Discovery of novel hydroxyamidine derivatives as indoleamine 2,3-dioxygenase 1 inhibitors with in vivo anti-tumor efficacy

Article abstract of DOI:10.1016/j.bmcl.2020.127038

Indoleamine 2,3-dioxygenase 1 (IDO1) is closely associated with immune escape in many tumor tissues, and is considered to be a valuable therapeutic target in cancer immunotherapy. In this study, the modification of amino sidechain was performed with the hydroxyamidine core kept intact to optimize lead compound Epacadostat. 19 new compounds with hydrazide, thietane or sulfonamide moiety as polar capping group in sidechain were prepared and their IDO1 inhibitory activities were evaluated. Sulfonamide 3a showed potent IDO1 inhibition in both enzymatic and cellular assays with the IC₅₀ value of 71 nM and EC₅₀ value of 11 nM, respectively. Furthermore, in vivo Lewis lung cancer (LLC) allograft studies of 3a indicated that it handicapped the tumor growth with similar efficacy to Epacadostat. Molecular docking demonstrated that the change of polar capping group affords influence on the orientation of amino ethylene side chain and forms new hydrogen bonding.

Full text of DOI:10.1016/j.bmcl.2020.127038

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel hydroxyamidine derivatives as indoleamine
2,3-dioxygenase 1 inhibitors with in vivo anti-tumor efficacy
Chang Liua, Yanyang Nanb, Zhifeng Xiaa, Kedan Gua, Cheng Chena, Xiaochun Donga,
⁎
⁎
Dianwen Jub, , Weili Zhaoa,
a Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, PR China
b Department of Microbiological and Biochemical Pharmacy, School of Pharmacy, Fudan University, Shanghai 201203, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Indoleamine 2,3-dioxygenase 1 (IDO1) is closely associated with immune escape in many tumor tissues, and is
considered to be a valuable therapeutic target in cancer immunotherapy. In this study, the modification of amino
sidechain was performed with the hydroxyamidine core kept intact to optimize lead compound Epacadostat. 19
new compounds with hydrazide, thietane or sulfonamide moiety as polar capping group in sidechain were
prepared and their IDO1 inhibitory activities were evaluated. Sulfonamide 3a showed potent IDO1 inhibition in
both enzymatic and cellular assays with the IC50 value of 71 nM and EC50 value of 11 nM, respectively.
Furthermore, in vivo Lewis lung cancer (LLC) allograft studies of 3a indicated that it handicapped the tumor
growth with similar efficacy to Epacadostat. Molecular docking demonstrated that the change of polar capping
group affords influence on the orientation of amino ethylene side chain and forms new hydrogen bonding.
Indoleamine 2,3-dioxygenase 1
Hydroxyamidine derivatives
Lewis lung cancer
Cancer immunotherapy
The indoleamine 2,3-dioxygenase 1 (IDO1), an extrahepatic cyto-
solic heme-containing dioxygenase, is the rate-limiting enzyme on the
tryptophan kynurenine pathway.1,2 Through this pathway, the trypto-
phan is oxidized to N-acetyl kynurenine, which is subsequently con-
verted to kynurenine, 3-hydroxykunurenine, quinolinic acid and other
metabolites.3 The overexpression of IDO1 in cancer cells leads to
tryptophan depletion and kynurenine pathway metabolites accumula-
tion. Tryptophan depletion induces effector T cells incompetence and
apoptosis through general control nonderepressible 2 (GCN2) activa-
tion4 and mechanistic target of rapamycin kinase (mTOR) inhibition5
pathway. Kynurenine pathway metabolites accumulation stimulates T
cell differentiate to regulatory T cells (Tregs) as a consequence of
binding to the aryl hydrocarbon receptor (AHR).6 Through above
mentioned approach, cancer cells evade immune surveillance in many
tumor tissues.7–12 Therefore, inhibition of IDO1 has become an attrac-
tive approach in cancer immunotherapy.
during combination with pembrolizumab in the recent phase III ECHO
301 trial,22 which may be related to insufficient drug exposure or
mismatched drug combination strategy,23,24 IDO1 related therapy is
still a promising area.
According to the co-crystal structure of IDO1 bound to
Epacadostat,25 the inhibitor forms vital coordination with heme iron
via the oxygen atom of the hydroxyamidine group. The halogenated
phenyl group occupies pocket A and was surrounded by a group of
hydrophobic residues. The Br and F atoms offer a favorable fluor-
ine–sulfur contact with C129 in the N-terminal domain. The central
furazan group is stabilized by F163, L234, and F226. Moreover, the
sulfamoylamino ethylene sidechain pointing into pocket B, with the
sulfamoylamino group stabilized by R231 through hydrogen bonding.
The halogenated phenyl region and coordinating group of Epacadostat
have been comprehensively optimized in Incyte’s previous reports.26,27
However, only a few types of polar sidechain were investigated,28–30
despite the fact that pocket B is not fully occupied and variable side part
with polar group may be tolerated.
Tremendous efforts have been made to discover potent small-mo-
lecule IDO1 inhibitors.13–15 Several IDO1 inhibitors with elucidated
molecular structure have entered various stages of clinical trials, in-
cluding Indoximod,16 Epacadostat,17 Navoximod,18 PF-068400319 and
Linrodostat.20 (Fig. 1) Among them, Epacadostat had increased re-
sponse when combined with nivolumab or pembrolizumab in phase I/II
trials.17,21 Although the enhancement of Epacadostat was not observed
In this study, lead optimization of Epacadostat was performed fo-
cusing on the modification of amino sidechain with the hydro-
xyamidine substituted furazan moiety kept intact to keep the two im-
portant intramolecular hydrogen bonds that forces amidine C]N
double bond to hold a cis conformation, which is critical for the
⁎ Corresponding authors.
E-mail addresses: dianwenju@fudan.edu.cn (D. Ju), zhaoweili@fudan.edu.cn (W. Zhao).
https://doi.org/10.1016/j.bmcl.2020.127038
Received 3 January 2020; Received in revised form 8 February 2020; Accepted 14 February 2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:ChangLiu,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2020.127038

C. Liu, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
reported methods.27 Oxidation of 6 with hydrogen peroxide afforded 4-
nitro-1,2,5-oxadiazole derivative (7). Upon hydrazide substitution, key
intermediate 4-hydrazinyl-1,2,5-oxadiazole 8 was generated in 63%
yield. After mesylation and further hydrolyzation, the target compound
methanesulfonyl hydrazide (1a) was obtained. It was noted that the
aminosulfonyl moiety in 1c was unstable under basic deprotection
condition and can’t be constructed by the similar route. To obtain 1c,
aminosulfonyl group was directly introduced into deprotected hy-
drazide derivative 1b, which was prepared from the hydrolysis of 8
under basic condition.
To fix the orientation of polar capping group, thietane analogues
(2a–d) were synthesized as described in Scheme 2. The substitution of
nitro compound 7 with thietan-3-amine generated thietane derivative
(10). After oxidation with IBX or m-CPBA and hydrolysis with NaOH,
the target compounds 2a and 2c were obtained, respectively. For the
preparation of target compound containing N-cyano sulfilimine (2b) or
sulfoximine (2d), 10 was oxidized with (diacetoxyiodo)benzene in the
presence of ammonium carbamate or cyanamide, and directly hydro-
lyzed without purification.
Fig. 1. Structures of representative IDO1 inhibitors in clinical stage.
inhibitory activity.27 Hydrazide derivative was initially constructed as
removal of ethyl linker and directly attaching the sulfamoylamine
component with amino group in sidechain. Moreover, thietane deri-
vatives with sulfoxide, sulphone, N-cyano sulfilimine or sulfoximine
moiety were designed to reduce the fixability of sidechain and fix the
orientation of polar capping group. Due to irrelevant interaction be-
tween the inner NH in the Epacadostat sulfamoylamine group and
pocket B of IDO1,25 sulfonamide and its substituted analogues were also
explored. Herein, the syntheses and SAR of novel hydroxyamidine de-
rivatives (Fig. 2) as new IDO1 inhibitors are communicated.
Sulfonamide derivative (3a) as removing inner nitrogen atom of
sulfamoylamine group in Epacadostat and its substituted analogues
(3b–i) were constructed according to Scheme 3. The above nitro com-
pound 7 was substituted with taurine to obtain sodium sulfonate (15)
with ethylene linker in sidechain. After activation with oxalyl chloride,
substitution with varied amines (for 3a–g), hydrazine (for 3h) or hy-
droxylamine (for 3i) and further hydrolyzation with 2 N NaOH in
MeOH, the desired target sulfonamide derivatives 3a–i were prepared.
Installation of the side chain with propylene linker was also achieved
with similar method to afford sulfonamide 4a and 4b. Moreover, linker
between oxadiazole core and polar capping group was changed from
The synthetic methods of all desired compounds were illustrated in
Schemes 1–4. As shown in Scheme 1, the syntheses of 1a–c started from
3-(4-amino-1,2,5-oxadiazol-3-yl)-4-(3-bromo-4-fluorophenyl)-1,2,4-ox-
adiazol-5(4H)-one (6) which was prepared according to previously
Fig. 2. Design concept of novel Epacadostat analogues.
Scheme 1. Synthesis of the compounds 1a–c. Reagents and conditions: (i) TFA, H2O2, 80 °C, 70%; (ii) N2H4, DMF, rt, 63%; (iii) MsCl, NEt3, rt; (iv) NaOH, MeOH, rt,
32%; (v) NaOH, MeOH, rt, 60%; (vi) ClSO2NCO, t-BuOH, NEt3; (vii) TFA, DCM, 82%.
2

C. Liu, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Scheme 2. Synthesis of the compounds 2a–d. Reagents and conditions: (i) thietan-3-amine, NEt3, THF, 63%; (ii) IBX, benzyltriethylammonium chloride, DCM, H2O;
(iii) NH2CN, (diacetoxyiodo)benzene, CH3CN; (iv) m-CPBA, DCM; (v) ammonium carbamate, (diacetoxyiodo)benzene, MeOH; (vi) NaOH, MeOH, 37–73%.
ethylamine to ethoxyamine and the compound 5 was prepared with
similar method to 3a–i (Scheme 4).
potency comparing to the target molecule 1c, but still lower than
Epacadostat. These results indicated that hydrazide and its derivatives
maybe weaken the interaction between the polar sidechain and pocket
B.
With all designed compounds in hand, we first tested their enzyme-
based IDO1 inhibition activity according to the reported procedure,32,33
and lead compound Epacadostat was used as positive control. As shown
in Table 1, replacement of amino sidechain with hydrazide, thietane or
sulfonamide played significant influences on IDO1 inhibition. Six of the
synthesized compounds exhibit the potent inhibition of IDO1-mediated
conversion of tryptophan to kynurenine with IC50 values less than
100 nM. The ethylene linker in sidechain is critical for the activities.
Removal the ethylene group is detrimental to inhibitory activity (1c vs
Epacadostat), with IC50 value of 218 nM. Replacing external amino
group with methyl further results 3-fold decrease of inhibitory activity
(1a vs 1c). The hydrazine intermediate 1b showed 2-fold higher
Fixing the orientation of polar capping group with thietane was
tolerated, with IC50 values in the range of 69–212 nM. Thietane deri-
vative containing sulphoxide moiety lead to 3-fold decrease of activity
(2a vs Epacadostat). But replacement of sulphoxide with N-cyano sul-
filimine (2b) exhibited comparable inhibitory potency (IC50 = 69 nM)
to Epacadostat. Thietane derivative with sulfone or sulfimide group
resulted in 1.5 to 3-fold loss in potency (2c/2d vs Epacadostat), with
IC50 value of 99 or 208 nM, respectively. It might suggest that sulfone
moiety afforded better H-bonding over sulfimide or sulphoxide (2c vs
2d/2a).
Scheme 3. Synthesis of the compounds 3a–i and 4a–b. Reagents and conditions: (i) 2-aminoethane-1-sulfonic acid, NaOH, H2O, DMF, 79%; (ii) 3-aminopropane-1-
sulfonic acid, NaOH, H2O, DMF, 37%; (iii) (COCl)2, DMF, THF; (iv) NR1R2, NEt3, THF; (v) NaOH, MeOH, H2O, 19–69%.
3

C. Liu, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Scheme 4. Synthesis of the compound 5. Reagents
and conditions: (i) 2-(aminooxy)ethane-1-sulfonic
acid,31 NaOH, H2O, DMF, 58%; (ii) (COCl)2, DMF,
THF; (iii) NH3, THF; (iv) NaOH, MeOH, H2O, 31%.
To our delight, the sulfonamide derivative 3a which removing inner
decreased enzymatic activity were observed (3f/3g vs 3d/3e). These
data suggested that hydrophobic substitution, especially large steric
substitution is not conducive for the sidechain of the compounds ex-
tending into the pocket B. N,N-Di-substituted sulfonamide derivatives
leaded to 1.5–2.7 fold drop in inhibitory activity (3b/3c vs 3a).
Hydrophilic hydroxyl (3 h) and amino (3i) substitution also exhibited
almost 2-fold less potency than 3a. Moreover, a change from ethylene
linker to propylene group resulted in slightly loss in potency (4a vs 3a),
with IC50 value of 126 nM. Alternatively, we changed the linker be-
tween oxadiazole core and polar capping group from ethylamine to
ethoxyamine and the obtained compound 5 showed slightly reduced
activity (IC50 = 87 nM).
NH in Epacadostat sulfamoylamine group displayed similar IDO1 in-
hibition activity (IC50 = 71 nM) comparing to the lead compound. The
hydrogen atom in sulfonamide was further replaced with a variety of
hydrophobic or hydrophilic substituents. When small hydrophobic
substitution ethyl or trifluoroethyl were introduced in sulfonamide
moiety, enzymatic activity slightly decreased (3d/3e vs 3a), with IC50
values of 95 nM and 140 nM, respectively. And introduction of large
substitution such as prop-2-yn-1-yl and cyclopropylmethyl, further
Table 1
IDO1 inhibitory activity of 1a–c, 2a–d, 3a–i, 4a–b and 5.
The obtained compounds were also evaluated for their inhibitory
activity for tryptophan depletion and simultaneous kynurenine pro-
duction in HeLa cell with stimulation of IFN-γ.32–33 As shown in
Table 1, most of compounds inhibit the IDO1-mediated conversion of
tryptophan to kynurenine in cell-based assay with EC50 values less than
200 nM. Hydrazine derivatives 1a–c showed decreased inhibitory effect
compared to Epacadostat, with EC50 values lager than 100 nM. Thie-
tane derivatives 2a–d exhibited better inhibitory activity than 1a–c, but
still lower than Epacadostat. Fortunately, non-substituted sulfonamide
3a displayed potent IDO1 inhibitory activity in cell (EC50 = 11 nM),
which is comparable with Epacadostat. Further hydrophobic mono- or
di-substitution in sulfonamide group all showed reduced inhibitory ef-
fect (3b–g vs 3a). And hydrophilic substitution (3 h and 3i) lead to
totally loss of inhibitory activity. Moreover, increasing chain length
from ethylene linker to propylene group resulted in slightly loss in
potency (4a/4b vs 3a), with EC50 values 15 nM and 20 nM, respec-
tively. Compound 5, which changing linker from ethylamine to
ethoxyamine, lead to 2-fold drop of inhibitory potency (EC50 = 27 nM)
compared with 3a. In general, most compounds showed a good corre-
lation between enzymatic and cellular assays.
Comp.
R
IDO1 IC50 (nM)a
HeLa EC50 (nM)a
> 800
1a
609
45
1b
1c
108
218
16
12
556
101
44
13
2a
2b
2c
212
69
47
79
73
59
16
7
6
16
7
99
2d
208
10
53
11
3a
3b
71
6
11
17
1
2
Encouraged by the potent inhibitory activity of 3a on enzyme-based
and cell-based assay, we further investigated the anti-tumor efficacy in
vivo. The LLC (Lewis lung cancer) allograft model in immunocompetent
mice was adopted34 and Epacadostat was used as positive control.
When the tumors diameter roughly reached 5 mm, the tested compound
3a or Epacadostat was administered with oral dosing (100 mg/kg)
twice a day for 14 days. As shown in Fig. 3, compared to the vehicle
control, both compound 3a and Epacadostat obviously inhibited the
tumor growth. The sulfonamide derivative 3a proved to show similar
efficacy compared to Epacadostat, which is consistent with their ten-
dency of cellar IDO1 inhibitory. In addition, mice were well tolerated to
3a with no significant body weight loss until the end of assay (Fig. S1).
To elucidate the binding model of 3a with IDO1, molecular docking
was preformed based on the crystal structure of IDO1 complexed with
Epacadostat (PDB code: 5WN8)25 using Schrodinger (Maestro suite). As
shown in Fig. 4, 3a (orange carbons) bind in almost the same location
as that of Epacadostat (cyan carbons). The phenyl group of occupies the
pocket A and was surrounded by hydrophobic residues with fluorine-
sulfur contact. The oxygen of hydroxyamidine coordinates to the fer-
rous iron of the heme. However, removing inner amino of sidechain
changes the orientation of amino ethylene side chain. The sidechain of
3a forms two hydrogen bonds between sulfonamide nitrogen atom and
L234/G262, nevertheless sulfonamide oxygen atom in Epacadostat in-
teracts with R231 and forms one hydrogen bond. All these
105
1
3c
3d
3e
3f
191
95
42
143
1
4
11
15
41
140
218
312
135
126
268
18
10
11
11
14
4
33
3
3g
3h
3i
268
18
> 800
> 800
4a
4b
126
97
15
20
1
1
7
5
87
66
3
9
27
1
Epacadostat
8
1
a
IC50/EC50 values are the mean of three independent assays.
4

C. Liu, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
sulfonamide (3a) is the most suitable type of sidechain with the enzy-
matic IC50 value of 71 nM and cellular EC50 value of 11 nM, respec-
tively. Efforts were made to optimize substitution in sulfonyl amide.
Unfortunately, no matter hydrophobic or hydrophilic substitution
leaded to a drop of inhibition efficacy. In vivo antitumor studies of 3a
indicated similar efficacy with Epacadostat. Moreover, molecular
docking demonstrated that the change of polar capping group affords
influence on the orientation of amino ethylene side chain and forms the
new hydrogen bonding. The SAR relationship of Epacadostat analogues
with varied sidechain provides the new information for drug design of
novel IDO inhibitors.
Acknowledgment
This work was supported by NSFC (21572037).
Appendix A. Supplementary data
Fig. 3. Tumor growth curve upon treatment with 3a, Epacadostat or vehicle in
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127038.
C57BL/6 mice bearing LLC allografts. Vehicle: 0.5% CMC suspension. Tumor
volume for indicated days was showed as means
than 0.05 compared with the vehicle group.
SEM (n = 6). (*) P less
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Fig. 4. Predicted binding modes for compound 3a (orange carbons) with the
active site of the human IDO1 enzyme in comparison with Epacadostat (cyan
carbons).
computational predictions explain the comparable potency of 3a with
Epacadostat.
In summary, a series of Epacadostat analogues with hydrazide,
thietane and sulfonamide as side part were designed and constructed as
IDO1 inhibitors. The in vitro IDO1 inhibition assay illustrated that
5