Discovery and structure-activity relationships of phenyl benzenesulfonylhydrazides as novel indoleamine 2,3-dioxygenase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.05.084

A novel class of phenyl benzenesulfonylhydrazides has been identified as potent inhibitors of indoleamine 2,3-dioxygenase (IDO), and their structure-activity relationship was explored. Coupling reactions between various benzenesulfonyl chlorides and phenylhydrazides were utilized to synthesize the sulfonylhydrazides bearing various substituents. Compound 3i exhibited 61 nM of IC₅₀ in enzymatic assay and 172 nM of EC₅₀ in the HeLa cell. The computational study of 3i suggested that the major interactions between 3i and IDO protein are the coordination of sulfone and heme iron, the hydrogen bonding and hydrophobic interactions between 3i and IDO. This novel class of IDO inhibitor provides a new direction to discover effective anti-cancer agents.

Full text of DOI:10.1016/j.bmcl.2014.05.084

Accepted Manuscript
Discovery and structure-activity relationships of phenyl benzenesulfonylhydra-
zides as novel indoleamine-2,3-dioxygenase inhibitors
Min-Fu Cheng, Ming-Shiu Hung, Jen-Shin Song, Shu-Yu Lin, Fang-Yu Liao,
Mine-Hsine Wu, Wenchi Hsiao, Chia-Ling Hsieh, Jian-Sung Wu, Yu-Sheng
Chao, Chuan Shih, Su-Ying Wu, Shau-Hua Ueng
PII:
S0960-894X(14)00593-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.084
BMCL 21698
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 April 2014
20 May 2014
22 May 2014
Please cite this article as: Cheng, M-F., Hung, M-S., Song, J-S., Lin, S-Y., Liao, F-Y., Wu, M-H., Hsiao, W., Hsieh,
C-L., Wu, J-S., Chao, Y-S., Shih, C., Wu, S-Y., Ueng, S-H., Discovery and structure-activity relationships of phenyl
benzenesulfonylhydrazides as novel indoleamine-2,3-dioxygenase inhibitors, Bioorganic & Medicinal Chemistry
Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.05.084
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Discovery
and
structure-activity
relationships
of
phenyl
benzenesulfonylhydrazides as novel indoleamine-2,3-dioxygenase inhibitors
Min-Fu Cheng,† Ming-Shiu Hung,† Jen-Shin Song, Shu-Yu Lin, Fang-Yu Liao, Mine-Hsine Wu,
Wenchi Hsiao, Chia-Ling Hsieh, Jian-Sung Wu, Yu-Sheng Chao, Chuan Shih, Su-Ying Wu,*
Shau-Hua Ueng*
Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli
County 35053, Taiwan, R. O. C.
Corresponding Authors
* Tel: +886-37-246166 ext. 35791. Fax: +886-37-586456. E-mail: shueng@nhri.org.tw
* Tel: +886-37-246166 ext. 35713. Fax: +886-37-586456. E-mail: suying@nhri.org.tw
Author contributions
† These authors contributed equally.

Abstract
A novel class of phenyl benzenesulfonylhydrazides has been identified as potent inhibitors of
indoleamine 2,3-dioxygenase (IDO), and their structure-activity relationship was explored.
Coupling reactions between various benzenesulfonyl chlorides and phenylhydrazides were utilized
to synthesize the sulfonylhydrazides bearing various substituents. Compound 3i exhibited 61 nM of
IC50 in enzymatic assay and 172 nM of EC50 in the HeLa cell. The computational study of 3i
suggested that the major interactions between 3i and IDO protein are the coordination of sulfone
and heme iron, the hydrogen bonding and hydrophobic interactions between 3i and IDO. This novel
class of IDO inhibitor provides a new direction to discover effective anti-cancer agents.
Keywords
Sulfonylhydrazide
Indoleamine 2,3-dioxygenase
Structure-activity relationship
Molecular docking

Cancer continues to be a leading cause of death around the world, and developing new
therapeutics is urgently needed.1 In addition to chemotherapeutic and targeted agents, cancer
immunotherapy is an emerging approach in anti-cancer therapy. The recently approved ipilimumab
along with sipuleucel-T has validated the principle, and both extend survival of cancer patients.
Several approaches including immune-checkpoint modulators are intensively under exploration and
development.2-6 Indoleamine 2,3-dioxygenase (IDO) is also a molecular target under investigation.
Indoleamine 2,3 dioxygenase (IDO) is a heme-containing enzyme which degrades tryptophan
in the kynurenine pathway. Depletion of tryptophan and production of bioactive metabolites upon
IDO induction exerts T cells to become more sensitive to apoptosis and cell cycle arrest. In addition,
T cells are prone to differentiate into immunosuppressive regulatory T cells under these
conditions.7–9 A mounting body of evidence indicates that overexpression of IDO is correlated with
tumor progression and invasiveness, and its expression is associated with poor prognosis.7,9 In
pre-clinical models, blocking IDO activity results in tumor regression in tumor-bearing syngeneic
mouse models.10,11
Several IDO inhibitors have been discovered. 1-methyl-DL-tryptophan (1-MT), with Ki ~ 34
μM, has been well studied, and its D form is now in clinical trials. Although it exhibits limited IDO
inhibitory activity in vitro, satisfactory anti-tumor activity in vivo has been reported.10 Natural
product-derived IDO inhibitors such as brassinin,12 MTH-trp,13 naphthaquinone,14 exiguamine A,15
tryptanthrins,16 and benzofuranquinones 17 have been reported too, and some of these exhibit potent
IDO inhibitory activity in vitro. Non-natural product-derived IDO inhibitors include
4-phenylimidazole,18 phenyl-triazoles,19,20 indol-2-yl ethanones,21 benzothiazole,22 candesartan,23
oxadiazolylcarboximidamides,24 and S-benzylisothioureas,25 some of which inhibited tumor growth
in vivo. Recently, the small molecule IDO inhibitor INCB024360 was reported to be well-tolerated
in patients and effective in highly refractory patients.26 These results support the validity of IDO
inhibition as a potential immunotherapeutic strategy for cancer treatment. In this study, a novel

structural scaffold of IDO inhibitors will be reported, its analogs synthesized, and its
structure-activity relationships discussed.
A high-throughput screening assay was carried out on an in-house library by measuring
kynurenine formation in an enzymatic assay with purified recombinant human IDO protein, 27 and
2-phenyl benzeneethanesulfonylhydrazide (1a) was identified as a potent IDO inhibitor (IC50 = 167
nM, figure 1). However, it did not show any cellular effect in inhibiting IDO activity. This result
suggests that 1a was not capable of entering into cells and modifications to improve cell
permeability would be required. This promising hit was further modified and a series of phenyl
benzenesulfonylhydrazides (1b–1f) were synthesized and assessed in the enzymatic assay (IC50)
and the Hela cell-based cellular assay (EC50).
Insert Figure 1
Methods for synthesis of phenyl benzenesulfonylhydrazides have been well developed.28–33
Coupling reactions of substituted phenyl hydrazines with commercially available sulfonyl chlorides
were used to prepare
a
series of phenyl benzenesulfonylhydrazides. Initially,
N'-(3-methylphenyl)benzenesulfonylhydrazides 1b–1f were synthesized and their biological
activities examined (Scheme 1). The resulting compounds 1b–1f inhibited IDO enzymatic activity
with IC50 values of less than 100 nM. Notably, compound 1b displayed cellular IDO inhibitory
activity in the HeLa cell line with an EC50 value of 241 nM.
Insert Scheme 1
Based on the data shown in scheme 1, the acetamido group at the para-position of the benzene
ring of the benzenesulfonyl moiety resulted a molecule with good cellular activity (1b, EC50 = 241
nM). In order to obtain even more potent compounds, the position of the acetamido group of the
benzenesulfonyl moiety was fixed, and various substituted phenyl hydrazines were introduced to

give the corresponding phenyl benzenesulfonylhydrazide derivatives. As shown in Table 1,
compounds 2a–2t were prepared by conditions similar to that for compounds 1b–1f. Compound 2b
(IC50 = 51 nM), in which R3 is a methyl group, inhibited IDO activity more potently than 1a.
p-Methoxy compound 2c (IC50 = 254 nM) was slightly less active than 2b against IDO. However,
2b and 2c showed poor or no cellular IDO inhibitory activity. Compounds 2d–2f, in which R3 is an
electron-withdrawing group exhibited poor inhibitory activity against IDO (IC50 = 1605 nM–5667
nM).
Next, the effect of halogen substituent on the phenyl ring of the phenylhydrazine was examined.
Compound 2g, with a fluoride at R3, was found to be a potent inhibitor of IDO in enzymatic assay
(IC50 = 79 nM), with modest inhibition of IDO in the HeLa cell line (EC50 = 609 nM). 4-Bromo
compound 2i exhibited potent cellular activity (EC50 = 85 nM) with an IC50 value comparable to 2g
(IC50 = 130 nM). Introducing a halogen at R2 resulted in a dramatic loss of IDO cellular potency,
even though the enzymatic activities of 2j and 2k were maintained (IC50 = 122 and 255 nM,
respectively). Addition of a p-fluoro substituent to these derivatives improved IDO cellular potency
slightly, without significantly affecting the IC50 value (2l, IC50 = 128 nM, EC50 = 2494 nM; 2m,
IC50 = 110 nM, EC50 = 2588 nM). Di-substituted compounds such as 4-bromo-3-fluoro (2n),
4-bromo-3-methyl (2o) and 4-chloro-3-fluoro (2p) exhibited good IDO enzymatic and cellular
potencies (IC50 = 50–194 nM; EC50 = 84–187 nM). A variety of other compounds bearing a
di-substituted phenyl hydrazine ring (2q–2t) were less active than 2n–2p in both enzymatic and
cellular assays.
Insert Table 1
As shown in table 1, bromo-substitution at the 4-position of the phenylhydrazinyl moiety
resulted in potent enzymatic activity as well as cellular activity (2i). To further investigate the role
played by substituents on the benzenesulfonyl moiety, the synthesis of a number of derivatives

(3a–3i) was carried out. The chemical structures and biological activities of these compounds are
shown in Table 2. Most were found to be potent IDO inhibitors with IC50 values of less than 180
nM, except 3c (IC50 = 1431 nM). Compounds 3b and 3i had IC50 values of less than 100 nM.
Insert Table 2
To investigate the role of free NH-group at N2-position of phenyl benzenesulfonylhydrazide,
the N2-methyl phenyl benzenzsulfonylhydrazide (4) was synthesized (figure 2). The inhibitory
activity of compound 4 (IC50 = 4055 nM) was greatly inferior to that of compound 2i (IC50 = 130
nM), suggesting that the NH-group at N2-position of phenyl benzenesulfonylhydrazide greatly
contributes to the inhibition of IDO.
Insert Figure 2
A computational study was performed to elucidate the interactions between the most potent
compound, 3i, and IDO (figure 3). Compound 3i was docked into the inhibitor binding site of IDO
by GOLD 5.1 using the structure of IDO in complex with the compound, 4-phenylimidazole (PDB
code 2D0T), as the template. One of the oxygen atoms of the sulfone group of 3i was found to
coordinate to the heme iron with a distance of 2.04 Å. The other oxygen atom of the sulfone group
formed a hydrogen bond with the main chain NH group of Ala264. The bound conformation of 3i,
which adopted a V-shape to bind to the protein, occupied both Pocket A and Pocket B. The benzoic
acid moiety of 3i formed a hydrogen bond with the thiol group of Cys129, and hydrophobic
contacts with the surrounding residues in Pocket A, including Tyr126, Cys129, Phe164, Ser167,

Gly262, Ser263, and Ala264. The bromine group of phenyl hydrazine moiety extended into the
hydrophobic cavity of Pocket B, formed by Phe163, Phe226, Arg231, Leu234, Ile354, and the heme
ring.
Insert Figure 3
In conclusion, a novel series of phenyl benzenesulfonylhydrazides has been synthesized and
identified as potent IDO inhibitors. A one step synthetic reaction of phenylhydrazines with sulfonyl
chlorides was used to efficiently synthesize the desired phenyl benzenesulfonylhydrazides. Most of
the analogs studied showed potent enzymatic IDO inhibitory activities. The computational study
performed on compound 3i suggests that the coordination formed between the oxygen of the
sulfone group and the heme of IDO together with the hydrogen bond formed between the benzoic
acid of 3i and the thiol group of Cys129 are the major interactions between 3i and the enzyme. In
addition, the hydrophobic interaction between the bromide group and phenyl ring of 3i with pocket
A and B of IDO also contribute to the activity. Two of the phenyl benzenesulfonylhydrazides
exhibited potent IDO inhibitory activities in the cell-based assay (2i and 2p). Both
halogen-containing phenylhydrazinyl and acetamido-substituted benzenesulfonyl moieties were
found to be necessary for potent inhibitory activity in the cellular assay. This study presents a novel
IDO inhibitor and its structure-activity relationships study is investigated. These results provide a
new direction to discover potent IDO inhibitors for the treatment of cancer.
Acknowledgements
We are grateful to National Health Research Institutes and National Science Council of the
Republic of China (100CA007) for financial support.

Supplementary data
Identification data of chemical compounds and biological assay protocols are available in the online
version.
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O
S O
hIDO IC = 167 nM
50
HN
NH
HeLa EC > 10 µM
50
1a
Figure 1. High-throughput screening hit

H
N
O
O
N
S
N
H
O
Br
hIDO IC50 = 4055 nM
Figure 2. Structure of compound 4.

Figure 3. Computational binding mode of 3i (magenta) at the IDO active site (PDB code 2D0T).
Carbon atoms are shown in gray/green/magenta, nitrogen in blue, oxygen in red, bromine in
crimson, sulfur in yellow, and iron in tan. Hydrogen atoms are omitted for clarity.

O
O
H
N
H
N
6
R
S
NH2
5
4
N
H
CH2Cl2, rt, 3 h
1
2
ClO2S
R
3
1b, R = NHAc (IC50 = 49 5 nM; EC50 = 241 52 nM)
1c, R = CONHCH2COOH (IC50 = 80 5 nM; EC50 = 571 65 nM)
1d, R = SO2CH3 (IC50 = 43 21 nM; EC50 = 7846 1029 nM)
1e, R = CN (IC50 = 59 2 nM; EC50 = 3404 607 nM)
O
N
(IC50 = 59 17 nM; EC50 = 296 59 nM)
1f, R =
CF3
Scheme 1. Synthesis and EC50 values of N’-(3-methylphenyl)benzenesulfonyl hydrazides 1b–1f.

Table 1. Structures and IDO inhibitory activities of phenyl benzenesulfonylhydrazides 2a–2t
O
O
H
N
R4
R3
S
N
H
O
R1
N
H
R2
2a−2t
compounds
R1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
R2
R3
H
R4
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
IC50 (nM)a EC50 (nM)a
H
H
71 7
51 6
>10000
1171 70
>10000
2a
2b
2c
2d
2e
2f
Me
OMe
CN
H
254 22
H
5667 1374 1393 159
H
SO2NH2
2475 524
1605 196
79 35
3118 509
181 17
609 16
142 12
85 4
H
CF3
F
H
2g
2h
2i
H
Cl
Br
H
155 24
130 27
122 31
255 28
128 21
110 26
205 1
H
F
>10000
7563 1324
2494 71
2588 136
187 7
2j
Br
Br
Cl
F
H
2k
2l
F
F
2m
2n
2o
2p
2q
2r
2s
Br
Br
Cl
H
Me
F
58 15
130 9
194 38
1746 15
608 105
247 57
352 70
84 15
F
2229 264
>10000
350 34
508 77
H
F
H
H
Cl
H
Cl
Cl
H
Cl
H
2t
a IC50 and EC50 values are the mean of at least three independent assays, presented as mean SD.

Table 2. Structures and IDO inhibitory activities of phenyl benzenesulfonylhydrazides 3a–3l
O
O
H
N
S
N
H
R
Br
3a−3l
compounds
R
H
IC50 (nM)a EC50 (nM)a
172 29
85 6
128 5
374 55
640 55
134 7
465 26
187 4
183 7
152 6
172 17
3a
3b
3c
3d
3e
3f
4-Br
4-OMe
4-CN
1431 346
176 3
4-(CH2)2CO2Me
4-NHCO(CH2)2CH3
4-(CH2)2CO2H
4-OCH2CO2H
CO2H
121 19
132 21
141 36
125 8
3g
3h
3i
61 12
a IC50 and EC50 values are the mean of at least three independent assays, presented as mean SD.

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Discovery and structure-activity relatLioeanvsehthiispasreaobflapnkhfeonr aybsltract info.
benzenesulfonylhydrazides as novel indoleamine-2,3-dioxygenas
inhibitors
Min-Fu Cheng, Ming-Shiu Hung, Jen-Shin Song, Shu-Yu Lin, Fang-Yu Liao, Mine-Hsine Wu, Wenchi Hsiao, Chia-Ling Hsieh, Jian-Su