A theranostic probe of indoleamine 2,3-dioxygenase 1 (IDO1) for small molecule cancer immunotherapy

Article abstract of DOI:10.1016/j.ejmech.2021.113163

Discovering novel small molecules for cancer immunotherapy represents a promising but challenging strategy in future cancer treatment. Herein, we designed the first theranostic fluorescent probes to efficiently detect and inhibit the enzymatic activity of

Full text of DOI:10.1016/j.ejmech.2021.113163

European Journal of Medicinal Chemistry 213 (2021) 113163
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
A theranostic probe of indoleamine 2,3-dioxygenase 1 (IDO1) for small
molecule cancer immunotherapy
,
,
,
, **
Ying Wu a 1, Yanhui Zhang b 1, Xi Chen b 1, Yulu Hu b, Guoqiang Dong a, Yuan Guo b
,
Chunquan Sheng a
,
*
a School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai, 200433, China
b Key Laboratory of Synthetic and Natural Functional Molecule of the Ministry of Education, College of Chemistry and Materials Science, Northwest
University, 1 Xuefu Avenue, Xi’an, 710127, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Discovering novel small molecules for cancer immunotherapy represents a promising but challenging
strategy in future cancer treatment. Herein, we designed the first theranostic fluorescent probes to
efficiently detect and inhibit the enzymatic activity of 2,3-dioxygenase 1 (IDO1). Probe 6b is a highly
active IDO1 inhibitor (IC50 ¼ 12 nM, Cellular IC50 ¼ 10 nM), which can sensitively and specifically detect
endogenous IDO1 in living cells. Furthermore, as a theranostic probe, 6b showed excellent in vivo
antitumor efficacy in the CT26 xenograft mouse model as well. Therefore, it can be applied as a valuable
chemical tool for better understanding the immunotherapy mechanism of IDO1 and improving the
therapeutic efficiency.
Received 6 October 2020
Received in revised form
30 December 2020
Accepted 4 January 2021
Available online 13 January 2021
Keywords:
IDO1
Theranostic probe
Cancer immunotherapy
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
an alternative way to improve clinical response and outcome of
cancer immunotherapy when either used alone or in combination
In recent years, there have been remarkable advances in cancer
immunotherapy [1]. Immune checkpoint antibodies targeting
programmed cell death protein 1 (PD-1), programmed death ligand
1 (PD-L1) and cytotoxic T-lymphocyte associated antigen 4 (CTLA-
4) have achieved great clinical success in cancer treatment with
superior efficacy and specificity [2,3]. However, despite the ad-
vantages, only a limited number of cancer patients have benefited
from these monoclonal antibodies’ treatments and immune-
related side effects are frequently observed. Moreover, checkpoint
antibodies generally suffer from difficult and expensive
manufacturing processes and unfavourable pharmacokinetic pro-
files such as long half-life, poor tissue and tumor penetration, and
low oral bioavailability [4]. In contrast, small-molecule immuno-
oncology agents generally have the advantages of reasonable
pharmacokinetics, convenient oral administration, flexible treat-
ment regimen and so on [5,6]. Therefore, small molecules provide
with antibodies.
Indoleamine 2,3-dioxygenase 1 (IDO1) is a well validated drug
target for the development of small-molecule cancer immuno-
therapeutics [7]. IDO1 is a key enzyme involved in the trypto-
phanekynurenineearyl hydrocarbon receptor (TrpeKyneAhR)
pathway which acts as an important mediator of immunosup-
pression in T celleinflamed tumors [8]. IDO1 catalyzes the con-
version of Trp to N-formyl-kynurenine (NFK), which is
subsequently hydrolyzed to Kyn. The depletion of Trp and the
accumulation of Kyn both contribute to the immune escape
mechanism in the tumor microenvironment [9]. Overexpression of
IDO1 leads to local degradation of Trp, which suppresses T cell
proliferation and activity. The generation of Kyn is also immuno-
suppressive because of the activation of AhR pathway and the
upregulation of the expansion of regulatory T cells [9]. Thus, inhi-
bition of IDO1 is capable to restore host immune responses to
eradicate cancer cells. Up to date, a large number of IDO1 inhibitors
have been discovered for cancer immunotherapy [10,11]. Several
IDO1 inhibitors (e.g. epacadostat, indoximod, BMS986205, navox-
imod, PF-06840003) have been developed to apply into different
stages of clinical trials [12,13]. However, phase III clinical trial of
epacadostat was terminated because the combination of epacado-
stat and PD-1 antibody pembrolizumab failed to exert therapeutic
* Corresponding author.
** Corresponding author.
E-mail addresses: guoyuan@nwu.edu.cn (Y. Guo), shengcq@hotmail.com,
shengcq@smmu.edu.cn (C. Sheng).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2021.113163
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

Y. Wu, Y. Zhang, X. Chen et al.
European Journal of Medicinal Chemistry 213 (2021) 113163
advantages over pembrozilumab alone [14]. Consequently, there is
an urgent need for better understanding the functions and mech-
anisms of IDO1 inhibition and discovery of similar new strategies.
Recently, the design of new IDO1 inhibitors remains an active
research area [15e19]. The IDO1 inhibition mechanisms were
further clarified by solving a series of X-ray crystal structures of
IDO1 in complex with various inhibitors [20e22]. Moreover, IDO1-
based multi-targeting strategies, such as combining IDO1/trypto-
phan 2,3-dioxygenase (TDO) dual inhibitors together [23] were
developed to achieve better efficacy in cancer immunotherapy.
Previously, we designed novel dual inhibitors for IDO1-DNA and
IDO1-histone deacetylase (HDAC), which showed improved anti-
tumor potency [24e26]. Inspired by these results, we propose that
a theranostic probe for simultaneous imaging and inhibiting IDO1
could have the advantage of diagnosing and treating patients with
IDO1 overexpressed cancer. In addition, theranostic IDO1 probes
will be helpful for elucidating the molecular mechanism beneath
immunotherapy and improving its efficacy. However, target-based
theranostic probes for small-molecule cancer immunotherapy have
rarely been reported. Herein, we designed the first theranostic
fluorescent probes to efficiently detect IDO1 protein.
compound 6b (Fig. 1C) also form two hydrogen bonds with the side
chains of Asn240 and Arg231, correspondingly. However, there is
no strong interaction between compound 6a (Fig. 1C) and IDO1.
These binding modes well explained why probes 4 and 6b inhibited
IDO1 better than probe 6a.
2.3. Spectral properties and selectivity of probes
To evaluate the polarity sensitivity of the three probes, we
explored their responsiveness to environmental polarity in the
mixed solutions of 1,4-dioxane and water with different polarities.
As displayed in Fig. 2AeC, when the 1,4-dioxane fraction (fd, by
volume %) in the mixture was increased from 0% to 80%, the fluo-
rescence intensities were enhanced by a factor of 46 (for 4, at
540 nm), 33 (for 6a, at 542 nm) and 37 (for 6b, at 540 nm), indi-
cating that all three compounds are sensitive to environmental
polarity, as expected.
Although the introduction of NBD endows 4, 6a and 6b with the
above sensitivities, their recognition of IDO1 in essence depends on
whether the inhibitor moiety introduced in the molecular skele-
tons can bind to IDO1 protein or not. Therefore, we first evaluated
their binding affinity to IDO1 in vitro. To facilitate this investigation,
we successfully cloned and expressed IDO1 in Escherichia coli BL21
(DE3), and purified the protein by Ni-NTA and gel-filtration (refer to
Supporting Information). Subsequently, the fluorescent responses
of 4, 6a and 6b towards various concentrations of IDO1 in the so-
lution of PBS were examined (Fig. 3, Fig. 4A and Fig. 2DeF). When
increasing the concentrations of IDO1, 4 and 6b showed progressive
enhancements in fluorescence intensity, especially 6b which
exhibited a 5-fold enhancement. Conversely, no significant changes
were observed when various concentrations of IDO1 were tested
with 6a. Furthermore, the selectivity of 4, 6a and 6b towards IDO1
was measured with a variety of enzyme species and biomolecules.
As depicted in Fig. 5, no distinct fluorescence changes were
observed for each compound in the absence of IDO1, and for 6a
even in the presence of IDO1, demonstrating that 4 and 6b possess
high selectivity towards IDO1. Afterwards, taking 6b as our pio-
neering probe, we investigated the reaction kinetics of 6b with
IDO1. As shown in Fig. 4B, the fluorescence intensity at 532 nm
gradually enhanced and reached a plateau within 60 min. Better
still, the fluorescence intensity of the 6b solution was linearly
2. Results and discussion
2.1. Chemistry
It has been reported that the meta-bromophenyl, oxadiazole and
hydroxyamidine groups in epacadostat are essential structural
features of the IDO1 inhibitors [27]. As shown in Fig. 1A (PDB:
5WN8), the aminoethyl-sulfamide substituent of epacadostat was
projected out of the active site of IDO1, which makes it suitable to
introduce the fluorophore here at the mouth of the substrate-
binding pocket without interfering with the catalytic activity of
this enzyme. Based on synthetic feasibility, the sulfamide substit-
uent was removed to form compound 2 as the pharmacophore of
IDO1 probes. Since low polarity is a unique feature of protein
interior microenvironment [20] we selected the environment-
sensitive chemical group 4-nitro-7-aminophenzoxadiazole (NBD)
[28] as the fluorophore because of its high quantum yield, specific
selectivity, excellent cell penetration ability and low toxicity
[29e32]. Various linkers were used to connect the pharmacophore
with the fluorophore, affording potential light-up fluorescent
probes 4, 6a and 6b.
The synthesis methods of the probes were shown in Scheme 1.
Compound 2a was synthesized according to our well established
protocols [24e26] which subsequently underwent nucleophilic
substitution and deprotection to expose the hydroxyamide group
to obtain target compound 4. Compound 3 underwent nucleophilic
substitution with different minocarboxylic acids to afford in-
termediates 5a or 5b. Target compounds 6a or 6b were synthetized
by amide condensation between 5a or 5b and compound 2a, fol-
lowed by the deprotection reaction.
related to the concentration of IDO1 in the range of 0e10 mM. The
detection limit of 6b for IDO1 was calculated to be 10 nM at a
signal-to-noise ratio of 3 (Fig. 4A), indicative of high-sensitivity to
changes in the quantities of IDO1.
2.4. Ido1 inhibitory and antiproliferative activities of probes
At first, the IDO1 inhibitory potency of probes 4, 6a and 6b was
assayed against human recombinant IDO1 [35]. As shown in Fig. 1B,
all the probes exhibited good inhibitory activity towards IDO1.
Especially, probes 4 (IC50 ¼ 28 nM) and 6b (IC50 ¼ 12 nM) were even
more effective than the positive control epacadostat (IC50 ¼ 47 nM).
To validate whether the introduction of NBD fluorophore might
cause cytotoxicity, in vitro antiproliferative activities were carried
out against human cervical carcinoma Hela cell line and mice
colorectal cancer CT26 cell line, which were easily stimulated by
2.2. Molecular docking studies
Molecular docking experiment was further carried out to
investigate the binding mode of probes with IDO1 (PDB code:
5WN8) [33] by the docking software Gold [34]. The results
demonstrate that the binding modes of these probes are similar to
IFN-
compounds showed no direct cytotoxicity towards these two cell
lines (IC50 > 100 M), suggesting that these probes had little effect
g to release endogenous IDO1 [36,37]. Fortunately, all of the
that of epacadostat. The phenyl group and oxadiazole form
pep
m
stacking interaction with Tyr126 and Phe163, respectively. The
oxygen atom in the hydroxyamidine group coordinates to heme
iron. Moreover, the oxadiazole and the nitro group of compound 4
(Fig. 1C) could form hydrogen bonds with the backbone of Gly236
and side chain of Arg231, respectively. Similarly, the nitro group of
on cell growth and were suitable for cellular IDO1 inhibition and
imaging. Furthermore, their inhibitory activities against IDO1 at the
cellular level were evaluated and the results showed that probes 4
and 6b reacted even better than epacadostat. Specially, probes 4
(Cellular IC50 ¼ 15 nM) and 6b (Cellular IC50 ¼ 10 nM) were capable
2

Y. Wu, Y. Zhang, X. Chen et al.
European Journal of Medicinal Chemistry 213 (2021) 113163
Fig. 1. Rational design, biological activity and binding mode of fluorescent probes. (A) The design rationale of IDO1 probes; (B) IDO1 enzyme inhibition and cellular potency of
probes (C) Binding mode of probes 4 (cyan), 6a (indigo) and 6b (orange) along with their superimposition in the active site of IDO1 (PDB: 5WN8). Hydrogen bonds (red) are
represented with dash lines. The figures were generated using PyMol (http://www. Pymol.org/).
of effectively transforming Trp to Kyn, whereas probe 6a was
inactive in this function.
green fluorescence enhancement signals after incubation with 4 or
6b. By contrast, Hela cells treated with IFN- and 6a presented dim
g
fluorescence signals in the green channel, which was consistent
with the experimental results in vitro. To further verify that IDO1 is
indeed responsible for the above fluorescence signals, the control
imaging experiment to compete with probes for the pocket of IDO1
in living cells was carried out. When Hela cells were treated with
2.5. In living cell imaging
Inspired by the favorable sensing properties of 4 and 6b to IDO1
in vitro, we further investigated their application for imaging of
endogenous IDO1 in living cells. As shown in Fig. 6, Hela cells
stained with probe 4 or probe 6b exhibited weak fluorescence. As
IFN-g and IDO1 inhibitor epacadostat, and further incubated with 4
or 6b, fluorescence enhancement signals were not observed, which
is the negative control as we anticipated. In short, these imaging
expected, after stimulated by IFN-g, Hela cells showed significant
3

Y. Wu, Y. Zhang, X. Chen et al.
European Journal of Medicinal Chemistry 213 (2021) 113163
Scheme 1. Reagents and Conditions: (a) i) NaHCO3, MeOH, H2O, 60 ꢀC, 2 h; ii) KOH, MeOH, H2O, rt, 0.5 h, yield 28%; (b) aminocarboxylic acid, NaHCO3, MeOH, H2O, 60 ꢀC, 2 h, yield
47e58%; (c) i) compound 2a, HATU, DIPEA, DMF, rt, 1 h; ii) KOH, MeOH, H2O, rt, 0.5 h, yield 14e21%.
Fig. 2. Spectral properties. Fluorescence spectra of 10
mM 4 (A), 10
mM 6a (B) and 10
mM 6b (C) in water/1,4-dioxane mixtures with different volume fractions of 1,4-dioxane (fd)
(
lex ¼ 465 nm, slit widths: Wex ¼ 5 nm, Wem ¼ 5 nm). Fluorescence spectra of 10
m
M 4 (D), 10
m
M 6a (E), and 10 M 6b (F) in the absence or presence IDO1 (10 M) in PBS buffer
m
m
solution (10 mM, pH ¼ 7.4, containing 1% DMSO, lex ¼ 465 nm, slit widths: Wex ¼ 5 nm, Wem ¼ 5 nm).
4

Y. Wu, Y. Zhang, X. Chen et al.
European Journal of Medicinal Chemistry 213 (2021) 113163
Fig. 3. Fluorescence spectra of 10
DMSO). lex ¼ 465 nm, slit widths: Wex ¼ 5 nm, Wem ¼ 5 nm.
m
M 4 (A) and 10
m
M 6a (B) upon the addition of various amounts of IDO1 (0, 1, 5, 10
mM) in PBS buffer solution (10 mM, pH ¼ 7.4, containing 1%
Fig. 4. (A) Fluorescence spectra of 6b (10
intensity of 6b (10 M) in the absence (black) and presence (green) of IDO1 (10
widths: Wex ¼ 5 nm, Wem ¼ 5 nm.
m
M) upon the addition of various amounts of IDO1 (0e10
mM, interval 2 mM) in PBS buffer solution. (B) Time course of fluorescence
m
mM) at 532 nm in PBS buffer solution (10 mM, pH ¼ 7.4, containing 1% DMSO). lex ¼ 465 nm, slit
results comprehensively suggested that both 4 and 6b could
sensitively and specifically detect endogenous IDO1 in living cells.
exhibit high expression of IDO1 [37]. After oral administration for
12 consecutive days at the dose of 100 mg/kg, compound 6b
showed good antitumor activity with the tumor growth inhibition
(TGI) rate of 46% (Fig. 7, P < 0.001), which was comparable to the
positive control epacadostat (TGI ¼ 49%). Furthermore, no signifi-
cant loss of body weight was observed in mice treated with com-
pound 6b, indicating low toxicity of this compound.
2.6. In vivo antitumor efficacy of 6b
The in vivo antitumor efficacy of compound 6b was further
evaluated in CT26 xenograft mouse model which was reported to
3. Conclusions
In conclusion, we rationally designed
a series of novel
environment-sensitive IDO1 probes, of which probe 6b displayed
excellent fluorescent properties for detection of this enzyme.
Further in vivo study showed that probe 6b exhibited good anti-
tumor activity in the CT26 xenograft mouse model, indicating that
it is a promising theranostic small-molecule probe. Such probes
provide good chemical tools for investigating the biological func-
tions of IDO1. We are looking forward to its application in better
understanding the immunotherapy mechanism of IDO1 based on
its visibility and diagnosis as well as clinical treatment of patients
with IDO1 overexpressed cancer based on its high sensitivity and
antitumor efficacy.
4. Experimental section
4.1. Chemistry
Fig. 5. Selectivity studies. Fluorescence intensity of 10
lem ¼ 531 nm), and 10 M 6b (lem ¼ 532 nm) with the addition of various species in
PBS buffer solution. 1, Blank; 2, trypsin; 3, bovine hemoglobin; 4, chymotrypsin; 5,
HSA; 6, BSA; 7, lysozyme; 8, E. coli -gal; 9, A. oryzae -gal; 10, GSH; 11, IDO1.
lex ¼ 465 nm, slit widths: Wex ¼ 5 nm, Wem ¼ 5 nm.
m
M 4 (lem ¼ 530 nm), 10
mM 6a
(
m
The materials used in the experiments were commercially
available. Column chromatography was performed on 200e300
mesh silica gel. 1H NMR and 13C NMR spectra were recorded on
b
b
5

Y. Wu, Y. Zhang, X. Chen et al.
European Journal of Medicinal Chemistry 213 (2021) 113163
Fig. 6. Imaging of endogenous IDO1 in living Hela cells. (A) Confocal images of living Hela cells stained with 4 (3.5 nM), 6a (5 nM) and 6b (5 nM). Column 1, 4 and 7: Hela cells
incubated with Hoechst (10 g/mL, 20 min) and different probes (5 min). Column 2, 5, and 8: Hela cells stimulated with IFN- (50 ng/mL, 12 h), followed by incubated with Hoechst
(10 g/mL, 20 min) and different probes (5 min). Column 3 and 6: IFN- -stimulated (50 ng/mL, 12 h) Hela cells treated with compound 1 (5 M, 30 min), followed by incubated with
Hoechst (10 g/mL, 20 min) and different probes (5 min). (BeD) Quantification of the relative fluorescence intensity from column 1e3 (B), column 4e6 (C) and column 7e8 (D). Blue
channel: lex ¼ 407 nm, lem ¼ 425e475 nm; green channel: lex ¼ 488 nm, lem ¼ 500e550 nm ***p < 0.001, N.S. ¼ not significant, two-sided Student’s t-test. Scale bar: 10 m.
m
g
m
g
m
m
m
Bruker AVANCE300 or AVANCE600 spectrometer (Bruker Company,
Germany) with DMSO‑d6 as the solvents and TMS as the internal
standard. Chemical shift (d) was given in ppm and the coupling
(263 mg, 3.13 mmol) were dissolved in MeOH (10 mL) and H2O
(5 mL), and the mixture was heated to 60 ꢀC. After stirred for 2 h,
the solution was cooled to room temperature, and KOH (292 mg,
5.22 mmol) was added. The mixture was stirred for another 0.5 h,
the organic solvent was removed under reduced pressure and the
remained mixture was adjusted by 1 M HCl aqueous solution until
pH reached 7. The precipitate was filtered, washed with water, and
dried to constant weight to obtain the intermediate 4 (380 mg,
constant (J) is reported in hertz (Hz). The intermediate 2a was
achieved according to literature [27]. 1H NMR (DMSO‑d6, 600 MHz)
d
: 8.12 (dd, J ¼ 2.28, 6.06 Hz, 1 H), 7.74e7.77 (m, 1H), 7.58(t,
J ¼ 8.64 Hz, 1H), 6.80e6.82 (m, 1H), 3.50 (d, J ¼ 5.64 Hz, 2H), 3.02 (t,
J ¼ 6.00 Hz, 2H).
28%). Purity >95%. 1H NMR (DMSO‑d6, 600 MHz)
d: 11.38 (s, 1H),
4.1.1. (Z)-N-(3-Bromo-4-fluorophenyl)-N0-hydroxy-4-((2-((7-
nitrobenzo
9.48 (s, 1H), 8.88 (s, 1H), 8.51 (d, J ¼ 8.67 Hz, 1 H), 7.13 (t, J ¼ 8.27 Hz,
1 H), 7.07 (dd, J ¼ 2.74, 6.07 Hz, 1 H), 6.74-6.71 (m, 1H), 6.45 (d,
J ¼ 9.00 Hz, 2 H), 3.69 (s, 2H), 3.60-3.54 (m, 2H); 13C NMR
4.1.1.1. [c][1,2,5]oxadiazol-4-yl)amino)ethyl)amino)-1,2,5-
oxadiazole-3-carboximidamide (4). The intermediate 2a (1 g,
2.61 mmol), compound 3 (517 mg, 2.61 mmol) and NaHCO3
(DMSO‑d6, 150 MHz)
d: 156.03, 155.79, 152.61, 145.31, 140.53,
139.55, 138.40, 138.37, 125.27, 121.82, 116.53, 116.37, 107.64, 107.35,
6

Y. Wu, Y. Zhang, X. Chen et al.
European Journal of Medicinal Chemistry 213 (2021) 113163
Fig. 7. Antitumor efficacy of compound 1 and compound 6b in the xenograft model. (A) The efficacy of compound 1 and compound 6b in the CT26 xenograft model at the dose of
100 mg/kg. (B) The change of body weight for mice that were treated with compound 1 and compound 6b. Data are presented as the mean SEM; n ¼ 5 BalB/C mice per group:
(***) P < 0.001 versus control group, determined with Student’s t-test.
99.70, 42.69, 42.38; HRMS (ESI, positive) m/z calcd for
C
4.1.5. (Z)-N-(2-((4-(N-(3-Bromo-4-fluorophenyl)-N0-
17H12BrFN9O5 (M ꢁ H)- 520.0134, found 522.0152.
hydroxycarbamim-idoyl)-1,2,5-oxa-diazol-3-yl)amino)ethyl)-2-((7-
nitrobenzo[c] [1,2,5]oxa-diazol-4-yl)amino)aceta-mide (6b)
The synthetic procedure of 6b was similar to that of 6b. Orange
solid, yield 14%. Purity >95%. 1H NMR (DMSO‑d6, 600 MHz)
d: 11.45
4.1.2. (7-Nitrobenzo[c] [1,2,5]oxadiazol-4-yl)glycine (5a)
Compound 3 (517 mg, 2.61 mmol), glycine (196 mg, 2.61 mmol)
and NaHCO3 (263 mg, 3.13 mmol) were dissolved in MeOH (10 mL)
and H2O (5 mL), and the mixture was heated to 60 ꢀC. The solvent
was removed under reduced pressure and the crude product was
purified by column chromatography on silica gel (dichloro-
methane: methanol ¼ 100: 8) to obtain the intermediate 5a
(s, 1 H), 9.43 (s, 1 H), 8.89 (s, 1H), 8.49 (d, J ¼ 9.80 Hz, 1H), 8.17 (s,
1 H), 7.17 (t, J ¼ 8.17 Hz 1H), 7.06e7.14 (m, 1H), 6.67e6.83 (m, 1H),
6.39 (d, J ¼ 7.80 Hz,1H), 6.20 (s,1H), 3.67 (s, 2H), 3.38 (s, 4H), 2.56 (t,
J ¼ 5.91 Hz, 2H). 13C NMR (DMSO‑d6, 150 MHz)
d: 170.59, 156.15,
154.95, 153.37, 144.81, 140.37, 139.72, 138.42, 138.24, 125.25, 121.91,
121.35, 116.33, 116.28, 107.54, 107.40, 99.67, 44.09, 37.81, 34.18;
HRMS (ESI, positive) m/z calcd for C20H19BrFN10O6 (M þ H)þ
593.0651, found 593.0657.
(292 mg, 47%) as an orange solid. 1H NMR (DMSO‑d6, 600 MHz)
d:
12.39 (s, 1 H), 9.44 (s, 1 H), 8.51 (d, J ¼ 8.06 Hz, 1H), 6.44 (d,
J ¼ 8.06 Hz, 1H), 3.67 (s, 2H), 2.72 (s, 2H).
4.2. Biological activity
4.1.3. 3-((7-Nitrobenzo[c] [1,2,5]oxadiazol-4-yl)amino)propanoic
acid (5b)
4.2.1. IDO1 enzymatic assay
The assays were performed according to the literature [35]. The
reaction mixture contained the test compounds at different con-
The synthetic procedure of 5b was similar to that of 5a. Orange
solid, yield 58%. 1H NMR (DMSO‑d6, 600 MHz)
d: 13.09 (s, 1 H), 9.40
centrations, 20 nM IDO1, 2 mM Trp, 3.5 mM methylene blue, 20 mM
(s, 1 H), 8.53 (d, J ¼ 8.90 Hz, 1H), 6.37 (d, J ¼ 8.90 Hz, 1H), 4.28 (s,
ascorbate, and 0.2 mg/mL catalase in 50 mM potassium phosphate
buffer (pH 6.5). The absorbance (OD) at 321 nm was read on the
WellscanMK-2 microplate reader (Labsystems). The concentration
triggering 50% inhibition (IC50) values were calculated using Prism
GraphPad software based on nonlinear regression with normalized
doseꢁresponse fit.
2H).
4.1.4. (Z)-N-(2-((4-(N-(3-Bromo-4-fluorophenyl)-N0-
hydroxycarbamim-idoyl)-1,2,5-oxa-diazol-3-yl)amino)ethyl)-2-((7-
nitrobenzo[c] [1,2,5]oxa-diazol-4-yl)amino)aceta-mide (6a)
The intermediate 5a (238 mg, 1 mmol), compound 2a (383 mg,
4.2.2. In vitro cytotoxicity assay
1 mmol), HATU (570 mg, 1.5 mmol) and DIPEA (265 mL, 1.5 mmol)
Human cervical cancer cell line HeLa or murine colon carcinoma
cell line CT26 in the logarithmic phase were harvested and seeded
at a density of 6 ꢂ 103/well in 96-well plates. After incubation for
24 h with 5% CO2 at 37 ꢀC, the cells were treated with test com-
pounds at different concentrations in three replicates for 72 h. Then
were dissolved in DMF (6 mL) and the mixture was stirred at room
temperature for 1 h. Then the solution was poured into saturated
NaCl aqueous solution (80 mL) and extracted with ethyl acetate
(3 ꢂ 15 mL). The organic phase was collected and concentrated
under reduced pressure to obtain the crude product, which was
subsequently dissolved in MeOH (10 mL) and H2O (5 mL) along
with KOH (78 mg, 2 mmol). The mixture was stirred at room
temperature for 0.5 h, and then the solvent was removed under
reduced pressure and the crude product was purified by column
chromatography on silica gel (dichloromethane: methanol ¼ 100:
1) to obtain the intermediate 6a (81 mg, 21%) as an orange solid.
they were replaced by100 mL of culture media containing 10% CCK8
kit and the plates were incubated for another 40 min. Then OD
values at 480 nm were read on the WellscanMK-2 microplate
reader (Labsystems). The concentration triggering 50% inhibition
(IC50) values were calculated using Prism GraphPad software based
on nonlinear regression with normalized doseꢁresponse fit.
Purity >95%. 1H NMR (DMSO‑d6, 600 MHz)
d: 9.43 (s, 1 H), 8.50 (t,
J ¼ 8.73 Hz, 1H), 8.32 (s, 1H), 8.08 (s, 1H), 7.72 (s, 1H), 7.49e7.66 (m,
4.2.3. Cell based IDO1 assay
1H), 6.55 (s, 1H), 6.24 (s, 1H), 5.56e5.88 (m, 1H), 4.12 (s, 2H), 3.53-
Cell based IDO1 assay was carried out according to the literature
[17,26]. HeLa cells originating from human cervical cancer in the
logarithmic phase were harvested and seeded at a density of
6 ꢂ 103/well in 96-well plates. After incubation for 24 h with 5% CO2
3.40 (m, 2H), 3.32 (s, 2H); 13C NMR (DMSO‑d6, 150 MHz)
d:170.59,
156.13, 154.22, 144.82, 140.39, 139.73, 138.44, 138.26, 125.27, 121.93,
116.36, 107.46, 99.68, 44.10, 37.83, 34.20; HRMS (ESI, positive) m/z
calcd for C19H15BrFN10O6 (M ꢁ H)- 577.0349, found 577.0336.
at 37 ꢀC, human IFN-
g (50 ng/mL), Trp (15 mg/mL) and test
7

Y. Wu, Y. Zhang, X. Chen et al.
European Journal of Medicinal Chemistry 213 (2021) 113163
compounds at different concentrations were added in three repli-
Discovery Studio 3.0 [34]. The ligands were also prepared and hy-
drogens were added to their structures. Then docking experiments
were carried out using Gold, in which the parameters were set by
default[ [34]]. The best conformation was chosen to analyze the
ligand-protein interactions between IDO1 and probes, respectively.
The figure demonstrating the best conformation was prepared us-
ing PyMOL.
cates. After 48 h, 140
mL of the supernatant was transferred to
another 96-well plate, and then 10
m
L of 6 N trichloroacetic acid was
added, separately. The mixture was incubated for 30 min at 50 ꢀC,
and then centrifuged at 2.5 ꢂ 103 rpm for 10 min. After that, 100
mL
of the supernatant was transferred to another 96-well plate, to
which 100 mL of 2% (w/v) p-dimethylaminobenzaldehyde in acetic
acid was added. Then OD values at 480 nm were read on the
WellscanMK-2 microplate reader (Labsystems). The concentration
triggering 50% inhibition (IC50) values were calculated using Prism
GraphPad software based on nonlinear regression with normalized
doseꢁresponse fit.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
4.2.4. General fluorescence spectra measurements
A stock solution of probes was prepared in DMSO. All fluores-
cence titration experiments were performed for 60 min at room
temperature using PBS buffer solution. Any changes in the fluo-
rescence intensity were monitored using a fluorescence spec-
trometer (lex ¼ 465 nm, slit widths: Wex ¼ 5 nm, Wem ¼ 5 nm).
Acknowledgements
This work was supported by the National Key R&D Program of
China (grant 2017YFA0506000 to C. S.), and National Natural Sci-
ence Foundation of China (grants 81725020 and 21738002 to C. S.,
grant 21977082 to Y. G.).
4.2.5. IDO1 cloning, protein expression and purification
The gene of human IDO1 was inserted into an engineered
pET21a vector (containing a sumo tag) with BamHI and XhoI as the
ligation sites. The resulted construct was transformed into Escher-
ichia coli BL21(DE3). The culture was grown in Luria Broth medium
to reach an OD600nm of 0.6, and the induction was performed by
adding isopropyl–D-galactoside to a final concentration of 0.4 mM.
The cells were cultured for another 18 h at 18 ꢀC. Cells were har-
vested by centrifugation at 6000 rpm for 20 min and then lysed by
sonication. After another round of centrifugation at 13,000 rpm for
45 min, the supernatant was collected and was loaded onto a 5-mL
HisTrap HP column (GE Healthcare Life Sciences). The IDO1 protein
was eluted by a gradient from 0 to 250 mM imidazole within
25 min and buffer exchanged to 50 mM sodium phosphate, pH 8.0
and 500 mM sodium chloride. Ulp1 cleavage of the sumo tag was
performed a 13:1 quantity ratio (protein: protease) overnight at
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113163.
References
[1] K.M. Mahoney, P.D. Rennert, G.J. Freeman, Combination cancer immuno-
therapy and new immunomodulatory targets, Nat. Rev. Drug Discov. 14
(2015) 561e584.
[2] Y. Liu, P. Zheng, Preserving the CTLA-4 checkpoint for safer and more effective
cancer immunotherapy, Nat. Immunol. Sci 41 (1) (2020) 4e42.
[3] L.P. Andrews, H. Yano, D.A.A. Vignali, Inhibitory receptors and ligands beyond
PD-1, PD-L1 and CTLA-4: breakthroughs or backups, Nat. Immunol. 20 (2019)
1425e1434.
[4] B. Cheng, W.E. Yuan, J. Su, Y. Liu, J. Chen, Recent advances in small molecule
based cancer immunotherapy, Eur. J. Med. Chem. 157 (2018) 582e598.
4
ꢀC. The cleaved sample was loaded to the HisTrap HP column
€
[5] B.R. Huck, L. Kotzner, K. Urbahns, Small molecules drive big improvements in
immuno-oncology therapies, Angew. Chem. Int. Ed. 57 (2018) 4412e4428.
[6] D. Dhanak, J.P. Edwards, A. Nguyen, P.J. Tummino, Small-molecule targets in
immuno-oncology, Cell Chem. Biol 24 (2017) 1148e1160.
[7] F. Li, R. Zhang, S. Li, J. Liu, Ido1: an important immunotherapy target in cancer
treatment, Int. Immunopharm. 47 (2017) 70e77.
[8] B. Ricciuti, G.C. Leonardi, P. Puccetti, F. Fallarino, V. Bianconi, A. Sahebkar,
S. Baglivo, R. Chiari, M. Pirro, Targeting indoleamine-2,3-dioxygenase in
cancer: scientific rationale and clinical evidence, Pharmacol. Ther. 196 (2019)
105e116.
again, the flow through was collected and EDTA was added to the
sample to the concentration of 2 mM. In the final step, the protein
was further purified by gel filtration on a Sperdex75 10/300 GL
column (GE Healthcare Life Sciences) in 20 mM Tris, pH ¼ 7.5,
150 mM sodium chloride and 1 mM EDTA.
4.2.6. In vivo studies
[9] L. Brochez, I. Chevolet, V. Kruse, The rationale of indoleamine 2,3-dioxygenase
inhibition for cancer therapy, Eur. J. Canc. 76 (2017) 167e182.
[10] J.E. Cheong, A. Ekkati, L. Sun, A patent review of Ido1 inhibitors for cancer,
Expert Opin. Ther. Pat. 28 (2018) 317e330.
CT 26 cells which were in the logarithmic phase were harvested
and resuspended in RPMI culture medium. Balb/c female mice (4e5
weeks) were inoculated subcutaneously into the right forelimb
with a density of 5 ꢂ 105 cells in 0.2 mL. Upon tumor volumes
reached approximately 50 mm3, they were assigned into groups
randomly and administered with compounds orally at a dosage of
100 mg/kg every day. The length (a), width (b) of the tumor and
body weight of mice were recorded every other day and the tumor
volumes were calculated according to V ¼ 1/2 ꢂ a ꢂ b2. When
tumor volume of control group approached approximately
2000 mm3, the mice were sacrificed. Tumor growth inhibition (TGI)
was calculated as TGI (%) ¼ (Vtcontrol -Vtcompound)/Vtcontrol (Vtcom-
pound refers to the tumor volume of the test compound group at the
last day, while Vtcontrol refers to the to the tumor volume of the
control group at the last day).
€
[11] U.F. Rohrig, S.R. Majjigapu, P. Vogel, V. Zoete, O. Michielin, Challenges in the
discovery of indoleamine 2,3-dioxygenase 1 (Ido1) inhibitors, J. Med. Chem.
58 (2015) 9421e9437.
[12] G.C. Prendergast, W.P. Malachowski, J.B. DuHadaway, A.J. Muller, Discovery of
Ido1 inhibitors: from bench to bedside, Canc. Res. 77 (2017) 6795e6811.
[13] B.W. Labadie, R. Bao, J.J. Luke, Reimagining Ido pathway inhibition in cancer
immunotherapy via downstream focus on the tryptophan-kynurenine-aryl
hydrocarbon Axis, Clin. Canc. Res. 25 (2019) 1462e1471.
[14] G.V. Long, R. Dummer, O. Hamid, T.F. Gajewski, C. Caglevic, S. Dalle, A. Arance,
M.S. Carlino, J.J. Grob, T.M. Kim, L. Demidov, C. Robert, J. Larkin, J.R. Anderson,
J. Maleski, M. Jones, S.J. Diede, T.C. Mitchell, Epacadostat plus pembrolizumab
versus placebo plus pembrolizumab in patients with unresectable or meta-
static melanoma (ECHO-301/KEYNOTE-252): a phase 3, randomised, double-
blind study, Lancet Oncol. 20 (2019) 1083e1097.
[15] S. Cai, X. Yang, P. Chen, X. Liu, J. Zhou, H. Zhang, Design, synthesis and bio-
logical evaluation of bicyclic carboxylic acid derivatives as Ido1 inhibitors,
Bioorg. Chem. (2019) 103356.
[16] H. Zhang, K. Liu, Q. Pu, A. Achab, M.J. Ardolino, M. Cheng, Y. Deng, A.C. Doty,
H. Ferguson, X. Fradera, I. Knemeyer, R. Kurukulasuriya, Y.H. Lam,
C.A. Lesburg, T.A. Martinot, M.A. McGowan, J.R. Miller, K. Otte, P.J. Biju,
N. Sciammetta, N. Solban, W. Yu, H. Zhou, X. Wang, D.J. Bennett, Y. Han,
4.3. Molecular docking simulations
The crystal structure of IDO1 in complex with epacadostat was
obtained from protein database bank (PDB code: 5WN8) [33] which
was then prepared for docking via the protein preparation tool in
Discovery of amino-cyclobutarene-derived indoleamine-2,3-dioxygenase
1
(Ido1) inhibitors for cancer immunotherapy, ACS Med. Chem. Lett. 10 (2019)
1530e1536.
8

Y. Wu, Y. Zhang, X. Chen et al.
European Journal of Medicinal Chemistry 213 (2021) 113163
[17] Y. Zou, Y. Hu, S. Ge, Y. Zheng, Y. Li, W. Liu, W. Guo, Y. Zhang, Q. Xu, Y. Lai,
Effective virtual screening strategy toward heme-containing proteins: iden-
tification of novel Ido1 inhibitors, Eur. J. Med. Chem. 184 (2019) 111750.
[18] Q. Du, X. Feng, Y. Wang, X. Xu, Y. Zhang, X. Qu, Z. Li, J. Bian, Discovery of
phosphonamidate Ido1 inhibitors for the treatment of non-small cell lung
cancer, Eur. J. Med. Chem. 182 (2019) 111629.
[19] Y. Zhou, J. Peng, P. Li, H. Du, Y. Li, L. Zhang, W. Sun, X. Liu, Z. Zuo, Discovery of
novel indoleamine 2,3-dioxygenase 1 (Ido1) inhibitors by virtual screening,
Comput. Biol. Chem. 78 (2019) 306e316.
J.S. Fridman, T. Emm, P.A. Scherle, B. Metcalf, A.P. Combs, INCB24360 (epa-
cadostat), a highly potent and selective indoleamine-2,3-dioxygenase 1 (Ido1)
inhibitor for immuno-oncology, ACS Med. Chem. Lett. 8 (2017) 486e491.
[28] Y.D. Zhuang, P.Y. Chiang, C.W. Wang, K.T. Tan, Environment-sensitive fluo-
rescent turn-on probes targeting hydrophobic ligand-binding domains for
selective protein detection, Angew. Chem. Int. Ed. 52 (2013) 8124e8128.
[29] R.P. Huijbregts, A.I. de Kroon, B. de Kruijff, Rapid transmembrane movement
of C6-NBD-labeled phospholipids across the inner membrane of Escherichia
coli, Biochim. Biophys. Acta 1280 (1996) 41e50.
[20] U.F. Rohrig, A. Reynaud, S.R. Majjigapu, P. Vogel, F. Pojer, V. Zoete, Inhibition
[30] K. Cui, Z. Chen, Z. Wang, G. Zhang, D. Zhang, A naked-eye visible and fluo-
rescence "turn-on" probe for acetyl-cholinesterase assay and thiols as well as
imaging of living cells, Analyst 136 (2011) 191e195.
mechanisms of indoleamine 2,3-dioxygenase
1 (Ido1), J. Med. Chem. 62
(2019) 8784e8795.
[21] K.N. Pham, A. Lewis-Ballester, S.R. Yeh, Structural basis of inhibitor selectivity
in human indoleamine 2,3-dioxygenase 1 and tryptophan dioxygenase, J. Am.
Chem. Soc. 141 (2019) 18771e18779.
[22] M.T. Nelp, P.A. Kates, J.T. Hunt, J.A. Newitt, A. Balog, D. Maley, X. Zhu, L. Abell,
A. Allentoff, R. Borzilleri, H.A. Lewis, Z. Lin, S.P. Seitz, C. Yan, J.T. Groves, Im-
mune-modulating enzyme indoleamine 2,3-dioxygenase is effectively
inhibited by targeting its apo-form, Proc. Natl. Acad. Sci. U. S. A 115 (2018)
3249e3254.
[23] D. Yang, S. Zhang, X. Fang, L. Guo, N. Hu, Z. Guo, X. Li, S. Yang, J.C. He, C. Kuang,
Q. Yang, N-Benzyl/Aryl substituted tryptanthrin as dual inhibitors of indole-
amine 2,3-dioxygenase and tryptophan 2,3-dioxygenase, J. Med. Chem. 62
(2019) 9161e9174.
[24] K. Fang, G. Dong, Y. Li, S. He, Y. Wu, S. Wu, W. Wang, C. Sheng, Discovery of
novel indoleamine 2,3-dioxygenase 1 (Ido1) and histone deacetylase (HDAC)
dual inhibitors, ACS Med. Chem. Lett. 9 (2018) 312e317.
[25] K. Fang, G. Dong, H. Wang, S. He, S. Wu, W. Wang, C. Sheng, Improving the
potency of cancer immunotherapy by dual targeting of Ido1 and DNA,
ChemMedChem 13 (2018) 30e36.
[26] K. Fang, S. Wu, G. Dong, Y. Wu, S. Chen, J. Liu, W. Wang, C. Sheng, Discovery of
Ido1 and DNA dual targeting antitumor agents, Org. Biomol. Chem. 15 (2017)
9992e9995.
[27] E.W. Yue, R. Sparks, P. Polam, D. Modi, B. Douty, B. Wayland, B. Glass,
A. Takvorian, J. Glenn, W. Zhu, M. Bower, X. Liu, L. Leffet, Q. Wang,
K.J. Bowman, M.J. Hansbury, M. Wei, Y. Li, R. Wynn, T.C. Burn, H.K. Koblish,
[31] W. Jiang, Q. Fu, H. Fan, J. Ho, W. Wang, A highly selective fluorescent probe for
thiophenols, Angew. Chem. Int. Ed. 46 (2007) 8445e8448.
[32] L. Yi, Z. Xi, Thiolysis of NBD-based dyes for colorimetric and fluorescence
detection of H(2)S and biothiols: design and biological applications, Org.
Biomol. Chem. 15 (2017) 3828e3839.
[33] A. Lewis-Ballester, K.N. Pham, D. Batabyal, S. Karkashon, J.B. Bonanno,
T.L. Poulos, S.R. Yeh, Structural insights into substrate and inhibitor binding
sites in human indoleamine 2,3-dioxygenase 1, Nat. Commun. 8 (2017) 1693.
[34] G. Jones, P. Willett, R.C. Glen, A.R. Leach, R. Taylor, Development and valida-
tion of a genetic algorithm for flexible docking, J. Mol. Biol. 267 (1997)
727e748.
[35] M.F. Cheng, M.S. Hung, J.S. Song, S.Y. Lin, F.Y. Liao, M.H. Wu, W. Hsiao,
C.L. Hsieh, J.S. Wu, Y.S. Chao, C. Shih, S.Y. Wu, S.H. Ueng, Discovery and
structure-activity relationships of phenyl benzenesulfonylhydrazides as novel
indoleamine 2,3-dioxygenase inhibitors, Bioorg. Med. Chem. Lett 24 (2014)
3403e3406.
[36] J.E. Cheong, L. Sun, Targeting the Ido1/TDO2-KYN-AhR pathway for cancer
immunotherapy - challenges and opportunities, Trends Pharmacol. Sci. 39
(2018) 307e325.
[37] H.K. Koblish, M.J. Hansbury, K.J. Bowman, G. Yang, C.L. Neilan, P.J. Haley,
T.C. Burn, P. Waeltz, R.B. Sparks, E.W. Yue, A.P. Combs, P.A. Scherle, K. Vaddi,
J.S. Fridman, Hydroxyamidine inhibitors of indoleamine-2,3-dioxygenase
potently suppress systemic tryptophan catabolism and the growth of Ido-
expressing tumors, Mol. Canc. Therapeut. 9 (2010) 489e498.
9