Trisulfides over disulfides: Highly selective synthetic strategies, anti-proliferative activities and sustained H2S release profiles

Article abstract of DOI:10.1039/c9cc05562b

Temperature-and solvent-induced selective synthesis of trisulfides and disulfides is demonstrated. A remarkable selectivity was achieved using Na₂S as a sulfur-Transfer agent under mild, greener, catalyst-free and additive-free conditions. This study reveals trisulfides as a better model than disulfides in general for a sustained release of H₂S and potent anti-cancer activities.

Full text of DOI:10.1039/c9cc05562b

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Trisulfides over disulfides: highly selective
synthetic strategies, anti-proliferative activities
and sustained H2S release profiles†
Cite this: Chem. Commun., 2019,
55, 13534
Received 18th July 2019,
Accepted 14th October 2019
Debojit Bhattacherjee,ab Abu Sufian,‡a Sulendar K. Mahato,‡a Samiyara Begum,‡a
Kaustav Banerjee,a Sharmistha De,a Hemant Kumar Srivastava *a and
ab
DOI: 10.1039/c9cc05562b
Krishna P. Bhabak
*
rsc.li/chemcomm
Temperature- and solvent-induced selective synthesis of trisulfides
and disulfides is demonstrated. A remarkable selectivity was achieved
using Na2S as a sulfur-transfer agent under mild, greener, catalyst-
free and additive-free conditions. This study reveals trisulfides as a
better model than disulfides in general for a sustained release of H2S
and potent anti-cancer activities.
Fig. 1 Structures of some naturally occurring trisulfides (A). Reported
schemes to organopolysulfides (B). Synthesis of trisulfides and disulfides
in the present work (C).
Organopolysulfides including organotrisulfides are very important
organosulfur compounds having notable pharmaceutical and
medicinal implications.1 Some naturally occurring trisulfides
include esperamicins (1), epipolythiodiketopiperazine alkaloids
such as (+)-Luteoalbusin B (2) as antitumor antibiotics,2 plant-
isolated trisulfides such as dibenzyl trisulfide (3a)3 and garlic- sulfur-transfer agents and electrophilic halide precursors have
based trisulfide (3b, DATS) as anti-cancer and chemopreventive become important (Fig. 1).
agents (Fig. 1A).4
In 1946, Na2S3 was used as the first nucleophilic sulfur-
DATS is extensively studied for its anti-cancer potential in transfer agent to prepare bis(2-hydroxyethyl)trisulfide. However,
organ-specific cancer cells and animal models.5 However, the the method resulted in a mixture of disulfides to pentasulfides.10
research impact of DATS and naturally occurring trisulfides has In 1960s, Na2S was utilized for trisulfides but in phosphate buffer
much evolved recently due to their performance as potent with highly toxic formaldehyde (35% w/v) as an additive.11 The
sources of H2S, which acts as a strong gasotransmitter2b,6 method was inconvenient and produced trisulfides with disul-
having implications on neurotransmission, vasodilation, cardio- fides and tetrasulfides. In 2001, a Cu(II)-catalyzed method was
protection, apoptosis and many other physiological processes.5,6a,7 reported utilizing SnCl2 and elemental sulfur in THF-DMSO
Therefore, a convenient and improved method for the synthesis solvent producing polysulfides.12 Subsequently, in 2013, TBAB-
of organotrisulfides in general including DATS is of utmost catalyzed flow-based synthesis using Na2S3 in an EtOH–H2O
importance for identifying efficient and sustained donors of mixture also ended up with polysulfides.13 A very recent report
H2S. Although conventional synthesis utilizes thiols as precursors utilized H2S and disulfides to generate trisulfides that equilibrate
with electrophilic sulfur-transfer reagents,8,9 these methods are among reactive sulfur species.14 Importantly, a selective synthesis
almost obsolete presently due to commercial unavailability, of trisulfides is much desired as the post-synthetic separation of
unpleasant smell of organomercaptans and incompatibility with polysulfides is very challenging due to similar polarities. However, to
unsaturated thiols. Therefore, methods utilizing nucleophilic the best of our knowledge, to date, there is no generalized method
for the synthesis of diorganotrisulfides with enhanced selectivity.
a Department of Chemistry, Indian Institute of Technology Guwahati,
Guwahati-781039, Assam, India. E-mail: kbhabak@iitg.ac.in, hemantkrsri@gmail.com
b Centre for the Environment, Indian Institute of Technology Guwahati, Guwahati-781039,
Assam, India
Herein, we report a highly selective synthesis of trisulfides under
mild, greener, catalyst-free and additive-free conditions. Additionally,
the methodology was tuned further to prepare the corresponding
disulfides exclusively. Further studies revealed trisulfides as potent
anti-cancer compounds with sustained H2S release profiles
compared to disulfides, indicating their enhanced pharmaceutical
implications.
† Electronic supplementary information (ESI) available. CCDC 1905587–1905589.
For ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c9cc05562b
‡ Authors contributed equally.
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Table 1 Optimization processes for 3a
Table 2 Synthesis of trisulfides 3a–3ua
Entry Na2S Temp. (1C) Time (h) Yielda of 3a (%) Yielda of 4a (%)
1
2
3
4
5
6
7
8
0.5
0.1
1.0
3.0
0.5
0.5
0.5
0.5
0.5
0.5
0.5
RT
RT
RT
RT
RT
RT
RT
RT
0
24
24
24
24
4
59
19
27
37
61
70
66
70
77
68
41
7
1
6
21
5
6
6
6
1
8
12
16
8
12
4
9
10
11
0
80
2
27
a
Calculated from the peak intensities of the –S–CH2– group in 1H NMR
spectra of the product mixture.
a
Yields are calculated from the peak intensities of the –S–CH2– group
in the 1H NMR spectra of the crude product mixture. *Isolated yield of
3m was 54%.
We attempted the optimization process with trisulfide 3a.
The synthesis of 3a needs an initial reaction of benzyl bromide
with Na2S2O3Á5H2O producing Bunte salt15 and a subsequent
reaction with Na2SÁ9H2O (Fig. 1C). As the preparation of Bunte
salt is well-established, the second step was optimized to decreased for 3g (59%) and 3h (49%) with traces of the
prepare 3a selectively (Table 1).
corresponding disulfides. In contrast, the yield of 3i–3l containing
The reaction was initiated using 0.5 equiv. Na2S with respect electron withdrawing groups was relatively higher (69–97%) with
to Bunte salt producing trisulfide 3a predominantly (59%) but better selectivity except 4-Br substituted compound 3k (47%).
with some disulfide 4a (7%). With decreased Na2S equivalence,
To understand the positional effect of substitutions, (3m–3q)
the overall yield was reduced and an increased equivalence were synthesized. Representative 2-substituted trisulfides 3m
rather produced higher disulfide content (entries 1–4). Water (56%) and 3n (77%) were synthesized with good selectivity and
was chosen as the best solvent as the investigation with organic yields. Particularly, trisulfide 3m (54%) could be separated from
solvents afforded disulfide 4a predominantly (Table S1, ESI†). disulfide 4m by column chromatography owing to a slight
To optimize the reaction time, reactions were carried out for difference in their relative polarity. The structure of 3m was
4–16 h. The formation of trisulfide 3a (61%) was significant unambiguously confirmed by X-ray studies (CCDC: 1905588†).
within 4 h and was improved further (70%) over 8–16 h, Similarly, 3-substituted trisulfides 3o–3q were synthesized in
however, with 6% of 4a (entries 5–8). Therefore, to minimize good yields with enhanced selectivity. Interestingly, a complete
the 4a content further, the reaction temperature was tuned selectivity with excellent yields was observed for disubstituted
(entries 9–11). Interestingly, the reaction at 0 1C produced 3a trisulfides 3r–3t (Table 2). Finally, the scope was expanded
exclusively (77%) with a trace amount of 4a (1%) within 8 h further with a naphthalene-based trisulfide 3u (96%) having
(entry 9). A longer reaction time at 0 1C or an increasing complete selectivity. Therefore, the proposed method has a
temperature (within 4 h) enhanced the formation of 4a (entries wider substrate scope with very good selectivity towards trisul-
10 and 11). Therefore, the reaction at a lower temperature (0 1C) fides. Moreover, many trisulfides (3b, 3d–3f, 3i, 3j, 3r–3u) can be
was considered optimized to synthesize 3a selectively (entry 9). prepared with complete selectivity and 3m can be easily purified.
As the normal phase column chromatography co-elutes 3a and The trace amount of co-produced disulfides (r5%) and higher
4a, the relative yields of 3a and 4a in the crude product mixture polysulfides (o1%) for a few trisulfides could be separated by
were calculated from the relative intensity of –SCH2– groups in reverse-phase HPLC. The method was validated further by a
1
the H NMR spectra.
gram-scale synthesis of 3f (3.24 g, 82%) with a complete
Optimized conditions were used for synthesizing DATS (3b) selectivity (Fig. S98, ESI†).
and various alkyl, alkenyl and substituted benzylic compounds
Dominance of 4a in non-aqueous solvents (Table S1, ESI†)
(Table 2). Although DATS was synthesized in good yield (50%) prompted us to explore disulfides 4a–4u selectively using the
with a complete selectivity, the yield was relatively lower for a same reagents. Considering the greener aspects, we retained
longer chain alkenyl trisulfide 3c (24%) and with a trace the water component and mixed EtOH to initiate optimization
amount of disulfide 4c (2%). However, complete selectivity with (Table S2, ESI†). As lower temperature favored trisulfide formation,
good yields was observed for alkyl trisulfides 3d (62%) and 3e the temperature was increased first to investigate disulfide
(44%). The scope was extended further to various substituted selectivity. Interestingly, a complete selectivity towards 4a was
benzyl trisulfides 3f–3u. For example, 4-substituted benzylic observed at 50 1C or above within 4 h in the EtOH–H2O (3 : 1)
trisulfides (3f–3l) were synthesized with significantly good system. This condition was utilized successfully to synthesize
yields. Although the yield was higher for 3f (78%), it was slightly alkenyl, alkyl and substituted benzylic disulfides 4a–4u in good
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yields with complete selectivity except 4e and 4s (Table S3, ESI†).
While preparing 4s at 50 1C in the EtOH–H2O system, the
corresponding trisulfide 3s was found to be predominant (41%).
The garlic-based disulfide DADS (4b) was synthesized exclusively in
65% yield. The structure of disulfide 4m was further confirmed by
an X-ray study (CCDC: 1905589†). These observations reveal that
the temperature and the solvent play very crucial roles for tuning
the selectivity between trisulfides and disulfides. To differentiate
disulfides from monosulfides, monosulfide (5m) corresponding
to 4m was also synthesized and characterized by NMR, ESI-MS and
X-ray studies (CCDC: 1905587, Fig. S89–S91, ESI†).
For the selective formation of trisulfides and disulfides, a
possible mechanistic pathway was proposed based on the
control experiments and computational studies (Scheme 1). While
trisulfides can be produced selectively via steps 1–3 at lower
Fig. 2 Energy profile diagram for the formation of 3a. Sodium ions are not
temperature, disulfides form at higher temperature (Z50 1C) via depicted here for a better clarity in representation; however, they are
present in calculations. All the reported energy values (kcal molÀ1) were
two possible pathways (steps 4, 5 and steps 6, 7). The involvement
of the perthiolate intermediate (step 2) was justified by reacting
two purified Bunte salts 6 and 7 (1 : 1) corresponding to the
calculated at the M06/6-31+G(d) level of DFT in water solvent (see
Fig. S120 and Table S4, ESI† for further details).
4-methylbenzyl and 4-isopropylbenzyl groups, respectively, with
Na2S at 0 1C and 50 1C (Scheme S1, ESI†). Interestingly, under corresponding reactants, indicating their clear feasibility at lower
both conditions, two symmetrical trisulfides/disulfides (3f + 3g/ temperature (Fig. 2). Moreover, a lower barrier and higher
4f + 4g) and a mixed trisulfide/disulfide (3v/4v) were obtained. product stability in the third step support a spontaneous trans-
The formation of mixed trisulfide and disulfide indicates the formation of perthiolate to trisulfide 3a at lower temperature.
intermediacy of perthiolate (Fig. S92–S97, ESI†). Interference by
Interestingly, the energy barriers for disulfide formation
the co-produced Na2SO3 to form disulfides (steps 4 and 6) was from trisulfide 3a (steps 4 and 5) were higher (16.48 and
studied further by its external addition during the synthesis of 0.75 kcal molÀ1, Fig. S120, ESI†) than those for trisulfide
trisulfides at lower and higher temperatures. Interestingly, the formation from perthiolate (step 3; 5.46 kcal molÀ1, Fig. 2),
formation of 4a was enhanced slightly at 0 1C but significantly at indicating the necessity of higher temperature to convert 3a to 4a.
50 1C after adding Na2SO3 (5.0 equiv.) during the synthesis of 3a. Similarly, the energy barriers for the conversion of perthiolate to
Similarly, Na2SO3 (5.0 equiv.) could convert some amount of disulfide 4a (steps 6 and 7) were higher (7.92 and 8.93 kcal molÀ1
,
pure 3a to 4a at 50 1C indicating relatively higher feasibility of Fig. S120, ESI†) than those for the formation of trisulfide 3a (step
disulfide formation at higher temperature (Fig. S99–S104, ESI†). 3, 5.46 kcal molÀ1), indicating the feasibility of trisulfide formation
This observation was utilized as a proof-of-concept to enhance at 0 1C. The computational studies support our experimental
selectivity towards disulfides 4b, 4e and 4u with an external findings and the proposed mechanistic pathways. In general, both
addition of Na2SO3 (5 equiv.) to the reaction mixture.
of these processes (steps 4, 5 and 6, 7) may work simultaneously at
Selectivity towards 3a and 4a was further studied using DFT an elevated temperature to form 4a exclusively.
calculations. Geometries of all the reactants, transition states
It is reported that DATS exhibits higher anti-cancer effects
(TS), intermediates and products were fully optimized at the than DADS in garlic.4,16,17 Therefore, the synthesized trisulfides
M06/6-31+G(d) level of theory using the Gaussian-09 program 3a–3u were screened for their anti-cancer activities (MCF-7 cells)
and water was chosen as the solvent (Scheme 1). Additionally, by morphological changes and MTT studies. The trisulfides that
single point calculations were carried out at various levels to significantly changed the cellular morphology were further purified
analyse the effect of methods and basis sets on the energy (HPLC) for final evaluation. Among the trisulfides, 3f, 3i–3k, 3m,
profile (Table S4, ESI†). The calculated energy barriers for steps 3o–3q and 3t exhibited very high anti-proliferative activities with
1–3 (14.01, 8.88 and 5.46 kcal molÀ1) were moderate to nominal IC50 values below 5 mM and 3f was the most potent trisulfide
and the products were significantly more stable than the (Fig. 3B and Fig. S116, S117, ESI†). However, the corresponding
disulfides were much less potent except 4k and 4m. A much
lower activity of DATS and most of the disulfides reveals
that the basic skeleton and functional group in trisulfides
and disulfides are important for developing better chemo-
therapeutic compounds.18
Inspired by the potent anti-proliferative activity of some
trisulfides, a representative trisulfide (3f) was studied further
for its H2S donation efficacy using MB assay and compared with
the corresponding disulfide 4f and DATS.19 While 4f (50 mM)
Scheme 1 Proposed selective pathways for trisulfides and disulfides.
13536 | Chem. Commun., 2019, 55, 13534--13537
was found to be inactive, a moderate and sustained H2S release
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respectively. We acknowledge the DST-FIST program and CIF,
PARAM-ISHAN, IIT Guwahati.
Conflicts of interest
There are no conflicts to declare.
Notes and references
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In summary, we show herein for the first time that the
selectivity between symmetrical trisulfides and disulfides can
be achieved using Na2S as the key sulfur-transfer agent by
tuning the temperature and the solvent under mild, greener,
catalyst-free and additive-free conditions with a wider substrate
scope. Mechanistic studies highlight the interference by the
co-produced Na2SO3 for the co-formation of disulfides along
with trisulfides. Furthermore, excellent anti-proliferative activities
of trisulfides along with a sustained H2S release profile open up
newer avenues for trisulfides as chemotherapeutic and cardio-
protective compounds for future studies compared to the corres-
ponding disulfides. However, further studies are warranted for a
detailed structure–activity correlation.
KPB and HKS acknowledge SERB for DST-INSPIRE (Ref:
IFA12-CH-68) and Ramanujan (SB/S2/RJN-004/2015) fellowships,
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