Discovery of imidazoleisoindole derivatives as potent IDO1 inhibitors: Design, synthesis, biological evaluation and computational studies

Article abstract of DOI:10.1016/j.ejmech.2017.09.025

Indoleamine-2,3-dioxygenase-1 (IDO1) is an attractive target for cancer immunotherapy. Herein, a series of novel imidazoleisoindole derivatives were prepared and evaluated for their ability to inhibit IDO1. Among these, derivative 11r was the most active compound with nanomolar potency in the Hela cell-based assay, while showed negligible cellular toxicity. UV-visible spectra study demonstrated that compounds 11p and 11r bound to IDO1 and coordinated with the heme iron. Furthermore, they could significantly promote T cell proliferation, increase IFN-γ production, and reduce the numbers of Foxp3⁺ regulatory T cells. Finally, induced fit docking (IFD) and quantum mechanics/molecular mechanics (QM/MM) calculation were performed to understand the interactions of these compounds to IDO1 protein, which provided a comprehensive guide for further structural modification and optimization.

Full text of DOI:10.1016/j.ejmech.2017.09.025

Accepted Manuscript
Discovery of imidazoleisoindole derivatives as potent IDO1 inhibitors: Design,
synthesis, biological evaluation and computational studies
Yi Zou, Fang Wang, Yan Wang, Qirui Sun, Yue Hu, Yuezhen Li, Wen Liu, Wenjie
Guo, Zhangjian Huang, Yihua Zhang, Qiang Xu, Yisheng Lai
PII:
S0223-5234(17)30739-0
10.1016/j.ejmech.2017.09.025
EJMECH 9740
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 March 2017
Revised Date: 20 August 2017
Accepted Date: 14 September 2017
Please cite this article as: Y. Zou, F. Wang, Y. Wang, Q. Sun, Y. Hu, Y. Li, W. Liu, W. Guo, Z. Huang,
Y. Zhang, Q. Xu, Y. Lai, Discovery of imidazoleisoindole derivatives as potent IDO1 inhibitors: Design,
synthesis, biological evaluation and computational studies, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.09.025.
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ACCEPTED MANUSCRIPT
Graphical abstract
Discovery of imidazoleisoindole derivatives as potent IDO1 inhibitors:
Design, synthesis, biological evaluation and computational studies
Yi Zou a,1, Fang Wang a,1, Yan Wang b,1, Qirui Sun a, Yue Hub, Yuezhen Li c, Wen Liu b, Wenjie
Guo b,*, Zhangjian Huang a, Yihua Zhang a, Qiang Xu b,*, Yisheng Lai a,**
aState Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for Metabolic
Diseases, Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, China
bState Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University,
Nanjing 210093, China
c Department of Organic Chemistry, School of Science, China Pharmaceutical University, Nanjing
211198, China

ACCEPTED MANUSCRIPT
Discovery of imidazoleisoindole derivatives as potent IDO1
inhibitors: Design, synthesis, biological evaluation and
computational studies
Yi Zou a,1, Fang Wang a,1, Yan Wang b,1, Qirui Sun a, Yue Hub, Yuezhen Li c, Wen Liu b,
Wenjie Guo b,*, Zhangjian Huang a, Yihua Zhang a, Qiang Xu b,*, Yisheng Lai a,**
a State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for Metabolic
Diseases, Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR China
b State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University,
Nanjing 210093, PR China
c Department of Organic Chemistry, School of Science, China Pharmaceutical University, Nanjing
211198, PR China
Author Contributions
1 Y.Z., F.W. and Y.W. contributed equally to this work.
* Corresponding author. State Key Laboratory of Pharmaceutical Biotechnology, School of Life
Sciences, Nanjing University, Nanjing 210093, PR China.
** Corresponding author. State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of
Drug Discovery for Metabolic Diseases, Center of Drug Discovery, China Pharmaceutical University,
Nanjing 210009, PR China.
E-mail addresses: guowj@nju.edu.cn (W. Guo), molpharm@163.com (Q. Xu), yslai@cpu.edu.cn (Y.
Lai).

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ABSTRACT
Indoleamine-2,3-dioxygenase-1 (IDO1) is an attractive target for cancer immunotherapy.
Herein, a series of novel imidazoleisoindole derivatives was prepared and evaluated for
their ability to inhibit IDO1. Among these, derivative 11r was the most active compound
with nanomolar potency in the Hela cell-based assay, while showed negligible cellular
toxicity. UV-visible spectra study demonstrated that compounds 11p and 11r bound to
IDO1 and coordinated with the heme iron. Furthermore, they could significantly promote
T cell proliferation, increase IFN-γ production, and reduce the numbers of Foxp3+
regulatory T cells. Finally, induced fit docking (IFD) and quantum mechanics/molecular
mechanics (QM/MM) calculation were performed to understand the interactions of these
compounds to IDO1 protein, which provided a comprehensive guide for further structural
modification and optimization.
Keywords:
Indoleamine 2,3-dioxygenase 1; Imidazoleisoindole; Induced fit docking; QM/MM
calculation

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1. Introduction
Immune escape is a fundamental trait of cancer growth and progression, and also a
significant barrier to clinical immunotherapy for cancer [1,2]. Growing evidence indicates
that the dysregulation of the kynurenine pathway of tryptophan metabolism is one of the
key molecular mechanisms that enables tumor cells to thwart the host immune response
[3,4]. The cytosolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is the most broadly
expressed “gatekeeper” enzyme that catalyzes the rapid degradation of tryptophan in the
initial step of the kynurenine pathway. Nonetheless, the overexpression of IDO1 by a wide
variety of tumor cells and antigen presenting cells has been observed in the tumor
microenvironment [5]. The resulting local depletion of tryptophan causes cell cycle arrest
of T lymphocytes and makes these cells more sensitive to apoptosis by activating the
amino acid-sensitive general control non-derepressible 2 (GCN2) stress kinase pathway
[6,7], and the accumulating kynurenine as well as its downstream metabolites can induce
the differentiation of naïve CD4+ T cells into immunosuppressive regulatory T cells (Tregs)
by binding to the aryl hydrocarbon receptor (AHR) [8,9]. Similar to other immune
checkpoints, IDO1 is therefore suggested to be an important target for immunotherapeutic
intervention [10].
In the past decade, many small molecular IDO1 inhibitors have been identified from
natural products [11-13], high-throughput screening [14-16], and structure-based design
[17-19]. However, most of them either showed low potency or failed in vivo assay, so
developing IDO1 inhibitors is still challenging [20,21]. To the best of our knowledge,
there is no drug approved as IDO1 inhibitor until now, and only a few of small molecule
IDO1 inhibitors entered clinical trials as a strategy for the treatment of cancer (e.g.,
indoximod, epacadostat and GDC-0919) (Fig. 1) [22]. Consequently, the discovery of a
novel and potent IDO1 inhibitor, which might be safe and efficacious in further
development stages, remains necessary.

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Fig. 1. The representative IDO1 inhibitors.
GDC-0919 (formerly known as NLG919, structure not disclosed), developed by
NewLink Genetics and later licensed to Genentech in 2014, is a potent and orally available
IDO1 inhibitor with values of Ki = 7.2 nM and EC50 = 75 nM in cell-free assays [23].
Preclinical studies demonstrated that GDC-0919 displayed a favorable pharmacologic and
toxicity profile, and showed significant T cell activation and antitumor activity alone and
in combination with chemotherapeutics or other checkpoint inhibitors in solid tumor
models [23,24]. The safety and preliminary efficacy of GDC-0919 employed as a
standalone therapeutic intervention and combination treatment with anti-PD-L1 antibody
atezolizumab are recently being assessed in patients with locally advanced or metastatic
solid tumors [25].
More recently, Wu and co-workers published a crystal structure of IDO1 bound to a
GDC-0919 analogue (PDB ID: 5EK3), which revealed that the imidazole nitrogen atom of
the compound coordinated with the heme iron, and the imidazoleisoindole core was
deeply placed into pocket A, while the 1-cyclohexylethanol moiety was extended towards
pocket B [26]. The hydroxyl group concurrently interacted with the 7-propionic acid of
the heme by an intermolecular hydrogen bond and the isoindole nitrogen by an
intramolecular hydrogen bond, which was found to be crucial to inhibit IDO1. Meanwhile,
the authors minimally modified the imidazoleisoindole core by other bioisosteric groups
that resulted in the dramatic loss of IDO1 inhibitory activity, which indicated the

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importance of this moiety for the inhibition of IDO1.
Through meticulous observation of the active site of this IDO1/ GDC-0919 analogue
co-crystal, it is found that the 1-cyclohexylethanol moiety stretches itself to the entrance
of the pocket B, not fully occupying it (Fig. 2). That’s mainly because the hydrogen
bonding network appears between the inhibitor and IDO1 protein that could control the
conformation of the molecules [27, 28]. Tojo et al. from Dainippon Sumitomo Pharma Co.
pointed out the generation of an induced fit and the resulting interaction with Phe226 and
Arg231 in pocket B were essential for potent IDO1 inhibitory activity through the
crystallization of IDO1/Amg-1 and IDO1/AMCL-13b complexes [29]. These results
provide new directions for developing novel IDO1 inhibitors that simultaneously occupy
pocket A and B.
Fig. 2. (A) The superposition of the active site of two IDO1 co-crystals. PDB ID: 4PK5 (colored in
green), 5EK3 (colored in grey). (B) Top view of two aligned ligands. Amg-1 is colored in green, and
GDC-0919 analogue is colored in grey. The red curve represents the shape of the binding pocket.
Therefore, for continuously identifying potent IDO1 inhibitors [30,31], in this study,
we reported the extensive structural modification of GDC-0919 analogue. Given that the
imidazoleisoindole core is a very important heterocyclic scaffold for IDO1 binding in the
structure of GDC-0919 analogue [32-34], we decided to take advantage of this skeleton as
a privileged scaffold and modify it on site P1 by using different linker length and
flexibility and site P2 by different substituted aromatic and aliphatic ring (Fig. 3), thereby
designing a new series of imidazoleisoindole derivatives. Interestingly, the resulting
compounds 11p and 11r proved to be potent inhibitors of IDO1 with no/negligible

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cytotoxicity. Subsequent experiments indicated that these compounds could promote T cell
proliferation, increase IFN-γ production, and reduce the conversion of naïve T cells into
Tregs. Furthermore, the binding mode analysis of them based on induced fit docking (IFD)
and quantum mechanics/molecular mechanics (QM/MM) calculation will give us a
direction for further structure optimization.
P2
R2
R1
R
N
Change the linker
and the P2 moiety
n
N
N
N
O
OH
OH
N
N
N
N
P1
11a-t
14a,b
n = 0, 1, 2
Fig. 3. Design strategy for novel imidazoleisoindole derivatives.
2. Results and discussion
2.1. Chemistry
Synthetic route for the target compounds 11a-t was shown in scheme 1.
4,5-Diiodo-1H-imidazole 2 was prepared from 1H-imidazole and iodine, and followed by
refluxing in ethanol in the presence of sodium sulfite to afford 4-iodo-1H-imidazole 3. The
resulting compound was subsequently protected with triphenylmethyl chloride to yield 4,
which was then converted into the key benzaldehyde 6 by Suzuki cross-coupling reaction
with phenylboronic acid 5. Ethyl acrylate 8 was obtained by Wittig reaction of 6 with the
reagent 7, and then cyclized by an intramolecular Michael reaction in refluxing acetic acid
to prepare imidazoleisoindole 9. The acetic acid 10 was obtained from 9 by hydrolysis
reaction, followed by esterification with various aromatic amines in the presence of
1-hydroxybenzotriazole (HOBT) and N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide
(EDCI) to form compounds 11a-t.

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B(OH)2
Trt
CHO
N
O
Br
N
Ph3P
I
I
O
I
N
N
N
7
a
b
N
c
CHO
5
I
N
N
H
e
N
H
N
H
d
Trt
2
1
3
4
6
O
R1
N
O
O
OH
h
i
R2
f
g
N
N
N
O
O
N
O
N
N
N
N
Trt
11a-t
9
8
10
o
Scheme 1. Reagents and conditions: (a) NaOH, I2, KI, H2O, 20 C, 1 h, 99.0%; (b) Na2SO3, EtOH,
o
reflux, 24 h, 48.1%; (c) TrtCl, TEA, DMF, 20 C, 48 h, 94.0%; (d) Pd(PPh3)4, K3PO4, DMF, H2O, N2,
90 oC, 66.7%; (e) PPh3, DCM, rt, 90.6%; (f) NaOH, H2O, DCM, rt, 77.5%; (g) HOAc, MeOH, reflux, 3
h, 76.0%; (h) NaOH, EtOH, H2O, 60 oC; (i) EDCI, HOBT, TEA, DMF, rt.
Synthetic route for the target compounds 14a and 14b was shown in scheme 2. The
direct aldol reaction was applied to 6 to give key intermediates 12a and 12b, which were
cyclized in refluxing acetic acid to afford imidazoleisoindoles 13a and 13b. Subsequent
condensation of hydroxylamine hydrochloride with 13a and 13b to yield compounds 14a
and 14b.
Scheme 2. Reagents and conditions: (a) Na, EtOH, rt, 3 h; (b) HOAc, MeOH, reflux, 3 h; (c)
NH2OH·HCl, EtOH, 50 oC, 12 h.
2.2. Inhibition of IDO1 activity in a cell-based assay
It is well-known that IDO1 enzymatic and cellular assays have greater biological
relevance and reported correlation issues [35]. Thus, HeLa cell line expressing native
human IDO1 induced with IFN-γ was chosen as the primary screen for investigating the
therapeutic potential of the newly synthesized compounds. This cellular assay is beneficial

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to drug development, as it not only evaluates the IDO1 inhibitory activity of the tested
compounds, but also measures their cytotoxicity on cell, and predicts their permeability
[11,15]. Both GDC-0919 analogue (GDC-0919 racemic mixture) and L-1-MT were used
as positive controls.
As shown in Table 1, we first investigated compounds 11a-11m with the linker having
four heavy atoms that may facilitate the P2 moiety sticking into pocket B. Quite
disappointingly, almost all compounds showed weak potency against IDO1 no matter what
R1 group was electron withdrawing (11a-11e, 11k-11m) or electron donating (11f, 11g, 11j)
aromatic nucleus. Meantime, 11h and 11i, which have a methyl group linked to amide and
methylene moieties, respectively, could not improve the inhibitory activity of 11a.
However, attempts at rigidifying the linker with three heavy atoms got some positive
results (11n-11o), which were more potent than compounds 11a-11m. Especially, 11p gave
a 13-fold improvement in inhibitory potency compared with the counterpart 11j, both of
which contained the same 1,3-benzodioxolyl moiety towards pocket B. Following these
encouraging results, we next explored compounds 11r-11t to see if the linker with five
heavy atoms could increase the potency. Surprisingly, their cell-based inhibitory activities
were much better than the corresponding series with three or four atoms linker (11r vs 11f;
11s vs 11a and 11n; 11t vs 11l). Among them, compound 11r was the most potent
derivative in the series, which was in the same order of magnitude as GDC-0919 analogue.
Finally, hydroxyl group in GDC-0919 analogue was replaced by hydroxyloxime, but
leading to 14a and 14b which exhibited poor activity with IC50 values higher than 20 µM
.
Furthermore, the results from 11n-11q and 11r-11t showed that the inhibitory activity
of compounds bearing an electron-donating aromatic ring was obviously greater than that
of one bearing an electron-withdrawing aromatic ring (11p vs 11o; 11r vs 11t), while the
activity of the latter was weaker than the corresponding benzene without substituent (11o
vs 11n; 11t vs 11s). At first glance, these results appear to be contradictory about the
correlation between the activity and the linker length of compounds (11a, 11n and 11s).
The discussion about this will be performed in the next section. Among these compounds,
11p and 11r were the two most active derivatives with IC50 values of 5.03 µM and 0.84

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µM, respectively, which were selected for further evaluation.
Table 1. Structures of the target compounds and their activity for inhibiting IDO1.
R1
R1
N
R2
N
N
N
O
OH
N
N
11a-t
14a,b
Compd.
R1
R2 IC50 (µM)a
Compd.
R1
R2
H
IC50 (µM)a
11a
H
60.56
69.44
58.11
53.93
50.9
11m
65.92
CN
11b
11c
11d
11e
11f
H
H
11n
11o
11p
11q
11r
11s
11t
H
H
H
H
H
H
H
25.93
33.1
H
5.03
H
39.04
0.84
O
H
77.54
71.7
11g
11h
H
14.98
21.93
CH3
64.47
N
11i
11j
H
H
74.39
66.73
14a
14b
20.37
22.72
O
O

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CO2H
11k
11l
H
H
95.07
82.12
L-1-MT
14.69
0.26
GDC0919b
N
a Results are mean of at least two experiments. b GDC-0919 analogue [26]
2.3. Effects of 11p, 11r and 11s on cell viability
Since the inhibition of tryptophan degradation could simply be an effect of the
cytotoxicity of the tested compounds, measurement of cell viability is indispensable when
reporting cellular IC50 values. In this study, for all compounds, the viability of Hela cells
was measured with the MTT method at the end of the IDO1 cellular assay. The results of
this assay indicated that most of the compounds displayed no/negligible level of toxicity
under the experimental conditions. The results of cell viability assay of 11p, 11r and 11s
were shown in Fig. 4A.
2.4. Effects of 11p, 11r and 11s on IDO1 expression
Since the suppression of IDO1 expression of the tested compounds could also be
responsible for the inhibitions of tryptophan degradation, Western blot analysis was
subsequently performed to investigate if compounds 11p, 11r and 11s could affect the
expression of IDO1 protein in the HeLa cell-based assay. The results showed that these
three compounds didn’t influence IDO1 protein level (Fig. 4B), which indicated that they
inhibited IDO1 enzymatic activity and not its expression in cells. Similarly, GDC-0919
analogue also did not affect IDO1 expression in Hela cells.
Fig. 4. (A) Measurement of cytotoxicity of compounds 11p, 11r and 11s. Cell viability was determined
by the MTT assay. Data are averages from at least three independent experiments. (B) HeLa cells were

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treated with IFN-γ with or without compounds 11p, 11r and 11s at their IC50 concentrations for 24
hours, and analyzed by Western blot using an anti-IDO antibody. GDC-0919 analogue was used as a
reference.
2.5. UV-visible spectra study of 11p and 11r
IDO1 has a heme in protein, and the specific UV absorption properties of porphyrins
are useful in studies of heme-contained proteins. Since the absorbance spectra of the heme
group is highly sensitive to the changes in the polarity of heme surroundings upon the
ligand binding, the changes in the absorbance spectra could be utilized to evaluate the
binding of the compound to IDO1 [36]. The UV−visible spectra of ferric IDO1 were
measured in the presence and absence of these two compounds in this study. As can be
seen from Fig.5, in the presence of compound 11p, the Soret peak shifted from 404 to 410
nm and compound 11r shifted the maxima from 404 to 412 nm, which indicated that these
compounds bound to IDO1 and coordinated with the iron of the heme group.
Fig. 5. UV spectra of ferric-IDO1 (black), compound 11p-IDO1 (red) and compound 11r-IDO1 (blue).
2.6. Effects of 11p and 11r on T cell proliferation and IFN-γ production
It is well-known that IDO1 protein is overexpressed by a variety of tumor cell types,
leading to the local depletion of tryptophan levels and the production of toxic tryptophan
metabolites [37]. This severely affects the proliferation of T lymphocytes and is thereby
profoundly immunosuppressive [38]. A proliferation assay was carried out to determine if

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inhibition of IDO1 activity could improve T cell activity in the presence of cancer cells.
B16F1 cells, which expressed IDO1 in high level, were thus used to co-culture with T
cells. As shown in Fig. 6A, compounds 11p, 11r and GDC-0919 analogue displayed a
significant augmentation of T cell stimulated with B16F1 cells. Especially, compound 11p
showed 48% on the rate of T cell proliferation. These results were in conformity with the
increase of IFN-γ levels compared with T cells and B16F1 co-culture system, which can
increase the growth of the neighboring T cells (Fig. 6B). These findings suggested that
compounds 11p and 11r could reverse the suppression of T lymphocytes caused by IDO1
to a great extent.
Fig. 6. T cell proliferation (A) and IFN-γ levels (B) assays in the B16F1-T cells co-culture system.
Compounds 11p and 11r were added to the system at the concentration of their triple IC50 values from
the HeLa-based assay (GDC-0919: 1.5 µM; 11p: 15 µM; 11r: 3 µM). Each bar of the graph indicates
the mean of three replicate wells with standard error of the mean.
2.7. Effects of 11p and 11r on Foxp3+ Tregs in B16F1 cells
It has been reported that IDO1-mediated tryptophan catabolism facilitates the
conversion of naïve T lymphocytes into CD4+ CD25+ Tregs, which can block T cell
activation and trigger T cell apoptosis, thus promotes immunoescape and favors tumor cell
growth [39]. Based on these reasons, compounds 11p and 11r were evaluated to elucidate
their effect on reversing the conversion. As shown in Fig. 7, when naïve T cells
co-cultured with B16F1 cells, it gave rise to an approximately 2.7-fold increase in the
number of Tregs. However, addition of 11p and 11r to the cultures partially reversed this
effect, as did treatment with GDC-0919 analogue. These results indicated that compounds

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11p and 11r could significantly reduce the numbers of Foxp3+ Tregs in B16F1 cells.
Fig. 7. Compounds 11p and 11r decreased differentiation of Tregs in B16F1-T cell co-culture system.
(A) A representative plot of FACS analysis is presented. CD4+ CD25+ FOXP3+ positive cells were
defined as Tregs. (B) Average values of 3 independent experiments are shown in the graph. Error bars
represent SD. *p < 0.05, **p < 0.01 vs T+B16F1.
2.8. Molecular modeling studies
Over the last decades, docking is becoming increasingly popular and has been
successfully applied in several lead-compound-discovery projects [40]. Most docking
algorithms rely on a rigid protein structure, which in many cases is sufficient to find some
active compounds. This is often referred to as “semi-flexible docking”. However, in the
case of IDO1 protein, high flexibility of side chain and backbone often poses a problem
for IDO1 docking, which could lead to the formation of different active site conformations.
Thus, the induced fit phenomenon should be considered here for IDO1 docking studies. In
our previous studies, docking that considered the flexibility of protein was found to
enhance the accuracy of predicted binding mode of IDO1 [31]. According to the induced
fit theory, proteins need not be rigid locks. They can accommodate the substrate by
flexibly adapting their substrate-binding site [41]. To explore the binding modes of the
target compounds with the active site of IDO1, compounds 11j, 11p and 11r were docked
into the binding site of IDO1 by the induced fit docking protocol in Schrödinger.
According to Wu’s research, GDC-0919 analogue contains two chiral centers and has four
stereoisomers, but only two of them display high affinity against IDO1 [26]. Thus, the

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absolute configuration of our chiral molecules with top rank was retained for further
analysis.
In addition to the protein flexibility, the iron-ligand interaction in the active site of
IDO1 is difficult to reliably describe by classical molecular force-field parameters, which
could not consider the charge transfer when the ligand coordinating to the metal [42].
Quantum mechanical calculations has been shown to increase computational accuracy but
it has some disadvantages including high computational cost and corresponding
intractability of large chemical systems. An alternative approach is hybrid QM/MM,
which was initially proposed by Warshel and Levitt in 1976 [43]. In a QM/MM simulation,
a small region of the system that is of particular chemical importance was chosen to be
treated using QM, while the remainder of the system is treated via MM, which offers a
good balance of physical accuracy and relatively low computational cost. Based on the
views mentioned above, QM/MM strategy will be performed to optimize the
IDO1-inhitbitor IFD-docking complex. In our system, the QM region included the full
heme ring, the iron, the coordinating His residue and the inhibitor, which maintained the
complex's sixfold coordinated system, and the remaining subsystem was treated with the
MM method.
As shown in Fig. 8, the overall structural features of QM/MM-optimized structures
were similar to those obtained by IFD docking. The main difference included the key
distance between iron atom and the corresponding coordinate atom and the orientation of
the polar atoms in ligand that faced the 7-propionate group of the heme. All of
imidazoleisoindole rings in the ligands were buried in hydrophobic pocket A like
GDC-0919 analogue in the co-crystals (Fig. 2A). The binding modes of compound 11j
presented the similar binding conformation with Amg-1. The benzyl group connected with
amide linker extended into the pocket B and its dioxomethylene oxygen formed a
hydrogen bond interaction with Arg231. The NH of amide unit made a hydrogen bond
with the backbone of Gly262. Unlike 11j and 11r, the linker length of 11p was so short
that the aniline moiety can’t stretch inside the pocket B. Nonetheless, the NH of amide
linker could interact with the 7-propionate group after QM/MM optimization and the

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dioxomethylene-substituted aniline ring of 11p formed a π-cation interaction with Arg231.
Although the linker length of 11j was longer than that of 11p, which could more stretch
into pocket B, the difference of IDO1 inhibitory activity between them might be attributed
to the formation of hydrogen bond interaction between the ligand and the heme
7-propionate. In the case of 11r, the m-methoxyl-phenyl group was bent sharply at the
methylene moiety and almost fully occupied pocket B, demonstrating the interaction with
Phe226 and Arg231 through π-π and π-cation interaction, respectively. In addition to that,
the amide NH in the linker was hydrogen-bonded with the heme 7-propionate like 11p.
This computational study finds that there are two dominating factors affecting IDO1
inhibitory activity: the occupation of pocket B and the resulting interaction with the
7-propionate group, which could be useful for further optimization.
A
B
C
Phe163
Gly262
Phe163
Phe226
Arg231
Phe226
Phe226
Arg231
Arg231
His346
His346
His346
Fig. 8. The superposition of IFD-based structure (colored in magenta) and QM/MM-optimized
structure (colored in green). Polar contacts were shown as red lines. (A) 11j-IDO1 complexes, (B)
11p-IDO1 complexes, (C) 11r-IDO1 complexes.
3. Conclusions
IDO1 plays a key role in tryptophan catabolism in the immune system and is also
considered as an important therapeutic target for the treatment of cancer and other diseases
that are linked with kynurenine pathway. In this study, a series of imidazoleisoindole
derivatives 11a-t and 14a-b were designed, synthesized and evaluated as novel IDO1
inhibitors. Structure and activity relationship analysis indicated that the compounds with
three or five atoms linker showed stronger inhibitory activity than those with four atoms
linker in an assay which measured IDO1 activity in cells, and the compound with an
electron-donating aromatic ring at site P2 could increase inhibitory potency. Among them,

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compounds 11p and 11r exhibited the highest inhibitory potency with no/negligible
cytotoxicity. UV spectra analysis revealed that these compounds could bind to IDO1 and
coordinate with the iron of the heme group. Importantly, they significantly promoted
proliferation of T lymphocytes and led to the dramatic decrease of Tregs in the B16F1 and
naïve T cells co-culture system. Finally, IFD and QM/MM calculation were carried out
and showed that the occupation of pocket B and the resulting interaction with the
7-propionate group would be essential for potent IDO1 inhibitory activity, which could
guide us for further structure optimization.
4. Experimental section
4.1. Chemistry
4.1.1. General procedures
Melting points were determined on a RDCSY-I capillary apparatus and were
uncorrected. All materials used were commercially available and used as supplied.
HG/T2354-92 silica gel 60 F254 sheets were used for analytical thin-layer chromatography
1
(TLC). Column chromatography was performed on silica gel (200-300 mesh). H NMR
spectra were recorded on a Bruker AV-300 spectrometer. Chemical shifts (δ) were given in
parts per million (ppm) relative to the solvent peak. IR spectra were recorded on shimadzu
IRTracer-100 in KBr with absorptions in cm-1. All the reagents and solvents were reagent
grade and were used without further purification unless otherwise specified. The purity of
compounds used for biological evaluations was determined to be greater than 95% using
Shimadzu HPLC system (Kyoto, Japan. see Supplementary material).
4.1.2. Preparation of intermediates 2-10
4.1.2.1 4,5-diiodo-1H-imidazole (2)
A mixture of KI (29.3 g, 176.0 mmol) and I2 (22.4 g, 88.0 mmol) in water (60 mL)
was added to a solution of 1H-imidazole (3.0 g, 44.1 mmol) in 2 M NaOH (10.6 g, 264.5
mmol). The mixture was stirred at 20 °C for 3 h and then neutralized with 6 M HCl (pH =
7). The resulting solid was collected by filtration, recrystallization from ethanol to give 2
as a grey solid (14.0 g, 99.0%). Mp 166-168 °C. 1H NMR (300 MHz, DMSO-d6) δ(ppm)

ACCEPTED MANUSCRIPT
12.75 (br s, 1H), 7.8 (br s, 1H). MS (EI) m/z 320.8 [M+H]+.
4.1.2.2 4-iodo-1H-imidazole (3)
A mixture of 2 (12.0 g, 37.5 mmol) and Na2SO3 (23.6 g, 187.3 mmol) in EtOH (120
mL) and H2O (20 mL) was refluxed for 72 h. The mixture was concentrated in vacuo and
the residue was extracted with ethyl acetate. The crude product was purified by
recrystallization from dichloromethane to afford 3 as a white solid (3.5 g, 48.1%). Mp
137-139 °C. 1H NMR (300 MHz, Chloroform-d) δ(ppm) 7.0 (s, 1H), 7.5 (s, 1H). MS (EI)
m/z 194.9 [M+H]+.
4.1.2.3 4-iodo-1-trityl-1H-imidazole (4)
To a solution of 3 (3.5 g, 18.0 mmol) in DMF (70 mL) was added TEA (3.0 mL, 21.7
mmol) and trityl chloride (5.5 g, 19.7 mmol). After stirring at 20 °C for 24 h, the solution
was poured into water. The solid was filtered to yield the crude compound that was
purified by flash column chromatography on silica gel to afford 4 as a white solid (7.4 g,
94.0%). Mp 148-150 °C. 1H NMR (300 MHz, Chloroform-d) δ(ppm) 6.9 (m, 1H), 7.0-7.2
(m, 6H), 7.25-7.4 (m, 10H). MS (EI) m/z 437.1 [M+H]+.
4.1.2.4 2-(1-trityl-1H-imidazol-4-yl)benzaldehyde (6)
A suspension of 4 (3.0 g, 6.9 mmol), the appropriate 2-formyl boronic acid (1.6 g, 10.7
mmol) and K3PO4 (4.4 g, 20.8 mmol) in DMF (30 mL) and water (6 mL) was purged with
nitrogen for 5 min, followed by the addition of Pd(PPh3)4 (0.6 g, 0.5 mmol) and the
mixture was purged with nitrogen for another 5 mins. The reaction mixture was stirred at
90 °C for 16 h under an atmosphere of N2. The solution was allowed to cool and was
filtered through a plug of celite. The mixture was diluted with water (50 mL) and was
extracted with ethyl acetate to afford the crude product which was purified by flash
column chromatography on silica gel to yield 6 as a white solid (1.9 g, 66.7%). Mp
147-149 °C. 1H NMR (300 MHz, Chloroform-d) δ(ppm) 7.97 (dd, J = 7.8, 1.4 Hz, 1H),
7.72-7.54 (m, 3H), 7.38 (h, J = 3.2 Hz, 11H), 7.25-7.19 (m, 6H), 7.07 (d, J = 1.3 Hz, 1H).
MS (EI) m/z 415.2 [M+H]+.
4.1.2.5 Wittig reactant ethyl 2-(triphenyl-λ5-phosphanylidene)acetate (7)

ACCEPTED MANUSCRIPT
The solution of triphenylphosphane (25.9 g, 98.8 mmol) in dichloromethane (300 mL)
was added ethyl 2-bromoacetate (15.0 g, 89.8 mmol) dropwise, and stirred at room
temperature for 3 days. After the evaporation of the solvent, the remaining residue was
collected and washed with dichloromethane three times to give the corresponding Wittig
reactant as a white solid (35.0 g, 90.6%).
4.1.2.6 An optical mixture of ethyl (Z)-3-(2-(1-trityl-1H-imidazol-4-yl)phenyl)acrylate and
ethyl (E)-3-(2-(1-trityl-1H-imidazol-4-yl)phenyl)acrylate (8)
To a suspension of NaOH (0.6 g, 15.4 mmol) in water (1 mL) at 0 °C was added 7 (3.3
g, 7.7 mmol) as a solution in dichloromethane (10 mL) and the mixture was stirred for 40
min. The intermediate 6 (3.2 g, 7.7 mmol) was added as a solution in dichloromethane (10
mL) drop wise over a period of 3 min. The reaction was allowed to room temperature and
stirred overnight. The mixture was diluted with water and was extracted with
dichloromethane to afford the crude product which was purified by flash column
chromatography on silica gel to yield 8 as a faint yellow solid (2.9 g, 77.5%). Mp
1
110-112 °C. H NMR (300 MHz, Chloroform-d) δ(ppm) 8.17 (d, J = 15.9 Hz, 1H), 7.77
(dd, J = 7.8, 1.4 Hz, 1H), 7.61-7.51 (m, 3H), 7.45-7.33 (m, 14H), 7.21 (td, J = 7.2, 6.6, 4.1
Hz, 8H), 7.06-6.89 (m, 2H), 6.32 (d, J = 15.9 Hz, 1H), 4.23 (q, J = 7.1 Hz, 2H), 4.07 (q, J
= 7.1 Hz, 1H), 1.29-1.24 (m, 3H), 1.15 (t, J = 7.1 Hz, 1H). MS (EI) m/z 485.3 [M+H]+.
4.1.2.7 ethyl 2-(5H-imidazo[5,1-a]isoindol-5-yl)acetate (9)
To a solution of 8 (5.0 g, 10.3 mmol) in methyl alcohol (50 mL) was added AcOH
(12.5 mL) and the mixture was stirred at 90 °C for 3 h. The mixture was allowed to cool to
room temperature and the pH was adjusted to 10 with saturated K2CO3 solution. The
mixture was evaporated in vacuo to remove methyl alcohol and was extracted with ethyl
acetate to afford the crude product which was purified by flash column chromatography
1
on silica gel to yield 9 as a grey solid (1.9 g, 76.0%). Mp 87-89 °C. H NMR (300 MHz,
Chloroform-d) δ(ppm) 7.81 (s, 1H), 7.57 (dt, J = 7.6, 1.0 Hz, 1H), 7.49-7.42 (m, 2H), 7.35
(dd, J = 1.7, 1.0 Hz, 1H), 7.21 (s, 1H), 5.58 (s, 1H), 4.30 (q, J = 7.1 Hz, 2H), 3.12 (dd, J =
17.3, 3.9 Hz, 1H), 2.73 (dd, J = 17.3, 9.7 Hz, 1H), 1.33 (t, J = 7.2 Hz, 3H). MS (EI) m/z
243.1 [M+H]+.

ACCEPTED MANUSCRIPT
4.1.2.8 2-(5H-imidazo[5,1-a]isoindol-5-yl)acetic acid (10)
To a solution of 9 (1.0 g, 4.1 mmol) in ethyl alcohol (50 mL) was added NaOH (0.8 g,
20.6 mmol) in water (2 mL) and the mixture was stirred at 60 °C for 3 h. After the reaction
mixture was allowed to cool to room temperature, the pH was adjusted to 5-6 with 10%
hydrochloric acid. Then, the mixture was evaporated in vacuo to remove solvent to afford
the crude product 10, which was used to the next step without any purification; MS (EI)
m/z 215.1 [M+H]+.
4.1.3. General preparation of compounds 11a-t
To the crude product 10 (1 mol equiv) in DMF (0.12 M) was added EDC (1.2 mol
equiv), HOBT (1.2 mol equiv) and TEA (3 mol equiv) and the mixture was stirred at room
temperature for 15 min. After that, the mixture was added various aromatic or aliphatic
amines (1.1 mol equiv), then it was stirred at room temperature for 12 h. The mixture was
diluted with water and was extracted with ethyl acetate to afford the crude product which
was purified by flash column chromatography on silica gel to yield the target products.
4.1.3.1. N-benzyl-2-(5H-imidazo[5,1-a]isoindol-5-yl)acetamide (11a)
1
Yield 67.2%. White powder. Mp 140-142 °C. H NMR (300 MHz, Chloroform-d)
δ(ppm) 7.61 (s, 1H), 7.51 (d, J = 7.5 Hz, 1H), 7.43-7.32 (m, 7H), 7.24 (d, J = 8.1 Hz, 1H),
7.08 (s, 1H), 6.55 (s, 1H), 5.67 (dd, J = 9.4, 4.5 Hz, 1H), 4.62-4.40 (m, 2H), 2.94 (dd, J =
15.4, 4.4 Hz, 1H), 2.50 (dd, J = 15.4, 9.5 Hz, 1H). MS (EI) m/z 304.2 [M+H]+. IR (KBr)
3207, 3030, 1652, 1615, 1504 cm-1.
4.1.3.2. N-(4-cyanobenzyl)-2-(5H-imidazo[5,1-a]isoindol-5-yl)acetamide (11b)
1
Yield 60.4%. Pale yellow powder. Mp 198-200 °C. H NMR (300 MHz, DMSO-d6)
δ(ppm) 8.70 (t, J = 5.9 Hz, 1H), 7.87-7.74 (m, 2H), 7.69-7.52 (m, 2H), 7.5-7.33 (m, 4H),
7.26 (td, J = 7.6, 1.2 Hz, 1H), 7.13 (s, 1H), 5.62 (dd, J = 8.7, 5.2 Hz, 1H), 4.60-4.37 (m,
2H), 3.07 (dd, J = 15.4, 5.2 Hz, 1H), 2.69 (dd, J = 15.5, 8.8 Hz, 1H). MS (EI) m/z 329.1
[M+H]+. IR (KBr) 3208, 3007, 2226, 1652, 1606, 1506 cm-1.
4.1.3.3. N-(3,4-difluorobenzyl)-2-(5H-imidazo[5,1-a]isoindol-5-yl)acetamide (11c)
1
Yield 78.0%. Pale yellow powder. Mp 88-90 °C. H NMR (300 MHz, Chloroform-d)

ACCEPTED MANUSCRIPT
δ(ppm) 7.60 (s, 1H), 7.51 (d, J = 7.6 Hz, 1H), 7.36 (dd, J = 21.6, 7.5 Hz, 2H), 7.24 (t, J =
7.7 Hz, 1H), 7.19-6.97 (m, 4H), 6.91 (s, 1H), 5.66 (dd, J = 9.2, 4.5 Hz, 1H), 4.53 (dd, J =
14.9, 5.9 Hz, 1H), 4.37 (dd, J = 14.9, 5.4 Hz, 1H), 2.96 (dd, J = 15.9, 4.2 Hz, 1H), 2.56
(dd, J = 15.5, 9.2 Hz, 1H). MS (EI) m/z 340.1 [M+H]+. IR (KBr) 3207, 3022, 1645, 1609,
1520 cm-1.
4.1.3.4. N-(3-chlorobenzyl)-2-(5H-imidazo[5,1-a]isoindol-5-yl)acetamide (11d)
Yield 66.0%. White powder. Mp 73-75 °C. 1H NMR (300 MHz, Chloroform-d) δ(ppm)
7.65 (s, 1H), 7.53 (d, J = 7.6 Hz, 1H), 7.44-7.29 (m, 4H), 7.29-7.16 (m, 3H), 7.12 (d, J =
1.7 Hz, 1H), 6.33 (s, 1H), 5.71 (dd, J = 9.4, 4.5 Hz, 1H), 4.59 (dd, J = 14.6, 5.8 Hz, 1H),
4.43 (dd, J = 15.0, 5.0 Hz, 1H), 3.08-2.86 (m, 1H), 2.66-2.41 (m, 1H). MS (EI) m/z 338.1
[M+H]+. IR (KBr) 3213, 3024, 1645, 1599, 1472 cm-1.
4.1.3.5. N-(3-chloro-4-fluorobenzyl)-2-(5H-imidazo[5,1-a]isoindol-5-yl)acetamide (11e)
Yield 67.2%. White powder. Mp 143-145 °C. 1H NMR (300 MHz, Chloroform-d)
δ(ppm) 7.67 (s, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.37 (dt, J = 14.1, 7.3 Hz, 2H), 7.30-7.22 (m,
2H), 7.14 (q, J = 8.2, 7.3 Hz, 2H), 6.29 (s, 1H), 5.79-5.63 (m, 1H), 4.54 (d, J = 5.9 Hz,
1H), 4.41 (d, J = 4.9 Hz, 1H), 2.99 (dd, J = 15.3, 4.5 Hz, 1H), 2.58 (dd, J = 15.4, 9.1 Hz,
1H). MS (EI) m/z 356.1 [M+H]+. IR (KBr) 3208, 3107, 1646, 1614, 1504 cm-1.
4.1.3.6. 2-(5H-imidazo[5,1-a]isoindol-5-yl)-N-(4-methoxybenzyl)acetamide (11f)
Yield 72.3%. White powder. Mp 74-76 °C. 1H NMR (300 MHz, DMSO-d6) δ(ppm)
8.52 (s, 1H), 7.77-7.55 (m, 2H), 7.47 (d, J = 7.6 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H),
7.32-7.18 (m, 3H), 7.13 (s, 1H), 6.97-6.84 (m, 2H), 5.62 (dd, J = 8.9, 5.2 Hz, 1H), 4.31 (d,
J = 5.8 Hz, 2H), 3.74 (s, 3H), 3.01 (dd, J = 15.3, 5.3 Hz, 1H), 2.61 (dd, J = 15.3, 9.0 Hz,
1H). MS (EI) m/z 334.2 [M+H]+. IR (KBr) 3275, 3050, 1651, 1612, 1512, 1248 cm-1.
4.1.3.7. N-(4-hydroxybenzyl)-2-(5H-imidazo[5,1-a]isoindol-5-yl)acetamide (11g)
Yield 65.8%. White powder. Mp 232-234 °C. 1H NMR (300 MHz, DMSO-d6) δ(ppm)
9.33 (d, J = 1.2 Hz, 1H), 8.46 (s, 1H), 7.70-7.55 (m, 2H), 7.47 (d, J = 7.6 Hz, 1H), 7.39 (t,
J = 7.4 Hz, 1H), 7.26 (dd, J = 7.5, 1.2 Hz, 1H), 7.17-7.01 (m, 3H), 6.79-6.63 (m, 2H), 5.62
(dd, J = 9.0, 5.2 Hz, 1H), 4.26 (d, J = 5.7 Hz, 2H), 3.00 (dd, J = 15.2, 5.3 Hz, 1H),

ACCEPTED MANUSCRIPT
2.74-2.56 (m, 1H). MS (EI) m/z 320.2 [M+H]+. IR (KBr) 3649, 3300, 3032, 1630, 1595,
1507, 1240 cm-1.
4.1.3.8. N-benzyl-2-(5H-imidazo[5,1-a]isoindol-5-yl)-N-methylacetamide (11h)
Yield 69.1%. White powder. Mp 208-210 °C. 1H NMR (300 MHz, Chloroform-d)
δ(ppm) 7.86 (d, J = 3.5 Hz, 1H), 7.66-6.99 (m, 10H), 5.79 (d, J = 9.3 Hz, 1H), 4.81-4.62
(m, 1H), 4.45 (s, 1H), 3.22-3.01 (m, 2H), 2.86 (s, 3H). MS (EI) m/z 318.2 [M+H]+. IR
(KBr) 3032, 2953, 2853, 1636, 1595, 1508 cm-1.
4.1.3.9. 2-(5H-imidazo[5,1-a]isoindol-5-yl)-N-(1-phenylethyl)acetamide (11i)
Yield 60.2%. White powder. Mp 144-146 °C. 1H NMR (300 MHz, Chloroform-d)
δ(ppm) 8.68-8.39 (d, J = 7.9 Hz, 1H), 7.86-7.44 (m, 3H), 7.44-7.27 (m, 6H), 7.27-7.07 (m,
2H), 5.76-5.47 (t, J = 7.1 Hz, 1H), 5.17-4.95 (m, 1H), 3.11-2.91 (td, J = 15.7, 5.3 Hz, 1H),
2.74-2.58 (dt, J = 16.1, 8.3 Hz, 1H), 1.47-1.34 (m, 3H). MS (EI) m/z 318.2 [M+H]+. IR
(KBr) 3273, 3030, 2966, 2853, 1645, 1622, 1520 cm-1.
4.1.3.10.
N-(benzo[d][1,3]dioxol-5-ylmethyl)-2-(5H-imidazo[5,1-a]isoindol-5-yl)acetamide (11j)
1
Yield 74.3%. White powder. Mp 88-90 °C. H NMR (300 MHz, DMSO-d6) δ(ppm)
7.76-7.64 (s, 1H), 7.57-7.41 (m, 2H), 7.37-7.11 (m, 6H), 7.08-7.01 (d, J = 6.7 Hz, 1H),
5.67-5.46 (m, 1H), 4.67-4.39 (d, J = 2.0 Hz, 2H), 3.34-3.26 (d, J = 4.0 Hz, 1H), 2.87-2.75
(d, J = 11.1 Hz, 3H). MS (EI) m/z 348.1 [M+H]+. IR (KBr) 3275, 3032, 1636, 1609, 1489,
1250 cm-1.
4.1.3.11. 4-((2-(5H-imidazo[5,1-a]isoindol-5-yl)acetamido)methyl)benzoic acid (11k)
Yield 79.2%. White powder. Mp 260-262 °C. 1H NMR (300 MHz, DMSO-d6) δ(ppm)
12.84 (s, 2H), 8.68 (s, 1H), 8.03-7.81 (m, 2H), 7.72-7.53 (m, 2H), 7.49 (d, J = 7.4 Hz, 1H),
7.39 (d, J = 7.5 Hz, 2H), 7.25 (t, J = 7.7 Hz, 1H), 7.13 (d, J = 2.3 Hz, 1H), 5.62 (d, J = 6.6
Hz, 1H), 4.45 (s, 2H), 3.07 (dd, J = 15.2, 5.3 Hz, 1H), 2.68 (dd, J = 15.5, 8.9 Hz, 1H). MS
(EI) m/z 348.1 [M+H]+. IR (KBr) 3065, 2920, 1653, 1616, 1508, 949 cm-1.
4.1.3.12. 2-(5H-imidazo[5,1-a]isoindol-5-yl)-N-(pyridin-3-ylmethyl)acetamide (11l)
1
Yield 59.1%. White powder. Mp 119-121 °C. H NMR (300 MHz, Chloroform-d)

ACCEPTED MANUSCRIPT
δ(ppm) 8.56-8.44 (m, 1H), 7.86 (d, J = 42.9 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.54 (d, J =
7.5 Hz, 1H), 7.46-7.23 (m, 5H), 7.14 (s, 2H), 5.75 (dd, J = 9.0, 4.4 Hz, 1H), 4.52 (dd, J =
20.6, 5.7 Hz, 2H), 3.09 (dd, J = 15.7, 4.8 Hz, 1H), 2.66 (s, 1H). MS (EI) m/z 305.1
[M+H]+. IR (KBr) 3275, 3065, 1653, 1595, 1508 cm-1.
4.1.3.13. 2-(5H-imidazo[5,1-a]isoindol-5-yl)-N-(pyrazin-2-ylmethyl)acetamide (11m)
Yield 64.1%. White powder. Mp 163-165 °C. 1H NMR (300 MHz, Chloroform-d)
δ(ppm) 8.65 (d, J = 4.1 Hz, 1H), 8.57-8.45 (m, 2H), 7.72 (s, 1H), 7.54 (d, J = 8.0 Hz, 1H),
7.38 (dd, J = 15.2, 7.7 Hz, 2H), 7.26 (dd, J = 11.4, 5.0 Hz, 1H), 7.13 (d, J = 4.4 Hz, 1H),
7.04 (s, 1H), 5.84-5.54 (m, 1H), 4.72 (t, J = 4.9 Hz, 2H), 3.13-2.95 (m, 1H), 2.63 (dd, J =
15.5, 9.7 Hz, 1H). MS (EI) m/z 306.1 [M+H]+. IR (KBr) 3273, 3033, 1684, 1593, 1506
cm-1.
4.1.3.14. 2-(5H-imidazo[5,1-a]isoindol-5-yl)-N-phenylacetamide (11n)
Yield 77.1%. Pale yellow powder. Mp 227-229 °C. 1H NMR (300 MHz, Chloroform-d)
δ(ppm) 7.95 (s, 1H), 7.74 (s, 1H), 7.56 (t, J = 7.9 Hz, 2H), 7.47-7.30 (m, 4H), 7.28 (d, J =
2.6 Hz, 2H), 7.17 (dd, J = 15.5, 7.7 Hz, 2H), 5.90-5.64 (m, 1H), 3.13 (d, J = 15.3 Hz, 1H),
2.73 (dd, J = 15.5, 9.5 Hz, 1H). MS (EI) m/z 290.2 [M+H]+. IR (KBr) 3273, 3030, 1653,
1601, 1506 cm-1.
4.1.3.15. 2-(5H-imidazo[5,1-a]isoindol-5-yl)-N-(1H-indazol-6-yl)acetamide (11o)
Yield 68.3%. White powder. Mp 280-282 °C. 1H NMR (300 MHz, DMSO-d6) δ(ppm)
8.23 (s, 1H), 7.99 (s, 1H), 7.67 (dd, J = 14.8, 8.3 Hz, 2H), 7.57 (d, J = 7.4 Hz, 1H), 7.40 (d,
J = 7.3 Hz, 1H), 7.31 (d, J = 7.5 Hz, 1H), 7.20-7.02 (m, 2H), 5.73 (s, 1H), 2.89 (dd, J =
16.0, 9.2 Hz, 1H). MS (EI) m/z 330.2 [M+H]+. IR (KBr) 3273, 3196, 3030, 1653, 1595,
1506 cm-1.
4.1.3.16.
N-(benzo[d][1,3]dioxol-5-yl)-2-(5H-imidazo[5,1-a]isoindol-5-yl)acetamide
(11p)
Yield 65.9%. White powder. Mp 210-212 °C. 1H NMR (300 MHz, DMSO-d6) δ(ppm)
8.64-8.46 (t, J = 5.9 Hz, 1H), 7.63-7.56 (s, 1H), 7.56-7.48 (d, J = 7.5 Hz, 1H), 7.43-7.34 (d,
J = 7.6 Hz, 1H), 7.33-7.23 (m, 3H), 7.21-7.11 (ddd, J = 7.5, 6.4, 1.4 Hz, 2H), 7.09-6.99 (s,

ACCEPTED MANUSCRIPT
1H), 5.79-5.41 (dd, J = 8.8, 5.4 Hz, 1H), 4.50-4.09 (m, 2H), 3.07-2.87 (dd, J = 15.4, 5.4
Hz, 1H), 2.74-2.54 (dd, J = 15.4, 8.8 Hz, 1H). MS (EI) m/z 334.1 [M+H]+. IR (KBr) 3275,
3078, 1636, 1609, 1489, 1198, 1034 cm-1.
4.1.3.17. N-cyclohexyl-2-(5H-imidazo[5,1-a]isoindol-5-yl)acetamide (11q)
Yield 69.3%. White powder. Mp 84-86 °C. 1H NMR (300 MHz, DMSO-d6) δ(ppm)
7.98 (d, J = 7.8 Hz, 1H), 7.68 (s, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.51 (d, J = 7.6 Hz, 1H),
7.39 (t, J = 7.4 Hz, 1H), 7.28 (td, J = 7.5, 1.2 Hz, 1H), 7.14 (s, 1H), 5.60 (dd, J = 9.1, 5.2
Hz, 1H), 2.94 (dd, J = 15.1, 5.2 Hz, 1H), 1.63 (ddd, J = 64.8, 31.0, 9.4 Hz, 6H), 1.18-1.10
(m, 2H). MS (EI) m/z 296.2 [M+H]+. IR (KBr) 3273, 3032, 1653, 1616, 1506, 1260, 1034
cm-1.
4.1.3.18. 2-(5H-imidazo[5,1-a]isoindol-5-yl)-N-(3-methoxyphenethyl)acetamide (11r)
1
Yield 78.2%. Yellow powder. Mp 94-96 °C. H NMR (300 MHz, Chloroform-d)
δ(ppm) 7.67 (s, 1H), 7.52 (d, J = 7.6 Hz, 1H), 7.43-7.17 (m, 4H), 7.12 (d, J = 1.5 Hz, 1H),
6.85-6.69 (m, 3H), 5.91 (d, J = 7.2 Hz, 1H), 5.68 (dd, J = 9.6, 4.3 Hz, 1H), 3.79 (s, 3H),
3.64 (qd, J = 6.9, 3.1 Hz, 2H), 2.93 (d, J = 4.4 Hz, 1H), 2.85 (td, J = 7.5, 7.0, 2.5 Hz, 2H),
2.42 (dd, J = 15.5, 9.5 Hz, 1H). MS (EI) m/z 348.2 [M+H]+. IR (KBr) 3273, 3030, 1684,
1595, 1506 cm-1.
4.1.3.19. 2-(5H-imidazo[5,1-a]isoindol-5-yl)-N-phenethylacetamide (11s)
Yield 77.2%. White powder. Mp 167-169 °C. 1H NMR (300 MHz, Chloroform-d)
δ(ppm) 7.70 (s, 1H), 7.53 (d, J = 7.6 Hz, 1H), 7.43-7.12 (m, 10H), 5.81 (s, 1H), 5.69 (dd, J
= 9.5, 4.3 Hz, 1H), 3.65 (tt, J = 6.7, 3.3 Hz, 2H), 2.92-2.85 (m, 2H), 2.43 (dd, J = 15.5, 9.5
Hz, 1H). MS (EI) m/z 318.2 [M+H]+. IR (KBr) 3273, 3032, 1653, 1593, 1506 cm-1.
4.1.3.20. 2-(5H-imidazo[5,1-a]isoindol-5-yl)-N-(2-(pyridin-2-yl)ethyl)acetamide (11t)
Yield 85.2%. White powder. Mp 140-142 °C. 1H NMR (300 MHz, DMSO-d6) δ(ppm)
8.50 (d, J = 4.3 Hz, 1H), 8.21 (s, 1H), 7.83-7.66 (m, 2H), 7.60 (d, J = 7.5 Hz, 1H), 7.47 (d,
J = 7.6 Hz, 1H), 7.38 (t, J = 7.4 Hz, 1H), 7.25 (dd, J = 14.8, 5.8 Hz, 3H), 7.15 (s, 1H),
5.59 (dd, J = 9.3, 5.6 Hz, 1H), 3.54 (q, J = 6.5 Hz, 2H), 2.93 (td, J = 8.0, 7.2, 3.5 Hz, 2H),
2.55 (d, J = 10.0 Hz, 2H). MS (EI) m/z 319.2 [M+H]+. IR (KBr) 3273, 3030, 1668, 1593,

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1506 cm-1.
4.1.4 General preparation of intermediates 12a-b
Different substituted ethan-1-one (1 mol equiv.) was added to a solution of NaOEt
(attained from Na (2 mol equiv.) dissolved in EtOH (1.4 M) at 0 °C), followed by the
addition of intermediate 6 (1 mol equiv.). The yellow liquid was stirred at room
temperature for 3 h. The solvent was removed under reduced pressure and the saturated
NH4Cl solution was added to the mixture to quench the reaction. The mixture was diluted
with water, and was extracted with dichloromethane to afford the crude product, which
was used to the next step without any further purification.
4.1.5 General preparation of intermediates 13a-b
To a solution of 12 (1 mol equiv.) in methyl alcohol (0.2 M) was added AcOH (0.8 M)
and the mixture was stirred at 90 °C for 3 h. The mixture was allowed to cool to room
temperature and the pH was adjusted to 10 with saturated K2CO3 solution. The mixture
was evaporated in vacuo to remove methyl alcohol and was extracted with ethyl acetate to
afford the crude product that was purified by flash column chromatography on silica gel to
yield 13a-b.
4.1.5.1 1-cyclohexyl-2-(5H-imidazo[5,1-a]isoindol-5-yl)ethan-1-one (13a)
Yield 50.8%. White powder. Mp 92-94 °C. 1H NMR (300 MHz, Chloroform-d) δ(ppm)
7.66 (s, 1H), 7.58-7.53 (dt, J = 7.6, 0.9 Hz, 1H), 7.43-7.36 (m, 1H), 7.28-7.21 (m, 2H),
7.28 (s, 1H), 5.73-5.59 (dd, J = 9.5, 3.6 Hz, 1H), 3.26-3.16 (dd, J = 18.4, 3.6 Hz, 1H),
3.04-2.76 (dd, J = 18.4, 9.5 Hz, 1H), 2.49-2.24 (m, 1H), 1.97-1.59 (m, 5H), 1.40-1.19 (m,
5H).
4.1.5.2 1-(4-chlorophenyl)-2-(5H-imidazo[5,1-a]isoindol-5-yl)ethan-1-one (13b)
Yield 59.9%. White powder. Mp 106-108 °C. 1H NMR (300 MHz, Chloroform-d)
δ(ppm) 8.01-7.85 (m, 2H), 7.66-7.25 (m, 8H), 6.30 (t, J = 7.0 Hz, 1H), 3.43 (dd, J = 12.5,
6.9 Hz, 1H), 3.20 (dd, J = 12.4, 7.0 Hz, 1H).
4.1.6 General preparation of compounds 14a-b
To a solution of 13 (1 mol equiv.) in EtOH at room temperature was added

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hydroxylamine hydrochloride (3 mol equiv.) and the solution was stirred at 50 °C
overnight. The solvent was removed under reduced pressure to afford the crude product,
which was purified by gel chromatography to afford the target products.
4.1.6.1 (Z)-1-cyclohexyl-2-(5H-imidazo[5,1-a]isoindol-5-yl)ethan-1-one oxime (14a)
Yield 54.2%. White powder. Mp 126-128 °C. 1H NMR (300 MHz, DMSO-d6) δ(ppm)
8.73 (s, 1H) , 7.81 (s, 1H), 7.70 (s, 1H) , 7.26-7.47 (m, 3H), 7.25 (s, 1H), 5.63-5.66 (m,
1H), 2.98-3.13 (dd, J = 11.4, 6.8 Hz, 2H), 2.87 (s, 1H). 1.64-1.70 (m, 4H), 1.48-1.55 (m,
3H), 1.32-1.42 (m, 3H). MS (EI) m/z 296.2 [M+H]+. IR (KBr) 3647, 3033, 1595, 1506
cm-1.
4.1.6.2 (Z)-1-(4-chlorophenyl)-2-(5H-imidazo[5,1-a]isoindol-5-yl)ethan-1-one oxime
(14b)
Yield 49.9%. White powder. Mp 89-91 °C. 1H NMR (300 MHz, Chloroform-d) δ(ppm)
9.51 (s, 1H), 7.81 (s, 1H), 7.68 (s, 1H), 7.51-7.41 (m, 7H), 7.35-7.26 (m, 1H), 5.89-5.83
(m, 1H), 3.60-3.52 (dd, J = 12.5, 6.7 Hz, 1H), 3.35-3.27 (dd, J = 11.8, 6.1 Hz, 1H). MS
(EI) m/z 324.1 [M+H]+. IR (KBr) 3648, 3030, 1595, 1506 cm-1.
4.2. Biology
4.2.1. Hela cell-based enzyme assays
HeLa cells were seeded in 96-well culture plates at a density of 5x
103 per well. On the
next day, human IFN-γ (100 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 6.1 N trichloroacetic acid
and the mixture was incubated for 30 min at 50 °C. The reaction mixture
was then centrifuged for 10 minutes at 4000 rpm to remove sediments. 100 µL
of the supernatant was mixed with 100 µL of 2% (w/v) p-dimethylaminobenzaldehyde in
acetic acid and measured at 480 nm. The initial wells containing the cells in the remaining
volume of 50 µL were used to estimate cell viability in a classical MTT assay. To that end,
50 µL of culture medium (Iscove medium with 10% FCS and amino acids) were added to
the wells together with 20 µL 4 mg/mL of MTT. After 4 h of incubation at 37 oC, 200 µL

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of DMSO were added to dissolve the crystals of formazan blue and the absorbance at 570
nm was measured after overnight incubation at 37 oC.
4.2.2. Western blot analysis
HeLa cells were seeded in 6-well culture plates at a density of 2x
105 per well. On the
next day, human IFN-γ (100 ng/mL) and compounds in a total volume of 2 mL culture
medium were added to the cells. After incubation for 24 hours, the cells were collected and
washed with PBS twice. Proteins were extracted from cells by lysis buffer consisting of 50
µM Tris–HCl, pH 8.0, 50 µM KCl, 5 µM DTT, 1 µM EDTA, 0.1% SDS, 0.5% Triton
X-100 (Sunshine Biotechnology Co. Ltd.,) and protease inhibitor cocktail tablets (Roche,
Indianapolis, IN). The protein lysates were separated by 10% SDS-PAGE and
subsequently electrotransferred onto a polyvinylidene difluoride membrane (Millipore,
Bedford, MA). The membrane was blocked with 5% nonfat milk for 1 h at room
temperature. The blocked membrane was incubated with the indicated primary Abs, and
then with a horseradish peroxidase-conjugated secondary Ab. Protein bands were
visualized using Western blotting detection system according to the manufacturer’s
instructions (Cell Signaling Technology, MA).
4.2.3. Measurement of IDO1 absorbance spectra
Absorbance spectra (370-600 nm) were measured immediately after addition of
compounds (500 µM) to rhIDO1 (3 µM) in phosphate buffer (pH: 6.5) using Safire
multifunctional microplate reader. Changes in the 404 nm maxima indicated binding to the
ferric iron of the heme.
4.2.4. T cell proliferation and cytokine assay
T lymphocytes prepared from splenocytes of C57/bl6 mice were resuspended in RPMI
1640 containing 10% FBS, L-glutamate, penicillin, and streptomycin. The B16F1 cells
were treated with mitomycin C at a final concentration of 25 mg/L and then incubated at
37 °C for 30 min. After being washed three times, the B16F1 cells were resuspended in
RPMI 1640 containing 10% FBS, L-glutamate, penicillin, and streptomycin. 1×105 T

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lymphocytes (responder cells) and 2×104 mitomycin C treated B16F1 cells (stimulator
cells) were added to each well of a 96-well plate in RPMI 1640 containing 10% FBS in
the presence of ConA (5 µg/mL). Cell proliferation was quantified by MTT assay. The
cells were incubated at 37 °C and 5% CO2 for 48 h; and 10 µL MTT (4 mg/mL) was added
to each well. MTT formazan production was dissolved by DMSO replacing the medium 4
h later. The absorbance at 570 nm (OD570) was measured by a microplate reader.
Supernatant collected from the co-culture system were subjected to ELISA analysis for
IFN-γ by using kits from Dakawe (Beijing, China).
4.2.5. Treg cell experiments
Tregs were analyzed by using an eBioscience intracellular staining kit according to the
manufacturer’s instructions. After co-cultured with B16F1 cells, T cells were collected.
Surface staining was performed with a CD4- FITC and CD25- PE for 15 min at 4 °C. After
this, the cells were fixed and permeabilized with fixation buffer and permeabilization
wash buffer. The intracellular staining was performed with FOXP3- APC for 20 min. The
cells were then analyzed by flow cytometry analysis.
4.3. Molecular modeling
4.3.1. Induced fit docking
In order to consider the flexibility of both ligand and protein, the induced fit docking
(IFD) protocol in Schrödinger was employed [44]. In IFD calculations, the ligands were
first docked into the rigid receptor using softened energy function in Glide. By default, a
maximum 20 poses per ligand were retained. Then, the protein degrees of freedom for
each complex were sampled and the protein-ligand complexes were minimized. The
protein structure in each pose now reflected an induced fit to the ligand structure and
conformation. The best protein-ligand complex was then identified based on the predicted
binding affinities of the docked ligand. Here, the residues within 5 Å of each of the 20
ligand poses were subjected to a conformational search and energy minimizations, and the
residues outside this range were fixed. Finally, the minimized ligand was rigorously
redocked into the induced-fit protein structure using Glide XP scoring mode, and metal

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constraints can be applied to both Glide docking stages in IFD protocol. The choice of the
best-docked structure for each ligand was made using a model energy score that combines
the energy grid score, the binding affinity predicted by GlideScore, and the internal strain
energy for the model potential used to direct the conformational-search algorithm.
4.3.2. QM/MM geometry optimization
QM/MM calculations were performed via a two-layer Qsite program in Schrödinger
[45]. QSite is a mixed mode QM/MM program used to study geometries and energies of
structures not parameterized for use with molecular mechanics, such as those that contain
metal. The reason for this may be complicated. For example, the partial charge of the
metal cation is far from being at least approximately constant, and its value might be
affected by many factors, for instance by switching the oxidation state of the metal, the
nature of the coordinated ligands, the solvation, and/or other environmental factors.
Nevertheless, the MM simulation can be basically performed with a reasonable accuracy
for a particular coordination sphere when only well-parametrized ligands of the metal
interact with the complex surrounding. This is especially the case of a combined QM/MM
method where the metal and its closest neighborhood are calculated at the QM level. QSite
is uniquely equipped to perform QM/MM calculations because it combines the superior
speed and power of Jaguar with the recognized accuracy of the OPLS force field. Jaguar
is used for the quantum mechanical part of the calculations [46], and Impact provides the
molecular mechanics simulation [47]. The QM region is the full heme ring, the Fe, the
coordinating His residue and the inhibitor. This subsystem was optimized by employing
density functional theory (DFT) using B3LYP with the lacvp* basis set and the
convergence criterion for geometry optimizations followed the original Qsite defaults [48].
The remaining subsystem was treated with the MM method, and was geometry-optimized
by using OPLS-2005 force field. The geometry optimization convergence criterion for
MM subsystem was set as RMSD of energy gradient 60 kcal mol-1Å-1, and cutoff value
was set to 10 Å for non-bonded interactions during the QM/MM calculations. Throughout
the QM/MM calculation, the cuts between the QM and MM regions were treated as
specially parameterized frozen-orbital boundaries, which was placed between Cα and Cβ of