploring the effect
of polysubstitution of the aromatic rings. Disubstituted indoles
were prepared from the corresponding dihalogenated nitrotolu-
enes 67 and 68 by using a known protocol (Scheme 5).19 The
reaction of 67 and 68 with N,N-dimethylformamide dimethyl
acetal (DMF-DMA) afforded a mixture of the enamine 69 with
the methoxy-substituted side-product 70. In this step, nucleo-
philic aromatic substitution can be avoided by replacement of
DMF-DMA with N,N-dimethylformamide diisopropyl acetal
(DMF-DIPA).20 A reductive cyclization provided the indoles
71 and 72.21 A variety of acetamides prepared from com-
mercially available or made in-house benzofuranones were used
in a coupling reaction with oxalates 73-76 to prepare the
maleimides 5-11. To attach an aromatic ring to the indole, a
Suzuki coupling reaction of the appropriate precursor, such as
5-fluoro-6-iodoindole (5), with p-chlorophenyl boronic acid was
employed to afford the final maleimide 12. In addition, 5,7-
dibromo, 5,6-methylenedioxy, and 1H-benzo[g]indole were used
to synthesize compounds 20, 26, and 38, respectively.
Results and Discussion
Chemical Synthesis. The synthesis of the benzofuran-3-
yl(indol-3-yl)maleimides (1-38) (Table 1) is straightforward and
based on the condensation of the appropriately substituted 3-in-
dolylglyoxylic acid esters and benzofuranyl-3-acetamides. The
general method is shown in Scheme 1 and follows our previously
published work.18 Preparation of indolyl-based glyoxalates com-
mences with N-alkylation of the indole 39 followed by acylation
of the resulting indole 40 with ethyl oxalyl chloride to afford the
precursor 41. The required benzofuranyl-3-acetamides 44 were
prepared from the appropriately substituted 3-benzofuranones 42.
A Horner-Emmons reaction of 42 with (carbethoxymethylene)t-
riphenylphosphorane afforded ethyl (1-benzofuran-3-yl)acetates 43,
which were subsequently converted into the acetamides 44.
Condensation of the acetamides 44 with the glyoxilic esters resulted
in formation of the maleimide core.
As we have already synthesized a small set of compounds
substituted in the 5-position,18 it was important to explore the
extent to which the introduction of different functionalities,
including H-bond donor and acceptor groups to the 6- or
7-position of the indole or benzofuran ring, could be used to
adjust kinase selectivity and potency. Based upon the above
considerations, we investigated the effects of adding a hydroxy,
alkoxy, hydroxymethyl, methoxymethyl, and carboxylic acid
group. Accordingly, commercially available 6-hydroxybenzo-
furan-3-one 45 was protected as its silyl ether 46 (Scheme 2).
Treatment of 46 with (carbethoxymethylene)triphenylphosphorane
Evaluation of the Maleimides to in Vitro Inhibit GSK-
3ꢀ. The new benzofuran-3-yl-(indol-3-yl)maleimides were
screened for their potency to inhibit GSK-3ꢀ. Commercially
available human GSK-3ꢀ was assayed for its ability to phos-
phorylate the primed peptide substrate (RRRPAS-
VPPSPSLSRHSS(P)HQRR; 10 µM) in the presence of 0-30
µM of the maleimides. For comparison purposes, we also
determined the IC50 values of the known GSK-3ꢀ inhibitors 77
(AR-A014418)22 and 78 (SB-216763)23 in the same assay. As
presented in Table 1, the IC50 values vary from poor to excellent
(less than 1 nM).
Many GSK-3ꢀ inhibitors are also quite potent toward CDK-2
since the ATP binding pockets of these two kinases are very
similar.24 To measure the selectivity of new maleimides, several
compounds with different structural features were tested against

Benzofuran-3-yl-(indol-3-yl)maleimides as GSK-3ꢀ Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1855
Table 1. GSK-3ꢀ Inhibition by Substituted Benzofuran-3-yl-(indol-3-yl)maleimides and Compounds 77 and 78
a
compd
X
Y
IC50 (nM)
77
78
1b
2
3
4
5
6
7
8
41.8 ( 6.4
50.0 ( 0.2
35 ( 9
H
5-F
5-F
5-F
H
6-CH2OH
6-CH2OCH3
6-OH
7-OCH3
0.35 ( 0.06
23.8 ( 1.7
3.5 ( 1.3
247 ( 28
184 ( 32
0.95 ( 0.19
870 ( 160
1290 ( 360
4090 ( 820
260 ( 32
7160 ( 1190
7.0 ( 3.0
7.5 ( 1.1
0.51 ( 0.05
25.3 ( 2.4
48.3 ( 7.0
335 ( 37
42 ( 8
88.7 ( 5.6
34.5 ( 3.8
180 ( 11
13.2 ( 3.2
235 ( 15
16.1 ( 3.9
710 ( 160
440 ( 22
223 ( 22
15 ( 3
5-F, 6-I
5-F, 6-Cl
5-F, 6-Cl
5-F, 6-Cl
5-F, 6-Cl-
5-F, 6-Cl-
5-F, 6-Cl
5-F, 6-p-Cl-Ph
5-Br
5-Br
5-Br
5-Br
5-Br
H
6-CH2OH
6-OCH3
6-cyclopropylmethoxy
6-cyclobutylmethoxy
7-OCH3
7-OCH3
H
7-OCH3
6-CH2OH
6-prop-2-ynyloxy
6-allyloxy
6-O-(p-CH3O)-Bn
5-F
7-OCH3
H
5-F
6-CH2OH
H
5-F
5-F
H
H
H
7-OCH3
H
6-CH2OH
H
6-CH2OH
H
H
H
9
10
11
12
13b
14
15
16
17
18
19b
20
21
22
23
24b
25
26
27
28
29b
30
31
32
33
34
35
36
37b
38
5-Br
5-Cl
5,7-dibromo
5-I
5-I
5-CN
5-cyclopropyl
5-cyclopropylethynyl
5,6-methylenedioxy
5-OCH3, 6-Cl
5-OCH3, 6-I
6-OH
6-CF3
830 ( 77
5.4 ( 0.7
5.1 ( 1.2
0.23 ( 0.04
0.73 ( 0.10
10.2 ( 3.6
1.2 ( 0.2
55 ( 8
7-CH2OH
7-CH2OH
7-CH2OMe
7-CH2OMe
7-CH2CH2COOEt
7-CH2CH2COOH
7-OH
1H-benzo[g]
5,6-difluoro
314 ( 19
a The ability of commercially available GSK-3ꢀ (21 nM, EMD Biosciences, Madison, WI) to phosphorylate the pGS peptide substrate (RRRPAS-
VPPSPSLSRHSS(P)HQRR) (10 µM final concentration) was assayed in the presence of 10 µM of ATP (specific activity 1.3 µCi of [γ-32P]ATP/nmol).
b Published in ref 18.
Scheme 1a
a Reagents and conditions: (a) MeI, NaH, DMF; (b) EtO2CCOCl, Et2O; (c) Ph3PdCH2CO2Et, toluene, 110 °C; (d) NH3/MeOH; (e) t-BuOK, THF.
CDK-2/CyclinE. The compounds 2, 7, 15, and 33 were found
to be selective toward GSK-3ꢀ, based on the ratios of IC50 values
found for these two kinases (Table 2). From this set, compounds
7, 11, 15, and 33 were selected for more extensive evaluation
against a panel of CDKs, and these exhibited a significant level
of selectivity (Table 1 in the Supporting Information).

1856 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Gaisina et al.
Scheme 2a
a Reagents and conditions: (a) TBDPSCl, imidazole, DMF-CH2Cl2; (b) Ph3PdCH2CO2Et, toluene, 110 °C; (c) NH3/MeOH; (d) TBDPSCl, imidazole,
DMF-CH2Cl2; (e) CeCO3, Na2CO3, RHal, DMF, 55 °C.
Scheme 3a
a Reagents and conditions: (a) Ac2O, Et3N, DMAP, CH2Cl2; (b) ClCH2COCl, AlCl3, CH2Cl2, reflux; (c) NaOAc, MeOH; (d) Ph3PdCH2CO2Et, toluene,
110 °C; (e) NH3/MeOH; (f) NaH, MeI, THF, 20 °C; (g) TBDPSCl, imidazole, DMF-CH2Cl2.
Scheme 4a
a Reagents and conditions: (a) MeI, NaH, DMF; (b) EtO2CCOCl, Et2O; (c) TBDPSCl, imidazole, DMF-CH2Cl2; (d) t-BuOK, THF; (e) TBAF, THF; (f)
PDC, THF; (g) Ph3PdCH2CO2Et, THF, reflux; (h) NiCl2 ·6H2O, NaBH4, THF-MeOH; (i) 1 M NaOH, EtOH.
SAR Analysis and Molecular Modeling Studies. Our SAR
studies revealed that the introduction of hydrophilic substituents
at position 6 or 7 of the indole part of this scaffold is favorable.
Indeed, compounds 29 and 31-36 demonstrated very good
GSK-3ꢀ inhibitory activity in an in vitro assay. Compound 33,
bearing a methoxymethyl group, was found to be the best
inhibitor with an IC50 ) 0.23 ( 0.04 nM. Previously, we
discovered that the presence of a halogen in the 5-position of
the indole is favorable.18 This finding was confirmed by the
remarkably high activities of compounds 2-4 and 13-15. On
the other hand, the introduction of an additional halogen led to
a reduction in the inhibitory activity of the corresponding

Benzofuran-3-yl-(indol-3-yl)maleimides as GSK-3ꢀ Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1857
Scheme 5a
a Reagents and conditions: (a) DMF-DMA, 125 °C; (b) DMF-DIPA, 125 °C; (c) Fe, AcOH, EtOH; (d) Pd(PPh3)4, 4-chlorophenylboronic acid, K2CO3,
DME/H2O, 85 °C.
Table 2. CDK-2/CyclinE Inhibition by Substituted Maleimidesa
substitution is that the incorporation of side chains with terminal
double (compound 17, IC50 48.3 nM) or triple bonds (compound
16, IC50 25.3 nM) generated compounds with comparable
activities, while the p-methoxybenzyl derivative 18 turned out
to be ca. 10-fold less potent. Compound 34, with methoxymethyl
and hydroxymethyl groups attached to the indole and benzofuran
rings, respectively, that was synthesized to further improve the
potency of the maleimide-containing scaffold, showed an IC50
value at 0.73 ( 0.10 nM. It was even slightly less potent than
33 (IC50 ) 0.23 ( 0.04 nM).
In addition, the hydroxymethyl substituent at the 6-position
of the benzofuran moiety provided an enhanced selectivity
against CDK-2, as can be seen upon comparing the pairs of
compounds 15 and 14 or 7 and 11 (Table 2). By contrast, the
same group attached to the 7-position of the indolyl counterpart
caused a considerable drop in selectivity (compounds 31 and
32).
IC50 (nM)
CDK-2/cyclinE
IC50 ratio CDK-2/cyclinE
compd
GSK-3ꢀ
staurosporine
3.8
1410
237
0.8
40
1
2
7
670
12300
410
150
660
2.6
30
130
27
11700
99000
1160
337
1840
1070
32000
149
11
14
15
19
21
24
31
32
33
27
3880
5
32300
a These compounds were tested at Reaction Biology, Inc. (http://
www.reactionbiology.com).
compounds. Thus, almost all of the 5,6 and 5,7 disubstituted
indoles (compounds 6, 8-11, and 20) were found to be less
potent than their monosubstituted analogs. The same effect
occurred with 5-methoxy-substituted indolylmaleimides. The
introduction of either Cl or I in the 6-position resulted in a ca.
2 (compound 28) or 3.5 (compound 27) fold decrease in activity
relative to the case of the monosubstituted 5-methoxyindolyl-
maleimide (IC50 125 nM).18 It should be noted that an even
more significant drop in activity was observed for compound
12 (IC50 > 7 µM), which contains a larger p-chlorophenyl
substituent in the 6-position. The SAR of the benzofuran
component is as follows: the hydroxymethyl group attached to
position 6 dramatically improved the potency of this scaffold;
thus, the 6-hydroxymethylbenzofuranyl analogues 2, 7, and 15
were found to be the most potent among all 6-substituted
benzofuranylmaleimides. This improvement is especially re-
markable in the polysubstituted series, as can be seen by
comparing maleimides 6 and 7. Also, the maleimide 2 was 10-
fold more potent than its 6-hydroxysubstituted analogue 4. In
contrast, the potency was significantly lower for compounds
having methoxy (compound 8, IC50 870 nM), cyclopropyl-
methoxy (compound 9, IC50 > 1 µM), or cyclobutylmethoxy
(compound 10, IC50 > 4 µM) attachments. Another observation
related to the size and nature of the benzofuran counterpart
To ascertain whether molecular modeling could possibly
explain the observed SAR results, compounds 2 and 31 were
docked into the ATP binding site of GSK-3ꢀ (1R0E) (Figure
1).25 Their binding poses were governed largely by two adjacent
intermolecular hydrogen bonds: the NH group of the maleimide
ring forms a hydrogen bond with the backbone carbonyl oxygen
of Asp133, and one of the carbonyl oxygen atoms of the
maleimide forms a hydrogen bond with the backbone NH of
Val135. In addition to these main interactions, the 7-hydroxym-
ethyl group of compound 31 appears to be within hydrogen
bonding distance of the side chain of Arg141 (IC50 ) 5.4 nM),
whereas the 6-hydroxymethyl group of compound 2 is within
hydrogen bonding distance of the side chains of Gln185,
Asn186, and Asp200 (IC50 ) 0.35 nM). In the case of compound
33, having a 7-methoxymethyl group at the 7-position of the
indole ring, its IC50 of 0.23 nM was better than that found for
31. While both the hydroxymethyl and methoxymethyl groups
at the 7-position of the indole ring are able to H-bond with the
nearby Arg residue, it is only the methoxymethyl group (as in
33) that can help drive the inhibitor into its binding site, whereas
the hydroxymethyl group of 31 may be less favorably oriented,
as it points into the open area where it is accessible to solvent
molecules. The modeling studies will find more solid ground

1858 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Gaisina et al.
Figure 1. (A, B) Views of compound 31 docked into the GSK-3ꢀ binding site in which the backbone is shown as ribbons (A) and the protein
surface is colored by lipophilic potential (blue ) hydrophilic, brown ) lipophilic) (B). (C) Docked pose of compound 31 in the GSK-3ꢀ binding
site. The 7-hydroxymethyl group is within the hydrogen bonding distance of the Arg141 side chain. (D) Docked pose of compound 2 in the
GSK-3ꢀ binding site. The 6-hydroxymethyl group is within hydrogen bonding distance of the Gln185 and Asn186 side chains.
Table 3. Growth Inhibition of Pancreatic Cancer Cells by Selected
Maleimides
once we complete X-ray cocrystal structures of some of these
inhibitors bound to human GSK-3ꢀ.
GSK-3 Inhibitors Decrease Proliferation and Survival
IC50 (µM) ( SD
compd
MiaPaCa-2a
BXPC3a
HupT3a
of Pancreatic Cancer Cells. A select few of the GSK-3ꢀ
inhibitors that have different structural features and demonstrate
varying potencies in the in vitro assay were examined for their
capacity to decrease pancreatic cancer cell proliferation and
survival. It was shown earlier that treatment of BXPC3, HupT3,
and MiaPaCa-2 cells with the known GSK-3ꢀ inhibitors 77 and
78, which we used as reference compounds, led to a significant
decrease in pancreatic cancer cell proliferation.16 Some of our
new compounds, such as 5, 6, 11, 20, and 26, showed activity
similar to the reference compounds but exhibited better efficacy
(Table 3).
The two most potent compounds, 5 and 26, and the inactive
compound 27 (Table 3) were tested for their ability to inhibit
GSK-3ꢀ in cell lines. Using Western immunoblotting, we
estimated GSK-3ꢀ activity by monitoring the level of phos-
phorylation of glycogen synthase (GS), a substrate of GSK-3ꢀ.
The two most potent compounds, 5 and 26, effectively inhibited
GSK-3ꢀ activity, as shown by a decrease in GS phosphorylation
(Figure 2A) leading to decreased proliferation and survival of
BXPC3 and HupT3 pancreatic cancer cells (Table 3; Figure
2A, B, D). Compound 27, however, did not affect GSK-3ꢀ
activity, proliferation, or survival of pancreatic cancer cells
(Table 3; Figure 2B, C). Consistent with the idea that GSK-3ꢀ
positively regulates NFκB-mediated pancreatic cancer cell
survival,16 we found that treatment with the GSK-3ꢀ inhibitors
77
78
5
6
7
11
13
20
21
22
25
26
27
30
33
38
29.0 ( 1.7
25.0 ( 0.9
0.6 ( 0.1
3.0 ( 0.4
7 ( 1
3.0 ( 0.2
30.0 ( 2.2
2.0 ( 0.2
8.0 ( 0.6
28.0 ( 4.1
34.0 ( 2.2
5.0 ( 0.9
>50
14.0 ( 0.6
22.0 ( 2.1
NTb
NTb
0.9 ( 0.2
2.0 ( 0.2
5.0 ( 0.7
1.0 ( 0.2
NTb
0.9 ( 0.1
33 ( 4
42.0 ( 7.6
>50
1.0 ( 0.2
>50
0.6 ( 0.1
2.0 ( 0.3
2.1 ( 1.5
1.0 ( 0.1
43.0 ( 6.3
2.0 ( 0.1
>50
42.0 ( 8.3
>50
0.6 ( 0.1
>50
6.0 ( 0.5
27.0 ( 4.2
6.0 ( 0.7
8.0 ( 1.2
>50
17.0 ( 3.2
6.0 ( 0.8
>50
7.0 ( 2.1
a The pancreatic cancer cell lines MiaPaCa-2, BXPC3, and HupT3 were
obtained from ATCC (Rockville, MD). MTS assay was carried out according
to the manufacturer protocol. 72 h post-treatment, cell viability was measured
and compounds’ IC50 values were determined b Not tested.
5 and 26 resulted in a pronounced decrease in NFκB-mediated
expression of XIAP, the most potent antiapoptotic protein,
leading to subsequent apoptosis in pancreatic cancer cells (Figure
2A, B, D). Again, compound 27 had no such effect (Figure
2B, C) even at concentrations up to 50 µM (Table 3). As
expected, compound 5 showed significant anticancer activity
at concentrations of 1-5 µM (Table 3). In addition, compound

Benzofuran-3-yl-(indol-3-yl)maleimides as GSK-3ꢀ Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1859
intracellular concentrations for some compounds that are
lipophilic and should show excellent ability to cross biological
membrane. In summary, because the maleimides 13, 21, 22,
25, 27, and 33 (Table 3) showed little or no inhibitory activity
in the cell proliferation studies, it is possible that these
compounds are being recognized and ejected by energy-
dependent transporters or detoxified by a CYP-mediated mech-
anism in the pancreatic cancer cells, a point worthy of further
investigation.
Conclusion
A library of benzofuran-3-yl-(indol-3-yl)maleimides was
designed, synthesized, and tested for their selectivity and potency
as GSK-3ꢀ inhibitors relative to other homologous kinases.
Selected compounds were examined in turn for their ability to
suppress pancreatic cancer cell proliferation and survival in vitro.
We have been able to show that some of these compounds,
namely compounds 5, 6, 11, 20, and 26, demonstrate good
antiproliferative activity against a panel of pancreatic cancer
cell lines, which consisted of MiaPaCa-2, BXPC3, and HupT3.
Most notable was our finding that the treatment of BXPC3 with
inhibitors 5 and 26 resulted in a distinct decrease in NFκB-
mediated expression of XIAP, the most potent antiapoptotic
protein, leading to subsequent apoptosis in these pancreatic
cancer cells. Because of these valuable results, we plan to
prepare a wider variety of GSK-3ꢀ inhibitors implementing
modifications aimed at improving compound druggability
(aqueous solubility and metabolic stability) through both sub-
stitutent modifications and alteration in the scaffold in order to
arrive at possible clinical candidates for pancreatic cancer
therapy.
Experimental Section
Synthesis of Benzofuran-3-yl-(indol-3-yl)maleimides. 1H NMR
and 13C NMR spectra were recorded on a Bruker spectrometer at
400 and 100 MHz, respectively, with TMS as an internal standard.
Standard abbreviations indicating multiplicity were used as follows:
s ) singlet, bs ) broad singlet, d ) doublet, t ) triplet, q )
quadruplet, and m ) multiplet. HRMS experiments were performed
on LTO-FTICR or Shimadzu IT-TOF mass spectrometers. TLC
was performed with Merck 250-mm 60F254 silica gel plates.
Preparative TLC was performed with Analtech 1000-mm silica gel
GF plates. Column chromatography was performed using Merck
silica gel (40-60 mesh). Analytical HPLC was carried out on an
Ace 3AQ column (100 mm × 4.6 mm), with a Shimadzu 10 VP
Series HPLC with a diode array detector; flow rate ) 2.0 mL/min;
from 10% acetonitrile in water to 50% in 10 min and to 100%
acetonitrile in 5 min with 0.05% TFA (method A) or from 30%
acetonitrile in water to 100% of acetonitrile in 15 min with 0.05%
TFA (method B) or from 30% acetonitrile in water to 100% of
acetonitrile in 30 min with 0.05% TFA (method C); a column Ace
AQ5 (250 mm × 10 mm) was used with this last method. The
purity of key target compounds determined by HPLC (methods A,
B, or C) was higher than 95% (Table 3 in the Supporting
Information).
Figure 2. Treatment with GSK-3ꢀ inhibitors induces apoptosis in
pancreatic cancer cells. (A) BXPC3 pancreatic cancer cells were treated
with GSK-3ꢀ inhibitors 5 (5 µM) and 26 (5 µM) for 6, 12, 24, and
36 h, as indicated. Whole cell lysates were prepared, separated by SDS-
PAGE (50 µg/well), transferred to a PVDF membrane, and probed with
indicated antibodies. pGS, phosphorylated glycogen synthase; GS,
glycogen synthase. (B) BXPC3 and HupT3 pancreatic cancer cells were
treated with 5 (5 µM), 26 (5 µM), or 27 (20 µM) for 36 h. The
percentage of apoptotic cells was determined by Hoechst staining as
previously described.26 Columns, mean; bars, SD; control, DMSO; 5;
26; 27. (C) BXPC3 pancreatic cancer cells were treated with different
concentrations of 27 which included 5, 10, and 20 µM. At 36 h post-
treatment, whole cell lysates were prepared and analyzed as in part A.
(D) Representative Hoechst staining pictures of BXPC3 and HupT3
pancreatic cancer cells treated with compounds 5 (5 µM) and 26 (5
µM) for 36 h.
5 was evaluated against a panel of kinases and found to be
selective (Table 2 in the Supporting Information).
Our results demonstrated that although some of these
compounds are potent GSK-3ꢀ inhibitors, as shown by the in
vitro kinase assay, they did not inhibit GSK-3ꢀ activity in cell
culture and, therefore, showed no anticancer effect in pancreatic
cancer cells. The discrepancy regarding the effect in the in vitro
kinase assay compared to the cell culture experiments is not
surprising. Chemoresistance is intrinsic to cancer. The common
reason for resistance to a broad range of drugs is overexpression
of permeability-glycoprotein (P-gp) and other drug transporters
(ATP-binding cassette) that detect and eject anticancer drugs
from cancer cells. This could result in failure to achieve effective
Compounds 1, 13, 19, 24, 29, and 37 were described in our
previous publication.18
General Procedures for the Preparation of Maleimides. The
following methods represent the typical procedures for the synthesis
of the benzofuranylindolylmaleimide-based compounds.
Synthesis of the Indole Moiety. To a solution of substituted
indole (1 equiv) in dry DMF (2 mL/mmol) cooled with an ice bath
was added NaH (55% suspension in mineral oil, 1.5 equiv),
followed by MeI (1.2 equiv), after which the reaction mixture was
allowed to warm to room temperature. The reaction was monitored
by TLC. After completion, the reaction mixture was poured into
ice-water and the solution was extracted with EtOAc. The organic

1860 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Gaisina et al.
phase was washed with water and brine, dried over anhydrous
Na2SO4, and concentrated in vacuo. The crude was purified by
column chromatography (from hexane to EtOAc-hexane; 1:3) or
filtered through silica gel (EtOAc-hexane; 1:3) and subjected to
further reaction without additional purification. To a solution of
substituted 1-methyl-1H-indole in Et2O (5 mL/mmol) cooled to 0
°C was added ethyl oxalyl chloride (6 equiv) dropwise. The reaction
mixture was stirred for 0.5 h at 0 °C, allowed to warm to room
temperature, and stirred until completion. The reaction was
quenched by addition of water, and the reaction mixture was diluted
with EtOAc. The organic layer was washed with saturated NaHCO3,
water, and brine and was dried over anhydrous Na2SO4 and
concentrated. The residue was purified by column chromatography
(EtOAc-hexane; 1:3) to give the substituted (1-methyl-1H-indol-
3-yl)oxo-acetic acid ethyl ester.
Formation of the Benzofuran Counterpart. Benzofuran-3-
ylacetic Acid Ethyl Esters. To a solution of substituted or
unsubstituted benzofuran-3-one (1 equiv) in toluene (35 mL/mmol)
was added (carbethoxymethylene)triphenylphosphorane (4 equiv),
and the mixture was refluxed overnight or until completion. When
needed, an additional amount (1 or 2 equiv) of (carbethoxymeth-
ylene)triphenylphosphorane was added and the mixture was refluxed
until completion. The reaction mixture was cooled to room
temperature and concentrated. The residue was purified by column
chromatography (hexane to ethyl acetate-hexane; 1:3) to give the
product.
for C23H17FN2O4 [M + H]+: 405.1245; found: 405.1243, calcd for
C22H15FN2O4 [M + Na]+: 427.1065; found: 427.1061.
3-(5-Fluoro-1-methyl-1H-indol-3-yl)-4-(6-hydroxybenzofuran-
3-yl)pyrrole-2,5-dione (4). 1H NMR (DMSO-d6, 400 MHz) δ 3.88
(s, 3H), 6.39 (dd, J ) 2.0, 8.5 Hz, 1H), 6.54 (dd, J ) 2.2, 10.5 Hz,
1H), 6.61 (d, J ) 8.5 Hz, 1H), 6.92 (d, J ) 1.8 Hz, 1H), 6.95 (dt,
J ) 2.0, 8.5 Hz, 1H), 7.49 (dd, J ) 4.5, 9.0 Hz, 1H), 7.98 (s, 1H),
8.09 (s, 1H), 9.58 (s, 1H), 11.16 (s, 1H); FAB-HRMS calcd for
C21H13N2O4F [M - H]-: 375.0781; found: 375.0773. (Partially
degradated after few weeks at room temperature).
3-(5-Fluoro-6-iodo-1-methyl-1H-indol-3-yl)-4-(7-methoxyben-
zofuran-3-yl)pyrrole-2,5-dione (5). 1H NMR (CDCl3, 400 MHz,)
δ 3.83 (s, 3H), 4.02 (s, 3H), 6.42 (d, J ) 7.9 Hz, 1H), 6.73 (d, J
) 7.5 Hz, 1H), 6.76 (d, J ) 9.0 Hz, 1H), 6.83 (t, J ) 7.9 Hz, 1H),
7.67 (m, 2H), 7.73 (s, 1H), 8.16 (s, 1H); FAB-HRMS calcd for
C22H14N2O4FI [M + Na]+: 538.9875; found: 538.9866.
3-Benzofuran-3-yl-4-(6-chloro-5-fluoro-1-methyl-1H-indol-3-
1
yl)pyrrole-2,5-dione (6). H NMR (CDCl3, 400 MHz) δ 3.81 (s,
3H), 6.75-6.80 (m, 2H), 6.87 (t, J ) 4.7 Hz, 1H), 7.20 (t, J ) 4.5
Hz, 1H), 7.31 (m, 2H), 7.50 (d, J ) 5.0 Hz, 1H), 7.73 (s, 1H),
8.14 (s, 1H); FAB-HRMS calcd for C21H12N2O3ClF [M + H]+:
395.0599; found: 395.0617.
3-(6-Chloro-5-fluoro-1-methyl-1H-indol-3-yl)-4-(6-hydroxym-
ethylbenzofuran-3-yl)pyrrole-2,5-dione (7). 1H NMR (CDCl3, 400
MHz) δ 3.83 (s, 3H), 4.67 (s, 2H), 6.75 (d, J ) 10 Hz, 1H), 6.77
(d, J ) 8.0 Hz, 1H), 6.87 (d, J ) 8.0 Hz, 1H), 7.33 (d, J ) 6.0 Hz,
1H), 7.52 (s, 1H), 7.77 (s, 1H), 8.14 (s, 1H); FAB-HRMS calcd
for C22H14N2O4FCl [M + H]+: 425.0699; found: 425.0713.
3-(6-Chloro-5-methoxy-1-methyl-1H-indol-3-yl)-4-(6-meth-
oxybenzofuran-3-yl)pyrrole-2,5-dione (8). 1H NMR (CD3CN, 400
MHz) δ 4.33 (s, 3H), 4.39 (s, 3H), 7.07 (dd, J ) 1.2, 5.2 Hz, 1H),
7.21 (d, J ) 5.2 Hz, 1H), 7.34 (d, J ) 6.5 Hz, 1H), 7.67 (d, J )
1.2 Hz, 1H), 8.12 (d, J ) 3.7 Hz, 1H), 8.37 (s, 1H), 8.64 (s, 1H),
9.25 (s, 1H); FAB-HRMS calcd for C22H14N2O4ClF [M - H]-:
423.0553; found: 423.0559.
3-(6-Chloro-5-methoxy-1-methyl-1H-indol-3-yl)-4-(6-cyclo-
propylmethoxybenzofuran-3-yl)pyrrole-2,5-dione (9). 1H NMR
(CD3CN, 400 MHz) δ 0.32 (m, 2H), 0.60 (m, 2H), 1.21 (m, 1H),
3.80 (d, J ) 7.0 Hz, 2H), 3.84 (s, 3H), 6.48 (dd, J ) 2.2, 8.7 Hz,
1H), 6.63 (d, J ) 8.7 Hz, 1H), 6.70 (d, J ) 10.5 Hz, 1H), 7.07 (d,
J ) 2.0 Hz, 1H), 7.56 (d, J ) 6.3 Hz, 1H), 7.82 (s, 1H), 8.08 (s,
1H), 8.70 (s, 1H); FAB-HRMS calcd for C25H18N2O4ClF [M +
H]+: 465.1012; found: 465.1010.
2-Benzofuran-3-ylacetamides. Product obtained from the previ-
ous step was added to liquid ammonia at -78 °C, and the reaction
flask was sealed and heated at 50-60 °C until completion. The
reaction mixture was cooled to -78 °C and allowed to reach room
temperature slowly so that the excess of ammonia was evaporated,
and residue was washed with hexane to afford the substituted or
unsubstituted 2-benzofuran-3-ylacetamide, which was used without
further purification.
Benzofuran-3-yl-(indol-3-yl)maleimides. To a suspension of
substituted or unsubstituted 2-benzofuran-3-ylacetamide (1 equiv)
and substituted or unsubstituted indolyl-3-glyoxylate (1 equiv) in
dry THF (10 mL/mmol) at 0 °C, a 1.0 M solution of tert-BuOK in
THF (4 equiv) was added dropwise, and the reaction mixture was
stirred at room temperature for several hours until completion. The
reaction mixture was quenched with water and diluted with EtOAc.
The organic phase was washed with brine and then dried over
Na2SO4 and evaporated in vacuo. When deprotection of the silyl
ether was required, the residue was dissolved in THF (20 mL/mmol)
and a 1.0 M solution of TBAF in THF (1.5 equiv) was added at
room temperature. The mixture was stirred for 2 h. The reaction
mixture was quenched with NH4Cl and stirred for 5 min. The
solvent was removed, and the residue was dissolved in CH2Cl2 and
washed twice with water and brine. The organic phase was dried
over Na2SO4 and concentrated. The residue was purified by
preparative TLC (EtOAc-hexane, 1:1, or MeOH-CH2Cl2, 5:95)
or column chromatography (EtOAc-hexane, 1:9 to 1:1, or
MeOH-CH2Cl2, 1:99 to 1:9) to afford product. The yields were in
the range 20-72%.
3-(5-Fluoro-1-methyl-1H-indol-3-yl)-4-(6-hydroxymethylben-
zofuran-3-yl)pyrrole-2,5-dione (2). 1H NMR (DMSO-d6, 400
MHz) δ 3.88 (s, 3H), 4.52 (d, J ) 5.7 Hz, 2H), 5.24 (t, J ) 5.7
Hz, 1H), 6.53 (dd, J ) 2.3, 10.3 Hz, 1H), 6.84 (d, J ) 8.1 Hz,
1H), 6.89 (d, J ) 8.2 Hz, 1H), 6.95 (dt, J ) 2.4, 9.1 Hz, 1H), 7.49
(dd, J ) 4.5, 9.5 Hz, 1H), 7.54 (s, 1H), 8.02 (s,1H), 8.24 (s,1H),
11.19 (s,1H); 13C NMR (DMSO-d6, 100 MHz) 33.7, 63.1, 104.27,
104.31, 103.1, 106.3, 109.4, 110.5, 110.8, 111.7, 112.2, 112.3,
121.5, 121.9, 122.9, 124.5, 126.4, 126.5, 132.6, 133.9, 136.2, 140.6,
147.4, 154.9, 156.5, 172.2, 172.5; FAB-HRMS calcd for
C22H15FN2O4 [M + H]+: 391.1089; found: 391.1089, calcd for
C22H15FN2O4 [M + Na]+: 413.0908; found: 413.0907.
3-(6-Chloro-5-methoxy-1-methyl-1H-indol-3-yl)-4-(6-cyclobu-
tylmethoxybenzofuran-3-yl)pyrrole-2,5-dione (10). 1H NMR
(CD3CN, 400 MHz) δ 1.84-1.88 (m, 4H), 1.95-1.99 (m, 2H),
2.75 (m, 1H), 3.83 (s, 3H), 3.93 (d, J ) 7.1 Hz, 2H), 6.47 (dd, J
) 2.1, 8.8 Hz, 1H), 6.62 (d, J ) 8.8 Hz, 1H), 6.77 (d, J ) 10.6
Hz, 1H), 7.08 (d, J ) 2.1 Hz, 1H), 7.54 (d, J ) 7.3 Hz, 1H), 7.81
(s, 1H), 7.81 (s, 1H), 8.08 (s, 1H), 8.7 (s, 1H); FAB-HRMS calcd
for C26H20N2O4ClF [M - H]-: 477.1023; found: 477.1024.
3-(6-Chloro-5-fluoro-1-methyl-1H-indol-3-yl)-4-(7-methoxy-
1
benzofuran-3-yl)pyrrole-2,5-dione (11). H NMR (CDCl3, 400
MHz) δ 3.82 (s, 3H), 4.00 (s, 3H), 6.38 (d, J ) 7.7 Hz, 1H), 6.71
(d, J ) 7.5 Hz, 1H), 6.80 (m, 2H), 7.31 (d, 1H), 7.52 (s, 1H), 7.74
(s, 1H), 8.16 (s, 1H); FAB-HRMS calcd for C22H14N2O4ClF [M -
H]-: 423.0553; found: 423.0557.
3-[6-(4-Chlorophenyl)-5-fluoro-1-methyl-1H-indol-3-yl]-4-(7-
methoxybenzofuran-3-yl)pyrrole-2,5-dione (12). 3-(5-Fluoro-6-
iodo-1-methyl-1H-indol-3-yl)-4-(7-methoxybenzofuran-3-yl)pyrrole-
2,5-dione (5) (0.020 g, 0.039 mmol), Pd(PPh3)4 (4.5 mg, 0.004
mmol), and 4-chlorophenylboronic acid (15.1 mg, 0.097 mmol)
were dissolved in dimethoxyethane (DME) (4 mL), and the mixture
was degassed for 1 min and stirred for 10 min at room temperature.
A 2 M K2CO3 solution (2 M, 0.049 mL, 0.098 mmol) was added.
The mixture was degassed again for 1 min and stirred at 85 °C
overnight. The resulting mixture was cooled to ambient temperature
and poured into a mixture of 0.1 N HCl/EtOAc (15 mL/15 mL).
After partitioning, the organic layer was washed with water, filtered,
and concentrated. The residue was purified by preparative TLC
using (EtOAc-hexane, 1:1) to afford 12 as an orange solid (10
3-(5-Fluoro-1-methyl-1H-indol-3-yl)-4-(6-methoxymethylben-
zofuran-3-yl)pyrrole-2,5-dione (3). 1H NMR (CDCl3, 400 MHz)
δ 3.33 (s, 3H), 3.86 (s, 3H), 4.48 (s, 2H), 6.64 (dd, J ) 2.2, 9.8
Hz, 1H), 6.81-6.90 (m, 3H), 7.21 (dd, J ) 4.5, 8.9 Hz, 1H), 7.39
(brs, 1H), 7.49 (s,1H), 7.79 (s,1H), 8.12 (s,1H); FAB-HRMS calcd

Benzofuran-3-yl-(indol-3-yl)maleimides as GSK-3ꢀ Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1861
mg, 51%). The sample was further purified by HPLC for biological
testing. H NMR (CDCl3, 400 MHz,) δ 3.89 (s, 3 H), 4.02 (s, 3
H), 6.53 (d, 1 H, J ) 7.8 Hz), 6.74 (d, 1 H, J ) 7.8 Hz), 6.90-6.82
(m, 2 H), 7.26 (m, 1 H), 7.48-7.39 (m, 4 H), 7.53 (s, 1 H), 7.79
(s, 1 H), 8.15 (s, 1H); FAB-HRMS calcd for C28H18N2O4FCl [M
+ Na]+: 523.0832; found: 523.0824.
3-(5-Cyclopropylethynyl-1-methyl-1H-indol-3-yl)-4-(5-fluo-
robenzofuran-3-yl)pyrrole-2,5-dione (25). 1H NMR (CDCl3, 400
MHz) δ 0.67 (m, 2H), 0.82 (m, 2H), 1.34 (m, 1H), 3.87 (s, 3H),
6.59 (d, J ) 8.7 Hz, 1H), 6.97-6.93 (m, 2H), 7.17 (d, J ) 8.3 Hz,
1H), 7.22 (d, J ) 8.4 Hz, 1H), 7.46-7.43 (m, 1H), 7.53 (br s, 1H),
7.81 (s, 1H), 8.10 (s, 1H); FAB-HRMS calcd for C26H16N2O3F [M
- H]-: 423.1150; found: 423.1147.
3-(5-Fluorobenzofuran-3-yl)-4-(5-methyl-5H-[1,3]dioxolo[4,5-
f]indol-7-yl)pyrrole-2,5-dione (26). 1H NMR (CDCl3, 400 MHz)
δ 3.79 (s, 3H), 5.80 (m, 2H), 6.29 (s, 1H), 6.59 (dd, J ) 2.4, 9.0
Hz, 1H), 6.74 (s, 1H), 6.91 (td, J ) 2.5, 9.0 Hz, 1H), 7.41 (dd, J
) 4.1, 8.0 Hz, 1H), 7.65 (s, 1H), 7.95 (s, 1H), 8.13 (s, 1H); FAB-
HRMS calcd for C22H13N2O5F [M + H]+: 405.0881; found:
405.0880.
1
3-(5-Bromo-1-methyl-1H-indol-3-yl)-4-(7-methoxybenzofuran-
1
3-yl)pyrrole-2,5-dione (14). H NMR (CDCl3, 400 MHz) δ 3.82
(s, 1H), 4.00 (s, 3H), 6.37 (d, J ) 7.8 Hz, 1H), 6.71 (d, J ) 7.8
Hz, 1H), 6.78 (t, J ) 7.8 Hz, 1H), 7.14-7.23 (m, 3H), 7.37 (bs,
1H), 7.70 (3, 1H), 8.14 (s, 1H); FAB-HRMS calcd for
C22H15N2O4Br [M + H]+: 451.0293; found: 451.0279.
3-(5-Bromo-1-methyl-1H-indol-3-yl)-4-(6-hydroxymethylben-
1
zofuran-3-yl)pyrrole-2,5-dione (15). H NMR (DMSO-d6, 400
3-Benzofuran-3-yl-4-(6-chloro-5-methoxy-1-methyl-1H-indol-
3-yl)pyrrole-2,5-dione (27). 1H NMR (DMSO-d6, 400 MHz) δ 3.11
(s, 3H), 3.83 (s, 3H), 6.30 (s, 1H), 6.93 (t, J ) 4.7 Hz, 1H), 7.04
(d, J ) 4.7 Hz, 1H), 7.22 (t, J ) 5.2 Hz, 1H), 7.30 (d, J ) 1.7 Hz,
1H), 7.47 (d, J ) 5.2 Hz, 1H), 7.86 (s, 1H), 8.01 (s, 1H); FAB-
HRMS calcd for C22H15N2O4Cl [M + H]+: 407.0793; found:
407.0793.
3-Benzofuran-3-yl-4-(6-iodo-5-methoxy-1-methyl-1H-indol-3-
yl)pyrrole-2,5-dione (28). 1H NMR (CDCl3, 400 MHz) δ 3.09 (s,
3H), 3.83 (s, 3H), 6.29 (s, 1H), 6.96 (t, J ) 4.7 Hz, 1H), 7.08 (d,
J ) 4.7 Hz, 1H), 7.21 (m, 2H), 7.47 (m, 2H), 7.69 (s, 1H), 7.86 (s,
1H), 8.00 (s, 1H); FAB-HRMS calcd for C22H15N2O4I [M + H]+:
441.0149; found: 441.0149.
3-(7-Methoxybenzofuran-3-yl)-4-(1-methyl-6-trifluoromethyl-
1H-indol-3-yl)pyrrole-2,5-dione (30). 1H NMR (CDCl3, 400 MHz)
δ 3.94 (s, 3H), 4.02 (s, 3H), 6.43 (d, J ) 7.8 Hz, 1H), 6.71 (d, J
) 8.0 Hz, 1H), 6.81 (t, J ) 8.0 Hz, 1H), 7.12 (m, 2H), 7.62 (s,
1H), 7.70 (s, 1H), 7.88 (s, 1H), 8.14 (s, 1H); FAB-HRMS calcd
for C23H15N2O4F3 [M + H]+: 441.1062; found: 441.1057.
3-Benzofuran-3-yl-4-(7-hydroxymethyl-1-methyl-1H-indol-3-
yl)pyrrole-2,5-dione (31). 1H NMR (CDCl3, 400 MHz) δ 1.77 (t,
J ) 6.0 Hz, 1H), 4.22 (s, 3H), 4.98 (d, J ) 6.0 Hz, 2H), 6.72 (t,
J ) 7.6 Hz, 1H), 6.86-6.95 (m, 2H), 6.98 (t, J ) 7.2 Hz, 2H),
7.19 (dt, J ) 1.4, 7.5 Hz, 1H), 7.47 (t, J ) 8.3 Hz, 1H), 7.56 (br
s, 1H), 7.72 (s, 1H), 8.09 (s, 1H); FAB-HRMS calcd for C22H16N2O4
[M - H]-: 371.1037; found: 371.1038.
3-(6-Hydroxymethylbenzofuran-3-yl)-4-(7-hydroxymethyl-1-
methyl-1H-indol-3-yl)pyrrole-2,5-dione (32). 1H NMR (DMSO-
d6, 400 MHz) δ 4.18 (s, 3H), 4.52 (d, J ) 5.6 Hz, 2H), 4.84 (d, J
) 5.6 Hz, 2H), 5.25 (t, J ) 6.0 Hz, 1H), 5.36 (t, J ) 6.0 Hz, 1H),
6.66 (t, J ) 7.2 Hz, 1H), 6.80 (d, J ) 8.0 Hz, 1H), 6.90 (s, 2H),
6.97 (d, J ) 6.8 Hz, 1H), 7.51 (s, 1H), 7.89 (s, 1H), 8.18 (s, 1H),
11.18 (s, 1H); FAB-HRMS calcd for C23H18N2O5 [M + H]+:
403.1289; found: 403.1287; calcd for C23H18N2O5 [M + Na]+:
425.1108; found: 425.1107.
MHz) δ 3.86 (s, 3H), 4.52 (d, J ) 5.6 Hz, 2H), 5.26 (t, J ) 6.0
Hz, 1H), 6.79 (d, J ) 8.0 Hz, 1H), 6.89 (d, J ) 8.0 Hz, 1H), 7.1
(d, J ) 2.0 Hz, 1H), 7.21 (dd, J ) 1.6, 8.8 Hz, 1H), 7.45 (d, J )
8.8 Hz, 1H), 7.55 (s, 1H), 7.96 (s, 1H), 8.26 (s, 1H), 11.20 (s, 1H);
FAB-HRMS calcd for C25H18BrN2O4 [M + H]+: 451.0288; found:
451.0288.
3-(5-Bromo-1-methyl-1H-indol-3-yl)-4-(6-prop-2-ynyloxyben-
zofuran-3-yl)pyrrole-2,5-dione (16). 1H NMR (CDCl3, 400 MHz)
δ 3.83 (s, 3H), 4.67 (s, 1H), 5.42 (d, J ) 3.9 Hz, 1H), 6.53-6.69
(m, 3H), 6.81 (t, J ) 4.5 Hz, 1H), 7.22-7.12 (m, 6H), 7.71 (s,
1H), 8.08 (s, 1H); FAB-HRMS calcd for C24H15BrN2O4 [M + H]+:
475.0288; found: 475.0297; calcd for C24H15BrN2O4 [M + Na]+:
497.0108; found: 497.0119. (Partially degraded after a few weeks
at room temperature).
3-(6-Allyloxybenzofuran-3-yl)-4-(5-bromo-1-methyl-1H-indol-
3-yl)pyrrole-2,5-dione (17). 1H NMR (DMSO-d6, 400 MHz) δ 3.84
(s, 3H), 4.99 (s, 2H), 6.59 (dd, J ) 1.8, 8.7 Hz, 1H), 6.69 (d, J )
8.7 Hz, 1H), 6.92 (d, J ) 1.8 Hz, 1H), 7.20 (dd, J ) 1.5, 8.5 Hz,
1H), 7.31-7.34 (m, 2H), 7.44 (d, J ) 8.5 Hz, 1H), 7.94 (s, 1H),
8.17 (s, 1H), 11.20 (s, 1H); FAB-HRMS calcd for C24H17N2O4Br
[M + H]+: 477.0445; found: 477.0445.
3-(5-Bromo-1-methyl-1H-indol-3-yl)-4-[6-(4-methoxybenzy-
loxy)benzofuran-3-yl]pyrrole-2,5-dione (18). 1H NMR (DMSO-
d6, 400 MHz) δ 3.75 (s, 3H), 3.84 (s, 3H), 4.99 (s, 2H), 6.61 (dd
J ) 2.0, 8.7 Hz, 1H), 6.70 (d, J ) 8.7 Hz, 1H), 6.91 (d, J ) 8.5
Hz, 1H), 7.04 (d, J ) 1.5 Hz, 1H), 7.04 (dd, J ) 1.5, 8.6 Hz, 1H),
7.31-7.36 (m, 3H), 7.45 (d, J ) 8.6 Hz, 1H), 7.94 (1s, 1H), 8.17
(s, 1H); FAB-HRMS calcd for C29H21N2O5Br [M - H]-: 579.0526;
found: 579.0521.
3-(5,7-Dibromo-1-methyl-1H-indol-3-yl)-4-(7-methoxybenzo-
1
furan-3-yl)pyrrole-2,5-dione (20). H NMR (CDCl3, 400 MHz)
δ 4.00 (s, 3H), 4.17 (s, 3H), 6.30 (d, J ) 7.8 Hz, 1H), 6.73 (d, J
) 7.8 Hz, 1H), 6.82 (t, J ) 7.8 Hz, 1H), 7.12 (d, J ) 1.7 Hz, 1H),
7.37 (d, J ) 1.7 Hz, 1H), 7.55 (s, 1H), 7.61 (s, 1H), 8.93 (s, 1H);
FAB-HRMS calcd for C22H14N2O4Br2 [M - H]-: 526.9248; found:
526.9247.
3-Benzofuran-3-yl-4-(7-methoxymethyl-1-methyl-1H-indol-3-
yl)pyrrole-2,5-dione (33). 1H NMR (DMSO-d6, 400 MHz) δ 3.26
(s, 3H), 4.10 (s, 3H), 4.73 (s, 2H), 6.63 (t, J ) 7.6 Hz, 1H), 6.83
(d, J ) 7.6 Hz, 1H), 6.86-6.93 (m, 2H), 6.96 (d, J ) 7.2 Hz, 1H),
7.20 (dt, J ) 1.4, 7.2 Hz, 1H), 7.57 (d, J ) 8.4 Hz, 1H), 7.88 (s,
1H), 8.25 (s, 1H), 11.19 (br s, 1H); FAB-HRMS calcd for
C23H18N2O4 [M + H]+: 387.1339; found: 387.1342; calcd for
C23H18N2O4 [M + Na]+: 409.1159; found: 409.1161.
3-Benzofuran-3-yl-4-(5-iodo-1-methyl-1H-indol-3-yl)pyrrole-
1
2,5-dione (21). H NMR (DMSO-d6, 400 MHz) δ 3.87 (s, 3H),
6.88 (d, J ) 7.8 Hz, 1H), 6.94 (t, J ) 7.8 Hz, 1H), 7.14 (s, 1H),
7.25 (t, J ) 8.0 Hz, 1H), 7.63 (d, J ) 8.2 Hz, 1H), 7.95 (s, 1H),
8.26 (s, 1H), 11.20 (s, 1H); FAB-HRMS calcd for C21H13N2O3I
[M + H]+: 469.0044; found: 469.0045.
3-(5-Fluorobenzofuran-3-yl)-4-(5-iodo-1-methyl-1H-indol-3-
yl)pyrrole-2,5-dione (22). 1H NMR (DMSO-d6, 400 MHz) δ 3.87
(s, 3H), 6.65 (dd, J ) 2.5, 9.0 Hz, 1H), 7.12 (m, 1H), 7.39 (m,
2H), 7.68 (dd, J ) 4.0, 9.0 Hz, 1H), 7.98 (s, 1H), 8.31 (s, 1H),
11.18 (s, 1H); FAB-HRMS calcd for C21H12N2O3FI [M - H]-:
484.9804; found: 484.9815.
3-[4-(6-Hydroxymethylbenzofuran-3-yl)-2,5-dioxo-2,5-dihydro-
1H-pyrrol-3-yl]-1-methyl-1H-indole-5-carbonitrile (23). 1H NMR
(DMSO-d6, 400 MHz) δ 3.91 (s, 3H), 4.51 (d, J ) 5.7 Hz, 2H),
5.25 (t, J ) 5.8 Hz, 1H), 6.68 (d, J ) 8.1 Hz, 1H), 6.85 (d, J ) 8.2
Hz, 1H), 7.40 (s, 1H), 7.47 (dd, J ) 1.2, 8.5 Hz, 1H), 7.55 (s, 1H),
7.68 (d, J ) 8.6 Hz, 1H), 8.05 (s, 1H), 8.33 (s, 1H), 11.27 (s, 1H);
FAB-HRMS calcd for C23H15N3O4 [M + Na]+: 420.0955; found:
420.0952.
3-(6-Hydroxymethylbenzofuran-3-yl)-4-(7-methoxymethyl-1-
methyl-1H-indol-3-yl)pyrrole-2,5-dione (34). 1H NMR (DMSO-
d6, 400 MHz) δ 3.27 (s, 3H), 4.11 (s, 3H), 4.50 (d, J ) 8.0 Hz,
2H), 4.73 (s, 2H), 5.23 (t, J ) 8.0 Hz, 1H), 6.66 (t, J ) 7.6 Hz,
1H), 6.80-6.89 (m, 3H), 6.97 (d, J ) 7.6 Hz, 1H), 7.50 (s, 1H),
7.88 (s, 1H), 8.21 (s, 1H), 11.19 (br s, 1H); FAB-HRMS calcd for
C24H20N2O5 [M - H]-: 415.1294; found: 415.1297.
3-[3-(4-Benzofuran-3-yl-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-
yl)-1-methyl-1H-indol-7-yl]propionic Acid Ethyl Ester (35). To
a solution of 3-[3-(4-benzofuran-3-yl-2,5-dioxo-2,5-dihydro-1H-
pyrrol-3-yl)-1-methyl-1H-indol-7-yl]acrylic acid ethyl ester (68) (45
mg, 0.10 mmol) in (MeOH-THF, 3:1) (20 mL) was added
successively at 0 °C NiCl2 ·6H2O (24 mg, 0.10 mmol) and NaBH4
(7.7 mg, 0.20 mmol), and the mixture was stirred at this temperature

1862 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Gaisina et al.
for 15 min and then at room temperature for 2 h. The resulting
mixture was diluted with EtOAc and washed with saturated aqueous
NaHCO3 solution. The aqueous phase was extracted with EtOAc,
and the combined organic phases were dried over Na2SO4 and
evaporated in vacuo. The residue was purified by preparative TLC
(EtOAc-hexane; 4:6) followed by a purification by HPLC with
Cytotoxicity Assay. Materials and Methods. Immunoblot
Analysis and Antibodies. For immunoblots, cells were lysed as
described previously.15 Protein sample concentration was quantified,
and an equal amount (50 µg of whole protein extract) of protein
was loaded in each well of SDS-polyacrylamide (PAGE) gel. Cell
or tissue extracts were separated by 10% SDS-PAGE, transferred
to polyvinylidene diflouride membrane (PVDF), and probed as
indicated. Antibodies for immunoblot analysis were obtained from
the following suppliers: PARP and XIAP from BD Biosciences
Pharmingen (San Diego, CA); phospho-glycogen synthase (Ser641)
and glycogen synthase from Cell Signaling Technologies (Beverly,
MA); ꢀ-actin from Novus (Littleton, CO). Bound antibodies were
detected as previously described.
1
method C to yield the product (20 mg, 45%). H NMR (CDCl3,
400 MHz) δ 1.25 (t, J ) 7.2 Hz, 3H), 2.67 (t, J ) 8.2 Hz, 2H),
3.40 (t, J ) 8.2 Hz, 2H), 4.08-4.18 (m, 5H), 6.68 (t, J ) 7.6 Hz,
1H), 6.81-6.93 (m, 4H), 7.18 (dt, J ) 1.5, 7.4 Hz, 3H), 7.45 (d,
J ) 8.3 Hz, 1H), 7.66 (s, 1H), 7.79 (s, 1H), 8.08 (s, 1H); FAB-
HRMS calcd for C26H22N2O5 [M + H]+: 443.1602; found:
443.1613.
MTSAssay.A3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-
phenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay from Promega
(Madison, WI) was carried out in the presence or absence of the
potential GSK-3 inhibitors according to the manufacturer’s protocol.
72 h post-treatment, cell viability was measured and compounds’
IC50 values were determined.
3-[3-(4-Benzofuran-3-yl-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-
yl)-1-methyl-1H-indol-7-yl]propionic Acid (36). The 3-[3-(4-
benzofuran-3-yl-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)-1-methyl-
1H-indol-7-yl]propionic acid ethyl ester (35) (8 mg, 0.018 mmol)
was dissolved in EtOH (2 mL), and 1 N NaOH (0.072 mL, 0.072
mmol) was added to the solution. The mixture was stirred at room
temperature overnight. The solvent was evaporated in vacuo, and
water and 1 N HCl were added to reach pH 2. The acidic solution
was extracted with EtOAc three times. The combined organic layers
were washed with water and brine, dried over Na2SO4, and
evaporated. The residue was first purified by preparative TLC
(MeOH-CH2Cl2; 1:9) followed by a purification by HPLC with
Acknowledgment. This work was supported in part by an
NIH grant (1R01 MH072940-01, Grantee Alan P. Kozikowski),
by a SPORE grant (P50 CA102701, Grantee Daniel D.
Billadeau), and by the Mayo Foundation (D.D.B.). We thank
Drs. Arsen Gaysin and Rong He for technical assistance, and
Dr. Aruna Mahesh for assistance with the GSK-3ꢀ kinase assay.
1
method C to yield the product (3 mg, 39%). H NMR (CD3OD,
400 MHz) δ 2.66 (t, J ) 7.8 Hz, 2H), 3.40 (t, J ) 7.8 Hz, 2H),
4.15 (s, 3H), 6.60 (t, J ) 7.8 Hz, 1H), 6.61-6.82 (m, 3H), 6.87 (d,
J ) 7.0 Hz, 3H), 7.15 (m, 1H), 7.45 (d, J ) 8.3 Hz, 3H), 7.73 (s,
1H), 8.13 (s, 1H); FAB-HRMS calcd for C24H18N2O5 [M + H]+:
415.1289; found: 415.1304.
Supporting Information Available: Molecular modeling meth-
ods, experimental procedures for some intermediates, 13C NMR
data, HPLC purity data for all final compounds, and selectivity data
for compounds 5, 7, 11, 15, and 33. These materials are available
free of charge via the Internet at http://pubs.acs.org.
3-(5,6-Difluorobenzofuran-3-yl)-4-(1-methyl-1H-benzo[g]in-
1
dol-3-yl)pyrrole-2,5-dione (38). H NMR (CDCl3, 400 MHz) δ
References
4.41 (s, 3H), 6.66 (dd, J ) 7.8, 10.2 Hz, 1H), 6.96 (d, J ) 8.7 Hz,
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1H), 8.17 (s, 1H), 8.47 (d, J ) 8.4 Hz, 1H); FAB-HRMS calcd for
C25H14N2O3F2 [M + H]+: 429.1050; found: 429.1059.
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