Aminoisoxazoles as Potent Inhibitors of Tryptophan 2,3-Dioxygenase 2 (TDO2)

Article abstract of DOI:10.1021/acsmedchemlett.7b00427

Tryptophan 2,3-dioxygenase 2 (TDO2) catalyzes the conversion of tryptophan to the immunosuppressive metabolite kynurenine. TDO2 overexpression has been observed in a number of cancers; therefore, TDO inhibition may be a useful therapeutic intervention for

Full text of DOI:10.1021/acsmedchemlett.7b00427

Subscriber access provided by Warwick University Library
Aminoisoxazoles as potent inhibitors of tryptophan 2,3-dioxygenase 2 (TDO2)
Zhonghua Pei, Rohan Mendonca, Lewis Gazzard, Richard Pastor, Leanne Goon, Amy
Gustafson, Erica C VanderPorten, Georgia Hatzivassiliou, Kevin Dement, Robert Cass, Po-
wai Yuen, Yamin Zhang, Guosheng Wu, Xingyu Lin, Yichin Liu, and Benjamin D. Sellers
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00427 • Publication Date (Web): 02 Apr 2018
Downloaded from http://pubs.acs.org on April 4, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 6
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
Aminoisoxazoles as potent inhibitors of tryptophan 2,3-dioxygenase 2
(
TDO2)
†
†
†
†
§
§
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Zhonghua Pei , Rohan Mendonca , Lewis Gazzard , Richard Pastor , Leanne Goon , Amy Gustafson ,
Erica VanderPorten , Georgia Hatzivassiliou , Kevin Dement , Robert Cass , Poꢀwai Yuen , Yamin
Zhang , Guosheng Wu , Xingyu Lin , Yichin Liu , Benjamin D. Sellers *
§
ß
∆
∆
ø
ø
ø
ø
§
†
†
Department of Discovery Chemistry, ∞Department of Neurosciences, §Department of Biochemical and Cellular
Pharmacology, ∆Department of Drug Metabolism and Pharmacokinetics, ßDepartment of Translational Oncology,
Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA
ø PharmaronꢀBeijing Co. Ltd., 6 Taihe Road, BDA, Beijing 100176, PR China
KEYWORDS:
ABSTRACT
CANCER
IMMUNOTHERAPY,
IDO,
TDO,
HEME,
ENZYME.
Tryptophan 2,3ꢀdioxygenase 2 (TDO2) catalyzes the conversion of tryptophan to the immunosuppressive metabolite kynurenine.
TDO2 overꢀexpression has been observed in a number of cancers; therefore, TDO inhibition may be a useful therapeutic
intervention for cancers. We identified an aminoisoxazole series as potent TDO2 inhibitors from a highꢀthroughput screen (HTS).
An extensive medicinal chemistry effort revealed that both the amino group and the isoxazole moiety are important for TDO2
inhibitory activity. Computational modeling yielded a binding hypothesis and provided insight into the observed structureꢀactivity
relationships. The optimized compound 21 is a potent TDO2 inhibitor with modest selectivity over indolamine 2,3ꢀdioxygenase 1
(
IDO1) and with improved human whole blood stability.
system, with concomitant inhibition of the effector T cell
Teff) compartment. IDO1ꢀdeficiency (gene ablation or small
molecule inhibition) has been shown to boost Teff and
INTRODUCTION
(
One of the critical functions of the immune system is
to detect and eliminate malignant cells as they arise and
develop antigenic mutations. For tumor cells to survive, the
host immune system must be rendered functionally tolerant to
decrease Treg function in several autoimmunity and in vivo
tumor models.
7, 8, 9
The physiological function of TDO2 includes the regulation
of systemic Trp levels as well as liver Kyn levels. In vitro and
in vivo studies suggest that TDO2 plays a role in promoting
tumor cell survival and motility, and that TDO2ꢀexpressing
otherwise immunogenic tumorꢀassociated antigens.
To
accomplish this, tumors have been shown to enlist an array of
1
endogenous host regulatory pathways.
One of these
pathways is mediated by catabolism of the essential amino
acid tryptophan (Trp) into kynurenine (Kyn), a process driven
tumors have a lower degree of T cell infiltrate that allows
6, 10
them to escape immune rejection.
Genetic knockout or
by the rateꢀlimiting enzymes indoleamineꢀ2,3ꢀdioxygenase 1
treatment of mice with a tool enzymatic inhibitor of TDO2
activity resulted in increased sensitivity of mice to endotoxinꢀ
2
(
IDO1) and Trpꢀ2,3ꢀdioxygenase 2 (TDO2). These enzymes
catalyze the oxidation of Lꢀtryptophan (Trp) to Nꢀformyl
kynurenine (NFK), which rapidly converts to kynurenine
induced shock, consistent with a role for TDO2 in suppressing
11
inflammation and maintaining immune homeostasis.
In
(
Kyn). Overꢀexpression of these enzymes has been observed
tumors, TDO2 activity is highly expressed in liver cancer and
in several cancers and is thought to mitigate the immune
6, 12
glioblastoma and upregulated in several other cancers.
3
4
response towards these tumors. ,
Thus IDO1 and TDO2 inhibitors may represent a useful
therapeutic intervention that restores the immune response
against tumors, especially when combined with immune
checkpoint inhibitors. Indeed, nonclinical data demonstrates
that IDO1 and antiꢀPDꢀL1 dual inhibition shows markedly
greater efficacy in activating the immune system and
inhibiting tumor growth than either treatment alone, with a
response associated with more pronounced activation of
The enzymatic functions of IDO1 and TDO2 lead to
multiple immuneꢀrelated responses. IDO1ꢀmediated Trp
5
depletion activates GCN2 kinase,
which in turn
phosphorylates and inactivates the transcription factor eIF2α
leading to the blockade of protein translation and inhibition of
cell proliferation. Kyn accumulation on the other hand
6
activates the aryl hydrocarbon receptor (AhR), a transcription
factor implicated in cancer progression, invasion and immune
suppression. The overall result of IDO1 activity is induction
intratumoral CD8+ T cells, including proliferation and
2
13, 14
cytokine production.
Preliminary data of a phase I/II
of the regulatory T cell (Treg) compartment of the immune
clinical trial in melanoma where the IDO1 inhibitor
1
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 6
epacadostat is being tested in combination with antiꢀPD1 has
shown promising results in terms of response rates and depth
of responses, compared to antiꢀPDꢀ1 data.
Table 1 TDO2 cellular and biochemical potencies of the
aminoisoxazole modifications
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
5
While several classes of IDO1 inhibitors have been
1
6 , 17 , 18 , 19 , 20
disclosed in the literature,
, at the onset of our
program, TDO2 inhibitors had been explored to a far lesser
21
extent. Several patent applications describing either TDOꢀ
selective or IDO/TDO dual inhibitors have appeared
2
2,23,24,25,26
since.
TDO2 inhibition alone or in combination with
1
IDO1 inhibition may allow fuller suppression of the
kynurenine pathway across a range of tumor types. Here we
report our efforts in identifying and optimizing
aminoisoxazoleꢀbased TDO2 inhibitors.
Compound R
TDO2 TDO2
EC50 a IC50 b
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(ꢀM)
(ꢀM)
1
0.085
0.14
RESULTS
A highꢀthroughput screen of the Genentech compound
library identified a number of related aminoisoxazoles as
inhibitors of TDO2. Inhibitory potency was measured in both
biochemical and cellular assays of IDO1 and TDO2. In the
biochemical assays, inhibitors were incubated with
recombinant enzymes in the presence of the substrate
tryptophan. The production of the Nꢀformylkynurenine (NFK)
was detected either in a fluorescenceꢀ or mass specꢀbased
assay and inhibitory potency was expressed as IC50 values. In
the cellular assays, inhibitors were incubated with either
SW48 cells (in which endogenous TDO2 is highly expressed)
or A172 cells (in which IDO1 is highly expressed upon
induction by IFNγ). The production of NFK released from
cells into the media was detected by a fluorescenceꢀbased
assay and the inhibitory potency was expressed as EC50
values. The cytotoxicity of compounds was also measured for
the same treated cells with a luminescenceꢀbased assay
detecting cellular ATP levels. Since TDO2 biochemical
potency was poorly predictive of cellular potency for the
aminoisoxazole compounds (see Figure S1 in supplemental
material), our medicinal chemistry designs were consequently
guided by cellular potency. As a counter screen to identify
inhibition as a result of cytotoxicity, we assessed cell viability
of SW48 and A172 cells by measuring intracellular ATP
concentration. We found that the aminoisoxazoles generally
do not affect intracellular ATP concentrations up to 25 µM
inhibitor concentration in a cell viability assay, indicating that
the cellular potency was not caused by cytotoxicity of the
inhibitors.
2
3
4
5
6
7
>25
0.67
>25
>25
18
>25
0.19
>25
>25
4.4
9.7
25
a
b
Cellular potency. Biochemical potency. IC50 and EC50 values
are the averages of at least 2 independent experiments.
Aminoisoxazole 1 was the most potent hit found hit
(cellular EC50 of 85 nM, Table 1). We first investigated the
importance of the amino group on the isoxazole moiety.
Removal of the amino group (2) resulted in complete loss of
potency. Addition of a methyl group on the nitrogen atom (3)
We next investigated substitution and replacement of the
phenyl moiety of compound 1. Introduction of pꢀCl (8), pꢀF
(9), mꢀCl (10), or mꢀmethoxy (11) on the phenyl ring reduced
TDO2 cell potency (Table 2). Replacing the phenyl group
with a cyclopentyl (12) resulted in ~4ꢀfold reduction of
potency while a cyclohexyl (13) reduced the potency by 18ꢀ
fold. Since more hydrophilic compounds tend to have better
metabolic stability, we were interested in introducing groups
that are more polar than the phenyl group. Pyridyl analogs (14
and 15) resulted in more than 10ꢀfold reduction of potency.
Between the two thiophenyl regioꢀisomers, cellular potency of
resulted in
a modest ~8ꢀfold reduction of potency.
Replacement with a primary amide (4), secondary amide (5),
hydroxymethyl group (6), or addition of a methyl group to the
isoxazole 3ꢀposition (7) all resulted in significant reduction of
TDO2 inhibitory potency.
3
ꢀthiophenyl 17 was much better than 2ꢀthiophenyl 16. The
unsubstituted pyrazole (18) was ~2.6ꢀfold less potent than the
corresponding phenyl analog 1, while addition of a methyl
group at either the C3ꢀ or C4ꢀ of the pyrazole (19 and 20)
resulted in further erosion of TDO2 potency. However,
introduction of a fluorine substituent at C4 of the pyrazole led
2
ACS Paragon Plus Environment

Page 3 of 6
ACS Medicinal Chemistry Letters
to the most potent TDO2 inhibitor 21 within the
aminoisoxazole series, with a cellular EC50 of 77 nM.
1
9
0.81
1.6
1
2
3
4
5
6
7
8
9
Table 2 TDO2 cellular and biochemical potencies of phenyl ring
substitution or replacements
20
21
0.34
1.6
0.077
0.34
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
Compound R
TDO2
TDO2
IC50
(ꢀM)
EC50
a
b
Cellular potency. Biochemical potency. IC50 and EC50 values
a
b
(
ꢀM)
are the averages of at least 2 independent experiments.
8
0.95
0.83
Because the isoxazole core was found to exhibit poor
stability in whole blood (vide infra), we investigated
replacement of the 5ꢀaminoisoxazole moiety with alternate
heterocycles. Given that the amino group is required in the 5ꢀ
aminoisoxazole for TDO2 potency (Table 1), the amino group
was retained in all the designed heterocyclic targets. However,
all azoles tested, including 4ꢀaminoisoxazole (22), 3ꢀ
aminopyrazoles (23 and 24), 3ꢀaminoꢀ1,2,4ꢀtriazole (25), 4ꢀ
aminoꢀ1,2,3ꢀtriazole (26), 4ꢀaminoisothiazole (27), 5ꢀ
aminoisothiazole (28), 5ꢀaminoꢀ1,2,3ꢀthiadiazole (29) and 4ꢀ
aminoꢀ1,2,3ꢀthiadiazole (30) were inactive against TDO2
(Table 3). We concluded that the 5ꢀaminoisoxazole is a
structurally unique core that is required for TDO2 inhibition.
9
0.21
0.58
1
0
0.25
1.5
0.50
3.1
11
1
1
2
3
0.36
1.5
0.64
7.8
14
15
1.4
2.3
1.7
0.82
1
1
1
6
7
8
0.81
0.099
0.23
0.029
0.028
0.29
3
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Table 3. TDO2 cellular and biochemical potencies of
Page 4 of 6
energy minimum using quantum mechanical calculations
(supplemental material Figure S2). The strong interaction
between the primary amine and the heme propionic acid in the
model of 21 could explain the requirement for a primary or
secondary amine as observed in Table 1. Small, hydrophobic
replacements of the 4ꢀfluoropyrazole in 21 would fit in the
pocket (Table 2) while larger substituents such as the methoxy
group in 11 would extend further than the tryptophan indole to
experience a steric clash, thus explaining the increase in both
IC50 and EC50 of 11. Finally, the model predicts important
interactions between the isoxazole heteroatoms and Arg144
which could explain the general loss of potency when either of
these atoms were replaced with hydrogenꢀbond donors or were
methylated as in 22-24 and 26-27. The predicted interactions
with Arg144 may also explain the measurable biochemical
activities in thiazole 28 or thiadiazole 30. Triazole 25
maintained the two hydrogen bond acceptors of the isoxazole
but the nitrogenꢀlinked ring was predicted to adopt an alternate
lowꢀenergy conformation (Figure S2) compared to 1 or 21.
The complete loss of cell potency of all isoxazole substitutions
cannot be fully explained by the model. However, this model
is consistent with our observation that the aminoisoxazole did
not cause perturbation of the maximal UV absorption
wavelength when incubated with IDO1 or TDO2 (data not
shown), suggesting that it does not bind the heme iron
directly. In addition, TDO2 biochemical IC50 values increased
when a higher tryptophan concentration was used, suggesting
that isoxazoles are competitive with the tryptophan substrate.
For example, biochemical IC50 of compound 8 shifted from
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
isoxazole replacement
R3
3
Compound R
TDO2
TDO2
IC50
(ꢀM)
EC50
a
b
(
ꢀM)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0.085
0.14
2
2
2
2
2
3
4
5
>25
>25
>25
>25
>25
>25
>25
>25
0
.83 µM to 4.21 µM when tryptophan concentration was
increased from 0.2 mM to 1 mM. IC50 of compound 10 shifted
from 0.50 µM to 2.94 µM when tryptophan concentration was
increased from 0.2 mM to 1 mM.
2
2
6
7
>25
>25
>25
>25
28
>25
>25
18
2
3
9
0
>25
>25
0.27
a
b
Cellular potency. Biochemical potency. IC50 and EC50 values
are the averages of at least 2 independent experiments.
To better understand the structureꢀactivity relationships and
develop a binding mode hypothesis, we constructed a model
of 21 bound to human TDO2 (Figure 1) using the Xꢀray
Figure 1. Binding mode hypothesis for compound 21. Xꢀray
structure (PDB: 5TI9) of human TDO2 (purple) is shown with
27
crystal structure, PDB 5TI9. Our model predicts a binding
mode with interactions similar to those of tryptophan. The
primary amine, isoxazole ring nitrogen and fluoropyrazole
moiety in 21 correspond with the ammonium, carboxylate, and
indole ring of tryptophan, respectively. In addition, the
internal strain energy in this pose was found to be at the global
heme (gray), Tryptophan substrate (gray), and O2 (red).
A
putative bound conformation for compound 21 (orange) was then
modeled (see methods in the supplement for details). Distances
are in angstroms.
4
ACS Paragon Plus Environment

Page 5 of 6
ACS Medicinal Chemistry Letters
Selected compounds were also tested in an IDO1 cellꢀbased
assay. As shown in Table 4, the compounds showed modest
TDO2 selectivity over IDO1, ranging from 6ꢀ to 14ꢀfold.
CONCLUSION
1
2
3
4
5
6
7
8
9
In summary, highꢀthroughput screening identified a series
of 5ꢀaminoisoxazole compounds as potent TDO2 inhibitors.
Medicinal chemistry investigations revealed that both the
amino group and the isoxazole moiety were important for
TDO2 inhibitory activity, and a hypothetical binding mode
was developed through molecular modeling, providing insight
into the observed SAR. Optimized compound 21 is a potent
TDO2 inhibitor with modest selectivity over IDO1, no
observed CYP inhibition, and improved human whole blood
stability over the original HTS hit. However, the mechanism
of whole blood instability for this series, and relationship with
structure, remained unclear. As a result, this series was
deprioritized for further optimization in light of alternative
more promising chemical matters.
Table 4. TDO2 selectivity over IDO1
a
Compound TDO2 selectivity
(fold)
1
3
13x
12x
14x
6x
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
7
1
ASSOCIATED CONTENT
Supporting Information
2
a
The Supporting Information is available free of charge on the
ACS Publications website and includes experimental procedures,
cell versus biochemical potencies and quantum mechanical
torsional energy analysis
Calculated as IDO1 EC /TDO2 EC .
5
0
50
As TDO2 is a hemeꢀcontaining enzyme, we tested the
compounds in cytochrome P450 (CYP) inhibition assays and
found that the 4ꢀaminoisoxazole analogs did not inhibit
common drugꢀmetabolizing CYP enzymes. For instance, the
most potent compounds 1, 17, 18 and 21 all had IC ’s greater
5
0
AUTHOR INFORMATION
Corresponding Author
than 10 uM for all the CYP isoforms that were tested
CYP3A4, CYP1A2, CYP2D6, CYP2C9 and CYP2C19).
(
*Tel. : 1(650)467ꢀ3995 Fax : 1(650)467ꢀ5155 Eꢀmail:
We also investigated the stability of select compounds in
whole blood to predict the in vivo stability (Table 5). The
original HTS hit 1, the Nꢀmethylated analog 3 and thiophene
7 demonstrated poor stability in rat, dog and human whole
blood. The 3ꢀmethylisoxazole analog 7 showed improved
sellers.benjamin@gene.com
Author Contributions
1
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
whole blood stability but with significantly reduced TDO2
potency.
However, pyrazole analog 21 demonstrated
improved human whole blood stability while maintaining
TDO2 potency. The cause of the observed whole blood
Notes
2
8
The authors declare no competing financial interest.
instability is not well understood at this time.
ACKNOWLEDGMENT
We thank the Genentech analytical chemistry group.
a
Table 5. Whole blood stability
ABBREVIATIONS
Compound %
%
%
IDO, indoleamine 2,3ꢀdioxegenase TDO, tryptophan 2,3ꢀ
dioxegenase; NFK, Nꢀformyl kynurenine, Kyn, kynurenine,
AhR, aryl hydrocarbon receptor; Treg, regulatory T cell; Teff,
T cell; P450, cytochrome P450
remaining remaining remaining
rat
0
dog
0
human
0
1
3
0.1
3
0.1
3
0.3
NT
84
b
1
7
7
50
NT
65
NT
2
1
91
a
Compounds (2 uL, 0.5 mM) were incubated in 100 µL of
fresh blood for 2 hours unless otherwise stated. Parent
remaining is analyzed by LCꢀMS/MS and reported as a
b
percentage. NT: not tested. Incubated for 30 minutes.
5
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 6
REFERENCES & NOTES
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
5 Gangadhar TC, Hamid O, Smith DC, Bauer TM, Wasser JS,
Olszanski AJ, Luke JJ, Balmanoukian AS, Kaufman DR, Zhao Y,
Maleski J, Jones MJ, Leopold L, Gajewski TF. Epacadostat plus
pembrolizumab in patients with advanced melanoma and select solid
tumors: Updated phase 1 results from ECHOꢀ202/KEYNOTEꢀ
37, Annals of Oncology, Volume 27, Issue suppl_6, 1 October
016,1110PD, https://doi.org/10.1093/annonc/mdw379.06.
16 For reviews, see a) Rohrig U F, Majjigapu SR, Vogel P, Zoete
V, Michielin O. Challenges in the Discovery of Indoleamine 2,3ꢀ
Dioxygenase 1 (IDO1) Inhibitors. J. Med. Chem. 2015, 58: 9421ꢀ
1
Chen L.; Flies, D. B. Molecular mechanisms of T cell coꢀ
stimulation and coꢀinhibition. Nat Rev Immunol. 2013, 13: 227ꢀ242.
van Baren N, Van den Eynde BJ. Tumoral Immune Resistance
2
Mediated by Enzymes That Degrade Tryptophan. Cancer Immunol
Res, 2015, 3: 978ꢀ985.
0
2
3
Lindstrom N, Aittoniemi J, Jylhava J, Eklund C, Hurme M,
Paavonen T, Oja S S, ItalaꢀRemes M, Sinisalo M. Indoleamine
,3ꢀDioxygenase Activity and Expression in Patients With Chronic
Lymphocytic Leukemia. Clinical Lymphoma Myeloma & Leukemia,
012, 12: 363ꢀ365.
Jia Y, Wang H, Wang Y, Wang T, Wang M, Ma M, Duan Y,
2
9
437; b) Qian, S.; Zhang, M.; He, Y.; Wang, W.; Wang, Z. IDO as a
drug target for cancer immunotherapy: recent developments in IDO
inhibitors discovery. RSC Advances. 2016, 6: 7575ꢀ81.
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
Meng X, Liu L. Low expression of Bin1, along with high expression
of IDO in tumor tissue and draining lymph nodes, are predictors of
poor prognosis for esophageal squamous cell cancer patients.
International J Cancer, 2015, 137: 1095ꢀ1106.
1
7 Yue EW, Douty B, Wayland B, Bower M, Liu X, Leffet L,
Wang Q,. Bowman KJ, Hansbury MJ, Liu C, Wei M, Li Y, Wynn R,
Burn TC, Koblish HK, Fridman JS, Metcalf B, Scherle PA, Combs
AP. J. Med. Chem. 2009, 52: 7364ꢀ7367.
18 Rohrig UF, Majjigapu SR, Grosdidier A, Bron S, Stroobant V,
Pilotte L, Colau D, Vogel P, Van den Eynde BJ, Zoete V, Michielin
O. Rational Design of 4ꢀArylꢀ1,2,3ꢀTriazoles for Indoleamine 2,3ꢀ
Dioxygenase 1 Inhibition. J. Med. Chem. 2012, 55: 5270ꢀ90.
5
Munn DH, Sharma MD, Baban B, Harding HP, Zhang Y, Ron
D, Mellor AL. GCN2 kinase in T cells mediates proliferative arrest
and anergy induction in response to indoleamine 2,3ꢀdioxygenase.
Immunity. 2005, 22: 633ꢀ642.
6 Opitz CA, Litzenburger UM, Sahm F, Ott M, Tritschler I, Trump
S, Schumacher T, Jestaedt L, Schrenk D, Weller M, Jugold M,
Guillemin GL, Miller CL, Lutz C, Radlwimmer B, Lehmann I, von
Deimling A, Wick W. Platten M. An endogenous tumourꢀpromoting
ligand of the human aryl hydrocarbon receptor. Nature. 2011, 478:
197ꢀ203.
1
9 Dolusic E, Larrieu P, Blanc S, Sapunaric F, Norberg B,
Moineaux L, Colette D, Stroobant V, Pilotte L, Colau D, Ferain T,
Fraser G, Galeni M, Frere JꢀM, Masereel B, Van den Eynde B,
Wouters J, Frederick R. Bioorg. Med. Chem. Lett, 2011, 19: 1550ꢀ
1
561.
0 Tojo S, Kohno T, Tanaka T, Kamioka S, Ota Y, Ishii T,
2
7
Koblish HK, Hansbury MJ, Bowman KJ, Yang G, Neilan CL,
Kamimoto K, Asano S, Isobe Y. Crystal Structures and
Structure−Activity Relationships of Imidazothiazole Derivatives as
IDO1 Inhibitors. ACS Med. Chem. Lett. 2014, 5: 1119ꢀ1123.
Haley PJ, Burn TC, Waeltz P, Sparks RB, Yue EW, Combs AP,
Scherle PA, Vaddi K, Fridman, JS. Hydroxyamidine inhibitors of
indoleamineꢀ2,3ꢀdioxygenase potently suppress systemic tryptophan
catabolism and the growth of IDOꢀexpressing tumors. Mol Cancer
Ther. 2010, 9: 489ꢀ498.
2
1 Pilotte L, Larrieu P, Stroobant V, Colau D, Dolusic E, Frederick
R, De Plaen E, Uyttenhove C, Wouters J, Masereel B, Van den Eynde
BJ. PNAS. 2012, 109, 2497.
22 Structurally similar aminoisoxazoles were disclosed in a patent
application: Cowley P, Wise A. Pharmaceutical Compounds. WO
8
Liu X, Shin N, Koblish HK, Yang G, Wang Q, Wang K, Leffet
L, Hansbury MJ, Thomas B, Rupar M, Waeltz P, Bowman KJ, Polam
P, Sparks RB, Yue EW,Li Y, Wynn R, Fridman JS,Burn TC, Combs
AP, Newton RC, Scherle PA. Selective inhibition of IDO1 effectively
regulates mediators of antitumor immunity. Blood. 2010, 115: 3520ꢀ
2
016026772 A1.
3 Cowley P, Wise A. Pharmaceutical Compounds. WO
015082499 A2.
4 Cowley P, Wise A. Pharmaceutical Compounds. WO
2016071293 A2.
5 Li Q, Gao D. Fusaedꢀring compounds, Pharmacentical
composition and uses thereof. WO 2016131381.
6 Cowley P, Wise A, Brown TJ, McGowan MA, Zhou H, Han Y.
2
2
3
530.
Yan Y, Zhang GꢀX, Gran B, Fallarino F, Yu S, Li H, Cullimore
ML, Rostami A, Xu H. IDO upregulates regulatory T cells via
tryptophan catabolite and suppresses encephalitogenic cell
2
9
2
T
responses in experimental autoimmune encephalomyelitis. J Immunol
. 2010, 185: 5953ꢀ5961.
2
Pharmaceutical Compounds. WO 2017007700 A1.
1
0 Novikov O, Wang Z, Stanford EA, Parks AJ, RamirezꢀCardenas
27 LewisꢀBallester A, Forouhar F, Kim SꢀM, Lew S, Wang Y,
Karkashon S, Seetharaman J, Batabyal D, Chiang BꢀY, Hussain M,
Correia MA, Yeh SꢀR, Tong L. Molecular Basis for Catalysis and
SubstrateꢀMediated Cellular Stabilization of Human Tryptophan 2,3ꢀ
Dioxygenase. Sci. Rep. 2016, 6: 35169ꢀ81.
A, Landesman E, Laklouk I, SaritaꢀReyes C, Gusenleitner D, Li A,
Monti S, Manteiga S, Lee K, Sherr DH. An Aryl Hydrocarbon
ReceptorꢀMediated Amplification Loop That Enforces Cell Migration
in ERꢀ/PRꢀ/Her2ꢀ Human Breast Cancer Cells. Mol Pharmacol. 2016,
9
0: 674ꢀ688.
1 Bessede A, Gargaro M, Pallotta MT, Matino D, Servillo G,
2
8 For an example of isoxazole instability, see Kalgutkar AS,
1
Nguyen HT, Vaz ADN, Doan A, Dalvie DK, McLeod DG, Murray
JC. In vitro metabolism studies on the isoxazole ring scission in the
antiꢀinflammatory agent leflunomide to its active –cyanoenol
metabolite A771726: mechanistic similarities with the cytochrome
p450ꢀcatalyzed dehydration of aldoximes. Drug Metab. Disp. 2003,
31: 1240ꢀ50. We could not verify that the mechanism proposed in the
paper was operative in our molecules.
Brunacci C, Bicciato S, Mazza EM, Macchiarulo A, Vacca C, Iannitti
R, Tissi L, Volpi C, Belladonna ML, Orabona C, Bianchi R, Lanz
TV, Platten M, Della Fazia MA, Piobbico D, Zelante T, Funakoshi H,
Nakamura T, Gilot D, Denison MS, Guillemin GJ, DuHadaway JB,
Prendergast GC, Metz R, Geffard M, Boon L, Pirro M, Iorio A,
Veyret B, Romani L, Grohmann U, Fallarino F, Puccetti P.. Aryl
hydrocarbon receptor control of a disease tolerance defence pathway.
Nature. 2014, 511: 184ꢀ190.
(TOC Image)
12 Pilotte L, Larrieu P, Stroobant V, Colau D, Dolusic E, Frédérick
R, De Plaen E, Uyttenhove C, Wouters J, Masereel B, Van den Eynde
BJ. PNAS, 2012, 109: 2497ꢀ502.
1
3 Holmgaard RB, Zamarin D, Munn DH, Wolchok JD, Allison
JP. Indoleamine 2,3ꢀdioxygenase is a critical resistance mechanism in
antitumor T cell immunotherapy targeting CTLAꢀ4. J Exp Med. 2013,
2
10: 1389ꢀ1402.
4 Spranger S, Koblish HK, Horton B, Scherle PA, Newton
1
R, Gajewski TF. Mechanism of tumor rejection with doublets of
CTLAꢀ4, PDꢀ1/PDꢀL1, or IDO blockade involves restored ILꢀ2
production and proliferation of CD8(+) T cells directly within the
tumor microenvironment. J Immunother Cancer. 2014, 2: 3ꢀ16.
6
ACS Paragon Plus Environment