Rational design of 4-aryl-1,2,3-triazoles for indoleamine 2,3-dioxygenase 1 inhibition

Article abstract of DOI:10.1021/jm300260v

Indoleamine 2,3-dioxygenase 1 (IDO1) is an important therapeutic target for the treatment of diseases such as cancer that involve pathological immune escape. Starting from the scaffold of our previously discovered IDO1 inhibitor 4-phenyl-1,2,3-triazole, we used computational structure-based methods to design more potent ligands. This approach yielded highly efficient low molecular weight inhibitors, the most active being of nanomolar potency both in an enzymatic and in a cellular assay, while showing no cellular toxicity and a high selectivity for IDO1 over tryptophan 2,3-dioxygenase (TDO). A quantitative structure-activity relationship based on the electrostatic ligand-protein interactions in the docked binding modes and on the quantum chemically derived charges of the triazole ring demonstrated a good explanatory power for the observed activities.

Full text of DOI:10.1021/jm300260v

Article
pubs.acs.org/jmc
Rational Design of 4-Aryl-1,2,3-Triazoles for Indoleamine 2,3-
Dioxygenase 1 Inhibition
Ute F. Rohrig,†,‡,# Somi Reddy Majjigapu,†,§,# Aurelien Grosdidier,‡ Sylvian Bron,‡,∥ Vincent Stroobant,⊥
́
̈
Luc Pilotte,⊥ Didier Colau,⊥ Pierre Vogel,§,† Benoît J. Van den Eynde,⊥ Vincent Zoete,*,‡
and Olivier Michielin∥,*,†,‡
†Ludwig Center for Cancer Research of the University of Lausanne, CH-1015 Lausanne, Switzerland
‡Swiss Institute of Bioinformatics, Molecular Modeling Group, CH-1015 Lausanne, Switzerland
§Laboratory of Glycochemistry and Asymmetric Synthesis, Ecole Polytechnique Fed
Switzerland
́ ́
erale de Lausanne (EPFL), CH-1015 Lausanne,
∥Pluridisciplinary Centre for Clinical Oncology (CePO), Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland
⊥Ludwig Institute for Cancer Research, Brussels Branch, and de Duve Institute, Universite
Belgium
́
Catholique de Louvain, B-1200 Brussels,
S
* Supporting Information
ABSTRACT: Indoleamine 2,3-dioxygenase 1 (IDO1) is an important therapeutic target
for the treatment of diseases such as cancer that involve pathological immune escape.
Starting from the scaffold of our previously discovered IDO1 inhibitor 4-phenyl-1,2,3-
triazole, we used computational structure-based methods to design more potent ligands.
This approach yielded highly efficient low molecular weight inhibitors, the most active
being of nanomolar potency both in an enzymatic and in a cellular assay, while showing no
cellular toxicity and a high selectivity for IDO1 over tryptophan 2,3-dioxygenase (TDO). A
quantitative structure−activity relationship based on the electrostatic ligand−protein
interactions in the docked binding modes and on the quantum chemically derived charges
of the triazole ring demonstrated a good explanatory power for the observed activities.
low Trp degradation activity, (ii) the presence of poly-
morphisms abolishing its enzymatic activity in about 50% of
Caucasians, and (iii) the presence of multiple splice
variants.21−23
INTRODUCTION
■
Many tumors develop the capacity to actively suppress a
potentially effective immune response.1 The enzyme indole-
amine 2,3-dioxygenase 1 (IDO1, EC 1.13.11.52) is one of the
key players in this pathological immune escape and has
therefore been selected as a therapeutic target for pharmaco-
logical interventions.2−4 IDO1 catalyzes the initial and rate-
limiting step in the catabolism of tryptophan (Trp) along the
kynurenine pathway.5,6 By depleting Trp and accumulating Trp
catabolites, IDO1 exerts a local immunosuppressive effect on T-
lymphocytes.7−10 The observations that many human tumors
constitutively express IDO111 and that increased IDO1
expression in tumor cells is correlated with poor prognosis
for survival in several cancer types12 led to the hypothesis that
its inhibition might enhance the efficacy of cancer treatments.
Indeed, results from in vitro and in vivo studies have suggested
that the efficacy of therapeutic vaccination or chemotherapy
may be improved by concomitant administration of an IDO1
inhibitor.11,13−16 Very recently, it has been shown that the
functionally related enzyme tryptophan 2,3-dioxygenase
(TDO) may be a complementary anticancer target.17,18
IDO1 is an extrahepatic heme-containing enzyme that
displays less substrate specificity than TDO.6 In the first step
of the catalytic cycle, IDO1 binds both the substrate and
molecular oxygen in the distal heme site. The enzyme catalyzes
the cleavage of the pyrrole ring of the substrate and
incorporates both oxygen atoms before releasing N-formyl
kynurenine, which is subsequently hydrolyzed to kynurenine by
a cytosolic formamidase.24 The two available crystal structures
of IDO1 include the heme-bound ligands cyanide and 4-
phenylimidazole (PIM), respectively.25 Mutant analyses
showed that none of the polar amino acid residues in the
distal heme site are essential for the activity of the enzyme,
suggesting a reaction mechanism involving only the substrate
and the dioxygen molecule.25−27 In the active form of IDO1,
the heme iron is in its ferrous state (Fe2+), while in its inactive
form, the heme iron is in the ferric (Fe3+) state. Formally, the
catalytic cycle of IDO1 does not alter the oxidation state of the
The IDO1-like protein Indoleamine 2,3-dioxygenase 2
(IDO2)19,20 shares 44% of sequence homology with IDO1.
However, its physiological role remains unclear due to (i) very
Received: February 24, 2012
Published: May 22, 2012
© 2012 American Chemical Society
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Figure 1. X-ray structure of IDO1: binding site with PIM ligand (green). (A) Two hydrophobic pockets and residues in hydrogen-bonding distance
are labeled. (B) Side view, highlighting the residues that form pocket B.
a
Scheme 1
a
Reagents, conditions, and yields: (a) TMSA, PdCl2(PPh3)2, Et3N, CuI, dioxane, 45 °C, 5 h, up to 95%; (b) KF, MeOH, rt, 3 h, up to 85%; (c)
TMSN3, CuI, DMF/MeOH (9:1), 100 °C, 10−12 h, 30−83%,
heme, but as IDO1 is prone to autoxidation, a reductant is
necessary in the enzymatic assay to maintain enzyme activity.
Cytochrome b5 has been suggested to be responsible for IDO1
reduction in vivo.28−30
(methylene blue and ascorbate) rather than through IDO1-
specific interactions.30 Other classes of inhibitors derived from
a target-based screen of the NCI diversity set,45 the 4-
phenylimidazole scaffold,46 the indole scaffold,47,48 the S-
benzylisothiourea scaffold,49 a high throughput screen,23 and
a virtual screen50 have been described. The most potent and
promising compounds known today are the competitive
hydroxyamidine inibitors developed by Incyte,15,51,52 with
enzymatic IC50 values of about 60 nM. Compound
INCB24360, whose structure has not been disclosed yet, is
currently undergoing a phase I clinical trial.
We have previously described a number of new IDO1
inhibitors based on a structure-based in silico design strategy.53
Among the new scaffolds, we discovered 4-phenyl-1,2,3-triazole
(1) to inhibit IDO1 with an IC50 value of 60 μM. Some
modifications of this scaffold have very recently been described
by another group,54 but improvement of the IC50 value was
limited to less than a factor of 2 compared to the parent
compound. The aim of the present work was to rationally
optimize the 4-phenyl-1,2,3-triazole scaffold relying on our in-
house drug design tools.55−61 Substituted 1,2,3-triazoles have
been found to be useful drug scaffolds62−66 and are syntheti-
cally accessible under mild conditions through copper(I)-
catalyzed azide−alkyne cycloaddition.67−69
Here, we computationally designed new ligands and docked
them into the IDO1 active site using EADock-DSS55−59 and
AttractingCavities70 in combination with the parameters
derived from SwissParam60 and a Morse-like metal binding
potential (MMBP).61 Candidates that displayed a good filling
of the A pocket and favorable heme interactions were selected
for synthesis and tested in an enzymatic assay for IDO1
inhibition. Bioisosteres of the 4-phenyl-1,2,3-triazole scaffold
were also investigated. Active ligands were subsequently tested
in cellular assays on murine and on human IDO1. Counter-
screening against TDO determined their selectivity for IDO1.
The two crystal structures of human IDO125 open the way
for the in silico design of new IDO1 inhibitors. In the PIM-
bound X-ray structure (PDB accession code 2D0T), the ligand
is bound in a deep binding site with its phenyl ring inside a
hydrophobic pocket (pocket A, Figure 1) and one imidazole
nitrogen coordinated to the heme iron at a distance of 2.1 Å.
The PIM binding site consists of residues Tyr126, Cys129,
Val130, Phe163, Phe164, Ser167, Leu234, Gly262, Ser263,
Ala264, and the heme ring. Possible hydrogen-bonding sites are
the hydroxyl group of Ser167, the CO group of Gly262, the
NH group of Ala264, and the heme 7-propionate group.
Ligands larger than PIM may also interact with Phe226,
Arg231, Ser235, Phe291, Ile354, and Leu384, which are located
at the binding site entrance. Here, additional hydrogen bonds
are possible with the side chain of Arg231. A hydrophobic
pocket in this region is provided by Phe163, Phe226, Arg231,
Leu234, Ile354, and the heme ring (pocket B, Figure 1). The
cyanide-bound structure (2D0U) differs from the PIM-bound
structure mainly in the access to pocket A, which is hindered by
an inward-movement of the backbone of loop 262−266 and the
side chain of Phe163, suggesting some flexibility in the active
site. In both structures, two buffer molecules (N-cyclo-
hexyltaurine) are bound at the entrance of the active site,
hydrogen-bonding to the heme propionate and interacting with
pocket B.
The oldest known IDO1 inhibitors with activities in the
micromolar range are Trp derivatives and β-carbolines.6,13,31−36
In recent years, many redox-active IDO1 inhibitors with IC50
values of up to 60 nM have been discovered.37−44 However,
there exists the possibility that these act through interference
with the chemical reducing agents in the enzymatic assay
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To rationalize the observed activities, we then developed a
quantitative structure−activity relationship (QSAR) based on
the docking results and on quantum chemical calculations.
Following this strategy, we obtained 39 triazole-based IDO1
inhibitors, the most potent having an IC50 value in the
nanomolar range both in the enzymatic and in the cellular
assays while being inactive on TDO and showing no detectable
toxicity on the cellular level.
Scheme 5
CHEMISTRY
RESULTS AND DISCUSSION
Compounds with Aryl Substitutions. Docking PIM into
■
■
The 4-phenyl-1,2,3-triazole derivatives were synthesized using
existing protocols or procedures adapted from the literature.
The N-unsubstituted 1,2,3-triazoles (1−42, 62−64, and 74)
were synthesized according to the method developed by
Yamamoto and co-workers,71 which involves reaction of
ethynyl derivatives with trimethylsilyl azide (Scheme 1). The
ethynyl derivatives were synthesized from iodo substrates by
the Sonogashira coupling reaction.72 Compound 33 was
synthesized by treatment of 74 with 6 N HCl at room
temperature.73 Compound 34 was derived from compound 30
by treatment with Meldrum’s acid in TEAF at 95−100 °C
(Scheme 2).74 Deprotection of the trifluoroacetamide of 41
the IDO1 active site using a Morse-like metal binding potential
(MMBP),61 we observe an excellent agreement with the X-ray
structure25 (rmsd 0.3 Å) and an iron−nitrogen bond of 2.1 Å.53
Our previously published triazoles53 (Table 1, compounds 1,
2, and 3) are structurally similar to PIM, with the imidazole ring
replaced by a triazole ring. The docked structure of the parent
compound 1 closely resembles that of PIM, with one of the
nitrogen atoms coordinated to the heme iron (Figure 2A−D).
As the triazole ring can occur in three different tautomers
(Figure 2A−C) and in a deprotonated form (Figure 2D), we
docked all four isomers into the IDO1 active site. Because the
MMBP parameters are the same for all nitrogen atoms with a
free electron pair,61 the MMBP cannot distinguish between the
binding energies of the deprotonated form and the neutral
triazole tautomers. All isomers show similar binding modes
(BM) with a conserved position of the phenyl ring, the only
difference being the orientation of the triazole ring. In the
following, we therefore show only the BM of the deprotonated
forms of the N-unsubstituted triazoles.
a
Scheme 2
a
The meta-pyridyl compound (3) is more than an order of
magnitude less active than the phenyl compound (1), while the
para-pyridyl compound (2) retains the activity of 1. The
docked BM of the three compounds do not directly explain this
difference in activity, as the pyridyl nitrogen is not making any
obvious interaction with the protein (Figure 2E,F).
Reagents, conditions, and yields: (a) TEAF, 95−100 °C, 3 h, 45%.
a
Scheme 3
In the following, we tested different derivatives of 1 with
substitutions in para, meta, and ortho positions and
combinations of these (Table 1, Figure 2G−O). All
substitutions in para position of the phenyl ring of the 4-
phenyl-1,2,3-triazole scaffold (1) reduce the IDO1 inhibitory
activity (Table 1, compounds 4, 5, 6, and 7). The fact that the
reduction in activity is correlated with the size of the
substituent (F < Cl < CH3 < CF3) as well as results from
docking suggest that the protein binding pocket is rather tight
in this region and does not provide space for additional groups
(Figure 2G).
Substitutions in the meta position generally increase the
ligand’s activity on IDO1, in agreement with the docking
results, which show the filling of a small subpocket in this
position (Figure 2H,I). Generally, electron-withdrawing and
hydrophobic substitutions yield better inhibitors. The only
meta-substituted compounds with a lower activity than
a
Reagents, conditions, and yields: (a) N2H4·H2O, dioxane, reflux, 2.5
h, 50%.
with N2H4·H2O provided 40 (Scheme 3).75 All other
compounds were synthesized according to literature procedures
[58,76 61,77 (54, 55, 56, and 57, Scheme 4),78,79 (43, 44, 45,
46, and 47, Scheme 5),80,81 (48, 49, and 50, Scheme 6),82−84
(51, 52, and 53, Scheme 6),82,85,86 and (59 and 60, Scheme
7)82] or were commercially available (compounds 65−73).
Scheme 4
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Scheme 6
Scheme 7
Table 1. Phenyl-Ring Substituted Derivatives
a
a
X1
X2
R
IC50 [μM]
X1
X2
R
IC50 [μM]
1
C
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
N
N
N
C
C
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
83
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
2-Br
6.4
15
2
85
2-OH
2-Cl
3
1000
190
530
1000
2300
1.2
32
4
4-F
2-CH3
2-NH2
2-F
39
5
4-Cl
60
6
4-CH3
4-CF3
3-Cl
80
7
2-C2H5
2-CF3
2-CHO
230
650
1300
1800
NI
8
9
3-Br
2.0
10
11
12
13
14
15
16
17
18
19
20
21
3-CN
3-CF3
3-C2H5
3-NO2
3-CH3
3-F
2.8
2-NHCH3
6.0
2-OCH3
13
2-CH2CH2NH2
2-CH2CH2COOH
2-OH,5-Cl
NI
15
NI
b
22
0.33
4.3
47
2-CH3,5-Cl
3-OH
3-NH2
3-Cl
310
1500
2.8
2,5-di-Cl
6.4
2,3-di-Cl
23
2-CH3,3-Cl
72
3-Br
3.3
2-NH2,6-CH3
2-NHCOCF3,6-CH3
2-CH3,5-NH2
1200
3000
3200
3-F
7.4
3-CF3
15
a
b
IC50 values are the mean of at least two independent assays. IC50 value of 0.071 μM at pH 7.4.
compound 1 are the ones with the hydrophilic hydroxyl (16,
IC50 = 310 μM) and amino (17, 1500 μM) substitutions.
Combination of a favorable meta substitution with the para-
pyridyl substitution (2) improves the activity only in the case of
the fluoro substitution, the least active compound (20), while
the activities of the other compounds (18, 19, and 21) remain
approximately constant.
Compounds substituted in the ortho position show a wide
variability of their IC50 values, ranging from 6.4 μM to no
detectable activity. Apparently, large substituents are not
allowed in the ortho position, even though docking results
suggest that these could extend to pocket B (Figure 2M).
Hydroxyl (23, IC50 = 15 μM) and amino (26, 60 μM)
substitutions in the ortho position are much better tolerated
than in the meta position, and they even lead to increased
activity. This could be due to (i) better solvation, (ii) an
intramolecular hydrogen bond formed between N3 of the
triazole and the o-OH or o-NH2 group (Figure 2K), or (iii) an
intermolecular hydrogen bond formed between the ligand and
Ser167. While the last hypothesis has been proposed for the o-
OH-substituted 4-phenylimidazole analogue46 and could also
explain the inactivity of the o-methoxyl substituted ligand (32),
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Figure 2. Proposed binding modes from docking results. The compound numbers are given in parentheses. Carbon atoms are shown in orange/gray,
nitrogen in blue, oxygen in red, bromine in pink, chlorine in green, fluorine in cyan, iron in magenta, and hydrogen in white.
Figure 3. UV spectra of ferric IDO1 without (black) and with 1 mM concentration of compound 35 (red). In the presence of 35, the Soret peak (A)
shifts from 404 to 416 nm and the Q-bands (B) from 499 and 633 nm to 538 and 566 nm, demonstrating a direct binding of the ligand to the heme
iron.
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it is in discrepancy with the docked BM of compounds 23 and
26 (Figure 2K,L).
Table 2. Triazole-Ring Substituted Derivatives
When testing doubly substituted compounds, we noted that
the substitution effects are not additive. Four of these
compounds, all featuring a meta-chloro substituent, showed a
better activity than the parent compound (1). The 2,5-
disubstituted compounds (36 and 37) were more potent than
the corresponding 2,3-disubstituted compounds (38 and 39),
which is probably due to steric constraints in the IDO1 binding
pocket. Docking results favor the 2,3-disubstituted compounds
(Figure 2O), as the 2,5-disubstituted compounds (36 and 37)
fail to bind to the heme, highlighting the lack of binding site
flexibility in our model. The only compound displaying a lower
IC50 value than the meta-chloro substitution alone (8, 1.2 μM)
is the 2-OH,5-Cl substituted compound (35, 330 nM, Figure
2N). Again, the ortho-hydroxyl substitution leads to an
especially favorable activity.
Kinetic experiments with IDO1 inhibitors often yield
uncompetitive or noncompetitive modes of inhibition even
when there exists strong evidence, as in the case of PIM,25,32
that they bind directly in the active site and should therefore be
competitive with respect to the substrate L-Trp.32,46,48,54,87
However, the interpretation of IDO1 inhibition kinetics may be
complicated by the preferential binding of some inhibitors to
the inactive ferric form of IDO1 and by the presence of a
second substrate, dioxygen. To obtain a more direct indication
of the binding site of our triazole compounds, we measured UV
spectra of ferric IDO1 with and without compound 35 (Figure
3). The absorption spectrum of substrate-free ferric IDO1
exhibits a Soret peak at 404 nm and Q-bands at 499 and 633
nm (plus a shoulder at 533 nm) as described in the literature.88
In presence of compound 35, the Soret peak strongly shifts to
416 nm together with a hypochromic effect, while the Q-bands
shift to 538 and 570 nm. This change in optical absorption is
similar to the changes induced by the binding of PIM (peaks at
412.5, 533, and 560 nm) and of pyridine (peaks at 419, 538,
and 570 nm) to IDO1, both of which have been confirmed to
bind directly to the heme iron.25,32
Compounds with Substituted Triazole Rings. Besides
the parent compound 4-phenyl-1,2,3-triazole (1), we docked its
isomer 1-phenyl-1,2,3-triazole (61) in the IDO1 active site
(Figure 2R) and observed a close similarity to PIM and
compound 1, as all three compounds are sterically almost
identical and provide a nitrogen atom with a free electron pair
for heme binding. However, 61 does not show any inhibitory
activity on IDO1 in the enzymatic assay (Table 2).
Additionally, we observed that any substitution of the triazole
ring in position R1, R2, R3, or R5 in combination with the
phenyl substitution in position R4 also completely abolished
activity (Table 2). This is a significant difference to the
imidazole analogues, where, for example, 1-benzyl-5-phenyl-
imidazole is more active than PIM.46 Taking these results
together, they hint at the necessity of an ionizable NH group in
the triazoles and at the hypothesis that the active form of the
triazole for IDO1 inhibition could be its deprotonated, anionic
form.
IC
50a
R1
R2
R3
R4
phenyl
R5
[μM]
1
H
H
H
H
H
83
NI
NI
NI
43
44
45
phenyl phenyl
phenyl 4-F-phenyl
phenyl 3-CH3-
phenyl
46
47
phenyl 3-Cl-phenyl
H
H
NI
NI
phenyl 2-OCH3-
phenyl
48
49
50
51
52
53
54
55
56
57
58
59
60
61
methyl phenyl
phenyl
H
H
H
H
H
H
H
H
H
H
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
methyl
methyl
ethyl
phenyl
ethyl
phenyl
phenyl
phenyl
ethyl
n-butyl phenyl
n-butyl
phenyl
benzyl
phenyl
phenyl
phenyl
benzyl
H
methyl
methyl 3-Cl-phenyl
H
H
H
methyl
phenyl
3-Cl-phenyl
H
a
IC50 values are the mean of at least two independent assays.
triazole (69), tetrazole (70), isoxazole (71), pyridine (72), or
pyrimidine (73) completely abolished IDO1 inhibitory activity
(Table 3). Also compounds with linkers between the aromatic
moieties (62 and 63), a cyclohexene analogue (64), and
benzotriazoles (65 and 66) failed to show any activity.
Steric reasons may play a role for some of these compounds.
For example, docking results for compounds 63 (filled volume
of pocket A: 85%) and 65 (78%) show that these do not fill the
IDO1 binding pocket as tightly as the other compounds (1:
96%). Compounds with two aromatic six-membered rings (72
and 73) apparently need an iron-binding nitrogen in 3-position,
as only 3-phenylpyridine has been shown to be active on IDO1
(IC50 = 161 μM).46
Other compounds (69, 70, and 71), however, are sterically
so similar to compound 1 that their inactivity on IDO1 must be
due to electronic effects. Compound 69 (Figure 2S) is a closely
related isomer of compound 1, however, with a higher pKa
value (9.29 vs 8.62), which may prevent its deprotonation
under experimental conditions. In contrast, the tetrazole (70,
Figure 2T) has a very low pKa value (4.53) and should
therefore be deprotonated under experimental conditions.
However, quantum chemical atomic point charges show that
its electron density is mainly concentrated on N1 and N4
(charge −0.36), which are next to the phenyl ring, while N2
and N3, which are available for heme iron binding, only carry
small negative charges (−0.17). In active compounds, the
negative charge on the iron-binding nitrogen atom ranges from
−0.33 to −0.46. Similarly to compound 70, in the inactive
isoxazole 71, the nitrogen atom available for iron binding is
The inactivity of the 4,5-disubstituted compound 58 (Figure
2Q), which preserves the ionizable NH group of the triazole
ring, could be related to its increased pKa value (9.11 vs 8.62 for
1, Table 4), due to the electron-donating substitution on the
triazole ring, or it could be due to steric effects.
Compounds with Modified Scaffolds. Any attempt to
replace the 1,2,3-triazole scaffold by bioisosteres such as 1,2,4-
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opposite is observed for compounds 23 and 35 featuring an
ortho-hydroxyl substitution, which display more acidic pKa
values than predicted from their σ value, suggesting that here
a hydrogen bond between the substituent and the anionic
triazole stabilizes the negatively charged form of the triazole.
This interpretation is further supported by the strong shift of
one pKa unit from 9.95 to 10.95 for the phenolic pKa of
compound 23, as this hydrogen now serves to stabilize the
monoanion.
Table 3. Modified Scaffolds
All measured pKa values are higher than the pH at which the
enzymatic assay is carried out (6.5). However, the pKa of a
ligand in water does not necessarily reflect its pKa in the
protein-bound state. It has been shown, e.g., that thiols binding
to CYP450-CAM show a pKa shift of 4 units as compared to
their values in water.91 It is therefore possible that the triazoles
bind to IDO1 in their deprotonated form. Taking into
consideration the inactivity of all triazole compounds lacking
an ionizable NH group (Table 2) or having a high pKa value,
this hypothesis becomes the most likely scenario.
We have further tested this hypothesis by carrying out an
enzymatic assay at pH 7.4 instead of pH 6.5 for compound 35.
As expected, 35 shows a higher activity (IC50 = 71 nM) at pH
7.4 than at pH 6.5 (IC50 = 330 nM, see Figure S2 in the
Supporting Information).
Quantitative Structure−Activity Relationship. Early on
during our efforts to optimize the potency of the triazole
ligands, we noticed a correlation between the activities of the
meta-substituted compounds and the Hammett σ constants (r =
0.62), a correlation that could be even improved by adding the
hydrophobicity constant π as a second parameter (r = 0.90,
Figure 4B).90 In this model, the more electron-withdrawing and
the more hydrophobic the substituent on the phenyl ring, the
better the potency of the triazole compound against IDO1. The
influence of the hydrophobicity is not surprising, as docking
suggests that the substituent in meta position will be placed
inside a hydrophobic subpocket of the IDO1 active site (Figure
2H−J). The electronic properties of the substituent, however,
control the electronic properties of the triazole ring, as
exemplified by our pKa measurements. Two possible
interpretations of the correlation between IDO1 inhibitory
activity and σ are therefore that (i) IDO1 inhibition of the
triazoles increases with decreasing electron density on the
triazole ring, possibly influencing the heme binding strength
(heme-binding strength hypothesis), or (ii) IDO1 inhibition
increases with decreasing pKa of the triazole ring, because the
ligand must be deprotonated in order to be active
(deprotonation hypothesis). A combination of both effects is
possible.
a
IC50 values are the mean of at least two independent assays.
very poor in electrons (charge −0.12) due to the electro-
negative neighboring oxygen atom.
pKa Measurements. To better understand the electronic
properties of the triazoles, we carried out pKa measurements of
selected compounds (Table 4). The parent compound (1) has
a pKa value of 8.62 for deprotonation of its triazole ring, 0.64
pKa units more acidic than the unsubstituted 1,2,3-triazole
(9.26).89 Compounds with electron-withdrawing substituents
on the phenyl ring are more acidic than 1, because these
substituents stabilize the negative charge on the triazole.
In Figure 4A, we plot the measured pKa values as a function
of the Hammett substituent constants σ,90 assuming (i) that the
influence of substituents is additive, σ = σortho + σmeta + σpara
which is a common assumption,90 and (ii) that the influence of
a substituent is equal in ortho and in para position, σortho = σpara
,
,
We investigated this question by quantum chemical
calculations. The direct calculation of binding energies of the
triazole compounds to a heme model system is a complex task
due to the existence of six possible ligand−heme orientations of
the three tautomeric forms and the deprotonated form of the
ligands, which, in combination with two possible iron redox
states (ferric and ferrous form), leads to 12 different binding
energies. Therefore, the complete set of calculations was only
carried out for the parent compound 1, from which favorable
ligand−heme orientations were obtained (details given in
Supporting Information).
which should be valid if steric hindrance is low and if no special
interactions arise in ortho position.
For all compounds with meta and para substitutions (black
stars in Figure 4A), a very good linear relationship is obtained:
pKa = 8.607 − 0.787 × σ
(1)
with a correlation coefficient of 0.997. Compounds with a
chloro substituent in ortho position (24, 37, and 38) follow this
correlation quite closely, but compounds with an oxygen-
containing substituent in ortho position (24, 37, and 38) are
clear outliers of this relationship.
Taking solvation effects into account by a polarizable
continuum model (PCM),92 the neutral parent compound
(1) binds more strongly to ferric heme (−4.5 kcal/mol, 3H
tautomer binding with N1 to iron) than to ferrous heme (−0.3
The ortho-methoxyl substituent of 32 leads to a more basic
pKa value than predicted from its σ value, suggesting a
hydrogen bond between the neutral triazole and the substituent
that renders deprotonation of the triazole less favorable. The
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Table 4. pKa Values and Hammett σ Values
a
b
c
Standard deviation given in parentheses. The definition of the Hammett σ values is given in the text. The acidic pKa value of 6.32 given in refs
89,134 goes back to a reference dating from the beginning of the 20th century and is obviously wrong.
kcal/mol, 2H tautomer binding with N1 to iron). These
binding energies are significantly lower than the binding
energies of the deprotonated form of 1 to both ferric (−17.0
kcal/mol, binding with N1 to iron) and to ferrous (−1.4 kcal/
mol, binding with N1 to iron) heme. The neutral form of
compound 8, which has an electron-withdrawing meta-chloro
substituent on the phenyl ring, binds even more weakly to ferric
heme (−3.7 kcal/mol) and just as weakly to ferrous heme
(−0.4 kcal/mol) than the parent compound. Its deprotonated
form shows similar binding energies for ferric heme (−16.7
kcal/mol) and for ferrous heme (−1.6 kcal/mol) as compound
1.
In summary, quantum chemical calculations suggest that
heme binding strength does not increase with decreasing
electron density on the triazole ring but that the deprotonated
form of the triazoles binds much more strongly to heme than
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Figure 4. (A) Correlation between the Hammett substituent constant and the measured pKa values, assuming (i) that the influence of substituents is
additive, and (ii) that the influence of a substituent is equal in ortho and in para position. Compounds with a substitution in ortho position are shown
as red circles. Compounds without a substitution in ortho position are shown as black stars, and the black line is a linear fit to these points (r =
0.997). (B) Correlation between experimental and calculated activities of meta-substituted compounds using the Hammett substituent constant
alone (black, r = 0.62) or in combination with the hydrophobicity parameter (red, r = 0.90). The black dotted line denotes a perfect correlation. (C)
QSAR relationship using the quantum chemical triazole charges and the electrostatic interaction/solvation term from the docking as descriptors (see
text). (D) Correlation between the IC50 values measured in the enzymatic assay and the IC50 values measured in the cellular assays on hIDO1
(black) and on mIDO1 (red).
the neutral form. They support thus the deprotonation
hypothesis.
bulky substituents in para or meta positions (compounds 5, 6,
7, 13, 36, and 37, see filtering conditions in the Experimental
Section) and were therefore removed from the test set. For the
remaining set of 33 compounds, we obtained the best QSAR
model when using two simple parameters in the regression
analysis: the sum of the ligand−protein electrostatic interaction
energy and the electrostatic desolvation energy on the one hand
(Velec), and the quantum chemical charge on the triazole
(CHtriz) on the other hand. With these two parameters a
correlation coefficient of 0.87 and a standard deviation of 0.73
kcal/mol is obtained (Figure 4C).
In the following, we developed a quantitative structure−
activity relationship (QSAR) for the triazole compounds based
on our docking results. All standard terms such as electrostatic
interactions, van der Waals interactions, and solvation terms are
calculated based on the CHARMM22 classical force field93,94
and the FACTS implicit solvent model95 (see Experimental
Section). However, this procedure does not include the
electronic effects described above. We therefore decided to
include an additional electronic term in the QSAR model. As
the Hammett constants are only available for a subset of our
compounds, we replaced them by an electronic parameter that
could be determined for all compounds using quantum
chemical calculations. After many trials, we chose to use the
sum of atomic charges on the deprotonated triazole ring as
calculated by natural population analysis with a large basis set
(CHtriz) because this parameter proved to be robust and yields a
good correlation (r = 0.96) with the Hammett σ constants for
15 compounds for which a σ is available90 (1, 4−17).
Of the 39 active triazoles described in this work, six could not
satisfactorily be docked into the IDO1 active site because of
Act = −38.79 + 0.60 × V − 34.48 × CHtriz
(2)
elec
where Act and Velec are given in kcal/mol and CHtriz is given in
elementary charge units. Compound 35 has a prominent
location on the correlation plot (Figure 4C). However, its
omittance from the test set only marginally changes the
correlation (r = 0.85, stdv = 0.73 kcal/mol). Addition of terms
for the van de Waals interactions and hydrophobic desolvation
does not improve the correlation, probably because these terms
show little variation between the described compounds, which
all tightly occupy only the A-pocket of the IDO1 active site.
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Figure 5. Ligand efficiency [kcal/mol/atom] of known IDO1 ligands (blue) and the compounds described in this work (red). Ligands that probably
interact with the cystein residues of IDO144,49 were not included. Indole-4,7-dione41 probably acts through a redox mechanism.
a
Table 5. Cellular Assays for IDO1 and TDO Inhibition
b
enzym IC50 [μM]
cell IC50 [μM]
hIDO1
mIDO1
hIDO1
mTDO
hTDO
LD50 [μM]
1
83
0.18 (0.05)
2.1 (1)
8.9 (0.8)
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
60 (30)
17
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
70 (20)
28
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
4
190
1.2
2.0
2.8
6.0
13
8
0.020 (0.003)
0.023 (0.009)
0.029 (0.006)
0.07 (0.03)
0.03 (0.02)
0.11 (0.02)
0.12 (0.04)
1.1 (0.4)
0.62 (0.01)
0.94 (0.07)
2.9 (0.6)
9 (4)
9
10
11
12
1.1 (0.1)
24 (0.1)
7 (3)
13
15
14
22
15
47
>100
16
310
1500
2.8
3.3
7.4
15
1.3 (0.6)
>100
17
4 (2)
>100
18
0.06 (0.02)
0.05 (0.03)
0.12 (0.01)
0.34 (0.05)
0.034 (0.001)
0.022 (0.001)
1.5 (0.3)
3.5 (0.5)
2.9 (0.4)
7 (1)
19
20
21
56 (2)
2.6 (0.1)
1.7 (0.1)
>100
22
6.4
15
23
24
32
25
39
1.7 (0.1)
>100
26
60
1.7 (0.4)
>100
27
80
0.63 (0.09)
13 (2)
70 (20)
>100
28
230
650
NI
29
20 (1)
>100
32
20 (1)
>100
35
0.33
4.3
6.4
23
0.002 (0.002)
0.12 (0.04)
0.15 (0.04)
1.7 (0.1)
0.08 (0.02)
14 (4)
34 (2)
>100
36
37
38
Incyte 5l51
LM1018
ND
ND
0.10 (0.07)
>100
0.06 (0.03)
>100
a
IC50 values of compounds tested in cells transfected with mouse IDO1 (mIDO1), human IDO1 (hIDO1), mouse TDO (mTDO), or human TDO
(hTDO). Standard deviations are given in parentheses. Cytotoxicity of the compounds was tested on Hep G2 cells (LD50). For comparison, also the
b
IC50 values of the enzymatic assay on hIDO1 are given again. ND: not determined. NI: no inhibition. IC50 values are the mean of at least two
independent assays.
It is important to notice that the QSAR model does not rely
on either the heme-binding strength or the deprotonation
hypothesis, but it is valid for both cases.
According to this model, compound 35 is highly active on
IDO1 because it displays very good electrostatic interactions
with the protein while carrying a low charge on the triazole
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ring. Interestingly, in the docked pose (Figure 2N), there is no
intramolecular hydrogen bond present, but the hydroxyl group
forms a hydrogen bond with Ser167 instead. As MMG-0358
(compound 35) is is very small, it shows an excellent ligand
efficiency96 of −0.72 kcal/mol/atom, ranking among the most
efficient IDO1 inhibitors known to date (Figure 5).
nanomolar range, with a good selectivity as no inhibition was
observed in TDO-expressing cells. Strikingly, we consistently
observed substantially higher inhibitory activities in the cellular
assay performed with murine IDO1 as compared to human
IDO1, with IC50 values on average 77-fold lower. This was not
related to the cell type because we used the same murine cell
line transfected with mIDO1 or hIDO1 for these assays. We
also used human cells HEK293 transfected with hIDO1 and
observed similar IC50 values as with the murine cells transfected
with hIDO1 (data not shown). These differences therefore
likely reflect genuine variation in the inhibition of murine
versus human IDO1 by this family of compounds. Murine
IDO1 shares 62% of sequence identity with human IDO1, and
the active site residues mentioned in the Introduction are 100%
conserved. However, the orthologues show some distinct
biochemical properties, as, for example, mIDO1 displays a 3-
fold lower Km for L-Trp than hIDO1.97 Despite these
differences, an excellent correlation between the cellular assays
on mIDO1 and on hIDO1 is observed (r = 0.95). Additionally,
the observed good correlation between enzymatic and cellular
assay results (Figure 4D, r = 0.71 for enzym. hIDO1/cell.
hIDO1, and r = 0.79 for enzym hIDO1/cell mIDO1) enables
application of the developed QSAR model also for optimization
of cellular activities.
Comparison to Imidazole and Pyrazole Analogues.
Although compound 1 is a close analogue of PIM and docks in
a similar manner to IDO1, the triazoles and imidazoles differ
substantially in their structure−activity relationship for IDO1
inhibition. For example, the disubstituted imidazole compound
1-benzyl-5-phenylimidazole shows a slightly better IC50 value
than PIM (32 vs 48 μM),46 while the direct analogue 1-benzyl-
5-phenyl-1,2,3-triazole (56) is inactive. Replacement of the
phenyl ring by a p-pyridyl ring in the triazole (2) leaves its
activity unaffected, while the same replacement in the imidazole
strongly reduces its activity (67). Despite these dissimilarities,
there are also similarities, as the general drop in activity when
introducing substituents in para position of the phenyl ring, and
the much higher activity of compounds with ortho-hydroxyl as
compared to meta-hydroxyl substituted phenyl rings.46 While
the similarities are probably due to the similar binding modes
and similar protein interactions in the IDO1 active site, the
differences can be explained by distinctive electronic structures
and heme interactions. PIM is a much stronger base and a
much weaker acid than compound 1 (Table 4), which
correlates with the higher charge density on the heme-binding
nitrogen of the imidazole (charge −0.46) as compared to 1,2,3-
triazole (−0.24 neutral, −0.35 deprotonated). It is therefore
active on IDO1 without deprotonation, so that the N3 position
is available for additional substitutions, and so that electron-
withdrawing substituents on the phenyl ring do not show the
same effect as in case of the triazoles.
A close analogue of compound 35 is the previously published
4-fluoro-2-(pyrazol-3-yl)phenol, featuring a pyrazole with a 2-
OH,5-F substituted phenyl ring and an IC50 value of 26 μM.46
The about 100-fold higher activity of compound 35 on IDO1
could be explained by the better protein interactions of chloride
vs fluoride and by the stronger heme bond of the triazole
(−14.8 kcal/mol) vs the pyrazole (−8.4 kcal/mol, see
Supporting Information).
Cellular Assays. We tested compounds with a low IC50 in
the enzymatic assay for their ability to inhibit tryptophan
degradation and kynurenine production in cells expressing
either murine (mIDO1) or human IDO1 (hIDO1). Such
cellular assay is informative for drug development, as it
evaluates not only the IDO1 inhibitory effect of the compounds
but also their capacity to permeate the cell, their potential
cytotoxicity, inhibition of tryptophan and kynurenine transport,
and interactions with other proteins as well as the effects of
their metabolites. To control specificity, we also tested the
inhibitory activity of the compounds on cells expressing murine
(mTDO) or human TDO (hTDO). The same murine cell
type, stably transfected with murine or human IDO1 or TDO,
was used for all assays. Additionally, cell viability was evaluated
using Hep G2 cells.
The most promising IDO1 inhibitor reported to date is
Incyte compound 5l,51 with IC50 values of 67 nM in an
enzymatic assay and 19 nM in a cellular assay on HeLa cells. In
our cellular assays, compound 35 displays a similar inhibitory
effect on hIDO1 as 5l (80 and 60 nM respectively, Table 5)
and therefore ranks among the most potent IDO1 inhibitors
described so far.
SUMMARY
■
In this work, we have used an in silico driven strategy for the
rational optimization of IDO1 inhibitors based on the 4-aryl-
1,2,3-triazole scaffold discovered in our earlier work.53
New ligands were designed computationally, docked into the
IDO1 active site, selected in case of favorable interactions,
synthesized, and evaluated for IDO1 inhibition potency in an
enzymatic assay. Successful candidates were subsequently
tested in cellular assays on mIDO1, hIDO1, mTDO, and
hTDO. Bioisosteres of the 4-phenyl-1,2,3-triazole scaffold were
also investigated. Using the results of the enzymatic assay, we
developed a QSAR model based on the docking results and on
quantum chemical calculations. Following this strategy, we
obtained 39 triazole-based IDO1 inhibitors, the most potent
having an IC50 value in the nanomolar range both in the
enzymatic and in the cellular assays while being inactive on
TDO and showing no detectable toxicity on the cellular level.
Some modifications of the 4-aryl-1,2,3-triazole scaffold have
very recently been described independently by another group.54
Among the 20 compounds described in their work, two were
described by us before (1 and 3) and seven were also
synthesized and tested in the present work (4, 5, 6, 22, 24, 32,
and 65), while the remaining 11 compounds are either only
weakly active or inactive. The enzymatic IC50 values measured
in our lab generally differ by a factor 2−3 from the values of
Huang et al., which is probably due to different experimental
conditions. The only outlier is compound 22, for which the
IC50 values differ significantly (14854 vs 7 μM here). The
inhibition mode of compounds 1, 22, and 24 was determined
to be uncompetitive.54 However, the UV spectra of IDO1 in
Most compounds active in the enzymatic assay were also
active in the cellular assay, usually with lower IC50 values (Table
5 and Figure 4D), in line with previous observations.51,54 In the
present case this might be due to the higher pH employed in
the cellular assays (7.4) as compared to the enzymatic assays
(6.5), favoring deprotonation of the triazole ligands. The IC50
values for the triazole compounds are in the micromolar to
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the A-pocket of at least 75%. Finally, for each compound the BM
having the lowest FullFitness and meeting all conditions was chosen
for analysis.
presence of compound 35 measured here (Figure 3) support
the notion that the triazoles bind directly to the heme iron.
As the compounds described in this work are very small, they
might suffer from a low selectivity profile. However, their low
cellular toxicity and inactivity on TDO, which shares a common
active site architecture with IDO1, support their further
development.
In summary, we have used our in-house docking tools to
successfully design new IDO1 inhibitors with a good efficacy in
enzymatic and in cellular assays. Our most potent compound
(MMG-0358, 35) shows IC50 values of 2 nM in a cellular assay
on mIDO1, 80 nM in a cellular assay on hIDO1, 330 nM in an
enzymatic assay on hIDO1 at pH 6.5, and 71 nM in an
enzymatic assay on hIDO1 at pH 7.4. This compound, which
additionally has an excellent ligand efficiency96 of −0.72 kcal/
mol/atom, will be evaluated in vivo in the future. The QSAR
model derived from the collection of our evaluated ligands will
be helpful in the future to design even more potent and specific
IDO1 inhibitors.
Density Functional Theory Calculations. Quantum chemical
geometry optimizations and charge calculations were carried out in the
density functional theory (DFT) framework with the PBE0 hybrid
functional99 using the Gaussian 09 code.100 Geometry optimizations
were carried out with standard settings and the TZVP basis set,101
while atomic point charges derived from a natural population analysis
(NPA)102 were calculated with the aug-cc-pVTZ basis set103−105 in
order to obtain converged charges.106 Solvation effects were taken into
account by the polarizable continuum model92 as implemented in
Gaussian 09.
For compound 1, the 2-H tautomer was the most stable both in
vacuo and in the PCM. For charge calculations for the QSAR,
compounds with an acidic pKa value of 9.5 or below were considered
in their deprotonated form.
The histidine-bound heme complex of IDO1 was modeled by an
iron−porphin−imidazole system. Binding energies were calculated by
subtracting the energy of the iron−porphin−imidazole system and the
energy of the isolated ligand from the energy of the 6-fold coordinated
system. For the ferric iron−porphin−imidazole system, the ground
state with the hybrid DFT functional PBE0 is a quartet, while for the
ferrous system it is a quintuplet. For the 6-fold coordinated systems, a
low-spin complex was always assumed, as it has been found
experimentally.32
Quantitative Structure−Activity Relationship. The binding
free energy ΔG of an inhibitor is related to its Ki value by the equation
ΔG = −RT ln Ki. As the Ki of an inhibitor is linearly related to its IC50
value and the prefactor only depends on enzyme concentration,
substrate concentration, and Km of the substrate,107,108 we can
calculate the activity of an inhibitor as Act = −RT ln IC50. Differences
in the calculated activities of different inhibitors will then be equal to
ΔΔG.
For the QSAR model, we carried out a linear regression with two
variables, using the activities as target values. The first variable is the
sum of the NPA charges of all atoms belonging to the deprotonated
triazole ring. The second variable, Velec, is
EXPERIMENTAL SECTION
■
Docking. A consensus docking procedure based on our in-house
docking algorithms EADock,55,56 EADock DSS,57 and AttractingCav-
ities70 was used. In the latter, in brief, an extended conformation of the
ligand is minimized in the cavities of the protein, starting from
different positions and orientations, in an approach similar to that of
MCSS.98 Minimized poses are finally clustered and ranked according
to the scoring function of EADock. A detailed description of this
algorithm will be the subject of a future communication.
All three algorithms rely on the physical scoring function of the
CHARMM22 force field,93,94 while solvation effects are taken into
account by the FACTS model,95 which has been shown to allow for
accurate docking results.59 Ligand force-field parameters were derived
with the SwissParam tool.60 For all compounds with an acidic pKa
value of 9.5 or below, the deprotonated species was considered. A
Morse-like metal binding potential (MMBP) was used to describe the
interactions between the heme iron of IDO1 and ligand atoms that
display a free electron pair for iron binding.61 The protein was kept
fixed during the docking. We use the PIM-bound X-ray structure
(2D0T, chain A) as a target because it has a higher resolution and
provides a more suitable induced fit for the triazole ligands than the
cyanide-bound structure (2D0U).25 The two molecules of N-
cyclohexyltaurine bound at the entrance of the binding site were
removed during setup.
For AttractingCavities, the standard parameters with a cubic search
space of (20 Å)3 around the IDO1 active site were used. For EADock
DSS, docking results were compared to EADock 2.0 results, and in
order to well reproduce the benchmarks, the following custom settings
were used: 30000 generated binding modes (BM), 12000 BM
evaluated with FullFitness, 100 steepest descent minimization steps,
1000 adopted basis Newton−Raphson minimization steps, 1.5 Å
clustering radius, and a cubic search space of (20 Å)3 around the IDO1
active site. Subsequently, the 250 best BM produced by EADock DSS
were fed as input to EADock 2.0, using the same clustering radius and
terminating the run when the first three clusters did not change over
the course of 100 generations of the evolutionary algorithm.
For analysis, the results of AttractingCavities and EADock 2.0 were
merged by comparing the best BM of each cluster and considering
them distinct if their mass-weighted rmsd was larger than 0.8 Å. The
resulting 10 best distinct BM were filtered to detect energetically
favorable poses with a direct bond to the heme iron and a good filling
of the A-pocket, criteria that were deemed necessary for IDO1
inhibition by the investigated scaffold. The following filters were
applied: (i) a FullFitness that is maximally 5 kcal/mol higher than the
one of the globally best BM, (ii) an iron-heteroatom (N, O, S) bond of
3 Å or shorter, (iii) a minimal distance between the ligand and the
sulfur atom of Cys129 of less than 5.2 Å, and (iv) a volume filling of
V
= ΔGsolv,elec + Einter,elec − Ecorr,elec
(3)
elec
with the electrostatic solvation term
complex
solv,elec
ligand
solv,elec
protein
solv,elec
ΔGsolv,elec = ΔG
− ΔG
− ΔG
(4)
as calculated by the FACTS solvation model.95 Einter,elec is the
electrostatic interaction energy between the ligand and the protein
calculated with the Coulomb equation and a dielectric constant of 2 as
appropriate for the use of FACTS. Ecorr,elec is a correction term
necessary to counterbalance the use of the MMBP and equals the
electrostatic interaction energy between the ligand on the one hand
and the iron and nitrogen atoms of the heme on the other hand.
Chemistry. General Remarks. Materials and reagents were
obtained from commercial suppliers and used without further
purification. For extraction and chromatography, all solvents were
distilled prior to use. Thin layer chromatography for reaction
monitoring was performed on silica gel plates (Merck 60 F254)
with detection by UV light (254 nm) and charring with KMnO4 or
Pancaldi reagent. Flash chromatography was conducted using silica gel
60 Å, 230−400 mesh (Merck 9385). Melting points were measured
with a Mettler FP52 apparatus and are uncorrected. IR spectra were
recorded on a Perkin-Elmer Paragon 1000 FT-IR spectrometer. Mass
spectra were recorded on a Nermag R 10−10C instrument in chemical
ionization mode. Electrospray mass analyses were recorded on a
Finnigan MAT SSQ 710C spectrometer in positive ionization mode.
1H and 13C NMR spectra were recorded on a Bruker-DPX-400 or
Bruker-ARX-400 spectrometer at 400 and 100.6 MHz, respectively.
Data for 1H NMR spectra are reported as follows: chemical shift,
multiplicity, coupling constant, and integration. Data for 13C NMR
spectra are reported in terms of chemical shifts. Chemical shifts are
given in parts per million, relative to an internal standard such as
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1
residual solvent signals. Coupling constants are given in hertz. High-
resolution mass spectra were recorded via ESI-TOF-HRMS or
MALDI-TOF-HRMS. The purity of all novel compounds was
confirmed to exceed 95% by NMR and high-resolution mass spectra.
Elemental analysis for C, H, and N was performed by Quantitative
Technologies Inc.
compound as an off-white solid in 85% yield. H NMR (400 MHz,
CDCl3): δ 10.55 (s, 1H), 7.94 (d, 1H, J = 7.6 Hz), 7.63 (dd, 1H, J =
7.2, Hz, 1.1 Hz), 7.58 (td, 1H, J = 7.5 Hz, 1.5 Hz), 7.49 (t, 1H, J = 7.6
Hz), 3.47 (s, 1H).
3-Ethynyl-4-methylaniline.118 Synthesized from 3-iodo-4-methyl-
aniline according to the representative procedure72 to afford the title
compound as a brown oil in 70% yield. 1H NMR (400 MHz, CDCl3):
δ 7.01 (d, 1H, J = 8.1 Hz), 6.85 (d, 1H, J = 2.5 Hz), 6.64 (dd, 1H, J =
5.5 Hz, 2.5 Hz), 3.67 (br s, 1H), 3.24 (s, 1H), 2.36 (s, 3H).
4-Chloro-2-ethynylphenol. Synthesized from 4-chloro-2-iodophe-
nol according to the representative procedure72 to afford the title
compound as a colorless oil in 70% yield. 1H NMR (400 MHz,
CDCl3): δ 7.35 (d, 1H, J = 2.5 Hz), 7.24 (dd, 1H, J = 8.7 Hz, 2.5 Hz),
6.90 (d, 1H, J = 8.8 Hz), 5.76 (s, 1H), 3.52 (s, 1H).
General Procedures. Representative Procedure for the Prepara-
tion of Phenylalkynes.72 To a stirred solution of iodo derivative
(Scheme 1, A, 1 mmol, 1 equiv) and Et3N (4 mmol, 4 equiv) in
dioxane (4 mL) were added trimethylsilyl acetylene (1.3 mmol, 1.3
equiv), PdCl2(PPh3)2 (0.01 mmol, 0.01 equiv), and CuI (0.02 mmol,
0.02 equiv). The reaction mixture was stirred at 45 °C for 5 h under
nitrogen. Diethyl ether (5 mL) and 0.1 N HCl (3 mL) were added,
and the organic layer was separated, neutralized with a saturated
NaHCO3 (3 mL, twice) solution, washed with brine (3 mL), dried
over Na2SO4, and evaporated. The residue (B) was added to KF (3.6
mmol, 3.6 equiv) dissolved in MeOH (5 mL), and the reaction
mixture was stirred for 3 h at room temperature. The reaction mixture
was concentrated, and CH2Cl2 (5 mL) and water (3 mL) were added.
The organic layer was collected, dried (MgSO4), and filtered through a
short silica plug to afford desired (ethynyl) derivative (C) compounds;
overall yields up to 85%.
4-Chloro-2-ethynyl-1-methylbenzene.119 Synthesized from 4-
chloro-2-iodo-1-methylbenzene according to the representative
procedure72 to afford the title compound as a yellow oil in 60%
yield. H NMR (400 MHz, CDCl3): δ 7.46 (d, 1H, J = 2.2 Hz), 7.24
(dd, 1H, J = 6.0 Hz, 2.3 Hz), 7.15 (d, 1H, J = 8.1 Hz), 3.33 (s, 1H),
2.44 (s, 3H).
1
General Procedure for N-Unsubstituted 1,2,3-Triazoles.71 To a
stirred solution of commercially available or synthetically prepared
ethynyl substrate (1 mmol, 1 equiv) and CuI (0.05 mmol, 0.05 equiv)
in DMF/MeOH solution (2 mL, 9:1) under an argon atmosphere,
trimethylsilyl azide was added (1.5 mmol, 1.5 equiv). The resulting
solution was stirred at 100 °C for 10−12 h. After consumption of the
ethynyl substrate, the mixture was cooled to room temperature and the
precipitate was filtered and concentrated under reduced pressure. The
crude residue was purified with silica gel column chromatography to
obtain the desired product.
2-Ethynylphenol.72 Synthesized from 2-iodophenol according to
the representative procedure72 to afford the title compound as a
1
yellowish oil in 85% yield. H NMR (400 MHz, CDCl3): δ 7.40 (dd,
1H, J = 7.5 Hz, 1.7 Hz), 7.33−7.30 (m, 1H), 6.98 (d, 1H, J = 8.5 Hz),
6.90 (td, 1H, J = 7.5 Hz, 1.0 Hz), 5.81 (s, 1H), 3.50 (s, 1H).
1-Ethyl-2-ethynylbenzene.109 Synthesized from 1-ethyl-2-iodoben-
zene according to the representative procedure72 to afford the title
compound as a yellow oil in 80% yield. 1H NMR (400 MHz, CDCl3):
δ 7.48 (d, 1H, J = 7.5 Hz, 1.7 Hz), 7.29 (td, 1H, J = 7.4 Hz, 1.5 Hz),
7.23 (d, 1H, J = 7.1 Hz), 7.15 (td, 1H, J = 7.5 Hz, 1.2 Hz), 3.25 (s,
1H), 2.84 (q, 2H, J = 7.5 Hz), 1.26 (t, 3H, J = 7.5 Hz).
General Procedure for the Preparation of Triazoles 48, 49, 50,
51, 52, and 53 by N-Alkylation of 4-Phenyl-1H-1,2,3-triazole.82−84
To a solution of 4-phenyl-1H-1,2,3-triazole (1 mmol, 1 equiv) in DMF
(3 mL), K2CO3 (0.25 mmol, 0.25 equiv) was added. The mixture was
stirred for 5 min at room temperature. Dimethyl or diethyl carbonate
(2.2 mmol, 2.2 equiv) was added, and the mixture was stirred at reflux
(around 145 °C) for 5 h. The resulting solution was filtered, and the
remaining solid material was washed with Et2O (5 mL). Evaporation
of the solvent afforded an oily residue. From the crude oil the different
regioisomers were separated by column chromatography on silica gel
to give compounds 48, 49, 50, 51, 52, and 53 in 7, 60, 20, 6, 19, and
55% yield respectively.
3-Ethynylbenzonitrile.110 Synthesized from 2-iodobenzonitrile
according to the representative procedure72 to afford the title
compound as a yellow oil in 80% yield. 1H NMR (400 MHz,
CDCl3): δ 7.79 (s, 1H), 7.73 (dt, 1H, J = 7.9 Hz, 1.3 Hz), 7.66 (dt,
1H, J = 7.9 Hz, 1.3 Hz), 7.48 (t, 1H, J = 7.9 Hz), 3.22 (s, 1H).
1-Ethyl-3-ethynylbenzene.111 Synthesized from 1-ethyl-3-iodoben-
zene according to the representative procedure72 to afford the title
compound as yellow oil in 75% yield. 1H NMR (400 MHz, CDCl3): δ
7.36−7.30 (m, 2H), 7.25−7.14 (m, 2H), 3.06 (s, 1H), 2.64 (q, 2H, J =
7.5 Hz), 1.24 (t, 3H, J = 7.6 Hz).
General Procedure for the Preparation of Triazoles 59 and 60 by
N-Methylation of 4-(3-Chlorophenyl)-1H-1,2,3-triazole.82 To a
solution of 8 (1 mmol, 1 equiv) in DMF (4 mL), K2CO3 (1 mmol,
1 equiv) was added. The mixture was stirred for 5 min at room
temperature. Iodomethane (1 mmol, 1 equiv) was added, and the
mixture was stirred at room temperature for 24 h. The resulting
solution was filtered, and the remaining solid material was washed with
Et2O (4 mL). Evaporation of the solvent afforded an oily residue.
From the crude oil, the different regioisomers were separated by
column chromatography on silica gel to give compounds 59 and 60 in
7 and 60% yield, respectively.
4-Ethynyl-2-fluoropyridine.112 Synthesized from 2-fluoro-4-iodo-
pyridine according to the representative procedure72 to afford the title
compound as colorless oil in 65% yield. 1H NMR (400 MHz, CDCl3):
δ 8.21 (d, 1H, J = 5.0 Hz), 7.24 (d, 1H, J = 5.0 Hz), 7.01 (s, 1H), 3.38
(s, 1H).
2-Chloro-4-ethynylpyridine.113 Synthesized from 2-chloro-4-iodo-
pyridine according to the representative procedure72 to afford the title
compound as a yellow solid in 60% yield. 1H NMR (400 MHz,
CDCl3): δ 8.38 (d, 1H, J = 5.0 Hz), 7.41 (s, 1H), 7.28 (dd, 1H, J = 5.0
Hz, 1.2 Hz), 3.37 (s, 1H).
4-Phenyl-1H-1,2,3-triazole (1).71 Synthesized from ethynylben-
zene71 and TMSN3 according to the general procedure to afford 1 as a
white solid in 65% yield. 1H NMR (400 MHz, CDCl3): δ 12.34 (br s,
1H), 8.00 (s, 1H), 7.84 (d, 2H, J = 7.6 Hz), 7.47 (t, 2H, J = 7.6 Hz),
7.43−7.37 (m, 1H).
1,2-Dichloro-3-ethynylbenzene.114 Synthesized from 1,2-dichloro-
3-iodobenzene according to the representative procedure72 to afford
1
the title compound as brown semisolid in 65% yield. H NMR (400
MHz, CDCl3): δ 7.46 (d, 2H, J = 8.0 Hz), 7.17 (t, 1H, J = 8.0 Hz),
3.38 (s, 1H).
4-(1H-1,2,3-Triazol-4-yl)pyridine (2).62 Synthesized from 4-ethy-
nylpyridine71 and TMSN3 according to the general procedure to afford
2 as a white solid in 40% yield. 1H NMR (400 MHz, CD3OD): δ 8.60
(d, 2H, J = 6.0 Hz), 8.41 (br s, 1H), 7.91 (d, 2H, J = 6.0 Hz).
3-(1H-1,2,3-Triazol-4-yl)pyridine (3).62 Synthesized from 3-ethy-
nylpyridine71 and TMSN3 according to the general procedure to afford
3 as a white solid in 60% yield. 1H NMR (400 MHz, CDCl3−
CD3OD): δ 8.96 (s, 1H), 8.51 (d, 1H, J = 4.8 Hz), 8.14 (d, 1H, J = 7.8
Hz), 7.99 (s, 1H), 7.42−7.38 (m, 1H).
1,4-Dichloro-2-ethynylbenzene.115 Synthesized from 1,4-dichloro-
2-iodobenzene according to the representative procedure72 to afford
1
the title compound as an off-white solid in 73% yield. H NMR (400
MHz, CDCl3): δ 7.52 (d, 1H, J = 2.52 Hz), 7.34 (d, 1H, J = 8.5 Hz),
7.26 (dd, 1H, J = 8.5 Hz, 2.5 Hz), 3.42 (s, 1H).
2-Ethynyl-N-methylaniline.116 Synthesized from 2-iodo-N-methyl-
aniline according to the representative procedure72 to afford the title
compound as a brown oil in 72% yield. 1H NMR (400 MHz, CDCl3):
δ 7.34 (dd, 1H, J = 7.6 Hz, 1.5 Hz), 7.28−7.23 (m, 1H), 6.65−6.58
(m, 2H), 4.68 (bs, 1H), 3.41 (s, 1H), 2.91 (s, 3H).
4-(4-Fluorophenyl)-1H-1,2,3-triazole (4).120 Synthesized from 1-
ethynyl-4-fluorobenzene71 and TMSN3 according to the general
2-Ethynylbenzaldehyde.117 Synthesized from 2-iodobenzaldehyde
according to the representative procedure72 to afford the title
1
procedure to afford 4 as a white solid in 62% yield. H NMR (400
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MHz, CDCl3): δ 11.65 (s, 1H), 7.95 (s, 1H), 7.85−7.80 (m, 2H),
7.21−7.14 (m, 2H).
2-Chloro-4-(1H-1,2,3-triazol-4-yl)pyridine (18). Synthesized from
2-chloro-4-ethynylpyridine71 and TMSN3 according to the general
procedure to afford 18 as a off-white solid in 60% yield, mp 211−213
4-(4-Chlorophenyl)-1H-1,2,3-triazole (5).62 Synthesized from 1-
chloro-4-ethynylbenzene71 and TMSN3 according to the general
1
°C. IR (film): ν 3121, 2171, 1604, 1454, 1070, 998, 838 cm−1. H
1
NMR (400 MHz, CD3OD): δ 8.45 (br s, 1H), 8.43 (d, 1H, J = 5.6
Hz), 7.96 (s, 1H), 7.86 (d, 1H, J = 5.38 Hz). 13C NMR (100 MHz,
CD3OD): δ 151.8, 149.9, 142.9, 142.0, 120.2, 119.0. ESI-TOF-HRMS:
m/z calcd for (M + H) C7H5ClN4, 181.0281; found, 181.0275.
2-Bromo-4-(1H-1,2,3-triazol-4-yl)pyridine (19). Synthesized from
2-bromo-4-ethynylpyridine71 and TMSN3 according to the general
procedure to afford 19 as a white solid in 61% yield, mp 206−209 °C.
procedure to afford 5 as a white solid in 65% yield. H NMR (400
MHz, CDCl3): δ 11.66 (s, 1H), 7.98 (s, 1H), 7.79 (d, 2H, J = 8.4 Hz),
7.46 (d, 2H, J = 8.4 Hz).
4-(p-Tolyl)-1H-1,2,3-triazole (6).62 Synthesized from 1-ethynyl-4-
methylbenzene71 and TMSN3 according to the general procedure to
afford 6 as a white solid in 72% yield. 1H NMR (400 MHz, CD3OD):
δ 8.07 (s, 1H), 7.69 (d, 2H, J = 7.9 Hz), 7.24 (d, 2H, J = 7.9 Hz), 2.35
(s, 3H).
1
IR (film): ν 3126, 2138, 1601, 1530, 1068, 834 cm−1. H NMR (400
4-(4-(Trifluoromethyl)phenyl)-1H-1,2,3-triazole (7).121 Synthe-
sized from 1-ethynyl-4-(trifluoromethyl)benzene71 and TMSN3
according to the general procedure to afford 7 as a white solid in
MHz, CD3OD): δ 8.44 (s, 1H), 8.39 (d, 1H, J = 5.4 Hz), 8.11 (s, 1H),
7.88 (d, 1H, J = 5.3 Hz). 13C NMR (100 MHz, DMSO): δ 151.6,
142.8, 142.6, 141.6, 124.0, 119.9. ESI-TOF-HRMS: m/z calcd for (M
+ H) C7H5BrN4, 224.9776; found, 224.9771.
1
55% yield. H NMR (400 MHz, CDCl3): δ 11.77 (s, 1H), 8.06 (s,
2-Fluoro-4-(1H-1,2,3-triazol-4-yl)pyridine (20). Synthesized from
4-ethynyl-2-fluoropyridine71 and TMSN3 according to the general
procedure to afford 20 as a off-white solid in 62% yield, mp 197−199
°C. IR (film): ν 3138, 2918, 2263, 1618, 1417, 1212, 1074, 830 cm−1.
1H NMR (400 MHz, CDCl3−CD3OD): δ 8.23 (d, 1H, J = 5.3 Hz),
8.03 (s, 1H), 7.62−7.51 (m, 1H), 7.37 (s, 1H). 13C NMR (100 MHz,
CD3OD): δ 164.5 (d, J = 237.2 Hz), 147.9, 147.7, 144.3 (d, J = 8.8
Hz), 118.1 (d, J = 3.7 Hz), 105.4, 105.0. ESI-TOF-HRMS: m/z calcd
for (M + H) C7H5FN4, 165.0576; found, 165.0583.
1H), 7.98 (d, 2H, J = 8.2 Hz), 7.74 (d, 2H, J = 8.2 Hz).
4-(3-Chlorophenyl)-1H-1,2,3-triazole (8).121 Synthesized from 1-
chloro-3-ethynylbenzene71 and TMSN3 according to the general
1
procedure to afford 8 as a white solid in 70% yield. H NMR (400
MHz, CDCl3): δ 11.79 (s, 1H), 8.0 (s, 1H), 7.86 (s, 1H), 7.74 (d, 2H,
J = 7.5 Hz), 7.45−7.36 (m, 2H).
4-(3-Bromophenyl)-1H-1,2,3-triazole (9).62 Synthesized from 1-
bromo-3-ethynylbenzene71 and TMSN3 according to the general
1
procedure to afford 9 as a white solid in 68% yield. H NMR (400
4-(1H-1,2,3-Triazol-4-yl)-2-(trifluoromethyl)pyridine (21). Synthe-
sized from 4-ethynyl-2-(trifluoromethyl)pyridine71 and TMSN3
according to the general procedure to afford 21 as a white solid in
51% yield, mp 211−213 °C. IR (film): ν 3147, 3098, 2885, 2160,
MHz, CDCl3): δ 11.87 (s, 1H), 8.02 (s, 1H), 8.0 (s, 1H), 7.78 (d, 1H,
J = 7.8 Hz), 7.53 (d, 1H, J = 8.1 Hz), 7.35 (t, 1H, J = 8.0 Hz).
3-(1H-1,2,3-Triazol-4-yl)benzonitrile (10).122 Synthesized from 3-
ethynylbenzonitrile71 and TMSN3 according to the general procedure
1
1
1618, 1321, 1112, 707 cm−1. H NMR (400 MHz, CD3OD): δ 8.76
to afford 10 as a off-white solid in 54% yield. H NMR (400 MHz,
(d, 1H, J = 5.0 Hz), 8.53 (br s, 1H), 8.29 (s, 1H), 8.11 (d, 1H, J = 5.2
Hz). 13C NMR (100 MHz, CD3OD): δ 150.4, 148.4 (q, J = 34.16),
143.1, 140.6, 122.7, 121.6 (q, J = 274.7, 116.6 (q, J = 2.89). ESI-TOF-
HRMS: m/z calcd for (M + H) C8H5F3N4, 215.0545; found,
215.0548.
CDCl3): δ 11.73 (s, 1H), 8.16 (s, 1H), 8.09 (d, 1H, J = 7.8 Hz), 8.04
(s, 1H), 7.71−7.65 (m, 1H), 7.60 (t, 1H, J = 7.8 Hz).
4-(3-(Trifluoromethyl)phenyl)-1H-1,2,3-triazole (11).123 Synthe-
sized from 1-ethynyl-3-(trifluoromethyl)benzene71 and TMSN3
according to the general procedure to afford 11 as a white solid in
4-(2-Bromophenyl)-1H-1,2,3-triazole (22).126 Synthesized from 1-
bromo-2-ethynylbenzene71 and TMSN3 according to the general
1
55% yield. H NMR (400 MHz, CDCl3): δ 11.85 (s, 1H), 8.12 (s,
1H), 8.06 (s, 1H), 8.04 (d, 1H, J = 7.8 Hz), 7.67 (d, 1H, J = 7.9 Hz),
7.61 (t, 1H, J = 7.8 Hz).
1
procedure to afford 22 as a white solid in 55% yield. H NMR (400
MHz, CD3OD): δ 8.26 (s, 1H), 7.76 (d, 1H, J = 7.4 Hz), 7.73 (d, 1H,
J = 8.0 Hz), 7.46 (t, 1H, J = 7.5 Hz), 7.31 (t, 1H, J = 7.8 Hz).
2-(1H-1,2,3-Triazol-4-yl)phenol (23).127 Synthesized from 2-
ethynylphenol71 and TMSN3 according to the general procedure to
afford 23 as a white solid in 32% yield. 1H NMR (400 MHz, CDCl3):
δ 11.77 (s, 1H), 8.10 (s, 1H), 7.63 (d, 1H, J = 7.6 Hz), 7.35−7.28 (m,
1H), 7.10 (d, 1H, J = 8.4 Hz), 7.0 (t, 1H, J = 7.8 Hz).
4-(3-Ethylphenyl)-1H-1,2,3-triazole (12). Synthesized from 1-ethyl-
3-ethynylbenzene71 and TMSN3 according to the general procedure to
afford 12 as a yellow oil in 71% yield. IR (film): ν 3136, 2964, 1613,
1476, 796 cm−1. 1H NMR (400 MHz, CDCl3): δ 11.90 (s, 1H), 8.0 (s,
1H), 7.70 (br s, 1H), 7.65 (d, 1H, J = 7.5 Hz), 7.40 (t, 1H, J = 7.7 Hz),
7.26 (d, 1H, J = 7.7 Hz). 13C NMR (100 MHz, CDCl3): δ 147.0,
145.2, 129.6, 129.1 128.5, 125.8, 123.6, 29.0, 15.6. ESI-TOF-HRMS:
m/z calcd for (M + H) C10H11N3, 174.1031; found, 174.1024.
4-(3-Nitrophenyl)-1H-1,2,3-triazole (13).124 Synthesized from 1-
ethynyl-3-nitrobenzene71 and TMSN3 according to the general
4-(2-Chlorophenyl)-1H-1,2,3-triazole (24).121 Synthesized from 1-
chloro-2-ethynylbenzene71 and TMSN3 according to the general
1
procedure to afford 24 as a white solid in 59% yield. H NMR (400
MHz, CDCl3): δ 12.10 (s, 1H), 8.29 (s, 1H), 7.95 (d, 1H, J = 5.9 Hz),
7.53 (dd, 1H, J = 7.6 Hz, 1.5 Hz), 7.43−7.33 (m, 2H).
1
procedure to afford 13 as a off-white solid in 58% yield. H NMR
4-(o-Tolyl)-1H-1,2,3-triazole (25).62 Synthesized from 1-ethynyl-2-
methylbenzene71 and TMSN3 according to the general procedure to
afford 25 as a white solid in 60% yield. 1H NMR (400 MHz, CDCl3):
δ 7.89 (s, 1H), 7.62 (d, 1H, J = 6.8 Hz), 7.37−7.29 (m, 3H), 2.51
(s,3H)
(400 MHz, CDCl3): δ 8.60 (s, 1H), 8.23−8.11 (m, 2H), 8.02 (s, 1H),
7.62 (t, 1H, J = 8.0 Hz).
4-(m-Tolyl)-1H-1,2,3-triazole (14).62 Synthesized from 1-ethynyl-3-
methylbenzene71 and TMSN3 according to the general procedure to
afford 14 as a white solid in 62% yield. 1H NMR (400 MHz, CDCl3):
δ 11.88 (s, 1H), 8.0 (s, 1H), 7.68 (s, 1H), 7.64 (d, 1H, J = 7.6 Hz),
7.37 (t, 1H, J = 7.7 Hz), 7.23 (d, 1H, J = 7.6 Hz), 2.45 (s, 3H).
4-(3-Fluorophenyl)-1H-1,2,3-triazole (15).125 Synthesized from 1-
ethynyl-3-fluorobenzene71 and TMSN3 according to the general
2-(1H-1,2,3-Triazol-4-yl)aniline (26). Synthesized from 2-ethynyla-
niline71 and TMSN3 according to the general procedure to afford 26 as
a white solid in 58% yield, mp 128−130 °C. IR (film): ν 3377, 3326,
2528, 1483, 1297, 748 cm−1. 1H NMR (400 MHz, CD3OD): δ 8.10 (s,
1H), 7.54 (d, 1H, J = 7.2 Hz), 7.10 (t, 1H, J = 7.8 Hz), 6.84 (d, 1H, J =
7.8 Hz), 6.73 (t, 1H, J = 7.8 Hz). 13C NMR (100 MHz, CD3OD): δ
145.1, 128.7, 127.8, 117.3, 116.5, 113.8. ESI-TOF-HRMS: m/z calcd
for (M + H) C8H8N4, 161.0827; found, 161.0834.
1
procedure to afford 15 as a white solid in 51% yield. H NMR (400
MHz, CD3OD): δ 8.21 (s, 1H), 7.67 (d, 1H, J = 7.8 Hz), 7.63−7.58
(m, 1H), 7.50−7.43 (m, 1H), 7.10 (td, 1H, J = 8.5 Hz, 2.5 Hz).
3-(1H-1,2,3-Triazol-4-yl)phenol (16).62 Synthesized from 3-ethy-
nylphenol71 and TMSN3 according to the general procedure to afford
16 as a light-yellow powder in 20% yield. 1H NMR (400 MHz,
CD3OD): δ 8.08 (s, 1H), 7.32−7.22 (m, 3H), 6.83−6.77 (m 1H).
3-(1H-1,2,3-Triazol-4-yl)aniline (17).62 Synthesized from 3-ethyny-
laniline71 and TMSN3 according to the general procedure to afford 17
as a light-yellow powder in 55% yield. 1H NMR (400 MHz, CD3OD):
δ 8.06 (s, 1H), 7.22−7.12 (m, 3H), 6.77−6.72 (m, 1H).
4-(2-Fluorophenyl)-1H-1,2,3-triazole (27).121 Synthesized from 1-
ethynyl-2-fluorobenzene71 and TMSN3 according to the general
1
procedure to afford 27 as a white solid in 52% yield. H NMR (400
MHz, CDCl3): δ 12.24 (s, 1H), 8.19−8.05 (m, 2H), 7.43−7.36
(m,1H), 7.32−7.26 (m,1H), 7.25−7.18 (m, 1H).
4-(2-Ethylphenyl)-1H-1,2,3-triazole (28). Synthesized from 1-ethyl-
2-ethynylbenzene71 and TMSN3 according to the general procedure to
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afford 28 as yellow oil in 73% yield. IR (film): ν 3139, 2965, 2931,
4-(5-Chloro-2-methylphenyl)-1H-1,2,3-triazole (36). Synthesized
from 4-chloro-2-ethynyl-1-methylbenzene71 and TMSN3 according to
the general procedure to afford 36 as a white solid in 52% yield, mp
122−124 °C. IR (film): ν 3091, 2869, 2752, 2604, 1480, 1074, 880,
1
2873, 1465, 1443, 968, 761 cm−1. H NMR (400 MHz, CDCl3): δ
11.70 (br s, 1H), 7.86 (s, 1H), 7.53 (d, 1H, J = 7.6 Hz), 7.42−7.35 (m,
2H), 7.33−7.29 (m, 1H), 2.83 (q, 2H, J = 7.5 Hz), 1.23 (t, 3H, J = 7.5
Hz). 13C NMR (100 MHz, CDCl3): δ 142.6, 129.7, 129.3, 129.1,
128.5, 126.1, 26.6, 15.5. ESI-TOF-HRMS: m/z calcd for (M + H)
C10H11N3, 174.1031; found, 174.1039.
1
802 cm−1. H NMR (400 MHz, CDCl3−CD3OD): δ 7.80 (s, 1H),
7.60 (br s, 1H), 7.25−7.16 (m, 2H), 2.40 (s, 3H). 13C NMR (100
MHz, CD3OD): δ 134.5, 132.1, 131.3, 128.2, 127.8, 19.5. ESI-TOF-
HRMS: m/z calcd for (M + H) C9H8ClN3, 194.0485; found,
194.0480.
4-(2-(Trifluoromethyl)phenyl)-1H-1,2,3-triazole (29).63 Synthe-
sized from 1-ethynyl-2-(trifluoromethyl)benzene71 and TMSN3
according to the general procedure to afford 29 as a white solid in
4-(2,5-Dichlorophenyl)-1H-1,2,3-triazole (37). Synthesized from
1,4-dichloro-2-ethynylbenzene71 and TMSN3 according to the general
procedure to afford 37 as a white solid in 70% yield, mp 175−176 °C.
1
46% yield. H NMR (400 MHz, CDCl3): δ 11.83 (s, 1H), 7.94 (s,
1H), 7.83 (d, 1H, J = 8.0 Hz), 7.75 (br s, 1H), 7.67 (t, 1H, J = 7.6 Hz),
7.57 (t, 1H, J = 7.7 Hz).
1
IR (film): ν 3161, 3061, 2095, 1466, 1054, 803 cm−1. H NMR (400
MHz, CDCl3−CD3OD): δ 8.35 (s, 1H), 7.96 (s, 1H), 7.49 (d, 1H, J =
8.6 Hz), 7.35 (dd, 1H, J = 7.3 Hz, 2.3 Hz). 13C NMR (100 MHz,
CD3OD): δ 142.1, 132.7, 131.4, 130.7, 129.9, 129.3, 129.0. ESI-TOF-
HRMS: m/z calcd for (M + H) C8H5F3Cl2N3, 213.9939; found,
213.9945.
2-(1H-1,2,3-Triazol-4-yl)benzaldehyde (30). Synthesized from 2-
ethynylbenzaldehyde71 and TMSN3 according to the general
procedure to afford 30 as a off-white solid in 65% yield, mp 134−
137 °C. IR (film): ν 3132, 3089, 2877, 2168, 1684, 1075, 758 cm−1. 1H
NMR (400 MHz, CDCl3): δ 12.32 (br s, 1H), 10.40 (s, 1H), 8.09 (d,
1H, J = 7.8 Hz), 8.02 (s, 1H), 7.76−7.69 (m, 2H), 7.63−7.57 (m, 1H).
13C NMR (100 MHz, CD3OD): δ 192.3, 134.1, 133.6, 130.0, 129.2,
128.5, 128.1, 127.6, 125.9. ESI-TOF-HRMS: m/z calcd for (M + H)
C9H7N3O, 174.0667; found, 174.0675.
4-(2,3-Dichlorophenyl)-1H-1,2,3-triazole (38). Synthesized from
1,2-dichloro-3-ethynylbenzene71 and TMSN3 according to the general
procedure to afford 38 as a white solid in 71% yield, mp 168−170 °C.
IR (film): ν 3170, 3071, 2642, 2112, 1444, 1073, 779 cm−1. 1H NMR
(400 MHz, CD3OD): δ 8.32 (s, 1H), 7.83 (d, 1H, J = 7.7 Hz), 7.56 (d,
1H, J = 7.7 Hz), 7.38 (t, 1H, J = 7.7 Hz). 13C NMR (100 MHz,
CD3OD): δ 142.6, 133.5, 131.5, 129.9, 128.5, 127.6. ESI-TOF-HRMS:
m/z calcd for (M + H) C8H5Cl2N3, 213.9939; found, 213.9933.
4-(3-Chloro-2-methylphenyl)-1H-1,2,3-triazole (39). Synthesized
from 1-chloro-3-ethynyl-2-methylbenzene71 and TMSN3 according to
the general procedure to afford 39 as a white solid in 51% yield, mp
135−137 °C. IR (film): ν 3162, 2907, 2139, 1572, 1438, 1067, 801
N-Methyl-2-(1H-1,2,3-triazol-4-yl)aniline (31). Synthesized from 2-
ethynyl-N-methylaniline71 and TMSN3 according to the general
procedure to afford 31 as a off-white solid in 72% yield, mp 129−
131 °C. IR (film): ν 3405, 3133, 2901, 2268, 1493, 1285, 751 cm−1. 1H
NMR (400 MHz, CD3OD): δ 8.11 (s, 1H), 7.56 (d, 1H, J = 7.3 Hz),
7.23 (t, 1H, J = 7.7 Hz), 6.76 (d, 1H, J = 8.3 Hz), 6.71 (t, 1H, J = 7.7
Hz), 2.93 (s, 3H). 13C NMR (100 MHz, CD3OD): δ 146.8, 129.2,
127.7, 115.6, 113.3, 110.2, 29.4. ESI-TOF-HRMS: m/z calcd for (M +
H) C9H10N4, 175.0984; found, 175.0990.
1
cm−1. H NMR (400 MHz, CDCl3−CD3OD): δ 7.80 (s, 1H), 7.44−
7.38 (m, 2H), 7.20 (t, 1H, J = 7.9 Hz). 13C NMR (100 MHz,
CD3OD): δ 135.1, 134.1, 132.8, 129.1, 128.0, 126.6, 16.5. ESI-TOF-
HRMS: m/z calcd for (M + H) C9H8ClN3, 194.0485; found,
194.0494.
4-(2-Methoxyphenyl)-1H-1,2,3-triazole (32).62 Synthesized from 1-
ethynyl-2-methoxybenzene71 and TMSN3 according to the general
1
procedure to afford 32 as a white solid in 38% yield. H NMR (400
MHz, CDCl3): δ 12.52 (s, 1H), 8.15 (s, 1H), 7.90 (br s, 1H), 7.43−
7.37 (m, 1H), 7.14−7.05 (m, 2H), 4.04 (s, 3H).
3-Methyl-2-(1H-1,2,3-triazol-4-yl)aniline (40). To a solution of 41
(40 mg, 0.15 mmol) in dioxane (2 mL), N2H4·H2O75 (38 mg, 0.75
mmol) was added dropwise at room temperature. The mixture was
refluxed for 2.5 h, cooled to room temperature, filtered, and
evaporated. The residue was dissolved in CH2Cl2 (5 mL), and the
solution was washed with brine (2 × 4 mL) and dried over Na2SO4.
Evaporation gave 40 as a white solid in 50% yield, mp 146−148 °C. IR
(film): ν 3354, 3123, 2922, 1583, 1466, 962, 771 cm−1. 1H NMR (400
MHz, CDCl3): δ 7.82 (s, 1H), 7.13 (t, 1H, J = 7.8 Hz), 6.74 (d, 1H, J
= 7.4 Hz), 6.70 (d, 1H, J = 8.0 Hz), 2.21 (s, 3H). 13C NMR (100
MHz, CD3OD): δ 146.3, 137.8, 129.2, 119.5, 113.4, 19.4. ESI-TOF-
HRMS: m/z calcd for (M + H) C9H10N4, 175.0984; found, 175.0978.
2,2,2-Trifluoro-N-(3-methyl-2-(1H-1,2,3-triazol-4-yl)phenyl)-
acetamide (41). Synthesized from N-(2-ethynyl-3-methylphenyl)-
2,2,2-trifluoroacetamide71 and TMSN3 according to the general
procedure to afford 41 as a white solid in 45% yield, mp 181−183
°C. IR (film): ν 3162, 2938 1698, 1555, 1458, 1213, 1184, 1135, 1066,
771 cm−1. 1H NMR (400 MHz, CD3OD): δ 7.90 (s, 1H), 7.53 (d, 1H,
J = 7.7 Hz), 7.40 (t, 1H, J = 7.7 Hz), 7.33 (d, 1H, J = 7.7 Hz), 2.30 (s,
3H). 13C NMR (100 MHz, CD3OD): δ 156.0 (q, J = 40.1), 138.7,
133.8, 129.1, 128.9, 125.3, 122.9, 116.0, (q, J = 287.5), 19.7. ESI-TOF-
HRMS: m/z calcd for (M + H) C11H9F3N4O, 271.0807; found,
271.0808.
2-(2-(1H-1,2,3-Triazol-4-yl)phenyl)ethanamine Hydrochloride
(33). Compound 74 (0.20 mmol) was dissolved in diethyl ether (1
mL), and 1 mL of HCl solution (5 N−6 N in isopropyl alcohol)73 was
added dropwise to the stirred solution. The mixture was stirred at
ambient temperature for 36 h. The resulting crystals were filtered and
washed with diethyl ether to give 33 as a white solid in quantitive yield,
mp 202−205 °C, 90% yield. IR (film): ν 3121, 2897, 2248, 1474,
1
1142, 763 cm−1. H NMR (400 MHz, CD3OD): δ 8.11 (s, 1H), 7.60
(d, 1H, J = 6.9 Hz), 7.47−7.39 (m, 3H), 3.28−3.22 (m, 2H), 3.21−
3.16 (m, 2H). 13C NMR (100 MHz, CD3OD): δ 143.5, 135.3, 130.4,
130.1, 129.8, 127.5, 127.4, 127.3, 40.2, 30.9. ESI-TOF-HRMS: m/z
calcd for (M + H) C10H12N4, 189.1140; found, 189.1145.
3-(2-(1H-1,2,3-Triazol-4-yl)phenyl)propanoic Acid (34). The
mixture of aldehyde 30 (80 mg, 0.46 mmol) and Meldrum’s acid74
(67 mg, 0.46 mmol) in TEAF (1.0 mL) was stirred at 95−100 °C for 3
h and then cooled to room temperature. Some ice and water (2−3 g)
was added to the cold reaction mixture before adjusting its pH to 1
with 6 N HCl (1−2 mL) and letting it crystallize at 5−10 °C for a day.
After filtering, the crystals were washed with cold water and pentane to
afford 34 as a white solid in 45% yield, mp 119−121 °C. IR (film): ν
1
3124, 2857, 1704, 1407, 1212, 975, 765 cm−1. H NMR (400 MHz,
CD3OD): δ 7.97 (br s, 1H), 7.54−7.47 (m,1H), 7.41−7.27 (m, 3H),
3.14−3.03 (m, 2H), 2.59−2.51 (m, 2H). 13C NMR (100 MHz,
CD3OD): δ 175.2, 139.0, 129.5, 126.2, 34.8, 28.5. ESI-TOF-HRMS:
m/z calcd for (M + H) C11H11N3O2, 218.0930; found, 218.0940.
4-Chloro-2-(1H-1,2,3-triazol-4-yl)phenol (35). Synthesized from 4-
chloro-2-ethynylphenol71 and TMSN3 according to the general
procedure to afford 35 as a off-white solid in 60% yield, mp 212−
4-Methyl-3-(1H-1,2,3-triazol-4-yl)aniline (42). Synthesized from 3-
ethynyl-4-methylaniline71 and TMSN3 according to the general
procedure to afford 42 as yellow semisolid in 40% yield. IR (film):
ν 3349, 3136, 2925, 1619, 1493, 1242, 1124, 817 cm−1. 1H NMR (400
MHz, CD3OD): δ 7.86 (s, 1H), 7.11 (d, 1H, J = 8.1 Hz), 7.0 (d, 1H, J
= 2.4 Hz), 6.69 (dd, 1H, J = 5.6 Hz, 2.5 Hz), 2.38 (s, 3H). 13C NMR
(100 MHz, CD3OD): δ 144.6, 131.3, 128.6, 125.6, 116.2, 116.0, 18.8.
ESI-TOF-HRMS: m/z calcd for (M + H) C9H10N4, 175.0984; found,
175.0980.
1
214 °C. IR (film): ν 3140, 2923, 2280, 1474, 1230, 813 cm−1. H
NMR (400 MHz, CD3OD): δ 8.30 (s, 1H), 7.90 (s, 1H), 7.20 (dd,
1H, J = 7.1 Hz, 2.5 Hz), 6.92 (d, 1H, J = 8.8 Hz). 13C NMR (100
MHz, CD3OD): δ 153.3, 128.6, 126.3, 124.1, 117.3. ESI-TOF-HRMS:
m/z calcd for (M + H) C8H6ClN3O, 196.0278; found, 196.0277.
1,5-Diphenyl-1H-1,2,3-triazole (43).80,81 To a solution of EtMgBr
in THF (1.0 M, 2 mL), phenylacetylene (0.22 mL, 2 mmol) was added
at room temperature. The reaction mixture was heated to 50 °C for 15
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min. After cooling the mixture to room temperature, a solution of
phenylazide (0.24 g, 2 mmol) in THF (0.8 mL) was added. The
resulting solution was stirred at room temperature for 30 min and then
heated to 50 °C for 1 h before quenching with saturated NH4Cl (2
mL). The layers were separated, and the aqueous layer was extracted
with CH2Cl2 (3 × 3 mL). The combined organic layers were dried
over Na2SO4 and concentrated under reduced pressure. The residue
was purified by flash column chromatography on silica gel to afford 43
as a white solid in 60% yield. 1H NMR (400 MHz, CDCl3): δ 7.89 (s,
1H), 7.48−7.44 (m, 3H), 7.42−7.35 (m, 5H), 7.27−7.24 (m, 2H).
5-(4-Fluorophenyl)-1-phenyl-1H-1,2,3-triazole (44). Using the
same procedure80,81 as that used for 43, starting from 1-ethynyl-4-
fluorobenzene (115 mg, 1 mmol) in the presence of a solution of
EtMgBr (1.0 M, 1 mL), product 44 was obtained as a white solid in
55% yield, mp 100−103 °C. IR (film): ν 3122, 1893, 1492, 1229,
2-Ethyl-4-phenyl-2H-1,2,3-triazole (52).86 Synthesized from 4-
phenyl-1H-1,2,3-triazole and diethyl carbonate according to the
general procedure of N-alkylation82 to afford 51 as a colorless oil,
19% yield. 1H NMR (400 MHz, CDCl3): δ 7.85 (s, 1H), 7.81 (d, 2H, J
= 7.5 Hz), 7.45 (t, 2H, J = 7.5 Hz), 7.40−7.34 (m, 1H), 4.54 (q, 2H, J
= 7.3 Hz), 1.63 (t, 3H, J = 7.4 Hz).
1-Ethyl-4-phenyl-1H-1,2,3-triazole (53).86 Synthesized from 4-
phenyl-1H-1,2,3-triazole and diethyl carbonate according to the
general procedure of N-alkylation82 to afford 52 as a white solid,
1
55% yield. H NMR (400 MHz, CDCl3): δ 7.86 (d, 2H, J = 7.5 Hz),
7.78 (s, 1H), 7.45 (t, 2H, J = 7.6 Hz), 7.36 (t, 1H, J = 7.5 Hz), 4.49 (q,
2H, J = 7.4 Hz), 1.67 (t, 3H, J = 7.4 Hz).
1-Butyl-5-phenyl-1H-1,2,3-triazole (54).78,79 Synthesized accord-
ing to a previously reported procedure.78,79 Oil, 13% yield. H NMR
1
(400 MHz, CDCl3): δ 7.71 (s, 1H), 7.55−7.49 (m, 3H), 7.42−7.39
(m, 2H), 4.37 (t, 2H, J = 7.4 Hz), 1.84 (quintet, 2H, J = 7.4 Hz), 1.30
(sextet, 2H, J = 7.5 Hz), 0.88 (t, 3H, J = 7.4 Hz).
1
1160, 830, 768 cm−1. H NMR (400 MHz, CD3OD): δ 7.97 (s, 1H),
7.54−7.47 (m, 3H), 7.39−7.35 (m, 2H), 7.33−7.28 (m, 2H), 7.13−
7.07 (m, 2H). 13C NMR (100 MHz, CD3OD): δ 163.3 (d, J = 249.7
Hz), 137.5, 136,3, 132.7, 130.7 (d, J = 8.7 Hz), 129.5, 129.3, 125.4,
122.6 (d, J = 3.6 Hz), 115.6 (d, J = 22.4 Hz). ESI-TOF-HRMS: m/z
calcd for (M + H) C14H10FN3, 240.0937; found, 240.0931.
1-Phenyl-5-(m-tolyl)-1H-1,2,3-triazole (45). Using the same
procedure80,81 as that used for 43, starting from 1-ethynyl-3-
methylbenzene (116 mg, 1 mmol) in the presence of a solution of
EtMgBr (1.0 M, 1 mL), product 45 was obtained as a white solid in
35% yield, mp 109−113 °C. IR (film): ν 2920, 1597, 1499, 1456,
1234, 1050, 763 cm−1. 1H NMR (400 MHz, CD3OD): δ 7.97 (s, 1H),
7.56−7.48 (m, 3H), 7.41−7.36 (m, 2H), 7.25−7.20 (m, 2H), 7.14 (br
s, 1H), 7.06−7.01 (m, 1H), 2.28 (s, 3H). 13C NMR (100 MHz,
CD3OD): δ 138.7, 138.6, 136.5, 132.5, 129.8, 129.4, 129.2, 128.9,
128.4, 126.2, 125.5, 125.3, 19.8. ESI-TOF-HRMS: m/z calcd for (M +
H) C15H13N3, 236.1188; found, 236.1184.
1-Butyl-4-phenyl-1H-1,2,3-triazole (55).78,79 Synthesized accord-
ing to a previously reported procedure.78,79 White solid, 40% yield. 1H
NMR (400 MHz, CDCl3): δ 7.88−7.84 (m, 2H), 7.77 (s, 1H), 7.48−
7.42 (m, 2H), 7.38−7.33 (m, 1H), 4.44 (t, 2H, J = 7.5 Hz), 1.97
(quintet, 2H, J = 7.5 Hz), 1.43 (sextet, 2H, J = 7.6 Hz), 1.0 (t, 3H, J =
7.5 Hz).
1-Benzyl-5-phenyl-1H-1,2,3-triazole (56).78,79 Synthesized accord-
ing to a previously reported procedure.78,79 Off-white solid, 15% yield.
1H NMR (400 MHz, CDCl3): δ 7.77 (s, 1H), 7.48−7.41 (m, 3H),
7.35−7.25 (m, 5H), 7.13−7.08 (m, 2H), 5.58 (s, 2H).
1-Benzyl-4-phenyl-1H-1,2,3-triazole (57).78,79 Synthesized accord-
ing to a previously reported procedure.78,79 White solid, 45% yield. 1H
NMR (400 MHz, CDCl3): δ 7.85−7.80 (m, 2H), 7.68 (s, 1H), 7.45−
7.38 (m, 5H), 7.37−7.31 (m, 3H), 5.61 (s, 2H).
4-Methyl-5-phenyl-1H-1,2,3-triazole (58).76 Synthesized according
to a previously reported procedure.76 White solid, 30% yield. 1H NMR
(400 MHz, CDCl3): δ 11.53 (s, 1H), 7.74 (d, 2H, J = 7.7 Hz), 7.52−
7.47 (m, 2H), 7.44−7.39 (m, 1H), 2.57 (s,3H)
5-(3-Chlorophenyl)-1-phenyl-1H-1,2,3-triazole (46). Using the
same procedure80,81 as that used for 43, starting from 1-chloro-3-
ethynylbenzene (123 mg, 1 mmol) in the presence of a solution of
EtMgBr (1.0 M, 1 mL), product 46 was obtained as a white solid in
70% yield, mp 85−87 °C. IR (film): ν 2920, 1597, 1499, 1456, 1234,
1050, 763 cm−1. 1H NMR (400 MHz, CD3OD): δ 8.01 (s, 1H), 7.55−
7.47 (m, 3H), 7.40−7.35 (m, 3H), 7.34−7.29 (m, 2H), 7.19−7.15 (m,
1H). 13C NMR (100 MHz, CD3OD): δ 137.0, 136.2, 134.4, 133.0,
130.1, 129.7, 129.4, 129.1, 128.3, 126.9, 125.4. ESI-TOF-HRMS: m/z
calcd for (M + H) C14H10ClN3, 256.0641; found, 256.0644.
5-(3-Chlorophenyl)-1-methyl-1H-1,2,3-triazole (59). Synthesized
from 8 and iodomethane according to the general procedure of N-
methylation82 to afford 59 as a yellow oil, 7% yield. IR (film): ν 2926,
1
2854, 1732, 1571, 1473, 1454, 1245, 1098, 790 cm−1. H NMR (400
MHz, CD3OD): δ 7.86 (s, 1H), 7.64−7.62 (m, 1H), 7.56−7.49 (m,
3H), 4.12 (s, 3H). 13C NMR (100 MHz, CD3OD): δ 136.0, 133.7,
131.7, 130.6, 129.8, 129.6, 128.1, 30.6. ESI-TOF-HRMS: m/z calcd for
(M + H) C9H8ClN3, 194.0485; found, 194.0478.
5-(2-Methoxyphenyl)-1-phenyl-1H-1,2,3-triazole (47).80,81 Using
the same procedure80,81 as that used for 43, starting from 1-ethynyl-2-
methoxybenzene (131 mg, 1 mmol) in the presence of a solution of
EtMgBr (1.0 M, 1 mL), product 47 was obtained as a white solid in
4-(3-Chlorophenyl)-1-methyl-1H-1,2,3-triazole (60). Synthesized
from 8 and iodomethane according to the general procedure of N-
methylation82 to afford 60 as a white solid, mp 104−107 °C, 49%
1
1
yield. IR (film): ν 3142, 2949, 2158, 1421, 1078, 887 cm−1. H NMR
50% yield. H NMR (400 MHz, CDCl3): δ 7.85 (s, 1H), 7.44−7.33
(m, 5H), 7.28−7.24 (m, 2H), 7.01 (td, 1H, J = 7.5 Hz, 1.0 Hz), 6.86
(d, 1H, J = 8.4 Hz), 3.44 (s, 3H).
(400 MHz, CD3OD): δ 8.26 (s, 1H), 7.80 (br s, 1H), 7.69 (d, 1H, J =
7.7 Hz), 7.38 (t, 1H, J = 7.9 Hz), 7.31 (br.d, 1H, J = 7.9 Hz), 4.13 (s,
3H). 13C NMR (100 MHz, CD3OD): δ 146.1, 134.5, 132.4, 130.2,
127.7, 125.0, 123.5, 122.3, 35.7. ESI-TOF-HRMS: m/z calcd for (M +
H) C9H8ClN3, 194.0485; found, 194.0484.
1-Methyl-5-phenyl-1H-1,2,3-triazole (48).83,84 Synthesized from 4-
phenyl-1H-1,2,3-triazole and dimethyl carbonate according to the
general procedure of N-alkylation82 to afford 48 as a light yellow oil,
7% yield. 1H NMR (400 MHz, CDCl3): δ 7.75 (s, 1H), 7.56−7.49 (m,
3H), 7.46−7.43 (m, 2H), 4.11(s, 3H).
1-Phenyl-1H-1,2,3-triazole (61).77 Synthesized according to a
previously reported procedure.77 Off-white solid, 71% yield. 1H
NMR (400 MHz, CDCl3): δ 8.02 (d, 1H, J = 1.0 Hz), 7.88 (d, 1H, J =
1.0 Hz), 7.80−7.76 (m, 2H), 7.59−7.54 (m, 2H), 7.50−7.45 (m, 1H).
4-Benzyl-1H-1,2,3-triazole (62).128 Synthesized from prop-2-yn-1-
ylbenzene71 and TMSN3 according to the general procedure to afford
62 as a light-yellow solid in 45% yield. 1H NMR (400 MHz, CDCl3): δ
11.78 (s, 1H), 7.48 (s, 1H), 7.37−7.31 (m, 2H), 7.30−7.24 (m, 3H),
4.13 (s, 2H).
1-Methyl-4-phenyl-1H-1,2,3-triazole (49).83,84 Synthesized from 4-
phenyl-1H-1,2,3-triazole and dimethyl carbonate according to the
general procedure of N-alkylation82 to afford 49 as a white solid, 50%
1
yield. H NMR (400 MHz, CDCl3): δ 7.86−7.83 (m, 2H), 7.77 (s,
1H), 7.47−7.43 (m, 2H), 7.38−7.33 (m, 1H), 4.18 (s, 3H).
2-Methyl-4-phenyl-2H-1,2,3-triazole (50).83,84 Synthesized from 4-
phenyl-1H-1,2,3-triazole and dimethyl carbonate according to the
general procedure of N-alkylation82 to afford 50 as a white solid, 20%
4-((Phenylthio)methyl)-1H-1,2,3-triazole (63).120 Synthesized from
yield. H NMR (400 MHz, CDCl3): δ 7.85 (s, 1H), 7.80 (d, 2H, J =
phenyl(prop-2-yn-1-yl)sulfane71 and TMSN3 according to the general
1
1
7.5 Hz), 7.45 (t, 2H, J = 7.5 Hz), 7.40−7.35 (m, 1H), 4.27 (s, 3H).
1-Ethyl-5-phenyl-1H-1,2,3-triazole (51).86 Synthesized from 4-
phenyl-1H-1,2,3-triazole and diethyl carbonate according to the
general procedure of N-alkylation82 to afford 51 as a yellow oil, 6%
procedure to afford 63 as an off-white solid in 55% yield. H NMR
(400 MHz, CDCl3): δ 11.73 (s, 1H), 7.57 (s, 1H), 7.40−7.35 (m, 2H),
7.33−7.27 (m, 2H), 7.26−7.22 (m, 1H), 4.25 (s, 2H).
4-(Cyclohex-1-en-1-yl)-1H-1,2,3-triazole (64).129 Synthesized from
1-ethynylcyclohex-1-ene71 and TMSN3 according to the general
1
yield. H NMR (400 MHz, CDCl3): δ 7.72 (s, 1H), 7.56−7.48 (m,
1
3H), 7.44−7.39 (m, 2H), 4.43 (q, 2H, J = 7.3 Hz), 1.51 (t, 3H, J = 7.3
procedure to afford 64 as a white solid in 65% yield. H NMR (400
Hz).
MHz, CDCl3): δ 11.65 (s, 1H), 7.70 (s, 1H), 6.43 (br s, 1H), 2.51−
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2.44 (m, 2H), 2.28−2.21 (m, 2H), 1.85−1.77 (m, 2H), 1.76−1.67 (m,
2H).
Assay. The assay was performed in 96-well flat bottom plates
seeded with 2 × 105 cells in a final volume of 200 μL of IMDM
(Iscove’s Modified Dulbecco’s Medium, Invitrogen) (80 μM L-
tryptophan) supplemented with 2% FCS and a titration of the
compound ranging from 0.001 to 100 μM. The cells were incubated at
37 °C. Kynurenine production in control wells without inhibitor was
monitored periodically during the assay using 4-(dimethylamino)-
benzaldehyde (pDMAB) as described below. The reaction was
stopped before 50% of the initial amount of tryptophan was converted
into kynurenine to ensure linearity of the reaction. The plates were
then centrifuged for 10 min at 300g, and 150 μL of the supernatant
were collected for HPLC analysis of tryptophan and kynurenine
concentrations. Proteins were precipitated by mixing 50 μL of
supernatant with an equal volume of TCA 6%. After centrifugation, 70
μL of supernatant were collected, diluted in an equal volume of water,
and injected into the HPLC system (C18 column). Tryptophan was
detected at an absorption wavelength of 280 nm and kynurenine at
360 nm.
tert-Butyl N-{2-[2-(1H-1,2,3-Triazol-4-yl)phenyl]ethyl}carbamate
(74). Synthesized from tert-butyl 2-ethynylphenethylcarbamate71 and
TMSN3 according to the general procedure to afford 74 as yellow oil
in 45% yield. IR (film): ν 3137, 2977, 2932, 1681, 1514, 1366, 1167,
1
765 cm−1. H NMR (400 MHz, CDCl3): δ 7.90 (s, 1H), 7.56−7.52
(m, 1H), 7.42−7.31 (m, 3H), 4.98 (br s, 1H) 3.40 (q, 2H, J = 6.8 Hz),
2.99 (t, 2H, J = 7.1 Hz), 1.45 (s, 9H). 13C NMR (100 MHz, CDCl3): δ
156.7, 137.2, 130.8, 129.9, 128.8, 126.8, 79.7, 41.8, 34.0, 28.5. ESI-
TOF-HRMS: m/z calcd for (M + H) C15H20N4O2, 289.1664; found,
289.1655.
Protein Expression and Purification of Recombinant Human
IDO1. The coding region for human IDO1 (Ala2-Gly403) was cloned
into a derivative of plasmid pET9 (Novagen). The recombinant
plasmid, pETIDO, encodes a histidine tag at the N-terminus of IDO1.
The bacterial strain BL21 AI (Invitrogen) was used for overexpression
of IDO1 and transformed with the plasmid pETIDO. The transformed
cells were grown on a rotary shaker at 37 °C and 220 rpm to an
OD600 of 1.2, in LB medium supplemented with kanamycin 25 μg/
mL, L-tryptophan 50 μg/mL, and bovine hemin (Sigma) 10 μM. The
culture was cooled in a water/ice bath and supplemented again with L-
tryptophan 50 μg/mL, bovine hemin 10 μM. The expression of his-
tagged IDO1 was induced by the addition of 1% (w/v) arabinose.
Induced cells were grown at 20 °C, 60 rpm for 20 h. Cells (1 L
culture) were collected by centrifugation, resuspended in 40 mL of
Mes 25 mM, KCl 150 mM, imidazole 10 mM, protease inhibitors
(complete EDTA free, Roche Applied Science), pH 6.5, and disrupted
with a French press. The extract was clarified by centrifugation and
filtration on a 0.22 μm filter. The enzyme was purified by IMAC using
Ni2+ as ligand and an IMAC HITRAP column (5 mL; GE Healthcare).
Briefly, the extract was loaded on the column with buffer Mes 25 mM,
KCl 150 mM, imidazole 10 mM, pH 6.5. The column was washed with
50 mL of the same buffer with imidazole adjusted to 100 mM. Finally,
the protein was eluted with Mes 25 mM, KCl 150 mM, EDTA 50 mM,
pH 6.5. The buffer was then exchanged to Mes 25 mM, KCl 150 mM,
pH 6.5 using a HITRAP desalting column (GE Healthcare). The
purity of the enzyme was estimated to be >95% based on SDS PAGE
gel and Coomassie blue staning. The ratio of absorbance 404 nm/280
nm of the protein was around 1.9.
Enzymatic Assay. The enzymatic inhibition assays were
performed as described by Takikawa et al.130 with some modifications.
Briefly, the reaction mixture (100 μL) contained potassium phosphate
buffer (100 mM, pH 6.5) ascorbic acid (20 mM), catalase (400 units/
mL), methylene blue (10 μM), purified recombinant IDO1 (2.5 ng/
μL), L-Trp (100 μM), and DMSO (5 μL). The inhibitors were serially
diluted 10-fold from 1000 to 0.1 μM, or, if not soluble at 1000 μM, by
4 orders of magnitude from their highest soluble concentration. The
reaction was carried out at ambient temperature for 60 min and
stopped by addition of 30% (w/v) trichloroacetic acid (40 μL). To
convert N-formylkynurenine to kynurenine, the samples were
incubated at 50 °C for 30 min, followed by centrifugation at 2500g
for 20 min. Lastly, 100 μL of supernatant from each probe was used
for HPLC analysis. The mobile phase for HPLC measurements
consisted of 50% sodium citrate buffer (40 mM, pH 2.35) and 50%
methanol with 400 μM SDS. The flow rate through the S5-ODS1
column was 1 mL/min, and kynurenine was detected at a wavelength
of 365 nm. A compound was defined to be “inactive” if it did not show
a clear signal on the dose−response curve, typically at a maximal
concentration of 1 mM
To measure kynurenine production during the assay, 200 μL of
supernatant from control wells were mixed with 40 μL TCA 30% and
centrifuged at 3000g for 5 min. Then 100 μL of supernatant were
mixed with an equal amount of pDMAB 2% in acetic acid and
incubated for 10 min at room temperature. Kynurenine concentration
was determined by measuring absorbance at 480 nm. A standard curve
was made with pure kynurenine (ICN Biochemicals).
To estimate cytotoxicity (LD50), 2 × 104 Hep G2 cells were plated
in 96-well flat bottom plates in a final volume of 200 μL of IMDM
(Invitrogen) supplemented with 10% FCS and a titration of the
compound ranging from 0.001 to 100 μM. After 24 h of incubation at
37 °C, the number of living cells was evaluated with a MTS/PMS
assay (Promega) as described by the manufacturer.
Determination of pKa Values. pKa values were determined
spectroscopically.131 About 1 mg of triazole compound were dissolved
in 100 mL of water under strong agitation and sonication. Then 75 mg
of KCl were added to keep the ionic strength weak but approximately
constant (10 mM). The filtered solution was brought to pH 2−3 by
addition of few drops of 1 M HCl. Then 300 μL were transferred to a
Greiner UV-Star 96-well plate, and its absorption spectra was recorded
immediately in 1 nm steps between 230 and 350 nm on a Tecan
Safire2 microplate reader. A few drops of 1 M NaOH were added to
bring the bulk solution to pH 11−12, and another sample of 300 μL
was transferred to the plate for an absorbance scan. If the spectra
allowed to differentiate between different species, in the following the
pH was varied in the region of the expected pKa values 1 pH unit in
steps of roughly 0.2 pH units by addition of a few drops of a diluted
HCl solution. At each step, a spectra was measured in above-
mentioned wavelength interval. As no buffer was used, care was taken
to measure the spectra immediately after stabilization of the pH in
order to avoid pH drifts due to the dissolution of CO2. The
wavelengths with the largest change in optical density A at different
pH values were chosen for analysis and fitted with the function
A − AI
AM − A
pH = pKa + log
(5)
where AI is the absorbance of the ionized species, and AM is the
absorbance of the molecular species. As the pKa values of the phenol
moiety present in some compounds differs by more than 1.6 pKa units
from the pKa values of the triazole moiety, and as both moieties give
rise to different absorption peaks, these pKa values could be
determined independently.
UV Spectra. UV spectra were recorded on a Tecan Safire2
microplate reader with 10 μM IDO1 in 100 mM phosphate buffer (pH
6.5) and 5% DMSO at room temperature.
Cellular Assay. Cell Lines. For the assay evaluating inhibition of
murine IDO1 and murine TDO, we used cell lines P815-mIDO1 clone
6 and P815-mTDO clone 12, respectively, as described in Rohrig et
̈
al.53 For human IDO1, we transfected mouse mastocytoma line P815B
with a plasmid construct encoding human IDO1 and selected clone
P815B-hIDO1 clone 6, which we used in the assay. For human TDO,
we transfected mouse mastocytoma line P815B with a plasmid
construct encoding human TDO and selected clone P815B-hTDO
clone 19, which overexpressed hTDO and was used in the cellular
assay. To estimate cytotoxicity (LD50), we used Hep G2 cells.
ASSOCIATED CONTENT
* Supporting Information
■
S
Figures of possible binding modes of selected compounds to
heme, ligand−heme binding energies calculated with DFT, the
values of the terms entering into the QSAR model, results of
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Journal of Medicinal Chemistry
Article
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the elemental analysis for compounds 12, 18, 19, 20, 21, 26,
28, 30, 31, 35, 36, 37, 38, 39, 41, 44, 46, and 60, and dose−
response curves of all active compounds in the enzymatic assay.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(7) Munn, D. H.; Zhou, M.; Attwood, J. T.; Bondarev, I.; Conway, S.
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AUTHOR INFORMATION
Corresponding Author
■
*For V.Z.: E-mail, vincent.zoete@isb-sib.ch. Phone, +41-21-692
4082. Fax, +41-21-692 4065. For O.M.: E-mail, olivier.
michielin@isb-sib.ch; Phone, +41-21-692 4053. Fax, +41-21-
692 4065.
(9) Terness, P.; Bauer, T. M.; Rose, L.; Dufter, C.; Watzlik, A.;
̈
Simon, H.; Opelz, G. Inhibition of allogeneic T cell proliferation by
indoleamine 2,3-dioxygenase-expressing dendritic cells: mediation of
suppression by tryptophan metabolites. J. Exp. Med. 2002, 196, 447−
457.
Author Contributions
(10) Fallarino, F.; Grohmann, U.; You, S.; McGrath, B. C.; Cavener,
D. R.; Vacca, C.; Orabona, C.; Bianchi, R.; Belladonna, M. L.; Volpi,
C.; Santamaria, P.; Fioretti, M. C.; Puccetti, P. The combined effects of
tryptophan starvation and tryptophan catabolites down-regulate T cell
receptor zeta-chain and induce a regulatory phenotype in naive T cells.
J. Immunol. 2006, 176, 6752−6761.
#Both authors contributed equally to this work (U.F.R. and
S.R.M.).
Notes
The authors declare no competing financial interest.
́
(11) Uyttenhove, C.; Pilotte, L.; Theate, I.; Stroobant, V.; Colau, D.;
ACKNOWLEDGMENTS
■
Parmentier, N.; Boon, T.; Van den Eynde, B. J. Evidence for a tumoral
immune resistance mechanism based on tryptophan degradation by
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Indoleamine 2,3-dioxygenase expression in human cancers: clinical and
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Enhancement of dendritic cell-tumor fusion vaccine potency by
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We are much obliged to the Center of Integrative Genomics
(University of Lausanne), especially to Gilles Boss, for
assistance with the enzymatic assay. For computational
resources and support, we thank the Vital-IT team at the
Swiss Institute for Bioinformatics and the Computing services
at the University of Lausanne, especially Hamid Hussain-Khan.
This work was supported by the Clinical Discovery Program of
the Ludwig Institute for Cancer Research, by fund 310030-
130857 to O.M. and V.Z. from the Swiss National Science
Foundation, and by CANTOL, a project of BioWin, the Health
Cluster of Wallonia (Belgium). Molecular graphics and analyses
were performed with the UCSF Chimera package from the
Resource for Biocomputing, Visualization, and Informatics at
the University of California, San Francisco, with support from
the National Institutes of Health (National Center for Research
Resources grant 2P41RR001081, National Institute of General
Medical Sciences grant 9P41GM103311).132 Instant JChem
was used for structure database management, search, and
prediction, Instant JChem 5.9, 2012, ChemAxon (http://www.
chemaxon.com). Marvin was used for drawing, displaying, and
characterizing chemical structures, Marvin 5.5, 2011, ChemAx-
on (http://www.chemaxon.com).
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ABBREVIATIONS USED
■
IDO1, indoleamine 2,3-dioxygenase 1; BM, binding mode;
MMBP, Morse-like metal binding potential; PCM, polarizable
continuum model; PIM, 4-phenylimidazole; TDO, tryptophan
2,3-dioxygenase
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