Synthesis and anticancer activity of novel bisindolylhydroxymaleimide derivatives with potent GSK-3 kinase inhibition

Article abstract of DOI:10.1016/j.bmc.2018.07.012

Synthesis and biological evaluation of a series of novel indole derivatives as anticancer agents is described. A bisindolylmaleimide template has been derived as a versatile pharmacophore with which to pursue chemical diversification. Starting from maleimide, the introduction of an oxygen to the headgroup (hydroxymaleimide) was initially investigated and the bioactivity assessed by screening of kinase inhibitory activity, identifying substituent derived selectivity. Extension of the hydroxymaleimide template to incorporate substitution of the indole nitrogens was next completed and assessed again by kinase inhibition identifying unique selectivity patterns with respect to GSK-3 and CDK kinases. Subsequently, the anticancer activity of bisindolylmaleimides were assessed using the NCI-60 cell screen, disclosing the discovery of growth inhibitory profiles towards a number of cell lines, such as SNB-75 CNS cancer, A498 and UO-31 renal, MDA MB435 melanoma and a panel of leukemia cell lines. The potential for selective kinase inhibition by modulation of this template is evident and will inform future selective clinical candidates.

Full text of DOI:10.1016/j.bmc.2018.07.012

Accepted Manuscript
Synthesis and anticancer activity of novel bisindolylhydroxymaleimide deriv-
atives with potent GSK-3 kinase inhibition
Hannah J. Winfield, Michael M. Cahill, Kevin D. O'Shea, Larry T. Pierce,
Thomas Robert, Sandrine Ruchaud, Stéphane Bach, Pascal Marchand, Florence
O. McCarthy
PII:
S0968-0896(18)30898-8
https://doi.org/10.1016/j.bmc.2018.07.012
BMC 14449
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Accepted Date:
9 May 2018
7 July 2018
Please cite this article as: Winfield, H.J., Cahill, M.M., O'Shea, K.D., Pierce, L.T., Robert, T., Ruchaud, S., Bach,
S., Marchand, P., McCarthy, F.O., Synthesis and anticancer activity of novel bisindolylhydroxymaleimide
derivatives with potent GSK-3 kinase inhibition, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/
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0.1016/j.bmc.2018.07.012
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Bioorganic & Medicinal Chemistry
Synthesis and anticancer activity of novel bisindolylhydroxymaleimide derivatives
with potent GSK-3 kinase inhibition
a
a
a
a
b
Hannah J. Winfield , Michael M. Cahill , Kevin D. O’Shea , Larry T. Pierce , Thomas Robert , Sandrine
b
b
c
a
Ruchaud , Stéphane Bach , Pascal Marchand and Florence O. McCarthy 
a
School of Chemistry, Analytical and Biological Chemistry Research Facility, University College Cork, Western Road, Cork, Ireland
Sorbonne Universités, UPMC Univ Paris 06, CNRS USR3151, Kinase Inhibitor Specialized Screening facility, KISSf, Station Biologique, Place Georges
b
Teissier, Roscoff, France
c
Département de Chimie Thérapeutique, EA1155 - IICiMed, Université de Nantes, Institut de Recherche en Santé 2, Nantes, France
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Synthesis and biological evaluation of a series of novel indole derivatives as anticancer agents is
described. A bisindolylmaleimide template has been derived as a versatile pharmacophore with
which to pursue chemical diversification. Starting from maleimide, the introduction of an
oxygen to the headgroup (hydroxymaleimide) was initially investigated and the bioactivity
assessed by screening of kinase inhibitory activity, identifying substituent derived selectivity.
Extension of the hydroxymaleimide template to incorporate substitution of the indole nitrogens
was next completed and assessed again by kinase inhibition identifying unique selectivity
patterns with respect to GSK-3 and CDK kinases. Subsequently, the anticancer activity of
bisindolylmaleimides were assessed using the NCI-60 cell screen, disclosing the discovery of
growth inhibitory profiles towards a number of cell lines, such as SNB-75 CNS cancer, A498
and UO-31 renal, MDA MB435 melanoma and a panel of leukemia cell lines. The potential for
selective kinase inhibition by modulation of this template is evident and will inform future
selective clinical candidates.
Available online
Keywords:
Bisindolylmaleimide
Kinase screening
Maleimide substitution
Drug discovery
NCI anticancer screen
2
009 Elsevier Ltd. All rights reserved.
1
. Introduction
role in cell signalling, makes them an object of study for drug
4,5
design.
Cancer is one of the leading causes of morbidity and mortality
worldwide, causing about 13% of all human deaths and at least
Deregulated kinase activity has been implicated in a variety of
human health conditions including disorders of the immune
system, neurodegenerative disorders and diabetes, as well as
cancer. Since the discovery that staurosporine 1 (Figure 1) was a
1,2
one fifth of all deaths in Europe and North America. Classical
cancer chemotherapy is associated with significant adverse
effects due to the nature of the treatment: all rapidly dividing
cells are treated the same leading to the well-known effects of
myelosuppression, alopecia and many others.
6
nanomolar inhibitor of PKC in 1986, great interest has been
generated into obtaining highly active and selective protein
kinase inhibitors either through chemical synthesis or screening
of new natural products.
More recently, targeted therapies have emerged using drugs that
interfere with specific molecular targets involved in cancer
progression. One such target is a series of enzymes responsible
for signal transduction called protein kinases which modify other
proteins by transferring phosphate groups from a high-energy
donor molecule, such as adenosine triphosphate (ATP), to a
specific substrate amino acid on the target protein. This
phosphorylation results in a conformational change in the
protein, affecting its function in areas such as enzyme activity,
The first kinase inhibitor to be approved by the FDA was
imatinib 2 in 2001 (Figure 1), as a treatment for chronic myeloid
leukemia. By April 2015, a total of 28 small-molecule kinase
inhibitors have been approved for clinical use by the FDA, half
7
of which were approved in the past 3 years. These include
erlotinib 3, which is marketed as a treatment for non-small cell
lung cancer, and sunitinib 4, which is marketed as a treatment for
renal cell carcinoma and imatinib-resistant gastrointestinal
3
association with other proteins or cellular location. In this way
8,9
kinases regulate other proteins and the activities of cells, playing
an important role in many intracellular signalling pathways
including those that control cell growth and cell division. This
stromal tumour. A large number are also currently undergoing
human clinical trials for cancer and other conditions.
—
——

Corresponding author. Tel.: +353 21 4901695; e-mail: f.mccarthy@ucc.ie

Figure 1 Structures of common kinase inhibitors 1-4
Many biologically relevant kinase inhibitors, including
Figure 2 The bisindolylmaleimide class of kinase inhibitors 5-8
staurosporine 1, have been shown to work by competing with
1
0-12
Although the bisindolyl analogue 9a was highly active against
GSK-3β (IC50 = 22 nM), it was not specific and was also active
against several other kinases such as CDK1 and CDK2, VEGF-R
and several PKC isoforms. Replacing one of the indole rings with
ATP.
The interaction of staurosporine with the ATP-binding
site has been proven through the resolution of crystal structures
of staurosporine bound to CDK2 and PKA. These crystal
structures show the indolocarbazole derivative being located in
the ATP binding site, in the cleft between the two lobes of the
protein kinase. The lactam/maleimide group mimics the
hydrogen bonding pattern of the adenine base of ATP by forming
two hydrogen bonds to the backbone of the hinge between the N-
terminal and C-terminal domains of the kinase. The sugar moiety
then forms hydrogen bonds within the ribose binding site. Due to
the similarity between ATP binding sites in different kinases,
staurosporine is a highly potent but unspecific kinase inhibitor.
However, by structural modification it has been possible to
obtain some selectivity by exploiting differences in the mode of
7
-azaindole (compound 9b) had little effect on the potency or
selectivity at GSK-3β (IC50 = 17 nM) but the replacement of both
indoles with 7-azaindole (compound 9c) significantly increased
the selectivity. The bis-7-azaindolylmaleimide 9c showed limited
activity against a panel of 50 other kinases indicating a specific
inhibitor of GSK-3β (IC50 = 34 nM). Subsequent extension of this
replacing one of the azaindoles with other aryl substituents
yielded good potency but were compromised by CDK2/5 activity
2
3,24
in a full kinase screen.
13
ligand interaction in the ATP-binding pocket.
Removal of the central ring leads to a group of compounds called
the bisindolylmaleimides. Due to their increased conformational
flexibility, the binding mode of bisindolylmaleimides is more
14
complex than that of staurosporine. Initially, GF 109203 X 5
and Ro 318220 6 (Figure 2) were reported to be potent and
selective inhibitors of protein kinase C (IC50 = 10 nM and 20 nM,
15,16
respectively).
However, they were later found to also potently
Figure 3 Azaindole/indole bisindolylmaleimides with selectivity
inhibit several other kinases, such as MAPKAP kinase-1β (IC50
=
of kinase inhibition
5
6
0 nM and 3 nM, respectively) and GSK-3 (IC50 = 360 nM and
17,18
It is therefore evident that indole substitution and azaindole
incorporation can affect potent kinase inhibition of this
pharmacophore. However, one key unexplored area in the SAR is
the headgroup maleimide. We hereby describe the discreet
exploration of this space via simple conversion to
hydroxymaleimide coupled with novel indole substituents to
probe the relationship of steric bulk on kinase binding and
selectivity using a scope of kinase inhibition and cellular growth.
A panel of 10 serine/threonine kinases was selected for initial
assay to complement screening via the NCI 60 cell line screen for
cancer cell growth.
.8 nM, respectively).
Another analogue, ruboxistaurin 7 (Figure 2), has been reported
to be a specific inhibitor of the PKC isoforms, PKCβ1 (IC50 = 4.7
19
nM) and PKCβ2 (IC50 = 5.9 nM). It displays selectivity over
other protein kinases and PKC isoforms such as PKCα (IC50
60 nM), PKCγ (IC50 = 300 nM), PKCε (IC50 = 600 nM) and
PDK1 (IC50 = 750 nM) but has since been identified with PIM
=
3
20
kinase inhibition (IC50 = 200 nM). Ruboxistaurin entered
clinical trials for the treatment of diabetic peripheral retinopathy.
Enzastaurin 8 (Figure 2) is a highly potent inhibitor of PKCβ
2
1
(IC50 = 6 nM) and the PI3K/Akt pathway. It shows moderate
2. Results and Discussion
selectivity over other PKC isoforms including PKCα (IC50 = 39
nM), PKCγ (IC50 = 83 nM) and PKCε (IC50 = 110 nM).
Enzastaurin also underwent clinical trials for cancers such as
glioblastoma, non-small cell lung cancer and colorectal cancer.
2.1 Synthesis of azaindole indole maleimide and
hydroxymaleimide
Our initial approach envisaged a bisindolylmaleimide and
corresponding bisindolylhydroxymaleimide in order to probe
whether atomic incorporation would differentiate activity and our
starting point incorporated an indole, 7-azaindole and maleimide
components. On initial scope of the literature of
Kuo et al. also investigated the synthesis of a panel of novel
indole substituted bisindolylmaleimide derivatives which were
found to be potent inhibitors of GSK-3β (Figure 3).
2
2

O
O
bisindolylmaleimides, it is surprising that the hydroxymaleimide
model has very little precedent. To date there exist only three
reported compounds with this substructure and none when a 7-
azaindole is incorporated (although it is more common for
indolocarbazoles). Hence this is a field ripe for discovery
especially since the reported compounds possess bioactivity
O
O
O
Ac2O, 80°C, 24h
O-
ClCOCO2Et, AlCl3
K2CO3, H2O/MeOH
rt, 5h, 70%
K+
O
42%
N
N
H
DCM, rt, overnight
53%
N
N
H
N
N
H
HO
19
N
1
6
17
18
25
SO2Ph
(micromolar PKC and PKA inhibition).
OH
O
O
O
O
O
O
N
O
O
HO-NH2.HCl, NEt3
The application of Perkin-type condensation methodology
towards the synthesis of novel maleic anhydride 14 proved to be
relatively straightforward (Scheme 1). Initially, N-methylindole-
KOH, MeOH/H O
2
o
DMF, 80 C, 24h
N
N
N
reflux, 24h, 27%
N
N
H
N
83%
N
N
N
Ac
SO2Ph
H
H
22
H
2
0
21
3
-acetic acid 13 was formed by alkylation of indole-3-acetic acid
26
1
2, with acid 13 formed in 84% yield. Separately, a stirred Scheme 2 Synthesis of maleimide 22 via Perkin-type condensation
solution of N-methyl-7-azaindole 10 was treated with oxalyl
chloride and corresponding glyoxyl chloride 11 was isolated as
an off-white solid in quantitative yield. Glyoxyl chloride 11 was
then added to a stirred solution containing N-methylindole-3-
acetic acid 13 and triethylamine in DCM, with isolation of maleic
anhydride 14 as a bright red solid in 37% yield achieved by flash
column chromatography (Scheme 1). Conversion of maleic
anhydride 14 to maleimide 15 occurred in excellent yield of 88%
via treatment with hexamethyldisilazane. Hence, compound 15
was formulated as the reference compound with some known
GSK-3β has been shown to reduce apoptosis signals it may be
useful for the treatment of Alzheimer’s disease and protection
29,30
against cell death.
In addition to the data above, there are
reports of bisindolylmaleimide involvement with GSK-3β in the
regulation of murine embryonic stem cell self-renewal so it is an
27
obvious starting point. Interestingly, inhibitors of GSK-3 have
also been shown to enhance apoptotic signal transduction
induced by TRAIL (TNF-related apoptosis inducing ligand
3
1
27
TRAIL).
bioactivity.
The evaluation of derivatives for their inhibition of cancer-related
protein kinases is also of relevance: HsHaspin, HsAurora kinase
B, HsRIPK3, HsCDK2, HsCDK5, HsCDK9, RnDYRK1A,
HsPIM1 and SscCK1δ/ε. Haspin is expressed in a variety of
tissues (e.g., testis, bone narrow, thymus, and spleen) and in
32,33,34
proliferating cells, including in a number of neoplasms.
Aurora-B is a key component of the spindle assembly checkpoint
35
and also functions in cytokinesis. The receptor-interacting
protein kinase-3 (RIPK3 or RIP3) is a critical regulator of
36-38
necroptosis.
A recent study showed the important role of
RIPK3 in maintaining in vivo tumor growth with RIPK3
knockout breast tumor cell growing at a significantly slower rate
39
than vector control cells. The Cyclin-Dependant Kinases
CDKs) play a direct role in the regulation of the cell cycle
(
controlling cell proliferation. CDK2 has been shown to have a
direct role in the cell cycle progression, while CDK subtypes 7–9
have been described to control RNA polymerase II mediated
40
transcription and CDK5 is involved in neuronal function. Dual-
specificity tyrosine phosphorylation-regulated kinase 1A
4
1
(
DYRK1A) is involved in many major diseases, including
Scheme
1
Synthesis of maleimide 15 via Perkin-type
41-43
cancer and neurodegenerative disorders.
PIM1 is
a
condensation
constitutively active enzyme and has been shown to have diverse
biological roles in cell survival, proliferation, differentiation and
44
apoptosis. Overexpression of PIM-1 has been found in various
hematopoietic malignancies as well as in solid tumors including
Synthesis of a related hydroxymaleimide was undertaken in order
to probe the effect of oxygen insertion into the headgroup and the
effect of indole substitution (Scheme 2). 7-Azaindole 16 was
converted to the ethyl glyoxylate 17 followed by hydrolysis to
the glyoxylate salt 18. Perkin condensation of this with
benzenesulfonyl protected indole-3-acetic acid 19 yielded the
protected maleic anhydride 20.
deprotected and converted to the hydroxymaleimide 22 via
treatment with hydroxylamine.
45
colon, prostate and pancreas. CK1 (casein kinase 1), when
deregulated, is responsible for non-regulation of growth,
proliferation, and apoptosis which may result in pathological
46
conditions, such as tumorigenesis or neurological diseases.
2
6
Results from one dose assay at 10 M concentration confirmed
that the maleimide was significantly more active than the
hydroxymaleimide (see Table 1, Supporting Information).
Significant kinase inhibition was seen for the maleimide 15 in all
the CDKs (CDK2, CDK5 and CDK9) in addition to GSK-3 and
PIM1 kinases. By contrast, the introduction of an oxygen into the
headgroup and removal of the indole methyl substituents reduced
the CDK activity completely with significant kinase inhibition
only evident in PIM1 kinase and to a lesser extent in CK1 and
GSK-3. This intriguing disparity and potential selectivity led to
an exploration of the molecular space occupied by the headgroup
and the indole substituents.
This was subsequently
2.2 Evaluation of the kinase inhibitory activity of lead
compounds 15 and 22
Due to the common reports of bisindolylmaleimides as kinase
inhibitors and the reported polypharmacology of the class, as
discussed in the introduction, the standard maleimide 15 and
novel hydroxymaleimide 22 were evaluated by a preliminary
2
8
kinase screen.

2.3 Synthesis of novel bisindolylmaleic anhydrides with
symmetrical N-substituents
At the outset we decided to base our study on the novel
bisindolylhydroxymaleimide series as this would probe the
indole substituent effects without any competing/conflicting
effects from the nitrogen of 7-azaindole. Initial synthesis begins
from indole which is converted to the potassium salt of indole-3-
47
glyoxylic acid 23 in excellent yield (Scheme 3). After a
deprotonation step, subsequent Perkin condensation in the
presence of acetic anhydride with benzenesulfonyl protected
indole-3-acetic acid 19 renders the fully protected maleic
anhydride 25 in moderate yield. Complete hydrolysis can be
achieved with aqueous base allowing for derivatisation of both
48
indole nitrogens simultaneously. To this end, the novel hexane
bisindolyl maleic anhydride 27 was generated as a test
macrocycle. Reaction of the same unsubstituted 26 with 6-
bromohexanenitrile yielded both the mono- and bis-substituted
hexanenitrile anhydrides 28 and 29. Hydrolysis of 29 yields the
monohexanoic acid derivative.
Scheme 4 Synthesis of maleic anhydrides 39-40
.5 Synthesis of novel bisindolylhydroxymaleimides
2
Formation of the ultimate hydroxymaleimides 47-58 was
achieved by condensation with hydroxylamine hydrochloride salt
in the presence of trimethylamine and yields were generally
excellent (Scheme 5 and Table 1).
Scheme 5 Synthesis of hydroxymaleimides 47-58 via novel
maleic anhydrides 27, 28, 30, 39-46
Table 1 Structure and synthetic yields of 41-58 via alkylation (A)
and hydroxymaleimide formation (B)
Precursor
maleic
anhydride
1
2
Yield
Yield
(B)
Compound
R
R
(A)
4
4
7
8
39
39
39
39
43
40
40
40
27
28
30
28
Me
Me
Me
Me
Me
iPr
iPr
iPr
H
Me
Et
-
83%
84%
69%
82%
79%
77%
93%
80%
82%
87%
86%
90%**
Scheme 3 Synthesis of key maleic anhydrides 27-30
8
(
0%
41)
7
(
2%
42)
2.4 Synthesis of novel bisindolyl maleic anhydrides with
49
unsymmetrical N-substituents
4
0%
(43)
5
5
5
5
5
5
5
5
5
0
1
2
3
4
5
6
7
8
(CH
(CH
2
)
5
CN
In order to probe the influence of indole nitrogen substitution on
the anticancer and kinase activity of these compounds a synthetic
route was devised to incorporate individual functionality on the
indoles (Scheme 4). To achieve selective substitution of either
nitrogen the strategy was adapted to incorporate an alkylation of
the indole at an earlier stage and this was brought through a
similar sequence. 1-Methyl (in order to correspond to 15) and 1-
isopropyl (to probe steric bulk) indole 31-32, were generated
from the starting indole and the corresponding potassium
glyoxylate salts 35-36 were generated as above. These undergo
Perkin condensation in the presence of acetic anhydride and
indole acetic acid 19, and subsequent base mediated deprotection
yielding the mono-alkylated bisindolyl maleic anhydrides 39-
7
6%
(44)*
2
)
5
COOH
H
-
4
6%
(45)
Me
Et
5
1%
(46)
-CH
2
(CH
2
)
4
CH
2
-
-
(CH
(CH
2
)
5
CN
(CH
2
)
5
CN
CN
-
-
-
49
4
0. These are then available for further alkylation to form fully
functionalised maleic anhydrides (Scheme 5 or in the case of 44
base hydrolysis) to form anhydrides 41-46. From observation of
2
)
5
COOH
H
1
(CH
2
)
5
CN
(CH
2 5
)
the H NMR data, there is an interesting correlation between the
substitution on the indole nitrogen and the chemical shift values
of the representative maleic anhydrides (see Supplementary
Information).
*
Using the method as per the formation of 30 in Scheme 3; **
Using HMDS rather than hydroxylamine form the corresponding
maleimide for reference purposes.

5
2
2
.6 Kinase screen
inhibition. The compounds were initially screened (again at 10
M) in a cellular based assay against the NCI 60-cell line panel
with some interesting results compared with other headgroups

The full library of novel substituted bisindolylmaleimides was
initially probed for kinase inhibitory activity against 10 kinase
enzymes at a concentration of 10 M (see Table 1, Supporting
Information). It is very evident that small changes in the
substitution pattern affect the ability to modulate phosphorylation
in a discreet fashion. As mentioned above, there is a striking
difference in the activity of related bisindolylmaleimides 15 and
53,54
(Table 3).
The majority gave high mean growth across the 60
cell lines at 10 M (70-100%) but intriguingly the restriction of
cellular growth appears related to kinase inhibition given the
potencies seen in Table 3. Specific growth percentages for two
renal, three melanoma and one leukaemia cell lines are given and
it is evident that the indole substitution affects activity.
22 and again it is evident that extension of the pharmacophore at
the indole nitrogens influences inhibition. From the heat map it
can be seen that CDK9, GSK-3 and, to a lesser extent, PIM1
kinase are all substantially affected by the panel, aside from 55
which is interesting given that it is the macrocyclic
bisindolylmaleimide. This suggests that restricting the
conformation in this way reduces the kinase activity (in contrast
to the known kinase inhibitor ruboxistaurin 7, see Figure 2). It is
also seen that 51 and 53 have a similar profile to 15.
Evaluation of compound 47 with one methyl N-substituent and
one unsubstituted indole identified an average mean growth of
78.0% across all 60 cell lines. Inhibition was observed for
leukaemia cell lines (growth reduced to 31% in SR) and also for
melanoma cell line MDA-MB-435 (17%), renal cancer cell line
UO-31 (41%) and breast cancer cell line MCF7 (31%). Addition
of a second methyl substituent significantly reduced activity with
compound 48 being almost completely inactive; although some
growth inhibition was observed for leukaemia cell line SR (75%),
melanoma cell line LOX IMVI (73%) and renal cancer cell line
UO-31 (74%). Compounds with an isopropyl substituent (52 and
53) were mostly inactive apart from a similar selectivity towards
LOX IMVI (59% and 62%, respectively) and UO-31 (68% and
56%, respectively) and for most leukaemia cell lines. Compounds
with one extended alkyl chain with a nitrile or carboxylic acid
functional group displayed little activity with nitrile 50 being
slightly more active than acids 51 and 57. These compounds
again displayed moderate inhibition of LOX IMVI and UO-31.
2.7 Kinase assay
Given the excellent selectivity and potency evident in the kinase
screen, the full library of novel substituted bisindolylmaleimides
5
0
progressed to kinase inhibitory assay (Table 2). The most potent
compound of the panel was the lead maleimide 15 with activity
against GSK-3 30 nM, CDK2/5/9 80-800 nM and PIM1 2 M.
On evaluating GSK-3 inhibition, the hydroxymaleimides are
active irrespective of indole substituent (save for the inactive
macrocyclic 55) which suggests a mode of binding for this
template whereby incorporation of anchoring subunits on the
indole nitrogens (as in 5, 6 and 8, Figure 2) may be fruitful.
Table 3 Summary of activity of selected bisindolylmaleimides in
the NCI 60 cell line screen.
Comp
The removal of indole substitution and incorporation of the
hydroxymaleimide headgroup rendered GSK-3 and PIM1
selective inhibition albeit at 4-4.5 M (15 vs 22). Comparing 47
and 48 which differ in only by the presence of a methyl
substituent, the influence of the indole N-H gives rise to more
potency against GSK-3 (200 nM). However this effect is not a
constant as on extension of the alkyl substituent further potency
is seen for 49, 50, 53 and 54 showing the positive influence of at
least one bulky substituent. Interestingly 51 and 54 have
submicromolar inhibition of CDK-9 kinase which could be
Mean
Growth
at 10
Growth of selected cell lines (%)
No.
NSC)
15*
A498
SK-
MEL-
2
MDA-
MB-
435
LOX
IMVI
UO-
31
SR

M (%)
(
2
7
9
8
-20
79
86
77
92
94
99
88
-13
76
102
94
-
13
17
41
51
73
60
80
59
62
79
30
80
70
23
41
74
51
63
68
56
80
44
61
73
12
31
75
67
90
86
-
(762129)
47
775309)
(
4
8
98
95
5
1
(774887)
exploited as a cancer target.
5
0
9
1
68
(
(
776694)
In comparing the methyl against the propyl subsets (47-49 Vs 52-
5
1
54) it is evident that the increased steric bulk of the propyl
96
102
102
104
111
16
776691)
substituent is tolerated by GSK-3. Extending the alkyl chain
further to hexanenitrile 50, maintains potency against GSK-3 and
kinase selectivity over CDKs. Conversion of 50 to carboxylic
acid 51 improves the potency to CDK-9 rather than GSK-3 with
the introduction of DYRK inhibition while removal of a methyl
from 51 to yield 57 reverses this effect. Interestingly, the
bishexanenitrile substitution pattern of 56 imbues selectivity of
action over CDKs and with inhibition of GSK kinase at 750 nM
and is a lead for future studies.
5
2
9
9
4
1
87
87
120
68
99
96
(775308)
53
775307)
(
(
(
5
5
103
68
92
781334)
6*
776693)
5
6
9
18
5
7
100
9
8
8
7
89
54
102
105
(776692)
5
8
-
Finally, in order to confirm the selectivity imposed by the N-OH
group extension of the maleimide, a direct comparison with the
corresponding maleimide 58 returns the potency with consequent
polykinase inhibition (GSK, DYRK, CDK-2/9). It is clearly
evident that the incorporation of the oxygen limits the CDK and
DYRK inhibition seen with maleimides.
(
781333)
*
Compounds taken forward to five dose assay by NCI.
Interestingly, addition of a second nitrile chain significantly
increased activity with compound 56 displaying a mean growth
of 68% and being selected to progress to five dose assay. This
derivative exhibited considerable activity towards a number of
individual cell lines, including leukaemia cell lines HL-60(TB)
15%) and SR (18%), melanoma cell lines LOX IMVI (30%) and
MDA-MB-435 (16%), renal cancer cell line UO-31 (44%) and
breast cancer cell lines MCF7 (40%) and MDA-MB-231/ATCC
40%). Replacement of the hydroxymaleimide moiety with an
2.8 Full anticancer evaluation.
Selected compounds were identified for further studies into their
effect on cancer cells through the NCI anticancer screening
programme. Compound 15, 47-48, 50-53, and 55-58 were chosen
in order to scope the breadth of structural diversity and kinase
(
(

unsubstituted maleimide surprisingly reduced activity, with
3. Conclusions
compound 58 showing a mean growth of 87%.
In summary, we have explored the molecular space around the
bisindolylmaleimide pharmacophore, generating 28 novel
compounds and the development of novel anticancer leads. Our
initial test compounds identified a dichotomy of activity with the
maleimide headgroup 15 offering potent kinase inhibitory
activity against all CDKs and GSK-3 while the
hydroxymaleimide and removal of the indole methyl groups 22
offers some selectivity of action albeit at lower potency.
The derivative with both indole nitrogens linked by a 6-carbon
chain 55 was found to be almost completely inactive with a mean
growth of 103% and little deviation from the mean growth across
the panel. This was a rather disappointing result as maleimide
analogue has been reported to inhibit of a number of kinases
including PKC(Ki = 0.3 μM), p70 S6K (Ki = 0.9 μM) and
19,55
GSK-3 (Ki = 0.7 μM), and is analogous to ruboxistaurin.
However, the most active anticancer agent in the screen was
compound 15 with a mean growth response of just 29%. This
compound was taken forward to five dose assays (along with 56).
In order to assess the effect of substitution on potency and
selectivity, a series of novel bisindolylhydroxymaleimides
functionalized at the indole nitrogens were consequently
synthesised and assessed for kinase inhibitory and anticancer
activity. The move from azaindole to indole does not
significantly affect potency or the GSK-3 kinase activity but does
remove all CDK2 and CDK5 inhibition (but not CDK9).
Converging towards a ruboxistaurin macrocyclic system is
disappointingly associated with a complete loss of kinase activity
and indeed anticancer activity as seen in the NCI screen but the
majority of other substitutions are well tolerated. In addition to
the significant potency of 15, bishexanitrile substituted 56 is a
key lead compound for future development with evident GSK-3
inhibition in the absence of other kinase activity in the screen.
The anticancer activity of 15 in the NCI 60 cell line screen has
uncovered sub-micromolar growth inhibition of cancer cells
across the full panel and significant inhibition of SNB-75 CNS
cancer, A498 and UO-31 renal and MDA-MB-435 melanoma
cell lines. The fact that 56 progressed to five dose assay suggests
that GSK-3 kinase has a likely role in its effect on cancer cell
growth.
A summary of the effects of compound 15 on the NCI 60 cell
line panel identifies that the mean GI50 of the compound is 5 M
and that the anticancer effects are not particularly specific for any
cancer sub-type or cell phenotype (see Supplementary
Information). It is clearly evident that there is a cytostatic effect
at concentrations around 10 M for all cell lines but this does not
translate into a cytotoxic effect at higher concentrations in the
majority of cases. Specific cell lines with sub-micromolar growth
inhibition values include: SNB-75 (GI50 = 291 nM); MDA-MB-
4
8
35 (GI50 = 648 nM); A498 (GI50 = 320 nM); HS 578T (GI50
47 nM). From this it can be seen that the spread of activity over
=
CNS, Melanoma, Renal and Breast cancer cell lines and the
activity against SNB-75 and A498 is of particular interest for
future studies.
Cancer
subtype
Cell line
GI50 M
TGI M
LC50 M
1
5
56
15
56
15
56
Overall, our findings confirm that the insertion of an oxygen into
the bisindolylmaleimide pharmacophore at the key headgroup is
well tolerated and capable of low nanomolar kinase inhibition in
particular with GSK-3 kinase. It is evident that this new binding
platform is capable of imbuing discrete inhibitory activity on
related target kinases and will be the focus of our future research.
Leukaemia
Melanoma
HL-
4.05
2.54
17.9
6.44
>100
>100
6
0(TB)
SR
1.04
3.55
2.74
18.4
19.3
9.48
8.31
>100
>100
>100
>100
MDA-
0.648
MB-435
4
. Experimental Section
.1 General procedures
Solvents were distilled prior to use as follows:
4
SK-
2.15
4.22
2.92
3.23
5.80
22.0
10.6
16.0
21.8
96.7
42.8
84.4
MEL-5
dichloromethane was distilled from phosphorous pentoxide; ethyl
acetate was distilled from potassium carbonate; ethanol and
methanol were distilled from magnesium in the presence of
iodine; toluene was distilled from sodium and benzophenone;
hexane was distilled prior to use; tetrahydrofuran was freshly
distilled from sodium and benzophenone. Diethyl ether was
obtained pure from Riedel-de Haën. Organic phases were dried
using anhydrous magnesium sulfate. All commercial reagents
were used without further purification unless otherwise stated.
Infrared spectra were recorded as a thin film on sodium chloride
plates for liquids or potassium bromide (KBr) disc for solids on a
Perkin Elmer Spectrum 100 FT-IR spectrometer. H (300 MHz)
and C (75 MHz) NMR spectra were recorded on a Bruker
Avance 300 NMR spectrometer. H (400 MHz) NMR spectra
were recorded on a Bruker Avance 400 NMR spectrometer. H
(600 MHz) and C (150 MHz) NMR spectra were recorded on a
Bruker Avance III 600 MHz NMR spectrometer equipped with a
dual CH cryoprobe. All spectra were recorded at room
temperature (~20 ºC) in deuterated chloroform (CDCl ) with
tetramethylsilane (TMS) as an internal standard, or deuterated
UACC-
6
2
CNS
Breast
Renal
SNB-75
0.291
2.84
3.97
2.36
7.97
4.06
2.34
20.0
>100
86.0
94.6
>100
>100
>100
>100
>100
HS-578T 0.847
A498 0.320
1
Table 4 Selected GI50, TGI and LC50 data for 15 and 56
13
1
1
Bishexanenitrile derivative 56 had a relatively high mean growth
on one dose screening but low micromolar GI50 values were
observed for a number of cell lines, especially in leukaemia and
melanoma cancer types. It is of interest to note that a number of
values are lower for 56 over 15 on transfer to cellular assay. Most
cell lines required a concentration of greater than 100 μM for the
death of 50% of cell population (LC50), with only 2 cell lines,
SK-MEL-5 and UACC-62, showing any appreciable cytotoxicity.
13
3
1
dimethylsulfoxide (DMSO-d6). H NMR spectra recorded in
deuterated dimethylsulfoxide (DMSO-d ) were assigned using
6

the DMSO-d
) are expressed in parts per million (ppm) relative to the
reference peak. Coupling constants (J) are expressed in Hertz
6
peak as the reference peak. Chemical shifts (δ
H
and
YSPTSPSYSPTSPSYSPTSPSKKKK,
as
substrate;
(vi)
δ
C
HsCDK5/p25 (human, recombinant, expressed in bacteria) was
assayed in buffer B, with 0.8 µg/µl of histone H1 as substrate;
(vii) HsAuroraB (human, recombinant, expressed by baculovirus
in Sf9 insect cells, SignalChem, product #A31-10G) was assayed
in buffer D with 0.2 µg/µl of MBP as substrate; (viii) SscGSK-
3 (Sus scrofa domesticus, glycogen synthase kinase-3, affinity
purified from porcine brain) was assayed in buffer A
(supplemented with 0.15 mg/ml BSA and 0.23 mg/ml DTT), with
0.010 µg/µl of GS-1 peptide, a GSK-3-selective substrate
(YRRAAVPPSPSLSRHSSPHQSpEDEEE, “Sp” stands for
phosphorylated serine); (ix) SscCK1δ/ε (Sus scrofa domesticus,
casein kinase 1δ/ε, affinity purified from porcine brain) was
assayed in buffer B, with 0.022 µg/µl of the following peptide:
RRKHAAIGSpAYSITA as CK1-specific substrate; (x)
RnDYRK1A-kd (Rattus norvegicus, amino acids 1 to 499
including the kinase domain, recombinant, expressed in bacteria,
DNA vector kindly provided by Dr. W. Becker, Aachen,
Germany) was assayed in buffer A (supplemented with 0.5
mg/mL BSA and 0.23 mg/ml DTT) with 0.033 μg/µl of the
following peptide: KKISGRLSPIMTEQ as substrate.
1
(Hz). Splitting patterns in H NMR spectra are designated as s
(singlet), br s (broad singlet), d (doublet), br d (broad doublet), t
(triplet), q (quartet), dd (doublet of doublets), dt (doublet of
triplets), ddd (doublet of doublet of doublets), ddt (doublet of
doublet of triplets) and m (multiplet). Low resolution mass
spectra were recorded on a Waters Quattro Micro triple
quadrupole spectrometer (QAA1202) in electrospray ionisation
(ESI) mode using 50% acetonitrile – water containing 0.1%
formic acid as eluent. High resolution mass spectra (HRMS)
were recorded on a Waters LCT Premier Time of Flight
spectrometer (KD160) in electrospray ionisation (ESI) mode
using 50% acetonitrile – water containing 0.1% formic acid as
eluent. All synthetic compounds were confirmed to be >95%
pure by LCMS analysis using a 14 minute gradient method
(90:10 to 10:90 water:acetonitrile with 0.1% formic acid as
additive) on a Waters Alliance 2695 HPLC and a Waters Xterra
Phenyl 3.5m (2.1 x 100mm) HPLC column. Melting points
were measured in a uni-melt Thomas Hoover capillary melting
point apparatus and are uncorrected. Thin layer chromatography
(
TLC) was carried out on precoated silica gel plates (Merck 60
PF254), and visualisation was achieved by UV light detection
254 nm).
5
6
4.3 NCI-60 anticancer screening
(
The experimental methodology involves initial growth of the
tumour cell lines in RPMI 1640 medium containing 5% foetal
bovine serum and 2 mM L-glutamine. For a typical screening
experiment, cells are inoculated into 96 well microtiter plates in
100 µL of medium at plating densities ranging from 5,000 to
40,000 cells/well, depending on the doubling time of individual
cell lines. After cell inoculation, the microtiter plates are
4
.2 Kinase inhibition assays
Kinase activities were assayed in appropriate kinase buffer, with
either protein or peptide as substrate in the presence of 15µM [-
P] ATP (3,000 Ci/mmol; 10 mCi/ml) in a final volume of 30 µl
33
2
8,50
following the assay described.
Controls were performed with
2
incubated at 37 °C, 5% CO , 95% air and 100% relative humidity
appropriate dilutions of dimethylsulfoxide. Full-length kinases
are used unless specified. Peptide substrates were obtained from
ProteoGenix (Schiltigheim, France).
for 24 hours prior to addition of candidate compounds.
After 24 hours, two plates of each cell line are fixed in situ
with trichloroacetic acid (TCA), to represent a measurement of
the cell population for each cell line at the time of drug addition
(Tz). Candidate compounds are dissolved in DMSO at 400-fold
the desired final maximum test concentration and stored frozen
prior to use. The single dose screen is carried out at a
concentration of 10 µM.
The buffers used for kinase assays are the following: (A) 10 mM
MgCl , 1 mM EGTA, 1 mM DTT, 25 mM Tris-HCl pH 7.5, 50
2
µg/mL heparin; (B) 60 mM -glycerophosphate, 30 mM p-
nitrophenyl-phosphate, 25 mM MOPS (pH 7), 5 mM EGTA, 15
mM MgCl
mM MOPS, pH7.2, 12.5 mM -glycerolphosphate, 25 mM
MgCl , 5 mM EGTA, 2 mM EDTA, 0.25 mM DTT; (H) MOPS
5 mM pH 7.5, 10 mM MgCl ; (R) 1.67 mM MOPS pH 7.2, 0.83
2
, 1 mM DTT, 0.1 mM sodium orthovanadate; (D) 25
Following drug addition, the plates are incubated for an
additional 48 hours at 37 °C, 5% CO , 95% air, and 100%
2
2
2
2
relative humidity. For adherent cells, the assay is terminated by
the addition of cold TCA. Cells are fixed in situ by the gentle
addition of 50 µL of cold 50% (w/v) TCA and incubated for 60
minutes at 4 °C. Sulforhodamine B (SRB) solution (100 µL) at
0.4% (w/v) in 1% acetic acid is added to each well, and plates are
incubated for 10 minutes at room temperature. Absorbance is
read on an automated plate reader at a wavelength of 515 nm, and
using the absorbance measurements of time zero (Tz), control
growth (C), and test growth in the presence of the drug at a
mM -glycerophosphate, 1.33 mM MgCl , 0.83 mM MnCl , 0.33
2
2
mM EGTA, 0.13 mM EDTA, 16.67 g/ml BSA, 0.017 mM
DTT.
A panel of ten native or recombinant protein kinases was used
during this study: (i) HsRIPK3 (human, recombinant, expressed
by baculovirus in Sf9 insect cells) was assayed in buffer R with
0.1 µg/µl of MBP as substrate; (ii) HsPIM1 (human proto-
oncogene, recombinant, expressed in bacteria) was assayed in
buffer B with 0.8 µg/µl of histone H1 (Sigma #H5505) as
substrate; (iii) HsHaspin-kd (human, kinase domain, amino acids
52
concentration of 10 µM, the percentage growth is calculated.
4
70 to 798, recombinant, expressed in bacteria) was assayed in
buffer H with 0.007 µg/µl of Histone H3 (1-21) peptide
ARTKQTARKSTGGKAPRKQLA) as substrate; (iv)
4.4 Chemical Data
(
For the synthesis of compounds (13), (15-19), (23-24), (26-27),
31-35), (37), (39) and (41) see the Supplementary Information.
HsCDK2/CyclinA (human, cyclin-dependent kinase-2, kindly
provided by Dr. A. Echalier-Glazer, Leicester, UK) was assayed
in buffer A (supplemented with 0.15 mg/ml BSA and 0.23 mg/ml
DTT) with 0.8 µg/µl of histone H1 as substrate; (v)
HsCDK9/CyclinT (human, recombinant, expressed by
baculovirus in Sf9 insect cells) was assayed in buffer A
(
3
-(1-Methyl-1H-indol-3-yl)-4-(1-methyl-1H-pyrrolo[2,3-
b]pyridin-3-yl)furan-2,5-dione (14)
A solution of 1-methyl-1H-pyrrolo[2,3-b]pyridine 10 (1.808 g,
o
1
3.7 mmol) in diethyl ether (80 mL) was cooled to 0 C in an ice
(supplemented with 0.15 mg/ml BSA and 0.23 mg/ml DTT) with
bath. Oxalyl chloride (1.39 mL, 16.4 mmol) was then added in a
0.27 µg/µl of the following peptide:
57
dropwise manner over a period of 20 minutes. The resulting

mixture was stirred for 3 hours, whilst being allowed to slowly
return to room temperature in that time. The solvent and excess
oxalyl chloride was removed under reduced pressure and the
solid residue was dissolved in DCM (60 mL). This was then
added to a stirred solution containing 2-(1-methyl-1H-indol-3-
yl)acetic acid 13 (2.592 g, 13.7 mmol) and triethylamine (3.82
mL, 27.4 mmol) in DCM (30 mL). The reaction mixture was
stirred at room temperature for 14 hours, after which time the
solvent was removed under reduced pressure. The dark red
residue was subjected to flash column chromatography
Purification by flash column chromatography (70:30 – 60:40,
hexane/ethyl acetate gradient elution) to yield the deprotected
maleic anhydride as an orange solid (0.283 g, 27%): m.p. 299 –
o
-1
301 C; νmax/cm (KBr) 3435, 3020, 2882, 1839, 1820, 1751,
1631, 1532; δ (400 MHz, DMSO-d ) 6.71-6.75 (m, 2H, C-H5’
C-H ), 6.79 (q, 1H, J = 8.0, 4.7 Hz, C-H ), 7.05 (ddd, 1H, J = 8.2,
1.3 Hz, C-H6’), 7.24 (dd, 1H, J = 8.1, 1.7 Hz, C-H7’), 7.45 (d, 1H,
J = 8.0 Hz, C-H4’), 7.91 (s, 1H, C-H2’), 7.95 (s, 1H, C-H ), 8.17
(dd, 1H, J = 4.6, 1.5 Hz, C-H ), 12.02 (brs, 1H, indole N-H),
6
12.39 (brs, 1H, 7-azaindole N-H); δ (100 MHz, DMSO-d )
H
6
,
4
5
2
6
C
(
hexane/ethyl acetate, 70:30), yielding maleic anhydride 14 as a
103.7 (C, aromatic C), 104.6 (C, aromatic C), 112.2 (CH,
aromatic CH), 116.1 (CH, aromatic CH), 117.5 (C, aromatic C),
120.0 (CH, aromatic CH), 121.0 (CH, aromatic CH), 122.2 (CH,
aromatic CH), 124.6 (C, aromatic C), 127.1 (C, aromatic C),
129.1 (C, aromatic C), 129. 3 (CH, aromatic CH), 130.5 (CH,
aromatic CH), 130.8 (CH, aromatic CH), 136.2 (C, aromatic C),
143.6 (CH, aromatic CH), 148.4 (C, aromatic C), 166.2 (C,
o
-1
red solid (1.796 g, 37%): m.p. 222-224 C; vmax/cm (KBr) 2919,
819, 1747, 1523, 1371, 1254; δ (300 MHz, CDCl ) 3.91 [3H, s,
NCH ], 3.94 [3H, s, NCH ], 6.75 [1H, q, J 8.0, 4.7, C-H5’], 6.77-
.79 [2H, m, C-H5,6], 7.13-7.19 [1H, m, C-H ], 7.30 [1H, dd, J
.0, 1.5, C-H4’], 7.34 [1H, d, J 8.3, C-H ], 7.85 [1H, s, C-H ],
.86 [1H, s, C-H2’], 8.25 [1H, dd, J 4.7, 1.5, C-H6’]; δ (75 MHz,
) 31.9 (CH , NCH ), 33.6 (CH , NCH ), 103.6 (C, aromatic
1
H
3
3
3
6
8
7
7
4
2
C
+
CDCl
3
3
3
3
3
C=O), 166.4 (C, C=O); m/z (ES+) 330.2 (M+H ) 100%; HRMS
+
C), 104.8 (C, aromatic C), 109.9 (CH, aromatic CH), 116.5 (CH,
aromatic CH), 118.6 (C, aromatic C), 120.9 (CH, aromatic CH),
(ES+): Exact mass calculated for (C19
378.0871.
12
H N
3
O
3
) 330.0879. Found
1
22.3 (CH, aromatic CH), 123.0 (CH, aromatic CH), 125.5 (C,
aromatic C), 126.4 (C, aromatic C), 128.1 (C, aromatic C), 130.5
CH, aromatic CH), 133.6 (CH, aromatic CH), 134.0 (CH,
aromatic CH), 137.0 (C, aromatic C), 144.0 (CH, aromatic CH),
47.8 (C, aromatic C), 166.6 (C, C=O), 166.8 (C, C=O); m/z
1
-Hydroxy-3-(1H-indol-3-yl)-4-(1H-pyrrolo[2,3-b]pyridin-3-
yl)-1H-pyrrole-2,5-dione 22
(
To a mixture of 3-(1H-indol-3-yl)-4-(1H-pyrrolo[2,3-b]pyridin-
3-yl)furan-2,5-dione 21 (0.132 g, 0.4 mmol) and
hydroxylammonium hydrochloride (0.109 g, 1.57 mmol) in DMF
(1 mL) was added triethylamine (0.21 mL, 1.51 mmol). The
mixture was allowed to stir for 24 hours at 80 C. Following this,
1
1
+
(ES+) 358.1 [M+H] (100%); HRMS (ES+): Exact mass
+
calculated for (C21
16 3 3
H N O ) 358.1192. Found 358.1185.
o
3
-(1-Acetyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-(1-
M HCl (10 mL) was added. The mixture was extracted with
(phenylsulfonyl)-1H-indol-3-yl)furan-2,5-dione 20
ethyl acetate (3 x 5 mL). The combined organic extracts were
washed with water (2 x 10 mL), brine (1 x 10 mL), dried and
concentrated under reduced pressure to yield the
hydroxymaleimide as a deep red solid (0.112 g, 83%): m.p.
>300 C; νmax/cm (KBr) 3340, 1777, 1721, 1703, 1645, 1539,
1419, 1133, 1100, 1026, 726; δ (400 MHz, DMSO-d ) 6.65 (t,
1H, J = 7 Hz, C-H5’), 6.70 (s, 1H, C-H7’), 6.72 (q, 1H, J = 8.1,
4.6 Hz, C-H ), 7.00 (t, 1H, J = 7.6 Hz, C-H6’), 7.15 (dd, 1H, J =
8.0, 1.2 Hz, C-H ), 7.41 (d, 1H, J = 8.0 Hz, C-H4’), 7.86 (s, 1H,
C-H2’), 7.87 (s, 1H, C-H ), 8.11 (dd, 1H, J = 4.5, 1.2 Hz, C-H ),
10.48 (brs, 1H, indole N-H), 11.81 (brs, 1H, 7-azaindole N-H),
12.24 (brs, 1H, N-OH); δ (100 MHz, DMSO-d ) 104.3 (C,
aromatic C), 105.1 (C, aromatic C), 112.0 (CH, aromatic CH),
115.1 (CH, aromatic CH), 117.7 (C, aromatic C), 119.6 (CH,
aromatic CH), 120.7 (CH, aromatic CH), 121.9 (CH, aromatic
CH), 123.4 (C, aromatic C), 125.0 (C, aromatic C), 125.2 (C,
aromatic C), 129.0 (CH, aromatic CH), 129.5 (CH, aromatic
CH), 129.7 (CH, aromatic CH), 136.0 (C, aromatic C), 143.2
(CH, aromatic CH), 148.3 (C, aromatic C), 168.2 (C, C=O),
A suspension of potassium 2-oxo-2-(1H-pyrrolo[2,3-b]pyridin-3-
yl)acetate 18 (4.05 g, 17.7 mmol) and 2-(1-(phenylsulfonyl)-1H-
indol-3-yl)acetic acid 19 (5.56 g, 17.63 mmol) were stirred in
o
o
-1
acetic anhydride (40 mL) at 80 C for 24 hours. Following this,
the mixture was vacuum filtered. The collected solid was stirred
in boiling ethyl acetate (100 mL) for 20 minutes before being
filtered hot to remove insoluble impurities and the protected
maleic anhydride recrystallized from ethyl acetate/hexane as a
bright yellow solid (3.292 g). Further work-up of the mother
liquor yielded a yellow/brown solid which was purified by flash
column chromatography (80:20, hexane/ethyl acetate) to yield a
further batch of the protected maleic anhydride (0.532 g,
H
6
5
4
2
6
C
6
o
-1
combined yield 3.824 g, 42%): m.p. 123 – 125 C; νmax/cm
(
(
4
KBr) 3432, 3139, 2929, 1831, 1764, 1724, 1645, 1547, 1375; δ
300 MHz, DMSO-d ) 3.01 (s, 3H, OAc), 6.50 (q, 1H, J = 8.0,
.7 Hz, C-H ), 6.89 (dd, 1H, J = 8.0, 1.7 Hz, C-H6’), 6.99 (m, 2H,
C-H3’’, H5’’), 7.28 (ddd, 1H, J = 8.5, 1.8 Hz, C-H7’), 7.67 (t, 2H, J
8.0 Hz, C-H4’, H5’), 7.80 (d, 1H, J = 7.5 Hz, C-H4’’), 7.96 (d,
H, J = 8.5 Hz, C-H ), 8.06 (m, 2H, C-H2’’, H6’’), 8.23 (s, 1H, C-
2’), 8.25 (dd, 1H, J = 4.7, 1.5 Hz, C-H ), 8.47 (s, 1H, C-H ); m/z
HRMS (ES+): Exact mass
S) 468.0654. Found 468.0661.
H
6
5
=
1
H
-
4
168.3 (C, C=O); m/z (ES-) 343.3 (M-H ) 100%; HRMS (ES+):
Exact mass calculated for (C19
345.0981.
+
6
2
H
13
N
4
O
3
)
345.0988. Found
-
(ES-) 468.2 (M-AcO ) 90%;
+
calculated for (C25
15 3 5
H N O
3
-(1-Acetyl-1H-indol-3-yl)-4-[1-(phenylsulfonyl)-1H-indol-3-
3
-(1H-Indol-3-yl)-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)furan-2,5-
yl]furan-2,5-dione 25
dione 21
A mixture of potassium 2-(1H-indol-3-yl)-2-oxoacetate 24
(3.677 g, 15.8 mmol), 2-[1-(phenylsulfonyl)-1H-indol-3-yl]acetic
acid 19 (5.020 g, 15.8 mmol) and acetic anhydride (30 mL) was
stirred at 80 °C for 24 hours. The yellow suspension was then
cooled to room temperature and filtered. The solid collected was
stirred in boiling ethyl acetate for 30 minutes before hot filtration
removed a white side product. The yellow filtrate was
concentrated to dryness giving a yellow crystalline solid (2.426
g). The acetic anhydride solution from the initial filtration was
concentrated under reduced pressure. The residue was then
washed with saturated sodium bicarbonate solution (50 mL) and
extracted into ethyl acetate (3 × 30 mL). The organic layer was
A suspension of 3-(1-acetyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-(1-
(phenylsulfonyl)-1H-indol-3-yl)furan-2,5-dione 20 (1.64 g, 3.21
mmol) and potassium hydroxide (1.07 g, 19.2 mmol) in aqueous
methanol (4:1 MeOH:Water, 50 mL) was heated to reflux for 24
hours. Following this, the mixture was concentrated under
reduced pressure, water (20 mL) was added to the residue, the pH
was adjusted to 5 using 2 M HCl and the mixture was extracted
with ethyl acetate (3 x 30 mL). The combined organic extracts
were washed with saturated aqueous sodium bicarbonate solution
(1 x 15 mL), water (2 x 20 mL), brine (1 x 30 mL), dried and
concentrated under reduced pressure to yield a glassy red solid.

+
filtered before being washed with saturated sodium bicarbonate
solution (30 mL), followed by water (30 mL) and brine (30 mL)
and then dried over anhydrous magnesium sulfate, filtered and
the solvent evaporated under reduced pressure. The crude residue
was recrystallised from ethyl acetate to give additional product
Exact mass calculated for (C32H31N4O3) 519.2396. Found
519.2401.
6
-{3-[4-(1H-Indol-3-yl)-2,5-dioxo-2,5-dihydrofuran-3-yl]-1H-
indol-1-yl}hexanenitrile 29 was isolated as a red solid (0.953 g,
5%): m.p. 102-104 °C; νmax/cm (KBr) 3391, 2935, 2246,
1819, 1748, 1529, 1251, 743; δH (300 MHz, DMSO-d6)
-1
1
(
ν
0.544 g, combined yield 2.970 g, 40%): m.p. 223-225 °C;
-1
max/cm (KBr) 3139, 1830, 1762, 1718, 1373, 1176; δ
) 2.69 (3H, s, CH ), 6.44 [1H, t, J 7.4, C(6′)-H],
.59 [1H, d, J 7.8, C(7′)-H], 6.95 [1H, t, J 7.3, C(6)-H], 7.03 [1H,
H
(300
1
1
4
.32 [2H, m, C(4′′)-H2], 1.57 [2H, quin, J 7.3, C(3′′)-H2],
.74 [2H, quin, J 7.3, C(5′′)-H2], 2.45 [2H, t, J 7.1, C(2′′)-H2],
.26 [2H, t, J 7.0, C(6′′)-H2], 6.69 [1H, ddd, J 7.9, 6.9, 0.9,
C(6)-H], 6.74-6.82 [2H, m, C(6′, 7)-H], 6.95 [1H, d, J 7.8,
C(7′)-H], 7.04 [1H, ddd, J 8.2, 7.1, 1.2, C(5)-H], 7.10 [1H,
ddd, J 8.2, 7.2, 1.1, C(5′)-H], 7.43 [1H, d, J 8.2, C(4)-H], 7.55
1H, d, J 8.2, C(4′)-H], 7.86 [1H, s, C(2′)-H], 7.89 [1H, s, C(2)-
H], 11.92 (1H, bs, NH); δc (75 MHz, DMSO-d6) 16.0 (CH2),
4.3 (CH2), 25.2 (CH2), 28.7 (CH2), 45.6 (CH2), 104.2 (C,
aromatic C), 104.9 (C, aromatic C), 110.5 (CH, aromatic
CH), 112.1 (CH, aromatic CH), 119.8 (CH, aromatic CH),
MHz, DMSO-d
6
6
3
d, J 7.6, C(7)-H], 7.16 [1H, t, J 7.8, C(5′)-H], 7.27 [1H, t, J 7.7,
C(5)-H], 7.61-7.71 [2H, m, C(3′′, 5′′)-H], 7.75-7.85 [1H, t, J 7.4,
C(4′′)-H], 7.93 [1H, d, J 8.6, C(4)-H], 7.98-8.06 [2H, m, C(2′′,
6
[
′′)-H], 8.19 [1H, s, C(2′)-H], 8.26 [1H, d, J 8.3, C(4′)-H], 8.30
1H, s, C(2)-H]; δ (75 MHz, DMSO-d ) 23.9 (CH ), 109.3 (C,
aromatic C), 111.2 (C, aromatic C), 113.1 (CH, aromatic CH),
15.9 (CH, aromatic CH), 120.5 (CH, aromatic CH), 121.6 (CH,
[
C
6
3
2
1
aromatic CH), 123.1 (CH, aromatic CH), 123.7 (CH, aromatic
CH), 125.4 (CH, aromatic CH), 125.7 (CH, aromatic CH), 126.6
1
20.0 (CH, aromatic CH), 120.5 (CN), 121.2 (CH, aromatic
CH), 121.4 (CH, aromatic CH), 122.1 (CH, aromatic CH),
22.2 (CH, aromatic CH), 124.7 (C, aromatic C), 125.5 (C,
aromatic C), 127.4 (C, aromatic C), 128.2 (C, aromatic C),
30.6 (CH, aromatic CH), 133.0 (CH, aromatic CH), 135.9
(C, aromatic C), 136.2 (C, aromatic C), 166.36 (C=O),
(C, aromatic C), 126.9 (2CH, 2 × aromatic CH), 127.6 (C,
aromatic C), 129.6 (CH, aromatic CH), 130.1 (2CH, 2 × aromatic
CH, C, aromatic C), 130.8 (CH, aromatic CH), 131.4 (C,
aromatic C), 133.6 (C, aromatic C), 134.7 (C, aromatic C), 135.2
1
1
(
(
(
CH, aromatic CH), 136.4 (C, aromatic C), 164.9 (C=O), 165.1
+ +
C=O), 169.7 (C=O); m/z (ESI ) 511.1 [(M+H) 10%]; HRMS
+
+
+
+
166.43 (C=O); m/z (ESI ) 424.2 [(M+H) 100%]; HRMS
ESI ): Exact mass calculated for (C28
H
19
N
2
O
6
S) 511.0964.
+
+
(
ESI ): Exact mass calculated for (C26H22N3O3)
Found 511.0965.
424.1661. Found 424.1659.
6
3
,6'-(3,3'-(2,5-Dioxo-2,5-dihydrofuran-3,4-diyl)bis(1H-indole-
,1-diyl))dihexanenitrile 28
6
1
-{3-[4-(1H-Indol-3-yl)-2,5-dioxo-2,5-dihydrofuran-3-yl]-
H-indol-1-yl}hexanoic acid 30
To a solution of 3,4-di(1H-indol-3-yl)furan-2,5-dione 26
5.004 g, 15.2 mmol) in anhydrous DMF (80 mL) at 0°C
under a nitrogen atmosphere was added sodium hydride (60
wt. % oil dispersion, 0.640 g, 16.0 mmol). The dark purple
solution was stirred for 30 minutes while warming to room
temperature before 6-bromohexanenitrile (2.62 mL, 3.47 g,
6
-{3-[4-(1H-Indol-3-yl)-2,5-dioxo-2,5-dihydrofuran-3-yl]-1-
(
indol-1-yl}hexanenitrile 29 (0.402 g, 0.950 mmol) was
dissolved in a mixture of methanol (20 mL) and 10% aqueous
potassium hydroxide solution (20 mL). The solution was heated
to reflux for 16 hours before being allowed to cool and the
solvent evaporated under reduced pressure. The residue was
dissolved in water (20 mL) and acidified to pH 2 using 20%
aqueous HCl, and then extracted with ethyl acetate (20 mL × 2).
The organic layer was washed with water (20 mL × 2) followed
by brine (20 mL) before being dried over anhydrous magnesium
sulfate, filtered and the solvent evaporated under reduced
19.7 mmol) was added. The solution was stirred for 16 hours
before being poured into water (200 mL) and extracted with
ethyl acetate (3 × 60 mL). The combined organic layers were
washed with 1 M aqueous HCl (2 × 60 mL) followed by water
(60 mL) before being dried over anhydrous magnesium
sulfate, filtered and the solvent evaporated under reduced
pressure. The crude material was purified using column
chromatography on silica gel with 20:80 – 30:70 ethyl
acetate/hexane to give 2 fractions found to be the mono
alkylated product 29 and also dialkylated 6,6'-[3,3'-(2,5-
pressure to give desired compound as a red solid (0.335 g,
-1
8
1
7
4%): 198-199 °C; νmax/cm (KBr) 3386, 2924, 1816, 1740,
699, 1531, 1261; δH (300 MHz, DMSO-d6) 1.23 [2H, quin, J
.5, C(4′′)- H2], 1.51 [2H, quin, J 7.5, C(3′′)-H2], 1.72 [2H,
quin, J 7.2, C(5′′)-H2], 2.17 [2H, t, J 7.3, C(2′′)-H2], 4.24 [2H,
t, J 7.0, C(6′′)-H2], 6.67 [1H, t, J 7.5, C(6)-H], 6.76 [1H, t, J
dioxo-2,5-dihydrofuran-3,4-
diyl)bis(1H-indole-3,1-
diyl)]dihexanenitrile 28.
7.8, C(6′)-H], 6.77 [1H, d, J 7.5, C(7)-H], 6.93 [1H, d, J 8.0,
6
3
,6'-(3,3'-(2,5-Dioxo-2,5-dihydrofuran-3,4-diyl)bis(1H-indole-
,1-diyl))dihexanenitrile 28 was isolated as a red solid (1.658
C(7′)-H], 7.04 [1H, t, J 8.0, C(5)-H], 7.09 [1H, t, J 7.8, C(5′)-
H], 7.43 [1H, d, J 8.1, C(4)-H], 7.53 [1H, d, J 8.3, C(4′)-H],
7.84 [1H, s, C(2′)-H], 7.89 [1H, s, C(2)-H], 11.92 (1H, bs, NH),
11.99 (1H, bs, COOH); δc (75 MHz, DMSO-d6) 24.0 (CH2),
25.6 (CH2), 29.3 (CH2), 33.5 (CH2), 45.7 (CH2), 104.2 (C,
aromatic C), 104.9 (C, aromatic C), 110.5 (CH, aromatic CH),
112.1 (CH, aromatic CH), 119.8 (CH, aromatic CH), 120.0
(CH, aromatic CH), 121.2 (CH, aromatic CH), 121.4 (CH,
aromatic CH), 122.07 (CH, aromatic CH), 122.12 (CH,
aromatic CH), 124.7 (C, aromatic C), 125.5 (C, aromatic C),
127.4 (C, aromatic C), 128.2 (C, aromatic C), 130.6 (CH,
aromatic CH), 133.0 (CH, aromatic CH), 135.9 (C, aromatic C),
136.1 (C, aromatic C), 166.4 (C=O), 166.5 (C=O), 174.3
-1
g, 21%): m.p. 120-122 °C; νmax/cm (KBr) 3435, 2939, 2243,
1
1
815, 1749, 1525, 1257, 747; δH (300 MHz, DMSO-d6)
.27-1.40 [4H, m, 2 × C(4′′)-H2], 1.57 [4H, quin, J 7.4, 2 ×
C(3′′)-H2], 1.76 [4H, quin, J 7.3, 2 × C(5′′)-H2], 2.45 [4H, t, J
7
7
7
.0, 2 ×C(2′′)-H2], 4.27 [4H, t, 2 × C(6′′)-H2], 6.75 [2H, t, J
.4, C(6, 6′)-H], 6.87 [2H, d, J 7.9, C(7, 7′)-H], 7.10 [2H, t, J
.7, C(5, 5′)-H], 7.55 [2H, d, J 8.3, C(4, 4′)-H], 7.90 [2H, s,
C(2, 2′)-H]; δc (75 MHz, DMSO-d6) 16.0 (2 × CH2), 24.3
(2 × CH2), 25.1 (2 × CH2), 28.7 (2 × CH2), 45.6 (2 × CH2),
1
1
2
04.2 (2C, 2 × aromatic C), 110.6 (2CH, 2 × aromatic CH),
20.0 (2CH, 2 × aromatic CH), 120.5 (2 × CN), 121.5 (2CH,
× aromatic CH), 122.2 (2CH, 2 × aromatic CH), 125.3 (2C, 2
(COOH); m/z (ESI-) 441.2 [(M-H)- 100%]; HRMS (ESI+):
+
×
aromatic C), 127.6 (2C, 2 × aromatic C), 133.1 (2CH, 2 ×
Exact mass calculated for (C26
H
23
N
2
O
5
)
443.1607. Found
aromatic CH), 136.0 (2C, 2 × aromatic C), 166.3 (2 ×
443.1611.
+
+
+
C=O); m/z (ESI ) 519.2 [(M+H)
10%]; HRMS (ESI ):

Synthesis of N-isopropyl maleic anhydride intermediate
Potassium 2-(1-isopropyl-1H-indol-3-yl)-2-oxoacetate 36
A solution of 2-(1-isopropyl-1H-indol-3-yl)-2-oxoacetic acid 34
3-(1H-Indol-3-yl)-4-(1-isopropyl-1H-indol-3-yl)furan-2,5-
dione 40
To a stirred suspension of 3-(1-isopropyl-1H-indol-3-yl)-4-[1-
(phenylsulfonyl)-1H-indol-3-yl]furan-2,5-dione 38 (1.353 g,
(3.499 g, 15.1 mmol) in ethanol (50 mL) was treated with
2.37 mmol) in a mixture of methanol (60 mL) and water (15
potassium hydroxide (85 wt. %, 0.993 g, 15.1 mmol) and the
mixture was stirred at room temperature for 4 hours. The
solvent was removed under reduced pressure and the residue
dried overnight at 40 °C to give the desired product as a pale
mL) was added potassium hydroxide (85 wt. %, 1.053 g, 15.9
mmol). The mixture was then heated to reflux for 24 hours
before being allowed to cool and the solvent evaporated under
reduced pressure. The residue was acidified to pH 2 using 10%
aqueous HCl and extracted with ethyl acetate (3 × 25 mL). the
combined organic layers were washed with water (3 × 30 mL)
and brine (30 mL) before being dried over anhydrous
magnesium sulfate, filtered and the solvent evaporated under
-1
pink solid (3.961 g, 97%): m.p. 80-84 °C; νmax/cm (KBr) 3456,
2
1
7
1
8
981, 1649, 1607, 1301, 1203, 738; δ
H 6
(300 MHz, DMSO-d )
.48 [d, 6H, J 6.6, CH(CH ], 4.80 [1H, sept, J 6.7, CH(CH ) ],
3
)
2
3 2
.17 [1H, ddd, J 8.2, 7.2, 1.1, C(5)-H], 7.22 [1H, ddd, J 8.5, 7.1,
.4, C(6)-H], 7.58 [1H, d, J 7.6, C(7)-H], 8.14 [1H, s, C(2)-H],
reduced pressure to give the desired product as red solid
.19 [1H, d, J 7.3, C(4)-H]; δ
c
(75 MHz, DMSO-d
6
) 22.2 (2 ×
-1
(
0.764g, 87%): m.p. 186-188 °C; νmax/cm (KBr) 3118, 2971,
CH
3
), 47.2 [CH(CH ], 110.5 (CH, aromatic CH), 113.4 (C,
3 2
)
1
[
6
812, 1745, 1627, 1529, 1247; δ
6H, d, J 6.7, CH(CH ], 4.81 [1H, sept, J 6.6, CH(CH
.68-6.71 [2H, m, C(6, 7)-H], 6.85 [1H, ddd, J 8.1, 6.9, 0.7,
H
(300 MHz, DMSO-d
6
) 1.38
)
3 2
aromatic C), 121.4 (CH, aromatic CH), 121.5 (CH, aromatic
CH), 122.2 (CH, aromatic CH), 126.5 (C, aromatic C), 134.0
)
3 2
],
(
CH, aromatic CH), 135.8 (C, aromatic C), 169.8 (C=O), 193.1
+
+
C(6′)-H], 7.03-7.17 [3H, m, C(5, 5′, 7′)-H], 7.44 [1H, d, J 8.1,
C(4)-H], 7.59 [1H, d, J 8.2, C(4′)-H], 7.75 [1H, s, C(2′)-H],
(C=O); m/z (ESI ) 232.3 [(M+2H) , 100%]; HRMS (ESI+):
+
Exact mass calculated for (C13
H
17
N
2
O
3
)
249.1239
+
7.92 [1H, d, J 2.9, C(2)-H], 11.94 (1H, bs, N-H); δ
c
(75 MHz,
[(M+NH
4
+H) ]. Found 249.1239.
DMSO-d ) 22.1 (2 × CH ), 47.2 (CH), 104.4 (C, aromatic C),
6
3
3
-(1-Isopropyl-1H-indol-3-yl)-4-[1-(phenylsulfonyl)-1H-
104.8 (C, aromatic C), 110.6 (CH, aromatic CH), 112.2 (CH,
aromatic CH), 119.9 (CH, aromatic CH), 120.2 (CH, aromatic
CH), 121.4 (CH, aromatic CH), 121.6 (CH, aromatic CH),
indol-3-yl]furan-2,5-dione 38
A
mixture of potassium 2-(1-isopropyl-1H-indol-3-yl)-2-
1
22.1 (2CH, 2 × aromatic CH), 124.2 (C, aromatic C), 125.6 (C,
aromatic C), 127.2 (C, aromatic C), 128.5 (C, aromatic C),
29.5 (CH, aromatic CH), 130.9 (CH, aromatic CH), 135.5 (C,
oxoacetate 36 (3.502 g, 13.0 mmol), 2-[1-(phenylsulfonyl)-1H-
indol-3-yl]acetic acid 19 (4.096 g, 13.0 mmol) and acetic
anhydride (30 mL) was stirred at 80 °C for 24 hours. The
yellow suspension was then allowed to cool to room
temperature and the solvent was evaporated under reduced
pressure. The residue was washed with saturated aqueous
sodium bicarbonate (50 mL) and extracted into ethyl acetate (2
1
aromatic C), 136.3 (C, aromatic C), 166.3 (C=O), 166.5 (C=O);
m/z (ESI-) 369.3 [(M-H)- 100%]; HRMS (ESI+): Exact mass
+
calculated for (C23
H
19
N
2
O
3
) 371.1396. Found 371.1395.
×
30 mL). The organic layer was washed with saturated
Substitutions on methyl intermediate
aqueous sodium bicarbonate (30 mL), followed by water (30
mL) and brine (30 mL) before being dried over anhydrous
magnesium sulfate, filtered and the solvent evaporated under
reduced pressure. The crude product was then purified using
column chromatography on silica gel with 10% ethyl
acetate/hexane to give a yellow foamy residue which was
dissolved in chloroform (15 mL) and diethyl ether was added
until a yellow solid precipitated which was collected by vacuum
3-(1-Ethyl-1H-indol-3-yl)-4-(1-methyl-1H-indol-3-yl)furan-
2,5-dione 42
To a solution of 3-(1H-indol-3-yl)-4-(1-methyl-1H-indol-3-
yl)furan-2,5-dione 39 (0.262 g, 0.76 mmol) in anhydrous DMF
(15 mL) under nitrogen was added sodium hydride (60 wt. % oil
dispersion, 0.037 g, 1.1 mmol) and the mixture was allowed to
stir for 40 minutes at room temperature. Iodoethane (0.09 mL,
-1
filtration (1.582 g, 24%): m.p. 102-105 °C; νmax/cm (KBr)
561, 3135, 2977, 1821, 1755, 1631, 1536, 1372, 1185; δ (300
MHz, DMSO-d ) 1.35 [6H, d, J 6.6, CH(CH ], 4.80 [1H, sept,
J 6.6, CH(CH ], 6.39 [1H, t, J 7.6, C(6′)-H], 6.70 [1H, d, J
.0, C(7′)-H], 6.83-6.93 [2H, m, C(6, 7)-H], 7.05 [1H, t, J 7.7,
C(5′)-H], 7.26 [1H, ddd, J 8.3, 6.9, 1.5, C(5)-H], 7.54 [1H, d, J
.4, C(4′)-H], 7.63-7.69 [2H, m, C(3′′, 5′′)-H], 7.80 [1H, m,
C(4′′)-H], 7.91 [1H, s, C(2′)-H], 7.95 [1H, d, J 8.3, C(4)-H],
.04-8.07 [2H, m, C(6′′, 2′′)-H], 8.15 [1H, s, C(2)-H]; δ (75
MHz, DMSO-d ) 22.0 (2 × CH ), 47.5 [CH(CH ], 104.1 (C,
aromatic C), 110.9 (CH, aromatic CH), 112.0 (C, aromatic C),
13.2 (CH, aromatic CH), 120.7 (CH, aromatic CH), 121.0
CH, aromatic CH), 121.7 (CH, aromatic CH), 122.5 (CH,
aromatic CH), 122.8 (C, aromatic C), 123.4 (CH, aromatic CH),
25.0 (C, aromatic C), 125.4 (CH, aromatic CH), 126.9 (2CH, 2
aromatic CH), 127.6 (C, aromatic C), 128.6 (CH, aromatic
CH), 130.0 (2CH, 2 × aromatic CH), 130.9 (CH, aromatic CH),
33.7 (C, aromatic C), 134.2 (C, aromatic C), 135.0 (CH,
aromatic CH), 135.7 (C, aromatic C), 136.6 (C, aromatic C),
65.5 (C=O), 165.8 (C=O); m/z (ESI+) 511.1 [(M+H)+ 50%];
0
.17 g, 1.1 mmol) was then added and the reaction was stirred for
3
H
a further 16 hours. Water (30 mL) was then added and the
mixture was extracted with ethyl acetate (3 × 20 mL). The
combined organic layers were then washed with 1 M aqueous
HCl (2 × 20 mL), followed by water (20 mL) and brine (20 mL)
before being dried over anhydrous magnesium sulfate, filtered
and the solvent evaporated under reduced pressure. The crude
residue was purified using column chromatography on silica gel
with 20% ethyl acetate/hexane to give desired product as a red
6
3 2
)
3 2
)
8
8
8
c
6
3
)
3 2
-1
solid (0.204 g, 72%): m.p. = 214-216 °C; νmax/cm (KBr) 3420,
3
d
7
133, 1816, 1758, 1626, 1525, 1219, 737; δ
) 1.31 (3H, t, J 7.2, CH CH ), 3.89 (3H, s, CH
.2, CH CH ), 6.67-6.74 [2H, m, C(6′, 7′)-H], 6.81 [1H, t, J 7.9,
H
(300 MHz, DMSO-
1
(
6
2
3
3
), 4.26 (2H, q, J
2
3
C(6)-H], 7.04 [1H, d, J 8.0, C(7)-H], 7.08-7.16 [2H, m, (5, 5′)-
H], 7.50 [1H, d, J 8.4, C(4′)-H], 7.54 [1H, d, J 8.4, C(4)-H], 7.81
1
×
[1H, s, C(2)-H], 7.97 [1H, s, C(2′)-H]; δ (75 MHz, DMSO-d
c
6
)
15.1 (CH CH ), 33.0 (CH ), 40.8 (CH CH ), 103.9 (C, aromatic
2
3
3
2
3
1
C), 104.2 (C, aromatic C), 110.4 (CH, aromatic CH), 110.5 (CH,
aromatic CH), 120.05 (CH, aromatic CH), 120.08 (CH, aromatic
CH), 121.5 (CH, aromatic CH), 121.6 (CH, aromatic CH), 122.2
1
+
HRMS (ESI+): Exact mass calculated for (C29
11.1328. Found 511.1310.
23 2 5
H N O S)
(
2CH, 2 × aromatic CH), 124.9 (C, aromatic C), 125.7 (C,
aromatic C), 127.1 (C, aromatic C), 127.7 (C, aromatic C), 132.5
CH, aromatic CH), 134.4 (CH, aromatic CH), 135.6 (C,
5
(

aromatic C), 136.8 (C, aromatic C), 166.3 (C=O), 166.5 (C=O);
aromatic C), 104.2 (C, aromatic C), 110.48 (CH, aromatic CH),
110.52 (CH, aromatic CH), 120.0 (2CH, 2 × aromatic CH),
121.4 (2CH, 2 × aromatic CH), 122.1 (2CH, 2 × aromatic CH),
+
+
+
m/z (ESI ) 371.1 [(M+H) 100%]. HRMS (ESI ): Exact mass
+
calculated for (C23
19 2 3
H N O ) 371.1396. Found 371.1389.
1
25.0 (C, aromatic C), 125.7 (C, aromatic C), 127.0 (C,
aromatic C), 127.8 (C, aromatic C), 132.9 (CH, aromatic CH),
34.3 (CH, aromatic CH), 135.9 (C, aromatic C), 136.7 (C,
6
-{3-[4-(1-Methyl-1H-indol-3-yl)-2,5-dioxo-2,5-
dihydrofuran-3-yl]-1H-indol-1-yl}hexanenitrile 43
1
To a solution of 3-(1H-indol-3-yl)-4-(1-methyl-1H-indol-3-
yl)furan-2,5-dione 39 (0.505 g, 1.48 mmol) in anhydrous DMF
aromatic C), 166.3 (C=O), 166.5 (C=O), 174.5 (COOH); m/z
(ESI+) 457.3 [(M+H)+ 100%]; HRMS (ESI+): Exact mass
+
(20 mL) under nitrogen was added sodium hydride (60 wt. %
calculated for (C27
H
25
N
2
O
5
) 457.1763. Found 457.1755.
oil dispersion, 0.070 g, 1.77 mmol) and the mixture was stirred
for 40 minutes at room temperature. 6-Bromohexane nitrile
(
0.30 mL, 0.38 g, 2.2 mmol) was then added and the reaction
Substitutions on isopropyl intermediate
-(1-Isopropyl-1H-indol-3-yl)-4-(1-methyl-1H-indol-3-
was stirred for a further 16 hours. Water (40 mL) was then
added and the mixture was extracted with ethyl acetate (3 × 30
mL). The combined organic layers were then washed with 1 M
aqueous HCl (2 × 30 mL), followed by water (30 mL) and brine
3
yl)furan-2,5-dione 45
To a solution of 3-(1H-indol-3-yl)-4-(1-isopropyl-1H-indol-3-
yl)furan-2,5-dione 40 (0.302 g, 0.81 mmol) in anhydrous DMF
(15 mL) under nitrogen was added sodium hydride (60 wt. % oil
dispersion, 0.037 g, 1.5 mmol) and the mixture was stirred for 40
minutes at room temperature. Iodomethane (0.06 mL, 0.14 g,
0.97 mmol) was then added and the reaction was allowed to stir
for a further 16 hours. Water (30 mL) was then added and the
mixture was extracted with ethyl acetate (3 × 20 mL). The
combined organic layers were then washed with 1 M aqueous
HCl (2 × 20 mL), followed by water (20 mL) and brine (20 mL)
before being dried over anhydrous magnesium sulfate, filtered
and the solvent evaporated under reduced pressure. The crude
residue was purified using column chromatography on silica gel
with 20% ethyl acetate/hexane to give desired product as a red
(30 mL) before being dried over anhydrous magnesium sulfate,
filtered and the solvent evaporated under reduced pressure. The
crude residue was purified using column chromatography on
silica gel with 30% ethyl acetate/hexane to give desired product
-1
as a red solid (0.258 g, 40%): m.p. 78-79 °C; νmax/cm (KBr)
411, 2936, 2244, 1816, 1750, 1529, 1254, 1100, 741; δH (400
3
MHz, DMSO-d6) 1.27-1.35 [2H, m, C(4′′)-H2], 1.56 [2H, quin,
J 7.4, C(3′′)-H2], 1.73 [2H, quin, J 7.4, C(5′′)-H2], 2.44 [2H, t,
J 6.9, C(2′′)-H2], 3.89 (3H, s, CH3), 4.24 [2H, t, J 6.9, C(6′′)-
H2], 6.69-6.72 [2H, m, C(6′, 7′)-H], 6.79 [1H, t, J 7.6, C(6)-H],
7
[
.01 [1H, d, J 8.15, C(7)-H], 7.07-7.13 [2H, m, (5, 5′)-H], 7.49
1H, d, J 8.15, C(4′)-H], 7.54 [1H, d, 8.49, C(4)-H], 7.82 [1H, s,
C(2)-H], 7.97 [1H, s, C(2′)-H]; δc (75 MHz, DMSO-d6) 16.0
-1
(CH2), 24.3 (CH2), 25.1 (CH2), 28.7 (CH2), 33.0 (CH3), 45.6
(CH2), 103.9 (C, aromatic C), 104.3 (C, aromatic C), 110.50
(CH, aromatic CH), 110.54 (CH, aromatic CH), 120.0 (CH,
solid (0.140 g, 46%): m.p. 164-167 °C; νmax/cm (KBr) 3118,
3048, 2970, 1812, 1745, 1627, 1529, 1247, 739; δ (300 MHz,
H
DMSO-d ) 1.37 [6H, d, J 6.7, 2 × CH ], 3.90 (3H, s, CH ), 4.81
6
3
3
aromatic CH), 120.1 (CH, aromatic CH), 120.5 (CN), 121.4
CH, aromatic CH), 121.5 (CH, aromatic CH), 122.1 (CH,
aromatic CH), 122.2 (CH, aromatic CH), 125.0 (C, aromatic C),
25.7 (C, aromatic C), 127.0 (C, aromatic C), 127.9 (C,
3 2
[1H, sept, J 6.6, CH(CH ) ], 6.60 [1H, d, J 8.0, C(7)-H], 6.72
[1H, t, J 8.0, C(6)-H], 6.87 [1H, t, J 7.8, C(6′)-H], 7.09-7.19 [3H,
m, C(5, 5′, 7′)-H], 7.50 [1H, d, J 8.4, C(4)-H], 7.59 [1H, d, J 8.4,
(
1
C(4′)-H], 7.73 [1H, s, C(2′)-H], 8.01 [1H, s, C(2)-H]; δ (75
c
aromatic C), 132.9 (CH, aromatic CH), 134.3 (CH, aromatic
CH), 135.9 (C, aromatic C), 136.7 (C, aromatic C), 166.3
MHz, DMSO-d ) 22.0 (2 × CH ), 33.0 [CH(CH ) ], 47.2 (CH ),
6
3
3 2
3
103. 9 (C, aromatic C), 104.5 (C, aromatic C), 110.5 (CH,
aromatic CH), 110.6 (CH, aromatic CH), 120.1 (CH, aromatic
CH), 120.2 (CH, aromatic CH), 121.5 (CH, aromatic CH), 121.6
(CH, aromatic CH), 122.15 (CH, aromatic CH), 122.18 (CH,
aromatic CH), 124.5 (C, aromatic C), 125.7 (C, aromatic C),
(
C=O), 166.5 (C=O); m/z (ESI+) 438.2 [(M+H)+ 12%];
+
24 3 3
HRMS (ESI+): Exact mass calculated for (C27H N O )
438.1818. Found 438.1802.
6
-{3-[4-(1-Methyl-1H-indol-3-yl)-2,5-dioxo-2,5-
126.9 (C, aromatic C), 128.1 (C, aromatic C), 129.1 (CH,
dihydrofuran-3-yl]-1H-indol-1-yl}hexanoic acid 44
aromatic CH), 134.65 (CH, aromatic CH), 134.69 (C, aromatic
+
6
3
-{3-[4-(1-Methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydrofuran-
-yl]-1H-indol-1-yl}hexanenitrile 43 (0.103 g, 0.235 mmol)
C), 136.9 (C, aromatic C), 166.2 (C=O), 166.5 (C=O); m/z (ESI )
+
385.3 [(M+H) , 100%]; HRMS (ESI+): Exact mass calculated for
+
was dissolved in a mixture of methanol (8 mL) and 10%
aqueous potassium hydroxide solution (8 mL). The solution was
heated to reflux for 16 hours before being allowed to cool and
the solvent evaporated. The residue was dissolved in water (10
mL) and acidified to pH 2 using 20% aqueous HCl, and was
then extracted with ethyl acetate (10 mL × 2). The organic layer
was washed with water (10 mL × 2) followed by brine (10 mL)
before being dried over anhydrous magnesium sulfate, filtered
and the solvent evaporated under reduced pressure to give
desired compound as a red solid (0.082 g, 76%): m.p. 95-97 °C;
(C24H
21
N
2
O
3
) 385.1552. Found 385.1540.
3
2
-(1-Ethyl-1H-indol-3-yl)-4-(1-isopropyl-1H-indol-3-yl)furan-
,5-dione 46
To a solution of 3-(1H-indol-3-yl)-4-(1-isopropyl-1H-indol-3-
yl)furan-2,5-dione 40 (0.167 g, 0.45 mmol) in anhydrous DMF
(10 mL) under nitrogen was added sodium hydride (60 wt. % oil
dispersion, 0.019 g, 0.83 mmol) and the mixture was stirred for
40 minutes at room temperature. Iodoethane (0.06 mL, 0.12 g,
0.69 mmol) was then added and the reaction was allowed to stir
for a further 16 hours. Water (20 mL) was then added and the
mixture was extracted with ethyl acetate (3 × 15 mL). The
combined organic layers were then washed with 1 M aqueous
HCl (2 × 15 mL), followed by water (15 mL) and brine (15 mL)
before being dried over anhydrous magnesium sulfate, filtered
and the solvent evaporated under reduced pressure. The crude
residue was purified using column chromatography on silica gel
with 20% ethyl acetate/hexane to give desired product as an
-1
ν
max/cm (KBr) 3050, 2937, 1816, 1751, 1705, 1611, 1528,
1255, 740; δH (300 MHz, DMSO-d6) 1.18-1.29 [2H, m, C(4′′)-
H2], 1.51 [2H, quin, J 7.4, C(3′′)-H2], 1.71 [2H, quin, J 7.3,
C(5′′)-H2], 2.17 [2H, t, J 7.4, C(2′′)-H2], 3.90 (3H, s, CH3),
4.23 [2H, t, J 6.7, C(6′′)-H2], 6.68 [2H, m, C(6′, 7′)-H], 6.78
[1H, t, J 7.6, C(6)-H], 7.00 [1H, d, J 8.0, C(7)-H], 7.06-7.14
[2H, m, (5, 5′)-H], 7.48 [1H, d, J 8.3, C(4′)-H], 7.53 [1H, d, J
8
(
2
.3, C(4)-H], 7.81 [1H, s, C(2)-H], 7.99 [1H, s, C(2′)-H], 11.99
COOH); δc (75 MHz, DMSO-d6) 24.1 (CH2), 25.9 (CH2),
9.3 (CH2), 33.0 (CH3), 33.8 (CH2), 45.7 (CH2), 103.9 (C,
-1
orange solid (0.091 g, 51%): m.p. 165-167 °C; νmax/cm (KBr)
2978, 1817, 1746, 1524, 1211, 741; δ (400 MHz, DMSO-d )
H
6

1
(
6
.34 (3H, t, J 7.1, CH
2H, q, J 7.2, CH CH
.86 [3H, m, C(6, 6′, 7)-H), 7.04 [1H, d, J 7.7, C(7′)-H] 7.10-7.16
2
CH
3
) 1.40 [6H, d, J 6.7, 2 × CH
3
], 4.30
[2H, t, J 7.4, C(5, 5′)-H], 7.44 [2H, d, J 8.2, C(4, 4′)-H], 7.84
(2H, s, C(2, 2′)-H], 10.44 (1 H, s, N-OH); δ (75 MHz, DMSO-
) 32.9 (2 × CH ), 104.6 (2C, 2 × aromatic C), 110.2 (2CH, 2 ×
2
3
), 4.82 [1H, sept, J 6.6 CH(CH
3
2
) ], 6.75-
c
d
6
3
[
8
2H, m, C(5, 5′)-H), 7.56 [1H, d, J 8.1, C(4)-H], 7.60 [1H, d, J
.3, C(4′)-H], 7.80 [1H, s, C(2′)-H], 7.93 [1H, s, C(2)-H]; δ (75
MHz, DMSO-d ) 15.1 (CH ), 22.1 (2 × CH ), 40.9 (CH ), 47.2
CH), 104.1 (C, aromatic C), 104.4 (C, aromatic C), 110.5 (CH,
aromatic CH), 119.6 (2CH, 2 × aromatic CH), 121.1 (2CH, 2 ×
aromatic CH), 121.8 (2CH, 2 × aromatic CH), 123.7 (2C, 2 ×
aromatic C), 125.7 (2C, 2 × aromatic C), 133.2 (2CH, 2 ×
aromatic CH), 136.5 (2C, 2 × aromatic C), 168.4 (2 × C=O); m/z
c
6
3
3
2
(
+
+
+
aromatic CH), 110.7 (CH, aromatic CH), 120.1 (CH, aromatic
CH), 120.2 (CH, aromatic CH), 121.65 (CH, aromatic CH),
(ESI ) 372.1 [(M+H) 100%]. HRMS (ESI ): Exact mass
+
calculated for (C22
18 3 3
H N O ) 372.1348. Found 372.1348.
1
21.73 (CH, aromatic CH), 122.1 (CH, aromatic CH), 122.2
3
-(1-Ethyl-1H-indol-3-yl)-1-hydroxy-4-(1-methyl-1H-indol-3-
(CH, aromatic CH), 125.0 (C, aromatic C), 125.3 (C, aromatic
yl)-1H-pyrrole-2,5-dione 49
C), 127.4 (C, aromatic C), 127.9 (C, aromatic C), 129.3 (CH,
aromatic CH), 132.9 (CH, aromatic CH), 135.5 (C, aromatic C),
Following general procedure (i) starting from 3-(1-ethyl-1H-
indol-3-yl)-4-(1-methyl-1H-indol-3-yl)furan-2,5-dione 42 (0.103
g, 0.27 mmol) in anhydrous DMF (10 mL) with hydroxylamine
hydrochloride (0.094 g, 1.3 mmol) and triethylamine (0.2 mL,
+
1
3
35.8 (C, aromatic C), 166.3 (C=O), 166.4 (C=O); m/z (ESI )
99.3 [(M+H) , 100%]; HRMS (ESI+): Exact mass calculated for
+
+
(C
25
H
23
N
2
O
3
) 399.1709. Found 399.1706.
0
.14 g, 1.3 mmol) gave desired product as a dark red solid (0.072
-1
g, 69%): m.p. = 236-238 °C; νmax/cm (KBr) 3239, 2932, 1763,
1
699, 1523, 1219, 739; δ
H
(300 MHz, DMSO-d
6
) 1.30 (3H, t, J
CH ),
Synthesis of bisindolyl hydroxymaleimides
7.6, CH CH ), 3.87 (3H, s, CH
2
3
3
), 4.24 (2H, q, J 7.1 CH
2
3
General procedure (i): To a solution of maleic anhydride (1 eq.)
in anhydrous DMF (30 mL/mmol) was added hydroxylamine
hydrochloride (5 eq.) followed by triethylamine (5 eq.). The
mixture was then heated to 70 °C and stirred at this temperature
for 24 hours. After cooling, water (100 mL/mmol) was added and
the mixture was extracted with ethyl acetate (3 × 40 mL/mmol).
The organic layer was washed with 1 M aqueous HCl (2 × 50
mL/mmol), followed by water (50 mL/mmol) and brine (50
mL/mmol) before being dried over anhydrous magnesium
sulfate, filtered and the solvent evaporated to give a dark red
solid.
6.62-6.69 [2H, m, C(6′, 7′)-H], 6.75 [1H, t, J 7.6, C(6)-H], 6.96
[1H, d, J 8.15, C(7)-H], 7.03-7.11 [2H, m, (5, 5′)-H], 7.45 [1H, d,
J 8.15, C(4′)-H], 7.50 [1H, d, 8.49, C(4)-H], 7.74 [1H, s, C(2)-H],
7.90 [1H, s, C(2′)-H], 10.47 (N-OH); δ (75 MHz, DMSO-d
c
6
)
15.2 (CH CH ), 32.9 (CH ), 40.7 (CH CH ), 104.4 (C, aromatic
2
3
3
2
3
C), 104.8 (C, aromatic C), 110.18 (CH, aromatic CH), 110.23
(CH, aromatic CH), 119.65 (CH, aromatic CH), 119.67 (CH,
aromatic CH), 121.2 (CH, aromatic CH), 121.3 (CH, aromatic
CH), 121.8 (2CH, 2 × aromatic CH), 123.4 (C, aromatic C),
124.1 (C, aromatic C), 125.2 (C, aromatic C), 126.0 (C, aromatic
C), 131.6 (CH, aromatic CH), 133.4 (CH, aromatic CH), 135.4
(
C, aromatic C), 136.6 (C, aromatic C), 168.37 (C=O), 168.41
1
-Hydroxy-3-(1H-indol-3-yl)-4-(1-methyl-1H-indol-3-yl)-1H-
+ + +
(C=O); m/z (ESI ) 386.2 [(M+H) 100%]. HRMS (ESI ): Exact
pyrrole-2,5-dione 47
+
mass calculated for (C23
20 3 3
H N O ) 386.1505. Found 386.1509.
Following general procedure (i) starting from 3-(1H-indol-3-yl)-
6
-{3-[1-Hydroxy-4-(1-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-
4
-(1-methyl-1H-indol-3-yl)furan-2,5-dione 39 (0.104 g, 0.29
dihydro-1H-pyrrol-3-yl]-1H-indol-1-yl}hexanenitrile 50
mmol) in anhydrous DMF (8 mL) with hydroxylamine
hydrochloride (0.101 g, 1.5 mmol) and triethylamine (0.20 mL,
Following general procedure (i) starting from 6-(3-(4-(1-
methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydrofuran-3-yl)-1H-
indol-1-yl)hexanenitrile 43 (0.103 g, 0.235 mmol) in anhydrous
DMF (8 mL) with hydroxylamine hydrochloride (0.079 g, 1.18
mmol) and triethylamine (0.16 mL, 0.12 g, 1.2 mmol) gave
desired product as a dark red solid (0.087 g, 82%): m.p. 123-
125 °C; νmax/cm (KBr) 3229, 2935, 2244, 1769, 1708, 1529,
1099, 742; δH (300 MHz, DMSO-d6) 1.22-1.39 [2H, m, C(4′′)-
H2], 1.57 [2H, quin, J 7.27, C(3′′)- H2], 1.73 [2H, quin, J 7.04,
C(5′′)-H2], 2.45 [2H, t, J 7.2, C(2′′)-H2], 3.88 (3H, s, CH3),
4.23 [2H, t, J 6.9, C(6′′)-H2], 6.62-6.68 [2H, m, C(6′, 7′)-H],
6.73 [1H, t, J 7.7, C(6)-H], 6.94 [1H, d, J 7.8, C(7)-H], 7.01-
7.10 [2H, m, (5, 5′)-H], 7.45 [1H, d, J 8.0, C(4′)-H], 7.50 [1H,
d, J 8.2, C(4)-H], 7.75 [1H, s, C(2)-H], 7.90 [1H, s, C(2′)-H],
10.43 (1H, s, NOH); δc (75 MHz, DMSO-d6) 16.0 (CH2), 24.3
(CH2), 25.2 (CH2), 28.8 (CH2), 32.9 (CH3), 45.9 (CH2), 104.4
(C, aromatic C), 104.9 (C, aromatic C), 110.2 (CH, aromatic
CH), 110.3 (CH, aromatic CH), 119.6 (2 × CH, 2 aromatic CH),
0
.15 g, 1.5 mmol) gave desired product as a dark red solid (83%):
-1
m.p. 137-139 °C; νmax/cm (KBr) 3332, 2930, 1767, 1706, 1612,
1
6
6
H 6 3
529, 1460, 1023; δ (300 MHz, DMSO-d ) 3.87 (3H, s, CH ),
.61-6.74 [3H, m, C(6, 6′, 7′)-H], 6.83 [1H, d, J 8.0, C(7)-H],
.99 [1H, t, J 7.5, C(5)-H], 7.04 [1H, t, J 7.5, C(5′)-H], 7.39 [1H,
-1
d, J 8.0, C(4)-H], 7.44 [1H, d, J 8.1, C(4′)-H], 7.77 [1H, d, J 7.8,
C(2)-H], 7.87 [1H, s, C(2′)-H], 10.45 (1H, s, N-OH), 11.74 (1H,
bs, NH); δ (75 MHz, DMSO-d ) 32.9 (CH ), 104.5 (C, aromatic
c 6 3
C), 105.5 (C, aromatic C), 110.2 (CH, aromatic CH), 111.9 (CH,
aromatic CH), 119.4 (CH, aromatic CH), 119.7 (CH, aromatic
CH), 120.8 (CH, aromatic CH), 121.0 (CH, aromatic CH), 121.7
(
CH, aromatic CH), 121.8 (CH, aromatic CH), 123.9 (C,
aromatic C), 124.0 (C, aromatic C), 125.4 (C, aromatic C), 125.6
C, aromatic C), 129.3 (CH, aromatic CH), 133.2 (CH, aromatic
CH), 135.9 (C, aromatic C), 136.5 (C, aromatic C), 168.5 (2 ×
(
-
-
-
C=O); m/z (ESI ) 356.2 [(M-H) 50%]. HRMS (ESI ): Exact mass
calculated for (C21
-
14 3 3
H N O ) 356.1035. Found 356.1025.
1
20.6 (CN), 121.1 (CH, aromatic CH), 121.2 (CH, aromatic
CH), 121.77 (CH, aromatic CH), 121.81 (CH, aromatic CH),
23.5 (C, aromatic C), 124.2 (C, aromatic C), 125.3 (C,
aromatic C), 126.0 (C, aromatic C), 132.0 (CH, aromatic CH),
33.3 (CH, aromatic CH), 135.7 (C, aromatic C), 136.6 (C,
1
-Hydroxy-3,4-bis(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-
dione 48
1
Following general procedure (i) starting from 3,4-bis(1-methyl-
1
1
H-indol-3-yl)furan-2,5-dione 41 (0.092 g, 0.26 mmol) in
anhydrous DMF (10 mL) with hydroxylamine hydrochloride
0.090 g, 1.3 mmol) and triethylamine (0.18 mL, 0.13 g, 1.3
mmol) gave desired product as a dark red solid (0.081 g, 84%):
aromatic C), 168.3 (C=O), 168.4 (C=O); m/z (ESI-) 451.3 [(M-
H)- 50%]; HRMS (ESI-): Exact mass calculated for
(
-
(C
27
H
23
N
4
O
3
) 451.1770. Found 451.1783.
-1
m.p. >300 °C; νmax/cm (KBr) 3224, 1764, 1694, 1610, 1519,
367, 1097; δ (300 MHz, DMSO-d ) 3.86 (6H, s, 2 × CH ), 6.67
2H, t, J 7.4, C(6, 6′)-H], 6.76 [2H, d, J 7.9, C(7, 7′)-H], 7.05
6
-{3-[1-Hydroxy-4-(1-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-
1
[
H
6
3
dihydro-1H-pyrrol-3-yl]-1H-indol-1-yl}hexanoic acid 51

Following general procedure (i) starting from 6-{3-[4-(1-
methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydrofuran-3-yl]-1H
s, N-OH); δ
c
(75 MHz, DMSO-d
6
)
3
22.1 (2 × CH ), 32.9
-
[CH(CH ], 47.0 (CH
)
3 2
3
), 104.3 (C, aromatic C), 105.1 (C,
indol-1-yl}hexanoic acid 44 (0.101 g, 0.221 mmol) in
anhydrous DMF (8 mL) with hydroxylamine hydrochloride
aromatic C), 110.2 (CH, aromatic CH), 110.3 (CH, aromatic
CH), 119.7 (CH, aromatic CH), 119.8 (CH, aromatic CH), 121.3
(2CH, 2 × aromatic CH), 121.75 (CH, aromatic CH), 121.77
(CH, aromatic CH), 123.4 (C, aromatic C), 124.6 (C, aromatic
C), 124.9 (C, aromatic C), 126.0 (C, aromatic C), 128.2 (CH,
aromatic CH), 133.6 (CH, aromatic CH), 135.3 (C, aromatic C),
(0.076 g, 1.11 mmol) and triethylamine (0.15 mL, 0.11 g, 1.1
mmol) gave desired compound as a dark red solid (0.085 g,
-1
7
9%): m.p. 196-197 °C; νmax/cm (KBr) 3114, 2935, 1710,
1609, 1529, 1098, 741; δH (300 MHz, DMSO-d6) 1.16-1.29
+
[2H, m, C(4′′)-H2], 1.51 [2H, quin, J 7.4, C(3′′)-H2], 1.70 [2H,
136.7 (C, aromatic C), 168.3 (C=O), 168.4 (C=O); m/z (ESI )
+
quin, J 7.1, C(5′′)-H2], 2.17 [2H, t, 7.3, C(2′′)-H2], 3.87 (3H, s,
CH3), 4.21 [2H, t, J 6.7, C(6′′)-H2], 6.58-6.66 [2H, m, C(6′, 7′)-
H], 6.72 [1H, t, J 7.4, C(6)-H], 6.93 [1H, d, J 8.0, C(7)-H],
400.2 [(M+H) , 100%]; HRMS (ESI+): Exact mass calculated for
+
(C24H
22
N
3
O
3
) 400.1661. Found 400.1651.
3
3
-(1-Ethyl-1H-indol-3-yl)-1-hydroxy-4-(1-isopropyl-1H-indol-
-yl)-1H-pyrrole-2,5-dione 54
7
[
.00-7.09 [2H, m, (5, 5′)-H], 7.43 [1H, d, J 8.3, C(4′)-H], 7.48
1H, d, J 8.3, C(4)-H], 7.73 [1H, s, C(2)-H], 7.90 [1H, s, C(2′)-
H], 10.43 (1H, bs, NH), 11.99 (1H, bs, COOH); δc (75 MHz,
DMSO-d6) 24.0 (CH ), 25.6 (CH ), 29.3 (CH ), 32.9 (CH ),
3.5 (CH ), 45.6 (CH ), 104.4 (C, aromatic C), 104.8 (C,
Following general procedure (i) starting from 3-(1-ethyl-1H-
indol-3-yl)-4-(1-isopropyl-1H-indol-3-yl)furan-2,5-dione 46
2
2
2
3
3
2
2
(0.068 g, 0.17 mmol) in anhydrous DMF (8 mL) with
hydroxylamine hydrochloride (0.059 g, 0.85 mmol) and
triethylamine (0.12 mL, 0.87 g, 0.85 mmol) gave desired product
aromatic C), 110.17 (CH, aromatic CH), 110.24 (CH, aromatic
CH), 119.57 (CH, aromatic CH), 119.59 (CH, aromatic CH),
-1
1
(
21.10 (CH, aromatic CH), 121.12 (CH, aromatic CH), 121.8
2CH, 2 × aromatic CH), 123.5 (C, aromatic C), 124.3 (C,
aromatic C), 125.3 (C, aromatic C), 126.0 (C, aromatic C),
32.0 (CH, aromatic CH), 133.3 (CH, aromatic CH), 135.7 (C,
aromatic C), 136.6 (C, aromatic C), 168.4 (C=O), 168.4 (C=O),
74.3 (COOH); m/z (ESI+) 472.2 [(M+H)+ 100%]; HRMS
as a dark red solid (0.056 g, 80%): m.p. 83-85 °C; νmax/cm
(KBr) 3125, 2975, 1769, 1713, 1527, 1214, 741; δ (300 MHz,
H
DMSO-d ,) 1.34 (3H, t, J 7.2, CH CH ) 1.39 (6H, d, J 6.6, 2 ×
6
2
3
1
3 2 3
CH ), 4.27 (2H, q, J 7.2, CH CH ), 4.79 [1H, sept, J 6.6
CH(CH ) ], 6.66-6.72 [2H, m, C(6, 7)-H], 6.76 [1H, t, J 7.6,
3
2
1
C(6′)-H], 6.95 [1H, d, J 7.8, C(7′)-H], 7.04-7.11 [2H, m, C(5, 5′)-
H], 7.50 [1H, d, J 8.3, C(4)-H], 7.55 [1H, d, J 8.4, C(4′)-H], 7.74
[1H, s, C(2′)-H], 7.85 [1H, s, C(2)-H], 10.47 (1H, s, N-OH), δc
+
26 3 5
(ESI+): Exact mass calculated for (C27H N O ) 472.1872.
Found 472.1864.
(
4
(
75 MHz, DMSO-d
7.0 (CH), 104.6 (C, aromatic C), 105.0 (C, aromatic C), 110.2
CH, aromatic CH), 110.4 (CH, aromatic CH), 119.7 (CH,
6 3 3 2
) 15.2 (CH ), 22.1 (2 × CH ), 40.7 (CH ),
1
1
-Hydroxy-3-(1H-indol-3-yl)-4-(1-isopropyl-1H-indol-3-yl)-
H-pyrrole-2,5-dione 52
Following general procedure (i) starting from 3-(1H-indol-3-yl)-
-(1-isopropyl-1H-indol-3-yl)furan-2,5-dione 40 (0.120 g, 0.31
mmol) in anhydrous DMF (8 mL) with hydroxylamine
hydrochloride (0.11 g, 1.6 mmol) and triethylamine (0.23 mL,
aromatic CH), 119.8 (CH, aromatic CH), 121.38 (CH, aromatic
CH), 121.44 (CH, aromatic CH), 121.7 (CH, aromatic CH),
121.8 (CH, aromatic CH), 123.9 (C, aromatic C), 124.3 (C,
aromatic C), 125.3 (C, aromatic C), 125.7 (C, aromatic C), 128.4
(CH, aromatic CH), 131.9 (CH, aromatic CH), 135.4 (C,
aromatic C), 135.6 (C, aromatic C), 168.3 (C=O), 168.4 (C=O);
4
0
.17 g, 1.6 mmol) gave desired product as a dark red solid (0.097
-1
g, 77%): m.p. 106-109 °C; νmax/cm (KBr) 3118, 3135, 2970,
812, 1745, 1627, 1529, 1247; δ (300 MHz, DMSO-d ) 1.38
6H, d, J 6.6, 2 × CH ), 4.78 [1H, sept, J 6.7 CH(CH ], 6.63
2H, m, C(6, 7)-H], 6.78 [1H, t, J 7.2, C(6′)-H], 6.96-7.10 [3H,
+
+
1
H
6
m/z (ESI ) 414.2 [(M+H) , 100%]; HRMS (ESI+): Exact mass
+
(
(
3
)
3 2
calculated for (C25
H
24
N
3
O
3
) 414.1818. Found 414.1814.
1
1
1
9-Hydroxy-6,7,8,9,10,11-hexahydro-5,21:12,17-dimetheno-
8H-dibenzo[i,o]pyrrolo[3,4-l][1,8]diazacyclohexadecine-
8,20(19H)-dione 55
m, C(5, 5′, 7′)-H], 7.39 [1H, d, J 8.1, C(4)-H], 7.53 [1H, d, J 8.3,
C(4′)-H], 7.69 [1H, s, C(2′)-H], 7.83 [1H, d, J 2.8, C(2)-H], 10.42
(1H, s, N-OH), 11.74 (1H, s, N-H); δ
c 6
(75 MHz, DMSO-d ) 22.1
[
CH(CH ], 47.0 [CH(CH ], 105.0 (C, aromatic C), 105.2 (C,
3
)
2
3
)
2
Following general procedure (i) starting from 6,7,8,9,10,11-
hexahydro-5,21:12,17-dimethenodibenzo[i,o]furo[3,4-
aromatic C), 110.3 (CH, aromatic CH), 111.9 (CH, aromatic
CH), 119.5 (CH, aromatic CH), 119.8 (CH, aromatic CH), 121.1
l][1,8]diazacyclohexadecine-18,20-dione 27 (0.151 g, 0.37
mmol) in anhydrous DMF (10 mL) with hydroxylamine
hydrochloride (0.127 g, 1.8 mmol) and triethylamine (0.26 mL,
0.19 g, 1.8 mmol) gave desired product as a purple solid (0.129
(
CH, aromatic CH), 121.3 (CH, aromatic CH), 121.71 (CH,
aromatic CH), 121.73 (CH, aromatic CH), 123.6 (C, aromatic C),
24.6 (C, aromatic C), 124.9 (C, aromatic C), 125.9 (C, aromatic
C), 128.2 (CH, aromatic CH), 129.8 (CH, aromatic CH), 135.3
1
-1
g, 82%): m.p. >300 °C; νmax/cm (KBr) 3373, 2924, 1768, 1712,
1619, 1534, 1470, 1390, 735; δ (300 MHz, DMSO-d ) 1.06 [4H,
(
C, aromatic C), 136.1 (C, aromatic C), 168.37 (C=O), 168.44
H
6
+ +
(C=O); m/z (ESI ) 386.4 [(M+H) , 100%]; HRMS (ESI+): Exact
2 2
bs, C(8, 9)-H ], 1.80 [4H, bs, C(7, 10)-H ], 4.12-4.22 [4H, m,
C(6, 11)-H ], 7.14 [2H, ddd, J 7.9, 7.2, 1.0, C(2, 15)-H], 7.21
2
+
mass calculated for (C23
20
H N
3
O
3
) 386.1505. Found 386.1495.
[
7
2H, ddd, J 8.2, 7.1, 1.2, C(3, 14)-H], 7.39 [2H, s, C(22, 23)-H],
.51 [2H, d, J 7.8, C(4, 13)-H], 7.81 [2H, dd, J 7.1, 0.9, C(1, 16)-
H], 10.47 (N-OH); δ (75 MHz, DMSO-d ) 22.7 (2 × CH ), 27.3
(2 × CH ), 44.2 (2 × CH ), 102.5 (2C, 2 × aromatic C), 110.5
1
-Hydroxy-3-(1-isopropyl-1H-indol-3-yl)-4-(1-methyl-1H-
Indol-3-yl)-1H-pyrrole-2,5-dione 53
c
6
2
Following general procedure (i) starting from 3-(1-isopropyl-1H-
indol-3-yl)-4-(1-methyl-1H-indol-3-yl)furan-2,5-dione 45 (0.099
g, 0.26 mmol) in anhydrous DMF (8 mL) with hydroxylamine
hydrochloride (0.089 g, 1.29 mmol) and triethylamine (0.18 mL,
2
2
(2CH, 2 × aromatic CH), 120.2 (2CH, 2 × aromatic CH), 121.4
(2CH, 2 × aromatic CH), 121.7 (2CH, 2 × aromatic CH), 126.8
(2C, 2 × aromatic C), 128.6 (2C, 2 × aromatic C), 131.9 (2CH, 2
× aromatic CH), 135.2 (2C, 2 × aromatic C), 167.7 (2 × C=O);
m/z (ESI+) 426.2 [(M+H)+ 10%]; HRMS (ESI+): Exact mass
calculated for (C26
0.13 g, 1.29 mmol) gave desired product as a dark red solid
-1
(
1
[
[
0.096 g, 93%): m.p. 84-86 °C; νmax/cm (KBr) 3118, 2930, 1712,
654; δ (300 MHz, DMSO-d ) 1.37 (6H, d, J 6.6, 2 × CH ), 4.78
1H, sept, J 6.6, CH(CH ], 6.52 [1H, d, J 7.7, C(7)-H] 6.63
1H, t, J 7.2, C(6)-H], 6.80 [1H, t, J 7.3, C(6′)-H], 7.03-7.11 [3H,
+
H
6
3
H
24
N
3
O
3
) 426.1818. Found 426.1827.
3 2
)
6
,6'-[3,3'-(1-Hydroxy-2,5-dioxo-2,5-dihydro-1H-pyrrole-3,4-
diyl)bis(1H-indole-3,1-diyl)]dihexanenitrile 56
m, C(5, 5′, 7)-H], 7.45 [1H, d, J 8.2, C(4)-H], 7.54 [1H, d, J 8.5,
C(4′)-H], 7.67 [1H, s, C(2′)-H], 7.93 [1H, s, C(2)-H], 10.48 (1H,

-
1
Following general procedure (i) starting from 6,6'-[3,3'-(2,5-
dioxo-2,5-dihydrofuran-3,4-diyl)bis(1H-indole-3,1-
(1.165 g, 90%): m.p. 82-84 °C; νmax/cm (KBr) 3274, 2936,
2244, 1704, 1533, 1334, 1160, 743; δH (300 MHz, CDCl
1.39-1.51 [4H, m, 2 × C(4′′)-H ], 1.65 [4H, quin, J 7.3, 2 ×
C(3′′)-H ], 1.87 [4H, quin, J 7.3, 2 × C(5′′)-H ], 2.30 [4H, t, J
7.0, 2 × C(2′′)-H ], 4.15 [4H, t, J 7.0, 2 × C(6′′)-H ], 6.76 [2H,
3
)
diyl)]dihexanenitrile 28 (0.084g, 0.162 mmol) in anhydrous
DMF (8 mL) with hydroxylamine hydrochloride (0.056 g, 0.809
mmol) and triethylamine (0.11 mL, 0.08 g, 0.81 mmol) gave
desired product as a dark red solid (0.076 g, 87%): m.p. 83-84
2
2
2
2
2
t, J 7.4, C(6, 6′)-H], 6.99 [2H, d, J 8.0, C(7, 7′)- H], 7.11 [2H, t,
J 7.6, C(5, 5′)-H], 7.28 [2H, d, J 8.3, C(4, 4′)-H], 7.43 (1H, s,
NH), 7.63 [1H, s, C(2, 2′)-H]; δc (75 MHz, CDCl
CH ), 25.0 (2 × CH ), 25.9 (2 × CH ), 29.2 (2 × CH
CH ), 105.9 (2C, 2 × aromatic C), 109.5 (2CH, 2 × aromatic
-1
°
1
C; νmax/cm (KBr) 3257, 2936, 2244, 1771, 1713, 1530, 1392,
159, 743; δH (300 MHz, DMSO-d6) 1.25-1.38 [4H, m, 2 ×
3
) 17.0 (2 ×
), 46.3 (2 ×
C(4′′)-H2], 1.57 [4H, quin, J 7.4, 2 × C(3′′)-H2], 1.75 [4H, quin,
J 7.2, 2 × C(5′′)-H2], 2.45 [4H, t, J 7.1, 2 × C(2′′)-H2], 4.25
2
2
2
2
2
[
[
[
4H, t, J 6.9, × C(6′′)-H2], 6.68 [2H, t, J 7.4, C(6, 6′)-H], 6.80
2H, d, J 7.7, C(7, 7′)-H], 7.05 [2H, t, J 7.6, C(5, 5′)-H], 7.50
2H, d, J 8.3, C(4, 4′)-H], 7.82 [2H, s, C(2, 2′)-H], 10.43 (N-
CH), 119.4 (2 × CN), 120.1 (2CH, 2 × aromatic CH), 122.3
(2CH, 2 × aromatic CH), 122.4 (2CH, 2 × aromatic CH), 126.2
(2C, 2 × aromatic C), 127.8 (2C, 2 × aromatic C), 131.7 (2CH,
2 × aromatic CH), 136.2 (2C, 2 × aromatic C), 172.3 (2 ×
OH); δc (75 MHz, DMSO-d6) 16.0 (2 × CH
2
), 24.3 (2 × CH
2
),
2
5.2 (2 × CH ), 28.7 (2 × CH ), 45.4 (2 × CH
2
2
2
), 104.7 (2C, 2 ×
C=O); m/z (ESI+) 518.2 [(M+H)+ 100%]; HRMS (ESI+):
+
aromatic C), 110.3 (2CH, 2 × aromatic CH), 119.6 (2CH, 2 ×
aromatic CH), 120.5 (2 × CN), 121.2 (2CH, 2 × aromatic CH),
Exact mass calculated for (C32
H
32
N
5
O
2
)
518.2556. Found
518.2554.
1
1
1
21.8 (2CH, 2 × aromatic CH), 124.0 (2C, 2 × aromatic C),
25.6 (2C, 2 × aromatic C), 132.1 (2CH, 2 × aromatic CH),
35.8 (2C, 2 × aromatic C), 168.3 (2 × C=O); m/z (ESI+) 534.3
Acknowledgments
[
(M+H)+ 40%]; HRMS (ESI+): Exact mass calculated for
The authors would like to acknowledge the Irish Research
Council for Science, Engineering and Technology and the
Ulysses scheme for funding this research, the National Cancer
Institute (NCI) screening program for 60-cell line testing. The
authors also thank the Cancéropôle Grand-Ouest (axis: Natural
sea products in cancer treatment), the Ligue Contre le Cancer
(CD29, 35, 22 and 75), GIS IBiSA (Infrastructures en Biologie
Santé et Agronomie, France) and Biogenouest (Western France
life science and environment core facilty network) for supporting
KISSf screening facility (Roscoff, France).
+
32 32 5 3
(C H N O ) 534.2505. Found 534.2496.
6
1
-{3-[1-Hydroxy-4-(1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-
H-pyrrol-3-yl]-1Hindol-1-yl}hexanoic acid 57
Following general procedure (i) starting from 6-{3-[4-(1Hindol-
-yl)-2,5-dioxo-2,5-dihydrofuran-3-yl]-1H-indol-1-yl}hexanoic
3
acid 30 (0.120 g, 0.271 mmol) in anhydrous DMF (10 mL) with
hydroxylamine hydrochloride (0.094 g, 1.36 mmol) and
triethylamine (0.19 mL, 0.14 g, 1.4 mmol) gave desired product
-1
as a dark red solid (0.107 g, 86%): m.p. 133-135 °C; νmax/cm
KBr) 3372, 2935, 1768, 1703, 1612, 1527, 1391, 1097, 735;
δH (400 MHz, DMSO-d6) 1.18-1.27 [2H, m, C(4′′)-H2], 1.51
2H, quin, J 7.5, C(3′′)-H2], 1.71 [2H, quin, J 7.3, C(5′′)-H2],
.18 [2H, t, J 7.4, C(2′′)-H], 4.22 [2H, t, J 6.9, C(6′′)-H], 6.60
1H, t, J 7.6, C(6)-H], 6.67-6.73 [2H, m, C(6′, 7)-H], 6.87 [1H,
(
This article is based upon work from COST Action CA15135,
supported by COST.
[
2
[
Author Contributions
d, J 8.2, C(7′)-H], 6.98 [1H, t, J 7.6, C(5)-H], 7.04 [1H, t, J 7.7,
C(5′)-H], 7.38 [1H, d, J 8.2, C(4)-H], 7.48 [1H, d, J 8.3, C(4′)-
H], 7.77 [1H, s, C(2′)-H], 7.82 [1H, d, J 2.9, C(2)-H], 10.45
HMW/KOS/MMC/LTP performed the research in relation to the
synthesis and characterization on novel compounds. TR/SB/SR
designed and performed the kinase assays. PM reviewed the draft
manuscript; FOM designed the research and drafted the
manuscript.
(
1H, s, NOH), 11.75 (1H, bd, J 2.4, NH), 12.02 (1H, bs,
COOH); δc (75 MHz, DMSO-d6) 24.0 (CH ), 25.6 (CH ), 29.3
CH ), 33.5 (CH ), 45.6 (CH ), 104.7 (C, aromatic C), 105.4 (C,
2
2
(
2
2
2
aromatic C), 110.2 (CH, aromatic CH), 111.8 (CH, aromatic
CH), 119.4 (CH, aromatic CH), 119.6 (CH, aromatic CH),
References and notes
1
(
20.9 (CH, aromatic CH), 121.1 (CH, aromatic CH), 121.7
CH, aromatic CH), 121.8 (CH, aromatic CH), 123.8 (C,
aromatic C), 124.6 (C, aromatic C), 125.0 (C, aromatic C),
25.9 (C, aromatic C), 129.5 (CH, aromatic CH), 132.0 (CH,
aromatic CH), 135.8 (C, aromatic C), 136.0 (C, aromatic C),
68.39 (C=O), 168.44 (C=O), 174.3 (COOH); m/z (ESI+) 458.2
(M+H)+ 100%]; HRMS (ESI+): Exact mass calculated for
1
2.
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[
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,6'-[3,3'-(2,5-Dioxo-2,5-dihydro-1H-pyrrole-3,4-
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0-72.

Ssc
GSK-
Compound
Hs
Hs
Hs
Hs
Hs
Hs
Hs
Hs
Ssc
HASPIN AURKB RIPK3 CDK2/CyclinA CDK5/p25 CDK9/CyclinT DYRK1A
PIM1 CK1δ/ε
3
α/β
1
2
4
4
4
5
5
5
5
5
5
5
5
5
5
2
7
8
9
0
1
2
3
4
5
6
7
8
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.10
>10
>10
>10
>10
>10
4.0
0.80
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.08
>10
2.0
>10
>10
>10
>10
>10
5.00
1.00
>10
>10
>10
>10
>10
2.00
0.62
0.03
4.0
2.0
4.5
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.20
3.0
5.0
9.0
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
1.5
0.30
0.27
3.20
1.5
2.10
0.70
>10
1.5
>10
7.0
0.20
0.40
>10
0.75
0.75
0.12
>10
>10
>10
>10
2.50
0.70
>10
>10
2.00
0.42
Table 2
IC50 values from the kinase inhibition assay (values quoted in M)

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Synthesis and anticancer activity of novel
bisindolylhydroxymaleimide derivatives with
potent GSK-3 kinase inhibition
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a
a
a
a
b
Hannah J. Winfield , Michael M. Cahill , Kevin D. O’Shea , Larry T. Pierce , Thomas Robert , Sandrine
b
b
c
a
Ruchaud , Stéphane Bach , Pascal Marchand and Florence O. McCarthy *
a
School of Chemistry, Analytical and Biological Chemistry Research Facility, University College Cork,
Western Road, Cork, Ireland
b
Sorbonne Universités, UPMC Univ Paris 06, CNRS USR3151, Kinase Inhibitor Specialized Screening facility,
KISSf, Station Biologique, Place Georges Teissier, Roscoff, France
c
Département de Chimie Thérapeutique, EA1155 - IICiMed, Université de Nantes, Institut de Recherche en
Santé 2, Nantes, France