Design, synthesis, and biological evaluation of novel derivatives of dithiodiglycolic acid prepared via oxidative coupling of thiols

Article abstract of DOI:10.1080/14756366.2019.1575372

Human thioredoxin reductase 1 (TrxR1) is a selenocysteine-containing enzyme which plays a crucial role in regulating numerous redox signalling pathways within the cell. While its functioning is important in all cells, levels of TrxR1 expression are higher

Full text of DOI:10.1080/14756366.2019.1575372

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis, and biological evaluation of
novel derivatives of dithiodiglycolic acid prepared
via oxidative coupling of thiols
Olga Bakulina, Anton Bannykh, Mirna Jovanović, Ilona Domračeva, Ana
Podolski-Renić, Raivis Žalubovskis, Milica Pešić, Dmitry Dar’in & Mikhail
Krasavin
To cite this article: Olga Bakulina, Anton Bannykh, Mirna Jovanović, Ilona Domračeva, Ana
Podolski-Renić, Raivis Žalubovskis, Milica Pešić, Dmitry Dar’in & Mikhail Krasavin (2019) Design,
synthesis, and biological evaluation of novel derivatives of dithiodiglycolic acid prepared via
oxidative coupling of thiols, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 665-671,
DOI: 10.1080/14756366.2019.1575372
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 665–671
https://doi.org/10.1080/14756366.2019.1575372
SHORT COMMUNICATION
Design, synthesis, and biological evaluation of novel derivatives of dithiodiglycolic
acid prepared via oxidative coupling of thiols
a
a
c
c,d
ꢀb
ꢁ
ꢀb
ꢁ
Olga Bakulina , Anton Bannykh , Mirna Jovanovic , Ilona Domraceva , Ana Podolski-Renic , Raivis Zalubovskis
,
a
a
ꢁ ꢀb
Milica Pesic , Dmitry Dar’in and Mikhail Krasavin
a
b
ꢁ
ꢀ
Saint Petersburg State University, Saint Petersburg, Russian Federation; Institute for Biological Research “Sinisa Stankovic”, University of
c
d
Belgrade, Belgrade, Serbia; Latvian Institute of Organic Synthesis, Riga, Latvia; Faculty of Materials Science and Applied Chemistry, Institute of
Technology of Organic Chemistry, Riga Technical University, Riga, Latvia
ABSTRACT
ARTICLE HISTORY
Received 20 December 2018
Revised 21 January 2019
Accepted 22 January 2019
Human thioredoxin reductase 1 (TrxR1) is a selenocysteine-containing enzyme which plays a crucial role in
regulating numerous redox signalling pathways within the cell. While its functioning is important in all
cells, levels of TrxR1 expression are higher in cancer cells, possibly as an adaptation to much higher levels
of reactive oxygen species and the need for more extensive DNA synthesis. This makes TrxR1 an attractive
target for cancer therapy development. Inspired by the structure of disulphide compounds which have
advanced through various stages of clinical development, we designed a series of dithiodiglycolic acid
derivatives. These were prepared from respective thiol synthons using an iodine- or benzotriazolyl chlor-
ide-promoted oxidative disulphide bond formation. Inhibition of TrxR present in cell lysates from human
neuroblastoma cells (SH-SY5Y) and rat liver cells indicated several compounds with a potential for TrxR
inhibition. Some of these compounds were also tested for growth inhibition against two human cancer
cell lines and normal human keratinocytes.
KEYWORDS
TrxR; disulphide inhibitors;
dithiodiglycolic acid;
anticancer activity
Introduction
electrophilicity – Michael acceptors, disulphide and diselenide sub-
strate mimics, metal-based inhibitors as well as compounds of
miscellaneous structure – have been recently reviewed in compre-
hensive accounts by Bellelli5 and Fang6,7. Particularly relevant to
the present study are the disulphide substrate analogues: e.g. alkyl
2-imidazolyl disulphides 1–38 (2, whose Ki value toward the
enzyme was determined as 31 mM, is also known as PX-12 and has
been in Phase 1 clinical trials for advanced metastatic cancer and
was stopped due to safety concerns6), disodium 2,20-dithio-bis-
ethane sulphonate 49 (also known as TravoceptTM which advanced
through phase III clinical trials in patients with non-small cell lung
cancer and has a Km value of 72 mM6) as well as bicyclic disul-
phide-containing natural products chaetocin (5), gliotoxin (6) and
chaetomin (7) which have been shown to possess Km values of
4.6 mM, 16.9 mM, and 16.1 mM, respectively – and have demon-
strated a substrate-competitive inhibition profile toward TrxR1 and
Thioredoxin reductase enzymes (TrxR, EC 1.8.1.9), which are the
focus of this work, belong to thioredoxin system along with
NADPH and thioredoxin (Trx). The system is highly conserved
among species and responsible for regulating redox processes,
gene transcription, and protection against reactive oxygen species
(ROS)1. TrxR maintains Trx in its reduced bis-sulfhydryl state that,
in turn, interacts with antioxidant enzymes and transcription fac-
tors2. TrxR1 is a cytosolic isoform of the enzyme (along with mito-
chondrial TrxR2 and TrxR3 expressed primarily in the testes). TrxR
enzymes are overexpressed in cancer cells contributing to their
resistant phenotype characterised by higher levels of ROS3.
Targeting TrxR specifically with various, mostly electrophilic, inhibi-
tors have thus become an attractive new approach for the devel-
opment of anticancer therapy4.
The various types of inhibitors targeting the catalytic
selenocysteine residue of TrxR with varying degree of pro-apoptotic efficacy toward cancer cells10. Other disulphide
CONTACT Mikhail Krasavin
mkrasavin@hotmail.com
Saint Petersburg State University, Saint Petersburg 199034, Russian Federation
Supplemental data for this article can be accessed here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

666
O. BAKULINA ET AL.
Figure 1. Known disulphide TrxR inhibitors (1–7) and fluorescent probes (8–10) as well as compounds explored in this work (11–13).
compounds – 5,50-dithiobis(2-nitrobenzoic acid) (8 also known as trityl chloride according to literature procedure14. Dimethyl 2,20-
DTNB or Ellman’s reagent)11, thioredoxin reductase fluorogenic
disulfanediyldiacetate (17) was prepared by oxidative dimerisation
substrate (9 or TRFS-green)12 and mitochondria-targeted “mito-
TRFS” (10)13 have not been employed as TrxR inhibitors but rather
as fluorescent probes, also due to their being substrate (disul-
phide-linked Trx) analogues. Based on the structure of these disul-
phide TrxR ligands, we designed a series of symmetric (11) and
non-symmetric (12–13) derivatives of dithiodiglycolic acid (Figure
1). Herein, we report the results of their synthetic exploration and
biological testing for TrxR inhibition and cytotoxicity toward two
human cancer cell lines and normal human keratinocytes.
of methyl thioglycolate15. 1-Chlorobenzotriazole was prepared
according to known procedure16. DCM was distilled from P2O5
and stored over MS 4 Å. THF was distilled from sodium benzophe-
none under Ar. All synthesised thiols and disulphides are air-
sensitive, and therefore were stored in sealed vials at ꢀ20 ꢁC.
Characterisation data are provided only for one representative
compound in each group. The rest of the compounds are
described in the Supplementary Data.
General procedure 1: preparation of 2-(tritylthio)
acetamides 18a–l
Materials and methods
Chemical syntheses – general
To a solution of 2-(tritylthio)acetic acid 16 (1.5–4.5 mmol) in dry
dichloromethane (DCM; 5 ml/mmol) 1,10-carbonyldiimidazole (CDI;
1.0–1.1 equiv.) was added portionwise (gas evolution!). After stir-
ring for 50 min, amine (1.0–1.1 equiv.) was added and the reaction
mixture was left stirring at r.t. overnight. The reaction mixture was
washed with citric acid (2 N aq. solution), saturated aq. NaHCO3
and water. Organic layer was dried (over Na2SO4), filtered, and
concentrated in vacuo to give pure title amides.
1-(Pyrrolidin-1-yl)-2-(tritylthio)ethan-1-one (18a) was pre-
pared according to General Procedure 1 from 2-(tritylthio)acetic
acid (668 mg, 2 mmol), pyrrolidine (150 mg, 2.1 mmol) and CDI
(340 mg, 2.1 mmol). Yield 584 mg, 75%. White solid. 1H NMR
(400 MHz, CDCl3) d 7.57–7.43 (m, 6H), 7.37–7.28 (m, 6H), 7.28–7.17
(m, 3H), 3.40 (t, J ¼ 6.6 Hz, 2H), 3.07 (t, J ¼ 6.4 Hz, 2H), 2.93 (s, 2H),
NMR spectroscopic data were recorded with Bruker Avance 400
spectrometer (400.13 MHz for 1H and 100.61 MHz for 13C) and
Bruker Avance 500 spectrometer (500.03 for 1H and 125 MHz for
13C) in DMSO-d6 and in CDCl3 and were referenced to residual
solvent proton signals (dH ¼ 2.50 and 7.26 ppm, respectively) and
solvent carbon signals (dC ¼ 39.5 and 77.0 ppm, respectively). Mass
spectra were recorded with a Bruker Maxis HRMS-ESI-qTOF spec-
trometer (electrospray ionisation mode). Merck silica gel 60 mesh
was used for column chromatography. TLC was performed with
Macherey-Nagel Alugram Sil G/UV254 plates. Thioacetic acid, trityl
chloride, methyl thioglycolate, amines, and 1H-benzotriazole, 2,20-
disulfanediyldiacetic acid (14) and 2,20-disulfanediyldiacetic acid
disodium salt (15) were obtained from commercial sources. 2-
(Tritylthio)acetic acid (16) was prepared from thioacetic acid and 1.85–1.77 (m, 4H).13C NMR (101 MHz, CDCl3) d 166.8, 144.3, 129.6,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
667
128.0, 126.8, 66.8, 46.5, 46.0, 36.1, 26.0, 24.3. HRMS (ESI), m/z calcd
for C25H25NOSNa [M þ Na]þ 410.1549, found 410.1553.
General procedure 5: preparation of non-symmetric
disulphides 12(13)a
A mixture of 1-chlorobenzotriazole (BtCl; 1.1 equiv.) and 1H-benzo-
triazole (BtH; 1 equiv.) was placed into a Schlenk tube, which was
then evacuated and filled with argon three times followed by add-
ition of dry DCM (10 ml) and cooling to ꢀ78 ꢁC. First thiol
(0.5–0.6 mmol) dissolved in 5 ml DCM was added dropwise during
15–20 min to form a yellow solution. Then the reaction mixture
was slowly warmed to ꢀ20 ꢁC and the second thiol (1.1 equiv.)
dissolved in 5 ml DCM was added dropwise during 15–20 min. The
resulting solution was left stirring at 0 ꢁC for 30 min. The reaction
mixture was then quenched with aqueous Na2S2O3, washed with
saturated aq. NaHCO3 (20 ml) and extracted with DCM (3 ꢂ 50 ml).
Combines organic layer was dried over Na2SO4, filtered and con-
centrated to give crude material, which was purified by silica gel
column chromatography using DCM/MeOH mixture (100:1)
as eluent20.
General procedure 2: preparation of thiols 19a–c
To a stirred solution of protected amide 18a–c (2 mmol) in TFA
(5 ml) triethylsilane (TES; 1.1 equiv.) was added at room tempera-
ture. Reaction progress was monitored by TLC and reaction mix-
ture turned colourless from deep yellow. After 5–30 min, reaction
mixture was washed, diluted with water (15 ml), and extracted
with hexane (3 ꢂ 15 ml). The aqueous layer was then extracted
with DCM (3 ꢂ 15 ml). The combined organic layer was dried over
Na2SO4, filtered and concentrated to provide pure title
compounds17.
2-Mercapto-1-(pyrrolidin-1-yl)ethan-1-one (19a) was pre-
pared according to General Procedure 2 from 18a (774 mg,
2 mmol) and TES (254 mg, 2.2 mmol). Yield 178 mg, 61%. 1H NMR
(400 MHz, CDCl3) d 3.57 (q, J ¼ 7.3 Hz, 4H), 3.32 (s, 2H), 2.23 (s, 1H),
2.07 (p, J ¼ 6.8 Hz, 2H), 1.96 (p, J ¼ 6.8 Hz, 2H). 13C NMR (101 MHz,
CDCl3) d 170.7, 47.4, 47.0, 26.3, 25.9, 24.3. HRMS (ESI), m/z calcd
for C6H12NOS [M þ H]þ 146.0634, found 146.0634.
2-((2-Morpholino-2-oxoethyl)disulfanyl)-N-propylacetamide
(12a) was prepared according to General Procedure 5 from 19d
(84 mg, 0.63 mmol) as the first thiol, 19 b (102 mg, 0.63 mmol) as
the second thiol, BtCl (106 mg, 0.69 mmol) and BtH (75 mg,
1
0.63 mmol). Yield 70 mg, 38%. H NMR (400 MHz, CDCl3) d 3.73 (dt,
J ¼ 6.4, 3.8 Hz, 4H), 3.69 (d, J ¼ 6.2 Hz, 2H), 3.67 (s, 2H), 3.53 (t,
J ¼ 4.9 Hz, 2H), 3.50 (s, 2H), 3.35–3.26 (m, 2H), 1.62 (h, J ¼ 7.4 Hz,
2H), 0.98 (t, J ¼ 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) d 167.8,
167.0, 66.8, 66.6, 46.8, 42.9, 42.5, 41.8, 39.9, 22.8, 11.5. HRMS (ESI),
General procedure 3: preparation of thiols 19d–k
[M þ Na]þ
315.0808,
To a stirred solution of protected amide 18d–k (1.2–2.1 mmol) in
DCM (10 ml) trifluoroacetic acid (TFA; 5 equiv.) was added fol-
lowed by triethylsilane (TES; 2 equiv.). Reaction progress was
monitored by TLC. After 2–16 h, reaction mixture was washed with
water (3 ꢂ 30 ml), dried and concentrated in vacuo. The residue
was suspended in hexane (10 ml) and sonicated using ultrasonic
bath, cooled and filtered to give corresponding thiol18.
m/z
calcd
for
C11H20N2O3S2Na
found 315.0815.
Methyl 2-((2-((4-fluorophenyl)amino)-2-oxoethyl)disulfany-
l)acetate (13a) was prepared according to General Procedure 5
from 19i (110 mg, 0.59 mmol) as the first thiol, methyl thioglycoate
(69 mg, 0.65 mmol) as the second thiol, BtCl (100 mg, 0.65 mmol)
and BtH (71 mg, 0.6 mmol). Yield 68 mg, 40%. 1H NMR (400 MHz,
CDCl3) d 8.71 (s, 1H), 7.67–7.43 (m, 2H), 7.05 (m, 2H), 3.84 (s, 3H),
3.65 (s, 2H), 3.60 (s, 2H). 13C NMR (101 MHz, CDCl3) d 171.31,
166.03, 159.51 (d, JCF ¼ 243.7 Hz), 133.79 (d, JCF ¼ 2.9 Hz), 121.69 (d,
JCF ¼ 7.8 Hz), 115.63 (d, JCF ¼ 22.5 Hz), 53.17, 42.85, 41.87. HRMS
N-Cyclopropyl-2-mercaptoacetamide (19d) was prepared
according to General Procedure 3 from 18d (746 mg, 2 mmol), TFA
(1.27 g, 5.9 mmol) and TES (514 mg, 4.4 mmol). Yield 233 mg, 89%
1
(85% NMR purity). H NMR (400 MHz, CDCl3) d 6.72 (s, 1H), 3.23 (d,
J ¼ 9.1 Hz, 2H), 2.76 (dq, J ¼ 7.1, 3.5 Hz, 1H), 1.87 (t, J ¼ 9.1 Hz, 1H), (ESI), m/z calcd for C11H12FNO3S2Na [M þ Na]þ 312.0135,
found 312.0139.
0.88–0.79 (m, 2H), 0.60–0.52 (m, 2H). HRMS (ESI), m/z calcd for
C5H9NOSNa [M þ Na]þ 154.0297, found 154.0299.
Cell culture
Human neuroblastoma cells (SH-SY5Y) were grown in 1:1 mixture
of Eagle’s Minimum Essential Medium and F12 Medium (Sigma)
supplemented with 10% foetal bovine serum (Sigma). Human ker-
atinocytes (HaCaT) were grown in DMEM, supplemented with 10%
foetal bovine serum, 2 mM L-glutamine, 5000 U/ml penicillin, and
5 mg/ml streptomycin. Human glioblastoma cell (U87-MG) were
grown in Minimum Essential Media (MEM), containing 10% foetal
bovine serum, 2 mM L-glutamine, 5,000 U/ml penicillin, and 5 mg/
ml streptomycin. All cell lines were obtained from American Type
Culture Collection (ATCC, Rockville, MD). All cell lines were grown
at 37 ꢁC in a humidified 5% CO2 atmosphere.
General procedure 4: preparation of symmetric
disulphides 11a–k
To the solution of thiol 19a–k (0.3–0.9 mmol) in DCM and triethyl-
amine (TEA; 1.5–3 equiv.) solid I2 was added portionwise until per-
sistent deep yellow colour appeared. After stirring for 5 min, the
conversion of thiol was checked by TLC. In case of compounds
11a–d, f, g, k the reaction mixture was concentrated and the resi-
due was crystallised from hexane-ethyl acetate to give pure disul-
phides. Compounds 11e, h, i, j precipitated from the reaction
mixture and were isolated by filtration and washing with
DCM (2 ꢂ 5 ml)19.
2,20-Disulfanediylbis(1-(pyrrolidin-1-yl)ethan-1-one)
(11a)
TrxR inhibition assay
was prepared according to General Procedure 4 from 19a (90 mg,
0.31 mmol) and TEA (66 mg, 0.93 mmol). Yield 66 mg, 75%. 1H
NMR (400 MHz, CDCl3) d 3.69 (s, 4H), 3.57 (t, J ¼ 6.8 Hz, 4H), 3.51
(t, J ¼ 6.9 Hz, 4H), 2.04–1.96 (m, 4H), 1.95–1.84 (m, 4H). 13C NMR
(126 MHz, CDCl3) d 166.7, 47.2, 46.2, 42.3, 26.2, 24.4. HRMS
(ESI), m/z calcd for C12H20N2O2S2Na [M þ Na]þ 311.0858,
found 311.0873.
For TrxR assay, lysate from SH-SY5Y cells and rat liver were used.
SHSY5Y cells were washed twice with phosphate buffer solution
(PBS) and harvested from the flask with a scraper. Cells were
homogenised in ice-cold 50 mM potassium phosphate buffer (pH
7.4), containing 1 mM EDTA, with Ultra Turrax T25 (Ikawerk, Janke
and Kunkel Inc., Staufen, Germany). The homogenates were

668
O. BAKULINA ET AL.
centrifuged at 10,000 ꢂ g for 15 min at 4 ꢁC. The protein concen- Trityl-protected thioacetic acid (16)14 was activated by 1,10-carbon-
trations of the supernatants were determined using a Bio-Rad pro- yldiimidazole (CDI) to give amides 18a–k in good to excellent
yields. The trityl group was removed by the action of trifluoroace-
tic acid (TFA) used as a solvent (18a–c)18 or as a reagent (5 equiv.)
in DCM (18d–k)19, in the presence of triethylsilane. The reaction
was completed in 5–30 min in the former case and took 2–16 h –
in the latter. The free thiols 19a–k thus obtained (in moderate to
excellent yield) were homo-coupled on the action of molecular
iodine in the presence of triethylamine19 to give target com-
pounds 11a–k in moderate to excellent yield.
For the preparation of non-symmetric disulphides 12(13)a, one
of the thiols 19 was activated at ꢀ78 ꢁC by the action of a
roughly equimolar mixture of N-chlorobenzotriazole (BtCl) and
benzotriazole (BtH), and the resulting intermediate was treated
with a second thiol 19 (12a) or methyl mercaptoacetate (13a) to
give, on stirring at 0 ꢁC for 30 min, respective disulphides
(Scheme 2).
Biological evaluation of compounds 11a–k, 12a, and 13a as
well as commercially available dithiodiglycolic acid (14), its diso-
dium salt (15) and dimethyl ester (17) against human neuroblast-
oma (SHSY5Y) as well as rat liver cell lysates using DTNB assay21
revealed that a number of compounds (11a–d, 11 g, 11j–k, 13a,
14, and 17) possessed double-digit micromolar activity against
liver cell lysate while their inhibitory activity measured against
neuroblastoma (SHSY5Y) cell lysate was significantly lower (com-
pounds 11a–b, 11 g, 11j, 14, and 17 displaying virtually no activ-
ity). Considering that TrxR1 expression levels in hepatocytes are
significantly higher compared to SHSY5Y cells24,25, these results
may suggest higher activity of our novel compounds towards
lysates with higher TrxR1 content. Notably, compound 15 which is
a close analogue (double homolog) of compound 4 (TravoceptTM
which entered Phase III clinical trials in 20156) is completely
devoid of inhibitory activity toward TrxR.
Selected compounds from the active cohort (11 b, 11d, 11g)
and also compound 12a (which displayed identical inhibitory
potency with IC50 around 45 mM against SHSY5Y and hepatocyte
lysates) were tested for cytotoxicity against neuroblastoma
(SHSY5Y), glioblastoma (U87), and immortal keratinocyte (HaCaT)
cells. Interestingly, compound 11 b was found to be 2–3 times
more active against U87 cell line compared to the other three
compounds (Table 1).
tein assay kit (Bio-Rad Laboratories, USA). TrxR activity was
assayed using 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB, Sigma-
Aldrich, USA) as substrate21. The reactions were run on a 96-well
plates, final volume of 100 ml in 50 mM potassium phosphate buf-
fer (pH 7.0), containing 50 mg of the cell lysate proteins, 1-mM
EDTA, 50 mM KCl, 0.2 mg/ml bovine serum albumin, 0.25 mM
NADPH (Acros Organics, USA). Reaction mixture was incubated for
15 min at room temperature in plate shaker (PST-60HL-4, BioSan,
Riga, Latvia); afterwards DTNB was added to the total concentra-
tion of 2.5 mM. Enzyme kinetics was monitored on a Tecan
Infinite1000 microplate reader, by measuring the increase in
absorbance at 412 nm for 20 min. The background TrxR-independ-
ent reduction of DTNB in the cell lysates, as determined in the
presence of aurothiomalate (200 mM), was subtracted from each
value22. Reaction mixture with rat liver lysate contained 35 mg of
proteins, 1 mM EDTA, 50 mM KCl, 0.2 mg/ml bovine serum albu-
min, 0.25 mM NADPH, and 2.5 mM DTNB. Reactions with rat liver
lysate were run in the same manner as cell lysates.
Cytotoxicity assay
Cells grown in 25 cm2 tissue flasks were trypsinized, seeded into
flat-bottomed adherent 96-well cell culture plates in appropriate
medium (SH-SY5Y 12,000 cells/well, U87-MG 4000 cells/well,
HaCaT 2000 cells/well) and incubated overnight; afterwards the
cells were exposed to different concentrations of the tested com-
pounds for 72 h. Cell viability was measured by MTT assay23
.
Briefly, after incubation with the compounds, medium containing
0.2 mg/ml MTT was added. After incubation (3 h, 37 ꢁC, 5% CO2),
the MTT-containing medium was removed, and 200 ml of dimethyl
sulfoxide (DMSO) was immediately added to each sample. The
samples were assessed at 540 nm on LKB 5060–006 Microplate
Reader (Vienna, Austria) microplate reader. The half-maximal
inhibitory concentration (IC50) of each compound was calculated
R
V
using Graph Pad Prism 6.0 (GraphPad Software, Inc., USA).
Results and discussion
Chemistry
Initial attempts to elaborate commercially available dithiodiglycolic
acid (14) into compounds 11–13 failed, since either HATU- or
DCC-mediated amidation of it resulted in a difficult-to separate
mixture of mono- and symmetric bis-amides of 14 (also contain-
Conclusions
We have described design, synthesis, and biological evaluation of
a series of dithiodiglycolic acid derivatives. Symmetric bis-amides
ing 14 itself). Therefore, for the preparation of symmetric were prepared via molecular iodine-promoted bis-homocoupling
bis-amides 11a–k we adopted the route depicted in Scheme 1. of thioglycolic acid amides, while non-symmetric diamide and
Scheme 1. Preparation of symmetric disulphides 11a–k.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
669
Scheme 2. Preparation of non-symmetric disulphides 12(13)a.
Table 1. Inhibition profile of compounds prepared in this work against TrxR1 measured in neuroblastoma (SHSY5Y) and hepatocyte lysates; cytotoxicity profile of
selected compounds determined against SHSY5Y, U87, and HaCaT cells.
TrxR IC50 (lM)a
Hepatocyte lysate
CC50 (lM)a
Compound
Structure
SHSY5Y lysate
SHSY5Y
nd
U87
nd
HaCaT
nd
11a
>200
170.4 54.2
11b
11c
>200
26.5 2.9
219.7 15.3
54.6 3.4
58.4 5.0
189.5 78.4
182.5 32.2
nd
nd
nd
11d
63.6 27.5
34.7 9.1
202.3 12.0
179.6 12.6
69.0 3.4
11e
11f
>200
>200
>200
>200
nd
nd
nd
nd
nd
nd
11g
11h
>200
>200
22.2 3.1
174 18.3
125.2 15.6
43.0 2.9
>200
nd
nd
nd
(continued)

670
O. BAKULINA ET AL.
Table 1. Continued.
TrxR IC50 (lM)a
Hepatocyte lysate
CC50 (lM)a
Compound
Structure
SHSY5Y lysate
SHSY5Y
nd
U87
nd
HaCaT
nd
11i
>200
>200
11j
>200
90.4 18.7
nd
nd
nd
11k
12a
>200
24.0 11.5
45.6 6.8
nd
nd
nd
44.4 15.1
176.3 13.4
186.8 15.9
71.8 6.9
13a
186.4 83.8
>200
nd
nd
nd
14
15
17
>200
>200
>200
>200
>200
>200
nd
nd
nd
nd
nd
nd
nd
nd
nd
aThe values provided are mean SEM (n ¼ 3).
“nd”: not determined; IC: inhibitory concentration; CC: cytotoxicity concentration.
amide-ester were prepared via bis-thiol heterocoupling promoted [project III41031] as well as by ERA.Net RUS plus joint programme
by N-chorobenzotriazole/benzotriazole mixture. The compound set grant RUS_ST2017-309 and State Education Development Agency
tested against human neuroblastoma (SHSY5Y) and rat liver cell
lysates for TrxR inhibition using DTNB assay revealed a promising
inhibitory profile for 6 out 16 compounds tested, namely, higher
inhibitory potency toward hepatocyte lysate TrxR1 compared to
SHSY5Y lysate. Several of active compounds also showed cytotoxic
effect toward human neuroblastoma (SHSY5Y) and glioblastoma
(U87), as well as, immortal human keratinocyte (HaCaT) cell lines.
Thus, the derivatives studied in this work represent a novel lead
chemotype that could be further developed into more potent and
more specific TrxR1 inhibitors valuable for anticancer therapy.
of Republic of Latvia (“THIOREDIN”). This work was also supported
by a Short-Term Scientific Mission (STSM) Grant from COST Action
CM1407 “Challenging organic syntheses inspired by nature - from
natural products chemistry to drug discovery and European
Cooperation in Science and Technology.
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We thank the Educational Resource Centre of Chemistry, Research
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Analysis and Materials Research of Saint Petersburg State
University Research Park for obtaining the analytical data.
Disclosure statement
No potential conflict of interest was reported by the authors.
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