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

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.¹The 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.²⁻⁴IDO1 catalyzes the initial and rate-

limiting step in the catabolism of tryptophan (Trp) along the

kynurenine pathway.^5,6By depleting Trp and accumulating Trp

catabolites, IDO1 exerts a local immunosuppressive eﬀect on T-

lymphocytes.⁷⁻¹⁰The observations that many human tumors

constitutively express IDO1¹¹and that increased IDO1

expression in tumor cells is correlated with poor prognosis

for survival in several cancer types¹²led 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.^11,13−16Very 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.⁶In 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.²⁴The two available crystal structures

of IDO1 include the heme-bound ligands cyanide and 4-

phenylimidazole (PIM), respectively.²⁵Mutant 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.²⁵⁻²⁷In 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)^19,20shares 44% of sequence homology with IDO1.

However, its physiological role remains unclear due to (i) very

Received: February 24, 2012

Published: May 22, 2012

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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.³⁰Other classes of inhibitors derived from

a target-based screen of the NCI diversity set,⁴⁵the 4-

phenylimidazole scaﬀold,⁴⁶the indole scaﬀold,^47,48the S-

benzylisothiourea scaﬀold,⁴⁹a high throughput screen,²³and

a virtual screen⁵⁰have been described. The most potent and

promising compounds known today are the competitive

hydroxyamidine inibitors developed by Incyte,^15,51,52with

enzymatic IC₅₀values 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 IC₅₀value of 60 μM. Some

modiﬁcations of this scaﬀold have very recently been described

by another group,⁵⁴but improvement of the IC₅₀value 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.⁵⁵⁻⁶¹Substituted 1,2,3-triazoles have

been found to be useful drug scaﬀolds⁶²⁻⁶⁶and 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-DSS⁵⁵⁻⁵⁹and

AttractingCavities⁷⁰in combination with the parameters

derived from SwissParam⁶⁰and a Morse-like metal binding

potential (MMBP).⁶¹Candidates 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 IDO1²⁵open 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.³⁷⁻⁴⁴However,

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 IC₅₀value 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,⁷¹which involves reaction of

ethynyl derivatives with trimethylsilyl azide (Scheme 1). The

ethynyl derivatives were synthesized from iodo substrates by

the Sonogashira coupling reaction.⁷²Compound 33 was

synthesized by treatment of 74 with 6 N HCl at room

temperature.⁷³Compound 34 was derived from compound 30

by treatment with Meldrum’s acid in TEAF at 95−100 °C

(Scheme 2).⁷⁴Deprotection of the triﬂuoroacetamide of 41

the IDO1 active site using a Morse-like metal binding potential

(MMBP),⁶¹we 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,⁶¹the 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).⁷⁵All other

compounds were synthesized according to literature procedures

[58,⁷⁶61,⁷⁷(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),^82,85,86and (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

X₁

X₂

R

IC₅₀[μM]

X₁

X₂

R

IC₅₀[μM]

1

C

N

C

N

C

N

C

83

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

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

IC₅₀values are the mean of at least two independent assays. IC₅₀value 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 IC₅₀values, 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-NH₂group (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 analogue⁴⁶and 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

IC₅₀value 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.⁴⁶Taking 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

83

NI

43

44

45

phenyl phenyl

phenyl 4-F-phenyl

phenyl 3-CH₃-

phenyl

46

47

phenyl 3-Cl-phenyl

H

NI

phenyl 2-OCH₃-

phenyl

48

49

50

51

52

53

54

55

56

57

58

59

60

61

methyl phenyl

phenyl

H

NI

methyl

ethyl

phenyl

ethyl

phenyl

ethyl

n-butyl phenyl

n-butyl

phenyl

benzyl

phenyl

benzyl

H

methyl

methyl 3-Cl-phenyl

H

methyl

phenyl

3-Cl-phenyl

H

a

IC₅₀values 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 pK_a

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 pK_avalue (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 pK_avalue (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 pK_a

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 pK_aunit from 9.95 to 10.95 for the phenolic pK_aof

compound 23, as this hydrogen now serves to stabilize the

monoanion.

Table 3. Modiﬁed Scaﬀolds

All measured pK_avalues are higher than the pH at which the

enzymatic assay is carried out (6.5). However, the pK_aof a

ligand in water does not necessarily reﬂect its pK_ain the

protein-bound state. It has been shown, e.g., that thiols binding

to CYP450-CAM show a pK_ashift of 4 units as compared to

their values in water.⁹¹It 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 pK_avalue,

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).⁹⁰In 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 pK_ameasurements. 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 pK_aof the triazole ring, because the

ligand must be deprotonated in order to be active

(deprotonation hypothesis). A combination of both eﬀects is

possible.

a

IC₅₀values are the mean of at least two independent assays.

very poor in electrons (charge −0.12) due to the electro-

negative neighboring oxygen atom.

pK_aMeasurements. To better understand the electronic

properties of the triazoles, we carried out pK_ameasurements of

selected compounds (Table 4). The parent compound (1) has

a pK_avalue of 8.62 for deprotonation of its triazole ring, 0.64

pK_aunits more acidic than the unsubstituted 1,2,3-triazole

(9.26).⁸⁹Compounds 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 pK_avalues as a function

of the Hammett substituent constants σ,⁹⁰assuming (i) that the

inﬂuence of substituents is additive, σ = σ_ortho+ σ_meta+ σ_para

which is a common assumption,⁹⁰and (ii) that the inﬂuence of

a substituent is equal in ortho and in para position, σ_ortho= σ_para

,

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:

pK_a= 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),⁹²the 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

pK_avalue 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. pK_aValues 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 pK_avalue 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 pK_avalues, 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 IC₅₀values measured in the enzymatic assay and the IC₅₀values 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

(V_elec), and the quantum chemical charge on the triazole

(CH_triz) 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 ﬁeld^93,94

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

(CH_triz) 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 × CH_triz

(2)

elec

where Act and V_elecare given in kcal/mol and CH_trizis 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 IDO1^44,49were not included. Indole-4,7-dione⁴¹probably 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

60 (30)

17

>100

70 (20)

28

>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

0.10 (0.07)

>100

0.06 (0.03)

>100

a

IC₅₀values 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

IC₅₀values of the enzymatic assay on hIDO1 are given again. ND: not determined. NI: no inhibition. IC₅₀values 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

eﬃciency⁹⁶of −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 IC₅₀values 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 IC₅₀values 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 K_mfor L-Trp than hIDO1.⁹⁷Despite 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 IC₅₀value

than PIM (32 vs 48 μM),⁴⁶while 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.⁴⁶While

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 IC₅₀value 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 IC₅₀in

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,⁵¹with IC₅₀values 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 IC₅₀value 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 IC₅₀values 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

IC₅₀values diﬀer signiﬁcantly (148⁵⁴vs 7 μM here). The

inhibition mode of compounds 1, 22, and 24 was determined

to be uncompetitive.⁵⁴However, the UV spectra of IDO1 in

Most compounds active in the enzymatic assay were also

active in the cellular assay, usually with lower IC₅₀values (Table

5 and Figure 4D), in line with previous observations.^51,54In 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 IC₅₀values 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 eﬃciency⁹⁶of −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

functional⁹⁹using the Gaussian 09 code.¹⁰⁰Geometry optimizations

were carried out with standard settings and the TZVP basis set,¹⁰¹

while atomic point charges derived from a natural population analysis

(NPA)¹⁰²were calculated with the aug-cc-pVTZ basis set^103−105in

order to obtain converged charges.¹⁰⁶Solvation eﬀects were taken into

account by the polarizable continuum model⁹²as 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 pK_avalue 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 K_ivalue by the equation

ΔG = −RT ln K_i. As the K_iof an inhibitor is linearly related to its IC₅₀

value and the prefactor only depends on enzyme concentration,

substrate concentration, and K_mof the substrate,^107,108we 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, V_elec, is

EXPERIMENTAL SECTION

■

Docking. A consensus docking procedure based on our in-house

docking algorithms EADock,^55,56EADock DSS,⁵⁷and AttractingCav-

ities⁷⁰was 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.⁹⁸Minimized 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,^93,94while solvation eﬀects are taken into

account by the FACTS model,⁹⁵which has been shown to allow for

accurate docking results.⁵⁹Ligand force-ﬁeld parameters were derived

with the SwissParam tool.⁶⁰For all compounds with an acidic pK_a

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.⁶¹The 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).²⁵The 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)³around 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)³around 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

= ΔG_solv,elec+ E_inter,elec− E_corr,elec

(3)

elec

with the electrostatic solvation term

complex

solv,elec

ligand

solv,elec

protein

solv,elec

ΔG_solv,elec= ΔG

− ΔG

(4)

as calculated by the FACTS solvation model.⁹⁵E_inter,elecis 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. E_corr,elecis 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 KMnO₄or

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.¹¹⁸Synthesized from 3-iodo-4-methyl-

aniline according to the representative procedure⁷²to 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 procedure⁷²to 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.⁷²To 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.¹¹⁹Synthesized from 4-

chloro-2-iodo-1-methylbenzene according to the representative

procedure⁷²to 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.⁷¹To 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.⁷²Synthesized from 2-iodophenol according to

the representative procedure⁷²to 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.¹⁰⁹Synthesized from 1-ethyl-2-iodoben-

zene according to the representative procedure⁷²to 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.¹¹⁰Synthesized from 2-iodobenzonitrile

according to the representative procedure⁷²to 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.¹¹¹Synthesized from 1-ethyl-3-iodoben-

zene according to the representative procedure⁷²to 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.⁸²To 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.¹¹²Synthesized from 2-ﬂuoro-4-iodo-

pyridine according to the representative procedure⁷²to 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.¹¹³Synthesized from 2-chloro-4-iodo-

pyridine according to the representative procedure⁷²to 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).⁷¹Synthesized from ethynylben-

zene⁷¹and TMSN₃according 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.¹¹⁴Synthesized from 1,2-dichloro-

3-iodobenzene according to the representative procedure⁷²to 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).⁶²Synthesized from 4-ethy-

nylpyridine⁷¹and TMSN₃according 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).⁶²Synthesized from 3-ethy-

nylpyridine⁷¹and TMSN₃according 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.¹¹⁵Synthesized from 1,4-dichloro-

2-iodobenzene according to the representative procedure⁷²to 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.¹¹⁶Synthesized from 2-iodo-N-methyl-

aniline according to the representative procedure⁷²to 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).¹²⁰Synthesized from 1-

ethynyl-4-ﬂuorobenzene⁷¹and TMSN₃according to the general

2-Ethynylbenzaldehyde.¹¹⁷Synthesized from 2-iodobenzaldehyde

according to the representative procedure⁷²to aﬀord the title

1

procedure to aﬀord 4 as a white solid in 62% yield. H NMR (400

5282

dx.doi.org/10.1021/jm300260v | J. Med. Chem. 2012, 55, 5270−5290

Journal of Medicinal Chemistry

Article

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-ethynylpyridine⁷¹and TMSN₃according 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).⁶²Synthesized from 1-

chloro-4-ethynylbenzene⁷¹and TMSN₃according 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-ethynylpyridine⁷¹and TMSN₃according 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).⁶²Synthesized from 1-ethynyl-4-

methylbenzene⁷¹and TMSN₃according 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).¹²¹Synthe-

sized from 1-ethynyl-4-(triﬂuoromethyl)benzene⁷¹and 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-ﬂuoropyridine⁷¹and TMSN₃according 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).¹²¹Synthesized from 1-

chloro-3-ethynylbenzene⁷¹and TMSN₃according 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).⁶²Synthesized from 1-

bromo-3-ethynylbenzene⁷¹and TMSN₃according 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)pyridine⁷¹and 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).¹²²Synthesized from 3-

ethynylbenzonitrile⁷¹and TMSN₃according to the general procedure

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).¹²³Synthe-

sized from 1-ethynyl-3-(triﬂuoromethyl)benzene⁷¹and TMSN₃

according to the general procedure to aﬀord 11 as a white solid in

4-(2-Bromophenyl)-1H-1,2,3-triazole (22).¹²⁶Synthesized from 1-

bromo-2-ethynylbenzene⁷¹and TMSN₃according 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).¹²⁷Synthesized from 2-

ethynylphenol⁷¹and TMSN₃according 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-ethynylbenzene⁷¹and TMSN₃according 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).¹²⁴Synthesized from 1-

ethynyl-3-nitrobenzene⁷¹and TMSN₃according to the general

4-(2-Chlorophenyl)-1H-1,2,3-triazole (24).¹²¹Synthesized from 1-

chloro-2-ethynylbenzene⁷¹and TMSN₃according 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).⁶²Synthesized from 1-ethynyl-2-

methylbenzene⁷¹and TMSN₃according 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).⁶²Synthesized from 1-ethynyl-3-

methylbenzene⁷¹and TMSN₃according 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).¹²⁵Synthesized from 1-

ethynyl-3-ﬂuorobenzene⁷¹and TMSN₃according to the general

2-(1H-1,2,3-Triazol-4-yl)aniline (26). Synthesized from 2-ethynyla-

niline⁷¹and TMSN₃according 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).⁶²Synthesized from 3-ethy-

nylphenol⁷¹and TMSN₃according 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).⁶²Synthesized from 3-ethyny-

laniline⁷¹and TMSN₃according 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).¹²¹Synthesized from 1-

ethynyl-2-ﬂuorobenzene⁷¹and TMSN₃according 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-ethynylbenzene⁷¹and TMSN₃according 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-methylbenzene⁷¹and TMSN₃according 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).⁶³Synthe-

sized from 1-ethynyl-2-(triﬂuoromethyl)benzene⁷¹and 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-ethynylbenzene⁷¹and TMSN₃according 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-

ethynylbenzaldehyde⁷¹and TMSN₃according 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-ethynylbenzene⁷¹and TMSN₃according 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-methylbenzene⁷¹and TMSN₃according 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-methylaniline⁷¹and TMSN₃according 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).⁶²Synthesized from 1-

ethynyl-2-methoxybenzene⁷¹and TMSN₃according 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-triﬂuoroacetamide⁷¹and TMSN₃according 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)⁷³was

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-ethynylphenol⁷¹and TMSN₃according 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-methylaniline⁷¹and TMSN₃according 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).^80,81To 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 Na₂SO₄and 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 procedure^80,81as 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).⁸⁶Synthesized from 4-

phenyl-1H-1,2,3-triazole and diethyl carbonate according to the

general procedure of N-alkylation⁸²to 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).⁸⁶Synthesized from 4-

phenyl-1H-1,2,3-triazole and diethyl carbonate according to the

general procedure of N-alkylation⁸²to 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).^78,79Synthesized accord-

ing to a previously reported procedure.^78,79Oil, 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

procedure^80,81as 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).^78,79Synthesized accord-

ing to a previously reported procedure.^78,79White 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).^78,79Synthesized accord-

ing to a previously reported procedure.^78,79Oﬀ-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).^78,79Synthesized accord-

ing to a previously reported procedure.^78,79White 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).⁷⁶Synthesized according

to a previously reported procedure.⁷⁶White 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 procedure^80,81as 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-

methylation⁸²to 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).^80,81Using

the same procedure^80,81as 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-

methylation⁸²to aﬀord 60 as a white solid, mp 104−107 °C, 49%

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).^83,84Synthesized from 4-

phenyl-1H-1,2,3-triazole and dimethyl carbonate according to the

general procedure of N-alkylation⁸²to 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).⁷⁷Synthesized according to a

previously reported procedure.⁷⁷Oﬀ-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).¹²⁸Synthesized from prop-2-yn-1-

ylbenzene⁷¹and TMSN₃according 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).^83,84Synthesized from 4-

phenyl-1H-1,2,3-triazole and dimethyl carbonate according to the

general procedure of N-alkylation⁸²to 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).^83,84Synthesized from 4-

phenyl-1H-1,2,3-triazole and dimethyl carbonate according to the

general procedure of N-alkylation⁸²to aﬀord 50 as a white solid, 20%

4-((Phenylthio)methyl)-1H-1,2,3-triazole (63).¹²⁰Synthesized from

yield. H NMR (400 MHz, CDCl₃): δ 7.85 (s, 1H), 7.80 (d, 2H, J =

phenyl(prop-2-yn-1-yl)sulfane⁷¹and TMSN₃according to the general

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).⁸⁶Synthesized from 4-

phenyl-1H-1,2,3-triazole and diethyl carbonate according to the

general procedure of N-alkylation⁸²to 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).¹²⁹Synthesized from

1-ethynylcyclohex-1-ene⁷¹and TMSN₃according 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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Journal of Medicinal Chemistry

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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 × 10⁵cells 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-ethynylphenethylcarbamate⁷¹and

TMSN₃according 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.¹³⁰with 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 × 10⁴Hep 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 pK_aValues. pK_avalues were determined

spectroscopically.¹³¹About 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 pK_avalues 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 − A_I

A_M− A

pH = pK_a+ log

(5)

where A_Iis the absorbance of the ionized species, and A_Mis the

absorbance of the molecular species. As the pK_avalues of the phenol

moiety present in some compounds diﬀers by more than 1.6 pK_aunits

from the pK_avalues of the triazole moiety, and as both moieties give

rise to diﬀerent absorption peaks, these pK_avalues 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.⁵³For 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

(6) Sono, M.; Roach, M.; Coulter, E.; Dawson, J. Heme-Containing

Oxygenases. Chem. Rev. 1996, 96, 2841−2888.

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.

J.; Marshall, B.; Brown, C.; Mellor, A. L. Prevention of allogeneic fetal

rejection by tryptophan catabolism. Science 1998, 281, 1191−1193.

(8) Hwu, P.; Du, M. X.; Lapointe, R.; Do, M.; Taylor, M. W.; Young,

H. A. Indoleamine 2,3-dioxygenase production by human dendritic

cells results in the inhibition of T cell proliferation. J. Immunol. 2000,

164, 3596−3599.

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

indoleamine 2,3-dioxygenase. Nature Med. 2003, 9, 1269−1274.

(12) Godin-Ethier, J.; Hanaﬁ, L.-A.; Piccirillo, C. A.; Lapointe, R.

Indoleamine 2,3-dioxygenase expression in human cancers: clinical and

immunologic perspectives. Clin. Cancer Res. 2011, 17, 6985−6991.

(13) Muller, A. J.; DuHadaway, J. B.; Donover, P. S.; Sutanto-Ward,

E.; Prendergast, G. C. Inhibition of indoleamine 2,3-dioxygenase, an

immunoregulatory target of the cancer suppression gene Bin1,

potentiates cancer chemotherapy. Nature Med. 2005, 11, 312−319.

(14) Ou, X.; Cai, S.; Liu, P.; Zeng, J.; He, Y.; Wu, X.; Du, J.

Enhancement of dendritic cell-tumor fusion vaccine potency by

indoleamine-pyrrole 2,3-dioxygenase inhibitor, 1-MT. J. Cancer Res.

Clin. Oncol. 2008, 134, 525−533.

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).¹³²Instant 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).

(15) Liu, X.; et al. Selective inhibition of IDO1 eﬀectively regulates

mediators of antitumor immunity. Blood 2010, 115, 3520−3530.

(16) Ino, K. Indoleamine 2,3-dioxygenase and immune tolerance in

ovarian cancer. Curr. Opin. Obstet. Gynecol. 2011, 23, 13−18.

(17) Opitz, C. A.; et al. An endogenous tumour-promoting ligand of

the human aryl hydrocarbon receptor. Nature 2011, 478, 197−203.

(18) Pilotte, L.; Larrieu, P.; Stroobant, V.; Colau, D.; Dolusic, E.;

́ ́

Frederick, R.; Plaen, E. D.; Uyttenhove, C.; Wouters, J.; Masereel, B.;

den Eynde, B. J. V. Reversal of tumoral immune resistance by

inhibition of tryptophan 2,3-dioxygenase. Proc. Natl. Acad. Sci. U.S.A.

2012, 109, 2497−2502.

(19) Metz, R.; Duhadaway, J. B.; Kamasani, U.; Laury-Kleintop, L.;

Muller, A. J.; Prendergast, G. C. Novel tryptophan catabolic enzyme

IDO2 is the preferred biochemical target of the antitumor indoleamine

2,3-dioxygenase inhibitory compound ^D-1-methyl-tryptophan. Cancer

Res. 2007, 67, 7082−7087.

(20) Ball, H. J.; Sanchez-Perez, A.; Weiser, S.; Austin, C. J. D.;

Astelbauer, F.; Miu, J.; McQuillan, J. A.; Stocker, R.; Jermiin, L. S.;

Hunt, N. H. Characterization of an indoleamine 2,3-dioxygenase-like

protein found in humans and mice. Gene 2007, 396, 203−213.

(21) Witkiewicz, A. K.; Costantino, C. L.; Metz, R.; Muller, A. J.;

Prendergast, G. C.; Yeo, C. J.; Brody, J. R. Genotyping and expression

analysis of IDO2 in human pancreatic cancer: a novel, active target. J.

Am. Coll. Surg. 2009, 208, 781−787.

(22) Yuasa, H. J.; Ball, H. J.; Austin, C. J. D.; Hunt, N. H. 1-^L-

Methyltryptophan is a more eﬀective inhibitor of vertebrate IDO2

enzymes than 1-^D-methyltryptophan. Comp. Biochem. Physiol., Part B:

Biochem. Mol. Biol. 2010, 157, 10−15.

(23) Meininger, D.; Zalameda, L.; Liu, Y.; Stepan, L. P.; Borges, L.;

McCarter, J. D.; Sutherland, C. L. Puriﬁcation and kinetic character-

ization of human indoleamine 2,3-dioxygenases 1 and 2 (IDO1 and

IDO2) and discovery of selective IDO1 inhibitors. Biochim. Biophys.

Acta 2011, 1814, 1947−1954.

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

REFERENCES

■

(1) Du, C.; Wang, Y. The immunoregulatory mechanisms of

carcinoma for its survival and development. J. Exp. Clin. Cancer Res.

2011, 30, 12.

(2) Zamanakou, M.; Germenis, A. E.; Karanikas, V. Tumor immune

escape mediated by indoleamine 2,3-dioxygenase. Immunol. Lett. 2007,

111, 69−75.

(3) Katz, J. B.; Muller, A. J.; Prendergast, G. C. Indoleamine 2,3-

dioxygenase in T-cell tolerance and tumoral immune escape. Immunol.

Rev. 2008, 222, 206−221.

(4) Prendergast, G. C. Immune escape as a fundamental trait of

cancer: focus on IDO. Oncogene 2008, 27, 3889−3900.

(5) Yamamoto, S.; Hayaishi, O. Tryptophan pyrrolase of rabbit

intestine. ^D- and L-tryptophan-cleaving enzyme or enzymes. J. Biol.

Chem. 1967, 242, 5260−5266.

5287

dx.doi.org/10.1021/jm300260v | J. Med. Chem. 2012, 55, 5270−5290

Journal of Medicinal Chemistry

Article

(24) Wogulis, M.; Chew, E. R.; Donohoue, P. D.; Wilson, D. K.

Identiﬁcation of formyl kynurenine formamidase and kynurenine

aminotransferase from Saccharomyces cerevisiae using crystallographic,

bioinformatic and biochemical evidence. Biochemistry 2008, 47, 1608−

1621.

(25) Sugimoto, H.; Oda, S.; Otsuki, T.; Hino, T.; Yoshida, T.; Shiro,

Y. Crystal structure of human indoleamine 2,3-dioxygenase: catalytic

mechanism of O2 incorporation by a heme-containing dioxygenase.

Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 2611−2616.

(26) Chauhan, N.; Basran, J.; Eﬁmov, I.; Svistunenko, D. A.; Seward,

H. E.; Moody, P. C. E.; Raven, E. L. The role of serine 167 in human

indoleamine 2,3-dioxygenase: a comparison with tryptophan 2,3-

dioxygenase. Biochemistry 2008, 47, 4761−4769.

(27) Eﬁmov, I.; Basran, J.; Thackray, S. J.; Handa, S.; Mowat, C. G.;

Raven, E. L. Structure and reaction mechanism in the heme

dioxygenases. Biochemistry 2011, 50, 2717−2724.

(28) Vottero, E.; Mitchell, D. A.; Page, M. J.; MacGillivray, R. T. A.;

Sadowski, I. J.; Roberge, M.; Mauk, A. G. Cytochrome b(5) is a major

reductant in vivo of human indoleamine 2,3-dioxygenase expressed in

yeast. FEBS Lett. 2006, 580, 2265−2268.

(29) Maghzal, G. J.; Thomas, S. R.; Hunt, N. H.; Stocker, R.

Cytochrome b5, not superoxide anion radical, is a major reductant of

indoleamine 2,3-dioxygenase in human cells. J. Biol. Chem. 2008, 283,

12014−12025.

(30) Pearson, J. T.; Siu, S.; Meininger, D. P.; Wienkers, L. C.; Rock,

D. A. In vitro modulation of cytochrome P450 reductase supported

indoleamine 2,3-dioxygenase activity by allosteric eﬀectors cytochrome

b(5) and methylene blue. Biochemistry 2010, 49, 2647−2656.

(31) Watanabe, Y.; Fujiwara, M.; Hayaishi, O. 2,5-Dihydro-^L-

phenylalanine: a competitive inhibitor of indoleamine 2,3-dioxygenase.

Biochem. Biophys. Res. Commun. 1978, 85, 273−279.

(32) Sono, M.; Cady, S. G. Enzyme kinetic and spectroscopic studies

of inhibitor and eﬀector interactions with indoleamine 2,3-

dioxygenase. 1. Norharman and 4-phenylimidazole binding to the

enzyme as inhibitors and heme ligands. Biochemistry 1989, 28, 5392−

5399.

(33) Cady, S. G.; Sono, M. 1-Methyl-^DL-tryptophan, beta-(3-

benzofuranyl)-^DL-alanine (the oxygen analog of tryptophan), and

beta-[3-benzo(b)thienyl]-^DL-alanine (the sulfur analog of tryptophan)

are competitive inhibitors for indoleamine 2,3-dioxygenase. Arch.

Biochem. Biophys. 1991, 291, 326−333.

(34) Peterson, A. C.; Loggia, A. J. L.; Hamaker, L. K.; Arend, R. A.;

Fisette, P. L.; Okazi, Y.; Will, J. A.; Brown, R. R.; Cook, J. M.

Evaluation of Substituted β-Carbolines as Noncompetitive Indole-

amine 2,3-Dioxygenase Inhibitors. Med. Chem. Res. 1993, 3, 473−482.

(35) Peterson, A. C.; Migawa, M. T.; Martin, M. J.; Hamaker, L. K.;

Czerwinski, K. M.; Zhang, W.; Arend, R. A.; Fisette, P. L.; Okazi, Y.;

Will, J. A.; Brown, R. R.; Cook, J. M. Evaluation of Functionalized

Tryptophan Derivatives and Related Compounds as Competitive

Inhibitors of Indoleamine 2,3-Dioxygenase. Med. Chem. Res. 1994, 3,

531−544.

(36) Gaspari, P.; Banerjee, T.; Malachowski, W. P.; Muller, A. J.;

Prendergast, G. C.; DuHadaway, J.; Bennett, S.; Donovan, A. M.

Structure-activity study of brassinin derivatives as indoleamine 2,3-

dioxygenase inhibitors. J. Med. Chem. 2006, 49, 684−692.

(37) Brastianos, H. C.; Vottero, E.; Patrick, B. O.; Soest, R. V.;

Matainaho, T.; Mauk, A. G.; Andersen, R. J.; Exiguamine, A an

indoleamine-2,3-dioxygenase (IDO) inhibitor isolated from the marine

sponge Neopetrosia exigua. J. Am. Chem. Soc. 2006, 128, 16046−16047.

(38) Andersen, R.; Pereira, A.; Huang, X.-H.; Mauk, G.; Vottero, E.;

Roberge, M.; Balgi, A. Indoleamine 2,3-Dioxygenase (IDO) Inhibitors.

Patent WO 2006/005185, 2006.

Dioxygenase (IDO) Inhibitors and Synthesis and Uses Thereof. Patent

WO 2008/052352, 2008.

(41) Carr, G.; Chung, M. K. W.; Mauk, A. G.; Andersen, R. J.

Synthesis of indoleamine 2,3-dioxygenase inhibitory analogues of the

sponge alkaloid exiguamine A. J. Med. Chem. 2008, 51, 2634−2637.

(42) Kumar, S.; Malachowski, W.; Duhadaway, J.; Lalonde, J.;

Carroll, P.; Jaller, D.; Metz, R.; Prendergast, G.; Muller, A.

Indoleamine 2,3-Dioxygenase Is the Anticancer Target for a Novel

Series of Potent Naphthoquinone-Based Inhibitors. J. Med. Chem.

2008, 51, 1706−1718.

(43) Carr, G.; Tay, W.; Bottriell, H.; Andersen, S. K.; Mauk, A. G.;

Andersen, R. J. Plectosphaeroic acids A, B, and C, indoleamine 2,3-

dioxygenase inhibitors produced in culture by a marine isolate of the

fungus Plectosphaerella cucumerina. Org. Lett. 2009, 11, 2996−2999.

(44) Terentis, A. C.; Freewan, M.; Plaza, T. S. S.; Raftery, M. J.;

Stocker, R.; Thomas, S. R. The selenazal drug ebselen potently inhibits

indoleamine 2,3-dioxygenase by targeting enzyme cysteine residues.

Biochemistry 2010, 49, 591−600.

(45) Vottero, E.; Balgi, A.; Woods, K.; Tugendreich, S.; Melese, T.;

Andersen, R. J.; Mauk, A. G.; Roberge, M. Inhibitors of human

indoleamine 2,3-dioxygenase identiﬁed with a target-based screen in

yeast. Biotechnol. J. 2006, 1, 282−288.

(46) Kumar, S.; Jaller, D.; Patel, B.; LaLonde, J. M.; DuHadaway, J.

B.; Malachowski, W. P.; Prendergast, G. C.; Muller, A. J. Structure

based development of phenylimidazole-derived inhibitors of indole-

amine 2,3-dioxygenase. J. Med. Chem. 2008, 51, 4968−4977.

̆ ́

(47) Dolusic, E.; et al. Indol-2-yl ethanones as novel indoleamine 2,3-

dioxygenase (IDO) inhibitors. Bioorg. Med. Chem. 2011, 19, 1550−

1561.

̆ ́

(48) Dolusic, E.; et al. Discovery and preliminary SARs of keto-

indoles as novel indoleamine 2,3-dioxygenase (IDO) inhibitors. Eur. J.

Med. Chem. 2011, 46, 3058−3065.

(49) Matsuno, K.; Takai, K.; Isaka, Y.; Unno, Y.; Sato, M.; Takikawa,

O.; Asai, A. S-Benzylisothiourea derivatives as small-molecule

inhibitors of indoleamine-2,3-dioxygenase. Bioorg. Med. Chem. Lett.

2010, 20, 5126−5129.

(50) Smith, J. R.; Evans, K. J.; Wright, A.; Willows, R. D.; Jamie, J. F.;

Griﬃth, R. Novel indoleamine 2,3-dioxygenase-1 inhibitors from a

multistep in silico screen. Bioorg. Med. Chem. 2012, 20, 1354−1363.

(51) Yue, E. W.; et al. Discovery of potent competitive inhibitors of

indoleamine 2,3-dioxygenase with in vivo pharmacodynamic activity

and eﬃcacy in a mouse melanoma model. J. Med. Chem. 2009, 52,

7364−7367.

(52) Koblish, H. K.; Hansbury, M. J.; Bowman, K. J.; Yang, G.;

Neilan, C. L.; Haley, P. J.; Burn, T. C.; Waeltz, P.; Sparks, R. B.; Yue,

E. W.; Combs, A. P.; Scherle, P. A.; Vaddi, K.; Fridman, J. S.

Hydroxyamidine inhibitors of indoleamine-2,3-dioxygenase potently

suppress systemic tryptophan catabolism and the growth of IDO-

expressing tumors. Mol. Cancer Ther. 2010, 9, 489−498.

(53) Rohrig, U. F.; Awad, L.; Grosdidier, A.; Larrieu, P.; Stroobant,

̈

V.; Colau, D.; Cerundolo, V.; Simpson, A. J. G.; Vogel, P.; Van den

Eynde, B. J. V.; Zoete, V.; Michielin, O. Rational design of indoleamine

2,3-dioxygenase inhibitors. J. Med. Chem. 2010, 53, 1172−1189.

(54) Huang, Q.; Zheng, M.; Yang, S.; Kuang, C.; Yu, C.; Yang, Q.

Structure−activity relationship and enzyme kinetic studies on 4-aryl-

1H-1,2,3-triazoles as indoleamine 2,3-dioxygenase (IDO) inhibitors.

Eur. J. Med. Chem. 2011, 46, 5680−5687.

(55) Grosdidier, A.; Zoete, V.; Michielin, O. EADock: docking of

small molecules into protein active sites with a multiobjective

evolutionary optimization. Proteins 2007, 67, 1010−1025.

(56) Grosdidier, A.; Zoete, V.; Michielin, O. Blind docking of 260

protein−ligand complexes with EADock 2.0. J. Comput. Chem. 2009,

30, 2021−2030.

(57) Grosdidier, A.; Zoete, V.; Michielin, O. SwissDock, a protein−

small molecule docking web service based on EADock DSS. Nucleic

Acids Res. 2011, 39, W270−W277.

(39) Pereira, A.; Vottero, E.; Roberge, M.; Mauk, A. G.; Andersen, R.

J. Indoleamine 2,3-dioxygenase inhibitors from the Northeastern

Paciﬁc Marine Hydroid Garveia annulata. J. Nat. Prod. 2006, 69,

1496−1499.

(40) Andersen, R.; Leblanc, M.; Brastianos, H.; Vottero, E.; Roberge,

M.; Mauk, G.; Carr, G. Substituted Quinone Indoleamine 2,3-

(58) Grosdidier, A.; Zoete, V.; Michielin, O. Fast docking using the

CHARMM force ﬁeld with EADock DSS. J. Comput. Chem. 2011, 32,

2149−2159.

5288

dx.doi.org/10.1021/jm300260v | J. Med. Chem. 2012, 55, 5270−5290

Journal of Medicinal Chemistry

Article

(59) Zoete, V.; Grosdidier, A.; Cuendet, M.; Michielin, O. Use of the

FACTS solvation model for protein−ligand docking calculations.

Application to EADock. J. Mol. Recognit. 2010, 23, 457−461.

(60) Zoete, V.; Cuendet, M. A.; Grosdidier, A.; Michielin, O.

SwissParam: a fast force ﬁeld generation tool for small organic

molecules. J. Comput. Chem. 2011, 32, 2359−2368.

(80) Liu, D.; Gao, W.; Dai, Q.; Zhang, X. Triazole-based

monophosphines for Suzuki−Miyaura coupling and amination

reactions of aryl chlorides. Org. Lett. 2005, 7, 4907−4910.

(81) Kadaba, P. K. Triazolines. 14. 1,2,3-Triazolines and triazoles, a

new class of anticonvulsants. Drug design and structure−activity

relationships. J. Med. Chem. 1988, 31, 196−203.

(82) Jiang, X.; Tiwari, A.; Thompson, M.; Chen, Z.; Cleary, T. P.;

Lee, T. B. K. A Practical Method for N-Methylation of Indoles Using

Dimethyl Carbonate. Org. Process Res. Dev. 2001, 5, 604−608.

(83) Kozima, S.; Itano, T.; Mihara, N.; Sisido, K.; Isida, T. Formation

of organotin-nitrogen bonds IV. N-Trialkyltin derivatives of 4-mono-

or 4,5-disubstituted 1,2,3-triazoles, 3-phenyl-1,2,4-triazole, 3-phenyl-

pyrazole and 4-phenylimidazole. J. Organomet. Chem. 1972, 44, 117−

126.

(61) Rohrig, U. F.; Grosdidier, A.; Zoete, V.; Michielin, O. Docking

̈

to Heme Proteins. J. Comput. Chem. 2009, 30, 2305−2315.

(62) Kallander, L. S.; et al. 4-Aryl-1,2,3-triazole: a novel template for

a reversible methionine aminopeptidase 2 inhibitor, optimized to

inhibit angiogenesis in vivo. J. Med. Chem. 2005, 48, 5644−5647.

(63) Oh, S.; Shin, W.-S.; Ham, J.; Lee, S. Acid-catalyzed synthesis of

10-substituted triazolyl artemisinins and their growth inhibitory

activity against various cancer cells. Bioorg. Med. Chem. Lett. 2010,

20, 4112−4115.

(64) Agalave, S. G.; Maujan, S. R.; Pore, V. S. Click chemistry: 1,2,3-

triazoles as pharmacophores. Chem. Asian J. 2011, 6, 2696−2718.

(65) Siddiquia, N.; Ahsana, W.; Alama, M. S.; Alia, R.; Jainb, S.;

Azada, B.; Akhtara, J. Trizaoles: as potential bioactive agents. Int. J.

Pharm. Sci. Rev. Res. 2011, 8, 161−169.

(66) Gubbins, P. O. Triazole antifungal agents drug−drug

interactions involving hepatic cytochrome P450. Expert Opin. Drug

Metab. Toxicol. 2011, 7, 1411−1429.

(67) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click Chemistry:

Diverse Chemical Function from a Few Good Reactions. Angew.

Chem., Int. Ed. Engl. 2001, 40, 2004−2021.

(68) Tornøe, C. W.; Christensen, C.; Meldal, M. Peptidotriazoles on

solid phase: [1,2,3]-triazoles by regiospeciﬁc copper(I)-catalyzed 1,3-

dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem.

2002, 67, 3057−3064.

(84) Butler, R. N. A Study of the Proton Nuclear Magnetic

Resonance Spectra of Aryl and Mono- and Disubstituted N-

Methylazoles. Can. J. Chem. 1973, 51, 2315−2322.

́ ́

(85) Beneteau, V.; Olmos, A.; Boningari, T.; Sommer, J.; Pale, P.

Zeo-click synthesis: CuI-zeolite-catalyzed one-pot two-step synthesis

of triazoles from halides and related compounds. Tetrahedron Lett.

2010, 51, 3673−3677.

(86) Wang, X.-J.; Sidhu, K.; Zhang, L.; Campbell, S.; Haddad, N.;

Reeves, D. C.; Krishnamurthy, D.; Senanayake, C. H. Bromo-directed

N-2 alkylation of NH-1,2,3-triazoles: eﬃcient synthesis of poly-

substituted 1,2,3-triazoles. Org. Lett. 2009, 11, 5490−5493.

(87) Eguchi, N.; Watanabe, Y.; Kawanishi, K.; Hashimoto, Y.;

Hayaishi, O. Inhibition of indoleamine 2,3-dioxygenase and

tryptophan 2,3-dioxygenase by beta-carboline and indole derivatives.

Arch. Biochem. Biophys. 1984, 232, 602−609.

(88) Lu, C.; Lin, Y.; Yeh, S.-R. Spectroscopic studies of ligand and

substrate binding to human indoleamine 2,3-dioxygenase. Biochemistry

2010, 49, 5028−5034.

(89) Schoﬁeld, K.; Grimmett, M. R.; Keene, B. R. T. Heteroaromatic

Nitrogen Compounds: The Azoles; Cambridge University Press: New

York, 1976.

(69) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. A

stepwise Huisgen cycloaddition process: copper(I)-catalyzed regiose-

lective “ligation” of azides and terminal alkynes. Angew. Chem., Int. Ed.

Engl. 2002, 41, 2596−2599.

(70) Zoete, V.; Grosdidier, A.; Schuepbach, T.; Michielin, O.

manuscript in preparation.

(71) Jin, T.; Kamijo, S.; Yamamoto, Y. Copper-Catalyzed Synthesis

of N-Unsubstituted 1,2,3-Triazoles from Nonactivated Terminal

Alkynes. Eur. J. Org. Chem. 2004, 18, 3789−3791.

(90) Hansch, C.; Leo, A. Substituent Constants for Correlation Analysis

in Chemistry and Biology; Wiley: New York, 1979.

(91) Sono, M.; Andersson, L. A.; Dawson, J. H. Sulfur donor ligand

binding to ferric cytochrome P-450-CAM and myoglobin. Ultraviolet−

visible absorption, magnetic circular dichroism, and electron para-

magnetic resonance spectroscopic investigation of the complexes. J.

Biol. Chem. 1982, 257, 8308−8320.

(92) Tomasi, J.; Mennucci, B.; Cammi, R. Quantum mechanical

continuum solvation models. Chem. Rev. 2005, 105, 2999−3093.

(93) Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.;

Swaminathan, S.; Karplus, M. CHARMM: A Program for Macro-

molecular Energy, Minimization, and Dynamics Calculations. J.

Comput. Chem. 1983, 4, 187−217.

(72) Arcadi, A.; Cacchi, S.; Del Rosario, M.; Fabrizi, G.; Marinelli, F.

Palladium-Catalyzed Reaction of o-Ethynylphenols, o-

((Trimethylsilyl)ethynyl)phenyl Acetates, and o-Alkynylphenols with

Unsaturated Triﬂates or Halides: A Route to 2-Substituted-, 2,3-

Disubstituted-, and 2-Substituted-3-acylbenzo[b]furans. J. Org. Chem.

1996, 61, 9280−9288.

(73) Han, G.; Tamaki, M.; Hruby, V. J. Fast, eﬃcient and selective

deprotection of the tert-butoxycarbonyl (Boc) group using HCl/

dioxane (4 m). J. Pept. Res. 2001, 58, 338−341.

́ ́

(74) Toth, G.; Kover, K. E. Simple, Safe, Large Scale Synthesis of 5-

̈

(94) MacKerell, A. D., Jr.; et al. All-Atom Empirical Potential for

Molecular Modeling and Dynamics Studies of Proteins. J. Phys. Chem.

B 1998, 102, 3586−3616.

Arylmethyl-2,2-dimethyl-1,3-dioxane-4,6-diones and 3-Arylpropanoic

Acids. Synth. Commun. 1995, 25, 3067−3074.

(75) Pak, J.; Hesse, M. Regioselective Deprotection and Acylation of

Penta-N-Protected Thermopentamine. Helv. Chim. Acta 1998, 81,

2300−2313.

(95) Haberthur, U.; Caﬂisch, A. FACTS: Fast analytical continuum

̈

treatment of solvation. J. Comput. Chem. 2008, 29, 701−715.

(96) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand eﬃciency: a

useful metric for lead selection. Drug Discovery Today 2004, 9, 430−

431.

(76) Kawai, N.; Shiori, T. New Methods and Reagents in Organic

Synthesis. 34. Diphenyl Phosphorazidate (DPPA) as a 1,3-Dipole. A

Simple, Eﬃcient Conversion of Akyl Phenyl Ketones to 2-Phenyl-

alkanoic Acids. Chem. Pharm. Bull. 1983, 31, 2564−2573.

(77) Jiang, Y.; Kuang, C.; Yang, Q. The Use of Calcium Carbide in

the Synthesis of 1-Monosubstituted Aryl 1,2,3-Triazole. Synlett 2009,

2009, 3163−3166.

(97) Austin, C. J. D.; Astelbauer, F.; Kosim-Satyaputra, P.; Ball, H. J.;

Willows, R. D.; Jamie, J. F.; Hunt, N. H. Mouse and human

indoleamine 2,3-dioxygenase display some distinct biochemical and

structural properties. Amino Acids 2009, 36, 99−106.

(98) Miranker, A.; Karplus, M. Functionality maps of binding sites: a

multiple copy simultaneous search method. Proteins 1991, 11, 29−34.

(99) Adamo, C.; Barone, V. Toward reliable density functional

methods without adjustable parameters: The PBE0 model. J. Chem.

Phys. 1999, 110, 6158−6169.

(78) Li, P.; Wang, L. One-Pot Synthesis of 1,2,3-Triazoles from

Benzyl and Alkyl Halides, Sodium Azide and Alkynes in Water Under

Transition-Metal-Catalyst Free Reaction Conditions. Lett. Org. Chem.

2007, 4, 23−26.

(79) George, S. R. D.; Edwards, G. L.; Harper, J. B. The eﬀects of

ionic liquids on azide−alkyne cycloaddition reactions. Org. Biomol.

Chem. 2010, 8, 5354−5358.

(100) Frisch, M. J. et al. Gaussian 09, revision A.1; Gaussian Inc.:

Wallingford, CT, 2009.

5289

dx.doi.org/10.1021/jm300260v | J. Med. Chem. 2012, 55, 5270−5290

Journal of Medicinal Chemistry

Article

(101) Schaefer, A.; Huber, C.; Ahlrichs, R. Fully optimized

contracted Gaussian-basis sets of triple zeta valence quality for

atoms Li to Kr. J. Chem. Phys. 1994, 100, 5829−5835.

(102) Reed, A. E.; Weinstock, R. B.; Weinhold, F. Natural-population

analysis. J. Chem. Phys. 1985, 83, 735−746.

(103) Dunning, T. H., Jr. Gaussian basis sets for use in correlated

molecular calculations. I. The atoms boron through neon and

hydrogen. J. Chem. Phys. 1989, 90, 1007−1023.

(104) Kendall, R. A.; Dunning, T. H., Jr.; Harrison, R. J. Electron

aﬃnities of the ﬁrst-row atoms revisited. Systematic basis sets and

wave functions. J. Chem. Phys. 1992, 96, 6796−6806.

(105) Woon, D. E.; Dunning, T. H., Jr. Gaussian-basis sets for use in

correlated molecular calculations. 3. The atoms aluminum through

argon. J. Chem. Phys. 1993, 98, 1358−1371.

(106) Martin, F.; Zipse, H. Charge distribution in the water

moleculea comparison of methods. J. Comput. Chem. 2005, 26, 97−

105.

(107) Cheng, Y.; Prusoﬀ, W. H. Relationship between the inhibition

constant (K1) and the concentration of inhibitor which causes 50%

inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 1973,

22, 3099−3108.

(108) Cer, R. Z.; Mudunuri, U.; Stephens, R.; Lebeda, F. J. IC50-to-

Ki: a web-based tool for converting IC50 to Ki values for inhibitors of

enzyme activity and ligand binding. Nucleic Acids Res. 2009, 37,

W441−W445.

(109) Koumbis, A. E.; Kyzas, C. M.; Savva, A.; Varvoglis, A.

Formation of new alkynyl(phenyl)iodonium salts and their use in the

synthesis of phenylsulfonyl indenes and acetylenes. Molecules 2005, 10,

1340−1350.

(110) Kempson, J.; et al. Synthesis, initial SAR and biological

evaluation of 1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-4-amine

derived inhibitors of IkappaB kinase. Bioorg. Med. Chem. Lett. 2009, 19,

2646−2649.

(111) Malamas, M. S.; Robichaud, A. J.; Porte, A. M.; Morris, K. M.;

Solvibile, W. R.; Kim, J.-i. Amino-5-[4-(diﬂuoromethoxy)phenyl]-5-

phenylimidazolone Compounds for the Inhibition of β-Secretase.

Patent WO/2008/118379, 2008.

(112) Fischer, C.; Methot, J.; Zhou, H.; Schell, A. J.; Munoz, B.;

Rivkin, A. A.; Ahearn, S. P.; Chichetti, S.; MacCoss, R. N.; Kattar, S.

D.; Christopher, M.; Li, C.; Rosenau, A.; Brown, W. C. Triazole

Derivatives For Treatment of Alzheimer’s Disease. Patent WO/2010/

071741, 2010.

(113) Conte, A.; Kuehne, H.; Luebbers, T.; Mattei, P.; Maugeais, C.;

Mueller, W.; Pﬂieger, P. Anthranilamide/2-amino-heteroarene Carbox-

amide Derivatives. Patent WO/2007/090752, 2007.

(114) Carroll, W. A.; Kalvin, D. M.; Medrano, A. P.; Florjancic, A. S.;

Wang, Y.; Donnelly-Roberts, D. L.; Namovic, M. T.; Grayson, G.;

(121) Jiang, Y.; Kuang, C.; Yang, Q. Copper(I) Iodide-Catalyzed

Synthesis of 4-Aryl-1H-1,2,3-triazoles from anti-3-Aryl-2,3-dibromo-

propanoic Acids and Sodium Azide. Synthesis 2010, 2010, 4256−4260.

(122) Roppe, J.; Smith, N. D.; Huang, D.; Tehrani, L.; Wang, B.;

Anderson, J.; Brodkin, J.; Chung, J.; Jiang, X.; King, C.; Munoz, B.;

Varney, M. A.; Prasit, P.; Cosford, N. D. P. Discovery of novel

heteroarylazoles that are metabotropic glutamate subtype 5 receptor

antagonists with anxiolytic activity. J. Med. Chem. 2004, 47, 4645−

4648.

(123) Lyga, J. W.; Zawacki, F.; Zhang, L. Y. Phenylalkyl Substituted

Heteroaryl Derivatives. Patent WO/2006/130403, 2006.

(124) Buckler, R. T.; Hartzler, H. E.; Kurchacova, E.; Nichols, G.;

Phillips, B. M. Synthesis and antiinﬂammatory activity of some 1,2,3-

and 1,2,4-triazolepropionic acids. J. Med. Chem. 1978, 21, 1254−1260.

(125) Coelho, A.; Diz, P.; Caamano, O.; Sotelo, E. Polymer-

̃

Supported 1,5,7-Triazabicyclo[4.4.0]dec-5-ene as Polyvalent Ligands

in the Copper-Catalyzed Huisgen 1,3-Dipolar Cycloaddition. Adv.

Synth. Catal. 2010, 352, 1179−1192.

(126) Zhang, W.; Kuang, C.; Yang, Q. Palladium-Catalyzed One-Pot

Synthesis of 4-Aryl-1H-1,2,3-triazoles from anti-3-Aryl-2,3-dibromo-

propanoic Acids and Sodium Azide. Synthesis 2010, 2010, 287−283.

(127) Kallander, L. S.; Ryan, D. M.; Thompson, S. K. Compounds

And Methods. Patent WO/2003/031434, 2003.

(128) Makriyannis, A.; Pandarinathan, L.; Zvonok, N.; Parkkari, T.;

Chapman, L. Inhibitors of fatty acid amide hydrolase and

monoacylglycerol lipase for modulation of cannabinoid receptors.

Patent WO/2009/117444, 2009.

(129) Lu, L.-H.; Wu, J.-H.; Yang, C.-H. Preparation of 1H-1,2,3-

triazoles by cuprous ion mediated cycloaddition of terminal alkyne and

sodium azide. J. Chinese Chem. Soc. 2008, 55, 414−417.

(130) Takikawa, O.; Kuroiwa, T.; Yamazaki, F.; Kido, R. Mechanism

of interferon-gamma action. Characterization of indoleamine 2,3-

dioxygenase in cultured human cells induced by interferon-gamma and

evaluation of the enzyme-mediated tryptophan degradation in its

anticellular activity. J. Biol. Chem. 1988, 263, 2041−2048.

(131) Albert, A.; Serjeant, E. The Determination of Ionization

Constants, 3rd ed.; Chapman and Hall: New York, 1984.

(132) Pettersen, E. F.; Goddard, T.; Huang, C.; Couch, G.;

Greenblatt, D.; Meng, E.; Ferrin, T. E. UCSF ChimeraA

Visualization System for Exploratory Research and Analysis. J. Comput.

Chem. 2004, 25, 1605−1612.

(133) Trifonov, R.; Shcherbinin, M.; Ostrovskii, V. A. Behavior of 4-

Phenyl-1,2,3-triazole in Media of Various Acidities. Russ. J. Org. Chem.

1997, 33, 1046−1047.

(134) Abboud, J.-L. M.; Foces-Foces, C.; Notario, R.; Trifonov, R. E.;

Volovodenko, A. P.; Ostrovskii, V. A.; Alkorta, I.; Elguero, J. Basicity of

N-H− and N-Methyl-1,2,3-triazoles in the Gas Phase, in Solution, and

in the Solid StateAn Experimental and Theoretical Study. Eur. J.

Org. Chem. 2001, 2001, 3013−3024.

(135) CRC Handbook of Chemistry and Physics, 92nd ed.; Haynes, W.

M., Ed.; CRC Press: Boca Raton, FL, 2011−2012.

́

Honore, P.; Jarvis, M. F. Novel and potent 3-(2,3-dichlorophenyl)-4-

(benzyl)-4H-1,2,4-triazole P2X7 antagonists. Bioorg. Med. Chem. Lett.

2007, 17, 4044−4048.

(115) Musso, D. L.; Clarke, M. J.; Kelley, J. L.; Boswell, G. E.; Chen,

G. Novel 3-phenylprop-2-ynylamines as inhibitors of mammalian

squalene epoxidase. Org. Biomol. Chem. 2003, 1, 498−506.

(116) Yoo, E. J.; Chang, S. A new route to indolines by the Cu-

catalyzed cyclization reaction of 2-ethynylanilines with sulfonyl azides.

Org. Lett. 2008, 10, 1163−1166.

(117) Park, J. H.; Bhilare, S. V.; Youn, S. W. NHC-catalyzed oxidative

cyclization reactions of 2-alkynylbenzaldehydes under aerobic

conditions: synthesis of O-heterocycles. Org. Lett. 2011, 13, 2228−

2231.

(118) Jones, C. D.; Luke, R. W. A.; McCoull, W. Saminopyrimidine

Derivates With TIE2 Inhibiting Activity. Patent WO/2006/103449,

2006.

(119) Yates, K.; Mandrapilias, G. Vinyl cation intermediates in

solvolytic and electrophilic reactions. 2. Bromination of arylacetylenes.

J. Org. Chem. 1980, 45, 3902−3906.

(120) Cohrt, A. E.; Jensen, J. F.; Nielsen, T. E. Traceless azido linker

for the solid-phase synthesis of NH-1,2,3-triazoles via Cu-catalyzed

azide−alkyne cycloaddition reactions. Org. Lett. 2010, 12, 5414−5417.

5290

dx.doi.org/10.1021/jm300260v | J. Med. Chem. 2012, 55, 5270−5290

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