Synthesis of 5-[(1H-indol-3-yl)methyl]-1,3,4-oxadiazole-2(3H)-thiones and their protective activity against oxidative stress

Article abstract of DOI:10.1002/ardp.202100001

A small library of 2-[(1H-indol-3-yl)methyl]-5-(alkylthio)-1,3,4-oxadiazoles was prepared, starting from indole-3-acetic acid methyl ester and its 5-methyl-substituted derivative. The synthetic route involved the formation of intermediate hydrazides, their condensation with carbon disulfide, and intramolecular cyclization to corresponding 5-[(1H-indol-3-yl)methyl]-1,3,4-oxadiazole-2(3H)-thiones. The latter were then S-alkylated, and in case of ester derivatives, they were further hydrolyzed into corresponding carboxylic acids. All 5-[(1H-indol-3-yl)methyl]-1,3,4-oxadiazole-2(3H)-thiones and their S-alkylated derivatives were then screened for their protective effects in vitro and in vivo. Methyl substitution on the indole ring and propyl, butyl, or benzyl substitution on sulfhydryl group-possessing compounds were revealed to protect Friedreich's ataxia fibroblasts against the effects of glutathione depletion induced by the γ-glutamylcysteine synthetase inhibitor, buthionine sulfoximine. Two of the active compounds also reproducibly increased the survival of Caenorhabditis elegans exposed to juglone-induced oxidative stress.

Full text of DOI:10.1002/ardp.202100001

Received: 1 January 2021
Revised: 8 February 2021
Accepted: 12 February 2021
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DOI: 10.1002/ardp.202100001
F U L L P A P E R
Synthesis of 5‐[(1H‐indol‐3‐yl)methyl]‐1,3,4‐
oxadiazole‐2(3H)‐thiones and their protective activity
against oxidative stress
Monika Iškauskienė1
Vida Malinauskienė1
| Alena Kadlecová2
| Asta Žukauskaitė6
| Jiří Voller3
| Lucie Janovská4,5
| Algirdas Šačkus1
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1Department of Organic Chemistry, Kaunas University of Technology, Kaunas, Lithuania
2Department of Experimental Biology, Faculty of Science, Palacký University, Olomouc, Czech Republic
3Department of Clinical and Molecular Pathology, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University,
Olomouc, Czech Republic
4Department of Microbiology, Faculty of Medicine and Dentistry, Palacký University, Olomouc, Czech Republic
5Laboratory of Growth Regulators, Institute of Experimental Botany of the Czech Academy of Sciences & Faculty of Science, Palacký University, Olomouc, Czech
Republic
6Department of Chemical Biology, Faculty of Science, Palacký University, Olomouc, Czech Republic
Correspondence
Asta Žukauskaitė, Department of Chemical
Biology, Faculty of Science, Palacký University,
Šlechtitelů 27, CZ‐78371 Olomouc, Czech
Republic.
Abstract
A small library of 2‐[(1H‐indol‐3‐yl)methyl]‐5‐(alkylthio)‐1,3,4‐oxadiazoles was pre-
pared, starting from indole‐3‐acetic acid methyl ester and its 5‐methyl‐substituted
derivative. The synthetic route involved the formation of intermediate hydrazides,
their condensation with carbon disulfide, and intramolecular cyclization to corre-
sponding 5‐[(1H‐indol‐3‐yl)methyl]‐1,3,4‐oxadiazole‐2(3H)‐thiones. The latter were
then S‐alkylated, and in case of ester derivatives, they were further hydrolyzed into
corresponding carboxylic acids. All 5‐[(1H‐indol‐3‐yl)methyl]‐1,3,4‐oxadiazole‐
2(3H)‐thiones and their S‐alkylated derivatives were then screened for their pro-
tective effects in vitro and in vivo. Methyl substitution on the indole ring and propyl,
butyl, or benzyl substitution on sulfhydryl group‐possessing compounds were re-
vealed to protect Friedreich's ataxia fibroblasts against the effects of glutathione
depletion induced by the γ‐glutamylcysteine synthetase inhibitor, buthionine sul-
foximine. Two of the active compounds also reproducibly increased the survival of
Caenorhabditis elegans exposed to juglone‐induced oxidative stress.
Email: asta.zukauskaite@upol.cz
Vida Malinauskienė, Department of Organic
Chemistry, Kaunas University of Technology,
Radvilėnų pl. 19, LT‐50254 Kaunas, Lithuania.
Email: vida.malinauskiene@ktu.lt
Funding information
Ministry of Education, Youth and Sports of the
Czech Republic (program INTER‐COST),
Grant/Award Numbers: LTC18078, LTC19030;
European Regional Development Fund,
Grant/Award Number: CZ.02.1.01/0.0/0.0/
16_019/0000868
K E Y W O R D S
Caenorhabditis elegans, Friedreich's ataxia, indole, oxadiazole, oxidative stress
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1
INTRODUCTION
research for a variety of reasons: They can serve as a part of the
pharmacophore, as flat aromatic linkers, or as moieties modulating
Nitrogen‐containing compounds, such as oxadiazoles and indoles, are
of great importance, both medically and industrially. Oxadiazoles,
which exist in four isomeric forms, are used in drug discovery
molecular properties.[1] In recent years, interest in 1,3,4‐oxadiazoles
has especially risen[2] as apparent from their wide use in
development of antiparasitic,[3] antioxidant,[4–8] anticonvulsant,[9]
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Arch Pharm. 2021;e2100001.
wileyonlinelibrary.com/journal/ardp
© 2021 Deutsche Pharmazeutische Gesellschaft
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antimicrobial,[4] anticancer,[10–12] anti‐HIV,[10] anti‐inflammatory,[13]
disulfide was carried out in the presence of methanolic potassium
hydroxide solution overnight, followed by in situ acidification with
diluted hydrochloric acid to give rise to 1,3,4‐oxadiazole derivatives
5 and 6 in 72% and 63% overall yield, respectively (Scheme 1).
Subsequently, S‐alkylation of 1,3,4‐oxadiazole derivatives 5 and
6 was undertaken. Several cases of compound 5 alkylation reactions
have been reported using NaH[47] or TEA[45] as a base. In this study,
regardless of a halogen type in an alkylating agent, all alkylation
reactions were successfully accomplished at ambient temperature
using K2CO3 as a base giving the desired products 7–20 in high
yields (63–91%). Finally, using simple alkaline conditions, esters 15
and 20 were hydrolyzed into the corresponding carboxylic acids 21
and 22 in 71% and 47% yield, respectively (Scheme 2 and Table 1).
[14]
analgesic,
and antitubercular agents[15] as well as α‐glucosidase
inhibitors.[16,17] Moreover, 1,3,4‐oxadiazole heterocycles are good
bioisosteres of esters and amides, which can substantially contribute
to hydrogen‐bonding interactions with receptors.[18]
However, indole is one of the most important interspecies and
interkingdom signaling molecules.[19] It is built into proteins in the
form of the amino acid tryptophan and it provides the skeleton to
neurotransmitters serotonin and melatonin,[20] as well as a number
of phytoconstituents such as the plant hormone auxin,[21] per-
fumes,[22] and indole alkaloids.[23,24] Moreover, it is a common
structural motif in a variety of natural or synthetic biologically active
or otherwise relevant compounds, for instance, antioxidants,[25–28]
anticancer agents,[29–31] photosensitizers for photodynamic ther-
apy,[32–34] dyes,[35,36] colorimetric sensors,[37–39] or fluorescent
probes.[40–42]
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2.2
Biology
Therefore, it might be of advantage to design compounds that
would offer the opportunity of combining indole and 1,3,4‐
oxadiazole systems. It is already known that 5‐alkyl/aryl‐substituted
indole‐containing 1,3,4‐oxadiazoles I demonstrated antifungal activ-
ity,[43] whereas amino bridge‐bearing analogs II showed antic-
onvulsant activity.[44] Biological activities of analogous sulfur‐bridged
compounds are understudied, with the exception of several mild
antifungal[45] and antibacterial[46] activity‐possessing compounds as
well as anticancer activity‐possessing 2‐[(1H‐indol‐3‐yl)methyl]‐5‐
[(ethoxymethyl)thio]‐1,3,4‐oxadiazole.[47] To fill this gap, in the pre-
sent work, we present a fast and effective route toward a collection
of new sulfur bridge‐containing 1,3,4‐oxadiazoles III, starting from
indole‐3‐acetic acid (IAA) esters and assessment of their biological
activities (Figure 1).
Production of free oxygen radicals is an inevitable consequence of a
normal aerobic cellular metabolism.[56] Reactive oxygen species (ROS)
play a complex role in the organism—for example, they act as essential
signaling molecules or influence epigenetic regulation of gene expres-
sion.[57] However, excessive amounts of ROS can be detrimental and
oxidative stress is a hallmark of many age‐related or hereditary diseases,
including cancer, diabetes, neurodegenerative disorders, and so forth.[58]
Compounds that have the ability to reduce oxidative stress can, there-
fore, find application in medicine.[59,60]
As discussed above, both indoles[25–28] and 1,3,4‐oxadiazoles[4–8]
are known as antioxidants, and 2‐(indol‐2‐yl)‐1,3,4‐oxadiazoles were
also reported to possess antioxidant properties.[61] Moreover, com-
pound 16 emerged in a recent high‐throughput screening whose
purpose was to find potential treatments to a rare hereditary mi-
tochondrial disease, Friedreich's ataxia (FA) (Voller et al., un-
published). FA fibroblasts are known to be sensitive to oxidative
stress.[62] Inspired by these results, we decided to evaluate the
protective effect of the compounds against chemically induced oxi-
dative stress in a model nematode Caenorhabditis elegans and in fi-
broblasts derived from patients suffering from FA.
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
Besides the prominent application in plant sciences, IAA and its de-
rivatives are frequently used as key intermediates in the synthesis of
drugs and other biologically relevant substances, that is, in-
domethacin,[48] tivantinib,[49] various paullones,[50] and so forth. For
our study, IAA methyl ester 1 and its 5‐methyl substituted derivative
2, which are both commercially available or readily preparable,[51]
were chosen as starting materials. There are several methods de-
scribed in literature of ester 1 conversion to hydrazide 3 in 80–90%
yields by treating it with hydrazine hydrate in methanol[52] or
ethanol.[31,45,53–55] Mamdouh et al.[45,47] showed that hydrazide 3
can be reacted with carbon disulfide in the presence of ethanolic
potassium hydroxide, followed by acidification with dilute hydro-
chloric acid to give the 1,3,4‐oxadiazoles derivative 4. In this study, a
similar path has been chosen with minor changes. Esters 1 and 2
were reacted with hydrazine hydrate in methanol to yield inter-
mediate hydrazides 3 and 4, which were used in the next step
without purification. Reaction of hydrazides 3 and 4 with carbon
FIGURE 1 Biologically relevant indole and 1,3,4‐oxadiazole
motifs combining compounds

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SCHEME 1 Synthesis of the intermediate 5‐[(1H‐indol‐3‐yl)methyl]‐1,3,4‐oxadiazole‐2(3H)‐thione derivatives 5 and 6
SCHEME 2 Synthesis of the alkylated 5‐[(1H‐indol‐3‐yl)methyl]‐1,3,4‐oxadiazole‐2‐thiol derivatives 7–22
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2.2.1
Toxic and protective effects in
5–22, their toxicity was evaluated by chitinase assay and microscopic
evaluation.[64] In the initial screening, all compounds, 5–22, were
tested at 20 and 200 μM concentrations. These concentrations were
selected on the basis of the solubility of the compounds in the test
medium. The standard anthelmintic drug ivermectin was used as a
positive control. Five of the screened compounds, namely 7, 8, 9, 13,
and 19, reduced the signal in the chitinase assay (quantifying egg
hatching) by at least 60% at 200 μM concentration (Figure S1) and
were thus further tested to more precisely assess their toxicity to
C. elegans (Table S1). Microscopic evaluation revealed that the
compounds did not kill the worms even in the highest concentrations
tested but negatively influenced the overall health of the popula-
tions, delaying the worms’ development and reducing their re-
productive capacity. It is worth noting that even the most toxic
compound 9 had only a mild effect on the growth and development
of the worms even at 200 µM concentration (Figure S2).
vivo—evaluation in Caenorhabditis elegans
C. elegans is a free‐living soil nematode and a popular model organism
in various biological fields, owing to, among other features, its small
size, transparency, rapid life cycle, and short lifespan.[63] Before the
evaluation of the protective capacity of the prepared compounds
TABLE 1 Overview of alkylated 5‐[(1H‐indol‐3‐yl)methyl]‐1,3,4‐
oxadiazole‐2‐thiols 7–20
Entry
R
R1
X
Yield (%)
7 (71)
1
2
3
4
5
6
7
8
H
H
H
H
H
H
H
H
I
I
8 (66)
Br
Br
Br
Br
I
9 (88)
10 (65)
11 (77)
12 (79)
13 (63)
14 (90)
C. elegans is also frequently used to model and study various
aspects of human diseases[65] and is routinely used for drug
screenings.[66] The worm is a eukaryote; therefore, a significant
portion of its genes have homologs in humans. These include
many disease‐relevant genes and pathways.[67] Owing to the
worms' small size and the possibility to cultivate them in liquid‐
based media, C. elegans enables whole‐organism in vivo high‐
throughput approaches.
Br
9
H
Br
15 (91)
The ability of compounds 5–22 to increase survival of nema-
todes under oxidative stress, induced by exposure to 500 µM of
the ROS generator 5‐hydroxy‐1,4‐naphthoquinone (juglone),[68]
was evaluated by measurement of worms’ motility using wMi-
croTracker. Curcumin (100 µM), a compound with a well‐described
protective effect in worms,[69,70] was used as a positive control.
The concentrations selected for this experiment were 5 and
50 µM, as in these concentrations, none of the compounds showed
toxic effect in the chitinase assay (Table S1). Worms were
10
11
12
13
Me
Me
Me
Me
Br
Br
Br
Br
16 (90)
17 (85)
18 (68)
19 (64)
14
Me
Br
20 (83)

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2.2.2
Toxic and protective effects in FA fibroblasts
FA is a rare mitochondrial disease affecting the heart and nervous
system due to a homozygotic mutation in the gene for frataxin (FXN),
a protein essential for iron–sulfur cluster assembly. Consequently,
aconitase and several proteins of the electron transport chain are
affected, as reviewed by Bürk.[72] Unfortunately, the therapies for FA
are currently limited to the treatment of the symptoms.
Initially, we assessed the cytoprotective effect of the compounds
5–22 on skin fibroblasts GM03816 and GM04078 derived from FA
patients against glutathione synthesis inhibitor L‐buthionine sulfox-
imine (BSO) at 10 and 50 µM concentrations. Protocols based on a
similar principle were used by Richardson et al.[73] as well as
Richardson and Simpkins.[74] An 8‐h pretreatment with compounds
5–22, followed by a 24‐h exposure to 100 µM BSO and a subsequent
resazurin test, was performed. These pilot experiments (data not
shown) revealed the activity of compounds 16, 17, and 19, which
were then subjected to experiments in a wider concentration range.
Figure 3 shows a dose‐dependent protective activity of compounds
16, 17, and 19 against BSO, which was used at 10 and 30 µM con-
centrations for GM04078 and GM30816, respectively. It is worth
noting that the protective activity of compound 19 was comparable
with that of R‐lipoic acid.
FIGURE 2 The effect of the compounds on the motility (as a
marker of survival) of Caenorhabditis elegans populations exposed to
500 µM of the reactive oxygen species generator juglone (measured
by wMicroTracker). Each dot represents an average value from an
independent experiment. Horizontal bars designate the average
values
pretreated with the compounds for 3 days, after which the com-
pounds were washed away, and the stressor was added. Six
compounds, namely, 7, 10, 11, 13, 17, and 19 were able to increase
the survival of worms by more than 5% at 5 µM concentration in
three independent experiments (Figure 2 and Table S2). The fact
that some of these compounds showed a minor toxic effect to the
worms in our previous assays and performed better at the lower
concentration in the stress assay suggests a possible biphasic dose
response typical for hormetins.[71]
To get an initial estimation of the therapeutic window, the
toxicity of the active compounds 16, 17, and 19 was tested not only
against FA fibroblasts but also against several other noncancer hu-
man cell lines, that is, skin fibroblasts BJ, retinal pigment epithelial
cells ARPE‐19, and keratinocytes HaCaT, using resazurin test after
48‐h exposure. The test compounds did not have any or only mar-
ginal toxic effects up to 30 µM. However, toxicity was apparent at
FIGURE 3 Protective effects of the selected compounds against L‐buthionine sulfoximine (BSO) in Friedreich's ataxia fibroblasts. The
concentrations of the compounds during 6‐h pretreatment are shown. In the 24‐h BSO treatment, the compound concentrations were
two‐thirds of the initial value due to dilution after BSO addition. Error bars are SEM. C−, control without BSO, C+, control with BSO, LA, R‐lipoic acid

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60 µM, the highest concentration tested (Figure S3). However,
samples. High‐resolution mass spectrometry (HRMS) spectra were re-
corded with a micrOTOF‐Q III Bruker spectrometer in electron spray
ionization mode.
R‐lipoic acid was not toxic at this concentration (data not shown).
The InChI codes of the investigated compounds, together
with some biological activity data, are provided as Supporting
Information.
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3
CONCLUSION
A small library of 2‐[(1H‐indol‐3‐yl)methyl]‐5‐(alkylthio)‐1,3,4‐oxadiazoles
was prepared, starting from indole‐3‐acetic acid methyl ester and its
5‐methyl substituted derivative via formation of corresponding inter-
mediate hydrazides. The immediate base‐mediated treatment of crude
hydrazides with carbon disulfide, followed by in situ acidification, gave
rise to corresponding 5‐[(1H‐indol‐3‐yl)methyl]‐1,3,4‐oxadiazole‐2(3H)‐
thione derivatives. The latter were then S‐alkylated and in the case of
ester derivatives and further hydrolyzed into corresponding carboxylic
acids. All 5‐[(1H‐indol‐3‐yl)methyl]‐1,3,4‐oxadiazole‐2(3H)‐thiones and
their S‐alkylated derivatives were screened for their protective effects in
vitro and in vivo. Three of the prepared compounds, namely, 16, 17, and
19, protected FA fibroblasts against glutathione depletion induced by γ‐
glutamylcysteine synthetase inhibitor BSO. It is worth noting that all
active compounds share methyl substitution on the indole ring and a
hydrophobic alkyl or benzyl substitution on the sulfhydryl group. Two of
the active compounds, namely 17 and 19, also reproducibly increased the
survival of C. elegans exposed to juglone‐induced oxidative stress. How-
ever, subsequent toxicity evaluation on a panel of human cells showed
that compounds 16, 17, and 19 decrease the viability of cultures at
60 µM concentration. Therefore, their possible use in FA, where cell
homeostasis is severely perturbed at multiple levels, requires caution.
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4.1.2
General procedure for the synthesis of
1,3,4‐oxadiazole‐2(3H)‐thiones 5 and 6
To a solution of 1 or 2 (8 mmol) in MeOH (16 ml), 56% aqueous
.
NH2NH2 H2O solution (8 ml) was added, and the resulting mixture
was stirred at reflux for 16 h. Upon completion, the reaction mixture
was poured into water (80 ml) and extracted with EtOAc (3 × 40 ml).
The combined organic layers were washed with brine (40 ml), dried
over anhydrous Na2SO4, filtered, and concentrated under reduced
pressure to give the corresponding hydrazide 3 or 4, which were
used for the next step without purification. To a solution of isolated
compound 3 or 4 in MeOH (20 ml), freshly ground KOH (762 mg,
13.6 mmol) and CS2 (1.1 ml, 18.4 mmol) were added and the reaction
mixture was stirred at room temperature (rt) for 1 h, followed by
refluxing for additional 16 h. Upon completion, the reaction mixture
was cooled to rt, aqueous HCl solution (HCl/H2O 1:4 v/v, 28 ml) was
added, and stirring was continued at rt for an additional 1 h. The
reaction mixture was poured into water (60 ml) and extracted with
EtOAc (3 × 40 ml). The combined organic layers were washed with
brine (40 ml), dried over anhydrous Na2SO4, filtered, and con-
centrated under reduced pressure. The residue was purified by col-
umn chromatography on silica gel using dichloromethane methanol
(DCM/MeOH) (100:1 v/v).
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4
EXPERIMENTAL
Chemistry
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4.1
5‐[(1H‐Indol‐3‐yl)methyl]‐1,3,4‐oxadiazole‐2(3H)‐thione (5),
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4.1.1
General
previously reported by Song et al.[45]
Yield 1.33 g (72%), yellow solid. Rf = 0.43 (DCM/MeOH 20:1 v/v). 1
H
The reagents and solvents were purchased from commercial suppliers
and used without further purification, unless otherwise indicated. The
purification of the reaction mixtures was performed using flash chro-
matography on a glass column with silica gel (high‐purity grade, Merck
grade 9385, pore size 60 Å, 230–400 mesh particle size). For thin‐layer
chromatography (TLC), Merck precoated TLC plates (Silica gel 60 F254)
were employed. The melting points (mp) were determined on a MEL‐
TEMP (capillary melting point apparatus) and were uncorrected. The 1H
NMR (nuclear magnetic resonance) and 13C NMR spectra (see the
Supporting Information) were recorded in deuterated dimethyl sulf-
oxide (DMSO‐d6) solutions at 25°C on a Bruker Avance III 400 in-
strument (400 MHz for 1H, 100 MHz for 13C). The chemical shifts,
expressed in ppm, were relative to tetramethylsilane. The assignments
of the 1H and 13C NMR resonances were achieved using standard
Bruker software and a combination of standard NMR spectroscopic
techniques, such as distortionless enhancement by polarization transfer
and heteronuclear single quantum coherence. The infrared (IR) spectra
were recorded on a Bruker Vertex 70 v FT‐IR spectrometer using neat
NMR (400 MHz, DMSO‐d6): δ 4.21 (s, 2H, CH2), 7.01 (t, J = 7.5 Hz,
1H, CH), 7.11 (t, J = 7.5 Hz, 1H, CH), 7.35 (d, J = 1.5 Hz, 1H, CH), 7.38
(d, J = 8.1 Hz, 1H, CH), 7.51 (d, J = 7.9 Hz, 1H, CH), 11.08 (br s, 1H,
NH), and 14.33 (br s, 1H, NH). 13C NMR (100 MHz, DMSO‐d6): δ 14.7
(CH3), 21.9 (CH2), 107.1 (C), 112.1 (CH), 118.7 (CH), 119.2 (CH),
121.8 (CH), 124.7 (CH), 127.1 (C), 136.7 (C), 164.6 (C), and 167.5 (C).
IR (neat, νmax, cm−1): 3393 (NH). HRMS (electrospray Ionization time‐
of‐flight [ESI TOF]): for [C11H9N3OS+Na]+ [M+Na]+: Calcd. 254.0359,
found 254.0359.
5‐[(5‐Methyl‐1H‐indol‐3‐yl)methyl]‐1,3,4‐oxadiazole‐2(3H)‐
thione (6)
Yield 1.23 g (63%), yellowish solid, mp 188–189°C. Rf = 0.26 (DCM/
MeOH 20:1 v/v). 1H NMR (400 MHz, DMSO‐d6): δ 2.36 (s, 3H, CH3),
4.17 (s, 2H, CH2), 6.93 (d, J = 8.3 Hz, 1H, CH), 7.25–7.28 (m, 2H,
2 × CH), 10.94 (br s, 1H, NH), and 14.33 (br s, 1H, NH). 13C NMR
(100 MHz, DMSO‐d6): δ 21.3 (CH3), 21.7 (CH2), 105.2 (C), 111.4 (CH),
117.6 (CH), 123.0 (CH), 124.5 (CH), 126.8 (C), 127.3 (C), 134.5 (C),

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163.5 (C), and 177.8 (C). IR (neat, νmax, cm−1): 3359 (NH). HRMS (ESI
TOF): for [C12H11N3OS+Na]+ [M+Na]+: Calcd. 268.0515, found
268.0515.
124.2 (CH), 126.6 (C), 136.2 (C), 163.2 (C), and 167.0 (C). IR (neat,
ν
max, cm−1): 3316 (NH). HRMS (ESI TOF): for [C14H15N3OS+Na]+ [M
+Na]+: Calcd. 296.0828, found 296.0828.
5‐(Butylthio)‐2‐[(1H‐indol‐3‐yl)methyl]‐1,3,4‐oxadiazole (10),
previously reported by Song et al.[45]
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4.1.3
General procedure for the alkylation of
1,3,4‐oxadiazole‐2‐thiones 5, 6 with alkyl halides
Purified by column chromatography on silica gel with Hex/acetone
(4:1 v/v). Yield 93 mg (65%), brownish solid, mp 78–80°C. Rf = 0.38
(Hex/acetone 3:1 v/v). 1H NMR (400 MHz, DMSO‐d6): δ 0.84 (t,
J = 7.4 Hz, 3H, CH3), 1.19–1.42 (m, 2H, CH2), 1.55–1.83 (m, 2H, CH2),
3.16 (t, J = 7.3 Hz, 2H, SCH2), 4.34 (s, 2H, CH2), 7.00 (t, J = 7.4 Hz, 1H,
CH), 7.10 (t, J = 7.5 Hz, 1H, CH), 7.34 (d, J = 1.3 Hz, 1H, CH), 7.38 (d,
J = 8.1 Hz, 1H, CH), 7.51(d, J = 7.9 Hz, 1H, CH), and 11.05 (br s, 1H,
NH). 13C NMR (100 MHz, DMSO‐d6): δ 13.8 (CH3), 21.5 (CH2), 21.9
(CH2), 31.5 (CH2), 32.1 (SCH2), 107.1 (C), 112.1 (CH), 118.6 (CH),
119.2 (CH), 121.8 (CH), 124.7 (CH), 127.1 (C), 136.7 (C), 163.8 (C),
and 167.5 (C). IR (neat, νmax, cm−1): 3281 (NH). HRMS (ESI TOF): for
[C15H17N3OS+Na]+ [M+Na]+: Calcd. 310.0984, found 310.0985.
To a solution of 5 or 6 (0.5 mmol) in DMF (3 ml), an appropriate alkyl
halide (0.55 mmol) and freshly ground K2CO3 (83 mg, 0.6 mmol) were
added, and the resulting mixture was stirred at rt for 16 h. Upon
completion, the reaction mixture was poured into water (20 ml) and
extracted with EtOAc (3 × 20 ml). The combined organic layers were
washed with brine (20 ml), dried over anhydrous Na2SO4, filtered,
and concentrated under reduced pressure. The residue was purified
by column chromatography using an appropriate eluent.
2‐[(1H‐Indol‐3‐yl)methyl]‐5‐(methylthio)‐1,3,4‐oxadiazole (7)
Purified by column chromatography on silica gel with Hex/EtOAc
(4:1 v/v). Yield 87 mg (71%), yellowish solid, mp 114–115°C. Rf =
0.26 (Hex/EtOAc 2:1 v/v). 1H NMR (400 MHz, DMSO‐d6): δ 2.65 (s,
3H, CH3), 4.34 (s, 2H, CH2), 7.01 (t, J = 7.4 Hz, 1H, CH), 7.11 (t,
J = 7.5 Hz, 1H, CH), 7.35 (d, J = 1.9 Hz, 1H, CH), 7.38 (d, J = 8.1 Hz,
1H, CH), 7.51 (d, J = 7.9 Hz, 1H, CH), and 11.06 (br s, 1H, NH). 13C
NMR (100 MHz, DMSO‐d6): δ 14.7 (CH3), 21.9 (CH2), 107.1 (C),
112.1 (CH), 118.7 (CH), 119.2 (CH), 121.8 (CH), 124.7 (CH), 127.1
(C), 136.7 (C), 164.6 (C), and 167.5 (C). IR (neat, νmax, cm−1): 3277
(NH). HRMS (ESI TOF): for [C12H11N3OS+Na]+ [M+Na]+: Calcd.
268.0515, found 268.0515.
2‐[(1H‐Indol‐3‐yl)methyl]‐5‐[(2‐methoxyethyl)thio]‐1,3,4‐
oxadiazole (11)
Purified by column chromatography on silica gel with Hex/acetone
(3:1 v/v). Yield 111 mg (77%), brownish solid, mp 83–84°C. Rf = 0.27
(Hex/acetone 3:1 v/v). 1H NMR (400 MHz, DMSO‐d6): δ 3.21 (s, 3H,
CH3), 3.37 (t, J = 6.0 Hz, 2H, SCH2), 3.60 (t, J = 6.0 Hz, 2H, OCH2),
4.34 (s, 2H, CH2), 7.01 (t, J = 7.4 Hz, 1H, CH), 7.11 (t, J = 7.5 Hz, 1H,
CH), 7.34 (s, 1H, CH), 7.38 (d, J = 8.1 Hz, 1H, CH), 7.51 (d, J = 7.9 Hz,
1H, CH), and 11.06 (br s, 1H, NH). 13C NMR (100 MHz, DMSO‐d6): δ
21.9 (CH2), 32.1 (SCH2), 58.3 (OCH3), 70.2 (OCH2), 107.1 (C), 112.1
(CH), 118.6 (CH), 119.2 (CH), 121.8 (CH), 124.7 (CH), 127.1 (C),
136.7 (C), 163.7 (C), and 167.6 (C). IR (neat, νmax, cm−1): 3266 (NH).
HRMS (ESI TOF): for [C14H15N3O2S+Na]+ [M+Na]+: Calcd. 312.0777,
found 312.0777.
5‐(Ethylthio)‐2‐[(1H‐indol‐3‐yl)methyl]‐1,3,4‐oxadiazole (8)
Purified by column chromatography on silica gel with DCM. Yield 85 mg
(66%), brownish amorphous solid. Rf = 0.26 (DCM/MeOH 20:1 v/v). 1H
NMR (400 MHz, DMSO‐d6): δ 1.32 (t, J = 7.3 Hz, 3H, CH3), 3.17 (q,
J = 7.3 Hz, 2H, SCH2), 4.33 (s, 2H, CH2), 7.00 (t, J = 7.4 Hz, 1H, CH), 7.10
(t, J = 7.5 Hz, 1H, CH), 7.33 (d, J = 2.0 Hz, 1H, CH), 7.37 (d, J = 8.1 Hz, 1H,
CH), 7.50 (d, J = 7.9 Hz, 1H, CH), and 11.04 (br s, 1H, NH). 13C NMR
(100 MHz, DMSO‐d6): δ 14.8 (CH3), 21.5 (CH2), 26.5 (SCH2), 106.7 (C),
111.6 (CH), 118.2 (CH), 118.8 (CH), 121.4 (CH), 124.2 (CH), 126.6 (C),
136.2 (C), 163.2 (C), and 167.1 (C). IR (neat, νmax, cm−1): 3267 (NH).
HRMS (ESI TOF): for [C13H13N3OS+Na]+ [M+Na]+: Calcd. 282.0672,
found 282.0672.
2‐({5‐[(1H‐Indol‐3‐yl)methyl]‐1,3,4‐oxadiazol‐2‐yl}thio)ethanol (12)
Purified by column chromatography on silica gel with DCM/MeOH
(100:1 v/v). Yield 652 mg (79%), brown resin. Rf = 0.28 (DCM/MeOH
100:5 v/v). 1H NMR (400 MHz, DMSO‐d6): δ 3.27 (t, J = 6.2 Hz, 2H,
SCH2), 3.66 (q, J = 6.0 Hz, 2H, OCH2), 4.32 (s, 2H, CH2), 5.11 (t,
J = 5.5 Hz, 1H, OH), 7.00 (t, J = 7.4 Hz, 1H, CH), 7.10 (t, J = 7.5 Hz, 1H,
CH), 7.33 (d, J = 2.1 Hz, 1H, CH), 7.38 (d, J = 8.1 Hz, 1H, CH), 7.50 (d,
J = 7.9 Hz, 1H, CH), and 11.04 (br s, 1H, NH). 13C NMR (100 MHz,
DMSO‐d6): δ 21.5 (CH2), 34.9 (SCH2), 59.3 (OCH2), 106.7 (C), 111.6
(CH), 118.2 (CH), 118.8 (CH), 121.4 (CH), 124.2 (CH), 126.6 (C),
136.2 (C), 163.5 (C), and 167.0 (C). IR (neat, νmax, cm−1): 3276 (NH,
OH). HRMS (ESI TOF): for [C13H13N3O2S+H]+ [M+H]+: Calcd.
276.0801, found 276.0801.
2‐[(1H‐Indol‐3‐yl)methyl]‐5‐(propylthio)‐1,3,4‐oxadiazole (9)
Purified by column chromatography on silica gel with DCM/MeOH
(100:1 v/v). Yield 721 mg (88%), brownish solid, mp 73–74°C. Rf =
0.20 (DCM/MeOH 50:1 v/v). 1H NMR (400 MHz, DMSO‐d6): δ 0.92
(t, J = 7.3 Hz, 3H, CH3), 1.61–1.72 (m, 2H, CH2), 3.14 (t, J = 7.1 Hz, 2H,
SCH2), 4.33 (s, 2H, CH2), 7.00 (t, J = 7.4 Hz, 1H, CH), 7.09 (t,
J = 7.5 Hz, 1H, CH), 7.33 (d, J = 2.0 Hz, 1H, CH), 7.37 (d, J = 8.1 Hz, 1H,
CH), 7.50 (d, J = 7.9 Hz, 1H, CH), and 11.04 (br s, 1H, NH). 13C NMR
(100 MHz, DMSO‐d6): δ 12.8 (CH3), 21.6 (CH2), 22.4 (CH2), 33.8
(CH2), 106.7 (C), 111.6 (CH), 118.2 (CH), 118.8 (CH), 121.3 (CH),
5‐[(3‐Fluoropropyl)thio]‐2‐[(1H‐indol‐3‐yl)methyl]‐1,3,4‐
oxadiazole (13)
Purified by column chromatography on silica gel with Hex/acetone
(3:1 v/v). Yield 92 mg (63%), brown solid, mp 59–61°C. Rf = 0.29
(Hex/acetone 3:1 v/v). 1H NMR (400 MHz, DMSO‐d6): δ 1.90–2.20

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(m, 2H, CH2), 3.27 (t, J = 7.2 Hz, 2H, SCH2), 4.34 (s, 2H, CH2), 4.45 (t,
J = 5.7 Hz, 1H, FCHaHb), 4.45 (t, J = 5.7 Hz, 1H, FCHaHb), 7.01 (t,
J = 7.4 Hz, 1H, CH), 7.11 (t, J = 7.5 Hz, 1H, CH), 7.35 (s, 1H, CH), 7.39
(d, J = 8.1 Hz, 1H, CH), 7.51 (d, J = 7.8 Hz, 1H, CH), and 11.06 (br s,
1H, NH). 13C NMR (100 MHz, DMSO‐d6): δ 21.9 (CH2), 28.5 (d, 3J
2‐(Butylthio)‐5‐[(5‐methyl‐1H‐indol‐3‐yl)methyl]‐1,3,4‐
oxadiazole (17)
Purified by column chromatography on silica gel with DCM. Yield
128 mg (85%), brown amorphous solid. Rf = 0.50 (DCM/MeOH 20:1
v/v). 1H NMR (400 MHz, DMSO‐d6): δ 0.84 (t, J = 7.4 Hz, 3H, CH3),
1.24–1.41 (m, 2H, CH2), 1.57–1.67 (m, 2H, CH2), 2.35 (s, 3H, CH3),
3.16 (t, J = 7.3 Hz, 2H, SCH2), 4.28 (s, 2H, CH2), 6.92 (dd, J = 8.3,
0.9 Hz, 1H, CH), 7.23–7.29 (m, 3H, 3 × CH), and 10.90 (br s, 1H, NH).
13C NMR (100 MHz, DMSO‐d6): δ 13.3 (CH3), 21.0 (CH3), 21.3 (CH2),
21.4 (CH2), 31.0 (CH2), 31.6 (SCH2), 106.1 (C), 111.3 (CH), 117.7
(CH), 123.0 (CH), 124.2 (CH), 126.8 (C), 127.2 (C), 134.6 (C), 163.3
(C), and 167.1 (C). IR (neat, νmax, cm−1): 3265 (NH). HRMS (ESI TOF):
for [C16H19N3OS+Na]+ [M+Na]+: Calcd. 324.1141, found 324.1141.
(C,F) = 5.2 Hz, SCH2CH2), 30.3 (d, 2J(C,F) = 19.8 Hz, SCH2), 82.6 (d, 1
J
(C,F) = 162.9 Hz, FCH2), 107.1 (C), 112.1 (CH), 118.6 (CH), 119.2
(CH), 121.8 (CH), 124.7 (CH), 127.1 (C), 136.7 (C), 163.5 (C), and
167.6 (C). IR (neat, νmax, cm−1): 3286 (NH). HRMS (ESI TOF): for
[C14H14FN3OS+Na]+ [M+Na]+: Calcd. 314.0734, found 314.0734.
5‐(Benzylthio)‐2‐[(1H‐indol‐3‐yl)methyl]‐1,3,4‐oxadiazole (14),
previously reported by Song et al.[45]
Purified by column chromatography on silica gel with DCM/MeOH
(100:1 v/v). Yield 144 mg (90%), brownish solid, mp 121–122°C.
Rf = 0.16 (DCM/MeOH 100:2 v/v). 1H NMR (400 MHz, DMSO‐d6): δ
4.33 (s, 2H, CH2), 4.40 (s, 2H, SCH2), 7.00 (t, J = 7.4 Hz, 1H, CH), 7.10
(t, J = 7.2 Hz, 1H, CH), 7.19–7.24 (m, 3H, 3 × CH), 7.26–7.28 (m, 2H,
2 × CH), 7.32 (d, J = 2.2 Hz, 1H, CH), 7.38 (d, J = 8.1 Hz, 1H, CH), 7.49
(d, J = 7.9 Hz, 1H, CH), and 11.05 (br s, 1H, NH). 13C NMR (100 MHz,
DMSO‐d6): δ 21.5 (CH2), 35.8 (SCH2), 106.6, 111.6 (CH), 118.2 (CH),
118.8 (CH), 121.3 (CH), 124.2 (CH), 126.6 (C), 127.7 (CH), 128.5
(2 × CH), 128.9 (2 × CH), 136.2 (C), 136.5 (C), 162.7 (C), 167.3 (C),
and 169.3 (C═O). IR (neat, νmax, cm−1): 3274 (NH). HRMS (ESI TOF):
for [C18H15N3OS+Na]+ [M+Na]+: Calcd. 344.0828, found 344.0827.
2‐({5‐[(5‐Methyl‐1H‐indol‐3‐yl)methyl]‐1,3,4‐oxadiazol‐2‐yl}thio)‐
ethanol (18)
Purified by column chromatography on silica gel with DCM/MeOH
(100:1 v/v). Yield 98 mg (68%), brownish solid, mp 90–91°C. Rf = 0.13
(DCM/MeOH 20:1 v/v). 1H NMR (400 MHz, DMSO‐d6): δ 2.36 (s, 3H,
CH3), 3.27 (t, J = 6.2 Hz, 2H, SCH2), 3.66 (q, J = 5.9 Hz, 2H, OCH2),
4.28 (s, 2H, CH2), 5.10 (t, J = 5.0 Hz, 1H, OH), 6.92 (d, J = 8.3 Hz, 1H,
CH), 7.24–7.28 (m, 3H, 3 × CH), and 10.90 (br s, 1H, NH). 13C NMR
(100 MHz, DMSO‐d6): δ 21.3 (CH3), 21.4 (CH2), 34.9 (SCH2), 59.3
(OCH2), 106.1 (C), 111.3 (CH), 117.7 (CH), 123.0 (CH), 124.2 (CH),
126.8 (C), 127.2 (C), 134.6 (C), 163.4 (C), and 167.0 (C). IR (neat, νmax
,
cm−1): 3244 (NH, OH). HRMS (ESI TOF): for [C14H15N3O2S+Na]+ [M
+Na]+: Calcd. 312.0777, found 312.0777.
Methyl 2‐({5‐[(1H‐indol‐3‐yl)methyl]‐1,3,4‐oxadiazol‐2‐yl}thio)‐
acetate (15)
Purified by column chromatography on silica gel with DCM/MeOH
(100:1 v/v). Yield 138 mg (91%), white solid, mp 106–107°C. Rf = 0.65
(DCM/MeOH 20:1 v/v). 1H NMR (400 MHz, DMSO‐d6): δ 3.62 (s, 3H,
CH3), 4.17 (s, 2H, SCH2), 4.33 (s, 2H, CH2), 7.00 (t, J = 7.4 Hz, 1H, CH),
7.10 (t, J = 7.5 Hz, 1H, CH), 7.33 (d, J = 2.1 Hz, 1H, CH), 7.37 (d, J = 8.1 Hz,
1H, CH), 7.50 (d, J = 7.9 Hz, 1H, CH), and 11.05 (br s, 1H, NH). 13C NMR
(100 MHz, DMSO‐d6): δ 21.4 (CH2), 33.5 (SCH2), 52.6 (CH3), 106.5 (C),
111.6 (CH), 118.2 (CH), 118.8 (CH), 121.4 (CH), 124.2 (CH), 126.6 (C),
136.2 (C), 162.4 (C), 167.3 (C), and 168.1 (C═O). IR (neat, νmax, cm−1):
1731 (C═O) and 3293 (NH). HRMS (ESI TOF): for [C14H13N3O3S+H]+ [M
+H]+: Calcd. 304.0750, found 304.0750.
2‐(Benzylthio)‐5‐[(5‐methyl‐1H‐indol‐3‐yl)methyl]‐1,3,4‐
oxadiazole (19)
Purified by column chromatography on silica gel with Hex/EtOAc (4:1 v/
v). Yield 107 mg (64%), brown resin. Rf = 0.43 (DCM/MeOH 20:1 v/v). 1H
NMR (400 MHz, DMSO‐d6): δ 2.35 (s, 3H, CH3), 4.29 (s, 2H, CH2), 4.40 (s,
2H, SCH2), 6.91–6.96 (m, 1H, CH), 7.19–7.24 (m, 3H, 3 × CH), 7.24–7.30
(m, 5H, 5 × CH), and 10.92 (br s, 1H, NH). 13C NMR (100 MHz, DMSO‐
d6): δ 21.3 (CH3), 21.4 (CH2), 35.8 (SCH2), 106.0 (C), 111.3 (CH), 117.7
(CH), 123.0 (CH), 124.3 (CH), 126.8 (C), 127.2 (C), 127.7 (CH), 128.5
(2 × CH), 128.9 (2 × CH), 134.6 (C), 135.5 (C), 162.6 (C), and 167.3 (C). IR
(neat, νmax, cm−1): 3265 (NH). HRMS (ESI TOF): for [C19H17N3OS
+Na]+ [M+Na]+: Calcd. 358.0985, found 358.0984.
2‐[(5‐Methyl‐1H‐indol‐3‐yl)methyl]‐5‐(propylthio)‐1,3,4‐
oxadiazole (16)
Methyl 2‐({5‐[(5‐methyl‐1H‐indol‐3‐yl)methyl]‐1,3,4‐oxadiazol‐2)‐
yl}thio)acetate (20)
Purified by column chromatography on silica gel with DCM/MeOH
(20:1 v/v). Yield 129 mg (90%), brown solid, mp 77–78°C (methanol).
Rf = 0.29 (DCM/MeOH 20:1 v/v). 1H NMR (400 MHz, DMSO‐d6): δ
0.92 (t, J = 7.3 Hz, 3H, CH3), 1.62–1.73 (m, 2H, CH2), 2.35 (s, 3H,
CH3), 3.14 (t, J = 7.3 Hz, 2H, SCH2), 4.28 (s, 2H, CH2), 6.92 (d,
J = 8.2 Hz, 1H, CH), 7.23–7.29 (m, 3H, 3 × CH), and 10.90 (br s, 1H,
NH). 13C NMR (100 MHz, DMSO‐d6): δ 12.7 (CH3), 21.3 (CH3), 21.4
(CH2), 22.4 (CH2), 33.8 (SCH2), 106.1 (C), 111.3 (CH), 117.7 (CH),
123.0 (CH), 124.2 (CH), 126.8 (C), 127.2 (C), 134.6 (C), 163.3 (C), and
167.1 (C). IR (neat, νmax, cm−1): 3267 (NH). HRMS (ESI TOF): for
[C15H17N3OS+Na]+ [M+Na]+: Calcd. 310.0985, found 310.0985.
Purified by column chromatography on silica gel Hex/EtOAc (2:1 v/v).
Yield 132 mg (83%), brownish solid, mp 85–86°C. Rf = 0.48 (DCM/MeOH
20:1 v/v). 1H NMR (400 MHz, DMSO‐d6): δ 2.36 (s, 3H, CH3), 3.62 (s, 3H,
OCH3), 4.17 (s, 2H, CH2), 4.29 (s, 2H, SCH2), 6.92 (d, J = 8.3 Hz, 1H, CH),
7.23–7.30 (m, 3H, 3 × CH), and 10.91 (br s, 1H, NH). 13C NMR (100 MHz,
DMSO‐d6): δ 21.7 (CH3), 21.9 (CH2), 34.0 (SCH2), 53.1 (OCH3), 106.4 (C),
111.8 (CH), 118.2 (CH), 123.4 (CH), 124.7 (CH), 127.3 (C), 127.7 (C),
135.0 (C), 162.9 (C), 167.8 (C), and 168.6 (C═O). IR (neat, νmax, cm−1):
1732 (C═O), 3311 (NH). HRMS (ESI TOF): for [C15H15N3O3S+Na]+ [M
+Na]+): Calcd. 340.0726, found 340.0726.

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4.1.4
General procedure for the hydrolysis of
under standard cell culture conditions (37°C, 5% CO2, humid environ-
ment) and subcultured two or three times a week.
1,3,4‐oxadiazol‐2‐yl‐thioacetates 15 and 20
To a solution of a compound 15 or 20 (0.3 mmol) in dioxane (4 ml)
and water (1 ml), LiOH (22 mg, 0.3 mmol) was added, and the re-
sulting mixture was stirred at rt for 2 h. Upon completion, the re-
action mixture was diluted with 5% KHSO4 aqueous solution (20 ml)
and extracted with EtOAc (3 × 20 ml). The combined organic layers
were washed with brine (20 ml), dried over anhydrous Na2SO4, fil-
tered, and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel with DCM/MeOH
(10:1 v/v).
|
4.2.2
Cytotoxicity evaluation in human cell lines
The effect of 48 h treatments with the compounds on cell viability was
evaluated using a resazurin reduction assay.[75] The cells were trypsinized
and diluted in DMEM (or DMEM/F12 in case of ARPE‐19) with 10% FBS.
About 1500 cells in 30 μl of culture medium were seeded into the wells
of a 384‐well plate. After 24 h incubation, the cells were treated by the
test compounds dissolved in DMSO using an Echo 555 system (Labcyte).
The final concentrations were 15, 30, and 60 μM. Five technical re-
plicates were used for each condition. The control wells were treated
with a DMSO vehicle (16 replicates). After another 48 h, 15 μl of a three‐
fold concentrated solution of resazurin (Sigma) in the culture medium
was added (final concentration of 0.0125 mg/ml). Fluorescence was read
after 3‐h incubation using an M2 reader (Biotek) with excitation and
emission wavelengths of 570 and 610 nm, respectively.
2‐({5‐[(1H‐Indol‐3‐yl)methyl]‐1,3,4‐oxadiazol‐2‐yl}thio)acetic
acid (21)
Yield 63 mg (73%), brown solid, mp 119–120°C. Rf = 0.01 (DCM/
MeOH 10:1 v/v). 1H NMR (400 MHz, DMSO‐d6): δ 4.09 (s, 2H, CH2),
4.33 (s, 2H, SCH2), 7.00 (t, J = 7.5 Hz, 1H, CH), 7.10 (t, J = 7.5 Hz, 1H,
CH), 7.32 (d, J = 1.6 Hz, 1H, CH), 7.37 (d, J = 8.1 Hz, 1H, CH), 7.50 (d,
J = 7.9 Hz, 1H, CH), and 11.04 (br s, 1H, NH). 13C NMR (100 MHz,
DMSO‐d6): δ 21.9 (CH2), 34.6 (SCH2), 107.0 (C), 112.1 (CH), 118.7
(CH), 119.3 (CH), 121.8 (CH), 124.6 (CH), 127.0 (C), 136.6 (C), 163.2
(C), 167.6 (C), and 169.3 (C═O). IR (neat, νmax, cm−1): 1700 (C═O),
3298 (NH, OH). HRMS (ESI TOF): for [C13H11N3O3S+Na]+ [M+Na]+:
Calcd. 312.0413, found 312.0413.
|
4.2.3
Fibroblast stress assays
Fibroblasts GM04078 and GM03816 were trypsinized, diluted in DMEM
medium with 10% FBS, and seeded into 384‐well plates (1500 cells in
30 µl medium/well). After 24 h incubation, the cells were pretreated by
the test compounds dissolved in DMSO using an Echo 555 system
(Labcyte). The final concentrations were in the range of 0.31–10 µM. At
least five technical replicates were used for each condition.
2‐({5‐[(5‐Methyl‐1H‐indol‐3‐yl)methyl]‐1,3,4‐oxadiazol‐2‐yl}thio)‐
acetic acid (22)
Yield 43 mg (47%), brown solid, mp 68–69°C. Rf = 0.03 (DCM/MeOH
100:5 v/v). 1H NMR (400 MHz, DMSO‐d6): δ 2.36 (s, 3H, CH3), 3.97
(s, 2H, CH2), 4.26 (s, 2H, SCH2), 6.92 (d, J = 8.2 Hz, 1H, CH),
7.24–7.28 (m, 3H, 3 × CH), and 10.92 (br s, 1H, NH). 13C NMR
(100 MHz, DMSO‐d6): δ 21.3 (CH3), 21.4 (CH2), 36.6 (SCH2), 106.1
(C), 111.4 (CH), 117.7 (CH), 123.0 (CH), 124.2 (CH), 126.9 (C), 127.2
Control wells were treated with DMSO vehicles (16 replicates). R‐
lipoic acid was used as a positive control. BSO was added in 15 µl of the
DMEM medium with 10% FBS after 8 h. After another 48 h, 15 μl of a
four‐fold concentrated solution of resazurin (Sigma‐Aldrich) in the culture
medium was added (final concentration of 0.0125 mg/ml). Fluorescence
was read after 3‐h incubation using an M2 reader (Biotek) with excitation
and emission wavelengths of 570 and 610 nm, respectively.
(C), 134.6 (C), 163.6 (C), 166.8 (C), and 170.0 (C═O). IR (neat, νmax
,
cm−1): 1714 (C═O), 3398 (NH, OH). HRMS (ESI TOF): for
[C14H13N3O3S+H]+ [M+H]+): Calcd. 304.0750, found 304.0750.
|
4.2.4
C. elegans strains and maintenance
|
4.2
Pharmacological/biological assays
The C. elegans strains used in this study were wild‐type N2 (Bristol)
and BA17 (fem‐1) obtained from the Caenorhabditis genetic center
(CGC). Worms were maintained on nematode growth media plates
seeded with Escherichia coli OP50 (obtained from CGC) under stan-
dard cultivation conditions.[76,77]
|
4.2.1
Origin and maintenance of human cell lines
Skin fibroblasts (BJ) and retinal pigment epithelium cells (ARPE‐19)
were obtained from the American Type Culture Collection. HaCaT
keratinocytes were obtained from the German Cancer Research
Center (DKFZ). Skin fibroblasts GM04078 and GM03816 homo-
zygous for the GAA expansion in the FXN gene were obtained from
the Coriell repository.
|
4.2.5
Toxicity evaluation in C. elegans
The cells were maintained in standard Dulbecco's modified Eagle's
medium (DMEM) (BJ, HaCaT, GM04078, GM03816) or DMEM/F12
(ARPE‐19) (Sigma) supplemented with fetal bovine serum (10%), glu-
tamine (2 mM), penicillin (100 U/ml), and streptomycin (100 μg/ml)
The toxicity of the compounds was tested in wild‐type N2 (Bristol)
Caenorhabditis elegans worms via a chitinase assay as well as by visual
evaluation of the worms under a microscope.[64] In short, worms were
age‐synchronized by sodium hypochlorite treatment and eggs in S

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medium were left to hatch overnight on a shaker. Starved L1 larvae were
diluted to a final concentration of 200–300 worms/ml, fed with 3 mg/ml
bacteria, and distributed on 96‐well plates. Larvae were then treated
with the test compounds or vehicle alone (DMSO). Standard anthelmintic
drug ivermectin was used as positive control. Populations were left to
grow at 20°C for 4 days, in which time healthy worms would reach
adulthood and start reproducing. The experimental plates were then
exposed to 20 µM fluorogenic chitinase substrate (4‐methylumbelliferyl
β‐D‐N,N′,N″‐triacetylchitotrioside) and incubated at 37°C for 1 h, after
which the reaction was stopped by the addition of alkaline buffer (1 M
glycine/1 M NaOH, pH 10.6), and the intensity of the fluorescence was
measured on a plate reader. Before the assay, the plates were visually
checked under a microscope and abnormalities, including toxicity or
developmental impairments, were noted. Initial screening of the com-
pounds was performed at 200 and 20 µM concentrations. Compounds
showing toxic effect in the initial tests were then retested over a wider
concentration range. IC50 values were calculated using GraphPad Prism.
All experiments were performed at least three times.
Czech Republic (program INTER‐COST, grant number LTC18078
and LTC19030) and by the European Regional Development Fund
(Project ENOCH, No. CZ.02.1.01/0.0/0.0/16_019/0000868).
CONFLICTS OF INTEREST
The authors declared there are no conflicts of interest.
ORCID
Monika Iškauskienė
https://orcid.org/0000-0002-7946-6780
https://orcid.org/0000-0003-2200-3977
Alena Kadlecová
Jiří Voller
https://orcid.org/0000-0001-7378-8941
Lucie Janovská
https://orcid.org/0000-0001-6198-892X
https://orcid.org/0000-0002-1342-9629
https://orcid.org/0000-0001-6759-8789
https://orcid.org/0000-0002-1514-4934
Vida Malinauskienė
Asta Žukauskaitė
Algirdas Šačkus
REFERENCES
[1] J. Boström, A. Hogner, A. Llinàs, E. Wellner, A. T. Plowright, J. Med.
Chem. 2012, 55, 1817.
For taking the photographs, worms were cultivated analogously
as for the chitinase assay. Worms were then washed three times with
M9 buffer to remove the bacteria and were anesthetized with 5 mM
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4.2.6
Oxidative stress assays in C. elegans
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The authors are grateful to Tomáš Jirsa and Kateřina Faksová for
their help with biological testing. The strains used in this study were
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How to cite this article: M. Iškauskienė, A. Kadlecová,
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