Discovery of new small molecule inhibitors targeting isocitrate dehydrogenase 1 (IDH1) with blood-brain barrier penetration

Article abstract of DOI:10.1016/j.ejmech.2019.111694

Isocitrate dehydrogenase 1 (IDH1), which catalyzes the conversion of isocitrate to α-ketoglutarate, is one of key enzymes in the tricarboxylic acid cycle (TCA). Hotspot mutation at Arg¹³² in IDH1 that alters the function of IDH1 by further converting the α-ketoglutarate(α-KG) to 2-hydroxyglutarate (2-HG) have been identified in a variety of cancers. Because the IDH1 mutations occur in a significant portion of gliomas and glioblastomas, it is important that IDH1 inhibitors have to be brain penetrant to treat IDH1-mutant brain tumors. Here we report the efforts to design and synthesize a novel serial of mutant IDH1 inhibitors with improved activity and the blood-brain barrier (BBB) penetration. We show that compound 5 exhibits good brain exposure and potent 2-HG inhibition in a HT1080-derived mouse xenograft model, which makes it a potential preclinical candidate to treat IDH1-mutant brain tumors.

Full text of DOI:10.1016/j.ejmech.2019.111694

Journal Pre-proof
Discovery of new small molecule inhibitors targeting isocitrate dehydrogenase 1
(IDH1) with blood-brain barrier (BBB) penetration
Hengyi Cao, Guangya Zhu, Lin Sun, Ge Chen, Xinxin Ma, Xiao Luo, Jidong Zhu
PII:
S0223-5234(19)30844-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.111694
EJMECH 111694
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 June 2019
Revised Date: 8 September 2019
Accepted Date: 10 September 2019
Please cite this article as: H. Cao, G. Zhu, L. Sun, G. Chen, X. Ma, X. Luo, J. Zhu, Discovery of
new small molecule inhibitors targeting isocitrate dehydrogenase 1 (IDH1) with blood-brain barrier
(BBB) penetration, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.111694.
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2019 Published by Elsevier Masson SAS.

Discovery of new small molecule inhibitors
targeting isocitrate dehydrogenase 1 (IDH1)
with blood-brain barrier (BBB) penetration
Hengyi Cao, a,b,†Guangya Zhu, a,b, Lin Sun, Ge Chen, Xinxin Ma, Xiao Luo, Jidong Zhu
a
a
a
a
†
a,*
a
Interdisciplinary Research Center on Biology and Chemistry, Center for Excellence in
Molecular Synthesis, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences,
Shanghai 201203, China.
b
University of the Chinese Academy of Sciences, Beijing 100049, China.
†
These authors contributed equally.
*
Email: zhujd@sioc.ac.cn
KEYWORDS: Isocitrate dehydrogenase 1, small molecule inhibitors, glioblastoma, acute
myeloid leukemia, blood-brain barrier, cancer therapy
GRAPHIC ABSTRACT

ABSTRACT
Isocitrate dehydrogenase (IDH), which catalyzes the conversion of isocitrate to
α-ketoglutarate, is one of key enzymes in the tricarboxylic acid cycle (TCA). Abundant
evidence has shown that IDH mutations occur in various tumors. Substitution of specific
amino acids, such as R132 in IDH1 or R172 in IDH2, in the active site alters the function of
IDH, converting the α-ketoglutarate(α-KG) to 2-hydroxyglutarate (2-HG) and leading to
abnormal cell metabolism. Because the IDH mutations involve multiple brain tumors, IDH
inhibitors have to breach the blood-brain barrier (BBB) and reach the site of action at the
minimal effective concentration in order to treat brain tumors. The requirement may limit the
clinical application in glioblastoma of the current IDH1 inhibitor, for example, AG-120.
Consequently, we have designed and synthesized a novel serial of mutant IDH1 inhibitors
with improved activity and BBB penetration. Compound 5 exhibits excellent inhibitory
activity both in the enzyme (R132C IC = 8.2 nM; R132H IC = 4.0 nM) and in the HT1080
5
0
50
cell line (IC = 15.9 nM). It also shows good BBB penetration with satisfactory PK and PD
5
0

profiling in a CDX model, which makes it a potential preclinical candidate to treat
glioblastoma.
INTRODUCTION
Isocitrate dehydrogenase (IDH) is a key enzyme in the tricarboxylic acid (TCA) cycle and
plays an important role in energy metabolism.[1] In humans, there are three isoforms of IDH
enzyme (IDH1, IDH2, IDH3).[2,3] IDH1, present mainly in the cytosol, is NADP-dependent
and performs the conversion of isocitrate to α-ketoglutarate (α-KG) through an oxidative
decarboxylation process.[4] With hotspot mutations in the active site, such as R132H and
R132C, IDH1 performs an erroneous catalytic conversion and accumulates the
oncometabolite (R)-2-hydroxyglutatrate (R-2-HG).[5–9]
Increased R-2-HG levels not only act as a biomarker for accurate diagnosis of the IDH
mutation,[10,11] but also promote tumor formation through a variety of mechanisms.
Numerous studies have confirmed that R-2-HG can affect many cellular enzymes that
regulate histone and DNA methylation levels, and this in turn is highly associated with
tumorigenesis.[12–14] Most recently, the crucial role of R-2-HG in immunity has been
reported.[15,16] R-2-HG may also acts as immune suppressor in the tumor
microenvironment,[17] which suggests a significant antitumor potential stemming from
inhibition of mutant IDH.
In the last decade, with the development of large-scale tumor genetic sequencing and the
development of precision medicine, IDH mutations with the neomorphic activity have been
identified as the important driving mutations in a variety of cancers, including malignancies

such as acute myeloid leukemia (AML),[6,18–20] gliomas,[8,10,21,22] and intrahepatic
cholangiocarcinoma.[23] Secondary glioblastoma multiforme (secondary GBM) is an
extremely malignant cancer that begins in the brain. At present, the treatment of GBM is
limited to surgery and radiotherapy.[24] Because most antitumor drugs have difficulty
passing the blood-brain barrier, there are few effective drugs in this context and the
application of some tyrosine kinase inhibitors as second-line treatment is also apparently
subject to certain obstacles.[25] An oral alkylating agent, temozolomide, widely used in
GBM chemotherapy, has been shown to be more effective in patients with MGMT
methylation.[26–28] Despite intense treatment with temozolomide, the cancer usually recurs
and the median survival time is only 15 months.[29] There is therefore an urgent need for
new drugs that can effectively control this serious disease. Since IDH1 mutations are present
in more than 70% of low-grade gliomas and secondary glioblastoma,[30] IDH inhibitors
could bring new breakthroughs in the treatment of this type of cancer. Acute myeloid
leukemia (AML) is another fatal leukemia caused by arrest of differentiation of the immature
myeloblast and can progress very rapidly. With the help of tumor genomic analysis, some
specific mutations that lead to AML have been identified and mutant IDH has been shown to
block cell differentiation.[31,32] Thus inhibitors against IDH mutations are designed for use
in AML patients with IDH mutations, a group which accounts for 20% of all AML
patients.[32] At present, with the AG-221 (enasidenib), a mutant IDH2 inhibitor, approved by
the US FDA, this treatment concept targeting mutant IDH has been affirmed.[33]
Currently, some IDH1 inhibitors are undergoing clinical trials, among which AG-120
(ivosidenib) and IDH-305 have progressed considerably. Both of these compounds can

significantly reduce D-2-HG levels in mice with xenograft tumors.[34,35] Compared to
AG-120, IDH-305 appears to be superior in respect of BBB penetration. Therefore, based on
IDH-305, we carried out further chemical modification by means of computer-assisted drug
design (CADD). In order to enhance the affinity of the compounds to the mutant IDH1, we
attempted to link the bipyridyl moiety of IDH-305 and change the dihedral angles in the
structure to produce a new π-π stacking with the W267 residue in IDH-305. Compound 5
exhibits great biochemical and cellular activities. The pharmacokinetic (PK) and
pharmacodynamics (PD) profiles were also evaluated in mouse models. Compound 5
effectively lower the 2-HG level in vivo. Notably, 5 is brain permeable and holds great
potential as a preclinical candidate to treat brain tumors with IDH1 mutations.
RESULTS AND DISCUSSION
Design strategies
In the initial stage of structural modification, we used the Maestro program in Schrödinger
software to explore the appropriate transformation method. According to the reported X-ray
co-crystal structure (PDB: 6b0z), consisting of compound IDH-305 and protein mutant IDH1,
there are 4 hydrogen bonds within the pocket and π-π stacking between the W124 and the
pyrimidine ring (Figure 1A).[35] The red area shows key atoms that participate in hydrogen
bonds and these core areas were retained before beginning further modification. Docking to
the protein template of some hypothetical small molecular structures was then examined. The
first alternative began with a connection to the bipyridyl moiety. A six-membered alkyl ring
was thought to be the most suitable ring with which to create a new π-π stacking between the

terminal pyridyl ring and W267. However, the calculated results show that this may conflict
with W124 and cause a conformational change (Figure 1B and 1C). We then resized the
internal pyridyl ring to a five-membered imidazole and found that the calculated results
support a new π-π stacking without such a clash (Figure 1E). We also tried to change the
trifluoromethyl group to chlorine and a carbon to oxygen. The product docked in the pockets
with a similar pose to that of IDH-305 (Figure 1D). These changes created a smart dihedral
angle (12.7° in compound 1 and 16.8° in compound 5), which are both much smaller than the
angle in IDH-305 (55.3°). The saturated heterocyclic ring can effectively reduce the dihedral
angle between the two aromatic rings while retaining a certain flexibility. This new π-π
stacking appears to lead to improvement in the activity of compounds.

Figure 1. (A) Conceptual design of new compounds. (B, C) Docking and possible binding
modes shown with the aid of Maestro software. A six-member alkyl ring between the
bipyridyl moiety may conflict with W124 and cause conformational change. All molecule
backbones are shown in green. The R132H IDH1 backbone comes from PDB bank (PDB:
6
b0z) and is grey. The π-π stacking is shown with blue dashed lines. Steric clashes are shown
as red dashed lines. Hydrogen bonds are shown as yellow dashed lines. (D) Compound 1
docks with mutant IDH1. (E) Compound 5 docks with mutant IDH1.

Chemistry
Compounds 1-24 were synthesized and subjected to structure-activity relationship (SAR)
studies. All these compounds consist of different amino side chains with a heterocyclic-fused
structure and, in addition, a 4-oxazolidinone pyrimidine moiety. Both fragments were
synthesized separately following the general route described in Scheme 1. For modification
of the amino side chains, we used different commercially available materials to synthesize
lactams as the starting substrates. Imidazole rings were then formed with diethyl
chlorophosphite and methyl isocyanoacetate. After DIBALH reduction to aldehydes
(
Dess-Martin periodinane was used to counter overreduction), a methyl group was introduced,
creating a chiral carbon, by a Grignard reagent with the help of chiral induction of
S)-tert-butylsulfinamide. Amino side chains were successfully formed after alcoholysis of
(
the inductive group. The synthesis of different substituted oxazolidinones uses the same
published route as was used for IDH-305 and its derivatives [35]. Finally, the oxazolidinone
was linked to the halogenated pyrimidine and the two fragments were combined to obtain the
desired compounds 1-24 (Table 1-4).

Scheme 1. General route for synthesis of compounds 1-25. Reagents and reaction conditions:
(
I) NaHMDS, diethyl chlorophosphite, methyl isocyanoacetate, THF, -30℃ , 2 h (II) DIBALH
DMP for overreduction), DCM, -78℃ , 40 min (III) (S)-(-)-2-Methyl-2-propanesulfinamide,
(
Ti(OEt) , THF, RT, overnight (IV) MeMgBr, THF, -78℃ , 1 h (V) HCl, MeOH, rt, 2 h (VI)
4
NaH, DMF, RT, 2h (VII)DIPEA, DMSO, 100℃ , 1.5 h.
SAR studies
A recombinant IDH1 enzyme assay was used to assess the ability of all compounds to inhibit
the enzymatic activity of mutant IDH1. Diaphorase and resazurin can induce a secondary
reaction for accurate quantification of NADPH residuals. Cellular activity was determined in
HT1080 cells by a commercially available kit. The amount of 2-HG present in the samples
can be measured directly. A selection of compounds with good enzymatic inhibitory activity

(
IC <20 nM) were tested for cellular activity.
50
Compounds 1-5 were synthesized and tested in the enzyme assay and all were found to have
good potency (Table 1). The results also met the expectations of the computer-aided design.
Some of them have even lower IC values than AG-120 and they are all better than the
5
0
prototype compound IDH-305. Compound 3, with IC = 1.0 nM has the highest enzyme
5
0
inhibitory activity. The structural changes among compounds 1, 2, 4, 5 do not cause
significant changes in enzyme inhibitory activity. However, substitution of a trifluoromethyl
group leads to better cellular activity than chlorine substitution. The oxygen substitution in
heterocyclic ring has less impact. Further structural modification and screening based on
compound 1-5 continued. The structural modification area is mainly divided into the four
groups, X, R , R and R .
1
2
3
Table 1. Recombinant IDH1 Enzyme Assay Activity and 2-HG Inhibition Assay Activity of
a
compound 1-5
Recombinant IDH1
2
-HG
Enzyme Assay
resazurin/diaphorase/NA
inhibiton in
HT1080
(
b
Compou
nd
b
Structure
DPH system)
R132C IC50 R132H IC50
IC (nM)
5
0
(nM)
(nM)
1
14.4
2.2
60.3

2
3
4
5
9.4
9.3
15.3
8.2
2.7
1.0
2.3
4.0
12.0
12.5
53.7
15.9
IDH-305
AG-120
50.2
23.0
49.6
3.5
52.7
25.4
a
b
See the experimental section for assay details. The given IC values are mean values of at
5
0
least three experiments. The deviations were within 10%.
In group X (Table 2), the ethylene group was replaced by a methyl group (10, 14) or by a
suitable bioisostere group (8, 13, 21). Due to the steric limitations imposed by I128 and
W124 (Figure 1B), any expansion results in loss of potency, which is consistent with the
results of the CADD. Compound 8 shows a slightly increased cellular activity with a smaller
sulfur atom linker. This may result from the enhanced planarity of its amino side chain (8:
ClogP=3.69 vs. 5: ClogP=3.22). However, the structural rigidity may be too strong to create a
new π-π stacking with W267 and this will finally limit its enzyme activity (Figure 2A).
Compound 13 has a more flexible seven-membered ring, which may result in triple π-π

stacking with W124 (Figure 2B). Therefore, a saturated six-membered ring was determined
to be the most suitable linking group for this spatial region. It is flexible, producing a suitable
dihedral angle (1: 12.7°, 5: 16.8° π-π stacking with W267).
Table 2. Recombinant IDH1 Enzyme Assay Activity and 2-HG Inhibition Assay Activity and
a
calculated dihedral angles of compound 8, 10, 13, 14, 21
2
-HG
Recombinant IDH1
Enzyme Assay
Inhibiton
in
Calculate
d
Dihedral
Angle
Calculate
d π-π
stacking
(
resazurin/diaphorase/
Comp X Group
HT1080
b
NADPH system)
b
ound
Structure
R132C
R132H
IC (nM)
5
0
IC (nM) IC (nM)
5
0
50
8
-S-
-0.3°
None
None
W124
None
17.3
93.5
43.5
119.1
24.1
19.2
9.7
-
-
17.7° or
18.5°
1
1
1
2
0
3
4
1
-CH(CH
3
)CH
2
-
128.5
44.3
-CH
-CH
CH
CH
-
-49.5°
35.5
17.0
2
2
2
-
-
15.2° or
19.5°
CH(CH
)-
119.8
23.8
2
3
-SCH
-
23.0°
W267
2
a
b
See the experimental section for assay details. The given IC values are mean values of at
5
0
least three experiments. The deviations were within 10%.

Figure 2. (A) Compound 8 docks with mutant IDH1. (B) Compound 13 docks with mutant
IDH1.
Then we also tested different substituents on the terminal benzene ring. According to the
SAR of IDH-305, the group R is critical to the physicochemical properties of the compound.
1
Low polarity substituents, such as tert-butyl, will increase the penetration to brain but such
compounds will be easily cleared [35]. Therefore, some other hydrophobic groups were tried
(Table 3). When compounds 1, 5, 23, 24 were compared, it was found that, in addition to
hydrophobic interactions, strong electron-withdrawing effects are also important contributors
to the activity of the compound and it was suspected that the trifluoromethyl group reduces
the electron density of the benzene ring and favors the formation of the π-π stacking with
W267. Compound 11, 12, 22 were synthesized as compounds with rich or medium electronic

substitution but they failed to show the same level of activity as compound 2. The change in
the position of the trifluoromethyl group (15) leads to maintenance of the activity level but
substitution by the 4-chlorine (9) decreased the activity.
Table 3. Recombinant IDH1 Enzyme Assay Activity and 2-HG Inhibition Assay Activity of
a
compounds 9, 11, 12, 15, 23-25
Recombinant IDH1
Enzyme Assay
2-HG inhibiton
Structure
R1
b
(
resazurin/diaphorase/NA in HT1080
Compou
nd
b
DPH system)
R132C
R132H
X
IC (nM)
50
IC (nM)
IC (nM)
5
0
50
9
4-Cl
-CH
2
CH
2
-
81.9
24.8
393.9
8.0
24.1
33.4
478.5
18.4
61.9
42.7
47.8
11
3-cyclopropyl
3-cyano
-OCH
2
2
-
-
166.6
13.2
21.9
1
1
2
2
2
2
5
2
3
4
-OCH
2-CF
3-OCH
-CH
-OCH
-CH CH
-OCH
CH
-
-
3
2
2
-
153.8
16.3
44.5
3
2
3-CH
3-F
3
2
2
-
2
a
b
See the experimental section for assay details. The given IC values are mean values of at
5
0
least three experiments. The deviations were within 10%.

Compounds 6 and 7 with smaller halogen substitution of R on the pyrimidine ring (Table 4),
2
such as fluorine or chlorine, are both tolerated. At group R , most other substitution on
3
oxazolidinones failed to improve the potency. Surprisingly, compound 20 with a phenyl
substituent has the highest potency in cells (IC = 6.6 nM). This may be due to the further
5
0
improvement of ClogP (3.86). Although current research has difficulty providing an accurate
of its conformation in the pocket, this may provide new ideas for further structural
modification.
Table 4. Recombinant IDH1 Enzyme Assay Activity and 2-HG Inhibition Assay Activity of
a
compound 6, 7, 16-20
6
N
R2
N
HN
5
N
N
O
N
F
F
O
F
R3
2
-HG
Recombinant IDH1
Enzyme Assay
inhibiton
in
Structure
(
resazurin/diaphorase/N
Compou
nd
HT1080
b
ADPH system)
b
R132C
R132H
R2
R3
IC (nM)
50
IC (nM)
IC (nM)
5
0
50
6
7
5-F
17.0
23.6
2.1
20.3
34.6
6-Cl
2.12
1
6
9.7
33.8
140.5

1
1
1
7
8
9
0
15.0
18.8
5858
14.2
42.0
86.8
26.3
50.8
>10000
15.5
2
6.6
a
b
See the experimental section for assay details. The given IC values are mean values of at
5
0
least three experiments. The deviations were within 10%.
DMPK results
Because of the excellent activity in the recombinant IDH1 enzyme assay of both R132C and
R132H, compounds 1-6 were selected for further in vivo pharmacokinetic testing and
comparison with AG-120 (Table 5). Trifluoromethyl-substituted compounds (2, 5) not only
have better R132C inhibitory activity and 2-HG inhibitory activity than chlorine-substituted
compounds (1, 4) (Table 1), but also have lower clearance, higher exposure and
bioavailability. Despite its isopropyl substitution in the R region (3) has a more enhanced
3
ability to inhibit the R132H IDH1 enzyme than the fluoroethyl group, but its clearance is
increased significantly and its AUC is extremely low. This is consistent with the study of
IDH-305.[35] Compound 6, with a fluorine substituent on the pyrimidine, has a similar

pharmacokinetic profile to that of compound 5. After 1 mg/kg IV administration in mice,
-1
-1
compound 5 showed a moderate plasma clearance (23±1 mL min kg ), T (1.5 ± 0.1 hr)
1
/2
-1
and MRT (1.6 ± 0.1 hr). Its mean Vss was 2.3 ± 0.2 L kg . About 30 min after PO
administration of 5 mg/kg (Tmax 0.4 ± 0.1 hr), the plasma concentration of compound 5
reached Cmax (1690 ± 241 nM). The PO exposure (AUClast 11041 ± 2593 nM*hr) was close
to AG-120 in our study. Compound 5 was also considered to be the most promising
compound for higher dosage PK profiling. A xenograft model derived from HT1080 cells was
established for PD testing. We measured the concentration of compound 5 in plasma, brain
and tumor tissue and compared them with those of AG-120 (Figure 3A-3C). Although the
plasma exposures of compound 5 and AG-120 in plasma were relatively close, the exposure
of compound 5 was significantly higher than that of AG-120 in the brain and the tumor. This
indicates that compound 5 has better targeting ability and has greater potential for use in the
treatment of brain tumors. The same CDX model was used for PD testing. It was found that
compound 5 could achieve a more effective R-2-HG reduction than AG-120 within 3-20 h
after administration (Figure 3D).
a
Table 5. In vivo mouse PK profiles of compound 1-6 and AG-120.
Compound
Cl(mL min
1
2
3
4
5
6
AG-120
9
-1
3
9
30
109
50
23
21
-1
kg )
-
1
Mouse Vss(L kg )
IV
2.3
1.0
1.0
5.2
2.1
2.9
3.0
0.5
0.4
4.3
1.7
1.4
2.3
1.5
1.6
2.1
1.4
1.6
1.5
2.5
2.8
T (hr)
1
/2
MRTinf(hr)

AUClast(hr*
nM)
9
17
30
1022
1113
312
323
717
735
1439
1472
1520
1543
2972
3258
AUCinf(hr*n
M)
9
Tmax(hr)
Cmax(nM)
T (hr)
0.4
1.7
0.3
730
2.0
0.3
932
3.4
0.4
0.9
0.4
1206
2.4
1122
2.5
1690
2.5
1703
2.5
2264
3.5
1
/2
Mouse
PO
AUClast(hr*
nM)
5
008
048
8464
1650
4019
11041 12700 11162
11057 12719 12952
AUCinf(hr*n
M)
5
9237
152
1761
109
4673
117
F(%)
109
150
165
80
a
Data represents the mean of three replicates.
Figure 3. (A-C) Drug concentration in plasma, brain and tumor tissues tested in HT1080
xenograft mouse model after single dose. (D) Free and estimated total plasma compound 5
concentration (mean±SD) was presented following a single 50mg/kg dose of compound 5.
Percent inhibition of 2-HG (mean±SEM) in HT1080 xenograft tumor samples in the case of

the single dose of 50 mg/kg and 150 mg/kg respectively. AG-120 was compared at a single
0 mg/kg dose.
5
CONCLUSION
AML and secondary GBM are two very difficult tumors and current clinical drug treatment
options are very limited. IDH1 and IDH2 are both considered to be promising drug targets for
the treatment of these two malignancies and the IDH2 targeted therapeutic drug, enasidenib,
has been successfully marketed. Through computer aided drug design, a promising π-π
stacking with W267 was revealed that may enhance the activity in the co-crystal structure of
IDH-305. To verify this hypothesis, we designed and synthesized compounds 1-24, analogs
with saturated heterocyclic linking groups. All compounds were screened by the recombinant
IDH1 enzyme assay and the best were selected for cellular activity and PK profiling
comparison. The modelling studies assisted the design of a first group of compounds with
linkers in six-membered rings (compounds 1-5). All five compounds exhibit excellent
inhibitory potency against mutant IDH1. Compound 3 has the best activity but is readily
metabolized in vivo. Another 20 compounds were designed for in-depth exploration of their
SARs. A trifluoromethyl group on the benzene ring may produce a more favorable π-π
stacking and a moderately flexible linking group also contributes to the proper dihedral angle.
Further structural modifications can be made with compound 6 and compound 20 because
they maintain relatively good activity and have more substitutable positions.
Compound 5 demonstrated good pharmacokinetic properties in the single-dose PK assay.
Subsequently the pharmacokinetic profile with higher dosages and pharmacodynamics profile

were realized in the CDX model. Compound 5 exhibits higher exposure than AG-120 in the
brain and tumor and significantly reduces R-2-HG in tumors, suggesting good blood-brain
barrier permeability and therapeutic value in brain tumors with IDH1 mutation. More
experiments for toxicity and selectivity will be conducted and verified in the future.
EXPERIMENTAL SECTION
General methods for Chemistry All reagents and starting materials were either
commercially available or prepared according to literature reported procedures. All reactions
were monitored by thin layer chromatography (TLC) on silica gel 60 F254 plates and
visualized under UV light or used KMnO staining. NMR spectra were obtained on a
4
1
13
19
Bruker AV-400 spectrometer ( H NMR at 400 MHz, C NMR at 101 MHz, and F NMR at
3
76 MHz). Chemicals shifts (δ) were reported in parts per million and referenced to the
1
13
residual solvent peaks (CDCl 7.26 ppm in H NMR spectra and 77.16 ppm in C NMR).
3
Coupling constants (J) values were in hertz, and the splitting patterns were abbreviated as
follows: s for singlet; b for broad; d for doublet; t for triplet; q for quartet; and m for multiplet.
Yields were of purified product and were not optimized. Purity of all tested compounds was
higher than 95%. Purity and low resolution electrospray ionization (ESI) mass spectra were
recorded on an Agilent 1260/6120 ES-LCMS system. The column was a Phenomenex Luna
C18, 100A, 2.0 50 mm, 5 µm. Mobile phase: A (H O + 0.1% HCOOH); B (ACN). Gradient:
2
3
0% B increase to 95% B within 1 min, 95% B to 100% B within 0.1 min; 100% B for 2.4

min. Flow rate 0.50 mL/min. Column Temperature: 40 ℃ . Sig=254nm. High resolution
ESI-MS were performed on an Thermo Fisher Scientific LTQ FTICR-MS instrument.
General method for combination of amino side chains and oxazolidinone fragments.
a (less than 100mg, 1.0 equiv) and g (1.2 equiv) were dissolved in anhydrous DMSO (1.5mL).
DIPEA (3 equiv) was added and the mixture was heated to 100 ℃ for 1.5 h. The mixture was
then extracted with EtOAc and H O. The organic layer was washed with H O and saturated
2
2
NaCl solution and dried over Na SO . The mixture was then concentrated and purified via
2
4
flash chromatography on silica gel to get the desired compounds (1-24). All final products
were lyophilized to give out white powder.
(R)-3-(2-(((S)-1-(7-chloro-4H-benzo[b]imidazo[1,5-d][1,4]oxazin-3-yl)ethyl)amino)pyrimidi
n-4-yl)-4-((S)-1-fluoroethyl)oxazolidin-2-one (1)
Compound 1 was synthesized from 1a and 1g. 1a was synthesized from
7
-chloro-2H-benzo[b][1,4]oxazin-3(4H)-one in a similar progress to that described for the
preparation of 2a.
1
H NMR (400 MHz, Chloroform-d) δ 8.19 (d, J = 5.8 Hz, 1H), 7.89 (s, 1H), 7.46 (d, J = 5.8
Hz, 1H), 7.33 (d, J = 8.5 Hz, 1H), 7.07 (d, J = 2.1 Hz, 1H), 7.01 (dd, J = 8.5, 2.2 Hz, 1H),
5
.68 (br, 1H), 5.37 – 4.78 (m, 4H), 4.74 – 4.58 (m, 1H), 4.51 (d, J = 3.3 Hz, 1H), 4.36 (t, J =
1
9
9
.0 Hz, 1H), 1.60 (d, J = 6.9 Hz, 3H), 1.24 (br, 3H). F NMR (376 MHz, Chloroform-d) δ

1
3
-
197.63 . C NMR (101 MHz, Chloroform-d) δ 160.78 , 159.04 , 157.20 , 154.21 , 146.60 ,
1
38.91 , 132.05 , 130.52 , 122.80 , 119.01 , 117.57 , 116.26 , 99.53 , 87.25 (d, J = 174.4 Hz),
6
2.45 , 61.86 (d, J = 6.3 Hz), 57.70 (d, J = 19.6 Hz), 44.62 , 21.39 , 16.83 (d, J = 21.6 Hz).
+
ES-LCMS m/z: 459.0 (M+H) . HRMS m/z: calcd for C21H21ClFN6O3 459.1342; found,
4
59.1341. 99.63% purity.
(R)-4-((S)-1-fluoroethyl)-3-(2-(((S)-1-(7-(trifluoromethyl)-4H-benzo[b]imidazo[1,5-d][1,4]ox
azin-3-yl)ethyl)amino)pyrimidin-4-yl)oxazolidin-2-one（2）
Compound 2 was synthesized from 2a and 1g.
1
H NMR (400 MHz, Chloroform-d) δ 8.11 (d, J = 5.8 Hz, 1H), 7.90 (s, 1H), 7.44 (d, J = 8.1
Hz, 1H), 7.38 (d, J = 5.8 Hz, 1H), 7.27 – 7.19 (m, 2H), 6.70 – 5.40 (br, 1H), 5.34 – 4.78 (m,
4
1
H), 4.59 (dd, J = 26.7, 8.5 Hz, 1H), 4.42 (dd, J = 8.9, 3.3 Hz, 1H), 4.29 (t, J = 9.0 Hz, 1H),
1
9
.55 (d, J = 6.9 Hz, 3H), 1.17 (d, J = 17.8 Hz, 3H). F NMR (376 MHz, Chloroform-d) δ
13
-
62.57 , -196.92 . C NMR (101 MHz, Chloroform-d) δ 160.80 , 158.92 , 157.17 , 154.15 ,
45.89 , 139.37 , 130.85 , 128.98 (q, J = 33.3 Hz), 126.55 , 123.41 (q, J = 272.2 Hz), 119.59
q, J = 3.9 Hz), 117.61 , 115.98 (q, J = 3.8 Hz), 115.73 , 99.37 , 87.24 (d, J = 174.2 Hz),
1
(
6
2.41 , 61.80 (d, J = 6.2 Hz), 57.66 (d, J = 19.5 Hz) , 44.10 , 21.25 , 16.74 (d, J = 21.6 Hz).
+
ES-LCMS m/z: 493.0 (M+H) . HRMS m/z: calcd for C22H21F4N6O3 493.1606; found,
4
93.1606. 98.41% purity.

(S)-4-isopropyl-3-(2-(((S)-1-(7-(trifluoromethyl)-4,5-dihydroimidazo[1,5-a]quinolin-3-yl)ethy
l)amino)pyrimidin-4-yl)oxazolidin-2-one (3)
Compound 3 was synthesized from 3a and 3g. 3g was synthesized from
2
,4-dichloropyrimidine and (S)-4-isopropyloxazolidin-2-one in a similar progress to that
described for the preparation of 1g.
1
H NMR (400 MHz, Chloroform-d) δ 8.18 (d, J = 5.8 Hz, 1H), 7.99 (s, 1H), 7.59 – 7.46 (m,
3
H), 7.43 (d, J = 5.8 Hz, 1H), 5.70 (br, 1H), 5.19 (br, 1H), 4.68 (br, 1H), 4.35 – 4.20 (m, 2H),
.18 – 2.82 (m, 4H), 2.51 (br, 1H), 1.59 (d, J = 6.8 Hz, 3H), 0.90 (d, J = 6.9 Hz, 3H), 0.81 (d,
3
1
9
13
J = 6.5 Hz, 3H). F NMR (376 MHz, Chloroform-d) δ -62.31 . C NMR (101 MHz,
Chloroform-d) δ 161.03 , 158.83 , 157.42 , 154.91 , 139.09 , 136.64 , 131.09 , 128.71 ,
1
27.82 (q, J = 32.9 Hz), 126.72 (q, J = 3.6 Hz), 125.10 (q, J = 3.5 Hz), 123.89 (q, J = 271.9
Hz), 122.37 , 115.94 , 99.30 , 63.13 , 58.65 , 43.93 , 27.75 , 26.65 , 21.67 , 19.01 , 18.24 ,
+
1
4.34 . ES-LCMS m/z: 487.0 (M+H) . HRMS m/z: calcd for C24H26F3N6O2 487.2064;
found, 487.2063. 100% purity.
(R)-3-(2-(((S)-1-(7-chloro-4,5-dihydroimidazo[1,5-a]quinolin-3-yl)ethyl)amino)pyrimidin-4-y
l)-4-((S)-1-fluoroethyl)oxazolidin-2-one (4)
Compound 4 was synthesized from 4a and 1g. 4a was synthesized from
(2,5-dichlorophenyl)boronic acid in a similar progress to that described for the preparation of

3
a.
H
N
N
N
N
N
O
N
Cl
O
F
1
H NMR (400 MHz, Chloroform-d) δ 8.21 (d, J = 5.7 Hz, 1H), 7.93 (s, 1H), 7.46 (d, J = 5.7
Hz, 1H), 7.35 – 7.24 (m, 3H), 5.75 (br, 1H), 5.17 (br, 2H), 4.82 – 4.61 (m, 1H), 4.54 (dd, J =
.8, 3.4 Hz, 1H), 4.39 (t, J = 8.9 Hz, 1H), 3.20 – 2.75 (m, 4H), 1.58 (d, J = 6.8 Hz, 3H), 1.33
8
1
9
13
(
d, J = 19.1 Hz, 3H). F NMR (376 MHz, Chloroform-d) δ -197.81 . C NMR (101 MHz,
Chloroform-d) δ 160.82 , 159.07 , 157.12 , 154.28 , 138.62 , 132.54 , 131.09 , 130.66 ,
29.85 , 129.49 , 127.82 , 122.29 , 116.93 , 99.12 , 87.28 (d, J = 174.0 Hz), 61.85 (d, J = 6.3
1
Hz), 57.74 (d, J = 19.6 Hz) , 43.64 , 26.52 , 21.70 , 19.07 , 16.90 (d, J = 21.7 Hz). ES-LCMS
+
m/z: 457.0 (M+H) . HRMS m/z: calcd for C22H23ClFN6O2 457.1550; found, 457.1549.
9
8.80% purity.
(R)-4-((S)-1-fluoroethyl)-3-(2-(((S)-1-(7-(trifluoromethyl)-4,5-dihydroimidazo[1,5-a]quinolin
-3-yl)ethyl)amino)pyrimidin-4-yl)oxazolidin-2-one (5)
Compound 5 was synthesized from 3a and 1g.
1
H NMR (400 MHz, Chloroform-d) δ 8.19 (s, 1H), 7.98 (s, 1H), 7.60 – 7.39 (m, 4H), 5.72 (br,
1
H), 5.16 (br, 2H), 4.76 – 4.61 (m, 1H), 4.52 (d, J = 8.4 Hz, 1H), 4.37 (t, J = 8.6 Hz, 1H),
19
3
.15 – 2.80 (d, J = 63.1 Hz, 4H), 1.57 (d, J = 6.6 Hz, 3H), 1.32 (d, J = 21.3 Hz, 3H). F

1
3
NMR (376 MHz, Chloroform-d) δ -62.35 , -197.86 . C NMR (101 MHz, Chloroform-d) δ
1
60.84 , 159.08 , 157.16 , 154.25 , 139.03 , 136.58 , 131.01 , 128.70 , 127.83 (q, J = 32.8,
2.2 Hz), 126.81 – 126.59 (m), 125.08 (q, J = 3.4 Hz), 123.87 (q, J = 271.9 Hz), 122.39 ,
3
115.92 , 99.18 , 87.27 (d, J = 174.7 Hz), 61.84 (d, J = 6.3 Hz), 57.74 (d, J = 19.6 Hz) , 43.57
+
2
6.60 , 21.63 , 19.02 , 16.89 (d, J = 21.7 Hz). ES-LCMS m/z: 491.0 (M+H) . HRMS m/z:
calcd for C23H23F4N6O2 491.1813; found, 491.1811. 100% purity.
(R)-3-(5-fluoro-2-(((S)-1-(7-(trifluoromethyl)-4,5-dihydroimidazo[1,5-a]quinolin-3-yl)ethyl)a
mino)pyrimidin-4-yl)-4-((S)-1-fluoroethyl)oxazolidin-2-one (6)
Compound 6 was synthesized from 3a and 6g. 6g was synthesized from 1h and
2
,4-dichloro-5-fluoropyrimidine in a similar progress to that described for the preparation of
1
g.
1
H NMR (400 MHz, Chloroform-d) δ 8.20 (d, J = 2.8 Hz, 1H), 7.98 (s, 1H), 7.58 – 7.46 (m,
H), 5.86 (br, 1H), 5.15 – 4.87 (m, 2H), 4.75 – 4.61 (m, 1H), 4.50 (p, J = 8.6 Hz, 2H), 3.13 –
3
1
9
2
.84 (m, 4H), 1.56 (d, J = 6.8 Hz, 3H), 1.27 (dd, J = 23.2, 6.3 Hz, 3H). F NMR (376 MHz,
1
3
Chloroform-d) δ -62.31 , -152.16 , -194.12 . C NMR (101 MHz, Chloroform-d) δ 157.54 (d, J =
2
.4 Hz), 153.69 , 147.88 (d, J = 11.8 Hz), 145.39 (d, J = 12.3 Hz), 143.94 (d, J = 254.2 Hz), 138.77 ,
36.56 , 131.08 , 128.68 , 127.82 (q, J = 32.9 Hz), 126.71 (q, J = 3.7 Hz), 123.87 (q, J = 271.9 Hz), 125.08
1
(q, J = 3.8 Hz), 122.55 , 115.96 , 86.18 (d, J = 176.8 Hz), 62.90 (d, J = 5.9 Hz), 58.40 (d, J = 20.3 Hz),
+
4
4.57 , 26.58 , 21.42 , 19.00 , 16.58 (d, J = 21.9 Hz). ES-LCMS m/z: 509.0 (M+H) . HRMS m/z:

calcd for C23H22F5N6O2 509.1719; found, 507.1718. 97.20% purity.
(R)-3-(6-chloro-2-(((S)-1-(7-(trifluoromethyl)-4,5-dihydroimidazo[1,5-a]quinolin-3-yl)ethyl)a
mino)pyrimidin-4-yl)-4-((S)-1-fluoroethyl)oxazolidin-2-one (7)
Compound 7 was synthesized from 3a and 7g. 7g was synthesized from 1h and
2
,4,6-trichloropyrimidine in a similar progress to that described for the preparation of 1g.
1
H NMR (400 MHz, Chloroform-d) δ 7.97 (s, 1H), 7.60 – 7.45 (m, 4H), 5.92 (d, J = 7.3 Hz,
H), 5.40 – 4.87 (m, 2H), 4.81 – 4.28 (m, 3H), 3.39 – 2.77 (m, 4H), 1.59 (d, J = 6.5 Hz, 3H),
1
1
9
1
.42 – 1.23 (m, 3H). F NMR (376 MHz, Chloroform-d) δ -62.30 , -197.78 (dp, J = 47.9,
1
3
2
4.0, 23.5 Hz). C NMR (101 MHz, Chloroform-d) δ 162.11 , 160.24 , 157.93 , 153.96 , 138.25 ,
1
36.55 , 131.16 , 129.04 , 127.83 (q, J = 33.4 Hz), 126.74 (q, J = 3.7 Hz), 125.01 , 123.86 (q, J = 271.9
Hz), 123.57 , 115.91 , 98.09 , 87.15 (d, J = 174.1 Hz), 61.89 (d, J = 6.0 Hz), 57.99 (d, J = 19.4 Hz), 43.25 ,
+
2
6.69 , 20.95 , 18.93 , 16.77 (d, J = 21.4 Hz). ES-LCMS m/z: 525.0 (M+H) . HRMS m/z: calcd for
C23H22ClF4N6O2 525.1423; found, 525.1435. 100% purity.
(R)-4-((S)-1-fluoroethyl)-3-(2-(((S)-1-(6-(trifluoromethyl)benzo[d]imidazo[5,1-b]thiazol-3-yl)
ethyl)amino)pyrimidin-4-yl)oxazolidin-2-one (8)
Compound 8 was synthesized from 8a and 1g.

1
H NMR (400 MHz, Chloroform-d) δ 8.29 (s, 1H), 8.24 (d, J = 5.8 Hz, 1H), 7.78 (s, 1H),
7
5
1
.73 (d, J = 8.4 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.51 (d, J = 5.8 Hz, 1H), 6.21 (br, 1H),
.17 (br, 1H), 4.65 (dd, J = 26.7, 7.4 Hz, 1H), 4.58 – 4.40 (m, 2H), 4.36 (t, J = 8.9 Hz, 1H),
1
9
.67 (d, J = 6.9 Hz, 3H), 1.06 (br, 3H). F NMR (376 MHz, Chloroform-d) δ -61.85 ,
13
-
197.40 . C NMR (101 MHz, Chloroform-d) δ 161.11 , 159.32 , 157.18 , 154.15 , 134.87 ,
34.58 , 133.17 , 128.33 (q, J = 33.3 Hz), 126.34 , 123.82 , 123.62 (q, J = 272.5 Hz), 123.52
q, J = 3.7 Hz), 121.94 (q, J = 3.9 Hz), 112.80 , 99.59 , 87.26 (d, J = 174.3 Hz), 61.80 (d, J =
1
(
6
.5 Hz), 57.75 (d, J = 19.5 Hz), 46.29 , 21.08 , 16.80 (d, J = 21.5 Hz). ES-LCMS m/z: 495.1
+
(
M+H) . HRMS m/z: calcd for C21H19F4N6O2S 495.1221; found, 495.1220. 100% purity.
(R)-3-(2-(((S)-1-(6-chloro-4,5-dihydroimidazo[1,5-a]quinolin-3-yl)ethyl)amino)pyrimidin-4-y
l)-4-((S)-1-fluoroethyl)oxazolidin-2-one (9)
Compound 9 was synthesized from 9a and 1g.
1
H NMR (400 MHz, Chloroform-d) δ 8.21 (d, J = 5.7 Hz, 1H), 7.95 (s, 1H), 7.46 (d, J = 5.7
Hz, 1H), 7.34 (dd, J = 7.0, 2.2 Hz, 1H), 7.31 – 7.19 (m, 2H), 5.81 (br, 1H), 5.18 (br, 2H), 4.85
–
4.60 (m, 1H), 4.54 (dd, J = 8.9, 3.4 Hz, 1H), 4.40 (t, J = 8.9 Hz, 1H), 3.25 – 2.73 (m, 4H),

1
9
1
.59 (d, J = 6.8 Hz, 3H), 1.47 – 1.18 (m, 3H). F NMR (376 MHz, Chloroform-d) δ -197.64 .
C NMR (101 MHz, Chloroform-d) δ 160.80 , 159.03 , 157.08 , 154.24 , 138.43 , 135.13 ,
34.70 , 130.92 , 128.17 , 126.65 , 126.32 , 122.23 , 114.21 , 99.05 , 87.27 (d, J = 174.1 Hz),
1
3
1
6
1.82 (d, J = 6.3 Hz), 57.71 (d, J = 19.6 Hz), 43.60 , 23.57 , 21.65 , 18.59 , 16.86 (d, J = 21.6
+
Hz). ES-LCMS m/z: 457.1 (M+H) . HRMS m/z: calcd for C22H23ClFN6O2 457.1550;
found, 457.1548. 100% purity.
(4R)-4-((S)-1-fluoroethyl)-3-(2-(((1S)-1-(5-methyl-7-(trifluoromethyl)-4,5-dihydroimidazo[1,5
-a]quinolin-3-yl)ethyl)amino)pyrimidin-4-yl)oxazolidin-2-one (10)
Compound 10 was synthesized from 10a and 1g.
1
H NMR (400 MHz, Chloroform-d) δ 8.20 (dd, J = 5.7, 2.0 Hz, 1H), 7.99 (s, 1H), 7.55 (d, J =
5
2
2
.6 Hz, 2H), 7.49 (d, J = 8.8 Hz, 1H), 7.45 (dd, J = 5.7, 4.0 Hz, 1H), 5.72 (br, 1H), 5.17 (br,
H), 4.69 (dd, J = 26.7, 7.1 Hz, 1H), 4.59 – 4.44 (m, 1H), 4.37 (t, J = 8.9 Hz, 1H), 3.19 –
1
9
.68 (m, 3H), 1.57 (dd, J = 6.8, 2.4 Hz, 3H), 1.45 – 1.10 (m, 6H). F NMR (376 MHz,
1
3
Chloroform-d) δ -62.25 , -197.47 . C NMR (101 MHz, Chloroform-d) δ 160.81 (d, J = 3.7
Hz), 159.06 , 157.15 (d, J = 3.7 Hz), 154.28 , 140.07 , 135.73 , 133.58 (d, J = 8.6 Hz),
1
2
1
1
30.84 , 128.10 (q, J = 32.8 Hz), 125.62 – 125.30 (m), 125.03 (q, J = 3.8 Hz), 123.91 (q, J =
72.0 Hz), 121.42 , 116.06 , 99.20 , 89.02 – 85.30 (m), 61.83 (d, J = 5.7 Hz), 57.74 (d, J =
9.4 Hz), 43.65 , 31.49 (d, J = 17.1 Hz), 26.68 , 21.81 (d, J = 23.0 Hz), 19.86 (d, J = 51.6 Hz),
+
6.94 (dd, J = 21.6, 5.5 Hz). ES-LCMS m/z: 505.1 (M+H) . HRMS m/z: calcd for

C24H25F4N6O2 505.1970; found, 505.1966. 98.66% purity.
(R)-3-(2-(((S)-1-(7-cyclopropyl-4H-benzo[b]imidazo[1,5-d][1,4]oxazin-3-yl)ethyl)amino)pyri
midin-4-yl)-4-((S)-1-fluoroethyl)oxazolidin-2-one (11)
Compound 11 was synthesized from 11a and 1g.
1
H NMR (400 MHz, Chloroform-d) δ 8.19 (d, J = 5.7 Hz, 1H), 7.87 (s, 1H), 7.46 (d, J = 5.7
Hz, 1H), 7.27 (d, J = 8.0 Hz, 1H), 6.79 – 6.70 (m, 2H), 5.71 (br, 1H), 5.31 – 4.89 (m, 4H),
4
.65 (dd, J = 25.8, 7.7 Hz, 1H), 4.50 (dd, J = 8.7, 2.9 Hz, 1H), 4.36 (t, J = 9.0 Hz, 1H), 1.84
(
(
tt, J = 8.5, 5.1 Hz, 1H), 1.60 (d, J = 6.8 Hz, 3H), 1.24 (br, 3H), 0.96 (q, J = 6.3 Hz, 2H), 0.66
1
9
13
q, J = 4.9 Hz, 2H). F NMR (376 MHz, Chloroform-d) δ -197.72 . C NMR (101 MHz,
Chloroform-d) δ 160.84 , 159.08 , 157.17 , 154.22 , 146.06 , 143.82 , 138.40 , 130.28 ,
21.64 , 120.14 , 117.91 , 115.29 , 115.19 , 99.43 , 87.27 (d, J = 174.2 Hz), 62.19 , 61.84 (d, J
1
=
6.4 Hz), 57.70 (d, J = 19.6 Hz), 44.46 , 21.56 , 16.82 (d, J = 21.6 Hz), 15.31 , 9.56 .
+
ES-LCMS m/z: 465.1 (M+H) . HRMS m/z: calcd for C24H26FN6O3 465.2045; found,
4
65.2043. 98.96% purity.
3
-((S)-1-((4-((R)-4-((S)-1-fluoroethyl)-2-oxooxazolidin-3-yl)pyrimidin-2-yl)amino)ethyl)-4H-
benzo[b]imidazo[1,5-d][1,4]oxazine-7-carbonitrile (12)
Compound 12 was synthesized from 12a and 1g.