Characterization of maleimide-based glycogen synthase kinase-3 (GSK-3) inhibitors as stimulators of steroidogenesis

Article abstract of DOI:10.1021/jm400511s

Inhibition of GSK-3β has been well documented to account for the behavioral actions of the mood stabilizer lithium in various animal models of mood disorders. Recent studies have showed that genetic or pharmacological inhibition of GSK-3β resulted in anxiolytic-like and pro-social behavior. In our ongoing efforts to develop GSK-3β inhibitors for the treatment of mood disorders, SAR studies on maleimide-based compounds were undertaken. We present herein for the first time that some of these GSK-3β inhibitors, in particular analogues 1 and 9, were able to stimulate progesterone production in the MA-10 mouse tumor Leydig cell model of steroidogenesis without any significant toxicity. These two compounds were tested in the SmartCube behavioral assay and showed anxiolytic-like signatures following daily dose administration (50 mg/kg, ip) for 13 days. Taken together, these results support the hypothesis that GSK-3β inhibition could influence neuroactive steroid production thereby mediating the modulation of anxiety-like behavior in vivo.

Full text of DOI:10.1021/jm400511s

Article
pubs.acs.org/jmc
Characterization of Maleimide-Based Glycogen Synthase Kinase‑3
(GSK-3) Inhibitors as Stimulators of Steroidogenesis
Hendra Gunosewoyo,† Andrew Midzak,‡ Irina N. Gaisina,† Emily V. Sabath,§ Allison Fedolak,§
Taleen Hanania,§ Dani Brunner,§ Vassilios Papadopoulos,‡ and Alan P. Kozikowski*,†
†Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois
60612, United States
‡The Research Institute of the McGill University Health Centre and Departments of Medicine, Biochemistry and Pharmacology &
Therapeutics, McGill University, Montreal General Hospital, 1650 Cedar Avenue, Montreal, Quebec H3G 1A4, Canada
§Psychogenics, Inc., 765 Old Saw Mill River Road, Tarrytown, New York 10591, United States
S
* Supporting Information
ABSTRACT: Inhibition of GSK-3β has been well documented to
account for the behavioral actions of the mood stabilizer lithium in
various animal models of mood disorders. Recent studies have
showed that genetic or pharmacological inhibition of GSK-3β
resulted in anxiolytic-like and pro-social behavior. In our ongoing
efforts to develop GSK-3β inhibitors for the treatment of mood
disorders, SAR studies on maleimide-based compounds were
undertaken. We present herein for the first time that some of
these GSK-3β inhibitors, in particular analogues 1 and 9, were able to
stimulate progesterone production in the MA-10 mouse tumor
Leydig cell model of steroidogenesis without any significant toxicity.
These two compounds were tested in the SmartCube behavioral
assay and showed anxiolytic-like signatures following daily dose
administration (50 mg/kg, ip) for 13 days. Taken together, these results support the hypothesis that GSK-3β inhibition could
influence neuroactive steroid production thereby mediating the modulation of anxiety-like behavior in vivo.
genesis, and cell survival/apoptosis.11,12 Two homologues of
GSK-3 exist, GSK-3α (51 kDa) and GSK-3β (47 kDa), which
INTRODUCTION
■
The mood stabilizer lithium, commonly available in the
share more than 95% amino acid sequence homology in the
carbonate form (Eskalith), has remained as a primary treatment
catalytic site, with the latter being predominantly expressed in
of choice for manic-depressive illness with demonstrated
antisuicidal and neuroprotective efficacies.1,2 Although the
exact molecular mechanisms underlying the actions of lithium
are still not completely understood, it has been reported to
the nervous system. GSK-3 is constitutively active in basal
nonstimulated cells, thereby catalyzing the phosphorylation of
numerous signaling molecules such as glycogen synthase, β-
catenin transcription factor, and tau proteins, leading to their
directly inhibit a series of biologically relevant enzymes
degradation.13,14 Phosphorylation of the Tyr279 and Tyr 216
including, but not limited to, glycogen synthase kinase-3
on GSK-3α and GSK-3β, respectively, is required for their
activation, while Ser21/Ser9 phosphorylation at GSK-3α/β is
necessary for their enzymatic inhibition. Significantly, GSK-3
(GSK-3), inositol monophosphatase, phosphoglucomutase,
other inositol polyphosphatases, and monophosphoesterases.3
Other proposed mechanisms of lithium action in psychiatric
has also been shown to be regulated not only by the mood
disorders also emerged, such as the ionic hypothesis,
stabilizers lithium and valproate but also by antidepressants and
modulation of neurotransmitter signaling, arachidonic acid
antipsychotics that modulate the key neurotransmitters signal-
metabolism, and modulation of the hypothalamus−pituitary−
ing in the brain such as dopamine and serotonin.15,16 Taken
together, these findings support the role of GSK-3 inhibition as
a key convergence point for at least some neuropsychiatric
drugs routinely used for the treatment of mental disorders
although the molecular mechanisms by which GSK-3 inhibition
elicits mood stabilization remains elusive.17,18
adrenal (HPA) axis activity.4,5 Among these reported possible
molecular mechanisms of lithium’s action, the inhibition of
GSK-3, either directly or through the destabilization of the Akt/
β-arrestin/protein phosphatase 2A complex, has been well
documented to account for the behavioral actions of lithium in
animal models.6−10 GSK-3 is a multifunctional serine/threonine
kinase responsible for the control of a number of central
physiological events, such as cell membrane signal-to-gene
transcription, metabolic homeostasis, cell development, neuro-
Received: April 8, 2013
Published: May 31, 2013
© 2013 American Chemical Society
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Recently, selective deletion of forebrain GSK-3β in mice was
reported to exert anxiolytic-like and pro-social behavioral
effects.19 In line with this study, lithium per se or in
combination with other drugs has been demonstrated to
suppress anxiety-like symptoms in different behavioral para-
digms using mouse models of Fragile X syndrome,
Huntington’s disease, and cerebral ischemia.20−24 In the context
of anxiety disorders, alteration of the levels of neuroactive
steroids such as pregnenolone and progesterone has been
implicated in the disease pathophysiology.25 The anxiolytic
effects of these neuroactive steroids are attributed to the
binding to GABAA receptors which manifests in the
potentiation of GABA-induced Cl− currents. A significant
body of research in stress physiology has revealed the important
roles of progesterone and its metabolite allopregnanolone in
the modulation of HPA axis.26 Of particular relevance to the
pathophysiology of anxiety disorders, dysregulation of the HPA
system has been observed in patients and normalization with
lithium therapy suggested that interactions of lithium with the
HPA axis may contribute to its therapeutic effects.5
Interestingly, inhibition of GSK-3β (increased Ser9 phosphor-
ylation) and the concomitant β-catenin accumulation has been
reported to be an important signaling pathway for the ability of
luteal cells to induce progesterone secretion when exposed to
luteinizing hormone.27 Our group previously identified
maleimide 1 (Figure 1) as a potent and selective, brain-
upon potency and water solubility (Figure 1) following the
synthetic routes depicted in Schemes 1−4. Commercially
available indoles 32a and 32b were initially alkylated with O-
trityl-3-bromopropan-1-ol or O-trityl-2-bromoethan-1-ol in
N,N-dimethylformamide (DMF) as a solvent and sodium
hydride as a base (Scheme 1). Treatment of the alkylated
indoles 33 with ethyl chlorooxoacetate in diethyl ether afforded
the glyoxalates 34. Perkin-type condensation30 between the
glyoxalates and benzofuran-3-yl-acetamide28 generated the
desired maleimides 2−4. Alkylation of 32a and 32b with tert-
butyl (2-bromoethyl)carbamate followed by treatment of the
intermediary indoles 35a and 35b with ethyl chlorooxoacetate
gave the glyoxalates 36a and 36b. Maleimide formation with
benzofuran-3-yl-acetamide and subsequent Boc deprotection
using trifluoroacetic acid (TFA) in chloroform yielded the
target compounds 5 and 6. The N-methylaminopropyl (7, 8)
and 2-methoxyethyl analogues (9−12) were synthesized in a
similar fashion. Commercially available 7-azaindoles 37a and
37b were subjected to the sequential N-alkylation, glyoxylation,
and condensation with benzofuran-3-yl-acetamide to give
maleimides 13−16 (Scheme 2).31,32 6-Aza-5-methoxyindole
was used as a starting material for target compounds 17 and 18.
2-(Imidazo[1,2-a]pyridin-3-yl)acetamide33 38 was condensed
with the appropriate glyoxalates to afford the final maleimides
19 and 20. For compounds 21−23, indole-7-carboxaldehyde 39
was first alkylated with the appropriate alkyl bromides and
subjected to sequential Wittig reaction with methylenetriphe-
nylphosphorane and then hydroboration. Alkylation of the
intermediary alcohols 40a and 40b followed by treatment with
ethyl chlorooxoacetate resulted in the formation of the
glyoxalates 41a−c, which upon condensation with benzofur-
an-3-yl-acetamide gave the desired maleimides 21−23.
To access target compounds 24, 25, and 27−31 (Scheme 3),
indole-7-carboxaldehyde 39 or 5-fluoroindole-7-carboxaldehyde
42 were initially subjected to a reductive amination protocol
using ethanolamine and hydrogen gas with palladium(0)
catalyst following a previously reported literature protocol.34
Sequential Boc protection of the secondary amine, tosylation of
the hydroxyl group and sodium hydride-assisted cyclization34
gave the intermediary 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-
hi]indoles 43a and 43b. Glyoxylation with ethyl chloroox-
oacetate, condensation with benzofuran-3-yl-acetamide and
subsequent Boc deprotection afforded the maleimides 24 and
25. Amide coupling of the maleimides with the appropriate
carboxylic acids using N-(3-dimethylaminopropyl)-N′-ethyl-
carbodiimide hydrochloride (EDC·HCl) and HOBt as the
coupling reagent or treatment with carbamoyl chlorides gave
the target compounds 27−31. For the bromo analogue 26
(Scheme 4), 5-bromoindoline 44 was first alkylated with 2-
chloroethylamine hydrochloride using potassium carbonate as a
base in DMF in a pressure tube and treated with aqueous
formaldehyde in acetic acid and sulfuric acid,35 followed by Boc
protection. The resultant (tert-butyl 9-bromo-3,4,6,7-tetrahy-
dro-[1,4]diazepino[6,7,1-hi]indole-2(1H)-carboxylate) 45 was
subsequently treated with DDQ, followed by glyoxylation,
condensation with benzofuran-3-yl-acetamide, and Boc depro-
tection to yield the final maleimide 26.
Figure 1. Synthetic modifications to improve the potency and water
solubility of 3-(benzofuran-3-yl)-4-(5-bromo-1-methyl-1H-indol-3-yl)-
1H-pyrrole-2,5-dione 1.
penetrant, GSK-3β inhibitor which attenuates hyperactivity in a
mouse model of mania induced by amphetamine and
chlordiazepoxide.28 On the basis of the hypothesis that GSK-
3 inhibition could induce the production of neurosteroids
which in turn modulate the anxiety-like behavior in vivo, we
sought to investigate the ability of these ATP-competitive
maleimide-based GSK-3β inhibitors to control steroid for-
mation in a steroidogenic cell model.
CHEMISTRY
■
Upon the basis of a body of SAR information accumulated over
the years for the development of benzofuran-3-yl-(indol-3-
yl)maleimides as GSK-3β inhibitors, 6-substitution on the
indole ring has generally been shown to decrease GSK-3β
inhibitory activity. On the other hand, substitution at the 5- or
7-position of the indole ring is generally well tolerated,29 as
exemplified by 5-halogen bearing analogues which include the
previously reported maleimide 1. A new generation of
maleimides 2−31 was synthesized with the aim to improve
RESULTS AND DISCUSSION
■
GSK-3 Inhibition by Maleimides 2−31. Testing of all the
newly synthesized compounds for their ability to inhibit GSK-
3β was carried out by Reaction Biology, Inc. using their
standard protocol (Tables 1 and 2). The hydroxyethyl analogue
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a
Scheme 1. Synthesis of Target Compounds 2−12
a
Reagents and conditions: (a) (i) NaH (1.5 equiv), DMF, rt, 0.5 h, (ii) O-trityl-3-bromopropan-1-ol or O-trityl-2-bromoethan-1-ol (1.2 equiv), 60
°C, 16 h, 80−99%; (b) ethyl chlorooxoacetate (5 equiv), Et2O, 0 °C to rt, 16 h, 42−74%; (c) benzofuran-3-yl-acetamide (1.1 equiv), t-BuOK (2.5
equiv), THF or DMF, 0 °C to rt, 16 h, 22−51%; (d) (i) NaH (1.5 equiv), DMF, rt, 0.5 h, (ii) tert-butyl (2-bromoethyl)carbamate (1.2 equiv), 60 °C,
16 h, 70−90%; (e) trifluoroacetic acid, CHCl3, rt, 2 h; (f) (i) NaH (1.5 equiv), DMF, rt, 0.5 h; (ii) tert-butyl N-(3-bromopropyl)-N-methylcarbamate
(1.2 equiv), 60 °C, 16 h, 76−99%.
2 was found to have a similar potency compared to the parent
maleimide 1 (IC50 value of 21 nM).36 Substitution of the
bromine by a fluorine atom (2 vs 3) at the 5-position of the
indole ring resulted in an approximately 4-fold increase in the
potency, whereas lengthening of the hydroxyethyl to a
hydroxypropyl moiety (3 vs 4) gave a 3-fold decrease in
potency. Subsequent chemical modifications were carried out
to improve upon the water solubility of these maleimides,
which is commonly achieved by the addition of an ionizable
nitrogen group. The aminoethyl substitution at the indole
nitrogen (5 and 6) diminished the GSK-3β inhibition by
approximately 6−8-fold in comparison with the hydroxyethyl
group (compounds 2 and 3), with the 5-bromo analogue 5
being 3-fold less potent than the 5-fluoro analogue 6. This
trend was also observed with the N-methylaminopropyl
analogues 7 and 8. As a primary hydroxyl group generally
poses a potential metabolic liability that arises from oxidation
and bioconjugation processes, the hydroxyethyl moiety in
analogues 2 and 3 was replaced with a 2-methoxyethyl group
(compounds 9−12). Within this series, the unsubstituted
indole analogue 9 was identified as the most potent GSK-3β
inhibitor with an IC50 value of 20 nM. The 5-fluoro and 5-
bromo analogues 10 and 11 were found to be 2- and 4-fold less
potent, with IC50 values of 40 and 82 nM, respectively. Direct
comparison of the methoxy analogues 10 and 11 vs the
hydroxyl analogues 3 and 2, respectively, showed that the
hydroxyl analogues were approximately 4−8-fold more potent.
The 5,6-difluoro analogue 12 had a similar potency to the 5-
fluoro analogue with an IC50 value of 36 nM.
The effects of: (i) introduction of an azaindole ring in place
of the indole ring (analogues 13−18) and (ii) replacement of
the benzofuran ring with an imidazopyridine (analogues 19 and
20) on the GSK-3β inhibition were examined.31,32 To our
delight, the azaindole analogue 13 had an IC50 value of 5 nM
while the extension of the methyl group to 2-methoxyethyl
(compound 14) resulted in a 5-fold reduction in potency
compared to 13. Deletion of the 5-bromo group gave a further
∼3-fold reduction in GSK-3β inhibitory activity (15, 16 vs 13,
14), while the 5-methoxy-6-azaindoles 17 and 18 had IC50
values of 66 and 127 nM, respectively. Replacement of the
benzofuran ring with an imidazopyridine (19 and 20) also
resulted in a further increase in potency (IC50 values of 5 and
0.9 nM, respectively).
The next set of SAR modifications focused on the
introduction of a 2-methoxyethyl group at the 7-position of
the indole ring. Previous SAR studies had shown that
introduction of hydroxyalkyl groups at the 7-position resulted
in single-digit nanomolar GSK-3β inhibitors.29 Toward this
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a
Scheme 2. Synthesis of Target Compounds 13−23
a
Reagents and conditions: (a) (i) NaH (1.5 equiv), DMF, rt, 0.5 h; (ii) R2Br (1.2 equiv), 60 °C, 16 h, 85−99%; (b) ethyl chlorooxoacetate (5
equiv), Et2O, 0 °C to rt, 16 h or (i) AlCl3 (5 equiv), CH2Cl2, rt, 1 h; (ii) ethyl chlorooxoacetate (5 equiv), Et2O, 0 °C to rt, 16 h, 31−83%; (c)
benzofuran-3-yl-acetamide (1.1 equiv), t-BuOK (2.5 equiv), THF or DMF, 0 °C to rt, 16 h, 42−60%; (d) ethyl 2-(5-bromo-1-methyl-1H-indol-3-yl)-
2-oxoacetate or ethyl 2-(5-methyl-5H-[1,3]dioxolo[4,5-f ]indol-7-yl)-2-oxoacetate, t-BuOK (2.5 equiv), THF or DMF, 0 °C to rt, 16 h, 30−40%; (e)
Ph3PCH3Br (3 equiv), t-BuOK (3 equiv), THF, 0 °C to rt, 16 h, 80−90%; (f) (i) BH3·THF (1.1 equiv), THF, rt, 2.5 h; (ii) 10% aq NaOH (1.3
equiv), 35% H2O2 (1.3 equiv), rt, 3.5 h, 55−70%; (g) (i) NaH (1.5 equiv), DMF, rt, 0.5 h; (ii) MeI (1.2 equiv), 60 °C, 16 h, 90−99%; (h) tert-butyl
2,2,2-trichloroacetimidate (1.1 equiv), cyclohexane, CH2Cl2, cat. BF3·OEt2, rt, 16 h, 53%.
end, the 2-methoxyethyl attached at the 7-position of the indole
ring of analogue 21 led to a potent inhibitor with an IC50 value
of 11 nM (Table 2). Replacement of the methoxy group by a
tert-butoxy was found to be deleterious for activity (22 vs 21).
The bisalkylated analogue 23 had similar potency to that of
compound 22. Inspired by the diazepinoindole scaffold
originally reported by Eli Lilly & Co. in 2004,34 cyclization of
the two alkyl chains to a diazepinoindole scaffold exemplified
by compounds 24−31 resulted in highly potent GSK-3β
inhibitors. The diazepinoindole analogues 24−26 all possessed
IC50 values between 4 and 12 nM, and further extension of the
diazepinoindole nitrogen atom to an amide moiety gave even
more potent GSK-3β inhibitors 27−31. Of a particular note,
compound 31 was identified as the most potent GSK-3β
inhibitor from this series with an IC50 value of 13 pM,
representing the most potent compound to date in our library
of GSK-3 inhibitors. In addition, the aqueous solubility of
compounds 5, 20, and 24 was determined to be 180, 35, and
240 mg/L using an HPLC-based method.38
(Tables 1 and 2) were screened for their ability to induce
progesterone expression in MA-10 mouse tumor Leydig cells.
This cell line is a hormone-responsive steroidogenic cell model
which has been extensively used to study the regulation of
steroidogenesis whereby the biosynthesis of progesterone, the
main steroid formed, is measured.39 Initially, MA-10 cells were
incubated in the presence of the GSK-3β inhibitors for 2 h at a
concentration of 100 μM, and at the end of the incubation
period, progesterone formation was measured by radio-
immunoassay (RIA). As shown in Figure 2A, 7 of the 17
tested GSK-3β inhibitors, namely analogues 1, 9, 12, 13, 14, 21,
and 26, were bioactive in stimulating progesterone production.
Dose−response experiments were next carried out for all the
tested compounds ranging from 0.1 to 100 μM, in which a
statistically significant induction of progesterone synthesis was
observed starting at a concentration of approximately 50 μM
(Figure 2B). As the high micromolar/low millimolar
concentrations posed significant concern regarding cytotoxicity,
and as steroid production can be facilitated by disruption of
mitochondrial membrane integrity,40 additional cytotoxicity
profiling was performed. MA-10 Leydig cells were exposed to
GSK-3 inhibitors for 2 h at 100 μM concentration and cellular
toxicity assessed by the MTT assay (Figure 2C). Some of the
tested analogues showed low, but observable signs of
Characterization of GSK-3β Inhibitors as Stimulators
of Steroidogenesis. To test the hypothesis that GSK-3β
inhibition could induce the production of neuroactive steroids,
thereby mediating the modulation of anxiety-like behavior, 17
of the most potent, newly synthesized GSK-3β inhibitors
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were incubated with MA-10 cells at a concentration of 50 μM
for 0−120 min, media collected, and progesterone production
was then assessed by RIA (Figure 3). There appears to be a
biphasic time response to these two compounds, with the first
maximum steroid production appearing within 15 min and
remaining until 60 min, followed by a linear increase in steroid
production between 60 and 120 min. These data altogether
demonstrated for the first time that some of the maleimide-
based GSK-3β inhibitors, namely 1, 9, 13, 14, and 21, were able
to stimulate progesterone production in MA-10 tumor Leydig
cells in a dose-dependent manner with either low or no
apparent signs of toxicity as assessed by the MTT assay.
In Vivo SmartCube Assay for GSK-3 Inhibitors 1 and 9.
Encouraged by the demonstrated ability of some of the newly
synthesized GSK-3β inhibitors to stimulate steroidogenesis in
vitro, two of the most potent, nontoxic stimulators 1 and 9
were administered to C57BL/6 mice in the proprietary
SmartCube system developed by Psychogenics, Inc.41,42 This
behavioral screening platform provides a sequence of challenges
to mice and captures more than 1400 features during the 45
min testing session. These features were analyzed with
computer algorithms and data mining approaches to automate
the study of mice behavior and compared to a database of
behavioral signatures obtained using a set of diverse reference
compounds, including antipsychotics, anxiolytics, and anti-
depressants.41 In this assay, mice were injected once a day with
either compound 1 or 9 (50 mg/kg, ip) for 13 days, and the
SmartCube signatures were depicted in Figure 4. Neither
compound demonstrated any classified behavioral activity after
a single dose (15 min, day 1 test, Figure 4A). In contrast, a
robust signature was obtained after chronic administration of
both compounds over 13 days (Figure 4B). The signature
classes for both compounds were found to be relatively similar,
with the predominant portion of the signatures predicting
anxiolytic-like activity, as well as some cognitive enhancer
effects. In accordance with the ability of compounds 1 and 9 to
stimulate steroidogenesis in MA-10 cells (Figure 2), the in vitro
and in vivo data presented herein supported the hypothesis that
GSK-3 inhibition could influence the formation of neuroactive
steroids, which in turn mediate the anxiolytic-like behavior in
living systems.
Scheme 3. Synthesis of Target Compounds 24, 25, and 27−
a
31
a
Reagents and conditions: (a) (i) ethanolamine (2 equiv), 10% Pd/C
(cat.), MeOH, rt, 1 h; (ii) H2, 1 atm, rt, 3 h, 84−95%; (b) (Boc)2O
(1.2 equiv), THF, aq K2CO3, 0 °C to rt, 5 h, 85−95%; (c) MsCl (1.2
equiv), N,N-diisopropylethylamine, THF, 0 °C to rt, 16 h, 72−96%;
(d) NaH (1.5 equiv), DMF, 0 °C, 1 h, 84−90%; (e) ethyl
chlorooxoacetate (5 equiv), Et2O, 0 °C to rt, 16 h, 42−49%; (f)
benzofuran-3-yl-acetamide (1.1 equiv), t-BuOK (2.5 equiv), THF or
DMF, 0 °C to rt, 16 h, 30−40%; (g) trifluoroacetic acid, CHCl3, rt, 2
h; (h) R4CO2H (1.2 equiv), EDC·HCl (1.3 equiv), HOBt (1.3 equiv),
DMF, N-methylmorpholine (3 equiv), rt, 16 h, 30−50%; (i) R4COCl
(1.5 equiv), N,N-diisopropylethylamine (3 equiv), MeOH, rt, 3 h, 30−
50%.
a
Scheme 4. Synthesis of Target Compound 26
a
Reagents and conditions: (a) 2-chloroethylamine hydrochloride (1.1
equiv), K2CO3, DMF, sealed tube, 110 °C, 16 h, 25%; (b) 36% aq
HCHO (1.2 equiv), AcOH, H2SO4, 70 °C, 16 h; (c) (Boc)2O (1
equiv), THF, aq K2CO3, 0 °C to rt, 5 h, 65% over 2 steps; (d) DDQ
(1.2 equiv), Et2O, PhMe, rt, 3 h, 50%; (e) ethyl chlorooxoacetate (5
equiv), Et2O, 0 °C to rt, 16 h, 39%; (f) benzofuran-3-yl-acetamide (1.1
equiv), t-BuOK (2.5 equiv), THF or DMF, 0 °C to rt, 16 h, 30%; (g)
trifluoroacetic acid, CHCl3, rt, 2 h.
CONCLUSIONS
■
In this study, we presented our findings on the SAR exploration
of the benzofuran-3-yl-(indol-3-yl)maleimides with the aim to
improve their potency at GSK-3β inhibitory activity as well as
their aqueous solubility. The replacement of indole ring in the
original parent maleimide 1 with an azaindole or diazepinoin-
dole moiety resulted in highly potent GSK-3β inhibitors with
improved aqueous solubility. Compound 31 has been identified
as the most potent GSK-3β inhibitor in our library thus far with
an IC50 value of 13 pM. To test the hypothesis that GSK-3β
inhibition could indeed stimulate steroid production thereby
mediating the modulation of anxiety-like behavior in vivo, 17
newly synthesized analogues were screened for their ability to
induce steroidogenesis in a steroidogenic cell model, the MA-
10 mouse tumor Leydig cell lines. Following dose−response
experiments measuring the progesterone induction and
cytotoxicity profiles, compounds 1, 9, 13, 14, and 21 were
identified for the first time to be the most potent steroido-
genesis stimulators in this assay without any significant toxicity
at the tested concentration of 100 μM. The two most potent,
nontoxic steroidogenesis stimulators 1 and 9 were subsequently
cytotoxicity at the 100 μM concentrations, for example,
compounds 16 and 23, whereas maleimides 12, 25−28, 30,
and 31 were found to be significantly more cytotoxic at the
tested concentration. Dose dependency curves for the
cytotoxicity were also obtained for all the tested compounds,
ranging from 0.1 to 100 μM concentrations (Figure 2D).
Examination of the overall progesterone production and
cytotoxicity data presented in Figure 2A−D revealed that out
of the seven compounds that stimulated progesterone
production (Figure 2A), analogues 12 and 26 were also
found to be toxic to the MA-10 cells. However, the remaining
five GSK-3 inhibitors that stimulated steroidogenesis (com-
pounds 1, 9, 13, 14, and 21) showed either low or no apparent
toxicity at a concentration of 100 μM. To examine the time-
dependency of these GSK-3β inhibitors in stimulating steroido-
genesis, two of the most potent, nontoxic stimulators 1 and 9
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Table 1. Inhibition of GSK-3β by Maleimides 2−20
a
compd
R1
X
R2
IC50 (nM)
1
5-Br
5-Br
5-F
5-F
5-Br
5-F
5-Br
5-F
H
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
−CH3
21.1 1.8
21.4 3.1
4.8 0.2
15.0 2.7
2
−(CH2)2OH
−(CH2)2OH
−(CH2)3OH
−(CH2)2NH2
−(CH2)2NH2
−(CH2)3NHMe
−(CH2)3NHMe
−(CH2)2OMe
−(CH2)2OMe
−(CH2)2OMe
−(CH2)2OMe
Me
3
4
5
116
9
6
41.1 0.7
404 23
7
8
202
5
9
19.8 5.8
39.7 0.6
82.3 5.7
35.7 1.9
5.3 0.8
10
11
12
13
14
15
16
17
18
19
20
5-F
5-Br
5,6-difluoro
5-Br
5-Br
H
N
−(CH2)2OMe
Me
25.2 1.2
17.6 1.0
58.2 3.5
65.9 3.6
N
H
N
−(CH2)2OMe
Me
−(CH2)2OMe
Me
127
5.3 1.0
0.87 0.11
5
5,6-methylenedioxy
5-Br
Me
a
The synthesized maleimides were evaluated for their ability to inhibit phosphorylation of primed substrate (YRRAAVPPSPSLSRHSSPHQ(pS)-
EDEEE; 20 μM) by human GSK-3β in the presence of 10 μM ATP concentration. These compounds were tested at Reaction Biology Inc.,37 and
IC50 values are presented as average of duplicates SEM.
Table 2. Inhibition of GSK-3β by Maleimides 21−31
a
compd
R1
R2
R3
R4
IC50 (nM)
21
22
23
24
25
26
27
28
29
30
31
H
H
H
H
F
Me
Me
Me
t-Bu
Me
11.2 3.1
148 15
−(CH2)2OMe
114 16
4.2 0.7
3.7 0.5
Br
H
H
F
11.5 2.2
0.20 0.06
0.59 0.11
1.9 0.4
2-pyrazinyl
4-pyridylmethyl
1-piperidinyl
F
4-piperidinopiperidin1-yl
4-morpholinyl
0.53 0.01
0.013 0.005
F
a
The synthesized maleimides were evaluated for their ability to inhibit phosphorylation of primed substrate (YRRAAVPPSPSLSRHSSPHQ(pS)-
EDEEE; 20 μM) by human GSK-3β in the presence of 10 μM ATP concentration. These compounds were tested at Reaction Biology Inc.,37 and
IC50 values are presented as either average of duplicates SEM.
tested in a proprietary SmartCube assay. Daily administration
of each compound at 50 mg/kg intraperitoneally resulted in
anxiolytic-like signatures after 13 days. Taken together, these
data supported the hypothesis that GSK-3β inhibition could be
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Figure 2. Steroidogenesis stimulation of maleimide-based GSK-3β inhibitors in MA-10 tumor Leydig cells. MA-10 tumor Leydig cells were
incubated in the presence or absence of GSK-3β inhibitor, and at the end of incubation, steroid (progesterone) production and cellular toxicity were
evaluated by RIA and MTT assay, respectively. (A) Steroid production by MA-10 cells exposed to 100 μM GSK-3 inhibitors. MA-10 Leydig cells
were exposed to GSK-3 inhibitor for 2 h and steroid production assessed by RIA. ***, p < 0.001 as assessed by one-way ANOVA with Newman−
Keuls post-test. (B) Dose-dependent steroid production by MA-10 cells exposed to 0−100 μM GSK-3 inhibitors. MA-10 Leydig cells were exposed
to GSK-3 inhibitor for 2 h and steroid production assessed by RIA. Only those that showed significant stimulation of steroid production are
presented; the remaining tested compounds (16, 17, 20, 23, 25, 27−31) showed <30 ng progesterone/mg protein at all the tested concentrations
after 2 h incubation. (C) Cellular toxicity of MA-10 cells exposed to 100 μM GSK-3 inhibitors. MA-10 Leydig cells were exposed to GSK-3 inhibitor
for 2 h and cellular toxicity assessed by MTT assay. *, p < 0.05 (orange); ***, p < 0.001 (red) as assessed by one-way ANOVA with Newman−Keuls
post-test. (D) Dose-dependent cellular toxicity of MA-10 cells exposed to 0−100 μM GSK-3 inhibitors. MA-10 Leydig cells were exposed to GSK-3
inhibitor for 2 h and cellular toxicity assessed by MTT assay (red = toxic, orange = slightly toxic, gray = nontoxic). Results are presented as the mean
SEM (n = 3) for (A) and (C) and presented as nonlinear regression curve fit of data points for (B) and (D).
Anhydrous THF and CH2Cl2 were obtained by distillation over
sodium wire or CaH2, respectively. All nonaqueous reactions were run
under an argon atmosphere with exclusion of moisture from reagents.
The progress of reactions was monitored by TLC on SiO2. For flash
chromatography, 230−400 mesh particle size SiO2 was used. Melting
points were determined on a Thomas−Hoover Unimelt apparatus and
are uncorrected. Infrared absorption spectra were recorded on a
Nicolet 6700 FT-IR spectrometer equipped with a Ge-on-KBr
involved in the induction of steroidogenesis and thus
neuroactive steroid production, thereby mediating the modu-
lation of anxiety-like behavior in vivo. Continued efforts are
directed toward the elucidation of the role of GSK-3β
inhibition on the stimulation of steroidogenesis.
EXPERIMENTAL SECTION
■
1
beamsplitter. H NMR and 13C NMR spectra were recorded on a
Chemical Synthesis. General Methods. Starting materials,
reagents, and solvents were purchased from commercial suppliers
and used without further purification unless otherwise stated.
Bruker spectrometer at 400 and 100 MHz, respectively, and were
referenced to the residual peaks of CDCl3 at 7.26 ppm or DMSO-d6 at
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Figure 3. Steroidogenic time-response of GSK-3 inhibitors 1 and 9. MA-10 cells were cultured with 50 μM of 1 (A) or 9 (B) for 0−120 min and
progesterone production was assessed by RIA. Results are presented as the mean SEM (n = 2).
Figure 4. Smartcube signatures of GSK-3 inhibitors 1 and 9 after 1 (acute) or 13 days (chronic) of treatment, 15 min following one dose of each
compound at 50 mg/kg i.p.
2.50 ppm (1H NMR) and CDCl3 at 77.2 ppm or DMSO-d6 at 39.51
ppm (13C NMR). Chemical shifts were reported in parts per million
(ppm) downfield of tetramethylsilane (TMS) and the following
abbreviations used to denote coupling patterns: s = singlet; d =
doublet, t = triplet, q = quartet, br = broad. 13C NMR chemical shifts
are reported in ppm relative to the internal perdeuteriated solvent
resonance and are specified as quarternary (C), tertiary (CH),
secondary (CH2), or primary (CH3) where appropriate, using
additional DEPT experiments. Mass spectra were measured in the
ESI mode at an ionization potential of 70 eV with an LC-MS MSD
(Hewlett-Packard). HRMS experiments were performed on a
Micromass Q-TOF-2 tandem mass instrument. Purities of all final
compounds (>96%) were established by analytical HPLC, which was
carried out on an Agilent 1100 HPLC system with a Synergi 4 μ
Hydro-RP 80A column, with detection at 254 and 280 nm on a
Shimadzu SPD-10A VP detector; flow rate =1.4 mL/min; gradient of
0−100% acetonitrile in water (both containing 0.05 vol % of TFA) in
25 min. Final products were purified by preparative HPLC under the
following conditions: ACE 5 AQ column, 150 mm × 21.2 mm; flow
rate =17 mL/min; all solvents containing 0.05 vol % TFA; UV
detection at 254 and 280 nm. Gradient I: 40−100% MeOH in water
over 35 min. Gradient II: 8−100% MeOH in water over 40 min.
General Procedure for the Indole or Azaindole N-Alkylation
(Method A). To a solution of substituted indole or azaindole (1 equiv)
in dry DMF (3 mL/mmol) cooled with an ice bath was added NaH
(55% suspension in mineral oil, 1.5 equiv). After 30 min stirring at 0 to
5 °C, an alkyl bromide (1.2 equiv) or methyl iodide (1.2 equiv) was
added in one portion. The reaction mixture was then heated to 60 °C
and stirred overnight. Upon completion of the reaction as indicated by
TLC, the reaction mixture was poured into ice−water and extracted
with EtOAc (3 × 20 mL). The combined organic phases were washed
with water (50 mL), brine (50 mL), dried over anhydrous Na2SO4,
and concentrated in vacuo. The crude product was either purified by
flash chromatography (hexane to EtOAc−hexane, 1:3) or directly used
for further reaction without additional purification.
General Procedure for the Indole or Azaindole Glyoxylation
(Method B). To a solution of the substituted N-alkylindole (1 mmol)
in Et2O (5 mL/mmol) cooled with an ice bath was added ethyl
chlorooxoacetate (5 equiv) dropwise. The reaction mixture was
allowed to slowly warm to room temperature overnight until
completion. Upon reaction completion as indicated by TLC, the
reaction mixture was cooled with an ice bath and carefully quenched
by slow addition of saturated aqueous NaHCO3. The reaction mixture
was extracted with CH2Cl2 (3 × 20 mL), and the combined organic
phases were washed with saturated aqueous NaHCO3 (2 × 40 mL),
water (40 mL), brine (40 mL), dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was purified by flash
chromatography (EtOAc−hexane, 1:2) to give the substituted
glyoxalates. For N-alkylazaindoles, a solution of the azaindole (1
equiv) in CH2Cl2 was treated with AlCl3 (5 equiv) and stirred at
ambient temperature for 1 h prior to the addition of ethyl
chlorooxoacetate (5 equiv). Quenching with MeOH and evaporation
in vacuo gave the residue which was purified by flash chromatography
(EtOAc−hexane. 2:3).
General Procedure for the Perkin-Type Condensation to Give
Maleimides (Method C). To a suspension of indolyl-3-glyoxylate or
azaindolyl-3-glyoxylate (1 equiv) and benzofuran-3-yl-acetamide (1.1
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equiv) in dry THF or DMF (10 mL/mmol) at 0−5 °C was added a
1.0 M solution of t-BuOK in THF (2.5 equiv) in a dropwise manner.
The reaction mixture was stirred at room temperature overnight. Upon
completion of the reaction as ascertained by TLC, the reaction mixture
was quenched with saturated aqueous NH4Cl and extracted with
EtOAc (3 × 20 mL). The combined organic phases were washed with
water (50 mL), brine (50 mL), dried over Na2SO4, and evaporated in
vacuo. The residue was purified by flash chromatography (1:2 to 1:1
EtOAc−hexane eluent) prior to further HPLC purification.
General Procedure for the Deprotection of N-Boc Amines
(Method D). To a solution of the N-Boc protected precursor (1
mmol) in CHCl3 (5 mL) was added TFA (1 mL) at ambient
temperature. The mixture was stirred for 2 h and concentrated in
vacuo. The crude product was dissolved in DMF and filtered over a
syringe filter (polytetrafluoroethylene, 17 mm diameter, 0.45 μm pore
size) prior to the preparative HPLC.
General Procedure for the Wittig Reaction (Method E). To a
suspension of the indole carboxaldehyde (1 equiv) and methyene-
triphenylphosphorane (3 equiv) in dry THF (15 mL/mmol) was
slowly added a 1.0 M solution of t-BuOK (3 equiv) at 0−5 °C. The
reaction mixture was slowly warmed to room temperature overnight
with stirring and concentrated in vacuo. The residue was purified by
flash chromatography (1:4 to 1:2 EtOAc−hexane eluent).
General Procedure for the Hydroboration (Method F). To a
solution of the vinyl indole (1 equiv) in dry THF (6 mL/mmol) was
added a solution of borane−tetrahydrofuran complex (1.0 M in THF,
1.1 equiv) in a dropwise manner at ambient temperature. After 2.5 h
stirring, 10% aqueous NaOH (1.3 equiv) was added in one portion
(effervescence was observed), followed by a hydrogen peroxide
solution (35%, 1.3 equiv). After 3.5 h stirring at ambient temperature,
the reaction mixture was quenched by the addition of saturated
aqueous NH4Cl and diluted with water. Following extraction with
EtOAc (3 × 50 mL), the combined organic phases were washed with
brine, dried over Na2SO4, and concentrated in vacuo. The residue was
purified by flash chromatography (1:4 to 1:1 EtOAc−hexane eluent).
General Procedure for the tert-Butyl Ether (Method G). To a
solution of 2-(1-methyl-1H-indol-7-yl)ethanol (1 mmol) in CH2Cl2 (1
mL/mmol) and cyclohexane (2 mL/mmol) was added tert-
butyltrichloroacetimidate (1.1 mmol) at ambient temperature in one
portion, followed by 30 μL of boron trifluoride diethyletherate. After
stirring overnight, the reaction mixture was diluted with CH2Cl2 and
directly purified by flash chromatography (1:7 to 1:3 EtOAc−hexane
eluent) to give the desired product.
General Procedure for the Amide Coupling (Method H). To a
solution of the free amine (1 equiv), carboxylic acid (1.2 equiv), N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1.3
equiv), and HOBt (1.3 equiv) in DMF (5 mL/mmol) was added N-
methylmorpholine (3 equiv) in one portion at ambient temperature.
After overnight stirring, saturated aqueous NaHCO3 was added,
followed by extraction with CH2Cl2 (3 × 15 mL). The combined
organic phases were washed with brine (40 mL), dried over anhydrous
Na2SO4, and concentrated in vacuo. The residue was purified by flash
chromatography (1:2 to 1:1 EtOAc−hexane or 3:97 MeOH−CH2Cl2
eluent) prior to further HPLC purification.
= 8.4 Hz, 1H), 7.50 (d, J = 8.8 Hz, 1H), 7.25 (t, J = 7.2 Hz, 1H), 7.18
(dd, J = 8.8, 2.0 Hz, 1H), 7.06 (d, J = 2.0 Hz, 1H), 6.94 (t, J = 7.4 Hz,
1H), 6.85 (d, J = 7.6 Hz, 1H), 4.93−4.89 (m, 1H), 4.27 (t, J = 5.2 Hz,
2H), 3.69−3.65 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 172.1
(CO), 171.8 (CO), 154.4 (C), 147.2 (CH), 135.3 (C), 135.1
(CH), 132.3 (C), 127.2 (C), 125.2 (C), 125.0 (CH), 124.4 (CH),
123.5 (CH), 123.0 (C), 122.9 (CH), 121.7 (CH), 112.9 (CH), 112.7
(C), 111.5 (CH), 111.4 (C), 103.7 (C), 60.1 (CH2), 48.9 (CH2).
HRMS (ESI): m/z [M + H]+ calculated for C22H16BrN2O4, 451.0288;
found, 451.0278. HPLC purity: 97.6%.
3-(Benzofuran-3-yl)-4-(5-fluoro-1-(2-hydroxyethyl)-1H-
indol-3-yl)-1H-pyrrole-2,5-dione (3). The title compound was
synthesized from commercially available 5-fluoroindole (32b) and O-
trityl-2-bromoethan-1-ol according to general procedures A−C,
purified using gradient I preparative HPLC, and isolated as an orange
solid in 15% yield over 3 steps; mp 107−112 °C. IR (ATR): νmax 3232,
1
1699, 1695, 1541, 1478, 1450, 1334, 741 cm−1. H NMR (400 MHz,
DMSO-d6) δ 11.19 (s, 1H), 8.28 (s, 1H), 7.98 (s, 1H), 7.62 (d, J = 8.0
Hz, 1H), 7.59−7.51 (m, 1H), 7.24 (t, J = 8.2 Hz, 1H), 6.97−6.85 (m,
3H), 6.58 (dd, J = 9.2, 2.0 Hz, 1H), 4.92 (br s, 1H), 4.29 (t, J = 5.2 Hz,
2H), 3.68 (t, J = 5.4 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ
1
172.0 (CO), 171.7 (CO), 157.0 (d, JC−F = 232.6 Hz, C), 154.2
(C), 147.1 (CH), 135.4 (CH), 133.1 (C), 132.3 (C), 126.1 (d, 3JC−F
=
11.0 Hz, C), 125.2 (C), 124.8 (CH), 122.8 (CH), 122.3 (C), 121.7
(CH), 112.0 (d, 3JC−F = 10.2 Hz, CH), 111.4 (CH), 111.3 (C), 110.0
2
2
(d, JC−F = 26.4 Hz, CH), 105.8 (d, JC−F = 24.9 Hz, CH), 104.0 (d,
4JC−F = 4.4 Hz, C), 60.0 (CH2), 48.9 (CH2). HRMS (ESI): m/z [M +
H]+ calculated for C22H16FN2O4, 391.1089; found, 391.1104. HPLC
purity: 99.1%.
3-(Benzofuran-3-yl)-4-(5-fluoro-1-(3-hydroxypropyl)-1H-
indol-3-yl)-1H-pyrrole-2,5-dione (4). The title compound was
synthesized from commercially available 5-fluoroindole (32b) and O-
trityl-3-bromopropan-1-ol according to general procedures A−C,
purified using gradient I preparative HPLC, and isolated as an orange
solid in 25% yield over 3 steps; mp 101−106 °C. IR (ATR): νmax 3140,
1
2944, 1704, 1699, 1545, 1479, 1451, 1336, 742 cm−1. H NMR (400
MHz, DMSO-d6) δ 11.20 (s, 1H), 8.32 (s, 1H), 7.93 (s, 1H), 7.61 (d, J
= 8.0 Hz, 1H), 7.55−7.50 (m, 1H), 7.22 (t, J = 8.4 Hz, 1H), 6.97−6.85
(m, 2H), 6.78 (d, J = 7.6 Hz, 1H), 6.64 (dd, J = 10.4, 2.4 Hz, 1H), 4.63
(br s, 1H), 4.30 (t, J = 6.8 Hz, 2H), 3.34 (t, J = 6.0 Hz, 2H), 1.85−1.79
(m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 172.0 (CO), 171.7
1
(CO), 157.2 (d, JC−F = 233.5 Hz, C), 154.2 (C), 147.1 (CH),
3
134.8 (CH), 132.7 (C), 132.2 (C), 126.1 (d, JC−F = 11.0 Hz, C),
125.1 (C), 124.8 (CH), 122.8 (CH), 122.7 (C), 121.5 (CH), 111.7 (d,
2
3JC−F = 10.3 Hz, CH), 111.4 (CH), 111.2 (C), 110.2 (d, JC−F = 26.4
2
4
Hz, CH), 105.8 (d, JC−F = 26.4 Hz, CH), 104.1 (d, JC−F = 4.4 Hz,
C), 57.4 (CH2), 43.1 (CH2), 32.7 (CH2). HRMS (ESI): m/z [M +
H]+ calculated for C23H18FN2O4, 405.1245; found, 405.1249. HPLC
purity: 97.9%.
3-(1-(2-Aminoethyl)-5-bromo-1H-indol-3-yl)-4-(benzofuran-
3-yl)-1H-pyrrole-2,5-dione trifluoroacetate (5). The title com-
pound was synthesized from commercially available 5-bromoindole
(32a) and tert-butyl (2-bromoethyl)carbamate according to general
procedures A−D, purified using gradient II preparative HPLC, and
isolated as an orange solid in 12% yield over 3 steps; mp 153−157 °C.
IR (ATR): νmax 3054, 1704, 1699, 1545, 1451, 1343, 1185, 1119, 743,
General Procedure for the Urea Formation (Method I). To a
solution of the free amine (1 equiv) in MeOH (10 mL/mmol) was
added carbamoyl chloride (1.5 equiv) at ambient temperature,
followed by N,N-diisopropylethylamine (3 equiv) in one portion.
After 3 h stirring, the reaction mixture was diluted with aqueous 1 M
NaOH and extracted with EtOAc (3 × 15 mL). The combined organic
phases were washed with brine (40 mL), dried over anhydrous
Na2SO4, and concentrated in vacuo prior to purification by HPLC.
3-(Benzofuran-3-yl)-4-(5-bromo-1-(2-hydroxyethyl)-1H-
indol-3-yl)-1H-pyrrole-2,5-dione (2). The title compound was
synthesized from commercially available 5-bromoindole (32a) and O-
trityl-2-bromoethan-1-ol according to general procedures A−C,
purified using gradient I preparative HPLC, and isolated as an orange
solid in 15% yield over 3 steps; mp 152−157 °C. IR (ATR): νmax 3140,
1
722 cm−1. H NMR (400 MHz, DMSO-d6) δ 11.26 (s, 1H), 8.28 (s,
1H), 7.99 (br s, 3H), 7.94 (s, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.56 (d, J
= 8.8 Hz, 1H), 7.31−7.24 (m, 2H), 7.18 (s, 1H), 6.98 (d, J = 7.6 Hz,
1H), 6.91 (d, J = 8.0 Hz, 1H), 4.44 (t, J = 6.4 Hz, 2H), 3.17 (t, J = 5.6
Hz, 2H). 13C NMR (100 MHz, DMSO-d6) 171.9 (CO), 171.6
2
(CO), 158.3 (q, JC−F = 36.1 Hz, TFA), 154.4 (C), 147.4 (CH),
134.9 (C), 134.6 (CH), 131.8 (C), 127.3 (C), 125.0 (CH), 124.9
(CH), 123.9 (CH), 123.8 (C), 122.9 (CH), 121.8 (CH), 117.3 (d,
1JC−F = 298.2 Hz, TFA), 113.1 (C), 112.4 (CH), 111.5 (CH), 111.1
(C), 104.5 (C), 104.5 (C), 43.6 (CH2), 38.5 (CH2). HRMS (ESI): m/
z [M + H]+ calculated for C22H17BrN3O3, 450.0448; found, 450.0458.
HPLC purity: 98.2%.
3-(1-(2-Aminoethyl)-5-fluoro-1H-indol-3-yl)-4-(benzofuran-
3-yl)-1H-pyrrole-2,5-dione trifluoroacetate (6). The title com-
1
3057, 2944, 1702, 1700, 1544, 1356, 1114, 744 cm−1. H NMR (400
MHz, DMSO-d6) δ 11.20 (s, 1H), 8.29 (s, 1H), 7.94 (s, 1H), 7.63 (d, J
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pound was synthesized from commercially available 5-fluoroindole
(32b) and tert-butyl (2-bromoethyl)carbamate according to general
procedures A−D, purified using gradient II preparative HPLC, and
isolated as an orange solid in 16% yield over 3 steps; mp 144−148 °C.
IR (ATR): νmax 3052, 1704, 1700, 1544, 1480, 1451, 1342, 1198, 1116,
742, 721 cm−1. 1H NMR (400 MHz, DMSO-d6) δ 11.25 (s, 1H), 8.28
(s, 1H), 7.97 (s, 1H), 7.91 (br s, 3H), 7.64 (d, J = 8.0 Hz, 1H), 7.62−
56 (m, 1H), 7.28 (t, J = 7.6 Hz, 1H), 7.05−6.91 (m, 3H), 6.69 (dd, J =
7.6, 2.5 Hz, 1H), 4.45 (t, J = 6.4 Hz, 2H), 3.19 (br s, 2H). 13C NMR
(100 MHz, DMSO-d6) δ 172.0 (CO), 171.7 (CO), 158.4 (q,
d6) δ 172.1 (CO), 171.8 (CO), 154.2 (C), 147.0 (CH), 136.2
(C), 133.6 (CH), 132.6 (C), 125.6 (C), 125.3 (C), 124.7 (CH), 122.7
(CH), 122.6 (C), 122.1 (CH), 121.7 (CH), 120.9 (CH), 120.1 (CH),
111.4 (C), 111.4 (CH), 110.7 (CH), 104.2 (C), 70.7 (CH2), 58.2
(CH3), 45.8 (CH2). HRMS (ESI): m/z [M + H]+ calculated for
C23H19N2O4, 387.1339; found, 387.1351. HPLC purity: 98.4%.
3-(Benzofuran-3-yl)-4-(5-fluoro-1-(2-methoxyethyl)-1H-
indol-3-yl)-1H-pyrrole-2,5-dione (10). The title compound was
synthesized from commercially available 5-fluoroindole (32b) and 2-
bromoethyl methyl ether according to general procedures A−C,
purified using gradient I preparative HPLC, and isolated as an orange
solid in 36% yield over 3 steps; mp 83−88 °C. IR (ATR): νmax 3189,
1
2JC−F = 35.9 Hz, TFA), 157.4 (d, JC−F = 233.5 Hz, C), 154.3 (C),
147.4 (CH), 135.1 (CH), 132.8 (C), 131.9 (C), 126.2 (d, 3JC−F = 10.6
1
2933, 1701, 1696, 1542, 1479, 1451, 1333, 1113, 742 cm−1. H NMR
Hz, C), 125.0 (CH), 125.0 (C), 123.3 (C), 122.9 (CH), 121.9 (CH),
1
3
(400 MHz, DMSO-d6) δ 11.19 (s, 1H), 8.31 (s, 1H), 7.92 (s, 1H),
7.62 (d, J = 8.4 Hz, 1H), 7.57−7.53 (m, 1H), 7.23 (t, J = 8.0 Hz, 1H),
6.96−6.87 (m, 2H), 6.80 (d, J = 8.0 Hz, 1H), 6.65 (dd, J = 9.2, 2.6 Hz,
1H), 4.40 (t, J = 4.8 Hz, 2H), 3.59 (t, J = 5.2 Hz, 2H), 3.18 (s, 3H).
13C NMR (100 MHz, DMSO-d6) δ 172.0 (CO), 171.7 (CO),
117.1 (d, JC−F = 299.3 Hz, TFA), 111.6 (d, JC−F = 9.5 Hz, CH),
2
111.5 (CH), 111.2 (C), 110.6 (d, JC−F = 26.4 Hz, CH), 106.4 (d,
2JC−F = 24.9 Hz, CH), 105.0 (d, JC−F = 2.9 Hz, C), 43.7 (CH2), 38.5
4
(CH2). HRMS (ESI): m/z [M + H]+ calculated for C22H17FN3O3,
390.1249; found, 390.1256. HPLC purity: 98.0%.
1
157.3 (d, JC−F = 233.5 Hz, C), 154.2 (C), 147.1 (CH), 135.1 (CH),
3-(Benzofuran-3-yl)-4-(5-bromo-1-(3-(methylamino)propyl)-
1H-indol-3-yl)-1H-pyrrole-2,5-dione trifluoroacetate (7). The
title compound was synthesized from commercially available 5-
bromoindole (32a) and tert-butyl (3-bromopropyl)(methyl)carbamate
according to general procedures A−D, purified using gradient II
preparative HPLC, and isolated as an orange solid in 14% yield over 4
steps; mp 126−129 °C. IR (ATR): νmax 3041, 1713, 1671, 1547, 1452,
133.0 (C), 132.2 (C), 126.1 (d, 3JC−F = 11.0 Hz, C), 125.1 (C), 124.9
3
(CH), 122.8 (CH), 122.7 (C), 121.6 (CH), 112.0 (d, JC−F = 9.5 Hz,
2
CH), 111.4 (CH), 111.2 (C), 110.1 (d, JC−F = 25.9 Hz, CH), 105.8
2
4
(d, JC−F = 24.7 Hz, CH), 104.4 (d, JC−F = 4.4 Hz, C), 70.8 (CH2),
58.2 (CH3), 46.1 (CH2). HRMS (ESI): m/z [M + H]+ calculated for
C23H18FN2O4, 405.1245; found, 405.1264. HPLC purity: 98.6%.
3-(Benzofuran-3-yl)-4-(5-bromo-1-(2-methoxyethyl)-1H-
indol-3-yl)-1H-pyrrole-2,5-dione (11). The title compound was
synthesized from commercially available 5-bromoindole (32a) and 2-
bromoethyl methyl ether according to general procedures A−C,
purified using gradient I preparative HPLC, and isolated as an orange
solid in 30% yield over 3 steps; mp 92−96 °C. IR (ATR): νmax 3213,
1
1337, 1202, 1127, 742, 721 cm−1. H NMR (400 MHz, DMSO-d6) δ
11.25 (s, 1H), 8.33 (s, 1H), 8.29 (br s, 2H), 7.95 (s, 1H), 7.64 (d, J =
8.0 Hz, 1H), 7.55 (d, J = 8.8 Hz, 1H), 7.29−7.21 (m, 2H), 7.13 (d, J =
1.2 Hz, 1H), 6.93 (t, J = 7.4 Hz, 1H), 6.80 (d, J = 8.0 Hz, 1H), 4.33 (t,
J = 6.4 Hz, 2H), 2.82−2.78 (m, 2H), 2.54 (s, 3H), 2.04−2.00 (m, 2H).
13C NMR (100 MHz, DMSO-d6) 171.9 (CO), 171.6 (CO),
1
2931, 1704, 1695, 1541, 1451, 1333, 1113, 742 cm−1. H NMR (400
2
158.2 (q, JC−F = 36.1 Hz, TFA), 154.3 (C), 147.2 (CH), 134.6 (C),
MHz, DMSO-d6) δ 11.21 (s, 1H), 8.30 (s, 1H), 7.87 (s, 1H), 7.61 (d, J
= 8.4 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.23 (t, J = 7.8 Hz, 1H), 7.17
(dd, J = 8.8, 1.6 Hz, 1H), 7.13 (d, J = 1.6 Hz, 1H), 6.89 (t, J = 7.8 Hz,
1H), 6.76 (d, J = 7.6 Hz, 1H), 4.36 (t, J = 5.0 Hz, 2H), 3.56 (t, J = 5.0
Hz, 2H), 3.15 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 172.1 (C
O), 171.7 (CO), 154.4 (C), 147.2 (CH), 135.1 (C), 134.7 (CH),
132.2 (C), 127.3 (C), 125.1 (C), 125.0 (CH), 124.6 (CH), 123.5
(CH), 123.4 (C), 122.8 (CH), 121.6 (CH), 112.8 (C), 112.8 (CH),
111.5 (CH), 111.3 (C), 103.9 (C), 70.8 (CH2), 58.2 (CH3), 46.1
(CH2). HRMS (ESI): m/z [M + H]+ calculated for C23H18BrN2O4,
465.0444; found, 465.0458. HPLC purity: 99.2%.
3-(Benzofuran-3-yl)-4-(5,6-difluoro-1-(2-methoxyethyl)-1H-
indol-3-yl)-1H-pyrrole-2,5-dione (12). The title compound was
synthesized from commercially available 5,6-difluoroindole (32d) and
2-bromoethyl methyl ether according to general procedures A−C,
purified using gradient I preparative HPLC, and isolated as an orange
solid in 37% yield over 3 steps; mp 98−103 °C. IR (ATR): νmax 3231,
3061, 2931, 1704, 1700, 1544, 1482, 1451, 1335, 1113, 854, 745 cm−1.
1H NMR (400 MHz, DMSO-d6) δ 11.22 (s, 1H), 8.34 (s, 1H), 7.88 (s,
134.1 (CH), 131.9 (C), 127.3 (C), 125.0 (C), 124.9 (CH), 124.7
(CH), 123.6 (CH), 123.6 (C), 122.8 (CH), 121.5 (CH), 117.2 (d,
1JC−F = 298.2 Hz, TFA), 112.9 (C), 112.5 (CH), 111.5 (CH), 111.1
(C), 104.0 (C), 45.6 (CH2), 43.2 (CH2), 32.5 (CH3), 26.2 (CH2).
HRMS (ESI): m/z [M + H]+ calculated for C24H21BrN3O3, 478.0761;
found, 478.0768. HPLC purity: 99.1%.
3-(Benzofuran-3-yl)-4-(5-fluoro-1-(3-(methylamino)propyl)-
1H-indol-3-yl)-1H-pyrrole-2,5-dione trifluoroacetate (8). The
title compound was synthesized from commercially available 5-
fluoroindole (32b) and tert-butyl (3-bromopropyl)(methyl)carbamate
according to general procedures A−D, purified using gradient II
preparative HPLC, and isolated as an orange solid in 16% yield over 4
steps; mp 120−125 °C. IR (ATR): νmax 3041, 1713, 1670, 1547, 1480,
1452, 1338, 1199, 1123, 745, 721 cm−1. 1H NMR (400 MHz, DMSO-
d6) δ 11.23 (s, 1H), 8.32 (s, 1H), 8.30 (br s, 2H), 7.99 (s, 1H), 7.65−
7.56 (m, 2H), 7.25 (t, J = 8.2 Hz, 1H), 7.01−6.90 (m, 2H), 6.82 (d, J =
7.6 Hz, 1H), 6.65 (dd, J = 10.4, 2.6 Hz, 1H), 4.34 (t, J = 7.0 Hz, 2H),
2.85−2.79 (m, 2H), 2.54 (s, 3H), 2.07−2.01 (m, 2H). 13C NMR (100
2
MHz, DMSO-d6) 172.0 (CO), 171.7 (CO), 158.2 (q, JC−F
=
31.5 Hz, TFA), 157.2 (d, 1JC−F = 234 Hz, C), 154.2 (C), 147.2 (CH),
1H), 7.74−7.67 (m, 1H), 7.63 (d, J = 8.6 Hz, 1H), 7.24 (t, J = 8.3 Hz,
1H), 6.92−6.84 (m, 2H), 6.73 (d, J = 8.1 Hz, 1H), 4.37 (t, J = 5.1 Hz,
2H), 3.57 (t, J = 4.8 Hz, 2H), 3.17 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 171.9 (CO), 171.6 (CO), 154.2 (C), 147.3 (CH),
147.1 (dd, J = 237, 16.1 Hz, C), 144.8 (dd, J = 234, 14.6 Hz, C), 134.9
(C), 134.8 (CH), 131.8 (C), 131.7 (C), 124.9 (CH), 123.6 (C), 122.8
3
134.6 (CH), 132.5 (C), 132.1 (C), 126.3 (d, JC−F = 10.5 Hz, C),
125.1 (C), 124.9 (CH), 123.1 (C), 122.8 (CH), 121.6 (CH), 117.2 (d,
3
1JC−F = 298.2 Hz, TFA), 111.8 (d, JC−F = 9.8 Hz, CH), 111.5 (CH),
111.1 (C), 110.4 (d, 2JC−F = 25.8 Hz, CH), 106.1 (d, 2JC−F = 24.9 Hz,
4
CH), 104.5 (d, JC−F = 4.4 Hz, C), 45.7 (CH2), 43.3 (CH2), 32.6
3
(CH3), 26.2 (CH2). HRMS (ESI): m/z [M + H]+ calculated for
C24H21FN3O3, 418.1561; found, 418.1570. HPLC purity: 98.4%.
3-(Benzofuran-3-yl)-4-(1-(2-methoxyethyl)-1H-indol-3-yl)-
1H-pyrrole-2,5-dione (9). The title compound was synthesized from
commercially available indole (32c) and 2-bromoethyl methyl ether
according to general procedures A−C, purified using gradient I
preparative HPLC, and isolated as a yellow solid in 35% yield over 3
steps; mp 147−150 °C. IR (ATR): νmax 3144, 2928, 1710, 1700, 1548,
1451, 1326, 1099, 1072, 734 cm−1. 1H NMR (400 MHz, DMSO-d6) δ
11.17 (s, 1H), 8.27 (s, 1H), 7.90 (s, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.52
(d, J = 8.0 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.06 (t, J = 7.8 Hz, 1H),
6.96−6.79 (m, 3H), 6.74 (t, J = 7.6 Hz, 1H), 4.41 (t, J = 5.2 Hz, 2H),
3.62 (t, J = 5.2 Hz, 2H), 3.19 (s, 3H). 13C NMR (100 MHz, DMSO-
(CH), 121.5 (CH), 121.2 (d, JC−F = 8.8 Hz, C), 111.5 (CH), 111.0
(C), 107.6 (d, 2JC−F = 20.5 Hz, CH), 104.5 (d, 4JC−F = 3.7 Hz, C), 99.4
2
(d, JC−F = 22.0 Hz, CH), 70.7 (CH2), 58.1 (CH3), 46.2 (CH2).
HRMS (ESI): m/z [M − H]− calculated for C23H15F2N2O4:,
421.1005; found, 421.1010. HPLC purity: 96.5%.
3-(Benzofuran-3-yl)-4-(5-bromo-1-methyl-1H-pyrrolo[2,3-b]-
pyridin-3-yl)-1H-pyrrole-2,5-dione (13). The title compound was
synthesized from commercially available 5-bromo-7-azaindole (37a)
according to general procedures A−C, purified using gradient I
preparative HPLC, and isolated as an orange solid in 25% yield over 3
steps; mp 244−248 °C. IR (ATR): νmax 3136, 3039, 2751, 1707, 1538,
1452, 1338, 1120, 744 cm−1. 1H NMR (400 MHz, DMSO-d6) δ 11.28
(s, 1H), 8.36 (s, 1H), 8.27 (s, 1H), 8.13 (s, 1H), 7.65 (d, J = 8.2 Hz,
5124
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1H), 7.41 (s, 1H), 7.26 (t, J = 7.7 Hz, 1H), 6.96 (t, J = 7.2 Hz, 1H),
6.83 (d, J = 7.8 Hz, 1H), 3.85 (s, 3H). 13C NMR (100 MHz, DMSO-
d6) 171.8 (CO), 171.5 (CO), 154.3 (C), 147.4 (CH), 145.9 (C),
143.3 (CH), 135.6 (CH), 131.4 (CH), 131.4 (C), 125.0 (CH), 124.9
(C), 123.8 (C), 122.9 (CH), 121.4 (CH), 119.4 (C), 111.5 (CH),
111.5 (C), 110.9 (C), 102.0 (C), 31.6 (CH3). HRMS (ESI): m/z [M
+ H]+ calculated for C20H13BrN3O3, 422.0135; found, 422.0124.
HPLC purity: 98.3%.
3-(Benzofuran-3-yl)-4-(5-bromo-1-(2-methoxyethyl)-1H-
pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-dione (14). The title
compound was synthesized from commercially available 5-bromo-7-
azaindole (37a) and 2-bromoethyl methyl ether according to general
procedures A−C, purified using gradient I preparative HPLC, and
isolated as a yellow solid in 25% yield over 3 steps; mp 142−145 °C.
IR (ATR): νmax 3133, 3040, 2752, 1714, 1547, 1455, 1335, 1108, 744
cm−1. 1H NMR (400 MHz, DMSO-d6) δ 11.28 (s, 1H), 8.37 (s, 1H),
8.28 (d, J = 1.8 Hz, 1H), 8.02 (s, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.60
(d, J = 8.0 Hz, 1H), 7.26 (t, J = 8.1 Hz, 1H), 6.93 (t, J = 7.4 Hz, 1H),
6.75 (d, J = 7.8 Hz, 1H), 4.42 (t, J = 5.2 Hz, 2H), 3.60 (t, J = 5.2 Hz,
2H), 3.14 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.8 (CO),
171.5 (CO), 154.4 (C), 147.5 (CH), 145.6 (C), 143.3 (CH), 134.8
(CH), 131.6 (CH), 131.4 (C), 125.0 (CH), 124.6 (C), 124.3 (C),
122.9 (CH), 121.5 (CH), 119.6 (C), 111.6 (C), 111.5 (CH), 110.7
(C), 102.3 (C), 70.2 (CH2), 57.9 (CH3), 44.0 (CH2). HRMS (ESI):
m/z [M + H]+ calculated for C22H17BrN3O4, 466.0397; found,
466.0397. HPLC purity: 99.2%.
131.5 (C), 130.4 (C), 128.6 (CH), 125.5 (CH), 125.0 (C), 123.1 (C),
123.0 (CH), 121.5 (CH), 111.5 (CH), 111.2 (C), 103.0 (C), 97.3
(CH), 54.2 (CH3), 33.7 (CH3). HRMS (ESI): m/z [M + H]+
calculated for C21H16N3O4, 374.1135; found, 374.1138. HPLC purity:
99.7%.
3-(Benzofuran-3-yl)-4-(5-methoxy-1-(2-methoxyethyl)-1H-
pyrrolo[2,3-c]pyridin-3-yl)-1H-pyrrole-2,5-dione (18). The title
compound was synthesized from commercially available 6-aza-5-
methoxyindole and 2-bromoethyl methyl ether according to general
procedures A−C, purified using gradient I preparative HPLC, and
isolated as an orange solid in 15% yield over 3 steps; mp 183−188 °C.
IR (ATR): νmax 3046, 2941, 2724, 1712, 1695, 1649, 1549, 1451, 1338,
1
1179, 1115, 743 cm−1. H NMR (400 MHz, DMSO-d6) δ 11.24 (s,
1H), 8.54 (s, 1H), 8.29 (s, 1H), 8.12 (s, 1H), 7.64 (d, J = 8.2 Hz, 1H),
7.26 (t, J = 8.2 Hz, 1H), 6.96−6.87 (m, 2H), 6.19 (s, 1H), 4.47 (d, J =
5.2 Hz, 2H), 3.63 (d, J = 5.2 Hz, 2H), 3.53 (s, 3H), 3.20 (s, 3H). 13C
NMR (100 MHz, DMSO-d6) δ 171.8 (CO), 171.5 (CO), 156.3
(C), 154.2 (C), 147.4 (CH), 140.5 (CH), 135.8 (C), 131.3 (C), 129.9
(C), 128.6 (CH), 125.2 (CH), 125.0 (C), 123.9 (C), 123.0 (CH),
121.4 (CH), 111.6 (CH), 111.0 (C), 103.6 (C), 97.4 (CH), 70.8
(CH2), 58.2 (CH3), 54.4 (CH3), 46.6 (CH2). HRMS (ESI): m/z [M +
H]+ calculated for C23H20N3O5, 418.1398; found, 418.1406. HPLC
purity: 99.9%.
3-(Imidazo[1,2-a]pyridin-3-yl)-4-(5-methyl-5H-[1,3]dioxolo-
[4,5-f ]indol-7-yl)-1H-pyrrole-2,5-dione Trifluoroacetate (19).
The title compound was synthesized from 5,6-methylenedioxyindole
and 2-(imidazo[1,2-a]pyridin-3-yl)acetamide 38 according to general
procedures A−C, purified using gradient II preparative HPLC, and
isolated as a red solid in 15% yield over 3 steps; mp 134−139 °C. IR
(ATR): νmax 3129, 2725, 1711, 1671, 1471, 1334, 1182, 1100, 755
cm−1. 1H NMR (400 MHz, CDCl3/CD3OD) δ 11.25 (s, 1H), 8.16 (s,
1H), 7.91 (s, 1H), 7.80−7.84 (m, 2H), 7.70 (t, J = 7.8 Hz, 1H), 7.04
(s, 1H), 6.92 (t, J = 6.9 Hz, 1H), 5.73 (s, 2H), 5.54 (s, 1H), 3.74 (s,
3H). 13C NMR (100 MHz, CDCl3/CD3OD) δ 170.7 (CO), 170.5
(CO), 145.6 (C), 144.1 (C), 135.0 (C), 134.3 (CH), 132.3 (C),
131.8 (CH), 127.2 (CH), 126.7 (C), 119.3 (C), 117.5 (C), 115.7
(CH), 112.9 (CH), 111.9 (C), 104.6 (C), 100.7 (CH), 100.7 (CH2),
96.9 (CH), 91.2 (CH), 33.1 (CH3). HRMS (ESI): m/z [M + H]+
calculated for C21H15N4O4, 387.1088; found, 387.1087. HPLC purity:
99.0%.
3-(Benzofuran-3-yl)-4-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-
yl)-1H-pyrrole-2,5-dione (15). The title compound was synthesized
from commercially available 7-azaindole (37b) according to general
procedures A−C, purified using gradient I preparative HPLC, and
isolated as an orange solid in 14% yield over 3 steps; mp 219−223 °C.
IR (ATR): νmax 3138, 3020, 2734, 1717, 1700, 1545, 1452, 1332, 1110,
978, 742 cm−1. 1H NMR (400 MHz, DMSO-d6) δ 11.25 (s, 1H), 8.33
(s, 1H), 8.11 (d, J = 4.5 Hz, 1H), 8.13 (s, 1H), 7.62 (d, J = 8.3 Hz,
1H), 7.26−7.19 (m, 2H), 6.92 (t, J = 7.2 Hz, 1H), 6.87−6.78 (m, 2H),
3.88 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 172.0 (CO), 171.7
(CO), 154.2 (C), 147.4 (C), 147.3 (CH), 143.3 (CH), 134.3 (CH),
131.9 (C), 129.1 (CH), 125.2 (C), 124.9 (CH), 123.0 (C), 122.8
(CH), 121.6 (CH), 117.9 (C), 116.3 (CH), 111.5 (CH), 111.1 (C),
102.4 (C), 31.4 (CH3). HRMS (ESI): m/z [M + H]+ calculated for
C20H14N3O3, 344.1030; found, 344.1041. HPLC purity: 99.9%.
3-(Benzofuran-3-yl)-4-(1-(2-methoxyethyl)-1H-pyrrolo[2,3-
b]pyridin-3-yl)-1H-pyrrole-2,5-dione (16). The title compound
was synthesized from commercially available 7-azaindole (37b) and 2-
bromoethyl methyl ether according to general procedures A−C,
purified using gradient I preparative HPLC, and isolated as an orange
solid in 15% yield over 3 steps; mp 180−183 °C. IR (ATR): νmax 3136,
3-(5-Bromo-1-methyl-1H-indol-3-yl)-4-(imidazo[1,2-a]-
pyridin-3-yl)-1H-pyrrole-2,5-dione Trifluoroacetate (20). The
title compound was synthesized from 5-bromoindole and 2-(imidazo-
[1,2-a]pyridin-3-yl)acetamide 38 according to general procedures A−
C, purified using gradient II preparative HPLC, and isolated as a
yellow solid in 15% yield over 3 steps; mp 230−235 °C. IR (ATR):
νmax 3163, 3081, 2940, 2708, 1717, 1656, 1552, 1330, 1201, 1178,
1126, 760 cm−1. 1H NMR (400 MHz, CD3OD/DMSO-d6) δ 11.24 (s,
1H), 8.33 (s, 1H), 8.17 (s, 1H), 7.92 (d, J = 9.0 Hz, 1H), 7.85 (d, J =
6.9 Hz, 1H), 7.70 (t, J = 8.0 Hz, 1H), 7.36 (d, J = 8.7 Hz, 1H), 7.13
(dd, J = 8.7, 1.7 Hz, 1H), 6.95 (t, J = 6.9 Hz, 1H), 6.21 (d, J = 1.7 Hz,
1H), 3.85 (s, 3H). 13C NMR (100 MHz, CD3OD/DMSO-d6) δ 171.7
(CO), 171.3 (CO), 158.2 (q, 2JC−F = 36.1 Hz, TFA), 141.7 (C),
137.9 (CH), 136.4 (CH), 135.5 (C), 133.0 (CH), 128.3 (CH), 128.1
1
2993, 2731, 1712, 1547, 1331, 1109, 982, 744 cm−1. H NMR (400
MHz, DMSO-d6) δ 11.25 (s, 1H), 8.35 (s, 1H), 8.19 (d, J = 4.8 Hz,
1H), 8.04 (s, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H),
7.23 (t, J = 7.3 Hz, 1H), 6.93−6.84 (m, 2H), 6.77 (d, J = 7.9 Hz, 1H),
4.47 (t, J = 5.3 Hz, 2H), 3.65 (t, J = 5.3 Hz, 2H), 3.18 (s, 3H). 13C
NMR (100 MHz, DMSO-d6) δ 171.9 (CO), 171.6 (CO), 154.3
(C), 147.3 (CH), 147.2 (C), 143.3 (CH), 133.6 (CH), 131.9 (C),
129.3 (CH), 124.9 (CH), 124.9 (C), 123.6 (C), 122.8 (CH), 121.6
(CH), 118.0 (C), 116.5 (CH), 111.5 (CH), 110.9 (C), 102.7 (C),
70.3 (CH2), 58.0 (CH3), 43.8 (CH2). HRMS (ESI): m/z [M + H]+
calculated for C22H18N3O4, 388.1292; found, 388.1298. HPLC purity:
98.6%.
(C), 127.6 (C), 125.9 (CH), 122.1 (CH), 117.8 (C), 117.2 (d, 1JC−F
=
298.2 Hz, TFA), 116.6 (C), 115.0 (C), 114.5 (CH), 114.0 (CH),
113.4 (CH), 104.2 (C), 33.7 (CH3). HRMS (ESI): m/z [M + H]+
calculated for C20H14BrN4O2, 421.02946; found, 421.0300. HPLC
purity: 96.8%.
3-(Benzofuran-3-yl)-4-(5-methoxy-1-methyl-1H-pyrrolo[2,3-
c]pyridin-3-yl)-1H-pyrrole-2,5-dione (17). The title compound
was synthesized from commercially available 6-aza-5-methoxyindole
according to general procedures A−C, purified using gradient I
preparative HPLC, and isolated as an orange solid in 13% yield over 3
steps; mp 170−175 °C. IR (ATR): νmax 3039, 2732, 1717, 1678, 1652,
1547, 1186, 1119, 722 cm−1. 1H NMR (400 MHz, DMSO-d6) δ 11.24
(s, 1H), 8.49 (s, 1H), 8.26 (s, 1H), 8.21 (s, 1H), 7.64 (d, J = 8.3 Hz,
1H), 7.28−7.24 (m, 1H), 7.00 (s, 2H), 6.03 (s, 1H), 3.94 (s, 3H), 3.49
(s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.9 (CO), 171.6
(CO), 156.6 (C), 154.2 (C), 147.3 (CH), 140.9 (CH), 135.4 (C),
3-(Benzofuran-3-yl)-4-(7-(2-methoxyethyl)-1-methyl-1H-
indol-3-yl)-1H-pyrrole-2,5-dione (21). The title compound was
synthesized from alcohol 40a (generated from indole-7-carboxalde-
hyde 39 employing general methods E and F) and methyl iodide
according to general procedures A−C, purified using gradient I
preparative HPLC, and isolated as an orange solid in 26% yield over 3
steps; mp 91−96 °C. IR (ATR): νmax 3135, 2928, 1704, 1699, 1695,
1545, 1450, 1320, 1102, 743 cm−1. 1H NMR (400 MHz, DMSO-d6) δ
11.17 (s, 1H), 8.22 (s, 1H), 7.85 (s, 1H), 7.58 (d, J = 8.4 Hz, 1H),
7.25−7.19 (m, 1H), 6.94−6.89 (m, 2H), 6.85 (d, J = 7.6 Hz, 1H), 6.70
(d, J = 8.0 Hz, 1H), 6.61 (t, J = 7.6 Hz, 1H), 4.11 (s, 3H), 3.59 (t, J =
5125
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7.0 Hz, 2H), 3.28 (t, J = 7.2 Hz, 2H), 3.24 (s, 3H). 13C NMR (100
MHz, DMSO-d6) δ 172.1 (CO), 171.9 (CO), 154.1 (C), 146.9
(CH), 136.0 (CH), 134.9 (C), 132.4 (C), 126.9 (C), 125.5 (C), 124.7
(CH), 124.5 (CH), 123.3 (C), 122.7 (CH), 122.4 (C), 121.7 (CH),
120.1 (CH), 119.0 (CH), 111.6 (C), 111.3 (CH), 103.5 (C), 73.3
(CH2), 58.0 (CH3), 37.2 (CH3), 31.5 (CH2). HRMS (ESI): m/z [M +
H]+ calculated for C24H21N2O4, 401.1496; found, 401.1510. HPLC
purity: 98.1%.
3-(Benzofuran-3-yl)-4-(7-(2-(tert-butoxy)ethyl)-1-methyl-1H-
indol-3-yl)-1H-pyrrole-2,5-dione (22). The title compound was
synthesized from alcohol 40a (generated from indole-7-carboxalde-
hyde 39 employing general methods E and F) and tert-butyl 2,2,2-
trichloroacetimidate (method G) according to general procedures A−
C, purified using gradient I preparative HPLC, and isolated as an
orange solid in 16% yield over 3 steps; mp 107−112 °C. IR (ATR):
νmax 3211, 2971, 1704, 1699, 1545, 1451, 1322, 1106, 1077, 743 cm−1.
1H NMR (400 MHz, DMSO-d6) δ 11.16 (s, 1H), 8.22 (s, 1H), 7.85 (s,
steps; mp 152−157 °C. IR (ATR): νmax 3136, 3050, 2738, 1710, 1670,
1547, 1452, 1340, 1181, 1120, 746 cm−1. 1H NMR (400 MHz,
DMSO-d6) δ 11.29 (s, 1H), 9.65 (br s, 2H), 8.30 (s, 1H), 7.99 (s, 1H),
7.63 (d, J = 8.4 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 7.04−6.94 (m, 2H),
6.90 (d, J = 7.6 Hz, 1H), 6.63 (d, J = 9.2 Hz, 1H), 4.66 (br s, 4H), 3.71
(br s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 171.9 (CO), 171.6
(CO), 158.2 (q, 2JC−F = 36.1 Hz, TFA), 156.6 (d, 1JC−F = 233.4 Hz,
C), 154.2 (C), 147.4 (CH), 135.9 (CH), 132.1 (C), 131.6 (C), 127.5
3
(d, JC−F = 10.6 Hz, C), 125.1 (C), 125.0 (CH), 124.2 (C), 123.0
3
1
(CH), 121.6 (CH), 119.8 (d, JC−F = 9.6 Hz, C), 117.3 (d, JC−F
=
2
298.2 Hz, TFA), 111.6 (CH), 111.1 (C), 110.9 (d, JC−F = 27 Hz,
2
4
CH), 105.9 (d, JC−F = 24.4 Hz, CH), 105.1 (d, JC−F = 4.4 Hz, C),
46.7 (CH2), 46.6 (CH2), 45.6 (CH2). HRMS (ESI): m/z [M + H]+
calculated for C23H17FN3O3, 402.1248; found, 402.1260. HPLC
purity: 97.9%.
3-(Benzofuran-3-yl)-4-(9-bromo-1,2,3,4-tetrahydro-[1,4]-
diazepino[6,7,1-hi]indol-7-yl)-1H-pyrrole-2,5-dione Trifluoroa-
cetate (26). The title compound was synthesized from 5-bromoindo-
line 45 (see Supporting Information), purified using gradient II
preparative HPLC, and isolated as an orange solid; mp 243−248 °C.
IR (ATR): νmax 3138, 3050, 1709, 1700, 1695, 1623, 1545, 1336, 1179,
746 cm−1. 1H NMR (400 MHz, DMSO-d6) δ 11.29 (s, 1H), 9.55 (br s,
2H), 8.31 (s, 1H), 7.96 (s, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.30−7.25
(m, 2H), 7.08 (s, 1H), 6.99 (t, J = 7.6 Hz, 1H), 6.88 (d, J = 8.8 Hz,
1H), 4.66 (br s, 4H), 3.70 (br s, 2H). 13C NMR (100 MHz, DMSO-
d6) δ 171.8 (CO), 171.5 (CO), 158.2 (q, 2JC−F = 30.9 Hz, TFA),
154.4 (C), 147.4 (CH), 135.4 (CH), 134.1 (C), 131.5 (C), 128.4 (C),
125.0 (CH), 124.9 (C), 124.9 (CH), 124.6 (C), 123.6 (CH), 123.0
1H), 7.58 (d, J = 8.4 Hz, 1H), 7.24−7.18 (m, 1H), 6.93−6.88 (m,
2H), 6.85 (d, J = 7.6 Hz, 1H), 6.71 (dd, J = 8.0, 1.0 Hz, 1H), 6.61 (t, J
= 7.6 Hz, 1H), 4.12 (s, 3H), 3.55 (t, J = 7.0 Hz, 2H), 3.18 (t, J = 6.8
Hz, 2H), 1.06 (s, 9H). 13C NMR (100 MHz, DMSO-d6) δ 172.2 (C
O), 171.9 (CO), 154.1 (C), 146.9 (CH), 136.0 (CH), 135.0 (C),
132.4 (C), 126.9 (C), 125.5 (C), 124.7 (CH), 124.6 (CH), 123.7 (C),
122.7 (CH), 122.2 (C), 121.7 (CH), 120.1 (CH), 119.0 (CH), 111.5
(C), 111.3 (CH), 103.5 (C), 72.3 (CH2), 62.9 (C), 37.1 (CH3), 32.6
(CH2), 27.2 (CH3). HRMS (ESI): m/z [M + H]+ calculated for
C27H27N2O4, 443.1965; found, 443.1983. HPLC purity: 96.9%.
3-(Benzofuran-3-yl)-4-(1,7-bis(2-methoxyethyl)-1H-indol-3-
yl)-1H-pyrrole-2,5-dione (23). The title compound was synthesized
from alcohol 40b (generated from indole-7-carboxaldehyde 39
employing general methods E and F) and 2-bromoethyl methyl
ether according to general procedures A−C, purified using gradient I
preparative HPLC, and isolated as an orange solid in 16% yield over 3
steps; mp 178−182 °C. IR (ATR): νmax 3146, 2892, 1697, 1559, 1337,
1
(CH), 121.6 (CH), 120.1 (C), 116.3 (d, JC−F = 294.3 Hz, TFA),
112.5 (C), 111.5 (CH), 111.1 (C), 104.6 (C), 46.5 (CH2), 46.3
(CH2), 45.3 (CH2). HRMS (ESI): m/z [M + H]+ calculated for
C23H17BrN3O3, 462.0448; found, 462.0448. HPLC purity: 98.9%.
3-(Benzofuran-3-yl)-4-(9-fluoro-2-(2-(pyridin-4-yl)acetyl)-
1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-1H-pyr-
role-2,5-dione (27). The title compound was synthesized from
maleimide 24 and pyrazine-2-carboxylic acid according to general
procedure H, purified using gradient II preparative HPLC, and isolated
as an orange solid (30%); mp 167−172 °C. IR (ATR): νmax 3138,
1
1109, 747 cm−1. H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H),
8.29 (s, 1H), 7.78 (s, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.20 (t, J = 7.6 Hz,
1H), 6.89−6.81 (m, 3H), 6.77 (d, J = 8.0 Hz, 1H), 6.67 (t, J = 7.6 Hz,
1H), 4.55 (t, J = 5.0 Hz, 2H), 3.63−3.54 (m, 4H), 3.25−3.15 (m, 8H).
13C NMR (100 MHz, DMSO-d6) δ 172.0 (CO), 171.8 (CO),
1
3053, 1708, 1627, 1545, 1451, 1332, 1017, 744 cm−1. H NMR (400
MHz, DMSO-d6, two rotamers observed) δ 11.22 (s, 1H), 11.21 (s,
1H), 8.87 (d, J = 1.5 Hz, 1H), 8.77 (d, J = 2.5 Hz, 1H), 8.75 (d, J = 2.5
Hz, 1H), 8.69 (dd, J = 2.5, 1.5 Hz, 1H), 8.66 (dd, J = 2.5, 1.5 Hz, 1H),
8.59 (d, J = 1.5 Hz, 1H), 8.25 (s, 2H), 7.96 (s, 1H), 7.85 (s, 1H), 7.61
(s, 1H), 7.59 (s, 1H), 7.27−7.21 (m, 2H), 7.03 (d, J = 6.8 Hz, 1H),
6.97−6.93 (m, 4H), 6.82−6.69 (m, 3H), 6.60 (t, J = 7.6 Hz, 1H), 6.52
(d, J = 6.8 Hz, 1H), 5.17 (s, 2H), 4.98 (s, 2H), 4.73−4.69 (m, 2H),
4.61−4.57 (m, 2H), 4.23−4.18 (m, 2H), 4.10−4.05 (m, 2H). 13C
NMR (100 MHz, DMSO-d6, two rotamers observed) δ 172.0 (C
O), 171.8 (CO), 166.1 (CO), 165.9 (CO), 154.2 (C), 149.3
(C), 149.2 (C), 147.2 (CH), 145.7 (CH), 145.6 (CH), 144.6 (CH),
143.8 (CH), 143.2 (CH), 143.0 (CH), 135.6 (C), 135.2 (C), 134.4
(CH), 134.3 (CH), 132.2 (C), 126.7 (C), 125.3 (C), 125.3 (C), 124.8
(CH), 123.3 (C), 123.3 (C), 123.2 (C), 122.8 (CH), 121.8 (CH),
121.7 (CH), 120.6 (CH), 120.3 (CH), 120.1 (CH), 120.0 (CH),
111.4 (CH), 104.7 (C), 104.5 (C), 50.3 (CH2), 50.1 (CH2), 48.7
(CH2), 47.3 (CH2), 46.9 (CH2), 40.5 (CH2). HRMS (ESI): m/z [M +
H]+ calculated for C28H20N5O4, 490.1510; found, 490.1523. HPLC
purity: 98.2%.
154.1 (C), 147.1 (CH), 135.1 (CH), 134.2 (C), 132.2 (C), 127.3 (C),
125.2 (C), 124.8 (CH), 124.7 (CH), 123.4 (C), 122.8 (C), 122.6
(CH), 121.7 (CH), 120.1 (CH), 119.2 (CH), 111.4 (CH), 111.3 (C),
104.3 (C), 72.8 (CH2), 71.7 (CH2), 58.3 (CH3), 58.1 (CH3), 48.2
(CH2), 32.0 (CH2). HRMS (ESI): m/z [M + H]+ calculated for
C26H25N2O5, 445.1758; found, 445.1777. HPLC purity: 99.4%.
3-(Benzofuran-3-yl)-4-(1,2,3,4-tetrahydro-[1,4]diazepino-
[6,7,1-hi]indol-7-yl)-1H-pyrrole-2,5-dione Tifluoroacetate (24).
The title compound was synthesized from diazepinoindole 43a
(generated from indole-7-carboxaldehyde 39) according to general
procedures B−D, purified using gradient II preparative HPLC, and
isolated as an orange solid in 12% yield over 3 steps; mp 160−165 °C.
IR (ATR): νmax 3038, 2731, 1706, 1695, 1669, 1545, 1451, 1340, 1180,
1
1116, 745 cm−1. H NMR (400 MHz, DMSO-d6) δ 11.26 (s, 1H),
9.44 (br s, 2H), 8.26 (s, 1H), 7.97 (s, 1H), 7.61 (d, J = 8.4 Hz, 1H),
7.25 (t, J = 8.0 Hz, 1H), 7.05 (d, J = 7.2 Hz, 1H), 7.02−6.86 (m, 3H),
6.77 (t, J = 8.0 Hz, 1H), 4.68 (br s, 4H), 3.72 (br s, 2H). 13C NMR
(100 MHz, DMSO-d6) δ 172.0 (CO), 171.7 (CO), 158.2 (q,
2JC−F = 36.1 Hz, TFA), 154.2 (C), 147.3 (CH), 135.3 (C), 134.4
3-(Benzofuran-3-yl)-4-(9-fluoro-2-(2-(pyridin-4-yl)acetyl)-
1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-1H-pyr-
role-2,5-dione Trifluoroacetate (28). The title compound was
synthesized from maleimide 24 and 2-(pyridin-4-yl)acetic acid
according to general procedure H, purified using gradient II
preparative HPLC, and isolated as an orange solid (30%); mp 142−
147 °C. IR (ATR): νm1ax 3063, 2725, 1711, 1645, 1548, 1451, 1338,
1180, 1127, 748 cm−1. H NMR (400 MHz, DMSO-d6, two rotamers
observed) δ 11.23 (s, 1H), 11.23 (s, 1H), 8.80 (s, 1H), 8.78 (s, 1H),
8.72 (s, 1H), 8.71 (s, 1H), 8.25 (s, 1H), 8.22 (s, 1H), 7.99 (s, 1H),
7.90 (s, 1H), 7.82 (s, 1H), 7.81 (s, 1H), 7.71 (s, 1H), 7.70 (s, 1H),
7.61 (s, 1H), 7.59 (s, 1H), 7.29−7.21 (m, 2H), 7.05−6.60 (m, 12H),
(CH), 131.9 (C), 126.8 (C), 125.3 (C), 124.9 (CH), 123.9 (C), 122.9
(CH), 122.8 (CH), 121.8 (CH), 121.3 (CH), 120.5 (CH), 118.0 (C),
117.2 (d, 1JC−F = 298.2 Hz, TFA), 111.5 (CH), 111.3 (C), 104.9 (C),
46.9 (CH2), 46.6 (CH2), 45.2 (CH2). HRMS (ESI): m/z [M + H]+
calculated for C23H18N3O3, 384.1343; found, 384.1362. HPLC purity:
98.2%.
3-(Benzofuran-3-yl)-4-(9-fluoro-1,2,3,4-tetrahydro-[1,4]-
diazepino[6,7,1-hi]indol-7-yl)-1H-pyrrole-2,5-dione Trifluoroa-
cetate (25). The title compound was synthesized from diazepinoin-
dole 43b (generated from 5-fluoroindole-7-carboxaldehyde 42)34
according to general procedures B−D, purified using gradient II
preparative HPLC, and isolated as an orange solid in 14% yield over 3
5126
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Journal of Medicinal Chemistry
Article
5.09 (s, 2H), 5.03 (s, 2H), 4.76 (br s, 2H), 4.60 (br s, 2H), 4.22−4.15
(m, 4H), 4.11 (s, 2H), 4.03 (s, 2H). 13C NMR (100 MHz, DMSO-d6,
4H), 3.09−3.03 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 171.9
1
(CO), 171.7 (CO), 163.0 (CO), 156.8 (d, JC−F = 234.9 Hz,
C), 154.2 (C), 147.2 (CH), 135.7 (CH), 132.2 (C), 131.9 (C), 127.2
(d, 3JC−F = 10.7 Hz, C), 126.6 (d, 3JC−F = 8.6 Hz, C), 125.2 (C), 124.9
(CH), 123.3 (C), 122.7 (CH), 121.7 (CH), 111.4 (CH), 111.2 (C),
109.5 (d, 2JC−F = 26.4 Hz, CH), 104.7 (d, 4JC−F = 4.4 Hz, C), 104.3 (d,
2JC−F = 24.9 Hz, CH), 65.7 (CH2), 50.3 (CH2), 49.0 (CH2), 48.8
(CH2), 47.2 (CH2). HRMS (ESI): m/z [M − H]− calculated for
C28H22FN4O5, 513.1580; found, 513.1605. HPLC purity: 97.3%.
Steroidogenesis Assay. Cell Culture. MA-10 mouse Leydig
tumor cells were a gift from Dr. Mario Ascoli (University of Iowa).
Cells were cultured in 75 cm2 cell culture flasks with Dulbecco’s
Modified Eagle’s Medium (DMEM)/nutrient mixture F-12 Ham
supplemented with 5% fetal bovine serum and 2.5% heat-inactivated
horse serum, as previously described.43
two rotamers observed) δ 172.1 (CO), 172.1 (CO), 171.8 (C
2
O), 171.8 (CO), 168.0 (CO), 167.9 (CO), 158.5 (q, JC−F
=
35.1 Hz, TFA), 155.2 (C), 154.3 (C), 154.2 (C), 154.2 (C), 147.2
(CH), 147.1 (CH), 142.5 (CH), 142.1 (CH), 135.6 (C), 135.3 (C),
134.3 (CH), 132.3 (C), 132.2 (C), 128.2 (C), 127.7 (CH), 126.8
(CH), 126.5 (C), 125.5 (C), 125.3 (C), 124.8 (CH), 123.7 (C), 123.4
(C), 123.3 (C), 123.0 (C), 122.9 (CH), 122.7 (CH), 121.8 (CH),
121.4 (CH), 120.8 (CH), 120.2 (CH), 120.2 (CH), 119.9 (CH),
116.2 (d, 1JC−F = 294.3 Hz, TFA), 111.4 (CH), 111.4 (C), 104.6 (C),
104.5 (C), 49.2 (CH2), 48.7 (CH2), 48.1 (CH2), 47.3 (CH2), 47.0
(CH2), 45.8 (CH2), 39.4 (CH2). HRMS (ESI): m/z [M + H]+
calculated for C30H23N4O4, 503.1714; found, 503.1727. HPLC purity:
99.4%.
Steroid Biosynthesis. For steroid synthesis experiments in the
presence of medium with the compounds under investigation, MA-10
cells were plated into 96-well plates at 2.5 × 104 cells per well. After
allowing cells to adhere for 18 h, the cells were washed with PBS and
exposed to the respective treatment (0−100 μM tested compound in
medium). All media conditions were serum-free. Compounds were
prepared as DMSO stock solutions and used at a final DMSO
concentration of <0.02% in media. Controls contained the same
amount of DMSO. At the end of the incubation, culture medium was
collected and tested for progesterone production using RIA with
antiprogesterone antisera (MP Biomedicals, Solon, OH), following the
manufacturer’s recommended conditions. Progesterone production
was normalized for the amount of protein in each well. RIA data were
analyzed using Prism 5.03 from GraphPad.
3-(Benzofuran-3-yl)-4-(9-fluoro-2-(piperidine-1-carbonyl)-
1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-1H-pyr-
role-2,5-dione (29). The title compound was synthesized from
maleimide 25 and piperidine-1-carbonyl chloride according to general
procedure I, purified using gradient II preparative HPLC, and isolated
as an orange solid (40%); mp 129−134 °C. IR (ATR): νmax 3136,
3053, 2934, 2855, 2731, 1714, 1614, 1548, 1451, 1336, 1116, 746
cm−1. 1H NMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H), 8.28 (s, 1H),
7.90 (s, 1H), 7.61 (d, J = 7.8 Hz, 1H), 7.25 (t, J = 8.0 Hz, 1H), 6.93 (t,
J = 7.2 Hz, 1H), 6.89−6.82 (m, 2H), 6.45 (d, J = 8.4 Hz, 1H), 4.65 (s,
2H), 4.50−4.44 (m, 2H), 3.87−3.81 (m, 2H), 3.05−2.97 (m, 4H),
1.51−1.43 (m, 6H). 13C NMR (100 MHz, DMSO-d6) δ 172.0 (C
1
O), 171.7 (CO), 163.3 (CO), 156.8 (d, JC−F = 245.9 Hz, C),
154.3 (C), 147.3 (CH), 135.7 (CH), 132.3 (C), 132.0 (C), 127.2 (d,
3
3JC−F = 10.7 Hz, C), 126.9 (d, JC−F = 8.6 Hz, C), 125.2 (C), 125.0
Protein Measurement. Proteins were quantified using the dye-
binding assay of Bradford44 with bovine serum albumin (BSA) as the
standard.
(CH), 123.3 (C), 122.8 (CH), 121.7 (CH), 111.5 (CH), 111.3 (C),
109.3 (d, 2JC−F = 30.0 Hz, CH), 104.8 (d, 4JC−F = 4.4 Hz, C), 104.2 (d,
2JC−F = 22.0 Hz, CH), 50.9 (CH2), 49.1 (CH2), 49.0 (CH2), 47.7
SmartCube Assay. Drugs were injected 15 min before the test,
during which multiple challenges were presented over the course of
the test session. At least 12 mice were used in each treatment group.
Digital videos of the subjects were processed with computer vision
algorithms to extract over 1400 dependent measures including
frequency and duration of behavioral states such as grooming and
rearing and many other features obtained during the test session.
These data were compared against a standard database of therapeutic
class signatures obtained with the same experimental protocol. The
database comprises 14 classes of drugs with some of the major classes,
such as the antidepressant class, comprising several subclasses with
representatives of most of the drugs on the market. Using machine
learning techniques, the reference database was combed to find
reference drug class signatures. The best performing techniques were
chosen from our evaluation tests and used to build classifiers that
capture the therapeutic class signatures. The behavioral signatures of
the test compound were then assessed quantitatively with these
classifiers to predict total and specific potential therapeutic utility.41,42
(CH2), 25.2 (CH2), 24.2 (CH2). HRMS (ESI): m/z [M + H]+
calculated for C29H26FN4O4, 513.1933; found, 513.1955. HPLC
purity: 97.3%.
3-(2-([1,4′-Bipiperidine]-1′-carbonyl)-9-fluoro-1,2,3,4-tetra-
hydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-4-(benzofuran-3-yl)-
1H-pyrrole-2,5-dione trifluoroacetate (30). The title compound
was synthesized from maleimide 25 and 4-piperidino-1-piperidine-
carbonyl chloride according to general procedure I, purified using
gradient II preparative HPLC, and isolated as a yellow solid (40%);
mp 190−195 °C. IR (ATR): νmax 3138, 3046, 2946, 2725, 1712, 1695,
1616, 1451, 1338, 1180, 1112, 748 cm−1. 1H NMR (400 MHz,
DMSO-d6) δ 11.23 (s, 1H), 8.97 (br s, 1H), 8.29 (s, 1H), 7.93 (s, 1H),
7.63 (d, J = 8.1 Hz, 1H), 7.26 (t, J = 6.3 Hz, 1H), 6.98−6.85 (m, 3H),
6.47 (d, J = 9.9 Hz, 1H), 4.71 (s, 2H), 4.53−4.49 (m, 2H), 3.90−3.86
(m, 2H), 3.60−3.46 (m, 5H), 2.96−2.89 (m, 2H), 2.77−2.67 (m, 2H),
1.95−1.33 (m, 10H). 13C NMR (100 MHz, DMSO-d6) δ 172.0 (C
2
O), 171.7 (CO), 162.6 (CO), 158.2 (q, JC−F = 31.6 Hz, TFA),
1
156.7 (d, JC−F = 233.4 Hz, C), 154.2 (C), 147.2 (CH), 135.8 (CH),
132.2 (C), 131.9 (C), 127.3 (d, 3JC−F = 10.8 Hz, C), 126.6 (d, 3JC−F
=
ASSOCIATED CONTENT
■
8.6 Hz, C), 125.2 (C), 124.9 (CH), 123.4 (C), 122.8 (CH), 121.7
1
S
* Supporting Information
(CH), 117.0 (d, JC−F = 294.8 Hz, TFA), 111.4 (CH), 111.2 (C),
109.3 (d, 2JC−F = 25.6 Hz, CH), 104.8 (d, 4JC−F = 4.3 Hz, C), 104.4 (d,
2JC−F = 23.4 Hz, CH), 62.6 (CH2), 50.7 (CH2), 49.0 (CH2), 48.9
Experimental procedures and analytical data for all the
compounds not included in the main text; kinetic aqueous
solubility measurements for compounds 5, 20, and 24. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(CH2), 48.9 (CH2), 45.4 (CH2), 25.5 (CH2), 22.8 (CH2), 21.4 (CH2).
HRMS (ESI): m/z [M − H]− calculated for C34H33FN5O4, 594.2522;
found, 594.2531. HPLC purity: 99.0%.
3-(Benzofuran-3-yl)-4-(9-fluoro-2-(morpholine-4-carbonyl)-
1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-1H-pyr-
role-2,5-dione (31). The title compound was synthesized from
maleimide 25 and morpholine-4-carbonyl chloride according to
general procedure I, purified using gradient I preparative HPLC, and
isolated as an orange solid (50%); mp 157−160 °C. IR (ATR): νmax
3139, 3058, 2962, 2856, 1711, 1700, 1615, 1547, 1458, 1334, 1111,
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +1-312-996-7577. Fax: +1-312-996-7107. E-mail:
kozikowa@uic.edu.
1
746 cm−1. H NMR (400 MHz, DMSO-d6) δ 11.22 (s, 1H), 8.28 (s,
Author Contributions
H.G., A.M., and I.N.G. contributed equally to this work. The
manuscript was written through contributions of all authors. All
1H), 7.91 (s, 1H), 7.62 (d, J = 8.1 Hz, 1H), 7.26 (t, J = 8.3 Hz, 1H),
6.94 (t, J = 7.3 Hz, 1H), 6.89−6.85 (m, 2H), 6.46 (d, J = 9.4 Hz, 1H),
4.71 (s, 2H), 4.53−4.49 (m, 2H), 3.90−3.86 (m, 2H), 3.60−3.54 (m,
5127
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Journal of Medicinal Chemistry
Article
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authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Institute of Mental
Health (R01MH079400 to A.K.), the Canadian Institutes of
Health Research (MOP102647to V.P.), and a Canada Research
Chair in Biochemical Pharmacology (V.P.). A.M. was supported
in part by a postdoctoral fellowship from the Le Fonds de la
Recherche du Queb
was supported by a Center grant from Le Fonds de la
Recherche du Quebec-Sante. The funding agencies had no role
́ ́
ec-Sante. The Research Institute of MUHC
́
́
in the study design, collection, analysis, and interpretation of
data, writing the manuscript, and decision to submit the
manuscript for publication.
(17) Beaulieu, J. M. A role for Akt and glycogen synthase kinase-3 as
integrators of dopamine and serotonin neurotransmission in mental
health. J. Psychiatry Neurosci. 2012, 37, 7−16.
ABBREVIATIONS USED
■
GSK-3, glycogen synthase kinase 3; HPA, hypothalamus
pituitary adrenal; SAR, structure−activity relationship; TLC,
thin layer chromatography; HPLC, high performance liquid
chromatography; rt, room temperature; TFA, trifluoroacetic
acid; RIA, radioimmunoassay; MTT, 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide; ATR, attenuated total
reflectance
(18) Beurel, E.; Song, L.; Jope, R. S. Inhibition of glycogen synthase
kinase-3 is necessary for the rapid antidepressant effect of ketamine in
mice. Mol. Psychiatry 2011, 16, 1068−1070.
(19) Latapy, C.; Rioux, V.; Guitton, M. J.; Beaulieu, J. M. Selective
deletion of forebrain glycogen synthase kinase 3beta reveals a central
role in serotonin-sensitive anxiety and social behaviour. Philos. Trans.
R. Soc., B 2012, 367, 2460−2474.
(20) Mines, M. A.; Yuskaitis, C. J.; King, M. K.; Beurel, E.; Jope, R. S.
GSK3 influences social preference and anxiety-related behaviors during
social interaction in a mouse model of fragile X syndrome and autism.
PLoS One 2010, 5 (e9706), 1−12.
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