3-(7-Azaindolyl)-4-indolylmaleimides as a novel class of mutant isocitrate dehydrogenase-1 inhibitors: Design, synthesis, and biological evaluation

Article abstract of DOI:10.1002/ardp.201800039

A series of 3-(7-azainodyl)-4-indolylmaleimides was designed, synthesized, and evaluated for their isocitrate dehydrogenase 1 (IDH1)/R132H inhibitory activities. Many compounds such as 11a, 11c, 11e, 11g, and 11s exhibited favorable inhibitory effects on IDH1/R132H and were highly selective against the wild-type IDH1. Evaluation of the biological activities at the cellular level showed that compounds 11a, 11c, 11e, 11g, and 11s could effectively suppress the production of 2-hydroxyglutaric acid in U87MG cells expressing IDH1/R132H. Preliminary structure–activity relationship (SAR) and molecular modeling studies were discussed based on the experimental data obtained. These findings may provide new insights into the development of novel IDH1/R132H inhibitors.

Full text of DOI:10.1002/ardp.201800039

Received: 14 February 2018
Revised: 3 July 2018
Accepted: 11 July 2018
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DOI: 10.1002/ardp.201800039
FULL PAPER
3-(7-Azaindolyl)-4-indolylmaleimides as a novel class of
mutant isocitrate dehydrogenase-1 inhibitors: Design,
synthesis, and biological evaluation
Yuanyuan Hu1
Anhui Gao2
Honghui Liao3
Mengmeng Zhang2
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Gaoya Xu2
Qing Ye1
Lixin Gao2
Jia Li2
Lei Xu2
Yubo Zhou2
Jianrong Gao1
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1 State Key Laboratory Breeding Base of Green
Chemistry-Synthesis Technology, Zhejiang
University of Technology, Hangzhou, China
Abstract
A series of 3-(7-azainodyl)-4-indolylmaleimides was designed, synthesized, and
evaluated for their isocitrate dehydrogenase 1 (IDH1)/R132H inhibitory activities.
Many compounds such as 11a, 11c, 11e, 11g, and 11s exhibited favorable inhibitory
effects on IDH1/R132H and were highly selective against the wild-type IDH1.
Evaluation of the biological activities at the cellular level showed that compounds 11a,
11c, 11e, 11g, and 11s could effectively suppress the production of 2-hydroxyglutaric
acid in U87MG cells expressing IDH1/R132H. Preliminary structure–activity
relationship (SAR) and molecular modeling studies were discussed based on the
experimental data obtained. These findings may provide new insights into the
development of novel IDH1/R132H inhibitors.
2 National Center for Drug Screening, State
Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy
of Sciences, Shanghai, China
3 The Second People Hospital of Xihu District,
Hangzhou, China
Correspondence
Dr. Qing Ye, State Key Laboratory Breeding
Base of Green Chemistry-Synthesis
Technology, Zhejiang University of
Technology, Hangzhou 310032, China.
Email: yeqing1975@zjut.edu.cn
Prof. Jia Li, National Center for Drug Screening,
State Key Laboratory of Drug Research, Shang-
hai Institute of Materia Medica, Chinese Acad-
emy of Sciences, Shanghai 201203, China.
Email: jli@simm.ac.cn
K E Y W O R D S
2-hydroxyglutaric acid, 3-(7-azaindoyl)-4-indolylmaleimides, isocitrate dehydrogenase 1
inhibitors, tumor
Funding information
China Postdoctoral Science Foundation,
Grant number: 2014M550256; Strategic
Priority Research Program of the Chinese
Academy of Sciences, Grant number:
XDA12020328; Open Fund of State Key
Laboratory of New Drugs, Shanghai Institute
of Materia Medica, Chinese Academy of
Sciences, Grant number: SIMM1601KF-04;
Personalized Medicines-Molecular Signature-
Based Drug Discovery and Development;
Natural Science Foundation of Zhejiang
Province, Grant number: LY18H300009;
National Natural Science Foundation of China,
Grant number: 8150131067
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1
INTRODUCTION
Isocitrate dehydrogenase 1 (IDH1) is a key rate-limiting enzyme in the
tricarboxylic acid cycle, which mainly exists in the cytoplasm and
Yuanyuan Hu and Anhui Gao contributed equally to this work.
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Arch Pharm Chem Life Sci. 2018;e1800039.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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peroxisomes.[1] IDH1 can catalyze the oxidative decarboxylation of
isocitric acid to produce α-ketoglutaric acid (α-KG) and reduce NADP+
to NADPH, using nicotinamide adenine dinucleotide phosphate
(NADP+) as the electron acceptor (Scheme 1).[2–4]
Recent studies have demonstrated a high mutation rate of IDH1 in
many malignant tumors.[5] For example, the mutation rate of IDH1
reached 56, 70, and 5.5–10.4% in sarcoma,[6] glioma,[7,8] and acute
myeloid leukemia (AML),[9–11] respectively. To date, all the IDH1
mutations were found at the R132 codon, including R132H, R132C,
R132L, R132S, R132G, and R132Q, among which R132H was the
predominant one, accounting for about 90%.[12–15] The IDH1 mutation
causes a dramatic decrease in the production of α-KG following the
oxidative decarboxylation of isocitric acid. Furthermore, IDH1
mutation exerts a new catalytic function that causes the reduction
of α-KG to 2-hydroxyglutaric acid (2-HG), leading to massive
accumulation of 2-HG in cells (Scheme 2).[13,16–18]
SCHEME 2 Reduction of α-KG to 2-HG
alcoholysis with sodium methoxide. N-Alkylation of 2a–i with different
alkyl halides using NaH as a base in dry N,N-Dimethylformamide (DMF)
afforded key intermediates 3a–p. The acylation of 7-azaindole 4 with
ClCOCO2Et using anhydrous AlCl3 as a catalyst gave compound 5,
which was reacted with (Boc)2O in the presence of a catalytic amount of
DMAP affording the key intermediate 6a. N-Alkylation of 5 with
bromoethane and 2-bromopropane resulted in other key intermediates
6b and 6c respectively. N-Methylation of 4 with iodomethane afforded
compound 7, which can be easily converted to compound 8 following
the same procedure as described for preparing compound 5. Compound
8 was reduced with triethylsilane in trifluoroacetic acid (TFA) to provide
acetic ether derivatives 9, which was ammonolyzed by saturated
solution of ammonia in methanol to give the key intermediate 10.
Maleimide condensation of 10 with 3a–p or 2a in the presence of
t-BuOK afforded the target compounds 11a–q, respectively.[34] Target
compounds 11r–t were prepared by the maleimide condensation of
6a–c with 12 using the same procedure as described above.
Because 2-HG and α-KG have
a similar structure, they
compete each other. For these two reasons, the activity of some
α-KG-dependent dioxygenases including proline hydroxylase, Tet
family DNA hydroxylase, and histone lysine methyltransferase
decreases, leading to excessive DNA methylation and histone
methylation, which can cause epigenetic disorders, cell differentiation
inhibition, and eventually tumorigenesis.[19–23] Because of the key
roles of IDH1 mutation in the development and progression of tumors,
the mutated IDH1 (including IDH1/R132H) has become a potentially
attractive target for anti-tumor therapies.[24] The search for their
inhibitors has become a hot spot in the research and development of
new anti-tumor drugs. During the past few years, many IDH1/R132H
inhibitors with varied structures have been reported (Figure 1).[25–33]
Among them, AGI-120 (developed by Agios Pharmaceuticals,
Cambridge, MA, USA) and IDH305 (Novartis Pharmaceuticals, Basel,
Switzerland) have entered clinical trials.
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2.2 Biological activity
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2.2.1 Enzymatic activity
All the prepared compounds 11a–t were tested for their IDH1/R132H
inhibitory activity. A well-known kinase inhibitor AG-120 developed
by Agios Pharmaceuticals was used as the positive control. The results
are presented in Table 1.
In our current study, a series of 3-(7-azaindolyl)-4-indolylmalei-
mide IDH1/R132H inhibitors was developed by carrying out high-
throughput screening followed by structural modification of lead
compounds. Their structure–activity relationship (SAR) and in silico
molecular modeling study were discussed as well.
As shown in Table 1, most of the tested compounds displayed
moderate to potent inhibitory activity against IDH1/R132H. Among
them, compound 11e (IC50 = 0.16 µM) and 11g (IC50 = 0.28 µM)
exhibited more potent inhibition of IDH1/R132H than other
analogues. SAR analysis showed that introduction of a suitable
hydrophilic side chain, which may form hydrogen bond interactions
with the enzyme, at the N1-position of the indole ring is necessary
for the activity. For example, compound 11a (IC50 = 0.40 µM) with a
3-(1H-imidazol-l-yl)propyl on the indole nitrogen was about 48-fold
more potent than N1-position no substituted compound 11q
(IC50 = 19.39 µM). However, compounds 11m–p, with a hydrophilic
3-(piperidin-1-yl)propyl, 3-morpholinopropyl, 3-(1H-1,2,4-triazol-1-
yl)-propyl, and 3-(pyrrolidin-1-yl)propyl, respectively, exhibited
weaker inhibitory activities than 11a, which suggested that these
hydrophilic side chains may not effectively form hydrogen bond
interactions with the protein. In addition, comparing the inhibitory
activity of 11a, 11j, and 11l revealed that the length of the N1-alkyl
linker affected IDH1/R132H inhibitory potency. For example,
compound 11a (IC50 = 0.40 µM) with a (CH2)3 linker showed better
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2
RESULTS AND DISCUSSION
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2.1 Chemistry
The synthesis routes of target compounds 11a–t are summarized in
Scheme 3. Compounds 2a–i were prepared from indole derivatives
1a–i by acylation with oxalyl chloride in dry Et2O, followed by
SCHEME 1 Oxidative decarboxylation of isocitric acid to produce
α-KG

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FIGURE 1 IDH1/R132H inhibitors
inhibitory activity than compound 11j (IC50 = 0.86 µM) with a (CH2)4
linker and 11l (IC50 = 14.50 µM) with a (CH2)2 linker. The same
conclusion could also be drawn from comparison of the inhibitory
potency of 11e and 11k.
accumulates in cancer cells. Compounds 11a, 11c, 11e, 11g, and 11s
were tested for their ability to reduce the production of 2-HG using
sensitive and specific LC/MS/MS methods. AGI-120, an IDH1/R132H
inhibitor, was used as a reference compound in this assay. As shown in
Figure 2, compounds 11a, 11c, 11e, 11g, and 11s effectively inhibited
2-HG production in IDH1/R132H-expressing U87MG cells in a dose-
independent manner, which correlates well with their inhibitory
activity toward IDH1/R132H.
When comparing the inhibitory activity of 11b–i with 11a, it
suggested that different substituents (R1) on the indole ring affected
the inhibitory potency for IDH1/R132H. Introduction of a chlorine
(11d, IC50 = 0.16 µM) or bromine (11f, IC50 = 0.28 µM) atom at the
6-position of the indole ring led to about twofold enhancement of the
activity as compared to 11a. Fluorine at 6-position of the indole ring
(11d, IC50 = 0.36 µM) or benzyloxy group at 5-position of the indole
ring (11i, IC50 = 0.47 µM) was tolerated for the activity, while halogen
atom at 5-position (i.e., 11b, 11d, and 11f) or methyl at the 7-position
(11h) showed less inhibitory potency.
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2.3 Molecular modeling study
In order to explore the possible binding mode of the target compounds,
a molecular modeling study of 11e was performed using CDOCKER
module in Discovery Studio 2.5, based on the published IDH1/R132H
crystal structure (5DE1).[30] As seen in Figure 3, the amino and
carbonyl in maleimide ring of 11e can tightly interact with Arg109,
Ala111, and Ile128 of IDH1/R132H via the hydrogen bonding
network. The 7-position nitrogen of the 7-azaindazole ring acting as
a hydrogen bond acceptor forms another H-bond with Arg119. The
7-azaindole and indole moiety of compound 11e both have π–π
interaction (T-shaped) with Trp124 residue. In addition, the chlorine
atom (in green) of compound 11e can fit in the hydrophobic pocket
composed of Val255 and Met259, which is similar to that reported by
Refs. [30,31]. The 3-position nitrogen of imidazole ring is about 3.7 and
3.4 Å from the NH of Arg119 and Leu120, respectively, and may keep
water-mediated interaction with them.
Comparison of IDH1/R132H inhibitory data of compounds with
various substituents at the N1-position of the 7-azaindole ring revealed
that compounds with
a Me (11a, IC50 = 0.40 µM) or Et (11s,
IC50 = 0.34 µM) on N1-position of the 7-azaindole ring were tolerated
for IDH1/R132H inhibitory activity. However, replacement of Me with
H (11r, IC50 = 0.83 µM) or i-Pr (11t, IC50 = 1.42 µM) led to about 2- and
3.5-fold loss in potency as compared to the case of compound 11a,
respectively.
Because of the important physiological role of wild-type IDH1
(IDH1/WT), compounds which also strongly inhibit IDH1/WT catalytic
activity are disadvantageous for their clinical application. The assay of
inhibitory activity of selected compounds 11a, 11c, 11e, 11g, and 11s
toward IDH1/WT was also conducted to determine the enzyme
selectivity. As shown in Table 2, all the tested compounds displayed
high selectivity for IDH1/R132H over IDH1/WT.
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CONCLUSION
In summary, a series of 3-(7-azaindolyl)-4-indolylmaleimides was
synthesized and biologically evaluated as IDH1/R132H inhibitors.
Among them, compounds 11a, 11c, 11e, 11g, and 11s exhibited high
inhibitory potencies against IDH1/R132H and were highly selective
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2.2.2 Cellular activity
IDH1 mutation gains a catalytic function that causes the reduction of
α-KG to 2-HG, resulting in a high concentration of 2-HG that

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against the wild-type IDH1, which inhibited the cellular 2-HG
production in a dose-dependent manner. Preliminary SAR study based
on the experimental data and molecular modeling study provided
information that could be useful for the design of novel IDH1/R132H
inhibitors as antitumor agents.
Methyl 2-(1-(3-(1H-imidazol-1-yl)propyl)-6-chloro-1H-indol-3-
yl)-2-oxoacetate (3e)
According to the procedure used to prepare 3b, reaction of 2e with
1-(3-chloropropyl)-1H-imidazole provided 3e in 46.5% yield as a light
yellow solid, mp: 135–136°C. 1H NMR (500 MHz, CDCl3) δ: 8.39 (d,
J = 8.5 Hz, 1H), 8.35 (s, 1H), 7.55 (s, 1H), 7.35 (dd, J = 8.5, 2.0 Hz, 1H),
7.29 (d, J = 2.0 Hz, 1H), 7.17 (s, 1H), 6.97 (s, 1H), 4.15 (t, J = 7.0 Hz, 2H),
4.02 (t, J = 7.0 Hz, 2H), 3.98 (s, 3H), 2.47–2.41 (m, 2H). ESI-MS:
m/z = 346 [M+H]+.
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4
EXPERIMENTAL
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4.1 Chemistry
Methyl 2-(1-(3-(1H-imidazol-1-yl)propyl)-6-bromo-1H-indol-3-
yl)-2-oxoacetate (3g)
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4.1.1 General
All reagents used in the synthesis were obtained commercially and
used without further purification, unless otherwise specified.
Melting points were determined with a BÜCHI Melting Point
B-450 apparatus (Büchi Labortechnik, Flawil, Switzerland) and are
uncorrected. 1H NMR spectra were recorded using TMS as the
internal standard at 500 MHz and the coupling constants are
reported in hertz. The reactions were followed by thin-layer
chromatography (TLC) on glass-packed precoated silica gel plates
and visualized in an iodine chamber or with a UV lamp. Tetrahydro-
furan (THF) was distilled from sodium-benzophenone. DMF was
distilled from calcium hydride. Compounds 2a–i, 3a, 3d, 3f, 3l–p, and
12 were synthesized according to literature procedures.[35–38]
The InChI codes together with some biological activity data are
provided as Supporting Information.
According to the procedure used to prepare 3b, reaction of 2g with
1-(3-chloropropyl)-1H-imidazole provided 3g in 29.6% yield as a light
yellow solid, mp: 139–141°C. 1H NMR (500 MHz, CDCl3) δ: 8.33–8.32
(m, 2H), 7.56 (s, 1H), 7.48 (dd, J = 8.5, 1.5 Hz, 1H), 7.45 (d, J = 1.5 Hz,
1H), 7.17 (s, 1H), 6.96 (s, 1H), 4.15 (t, J = 7.0 Hz, 2H), 4.02 (t, J = 7.0 Hz,
2H), 3.97 (s, 3H), 2.50–2.38 (m, 2H). ESI-MS: m/z = 390 and 392
[M+H]+.
Methyl 2-(1-(3-(1H-imidazol-1-yl)propyl)-7-methyl-1H-indol-3-
yl)-2-oxoacetate (3h)
According to the procedure used to prepare 3b, reaction of 2h with
1-(3-chloropropyl)-1H-imidazole provided 3h in 47.3% yield as a light
yellow solid, mp: 106–107°C. 1H NMR (500 MHz, CDCl3) δ: 8.37
(d, J = 7.9 Hz, 1H), 8.29 (s, 1H), 7.56 (s, 1H), 7.25 (t, J = 7.6 Hz, 1H), 7.16
(s, 1H), 7.09 (d, J = 7.3, 1.1 Hz, 1H), 6.98 (s, 1H), 4.39 (t, J = 7.1 Hz, 2H),
4.04 (t, J = 6.7 Hz, 2H), 3.98 (s, 3H), 2.61 (s, 3H), 2.50–2.39 (m, 2H). ESI-
MS: m/z = 326 [M+H]+.
Methyl 2-(1-(3-(1H-imidazol-1-yl)propyl)-5-fluoro-1H-indol-3-
yl)-2-oxoacetate (3b)
To a solution of 2b (2.4 g, 7.0 mmol) in anhydrous DMF (15 mL), 70%
NaH (0.32 g, 8.0 mmol) was added portionwise at 0–5°C. After stirring
for 30 min at room temperature, 1-(3-chloropropyl)-1H-imidazole
(1.59 g, 11.0 mmol) was added. Then the mixture was stirred for 6 h at
60°C. After cooling, the mixture was then poured into water (100 mL)
and extracted with ethyl acetate (50 mL × 3). The organic phase was
combined and washed with brine (150 mL × 3), dried over Na2SO4, and
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel using dichloromethane/methanol (50:1,
v/v) as eluent to afford 3b (33.2% yield) as a light yellow solid, mp:
108–110°C. 1H NMR (500 MHz, CDCl3) δ: 8.37 (d, J = 8.0 Hz, 1H), 8.29
(s, 1H), 7.56 (s, 1H), 7.25 (t, J = 7.5 Hz, 1H), 7.16 (s, 1H), 7.09 (d,
J = 7.5 Hz, 1H), 6.98 (s, 1H), 4.38 (t, J = 7.0 Hz, 2H), 4.04 (t, J = 7.0 Hz,
2H), 3.97 (s, 3H), 2.42–2.36 (m, 2H). ESI-MS: m/z = 330 [M+H]+.
Methyl 2-(1-(3-(1H-imidazol-1-yl)propyl)-5-(benzyloxy)-1H-
indol-3-yl)-2-oxoacetate (3i)
According to the procedure used to prepare 3b, reaction of 2i with
1-(3-chloropropyl)-1H-imidazole provided 3i in 43.8% yield as a light
yellow solid, mp: 147–148°C. 1H NMR (500 MHz, CDCl3) δ: 8.30
(s, 1H), 8.07 (d, J = 2.5 Hz, 1H), 7.55–7.49 (m, 3H), 7.44–7.38 (m, 2H),
7.34 (t, J = 7.5 Hz, 1H), 7.17 (d, J = 9.0 Hz, 1H), 7.16 (s, 1H), 7.07 (dd,
J = 9.0, 2.5 Hz, 1H), 6.95 (s, 1H), 5.18 (s, 2H), 4.14 (t, J = 7.0 Hz, 2H),
4.00–3.95 (m, 5H), 2.46–2.39 (m, 2H). ESI-MS: m/z = 418 [M+H]+.
Methyl 2-(1-(4-(1H-imidazol-1-yl)butyl)-1H-indol-3-yl)-2-
oxoacetate (3j)
According to the procedure used to prepare 3b, reaction of 2a with
1-(4-chlorobutyl)-1H-imidazole provided 3j in 43.9% yield as a light
yellow solid, mp: 97–98°C. 1H NMR (500 MHz,CDCl3) δ: 8.51–8.44
(m, 1H), 8.38 (s, 1H), 7.45 (s, 1H), 7.42–7.31 (m, 3H), 7.08 (t, J = 1.1 Hz,
1H), 6.86 (t, J = 1.3 Hz, 1H), 4.21 (t, J = 6.8 Hz, 2H), 3.97 (s, 3H), 3.95
(t, J = 6.7 Hz, 2H), 1.96–1.79 (m, 4H). ESI-MS: m/z = 326 [M+H]+.
Methyl 2-(1-(3-(1H-imidazol-1-yl)propyl)-6-fluoro-1H-indol-3-
yl)-2-oxoacetate (3c)
According to the procedure used to prepare 3b, reaction of 2c with
1-(3-chloropropyl)-1H-imidazole provided 3c in 39.5% yield as a light
yellow solid, mp: 113–115°C. 1H NMR (500 MHz, CDCl3) δ: 8.41
(dd, J = 8.8, 5.4 Hz, 1H), 8.34 (s, 1H), 7.53 (s, 1H), 7.17 (s, 1H), 7.13 (td,
J = 9.0, 2.2 Hz, 1H), 6.98–6.95 (m, 2H), 4.14 (t, J = 7.0 Hz, 2H), 4.01 (t,
J = 6.8 Hz, 2H), 3.98 (s, 3H), 2.48–2.38 (m, 2H). ESI-MS: m/z = 330
[M+H]+.
Methyl 2-(1-(4-(1H-imidazol-1-yl)butyl)-6-chloro-1H-indol-3-
yl)-2-oxoacetate (3k)
According to the procedure used to prepare 3b, reaction of 2e with
1-(4-chlorobutyl)-1H-imidazole provided 3k in 39.7% yield as a light

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SCHEME 3 Synthetic route to compounds 11a–t. Reagents and conditions: (a) (i) oxalyl chloride, diethyl ether; (ii) CH3ONa, CH3OH; (b)
NaH, DMF, R2X; (c) AlCl3, ClCOCO2Et; (d) (Boc)2O, DMAP, THF; (e) cesium carbonate, DMF, R3Br; (f) NaH, DMF, CH3I; (g) triethylsilane,
trifluoroacetic acid; (h) NH3, methanol; (i) t-BuOK, THF; (ii) concentrated HCl
yellow solid, mp: 68–69°C. 1H NMR (500 MHz, CDCl3) δ: 8.38–8.33
(m, 2H), 7.46 (s, 1H), 7.36–7.29 (m, 2H), 7.08 (s, 1H), 6.89–6.87 (m, 1H),
4.15 (t, J = 6.7 Hz, 2H), 4.02–3.93 (m, 5H), 1.94–1.75 (m, 4H). ESI-MS:
m/z = 360 [M+H]+.
Ethyl 2-oxo-2-(1H-pyrrolo[2,3-b]pyridin-3-yl)acetate (5)
To a mixture of 4 (1.2 g, 10 mmol), anhydrous aluminum chloride
(18.0 g, 135 mmol) and 200 mL of anhydrous dichloromethane, ethyl
oxalyl chloride (8.2 g, 60 mmol) in 30 mL anhydrous dichloromethane

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TABLE 1 IDH1/R132H inhibitory activities of target compounds
IC50 (µM) SE or
Compd.
AG-120
11a
R1
R2
R3
inhibition% at ∼20 µg/mL
0.20 0.08
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
0.40 0.13
11b
11c
11d
11e
11f
11g
11h
11i
5-F
0.74 0.06
0.36 0.04
1.54 0.37
0.16 0.04
1.75 0.43
0.28 0.018
0.62 0.04
0.47 0.07
0.86 0.31
0.63 0.05
14.50 2.43
20.33%
6-F
5-Cl
6-Cl
5-Br
6-Br
7-Me
5-OCH2Ph
H
11j
11k
11l
6-Cl
H
11m
H
11n
11o
11p
H
H
H
CH3
CH3
CH3
27.51%
45.81%
14.80%
11q
11r
H
H
H
CH3
H
19.39 2.86
0.83 0.24
11s
11t
H
H
Et
0.34 0.06
1.42 0.18
i-Pr
SE, standard error mean.
was added dropwise. After addition, the mixture was stirred at room
temperature for 6 h. The supernatant was abandoned and 20%
ammonium acetate aqueous was added to the reaction mixture at
0–5°C. The mixture was stirred sufficiently and extracted with
dichloromethane (50 mL × 3). The organic phase was combined, dried
over Na2SO4, and concentrated in vacuo. The residue was purified by
flash column chromatography on silica gel using petroleum ether/ethyl
acetate (3:1, v/v) as eluent to afford 5 in 49.1% yield as a light yellow

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TABLE 2 IDH1/WT inhibitory activity of selected compounds
acetate (2:1, v/v) as eluent to afford 6b in 55.3% yield as a light yellow
solid, mp: 72–73°C. 1H NMR (500 MHz, CDCl3) δ: 8.68 (dd, J = 8.0,
1.5 Hz, 1H), 8.55 (s, 1H), 8.43 (dd, J = 4.5, 1.5 Hz, 1H), 7.32–7.27 (m,
1H), 4.47–4.40 (m, 4H), 1.58 (t, J = 7.5 Hz, 3H), 1.46 (t, J = 7.0 Hz, 3H).
ESI-MS: m/z = 247 [M+H]+.
Compd.
11a
IC50 (µM)
≥10
11c
≥10
11e
≥10
11g
≥10
Ethyl 2-(1-isopropyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-
oxoacetate (6c)
11s
≥10
According to the procedure used to prepare 6b, reaction of 5 with
2-bromopropane provided 6c in 52.1% yield as a light yellow solid.
1H NMR (500 MHz,CDCl3) δ: 8.68 (dd, J = 7.9, 1.7 Hz, 1H), 8.60 (s, 1H),
8.43 (dd, J = 4.7, 1.7 Hz, 1H), 7.30 (dd, J = 7.9, 4.7 Hz, 1H), 5.27–5.21
(m, 1H), 4.44 (q, J = 7.2 Hz, 2H), 1.62 (d, J = 6.8 Hz, 6H), 1.46
(t, J = 7.2 Hz, 3H). ESI-MS: m/z = 261 [M+H]+.
solid, mp: 98–99°C. 1H NMR (500 MHz, CDCl3) δ: 12.27 (s, 1H), 8.78
(dd, J = 7.9, 1.6 Hz, 1H), 8.72 (s, 1H), 8.47 (dd, J = 4.8, 1.6 Hz, 1H), 7.37
(dd, J = 7.9, 4.8 Hz, 1H), 4.46 (q, J = 7.1 Hz, 2H), 1.47 (t, J = 7.1 Hz, 3H).
ESI-MS: m/z = 219 [M+H]+.
1-Methyl-1H-pyrrolo[2,3-b]pyridine (7)
tert-Butyl 3-(2-ethoxy-2-oxoacetyl)-1H-pyrrolo[2,3-b]pyridine-
1-carboxylate (6a)
To a solution of 1H-pyrrolo[2,3-b]pyridine 4 (1.6 g, 10 mmol) in 30 mL
anhydrous DMF, 70% NaH (0.96 g, 28 mmol) was added portionwise at
0–5°C. After stirring for 30 min, a solution of iodomethane (4.3 g,
30 mmol) in 5 mL anhydrous DMF was added dropwise. After addition,
the mixture was stirred for 1 h at 0–5°C and then 1 h at room
temperature. The mixture was then poured into water (100 mL) and
extracted with dichloromethane (40 mL × 3). The organic phase was
combined and washed with brine (120 mL × 3), dried over Na2SO4, and
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel using petroleum ether/ethyl acetate (3:1,
v/v) as eluent to afford 7 in 85.0% yield as a light yellow oil. 1H NMR
(500 MHz, CDCl3) δ: 8.34 (dd, J = 4.7, 1.6 Hz, 1H), 7.90 (dd, J = 7.8,
1.6 Hz, 1H), 7.17 (d, J = 3.4 Hz, 1H), 7.05 (dd, J = 7.8, 4.7 Hz, 1H), 6.44
(d, J = 3.4 Hz, 1H), 3.89 (s, 3H). ESI-MS: m/z = 133 [M+H]+.
A solution of 5 (0.50 g, 2.3 mmol), DMAP (14 mg, 0.01 mmol), and
(Boc)2O (0.63 g, 2.9 mmol) in 30 mL dry THF was stirred at room
temperature for 1 h. The mixture was then concentrated in vacuo. The
residue was purified by flash column chromatography on silica gel
using petroleum ether/ethyl acetate (4:1, v/v) as eluent to afford 6a in
80.6% yield as a light yellow solid, mp: 79–81°C. 1H NMR (500 MHz,
CDCl3) δ: 8.89 (s, 1H), 8.69 (dd, J = 7.9, 1.7 Hz, 1H), 8.61 (dd, J = 4.8,
1.7 Hz, 1H), 7.38 (dd, J = 7.9, 4.8 Hz, 1H), 4.47 (q, J = 7.2 Hz, 2H), 1.73
(s, 9H), 1.48 (t, J = 7.2 Hz, 3H). ESI-MS: m/z = 319 [M+H]+.
Ethyl 2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-oxoacetate
(6b)
A mixture of 5 (0.50 g, 2.3 mmol), cesium carbonate (1.6 g, 5 mmol), and
bromoethane (0.35 g, 3.0 mmol) in 25 mL of anhydrous DMF was
stirred at 50°C for 2 h. After cooling, the mixture was then poured into
water (100 mL) and extracted with ethyl acetate (60 mL × 3). The
organic phase was combined and washed with brine (180 mL × 3), dried
over Na2SO4, and concentrated in vacuo. The residue was purified by
flash column chromatography on silica gel using petroleum ether/ethyl
Ethyl 2-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-oxoacetate
(8)
According to the procedure used to prepare 5, reaction of 7 with ethyl
oxalyl chloride provided 8 in 31.1% yield as a light yellow solid, mp:
96–98°C. 1H NMR (500 MHz, DMSO-d6) δ: 8.71 (s, 1H), 8.52–8.43
FIGURE 2 Compounds 11a, 11c, 11e, 11g, and 11s inhibited 2-HG production in IDH1/R132H expressing U87MG cells

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FIGURE 3 Compound 11e docked into IDH1/R132H crystal structure. (A) Binding mode of compound 11e (in yellow); (B) compound 11e
bound to IDH1/R132H after surfacing receptor
(m, 2H), 7.39 (dd, J = 7.8, 4.7 Hz, 1H), 4.38 (q, J = 7.1 Hz, 2H), 3.94
(s, 3H), 1.36 (t, J = 7.1 Hz, 3H). ESI-MS: m/z = 233 [M+H]+.
ice water (100 mL), alkalized with sodium bicarbonate, and extracted
with ethyl acetate (100 mL × 3). The combined organic phase was dried
over anhydrous sodium sulfate and concentrated in vacuo. The residue
was then purified by flash column chromatography on silica gel using
DCM/MeOH (60:1, v/v) as eluent to afford 11a in 11.3% yield as a red
solid, mp: 207–209°C. 1H NMR (500 MHz, DMSO-d6) δ: 11.02 (s, 1H),
8.12 (dd, J = 4.5, 1.5 Hz, 1H), 8.03 (s, 1H), 7.80 (s, 1H), 7.62 (s, 1H), 7.42
(d, J = 8.5 Hz, 1H), 7.20 (s, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.02 (d,
J = 7.5 Hz, 1H), 6.93 (s, 1H), 6.85 (d, J = 7.5 Hz, 1H), 6.72 (t, J = 7.5 Hz,
1H), 6.67 (dd, J = 8.2, 4.7 Hz, 1H), 4.23 (t, J = 7.0 Hz, 2H), 3.97 (t,
J = 7.0 Hz, 2H), 3.88 (s, 3H), 2.30–2.18 (m, 2H). HRMS (ESI) m/z [M+H]+
for C26H23N6O2 calcd. 451.1877. Found 451.1873.
Ethyl 2-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)acetate (9)
A mixture of 8 (3.8 g, 16.5 mmol) and triethylsilane (10.4 g, 67.2 mmol)
in 75 mL of trifluoroacetic acid was stirred at 55°C for 10 h. After
cooling, the mixture was then poured into water (200 mL), alkalized
with sodium bicarbonate, and extracted with ethyl acetate
(100 mL × 3). The organic phase was combined, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel using petroleum ether/ethyl acetate (6:1,
v/v) as eluent to afford 9 in 73.0% yield as a light yellow solid, mp:
107–108°C. 1H NMR (500 MHz, CDCl3) δ: 8.29 (dd, J = 4.5, 1.5 Hz,
1H), 7.86 (dd, J = 8.0, 1.5 Hz, 1H), 7.09 (s, 1H), 7.00 (dd, J = 7.8, 4.7 Hz,
1H), 4.12 (q, J = 7.0 Hz, 2H), 3.79 (s, 3H), 3.68 (s, 2H), 1.21 (t, J = 7.0 Hz,
3H). ESI-MS: m/z = 219 [M+H]+.
3-(1-(3-(1H-Imidazol-1-yl)propyl)-5-fluoro-1H-indol-3-yl)-4-(1-
methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-dione
(11b)
According to the procedure used to prepare 11a, reaction of 3b with
10 provided 11b in 12.1% yield as a red solid, mp: 172–174°C. 1H NMR
(500 MHz, DMSO-d6) δ: 11.04 (s, 1H), 8.15 (dd, J = 4.7, 1.6 Hz, 1H),
8.06 (s, 1H), 7.84 (s, 1H), 7.63 (s, 1H), 7.46 (dd, J = 9.0, 4.5 Hz, 1H), 7.19
(s, 1H), 7.00 (dd, J = 7.9, 1.6 Hz, 1H), 6.99–6.89 (m, 2H), 6.71 (dd,
J = 8.0, 4.7 Hz, 1H), 6.56 (dd, J = 10.2, 2.6 Hz, 1H), 4.22 (t, J = 7.1 Hz,
2H), 3.95 (t, J = 7.2 Hz, 2H), 3.90 (s, 3H), 2.24–2.17 (m, 2H). HRMS (ESI)
m/z [M+H]+ for C26H22FN6O2 calcd. 469.1783. Found 469.1781.
2-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)acetamide (10)
A mixture of 9 (1.76 g, 8 mmol) and saturated solution of ammonia in
methanol (10 mL) was stirred at 90°C in a sealed tube for 2 h. After
cooling, the mixture was concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel using ethyl
acetate/methanol (50:1, v/v) as eluent to afford 10 in 36.7% yield as a
white solid, mp: 161–163°C. 1H NMR (500 MHz, DMSO-d6) δ: 8.24
(dd, J = 4.5, 1.5 Hz, 1H), 7.97 (dd, J = 8.0, 1.5 Hz, 1H), 7.39 (brs, 1H),
7.35 (s, 1H), 7.07 (dd, J = 7.8, 4.6 Hz, 1H), 6.87 (brs, 1H), 3.79 (s, 3H),
3.48 (s, 2H). ESI-MS: m/z = 190 [M+H]+.
3-(1-(3-(1H-Imidazol-1-yl)propyl)-6-fluoro-1H-indol-3-yl)-4-(1-
methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-dione
(11c)
3-(1-(3-(1H-Imidazol-1-yl)propyl)-1H-indol-3-yl)-4-(1-methyl-
1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-dione (11a)
To a solution of 3a (0.37 mmol) and 10 (0.28 mmol) in anhydrous THF
(20 mL), 1M t-BuOK in t-butanol (0.84 mL, 0.84 mmol) was added
dropwise at −10 to 0°C. After stirring for 2 h at room temperature,
concentrated hydrochloric acid (5 mL) was added and the resultant
mixture was stirred for 30 min at room temperature, then poured into
According to the procedure used to prepare 11a, reaction of 3c with 10
provided 11c in 14.6% yield as a red solid, mp: 225–226°C. 1H NMR
(500 MHz, DMSO-d6) δ: 11.05 (s, 1H), 8.14 (dd, J = 4.6, 1.7 Hz, 1H),
8.06 (s, 1H), 7.78 (s, 1H), 7.63 (s, 1H), 7.35 (dd, J = 10.0, 2.0 Hz, 1H),
7.20 (s, 1H), 6.98 (dd, J = 8.0, 1.5 Hz, 1H), 6.93 (s, 1H), 6.84 (dd, J = 8.8,
5.4 Hz, 1H), 6.70 (dd, J = 8.2, 4.6 Hz, 1H), 6.61 (td, J = 9.5, 2.0 Hz, 1H),
4.20 (t, J = 7.0 Hz, 2H), 3.96 (t, J = 7.0 Hz, 2H), 3.89 (s, 3H), 2.25–2.18

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(m, 2H). HRMS (ESI) m/z [M+H]+ for C26H22FN6O2 calcd. 469.1783.
3-(1-(3-(1H-Imidazol-1-yl)propyl)-7-methyl-1H-indol-3-yl)-4-(1-
methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-dione
(11h)
Found 469.1779.
3-(1-(3-(1H-Imidazol-1-yl)propyl)-5-chloro-1H-indol-3-yl)-4-(1-
methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-dione
(11d)
According to the procedure used to prepare 11a, reaction of 3h with
10 provided 11h in 10.0% yield as an orange solid, mp: 140–142°C.
1H NMR (500 MHz, DMSO-d6) δ: 11.01 (s, 1H), 8.13 (dd, J = 4.6, 1.5 Hz,
1H), 8.02 (s, 1H), 7.70 (s, 1H), 7.67 (s, 1H), 7.23 (s, 1H), 7.02 (dd, J = 8.0,
1.6 Hz, 1H), 6.93 (s, 1H), 6.77 (d, J = 6.8 Hz, 1H), 6.73–6.65 (m, 2H),
6.61–6.54 (m, 1H), 4.38 (t, J = 7.5 Hz, 2H), 4.03 (t, J = 7.0 Hz, 2H), 3.87
(s, 3H), 2.26–2.16 (m, 2H), 1.24 (s, 3H). HRMS (ESI) m/z [M+H]+ for
According to the procedure used to prepare 11a, reaction of 3d
with 10 provided 11d in 16.9% yield as a red solid, mp: 113–115°C.
1H NMR (500 MHz, DMSO-d6) δ: 11.07 (s,1H), 8.15 (dd, J = 4.5,
1.5 Hz, 1H), 8.05 (s, 1H), 7.93 (s, 1H), 7.81 (s, 1H), 7.49
(d, J = 8.5 Hz, 1H), 7.30 (s, 1H), 7.11–7.07 (m, 2H), 6.99 (d,
J = 7.0 Hz, 1H), 6.88 (d, J = 2,0 Hz, 1H), 6.73 (dd, J = 8.0, 4.7 Hz, 1H),
4.23 (t, J = 7.0 Hz, 2H), 3.98 (t, J = 7.0 Hz, 2H), 3.90 (s, 3H), 2.24–
2.18 (m, 2H). HRMS (ESI) m/z [M+H]+ for C26H22ClN6O2 calcd.
485.1487. Found 485.1478.
C27H25N6O2 calcd. 465.2034. Found 465.2042.
3-(1-(3-(1H-Imidazol-1-yl)propyl)-5-(benzyloxy)-1H-indol-3-yl)-
4-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-
dione (11i)
According to the procedure used to prepare 11a, reaction of 3i with 10
provided 11i in 21.1% yield as an orange solid, mp: 121–123°C.
1H NMR (500 MHz, DMSO-d6) δ: 11.02 (s, 1H), 8.18 (dd, J = 4.5, 1.5 Hz,
1H), 7.96 (s, 1H), 7.91 (s, 1H), 7.64 (s, 1H), 7.37–7.28 (m, 4H), 7.23–
7.19 (m, 2H), 7.16 (d, J = 7.0 Hz, 2H), 6.93 (s, 1H), 6.75 (dd, J = 8.0,
4.6 Hz, 1H), 6.71 (dd, J = 9.0, 2.5 Hz, 1H), 6.17 (d, J = 2.5 Hz, 1H), 4.22
(t, J = 7.0 Hz, 2H), 4.16 (s, 2H), 3.98 (t, J = 7.0 Hz, 2H), 3.86 (s, 3H),
2.30–2.22 (m, 2H). HRMS (ESI) m/z [M+H]+ for C33H29N6O3 calcd.
557.2296. Found 557.2292.
3-(1-(3-(1H-Imidazol-1-yl)propyl)-6-chloro-1H-indol-3-yl)-4-(1-
methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-dione
(11e)
According to the procedure used to prepare 11a, reaction of 3e with 10
provided 11e in 17.5% yield as a red solid, mp: 144–146°C. 1H NMR
(500 MHz, DMSO-d6) δ: 11.07 (s, 1H), 8.14 (dd, J = 4.5, 1.5 Hz, 1H),
8.08 (s, 1H), 7.79 (s, 1H), 7.63 (s, 1H), 7.61 (d, J = 1.5 Hz, 1H), 7.20
(s, 1H), 6.96 (dd, J = 8.0, 1.5 Hz, 1H), 6.93 (s, 1H), 6.87 (d, J = 8.5 Hz,
1H), 6.76 (dd, J = 8.5, 1.5 Hz, 1H), 6.71 (dd, J = 8.0, 4.6 Hz, 1H), 4.22
(t, J = 7.0 Hz, 2H), 3.96 (t, J = 7.0 Hz, 2H), 3.89 (s, 3H), 2.23–2.18
(m, 2H). HRMS (ESI) m/z [M+H]+ for C26H22ClN6O2 calcd. 485.1487.
Found 485.1490.
3-(1-(4-(1H-Imidazol-1-yl)butyl)-1H-indol-3-yl)-4-(1-methyl-1H-
pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-dione (11j)
According to the procedure used to prepare 11a, reaction of 3j with 10
provided 11j in 26.1% yield as an orange solid, mp: 133–135°C.
1H NMR (500 MHz, DMSO-d6) δ: 11.01 (s, 1H), 8.14–8.10 (m, 2H), 8.04
(s, 1H), 7.82 (s, 1H), 7.50 (d, J = 8.3 Hz, 1H), 7.34 (s, 1H), 7.16 (s, 1H),
7.09–7.01 (m, 1H), 6.95 (dd, J = 7.9, 1.6 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H),
6.70 (t, J = 7.6 Hz, 1H), 6.61 (dd, J = 8.0, 4.6 Hz, 1H), 4.29 (t, J = 6.1 Hz,
2H), 4.06 (t, J = 6.0 Hz, 2H), 3.88 (s, 3H), 1.74–1.67 (m, 4H). HRMS (ESI)
m/z [M+H]+ for C27H25N6O2 calcd. 465.2034. Found 465.2051.
3-(1-(3-(1H-Imidazol-1-yl)propyl)-5-bromo-1H-indol-3-yl)-4-(1-
methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-dione
(11f)
According to the procedure used to prepare 11a, reaction of 3f with 10
provided 11f in 14.3% yield as a red solid, mp: 135–137°C. 1H NMR
(500 MHz, DMSO-d6) δ: 11.07 (s, 1H), 8.16 (dd, J = 4.5, 1.5 Hz 1H),
8.10 (s, 1H), 8.03 (s, 1H), 7.81 (s, 1H), 7.45 (d, J = 8.5 Hz, 1H), 7.37 (s,
1H), 7.21–7.15 (m, 2H), 7.02–6.98 (m, 2H), 6.73 (dd, J = 8.0, 4.6 Hz,
1H), 4.24 (t, J = 7.0 Hz, 2H), 4.02 (t, J = 7.0 Hz, 2H), 3.90 (s, 3H), 2.25–
2.20 (m, 2H). HRMS (ESI) m/z [M+H]+ for C26H22BrN6O2 calcd.
529.0982. Found 529.0971.
3-(1-(4-(1H-Imidazol-1-yl)butyl)-6-chloro-1H-indol-3-yl)-4-(1-
methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-dione
(11k)
According to the procedure used to prepare 11a, reaction of 3k with 10
provided 11j in 24.7% yield as a red solid, mp: 129–131°C. 1H NMR
(500 MHz, DMSO-d6) δ: 11.04 (s, 1H), 8.15 (dd, J = 4.7, 1.6 Hz, 1H),
8.08 (s, 1H), 7.81 (s, 1H), 7.68 (d, J = 1.8 Hz, 1H), 7.61 (s, 1H), 7.14
(s, 1H), 6.90–6.87 (m, 2H), 6.84 (d, J = 8.6 Hz, 1H), 6.74 (t, J = 8.5 Hz,
1H), 6.64 (dd, J = 8.0, 4.6 Hz, 1H), 4.29 (t, J = 6.1 Hz, 2H), 4.06
(t, J = 6.0 Hz, 2H), 3.89 (s, 3H), 1.73–1.61 (m, 4H). HRMS (ESI) m/z
[M+H]+ for C27H24ClN6O2 calcd. 499.1644. Found 499.1631.
3-(1-(3-(1H-Imidazol-1-yl)propyl)-6-bromo-1H-indol-3-yl)-4-(1-
methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-dione
(11g)
According to the procedure used to prepare 11a, reaction of 3g with
10 provided 11g in 12.9% yield as a red solid, mp: 204–206°C.
1H NMR (500 MHz, DMSO-d6) δ: 11.06 (s, 1H), 8.14 (dd, J = 4.5,
1.5 Hz, 1H), 8.08 (s, 1H), 7.78 (s, 1H), 7.75 (d, J = 1.5 Hz, 1H), 7.67
(s, 1H), 7.22 (s, 1H), 6.99–6.93 (m, 2H), 6.88 (dd, J = 8.5, 1.5 Hz, 1H),
6.82 (d, J = 8.5 Hz, 1H), 6.71 (dd, J = 8.0, 4.7 Hz, 1H), 4.23
(t, J = 7.0 Hz, 2H), 3.97 (t, J = 7.0 Hz, 2H), 3.89 (s, 3H), 2.24–2.18
(m, 2H). HRMS (ESI) m/z [M+H]+ for C26H22BrN6O2 calcd. 529.0982.
Found 529.0994.
3-(1-(2-(1H-Imidazol-1-yl)ethyl)-1H-indol-3-yl)-4-(1-methyl-1H-
pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-dione (11l)
According to the procedure used to prepare 11a, reaction of 3l with 10
provided 11l in 14.3% yield as a red solid, mp: 150–152°C. 1H NMR
(500 MHz, DMSO-d6) δ 11.02 (s, 1H), 8.16–8.10 (m, 1H), 8.04 (s, 1H),

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7.68 (s, 1H), 7.47–7.41 (m, 2H), 7.19 (s, 1H), 7.01–6.90 (m, 2H), 6.88
(s, 1H), 6.76 (dd, J = 8.0, 4.6 Hz, 1H), 6.65–6.55 (m, 2H), 4.68
(t, J = 5.9 Hz, 2H), 4.47 (t, J = 5.9 Hz, 2H), 3.90 (s, 3H). HRMS (ESI)
m/z [M+H]+ for C25H21N6O2 calcd. 437.1721. Found 437.1736.
(500 MHz, DMSO-d6) δ: 11.75 (s, 1H), 10.98 (s, 1H), 8.14 (dd, J = 4.5,
1.5 Hz, 1H), 7.99 (s, 1H), 7.80 (d, J = 3.0 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H),
7.05 (dd, J = 8.0, 1.5 Hz, 1H), 6.99 (t, J = 7.5 Hz, 1H), 6.74 (d, J = 8.0 Hz,
1H), 6.71 (dd, J = 8.0, 4.6 Hz, 1H), 6.64 (t, J = 7.5 Hz, 1H), 3.87 (s, 3H).
HRMS (ESI) m/z [M+H]+ for C20H15N4O2 calcd. 343.1190. Found
343.1189.
3-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-(1-(3-(pyrrolidin-
1-yl)propyl)-1H-indol-3-yl)-1H-pyrrole-2,5-dione (11m)
According to the procedure used to prepare 11a, reaction of 3m with
10 provided 11m in 12.3% yield as a red solid, mp: >250°C. 1H NMR
(500 MHz, DMSO-d6) δ: 11.03 (s, 1H), 8.15 (dd, J = 4.5, 1.5 Hz, 1H),
8.04 (s, 1H), 7.84 (s, 1H), 7.56 (d, J = 7.5 Hz, 1H), 7.07 (t, J = 7.0 Hz, 1H),
6.99 (dd, J = 8.0, 1.5 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.74–6.69 (m, 2H),
4.35 (t, J = 7.0 Hz, 2H), 3.88 (s, 3H), 3.10–3.01 (m, 6H), 2.13–2.03 (m,
2H), 1.90–1.80 (m, 4H). HRMS (ESI) m/z [M+H]+ for C27H28N5O2 calcd.
454.2238. Found 454.2245.
3-(1-(3-(1H-Imidazol-1-yl)propyl)-1H-indol-3-yl)-4-(1H-
pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-dione (11r)
According to the procedure used to prepare 11a, reaction of 6a with 12
provided 11r in 12.3% yield as a red solid, mp: 107–109°C. 1H NMR
(500 MHz, DMSO-d6) δ: 12.24 (s 1H), 11.02 (s, 1H), 8.62 (s, 1H), 8.08
(dd, J = 4.5, 1.5 Hz, 1H), 7.86–7.81 (m, 2H), 7.59 (s, 1H), 7.47
(d, J = 8.2 Hz, 1H), 7.43 (s, 1H), 7.14 (d, J = 7.9 Hz, 1H), 7.06
(t, J = 7.6 Hz, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.74–6.66 (m, 2H), 4.31
(t, J = 7.2 Hz, 2H), 4.15 (t, J = 7.2 Hz, 2H), 2.38–2.28 (m, 2H). HRMS
(ESI) m/z [M+H]+ for C25H21N6O2 calcd. 437.1721. Found 437.1733.
3-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-(1-(3-
morpholinopropyl)-1H-indol-3-yl)-1H-pyrrole-2,5-dione (11n)
According to the procedure used to prepare 11a, reaction of 3n with
10 provided 11n in 16.1% yield as a red solid, mp: 86–88°C. 1H NMR
(500 MHz, DMSO-d6) δ: 11.00 (s, 1H), 8.14 (dd, J = 4.5, 1.5 Hz, 1H),
8.03 (s, 1H), 7.81 (s, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.04 (t, J = 8.0 Hz, 1H),
6.98 (dd, J = 8.0, 1.5 Hz, 1H), 6.81 (d, J = 8.0 Hz, 1H), 6.71–6.63 (m, 2H),
4.30 (t, J = 6.5 Hz, 2H), 3.88 (s, 3H), 3.63–3.54 (t, J = 4.5 Hz, 4H),
2.4–2.23 (m, 4H), 2.15 (t, J = 6.5 Hz, 2H), 1.92–1.85 (m, 2H). HRMS
(ESI) m/z [M+H]+ for C27H28N5O3 calcd. 470.2187. Found 470.2196.
3-(1-(3-(1H-Imidazol-1-yl)propyl)-1H-indol-3-yl)-4-(1-ethyl-1H-
pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-dione (11s)
According to the procedure used to prepare 11a, reaction of 6b with
12 provided 11s in 13.8% yield as a red solid, mp: 199–201°C. 1H NMR
(500 MHz, DMSO-d6) δ: 11.03 (s, 1H), 8.14 (dd, J = 4.5, 1.5 Hz, 1H),
7.94 (s, 1H), 7.84 (s, 1H), 7.75 (s, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.26 (s,
1H), 7.21 (d, J = 8.0 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 6.99 (s, 1H),
6.77–6.67 (m, 3H), 4.32 (q, J = 7.2 Hz, 2H), 4.25 (t, J = 7.0 Hz, 2H), 4.00
(t, J = 7.0 Hz, 2H), 2.29–2.24 (m, 2H), 1.33 (t, J = 7.2 Hz, 3H). HRMS
(ESI) m/z [M+H]+ for C27H25N6O2 calcd. 465.2034. Found 465.2051.
3-(1-(3-(1H-1,2,4-Triazol-1-yl)propyl)-1H-indol-3-yl)-4-(1-methyl-
1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-dione (11o)
According to the procedure used to prepare 11a, reaction of 3o with
10 provided 11o in 30.3% yield as a red solid, mp: 121–123°C. 1H NMR
(500 MHz, DMSO-d6) δ: 11.00 (s, 1H), 8.51 (s, 1H), 8.11 (dd, J = 4.5,
1.5 Hz, 1H), 8.02 (s, 1H), 8.00 (s, 1H), 7.83 (s, 1H), 7.45 (d, J = 8.5 Hz,
1H), 7.06 (t, J = 7.5 Hz, 1H), 7.01 (dd, J = 8.0, 1.5 Hz, 1H), 6.84
(d, J = 8.0 Hz, 1H), 6.74–6.66 (m, 2H), 4.30 (t, J = 7.0 Hz, 2H), 4.17
(t, J = 7.0 Hz, 2H), 3.87 (s, 3H), 2.32–2.25 (m, 2H). HRMS (ESI)
m/z [M+H]+ for C25H22N7O2 calcd. 452.1829. Found 452.1844.
3-(1-(3-(1H-Imidazol-1-yl)propyl)-1H-indol-3-yl)-4-(1-isopropyl-
1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-dione (11t)
According to the procedure used to prepare 11a, reaction of 6c with 12
provided 11t in 11.8% yield as a red solid, mp: 165–167°C. 1H NMR
(500 MHz, DMSO-d6) δ: 11.04 (s, 1H), 8.16 (dd, J = 4.5, 1.5 Hz, 1H),
7.87 (s, 1H), 7.86 (s, 1H), 7.77 (s, 1H), 7.44 (d, J = 8.5 Hz, 1H), 7.36 (dd,
J = 8.0, 1.5 Hz, 1H), 7.27 (s, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.00 (s, 1H), δ
6.84–6.78 (m, 1H), 6.68 (t, J = 7.5 Hz, 1H), 6.64 (d, J = 8.0 Hz, 1H),
5.11–5.04 (m, 1H), 4.26 (t, J = 7.0 Hz, 2H), 4.01 (t, J = 7.0 Hz, 2H),
2.31–2.23 (m, 2H), 1.39 (d, J = 6.5 Hz, 6H). HRMS (ESI) m/z [M+H]+ for
3-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-(1-(3-(piperidin-1-
yl)propyl)-1H-indol-3-yl)-1H-pyrrole-2,5-dione (11p)
C28H27N6O2 calcd. 479.2190. Found 479.2173.
According to the procedure used to prepare 11a, reaction of 3p with 10
provided 11p in 15.4% yield as a red solid, mp: 206–207°C. 1H NMR
(500 MHz, DMSO-d6) δ: 11.00 (s, 1H), 8.14 (dd, J = 4.5, 1.5 Hz, 1H), 8.03
(s, 1H), 7.79 (s, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.04 (t, J = 7.5 Hz, 1H), 6.97
(dd, J = 8.0, 1.5 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.71–6.63 (m, 2H), 4.27
(t, J = 6.5 Hz, 2H), 3.88 (s, 3H), 2.29–2.18 (m, 4H), 2.11 (t, J = 7.0 Hz, 2H),
1.91–1.80 (m, 2H), 1.52–1.47 (m, 4H), 145–1.32 (m, 2H). HRMS
(ESI) m/z [M+H]+ for C28H30N5O2 calcd. 468.2394. Found 468.2382.
|
4.2 Biological activity assays
|
4.2.1 IDH1/R132H inhibition assay
Full-length human IDH1/R132H was expressed as N-terminal GST-
fusion protein using Escherichia coli expression system. Enzyme
inhibition assay for IDH1(R132H) was performed in a 384-well
microplate in 25 mM Tris-HCl buffer (pH 7.0) containing the enzyme
(27 nM), 8 mM MnCl2, 12 µM NADPH, and increasing concentrations
of an inhibitor. Upon incubation for 15 min, α-KG (1 mM) was added to
initiate the reaction. The NADPH of probe was detected by monitoring
the increase of fluorescence with Envision, at 355 nm excitation and
3-(1H-Indol-3-yl)-4-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-
1H-pyrrole-2,5-dione (11q)
According to the procedure used to prepare 11a, reaction of 2a with 10
provided 11q in 9.1% yield as a red solid, mp: >250°C. 1H NMR

HU ET AL.
11 of 12
|
460 nm emission (PerkinElmer). The IC50 data were calculated using
the software GraphPad Prism and the equation “sigmoidal dose-
response (variable slope)” was chosen for curve fitting.
China (2014M550256), the State Key Laboratory of Drug Research
(SIMM1601KF-04), National Natural Science Foundation of China
(8150131067), “Personalized Medicines-Molecular Signature-based
Drug Discovery and Development,” Strategic Priority Research
Program of the Chinese Academy of Sciences (XDA12020328).
|
4.2.2 IDH1/WT inhibition assay
Full-length human IDH1/WT was expressed as N-terminal GST-fusion
protein using Escherichia coli expression system. The inhibition assay of
IDH1/WT was carried out in 25 mM Tris-HCl buffer (pH 7.0) containing
the enzyme (30 nM), 1 mM MgCl2, 75 µM NADP, an inhibitor, and
100 µM sodium (D)-isocitrate. And the NADPH of probe was detected
by monitoring the increase of fluorescence with Envision (PerkinElmer),
at 355 nm excitation and 460 nm emission. The IC50 data were
calculated using the software GraphPad Prism and the equation
“sigmoidal dose-response (variable slope)” was chosen for curve fitting.
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
ORCID
Qing Ye
http://orcid.org/0000-0002-8487-2118
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How to cite this article: Hu Y, Gao A, Liao H, et al.
3-(7-Azaindolyl)-4-indolylmaleimides as a novel class of
mutant isocitrate dehydrogenase-1 inhibitors: Design,
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