Novel selective ido1 inhibitors with isoxazolo[5,4-d]pyrimidin-4(5h)-one scaffold

Article abstract of DOI:10.3390/ph14030265

Indoleamine 2,3-dioxygenase 1 (IDO1) is a promising target in immunomodulation of several pathological conditions, especially cancers. Here we present the synthesis of a series of IDO1 inhibitors with the novel isoxazolo[5,4-d]pyrimidin-4(5H)-one scaffold. A focused library was prepared using a 6-or 7-step synthetic procedure to allow a systematic investigation of the structure-activity relationships of the described scaffold. Chemistry-driven modifications lead us to the discovery of our best-in-class inhibitors possessing p-trifluoromethyl (23), p-cyclohexyl (32), or p-methoxycarbonyl (20, 39) substituted aniline moieties with IC₅₀ values in the low micromolar range. In addition to hIDO1, compounds were tested for their inhibition of indoleamine 2,3-dioxygenase 2 and tryptophan dioxygenase, and found to be selective for hIDO1. Our results thus demon-strate a successful study on IDO1-selective isoxazolo[5,4-d]pyrimidin-4(5H)-one inhibitors, defining promising chemical probes with a novel scaffold for further development of potent small-molecule immunomodulators.

Full text of DOI:10.3390/ph14030265

pharmaceuticals
Article
Novel Selective IDO1 Inhibitors with Isoxazolo[5,4-d]pyrimidin-
4(5H)-one Scaffold
1
1
Ana Dolšak 1, Tomaž Bratkovicˇ
, Larisa Mlinaricˇ
, Eva Ogorevc 1, Urban Švajger 1,2, Stanislav Gobec 1
and Matej Sova 1,
*
1
Faculty of Pharmacy, University of Ljubljana, Aškercˇeva 7, 1000 Ljubljana, Slovenia;
ana.dolsak@ffa.uni-lj.si (A.D.); tomaz.bratkovic@ffa.uni-lj.si (T.B.); larisa.mlinaric@gmail.com (L.M.);
eva.ogorevc@ijs.si (E.O.); urban.svajger@ztm.si (U.Š.); stanislav.gobec@ffa.uni-lj.si (S.G.)
Blood Transfusion Centre of Slovenia, Šlajmerjeva 6, 1000 Ljubljana, Slovenia
Correspondence: matej.sova@ffa.uni-lj.si; Tel.: +386-1-476-9577
2
*
Abstract: Indoleamine 2,3-dioxygenase 1 (IDO1) is a promising target in immunomodulation of
several pathological conditions, especially cancers. Here we present the synthesis of a series of
IDO1 inhibitors with the novel isoxazolo[5,4-d]pyrimidin-4(5H)-one scaffold. A focused library
was prepared using a 6- or 7-step synthetic procedure to allow a systematic investigation of the
structure-activity relationships of the described scaffold. Chemistry-driven modifications lead us to
the discovery of our best-in-class inhibitors possessing p-trifluoromethyl (23), p-cyclohexyl (32), or
p-methoxycarbonyl (20, 39) substituted aniline moieties with IC50 values in the low micromolar range.
In addition to hIDO1, compounds were tested for their inhibition of indoleamine 2,3-dioxygenase
2 and tryptophan dioxygenase, and found to be selective for hIDO1. Our results thus demon-
strate a successful study on IDO1-selective isoxazolo[5,4-d]pyrimidin-4(5H)-one inhibitors, defining
promising chemical probes with a novel scaffold for further development of potent small-molecule
immunomodulators.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Dolšak, A.; Bratkovicˇ, T.;
Mlinaricˇ, L.; Ogorevc, E.; Švajger, U.;
Gobec, S.; Sova, M. Novel Selective
IDO1 Inhibitors with Isoxazolo[5,4-
d]pyrimidin-4(5H)-one Scaffold.
Pharmaceuticals 2021, 14, 265.
Keywords: indoleamine 2,3-dioxygenase 1; selective inhibitors; isoxazolo[5,4-d]pyrimidin-4(5H)-one;
immunomodulation; cancer
https://doi.org/10.3390/ph14030265
1. Introduction
Academic Editor: Thierry Besson
Tryptophan (Trp) is the least abundant essential amino acid with significant and vari-
ous roles in the human body. Since its only source is dietary intake, its plasma concentration
must be precisely regulated. Most of the Trp is metabolized via the kynurenine (Kyn) path-
Received: 12 February 2021
Accepted: 11 March 2021
Published: 15 March 2021
way, with only 5% being used for protein synthesis, or converted into serotonin [1]. The
first and rate-limiting step of Trp conversion to N-formylkynurenine, which spontaneously
hydrolyses to Kyn, is catalyzed by one of three isoforms of heme-containing dioxygenases
(indoleamine 2,3-dioxygenase 1 (IDO1), indoleamine 2,3-dioxygenase 2 (IDO2), and tryp-
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
tophan dioxygenase (TDO)) [
responsible for the metabolism of dietary Trp and is therefore mainly located in the liver [
In contrast, IDO2 has only been known for 13 years, and its biological role has not yet
been clarified in detail [ ]. Among the three isoforms, the most studied one is IDO1,
which is widely distributed in many tissues. It has been reported that its overexpression in
various cancers correlates with poor disease prognosis [ ]. IDO1, TDO, and IDO2 have been
studied primarily for their pivotal role in cancer evasion and immunosuppression [ 10],
yet Kyn and its metabolites are involved in several other pathological conditions (e.g., viral
2]. TDO, which was first described in 1936 [3], is primarily
4].
5,6
7
Copyright:
© 2021 by the authors.
8
–
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
and bacterial infections-related immunosuppression [11], neurological disorders [12
,13],
and osteoporosis [14]).
In the last decade, a considerable number of IDO1 inhibitors have been developed
(thoroughly reviewed in our recent paper [15]), possessing several different scaffolds, i.e.,
hydroxyamidine (e.g., epacadostat 1) [16,17], phenylimidazole (e.g., navoximod 2) [18],
Pharmaceuticals 2021, 14, 265. https://doi.org/10.3390/ph14030265
https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2021, 14, 265
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indole (e.g., PF-06840003
3) [19], and 1-(4-arylcyclohex-1-yl)propanamide (e.g., linrodostat
4) [20–22], which are presented in Figure 1. To date, however, none of the IDO inhibitors
have successfully completed clinical trials. A design of dual or multiple inhibitors of
isoforms could contribute to the improved clinical efficacy of Kyn pathway blockade [23].
A detailed discussion on differences and selectivity determinants of IDO1, IDO2, and TDO
is presented in our recent review paper [15]. Compensatory up-regulation of IDO2 and
TDO as a consequence of IDO1 inhibition is possible; however, the exact mechanisms have
not been elucidated yet. Multitargeting has not been extensively researched yet, so little is
known about possible benefits and/or negative side effects. One of the possible drawbacks
of multitargeting could be (potentially toxic) accumulation of Trp if both IDO1 and TDO
were inhibited since they represent the two enzymes physiologically important for Trp
metabolism. Taken together, until some new revolutionary findings of multitargeting are
uncovered, selective inhibition of IDO1 is still of major importance.
Figure 1. Structures of representative IDO1 inhibitors.
Herein, we report the synthesis of selective IDO1 inhibitors with novel isoxazolo[5,4-
d]pyrimidin-4(5H)-one scaffold identified in our hit compound 12 [24]. The aim of this
study was to develop the appropriate synthetic procedures, synthesize a focused library
of isoxazolo[5,4-d]pyrimidin-4(5H)-ones along with 12 (Scheme 1, Table 1), explore the
structure-activity relationships (SARs) of synthesized compounds and determine the selec-
tivity among three enzyme isoforms, i.e., IDO1, TDO, and IDO2. For the determination of
inhibitory potency, hIDO1 was expressed in Escherichia coli and purified by immobilized-
metal affinity chromatography (IMAC). The selectivity of the compounds for hIDO1 was
evaluated by comparative determination of inhibitory potencies against hIDO2 and hTDO
using newly established highly sensitive fluorescence assays. The fluorescence-based assay
for the determination of IDO1 activity was established in 2013 [25]; however, it has not
been used for TDO and IDO2 yet.
Table 1. Inhibitory potencies of synthesized compounds 12–37 with 3-(4-fluorophenyl)isoxazolo[5,4-
d]pyrimidin-4(5H)-one scaffold against IDO1, IDO2, and TDO.
3-(4-fluorophenyl)isoxazolo[5,4-d]pyrimidin-4(5H)-one scaffold
a,b
% of Inhibition @ 100 µM or IC50
R2
Compound
12
IDO1
IDO2
TDO
IC50
,
c
n.a.
n.a.
50 ± 1 µM

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Table 1. Cont.
a,b
% of Inhibition @ 100 µM or IC50
R2
Compound
IDO1
IDO2
TDO
13
14
21 ± 5%
n.a.
n.a.
27 ± 4%
n.a.
n.a.
IC50
88 ± 4 µM
,
15
16
n.a.
n.a.
n.a.
15 ± 4%
28 ± 9%
17
18
19
33 ± 2%
32 ± 4%
n.a.
15 ± 1%
n.a.
n.a.
n.a.
n.a.
IC50
,
55 ± 2 µM
IC50
27 ± 1 µM
,
20
21
n.a.
n.a.
n.a.
n.a.
27 ± 3%
IC50
55 ± 2 µM
,
22
23
24
n.a.
n.a.
n.a.
n.a.
IC50
23 ± 1 µM
,
30 ± 2%
39 ± 5%
25 ± 8%

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Table 1. Cont.
a,b
% of Inhibition @ 100 µM or IC50
R2
Compound
IDO1
IDO2
TDO
IC50
98 ± 5 µM
,
25
28 ± 0%
24 ± 7%
26
27
28
11 ± 2%
n.a.
n.a.
n.a.
n.a.
11 ± 4%
31 ± 3%
17 ± 2%
18 ± 4%
IC50
30 ± 1 µM
,
29
n.a.
29 ± 3%
30
31
n.a.
n.a.
n.a.
17 ± 2%
IC50
87 ± 3 µM
,
n.a.
IC50
22 ± 1 µM
,
32
33
34
n.a.
n.a.
n.a.
n.a.
22 ± 4%
n.a.
n.a.
n.a.

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Table 1. Cont.
a,b
% of Inhibition @ 100 µM or IC50
R2
Compound
IDO1
IDO2
TDO
35
36
20 ± 6%
n.a.
n.a.
IC50
,
13 ± 2%
n.a.
46 ± 2 µM
37
17 ± 5%
n.a.
n.a.
a
IC50 values are the means of at least two independent determinations, presented as mean
±
SD; b Positive
control: epacadostat (
1), IC50 values obtained for IDO1, IDO2, and TDO were 53, 580, and 9620 nM, respectively;
c
n.a., not active.
Scheme 1. Synthetic procedure for the preparation of isoxazolo[5,4-d]pyrimidin-4(5H)-one derivatives. Reagents, conditions
and yields for each step: (a) hydroxylamine hydrochloride, THF/EtOH/H2O (2/5/1, v/v), r.t., 30 min; 71–98%; (b) NCS,
◦
DMF, r.t., 18 h; (c) Procedure I: (i) NaOEt/EtOH, 2-cyanoacetamide, 50 C; (ii) dropwise addition of hydroxyimidoyl
chlorides 7a
-
k
at 0 ◦C; (iii) r.t., 30 min; then reflux, 18 h; or procedure II: (i) NaH, anh. DMF, 2-cyanoacetamide, r.t., 2 h;
◦
(ii) 70 C, 18 h; 6–71%; (d) triethyl orthoformate, Ac2O, reflux, 18 h; 8–85%; (e) Procedure III: (i) chloroacetyl chloride, glacial
◦
◦
acid, 10–15 C, 30 min; (ii) NaOAc, H2O, rt, 1h; 24–100%; or Procedure IV: chloroacetyl chloride, DCM, 10–15 C, 2 h;
48–100%; or Procedure V: bromoacetyl bromide in 1,4-dioxane, DMF, r.t., 18 h; 61–71%; (f) (i) R2COCH2X, NaH, BTEAC, Ar;
(ii) KI, anh. MeCN, r.t., 18 h; 6–82%; (g) (i) NaN3, ZnBr2, H2O, reflux, 18 h; (ii) NaOH, 1 h; 15–80%. Abbreviations: THF,
tetrahydrofuran; NCS, N-chlorosuccinimide; DMF, N,N-dimethylformamide; r.t., room temperature; DCM, dichloromethane;
BTEAC, benzyltriethylammonium chloride.

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2. Results and Discussion
2.1. Chemistry
First of all, the appropriate synthetic procedures for the synthesis of the main isoxazolo-
[5,4-d]pyrimidin-4(5H)-one scaffold were established. To examine the chemical space
around this scaffold and investigate the SAR, a focused library of substituted isoxazolo[5,4-
d]pyrimidin-4(5H)-ones was prepared. Modifications were implemented in two positions,
namely R1 and R2 (as depicted in Scheme 1), where different aromatic rings (R1) and amines
(R2H) were introduced. Isoxazolo[5,4-d]pyrimidin-4(5H)-one scaffold was prepared in four
synthetic steps according to previously published procedures [26–29]. Some steps were
also optimized during the synthesis. Substitutions in positions R1 and R2 were introduced
in the first or the last step of the synthesis, respectively.
In the first two steps, arylaldehydes (5a
drochloride to yield corresponding oximes (6a
chlorosuccinimide (NCS) afforded hydroxyimidoyl chlorides (7a
–
k
) were subjected to hydroxylamine hy-
). The following treatment with N-
). These nitrile oxide
–
k
–
k
precursors, which were used immediately without further purification, easily underwent
1,3-dipolar cycloaddition in the third synthetic step. The published procedure [26] em-
ployed NaOEt as a strong base to convert 2-cyanoacetamide to a nucleophilic moiety. How-
ever, this required additional time to prepare fresh NaOEt in situ and in some cases low
yields in the following reaction were obtained. Thus, we optimized and shortened reaction
time by using NaH in DMF instead of NaOEt. The obtained 3-aryl-5-aminoisoxazole-
4-carboxamides (8a
isoxazolo[5,4-d]pyrimidin-4(5H)-ones (9a
11a ) were prepared via three different procedures, based on their physicochemical prop-
erties (e.g., solubility, reactivity). In the last step, isoxazolo[5,4-d]pyrimidin-4(5H)-one core
was subjected to N-alkylation to yield the final compounds 12 53. To improve the reaction,
–
k) were further cyclized with triethyl orthoformate to yield aryl-
–
k). Concurrently, 2-chloro or 2-bromoacetamides
(
–y
–
benzyltriethylammonium chloride (BTEAC) as a phase-transfer catalyst, and potassium
iodide (conversion to a better leaving group) were used. The compounds with cyano
substituent (i.e., 27
prepare the corresponding tetrazolo substituted derivatives (i.e., 30
compounds (12 53) were prepared via the described synthetic procedures. For twenty-five
,
44) were reacted with NaN3 in the presence of ammonium chloride to
,
45). Forty-two final
–
of them, modifications in the form of various 2-halogenoacetamides were introduced in
the last step, ranging from aliphatic amines to differently substituted anilines. Instead of
4-fluorobenzaldehyde, which was present in compound 12, several other arylaldehydes
were introduced in the first synthetic step to yield additional sixteen final compounds for
biological evaluation.
2.2. hIDO1 Expression
Expression and purification of hIDO1 was essentially performed as previously de-
scribed [20], with minor modifications. Relatively high cell density prior to induction of
recombinant protein expression was necessary for optimal yield. The glucose present in the
induction medium suppresses the lacUV5 promoter, allowing gentle induction, providing
it is gradually consumed by the expression strain growing in the buffered medium at
low temperature. The induction medium was supplemented with 5-aminolevulinic acid
(heme precursor) to boost heme synthesis. The addition of strong reducing agents (i.e.,
tris(2-carboxyethyl)phosphine (TCEP) and 1,4-dithiothreitol) in all buffers during hIDO1
purification was essential to ensure high heme occupancy by preventing oxidation of
cysteine residues (i.e., formation of disulfide bonds), thus retaining the enzyme in its native
conformation. Single-step chromatographic purification afforded highly pure hIDO1 as
confirmed by SDS-PAGE electrophoresis of eluted material (Figure 2).

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Figure 2. Recombinant hIDO1 purification: (a) IMAC chromatogram; (b) SDS-PAGE analysis of purified protein: L—
bacterial lysate; FT—flow-through; M—protein standard; E1 & E2—imidazole-eluted fractions.
2.3. Determination of Inhibitory Potency
All the synthesized compounds were evaluated for their inhibitory potencies against
hIDO1 (Table 1) according to the published protocol [25]. Our synthesized starting com-
pound 12 exhibited IDO1 inhibitory activity of 50 µM. To investigate SAR, two series of
compounds were prepared, the first focusing on amine substitution, whereas the second
series possessed various modifications of 4-fluorophenyl fragment. Regarding the first
series, the inhibition of IDO1 was only achieved with bulkier substituents, which can be
clearly seen by the active compound 15 (IC50 value of 88 µM), contrary to significantly
less potent 13 and 14 with smaller amine substituents. Concerning aromatic amines, the
appropriate substitution on aniline is mandatory for the potency, since 16 and 17 with
aniline and p-fluoroaniline, respectively, showed only weak inhibition. Our aim to identify
the preferred position and nature of aniline substitution encouraged us to prepare several
aniline-substituted isomers (compounds 17–34, Scheme 1, Table 1) On the trio of isomers 18,
19 and 20, substituted with methoxycarbonyl group, the ortho substitution corresponding
to 18 was found less appropriate in comparison with para in 20, which emerged as superior
in potency with IC50 of 27 µM. The following observation was verified and confirmed with
two additional sets of meta vs. para isomers (22 vs. 23 and 24 vs. 25). In accordance with
obtained results, we therefore focused solely on para aniline substitution to further explore
the chemical space around the aniline fragment. The introduction of an additional methy-
lene group in the aniline moiety affording p-methoxycarbonyl substituted benzylamine
fragment resulted in a decrease of inhibitory potency (21 compared to 20).
Most of the active compounds possess hydrogen bond acceptors incorporated in the
p-aniline substituent. The only exceptions are compounds 22 and 23 with trifluoromethyl
group, as well as 31 and 32 possessing lipophilic fragments (Table 1). The introduction of
polar dimethylamino group (as in 28) or acidic tetrazol moiety (as in 30), which mimics the
carboxyl group led to weak inhibition or inactive compounds, respectively. Substitution
with methylsulfonyl (26), cyano (27), 3,4,5-trimethoxy (34), or longer butoxy group (33
)
also resulted in inactive compounds. Interestingly, the attachment of isoxazolo ring in

Pharmaceuticals 2021, 14, 265
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compound 29 on the para position of aniline afforded a potent IDO1 inhibitor with IC50
value of 30 µM.
The next step was the replacement of the aniline ring with the naphthalene or tetrahy-
droisoquinoline moiety (Table 1, compounds 35, 36, and 37). 2-naphthyl and tetrahydro-
quinoline substitutions (as in 12) were more appropriate compared to 1-naphthyl and
tetrahydroisoquinoline, respectively. With the second series of compounds, we tried to
investigate the chemical space around the substitution at the other (left) side of the se-
lected isoxazolo[5,4-d]pyrimidin-4(5H)-one scaffold (Table 2). Different modifications of 4-
fluorophenyl fragment were introduced in the first step of the synthesis (Scheme 1), whereas
the tetrahydroquinoline moiety as the R2 fragment, originating from 12, was preserved in
most of the compounds. Interestingly, in combination with tetrahydroquinoline as R2 sub-
stituent, the optimal R1 substituent was found to be the original 4-fluorophenyl fragment
since none of the other aryl analogs seemed to improve the potency of 12. However, the
replacement of tetrahydroquinoline with p-nitroaniline or methyl ester of p-aminobenzoic
acid in the R2 position also afforded potent inhibitors. Inhibitory potencies of 48 and 49
,
possessing 3,4-dihalogenophenyl fragment, and 40 with phenyl fragment were comparable
to their 4-fluorophenyl analog 25. Another heteroaromatic substituent in the form of thio-
phene was found active, when combined with p-nitroaniline moiety as R2. Two compounds
with different thiophene orientations were prepared, both 51 with tiophen-2-yl fragment
retained the potency (IC50 of 53 µM) and 53 with tiophene-3-yl fragment (IC50 of 40 µM)
exhibited superior potency than their analog 25. Compound 39 with phenyl substituent as
R1 and methyl ester of p-aminobenzoic acid in the R2 position retained a similar potency to
its 4-fluorophenyl analog 20. Taken together, the R1 position is more liable to modifications,
when combined to p-substituted anilines with hydrogen bond acceptors as R2 substituents.
Table 2. Inhibitory potencies of synthesized compounds 38–53 with 3-arylisoxazolo[5,4-d]pyrimidin-4(5H)-one scaffold
against IDO1, IDO2, and TDO.
3-arylisoxazolo[5,4-d]pyrimidin-4(5H)-one scaffold
a,b
% of Inhibition @ 100 µM or IC50
R1
R2
Compound
38
IDO1
IDO2
TDO
c
n.a.
16 ± 4%
n.a.
n.a.
IC50
,
39
11 ± 7%
22 ± 2 µM
IC50
127 ± 5 µM
,
40
41
16 ± 5%
n.a.
n.a.
32 ± 1%
n.a.

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Table 2. Cont.
a,b
% of Inhibition @ 100 µM or IC50
R1
R2
Compound
42
IDO1
IDO2
TDO
d
15 ± 4%
n.a.
n.d.
n.d.
n.a.
n.d.
43
44
n.d.
n.a.
n.a.
45
n.a.
n.a.
n.a.
46
47
14 ± 3%
34 ± 1%
n.a.
n.a.
n.a.
n.a.
IC50
75 ± 4 µM
,
48
n.a.
10 ± 6%
IC50
96 ± 5 µM
,
49
50
51
52
n.a.
n.a.
n.a.
10 ± 2%
n.a.
n.a.
IC50
53 ± 3 µM
,
n.a.
23 ± 5%
n.a.
n.a.
n.a.
IC50
40 ± 2 µM
,
53
21 ± 6%
a
IC50 values are the means of at least two independent determinations, presented as mean
±
SD; b Positive control: epacadostat (
1),
IC50 values obtained for IDO1, IDO2, and TDO were 53, 580, and 9620 nM, respectively; c n.a., not active; d n.d., not determined.

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Additionally, to determine IDO1 selectivity, all final compounds were assayed for their
inhibitory potencies towards hIDO2 and hTDO. For this purpose, two new protocols were
developed based on the published fluorescence assay for hIDO1 [25]. Sodium ascorbate,
methylene blue, and bovine catalase were used in the same concentrations as for hIDO1.
50 mM phosphate buffers with pH values of 7.5 and 7.0 were used for hIDO2 and hTDO,
respectively, as has been already mentioned before [30–32]. First, the activities of hIDO2
and hTDO were determined at four different concentrations. Enzyme concentrations of
600 nM and 200 nM for hIDO2 and hTDO, respectively, exhibited sufficient enzyme activity
that could be measured in the assay. Additionally, the effect of DMSO concentration on
enzyme activity was investigated. Up to 5%, v/v of DMSO did not show any effect on the
assay performance. The optimal hIDO2 and hTDO assay concentrations of the substrate
L-Trp were determined to be 4 mM and 500
potency of epacadostat ( ) was determined, with IC50 values of 580 nM and 9620 nM for
hIDO2 and hTDO, respectively, corresponding to those published in the literature (see
Figure S1) [32 33]. All compounds tested were found to be selective for hIDO1, since none
µM, respectively. To validate both assays, the
1
,
of them exhibited significant inhibition of hIDO2 or hTDO.
All active compounds were additionally evaluated on a cellular level, starting with
the determination of cytotoxicity on Hep G2 cell line using the MTS assay (Figure 3), where
inner salt of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium is converted to formazan product, its quantity is directly proportional to
the number of living cells. Since the majority of compounds were only partially soluble in
a cell medium at concentrations above 100 µM, their effect on cell viability was measured
at 50
against Hep G2 cells at 50 µM. Compounds 39 and 49 significantly impacted cell viability
even at 10 M, especially 39 was thus not considered as a perspective inhibitor despite
µM, 10 µM, and 1 µM. Most of the compounds displayed little or no cytotoxicity
µ
its potent IDO1 inhibition in an enzymatic assay. The most promising compound 23 was
further evaluated in the cell-based IDO1 inhibition assay on HeLa cells, where no activity
was detected, possibly due to poor physicochemical properties, such as solubility in the
culture medium. Taken together, the results obtained from cellular tests reminded us of
the necessity to consider the physicochemical properties of compounds even at the early
stages of design.
Figure 3. Cytotoxicity of active compounds on Hep G2 cell line was evaluated at three different
concentrations. Data are represented as mean values ± standard deviation.

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To predict the binding mode of our inhibitors based on isoxazolo[5,4-d]pyrimidin-
4(5H)-one scaffold we selected 23 as the most promising compound (since it showed
potent hIDO1 activity with IC50 of 23 µM, no activity on hIDO2 and hTDO, and satisfying
cytotoxicity profile) and docked it into the active site of IDO1 (Figure 4), where in addition
to a coordinate bond between heme iron and the oxygen atom in the isoxazole ring, H-
bonds,
compare the binding mode of 23 to the one of the most potent inhibitors from clinical trials,
the superposition of 23 and epacadostat ( ) was also performed (Figure 4c). In accordance
with interactions with Cys129 via fluorine and bromine atoms on the phenyl ring of
π-π stacking and hydrophobic interactions (Figure 4a) were also observed. To
1
1,
which were observed in the literature [34], a similar interaction between fluorine atom on
the phenyl ring of 23 and Cys129 can be predicted.
Figure 4. Comparison of predicted binding mode for 23, crystal structure of IDO1 with
1 and
superposition of and 23. ( ) Compound 23 (purple) was docked into the active site of IDO1
1
a
(pdb code 5WN8, cyan) with protein ligand docking software GOLD 2020.0 (CCDC, UK). Possible
interactions formed were additionally predicted with Protein-Ligand Interaction Profiler [35] and
are: a coordinate bond (orange) with heme iron; H-bonds (blue) with Gly262 and Ala264;
π-π
stacking (grey) with Tyr126 and Phe163 and several hydrophobic interactions (not shown) with
lipophilic milieu. 4-fluorophenyl ring of 23 was positioned similarly to 3-bromo-4-fluorophenyl ring
of
1
; therefore, we additionally predicted another interaction (olive) between halogen and Cys129.
) Crystal structure of (orange) with IDO1 (cyan, 5WN8) with reported interactions: a coordinate
bond (orange) with heme iron; H-bonds (blue) with Ala264 and Arg231; stacking and cation-
interaction (both grey) between heme and , and interaction between halogens of F and Br with
(
b
1
π-
π
π
1
Cys129. (c) Superposition of 1 (orange) and 23 (purple) shows similar binding into the active site of
IDO1 (heme, grey; amino acid residues responsible for interactions, cyan).
3. Materials and Methods
3.1. Chemistry and Chemical Characterization of Compounds
The reagents and solvents were purchased from commercial sources (Sigma-Aldrich,
Acros Organics, Alfa Aesar, TCI) and used without further purification. Monitoring of
the reactions was done by thin-layer chromatography on silica-gel plates (Merck DC
Fertigplatten Kieselgel 60 GF254) and visualized under UV light and/or stained with the
relevant reagents. Flash column chromatography was performed on Merck silica gel 60
(mesh size, 70–230), using the indicated solvents. Yields refer to the purified products and
were not optimized. All of the melting points were determined on a Reichert hot-stage
1
apparatus, and are uncorrected. H and 13C NMR spectra were recorded at 295 K in CDCl3,
DMSO-d6, (CD3)2CO, or MeOD on a Bruker Avance III NMR spectrometer equipped
1
with a Broadband decoupling inverse H probe. The coupling constants (J) are in Hz,
and the splitting patterns are designated as s, singlet; br s, broad singlet; d, doublet; dd,
double doublet; td, triple doublet; t, triplet; dt, double triplet; ddd, double of doublet of
doublet; and m, multiplet. Mass spectra and high-resolution mass measurements were
performed at the Faculty of Pharmacy, University of Ljubljana, on ADVION Expression
CMSL mass spectrometer (Advion Inc., Ithaca, NY, USA) and ExactiveTM Plus Orbitrap
mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA), respectively. The

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analytical purities of the assayed compounds were
≥
95%, unless stated otherwise, as
1
◦
determined by H NMR and HPLC analyses. HPLC analyses were run at 50 C on a
Thermo Scientific Dionex UltiMate 3000 System (Thermo Fisher Scientific Inc., Waltham,
MA, USA) equipped with a quaternary pump and a multiple wavelength detector. An
Agilent Extend-C18 column was used (4.6 mm
×
150 mm, 3.6 µm), with a flow rate of
1.0 mL/min, injection volume of 5 L, detection at 254 nm, and an eluent system of A, 0.1%
µ
aqueous TFA; B, CH3CN. The following gradients were applied: 0–12 min, 10%
→
90% B;
12–14 min 90% B; 14–15 min, 90% → 10% B.
Analytical data of synthesized compounds as well as representative 1H and 13C NMR
spectra are available in the Supplementary Materials related to this manuscript.
3.2. General Procedure for the Synthesis of Aldehyde Oximes (6a–k)
Appropriate aldehyde 5a
–k (1.0 equiv.) was added to a stirred solution of hydroxy-
lamine HCl (1.1 equiv.) in THF-EtOH-H2O (2:5:1, v/v), with the mixture being stirred at
×
room temperature for approximately 30 min. THF and EtOH were then evaporated under
reduced pressure. The remaining residue was extracted with Et2O, brine, and dried over
anhydrous Na2SO4. Solvents were removed under reduced pressure to yield the product
(6a–k), which was used in the next step without further purification.
3.3. General Procedure for the Synthesis of N-Hydroxyimidoyl Chlorides (7a–k)
Appropriate aldehyde oxime (6a–k) (1.0 equiv.) and NCS (1.0 equiv.) were dissolved
in anhydrous DMF, then the reaction mixture was stirred at room temperature overnight
(16 h). The next day, the mixture of n-Hex/Et2O (2:1, v/v) was added and extracted with
H2O. The organic phase was washed with brine, dried over anhydrous Na2SO4, and the
solvents were removed under reduced pressure to yield the N-hydroxyimidoyl chloride
(7a–k), which was immediately used in the next step without any purification.
3.4. General Procedure for the Synthesis of 5-Aminoisoxazole-4-Carboxamides (8a–k)
3.4.1. Procedure I
A fresh solution of NaOEt in EtOH, prepared at room temperature from sodium
(1.5 equiv.) and absolute EtOH (~40 equiv.), was ad◦ded dropwise to a stirred solution of
2-cyanoacetamide (1.0 equiv.) in absolute EtOH at 50 C. The resulting suspension was then
cooled in an ice bath, and the solution of N-hydroxyimidoyl chloride (7a–k) (1.0 equiv.) in
EtOH was added dropwise. The mixture was stirred for an additional 30 min at 0 ◦C and
then refluxed for 18 h. After completion of the reaction, EtOH was removed under reduced
pressure. If possible, the resulting solid was washed with H2O and recrystallized from
MeOH to yield a crystalline solid (8a–k). Otherwise, the solid was dissolved in EtOAc,
washed with brine, and dried over anhydrous Na2SO4. EtOAc was removed under reduced
pressure and the remaining solid was purified using flash column chromatography with
gradient EtOAc/n-Hex as an eluent.
3.4.2. Procedure II
NaH (1.2 equiv.) was added to a stirred solution of 2-cyanoacetamide (1.0 equiv.) in
anhydrous DMF. After 15 min, a solution of appropriate N-hydroxyimidoyl chloride (7a–k)
in anhydrous DMF was added dropwise. The reaction mixture was stirred under an inert
atmosphere for 2 h at room temperature and then at 70 ◦C for 18 h. After completion of
the reaction H2O was added and extracted with EtOAc. Combined organic phases were
washed with brine, dried over anhydrous Na2SO4, and the solvents were removed under
reduced pressure to yield 5-aminoisoxazole-4-carboxamides (8a–k), which were used in
the next step without any purification.
3.5. General Procedure for the Synthesis of Isoxazolo[5,4-d]pyrimidin-4(5H)-ones (9a–k)
Isoxazolo[5,4-d]pyrimidin-4(5H)-ones (9a ) were synthesized from appropriate 5-
aminoisoxazole-4-carboxamide (8a ) (1.0 equiv.) and triethylorthoformate (1.0 equiv.)
–
k
–
k

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dissolved in Ac2O and refluxed for 18 h. After completion, the reaction mixture was cooled
down and stored in the fridge long enough for the precipitate to form, which was then
collected by filtration and used in the next step without further purification.
3.6. General Procedures for the N-Acylation (Preparation of Alkyl Halogenides 11a–y)
3.6.1. Procedure III
To a solution of appropriate aromatic amine 10d–e, 10i–y (1.0 equiv.) in glacial acid
(14.0 equiv.) chloroacetyl chloride (1.3 equiv.) was added dropwise. The reaction mixture
◦
was stirred at 10–15 C for 30 min. After the addition of sodium acetate (3.7 equiv.) solution
in H2O, the suspension was stirred at room temperature for 1.5 h. If the precipitate was
formed, it was collected by filtration and used in the next step without further purification.
Otherwise, EtOAc was added to the reaction mixture and extracted with H2O, brine, and
dried over anhydrous Na2SO4. Then the solvents were evaporated under reduced pressure
to yield a brown oily residue.
3.6.2. Procedure IV
To a solution of aliphatic amine 10a
–
c
(1.0 equiv.) in DCM, chloroacetyl chloride
◦
(1.3 equiv.) was added dropwise. The reaction mixture was stirred at 10–15 C for 2 h. After
completion, the reaction mixture was extracted with saturated NaHCO3 and brine. The
organic phase was dried over anhydrous Na2SO4 and the solvents were removed under
reduced pressure to yield an oily residue.
3.6.3. Procedure V
To a solution of appropriate aromatic amine 10f–h (1.0 equiv.) in anhydrous DMF on
the ice bath and under argon atmosphere bromoacetyl bromide (1.1 equiv.) in 1,4-dioxane
(6 mL) was added dropwise. The reaction mixture was then stirred overnight at room
temperature. The resulting precipitate was collected by filtration and used in the next step
without further purification.
3.7. General Procedure for the Synthesis of Final Compounds (12–53)
To a solution of appropriate isoxazolo[5,4-d]pyrimidin-4(5H]-one (9a–k) (1.0 eqiv.) in
anhydrous MeCN (10 mL) and anhydrous DMF (3 mL), NaH (60% dispersion in mineral
oil, 1.7 equiv.) and BTEAC (10%, m/m) were added and the reaction mixture was stirred at
0 ◦C for 15 min. KI (1.1 equiv.) and the selected alkyl halogenide (11a
–y, 1.3 equiv.) were
added at room temperature and the reaction mixture was stirred for 18 h. After completion,
the solvent was removed under reduced pressure, and the product was purified by flash
column chromatography with EtOAc/n-Hex as the eluent.
3.8. Cloning, Expression and Isolation of rhIDO1
Synthetic codon-optimized gene encoding human IDO1 residues 6-403 His-tagged at
the N-terminus (gBlock, IDT) was PCR amplified and subcloned into pET28 plasmid be-
tween the NcoI and XhoI restriction sites. A single bacterial colony of Escherichia coli NiCo21
harboring the expression vector was inoculated into 10 mL TB medium with 30
kanamycin and the culture was grown overnight at 37 C with vigorous shaking. The
µ
g/mL
◦
next day, the culture was diluted 1:100 in 200 mL TB medium with 30
and grown at 37 ◦C until OD600 reached ~2.5, at which point the cells were harvested by
centrifugation and resuspended in 500 mL pre-warmed medium composed of 1 M9 salts,
0.5% (wt/vol) D-glucose, 0.5% (wt/vol) casamino acids, 1 trace minerals, 1 mM MgSO4,
0.3 mM CaCl2, 1 g/mL thiamine, 1 g/mL biotin, 100 g/mL L-tryptophan, 30 g/mL
µg/mL kanamycin
×
×
µ
µ
µ
µ
kanamycin, and 1 mM aminolevulinic acid. The culture in a 3L baffled flask was shaken
◦
at 250 rpm at 37 C for 45 min, and cooled on ice for 15 min. The chilled culture was
supplemented with 0.5 mM isopropyl β-D-1-thiogalactopyranoside to induce recombinant
protein expression, and incubated at 20 ◦C for 28 h, shaking at 250 rpm. The cells were
harvested by centrifugation, washed with cold TBS buffer, and frozen at −80 ◦C.

Pharmaceuticals 2021, 14, 265
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The frozen cell paste was suspended in 10 volumes of cold IMAC buffer A (50 mM
HCl, pH 7.4; 300 mM KCl, 25 mM imidazole, 5% glycerol, 1 mM TCEP) and disrupted
by sonication (30% amplitude, 5 s pulses followed by 10 s pause) over 2 min. The lysate
Tris
·
was supplemented with 10
µ
g/mL DNase I, incubated on ice for 20 min, and centrifuged at
◦
10,000
×
g for 10 min at 4 C. The supernatant was loaded on a 1 mL HiTrap IMAC column
connected to ÄKTAexplorer 10 FPLC system (GE Healthcare, Wauwatosa, WI, USA) pre-
equilibrated with IMAC buffer A at 0.5 mL/min. Absorbance was monitored at 280 nm
and 404 nm. The column was washed at 1 mL/min with 15 mL IMAC buffer A, followed
by 10 mL 87.3% IMAC buffer A + 12.7% IMAC buffer B (50 mM Tris HCl, pH 7.4; 300 mM
·
KCl, 300 mM imidazole, 5% glycerol, 1 mM TCEP), and the intensely red recombinant
protein was eluted with IMAC buffer B. Eluates were immediately supplemented with
2 mM EDTA, combined and concentrated to 1 mL by ultrafiltration. Elution buffer was
exchanged for 20 mM Tris HCl, pH 8.0; 150 mM NaCl, 1 mM EDTA, 1 mM TCEP using a
·
5 mL HiTrap desalting column (GE Healthcare, Wauwatosa, WI, USA). The final rhIDO1
solution (3.5 mg/mL) was supplemented with 2 mM dithiotreitol, aliquoted and frozen at
−80 ◦C. SDS-PAGE analysis was performed to confirm a high level of rhIDO1 purity.
3.9. hIDO1 Inhibitory Potency Evaluation
The activity of the compounds on hIDO1 was determined according to the previously
reported fluorescence protocol [25]. Recombinant hIDO1 was prepared in-house as de-
scribed above, and bovine catalase was purchased from Sigma-Aldrich (Sigma-Aldrich,
St. Louis, MO, USA). Briefly, the reagents needed were dissolved in 50 mM potassium phos-
phate buffer (pH 6.5, 0.01%, v/v Tween 20), the reaction was performed in flat-bottomed,
black 96-well microplates. After the addition of 10
tralized with an equimolar amount of 1 M NaOH, 10
1 mg/mL bovine catalase, 2 L of DMSO (negative control) or solution of a compound
in DMSO, 50 L 20 nM rhIDO1 and 18 L 444 ML-Trp were added and the microplate
was incubated for 30 min at 37 ◦C. The reaction was quenched with the addition of 100
µL 100 mM sodium ascorbate neu-
µ
L 100 M methylene blue, 10
µ
µL
µ
µ
µ
µ
µL
400 mM piperidine solution in MilliQ water and the formation of the examined fluorophore
◦
began following incubation at 65 C for 20 min. The production of NFK-derived fluo-
rophore (λEX = 400 nm
H4; BioTek Instruments, Inc., Winooski, VT, USA) after 60 min post-incubation at room
temperature. In the preliminary testing compounds were tested at 100 M with activities
determined as residual activities—RA, which were calculated to % of inhibition, presented
in Tables 1 and 2 (% of inhibition = 100% RA). For compounds with RA values lower
than 50% (higher inhibition than 50% at 100 M), IC50 values were then calculated from
, λEM = 500 nm) was measured using a microplate reader (Synergy
µ
−
µ
determined RAs at 7 different concentrations with the help of GraphPad Prism 8 soft-
ware (GraphPad Software, San Diego, CA, USA). All determinations were carried out in
duplicates, with IC50 values defined with at least two independent determinations.
3.10. hIDO2 Inhibitory Potency Evaluation
Activity of the compounds on hIDO2 was determined according to the previously
reported fluorescence protocol for hIDO1 [25] and reported absorbance protocols for
hIDO2 [30,36] with several modifications. Recombinant hIDO2 was purchased from Vinci-
Biochem (Vinci-Biochem, Srl, Vinci, Italy), bovine catalase was purchased from Sigma-
Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Prior to the measurement of activity for
compounds, the enzyme activity was determined and optimal assay enzyme concentration
was selected. The amounts of reagents needed to perform the assay were optimized.
Briefly, the reagents needed were dissolved in 50 mM potassium phosphate buffer (pH 7.5,
0.01%, v/v Tween 20), the reaction was performed in black 384-well microplates. After the
addition of 1
NaOH, 1 L of 100
(negative control) or solution of a compound in DMSO, 5
of 26.7 mM L-Trp were added and the microplate was incubated for 60 min at 37 ◦C. The
µ
L of 100 mM sodium ascorbate neutralized with an equimolar amount of 1M
M methylene blue, 1 L of 1 mg/mL bovine catalase, 0.5 L of DMSO
L of 1.2 mM hIDO2 and 1.5 µL
µ
µ
µ
µ
µ

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reaction was quenched with the addition of 10
µ
L of 400 mM piperidine solution in MilliQ
◦
water and the formation of the examined fluorophore began following incubation at 65 C
for 20 min. The production of NFK-derived fluorophore (λEX = 400 nm, λEM = 500 nm)
was measured using a microplate reader (Synergy H4; BioTek Instruments, Inc., Winooski,
VT, USA) after 60 min post-incubation at room temperature. In the preliminary testing
compounds were tested at 100 µM with activities determined as residual activities—RA,
which were calculated to% of inhibition, presented in Tables 1 and 2. (% of inhibition =
100% − RA). All determinations were carried out in duplicates.
3.11. hTDO Inhibitory Potency Evaluation
The activity of the compounds on hTDO was determined according to the previously
reported fluorescence protocol for hIDO1 [25] and reported absorbance protocols for
hTDO [30] with several modifications. Recombinant hTDO was purchased from Vinci-
Biochem (Vinci-Biochem, Vinci, Italy), bovine catalase was purchased from Sigma-Aldrich
(Sigma-Aldrich, St. Louis, MO, USA). Prior to the measurement of activity for compounds,
the enzyme activity was determined, and optimal assay enzyme concentration was selected.
The amounts of reagents needed to perform the assay were optimized. Briefly, the reagents
needed were dissolved in 50 mM potassium phosphate buffer (pH 7.0, 0.01%, v/v Tween
20), the reaction was performed in black 384-well microplates. After the addition of 1
of 100 mM sodium ascorbate neutralized with equimolar amount of 1 M NaOH, 1 L of
100 M methylene blue, 1 L of 1 mg/mL bovine catalase, 0.5 L of DMSO (negative
control) or solution of a compound, 5 L of 300 nM hTDO and 1.5 L of 3.3 mM L-Trp
were added and the microplate was incubated for 60 min at 37 C. The reaction was
µL
µ
µ
µ
µ
µ
µ
◦
quenched with the addition of 10
µ
L of 400 mM piperidine solution in Mill◦iQ water and
the formation of the examined fluorophore began following incubation at 65 C for 20 min.
The production of NFK-derived fluorophore (λEX = 400 nm, λEM = 500 nm) was measured
using a microplate reader (Synergy H4; BioTek Instruments, Inc., Winooski, VT, USA)
after 60 min post-incubation at room temperature. In the preliminary testing compounds
were tested at 100 µM with activities determined as residual activities—RA, which were
calculated to% of inhibition, presented in Tables 1 and 2 (% of inhibition = 100%
determinations were carried out in duplicates.
−
RA). All
3.12. Cytotoxicity
Cytotoxicity of the compounds was determined on HepG2 cell line using the MTS cell
proliferation assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega,
Madison, WI, USA). The cells were seeded in 96-well plates (3
maintained in Dulbecco’s Modified Eagle’s Medium, supplemented with 10% fetal bovine
serum (both from Thermo Fisher Scientific, Waltham, MA, USA) and 100 g/mL gentamicin
×
104 cells/well) and
µ
(Sigma-Aldrich, St. Louis, MO, USA). Following overnight incubation in a humidified
chamber at 37 ◦C and 5% CO2 cells were treated with the compounds of interest at final
concentrations of 1
under the same conditions, 100
with 10 L of MTS solution. Following 3h incubation, the absorbance (λABS = 490 nm)
was measured using a microplate reader (Synergy H4; BioTek Instruments, Inc., Winooski,
µM, 10
µM, 50
µM, 100 µM, 150 µM, and 200 µM. After 24h incubation
µL of the medium from each well were collected and mixed
µ
VT, USA). The results are expressed as means of triplicates
independent experiments.
±
standard deviation of three
3.13. Cell-Based IDO1 Inhibitory Assay
HeLa cells were seeded in 96-well plates (2
Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum (both
from Thermo Fisher Scientific, Waltham, MA, USA) and 100 g/mL gentamicin (Sigma-
Aldrich, St. Louis, MO, USA). Following overnight incubation in a humidified chamber at
37 ◦C and 5% CO2 cells were treated with human IFN-
(50 ng/mL final concentration).
After 24h incubation cells were treated with compounds (epacadostat at 3 M to 0.3 nM was
×
104 cells/well) and maintained in
µ
γ
µ

Pharmaceuticals 2021, 14, 265
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used as a positive control) in a total volume of 200
overnight incubation, 140
96-well plate, mixed with 10
µ
L culture medium per well. Following
µ
L of the supernatant from each well were transferred to a new
µ
L of 6.1 N trichloroacetic acid and incubated at 50 ◦C for
30 min to hydrolize N-formylkynurenine, produced by uninhibited IDO1, to kynurenine.
The reaction mixture was then centrifuged for 10 min at 2500 rpm to remove sediments.
100
with 100
λABS = 480 nm) of the yellow color derivate formed after 10 min was measured using a
µL of supernatant from each well were transferred to a new 96-well plate and mixed
µL of 2%, w/v p-dimethylaminobenzaldehyde in acetid acid. The absorbance
(
microplate reader (Synergy H4; BioTek Instruments, Inc., Winooski, VT, USA).
3.14. Molecular Docking
Binding mode of compound 23 into the active site of IDO1 was predicted using the
protein-ligand docking software GOLD 2020.0 (CCDC, Cambridge, UK). Prior to the dock-
ing, crystal structure 5WN8, retrieved from the Protein Data Bank, was visually examined
in PyMOL 2.4.0 (Schrödinger, Munich, Germany) and subjected to crystal structure refine-
ment (removal of water molecules and the addition of hydrogen atoms). The parameters
used for docking are: the binding site was defined as all amino acid residues within 10 Å of
the bound ligand, with residues being kept rigid during the docking process; coordination
to the ferrous atom with default coordination geometry was included; the scoring function
used for the docking was CHEMPLP, with search efficiency set to 200%. Docking poses of
ligands were minimized using the MM2 force field implemented in Chem3D (PerkinElmer,
Waltham, MA, USA).
4. Conclusions
In summary, a series of isoxazolo[5,4-d]pyrimidin-4(5H)-one-based selective IDO1 in-
hibitors was synthesized in the optimized 6- or 7-step synthetic procedure. Forty-two final
compounds were obtained and key information about SARs of isoxazolo[5,4-d]pyrimidin-
4(5H)-one scaffold were provided. The chemistry-driven optimization afforded modi-
fications at two positions, on the left-hand (R1) and right-hand (R2) site of the central
heterocyclic core. In general, the optimal R1 substituent was found to be 4-fluorophenyl,
whereas most of the potent inhibitors contained p-substituted aniline as the R2 substituent,
with 20, 23, and 32 as best-in-class inhibitors with IC50 values between 22 µM and 27 µM.
The optimization of the fluorescence-based assay on hIDO2 and hTDO enabled the screen-
ing of all compounds against these two enzymes. None of the synthesized IDO1 inhibitors
showed any significant activity in hIDO2 and hTDO assay. Therefore, we propose that our
micromolar hIDO1-selective inhibitors with a novel chemical scaffold represent appealing
chemical probes for further development of potent immunomodulators.
Supplementary Materials: The following are available online at https://www.mdpi.com/1424-824
7/14/3/265/s1, Analytical data for synthesized compounds; Representative 1H NMR, 13C NMR and
HPLC spectra for compounds 20, 23, 32, 39, and 53; Determination of inhibitory potencies—Figure
S1: Inhibitory potencies of epacadostat on hIDO1, hIDO2, and hTDO, Figure S2: Inhibitory potencies
of most potent compounds 20, 23, 29, 32, and 39.
Author Contributions: Conceptualization, A.D. and M.S.; methodology, A.D., M.S., U.Š. and T.B.;
validation, A.D. and E.O.; formal analysis, A.D., M.S., U.Š. and T.B.; investigation, A.D. and L.M.;
resources, M.S. and S.G.; writing—original draft preparation, A.D. and T.B.; writing—review and
editing, M.S. and S.G.; supervision, M.S.; project administration, M.S. and S.G.; funding acquisition,
S.G. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Slovenian Research Agency (research core funding No.
P1-0208 and junior researcher’s program for A.D.).
Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing not applicable.

Pharmaceuticals 2021, 14, 265
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Acknowledgments: The authors thank Master students (Aljaž Abe, Eva Kožuh, Teja Novak, Eva
Poljanšek Bitenc, Špela Šmon) for their contribution in synthesis.
Conflicts of Interest: The authors declare no conflict of interest.
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