Improving the Potency of Cancer Immunotherapy by Dual Targeting of IDO1 and DNA

Article abstract of DOI:10.1002/cmdc.201700666

Herein we report the first exploration of a dual-targeting drug design strategy to improve the efficacy of small-molecule cancer immunotherapy. New hybrids of indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors and DNA alkylating nitrogen mustards that respectively target IDO1 and DNA were rationally designed. As the first-in-class examples of such molecules, they were found to exhibit significantly enhanced anticancer activity in vitro and in vivo with low toxicity. This proof-of-concept study has established a critical step toward the development of a novel and effective immunotherapy for the treatment of cancers.

Full text of DOI:10.1002/cmdc.201700666

Accepted Article
Title: Improving the Potency of Cancer Immunotherapy by Dual
Targeting IDO1 and DNA
Authors: kun Fang, Guoqiang Dong, Hongyu Wang, Shipeng He,
Shanchao Wu, Wei Wang, and Chunquan Sheng
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201700666
Link to VoR: http://dx.doi.org/10.1002/cmdc.201700666
A Journal of
www.chemmedchem.org

10.1002/cmdc.201700666
ChemMedChem
COMMUNICATION
Improving the Potency of Cancer Immunotherapy by Dual
Targeting IDO1 and DNA
Kun Fang,†[a][b] Guoqiang Dong,†[b] Hongyu Wang,[b] Shipeng He,[a] Shanchao Wu,[b] Wei Wang,*[a][c] and
Chunquan Sheng*[b]
[a]
[b]
Dr. K. Fang, S. He and Prof. Dr. W. Wang
School of Pharmacy, East China University of Science and Technology
Shanghai 200237, P. R. China.
E-mail: wwang@unm.edu
G. Dong, H. Wang, S. Wu, and Prof. Dr. C. Sheng
Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University
325 Guohe Road, Shanghai 200433, P. R. China
E-mail: shengcq@smmu.edu.cn
[c]
Prof. Dr. W. Wang
Department of Chemistry and Chemical Biology, University of New Mexico
Albuquerque, NM87131-0001, USA
Dr. K. Fang and G. Dong contributed equally to this work.
†
Supporting information for this article is given via a link at the end of the document.
Abstract: Here we report the first effort on exploration of the dual-
targeting drug design strategy to improve the efficacy of small
molecule cancer immunotherapy. New hybrids of IDO1 (indoleamine
2, 3-dioxygenase 1) inhibitors and DNA alkylating nitrogen mustards
targeting respective IDO1 and DNA are rationally designed. As the
first-in-class example of such molecules, they exhibited significantly
enhanced anticancer activity in vitro and in vivo with low toxicity. The
proof-of-concept by the studies has established a critical step toward
developing novel and effective immunotherapy for the treatment of
cancers.
synergized with traditional chemo-therapeutics to enhance
antitumor efficacy both in vitro and in vivo.[11] IDO1
inhibitors in combination with cytotoxic chemotherapeutic
agents have been identified as an effective remedy in
cancer therapy. Moreover, synergistic antitumor effects are
observed in combination IDO1 inhibitors with temozolomide
11c]
or cyclophosphamide.[11b,
Several clinical trials are
underway to determine the potential synergistic effects
combining indoximod or epacadostat with traditional cancer
chemotherapies.[10, 12]
Classic DNA alkylating agents (Figure S1) such as nitrogen
mustards are widely used clinically for the treatment of a
variety of cancers.[13] However, the poor drug potency,
dose-limiting toxicities, drug resistance, substantial
pharmacokinetic and response variability remain significant
hurdles in the use of these drugs. The search of highly
potent and safe DNA alkylation agents represents a long-
standing interest in drug discovery.
Cancer immunotherapy by the use of the immune system to
treat cancer has been recognized as a pillar of cancer therapy
by joining the ranks of surgery, radiation, chemotherapy, and
targeted therapy.[1] However, the overarching challenge in
current clinically used immunotherapy is that it is only effective
for a fraction of cancer patients whose immunogenic tumor
microenvironment can respond to activated immune cells.[1-2]
This is largely because tumor cells are capable of escaping
immune surveillance and suppress antitumor immunity.[3] Among
them, kynurenine (Kyn) pathway of tryptophan (Trp) catabolism
plays a key role in immune escape,[4] in which indoleamine 2,3-
dioxygenase 1 (IDO1) catalyzes the first and rate-limiting step.[5]
IDO1 is a heme-containing enzyme that oxidizes Trp to N-
formylkynurenine (NFK) and subsequent deformylation by
formamidase produces Kyn.[6] Both Trp degradation and Kyn
accumulation are associated with immune tolerance by affecting
T cell activity and altering the tumor microenvironment.[7]
Although studies have showed that the co-administration of
IDO1 inhibitors indoximod or epacadostat with traditional
cancer chemotherapies exhibits promising anti-cancer
synergistic effects, the simple combination of IDO1
inhibitors indoximod or epacadostat with traditional cancer
chemotherapies cannot overcome severe side effects
caused by these highly toxic compounds and possible drug-
drug interactions. In addition, the different pharmacokinetics,
bio-distributions and metabolisms of two individual drugs
make them difficult to achieve optimal therapeutic effects,
particularly synergistic actions. Ideally, one drug entity with
the capacity of targeting two targets simultaneously may
overcome these inherently challenging problems. Toward
this end, herein we rationally designed new hybrid
molecules by incorporation of the pharmacophores of IDO1
inhibitors and DNA alkylating agents. We found that they
exhibited highly inhibitory activity against IDO1 with
significantly reduced side effects caused by the cytotoxic
alkylating agents.
IDO1 has become an attractive target for cancer
immunotherapy. Currently, three small molecule IDO1
inhibitors are under clinical development (Figure S1):
indoximod (1-D-MT), epacadostat (INCB024360) and GDC-
0919.[8] However, indoximod is a weak IDO1 inhibitor with
the IC50 value greater than 100 μM.[9] GDC-0919
demonstrated a high dose tolerance, but failed to induce
tumor eradication and prevent disease progression in
phase I clinical trial.[8c] Hydroxyamidine IDO1 inhibitor
epacadostat is still in active clinical development.[10]
Mechanistically, IDO1 inhibitors do not destroy tumor cells
directly. A number of studies showed that IDO1-blockade
We chose IDO1 inhibitor epacadostat and DNA alkylating
agents[14] chlorambucil and melphalan as the starting points in
1
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700666
ChemMedChem
COMMUNICATION
Figure 1. (A) Model of epacadostat bound to IDO1 (PDB ID: 4PK5). (B)
Design strategy of the dual targeting agents.
the design of dual targeting molecules (Figure 1). Human IDO1
contains a binding pocket in the distal heme site (pocket A) and
a second pocket toward the entrance of the active site (pocket B,
Figure 1A). Molecular docking was performed to investigate the
binding mode of epacadostat with IDO1 (Figure 1A).[15] The
meta-bromophenyl, hydroxyamidine and oxadiazole groups in
epacadostat are essential for the IDO1 inhibition.[16] The phenyl
and hydroxyamidine groups are bound to the pocket A in the
active site of IDO1. The side chain projected out of the active
site in pocket B toward solvent, which could be modified to
optimize IDO1 inhibitory activity and physicochemical properties.
Guided by the binding mode, the aminoethyl-sulfamide
substituent of epacadostat is replaced by the alkylating
The newly designed dual targeting molecules are
synthesized using the methods depicted in Scheme 1. The
IDO1 inhibitory activity of the synthesized compounds
4-13
was investigated against human recombinant IDO1 and
IDO1 in HeLa cells accordingly.[16] As shown in Table 1,
most compounds showed moderate to good activity at the
molecular and cellular level. For the compounds conjugated
with chlorambucil or phenyl carboxylic acid mustard, N,N-
bis(2-chloroethyl)-amine
(compounds and ) resulted in better enzyme inhibition
than those of the corresponding para-position derivatives
(compounds and ). Moreover, it was observed that the
group
at
meta-position
4
,
5
6
pharmacophore
N,N-bis(2-chloroethyl)-amine
using
the
pharmacophore fusion strategy. As a result, novel dual targeting
molecules 4-13 are rationally designed (Figure 1B), in which
various spacers will be investigated to probe IDO1 inhibitory
activity and antitumor potency.
7
,
8
9
cellular activity was decreased with the increasing of the
carbon chain length with the exception of compound 10. In
particular, compounds
4 (IC50 = 0.13 μM, EC50 = 0.27 μM),
8
(IC50 = 0.94 μM, EC50 = 0.30 μM) and 13 (IC50 = 0.94 μM,
EC50 = 0.26 μM) displayed excellent enzyme and cellular
inhibitory activity.
To investigate whether the cellular activity was caused by IDO1
inhibition or solely by cytotoxicity, cell-based assay was also
performed to determine LC50 values of the target compounds
against HeLa cells and the ratio (LC50/EC50) was calculated. As
shown in Table 1, the ratios were generally larger than 30,
indicating that the cellular activities were caused by IDO1-
blockade. However, compounds 10-12 showed LC50 values that
were only 11 to 23-fold higher than their corresponding EC50
values in the cell-based assay, suggesting that the cellular
activities of these compounds were mainly resulted from their
cytotoxicity instead of IDO1 inhibition.
2
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700666
ChemMedChem
COMMUNICATION
Scheme 1. Synthesis of Compounds
4-
13. Reagents and Conditions: (a) HATU, DIPEA, DMF, rt, 12 h, yield 42-66%; (b) CF3COOH, DCM, 40 °C, 3 h,
yield 74%; (c) Ethylene oxide, MeOH, 50 °C, overnight, yield 78%; (d) SOCl2, DCM, 40 °C, 3 h, yield 85%; (e) NaOH, MeOH, rt, 2 h, yield 54-93%.
Table 1. IDO1 enzyme inhibition and cellular potency of
dependent manner. After exposed with compounds
4 and 8
target compoundsa.
for 48 h, the ratios in G2/M phase of the cell cycle were
29.23% and 69.72%, respectively. In contrast, the ratio of
cells with vehicle in G2/M phase of the cell cycle was
10.24%. The data suggested that the CT-26 cells treated
rhIDO1 IC50
Ratio
LC50
EC50
(μM) or
%Inhibition at 1
μM
HeLa
(EC50, μM)
HeLa
(LC50, μM)
Compounds
/
with compound
In vivo antitumor efficacy of compounds
evaluated in CT-26 tumor growth model in
4
or
8
showed G2/M phase arrest.
Epacadostat
0.066 ± 0.008
0.13 ±0.02
0.83 ± 0.007
0.85 ± 0.005
26%
0.029 ± 0.005
0.27 ± 0.059
0.47 ± 0.052
0.86 ± 0.080
0.21 ± 0.034
0.30 ± 0.076
0.86 ± 0.073
0.79 ± 0.068
1.5 ± 0.15
>100
>100
>100
>100
63
4
and were firstly
8
4
5
39 ± 4.33
52 ± 5.25
54 ± 2.21
32 ± 1.22
27 ± 2.46
27 ± 3.21
18 ± 1.63
21 ± 0.54
25 ± 0.87
27 ± 1.22
a
immunocompetent mice by measuring tumor growth
inhibition (TGI). When implanted tumors had reached a
6
volume of about 100 mm3, compounds
4 and 8 were
7
>100
91
administered orally (po) at 100 mg/kg twice a day for 14
consecutive days. Epacadostat (100 mg/kg, po, bid) was
used as the positive control. As shown in Figure 2A, both
8
0.94 ± 0.01
31%
9
32
10
11
12
13
23%
23
compounds
(P < 0.001) with dramatic reduction in tumor growth.
Compound showed comparable antitumor activity (TGI =
47.9%) to that of epacadostat (TGI = 47.5%). Notably,
compound displayed superior antitumor activity (TGI =
58.2%). Importantly, mice treated with a high dose of and
(100 mg/kg, po, bid) showed no significant body weight
loss (Figure S5) and no adverse effects were observed.
Furthermore, compound was also evaluated for in vivo
4 and 8 produced significant antitumor activity
1.3 ± 0.05
10%
14
8
2.3 ± 0.13
11
0.94 ± 0.02
0.26 ± 0.016
>100
4
aIC50, EC50 and LC50 values are the mean of at least three independent assays,
presented as mean ± SD.
4
8
The target compounds were further screened for their in
vitro antiproliferative activities against four cancer cell lines
(murine colon adenocarcinoma CT-26 cells, human lung
cancer A549 cells, colon cancer HCT116 and HT-29 cells)
by the standard CCK-8 methods (Table S1). The IC50
values revealed that all compounds showed better
antiproliferative activities than positive DNA alkylating drug
chlorambucil for all the tested cell lines whereas IDO1
inhibitor epacadostat was totally inactive. All the target
compounds exhibited moderate to good antiproliferative
potencies with a broad spectrum against all the four cell
4
efficacy of tumor xenograft in nude mice bearing human
CT-26 tumors. As shown in Figure 2B, it exhibited
significant antitumor activity (P < 0.01) and reduced 28.6%
of the tumor volume. On the basis of the in vivo antitumor
activity in immunocompetent and immunodeficient mice, it
could be reached that the antitumor effect of compound
4
resulted from the synergistic effect of antitumor immunity
and cytotoxicity to the tumor cell.
lines. Finally, compounds
4 and 8 were chosen for the
further study because of their potent IDO1 inhibitory activity,
highly cellular potency (Figure S2) and moderate in vitro
cytotoxicity.
To clarify whether compounds
4 and 8 can lead to cell
death by apoptosis, an annexin VFITC/propidium iodide (PI)
binding assay was performed. CT-26 cells were treated with
vehicle alone or various concentrations of the tested
compounds for 48 h, then stained with Annexin V-FITC and
PI, and analyzed by flow cytometry. Compounds
generated significant induction of apoptosis in the CT-26
cell line in a dose-dependent manner (Figure S3). The
4 and 8
Figure 2. Compounds 4 and 8 effectively suppressed the tumor growth. (A)
Antitumor efficacy of compounds 4 and 8 in murine CT-26 tumor allograft
model. Data are expressed as the mean ± SEM (n = 6 mice/group). *** P <
0.001 compared with the control group; * P < 0.05 compared between group
epacadostat and 4, determined with Student’s t test. (B) In vivo efficacy of
compound 4 in CT-26 tumor xenograft model. Data are expressed as the
mean ± SEM (n = 6 mice/group). ** P < 0.01 compared with the control group,
determined with Student’s t test.
percentage of apoptotic cells for compounds
and (at 40 μM) were 68.69% and 91.04%, respectively.
Therefore, it is concluded that the antiproliferative activity of
compounds and could be ascribed to the induction of
4 (at 50 μM)
8
4
8
apoptosis in CT-26 cells.
Cell cycle distribution of compounds
4 and 8 were further
examined in CT-26 cells (Figure S4) to investigate whether
the cell growth suppression was induced by cell cycle arrest.
CT-26 cells were treated with vehicle alone or the tested
Overexpression of IDO1 is a common feature in many
tumor types, which lead to Kyn production and
accumulation and Trp degradation. Thus, to investigate the
IDO1 activity in vivo, pharmacodynamic assay[16] was
performed. Wild-type female C57BL/6 mice were
compounds (compound
for 12 h, 36 h and 48 h, respectively. Initially, both
compounds and induced an increase in cells at G2/M
4 at 25 μM, compound 8 at 15 μM)
4
8
administrated orally with
4 (100 mg/kg) and then blood was
phase after 12 h of the drug administration. The results
showed a steady increase in cells at G2/M phase in a time-
collected at the indicated times. Blood samples were
3
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700666
ChemMedChem
COMMUNICATION
prepared for docking using Protein Preparation Wizard. During this
process, waters were removed and hydrogens were added to the
structure. The resulting structure was refined by OPLS3 force-field with
the hydrogens only. Then, Receptor Grid Preparation was used to
generate the protein grid for docking experiments. Amg-1 was picked for
ligand identification. Fe in heme was selected which could participate in
metal-ligand interactions. Other parameters were set default. Ligand
Docking option was used for docking performance. Under Setting, SP
(standard precision), “Dock flexibly”, “Sample nitrogen inversions”,
“Sample ring conformation” and “Epik state penalties” to docking score
were selected.
analyzed by LC/MS/MS for Kyn level detection. As shown in
Figure 3A, compound
4 significantly reduced plasma Kyn
level after 0.5 h oral exposure and the effects maintained
and enhanced in the next 8 h. Additionally, reduction of the
tumor Kyn to Trp (Kyn/Trp) ratio caused by
4 and 8 was
also measured in CT-26 tumor allograft model. Tumor
samples were collected at 3 h after last dosing after 14
days of treatment (100 mg/kg, po, bid). The Kyn and Trp
levels were determined by LC/MS/MS, and the ratios of
Kyn/Trp were calculated. It was found that the Kyn/Trp ratio
was significantly lower in compound
that in the control (Figure 3B). The results indicated that
compound significantly inhibited IDO1 activity in tumor
tissues and reduced Kyn level in plasma.
4 treated mice than
Conformations were generated and scored using glide score as fitness
function. The best conformation was chosen to analyse the ligand–
protein interaction.
4
IDO1 Enzyme Activity Assay[18]
IDO1 catalyzes the oxidative cleavage of the pyrrole ring of the indole
nucleus of tryptophan to yield NFK. The assays were performed at room
temperature as described in the literature using 20 nM IDO1 (NOVUS
BioscienceInc. # H00003620-P01) and 2 mM D-Trp in the presence of 20
mM ascorbate, 3.5 uM methylene blue and 0.2 mg/mL catalase in 50 mM
potassium phosphate buffer (pH 6.5). The initial reaction rates were
recorded by continuously following the absorbance increase at 321 nm
due to the formation of NFK. The IC50 values were calculated using
nonlinear regression with normalized dose-response fit using Prism
GraphPad software.
IDO1 Cellular Assay[18]
Figure 3. Compound
Mean (± SD) plasma concentration-time profiles of Kyn in wild-type
female C57BL/6 mice (n = 3) following po with (100 mg/kg). (B) Mean
4 reduced Kyn and Kyn/Trp ratio efficiently. (A)
HeLa cells were seeded in 96-well culture plates at a density of 6 × 103
per well. On the next day, human IFN-γ (Sangon Biotech # C600039, 50
ng/mL) and compounds in a total volume of 200 L culture medium
containing 15 g/mL of Try were added to the cells. After incubation for
48 hours, 140 L of the supernatant was mixed with 10 L of 6.1N
trichloroacetic acid and the mixture was incubated for 30 min at 50 °C to
hydrolyze NFK produced by IDO1 to Kyn. The reaction mixture was then
centrifuged for 10 minutes at 2500 rpm to remove sediments. 100 L of
the supernatant was mixed with 100 L of 2% (w/v) p-
dimethylaminobenzaldehyde in acetic acid and measured at 480 nm. The
data was processed using nonlinear regression to generate EC50 values
(Prism Graphpad).
4
(± SD) ratios of Kyn/Trp in CT-26 tumor allograft model. **, P <0.01,
determined with Student’s t test.
In conclusion, we have demonstrated that single agents with the
dual targeting capacity are a feasible and powerful tool to
improve cancer immunotherapy, an emerging field in oncology.
In these studies, hybrid molecules as IDO1/DNA dual-targeting
agents are rationally designed, synthesized and evaluated. As
the first-in-class example of such molecules, they exhibited
significantly enhanced anticancer activity in vitro and in vivo with
low toxicity. Among them, compound 4 showed the most potent
IDO1 inhibitory activity (IC50 = 0.13 μM) and good cellular
potency (EC50 = 0.27 μM), which significantly inhibits IDO1
activity in tumor tissues and reduces Kyn level in plasma.
Importantly, it exhibits better in vivo antitumor efficacy than
clinical candidate IDO1 inhibitor epacadostat (58.2% versus
47.5%, 100 mg/kg, po, bid) in the CT-26 allograft models without
significant body weight loss and adverse effects. To the best our
knowledge, the study represents the first case of proof-of-
concept of dual targeting strategy in emerging cancer
immunotherapy. Systematic development of similar dual-
targeting agents may create a general and fruitful approach to
improving cancer immunotherapy.
In Vitro Cytotoxicity Assay
This assay was determined by the Cell Counting Kit-8 (CCK-8) method.
Cells were plated in 96-well microtiter plates at a density of 6~10 ×
103/well and incubated in a humidified atmosphere with 5% CO2 at 37 °C
for 24 h. Test compounds were added onto triplicate wells with different
concentrations and 0.1% DMSO for control. After they had been
incubated for 48 h, 10 L of the CCK-8 solution was added to each well
and the plate was incubated for additional 1-4 h in the incubator. The
absorbance (OD) was read on a WellscanMK-2 microplate reader
(Labsystems) at 450 nm. The concentration causing 50% inhibition of cell
growth (IC50) was determined by the Logit method. All experiments were
performed three times.
In Vitro Cell Apoptosis Assay
CT-26 (obtained from Shcellbank, 3 × 105 /well) cells were incubated in
six-well plates for 24 h, and then treated with 0.1% DMSO (as control),
various concentrations of compounds 4 or 8 in fresh growth medium.
After 48 h, the cells were then harvested by trypsinization and washed
twice with cold PBS. After centrifugation and removal of the
supernatants, cells were resuspended in 400 μL of 1 × binding buffer,
which was then added to 5 μL of annexin V-FITC and incubated at room
Experimental Section
Molecular Modeling
Molecular modeling experiments were carried out with the Schrӧdinger
Maestro 9.0 package. The crystal structure of IDO1 in complex with Amg-
1 was obtained from protein database bank (PDB ID 4PK5)[17] and
4
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700666
ChemMedChem
COMMUNICATION
temperature for 15 min. After adding 10 μL of PI, the cells were incubated
at room temperature for another 15 min in the dark. The stained cells
were analyzed by a flow cytometer (BD Accuri C6).
layers was washed with water, brine, dried over sodium sulfate, purified
by column chromatography on SiO2 (DCM: MeOH = 100: 1) to afford 3e
(0.15 g, 58%) as a white solid. 1H NMR (DMSO-d6, 600 MHz) δ : 8.09 (dd,
J = 6.2, 2.5 Hz, 1H), 8.05 (t, J = 5.7 Hz, 1H), 7.71 (m, 1H), 7.60 (t, J = 8.5
Hz, 1H), 7.08 (d, J = 8.6 Hz, 2H), 6.67 (d, J = 8.6 Hz, 2H), 6.55 (t, J = 5.7
Hz, 1H), 3.70 (s, 8H), 3.31 (m, 4H), 3.23 (s, 2H).
In Vitro Cell Cycle Assay
CT-26 (3 × 105 /well) cells were incubated in six-well plates (Corning) for
24 h, and then treated with 0.1% DMSO (as control), various
concentrations of compounds 6 or 10 for 0-48 h. The treated cells were
collected, resuspended, and incubated for 30 min at 37 °C with 25 μg/mL
PI and 10 μg/mL RNase buffer. For each sample, at least 1 × 104 cells
were analyzed using flow cytometry (BD Accuri C6).
The synthetic methods for intermediates 3a-3d and 3f-3i were similar to
the synthesis of intermediate 3e.
3-(4-((2-(bis(2-hydroxyethyl)amino)ethyl)amino)-1,2,5-oxadiazol-3-
yl)-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one
(3j).
Intermediate 1 (1.0 g, 2.0 mmol), water (75 mL) and acetic acid (25 mL)
were added into a sealed tube in an ice water bath. Then ethylene oxide
(3 mL) was slowly dropwise added with violent stirring. The mixture was
further stirred for 24 h at room temperature. Then the pH value of the
solution was adjusted to 7 by 2M NaOH aqueous solution. The aqueous
phase was extracted with ethyl acetate (2 × 100 mL). The combined
organic layers was washed with water, brine, dried over sodium sulfate,
purified by column chromatography on SiO2 (DCM: MeOH = 100: 5) to
afford 3j (0.75 g, 78%) as a white solid. 1H NMR (DMSO-d6, 600 MHz) δ :
8.10 (dd, J = 6.2, 2.4 Hz, 1H), 7.74 (m, 1H), 7.61 (t, J = 8.5 Hz, 1H), 6.45
(s, 1H), 4.41 (s, 2H), 3.45 (s, 4H), 3.31 (s, 2H), 2.74 (s, 2H), 2.60 (s, 4H).
In Vivo Antitumor Assay and Analysis of the Ratio of Kyn to Try in Tumor
Tissues
Balb/c male mice or Balb/c nude male mice (certificate SCXK-2013-0016,
weighing 18~20 g) were purchased from Shanghai Laboratory Animal
Center, SLAC. The CT-26 colorectal cancer cells used for implantation
were harvested during log phase growth and resuspended in phosphate-
buffer saline. Each mice were inoculated s.c. into the right forelimb with 5
× 105 tumor cells. When tumor volumes approached ~50 mm3, the mice
were assigned into groups randomly and treated orally with compound 4,
compound 8, or epacadostat at doses 100 mg/kg twice every day, and
the blank control group received an equal volume of water containing
0.5% carboxyl methyl cellulose and 0.5% Tween 80 twice every day.
Compounds were reconstituted in 0.5% carboxyl methyl cellulose and
0.5% Tween 80 in water. Tumor volumes were monitored by caliper
measurement of the length and width and calculated using the formula of
TV = 1/2 × a × b2, where a is the tumor length and b is the width. Tumor
volumes and body weights were monitored every 4 days over the course
of treatment. After 14 days of treatment, the mice were sacrificed and the
3-(4-((2-(bis(2-chloroethyl)amino)ethyl)amino)-1,2,5-oxadiazol-3-yl)-
4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one
(3k).
Intermediate 3j (0.5 g, 1.1 mmol) was dissolved in dichloromethane (40
mL). SOCl2 (0.24 mL, 3.3 mmol) was added dropwise under the ice water
bath. The reaction was stirred at 40 °C for 3 h. After cooling to room
temperature, the mixture was concentrated in vacuo to give the desired
product (0.46 g, 85 %) as a crude brown oil, which was used directly for
the next step without further purification. 1H NMR (DMSO-d6, 600 MHz) δ :
8.09 (dd, J = 6.2, 2.6 Hz, 1H), 7.23 (m, 1H), 7.59 (t, J = 8.7 Hz, 1H), 6.36
(t, J = 5.8 Hz, 1H), 3.61 (t, J = 6.7 Hz, 4H), 3.30 (s, 2H), 2.90 (t, J = 6.9
Hz, 4H), 6.6 (t, J = 6.6 Hz, 2H).
tumor samples were collected at
3 h. The tumor samples were
homogenized with 9 volumes (v/w) of PBS for 30 s. An aliquot of 12 µL
sample was added with 48 µL PBS (pH7.4), mixed well, and the sample
dilution factor is 5. Then added with 240ul IS (500 ng/mL) in MeOH-
1%FA. The mixture was vortexed for 5min and centrifuged at 5800 rpm
for 10 min. Aliquots (50 µL) of supernatant were diluted with aliquots (50
µL) H2O and then an aliquot of 2 µL supernatant was injected onto the
LC-MS/MS (API 6500) system. The levels of Kyn and Try were measured
and the Kyn/Trp ratios were calculated.
3-(bis(2-chloroethyl)amino)-N-(2-((4-(N-(3-bromo-4-fluorophenyl)-N'-
hydroxycarbamimidoyl)-1,2,5-oxadiazol-3-yl)amino)ethyl)benzamide
(4). A solution of intermediate 3a (0.15 g, 0.2 mmol) in methanol (10 mL)
was treated with 2 M NaOH (0.5 mL, 1.0 mmol) and stirred at 40 °C for 2
h. The reaction mixture was quenched with 6 N HCl to pH ~ 7 and the
methanol was removed under reduced pressure. The solid that
precipitated was filtered and washed with water to give the desired
In Vivo Pharmacodynamic Assay
1
product (0.13 g, 93 %) as a white solid. H NMR (DMSO-d6, 600 MHz) δ :
11.41 (s, 1H), 8.84 (s, 1H), 8.50 (t, J = 5.6 Hz, 1H), 7.26 (t, J = 8.1 Hz,
1H), 7.12 (m, 4H), 6.88 (m, 1H), 6.74 (m, 1H), 6.30 (t, J = 6.0 Hz, 1H),
3.73 (m, 8H), 3.47 (m, 2H), 3.40 (m, 2H); 13C NMR (DMSO-d6, 150 MHz)
δ : 167.61, 156.20, 154.98, 153.40, 146.84, 140.45, 139.71, 138.49,
136.24, 129.80, 125.28, 121.92, 116.38, 116.09, 115.01, 110.88, 107.48,
52.39, 44.17, 41.51, 38.46; HRMS (ESI, positive) m/z calcd for
C22H24BrCl2FN7O3 (M + H):604.0460, found 604.0463.
Compound 4 at a dose of 100 mg/kg were administered by oral gavage
to C57BL/6 male mice (certificate SCXK-2013-0018,
n
=
4
animals/group). Food will be removed for 2 h prior to study start and
animals will be kept off food for the duration of the study. At various time
points, mice were euthanized and blood was collected via retro-orbital
puncture (under anesthesia with Isoflurane) into pre-cold EDTA-3K
tubes. Blood sample was centrifuged at 4 °C (2000 g, 5 min) to obtain
plasma within 15 min after sample collection. Then 60 µL homogenized
solution added with 240ul IS (500 ng/mL) in MeOH-1% trifluoroacetic
acid. The mixture was vortexed for 5 min and centrifuged at 5800 rpm for
10 min. Aliquots (50 µL) of supernatant were diluted with aliquots (50 µL)
H2O and then an aliquot of 2 µL supernatant was injected onto the LC-
MS/MS (API 6500) system. The levels of Kyn were measured.
The synthetic methods for compounds 5-13 were similar to the synthesis
of compound 4.
2-(3-(bis(2-chloroethyl)amino)phenyl)-N-(2-((4-(N-(3-bromo-4-
fluorophenyl)-N'-hydroxycarbamimidoyl)-1,2,5-oxadiazol-3-
yl)amino)ethyl)acetamide (5). White solid (0.12 g, 77%). 1H NMR
(DMSO-d6, 600 MHz) δ : 11.47 (s, 1H), 8.88 (s, 1H), 8.15 (t, J = 5.0 Hz,
1H), 7.15 (t, J = 9.0 Hz, 1H), 7.10 (dd, J = 5.8, 2.7 Hz, 1H), 7.07 (t, J =
8.1 Hz, 1H), 6.74 (m, 1H), 6.61 (s, 1H), 6.56 (m, 2H), 6.24 (t, J = 5.7 Hz,
1H), 3.68 (s, 8H), 3.31 (s, 2H), 3.25 (m, 4H); 13C NMR (DMSO-d6, 150
MHz) δ : 171.11, 156.07, 154.95, 153.36, 146.61, 140.37, 139.65, 138.43,
137.83, 129.69, 125.24, 121.90, 117.92, 116.36, 112.98, 110.36, 107.45,
52.55, 44.13, 43.29, 41.43, 37.95; ESI-MS (m/s): 618.27 [M + H].
Synthesis
2-(4-(bis(2-chloroethyl)amino)phenyl)-N-(2-((4-(4-(3-bromo-4-
fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1,2,5-
oxadiazol-3-yl)amino)ethyl)acetamide (3e). A mixture of intermediate 1
(0.20 g, 0.4 mmol), intermediate 2e (0.12 g, 0.4 mmol), HATU (0.16 g,
0.4 mmol), DIPEA (20 μmol, 1 mmol) and DMF (10 mL) was stirred for 24
h at room temperature. The reaction was poured into ice/water (100 mL),
and extracted with ethyl acetate (2 × 100 mL). The combined organic
3-(3-(bis(2-chloroethyl)amino)phenyl)-N-(2-((4-(N-(3-bromo-4-
fluorophenyl)-N'-hydroxycarbamimidoyl)-1,2,5-oxadiazol-3-
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700666
ChemMedChem
COMMUNICATION
yl)amino)ethyl)propanamide (6). White solid (0.23 g, 85%). 1H NMR
(DMSO-d6, 600 MHz) δ : 11.44 (s, 1H), 8.85 (s, 1H), 7.96 (s, 1H), 7.14 (t,
J = 8.8 Hz, 1H), 7.10 (dd, J = 6.0, 2.7 Hz, 1H), 7.06 (t, J = 8.0 Hz, 1H),
6.76 (m, 1H), 6.54 (s, 1H), 6.50 (m, 2H), 6.20 (t, J = 5.6 Hz, 1H), 3.68 (s,
8H), 3.25 (s, 4H), 2.72 (t, J = 7.4 Hz, 2H), 2.48 (q, J = 1.7 Hz, 2H), 2.34 (t,
J = 8.2 Hz, 2H); 13C NMR (DMSO-d6, 150 MHz) δ : 172.36, 156.13,
154.99, 153.41, 146.77, 143.09, 140.43, 139.72, 138.51, 129.79, 125.28,
121.94, 117.28, 116.39, 112.20, 110.02, 107.51, 52.60, 44.22, 40.55,
41.58, 37.72, 31.99; ESI-MS (m/s): 632.27 [M + H].
4-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)-N-
(2-((4-(N-(3-bromo-4-fluorophenyl)-N'-hydroxycarbamimidoyl)-1,2,5-
oxadiazol-3-yl)amino)ethyl)butanamide (12). White solid (0.03 g, 66%).
1H NMR (DMSO-d6, 600 MHz) δ : 11.90 (s, 1H), 8.87 (s, 1H), 7.98 (s, 1H),
7.32 (d, J = 8.9 Hz, 1H), 7.14 (t, J = 8.9 Hz, 1H), 7.09 (m, 1H), 6.87 (d, J
= 2.5 Hz, 1H), 6.76 (m, 2H), 6.20 (s, 1H), 3.68 (s, 8H), 3.65 (s, 3H), 3.26
(m, 4H), 2.80 (t, J = 7.5 Hz, 2H), 2.20 (t, J = 7.5 Hz, 2H), 1.97 (t, J = 7.5
Hz, 2H); 13C NMR (DMSO-d6, 150 MHz) δ : 172.66, 156.10, 155.12,
154.96, 153.37, 142.93, 140.45, 139.74, 138.53, 125.22, 121.88, 116.37,
110.73, 107.49, 102.41, 53.90, 44.23, 41.88, 37.68, 35.10, 29.98, 26.37,
23.47; ESI-MS (m/s): 700.29 [M + H].
4-(bis(2-chloroethyl)amino)-N-(2-((4-(N-(3-bromo-4-fluorophenyl)-N'-
hydroxycarbamimidoyl)-1,2,5-oxadiazol-3-yl)amino)ethyl)benzamide
(7). White solid (0.08 g, 64%). 1H NMR (DMSO-d6, 600 MHz) δ : 11.43 (s,
1H), 8.86 (s, 1H), 8.29 (t, J = 6.0 Hz, 1H), 7.71 (s, 1H), 7.70 (s, 1H), 7.13
(t, J = 8.3 Hz, 1H), 7.10 (dd, J = 6.0, 2.7 Hz, 1H), 6.77 (s, 1H), 6.75 (s,
1H), 6.73 (m, 1H), 6.29 (t, J = 6.0 Hz, 1H), 3.75 (m, 8H), 3.43 (t, J = 5.9
Hz, 2H), 3.37 (t, J = 5.8 Hz, 2H); 13C NMR (DMSO-d6, 150 MHz) δ :
166.79, 156.18, 154.98, 153.39, 149.15, 140.47, 139.69, 138.51, 129.42,
125.27, 122.68, 121.91, 116.37, 111.33, 107.50, 52.27, 44.40, 41.48,
38.34; ESI-MS (m/s): 604.26 [M + H].
4-((2-(bis(2-chloroethyl)amino)ethyl)amino)-N-(3-bromo-4-
fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-carboximidamide (13).
Light yellow solid (0.23 g, 74%). 1H NMR (DMSO-d6, 600 MHz) δ : 11.40
(s, 1H), 8.83 (s, 1H), 7.16 (t, J = 8.7 Hz, 1H), 7.07 (dd, J = 6.0, 2.5 Hz,
1H), 6.74 (m, 1H), 6.14 (t, J = 5.6 Hz, 1H), 3.60 (t, J = 6.8 Hz, 4H), 3.24
(q, J = 6.0 Hz, 2H), 2.88 (t, J = 6.8 Hz, 4H), 2.76 (t, J = 6.8 Hz, 2H); 13C
NMR (DMSO-d6, 150 MHz) δ : 155.57, 154.49, 152.90, 139.92, 139.22,
138.05, 124.70, 121.31, 107.05, 55.49, 51.76, 42.29, 42.05; MS (ESI,
positive) m/z calcd for C15H17BrCl2FN6O2 (M - H): 482.9936; found
482.9941.
2-(4-(bis(2-chloroethyl)amino)phenyl)-N-(2-((4-(N-(3-bromo-4-
fluorophenyl)-N'-hydroxycarbamimidoyl)-1,2,5-oxadiazol-3-
yl)amino)ethyl)acetamide (8). White solid (0.16 g, 79%). 1H NMR
(DMSO-d6, 600 MHz) δ : 11.46 (s, 1H), 8.86 (s, 1H), 8.04 (t, J = 5.6 Hz,
1H), 7.17 (t, J = 8.8 Hz, 1H), 7.13 (dd, J = 6.2, 2.6 Hz, 1H), 7.09 (s, 1H),
7.07 (s, 1H), 6.78 (m, 1H), 6.67 (s, 1H), 6.65 (s, 1H), 6.23 (t, J = 5.5 Hz,
1H), 3.69 (s, 8H), 3.28 (m, 6H); 13C NMR (DMSO-d6, 150 MHz) δ :
171.56, 156.11, 154.99, 153.41, 145.31, 140.42, 139.73, 138.49, 130.44,
125.28, 124.88, 121.92, 116.40, 112.22, 107.52, 52.63, 44.19, 41.82,
41.59, 37.92; HRMS (ESI, positive) m/z calcd for C23H26BrCl2FN7O3 (M +
H):618.0618, found 618.0614.
Acknowledgements
This work was supported by the National Key R&D Program
of China (2017YFA0506000 to C. S.), National Natural
Science Foundation of China (21738002 to W. W and C. S
and 81725020 to C. S.)
.
3-(4-(bis(2-chloroethyl)amino)phenyl)-N-(2-((4-(N-(3-bromo-4-
fluorophenyl)-N'-hydroxycarbamimidoyl)-1,2,5-oxadiazol-3-
yl)amino)ethyl)propanamide (9). White solid (0.11 g, 69%). 1H NMR
(DMSO-d6, 600 MHz) δ : 11.46 (s, 1H), 8.88 (s, 1H), 7.96 (t, J = 5.1 Hz,
1H), 7.18 (t, J = 8.6 Hz, 1H), 7.13 (dd, J = 6.2, 2.8 Hz, 1H), 7.03 (s, 1H),
7.02 (s, 1H), 6.79 (m, 1H), 6.65 (s, 1H), 6.64 (s, 1H), 6.23 (t, J = 5.4 Hz,
1H), 3.69 (s, 8H), 3.27 (m, 4H), 2.69 (t, J = 7.2 Hz, 2H), 2.31 (t, J = 8.1
Hz, 2H); 13C NMR (DMSO-d6, 150 MHz) δ : 172.44, 156.13, 155.10,
153.39, 144.95, 140.42, 139.71, 138.51, 129.95, 129.63, 125.27, 121.93,
116.40, 112.34, 107.53, 52.66, 44.26, 41.61, 37.97, 37.74, 30.56; ESI-
MS (m/s): 632.27 [M + H].
Conflict of interest
The authors declare no conflict of interest.
Keywords: Cancer immunotherapy • IDO1 • Multi-targeting drug
design • Antitumor activity
[1]
[2]
[3]
P. Sharma, J. P. Allison, Science 2015, 348, 56-61.
J. A. Joyce, D. T. Fearon, Science 2015, 348, 74-80.
a) T. Boon, P. van der Bruggen, J Exp Med 1996, 183, 725-729; b) R. D.
Schreiber, L. J. Old, M. J. Smyth, Science 2011, 331, 1565-1570.
A. B. Dounay, J. B. Tuttle, P. R. Verhoest, J Med Chem 2015, 58, 8762-
8782.
4-(4-(bis(2-chloroethyl)amino)phenyl)-N-(2-((4-(N-(3-bromo-4-
fluorophenyl)-N'-hydroxycarbamimidoyl)-1,2,5-oxadiazol-3-
[4]
yl)amino)ethyl)butanamide (10). White solid (0.04 g, 71%). 1H NMR
(DMSO-d6, 600 MHz) δ : 11.48 (s, 1H), 8.90 (s, 1H), 7.94 (s, 1H), 7.15 (t,
J = 8.5 Hz, 1H), 7.10 (m, 1H), 7.00 (d, J = 7.7 Hz, 2H), 6.75 (m, 1H), 6.63
(d, J = 8.5 Hz, 2H), 6.23 (s, 1H), 3.68 (s, 8H), 3.27 (s, 4H), 2.41 (t, J = 7.1
Hz, 2H), 2.06 (t, J = 7.1 Hz, 2H), 1.72 (m, 2H); 13C NMR (DMSO-d6, 150
MHz) δ : 172.84, 156.12, 154.96, 153.38, 144.85, 140.38, 139.70, 138.49,
130.38, 129.75, 125.24, 121.89, 116.44, 112.34, 107.49, 52.69, 44.32,
41.61, 37.74, 35.31, 34.05, 27.67; HRMS (ESI, positive) m/z calcd for
C25H30BrCl2FN7O3 (M + H): 646.0925; found 646.0929.
[5]
[6]
C. J. Austin, L. M. Rendina, Drug Discov Today 2015, 20, 609-617.
T. Jiang, Y. Sun, Z. Yin, S. Feng, L. Sun, Z. Li, Future Med Chem 2015,
7, 185-201.
[7]
[8]
Y. W. Moon, J. Hajjar, P. Hwu, A. Naing, J Immunother Cancer 2015, 3,
51.
a) R. Metz, S. Rust, J. B. Duhadaway, M. R. Mautino, D. H. Munn, N. N.
Vahanian, C. J. Link, G. C. Prendergast, Oncoimmunology 2012, 1,
1460-1468; b) 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, Mol Cancer Ther
2010, 9, 489-498; c) S. Qian, M. Zhang, Q. Chen, Y. He, W. Wang, Z.
Wang, RSC Advances 2016, 6, 7575-7581.
2-amino-3-(4-(bis(2-chloroethyl)amino)phenyl)-N-(2-((4-(N-(3-bromo-
4-fluorophenyl)-N'-hydroxycarbamimidoyl)-1,2,5-oxadiazol-3-
yl)amino)ethyl)propanamide (11). White solid (0.02 g, 54%). 1H NMR
(CD3OD-d6, 600 MHz) δ : 7.12 (dd, J = 6.0, 2.6 Hz, 1H), 7.07 (s, 1H),
7.06 (s, 1H), 7.02 (t, J = 8.6 Hz, 1H), 6.84 (m, 1H), 6.64 (s, 1H), 6.62 (s,
1H), 3.70 (t, J = 7.3 Hz, 4H), 3.62 (t, J = 6.7 Hz, 4H), 3.50 (t, J = 7.1 Hz,
1H), 3.43 (m, 2H), 3.27 (m, 2H), 2.85 (dd, J = 13.4, 6.5 Hz, 1H), 2.75 (dd,
[9]
S. Yang, X. Li, F. Hu, Y. Li, Y. Yang, J. Yan, C. Kuang, Q. Yang, J Med
Chem 2013, 56, 8321-8331.
[10] D. H. Munn, A. L. Mellor, Trends Immunol 2016, 37, 193-207.
[11] a) A. J. Muller, J. B. DuHadaway, P. S. Donover, E. Sutanto-Ward, G. C.
Prendergast, Nat Med 2005, 11, 312-319; b) D. Y. Hou, A. J. Muller, M.
D. Sharma, J. DuHadaway, T. Banerjee, M. Johnson, A. L. Mellor, G. C.
Prendergast, D. H. Munn, Cancer Res 2007, 67, 792-801; c) M. Li, A. R.
Bolduc, M. N. Hoda, D. N. Gamble, S. B. Dolisca, A. K. Bolduc, K.
J
=
13.7,
7
Hz, 1H); HRMS (ESI, positive) m/z calcd for
C24H29BrCl2FN8O3 (M + H): 647.3393 found 647.0894.
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700666
ChemMedChem
COMMUNICATION
Hoang, C. Ashley, D. McCall, A. M. Rojiani, B. L. Maria, O. Rixe, T. J.
MacDonald, P. S. Heeger, A. L. Mellor, D. H. Munn, T. S. Johnson, J
Immunother Cancer 2014, 2, 21.
[12] S. R. Selvan, J. P. Dowling, W. K. Kelly, J. Lin, Curr Cancer Drug
Targets 2016, 16, 755-764.
[13] S. R. R. And, R. M. Williams, Chemical Reviews 1998, 98, 2723-2796.
[14] a) L. F. Povirk, D. E. Shuker, Mutat Res 1994, 318, 205-226; b) W. U.
Knauf, T. Lissitchkov, A. Aldaoud, A. M. Liberati, J. Loscertales, R.
Herbrecht, G. Juliusson, G. Postner, L. Gercheva, S. Goranov, M.
Becker, H. J. Fricke, F. Huguet, I. Del Giudice, P. Klein, K. Merkle, M.
Montillo, Br J Haematol 2012, 159, 67-77.
[15] S. Tojo, T. Kohno, T. Tanaka, S. Kamioka, Y. Ota, T. Ishii, K. Kamimoto,
S. Asano, Y. Isobe, ACS Med Chem Lett 2014, 5, 1119-1123.
[16] 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, J. S. Fridman, T. Emm, P. A. Scherle, B. Metcalf, A.
P. Combs, ACS Med Chem Lett 2017, 8, 486-491.
[17] S. Tojo, T. Kohno, T. Tanaka, S. Kamioka, Y. Ota, T. Ishii, K. Kamimoto,
S. Asano, Y. Isobe, ACS Med Chem Lett 2014, 5, 1119-1123.
[18] 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, Bioorganic & Medicinal
Chemistry Letters 2014, 24, 3403-3406.
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700666
ChemMedChem
COMMUNICATION
Entry for the Table of Contents
Kill two birds with one stone. The first-in-class IDO1-DNA dual targeting inhibitors were designed to improve the efficacy of small
molecule cancer immunotherapy.
8
This article is protected by copyright. All rights reserved.