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

pharmaceuticals

Article

Novel Selective IDO1 Inhibitors with Isoxazolo[5,4-d]pyrimidin-

4(5H)-one Scaffold

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 IC₅₀values 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-

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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:

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://ww w .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.

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.

27 ± 4%

n.a.

IC₅₀

88 ± 4 µM

,

15

16

n.a.

15 ± 4%

28 ± 9%

17

18

19

33 ± 2%

32 ± 4%

n.a.

15 ± 1%

n.a.

IC₅₀

,

55 ± 2 µM

IC₅₀

27 ± 1 µM

,

20

21

n.a.

27 ± 3%

IC₅₀

55 ± 2 µM

,

22

23

24

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.

11 ± 4%

31 ± 3%

17 ± 2%

18 ± 4%

IC₅₀

30 ± 1 µM

,

29

n.a.

29 ± 3%

30

31

n.a.

17 ± 2%

IC₅₀

87 ± 3 µM

,

n.a.

IC₅₀

22 ± 1 µM

,

32

33

34

n.a.

22 ± 4%

n.a.

Pharmaceuticals 2021, 14, 265

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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.

IC₅₀

,

13 ± 2%

n.a.

46 ± 2 µM

37

17 ± 5%

n.a.

a

IC₅₀values are the means of at least two independent determinations, presented as mean

±

SD; ^bPositive

control: epacadostat (

1), IC₅₀values 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 R¹and 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 R¹and R²were 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 NaN₃in 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 (IC₅₀value 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 IC₅₀of 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

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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 R²fragment, originating from 12, was preserved in

most of the compounds. Interestingly, in combination with tetrahydroquinoline as R²sub-

stituent, the optimal R¹substituent 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 R²position 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 (IC₅₀of 53 µM) and 53 with tiophene-3-yl fragment (IC₅₀of 40 µM)

exhibited superior potency than their analog 25. Compound 39 with phenyl substituent as

R¹and methyl ester of p-aminobenzoic acid in the R²position retained a similar potency to

its 4-ﬂuorophenyl analog 20. Taken together, the R¹position is more liable to modiﬁcations,

when combined to p-substituted anilines with hydrogen bond acceptors as R²substituents.

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.

IC₅₀

,

39

11 ± 7%

22 ± 2 µM

IC₅₀

127 ± 5 µM

,

40

41

16 ± 5%

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.a.

n.d.

43

44

n.d.

n.a.

45

n.a.

46

47

14 ± 3%

34 ± 1%

n.a.

IC₅₀

75 ± 4 µM

,

48

n.a.

10 ± 6%

IC₅₀

96 ± 5 µM

,

49

50

51

52

n.a.

10 ± 2%

n.a.

IC₅₀

53 ± 3 µM

,

n.a.

23 ± 5%

n.a.

IC₅₀

40 ± 2 µM

,

53

21 ± 6%

a

IC₅₀values are the means of at least two independent determinations, presented as mean

±

SD; ^bPositive control: epacadostat (

1),

IC₅₀values obtained for IDO1, IDO2, and TDO were 53, 580, and 9620 nM, respectively; ^cn.a., not active; ^dn.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 IC₅₀values 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 IC₅₀of 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

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 Exactive^TMPlus 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 NaHCO₃and brine. The

organic phase was dried over anhydrous Na₂SO₄and 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 OD₆₀₀reached ~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

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

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 (λ_EX= 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), IC₅₀values were then calculated from

, λ_EM= 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 IC₅₀values 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

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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 (λ_EX= 400 nm, λ_EM= 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

◦

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 (λ_EX= 400 nm, λ_EM= 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 AQ_ueousOne 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

×

10⁴cells/well) and

µ

(Sigma-Aldrich, St. Louis, MO, USA). Following overnight incubation in a humidiﬁed

chamber at 37 ^◦C and 5% CO₂cells 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 (λ_ABS= 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% CO₂cells 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

×

10⁴cells/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

λ_ABS= 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 R¹substituent was found to be 4-ﬂuorophenyl,

whereas most of the potent inhibitors contained p-substituted aniline as the R²substituent,

with 20, 23, and 32 as best-in-class inhibitors with IC₅₀values 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://ww w .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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Article Doi

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

DOI: 10.3390/ph14030265

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Full text of DOI:10.3390/ph14030265

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