Azole-Based Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors

pubs.acs.org/jmc

Article

̈

Ute F. Rohrig,* Somi Reddy Majjigapu, Aline Reynaud, Florence Pojer, Nahzli Dilek,

Patrick Reichenbach, Kelly Ascencao, Melita Irving, George Coukos, Pierre Vogel, Olivier Michielin,*

and Vincent Zoete *

Cite This: J. Med. Chem. 2021, 64, 2205 −2227

Read Online

sı

*

Metrics & More

Article Recommendations

Supporting Information

ACCESS

ABSTRACT: The heme enzyme indoleamine 2,3-dioxygenase 1

(IDO1) plays an essential role in immunity, neuronal function, and

aging through catalysis of the rate-limiting step in the kynurenine

pathway of tryptophan metabolism. Many IDO1 inhibitors with

diﬀerent chemotypes have been developed, mainly targeted for use

in anti-cancer immunotherapy. Lead optimization of direct heme

iron-binding inhibitors has proven diﬃcult due to the remarkable

selectivity and sensitivity of the heme−ligand interactions. Here,

we present experimental data for a set of closely related small azole

compounds with more than 4 orders of magnitude diﬀerences in

their inhibitory activities, ranging from millimolar to nanomolar

levels. We investigate and rationalize their activities based on

structural data, molecular dynamics simulations, and density

functional theory calculations. Our results not only expand the presently known four conﬁrmed chemotypes of sub-micromolar

heme binding IDO1 inhibitors by two additional scaﬀolds but also provide a model to predict the activities of novel scaﬀolds.

There is an ongoing interest for IDO1 inhibitors capable of

modulating these diﬀerent pathways and processes.

1. INTRODUCTION

Recent developments in cancer immunotherapy have greatly

improved the prognosis of cancer patients, but low response

rates caused by tumoral immune resistance remain an unsolved

issue. Tryptophan catabolism along the kynurenine pathway

has emerged as an important mechanism in this ﬁeld,

motivating the development of small-molecule inhibitors of

indoleamine 2,3-dioxygenase 1 (IDO1) and of tryptophan 2,3-

dioxygenase (TDO), the enzymes catalyzing the rate-limiting

step in this pathway. However, despite compelling pre-clinical

and early clinical data, a phase 3 clinical trial (ECHO-301) of

the IDO1 inhibitor epacadostat (INCB024360) in combina-

tion with pembrolizumab failed to increase the overall and

progression-free survival when compared to pembrolizumab

alone.^1,2Potential reasons for the negative outcome of this

clinical trial and possible solutions have been suggested, such

as the use of more potent and more speciﬁc IDO1 inhibitors

and the use of dual IDO1/TDO inhibitors in combination

with intratumoral kynurenine level monitoring.³Alternative

targeting of the downstream eﬀector aryl hydrocarbon receptor

(AHR)⁴⁻⁶or the AHR-activating enzyme IL4I1 has also been

suggested.⁷Novel aspects of IDO1 biology have recently come

to light and might inﬂuence its role as an anti-cancer target,

such as its signaling activity,⁸⁻¹⁰regulation by heme

availability,¹¹nitrite reductase activity in hypoxic tissues,¹²

involvement in the redox signaling pathways of hydrogen

peroxide and singlet oxygen,¹³and activation by polysulﬁdes.¹⁴

We have previously classiﬁed the available IDO1 inhibitors

into four types according to their preferential binding and

inhibition mechanism.¹⁵Type I inhibitors preferentially bind

to oxygen-bound holoIDO1 (e.g., 1-methyl-^L-tryptophan), type

II inhibitors to free ferrous holoIDO1 (e.g., epacadostat), type

III to free ferric holoIDO1 (e.g., navoximod), and type IV to

apoIDO1 (e.g., linrodostat). Since L-trp competitive type I

inhibitors display the same relatively low (micromolar) aﬃnity

for IDO1 as the substrate, and type IV inhibitors were for the

ﬁrst time described in 2018,^11,16most of the inhibitor design

eﬀorts of the last 15 years concentrated on the heme−iron

binding type II and type III inhibitors.

Lead optimization of these inhibitors has proven diﬃcult in

the past due to the remarkable selectivity and sensitivity of the

heme−ligand interactions to changes in the electronic

structure of the ligand and due to the small size of the distal

heme pocket (pocket A, Figure 1).¹⁷In fact, there are only four

conﬁrmed chemotypes of sub-micromolar type II or type III

Received: November 13, 2020

Published: February 9, 2021

American Chemical Society

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

2205

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 1. Binding pockets A and B in IDO1-active site (PDB ID 6e40; ligand: epacadostat) and examples of nanomolar inhibitors with diﬀerent

heme-binding scaﬀolds. The iron-binding atom, as conﬁrmed by X-ray crystallography, is marked in blue.

IDO1 inhibitors, consisting of imidazoles, hydroxyamidines,

indazoles, and 1,2,3-triazoles (Figure 1).

providing a hitherto unknown heme-binding group. In the

following, many groups described N-hydroxyamidine deriva-

tives as IDO1 inhibitors, modifying mainly the extension in

pocket B and less frequently also the extension in pocket

A.^39,41−45

More recently, indazole has emerged as another potentially

high-aﬃnity heme-binding scaﬀold. In the patent literature,

they were ﬁrst described as TDO inhibitors, but over the last

years, a few sub-micromolar IDO1 inhibitors have also been

disclosed.⁴⁶⁻⁴⁹In addition, it has been suggested that they

preferentially bind to ferrous IDO1⁴⁸(type II inhibitors). This

scaﬀold might provide a good route to dual IDO1/TDO

inhibitors (Figure 1, compound 35).⁴⁹Structural data is only

available for an indazole bound to hTDO (PDB ID 6a4i) and

reveals a direct N2−Fe bond.

Finally, we have previously discovered 1,2,3-triazole as a

heme-binding scaﬀold yielding nanomolar IDO1 inhibi-

tors.^17,50The X-ray structure of the potent 4-phenyl-1,2,3-

triazole MMG-0358 (PDB ID 6r63)¹⁵conﬁrmed the predicted

binding mode with a direct iron-1,2,3-triazole bond. Such a

bond also exists in the case of 4-amino-1,2,3-triazole bound to

IDO1 (PDB ID 6f0a),⁵¹although this compound otherwise

shows a diﬀerent inhibition mechanism.¹⁵Recently, nanomolar

4,5-disubstituted 1,2,3-triazole IDO1 inhibitors occupying

both pockets A and B have also been described without

providing structural data.⁵²In general, structural data of direct

iron-1,2,3-triazole bonds is extremely scarce but has been

documented for seviteronel binding to human cytochrome

P450 17A1 (PDB ID 5irv).⁵³

1,2,4-Triazole is a common heme-binding scaﬀold present in

many antifungal drugs, and its direct iron binding in sterol

14α-demethylase (CYP51) is well documented by structural

data. However, until now, eﬀorts to develop IDO1 inhibitors

based on this scaﬀold were unsuccessful. We demonstrated

that imidazole antifungal drugs such as miconazole and

sulconazole consistently inhibited IDO1 at micromolar levels,

while all tested 1,2,4-triazole antifungals were inactive.⁵⁴

Bioisosteric replacement of imidazole by 1,2,4-triazole in 4-

phenylimidazole also yielded an inactive compound.⁵⁰The

only known IDO1 inhibitor with a 1,2,4-triazole scaﬀold is the

fused thiazolo-1,2,4-triazole Amg-1,⁵⁵which has been co-

Imidazoles are classical heme binders, and 4-phenyl-

imidazole is a known IDO1 ligand¹⁸and the ﬁrst co-

crystallized IDO1 inhibitor.¹⁹Structure-based optimization of

this chemotype yielded the 2-hydroxy-substituted phenyl

derivative (IC₅₀4.8 μM) as the most active compound.²⁰

Subsequent development also used pocket B (Figure 1) and

led to the discovery of navoximod (Figure 1), ﬁrst disclosed in

the patent literature and more recently in the scientiﬁc

literature.²¹Based on the pioneering work of New Link

Genetics, other groups developed imidazole-based IDO1

inhibitors.²²⁻²⁷While there are very few examples of 4-

phenylimidazoles of nanomolar potency, rigidiﬁcation of the

scaﬀold to imidazoleisoindole by a methylene linker fusing the

N-1 of the imidazole with the 2′-position of the phenyl ring

greatly improved the activity and ligand eﬃciency.²¹X-ray

structures of IDO1 in complex with imidazoleisoindoles

including navoximod have been reported (PDB IDs 5ek2,

5ek3, 5ek4, 6o3i).^21,22Recently, two nanomolar 1-benzyl-

imidazoles have also been described.²⁸In the absence of

structural data and in vitro inhibitory activities, the reported

benzimidazole IDO1 inhibitors with low nanomolar activities

in cellular assays²⁹might in fact be apoIDO1 binding type IV

inhibitors.

Providing a variation of the imidazole scaﬀold, nanomolar

imidazothiazole-based IDO1 inhibitors were disclosed in 2014

by researchers from Sumitomo Dainippon Pharma, including

both structural and functional data (Figure 1, compound

17g).³⁰Subsequent works on this scaﬀold, however, showed

that these compounds do not inhibit IDO1 in a cellular

context.^31,32Replacement of the phenyl ring in pocket A with a

cyclopropylethynyl group and of the urea function by a

thiourea function led to a compound with increased in vitro

activity but still lacking cellular activity.³³

The second high-aﬃnity heme iron-binding scaﬀold, the N-

hydroxyamidines including epacadostat (Figure 1), has been

pioneered by researchers from Incyte.^34,35Structural data

(PDB IDs 5xe1, 5wn8, 6e40, 6e41, and 6pu7)³⁶⁻³⁹and

quantum chemical computations⁴⁰established that this

scaﬀold binds through an oxygen atom to the heme iron,

2206

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Table 1. Enzymatic IC₅₀Values [μM] for rhIDO1 Inhibition, Mean of at Least Two Independent Assays

a

b

Standard deviations are given in parentheses. For more details, see the Supporting Information, Table S1. Only one assay.

crystallized with IDO1 (PDB ID 4pk5)³⁰and displays an

activity in the low micromolar range.

magnitude.⁵⁰Here, we systematically investigated the inﬂuence

of these two substitutions separately and in combination upon

a series of six diﬀerent heme-binding azoles. We added the 2-

amino substitution and its combination with the 5-chloro

substitution to the investigation because it showed a diﬀerent

inﬂuence upon inhibition activities than the 2-hydroxy

substitution and led to some highly active compounds

(Table 1). All compounds in Table 1, combining six diﬀerent

heme-binding azoles with six diﬀerent phenyl ring substitu-

tions, were bought or synthesized and tested for their IDO1

inhibitory activity. Surprisingly, they displayed activities

ranging from 3 mM to 35 nM, spanning more than 4 orders

of magnitude. Large activity diﬀerences are seen both between

diﬀerent azoles and between diﬀerent phenyl ring substitu-

tions. Interestingly, the inﬂuence of the phenyl ring

substitutions and of the azole ring upon the activity depend

on each other and show little correlation (Figure 2A,B).

We decided to look at this intriguing data set of highly

similar small compounds with highly disparate activities in

more detail with the aim to understand favorable and

unfavorable factors for IDO1 inhibitory activity and to apply

this knowledge to future inhibitor design.

2.2. Chemical Synthesis. The imidazole, triazole, and

tetrazole derivatives were synthesized using the existing

protocols or procedures adapted from the literature.

Compounds 4a−4f have been described previously by us.⁵⁰

Other compounds were synthesized according to literature

procedures [(1b, 1c, 1e),²⁰(1d, 1f),^20,59(2a−f),⁵⁹⁻⁶¹(3c,

3e),^61,62(3f),⁶²(5b−c, 5e−g),⁶³(6c, 6e−f),⁶⁴9,⁶⁵15,⁶⁶17,⁶⁷

20,⁶⁸21,⁶⁹and 23].⁷⁰Brieﬂy, the 4-phenylimidazole

derivatives 1b−c and 1e were obtained by the reaction of α-

There are very few examples of heme-binding tetrazoles in

the literature. However, they have been exploited by Viamet

Pharmaceuticals with the rationale to replace the imidazole or

1,2,4-triazole iron binding group in antifungals by a weaker

iron-binding group in combination with potency-enhancing

modiﬁcations in other parts of the compounds to achieve a

better selectivity for CYP51B.^56,57No nanomolar tetrazole

IDO1 inhibitor has been reported to date.

Here, we report IDO1 enzymatic inhibition data for 36

closely related small heterocyclic compounds, including 12

known^{17,20,21,50,54,58}and 24 new IDO1 ligands, spanning more

than 4 orders of magnitude of activities on IDO1. They include

the ﬁrst nanomolar 1,2,4-triazole and tetrazole inhibitors. For

the most active compounds, we determined cellular IC₅₀and

toxicity values. We provide three new X-ray structures of azole-

bound IDO1 to support our structural hypotheses and

rationalize the observed activities with the support of classical

molecular dynamics (MD) simulations and static quantum

mechanical (QM) calculations. The resulting model can be

applied to predict activities of novel scaﬀolds and to meet the

challenge of discovering novel high-aﬃnity IDO1 inhibitor

scaﬀolds.

2. RESULTS AND DISCUSSION

2.1. Azole Compounds with Chloro-, Hydroxy-, and

Amino-Substituted Phenyl Rings. For the 4-phenyl-1,2,3-

triazole scaﬀold, we have previously shown that introduction of

two substituents on the phenyl ring, namely, 2-hydroxy and 5-

chloro, improved the IC₅₀values by more than 2 orders of

2207

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 2. Experimentally determined enzymatic activities. The activity Act of each compound was calculated as Act = −RT ln IC₅₀in analogy to the

relation between the binding free energy and measured K_ivalues. (A) Absolute activites. (B) Change in activities due to phenyl ring substitutions.

(C) Additivity of substituent eﬀects. Calc. stands for the calculated sum of substituent eﬀects (ΔAct(Cl) + ΔAct(OH/NH₂)), while exp. stands for

the experimentally measured eﬀect of the di-substituted compounds (ΔAct(Cl + OH/NH₂)). Left side: additivity of chloro/hydroxy eﬀects; right

side: additivity of chloro/amino eﬀects.

a

Scheme 1

a

Reagents and conditions: (a) formamide, 170−180 °C, rt, and 4 h and (b) FeCl₃, N₂H₄·H₂O, activated carbon, MeOH, rt−70 °C, and 48 h.

bromoketone derivatives (36b−c, 36e) with formamide

(Scheme 1). The amino derivatives 1d and 1f were obtained

from the nitro arenes 37 and 38 through reaction with

formamide, followed by reduction with N₂H₄·H₂O/FeCl₃

(Scheme 1). Compounds 2a and 2b were obtained by the

reaction of the corresponding aryl iodides 39a and 39b with N-

methyl imidazole in the presence of tricyclohexyl phospho-

nium tetraﬂuoroborate, Cs₂CO₃, and Pd(OAc)₂in DMF at

120 °C. The same procedure applied to aryl iodides 42 and 43

produced the corresponding nitro derivatives that were then

reduced with N₂H₄·H₂O/FeCl₃to produce 2d and 2f.

Similarly, the corresponding aryl methyl ethers were obtained

from aryl iodides 40 and 41. Demethylation with 48% HBr in

H₂O at 100 °C yielded compounds 2c and 2e (Scheme 2).

Aryl iodide 39f reacted with imidazole in the presence of N,N′-

dimethylethylenediamine, Cs₂CO₃, and CuI in DMF at 140 °C

to produce 3f. In the same way, the aryl methyl ethers 40 and

41 were converted to the corresponding N-aryl imidazoles,

followed by demethylation with 48% HBr in H₂O at 100 °C to

produce phenols 3c and 3e, respectively (Scheme 3). The

2208

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 2

a

Reagents and conditions: (a) tricyclohexylphosphonium tetraﬂuoroborate, Cs₂CO₃, Pd(OAc)₂, DMF, 120 °C, and 15 h; (b) 48% HBr in H₂O,

100 °C, and 14 h; and (C) FeCl₃, N₂H₄·H₂O, activated carbon, MeOH, rt−70 °C, and 48 h.

a

Scheme 3

a

Reagents and conditions: (a) N,N′-dimethylethylenediamine, Cs₂CO₃, Cul, DMF, 140 °C, and 12 h and (b) 48% HBr in H₂O, 100 °C, and 14 h.

a

Scheme 4

a

Reagents and conditions: (a) N,N-dimethyl formamide-diethyl acetal (DMF-DMA), reﬂux, and 1 h and (b) N₂H₄·H₂O, AcOH, 90 °C, and 1.5 h.

triazoles 5b−c and 5e−g were obtained from benzamides

44b−c and 44e−g through condensation with N,N-dimethyl

formamide-dimethyl acetal (DMF-DMA), followed by cycliza-

tion with hydrazine hydrate in acetic acid at 90 °C (Scheme 4).

Tetrazoles 6c and 6e−f were prepared by reaction of the

corresponding arene carbonitriles 45c and 45e−f with sodium

azide and triethylamine hydrochloride in toluene at 99 °C

(Scheme 5).

Compound 9 was synthesized by N-arylation of pyrazole

with phenyl iodide, pyrazole, and Cs₂CO₃in DMF at 110 °C

(Scheme 6). For compound 15, phenylacetic acid underwent a

double Vilsmeier−Haack reaction, followed by hydrolysis with

NaOH and treatment with SOCl₂in DCM to yield the

2209

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 5

a

Reagents and conditions: (a) NaN₃, Et₃N·HCl, toulene, rt−90 °C, and overnight.

a

Scheme 6

a

Reagents and conditions: (a) Cu(OAc)₂·H₂O, Cs₂CO₃, DMF, 110 °C, and 24 h.

chloroacrolein, which was heated in DMF at 70 °C with

NH₄SCN to yield compound 15 (Scheme 7). Compound 17

stopping the reaction in its linear phase. Kynurenine and L-trp

concentrations were determined by HPLC through UV

detection. The measured IC₅₀values are given in Table 1,

while dose−response curves and Hill slopes can be found in

the Supporting Information (Figure S1). We chose to test

compounds at maximal concentrations of 1 mM to be able to

also detect weak binders. The most active compound, 5f, is a

tight binder with the IC₅₀value in the range of the enzyme

concentration. We therefore determined its apparent K_ivalue

using the Morrison equation.^72,73

From Table 1 and Figure 2A,B, it is clear that for the

unsubstituted parent compounds (a), the 1,2,3-triazole hetero-

cycle is the most potent, the imidazoles are of intermediate

activity, and the tetrazole performs the worst. The 5-chloro

substitution (b) is favorable for all scaﬀolds and does not

change this ordering. The 2-hydroxy substitution (c) is also

favorable for all scaﬀolds, but in a more diverse way, changing

also the ordering. This is even more strikingly the case for the

2-amino substitution (d), which is very favorable for the 1,2,4-

triazole and the tetrazole but neutral or unfavorable for the

other scaﬀolds. The combination of 2-hydroxy or 2-amino

substitution with the 5-chloro substitution (Figure 2C)

generally produces additive results, except for the 1-methyl-

imidazole scaﬀold (red bar), where the eﬀect of combining the

two substituents is smaller than the sum of the eﬀects of the

single substituents. The strongest substitution eﬀects are in all

cases seen for the 1,2,4-triazole scaﬀold, yielding IC₅₀values

ranging from 1.8 mM to 35 nM, while the biggest dependence

on the character of the azole ring was detected for the 2-

amino-5-chloro substitution (IC₅₀values of 1.2 mM to 35

nM).

a

Scheme 7

a

Reagents and conditions: (a) POCl₃, DMF, 85 °C, and 2 h; (b)

NaOH, EtOH/H₂O, reﬂux, and 1 h; (c) SOCl₂, CH₂Cl₂, 0 °C, and 1

h; and (d) NH₄SCN, DMF, 70 °C, and 16 h.

was obtained from the treatment of methyl benzenecarbo-

thioate with TMSCHN₂and n-BuLi at −78 °C (Scheme 8).

Compound 20 was synthesized by the coupling reaction of

benzaldehyde with TosMIC in the presence of K₂CO₃in

MeOH (Scheme 9). The 4-phenylisoxazole 21 was obtained

from 2-phenylmalonaldehyde and NH₂OH·HCl in EtOH at

80 °C (Scheme 10). Compound 23 was prepared by the

reaction of benzohydrazide with triethyl orthoformate and p-

TsOH in toluene at reﬂux (Scheme 11). Compounds 1a, 3a,

3b, 3d, 5a, 5d, 6a, 6b, 6d, 22, and 29 were bought from Sigma-

Aldrich and compound 7 from Fluorochem.

2.3. Enzymatic Activities. The enzymatic IC₅₀values were

measured as previously described.⁷¹Brieﬂy, ascorbate and

methylene blue were used as reductants and catalase to remove

hydrogen peroxide. Triton-X was added to inhibit compound

aggregation, and a substrate concentration of 100 μM L-trp

was chosen to minimize substrate inhibition. With an

estimated IDO1 concentration of about 40 nM (2.5 μg/mL),

an incubation time of 20 min at room temperature allowed for

a

Scheme 8

a

Reagents and conditions: (a) n-BuLi, Et₂O/H₂O, -78 °C (30 min), 0 °C (30 min), and rt (1 h).

2210

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 9

a

Reagents and conditions: (a) K₂CO₃, MeOH, 80 °C, and 2 h.

imidazole (compound 1b, PDB ID 6e42),³⁸and MMG-0358

(compound 4e, PDB ID 6r63).¹⁵All the three compounds

bind similarly to the heme iron of IDO1 through an azole ring

nitrogen (Figure 4A−C).

a

Scheme 10

The chloro substituent in the latter two is oriented toward a

small hydrophobic cavity between Cys129, Leu234, and

Gly262 (Figure 4B,C). The closest contact (3.2−3.4 Å) exists

with the sulfur atom of Cys129 and is smaller than the sum of

their van der Waals radii. However, the angle C−Cl···S of 95−

112° is in disagreement with a halogen bond, where this angle

should be close to 180° due to the σ-hole of the C−Cl

bond.^74,75Another contact exists between the chloro

substituent and the backbone amide carbon of Gly262 (3.6

Å), forming a characteristic CO···Cl angle of about 60°. This

“side-on carbon interaction” between the electronegative belt

around halogen atoms and the positively polarized amide

carbon has been found in the crystallographic structures of

diﬀerent small molecules⁷⁶and ligand−protein complexes.⁷⁷

Hydrophobic contacts, which are frequently observed for

chlorine,⁷⁷exist mainly between the Cl and the side chain of

Leu234 (Figure 4C). Taking all evidence into account, we

suggest that the chloro primarily interacts via van der Waals

and dipolar interactions with the IDO1 binding pocket, in

addition to modulating the electronic properties of the azole

ring. This is at variance with earlier suggestions of a chlorine−

cysteine halogen bond,³⁶which are known to be extremely

rare.⁷⁷

The hydroxy group of MMG-0358 forms a hydrogen bond

with the side chain of Ser167, probably donating the hydrogen,

because the side chain of Ser167 simultaneously donates a

hydrogen bond to the backbone oxygen of Phe163 (Figure

4C). The importance of this interaction has been demon-

strated previously by the strongly increased IC₅₀value of

compound 1c in the Ser167Ala hIDO1 mutant (41 μM) as

compared to the wild type (1.2 μM).⁷⁸

Here, we co-crystallized compounds 4f and 5f with rhIDO1

and resolved the X-ray structures of the complexes. Addition-

ally, we co-crystallized an analogue of 5f, where the chloro

substituent is replaced by a bromo substituent (5g).

Compound 5g showed an even better enzymatic IC₅₀value

(0.020 0.001 μM) than 5f, and the bromo substituent is very

amenable to X-ray crystallography because of its high electron

density. The 2F_o− F_cmaps of the bound ligands are shown in

the Supporting Information (Figure S4).

a

Reagents and conditions: (a) NH₂OH·HCl, EtOH, reﬂux, and 2 h.

In the following, we ﬁrst describe the cellular activities of

compounds 1a to 5f before rationalizing the observed

enzymatic activities based on structural and theoretical data.

2.4. Cellular Activities. We tested the cellular hIDO1

inhibitory activity (Figure 3A) and toxicity (Figure 3B) of all

active compounds at a concentration of 200 μM (Table S1) to

be able to also detect weak cellular inhibitors. As some aniline

compounds reacted with Ehrlich’s reagent (p-dimethylamino-

benzaldehyde, p-DMAB), the kynurenine content of these

samples had to be determined by HPLC (Figure S3). In

general, the compounds displayed very little cytotoxicity under

the experimental conditions; only the amino-substituted 1,2,3-

triazoles showed up to 30% of toxicity.

Cellular inhibition was observed to be closely related to

enzymatic activity, showing a sigmoidal dependence (Figure

3C). The 1-phenyl-imidazole 3e is an outlier of this

dependence, showing much lower cellular activity than

expected for an unknown reason. We proceeded to determine

cellular IC₅₀values for the 10 most active compounds

(Supporting Information, Table S1, Figure S2). Figure 3D

shows the correlation between cellular and enzymatic activities,

including our previously published cellular data for compounds

4a, 4b, and 4c.⁵⁰There is generally a good correlation, except

for the tetrazole 6f, which displays a 16 times higher enzymatic

than cellular activity. This might be due to the presence of a

negative charge on the tetrazole under physiological con-

ditions, hampering cellular uptake. Compounds 1e (0.88

0.16 μM), 4b (0.62 0.01 μM),⁵⁰4e (0.11 0.04 μM), and

5f (0.034 0.011 μM) show nanomolar IC₅₀values also in the

cellular assay (Figure 3E). In summary, the cellular assay data

shows that most azoles are as active in the cellular environment

as in the in vitro assay without signiﬁcant toxicity, supporting

their interest for drug development.

2.5. Structural Data for Azole Compounds. Structural

data was available for three of the compounds, 4-phenyl-

imidazole (compound 1a, PDB ID 2d0t),¹⁹4-(5-Cl-phenyl)-

a

Scheme 11

a

Reagents and conditions: (a) p-TsOH, toulene, reﬂux, and 2 h.

2211

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 3. Cellular inhibition data and its comparison to enzymatic inhibition data. (A) Cellular inhibition of kynurenine production at a compound

concentration of 200 μM. (B) Cellular toxicity under the same conditions. (C) Cellular inhibition compared to enzymatic activity. The orange line

is a sigmoidal ﬁt to all data points except for the outlier of compound 3e. (D) Correlation between cellular activity and enzymatic activity. Color

code given in the legend of part B. (E) Cellular dose−response curves of compounds 1e, 4e, and 5f.

The 1,2,3-triazole 4f (PDB ID 7ah4, Figure 4D) binds to

IDO1 in the same way as 4e, with the triazole ring directly

bound to the heme iron, the chloro substituent in the

hydrophobic cavity close to Cys129 (distance 3.4 Å) and

Gly262 (distance 3.7 Å), and the amino instead of the hydroxy

group making a hydrogen bond to Ser167 in the back of

pocket A. The orientation and protonation state of the 1,2,3-

triazole ring cannot be unequivocally determined from the

electron density. In analogy to the structure of 4e, the

conformation shown in Figure 4D was selected based on the

proximity between the N3 atom of the triazole ring and the

backbone amide nitrogen of Ala264 (3.0 and 2.7 Å in chains A

and B, respectively). In this conformation, the negatively

charged triazole could make a favorable electrostatic

interaction with the NH hydrogen. In both asymmetric

subunits, a second ligand is bound to pocket D on the

proximal heme side, roughly parallel to the heme plane. This

ligand binding site, which has been observed by several groups

since 2017,^15,37,38is opened by a conformational change of

Phe270, clearly seen here in the electron density. In the case of

ligand 4f, there seem to be two orientations of the ligand in

pocket D present (Figure 4G), the slightly higher populated

one (shown in magenta) similar to the one of compounds 4e

and 1b, with the triazole pointing toward His346 and the

chloro substituent toward a hydrophobic aliphatic pocket

formed by Val170, Ala174, Leu207, and Ala210. The other

ligand pose (dark green) is roughly rotated by 180°, with the

phenyl ring pointing toward the heme and the chloro

substituent toward His346. This ligand pose makes a hydrogen

bonding interaction with Asp274 and Phe270 bridged by a

solvent molecule.

of ferric heme but negative in the case of ferrous heme. This

supports the earlier formulated hypothesis that the 1,2,3-

triazoles bind in their deprotonated form to ferric heme. On

the other hand, the ligands in pocket D are most likely present

in their neutral form.

The X-ray structure of 5f bound to IDO1 demonstrates that

the 1,2,4-triazole binds very similar to pocket A as the 1,2,3-

triazole (PDB ID 7ah5, Figure 4E) with the chloro substituent

close to Cys129 and Gly262 and the amino group pointing to

the back of pocket A. The amino group simultaneously donates

two hydrogen bonds to Ser167 and to the N2 atom of the

triazole ring. Compound 5f is also detected in pocket D,

adopting a distinct binding mode, with the phenyl ring

pointing toward the heme, but at variance with compound 4f

pointing the chloro substituent toward Phe214 and the amino

group toward Arg343 (Figure 4H).

Compound 5g (PDB ID 7ah6) binds very similarly to

pocket A as compound 5f. The bromo substituent is located at

a distance of 3.6 Å from the sulfur atom of Cys129 and at 3.5 Å

from the backbone amide carbon of Gly262. Inside pocket D

(Figure 4I), it adopts yet another binding mode, occupying the

same plane but pointing the triazole toward Phe214 and the

amino group toward the heme propionate donating a hydrogen

bond to it (distance 2.8 Å).

In summary, ligands binding to pocket D all adopt a planar

conformation with respect to the heme plane but can adopt

diﬀerent conformations inside this plane depending on the

scaﬀold and the substituents. The halogen substituent is

located either in a hydrophobic aliphatic pocket or close to the

His346 or Phe214 aromatic rings. These interactions could

contribute to ligand binding in pocket D, as the unsubstituted

compound 1a was not detected in this pocket. However, the

absence of speciﬁc interactions suggests a low-aﬃnity site

potentially only accessible at the very high ligand concen-

trations used for crystallization.

Interestingly, the electrostatic potential created by the

protein and the heme cofactor in pocket D is negative,

regardless of the oxidation state of the heme iron (Figure 5).

However, the electrostatic potential in pocket A strongly

depends on the iron charge, being slightly positive in the case

2212

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 4. X-ray crystal structures of hIDO1 in complex with (A) compound 1a (PDB ID 2d0t), (B) compound 1b (PDB ID 6e42), (C) compound

4e (PDB ID 6r63), (D,G) compound 4f (PDB ID 7ah4), (E,H) compound 5f (PDB ID 7ah5), and (F,I) compound 5g (PDB ID 7ah6). The

structures of 4f, 5f, and 5g have been resolved for the present work. Ligand carbon atoms in pocket A are highlighted in orange, in pocket D in

magenta/dark green. Hydrogen bonds are displayed as green lines. (A−F) Pocket A: Distances of halogen substituents with the closest protein

contacts are indicated and the carbon−halogen−sulfur angle is given. (G−I) Ligand binding poses in pocket D are shown. For 4f, two alternative

conformations are detected and depicted in magenta and dark green, respectively.

Figure 5. Electrostatic potential generated by the protein and the heme cofactor in pockets A and D of holoIDO1 (PDB ID 6r63). (A) Ferric

heme. (B) Ferrous heme.

2213

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 6. Enzymatic dose−response curves measured at diﬀerent pH values for an imidazole (1a), a 1,2,3-triazole (4e), and a 1,2,4-triazole (5a).

While the activity of the imidazole is pH-insensitive, both the 1,2,3-triazole and the 1,2,4-triazole are more active at higher pH.

Figure 7. DFT calculations. (A) Correlation between the calculated Hirshfeld charge on the iron-binding nitrogen atom and the experimentally

measured activity for unsubstituted parent compounds. For 1,2,4-triazole 5a, the charges for the negatively charged compound (orange left

triangle), for the neutral compound (purple left triangle), and their average (black left triangle) are shown. (B) The same correlation for m-chloro-

substituted compounds (red). (C) Iron−nitrogen bond lengths in a ferric heme model system relative to the unsubstituted parent compounds. (D)

Optimized geometries of 2-amino-substituted compounds in ferric heme models.

2.6. Protonation States of Active Species. It is known

that ligand−protein binding,⁷⁹and especially ligand binding to

ferric hemoproteins,⁸⁰⁻⁸²induces large pK_ashifts in ligands. In

our earlier works on 4-phenyl-1,2,3-triazole IDO1 inhibitors,

we concluded that this scaﬀold most likely binds in its

deprotonated, negatively charged form to the ferric heme

cofactor.⁵⁰More recently, we corroborated preferential binding

of compounds 1a and 4e to ferric IDO1 by experimental

techniques and reported density functional theory (DFT)

calculations, suggesting that the deprotonation of compound

4e is energetically much more favorable when bound to ferric

heme than in solution.¹⁵

Here, we tested compounds 1a, 4e, and 5a in the enzymatic

assay at two diﬀerent pH values, namely, at pH 6.5 and at pH

7.4 (Figure 6). We did not test other imidazoles for their pH

dependence, as they can only bind to holoIDO1 in their

neutral form. We also did not test the tetrazoles, as their low

pK_avalue in solution (4.53)⁸³ensures their prevalent existence

in their negatively charged form at physiological pH values. At

both pH values, the enzymatic reaction is about equally

eﬃcient. While the IC₅₀value of imidazole 1a remains

constant, the IC₅₀of 1,2,3-triazole 4e shifts to a lower value

at higher pH, as shown previously.⁵⁰The same is true for the

1,2,4-triazole 5a, supporting the hypothesis that the 1,2,4-

triazoles bind stronger to IDO1 in their deprotonated form.

In summary, our results suggest that the imidazoles bind in

their neutral form to ferric holoIDO1, while the 1,2,3-triazoles

and tetrazoles bind in their anionic form to it. The activity of

the 1,2,4-triazole 5a shows a pH dependence and is likely to

bind to IDO1 in its deprotonated form.

2.7. Molecular Modeling. The activity of the azole

compounds is determined by their binding free energies to

holoIDO1, which are inﬂuenced by the ligand−protein and the

ligand−heme interaction energies, the changes in conforma-

tional energies, the changes in solvation energies, and the

entropic changes due to ligand binding. For an in-depth

investigation of all of these factors, the dynamics and the

electronic details of the systems should be taken into account

at a high level of theory to accurately describe transition metal

interactions. As this would be prohibitively computing

intensive, we tried instead to deduce trends from static QM

calculations and from classical MD simulations.

2.8. QM Calculations. To investigate the interaction

energy between the ligands and the heme cofactor, we

optimized the structures of the 36 azole compounds using

DFT calculations. For all ligands with more than one local

2214

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 8. MM-GBSA calculations for azole compounds. (A) Non-polar contributions of substituted compounds relative to their parent compound.

(B) Sum of all polar contributions of substituted compounds relative to their parent compound. Standard errors are indicated by error bars. The

absolute values can be found in the Supporting Information, Figure S7.

energy minimum, we assumed the conformation where the o-

hydroxy and o-amino groups could donate a hydrogen bond to

Ser167 and the m-chloro substituent would be located in the

identiﬁed hydrophobic subpocket (Supporting Information

Figure S5). After careful investigation of many variables and

diﬀerent charge partitioning schemes, we chose to use the

atomic Hirshfeld charge on the iron-binding nitrogen atom as a

proxy for the basicity of the site and its heme binding strength

because this was the only observable that correlated with

known basicities of diﬀerent scaﬀolds and that reproduced the

ordering of the activities of the six parent compounds.

As shown in Figure 7A, there is a correlation between the

charge on that atom and the activity of the compound. The

more basic or electron-rich the nitrogen atom is, the higher the

activity. The deprotonated 1,2,3-triazole 4a has the most

negative charge (−0.245), the tetrazole 6a the least negative

charge (−0.186), and the imidazoles display charges in

between these values. For the 1,2,4-triazole 5a, we calculated

the charge both for the neutral (purple left triangle) and for the

negatively charged (orange left triangle) compound. Interest-

ingly, neither value obeys the correlation found for the other

compounds, but the average of both values (black left triangle)

provides the best correlation (correlation coeﬃcient 0.862,

standard deviation 0.69 kcal/mol). This shows that the

deprotonated form of 5a has a very high aﬃnity for IDO1,

but the neutral form is prevalent under experimental

conditions. More sophisticated pK_a-based models may be

used to estimate the ratio of deprotonated to neutral

compound but are unlikely to yield additional insights. The

activity of the 4-phenyl-imidazole 1a is underestimated by the

regression for an unknown reason.

compound activity becomes less clear (Supporting Informa-

tion, Figure S6).

In order to better understand the eﬀects of the ortho

substituents, we optimized the structures of the azoles bound

to a ferric iron−porphin−imidazole system as a model of the

heme cofactor. Solvation eﬀects were taken into account via a

polarizable continuum model. We chose to use the ligand

nitrogen to heme iron bond distance as a proxy for the binding

strength because this avoids the use of an unbound ligand

reference structure, which is challenging to unambiguously

deﬁne. All optimized structures are shown in the Supporting

Information Figure S7, and the calculated absolute bond

lengths are shown in Figure S8.

Not surprisingly, the results of the calculations (Figure S7)

show no correlation to the IDO1 inhibitory activities (Figure

2A) because all protein interactions are neglected. However, a

few things can be deduced from the results. For all compounds,

the 5-chloro substitution leads to a decrease in binding

strength, while the 2-hydroxy substitution leads to an increase

in binding strength (Figure 7C). Interestingly, the 2-amino

substitution is favorable only for the 1,2,4-triazole and the

tetrazole and unfavorable for all other scaﬀolds. This is

probably related to the fact that only in the 1,2,4-triazole and

the tetrazole, the 2-amino substituent can make a favorable

electrostatic interaction with a nitrogen atom (leading to an

essentially planar conformation), while in all other scaﬀolds, it

is oriented toward a CH group, leading to a twisted

conformation (Figure 7D), unfavorable in the protein

environment.

2.9. Molecular Mechanics-Generalized Born Surface

Area Calculations. Classical MD simulations suﬀer from

shortcomings in the ligand force ﬁelds and in describing the

electronic interactions with the heme cofactor, but they give

insight into the dynamics of ligand−protein interactions. The

molecular mechanics-Generalized Born surface area (MM-

GBSA) is a popular method for free energy prediction, where

the binding free energy ΔG_bindis decomposed into the sum of

the gas-phase binding enthalpy, the desolvation free energy,

and an entropic contribution.⁸⁴The electrostatic part of the

desolvation free energy is estimated using a Generalized Born

(GB) model, while the non-polar part is assumed to be

proportional to the solvent-accessible surface area. Because of

Figure 7B shows the same data as part A but for the activities

of the m-chloro-substituted compounds. A very similar

correlation emerges between the diﬀerent azole scaﬀolds, but

all values are shifted toward a better activity, although the

magnitude of charges decreases for each scaﬀold due to the

electron-withdrawing substituent. This demonstrates that the

m-chloro substituent increases activity mainly by interaction

with the protein environment, as supported by the structural

data. Again, the average charge of the 1,2,4-triazole 5b ﬁts well

with the regression. For the compounds with ortho

substituents, the correlation between atomic charge and

2215

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 9. QSAR relationships. (A) Using four binary variables as descriptors (see text). (B) Using three binary variables and the charge density on

the iron-binding nitrogen atom as descriptors.

its computational eﬃciency and accuracy, MM-GBSA has been

widely used in biomolecular studies and in drug design.^85,86

Here, we carried out MM-GBSA calculations for all

compounds of Table 1. During the MD simulations, a covalent

iron−ligand nitrogen bond was introduced, but for analysis, all

ligand interactions with the heme porphyrin and His346 were

excluded because they are badly described within the classical

approach and introduce high ﬂuctuations. The largest

contributions to the binding free energies stem from residues

Ala264, Ser167, Ser263, Tyr126, Gly262, and Val130

(Supporting Information, Figure S9C).

the presence of a o-amino substituent making an intra-

molecular interaction with a nitrogen atom, and (iv) the

presence of a 4-phenyl-imidazole or a 4-phenyl-1,2,3-triazole

scaﬀold. The last variable captures the observed preference of

hIDO1 to bind to these scaﬀolds, which can be explained by

the basicity or negative charge density on the iron-binding

nitrogen atom (Figure 7A). In fact, the fourth variable can be

replaced by the Hirshfeld charge on the iron-binding nitrogen

atom of the parent compound for each scaﬀold. This provides

a correlation with a correlation coeﬃcient of 0.893 (sd 0.821

kcal/mol) and a correlation coeﬃcient of 0.865 (sd 0.917 kcal/

mol) with the leave-one-out method (Figure 9B).

The electrostatic interactions favor the neutral ligands over

the negatively charged ones (Figure S9A) and are slowly

converging, especially for the charged ligands. The relative

ordering of the binding free energies of the diﬀerent azole

scaﬀolds does not correlate with the experimental data as

expected from the neglect of part of the interactions. We

therefore display just the diﬀerences due to the phenyl

substituents in Figure 8. It can be concluded from the results

that (i) the 5-chloro substituent makes favorable interactions

with the protein, mainly through non-polar (van der Waals)

forces; (ii) the 2-hydroxy substituent makes favorable

interactions with the protein, mainly through electrostatic

interactions; and (iii) the 2-amino substituent is favorable for

some compounds and detrimental for others. As expected, the

2-hydroxy and the 2-amino substituent mainly inﬂuence the

interaction with Ser167 in the active site (Figure S9B).

2.10. Quantitative Structure−Activity Relationship

and Application to Other Compounds. With the

information gathered by classical and quantum chemical

calculations, we developed a new quantitative structure−

activity relationship (QSAR) model, as the one we previously

developed for 1,2,3-triazoles⁵⁰proved not to be transferable to

other heterocycles. Despite many trials, QSAR models taking

into account the MM-GBSA and DFT energies and

combinations thereof did not display a good correlation with

the experimentally measured values. However, a multiple linear

regression on a simple binary matrix provided a correlation

coeﬃcient of 0.922 with a standard deviation of 0.706 kcal/

mol (Figure 9A) and a correlation coeﬃcient of 0.896

(standard deviation 0.812 kcal/mol) with the leave-one-out

method. The four variables are (i) the presence of a m-chloro

substituent, (ii) the presence of a o-hydroxy substituent, (iii)

From the graphs, it is clear that the second model

underestimates the 4-imidazole activities (black) and over-

estimates the activities of the other imidazoles (green and red),

which is probably due to steric factors unaccounted for in the

model.

We applied this last model to other unsubstituted scaﬀolds

of similar shape and type, compounds 7−35 shown in Figure

10. For each compound, we optimized the structure and

calculated the Hirshfeld charge on the hypothetically iron-

binding nitrogen atom, circled in blue. We tested compounds

7, 9, 15, 17, 20−23, and 29 in the enzymatic assay. In Figure

10, enzymatic activity is indicated by a green frame even if the

IC₅₀value is the millimolar range, inactivity by a red frame, and

absence of experimental data by no frame (for details, see

Table S3 in the Supporting Information). It is clear that the

Hirshfeld charges, which are related to the basicity of the

nitrogen atom, allow to distinguish between active and inactive

scaﬀolds. With two exceptions (compounds 15 and 34), all

compounds with a charge smaller than −0.18 are inactive.

With one exception (compound 28), all compounds with a

charge larger than −0.18 are active. This could indicate that

sterically the benzo-compounds 26 to 29 are less favorable

than the phenyl compounds because of suboptimal ﬁlling of

pocket A. Scaﬀolds 30 and 31 have been used before in IDO1

inhibitors, and interestingly, a large increase in activity was

observed when passing from scaﬀold 31 to 30, in agreement

with the higher charge density on the nitrogen.^30−33,55

Interestingly, our results suggest testing compound 10, as it

is predicted to be active on IDO1.

2216

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 10. Other potential IDO1 inhibitor scaﬀolds. Hirshfeld charges on the nitrogen atom circled in blue are indicated. Inactive compounds are

framed in red, active ones in green.

3. CONCLUSIONS

4. EXPERIMENTAL SECTION

4.1. Chemistry. 4.1.1. General Remarks. All commercially

available reagents and solvents (Fluka, Aldrich, Acros) were used

without further puriﬁcation. For reactions requiring anhydrous

conditions, dry solvents were bought (Fluka, Aldrich). Reactions

were monitored by TLC (Merck; silica gel 60 F254 plates) and

detected with UV light, KMnO₄. Puriﬁcations were performed using

ﬂash chromatography (FC) on silica gel (SiO₂; Merck no. 9385 silica

gel 60 Å, 230−400 mesh). IR Spectra: Perkin-Elmer Paragon 1000

FT-IR spectrometer. Mass spectra were recorded on a Nermag R 10−

10C instrument in the chemical ionization mode. Electrospray mass

analyses were recorded on a Finnigan MAT SSQ 710C spectrometer

in the positive ionization mode. ¹H and ¹³C NMR spectra were

recorded on a Bruker-DPY-400 or a Bruker-ARX-400 spectrometer at

400 and 100.6 MHz, respectively. Data for ¹H NMR spectra are

reported as follows: chemical shift, multiplicity, apparent coupling

Despite the clinical failure of epacadostat, recent ﬁndings

suggest that IDO1 is a player in many diﬀerent pathways. Well-

characterized and potent IDO1 inhibitors will be instrumental

in deciphering its precise biological role. Here, we have

presented experimental data for a set of closely related small

heterocyclic compounds with more than 4 orders of magnitude

diﬀerences in their IDO1 inhibitory activities ranging from

millimolar to nanomolar levels. Based on structural data,

classical MD simulations, and density functional theory

calculations, we developed a model that rationalizes the

observed activities and that can be applied to predict the

activities of novel scaﬀolds. This may be important for

expanding the currently very limited number of known high-

aﬃnity heme-binding IDO1 inhibitor scaﬀolds.

2217

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

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 residual solvent signals.

Coupling constants are given in hertz (Hz). High-resolution mass

spectra were recorded via ESI−TOF−HRMS or MALDI−TOF−

HRMS. The purity of all novel compounds was assessed by ¹H NMR

and analytical HPLC (UV 220 nm) using a PFP propyl column

(RESTEK Allure HPLC column, particle size 5 μm, pore size 60 Å,

dimensions 150 × 4.6 mm) with a linear gradient of solvent B

(acetonitrile, 0.1% TFA) over solvent A (H₂O, 0.1% TFA) from 0 to

100% in 30 min at a ﬂow rate of 1 mL/min. Typically, 5 μL solution

(0.5 mg/mL in 50% ACN/H₂O) was injected for each compound.

The measured purity of all compounds was 95% or higher.

4.1.2. Procedures. 4.1.2.1. General Procedure for the Synthesis of

Imidazole Derivatives (1b−1f).^20,59A solution of α-bromoacetophe-

none derivatives (2 mmol, 1 equiv) was heated to 170−180 °C in

formamide (12 mL) for 3−8 h. The solution was allowed to cool to

room temperature (r.t) and the mixture was diluted with a saturated

aq. soln. of NaHCO₃(8 mL). The aqueous phase was extracted with

EtOAc (3 × 10 mL). The combined organic extracts were washed

successively with water and brine, then dried (Na₂SO₄), and

concentrated in vacuo to aﬀord a residue which was puriﬁed by

ﬂash column chromatography on silica gel to aﬀord compounds 1b−

1f.

4-Chloro-2-(1H-imidazole-4-yl)aniline (1f). Synthesized from 2-

bromo-1-(5-chloro-2-nitrophenyl)ethan-1-one⁸⁸(38) and formamide

according to the general procedure to aﬀord 5-(5-chloro-2-nitro-

phenyl)-1H-imidazole. ¹H NMR (400 MHz, CD₃OD): δ 9.09 (d, J =

1.4 Hz, 1H), 8.31 (d, J = 8.8 Hz, 1H), 7.90−7.80 (m, 3H). 5-(5-

Chloro-2-nitrophenyl)-1H-imidazole (1 equiv) was dissolved in

methanol and treated with ferric chloride (0.33 equiv), activated

carbon (1.70 equiv), and hydrazine hydrate (20 equiv). The resulting

mixture was stirred at r.t. for 5 min and then heated to 70 °C for 48 h.

The mixture was cooled to r.t. and the solid was ﬁltered oﬀ and

washed with methanol. The liquids were combined and the solvent

was removed under reduced pressure. The residue was puriﬁed by

ﬂash chromatography to aﬀord 1f as a white solid in 30% yield (over

1

two steps). H NMR (400 MHz, CD₃OD): δ 7.83 (d, J = 2.3 Hz,

1H), 7.80−7.76 (m, 2H), 7.50 (dd, J = 8.6, 2.3 Hz, 1H), 7.37 (d, J =

1.1 Hz, 1H), ¹³C NMR (100 MHz, CD₃OD): δ 146.39, 139.66,

134.80, 132.75, 131.32, 128.34, 126.93, 123.30, 118.39; ESI−TOF−

HRMS m/z: calcd for (M + H) C₉H₁₀N₃, 194.0480; found, 194.0476.

4.1.2.2. General Procedure for the C5 Arylation of N-

Methylimidazoles (2a−f).⁵⁹⁻⁶¹The aryl iodide (1 mmol, 1 equiv)

was combined with 1-methyl-1H-imidazole (4 mmol, 4 equiv), Pd

(OAc)₂(5 mol %, 0.05 equiv), tricyclohexylphosphonium tetra-

ﬂuoroborate (10 mol %, 0.1 equiv), and Cs₂CO₃(1 mmol, 1 equiv) in

DMF (4 mL). The mixture was heated to 120 °C for 15 h. After

cooling to r.t., the solids were ﬁltered oﬀ and the solution was

partitioned between EtOAc and H₂O. The organic layer was

collected, and the H₂O layer was extracted with EtOAc (2 × 5

mL). The combined extracts were washed with a saturated aq. soln. of

NaHCO₃and dried over Na₂SO₄. The solvent was removed under

vacuum and the residue was puriﬁed by column chromatography on

silica gel to aﬀord 2a−2f.

4-(3-Chlorophenyl)-1H-imidazole (1b).⁸⁷Synthesized from 2-

bromo-(3-chlorophenyl)ethanone (36b) and formamide according

to the general procedure to aﬀord 1b as a white solid in 70% yield. ¹H

NMR (400 MHz, CDCl₃): δ 7.78 (t, J = 1.9 Hz, 1H), 7.74 (s, 1H),

7.69−7.64 (m, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.25 (ddd, J = 8.0, 2.1,

1.1 Hz, 1H), ¹³C NMR (100 MHz, CD₃OD): δ 137.65, 136.07,

135.32, 134.32, 129.81, 126.22, 124.30, 122.70, 115.19, ESI−TOF−

HRMS m/z: calcd for (M + H) C₉H₈ClN₂, 179.0371; found,

179.0367.

1-Methyl-5-phenyl-1H-imidazole (2a).⁸⁹Synthesized from 1-

iodobenzene (39a) and N-methylimidazole according to the general

1

procedure to aﬀord 2a as a white solid in 35% yield. H NMR (400

2-(1H-Imidazole-4-yl)phenol (1c).²⁰Synthesized from 2-(2-

bromoacetyl)phenol (36c) and formamide according to the general

MHz, CDCl₃): δ 7.59 (s, 1H), 7.51−7.36 (m, 5H), 7.17 (s, 1H), 3.70

(s, 3H). ¹³C NMR (100 MHz, CD₃COCD₃): δ 139.65, 130.42,

128.70, 128.08, 127.76, 127.48, 31.92; ESI−TOF−HRMS m/z: calcd

for (M + H) C₁₀H₁₁N₂, 159.0917; found, 159.0913.

1

procedure to aﬀord 1c as a white solid in 60% yield. H NMR (400

MHz, CD₃OD): δ 7.75 (d, J = 1.3 Hz, 1H), 7.63 (dd, J = 7.7, 1.7 Hz,

1H), 7.54 (d, J = 1.2 Hz, 1H), 7.13−7.04 (m, 1H), 6.90−6.80 (m,

2H), ¹³C NMR (100 MHz, CD₃OD): δ 154.71, 133.64, 127.36,

125.13, 118.99, 117.80, 115.95, 114.02, ESI−TOF−HRMS m/z:

calcd for (M + H) C₉H₉N₂O, 161.0709; found, 161.0705.

1-Methyl-5-(3-chlorophenyl)-1H-imidazole (2b). Synthesized from

1-chloro-3-iodobenzene (39b) and N-methylimidazole according to

1

the general procedure to aﬀord 2b as an oil in 60% yield. H NMR

(400 MHz, CDCl₃): δ 7.52 (s, 1H), 7.39−7.26 (m, 3H), 7.25 (dt, J =

7.2, 1.7 Hz, 1H), 7.10 (s, 1H), 3.66 (s, 3H); ¹³C NMR (100 MHz,

CDCl₃): δ 139.51, 134.59, 132.06, 131.51, 130.03, 128.55, 128.25,

127.94, 126.45, 32.65, ESI−TOF−HRMS m/z: calcd for (M + H)

C₁₀H₁₀ClN₂, 193.0527; found, 193.0524.

2-(1H-Imidazole-4-yl) aniline (1d).⁵⁹Synthesized from 2-bromo-1-

(2-nitrophenyl)ethan-1-one (37) and formamide according to the

general procedure to aﬀord 5-(2-nitrophenyl)-1H-imidazole. ¹H

NMR (400 MHz, CD₃OD): δ 9.08 (d, J = 1.4 Hz, 1H), 8.29 (dd, J

= 8.1, 1.4 Hz, 1H), 7.94−7.80 (m, 2H), 7.78 (d, J = 1.4 Hz, 1H), 7.74

(dd, J = 7.5, 1.6 Hz, 1H). 5-(2-Nitrophenyl)-1H-imidazole (1 equiv)

was dissolved in methanol and treated with ferric chloride (0.33

equiv), activated carbon (1.70 equiv), and hydrazine hydrate (20

equiv). The resulting mixture was stirred at r.t. for 5 min and then

heated to 70 °C for 48 h. The mixture was cooled to r.t and the solid

was ﬁltered oﬀ and washed with methanol. The liquids were

combined and the solvent was removed under reduced pressure.

The residue was puriﬁed by ﬂash chromatography to aﬀord 1d as a

white solid in 25% yield for two steps. ¹H NMR (400 MHz, CD₃OD):

δ 7.77 (s, 1H), 7.37 (dd, J = 7.7, 1.5 Hz, 1H), 7.31 (s, 1H), 7.10−7.03

(m, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.74 (t, J = 7.5 Hz, 1H). ¹³C NMR

(100 MHz, CD₃OD): δ 148.06, 134.49, 133.77, 133.00, 131.54,

129.45, 125.15, 121.27, 117.80; ESI−TOF−HRMS m/z: calcd for (M

+ H) C₉H₉ClN₃, 160.0867; found, 160.0866.

2-(1-Methyl-1H-imidazole-5-yl)phenol (2c, CAS 1781522-02-8).

Synthesized from 2-iodoanisole (40) and N-methylimidazole

according to the general procedure to aﬀord crude 2-(1-methyl-1H-

imidazole-5-yl)anisole. ¹H NMR (400 MHz, CD₃OD): δ 7.73 (s,

1H), 7.46 (ddd, J = 8.3, 7.4, 1.8 Hz, 1H), 7.26 (dd, J = 7.5, 1.8 Hz,

1H), 7.13 (dd, J = 8.4, 1.1 Hz, 1H), 7.05 (td, J = 7.5, 1.1 Hz, 1H),

6.92 (s, 1H), 3.85 (s, 3H), 3.55 (s, 3H). 2-(1-Methyl-1H-imidazole-5-

yl)anisole (1.0 mmol) was dissolved in 48% hydrobromic acid (HBr)

in water (4 mL) and the orange solution was heated to 100 °C for 14

h under N₂. The solution was cooled to r.t., diluted with water, and

neutralized by the addition of a saturated aq. soln. of NaHCO₃until

the evolution of CO₂ceased. The organic product was extracted with

ethyl acetate. The extract was dried over sodium sulfate and

concentrated in vacuo to aﬀord a residue that was puriﬁed by ﬂash

column chromatography on silica gel to aﬀord 2c as a white solid in

60% yield (over two steps). ¹H NMR (400 MHz, CD₃OD): δ 7.78 (s,

1H), 7.29 (ddd, J = 8.2, 7.4, 1.7 Hz, 1H), 7.20 (dd, J = 7.5, 1.7 Hz,

1H), 6.97−6.86 (m, 3H), 3.62 (s, 3H), ¹³C NMR (100 MHz,

CD₃OD): δ 155.44, 131.71, 130.03, 126.38, 119.29, 116.44, 115.27,

31.13, ESI−TOF−HRMS m/z: calcd for (M + H) C₁₀H₁₁N₂O,

175.0866; found, 175.0862.

4-Chloro-2-(1H-imidazole-4-yl)phenol (1e).²¹Synthesized from 4-

chloro-2-(2-bromoacetyl) phenol (36e) and formamide according to

the general procedure to aﬀord 1e as a light yellow solid in 75% yield.

¹H NMR (400 MHz, CDCl₃): δ 12.23 (br s, −OH, 1H), 9.39 (br s,

−OH, 1H), 7.77 (d, J = 1.2 Hz, 1H), 7.45 (d, J = 2.6 Hz, 1H), 7.39

(d, J = 1.2 Hz, 1H), 7.12 (dd, J = 8.7, 2.6 Hz, 1H), 6.94 (d, J = 8.8 Hz,

1H), ¹³C NMR (100 MHz, CD₃OD): δ 153.57, 136.86, 133.95,

126.78, 124.58, 123.61, 119.65, 117.33, 114.43, ESI−TOF−HRMS

m/z: calcd for (M + H) C₉H₈ClN₂O, 195.0320; found, 195.0316.

2-(1-Methyl-1H-imidazole-5-yl)aniline (2d, CAS 1780550-29-9).

Synthesized from 2-iodonitrobenzene (42) and N-methylimidazole

according to the general procedure to aﬀord 1-methyl-5-(2-nitro-

2218

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1

phenyl)-1H-imidazole. H NMR (400 MHz, CDCl₃): δ 8.04−7.90

aﬀord 2-(1H-imidazole-1-yl)anisole. H NMR (400 MHz, CD₃OD):

δ 7.97 (s, 1H), 7.51−7.37 (m, 3H), 7.24 (d, J = 8.9 Hz, 1H), 7.13 (s,

1H), 6.96 (m, 1H), 3.90 (s, 3H). 2-(1H-Imidazole-1-yl)anisole was

dissolved in 48% HBr in water (4 mL) and the orange solution was

heated to 100 °C for 14 h under N₂. The mixture was cooled to r.t.,

then diluted with water, and neutralized by the addition of a saturated

aq. soln. of NaHCO₃until the evolution of CO₂ceased. The mixture

was extracted with ethyl acetate (2 × 5 mL), and the combined

extracts were dried over sodium sulfate and concentrated in vacuo to

aﬀord a residue that was puriﬁed by ﬂash column chromatography on

silica gel to aﬀord 3c as a white solid in 65% yield (over two steps).

¹H NMR (400 MHz, CD₃OD): δ 7.96 (d, J = 1.2 Hz, 1H), 7.40 (t, J =

1.4 Hz, 1H), 7.33 (dd, J = 7.9, 1.6 Hz, 1H), 7.26 (ddd, J = 8.2, 7.4, 1.6

Hz, 1H), 7.11 (s, 1H), 7.04 (dd, J = 8.2, 1.3 Hz, 1H), 6.97 (td, J = 7.7,

1.4 Hz, 1H), ¹³C NMR (100 MHz, CD₃OD): δ 150.54, 128.73,

127.04, 125.03, 124.73, 120.36, 119.66, 116.50, ESI−TOF−HRMS

m/z: calcd for (M + H) C₉H₉N₂O, 161.0709; found, 161.0706.

4-Chloro-2-(1H-imidazole-1-yl)phenol (3e).⁹¹Synthesized from 4-

chloro-2-iodoanisole (41) and 1H-imidazole according to the general

(m, 2H), 7.66−7.60 (m, 2H), 7.58−7.50 (m, 1H), 7.39 (dd, J = 7.5,

1.6 Hz, 1H), 3.42 (s, 3H). 1-Methyl-5-(2-nitrophenyl)-1H-imidazole

(1 equiv) was dissolved in methanol and treated with ferric chloride

(0.33 equiv), activated carbon (1.70 equiv), and hydrazine hydrate

(20 equiv). The resulting mixture was stirred at r.t. for 5 min before

heating to 70 °C for 48 h. The mixture was cooled to r.t., and then the

solid was ﬁltered oﬀ and washed with methanol. The liquids were

combined, and the solvent was removed under reduced pressure. The

residue was puriﬁed by ﬂash chromatography to aﬀord 2d as a brown

solid in 60% yield (over two steps). ¹H NMR (400 MHz, CD₃OD): δ

7.65 (s, 1H), 7.06 (ddd, J = 8.1, 7.3, 1.6 Hz, 1H), 6.90 (dd, J = 7.6, 1.6

Hz, 1H), 6.84 (s, 1H), 6.71 (dd, J = 8.2, 1.2 Hz, 1H), 6.61 (td, J = 7.4,

1.2 Hz, 1H), 3.41 (s, 3H); ¹³C NMR (100 MHz, CD₃OD): δ 146.86,

138.35, 131.30, 130.79, 129.91, 126.25, 117.17, 115.25, 113.67, 30.84;

ESI−TOF−HRMS m/z: calcd for (M + H) C₁₀H₁₁N₃, 174.1029;

found, 174.1040.

4-Chloro-2-(1-methyl-1H-imidazole-5-yl)phenol (2e, CAS

2107720-24-9). Synthesized from 2-iodo-4-chloroanisole (41) and

N-methylimidazole according to the general procedure to aﬀord the

crude 4-chloro-2-(1-methyl-1H-imidazole-5-yl)anisole. ¹H NMR (400

MHz, CD₃OD): δ 7.71 (s, 1H), 7.45 (dd, J = 8.8, 2.7 Hz, 1H), 7.27

(d, J = 2.7 Hz, 1H), 7.13 (d, J = 8.9 Hz, 1H), 6.94 (s, 1H), 3.85 (s,

3H), 3.56 (s, 3H). 4-Chloro-2-(1-methyl-1H-imidazole-5-yl)anisole

(1.0 mmol) was dissolved in 48% HBr in water (4 mL) and the

orange solution was heated to 100 °C for 14 h under N₂. The mixture

was cooled to r.t., diluted with water, and neutralized by the addition

of a saturated aq. soln. of NaHCO₃until the evolution of CO₂ceased.

The organic layer was extracted with ethyl acetate (2 × 5 mL), dried

over sodium sulfate, and concentrated in vacuo to aﬀord a residue that

was puriﬁed by ﬂash column chromatography on silica gel to aﬀord 2e

1

procedure to aﬀord 4-chloro-2-(1H-imidazole-1-yl)anisole. H NMR

(400 MHz, CDCl₃): δ 7.82 (d, J = 1.1 Hz, 1H), 7.38−7.29 (m, 2H),

7.25−7.14 (m, 2H), 7.01 (d, J = 8.8 Hz, 1H), 3.87 (s, 3H). 4-Chloro-

2-(1H-imidazole-1-yl)anisole was dissolved in 48% HBr in water (4

mL) and the orange solution was heated to 100 °C for 14 h under N₂.

The mixture was cooled to r.t, then diluted with water, and

neutralized by the addition of a saturated aq. soln. of NaHCO₃

until the evolution of CO₂ceased. The mixture was extracted with

ethyl acetate. The combined extracts were dried over Na₂SO₄and

concentrated in vacuo to aﬀord a residue that was puriﬁed by ﬂash

column chromatography on silica gel to aﬀord 3e as a white solid in

52% yield (over two steps). ¹H NMR (400 MHz, CD₃OD): δ 8.04 (s,

1H), 7.46 (s, 1H), 7.42 (d, J = 2.5 Hz, 1H), 7.27 (dd, J = 8.7, 2.6 Hz,

1H), 7.14 (s, 1H), 7.02 (d, J = 8.8 Hz, 1H), ¹³C NMR (100 MHz,

CD₃OD): δ 149.43, 128.47, 125.53, 124.77, 123.96, 117.69, ESI−

TOF−HRMS m/z: calcd for (M + H) C₉H₈ClN₂O, 195.0320; found,

195.0315.

2-(1H-Imidazole-1-yl)aniline (3f, CAS 190200-15-8). Synthesized

from 4-chloro-2-iodoaniline (39f) and 1H-imidazole according to the

general procedure to aﬀord 3f as a white solid in 75% yield. ¹H NMR

(400 MHz, CD₃OD): δ 7.82 (s, 1H), 7.30 (s, 1H), 7.21 (dd, J = 8.8,

2.4 Hz, 2H), 7.16 (d, J = 2.4 Hz, 1H), 6.90 (d, J = 8.7 Hz, 1H), ¹³C

NMR (100 MHz, CD₃OD): δ 142.27, 137.56, 129.37, 128.40, 126.38,

123.16, 121.06, 120.43, 117.28, ESI−TOF−HRMS m/z: calcd for (M

+ H) C₉H₉ClN₃, 194.0480; found, 194.0476.

1

as a white solid in 70% yield for two steps. H NMR (400 MHz,

CD₃OD): δ 7.58 (s, 1H), 7.15 (dd, J = 8.7, 2.7 Hz, 1H), 7.08 (d, J =

2.7 Hz, 1H), 6.83 (s, 1H), 6.79 (d, J = 8.7 Hz, 1H), 3.50 (s, 3H); ¹³C

NMR (100 MHz, CD₃OD): δ 154.37, 138.41, 131.01, 130.05, 129.67,

126.91, 123.68, 118.23, 116.70, 31.23; ESI−TOF−HRMS m/z: calcd

for (M + H) C₁₀H₁₀ClN₂O, 209.0476; found, 209.0472.

4-Chloro-2-(1-methyl-1H-imidazole-5-yl)aniline (2f, CAS

2106580-97-4). Synthesized from 2-iodo-4-chloronitrobenzene (43)

and N-methylimidazole according to the general procedure to aﬀord

5-(5-chloro-2-nitrophenyl)-1-methyl-1H-imidazole. ¹H NMR (400

MHz, CDCl₃): δ 8.75 (s, 1H), 8.12 (d, J = 8.8 Hz, 1H), 7.64 (dd,

J = 8.8, 2.2 Hz, 1H), 7.47 (d, J = 2.3 Hz, 1H), 7.18 (s, 1H), 3.61 (s,

3H). 5-(5-Chloro-2-nitrophenyl)-1-methyl-1H-imidazole (1 equiv)

was dissolved in methanol and treated with ferric chloride (0.33

equiv), activated carbon (1.70 equiv), and hydrazine hydrate (20

equiv). The resulting mixture was stirred at r.t. for 5 min before

heating to 70 °C for 48 h. The mixture was cooled to r.t., and then the

solid was ﬁltered oﬀ and washed with methanol. The liquids were

combined, and the solvent was removed under reduced pressure. The

residue was puriﬁed by ﬂash chromatography to aﬀord 2f as a white

4.1.2.4. General Procedure for the Synthesis of 1,2,4-Triazoles

(5b−c and 5e−g).⁶³A solution of amide (44b−c, 44e−g) (1.0

mmol, 1 equiv) and N,N-dimethylformamide−dimethyl acetal

(DMF−DMA) (1.5 mL) was heated under reﬂux for 1 h under N₂.

The mixture was concentrated under reduced pressure and the

residue was washed with hexane. After drying in vacuo, a white solid

was obtained that was used without puriﬁcation in the next step. The

crude product was dissolved in acetic acid (2 mL), and then hydrazine

hydrate (1.1 mmol, 1.1 equiv) was added dropwise to the stirred

solution. The mixture was stirred at 90 °C for 2 h. After cooling to r.t.,

the solvent was removed in vacuo. The mixture was taken with diethyl

ether (5 mL) and stirred at 0 °C for 30 min. The precipitate was

collected, dried under reduced pressure, and puriﬁed by ﬂash column

chromatography on silica gel to aﬀord the corresponding triazoles

(5b−c, 5e−g).

1

solid in 65% yield for two steps. H NMR (400 MHz, CD₃OD): δ

7.75 (s, 1H), 7.16 (dd, J = 8.7, 2.5 Hz, 1H), 7.04 (d, J = 2.5 Hz, 1H),

6.98 (s, 1H), 6.81 (d, J = 8.7 Hz, 1H), 3.56 (s, 3H); ¹³C NMR (100

MHz, CD₃OD): δ 145.96, 138.82, 130.58, 129.59, 126.99, 121.11,

116.29, 115.04, 30.80; ESI−TOF−HRMS m/z: calcd for (M + H)

C₁₀H₁₁ClN₃, 208.0641; found, 208.0643.

4.1.2.3. General Procedure for the Synthesis of N-Phenyl-

imidazole Derivatives (3c, 3e, and 3f).^61,62The aryl iodide (1

mmol, 1 equiv) was dissolved in DMF and treated with N,N-

dimethylethane-1,2-diamine (0.3 mmol, 0.3 equiv), Cs₂CO₃(2 mmol,

2.0 equiv), CuI (0.1 mmol, 0.1 equiv), and 1H-imidazole (2.0 mmol,

2.0 equiv) under an N₂atmosphere. The mixture was stirred at 140

°C for 12 h. After cooling to r.t., the solids were ﬁltered oﬀ and

washed with dichloromethane (2 × 5 mL). The combined organic

phases were concentrated under reduced pressure and the residue was

puriﬁed by ﬂash column chromatography on silica gel to aﬀord 3c, 3e,

and 3f.

3-(3-Chlorophenyl)-1H-1,2,4-triazole (5b).⁹²Synthesized from 2-

chlorobenzamide (44b) and hydrazine hydrate according to the

general procedure to aﬀord 5b as a white solid in 50% yield. ¹H NMR

(400 MHz, CDCl₃): δ 8.24 (s, 1H), 8.09 (q, J = 1.3 Hz, 1H), 7.97 (td,

J = 4.4, 1.6 Hz, 1H), 7.43−7.40 (m, 2H). ¹³C NMR (100 MHz,

CD₃OD): δ 134.49, 130.12, 129.24, 125.92, 124.28; ESI−TOF−

HRMS m/z: calcd for (M + H) C₈H₇ClN₃, 180.0323; found,

180.0319.

2-(1H-1,2,4-Triazol-3-yl)phenol (5c).⁹³Synthesized from 2-

hydroxybenzamide (44c) and hydrazine hydrate according to the

general procedure to aﬀord 5c as a white ﬂuﬀy powder in 75% yield.

2-(1H-Imidazole-1-yl)phenol (3c).⁹⁰Synthesized from 1-iodoani-

sole (40) and 1H-imidazole according to the general procedure to

2219

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

¹H NMR (400 MHz, CD₃OD): δ 8.34 (s, 1H), 7.97 (dd, J = 7.8, 1.7

Hz, 1H), 7.34 (ddd, J = 8.7, 7.3, 1.7 Hz, 1H), 7.04−6.93 (m, 2H), ¹³C

NMR (100 MHz, CD₃OD): δ 156.33, 131.15, 126.60, 119.25, 116.47,

112.72, ESI−TOF−HRMS m/z: calcd for (M + H) C₈H₈N₃O,

162.0662; found, 162.0658.

4-Chloro-2-(1H-1,2,4-triazol-3-yl)phenol (5e, CAS 814263-35-9).

Synthesized from 5-chloro-2-hydroxybenzamide (44e) and formic

acid according to the general procedure to aﬀord 5e as a white solid in

76% yield. ¹H NMR (400 MHz, CD₃OD): δ 8.44 (s, 1H), 7.99 (d, J =

2.6 Hz, 1H), 7.31 (dd, J = 8.8, 2.6 Hz, 1H), 6.99 (d, J = 8.8 Hz, 1H),

¹³C NMR (100 MHz, CD₃OD): δ 155.10, 130.65, 125.97, 123.92,

acetate. The combined organic layers were washed with brine, dried

over Na₂SO₄, and concentrated in vacuo. The residue was puriﬁed by

ﬂash chromatography on silica gel to aﬀord 4-phenyl-4H-1,2,4-triazole

(9) as a semi solid in 45% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.59

(s, 1H), 8.14 (s, 1H), 7.75−7.67 (m, 2H), 7.59−7.50 (m, 2H), 7.48−

7.39 (m, 1H), ¹³C NMR (101 MHz, CDCl₃): δ 152.60, 140.87,

137.01, 129.80, 128.26, 120.10, ESI−TOF−HRMS m/z: calcd for (M

+ H) C₈H₈N₃, 146.0713; found, 146.0710.

4-Phenylisothiazole (15).⁶⁶DMF (2.0 mL) was added dropwise to

POCl₃(9.0 mmol, 3.0 equiv) and cooled to 0 °C. Phenyl acetic acid

(3.0 mmol, 1.0 equiv) was then added and the mixture was stirred at

85 °C for 2 h. The reaction mixture was cooled to room temperature

and cracked ice was added to adjust the volume to ca. 15 mL. The

reaction mixture was extracted with dichloromethane, the combined

extracts were dried, and the solvents were evaporated to yield crude 2-

phenyl-3-N,N-dimethylaminoacrolein as a yellow oil. NaOH (25% aq.

soln., 6 mL) was added to crude 2-phenyl-3-N,N-dimethylaminoa-

crolein in EtOH (5.0 mL), and the reaction mixture was reﬂuxed

under stirring for 1 h. Ethanol was removed under vacuum, and the

residue was diluted by addition of crushed ice and acidiﬁed (pH < 3)

using aqueous HCl (1:1). The resulting mixtures were extracted with

diethyl ether, the combined extracts were dried, and the solvents were

evaporated to leave crude enolized 2-aryl malondialdehyde as a yellow

oil. SOCl₂(4.2 mL, 57.6 mmol) was added to crude 2-aryl

malondialdehyde in CH₂Cl₂(6 mL) and cooled to 0 °C, and the

reaction mixtures were stirred at 0 °C for 1 h. The solvents were

evaporated, and the crude product was used without puriﬁcation in

the next step. NH₄SCN (7.2 mmol, 4.0 equiv) was added to a solution

of chloroacrolein (1.8 mmol, 1.0 equiv) in DMF (3.0 mL), and the

reaction mixture was heated for 16 h at 70 °C under stirring. The

reaction mixture was diluted with brine (20 mL) and extracted with

ethyl acetate. The combined extract was dried, the solvent evaporated,

and the residue was subjected to ﬂash chromatography on silica gel to

118.28, 114.68, ESI−TOF−HRMS m/z: calcd for (M + H)

C₈H₇ClN₃O, 196.0272; found, 196.0267.

4-Chloro-2-(1H-1,2,4-triazol-3-yl)aniline (5f, CAS 55138-98-2).

Synthesized from 2-amino-5-chlorobenzamide (44f) and formic acid

according to the general procedure to aﬀord 5f as a white solid in 52%

yield. ¹H NMR (400 MHz, CD₃COCD₃): δ 7.81 (s, 1H), 7.57 (d, J =

2.5 Hz, 1H), 7.10 (dd, J = 8.7, 2.5 Hz, 1H), 6.74 (d, J = 8.6 Hz, 1H),

6.39 (s, 1H); ¹³C NMR (100 MHz, CD₃COCD₃): δ 151.43, 147.31,

141.10, 131.16, 123.33, 122.87, 116.63, 112.29; ESI−TOF−HRMS

m/z: calcd for (M + H) C₈H₈ClN₄, 195.0437; found, 195.0441.

4-Bromo-2-(1H-1,2,4-triazol-3-yl)aniline (5g).⁹⁴Synthesized from

2-amino-5-bromobenzamide (44g) and hydrazine hydrate according

to the general procedure to aﬀord 5g as a white solid in 35% yield. ¹H

NMR (400 MHz, CD₃OD): δ 8.50 (s, 1H), 8.04 (s, 1H), 7.24 (dd, J

= 8.8, 2.4 Hz, 1H), 6.78 (d, J = 8.7 Hz, 1H); ¹³C NMR (100 MHz,

CD₃OD): δ 145.55, 142.70, 132.04, 130.07, 117.80, 107.17; ESI−

TOF−HRMS m/z: calcd for (M + H) C₈H₈BrN₄, 238.9927; found,

238.9922.

4.1.2.5. General Procedure for the Synthesis of 1,2,3,4-Tetrazoles

(6c, 6e, and 6f).⁶⁴A mixture of the corresponding carbonitrile

(45c,e,f) (1 mmol, 1 equiv), NaN₃(2 mmol, 3 equiv), and Et₃N·HCl

(3.7 mmol, 3.7 equiv) in toluene (3 mL) was stirred at 99 °C

overnight. After cooling to r.t., the mixture was extracted with H₂O (2

mL) and the aqueous phase was acidiﬁed by dropwise addition of

conc. HCl aﬀording a precipitate. The precipitate was collected,

washed with H₂O (2 × 5 mL), dried under reduced pressure, and

puriﬁed by ﬂash column chromatography on silica gel to aﬀord the

corresponding products 6c, 6e, and 6f.

1

aﬀord compound 15 as a white solid in 70% yield. H NMR (400

MHz, CDCl₃): δ 8.81 (s, 1H), 8.74 (s, 1H), 7.66−7.59 (m, 2H),

7.51−7.45 (m, 2H), 7.48−7.34 (m, 1H), ¹³C NMR (100 MHz,

CDCl₃): δ 156.05, 142.69, 139.98, 132.59, 129.17, 128.07, 126.93,

ESI−TOF−HRMS m/z: calcd for (M + H) C₉H₈NS, 162.0372;

found, 162.0373.

5-Phenyl-1,2,3-thiadiazole (17).⁶⁷To a solution of TMSCHN₂(2

M ether solution, 0.6 mL, 1.2 mmol, 1.2 equiv) in diethylether (8

mL), n-BuLi (2.5 M hexane solution, 0.48 mL, 1.2 mmol, 1.2 equiv)

was added dropwise at −78 °C under nitrogen, and the mixture was

stirred at −78 °C for 20 min. A solution of methyl thiobenzoate (1.0

mmol, 1.0 equiv) in diethyl ether (3 mL) was then added dropwise at

−78 °C. The mixture was stirred at −78 °C for 30 min and then at 0

°C f or 30 min. After addition of water (1 mL) and methanol (5 mL),

the mixture was stirred at r.t. for 1 h and concentrated in vacuo. The

residue was suspended in water and extracted with ethyl acetate. The

organic extracts were washed with water, saturated aq. NaCl, dried

over anhydrous Na₂SO₄, and concentrated in vacuo. The residue was

puriﬁed by column chromatography to aﬀord 5-phenyl-1,2,3-

2-(1H-Tetrazol-5-yl)phenol (6c).⁶⁴Synthesized from 2-hydrox-

ybenzonitrile (45c) and NaN₃according to the general procedure to

aﬀord 6c as a white solid in 80% yield. ¹H NMR (400 MHz,

CD₃OD): δ 8.08 (dd, J = 7.8, 1.7 Hz, 1H), 7.44 (ddd, J = 8.3, 7.3, 1.7

Hz, 1H), 7.10−7.02 (m, 2H), ¹³C NMR (100 MHz, CD₃OD): δ

155.63, 152.42, 132.74, 128.51, 119.80, 116.00, 109.76, ESI−TOF−

HRMS m/z: calcd for (M + H) C₇H₇N₄O, 163.0614; found,

163.0612.

4-Chloro-2-(1H-tetrazol-5-yl)phenol (6e).⁹⁵Synthesized from 5-

chloro-2-hydroxybenzonitrile (45e) and NaN₃according to the

1

general procedure to aﬀord 6d as a white solid in 70% yield. H

NMR (400 MHz, CD₃OD): δ 8.08 (d, J = 2.7 Hz, 1H), 7.42 (dd, J =

8.8, 2.7 Hz, 1H), 7.07 (d, J = 8.9 Hz, 1H); ¹³C NMR (100 MHz,

CD₃OD): δ 155.23, 152.78, 133.16, 128.69, 125.27, 118.99, 112.15;

ESI−TOF−HRMS m/z: calcd for (M + H) C₇H₆ClN₄O, 197.0225;

found, 197.0221.

1

thiadiazole (17) as a semi solid in 75% yield. H NMR (400 MHz,

CDCl₃): δ 8.90 (s, 1H), 7.67-7-61 (m, 2H), 7.57−7.47 (m, 3H), ¹³

C

NMR (100 MHz, CDCl₃): δ 157.06, 144.18, 130.55, 129.60, 128.19,

127.45, ESI−TOF−HRMS m/z: calcd for (M + H) C₈H₇N₂S,

163.0324; found, 163.0323.

4-Chloro-2-(1H-tetrazol-5-yl)aniline (6f).⁹⁶Synthesized from 2-

amino-5-chlorobenzonitrile (45f) and NaN₃according to the general

procedure to aﬀord 6f as a white solid in 69% yield. H NMR (400

5-Phenyloxazole (20).⁶⁸To a solution of benzaldehyde (1.0 mmol,

1.0 equiv) in MeOH (5 mL) was added K₂CO₃(1.5 mmol, 1.5 equiv)

and toluenesulfonylmethyl isocyanide (TosMIC, 1.0 mmol, 1.0

equiv). The reaction mixture was stirred for 2 h at 80 °C, cooled

down to r.t., quenched with H₂O, and diluted with EtOAc. After

removal of the organic layer, the aqueous layer was extracted with

EtOAc. The organic layer was combined, dried over Na₂SO₄, ﬁltered,

and concentrated in vacuo. Flash chromatography aﬀorded 5-

1

MHz, CDCl₃): δ 7.71 (d, J = 2.5 Hz, 1H), 7.21 (dd, J = 8.8, 2.4 Hz,

1H), 6.78 (d, J = 8.8 Hz, 1H), ¹³C NMR (100 MHz, CD₃OD): δ

155.11, 146.09, 131.40, 126.70, 120.42, 117.94, 106.45, ESI−TOF−

HRMS m/z: calcd for (M + H) C₇H₇ClN₅, 196.0384; found,

196.0381.

4-Phenyl-4H-1,2,4-triazole (9).⁶⁵To a solution of Cu(OAc)₂·H₂O

(0.01 mmol, 0.01 equiv) in DMF (2 mL) were added phenyl iodide

(1.2 mmol, 1.2 equiv), 4H-1,2,4-triazole (1.0 mmol, 1.0 equiv), and

Cs₂CO₃(2.0 mmol, 2.0 equiv) under a nitrogen atmosphere. The

mixture was stirred at 110 °C for 24 h. After cooling to r.t., the

mixture was partitioned between water and ethyl acetate. The organic

layer was separated, and the aqueous layer was extracted with ethyl

1

phenyloxazole (20) as a white solid in 60% yield. H NMR (400

MHz, CDCl₃): δ 7.95 (s, 1H), 7.72−7.65 (m, 2H), 7.50−7.42 (m,

2H), 7.39 (s, 2H), ¹³C NMR (101 MHz, CD₃OD): δ 154.02, 147.65,

128.79, 128.61, 127.59, 126.00, 121.41, 121.39, ESI−TOF−HRMS

m/z: calcd for (M + H) C₉H₈NO, 146.0600; found, 146.0599.

2220

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

4-Phenylisoxazole (21).⁶⁹To a solution of 2-phenylmalonaldehyde

(1.0 mmol, 1.0 equiv) in EtOH (5 mL) was added hydroxylamine

hydrochloride (2.0 mmol, 2.0 equiv). The reaction mixture was stirred

for 2 h at 80 °C. After completion of the reaction, the solvents were

removed under reduced pressure, and the crude product was

dissolved in CH₂Cl₂(2 × 5 mL) and washed with water (10 mL).

The organic layers were combined, dried over Na₂SO₄, ﬁltered, and

concentrated in vacuo. Puriﬁcation was accomplished by column

chromatography on silica gel to aﬀord compound 21 as a white solid

analysis, a Mulliken population analysis, a Bader charge analysis, and

electrostatic potential-derived charges according to the Merz−Singh−

Kollman scheme. Solvation eﬀects were taken into account by the

polarizable continuum model¹⁰⁸as implemented in Gaussian09. 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.⁵⁸

4.5. MM-GBSA Calculations. Starting from the crystal structure

2d0t of the complex between hIDO1 and compound 1a,¹⁹we

removed chain B, the N-cyclohexyltaurine molecules, and the resolved

water molecules. For all other compounds, ligand 1a was replaced by

the docked conformation of the respective ligand. A covalent bond

with an equilibrium distance of 1.97 Å and a force constant of 250

kcal/mol/Å²was added between the heme iron and the coordinated

ligand nitrogen atom. The ligand force ﬁelds were generated with the

SwissParam algorithm.¹⁰⁹MD simulations were performed in the

CHARMM program¹¹⁰version 36b1 with the CHARMM27 force

ﬁeld.^111,112The systems were locally solvated in a sphere of TIP3P

water¹¹³of 25 Å surrounding the active site using the stochastic

boundary method.¹¹⁴A time step of 1 fs was used. After 220 ps of

initial heating and equilibration, data was collected for 2.5 ns.

Structural snapshots excluding water molecules were saved every 10

ps for a total of 250 frames. At least three simulations with

independent initial velocities were carried out for each ligand.

In the MM-GBSA approach,⁸⁴⁻⁸⁶the binding free energy is written

as the sum of the change in gas-phase enthalpy ΔH_bind,gas, the

solvation free energy ΔG_solv, and the entropic contribution −TΔS.

1

in 80% yield. H NMR (400 MHz, CDCl₃): δ 8.70 (s, 1H), 8.59 (s,

1H), 7.55−7.41 (m, 2H), 7.41−7.33 (m, 2H), 3.47 (s, 1H), ¹³C NMR

(100 MHz, CDCl₃): δ 153.38, 147.99, 129.21, 128.54, 128.12, 126.45,

121.41, ESI−TOF−HRMS m/z: calcd for (M + H) C₉H₈NO,

146.0600; found, 146.0597.

5-Phenyl-1,2,3-oxadiazole (23).⁹⁴To a solution of benzoic

hydrazide (2.0 mmol, 1 equiv) dissolved in toluene (3 mL) were

added triethylorthoformate (3.0 mL) and p-toluenesulfonic acid (0.2

mmol, 0.1 equiv). The reaction mixture was heated to reﬂux for 2 h,

cooled to r.t., and concentrated. The residue was dissolved in Et₂O

and washed with H₂O (2 × 10 mL), 0.1 N HCl (10 mL), and brine

(10 mL); dried over Na₂SO₄; ﬁltered; and concentrated. The crude

product was puriﬁed by column chromatography on silica gel to aﬀord

1

compound 23 as a light yellow solid in 65% yield. H NMR (400

MHz, CDCl₃): δ 8.50 (s, 1H), 8.16−8.08 (m, 2H), 7.64−7.51 (m,

3H), ¹³C NMR (101 MHz, CDCl₃): δ 164.79, 152.63, 132.02, 129.14,

127.12, 123.51, ESI−TOF−HRMS m/z: calcd for (M + H)

C₈H₇N₂O, 147.0553; found, 147.0551.

4.2. Protein Expression and Puriﬁcation. Full-length human

IDO1 gene was cloned into the pET-28a vector and expressed in E.

coli Rosetta (DE3) cells. Protein was produced in the LB medium

supplemented with 0.5 mM dALA (5-aminolevulinic acid) and was

induced by addition of 0.1 mM isopropylthio-β-galactoside overnight

at 16 °C. The culture was harvested and frozen at −80 °C until

further use. The frozen cells were lysed by sonication in 20 mM Tris

pH 7, 400 mM NaCl, 1 mM MgCl₂, 20 mM imidazole, 1 mM DTT,

0.5 mg/mL lysozyme, and protease inhibitor. After centrifugation, the

supernatant was loaded into a HisTrap HP column pre-equilibrated

with buﬀer containing 20 mM Tris pH 7, 500 mM NaCl, and 20 mM

imidazole and eluted with the same buﬀer containing 500 mM

imidazole. Fractions containing IDO1 were pooled, concentrated, and

injected on a size exclusion chromatography column (HiLoad

Superdex S75 16/60) to increase purity. hIDO1 fractions were

concentrated to 20 mg/mL in 20 mM Tris pH 7 and 150 mM NaCl.

4.3. Protein Crystallization, Data Collection, Structure

Determination, and Model Reﬁnement. hIDO1 protein at 20

mg/mL was mixed with compounds 4f, 5f, and 5g at a ﬁnal

concentration of 5 mM and co-crystallized by a hanging drop vapor

diﬀusion method. Crystals formed in a couple of days in 20% PEG

1500, 0.15 M potassium thiocyanate, and 0.1 M Tris pH 8.5. The

crystals were cryoprotected with 25% glycerol. Diﬀraction data were

collected at the Paul Scherrer Institute (SLS, Villigen) at PXII

X10SA beamline. Data were processed with the XDS Program

Package.⁹⁷Structures were solved by molecular replacement using

Phaser-MR and chain A of PDB entry 2d0t¹⁹as the model. Manual

model building and structure reﬁnement were carried out in Phenix

Suite⁹⁸using coot⁹⁹software and phenix-reﬁne, respectively. After

validation, ligand-bound IDO1 models were deposited in the PDB

database under PDB codes 7ah4 (4f), 7ah6 (5f), and 7ah6 (5g). Data

collection and reﬁnement statistics are summarized in the Supporting

Information (Table S2). The structures were displayed with PyMOL

(http://www.pymol.org/) and UCSF Chimera.¹⁰⁰

ΔG_bind= ΔH_bind,gas+ ΔG_solv− TΔS

in which

ΔH_bind,gas= ΔH_elec+ ΔH_vdW+ ΔH_intra

and

ΔG_solv= ΔG_solv,elec+ ΔG_solv,np= ΔG_solv,elec+ γΔSASA

where ΔH_elecis the electrostatic interaction energy, ΔH_vdWis the van

der Waals interaction energy, ΔH_intrais the change in internal energy

of the ligand and the receptor, ΔG_solv,elecis the electrostatic

contribution to the solvation free energy, ΔG_solv,npis the non-polar

contribution, and SASA is the solvent-accessible surface area. Here,

we neglected the entropic term and the change in internal energy.

ΔG_solv,elecwas calculated with the GB-MV2 model^115,116and a value of

0.0072 kcal/mol/Å²was used for the parameter γ.^117,118For each

neutral ligand, the values were averaged over a minimum of 750

structural snapshots extracted from three independent simulations,

while for charged ligands, 1500 structural snapshots were extracted

from six independent simulations to improve convergence. All ligand

interactions with the heme porphyrin ring and the His346 side chain

were excluded from the analysis. The standard errors were calculated

taking into account the autocorrelation within the data.¹¹⁹

4.6. Electrostatic Potential Calculation. Electrostatic potentials

were calculated with the UHBD code¹²⁰on the structure of hIDO1

extracted from its complex with compound MMG-0358 in structure

6r63.¹⁵The protein was described with the CHARMM27 force

ﬁeld,^111,112and the charge of the iron was left at +0.24 for the ferrous

complex and set to +1.24 for the ferric complex.

4.4. 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 functional¹⁰¹using the Gaussian09 code.¹⁰²Geometry

optimizations were carried out with standard settings and the

TZVP basis set,¹⁰³while atomic point charges derived from a

Hirshfeld population analysis¹⁰⁴were calculated with the aug-cc-

pVTZ basis set^105−107to obtain converged charges. We also tested the

use of atomic point charges derived from a natural population

4.7. Enzymatic Assays. The enzymatic inhibition assays were

performed as described by Takikawa et al.¹²¹with 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), Triton X-100 (0.01%), and DMSO (5 μL).

The inhibitors were serially diluted 3-fold for at least eight diﬀerent

2221

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

concentrations. After incubation at room temperature for 20 min, the

reaction was stopped in its linear phase by addition of trichloroacetic

acid solution (40 mL, 30% w/v), and the samples were incubated at

50 °C for 30 min. After centrifugation, 80 μL of supernatant from

each well was used for HPLC analysis.

The mobile phase for HPLC analysis was composed of 50% (v/v)

of methanol and 50% of sodium citrate buﬀer (40 mM, pH 2.4)

containing 400 μM sodium dodecyl sulfate. An Agilent Zorbax Eclipse

XDB C18 column (150 n 4.6 mm) was used at 23 °C with a ﬂow rate

of 1 mL/min and an injection volume of 20 μL. For detection of

kynurenine, absorption at 365 nm was measured, while the remaining

L-trp was detected at a wavelength of 280 nm. All assays were carried

out in duplicates. The activity Act of each compound was calculated

as Act = −RT ln IC₅₀in analogy to the relation between the binding

free energy and the measured K_ivalues.

4.8. Cellular Assays. The cDNA encoding hIDO1 (NCBI,

NM_0021464.3) was purchased from Origene as a pCMV6-Entry

vector (RC206592). The full ORF of hIDO1 was cloned into the

mammalian expression vector pcDNA6/myc-His from Invitrogen.

Human embryonic kidney 293T (HEK 293T) cells were transiently

transfected with the plasmid construct encoding hIDO1. A 75 cm²

ﬂask containing HEK 293T cells at a conﬂuency of 70% was

transfected with the jetPEI DNA transfection reagent (Polyplus

transfection) according to manufacturer’s protocol. A total of 20 μg of

pcDNA-IDO1 along with 5 μg of pcDNA-GFP plasmid was used for

the transfection; GFP was used to evaluate the transfection eﬃciency

prior to the cellular assays; 16−18 h post-transfection, the 293T cells

were detached with trypsin, centrifuged, and resuspended in 25 mL of

DMEM medium without phenol red, supplemented with penicillin

and streptomycin, 10% fetal bovine serum, and 1 mM pyruvate

(Gibco). Subsequently, 100 μL of cells were distributed to each well

of two 96-well round-bottom plates that had been pre-loaded with

100 μL DMEM and the small organic test molecule in DMSO (for a

ﬁnal DMSO concentration of 2%). The DMEM medium contains 78

μM L-trp and was supplemented with 500 μM of L-trp for hIDO1.

Cells transfected with hIDO1 were incubated for 7 h at 37 °C in a

CO₂incubator. The reaction was stopped in its linear phase by adding

trichloroacetic acid (TCA) to the medium at a ﬁnal concentration of

4%. Plates were centrifuged for 15 min at 4000 rpm. The supernatant

(100 μL) was added to 100 μL of 2% (w/v) p-DMAB in glacial acetic

acid. After 10 min, the absorbance was measured at a wavelength of

480 nm to detect kynurenine formation.¹²²Alternatively, kynurenine

content was determined by HPLC as done for the enzymatic assay.

4.9. Cell Viability. Following DMSO (negative control) or

inhibitor treatments, apoptotic cells were detected by 4′,6-diamidino-

2-phenylindole (DAPI) staining. Brieﬂy, after 7 h of incubation, the

cells were washed with PBS, resuspended in PBS with 1 μg/mL of

DAPI, and immediately analyzed using a BD LSR II cytometer. A 405

nm laser with a 450/50 nm band-pass ﬁlter was used to collect data.

structures of ligand/heme models of compounds 1a−

6f; DFT-calculated iron−nitrogen bond lengths includ-

ing neutral model structures; MM-GBSA results; ¹H and

¹³C NMR spectra for synthesized compounds; and

HPLC purity analysis of compound 5f (PDF)

Molecular formula strings for all compound (CSV)

Accession Codes

IDO1 in complex with compounds 4f (PDB ID 7ah4), 5f

(7ah5), and 5g (7ah6). Authors will release the atomic

coordinates and experimental data upon article publication.

AUTHOR INFORMATION

Corresponding Authors

■

̈

Ute F. Rohrig − Molecular Modeling Group, SIB Swiss

Institute of Bioinformatics, 1015 Lausanne, Switzerland;

orcid.org/0000-0002-4676-4087; Email: Ute.Roehrig@

sib.swiss

Olivier Michielin − Molecular Modeling Group, SIB Swiss

Institute of Bioinformatics, 1015 Lausanne, Switzerland;

Department of Oncology, Ludwig Cancer Research-Lausanne

Branch, University Hospital of Lausanne (CHUV), 1011

Lausanne, Switzerland; Email: Olivier.Michielin@chuv.ch

Vincent Zoete − Molecular Modeling Group, SIB Swiss

Institute of Bioinformatics, 1015 Lausanne, Switzerland;

Department of Oncology UNIL-CHUV, Ludwig Lausanne

Branch, University of Lausanne, 1066 Epalinges,

Switzerland; orcid.org/0000-0002-2336-6537;

Email: Vincent.Zoete@unil.ch

Authors

Somi Reddy Majjigapu − Molecular Modeling Group, SIB

Swiss Institute of Bioinformatics, 1015 Lausanne,

Switzerland; Laboratory of Glycochemistry and Asymmetric

Synthesis, École Polytechnique Fédérale de Lausanne (EPFL),

1015 Lausanne, Switzerland; orcid.org/0000-0001-

6422-5223

Aline Reynaud − Protein Production and Structure Core

Facility, School of Life Sciences, École Polytechnique Fédérale

de Lausanne (EPFL), 1015 Lausanne, Switzerland

Florence Pojer − Protein Production and Structure Core

Facility, School of Life Sciences, École Polytechnique Fédérale

de Lausanne (EPFL), 1015 Lausanne, Switzerland

Nahzli Dilek − Molecular Modeling Group, SIB Swiss Institute

of Bioinformatics, 1015 Lausanne, Switzerland

Patrick Reichenbach − Department of Oncology UNIL-

CHUV, Ludwig Lausanne Branch, University of Lausanne,

1066 Epalinges, Switzerland

Kelly Ascencao − Molecular Modeling Group, SIB Swiss

Institute of Bioinformatics, 1015 Lausanne, Switzerland

Melita Irving − Department of Oncology UNIL-CHUV,

Ludwig Lausanne Branch, University of Lausanne, 1066

Epalinges, Switzerland

George Coukos − Department of Oncology UNIL-CHUV,

Ludwig Lausanne Branch, University of Lausanne, 1066

Epalinges, Switzerland; Department of Oncology, Ludwig

Cancer Research-Lausanne Branch, University Hospital of

Lausanne (CHUV), 1011 Lausanne, Switzerland

Pierre Vogel − Laboratory of Glycochemistry and Asymmetric

Synthesis, École Polytechnique Fédérale de Lausanne (EPFL),

1015 Lausanne, Switzerland

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01968.

Enzymatic and cellular IDO1 inhibition and toxicity

data; data collection and reﬁnement statistics for X-ray

crystallography; calculated Hirshfeld charges and activity

information for scaﬀolds 7−35; enzymatic dose−

response curves and Hill slopes for compounds 1a−6f;

cellular dose−response curves and Hill slopes for

selected compounds; correlation between kynurenine

detection by Ehrlich reagent and by HPLC for cellular

assays; 2F_o− F_cand F_o− F_cmaps for active sites of 4f,

5f, and 5g bound to IDO1; DFT-optimized ligand

structures 1a−6f; correlation between calculated Hirsh-

feld charge on iron-binding nitrogen atom and

experimentally measured activities; DFT-optimized

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01968

2222

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

AHR Restricts a Treg-Macrophage Suppressive Axis Induced by L-

Author Contributions

Kynurenine. Nat. Commun. 2020, 11, 4011.

U.F.R. designed the compounds, performed the enzymatic

assays and the HPLC analysis of enzymatic and cellular

samples, performed QM and MM-GBSA calculations,

determined the X-ray crystallographic protein structures with

F.P., analyzed and interpreted the data, and wrote the

manuscript. S.M.R. and P.V. planned and carried out chemical

synthesis and characterization. A.R. and F.P. performed protein

expression and crystallography. N.D., P.R., and K.A. carried out

the cellular assays under supervision of M.I. and G.C. O.M.

and V.Z. initiated the project and provided critical feedback.

V.Z. also provided the MM-GBSA algorithm, contributed to

compound design, and edited the manuscript.

̈

(7) Sadik, A.; Somarribas Patterson, L. F.; Ozturk, S.; Mohapatra, S.

̈

R.; Panitz, V.; Secker, P. F.; Pfander, P.; Loth, S.; Salem, H.; Prentzell,

M. T.; Berdel, B.; Iskar, M.; Faessler, E.; Reuter, F.; Kirst, I.; Kalter,

̈

V.; Foerster, K. I.; Jager, E.; Guevara, C. R.; Sobeh, M.; Hielscher, T.;

Poschet, G.; Reinhardt, A.; Hassel, J. C.; Zapatka, M.; Hahn, U.; von

Deimling, A.; Hopf, C.; Schlichting, R.; Escher, B. I.; Burhenne, J.;

̈

Haefeli, W. E.; Ishaque, N.; Bohme, A.; Schauble, S.; Thedieck, K.;

Trump, S.; Seiffert, M.; Opitz, C. A. IL4I1 Is a Metabolic Immune

Checkpoint That Activates the AHR and Promotes Tumor

Progression. Cell 2020, 182, 1252−1270.

(8) Pallotta, M. T.; Orabona, C.; Volpi, C.; Vacca, C.; Belladonna,

M. L.; Bianchi, R.; Servillo, G.; Brunacci, C.; Calvitti, M.; Bicciato, S.;

Mazza, E. M. C.; Boon, L.; Grassi, F.; Fioretti, M. C.; Fallarino, F.;

Puccetti, P.; Grohmann, U. Indoleamine 2,3-Dioxygenase Is a

Signaling Protein in Long-Term Tolerance by Dendritic Cells. Nat.

Immunol. 2011, 12, 870−878.

(9) Albini, E.; Rosini, V.; Gargaro, M.; Mondanelli, G.; Belladonna,

M. L.; Pallotta, M. T.; Volpi, C.; Fallarino, F.; Macchiarulo, A.;

Antognelli, C.; Bianchi, R.; Vacca, C.; Puccetti, P.; Grohmann, U.;

Orabona, C. Distinct Roles of Immunoreceptor Tyrosine-Based

Motifs in Immunosuppressive Indoleamine 2,3-Dioxygenase 1. J. Cell

Mol. Med. 2017, 21, 165−176.

(10) Albini, E.; Coletti, A.; Greco, F.; Pallotta, M. T.; Mondanelli,

G.; Gargaro, M.; Belladonna, M. L.; Volpi, C.; Bianchi, R.; Grohmann,

U.; Macchiarulo, A.; Orabona, C. Identification of a 2-Propanol

Analogue Modulating the Non-Enzymatic Function of Indoleamine

2,3-Dioxygenase 1. Biochem. Pharmacol. 2018, 158, 286−297.

(11) Nelp, M. T.; Kates, P. A.; Hunt, J. T.; Newitt, J. A.; Balog, A.;

Maley, D.; Zhu, X.; Abell, L.; Allentoff, A.; Borzilleri, R.; Lewis, H. A.;

Lin, Z.; Seitz, S. P.; Yan, C.; Groves, J. T. Immune-Modulating

Enzyme Indoleamine 2,3-Dioxygenase Is Effectively Inhibited by

Targeting Its Apo-Form. Proc. Natl. Acad. Sci. U.S.A. 2018, 115,

3249−3254.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank Bili Seijo from the M.I. and G.C. laboratory for

providing the IDO1 construct and Jean Philippe Gaudry from

the Protein Production and Structure Core Facility for rhIDO1

production and puriﬁcation. We thank Ganesh Kumar

Mothukuri for help with the analytical HPLC analysis for

purity of the compounds. We acknowledge computational

resources from Vital-IT (SIB Swiss Institute of Bioinformatics)

and the Scientiﬁc Computing and Research Support Unit

(University of Lausanne). We are grateful to Michel Cuendet

for support with the MM-GBSA analysis and Antoine Daina

for fruitful discussions. MarvinSketch 19.11, 2019, ChemAxon

(http://www.chemaxon.com), was used for drawing, display-

ing, and characterizing chemical structures.

ABBREVIATIONS

■

(12) Lim, Y. J.; Foo, T. C.; Yeung, A. W. S.; Tu, X.; Ma, Y.; Hawkins,

C. L.; Witting, P. K.; Jameson, G. N. L.; Terentis, A. C.; Thomas, S. R.

Human Indoleamine 2,3-Dioxygenase 1 Is an Efficient Mammalian

Nitrite Reductase. Biochemistry 2019, 58, 974−986.

AHR, aryl hydrocarbon receptor; IDO1, indoleamine 2,3-

dioxygenase 1; MM-GBSA, molecular mechanics-Generalized

Born surface area; p-DMAB, p-dimethylaminobenzaldehyde;

QM, quantum mechanical; TDO, tryptophan 2,3-dioxygenase

(13) Stanley, C. P.; Maghzal, G. J.; Ayer, A.; Talib, J.; Giltrap, A. M.;

Shengule, S.; Wolhuter, K.; Wang, Y.; Chadha, P.; Suarna, C.;

Prysyazhna, O.; Scotcher, J.; Dunn, L. L.; Prado, F. M.; Nguyen, N.;

Odiba, J. O.; Baell, J. B.; Stasch, J.-P.; Yamamoto, Y.; di Mascio, P.;

Eaton, P.; Payne, R. J.; Stocker, R. Singlet Molecular Oxygen

Regulates Vascular Tone and Blood Pressure in Inflammation. Nature

2019, 566, 548−552.

REFERENCES

■

(1) Garber, K. A new cancer immunotherapy suffers a setback.

Science 2018, 360, 588.

(2) Long, G. V.; Dummer, R.; Hamid, O.; Gajewski, T. F.; Caglevic,

C.; Dalle, S.; Arance, A.; Carlino, M. S.; Grob, J.-J.; Kim, T. M.;

Demidov, L.; Robert, C.; Larkin, J.; Anderson, J. R.; Maleski, J.; Jones,

M.; Diede, S. J.; Mitchell, T. C. Epacadostat plus Pembrolizumab

versus Placebo plus Pembrolizumab in Patients with Unresectable or

Metastatic Melanoma (ECHO-301/KEYNOTE-252): A Phase 3,

Randomised, Double-Blind Study. Lancet Oncol. 2019, 20, 1083−

1097.

(14) Nelp, M. T.; Zheng, V.; Davis, K. M.; Stiefel, K. J. E.; Groves, J.

T. Potent Activation of Indoleamine 2,3-Dioxygenase by Polysulfides.

J. Am. Chem. Soc. 2019, 141, 15288−15300.

̈

(15) Rohrig, U. F.; Reynaud, A.; Majjigapu, S. R.; Vogel, P.; Pojer,

F.; Zoete, V. Inhibition Mechanisms of Indoleamine 2,3-Dioxygenase

1 (IDO1). J. Med. Chem. 2019, 62, 8784−8795.

(16) Ortiz-Meoz, R.; Wang, L.; Matico, R.; Rutkowska, A.; De la

Rosa, M.; Bedard, S.; Midgett, R.; Strohmer, K.; Thomson, D.; Zhang,

C.; Guss, J.; Totoritis, R.; Consler, T.; Campobasso, N.; Taylor, D.;

Lewis, T.; Weaver, K.; Mulbaier, M.; Seal, J.; Dunham, R.; Kazmierski,

W.; Favre, D.; Bergamini, G.; Shewchuk, L.; Rendina, A.; Zhang, G.

Characterization of Novel Inhibition of Indoleamine 2,3-Dioxygenase

by Targeting Its Apo Form. 2018, bioRxiv:324947.

(3) van den Eynde, B. J.; van Baren, N.; Baurain, J.-F. Is There a

Clinical Future for IDO1 Inhibitors After the Failure of Epacadostat

in Melanoma? Annu. Rev. Canc. Biol. 2020, 4, 241−256.

(4) Opitz, C. A.; Litzenburger, U. M.; Sahm, F.; Ott, M.; Tritschler,

I.; Trump, S.; Schumacher, T.; Jestaedt, L.; Schrenk, D.; Weller, M.;

Jugold, M.; Guillemin, G. J.; Miller, C. L.; Lutz, C.; Radlwimmer, B.;

Lehmann, I.; von Deimling, A.; Wick, W.; Platten, M. An Endogenous

Tumour-Promoting Ligand of the Human Aryl Hydrocarbon

Receptor. Nature 2011, 478, 197−203.

̈

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

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

Eynde, B. J.; Zoete, V.; Michielin, O. Rational Design of Indoleamine

2,3-Dioxygenase Inhibitors. J. Med. Chem. 2010, 53, 1172−1189.

(18) Sono, M. Enzyme Kinetic and Spectroscopic Studies of

Inhibitor and Effector Interactions with Indoleamine 2,3-Dioxygenase.

2. Evidence for the Existence of Another Binding Site in the Enzyme

for Indole Derivative Effectors. Biochemistry 1989, 28, 5400−5407.

(19) Sugimoto, H.; Oda, S.-i.; Otsuki, T.; Hino, T.; Yoshida, T.;

Shiro, Y. Crystal Structure of Human Indoleamine 2,3-Dioxygenase:

̈

(5) Platten, M.; Nollen, E. A. A.; Rohrig, U. F.; Fallarino, F.; Opitz,

C. A. Tryptophan Metabolism as a Common Therapeutic Target in

Cancer, Neurodegeneration and Beyond. Nat. Rev. Drug Discovery

2019, 18, 379−401.

(6) Campesato, L. F.; Budhu, S.; Tchaicha, J.; Weng, C.-H.; Gigoux,

M.; Cohen, I. J.; Redmond, D.; Mangarin, L.; Pourpe, S.; Liu, C.;

Zappasodi, R.; Zamarin, D.; Cavanaugh, J.; Castro, A. C.; Manfredi,

M. G.; McGovern, K.; Merghoub, T.; Wolchok, J. D. Blockade of the

2223

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Catalytic Mechanism of O2 Incorporation by a Heme-Containing

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

(20) 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.

(21) Kumar, S.; Waldo, J. P.; Jaipuri, F. A.; Marcinowicz, A.; van

Allen, C.; Adams, J.; Kesharwani, T.; Zhang, X.; Metz, R.; Oh, A. J.;

Harris, S. F.; Mautino, M. R. Discovery of Clinical Candidate (1R,4r)-

4-((R)-2-((S)-6-Fluoro-5H-Imidazo[5,1-a]Isoindol-5-Yl)-1-

Hydroxyethyl)Cyclohexan-1-Ol (Navoximod), a Potent and Selective

Inhibitor of Indoleamine 2,3-Dioxygenase 1. J. Med. Chem. 2019, 62,

6705−6733.

Chang, J.-Y.; Lee, L.-C.; Shih, C.; Shia, K.-S.; Yeh, T.-K.; Hung, M.-S.;

Kuo, C.-C.; Song, J.-S.; Wu, S.-Y.; Ueng, S.-H. Unique Sulfur-

Aromatic Interactions Contribute to the Binding of Potent

Imidazothiazole Indoleamine 2,3-Dioxygenase Inhibitors. J. Med.

Chem. 2020, 63, 1642−1659.

(34) Yue, E. W.; Douty, B.; Wayland, B.; Bower, M.; Liu, X.; Leffet,

L.; Wang, Q.; Bowman, K. J.; Hansbury, M. J.; Liu, C.; Wei, M.; Li, Y.;

Wynn, R.; Burn, T. C.; Koblish, H. K.; Fridman, J. S.; Metcalf, B.;

Scherle, P. A.; Combs, A. P. Discovery of Potent Competitive

Inhibitors of Indoleamine 2,3-Dioxygenase with in Vivo Pharmacody-

namic Activity and Efficacy in a Mouse Melanoma Model. J. Med.

Chem. 2009, 52, 7364−7367.

(35) Yue, E. W.; Sparks, R.; Polam, P.; Modi, D.; Douty, B.;

Wayland, B.; Glass, B.; Takvorian, A.; Glenn, J.; Zhu, W.; Bower, M.;

Liu, X.; Leffet, L.; Wang, Q.; Bowman, K. J.; Hansbury, M. J.; Wei,

M.; Li, Y.; Wynn, R.; Burn, T. C.; Koblish, H. K.; Fridman, J. S.;

Emm, T.; Scherle, P. A.; Metcalf, B.; Combs, A. P. INCB24360

(Epacadostat), a Highly Potent and Selective Indoleamine-2,3-

Dioxygenase 1 (IDO1) Inhibitor for Immuno-Oncology. ACS Med.

Chem. Lett. 2017, 8, 486−491.

(36) Wu, Y.; Xu, T.; Liu, J.; Ding, K.; Xu, J. Structural Insights into

the Binding Mechanism of IDO1 with Hydroxylamidine Based

Inhibitor INCB14943. Biochem. Biophys. Res. Commun. 2017, 487,

339−343.

(22) Peng, Y.-H.; Ueng, S.-H.; Tseng, C.-T.; Hung, M.-S.; Song, J.-

S.; Wu, J.-S.; Liao, F.-Y.; Fan, Y.-S.; Wu, M.-H.; Hsiao, W.-C.; Hsueh,

C.-C.; Lin, S.-Y.; Cheng, C.-Y.; Tu, C.-H.; Lee, L.-C.; Cheng, M.-F.;

Shia, K.-S.; Shih, C.; Wu, S.-Y. Important Hydrogen Bond Networks

in Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitor Design Revealed

by Crystal Structures of Imidazoleisoindole Derivatives with IDO1. J.

Med. Chem. 2016, 59, 282−293.

̀

(23) Fallarini, S.; Massarotti, A.; Gesu, A.; Giovarruscio, S.; Coda

Zabetta, G.; Bergo, R.; Giannelli, B.; Brunco, A.; Lombardi, G.; Sorba,

G.; Pirali, T. In silico-driven multicomponent synthesis of 4,5- and

1,5-disubstituted imidazoles as indoleamine 2,3-dioxygenase inhib-

itors. MedChemComm 2016, 7, 409−419.

(37) Lewis-Ballester, A.; Pham, K. N.; Batabyal, D.; Karkashon, S.;

Bonanno, J. B.; Poulos, T. L.; Yeh, S.-R. Structural Insights into

Substrate and Inhibitor Binding Sites in Human Indoleamine 2,3-

Dioxygenase 1. Nat. Commun. 2017, 8, 1693.

(24) Zou, Y.; Wang, F.; Wang, Y.; Sun, Q.; Hu, Y.; Li, Y.; Liu, W.;

Guo, W.; Huang, Z.; Zhang, Y.; Xu, Q.; Lai, Y. Discovery of

Imidazoleisoindole Derivatives as Potent IDO1 Inhibitors: Design,

Synthesis, Biological Evaluation and Computational Studies. Eur. J.

Med. Chem. 2017, 140, 293−304.

(25) Brant, M. G.; Goodwin-Tindall, J.; Stover, K. R.; Stafford, P.

M.; Wu, F.; Meek, A. R.; Schiavini, P.; Wohnig, S.; Weaver, D. F.

Identification of Potent Indoleamine 2,3-Dioxygenase 1 (IDO1)

Inhibitors Based on a Phenylimidazole Scaffold. ACS Med. Chem. Lett.

2018, 9, 131−136.

(26) Wen, H.; Liu, Y.; Wang, S.; Wang, T.; Zhang, G.; Chen, X.; Li,

Y.; Cui, H.; Lai, F.; Sheng, L. Design and Synthesis of Indoleamine

2,3-Dioxygenase 1 Inhibitors and Evaluation of Their Use as Anti-

Tumor Agents. Molecules 2019, 24, 2124.

(27) Tu, W.; Yang, F.; Xu, G.; Chi, J.; Liu, Z.; Peng, W.; Hu, B.;

Zhang, L.; Wan, H.; Yu, N.; Jin, F.; Hu, Q.; Zhang, L.; He, F.; Tao, W.

Discovery of Imidazoisoindole Derivatives as Highly Potent and

Orally Active Indoleamine-2,3-Dioxygenase Inhibitors. ACS Med.

Chem. Lett. 2019, 10, 949−953.

(28) Bai, Z.; Huang, H.; Chen, J.; Zhang, X.; Ding, Y. Identification

of novel imidazoles as IDO1 inhibitors through microwave-assisted

one-pot multicomponent reactions. Arch. Pharm. 2019, 352, 1900165.

(38) Luo, S.; Xu, K.; Xiang, S.; Chen, J.; Chen, C.; Guo, C.; Tong,

Y.; Tong, L. High-Resolution Structures of Inhibitor Complexes of

Human Indoleamine 2,3-Dioxygenase 1 in a New Crystal Form. Acta

Crystallogr., Sect. F: Struct. Biol. Commun. 2018, 74, 717−724.

(39) Zhang, H.; Liu, K.; Pu, Q.; Achab, A.; Ardolino, M. J.; Cheng,

M.; Deng, Y.; Doty, A. C.; Ferguson, H.; Fradera, X.; Knemeyer, I.;

Kurukulasuriya, R.; Lam, Y.-h.; Lesburg, C. A.; Martinot, T. A.;

McGowan, M. A.; Miller, J. R.; Otte, K.; Biju, P. J.; Sciammetta, N.;

Solban, N.; Yu, W.; Zhou, H.; Wang, X.; Bennett, D. J.; Han, Y.

Discovery of Amino-Cyclobutarene-Derived Indoleamine-2,3-Dioxy-

genase 1 (IDO1) Inhibitors for Cancer Immunotherapy. ACS Med.

Chem. Lett. 2019, 10, 1530−1536.

̈

(40) Rohrig, U. F.; Zoete, V.; Michielin, O. The Binding Mode of N-

Hydroxyamidines to Indoleamine 2,3-Dioxygenase 1 (IDO1).

Biochemistry 2017, 56, 4323−4325.

(41) Paul, S.; Roy, A.; Deka, S. J.; Panda, S.; Trivedi, V.; Manna, D.

Nitrobenzofurazan derivatives of N ′-hydroxyamidines as potent

inhibitors of indoleamine-2,3-dioxygenase 1. Eur. J. Med. Chem.

2016, 121, 364−375.

̀

(29) Serafini, M.; Torre, E.; Aprile, S.; Grosso, E. D.; Gesu, A.;

(42) Du, Q.; Feng, X.; Wang, Y.; Xu, X.; Zhang, Y.; Qu, X.; Li, Z.;

Bian, J. Discovery of Phosphonamidate IDO1 Inhibitors for the

Treatment of Non-Small Cell Lung Cancer. Eur. J. Med. Chem. 2019,

182, 111629.

(43) Chen, S.; Guo, W.; Liu, X.; Sun, P.; Wang, Y.; Ding, C.; Meng,

L.; Zhang, A. Design, Synthesis and Antitumor Study of a Series of N-

Cyclic Sulfamoylaminoethyl Substituted 1,2,5-Oxadiazol-3-Amines as

New Indoleamine 2, 3-Dioxygenase 1 (IDO1) Inhibitors. Eur. J. Med.

Chem. 2019, 179, 38−55.

(44) Song, X.; Sun, P.; Wang, J.; Guo, W.; Wang, Y.; Meng, L.-h.;

Liu, H. Design, Synthesis, and Biological Evaluation of 1,2,5-

Oxadiazole-3-Carboximidamide Derivatives as Novel Indoleamine-

2,3-Dioxygenase 1 Inhibitors. Eur. J. Med. Chem. 2020, 189, 112059.

(45) Steeneck, C.; Kinzel, O.; Anderhub, S.; Hornberger, M.; Pinto,

S.; Morschhaeuser, B.; Braun, F.; Kleymann, G.; Hoffmann, T.

Discovery of Hydroxyamidine Based Inhibitors of IDO1 for Cancer

Immunotherapy with Reduced Potential for Glucuronidation. ACS

Med. Chem. Lett. 2020, 11, 179−187.

(46) Qian, S.; He, T.; Wang, W.; He, Y.; Zhang, M.; Yang, L.; Li, G.;

Wang, Z. Discovery and preliminary structure-activity relationship of

1H-indazoles with promising indoleamine-2,3-dioxygenase 1 (IDO1)

inhibition properties. Bioorg. Med. Chem. 2016, 24, 6194−6205.

Griglio, A.; Colombo, G.; Travelli, C.; Paiella, S.; Adamo, A.;

Orecchini, E.; Coletti, A.; Pallotta, M. T.; Ugel, S.; Massarotti, A.;

Pirali, T.; Fallarini, S. Discovery of Highly Potent Benzimidazole

Derivatives as Indoleamine 2,3-Dioxygenase-1 (IDO1) Inhibitors:

From Structure-Based Virtual Screening to in Vivo Pharmacodynamic

Activity. J. Med. Chem. 2020, 63, 3047−3065.

(30) Tojo, S.; Kohno, T.; Tanaka, T.; Kamioka, S.; Ota, Y.; Ishii, T.;

Kamimoto, K.; Asano, S.; Isobe, Y. Crystal Structures and Structure-

Activity Relationships of Imidazothiazole Derivatives as IDO1

Inhibitors. ACS Med. Chem. Lett. 2014, 5, 1119−1123.

(31) Griglio, A.; Torre, E.; Serafini, M.; Bianchi, A.; Schmid, R.;

Coda Zabetta, G.; Massarotti, A.; Sorba, G.; Pirali, T.; Fallarini, S. A

Multicomponent Approach in the Discovery of Indoleamine 2,3-

Dioxygenase 1 Inhibitors: Synthesis, Biological Investigation and

Docking Studies. Bioorg. Med. Chem. Lett. 2018, 28, 651−657.

(32) Serafini, M.; Torre, E.; Aprile, S.; Massarotti, A.; Fallarini, S.;

Pirali, T. Synthesis, Docking and Biological Evaluation of a Novel

Class of Imidazothiazoles as IDO1 Inhibitors. Molecules 2019, 24,

1874.

(33) Peng, Y.-H.; Liao, F.-Y.; Tseng, C.-T.; Kuppusamy, R.; Li, A.-S.;

Chen, C.-H.; Fan, Y.-S.; Wang, S.-Y.; Wu, M.-H.; Hsueh, C.-C.;

2224

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(47) Pradhan, N.; Paul, S.; Deka, S. J.; Roy, A.; Trivedi, V.; Manna,

D. Identification of Substituted 1H -Indazoles as Potent Inhibitors for

Immunosuppressive Enzyme Indoleamine 2,3-Dioxygenase 1. Chem-

istrySelect 2017, 2, 5511−5517.

(62) Haddach, M.; Tran, J. A.; Pierre, F.; Regan, C. F.; Raﬀaele, N.

B.; Ravula, S.; Ryckman, D. M. Pyrazolopyrimidines and Related

Heterocycles as CK2 Inhibitors, WO/2011/068667.

WO2011068667A1, 2011.

(63) Sun, X.; Hong, Z.; Liu, M.; Guo, S.; Yang, D.; Wang, Y.; Lan,

T.; Gao, L.; Qi, H.; Gong, P.; Liu, Y. Design, Synthesis, and Biological

Activity of Novel Tetrahydropyrazolopyridone Derivatives as FXa

Inhibitors with Potent Anticoagulant Activity. Bioorg. Med. Chem.

2017, 25, 2800−2810.

(64) Xie, F.; Du, C.-C.; Dong, J.-L.; Du, J.-Q.; Han, Y.-Z.; Wang, D.-

Z. New Complexes Constructed Based on (1H-Tetrazol-5-Yl)Phenol:

Synthesis, Structures and Properties. Polyhedron 2018, 149, 17−24.

(65) Xu, Z.-L.; Li, H.-X.; Ren, Z.-G.; Du, W.-Y.; Xu, W.-C.; Lang, J.-

P. Cu(OAc)2 ·H2O-catalyzed N-arylation of nitrogen-containing

heterocycles. Tetrahedron 2011, 67, 5282−5288.

(66) Pedras, M. S. C.; Suchy, M. Design, Synthesis, and Antifungal

Activity of Inhibitors of Brassilexin Detoxification in the Plant

Pathogenic Fungus Leptosphaeria Maculans. Bioorg. Med. Chem.

2006, 14, 714−723.

(48) Tsujino, H.; Uno, T.; Yamashita, T.; Katsuda, M.; Takada, K.;

Saiki, T.; Maeda, S.; Takagi, A.; Masuda, S.; Kawano, Y.; Meguro, K.;

Akai, S. Correlation of Indoleamine-2,3-Dioxigenase 1 Inhibitory

Activity of 4,6-Disubstituted Indazole Derivatives and Their Heme

Binding Affinity. Bioorg. Med. Chem. Lett. 2019, 29, 126607.

(49) Yang, L.; Chen, Y.; He, J.; Njoya, E. M.; Chen, J.; Liu, S.; Xie,

C.; Huang, W.; Wang, F.; Wang, Z.; Li, Y.; Qian, S. 4,6-Substituted-

1H-Indazoles as Potent IDO1/TDO Dual Inhibitors. Bioorg. Med.

Chem. 2019, 27, 1087−1098.

̈

(50) Rohrig, U. F.; Majjigapu, S. R.; Grosdidier, A.; Bron, S.;

Stroobant, V.; Pilotte, L.; Colau, D.; Vogel, P.; van den Eynde, B. J.;

Zoete, V.; Michielin, O. Rational Design of 4-Aryl-1,2,3-Triazoles for

Indoleamine 2,3-Dioxygenase 1 Inhibition. J. Med. Chem. 2012, 55,

5270−5290.

(51) Alexandre, J. A. C.; Swan, M. K.; Latchem, M. J.; Boyall, D.;

Pollard, J. R.; Hughes, S. W.; Westcott, J. New 4-Amino-1,2,3-Triazole

Inhibitors of Indoleamine 2,3-Dioxygenase Form a Long-Lived

Complex with the Enzyme and Display Exquisite Cellular Potency.

ChemBioChem 2018, 19, 552−561.

(67) Aoyama, T.; Iwamoto, Y.; Shioiri, T. New Methods and

Reagents in Organic Synthesis. 59. Lithium Trimethylsilydiazo-

methane: A New Synthon for the Preparation of 5-Substituted

1,2,3-Thiadiazoles. Heterocycles 1986, 24, 589−592.

(52) Panda, S.; Pradhan, N.; Chatterjee, S.; Morla, S.; Saha, A.; Roy,

A.; Kumar, S.; Bhattacharyya, A.; Manna, D. 4,5-Disubstituted 1,2,3-

Triazoles: Effective Inhibition of Indoleamine 2,3-Dioxygenase 1

Enzyme Regulates T Cell Activity and Mitigates Tumor Growth. Sci.

Rep. 2019, 9, 18455.

(68) Yamamuro, D.; Uchida, R.; Ohtawa, M.; Arima, S.; Futamura,

Y.; Katane, M.; Homma, H.; Nagamitsu, T.; Osada, H.; Tomoda, H.

Synthesis and Biological Activity of 5-(4-Methoxyphenyl)-Oxazole

Derivatives. Bioorg. Med. Chem. Lett. 2015, 25, 313−316.

(69) Wakeﬁeld, B. J. Product Class 9: Isoxazoles. In Science of

Synthesis; Schaumann, E., Ed.; Thieme Verlagsgruppe: Stuttgart, New

York, Delhi, Rio, 2002; pp 229−288.

(53) Petrunak, E. M.; Rogers, S. A.; Aubé, J.; Scott, E. E. Structural

and Functional Evaluation of Clinically Relevant Inhibitors of

Steroidogenic Cytochrome P450 17A1. Drug Metab. Dispos. 2017,

45, 635−645.

(70) Ohmoto, K.; Yamamoto, T.; Okuma, M.; Horiuchi, T.;

Imanishi, H.; Odagaki, Y.; Kawabata, K.; Sekioka, T.; Hirota, Y.;

Matsuoka, S.; Nakai, H.; Toda, M.; Cheronis, J. C.; Spruce, L. W.;

Gyorkos, A.; Wieczorek, M. Development of Orally Active Non-

peptidic Inhibitors of Human Neutrophil Elastase. J. Med. Chem.

2001, 44, 1268−1285.

̈

(54) Rohrig, U. F.; Majjigapu, S. R.; Chambon, M.; Bron, S.; Pilotte,

L.; Colau, D.; van den Eynde, B. J.; Turcatti, G.; Vogel, P.; Zoete, V.;

Michielin, O. Detailed Analysis and Follow-up Studies of a High-

Throughput Screening for Indoleamine 2,3-Dioxygenase 1 (IDO1)

Inhibitors. Eur. J. Med. Chem. 2014, 84, 284−301.

̈

(71) Rohrig, U. F.; Majjigapu, S. R.; Vogel, P.; Zoete, V.; Michielin,

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

McCarter, J. D.; Sutherland, C. L. Purification and Kinetic

Characterization of Human Indoleamine 2,3-Dioxygenases 1 and 2

(IDO1 and IDO2) and Discovery of Selective IDO1 Inhibitors.

Biochim. Biophys. Acta, Proteins Proteomics 2011, 1814, 1947−1954.

(56) Hargrove, T. Y.; Friggeri, L.; Wawrzak, Z.; Qi, A.; Hoekstra, W.

J.; Schotzinger, R. J.; York, J. D.; Guengerich, F. P.; Lepesheva, G. I.

Structural analyses of Candida albicans sterol 14 α -demethylase

complexed with azole drugs address the molecular basis of azole-

mediated inhibition of fungal sterol biosynthesis. J. Biol. Chem. 2017,

292, 6728−6743.

(57) Hoekstra, W. J.; Garvey, E. P.; Moore, W. R.; Rafferty, S. W.;

Yates, C. M.; Schotzinger, R. J. Design and Optimization of Highly-

Selective Fungal CYP51 Inhibitors. Bioorg. Med. Chem. Lett. 2014, 24,

3455−3458.

(58) 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.

(59) Mautino, M. R.; Kumar, S.; Jaipuri, F.; Waldo, J.; Kesharwani,

T.; Zhang, X. Imidazole Derivatives as IDO Inhibitors, WO/2011/

056652. WO2011056652A1, 2011.

O. Challenges in the Discovery of Indoleamine 2,3-Dioxygenase 1

(IDO1) Inhibitors. J. Med. Chem. 2015, 58, 9421−9437.

(72) Morrison, J. F. Kinetics of the Reversible Inhibition of Enzyme-

Catalysed Reactions by Tight-Binding Inhibitors. Biochim. Biophys.

Acta, Enzymol. 1969, 185, 269−286.

̌

(73) Kuzmic, P. History, Variants and Usage of the “Morrison

Equation” in Enzyme Inhibition Kinetics. BioKin Technical Note;

BioKin Ltd., 2015; pp 1−15.

(74) Wilcken, R.; Zimmermann, M. O.; Lange, A.; Zahn, S.;

Kirchner, B.; Boeckler, F. M. Addressing Methionine in Molecular

Design through Directed Sulfur-Halogen Bonds. J. Chem. Theory

Comput. 2011, 7, 2307−2315.

(75) Wilcken, R.; Zimmermann, M. O.; Lange, A.; Joerger, A. C.;

Boeckler, F. M. Principles and Applications of Halogen Bonding in

Medicinal Chemistry and Chemical Biology. J. Med. Chem. 2013, 56,

1363−1388.

(76) Bissantz, C.; Kuhn, B.; Stahl, M. A Medicinal Chemist ’s Guide

to Molecular Interactions. J. Med. Chem. 2010, 53, 5061−5084.

(77) Shinada, N. K.; de Brevern, A. G.; Schmidtke, P. Halogens in

Protein-Ligand Binding Mechanism: A Structural Perspective. J. Med.

Chem. 2019, 62, 9341−9356.

(78) Tomek, P.; Palmer, B. D.; Flanagan, J. U.; Sun, C.; Raven, E. L.;

Ching, L.-M. Discovery and Evaluation of Inhibitors to the

Immunosuppressive Enzyme Indoleamine 2,3-Dioxygenase 1

(IDO1): Probing the Active Site-Inhibitor Interactions. Eur. J. Med.

Chem. 2017, 126, 983−996.

(79) Onufriev, A. v.; Alexov, E. Protonation and PK Changes in

Protein-Ligand Binding. Q. Rev. Biophys. 2013, 46, 181−209.

(80) George, P.; Hanania, G. I. H.; Irvine, D. H.; Abu-Issa, I. 1090.

The effect of co-ordination on ionization. Part IV. Imidazole and its

ferrimyoglobin complex. J. Chem. Soc. 1964, 5689−5694.

(60) Bondy, S. S.; Cannizzaro, C. E.; Chien-Hung, C.; Link, J. O.;

Qui, L.; Schroeder, S. D.; Winston, T. C.; Zhang, J. 5-Membered

Heteroaryls and Their Use as Antiviral Agents, WO/2014/110298.

WO2014110298A1, 2014.

(61) Pratt, D. A.; Pesavento, R. P.; van der Donk, W. A. Model

Studies of the Histidine-Tyrosine Cross-Link in CytochromecOxidase

Reveal the Flexible Substituent Effect of the Imidazole Moiety. Org.

Lett. 2005, 7, 2735−2738.

2225

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(81) Song, Y.; Mao, J.; Gunner, M. R. Electrostatic Environment of

Hemes in Proteins: pKas of Hydroxyl Ligands †. Biochemistry 2006,

45, 7949−7958.

(82) Erman, J. E.; Chinchilla, D.; Studer, J.; Vitello, L. B. Binding of

Imidazole, 1-Methylimidazole and 4-Nitroimidazole to Yeast

Cytochrome c Peroxidase (CcP) and the Distal Histidine Mutant,

CcP(H52L). Biochim. Biophys. Acta, Proteins Proteomics 2015, 1854,

869−881.

(99) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features

and development ofCoot. Acta Crystallogr., Sect. D: Biol. Crystallogr.

2010, 66, 486−501.

(100) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.;

Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera?A

Visualization System for Exploratory Research and Analysis. J.

Comput. Chem. 2004, 25, 1605−1612.

(101) Adamo, C.; Cossi, M.; Barone, V. An Accurate Density

Functional Method for the Study of Magnetic Properties: The PBE0

Model. J. Mol. Struct.: THEOCHEM 1999, 493, 145−157.

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

Nitrogen Compounds: The Azoles; Cambridge University Press:

Cambridge [England], 1976.

(84) Srinivasan, J.; Cheatham, T. E.; Cieplak, P.; Kollman, P. A.;

Case, D. A. Continuum Solvent Studies of the Stability of DNA, RNA,

and Phosphoramidate −DNA Helices. J. Am. Chem. Soc. 1998, 120,

9401−9409.

(85) Zoete, V.; Irving, M. B.; Michielin, O. MM-GBSA Binding Free

Energy Decomposition and T Cell Receptor Engineering. J. Mol.

Recognit. 2010, 23, 142−152.

(86) Wang, E.; Sun, H.; Wang, J.; Wang, Z.; Liu, H.; Zhang, J. Z. H.;

Hou, T. End-Point Binding Free Energy Calculation with MM/PBSA

and MM/GBSA: Strategies and Applications in Drug Design. Chem.

Rev. 2019, 119, 9478−9508.

(87) Beers, S.; Xia, C.; Brooks, J.; Kottas, G. Cyclometallated

Tetradentate Pt(Ii) Complexes Comprised of One or Two Imidazole

Rings with Twisted Aryl Group Bonded to N-1, JP/2013/079235.

JP2013079235A, 2013.

(88) Yang, J.; Chen, C.; Tang, X. Cholesterol-Modified Caged

SiRNAs for Photoregulating Exogenous and Endogenous Gene

Expression. Bioconjugate Chem. 2018, 29, 1010−1015.

(89) Bellina, F.; Cauteruccio, S.; di Fiore, A.; Rossi, R. Regioselective

Synthesis of 4,5-Diaryl-1-methyl-1H-imidazoles Including Highly

Cytotoxic Derivatives by Pd-Catalyzed Direct C-5 Arylation of 1-

Methyl-1H-imidazole with Aryl Bromides. Eur. J. Org Chem. 2008,

5436−5445.

(90) Kirsch, P.; Jakob, V.; Oberhausen, K.; Stein, S. C.; Cucarro, I.;

Schulz, T. F.; Empting, M. Fragment-Based Discovery of a Qualified

Hit Targeting the Latency-Associated Nuclear Antigen of the

Oncogenic Kaposi ’s Sarcoma-Associated Herpesvirus/Human Her-

pesvirus 8. J. Med. Chem. 2019, 62, 3924−3939.

(102) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;

Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci,

B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.

P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;

Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.;

Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.;

Montgomery, J. A., Jr; Peralta, J. E.; Ogliaro, F.; Bearpark, M.;

Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi,

R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar,

S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox,

J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.;

Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.;

Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.;

Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A.

̈

D.; Farkas, O.; Foresman, J. B.; Ortiz, J. v.; Cioslowski, J.; Fox, D. J.

Gaussian 09 Revision A.1.; Gaussian, Inc., 2009.

̈

(103) Schafer, 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.

(104) Hirshfeld, F. L. Bonded-Atom Fragments for Describing

Molecular Charge Densities. Theor. Chim. Acta 1977, 44, 129−138.

(105) Dunning, T. H. Gaussian Basis Sets for Use in Correlated

Molecular Calculations. I. The Atoms Boron through Neon and

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

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

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

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

(107) Woon, D. E.; Dunning, T. H. Gaussian Basis Sets for Use in

through Argon. J. Chem. Phys. 1993, 98, 1358−1371.

(108) Tomasi, J.; Mennucci, B.; Cammi, R. Quantum Mechanical

Continuum Solvation Models. Chem. Rev. 2005, 105, 2999−3094.

(109) 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.

(91) Strehlke, P. Antifungal Imidazole Derivatives. II. Ethers of (1-

Imidazolyl)-Phenols. Eur. J. Med. Chem. 1979, 14, 227−230.

(92) Shechter, S.; Kauﬀmann, M.; Sandanyaka, V. P.; Shacham, S.

Nuclear Transport Modulators and Uses Thereof, WO/2011/109799.

WO2011109799A1, 2011.

(110) Brooks, B. R.; Brooks, C. L.; Mackerell, A. D.; Nilsson, L.;

Petrella, R. J.; Roux, B.; Won, Y.; Archontis, G.; Bartels, C.; Boresch,

S.; Caflisch, A.; Caves, L.; Cui, Q.; Dinner, A. R.; Feig, M.; Fischer, S.;

Gao, J.; Hodoscek, M.; Im, W.; Kuczera, K.; Lazaridis, T.; Ma, J.;

Ovchinnikov, V.; Paci, E.; Pastor, R. W.; Post, C. B.; Pu, J. Z.;

Schaefer, M.; Tidor, B.; Venable, R. M.; Woodcock, H. L.; Wu, X.;

Yang, W.; York, D. M.; Karplus, M. CHARMM: The Biomolecular

Simulation Program. J. Comput. Chem. 2009, 30, 1545−1614.

(111) MacKerell, A. D.; Bashford, D.; Bellott, M.; Dunbrack, R. L.;

Evanseck, J. D.; Field, M. J.; Fischer, S.; Gao, J.; Guo, H.; Ha, S.;

Joseph-McCarthy, D.; Kuchnir, L.; Kuczera, K.; Lau, F. T. K.; Mattos,

C.; Michnick, S.; Ngo, T.; Nguyen, D. T.; Prodhom, B.; Reiher, W. E.;

Roux, B.; Schlenkrich, M.; Smith, J. C.; Stote, R.; Straub, J.;

Watanabe, M.; Wiórkiewicz-Kuczera, J.; Yin, D.; Karplus, M. All-Atom

Empirical Potential for Molecular Modeling and Dynamics Studies of

Proteins †. J. Phys. Chem. B 1998, 102, 3586−3616.

(93) Pagacz-Kostrzewa, M.; Sałdyka, M.; Wierzejewska, M.;

Khomenko, D. M.; Doroschuk, R. O. Theoretical DFT and Matrix

Isolation FTIR Studies of 2-(1,2,4-Triazolyl)Phenol Isomers. Chem.

Phys. Lett. 2016, 657, 156−161.

(94) Knust, H.; Thomas, A. W. Substituted Imidazole[1,5-

A][1,2,4]Triazolo[1,5-D][1,4]Benzodiazepine Derivatives. U.S. Pat-

ent 0,084,642 A1, 2006.

(95) Treitler, D. S.; Leung, S.; Lindrud, M. Development and

Demonstration of a Safer Protocol for the Synthesis of 5-

Aryltetrazoles from Aryl Nitriles. Org. Process Res. Dev. 2017, 21,

460−467.

(96) Antypenko, O. M.; Kovalenko, S. I.; Karpenko, O. v. Synthesis

and hydrolytic cleavage of tetrazolo[1,5-c]quinazolines. Synth.

Commun. 2016, 46, 551−555.

(97) Kabsch, W. XDS. Acta Crystallogr., Sect. D: Biol. Crystallogr.

2010, 66, 125−132.

(98) Adams, P. D.; Afonine, P. v.; Bunkóczi, G.; Chen, V. B.; Davis,

I. W.; Echols, N.; Headd, J. J.; Hung, L.-W.; Kapral, G. J.; Grosse-

Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R.

J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P.

H. PHENIX: a comprehensive Python-based system for macro-

molecular structure solution. Acta Crystallogr., Sect. D: Biol. Crystallogr.

2010, 66, 213−221.

(112) Mackerell, A. D.; Feig, M.; Brooks, C. L. Extending the

Treatment of Backbone Energetics in Protein Force Fields:

Limitations of Gas-Phase Quantum Mechanics in Reproducing

Protein Conformational Distributions in Molecular Dynamics

Simulations. J. Comput. Chem. 2004, 25, 1400−1415.

(113) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R.

W.; Klein, M. L. Comparison of Simple Potential Functions for

Simulating Liquid Water. J. Chem. Phys. 1983, 79, 926−935.

2226

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(114) Brooks, C. L.; Brunger, A.; Karplus, M. Active Site Dynamics

̈

in Protein Molecules: A Stochastic Boundary Molecular-Dynamics

Approach. Biopolymers 1985, 24, 843−865.

(115) Lee, M. S.; Salsbury, F. R.; Brooks, C. L. Novel Generalized

Born Methods. J. Chem. Phys. 2002, 116, 10606−10614.

(116) Lee, M. S.; Feig, M.; Salsbury, F. R.; Brooks, C. L. New

Analytic Approximation to the Standard Molecular Volume Definition

and Its Application to Generalized Born Calculations. J. Comput.

Chem. 2003, 24, 1348−1356.

(117) Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T.

Semianalytical Treatment of Solvation for Molecular Mechanics and

Dynamics. J. Am. Chem. Soc. 1990, 112, 6127−6129.

(118) Gohlke, H.; Kiel, C.; Case, D. a. Insights into Protein-Protein

Binding by Binding Free Energy Calculation and Free Energy

Decomposition for the Ras-Raf and Ras-RalGDS Complexes. J. Mol.

Biol. 2003, 330, 891−913.

(119) Zięba, A.; Ramza, P. Standard Deviation of the Mean of

Autocorrelation Function Estimated From the Data. Metrol. Meas.

Syst. 2011, 18, 529−542.

(120) Madura, J. D.; Briggs, J. M.; Wade, R. C.; Davis, M. E.; Luty,

B. a.; Ilin, A.; Antosiewicz, J.; Gilson, M. K.; Bagheri, B.; Scott, L. R.;

McCammon, J. A. Electrostatics and Diffusion of Molecules in

Solution: Simulations with the University of Houston Brownian

Dynamics Program. Comput. Phys. Commun. 1995, 91, 57−95.

(121) 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.

(122) Littlejohn, T. K.; Takikawa, O.; Skylas, D.; Jamie, J. F.;

Walker, M. J.; Truscott, R. J. W. Expression and Purification of

Recombinant Human Indoleamine 2,3-Dioxygenase. Protein Expres-

sion Purif. 2000, 19, 22−29.

2227

https://dx.doi.org/10.1021/acs.jmedchem.0c01968

J. Med. Chem. 2021, 64, 2205−2227

Article Doi

Azole-Based Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors

DOI: 10.1021/acs.jmedchem.0c01968

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.0c01968

Full text of DOI:10.1021/acs.jmedchem.0c01968

Products guided by the article

R&D Labs maybe for 61292-68-0

Relevant to this article

Hot Product