Discovery of secondary sulphonamides as IDO1 inhibitors with potent antitumour effects in vivo

Article abstract of DOI:10.1080/14756366.2020.1765165

Indoleamine 2,3-dioxygenase 1 (IDO1) as a key rate-limiting enzyme in the kynurenine pathway of tryptophan metabolism plays an important role in tumour immune escape. Herein, a variety of secondary sulphonamides were synthesised and evaluated in the HeLa cell-based IDO1/kynurenine assay, leading to the identification of new IDO1 inhibitors. Among them, compounds 5d, 5l and 8g exhibited the strongest inhibitory effect with significantly improved activity over the hit compound BS-1. The in vitro results showed that these compounds could restore the T cell proliferation and inhibit the differentiation of na?ve CD4⁺ T cell into highly immunosuppressive FoxP3⁺ regulatory T (Treg) cell without affecting the viability of HeLa cells and the expression of IDO1 protein. Importantly, the pharmacodynamic assay showed that compound 5d possessed potent antitumour effect in both CT26 and B16F1 tumours bearing immunocompetent mice but not in immunodeficient mice. Functionally, subsequent experiments demonstrated that compound 5d could effectively inhibit tumour cell proliferation, induce apoptosis, up-regulate the expression of IFN-γ and granzyme B, and suppress FoxP3⁺ Treg cell differentiation, thereby activate the immune system. Thus, compound 5d could be a potential and efficacious agent for further evaluation.

Full text of DOI:10.1080/14756366.2020.1765165

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Discovery of secondary sulphonamides as IDO1
inhibitors with potent antitumour effects in vivo
Shushan Ge, Haiqing Zhong, Xuewei Ma, Yingbo Zheng, Yi Zou, Fang Wang,
Yan Wang, Yue Hu, Yuezhen Li, Wen Liu, Wenjie Guo, Qiang Xu & Yisheng Lai
To cite this article: Shushan Ge, Haiqing Zhong, Xuewei Ma, Yingbo Zheng, Yi Zou, Fang Wang,
Yan Wang, Yue Hu, Yuezhen Li, Wen Liu, Wenjie Guo, Qiang Xu & Yisheng Lai (2020) Discovery
of secondary sulphonamides as IDO1 inhibitors with potent antitumour effects in vivo, Journal of
Enzyme Inhibition and Medicinal Chemistry, 35:1, 1240-1257
To link to this article: https://doi.org/10.1080/14756366.2020.1765165
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1240–1257
https://doi.org/10.1080/14756366.2020.1765165
RESEARCH PAPER
Discovery of secondary sulphonamides as IDO1 inhibitors with potent antitumour
effects in vivo
a
a
a
a
a
b
b
, Haiqing Zhongb , Xuewei Ma , Yingbo Zheng , Yi Zou , Fang Wang , Yan Wang , Yue Hu ,
ꢀ
ꢀ
ꢀ
Shushan Ge
Yuezhen Lic, Wen Liub, Wenjie Guob , Qiang Xub and Yisheng Laia
aState Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Center of Drug Discovery, China
b
Pharmaceutical University, Nanjing, PR China; State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing
c
University, Nanjing, PR China; Department of Organic Chemistry, School of Science, China Pharmaceutical University, Nanjing, PR China
ABSTRACT
ARTICLE HISTORY
Received 27 February 2020
Revised 29 April 2020
Accepted 30 April 2020
Indoleamine 2,3-dioxygenase 1 (IDO1) as a key rate-limiting enzyme in the kynurenine pathway of
tryptophan metabolism plays an important role in tumour immune escape. Herein, a variety of sec-
ondary sulphonamides were synthesised and evaluated in the HeLa cell-based IDO1/kynurenine assay,
leading to the identification of new IDO1 inhibitors. Among them, compounds 5d, 5l and 8g exhib-
ited the strongest inhibitory effect with significantly improved activity over the hit compound BS-1.
The in vitro results showed that these compounds could restore the T cell proliferation and inhibit
the differentiation of naïve CD4þ T cell into highly immunosuppressive FoxP3þ regulatory T (Treg)
cell without affecting the viability of HeLa cells and the expression of IDO1 protein. Importantly, the
pharmacodynamic assay showed that compound 5d possessed potent antitumour effect in both
CT26 and B16F1 tumours bearing immunocompetent mice but not in immunodeficient mice.
Functionally, subsequent experiments demonstrated that compound 5d could effectively inhibit
tumour cell proliferation, induce apoptosis, up-regulate the expression of IFN-c and granzyme B, and
suppress FoxP3þ Treg cell differentiation, thereby activate the immune system. Thus, compound 5d
could be a potential and efficacious agent for further evaluation.
KEYWORDS
Indoleamine 2,3-
dioxygenase 1; kynurenine
pathway; immune escape;
immune checkpoint;
secondary sulphonamides
1. Introduction
microenvironment have been demonstrated to induce effector T
cell anergy and enhance regulatory T (Treg) cell function, which
eventually lead to an immunosuppressive milieu that helps tumour
cells evade detection and killing by the immune system10.
Tryptophan depletion has been implicated in the activation of gen-
eral control non-derepressible 2 (GCN2) and suppression of the
With the increasing recognition of the interaction between the
immune system and tumour cells, immunotherapy has gained tre-
mendous attention in the field of cancer research1,2. The recent
clinical successes of immune checkpoint inhibitors and chimeric
antigen receptor T cell (CAR-T) therapy have dramatically changed
the landscape of the cancer treatment3–6. To date, the US Food
and Drug Administration (FDA) has approved six immune check-
point inhibitors blocking cytotoxic T lymphocyte-associated anti-
gen 4 (CTLA-4), programmed cell death protein 1 (PD-1) or
programmed cell death ligand 1 (PD-L1), and the second CAR-T
cell therapy for the treatment of solid and hematological malig-
nancies. However, their limited response rate and immune-related
adverse effects call for a need to develop other immunotherapy
mechanistic target of rapamycin kinase (mTOR) pathways11,12
.
Furthermore, kynurenine and other tryptophan metabolites can
activate the aryl hydrocarbon receptor (AHR)13. Indeed, high expres-
sion of IDO1 in tumour cells has been shown to correlate with
worse clinical prognosis in patients with a variety of cancers, includ-
ing colorectal, ovarian, endometrial, and hepatocellular carcino-
mas14–18. Moreover, preclinical studies have demonstrated that IDO
inhibitors can effectively restore antitumour immunity and have the
potential to synergize with chemotherapy, radiotherapy, and other
immunotherapies, including anti-CTLA-4, anti-PD-1 and anti-PD-L1
antibodies19–22. Hence, these findings have motivated interest in
developing IDO1 inhibitors for cancer immunotherapy.
So far, a number of IDO1 inhibitors have been identified from
high-throughput screening23,24, structure-based design25–27, and
natural product screening28,29. Several of them, including, navoxi-
mod, epacadostat, PF-06840003, linrodostat, LY-3381916 and
KHK2455 (structure not disclosed), have entered clinical develop-
options to solve those problems5–7
.
Indoleamine 2,3-dioxygenase 1 (IDO1) is a haem-containing
enzyme that catalyses the initial and rate-limiting step in the
catabolism of the essential amino acid tryptophan along the
kynurenine pathway8. IDO1 plays an important role in the regula-
tion of local inflammation and immune tolerance9. In cancer, aber-
rant activation of IDO1 results in suppression of antitumour
immunity. The depletion of tryptophan and the accumulation of
kynurenine and its downstream metabolites in the tumour ment as monotherapy and in combination with other immune
CONTACT Wenjie Guo
Nanjing University, Nanjing 210093, PR China; Yisheng Lai
guowj@nju.edu; Qiang Xu
molpharm@163.com
yslai@cpu.edu.cn
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences,
State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug
Discovery for Metabolic Diseases, Center of Drug Discovery, China Pharmaceutical University, Nanjing, 210009, PR China
ꢀ
These contributed equally to this article.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1241
Figure 1. Structure of representative IDO1 clinical candidates.
Figure 2. (A) The co-crystal of IDO1 (PDB ID: 4PK5). (B) Site maps generated using Sitemap: the ligand was Amg-1, white points represented the new identified active
region, as well as yellow, blue and red solid maps represented hydrophobic regions, hydrogen bond donors and acceptors, respectively.
therapies or chemotherapy (Figure 1)30. However, the FDA has not secondary sulphonamide compound BS-1 showed inhibitory activ-
yet approved IDO inhibitors for the treatment of malignancies to ity against IDO1 with an IC50 value of 48.42 mM. As well known,
date. We therefore need to continue to identify novel IDO1 inhibitors.
In recent years, we have been working on the development of
novel IDO1 inhibitors31–34. From our previous study on the inter-
actions between IDO1 and imidazole derivatives, it has been
found that metal chelation, hydrogen bonding, and hydrophobic
interactions between haem and the ligand contribute a large pro-
portion to the binding affinity of the inhibitor31. Furthermore, the
human IDO1 crystal structures in complex with the ligand Amg-1
(PDB ID: 4PK5) revealed that IDO1 is characterised by a small and
highly lipophilic active site including two pockets (A and B), imply-
ing that the hydrophobic interaction may account for a large part
of protein-ligand nonbonding interactions (Figure 2). In addition,
Muller’s team demonstrated that as the charge on the coordinat-
ing atom decreased, the binding affinity to the haem iron
increased, which would be possible to improve IDO1 inhibitory
activity35. These findings provide directions for improving the
binding affinity of novel IDO1 inhibitors.
bezenesulfonamide is a common pharmacophore that is the basis
of several groups of drugs, such as sulpha antibiotics, thiazide diu-
retics and nonsteroidal antiinflammatory drug (NSAID) cele-
coxib36,37. Moreover, the sulphonyl is a heme-binding function that
coordinates to the haem iron of IDO138. Therefore, the hit com-
pound BS-1 was further modified, as well as a variety of secondary
sulphonamides were synthesised and evaluated in the HeLa cell-
based IDO1/kynurenine assay. The resulting compounds 5d, 5l and
8g showed high inhibitory activity without affecting the cell viabil-
ity and the IDO1 protein expression. Subsequent in vitro and in vivo
experiments demonstrated that compound 5d could exert potent
antitumour effects by activating the mouse immune system.
2. Material and methods
2.1. Chemistry
Melting points were determined on a RDCSY-I capillary apparatus
In this study, we screened our in-house compound library using
the HeLa cell-based IDO1/kynurenine assay, and found that the and were uncorrected. Allmaterials used were commercially

1242
S. GE ET AL.
available and used as supplied. HG/T2354-92 silica gel 60 F254 2.1.1.8. N-(4-(N-(3-(Trifluoromethyl)benzyl)sulfamoyl) phenyl)aceta-
mide (3h). White solid. Yield 88%. Mp 154–156 ꢁC. 1H NMR
sheets were used for analytical thin-layer chromatography (TLC).
Column chromatography was performed on silica gel (300–400
(300 MHz, DMSO-d6) d 10.19 (s, 1H), 8.05 (t, J ¼ 6.3 Hz, 1H),
mesh). 1H NMR spectra were recorded on a Bruker AV-300 spec-
trometer. Chemical shifts (d) were given in parts per million (ppm)
7.61–7.54 (m, 4H), 7.46–7.35 (m, 4H), 3.95 (d, J ¼ 6.3 Hz, 2H), 1.95
(s, 3H). MS (EI) m/z 371.1 [M-H]ꢂ.
relative to the solvent peak. J values are in Hz. Chemical shifts are
expressed in ppm downfield from internal standard TMS. Mass
spectra (MS) were measured using a Thermo Scientific iCAP RQ
ICP-MS. All the reagents and solvents were reagent grade and
were used without further purification unless otherwise specified.
2.1.1.9. N-(4-(N-Phenethylsulfamoyl)phenyl)acetamide (3i). White
solid. Yield 85%. Mp 130–132 ꢁC. 1H NMR (300 MHz, DMSO-d6) d
10.19 (s, 1H), 7.59 (q, J ¼ 8.5 Hz, 4H), 7.46 (t, J ¼ 5.9 Hz, 1H),
7.16–7.00 (m, 5H), 2.82–2.75 (m, 2H), 2.52 (t, J ¼ 7.5 Hz, 2H), 1.95 (s,
3H). MS (EI) m/z 317.2 [M-H]ꢂ.
2.1.1. General preparation of compounds 3a-i
To a solution of substituted aniline (0.97 mmol) in DCM (15 ml)
2.1.2. General preparation of compounds 4a-f
To a solution of compounds 3 (0.68 mmol) in ethanol (15 ml) was
was added triethylamine (1.22 mmol)39. A solution of 4-acrylami-
dobenzenesulfonyl chloride (0.81 mmol) in DCM (10 ml) was added
added hydrochloric acid (1 ml)39. Then the mixture was stirred at
dropwise to the mixture at 0 ꢁC. The reaction was stirred at room
70 ꢁC for 12 h. After the reaction was completed, the solvent was
temperature for 4 h. The solvent was evaporated under reduced
evaporated under reduced pressure. Water was added and the pH
was adjusted to 7–8 with saturated NaHCO3 solution. The aqueous
pressure and the crude product was recrystallization to afford tar-
get compounds 3a-i.
phase was extracted with EtOAc (3 ꢃ 30 ml). The combined
organic layers were washed with water, brine, and dried. The solv-
2.1.1.1. N-(4-(N-Phenylsulfamoyl)phenyl)acetamide (3a). White
solid. Yield 90%. Mp 204–206 ꢁC. 1H NMR (300 MHz, DMSO-d6) d
10.17 (s, 1H), 10.04 (s, 1H), 7.54 (s, 4H), 7.08 (t, J ¼ 7.8 Hz, 2H),
6.94–6.84 (m, 3H), 1.91 (s, 3H). MS (EI) m/z 289.1 [M-H]ꢂ.
ent was removed in vacuo and the crude product was recrystal-
lization to afford target compounds 4a-f.
2.1.2.1. 4-Amino-N-phenylbenzenesulfonamide (4a). Light yellow
solid. Yield 89%. Mp 188–190 ꢁC. 1H NMR (300 MHz, DMSO-d6) d
9.81 (s, 1H), 7.34 (d, J ¼ 8.6 Hz, 2H), 7.15 (t, J ¼ 7.8 Hz, 2H), 7.01 (d,
J ¼ 7.7 Hz, 2H), 6.94 (d, J ¼ 7.3 Hz, 1H), 6.48 (d, J ¼ 8.7 Hz, 2H), 5.92
(s, 2H). MS (EI) m/z 247.1 [M-H]ꢂ.
2.1.1.2. N-(4-(N-(p-Tolyl)sulfamoyl)phenyl)acetamide (3b). White
solid. Yield 87%. Mp > 250 ꢁC. 1H NMR (300 MHz, DMSO-d6) d
10.45 (s, 1H), 9.89 (s, 1H), 7.69 (d, J ¼ 8.1 Hz, 2H), 7.57 (d, J ¼ 8.4 Hz,
2H), 7.08 (d, J ¼ 7.8 Hz, 2H), 6.73 (d, J ¼ 7.8 Hz, 2H), 2.19 (s, 3H),
1.92 (s, 3H). MS (EI) m/z 303.1 [M-H]ꢂ.
2.1.2.2. 4-Amino-N-(p-tolyl)benzenesulfonamide (4b). White solid.
1
Yield 89%. Mp 188–190 ꢁC. H NMR (300 MHz, DMSO-d6) d 9.55 (s,
2.1.1.3. N-(4-(N-(4-Isopropylphenyl)sulfamoyl)phenyl)acetamide (3c).
Light yellow solid, Yield 90%, Mp 186–188 ꢁC. 1H NMR (300 MHz,
DMSO-d6) d 10.16 (s, 1H), 9.91 (s, 1H), 7.54 (s, 4H), 6.95 (d,
J ¼ 8.1 Hz, 2H), 6.94 (d, J ¼ 8.1 Hz, 2H), 2.65–2.58 (m, 1H),1.92 (d,
J ¼ 2.4 Hz, 3H), 0.97 (dd, J ¼ 2.1, 2.7 Hz, 6H). MS (EI) m/z 331.1
[M-H]ꢂ.
1H), 7.21 (d, J ¼ 8.5 Hz, 2H), 6.83 (dd, J ¼ 8.0, 8.3 Hz, 4H), 6.37 (d,
J ¼ 8.6 Hz, 2H), 5.82 (s, 2H), 2.03 (s, 3H). MS (EI) m/z 261.1 [M-H]ꢂ.
2.1.2.3. 4-Amino-N-benzylbenzenesulfonamide (4c). White solid.
Yield 93%. Mp 116–118 ꢁC. 1H NMR (300 MHz, DMSO-d6) d 7.53 (t,
J ¼ 6.1 Hz, 1H), 7.32 (d, J ¼ 8.6 Hz, 2H), 7.09–7.18 (m, 5H), 6.49 (d,
J ¼ 8.6 Hz, 2H), 3.73 (d, J ¼ 5.9 Hz, 2H). MS (EI) m/z 261.1 [M-H]ꢂ.
2.1.1.4. N-(4-(N-(3-Isopropylphenyl)sulfamoyl)phenyl)acetamide (3d).
White solid. Yield 90%. Mp 158–160 ꢁC. 1H NMR (300 MHz, DMSO-
d6) d 10.17 (s, 1H), 9.95 (s, 1H), 7.55 (s, 4H), 6.98 (t, J ¼ 7.7 Hz, 1H),
6.79–6.72 (m, 3H), 2.63–2.58 (m, 1H), 1.92 (s, 3H), 0.95 (d,
J ¼ 6.9 Hz, 6H). MS (EI) m/z 331.1 [M-H]ꢂ.
2.1.2.4.
4-Amino-N-(4-chlorobenzyl)benzenesulfonamide
(4d).
1
White solid. Yield 95%. Mp 172–174 ꢁC. H NMR (300 MHz, DMSO-
d6) d 7.66 (t, J ¼ 6.2 Hz, 1H), 7.21–7.40 (m, 6H), 6.56 (d, J ¼ 8.4 Hz,
2H), 5.91 (s, 2H), 3.83 (d, J ¼ 6.2 Hz, 2H). MS (EI) m/z 295.1 [M-H]ꢂ.
2.1.1.5. N-(4-(N-Benzylsulfamoyl)phenyl)acetamide (3e). White
solid. Yield 90%. Mp 140–142 ꢁC. 1H NMR (300 MHz, DMSO-d6) d
10.20 (s, 1H), 7.89 (t, J ¼ 6.3 Hz, 1H), 7.64–7.57 (m, 4H), 7.18–7.07
(m, 5H), 3.81 (d, J ¼ 6.3 Hz, 2H), 1.96 (s, 3H). MS (EI) m/z 303.1
[M-H]ꢂ.
2.1.2.5.
4-Amino-N-(3-chlorobenzyl)benzenesulfonamide
(4e).
1
White solid. Yield 90%. Mp 119–121 ꢁC. H NMR (300 MHz, DMSO-
d6) d 7.70 (s, 1H), 7.42 (d, J ¼ 8.6 Hz, 2H), 7.36–7.11 (m, 4H), 6.60
(d, J ¼ 8.4 Hz, 2H), 5.92 (s, 2H), 3.90 (d, J ¼ 5.0 Hz, 2H). MS (EI) m/z
295.1 [M-H]ꢂ.
2.1.1.6. N-(4-(N-(4-Chlorobenzyl)sulfamoyl)phenyl)acetamide (3f).
White solid. Yield 90%. Mp 172–174 ꢁC. 1H NMR (300 MHz, DMSO-
d6) d 10.20 (s, 1H), 7.95 (t, J ¼ 6.5 Hz, 1H), 7.63–7.56 (m, 4H), 7.17
(dd, J ¼ 8.1, 8.4 Hz, 4H), 3.81 (d, J ¼ 6.3 Hz, 2H), 1.95 (s, 3H). MS (EI)
m/z 337.1 [M-H]ꢂ.
2.1.2.6. 4-Amino-N-phenethylbenzenesulfonamide (4f). White solid.
1
Yield 90%. Mp 138–140 ꢁC. H NMR (300 MHz, DMSO-d6) d 7.37 (d,
J ¼ 8.4 Hz, 2H), 7.14–7.25 (m, 5H), 6.56 (d, J ¼ 8.5 Hz, 2H), 2.82 (m,
2H), 2.60 (t, J ¼ 7.4 Hz, 2H). MS (EI) m/z 275.1 [M-H]ꢂ.
2.1.1.7. N-(4-(N-(4-(Trifluoromethyl)benzyl)sulfamoyl)phenyl) aceta-
2.1.3. General preparation of compounds 5a-m
mide (3g). White solid. Yield 89%. Mp 186–188 ꢁC. 1H NMR To a solution of compounds 4 (0.55 mmol) in DCM (15 ml) was
(300 MHz, DMSO-d6) d 10.19 (s, 1H), 8.04 (t, J ¼ 6.0 Hz, 1H), added TEA (1.1 mmol). Then acryloyl chloride (0.61 mmol)
7.62–7.56 (m, 4H), 7.50 (d, J ¼ 8.1 Hz, 2H), 7.33 (d, J ¼ 8.1 Hz, 2H), was added dropwise to the mixture at 0 ꢁC for 0.5 h. The reaction
3.93 (d, J ¼ 5.7 Hz, 2H), 1.95 (s, 3H). MS (EI) m/z 371.1 [M-H]ꢂ.
was stirred at rt overnight. After the reaction was completed, the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1243
water was added to quench the reaction. The mixture was 2H), 6.37–6.28 (m, 1H), 6.18 (d, J ¼ 17.1 Hz, 1H), 5.70 (d, J ¼ 9.9 Hz,
extracted with DCM to afford the crude product. The crude resi- 1H), 3.82 (d, J ¼ 6.0 Hz, 2H). MS (EI) m/z 357.1 [M þ Na]þ.
due was recrystallization to afford target compounds 5a-m.
2.1.3.9. N-(4-(N-(3-Fluorobenzyl)sulfamoyl)phenyl)acrylamide (5i).
1
2.1.3.1. N-(4-(N-Benzylsulfamoyl)phenyl)acrylamide (5a). White White solid. Yield 80%. Mp 152–154 ꢁC. H NMR (300 MHz, DMSO-
solid. Yield 75%. Mp 116–118 ꢁC. 1H NMR (300 MHz, DMSO-d6) d d6) d 10.39 (s, 1H), 8.01 (t, J ¼ 6.3 Hz, 1H), 7.70 (d, J ¼ 8.4 Hz, 2H),
10.50 (s, 1H), 8.02 (t, J ¼ 6.2 Hz, 1H), 7.81 (d, J ¼ 8.7 Hz, 2H), 7.72 (d, 7.61 (d, J ¼ 5.7 Hz, 2H), 7.22–7.15 (m, 1H), 6.95–6.89 (m, 3H),
J ¼ 8.7 Hz, 2H), 7.27–7.16 (m, 5H), 6.48–6.39 (m, 1H), 6.28 (d,
J ¼ 15.6 Hz, 1H), 5.79 (d, J ¼ 9.9 Hz, 1H), 3.92 (d, J ¼ 6.0 Hz, 2H). MS
(EI) m/z 315.1 [M-H]ꢂ.
6.37–6.28 (m, 1H), 6.18 (d, J ¼ 16.8 Hz, 1H), 5.69 (d, J ¼ 9.9 Hz, 1H),
3.87 (d, J ¼ 6.3 Hz, 2H). MS (EI) m/z 357.1 [M þ Na]þ.
2.1.3.10. N-(4-(N-(2-Chlorobenzyl)sulfamoyl)phenyl)acrylamide (5j).
1
White solid. Yield 78%. Mp 178–180 ꢁC. H NMR (300 MHz, DMSO-
2.1.3.2.
N-(4-(N-(4-Methoxybenzyl)sulfamoyl)phenyl)acrylamide
(5b). White solid. Yield 81%. Mp 156–158 ꢁC. 1H NMR (300 MHz,
DMSO-d6) d 10.38 (s, 1H), 7.82 (t, J ¼ 6.0 Hz, 1H), 7.70 (d, J ¼ 8.7 Hz,
2H), 7.61 (d, J ¼ 8.7 Hz, 2H), 7.00 (d, J ¼ 8.4 Hz, 4H), 6.69 (d,
J ¼ 8.7 Hz, 2H), 6.38–6.29 (m, 1H), 6.18 (d, J ¼ 17.1 Hz, 1H), 5.69 (d,
J ¼ 9.9 Hz, 1H), 3.75 (d, J ¼ 6.0 Hz, 2H), 3.57 (s, 3H). MS (EI) m/z
345.2 [M-H]ꢂ.
d6) d 10.41 (s, 1H), 8.00 (s, 1H), 7.71 (d, J ¼ 7.5 Hz, 2H), 7.64 (d,
J ¼ 8.7 Hz, 2H), 7.28–7.15 (m, 4H), 6.39–6.29 (m, 1H), 6.18 (d,
J ¼ 17.4 Hz, 1H), 5.69 (d, J ¼ 2.7 Hz, 1H), 3.91 (d, J ¼ 4.5 Hz, 2H). MS
(EI) m/z 349.1 [M-H]ꢂ.
2.1.3.11. N-(4-(N-(4-Bromobenzyl)sulfamoyl)phenyl)acrylamide (5k).
1
White solid. Yield 83%. Mp 194–196 ꢁC. H NMR (300 MHz, DMSO-
d6) d 10.44 (s, 1H), 7.98 (t, J ¼ 6.2 Hz, 1H), 7.71 (d, J ¼ 8.4 Hz, 2H),
7.61 (d, J ¼ 8.7 Hz, 2H), 7.34 (d, J ¼ 7.8 Hz, 2H), 7.07 (d, J ¼ 7.8 Hz,
2H), 6.38–6.29 (m, 1H), 6.17 (d, J ¼ 15.9 Hz, 1H), 5.69 (d, J ¼ 10.2 Hz,
1H), 3.80 (d, J ¼ 6.3 Hz, 2H). MS (EI) m/z 393.1, 395.1 [M-H]ꢂ.
2.1.3.3. N-(4-(N-(4-Chlorobenzyl)sulfamoyl)phenyl)acrylamide (5c).
White solid. Yield 80%. Mp 190–192 ꢁC. 1H NMR (300 MHz, DMSO-
d6) d 10.40 (s, 1H), 7.98 (t, J ¼ 6.3 Hz, 1H), 7.70 (d, J ¼ 8.4 Hz, 2H),
7.61 (d, J ¼ 8.4 Hz, 2H), 7.17 (dd, J ¼ 8.4, 8.1 Hz, 4H), 6.37–6.28 (m,
1H), 6.18 (d, J ¼ 16.5 Hz, 1H), 5.69 (d, J ¼ 2.7 Hz, 1H), 3.82 (d,
J ¼ 6.0 Hz, 2H). MS (EI) m/z 349.1 [M-H]ꢂ.
2.1.3.12. N-(4-(N-(3-Bromobenzyl)sulfamoyl)phenyl)acrylamide (5l).
1
White solid. Yield 81%. Mp 156–158 ꢁC. H NMR (300 MHz, DMSO-
d6) d 10.40 (s, 1H), 8.01 (t, J ¼ 6.0 Hz, 1H), 7.70 (d, J ¼ 8.4 Hz, 2H),
7.60 (d, J ¼ 8.7 Hz, 2H), 7.26 (s, 2H), 7.11 (d, J ¼ 3.9 Hz, 2H),
6.38–6.29 (m, 1H), 6.18 (d, J ¼ 15.9 Hz, 1H), 5.69 (d, J ¼ 10.2 Hz, 1H),
3.86 (d, J ¼ 6.0 Hz, 2H). MS (EI) m/z 393.1, 395.1 [M-H]ꢂ.
2.1.3.4. N-(4-(N-(3-Chlorobenzyl)sulfamoyl)phenyl)acrylamide (5d).
White solid. Yield 51%. Mp 157–159 ꢁC. 1H NMR (300 MHz, DMSO-
d6) d 10.58 (s, 1H), 8.15 (s, 1H), 7.83 (dd, J ¼ 9.0, 3.2 Hz, 2H),
7.78–7.65 (m, 2H), 7.47–7.06 (m, 4H), 6.44 (dd, J ¼ 9.8, 3.4 Hz, 1H),
6.37–6.22 (m, 1H), 5.88 –5.74 (m, 1H), 3.99 (d, J ¼ 3.3 Hz, 2H). 13C
NMR (126 MHz, DMSO) d 164.08, 142.99, 140.90, 135.28, 133.36,
131.94, 130.53, 128.38, 128.13, 127.79, 127.47, 126.65, 119.53,
45.86. MS (EI) m/z 349.1 [M-H]ꢂ.
2.1.3.13. N-(4-(N-(3,4-Dichlorobenzyl)sulfamoyl)phenyl)acrylamide
(5m). White solid. Yield 82%. Mp 148–150 ꢁC. 1H NMR (300 MHz,
DMSO-d6) d 10.41 (s, 1H), 8.05 (s, 1H), 7.65 (d, J ¼ 29.4 Hz, 4H), 7.34
(d, J ¼ 30.0 Hz, 2H), 7.11 (s, 1H), 6.38–6.15 (m, 2H), 5.70 (s, 1H),
3.82 (d, J ¼ 6.0 Hz, 2H). MS (EI) m/z 383.1 [M-H]ꢂ.
2.1.3.5. N-(4-(N-Phenethylsulfamoyl)phenyl)acrylamide (5e). White
solid. Yield 75%. Mp 154–156 ꢁC. 1H NMR (300 MHz, DMSO-d6) d
10.39 (s, 1H), 7.70 (d, J ¼ 8.1 Hz, 2H), 7.61 (d, J ¼ 7.5 Hz, 2H), 7.49 (s,
1H), 7.09 (m, 4H), 6.37–6.28 (m, 1H), 6.17 (d, J ¼ 15.6 Hz, 1H), 5.69
(d, J ¼ 8.7 Hz, 1H), 2.80 (s, 1H), 2.53 (s, 1H). MS (EI) m/z
353.1 [M þ Na]þ.
2.1.4. General preparation of N-phenylacrylamide 6
To a solution of aniline (0.043 mol) in DCM (50 ml) was added TEA
(0.086 mol). Then acryloyl chloride (0.047 mol) was added dropwise
to the mixture at 0 ꢁC for 0.5 h. The reaction was allowed to stir at
rt overnight. After the reaction was completed, the water was
added to quench the reaction. The mixture was extracted with
DCM to afford the crude product that was purified by flash col-
umn chromatography on silica gel to afford N-phenylacrylamide 6
(white solid, Yield 72.8%)40. 1H NMR (300 MHz, Chloroform-d) d
10.14 (s, 1H), 7.67 (d, J ¼ 8.1 Hz, 2H), 7.32 (t, J ¼ 7.8 Hz, 2H), 7.07 (t,
J ¼ 7.2 Hz, 1H), 6.44 (dd, J ¼ 16.8, 9.9 Hz, 1H), 6.26 (dd, J ¼ 16.8,
1.5 Hz, 1H), 5.75 (dd, J ¼ 9.9, 1.5 Hz, 1H).
2.1.3.6. N-(4-(N-(4-(Trifluoromethyl)benzyl)sulfamoyl)phenyl) acryl-
amide (5f). White solid. Yield 80%. Mp 198–200 ꢁC. 1H NMR
(300 MHz, DMSO-d6) d 10.38 (s, 1H), 8.07 (s, 1H), 7.69 (d, J ¼ 7.8 Hz,
2H), 7.61 (d, J ¼ 8.1 Hz, 2H), 7.50 (d, J ¼ 7.2 Hz, 4H), 7.33 (d,
J ¼ 7.2 Hz, 2H), 6.37–6.15 (m, 1H), 6.18 (d, J ¼ 16.5 Hz, 1H), 5.69 (d,
J ¼ 9.9 Hz, 1H), 3.94 (d, J ¼ 4.5 Hz, 2H). MS (EI) m/z 407.1 [M þ Na]þ.
2.1.3.7. N-(4-(N-(3-(Trifluoromethyl)benzyl)sulfamoyl)phenyl) acryl-
amide (5g). White solid. Yield 77%. Mp 152–154 ꢁC. 1H NMR
(300 MHz, DMSO-d6) d 10.55 (s, 1H), 8.19 (t, J ¼ 6.2 Hz, 1H), 7.79 (d,
J ¼ 8.7 Hz, 2H), 7.69 (d, J ¼ 8.7 Hz, 2H), 7.55–7.44 (m, 4H), 6.49–6.40
(m, 1H), 6.27 (d, J ¼ 16.8 Hz, 1H), 5.78 (d, J ¼ 3.9 Hz, 1H), 4.06 (d,
J ¼ 6.0 Hz, 2H). MS (EI) m/z 383.1 [M-H]ꢂ.
2.1.5. General preparation of 4-acrylamidobenzenesulfonyl chlor-
ide 7
N-phenylacrylamide (0.031 mol) was added slowly to sulphuryl
chloride (15 ml) at 0 ꢁC for more than 0.5 h. The reaction was
stirred at 70 ꢁC for 1 h. After the reaction was completed, the mix-
ture was poured into ice water slowly to quench the reaction. The
2.1.3.8. N-(4-(N-(4-Fluorobenzyl)sulfamoyl)phenyl)acrylamide (5h).
White solid. Yield 82%. Mp 178–180 ꢁC. 1H NMR (300 MHz, DMSO- mixture was stirred at rt for 1 h and the solid was filtered-off and
d6) d 10.40 (s, 1H), 7.95 (t, J ¼ 6.0 Hz, 1H), 7.70 (d, J ¼ 8.7 Hz, 2H), washed with water (2 ꢃ 50 ml). The crude solid was dissolved in
7.61 (d, J ¼ 8.4 Hz, 2H), 7.14 (t, J ¼ 6.8 Hz, 1H), 6.97 (t, J ¼ 8.6 Hz, CH2Cl2 (50 ml), the organic layer was washed with brine, and the

1244
S. GE ET AL.
solvent was evaporated under reduced pressure to afford 4-acryla- 128.44, 128.38, 122.42, 121.28, 121.22, 120.13 (d, JC–F ¼ 6.3 Hz,
midobenzenesulfonyl chloride as off-white solid (Yield 72.8%).
1 C), 119.60, 117.90 (d, JC–F ¼ 22.68 Hz, 1C). MS (EI) m/z 353.1
[M-H]ꢂ.
2.1.6. General preparation of compounds 8a-j
2.1.6.8. N-(4-(N-(Pyrimidin-2-yl)sulfamoyl)phenyl)acrylamide (8h).
To
a solution of substituted aniline or heterocyclic amine
1
White solid. Yield 70%. Mp 229–230 ꢁC. H NMR (300 MHz, DMSO-
(0.98 mmol) in DCM (15 ml) was added pyridine (1.22 mmol). A
solution of 4-acrylamidobenzenesulfonyl chloride (0.81 mmol) in
DCM (10 ml) was added dropwise to the mixture at 0 ꢁC. The reac-
tion was stirred at rt for 4 h. The solvent was evaporated under
reduced pressure and the crude product was recrystallization to
afford target compounds 8a-j.
d6) d 11.73 (s, 1H), 10.52 (s, 1H), 8.50 (d, J ¼ 4.9 Hz, 2H), 7.95 (d,
J ¼ 9.0 Hz, 2H), 7.93 (d, J ¼ 8.7 Hz, 2H), 7.05 (t, J ¼ 4.9 Hz, 1H), 6.45
(dd, J ¼ 17.0, 9.9 Hz, 1H), 6.30 (dd, J ¼ 16.9, 2.1 Hz, 1H), 5.82 (dd,
J ¼ 9.8, 2.0 Hz, 1H). MS (EI) m/z 327.1 [M þ Na]þ.
2.1.6.9. N-(4-(N-(3,4-Dimethylisoxazol-5-yl)sulfamoyl)phenyl)acryla-
mide (8i). White solid. Yield 81%. Mp 206–208 ꢁC. 1H NMR
(300 MHz, DMSO-d6) d 10.96 (s, 1H), 10.61 (s, 1H), 7.89–7.86 (d,
J ¼ 9.0 Hz, 2H), 7.74–7.71 (d, J ¼ 9.0 Hz, 2H), 6.51–6.42 (m, 1H),
6.34–6.29 (d, J ¼ 15.0 Hz, 1H), 5.85–5.82 (d, J ¼ 9.0 Hz, 1H), 2.08 (s,
3H), 1.63 (s, 3H). MS (EI) m/z 320.1 [M-H]ꢂ.
2.1.6.1. N-(4-(N-Phenylsulfamoyl)phenyl)acrylamide (8a). White
1
solid. Yield 72%. Mp 212–213 ꢁC. H NMR (300 MHz, Chloroform-d)
d 10.50 (s, 1H), 10.19 (s, 1H), 7.74 (dd, J ¼ 23.7, 8.7 Hz, 4H), 7.22 (t,
J ¼ 7.8 Hz, 2H), 7.09–6.98 (m, 3H), 6.42 (dd, J ¼ 16.8, 9.9 Hz, 1H),
6.28 (d, J ¼ 15.3 Hz, 1H), 5.81 (d, J ¼ 10.2 Hz, 1H). MS (EI) m/z 301.1
[M-H]ꢂ.
2.1.6.10. N-(4-(N-(5-Methylisoxazol-3-yl)sulfamoyl)phenyl)acrylamide
(8j). White solid. Yield 85%. Mp 232–233 ꢁC. 1H-NMR (300 MHz,
DMSO-d6) d 11.35 (s, 1H), 10.56 (s, 1H), 7.87–7.80 (dd, J ¼ 12.0,
9.0 Hz, 4H), 6.49–6.40 (m, 1H), 6.33–6.28 (d, J ¼ 15.0 Hz, 1H), 6.13 (s,
1H), 5.85–5.81 (dd, J ¼ 9.0, 3.0 Hz, 1H), 2.29 (s, 3H). MS (EI) m/z
306.1 [M-H]ꢂ.
2.1.6.2. N-(4-(N-(p-Tolyl)sulfamoyl)phenyl)acrylamide (8b). White
solid. Yield 79%. Mp 230–232 ꢁC. 1H NMR (300 MHz, DMSO-d6) d
10.46 (s, 1H), 9.98 (s, 1H), 7.75 (d, J ¼ 8.7 Hz, 2H), 7.65 (d, J ¼ 8.7 Hz,
2H), 6.96 (dd, J ¼ 19.5, 8.4 Hz, 4H), 6.40 (dd, J ¼ 17.1, 9.9 Hz, 1H),
6.26 (d, J ¼ 17.1 Hz, 1H), 5.82–5.70 (m, 1H), 2.15 (s, 3H). MS (EI) m/z
315.1 [M-H]ꢂ.
2.2. Biological assays
2.1.6.3. N-(4-(N-(4-Methoxyphenyl)sulfamoyl)phenyl)acrylamide (8c).
White solid. Yield 80%. Mp 234–235 ꢁC. 1H NMR (300 MHz, DMSO-
d6) d 10.46 (s, 1H), 9.77 (s, 1H), 7.75 (d, J ¼ 8.7 Hz, 2H), 7.60 (d,
J ¼ 8.7 Hz, 2H), 6.94 (d, J ¼ 9.0 Hz, 2H), 6.77 (d, J ¼ 8.7 Hz, 2H), 6.41
(dd, J ¼ 17.1, 9.9 Hz, 1H), 6.26 (d, J ¼ 15.3 Hz, 1H), 5.78 (d,
J ¼ 9.6 Hz, 1H), 3.64 (s, 3H). MS (EI) m/z 331.1 [M-H]ꢂ.
The main bioassays performed in the study, including HeLa cell-
based IDO1/kynurenine assay, Western blot analysis, T cell prolifer-
ation and Treg cell differentiation experiments, were carried out
as described previously32.
2.2.1. Cellular thermal shift assay
2.1.6.4. N-(4-(N-(3-Methoxyphenyl)sulfamoyl)phenyl)acrylamide (8d).
White solid. Yield 77%. Mp 197–199 ꢁC. 1H NMR (300 MHz, DMSO-
d6) d 10.38 (s, 1H), 10.09 (s, 1H), 7.63 (dd, J ¼ 21.3, 8.7 Hz, 4H), 6.98
(t, J ¼ 8.4 Hz, 1H), 6.53–6.51 (m, 2H), 6.45 (d, J ¼ 8.4 Hz, 1H), 6.29
(dd, J ¼ 16.8, 9.9 Hz, 1H), 6.15 (d, J ¼ 16.8 Hz, 1H), 5.67 (d,
J ¼ 9.9 Hz, 1H), 3.52 (s, 3H). MS (EI) m/z 331.1 [M-H]ꢂ.
HeLa cells were seeded in 6-well culture plates at a density of
2 ꢃ 105 per well. On the next day, human IFN-c (100 ng/mL) were
added and incubated for 24 h, and then the cells were treated
with 5d for 2 h. At the end of incubation, cells collected and sub-
jected to CETSA assay. Briefly, incubated cells were equally divided
into 10 parts, each part was heated for 3 min under different tem-
perature (43, 46, 49, 52, 55, 58, 61, 64, 67, and 70 ꢁC), and then
the heated cells were kept at ꢂ80 ꢁC for 12 h, then at room tem-
perature for 5 min, and the process repeated one more time. After
that, cell lysates were extracted by centrifugation at 20,000 ꢃ g,
20 min. Levels of IDO1 protein were assessed by Western
blot analysis.
2.1.6.5. N-(4-(N-(3-Fluorophenyl)sulfamoyl)phenyl)acrylamide (8e).
White solid. Yield 81%. Mp 212–215 ꢁC. 1H NMR (300 MHz, DMSO-
d6) d 10.39–10.37 (m, 2H), 7.68 (d, J ¼ 8.7 Hz, 2H), 7.60 (d,
J ¼ 8.7 Hz, 2H), 7.12 (t, J ¼ 8.1 Hz, 1H), 6.97–6.91 (m, 3H), 6.29 (dd,
J ¼ 16.8, 9.6 Hz, 1H), 6.15 (d, J ¼ 15.9 Hz, 1H), 5.68 (d, J ¼ 9.3 Hz,
1H). MS (EI) m/z 319.1 [M-H]ꢂ.
2.1.6.6. N-(4-(N-(3-Chlorophenyl)sulfamoyl)phenyl)acrylamide (8f).
White solid. Yield 83%. Mp 217–219 ꢁC. 1H NMR (300 MHz, DMSO-
d6) d 10.39 (d, J ¼ 8.1 Hz, 2H), 7.69 (d, J ¼ 9.0 Hz, 2H), 7.61 (d,
J ¼ 9.0 Hz, 2H), 7.12 (t, J ¼ 7.8 Hz, 1H), 6.97–6.92 (m, 3H), 6.30 (dd,
J ¼ 17.1, 10.2 Hz, 1H), 6.16 (d, J ¼ 16.8 Hz, 1H), 5.70–5.62 (m, 1H).
MS (EI) m/z 335.1 [M-H]ꢂ.
2.2.2. In vivo antitumour activity assay
BALB/c mice (6–8 weeks old, 18–22 g) were supplied by Model
Animal Research Centre of Nanjing University (Nanjing, China).
Mice were maintained under standard specific-pathogen-free (SPF)
conditions (21 2 ꢁC and 12-h light: dark cycle). Animal welfare
and experimental procedures were carried out strictly in accord-
ance with the Guide for the Care and Use of Laboratory Animals
(The Ministry of Science and Technology of China, 2006) and the
related ethical regulations of our university. All efforts were made
to minimise animals’ suffering and to reduce the number of ani-
mals used. Mouse colon cancer cells (CT26) and melanoma cells
(B16F1) were cultured and collected by centrifugation (1000 rpm,
5 min) and washed twice with ice-cold PBS. Then cells were
2.1.6.7. N-(4-(N-(3-Chloro-4-fluorophenyl)sulfamoyl)phenyl)acryla-
mide (8g). White solid. Yield 70%. Mp 224–226 ꢁC. 1H NMR
(300 MHz, DMSO-d6) d 10.42 (s, 1H), 7.66 (d, J ¼ 8.7 Hz, 2H), 7.56 (d,
J ¼ 8.4 Hz, 2H), 7.16 (t, J ¼ 9.1 Hz, 1H), 7.05 (d, J ¼ 4.5 Hz, 1H),
6.93–6.89 (m, 1H), 6.28 (dd, J ¼ 16.5, 9.6 Hz, 1H), 6.15 (d,
J ¼ 16.8 Hz, 1H), 5.68 (d, J ¼ 9.6 Hz, 2H). 13C NMR (126 MHz, DMSO)
d 164.19, 154.52 (d, JC–F ¼ 244.44 Hz, 1C), 143.66, 135.54, 133.30, diluted to 1 ꢃ 107/ml and 1 ꢃ 106 CT26/B16F1 cells (in 0.1 ml PBS)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1245
were injected subcutaneously into the right flanks of mice. All In IFD calculations, the ligands were first docked into the rigid
mice formed tumours three days after injection. Then mice were receptor using softened energy function in Glide. By default, a
maximum 20 poses per ligand were retained. Then, the protein
randomly distributed into five groups (n ¼ 10/8) according to
degrees of freedom for each complex were sampled and the pro-
tein-ligand complexes were minimised. The protein structure in
each pose now reflected an induced fit to the ligand structure
and conformation. The best protein-ligand complex was then
identified based on the predicted binding affinities of the docked
ligand. Here, the residues within 5 Å of each of the 20 ligand
poses were subjected to a conformational search and energy mini-
mizations, and the residues outside this range were fixed. Finally,
the minimised ligand was rigorously redocked into the induced-fit
protein structure using Glide XP scoring mode, and metal con-
straints can be applied to both Glide docking stages in IFD proto-
col. The choice of the best-docked structure for each ligand was
made using a model energy score that combines the energy grid
score, the binding affinity predicted by GlideScore, and the
internal strain energy for the model potential used to direct the
conformational-search algorithm.
tumour volumes. 5d (3.125, 6.5, 12.5 mg/kg) were administered
(i.g) to each group respectively every day. 5-FU (25 mg/kg) and
Cis-platinum (1 mg/kg) were administered (i.p) every three days.
Tumour length and tumour width were measured with a Vernier
calliper every three days. Tumour volumes were measured and
calculated using the equation volume ¼ a ꢃ b2/2, where “a” is the
maximal width and “b” is maximal orthogonal width. After the
administration were completed, mice were weighed, euthanized,
and tumours were removed and the weight were taken.
Eight-week-old male BALB/c nude mice, weighing 18–22 g,
were supplied by Model Animal Research Centre of Nanjing
University (Nanjing, China). 1 ꢃ 107/ml and 1 ꢃ 106 CT26 cells (in
0.1 ml PBS) were injected subcutaneously into the right flanks of
mice. After CT26 xenograft BALB/c nude mouse model was estab-
lished, all mice were randomly divided into three groups (n ¼ 8).
5d (12.5 mg/kg) were administered (i.g) every day and 5-FU
(25 mg/kg) awere administered (i.p) every three days. The follow-
ing experimental procedures were similar to the described above.
3. Results and discussion
3.1. Chemistry
2.2.3. Immunohistochemistry analysis
For immunohistochemistry staining, the sections were deparaffi-
nized, rehydrated, and washed in 1% PBS-Tween 20, and then
treated with 2% hydrogen peroxide, blocked with 3% goat serum
(Life Technology, 16210–064) and incubated for 2 h at room tem-
perature with specific primary antibodies. Then the slides were
incubated with streptavidin-HRP (Shanghai Gene Company,
The synthetic pathways for the target compounds were shown in
Scheme 1 and 2. N-acetyl secondary sulphonamides 3 were pre-
pared by the condensation reaction of substituted 4-acetamido-
benzenesulfonyl chloride 1 with various amines 2. Deacetylation
of compounds 3 with concentrated HCl in ethanol gave amino
secondary sulphonamides hydrochloride salts, and followed by
GK500705) for 40 min, then stained with DAB (Shanghai Gene neutralisation with saturated NaHCO3 solution to afford the corre-
Company, GK500705) substrate and counter-stained with haema- sponding amino secondary sulphonamides 4. Finally, the com-
toxylin. Images were acquired by microscopy (Olympus IX51).
pounds 5 were prepared via the condensation reaction of
compounds 4 with acryloyl chloride (Scheme 1).
Alternatively, N-phenylacrylamide 6 was synthesised by con-
densation of aniline with acryloyl chloride in the presence of trie-
2.3. Molecular modelling
In order to consider the flexibility of both ligand and protein, the thylamine, and followed by chlorosulfonation with chlorosulfonic
€
induced fit docking (IFD) protocol in Schrodinger was employed. acid to give 4-acetamidobenzenesulfonyl chloride 7. Subsequently,
Scheme 1. Reagents and conditions: (a) TEA, DCM, 0 ꢁC, rt; (b) HCl, ethanol, 70 ꢁC; (c) Acryloyl chloride, TEA, DCM, 0 ꢁC, rt.
Scheme 2. Reagents and conditions: (a) Acryloyl chloride, TEA, DCM, 0 ꢁC, rt; (b) HSO3Cl, 70 ꢁC, 1 h; (c) Substituted aniline, pyridine, DCM, 0 ꢁC, rt.

1246
S. GE ET AL.
the 4-acetamidobenzenesulfonyl chloride was treated with substi- remarkably increased by 8.4-fold as compared with the hit BS-1.
Inspired by N-(4-(N-(4-methoxybenzyl)sulfamoyl)phenyl)acetamide,
we further synthesised the analogues 3a-i bearing an electron-
donor group on the benzyl group, but they displayed only weak
activity. Subsequently, we tried to remove the acetyl group of N-
acetyl secondary sulphonamides, and found that all the obtained
aniline compounds 4a-f had no improvement in activity. These
results indicated that a free amino group on the phenyl ring with
strong electron-donating ability appeared to be unfavourable for
the inhibitory activity of these compounds. To this end, we tried
to introduce an acryloyl group instead of the acetyl group to the
amine group. As expected, compounds 5a-e bearing an acetyl
group showed moderate to good inhibitory activity. Especially,
compound 5d (HeLa IC50: 6.26 mM) exhibited significantly stronger
activity than the hit compound BS-1 (HeLa IC50: 49.42 mM) and its
corresponding acetyl compound 4e (HeLa IC50 > 50 mM).
We then further optimised the inhibitory activity of N-acryloyl
secondary sulphonamides by modifying the substituents, including
n, R2 and R3, while retaining the acryloyl group at R1. As shown in
Table 2, most of N-acryloyl secondary sulphonamides displayed
good inhibitory activity against IDO1. Among them, benzylamine
derivative 5l and aniline derivative 8g exhibited potent activity
with IC50 values of 6.64 mM and 3.76 mM, which were significantly
increased by 7.4-fold and 13.1-fold as compared with the hit BS-1,
respectively. It is worth noting that the compounds 5d, 5g, 5l,
and 8d with a substituent at 3-position on aniline or benzylamine
had higher activity than those compounds 5c, 5f, 5k and 8c with
a substituent at 4-position (5c vs 5d, 5f vs 5g, 5k vs 5l, 8c vs 8d).
However, compound 5i (IC50: 39.53 mM) with a fluoro substituent
tuted aniline in the presence of pyridine to form the target com-
pounds 8 (Scheme 2).
3.2. Effect of secondary sulphonamides on IDO1 activity
It is well known that IDO1 is broadly expressed in human cancers
and is induced by pro-inflammatory cytokines, such as interferon
gamma (IFN-c) and tumour necrosis factor alpha (TNF-a)41. Here
we utilised a human cervical cancer cell line HeLa that endogen-
ously expresses IDO1 upon IFN-c treatment to evaluate the IDO1
inhibitory activity of the synthesised compounds by measuring
kynurenine secreted into the media. This cell-based assay, as a
rapid and simple method for determination IDO1/kynurenine, is
closely related to the potency of the enzyme assay and has been
widely used to identify various IDO1 inhibitors19,23,29,42. In add-
ition, cytotoxic compounds can reduce cell viability, which will
decrease the IDO1 levels and then lower the conversion of trypto-
phan to kynurenine. Thus, we also employed this cellular assay to
detect false positive IDO1 inhibitors and cytotoxic compounds by
measuring HeLa cell viability. Clinical candidate navoximod was
used as an assay control. The results were shown in Table 1
and 2.
Our group had already synthesised a series of N-acetyl second-
ary sulphonamides as the analogs of compound BS-1 and eval-
uated their inhibitory activity43. To our surprise, although most of
these compounds had weak inhibitory activity against IDO1, N-(4-
(N-(4-methoxybenzyl)sulfamoyl)phenyl)acetamide
(HeLa
IC50:
5.88 mM) showed good inhibitory activity, and its potency was
Table 2. IDO1 inhibitory activity of the target compounds 5f-m and 8c-j.
Table 1. IDO1 inhibitory activity of the target compounds 3a-i, 4a-f, 5a-e and
8a-b.
ꢀ
Compd.
R1
n
R2
p-CF3
m-CF3
p-F
m-F
o-Cl
R3
Inhibition %
IC50 (lM)
5f
CH2¼CHCO
CH2¼CHCO
CH2¼CHCO
CH2¼CHCO
CH2¼CHCO
CH2¼CHCO
CH2¼CHCO
CH2¼CHCO
CH2¼CHCO
CH2¼CHCO
CH2¼CHCO
CH2¼CHCO
CH2¼CHCO
CH2¼CHCO
1
1
1
1
1
1
1
1
0
0
0
0
0
0
–
–
–
–
–
–
–
–
–
–
–
–
–
38.2%
49.5%
70.9%
37.8%
65.1%
37.5%
89.1%
44.0%
20.9%
74.8%
69.7%
89.5%
93.2%
16.4%
39.16
30.53
20.54
39.53
22.66
42.08
6.64
5g
5h
5i
5j
5k
5l
5m
8c
8d
8e
8f
8g
8h
ꢀ
Compd.
R1
n
R2
Inhibition %
IC50 (lM)
BS-1
3a
3b
3c
3d
3e
3f
3g
3h
3i
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
8a
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
NH
1
0
0
0
0
1
1
1
1
2
0
0
1
1
1
2
1
1
1
1
2
0
0
m-Cl
H
p-CH3
p-CH(CH3)2
m-CH(CH3)2
H
p-Cl
p-CF3
m-CF3
H
H
p-CH3
H
p-Cl
m-Cl
H
30.7%
3.9%
16.3%
5.9%
14.6%
2.5%
13.5%
14.5%
7.8%
49.42
N.D.
ꢀꢀ
p-Br
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
22.36
40.06
36.20
6.26
26.27
13.18
N.D.
0.41
m-Br
m,p-diCl
p-OCH3
m-OCH3
m-F
m-Cl
m-Cl-p-F
37.27
ꢀꢀ
N.D.
13.45
17.66
9.48
3.76
N.D.
5.9%
–
12.0%
10.8%
7.4%
–4.2%
2.3%
NH
NH
NH
NH
8i
8j
CH2¼CHCO
CH2¼CHCO
0
0
–
23.6%
22.7%
N.D.
N.D.
NH
3.1%
CH2¼CHCO
CH2¼CHCO
CH2¼CHCO
CH2¼CHCO
CH2¼CHCO
CH2¼CHCO
CH2¼CHCO
H
62.5%
36.0%
40.4%
91.1%
55.2%
85.9%
25.3%
98.6%
p-OCH3
p-Cl
m-Cl
H
H
p-CH3
–
8b
Navoximod
ꢀ
Navoximod
98.6%
0.41
ꢀ
Percentage inhibition at 30 lM.
N.D.: not determined.
Percentage inhibition at 30 lM.
ꢀꢀ
ꢀꢀ
N.D.: not determined.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1247
Figure 3. Compounds 3d, 5d, 5l and 8g did not affect the expression of IDO1 protein. (A) HeLa cells were treated with IFN-c (100 ng/mL) for 24 h, then incubated
with or without test compounds at their IC50 concentrations for 24 h, and expression of IDO was analysed by Western blot using an anti-IDO antibody. (B) The grey-
scale scanning statistical results of A. (C) HeLa cells were incubated with compound 5d (3, 10, 30 lM) for 24h. (D) The greyscale scanning statistical results of C. (E)
HeLa cells were incubated with compound 5d (3, 10, 30 lM) for 48 h. (F) The greyscale scanning statistical results of E. Data are presented as means SEM (n ¼ 3).
at 3-position on aniline showed lower inhibitory activity compared Figure 3, compared with the blank group, the expression of IDO1
with the corresponding compound 5h with a fluorine atom at 4- protein was significantly induced in IFN-c–treated HeLa cells.
position on aniline (IC50: 20.54 mM), which showed similar inhibi- Evidently, all tested compounds 5d, 5l, 8f and 8g did not influ-
tory activity with compound 5a without substituent on aniline ence IDO1 expression as compared with the control group. In
(IC50: 22.36 mM), likely due to the similar size of the fluorine and addition, we further confirmed that treatment of HeLa cells with
hydrogen atoms, Interestingly, compound 8g containing a m- different concentrations of compound 5d (3, 10, 30 mM) for 24 h or
chlorine and a p-fluorine atoms showed the strongest potency 48 h also did not affect IDO1 expression. The results demonstrated
(IC50: 3.76 mM), while compound 5m with m,p-di-chlorine atoms that these compounds blocked the kynurenine pathway by affect-
had only moderate activity (IC50: 37.27 mM). It might be duo to the ing IDO1 enzyme activity rather than its expression and/or HeLa
large volume of the 4-substituent on aniline or benzylamine that cell viability.
cause the bad clashes with the neighbouring residues in active
site of IDO1. In addition, we also tried to replace benzene or ben-
zyl rings with aromatic heterocycles (8 h-8j), including pyrimidine
and isoxazole, but they showed only weak activity.
3.4. Compounds 5d and 8g could effectively restore T cell
proliferation
Taken together, compounds 5d, 5l and 8g showed good
inhibitory activity in the HeLa cell-based IDO1/kynurenine assay.
Furthermore, the viability of HeLa cells was evaluated by the MTT
method at the end of the assay. The readouts showed that most
of the synthesised compounds did not affect cell viability under
the experimental conditions (data not shown), indicating that
IDO1 inhibitory activity of these compounds is not mediated by
their cytotoxicity.
T lymphocytes are exceedingly sensitive to low tryptophan levels.
Overactivation of the kynurenine pathway by high IDO1 expres-
sion leads to local tryptophan depletion, which can suppress T
cell proliferation by activating the GCN2 kinase pathway and
eventually result in T cell anergy44. Here, to determine whether
secondary sulphonamides could rescue IDO1-mediated blockade
of T cell proliferation, we used a co-culture of IDO1-expressing
B16F1 melanoma cells with naïve CD4þ T cells to mimic the
tumour microenvironment. Given that melanoma is a highly
immunogenic tumour and IDO1 constitutively expresses in melan-
oma cells40, B16F1 murine melanoma cell lines that express IDO1
3.3. Effect of 5d, 5 l, 8f and 8g on IDO1 protein expression
To exclude the possibility that these secondary sulphonamides were then used to co-culture with T cells. Naïve CD4þ T cells were
inhibit IDO1 activity by down-regulating IDO1 expression, we uti- isolated from spleens of C57BL/6 mice. As shown in Figure 4, com-
lised Western blot to measure the expression of IDO1 protein in pounds 5d, 8g and navoximod displayed a significant augmenta-
HeLa cells treated with different compounds. As depicted in tion on T cell activity stimulated with B16F1 cells. Especially,

1248
S. GE ET AL.
compound 5d showed better enhancement of T cell proliferation number of the FoxP3þ Treg cells increased approximately 4 times
compared with naïve T cells cultured alone, indicating that IDO1-
expressing B16F1 cells could significantly stimulate naïve T cell dif-
ferentiation into FoxP3þ Treg cells. However, treatment with 5d,
8g and navoximod could significantly reduce FoxP3þ Treg cell dif-
ferentiation. Especially, compound 5d displayed the best effect in
this test, and its FoxP3þ Treg cell number returned to the initial
level. These data evidenced that compounds 5d and 8g could
effectively suppress the differentiation of naïve CD4þ T cell into
FoxP3þ Treg cells, which would help reverse IDO1-mediated
immune suppression.
than navoximod. These findings demonstrated that compounds
5d and 8g could reverse the suppression of T lymphocytes caused
by IDO1.
3.5. Compounds 5d and 8g could effectively inhibit FoxP31 Treg
cell differentiation
Besides causing local tryptophan depletion, high IDO1 expression
can also accumulate metabolites of kynurenine pathway. The ele-
vated metabolites can not only induce effector T cell death dir-
ectly through their cytotoxicity, but also promote the
differentiation of naïve CD4þ T cells into CD4þ FoxP3þ Treg cells
by activating the AHR pathway10. As a highly immunosuppressive
subset of CD4þ T cells, FoxP3þ Treg cells are critical to the main-
tenance of immune homeostasis and are involved in tumour
immune escape, thereby contributing to tumour development
and progression45. Therefore, we also utilised co-culture of B16F1
cells with naïve CD4þ T cells to test whether the synthesised com-
pounds could suppress IDO1 mediated Treg cell differentiation. As
illustrated in Figure 5, when the naïve CD4þ T cells were co-cul-
tured with B16F1 cells under Con A stimulation for 48 h, the
3.6. Compound 5d could bind to IDO1 protein in HeLa cells
The cellular thermal shift assay (CETSA) is usually used to identify
compounds and their potential targets. CESTA can detect the tar-
get engagement of drugs in a cellular context based on the bio-
physical principle of ligand-induced thermal stabilisation of target
proteins46. To this end, we further employed the CETSA assay to
detect whether compound 5d could directly bind to the target
protein IDO1 in HeLa cells. As presented in Figure 6, treatment
with 5d could improve the thermal stability of IDO1 protein in
HeLa cells as compared with the vehicle control, and still main-
tained the protein stability at 55 ꢁC. These results suggested that
compound 5d could enter HeLa cells and then directly bind to
IDO1 protein.
3.7. Compound 5d could effectively inhibit colon carcinoma
growth in immunocompetent mice, but not in
immunodeficient mice
Given that it was the most prominent inhibitor in vitro, we chose
compound 5d for further pharmacodynamic evaluation in vivo.
Bowel cancer is the third leading cause of cancer in the world,
thus we first evaluated the efficacy of compound 5d on the
growth of murine CT26 colon carcinoma xenograft model in
immunocompetent BALB/c mice. Fluorouracil (5-FU) is one of the
most commonly used drugs to different cancers including bowel
cancer, and was used as the positive control. The CT26 tumour-
Figure 4. Compounds 5d and 8 g could promote the proliferation of T cells.
Based on the B16F1-T cell co-culture system, use MTT assay to test T cells prolifer-
ation. The Navoximod and compounds were added to the system at the concen-
tration of 1.5 and 18 lM. Data represent mean SEM (n ¼ 3).
Figure 5. Compounds 5d and 8 g inhibited Treg cell differentiation in the cell co-culture system. (A) After co-cultured with B16F1 cells, T cells were collected. Surface
staining was performed with CD4þ FTTC and CD25þ PE for 30min at 4 ꢁC. The cells were fixed and permeabilized with fixation buffer and permeabilization wash buf-
fer. The intracellular staining was performed with FoxP3þ APC for 20min. The cells were then analysed by flow cytometry analysis. (B) Statistical analysis of result A.
ꢀ
ꢀꢀ
Data are represented as SEM (n ¼ 3). p < 0.05, p < 0.01 vs T þ B16F1.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1249
bearing mice were treated with 5d (3.125, 6.25, 12.5 mg/kg, i.g, carcinoma xenograft were treated with compound 5d and 5-FU
every day) or 5-FU (25 mg/kg, i.p, every other day). Tumour vol-
umes were measured every two days for 14 days. As outlined in
Figure 7, treatment of 5d significantly suppressed the growth of
tumour in a dose-dependent manner as compared with the
vehicle group. Especially, 5d reduced tumour volume by 35.9%
and average tumour weight by 37.5% at the dose of 12.5 mg/kg,
which were comparable to that of 5-FU. Moreover, no significant
change in body weight of the mice was observed in administra-
tion groups compared with the vehicle group during the 14-
day treatment.
for 10 days. As introduced in Figure 8, administration of 5d did
not block tumour growth as compared with the vehicle group,
while classical cytotoxics 5-FU significantly suppressed tumour
growth. Taken together, these results implied that the in vivo anti-
tumour efficacy of compound 5d was achieved by activating
mouse immune system rather than its cytotoxicity.
3.8. Compound 5d could inhibit the growth of tumour cells by
curbing proliferation and inducing apoptosis in CT26
tumour tissues
Then, in order to clarify whether the in vivo antitumour efficacy
of compound 5d was related to the mouse immune system, we
repeated the above experiment in immunodeficient nude mice.
Briefly, BALB/c nude mice bearing established CT26 colon
In order to further observe morphological changes in tumour tis-
sues after administration of compound 5d, the tumour tissue sec-
tions obtained from the BALB/c mice bearing CT26 colorectal
tumours were stained with haematoxylin-eosin (H&E) to highlight
different tissue structures, such as the nucleus and cytoplasm. As
illustrated in Figure 9(A), the number of tumour cells in 5-FU-
treated group was significantly reduced as compared with the
vehicle group. Similarly, administration of 5d decreased the num-
ber of tumour cells, as well as led to the morphological changes
including cell shrinkage and chromatin condensation in a dose-
dependent manner. In addition, it was clearly observed from the
enlarged local image that there are more blood vessels in the
compound 5d group, and their morphology appears more normal,
suggesting that compound 5d might contribute to tumour vascu-
lar normalisation, thereby promoting antitumour effect of the
immune system47.
Figure 6. Compound 5d could bind to IDO1 protein in HeLa cells. HeLa cells
were treated with human IFN-c (100ng/mL) for 24h, and then treated with 5d
for 2 h. The cells were collected and subjected to CETSA assay. The levels of IDO1
protein were assessed by Western blot analysis.
Figure 7. Compound 5d suppressed colon cancer growth in immunocompetent mice. 1 ꢃ 106 CT26 cells were transplanted subcutaneously into the armpit of the
BALB/c mice. Three days after transplantation, mice were randomly allocated to vehicle control or treatment groups (n ¼ 10). Drugs were administered on days 1–14.
Tumour volume (A) and mice body weight (B) were recorded. After sacrifice, solid tumours were separated and weighed (C, D). Data represent mean SEM, n ¼ 10,
ꢀ
ꢀꢀ
p < 0.05, p < 0.01 vs vehicle.

1250
S. GE ET AL.
Figure 8. Compound 5d did not suppress the growth of CT26 tumour in nude mice. 1 ꢃ 106 CT26 cells were transplanted subcutaneously into the armpit of immuno-
deficient BALB/c nude mice. Three days after transplantation, mice were randomly allocated to vehicle control or treatment groups (n ¼ 8). Drugs were administered
on days 1–9. (A) Tumour volume and mouse body weight (B) were recorded. After sacrifice, solid tumours were separated and weighed (C, D). Data represent
ꢀ
ꢀꢀ
mean SEM, n ¼ 8, p < 0.05, p < 0.01 vs vehicle.
Then, to understand the mechanism of tumour cell death, we 3.9. Compound 5d facilitated immune system rejuvenation in
detected the level of proliferating cell nuclear antigen (PCNA)
protein and tumour cell apoptosis by immunohistochemistry
(IHC) and terminal deoxynucleotidyl transferase dUTP nick end
labelling (TUNEL) experiments, respectively. PCNA is known as a
molecular marker for assessing cell proliferation status, and
TUNEL assay is the most widely used method to identify apop-
totic cells. The results of IHC analysis showed that the expression
of PCNA protein in the 5-FU group was significantly reduced as
compared with the vehicle group. The similar results were
observed in the 5d treatment group and showed a dose-
response relationship (Figure 9(B)). In addition, the results of
TUNEL assay showed that the tumour cells had less apoptosis in
the vehicle group, while the apoptosis increased in the 5-FU
group. Likewise, treatment with 5d also significantly induced the
tumour cell apoptosis in a dose-dependent manner (Figure 9(C)).
These data revealed that compound 5d displayed in vivo antitu-
mour efficacy by inhibiting tumour cell proliferation and induc-
ing apoptosis.
To verify the above conclusions, we further performed H&E
and IHC staining on the nude mouse CT26 tumour tissues. As
depicted in Figure 10(A), the tumour cells in the 5d-treated group
remained intact morphology and were arranged in an orderly
manner as that in the vehicle-treated group, while the results of
5-FU treatment were just the opposite. Moreover, the results of
TUNEL assay showed that administration of 5d had no influence
on the expression of PCNA (Figure 10(B)). These findings further
supported the above conclusions.
CT26 tumour-bearing mice
To investigate the effect of compound 5d on the immune func-
tion in CT26 tumour-bearing mice, we carried out the IHC experi-
ment to detect the expression of IFN-c and granzyme B in the
tumour tissues. IFN-c is a key cytokine secreted primarily by acti-
vated T cells and NK cells, which is essential for both innate and
adaptive immunity. Therefore, IFN-c is an important sign of reju-
venation of the immune system. Moreover, cytotoxic T lympho-
cytes (CTLs) is the key effector cells of the adaptive antitumour
immune response. Granzyme B is the primary mediator of apop-
tosis by CTLs and NK cells. It can initiate the processing of cas-
pases and apoptosis when it is released from activated CTLs48. As
detailed in Figure 11, compared with the vehicle, administration
of compound 5d increased the expression of IFN-c in a dose-
dependent manner in the CT26 tumour tissues, suggesting that
5d treatment could facilitate T cell activation and the rejuvenation
of the immune system in the tumour microenvironment. Similarly,
all treatments of 5d elevated granzyme B levels, which was con-
sistent with the results of the TUNEL assay described above.
Furthermore, we also detected the content of FoxP3þ Treg
cells in the CT26 tumour-bearing mouse spleens by fluorescence
activated cell sorting (FACS) assay (Figure 12). The results showed
that 5d treatment decreased the number of FoxP3þ Treg cells in a
dose-dependent manner. Especially, the amount in the high dose
group was reduced 2.4-fold compared with the vehicle group.
Taken together, these results revealed that the compound 5d

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1251
Figure 9. Compound 5d dose-dependently reduced tumour cells and induced apoptosis in mice. The tumour tissues were infused in formaldehyde solution for paraffin
section. (A) H&E staining of tumour tissue. (B) Expression of PCNA. (C) TUNEL staining of tumour sections. Scale bar: 50 lm.
could promote T cell activation, decrease FoxP3þ Treg cell popula- Tumour volumes were measured every two days for 11 days. As
shown in Figure 13, treatment with 5d significantly suppressed
the growth of tumour in a dose-dependent manner both in
tumour volume and in tumour weight as compared with the
vehicle group without significant body weight loss. Especially at
high dose, the average volume of tumour was reduced by 34.3%
after 5d treatment, which were comparable to Cis-platinum.
Analogously, we also performed H&E, IHC and TUNEL experi-
ments on the B16F1 melanoma tissues (Figure 14 and 15). These
results indicated that compound 5d could effectively contribute
to the morphological changes of B16F1melanoma cells, reduce
the number of tumour cells by inhibiting proliferation and induc-
ing apoptosis, and facilitate tumour vascular normalisation.
Importantly, compound 5d also promoted T cell activation by up-
regulating IFN-c expression, and elevated CTLs function by stimu-
tion and heighten the function of CTLs.
3.10. Compound 5d could effectively suppress melanoma
growth in immunocompetent mice
In addition, we also utilised a B16F1 melanoma mouse xenograft
model to observe the efficacy of compound 5d on the other
tumours. Malignant melanoma is a highly invasive tumour and its
immunotherapy has been receiving attention in recent years. As
mentioned above, B16F1 is a murine melanoma cell line that
expresses IDO1. The mouse xenograft model was established by
subcutaneously implanting B16F1 melanoma cells into BALB/c
mice and treated with 5d (3.125, 6.25, 12.5 mg/kg, i.g, every day)
or the positive control Cis-platinum (1 mg/kg, i.p, every other day). lating granzyme B secretion. These results reaffirm that compound

1252
S. GE ET AL.
Figure 10. Compound 5d could not suppress the proliferation of tumour cells in immunodeficient mice. Tumour sections were infused in formaldehyde solution for
immunohistochemistry. (A) H&E staining of tumour tissue. (B) Expression of PCNA. Scale bar: 50 lm.
Figure 11. Compound 5d up-regulated the expression of IFN-c and granzyme B. Tumour sections were infused in formaldehyde solution for immune stain. (A)
Expression of IFN-c. (B) Expression of Granzyme B. Scale bar: 50 lm.
5d could inhibit tumour growth by activating the mouse compounds 5d with IDO1. Since the high flexibility of the side
chain and backbone of IDO1 protein, the classical semi-flexible
docking method often poses a problem for IDO1 docking, whose
algorithm relies on a rigid protein structure. Thus, the induced fit
docking (IFD) method was utilised to overcome the difficulty men-
tioned above. The cocrystallization of Amg-1 in complex with
human IDO1 protein (PDB ID: 4PK5) was employed as the tem-
immune system.
3.11. Molecular modelling studies
Based on their outstanding performance in biological activity, we
performed computational studies to elucidate the interactions of plate in the following docking calculations because of its relatively

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1253
Figure 12. Compound 5d decreased Treg cell differentiation in the CT26 tumour-bearing mouse. (A) Decreased differentiation of Treg cells in the spleen of tumour-
ꢀ
bearing mice treated by compounds. (B) Statistical analysis of result A. Data represent mean SEM, p < 0.05 vs vehicle.
Figure 13. Compound 5d suppressed melanoma growth in mice. 1 ꢃ 106 melanoma cells were transplanted subcutaneously into the armpit of the BALB/c mice. Three
days after transplantation, mice were randomly allocated to vehicle control or treatment groups (n ¼ 6). Drugs were administered on days 1–11. (A) Tumour volume
ꢀ
ꢀꢀ
and mouse body weight (B) were recorded. After sacrifice, solid tumours were separated and weighed (C, D). Data represent mean SEM, n ¼ 6, p < 0.05, p < 0.01
vs vehicle.
high resolution and more structural information at pocket B. As towards pocket B formed by Phe163, Phe226, Arg231, Leu234 and
shown in Figure 16, The benzsulfamide moiety situated deeply in Ile354. Specifically, one oxygen atom of the sulphonyl group bond
pocket A and their benzene ring was interacted with Phe163 directly to the haem iron of IDO1 while the other oxygen atom
through p-p interaction, while the benzylamine moiety extended formed a hydrogen bond with the main chain NH group of

1254
S. GE ET AL.
Figure 14. Compound 5d dose-dependently reduced tumour cells and induced apoptosis in mice. Tumour sections were infused in formaldehyde solution for immuno-
histochemistry. (A) H&E staining of tumour tissue. (B) Expression of PCNA. (C) TUNEL staining of tumour sections. Scale bar: 50lm.
Ala264. Acryloyl groups located at the top of pocket A, and the strongest inhibitory effect, and their activities were signifi-
cantly improved over the hit compound BS-1. The in vitro results
showed that these compounds could bind to IDO1 protein in
HeLa cells without affecting the cell viability and the IDO1 protein
expression under experimental conditions, indicating that these
compounds blocked the kynurenine pathway by influencing IDO1
enzyme activity rather than IDO1 expression and/or cell viability.
In addition, these compounds could promote the T cell prolifer-
ation and suppress the differentiation of naïve CD4þ T cell into
FoxP3þ Treg cells, suggesting that these sulphonamides could
reverse IDO1-mediated immunosuppression.
Based on its excellent performance in vitro, compound 5d was
selected for further evaluating the pharmacodynamic effects in
three types of tumour-bearing mouse models, including two
immunocompetent mouse xenografts and one immunodeficient
mouse xenograft. These in vivo experiments demonstrated that
ethenyl moiety of acryloyl group extended towards a small hydro-
phobic subpocket formed by Met88, Leu124, Cys129, Leu234 and
Gly262. This computational study finds that the occupation of the
small hydrophobic subpocket in pocket A may a key factor affect-
ing IDO1 inhibitory activity. These data could be useful for further
optimisation.
4. Conclusions
In this study, the hit compound BS-1 was found as an IDO1 inhibi-
tor in-house compound library. After structural optimisation, a ser-
ies of sulphonamide derivatives were designed, synthesised, and
evaluated in the HeLa cell-based IDO1/kynurenine assay, resulting
in the identification of secondary sulphonamides as novel IDO1
inhibitors. Among them, the compounds 5d, 5l and 8g displayed treatment with 5d could effectively inhibit the growth of murine

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1255
Figure 15. Compound 5d promoted T cells activation while reduced Foxp3þ Treg cell number in tumour tissue. Tumour sections were infused in formaldehyde solu-
tion for immune stain. (A) Expression of IFN-c. (B) Expression of Granzyme B. Scale bar: 50lm.
Figure 16. (A) Proposed binding mode of compound 5d docked into the crystal structure of human IDO1 (PDB: 4PK5) by IFD methods. (B) The interactions between
compound 5d and IDO1. Polar interactions were presented by red dotted line.
CT26 coloreatic carcinoma xenograft tumour in a dose-dependent and pathological analysis were utilised to detect these tumour tis-
manner in immunocompetent mice, but not in immunodeficient sues. The results from two immunocompetent mouse models indi-
mice, which implied that the in vivo efficacy of compound 5d was cated that compound 5d could induce the morphological
closely associated with the mouse immune system. Similarly, 5d changes of both CT26 and B16F1 tumour cells, decrease the num-
also significantly blocked the growth of B16F1 melanoma in ber of tumour cells by suppressing proliferation and inducing
immunocompetent mice. Functionally, biochemical experiment apoptosis. Importantly, compound 5d could contribute to T cell

1256
S. GE ET AL.
activation by stimulating IFN-c expression, improve CTLs function 11. Munn DH, Sharma MD, Baban B, et al. GCN2 kinase in T cells
by inducing granzyme B secretion, and inhibit FoxP3þ Treg cell
mediates proliferative arrest and anergy induction in
differentiation, which would help facilitate the rejuvenation of the
immune system. These promising results motivate us to further
develop this new type of IDO1 inhibitors.
response to indoleamine 2,3-dioxygenase. Immunity 2005;
22:633–42.
12. Frumento G, Rotondo R, Tonetti M, et al. Tryptophan-
derived catabolites are responsible for inhibition of T and
natural killer cell proliferation induced by indoleamine 2,3-
dioxygenase. J Exp Med 2002;196:459–68.
Disclosure statement
13. Mezrich JD, Fechner JH, Zhang X, et al. An interaction
between kynurenine and the aryl hydrocarbon receptor can
generate regulatory T cells. J Immunol 2010;185:3190–8.
14. Okamoto A, Nikaido T, Ochiai K, et al. Indoleamine 2,3-dioxy-
genase serves as a marker of poor prognosis in gene expres-
sion profiles of serous ovarian cancer cells. Clin Cancer Res
2005;11:6030–9.
15. Ino K, Yoshida N, Kajiyama H, et al. Indoleamine 2,3-dioxyge-
nase is a novel prognostic indicator for endometrial cancer.
Br J Cancer 2006;95:1555–61.
No potential conflict of interest was reported by the author(s).
Funding
This work was supported by grants from the National Natural
Science Foundation of China [No. 21772233, 21977117, 81730100,
21807104 and 81922067], the Natural Science Foundation of
Jiangsu Province [No. BK20190306], the Fundamental Research
Funds for the Central Universities [No. 2632020TD037 and
2181900125], and the Postgraduate Research
&
Practice
16. Pan K, Wang H, Chen MS, et al. Expression and prognosis
role of indoleamine 2,3-dioxygenase in hepatocellular carcin-
oma. J Cancer Res Clin Oncol 2008;134:1247–53.
Innovation Programme of Jiangsu Province [No. KYCX18_0790].
17. Folgiero V, Goffredo BM, Filippini P, et al. Indoleamine 2,3-
dioxygenase 1 (IDO1) activity in leukemia blasts correlates
with poor outcome in childhood acute myeloid leukemia.
Oncotarget 2014;5:2052–64.
18. Speeckaert R, Vermaelen K, van Geel N, et al. Indoleamine
2,3-dioxygenase, a new prognostic marker in sentinel lymph
nodes of melanoma patients. Eur J Cancer 2012;48:2004–11.
19. Liu X, Shin N, Koblish HK, et al. Selective inhibition of IDO1
effectively regulates mediators of antitumor immunity.
Blood 2010;115:3520–30.
20. Holmgaard RB, Zamarin D, Munn DH, et al. Indoleamine 2,3-
dioxygenase is a critical resistance mechanism in antitumor
T cell immunotherapy targeting CTLA-4. J Exp Med 2013;
210:1389–402.
21. Tu W, Yang F, Xu G, et al. Discovery of imidazoisoindole
derivatives as highly potent and orally active indoleamine-
2,3-dioxygenase inhibitors. ACS Med Chem Lett 2019;10:
949–53.
ORCID
Shushan Ge
Wenjie Guo
Yisheng Lai
http://orcid.org/0000-0003-2812-1376
http://orcid.org/0000-0001-8506-3905
http://orcid.org/0000-0002-8026-7949
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