Synthesis and preliminary evaluation of the anti-cancer activity on A549 lung cancer cells of a series of unsaturated disulfides

Article abstract of DOI:10.1039/C8MD00503F

We synthesized a series of small symmetrical unsaturated disulfides by a multi-step reaction starting from organic alcohols, and we performed a preliminary test to evaluate the effect of these compounds on the viability of A549 lung cancer cells. The garlic-derived natural compound diallyl disulfide, known for its anticancer activity, was used as the lead compound in this study. We synthesized five DADS analogues having different carbon chain lengths and different positions of the double bonds. Two analogues exhibited a promising antitumor activity in vitro, and the allylic double bond did not seem to be the main driving force.

Full text of DOI:10.1039/C8MD00503F

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RESEARCH ARTICLE
Synthesis and preliminary evaluation of the anti-
cancer activity on A549 lung cancer cells of a
series of unsaturated disulfides†
Cite this: DOI: 10.1039/c8md00503f
ad
Nicola Amodio,b Maria Luisa Di Gioia,c Monica Nardi,de
*
Fabrizio Olivito,
Manuela Oliverio,
a
a
Giada Juli,b Pierfrancesco Tassoneb and Antonio Procopio
We synthesized a series of small symmetrical unsaturated disulfides by a multi-step reaction starting from
organic alcohols, and we performed a preliminary test to evaluate the effect of these compounds on the
viability of A549 lung cancer cells. The garlic-derived natural compound diallyl disulfide, known for its anti-
cancer activity, was used as the lead compound in this study. We synthesized five DADS analogues having
different carbon chain lengths and different positions of the double bonds. Two analogues exhibited a
promising antitumor activity in vitro, and the allylic double bond did not seem to be the main driving force.
Received 4th October 2018,
Accepted 1st December 2018
DOI: 10.1039/c8md00503f
rsc.li/medchemcomm
Design, synthesis, and pharmacological assays of new
synthetic organic compounds are a common practice within
today's scientific community.1,2 Natural compounds present a
wide source of structurally and biologically interesting
molecules.3,4 In the past few decades, many efforts have been
conducted to rationalize the activity of a wide range of
natural molecules and to mimic them using synthetic
chemistry.5,6 For example, Allium genus plants have been
studied for centuries, due to their chemical complexity and
their unbelievable pharmacological potential, which still
makes this genus fascinating.7,8 Garlic and onion represent
its most important species, with tons of them being
consumed worldwide mostly for food or related products.9
These and other Allium genus plants are typically
characterized by the presence of different organosulfur
compounds, many of which still remain unknown. These
molecules are only produced when these plants are cut or
chopped.10 Organic disulfides constitute one of the most
abundant classes of molecules; for this reason, it is likely that
they provide an important contribution to their health-
benefiting effect.11 Allicin, which belongs to the class of or-
ganic thiosulfinates, is one of the most studied out of all
kinds of garlic molecules, with proven antimicrobial, antioxi-
dant and anticancer activities.12–14 The first report of the
in vivo anticancer activity of this molecule can be dated back
to 1958.15 In 2001, a study was conducted about the in vitro
anticancer activity of pure allicin on different cancer cell
lines.16 In a recent study, allicin was identified as an active
anticancer compound present in aqueous garlic extracts; the
activity was determined by an MTT cell viability assay on Mus
musculus colon carcinoma cells CT26.WT.17 One of the limita-
tions of this molecule is its instability at moderate tempera-
ture and under light sources, as well as in biological fluids.18
The main by-product of allicin decomposition is a small mol-
ecule called diallyl disulfide (DADS). Hence, it is another mol-
ecule that is abundantly detected in garlic extracts. With re-
spect to allicin, DADS has greater stability and preserves
many pharmacological activities.19–21 Recent studies were fo-
cused on its multi-target action against different types of can-
cer.22,23 This small molecule produces antiproliferative effects
by inducing inhibition of cell proliferation and apoptosis with
cell cycle arrest at the G0/G1 or G2/M phase.24 Moreover, DADS
induces cell cycle arrest at the G2/M phase and apoptosis in
human A549 lung cancer cells in a time- and dose-dependent
manner. The increase of intracellular reactive oxygen species
(ROS) after treatment was indicated as one of the most impor-
tant triggering factors.25 Beyond the in vitro tests, DADS
a Dipartimento di Scienze della Salute, Università Magna Græcia, Viale Europa,
Germaneto, Catanzaro, 88100, Italy. E-mail: fabrizioolivito@gmail.com
b Dipartimento di Medicina Sperimentale e Clinica, Università Magna Græcia,
Viale Europa, Germaneto, Catanzaro, 88100, Italy
c Dipartimento di Farmacia e Scienze della Salute e della Nutrizione, Università
della Calabria, Arcavacata di Rende, Cosenza, 87030, Italy
d Dipartimento di Chimica, Università della Calabria, Cubo 12C, Arcavacata di
Rende, Cosenza, 87030, Italy
Scheme 1 General multi-step reaction for the synthesis of disulfides
starting from alcohols. (a) 1 eq. of methanesulfonyl chloride and 1 eq.
of triethylamine in CH2Cl2. (b) 1.5 eq. of thioacetic acid and 3 eq. of
potassium carbonate in water. (c) Acidic methanol. (d) 5% iodine in
methanol.
e Dipartimento di Agraria, Università Telematica San Raffaele, Via di Val
Cannuta, 247, Roma, 00166, Italy
† Electronic supplementary information (ESI) available: Experimental details
and compound characterization. See DOI: 10.1039/c8md00503f
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reduces carcinogen-induced cancers in experimental animals,
as reported by Xia et al. in a recent review.26 Unfortunately,
this molecule is sold at just 80% purity due to the presence of
other sulfides that can affect its pharmacological activities. In
this work, we synthesized the natural molecule DADS as the
lead compound and a series of symmetrical unsaturated
disulfides with different lengths of the carbon chains and dif-
ferent positions of the double bonds, via a multistep reaction
(Scheme 1).
with terminal double bonds and different carbon chain
lengths (entries 2 and 6). Molecule D was newly synthesized.
We proved the acceptable purity of the products by 1H and
13C-NMR spectroscopy. We evaluated the viability of the A549
cell line 24 and 48 hours after the treatment. A comparison
between the activity of the lead compound and that of the
new analogues was made. We used the human A549 lung ad-
enocarcinoma cell line to assess the effects of the compounds
on tumor viability in vitro. The A549 non-small cell lung can-
cer cell line was purchased from ATCC-LGC Promochem
(South West London, UK) as previously reported (Amodio
et al., Am. J. Pathol. 2010).32 Cells were maintained in RPMI
1640 medium (Gibco-Invitrogen Cell Culture, Carlsbad, CA),
supplemented with 10% fetal bovine serum and 100 units
per ml penicillin–streptomycin (Life Technologies, Inc., Carls-
bad, CA). Cells were assayed using the Cell Titer-Glo Lumi-
nescent Cell Viability assay (Promega), after being treated
with increasing concentration of each molecule (50, 100, 250,
and 500 μM). Each measurement was repeated three times
and the mean value calculated with the relative standard de-
viation. Cell viability was analyzed by a luminometric assay,
and all values were calculated with respect to the control that
was labeled C.
After 24 hours of incubation (Fig. 1), molecules A and B
seem to follow the trend of the lead molecule X, with a de-
crease in cell viability in a concentration-dependent manner.
The results relative to the natural molecule X are in accor-
dance with the data reported in the literature.33 The activity
of DADS analogues A and B is found to be slightly weaker
compared to the reference molecule X, for all the four con-
centrations used. The presence of a double bond is signifi-
cant, but it doesn't have to be necessarily allylic, as proved by
molecule A that possesses a terminal double bond on its car-
bon chain. Molecules M and D with allylic double bonds in
Recently, our group developed a new methodology to syn-
thesize organic thioacetates, particularly unsaturated thio-
acetates, by an environmentally-friendly reaction in aqueous
media, starting with alcohols (Table 1).27 Green procedures
can be related to the synthesis of pharmacologically active
molecules by different strategies.28 In this work, unsaturated
thioacetates represent the starting point in the process of
obtaining symmetrical unsaturated disulfides in a one-pot
procedure, passing from thiols as the intermediate.29 The
garlic compound diallyl disulfide (DADS) was freshly pre-
pared with an acceptable level of purity (>95% by NMR) (en-
try 1). This and other unsaturated compounds play an impor-
tant role in the pharmacological activity of this type of
plant.30,31 Many studies have proved the stronger activity of
unsaturated compounds with respect to saturated ones.23 In
this direction, we synthesized similar allylic disulfides B, M,
and D, only with different carbon chain lengths (entries 3–5).
Starting from trans-5-hexen-1-ol, we stereospecifically
obtained the newly synthesized trans isomer (E)-1-[[(E)-hex-2-
henyl]disulfanyl]hex-2-ene D (entry 5). Starting from cis-5-
hexen-1-ol, we obtained the newly synthesized mixture of
trans and cis isomers M, in a percentage isomeric cis/trans ra-
tio of 65/35, probably due to a partial rearrangement of the
cis isomer to the more stable trans isomer (entry 4). Finally,
we synthesized two other symmetrical disulfides A and D
Table 1 Reagents, intermediates and final products of the multi-step reaction
Entry
1
Reagent
Intermediate 1
Intermediate 2
Final producta
X
1b
2b
3b
1a
2a
3a
2
A
B
3
4b
4a
4b
M
D
5
6
5a
6a
5b
6b
E
a
b
All the products were purified using flash column chromatography with alumina and stored at 0 °C. The final product is a mixture of the
two isomers in a cis/trans ratio of 65/35 estimated by 13C-NMR spectroscopy.
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lar to A, with no improvements at 100 or 250 μM. The inhibi-
tion of cell viability by B at 500 μM is weaker than that by
molecule A, which shows a value of 84% regarding the same
aspect. All other tested compounds do not show significant
effects 48 hours after the treatment. The isomeric mixture M
is more active than the trans isomer D at all the four used
concentrations, even after 48 hours from the treatment.
Finally, we performed a preliminary stability test on more
active molecules A and B and on the natural molecule X.
These compounds were allowed to stay in their pure form at
room temperature (an average of 298.15 K) for one week. Af-
1
ter this period the purity was checked and confirmed by H-
NMR spectroscopy. Small quantities of A, B, and X were
dissolved in deuterated chloroform and these samples were
left to stand at room temperature (an average of 298.15 K) for
one week. After this period, the acceptable purity was
Fig. 1 Percentage of cell viability 24 hours after the treatment.
1
checked and confirmed by H-NMR spectroscopy for the syn-
thetic compounds A and B, while for the natural molecule X
a significant change of purity was evident from the spectrum
(molecule X was estimated to be 40% pure by chromatogra-
phy). This test can be useful for more advanced studies with
medicinal interests. Unsaturated organosulfur compounds
are widespread in nature, mostly thanks to the plants of ge-
nus Allium. These compounds are observed as promising
pharmacologically active molecules, but the relationship be-
tween the structure and the activity is still not clear. In this
work, we synthetized and tested the in vitro anti-tumor poten-
tial of different unsaturated disulfides using human adeno-
carcinoma cells. From the data collected and reported above,
we can conclude that unsaturated disulfides hold promise for
the inhibition of the cell viability of the A549 cell line. For
the first time, the DADS analogues A and B have been proven
to be more active on a cancer cell line than the natural mole-
cule diallyl disulfide. In addition, the unsaturation site is not
the principal driving force, but other factors, like the size of
the molecule and the length of the carbon chain, can be im-
portant guidelines for more advanced studies in this field in
future.
Fig. 2 Percentage of cell viability 48 hours after the treatment.
different isomeric forms triggered a slight decrease in cell vi-
ability only at the highest concentration (500 μM). The iso-
meric mixture M is more active than the trans isomer D at all
the four used concentrations, probably due to the higher per-
centage of the cis isomer. The loss of the anti-tumor activity
was also observed for molecule E, which had a terminal dou-
ble bond on its carbon chain. These results indicate that the
carbon chain length is likely to play a more active role in the
potential anti-tumor activity of such unsaturated compounds.
After 48 hours of incubation (Fig. 2), molecules X, A, and
B show a significant and time-dependent in vitro anti-tumor
activity. These two synthetic derivatives of DADS generate
stronger effects at all the four used concentrations. The lead
molecule X induces 54% viability inhibition at 50 μM. Mole-
cule A induces a viability inhibition of 68% at the same con-
centration. Molecule B has a similar effect, with 70% viability
inhibition at 50 μM. For molecule X, the percentage inhibi-
tion doesn't change significantly from 50 to 250 μM, and
reaches almost the same activity as that of the synthetic ana-
logues at 500 μM, with 65% inhibition overall. Molecule A
shows a concentration-dependent activity while passing from
50 μM to 100 μM, while no significant changes are observed
at 250 μM. Almost complete inhibition was observed for mol-
ecule A at 500 μM. Compound B at 50 μM shows effects simi-
Conflicts of interest
There are no conflicts to declare.
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