Rational Design of Original Fused-Cycle Selective Inhibitors of Tryptophan 2,3-Dioxygenase

Article abstract of DOI:10.1021/acs.jmedchem.1c00323

Tryptophan 2,3-dioxygenase (TDO2) is a heme-containing enzyme constitutively expressed at high concentrations in the liver and responsible for l-tryptophan (l-Trp) homeostasis. Expression of TDO2 in cancer cells results in the inhibition of immune-mediated tumor rejection due to an enhancement of l-Trp catabolism via the kynurenine pathway. In the study herein, we disclose a new 6-(1H-indol-3-yl)-benzotriazole scaffold of TDO2 inhibitors developed through rational design, starting from existing inhibitors. Rigidification of the initial scaffold led to the synthesis of stable compounds displaying a nanomolar cellular potency and a better understanding of the structural modulations that can be accommodated inside the active site of hTDO2.

Full text of DOI:10.1021/acs.jmedchem.1c00323

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Article
Rational Design of Original Fused-Cycle Selective Inhibitors of
Tryptophan 2,3-Dioxygenase
Arina Kozlova, Léopold Thabault, Maxime Liberelle, Simon Klaessens, Julien R. C. Prévost,
Caroline Mathieu, Luc Pilotte, Vincent Stroobant, Benoît Van den Eynde, and Raphaël Frédérick*
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ABSTRACT: Tryptophan 2,3-dioxygenase (TDO2) is a heme-containing enzyme constitutively expressed at high concentrations in
the liver and responsible for L-tryptophan (L-Trp) homeostasis. Expression of TDO2 in cancer cells results in the inhibition of
immune-mediated tumor rejection due to an enhancement of L-Trp catabolism via the kynurenine pathway. In the study herein, we
disclose a new 6-(1H-indol-3-yl)-benzotriazole scaffold of TDO2 inhibitors developed through rational design, starting from existing
inhibitors. Rigidification of the initial scaffold led to the synthesis of stable compounds displaying a nanomolar cellular potency and a
better understanding of the structural modulations that can be accommodated inside the active site of hTDO2.
activation of the general control nonderepressible 2 (GCN2)
“stress” sensing kinase, further hindering CD8+ T-cell
proliferation.10 Finally, immunosuppression from L-Trp depri-
vation might also be linked to the inhibition of the mammalian
target of the rapamycin (mTOR) pathway.11
On the other hand, along with its catabolites, KYN favors
regulatory T cells (Tregs) through several mechanisms. One of
the currently proposed mechanisms resides in the interaction
between KYN and the aryl hydrocarbon receptor (AHR),
leading to the polarization of CD4+ T-cells into the Treg
phenotype.12−14
Overall, excessive KP activation promotes carcinogenesis and
tumor cell growth by reducing T-cell proliferation, suppressing
natural killer cells (NK), promoting Tregs differentiation, and
activating, recruiting, and expanding myeloid-derived suppres-
sor cells (MDSCs).15,16
This implication of the KP in tumor immune escape has led to
considerable efforts from the medicinal chemist community to
develop SMIs to interfere with this pathway. Most notably, the
development of SMIs for IDO1 has received tremendous
INTRODUCTION
■
Multiple immune resistance mechanisms are often involved
within a tumor microenvironment, hampering the immune
response and leading to the failure of currently available
treatments.1,2 As we progress toward an individualized treat-
ment approach, there is a need for new tools to overcome these
hurdles. Using small-molecule inhibitors (SMIs) to target
tolerogenic pathways is an attractive strategy for cancer
immunotherapy. The advantages of SMIs reside in their ability
to reach a wide array of intracellular targets, the possibility to
fine-tune their absorption, distribution, metabolism, and
excretion (ADME) properties, and their superior cost-
effectiveness compared to biologics.3,4 Within this context,
small-molecular scaffolds are a rapidly expanding area of
research, as exemplified by the increasing number of patent
applications and compounds currently under evaluation at
preclinical and clinical stages both as stand-alone or combina-
tion therapies.3−5
Enzymes of the tryptophan-kynurenine pathway (KP), such
as indoleamine-2,3-dioxygenase 1 (IDO1), tryptophan 2,3-
dioxygenase (TDO2), or kynurenine 3-monooxygenase
(KMO), are attractive drug targets for cancer immunother-
apy.6,7 Catabolism of L-Trp promotes an immunosuppressive
environment both through the depletion of this essential amino
acid and the accumulation of kynurenine (KYN) metabolites.8
First, a decrease in L-Trp levels results in the blockage of CD8+
and CD4+ T-cells, in the G1 cell cycle phase, due to their
susceptibility to amino acid shortage.9 Second, it leads to the
Received: February 21, 2021
Published: August 2, 2021
https://doi.org/10.1021/acs.jmedchem.1c00323
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Table 1. 680C91, LM10, and their Fused-Cycle Analogues (1−6) with their Inhibition Data
a
95% confidence intervals. Reported IC50 values are calculated from measurements performed in triplicates, in at least two separate experiments.
b
c
d
Toxicity was determined using an MTS assay on P815B cells. log D7.4 values were calculated using Chemicalize. Previously obtained data from
Pilotte et al.24 NI: no inhibition at 100 μM.
e
attention since the discovery of its implications in cancer
immune escape.17,18 Although the outcomes of IDO1 inhibitors
in clinical trials did not meet expectations for several possible
reasons discussed in a recently published review, the KP remains
a reservoir of attractive targets and an active research area.19,20
Much like IDO1, TDO2 independently catalyzes the first and
rate-limiting step of the KP: the oxidative cleavage of the L-Trp
pyrrole moiety to form N-formylkynurenine. TDO2 plays a
central role in the regulation of the systemic L-Trp
concentration.21 Despite sharing the same enzymatic reaction,
TDO2 only shares around 10% sequence identity with IDO1
and is much more specific for L-Trp.22,23 Numerous human
tumors, including glioblastoma, melanoma, and liver, bladder,
pancreatic, and colon carcinomas, express TDO2 at various
levels to exploit the KP as an immune escape mechanism.24,25
TDO2 inhibitors would be particularly relevant in hepatocarci-
noma, 100% of which express this enzyme.24,26
resulted in inactive or poorly active derivatives. Thus, it
appeared that TDO2 was particularly susceptible to small
modifications of the inhibitor. Recent crystallographic studies
revealed that the active site of hTDO2 was, in fact, more rigid
than that of hIDO1, which has a broader substrate specificity.32
Hence, our previous research eventually led to the
identification of LM10 (Figure 1), characterized by a trans-
This interest in TDO2 inhibition led to the development of
several families of TDO2 inhibitors, as exemplified by some
patents and few articles published to date.27−30 We previously
established initial structure−activity relationships (SARs) for a
series of TDO2-selective and competitive inhibitors charac-
terized by a 3-(2-(pyridyl)ethenyl)indole scaffold that demon-
strated several prerequisites for TDO2 inhibition and potential
for optimization.31 A thorough exploration of SARs around the
initial 680C91 inhibitor (Table 1) drew the first prerequisite for
TDO2 inhibition by catalytic-site inhibitors. To summarize, all
investigated replacements of the indole moiety led to inactive
compounds, even when changes were minor (5-azaindole or
indazole). Moreover, the indole nitrogen should remain free,
and only substitution in C5 or C6 with small lipophilic
substituents such as fluorine led to increased potency. As
suggested by molecular docking, the 3-pyridyl group of 680C91
is found in the vicinity of Arg-144 guanidinium. Accordingly, the
replacement of this 3-pyridyl with H-bond acceptors or
negatively charged groups provided more soluble compounds
that could be further stabilized in the TDO2 binding cleft
through ionic interactions.31 Replacement of the trans-vinyl
linker with a longer, shorter, or more flexible ethyl spacer
Figure 1. Molecular modeling of LM10 inside the hTDO2 active site
(PDB code 5TI9)39 and optimization strategy considered in this work.
vinyl tetrazole side chain in the 3-position of a 6-fluoroindole
scaffold and a low micromolar (μM) potency. The admin-
istration of LM10 restored the immune response and promoted
tumor rejection in a mouse model.24 LM10 was, to our
knowledge, the very first soluble and bioavailable TDO2
inhibitor demonstrating preclinical antitumor efficacy and thus
the proof-of-concept of TDO2 inhibition for anticancer
immunotherapy.24 Lately, inhibition of TDO2 was also
demonstrated to be beneficial for the efficacy of immune
checkpoint inhibitors such as anti-PD1 and anti-CTLA4
monoclonal antibodies.25 Interestingly, the benefit of a TDO2
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Figure 2. Molecular modeling of 6 inside the hTDO2 active site (PDB code 5TI9)39 and envisioned modulations around this scaffold.
blockade persisted even in the absence of its tumoral expression,
probably due to the increased availability of circulating L-Trp
levels.25
previously described assay using the hTDO2-expressing
P815B murine mastocytoma cell line.24
Compounds 1 to 6 all exhibited low-μM range enzymatic
IC50s and mid-nanomolar (nM) to low-μM activities in cells.
Interestingly, these compounds also presented very low cellular
toxicity, as assessed by our MTS assay (Table 1).
Our previous work provided useful data for TDO2 inhibitor
development, exemplified by numerous publications and patents
reporting TDO2 inhibitors based on the 6-F-indole pharmaco-
phore.27 However, the LM10 series suffered from several
drawbacks, which prevented its further therapeutic application.
First, the TDO2 inhibitory potency in this series could not be
much improved and could not reach a cellular efficacy below the
μM threshold. Furthermore, we could not find a replacement for
the electrophilic vinyl linker prone to chemical instability
(trans−cis isomerization), side reactions (Michael acceptor),
and metabolic liabilities. In the present work, to further expand
SARs data for TDO2 and provide a better molecular framework
for TDO2 inhibitors, we designed, synthesized, and evaluated a
series of original, highly stable, and selective TDO2 inhibitors.
To rank and compare these initial hits, we normalized their
respective affinities using ligand efficiency [LE: 1.4(−log IC50)/
(heavy atom count)] and lipophilic ligand efficiency (LLE:
pIC50 − c log D7.4) metrics.36,37 These values allow us to
consider both activity and lipophilicity,2 an equally important
attribute in drug design affecting ligand association and ADME
properties.38
Isoquinoline analogues 1 and 2 were the most active
compounds in cells, whereas quinazolines (3, 4) showed a loss
in their potency and LE/LLE values. Despite being less potent in
cells than 1 and 2, hit compound 6, bearing a triazole moiety,
had the lowest IC50(enz) on hTDO2 and overall more favorable
metrics due to its higher hydrophilicity and heteroatom count.
In addition to the absence of cell toxicity even at high
concentrations (>100 μM), incubation of cells with 6 did not
affect cell cycle distribution, which was determined by flow
cytometry with propidium iodide DNA staining (PI) (Figure
S1).
RESULTS AND DISCUSSION
■
Design of TDO2 Inhibitors. We first reasoned that the
rigidification of the lateral trans-vinyl in the 680C91 and LM10
scaffolds could improve the TDO2 inhibitory potency by
conformational restriction and provide a molecular template
devoid of chemical liabilities (Figure 1). Moreover, several
fused-cycle scaffolds were previously reported in a patent
regarding TDO2 inhibitors, suggesting that such rigidification
could lead to potent TDO2 inhibitors.33 To investigate the
impact of this replacement, we synthesized several fused-cycle
analogues of 680C91 and LM10 (1−6, Table 1). As indicated
by our previous results, fluorine substitution in the 6-position of
indole usually leads to a slight increase in the TDO2 inhibitory
potency.31 However, we first decided to avoid 6-fluorine
derivatives as their synthesis results, in our experience, in
lower yields compared to their nonfluorinated counterparts.
Evaluation of these compounds in enzyme and cell-based assays
on IDO1 and TDO2 demonstrated their ability to selectively
inhibit TDO2. Interestingly, inhibition data on isolated enzyme
assay are often lacking for reported TDO2 inhibitors. Indeed,
several reported series of TDO2 inhibitors were characterized
only by cell-based inhibition assays, which limits the SAR
understanding and hence the future development of inhib-
itors.27,28 We used the most commonly described assay for
IDO1/TDO2 activity which follows the formation of the
enzymatic product N′-formylkynurenine after hydrolysis with
trichloroacetic acid and reaction with p-dimethylaminobenzal-
dehyde.34,35 Regarding cell-based evaluation, we used a
Evaluation of hTDO2 inhibition by 6 demonstrated an
IC50(CI95%) of 2.7 μM in the enzymatic assay (IC50(enz)) and
0.8 μM in cell-based settings (IC50(cell)), while LM10 had an
IC50(enz) of 7.3 μM and an IC50(cell) of 2.8 μM. Consequently,
we decided to use 6 as a starting point for designing various N-
substituted original indole 3-benzotriazoles (Figure 2). How-
ever, compound 6 displayed worsened parameters (LLE = 2.56,
c log D7.4 = 3.01) compared to LM10 (LLE = 4.99, c log D7.4
=
0.15), due to the loss of the tetrazole function, ionized at a
physiological pH of 7.4. Thus, our optimization included
benzotriazole substitutions of various hydrophobicity ranges
aimed at probing TDO2 inhibition while considering their
potency and physicochemical characteristics. Then, we further
modulated the benzotriazole ring by (i) replacement with a
triazolo-pyridine scaffold and (ii) substitution on the 6-
membered ring of benzotriazole.
SARs of Benzotriazole Analogues. We next synthesized a
chemical library of benzotriazole analogues and evaluated the
synthesized compounds for their ability to inhibit TDO2 both in
vitro, using a purified truncated form of TDO2, and in cellulo
using TDO2-expressing P815B cells.23 To remain under the
initial velocity conditions, the assay was optimized so that the
cells consume less than 25% of the available L-Trp. The same
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Table 2. Inhibitory Affinities (IC50) of Compounds Against hTDO2 in the Spectrophotometric Assay and P815B Cells
a
Reported IC50 values are calculated from measurements performed in triplicates, in at least two separate experiments. 95% confidence intervals of
b
c
d
the values can be found in the Supporting Information. log D7.4 values were calculated using Chemicalize. Residual activity of ∼30%. NI: no
inhibition. Unless indicated otherwise, Y and R2 = H, X = CH.
evaluation was performed on IDO1 to assess the selectivity
toward TDO2 and demonstrated that none of these compounds
could inhibit IDO1 (data not shown).
The active site of TDO2 is highly hydrophobic and relatively
small, suggesting that an increase in potency could be achieved
by adding lipophilic substituents to the initial scaffold of 6
[IC50(enz): 2.7 μM/IC50(cell): 0.8 μM]. To that end, we
examined if the N-alkylation of the benzotriazole moiety would
result in more potent compounds. Interestingly, The N-methyl
analogues (7, 8, and 9) displayed a significant gain in IC50(cell)
(up to 5-fold for 7) compared to their parent compound 6, while
maintaining a similar IC50(enz) (Table 2). These results can be
explained by the increase in the log D7.4 value resulting from N-
methylation. For SAR purposes, we next took an interest in
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Figure 3. Epitope mapping for regioisomers 32 [IC50 52.6 μM (enz), 1.6 μM (cell)] and 31 [IC50 118.6 μM (enz), 3.7 μM (cell)]. One-dimensional
(1D) NMR 1H proton spectra (blue) overlaid with STD spectra (orange). Corresponding compounds are represented above each set of spectra. Blue
circles represent the protons at the designated position(s), with percentages representing STD intensity of the respective proton(s). Two of the
ethylene protons could not be seen as their shift coincide with the water signal.
further modifying the N-methyl analogues (7, 8, and 9). We thus
synthesized and evaluated compounds 10 to 16, modified on the
6-membered ring of benzotriazole. Our docking studies
suggested that molecules 10, 11, and 12 could form a H-bond
with heme propionate through their aniline. The most potent
molecule remained the N2 analogue 10. The affinity of these
compounds was not significantly different from their precursors,
but their lower c log D7.4 value resulted in a better LLE (Table 2).
Of note, the replacement of benzotriazole by 4-aza-benzo-
triazole in compounds 13, 14, and 15 did not result in better
activities.
Next, we investigated the impact of bulkier N-chain
substituents in N1 and N3 positions of benzotriazole
(compounds 17 to 40). As the N2-substitution was poorly
available for alkylation, which only occurred through direct
methylation of benzotriazole, we investigated only N1 and N3
substitutions. Lipophilic substituents such as trifluoroethyl (17
and 18) and ethyl-cyclopropyl (19 and 20) yielded potent
compounds, as expected from their higher c log D7.4. We quickly
noticed that N1-substitution was generally more favorable than
N3 for the activity. Interestingly, this trend was not apparent for
the smaller methyl group as compounds 7, 8, and 9 exhibit the
same activity. For example, N1-substitution with a trifluoroethyl
([8, IC50(enz): 1.9 μM/IC50(cell)l: 0.19 μM] resulted in a 3- to
7-fold increase in activity compared to N3-substitution of the
same moiety [17, IC50(enz): 15.0 μM/IC50(cell): 0.44 μM].
However, N-substitution by the less lipophilic acetamide moiety
(21, 22) resulted in less potent TDO2 inhibitors with similar
affinity, although the N1-substituted compound 22 appeared 2-
fold more potent in cells than its isomer. Surprisingly, their
hydrolyzed counterparts 23 and 24 displayed a loss of activity in
both cellular and biochemical assays, suggesting that a low
membrane permeability was not the only reason that could
account for their poor activity. Compounds 25 and 26,
displaying a 2-(dimethylamino)ethyl moiety, shared a similar
potency, perhaps due to the more flexible nature of their N-
substituent. A further increase in the N-substituent size with
physiologically nonionized moieties (27 to 32) led to a
systematic loss of activity and an increasing gap between cellular
and enzymatic potencies. Interestingly, the introduction of
longer N-substituents with H-bond donors such as piperidine
(33 and 34) and pyrrolidine (35 and 36) led to a better activity
for N3-substituted compounds. Of note, these compounds had
the best LLE values of all the studied compounds due to their
predominantly protonated state at physiological pH, resulting in
low c log D7.4 values.
We also investigated a substitution of the indole ring H6 to
fluorine, with the synthesis of compounds 8a and 37a. As
expected from previous results,40 substitution with fluorine in
this position of the indole was beneficial for the activity, and
compound 37a demonstrated a fivefold increase in activity in
both cellular and enzymatic assays [IC50(enz): 5.2 μM/
IC50(cell): 0.23 μM] compared to 37 [IC50(enz): 24.9 μM/
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IC50(cell): 0.74 μM]. The same gain in activity occurred for 8a
in cells [IC50(enz): 1.1 μM, IC50(cell): 0.02 μM] compared to 8.
STD-NMR Study of the Interaction between the Active
Site and the Inhibitor. To better understand the observed
SARs and notably how the nature and positioning of the lateral
N-sidechain influenced the binding of the inhibitor inside the
active pocket, we performed epitope mappings on several
couples of regioisomers using saturation transfer difference
NMR (STD-NMR). In STD-NMR, selective saturation of
hTDO2 in solution is transferred to the bound ligand through
intermolecular nuclear Overhauser effects (NOEs) between
protons of the inhibitor and the active site’s residues.
Consequently, the ligand’s protons interacting more closely
display intense STD signals, while a decrease in STD intensity
correlates with lower interaction.41 Due to solubility issues, we
focused our investigation on compounds bearing solubilizing
moieties to attain a high compound concentration (up to 200
μM) in aqueous buffer, suitable for the present evaluation. We
mapped the epitopes of regioisomers 25−26, 35−36, and 31−
32 (Figure 3). Epitope mapping of all six compounds suggested
that N3-substitutions led to an improper position of the indole
ring compared to N1-substitutions (data regarding compounds
25−26 and 35−36 can be found in Supporting Information,
Figure S2). Indeed, as pictured in Figure 3, epitope mapping on
the different compounds showed that the indole ring displays
the highest saturation transfer for protons H5, H6, and H7
(around 100%). Concurrently, cocrystallographic structures of
hTDO2 with L-Trp show that this part of the indole tightly
interacts with a cluster of several hydrophobic residues involving
L120, F5, Y24, Y27, and L28.39 In the case of L-Trp, the indole
H2 position is located very close to the heme.39 Similarly, 26, 36,
and 32 all exhibited a saturation transfer of around 90% for H2.
However, we observed that the less active N3-substituted
analogues 25, 35, and 31 systematically displayed a 20 to 30%
decrease in saturation transfer for H2 compared to their
regioisomers substituted in N1. Overall, these results suggest a
shift in the position of the indole core for N3-substituted
compounds, resulting in misplacement of H2. This apparent shift
of the indole position could, at least in part, explain the
difference in activity between N1- and N3-substituted com-
pounds.
Regioisomers displayed a similar binding pose, as shown in
Figure 4, for analogues 17 [IC50(enz): 15.0 μM/IC50(cell): 0.44
Figure 4. Molecular modeling of regioisomers 17 (pink) and 18
(green), bearing a trifluoroethyl from the front (a) and the side (b).
(PDB code: 5TI9)39
μM] and 18 [IC50(enz): 1.9 μM, IC50(cell): 0.19 μM] with a
particularly noticeable affinity difference. However, docking of
N1-substituted compounds resulted in a more favorable
geometry for the indole core placement. Indeed, N3-substituted
compounds display a shift of the indole core from the position
naturally adopted by the indole of L-Trp, with a shift of C2,
disturbing the interaction with the aromatic hotspots. Regarding
compounds bearing a longer sidechain with a H-bond donor
(compounds 33 to 40), our docking results suggested a possible
interaction with the propionate of the heme or with a H-
acceptor sidechain (such as Ser345) from the mobile loop. This
interaction could potentially explain the slightly better activity
observed for N3 analogues.
Phase I Metabolic Stability and Electrophilic Reac-
tivity. The potential reactivity of LM10 as a Michael acceptor
was assessed with a thiol-trapping assay using cysteamine as the
S-nucleophile and compared to that of the unsubstituted
benzotriazole 6.42−44 Following incubation with cysteamine,
the 1H NMR spectrum of LM10 quickly changed to reveal the
apparition of several new aromatic signals. In contrast, the
compound 6 spectrum stayed the same (Figure 5). Accordingly,
LC/MS analysis of LM10 ([M + H]+ = 230.1) confirmed the
apparition of a secondary product ([M + H]+ = 305.1) upon
cysteamine addition while 6 was not impacted.
Then, we evaluated the microsomal half-life of LM10 and
several representative benzotriazoles (Table 3). The recovery
percentage of LM10 after 24 h remained close to the initial
concentration. Regarding the present series, we noticed that the
nature of the N-substituent was determinant for the recovery
rate of the compounds. The presence of fluorine on the indole
did not impact the microsomal stability in compounds 8a and
37a compared to their nonfluorinated analogues. Furthermore,
in isomers 37 and 38, the localization of the N-substituent did
not impact the microsomal stability.
Interestingly, the sidechain of the different isomers always
showed a similar and low saturation transfer compared to the
aromatic protons. Therefore, the nature of the sidechains does
not appear to be useful for the interaction with hTDO2, which
opens the door to further modulation to fine-tune this family of
inhibitor physicochemical properties.
Molecular Docking. Our molecular docking studies
suggested two main hindrances for the accommodation of
different ligands in the active site of hTDO2. The first identified
hindrance was Arg144, as a shift in its sidechain was required for
the binding of the inhibitors. Displacement of this sidechain
increased with the size of the N-substituent and was more
important for N3 regioisomers. We noticed that wider Arg144
shifts correlated with lower enzymatic activity. The second
hindrance was the flexible 338−346 loop, suggesting that an
induced-fit-type binding was perhaps needed for larger
compounds. It was previously described that disordering of
this loop allows NFK to diffuse out and enables debinding of a
new L-Trp molecule.39 Its mobility was particularly noticeable
when hTDO2 was cocrystallized with an NLG919-type
analogue, widely opening the active site.28
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Figure 5. Aromatic regions of 1H NMR in DMSO-d6 spectra of 1 mM of LM10 (a) and 6 (c) before and after (b,d) addition of 10 equivalents of
cysteamine. Spectra were recorded 30 min after the addition of cysteamine. Spectrum (b) (LM10 + cysteamine) shows several new aromatic signals,
while proton integrations of compound 6 remain unchanged but displayed a slight shift in the presence of cysteamine.
Overall, these data demonstrate that the structural rigid-
ification results in a scaffold that does not exhibit Michael
acceptor behavior. Nonetheless, the microsomal stability studies
contrast our initial idea that this template rigidification would
necessarily lead to more metabolically stable compounds.
Indeed, the present series display considerable heterogeneity
in metabolic stability, which appears heavily dependent on the
nature of the N-substituent.
Chemistry. The target compounds were synthesized by
Suzuki−Miyaura cross-coupling between halogenated benzo-
triazole analogues and the appropriate indole boronic ester.
Compounds 1−5 were obtained following the reaction
between brominated isoquinolines or quinazolines and 41.
The synthesis of 680C91-type analogues was performed using
the same strategy, starting from 3 in the cross-coupling reactions
(Scheme 1).
Several routes were attempted to synthesize compound 6,
bearing an unsubstituted benzotriazole, leading to the synthetic
pathway depicted in Scheme 2. We used p-methoxybenzyl
(PMB) as a protecting group for 5-bromo-benzotriazole, which
led to the formation of two regioisomers. Then, we performed
the cross-coupling using the MIDA boronate 43 following a
procedure adapted from Knapp et al.45 The MIDA boronate
allows a continuous release of the boronic acid over several
hours. This unique capacity enabled us to obtain the cross-
coupled product with an almost quantitative yield. The cross-
coupled product was first deprotected from its phenylsulfonyl
(SO2Ph) using KOH, after which the PMB was removed in pure
trifluoroacetic acid (TFA) at reflux temperature for 16 h to
afford 6. We noted that no degradation of the scaffold occurred
despite the harsh basic and acidic conditions of both
deprotection.
Two different routes, depicted in Scheme 3 and Scheme 4,
were used to synthesize N-substituted benzotriazoles, either by
direct alkylation of commercially available 5-halo-benzotriazole
(Scheme 3) or by nucleophilic aromatic substitution starting
from bromofluoronitrobenzene derivatives (Scheme 4), fol-
lowed by reduction of the aromatic nitro group and treatment of
the resulting compounds by sodium nitrite in acetic acid to get
the final alkylated benzotriazoles (7b−23b and 17d−40d). To
assess the substitution influence on the 6-membered ring, we
took advantage of a highly electron-rich position between the
halogen and the carbon between the two cycles to perform
nitration using KNO3 in H2SO4. A reduction using Fe(s)/NH4Cl
was then performed to reduce the aromatic nitro group (Scheme
5).
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reference compound LM10. We evaluated these compounds
through a SAR study completed by STD epitope mapping and
stability evaluation. These compounds present a wide range of
lipophilicity and various chemical characteristics, showing the
possibility of fine-tuning these inhibitors’ molecular and
pharmacokinetic characteristics for potential ADME purposes.
Compared to existing potent TDO2 inhibitors (Figure 6a), the
present series shows similar or slightly better (8a) cellular
affinities, up to nanomolar range (Figure 6b). Previously, we
reported that the enzyme seemed to be susceptible to small
modifications of the inhibitor. However, we bring nuance to that
affirmation, as TDO2 appears to allow bulkier substitutions for
this series of compounds.
Table 3. Microsomal Stabilities (Half-Lives) of
Representative Compounds
a
Interestingly, this series of compounds showed a systemati-
cally better activity on cells than on the isolated enzyme. Such a
difference was previously observed for other indole-based
patented TDO2 inhibitors.33,46 Moreover, other previous
works preferred focusing on cellular activities and did not
include enzymatic inhibition values (i.e., preclinical compound
PF06845102/EOS200809, evaluated here in the biochemical
assay, Figure 6).25,27,28 Different parameters could account for
this discrepancy. First, the enzymatic assay that uses ascorbate,
methylene blue, and catalase to maintain a reduced state could
mimic poorly the physiological protein environment.47
Furthermore, some inhibitors could also bind to the apo form
of the protein, inhibiting its activity in a way that the enzymatic
assay would not detect.
Finally, the isoquinoline and quinazoline derivatives reported
in this work (1−5) also constitute attractive and highly
functionalizable scaffolds for the future development of TDO2
inhibitors.
a
The stability values were obtained in duplicates in two separate
experiments. Graphs of stability curves can be found in Supporting
Information (Figure S3).
EXPERIMENTAL SECTION
■
Chemistry. The following section comprises the general synthetic
procedures used for the synthesis of compounds reported in this
manuscript. Unless stated otherwise, reactions were performed under
atmospheric pressure. Detailed experimental information and spectro-
scopic data of synthesized compounds are supplied in the Supporting
Information. LC−MS analysis was performed on an Agilent (1100
series) HPLC single quadrupole (InfinityLab, ESI+) system equipped
with a Kinetex 5 μm EVO C18 (150 mm × 4,6 mm) using a gradient of
distilled water + 0.1% TFA (buffer A) and acetonitrile + 0.1% TFA
(buffer B). HPLC conditions for purity analysis were 5% B + 95% A at a
flow rate of 1 mL/min, followed by an increase of solution B to 80% in
15 min (5%/min). The wavelengths for UV detection were 254 and 210
nm. HPLC purities of final compounds that underwent biological
assessment were ≥95%.
Scheme 1. General Synthetic Procedure for Compounds 1−
a
5
a
Reagents and conditions: (a) 3 (1-(phenylsulfonyl)-3-(4,4,5,5-
The N-substituted benzotriazoles (18d−40d) were synthesized
using the following three-step general procedure. (A) To a 0.5 M
solution of 4-bromo-1-fluoro-2-nitrobenzene or 4-bromo-2-fluoro-1-
nitrobenzene (1 equiv) in ethanol was added the primary amine (1.5
equiv) and triethylamine (2 equiv). The resulting mixture was stirred
under reflux until complete conversion (30 min to 16 h), before being
evaporated in vacuo. The residue was extracted using EtOAc and water,
and the organic layer was dried with Na2SO4 and concentrated to afford
the desired compound, used in the next step without further
purification (unless stated otherwise). (B) The nitro group of N-
substituted 4-bromo-2-nitroaniline or 5-bromo-2-nitroaniline (1 equiv)
obtained in step A was reduced into an amine using iron powder (10
equiv) in a mixture of NH4Clsat./EtOH (1:4) stirred under reflux for 30
min to 16 h. Upon complete conversion, the reaction mixture was
filtrated over a celite pad, washed with EtOAc. After extraction, the
organic layer was dried over Na2SO4 and concentrated in vacuo. Unless
stated otherwise, the obtained residue was used for step C without
further purification. (C) The cyclization of mono-N-substituted 4-
bromobenzene-1, 2-diamine or 5-bromobenzene-1, 2-diamine was
tetramethyl-1,3,2-dioxaborolaN-2-yl)-1H-indole 42 (1.2 equiv), 5
mol % Pd(dppf)Cl2, 10 mol % SPhos, K3PO4 (2.0 equiv), dioxane/
H2O (5:1), 60 °C, 3−6 h, under N2. (b) KOH 2.5 M in MeOH/H2O
(1:1), reflux 30 min to 48 h.
The halogenated benzotriazoles (7b−23b and 17d−40d)
were reacted with indole derivatives bearing a pinacol or MIDA
boronate in the 3-position and a SO2Ph or a tert-
butyloxycarbonyl (Boc) as a protecting group to afford the
cross-coupled intermediates (Scheme 6). Finally, the target
compounds 7−40 were eventually obtained after appropriate
deprotection.
CONCLUSIONS
■
In this work, we designed a series of original TDO2 inhibitors,
devoid of the potentially problematic vinyl linker of the
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a
Scheme 2. Synthesis of Compound 5-(1H-Indol-3-yl)-1H-benzotriazole (6)
a
Reagents and conditions: (a) 5 mol % Pd(dppf)Cl2, 10 mol % SPhos, K3PO4 (2.0 equiv), dioxane/H2O (5:1), 60 °C, 6 h, under N2. (b) KOH 2.5
M in H2O/MeOH (1:1), reflux, 1 h. (c) 100% TFA, reflux, 16 h.
a
Scheme 3. Synthetic Routes for the N-Substituted 5- and 6-Halobenzotriazoles (7b−9b, 13b−15b, and 21b−22b)
a
Reagents and conditions: (a) Me2SO4 (1.6 equiv), NaOHaq 2 M, r.t., 1 h 30 (b) ClCH2CN (1.5 equiv), Et3N, DMF, reflux, 16 h.
a
Scheme 4. Synthetic Route for N-Substituted 5-Bromobenzotriazoles and 6-Bromobenzotriazoles (17d−40d)
a
Reagents and conditions: (a) primary amine derivative, Et3N (1.3 equiv), EtOH or THF, r.t. or reflux, 30 min to overnight; (b) Fe(s) (10 equiv),
EtOH/H2O (4:1), reflux, 30 min to overnight; (c) NaNO2 (2.5 equiv), glacial acetic acid, sonication, r.t., 1 h.
performed using NaNO2 in acetic acid using a sonication bath for 30
min to 1 h. The reaction mixture was then poured onto ice water and
extracted with EtOAc. The combined organic layers were washed with
saturated Na2CO3 solution and brine, dried with Na2SO4, and
concentrated in vacuo. The N-substituted benzotriazole was purified
using flash chromatography on silica gel with an EtOAc/cyclohexane
gradient.
General Procedures for Nitration (D) of Chloro-methyl-
benzotriazoles. (D) 2 mmol of the appropriate chloro-methyl-
benzotriazole (7b−9b) was dissolved in 3.5 mL of concentrated
H2SO4. To this solution, 6.5 mmol KNO3 in concentrated H2SO4 was
added dropwise. The resulting mixture was agitated for 1 h 30 min at 50
°C. After cooling down to room temperature, the reaction mixture was
poured on ice, resulting in precipitate formation (10b−12b). The
obtained powder was washed with water until neutrality. In accordance
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a
Scheme 5. Synthetic Route for Nitration and Reduction to Anilines of Chloro-methyl-benzotriazoles
a
Reagents and conditions: (a) KNO3 (3.0 equiv) in H2SO4, 50 °C, 1 h 30 (b) Fe(s) (10.0 equiv), EtOH/H2O (4:1), reflux, 30 min.
a
Scheme 6. General Synthetic Procedure for Cross-Coupling and Deprotection of 6-(1H-Indol-3-yl)-benzotriazole Derivatives
a
Reagents and conditions: (a) boronic ester derivative (41, 42, or 43) (1.2−1.5 equiv), 5 mol % Pd(dppf)Cl2, 10 mol % SPhos, K3PO4 (2.0 equiv),
dioxane/H2O (5:1), 60 °C, 3−6 h, under N2. (b) HCl 4.0 M in MeOH/H2O (1:1), reflux, 30 min to 5 h for Boc deprotection, (c) KOH 2.5 M in
MeOH/H2O (1:1) or 1 M TBAF in THF, reflux 30 min to 48 h. for SO2Ph deprotection.
with the literature, nitration of 5-chloro-2-methyl-2H-benzo[d][1,2,3]-
triazole (7b) led to the formation of a second dinitrated product, 5-
bromo-2-methyl-4,6-dinitro-2H-benzo[d][1,2,3]triazole, in addition to
the mononitrated 5-chloro-2-methyl-4-nitro-2H-benzo[d][1,2,3]-
triazole (10b).49 The general reduction procedure (C) was used to
reduce the nitrated chloro-methyl-benzotriazoles into N-methylbenzo-
triazol-4-amines (10c−12c).
General Procedures for the Suzuki−Miyaura Cross-Coupling
Reaction. Prior to the coupling procedures, the solvent 1,4-dioxane
and K3PO4 solution in water were degassed by atmosphere exchange
with nitrogen in a sonication bath for 45 min. The cross-coupling
procedure was adapted from Knapp et al.45 We found that the use of
Pd(dppf)Cl2 as a catalyst significantly increased the yield as compared
to Pd(OAc)2. (E) The coupling procedure was adapted for the use of a
pinacol boronic ester (41 and 42). Aryl-halide (1 mmol, 1 equiv) and
the boronic ester (1.6 equiv), Pd(dppf)Cl2 (5 mol %) and SPhos (10
mol %), were introduced into a 25 mL two-neck round-bottom flask
under an inert atmosphere. Then, 10 mL of degassed dioxane was
added, and the reaction mixture was left under agitation at r.t. for 10 min
until dissolution of reactants. Finally, 2 mL of degassed distilled water
containing K3PO4 (3.5 mmol) was added to the media, and the reaction
mixture was stirred for 3 to 16 h at 70 °C. When the conversion was
complete, the mixture was cooled to room temperature, diluted with 1
N NaOH (10 mL), and extracted twice with 10 mL of EtOAc. The
combined organic fractions were dried over Na2SO4 and concentrated
in vacuo. The obtained residue was purified by flash chromatography on
silica gel with the appropriate gradient of EtOAc in cyclohexane to
afford the cross-coupled protected intermediate. (F) A coupling
procedure was adapted for the use of a MIDA boronate (43). The
general procedure (E) was slightly adapted when couplings were
performed using MIDA boronate 43. In this modified procedure (F),
1.2 equiv of boronate and 7 equiv of K3PO4 were used.
General Phenylsulfonyl (SO2Ph) Deprotection Procedures of
Cross-Coupled Products. Intermediates obtained using procedures
(F) and (E), bearing an SO2Ph protecting group, were deprotected
using deprotection procedures (G) or (H), while Boc deprotection was
performed using the deprotection procedure (I).
Deprotection Procedure G. 0.5 mmol of starting compound was
dissolved in 4 mL of a 1:1 solution of MeOH/H2O containing 2.5 M of
KOH. The resulting mixture was refluxed until completion of the
reaction (30 min to 48 h) to afford compounds 1−20, 23−27, and 31−
40.
Deprotection Procedure H. 0.5 mmol of starting compound was
dissolved in 4 mL of a 1 M solution of TBAF in THF. The resulting
mixture was refluxed until completion of the reaction (2−48 h) to
afford compounds 21−22 and 28−30.
Deprotection Procedure I. 0.5 mmol of starting compound was
dissolved in 4 mL of a 1:1 solution of MeOH/H2O containing 4 M HCl.
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Figure 6. Examples of potent TDO2 inhibitors (a) and representative examples of the series developed in the present work (b) (8a and 37a). alog D7.4
48
b
values were calculated using Chemicalize. Compound 21 and its inhibition values are from Pei et al. 2018. cCompounds 20 and 30 and their
inhibition values are from Parr et al.28 dCellular potency of PF06845102/EOS200809 is from Schramme et al. 2020.25 eBiochemical IC50 was
measured in the present work.
The resulting mixture was refluxed until completion of the reaction (30
min to 48 h) to afford compounds 8a, 37a, and 17−18.
mM sodium phosphate, 100 mM NaCl, 20% glycerol, and 10 mM L-
Trp. To ensure of the quality and proper folding of the purified enzyme,
nano differential scanning fluorimetry experiments were performed on
a Tycho NT.6 device (NanoTemper Technologies). According to the
standard manufacturer’s procedures, samples were poured into
capillaries and heated up to 95 °C in 3 min while following fluorescence
emission at 330 and 350 nm. Melting temperatures were extracted from
the derivative of the 350/330 nm fluorescence ratios upon increasing
temperature.
Nuclear Magnetic Resonance. All experiments were performed at
277 K on a Bruker Ascend AVANCE III 600 MHz system equipped
with a broadband cryoprobe (Bruker). For 1D STD studies, samples
were prepared at 200 μM in PBS with 5% deuterated methanol. The
concentration of hTDO2tr was 15 μM. Ligand binding was detected
using an STD stddiffesgp.3 sequence with a 2 s saturation time. Water
signal suppression was achieved using an excitation-sculpting scheme,
and a 50 ms spinlock was used to suppress protein background signals.
For each experiment, 2048 scans were collected for on- and off-
resonance experiments (respectively at 0 and 40 ppm) to achieve a
sufficient signal to noise ratio for epitope mapping.
Enzymatic Assay. All the graphs were obtained with Graph-
PadPrism 8 software (San Diego, CA). The UV spectra were recorded
at room temperature on a Spectramax M2E spectrophotometer
(Molecular Devices, LLC, Sunnyvale, CA) in 96-well flat bottom
plates. The assay was performed in a pH 6.5 potassium phosphate buffer
(100 mM) at 37 °C. The final concentrations in each well were 100 μM
L-Trp, 20 mM L-ascorbate, 10 μM methylene blue, 150 nM catalase, and
3% of DMSO containing the inhibitor and (optionally) 0.01% v/v of
Triton X-100. The assay was initiated by adding 15 μL of buffer
containing hTDO2tr (no L-Trp, no DMSO) to 85 μL of buffer (with L-
Trp and the tested inhibitor concentration in DMSO). After 15 min, 20
μL of 30% m/v of trichloroacetic acid in water was added, and the plates
were heated at 65 °C for 15 min. Then, 100 μL of a 2% m/v p-
dimethylbenzaldehyde solution in pure acetic acid was added to each
well. After 10 min, absorbance reading was performed at 490 nM.
Cellular Assays. The assay was performed in 96-well flat bottom
plates seeded with 1 × 105 cells (P815B-hTDO, clone 19) in a final
volume of 200 μL of Iscove’s modified Dulbecco medium (GIBCO-
Thermo Fisher Scientific; which contains 80 μM L-Trp) supplemented
Molecular Docking. Molecular docking jobs were performed with
AutodockVina 1.1.2,50 using the disclosed tryptophan 2,3-dioxygenase
structure (PDB ID: 5TI9)39 with the orthosteric binding site occupied
by natural ligand tryptophan, heme, and dioxygen. This structure was
chosen as it displayed the full 338−346 loop that is part of this binding
site. Hydrogens and charges were added, and then, tryptophan and
dioxygen were removed, centering the searching box 15 Å around this
region. During the process, sidechain flexibility was allowed for several
residues of the binding site and later on only for Arg144 that appeared
critical. Binding poses were analyzed for their binding mode and
assessed in comparison with the crystallized analogue and with L-Trp.
Protein Expression and Purification. The sequence of the
truncated form of hTDO2 in which 1−17 and 389−406 fragments were
deleted (His6Thrb-TDO2tr),23 described by Batabyal and Yeh, was
inserted in a pET-28 plasmid, ordered from Genecust. The
recombinant plasmid was transferred into the Escherichia coli strain
BL21 (DE3) (Rosetta). The transformant was selected from a single
colony on a LB kanamycin agar plate and was used to inoculate in a LB
medium (50 μg/mL kanamycin and 34 μg/mL chloramphenicol) and
was cultured at 37 °C until an optical density of 0.6 was reached. The
expression of hTDO2 was induced by isopropyl 1-thio β galacto-
pyranoside (IPTG), with a final concentration of 1 mM. An aliquot of
hemin in NaOH 0.5 M, with a final concentration of 8−10 μM, was
added to the culture. The culture was grown at 20 °C for 20 h. Cells
were harvested by centrifugation at 5000 rpm, 4 °C for 25 min. Cell
pellets were resuspended in a lysis buffer (Tris−HCl 50 mM, pH 8.5,
MgCl2 10 mM, NaCl 300 mM, imidazole 5 mM, and glycerol 10%)
supplemented with protease inhibitors (Roche) and then disrupted by
sonication, followed by centrifugation at 4 °C and 10,000 rpm for 30
min. The supernatant was collected, and to 1 μL of β-mercaptoethanol
per mL of the supernatant was added, before loading onto 1 mL His-
trap FF-crude columns (GE Healthcare) according to the manufac-
turer’s instructions. Protein concentrations were measured using the
Bradford method with the Biorad protein assay kit, and sample
homogeneity was assessed using sulfate−polyacrylamide gel electro-
phoresis with Coomassie brilliant blue as a staining agent. Purified
TDO2tr was then dialyzed overnight in a pH 7.6 buffer containing 50
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with 2% FCS in the presence of the TDO inhibitor at different
concentrations (1−25,000 nM) for 7 h when substrate consumption
was below 25%. Cells were centrifuged 10 min at 300 rcf, after which 60
μL of supernatant was collected and mixed with 60 μL of 12% (wt/vol)
trichloroacetic acid. After centrifugation at 4 °C at 23,000 rcf, 70 μL of
supernatant was diluted with 70 μL of Milli-Q H2O. The resulting
solution was used to quantify L-Trp and Kyn concentrations by
ultraperformance liquid chromatography (UPLC) based on the
retention time and UV absorption. Briefly, samples were injected into
the Kinetex 2.6 μm EVO C18 100A (2,1 × 50) column (Phenomenex)
and were eluted using a 1,7 min linear gradient of acetonitrile in water
(5−50%) containing 0.1% trifluoroacetic acid at a flow rate of 0,6 mL/
min. The column eluent was monitored with a UV detector at 280 and
360 nm to detect L-Trp and Kyn, respectively.
For hIDO counter-screening (P815B-hIDO, clone 6), the same
protocol was applied, but the plates were seeded with 2 × 105 cells and
incubated for 24 h in order to achieve a similar substrate consumption.
In Cellulo Assessment of Toxicity. A solution (100 μL)
containing 80 μL of PBS, 20 μL of PMS, and 1 μL of MTS was
added to wells containing 1 × 105 cells in 100 μL of DMEM. After 2−4
h of incubation with MTS and PMS at 37 °C, absorbance at 490 nm was
measured.
Microsomal Stability Assay. Liver microsomes (20 mg protein/
mL), NADPH regenerating system solutions A and B, and 10 mM stock
compound solution (100% DMSO) were prepared. The reaction
mixture finally contains 713 μL of purified water, 200 μL of 0.5 M
potassium phosphate pH 7.4, 50 μL of NADPH regenerating system
solution A (BD Biosciences Cat. no. 451220), 10 μL of NADPH
regenerating system solution B (BD Biosciences Cat. no. 451200), and
2 μL of the compound stock solution (10 μM final concentration). A
control experiment was realized for each compound by substituting
NADPH regenerating solutions A and B for 60 μl of purified water. The
reaction mixture is warmed to 37 °C for 5 min in a water bath, and the
reaction is initiated by the addition of 25 μL of liver microsomes (0.5
mg protein/mL final concentration). At different time points (0 min; 60
min; 180 min; 360 min; 24 h), 100 μL is withdrawn and added to 400
μL of cold acetonitrile on ice. Then, the mixtures are centrifuged at
13,000 rpm for 5 min at 4 °C. Finally, 450 μL of the supernatant is
recovered, evaporated using SpeedVac, and evaluated by UPLC. 7-
Ethoxycoumarine was used as a positive control.
Phone: +32 2 764 73 41; Email: raphael.frederick@
uclouvain.be
Authors
Arina Kozlova − Louvain Drug Research Institute (LDRI),
Université Catholique de Louvain (UCLouvain), Brussels B-
1200, Belgium; Ludwig Institute for Cancer Research, Brussels
B-1200, Belgium; de Duve Institute, UCLouvain, Brussels B-
1200, Belgium
Léopold Thabault − Louvain Drug Research Institute (LDRI),
Université Catholique de Louvain (UCLouvain), Brussels B-
1200, Belgium; Pole of Pharmacology and Therapeutics,
Institut de Recherche Expérimentale et Clinique (IREC),
UCLouvain, Brussels B-1200, Belgium; orcid.org/0000-
0001-9501-6563
Maxime Liberelle − Louvain Drug Research Institute (LDRI),
Université Catholique de Louvain (UCLouvain), Brussels B-
1200, Belgium
Simon Klaessens − Ludwig Institute for Cancer Research,
Brussels B-1200, Belgium; de Duve Institute, UCLouvain,
Brussels B-1200, Belgium
Julien R. C. Prévost − Louvain Drug Research Institute
(LDRI), Université Catholique de Louvain (UCLouvain),
Brussels B-1200, Belgium; orcid.org/0000-0003-1569-
8125
Caroline Mathieu − Louvain Drug Research Institute (LDRI),
Université Catholique de Louvain (UCLouvain), Brussels B-
1200, Belgium
Luc Pilotte − Ludwig Institute for Cancer Research, Brussels B-
1200, Belgium; de Duve Institute, UCLouvain, Brussels B-
1200, Belgium; orcid.org/0000-0001-6761-6836
Vincent Stroobant − Ludwig Institute for Cancer Research,
Brussels B-1200, Belgium; de Duve Institute, UCLouvain,
Brussels B-1200, Belgium
Benoît Van den Eynde − Ludwig Institute for Cancer Research,
Brussels B-1200, Belgium; de Duve Institute, UCLouvain,
Brussels B-1200, Belgium; Walloon Excellence in Life Sciences
and Biotechnology, Brussels B-1200, Belgium
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00323.
■
sı
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00323
Notes
Atomic coordinates for compound 17 inside hTDO2
The authors declare no competing financial interest.
cavity (PDB)
Atomic coordinates for compound 18 inside hTDO2
cavity (PDB)
Atomic coordinates for compound 6 inside hTDO2 cavity
(PDB)
Inhibitory affinities; evaluation of influence of compound
6; epitope mapping; microsomal stability and half-life
curves; enzymatic IC50 curves; cellular IC50 curves;
HPLC traces; chemicals and reagents; synthesis of
boronic esters; and synthesis of N-substituted benzo-
triazoles (PDF)
ACKNOWLEDGMENTS
■
This work was supported by the Belgian Fonds National de la
Recherche Scientifique (F.R.S.-FNRS; Grants 3.05557.43,
28252254 and 32704190), the Belgian Fondation contre le
Cancer (Large Equipment grant), the French Community of
Belgium (ARC 14/19-058), the Fonds Spéciaux de recherche
(FSR) at UCLouvain, and a J. Maisin Foundation grant. A.K.
was a PhD fellow of the F.R.S.-FNRS. The authors thank Esra
Yildiz, Bénédicte Tollet, Lionel Pochet, and MSc students
Kamie Peters and Lucie Jacques for their involvement in organic
synthesis, technical assistance, and fruitful discussions. We
acknowledge the Nuclear & Electron Spin Technologies
Platform (NEST) platform for the access to the Bruker Ascend
AVANCE III 600 MHz system equipped with a broadband
cryoprobe (Bruker). The Center for Protein Engineering (CIP)
Molecular formula strings of the synthesized compounds
(CSV)
AUTHOR INFORMATION
Corresponding Author
Raphaël Frédérick − Louvain Drug Research Institute (LDRI),
Université Catholique de Louvain (UCLouvain), Brussels B-
1200, Belgium; orcid.org/0000-0001-8119-1272;
■
̀
of the University of Liege (ULg) is acknowledged for their
expertise in hTDO2 production and purification.
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ABBREVIATIONS
■
ADME, absorption, distribution, metabolism, and excretion;
AhR, aryl hydrocarbon receptor; GCN2, general control
nonderepressible 2; IDO1, indoleamine-2,3-dioxygenase 1;
KMO, kynurenine-3-monooxygenase; KP, tryptophan-kynur-
enine pathway; KYN, kynurenine; LE, ligand efficiency; LLE,
lipophilic ligand efficiency; L-Trp, L-tryptophan; MDSCs,
myeloid-derived suppressor cells; NK, natural killer cell;
NMR, nuclear magnetic resonance; NOE, nuclear Overhauser
effect; SMI, small-molecule inhibitor; STD, saturation transfer
difference; TDO2, tryptophan 2,3-dioxygenase; Treg, regulatory
T-cells
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