Design and synthesis of (aza)indolyl maleimide-based covalent inhibitors of glycogen synthase kinase 3β

Article abstract of DOI:10.1039/c8ob00642c

As an important kinase in multiple signal transduction pathways, GSK-3β has been an attractive target for chemical probe discovery and drug development. Compared to numerous reversible inhibitors that have been developed, covalent inhibitors of GSK-3β are

Full text of DOI:10.1039/c8ob00642c

Organic &
Biomolecular Chemistry
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Design and synthesis of (aza)indolyl maleimide-
based covalent inhibitors of glycogen synthase
kinase 3β†
Cite this: DOI: 10.1039/c8ob00642c
Zhimin Yang, Hui Liu, Botao Pan, Fengli He and Zhengying Pan
*
As an important kinase in multiple signal transduction pathways, GSK-3β has been an attractive target for
chemical probe discovery and drug development. Compared to numerous reversible inhibitors that have
been developed, covalent inhibitors of GSK-3β are noticeably lacking. Here, we report the discovery of a
series of covalent GSK-3β inhibitors by optimizing both non-covalent interactions and reactive groups.
Among these covalent inhibitors, compound 38b with a mild α-fluoromethyl amide reactive group
emerges as a selective and covalent inhibitor against GSK-3β, effectively inhibits the phosphorylation of
glycogen synthase and tau protein, and increases β-catenin’s levels in living cells. In addition, compound
38b is highly permeable and not a substrate of P-glycoprotein.
Received 15th March 2018,
Accepted 1st May 2018
DOI: 10.1039/c8ob00642c
rsc.li/obc
3
9
10
11
such as bisaryl (2 , 9 and 4 ), macrocyclic bisindolyl (5
Introduction
1
2
13
14
and 6 ), and monoindolyl compounds (7 and 8 ).
Due to the conserved nature of the ATP-binding site
Glycogen synthase kinase 3 (GSK-3) is a multifunctional
serine/threonine protein kinase that was identified more than throughout the kinome, the development of highly selective
forty years ago. In humans, two GSK-3 isoforms, GSK-3α and ATP-competitive kinase inhibitors is great challenge.
a
GSK-3β, share 98% amino acid sequence identity in the cata- Recently, the covalent inhibitor approach has gained much
1
lytic domain. GSK-3β-null mice were reported to die prenatally attention by targeting particular cysteines in or around the
1
8
with liver degeneration, while a phenotype for GSK-3α-null has ATP-binding site. However, only limited numbers of covalent
2
not yet been reported. GSK-3β was initially studied due to its inhibitors of GSK-3β have been developed. Compound 9 (tide-
role in glycogen biosynthesis, which inactivates glycogen glusib) was a compound in a Phase II clinical trial and is
1
9,20
synthase by phosphorylation. Later, GSK-3β was found to regu- claimed to irreversibly inhibit GSK-3β’s activity
(Fig. 2).
late many signaling pathways, such as the insulin signaling However, the inhibitory activity of this compound did not fully
pathway and the Wnt signaling pathway; it also plays a role in depend on Cys199, a cysteine residue located inside the ATP
the regulation of the functions of c-Jun, c-Myc and tau proteins binding pocket, and the role of this residue in tideglusib’s
3
,4
20
and the stability of microtubules. Dysfunction or abnormally effect is not clear. Covalent inhibitors have been reported by
high activity of GSK-3β is linked to type 2 diabetes, bipolar dis- utilizing α-bromomethyl carbonyl or α-chloromethyl carbonyl
orders, stroke, cancer, chronic inflammatory diseases and neu- reactive groups and reacting with Cys199 (Fig. 2). The thienyl
5
–7
rodegenerative diseases.
halomethylketone and phenyl halomethylketone inhibitors
2
1
A large number of GSK-3β inhibitors have been reported, show micromolar range inhibitory activity against GSK-3β.
including metal ions, marine natural products, short peptides Derivatives of adenine, benzimidazole and maleimide, con-
8
and small molecules. Several series of maleimide analogues taining the α-halomethyl carbonyl moiety, were also reported.
2
2
of a microbial alkaloid staurosporine (1) have been identified However, their selectivities have not been reported.
as potent, ATP-competitive GSK-3β inhibitors
9
–17
22
(Fig. 1), Compound 14 inhibited GSK-3β potently with single-digit
nanomolar IC50 using a highly reactive α-bromomethyl ketone
group.
In this work, a series of (aza)indolyl maleimide covalent
inhibitors were discovered by utilizing mild reactive groups
like acrylamido or α-fluoroacetamido onto the maleimide
scaffold. We describe the synthesis, structure–activity relation-
ship (SAR) and biological evaluation of these compounds as
GSK-3β covalent inhibitors.
State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical
Genomics, School of Chemical Biology and Biotechnology, Peking University
Shenzhen Graduate School, Xili University Town, PKU Campus F-311, Shenzhen,
518055, China. E-mail: panzy@pkusz.edu.cn; Tel: +86-755-26033072
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8ob00642c
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Fig. 1 Examples of ATP-competitive GSK-3β inhibitors.
Fig. 2 Reported covalent GSK-3β inhibitors.
Scheme 1 The synthetic routes for compounds 19a–e, 20a–g.
Reagents and conditions: (a) HOBt, EDCI, TEA, DCM, DMF, NH
methanol), r.t., 65–88%; (b) NaH, MeI, DMF, 0 °C to r.t., 91%; (c) t-BuOK
1 M in THF), THF, 0 °C to r.t., 42–70%; (d) various acyl chlorides, DIPEA,
THF, H O, 0 °C to r.t., 70–85%; or various carboxylic acids, DIPEA,
HATU, DMF, 0 °C to r.t., 22–55%.
3
(7 N in
(
2
Results and discussion
Chemistry
Multiple series of maleimides, 19a–e, 20a–g, 24a–b, 26a–b, elevated temperature, followed by deprotection and amide for-
8a–b, 33a–g, and 38a–b were synthesized following the syn- mation, furnished target compound 26b. The treatment of
thetic routes depicted in Schemes 1–4. Commercially available compound 23 with NH in methanol gave compound 27.
2
3
aryl acetic acids 15a–e were converted into amides 16a–e. Compounds 28a–b were prepared by reacting compound 27
Perkin-type condensation between the compound 18 and aryl with acryloyl chloride or fluoroacetyl chloride (Scheme 2).
2
3
acetamides generated the desired maleimides 19a–e. Amide
The treatment of commercially available indoles 29a–g with
25
bond formation between compound 19e and various car- methyl chlorooxoacetate afforded the glyoxalates 30a–g. The
boxylic acids or acyl chlorides yielded the target compounds alkylation of indole glyoxalates in DMF gave compounds 31a–
2
0a–g (Scheme 1).
g. Maleimides were formed by the condensation of compounds
Indole adduct 22 was readily synthesized from 3,4-dibromo- 31a–g with tert-butyl(3-(2-amino-2-oxoethyl)phenyl)carbamate
H-pyrrole-2,5-dione by a method described in the literature and subsequent Boc deprotection and nucleophilic substi-
1
2
4
with some modifications. The methylation of compound 22 tution yielded target compounds 33a–g (Scheme 3).
with iodomethane in DMF gave the N-methylation product 23. Azaindole was alkylated with iodopropane in the presence
Subsequent Suzuki cross-coupling catalyzed by Pd(PPh
afforded target compounds 24a–b. The substitution of a was prepared by AlCl
bromine atom on compound 23 with a secondary amine at 2-oxoacetate. Maleimide condensation proceeded smoothly in
2
6
3
)
4
of NaH in DMF, affording intermediate 35. Compound 36
activated acylation with methyl 2-chloro-
3
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Scheme 2 The synthetic routes for target compounds 24a–b, 26b, and 28a–b. Reagents and conditions: (a) EtMgBr (1 M in Et
2
O), THF, reflux 1 h,
then indole, 0 °C to r.t., overnight, 87%; (b) NaH, MeI, DMF, 0 °C to r.t., 70%; (c) 2-chloro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine or
-chloro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine, Pd(PPh , Cs CO , THF, H O, microwave, 117 °C, 1 h, 55–60%; (d) DMF, amine,
17 °C, overnight, 55%; (e) (1) TFA : DCM = 1 : 2, 2 h, (2) DIPEA, acryloyl chloride, THF, H O, 0 °C to r.t., 80%; (f) NH (7 N in methanol), microwave,
17 °C, 1 h, 85%; (g) acryloyl chloride or fluoroacetyl chloride, DIPEA, THF, H O, 0 °C to r.t., 32–50%. *26a (R = 1-acryloylpiperidin-3-yl) was syn-
2
3
)
4
2
3
2
1
1
2
3
5
2
thesized by the Perkin-type condensation reaction under conditions c and d, as shown in Scheme 1.
2
Scheme 3 The synthetic routes for target compounds 33a–g. Reagents and conditions: (a) methyl 2-chloro-2-oxoacetate, Et AlCl, DCM, 66%; (b)
7
7
Cs
2
CO
3
, R -Cl or R -I, DMF, 55 °C, 40–60%; (c) tert-butyl(3-(2-amino-2-oxoethyl)phenyl)carbamate or 2-(3-azidophenyl)acetamide, t-BuOK (1 M in
O, 0 °C to r.t., 50–62%.
THF), THF, 0 °C to r.t., 45–70%; (d) (1) TFA : DCM = 1 : 2, 2 h, (2) acryloyl chloride, DIPEA, THF, H
2
1
0,13–16,27–29
the presence of t-BuOK. Subsequent Boc deprotection and selectivity were well studied.
We first optimized
nucleophilic substitution yielded target compounds 38a–b this scaffold to achieve nanomolar affinity against GSK-3β.
(
Scheme 4).
Then, by structure-guided design, electrophilic reactive groups
were incorporated into the scaffold. The Cys199 of GSK-3β is
located at the position immediately before the DFG (Asp-Phe-
Biological studies
We applied a two-step strategy to develop covalent GSK-3β Gly) sequence, which marks the start of the activation
1
8,30,31
inhibitors. Indolyl maleimide was chosen as the initial loop.
This position of Cys199 is relatively deep inside
scaffold because its physical properties, potency and kinome the ATP binding pocket within a short distance from the hinge
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Scheme 4 The synthetic routes for target compounds 38a–b. Reagents and conditions: (a) 2-iodopropane, NaH, DMF, 0 °C to r.t., 88%; (b) methyl
-chloro-2-oxoacetate, AlCl , DCM, overnight, 70%; (c) tert-butyl(3-(2-amino-2-oxoethyl)phenyl)carbamate, t-BuOK (1 M in THF), THF, 0 °C to r.t.,
5%; (d) (1) TFA : DCM = 1 : 2, 2 h, (2) acryloyl chloride or fluoroacetyl chloride, DIPEA, THF, H O, 0 °C to r.t., 68–70%.
2
3
4
2
region, and the freedom for either the side chain of Cys199 or
Compound 19e was highly potent against GSK-3β and was
the reactive group extending from the scaffold is more restric- used as the template to introduce reactive groups. A panel of
tive than that for some other kinases.
potential covalent reactive groups was attached to the amino
group. Compound 20a, which incorporates the classic acrylamide
group, yielded an IC50 of 285 nM for GSK-3β (Table 1). The
addition of a methyl (20b) or dimethylamino methyl (20c) group
Inhibitory potency of compounds against GSK-3β
The compounds were tested for their inhibitory activity against to the terminal carbon of the acrylamide reduced the inhibitors’
GSK-3β in vitro by using a LanthaScreen™ Eu Kinase Binding potency and 2-butynoic amide (20f) also led to reduced potency.
Assay. Staurosporine was used as a positive control compound. The use of smaller reactive groups such as α-chloromethyl carbo-
The results are summarized in Tables 1 and 2.
nyl (20d) or α-fluoromethyl carbonyl groups (20e) resulted in an
As shown in Table 1, in the right region of the maleimide approximately 5-fold or 2-fold increase, respectively, in the
scaffold, compounds with amino-substituted (19c, 19d, 19e) potency compared to 20a. However, compound 20d has a very
phenyl groups show potent inhibitory activities against reactive group and is quickly consumed in a simple GSH stability
GSK-3β, while compound 19a with bulky substituents at the assay, and thus is not profiled in subsequent cellular studies (ESI
para position showed very weak activity against GSK-3β. Fig. S1†). Even though compound 20a does form a covalent bond
Bicyclic systems (19b) also caused a noticeable decrease in the with Cys199 of GSK-3β as identified by mass spectroscopy and
potency even though heteroatoms appeared to have some ben- discussed later, it is clear that further optimization is needed to
eficial influence.
improve the potency.
Table 1 Structure–activity relationship of the right region
1
2
a
1
2
a
1
2
a
Compd
R /R /R
IC50 (nM)
Compd
R /R /R
IC50 (nM)
Compd
R /R /R
IC50 (nM)
19a
>3000
20b
652
24a
6
1
1
1
1
2
a
9b
9c
9d
9e
0a
755
97
20c
20d
20e
20f
528
48
24b
26a
26b
28a
28b
41
1419
>3000
2393
914
30
102
456
340
6
285
20g
IC50 values are reported as means of duplicate measurements.
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Table 2 Structure–activity relationship of the left region
8
a
8
a
Compd
R
R
IC50 (nM)
Compd
R
R
IC50 (nM)
33a
Acryloyl
150
33f
Acryloyl
958
33b
Acryloyl
199
33g
Acryloyl
688
3
3
3c
Acryloyl
Acryloyl
690
280
38a
38b
Acryloyl
34
17
3d
2-Fluoroacetyl
33e
Acryloyl
>3000
a
IC50 values are reported as means of duplicate measurements.
1
1,32
We subsequently attempted to explore different linkers and
Inspired by previous studies,
we finally introduced the
reactive groups to better engage Cys199 (Table 1). Although 7-azaindole ring to replace the indole moiety. To our delight,
o-chloropyridinyl compounds 24a–b were potent against the 7-azaindole analogues with two types of reactive groups
GSK-3β, they could not block the labelling of GSK-3β by probe showed significant increases in potency, especially 7-azaindole
3
9 (ESI Fig. S2C†). Therefore, compounds 24a and 24b were (38b) with an α-fluoromethyl carbonyl group (Table 2).
not covalent irreversible inhibitors of GSK-3β. When saturated To understand the possible binding modes of compounds
heterocycles replaced the phenyl ring linkers, compounds 20a, 20e, 38a and 38b with GSK-3β, docking studies were per-
3
3
2
6a–b showed much weaker inhibitory activities against formed using MOE. The crystal structure of GSK-3β in a
1
4
GSK-3β. By removing the linker moiety entirely, compounds complex with a reversible inhibitor (PDB code: 1R0E) was
8a–b were approximately 4-fold or 10-fold less potent than selected as the template. As shown in Fig. 3, the compounds
2
compound 20a. The interaction between the phenyl ring and adopt non-planar conformations with the two substituents
amino acid residues in the binding pocket (e.g. Phe67) may rotated around the central maleimide moiety, which forms
also contribute to inhibitors’ potency. Noticeably, compounds hydrogen bonds with the residues in the hinge region (Val135
2
1
8a–b are quite similar to the literature reported compound and Asp133). An additional H-bond appears between the
2
2
4
in structure, but with much weaker activity, suggesting nitrogen on the azaindole and the side-chain guanidine
2
2
that the potency of compound 14 is mostly due to its reactive group of Arg141, which might contribute to the higher
group. potency of compound 38a. The introduced isopropyl group is
Next, we turned our attention to the indole region (Table 2). close to a small hydrophobic pocket formed by Ile62, Val70,
Substitution by a fluorine atom at the 5-(33a) or 6-(33b) posi- Phe67 and Leu88.
tion of the indole ring resulted in an approximately 1.5-fold or
Identification of covalent binding
2
-fold increase in the potency compared to compound 20a,
whereas methoxy substitution at the 6-(33c) position led to a Mass spectrometry was employed to determine whether com-
loss in potency. Small substitutions at the 5- or 6-position of pound 20a formed a covalent adduct with GSK-3β. After incu-
the indole ring increased the activities, while larger substi- bation with the compound, the protein was digested with
tutions were not tolerated. Bulky groups on the nitrogen atom trypsin, and mass spectrometry analysis of the digested
of the indole (33e–g) diminished the inhibition of GSK-3β peptide fragments indicated that the modification site was
activity by approximately 4–10 times compared to compound indeed Cys199 (Fig. 4a). Fluorescent probe 39 (IC50 = 417 nM)
2
0a, except for the substitution of an isopropyl group (33d).
was designed based on compound 20a, attaching
a
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completely blocked by excess compounds 20e, 38a and 38b
(
Fig. 4b). These experiments suggest that the binding of com-
pounds 20e, 38a and 38b with GSK-3β is irreversible. In con-
trast, reversible inhibitors could not block the labelling by
probe 39 (Fig. S2C†).
Kinase profiling
The initial identified covalent inhibitor compound 20a was
profiled against a panel of 44 protein kinases including the
representatives of major classes of kinases. At 1 μM, com-
pound 20a showed very weak inhibitory activity against most
of the kinases tested except for VEGFR and MST2 (Fig. 5a). We
also measured the inhibitory effects of four covalent inhibitors
against TAK1, which shares a cysteine residue at the structu-
3
1
Fig. 3 Docking of compounds 20a (green), 20e (yellow), 38a (purple) rally identical position to Cys199 in GSK-3β. In addition,
and 38b (cyan) into a GSK-3β crystal structure (PDB code: 1R0E).
CDK2 and GSK-3β share a highly homologous sequence and
were used as examples of kinases within the homological
3
4,35
CMGC protein kinase family.
Compounds 20a, 20e, 38a
BODIPY®FL fluorophore to 20a via an n-propylamino linker. and 38b showed much decreased inhibitory potency against
Probe 39 successfully labelled recombinant GSK-3β, demon- TAK1 and CDK2 compared to GSK-3β (Fig. 5b). These experi-
strating that it bound covalently with the kinase. In compe- ments indicated that the compounds have good selectivity
tition experiments, the labelling of GSK-3β by probe 39 was among these kinases.
Fig. 4 The verification of compounds 20a, 20e, 38a and 38b as irreversible covalent inhibitors. (a) Mass spectrum of the GSK-3β kinase digested
peptide fragment 198–205 of GSK-3β kinase, and Cys199 was modified by compound 20a; (b) compounds 20a, 20e, 38a and 38b effectively
blocked the labelling of GSK-3β by probe 39.
Fig. 5 (a) Selectivity of covalent inhibitor 20a (1 μM) against a panel of kinases; (b) selectivity of compounds 20a, 20e, 38a and 38b among GSK-3β,
TAK1 and CDK2.
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Fig. 6 Effects of GSK-3β covalent inhibitors in live cells. (a) The GSK-3β down-stream substrates were regulated after the incubation of compounds
0a, 20e, 38a and 38b with RS4;11 cells; (b) wash-out experiments with the irreversible inhibitors 20a, 20e, 38a, 38b and reversible inhibitors 20g
2
and 19e; (c) antiproliferative activity of covalent compounds against RS4;11; (d) compounds 38a and 38b reduced the phosphorylation of tau (S396)
protein in SK-N-SH cells.
Compounds 20a, 20e, 38a and 38b inhibited GSK-3β in RS4;11
cells irreversibly
the development of aberrant structures in the brain of patients
with several late-onset neurodegenerative disorders such as
Alzheimer’s disease (AD). Abnormal GSK-3β activity was found
to be responsible for the hyperphosphorylated tau, which was
GSK-3β has been implicated as a key regulator in multiple cel-
lular processes. The ability to phosphorylate glycogen synthase
3
8,39
polymerized into neurofibrillary tangles.
Therefore, com-
(
GS) and β-catenin (in the Wnt pathway) has been intensively
4
pounds 38a and 38b were further investigated for their effects
on inhibiting the hyperphosphorylation of tau in human neu-
roblastoma SK-N-SH cells. They significantly reduced tau phos-
phorylation at Ser396 in a dose-dependent manner (Fig. 6d).
studied. Therefore, cell-based assays examining GS’s phos-
phorylation and β-catenin’s levels represent direct functional
assays for measuring cellular activities of the GSK-3β covalent
inhibitors. As presented in Fig. 6a, the levels of phosphory-
lated GS were downregulated, and β-catenin was upregulated
in an inhibitor dose-dependent manner in the leukemia cell
3
6
RS4;11.
In vitro permeability and metabolic stability
Next, wash-out studies were conducted to confirm the irre-
versible inhibition of GSK-3β by compounds 20a, 20e, 38a and
8b in cells. RS4;11 cells were exposed to 20a, 20e, 38a and
8b or reversible analogues 20g and 19e. After the removal of
compounds with fresh culture media, the inhibitory effects of
0a, 20e, 38a and 38b were maintained compared to those of
compounds 20g and 19e (Fig. 6b, ESI Fig. S3†). Thus, these
data indicated that compounds 20a, 20e, 38a and 38b inhib-
ited GSK-3β in cells irreversibly.
A permeability assay was carried out in hMDRI-MDCK cells to
evaluate the compounds’ ability to pass through the blood–
3
3
40
brain barrier (BBB). Compounds 38a and 38b displayed high
permeability and are not P-glycoprotein (Pgp) substrates
(
Table 3), which suggest that both compounds are potential
2
small-molecule tools to study GSK-3β’s functions in CNS
models. These compounds showed a relatively high hepatic
clearance rate in a human liver microsome stability assay
(
Table 3). In animal studies, these covalent inhibitors would
benefit from the long-lasting irreversible inhibition of GSK-3β
and diminished non-covalent off-target effects due to quick
clearance.
Antiproliferative activity
The activity of GSK-3β contributes to the growth of leukemia
cell lines; upon GSK-3β inhibition, multiple leukemia cell lines
showed decreased proliferation.
activities of our covalent compounds against the MLL-associ-
ated leukemia cell RS4;11 were also examined (Fig. 6c).
Compounds 20a, 20e, 38a and 38b could effectively inhibit the
3
6,37
The antiproliferative
Table 3 Permeability and metabolic stability for compounds 38a and
3
8b
Compd
growth of RS4;11 cells at the low micromolar level, cause early
cell apoptosis and upregulate the levels of p21
Cip1
Parameters
38a
38b
and caspase
3
compared to the negative control (ESI Fig. S4 and S5†).
Permeability assay in hMDRI-MDCK data report
Papp (×10− cm s ) (N = 2)
Permeability classification
Pgp substrate
6
−1
32.9
High
No
41.9
High
No
Inhibition of tau protein phosphorylation by compounds
8a–b
3
Human liver microsomal stability
T
1/2 (min)
16.3
60.9
54.8
20.6
48.0
43.2
It is well established that the hyperphosphorylation of the
microtubule-associated protein tau is strongly correlated with CL
CL (μL min mg−1)
−1
liver
(mL min kg−1)
−1
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(
1
3
s, 1H), 7.99 (s, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.15 (t, J = 7.6 Hz,
Experimental
Chemistry
H), 6.96–6.79 (m, 4H), 6.47 (d, J = 8.0 Hz, 1H), 6.02 (s, 2H),
13
6
.90 (s, 3H). C NMR (100 MHz, DMSO-d ) δ 172.9, 172.6,
All of the reagents and starting materials were purchased com- 147.9, 147.3, 137.4, 135.0, 131.3, 128.9, 124.7, 124.6, 124.5,
mercially and were used without further purification, unless 122.5, 121.7, 120.5, 111.0, 110.0, 108.6, 103.5, 101.6, 33.4.
+
+
15 2 4
mentioned otherwise. Anhydrous DCM was distilled from HRMS (m/z): calculated for C20H N O [M + H] : 347.1026,
calcium hydride. Absolutely anhydrous solvents (THF, DMF, found 347.1029.
and dioxane) were purchased from J&K Scientific and pack-
3-(2-Aminophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-
aged under nitrogen in J&K Seal bottles. All reactions were dione (19c). 3-(2-Aminophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-
monitored by thin-layer chromatography (TLC) carried out on pyrrole-2,5-dione was prepared in a similar manner to com-
0
.2 mm Jiangyou silica gel plates (HSGF224) using UV light as pound 19a. Boc deprotection was subsequently operated in the
the visualizing agent or ethanolic solution of ninhydrin as the mixture of DCM and TFA to obtain compound 19c as an
1
developing agent. Flash column chromatography was carried orange solid (yield 65% over two steps). H NMR (400 MHz,
out using a Puke silica column (ZCX-II, 170–300 mesh). Dried DMSO-d ) δ 10.89 (s, 1H), 8.00 (s, 1H), 7.42 (d, J = 8.2 Hz, 1H),
6
argon was used to protect the reactions or purge the reaction 7.13–6.98 (m, 3H), 6.68 (td, J = 7.5, 6.9, 1.0 Hz, 1H), 6.64–6.49
1
13
13
flask. H NMR and C NMR spectra were recorded on Bruker (m, 3H), 4.77 (s, 2H), 3.86 (s, 3H). C NMR (100 MHz, DMSO-
Advance 400 MHz and 500 MHz spectrometers at room temp- d6) δ 173.1, 172.9, 147.2, 137.2, 134.7, 133.6, 131.4, 129.9,
erature. Chemical shifts in ppm relative to TMS, CDCl
CD OD, d -DMSO, or d -acetone serve as the internal refer- 104.8, 33.4. HRMS (m/z): calculated for C19
ences. Data are reported as follows: chemical shift, multiplicity 318.1237, found 318.1236.
s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = 3-(4-Aminophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-
3
,
128.7, 126.1, 122.3, 121.4, 120.5, 115.8, 115.6, 115.1, 110.6,
+
+
3
6
6
16 3 2
H N O [M + H] :
(
multiplet), coupling constants and number of protons. HRMS dione (19d). Compound 19d was synthesized with a similar
data were obtained using an AB Q-Star mass spectrometer. The procedure to that described for 19c and yielded an orange
1
purities of the final products were ≥95% and were determined solid (yield 65% over two steps). H NMR (400 MHz, acetone-
by using an Agilent 1260 HPLC system with an Agilent Eclipse
6
d ) δ 9.58 (s, 1H), 7.85 (s, 1H), 7.44 (dt, J = 8.3, 0.9 Hz, 1H),
Plus C18 4.6 × 250 mm column. HPLC analysis used ultrapure 7.38–7.31 (m, 2H), 7.15 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 6.82
water and acetonitrile as the gradient eluent, with acetonitrile (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 6.74 (dt, J = 8.1, 1.0 Hz, 1H),
1
3
varying from 30% to 95% in 10–15 min. Detection was per- 6.60–6.52 (m, 2H), 5.02 (s, 2H), 3.96 (s, 3H). C NMR
formed at 254 nm, and the average peak area was used to (100 MHz, acetone-d ) δ 172.4, 172.0, 149.4, 137.4, 133.5,
6
determine the purities.
131.1, 130.7, 125.1, 122.3, 121.8, 119.6, 118.7, 113.3, 113.2,
+
3
-(4-(tert-Butyl)phenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole- 109.7, 104.4, 32.4. HRMS (m/z): calculated for C19H N O
16 3 2
+
2
,5-dione (19a). To
a
solution of 2-(4-(tert-butyl)phenyl) [M + H] : 318.1237, found 318.1237.
3-(3-Aminophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-
-yl)-2-oxoacetate (0.11 g, 0.5 mmol) at 0 °C, t-BuOK (2.25 mL, dione (19e). Compound 19e was synthesized with a similar
acetamide (0.11 g, 0.6 mmol) and methyl 2-(1-methyl-1H-indol-
3
2
1
.2 mmol, 1 M in anhydrous THF) was added dropwise over procedure to that described for 19c and yielded an orange
7 min under an argon atmosphere. The reaction mixture was solid (yield 63% over two steps). H NMR (400 MHz, acetone-
1
stirred at 0 °C for 40 min, and then allowed to warm to
ambient temperature. After stirring for 4 h, the reaction 7.15 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 6.96 (t, J = 7.8 Hz, 1H), 6.86
mixture was quenched with saturated NH Cl solution, dis- (t, J = 2.0 Hz, 1H), 6.79 (ddd, J = 8.1, 7.0, 1.0 Hz, 1H), 6.70–6.59
solved in ethyl acetate (50 mL) and washed with brine (3 × (m, 3H), 4.63 (s, 2H), 3.96 (s, 3H). C NMR (100 MHz, acetone-
0 mL). The organic phase was dried over anhydrous Na SO d6) δ 172.3, 171.7, 148.1, 137.2, 134.4, 131.6, 130.1, 130.1,
and concentrated in vacuo. The residue was purified by flash 128.4, 125.2, 122.2, 122.0, 119.9, 118.5, 115.6, 114.7,
6
d ) δ 9.71 (s, 1H), 7.98 (s, 1H), 7.43 (dt, J = 8.2, 0.9 Hz, 1H),
4
1
3
2
2
4
+
column chromatography (gradient eluent: 15–35% ethyl 109.8, 104.2, 32.5. HRMS (m/z): calculated for C19
H
15
N
3
O
2
Na
+
acetate in petroleum ether), to give compound 19a as an [M + Na] : 340.1056, found 340.1057.
1
orange solid (0.13 g, yield 70%). H NMR (400 MHz, CDCl
3
) δ
N-(3-(4-(1-Methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-
7
7
.95 (s, 1H), 7.93 (s, 1H), 7.49–7.41 (m, 2H), 7.40–7.26 (m, 3H), 3-yl)phenyl)acrylamide (20a). To a solution of compound 19e
.17 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 6.74 (ddd, J = 8.1, 7.0, 1.0 (0.13 g, 0.4 mmol) in THF (with 2% H O v/v, 2 mL) at 0 °C was
2
Hz, 1H), 6.34 (d, J = 8.1 Hz, 1H), 3.89 (s, 3H), 1.31 (s, 9H). added DIPEA (0.13 mL, 0.8 mmol). The mixture was kept stir-
1
3
3
C NMR (100 MHz, CDCl ) δ 171.9, 171.8, 152.2, 137.3, 134.4, ring at 0 °C for 30 min, and acryloyl chloride (0.48 mL,
1
1
C
32.1, 129.7, 129.4, 127.4, 125.2, 124.9, 122.7, 122.5, 120.4, 0.6 mmol) was added dropwise. The reaction mixture was
09.5, 104.5, 34.8, 33.4, 31.1. HRMS (m/z): calculated for stirred until the completion of the reaction and then dissolved
+
+
23
H
22
N
2
O
2
Na [M + Na] : 381.1573, found 381.1577.
in ethyl acetate (25 mL). The organic layer was washed with
3
-(Benzo[d][1,3]dioxol-5-yl)-4-(1-methyl-1H-indol-3-yl)-1H- brine (3 × 20 mL), dried over anhydrous Na SO , evaporated in
2
4
pyrrole-2,5-dione (19b). Compound 19b was synthesized with vacuo and purified by flash column chromatography (gradient
a similar procedure to that described for 19a and yielded an eluent: 15–50% ethyl acetate in petroleum ether), to give com-
1
1
orange solid (yield 42%). H NMR (400 MHz, DMSO-d ) δ 11.02 pound 20a as an orange solid (0.12 g, yield 80%). H NMR
6
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(500 MHz, DMSO-d
6
) δ 11.08 (s, 1H), 10.18 (s, 1H), 8.06 (s, 1H), 132.7, 131.6, 129.0, 128.4, 125.6, 124.9, 122.5, 121.6, 120.7,
7
8
1
1
5
.82 (dt, J = 8.3, 1.5 Hz, 1H), 7.69 (t, J = 1.8 Hz, 1H), 7.48 (d, J = 120.5, 119.9, 111.0, 103.5, 43.9, 33.4. HRMS (m/z): calculated
+
+
.2 Hz, 1H), 7.24 (t, J = 8.0 Hz, 1H), 7.12 (ddd, J = 8.2, 7.1, for C21
.1 Hz, 1H), 7.00 (dt, J = 7.8, 1.3 Hz, 1H), 6.75 (ddd, J = 8.1, 7.1, 2-Fluoro-N-(3-(4-(1-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-
.0 Hz, 1H), 6.47–6.30 (m, 2H), 6.22 (dd, J = 17.0, 2.1 Hz, 1H), 1H-pyrrol-3-yl)phenyl)acetamide (20e). Compound 20e was syn-
16 3 3
H N O ClNa [M + Na] : 416.0772, found 416.0772.
1
3
.73 (dd, J = 10.1, 2.1 Hz, 1H), 3.90 (s, 3H). C NMR (125 MHz, thesized with a similar procedure to that described for 20a
1
DMSO-d
6
) δ 172.8, 172.6, 163.6, 139.2, 137.5, 135.3, 132.7, and yielded an orange solid (yield 75%). H NMR (400 MHz,
1
32.2, 131.6, 128.9, 128.6, 127.4, 125.3, 124.9, 122.5, 121.7, DMSO-d ) δ 11.07 (s, 1H), 10.12 (s, 1H), 8.09 (s, 1H), 7.84–7.73
6
1
20.7, 120.5, 119.9, 111.0, 103.5, 33.4. HRMS (m/z): calculated (m, 2H), 7.52 (d, J = 8.2 Hz, 1H), 7.28 (t, J = 8.3 Hz, 1H), 7.16 (t,
+
+
for C22
H
18
N
3
O
3
[M + H] : 372.1343, found 372.1344.
J = 7.6 Hz, 1H), 7.05 (d, J = 7.7 Hz, 1H), 6.78 (t, J = 7.6 Hz, 1H),
(
E)-N-(3-(4-(1-Methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H- 6.39 (d, J = 8.1 Hz, 1H), 5.03 (s, 1H), 4.91 (s, 1H), 3.90 (s, 3H).
1
3
pyrrol-3-yl) phenyl)but-2-enamide (20b). To a solution of com-
6
C NMR (100 MHz, DMSO-d ) δ 172.3, 172.1, 166.0, 137.9,
pound 19e (0.35 g, 1.1 mmol) and (E)-but-2-enoic acid (0.14 g, 137.0, 134.9, 132.3, 131.1, 128.4, 128.1, 125.2, 122.1, 121.2,
.6 mmol) in anhydrous DMF (2 mL) was added DIPEA 120.8, 120.1, 119.9, 110.5, 104.5, 103.1, 54.9, 33.0. HRMS (m/z):
1
+
+
16 3 3
(0.73 mL, 4.4 mmol) at 0 °C. The mixture was kept stirring at calculated for C21H N O FNa [M + Na] : 400.1068, found
0
°C for 30 min, and HATU (0.84 g, 2.2 mmol) was added in 400.1069.
N-(3-(4-(1-Methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-
completion of the reaction and then dissolved in ethyl acetate pyrrol-3-yl)phenyl)but-2-ynamide (20f). Compound 20f was
30 mL). The organic layer was washed with brine (2 × 20 mL), synthesized with a similar procedure to that described for 20b
small partitions. The reaction mixture was stirred until the
(
1
dried over anhydrous Na SO , evaporated in vacuo and purified and yielded an orange solid (yield 55%). H NMR (400 MHz,
by flash column chromatography (gradient eluent: 3–8% DMSO-d
2
4
1
6 6
) δ H NMR (400 MHz, DMSO-d ) δ 11.06 (s, 1H),
MeOH in DCM), to give compound 20b as an orange solid 10.58 (s, 1H), 8.05 (s, 1H), 7.66 (dd, J = 4.7, 2.8 Hz, 2H), 7.48
1
(
0.13 g, yield 30%). H NMR (500 MHz, DMSO-d ) δ 11.05 (s, (d, J = 8.2 Hz, 1H), 7.23 (t, J = 8.3 Hz, 1H), 7.13 (t, J = 7.6 Hz,
6
1
7
H), 9.95 (s, 1H), 8.04 (s, 1H), 7.78 (dd, J = 8.1, 2.1 Hz, 1H), 1H), 7.03 (d, J = 7.8 Hz, 1H), 6.75 (t, J = 7.6 Hz, 1H), 6.33 (d, J =
.64 (d, J = 1.8 Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.20 (t, J = 7.9 8.0 Hz, 1H), 3.90 (s, 3H), 2.01 (s, 3H). C NMR (100 MHz,
1
3
Hz, 1H), 7.11 (t, J = 7.6 Hz, 1H), 6.96 (d, J = 7.7 Hz, 1H), DMSO-d ) δ 172.3, 172.1, 150.4, 138.4, 137.0, 134.9, 131.1,
6
6
3
.80–6.69 (m, 2H), 6.34 (d, J = 8.2 Hz, 1H), 6.08–6.04 (m, 1H), 128.4, 128.0, 125.1, 125.0, 124.4, 122.1, 121.2, 120.4, 120.1,
1
3
.89 (s, 3H), 1.82 (dd, J = 6.9, 1.3 Hz, 3H). C NMR (125 MHz, 119.5, 110.5, 103.1, 84.3, 75.8, 33.0, 3.15. HRMS (m/z): calcu-
+
+
DMSO-d ) δ 172.8, 172.6, 163.9, 140.3, 139.6, 137.5, 135.3, lated for C H N O [M + H] : 384.1343, found 384.1343.
6
23 18 3 3
1
1
32.7, 131.5, 128.9, 128.8, 126.4, 125.0, 125.0, 122.5, 121.7,
N-(3-(4-(1-Methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-
20.7, 120.5, 119.9, 110.9, 103.7, 33.4, 17.9. HRMS (m/z): calcu- pyrrol-3-yl)phenyl)propionamide (20g). Compound 20g was
+
+
lated for C H N O [M + H] : 386.1499, found 386.1498.
synthesized with a similar procedure to that described for 20a
2
3
20 3 3
1
(
E)-4-(Dimethylamino)-N-(3-(4-(1-methyl-1H-indol-3-yl)-2,5- and yielded an orange solid (yield 70%). H NMR (400 MHz,
dioxo-2,5-dihydro-1H-pyrrol-3-yl)phenyl)but-2-enamide (20c). DMSO-d ) δ 11.05 (s, 1H), 9.88 (s, 1H), 8.04 (s, 1H), 7.72 (d, J =
6
Compound 20c was synthesized with a similar procedure to 8.4 Hz, 1H), 7.64 (d, J = 2.0 Hz, 1H), 7.48 (d, J = 8.2 Hz, 1H),
that described for 20b and yielded an orange solid (yield 22%). 7.23–7.08 (m, 2H), 6.93 (d, J = 7.7 Hz, 1H), 6.80–6.70 (m, 1H),
1
6
H NMR (400 MHz, DMSO-d ) δ 11.08 (s, 1H), 10.17 (s, 1H), 6.36 (d, J = 8.1 Hz, 1H), 3.90 (s, 3H), 2.27 (q, J = 7.6 Hz, 2H),
1
3
8
7
8
2
2
1
1
1
.05 (s, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.70 (t, J = 1.9 Hz, 1H), 1.04 (t, J = 7.5 Hz, 3H). C NMR (100 MHz, DMSO) δ 172.8,
.48 (d, J = 8.2 Hz, 1H), 7.23 (t, J = 8.0 Hz, 1H), 7.12 (ddd, J = 172.6, 172.4, 139.6, 137.4, 135.2, 132.6, 131.4, 128.8, 128.7,
.2, 6.9, 1.1 Hz, 1H), 6.99 (dt, J = 7.6, 1.3 Hz, 1H), 6.79–6.66 (m, 124.9, 124.7, 122.5, 121.7, 120.5, 120.4, 119.6, 110.9, 103.6,
+
H), 6.40–6.25 (m, 2H), 3.90 (s, 3H), 3.24 (d, J = 5.8 Hz, 2H), 33.4, 29.8, 10.0. HRMS (m/z): calculated for C H N O Na
2
2
19 3 3
1
3
+
.32 (s, 6H). C NMR (100 MHz, DMSO-d
63.3, 139.4, 137.5, 135.3, 132.7, 131.5, 128.9, 128.6, 127.9,
25.2, 124.9, 122.5, 121.7, 120.6, 120.5, 120.0, 111.0, 110.9, 2,5-dione (24a). To a mixture of compound 23 (0.30 g,
6
) δ 172.8, 172.6, [M + Na] : 396.1319, found 396.1314.
3-(2-Chloropyridin-3-yl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-
+
03.6, 59.2, 44.6, 33.4. HRMS (m/z): calculated for C25
H
25
N
4
O
3
1.0 mmol), Pd(PPh
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (0.48 g,
-Chloro-N-(3-(4-(1-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro- 2.0 mmol) in dioxane (with 10% H O v/v, 2 mL), Cs CO
3
3 4
) (0.23 g, 0.2 mmol) and 2-chloro-3-
+
[M + H] : 429.1921, found 429.1919.
2
2
2
1H-pyrrol-3-yl)phenyl)acetamide (20d). Compound 20d was syn- (0.65 g, 2 mmol) was added. Argon was bubbled through the
thesized with a similar procedure to that described for 20a mixture for 5 min and the mixture was microwaved at 140 °C
1
and yielded an orange solid (yield 85%). H NMR (400 MHz, for 1 h. The mixture was filtered using diatomite, dissolved in
DMSO-d
6
) δ 11.07 (s, 1H), 10.33 (s, 1H), 8.05 (s, 1H), 7.72 (dt, ethyl acetate (30 mL), washed with brine (2 × 20 mL), dried
SO , filtered and concentrated to give the crude
H), 7.23 (t, J = 7.9 Hz, 1H), 7.12 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H), product. The residue was purified by flash chromatography
.00 (dt, J = 7.7, 1.3 Hz, 1H), 6.75 (ddd, J = 8.1, 7.0, 1.0 Hz, 1H), (25% ethyl acetate in petroleum ether), to afford compound
J = 8.1, 1.7 Hz, 1H), 7.64 (t, J = 1.9 Hz, 1H), 7.48 (d, J = 8.3 Hz, over Na
2
4
1
7
6
1
3
1
.35 (d, J = 8.0 Hz, 1H), 4.21 (s, 2H), 3.90 (s, 3H). C NMR 24a as a pale-orange solid (0.20 g, yield 60%). H NMR
(
100 MHz, DMSO-d ) δ 172.7, 172.5, 165.1, 138.7, 137.5, 135.3, (400 MHz, acetone-d ) δ 10.00 (s, 1H), 8.46 (dd, J = 4.8, 2.0 Hz,
6
6
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1
2
1
H), 8.11 (s, 1H), 7.87 (dd, J = 7.6, 2.0 Hz, 1H), 7.53–7.41 (m, drous Na
2
SO
4
, evaporated in vacuo and purified by flash
H), 7.17 (ddd, J = 8.2, 7.1, 1.1 Hz, 1H), 6.80 (ddd, J = 8.2, 7.1, column chromatography (gradient eluent: 15–50% ethyl
1
3
.0 Hz, 1H), 6.47 (dt, J = 8.2, 1.0 Hz, 1H), 3.97 (s, 3H). C NMR acetate in petroleum ether), to give compound 26b as an
1
6
(100 MHz, acetone-d ) δ 172.0, 171.2, 151.5, 150.6, 141.8, orange solid (0.14 g, yield 80%). H NMR (400 MHz, DMSO-
1
1
C
38.4, 136.7, 136.2, 128.8, 126.5, 125.8, 123.3, 123.2, 121.6, d6) δ 10.37 (s, 1H), 8.22 (d, J = 6.6 Hz, 1H), 7.40 (dd, J = 14.8,
21.1, 111.3, 105.5, 33.5. HRMS (m/z): calculated for 8.0 Hz, 2H), 7.19 (s, 1H), 7.19–7.10 (m, 1H), 7.02 (dd, J = 11.0,
+
+
18
H
13ClN
3
O
2
[M + H] : 338.0691, found 338.0691.
3.8 Hz, 1H), 6.16 (dd, J = 14.1, 9.9 Hz, 1H), 6.05 (dd, J = 14.1,
3
-(6-Chloropyridin-2-yl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole- 2.4 Hz, 1H), 5.58 (dd, J = 9.9, 2.4 Hz, 1H), 4.17 (dd, J = 10.3,
2
,5-dione (24b). Compound 24b was synthesized with a similar 5.1 Hz, 1H), 3.80 (s, 3H), 3.70 (dd, J = 12.3, 6.1 Hz, 1H), 3.52
procedure to that described for 24a and yielded an orange (d, J = 6.6 Hz, 2H), 3.37 (d, J = 12.6 Hz, 1H), 1.94 (dq, J = 13.6,
1
13
solid (yield 55%). H NMR (400 MHz, DMSO-d ) δ 11.18 (s, 7.7 Hz, 1H), 1.70 (td, J = 12.1, 5.9 Hz, 1H). C NMR
6
1
H), 8.14 (s, 1H), 7.93 (t, J = 7.8 Hz, 1H), 7.64 (d, J = 7.4 Hz, (100 MHz, DMSO-d
6
) δ 172.8, 168.9, 164.8, 143.3, 136.2,
1
H), 7.49 (dd, J = 11.1, 8.1 Hz, 2H), 7.16 (t, J = 7.6 Hz, 1H), 6.81 131.7, 130.8, 130.1, 125.9, 121.6, 120.2, 119.5, 110.1, 104.4,
1
3
(t, J = 7.5 Hz, 1H), 6.23 (d, J = 8.0 Hz, 1H), 3.91 (s, 3H).
C
95.4, 55.3, 48.7, 48.6, 32.9, 30.5. HRMS (m/z): calculated for
+
+
NMR (100 MHz, DMSO-d
6
) δ 172.2, 172.0, 150.9, 150.2, 140.3,
C
20
H
21
N
4
O
3
[M + H] : 365.1608, found 365.1605.
1
1
C
37.5, 136.6, 135.5, 125.5, 125.4, 125.2, 124.1, 122.7, 121.1,
N-(4-(1-Methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-
20.9, 111.1, 104.2, 33.5. HRMS (m/z): calculated for 3-yl)acrylamide (28a). Compound 28a was synthesized with a
+
+
18
H
13ClN
3
O
2
[M + H] : 338.0691, found 338.0690.
similar procedure to that described for 20a and yielded an
1
3
-(1-Acryloylpiperidin-3-yl)-4-(1-methyl-1H-indol-3-yl)-1H- orange solid (yield 32%). H NMR (400 MHz, DMSO-d ) δ 10.95
6
pyrrole-2,5-dione (26a). Compound 26a was synthesized with (s, 1H), 10.22 (s, 1H), 7.90 (s, 1H), 7.50 (d, J = 8.2 Hz, 1H), 7.38
a similar procedure to that described for 20a and yielded a (d, J = 8.0 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 7.03 (t, J = 7.5 Hz,
1
pale-yellow solid (yield 80%). H NMR (400 MHz, DMSO-d
6
)
1H), 6.52 (dd, J = 14.1, 10.3 Hz, 1H), 6.02 (dd, J = 14.1, 1.6 Hz,
1
3
δ 10.93 (10.89) (s, 1H), 7.77 (7.52) (t, J = 8.7 Hz, 2H), 7.68–7.59 1H), 5.68 (dd, J = 10.3, 1.6 Hz, 1H), 3.88 (s, 3H). C NMR
(m, 1H), 7.25 (t, J = 7.4 Hz, 1H), 7.15–7.06 (m, 1H), 6.93–6.77 (100 MHz, DMSO-d
6
) δ 172.0, 170.0, 162.9, 137.0, 133.6, 130.7,
(
m, 1H), 6.13–6.08 (m, 1H), 5.75–5.65 (m, 1H), 4.51 (d, J = 128.2, 126.9, 126.8, 124.8, 122.3, 121.1, 120.6, 110.9, 104.4,
+
+
9
2
1
.5 Hz, 1H), 4.28 (4.09) (J = 13.4 Hz, 1H), 3.88 (s, 3H), 33.3. HRMS (m/z): calculated for C H N O [M + H] :
16 14 3 3
.86–2.80 (m, 1H), 2.57 (3.65) (t, J = 12.5 Hz, 1H), 2.22–2.03(m, 296.1030, found 296.1031.
1
3
H), 1.93–1.66 (m, 2H), 1.31–1.23 (m, 1H). C NMR (100 MHz,
2-Fluoro-N-(4-(1-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-
DMSO-d ) δ 173.1, 172.2, 164.7, 137.3, 135.0, 133.5, 128.7, 1H-pyrrol-3-yl)acetamide (28b). Compound 28b was syn-
6
1
3
27.8, 126.1, 122.6, 121.1, 120.8, 110.9, 105.1, 103.2, 46.1, 45.5, thesized with a similar procedure to that described for 20e and
+
1
6.5, 33.4, 28.9, 26.5. HRMS (m/z): calculated for C21
H
22
N
3
O
3
yielded a pale-orange solid (yield 50%). H NMR (400 MHz,
+
[M + H] : 364.1656, found 364.1652.
DMSO-d ) δ 10.99 (s, 1H), 10.23 (s, 1H), 7.94 (s, 1H), 7.51 (t, J =
6
N-(1-(4-(1-Methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H- 8.8 Hz, 2H), 7.24 (t, J = 7.3 Hz, 1H), 7.14 (t, J = 7.4 Hz, 1H), 5.02
1
3
pyrrol-3-yl)pyrrolidine-3-yl)acrylamide (26b). To a solution (s, 1H), 4.91 (s, 1H), 3.89 (s, 3H). C NMR (100 MHz, DMSO-
of compound 23 (0.42 g, 1.4 mmol) and triethylamine d6) δ 171.7, 169.8, 166.1, 165.9, 137.1, 134.0, 126.7, 126.5,
(
0.39 mL, 2.8 mmol) in DMF (2.5 mL), tert-butyl pyrrolidin-3- 125.7, 122.5, 121.0, 111.1, 104.0, 80.8, 79.0, 33.3. HRMS (m/z):
+
+
ylcarbamate (0.32 g, 1.7 mmol) was added. The reaction calculated for C15
mixture was stirred at 117 °C for 12 h and then quenched with 324.0758.
12 3 3
H N O FNa [M + Na] : 324.0755, found
water. The reaction mixture was dissolved in ethyl acetate
30 mL), washed with brine (2 × 20 mL), dried over anhydrous 1H-pyrrol-3-yl)phenyl)acrylamide (33a). Compound 33a was syn-
Na SO , evaporated in vacuo and purified by flash column thesized with a similar procedure to that described for 20a
N-(3-(4-(5-Fluoro-1-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-
(
2
4
1
chromatography (gradient eluent: 25–35% ethyl acetate in pet- and yielded an orange solid (yield 58%). H NMR (400 MHz,
roleum ether), to give tert-butyl (1-(4-(1-methyl-1H-indol-3-yl)- acetone-d ) δ 9.86 (s, 1H), 9.43 (s, 1H), 8.07 (s, 1H), 7.94 (d, J =
,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)carbamate 8.2 Hz, 1H), 7.81 (t, J = 2.1 Hz, 1H), 7.45 (dd, J = 8.9, 4.5 Hz,
25) as an orange solid (0.32 g, 0.77 mmol). To a solution of 1H), 7.29 (t, J = 7.9 Hz, 1H), 7.14 (dt, J = 7.7, 1.3 Hz, 1H), 6.93
6
2
(
compound 25 (0.21 g, 0.5 mmol) in DCM (2 mL) was added (td, J = 9.0, 2.5 Hz, 1H), 6.40 (dd, J = 16.9, 9.8 Hz, 1H), 6.31
TFA (1 mL) at ambient temperature. The reaction mixture was (dd, J = 16.9, 2.3 Hz, 1H), 6.14 (dd, J = 10.6, 2.5 Hz, 1H), 5.68
1
3
stirred for 2 h until the completion of the reaction and evap- (dd, J = 9.8, 2.3 Hz, 1H), 3.96 (s, 3H). C NMR (100 MHz,
orated under vacuum to dryness. The crude product was used acetone-d ) δ 171.5, 171.4, 163.1, 158.8, 156.5, 139.2, 136.2,
for further reaction directly. To a solution of the crude 134.1, 132.3, 131.7, 131.7, 131.4, 129.0, 128.4, 126.3, 125.7,
product in THF (with 2% H O v/v, 3 mL) was added DIPEA 125.6, 125.1, 120.2, 119.8, 119.7, 111.2, 111.1, 110.2, 110.0,
0.16 mL, 1 mmol) at 0 °C. The mixture was kept stirring at 107.1, 106.8, 103.9, 103.8, 32.9. HRMS (m/z): calculated for
6
2
(
0
+
+
°C for 30 min, and acryloyl chloride (0.06 mL, 0.75 mmol) C H N O F [M + H] : 390.1248, found 390.1249.
22 17 3 3
was added dropwise. The reaction mixture was stirred until
N-(3-(4-(6-Fluoro-1-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-
the completion of the reaction and dissolved in ethyl acetate. 1H-pyrrol-3-yl)phenyl)acrylamide (33b). Compound 33b was
The organic layer was washed with brine, dried over anhy- synthesized with a similar procedure to that described for 20a
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1
1
and yielded an orange solid (yield 50%). H NMR (400 MHz, and yielded an orange solid (yield 51%). H NMR (400 MHz,
acetone-d ) δ 9.86 (s, 1H), 9.42 (s, 1H), 8.01 (s, 1H), 7.94 (d, J = DMSO-d ) δ 11.09 (s, 1H), 10.17 (s, 1H), 8.03 (s, 1H), 7.84–7.72
6
6
8
1
6
9
.1 Hz, 1H), 7.78 (s, 1H), 7.31–7.19 (m, 2H), 7.13 (dt, J = 7.8, (m, 2H), 7.54 (d, J = 8.3 Hz, 1H), 7.24 (t, J = 7.9 Hz, 1H), 7.12 (t,
.3 Hz, 1H), 6.59 (td, J = 9.2, 2.4 Hz, 1H), 6.51–6.43 (m, 1H), J = 7.2 Hz, 1H), 7.00 (d, J = 7.8 Hz, 1H), 6.76 (t, J = 7.5 Hz, 1H),
.43–6.36 (m, 1H), 6.32 (dd, J = 16.9, 2.3 Hz, 1H), 5.69 (dd, J = 6.49–6.35 (m, 2H), 6.23 (dd, J = 14.0, 2.0 Hz, 1H), 5.77–5.69 (m,
1
3
.7, 2.3 Hz, 1H), 3.95 (s, 3H). C NMR (100 MHz, acetone-d
6
)
1H), 4.35 (t, J = 6.9 Hz, 2H), 2.89 (t, J = 2.6 Hz, 1H), 2.19 (td, J =
1
3
δ 171.5, 171.4, 163.1, 160.8, 158.5, 139.1, 137.8, 137.7, 135.3, 7.0, 2.5 Hz, 2H), 1.98 (p, J = 6.9 Hz, 2H). C NMR (100 MHz,
1
35.3, 132.3, 131.7, 131.3, 129.3, 128.4, 126.3, 125.1, 123.0, DMSO-d ) δ 172.7, 172.5, 163.6, 139.3, 136.7, 134.2, 132.6,
6
1
22.9, 121.5, 120.6, 119.8, 108.4, 108.1, 104.1, 96.6, 96.4, 32.7. 132.2, 131.5, 129.1, 128.9, 127.4, 125.2, 125.1, 122.6, 121.9,
+
+
17 3 3
HRMS (m/z): calculated for C22H N O F [M + H] : 390.1248, 120.7, 120.5, 119.9, 110.9, 103.9, 83.7, 72.4, 45.3, 29.0, 15.6.
found 390.1248.
+
+
HRMS (m/z): calculated for C H N O [M + H] : 424.1656,
26 22 3 3
N-(3-(4-(6-Methoxy-1-methyl-1H-indol-3-yl)-2,5-dioxo-2,5- found 424.1657.
dihydro-1H-pyrrol-3-yl)phenyl)acrylamide (33c). Compound tert-Butyl(3-(3-(4-(3-acrylamidophenyl)-2,5-dioxo-2,5-dihydro-
3c was synthesized with a similar procedure to that described 1H-pyrrol-3-yl)-1H-indol-1-yl)propyl)carbamate (33g). Compound
3
1
for 20a and yielded an orange solid (yield 55%). H NMR 33g was synthesized with a similar procedure to that described
1
(400 MHz, acetone-d
6
) δ 9.78 (s, 1H), 9.41 (s, 1H), 8.00–7.90 (m, for 20a and yielded an orange solid (yield 62%). H NMR
2
1
2
H), 7.75 (s, 1H), 7.26 (t, J = 7.9 Hz, 1H), 7.11 (d, J = 7.8 Hz, (400 MHz, DMSO-d ) δ 11.07 (s, 1H), 10.18 (s, 1H), 8.10 (s, 1H),
6
H), 6.99 (d, J = 2.2 Hz, 1H), 6.48–6.37 (m, 2H), 6.37–6.28 (m, 7.80 (d, J = 8.3 Hz, 1H), 7.73 (s, 1H), 7.51 (d, J = 8.3 Hz, 1H),
H), 5.69 (dd, J = 9.8, 2.3 Hz, 1H), 3.93 (s, 3H), 3.80 (s, 3H). 7.23 (t, J = 8.0 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 6.98 (d, J =
1
3
C NMR (100 MHz, acetone-d ) δ 172.5, 172.4, 163.9, 157.6, 7.7 Hz, 2H), 6.75 (t, J = 7.5 Hz, 1H), 6.49–6.33 (m, 2H),
6
1
1
40.0, 139.3, 134.8, 133.5, 132.6, 132.6, 132.5, 129.1, 127.1, 6.23 (dd, J = 17.0, 1.9 Hz, 1H), 5.81–5.65 (m, 1H), 4.29 (t, J = 6.9
26.0, 123.5, 121.5, 120.4, 119.7, 110.8, 105.0, 94.2, 55.6, 33.4. Hz, 2H), 2.98 (q, J = 6.1 Hz, 2H), 2.04–1.83 (m, 2H), 1.39 (s,
+
+
13
HRMS (m/z): calculated for C H N O [M + H] : 402.1448, 9H). C NMR (100 MHz, DMSO-d ) δ 172.7, 172.5, 163.6,
2
3
20
3
4
6
found 402.1449.
156.1, 139.3, 136.7, 134.5, 132.7, 132.2, 131.5, 128.9, 128.80,
N-(3-(4-(1-Isopropyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H- 127.4, 125.2, 125.10, 122.5, 121.9, 120.7, 120.5, 119.9, 111.0,
pyrrol-3-yl)phenyl)acrylamide (33d). Compound 33d was syn- 103.8, 78.1, 44.2, 37.8, 30.2, 28.7. HRMS (m/z): calculated for
+
+
thesized with a similar procedure to that described for 20a
and yielded an orange solid (yield 55%). H NMR (400 MHz,
C
29
H
31
N
4
O
5
[M + H] : 515.2289, found 515.2310.
1
N-(3-(4-(1-Isopropyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-2,5-dioxo-
acetone-d ) δ 9.85 (s, 1H), 9.44 (s, 1H), 8.14 (s, 1H), 7.95 (d, J = 2,5-dihydro-1H-pyrrol-3-yl)phenyl)acrylamide (38a). Compound
6
8
7
.1 Hz, 1H), 7.82 (s, 1H), 7.53 (d, J = 8.3 Hz, 1H), 7.23 (t, J = 28a was synthesized with a similar procedure to that described
.9 Hz, 1H), 7.18–7.04 (m, 2H), 6.78 (t, J = 7.6 Hz, 1H), 6.59 (d, for 20a and yielded an orange solid (yield 70%). H NMR
1
J = 8.1 Hz, 1H), 6.48–6.37 (m, 1H), 6.33 (dd, J = 16.9, 2.2 Hz, (400 MHz, DMSO-d ) δ 11.16 (s, 1H), 10.20 (s, 1H), 8.30–8.18
6
1
1
H), 5.69 (dd, J = 9.8, 2.2 Hz, 1H), 4.88 (septet, J = 6.7 Hz, 1H), (m, 2H), 7.82 (d, J = 8.2 Hz, 1H), 7.75 (s, 1H), 7.28 (t, J = 7.9 Hz,
1
3
.60 (d, J = 6.7 Hz, 6H). C NMR (100 MHz, acetone-d
6
)
1H), 7.02 (d, J = 7.8 Hz, 1H), 6.86 (dd, J = 8.0, 4.6 Hz, 1H),
δ 171.7, 171.7, 163.1, 139.1, 136.4, 132.8, 131.8, 131.6, 129.9, 6.77 (dd, J = 8.0, 1.5 Hz, 1H), 6.40 (dd, J = 14.0, 10.1 Hz, 1H),
1
1
28.7, 128.3, 126.2, 125.2, 125.1, 122.1, 122.0, 120.6, 120.2, 6.23 (dd, J = 14.0, 2.0 Hz, 1H), 5.73 (dd, J = 10.1, 2.0 Hz, 1H),
1
3
19.6, 110.2, 104.4, 47.6, 21.9. HRMS (m/z): calculated for 5.13 (septet, J = 6.6 Hz, 1H), 1.52 (d, J = 6.7 Hz, 6H). C NMR
+
+
C H N O [M + H] : 400.1656, found 400.1656.
(100 MHz, DMSO-d ) δ 172.5, 172.3, 163.6, 147.5, 143.4, 139.4,
6
2
4
22
3
3
N-(3-(4-(1-(4-Phenethyl)-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro- 132.3, 132.1, 131.1, 131.0, 130.1, 130.0, 129.1, 127.5, 125.2,
1
3
1
H-pyrrol-3-yl)phenyl)acrylamide (33e). Compound 33e was 120.7, 120.1, 117.8, 116.8, 102.6, 46.5, 22.6.
C NMR
synthesized with a similar procedure to that described for 20a (100 MHz, DMSO-d ) δ 172.5, 172.3, 163.6, 147.5, 143.4, 139.4,
6
1
and yielded a yellow solid (yield 52%). H NMR (400 MHz, 132.3, 132.1, 131.1, 131.0, 130.1, 130.0, 129.1, 127.5, 125.2,
acetone-d
6
) δ 9.79 (s, 1H), 9.42 (s, 1H), 7.92 (d, J = 10.7 Hz, 2H), 120.7, 120.1, 117.8, 116.8, 102.6, 46.5, 22.6. HRMS
+
+
7
7
.83 (s 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.36–7.15 (m, 6H), (m/z): calculated for C H N O [M + H] : 401.1608, found
23 21 4 3
.14–7.03 (m, 2H), 6.76 (ddd, J = 8.0, 7.2, 0.8 Hz, 1H), 6.55 (d, 401.1609.
J = 8.1 Hz, 1H), 6.44 (dd, J = 16.9, 9.9 Hz, 1H), 6.33 (dd, J =
2-Fluoro-N-(3-(4-(1-isopropyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-
1
2
6.9, 2.2 Hz, 1H), 5.69 (dd, J = 9.9, 2.2 Hz, 1H), 4.65–4.52 (m, 2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)phenyl)acetamide (38b).
1
3
6
H), 3.19 (t, J = 7.4 Hz, 2H). C NMR (100 MHz, acetone-d )
Compound 38b was synthesized with a similar procedure to
δ 171.6, 171.5, 163.1, 139.1, 138.4, 136.6, 133.8, 132.7, 131.8, that described for 20e and yielded a pale-orange solid (yield
1
1
31.7, 131.5, 128.8, 128.4, 128.2, 126.5, 126.2, 125.1, 125.1, 68%). H NMR (400 MHz, DMSO-d ) δ 11.16 (s, 1H), 10.17 (s,
6
1
22.1, 122.0, 120.7, 120.1, 119.6, 110.2, 104.0, 47.9, 36.1. 1H), 8.28–8.19 (m, 2H), 7.81–7.72 (m, 2H), 7.29 (t, J = 7.9 Hz,
+
+
HRMS (m/z): calculated for
C
29
H
23
N
3
O
3
Na [M
+
Na] : 1H), 7.04 (d, J = 7.8 Hz, 1H), 6.85 (dd, J = 8.0, 4.6 Hz, 1H),
4
84.1632, found 484.1629.
6.74 (dd, J = 8.0, 1.5 Hz, 1H), 5.12 (septet, J = 6.7 Hz, 1H),
1
3
N-(3-(2,5-Dioxo-4-(1-(pent-4-yn-1-yl)-1H-indol-3-yl)-2,5-dihydro- 5.00 (s, 1H), 4.89 (s, 1H), 1.52 (d, J = 6.7 Hz, 6H). C NMR
H-pyrrol-3-yl)phenyl)acrylamide (33f). Compound 33f was syn- (100 MHz, DMSO-d ) δ 172.1, 171.9, 165.9, 147.1, 143.0, 138.1,
1
6
thesized with a similar procedure to that described for 20a 131.9, 130.7, 130.6, 129.6, 128.6, 125.2, 120.8, 120.7, 120.2,
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1
17.4, 116.4, 102.1, 80.7, 78.9, 46.1, 22.2. HRMS (m/z): calcu- various concentrations of compounds, and incubated in a CO
2
+
+
lated for C H N O F [M + H] : 407.1514, found 407.1516.
N-(3-(4-(1-(3-(3-(5,5-Difluoro-7,9-dimethyl-5H-4λ ,5λ -dipyr- the culture mixture at ambient temperature. After 1 h, lumine-
rolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-3-yl)propanamido)propyl)- scence was read by using an Envision plate reader Envision
incubator for 72 h. The detection reagent (40 μL) was added to
2
2
20 4 3
4
4
1
H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)phenyl) 2104. GraphPad Prism was used to analyze the data, forming
acrylamide (probe 39). Compound 33g as a precursory com- dose–response curves. The GI50 values were means of triplicate
pound of probe 39. Probe 39 was prepared in a similar manner measurements.
to compound 20b using BODIPY®FL instead of (E)-but-2-enoic
Western blot analysis. 3 000 000 RS4;11 cells or 1 700 000
acid, yielding a dark red solid (yield 20%). H NMR (400 MHz, SK-N-SH cells were treated with various concentrations of the
DMSO-d ) δ 11.07 (s, 1H), 10.19 (s, 1H), 8.28–7.99 (m, 2H), 7.81 compounds and incubated in a CO incubator for 8 h. The
d, J = 7.6 Hz, 1H), 7.78–7.63 (m, 2H), 7.51 (d, J = 8.3 Hz, 1H), cells were collected and rinsed once in pre-ice-cold PBS (pH
1
6
2
(
7
1
5
4
.24 (t, J = 7.9 Hz, 1H), 7.19–7.05 (m, 2H), 6.99 (d, J = 6.9 Hz, 7.4, Gibco). The extracted cells were suspended in a lysis buffer
H), 6.85–6.70 (m, 1H), 6.56–6.35 (m, 3H), 6.36–6.18 (m, 2H), (150 mM sodium chloride, 1.0% NP40 or Triton X-100, 50 mM
.73 (d, J = 10.1 Hz, 1H), 4.30 (d, J = 6.0 Hz, 2H), 3.30–2.94 (m, Tris, 100 μM PMSF, 5 μM NaF, 100 × protease inhibitor cock-
H), 2.51–2.43 (m, 5H), 2.25 (s, 3H), 1.95 (d, J = 6.4 Hz, 2H). tail, 100 × phosphatase inhibitor cocktail 3) for 40 min. The
1
3
6
C NMR (100 MHz, DMSO-d ) δ 172.8, 172.6, 171.4, 165.2, protein concentrations of the supernatant were measured by
1
1
1
4
63.6, 159.7, 158.2, 139.3, 136.7, 134.9, 134.4, 134.0, 133.4, using a Nanodrop 1700 instrument after centrifugation at
32.7, 132.2, 131.5, 130.0, 129.3, 129.0, 128.9, 127.5, 125.8, 15 000g for 15 min at 4 °C. The proteins were separated by
25.3, 125.1, 122.6, 120.7, 120.5, 119.9, 117.0, 111.0, 103.9, 10% Tris SDS-PAGE and transferred to nitrocellulose mem-
4.3, 36.6, 34.3, 30.1, 24.5, 15.0, 11.4. HRMS (m/z): calculated branes (Millipore). The membranes were blocked with 5%
+
+
for C38
H
35BF
2
N
6
O
4
Na [M + Na] : 711.2673, found 711.2678.
(m/v) non-fat milk in TBST buffer for 1 h at ambient tempera-
ture. The membranes were incubated with a primary antibody
at 4 °C overnight. Subsequently, the membranes were rinsed
Biology
Kinase binding assay for GSK-3β. Human recombinant three times in a TBST buffer for 5 min and exposed to the anti-
GSK-3β was purchased from SignalChem (GST-tag, G09- rabbit or anti-mouse peroxidase-conjugated secondary anti-
1
0G-10). The inhibition of GSK-3β by synthetic compounds body for 1 h at ambient temperature. The blots were visualized
was determined by using a LanthaScreen™ Eu Kinase Binding by using an Automatic Imaging analyzer (Bio-Rad) using
Assay (Thermo Fisher Scientific). The assays for the evaluation Western HRP Substrate (Millipore). The primary antibodies
of the inhibitors were performed in 384-well plates included phospho-glycogen synthase antibody (3891, 1 : 1000,
(PerkinElmer, USA) by the addition of 3 components each at Cell Signaling Technology), β-catenin antibody (9587, 1 : 1000,
3
× the final desired concentration as follows: 5 µL of the test Cell Signaling Technology), glycogen synthase rabbit mAb
compound with twelve diluted concentrations, 5 µL of kinase/ (3886, 1 : 1000, Cell Signaling Technology), β-actin rabbit mAb
antibody (PV5594) mixture, and 5 µL of tracer236 (PV5592). (4970, 1 : 1000, Cell Signaling Technology), GAPDH rabbit mAb
The mixture was incubated for 1 h at ambient temperature and (2118, 1 : 1000, Cell Signaling Technology), tau mouse mAb
read by using an Envision plate reader Envision 2104 (4019, 1 : 1000, Cell Signaling Technology), anti-tau (phospho
(PerkinElmer, USA). Staurosporine, a well-known inhibitor of S396) antibody (ab109390, 1 : 10 000, Abcam), p21 Waf1/Cip1
GSK-3β, was used as the positive control compound and gave rabbit mAb (2947, 1 : 1000, Cell Signaling Technology), and
an IC50 of 54 nM under our assay conditions. All operations caspase-3 antibody (9662, 1 : 1000, Cell Signaling Technology).
were carried out in duplicate, and the results generated dose– The secondary antibodies included anti-rabbit IgG HRP-linked
response curves by GraphPad Prism.
(7074s, 1 : 1000, Cell Signaling Technology) and anti-mouse
Kinase selectivity. TAK1 and CDK2 were purchased from IgG HRP-linked (7076s, 1 : 1000, Cell Signaling Technology).
Thermo Fisher Scientific (PV4394, PV6295). The inhibition of The results were repeated by independent experiments.
the two kinases by synthetic compounds was determined by
Mass spectrometry study. Human recombinant GSK-3β
using a LanthaScreen™ Eu Kinase Binding Assay (Thermo (2 μg, approximately 4 μM), compound 20a (40 μM) and PBS
Fisher Scientific). Compound 20a (1 μM) against a series of constituted a 10 μL reaction system. The mixture was blended
representative 44 kinases was determined at the University of and incubated at the ambient temperature for 2 h. The protein
Dundee, MRC PPU International Centre of Kinase Profiling.
was separated by SDS-PAGE and stained by using a
Cell culture and antiproliferative activity assay. The leukemia SilverQuest™ kit (Thermo Fisher Scientific). The band was cut,
cell RS4;11 was obtained from ATCC (the American Type de-stained and digested by trypsin and then loaded onto an
Culture Collection). The cells were cultured in RPMI 1640 LC-MS/MS system (Q Exactive, Thermo Fisher Scientific). The
(ATCC modification, Gibco, A10491-01) containing 10% (v/v) results were analyzed by Mascot Search.
fetal bovine serum (Gibco) at 37 °C under a humid atmosphere Fluorescent probe labelling and competition assay. Human
and 5% CO . The antiproliferative activity of synthetic com- recombinant GSK-3β (0.6 μg, Invitrogen) was preincubated
2
pounds was determined by using a CellTiter-Glo® luminescent with and without compounds 20a, 20e, 38a and 38b at 50 μM
cell visibility assay (Promega, G7570). RS4;11 cells were seeded for 2 h at ambient temperature. Subsequently, the mixture was
in 96-well plates (5000 cells per well), treated with DMSO and incubated with probe 39 at 5 μM for 2 h. The incubation was
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stopped by the addition of 2 × loading buffer. The protein was
separated by 10% Tris SDS-PAGE, and the gel was scanned by
using a Pharos FXTM Plus Molecular imager (Bio-Rad) and
stained by using a SilverQuest™ kit (Thermo Fisher Scientific).
Docking study. Docking was performed with MOE™ 2015
7 S. Phukan, V. S. Babu, A. Kannoji, R. Hariharan and
V. N. Balaji, Br. J. Pharmacol., 2010, 160, 1–19.
8 H. Eldar-Finkelman and A. Martinez, Front. Mol. Neurosci.,
2011, 4, 32.
9 I. Hers, J. M. Tavare and R. M. Denton, FEBS Lett., 1999,
460, 433–436.
(Chemical Computing Group). The docking of synthetic com-
pounds into an X-ray crystal structure (PDB code: 1R0E) was 10 T. A. Engler, J. R. Henry, S. Malhotra, B. Cunningham,
carried out according to the operation manual. The top 17
ranked docking modes were further examined by the combi-
nation of scores and visual inspection.
K. Furness, J. Brozinick, T. P. Burkholder, M. P. Clay,
R. M. Campbell and R. Vaughn, J. Med. Chem., 2004, 47,
3934–3937.
1
1 G. H. Kuo, C. Prouty, A. DeAngelis, L. Shen, D. J. O’Neill,
C. Shah, P. J. Connolly, W. V. Murray, B. R. Conway,
P. Cheung, L. Westover, J. Z. Xu, R. A. Look, K. T. Demarest,
S. Emanuel, S. A. Middleton, L. Jolliffe, M. P. Beavers and
X. Chen, J. Med. Chem., 2003, 46, 4021–4031.
Conclusions
In summary, we have designed and synthesized a series of
indolyl maleimide inhibitors of GSK-3β and identified several 12 H. C. Zhang, L. V. Bonaga, H. Ye, C. K. Derian,
highly potent and selective GSK-3β covalent inhibitors with
low-nanomolar IC50 values. Structure–activity relationships
B. P. Damiano and B. E. Maryanoff, Bioorg. Med. Chem.
Lett., 2007, 17, 2863–2868.
and preliminary docking studies provided insight into the 13 M. P. Coghlan, A. A. Culbert, D. A. E. Cross, S. L. Corcoran,
interactions between the kinase and inhibitors to facilitate
optimization. Functional assays revealed that covalent inhibi-
tors 20a, 20e, 38a and 38b could reduce the phosphorylation
activity of GSK-3β upon its down-stream substrates in live cells.
J. W. Yates, N. J. Pearce, O. L. Rausch, G. J. Murphy,
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Acknowledgements
We thank funding support from 973 Program (2013CB910704),
Shenzhen Science and Technology Innovation Commission
(JCYJ20160226105227446), and Peking University.
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