Cyclic analogue of S-benzylisothiourea that suppresses kynurenine production without inhibiting indoleamine 2,3-dioxygenase activity

Article abstract of DOI:10.1016/j.bmcl.2018.07.034

Kynurenine is biosynthesised from tryptophan catalysed by indoleamine 2,3-dioxygenase (IDO). The abrogation of kynurenine production is considered a promising therapeutic target for immunological cancer treatment. In the course of our IDO inhibitor programme, formal cyclisation of the isothiourea moiety of the IDO inhibitor 1 afforded the 5-Cl-benzimidazole derivative 2b-6, which inhibited both recombinant human IDO (rhIDO) activity and cellular kynurenine production. Further derivatisation of 2b-6 provided the potent inhibitor of cellular kynurenine production 2i (IC₅₀ = 0.34 μM), which unexpectedly exerted little effect on the enzymatic activity of rhIDO. Elucidation of the mechanism of action revealed that compound 2i suppresses IDO expression at the protein level by inhibiting STAT1 expression in IFN-γ-treated A431 cells. The kynurenine-production inhibitor 2i is expected to be a promising starting point for a novel approach to immunological cancer treatment.

Full text of DOI:10.1016/j.bmcl.2018.07.034

Accepted Manuscript
Cyclic analogue of S-benzylisothiourea that suppresses kynurenine production
without inhibiting indoleamine 2,3-dioxygenase activity
Miwa Fukuda, Tomomi Sasaki, Tomoko Hashimoto, Hiroyuki Miyachi, Minoru
Waki, Akira Asai, Osamu Takikawa, Osamu Ohno, Kenji Matsuno
PII:
S0960-894X(18)30614-0
https://doi.org/10.1016/j.bmcl.2018.07.034
BMCL 25965
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
29 May 2018
18 July 2018
19 July 2018
Please cite this article as: Fukuda, M., Sasaki, T., Hashimoto, T., Miyachi, H., Waki, M., Asai, A., Takikawa, O.,
Ohno, O., Matsuno, K., Cyclic analogue of S-benzylisothiourea that suppresses kynurenine production without
inhibiting indoleamine 2,3-dioxygenase activity, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://
doi.org/10.1016/j.bmcl.2018.07.034
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Cyclic analogue of S-benzylisothiourea that
suppresses kynurenine production without
inhibiting indoleamine 2,3-dioxygenase
activity
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Miwa Fukuda, Tomomi Sasaki, Tomoko Hashimoto, Hiroyuki Miyachi, Minoru Waki, Akira Asai,
Osamu Takikawa, Osamu Ohno, and Kenji Matsuno*

Bioorganic & Medicinal Chemistry Letters
Cyclic analogue of S-benzylisothiourea that suppresses kynurenine production
without inhibiting indoleamine 2,3-dioxygenase activity
Miwa Fukudaa, Tomomi Sasakib, Tomoko Hashimotob, Hiroyuki Miyachia, †, Minoru Wakia, Akira Asaic,
Osamu Takikawad, Osamu Ohnob, and Kenji Matsunob, *
a Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.
b Laboratory of Medicinal Chemistry, Department of Chemistry and Life Science, School of Advanced Engineering, Kogakuin University,
2665-1 Nakano-machi, Hachi-oji, Tokyo 192-0015, Japan.
c Graduate School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.
d National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, 36-3 Gengo, Morioka, Obu, Aichi 474-8522, Japan.
†present address: Drug Discovery Initiative, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
ARTICLE INFO
ABSTRACT
Kynurenine is biosynthesised from tryptophan catalysed by indoleamine 2,3-
Article history:
Received
Revised
Accepted
Available online
dioxygenase (IDO). The abrogation of kynurenine production is considered a promising
therapeutic target for immunological cancer treatment. In the course of our IDO inhibitor
programme, formal cyclisation of the isothiourea moiety of the IDO inhibitor 1 afforded the 5-
Cl-benzimidazole derivative 2b-6, which inhibited both recombinant human IDO (rhIDO)
activity and cellular kynurenine production. Further derivatisation of 2b-6 provided the potent
inhibitor of cellular kynurenine production 2i (IC50 = 0.34 µM), which unexpectedly exerted
little effect on the enzymatic activity of rhIDO. Elucidation of the mechanism of action revealed
that compound 2i suppresses IDO expression at the protein level by inhibiting STAT1
expression in IFN-γ-treated A431 cells. The kynurenine-production inhibitor 2i is expected to be
a promising starting point for a novel approach to immunological cancer treatment.
2009 Elsevier Ltd. All rights reserved.
Keywords:
kynurenine
inhibitor
indoleamine 2,3-dioxygenase
signal transducer and activator of transcription 1
immunological cancer treatment
Catabolism of the essential amino acid tryptophan generates
several biologically active metabolites, such as the
neurotransmitter serotonin, the excitotoxin quinolinic acid,
kynurenic acid, and nicotinamide adenine dinucleotide.1,2 These
metabolites are predominantly formed through the kynurenine
pathway (>90% of tryptophan in mammals).3 The initial step in
this pathway is the oxidative cleavage of the C-2/C-3 bond of the
indole ring of tryptophan to afford Nʹ-formylkynurenine. This is
the rate-limiting step in the kynurenine pathway and is catalysed
by intracellular heme-containing dioxygenases, namely
indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-
dioxygenase.4 Nʹ-Formylkynurenine is then converted to
kynurenine by a formamidase, and the subsequent metabolism of
kynurenine provides many biological substances (vide supra).
regulatory T (Treg) cells.11 These effects influence the pathology
of autoimmune diseases and, in particular, cancer immune
tolerance. Furthermore, kynurenine has been identified as an
endogenous tumour-promoting ligand of the human aryl
hydrocarbon receptor (AhR).12 Cancer-cell-derived kynurenine
promotes tumour-cell survival and motility and suppresses the
antitumour immune response through AhR in an
autocrine/paracrine manner.12 With progress in understanding
cancer immunology, disruption of the PD-1/PD-L1 immune
checkpoint successfully led to the new cancer therapies
nivolumab and pembrolizumab.13,14 Consequently, the abrogation
of kynurenine production in cancer cells is considered a
promising approach to anticancer therapy.15
The importance of the kynurenine pathway in drug discovery
has prompted efforts to identify modulators for this pathway. In
particular, IDO has received attention as the regulatory enzyme
that catalyses the initial and rate-limiting step in the kynurenine
pathway. Several IDO inhibitors have been reported,16 such as
the S-benzylisothioureas17 and analogues of the anti-hypertensive
agent candesartan18 that we recently described. Although the S-
benzylisothiourea analogues inhibited both recombinant human
IDO (rhIDO) and cellular kynurenine production in A431 cells,
the isothiourea moiety is not a drug-like structure owing to its
extremely hydrophilic properties and potential toxicity associated
with substitution reactions involving nucleophilic groups of
amino acids.
Kynurenine and its metabolites play a variety of roles in the
central nervous system (CNS) and the innate and adaptive
immune responses.5–9 Most metabolites of the kynurenine
pathway are neuroactive and play essential roles in regulating the
function of the N-methyl-D-aspartate (NMDA) receptor. In
particular, increased levels of quinolinic acid cause excitotoxicity
by stimulating NMDA receptors in the CNS and have been
implicated in neurodegenerative disorders such as Parkinson’s
and Alzheimer’s diseases.10 In addition, kynurenine and its
metabolites are responsible for immunological tolerance. It has
been reported that kynurenine modulates T cells by suppressing
their proliferation and inducing apoptosis, and it also activates

Fig. 1. Synthetic concept: conversion of the isothiourea to drug-like heterocycles.
Scheme 2. (a) BnBr, K2CO3, DMF, 100 °C, 95%; (b) 1 mol/L NaOH (aq.),
Therefore, conversion of the isothiourea moiety to a drug-like
structure is essential for exploiting these promising compounds
as drugs for cancer immunotherapy. Herein, we describe the
design and synthesis of cyclic derivatives 2 of isothiourea 1 (Fig.
1), as well as the installation of substituents on the parent phenyl
ring. The biological activities of these compounds with respect to
rhIDO inhibition and cellular kynurenine production in A431
cells are also reported. In particular, one derivative exhibited
unexpectedly potent inhibition of cellular kynurenine production
with very low rhIDO inhibition, and the underlying mechanism
of action was elucidated.
EtOH, 50 °C, 76%; (c) BH3–THF, THF, 0 °C to RT, 86%; (d) phenol, K2CO3,
DMF, 120 °C, 77%; (e) NaBH4, MeOH, 0 °C, 87% for 3d, 90% for 3h; (f)
mCPBA (0.77 eq.), CH2Cl2, 58%; (g) mCPBA (2.3 eq.), CH2Cl2, 89%.
Unsaturation (2e–2g) and extension (2l) of the ethylene chain
in the 2-phenethyl moiety of compound 2b-6 (Table 1) were
achieved via palladium chemistry (Scheme 3). 2-
Bromobenzaldehyde (9) served as the starting material for benzyl
alcohols 3e–3g and 3l via the corresponding aldehydes.
Sonogashira–Hagihara cross-coupling with phenylacetylene
afforded 2-(phenylethynyl)benzaldehyde (7g) in excellent yield.
(Z)-2-Styrylbenzaldehyde (7e) was synthesised by partial
hydrogenation of 7g using Lindlar catalyst in MeOH. Following
unsuccessful attempts to directly convert the (Z)-olefin 7e to the
(E)-olefin 7f using I2 or light, the Mizoroki–Heck reaction of 9
with styrene selectively provided the (E)-styryl analogue 7f.
Reduction of the aldehydes 7g, 7e, and 7f with NaBH4 afforded
the corresponding benzyl alcohols 3g (-C≡C-), 3e ((Z)-CH=CH-),
and 3f ((E)-CH=CH-), respectively. The use of allylbenzene
instead of styrene in the Mizoroki–Heck reaction of 9 furnished a
mixture of alkene regioisomers (7l-1, 7l-2), which was reduced
using NaBH4 and subsequently hydrogenated to afford the sole
product 2-(3-phenylpropyl)benzyl alcohol (3l).
All of the target molecules were synthesised by the
condensation of a mercaptoheterocycle (Het-SH) and benzyl
alcohol 3 by activation of the hydroxyl group using MsCl
(method A) or PBr3 (method B), except for the commercially
available compound 2a (Scheme 1).
Scheme 1. (method A) MsCl, Et3N, 4-DMAP, CH2Cl2, 0 °C to RT; (method
B) PBr3, THF, 0 °C.
Commercially available 2-phenethylbenzyl alcohol (3b) was
used to prepare the compound 2b series (Table 1). The
preparation of the requisite benzyl alcohols (3c, 3d, 3h–3k) for
the target molecules 2c, 2d, and 2h–2k is illustrated in Scheme 2.
Reduction of carboxylic acids 5c and 5i with BH3–THF provided
the corresponding alcohols 3c (X = CH2) and 3i (X = SCH2) in
good yield. The starting material 2-benzylbenzoic acid (5c) was
commercially available, whereas 2-(benzylthio)benzoic acid (5i)
was obtained by benzylation of thiosalicylic acid 4 followed by
hydrolysis with aqueous NaOH. Compounds 3j (X = SOCH2)
and 3k (X = SO2CH2) were synthesised by the oxidation of 3i
with different stoichiometric amounts of mCPBA. Furthermore,
reduction of aldehydes 7d and 7h with NaBH4 provided the
corresponding alcohols 3d (X = O) and 3h (X = OCH2) in good
yield. The starting material 2-phenoxybenzaldehyde (7d) was
synthesised by SNAr reaction of 2-fluorobenzaldehyde (6) with
phenol, whereas 2-benzyloxybenzaldehyde (7h) was purchased
from a commercial supplier (Scheme 2).
Scheme 3. (a) PhC≡CH, Pd(PPh3)4, dppf, CuI, EtiPr2N, THF, reflux, 92%;
(b) H2, Lindlar catalyst, MeOH, RT, 60%; (c) PhCH=CH2, Pd(OAc)2,
BINAP, EtiPr2N, dioxane, reflux, 51%; (d) PhCH2CH=CH2, Pd(OAc)2,
BINAP, EtiPr2N, dioxane, reflux, quant.; (e) NaBH4, MeOH, 0 °C, 81% for
3e, quant. for 3f, 100% for 3g, 88% for 3l; (f) H2, 10% Pd/C, MeOH, 2.5 atm,
RT, 54%.
Table 1 lists the rhIDO inhibitory activities of the synthesised
compounds in which the isothiourea moiety had been formally
replaced with a heterocycle. Contrary to our expectations, the
simple benzimidazole derivative 2a, an analogue of compound
1a containing a cyclised isothiourea moiety, was completely
inactive with respect to rhIDO inhibition. However, the
introduction of a 2-phenethyl group into the benzene ring (2b-1)
led to obvious rhIDO inhibition (43% at 10 µM). Therefore, we
examined a variety of heterocyclic rings attached to the 2-
phenethylbenzylthio moiety in an effort to enhance the rhIDO
inhibition. Replacement of the benzimidazole ring with imidazole

(2b-2), imidazoline (2b-3), thiazole (2b-4), and thiazoline (2b-5)
rings reduced the activity. In contrast, the introduction of 5-Cl
into the benzimidazole ring (2b-6) retained the rhIDO inhibition.
Analogues with other Cl-containing heterocycles (2b-7, 2b-8)
were weak rhIDO inhibitors.
Table 1. Structures and synthetic methods for compounds 2a and 2b, and their inhibitory activities towards rhIDO and cellular kynurenine production.
rhIDO
KYN productionb
Synthetic
methoda
Compound R2
Het
% inhibition @10 µM
% inhibition @1 µM
1a
2a
H
H
(IC50 = 61 µM)
(44% @10 µM)
12%
−42%
2b-1
2b-2
CH2CH2Ph
CH2CH2Ph
A
B
43%
19%
43%
1%
2b-3
2b-4
2b-5
2b-6
CH2CH2Ph
CH2CH2Ph
CH2CH2Ph
CH2CH2Ph
B
B
A
A
17%
31%
0%
7%
7%
0%
47%
48%
2b-7
2b-8
CH2CH2Ph
CH2CH2Ph
B
B
21%
−18%
9%
−8%
a Synthetic method for final condensation with Het-SH.
b Kynurenine production in A431 cells.
In addition, we examined the cellular inhibition of
kynurenine production, which is mediated by IDO, by the
synthesised compounds, using interferon-γ (IFN-γ)-treated
human epithelial carcinoma A431 cells (Table 1). Among the
compounds that displayed obvious rhIDO inhibition, compounds
2b-1 and 2b-6 also exhibited cellular inhibition of kynurenine
production without cytotoxicity (data not shown).
kynurenine production in A431 cells (IC50 = 0.45 µM) without
substantial rhIDO inhibition, which occurred without cytotoxicity
even at 100 µM (data not shown). We therefore considered that
the potent inhibition of cellular kynurenine production could
possibly arise from a distinct mechanism of action.
Table 2. Structures and synthetic methods for compounds 2c-2l, and their
inhibitory activities towards rhIDO and cellular kynurenine production.a
We next examined the influence of the ethylene linker of the
2-phenethyl moiety of the 5-Cl-benzimidazole analogue 2b-6.
Table 2 lists the inhibitory activities of the resulting compounds
2c–2l. Replacement of the 2-phenethyl moiety with -CH2Ph (2c),
(Z)-CH=CHPh (2e), and -C ≡ CPh (2g) groups resulted in
comparable rhIDO inhibition as the parent compound 2b-6,
whereas the -OPh (2d), (E)-CH2=CHPh (2f), -OCH2Ph (2h), -
SCH2Ph (2i), -SOCH2Ph (2j), -SO2CH2Ph (2k), and -(CH2)3Ph
(2l) analogues displayed reduced activity. These results indicate
that the distance and orientation of the phenyl ring from the
benzimidazole moiety are important and that heteroatoms exert a
detrimental effect on the interaction with rhIDO. Furthermore,
we evaluated the cellular inhibition of kynurenine production in
IFN-γ-treated A431 cells by the synthesised compounds (Table
2). Among the compounds that exhibited obvious rhIDO
rhIDO
KYN productionb
Compound R2
% inhibition
@10 µM
% inhibition @1 µM
or IC50
2c
2d
2e
CH2Ph
46%
30%
49%
13%
39%
OPh
CH=CHPh (Z)
IC50 = 0.34 µM
2f
2g
CH=CHPh (E)
C≡CPh
−6%
52%
15%
6%
2h
2i
OCH2Ph
3%
19%
11%
24%
19%
30%
IC50 = 0.45 µM
30%
SCH2Ph
inhibition, compound 2e demonstrated potent inhibition (IC50
=
2j
SOCH2Ph
0.34 µM) of kynurenine production without causing cytotoxicity
even at the higher concentration of 100 µM (data not shown),
whereas compounds 2c and 2g showed no obvious cellular
activity. Surprisingly, compound 2i exhibited potent inhibition of
2k
2l
SO2CH2Ph
CH2CH2CH2Ph
1%
44%
a All compounds were synthesised by method A.
b Kynurenine production in A431 cells.

To investigate the mechanism of action of compound 2i,
namely its potent inhibition of kynurenine production in A431
cells without rhIDO inhibition, the IDO expression level of the
cells was measured by Western blotting. IFN-γ induced
upregulation of the IDO protein level, whereas compound 2i
suppressed IDO expression in a concentration-dependent manner
(Fig. 2). This phenomenon was only observed for the cyclic
analogue 2i, that is, we have previously reported that the parent
S-benzylisothiourea 1 does not affect the IDO expression level in
IFN-γ-treated A431 cells.17 Therefore, we next explored the
effect on Janus kinase/signal transducer and activator of
transcription 1 (JAK/STAT1) signalling, because IFN-γ induces
IDO expression through this pathway.19 As shown in Fig. 2,
compound 2i reduced STAT1 expression, which was upregulated
by IFN-γ treatment, in a concentration-dependent manner. Taken
together, these results indicate that compound 2i suppressed IDO
expression by inhibiting STAT1 expression in IFN-γ-treated
A431 cells.
1. Sono M, Roach M. P., Coulter E. D., Dawson J. H. Chem. Rev.
1996; 96: 2841-2887.
2. Botting N. P. Chem. Soc. Rev. 1995; 24: 401-412.
3. Leklem J. E. Am. J. Clin. Nutr. 1971; 24: 659–672.
4. Takikawa O. Biochem. Biophys. Res. Commun. 2005; 338: 12-
19.
5. Stone T. W., Darlington L. G. Nat. Rev. Drug Discov. 2002; 1:
609–620.
6. Munn D. H., Mellor A. L. Trends Mol. Med. 2004; 10: 15–18.
7. Munn D. H., Mellor A. L. J. Clin. Invest. 2007; 117: 1147–
1154.
8. Lob S., Konigsrainer A., Rammensee H. G., Opelz G., Terness
P. Nat Rev. Cancer 2009; 9: 445–452.
9. Vécsei L., Szalárdy L., Fülöp F., Toldi J. Nat. Rev. Drug
Discov. 2013; 12: 64-82.
10. Schwarcz R., Bruno J. P., Muchowski P. J., Wu H.-Q. Nat.
Rev. Neurosci. 2012; 13: 465-477.
11. Mándi Y., Vécsei L. J. Neural Transm. 2012; 119: 197–209.
12. Opitz C. A., Litzenburger U. M., Sahm F., Ott M., Tritschler
I., Trump S., Schumacher T., Jestaedt L., Schrenk D., Weller M.,
Jugold M., Guillemin G. J., Miller C. L., Lutz C., Radlwimmer
B., Lehmann I., Deimling A. v., Wick W., Platten M. Nature
2011; 478: 197–203.
13. Deeks E. D. Drugs 2014; 74: 1233-1239.
14. Ribas A., Puzanov I., Dummer R., Schadendorf D., Hamid
O., Robert C., Hodi F. S., Schachter J., Pavlick A. C., Lewis K.
D., Cranmer L. D., Blank C. U., O'Day S. J., Ascierto P. A.,
Salama A. K., Margolin K. A., Loquai C., Eigentler T. K.,
Gangadhar T. C., Carlino M. S., Agarwala S. S., Moschos S. J.,
Sosman J. A., Goldinger S. M., Shapira-Frommer R., Gonzalez
R., Kirkwood J. M., Wolchok J. D., Eggermont A., Li X. N.,
Zhou W., Zernhelt A. M., Lis J., Ebbinghaus S., Kang S. P.,
Daud A. Lancet Oncol. 2015; 16: 908-918.
Fig. 2. Western blotting analysis of the effects of compound 2i on IDO and
STAT1 expression in IFN-γ-treated A431 cells.
15. Weinmann H. ChemMedChem 2016; 11: 450-466.
16. Collin M. Expert Opin. Ther. Pat. 2016; 26: 555-564.
17. Matsuno K., Takai K., Isaka Y., Unno Y., Sato M., Takikawa
O., Asai A. Bioorg. Med. Chem. Lett. 2010; 20: 5126-5129.
18. Matsuno K., Yamazaki H., Isaka Y., Takai K., Ishikawa Y.,
Unno Y., Fujii S., Takikawa O., Asai A. MedChemComm 2012;
3: 475-479.
In summary, we synthesised cyclic analogues of our IDO
inhibitor S-benzylisothiourea in an effort to circumvent its
possible toxicity. The 5-Cl-benzimidazole derivative 2b-6 was a
moderate rhIDO inhibitor with cellular inhibition of kynurenine
production. Compound 2i was found to exhibit potent inhibition
of cellular kynurenine production despite its unexpectedly minor
effect on the enzymatic activity of rhIDO. Analysis of the
mechanism of action revealed that compound 2i suppresses IDO
expression at the protein level by inhibiting STAT1 expression in
IFN-γ-treated A431 cells. With its distinct mechanism of action,
the kynurenine-production inhibitor 2i is expected to be a novel
lead compound for immunological cancer treatment.
19. Du M. X., Sotero-Esteva W. D., Taylor M. W. J. Interferon
Cytokine Res. 2004; 20: 133-142.
Acknowledgements
The authors thank Ms. Masumi Namba, Mr. Takeo Makino,
Ms. Hiromi Yamamoto and Mr. Kanta Nakayama for their
technical assistance for compound synthesis and analysis. This
study was supported by the Japan Society for the Promotion of
Science (JSPS) Grants-in-Aid for Scientific Research (C) (grant
number JP25460149 to Kenji Matsuno) and by a grant from the
Strategic Research Foundation Grant-aided Project for Private
Universities (grant number S1411005 to Yasutada Imamura and
Kenji Matsuno) from the Ministry of Education, Culture, Sports,
Science and Technology, Japan.
Conflict of Interest (COI)
The authors declare no conflict of interest.
Supplementary Material
The online version of this article contains supplementary
material.
References





Modifying S-benzylisothiourea provided IDO
inhibitors with kynurenine production.
Cpd 2i demonstrated potent inhibition for
kynurenine production.
Cpd 2i had barely effect on the enzymatic
activity of IDO unexpectedly.
Cpd 2i suppressed the expression of IDO by
inhibiting STAT1 expression in cells.