Synthesis and in vitro biological evaluation of thiosulfinate derivatives for the treatment of human multidrug-resistant breast cancer

Article abstract of DOI:10.1038/aps.2016.170

Organosulfur compounds derived from Allium vegetables have long been recognized for various therapeutic effects, including anticancer activity. Allicin, one of the main biologically active components of garlic, shows promise as an anticancer agent; however, instability makes it unsuitable for clinical application. The aim of this study was to investigate the effect of stabilized allicin derivatives on human breast cancer cells in vitro. In this study, a total of 22 stabilized thiosulfinate derivatives were synthesized and screened for their in vitro antiproliferative activities against drug-sensitive (MCF-7) and multidrug-resistant (MCF-7/Dx) human adenocarcinoma breast cancer cells. Assays for cell death, apoptosis, cell cycle progression and mitochondrial bioenergetic function were performed. Seven compounds (4b, 7b, 8b, 13b, 14b, 15b and 18b) showed greater antiproliferative activity against MCF-7/Dx cells than allicin. These compounds were also selective towards multidrug-resistant (MDR) cells, a consequence attributed to collateral sensitivity. Among them, 13b exhibited the greatest anticancer activity in both MCF-7/Dx and MCF-7 cells, with IC50 values of 18.54±0.24 and 46.50±1.98 μmol/L, respectively. 13b altered cellular morphology and arrested the cell cycle at the G2/M phase. Additionally, 13b dose-dependently induced apoptosis, and inhibited cellular mitochondrial respiration in cells at rest and under stress. MDR presents a significant obstacle to the successful treatment of cancer clinically. These results demonstrate that thiosulfinate derivatives have potential as novel anticancer agents and may offer new therapeutic strategies for the treatment of chemoresistant cancers.

Full text of DOI:10.1038/aps.2016.170

Acta Pharmacologica Sinica 2017: 1–16
© 2017 CPS and SIMM All rights reserved 1671-4083/17
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Original Article
Synthesis and in vitro biological evaluation of
thiosulfinate derivatives for the treatment of human
multidrug-resistant breast cancer
Ariane ROSEBLADE1, Alison UNG2, Mary BEBAWY1,
*
1Discipline of Pharmacy, Graduate School of Health, The University of Technology Sydney, NSW, Australia; 2School of Mathematical and
Physical Sciences, Faculty of Science, The University of Technology Sydney, NSW, Australia
Abstract
Organosulfur compounds derived from Allium vegetables have long been recognized for various therapeutic effects, including
anticancer activity. Allicin, one of the main biologically active components of garlic, shows promise as an anticancer agent; however,
instability makes it unsuitable for clinical application. The aim of this study was to investigate the effect of stabilized allicin derivatives
on human breast cancer cells in vitro. In this study, a total of 22 stabilized thiosulfinate derivatives were synthesized and screened
for their in vitro antiproliferative activities against drug-sensitive (MCF-7) and multidrug-resistant (MCF-7/Dx) human adenocarcinoma
breast cancer cells. Assays for cell death, apoptosis, cell cycle progression and mitochondrial bioenergetic function were performed.
Seven compounds (4b, 7b, 8b, 13b, 14b, 15b and 18b) showed greater antiproliferative activity against MCF-7/Dx cells than allicin.
These compounds were also selective towards multidrug-resistant (MDR) cells, a consequence attributed to collateral sensitivity.
Among them, 13b exhibited the greatest anticancer activity in both MCF-7/Dx and MCF-7 cells, with IC50 values of 18.54±0.24 and
46.50±1.98 µmol/L, respectively. 13b altered cellular morphology and arrested the cell cycle at the G2/M phase. Additionally, 13b
dose-dependently induced apoptosis, and inhibited cellular mitochondrial respiration in cells at rest and under stress. MDR presents
a significant obstacle to the successful treatment of cancer clinically. These results demonstrate that thiosulfinate derivatives have
potential as novel anticancer agents and may offer new therapeutic strategies for the treatment of chemoresistant cancers.
Keywords: garlic; allicin; thiosulfinate derivatives; breast cancer; multidrug resistance; cell cycle arrest; apoptosis; mitochondrial
respiration
Acta Pharmacologica Sinica advance online publication, 31 Aug 2017; doi: 10.1038/aps.2016.170
in the cell membrane, such as P-glycoprotein (P-gp)[2, 3]. P-gp
Introduction
Cancer is one of the primary causes of mortality worldwide, facilitates ATP-dependent efflux of chemotherapeutic drugs,
accounting for approximately 8 million deaths per year[1]
.
allowing MDR cells to maintain sub-lethal intracellular con-
While significant advances in diagnostic screening, surgical centrations of these compounds, resulting in poor therapeutic
resection, and targeted therapies have been made in recent response and prognosis in many types of cancer[4–6]. A broad
years, many types of malignancy remain difficult to cure. range of synthetic and natural P-gp inhibitors have been inves-
Currently, the most common forms of treatment include radio- tigated, but due to lack of efficacy and dose-limiting toxicity,
therapy, surgery and chemotherapy; however, resistance to these have so far proven to be unsuitable for clinical applica-
chemotherapy, whether intrinsic or acquired, presents a sig- tion[7].
nificant obstacle to the successful treatment of cancer.
Phytochemicals have long been known to have numerous
Multidrug resistance (MDR) occurs when cancer cells health benefits in addition to showing potential as anticancer
become cross-resistant to a wide variety of structurally and agents and modulators of MDR[8–10]. Organosulfur compounds
functionally unrelated drugs and is predominately associated derived from Allium vegetables, such as onion (Allium cepa)
with increased expression of multidrug efflux transporters and garlic (Allium sativum), are well documented as having a
wide range of protective effects in numerous disease states.
*
The consumption of these vegetables is associated with many
health benefits, including a reduced risk of cancer[11–14]
Allicin, an allyl thiosulfinate, is the main biologically active
To whom correspondence should be addressed.
E-mail mary.bebawy@uts.edu.au
.
Received 2016-09-12 Accepted 2016-12-21

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Roseblade A et al
compound derived from garlic, and it has been shown to All other reagents and solvents were obtained from commer-
exhibit various biological effects including antioxidant[15]
,
cial suppliers and were used without further purification. All
antiparasitic[16, 17], antimicrobial[18], antibacterial[19], anti-inflam- thiosulfinates were synthesized via a disulfide intermediate,
matory[20, 21], and anticancer activity[22–24]. In vitro studies have except for allicin (1b), which was synthesized directly from
shown that allicin inhibits cancer cell proliferation through cell diallyl disulfide purchased from Sigma-Aldrich (NSW, Aus-
cycle arrest in gastric, breast and cervical cancer[25–27]. Studies tralia). Thin-layer chromatography (TLC) was performed on
have also shown that allicin induces apoptosis in leukemia- Merck pre-coated silica gel plates (60 F254), and spots were
derived cells through the depletion of cellular glutathione visualized by exposure to iodine vapor or short-wave UV light
(GSH) and modulation of the cellular redox state[28], as well (l254 nm). Whenever required, column chromatography was
as significantly increasing reactive oxygen species (ROS) performed using Scharlau silica gel 60 (230–400 mesh), with
production in liver cancer, leading to reduced mitochondrial n-hexane and ethyl acetate used as eluents.
membrane potential, apoptosis, and cell death[29]. Addition-
ally, allicin and saturated short-chain thiosulfinate analogs are Synthesis of reaction catalysts
known to act on intracellular calpain, inhibiting invasion and CsF-Celite
migration in cancer cells[30]. This activity inhibits the release of Celite 521 (3.20 g) was added to cesium fluoride (5.36 g, 32.0
microparticles (MPs), which have been shown to transfer P-gp mmol) dissolved in 80 mL of H2O. The mixture was stirred
from MDR cells to drug-sensitive cells[31, 32]. These findings for 30 min at RT, after which the H2O was removed under
make these compounds attractive leads in targeting prolifera- reduced pressure. The resultant solid was then washed twice
tion, metastasis and MDR in cancer[30, 32–34]. Importantly, allicin in CH3CN, filtered, and dried in a desiccator at room tempera-
and thiosulfinate derivatives have been shown to be cancer- ture.
specific while being non-toxic to normal cells and have been
reported to increase survival times in tumor-bearing mice[35, 36]
.
1-Chlorobenzotriazole
The anticancer activity of allicin is widely attributed to the An 8%–12% NaOCl solution (commercial bleach) was added
allylthio group; however, this group is highly unstable, and as dropwise to a stirred solution of 50% acetic acid (32 mL) and
a consequence, allicin is prone to decomposition under rela- benzotriazole (8 g, 67.2 mmol). Once the addition was com-
tively mild conditions[37–39]. To circumvent this instability and plete, the reaction was stirred for 3 h. The product was then
to increase potency and compound half-life, substitution of the filtered and washed until the pH of the filtrate was neutral.
labile allylthio group with saturated alkanes and/or benzyl The product was then dried under reduced pressure to obtain
moieties has been shown to prevent thiosulfinate degradation 1-chlorobenzotriazole (m-CBTA) as a white powder. The
in storage and under biological conditions[39, 40]
.
spectral data agree with values reported in the literature[41]
.
1
In the present work, we describe the synthesis and antican- Yield: 94%; H NMR δ (500 MHz, CDCl3): 8.09 (1H, d, J=8.5
cer activity of a series of aromatic and aliphatic thiosulfinates Hz), 7.61–7.47 (2H, m), 7.46–7.44 (1H, m) ppm; and 13C NMR δ
against both the human adenocarcinoma breast cancer cell line (500 MHz, CDCl3): 144.46, 134.35, 129.55, 125.35, 120.99, 109.54
MCF-7 and the MDR sub-line MCF-7/Dx. We demonstrate ppm.
that these thiosulfinate derivatives show specificity towards
MDR breast cancer cells and inhibit cell proliferation through General procedures for the synthesis of symmetrical disulfides
the disruption of mitochondrial respiration, leading to the CsF-Celite (1.59 g) was stirred in 10 mL of acetonitrile, the
induction of apoptosis and cell cycle arrest.
appropriate thiol (5 mmol) was then added, and the solution
was stirred for 5–48 h. The reaction progression was moni-
tored by TLC. Once the reaction was complete, the mixture
Methods
Materials
was filtered, and the filtrate evaporated under reduced pres-
1
IR spectra were recorded on an Agilent Cary 630 FTIR spec- sure. The resultant disulfide was characterized by H and 13C
trometer (Agilent Technologies, Santa Clara, CA, USA). 1H and used in thiosulfinate synthesis without further purifica-
and 13C NMR spectra were recorded on an Agilent 500 MHz tion.
NMR spectrometer in deuterated chloroform (CDCl3) unless
otherwise stated. Chemical shifts are quoted relative to resid- 1-(Ethyldisulfanyl)ethane (2a)[42]
1
1
ual chloroform (δ 7.26 for H NMR and δ 77.36 for 13C NMR) Yield: 91%, as a yellow oil; H NMR δ (500 MHz, CDCl3): 2.70
as an internal standard, and all chemical shifts (δ) are reported (4H, q, J=7.5 Hz, SCH2), 1.32 (6H, t, J=7.5 Hz, CH3) ppm; 13C
in parts per million (ppm). The coupling constants (J) are NMR δ (500 MHz, CDCl3): 33.27, 14.81 ppm.
quoted in Hertz (Hz). The type of signal detected is indicated
by the following abbreviations: s=singlet, d=doublet, t=triplet, 1-(propyldisulfanyl)propane (3a)[42]
q=quartet, tt=triplet of triplets, and m=multiplet. High-resolu- Yield: 97%, as a yellow oil; H NMR δ (500 MHz, CDCl3): 2.67
1
tion mass spectrometry (HRMS) data were obtained using an (4H, t, J=7.2 Hz, SCH2), 1.71 (4H, sextet, J=7.2 Hz, SCH2CH2),
Agilent 6510 Q-TOF LC/MS. DCM (dichloromethane), used 1.00 (6H, t, J=7.2 Hz, CH3) ppm; 13C NMR δ (500 MHz, CDCl3):
under N2 in the synthesis of asymmetric disulfides and the 41.54, 22.86, 13.47 ppm.
oxidation of thiosulfinates, was dried over molecular sieves.
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Roseblade A et al
3
1-(Butyldisulfanyl)butane (4a)[42]
J=7.0 Hz, CH2CH3) ppm; 13C NMR δ (500 MHz, CDCl3): 137.33,
1
Yield: 92%, as a yellow oil; H NMR δ (500 MHz, CDCl3): 2.69 134.57, 130.07, 128.69, 32.98, 21.37, 14.46 ppm.
(4H, t, J=7.5 Hz, SCH2), 1.65 (4H, quintet, J=7.5 Hz, SCH2CH2),
1.41 (4H, sextet, J=7.5 Hz, CH2CH3), 0.92 (6H, t, J=7.5 Hz, CH3) 1-(Propyldisulfanyl)-4-methylbenzene (8a)[40]
ppm; 13C NMR δ (500 MHz, CDCl3): 39.25, 31.67, 22.01, 14.04 Yield: 89%, as a yellow oil; H NMR δ (500 MHz, CDCl3): 7.43
1
ppm.
(2H, d, J=8.5 Hz, ArH-2), 7.13 (2H, d, J=8.5 Hz, ArH-3), 2.71
(2H, t, J=7.3 Hz, SCH2), 2.33 (3H, s, C6H4CH3), 1.70 (2H, sextet,
J=7.3 Hz, CH2CH3), 0.96 (3H, t, J=7.3 Hz, CH2CH3) ppm; 13C
1-(Hexyldisulfanyl)hexane (5a)[42]
1
Yield: 62%, as a yellow oil; H NMR δ (500 MHz, CDCl3): 2.68 NMR δ (500 MHz, CDCl3): 137.29, 134.55, 130.06, 128.64, 41.21,
(4H, t, J=6.8 Hz, SCH2), 1.67 (4H, quintet, J=6.8 Hz, SCH2CH2), 22.48, 21.37, 13.44 ppm.
1.41–1.26 (12H, m, CH2CH2CH2CH3), 0.89 (6H, t, J=6.8 Hz,
CH3), ppm; 13C NMR δ (500 MHz, CDCl3): 39.58, 31.79, 31.27, 1-(Tert-butyldisulfanyl)-4-methylbenzene (9a)[45]
1
29.54, 28.56, 14.37 ppm.
Yield: 92%, as a yellow oil; H NMR δ (500 MHz, CDCl3): 7.44
(2H, d, J=8.5 Hz, ArH-2), 7.10 (2H, d, J=8.5 Hz, ArH-3), 2.32
(3H, s, C6H4CH3), 1.30 (9H, s, C(CH3)3) ppm; 13C NMR δ (500
1-(4-Methylphenyldisulfanyl)-4-methylbenzene (6a)[43]
1
Yield: 90%, as a white solid; H NMR δ (500 MHz, CDCl3): MHz, CDCl3): 136.65, 135.71, 129.85, 127.76, 49.39, 30.22, 21.32
7.38 (4H, d, J=8.0 Hz, ArH-2), 7.10 (4H, d, J=8.0 Hz, ArH-3), ppm.
2.32 (6H, s, CH3) ppm; 13C NMR δ (500 MHz, CDCl3): 137.80,
134.26, 130.14, 128.90, 21.41 ppm.
1-(4-Tert-butylbenzyldisulfanyl)-4-methylbenzene (10a)
Yield: 54%, as a yellow oil; H NMR δ (500 MHz, CDCl3): 7.32
(2H, d, J=8.0 Hz, MeArH-2), 7.30 (2H, d, J=8.0 Hz, ArH-2), 7.21
1
1-(4-Methoxyphenyldisulfanyl)-4-methoxylbenzene (13a)[43]
1
Yield: 91%, as a yellow oil; H NMR δ (500 MHz, CDCl3): 7.40 (2H, d, J=8.0 Hz, ArH-3), 7.08 (2H, d, J=8.0 Hz, MeArH-3), 3.93
(4H, d, J=9.0 Hz, ArH-2), 6.83 (4H, d, J=9.0 Hz, ArH-3), 3.80 (2H, s, CH2), 2.32 (3H, s, C6H4CH3), 1.30 (9H, s, C(CH3)3) ppm;
(6H, s, OCH3) ppm; 13C NMR δ (500 MHz, CDCl3): 160.27, 13C NMR δ (500 MHz, CDCl3): 150.83, 137.32, 134.57, 134.09,
133.02, 128.80, 114.96, 55.72 ppm.
129.99, 129.45, 128.88, 125.75, 43.33, 34.89, 31.70, 21.37 ppm.
1-(4-Tert-butylbenzyldisulfanyl)-4-tert-butylbenzene-1-methane 1-(4-Methylphenyldisulfanyl)-4-methoxybenzene (11a)[46]
(20a)[43]
Yield: 67%, as a yellow oil; H NMR δ (500 MHz, CDCl3):
1
1
Yield: 82%, as an orange solid; H NMR δ (500 MHz, CDCl3): 7.41 (2H, d, J=8.5 Hz, MeOArH-2), 7.38 (2H, d, J=8.5 Hz,
7.34 (2H, d, J=8.0 Hz, ArH-2), 7.17 (2H, d, J=8.0 Hz, ArH-3), ArH-2), 7.11 (2H, d, J=8.5 Hz, ArH-3), 6.83 (2H, d, J=8.5 Hz,
3.60 (2H, s, CH2), 1.31 (9H, s, C(CH3)3) ppm; 13C NMR δ (500 MeOArH-3), 3.79 (3H, s, OCH3), 2.33 (3H, s, C6H4CH3) ppm;
MHz, CDCl3): 150.84, 134.57, 129.45, 125.76, 43.33, 34.90, 31.71 13C NMR δ (500 MHz, CDCl3): 160.14, 137.96, 134.41, 132.30,
ppm.
130.12, 128.88, 128.66, 114.00, 55.71, 21.43 ppm.
General procedures for the synthesis of asymmetric disulfides
1-(Ethyldisulfanyl)-4-methoxybenzene (14a)
1
m-CBTA (0.61 g, 4.0 mmol) and benzotriazole (0.36 g, 3.0 Yield: 76%, as a yellow oil; H NMR δ (500 MHz, CDCl3): 7.48
mmol) were dissolved in dry DCM and stirred under a N2 (2H, d, J=9.0 Hz, ArH-2), 6.86 (2H, d, J=9.0 Hz, ArH-3), 3.80
atmosphere at RT. The temperature was then reduced to (3H, s, OCH3), 2.74 (2H, q, J=7.5 Hz, SCH2), 1.31 (3H, t, J=7.5
-78 °C, and the appropriate thiol (R1, 3.0 mmol, dissolved in Hz, CH2CH3) ppm; 13C NMR δ (500 MHz, CDCl3): 159.84,
dry DCM) was slowly added dropwise. The reaction mixture 131.99, 128.90, 114.97, 55.74, 32.91, 14.42 ppm.
was then left to stir for 2 h. The second thiol (R2, 3.2 mmol,
dissolved in dry DCM) was then added slowly to the reaction. 1-(Butyldisulfanyl)-4-methoxybenzene (15a)[47]
1
Once the addition was complete, the reaction mixture was Yield: 69%, as a yellow oil; H NMR δ (500 MHz, CDCl3): 7.48
allowed to warm to 0°C and then stirred for a further 30 min. (2H, d, J=9.0 Hz, ArH-2), 6.86 (2H, d, J=9.0 Hz, ArH-3), 3.81
The reaction was then quenched with Na2S2O3 (0.5 g in 15 mL (3H, s, OCH3), 2.73 (2H, t, J=7.5 Hz, SCH2), 1.65 (2H, quintet,
H2O) and stirred for a further 10 min at 0 °C. The organic J=7.5 Hz, SCH2CH2), 1.38 (2H, sextet, J=7.5 Hz, CH2CH3), 0.89
fraction was then extracted with aqueous NaHCO3 and then (3H, t, J=7.5 Hz, CH2CH3) ppm; 13C NMR δ (500 MHz, CDCl3):
twice more with H2O. The product was dried over anhydrous 159.83, 131.97, 128.90, 114.97, 55.75, 38.87, 31.13, 21.97, 13.98
MgSO4, and the solvent was removed under reduced pressure. ppm.
The resultant disulfide was characterized by H and 13C and
used in thiosulfinate synthesis without further purification.
1
1-(Tert-butyldisulfanyl)-4-methoxybenzene (16a)[45]
Yield: 84%, as a yellow oil; H NMR δ (500 MHz, CDCl3): 7.49
(2H, d, J=9.0 Hz, ArH-2), 6.84 (2H, d, J=9.0 Hz, ArH-3), 3.79
1
1-(Ethyldisulfanyl)-4-methylbenzene (7a)[44]
1
Yield: 76%, as a yellow oil; H NMR δ (500 MHz, CDCl3): 7.43 (3H, s, OCH3), 1.29 (9H, s, C(CH3)3) ppm; 13C NMR δ (500
(2H, d, J=8.5 Hz, ArH-2), 7.13 (2H, d, J=8.5 Hz, ArH-3), 2.74 MHz, CDCl3): 159.27, 130.63, 130.11, 114.77, 55.71, 49.28, 30.25
(2H, q, J=7.0 Hz, CH2CH3), 2.33 (3H, s, C6H4CH3), 1.31 (3H, t, ppm.
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Roseblade A et al
(n-hexane/ethyl acetate). After purification, all thiosulfinates
were stored at -80°C until required. All stock solutions were
1-(Hexyldisulfanyl)-4-methoxybenzene (17a)[46]
1
Yield: 85%, as a yellow oil; H NMR δ (500 MHz, CDCl3): 7.48 prepared in DMSO and stored at -20°C.
(2H, d, J=9.0 Hz, ArH-2), 6.86 (2H, d, J=9.0 Hz, ArH-3), 3.81
(3H, s, OCH3), 2.73 (2H, t, J=7.5 Hz, SCH2), 1.66 (2H, quintet, Allicin, S-allyl prop-2-ene-1-sulfinothioate (1b)[39]
J=7.5 Hz, SCH2CH2), 1.38-1.23 (6H, m, CH2CH2CH2CH3), 0.87 Yield: 23%, as a yellow oil; IR vmax cm-1: 1060 (S=O); 1H NMR δ
(3H, t, J=7.5 Hz, CH3) ppm; 13C NMR δ (500 MHz, CDCl3): (500 MHz, D2O): 6.05–5.94 (m, 2H, SCH2CH), 5.53–4.79 (m, 4H,
159.83, 132.00, 128.93, 114.96, 55.75, 39.25, 31.72, 29.02, 28.50, CH=CH2), 4.05–3.82 (m, 4H, SCH2) ppm; 13C NMR δ (500 MHz,
22.85, 14.36 ppm.
D2O): 136.01, 128.07, 127.87, 121.00, 61.07, 38.47 ppm; HRMS
m/z: calcd. for C6H10OS2 (M+H)+: 163.0246; found: 163.0245.
1-(Cyclohexyldisulfanyl)-4-methoxybenzene (18a)[47]
Yield: 89%, as a yellow oil; H NMR δ (500 MHz, CDCl3): 7.47 S-ethyl ethanesulfinothioate (2b)[39]
(2H, d, J=9.0 Hz, ArH-2), 6.86 (2H, d, J=9.0 Hz, ArH-3), 3.80 Yield: 46%, as a yellow oil; IR vmax cm-1: 1074 (S=O); 1H NMR δ
(3H, s, OCH3), 2.80 (1H, tt, J=10.5, 3.7 Hz, H-1), 2.03-2.00 (2H, (500 MHz, CDCl3): 3.18–3.11 (4H, m, SCH2), 1.46 (3H, t, J=7.5
m, H-6), 1.78–1.75 (2H, m, H-2), 1.61–1.58 (1H, m, H-4a), 1.39- Hz, SCH2CH3), 1.40 (3H, t, J=7.5 Hz, S=OCH2CH3) ppm; 13C
1.20 (5H, m, H-3, H-5 and H-4b) ppm; 13C NMR δ (500 MHz, NMR δ (500 MHz, CDCl3): 50.26, 27.38, 16.58, 8.12 ppm; HRMS
CDCl3): 159.46, 131.03, 129.69, 114.85, 55.70, 49.99, 32.90, 26.32, m/z: calcd. for C4H10OS2 (M+H)+: 139.0246; found: 139.0243.
25.97 ppm.
1
S-propyl propane-1-sulfinothioate (3b)[39]
1
1-(4-Tert-butylbenzyldisulfanyl)-4-methoxybenzene (19a)
Yield: 51%, as a yellow oil; IR vmax cm-1: 1076 (S=O); H NMR
1
Yield: 64%, as a yellow oil; H NMR δ (500 MHz, CDCl3): δ (500 MHz, D2O): 3.26-3.19 (4H, m, SCH2), 1.86-1.80 (4H, m,
7.34 (2H, d, J=9.0 Hz, MeOArH-2), 7.30 (2H, d, J=9.0 Hz, SCH2CH2), 1.07 (3H, t, J=7.5 Hz, (SCH2CH2CH3), 1.02 (3H, t,
ArH-2), 7.21 (2H, d, J=9.0 Hz, ArH-3), 6.80 (2H, d, J=9.0 Hz, J=7.5 Hz, S=OCH2CH2CH3) ppm; 13C NMR δ (500 MHz, D2O):
MeOArH-3), 3.93 (2H, s, CH2), 3.80 (3H, s, CH3), 1.31 (9H, s, 59.65, 38.20, 26.59, 19.70, 15.07, 15.00 ppm; HRMS m/z: calcd.
C(CH3)3) ppm; 13C NMR δ (500 MHz, CDCl3): 159.81, 150.78, for C6H14OS2 (M+H)+: 167.0559; found: 167.0562.
134.06, 132.19, 129.44, 128.47, 125.79, 114.86, 55.70, 43.46, 34.87,
31.70 ppm.
S-butyl butane-1-sulfinothioate (4b)[39]
Yield: 57%, as a yellow oil; IR vmax cm-1: 1081 (S=O); 1H NMR δ
(500 MHz, CDCl3): 3.15–3.10 (4H, m, SCH2), 1.82–1.75 (4H, m,
4-(Butyldisulfanyl)phenol (21a)
1
Yield: 41%, as a yellow oil; H NMR δ (500 MHz, CDCl3): 7.43 SCH2CH2), 1.50–1.43 (4H, m, CH2CH3), 0.98–0.93 (6H, m, CH3)
(2H, d, J=8.5 Hz, ArH-2), 6.80 (2H, d, J=8.5 Hz, ArH-3), 2.73 ppm; 13C NMR δ (500 MHz, CDCl3): 56.33, 33.22, 32.95, 25.81,
(2H, t, J=7.5 Hz, SCH2), 1.73 (2H, quintet, J=7.5 Hz, SCH2CH2), 22.21, 22.08, 14.01, 13.86 ppm; HRMS m/z: calcd. for C8H18OS2
1.41 (2H, sextet, J=7.5 Hz, CH2CH3), 0.88 (3H, t, J=7.5 Hz, CH3) (M+H)+: 195.0872; found: 195.0873.
ppm; 13C NMR δ (500 MHz, CDCl3): 155.94, 132.17, 117.49,
116.46, 39.23, 31.65, 21.00, 14.03 ppm.
S-hexyl hexane-1-sulfinothioate (5b)[17]
1
Yield: 48%, as a yellow oil; IR vmax cm-1: 1079 (S=O); H NMR
4-(hexyldisulfanyl)phenol (22a)
δ (500 MHz, CDCl3): 3.16-3.08 (4H, m, SCH2), 1.84–1.77 (4H,
1
Yield: 35%, as a yellow oil; H NMR δ (500 MHz, CDCl3): 7.43 m, SCH2CH2), 1.46–1.41 (4H, m, S(CH2)2CH2), 1.35–1.29 (8H,
(2H, d, J=8.5 Hz, ArH-2), 6.81 (2H, d, J=8.5 Hz, ArH-3), 2.73 m, CH2CH2CH3), 0.91–0.88 (6H, m, CH3) ppm; 13C NMR δ (500
(2H, t, J=7.5 Hz, SCH2), 1.74 (2H, quintet, J=7.5 Hz, SCH2CH2), MHz, CDCl3): 56.62, 33.26, 31.67, 31.57, 31.17, 28.63, 28.61,
1.44-1.23 (6H, m, CH2CH2CH2CH3), 0.87 (3H, t, J=7.5 Hz, CH3) 23.78, 22.83, 22.73, 14.34, 14.30 ppm; HRMS m/z: calcd. for
ppm; 13C NMR δ (500 MHz, CDCl3): 155.90, 132.15, 117.46, C12H26OS2 (M+H)+: 251.1498; found: 251.1496.
116.43, 39.24, 31.70, 29.13, 28.54, 22.85, 14.35 ppm.
S-4-tolyl 4-methylbenzenesulfinothioate (6b)[48]
General procedures for the synthesis of symmetric and asym- Yield: 24%, as a white solid; IR vmax cm-1: 1075 (S=O); 1H NMR
metric thiosulfinates
δ (500 MHz, CDCl3): 7.46 (2H, d, J=8.0 Hz, ArH-2), 7.24 (2H, d,
The selected disulfide (3.0 mmol) was dissolved in dry DCM J=8.0 Hz, ArH-2’), 7.21 (2H, d, J=8.0 Hz, ArH-3), 7.14 (2H, d,
(5 mL) under a N2 atmosphere at -78°C. m-CPBA (0.52 g, 3.0 J=8.0 Hz, ArH-3’), 2.42 (3H, s, CH3’), 2.38 (3H, s, CH3-1) ppm;
mmol), dissolved in dry DCM (5 mL), was then slowly added 13C NMR δ (500 MHz, CDCl3): 144.91, 142.38, 140.84, 136.85,
dropwise. Once the addition was complete, the reaction was 130.54, 129.71, 127.96, 124.96, 22.01, 21.83 ppm; HRMS m/z:
left to stir for 3 h, slowly warming to 0°C. The reaction was calcd. for C14H14OS2 (M+H)+: 263.0559; found: 263.0557.
quenched with saturated NaHCO3, and the resulting aque-
ous solution was extracted 3 times with DCM. The combined S-4-tolyl ethanesulfinothioate (7b)
1
organic fractions were then dried over anhydrous MgSO4 Yield: 39%, as a yellow oil; IR vmax cm-1: 1079 (S=O); H NMR
and evaporated under reduced pressure. The crude product δ (500 MHz, CDCl3): 7.49 (2H, d, J=7.5 Hz, ArH-2), 7.22 (2H,
was then purified by silica gel flash column chromatography d, J=7.5 Hz, ArH-3), 3.10 (2H, q, J=7.5 Hz, S=OCH2), 2.38 (3H,
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5
s, C6H4CH3), 1.43 (3H, t, J=7.5 Hz, CH2CH3) ppm; 13C NMR δ d, J=8.5 Hz, ArH-2’), 6.98 (2H, d, J=8.5 Hz, ArH-3’), 6.89 (2H,
(500 MHz, CDCl3): 141.06, 135.73, 130.62, 125.58, 50.22, 21.70, d, J=8.5 Hz, ArH-3), 3.86 (3H, s, OCH3-2) 3.84 (3H, s, OCH3-
8.24 ppm; HRMS m/z: calcd. for C9H12OS2 (M+H)+: 201.0402; 1) ppm; 13C NMR δ (500 MHz, CDCl3): 162.56, 162.01, 137.88,
found: 201.0404.
135.55, 126.51, 120.31, 115.15, 114.71, 55.94, 55.80 ppm; HRMS
m/z: calcd. for C14H14O3S2 (M+H)+: 295.0457; found: 295.0457.
S-4-tolyl propane-1-sulfinothioate (8b)[40]
Yield: 41%, as a yellow oil; IR vmax cm-1: 1080 (S=O); 1H NMR δ S-4-methoxyphenyl ethanesulfinothioate (14b)
(500 MHz, CDCl3): 7.50 (2H, d, J=8.0 Hz, ArH-2), 7.23 (2H, d, Yield: 48%, as a yellow oil; IR vmax cm-1: 1076 (S=O); H NMR
J=8.0 Hz, ArH-3), 3.08 (2H, m, S=OCH2), 2.39 (3H, s, C6H4CH3), δ (500 MHz, CDCl3): 7.53 (2H, d, J=9.0 Hz, ArH-2), 6.94 (2H,
1.89 (2H, m, CH2CH3), 1.10 (3H, t, J=7.5 Hz, CH2CH3) ppm; d, J=9.0 Hz, ArH-3), 3.84 (3H, s, OCH3), 3.07 (2H, q, J=7.5 Hz,
13C NMR δ (500 MHz, CDCl3): 141.04, 135.66, 130.59, 125.83, CH2CH3), 1.42 (3H, t, J=7.5 Hz, CH2CH3) ppm; 13C NMR δ
58.16, 21.68, 17.59, 13.58 ppm; HRMS m/z: calcd. for C10H14OS2 (500 MHz, CDCl3): 161.95, 137.76, 119.28, 115.42, 55.80, 49.98,
1
(M+H)+: 215.0559; found: 215.0558.
8.22 ppm; HRMS m/z: calcd. for C9H12O2S2 (M+H)+: 217.0351;
found: 217.0350.
S-4-tolyl 2-methylpropane-2-sulfinothioate (9b)[49]
Yield: 17%, as a white solid; IR vmax cm-1: 1078 (S=O); H S-4-methoxyphenyl butane-1-sulfinothioate (15b)
1
NMR δ (500 MHz, CDCl3): 7.52 (2H, d, J=8.0 Hz, ArH-2), 7.20 Yield: 39%, as a yellow oil; IR vmax cm-1: 1080 (S=O); H NMR
(2H, d, J=8.0 Hz, ArH-3), 2.38 (3H, s, C6H4CH3), 1.46 (9H, s, δ (500 MHz, CDCl3): 7.53 (2H, d, J=9.0 Hz, ArH-2), 6.94 (2H,
C(CH3)3) ppm; 13C NMR δ (500 MHz, CDCl3): 140.52, 135.59, d, J=9.0 Hz, ArH-3), 3.84 (3H, s, OCH3), 3.06 (2H, t, J=7.5 Hz,
130.60, 126.51, 60.47, 24.50, 21.63 ppm; HRMS m/z: calcd. for SCH2), 1.82 (2H, m, SCH2CH2), 1.45 (2H, m, CH2CH3), 0.98
1
C11H16OS2 (M+H)+: 229.0715; found: 229.0715.
(3H, t, J=7.5 Hz, CH2CH3) ppm; 13C NMR δ (500 MHz, CDCl3):
161.94, 137.72, 119.57, 115.41, 55.94, 55.80, 25.84, 22.29, 14.05
ppm; HRMS m/z: calcd. for C11H16O2S2 (M+H)+: 245.0664;
S-4-tolyl (4-tert-butylphenyl)methanesulfinothioate (10b)
Yield: 33%, as a white solid; IR vmax cm-1: 1078 (S=O); 1H NMR found: 245.0665.
δ (500 MHz, CDCl3): 7.44 (2H, d, J=8.5 Hz, MeArH-2), 7.41
(2H, d, J=8.5 Hz, ArH-2), 7.30 (2H, d, J=8.5 Hz, ArH-3), 7.19 S-4-methoxyphenyl 2-methylpropane-2-sulfinothioate (16b)
(2H, d, J=8.5 Hz, MeArH-3), 4.37 (1H, d, J=13.0 Hz, CHa), 4.25 Yield: 27%, as a white solid; IR vmax cm-1: 1072 (S=O); H
(1H, d, J=13.0 Hz, CHb), 2.37 (3H, s, C6H4CH3), 1.33 (9H, s, NMR δ (500 MHz, CDCl3): 7.55 (2H, d, J=8.5 Hz, ArH-2),
C(CH3)3) ppm; 13C NMR δ (500 MHz, CDCl3): 152.09, 141.02, 6.92 (2H, d, J=8.5 Hz, ArH-3), 3.83 (3H, s, OCH3), 1.45 (9H, s,
135.80, 130.59, 130.44, 127.38, 126.21, 125.80, 62.50, 35.03, 31.62, C(CH3)3) ppm; 13C NMR δ (500 MHz, CDCl3): 161.62, 137.63,
21.69 ppm; HRMS m/z: calcd. for C18H22OS2 (M+H)+: 319.1185; 120.24, 115.44, 60.27, 55.78, 24.50 ppm; HRMS m/z: calcd. for
1
found: 319.1187.
C11H16O2S2 (M+H)+: 245.0664; found: 245.0660.
S-4-methoxyphenyl 4-methylbenzenesulfinothioate (11b)[48]
S-4-methoxyphenyl hexane-1-sulfinothioate (17b)
Yield: 20%, as a white solid; IR vmax cm-1: 1075 (S=O); 1H NMR Yield: 51%, as a yellow oil; IR vmax cm-1: 1078 (S=O); H NMR
δ (500 MHz, CDCl3): 7.53 (2H, d, J=8.0 Hz, MeOArH-2), 7.43 δ (500 MHz, CDCl3): 7.53 (2H, d, J=9.0 Hz, ArH-2), 6.94 (2H,
(2H, d, J=8.0 Hz, ArH-2), 7.29 (2H, d, J=8.0 Hz, ArH-3), 6.89 d, J=9.0 Hz, ArH-3), 3.84 (3H, s, OCH3), 3.05 (2H, t, J=7.5 Hz,
(2H, d, J=8.0 Hz, MeOArH-3), 3.84 (3H, s, OCH3) 2.42 (3H, s, SCH2), 1.84-1.81 (2H, m, SCH2CH2), 1.46-1.31 (6H, m, CH2CH-
C6H4CH3) ppm; 13C NMR δ (500 MHz, CDCl3): 161.90, 142.48, 2CH2CH3), 0.90 (3H, t, J=7.5 Hz, CH3) ppm; 13C NMR δ (500
135.80, 129.94, 129.74, 124.67, 120.32, 115.18, 55.80, 21.87 ppm; MHz, CDCl3): 161.94, 137.72, 119.59, 115.41, 56.21, 55.80, 31.69,
HRMS m/z: calcd. for C14H14O2S2 (M+H)+: 279.0508; found: 28.70, 23.80, 22.73, 14.31 ppm; HRMS m/z: calcd. for C13H20O2S2
1
279.0504.
(M+H)+: 273.0977; found: 273.0974.
S-4-tolyl 4-methoxybenzenesulfinothioate (12b)[48]
S-4-methoxyphenyl cyclohexanesulfinothioate (18b)
Yield: 20%, as a white solid; IR vmax cm-1: 1075 (S=O); 1H NMR Yield: 38%, as a white solid; IR vmax cm-1: 1077 (S=O); 1H NMR
δ (500 MHz, CDCl3): 7.60 (2H, d, J=8.5 Hz, MeOArH-2), 7.43 δ (500 MHz, CDCl3): 7.52 (2H, d, J=8.5 Hz, ArH-2), 6.92 (2H,
(2H, d, J=8.5 Hz, ArH-2), 7.19 (2H, d, J=8.5 Hz, ArH-3), 6.99 d, J=8.5 Hz, ArH-3), 3.82 (3H, s, OCH3), 2.99 (1H, tt, J=11.5, 3.9
(2H, d, J=8.5 Hz, MeOArH-3), 3.87 (3H, s, OCH3) 2.38 (3H, s, Hz, H-1), 2.17–2.14 (2H, m, H-6), 1.94–1.88 (2H, m, H-2), 1.71–
C6H4CH3) ppm; 13C NMR δ (500 MHz, CDCl3): 162.60, 141.08, 1.50 (3H, m, H-3 and H-4a), 1.41–1.25 (3H, m, H-5 and H-4b)
137.85, 130.42, 127.97, 126.53, 126.50, 114.76, 55.94, 21.73 ppm; ppm; 13C NMR δ (500 MHz, CDCl3): 161.71, 137.56, 119.86,
HRMS m/z: calcd. for C14H14O2S2 (M+H)+: 279.0508; found: 115.35, 63.74, 55.73, 27.50, 26.52, 25.86, 25.77, 25.69 ppm; HRMS
279.0504.
m/z: calcd. for C13H18O2S2 (M+H)+: 271.0821; found: 271.0824.
S-4-methoxyphenyl 4-methoxybenzenesulfinothioate (13b)[50]
S-4-methoxyphenyl (4-tert-butylphenyl)methanesulfinothioate
Yield: 74%, as a yellow solid; IR vmax cm-1: 1073 (S=O); 1H NMR (19b)
δ (500 MHz, CDCl3): 7.56 (2H, d, J=8.5 Hz, ArH-2), 7.42 (2H, Yield: 29%, as a yellow oil; IR vmax cm-1: 1074 (S=O); H NMR
1
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Roseblade A et al
δ (500 MHz, CDCl3): 7.48 (2H, d, J=8.5 Hz, MeOArH-2), 7.41 determined using the MTS assay. Cells were seeded in
3
×
(2H, d, J=8.5 Hz, ArH-2), 7.30 (2H, d, J=8.5 Hz, ArH-3), 6.91 96-well plates (5 10 cells per well) and, once adherent, were
(2H, d, J=8.5 Hz, MeOArH-3), 4.34, (1H, d, J=13.0 Hz, CHa), cultured for 72 h in the presence of selected thiosulfinates at
4.22 (1H, d, J=13.0 Hz, CHb), 3.82 (3H, s, OCH3), 1.33 (9H, s, the indicated concentrations. Due to the short half-life of alli-
C(CH3)3) ppm; 13C NMR δ (500 MHz, CDCl3): 161.92, 152.06, cin, cells were treated with fresh media supplemented with
137.84, 130.42, 127.42, 126.21, 119.54, 115.39, 62.35, 55.79, 35.03, the appropriate concentration of allicin every 24 h. Control
31.62 ppm; HRMS m/z: calcd. for C18H22O2S2 (M+H)+: 335.1134; cells were incubated with DMSO at 0.1% (v/v). Cell viability
found: 335.1137.
was determined using the CellTiter 96® AQueous One Solu-
tion Cell Proliferation Assay (Promega, USA) according to the
S-4-tert-butylphenyl (4-tert-butylphenyl)methanesulfinothioate manufacturer’s recommendation. Briefly, following 72 h of
(20b)
treatment, 20 µL of the MTS reagent 3-(4,5-dimethyl-2-yl)-5-
Yield: 67%, as a white solid; IR vmax cm-1: 1067 (S=O); 1H NMR (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
δ (500 MHz, CDCl3): 7.38 (2H, d, J=8.5 Hz, ArH-2), 7.33 (2H, d, was added to each well and then incubated for 2 h in the dark
J=8.5 Hz, ArH-2’), 7.25 (2H, d, J=8.5 Hz, ArH-3’), 7.24 (2H, d, at 37 °C. Absorbance values were then obtained at 490 nm
J=8.5 Hz, ArH-3), 4.29–4.25 (4H, m, CH2), 1.31 (9H, s, C(CH3)3- (infinite M1000Pro, Tecan), and blank-subtracted absorbance
1), 1.30 (9H, s, C(CH3)3-2) ppm; 13C NMR δ (500 MHz, CDCl3): values were normalized to the vehicle control, which was arbi-
152.04, 151.08, 133.82, 130.387, 129.17, 127.31, 126.11, 126.06, trarily assigned as 100%. IC50 values were defined as the drug
62.15, 36.14, 34.97, 34.88, 31.61, 31.59 ppm; HRMS m/z: calcd. concentration that prevented cell growth of more than 50%
for C22H30OS2 (M+H)+: 375.1811; found: 375.1811.
relative to the vehicle control and were determined using non-
linear regression analysis with Prism 7.0 (GraphPad Software,
CA, USA).
S-4-hydroxyphenyl butane-1-sulfinothioate (21b)
Yield: 17%, as a yellow oil; IR vmax cm-1: 1036 (S=O); H NMR
1
δ (500 MHz, CDCl3): 7.40 (2H, d, J=8.5 Hz, ArH-2), 6.75 (2H, Cell cycle analysis
d, J=8.5 Hz, ArH-3), 3.11 (2H, t, J=7.5 Hz, SCH2), 1.84 (2H, dq, Flow cytometric analysis was performed to calculate the distri-
J=7.5, 3.5 Hz, SCH2CH2), 1.51 (2H, ds, J=7.5, 3.5 Hz, CH2CH3), bution of the cell population through cell cycle phases. Cells
5
0.99 (3H, t, J=7.5 Hz, CH3) ppm; 13C NMR δ (500 MHz, CDCl3): were seeded in 6-well plates (5 10 cells per well) and, once
×
159.46, 138.09, 117.53, 117.39, 55.39, 25.84, 22.25, 14.03 ppm; adherent, were cultured for 24 h in the presence of selected
HRMS m/z: calcd. for C10H14O2S2 (M+H)+: 231.0508; found: thiosulfinates at the indicated concentrations. Colchicine was
231.0507.
used as a positive control at 1 µmol/L (Supplementary mate-
rial), and control cells were incubated with DMSO at 0.1%
(v/v). After incubation, the cells were harvested, washed twice
S-4-hydroxyphenyl hexane-1-sulfinothioate (22b)
Yield: 12%, as a yellow oil; IR vmax cm-1: 1038 (S=O); H NMR with PBS, and fixed with 70% ice-cold ethanol for at least 24 h.
δ (500 MHz, CDCl3): 7.40 (2H, d, J=8.5 Hz, ArH-2), 6.74 (2H, The fixed cells were then washed twice with PBS, resuspended
d, J=8.5 Hz, ArH-3), 3.10 (2H, t, J=7.5 Hz, SCH2), 1.85 (2H, in fresh PBS containing 0.1 mg/mL RNase A, 20 µg/mL
quintet, J=7.5 Hz, SCH2CH2), 1.49–1.46 (2H, m, SCH2CH2CH2), propidium iodide (PI) and 0.1% (v/v) Triton X-100, and then
1.36–1.32 (4H, m, CH2CH2CH3), 0.91 (3H, t, J=7.5 Hz, CH3) incubated in the dark at 37 °C for 30 min. After incubation,
1
ppm; 13C NMR δ (500 MHz, CDCl3): 159.37, 138.09, 117.60, the cells were analyzed by flow cytometry (LSRFortessa X-20,
4
×
117.37, 55.66, 31.65, 28.65, 23.80, 22.72, 14.30 ppm; HRMS m/z: BD Biosciences), with 1 10 events per sample acquired. Cell
calcd. for C12H18O2S2 (M+H)+: 259.0821; found: 259.0824.
cycle distribution was calculated using FlowJo version 10.1
(Tree Star, Inc, Australia).
Cell culture
The drug-sensitive human breast adenocarcinoma cell line Apoptosis studies
MCF-7 and its drug-resistant sub-line, MCF-7/Dx (designated The extent of apoptosis was quantitatively measured using
Dx for simplicity), were used in this study. Cells were grown the Annexin V binding assay according to the manufacturer’s
in RPMI-1640 media (Sigma-Aldrich, NSW, Australia) supple- recommendation (BD Biosciences, Sydney, NSW, Austra-
mented with 10% heat-inactivated fetal bovine serum (Invit- lia). Cells were seeded in 6-well plates (5×105 cells per well)
rogen, Life Technologies, Victoria, Australia) and maintained and, once adherent, were cultured for 24 h in the presence of
at 37°C and 5% CO2. Dx cells were initially developed from selected thiosulfinates at the indicated concentrations. Control
MCF-7 cells by incremental exposure to doxorubicin[51]. To cells were incubated with DMSO at 0.1% (v/v). After the incu-
maintain the MDR phenotype, Dx cells were cultured in the bation period, cells were harvested and washed twice with
presence of 1 μg/mL doxorubicin; however, during experi- PBS and then resuspended in 1×binding buffer (1×106 cells/
ments, cells were cultured in the absence of doxorubicin. Cells mL). Next, 5 µL of Annexin V and 5 µL of PI (BD Pharmin-
were routinely tested for mycoplasma contamination.
gen, North Ryde, NSW, Australia) were added to a 100-µL
aliquot of the cell suspension, which was then vortexed and
incubated at RT in the dark for 15 min. Subsequently, 200 µL
Cell antiproliferative activity assay
The anticancer activity of the synthesized compounds was of 1×binding buffer was added to the mixture, and the cells
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7
were analyzed immediately by flow cytometry (LSRFortessa of variance (ANOVA) followed by Tukey’s post hoc analysis.
X-20, BD Biosciences), with 1×104 events per sample acquired. P<0.05 were accepted as being statistically significant. All
The percentages of viable (Annexin V–/PI–), early apoptotic statistical analyses were performed using GraphPad Prism
(Annexin V+/PI–), late apoptotic (Annexin V+/PI+), and version 7.0 (GraphPad Software, CA, USA). The results are
necrotic (Annexin V–/PI+) cells were calculated using FlowJo expressed as the mean±SEM unless otherwise noted.
version 10.1 (Tree Star, Inc).
Results
Chemistry
Measurement of mitochondrial function
Mitochondrial function was measured by determining the The symmetric disulfides 2a–6a, 13a and 20a were synthesized
oxygen consumption rate (OCR) of cells with a Seahorse XF24 via oxidative coupling (Scheme 1, Eq 1) using CsF-Celite as
extracellular flux analyzer (Seahorse Bioscience, MA, USA) a solid base[54]. Synthesis of the short chain disulfides, 2a–4a,
according to the manufacturer’s protocol. Cells were seeded was carried out at room temperature, after which any remain-
4
×
in 24-well XF cell culture microplates (2 10 cells per well) ing starting materials were removed under reduced pressure
and, once adherent, were cultured with selected thiosulfinates to generate the pure disulfides at good yields (>90%). For
for 24 h at 37 °C and 5% CO2. Prior to analysis, cells were disulfides 5a–6a, 13a and 20a, it was found that at room tem-
washed, and the supernatant was replaced with buffered XF perature, only trace amounts of the product were formed after
Base Medium supplemented with 2 mmol/L L-glutamine, 10 a 48-h period. These compounds were instead synthesized
mmol/L glucose and 2 mmol/L sodium pyruvate at pH=7.4. under reflux, which improved yields.
The cells were incubated at 37 °C without CO2 for an hour,
and then, the OCR was measured utilizing an XF Cell Mito
Stress Test Kit (Seahorse Bioscience, MA, USA). Oligomycin
(final concentration 1 µmol/L), carbonylcyanide m-chloro-
phenylhydrazone (FCCP-final concentration 0.5 µmol/L), and
rotenone/antimycin A (final concentration 0.5 µmol/L) were
added to the sensor cartridge, and the OCR was measured
using the standard cycling program.
Cell imaging
Cells were treated with thiosulfinates to examine effects on
5
×
cellular morphology. Cells were seeded in 6-well plates (7 10
cells per well) and, once adherent, were cultured for 24 h in
the presence of selected thiosulfinates at the indicated con-
centrations. Control cells were incubated with DMSO at 0.1%
(v/v). After the incubation period, images were captured on a
digital camera (INFINITY 1, Lumenera, Ottawa).
Scheme 1. Synthetic methods for the preparation of symmetric thio-
sulfinates 1b–6b, 13b and 20b.
Trypan blue exclusion assay
Cells were harvested and stained with Trypan blue, and viable
The asymmetric disulfides 7a–11a, 14a–19a, and 21a–22a
cells were counted on a hemocytometer. Cell mortality was were synthesized by nucleophilic substitution (Scheme 2, Eq 3)
expressed as a percentage of viable cells relative to the vehicle via a sulfenyl chloride intermediate[46], and all symmetric and
control. The vehicle control DMSO concentration did not asymmetric disulfides were used in the next synthesis step
exceed 0.1% (v/v).
without any further purification. Symmetric and asymmetric
disulfides were converted to the corresponding thiosulfinate
(Eq 2 and 4) by oxidation, using m-chloroperoxybenzoic acid,
Calculation of drug-likeness properties and toxicity prediction
Toxicity risk parameters and drug-likeness properties were to provide our target thiosulfinates.
calculated using OSIRIS Property Explorer. OSIRIS analyzes
The formation of thiosulfonates (R1-S-SO2-R2) was totally
molecules with respect to Lipinski’s rule of five, generating avoided when the reactions were performed at -78°C[55]. TLC
both a drug-likeness score and an overall drug score in addi- and NMR analysis of the crude reaction mixtures confirmed
tion to identifying any toxicity risks[52, 53]. To comply with the absence of thiosulfonate formation at this temperature
Lipinski’s rule of five, compounds must have a molecular (data not shown). Crude thiosulfinate mixtures were puri-
weight of less than 500 g/mol, LogP≤5, hydrogen bond donors fied by flash column chromatography, with ethyl acetate
≤5, hydrogen bond acceptors ≤10, a solubility score between -5 and n-hexane used as an eluent. The structures of the final
and 1, and a polar surface area less than 120 Å2.
compounds 1b–22b (Figure 1) were confirmed by analysis of
their spectral and analytical data (Supplementary material).
Compounds 11b and 12b were produced from the oxidation
Statistical analysis
For in vitro assays, data were analyzed using one-way analysis of compound 11a, and due to their almost identical physical
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Roseblade A et al
Scheme 2. Synthetic methods for the preparation of asymmetric thiosulfinates 7b–12b, 14b–19b and 21b–22b.
Figure 1. Chemical structures of synthesised thiosulfinates.
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9
properties, separation was not possible during the purifica-
tion step. 11b and 12b, formed at a ratio of 4:3, were treated
as a mixture and will, for simplicity, henceforth be collectively
referred to as 11b. To evaluate degradation associated with
the storage conditions, compounds 7b and 8b were dissolved
in chloroform and stored at room temperature for 3 weeks.
Spectral analysis did not show decomposition, but allicin
stored under similar conditions showed a significant increase
in degradation by-products (data not shown). Compound
13b was also found to be significantly more stable than allicin.
Spectral analysis of pure 13b did not show signs of decompo-
sition after >12 months of storage at room temperature (data
not shown).
The half-life of allicin is drastically increased when stored
at -80°C, with no detectable loss of purity after 24 months of
storage at this temperature[56]. Consequently, all compounds
were stored under these conditions until required. The stabil-
ity of thiosulfinates is significantly improved when dissolved
in polar solvents; therefore, all working stock solutions were
dissolved in DMSO and stored at -20 °C[57]. To ensure that
these compounds were suitable for biological application, we
analyzed each with OSIRIS Property Explorer to determine
the toxicity risks and drug-likeness of each molecule (Supple-
mentary material, Table S1). Each compound was found to
be free of any toxicity risks, with all compounds free of muta-
genic, tumorigenic, reproductive or irritant-like effects. Of the
compounds tested, only 20b had Lipinski violations, due to a
high predicted LogP and poor solubility, indicating that poor
absorption or poor permeability is likely[58]. This compound
did show some anticancer activity against both MCF-7 and Dx
cells (Supplementary material); however, in agreement with
the OSIRIS prediction, it was poorly soluble in both DMSO
and aqueous solutions and as such was excluded from further
testing.
Figure 2. Antiproliferative activity of thiosulfinates. A) Specificity of
compounds 1b, 4b, 7b, 8b, 11b and 13b–19b at 50 µmol/L towards MDR
MCF-7/Dx cells, compared to drug sensitive MCF-7 cells. B) Comparison
of molecule potency at 50 µmol/L towards MCF/Dx cells, where side chain
R2 is conserved and electron donating functional group (R1) is altered.
Data represents the mean±SEM of at least 3 independent experiments.
In vitro anticancer activity
The in vitro antiproliferative activities of the prepared com-
pounds were evaluated against MCF-7 and MCF-7/Dx cells.
All thiosulfinates were initially screened at a single high dose
(50 µmol/L). Compounds that satisfied a predetermined level
of cellular viability (<50%), relative to a vehicle control, were
selected for further analysis.
It has previously been shown that after 24 h of incubation in
water at 37°C at a pH of 7.5, only 62% of pure allicin remains
in a test sample[59]. Therefore, for the 72-h viability assays,
the cell culture media was replaced daily with fresh allicin
medium. This considerably improved anticancer activity
against both cell lines compared to experiments in which cells
were treated in the same allicin stock for 72 h (Supplementary
material).
In preliminary screenings, compounds allicin, 4b, 7b, 8b,
11b, and 13b–19b showed pronounced growth inhibition
against MDR Dx cells at 50 µmol/L. However, at the same
concentration, only allicin and 13b reduced the cellular viabil-
ity of MCF-7 cells below 50%. The thiosulfinate derivatives
showed pronounced selectivity towards the MDR Dx cells
compared to the drug-sensitive MCF-7 cells (Figure 2A).
Amongst the aromatic thiosulfinates, there was a clear
correlation between anticancer activity and the substituent
attached to the sulfenyl sulfur (R1), with the more electron-
rich 4-methoxybenzyl group showing higher antiproliferative
effects compared to the 4-methylbenzyl group (Figure 2B).
Additionally, for the asymmetric thiosulfinates, the longer
and/or bulkier the substituent on the sulfinyl sulfur (R2), the
less potent the compound’s anticancer activity. This trend was
also apparent at lower concentrations (Supplementary mate-
rial). Accordingly, 4b, 7b, 8b, 11b, and 13a–19b were selected
for further analysis. The IC50 values of allicin, 4b, 7b, 8b, 11b,
and 13a–19b were determined and are summarized in Table
1. As with the initial screening, the compounds with the most
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Roseblade A et al
Table 1. In-vitro anticancer activity (IC50)a of selected active molecules.
MCF-7/Dx
IC50 (µmol/L)
MCF-7
IC50 (µmol/L)
Compound
b
Allicin
4b
7b
41.23±0.52
33.74±2.38
35.99±0.82
38.11±1.46
40.77±3.18
18.54±0.24
28.97±1.78
28.97±1.31
45.54±2.68
41.11±2.08
28.75±0.38
48.61±3.57
−
>50
>50
>50
8b
11b
13b
14b
15b
16b
17b
18b
19b
>50
46.50±1.98
>50
>50
>50
>50
>50
>50
a 50% inhibitory concentration after 72 h of treatment.
b Not reported.
Figure 3. Effects of thiosulfinate 13b on the cellular morphology and
viability of MCF-7/Dx cells. Images show morphological changes in MCF-
7/Dx cells treated for 24 h with A) vehicle control, B) 25 µmol/L, C) 37.5
µmol/L and D) 50 µmol/L. There was no decrease in cell viability at 12.5
µmol/L (98.33±1.86)%, a slight decrease at 25 µmol/L (91.75±4.31)%,
and significant decreases at 37.5 µmol/L and 50 µmol/L (43.13%±2.07%
and 27.29%±5.79%, respectively). Data and images represent the
mean±SEM of 3 independent experiments.
electron-rich group on the sulfenyl sulfur exhibited stronger
antiproliferative activity, with 13b (both R groups occupied by
the 4-methoxybenzyl moiety) exhibiting the greatest antican-
cer activity in both MCF-7 and Dx cells. Due to its promising
cytotoxic activity against both MCF-7 and Dx cells, 13b was
selected for further studies.
Cellular morphology
To observe the effects of thiosulfinate treatment on cellular
morphology, MCF-7/Dx cells were treated with various
concentrations of 13b for 24 h. The morphology of Dx cells
treated with 25 µmol/L 13b was not significantly affected after
24 h. However, at 37.5 and 50 µmol/L, cells displayed dis-
tinct membrane blebbing, cell shrinkage, detachment and an
apparent disaggregation (Figure 3). This reduction in viable
cells was confirmed using the Trypan blue exclusion assay,
which after treatment with 37.5 and 50 µmol/L 13b resulted in
a cell viability reduction to 43.13%±2.07% and 27.29%±5.79%,
respectively.
Cell cycle analysis
Many cytotoxic compounds inhibit cell growth by arrest-
ing cells at particular stages in the cell cycle. To investigate
whether the synthesized thiosulfinates had this effect, MCF-7/
Dx cells were incubated with various concentrations of 13b
for 24 h and then stained with PI as described above in the
Materials and Methods section. Consistent with its effect on
cell growth inhibition, 13b caused cell cycle arrest in Dx cells
in a dose-dependent manner (Figure 4). Cells treated with
the vehicle control showed a typical distribution of 44.30% of
cells in the G0/G1 phase, 38.13% in the S phase, and 16.70%
of cells in the G2/M phase (Table 2). Treatment of cells with
12.5 µmol/L 13b increased the proportion of cells in the G0/G1
phase to 50.54% and caused a small decrease in cells in the S
phase. At 37.5 µmol/L and 50 µmol/L, there were reductions
in the proportion of cells in the G0/G1 and S phases, while the
Figure 4. Effect of 13b on the cell cycle progression of MCF-7/Dx cells.
Cells were treated for 24 h with A) DMSO (vehicle control), B) 25 μmol/L,
C) 37.5 μmol/L and D) 50 μmol/L. Images are representative of 4
independent experiments.
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Table 2. Effect of compound 13b on the cell cycle progression of MCF-7/
Dx cellsa. *P<0.05, **P<0.01 compared with the indicated compound to
control cells in the corresponding cell cycle phase.
Conc
(μmol/L)
G0/G1 phase (%)
S phase (%)
G2/M phase (%)
Control
12.5
25
37.5
50
44.30±0.76
50.54±2.07
39.66±4.78
32.40±1.33*
29.37±3.37**
38.13±0.57
33.24±1.99
28.94±1.53*
26.90±3.56*
26.17±2.80**
16.70±0.77
15.42±0.77
30.46±6.06**
40.00±2.95**
44.83±3.19**
a MCF-7/Dx cells were treated with compound 13b then cell cycle distribu-
tion was analysed after staining with PI. Results are expressed as the
percentage of cells in each phase of the cell cycle, data represents the
mean±SEM of 4 independent experiments.
number of cells in G2/M increased significantly. After being
treated with 50 µmol/L, 29.37% of cells were found to be in
the G0/G1 phase, 26.17% in the S phase, and 44.83% arrested
in the G2/M phase. During the screening, many thiosulfinates
were also found to significantly arrest Dx cells in the G2/M
phase (Supplementary material). These data demonstrate that
synthesized thiosulfinates primarily arrest Dx cells in the G2/
Figure 5. Effect of compound 13b on the induction of apoptosis in MCF-
7/Dx cells. MCF-7/Dx cells were treated with various concentrations
M phase, significantly reducing the proportion of cells in the of 13b for 24 h, stained with Annexin V-450 and PI, and the apoptotic
effect was assessed by flow cytometry. Representative results are
shown, with quadrants indicating the proportion of cells that are necrotic:
G0/G1 and S phases.
Q1, late apoptotic: Q2, early apoptotic: Q3, and viable: Q4. Images are
representative of 3 independent experiments.
Induction of apoptosis
The translocation of phospholipid phosphatidylserine (PS)
to the outer leaflet of the cell plasma membrane is one of the
hallmarks of apoptosis[60]. This is followed by a loss of mem-
brane integrity and eventual cell death. To establish whether
thiosulfinates induce cell death via this mechanism, MCF-7/
Dx cells were treated with 12.5–50 µmol/L of 13b for 24 h and
then stained with Annexin V-450 (which binds to exposed
PS) and PI to determine the proportion of cells undergoing
early apoptosis (Annexin-positive, PI-negative), late apoptosis
and significant increases were observed in Dx cells treated
with 37.5 and 50 µmol/L 13b. The proportion of cells under-
going apoptosis at 25 µmol/L, 37.5 µmol/L and 50 µmol/L
was 7.06%±0.82%, 31.32%±8.06% and 57.52%±2.92%, respec-
tively, indicating that 13b induces apoptosis in a dose-depen-
dent manner (Table 3).
(Annexin-positive, PI-positive), and total apoptosis (early+late
apoptosis) (Figure 5).
At 12 µmol/L, there was no significant increase in the total
number of apoptotic cells relative to the solvent control. A
Measurement of mitochondrial function
Mitochondria play a central role in many biological processes,
especially in the initiation of apoptosis. To further investigate
the cytotoxic effects of thiosulfinates on MCF-7/Dx cells, we
small increase was observed in cells treated with 25 µmol/L,
sought to determine whether 13b disrupted mitochondrial
Table 3. Quantitative apoptosis assay of MCF-7/Dx cells treated with 13ba. **P<0.01 vs the % of apoptotic cells of the control.
Compound
(μmol/L)
Viable cells
Early apoptotic cells
Late apoptotic cells
Necrotic cells
Apoptotic cells
(Q4, %)
(Q3, %)
(Q2, %)
(Q1, %)
(Q2+Q3, %)
Control
13b
13b
13b
13b
0
12.5
25
37.5
50
96.89±0.48
94.65±0.65
91.88±0.81
62.58±7.34
36.85±3.33
1.48±0.22
2.16±0.10
3.93±0.09
18.97±6.88
33.35±1.87
1.03±0.23
1.95±0.27
3.13±0.87
12.35±1.85
24.17±2.92
0.60±0.11
1.24±0.25
1.05±0.26
6.07±2.15
5.61±0.66
2.51±0.45
4.10±0.36
7.06±0.82
31.32±8.06**
57.52±2.92**
a MCF-7/Dx cells were treated with various concentrations of 13b for 24 h, the apoptotic affect was assessed by flow cytometry after staining with
Annexin V-450 and PI. Data represents the mean±SEM of 3 independent experiments.
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Roseblade A et al
respiratory function, which was investigated using the XF24 lation/respiration, they often compensate by increasing gly-
extracellular flux analyzer. As allicin has previously been colysis to maintain ATP production[61]. Due to the decrease
shown to interfere with mitochondrial functions in various cell observed in the OCR, we investigated the effects of allicin and
lines, including MCF-7, we included it for comparison[25, 29]
.
13b on the ECAR, a surrogate marker for glycolysis[62]. The
Dx cells were treated with 10–50 µmol/L of allicin and 13b glycolytic potential was also measured by the addition of oli-
for 24 h, with both OCR and extracellular acidification rate gomycin, which forces the cells to compensate by increasing
(ECAR) measured using the “mitochondrial stress test” (Agi- glycolysis (Supplementary material). Despite decreases in
lent Technologies, CA, USA). Sequential addition of the mito- the OCR at all tested concentrations, there was no significant
chondrial complex inhibitors oligomycin, FCCP, antimycin A effect on basal ECAR or glycolytic potential.
and rotenone allowed for the determination of several mito-
chondrial function parameters. Treatment with allicin and
Discussion
13b inhibited the basal OCR relative to the vehicle control in a Allicin has numerous health benefits and has been shown to
dose-dependent manner (Figure 6). The addition of oligomy- be effective against many diseases. Use as a therapeutic agent
cin, an ATP synthase inhibitor, allows for the determination is limited, however, due to a propensity for allicin to degrade
of ATP-linked respiration, with both allicin and 13b inducing under relatively mild conditions. In considering this, we
a decrease. The addition of FCCP, an electron transport chain developed a library of allicin derivatives and assessed their
uncoupler, allows for the measurement of maximum cellu- anticancer activity against the breast adenocarcinoma cell line
lar respiration rate in response to increased energy demand. MCF-7 and the MDR sub-line MCF-7/Dx. In an effort to stabi-
There was a significant dose-dependent decrease observed for lize the functionality of the thiosulfinate group, we substituted
both compounds. Likewise, spare respiratory capacity was the labile allylthio moiety with various substituents. Spectral
significantly impaired after treatment with both thiosulfinates. analysis of aryl thiosulfinates stored in chloroform at room
When cells lose the ability to maintain oxidative phosphory- temperature for several weeks showed minimal decomposi-
Figure 6. Effect of thiosulfinates on mitochondrial function. Cells were seeded in XF24 well plates and treated for 24 h with A) 13b and B) allicin at the
indicated concentrations. Medium was then replaced with unbuffered XF assay medium, supplemented with sodium pyruvate, glucose and L-glutamine,
and allowed to equilibrate for 1 h. Baseline measurements were recorded then oligomycin, FCCP, antimycin A and rotenone were injected sequentially
at the indicated time points. Data represents the mean±SEM of at least 4 independent experiments. *P<0.05, **P<0.01, compared with the indicated
compound to the vehicle control.
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Roseblade A et al
13
tion, while allicin showed distinct increases in impurities (data malignant human brain epithelial cells (HBEC) (Supplemen-
not shown). Additionally, compound 13b was found to be sta- tary material). Interestingly, when treated with allicin, HBEC
ble after storage at room temperature for over a year, whereas cells showed levels of cytotoxicity similar to those observed
allicin is known to be incredibly unstable when stored in its in Dx cells (data not shown), despite the numerous reports of
pure state under these conditions[63]. These results reflect allicin’s cancer-specific activity. In light of this, we are cur-
previous findings that electron-rich aryl-substituted thiosul- rently undertaking further studies to improve the specificity of
finates are much more stable than allicin under similar condi- these molecules through modification of the pharmacophore.
tions[40, 57]. These results were also reflected in our 72-h antip- Additionally, it would be interesting to investigate the anti-
roliferative assay (Figure 2A). The majority of thiosulfinates cancer effect of these molecules on hematological cell lines, as
tested showed improved activity compared to cells incubated it has previously been shown that allicin is more toxic to cells
with allicin, which we propose is in part due to the increased in suspension compared to cells in monolayers[68]
stability of these compounds. To maintain activity for the Allicin has previously been shown to rapidly induce mor-
.
duration of the assay, allicin was replenished with fresh stock phological changes in colon carcinoma cells, gastric cancer
and media every 24 h. Conversely, when incubated with the cells and MCF-7 breast cancer cells, as well as reduce cell
same allicin stock for 72 h, cell viability remained largely unaf- proliferation and induce growth arrest[26, 69, 70]. Similarly,
fected in both cell lines (Supplementary material). This sug- the thiosulfinates described in this study altered Dx cellular
gests that the antiproliferative activity observed in this assay is morphology. Cell shrinkage, detachment, and fragmentation
due to allicin and not its degradation by-products.
were observed 24 h after treatment (Figure 3). This change
Increasing the chain length of the symmetric alkyl thiosul- in morphology was evident as early as 6 h after initial treat-
finates showed a corresponding reduction in the proportion of ment (Supplementary material). To further investigate the
viable cells in both cell lines. However, when one alkyl chain mechanisms behind this reduced viability and altered cellular
was replaced with a benzyl functional group, the compounds morphology, we tested for apoptosis, as allicin and saturated
with shorter and less bulky alkyl groups showed more favor- short-chain thiosulfinates have been shown to induce apopto-
able results, with compounds containing the 4-methoxybenzyl sis in a number of cell lines[26–28]. In the present study, thiosul-
moiety showing the greatest activity (Figure 2). We propose finates were found to induce apoptosis in Dx cells to varying
that this improved activity of the benzyl group over the degrees (Supplementary material), with 13b showing the most
straight chain substituents is due in part to the benzyl group activity, inducing apoptosis in a dose-dependent manner
acting as a bioisostere for the allyl group.
(Table 3).
Several thiosulfinate derivatives exhibited cytotoxic activ-
The effect of allicin on viability and apoptosis has been
ity towards Dx cells (Table 1). However, of the compounds linked to a reduction in intracellular GSH, which indicates an
tested, only 13b displayed an IC50 <50 µmol/L in MCF-7 cells. alteration in the cellular redox state[25, 28, 71]. GSH reduction
These results highlight the disparity in the anticancer activity occurs in response to sustained production of ROS upon expo-
(IC50) of the thiosulfinates against the drug-sensitive MCF-7 sure to allicin, which then leads to a decrease in mitochondrial
cells vs MDR Dx cells. Since Dx cells express P-gp, it was potential, a reduction in ATP generation and subsequent cell
expected that they would be less sensitive to treatment, as Dx death[16, 29, 72]. ROS are generated in cells under normal condi-
cells exhibit up to a 200-fold resistance to conventional chemo- tions; however, production is elevated when there is a disrup-
therapeutics in vitro[64]
.
tion either in the electron transport chain or ATP generation.
A possible explanation for this selectivity could be linked to Tumor cells inherently show elevated metabolic activity and
caspase-3 expression. MCF-7 cells do not express caspase-3, oxygen consumption and produce high levels of ROS under
a key player in the apoptosis cascade[65]. Conversely, Dx cells normal conditions, making them more susceptible to damage
do express caspase-3 and, in direct comparison with MCF-7 from excess ROS production[73, 74]. As mitochondria are major
cells, have been shown to be significantly more susceptible to mediators of apoptosis and cellular bioenergetics, modula-
certain apoptotic stimuli[64, 66]. Whether this accounts for the tion of mitochondrial respiratory function in an effort to kill
disparity in the anticancer activity shown in Table 1 is cur- cancer cells is attracting interest as a therapeutic strategy[74–76]
.
rently unknown. As allicin has been shown to initiate apopto- To investigate the possible mitochondrial effects of the syn-
sis through both caspase-dependent and caspase-independent thesized thiosulfinates on Dx cells, we used the XF24 extracel-
mechanisms in various cell lines[27, 67], further work is required lular flux analyzer, allowing for the measurement of multiple
to determine whether Dx cell death is executed via this path- parameters associated with mitochondrial function[77]
.
way.
In this study, we found that allicin and compound 13b cause
Allicin has previously been shown to be cancer specific, similar responses in Dx cells, with significant reductions in
inducing significantly lower levels of cytotoxicity against oxygen consumption both at basal levels and under stress, as
normal cells[18, 25, 35]. Additionally, short chain thiosulfinates, well as a decrease in ATP turnover (Figure 6). This suggests
containing both allyl and saturated chains, show good activity that these compounds reduce the cell’s ability to generate a
in vivo, significantly increasing the survival times of sarcoma- membrane potential and its capacity to synthesize ATP[77]
.
bearing mice[36]. Our most potent molecule, 13b, did not show Spare respiratory capacity was also significantly impaired
specificity toward cancer cells, with effects observed in non- after treatment with both thiosulfinates, which indicates a loss
Acta Pharmacologica Sinica

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14
Roseblade A et al
of mitochondrial membrane potential as well as a significantly breast cancer cell lines. Several compounds showed specificity
reduced capacity to respond to stressors, such as ROS[78, 79]. We for the MDR cells, and Dx cells showed collateral sensitivity
hypothesize that these compounds damage the mitochondrial to treatment with 13b. We report that these derivatives
electron transport chain through an elevation in ROS, which alter cellular morphology, induce apoptosis and cell cycle
initiates the apoptosis cascade leading to cell death. Our arrest, and inhibit mitochondrial bioenergetics, presenting
investigations are ongoing to determine the precise molecular a promising lead for future studies in the treatment of MDR
mechanisms underlying this process.
cancer.
These disruptions to mitochondrial respiration offer another
possible explanation for the observed sensitivity of MDR Dx
Author contribution
cells to thiosulfinate treatment relative to the drug-sensitive Mary BEBAWY and Alison UNG designed the research;
MCF-7 cells. Dx cells may be more susceptible to thiosulfinate Ariane ROSEBLADE performed the research; Ariane ROSE-
treatment due to the effects of a phenomenon known as col- BLADE, Mary BEBAWY and Alison UNG analyzed the data
lateral sensitivity. Collateral sensitivity is the ability of a com- and wrote the paper.
pound to preferentially kill MDR cells over drug-sensitive cells
and is generally associated with the over-expression of P-gp[80]
.
Supplementary information
Various mechanisms have been proposed for the effects of CS, Supplementary information is available at Acta Pharmaco-
such as the down-regulation of P-gp expression, disruption of logica Sinica’s website
oxygen consumption, elevated ROS, and depletion of cellular S1. Biological figures and tables.
ATP[81–83]. CS is represented as a ratio, which is determined by S2. 1H NMR, 13C NMR, 13C DEPT and 1H-13C HSQC spectra.
dividing the IC50 of the MDR sub-line by the IC50 of the drug-
sensitive parental line to produce a resistance ratio ≤0.5[84]
.
References
1
Torre LA, Bray F, Siegel RL, Ferlay J, Lortet Tieulent J, Jemal A. Global
-
For 13b, the IC50 was 46.50 µmol/L for MCF-7 cells and 18.54
mmol/L for Dx cells, providing a resistance ratio of 0.40. In
conjunction with the ability of 13b to reduce ATP-linked respi-
ration and to disrupt OCR under stress and at basal levels, this
result suggests that 13b is a potential CS agent.
cancer statistics, 2012. CA Cancer J Clin 2015; 65: 87–108.
Szakács G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM.
Targeting multidrug resistance in cancer. Nat Rev Drug Discov 2006; 5:
219–34.
2
3
Longley DB, Johnston PG. Molecular mechanisms of drug resistance.
J Pathol 2005; 205: 275–92.
Garlic-derived compounds, including allicin, are known to
inhibit cancer cell growth by disrupting the cell cycle, caus-
ing cells to accumulate predominately in the G2/M phase in
direct response to the overproduction of ROS and disruption
to mitochondrial respiration[85, 86]. Treatment with compound
13b resulted in a dose-dependent accumulation of cells in
G2/M (Table 2). Additionally, during initial screenings at 50
µmol/L, the cell cycle was found to be arrested at the G2/M
phase for most of the thiosulfinates tested (Supplementary
material). For allicin, we did not find any change in cell cycle
distribution at this concentration; however, higher concentra-
tions increased accumulation in G0/G1 and G2/M, with these
4
Sharom FJ, Yu X, Doige CA. Functional reconstitution of drug transport
and ATPase activity in proteoliposomes containing partially purified
P-glycoprotein. J Biol Chem 1993; 268: 24197–202.
Breuninger LM, Paul S, Gaughan K, Miki T, Chan A, Aaronson SA, et
al. Expression of multidrug resistance-associated protein in NIH/3T3
cells confers multidrug resistance associated with increased drug
efflux and altered intracellular drug distribution. Cancer Res 1995;
55: 5342–7.
5
6
7
Leonard GD, Fojo T, Bates SE. The role of ABC transporters in clinical
practice. Oncologist 2003; 8: 411–24.
Tiwari AK, Sodani K, Dai CL, Ashby CR, Chen ZS. Revisiting the
ABCs of multidrug resistance in cancer chemotherapy. Curr Pharm
Biotechnol 2011; 12: 570–94.
results reflecting a previous study on MCF-7 cells[25]
.
In addition to antiproliferative effects, thiosulfinates have
also shown promise as antimetastatic agents. It has recently
been reported that blocking mitochondrial ATP production
inhibits breast cancer cell migration and invasion. Likewise,
allicin has been shown to induce cell cycle arrest through
microtubule disruption and to suppress invasion and metas-
tasis of breast cancer in vitro[30, 70, 87]. As we have shown that
allicin and 13b inhibit ATP-linked mitochondrial respiration,
we hypothesize that these compounds are likely to influence
cellular migration and therefore warrant further investigation.
In conclusion, allicin has previously been shown to exhibit
many beneficial biological effects, including strong anticancer
activity. In light of the instability of allicin in vitro and in vivo,
we sought to improve its activity by developing a library of
thiosulfinate derivatives with increased half-life and improved
efficacy. We found that these thiosulfinate derivatives have
improved antiproliferative activity in drug-sensitive and MDR
8
Saab AM, Guerrini A, Zeino M, Wiench B, Rossi D, Gambari R, et
al. Laurus nobilis L. seed extract reveals collateral sensitivity in
multidrug-resistant p-glycoprotein-expressing tumor cells. Nutr Cancer
2015; 67: 664–75.
Huang TH, Bebawy M, Tran VH, Roufogalis BD. Specific reversal of
multidrug resistance to colchicine in CEM/VLB 100 cells by Gyno-
stemma pentaphyllum extract. Phytomedicine 2007; 14: 830–9.
9
10 Tran VH, Marks D, Duke RK, Bebawy M, Duke CC, Roufogalis BD.
Modulation of P-glycoprotein-mediated anticancer drug accumulation,
cytotoxicity, and ATPase activity by flavonoid interactions. Nutr Cancer
2011; 63: 435–43.
11 Block E. The organosulfur chemistry of the genus allium–implications
for the organic chemistry of sulfur. Angew Chem Int Ed Engl 1992;
31: 1135–78.
12 Park EK, Kwon KB, Park KI, Park BH, Jhee EC. Role of Ca2+ in diallyl
disulfide-induced apoptotic cell death of HCT-15 cells. Exp Mol Med
2002; 34: 250–7.
13 Herman-Antosiewicz A, Singh SV. Signal transduction pathways
Acta Pharmacologica Sinica

www.chinaphar.com
Roseblade A et al
15
leading to cell cycle arrest and apoptosis induction in cancer cells by 32 Rendu F, Brohard-Bohn B, Pain S, Bachelot-Loza C, Auger J. Thio-
Allium vegetable-derived organosulfur compounds: a review. Mutat
Res 2004; 555: 121–31.
sulfinates inhibit platelet aggregation and microparticle shedding at a
calpain-dependent step. Thromb Haemost 2001; 86: 1284–91.
14 Xu YS, Feng JG, Zhang D, Zhang B, Luo M, Su D, et al. S-allylcysteine, 33 Roseblade A, Luk F, Ung A, Bebawy M. Targeting microparticle
a garlic derivative, suppresses proliferation and induces apoptosis in
human ovarian cancer cells in vitro. Acta Pharmacol Sin 2014; 35:
267–74.
biogenesis: a novel approach to the circumvention of cancer multidrug
resistance. Curr Cancer Drug Targets 2015; 15: 205–14.
34 Roseblade A, Luk F, Rawling T, Ung A, Grau GE, Bebawy M. Cell-
derived microparticles: new targets in the therapeutic management of
disease. J Pharm Pharm Sci 2013; 16: 238–53.
35 Ghazanfari T, Yaraee R, Rahmati B, Hakimzadeh H, Shams J, Jalali-
Nadoushan MR. In vitro cytotoxic effect of garlic extract on malignant
and nonmalignant cell lines. Immunopharmacol Immunotoxicol 2011;
33: 603–8.
36 Park KW, Kim SY, Jeong IY, Byun MW, Park KH, Yamada K, et al.
Cytotoxic and antitumor activities of thiosulfinates from Allium
tuberosum L. J Agric Food Chem 2007; 55: 7957–61.
15 Chung LY. The antioxidant properties of garlic compounds: allyl
cysteine, alliin, allicin, and allyl disulfide. J Med Food 2006; 9: 205–
13.
16 Corral MJ, Benito-Peña E, Jiménez-Antón MD, Cuevas L, Moreno-
Bondi MC, Alunda JM. Allicin induces calcium and mitochondrial
dysregulation causing necrotic death in leishmania. PLoS Negl Trop
Dis 2016; 10: e0004525.
17 Waag T, Gelhaus C, Rath J, Stich A, Leippe M, Schirmeister T. Allicin
and derivates are cysteine protease inhibitors with antiparasitic
activity. Bioorg Med Chem Lett 2010; 20: 5541–3.
18 Ilić D, Nikolić V, Ćirić A, Soković M, Stanojković T, Kundaković T, et al.
Cytotoxicity and antimicrobial activity of allicin and its transformation
products. Med Plant Res 2012; 6: 59–65.
37 Block E, Ahmad S, Catalfamo JL, Jain MK, Apitz-Castro R. Antithro-
mbotic organosulfur compounds from garlic: structural, mechanistic,
and synthetic studies. J Am Chem Soc 1986; 108: 7045–55.
38 Cavallito CJ, Bailey JH. Allicin, the antibacterial principle of Allium
19 Cañizares P, Gracia I, Gómez LA, de Argila CM, Boixeda D, García A, et
al. Allyl-thiosulfinates, the bacteriostatic compounds of garlic against
Helicobacter pylori. Biotechnol Prog 2004; 20: 397–401.
20 Briggs WH, Xiao H, Parkin KL, Shen C, Goldman IL. Differential
sativum I. Isolation, physical properties and antibacterial action. J
Am Chem Soc 1944; 66: 1950–1.
39 Small LD, Bailey JH, Cavallito CJ. Alkyl thiolsulfinates. J Am Chem Soc
1947; 69: 1710–3.
inhibition of human platelet aggregation by selected allium thiosul- 40 Stellenboom N, Hunter R, Caira MR, Bourne SA, Cele K, Qwebani T,
finates. J Agric Food Chem 2000; 48: 5731–35.
et al. Synthesis and inclusion of S-aryl alkylthiosulfinates as stable
21 Dirsch VM, Kiemer AK, Wagner H, Vollmar AM. Effect of allicin and
allicin mimics. ARKIVOC 2007; 9: 53–63.
ajoene, two compounds of garlic, on inducible nitric oxide synthase. 41 Hughes TV, Hammond SD, Cava MP. A convenient new synthesis of
Atherosclerosis 1998; 139: 333–39.
22 Cha JH, Choi YJ, Cha SH, Choi CH, Cho WH. Allicin inhibits cell growth
1-cyanobenzotriazole and its use as a C-cyanating reagent. J Org
Chem 1998; 63: 401–2.
and induces apoptosis in U87MG human glioblastoma cells through 42 Natarajan P, Sharma H, Kaur M, Sharma P. Haloacid/dimethyl
an ERK-dependent pathway. Oncol Rep 2012; 28: 41.
sulfoxide-catalyzed synthesis of symmetrical disulfides by oxidation of
23 Wang Z, Liu Z, Cao Z, Li L. Allicin induces apoptosis in EL-4 cells
thiols. Tetrahedron Lett 2015; 56: 5578–82.
in vitro by activation of expression of caspase-3 and-12 and up- 43 Dong WL, Huang GY, Li ZM, Zhao WG. A convenient method for the
regulation of the ratio of Bax/Bcl-2. Nat Prod Res 2012; 26: 1033–7.
24 Lee J, Gupta S, Huang JS, Jayathilaka LP, Lee BS. HPLC–MTT assay:
aerobic oxidation of thiols to disulfides. Phosphorus Sulfur Silicon
2009; 184: 2058–65.
Anticancer activity of aqueous garlic extract is from allicin. Anal 44 Harpp DN, Smith RA. Reaction of trialkyl phosphites with organic
Biochem 2013; 436: 187–9.
trisulfides. Synthetic and mechanistic aspects. J Org Chem 1979; 44:
25 Hirsch K, Danilenko M, Giat J, Miron T, Rabinkov A, Wilchek M, et al.
4140–44.
Effect of purified allicin, the major ingredient offreshly crushed garlic, 45 Hunter R, Caira M, Stellenboom N. Inexpensive, one-pot synthesis of
on cancer cell proliferation. Nutr Cancer 2000; 38: 245–54.
26 Sun L, Wang X. Effects of allicin on both telomerase activity and
unsymmetrical disulfides using 1-chlorobenzotriazole. J Org Chem
2006; 71: 8268–71.
apoptosis in gastric cancer SGC-7901 cells. World J Gastroenterol 46 Stellenboom N, Hunter R, Caira MR. One-pot synthesis of
2003; 9: 1930–4.
unsymmetrical disulfides using 1-chlorobenzotriazole as oxidant:
Interception of the sulfenyl chloride intermediate. Tetrahedron 2010;
66: 3228–41.
27 Oommen S, Anto RJ, Srinivas G, Karunagaran D. Allicin (from garlic)
induces caspase-mediated apoptosis in cancer cells. Eur J Pharmacol
2004; 485: 97–103.
28 Miron T, Wilchek M, Sharp A, Nakagawa Y, Naoi M, Nozawa Y, et al.
Allicin inhibits cell growth and induces apoptosis through the mito-
47 Tanaka K, Ajiki K. Phosphine-free cationic rhodium (I) complex-
catalyzed disulfide exchange reaction: convenient synthesis of
unsymmetrical disulfides. Tetrahedron Lett 2004; 45: 5677–9.
chondrial pathway in HL60 and U937 cells. J Nutr Biochem 2008; 19: 48 Backer HJ, Kloosterziel H. Esters thiolsulfiniques. Recl Trav Chim
524–35. Pays-Bas Et Belg 1954; 73: 129–39.
29 Chu YL, Ho CT, Chung JG, Raghu R, Lo YC, Sheen LY. Allicin induces 49 Colonna S, Gaggero N, Carrea G, Pasta P, Alphand V, Furstoss R.
anti-human liver cancer cells through the p53 gene modulating
apoptosis and autophagy. J Agric Food Chem 2013; 61: 9839–48.
30 Lee CG, Lee HW, Kim BO, Rhee DK, Pyo S. Allicin inhibits invasion and
Enantioselective synthesis of tert-butyl tert-butanethiosulfinate
catalyzed by cyclohexanone monooxygenase. Chirality 2001; 13:
40–2.
migration of breast cancer cells through the suppression of VCAM- 50 McNeil NM, McDonnell C, Hambrook M, Back TG. Oxidation of
1: Regulation of association between p65 and ER-α. J Funct Foods
2015; 15: 172–85.
disulfides to thiolsulfinates with hydrogen peroxide and a cyclic
seleninate ester catalyst. Molecules 2015; 20: 10748–62.
31 Bebawy M, Combes V, Lee E, Jaiswal R, Gong J, Bonhoure A, et al. 51 Kars MD, Iseri OD, Gunduz U, Ural AU, Arpaci F, Molnar J. Develop-
Membrane microparticles mediate transfer of P-glycoprotein to drug
sensitive cancer cells. Leukemia 2009; 23: 1643–9.
ment of rational in vitro models for drug resistance in breast cancer
and modulation of MDR by selected compounds. Anticancer Res
Acta Pharmacologica Sinica

www.nature.com/aps
16
Roseblade A et al
2006; 26: 4559–68.
division via its direct effect on microtubules. Cell Motil Cytoskeleton
2007; 64: 321–37.
71 Filomeni G, Rotilio G, Ciriolo MR. Cell signalling and the glutathione
redox system. Biochem Pharmacol 2002; 64: 1057–64.
72 Marchetti P, Decaudin D, Macho A, Zamzami N, Hirsch T, Susin SA, et
al. Redox regulation of apoptosis: Impact of thiol oxidation status on
mitochondrial function. Eur J Immunol 1997; 27: 289–96.
73 Fath MA, Diers AR, Aykin-Burns N, Simons AL, Hua L, Spitz DR.
Mitochondrial electron transport chain blockers enhance 2-deoxy-
D-glucose induced oxidative stress and cell killing in human colon
carcinoma cells. Cancer Biol Ther 2009; 8: 1228–36.
52 Lipinski CA. Drug-like properties and the causes of poor solubility and
poor permeability. J Pharmacol Toxicol Methods 2000; 44: 235–49.
53 OSIRIS Property Explorer [10 Jun 2016]. Available from: http://www.
organic-chemistry.org/prog/peo/.
54 Shah STA, Khan KM, Fecker M, Voelter W. A novel method for the
syntheses of symmetrical disulfides using CsF-Celite as a solid base.
Tetrahedron Lett 2003; 44: 6789–91.
55 Hunter R, Caira M, Stellenboom N. Thiolsulfinate allicin from garlic:
inspiration for a new antimicrobial agent. Ann N Y Acad Sci 2005;
1056: 234–41.
56 Lawson LD, Gardner CD. Composition, stability, and bioavailability of 74 Pelicano H, Feng L, Zhou Y, Carew JS, Hileman EO, Plunkett W, et al.
garlic products used in a clinical trial. J Agric Food Chem 2005; 53:
6254–61.
57 Fujisawa H, Suma K, Origuchi K, Kumagai H, Seki T, Ariga T. Biological
Inhibition of mitochondrial respiration a novel strategy to enhance
drug-induced apoptosis in human leukemia cells by a reactive oxygen
species-mediated mechanism. J Biol Chem 2003; 278: 37832–9.
and chemical stability of garlic-derived allicin. J Agric Food Chem 75 Costantini P, Jacotot E, Decaudin D, Kroemer G. Mitochondrion as a
2008; 56: 4229–35.
58 Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and
novel target of anticancer chemotherapy. J Natl Cancer Inst 2000;
92: 1042–53.
computational approaches to estimate solubility and permeability in 76 Green DR, Reed JC. Mitochondria and apoptosis. Science 1998;
drug discovery and development settings. Adv Drug Deliv Rev 2012;
64: 4–17.
281: 1309.
77 Divakaruni A, Paradyse A, Ferrick D, Murphy A, Jastroch M. Analysis
and interpretation of microplate-based oxygen consumption and pH
data. Methods Enzymol 2013; 547: 309–54.
78 Pan J, Zhang Q, Liu Q, Komas SM, Kalyanaraman B, Lubet RA, et
al. Honokiol inhibits lung tumorigenesis through inhibition of mito-
chondrial function. Cancer Prev Res 2014; 7: 1149–59.
59 Freeman F, Kodera Y. Garlic chemistry: stability of S-(2-propenyl)-2-
propene-1-sulfinothioate (allicin) in blood, solvents, and simulated
physiological fluids. J Agric Food Chem 1995; 43: 2332–8.
60 Elmore S. Apoptosis: a review of programmed cell death. Toxicol
Pathol 2007; 35: 495–516.
61 Xu RH, Pelicano H, Zhou Y, Carew JS, Feng L, Bhalla KN, et al. 79 Dranka BP, Benavides GA, Diers AR, Giordano S, Zelickson BR, Reily C,
Inhibition of glycolysis in cancer cells: a novel strategy to overcome
drug resistance associated with mitochondrial respiratory defect and
hypoxia. Cancer Res 2005; 65: 613–21.
et al. Assessing bioenergetic function in response to oxidative stress
by metabolic profiling. Free Radic Biol Med 2011; 51: 1621–35.
80 Laberge RM, Ambadipudi R, Georges E. P-glycoprotein (ABCB1)
modulates collateral sensitivity of a multidrug resistant cell line to
verapamil. Arch Biochem Biophys 2009; 491: 53–60.
62 Radde BN, Alizadeh-Rad N, Price SM, Schultz DJ, Klinge CM.
Anacardic acid, salicylic acid, and oleic acid differentially alter cellular
bioenergetic function in breast cancer cells. J Cell Biochem 2016; 81 Pluchino KM, Hall MD, Goldsborough AS, Callaghan R, Gottesman
117: 2521–32.
MM. Collateral sensitivity as a strategy against cancer multidrug
resistance. Drug Resist Updat 2012; 15: 98–105.
63 Cavallito CJ, Bailey JH, Buck JS. The antibacterial principle of Allium
sativum. III. Its precursor and “Essential Oil of Garlic” 1. J Am Chem 82 Arora A, Seth K, Shukla Y. Reversal of P-glycoprotein-mediated
Soc 1945; 67: 1032–3.
multidrug resistance by diallyl sulfide in K562 leukemic cells and in
64 Chen JS, Konopleva M, Andreeff M, Multani AS, Pathak S, Mehta
mouse liver. Carcinogenesis 2004; 25: 941–9.
K. Drug-resistant breast carcinoma (MCF-7) cells are paradoxically 83 Höllt V, Kouba M, Dietel M, Vogt G. Stereoisomers of calcium anta-
sensitive to apoptosis. J Cell Physiol 2004; 200: 223–34.
65 Janicke RU. MCF-7 breast carcinoma cells do not express caspase-3.
Breast Cancer Res Treat 2009; 117: 219–21.
gonists which differ markedly in their potencies as calcium blockers
are equally effective in modulating drug transport by P-glycoprotein.
Biochem Pharmacol 1992; 43: 2601–8.
66 Leoni LM, Hamel E, Genini D, Shih H, Carrera CJ, Cottam HB, et al. 84 Hall MD, Handley MD, Gottesman MM. Is resistance useless? Multi-
Indanocine, a microtubule-binding indanone and a selective inducer
of apoptosis in multidrug-resistant cancer cells. J Natl Cancer Inst
2000; 92: 217–24.
drug resistance and collateral sensitivity. Trends Pharmacol Sci
2009; 30: 546–56.
85 Wu XJ, Kassie F, Mersch-Sundermann V. The role of reactive oxygen
species (ROS) production on diallyl disulfide (DADS) induced apoptosis
and cell cycle arrest in human A549 lung carcinoma cells. Mutat Res
2005; 579: 115–24.
67 Park SY, Cho SJ, Kwon HC, Lee KR, Rhee DK, Pyo S. Caspase-
independent cell death by allicin in human epithelial carcinoma cells:
involvement of PKA. Cancer Lett 2005; 224: 123–32.
68 Miron T, Arditti F, Konstantinovski L, Rabinkov A, Mirelman D, 86 Xiao D, Herman-Antosiewicz A, Antosiewicz J, Xiao H, Brisson M, Lazo
Berrebi A, et al. Novel derivatives of 6-mercaptopurine: synthesis,
characterization and antiproliferative activities of S-allylthio-mercapto-
purines. Eur J Med Chem 2009; 44: 541–50.
JS, et al. Diallyl trisulfide-induced G2–M phase cell cycle arrest in
human prostate cancer cells is caused by reactive oxygen species-
dependent destruction and hyperphosphorylation of Cdc25C. Onco-
gene 2005; 24: 6256–68.
69 Modem S, DiCarlo SE, Reddy TR. Fresh garlic extract induces growth
arrest and morphological differentiation of MCF7 breast cancer cells. 87 Zhao J, Zhang J, Yu M, Xie Y, Huang Y, Wolff DW, et al. Mitochondrial
Genes Cancer 2012; 3: 177–86.
dynamics regulates migration and invasion of breast cancer cells. On-
cogene 2013; 32: 4814–24.
70 Prager Khoutorsky M, Goncharov I, Rabinkov A, Mirelman D, Geiger
-
B, Bershadsky AD. Allicin inhibits cell polarization, migration and
Acta Pharmacologica Sinica