Design, synthesis and antitumor study of a series of N-Cyclic sulfamoylaminoethyl substituted 1,2,5-oxadiazol-3-amines as new indoleamine 2, 3-dioxygenase 1 (IDO1) inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.06.037

Indoleamine 2, 3-dioxygenase 1 (IDO1) plays a key role in tryptophan catabolism which is an important mechanism in immune tolerance. The small molecule epacadostat is the most advanced IDO1 inhibitor, but its phase III trials as a single agent or in combi

Full text of DOI:10.1016/j.ejmech.2019.06.037

Accepted Manuscript
Design, synthesis and antitumor study of a series of N-Cyclic sulfamoylaminoethyl
substituted 1,2,5-oxadiazol-3-amines as new indoleamine 2, 3-dioxygenase 1 (IDO1)
inhibitors
Shulun Chen, Wei Guo, Xiaohua Liu, Pu Sun, Yi Wang, Chunyong Ding, Linghua
Meng, Ao Zhang
PII:
S0223-5234(19)30564-1
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.037
EJMECH 11440
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 April 2019
Revised Date: 13 June 2019
Accepted Date: 13 June 2019
Please cite this article as: S. Chen, W. Guo, X. Liu, P. Sun, Y. Wang, C. Ding, L. Meng, A. Zhang,
Design, synthesis and antitumor study of a series of N-Cyclic sulfamoylaminoethyl substituted 1,2,5-
oxadiazol-3-amines as new indoleamine 2, 3-dioxygenase 1 (IDO1) inhibitors, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.06.037.
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.

ACCEPTED MANUSCRIPT
Graphic Abstract:
Design, Synthesis and Antitumor Study of a Series of N-Cyclic
Sulfamoylaminoethyl Substituted 1,2,5-Oxadiazol-3-amines as New
Indoleamine 2, 3-dioxygenase 1 (IDO1) Inhibitors
1
,3
2,3
1,3
2
2
1,3
Shulun Chen, Wei Guo, Xiaohua Liu, Pu Sun, Yi Wang, Chunyong Ding, Linghua
2
,3,*
1,3,4,*
Meng, Ao Zhang

ACCEPTED MANUSCRIPT
Highlights:
ò IDO1 plays a key role in tryptophan catabolism and in immune tolerance.
ò Epacadostat is the most advanced IDO1 inhibitor, but its phase III trial was failed.
ò A series of analogues of epacadostat was designed and further optimized.
ò 5a displayed significant potency against hIDO1 and hIDO1 expressed cancer
cells.
ò 5a was ineffective as single agent in the CT-26 syngeneic xenograft model.
ò Combination with mPD-1 showed elevated efficacy and prolonged median life
span.

ACCEPTED MANUSCRIPT
Design, Synthesis and Antitumor Study of a Series of N-Cyclic
Sulfamoylaminoethyl Substituted 1,2,5-Oxadiazol-3-amines as New
Indoleamine 2, 3-dioxygenase 1 (IDO1) Inhibitors
1
,3
2,3
1,3
2
2
1,3
Shulun Chen, Wei Guo, Xiaohua Liu, Pu Sun, Yi Wang, Chunyong Ding, Linghua
2
,3,*
1,3,4,*
Meng, Ao Zhang
1
CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica (SIMM),
Chinese Academy of Sciences, Shanghai 201203, China
2
Division of Anti-tumor Pharmacology, Shanghai Institute of Materia Medica (SIMM), Chinese
Academy of Sciences, Shanghai 201203, China.
3
University of Chinese Academy of Sciences, Beijing100049, China.
4
School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China

ACCEPTED MANUSCRIPT
Abstract
Indoleamine 2, 3-dioxygenase 1 (IDO1) plays a key role in tryptophan
catabolism which is an important mechanism in immune tolerance. The small
molecule epacadostat is the most advanced IDO1 inhibitor, but its phase III trials as a
single agent or in combinations with PD-1 antibody failed to show appreciable
objective responses. To gain more insight on the antitumor efficacy of IDO1 inhibitors,
we have designed a series of analogues of epacadostat by incorporating a cyclic
aminosulfonamide moiety as the sidechain capping functionality. Compound 5a was
found to display significant potency against recombinant hIDO1 and hIDO1
expressed HEK293 cancer cells. This compound has improved physico-chemical
properties, acceptable PK parameters as well as optimal cardiac safety. Similar to
epacadostat, 5a is ineffective as single agent in the CT-26 syngeneic xenograft model,
however, the combination of 5a with PD-1 antibody showed both elevated tumor
growth inhibition and prolonged median life span.
Keywords: Indoleamine 2,3-dioxygenase; Immunotherapy; Oxadiazole; PD-1; Life
span.

ACCEPTED MANUSCRIPT
1
. Introduction
The rapid advances in oncology immunotherapy, highlighted by checkpoint
inhibitors targeting cytotoxic T lymphocyte associated protein 4 (CTLA-4) and the
programmed death receptor 1/ligand 1 (PD-1/PD-L1), have substantially
1
-2
revolutionized our vision and treatment options on advanced human cancers.
Among which, the anti-CTLA-4 antibody (ipilimumab) and anti-PD1 antibody
pembrolizumab and nivolumab) approved by FDA, have demonstrated impressive
(
3
-5
therapeutic effects on patients. Compared to other anticancer modalities (irradiation,
chemotherapy, and targeted therapy) that exert their therapeutic effects via directly
suppressing/killing proliferating cancer cells, cancer immunotherapy can recognize
and eliminate tumor cells by restoring and even activating the patients’ native immune
6
system, thus affording better control of tumor growth with sustainable therapeutic
responses. However, clinic studies have shown that a significant majority of cancer
patients do not respond to the existing checkpoint inhibitors, and the emergence of
acquired resistance is more and more commonly observed in patients who initially
7
-8
responded. Therefore, there is a need to develop new immunotherapies either to be
used alone or in combination with existing checkpoint inhibitors.
In addition to CTLA-4 and PD-1/PD-L1, indoleamine 2, 3-dioxygenase (IDO1),
an enzyme with immunosuppressive property was found being overexpressed in a
9
wide variety of human tumors. IDO1 catalyzes the initial and rate-limiting step of
tryptophan (TRP) catabolism via the kynurenine (Kyn) pathway, an important
1
0-12
mechanism in immune tolerance
. IDO1 oxidizes the essential amino acid

ACCEPTED MANUSCRIPT
tryptophan to N-formyl kynurenine (NFK) which is further converted to Kyn and
11
other bioactive metabolites . Tryptophan depletion activates general control
non-derepressible 2 (GCN2) and represses mechanistic target of rapamycin kinase
(mTOR). Furthermore, biologically active metabolites in kynurenine pathway, such as
Kyn, activate the aryl hydrocarbon receptor (AhR). IDO1 is overexpressed in cancer
cell and the antigen presenting dendritic cell in tumor microenvironment. The cancer
cells overexpressing IDO1 can evade immune surveillance, and the transcriptional
factor AhR participates in cancer immune escape by binding to the IDO product
1
3-16
15
Kyn.
Considering the important role of IDO1 in immune tolerance , inhibition of
IDO1 has become an exciting approach to cancer immunotherapy. A number of
6
, 10, 17-25
small-molecule IDO1 inhibitors have been reported,
with a few being
6
extensively investigated in clinical trials, including 1 (epacadostat, INCB024360 ), 2
2
6
27
20
(
BMS-986205 ), 3 (navoximod, GDC-0919 ) and 4 (PF-06840003 ) (Figure 1).
Unfortunately, clinical trials of IDO1 inhibitors as monotherapy have suffered from
setbacks recently since the pronounced tumor reduction of epacadostat observed in
2
8-29
preclinical studies was not confirmed in phase I and II trials.
Alternatively,
combination regimens with other immunotherapies, chemo-therapies or
3
0-32
chemo-radiation are being extensively pursued.
Both IDO1 and PD-L1 are
co-expressed in tumor cells. A recent phase II trial demonstrated that inhibition of
PD-1 has limited activity in selected advanced soft-tissue sarcoma, which might be
associated with an immunosuppressive tumor microenvironment resulting from
3
3
macrophage infiltration and IDO1 pathway activation. In addition, the ratio of

ACCEPTED MANUSCRIPT
Kyn/tryptophan was found to be increased during PD-1 antibody treatment, indicating
that IDO1 activation might be related to the limited efficacy of PD-1/PD-L1 treatment.
Therefore, a synergistic effect is expected by combination of IDO1 inhibitors with
PD-1/PD-L1 blockades. Intriguingly, a recent phase III ECHO 301 trial testing the
combination of IDO1 inhibitor epacadostat with PD-1 antibody pembrolizumab in
melanoma failed to show superior objective responses compared to pembrolizumab
3
4
alone. Although the clinical readout from a single trial is not a definitive determinant
for the field, development of new IDO1 inhibitors and combinations with different
checkpoint inhibitors in well-defined patients together with precise trial design are
urgently needed.
To gain more insight on the tumor-suppressing effect of IDO1 inhibitors, we
recently conducted a structural modification campaign on the clinical IDO1 inhibitor
epacadostat, by cyclizing the sulfamoylamino component of the sidechain to explore
the electronic and steric effects on IDO1 activity. Herein, we report on the SAR and

ACCEPTED MANUSCRIPT
antitumor activity of these new IDO1 inhibitors both as monotherapy and in
combination with PD-1 antibody blockade.
2
. Results and discussion
2
.1. Structural analysis and new inhibitor design
6
1
,2,5-Oxadiazole 1 , developed by Incyte Corp is the most advanced IDO1
inhibitor in clinical trials. It has moderate inhibitory potency (IC , 73 nM) against
5
0
IDO1 enzyme and high inhibitory potency in HeLa cells (IC , 7.4 nM). The
5
0
3
5
co-crystal structure of hIDO1 in complex with 1 (PDB code: 5WN8) shows that the
oxime oxygen forms the key hexa-coordination with the heme iron while the
halogenated phenyl group forms a favorable fluorine–sulfur contact with C129 and
occupies the hydrophobic pocket A situating above the heme. The sulfamoylamino
ethylene sidechain projects out to reach the pocket B. The central 1,2,5-oxadiazole
moiety is stabilized by F163, L234, and F226. Although compound 1 shows
significant inhibitory effects on IDO1 catalytic activity, and appreciable tumor growth
inhibition in immunocompetent C57BL/6 mouse syngeneic models of several cancer
6
,36
types, its chemical structure lacks optimal drug-like properties (e.g. the number of
H-bond donor/acceptor >10; PSA > 140 Å) due to the co-existence of several polar
functional groups including hydroxamidine and sulfamoylamine. A close examination
of the interaction of the sulfamoylamino ethylene sidechain suggests that the proposed
allosteric pocket B is not fully occupied and the secondary NH in the sulfamoylamine
component is not involved in the H-bonding network (Figure 2). In this regard, we
decide to cyclize the sulfamoylamine component aiming to fully occupy the pocket B

ACCEPTED MANUSCRIPT
and to balance the PK properties by adjusting electronic and steric effects of the cyclic
congeners. In the meantime, the polar hydroxamidine functionality is left intact to
maintain the key intramolecular H-bonding network that forces the amidine C=N
double bond to hold a cis conformation (Figure 2).
2
.2. Structure-activity relationship analysis
All the new compounds were assayed for their activity against hIDO1 enzyme and
active compounds were further tested for their activity in HEK293 cells
over-expressing hIDO1. The clinical compound 1 was used as comparison. As shown
in Table 1, we first evaluated a series of analogues bearing a five- or six-membered
cyclic aminosulfonamide as the capping group of the sidechain. Compared to the
parent compound 1, compound 5a, formed by directly cyclization of the
sulfamoylamine moiety of 1 into a five-membered heterocycle, showed slightly lower
but still compatible potency against hIDO1 in both biochemical and cellular assays
with IC values of 62.5 and 28.2 nM, respectively. Further masking the terminal NH
5
0

ACCEPTED MANUSCRIPT
with a methyl group yielded compound 5b, showing nearly the same potency as that
of 5a. These results suggested that both the NH and NH groups in the
2
aminosulfonamide component of 1 are not substantially requested for the interaction
with hIDO1. However, a larger six-membered analog 5c lost activities against the
recombinant hIDO1 with an IC greater than 1 µM, suggesting that the size of the
5
0
heterocycle and the steric effect of the capping group are crucial to interact with
hIDO1. Interestingly, compound 5d lacking the cyclic NH moiety of 5a showed even
higher potency in both biochemical and cellular assays with IC values of 46.5 and
5
0
2
1.4 nM, respectively. This compound is slightly more potent than the reference
compound 1 (62.6 and 28.2 nM, respectively in both assays), though the difference is
not statistically significant. Replacing the sulfonyl group in 5a by carbonyl group led
to compound 5e, completely lost the activity against hIDO1. However, the analog 5f
retained good potency in both biochemical and cellular assays with IC values of
5
0
7
1.5 and 16.8 nM, respectively. Compared to the parent compound 1, the retained
good potency of 5d and 5f suggested that the H-bonding donor in the sidechain
terminal group is not necessary, which may benefit the PK-related physico-chemical
properties. This analysis was further confirmed by the isoxazolidinone 5g and
dihydroisoxazole 5f, both retained reasonable potency in both assays. The phenyl
fused cyclic analogs 5i and 5j were inactive to hIDO1, likely due to their steric and
electronic
effects.
Interestingly,
compound
6
bearing
a
4
-methoxycyclobut-3-ene-1,2-dion-3-amino moiety as the sidechain capping group
also retained moderate potency against hIDO1, only 3.3-fold less potent than 1 (138
vs 40.9 nM).
Table 1.
a
Biochemical and cellular potency of cyclic aminosulfonamides as new IDO1 inhibitors

ACCEPTED MANUSCRIPT
F
OH
Br
N
H
R
NH
N
N
N
O
IC (nM)
5
0
Compound
1
R
hIDO-1
HEK293
40.9
12.6 ± 1.0
5
a
62.5
56.2
28.2 ± 2.2
33.0 ± 0.8
5
b
b
5
c
>1000
46.5
NT
5
d
21.4 ± 0.8
O
b
5
e
>1000
71.5
NT
HN
N
5
f
16.8±2.0
O
5
5
g
115
72.9 ± 4.9
N
O
h
94.5
817
73.2 ± 1.4
b
5
i
NT
b
5
j
>1000
NT

ACCEPTED MANUSCRIPT
b
6
138
NT
a
b
The IC values are obtained from two separate experiments. NT = not tested.
5
0
Starting from cyclic aminosulfonamide 5a, we next screened a small series of
aryl groups with different substitution patterns to determine the electronic and steric
tolerance of the pocket A. As shown in Table 2, the 4-fluoro group of phenyl is critical
for IDO1 interaction and other substituents (e.g. OCH CF in 7a, SO CH CH in 7b,
2
3
2
2
3
SeCH CH in 7c) completely abolished the activity against IDO1 with IC values
2
3
50
greater than 1 µM. Keeping the 4-fluoro intact and replacement of the 3-bromo with
chloro or introducing an additional halogen atom on the 5-position of the phenyl led
to compounds 7d-f, all showing much reduced potency against hIDO1 with IC value
5
0
ranging between 112 nM to >1 µM.
Table 2.
a
The hIDO1 activity of cyclic aminosulfonamides with different aryl substituents
1
2
3
Compound
R
R
R
hIDO-1 IC (nM)
50
5
7
a
a
Br
Br
F
H
H
62.5
OCH
SO CH
SeCH
CF
CH
CH
>1000
>1000
>1000
112
2
3
7
b
Br
Br
Cl
Cl
2
2
3
H
H
7
c
2
3
7
d
F
F
H
7
e
Cl
304

ACCEPTED MANUSCRIPT
7
f
Br
F
Br
>1000
a
The IC values are obtained from two separate experiments.
5
0
We also used a few alkyl linkers bearing different length and steric effect to
replace the ethylene linkage in cyclic aminosulfonamide 5a. As shown in Table 3,
extending the ethylene linker in 5a to a three-methylene linker in 8a led to 3~ 4-fold
reduction in the potency against hIDO1 with an IC50 value of 217 nM. Further
reduced activity was observed for the steric linker as in 8b and 8c. The 3-fold higher
potency for 8c over 8b (221 vs 696 nM) further confirms the ethylene unit as the
optimum linkage.
Table 3.
a
The hIDO1 enzymatic activity of cyclic aminosulfonamides with different linkers
Compound
R
hIDO-1 IC (nM)
5
0
5
8
a
a
62.5
217
8b
696
8c
221
a
The IC values are obtained from two separate experiments.
5
0
Since no improvement was achieved by modifying the linker and the aryl
substituents, we then optimized the N-substituent of the cyclic aminosulfonamido
component in 5a. As shown in Table 4, we first introduced a series of water-soluble
groups as N-substituents. Compared to 5a, N-(2-MeO)ethyl substituted compound 9a

ACCEPTED MANUSCRIPT
showed slightly lower potency (78.5 vs 62.5 nM) against hIDO1 in the biochemical
assay, whereas the potency in IDO1-expressed HEK293 cells was slightly improved
(22.2 vs 28.2 nM). One methylene longer N-substituent in 9b led to 6-fold reduction
in potency. N-(2-OH)-ethyl substituted 9c showed nearly the same potency as 5a in
both biochemical and cellular assays with IC50 values of 74.5 and 26.6 nM,
respectively. No potency gain for the PEGlated analogue 9d (284 nM). Reduced
potency was observed as well for the N-((3-(hydroxymethyl)oxetan-3-yl)methyl)
analogue 9e (322 nM). Basic substituents as in 9f and 9g were introduced aiming to
increase the aqueous solubility. Compared to 5a, compound 9f bearing dimethylamino
ethyl as the N-substituent showed a 4-fold reduction in potency, whereas the
morpholin-4-ylethyl substituted analogue 9g showed compatible potency in both
biochemical and cell assays with IC values of 83.3 and 28.7 nM, respectively.
5
0
Various aryl, heteroaryl and alkyl substituents were also tested and the corresponding
compounds 9h-k were 4- to 11-fold less potent than 5a. The N-ethyl substituted
analogues 9l and 9m bearing a terminal cabamoyl moiety also showed reduced
potency against hIDO1.
Table 4.
N-Substituted cyclic aminosulfonamides as new IDO1 inhibitors
IC (nM)
5
0
Compound
1
R
hIDO-1
40.9
HEK 293
12.6 ± 1.0
28.2 ± 2.2
/
5
a
H
62.5
9
a
78.5
22.2 ± 6.2

ACCEPTED MANUSCRIPT
a
9
b
461
NT
9
c
74.5
284
26.6 ± 4.5
a
9
d
NT
a
9
e
322
NT
9
f
221
45.7 ± 8.3
28.7 ± 5.0
9
g
83.3
a
9
h
657
399
NT
a
9
i
NT
a
9
j
457
259
767
NT
a
9
k
NT
F
a
N
O
9
l
NT
O
a
9
m
354
NT
a
NT = not tested. The IC values are obtained from two separate experiments.
5
0
2
.3. hERG potassium channel test
By balancing the biochemical and cellular potency as well as the structural diversity,
compounds 5a, 5d, 5f and 9a were selected as the representative leads of
corresponding subseries for further evaluation (Table 5). Compared to the parent
compound 1, all these new compounds showed similar potency, but more favorable
polar surface area thus matching Veber’s permeability rules (tPSA <140 Å). Since
these compounds have high loading of N-atom, compared to most clinically approved

ACCEPTED MANUSCRIPT
drugs, we then tested their inhibitory effects against the hERG potassium channel to
exclude the potential effects on the cardiac arrhythmia. Fortunately, all these
compounds showed IC values greater than 15 µM, indicating their optimal cardiac
5
0
safety profile.
Table 5.
Inhibitory effects of new IDO1 inhibitors against hERG channel
Compound
hERG, IC (µM)
tPSA (Å)
5
0
X =
1
>40
162.8
140.0
128.0
5
a
>40
5
d
17.27
31.12
16.63
5
f
120.1
140.4
9
a
2
.4. Pharmacokinetic study
To further evaluate the developable potential of these new IDO1 inhibitors, the
pharmacokinetic profile (PK) of the four new compounds was tested in SD rats dosed
iv (1 mg/kg) and orally (3 mg/kg). As shown in Table 6, 5d and 9a showed poor oral
bioavailability of 6.55% and 1.21%, respectively. However, 5a showed a good oral
6
bioavailability of 22.6% (reported data for 1 : F(rat) = 11%). 5f also displayed an

ACCEPTED MANUSCRIPT
acceptable oral bioavailability of 13.9%. Both 5a and 5f showed moderate plasma
clearance (40.3 and 51.0 mL/min/kg), respectively.
Table 6.
a
Pharmacokinetic profile of four compounds in SD rats
T1/2
AUC
CLobs
Vssobs
Compound
（h）
(h*ng/mL)
F(%)
(
mL/min/kg)
(ml/kg)
AUClast
AUCINF_obs
356
p.o.
i.v
1.94
1.49
275
406
-
-
5
a
22.6
415
60.5
311
142
330
6.52
40.3
4295
p.o.
i.v
1.58
0.575
1.79
1.18
1.91
58.1
296
137
329
2.59
-
54.6
-
-
3016
-
5
d
6.55
13.9
1.21
p.o.
i.v
5
f
51.0
-
2557
-
p.o.
9
a
i.v
0.617
71.2
75.6
225
9835
a
Values are the average of three runs. Vehicle: PO: DMSO/0.5%HPMC (5/95, v/v); iv:
EtOH/PEG300/NaCl (10/40/50, v/v/v). CL, clearance; Vss, volume of distribution; T , half-life;
1
/2
AUC, area under the plasma concentration time curve; F, oral bioavailability.
2
.5. Antitumor activity of compound 5a in CT-26 murine xenograft model
On the basis of its high potency against hIDO1, and acceptable PK parameters,
compound 5a was further investigated in vivo for its antitumor efficacy both as single
agent and in combination with mPD-1 antibody (mouse PD-1 antibody, BioXCell,
3
7
Cat# BE0146)
in a CT26 xenograft model in immunocompetent mice.
Immunocompetent mice were treated with vehicle, compound 5a (100 mg/kg, BID),
clinical compound 1 (100 mg/kg, BID), anti-mPD-1 (10 mg/kg, BIW) or a
combination of 5a and antibody. As shown in Table 7 and Figure 3, treatment with 1
or 5a alone showed marginal inhibition on tumor growth (TGI =19.4% and 7.3%,

ACCEPTED MANUSCRIPT
respectively) indicating that monotherapy of either the clinical drug 1 or our new
inhibitor 5a is ineffective. Treatment with anti-mPD-1 showed moderate antitumor
efficacy (TGI = 37.0%), which is better than both small molecules 1 and 5a. As
expected, combination of 5a with the anti-PD1antibody significantly enhanced the
efficacy of the monotherapy, with a TGI of 47.4%. Moreover, no significant change in
body weight was observed in combinatorial group compared to each monotherapy
group during the 14-day treatment.
Table 7.
Inhibition of compound 5a on tumor growth in the CT-26 syngeneic model
Tumor Size
b
T/C
TGI
p value
3
a
Treatment
(mm )
c
(
one-way ANOVA)
at PG-D14
(%)
(%)
Vehicle
1
1557±256
--
--
--
1265±115
81.2
19.4
0.317
mPD-1
999±247
1447±55
842±148
64.2
92.9
54.1
37.0
7.3
0.140
0.681
0.030
5
a
5
a+mPD-1
47.4
a
Mean ± SEM.
b
Tumor growth inhibition is calculated by dividing the average tumor volume in the treated group
by that in the vehicle control group (T/C).
c
p value was calculated with one-way-ANOVA by comparing treated group with vehicle group.

ACCEPTED MANUSCRIPT
Figure 3. Tumor growth curve and change in body weights in female BALB/c mice bearing CT-26
xenografts. Data points represent mean of each group, error bars represent standard error of the mean
(
SEM).
Meanwhile, as shown in Table 8 and Figure 4, we further gauged the median
survival time (MST) of each treatment group. It was found that mice treated with both
small-molecule IDO1 inhibitors 1 and 5a showed similar survival time to those in

ACCEPTED MANUSCRIPT
control group (~20 d), while the mice treated with anti-PD1antibody showed a
slightly longer MST of 24 d. Significantly improved MST (> 26 d) was observed in
mice in the combination groups (P< 0.001) with the ratio of increase in life-span
(
ILS) > 30%, indicating that combination of 5a and anti-mPD-1 not only enhanced
the antitumor efficacy and also extended the survival time of tumor-bearing mice.
Table 8.
The effects of 5a or/and mPD-1 on the survival of tumor bearing mice
c
p value
ILS (%) (Kaplan-Meier
method)
MST
(
d
Group
Treatment
Vehicle
Mean (days)
days)
a
1
20.5 (16~24)
20
－
－
a
b
b
2
3
4
1
mPD-1
5a
21.5 (20~24)
20
24
20
0
20
0
0.754
0.036
0.744
a
23.5 (18~>26)
a
b
20.25 (18~22)
a
b
5
5a+mPD-1
25.25 (22~>26)
>26
>30
0.001
a
The range of survival times.
b
p value when comparing each group with Group 1.
c
The survival of all animals was followed and median survival time (MST) was calculated for
each group.
d
The increase in life-span (ILS) was calculated by dividing the MST of treatment group by the
MST of the control group and was expressed as the percent increase over the life-span of animals
in the control group.
3
Figure 4. Survival curve. Mouse was euthanized when its tumor volume reached 3000 mm separately.

ACCEPTED MANUSCRIPT
3
. Chemistry
The procedures to synthesize all new compounds were outlined in Schemes 1-4.
First, commercially available 2-(Boc-amino)ethyl bromide (10) was used as the
starting material and reacted with different cyclic fragments in the presence of cesium
carbonate to afford 11a-e and 11g-j in 76-84% yields, respectively (Scheme 1).
Bromide 10 was reacted with 3-isoxazolidinone to afford two isomers 11g and 11h.
Removal of the Boc-protecting group in 11a-d and 11f-j with trifluoroacetic acid
(TFA) delivered the corresponding amines 12a-d and 12f-j. Oxidation of the key
6
intermediate 13 with hydrogen peroxide and TFA gave intermediate 14 in 52% yield.
Treatment of amines 12a-j with 14 in the presence of 2.5 N NaOH produced target
6
compounds 5a-j in 25-30% overall yields (two steps). In addition, treatment of 15
with 3, 4-dimethoxy-3-cyclobutene-1,2-dione followed by deprotection with 2.5 N
NaOH solution afforded the final product 6 in 22% overall yield.

ACCEPTED MANUSCRIPT
The synthesis of compounds 7a-f was described in Scheme 2. Substitution of
fluorobenzene 16 with different nucleophiles afforded nitrobenzenes 17a-c in 72-91%
yields. Reduction of 17a-c with iron powder and ammonium chloride yielded anilines
3
8
1
8a-c in 74-88% yields. Condensation of 19 with differently substituted anilines
delivered oxime derivatives 20a-f in 68-80% yields. Protection of oxime derivatives
0a-f in the presence of CDI provided 1,2,4-oxadiazol-5(4H)-ones 21a-f in 98%
2
yields. Oxidation of 21a-f with hydrogen peroxide and TFA was found sluggish and
yielded nitro compounds 22a-f in 15-28% yields. It has to be mentioned that sulfane

ACCEPTED MANUSCRIPT
2
1b was oxidized to sulfone 22b during the reaction process. Amine 12a was reacted
with 22a-f in the presence of 2.5 N NaOH solution to afford target compounds 7a-f in
2
5-30% overall yields.
3
9-40
As shown in Scheme 3, substitution of alcohols 23b-c
with methanesulfonyl
chloride (MsCl) generated 24b-c in 65-72% yields. Reaction of tert-butyl
4
1
1
,2,5-thiadiazolidine-2-carboxylate 1,1-dioxide (25) with different linkers led to
compounds 26a-c in 32-72% yields. After N-deprotection with TFA of 26a-c,
subsequent reaction with the intermediate 14 in the presence of 2.5 N NaOH solution

ACCEPTED MANUSCRIPT
afforded the final compounds 8a-8c in 21-33% overall yields (two steps).
As shown in Scheme 4, N-benzylethane-1,2-diamine (27) was refluxed with
sulfamide in pyridine to provide heterocycle 28 in 72% yield. Substitution of 28 with
tert-butyl N-(2-bromoethyl)carbamate delivered the aminoethyl compound 29 in 75%
yield. Removal of the benzyl group in 29 through catalytic hydrogenation afforded the
key intermediate 30 in 87% yield. Compounds 32a-g and 32i-k were prepared from
3
0 via substitution reaction in the presence of cesium carbonate in 37-51% yields,
respectively. Substitution of 30 with 1,2-dibromoethane resulted in the intermediate
1 in 45% yield. Compounds 32l and 32m were synthesized by nucleophilic
3
displacement from bromide 31 in the presence of cesium carbonate in 37% and 51%
yields, respectively. Removal of the N-Boc protective group in 32a-g, 29 and 32i-m
with TFA followed by reaction with the intermediate 14 in the presence of 2.5 N
NaOH solution afforded target compounds 9a-m in 16-43% overall yields (two steps).

ACCEPTED MANUSCRIPT
4
. Conclusion
PD-1/PD-L1, CTLA-4, and IDO1 are the frontier oncology immuno-therapeutic
targets. However, unlike the cell-surface checkpoint receptor molecules that can be
effectively targeted by antibody-based inhibitors, IDO1 or its downstream effectors
are intracellular targets that are ideally targeted by small molecules. Epacadostat is the
most advanced IDO1 inhibitor, but its clinical trial as monotherapy is unsuccessful.
Recent failure of its combination with PD-1 antibody blockade in phase III trial in
melanoma further deterred many other clinical trials with IDO1 inhibitors. To gain

ACCEPTED MANUSCRIPT
more insight on the antitumor efficacy of IDO1 inhibitors, we have designed a series
of analogues of epacadostat by incorporating a cyclic aminosulfonamide moiety as the
sidechain capping functionality. Subsequent multidimensional optimization of the
heterocyclic skeleton, N-substituent as well as the linker led to identification of
compound 5a showing good potency compatible to that of epacadostat against hIDO1
and IDO1-expressing HEK293 cells. This compound has improved physico-chemical
properties, acceptable PK parameters as well as optimal cardiac safety. Furthermore,
in the CT-26 syngeneic xenograft model, treatment with our new IDO1 inhibitor 5a or
the clinical drug epacadostat produced marginal antitumor activity in 14 d - treatment
regimen, which is not statistically different from that of the vehicle group. However,
the combination of 5a with PD-1 antibody produced elevated antitumor activity.
Moreover, the life span of mice was also significantly extended in the combination
treatment group. Combining the outcomes of epacadostat and our only inhibitor 5a, it
might be more reasonable to pursuit the overall benefits of IDO1 inhibitors in a much
broader vision rather than only focusing on antitumor efficacy, for example, the
median survival time of tumor patients.
5
. Experimental
5
5
.1. Chemistry.
.1.1. General methods
All reactions were performed in glassware containing a Teflon coated stir bar.
Commercial solvents and reagents were obtained from sources Adamas-beta, Acros

ACCEPTED MANUSCRIPT
Organics, Bidepharm, Alfa Aesar, J&K, TCI, and Accela and used without further
1
13
purification. H and C NMR spectra were recorded with a Varian-MERCURY Plus
00, 400 or 500 MHz NMR spectrometer and referenced to deuterium dimethyl
sulfoxide (DMSO-d ), deuterium acetonitrile (CD CN), deuterium chloroform
3
6
3
(
CDCl ). Chemical shifts (δ) were reported in ppm downfield from an internal TMS
3
standard. Low- and high-resolution mass spectra were obtained in the ESI mode.
Flash column chromatography on silica gel (200−300 mesh) was used for the routine
purification of reaction products. The column outputs were monitored by TLC on
silica gel (200−300 mesh) precoated on glass plates (15 mm × 50 mm), and spots
were visualized by UV light at 254 or 365 nm. Some outputs were colored by basic
KMnO solution or ninhydrin regent. HPLC analysis was conducted for all
4
biologically evaluated compounds on an Agilent Technologies 1260 series LC system
with ultraviolet wavelengths in UV 254 to determine the chemical purity and optical
purity. The purities of all biologically evaluated compounds were above 95%.
5
.1.2. Synthesis of new IDO inhibitors
N-(3-Bromo-4-fluorophenyl)-4-((2-(1,1-dioxido-1,2,5-thiadiazolidin-2-yl)ethyl)
amino)-N'-hydroxy-1,2,5-oxadiazole-3-carboximidamide (5a). To the mixture of
the precursor 10 (2.26 g, 10.1 mmol, 1.5 equiv) and tert-butyl
1
,2,5-thiadiazolidine-2-carboxylate 1,1-dioxide (1.50 g, 6.75 mmol, 1.0 equiv) in dry
DMF (10 mL), was added Cs CO (6.60 g, 20.25 mmol, 3.0 equiv). The mixture was
2
3
stirred at room temperature overnight, and then extracted with ethyl acetate. The
organic phase was washed with saturated NaHCO and brine, dried over anhydrous
3

ACCEPTED MANUSCRIPT
Na SO , filtered and concentrated. The resulting residue was purified via silica gel
2
4
1
chromatography to give 11a as colorless oil (1.96 g, 79%). H NMR (300 MHz,
CDCl ) δ 4.89 (br, 1H), 3.80 (t, J = 6.5 Hz, 2H), 3.47 – 3.34 (m, 4H), 3.18 (t, J = 6.1
3
Hz, 2H), 1.54 (s, 9H), 1.44 (s, 9H).
The obtained compound 11a (730 mg, 2 mmol, 1.0 equiv) dissolved in DCM (5
mL). TFA (5 mL) was added and the mixture was stirred at room temperature until the
starting material was consumed completely. The solvent was removed under vacuum
to afford intermediate 12a, which was employed for use without further purification.
To the mixture of 13 (2.0 g, 5.85 mmol, 1.0 equiv) and TFA (23 mL), was added
o
3
0% hydrogen peroxide solution (15 mL). The reaction mixture was heated at 45 C
for 2 days. Ethyl acetate (20 mL) and saturated NaS O solution (50 mL) were added,
2
3
and the mixture was allowed to stir for 20 min. Additional ethyl acetate was added
and the reaction mixture was partitioned between ethyl acetate and water. The organic
phase was washed with water and brine, then dried over anhydrous Na SO , filtered
2
4
and concentrated. The resulting residue was further purified via silica gel
1
chromatography to give 14 as a yellow solid (1.12 g, 52%). H NMR (400 MHz,
CDCl ) δ 7.65 – 7.55 (m, 1H), 7.30 – 7.20 (m, 2H).
3
The intermediate 12a (42 mg, 0.25 mmol, 1.0 equiv) and 14 (93 mg, 0.25 mmol,
1
.0 equiv) were dissolved in THF (5 mL). 2.5 N NaOH solution (0.5 mL) was added
and the mixture was stirred at room temperature for 3 h. After extraction with ethyl
acetate, the combined organic phase was washed with water and brine, dried over
anhydrous Na SO , filtered and then concentrated. The resulting residue was purified
2
4

ACCEPTED MANUSCRIPT
via silica gel chromatography to give target compound 5a as white solid (29 mg,
1
2
5%). H NMR (400 MHz, CD CN) δ 9.08 (s, 1H), 7.51 (s, 1H), 7.25 (dd, J = 6.0, 2.5
3
Hz, 1H), 7.12 (t, J = 8.7 Hz, 1H), 6.99 – 6.92 (m, 1H), 6.07 (t, J = 7.6 Hz, 1H), 5.21 (s,
H), 3.54 (dd, J = 12.3, 6.2 Hz, 2H), 3.44 (dt, J = 11.5, 6.0 Hz, 4H), 3.25 (t, J = 6.1
1
13
Hz, 2H). C NMR (126 MHz, CD CN) δ 156.80, 156.57 (d, J = 241.9 Hz), 142.26,
3
1
1
40.18, 138.16 (d, J = 3.8 Hz), 128.51, 124.81 (d, J = 7.6 Hz), 116.87 (d, J = 22.7 Hz),
-
08.43 (d, J = 21.4 Hz), 50.48, 46.17, 43.33, 40.80. ESI-MS: m/z 462.3 [M-H] ;
-
HRMS-ESI: m/z [M-H] calcd for C H BrFN O S: 462.0001, found: 462.0006.
1
3
14
7
4
Compounds 5b-j were synthesized according to a similar protocol as that
described for 5a.
N-(3-Bromo-4-fluorophenyl)-N'-hydroxy-4-((2-(5-methyl-1,1-dioxido-1,2,5-th
iadiazolidin-2-yl)ethyl)amino)-1,2,5-oxadiazole-3-carboximidamide (5b). Yellow
1
foam (30%). H NMR (400 MHz, CD CN) δ 9.14 (s, 1H), 7.48 (s, 1H), 7.21 (dd, J =
3
6
3
.0, 2.7 Hz, 1H), 7.08 (t, J = 8.7 Hz, 1H), 7.02 – 6.88 (m, 1H), 6.03 (t, J = 6.0 Hz, 1H),
13
.50 (q, J = 6.1 Hz, 2H), 3.41 – 3.34 (m, 2H), 3.29 – 3.23 (m, 4H), 2.65 (s, 3H). C
NMR (126 MHz, CD CN) δ 155.86, 155.62 (d, J = 241.9 Hz), 141.31, 139.26, 137.23
3
(d, J = 2.5Hz), 127.54, 123.83 (d, J = 6.3 Hz), 115.93 (d, J = 23.9 Hz), 107.49 (d, J =
-
2
2.7 Hz), 47.46, 46.17, 45.78, 42.20, 33.29. ESI-MS: m/z 476.4 [M-H] ; HRMS-ESI:
-
m/z [M-H] calcd for C H BrFN O S: 476.0157, found: 476.0165.
1
4
16
7
4
N-(3-Bromo-4-fluorophenyl)-N'-hydroxy-4-((2-(6-methyl-1,1-dioxido-1,2,6-th
iadiazinan-2-yl)ethyl)amino)-1,2,5-oxadiazole-3-carboximidamide (5c). Colorless
1
foam (28%). H NMR (400 MHz, CD CN) δ 9.12 (s, 1H), 7.51 (s, 1H), 7.24 (dd, J =
3