Identification and preliminary structure–activity relationships of 1-Indanone derivatives as novel indoleamine-2,3-dioxygenase 1 (IDO1) inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.05.017

Indoleamine 2,3-dioxygenase 1 (IDO1) plays a vital role in the catabolism of tryptophan along with the kynurenine pathway which is involved in many human diseases including cancer, Alzheimer's disease, etc. In this study, compound 1 bearing a 1-Indanone scaffold was identified as a novel IDO1 inhibitor by structure-based virtual screening, with moderate to good enzymatic and cellular inhibitory activities. Also, surface plasmon resonance analysis validated the direct interaction between compound 1 and IDO1 protein. The preliminary SAR was further explored and the binding mode with IDO1 protein was predicted by experiment along with molecular docking. Subsequent ADME properties of these active compounds were analyzed in silico, and the results showed good pharmacokinetic efficiencies. We believe this study contributes a lot to the structural diversity for the future development of highly potent IDO1 inhibitors.

Full text of DOI:10.1016/j.bmc.2017.05.017

Accepted Manuscript
Identification and preliminary structure–activity relationships of 1-Indanone
derivatives as novel indoleamine-2, 3-dioxygenase 1 (IDO1) inhibitors
Dingding Gao, Yingxia Li
PII:
S0968-0896(17)30411-X
http://dx.doi.org/10.1016/j.bmc.2017.05.017
BMC 13735
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 March 2017
5 May 2017
8 May 2017
Please cite this article as: Gao, D., Li, Y., Identification and preliminary structure–activity relationships of 1-
Indanone derivatives as novel indoleamine-2, 3-dioxygenase 1 (IDO1) inhibitors, Bioorganic & Medicinal
Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.05.017
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Identification and preliminary structure–activity relationships of
1-Indanone derivatives as novel indoleamine-2, 3-dioxygenase 1
(IDO1) inhibitors
Dingding Gao and Yingxia Li*
School of Pharmacy, Fudan University, Shanghai, 201203, China
Abstract: Indoleamine 2, 3-dioxygenase 1 (IDO1) plays a vital role in the catabolism of tryptophan along with the
kynurenine pathway which is involved in many human diseases including cancer, Alzheimer’s disease, etc. In this
study, compound 1 bearing a 1-Indanone scaffold was identified as a novel IDO1 inhibitor by structure-based
virtual screening, with moderate to good enzymatic and cellular inhibitory activities. Also, surface plasmon
resonance analysis validated the direct interaction between compound 1 and IDO1 protein. The preliminary SAR
was further explored and the binding mode with IDO1 protein was predicted by experiment along with molecular
docking. Subsequent ADME properties of these active compounds were analyzed in silico, and the results showed
good pharmacokinetic efficiencies. We believe this study contributes a lot to the structural diversity for the future
development of highly potent IDO1 inhibitors.
Keywords: Indoleamine 2, 3-dioxygenase 1; inhibitors; 1-Indanone derivatives; structure–activity relationships.
* Corresponding authors. Y. Li, Tel.: +86-21-51980127; E-mail: liyx417@fudan.edu.cn.
1

1. Introduction
Tryptophan (Trp) is the least abundant essential amino acid in mammals and about 95% of dietary Trp is
metabolized in the liver through kynurenine pathway (KP).1 Indoleamine 2,3-dioxygenase 1 (IDO1) is a
heme-containing enzyme that catalyzes the oxidative cleavage of L-Trp to N-formyl kynurenine (NFK) in the
initial and rate limiting step of kynurenine pathway.2 Dysregulation of the kynurenine pathway is mainly due to the
elevation of IDO1 and the metabolites have been involved in many human diseases including cancer,
neuroinflammatory disorders, Alzheimer’s disease, age-related cataracts, and HIV encephalitis.3-7 Nowadays,
IDO1-mediated immune tolerance and immune evasion have been wildly accepted as one of the most important
mechanisms in the process of immune evasion by tumors.8-11 The high IDO1 expression causes the depletion of the
local tryptophan levels and the accumulation of the toxic tryptophan metabolites, which lead to the suppression of
T-cell response and the enhancement of immunosuppression mediated by regulatory T cells (Tregs).12, 13 Hence,
IDO1 is an attractive target for cancer immunotherapy.
Structure-based drug design was dramatically utilized to identify novel scaffold IDO1 inhibitors as its crystal
structure was reported. Compound 4-phenylimidazole (PIM) is the first IDO1 inhibitor co-crystalized with the
IDO1 protein (PDB code: 2D0T), with an IC50 value of 48 µM.14 The complex crystal structure shows that the
phenyl group of PIM interacts with Phe-163 of IDO1 via π-π stacking, while the distal nitrogen of the imidazole
interacts directly with the heme iron. MMG-0385 is another highly efficient (ligand efficiency=0.72) low
molecular weight inhibitor with potent inhibitory activities both in an enzymatic and cellular assay.15 Amg-1 was
identified via high throughput screening by Amgen with an IC50 value of 3 µM and its crystal structure with IDO1
protein was reported by Yoshiaki Isobe in 2014 (PDB code: 4PK5).16 Guided by the cocrystal structure, they
developed the compound 17g as a novel potent IDO1 inhibitor. Recently, several classes of IDO1 inhibitors have
been reported in the literatures.17 To the best of our knowledge, only three IDO1 inhibitors are in clinical trials
(Indoximod, NLG919 and Epacadostat) and no drug has been approved by FDA up to now (Fig. 1). Indoximod
is the D-enantiomer of 1-methyltryptophan, developed by NewLink Genetics, and currently is undergoing
evaluation in multiple human clinical trials.18-22 NewLink Genetics reported another compound GDC-0919 which
currently is in phase I clinical trial for adult patients with recurrent advanced solid tumors.23 Incyte Corporation
developed a clinical candidate Epacadostat (INCB024360) which was under phase 3 development in combination
with pembrolizumab for unresectable or metastatic melanoma.24-29
2

Fig. 1 Structures of the representative IDO1 inhibitors
In this study, a novel IDO1 inhibitor (Compound 1) bearing 1-Indanone scaffold (Fig. 2) was identified via
structure-based virtual screening. In vitro enzymatic assay with purified recombinant human IDO1 protein
showed that compound 1 exhibited potent IDO1 inhibitory activity (IC50 = 2.780 µM). Furthermore, compound 1
displayed cellular IDO 1 inhibitory activity in the HeLa cell line with an EC50 value of 9.171 µM (Fig. 3). 30, 31
Consequently, compound 1 was considered as a potential hit compound and a series of structural optimizations was
performed. We focused on the studies of the ring C (3a-3j, 6), ketone group (3k-3l) and the linker between ring B
and ring C (24, 27) to explore its preliminary SAR. Besides, scaffold hopping strategy was used to replace the
thiazoleyl with carbonyl group generating hydrazide derivatives (9, 15) (Fig. 2).
Fig. 2 Structures of hit compound 1 and the design of target compounds.
Hela cell
Compound 1
IDO1
Compound 1
150
100
50
150
100
50
0
0
-3
-2
-1
0
1
2
-3
-2
-1
0
1
2
3
Log 10 concentration (µM)
Log 10 concentration (µM)
IC50/ 2.780 µM
EC50/9.171 µM
Fig. 3 Dose-dependent inhibition of compounds 1 on IDO1 enzymatic assay and cellular IDO1 inhibitory activity in the HeLa
cell line. The data were evaluated by Prism 5.
2. Results and discussion
3

2.1. Chemistry
Methods for the synthesis of compounds 3a-3l have been well developed.32, 33 Commercially available 1a-1c
was condensed with thiosemicarbazide in ethanol with catalytic amounts of acetic acid to afford
thiosemicarbazones (2a-2c). Then the desired compounds (3a-3l) were obtained through Hantzsch reaction from
corresponding thiosemicarbazones (2a-2c) and substituted α-bromo ketone (Scheme 1). To access compound 6,
α-bromo ketone 5 which was prepared from cyclohexyl methyl ketone (4) by bromination underwent a cyclization
with intermediate 2a in ethanol /water under sealed tube (Scheme 2).36
Scheme 1 Synthesis of target compounds 3a– 3l. Reagents and conditions: (a) thiosemicarbazide, C2H5OH, AcOH, reflux, 2-4h,
82-88%; (b) substituted α-bromo ketone, C2H5OH, reflux, 4h, 85-95%.
Scheme 2 Synthesis of target compound 6. Reagents and conditions: (a) Br2, C2H5OH/H2O, rt, 6h; (b) 2a, C2H5OH, sealed tube,
130° C, overnight, 65%.
The synthetic route of hydrazide derivatives is provided in Scheme 3. The reaction of commercially available
methyl 3-hydroxybenzoate (7) with hydrazine hydrate in ethanol led to the formation of the corresponding acyl
hydrazides 8,37 which was further reacted with 1a in ethanol under the catalysis of acetic acid to give compound 9
(Scheme 3A). Compound 15 was synthesized as described in Scheme 3B. Treatment of methyl 3-aminobenzoate
with di-tert-butyl dicarbonate in dichloromethane at room temperature resulted in the formation of 11. Then it was
methylated with iodomethane in DMF, providing intermediate 12. After removing the protecting group by
CF3COOH, the corresponding product 13 was treated with hydrazine hydrate in ethanol to give 14.37,38 Finally, the
compound 15 was obtained using the same method as the synthesis of compounds 2a-2c.
4

Scheme 3 Synthesis of target compounds 9 and 15. Reagents and conditions: (a) NH2NH2.H2O, C2H5OH, reflux, 10h, 80%; (b)
1a, C2H5OH, AcOH, reflux, 2h, 85%; (c) (BOC)2O, CH2Cl2, Et3N, rt, 10h, 51%; (d) CH3I, NaH, DMF, 0° C, overnight; (e)
CF3COOH, CH2Cl2, rt, 12h, 85%; (f) NH2NH2.H2O, C2H5OH, reflux,10h, 65%; (g) 1a, C2H5OH, AcOH, reflux, 4h, 45%;
The preparation of target compounds 24 and 27 are described in Scheme 4 and Scheme 5. Firstly,
3-Carboxy-1-indanone was esterified with concentrated sulfuric acid in CH3OH to obtain the methyl ester 17.
Reaction of 17 with ethylene glycol in the presence of p-toluene sulfonic acid and trimethyl orthoformate gave
ketal 18. Reduction of the ester with lithium aluminum hydride gave alcohol 19, which was hydrolyzed with 1M
HCl to obtain 20.39 Treatment of alcohol 20 with Diphenylphosphoryl azide gave 80% of azide 21, which was
reduced with 10% Pd/C and H2 to afford amine 22.40 The amine 22 was then treated with
1-isocyanato-3-methoxybenzene to form intermediate 23 which was demethylated by BBr3 (17% in CH2Cl2) to
give final compound 24. A two-step procedure was chosen whereby acylthiourea intermediate 25 was obtained
from 22 by treatment with benzoyl isothiocyanate and subsequently solvolyzed to yield 26 (Scheme 5).41 Finally,
mixing 26 with 2-bromo-1-(3-hydroxyphenyl)ethan-1-one in ethanol under reflux provided compound 27. The
purity of all the designed compounds was analyzed with the Agilent 1200 HPLC system.
Scheme 4 Synthesis of target compound 24. Reagents and conditions: (a) H2SO4, CH3OH, reflux, 4h, 92%; (b) p-TsOH,
anhydrous benzene, ethylene glycol, trimethyl orthoformate, 50° C, 2h, 95%; (c) LiAlH4, anhydrous THF, reflux, 100%; (d)
HCl(1M), 100%; (e) DPPA, DBU, DMF, 90 °C, 6h, 80%; (f) 10% Pd/C, H2; (g) 1-isocyanato-3-methoxybenzene, DIPEA, THF,
2h,80%; (h) BBr3 (17% in CH2Cl2), CH2Cl2, 0° C, 50%.
5

Scheme 5 Synthesis of target compound 27. Reagents and conditions: (a) benzoyl isothiocyanate, trimethylamine, THF, 2h; (b)
1M NaOH, CH3OH,2h; (c) 2-bromo-1-(3-hydroxyphenyl)ethan-1-one, CH3CH2OH, reflux, 4h, 85%.
2.2. IDO1 inhibitory activities
All of the designed compounds were tested for IDO1 inhibitory activities at 10 µM (L-1MT was used as a
positive control). Compounds with higher inhibition rates were selected to test IC50 values. As shown in Table 1
and Table 2, the hydroxyl group at C-3 position in ring C was critical for the activities. Changing the position of
hydroxyl group or replacing it with hydrogen, methoxy and halogen led to decreased inhibitory activity (1 vs
3a-3j). Moreover, the replacement of a cyan group at C-3 position in ring C reduced the activity from 2.780 µM to
9.784 µM (1 vs 3h), and moving the cyan group from C-3 position to C-4 position resulted in a significant drop of
inhibitory activity (3h vs 3i). Incorporation of methoxy both at C-3 and C-4 position restored the inhibitory
activity with an IC50 value of 5.255 µM. Changing the aromatic ring C to cyclohexyl displayed similar activity to
compound 3d with IC50 values 10.39 µM and 10.7 µM respectively (6 vs 3d). Intriguingly, removing or changing
the position of the ketone in ring B were detrimental for enzyme inhibition which indicated that it may coordinate
with the heme iron atom (1 vs 3k, 3l). Additionally, the hydrazide derivatives 9 and 15 showed weaker activities
than the hit compound 1. This may be due to the binding position of ring C was greatly changed.
Table 1 Structures and IDO1 inhibitory activities of the test compounds
Compound
R1
hIDO1 10 µM (%)
17.51
Compound
R1
hIDO1 10 µM (%)
59.47
3a
3b
3c
3d
3j
3k
3l
30.13
8.56
35.38
-0.027
44.32
25.36
9
6

33.39
33.37
33.37
50.96
43.9
70.82
<10
3e
3f
15
1
3g
3h
24
<10
27
CN
19.21
40.25
30.5
3i
6
L-1MT
Table 2 Enzymatic and cellular assays for IDO1 inhibition
Compound
hIDO1 IC50 (µM) a
95.6 5.3
Hela-IDO1 EC50 (µM) b
110 10
L-1MT
1
2.780 0.408
14.97 0.815
10.39 0.318
9.784 0.592
5.255 0.179
10.7 1.205
9.171 0.356
17.54 0.745
13.26 0.636
12.52 0.721
9.703 0.518
15.31 0.472
3c
3d
3h
3j
6
a IC50 values are the mean of at least two independent assays.
b EC50 values are the mean of at least three independent assays.
2.3. Cellular IDO1 inhibitory activity
Some selected compounds were further tested the cellular IDO1 inhibition. As illustrated in Table2, all of these
compounds show micromole EC50 values in Hela cell lines where native human IDO1 is overexpressed by the
stimulation of IFN-γ. Compounds 1 and 3j with higher enzymatic inhibitions display better cellular inhibitory
activities with EC50 values of 9.171 µM and 9.703 µM respectively. More importantly, a good correlation between
the two assays confirmed that 1-Indanone derivatives can be developed to potent IDO1 inhibitors.
2.4. Molecular docking
7

Molecular docking (Schrodinger, Maestro suite) models of compound 1 and compound 9 binding with IDO1 were
generated using the crystal structure of IDO1 complexed with Amg-1 (PDB code: 4PK5). As illustrated in Figure 4,
the ketone group in ring B of compound 1 directly binds to the heme iron and ring A is sitting at a pocket made of
Tyr126, Cys129, Val130, Phe163, Phe164, Ser167, and Leu234 (Pocket A). So removing or changing the position
of the ketone in ring B are detrimental for enzyme inhibition (1 vs 3k, 3l). Furthermore, it is predicted that the NH
participates in a hydrogen bond interaction with the heme 7-propionate group. Also, the thiazole and ring C
moieties extend into the active site entrance region (Pocket B) formed by Phe163, Phe226, Leu234, Ile354, and
Arg231. More importantly, the hydroxyl group at 3-position in ring C formed a critical H-bond interaction with
Phe226. It may be the reason that changing the position or replacing the hydroxyl group led to the decrease of
activities (1 vs 3a-3j). Additionally, ring B and ring C interacted with the electron-rich benzene ring of Phe163 and
Phe226 via π-π stacking respectively. For compound 9, the binding position of ring C was greatly changed.
Therefore, the important π-π stacking and H-bond interaction with Phe226 were missing. Also, the NH cannot
form H-bond interaction with the heme 7-propionate group. All of these reasons led to the decrease of the activities
(1 vs 9, 15).
Fig. 4 Predicted binding modes for compound 1 (A1, A2) and compound 9 (B1, B2) with IDO1. The pictures were generated
using Pymol.
2.5. SPR Analysis
To further validate the interaction between compound 1 and IDO1 protein, surface plasmon resonance (SPR) assay
was conducted using a Biacore T200 optical biosensor. As shown in figure 5, the response unit (RU) values were
proportional to compound concentrations within the selected ranges. The equilibrium dissociation constant (KD)
8

between compound 1 and IDO1 was 9.828 µM, which demonstrated its binding affinity to the target IDO1 protein.
Fig. 5 SPR analysis of compound 1 with IDO1 protein. Concentration from top to bottom: 20µM, 12.5µM, 10 µM, 6.25 µM,
1.5625 µM, 0.78125 µM. The equilibrium dissociation constant (KD) value is 9.828 µM.
2.6. ADME properties
The values of ADME properties were predicted using Qikprop (Schrödinger, LLC, NewYork, USA) and presented
in Table 3. All the selected compounds showed a good percentage human oral absorption ranging from 87.46 to
100%. Additionally, these compounds fulfilled Lipinski’s drug-like 34,35properties and no more than one Lipinski
violations had been observed: QPLogP o/w ≤5, molecular weight ≤ 500, number of hydrogen bond acceptors≤ 10
and number of hydrogen bond donors≤5. What’s more, the blockage of HERG K+ channels (QPLogHERG) was
also calculated. The predicted Caco2 cell permeability presented as QPPCaco was found between 322 and 1877
that are in the acceptable range for 95% of oral drugs. The predicted brain/blood partition coefficient (QP logBB)
is also within acceptable range (-3.0 to 1.2 for 95% of oral drugs). To obtain solid evidence for further
development of our compounds, 3j was selected to evaluate the in vitro liver microsomal stability and Caco-2
permeability (see supplementary material). For liver microsomal stability assay (Table S1), compound 3j showed
differences between species (mouse, rat, and human). 46.7% of parent compound was remaining after metabolism
in human liver microsome which indicated moderate metabolic stabilities. Furthermore, compound 3j metabolized
slowly with a T1/2 value of 93.8 min and a low Clint (22.4 mL/min/gprot) in human liver microsome. The
permeability coefficients for compound 3j were summarized in Table S2. According to the previous study,
compounds with permeability coefficients Papp < 1×10-6cm/s, 1-10×10-6cm/s, and > 10×10-6cm/s were defined
as poorly, moderately and well absorbed, respectively. The parental compound 3j exhibited moderate permeation
through the Caco-2 cell monolayer (Apical to Basal:1.63×10-6cm/s, Basal to Apical: 2.33×10-6cm/s). All of these
results can be used as important references for further development of our compounds.
Table 3 Pharmacokinetic parameters important for good oral bioavailability of the selected compounds
Compd
% ABS
MW
QPLogP
o/w
DonorHB
AccptHB
QPLogHERG
QPPcaco-2
QPLogBB
9

90.76
100
335.3
349.4
319.3
344.3
379.4
325.4
2.74
3.47
3.46
2.67
3.54
3.61
2
1
1
1
1
1
6
6
5
7
7
5
-6.53
-6.46
-6.66
-6.71
-6.41
-5.70
465
1521
1521
322
-0.995
-0.437
-0.366
-1.21
1
3c
3d
3h
3j
6
100
87.46
100
1521
1877
-0.517
-0.267
100
%ABS: percentage human oral absorption; MW: molecular weight; QPLogP o/w: Log of the octanol/water partition coefficient;
DonorHB: number of hydrogen bonds donors; AccptHB: number of hydrogen bond acceptors; QPLogHERG: Predicted IC50 value for
blockage of HERG K+ channels; QPPcaco-2: Caco-2 cell permeability in nm/s, range 95% of drugs (<25 poor, >500 great); QPLogBB:
Brain/blood partition coefficient.
3. Conclusions
In summary, a novel IDO1 inhibitor 1 bearing a 1-Indanone scaffold was discovered via structure-based virtual
screening, with the enzymatic IC50 value of 2.780 µM and cellular EC50 value of 9.171 µM respectively. Then,
surface plasmon resonance analysis validated the interaction between compound 1 and IDO1 protein with an
equilibrium dissociation constant (KD) value of 9.828 µM. Subsequently, preliminary structure–activity
relationships and scaffold hopping strategy were carried out. Molecular docking along with the experiment
validated the important position of hydroxyl group in ring C and the key role of ketone group in ring B. Further
studies of ADME properties were predicted in silico and all of the selected compounds followed the criteria for an
orally active drug. This study may provide more structural reference for the development of IDO1 inhibitors. Also,
the extensive research to find more potent inhibitors are currently undergoing and will be reported in due course.
4. Experiment
4.1. Virtual screening
The crystal structure of IDO1 in complex with Amg-1 was retrieved from Protein Data Bank (PDB code 4PK5) for
docking simulation. After removing the chain B and all the crystallographic waters, the missing hydrogen atoms
were added. Then the complex was submitted to a series of restrained minimization to relieve static clashes using
OPLS2005 force field. Finally, the protein were adjusted and minimized up to 0.30 Å RMSD. Then, the grid box
with 20×20×20 was used to construct the grid for docking screening. Before screening the chemical libraries, the
docking model was validated with compound Amg-1 docked into the designated target using the default glide
settings. Then, the 200,000 compounds from Specs database were prepared and filtered according with the criteria
(200 Da<MW<650 Da) using the LigPrep module. All output structures were initially docked and scored by the
Glide high-throughput virtual screening (HTVS) mode. The top 10% ranked compounds were chosen and
redocked by the Glide standard precision scoring mode (SP), and the top 5% ranked compounds were subjected to
10

another round of docking by the extra precision (XP) scoring mode. Finally, according to the scores, the binding
pose and chemical diversity, a total of 150 compounds were prioritized and submitted to biological assays.
4.2. Biological evaluation
4.2.1. Enzyme-based IDO1 activity assay
The human IDO1 protein, His-tag was purchased from BPS Bioscience Company. The standard assay medium
contained 50 mM sodium phosphate buffer (pH 6.5), 10 mM ascorbic acid (neutralized with 1M NaOH solution),
10 µM methylene blue, 100 µg/mL catalase, 200 µM L-tryptophan. The IDO1 protein, His-tag was diluted to a
concentration of 25 µg/mL and the test compounds were prepared to a concentration of 100 µM in sodium
phosphate buffer. 10 µL IDO1 enzyme was added to 80 µL standard assay medium in 96-well black plate.
Subsequently, 10 µL test compounds were added to 96-well black plate. This mixture was incubated at 37 oC for 1
hr. 20 µL of 1M NaOH was finally added and incubated at 60 oC for 4 hr. The mixture was cooled at 4oC for 10
min and then centrifuged at 800 g for 3 min at room temperature. The IDO1 activity is associated with the amount
of kynurenine production. The amount of kynurenine production was determined by the emission of fluorescence
(λ (ex) = 355 nm, λ (em) = 460 nm), which was detected by MD-SpectraMax M5. Graphs of inhibition curves with
IC50 values were generated using Prism v.5.0 (GraphPad Software, Inc.).30, 31
4.2.2. Cell-based IDO1 activity assay
HeLa cells were seeded in 96-well culture plates at a density of 1 x 104 per well. On the next day, human IFN-γ (20
ng/mL) and compounds in a total volume of 200 µL culture medium containing 15 µg/mL of L-tryptophan were
added to the cells. After incubation for 24 hours, 140 µL of the supernatant was mixed with 10 µL of 6.1N
trichloroacetic acid and the mixture was incubated for 30 min at 50°C. The reaction mixture
was then centrifuged for 10 minutes at 2500 rpm to remove sediments. 100 µL of the supernatant was mixed
with 100µL of 2% (w/v) p-dimethylaminobenzaldehyde in acetic acid and measured at 480 nm using
MD-SpectraMax M5. Graphs of inhibition curves with IC50 values were generated using Prism v.5.0 (GraphPad
Software, Inc.). 30, 31
4.3. Surface plasmon resonance analysis
The interaction between compound 1 and the IDO1 protein was determined using surface plasmon resonance
(SPR). Purified human IDO1 protein was immobilized on sensor chip CM5 (GE Healthcare) and carried out at
25°C on Biacore T200 instruments. HBS-EP buffer (0.01 M HEPES, 0.15 M NaCl, 3mM EDTA, 0.05%v/v
Surfactant P20, pH 7.4) was used as running buffer. The ratio of the association and dissociation rate constants was
11

determined as the affinity (KD).
4.4. Chemistry
4.4.1. General information
All the reagents were reagent grade and purchased from companies such as Adamas-beta®, TCI etc.
1
Characterization of compounds were established by a combination of LC–MS and NMR. H NMR spectra were
recorded with a 400 MHz Varian spectrometer and 13C NMR spectra were recorded with a 600 MHz Bruker
spectrometer. Chemical shifts (δ) are expressed in ppm, and coupling constants (J) are expressed in Hz. High
resolution mass spectrometry data were given by AB 5600+Q TOF. The purity of all the designed compounds was
analyzed using an Agilent 1200 HPLC system with a G1311B quaternary pump, a G1329B ALS and a G4212B
DAD detector. The HPLC method consisted of the following: Agilent C18 RP column (250 mm×4.6 mm, 5µm);
Column temperature 25° C; Inject volume 5.0 µL; HPLC solvent H2O (0.1% TFA)/CH3OH = 60/40 (v/v); flow
rate of 1.2 mL/min; detector wavelength of 254 nm.
4.4.2. General Procedure for the synthesis of compounds 2a-2c.
To a solution of 1a-1c (1.1 mmol) in ethanol (5 mL) were added thiosemicarbazide (1.0 mmol) and acetic acid (0.1
mmol). After stirring at reflux for about 2-4h, the reaction was cooled to room temperature and the formed solid
was filtered off, dried and recrystallized to afford 2a-2c.
4.4.2.1. (E)-2-(3-oxo-2,3-dihydro-1H-inden-1-ylidene)hydrazine-1-carbothioamide (2a).
Yellow solid, yield 84.4%. 1H NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H), 8.40 (s, 1H), 8.26 (d, J = 7.9 Hz, 2H),
7.77 (t, J = 7.5 Hz, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.59 (t, J = 7.4 Hz, 1H), 3.50 (s, 2H). ESI-MS: calcd for [M+H]+
m/z 220.1, found:220.1.
4.4.2.2. (Z)-2-(2-oxoindolin-3-ylidene) hydrazine-1-carbothioamide (2b).
Yellow solid, yield 87.6%. 1H NMR (400 MHz, DMSO-d6) δ 12.47 (s, 1H), 11.20 (s, 1H), 9.03 (s, 1H), 8.68 (s,
1H), 7.65 (d, J = 7.5 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.08 (t, J = 7.6 Hz, 1H), 6.92 (d, J = 7.8 Hz, 1H). ESI-MS:
calcd for [M+H]+ m/z 221.0, found:221.0
4.4.2.3. (E)-2-(2,3-dihydro-1H-inden-1-ylidene)hydrazine-1-carbothioamide (2c).
Yellow solid, yield 82.3%. 1H NMR (400 MHz, DMSO-d6) δ 10.19 (s, 1H), 8.14 (s, 1H), 7.89 (s, 1H), 7.84 (d, J =
7.6 Hz, 1H), 7.35 – 7.31 (m, 2H), 7.27 – 7.21 (m, 1H), 3.05 – 2.99 (m, 2H), 2.86 – 2.81 (m, 2H). ESI-MS: calcd
12

for [M+H]+ m/z 206.1, found:206.1.
4.4.3. General Procedure for the synthesis of compounds 3a-3l.
To a solution of 2a-2c (1.0 mmol) in ethanol (5 mL) was added substituted α-bromo ketone (1.0 mmol). After
stirring at reflux for about 4h, the reaction was cooled to room temperature and the formed solid was filtered off,
dried and recrystallized to afford 3a-3l.
4.4.3.1. (E)-3-(2-(4-(2-hydroxyphenyl)thiazol-2-yl)hydrazono)-2,3-dihydro-1H-inden-1-one (3a).
Brown solid, yield 89.5%. 1H NMR (400 MHz, DMSO-d6) δ 7.96 (d, J = 7.8 Hz, 1H), 7.87 – 7.75 (m, 3H), 7.62 (t,
J = 7.5 Hz, 1H), 7.48 (s, 1H), 7.21 – 7.15 (m, 1H), 6.92 (d, J = 8.1 Hz, 1H), 6.87 (t, J = 7.5 Hz, 1H), 3.54 (s, 2H).
13C NMR (150 MHz, DMSO-d6) δ 198.15, 167.82, 154.98, 146.09, 137.68, 135.36, 130.58, 128.96, 127.04, 122.84,
121.09, 118.98, 118.47, 116.62, 104.61, 38.74. ESI-HRMS [M + H]+ calcd for C18H13N3O2S: 336.0801, found:
336.0800. HPLC purity 97.1% (tR =4.554 min).
4.4.3.2. (E)-3-(2-(4-(4-hydroxyphenyl)thiazol-2-yl)hydrazono)-2,3-dihydro-1H-inden-1-one (3b). Brown solid,
yield 92.5%. 1H NMR (400 MHz, DMSO-d6) δ 7.94 (d, J = 7.8 Hz, 1H), 7.80 (t, J = 7.5 Hz, 1H), 7.75 (d, J = 7.6
Hz, 1H), 7.69 (d, J = 8.6 Hz, 2H), 7.60 (t, J = 7.4 Hz, 1H), 7.11 (s, 1H), 6.81 (d, J = 8.6 Hz, 2H), 3.51 (s, 2H). 13
C
NMR (150 MHz, DMSO-d6) δ 198.40, 168.45, 157.02, 146.36, 137.51, 135.32, 130.28, 126.83, 122.80, 120.97,
115.19, 101.06, 38.62. ESI-HRMS [M + H]+ calcd for C18H13N3O2S: 336.0801, found: 336.0800. HPLC purity
99.0% (tR =3.802 min).
4.4.3.3. (E)-3-(2-(4-(3-methoxyphenyl)thiazol-2-yl)hydrazono)-2,3-dihydro-1H-inden-1-one (3c).
1
Brown solid, yield 91.3%. H NMR (400 MHz, DMSO-d6) δ 7.94 (d, J = 7.7 Hz, 1H), 7.81 (t, J = 7.4 Hz, 1H),
7.75 (d, J = 7.5 Hz, 1H), 7.60 (t, J = 7.2 Hz, 1H), 7.50 – 7.41 (m, 3H), 7.33 (t, J = 7.8 Hz, 1H), 6.89 (d, J = 8.3 Hz,
1H), 3.80 (s, 3H), 3.52 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ 198.44, 168.54, 159.36, 146.29, 137.50, 135.35,
130.33, 129.56, 122.82, 120.97, 117.76, 113.37, 110.55, 104.56, 54.93, 38.63. ESI-HRMS [M + H]+ calcd for
C19H15N3O2S: 372.0777, found: 372.0772. HPLC purity 96.2% (tR =4.968 min).
4.4.3.4. (E)-3-(2-(4-phenylthiazol-2-yl)hydrazono)-2,3-dihydro-1H-inden-1-one (3d).
Brown solid, yield 95.0%. 1H NMR (400 MHz, DMSO-d6) δ 7.93 (d, J = 7.8 Hz, 1H), 7.88 (d, J = 7.5 Hz, 2H),
7.80 (t, J = 7.5 Hz, 1H), 7.75 (d, J = 7.7 Hz, 1H), 7.59 (t, J = 7.4 Hz, 1H), 7.46 – 7.39 (m, 3H), 7.31 (t, J = 7.2 Hz,
1H), 3.51 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ 198.44, 168.65, 146.30, 137.51, 135.35, 130.32, 128.51,
13

127.48, 125.38, 122.83, 120.97, 104.15, 38.63. ESI-HRMS [M + H]+ calcd for C18H13N3OS: 320.0852, found:
320.0852. HPLC purity 98.3% (tR =4.509 min).
4.4.3.5. (E)-3-(2-(4-(3-fluorophenyl)thiazol-2-yl)hydrazono)-2,3-dihydro-1H-inden-1-one (3e).
1
Brown solid, yield 92.0%. H NMR (400 MHz, DMSO-d6) δ 7.93 (d, J = 7.8 Hz, 1H), 7.81 (t, J = 7.5 Hz, 1H),
7.75 (t, J = 7.7 Hz, 2H), 7.67 (d, J = 10.6 Hz, 1H), 7.60 (t, J = 7.4 Hz, 1H), 7.54 (s, 1H), 7.51-7.43 (m, 1H),
7.18-7.11 (m, 1H), 3.52 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ 198.33, 168.73, 163.18, 161.57, 146.26, 145.24,
137.53, 135.32, 130.52, 130.47, 130.34, 122.81, 121.40, 120.96, 114.07, 111.90, 105.66, 38.62. ESI-HRMS [M +
H]+ calcd for C18H12FN3OS: 338.0758, found: 338.0757. HPLC purity 96.4% (tR =4.633 min).
4.4.3.6. (E)-3-(2-(4-(3-chlorophenyl)thiazol-2-yl)hydrazono)-2,3-dihydro-1H-inden-1-one (3f).
Brown solid, yield 90.3%. 1H NMR (400 MHz, DMSO-d6) δ 8.09 (s, 1H), 7.93 (d, J = 7.8 Hz, 1H), 7.89 (d, J = 8.0
Hz, 1H), 7.81 (t, J = 7.7 Hz, 1H), 7.75 (d, J = 7.7 Hz, 1H), 7.61 (d, J = 7.3 Hz, 1H), 7.56 (s, 1H), 7.51 (d, J = 8.0
Hz, 1H), 7.39 (t, J = 7.9 Hz, 1H), 3.51 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ 198.31, 168.73, 146.25, 145.25,
137.53, 136.66, 135.32, 130.68, 130.35, 130.00, 128.04, 124.22, 122.81, 121.95, 120.96, 105.74, 38.61.
ESI-HRMS [M + H]+ calcd for C18H12ClN3OS: 354.0462, found: 354.0463. HPLC purity 97.4% (tR =4.558 min).
4.4.3.7. (E)-3-(2-(4-(3-bromophenyl)thiazol-2-yl)hydrazono)-2,3-dihydro-1H-inden-1-one (3g).
Brown solid, yield 85.0 %. 1H NMR (400 MHz, DMSO-d6) δ 7.93 (d, J = 7.6 Hz, 2H), 7.86 – 7.78 (m, 2H), 7.76 (d,
J = 7.7 Hz, 1H), 7.60 (t, J = 7.4 Hz, 1H), 7.56 (s, 1H), 7.46 (t, J = 7.9 Hz, 1H), 7.37 (d, J = 7.9 Hz, 1H), 3.52 (s,
2H). 13C NMR (150 MHz, DMSO-d6) δ 198.32, 168.75, 146.25, 145.25, 137.54, 136.44, 135.33, 133.34, 130.40,
130.36, 127.12, 125.11, 123.87, 122.81, 120.96, 105.75, 38.61. ESI-HRMS [M + H]+ calcd for C18H12BrN3OS:
397.9957, found: 397.9954. HPLC purity 97.8% (tR =4.886 min).
4.4.3.8. (E)-3-(2-(2-(3-oxo-2,3-dihydro-1H-inden-1-ylidene)hydrazinyl)thiazol-4-yl)benzonitrile (3h).
Brown solid, yield 93.0%. 1H NMR (400 MHz, DMSO-d6) δ 8.30 (s, 1H), 8.20 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 7.8
Hz, 1H), 7.83-7.73 (m, 3H), 7.67 – 7.57 (m, 3H), 3.51 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ 198.29, 168.98,
146.22, 145.40, 137.56, 135.51, 135.33, 130.76, 130.39, 129.88, 129.83, 128.74, 122.81, 120.97, 118.59, 111.68,
106.47, 38.63. ESI-HRMS [M + H]+ calcd for C19H12N4OS: 345.0805, found: 345.0803. HPLC purity 97.3% (tR
=4.313 min).
4.4.3.9. (E)-4-(2-(2-(3-oxo-2,3-dihydro-1H-inden-1-ylidene)hydrazinyl)thiazol-4-yl)benzonitrile (3i).
14

1
Brown solid, yield 87.4%. H NMR (400 MHz, DMSO-d6) δ 8.02 (d, J = 8.3 Hz, 2H), 7.90 (d, J = 7.7 Hz, 1H),
7.85 (d, J = 8.3 Hz, 2H), 7.77 (t, J = 7.5 Hz, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.68 (s, 1H), 7.57 (t, J = 7.4 Hz, 1H),
3.48 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ 198.29, 168.99, 146.21, 145.46, 138.59, 137.57, 135.33, 132.58,
130.41, 125.97, 122.82, 120.98, 118.79, 109.49, 108.03, 38.64. ESI-HRMS [M + H]+ calcd for C19H12N4OS:
345.0805, found: 345.0802. HPLC purity 98.9% (tR =4.228 min).
4.4.3.10. (E)-3-(2-(4-(3,4-dimethoxyphenyl)thiazol-2-yl)hydrazono)-2,3-dihydro-1H-inden-1-one (3j).
1
Brown solid, yield 91.2%. H NMR (400 MHz, DMSO-d6) δ 7.89 (d, J = 7.8 Hz, 1H), 7.76 (t, J = 7.5 Hz, 1H),
7.71 (d, J = 7.7 Hz, 1H), 7.55 (t, J = 7.4 Hz, 1H), 7.43 – 7.35 (m, 2H), 7.23 (s, 1H), 6.95 (d, J = 8.2 Hz, 1H), 3.77
(s, 3H), 3.74 (s, 3H).3.47 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ 198.40, 168.48, 148.59, 148.39, 146.34,
137.51, 135.31, 130.28, 122.80, 120.95, 117.89, 111.71, 109.19, 102.27, 55.39, 55.35, 38.61.ESI-HRMS [M + H]+
calcd for C20H17N3O3S: 380.1063, found: 380.1061. HPLC purity 97.5% (tR =4.227 min).
4.4.3.11. (Z)-3-(2-(4-(3-hydroxyphenyl)thiazol-2-yl)hydrazono)indolin-2-one (3k).
Yellow solid, yield 75.0 %.1H NMR (400 MHz, DMSO-d6) δ 11.26 (s, 1H), 7.54 (m, 2H), 7.33 (m, 3H), 7.21 (t, J =
8.1 Hz, 1H), 7.09 (t, J = 7.6 Hz, 1H), 6.97 (d, J = 7.7 Hz, 1H), 6.73 (d, J = 8.3 Hz, 1H). 13C NMR (150 MHz,
DMSO-d6) δ 165.67, 163.04, 157.47, 150.87, 141.11, 134.99, 131.90, 130.35, 129.57, 122.28, 119.71, 119.54,
116.33, 114.86, 112.44, 110.92, 106.54. ESI-HRMS [M + H]+ calcd for C17H12N4O2S: 337.0754, found: 337.0757.
HPLC purity 99.3% (tR =4.929 min).
4.4.3.12. (E)-3-(2-(2-(2, 3-dihydro-1H-inden-1-ylidene)hydrazinyl)thiazol-4-yl)phenol (3l).
Brown solid, yield 86.0 %.1H NMR (400 MHz, DMSO-d6) δ 7.62 (d, J = 7.4 Hz, 1H), 7.41-7.25 (m, 5H), 7.22 –
7.15 (m, 2H), 6.70 (d, J = 8.0 Hz, 1H), 3.13 – 3.06 (m, 2H), 2.92 – 2.85 (m, 2H). 13C NMR (150 MHz, DMSO-d6)
δ 169.07, 157.38, 147.96, 137.49, 130.01, 129.44, 126.88, 125.60, 120.63, 116.40, 114.65, 112.43, 103.60, 28.00,
27.42. ESI-HRMS [M + H]+ calcd for C18H15N3OS: 322.1009, found: 322.1010. HPLC purity 99.2% (tR =4.079
min).
4.4.4. Synthesis of compound 6
4.4.4.1. 2-bromo-1-cyclohexylethan-1-one (5).
Cyclohexyl methyl ketone (300µL, 2.4mmol) was dissolved in methanol (3.0 mL) cooled in an ice bath and
bromine (380mg, 2.4mmol) was added dropwise. The mixture was stirred for 2h and the water (3.0 mL) was added
15

and the reaction mixture was allowed to stir for 4h. The reaction mixture was extracted with ethyl acetate. The
combined organic layer was washed with water saturated potassium carbonate, brine, dried over Na2SO4 and
concentrated to give 2-bromo-1-cyclohexylethan-1-one (5) as a clear oil which was used directly for the following
synthesis. ESI-MS: calcd for [M+H]+ m/z 205,0、207.0, found:205.0、207.0.
4.4.4.2. (E)-3-(2-(4-cyclohexylthiazol-2-yl)hydrazono)-2,3-dihydro-1H-inden-1-one (6).
To a solution of compound 5 (110 mg, 0.537 mmol) in ethanol (5 mL) was added 2a (353 mg, 1.61 mmol). The
mixture was stirred in sealed tube at 130°C overnight. Then the reaction mixture was extracted with ethyl acetate.
The combined organic layer was washed with water, brine, dried over Na2SO4 and concentrated under reduced
pressure. The residue was purified by silica gel column chromatography to give compound 6 as a brown solid
(113mg, 65.0%). 1H NMR (400 MHz, DMSO-d6) δ 11.40 (s, 1H), 7.86 (d, J = 7.2 Hz, 1H), 7.79 – 7.66 (m, 2H),
7.54 (t, J = 7.2 Hz, 1H), 6.37 (s, 1H), 3.41 (s, 2H), 1.91 (d, J = 9.9 Hz, 2H), 1.78-1.60 (m, 3H), 1.39 – 1.18 (m, 6H).
13C NMR (150 MHz, DMSO-d6) δ 198.73, 146.59, 137.45, 135.27, 130.03, 122.76, 120.88, 38.57, 31.77, 25.63.
ESI-HRMS [M + H]+ calcd for C18H19N3OS: 326.1322, found: 326.1320. HPLC purity 99.8% (tR =4.162 min).
4.4.5. Synthesis of compound 9
4.4.5.1. 3-hydroxybenzohydrazide (8).
An 80% solution of hydrazine hydrate (1.52 mL, 25 mmol) was added dropwise to a solution of methyl
3-hydroxybenzoate (760 mg, 5 mmol) in ethanol (25 mL). The reaction mixture was refluxed for 10 h until
completion, as determined by TLC. After evaporating approximately half of the solvent, water (7 mL) was added
and the precipitated product was filtered and washed with a small amount of ethanol and water. White solid, yield
80.0%. 1H NMR (400 MHz, DMSO-d6) δ 9.61 (s, 1H), 9.60 (s, 1H), 7.21 – 7.15 (m, 3H), 6.88-6.81 (m, 1H), 4.41
(s, 2H). ESI-MS: calcd for [M+H]+ m/z 153.1, found:153.2.
4.4.5.2. (E)-3-hydroxy-N'-(3-oxo-2,3-dihydro-1H-inden-1-ylidene)benzohydrazide (9).
1
Following the preparation protocol of compounds of 2a-2c. Off-white solid, yield 85.0%. H NMR (400 MHz,
DMSO-d6) δ 10.84 (s, 1H), 9.79 (s, 1H), 8.06-7.98 (m, 1H), 7.84 (t, J = 7.3 Hz, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.67
(t, J = 7.2 Hz, 1H), 7.36-7.29 (m, 2H), 7.27 (s, 1H), 7.02-6.95 (m, 1H), 3.68 (s, 2H). 13C NMR (150 MHz,
DMSO-d6) δ 198.36, 157.12, 146.30, 138.29, 135.40, 134.82, 131.23, 129.29, 122.76, 121.76, 118.54, 114.64,
38.55. ESI-HRMS [M + H]+ calcd for C16H12N2O3: 281.0921, found: 281.0918. HPLC purity 99.3% (tR =4.929
min).
16

4.4.6. Synthesis of compound 15
4.4.6.1. Methyl 3-((tert-butoxycarbonyl)amino)benzoate (11).
To a solution of methyl 3-aminobenzoate (10) (2 g, 13.19 mmol) in dry CH2Cl2 (100 mL) was added Et3N (3.66
mL, 26.38 mmol), and then the solution was cooled to 0oC, followed by addition of di-tert-butyl dicarbonate (3.45
g, 15.83 mmol). The mixture was stirred at room temperature for 10 h, then the reaction was quenched with water
and CH2Cl2. The combined organic layers were washed with 1 M HCl, water and brine, and dried over anhydrous
Na2SO4. The solvent was removed in vacuo and the residue was purified by flash chromatography on silica gel to
give compound 11 as a white solid (1.70 g, yield 51.3%). 1H NMR (400 MHz, DMSO-d6) δ 9.57 (s, 1H), 8.16 (s,
1H), 7.60 (d, J = 8.1 Hz, 1H), 7.52 (d, J = 7.7 Hz, 1H), 7.36 (t, J = 7.9 Hz, 1H), 3.80 (s, 3H), 1.45 (s, 9H). ESI-MS:
calcd for [M+H]+ m/z 252.1, found:252.2.
4.4.6.2. Methyl 3-((tert-butoxycarbonyl)(methyl)amino)benzoate (12).
Into a suspension of NaH (115 mg, 4.78 mmol) in dry DMF (80 mL) was added a solution of 11 (1.0 g, 3.98 mmol)
in dry DMF (20 mL) at 0oC. The mixture was stirred at 0oC for 30 min and then iodomethane (496 µL, 7.96 mmol)
was added. The mixture was stirred at 0oC overnight, then the reaction was quenched with water, followed by
extraction with ethyl acetate. The combined organic layers were washed with 1 M HCl, water and brine, and dried
over anhydrous Na2SO4. The solvent was removed in vacuo and the residue was used directly for the following
synthesis. ESI-MS: calcd for [M+H]+ m/z 266.1, found:266.2.
4.4.6.3. Methyl 3-(methylamino)benzoate (13).
Into a solution of 12 (530 mg, 2 mmol) in dry CH2Cl2 (10 mL) was added trifluoroacetic acid (1 mL) at 0oC. The
mixture was stirred at room temperature for 12 h, then the reaction was quenched with sat. NaHCO3 and the whole
was extracted with CH2Cl2. The combined organic layers were washed with water and dried over anhydrous
Na2SO4. The solvent was removed in vacuo and the residue was purified by flash chromatography on silica gel to
give compound 13 as a colorless oil. (280 mg, yield 85.0 %). 1H NMR (400 MHz, DMSO-d6) δ 7.18 (t, J = 7.7 Hz,
1H), 7.10 (d, J = 7.7 Hz, 2H), 6.77 – 6.72 (m, 1H), 5.98-5.90 (m, 1H), 3.77 (s, 3H), 2.66 (d, J = 5.1 Hz, 3H).
ESI-MS: calcd for [M+H]+ m/z 166.1, found:166.2.
4.4.6.4. 3-(methylamino)benzohydrazide (14).
Following the preparation protocol of compounds of 8. After purified by flash chromatography on silica gel to give
compound 14. White solid, yield 65.0%. 1H NMR (400 MHz, DMSO-d6) δ 9.54 (s, 1H), 7.07 (t, J = 8.0 Hz, 1H),
17

6.95-6.89 (m, 2H), 6.60 (d, J = 8.1 Hz, 1H), 5.80-5.71 (m, 1H), 4.32 (s, 2H), 2.64 (d, J = 4.5 Hz, 3H). ESI-MS:
calcd for [M+H]+ m/z 166.1, found:166.2.
4.4.6.5. (E)-3-(methylamino)-N'-(3-oxo-2,3-dihydro-1H-inden-1-ylidene)benzohydrazide (15).
Following the preparation protocol of compounds of 2a-2c. After purified by flash chromatography on silica gel to
give compound 15. Brown solid, yield 45.0 %. 1H NMR (400 MHz, DMSO-d6) δ 10.76 (s, 1H), 8.07-7.97 (m, 1H),
7.84 (t, J = 7.3 Hz, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.66 (t, J = 7.4 Hz, 1H), 7.22 (t, J = 7.8 Hz, 1H), 7.04 (d, J = 7.5
Hz, 1H), 7.00 (s, 1H), 6.74 (d, J = 8.0 Hz, 1H), 5.93 (s, 1H), 3.67 (s, 2H), 2.72 (d, J = 4.9 Hz, 3H). 13C NMR (150
MHz, DMSO-d6) δ 198.38, 149.69, 146.38, 138.27, 135.38, 134.28, 131.14, 128.65, 122.75, 121.71, 114.67,
110.70, 38.50, 29.53. ESI-HRMS [M + H]+ calcd for C17H15N3O2: 294.1237, found: 294.1235. HPLC purity 97.3%
(tR =1.029 min).
4.4.7. Synthesis of compounds 24 and 27
4.4.7.1. Methyl 3-oxo-2,3-dihydro-1H-indene-1-carboxylate (17).
A solution of 3-Carboxy-1-indanone (3 g, 17.03 mmol) and concentrated sulfuric acid (100 µL) in methanol (20
mL) was heated to reflux for 4 h. After cooling to room temperature, the solvent was removed on a rotary
evaporator. The residue was dissolved in ethyl acetate, and washed with saturated NaHCO3 and water. Then the
organic phase dried with sodium sulfate and concentrated in vacuo. The crude compound was purified by Silica gel
column chromatography to give 17 as a white solid（2.98 g，92.0%）. 1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 7.6
Hz, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.65 – 7.59 (m, 1H), 7.44 (t, J = 7.4 Hz, 1H), 4.29 (dd, J = 8.1, 3.5 Hz, 1H), 3.77
(s, 3H), 3.14 (dd, J = 19.1, 3.5 Hz, 1H), 2.87 (dd, J = 19.1, 8.1 Hz, 1H). ESI-MS: calcd for [M+H]+ m/z 191.1,
found:191.1.
4.4.7.2. Methyl 2,3-dihydrospiro[indene-1,2'-[1,3]dioxolane]-3-carboxylate (18).
To a solution of 17 (2 g, 10.5mmol) in anhydrous benzene (20 mL) was added ethylene glycol(3.26 mL, 52.5
mmol)、trimethyl orthoformate (5.12g, 48.3 mmol) and p-TsOH(180 mg, 1.05 mmol). After stirring at 50℃ for 2h,
the reaction was cooled to room temperature and concentrated in vacuo. The crude compound was purified by
Silica gel column chromatography to give 18 as a colorless oi（l 2.33 g，94.8%）. 1H NMR (400 MHz, CDCl3) δ 7.45
– 7.42 (m, 1H), 7.40-7.36 (m, 2H), 7.36 – 7.32 (m, 1H), 4.26 – 4.04 (m, 5H), 3.76 (s, 3H), 2.70 (dd, J = 13.8, 6.6
Hz, 1H), 2.56 (dd, J = 13.8, 8.2 Hz, 1H). ESI-MS: calcd for [M+H]+ m/z 235.1, found:235.1.
4.4.7.3. 3-(hydroxymethyl)-2,3-dihydro-1H-inden-1-one (20).
18

A solution of 18 (1.5g, 6.41mmol ) in anhydrous THF was added dropwise to a vigorously stirred suspension of
LiAlH4 (0.9 g, 24 mmol) in anhydrous THF (15 mL) under argon.The reaction mixture was heated under reflux for
6 h, cooled to 0 °C in an ice bath, and quenched by the sequential dropwise addition of H2O (1.5 mL), NaOH (1.5
mL), and H2O (1.5 mL). The coarse precipitate formed was filtered off and thoroughly washed with ethyl acetate.
The organic extracts were washed with aqueous HCl (1M) and water several times and dried with sodium sulfate
and concentrated in vacuo. The crude compound was purified by Silica gel column chromatography to give 20 as a
colorless oil（1.04 g，100 %）. 1H NMR (400 MHz, CDCl3) δ 7.73 (d, J = 7.6 Hz, 1H), 7.63 – 7.56 (m, 2H),
7.43-7.35 (m, 1H), 3.91-3.80 (m, 2H), 3.60-3.52 (m, 1H), 2.81 (dd, J = 19.2, 7.6 Hz, 1H), 2.56 (dd, J = 19.2, 2.6
Hz, 1H). ESI-MS: calcd for [M+H]+ m/z 163.1, found:163.1.
4.4.7.4. 3-(azidomethyl)-2,3-dihydro-1H-inden-1-one (21).
To a solution of 20 (950 mg, 5.86 mmol) in anhydrous DMF were added DPPA(2.53 mL, 11.72 mmol) and
DBU(1.75 mL, 11.72 mmol). The mixture was stirred at 90 °C for about 6h. After cooling to room temperature,
saturated NaHCO3 was added and extracted with ethyl acetate. The combined organics were washed with water
and saturated NaCl. Then the organic phase was dried with sodium sulfate and concentrated in vacuo. The crude
compound was purified by Silica gel column chromatography to give 21 as a brown oi（l 877 mg，80.0%）. 1H NMR
(400 MHz, CDCl3) δ 7.78 (d, J = 7.7 Hz, 1H), 7.65 (t, J = 7.4 Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.45 (t, J = 7.3 Hz,
1H), 3.72 – 3.58 (m, 3H), 2.90 (dd, J = 19.1, 7.3 Hz, 1H), 2.52 (dd, J = 19.1, 2.3 Hz, 1H). ESI-MS: calcd for
[M+H]+ m/z 188.1, found:188.1.
4.4.7.5. 3-(aminomethyl)-2,3-dihydro-1H-inden-1-one (22).
A solution of the 21 (100 mg, 0.534 mmol) in methanol (3 mL) was hydrogenated in the presence of 10% Pd/C (10
mg) and 1 M/L aqueous HCl (3 mL) at room temperature for 4 h. The mixture was then filtered through a celite
pad, and the filtrate was added 1M aqueous NaOH to PH=8-9. The mixture was extracted with ethyl acetate twice
and the organic phase was dried with sodium sulfate. After concentrated in vacuo, the crude product was used
directly for the next step without further purification. ESI-MS: calcd for [M+H]+ m/z 162.1, found:162.1.
4.4.7.6. 1-(3-methoxyphenyl)-3-((3-oxo-2,3-dihydro-1H-inden-1-yl)methyl)urea (23).
To a solution of 22 (16.1 mg, 0.1 mmol) in anhydrous THF (5 mL) were added 1-isocyanato-3-methoxybenzene
(14.9 mg, 0.1 mmol) and DIPEA (22.4 µL,0.13 mmol). The mixture was stirred at room temperature for 2h. Then
saturated NaHCO3 was added and the mixture was extracted with ethyl acetate twice. The combined organic
19

fractions were dried with sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel
column chromatography to give 23 as a white solid (25 mg, 80.0%). 1H NMR (400 MHz, DMSO-d6) δ 8.52 (s, 1H),
7.71 (d, J = 3.7 Hz, 2H), 7.64 (d, J = 7.7 Hz, 1H), 7.50-7.42 (m, 1H), 7.14 – 7.03 (m, 2H), 6.82 (d, J = 8.1 Hz, 1H),
6.45 (dd, J = 8.2, 1.5 Hz, 1H), 6.24 (s, 1H), 3.68 (s, 3H), 3.65-3.55 (m, 2H), 3.31 – 3.24 (m, 1H), 2.78 (dd, J = 19.1,
7.3 Hz, 1H), 2.44 (dd, J = 19.3, 2.4 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ 205.18, 159.45, 155.93, 155.20,
141.43, 136.82, 134.70, 129.23, 127.78, 126.12, 122.74, 109.76, 106.38, 103.12, 54.65, 42.99, 40.29, 38.35.
ESI-MS: calcd for [M+H]+ m/z 311.1, found:311.1.
4.4.7.7. 1-(3-hydroxyphenyl)-3-((3-oxo-2,3-dihydro-1H-inden-1-yl)methyl)urea（24）.
To a solution of 23 (20 mg, 0.064 mmol) in anhydrous dichloromethane(1 mL) was added Boron Tribromide (17%
in CH2Cl2,50 µL) at 0℃.Water (1 mL) was added after the reaction mixture were stirred for 3h. Then the mixture
was stirred for another 30min before extract with dichloromethane. The organic layer was combined and dried with
sodium sulfate. The solution was evaporated to give the crude product, which was purified by silica gel column
chromatography to give 24 as an off-white solid (9.6 mg, 50.0%).1H NMR (400 MHz, DMSO-d6) δ 9.18 (s, 1H),
8.32 (s, 1H), 7.71-7.65 (m, 2H), 7.61 (d, J = 7.6 Hz, 1H), 7.47-7.40 (m, 1H), 6.95 – 6.88 (m, 2H), 6.63 (d, J = 7.9
Hz, 1H), 6.23 (d, J = 7.9 Hz, 1H), 6.10 (d, J = 6.1 Hz, 1H), 3.60-3.50 (m, 2H), 3.30-3.21 (m, 1H), 2.75 (dd, J =
19.2, 7.5 Hz, 1H), 2.41 (dd, J = 19.3, 2.3 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ 205.09, 157.50, 155.94,
155.17, 141.24, 136.85, 134.66, 129.09, 127.75, 126.09, 122.71, 108.26, 108.13, 104.60, 42.98, 40.31, 38.40.
ESI-HRMS [M + Na]+ calcd for C17H16N2O3Na: 319.1053, found: 319.1050. HPLC purity 99.3% (tR =1.951 min).
4.4.7.8. N-(((3-oxo-2,3-dihydro-1H-inden-1-yl)methyl)carbamothioyl)benzamide (25).
To a solution of 22 (161 mg, 1.0 mmol) in anhydrous THF (10 mL) were added benzoyl isothiocyanate (134 µL,
1.0 mmol) and trimethylamine (279 µL,2.0 mmol). The mixture was stirred at room temperature for 2h. Then
saturated NaHCO3 was added and the mixture was extracted with ethyl acetate twice. The combined organic
fractions were dried with sodium sulfate, filtered and concentrated in vacuo to give 25. The crude product was
used directly for the next step without further purification. ESI-MS: calcd for [M+H]+ m/z 325.1, found:325.1.
4.4.7.9. 1-((3-oxo-2,3-dihydro-1H-inden-1-yl)methyl)thiourea (26).
To a solution of 25 (162 mg, 0.5mmol) in methanol (5 mL) was added 1M aqueous NaOH (2 mL). The mixture
was stirred at room temperature for 2h and extracted with ethyl acetate twice. The combined organic fractions were
dried with sodium sulfate, filtered and concentrated in vacuo to give 26. The crude product was used directly for
20

the next step without further purification. ESI-MS: calcd for [M+H]+ m/z 221.1, found:221.1.
4.4.7.10. 3-(((4-(3-hydroxyphenyl)thiazol-2-yl)amino)methyl)-2,3-dihydro-1H-inden-1-one (27).
To a solution of 26 (110 mg, 0.5 mmol) in ethanol (3 mL) was added 2-bromo-1-(3-hydroxyphenyl)ethan-1-one
(107 mg, 0.5 mmol). After stirring at reflux for 4 h, the reaction was determined to be complete by TLC. Then the
solvent was evaporated under reduce pressure. The residue was dissolved in ethyl acetate and washed with distilled
water. The organic phase was separated from the aqueous phase and washed with brine, dried over Na2SO4, and
evaporated under reduced pressure. The crude compound was purified by Silica gel column chromatography to
give 27 as an off-white solid (143 mg, 85.0%).1H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 7.6 Hz, 1H), 7.64 – 7.58
(m, 2H), 7.46 – 7.40 (m, 1H), 7.36-7.33 (m, 1H), 7.28 (d, J = 7.7 Hz, 1H), 7.22 (t, J = 7.8 Hz, 1H), 6.75 (dd, J =
7.9, 1.5 Hz, 1H), 6.67 (s, 1H), 3.84-3.77 (m, 1H), 3.68 (dd, J = 13.2, 5.6 Hz, 1H), 3.55 (dd, J = 13.2, 7.1 Hz, 1H),
2.90 (dd, J = 19.1, 7.6 Hz, 1H), 2.56 (dd, J = 19.1, 3.0 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 204.43, 168.62,
155.35, 154.27, 150.51, 136.72, 135.53, 134.41, 129.25, 127.82, 125.29, 123.46, 117.74, 114.39, 112.65, 100.96,
49.87, 40.51, 37.43. ESI-HRMS [M + H]+ calcd for C19H17N2O2S: 337.1005, found: 337.0999. HPLC purity 97.5%
(tR =2.623 min).
Acknowledgments
This work was financially supported by the National Natural Science Foundation of China (Grant No.
81473075).
A. Supplementary material
Supplementary material is available on the publishers Web site along with the published article.
References and notes
1. Hayaishi O. My life with tryptophan--never a dull moment. Protein Sci. 1993; 2: 472-475.
2. Takikawa O, Yoshida R, Kido R, Hayaishi O. Tryptophan degradation in mice initiated by indoleamine 2,
3-dioxygenase. J Biol Chem. 1986; 261: 3648-3653.
3. Holmgaard RB, Zamarin D, Munn DH, Wolchok JD, Allison JP. Indoleamine 2,3-dioxygenase is a critical
resistance mechanism in antitumor T cell immunotherapy targeting CTLA-4. J Exp Med. 2013; 210: 1389-1402.
4. Heyes MP, Saito K, Crowley JS, Davis LE, Demitrack MA, Der M, Dilling LA, Elia J, Kruesi MJ, Lackner A, et
al. Quinolinic acid and kynurenine pathway metabolism in inflammatory and non-inflammatory neurological
21

disease. Brain. 1992; 115: 1249-1273.
5. Guillemin GJ, Brew BJ, Noonan CE, Takikawa O, Cullen KM. Indoleamine 2,3 dioxygenase and quinolinic acid
immunoreactivity in Alzheimer's disease hippocampus. Neuropathol Appl Neurobiol. 2005; 31: 395-404.
6. Wichers MC, Maes M. The role of indoleamine 2,3-dioxygenase (IDO) in the pathophysiology of
interferon-alpha-induced depression. J Psychiatry Neurosci. 2004; 29: 11-17.
7. Sardar AM, Reynolds GP. Frontal cortex indoleamine-2,3-dioxygenase activity is increased in HIV-1-associated
dementia. Neurosci Lett.1995; 187: 9-12.
8. Munn DH, Mellor AL. Indoleamine 2,3 dioxygenase and metabolic control of immune responses. Trends
Immunol. 2013; 34: 137-143.
9. Godin-Ethier J, Hanafi LA, Piccirillo CA, Lapointe R. Indoleamine 2,3-dioxygenase expression in human
cancers: clinical and immunologic perspectives. Clin Cancer Res. 2011; 17: 6985-6991.
10. van Baren N, Van den Eynde BJ. Tryptophan-degrading enzymes in tumoral immune resistance. Front
Immunol. 2015; 6: 34.
11. Uyttenhove C, Pilotte L, Théate I, Stroobant V, Colau D, Parmentier N, Boon T, Van den Eynde BJ. Evidence
for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase.
Nat Med. 2003; 9: 1269-1274.
12. Gajewski TF1, Meng Y, Blank C, Brown I, Kacha A, Kline J, Harlin H. Immune resistance orchestrated by the
tumor microenvironment. Immunol Rev. 2006; 213: 131-145.
13. Bauer TM, Jiga LP, Chuang JJ, Randazzo M, Opelz G, Terness P. Studying the immunosuppressive role of
indoleamine 2,3-dioxygenase: tryptophan metabolites suppress rat allogeneic T-cell responses in vitro and in vivo.
Transpl Int. 2005; 18: 95-100.
14. Sugimoto H, Oda S, Otsuki T, Hino T, Yoshida T, Shiro Y. Crystal structure of human indoleamine
2,3-dioxygenase: catalytic mechanism of O2 incorporation by a heme-containing dioxygenase. Proc Natl Acad Sci
U S A. 2006; 103: 2611-2616.
15. Röhrig UF, Majjigapu SR, Grosdidier A, Bron S, Stroobant V, Pilotte L, Colau D, Vogel P, Van den Eynde BJ,
Zoete V, Michielin O. Rational design of 4-aryl-1,2,3-triazoles for indoleamine 2,3-dioxygenase 1 inhibition. J
Med Chem. 2012; 55: 5270-5290.
16. (a) Meininger D, Zalameda L, Liu Y, Stepan LP, Borges L, McCarter JD, Sutherland CL. Purification and
kinetic characterization of human indoleamine 2,3-dioxygenases 1 and 2 (IDO1 and IDO2) and discovery of
selective IDO1 inhibitors. Biochim Biophys Acta. 2011; 1814: 1947-1954.
22

(b)Tojo S, Kohno T, Tanaka T, Kamioka S, Ota Y, Ishii T, Kamimoto K, Asano S, Isobe Y. Crystal Structures
and Structure-Activity Relationships of Imidazothiazole Derivatives as IDO1 Inhibitors. ACS Med Chem Lett.
2014; 5: 1119-1123.
17 (a) Tomek P, Palmer BD, Flanagan JU, Sun C, Raven EL, Ching LM. Discovery and evaluation of inhibitors to
the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1): Probing the active site-inhibitor
interactions. Eur J Med Chem. 2017; 126: 983-996.
(b) Wu Y, Xu T, Liu J, Ding K, Xu J. Structural insights into the binding mechanism of IDO1 with
hydroxylamidine based inhibitor INCB14943. Biochem Biophys Res Commun. 2017; 487: 339-343.
(c) Markwalder JA, Seitz SP, Blat Y, Elkin L, Hunt JT, Pabalan JG, Jure-Kunkel MN, Vite GD, Covello K.
Identification and optimization of a novel series of indoleamine 2,3-dioxygenase inhibitors. Bioorg Med
Chem Lett. 2017; 27: 582-585.
(d) Lin SY, Yeh TK, Kuo CC, Song JS, Cheng MF, Liao FY, Chao MW, Huang HL, Chen YL, Yang CY, Wu
MH, Hsieh CL, Hsiao W, Peng YH, Wu JS, Lin LM, Sun M, Chao YS, Shih C, Wu SY, Pan SL, Hung MS,
Ueng SH. Phenyl Benzenesulfonylhydrazides Exhibit Selective Indoleamine 2,3-Dioxygenase Inhibition
with Potent in Vivo Pharmacodynamic Activity and Antitumor Efficacy. J Med Chem. 2016; 59: 419-430.
18. Sheridan C. IDO inhibitors move center stage in immuno-oncology. Nat Biotechnol. 2015; 33: 321-322.
19. http://clinicaltrials.gov/show/NCT01191216 (accessed February 27, 2017).
20. http:// clinicaltrials.gov/show/NCT00567931 (accessed February 27, 2017).
21. Hou DY, Muller AJ, Sharma MD, DuHadaway J, Banerjee T, Johnson M, Mellor AL, Prendergast GC, Munn
DH. Inhibition of indoleamine 2,3-dioxygenase in dendritic cells by stereoisomers of 1-methyl-tryptophan
correlates with antitumor responses. Cancer Res. 2007; 67: 792-801.
22. Cady SG, Sono M. 1-Methyl-DL-tryptophan, beta-(3-benzofuranyl)-DL-alanine (the oxygen analog of
tryptophan), and beta-[3-benzo(b)thienyl]-DL-alanine (the sulfur analog of tryptophan) are competitive inhibitors
for indoleamine 2,3-dioxygenase. Arch Biochem Biophys. 1991; 291: 326-333.
23. Mautino MR, Jaipuri FA, Waldo J, Kumar S, Adams J, van Allen C, Marcinowicz-Flick A, Munn D, Vahanian
NN, Link CJ. Washington, DC, Apr 6−10, 2013; Cancer Research: Philadelphia, PA, 2013.
24. Combs A, Zhu W, Sparks RB. Patent WO2008/058178, 2008.
25. Combs A, Yue E, Sparks R, Zhu W, Zhou J, Lin Q, Weng L, Yue T, Liu P. Patent WO2010/005958, 2010.
26. Liu X, Shin N, Koblish HK, Yang G, Wang Q, Wang K, Leffet L, Hansbury MJ, Thomas B, Rupar M, Waeltz P,
Bowman KJ, Polam P, Sparks RB, Yue EW, Li Y, Wynn R, Fridman JS, Burn TC, Combs AP, Newton RC,
23

Scherle PA. Selective inhibition of IDO1 effectively regulates mediators of antitumor immunity. Blood. 2010;
115: 3520-3530.
27. Yue EW, Douty B, Wayland B, Bower M, Liu X, Leffet L, Wang Q, Bowman KJ, Hansbury MJ, Liu C, Wei M,
Li Y, Wynn R, Burn TC, Koblish HK, Fridman JS, Metcalf B, Scherle PA, Combs AP. Discovery of potent
competitive inhibitors of indoleamine 2,3-dioxygenase with in vivo pharmacodynamic activity and efficacy in
a mouse melanoma model. J Med Chem. 2009; 52: 7364-7367.
28. 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. Hydroxyamidine inhibitors of
indoleamine-2,3-dioxygenase potently suppress systemic tryptophan catabolism and the growth of
IDO-expressing tumors. Mol Cancer Ther. 2010; 9: 489-498.
29. https://clinicaltrials.gov/ct2/show/NCT02752074?term=Epacadostat&rank=3 (accessed February 27, 2017).
30. Cheng MF, Hung MS, Song JS, Lin SY, Liao FY, Wu MH, Hsiao W, Hsieh CL, Wu JS, Chao YS, Shih C, Wu
SY, Ueng SH. Discovery and structure-activity relationships of phenyl benzenesulfonylhydrazides as novel
indoleamine 2,3-dioxygenase inhibitors. Bioorg Med Chem Lett. 2014; 24: 3403-3406.
31. Peng YH, Ueng SH, Tseng CT, Hung MS, Song JS, Wu JS, Liao FY, Fan YS, Wu MH, Hsiao WC, Hsueh CC,
Lin SY, Cheng CY, Tu CH, Lee LC, Cheng MF, Shia KS, Shih C, Wu SY. Important Hydrogen Bond
Networks in Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitor Design Revealed by Crystal Structures of
Imidazoleisoindole Derivatives with IDO1. J Med Chem. 2016; 59: 282-293.
32. Caputto ME, Ciccarelli A, Frank F, Moglioni AG, Moltrasio GY, Vega D, Lombardo E, Finkielsztein LM.
Synthesis and biological evaluation of some novel 1-indanone thiazolylhydrazone derivatives as
anti-Trypanosoma cruzi agents. Eur J Med Chem. 2012; 55:155-163.
33. Makam P, Kankanala R, Prakash A, Kannan T. 2-(2-Hydrazinyl)thiazole derivatives: design, synthesis and in
vitro antimycobacterial studies. Eur J Med Chem. 2013; 69: 564-576.
34. Lipinski CA. Lead- and drug-like compounds: the rule-of-five revolution. Drug Discov Today Technol. 2004; 1:
337-341.
35. Voet AR, Kumar A, Berenger F, Zhang KY. Combining in silico and in cerebro approaches for virtual screening
and pose prediction in SAMPL4. J Comput Aided Mol Des. 2014; 28: 363-373.
36. Kim J, Ok T, Park C, So W, Jo M, Kim Y, Seo M, Lee D, Jo S, Ko Y, Choi I, Park Y, Yoon J, Ju MK, Ahn J,
Kim J, Han SJ, Kim TH, Cechetto J, Nam J, Liuzzi M, Sommer P, No Z. A novel 3,4-dihydropyrimidin-2(1H)-one:
HIV-1 replication inhibitors with improved metabolic stability. Bioorg Med Chem Lett. 2012; 22: 2522-2526.
24

37. Karabanovich G, Zemanová J, Smutný T, Székely R, Šarkan M, Centárová I, Vocat A, Pávková I, Čonka P,
Němeček J, Stolaříková J, Vejsová M, Vávrová K, Klimešová V, Hrabálek A, Pávek P, Cole ST, Mikušová K, Roh
J. Development of 3,5-Dinitrobenzylsulfanyl-1,3,4-oxadiazoles and Thiadiazoles as Selective Antitubercular
Agents Active Against Replicating and Nonreplicating Mycobacterium tuberculosis. J Med Chem. 2016; 59:
2362-2380.
38. Ohta Y, Fujii S, Yokoyama A, Furuyama T, Uchiyama M, Yokozawa T. Synthesis of well-defined
hyperbranched polyamides by condensation polymerization of AB2 monomer through changed substituent effects.
Angew Chem Int Ed Engl. 2009; 48: 5942-5945
39. Raviña E, Negreira J, Cid J, Masaguer CF, Rosa E, Rivas ME, Fontenla JA, Loza MI, Tristán H, Cadavid MI,
Sanz F, Lozoya E, Carotti A, Carrieri A. Conformationally constrained butyrophenones with mixed dopaminergic
(D(2)) and serotoninergic (5-HT(2A), 5-HT(2C)) affinities: synthesis, pharmacology, 3D-QSAR, and molecular
modeling of (aminoalkyl)benzo- and -thienocycloalkanones as putative atypical antipsychotics. J Med Chem. 1999;
42: 2774-2797.
40. Spencer TA, Li D, Russel JS, Collins JL, Bledsoe RK, Consler TG, Moore LB, Galardi CM, McKee DD,
Moore JT, Watson MA, Parks DJ, Lambert MH, Willson TM. Pharmacophore analysis of the nuclear oxysterol
receptor LXRalpha. J Med Chem. 2001; 44: 886-897.
41. Tamura Y, Suzuki S, Matsumoto S, Hori A, Kooriyama Y. Patent US2012/0015961 A1, 2012.
25

Graphical Abstract
Identification and preliminary structure–activity relationships of
1-Indanone derivatives as novel indoleamine-2, 3-dioxygenase 1
(IDO1) inhibitors
Dingding Gao, Yingxia Li*
School of Pharmacy, Fudan University, Shanghai, 201203, China
Novel IDO1 inhibitor 1 bearing a 1-Indanone scaffold was discovered via structure-based virtual screening, with
enzymatic and cellular IC50 values of 2.780 µM and 9.171 µM respectively. The preliminary SAR was discussed
and the binding mode with IDO1 protein was predicted by experiment along with molecular docking.
*Corresponding authors. Y. Li, Tel.: +86-21-51980127; E-mail: liyx417@fudan.edu.cn.
26