Design, synthesis, and biological evaluation of 1,2,5-oxadiazole-3-carboximidamide derivatives as novel indoleamine-2,3-dioxygenase 1 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112059

Indoleamine 2,3-dioxygenase 1 (IDO1) is the enzyme catalyzing the oxidative metabolism of tryptophan, which accounts for cancer immunosuppression in tumor microenvironment. Several compounds targeting IDO1 have been reported and epacadostat shows strong i

Full text of DOI:10.1016/j.ejmech.2020.112059

Journal Pre-proof
Design, synthesis, and biological evaluation of 1,2,5-oxadiazole-3-carboximidamide
derivatives as novel indoleamine-2,3-dioxygenase 1 inhibitors
Xiaohan Song, Pu Sun, Jiang Wang, Wei Guo, Yi Wang, Ling-hua Meng, Hong Liu
PII:
S0223-5234(20)30026-X
DOI:
https://doi.org/10.1016/j.ejmech.2020.112059
EJMECH 112059
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 October 2019
Revised Date: 16 December 2019
Accepted Date: 8 January 2020
Please cite this article as: X. Song, P. Sun, J. Wang, W. Guo, Y. Wang, L.-h. Meng, H. Liu, Design,
synthesis, and biological evaluation of 1,2,5-oxadiazole-3-carboximidamide derivatives as novel
indoleamine-2,3-dioxygenase 1 inhibitors, European Journal of Medicinal Chemistry (2020), doi: https://
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Title:
Design, synthesis, and biological evaluation of
1
,2,5-oxadiazole-3-carboximidamide derivatives as novel
indoleamine-2,3-dioxygenase 1 inhibitors

Graphical Abstract
*
**

Highlights:
‹
‹
‹
Novel 1,2,5-oxadiazole-3-carboximidamide derivatives as IDO1 inhibitors were
designed, synthesized, and evaluated.
The structure-activity relationship (SAR) of this novel series of 27 compounds
was demonstrated.
Compound 23, 25 and 26 demonstrated potent in vitro inhibitory activity against
hIDO1 (IC = 108.7, 178.1 and 139.1 nM respectively) and compound 25
5
0
showed improved PK profiles (t1/2 = 3.81 h, F = 33.6 %) compared with
epacadostat.
‹
Compound 25 exhibited the similar anti-tumor efficacy with epacadostat without
inducing significant change in body weight compared to the control group.

Design, synthesis, and biological evaluation of
,2,5-oxadiazole-3-carboximidamide derivatives as novel
indoleamine-2,3-dioxygenase 1 inhibitors
1
†
,‡,
║
#,‡,
║
†,‡
#,‡
#
Xiaohan Song,
Pu Sun,
Jiang Wang, Wei Guo, Yi Wang, Ling-hua Meng,
*,#,‡
*,†,‡
and Hong Liu
†
State Key Laboratory of Drug Research and CAS Key Laboratory of Receptor
Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555
Zu Chong Zhi Road, Shanghai, 201203, China.
#
Division of Anti-Tumor Pharmacology, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, 501 Haike Road, Shanghai, 201203, China.
‡
University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049,
China.
║These authors contributed equally to this study.
Corresponding Authors
*
*
H. Liu: Phone, +86-21-50807042; E-mail, hliu@simm.ac.cn.
L. Meng: Phone, +86-21-50805315; E-mail, lhmeng@simm.ac.cn

Abstract:
Indoleamine 2,3-dioxygenase 1 (IDO1) is the enzyme catalyzing the oxidative
metabolism of tryptophan, which accounts for cancer immunosuppression in tumor
microenvironment. Several compounds targeting IDO1 have been reported and
epacadostat shows strong inhibitory activity against IDO1, which is further studied in
clinic trails. However, its pharmacokinetic profiles are not satisfactory. The half-life
of epacadostat is 2.4 h in human and dosage is 50 mg BID in the phase III clinic trial.
To overcome the shortcomings of epacadostat, structure-based drug design was
performed to improve the pharmacokinetic profiles via changing the metabolic
pathway of epacadostat and to enhance anti-tumor potency. A novel series of
1
,2,5-oxadiazole-3-carboximidamide derivatives bearing cycle in the side chain were
designed, synthesized, and biologically evaluated for their anti-tumor activity. Most
of them exhibited potent activity against hIDO1 in enzymatic assays and in HEK293T
cells over-expressing hIDO1. Among them, compound 23, 25 and 26 showed
significant inhibitory activity against hIDO1 (IC = 108.7, 178.1 and 139.1 nM
50
respectively) and in HEK293T cells expressing hIDO1 (cellular IC = 19.88, 68.59
5
0
and 57.76 nM respectively). Moreover, compound 25 displayed improved PK
property with longer half-life (t1/2 = 3.81 h in CD-1 mice) and better oral
bioavailability (F = 33.6%) compared with epacadostat. In addition, compound 25
showed similar potency to inhibit the growth of CT-26 syngeneic xenograft compared
to epacadostat, making it justifiable for further investigation.
Keywords: IDO1 inhibitors, immunotherapy, anti-tumor

INTRODUCTION
Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing protein catalyzing
the oxidative metabolism of tryptophan to produce N-formylkynurenine [1, 2]. This
reaction is the rate limiting step in the kynurenine pathway of tryptophan. It has been
reported that tumor cells and immune cells that constitute the tumor
microenvironment overexpress IDO1, which is considered to be one of the key factors
that result in cancer immunosuppression [3, 4]. Tumor cells and suppressive immune
cells could deplete tryptophan that is essential for the T cell differentiation in the
tumor microenvironment [5]. As a result, tryptophan depletion activates GCN2 kinase
and induces the mid-G1 cell cycle arrest in T cells. However, tumor cells are able to
maintain proliferation in the presence of low level of tryptophan [6]. Moreover,
accumulation of IDO metabolites also inhibits the function and proliferation of T cell
in the tumor microenvironment [7]. For example, kynurenine is a metabolite of
tryptophan and it also serves as a ligand of the aryl hydrocarbon receptor (AhR)，
which in turn promotes the differentiation of Tregs and suppresses antitumor immune
responses [8]. As high expression of IDO1 is associated with poor prognosis in
varieties of cancer types, and its important role in immune tolerance, a lot of research
has been done towards IDO1 and experimental results indicate that IDO1 inhibition
could restore the immune responses in the tumor microenvironment and improve the
therapeutic effects of tumor treatment in combination with immune checkpoint
inhibitors [5, 9-14].
To date, several types of IDO1 inhibitors, such as epacadostat [15], navoximod
[
16], BMS-986205 [17] and PF-06840003 [18], have been reported and tested in
clinic trials. Compounds 5 [19] and 6 [20] were also proved to be effective IDO1
inhibitors tested in the in vivo assays. However, the development of IDO1 inhibitors
suffered a setback that the phase III clinical trial of epacadostat in combination with
keytruda for advanced melanoma treatment did not reach the primary end point in
2
018 [21]. Therefore, pharmaceutical companies reappraise their clinical trials and try
to look for a suitable treatment strategy for IDO1 inhibitors. Although the clinical

outcome from a single trail is not the definitive determinant for the field, further
exploration for the role of IDO1 serving in the process of tumor immune escape
should be done and the development of novel IDO-1 inhibitors is highly needed.
Figure 1. Representative structures of IDO1 inhibitors
Although epacadostat is a well-developed IDO1 inhibitor, its pharmacokinetic
profile is not satisfactory. Herein, a series of 1,2,5-oxadiazole-3-carboximidamide
derivatives bearing cycle in the side chain were synthesized and evaluated to improve
the half-life and oral bioavailability of IDO1 inhibitors by increasing the steric
hindrance and lipid solubility. The SAR of new IDO inhibitors was explored and
active compounds were tested in the immune-competent animal models.
RESULTS AND DISCUSSION
Design
Epacadostat is quickly consumed by the metabolic enzymes in human, which
account for its poor half-life (t1/2 = 2.4 h in human). The metabolic study revealed that
epacadostat was mainly transformed to O-glucuronic acid conjugate compound M9
by UGT1A9 (Figure 2) [22]. After a single dose of epacadostat in human, the AUC
value of M9 was more than 8-fold greater than that of epacadostat, which lead to poor
half-life of epacadostat. Moreover, M11 was a minor metabolite of epacadostat,

which was produced by gut microbiota and M12 was a secondary metabolite of
epacadostat formed from M11. On the other hand, the oral bioavailability of
epacadostat is unsatisfactory (F = 11% in rat and F = 33% in cynomolgus), which
might result from its poor hydrophobicity (cLogP = 0.09). To overcome these
shortcomings, further structural modification of epacadostat is highly desired.
Figure 2. Metabolic pathways of epacadostat in human.
In 2017, Yeh and his coworkers reported the crystal structure of hIDO1 enzyme in
complex with epacadostat [23]. As shown in Figure 3, the 3-Br-4-F-phenylamine
fragment of epacadostat fulfilled the pocket 1 and the fluoride and bromine group
formed halogen bonds with Cys129. The amidoxime group coordinates to the heme
iron via the oxygen atom, which was of vital importance for stabilizing protein–
inhibitor interactions. The furazan and side chain of epacadostat extend into the
pocket 2 and the sulfamide group forms electrostatic interaction with Arg231.

Figure 3. Crystal structure of hIDO1 enzyme in complex with epacadostat
IDO1 (PDB ID: 5WN8, gray) binding pocket for epacadostat (blue carbons), some
key interactions are shown as yellow dashed lines.
As aforementioned, the amidoxime group of epacadostat is essential for its
inhibitory activity against IDO1 enzyme and the modification of amidoxime group
would lead to the decrease of the inhibitory activity against IDO1. As a result,
replacing the amidoxime group by bioisosteric groups to avoid the metabolism by
UGT1A9 is impracticable. It has been reported that introducing a side chain on the C3
position of furazan could reduce the phase II metabolism, which may cause the collide
between the compound and metabolic enzymes [15]. We noticed that the pocket 2 was
not fulfilled by epacadostat and the amino group of sulfamide didn’t form any
hydrogen bond with hIDO1 enzyme, which may be a suitable site to be modified.
Therefore, we retained the 1,2,5-oxadiazole-3-carboximidamide fragment and
introduced a cycle in the side chain to increase the steric hindrance and lipid solubility,
which may improve the PK profiles and biological activity of IDO1 inhibitors (Figure
4
). As a result, 1,2,5-oxadiazole-3-carboximidamide derivatives 7-33 were
synthesized and evaluated as novel IDO1 inhibitors.

Figure 4. Design of novel IDO inhibitors.
Synthesis of 1,2,5-oxadiazole-3-carboximidamide derivatives
The synthetic route of compounds 7-9, 19, 20 and 23-26 is outlined in Scheme 1.
Malononitrile 34 was treated with sodium nitrite, hydroxylamine, and hydrochloric
acid to afford hydroxyamidine 35. Then, it was diazotized under acidic condition and
reacted with sodium chloride to provide the hydroximoyl chloride 36, which was
further transferred to compound 37 by coupling with 3-Br-4-F-phenylamine. After
that, compound 37 was treated with carbonyl diimidazole (CDI) to protect the oxime
group, which resulted compound 38. To introduce the side chain at oxadiazole,
compound 38 was oxidized to nitro compound 39. It was then substituted by a variety
of amino compounds (40a-40k) and hydrolyzed under acidic condition to afford
compounds 41a-41k. These compounds were further hydrolyzed under acidic
condition and reacted with tert-butyl (chlorosulfonyl)carbamate to provide
compounds 42a-42k. Finally, target products were obtained via a two-step procedure
containing acidic and basic hydrolysis.
Scheme 1. Synthesis of the compounds 7-9, 19, 20, and 23-26.

o
Reagents and conditions: (a) NaNO , HCl, NH OH, H O, 100 C (b) NaNO , HCl,
2
2
2
2
o
o
CH COOH, NaCl, H O, 0 C (c) 3-Br-4-F-phenylamine, NaHCO , EtOH/H O, 60 C
3
2
3
2
o
o
(
d) CDI, THF, 65 C (e) H O , TFA, 55 C (f) TEA, THF, r.t. (g) 4 N HCl in
2 2
o
1
,4-dioxane, CH Cl , r.t. (h) tert-butyl (chlorosulfonyl)carbamate, TEA, THF, 0 C (i)
2 2
TFA, r.t. (j) NaOH, THF/H O, r.t.
2
Synthetic routes of compounds 10-17, 21, 22, and 27-33 are shown in Scheme 2.
Intermediate 39 was substituted by a variety of amino compounds (44a-44j) and
hydrolyzed under acidic condition to afford compounds 45a-45j. Then they reacted
with alkyl sulfonyl chloride (46a-46f) to provide compounds 47aa-47df. Desired
compounds 10-17, 21, 22, 27-33 were obtained after a simple basic hydrolysis of
compounds 47aa-47df.

Scheme 2. Synthesis of the compounds 10-17, 21, 22, and 27-33
Reagents and conditions: (a) TEA, THF, r.t. (b) 4 N HCl in 1,4-dioxane, CH Cl , r.t. (c)
2
2
o
TEA, THF, 0 C (d) NaOH, THF/H O, r.t.
2
The synthetic route of compound 18 is very similar to the protocol described in
Scheme 1 by replacing compound 40a-40k with 48.
Scheme 3. Synthesis of the compound 18.
Reagents and conditions: (a) TEA, THF, r.t. (b) 4 N HCl in 1,4-dioxane, CH Cl , r.t. (c)
2
2
o
tert-butyl (chlorosulfonyl)carbamate, TEA, THF, 0 C (d) NaOH, THF/H O, r.t.
2
Structure−Activity Relationships of 1,2,5-oxadiazole-3-carboximidamides
derivatives

All the synthesized compounds were screened for their activity to inhibit the
enzymatic activity of the purified recombinant hIDO1. Dozens of
1
,2,5-oxadiazole-3-carboximidamides derivatives were designed and synthesized to
explore their structure−activity relationship. The preliminary SAR at the side chain
bearing different six-membered cycle was first investigated (compound 7-9). Among
them, compound 8 of which the sulfamide was on the para-position exhibited a
moderate inhibitory activity (IC50 = 142 nM) and improvement in lipid solubility
(cLogP = 0.82). Analogs 10 and 11 were obtained by replacing the amino of sulfamide
with methyl. However, these two compounds showed decreased inhibitory activity
against hIDO1 enzyme, which may be due to the lack of interaction with the solvent.
Then, difluoromethyl substituted analog 12 were synthesized, which might form
electrostatic interaction with Arg231 residue in pocket 2 and possessed better lipid
solubility (12, cLogP = 2.88). To our disappointment, it displayed less potent
inhibitory activity than compound 8 (12, IC = 489.6 nM). Then different substitution
5
0
of sulfamide which might filled the pocket 2 were investigated. Extending the chain
of sulfonyl to yield compounds 13 and 14 resulted in decreased inhibitory activities
(
13, IC = 517.2 nM; 14, IC > 1000 nM). And analogs containing two cycles were
50 50
prepared for further exploration of SAR. Replacing the amino group of compound 8
by cyclopropyl, pyrroline, or phenyl gave compounds 15-17, however, none of them
displayed satisfactory inhibitory activity (15, IC = 476.9 nM; 16, IC = 601.9 nM;
5
0
50
1
7, IC = 585.2 nM). Prolonging the side chain gave compounds 18 and 19, which
50
were also inferior IDO inhibitors than compound 8 (18, IC = 425.7 nM; 19, IC =
5
0
50
3
59.7 nM). Replacing the piperidine by pyrroline gave compounds 20-22. Among
them, compounds 20 and 22 exhibited similar activities with compound 8 (20, IC =
5
0
1
22.8 nM; 22, IC = 188.0 nM). Then, enantiomers of compounds 7, 8, 10, 20, 21,
50
and 22 were synthesized and evaluated. As shown in Table 1, the R enantiomers (26,
IC50 = 139.1 nM; 29, IC50 = 443.3 nM; 31, IC50 = 150.1 nM) demonstrated
improvement in inhibitory activity against IDO1 over the S enantiomers (25, IC =
5
0
1
78.1 nM; 28, IC = 716.8 nM; 30, IC = 529.0 nM). However, enantiomer 23 (S
50 50
configuration, IC = 108.7 nM) is more effective than 24 (R configuration, IC =
5
0
50

1
46.4 nM) and the two enantiomers of 10 showed similar inhibitory activity (32, IC50
360.3 nM; 33, IC = 363.2 nM). The difference in the inhibitory activity might be
=
5
0
related to the distance between sulfamide group of compound and Arg231, which
formed hydrogen bond to stabilize protein–inhibitor interactions. And enantiomer 24
(
IC = 108.7 nM) showed potent inhibitory activity similar to epacadostat.
50
Table 1. Inhibitory activity against IDO1 of 1,2,5-oxadiazole-3-carboximidamide
derivatives.
Compd.
R
IC (nM)
cLogP
Compd.
21
R
IC (nM)
cLogP
5
0
50
7
8
9
245.2±9.7
142.5±53.1
357.8±25.0
364.1±134.9
959.5±276.5
489.6±58.5
517.2±9.3-
>1000
1.22
480.1±65.2
188.0±4.9
1.23
0.82
0.78
1.28
1.68
2.88
1.79
1.05
22
0.83
0.38
0.38
0.82
0.82
1.22
1.23
23
108.7±6.9
10
24
146.4±29.8
178.1±32.2
139.1±4.6
11
25
12
13
14
26
H2N
O
S
O
27
N
261.7±112.6
716.8±286.0
28

1
1
5
6
476.9±9.3
601.9±0.1
2.05
1.68
29
30
443.3±35.9
1.23
0.83
529.0±237.5
17
585.2±96.6
3.39
31
150.1±26.9
0.83
18
19
20
425.7±41.6
359.7±19.5
122.8±10.5
1.51
1.23
0.38
32
33
1
360.3±41.6
363.2±38.8
87.4±3.1
1.28
1.28
0.09
(
rac)
-
3
.2 Inhibitory Activities of the Selected Compounds in HEK 293T cells
over-expressing hIDO1
Active compounds (12, 15, 18, 19, 23-30, 32, and 33) were further evaluated for
their activity in HEK 293T cells over-expressing hIDO1. As shown in Table 2,
compounds 12 and 15 displayed weak inhibitory activity in the cell-based assay,
indicating that introducing large group might be unbefitting. Compound 23 showed a
potent inhibitory activity in the cell-based assay (cellular IC = 19.88 nM), which
5
0
was close to the inhibitory activity of epacadostat (cellular IC50 = 9.63 nM).
Compounds 24, 25, 26, 32, and 33, of which the substitution was located on the
3
-position of pyrroline or piperidine, also exhibited good potency (cellular IC50
=
4
2.58, 68.59, 57.76, 42.99, and 49.83 nM respectively). Notably, most compounds
including epacadostat displayed more potent inhibitory activities in cellular system
than those against purified enzyme, which is consistent with other published results
[
19, 24]. Multiple reasons mentioned in the review by Schwaid and Cornella-Taracido
may explain the increased potency in cell-based assay, such as cellular localization,
contacts with other proteins or nucleic acids, translational modifications of the protein

and more active metabolites formed in cells [25].
Table 2. Inhibitory activity of derivatives in HEK 293T cells over-expressing hIDO1.
Compd.
R
IC (nM)
Compd.
26
R
IC (nM)
5
0
50
12
15
18
19
23
24
25
＞1000
＞1000
57.76 ± 25.69
177.35 ± 17.32
186.20 ± 11.31
333.65 ± 43.20
42.99 ± 5.78
H N
O
2
S
O
N
27
137.95 ± 16.05
153.75 ± 5.44
19.88 ± 5.35
42.58 ± 14.81
68.59 ± 21.47
9.63 ± 4.58
28
29
30
32
49.83 ± 13.26
125.44 ± 79.29
33
1
-
3
.3 PK profiles of compounds 23, 25, 26, and epacadostat
To further investigate the effectiveness of design strategy, the pharmacokinetic
evaluation of compounds 23, 25, 26, and epacadostat was carried out via intravenous
10 mg/kg) and oral administration (20 mg/kg) in CD-1 mice. As shown in table 3,
compounds 23 and 26 exhibited similar half-lives (2.81 h and 3.17 h, respectively).
(

However, these compounds displayed lower bioavailability than that of epacadostat.
Compound 25 showed longer half-life (T1/2 3.81 h) than epacadostat and acceptable
=
oral bioavailability (F = 33.6 %). Meanwhile, it is noteworthy that compound 25
possessed four-fold steady-state volume of distribution than epacadostat (24277
mL/kg and 6319 mL/kg, respectively), which indicated the remarkable enrichment of
2
5 in extravascular space (e.g., the target tumor tissue).
Table 3. Pharmacokinetic profiles of compounds 23, 25, 26, and epacadostat in CD-1
mice.
Compd.
T1/2
h)
Tmax
Cmax
AUClast AUCINF_obs
Cl_obs
MRTINF_obs Vss_obs
F
(
(h) (ng/mL) (h*ng/mL) (h*ng/mL) (mL/min/kg)
(h)
(mL/kg) (%)
2
3
2.81
3.81
0.25
0.33
622
358
392
-
2.65
-
9.70
2
2
5
6
261
118
343
588
244
672
764
304
971
5.10
4.91
3.34
33.6
9.84
22.1
PO
3.17 0.333
-
-
-
-
Epacadostat 2.59
1.17
-
2
3
1.19
-
1846
1858
90.0
0.622
3356
-
2
2
5
6
4.09
2.82
875
1241
1524
888
1265
1646
195
139
104
2.05
1.71
1.04
24277
12881
6319
-
-
-
IV
-
-
-
-
Epacadostat 0.903
3
.4 Compound 25 abrogated IDO1-mediated suppression of T cell proliferation
and inhibited the growth of colorectal carcinoma CT26 xenografts
It is reported that IDO1 mediate immunosuppression through its capacity to block
CD8+T cell proliferation by depleting tryptophan locally [26]. Due to the high
potency against hIDO1 in vitro and acceptable pharmacokinetic parameters,
compound 25 was further evaluated for its effect on T cell proliferation in the
presence of tumor cells over-expressing IDO1 by a co-culture assay. Briefly, mouse
pancreatic cancer Pan02 cells transfected with pcDNA3.1-IDO1 (Pan02-IDO1) or the

empty plasmid (Pan02-mock) were co-cultured with CD8+T cells labeled with CFSE.
T cells were stimulated with IL-2 and dynabeads coated with anti-CD3/anti-CD28.
The CD8+T cells were collected and analyzed for proliferation by flow cytometry
after 3 days of incubation. Compared to Pan02-mock cells, co-culture of Pan02-IDO1
cells partially suppressed T cell proliferation, which could be completely abrogated in
the presence of 100 nM epacadostat or compound 25 (Figure 5a). This result indicated
+
that compound 25 prevented inhibition of CD8 T cell proliferation by tumor cells
overexpressing IDO1.
The anti-tumor efficacy of compound 25 was further evaluated in vivo. Female
BALB/c mice bearing established CT26 colorectal tumors were administered orally
with compound 25 for 16 days (100 mg/kg, BID) and antitumor efficacy was
expressed as tumor growth inhibition (TGI). As shown in Figure 5b and 5c,
compound 25 (TGI = 24.5%) exhibited the similar anti-tumor efficacy with
epacadostat (TGI = 23.4%) without inducing significant change in body weight
compared to the control group.
(a)
(b)

*
**
(c)
+
Figure 5. (a) Overlays of CFSE dilution in CD8 T cells after co-culture with Pan02
cells in the presence of 100 nM Compound 25 or epacadostat. Flow cytometry
histograms are representatives of two independent experiments. (b)Tumor volume of
CT26 xenograft in female BALB/c mice. (Data points represent mean ± SEM.) (c)
Body weight of female BALB/c mice.
4
. Conclusion
Although epacadostat shows strong inhibitory activity against IDO1 and is further
studied in clinic trails., its pharmacokinetic character is not satisfactory. To that point,
a cycle in the side chain of epacadostat was introduced aiming to increase the steric
hindrance and improve the lipid solubility of compound. Sixteen analogs were
synthesized and evaluated to explore the structure-activity relationship and six

analogs of them displayed potent inhibitory activities. The enantiomers of these IDO
inhibitors were also evaluated. Compounds 23, 25, and 26 exhibited good potency
against hIDO1 and IDO1-expressing HEK 293T cells, which were further
investigated for their PK profiles. Compound 25 showed improved PK properties with
longer half-life and better oral bioavailability compared with epacadostat. Finally, oral
administration of compound 25 showed similar therapeutic efficacy with epacadostat
in the CT-26 syngeneic xenograft model, which demonstrated that it was suitable for
further development as a lead compound.
Experiment Section
hIDO1 enzymatic assay
The effect of the tested compounds on the enzymatic activity of IDO1 was
determined as previously described with minor modifications [27]. Briefly, the
standard reaction mixture (30 µL) containing potassium phosphate (100 mmol/L, pH
6
.5), ascorbic acid neutralized with NaOH (40 mmol/L), catalase (200 µg/mL),
methylene blue (20 mmol/L), 0.01% Triton X-100 and rhIDO-1 (0.05 µmol/L) were
added to a solution (60 µL) containing the substrate L-tryptophan (250 µmol/L) and
test compounds at the desired concentration. The reaction was carried out at 37 ℃ for
3
0 min and stopped by adding 45 µL of 30% (w/v) trichloroacetic acid. After being
heated at 65 ℃ for 15 min, the reaction mixture was centrifuged at 12000 rpm for 10
min. The supernatant (100 µL) was transferred into a 96-well microplate and mixed
with 100 µL of 2% (w/v) p-dimethylaminobenzaldehyde (pDMAB) in acetic acid. The
yellow pigment derived from kynurenine was measured at 492 nm using a
SpectraMax Plus 384 microplate reader (Molecular Devices, Sunnyvale, CA). The
IC values were determined by nonlinear regression analysis with GraphPad Prism 7
5
0
software (San Diego, CA, USA).
Cell-based assay of IDO1 activity
The cellular activity of IDO1 was detected as described previously [24]. HEK
5
2
93T cells were seeded in a 6-well culture plate at a density of 5 × 10 cells/well. On
the second day, HEK 293T cells were transfected with pcDNA3.1-hIDO1 using

Lipofectamine 2000 according to the manufacturer’s instructions. The cells were
4
seeded in 96-well microplates at a density of 2.5 × 10 cells/well 24 h after
transfection and treated with serially diluted tested compounds. After additional 12-h
incubation, 200 µL of the culture medium from each well was transferred to a new
9
6-well plate and mixed with 100 µL of 30% (w/v) trichloroacetic acid. The plate was
incubated at 65 ℃ for 15 min to hydrolyze N-formylkynurenine produced by the
catalytic reaction of IDO1. The reaction mixture was then centrifuged at 12000 rpm
for 10 min and 100 µL of the supernatant was transferred to another 96-well plate and
mixed with 100 µL of 2% (w/v) p-dimethylaminobenzaldehyde in acetic acid. The
yellow pigment derived from kynurenine was measured at 492 nm using a
SpectraMax Plus 384 microplate reader (Molecular Devices, Sunnyvale, CA). The
IC values were calculated by using GraphPad Prism 7 software (San Diego, CA,
5
0
USA).
T cell proliferation assay
Pan02 cells were transfected with pcDNA3.1-hIDO1 or the empty plasmid using
Lipofectamine 3000 according to the manufacturer’s instructions. After 24 hours,
Pan02-IDO1 and Pan02-mock cells were seeded into 6-well plate at a density of 2.5 ×
5
1
0 cells/well. Splenic CD8+T cells were purified by magnetic negative selection
(Stem Cell Technologies) and labeled with CFSE (BD Pharmingen). CD8+T cells
5
were co-cultured with Pan02-IDO1 and Pan02-mock cells at a density of 5 × 10
cells/well and stimulated with IL-2 (5 ng/ml) and dynabeads coated with
anti-CD3/anti-CD28. After 3 days of incubation, CD8+T cells were collected and
stained with CD45 (clone 30-F11)-APC-Cy7, CD8 (clone 53-6.7)-APC (BD
TM
Pharmingen ) and 7-amino-actinomycin D (7-AAD; Biolegend) prior to analysis on
Fortessa flow cytometer. Data were analyzed with FlowJo.
In vivo antitumor activity assay
Female BALB/c mice (4-6 weeks old) were housed and maintained under specific
pathogen-free conditions. Animal procedures were performed according to
institutional ethical guidelines of animal care. The murine CT26 colon carcinoma
cells were maintained as a monolayer culture in RPMI-1640 medium supplemented

with 10% heat inactivated fetal bovine serum (Gibco product), 100 U/mL penicillin
and 100 µg/mL streptomycin at 37 ℃ in an atmosphere of 5% CO . Cells growing in
2
an exponential phase were harvested for tumor inoculation. Each mouse was
6
inoculated subcutaneously at the right lower flank with CT26 cells (0.4 × 10 /mouse)
3
in 0.1 mL of PBS. When the tumor volume reached approximately 50 mm , the
animals were randomly grouped to be administered orally with vehicle or indicated
tested compounds twice daily. The tumor sizes and animal weights were measured
twice per week using a caliper and weight scale, respectively. The tumor volume (V)
2
2
was calculated using the formula: V= 0.5 × [length (mm) × width (mm )].
Chemistry
All reagents (chemicals) were commercially available and used without further
purification. Analytical thin-layer chromatography (TLC) was performed on HSGF
2
3
54 (0.1 mm thickness). Column chromatography was performed on silica gel 200–
00 mesh to purify the compounds. NMR spectra were recorded on
Varian-MERCURY Plus-400 and AVANCE III 500 in CDCl , Methanol-d , DMSO-d ,
3
4
6
Acetone-d . Chemical shifts were reported in parts per million (ppm, δ) downfield
6
from tetramethylsilane. Proton coupling patterns were described as s = singlet, d =
doublet, dd = doublet of doublet, t = triplet, q = quartet, m = multiplet. Low- and
high-resolution mass spectra (LRMS and HRMS) were given with electrospray
ionization (ESI).
All target compounds 7-33 were confirmed with over 95% purity which were
determined by Agilent 1260 HPLC with binary pump, photodiode array detector
(
DAD), using Agilent Extend-C18 (4.6 × 250 mm, 5 µm), MeOH/H O = 70/30 (v/v)
2
at 1.0 mL/min, or Agilent Extend-C18 (4.6 × 250 mm, 5 µm), MeOH/H O = 60/40
2
(v/v) at 1.0 mL/min and calculated the peak areas at 254 nm.
Synthesis of compound 8
Malononitrile (10 g, 151 mmol) was added to water (200 mL) and stirred for 5
min. The resulting solution was cooled in an ice bath and sodium nitrite (11.8 g, 171

mmol) was added. Then, 10 N hydrochloric acid (10 mL) was added slowly. After 15
min the cold bath was removed and the reaction mixture was stirred for 1.5 h at room
temperature. The reaction mixture was cooled to 0°C and 50% aqueous
hydroxylamine (30.9 g, 468 mmol) was added all at once. The resulting mixture was
stirred for 1 h at room temperature, then it was slowly brought to reflux. Reflux was
maintained for 2 h and then the reaction mixture was cooled to room temperature. The
reaction mixture was stirred in an ice bath and 6 N hydrochloric acid was added in
portions to pH 7.0. Stirring was continued in the ice bath at 5 °C. The precipitate was
collected by filtration, washed well with water and dried under vacuum to give the
1
3
desired product 35 (20 g, 92%). C NMR (100 MHz, Methanol-d ) δ 154.5, 144.4,
4
1
39.7.
4
-amino-N-hydroxy-1,2,5-oxadiazole-3-carbimidoyl chloride (36)
Compound 35 (20 g, 140 mmol) was added to a mixture of water (240 mL),
acetic acid (140 mL) and 12 N hydrochloric acid (70 mL) and this suspension was
stirred at room temperature until complete solution was achieved. Sodium chloride
(23.7 g, 400 mmol) was added and this solution was cool to 0 °C in an ice bath. A
solution of sodium nitrite (9.5 g, 140 mmol) in water (30 mL) was added over 1 h.
After that, the reaction mixture was stirred in the ice bath for another 1.5 h and then
the reaction mixture was warmed to room temperature. The precipitate was collected
by filtration, washed well with water and dried under vacuum to give the desired
1
3
product 36 as a white solid (11 g, 48%). C NMR (100 MHz, Methanol-d ) δ 153.8,
4
1
41.6, 127.9.
4
-amino-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-carboximid
amide (37)
Compound 36 (11 g, 68 mmol) was dissolved in 150 mL EtOH/H O (v/v = 5:1).
2
3
-Bromo-4-fluoroaniline (13 g, 68 mmol) and sodium bicarbonate (4.3 g, 41 mmol)
were added and stirred for 10 min. Then the mixture was heated to 60 °C and was
stirred at 60 °C for 2 hrs. The reaction solution was cooled to room temperature and
extracted with ethyl acetate. The combined ethyl acetate solution was dried over

sodium sulfate and concentrated to give the desired product 37 as a white solid (20 g,
1
3
9
1
3
3%). C NMR (100 MHz, DMSO-d ) δ 155.6, 154.1 (d, J = 239.1 Hz), 140.8, 139.7,
6
38.4, 125.2, 121.8 (d, J = 7.3 Hz), 116.4 (d, J = 23.4 Hz), 107.5 (d, J = 22.2 Hz).
-(4-amino-1,2,5-oxadiazol-3-yl)-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4
H)-one (38)
Compound 37 (20 g, 63 mmol) was dissolved in THF. CDI (12 g, 76 mmol) was
added to the mixture and stirred for 10 min. The reaction was stirred at 65 °C for 2 hrs.
After that, the reaction was concentrated under vacuum. The resulting solid was
treated with 1N HCl (100 mL) and stirred for 10 min. The precipitate was collected by
filtration, washed well with EtOAc and dried under vacuum to give the desired
1
3
product 38 as a light yellow solid (16 g,74%). C NMR (100 MHz, DMSO-d ) δ
6
1
60.7, 157.2, 156.9 (d, J = 256.1 Hz), 149.3, 134.7, 133.8, 130.5, 129.1, 117.9 (d, J =
3.7 Hz), 108.6 (d, J = 22.6 Hz).
2
4
-(3-bromo-4-fluorophenyl)-3-(4-nitro-1,2,5-oxadiazol-3-yl)-1,2,4-oxadiazol-5
4H)-one (39) Compound 38 (16 g, 47 mmol) was dissolved in TFA (100 mL). 30%
H O solvent (20 mL) was added and stirred for 10 min. Then the mixture was stirred
(
2
2
at 55 °C for 48 h. After that, water was added to the mixture and precipitate was
collected by filtration. The crude product was purified by silica gel column
1
chromatography to afford product 39 as a yellow solid (5.6 g, 32%). H NMR (400
MHz, Chloroform-d) δ 7.68 – 7.55 (m, 1H), 7.39 – 7.19 (m, 2H).
4
-(4-bromo-3-fluorophenyl)-3-(4-((piperidin-3-ylmethyl)amino)-1,2,5-oxadiazol-3
-yl)-1,2,4-oxadiazol-5(4H)-one hydrochloride (41d)
Compound 39 (330 mg, 0.88 mmol) was dissolved in THF (5 mL). Tert-butyl
3
1
-(aminomethyl)piperidine-1-carboxylate (225 mg, 1.05 mmol) and TEA (106 mg,
.05 mmol) were added to the mixture and the reaction was stirred at room
temperature for 4 h. After that, 4 N HCl in dioxane (2 mL) was added to the mixture
and the reaction was stirred for another 1 h. Then the reaction mixture was
concentrated under vacuum and the crude product was purified by silica gel column
1
chromatography to afford product 41d as a white solid (380 mg, 90%). H NMR (400
MHz, Methanol-d ) δ 7.92 (dd, J = 6.0, 2.5 Hz, 1H), 7.60 (ddd, J = 8.8, 4.2, 2.6 Hz,
4

1
H), 7.44 (t, J = 8.6 Hz, 1H), 3.44 (dd, J = 12.6, 4.0 Hz, 1H), 3.40 – 3.31 (m, 4H),
.94 (td, J = 12.8, 3.2 Hz, 1H), 2.78 (t, J = 12.2 Hz, 1H), 2.30 (ddt, J = 11.0, 7.3, 4.0
2
Hz, 1H), 1.96 (d, J = 12.8 Hz, 1H), 1.84 – 1.69 (m, 1H), 1.42 – 1.30 (m, 1H).
Tert-butyl
((3-(((4-(4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1,2,
5
-oxadiazol-3-yl)amino)methyl)piperidin-1-yl)sulfonyl)carbamate (42d)
Compound 41d (380 mg, 0.80 mmol) was dissolved in 5 mL dichloromethane
followed by the adding of triethylamine (0.148 mL, 1.08 mmol). After cooling to 0 °C,
the solution of tert-butyl [chlorosulfonyl]carbamate (1 mL, 0.9 mmol) was added to
the mixture slowly. After that, the reaction was allowed to warm to 10 °C and stirred
at 10 °C for 30 min. Then water (10 mL) was added to the mixture and the layers
were separated. The organic layer was washed with brine (10 mL) and the solvents
was removed in vacuum. The crude product was purified by silica gel column
1
chromatography to afford product 42d as a white solid (405 mg, 81%). H NMR (400
MHz, DMSO-d ) δ 10.95 (s, 1H), 8.10 (s, 0H), 7.73 (ddd, J = 8.9, 4.4, 2.5 Hz, 1H),
6
7
.61 (t, J = 8.7 Hz, 1H), 6.72 (t, J = 5.9 Hz, 1H), 3.60 (dd, J = 12.3, 3.9 Hz, 1H), 3.50
(d, J = 12.0 Hz, 1H), 3.24 – 3.11 (m, 2H), 2.87 – 2.74 (m, 1H), 2.61 (dd, J = 12.4,
1
0.1 Hz, 1H), 1.94 (d, J = 3.6 Hz, 1H), 1.70 (t, J = 17.8 Hz, 2H), 1.43-1.46 (m,
H)1.42 (s, 9H), 1.14 – 0.99 (m, 1H).
1
Preparation of tert-butyl [chlorosulfonyl]carbamate solution : A 100 mL round
bottom flask was charged with chlorosulfonyl isocyanate (1 mL, 11 mmol) and
dichloromethane (10 mL). The mixture was cooled to 2 °C and tert-Butanol (1 mL, 11
mol) in dichloromethane (1 mL) was added dropwise at a rate so that the temperature
did not exceed 10 °C. The resulting solution was stirred at room temperature for
3
0-60 min to provide tert-butyl [chlorosulfonyl]carbamate solution.
N-(3-bromo-4-fluorophenyl)-N'-hydroxy-4-(((1-sulfamoylpiperidin-3-yl)methyl)a
mino)-1,2,5-oxadiazole-3-carboximidamide (8) Compound 42d (405 mg, 0.78
mmol) was dissolved in 5 mL THF and 5M NaOH solution (1 mL, 5.5 mmol) was
added. The reaction was stirred for 3h at room temperature. Then 20 mL EtOAc was
poured into the mixture and the organic layer was washed by brine for three times.

The desired product 8 was obtained after the purification by silica gel column
chromatography as a white solid (265 mg, 69%).
N-(3-bromo-4-fluorophenyl)-N'-hydroxy-4-(((1-sulfamoylpiperidin-2-yl)methyl)a
mino)-1,2,5-oxadiazole-3-carboximidamide (7)
o
1
White solid (yield: 53%). HPLC purity: 98.28%. m.p.：70 - 72 C. H NMR (500 MHz,
DMSO-d ) δ 11.42 (s, 1H), 8.86 (s, 1H), 7.21 – 7.07 (m, 2H), 6.79 (dt, J = 8.9, 3.5 Hz,
6
1
H), 6.66 (s, 2H), 6.18 (dd, J = 7.2, 5.1 Hz, 1H), 4.07 (q, J = 6.9 Hz, 1H), 3.52 - 3.59
(m, 2H), 3.38 - 3.46 (m, 1H), 2.99 (td, J = 10.7, 4.9 Hz, 1H), 1.69 (dt, J = 13.6, 6.8 Hz,
1
3
1
H), 1.54 (q, J = 13.9, 13.3 Hz, 5H). C NMR (125 MHz, DMSO-d ) δ 156.2, 154.3
6
(d, J = 237.5 Hz), 140.1, 139.8, 138.4 (d, J = 2.8 Hz), 125.3, 122.0 (d, J = 7.1 Hz),
116.4 (d, J = 23.2 Hz), 107.5 (d, J = 22.0 Hz), 51.5, 43.3, 41.3, 25.3, 24.2, 19.0.
-
+
ESI-MS: m/z 489.7 [M-H] ; HRMS (ESI) calculated for C15H19BrFN7O4S [M-H ]:
90.0314; found: 490.0311.
4
N-(3-bromo-4-fluorophenyl)-N'-hydroxy-4-(((1-sulfamoylpiperidin-3-yl)methyl)a
mino)-1,2,5-oxadiazole-3-carboximidamide (8)
o
1
White solid (yield: 49%). HPLC purity: 98.12%. m.p.：98 - 101 C. H NMR (400
MHz, DMSO-d ) δ 11.45 (s, 1H), 8.90 (s, 1H), 7.17 (t, J = 8.8 Hz, 1H), 7.11 (dd, J =
6
6
.0, 2.7 Hz, 1H), 6.77-6.71 (m, 1H), 6.70 (s, 2H), 6.27 (t, J = 5.9 Hz, 1H), 3.39 (dd, J
11.9, 3.3 Hz, 1H), 3.35-3.25 (m ,1H), 3.12 (t, J = 6.6 Hz, 2H), 2.53-2.45 (m, 1H),
.28 (t, J = 10.8 Hz, 1H), 1.95-1.83 (m, 1H), 1.77 – 1.65 (m, 1H), 1.65 – 1.57 (m, 1H),
=
2
1
1
1
3
.44 (q, J = 12.0 Hz, 1H), 1.07 – 0.91 (m, 1H). C NMR (125 MHz, DMSO-d ) δ
6
56.3, 154.3 (d, J = 237.5 Hz), 140.4, 139.6, 138.4, 125.4, 122.0 (d, J = 6.9 Hz),
116.5 (d, J = 23.4 Hz), 107.6 (d, J = 22.1 Hz), 50.2, 47.8, 46.9, 35.1, 27.7, 24.0.
-
ESI-MS: m/z 489.8 [M-H] ; HRMS (ESI) calculated for C15H19BrFN7O4S
+
[
M-H ]:490.0314; found: 490.0319.
N-(3-bromo-4-fluorophenyl)-N'-hydroxy-4-(((1-sulfamoylpiperidin-4-yl)methyl)a
mino)-1,2,5-oxadiazole-3-carboximidamide (9)

o
1
White solid (yield: 62%). HPLC purity: 95.66%. m.p.：147 - 149 C. H NMR (500
MHz, DMSO-d ) δ 11.46 (s, 1H), 8.89 (s, 1H), 7.19 (t, J = 8.7 Hz, 1H), 7.12 (dd, J =
6
6
1
1
.1, 2.7 Hz, 1H), 6.78 (ddd, J = 8.9, 4.2, 2.7 Hz, 1H), 6.69 (s, 2H), 6.28 (t, J = 5.9 Hz,
H), 3.47 (d, J = 11.7 Hz, 2H), 3.11 (t, J = 6.2 Hz, 2H), 2.50 – 2.43 (m, 2H), 1.74 –
13
.63 (m, 2H), 1.31 – 1.07 (m, 3H). C NMR (125 MHz, DMSO-d ) δ 156.2, 154.3 (d,
6
J = 237.5 Hz), 140.5, 139.6, 138.4, 125.5, 122.0 (d, J = 7.2 Hz), 116.4 (d, J = 23.2
-
Hz), 107.6 (d, J = 22.0 Hz), 49.5, 46.2, 34.6, 29.0. ESI-MS: m/z 489.8 [M-H] ; HRMS
+
(
ESI) calculated for C15H19BrFN7O4S [M+H ]: 492.0459; found: 492.0466.
N-(3-bromo-4-fluorophenyl)-N'-hydroxy-4-(((1-(methylsulfonyl)piperidin-3-yl)m
ethyl)amino)-1,2,5-oxadiazole-3-carboximidamide (10)
o
1
White solid (yield: 67%). HPLC purity: 99.05%. m.p.：140 - 142 C. H NMR (600
MHz, DMSO-d ) δ 11.44 (s, 1H), 8.91 (s, 1H), 7.17 (t, J = 8.8 Hz, 1H), 7.11 (dd, J =
6
6
.1, 2.7 Hz, 1H), 6.74 (ddd, J = 8.9, 4.1, 2.7 Hz, 1H), 6.30 (t, J = 6.0 Hz, 1H), 3.48
(dd, J = 11.6, 3.8 Hz, 1H), 3.41 (dt, J = 11.6, 4.1 Hz, 1H), 3.05 - 3.19 (m, 2H), 2.81 (s,
3
1
1
H) 2.68 (td, J = 11.5, 3.1 Hz, 1H), 2.46 (dd, J = 11.6, 10.1 Hz, 1H), 1.89 (ddq, J =
0.4, 7.2, 3.5 Hz, 1H), 1.72 (dp, J = 13.2, 3.5 Hz, 1H), 1.68 – 1.61 (m, 1H), 1.50 –
1
3
.38 (m, 1H), 1.03 (dtd, J = 12.9, 11.2, 3.8 Hz, 1H). C NMR (150 MHz, DMSO-d )
6
δ 156.4, 154.3 (d, J = 237.5 Hz), 140.5, 139.5, 138.3 (d, J = 2.8 Hz), 125.4 , 122.0 (d,
J = 7.1 Hz), 116.5 (d, J = 23.2 Hz), 107.6 (d, J = 22.0 Hz), 49.5, 47.5, 46.4, 35.3, 34.3,
-
2
7.6, 24.3. ESI-MS: m/z 489.7 [M-H] ; HRMS (ESI) calculated for
+
C16H20BrFN6O4S [M+H ]: 491.0507; found: 491.0513.
N-(3-bromo-4-fluorophenyl)-N'-hydroxy-4-(((1-(methylsulfonyl)piperidin-2-yl)m
ethyl)amino)-1,2,5-oxadiazole-3-carboximidamide (11)
o
1
White solid (yield: 43%). HPLC purity: 96.91%. m.p.：85 - 87 C. H NMR (600 MHz,
DMSO-d ) δ 11.60 – 11.32 (m, 1H), 8.87 (s, 1H), 7.14 (t, J = 8.7 Hz, 1H), 7.10 (dd, J
6
=
–
6.1, 2.7 Hz, 1H), 6.74 (ddd, J = 8.9, 4.1, 2.7 Hz, 1H), 6.27 (t, J = 6.3 Hz, 1H), 4.22
3.99 (m, 1H), 3.53-3.59 (m, 2H), 3.35 (dt, J = 13.5, 6.6 Hz, 1H), 3.13 – 3.03 (m,
1
3
1
H), 2.91 (s, 3H), 1.50-1.60 (m, 5H), 1.39 (ddt, J = 19.9, 12.2, 5.6 Hz, 1H). C NMR
(
150 MHz, DMSO-d ) δ 156.1, 154.2 (d, J = 237.5 Hz), 140.4, 139.7, 138.4 (d, J =
6

2
5
.8 Hz), 125.3, 121.9 (d, J = 6.9 Hz), 116.4 (d, J = 23.2 Hz), 107.6 (d, J = 22.0 Hz),
-
0.7, 43.6, 40.8, 40.6, 25.9, 25.0, 18.9. ESI-MS: m/z 489.7 [M-H] ; HRMS (ESI)
+
calculated for C16H20BrFN6O4S [M+H ]: 491.0507; found: 491.0497.
N-(3-bromo-4-fluorophenyl)-4-(((1-((difluoromethyl)sulfonyl)piperidin-3-yl)meth
yl)amino)-N'-hydroxy-1,2,5-oxadiazole-3-carboximidamide (12)
o
1
White solid (yield: 47%). HPLC purity: 95.10%. m.p.：65 - 67 C. H NMR (600 MHz,
DMSO-d ) δ 11.46 (s, 1H), 8.92 (s, 1H), 7.15 (t, J = 8.7 Hz, 1H), 7.11 (dd, J = 6.1, 2.7
6
Hz, 1H), 7.08 (t, J = 52.8 Hz), 6.73 (ddd, J = 8.9, 4.1, 2.7 Hz, 1H), 6.34 (t, J = 6.1 Hz,
1
H), 3.67 (dd, J = 12.9, 4.0 Hz, 1H), 3.62 (dd, J = 12.6, 3.8 Hz, 1H), 3.12 (h, J = 7.1
Hz, 2H), 3.07 – 3.00 (m, 1H), 2.83 (dd, J = 12.8, 10.3 Hz, 1H), 1.87 (ddp, J = 10.4,
7
5
1
2
.1, 3.3 Hz, 1H), 1.77 – 1.64 (m, 2H), 1.48 – 1.34 (m, 1H), 1.14 (dddt, J = 16.5, 12.4,
13
.3, 3.3 Hz, 1H). C NMR (150 MHz, DMSO-d ) δ 156.3, 154.3(d, J = 237.5 Hz),
6
40.5, 139.4, 138.3, 125.4, 121.9 (d, J = 7.1 Hz), 114.7 (t, J = 277.5 Hz), 113.8 (d, J =
1
9
78.0 Hz), 107.6 (d, J = 22.1 Hz), 49.8, 47.1, 46.9, 35.7, 27.2, 24.9. F NMR (470
-
MHz, Methenol-d ) δ -117.7, -124.11. ESI-MS: m/z 542.8 [M-H] ; HRMS (ESI)
4
+
calculated for C16H17BrF4N6O4S [M+H ]: 545.0224; found: 545.0223.
N-(3-bromo-4-fluorophenyl)-4-(((1-(ethylsulfonyl)piperidin-3-yl)methyl)amino)-
N'-hydroxy-1,2,5-oxadiazole-3-carboximidamide (13)
o
1
White solid (yield: 63%). HPLC purity: 96.80%. m.p.：139 - 141 C. H NMR (500
MHz, DMSO-d ) δ 11.48 (s, 1H), 8.89 (s, 1H), 7.18 (t, J = 8.8 Hz, 1H), 7.13 (dd, J =
6
6
.1, 2.7 Hz, 1H), 6.77 (dt, J = 9.0, 3.4 Hz, 1H), 6.31 (t, J = 6.0 Hz, 1H), 3.53 (dd, J =
1
2
3
3
1.9, 3.8 Hz, 1H), 3.46 – 3.48 (m, 1H) 3.21 – 3.10 (m, 2H), 3.00 (q, J = 7.3 Hz, 2H),
,
.80 (td, J = 11.4, 2.7 Hz, 1H), 2.59 (dd, J = 11.9, 9.9 Hz, 1H), 1.88 (dtq, J = 13.8, 7.0,
.5 Hz, 1H), 1.79 – 1.59 (m, 2H), 1.43 (qd, J = 10.8, 4.1 Hz, 1H), 1.20 (t, J = 7.4 Hz,
1
3
H), 1.09 (td, J = 10.8, 7.7 Hz, 1H). C NMR (125 MHz, DMSO-d6) δ 156.3, 154.3 (d,
J = 237.5 Hz), 140.5, 139.6, 138.3 (d, J = 2.8 Hz), 125.4, 122.0 (d, J = 6.9 Hz), 116.4
d, J = 23.1 Hz), 107.6 (d, J = 22.0 Hz), 49.3, 47.4, 46.3, 43.0, 35.6, 27.6, 24.6, 8.0.
(

-
+
ESI-MS: m/z 502.9 [M-H] ; HRMS (ESI) calculated for C16H20BrFN6O4S [M-H ]:
5
03.0518; found: 503.0522.
N-(3-bromo-4-fluorophenyl)-N'-hydroxy-4-(((1-(N-methylsulfamoyl)piperidin-3-
yl)methyl)amino)-1,2,5-oxadiazole-3-carboximidamide (14)
o
1
White solid (yield: 62%). HPLC purity: 98.27%. m.p.：160 – 162 C. H NMR (400
MHz, DMSO-d ) δ 11.48 (s, 1H), 8.94 (s, 1H), 7.18 (t, J = 8.8 Hz, 1H), 7.13 (dd, J =
6
6
.1, 2.7 Hz, 1H), 7.06 (q, J = 4.9 Hz, 1H), 6.77 (ddd, J = 8.9, 4.2, 2.7 Hz, 1H), 3.45
dd, J = 11.9, 3.8 Hz, 1H), 3.33 – 3.39 (m, 1H), 3.20 – 3.08 (m, 2H), 2.67 (td, J = 11.4,
.7 Hz, 1H), 2.49 (d, J = 4.9 Hz, 4H), 2.47 - 2.52 (m, 1H), 1.89 (ddt, J = 10.2, 6.7, 3.4
(
2
1
3
Hz, 1H), 1.76 – 1.59 (m, 2H), 1.52 – 1.38 (m, 1H), 1.12 – 0.98 (m, 1H). C NMR
(
100 MHz, DMSO-d ) δ 156.3, 154.2 (d, J = 239.5 Hz), 140.4, 139.5, 138.3, 125.4 ,
6
1
3
21.9 (d, J = 6.8 Hz), 116.4 (d, J = 23.2 Hz), 107.5 (d, J = 22.1 Hz), 49.6, 47.5, 46.5,
-
5.2, 29.4, 27.6, 24.2. ESI-MS: m/z 503.9 [M-H] ; HRMS (ESI) calculated for
+
C16H21BrFN7O4S [M-H ]: 504.0470; found: 504.0487.
N-(3-bromo-4-fluorophenyl)-4-(((1-(cyclopropylsulfonyl)piperidin-3-yl)methyl)a
mino)-N'-hydroxy-1,2,5-oxadiazole-3-carboximidamide (15)
o
1
White solid (yield: 49%). HPLC purity: 95.45%. m.p.：128 - 130 C. H NMR (500
MHz, DMSO-d ) δ 11.43 (s, 1H), 8.91 (s, 1H), 7.19 (t, J = 8.8 Hz, 1H), 7.13 (dd, J =
6
6
.0, 2.7 Hz, 1H), 6.77 (ddd, J = 9.0, 4.2, 2.7 Hz, 1H), 6.32 (t, J = 6.0 Hz, 1H), 3.55
(dd, J = 11.9, 3.7 Hz, 1H), 3.49 (dd, J = 10.0, 5.9 Hz, 1H), 3.15 (td, J = 6.6, 3.7 Hz,
2
H), 2.82 (td, J = 11.5, 2.8 Hz, 1H), 2.60 (dd, J = 11.8, 10.1 Hz, 1H), 2.56 – 2.52 (m,
H), 1.91 (ddd, J = 10.4, 6.8, 3.6 Hz, 1H), 1.71 (ddt, J = 23.8, 13.2, 3.8 Hz, 2H), 1.46
1
(tdt, J = 10.9, 7.1, 3.6 Hz, 1H), 1.09 (qd, J = 11.3, 3.5 Hz, 1H), 0.96 (dt, J = 8.0, 3.1
1
3
Hz, 2H), 0.90 (tt, J = 4.8, 2.6 Hz, 2H). C NMR (125 MHz, DMSO-d ) δ 156.4,
6
1
54.3 (d, J = 239.5 Hz), 140.5, 139.5, 138.4, 125.4, 122.0 (d, J = 7.0 Hz), 116.5 (d, J
=
23.5 Hz), 107.6 (d, J = 22.3 Hz), 49.9, 47.5, 46.8, 35.5, 27.6, 25.6, 24.5, 4.3.
+
ESI-MS: m/z 515.0 [M-H ]. HRMS (ESI) calculated for C18H22BrFN6O4S [M+H+]: