Chiral resolution, absolute configuration determination, and stereo-activity relationship study of IDO1 inhibitor NLG919

Article abstract of DOI:10.1016/j.tet.2018.05.005

NLG919 (1) with two chiral carbon atoms on its chemical structure is a potent indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor. We developed an effective way to prepare all stereoisomers of 1, the key step being the chiral resolution of racemic intermediate 2. The optimal resolution solvent system was identified as dichloromethane and n-pentane or petroleum ether. Using (?)-di-p-toluoyl-D-tartaric acid as resolution reagent, optical pure (R)-2 (e.e. > 99%, yield = 70%) was obtained. The mechanism of chiral resolution was clarified through single-crystal X-ray diffraction of the diastereomeric salt. The absolute configurations of four stereoisomers of 1 were established through electronic circular dichroism spectra, quantum chemical calculation and transition metal method. Their IDO1 inhibitory activity was assessed by pharmacological experiments in vitro and in mouse, demonstrating that S configuration of C5 played an important role on the inhibition of IDO1, while the stereochemistry on C2′ exerted little effect on the IDO1 inhibitory activity in mouse.

Full text of DOI:10.1016/j.tet.2018.05.005

Accepted Manuscript
Chiral resolution, absolute configuration determination, and stereo-activity relationship
study of IDO1 inhibitor NLG919
Wen-Qiang Liu, Fang-Fang Lai, Jie Zhang, Li Sheng, Yan Li, Li Li, Xiao-Guang Chen
PII:
S0040-4020(18)30513-1
10.1016/j.tet.2018.05.005
TET 29512
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 17 March 2018
Revised Date: 26 April 2018
Accepted Date: 2 May 2018
Please cite this article as: Liu W-Q, Lai F-F, Zhang J, Sheng L, Li Y, Li L, Chen X-G, Chiral resolution,
absolute configuration determination, and stereo-activity relationship study of IDO1 inhibitor NLG919,
Tetrahedron (2018), doi: 10.1016/j.tet.2018.05.005.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Chiral resolution, absolute configuration
determination, and stereo-activity
relationship study of IDO1 Inhibitor
NLG919
Leave this area blank for abstract info.
Wen-Qiang Liu#, Fang-Fang Lai#, Jie Zhang, Li Sheng, Yan Li, Li Li*, Xiao-Guang Chen*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica,
Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China

1
ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Chiral resolution, absolute configuration determination, and stereo-activity
relationship study of IDO1 inhibitor NLG919
Wen-Qiang Liu#, Fang-Fang Lai#, Jie Zhang, Li Sheng, Yan Li, Li Li , Xiao-Guang Chen*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking
Union Medical College, Beijing 100050, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
NLG919 (1) with two chiral carbon atoms on its chemical structure is a potent indoleamine 2,3-
dioxygenase 1 (IDO1) inhibitor. We developed an effective way to prepare all stereoisomers of
1, the key step being the chiral resolution of racemic intermediate 2. The optimal resolution
solvent system was identified as dichloromethane and n-pentane or petroleum ether. Using (−)-
di-p-toluoyl-D-tartaric acid as resolution reagent, optical pure (R)-2 (e.e. > 99%, yield = 70%)
was obtained. The mechanism of chiral resolution was clarified through single-crystal X-ray
diffraction of the diastereomeric salt. The absolute configurations of four stereoisomers of 1
were established through electronic circular dichroism spectra, quantum chemical calculation
and transition metal method. Their IDO1 inhibitory activity was assessed by pharmacological
experiments in vitro and in mouse, demonstrating that S configuration of C5 played an important
role on the inhibition of IDO1, while the stereochemistry on C2’ exerted little effect on the
IDO1 inhibitory activity in mouse.
Available online
Keywords:
Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor
Single crystal X-ray diffraction
Optical resolution
Absolute configuration
Electronic circular dichroism
2018 Elsevier Ltd. All rights reserved
.
1. Introduction
The classical enantiomer separation through the diastereomeric
salt formation is a useful approach for large scale preparation of
chiral molecules8. The synthetic intermediate (2, Fig. 1) contains
an alkaline imidazole fragment and a carbonyl group, which
could interact with resolution agents or solvents. Thus, it was
possible to realize optical resolution of rac-2 through the
formation of diastereomeric salts with chiral organic acids.
Immune checkpoint therapies have become a research hotspot
in cancer treatment1. Several immune checkpoint inhibitors have
been approved by FDA and applied clinically2. Indoleamine 2,3-
dioxygenase 1 (IDO1) is an intracellular heme-containing
enzyme, which initiates the first and rate-limiting step of the L-
tryptophan (L-Trp) degradation along the kynurenine (Kyn)
pathway3. The depletion of L-Trp in tumor microenvironment
would lead to an immunosuppressive effect, causing the immune
escape of tumors4. Some IDO1 inhibitors have been reported,
such as epacadostat, indoximod, and NLG919 (1, Fig. 1)5.
By using optical pure 7 as resolution agent, we achieved
highly effective resolution of rac-2. Four diastereoisomers of 1
were then prepared by the reduction of
2 and column
chromatography. The absolute configurations of stereoisomers
were assigned by electronic circular dichroism (ECD) spectra
combined with quantum-chemical calculations, and transition
metal method. The chiral resolution mechanism was elucidated
by the analysis of the diastereomeric salt crystal of (S)-2:L-7
(CCDC 1570864). The pharmacological evaluation of
stereoisomers of 1 in vitro and in vivo was also carried out.
Herein, the results of our study will be reported.
Fig. 1. Chemical structures of 1 and 2.
2. Result and discussion
It is reported that stereochemistry might affect the IDO1
inhibitory activity of NLG9196. Meanwhile, the preparation of
stereoisomers of 1 need harsh reaction condition and give low
yield7.Therefore, it is necessary to develop an effective way to
prepare optical pure stereoisomers of 1 for pharmacology and
2.1. Synthesis and chiral resolution of rac-2
The synthesis route of optical pure diastereomers of 1 is
displayed in Scheme 1. Firstly, rac-2 was prepared through
Suzuki reaction, condensation and cyclization reactions.
pharmacokinetics study.
———
# W.-Q. Liu and F.-F. Lai contributed equally to this work. Corresponding authors. Tel.: +86-10-63165247; e-mail: annaleelin@imm.ac.cn (L.
Li); e-mail: chxg@imm.ac.cn (X.-G. Chen).

2
Tetrahedron
ACCEPTED MANUSCRIPT
Scheme 1. Synthesis route of optical pure diastereomers of 1 through chiral resolution of rac-2.
Five commercially available and commonly used acidic
resolving agents 3-7 (Fig. 2) were screened for the enantiomer
resolution of rac-2. Firstly, Rac-2 and resolving agents were
mixed and dissolved in ethanol (EtOH) for crystallization (Table
1, entries 1-5). Only when using D-7, a small amount of needle-
like precipitation appeared with low enantiomeric excess (entry
5, (R)-2, e.e. = 15.62%). However, when the mixture solvent
based on EtOH was used, no precipitation appeared (entry 6).
Other resolving agents could not form solid with rac-2, even
after removing the solvent. It is thus deduced that the resolution
agents with large aromatic group are suitable for the resolution of
rac-2, and the methyl group of 7 might play an important role in
the process of chiral recognition.
Table 2. Optimization of the resolution conditions of rac-2 with
D-7a.
Entry Resolving agent Conditionsb
e.e. (%)e
d
1
D-7 (2.0 equiv.) MeOH (4.0 L/mol)c
D-7 (2.0 equiv.) DMSO (4.0 L/mol)c
D-7 (2.0 equiv.) EG (4.0 L/mol)c
D-7 (2.0 equiv.) i-PrOH (4.0 L/mol)c
D-7 (2.0 equiv.) EA (4.0 L/mol)c
D-7 (2.0 equiv.) THF (10.0 L/mol)c
-
d
-
2
d
-
3
d
-
4
d
-
5
d
-
6
d
-
7
D-7 (2.0 equiv.) DCM (10.0 L/mol)c
d
-
8
D-7 (2.0 equiv.) DCM:EtOH = 4:1 (8.3 L/mol)c
D-7 (2.0 equiv.) DCM:i-PrOH = 4:1 (8.3 L/mol)c
D-7 (2.0 equiv.) DCM:EA = 4:1 (8.3 L/mol)c
D-7 (2.0 equiv.) DCM:PE = 4:1 (8.3 L/mol)c
D-7 (2.0 equiv.) DCM:PE = 8:1 (11.3 L/mol)c
D-7 (2.0 equiv.) DCM:PE = 13:1 (14.3 L/mol)c
D-7 (2.0 equiv.) DCM:PE = 17:1 (18 L/mol)c
D-7 (1.5 equiv.) DCM:PE = 8:1 (11.3 L/mol)c
D-7 (1.5 equiv.) DCM:PE = 13:1 (14.3 L/mol)c
D-7 (1.5 equiv.) Wash once
d
-
9
d
-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
19.54 (R)
55.78 (R)
82.43 (R)
71.57 (R)
64.93 (R)
87.21 (R)
96.32 (R)
99.75 (R)
Fig. 2. Acidic resolving agents used in this study.
Table 1. Chiral resolution of rac-2 with acidic resolving agentsa
Entry Resolving agent Conditionsb
e.e. (%)e
d
1
2
3
4
5
L-3 (2.0 equiv.)
D-4 (2.0 equiv.)
L-5 (2.0 equiv.)
D-6 (2.0 equiv.)
D-7 (2.0 equiv.)
D-7 (2.0 equiv.)
EtOH (2.0 L/mol)c
EtOH (2.0 L/mol)c
EtOH (2.0 L/mol)c
EtOH (2.0 L/mol)c
EtOH (2.0 L/mol)c
EtOH:H2O = 6:1 (4.2 L/mol)c
-
d
-
D-7 (1.5 equiv.) Wash twice
d
d
D-7 (1.0 equiv.) DCM:PE = 13:1 (14.3 L/mol)c
D-7 (0.5 equiv.) DCM:PE = 13:1 (14.3 L/mol)c
D-7 (1.5 equiv.) DCM:n-Hex = 13:1 (14.3 L/mol)c
D-7 (1.5 equiv.) Wash once
-
-
d
d
-
-
15.62 (R)
80.21 (R)
76.54 (R)
d
6
-
a
The amount of rac-2 was 0.30 mmol. b The amount of solvent based on the
D-7 (1.5 equiv.) DCM:n-pentane = 13:1 (14.3 L/mol)c 92.52 (R)
c
amount of rac-2 is shown within the parenthesis. The process of heating to
d
complete dissolution and cooling to room temperature for 2−24 h. No
D-7 (1.5 equiv.) Wash once
D-6 (1.5 equiv.) DCM:PE = 13:1 (14.3 L/mol)c
D-6 (2.0 equiv.) DCM:PE = 13:1 (14.3 L/mol)c
99.93 (R)
d
crystallization. e e.e. was determined by chiral HPLC analysis.
-
d
-
a The amount of rac-2 was 0.30 mmol. b The amount of solvent relative to the
2.2. Optimization of resolution conditions
c
amount of rac-2 is shown within the parenthesis. The process of heating to
d
By using other solvents such as methanol (MeOH), dimethyl
sulfoxide (DMSO), ethylene glycol (EG), isopropanol (i-PrOH),
ethyl acetate (EA), tetrahydrofuran (THF) and dichloromethane
(DCM) (Table 2, entries 1-7), it was found that when polar
solvents (MeOH, DMSO, EG, i-PrOH) was used, there was no
precipitate (entries 1-4). However, when using low polar solvents
(EA, THF, and DCM), there was still no resolution effect (entries
5-7). But, it was noticed that rac-2 and D-7 tended to form a
solid without enantiomeric excess after removal of DCM under
reduced pressure. Considering this unique phenomenon, further
optimization of the resolution solvent was carried out based on
the solvent of DCM.
complete dissolution and cooling to room temperature for 2−24 h. No
crystallization. e e.e. was determined by chiral HPLC analysis.
When adding EtOH, i-PrOH, or EA in DCM (entries 8-10),
there were no resolution effect. Presumably, the polar solvent
might inhibit the interaction between rac-2 and the resolution
agents. Fortunately, when added petroleum ether (PE, boiling
range is 60-90°C) in DCM (entry 11), large amounts of white
precipitate appeared with low enantiomeric excess ((R)-2, e.e. =
19.54%).
After the optimization of the resolution conditions (entry 12-
20), through adjustment of the ratio of DCM to PE and the initial
molar ratio of rac-2 to D-7, obvious resolution effect was

3
achieved (entry 16, e.e. = 87.21%). The enantiomeric excess
(entry 18, e.e. = 99.75%) could be further improved by washing
the precipitate twice with the corresponding solvents. The result
showed that DCM was necessary to increase the solubility of the
salt and provide a suitable environment for the interaction of D-7
and rac-2. Meanwhile, PE in the resolution solvent mixture
might balance the solubility of diastereomeric salt and facilitate it
to deposit.
antiparallel chain (the CHꢀꢀꢀO distance was 2.478 Å) (Fig. S5).
ACCEPTED MANUSCRIPT
In addition, the hydrogen atom of the phenyl fragment of (S)-2
formed a CHꢀꢀꢀO interaction with the ester oxygen atom (the
CHꢀꢀꢀO distance was 2.419 Å). All three CHꢀꢀꢀO interactions were
shorter than the sum of the van der Walls radii of hydrogen and
oxygen atoms 2.80 Å8c, indicating their contribution to the chiral
recognition of (S)-2. The phenomenon that tolyl group of L-7
involved in the chiral recognition process might be the reason for
the striking difference of resolution capability between D-6 and
D-7. Similar situation also occurred in the optical resolution of
rac-2-[amino(phenyl)methyl] phenol8d.
Since PE contains mainly hexane and pentane, a mixture of
DCM and n-hexane (n-Hex) was used as resolution solvent, and a
fairly good resolution effect (entry 21, e.e. = 80.21%) was
attained. But the optical purity could not be further raised by
washing of the diastereomeric salt (entry 22). When using the
DCM and n-pentane, a high resolution effect was obtained (e.e. =
92.52%) (entry 23). After simple washing, the e.e. value was
remarkably increased to 99.93% (entry 24). The process of
washing might just remove the residue of (S)-2ꢀD-7 salt exiting in
the less-soluble salt of (R)-2ꢀD-7. From this result, it might be
inferred that the five-carbon chain of n-pentane is more suitable
for the resolution of rac-2.
Under the optimized condition, D-6 was tried again as
resolving agent, but still no precipitate appeared (entries 25, 26).
Thus, it could be deduced that the methyl group of D-7 is
essential for the optical resolution of rac-2.
2.3. Crystallographic analysis of (S)-2·L-7
To elucidate the mechanism underlying the optical resolution
of rac-2, the crystallographic analysis of the less-soluble salt (S)-
2ꢀL-7 was performed. The single crystal of the (S)-2ꢀL-7 salt
tended to grow in ethanol after recrystallization of the
diastereomeric salt isolated from DCM-PE mixture solvents. The
crystallized salt consisted of (S)-2, L-7 and EtOH in a ratio of
1:1:2. They composed a one-dimensional 21 screw linear
structure along the a axis supported by salt bond, hydrogen bond,
CH-π interaction, and π-π interaction (Fig. 3, Fig. S1-4).
It was observed that one carboxyl group of L-7 was ionized
and a salt bond between (S)-2 and L-7 was thus formed. The
same carboxyl group bearing negative charge also formed an
intermolecular hydrogen bond with another L-7 molecule (H-
bond, the OꢀꢀꢀO distance was 2.566 Å). The methyl group of L-7
formed CH-π interaction with the phenyl fragment of (S)-2 (The
distance of C π-plane was 3.338 Å as shown in Fig. S2). The π-
stacking distance is typically 3.4-3.6 Å that represents the
shortest interplanar distance9. In this study, the distance between
two benzyl groups of adjacent L-7 molecules was 3.441 Å, and
the dihedral angle of two π-planes was 4.29° (Fig. S3), indicating
that the π-π interaction appeared between adjacent L-7
molecules. Meanwhile, the benzyl group of L-7 and the
imidazole group of (S)-2 were located closely in space, with the
distance between two π-planes being 3.492 Å (Fig. S4). This
implied that the π-π interaction stabled the crystal structure of
(S)-2ꢀL-7 salt.
Fig. 3. Crystal structure of (S)-2ꢀL-7ꢀ2EtOH. (a) Top view of the four linear
structures viewed from a axis. Hydrogen atoms are omitted for clarity. (b)
Side view of the linear structure viewed along a axis. The dotted lines and
arrows show hydrogen bonds, CHꢀꢀꢀO interactions and πꢀꢀꢀπ interactions,
respectively.
When considering the conformation of the salt unit (S)-2ꢀL-7
(Fig. 4a) in the crystal, the total binding energy of (S)-2 and L-7
was calculated to be −387.15 kJ/mol, adopting time-dependent
density functional theory (TDDFT) methodology at the
B3LYP/6-31+G(d,p) basis set. Meanwhile, the calculated binding
energy of (R)-2ꢀL-7 salt was slightly higher (−381.10 kJ/mol),
illustrating that (S)-2ꢀL-7 salt was more stable than the
diastereomeric counterpart.
Each one-dimensioned chain was antiparallel with their
neighboring chains (Fig. 3a). For this chiral resolution, the
intermolecular interactions between (S)-2 and the L-7 from the
neighboring antiparallel one-dimensioned chain were crucial.
There were three CHꢀꢀꢀO interactions between antiparallel chains.
The hydrogen atom of the chair carbon of (S)-2 formed a CHꢀꢀꢀO
interaction with the ester carbonyl oxygen atom of L-7 from the
same neighboring antiparallel one-dimensioned chain (the
CHꢀꢀꢀO distance was 2.319 Å, Fig. S5). The keto carbonyl
oxygen atom of (S)-2 also formed a CHꢀꢀꢀO interaction with the
methyl hydrogen atom of L-7 from the same neighboring
a)
b)
Fig. 4. Geometries of the diastereomeric salts. (a) (S)-2ꢀL-7 and (b) (R)-2ꢀL-7.
2.4. Effects of solvent on the optical resolution

4
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ACCEPTED MANUSCRIPT
Suitable resolution solvents could enhance the interaction
spectra combined with TDDFT calculation has become a
between the racemic substrate and the resolution agent. In this
case, the DCM-PE solvent system might provide a hydrophobic
environment for the highly selective interaction between L-7 and
rac-2. Two adjacent parallel chains might form a hydrophobic
channel by the cyclohexyl group of (S)-2 (Fig. S6). The distance
between two cyclohexyl groups of (S)-2 was 6.901 Å.
Coincidentally, the molecular lengths of n-pentane and n-Hex
were 6.793 Å and 8.047 Å, respectively (Fig. S7). It was thus
inferred that the n-pentane molecule might connect cyclohexyl
group through hydrophobic interaction and promote the
formation and enhance the stabilization of the channel. These
might also be the reason for that the PE or n-pentane could
trigger the formation of the less-soluble salt in DCM, but the
longer n-Hex exerted low resolving capability.
powerful tool to stereochemical study10. In this work, this method
was also adopted to confirm the absolute configurations of
enantiopure 2 (Fig. 5). Albeit slightly blue-shifted by 5 nm in the
range of 280-200 nm, the calculated ECD spectra reproduced all
the Cotton effects (CEs) appeared in the experimental ECD of the
corresponding stereoisomer of 2.
2.6. Preparation and absolute configuration assignments of four
stereoisomers of 1
Four diastereoisomers of 1 were prepared by the reduction of
enantiopure 2, followed by the separation of the silica gel column
chromatography. Stereo-selectivity was observed to occur during
the reduction by NaBH4, since (S)-2 tended to give (5S,2’R)-1
and (5S,2’S)-1 at a ratio of 4:1. This priority might be due to the
difference between steric hindrances encountered by the
reduction agent from two sides of the large planar aromatic ring.
Chiral HPLC analysis of rac-1 showed four peaks at 9.78 min,
11.93 min, 18.71 min, and 21.15 min (Fig. S9).
In the crystal of diastereomeric salt, EtOH only formed an H-
bond with the carboxyl group of L-7. Moreover, when only EtOH
was used to dissolve L-7 and (S)-2 for the recrystallization, no
precipitate appeared. It is thus regarded that EtOH might attribute
little to the chiral recognition but could promote the crystal
growth of the less-soluble salt.
ECD method and quantum chemical calculation were firstly
tried to establish the absolute configurations of all the
stereoisomers of 1. Nevertheless, since the C2’ atom is far from
the aromatic chromophore and exerts little effect on the ECD
spectra, two isomers with the same C5 configuration would give
quite similar ECD spectra. Both experimental and theoretical
ECD spectra verified the presumption (Fig. 6). When comparing
the ECD spectra of optical pure 1 and 2 with the same C5
configuration, it was obvious that the C2’ chirality changed the
sign of the CE at 300 nm.
2.5. Large scale optical resolution and absolute configuration
assignments of enantiopure 2
With the optimized condition in hand, we applied it to prepare
the optical pure 2 in a large scale. By using two commercially
available resolving agents D-7 and L-7, two enantiomers of rac-2
was obtained in a good efficiency in a gram scale (Table 3).
Compared with DCM-PE solvent, using DCM of n-pentane
solvent as resolution solvent could get better yield in a short time
and with great resolution effect (e.e. > 99%, eff. up to 0.7).
Fig. 6. Comparison of experimental and theoretical ECD spectra
for four stereoisomers of 1.
Fig. 5. Comparison of experimental and theoretical ECD spectra
for four stereoisomers of 2.
The establishment of C2’ configuration fell back on transition
metal method developed for the chiral secondary alcohols11. The
absolute configuration at the C2’ atom could be deduced
unambiguously from the induced ECD (ICD) data of the in situ-
formed Rh2(CF3COO)4 complexes of enantiopure 1.
Although the optical pure stereoisomer of 2 obtained using L-
7 could be assigned as S configuration according to the single
crystal of diastereomeric salt, it is more convincing to use
another independent method to verify the stereochemical
characterization. Nowadays, electronic circular dichroism (ECD)
Table 3. Enantioseparation of rac-2 with acidic resolving agents.
Entry
1
rac-2
Resolving agent
Conditionsa
DCM:PE = 13:1 (14.3 L/mol) b
e.e. (%)d
Yield (%)
61
effe
2.14 mmol
D-7 (1.5 equiv.)
98.75 (R)
0.60
2
3
4
7.85 mmol
1.0 mmol
2.85 mmol
L-7 (1.5 equiv.)
D-7 (1.5 equiv.)
L-7 (1.5 equiv.)
DCM:PE = 13:1 (14.3 L/mol) b
DCM:n-pentane = 13:1 (14.3 L/mol) c
DCM:n-pentane = 13:1 (14.3 L/mol) c
99.61 (S)
99.98 (R)
99.93 (S)
56
70
68
0.55
0.69
0.67
a The amount of solvent relative to the amount of rac-2 is shown within the parenthesis. b The process of keeping slow volatilization in room temperature for 3 to
c
24 h, and the precipitation was washed twice. The process of keeping slow volatilization in room temperature for 2 to 6 h, and the precipitation was washed
once or twice. d e.e. was determined by chiral HPLC analysis. e eff = e.e.% ×yield%/10000.

5
ACCEPTED MANUSCRIPT
The ICD of components at 11.93 min and 18.71 min displayed
a positive CE at around 340 nm (Fig. 7), indicating that C2’ atom
on their structures could be assigned as S configuration. Both
other peaks gave negative CEs at around 340 nm in the ICD
spectra, leading to the assignments of their C2’ atoms as R
configuration. Thus, four stereoisomers were obtained, and their
absolute configurations were firmly established.
Thinking of the complexity of biological metabolism system
and the original function of IDO1 enzyme, it might be reasonable
and acceptable that the tremendous difference of IDO1 inhibitory
activity between these stereoisomers reduced greatly in mouse
(Table 5). Nevertheless, both isomers with S configuration at C5
position showed significant IDO1 inhibitory activity compared
with vehicle group and two other isomers’ groups at 1.5 h. It is
thus inferred that S configuration at C5 position is crucial for the
in vivo inhibitory activity against IDO1, while the C2’
stereochemistry shows less affection.
3. Conclusion
In this study, we carried out the direct enantiomer separation
of rac-2 by using di-p-toluoyl-L-tartaric acid (L-7) in
dichloromethane of petroleum ether or n-pentane solvent. The
special solvent system provided a suitable condition for the
interaction between rac-2 and L-7, including salt bond, H-bond,
CHꢀꢀꢀπ interaction, CHꢀꢀꢀO interaction and πꢀꢀꢀπ interaction, which
caused the highly selective combination and the effective
resolution. Absolute configurations of all stereoisomers of 1 and
2 were assigned by single crystal X-ray diffraction, comparison
of experimental and theoretical ECD spectra, and transition metal
methods. The IDO inhibitory activity of each stereoisomer of 1 in
vitro and in mouse plasma demonstrated that S configuration of
C5 played great role on the IDO1 inhibitory activity, while the
stereochemistry on C2’ exerted little effect on the inhibitory
activity of IDO1 in mouse plasma.
Fig. 7. ICD spectra induced by Rh2(CF3COO)4 complexation
with four stereoisomers of 1.
2.7. Effect of stereochemistry on IDO1 inhibitory activity
The inhibitory activity assessment results of rac-1 and its four
stereoisomers against IDO1 enzymatically and in HEK293 cells
are listed in Table 4. It is obvious that (5S,2’R)-1 possessed the
most potent IDO1 inhibitory activity with IC50 < 0.001 µM in
both assessments. Another isomer with the same C5
configuration displayed moderate activity against IDO1, while
the inhibitory activity was totally lost in the other two isomers
with C5 position as R configuration. This result is in coincidence
with Peng’s report against IDO1 at the level of isolated enzyme,
which showed that (5S,2’R)- and (5S,2’S)- isomers significantly
contributed to the IDO1 inhibitory activity 6.
4. Experimental section
4.1. Materials and methods
Reagents and solvents were purchased from Energy
Chemistry, and J&K Chemical. All commercial products were
used without further purification. The reactions were monitored
by thin layer chromatography (TLC). The silica gel column
chromatography was carried out with 300–400 mesh silica gel.
Melting points were measured on a RY-2 digital melting point
apparatus. Chiral HPLC analysis was performed using a Jasco
LC-2000 HPLC instrument with a UV detector (MD-2010) and
an auto sampler (AS-2055). Separation was implemented on
Daicel Chiralpak AD-H column (4.6 × 250 mm, 5ꢁm) or Daicel
Chiralcel OD-H column (4.6 × 250 mm, 5ꢁm). NMR spectra
were recorded on Joel ECZ-400S NMR System or Bruker
Avance-III 500 NMR spectrometer in CDCl3 or DMSO-d6. ESI-
HRMS data were collected on the Thermo Exactive Orbitrap
mass spectrometer. Optical rotations were obtained using an
INESA SGW-5 automatic polarimeter at D line at room
temperature. ECD spectra were recorded on a Jasco J-815
spectrometer in methanol. X-ray crystallographic data were
collected on an Oxford Gemini E diffractometer with graphite
monochromated Cu-Kα radiation.
Table 4. IDO1 inhibitory activity of rac-1 and its stereoisomers in vitro.
IC50 (µM)
Entry
rhIDO1
0.517
HEK293T cell
0.76
rac-1
(5S,2’S)-1
(5S,2’R)-1
(5R,2’R)-1
(5R,2’S)-1
0.329
0.29
< 0.001
> 100
< 0.001
> 100
> 100
> 100
Table 5. IDO1 inhibitory activity of rac-1 and its stereoisomers in mouse.
Specific metabolic rate (%)a
Entry
4.2. Synthesis of rac-1-cyclohexyl-2-(5H-imidazo[5,1-a]isoindol-
0 h
1.5 h
5-yl)ethanone (rac-2)
vehicle
1.81±0.64
1.42±0.22
1.66±0.27
1.45±0.10
1.33±0.35
1.61±0.26
2.11±0.51
1.24±0.08**
0.79±0.15***
1.01±0.07**
1.36±0.20*
1.53±0.27
The synthesis of rac-1-cyclohexyl-2-(5H-imidazo[5,1-a]
isoindol-5-yl)ethanone (rac-2) followed the reported procedure
with necessary modifications7. Rac-2 was purified by silica gel
column chromatography using PE and EA as elute to give rac-2
rac-1
(5S,2’S)-1
(5S,2’R)-1
(5R,2’R)-1
(5R,2’S)-1
1
with a yield of 51%. Yellow solid, m.p.: 80 − 81.9°C. H NMR
(400 MHz, DMSO-d6): δ (ppm) 7.56 (d, 2H, J = 6.0 Hz), 7.45 (d,
1H, J = 7.6 Hz), 7.34 (t, 1H, J = 7.5 Hz), 7.23 (t, 1H, J = 7.5
Hz), 7.08 (s, 1H), 5.54 (dd, 1H, J = 8.4, 4.0 Hz), 3.42 (dd, 1H, J
= 18.8, 4.0 Hz), 3.04 (dd, 1H, J = 17.6, 8.0 Hz), 2.40 (m, 1H),
a
Specific metabolic rate (%) = CKyn/CTrp × 100%, and values are represented
as means ± S.D. Compared with vehicle group, * P < 0.05, ** P < 0.01, ***
< 0.001.
P

6
Tetrahedron
1.84 – 1.52 (m, 5H), 1.28 – 1.12 (m, 5H). ESI-HRMS: calcd for
n-Hex/EtOH = 85:15, 20°C, 1.0 mL/min, t (5R,2’R) = 9.78 min;
ACCEPTED MANUSCRIPT
R
C18H21N2O [M+H]+ 281.1648, found 281.1646.
tR(5R,2’S) = 11.93 min; tR(5S,2’S) = 18.71 min; tR(5S,2’R) =
21.15 min).
4.3. Synthesis of rac-5-(2-hydroxylcyclohexylethyl)-5H-imidazo
[5,1-a]isoindole (rac-1)
4.5.1. (R)-5-((R)-2-hydroxylcyclohexylethyl)-5H-
imidazo[5,1-a]isoindole ((5R,2’R)-1)
The synthesis of rac-5-(2-hydroxylcyclohexylethyl)-5H-
imidazo[5,1-a]isoindole (rac-1) followed the reported procedure
with necessary modifications7. Rac-1 was purified by silica gel
column chromatography using PE and EA as elute with a yield of
1
White solid, m.p.: 142 − 143°C. H NMR (400 MHz, CDCl3): δ
(ppm) 7.88 (s, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.36 – 7.29 (m, 2H),
7.24 – 7.19 (m, 1H), 7.15 (s, 1H), 5.49 (dd, J = 10.8, 2.8 Hz, 1H),
3.79-3.74 (m, 1H), 3.40 (s, 1H), 2.23 (m, 1H), 1.90 (m, 1H), 1.81
– 1.59 (m, 5H), 1.29 – 0.92 (m, 6H). 13C NMR (125 MHz,
CDCl3): δ (ppm) 145.50, 137.60, 132.66, 130.02, 128.56, 126.60,
124.15, 120.25, 117.89, 73.26, 59.35, 44.76, 40.96, 29.20, 28.21,
26.61, 26.38, 26.29. ESI-HRMS: calcd for C18H23N2O [M+H]+
1
80%. White solid, m.p.: 158 − 159°C. H (500 MHz, CDCl3 for a
mixture of two pairs of diastereoisomersm, major/minor = 4/1) δ
(ppm) 7.94 (s, 1H, minor), 7.89 (s, 1H, major), 7.58 (d, J = 7.5
Hz, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H, major),
7.38 (s, 1H, minor), 7.27 (s, 1H), 7.21 (s, 1H), 5.55 (d, J = 12.0
Hz, 1H, minor), 5.41 (t, J = 6.0 Hz, 1H, major), 3.86 − 3.80
(m,1H, minor), 3.80 − 3.76 (m, 1H, major), 2.28(s,1H), 2.20 −
2.18 (m, 1H), 1.80 − 1.69 (m, 5H), 1.30-1.04 (m, 6H). ESI-
HRMS: calcd for C18H23N2O [M+H]+ 283.1805, found 283.1805.
23
283.1805, found 283.1805. [α]D = −20.9 (c = 0.3, EtOH). ECD
(CH3OH): ∆ε 267.5 (−2.41), 246.5 (−1.39), 231.5 (−2.51), 203.0
(+6.05).
4.5.2. (S)-5-((S)-2-hydroxylcyclohexylethyl)-5H-
imidazo[5,1-a]isoindole ((5S,2’S)-1)
4.4. Chiral resolution of rac-2
1
White solid, m.p.: 141-142°C. H NMR (400 MHz, CDCl3): δ
(ppm) 7.91 (s, 1H), 7.52 (d, J = 7.2 Hz, 1H), 7.34 (t, J = 8.0 Hz,
2H), 7.23 (t, J = 8.0 Hz, 1H), 7.17 (s, 1H), 5.49 (dd, J = 10.4, 2.8
Hz, 1H), 3.78 (m, 1H), 2.95 (s, 1H), 2.27-2.19 (m,1H), 1.89-1.86
(m,1H), 1.83 – 1.59 (m, 5H), 1.30 – 0.93 (m, 6H). 13C NMR (125
MHz, CDCl3): δ (ppm) 146.05, 137.91, 133.37, 129.80, 128.50,
126.79, 123.35, 120.26, 117.78, 71.19, 58.52, 44.77, 39.72,
29.04, 28.51, 26.57, 26.25,18.66. ESI-HRMS: calcd for
C18H23N2O [M+H]+ 283.1805, found 283.1805. [α]D23 = +21.1 (c
= 0.6, EtOH). ECD (CH3OH): ∆ε 265.0 (+2.28), 245.5 (+1.37),
229.5 (+2.52), 204.0 (−4.15).
Rac-2 (2.20 g, 7.85 mmol) and resolving agent D-7 (4.55 g,
11.78 mmol) were dissolved in DCM (104 mL). PE (7.8 mL) was
added to the mixture and stirred at room temperature for 24h. The
precipitated salt was collected by filtration and washed by 20 mL
DCM. The residue was then suspended in 20 mL water, and
saturated Na2CO3 aqueous solution was added to adjust pH value
to 9-10. The solution was extracted with DCM (20 mL × 3), and
dried over anhydrous Na2SO4. After removal of the solvent, (R)-2
(0.62 g, 2.21 mmol, 99% ee, 56% yield) was obtained as yellow
oil. The yield was calculated based on a half amount of rac-2
initially used. The enantiomer excess was determined by chiral
HPLC analysis (Daicel Chiralcel OD-H, 20°C, n-Hex:2-propanol
4.5.3. (S)-5-((R)-2-hydroxylcyclohexylethyl)-5H-
imidazo[5,1-a]isoindole ((5R,2’S)-1)
1
= 90:10, 1.0 mL/min, tr(R) = 28.71 min, tr(S) = 20.31 min). H
1
White solid, m.p.: 139 − 140°C. H NMR (400 MHz, CDCl3): δ
NMR (400 MHz, DMSO-d6): δ (ppm) 7.55 (d, 2H, J = 7.2 Hz),
7.44 (d, 1H, J = 7.6 Hz), 7.34 (t, 1H, J = 7.6 Hz), 7.22 (t, 1H, J =
7.6 Hz), 7.08 (s, 1H), 5.54 (dd, 1H, J = 8.4, 4.0 Hz), 3.41 (dd,
1H, J = 18.4, 4.4 Hz), 3.03 (dd, 1H, J = 18.4, 8.8 Hz), 2.40 (m,
1H), 1.83 – 1.51 (m, 5H), 1.32 – 1.05 (m, 5H). 13C NMR (125
MHz, DMSO-d6): δ (ppm) 212.11, 145.53, 137.66, 133.35,
130.05, 129.02, 124.51, 120.23, 118.59, 56.68, 56.33, 50.15,
46.72, 40.31, 28.37, 26.05, 25.66. ESI-HRMS: calcd for
(ppm) 7.81 (s, 1H), 7.49 (d, J = 7.2 Hz, 1H), 7.42 (d, J = 7.6 Hz,
1H), 7.32 (t, J = 7.6 Hz, 1H), 7.20 (t, J = 7.6 Hz, 1H), 7.11 (s,
1H), 5.35 (t, J = 6.4 Hz, 1H), 3.79 – 3.70 (m, 1H), 3.15 (s, 1H),
2.13 (m, 1H), 2.01 (m, 1H), 1.74 (m, 5H), 1.29 – 0.88 (m, 6H).
13C NMR (125 MHz, CDCl3): δ (ppm) 145.50, 137.60, 132.66,
130.02, 128.56, 126.60, 124.15, 120.25, 117.89, 73.26, 59.35,
44.76, 40.96, 29.20, 28.21, 26.61, 26.38, 26.29. ESI-HRMS:
23
calcd for C18H23N2O [M+H]+ 283.1805 found 283.1805. [α]D
=
23
C18H21N2O [M+H]+ 281.1648, found 281.1646. [α]D = +81.0 (c
−38.2 (c = 0.6, EtOH). ECD (CH3OH): ∆ε 268.0 (−2.73), 245.0
(−1.35), 231.5 (−2.78), 202.0 (+8.21).
= 0.5, CH3OH). ECD(CH3OH): ∆ε 296.5 (+2.03), 265.5 (−2.30),
226.5 (−3.81), 200.0 (+19.58).
4.5.4. (R)-5-((S)-2-hydroxylcyclohexylethyl)-5H-
By the same way, using L-7 as resolving agent to resolved rac-
2 (1 g, 3.57 mmol) obtained (S)-2 (0.28 g, 1.00 mmol, 99% e.e.,
56% yield). Yellow oil. 1H NMR (400 MHz, DMSO-d6): δ (ppm)
7.56 (t, 2H, J = 4.8 Hz), 7.45 (d, 1H, J = 4.4 Hz), 7.34 (t, 1H, J =
8.0 Hz), 7.22 (t, 1H, J = 8.0 Hz), 7.08 (s, 1H), 5.54 (dd, 1H, J =
9.2, 4.8 Hz), 3.41 (dd, 1H, J = 18.4, 4.4 Hz), 3.04 (dd, 1H, J =
18.8, 8.8 Hz), 2.40 (m, 1H), 1.87 – 1.45 (m, 5H), 1.33 – 1.02 (m,
5H). 13C NMR (125 MHz, DMSO-d6): δ (ppm) 212.90, 145.35,
137.38, 133.16, 129.86, 128.85, 126.85, 124.33, 120.05, 118.41,
imidazo[5,1-a]isoindole ((5S,2’R)-1)
1
White solid, m.p.: 139 − 141°C. H NMR (400 MHz, CDCl3): δ
(ppm) 7.95 (s, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.43 (d, J = 7.6 Hz,
1H), 7.36 (t, J = 7.2 Hz, 1H), 7.27-7.23 (m, 1H), 7.17 (s, 1H),
5.38 (t, J = 6.0 Hz, 1H), 3.77 – 3.69 (m, 1H), 2.21 – 1.94 (m,
2H), 1.85 – 1.59 (m, 5H), 1.46 – 0.82 (m, 6H). 13C NMR (125
MHz, CDCl3): δ (ppm) 145.25, 137.64, 132.68, 129.92, 128.76,
126.84, 124.12, 117.47, 120.45, 77.49, 73.60, 59.59, 44.63,
40.62, 29.11, 28.01, 26.55, 26.20, 18.65. ESI-HRMS: calcd for
56.14, 49.97, 46.53, 39.96, 28.21, 25.86, 25.48. ESI-HRMS:
C18H23N2O [M+H]+ 283.1805, found 283.1805. [α]D = +38.8 (c
23
23
calcd for C18H21N2O [M+H]+ 281.1648, found 281.1646. [α]D
=
=0.7, EtOH). ECD (CH3OH): ∆ε 267.5 (+2.51), 246.0 (+1.42),
231.5 (+2.46), 203.0 (−7.43).
−85.6 (c = 0.9, CH3OH). ECD (CH3OH): ∆ε 296.5 (−2.03), 267.0
(+2.47), 228.5 (+3.96), 200.0 (−20.67).
4.6. Single crystal X-ray analysis
4.5. General procedure for the preparation of enantiopure 5-(2-
hydroxylcyclohexylethyl)-5H-imidazo[5,1-a]isoindole (1)
X-ray crystallographic data were collected on the Oxford
Gemini E diffractometer with graphite monochromated Cu-Kα
radiation. The structure was solved by a direct method using
Shelx97 and refined by SHELXL-2014/7 program. Crystal data
for (S)-2ꢀL-7ꢀ2EtOH: C42H50N2O11, M = 758.84, orthorhombic, a
Enantiopure 2 was treated in the same way as its racemate two
give four enantiopure stereoisomers of 1 after silica gel column
chromatography. The enantiomeric excess of each optical pure 1
was determined by an HPLC analysis (Daicel Chiralpak AD-H,

7
= 13.3264(7) Å, b = 15.6600(11) Å, c = 19.3842(10) Å, U =
4045.3(4) Å3, T = 106.5 K, space group P212121, ꢁ(Cu Kα) =
mM using 50% acetonitrile. The pure water with different
ACCEPTED MANUSCRIPT
concentration of Trp and Kyn solution (10 ꢁL) and the internal
standard (Inderal, 0.2 ꢁg/mL, 170 ꢁL) were mixed, followed by
the centrifugation (14000 rpm × 5 min) twice. The supernate (3
ꢁL) was analyzed by LC/MS/MS, the condition of which was as
follows: Zobax C18 (2.1 mm × 100 mm, 3.5 ꢁm) column, 37°C,
acetonitrile/water (0.1% formic acid), 0.2 mL/min, m/z
204.9→188 (Trp), m/z 208.8→192.1 (Kyn), m/z 260→183
(Inderal as internal standard).
0.741, Z = 4, 13929 reflections measured, 7637 unique (Rint
=
0.0221) which were used in all calculations. The final wR(F2)
was 0.1004 (all data). Crystallographic data for the structures in
this paper with deposition numbers CCDC 1570864 can be
obtained free of charge from the Cambridge Crystallographic
Data Centre via https://www.ccdc.cam.ac.uk/structures/.
4.7. Quantum-chemical calculation
Thirty mice were divided randomly into six groups. Five
groups were fed respectively by 9 mg/mL 0.5% CMC suspension
of five tested samples, in which tween was used for the assisted
dissolution. Orbital blood collection was carried out immediately
and after 1.5 h. The serum samples (10 ꢁL), 20 ꢁL acetonitrile
and the internal standard (Inderal, 0.2 ꢁg/mL, 170 ꢁL) were
mixed, followed by the centrifugation (14000 rpm × 5 min)
twice. The supernate (3 ꢁL) was analyzed by LC/MS/MS.
Absolute configurations at C5 atom on enantiopure 1 and 2
were assigned by comparison of experimental ECD spectra with
their theoretical ECD spectra obtained by quantum-chemical
calculations using Gaussian 09. Conformational analysis and
ECD calculations were carried out according to the previously
reported protocols12. For each main stable conformer (Boltzmann
distribution > 1%), sixty lowest electronic transitions were
obtained using combinations of hybrid functionals and basis sets
including B3LYP/6-31+G(d,p) and Cam-B3LYP/6-31+G(d,p)
approaches. Solvent effects were taken into consideration by
using polarizable continuum model (PCM) for methanol using
the dielectric constants of 32.613. The overall ECD spectra were
then gained at the bandwidth σ = 0.35 eV according to the
Boltzmann average of each conformer.
Supplementary Materia
Single crystal X-ray diffraction, chiral HPLC analysis, NMR, and
ESI-HRMS spectra for compounds 1-2 are included. This
material is available free of charge via the Internet at https://.
Acknowledgments
4.8. IDO1 inhibitory activity assessment
4.8.1. Enzyme-based IDO1 inhibitory activity assay
This study was financially supported by the CAMS Innovation
Fund for Medical Sciences (CIFMS, No. 2016-I2M-3-009).
The inhibitory activity of the compounds against IDO-1 was
determined as previously reported13. A standard reaction mixture
(100 ꢁL) containing 0.05 ꢁM rhIDO-1, 200 ꢁg/mL catalase, 20
ꢁM methylene blue, 100 mM potassium phosphate buffer (pH
6.5), and 40 mM ascorbic acid (neutralized with NaOH) was
added to the solution containing the substrate L-Trp and the test
References and notes
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sample at
a determined concentration. The reaction was
2.
3.
Sharma P, Allison JP. Science. 2015; 348: 56−61.
Sugimoto H, Oda S, Otsuki T, Hino T, Yoshida T, Shiro Y. P Natl
o
performed at 37 C for 45 min and stopped by adding 20 ꢁL of
30% (w/v) trichloroacetic acid. After heating at 65°C for 15 min,
100 ꢁL of 2% (w/v) p-dimethylaminobenzaldehyde (DMAB) in
acetic acid was added to each well. The yellow pigment derived
from Kyn was measured at 492 nm using a Synergy-H1
microplate reader (BioTek). IC50 was analyzed using the
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4.8.2. HEK293T cell-based IDO1 inhibitory assay
6.
Peng YH, Ueng SH, Tseng CT, Hung MS, Song JS, Wu JS, Liao FY,
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The assay was performed according to a reported method13.
HEK293 cells were seeded in a 96-well culture plate at a density
of 2×104 cells/well and cultured overnight. The HEK293 cells
were then transfected with pcDNA3.1-hIDO-1 using
Lipofectamin 3000 according to the manufacturer’s instructions.
At 16 h after transfection, a serial dilution of the tested
compounds in 200 ꢁL culture medium was added to the cells.
After an additional 24 h incubation, 100 ꢁL of the supernatant
per well was transferred to a new 96-well plate and mixed with
20 ꢁL of 30% trichloroacetic acid in each well. The plate was
incubated at 65oC for 15 min to hydrolyze N-formyl Kyn
produced by the catalytic reaction of IDO-1. Then, 100 ꢁL of 2%
(w/v) DMAB in acetic acid was added to each well. The yellow
color derived from Kyn was measured at 492 nm using a
Synergy-H1 microplate reader (BioTek).
7.
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Mautino M, Kumar S, Jaipuri F, Waldo J, Kesharwani T, Zhang X.
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4.8.3. IDO1 inhibitory activity assay in mouse
plasma
Male C57 mice (14-16g) were bought from Beijing Vital River
Laboratory Animal Technology Co. Ltd. The assay was
performed as literature14. The establishment of standard curve
was carried out by different concentration of working liquid of
Trp and Kyn. The solution of Trp and Kyn were prepared as 20
13. Yu CJ, Zheng MF, Kuang CX, Huang WD, Yang Q. J. Alzheimers.
Dis. 2010; 22: 257–266.
14. Koblish HK, Hansbury MJ, Bowman KJ, Yang G, Neilan CL, Haley
PJ, Burn TC, Waeltz P, Sparks RB, Yue EW, Combs AP, Scherle PA,
Vaddi K, Fridman JS. Mol Cancer Ther. 2010; 9: 489−498.