1,2,4-Thiadiazolidin-3,5-diones as novel hydrogen sulfide donors

Article abstract of DOI:10.1016/j.ejmech.2017.10.068

Hydrogen sulfide (H₂S) is an endogenous modulator that plays significant physio-pathological roles in several biological systems. In this research field there is a large interest in developing selective CBS and CSE inhibitors and H₂S

Full text of DOI:10.1016/j.ejmech.2017.10.068

European Journal of Medicinal Chemistry xxx (2017) 1e10
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
1,2,4-Thiadiazolidin-3,5-diones as novel hydrogen sulfide donors
,
*,
,
Beatrice Severino a 1, Angela Corvino a 1, Ferdinando Fiorino a, Paolo Luciano a,
Francesco Frecentese a, Elisa Magli a, Irene Saccone a, Paola Di Vaio a, Valentina Citi b,
Vincenzo Calderone b, Luigi Servillo c, Rosario Casale c, Giuseppe Cirino a,
Valentina Vellecco a, Mariarosaria Bucci a, Elisa Perissutti a, Vincenzo Santagada a,
Giuseppe Caliendo a
a Department of Pharmacy, School of Medicine, University of Naples
«Federico II», Via D. Montesano, 49, 80131 Napoli, Italy
b Department of Pharmacy, University of Pisa, Via Bonanno, 6, I-56126 Pisa, Italy
c
ꢀ
Department of Biochemistry, Biophysics and General Pathology, Universita della Campania “Luigi Vanvitelli”, Via L. De Crecchio 7, 80138 Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Hydrogen sulfide (H2S) is an endogenous modulator that plays significant physio-pathological roles in
several biological systems. In this research field there is a large interest in developing selective CBS and
CSE inhibitors and H2S releasing moieties, that could be either used as therapeutic agents or linked to
known drugs. One of the major problem is the limited availability of chemicals that ensure a controlled
release of H2S in vitro as well in vivo. Aiming to obtain novel H2S donors, whose release properties could
be appropriately modulated, we have synthesized a series of 1,2,4-thiadiazolidine-3,5-diones (THIA 1
e10) as innovative donors that could release H2S in controlled manner. All the synthesized compounds
were evaluated for their H2S releasing properties by an amperometric approach and for their vaso-
relaxant ability on aorta rings. In order to rationalize the obtained results, a detailed study on the release
Received 8 September 2017
Received in revised form
3 October 2017
Accepted 24 October 2017
Available online xxx
Keywords:
Hydrogen sulfide
H2S donors
1,2,4-Thiadiazolidine-3,5-diones
Release mechanism
mechanism has been performed using the most efficient H2S donor, THIA 3 (Cmax 65.4
1.7 M).
mM and EC50
m
© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
controlled release and on the discovery of more selective enzyme
inhibitors [4,5]. As concerns H2S donors, the research has been
focused on the development of novel releasing moieties [6], whose
properties could be modulated by means of appropriate chemical
modifications. The slow kinetic of H2S release is a relevant phar-
macological feature already attributed to naturally occurring
compounds, such as the diallylpolysulfides of garlic [7] (Allium
sativum) and the isothiocyanates typical of Brassicaceae [8]. Garlic
has long been felt beneficial as an antioxidant, and recent evidence
suggests that a number of beneficial effects of garlic derive from
H2S production. The best characterized naturally occurring H2S-
donating compound from garlic is allicin (diallylthiosulfinate)
which decomposes in water to a number of compounds, such as
diallyldisulfide (DADS) and diallyltrisulfide (DATS). In particular,
H2S release from DATS was suggested to mediate the vasoactivity of
garlic [9]. Sulforaphane, an isothiocyanate, has proved to be an
effective chemoprotective agent in several cancer models [10].
Currently available H2S sources and/or donors are limited. NaHS,
although widely used as a research tool, causes a fast burst of H2S
that rapidly declines, as clearly reported in both in vitro and in vivo
Hydrogen sulfide (H2S) is a gasotransmitter acting as an
endogenous modulator that plays significant physio-pathological
roles in several biological systems [1]. H2S is mainly derived from
two pyridoxal-50-phosphate (PLP)-dependent enzymes, cys-
tathionine-b-synthase (CBS) and cystathionine-g-lyase (CSE) [2], or
from a third pathway (PLP-independent) that combines the actions
of 3-mercaptopyruvate sulfurtransferase (3-MST) and cysteine
aminotransferase (CAT) [3].
Inorganic source of H2S, such as NaHS, and relatively selective
inhibitors of either CBS or CSE have been used for evaluating the
physiopathological involvement of H2S pathway in the regulation
of several biological functions. In more recent years the research
has been focused on the development of organic donors with
* Corresponding author.
E-mail address: bseverin@unina.it (B. Severino).
B.S. and A.C. equally contributed to the paper.
1
https://doi.org/10.1016/j.ejmech.2017.10.068
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: B. Severino, et al., 1,2,4-Thiadiazolidin-3,5-diones as novel hydrogen sulfide donors, European Journal of
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2
B. Severino et al. / European Journal of Medicinal Chemistry xxx (2017) 1e10
experimental studies [11e13]. Among the organic H2S donors, the
most widely studied and characterized is GYY4137. This synthetic
H2S donor decomposes spontaneously in aqueous buffers to release
H2S over a long time period. Some other H2S-releasing moieties,
such as dithiolethiones and thioamides, have been largely used for
synthesizing hybrids with several pharmacologically active com-
pounds [14]. Moreover, thiol-triggered H2S donors, including N-
(benzoylthio)benzamides [15,16], S-aroylthiooximes [17] and
arylthioamides [18] have been described. However there is still the
necessity to develop new H2S donors that can ensure a well defined
kinetic of release in order to better address and exploit the potential
therapeutic of H2S. Indeed, substantial differences are present in
the relevant preclinical literature between NaHS and GYY4137. In
order to address this issue our attention was devoted to the 1,2,4-
thiadiazolidine-3,5-diones, already described in literature as able
Both the compounds promoted a modest, but appreciable,
generation of H2S even in the absence of -cysteine (Cmax of about
5.5 and 2.4 M, respectively). Then several derivatives were
designed and synthesized, aiming to have a more controllable
release (THIA 3e10, Table 1); moreover their biological effect was
assessed by evaluating the vasorelaxant ability on isolated aorta
rings. Finally, in order to rationalize the obtained results, a detailed
study on the H2S release mechanism has been performed.
L
m
2. Results and discussion
The synthetic strategies for compounds THIA 1 [20,21], 2 [20], 3
[22], 4 [23] and 9 [23] have been previously reported. Noteworthy,
for the first time, we describe the ability of this scaffold to release
H2S in presence of thiols. All the compounds were firstly synthe-
sized through oxidative condensation of isothiocyanates with iso-
cyanates in the presence of SO2Cl2, following the strategy already
described in literature (Scheme 1) [21].
Moreover, the synthetic procedure has been opportunely
modified using microwave irradiation in order to get better yields
and to reduce reaction times. Microwave assisted organic synthesis
(MAOS), in fact, has shown a great ability to rapidly synthesize
organic compounds and generate library with a great potential for
drug-discovery programs [24,25]. A microwave oven, especially
to inhibit glycogen synthase kinase 3b (GSK3b) [19] and regulators
of G protein signalling (RGS) proteins [20]. Several authors have
hypothesized that their molecular mechanism involves the for-
mation of a disulfide adduct with an enzymatic cysteine residue
[19,20]. This observation suggested us that the 1,2,4-
thiadiazolidine-3,5-dione scaffold could act as a thiol-triggered
H2S donor. In order to verify this hypothesis we synthesized THIA
1 and 2 [20] (Table 1), that were evaluated in vitro for their ability to
release H2S.
Table 1
Structures of the 1,2,4-thiadiazolidine-3,5-diones THIA 1e10 and yields obtained with standard procedure versus microwave irradiation.
Compound
R
R0
Standard procedure
Microwave irradiation
Yield (%)
63
Time
ꢀC
Yield (%)
75
Time
ꢀC
THIA 1
24 h
25
1 h
50
30 min
100
30 min
120
THIA 2
66
24 h
25
82
1 h
50
30 min
100
30 min
120
THIA 3
THIA 4
THIA 5
THIA 6
THIA 7
THIA 8
THIA 9
THIA 10
64
62
65
62
69
64
65
60
24 h
30 min
25
100
80
76
79
78
82
81
77
74
1 h
30 min
50
120
24 h
30 min
25
100
1 h
30 min
50
120
24 h
30 min
25
100
1 h
30 min
50
120
24 h
30 min
25
100
1 h
30 min
50
120
24 h
30 min
25
100
1 h
30 min
50
120
24 h
30 min
25
100
1 h
30 min
50
120
24 h
30 min
25
100
1 h
30 min
50
120
24 h
25
1 h
50
30 min
100
30 min
120
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B. Severino et al. / European Journal of Medicinal Chemistry xxx (2017) 1e10
3
Scheme 1. Method A: (i) SO2Cl2/diethyl ether, 24 h; (ii) H2O, reflux, 30 min; Method B: (i) SO2Cl2/diethyl ether/MW, 500 W, 50 ꢀC, 1 h,; (ii) H2O, MW, 500 W, 120 ꢀC, 30 min.
designed for organic synthesis, was used (ETHOS 1600, Milestone)
and all reactions were performed in closed vessels, monitoring the
temperature of the reaction mixtures directly by a microwave-
transparent fluoroptic probe. We found the optimal microwave
conditions to be a ramp of 1 min of irradiation at 500 W and 50 ꢀC,
followed by 60 min at the same conditions. The obtained chlori-
nated intermediate, isolated by filtration, was then irradiated for
30 min at 500 W and 120 ꢀC. The microwave approach afforded the
desired compounds in slightly better yields than those obtained
both by standard described procedure and by conventional heat-
ing; moreover the overall times for the synthesis were considerably
reduced from more than 24 h to 1 and half hour. The obtained re-
sults are summarized in Table 1.
micromolar range (see Table 2). In this experimental setup, exog-
enous thiols were not added because the intracellular concentra-
tions of GSH and Cys, that are respectively 1e10 mM and
30e200
Considering the results obtained from both chemical and biological
experiments, compound THIA 3 (Cmax 65.4 1.1 M and EC50
1.7 M) was selected for further studies. In particular, THIA 3-
mM [26], are sufficient to promote the release of H2S.
m
m
induced vasorelaxation has been tested also in isolated aortic rings
deprived of the endothelium. As shown in Fig. 1C, the removal of
endothelium did not modify THIA 3-induced relaxation suggesting
that its vasodilating activity was due exclusively to its ability to
release H2S, that in turn acts on smooth muscle component of the
vessels.
In order to characterize the potential H2S-releasing properties of
1,2,4-thiadiazolidine-3,5-diones, all compounds were added at
1 mM concentration to the assay buffer. The generation of H2S was
evaluated by an amperometric approach, allowing to have a real-
time determination of the H2S-release and thus to perform a
qualitative/quantitative description of the process. The experi-
On the basis of these data, the series here described, even if too
small to perform a complete structure-activity relationships study,
allowed to do some general considerations. In particular, the elec-
tronic effect of the R0 substituent seems to be more important than
that of the R substituent. This might be related to the close prox-
imity to the sulfur atom that has been hypothesized to be primarily
involved in the H2S release molecular mechanism. All the de-
rivatives embodying electron releasing substituents in R’ (THIA
4e9) showed in fact a slower H2S release (TCM50 ranging between
13.25 and 15.36 min) when compared to the parent donor (THIA 3;
TCM50 12.01 min); on the contrary, the 4-F substituted derivative,
THIA 10, showed a faster H2S release (TCM50 9.52 min). Our results
are in accordance with those obtained by Zhao et al. with N-mer-
capto based H2S donors [15,16].
ments were also performed in the presence of 4 mM L-cysteine. The
parameters of Cmax (maximal concentration of H2S at the steady
state) and TCM50 (time required to reach a concentration ¼ 50% of
Cmax) of the tested compounds are listed in Table 2. Differently from
THIA 1 and 2, the other synthesized compounds were found to be
stable in buffers not releasing H2S spontaneously. The incubation of
THIA 3e10, in fact, in the assay buffer led to a negligible release of
H2S (Cmax < 2
mM; Table 2). In contrast, in the presence of L-cysteine,
all these compounds exhibited a slow and significant release of H2S
Furthermore, to investigate the mechanism of H2S generation, a
mass spectrometric approach was employed to identify the reac-
tion products of THIA 3 and thiols. Glutathione (GSH) is the most
abundant among the cellular thiols, reaching millimolar concen-
trations in most cells. Therefore, we thought of prime interest to
study the reaction between THIA 3 and GSH. However, because of
the structural complexity of reaction product, we sought to pre-
liminarily study the reaction between THIA 3 and cysteine to
assess, with a simpler product, the mechanistic features of the re-
action, and successively address by way of analogy those of the
with Cmax parameters ranging between 13 and 65
values ranging between 12 and 16 min (Table 2).
mM and TCM50
The evaluation of the vasodilating properties of 1,2,4-
thiadiazolidine-3,5-diones has been performed by a cumulative
concentration response curves of THIA 3e10 (10 nM - 300 mM) on
isolated aortic rings. All the compounds exerted vasodilating ac-
tivity in a concentration dependent manner (Fig. 1AeB).
In particular all the compounds reached an Emax of about 100% at
the concentration of 100
mM and showed EC50 values within
Table 2
Parameters of Cmax and TCM50 of the compounds THIA 1e10 (1 mM) were determined after incubation in the assay buffer at physiological pH and temperature, in the absence
or in presence of 4 mM
L
-Cysteine; nc ¼ Not Calculated. Emax represents the maximal effect obtained. EC50 was calculated after performing cumulative concentration-curves for
each compound (10nM-300
m
M) or vehicle (DMSO) on isolated aortic rings precontracted with PE (1
mM).
Compound
H2S-release H2S-release
Emax
EC50 (mM)
without -cysteine -cysteine 4 mM
L
þ
L
Cmax M) Cmax M)
(
m
TCM50 (min)
(
m
TCM50 (min)
THIA 1
THIA 2
THIA 3
THIA 4
THIA 5
THIA 6
THIA 7
THIA 8
THIA 9
THIA 10
2.38 0.36
5.49 0.03
9.56 0.84
13.46 0.72
2.43 0.34
5.91 0.72
65.39 1.14
37.49 3.09
38.81 0.80
31.07 0.26
35.70 3.85
36.26 5.27
19.43 1.20
13.44 1.30
11.40 0.52
15.75 1.42
12.01 0.63
13.25 0.78
13.25 0.02
14.80 0.77
15.36 1.27
13.93 1.26
13.84 0.83
9.52 0.32
e
e
e
e
<2
<2
<2
<2
<2
<2
<2
<2
n.c.
n.c.
n.c.
n.c.
n.c.
n.c.
n.c.
n.c.
99.7 0.33
100 0.10
99.0 1.0
95.0 5.0
98.6 1.4
97.7 1.2
94.5 4.3
94.3 5.7
1,7
1,6
1,5
3,0
2,1
1,9
1,3
3,9
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B. Severino et al. / European Journal of Medicinal Chemistry xxx (2017) 1e10
A
vehicle
0
25
3
4
5
6
50
75
100
***
-8
-7
-6
-5
-4
-3
THIA (log M)
B
0
25
vehicle
7
8
9
50
10
75
***
100
-8
-7
-6
-5
-4
-3
THIA (log M)
C
0
25
50
75
vehicle
with endothelium
***
100
without endothelium
-8
-7
-6
-5
-4
-3
THIA 3 (log M)
Fig. 1. A-B Vasorelaxing effect of THIAs on endothelium-intact aortic rings (n ¼ 4; *** ¼ p < 0.001 vs. vehicle). C Vasorelaxing effect of THIA 3 on aortic rings in presence and absence
of endothelium (n ¼ 4; *** ¼ p < 0.001 vs vehicle).
reaction between THIA 3 and GSH.
Stopping the reaction after 30 min, we isolated by HPLC compound
a that was thoroughly characterized by 1H and 13C NMR, and ESI-
MS (for the complete characterization see Supplementary data).
The MS analysis has been particularly useful in elucidating the
chemical structure of intermediate a. Its MS2 fragmentation pattern
shows various fragments of which the most intense were at m/z 273
and 271, originating by neutral losses of 119 amu (likely PhNCO)
and 121 amu (Cys), respectively (Fig. 3, panel A). The structure of
MS2 fragment at m/z 273 may be PhNHCOSSCys$Hþ. Such possi-
bility is supported by the MS3 pattern of the MS2 fragment at m/z
273 (Fig. 3, panel B), showing three fragments at m/z 227, 154, 152,
which were further subjected to MS4 fragmentation in order to
have an insight of their structures (Fig. S1, Supplementary data).
The MS3 fragmentation pattern of the MS2 fragment at m/z 271
A model reaction using THIA 3 and cysteine (1 eq) was studied.
Using a combination of HPLC, MS and NMR we were able to isolate
and characterize three reaction products: 2-amino-3-((phenyl(-
phenylcarbamoyl)carbamoyl)disulfanyl)propanoic acid (interme-
diate a, Scheme 2), cystine (b) and 1,3-diphenylurea (d). Based on
these isolated compounds, H2S release mechanism was defined as
reported in Scheme 2. The reaction starts with the thiol group of
cysteine that gives a nucleophilic attack on the electron deficient
sulfur atom of the 1,2,4-thiadiazolidine-3,5-dione nucleus opening
the ring and producing intermediate a.
Monitoring the reaction by HPLC/MS we found that after 5 min,
there was a 13% of intermediate a that became 74% after 30 min
(Fig. 2). A complete conversion of a in d was observed after 60 min.
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B. Severino et al. / European Journal of Medicinal Chemistry xxx (2017) 1e10
5
Scheme 2. Proposed mechanism of H2S production.
(Fig. 3, panel C) turned out to be identical to the MS2 fragmentation
pattern of THIA 3. No reaction was observed when THIA 3 was
treated in the same experimental conditions but with S-methyl-
cysteine in place of cysteine, proving the selectivity of the thiol
nucleophilic attack. When intermediate a was treated with water,
no reaction was observed (Scheme 2); on the contrary, when
cysteine was added to the compound, this latter evolved to 1,3-
diphenylurea, as verified by HPLC-MS. Thus, we can state that a
second molecule of cysteine, through a classical thioledisulfide
interchange mechanism, reacts with intermediate a and generates
phenyl(phenylcarbamoyl)carbamothioic S-acid (c, not isolated) and
cystine, b. The instable intermediate c is hydrolyzed yielding 1,3-
diphenylurea (d), CO2 and H2S.
signals related to cysteine and diphenylureic residues linked
through the disulfide bridge; in fact the signals at 4.19, 3.41 and
3.24 of the cysteine residue in the HMBC spectrum (Fig. S5, Sup-
plementary data) in CD3OD correlated exclusively with the
d
carbonyl at
not detect any correlation with carbonyl at
Additionally, when the proton spectrum of a was recorded in
DMSO-d6, a signal at 10.25 (bs, NH) appeared (data not shown),
d
170.0 (carboxylic group of cysteine) whereas we did
d
173.4 (CO-S-S-Cys).
d
which did not display any correlations in the HSQC spectrum. These
NMR data further supported the nucleophilic attack that the thiol
group of cysteine gives on the sulfur atom of the 1,2,4-
thiadiazolidine-3,5-dione nucleus resulting in ring opening and
intermediate a producing.
Similarly, glutathione reacts with THIA 3 forming a compound
with a molecular weight of 577 amu. This product was subjected to
mass spectrometric analysis highlighting that glutathione, in
analogy with cysteine, reacts with THIA 3 forming a disulfide
bridge between the sulfur atoms of THIA 3 and that of the cysteine
residue of glutathione (Fig. S2, Supplementary data).
The structure of the intermediate a, isolated by HPLC, has been
confirmed also by NMR experiments. The 13C NMR (Fig. S3, Sup-
plementary data) and HSQC (Fig. S4, Supplementary data) spectra
of compound a in CD3OD revealed 17 carbons, thus confirming the
mass data, and indicated the presence of one sp3 methylene, one
sp3 methine, ten sp2 methines and five unprotonated carbons (two
quaternary carbons, dC 136.5 and 138.4, and three carbonyl groups
dC 151.6, 170.0 and 173.4). The combined NMR and mass spectral
data supported a molecular formula of C17H17N3O4S2 for interme-
diate a, which implied 11 degrees of unsaturation. Analysis of NMR
spectra of the intermediate a revealed the presence of two set of
3. Conclusion
In this work, we have described the 1,2,4-thiadiazolidine-3,5-
dione scaffold as a novel thiol-triggered H2S-releasing moiety.
The observation that THIA 1 and 2 were able to spontaneously
release H2S, prompted us to prepare other derivatives aiming to
obtain a more controllable H2S production. Compounds THIA 3e10
were stable in buffer and showed a cysteine-dependent ability to
release H2S as measured by an amperometric approach. THIA 3
resulted as the best H2S donor, in terms of Cmax, and all the
attempted substitutions determined a reduction in the H2S pro-
duction. The R0 substituent influences the rate of release; in
particular, the introduction of electron donating groups (-OCH3,
-CH3), compared to an electron withdrawing group (-F) or to the
unsubstituted derivative (THIA 3), slowed the H2S production.
Then, the influence of THIAs on the vasoconstrictor effects of PE
Please cite this article in press as: B. Severino, et al., 1,2,4-Thiadiazolidin-3,5-diones as novel hydrogen sulfide donors, European Journal of
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B. Severino et al. / European Journal of Medicinal Chemistry xxx (2017) 1e10
Fig. 2. HPLC and MS profiles of the products derived from THIA 3. Panel A shows the HPLC profiles of the reaction among THIA 3 and cysteine at to (black), t5 (green), t30 (blue) and
60 (red). Panel B shows the MS spectrum of the isolated intermediate a (Scheme 2), 2-amino-3-((phenyl(phenylcarbamoyl)carbamoyl)disulfanyl)propanoic acid. Panel C shows the
t
MS spectrum of the final product d (Scheme 2), 1,3-diphenylurea. Panel D shows the MS spectrum of the starting compound, THIA 3. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
was tested in isolated mouse aortic rings (Fig.1, panels A and B). The
presence of THIA 3e10 (1 mM) completely abolished the ability of
PE to evoke any vasoconstriction (Emax > 94%).
Aldrich. Melting points, determined using a Buchi Melting Point B-
540 instrument, are uncorrected and represent values obtained on
recrystallized or chromatographically purified material. 1H and 13
C
An original part of this paper is represented by the investigation
on the molecular mechanism responsible for the H2S release. We
have clearly demonstrated the route depicted in Scheme 2 isolating
by HPLC and characterizing by MS and NMR the intermediate a and
the final compound d. These results propose 1,2,4-thiadiazolidine-
3,5-dione, an already known scaffold present in several pharma-
cologically active compounds [19,20], as a novel H2S releasing
moiety whose properties could be modulated by opportune sub-
stitutions on the aromatic rings.
NMR spectra were recorded on Varian Mercury Plus 400 MHz in-
strument. Unless otherwise stated, all spectra were recorded in
CDCl3. Chemical shifts are reported in ppm. The following abbre-
viations are used to describe peak patterns when appropriate: s
(singlet), d (doublet), t (triplet), m (multiplet). Mass spectra of the
final products were performed on API 2000 Applied Biosystem
mass spectrometer. All reactions were followed by TLC, carried out
on Merck silica gel 60 F254 plates with fluorescent indicator and
the plates were visualized with UV light (254 nm). Preparative
chromatographic purifications were performed using silica gel
column (Kieselgel 60). Solutions were dried over Na2SO4 and
concentrated with a Buchi R-114 rotary evaporator at low pressure.
Homogeneity of the products was assessed by analytical reversed-
phase HPLC on a Shimadzu-10 ADsp HPLC system using a Phe-
4. Experimental
4.1. Chemistry
nomenex Kinetex XB-C18 column, 5
m
m, 4.6 ꢁ 250 mm and
All reagents were commercial products purchased from Sigma
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B. Severino et al. / European Journal of Medicinal Chemistry xxx (2017) 1e10
7
Fig. 3. Mass spectra of the compound formed by reaction between THIA 3 and cysteine. (A) MS2 fragmentation pattern of the protonated compound at m/z 392.1 (intermediate a,
Scheme 1). The putative compound structure is reported with indication of the fragmentation points generating the two MS2 major fragments at m/z 271 and m/z 273. (B) MS3
fragmentation pattern by isolating the MS2 fragment at m/z 273. (C) MS3 fragmentation pattern by isolating the MS2 fragment at m/z 271.
applying the following gradient of CH3CN in 0.1% aqueous TFA, from
0% to 100% in 25 min, at a flow rate of 1 mL/min. Analytical RP-HPLC
indicated a purity >98%. Analyses indicated by the symbols of the
elements were within 0.4% of the theoretical values.
4.1.3. 4-Benzyl-2-phenyl-1,2,4-thiadiazolidine-3,5-dione (THIA 1)
[20,21]
Compound THIA 1 was prepared from benzyl-isocyanate (1 g,
6.7 mmol) and phenyl-isothiocyanate (0.905 g, 6.7 mmol) accord-
ing to the procedures described above: mp 119.1e120 ꢀC;1H NMR
(400 MHz, CDCl3)
d
4.91 (s, 2H, N-CH2), 7.28 (d, 1H, Ar-H, J ¼ 7.43),
4.1.1. General procedure for the preparation of 1,2,4-thiadiazoline-
3,5-diones (THIA 1e10)
7.34e7.38 (m, 3H, Ar-H), 7.41 (t, 2H, Ar-H, J ¼ 7.43, J ¼ 8.61), 7.50 (d,
4H, Ar-H, J ¼ 7.82);13C NMR (400 MHz, CDCl3)
d 46.2, 123.4, 127.0,
A three-necked flask was equipped with a mechanical stirrer
and charged with an ice-cold solution of corresponding isothiocy-
anate (1eq) and isocyanate (1eq) in anhydrous diethyl ether. Sul-
furyl chloride (1eq), dissolved in a minimum volume of anhydrous
diethyl ether, was added dropwise under inert atmosphere, keep-
ing the mixture at 0 ꢀC. After completion of the addition, the so-
lution was stirred at room temperature for 24 h. The obtained solid
was filtered, dissolved in 10 mL of water and vigorously stirred
under reflux for 30 min. The reaction mixture was gradually cooled
to 0 ꢀC and the solid was recrystallized from water, filtered and
dried to yield the pure product as a white solid. The synthetic
procedure is depicted in Scheme 1.
128.4, 128.8, 129.1, 129.5, 135.0, 135.7, 151.0, 165.1.
ESI-MS (M þ H)þ m/z calcd 284.3 for C15H12N2O2S; found 285.3.
Anal. (C15H12N2O2S), C, H, N.
4.1.4. 2-Benzyl-4-(4-methoxyphenyl)-1,2,4-thiadiazoline-3,5-dione
(THIA 2) [20]
Compound THIA 2 was prepared from benzyl-isothiocyanate
(1 g, 6.7 mmol) and 4-methoxyphenyl-isocyanate (1 g, 6.7 mmol)
according to the procedures described above: mp 113.7e114.5 ꢀC;
1H NMR (400 MHz, CDCl3)
d
3.81 (s, 3H, OCH3), 4.90 (s, 2H, N-CH2),
6.92 (d, 2H, OCH3, J ¼ 8.60), 7.34e7.39 (m, 5H, Ar-H), 7.50 (d, 2H, Ar-
H); 13C NMR (400 MHz, CDCl3)
46.2, 55.6, 114.7, 126.2, 128.1, 128.4,
d
128.8, 129.1, 135.1, 151.4, 158.8, 165.4.
4.1.2. General procedure for the microwave assisted preparation of
1,2,4-thiadiazoline-3,5-diones (THIA 1e10)
ESI-MS (M þ H)þ m/z calcd 314.36 for C16H14N2O3S; found 315.0.
Anal. (C16H14N2O3S), C, H, N.
A solution of the corresponding isothiocyanate (1eq) and iso-
cyanate (1eq) in anhydrous diethyl ether was placed in a closed
reaction vessel and treated with sulfuryl chloride (1eq) at room
temperature. The mixture was then irradiated according to the
following parameters: initial power, 500 W; initial time, 1 min
(ramping); final power, 500 W; T, 50 ꢀC; reaction time, 1 h. After
cooling, the obtained solid was filtered, dissolved in 10 mL of water
and placed in a closed reaction vessel applying the following pa-
rameters: initial power, 500 W; initial time, 1 min (ramping); final
power, 500 W; T, 120 ꢀC; reaction time, 30 min.
4.1.5. 2,4-Diphenyl-1,2,4-thiadiazoline-3,5-dione (THIA 3) [22]
Compound THIA 3 was prepared from phenyl-isothiocyanate
(1 g, 7.4 mmol) and phenyl-isocyanate (0.881 g, 7.4 mmol) ac-
cording to the procedures described above: mp 119.1e120 ꢀC; 1H
NMR (400 MHz, CDCl3)
d
7.31 (t, 2H, Ar-H, J ¼ 7.04, J ¼ 6.65), 7.46 (t,
4H, Ar-H, J ¼ 12.52, J ¼ 13.78), 7.55 (d, 2H, Ar-H, J ¼ 7.82), 7.58 (d,
2H, Ar-H, J ¼ 7.43); 13C NMR (400 MHz, CDCl3)
d 123.5, 127.2, 127.4,
The reaction mixture was gradually cooled to 0 ꢀC and the ob-
tained solid was recrystallized from water, filtered and dried to
yield the pure product as a white solid.
129.4, 129.5, 129.6, 132.6, 135.8, 150.4, 164.7.
ESI-MS (M þ H)þ m/z calcd 270.31 for C14H10N2O2S; found 271.1.
Anal. (C14H10N2O2S), C, H, N.
Please cite this article in press as: B. Severino, et al., 1,2,4-Thiadiazolidin-3,5-diones as novel hydrogen sulfide donors, European Journal of
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8
B. Severino et al. / European Journal of Medicinal Chemistry xxx (2017) 1e10
4.1.6. 4-(4-methoxyphenyl)-2-phenyl-1,2,4-thiadiazoline-3,5-dione
(THIA 4) [23]
4.1.11. 2-(4-methylphenyl)-4-phenyl-1,2,4-thiadiazolidine-3,5-
dione (THIA 9) [23]
Compound THIA 4 was prepared from phenyl-isothiocyanate
(1 g, 7.4 mmol) and 4-methoxyphenyl-isocyanate (1.103 g,
7.4 mmol) according to the procedures described above: mp
Compound THIA 9 was prepared from phenyl-isocyanate (1 g,
7.4 mmol) and p-tolyl-isothiocyanate (0.985 g, 7.4 mmol) according
to the procedures described above: mp 117.3e118.9 ꢀC; 1H NMR
155.3e155.4 ꢀC; 1H NMR (400 MHz, CDCl3)
d
3.83 (s, 3H, OCH3),
(400 MHz, CDCl3)
d
4.91 (s, 3H, CH3), 7.26 (d, 1H, Ar-H, J ¼ 6.65),
6.95 (d, 2H, Ar-H, J ¼ 9.0), 7.43e7.47 (m, 5H, Ar-H), 7.52 (d, 2H, Ar-H,
7.29e7.43 (m, 4H, Ar-H), 7.51 (d, 4H, Ar-H, J ¼ 7.43); 13C NMR
J ¼ 7.43); 13C NMR (400 MHz, CDCl3)
d
55.6,114.8,126.3,127.3,128.1,
(400 MHz, CDCl3) d 21.04, 123.9, 127.4, 129.3, 129.4, 130.1, 132.6,
129.3, 129.4, 132.7, 150.8, 158.9, 165.0.
133.0, 137.5, 150.5, 164.9.
ESI-MS (M þ H)þ m/z calcd 300.33 for C15H12N2O3S; found 301.1.
ESI-MS (M þ H)þ m/z calcd 284.3 for C15H12N2O2S; found 285.2.
Anal. (C15H12N2O2S), C, H, N.
Anal. (C15H12N2O3S), C, H, N.
4.1.12. 2-(4-fluorophenyl)-4-phenyl-1,2,4-thiadiazolidine-3,5-dione
(THIA 10)
Compound THIA 10 was prepared from phenyl-isocyanate (1 g,
7.4 mmol) and p-fluoro-phenyl-isothiocyanate (1.02 g, 7.4 mmol)
4.1.7. 4-(4-methoxyphenyl)-2-p-tolyl-1,2,4-thiadiazoline-3,5-dione
(THIA 5)
Compound THIA 5 was prepared from p-tolyl-isothiocyanate
(1 g, 6.7 mmol) and 4-methoxyphenyl-isocyanate (1 g, 6.7 mmol)
according to the procedures described above: mp 156.3e156.5 ꢀC;
according
143.8e145.0 ꢀC;1H NMR (400 MHz, CDCl3)
J ¼ 8.61), 7.43 (d, 2H, Ar-H, J ¼ 8.61), 7.48 (d, 2H, Ar-H, J ¼ 7.04);
7.52e7.56 (m, 4H, Ar-H); 13C NMR (400 MHz, CDCl3)
116.5, 116.7,
to
the
procedures
described
above:
mp
1H NMR (400 MHz, CDCl3)
d 2.41 (s, 3H, -CH3), 3.83 (s, 3H, -OCH3),
d
7.14 (t, 1H, Ar-H,
6.95 (d, 2H, Ar-H, J ¼ 9.0), 7.32 (s, 4H, Ar-H), 7.45 (d, 2H, Ar-H,
J ¼ 9.0); 13C NMR (400 MHz, CDCl3)
d 21.2, 55.6, 114.8, 126.2,
d
126.0, 126.1, 127.3, 129.5, 131.5, 132.5, 150.6, 160.0, 162.5.
ESI-MS (M þ H)þ m/z calcd 288.3 for C14H9FN2O2S; found 289.0.
Anal. (C14H9FN2O2S), C, H, N.
127.1, 128.2, 130.1, 139.5, 150.9, 158.8.
ESI-MS (M þ H)þ m/z calcd 314.36 for C15H12N2O3S; found 315.1.
Anal. (C15H12N2O3S), C, H, N.
4.2. Determination of H2S
4.1.8. 2,4-bis(4-methoxyphenyl)-1,2,4-thiadiazoline-3,5-dione
(THIA 6)
The characterization of the potential H2S-generating properties
of the tested compounds has been carried out by amperometric
approaches, through an Apollo-4000 Free Radical Analyzer (WPI)
detector and H2S-selective minielectrodes (ISO-H2S-2, WPI)
endowed with gas-permeable membranes. The experiments were
carried out at room temperature. Following the manufacturer's
instructions, a “PBS buffer 10x” was prepared (NaH2PO4.H2O 1.28 g,
Na2HPO4.12H2O 5.97 g, NaCl 43.88 g in 500 mL H2O) and stocked at
4 ꢀC. Immediately before the experiments, the “PBS buffer 10x” was
diluted in distilled water (1:10), to obtain the assay buffer (AB); pH
was adjusted to 7.4. The H2S-selective minielectrode was equili-
brated in 2 mL of the AB, until the recovery of a stable baseline.
Compound THIA 6 was prepared from 4-methoxyphenyl-iso-
thiocyanate (1 g, 6.1 mmol) and 4-methoxyphenyl-isocyanate
(0.902 g, 6.1 mmol) according to the procedures described above:
mp 177.8e177.3 ꢀC; 1H NMR (400 MHz, CDCl3)
d 3.83 (s, 3H, -OCH3),
3.84 (s, 3H, -OCH3), 6.95 (d, 2H, Ar-H, J ¼ 7.04), 7.01 (d, 2H, Ar-H,
J ¼ 7.43), 7.34 (d, 2H, Ar-H, J ¼ 6.65), 7.45 (d, 2H, Ar-H, J ¼ 7.05);
13C NMR (400 MHz, CDCl3)
d 55.5, 55.6, 114.7, 114.8, 125.3, 126.2,
128.2, 128.6, 151.0, 158.8, 160.0, 165.2.
ESI-MS (M þ H)þ m/z calcd 330.36 for C16H14N2O4S; found 331.1.
Anal. (C16H14N2O4S), C, H, N.
4.1.9. 2-(4-chlorophenyl)-4-(4-methoxyphenyl)-1,2,4-
thiadiazoline-3,5-dione (THIA 7)
Then, 20 mL of a dimethyl sulfoxide (DMSO) solution of the tested
H2S-releasing compounds was added (final concentration of the
tested H2S-donors 1 mM; final concentration of DMSO in the AB
1%). The eventual generation of H2S was observed for 30 min. When
Compound THIA 7 was prepared from 4-chlorophenyl-isothio-
cyanate (1 g, 5.9 mmol) and 4-methoxyphenyl-isocyanate (0.880 g,
5.9 mmol) according to the procedures described above: mp
required by the experimental protocol, L-Cysteine 4 mM was added,
182.7e183.4 ꢀC; 1H NMR (400 MHz, CDCl3)
d 3.83 (s, 3H, OCH3),
before the H2S-donors. The correct relationship between the
amperometric currents (recorded in pA) and the corresponding
concentrations of H2S was determined by opportune calibration
curves, which were previously obtained by the use of increasing
6.95 (d, 2H, Ar-H, J ¼ 8.61), 7.40 (d, 2H, Ar-H, J ¼ 8.61), 7.44 (d, 2H,
Ar-H, J ¼ 7.04), 7.49 (d, 2H, Ar-H, J ¼ 8.61); 13C NMR (400 MHz,
CDCl3)
d 55.6, 114.9, 126.4, 127.8, 128.6, 129.6, 131.1, 135.2, 150.5,
159.0, 164.8.
concentrations of NaHS (1 mM, 3 mM, 5 mM, 10 mM) at pH 4.0.
ESI-MS (M þ H)þ m/z calcd 334.78 for C15H11ClN2O3S; found
The curves relative to the progressive increase of H2S vs time,
following the incubation of the tested compounds, were analysed
by computer fitting procedure (software: GraphPad Prism 6.0). The
parameter of Cmax (the highest concentration recorded during the
335.8.
Anal. (C15H11ClN2O3S), C, H, N.
4.1.10. 2-Cyclohexyl-4-(4-methoxyphenyl)-1,2,4-thiadiazoline-3,5-
recording time) and TCM50 (time required to reach
a
dione (THIA 8)
concentration Cmax were calculated and expressed as
¼
½
)
Compound THIA
8
was prepared from cyclohexyl-
mean standard error from 5 different experiments. ANOVA and
Student t-test were selected as statistical analysis, P < 0.05 was
considered representative of significant statistical differences.
isothiocyanate (1 g, 7.1 mmol) and 4-methoxyphenyl-isocyanate
(1.06 g, 7.1 mmol) according to the procedures described above: mp
98.8e99.7 ꢀC; 1H NMR (400 MHz, CDCl3)
d 1.18e1.41 (m, 5H,
cyclohexane: (CH2)3), 1.67 (d, 1H, cyclohexane), 1.78e1.88 (dd, 2H,
cyclohexane: CH2), 2.23e2.32 (q, 2H, cyclohexane: CH2), 3.81 (s, 3H,
OCH3), 4.17e4.23 (qt, 1H, cyclohexane-CH), 6.92 (d, 2H, Ar-H,
J ¼ 11.7), 7.37 (d, 2H, Ar-H, J ¼ 8.7); 13C NMR (400 MHz, CDCl3)
4.3. Tissue preparation
Male CD-1 mice (8e10 weeks of age) were purchased from
Charles River (Italy). Animals were kept in animal care facility un-
der controlled temperature, humidity and light/dark cycle and with
food and water ad libitum. Animal were sacrificed and thoracic
aorta was rapidly dissected and cleaned from fat and connective
d
24.9, 25.9, 28.8, 55.5, 55.6, 114.6, 126.2, 128.3, 151.5, 158.7, 165.4.
ESI-MS (M þ H)þ m/z calcd 306.38 for C15H18N2O3S; found 307.1.
Anal. (C15H18N2O3S), C, H, N.
Please cite this article in press as: B. Severino, et al., 1,2,4-Thiadiazolidin-3,5-diones as novel hydrogen sulfide donors, European Journal of
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B. Severino et al. / European Journal of Medicinal Chemistry xxx (2017) 1e10
9
tissue. Rings of 1.5e2 mm length were cut and mounted in isolated
organ bath filled with oxygenated Krebs solution (95% O2þ 5% CO2)
at 37 ꢀC. The rings were connected to an isometric transducer (type
7006, Ugo Basile, Comerio, Italy) and changes in tension were
recorded continuously with a computerized system (Data Capsule
17400, Ugo Basile, Comerio, Italy). The composition of the Krebs
solution was as follows (mol/l): NaCl 0.118, KCl 0.0047, MgCl2
0.0012, KH2PO4 0.0012, CaCl2 0.0025, NaHCO3 0.025 and glucose
0.010. Rings were initially stretched until a resting tension of 1.5 g
was reached and allowed to equilibrate for at least 40 min during
which tension was adjusted, when necessary, to 1.5 g and bathing
solution was periodically changed. In a preliminary study a resting
tension of 1.5 g was found to develop the optimal tension to
stimulation with contracting agents.
drying gas under the following conditions: nebulizer pressure, 30
psi; drying temperature, 350 ꢀC and drying gas, 7 L/min. The ion
charge control (ICC) was applied with the target set at 30000 and
maximum accumulation time at 20 ms. Optimization of instru-
mental parameters for analyses of the compounds under study was
performed by continuous infusion of 5 mM standard solution in 0.1%
formic acid. Mass cutoff and amplitude were optimized to obtain
the most efficient MS/MS transitions from the positively charged
precursor ion [MþH]þ to the fragment ions. Measurements were
performed from the peak area of the extracted ion chromatogram
(EIC). Retention times and peak areas of the monitored fragment
ions were determined by the Agilent software Chemstation version
4.2.
4.7. NMR analysis
4.4. Isolated aorta rings
1H (500 MHz) and 13C (125 MHz) NMR spectra were recorded on
a Agilent INOVA spectrometer; chemical shifts were referenced to
the residual solvent signal (CD3OD: dH ¼ 3.31, dC ¼ 49.0). HSQC
In each experiment rings were firstly challenged with
L-phen-
ylephrine (PE) 1 M and bathes solution changed until the con-
m
1
tractile response was reproducible. To test the integrity of the
endothelium, acetylcholine (Ach) cumulative concentration-curve
experiments, multiplicity-edited, optimized for a JC,H of 140 Hz.
Two and three bonds 1H-13C connectivities were determined by
gradient 2D HMBC experiments optimized for a2,3J of 8 Hz.
(10 nM-30
mM) was performed on stable tone induced by PE
1
mM. When necessary, to obtain endothelium-denuded rings, the
endothelial layer was removed by gently rubbing the internal sur-
face of the vascular lumen. To test the selected donors, rings, that
Acknowledgment
were stabilized as described above, were contracted with PE (1
and once a stable tone was reached, cumulative concentration-
mM)
This work was supported by the Italian Ministry of University
and Research (MIUR, PRIN 2010e2011 prot. nꢀ 2010Y4WMCR_002).
curves for each compound (10nM-300mM) or vehicle (DMSO)
were performed.
Appendix A. Supplementary data
4.5. Product analysis
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2017.10.068.
THIA 3 (150 mg, 0.5 mmol) in 2 mL of MeOH was added to a
stirred solution of -cysteine (60.5 mg, 0.5 mmol) in 50 mL of PBS
L
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Please cite this article in press as: B. Severino, et al., 1,2,4-Thiadiazolidin-3,5-diones as novel hydrogen sulfide donors, European Journal of
Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.068