Design, synthesis and biological activity of 3-pyrazine-2-yl-oxazolidin-2-ones as novel, potent and selective inhibitors of mutant isocitrate dehydrogenase 1

Article abstract of DOI:10.1016/j.bmc.2017.10.009

Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitrate to alpha-ketoglutarate (α-KG) generating carbon dioxide and NADPH/NADH. Evidence suggests that the specific mutations in IDH1 are critical to the growth and reproduction of some tumor cells such as gliomas and acute myeloid leukemia, emerging as an attractive antitumor target. In order to discovery potent new mutant IDH1 inhibitors, we designed, synthesized and evaluated a series of allosteric mIDH1 inhibitors harboring the scaffold of 3-pyrazine-2-yl-oxazolidin-2-ones. All tested compounds effectively suppress the D-2-hydroxyglutarate (D-2-HG) production in cells transfected with IDH1-R132H and IDH1-R132C mutations at 10 μM and 50 μM. Importantly, compound 3g owns the similar inhibitory activity to the positive agent NI-1 and shows no significant toxicity at the two concentrations. The parallel artificial membrane permeation assay of the blood–brain barrier (PAMPA-BBB) identified 3g with a good ability to penetrate the blood–brain barrier (BBB). These findings indicate that 3g deserves further optimization as a lead compound for the treatment of patients with IDH1 mutated brain cancers.

Full text of DOI:10.1016/j.bmc.2017.10.009

Accepted Manuscript
Design, Synthesis and Biological Activity of 3–Pyrazine-4-yl-oxazolidin-2-
ones as Novel, Potent and Selective Inhibitors of Mutant Isocitrate Dehydro-
genase 1
Tianfang Ma, Fangxia Zou, Stefan Pusch, Lijun Yang, Qihua Zhu, Yungen Xu,
Yueqing Gu, Andreas von Deimling, Xiaoming Zha
PII:
S0968-0896(17)31543-2
https://doi.org/10.1016/j.bmc.2017.10.009
BMC 14014
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 July 2017
8 October 2017
12 October 2017
Please cite this article as: Ma, T., Zou, F., Pusch, S., Yang, L., Zhu, Q., Xu, Y., Gu, Y., von Deimling, A., Zha, X.,
Design, Synthesis and Biological Activity of 3–Pyrazine-4-yl-oxazolidin-2-ones as Novel, Potent and Selective
Inhibitors of Mutant Isocitrate Dehydrogenase 1, Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/
10.1016/j.bmc.2017.10.009
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design, Synthesis and Biological Activity of
3-Pyrazine-4-yl-oxazolidin-2-ones as Novel, Potent and
Selective Inhibitors of Mutant Isocitrate Dehydrogenase
1
Tianfang Ma a, b, #, Fangxia Zou a, b, #, Stefan Pusch c, d, #, Lijun Yang a, b, Qihua Zhub, Yungen Xu b,
Yueqing Gu a, Andreas von Deimling c, d,*, Xiaoming Zha a, b,*
a
Department of Pharmaceutical Engineering & Department of Biochemical Engineering, China
Pharmaceutical University, 639 Longmian Avenue, Nanjing 211198, China.
b
Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing
210009, PR China.
c
German Consortium of Translational Cancer Research (DKTK), Clinical Cooperation Unit
Neuropathology, German Cancer Research Center (DKFZ), INF 280, Heidelberg D-69120,
Germany.
d
Department of Neuropathology, Institute of Pathology, INF 224, Ruprecht-Karls-University
Heidelberg, Heidelberg D-69120, Germany.
ABSTRACT: Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitrate to
alpha-ketoglutarate (α-KG) generating carbon dioxide and NADPH/NADH. Evidence suggests that the
specific mutations in IDH1 are critical to the growth and reproduction of some tumor cells such as
gliomas and acute myeloid leukemia, emerging as an attractive antitumor target. In order to discovery
potent new mutant IDH1 inhibitors, we designed, synthesized and evaluated a series of allosteric
mIDH1 inhibitors harboring the scaffold of 3-pyrazine-4-yl-oxazolidin-2-ones. All tested compounds
1

effectively suppress the D-2-hydroxyglutarate (D-2-HG) production in cells transfected with
IDH1-R132H and IDH1-R132C mutations at 10 µM and 50 µM. Importantly, compound 3g owns the
similar inhibitory activity to the positive agent NI-1 and shows no significant toxicity at the two
concentrations. The parallel artificial membrane permeation assay of the blood-brain barrier
(PAMPA-BBB) identified 3g with a good ability to penetrate the blood-brain barrier (BBB). These
findings indicate that 3g deserves further optimization as a lead compound for the treatment of patients
with IDH1 mutated brain cancers.
Key words: Mutant IDH1; inhibitors; D-2-HG; allosteric; BBB.
1. Introduction
Isocitrate dehydrogenases (IDHs), a key enzyme family associated with the tricarboxylic acid
(TCA), catalyze oxidative decarboxylation of isocitrate acid to α-ketoglutaric (α-KG) using divalent
magnesium ion and NADP+ (or NAD+) as cofactors, which is critical to biosynthesis and energy
metabolism [1]. Isocitrate dehydrogenase 1 (IDH1), one of the IDHs, locates in the cytosol and
peroxisome, while IDH2 and IDH3 in mitochondria. IDH1 is the only NADPH producer in the
biosynthesis and the thiolated antioxidant system and plays a vital role in the process of adipogenesis
and phospholipid metabolism [2].
Recent studies revealed that IDH1 is a metabolic enzyme associated with the progression of cancers,
and mutant IDH1 (mIDH1) primarily exists in gliomas, acute myeloid leukemia and other solid tumors
[3,4]. Substantial data suggests that more than 70% low-grade gliomas and up to 20% secondary
glioblastoma multiform are related to IDH1 mutations [5]. In addition, mutant IDH1 is also found in
about 10% of AML cases and 10% of cholangiocarcinomas, as well as melanomas and chondrosarcomas
2

[5]. The most common mutation in IDH1 is a key amino acid residue Arg132 located in the active site,
in which R132H is the predominant [6].
Mutations in IDH1 are heterozygous missense mutations which lead to an arginine at amino acid
132 replaced by different amino acid residues, including R132H, R132C, R132L, R132G, R132S and
R132W [2]. mIDH1 exhibits a neomorphic catalytic activity to catalyze the conversion of α-KG to
D-2-hydroxyglutarate (D-2-HG) [7]. Comparably, IDH1 mutated cells show higher concentrations of
D-2-HG than normal cells, which can be as a significant evaluation of certain tumors. D-2-HG, an
oncometabolite related to tumorigenesis, induces hypermethylation of histone and chromatin and blocks
cell differentiation through competitive inhibition with relevant α-KG-dependent dioxygenases, such as
methylases and histone demethylases [8,9]. Therefore, high level of D-2-HG from IDH1 mutations are
sufficient to promote the initiation and progression of cancers, such as gliomas and acute myeloid
leukemia [10]. Taken these findings into consideration, it is demonstrated that potent drugs targeting
mutant IDH1 should be developed for the treatment of IDH1 mutated tumors, including brain tumors.
To date, a number of mutant IDH1 inhibitors have been reported and only IDH-305 (structure not
disclosed), FT-2102 (structure not disclosed), BAY-1436032 and AG-120 have entered into clinical
trials, of which AG-120 in Phase III clinical trial [11,12]. According to the binding modes, the active
sites of mIDH1 inhibitors could be classified as four types: substrate-based pocket, Seg-2 allosteric
pocket, Mg2+ pocket and other type. SYC-435 (1), a representative agent based on substrate-based
pocket (Ki = 120 nM), reveals a significant selectivity for mIDH1 over WT-IDH1 [13]. GSK321 (2),
binding to the Seg-2 allosteric pocket and locking the mutant proteins into a catalytically inactive “open”
conformation to prevent mIDH1 turnover, inhibits the D-2-HG production and the growth and
differentiation of mIDH1 cells [14,15]. VVS (3), with IC50 values of 11 nM against IDH1-R132H and
3

259 nM against IDH1-R132C, is noncompetitive with NADPH and can selectively inhibit mIDH1 via
direct interaction with Asp279 in the metal-binding pocket [16]. AGI-5198 (4), developed by Agios
Pharmaceuticals, is a potent and selective mIDH1 inhibitor that reduces levels of D-2-HG in tumor cells
with the IC50 values of 70 nM and 160 nM against IDH1-R132H and IDH1-R132C, respectively[17,18].
AG-120 (5, IC50 = 8 nM) is under a Phase III clinical trial and early evidence suggested it is well
tolerated with majority of mild to moderate adverse events [11,12]. BAY-1436032 (6) is a novel
pan-inhibitor of IDH1 with different codon 132 mutations [19]. Recently, it has been entered into Phase
I to evaluate its safety, tolerability, pharmacokinetics, and pharmacodynamics in patients with
IDH1-R132 mutated tumors [20,21]. BRD-2879 (7) and 8, discovered by high-throughput screen (HTS)
and further optimization, are potent and selective mIDH1 inhibitors [22,23]. Notably, Novartis reported
a novel class of mIDH1 inhibitors with 3-pyrimidin-4-yl-oxazolidin-2-ones motif, such as NI-1 (9, IC50
= 0.094 µM, IDH1-R132H) [24]. In this article, to obtain more active and drug-like compounds with
new scaffolds, we used NI-1 as
a
template compound and designed
a
series of
3-pyrazine-4-yl-oxazolidin-2-ones. Interestingly, during the period of biological evaluation of our target
compounds, IDH889 (10, IC50 = 20 nM), an analog of NI-1, was reported its co-crystal complex with
IDH1-132H [25]. The disclosed structures of 1-10 are presented (Fig. 1).
4

Fig. 1. Representative structures of reported mIDH1 inhibitors.
Based on the analysis of these findings, we successfully designed and synthesized a series of
3-pyrazine-4-yl-oxazolidin-2-ones allosteric mIDH1 inhibitors. All compounds display inhibitory
activity against D-2-HG production in tumor cells harboring IDH1-R132H and IDH1-R132C at 10 µM
and 50 µM. The most active one 3g displays weak inhibition against WT-IDH1, indicating its good
selectivity for mIDH1 inhibition. Molecular docking was also studied to predict the binding mode of 3g
at the allosteric site of mIDH1. Importantly, 3g can effectively cross BBB in PAMPA model. Taken
together, our work identified 3-pyrazine-4-yl-oxazolidin-2-ones as a novel class of selective and potent
allosteric mIDH1 inhibitors, and 3g deserves further optimization as an antitumor agent to treat patients
with IDH1 mutated brain cancers.
5

2. Results and Discussion
2.1. Chemistry.
The general route for the synthesis of the target compounds 3a-3m is depicted in Schemes 1. It
started from 2,6-dichloropyrazine, selectively substituted by (S)-4-isopropyloxazolidin-2-one at an ice
bath to afford the intermediate I. (S)-1-(4-bromophenyl)ethan-1-amine was reacted with 1a-1m under
Pd[P(Ph)3]4/K2CO3 at 80°C to afford the intermediates 2a-2m. Then I was reacted with 2a-2m using
tris(dibenzylideneacetone)dipalladium and BINAP as the catalysts in anhydrous toluene at 90°C under
nitrogen to give the target compounds 3a-3m.
Scheme 1. Synthesis of target compounds 3a-3m. Reagents and conditions: (i)
(S)-4-isopropyloxazolidin-2-one, NaH, DMF, ice bath, 2h; (ii) (S)-1-(4-bromophenyl)ethan-1-amine,
Pd[P(Ph)3]4, 2N K2CO3 solution, MeCN, reflux, overnight; (iii) tris(dibenzylideneacetone)dipalladium,
BINAP, K2CO3, toluene, reflux, overnight.
2.2. Biological Evaluation
Effects of 3a-3m on D-2-HG production in HEK-293T cells expressing IDH1-R132H and
IDH1-R132C. First, we employed HEK-293T cell lines expressing IDH1-R132H and IDH1-R132C to
6

test the inhibitory effects of 3a-3m at two concerntrations (10 µM and 50 µM). NI-1, a known mIDH1
inhibitor, was chosen as a positive control. Then the D-2-HG concentration in the cells was measured
after 3 days of incubation (Table 1). To our surprise, all the tested compounds could inhibit the D-2-HG
production in cells carrying IDH1-R132H or IDH1-R132C at 50 µM (Fig. 2). 3b, 3g, 3j and 3k showed
relatively strong inhibitory effects at both 10 µM and 50 µM. However, 3c, 3d, 3f, 3h and 3l exhibited
relatively weak inhibitory effects on the production of D-2-HG at 10 µM. As with NI-1, 3g and 3k
strongerly reduced the D-2-HG levels in the cells than other tested compounds at the two
concentrations, demostrating that the compounds with 3-pyrazine-4-yl-oxazolidin-2-ones have a bit
better inhibitory effects on mutant IDH1 and deserve further optimization.
HEK-293T (IDH-R132C)
150
10
M
50
M
100
50
0
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m NI-1
Compound code
Fig. 2. The inhibitory activities of 3a-3m and NI-1 against IDH1-R132H and IDH1-R132C
7

Table 1. Structures and LogPs of 3a-3m and their inhibitory effect at 10 µM and 50 µM in HEK-293T
cells transfected with IDH1-R132H and IDH1-R132C.
D2HG levels
(%, IDH1 R132H)
D2HG levels
(%, IDH1 R132C)
LogPa
Compd
R
10 µM
50 µM
10 µM
50 µM
Ph
4-F-Ph
50.0
34.3
97.2
78.4
47.7
59.8
31.9
70.5
49.7
39.2
28.0
85.4
47.9
33.9
38.8
33.0
34.7
28.0
31.9
33.9
24.6
38.7
28.5
28.3
30.7
39.5
21.5
24.7
50.4
39.0
115.5
89.9
51.3
86.0
40.6
74.9
51.1
44.6
39.9
71.3
54.1
/
108.0
51.3
50.6
44.1
51.7
41.7
28.2
54.8
29.7
29.1
27.4
73.9
30.2
29.7
4.38
4.39
4.37
4.48
4.43
4.41
4.40
4.63
4.62
4.61
5.01
5.27
4.25
3.71
3a
3b
3c
3-F-Ph
2-F-Ph
3d
3e
4-OMe-Ph
3-OMe-Ph
2-OMe-Ph
4-Me-Ph
3f
3g
3h
3i
3-Me-Ph
2-Me-Ph
3j
4-CF3-Ph
2-naphthyl
4-dimethylisoxazole
2-pyridine
3k
3l
3m
NI-1b
a LogP values were predicted at http://www.vcclab.org/.
b NI-1 as a positive control.
8

Cytotoxicity towards HEK-293T WT-IDH1. Owning weak or no effects on WT-IDH1 is essential
for an ideal mIDH1 inhibitor. Thus, all the compounds were tested to verify the selectivity of mIDH1
over WT-IDH1. Unexpectedly, 3b, 3k and 3m exhibited relatively strong toxicity compared to other
tested compounds at 50 µM. However, 3a and 3h were found to retain a better selectivity between
mIDH1 and wt-IDH1 at 10 µM and 50 µM. Importantly, 3g had a controlled toxicity with the values of
89.8% at 10 µM and 74.5% at 50 µM. All the compounds showed no significant toxicity at 10 µM (Fig.
3).
Fig. 3. Cytotoxicity of all tested compounds at 10 µM and 50 µM in HEK-293T WT-IDH1 cells.
PAMPA-BBB assays. Blood-brain barrier (BBB) permeability is essential for successful central
nervous system (CNS) drugs. To measure the possible BBB permeability of 3g in vivo, the parallel
artificial membrane permeation assay of the blood-brain barrier (PAMPA-BBB) was performed. On the
basis of the measured permeability, 3g could cross the BBB (Table 2).
Table 2. Permeability (Pe, ×10-6 cm/s) results in the PAMPA-BBB assay for 3g and its predictive
penetration in the CNS.
9

Compd
Pe (ꢀ10-6 cm/s) a
Prediction b
6.65±0.42
8.82±0.29
0.49±0.15
CNS+
CNS+
CNS-
3g
Desipramine
Atenolol
a Values are expressed as the mean SD of three independent experiments. b Standards of test results are
described as follows: i) CNS+ (high BBB permeation predicted): Pe > 4.0; ii) CNS- (low BBB
permeation predicted): Pe < 2.0; iii) CNS+/- (BBB permeation uncertain): Pe = 2.0 ~ 4.0.
2.3. The preliminary structure-activity relationship (SAR) analysis.
As shown in Table 1, when R group is the phenyl ring, different substituents are introduced in all
positions of R, which has a significant effect on the inhibitory activities of the compounds. For the
para-positon, 3b (with -F) and 3k (with –CF3) perform much better, while for meta- and ortho-position,
3g (with -OCH3) and 3i-3j (with -CH3) have better inhibitory activities, suggesting that
electon-withdrawing groups in the para-positon or electron-donating substituents in the meta- and
ortho-position are more favorable for R. For the effects of R group’s size on the activity, 3m and 3a
exhibits relatively stronger reduction of D-2-HG levels compared to 3l, indicating that smaller groups
are more suitable for R, but larger groups are limited in this cavity.
2.4. Docking analysis of potential binding modes of 3g and NI-1.
To understand the binding mode of the most active compound 3g in this newly reported allosteric
pocket, optimal molecular docking (CDOCKER, Discovery Studio 3.0) was performed. The C2-amine
group of 3g forms a donor-acceptor polar interaction with Ile128 and a hydrogen bond interaction is
observed between the carbonyl of the oxazolidinone and the amine of Leu120. The alpha-methyl fits
into a specific cavity. In addition, the isopropyl and the phenyl ring of the amine side chain are in a
10

hydrophobic cavity. From the superimposed complex conformation between 3g and NI-1, we find that
the molecular streching direction of 3g and NI-1 is identical. However, 4-N of the pyrazine of 3g can’t
interact with Ile128 to form a hydrogen bond. This may be the reason why 3g shows decreased
inhibitory activity compared to NI-1 (Fig. 4).
Fig. 4. Representation of the binding modes of 3g (A) and the molecular overlap of 3g and NI-1 (B)
with mIDH1(PDB code: 5TQH). Key residues in yellow and the hydrogen bonds in dashed red lines.
3. Conclusion
In conclusion, a novel series of 3-pyrazine-4-yl-oxazolidin-2-ones as allosteric mIDH1 inhibitors
were synthesized. Most of the test compounds exhibit selectivity and inhibitory effects on HEK293 cell
lines harboring IDH1-R132H and IDH1-R132C and among which, 3g is the most potent one. The
molecular docking study illustrates its potential allosteric binding mode. Furthermore, 3g also owns the
ability to penetrate BBB which is of great importance for CNS drugs. Our study provides preliminary
data that 3-pyrazine-4-yl-oxazolidin-2-ones may be beneficial to the design of potent and selective
mIDH1 inhibitors, and the encouraging compound 3g deserves further optimization as a lead compound
to find more low-toxic and potent allosteric inhibitors for the IDH1 mutated brain cancers therapy.
11

4. Experimental section
4.1. Chemistry.
All chemicals with the highest purity were obtained from Shanghai Bide Pharmaceutical Technology
Co., Ltd. Organic solvents (reagent grade) were dried over anhydrous CaCl2 or molecular sieves.
Thin-layer chromatography (TLC) was performed on silica gel GF254 plates (10ꢀ10 cm) from Qingdao
Haiyang Chemical Plant (Qingdao, Shandong, China). The spots of reactions were visualized by a ZF-1
type ultraviolet analyzer (λ = 254 nm) purchased from Hangzhou David Science and Education
Instrument Co. Ltd. Column chromatography silica gel with 100-200 or 200-300 mesh was obtained
from Qingdao Ocean Chemical Plant Factory. Flash column chromatography was carried out on 90-150
µm silica gel columns from Qingdao Marine Chemical Inc. Melting points of the targeted compounds
1
13
were determined on XT-4 melting-point facility from Beijing Tech Instrument Corp. H NMR and C
NMR spectra were recorded on 300MHz or 500MHz and 75 MHz Bruker ACF-300 spectrometers at
1
ambient temperature. H NMR and 13C NMR data were indicated with chemical shifts (ppm, δ)
referenced to tetramethylsilane (TMS) as an internal standard, splitting patterns (s, singlet; d, doublet; t,
triplet; m, multiplet; dd, double of doublets, etc.) and the coupling constant (J) in Hz. Mass spectrometry
was performed on an Agilent 1100 Series (Agilent Technologies, Santa Clara, CA, USA), LC/MSD high
performance ion trap mass spectrometer and a Mariner ESI-TOF spectrometer. Reverse phase
chromatography with a Shimadzu Shim-Pack VP-ODS (150×4.6 mm i.d, 5 µm particle size, Shimadzu
Corp., Kyoto, Japan) column and DAD detection were used to determine the purity of all the new
compounds. All the detailed data including H NMR, 13C NMR, HRMS and HPLC purity is shown in the
supporting information.
12

4.1.1. 3-(6-chloropyrazin-2-yl)-4-isopropyloxazolidin-2-one (I)
To a solution of 2,6-dichloropyrazine (100 mg, 0.671 mmol) and (S)-4-isopropyloxazolidin-2-one
(86.7 mg, 0.671 mmol) in 5 mL DMF was slowly added with a solution of NaH (35 mg, 1.34 mmol) in
1 mL DMF at an ice bath. The mixture was stirred at 0°C for about 2 hours. After the completion of the
reaction monitored by TLC, the mixture was added dropwise to ice water. Then the mixture was
extracted with EtOAc. The combined EtOAc extracts were washed with brine, dried over anhydrous
Na2SO4 and evaporated to afford the white crude product. Purification of the crude product with flash
1
column chromatography (PE : EA = 8:1, v:v) gave 150 mg of I as a white solid, yield 92%. H NMR
(300 MHz, DMSO-d6) δ 9.30 (s, 1H), 8.52 (s, 1H), 4.69-4.67 (m, 1H), 4.49–4.46 (m, 2H), 2.38-2.33 (m,
1H), 0.92–0.78 (dd, J = 6.0 Hz, J = 9.0 Hz, 6H).
4.1.2. (S)-1-([1,1'-biphenyl]-4-yl)ethan-1-amine (2a)
To a solution of 1a (183 mg, 1.50 mmol), (S)-1-(4-bromophenyl)ethan-1-amine (200 mg, 1 mmol),
Pd[P(Ph)3]4 (57.8 mg, 0.05 mmol) and 2N K2CO3 solution (1.00 mL, 2.00 mmol) in anhydrous MeCN
(12 mL) was purged with N2 and refluxed for overnight. After the completion of the reaction monitored
by TLC, the solvent was removed under vacuum and the crude mixture was dissolved by EtOAc. Then
the solution was filtered. The filtrate was washed by water and dried over anhydrous Na2SO4. After
about two hours, the concentrated hydrochloric acid was added dropwise to the solution to adjust pH to
1. The mixture was stirred for 1.5 hours. Then the mixture was washed by water and the combined water
layer was added with 5 mol/L NaOH to adjust pH to 10. Finally, the mixture was extracted with CH2Cl2
and the organic layer was removed under vacuum to give the crude product, which can be used for the
next step without further purification.
4.1.3. (S)-1-(4'-fluoro-[1,1'-biphenyl]-4-yl)ethan-1-amine (2b)
13

1
According to the preparation procedure of 2a, 2b were prepared as a yellow oil, yield 47.2%. H
NMR (300 MHz, DMSO-d6) δ 7.62 (d, J = 7.3 Hz, 2H), 7.55 (t, J = 10.0 Hz, 2H), 7.48-7.37 (m, 4H),
7.32 (t, J = 7.2 Hz, 1H), 4.01 (q, J = 6.4 Hz, 1H), 1.25 (t, J = 7.1 Hz, 3H).
4.1.4. (S)-1-(R)ethan-1-amines (2c-2m)
According to the preparation procedure of 2a, 2c-2m were prepared as a yellow oil.
4.1.5. 3-(6-((1-([1,1'-biphenyl]-4-yl)ethyl)amino)pyrazin-2-yl)-4-isopropyloxazolidin-2-one (3a)
To
a
solution of 2a (144 mg, 0.73 mmol),
I
(126 mg, 0.52 mmol),
tris(dibenzylideneacetone)dipalladium (4.76 mg, 0.0052 mmol), BINAP (13.0 mg, 0.021 mmol ) and
K2CO3 (100 mg, 0.73 mmol) in anhydrous toluene (12 mL) was purged with N2 and heated at 90°C for
overnight. After the completion of the reaction monitored by TLC, the mixture was added with water
and extracted with EtOAc. The EtOAc extracts were washed by saturated brine and dried over
anhydrous Na2SO4. Purification of the crude product with flash column chromatography (PE : EA = 3:1,
1
v:v) gave 3a as a white solid (100 mg, 38.1%). Mp: 175-178°C. H NMR (300 MHz, CDCl3) δ 8.66 (s,
1H), 7.76 (s, 1H), 7.54 (d, J = 6.4 Hz, 4H), 7.48–7.29 (m, 5H), 5.20 (s, 1H), 5.02–4.85 (m, 1H),
4.61–4.48 (m, 1H), 4.32 (t, J = 8.7 Hz, 1H), 4.25–4.13 (m, 1H), 1.87 (s, 1H), 1.62 (d, J = 6.8 Hz, 3H),
0.64 (d, J = 6.3 Hz, 6H). 13C NMR (75 MHz, DMSO-d6) δ 154.35, 152.41, 144.74, 144.66, 140.13,
138.40, 128.83, 127.32, 127.12, 126.62, 126.46, 125.95, 120.76, 62.87, 57.79, 49.50, 26.93, 23.54,
17.58, 13.68. HRMS (m/z) 403.2131 [M + H] +. HPLC purity 98.69 %.
4.1.6. (S)-3-(6-(((S)-1-(4'-fluoro-[1,1'-biphenyl]-4-yl)ethyl)amino)pyrazin-2-yl)-4-isopropyloxazolidin
-2-one (3b)
According to the preparation procedure of 3a, 3b was prepared as a white solid, yield 38.2%. Mp:
174-176°C. 1H NMR (300 MHz, DMSO-d6) δ 8.28 (s, 1H), 7.82–7.72 (m, 2H), 7.68–7.59 (m, 2H), 7.55
14

(d, J = 8.2 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.26 (t, J = 8.8 Hz, 2H), 4.99–4.88 (m, 1H), 4.50 (d, J =
8.7 Hz, 1H), 4.34 (t, J = 8.8 Hz, 1H), 4.23 (d, J = 8.9 Hz, 1H), 1.63 (s, 1H), 1.48 (d, J = 6.9 Hz, 3H),
13
0.56 (d, J = 6.9 Hz, 3H), 0.45 (d, J = 6.8 Hz, 3H). C NMR (75 MHz, DMSO-d6) δ 163.29, 154.32,
152.37, 144.71, 144.64, 137.33, 136.53, 128.40, 128.30, 127.28, 126.54, 125.95, 120.75, 115.73, 115.44,
+
62.84, 57.76, 49.45, 26.91, 23.50, 17.55, 13.66. HRMS (m/z) 421.2036 [M + H] . HPLC purity
99.56%.
4.1.7. (S)-3-(6-(((S)-1-(3'-fluoro-[1,1'-biphenyl]-4-yl)ethyl)amino)pyrazin-2-yl)-4-isopropyloxazolidin
-2-one (3c)
According to the preparation procedure of 3a, 3c was prepared as a white solid, yield 39.2%. Mp:
184-186°C. 1H NMR (300 MHz, DMSO-d6) δ 8.29 (s, 1H), 7.79 (s, 2H), 7.62 (d, J = 8.1 Hz, 2H),
7.49-7.36 (m, 5H), 7.16 (s, 1H), 4.95 (s, 1H), 4.51-4.45 (m, 1H), 4.33 (d, J = 9.0 Hz, 1H), 4.25-4.20 (m,
1H), 1.65-1.60 (m, 1H), 1.48 (d, J = 6.9 Hz, 3H), 0.56 (d, J = 6.9 Hz, 3H), 0.45 (d, J = 6.8 Hz, 3H). 13C
NMR (75 MHz, DMSO-d6) δ 164.23, 154.32, 152.36, 145.40, 144.71, 136.91, 130.78, 130.67, 127.28,
126.72, 126.00, 122.47, 120.78, 113.94, 113.66, 113.20, 112.91, 62.84, 57.76, 49.47, 26.91, 23.48, 17.54,
13.65. HRMS (m/z) 421.2041 [M + H] +. HPLC purity 99.34 %.
4.1.8. (S)-3-(6-(((S)-1-(2'-fluoro-[1,1'-biphenyl]-4-yl)ethyl)amino)pyrazin-2-yl)-4-isopropyloxazolidin
-2-one (3d)
According to the preparation procedure of 3a, 3d was prepared as a white solid, yield 39.1%. Mp:
176-178°C. 1H NMR (300MHz, DMSO-d6) δ 8.30 (s, 1H), 7.78 (d, J = 9.3 Hz, 2H), 7.47 (d, J = 7.1 Hz,
3H), 7.39 (d, J = 8.2 Hz, 3H), 7.28 (t, J = 7.9 Hz, 2H), 5.10-4.86 (m, 1H), 4.57-4.43 (m, 1H), 4.34 (t, J =
8.8 Hz, 1H), 4.29-4.17 (m, 1H), 1.63 (s, 1H), 1.49 (d, J = 6.9 Hz, 3H), 0.56 (d, J = 6.9 Hz, 3H), 0.46 (d,
J = 6.8 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 160.66, 154.32, 152.36, 145.10, 144.73, 133.14,
15

130.55, 130.50, 129.28, 129.17, 128.70, 128.66, 127.29, 125.58, 124.82, 124.77, 120.71, 116.13, 115.83,
+
62.85, 57.76, 49.49, 26.90, 23.49, 17.50, 13.61. HRMS (m/z) 421.2033 [M + H] . HPLC purity
99.36 %.
4.1.9. (S)-4-isopropyl-3-(6-(((S)-1-(4'-methoxy-[1,1'-biphenyl]-4-yl)ethyl)amino)pyrazin-2-yl)
oxazolidin-2-one (3e)
According to the preparation procedure of 3a, 3e was prepared as a white solid, yield 45.2%. Mp:
138-141°C. 1H NMR (300 MHz, DMSO-d6) δ 8.28 (s, 1H), 7.81–7.71 (m, 2H), 7.58–7.47 (m, 4H), 7.34
(d, J = 8.1 Hz, 2H), 7.00 (d, J = 8.4 Hz, 2H), 4.92 (d, J = 7.2 Hz, 1H), 4.48 (s, 1H), 4.34 (t, J = 8.9 Hz,
1H), 4.23 (d, J = 9.0 Hz, 1H), 3.78 (s, 3H), 1.64 (s, 1H), 1.48 (d, J = 6.8 Hz, 3H), 0.57 (d, J = 6.9 Hz,
3H), 0.46 (d, J = 6.9 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 158.59, 152.38, 144.71, 143.82, 138.04,
131.07, 127.57, 127.48, 127.27, 126.08, 125.86, 120.68, 114.28, 62.83, 57.76, 55.08, 49.44, 26.89,
23.51, 17.56, 13.66. HRMS (m/z) 433.2236 [M + H] +. HPLC purity 99.08 %.
4.1.10. (S)-4-isopropyl-3-(6-(((S)-1-(3'-methoxy-[1,1'-biphenyl]-4-yl)ethyl)amino)pyrazin-2-yl)
oxazolidin-2-one (3f)
According to the preparation procedure of 3a, 3f was prepared as a white solid, yield 46.0%. Mp:
170-173°C. 1H NMR (300 MHz, DMSO-d6) δ 8.29 (s, 1H), 7.77 (d, J = 11.0 Hz, 2H), 7.57 (d, J = 8.0
Hz, 2H), 7.35 (t, J = 8.7 Hz, 3H), 7.23-7.03 (m, 2H), 6.90 (d, J = 8.1 Hz, 1H), 5.07-4.86 (m, 1H),
4.57-4.41 (m, 1H), 4.34 (t, J = 8.8 Hz, 1H), 4.25-4.15 (m, 1H), 3.80 (s, 3H), 1.61 (s, 1H), 1.48 (d, J =
6.9 Hz, 3H), 0.56 (d, J = 6.9 Hz, 3H), 0.45 (d, J = 6.8 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 159.64,
154.33, 152.38, 144.80, 144.71, 141.63, 138.27, 129.86, 127.29, 126.70, 125.87, 120.75, 118.78, 112.58,
112.12, 62.84, 57.76, 54.99, 49.49, 26.90, 23.52, 17.57, 13.67. HRMS (m/z) 433.2235 [M + H] +. HPLC
purity 98.80 %.
16

4.1.11. (S)-4-isopropyl-3-(6-(((S)-1-(2'-methoxy-[1,1'-biphenyl]-4-yl)ethyl)amino)pyrazin-2-yl)
oxazolidin-2-one (3g)
According to the preparation procedure of 3a, 3g was prepared as a white solid, yield 46.3%. Mp:
153-155°C. 1H NMR (300 MHz, DMSO-d6) δ 8.31 (s, 1H), 7.83–7.72 (m, 2H), 7.39 (d, J = 8.1 Hz, 2H),
7.30 (dd, J = 10.4, 4.7 Hz, 3H), 7.25–7.19 (m, 1H), 7.08 (d, J = 8.1 Hz, 1H), 7.00 (t, J = 7.4 Hz, 1H),
4.96 (p, J = 6.9 Hz, 1H), 4.60–4.48 (m, 1H), 4.35 (t, J = 8.8 Hz, 1H), 4.24 (dd, J = 9.0, 3.4 Hz, 1H), 3.73
(s, 3H), 1.71 (d, J = 3.2 Hz, 1H), 1.49 (d, J = 6.9 Hz, 3H), 0.61 (d, J = 6.9 Hz, 3H), 0.50 (d, J = 6.9 Hz,
3H). 13C NMR (75 MHz, DMSO-d6) δ 156.06, 154.33, 152.38, 144.75, 143.85, 136.25, 130.16, 129.63,
129.08, 128.57, 127.24, 125.01, 120.65, 120.57, 111.61, 62.86, 57.82, 55.29, 49.46, 26.89, 23.50, 17.52,
13.63. HRMS (m/z) 433.2233 [M + H] +. HPLC purity 99.72%.
4.1.12. (S)-4-isopropyl-3-(6-(((S)-1-(4'-methyl-[1,1'-biphenyl]-4-yl)ethyl)amino)pyrazin-2-yl)
oxazolidin-2-one (3h)
According to the preparation procedure of 3a, 3h was prepared as a white solid, yield 50.1%. Mp:
165-170°C. 1H NMR (300 MHz, DMSO-d6) δ 8.28 (s, 1H), 7.89-7.65 (m, 2H), 7.52 (dd, J = 15.6, 8.1
Hz, 4H), 7.35 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 7.9 Hz, 2H), 4.94 (t, J = 6.9 Hz, 1H), 4.61-4.43 (m, 1H),
4.34 (t, J = 8.8 Hz, 1H), 4.22 (dd, J = 9.0, 3.4 Hz, 1H), 2.32 (s, 3H), 1.63 (s, 1H), 1.48 (d, J = 6.9 Hz,
3H), 0.56 (d, J = 6.9 Hz, 3H), 0.45 (d, J = 6.8 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 154.32, 152.39,
144.71, 144.29, 138.28, 137.21, 136.37, 129.40, 127.30, 126.31, 126.24, 125.88, 120.72, 62.83, 57.76,
49.48, 26.88, 23.52, 20.54, 17.55, 13.64. HRMS (m/z) 417.2292 [M + H] +. HPLC purity 99.18%.
4.1.13. (S)-4-isopropyl-3-(6-(((S)-1-(3'-methyl-[1,1'-biphenyl]-4-yl)ethyl)amino)pyrazin-2-yl)
oxazolidin-2-one (3i)
According to the preparation procedure of 3a, 3i was prepared as a white solid, yield 50.1%. Mp:
17

145-149°C. 1H NMR (300 MHz, DMSO-d6) δ 8.29 (s, 1H), 7.84-7.70 (m, 2H), 7.55 (d, J = 8.1 Hz, 2H),
7.35 (dt, J = 18.6, 7.6 Hz, 5H), 7.14 (d, J = 7.2 Hz, 1H), 4.95 (t, J = 6.8 Hz, 1H), 4.56-4.45 (m, 1H),
4.34 (t, J = 8.8 Hz, 1H), 4.23 (dd, J = 9.0, 3.4 Hz, 1H), 2.35 (s, 3H), 1.63 (s, 1H), 1.48 (d, J = 6.9 Hz,
3H), 0.56 (d, J = 6.9 Hz, 3H), 0.45 (d, J = 6.8 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 154.32, 152.38,
144.72, 144.53, 140.07, 138.48, 137.89, 128.69, 127.76, 127.27, 127.09, 126.57, 125.88, 123.57, 120.71,
+
62.85, 57.77, 49.46, 26.91, 23.51, 21.05, 17.56, 13.67. HRMS (m/z) 417.2293 [M + H] . HPLC purity
98.94%.
4.1.14. (S)-4-isopropyl-3-(6-(((S)-1-(2'-methyl-[1,1'-biphenyl]-4-yl)ethyl)amino)pyrazin-2-yl)
oxazolidin-2-one (3j)
According to the preparation procedure of 3a, 3j was prepared as a white solid, yield 49.1%. Mp:
176-179°C. 1H NMR (300 MHz, DMSO-d6) δ 8.32 (s, 1H), 7.78 (d, J = 6.3 Hz, 2H), 7.44-7.31 (m, 2H),
7.24 (t, J = 7.8 Hz, 5H), 7.14 (d, J = 4.7 Hz, 1H), 5.00 (t, J = 6.9 Hz, 1H), 4.64-4.50 (m, 1H), 4.36 (t, J =
8.7 Hz, 1H), 4.31-4.15 (m, 1H), 2.20 (s, 3H), 1.73 (s, 1H), 1.50 (d, J = 6.9 Hz, 3H), 0.62 (d, J = 6.9 Hz,
3H), 0.51 (d, J = 6.8 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 152.37, 144.77, 144.05, 141.08, 139.32,
134.56, 130.23, 129.36, 128.81, 127.24, 127.08, 125.83, 125.24, 120.50, 62.97, 57.78, 49.34, 27.01,
23.58, 20.06, 17.65, 13.77. HRMS (m/z) 417.2286 [M + H] +. HPLC purity 98.71 %.
4.1.15. (S)-4-isopropyl-3-(6-(((S)-1-(4'-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)ethyl)amino)pyrazin-2-yl)
oxazolidin-2-one (3k)
According to the preparation procedure of 3a, 3k was prepared as a white solid, yield 53.1%. Mp:
96-98°C. 1H NMR (300 MHz, DMSO-d6) δ 8.30 (s, 1H), 7.81 (q, J = 8.3 Hz, 6H), 7.66 (d, J = 7.6 Hz,
2H), 7.43 (d, J = 7.8 Hz, 2H), 5.02–4.90 (m, 1H), 4.53–4.42 (m, 1H), 4.34 (t, J = 8.7 Hz, 1H), 4.26–4.18
(m, 1H), 1.59 (s, 1H), 1.50 (d, J = 6.8 Hz, 3H), 0.55 (d, J = 6.7 Hz, 3H), 0.44 (d, J = 6.7 Hz, 3H). 13C
18

NMR (75 MHz, DMSO-d6) δ 154.33, 152.35, 145.87, 144.65, 144.04, 136.67, 127.30, 127.17, 126.96,
126.12, 125.72, 125.67, 120.80, 62.84, 57.74, 49.49, 26.91, 23.48, 17.55, 13.66. HRMS (m/z) 471.2003
[M + H] +. HPLC purity 97.79 %.
4.1.16. (S)-4-isopropyl-3-(6-(((S)-1-(4-(naphthalen-2-yl)phenyl)ethyl)amino)pyrazin-2-yl)oxazolidin
-2-one (3l)
According to the preparation procedure of 3a, 3l was prepared as a white solid, yield 52.3%. Mp:
210-214°C. 1H NMR (500 MHz, CDCl3) δ 8.67 (s, 1H), 7.99 (s, 1H), 7.94–7.84 (m, 3H), 7.68 (dd, J =
15.2, 9.4 Hz, 4H), 7.56–7.46 (m, 2H), 7.40 (d, J = 8.0 Hz, 2H), 5.01–4.89 (m, 2H), 4.60–4.51 (m, 1H),
4.32 (t, J = 8.9 Hz, 1H), 4.20 (dd, J = 9.0, 3.6 Hz, 1H), 1.88 (dd, J = 10.6, 6.7 Hz, 1H), 1.63 (d, J = 6.5
Hz, 3H), 0.64 (t, J = 6.1 Hz, 6H). 13C NMR (75 MHz, DMSO-d6) δ 154.33, 152.40, 144.82, 138.12,
137.40, 133.28, 132.07, 128.35, 128.03, 127.39, 127.32, 126.87, 126.30, 126.03, 125.92, 124.96, 124.82,
120.78, 62.84, 57.78, 49.52, 28.91, 26.93, 23.53, 17.59, 13.67. HRMS (m/z) 453.2290 [M + H] +. HPLC
purity 99.01 %.
4.1.17. (S)-3-(6-(((S)-1-(4-(3,5-dimethylisoxazol-4-yl)phenyl)ethyl)amino)pyrazin-2-yl)-4-isopropyl-
oxazolidin-2-one (3m)
According to the preparation procedure of 3a, 3m was prepared as a white solid, yield 49.8%. Mp:
1
175-178°C. H NMR (500 MHz, CDCl3) δ 8.67 (d, J = 13.4 Hz, 1H), 7.67 (s, 1H), 7.37 (d, J = 8.0 Hz,
2H), 7.21 (d, J = 8.0 Hz, 2H), 4.96–4.88 (m, 2H), 4.61–4.54 (m, 1H), 4.33 (t, J = 8.9 Hz, 1H), 4.23 (dd,
J = 9.0, 3.5 Hz, 1H), 2.39 (s, 3H), 2.26 (s, 3H), 1.99 (dd, J = 10.8, 6.7 Hz, 1H), 1.60 (d, J = 6.7 Hz, 3H),
0.68 (d, J = 6.6 Hz, 6H). 13C NMR (75 MHz, DMSO-d6) δ 164.75, 157.98, 154.31, 152.32, 144.71,
128.69, 127.87, 127.23, 125.85, 120.59, 115.69, 62.96, 57.75, 49.36, 27.05, 23.50, 17.57, 13.78, 11.19,
10.37. HRMS (m/z) 422.2190 [M + H] +. HPLC purity 98.22 %.
19

4.2. Biological Assays.
The relevant biological assays were performed according to the previous methods [26].
4.2.1. IDH1 mutant generation and cloning.
IDH1 mutants were generated using the site directed mutagenesis and validated as described before
[27]. The pDONR221 clones were used for all further LR-reactions into the described destination
vectors. LR-reactions were performed following the manufacturers protocol (Invitrogen, Carlsbad,
USA).
4.2.2. Cell culture.
Human embryonic kidney cell line HEK293 was obtained from the ATCC and cultured under
standard culture conditions (37 °C, 5% CO2) in DMEM medium with 1% penicillin and streptomycin
and 10% fetal calf serum (all obtained from Gibco® Invitrogen, Carlsbad, USA). To generate HEK293
cell lines which express IDH1 WT and mutant proteins, we used cDNAs in pMXs-GW-IRES-BsdR
transfected with FuGene®HD (Promega, Madison, USA). Cells were subsequently put under selection
pressure, by adding 4 mg mL-1 Blasticidine S (Sigma Aldrich, St. Louis, USA).
4.2.3. In vitro 2-HG reduction.
2 ꢀ 105 cells per well in 1 mL of the medium were seeded in a 12-well plate and treated with an
appropriate dilution of the compound in DMSO. DMSO was used as solvent control (final concentration
0.01%). The cells were incubated at standard conditions (37 °C, 5% CO2) for 72 h. There after
supernatant was removed and subsequently analyzed with 2-HG assay. The cells were washed with PBS
(Gibco® Invitrogen, Carlsbad, USA) and treated with 150 µL NP-40 lysis buffer; 50 mM Tris–HCl, pH
8.0 (Sigma Aldrich, St.Louis, USA), 150 mM NaCl (Sigma Aldrich, St. Louis, USA), 0.1% NP-40
20

(USBiologicals, Salem, MA, USA) and complete protease inhibitor (Roche, Basel, Switzerland). This
suspension was recovered from the plate and lysate was progressed in three freeze & thaw cycles with
liquid nitrogen. There after the lysate was centrifuged at 16.1g for 15 min, the supernatant was
transferred to another tube and protein concentration of it was determined with BCA assay kit (Thermo
Fisher Scientific, Waltham, USA) following the manufacturer's protocol.
4.2.4. 2-HG assay.
The 2-HG assay has been described previously [28]. In brief, probe material was treated with a
deproteinization kit (Biovision, Mountain View, CA, USA). Supernatants were then collected and stored
at -20 °C. The total enzymatic reaction volume was 100 ml. Ten milliliter of assay solution was freshly
prepared for each 96-well plate subjected to D2HG assay. The assay solution contained 100 mM HEPES
pH 8.0, 100 µM NAD+ (Applichem, Darmstadt, Germany), 0.1 µg HGDH, 5 µM resazurin (Applichem,
Darmstadt, Germany) and 0.01 U mL-1 diaphorase (0.01 U mL-1; MP Biomedical, Irvine, USA).
Immediately before use, 25 µL sample volume was added to 75 µL of assay solution and incubated at
room temperature for 30 min in black 96-well plates (Thermo Fisher Scientific, Waltham, USA) in the
dark. Fluorometric detection was performed in triplicate with 25 µL deproteinized sample being
analyzed in each reaction with excitation at 540±10 nm and emission of 580±10 nm (FLUOstar
Omega, BMG Labtech, Offenburg, Germany).
4.2.5. Cell viability assay.
HEK-293T cells carrying pMXs-IDH1wtIRES-BsdRs were seeded in white opaque 96-well plates
(Falcon®, Corning, New York, USA) at a density of 10 000 cells per well. Subsequently they were
treated with compound at the depicted concentration and incubated for 72 h. There after the cell viability
was analyzed using CellTiterGlo (Promega, Madison, USA) following the manufacturers protocol.
21

4.3. Molecular docking studies
4.3.1. Preparation of protein structure
The X-ray structure of IDH1-R132H homodimer in a complex with SYC-435 (PDB code 4XRX)
and IDH889 (PDB code 5TQH) were obtained from Protein Data Bank and were reserved for
docking-based studies. All water molecules were cleaned utilizing the Prepare Protein utility in Maestro
14.0. Briefly, all hydrogen atoms were added properly to the complexes and hydrogen bond formations
were well maximized by the optimization of key residues.
4.3.2. Preparing the ligand database
Compounds were sketched in ChemBioDraw14.0 and converted into 3D structures followed by a
local minimization using MMFF force field. The resulting structures were used for the following
docking simulations.
4.3.3. Docking procedure
First, protein’s geometry was optimized utilizing a fast Dreiding-like force field. And the targeted
protein was completely checked by the “Clean Geometry” toolkit of DS 3.0. Then, 10 replicas for each
described compound were produced as a spherical scope with a diameter of 18 Å and centered on
IDH1-R132H inhibitor adduct. Besides, random conformation was limited as 10, simulated annealing
methods were set to true and other parameters were retained by default. Finally, ten best conformations
were saved for further analysis and molecular docking.
4.4. PAMPA-BBB assays.
The permeability of the compounds was determined by a PAMPA-BBB assay [29]. The tested drugs
(3g, Desipramine as a positive control and Atenolol as a negative control) were dissolved in DMSO to
22

5 mg/ml stock solutions, and 10 uL sample mother liquor was diluted with PBS into secondary mother
liquor (final concentration: 25 ug/ml). All solutions were filtered through a filter. Then 300uL secondary
mother liquors were added into every donor well. The porcine polar brain lipid (PBL) was dissolved in
twelve burns (20 mg/mL) and 4 µL of the mixture was added dropwise to simulate the biofilm on the
lipophilic membrane of the recipient wells. 150 µL PBS buffer was taken to the acceptor well. In
addition, the phospholipid membrane was carefully contacted with the donor fluid. Then the formation
of sandwich structure was formed that the donor plate containing the test drugs was in the bottom, the
artificial phospholipid membrane was in the middle, acceptor plate was in the top. The “sandwich” was
left for 18h at room temperature. After incubation, the concentration of the test drugs in the reference
solution and the acceptor plate was determined using a UV plate reader. Finally, the effective
transmittance (Pe) of the compounds was calculated by a formula.
VdnVac
[drug]ac
[drug]ref
P = −
ln{1−
}
e
st(Vdn +Vac )
Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://
Author Information
Corresponding Authors
*For X. Zha: phone, +86-25-86185474; fax, +86-25-86185474; e-mail, xmzha@cpu.edu;
*For A. von Deimling: phone, +49-6221-56-4650; fax: +49-6221-56-4655; e-mail,
andreas.vondeimling@med.uni-heidelberg.de;
23

# These authors contributed equally to this work.
Notes
Prof. Andreas von Deimling and Dr. Stefan Pusch are patent holders for the enzymatic 2-HG detection
assay. All other authors declare no competing financial interest.
Acknowledgments
This work was supported by the Natural Science Foundation of Jiangsu Province (BK20161458), “Six
Talent Peaks” Project of Jiangsu Province (2016-YY-042) and Postgraduate Research & Practice
Innovation Program of Jiangsu Province (KYCX17_0721).
Abbreviations
IDH, Isocitrate dehydrogenase; TCA, the tricarboxylic acid; a-KG, a-Ketoglutaric acid; D2HG,
D-2-Hydroxyglutaric acid; mIDH1, Mutant IDH1; mIDH2, Mutant IDH2; R132H, Arg132 mutation to
His; R132C, Arg132 mutation to Cys; R140Q, Arg140 mutation to Gln; WT, wild type; BBB, Blood
Brain Barrier;
References
[1] J. Chen, J. Yang, P. Cao, The Evolving Landscape in the Development of Isocitrate Dehydrogenase
Mutant Inhibitors, Mini-Rev. Med. Chem. 16 (2016) 1344-1358.
24

[2] L. Dang, S.M. Su, Isocitrate Dehydrogenase Mutation and (R)-2-Hydroxyglutarate: From Basic
Discovery to Therapeutics Development, Annu. Rev. Biochem. 2017.
[3] Z.J. Reitman, H. Yan, Isocitrate Dehydrogenase 1 and 2 Mutations in Cancer: Alterations at a
Crossroads of Cellular Metabolism, JNCI J. Natl. Cancer Inst. 102 (2010) 932–941.
[4] L. Dang, K. Yen, E. C. Attar, IDH mutations in cancer and progress toward development of targeted
therapeutics, Ann Oncol. 27 (2016) 599-608.
[5] P.L. Sulkowski, C.D. Corso, N.D. Robinson, et al, 2-Hydroxyglutarate produced by neomorphic
IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity, Sci.
Transi. Med. 9 (2017) 1-15.
[6] C. Hartmann, J. Meyer, J. Balss, D. Capper, W. Mueller, A. Christians, J. Felsberg, M. Wolter, C.
Mawrin, W. Wick, M. Weller, C. Herold-Mende, A. Unterberg, J.W.M. Jeuken, P. Wesseling, G.
Reifenberger, A. von Deimling, Type and frequency of IDH1 and IDH2 mutations are related to
astrocytic and oligodendroglial differentiation and age: a study of 1,010 diffuse gliomas, Acta
Neuropathol. 118 (2009) 469–474.
[7] L. Dang, D.W. White, S. Gross, B.D. Bennett, M.A. Bittinger, E.M. Driggers, V.R. Fantin, H.G.
Jang, S. Jin, M.C. Keenan, K.M. Marks, R.M. Prins, P.S. Ward, K.E. Yen, L.M. Liau, J.D. Rabinowitz,
L.C. Cantley, C.B. Thompson, M.G. Vander Heiden, S.M. Su, Cancer associated IDH1 mutations
produce 2-hydroxyglutarate, Nature. 462 (2009) 739-744.
[8] E. Baader, G. Tschank, K.H. Baringhaus, et al, Inhibition of prolyl 4-hydroxylase by oxalyl amino
acid derivatives in vitro, in isolated microsomes and in embryonic chicken tissues, Biochem. J. 300
(1994) 525.
25

[9] D. Ye, Y. Xiong, K.L. Guan, The mechanisms of IDH mutations in tumorigenesis, Cell Res. 22
(2012) 1102-1104.
[10] J. A. Losman, R. E. Looper, P. Koivunen, S. Lee, R.K. Schneider, C. McMahon, G. S. Cowley,
D.E. Root, B.L. Ebert, W.G.Jr. Kaelin, (R)-2-hydroxyglutarate is sufficient to promote leukemogenesis
and its effects are reversible, Science. 339 (2013) 1621-1625.
[11] E. Hansen, C. Quivoron, K. Straley, R.M. Lemieux, J. Popovici-Muller, H. Sadrzadeh, A.T. Fathi,
C. Gliser, M, David, V. Saada, J.B. Micol, O. Bernard, M. Dorsch, H. Yang, M. Su, S. Agresta, S. de
Botton, V. Penard-Lacronique, K. Yen, AG-120, an Oral, Selective, First-in-Class, Potent Inhibitor of
Mutant IDH1, Reduces Intracellular 2HG and Induces Cellular Differentiation in TF-1 R132H Cells and
Primary Human IDH1 Mutant AML Patient Samples Treated Ex Vivo, Blood. 124 (2014) 3734.
[12] Agios Announces First Patient Dosed with AG-881 in Phase 1 Study in Patients with Advanced
Solid Tumors with an IDH Mutation, Agios Pharmaceuticals Press Release. 2015.
[13] B. Zheng, Y. Yao, Z. Liu, L. Deng, J. L. Anglin, H. Jiang, B.V.V. Prasad, Y. Song,
Crystallographic investigation and selective inhibition of mutant isocitrate dehydrogenase, Med. Chem.
Lett. 4 (2013) 542–546.
[14] U.C. Okoye-Okafor, B. Bartholdy, J. Cartier, E.N. Gao, B. Pietrak, A. R. Rendina, C. Rominger, C.
Quinn, A. Smallwood, K.J. Wiggall, A.J. Reif, S.J. Schmidt, H. Qi, H. Zhao, G. Joberty, M.
Faelth-Savitski, M. Bantscheff, G. Drewes, C. Duraiswami, P. Brady, A. Groy, S.R. Narayanagari, I.
Antony-Debre, K. Mitchell, H.R. Wang, Y.R. Kao, M. Christopeit, L. Carvajal, L. Barreyro, E.
Paietta, H. Makishima, B. Will, N. Concha, N.D. Adams, B. Schwartz, M.T. McCabe,
J.
Maciejewski, A. Verma, U. Steidl, New IDH1 mutant inhibitors for treatment of acute myeloid
leukemia, Nat. Chem. Biol. 11 (2015) 878-886.
26

[15] B. Yang, C. Zhong, Y. Peng, Z. Lai, J. Ding, Molecular mechanisms of “off-on switch” of
activities of human IDH1 by tumor-associated mutation R132H, Cell. Res, 20 (2010) 1188-1200.
[16] G. Deng, J. Shen, M. Yin, J. McManus, M. Mathieu, P. Gee, T. He, C. Shi, O. Bedel, L.R. McLean,
F. Le-Strat, Y. Zhang, J.P. Marquette, Q. Gao, B. Zhang, A. Rak, D. Hoffmann, E. Rooney, A. Vassort,
W. Englaro, Y. Li, V. Patel, F. Adrian, S. Gross, D. Wiederschain, H. Cheng, S. Licht, Selective
inhibition of mutant isocitrate dehydrogenase 1 (IDH1) via disruption of a metal binding network by an
allosteric small molecule, J. Biol. Chem. 290 (2015) 762-774.
[17] D. Rohle, J. Popovici-Muller, N. Palaskas, S. Turcan, C. Grommes, C. Campos, J. Tsoi, O. Clark,
B. Oldrini, E. Komisopoulou, K. Kunii, A. Pedraza, S. Schalm, L. Silverman, A. Miller, F. Wang, H.
Yang, Y. Chen, A. Kernytsky, M.K. Rosenblum, W. Liu, S.A. Biller, S.M. Su, C.W. Brennan, T.A.
Chan, T.G. Graeber, K.E. Yen, I. K. Mellinghoff, An inhibitor of mutant IDH1 delays growth and
promotes differentiation of glioma cells, Science. 340 (2013) 626–630.
[18] L. Li, A.C. Paz, B.A. Wilky, B. Johnson, K. Galoian, A. Rosenberg, G. Hu, G. Tinoco, O. Bodamer,
J.C. Trent, Treatment with a Small Molecule Mutant IDH1 Inhibitor Suppresses Tumorigenic Activity
and Decreases Production of the Oncometabolite 2-Hydroxyglutarate in Human Chondrosarcoma Cells,
PLos One. 10 (2015) e0133813.
[19] S. Pusch, S. Krausert, V. Fischer, J. Balss, M. Ott, D. Schrimpf, D. Capper, F. Sahm, J. Eisel, A.C.
Beck, M. Jugold, V. Eichwald, S. Kaulfuss, O. Panknin, H. Rehwinkel, K. Zimmermann, R.C. Hillig, J.
Guenther, L. Toschi, R. Neuhaus, A. Haegebart, H. Hess-Stumpp, M. Bauser, W. Wick, A. Unterberg, C.
Herold-Mende, M. Platten, A. von Deimling, Pan-mutant IDH1 inhibitor BAY 1436032 for effective
treatment of IDH1 mutant astrocytoma in vivo, Acta Neuropathol. 133 (2017) 629-644.
27

[20] A. Chaturvedi, L. Herbst, S. Pusch, L. Klett, R. Goparaju, D. Stichel, S. Kaulfuss, O. Panknin, K.
Zimmermann, L. Toshi, R. Neuhaus, A. Haegebarth, H. Rehwinkel, H. Hess-Stumpp, M. Bauser, T.
Bochtler, E.A. Struys, A. Sharma, A. Bakkali, R. Geffers, M.M. Araujo-Cruz, F. Thol, R. Gabdoulline,
A. Ganser, A.D. Ho, A. von Deimling, K. Rippe, M. Heuser, A. Krämer, Pan-mutant-IDH1 inhibitor
BAY1436032 is highly effective against human IDH1 mutant acute myeloid leukemia in vivo,
Leukemia. (2017) 1-9.
[21] M. Heuser, L. Herbst, S. Pusch, L. Klett, R. Goparaju, D. Stichel, S. Kaulfuss, O. Panknin, K.
Zimmermann, L. Toschi, R. Neuhaus, A. Haegebarth, H. Rehwinkel, H. Hess-Stumpp, M. Bauser, T.
Bochtler, E.A. Struys, A. Sharma, A. Abdellatif, R. Ceffers, M.M.A. Gruz, F. Thol, R. Gaboulline, A.
Ganser, A.D. Ho, A. von Deimling, K. Rippe, A. Chaturvedi, A. Kraemer, Pan-Mutant-IDH1 Inhibitor
Bay-1436032 Is Highly Effective Against Human IDH1 Mutant Acute Myeloid Leukemia In Vivo,
Blood. 128 (2016) 745.
[22] J.M. Law, S.C. Stark, L. Ke, et al, Discovery of 8-Membered Ring Sulfonamides as Inhibitors of
Oncogenic Mutant Isocitrate Dehydrogenase 1, Med. Chem. Lett. 7 (2016) 944-949.
[23] S. Jones, J. Ahmet, K. Ayton, et al, Discovery and Optimization of Allosteric Inhibitors of Mutant
Isocitrate Dehydrogenase 1 (R132H IDH1) Displaying Activity in Human Acute Myeloid Leukemia
Cells, J. Med. Chem. 59 (2016) 11120-11137.
[24] T.R. Caferro, Z. Chen, Y.S. Cho, et al, 3-pyrimidin-4-yl-oxazolidin-2-ones as inhibitors of mutant
IDH: WO 2013046136 (A1) [P]. 2013.
[25] J.R. Levell, T. Caferro, G. Chenail, et al, Optimization of 3-Pyrimidin-4-yl-oxazolidin-2-ones as
Allosteric and Mutant Specific Inhibitors of IDH1, Med. Chem. Lett, 8 (2016) 151-156.
28

[26] F. Zou, S. Pusch, J. Eisel, T. Ma, Q. Zhu, D. Deng, Y. Gu, Y. Xu, A. von Deimling, X. Zha,
Identification of a novel selective inhibitor of mutant isocitrate dehydrogenase 1 at allosteric site by
docking-based virtual screening, RSC Adv. 6 (2016) 96735–96742.
[27] S. Pusch, L. Schweizer, A.C. Beck, J.M. Lehmler, S. Weissert, J. Balss, A.K. Miller, A. von
Deimling, D-2-Hydroxyglutarate producing neo-enzymatic activity inversely correlates with frequency
of the type of isocitrate dehydrogenase 1 mutations found in glioma, Acta Neuropathol. Commun. 2
(2014) 19.
[28] J. Balss, S. Pusch, A.C. Beck, C. Herold-Mende, A. Krämer, C. Thiede, W. Buckel, C.D. Langhans,
J.G. Okun, A. von Deimling, Enzymatic assay for quantitative analysis of (d)-2-hydroxyglutarate, Acta
Neuropathol. 124 (2012) 883–891.
[29] L. Di, E.H. Kerns, K. Fan, O.J. McConnell, G.T. Carter, High throughput artificial membrane
permeability assay for blood–brain barrier, Eur. J. Med. Chem. 38 (2003) 223–232.
Highlights
ꢀ
ꢀ
Synthesis of novel selective mIDH1 inhibitors with 3-pyrazine-4-yl-oxazolidin-2-ones scaffold.
3g shows potent inhibition against IDH1-132H and IDH1-132C as well as high selectivity over
29