Structure-activity relationship and enzyme kinetic studies on 4-aryl-1H-1,2,3-triazoles as indoleamine 2,3-dioxygenase (IDO) inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.08.044

Previously, we have reported the design and synthesis of 4-aryl-1H-1,2,3-triazoles as inhibitors of indoleamine 2,3-dioxygenase (IDO), a promising therapeutic target of cancer. Here, we present the structure-activity relationship and enzyme kinetic studie

Full text of DOI:10.1016/j.ejmech.2011.08.044

European Journal of Medicinal Chemistry 46 (2011) 5680e5687
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Short communication
Structureeactivity relationship and enzyme kinetic studies on 4-aryl-1H-1,2,
3-triazoles as indoleamine 2,3-dioxygenase (IDO) inhibitors
,
1
,
1
,
*
Qiang Huanga , Maofa Zhenga , Shuangshuang Yanga, Chunxiang Kuangb, Cunjing Yua, Qing Yanga
a State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Handan Road 220, Shanghai 200433, China
b Department of Chemistry, Tongji University, Siping Road 1239, Shanghai 200092, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Previously, we have reported the design and synthesis of 4-aryl-1H-1,2,3-triazoles as inhibitors of
indoleamine 2,3-dioxygenase (IDO), a promising therapeutic target of cancer. Here, we present the
structureeactivity relationship and enzyme kinetic studies on a series of 4-aryl-1H-1,2,3-triazoles. Three
compounds (1, 6, 8) were found to possess more IDO inhibitory potency than the most commonly used
1-methyltryptophan. The results from the structureeactivity relationship and molecular docking studies
indicated that an electron-withdrawing group with low steric hindrance near the NH group of triazoles
was necessary for the IDO inhibition.
Received 20 June 2011
Received in revised form
29 August 2011
Accepted 31 August 2011
Available online 7 September 2011
Keywords:
Ó 2011 Elsevier Masson SAS. All rights reserved.
Indoleamine 2,3-dioxygenase
4-Aryl-1H-1,2,3-triazoles
Enzyme kinetics
Structureeactivity relationship
1. Introduction
molecular target of new therapeutic agents for treating neurolog-
ical disorders, cancer as well as other diseases characterized by
Indoleamine 2,3-dioxygenasee (IDO; EC 1.13.11.42) is a mono-
meric 45 kDa heme-containing dioxygenase that catalyzes the
initial and rate limiting step of L-tryptophan (L-Trp) catabolism
pathological tryptophan metabolism. Several studies have provided
proof-of-principle demonstration for the potential value of IDO
inhibitors in cancer treatment [13,14].
along the kynurenine pathway (KP) leading to the production of
N-formylkynurenine [1]. N-formylkynurenine is readily hydrolyzed
to kynurenine and subsequently converted to a range of metabo-
lites including excitotoxin quinolinic acid (QUIN) [2]. Dysregulation
of the KP, mainly associated with the elevation of IDO activity and
QUIN production, has been implicated in the pathogenesis of
neuroinflammatory, neurodegenerative disorders (Alzheimer’s
disease), depression, age-related cataract and HIV encephalitis
[3e7]. In addition, IDO is associated with immunosuppression and
immunotolerance [8]. IDO expressing cancer cells use KP trans-
formation to reduce tryptophan concentrations in their microen-
vironments to the levels that prevent T-lymphocyte proliferation
[9]. Some catabolites in the KP are toxic to T-cells and further
inhibit their action [10]. A growing body of clinical data has shown
that many primary tumor cell lines obtained from patients over-
express IDO, and this strongly correlates with a poor prognosis
for survival [11,12]. Thus, IDO has emerged as a promising
Although the potential of the IDO inhibitors as pharmaceutical
agents has aroused great interest in scientific communities, the
development of IDO inhibitors during a long period of time since
1970’s has been mainly focused on synthesis of Trp analogs, but not
the discovery of molecules bearing novel structural skeleton. Even
a few years ago, the best known IDO inhibitors were mainly
comprised of competitive Trp derivatives and noncompetitive
b-carbolines, which displayed affinities in the micromolar range
[15,16]. For example, 1-methyl-tryptophan (1-MT) is the most
frequently used competitive inhibitor, and has a reported Ki of
34 mM [17]. Since 2006, some potent nanomolar IDO inhibitors with
novel structural skeleton have been discovered by natural product
isolation and the optimization of the core pharmacophores in the
structures [18e20]. Especially, the X-ray crystal structures of
human IDO in complex with the ligand inhibitors
4-phenylimidazole (PI) and cyanide have opened the doors for the
design of new IDO inhibitors [21]. The efforts of PI analog design
and synthesis, which were guided by computational docking, have
led to the discovery of IDO inhibitors that possess low micromolar
activities and are approximately 10-fold more potent than PI [22].
In silico design using an evolutionary docking algorithm yielded
several new inhibitor scaffolds, which showed the nanomolar level
* Corresponding author. Tel.: þ86 21 65643446.
E-mail address: yangqing68@fudan.edu.cn (Q. Yang).
1
Contributed equally.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.044

Q. Huang et al. / European Journal of Medicinal Chemistry 46 (2011) 5680e5687
5681
M)
Table 1
Table 1 (continued )
IDO inhibitory activity of triazoles.
Compound
Structure
IC50 (m
Cl
I
Compound
Structure
IC50 (mM)
N
N
13
14
15
N.I.
N
N
N
H
1
143
N
H
N
N
N
1371
1028
N
H
N
2
N.I.
N
H
Br
N
N
N
N
N
3
6000
1028
1256
148
817
86
N
H
H
Me
MeO
O N
2
N
N
N
N
16
533
N
4
N
H
H
O N
2
OMe
N.I.: no inhibition.
N
N
5
N
H
activities [23]. However, few structural classes of small molecules
are known to be IDO inhibitors, and the most of them either
showed low potency or was failed in vivo assay, and therefore are
marginal drug candidates. Hence, it is still of great interest to find
new potent IDO inhibitors.
Br
N
N
6
N
H
For several years, we have concentrated on the development of
novel potent IDO inhibitors under the guidance of in vitro enzy-
matic assays. Our studies included the identification of novel
candidates, which were isolated from natural medicines with
therapeutic potential in Alzheimer’s disease or cancer, the design
and synthesis of novel compounds. Several constituents comprising
berberine, baicalein, jatrorrhizine and palmatine have been iden-
tified as IDO inhibitors from the traditional Chinese medicinal
prescription of Oren-gedoku-to [24]. Using the valuable structural
information revealed by the co-crystal structure of IDO with PI (PDB
code: 2D0T), we discovered that 1H-1,2,3-triazole might be a new
key pharmacophore of potent IDO inhibitors, because 1H-1,2,3-
triazole shares structural similarity with PI except that the imid-
azole ring is replaced by a triazole ring, which have widespread
applications in pharmaceuticals and agrochemicals. For such
a reason, we sought to synthesize different series of 1,2,3-triazoles
[25] for screening high efficacy IDO inhibitors [26]. Here, we
present the recent studies on the IDO inhibitory activity and the
structureeactivity relationship (SAR) of the 4-aryl-1H-1,2,3-
triazole derivatives.
Br
N
N
7
N
H
Cl
N
N
8
N
H
Cl
N
N
9
580
N.I.
N
H
F
N
N
10
N
H
MeO2C
2. Results and discussion
N
N
2.1. IDO inhibitory activity
11
12
708
N
H
Sixteen synthesized 4-aryl-1H-1,2,3-triazoles were subjected to
N
IDO inhibition assay and their IC50 values were shown in Table 1.
Under the conditions, the IC50 value of 1-MT was 328
mM, which
was consistent with previous value in Ref. [17] (IC50 ¼ 380
m
M). As
Br
N
expected, some compounds displayed IDO inhibitory activities,
especially, compounds 1, 6 and 8 exhibited stronger IDO inhibitory
activity than 1-MT. The IC50 values of these 4-aryl-1H-1,2,3-
N
N.I.
N
H
triazoles (1, 6, 8) were 143
mM, 148 mM and 86 mM, respectively.
Compounds 12e14 with a substituent on the triazole showed none

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Q. Huang et al. / European Journal of Medicinal Chemistry 46 (2011) 5680e5687
Table 2
shown in Fig. 1B, all three showed good graphical match to the
mode of an uncompetitive inhibition. Their inhibitory kinetics were
evaluated by plotting [S]/[V] against inhibitor concentration, where
[S] represents the substrate concentration and [V] represents the
IDO inhibitory activity of nitrogen-containing heterocyclic compounds.
Compound
Structure
IC50 (mM)
N
N
N
reaction velocity. The Ki values of 1, 6 and 8 were 22.5
and 14.5 M, respectively (Table 3). Under the same conditions, the
Ki value of 1-MT was 34 M.
mM, 18 mM
CO Et
m
2
17
2000
m
Cl
2.3. Cellular IDO inhibitory activity
N
To study the cellular IDO inhibition, the HEK 293 cells
expressing human IDO were created and used for the assay with
compounds 1, 6 and 8. The kynurenine levels released from the
assays were measured spectrophotometrically (Fig. 2). Compounds
6 and 8 displayed 3e4 times higher cellular inhibition activities
N
N
18
19
2570
N.I.
N
H
N
N
OH
(IC50: 31.6
activities (IC50: 148
exhibited a less cellular inhibition (IC50: 251.2
IDO inhibition (IC50: 143 M, Table 1). This might be resulted from
m
M and 19.3
m
M) than their in vitro IDO inhibition
M, Table 1). However, compound 1
M) than its in vitro
mM and 86
m
m
N
N
m
N
20
N.I.
N
the complexity of enzyme assays, which utilized a methylene blue
ascorbate regeneration system to maintain IDO in an active reduced
form, or from unidentified differences between the recombinant
IDO used in the enzyme assay and HEK 293 cells. Nevertheless, we
observed a good correlation between the two assays and confirmed
that 4-aryl-1H-1,2,3-triazoles containing an electron-withdrawing
group at 2-position of the phenyl were good IDO inhibitors.
H
Br
N.I.: no inhibition.
or poor activity; while compounds 15 and 16 with an additional
substituent on the 4-aryl ring exhibited improved inhibitory
potency. Without considering any substituents on triazole cycle,
the electronegative and the position of the substituent on the 4-aryl
appeared to be very important for the IDO inhibitory activity of
these 4-aryl-1H-1,2,3-triazoles (compounds 1e11). It showed that
an electron-withdrawing group with low steric hindrance near the
free NH of triazoles was essential for IDO inhibition. For further
comparison, the IDO inhibition assays were carried out with several
nitrogen-containing heterocyclic compounds (Table 2). The data of
compounds 18 and 1 revealed that 4-aryl triazole preferred over
other aryl triazole to have inhibitory potency. Compound 20 that
resembles compound 5 except the nitrogen heterocycle had no IDO
inhibitory activity. Compounds 17 and 19 containing a triazole
group but lacking the free NH group also showed poor or none
activity.
As we were concentrating on our project to develop 4-aryl tri-
azoles as potent IDO inhibitors, an independent work from Michi-
elin’s group also revealed that triazole 1 bore IDO inhibitory activity
[23]. Based on the docked conformations of known IDO inhibitors,
they developed a pharmacophore model for designing new IDO
inhibitors. They discovered three families with IDO inhibitory
activity: benzothiazoles, phenylthiazoles, and triazoles. 4-phenyl-
1H-1,2,3-triazole (1) was one of the three triazoles being examined.
However, they evaluated the compounds only by IC50 at the enzy-
matic and cellular activity levels, not by the kinetic studies on
inhibition type and kinetic parameters. Under their conditions, the
2.4. T cell proliferation assay
As an immune tolerance-promoting enzyme, IDO contributes to
the progression of tumors through its capacity to inhibit
T-lymphocyte proliferation. It has been reported that 1-MT can
augment T cell function stimulated by LLC cells [27]. Here,
a proliferation assay was carried out to determine if compounds 1, 6
and 8 could improve T cell proliferation in the presence of LLC cells.
Each compound was applied at three different concentrations
(62.5 mM, 125 mM and 250 mM). As shown in Fig. 3, the three
compounds and1-MT enhanced the proliferation of T cells stimu-
lated with LLC tumor cells in a dose-dependent mode. In accor-
dance with their IDO inhibitory activities, these three compounds
also showed more enhancement of T cell proliferation than that of
1-MT at all three concentrations. Compound 8 increased T cell
proliferation 2e3 folds more than that of 1-MT at the same
concentrations.
Compared to other antitumor agents such as cytotoxic and
antiangiogenic agents, IDO inhibitors have an appealing feature,
which can modulate immunity. So it is important to evaluate the
ability of potent IDO inhibitors for increasing T cell function.
However, such study was seldom described in the references of
developing IDO inhibitors. The present study clearly demonstrates
that the novel IDO inhibitors 4-aryl-1H-1,2,3-triazoles (1, 6 and 8)
can augment T cell proliferation.
IC50 of compound 1 was 60
cellular levels, respectively, which were much lower than those
(143 M and 251 M) obtained from our assays. Under the present
conditions, the IC50 of 1-MT was determined to be 328 M, which
was consistent with the reference value of 380 M [17]. However,
mM and 70 mM at the enzymatic and
2.5. Molecular docking
m
m
m
To elucidate the binding modes of compounds 1, 6 and 8 to the
human IDO, we performed molecular docking for these compounds
using the program AutoDock 4.2 [28,29] and the three-dimensional
(3D) structure of IDO in complex with the ligand inhibitor of
4-phenylimidazole (PI) (PDB code: 2D0T) [21] (Fig. 4A). The ob-
tained lowest-energy poses of three compounds are illustrated in
Fig. 4BeE.
As described in Experimental section, for each compound 200
independent docking runs and the clustering analysis of the lowest-
energy poses from the runs were performed. For compound 6 or 8
m
the IC50 of 1-MT was not determined in Michielin’s report [23].
2.2. Enzyme kinetics
More detailed kinetic analysis was performed on the most
potent 4-aryl-1H-1,2,3-triazoles, 1, 6 and 8. Based on plotting
a function of rate ([V]) against enzyme amount ([E]), the three
compounds were categorized as reversible inhibitors (Fig. 1A). As

Q. Huang et al. / European Journal of Medicinal Chemistry 46 (2011) 5680e5687
5683
Fig. 1. Determination of the inhibition type and kinetic parameters of compounds 1, 6 and 8. (A) Plot of reaction rate [V] against enzyme amount [E]. The concentrations of L-
tryptophan and the inhibitor were 300 M and 200 M, respectively. (B) [S]/[V] against [I] was plotted. Concentration of L-tryptophan varied from 100 M to 400 M. The inhibitor
concentration varied over a three-fold range above and below the concentration yielding approximately 50% inhibitory activity.
m
m
m
m
all 200 lowest-energy poses were almost the same (Fig. 4C or D). For
compound 1 the lowest-energy poses from 199 runs were similar to
those of compounds 6 and 8 (Fig. 4A) except for one pose, which
was also the global lowest-energy of that docking run. Thus, these
three compounds (1, 6, 8) could be considered to have the similar
global lowest-energy binding modes (Fig. 4BeD). Very likely, these
predicted binding modes are the optimal modes of the compounds
to the IDO active site. As illustrated in Fig. 4BeD, the binding modes
revealed that the NH group of the compounds could form
hydrogen-bonds with the two O atoms of the 7-propionate of the
heme, which points upward from the heme plane, and the 3-N atom
of the triazole ring, which points toward the heme iron and appears
to coordinate with the heme iron. At the same time, the phenyl
group of the compounds is located in the hydrophobic pocket above
the heme plane and very close to two IDO aromatic residues of
Phe163 and Phe164. Taken together, these modes and analysis
indicate that hydrophobic interactions, to some extent, have
contributed to the binding affinities of the compounds to the IDO
active site.
Table 3
For compound 8 in Fig. 4D, the distances from the H atom of the
Kinetic parameters of the three most potent IDO inhibitors (1, 6, and 8).
ꢀ
NH group to two O atoms of the 7-propionate are 1.91 and 3.11 A,
Compound
Structure
Inhibition type
Ki (mM)
respectively, and that from the 3-N atom of the triazole ring to the
ꢀ
N
heme iron is 2.88 A. Corresponding distances of compound 6 in
ꢀ
N
Fig. 4C are slightly greater, namely, 1.96, 3.14 and 2.89 A, respec-
1
Uncompetitive
22.5
N
tively. Since the hydrogen bonding and N-iron coordination are
strongly attractive interactions, the slightly shorter distances of
compound 8 indicate that the interactions between compound 8
and the heme are also slightly stronger than those of compound 6.
This might explain why compound 8 was found to be slightly
stronger in the binding affinity to the IDO active site. It becomes
more obvious that the corresponding distances of compound 1,
lacking any substituents on 4-aryl of the triazole, are also greater
H
N
N
6
8
Uncompetitive
Uncompetitive
18
N
H
Br
Cl
N
N
ꢀ
(2.01, 3.13, and 3.14 A, respectively). Thus, the halogen atoms on
14.5
N
H
4-aryl may affect the positions of the compounds bound to the IDO
active site, especially the relative position to the heme, and increase
their binding affinities.

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Q. Huang et al. / European Journal of Medicinal Chemistry 46 (2011) 5680e5687
Fig. 2. Cell-based assay of IDO inhibition of compounds 1, 6 and 8 (see A, B and C, respectively). HEK 293 cells transiently transfected with human IDO were exposed to a range of
test compound concentrations. Percent inhibition against log [I] was plotted and IC50 value was determined. The data were evaluated by OriginPro 8.0.
As mentioned, one of the 200 docking runs for compound 1
resulted in a lowest-energy pose similar to that of PI (Fig. 4E).
However, in this pose the hydrogen bonding between the
compound and the 7-propionate of the heme was disrupted. Thus,
its binding energy was slightly higher than that in Fig. 4B. Inter-
estingly, no such kind of lowest-energy poses was observed for
compounds 6 and 8, in which there is a halogen substituent on
4-aryl of the triazole. This might imply that the volume of the
substituent on 4-aryl is crucial for the binding. A substituent group
with a large volume may result in certain steric hindrance with the
nearby helix, and then reduce the binding affinity of the compound.
This is consistent with the previous observation that an electron-
withdrawing group with low steric hindrance near the NH group
of triazole was essential for IDO inhibition. In contrast, for the poses
shown in Fig. 4C and D, the substituent on 4-aryl is just located in
the center of the hydrophobic pocket, and thus the steric hindrance
could be avoided. However, due to the limit space of the hydro-
phobic pocket, one may image that the substituent group on 4-aryl
is not allowed to be very large. Therefore, the present study and
discussion may provide useful clues for future design and devel-
opment of more potent IDO inhibitors.
3. Conclusion
In this study, we have investigated the structureeactivity rela-
tionship and enzyme kinetics for a series of 4-aryl-1H-1,2,3-
triazoles, and discovered three potent IDO inhibitors (compounds
1, 6 and 8) that showed stronger IDO inhibitory activity than that of
the most commonly used inhibitor 1-MT. Further cellular assay and
T cell proliferation tests also confirmed the potential biological
relevance of these three IDO inhibitors. The inhibitor structures
suggested that an electron-withdrawing group with low steric
hindrance near the NH group of the triazoles was necessary for the
IDO inhibition. The binding modes of these inhibitors to the IDO
active site were studied by molecular docking. Our results showed
that the O and N atoms of the triazole ring could interact with the
heme by hydrogen-bonds and N-iron coordination; and these
interactions led the compounds to a specific binding orientation in
which the substituent on 4-aryl is located in the center of the
hydrophobic pocket above the heme plane. Due to the limited space
of the pocket, the substituent group on 4-aryl should not be too
large. Therefore, our finding would provide very useful clues for the
design and development of more potent human IDO inhibitors.
Fig. 3. Compounds 1, 6 and 8 enhanced the proliferation of T cells stimulated with LLC
tumor cells. Each bar on the graph indicates the mean of 3 replicate wells with the
standard error of the means shown. The experiment shown is representative of 2
experiments carried out separately.

Q. Huang et al. / European Journal of Medicinal Chemistry 46 (2011) 5680e5687
5685
Fig. 4. Predicted binding modes of compounds 1, 6 and 8 to the human IDO active site. (A) The crystal structure of PI-IDO complex (PDB code: 2D0T) used as in the molecular
docking. (B) The predicted binding mode for compound 1. (C) The predicted binding mode for compound 6. (D) The predicted binding mode for compound 8. (E) Two binding modes
for compound 1: the global lowest-energy pose (yellow) and the pose (orange) which is similar to that of PI (green) bound to the IDO active site. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
4. Experimental section
from the commercial providers and used without further purifi-
cation. All chemicals were dissolved in DMSO to make a concen-
tration of 10 mM, if not mentioned specially.
4.1. Materials
Human embryonic kidney 293 (HEK 293) cells and LLC (Lewis
Lung Carcinoma) cells were purchased from the American Type
Culture Collection. Dulbecco’s Modified Eagle’s Medium (DMEM),
fetal bovine serum (FBS) and the transfection reagent Lipofectamin
2000 were purchased from Invitrogen (Carlsbad, CA). The plasmids
pET28a-hIDO and pcDNA3.1-hIDO were generous gifts from Dr. A.
Grant Mauk from the Department of Biochemistry and Molecular
Biology, University of British Columbia, and Dr. O. Takikawa from
the National Institute for Longevity Sciences, National Center for
Geriatrics and Gerontology, Japan, respectively.
Details of synthetic procedures used to prepare the test
compounds as well as structural and analytical characterizations
can be found in corresponding literatures. 4-Aryl-1H-1,2,3-triazoles
1e11 were synthesized by CuI-catalyzed synthesis from anti-3-
aryl-2,3-dibromopropanoic acid and sodium azide (Scheme 1) [25].
Compounds 12e15 were synthesized according to Ref. [30] as
shown in Scheme 2. Compounds 16, 17 and 20 were synthesized
according to the corresponding literatures [26,31,32] as described
in Schemes 3e5, respectively. Compounds 18 and 19 were obtained
4.2. Expression of recombinant human IDO
N
Br
CuI, Na ascorbate, Cs2CO3
DMSO, N2, 110oC
N
CO2H
NaN3
MeO
+
N
Ar
Ar
Escherichia coli cells were routinely grown at 37 ꢀC in Luria-
H
Br
Bertani (LB) medium containing 50
mg/mL kanamycin. A single
Ar:
X
N
OMe
Br
Br
Me
N
N
N
2
3
4
5
6
1
NXS
N
H
N
H
DMF, rt
R
MeO2C
Cl
Cl
F
N
R = H, X = Br 12
R = H, X = Cl 13
R = H, X = I 14
R = Me, X = Br 15
11
10
8
9
7
Scheme 1. CuI-catalyzed synthesis of 4-aryl-1H-1,2,3-triazoles 1e11 from anti-3-aryl-
2,3-dibromopropanoic acids and sodium azide. Reagents and conditions were as
follows: substrate anti-3-aryl-2,3-dibromopropanic acid (0.5 mmol), NaN3
(0.65 mmol), Cs2CO3 (1.25 mmol), CuI (0.05 mmol), Na ascorbate (0.1 mmol), DMSO
(3 mL), 110 ꢀC, 4 h, N2 atmosphere.
Scheme 2. Synthesis of 4-aryl-1H-1,2,3-triazoles 12e15. Reagents and conditions were
as follows: substrate 4-phenyl-1H-1,2,3-triazoles (1.0 mmol), N-halosuccinimide
(1.0 mmol), DMF (2 mL), rt, 2.5e16 h.

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N
O2N
N
N
N
CN
NH4Cl, DMF
100 oC, 20 h
N
N
N
H2SO4, HNO3
DMF, 0 oC
H
N
N
H
+
NaN3
Br
N
Br
H
O2N
20
16
Scheme 5. Synthesis of 4-aryl-1H-1,2,3-triazole 20. Reagents and conditions were as
follows: NaN3, NH4Cl/DMF, 100 ꢀC, 20 h.
Scheme 3. Synthesis of 4-aryl-1H-1,2,3-triazole 16. Reagents and conditions were as
follows: substrate 4-phenyl-1H-1,2,3-triazoles (0.6 mmol), 1 g H2SO4 (10.2 mmol),
189 mg HNO3 (3 mmol), DMF (2 mL), stirred at 0 ꢀC for 1 h.
4.5. Determination of the inhibition type
colony of BL21 containing the plasmid pET28a-hIDO was inoculated
in 10 mL LB medium and cultured overnight. The 10 mL culture was
added to 500 mL of the same medium and incubated at 37 ꢀC to
a density of 0.6 OD at 600 nm. The culture temperature was then
reduced to 30 ꢀC, and alpha-aminolevulinic acid was added to
a final concentration of 0.5 mM. The culture was shaken at 120 rpm
for 30 min and cooled down before IPTG was added to a final
concentration of 0.5 mM. The culture was incubated at 30 ꢀC for 6 h.
The cell pellets were collected by centrifugation at 5000 rpm for
20 min at 4 ꢀC. The pellets were stored at ꢁ20 ꢀC.
The inhibition type was determined using the method previ-
ously described by Voet and Voet [34]. First, the IDO activity assay
was performed with 300 mM L-tryptophan, a 100 mM inhibitor
solution in DMSO and a series amount of rhIDO according to the
procedure described above. Based on the assay results, a function of
rate ([V]) against enzyme amount ([E]) was plotted. The inhibition
type was determined according to the plot of the enzyme kinetics.
4.6. Evaluation of kinetic parameters and IC50
Ki and IC50 values were determined based on the IDO activity
4.3. Purification of recombinant human IDO
assay in which the concentration of
L-tryptophan varied from
100 M to 400 M, and the inhibitor concentration varied over
m
m
The pellet from 500 mL of bacterial culture, obtained as described
above, was suspended in 10 mL of PBS containing 1 mM PMSF,
sonicated on ice using a Branson Sonifier 250 for 15 min at the
maximum power and centrifuged at 12000 rpm for 10 min. The clear
supernatant (10 mL) was then applied to a 1 mL Hi-Trap chelating
column charged with nickel ions that had been equilibrated with
10 mM imidazole in phosphate buffer (20 mM, pH 7.4) containing
0.5 M NaCl. After washing with 20 mL of basal buffer (Tris 25 mM,
pH 7.4, 40 mM imidazole), rhIDO was eluted with the imidazole
solution (250 mM) that was prepared in the same phosphate/NaCl
buffer. The protein collected at the elution step was then buffer-
exchanged into 50 mM Tris, pH 7.4, using a Sephadex G25 column.
a three-fold range above and below the concentration yielding
approximately 50% inhibitory activity. Otherwise, the reaction
conditions were exactly the same as those described above. Data
were analyzed according to the method previously described by
Cornish-Bowden [35].
4.7. Cellular IDO inhibition assay
HEK 293 cells were maintained in DMEM with 4500 mg/L
glucose supplemented with 50 U/mL penicillin, 50 mg/mL strep-
tomycin and 10% FBS. The cells were cultured at 37 ꢀC with 5% CO2
and 95% humidity. The assay was performed as follows: HEK 293
cells were seeded in a 96-well culture plate at a density of
2.5 ꢂ 104 cells/well and cultured overnight. After 24 h, HEK 293
cells were transfected with pcDNA3.1-hIDO using Lipofectamin
2000 according to the manufacturer’s instructions; 24 h after
4.4. IDO inhibition assay
The effects of testcompounds on IDO activity weredetermined as
previously described by Littlejohn et al. with minor modifications
[33]. In brief, a standard reaction mixture (0.5 mL) containing
100 mM potassium phosphate buffer (pH 6.5), 40 mM ascorbic acid
transfection, a serial dilution of the tested compounds in 200
culture medium was added to the cells. After an additional 5-h
incubation, 140 L of the supernatant per well was transferred to
a new 96-well plate and mixed with 10 L of 30% trichloroacetic
mL
m
(neutralized with NaOH), 200
blue and 0.05 M rhIDO was added to the solution containing the
substrate -tryptophan and the test sample at a determined
concentration. The reaction was carried out at 37 ꢀC for 30 min and
stopped by adding 200 L of 30% (w/v) trichloroacetic acid. After
heating at 65 ꢀC for 15 min, the reaction mixture was centrifuged at
12000 rpm for 10 min. The supernatant (100 L) was transferred
into awell of a 96-well microplate and mixed with 100 L of 2% (w/v)
mg/mL catalase, 20 mM methylene
m
acid in each well. The plate was incubated at 65 ꢀC for 15 min to
hydrolyze N-formylkynurenine produced by the catalytic reaction
of IDO. The reaction mixture was then centrifuged for 10 min at
m
L
m
12000 rpm to remove the sediments. Then, 100
natant per well were transferred to another new 96-well plate and
mixed with 100 L of 2% (w/v) p-dimethylaminobenzaldehyde in
mL of the super-
m
m
m
acetic acid. The yellow color derived from kynurenine was
measured at 492 nm using a SPECTRAmax 250 microplate reader.
p-dimethylaminobenzaldehyde in acetic acid. The yellow pigment
derived from kynurenine was measured at 492 nm using a SPEC-
TRAmax 250 microplate reader (Molecular Devices, Sunnyvale, CA).
The data were analyzed using the Enzyme Kinetics module in Sig-
maPlot, version 10.
4.8. T cell proliferation assay
T cell proliferation assay was performed as previously described
[25] with some modification. Briefly, T lymphocytes prepared from
splenocytes of BALB/c mice were resuspended in RPMI1640 contain-
N
N
N
CO2Et
N3
ing 10% FBS, L-glutamate, penicillin and streptomycin. To the LLC cell
CuI / Na ascorbate
MeCN-H2O
CO2Et
+
culture mitomycin C was added at a final concentration of 25 mg/L.
The culture was incubated at 37 ꢀC for 30 min, and the cells were
washed three times with RPMI1640 and resuspended in RPMI1640
Cl
Cl
17
containing 10% FBS,
L
-glutamate, penicillinand streptomycin.1 ꢂ105 T
Scheme 4. Synthesis of 4-aryl-1H-1,2,3-triazole 17. Reagents and conditions were as
follows: CuI/Na ascorbate, MeCNeH2O, 85 ꢀC, 24 h.
lymphocytes (responder cells) and 2 ꢂ 104 mitomycin C treated LLC

Q. Huang et al. / European Journal of Medicinal Chemistry 46 (2011) 5680e5687
5687
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RPMI1640 containing 10% FBS. Cell proliferation was quantified by
WST-1 (KenGEN, Nanjing, China) method, which is based on the
cleavage of the tetrazolium salt WST-1 by mitochondrial dehydroge-
nases in viable cells to a water-soluble formazen dye. The cells were
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The AutoDock program (Version 4.2) with the Lamarckian
genetic algorithm [28,29] was used for the molecular docking to
predict the binding modes of compounds 1, 6 and 8 to the human
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were conducted for each compound. To perform the Lamarckian
genetic algorithm, a population of 150 random ligand conformations
in random orientations and at random translations was first
generated, and then the population evolved according to the algo-
rithm and terminated after 27,000 generations and a maximum of
1,500,000 energy evaluations. Other parameters for running the
program were set to the default values in the AutoDock program.
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Acknowledgments
[22] S. Kumar, D. Jaller, B. Patel, J.M. LaLonde, J.B. DuHadaway, W.P. Malachowski,
G.C. Prendergast, A.J. Muller, Structure based development of
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This work was sponsored by the National Natural Science
Foundation of China (No. 30873153), the National Drug Innovative
Program (No. 2009ZX09301-011) and the Shanghai Pujiang
Program. We thank Dr. Y. Hefner (1278 Avenida Miguel, Encinitas,
CA 92024, U.S.A.) for critical reading and editing this manuscript.
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triazoles from anti-3-aryl-2,3-dibromopropanoic acids and sodium azide,
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