I-8, a novel inhibitor of mutant IDH1, inhibits cancer progression in vitro and in vivo

Article abstract of DOI:10.1016/j.ejps.2019.105072

Isocitrate dehydrogenase 1 mutations have been discovered in an array of hematologic malignancies and solid tumors. These mutations could cause the production of high levels of 2-hydroxyglutarate, which in turn implicated in epigenetic changes and impaire

Full text of DOI:10.1016/j.ejps.2019.105072

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I-8, a novel inhibitor of mutant IDH1, inhibits cancer progression
in vitro and in vivo
Panli Jia, Yao Wu, Hongzhi Du, Lijun Yang, Zhibo Zhang,
Tianfang Ma, Li Sun, Shengtao Yuan, Ligong Lu, Xiaoming Zha
PII:
S0928-0987(19)30343-4
DOI:
https://doi.org/10.1016/j.ejps.2019.105072
PHASCI 105072
Reference:
To appear in:
European Journal of Pharmaceutical Sciences
Received date:
Revised date:
Accepted date:
15 May 2019
1 September 2019
9 September 2019
Please cite this article as: P. Jia, Y. Wu, H. Du, et al., I-8, a novel inhibitor of mutant IDH1,
inhibits cancer progression in vitro and in vivo, European Journal of Pharmaceutical
Sciences(2018), https://doi.org/10.1016/j.ejps.2019.105072
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2018 Published by Elsevier.

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I-8, a novel inhibitor of mutant IDH1, inhibits cancer
progression in vitro and in vivo
a,1
a,1
c,1
b,1
a
b
Panli Jia , Yao Wu , Hongzhi Du , Lijun Yang , Zhibo Zhang , Tianfang Ma , Li
a
a,*
d,*
b, 
Sun , Shengtao Yuan , Ligong Lu , Xiaoming Zha
a
Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing, China
b
Department of Pharmaceutical Engineering and Department of Biochemical Engineering, China
Pharmaceutical University, Nanjing, China
c
School of Pharmacy, Hubei University of Chinese Medicine, Hubei, China
d
Zhuhai Interventional Medical Center, Zhuhai Precision Medical Center, Zhuhai People's
Hospital, Zhuhai Hospital Affiliated with Jinan University, Zhuhai, China

Corresponding author
E-mail addresses: xmzha@cpu.edu.cn (X. Zha), luligong1969@126.com (L. Lu),
yuanst@cpu.edu.cn (S. Yuan)
1
These authors contributed equally to this work.
ARTICLE INFO
Key words: Isocitrate dehydrogenase 1; mutant IDH1 inhibitor; compound I-8;
2
-hydroxyglutarate; differentiation; histone methylation
Abbreviations: NADPH, nicotinamide adenine dinucleotide phosphate; AML, acute
myelocytic leukemia; DATAN, (+)-O, O’-Diacetyl-L-tartaric anhydride; MTT, methyl
thiazolyl tetrazolium; PAGE, polyacrylamide gelelectrophoresis; BSA, bovine serum
albumin; PVDF, Polyvinylidene Fluoride; ECL, Electro-chemiluminescence
Immunoassay
F-actin,
Filamentous
actin;
LC/MS/MS,
liquid
chromatograph-mass-mass spectrometer; DMF, Dimethyl Formamide; DIPEA,
N,N-Diisopropylethylamine.

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Abstract
Isocitrate dehydrogenase 1 mutations have been discovered in an array of hematologic
malignancies and solid tumors. These mutations could cause the production of high
levels of 2-hydroxyglutarate, which in turn implicated in epigenetic changes and
impaired cell differentiation. Here, we described the characterization of compound I-8,
a novel mutant IDH1 inhibitor, both in vitro and in vivo. Compound I-8 specifically
inhibited 2-HG production, reduced histone methylation levels, induced
differentiation and depleted stem characteristics in engineered and endogenous IDH1
mutant cells. In addition, oral administration of I-8 also significantly suppressed 2-HG
production and histone methylation with dose of 150 mg/kg. And I-8 treatment also
could induce differentiation and attenuate stem characteristics in tumor tissue.
Together, these studies indicated that compound I-8 has clinical potential in tumor
therapies as a effective mutant IDH1 inhibitor, and provided scientific guidance for
the development of mutant IDH1 inhibitor in the future.
Introduction
In 2011, Douglas Hanahan and Robert A. Weinberg summarized ten
characteristics of tumor cells, including deregulating cellular energetics (Hanahan and
Weinberg, 2011). Abnormal energy metabolism plays an important role in the
development and progression of tumors, and targeting tumor metabolism has become
a hot spot for anti-tumor drug development (Vander Heiden and DeBerardinis, 2017).
Particularly, isocitrate dehydrogenases (IDHs) are key rate-limiting enzyme that
catalyzes the conversion of isocitrate to alpha-ketoglutarate (α-KG) using divalent
+
+
magnesium ion and NADP (or NAD ) as cofactors in the Krebs cycle, which are
widely found in humans and eukaryotes (Stoddard et al., 1993). The IDHs family has
three subtypes, including IDH1, IDH2 and IDH3, of which IDH1 and IDH2 could be

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mutated (Dang and Su, 2017). IDH mutations have been frequently found in IDH1
R132H and IDH1 R132C or IDH2 R140Q and IDH2 R172K, which are located in the
catalytic pocket of these enzymes (Saha et al., 2014). IDHs are metabolic enzyme
associated with the progression of cancers and its mutations (including IDH1 and
IDH2) mainly occur in acute myeloid leukemia (Mardis et al., 2009) (AML) and solid
tumors such as glioma (Rohle et al., 2013), osteosarcoma (Liu et al., 2013) and
intrahepatic cholangiocarcinoma (Farshidfar et al., 2017).
Significantly, substantial datas revealed that approximately 70% of grade II and
III astrocytomas, oligodendrogliomas, and malignant gliomas that have evolved from
these low-grade tumors have related to IDH1 R132H mutations, which its active site a
key amino acid residue arginine 132 mutated to histidine (Hartmann et al., 2009).
Biochemically, these mutations allow IDH enzymes to gain a neomorphic activity that
α-KG is converted to 2-hydroxyglutarate (2-HG) by using NADPH (Dang et al.,
2
010). Notably, high intracellular levels of 2-HG competitively binds to
α-KG-dependent dioxygenase, which could affect the methylation status of histones
and DNA and blocks cell differentiation (Ye et al., 2012). Epigenetic changes could
induce normal cells to transform into tumor cells or promote initiation and
progression of tumors. Losman et al found that adding 2-HG or over-expressing IDH
mutation in TF-1 cells could inhibit the normal differentiation of hematopoietic cells
and promote tumor cell proliferation (Losman et al., 2013). Sasaki et al showed that
mouse brain-specific knock-in IDH1 R132H mutation could cause cerebral
hemorrhage and perinatal mortality (Masato et al., 2012b). In addition, IDH1 mutant
cells also showed high levels of 2-HG, which mediates collagen maturation damage,
causes basement membrane aberrations, and promotes glioma progression (Masato et
al., 2012a). These findings demonstrated that reduction of 2-HG production by IDH1
inhibitors could alter the epigenetic.
To date, as novel class of targeted cancer metabolism therapy, many inhibitors of
IDH1 have been reported (Luengo et al., 2017). As our published review has updated
and prospected the research progress of mutant IDH1/2 inhibitors (Ma et al., 2018).
The first prototype mIDH1 inhibitor AGI-5198 was reported that could inhibit both

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biochemical and cellular production of 2-HG, cause histone H3K9Me3 demethylation
and promote differentiation both in vivo and in vitro (Rohle et al., 2013). Subsequently,
pharmacodynamic and structural optimized IDH1 inhibitors, such as IDH-305 (Cho et
al., 2017), FT-2102 (NCT03684811, Recruiting), BAY-1436032 (NCT03127735,
Active ) and AG-120 (Popovicimuller et al., 2018), were discovered and entered into
clinical trials. And AG-120 (Ivosidenib, Agios and Celgene) has been approved by the
US FDA for the treatment of susceptible IDH1 mutation in patients with relapsed or
refractory AML in 2018 (Dhillon, 2019). Therefore, these reports indicate that studies
targeting IDH mutations and the development of mutant IDH inhibitors have become
new research directions. However, the high price of Ivosidenib limits its clinical
application, and it is now urgent to identify new chemical substances for development
as inhibitors to meet the requirements of IDH mutant patients.
Therefore, it is noted that studying the relationship between IDH1 mutations and
tumorigenesis development could provide new ideas for the clinical treatment of
malignant tumors and the development of drugs for mutant IDH1 (Sharma, 2018). In
the present study, we got a novel potential IDH1 inhibitors I-8 with the granted patent.
The effect of I-8 on three different IDH1 mutant cell lines was investigated in vitro,
which mainly reduced the production of 2-HG, inhibited histone methylation, and
induced cell differentiation. Ultimately, the pharmacodynamics of I-8 against human
fibrosarcoma HT1080 subcutaneously xenograft in vivo were explored. Obviously, the
novel inhibitor of mutant IDH1 may be a potential therapeutic strategy and will
provide scientific references for development of mutant IDH1 inhibitor in the future.
Materials and Methods
2
.1 Chemicals and reagents
Details of compound I-8 characterization could be found in Supporting
Information. Compound I-8 was dissolved in DMSO (10 mM) and AG-120 was
obtained from MedChem Express (Monmouth, NJ, USA). IDH1 R132H/C

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recombinant enzyme were purchased from Raction Biology Corporation (San Diego,
CA, USA). NADPH•4Na, diaphorase, resazurin sodium and DATAN were bought
from Sigma-Aldrich (St. Louis, MO, USA). Protease and phosphatase inhibitor
complex was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Rabbit
anti-H3, anti-H3K4me3, anti-H3K9me3, anti-H3K27me3, anti-α-SMA and RIPA
buffer were obtained from Cell Signaling Technology (Beverly, MA, USA). Rabbit
anti-GFAP was bought from Proteintech (Chicago, MA, USA). Rabbit anti-MyoD1,
anti-Vimentin, anti-AFP, anti-DLK1, anti-SOX2, anti-Nanog, anti-Oct3/4 were
obtained from ABclonal Biotech (Wuhan, China). Rabbit anti-Myf-6 was purchased
from Biogot technology (Nanjing, China). The BCA kit was provided by Beyotime
Biotech (Shanghai, China). Hoechst and Actin-Red dyes were obtained from KeyGEN
Biotech (Nanjing, China). All other reagents were purchased from commercial
sources and were of chromatographic grade.
2
.2 Cell culture
Human fibrosarcoma HT1080 cells (IDH1 R132C) and human
cholangiocarcinoma RBE cells (IDH1 R132S) were obtained from Cell Bank of
Shanghai Institute for Biological Sciences, University of Chinese Academy of
Sciences. Stable U87 MG-IDH1 R132H cells were obtained as a gift from Prof. Qinxi
Li (Xiamen University, Fujian, China). HT1080 cells and U87 MG-IDH1 R132H cells
were cultured in MEM containing 10% fetal bovine serum (FBS, PAN Biotech,
Germany) and 100 U/ml penicillin and 100 U/ml streptomycin. RBE cells were
cultured in RPMI-1640 supplemented with 10% fetal bovine serum and 100 U/ml
penicillin and 100 U/ml streptomycin. All cultures were incubated at 37 °C in a
humidified atmosphere containing 5% CO2.
2
.3 Cell viability assay
Effect of compound I-8 treatment on HT1080 cells, U87 MG-IDH1 R132H cells
and RBE cells were evaluated by MTT assay. Cell suspensions were seeded into

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3
9
6-well plate (2.5×10 cells/well). After incubation for 24 h, cells were treated with
different concentrations of I-8 for 72 h. Afterwards, 20 μL MTT (5 mg/ml) was added
into each well in the dark and cells were incubated for another 4 h. The supernatant
was removed carefully, 100 μL of DMSO was added to each well to dissolve the
formazan crystals. The absorbance was measured at a wavelength of 570 nm, and the
inhibition rate of compound I-8 was calculated by the formula: Inhibition rate (%) =
(1 - absorbance of the treated group/absorbance of the control group) × 100%, and the
inhibition rate curve was plotted.
2
.4 Enzyme activity assay
The activity of the isocitrate dehydrogenase 1 assay has been described
previously (Pusch et al., 2017). In brief, assays were performed in 384-well plates in a
reaction volume of 10 μL, which containing a mixture of isocitrate dehydrogenase 1
(R132H, 10 nM or R132C, 5 nM), α-KG (4 mM or 1 mM) and NADPH (25 μM).
After knocking the reagent to the bottom of plate, the plate was sealed and incubated
in a 37 °C incubator for 60 min. 2.5 μL of resazurin solution and 2.5 μL of 4 UN/mL
diaphorase (final concentrations of resazurin and diaphorase 4 μM and 0.01 UN,
respectively) were added to each well. Incubated in a 37 °C incubator for 10 min to
stop the reaction. Fluorescence Intensity (FI) was measured at excitation and emission
wavelengths of 544 nm and 590 nm (E /E = 544/590 nm) using the BioTek Synergy
x
m
2
multimode plate reader. The greater degree of inhibition of enzyme activity, the
stronger the fluorescent signal. According to the principle of the reaction, the
fluorescence signal of the well without IDH R132H/C was the strongest and was
recorded as FImax, and the fluorescence signal of the solvent control well was the
weakest was recorded as FI . Therefore, the enzyme activity inhibition rate is
min
calculated as: enzyme activity inhibition rate (%) = [1- (FImax - FIx)/(FImax- FI )]×
min
1
00%.

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2
.5 Molecular docking
The crystal structure of mutant IDH1 was searched on the RCBS Protein Data
Bank (RCSB PDB). Autodock Vina software was used to process and optimize the
protein structure in the crystal, including the removal of water molecules, the addition
of hydrogen atoms, and so on. Further, the active pocket of the protein was defined.
After completion of molecular flexible docking, the molecular dynamics parameters
of the compounds were evaluated using GROMACS. Finally, the lowest-energy and
the most favorable orientation of the ligand were selected.
2
.6 Western blotting
Cells were lysed with RIPA buffer containing protease and phosphatase
inhibitors for protein extraction. Equal volumes of protein lysates were separated
using SDS-PAGE. After electrophoresis, proteins in the gel were transferred to PVDF
membrane (Millipore, United States). The membranes were blocked with 5% BSA in
TBS buffer containing Tween-20 (TBST) for 1 h, and then incubated with appropriate
primary antibodies diluted in 5% BSA buffer overnight at 4 °C. Next, they were
washed with TBST buffer for three times every 10 min and incubated with secondary
antibodies for 1 h at room temperature. Finally, the immunoblots were visualized by
ECL reagents (Millipore) via Gel 2000 image analyzer (Bio-Rad Laboratories, USA).
2
.7 Cellular F‑actin measurement
HT1080 cells, U87 MG-IDH1 R132H cells and RBE cells were seeded in
4
2
4-well plates at a density of 2×10 cells/mL for cell climbing. These cells were
treated with I-8 (0.5, 1.0, 2.0 μM) and AG-120 (1.0 μM) for 72 h, fixed with 4%
paraformaldehyde for 60 min. 1 mL of 0.3%-0.5% Triton X-100 containing 3% BSA
was added to permeabilize cells for 30 minutes at room temperature. Afterwards,
coverslips were stained with Hoechst 33342 (1:2000) and Actin-Red (1:40) for 60 min
in the dark. Finally, they were washed with PBS for three times every 10 min and

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added appropriate amount of anti-fade fluorescence mounting medium. Images were
captured by using Olympus IX71 (Olympus, Tokyo, Japan).
2
.8 Immunofluorescence
HT1080 cells, U87 MG-IDH1 R132H cells and RBE cell were seeded in 24-well
4
plates at a density of 2×10 cells/mL for cell climbing. After 24 h, the supernatant was
removed and washed twice with pre-cooled PBS. Those cells were treated with I-8
(0.5, 1.0, 2.0 μM), and AG-120 (1.0 μM) for 72 h, fixed with 4% paraformaldehyde
for 60 min. Added 1 mL of 0.3%-0.5% Triton X-100 containing 3% BSA and
permeabilized cells for 30 min at room temperature. Subsequently, they were blocked
with 5% bovine serum albumin for 1 h and incubated with primary antibodies at 4 °C
for overnight. Next, they were washed with PBS for three times every 10 min and
incubated with Alexa Fluor 488 labeled goat anti-rabbit IgG (1:500) for 1 h at room
temperature. Finally, coverslips were stained with Hoechst 33342 (1:2000) for 30 min
and added appropriate amount of anti-fade fluorescence mounting medium. Images
were captured by using Olympus IX71.
2
.9 Animal study
BALB/c nude mice (body weight ∼18 g) were purchased from Nanjing
Biomedical Research Institute of Nanjing University. Throughout the study, all mice
were maintained in a light, temperature, and humidity controlled environment (12 h
light/dark cycle, 23 ± 1 °C, 50 ± 5%) with access to food and water ad libitum
(Permission Number: SYXK 2019-0019). Ethical approvals for the animal
experiments were obtained from the Animal Ethics Committee of China
6
Pharmaceutical University (Nanjing, China). HT1080 cells (2×10 ) were transplanted
1
1
cm below the forelimb of the mice. After tumor volumes reached approximatively
3
00 mm , tumor-bearing mice were treated with different doses (75 and 150 mg/kg)
of I-8 (i.g.) once a day for 10 consecutive days. The vernier caliper was used to
measure the maximum diameter a (mm) and transverse diameter b (mm) of the tumor,

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2
and tumor volume was calculated according to the formula of V=ab /2. The body
weight of the mice was recorded and the changes in body weight of the mice were
observed. After 10 days, the mice were sacrificed, serum and subcutaneous tumor
tissues were collected
2
.10 Measurement of 2-HG level
HT1080 cells, U87 MG-IDH1 R132H cells and RBE cell were seeded in 24-well
4
plates at a density of 2×10 cells/mL, and were treated with compound I-8 for 72 h.
Supernatants were then collected and stored at -80 °C. Serum and tumor tissues were
collected and stored at -80 °C. Accurately weigh 50 mg of tumor tissue, and grind it
thoroughly by adding 200 μL of physiological saline in a medium flux grinder. In
addition, 400 μL of normal saline was added to make the system to 600 μL. The
system was centrifuged at 13,000 rpm for 5 min and collected supernatant. Finally, the
supernatant sample was accurately transferred 20 μL into a centrifuge tube, and 230
μL of precipitant was added. And medium and serum sample were accurately
transferred 20 μL into a centrifuge tube, and 230 μL of a precipitant was added.
All mixture were vortexed for 3 min and then centrifuged at 13,000 rpm for 5
min. The supernatant was taken 230 μL in a vacuum apparatus and dried at 50 °C.
Next, The residue was subjected to a derivatization reaction with DATAN (50 mg/mL).
After the derivatization, residue was reconstituted with 1 mL of internal standard
(
chloramphenicol, 500 ng/mL) solution. The supernatant was collected and subjected
to LC-MS/MS to separate and quantitate the amount of 2-HG. Briefly, separation of
-HG was performed using high pressure liquid chromatography with a Hanbon
2
Hedera ODS-2 5 μm column (150 × 2.1 mm) (Jiangsu Hanbang Technology, Huaian,
China). Acetonitrile and 0.05% aqueous formic acid were used as the mobile phase,
the injection volume was 5 μL, the flow rate was 400 μL/min, and the elution method
was gradient elution (Table 1). Mass spectrometry detection was made using
electrospray ion source in negative ion mode with multiple reaction monitoring mass
ion transitions 363.13/146.9 (2-HG) and 321.2/152.2 (chloramphenicol, IS).

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Table 1 Details of gradient elution
B: 0.05%
Time
min）
A: acetonitrile
aqueous formic
（
（%）
acid（%）
0
3
3
6
9
.00
.30
.40
.50
.50
6
6
94
94
20
20
94
94
80
80
6
1
2.00
.11 Statistical analysis
Data were shown as the mean ± SD and statistical analysis was performed by a
6
2
Student's t-test or one-way ANOVA using the GraphPad Prism 5.0 software. A value
of P < 0.05 was accepted as a significant difference, and values of P < 0.01 and P <
0
.001 were considered highly significant difference.
Results
3
.1 Chemical synthesis and biochemical activity of compound I-8
3
.1.1 Synthesis of (S)-3-(2-chloropyrimidin-4-yl)-4-isopropyloxazolidin-2-one (3)
To a mixture of 2,4-dichloropyrimidine (1.00 g, 6.71 mmol) and
(S)-4-isopropyl-oxazolidin-2-one (867 mg, 6.71 mmol) in 7 ml anhydrous DMF, a
solution of NaH (322 mg, 13.43 mmol) in DMF was added dropwise under ice bath.
The mixture was stirred for 4 h at 0°C till its completion monitored by TLC. The
reaction mixture was poured into ice water and the organic layer was collected with
EtOAc, washed with saturated NaHCO solution and dried with anhydrous Na SO .
3
2
4
Purification of the crude product by rapid column chromatography (Petroleum :
1
EtOAc = 15:1, v:v) to afford the intermediate 3 as white solid (1.2 g, yield 74.1%). H
NMR (500 MHz, CDCl ) δ 8.52 (d, J = 5.8 Hz, 1H), 8.23 (d, J = 5.8 Hz, 1H),
3

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4
0
.94-4.77 (m, 1H), 4.50-4.26 (m, 2H), 2.72-2.55 (m, 1H), 1.04 (d, J = 7.0 Hz, 3H),
.93 (s, 3H).
Synthesis of (S)-1-(4'-fluoro-3'-methyl-[1,1'-biphenyl]-4-yl)ethan-1-amine (6)
To a mixture of 4-fluoro-3-methylbenzeneboronic acid (1 g, 5 mmol),
(S)-1-(4-bromophenyl)ethylamine (846 mg, 5.50 mmol), tetratriphenylphosphine
palladium (275 mg, 0.249 mmol) and cesium carbonate (3.26 g, 10 mmol) was added
with 3 ml of water and 15 ml of 1,4-dioxane. The reaction mixture was stirred at
9
0 °C under nitrogen for 5 h till its completion monitored by TLC. After cooling to
room temperature, the solvent was evaporated under vacuum to afford yellow oil and
dissolved by 50 ml EtOAc and stirred for 10 min at rt. The reaction mixture was
washed with 2 N NaOH solution and brine and dried over anhydrous Na SO . The
2
4
crude product was condensed under vacuum to afford yellow solid for the next step
without purification.
3
.1.2
Synthesis
of
(S)-3-(2-(((S)-1-(4'-fluoro-3'-methyl-(1,1'-biphenyl)-4-yl)ethyl)amino)pyrimidin-4-yl)-
4
-isopropyloxazolidin-2-one (I-8)
To a mixture of 3 (1.68 g, 7.33 mmol), 6 (885 mg, 3.66 mmol) and DIPEA (4.34
g, 36 mmol) was dissolved in 15 mL DMSO and then heated to 110 °C for 3 h. After
the reaction was completed, the mixture was cooled to rt and was added with 50 mL
of EtOAc. The mixture was washed twice with brine and dried over anhydrous
Na SO to give the target compound I-8 as pale yellow solid (412 mg, yield 40%),
2
4
1
purity 97% (HPLC). H NMR (600 MHz, CDCl3) δ 8.22 (d, J = 5.7 Hz, 1H), 7.48 (m,
J = 9.5, 7.0 Hz, 4H), 7.37 (m, J = 9.8, 4.5 Hz, 4H), 7.07 (d, J = 17.8 Hz, 1H), 5.57 (s,
1
1
H), 5.13-5.00 (m, 1H), 4.60 (s, 1H), 4.30 (d, J = 17.6 Hz, 1H), 4.22 (d, J = 7.6 Hz,
1
3
H), 2.38-2.33 (d, 3H), 1.60 (d, J = 6.9 Hz, 3H), 0.75-0.56 (m, 6H). C NMR (150
MHz, CDCl3) δ 161.83, 160.96, 160.20, 158.80, 157.38, 154.75, 143.82, 139.06,
1
5
4
36.70, 132.09, 130.06, 128.58, 127.15, 125.80, 125.05, 115.38, 99.40, 62.93, 58.52,
+
0.69, 27.34, 18.20, 14.75, 13.99. HRMS m/z: calc. 435.2196[M+H] , found
+
35.2186[M+H] .

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To better understand the molecular biological basis of I-8 inhibitory activity
against IDH1-R132H, we carried out the molecular docking of I-8 and IDH1-R132H
(PDB ID: 5TQH). As shown in Figure 1B, compound I-8 binds to an allosteric site of
mIDH1-R132H. Its aminopyrimidine fragment forms two hydrogen bonds with Ile128.
Meanwhile, its oxazolidinone group extends into the interior of the cavity, and its
carbonyl group forms a hydrogen bond with Leu120. The α-methyl group of I-8
protrudes into the cavity of the keratin methyl group to form a hydrophobic action.
To investigate whether compound I-8 owns a directly impact on IDH1 R132H/C,
we performed enzymatic activity assay. The results demonstrated that compound I-8
exhibited high inhibitory activity against both IDH1 R132C and IDH1 R132H (Figure
1
C-D), and that I-8 inhibited IDH1 R132C and IDH1 R132H with IC value of 174.2
50
±
22.1 nM and 135.6 ± 17.9 nM (Figure 1E-F). However, compound I-8 had weak
inhibitory effect on wild-type IDH1 and the IC was 2.67 ± 0.82 mM (Figure 1G).
5
0
Notably, when the substrate α-KG concentration was increased to 10 μM, the IC50
value of I-8 inhibiting the IDH1 R132C enzyme increased to 406.5 nM, and inhibition
rate of enzyme activity decreased, which indirectly suggested that the binding of the
substrate/compound to the IDH1 R132C enzyme was competitively bound (Figure
1
H). These results indicated that compound I-8 could significantly inhibit the mutant
IDH1.
3
.2 Compound I-8 inhibits 2-HG production and suppress histone methylation in
IDH1 mutant cells
Three different IDH1 mutant cells, including HT1080 cells, U87 MG-IDH1
R132H cells and, RBE cells were used to investigate the fundamental anti-tumor role
of compound I-8. As shown in Figure 2A, MTT results revealed that compound I-8
had no significant inhibition on the growth of all IDH1 mutant cells. We next explored
the effect of compound I-8 on 2-HG production in IDH1 mutant cells. A LC-MS/MS
method was established to measure the concentration of 2-HG in cellular supernatant.

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As shown in Figure 2B, a dose-dependent decrease in 2-HG levels was observed, and
the IC values of I-8 for HT1080 cells, U87 MG-IDH1 R132H cells and RBE cells
5
0
were 0.428 ± 0.13 μM, 0.680 ± 0.20 μM and 1.341 ±0.32 μM, respectively. These
results suggested that mutant IDH1 potential inhibitor I-8 reduces 2-HG production
effectively in engineered and endogenous mutated cells.
As mentioned above, 2-HG is an inhibitor of histone demethylases, which could
affect the methylation status of histones. Therefore, the methylation level of histones
in all IDH1 mutant cells were measured by western blot analysis. Compared to the
control group, compound I-8 treatment, either at low or high concentrations, markedly
reduced histone methylation levels (including H3k4me3, H3K9me3 and H3K27me3)
in HT1080 and, to a certain extent, was superior to the positive control AG-120
(Figure 2C-D ). Also compound I-8 intervention could significantly decrease the level
of histone methylation in U87 MG-IDH1 R132H cells (Figure 2E-2F). Particularly,
compound I-8 treatment could significantly decrease the level of histone methylation
in RBE cells with a concentration-dependent manner (Figure 2G-2H). These data
suggested that I-8 could restore the activities of histone demethylases and avoid the
wide hypermethylation of histones, due to the result of reducing the cellular
concentration of 2-HG.
3
.3 Compound I-8 induces differentiation in IDH1 mutant cell lines
As previously reported, IDH1 mutations caused increase in intracellular 2-HG
levels, which leads to epigenetic regulation abnormalities and block cell
differentiation (Birendra and DiNardo, 2016). Mutant IDH1 inhibitors could inhibit
the production of 2-HG and reverse the physiological effects of 2-HG. Therefore, we
assessed the effect of I-8 on differentiation of three different IDH1 mutant cells. After
cells were induced to differentiate, the morphology of cells changes and appears as
shuttle type. As shown in Figure 3, the cytoskeleton, utilizing the phalloidin chimeric
rhodamine dye (Actin-Red), showed that the morphology of three IDH1 mutant cell

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lines were significantly changed (irregular rounds become fusiform) after treatment
with I-8 for 72 h and this potential role in differentiation was concentration
dependent.
In addition, to verify the induction of differentiation in detail, markers associated
with HT1080 cells differentiation, including MyoD (Rudnicki et al., 1992), Myf-6,
and α-SMA, were analyzed by using western blotting. The results indicated that
compound I-8 treatment could significantly promote the expression of marker proteins
(Figure 4A). And Immunofluorescence assay showed that the fluorescence intensity
of MyoD1 protein increased with the concentration of compound I-8 (Figure 4C).
Similarly, the astrocytic differentiation markers glial fbrillary acidic protein (Chen et
al., 2013b) (GFAP) and Vimentin (Chen et al., 2006) in U87 MG-IDH1 R132H cells
were examined. After treatment with I-8 for 72 h, it was able to promote the
expression of GFAP protein and reduce the level of Vimentin protein with a
concentration-dependent manner (Figure 4D and 4F). For RBE cells, the higher
degree of differentiation, the more expression of the DLK1 protein (Cai et al., 2016)
and alpha fetal protein (Seino et al., 2018) (AFP). Compound I-8 intervention
improved the expression of DLK1 and AFP, which is consistent with the results of IF
detection of AFP expression (Figure 4G and 4I). In short, these results demonstrated
that compound I-8 could strongly induces differentiation in IDH1 mutant cell lines.
3
.4 Compound I-8 attenuates tumor stem cell characteristics of IDH1 mutant cell
lines
After tumor cells differentiate, the characteristics of tumor stem cells (TSC) are
weakened, and their ability to self-renew and replicate is also reduced (Chen et al.,
013a). Mutant IDH1/2 inhibitors induce cell differentiation while down-regulating
2
the expression of tumor stem cell-associated genes SOX2 (Pusch et al., 2017) and
Nestin (Rohle et al., 2013). Similarly, compound I-8 treatment significantly reduced
the expression of TSC-associated marker proteins SOX2, Nanog and Oct3/4 in
HT1080 cells (Figure 5A). Among them, the expression level of Oct3/4 was further

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detected by immunofluorescence, and the visualized results showed that the
fluorescence intensity of Oct3/4 was decreased after I-8 treatment (Figure 5C). Next,
we evaluated TSC-associated marker proteins in additional endogenous IDH1 mutant
RBE cells. The results showed that after treatment with I-8 for 72 h, the expression
levels of the marker proteins, including SOX2, Nanog and Oct3/4, and fluorescence
intensity of Oct3/4 were also significantly reduced (Figure 5G and Figure 5I). To
further confirm this finding, the engineered IDH1 R132H mutant U87 MG cells was
used to investigate the characteristics of TSC. As expected, compound I-8
significantly reduced the expression of the marker proteins SOX2, Nanog, and Oct3/4,
consistent with immunofluorescence results (Figure 5D and Figure 5F). In summary,
these results revealed that I-8 could significantly attenuates TSC characteristics of
IDH1 mutant cell lines.
3
.5 Compound I-8 represses Human Fibrosarcoma HT1080 xenograft progression in
vivo
All above results suggested compound I-8 was an effective inhibitor of mutant
IDH1 in vitro, and the antitumor efficacy of compound I-8 in vivo was further
evaluated. Balb/C mice were transplanted with human fibrosarcoma HT1080 line
carrying a native IDH1 R132C mutation. The tumor-bearing mice were then divided
into three matched groups including the vehicle control and two compound I-8
treating groups (75, 150 mg/kg) and administrated with I-8 or vehicle via gavage once
a day for 10 consecutive days. As shown in Figure 6A, no significant differences of
average body weights between three groups were observed throughout the study,
which implied that the dosage of I-8 used in this experiment had no obvious side
effects on the growth of mice. However, the unexpected results was that the tumor
volume and tumor weight are also unaffected by the compound I-8 (Figure 6B-6D).
Next, 2-HG concentrations in tumors and serum were examined. As shown in Figure
6
E, compound I-8 intervention significantly reduced intratumoral 2-HG levels
compared to the control group. Compound I-8 at 75 and 150 mg/kg could induce 10%

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and 30% inhibitory of 2-HG production respectively, meanwhile, a similar inhibition
was measured in serum (Figure 6F). Compared to the control group, I-8 treatment
markedly reduced histone methylation levels (including H3k4me3, H3K9me3 and
H3K27me3) in tumor tissue (Figure 6G and 6H). In addition, compound I-8
administration could promote the expression of marker proteins associated with
differentiation (Figure 6I and 6J) and reduce the expression of marker proteins
associated with characteristics of TSC (Figure 6K and 6L). Taken together, these
results suggested that compound I-8 safely exerts an anti-tumor effect by inhibiting
mutant IDH1 in vivo.
Discussion
Mutations in the IDH1 are known to be associated with several human tumor
types, and IDH1 mutant cells produce higher levels of 2-HG relative to WT cells
(Dang et al., 2010). The inhibition of mutant IDH1 enzyme activity to reduce 2-HG
levels may be a potential pathway for therapeutic treatment. Several potent and
specific mutant IDH1 inhibitors have been presented since 2012, which could block
2
-HG production and inhibit tumorigenesis both in vitro and in vivo. Peculiarly,
AG-120 was a selective mutant IDH1 inhibitor and currently approved by the US
FDA for the treatment of AML with IDH1 mutation (Dhillon, 2019). Similarly, we
have got a novel mutant IDH1 inhibitor (I-8) and evaluated the anti-tumor effect.
Compound I-8 inhibited 2-HG production and suppressed histone methylation, and
also induced differentiation and attenuated tumor stem cell characteristics in vitro and
in vivo. Obviously, the novel inhibitor of mutant IDH1, compound I-8, may be a
potential therapeutic strategy in the future.
Initially, we found that compound I-8 had strong inhibitory effect on mIDH1
enzyme activity. Although the IC50 values of I-8 were much higher than the IC50
values of AG-120 inhibition of IDH1 R132H/C (12 and 13 nM) (Popovici-Muller et
al., 2018), compound I-8 has a new chemical structure. Besides, molecular docking
predicted that compound I-8 bind with mutant IDH1 in the ligand binding pocket and

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still have a guiding role in the development of mutant IDH1 inhibitors (Chen et al.,
2
016).
It has been shown that elevated levels of 2-HG inhibits dioxygenases enzymes
associated with regulating epigenetic mechanisms, such as inducing hypermethylation
of histone and chromatin and blocking cell differentiation, and thereby promotes
tumorigenesis (Xu et al., 2011). We found that I-8 could inhibit 2-HG production with
IC value less than 1.5 μM in three different IDH1 mutant cells. These data were
5
0
consistent with previous studies involving inhibitors to reduce 2-HG levels in IDH1
mutant cells (Jones et al., 2016). In addition, we observed that treatment with I-8 led
to significantly decreased histone methylation marker levels in all IDH1 mutant cells.
These fndings were analogous to what has been described for H3K9me2 by GSK321
in IDH1 R132C expressing HT1080 cells (Okoyeokafor et al., 2015) and H3K4me3,
H3K9me3 and H3K27me3 marks by BAY1436032 in primary IDH1 mutant AML
(Chaturvedi et al., 2017). It is conceivable that I-8 could restores the activities of
histone demethylases and avoids the wide hypermethylation of histones through
reducing the level of 2-HG.
To a certain extent, the differentiation state of tumor cells is negatively correlated
with the degree of malignancy (Kaminska et al., 2019; (Swartling et al., 2015).
Therefore, inducing differentiation may be an effective strategy to reduce the
malignant degree of tumors. As expected, compound I-8 induced differentiation in
three IDH1 mutant cells cultured in vitro. After treatment with I-8 for 72 h, the
expression levels of differentiation-associated marker proteins (such as Myf-6, GFAP
and DLK1) were up-regulated in three different IDH mutant cells, and the
morphology of those cells could also be changed into shuttle type. These findings
were similar to the results seen in previous studies that other IDH1 inhibitors,
AGI-5198 (Rohle et al., 2013), GSK321 (Okoyeokafor et al., 2015) and AG-120
(Birendra and DiNardo, 2016), have been shown to induce differentiation of IDH1
mutant glioma or AML cells. What’s more, the characteristics of TSC were gradually
weakened, and their ability to self-renew and replicate was also reduced with the
differentiation of tumor cells. Several marker proteins associated with TSC, including

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SOX2, Nanog and Oct3/4, were decreased after treatment with I-8 in IDH1 mutant
cells. These results further confirm that, similar to inhibition of BAY 1436032 (Pusch
et al., 2017), compound I-8 could induce tumor cell differentiation, impair TSC
characteristics, and repress tumor progression.
We used BALB/c mice to establish a Human Fibrosarcoma HT1080 xenograft
model to evaluate the anti-tumor effect of I-8 in vivo. However, just as the compound
I-8 did not inhibit the proliferation of the IDH1 mutant cell line in vitro, the
compound I-8 did not change the tumor volume and tumor weight in vivo, whether it
was a dose of 75 mg/kg or 150 mg/kg. This result may suggested that the I-8 is not
toxic for cells or animals. Besides, IDH1 mutation inhibitors AGI-5198(10μM)
(Johannessen et al., 2016) did not inhibit cell proliferation and BAY-1436032(150
mg/kg) (Pusch et al., 2017) also had no significant inhibitory effect on tumor
proliferation, and I-8 had similar effects. On the one hand, the highest dose of I-8 was
1
50 mg/kg in our study, we speculate that dose is low or administration time is short.
A better anti-tumor effect may be obtained if increase the dose or prolong the time of
administration in subsequent studies. On the other hand, we hypothesize that due to
the poor solubility of compound I-8 in water, the plasma drug concentration after
intragastric administration is too low to inhibit tumor growth. So pharmacokinetic
experiments will need to be implemented in the future, and the structural optimization
of compound I-8 should also be carried out to improve its physical and chemical
characteristics. It is worth noting that I-8 treatment could significantly reduce the
level of 2-HG in tumor tissue and serum, which could be an important indicator for
clinical drug development (Kopinja et al., 2017).
In summary, we have developed a novel compound, I-8, which specifically
inhibits the IDH1 mutation mediated reduction of α-KG to 2-HG, and demonstrated its
pharmacological inhibition on mutant IDH1 enzyme both in vitro and in vivo. In
future study, more detailed action mechanisms of compound I-8 will be investigated
and more derivatives of compound I-8 will be synthesized followed by structure
guided lead optimization.

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Conflict of interest
All authors declare no conflicts of interest.
Acknowledgements
This work were supported by the National Natural Science Foundation of China
(
No. 81773766 and No. 81571785), the Natural Science Foundation of Jiangsu
Province (BK20161458), Six Talent Peaks Project in Jiangsu Province (No.
016-YY-042), the "Double First-Class" University project (No. CPU2018GY38), the
2
TCM research project of Health Commission of Hubei Province (No. ZY2019Q005),
the National Key Research and Development Program of China (No.
2
017YFA0205200), Graduate Student Research Innovation Projects in Jiangsu
Province (KYCX17_0691). We would also like to thank Prof. Qinxi Li for kindly gift
of U87 MG-IDH1 R132H.
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Figure Legends
Figure 1 The synthetic route and biochemical activity of compound I-8.. (A). Synthetic route
of compound I-8. Reagents and conditions: (i) NaH, DMF, 1 h; (ii) Pd[P(Ph) ] , Cs CO ,
3
4
2
3
1
,4-dioxane/H O, 90 °C, 5 h; (iii) DIPEA, DMSO, 110 °C, 3 h. (B) Molecular docking analysis of
2
compound I-8 binding to IDH1 R132H (PDB ID: 5TQH). (C) and (D) Effect of compound I-8
inhibiting IDH1 R132C/H were assessed with different concentration (0.01 μM, 0.1 μM, and 1
μM). (E), (F) and (G) IC50 values of compound I-8 on the IDH1 R132C/H and IDH1 wild-type
(IDH1 wt) were measured. (H) The binding mode of compound I-8 with IDH1 R132C was
competitively. Substrate α-KG concentration was increased to 10 μM from 1 μM and IC value of
5
0
compound I-8 inhibiting the IDH1 R132C was measured. Data were shown as the mean ± SD of
three independent experiments.
Figure 2 Compound I-8 inhibits 2-HG production and inhibits histone methylation in IDH1
mutant cells. (A) HT1080 cells, U87 MG-IDH1 R132H cells and RBE cells were treated with I-8
for 72 h, and MTT assay was performed to analyze the cell viability. (B) IDH1 mutant cells treated
with I-8 for 72 h and extracellular 2-HG level was detected by LC-MS/MS. (C-H) Effect of
compound I-8 on the expression of H3K4me3, H3K9me3, H3K27me3 in HT1080 cells (C), U87
MG-IDH1 R132H cells (E) and RBE cells (G) at 72 h were detected by western blotting and the
relative protein expression was quantified by densitometric analysis. Data were expressed as
means ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control
group.
Figure 3 Compound I-8 induced IDH1 mutant cell lines differentiated-appearing
morphology. Cells were seeded into 24-well plate with coverslips and treated with or without
compound I-8 and AG-120 for 72 h. The morphology was detected by confocal microscopy. (A)
HT1080 cells, (B) U87 MG-IDH1 R132H cells and (C) RBE cells.
Figure 4 Compound I-8 induces differentiation in IDH1 mutant cell lines. Cells were seeded
into 6-well plate and treated with or without compound I-8 and AG-120 for 72 h. The expression

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of Myf-6, MyoD1 and α-SMA in HT1080 cells (A), GFAP and Vimenrin in U87 MG-IDH1
R132H cells (D), AFP and DLK1 in RBE cells (G) were detected by western blotting. The
differentiation- related proteins in HT1080 cells (C), U87 MG-IDH1 R132H cells (F) and RBE
cells (I) were analyzed by immunofluorescence. Data were expressed as means ± SD of three
independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group.
Figure 5 Compound I-8 attenuates tumor stem cell characteristics of IDH1 mutant cell lines.
Cells were seeded into 6-well plate and exposed with or without compound I-8 and AG-120 for 72
h. Effect of compound I-8 on the expression of SOX2, Nanog and Oct3/4 in HT1080 cells (A),
U87 MG-IDH1 R132H cells (D), and RBE cells (G) were detected by western blotting. The
TSC-related proteins Oct3/4 in HT1080 cells (C), U87 MG-IDH1 R132H cells (F) and RBE
cells (I) were analyzed by immunofluorescence. Data were expressed as means ± SD of three
independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group.
Figure 6 Compound I-8 represses Human Fibrosarcoma HT1080 xenograft progression in
vivo. Balb/C mice were transplanted with human fibrosarcoma HT1080 line carrying a native
IDH1 R132C mutation. Compound I-8 (75, 150 mg/kg) or vehicle control was administered once
a day for 10 consecutive days. (A) Body weight change was analyzed. (B-D) Tumor volume and
weight were recorded. (E) and (F) The level of 2-HG in tumor tissue and serum was measured by
LC-MS/MS. (G) and (H) The protein levels of H3K4me3, H3K9me3, H3K27me3 in tumor tissue
were analyzed by western blotting and the relative protein expression was quantified by
densitometric analysis. (I) and (J) Markers associated with differentiation in tumor tissue,
including MyoD1 and Myf-6, were analyzed by using western blotting. (K) and (L) The protein
levels of Oct3/4 and Nanog were associated with characteristics of TSC were analyzed by western
blotting. Data were expressed as means ± SD of six mice in each group. *P < 0.05, **P < 0.01 vs.
control group.

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