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K. Matsuno et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5126–5129
7
6
with SARs that were distinct from those of the benzylisothioure-
as. Most of the potent IDO inhibitors also suppressed kynurenine
production in A431 cells, with the exception of benzenethiol
analogue 10i. Combined with their excellent ligand
efficiencies, benzylthioureas could potentially serve as lead com-
pounds for further drug design such as structure-based drug
design.
5
4
3
2
Acknowledgements
1
0
The authors thank Ms. Yasuko Watanabe for her excellent tech-
nical assistance in chemical synthesis. This work was supported by
the grant entitled, ‘‘Drug Discovery Program” from Pharma Valley
Center, Shizuoka Prefecture, Japan.
-0.1
-0.05
0
0.05
0.1
0.15
-1
1/[S] (μM)
Figure 2. Steady-state kinetic analysis of recombinant human IDO by Lineweaver–
Burk plot. ꢀ No inhibition, j -1-MT (100 M), and N compound 3d (1 M).
L
l
l
Supplementary data
and 8b) and hydroxyl (9) groups resulted in total loss of activity.
On the contrary, the benzylthiols (10a–10h) showed potent inhib-
itory activity both for IDO and kynurenine production in A431
cells. With respect to the substituents on the phenyl ring, the SARs
differed from those of the benzylisothioureas 3; 4-Cl (10a) was also
a potent inhibitor, and 2-Cl (10b), H (10c), 4-F (10d), 4-Me (10e),
and 4-OMe (10f) retained similarly potent inhibitory activity, un-
like the corresponding benzylisothioureas 3. For incorporation of
an additional Cl atom (10g and 10h), 2,4-Cl2 (10g) was equipotent
to the parent 4-Cl (10a), and 3,4-Cl2 (10h) exhibited the most po-
Supplementary data associated with this article can be found, in
References and notes
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in Figure 2, the Lineweaver–Burk plot was found to be consistent
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same as reported in the literature.27
The distinct SARs between benzylisothioureas 3 and benzylthi-
ols 10 might be due to differing binding modes in the heme region
at the active site of IDO, since noncompetitive inhibitors could also
bind at alternative pockets around the active site of IDO.28,29 Given
the well-known metal coordinating properties of SH, it is likely
that the SH moiety of benzylthiol chelates to the heme iron. In sim-
ilar fashion, the isothiourea may bind to heme itself. Alternatively,
the isothiourea could be a good bidentate acceptor of carboxylic
acid. Since there are propionic acid in the heme structure, and
some amino acid residues of carboxylic acid such as Asp274 and
Glu171 around the active site,30 we speculate that the isothiourea
moiety interacts with such carboxylic acid. This speculation is sup-
ported by the reduction in IDO inhibition for bis-methylated S-isot-
hioureas (4) and N-isothioureas (5a and 5b). Further analysis to
clarify the molecular interaction of those compounds with IDO is
in progress.
21. Zheng, X.; Koropatnick, J.; Li, M.; Zhang, X.; Ling, F.; Ren, X.; Hao, X.; Sun, H.;
Vladau, C.; Franek, J. A.; Febg, B.; Urquhart, B. L.; Zhong, R.; Freeman, D. J.;
Garcia, B.; Min, W.-P. J. Immunol. 2006, 177, 5639.
22. IDO activity was determined as follow. In brief, the standard reaction mixture
(200
with NaOH and HCl), 100
tryptophan, 5 nM recombinant human IDO, and DMSO solution of the
compound (4 L). The reaction was carried out at 37 °C for 60 min and
stopped by the addition of 40 L of 30% (w/v) CCl3COOH. After heating at 50 °C
for 15 min, the reaction mixture was centrifuged at 15000g for 5 min. The
supernatant (150 L) was transferred into a well of a 96-well microplate and
mixed with 150 L of 2% (w/v) p-dimethylaminobenzaldehyde in acetic acid.
l
L) contained 50 mM KPB (pH 6.5), 20 mM ascorbic acid (neutralized
l
g/mL catalase, 10 M methylene blue, 200
l
lM L-
l
l
l
l
In conclusion, we have identified the benzylisothiourea ana-
logues 3 as novel noncompetitive IDO inhibitors by our screen-
ing campaign, derivatization and kinetic analysis. The 4-Cl/Br
atom on the phenyl ring and the distance from the isothiourea
moiety were important for potent IDO inhibition. Replacements
of the isothiourea moiety afforded potent benzylthiol analogues
The yellow pigment derived from kynurenine was measured at 490 nm using a
SPECTRAmax M5SK microplate reader (Molecular Devices).
23. Matter, H.; Nazaré, M.; Güssregen, S.; Will, D. W.; Schreuder, H.; Bauer, A.;
Urmann, M.; Ritter, K.; Wagner, M.; Wehner, V. Angew. Chem., Int. Ed. 2009, 48,
2911.
24. Hopkins, A. L.; Groom, C. R.; Hopkins, A. A. Drug Discovery Today 2004, 9, 430.
Matsuno, Kenji
